Article pubs.acs.org/jmc
Synthesis and Antiviral Evaluation of Octadecyloxyethyl Benzyl 9‑[(2Phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG), a Potent Inhibitor of Transient HPV DNA Amplification James R. Beadle,† Nadejda Valiaeva,† Guang Yang,‡ Jei-Hwa Yu,‡ Thomas R. Broker,‡ Kathy A. Aldern,† Emma A. Harden,‡ Kathy A. Keith,‡ Mark N. Prichard,‡ Tracy Hartman,§ Robert W. Buckheit, Jr.,§ Louise T. Chow,‡ and Karl Y. Hostetler*,†,∥ †
University of California, San Diego, La Jolla, California 92093, United States University of Alabama at Birmingham, Birmingham, Alabama 35233, United States § ImQuest BioSciences, Frederick, Maryland 21704, United States ∥ Antiva Biosciences, Inc., South San Francisco, California 94080, United States ‡
S Supporting Information *
ABSTRACT: Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virusassociated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origincontaining plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.
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INTRODUCTION Persistent infection with high-risk human papillomavirus (HPV) genotypes such as HPV-16 and -18 is the cause of 70% of cervical cancers.1 HPV DNA is detectable in more than 99% of cervical cancers.2 Cervical cancer is the third ranked cause of gynecological cancer in the United States, both in diagnosis and in death, with over 12,000 new cases of invasive cervical cancer and 4,000 deaths annually.3 Worldwide cervical cancer-related deaths rank second overall among all cancers in women,4 and an estimated 528,000 new cervical cancers and 266,000 deaths from cervical cancer occurred in 2012.5 It is estimated that 10% of adult women worldwide have an active HPV infection of the cervix (>300 million). The disease burden attributable to HPV in the anogenital tract additionally includes anogenital warts caused primarily by low-risk HPV-6 and HPV11 and high-risk HPV associated anal intraepithelial neoplasia (AIN), vulvar intraepithelial neoplasia (VIN), as well as cancer at these anatomical sites.6−8 HPVs also infect the oropharyngeal epithelia, causing all the recurrent respiratory papillomatosis9 and a fraction of the head and neck cancers.10 Three vaccines have been approved to prevent HPV infection, but these prophylactic vaccines lack efficacy in women who have already been infected with high-risk HPV types. Notably, © 2016 American Chemical Society
there are no approved antiviral therapeutic agents to treat HPV infections of the cervix or lower anogenital tract, which could prevent progression to cancer.11,12 A very significant unmet medical need is to develop more effective antiviral treatment to reduce or eradicate persistent HPV infections. For cervical intraepithelial neoplasia (CIN 1), the earliest stage of high-risk HPV cervical infection, current recommendations call for “watchful waiting”, consisting of repeat PAP smears, colposcopy, and biopsy. Ninety percent of untreated cervical HPV infections resolve in 2 years. However, if not cleared by the immune system, persistent infection by the highrisk HPVs may lead to CIN 2/3 and invasive cervical cancer.13,14 For CIN 2/3, the standard of care involves surgical removal of the lesion via conization of the cervix with LEEP or other extirpative techniques; topical treatments with podofilox or imiquimod, neither of which is an antiviral agent, are only marginally effective.15 Topical cidofovir gel has shown effectiveness in HPV infection of the cervix16 and vulva.17 HPVs do not code for a DNA polymerase, and viral DNA replication relies on host DNA polymerases and other Received: April 28, 2016 Published: November 11, 2016 10470
DOI: 10.1021/acs.jmedchem.6b00659 J. Med. Chem. 2016, 59, 10470−10478
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Figure 1. Structures of PMEG (1) and its monoester and diester prodrugs (2−4).
Scheme 1. Synthesis of PMEG Diester Prodrugs 3 and 4a
a
Reagents and conditions: (a) (1) bromotrimethylsilane, CH3CN, (2) H2O, (3) tributylamine, toluene; (b) 2-octadecyloxyethan-1-ol, PyBOP, DIEA, DMF, rt, 18 h; (c) 80% aq acetic acid, reflux, 5 h; (d) benzyl alcohol (3) or phenol (4), PyBOP, DIEA, rt, 18 h.
replication and a NanoLuc reporter gene. This reporter system greatly facilitated detection of effects on HPV ori-plasmid replication. This paper describes the synthesis of 3 and 4, evaluation of their effects on HPV-11, HPV-16, HPV-18, HIV1, and HSV-2 DNA replication, and characterization of the cellular uptake and activation of compound 3 in human foreskin fibroblast (HFF) cells. Additionally, the P-chiral enantiomers of 3 were obtained by enantioselective HPLC to investigate the impact of chirality on antiviral activity.
replication proteins. The virus encodes protein E2, which binds to the origin of HPV DNA replication (ori) and helps recruit the virus encoded replicative DNA helicase E1 to replicate the double-stranded, circular DNA genome in the nucleus. Transfection into adenovirus E1 transformed-human embryonic kidney (HEK-293) cells of expression vectors of typematched HPV E1 and E2 proteins can support replication and transient amplification (i.e., increase in copy number) of a cotransfected HPV ori-containing plasmid,18 and this system can be used to identify small molecule inhibitors of HPV replication. Using this transient HPV DNA amplification assay, we evaluated several new acyclic nucleoside phosphonate (ANP) diester derivatives for anti-HPV-11, HPV-16, and HPV18 efficacy. Among the screened compounds, two diester analogs of 9-[(2-phosphonomethoxy)ethyl]guanine (PMEG) were found to be potent, selective inhibitors of HPV-11 and -16 DNA amplification in HEK-293 cells. The structures of PMEG and the two active diester analogs, ODE-Bn-PMEG (3) and ODE-Ph-PMEG (4), are shown in Figure 1. The HPVinhibitory effects of compounds 3 and 4 were then confirmed in a separate transient HPV DNA amplification assay utilizing HPV-negative cervical cancer cells (C33A) containing HPV-11, HPV-16, or HPV-18 plasmids containing the origin of
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RESULTS AND DISCUSSION Chemistry. Synthesis of the two diester PMEG prodrugs (3 and 4) is outlined in Scheme 1. The parent phosphonate PMEG and several of its prodrugs have previously been synthesized starting from diisopropyl 9-(2phosphonomethoxy)ethyl]-2-amino-6-chloropurine (5), which is readily available by the method of Holý et al.19 In contrast to earlier descriptions of its conversion to the phosphonic acid, we observed that treatment of diisopropyl phosphonate compound 5 with bromotrimethylsilane yields a mixture of the 6-chloro and 6-bromo purine phosphonates rather than the 6-Cl HBr salt.19 Addition of tributylamine to the 6-Br/Cl mixture followed by recrystallization from toluene provided phospho10471
DOI: 10.1021/acs.jmedchem.6b00659 J. Med. Chem. 2016, 59, 10470−10478
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Table 1. Effect of Compounds on HPV-11 and HPV-16 DNA Amplification in HEK-293 Cells cidofovir
3, ODE-Bn-PMEG (rac)
3-fast
3-slow
4, ODE-Ph-PMEG
HPV-11
EC50, μMa CC50, μMa SI50b
150c >300c >2
0.4 (0.23−0.65) 17 (16-18) 42
0.4 (0.33−0.59) 91 (70−100) 230
0.5 (0.32−0.59) 83 (49−100) 170
9.5c n.d.b n.d.
HPV-16
EC50, μMa SI50b
150c >2c
0.2 (0.20−0.23) 85c
0.39c 230c
0.69c 120c
5.2c n.d.
a Values shown represent the average and the range from two independent experiments, except where noted. bSelectivity = CC50/EC50; n.d. = not determined cValue represents the result from a single determination.
Table 2. Effect of Compounds on HPV-11, HPV-16, and HPV-18 DNA Amplification in C-33 A Cells
a
cidofovir
3, ODE-Bn-PMEG (rac)
3-fast
3-slow
4, ODE-Ph-PMEG
HPV-11
EC50, μM EC90, μMa CC50, μMa SI50b SI90b
>300 ± 0 >300 ± 0 >300 ± 0 1 1
0.18 ± 0.11 0.72 ± 0.26 11 ± 3.5 61 15
0.41 ± 0.21 1.4 ± 0.4 >10 ± 0 > 24 >7
0.71 ± 0.49 2.5 ± 1.2 >10 ± 0 >14 >4
0.10 ± 0.05 0.61 ± 0.14 49 ± 10 490 80
HPV-16
EC50, μM EC90, μM CC50, μM SI50 SI90
220 ± 97 >300 ± 0 >300 ± 0 >1 1
0.04 ± 0.02 0.64 ± 0.55 >10 ± 0 >250 >16
0.14 ± 0.07 0.89 ± 0.47 >10 ± 0 >71 >11
0.22 ± 0.13 1.5 ± 0.20 >10 ± 0 >45 >7
0.09 ± 0.03 0.49 ± 0.06 60 ± 11 670 120
HPV-18
EC50, μM EC90, μM CC50, μM SI50 SI90
260 ± 42 >300 ± 0 >300 ± 0 >1 1
0.10 ± 0.07 0.59 ± 0.48 >10 ± 0 >100 >17
0.08 ± 0.02 0.72 ± 0.34 >10 ± 0 >130 >14
0.27 ± 0.03 1.4 ± 0.11 >10 ± 0 >37 >7
0.06 ± 0.05 0.28 ± 0.40 59 ± 19 980 210
a
Values shown represent the average and SD from at least three independent experiments. bSI50 = CC50/EC50; SI90 = CC50/EC90
HPV-11 and -16 Assays in HEK-293 Cells. PMEG diesters 3 and 4 were initially evaluated for anti-HPV-11 and -16 effects in transiently transfected HEK-293 cells expressing proteins E1 and E2 and harboring HPV ori-plasmids. Briefly, HPV-11 or HPV-16 origin-containing plasmids were cotransfected into HEK-293 cells with HPV-11 or HPV-16 E1 and E2 protein expression vectors. After treatment with drug dilutions and incubation, effects of the test compounds on ori-plasmid amplification were measured by quantitative real time PCR (qPCR), and EC50 values were calculated. Cytotoxity (CC50) of test compounds was also assessed in HEK-293 cells using CellTiter-Glo reagent (Promega), and selectivity indexes (CC50/EC50) were calculated. The resulting antiviral data are summarized in Table 1. Cidofovir, an active control, showed weak anti-HPV activity with EC50 value of 150 μM against both HPV-11 and HPV-16. ODE-Bn-PMEG (compound 3) was over 300 times more potent than cidofovir with EC50 values of 0.40 ± 0.22 μM and 0.22 ± 0.02 μM versus HPV-11 and -16, respectively. Selectivity indexes for compound 3 were 44 (HPV-11) and 80 (HPV-16). The antiviral activity of the resolved P-chiral enantiomers, 3-fast and 3-slow, did not differ signicantly (Student’s t test, P > 0.05) from the racemate in the anti-HPV11 assay. The corresponding PMEG diester with a phenyl substitution in place of benzyl, compound 4, was a slightly less potent inhibitor of HPV DNA amplification with EC50 values of 9.5 and 5.3 μM versus HPV-11 and HPV-16, respectively. HPV-11, HPV-16, and HPV-18 Assays in C33A Cells. To provide more robust estimates of the efficacy of these new
nate 6, a convenient intermediate for elaboration with diverse ester promoieties. A variety of coupling reagents have been employed to prepare phosphonate-masked ANP prodrugs.20 Notably, Campagne et al.21 reported the preparation of asymmetric phosphonate diesters using BOP or PyBOP for the second esterification step. Hoping to devise an efficient process for PMEG diesters, we attempted to utilize a single reagent, PyBOP, for both esterification steps. Treatment of a mixture of compound 6 and 2-octadecyloxyethan-1-ol in DMF with PyBOP/diisopropylethylamine/HOBt resulted in efficient esterification as well as complete displacement of the 6-halo group with 1-hydroxybenzotriazole to give compound 7, as shown in Scheme 1. Refluxing compound 7 with 80% acetic acid gave the PMEG monoester, ODE-PMEG (2), in overall better yield than our previously reported process.22 Further esterification of compound 2 with either benzyl alcohol or phenol in the presence of PyBOP/DIEA provided diesters 3 and 4 in 58 and 31% yields, respectively. Due to the asymmetric center formed at phosphorus, diesters 3 and 4 were obtained as racemates. To investigate the effect of phosphorus chirality on the antiviral potency of compound 3, small quantities of the enantiomers were obtained by chiral preparative HPLC on a Lux Cellulose-1 column (Phenomenex, Torrance, CA U.S.A.) using reverse phase conditions. The absolute stereochemistry of the P-chiral enantiomers was not determined, therefore, the resolved enantiomers were designated as 3-fast (first eluting enantiomer) and 3-slow (second eluting enantiomer). 10472
DOI: 10.1021/acs.jmedchem.6b00659 J. Med. Chem. 2016, 59, 10470−10478
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analogs, antiviral activity was also evaluated in a series of antiHPV assays that, in addition to HPV-11, -16, and -18 E1 and E2 viral protein expression vectors, utilized a NanoLuc reporter gene in the ori-plasmid to facilitate its quantification in transiently transfected C33A cells. Thus, after drug treatment and incubation, replication of the NanoLuc reporter vector was quantified by measuring the corresponding increase in luciferase activity, and EC50 values were determined. Cytotoxicity (CC50) values were also determined in C33A cells using CellTiter-Glo reagent (Promega). Results are summarized in Table 2. Consistent with the screening assay, cidofovir was not very active with EC50 values ranging from 220 μM (HPV-16) to >300 μM for HPV-11. Compound 3 (rac) was much more active and selective with EC50 values of 0.04 μM (HPV-16), 0.10 μM (HPV-18), and 0.18 μM (HPV-11) and selectivity indexes of 61, >250, and >100 for HPV-11, -16, and -18, respectively. Note that EC90 values were also determined to assess ability of the test compounds to more fully suppress HPV ori replication. Enantiomers 3-fast and 3-slow again provided EC50s not significantly different from the racemate, suggesting that antiviral activity is independent of the prodrug’s stereoconfiguration at phosphorus. In contrast to the assay in HEK-293 cells, compound 4 was slightly more active and significantly more selective in the C33A assays, particularly against high-risk HPV-type 18. HIV-1 Antiviral Assay. Compounds 3, 3-fast, and 3-slow were also assessed for antiviral activity in human PBMCs infected with a clinical HIV-1 isolate, and AZT was used as a positive control (Table 3). Racemic ODE-Bn-PMEG (3) was
Table 4. Effect of Compounds on HSV-2 Replication in HFF
a
compound
EC50, μMa
CC50, μM
selectivity index
acyclovir 3, ODE-Bn-PMEG 3-fast, ODE-Bn-PMEG 3-slow, ODE-Bn-PMEG
0.99 ± 0.69 0.022 ± 0.011 0.033 ± 0.036 0.032 ± 0.022
>300 ± 0 5.2 ± 0.42 5.4 ± 0 6.1 ± 0.35
>300 240 160 190
Results are the mean ± standard deviation (three replicates).
either normal or heat inactivated 10% fetal bovine serum (FBS). In both experiments, reverse phase HPLC analysis after 38 days showed the presence of 83% of 3 starting concentration and appearance of ODE-PMEG (2, ca. 17% total peak area), indicating a slow loss of the benzyl promoiety under both sets of conditions. PMEG (1) was not detected as a degradation product in either assay. The cellular uptake of 3, monoester ODE-PMEG (2), and unmodified PMEG (1) into HFF cells was assessed using radiolabeled analogs (Figure 2). Cells were exposed to 1 μM
Table 3. Effect of Compounds on HIV-1 Replication in Human Peripheral Blood Mononuclear Cells compound AZT 3, ODE-Bn-PMEG (rac) 3-fast 3-slow
EC50, μMa
CC50, μM
selectivity index
0.004 0.002 ± 0.0004
>1.0 0.020 ± 0.020
>250 11.0
0.004 ± 0.0021 0.003 ± 0.0010
0.070 ± 0.040 0.040 ± 0.020
16.3 16.3
Results are mean ± standard deviation of three replicates. AZT was a single determination. HIV-1, subtype B clinical isolate 92US727; ImQuest BioSciences Inc., Frederick, MD.
a
Figure 2. Uptake of 8-14C-labeled PMEG, ODE-PMEG, or ODE-BnPMEG into HFF. Data are the average of two determinations; ◆ indicates PMEG, ● indicates ODE-PMEG, and ■ indicates ODE-BnPMEG.
highly active, and the enantiomeric compounds 3-fast and 3slow also showed similar antiviral activity with 2 ± 0.4 to 4 ± 2 nM EC50 values. Cytotoxicity values of the three compounds were also similar to CC50 values ranging from 20 to 70 nM. The selectivity index of racemic 3 was somewhat lower, 11.0, than the more highly purified enantiomers, 16.3. These compounds have antiviral activity against HIV-1 similar to that of AZT, but are more cytotoxic with narrower selectivity indexes. HSV-2 Antiviral Assay. ODE-Bn-PMEG, 3, and its P-chiral enantiomers were also tested in vitro in HFF cells infected with HSV-2 (Table 4). Acyclovir, an active control, had an EC50 of 0.99 μM. Compound 3 (rac) and the two enantiomers, 3-fast and 3-slow, had essentially identical EC50 values of 0.022−0.033 μM. Cytotoxicity ranged from CC50s of 5.2 to 6.1 μM, and the selectivity indexes for 3, 3-fast, and 3-slow ranged from 160 to 240. Compounds 3, 3-fast, and 3-slow were 30−45 times more active against HSV-2 than acyclovir in vitro (Table 4). ODE-Bn-PMEG Stability, Intracellular Uptake, and Metabolism. PMEG diester 3 was evaluated for stability at 37 °C in minimum essential media (MEM) supplemented with
[14C]-1, -2, or -3 for times ranging from 1 to 48 h, and uptake was assessed in duplicate using a previously reported method.23 As shown in Figure 3, cellular drug levels of the lipophilic PMEG analogs 2 and 3 increased progressively for 48 h. In contrast, cells exposed to [14C]-1 contained much lower levels of radioactivity that remained essentially constant during the incubation. After 48 h, uptake of either 2 or 3 was more than 100-fold higher than 1, consistent with previously observed increases in cell membrane penetration afforded by alkoxyalkyl modification of acyclic nucleoside phosphonates.24 Activation of 3 in HFF cells was also evaluated using a previously reported approach (Figure 3).22,23 Intracellular levels of PMEG, PMEG monophosphate (PMEGp), and PMEG diphosphate (PMEGpp) were measured following 48 h of exposure to 1 μM [14C]-3 and removal of the drug. The results are the average of two separate replicates. Compound 3 generated high intracellular levels of PMEGpp, the active antiviral metabolite at day 0 following drug removal, and levels continued to increase steadily for at least 14 days. As expected, 10473
DOI: 10.1021/acs.jmedchem.6b00659 J. Med. Chem. 2016, 59, 10470−10478
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293 and C33A cells. This compound could address an important unmet medical need as a treatment for cervical and other anogenital high-risk HPV infections.
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EXPERIMENTAL SECTION
All reagents were of commercial quality and used without further purification unless indicated otherwise. Chromatographic purification was done using the flash method with silica gel 60 (EMD Chemicals, Inc., 230−400 mesh). 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectra were recorded on either a Varian VX-500 or a Varian HG-400 spectrometer and are reported in units of ppm relative to internal tetramethylsilane at 0.00 ppm. Electrospray ionization mass spectra (ESI-MS) were recorded on a Finnigan LCQDECA mass spectrometer at the small molecule facility in the Department of Chemistry at University of California, San Diego. Purity of the target compounds was characterized by high-performance liquid chromatography (HPLC) using a Beckman Coulter System Gold chromatography system. The analytical column was Phenomenex Synergi PolarRP (4.6 × 150 mm) equipped with a SecurityGuard protection column. Mobile phase A was 95% water/5% methanol and mobile phase B was 95% methanol/5% water. At a flow rate of 0.8 mL/min, gradient elution was as follows: 10% B (0−3 min.); 10−95% B (3−20 min.); 95% B (20−25 min.); 95% to 10% B (25−34 min.). Compounds were detected by ultraviolet light (UV) absorption at 274 nm. Purity of compounds was also assessed by thin-layer chromatography (TLC) using Analtech silica gel-GF (250 μm) plates and the solvent system: CHCl 3 /MeOH/conc NH 4 OH/H 2 O (70:30:3:3 v/v). TLC results were visualized with UV light, phospray (Supelco, Bellefonte, PA, U.S.A.) and charring at 400 °C. Purity of compounds was ≥95% or as indicated (see Supporting Information). Octadecyloxyethyl benzyl [8-14C]-PMEG (specific activity = 55 mCi/ mmol), ODE-[8-14C]-PMEG (specific activity = 51.1 mCi/mmol), and [8-14C]-PMEG (specific activity = 51.6 mCi/mmol were purchased from Moravek Biochemicals (Brea, CA). 2-(Octadecyloxy)ethyl Hydrogen ((2-(2-Amino-6-oxo-1,6dihydro-9H-purin-9-yl)ethoxy)methyl)phosphonate (ODEPMEG, Compound 2). A 1 L flask was equipped with a magnetic stirrer, then 6 (21.7 g, 44 mmol), 2-octadecyloxyethanol (ODE−OH, 14.2 g, 45 mmol), and anhydrous DMF (300 mL) were added. The mixture was stirred, and (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 35.0 g, 67.5 mmol) was subdivided in five equal portions (7 g each), and each portion was then added at 30 min intervals. After the addition of PyBOP, diisopropylethylamine (DIEA, 5.8 g, 45 mmol), and 1-hydroxybenzotriazole (HOBt, 3.0 g, 22.5 mmol) were added. The resulting mixture was stirred at 22−25 °C. After 18 h, the reaction mixture was slowly poured into a stirred acidic mixture comprised of conc. HCl (10 mL), water (750 mL), and crushed ice (750 mL). Stirring was continued for 10 min. The precipitated solid was collected by filtration, washed with cold water (2 × 100 mL), and dried under vacuum to give crude 7. The crude product was purified by silica gel column chromatography with elution of the product by CH2Cl2:MeOH 90:10 to yield 7 (9.5 g, 30.7% yield). 1H NMR (500 MHz, CDCl3 + CD3OD) δ 8.19 (s, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.61−7.46 (m, 3H), 4.31 (t, J = 5.0 Hz, 2H), 3.97 (d, J = 5.3 Hz, 2H), 3.86 (t, J = 5.1 Hz, 2H), 3.67 (d, J = 9.1 Hz, 2H), 3.52 (t, J = 5.0 Hz, 2H), 3.42 (t, J = 6.9 Hz, 2H), 1.52 (t, J = 6.9 Hz, 2H), 1.24 (m, 30H), 0.88 (t, 3H). 13C NMR (126 MHz, CDCl3 + CD3OD) δ 159.21, 159.12, 156.27, 143.81, 143.30, 129.10, 129.02, 125.37, 120.12, 111.76, 109.06, 71.65, 70.65, 70.57, 70.51, 66.74 (d, J = 162.6 Hz), 64.12 (d, J = 5.5 Hz), 43.57, 32.06, 29.85, 29.84, 29.82, 29.79, 29.69, 29.64, 29.50, 26.13, 22.82, 14.18. 31P NMR (162 MHz, CDCl3 + CD3OD) δ 13.23. ESI MS: 701.32 [M − H]−. A 1 L reaction flask was equipped with a magnetic stirrer and a condenser. Compound 7 (9.52 g, 13.5 mmol), acetic acid (240 mL), and water (60 mL) were added. The resulting mixture was stirred and heated to reflux. After the reaction was complete (3.5 h), the reaction mixture was cooled to 5 °C, stirred for 2 h, and filtered. The product was dried under vacuum and then recrystallized (80:20 isopropanol/ water, decolorizing carbon), the filtrate was cooled in an ice-bath, and
Figure 3. Persistence of metabolites in HFF cells after 48 h treatment with 1 μM [14C]-ODE-Bn-PMEG followed by drug removal. The results are the average of two separate determinations; ● indicates PMEG, ■ indicates PMEGp, and ◆ indicates PMEGpp.
lower levels of PMEGp and PMEG were also detectable. Coupled with enhanced cellular penetration, maintenance of high PMEGpp levels in HFF cells suggests potential utility for ODE-Bn-PMEG as a long-lasting topical antiviral therapy. ODE-Bn-PMEG is a novel double prodrug of PMEG. The compound is a potent inhibitor of transient amplification of HPV-11 and HPV-16 DNA ori-containing plasmids in HEK293 cells when cotransfected with matched HPV-11 and HPV16 expression vectors of the E1 and E2 replication proteins. In this system, EC50 values for HPV-11 and HPV-16 were
