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. 2020 Jun 24;11(7):1410–1415. doi: 10.1021/acsmedchemlett.0c00090

Amidate Prodrugs of O-2-Alkylated Pyrimidine Acyclic Nucleosides Display Potent Anti-Herpesvirus Activity

Min Luo †, Elisabetta Groaz †, Robert Snoeck §, Graciela Andrei §, Piet Herdewijn †,*
PMCID: PMC7359042  PMID: 32676147

Abstract

graphic file with name ml0c00090_0006.jpg

Three series of amidate prodrugs of O-2-alkylated acyclic nucleosides of the 3-fluoro-2-(phosphonomethoxy)propyl (FPMP), cyclic 3-hydroxy-2-(phosphonomethoxypropyl) (cHPMP), and 2-(phosphonomethoxypropyl) (PMP)-type featuring cytosine and 5-fluorocytosine as nucleobases were readily synthesized. Both the aspartic acid ester and valine ester prodrugs of (R)-O-2-alkylated FPMPC exhibited potent anti-HCMV and VZV activity in the micromolar range. In addition, the valine ester prodrugs of 5-fluorocytosine (R)-O-2-alkylated FPMP and (R)-O-2-alkylated cHPMPC showed inhibitory activity at molar concentrations against these viruses.

Keywords: Acyclic nucleoside phosphonates, prodrugs, human cytomegalovirus, varicella zoster virus, antiviral activity

Acyclic nucleoside phosphonates (ANPs) have gained increasing attention in nucleos(t)ide research due to a number of significant advantages, including potent antiviral activity, easy and economical chemical synthesis, and potential as genetic materials.1−4 Extensive investigation of their synthesis and therapeutic properties has led to the marketing approval of three ANPs as antiviral agents (Figure 1).5,6 Cidofovir (HPMPC, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine) is currently being used for the treatment of human cytomegalovirus (HCMV) retinitis in acquired immune deficiency syndrome (AIDS) patients, while adefovir (PMEA, 9-(2-phosphonylmethoxyethyl)adenine) was approved as antihepatitis B virus (HBV) agent. Upon derivatization of the active drug tenofovir (PMPA, (R)-9-(2-phosphonylmethoxypropyl)adenine), two nucleoside phosphonate prodrugs, i.e., tenofovir disoproxil and alafenamide, have been developed and licensed for the treatment of HBV as well as human immunodeficiency virus (HIV) infections.

Figure 1.

Figure 1

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Chemical structures of cidofovir (HPMPC), adefovir dipivoxil, tenofovir alafenamide, (S)-cHPMPA, (S)-cHPMPC, FPMPA, and FPMPC.

The generation of structurally modified ANPs remains an attractive platform for the development of new bioactive compounds (Figure 1).7 Among these, the cyclic forms of HPMP-type nucleosides such as (S)-cHPMPA and (S)-cHPMPC, which were prepared aiming to reduce the toxic side effects of the parent ANPs, retained a remarkable antiviral potency against DNA viruses.8,9 On the other hand, the replacement of the hydroxyl group of HPMC derivatives with a fluorine atom led to 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) nucleoside analogues that lacked anti-DNA virus activity, while being moderately active inhibitors of HIV and HBV.10−12 Similarly, the PMP series characterized by the presence of a methyl group at the acyclic side chain, as exemplified by tenofovir, showed potent activity against retroviruses but were completely devoid of anti-DNA virus activity.12

It should be noted that the majority of reported nucleoside phosphonate analogues belong to the N-nucleoside series, i.e., where a heterocyclic base (purine or pyrimidine) is linked to an acyclic chain through a nitrogen atom (Figure 1). On the other hand, O-alkylated nucleosides, especially O-alkylated cytosine derivatives, were not widely investigated and never commercialized as either anticancer or antiviral agents. The best studied class of O-alkylated acyclic phosphonate nucleosides comprises substituted 4-amino-6-hydroxypyrimidine derivatives as nucleobase moiety (Figure 2).13,14 For instance, 5-methyl derivative B (Figure 2) showed excellent antiviral activity against HIV and Moloney murine sarcoma virus in vitro (EC50 = 0.00016–0.00043 μmol/mL). In addition, 5-halogen-substituted derivatives C (Figure 2) were endowed with pronounced antiretroviral activity with EC50 values in the 0.0023–0.0110 μmol/mL range.

Figure 2.

Figure 2

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4-Amino-6-hydroxypyrimidine (A) and selected examples of 6-O-alkylated acyclic nucleosides (B and C).

Among the various available strategies for nucleotide prodrug design,15 amidate prodrugs have been shown on many occasions to lead to superior antiviral activities by masking the negatively charged phosphate or phosphonate group, which commonly limits the ability of the parent compounds to penetrate the lipid-rich cell membrane, consequently contributing to higher levels of active cellular metabolites.16−19 In particular, we previously demonstrated that the use of l-aspartic acid diamyl ester18 and l-valine amyl ester20 moieties afforded analogues with an enhanced antiviral efficacy.

Herein, we report the discovery of a new family of O-alkylated acyclic nucleoside phosphonates and their amidate prodrugs. For this specific class of molecules, various aliphatic pseudosugar side chains (i.e., FPMP, cHPMP, and PMP) were linked to the 2-O position of a pyrimidine base rather than the 4-O-position.

As shown in Scheme 1, N4-acetylcytosine 1 was successfully condensed under Mitsunobu conditions (Ph3P, DIAD) with 3a and 3b to provide compounds 4a and 4b. The initial enantiomeric fluorinated acyclic phosphonate ester synthons 3a and 3b were synthesized according to a previously reported method.18,21 Subsequent removal of the N4-acetyl protecting group using methanolic ammonia furnished compounds 6a and 6b in 48 and 50% yield, respectively. On the other hand, the alkylation of 5-fluorocytosine 2 with 3a and 3b generated the corresponding triphenylphosphine adducts 5a and 5b in agreement with a previous report,22 which could be successfully deprotected by treatment with 1 M HCl at room temperature to afford the desired products 7a and 7b. All phosphonate esters underwent hydrolysis in the presence of TMSBr to give compounds 8a, 8b, 9a, and 9b in good yields. Phosphonic acids 8a, 8b, 9a, and 9b were then converted to their aryloxyphosphonamidates 10a, 10b, 11a, 11b, 12a, and 12b upon reacting with either l-aspartic acid amyl diester HCl salt or l-valine amyl ester HCl salt and phenol using 2,2′-dithiodipyridine and triphenylphosphine as activating agents. All compounds were isolated as diastereoisomeric mixtures (ratios in the 1/1–1/1.5 range).

Scheme 1. Synthesis of (S)/(R)-Phosphonamidates 10a, 10b, 11a, 11b, 12a, and 12b.

Scheme 1

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Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/MeOH, 45 °C, 15 h, 48–50% over 2 steps; (c) 1 M HCl, CH3CN/H2O 12 h, 30–31% over 2 steps; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 12 h, 65–70%; (e) l-Aspartic acid amyl diester HCl salt or l-valine amyl ester HCl salt, PhOH, 2,2′-dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h, 28–54%.

Subsequent efforts were directed toward the synthesis of phosphonamidates 22a, 22b, 23a, and 23b, as illustrated in Scheme 2. In this case, compounds 13a and 13b were prepared according to the same method used for the synthesis of diethyl alcohols (Scheme S-1, Supporting Information).23 Under Mitsunobu conditions (Ph3P, DIAD), the condensation reaction between 13a and 13b and either nucleobase 1 or 2 occurred smoothly to afford nucleoside phosphonates 14a, 14b and 15a, 15b, respectively. After deprotection of either the acetyl group or triphenylphosphine adduct, standard TMSBr-promoted hydrolysis of the phosphonate diesters afforded compounds 18a, 18b, 19a, and 19b in 70–75% yields. The key aryloxyphosphonamidate intermediates 20a, 20b, 21a, and 21b were then obtained as described above. Lewis-acid assisted removal of the benzyl moiety using boron trichloride and sequential treatment with triethylamine in DCM led to a concomitant cyclization, affording the desired cyclic l-valine amyl ester containing (S)/(R)-phosphonamidates 22a, 22b, 23a, and 23b as diastereoisomeric mixtures (ratios in the 1/1.1–1/1.2 range) in 25–30% yields.

Scheme 2. Synthesis of (S)/(R)-Phosphonamidates 22a, 22b, 23a, and 23b.

Scheme 2

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Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/MeOH, 45 °C, 15 h, 42–53% over 2 steps; (c) 1 M HCl, CH3CN/H2O, 12 h, 31–46% over 2 steps; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 12 h, 70–75%; (e) l-Valine amyl ester HCl salt, PhOH, 2,2′-dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h; (f) (i) BCl3, DCM, −78 to 0 °C, 2 h; (ii) Et3N, DCM, rt, 1 h, 25–30% over 2 steps.

Lastly, phosphonamidates 31a, 31b, 32a, and 32b were easily accessible from nucleobases 1 and 2 and compounds 24a(24) and 24b (Scheme S-2, Supporting Information), as illustrated in Scheme 3. Compounds 27a, 27b, 28a, and 28b were obtained under the established Mitsunobu conditions as diastereoisomeric mixtures (ratios in the 1/1–1/1.3 range), followed by hydrolysis of either the acetyl moiety or triphenylphosphine adduct. Cleavage of the phosphonate ester groups furnished the corresponding phosphonic acids, which were converted to the desired prodrugs.

Scheme 3. Synthesis of (S)/(R)-Phosphonamidates 31a, 31b, 32a, and 32b.

Scheme 3

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Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/MeOH, 45 °C, 15 h, 35% over 2 steps; (c) 1 M HCl, CH3CN/H2O, 12 h, 30–35% over 2 steps; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 12 h, 65–70%; (e) l-Valine amyl ester HCl salt, PhOH, 2,2′-dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h, 40–45%.

Next, we proceeded with the antiviral activity evaluation of all synthesized phosphonamidate prodrugs and their parent phosphonates against HCMV (strains AD-169 and Davis) and VZV [strains (TK+) Oka and thymidine kinase deficient (TK–) 07-1] in human embryonic lung (HEL) cells, along with the assessment of their toxic effects on the same cell line (Table 1). Ganciclovir, cidofovir, acyclovir, and brivudin were also included in this study as reference drugs.

Table 1. Antiviral Activity and Cytotoxicity of Acyclic Nucleoside Phosphonates and Phosphonamidates against HCMV and VZV in HEL Cells.

  Antiviral activity EC50a (μM)
 Cytotoxicity (μM)
  HCMV
 VZV
 Cell morphology (MCC)b Cell growth (CC50)c  
Compound AD-169 strain Davis strain TK+ VZV strain (OKA) TK– VZV strain (07-1) HEL HEL clogPd
8a >100 >100 >100 >100 >100 >100 –0.70
8b 2.36 ± 0.24 0.80 3.79 ± 0.30 25.3 ± 9.75 >100 >100 –0.70
10a 62.8 ± 4.20 48.2 ± 7.60 38.2 ± 7.04 24.7 ± 12.9 >100 >100 6.28
10b 0.54 ± 0.37 0.094 0.36 ± 0.16 0.098 ± 0.003 >100 51.84 6.28
11a >100 7.61 38.6 35.9 >100 NDe 5.25
11b 0.26 ± 0.14 0.097 ± 0.004 0.36 ± 0.035 0.18 ± 0.014 >100 79.3 5.25
12a >100 >100 >100 >100 >100 NDe 5.34
12b 11.2 ± 3.64 4.70 ± 2.76 6.73 ± 3.64 2.94 ± 1.76 >100 >100 5.34
22a >100 >100 >100 >100 >100 NDe 4.00
22b 14.0 ± 1.88 4.76 ± 1.07 53.6 ± 25.1 35.7 ± 13.5 >100 >100 4.00
23a >100 >100 >100 >100 >100 NDe 4.09
23b >100 >100 >100 >100 >100 NDe 4.09
31a >100 >100 >100 >100 >100 NDe 5.38
31b >100 >100 >100 >100 >100 NDe 5.38
32a >100 >100 >100 >100 >100 NDe 5.47
32b >100 >100 >100 >100 >100 NDe 5.47
Ganciclovir 13.0 ± 3.40 4.44 ± 2.23 NDe NDe >394 >394  
Cidofovir 1.74 ± 0.77 0.94 ± 0.86 NDe NDe >317 >317 –2.39
Acyclovir NDe NDe 7.06 ± 3.56 42.7 ± 13.7 >444 >444  
Brivudin NDe NDe 0.089 ± 0.058 0.18 >300 >300  
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a

Effective concentration required to reduce virus-induced cytopathicity (HCMV) or plaque formation (VZV) by 50%.

b

Minimum concentration required to cause a microscopically detectable alteration of cell morphology.

c

Cytotoxic concentration required to reduce cell viability by 50%.

d

cLogP values were calculated using ChemBioDraw Ultra version 14.0 from CambridgeSoft.

e

Not determined.

As discussed earlier, N-alkylated FPMP nucleoside derivatives bearing canonical and modified nucleobases showed no or very weak antiviral activity against HCMV and VZV at subtoxic concentrations.18,25 However, we previously established that the use of a diamyl aspartate phenoxyamidate group as phosphonate prodrug moiety was beneficial for enhancing the antiviral activity of FPMP nucleosides and particularly enlarging their spectrum of activity against herpesviruses.18 In particular, amyl aspartate phosphonamidate prodrugs of purine containing analogues exhibited submicromolar anti-VZV potency, while (S)-aspartate-FPMPC displayed an EC50 value of 0.76 μM against HCMV.

Interestingly, for the O-2-alkylated counterparts, (R)-O-2-alkylated FPMPC 8b showed moderate antiviral activity against both HCMV and VZV with EC50 values in the 0.80–2.36 and 3.79–25.3 μM range, respectively, while its (S)-enantiomer 8a was found to be inactive.

Further derivatization of (R)-O-2-alkylated FPMPC 8b afforded amidate prodrugs 10b and 11b, which exhibited potent antiviral activity against different strains of HCMV and VZV (including a thymidine kinase mutant virus) with EC50 values ranging between 0.094 and 0.54 μM and concomitantly low cytotoxicity and cytostatic effects. These compounds displayed interesting selectivity indices (ratio CC50/EC50), i.e., 96–551 (10b) and 220–818 (11b). In contrast, the corresponding (S)-enantiomers 10a and 11a showed minimal antiviral activity against HCMV and VZV with EC50 values in the range of 7.61 to >100 μM. The l-valine amyl ester prodrug of (R)-O-2-alkylated FPMP carrying 5-fluorocytosine (12b) was found to be moderately active against both HCMV strains with EC50 values in the 2.94–11.2 μM range, while no activity was observed for its (S)-counterpart 12a. It is worth mentioning that the lack of anti-herpesvirus activity was associated with the opposite configuration at the pseudosugar chiral center for the N- and O-alkylated series of prodrug analogues [(R) and (S), respectively]. Moreover, when compared to (R)-O-2-alkylated FPMPC 8b, the corresponding prodrugs 10b and 11b displayed a dramatically improved cellular permeability due to increased lipophilicity, as evidenced by their clogP values (Table 1).

In previous studies, N-alkylated (S)-cHPMPC and (S)-cHPMPA (Figure 1) were synthesized and displayed good antiviral potency against DNA viruses.26,27 It was also demonstrated that their corresponding amidate prodrugs showed excellent antiviral potency against DNA viruses.20 However, the alkylation of cytosine or 5-fluorocytosine at the 2-O-position as in the case of compounds 22a, 22b, 23a, and 23b generally resulted in loss of antiviral activity, except for the prodrug of (R)-O-2-cHPMPC (22b) that proved to be moderately active against both VZV strain mutants expressing either a functional or deficient thymidine kinase with EC50 values of 14.0 and 4.76 μM, respectively.

With regard to the O-2-alkylated nucleoside phosphonates and prodrug analogues of the PMP-type, i.e., compounds 29a, 29b, 30a, 30b, 31a, 31b, 32a, and 32b, no inhibition of HCMV and VZV replication was observed.

In addition, the ability of all synthesized amidate prodrugs to inhibit the replication of HBV was also assessed in the human hepatoblastoma cell line HepG2.2.15, which is a widely used cell line containing two copies of the HBV wild-type strain ayw1 genome and constitutively produces high levels of HBV.28 Real-time qPCR (TaqMan) was used to measure the extracellular HBV DNA copy number associated with virions released from HepG2 2.2.15 cells. A tetrazolium dye uptake assay was employed to measure cell viability and calculate the CC50 values. Only compounds 10b, 11b, and 12b proved moderately active against HBV with EC50 values in the range of 2.10–8.71 μM (Table S1).

The amidate prodrugs of (R)-O-2-alkylated FPMPC, i.e., compounds 10b and 11b, showed promising antiviral activity against HCMV and VZV in vitro. To achieve this efficacy, prodrugs 10b and 11b must efficiently penetrate the target cells and undergo further intracellular metabolism. Previously, we suggested that deoxythreosyl and 3-fluoro-2-(phosphonomethoxy)propyl aryloxyphosphonamidate prodrugs containing a l-aspartate acid diester could be hydrolyzed to afford the nucleoside phosphonates in the cell, which then formed the pharmacologically active diphosphates of phosphonates.18,19 Therefore, these two prodrugs can be expected to generate intracellularly phosphonate 8b, which then undergoes activation to its diphosphate species.

In summary, a number of amidate prodrugs of O-2-alkylated acyclic nucleosides bearing cytosine or 5-fluorocytosine as nucleobase in combination with three structurally diverse aliphatic side chains were synthesized. Such modification proved to be especially successful within the FPMP series, as demonstrated by the potent activity exhibited by the prodrugs of (R)-O-2-alkylated FPMPC against both HCMV and VZV. Furthermore, prodrugs of (R)-O-2-alkylated FPMP containing 5-fluorocytosine and (R)-O-2-cHPMPC showed a moderate ability to inhibit HCMV replication or VZV plaque formation. These results can help guide the development of novel potential drug candidates against DNA viruses.

Acknowledgments

We wish to thank Raj Kalkeri and Roger G. Ptak at the Southern Research Institute for testing against HBV. Testing was conducted using federal funds from the Division of Microbiology and Infectious Diseases (DMID), NIAID/NIH under contract IV19VB12839 entitled “Anti-HBV screening of (R)-O-2-alkylated cytosine Asp-prodrug analogues”. In addition, we are grateful to Ellen De Waegenaere, Brecht Dirix, and Luc Baudemprez for excellent technical assistance.

Glossary

Abbreviations

ANPs

acyclic nucleoside phosphonates

HPMPC

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine)

PMEA

9-(2-phosphonylmethoxyethyl)adenine

PMPA

(R)-9-(2-phosphonylmethoxypropyl)adenine

FPMP

3-fluoro-2-(phosphonomethoxy)propyl

HIV

human immunodeficiency virus

HCMV

human cytomegalovirus

VZV

varicella zoster virus

cHPMPC

cyclic cidofovir

TMSBr

bromotrimethylsilane

HEL

human embryonic lung

TK

thymidine kinase

Val

valine

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00090.

  • Experimental details and characterization data for the reported compounds, NMR spectra, and biological assays (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml0c00090_si_001.pdf (5.2MB, pdf)

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