Synthesis and Antiviral Evaluation of (1,4-Disubstituted-1,2,3-Triazol)-(E)-2-Methyl-but-2-Enyl Nucleoside Phosphonate Prodrugs

A series of hitherto unknown (1,4-disubstituted-1,2,3-triazol)-(E)-2-methyl-but-2-enyl nucleosides phosphonate prodrugs bearing 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as the key synthetic step. All novel compounds were evaluated for their antiviral activities against HBV, HIV and SARS-CoV-2. Among these molecules, only compound 15j, a hexadecyloxypropyl (HDP)/(isopropyloxycarbonyl-oxymethyl)-ester (POC) prodrug, showed activity against HBV in Huh7 cell cultures with 62% inhibition at 10 μM, without significant cytotoxicity (IC50 = 66.4 μM in HepG2 cells, IC50 = 43.1 μM in HepG2 cells) at 10 μM.


Chemistry
First, we synthetized the bis(POC)allylphosphonate 7 from dimethylallylphosphonate according our previously reported method [14]. Then, optimized olefin cross-metathesis reaction between 7 and 2-methylprop-2-en-1-ol (8) was performed in dry dichloromethane with a Hoveyda-Grubbs catalyst (15 mol%) under ultrasonic irradiation at 55 °C during 24 h [17], (Scheme 1). The desired compound 9 was obtained in an excellent 94% yield with only E-isomer; no trace of Z-isomer was detected (NOESY NMR, see Supplementary Materials). Compound 9 was converted to the corresponding mesylate and the obtained sulfone was used directly in the next step without further purification. The introduction of the azido group on the mesylated compound was realized with sodium azide in DMF at room temperature for 5 h to afford compound 10 in excellent 93% yield.  For nucleosides, it appears that chemical alterations (such as a methyl group at 2 position) at the sugar (or acyclic side-chain) moiety or at the heterocycle such as 1,2,3triazoles [16] often lead to marked differences in antiviral activity. Based on these findings and on our data for compounds 5 and 6, we were interested in the synthesis of hitherto unknown (1,4-disubstituted-1,2,3-triazol)-(E)-2-methyl-but-2-enyl nucleosides phosphonate prodrugs and we wish to compare the impact of bis(POC) with the mixed HDP/POC biolabile moiety on the antiviral activity. All compounds were evaluated against hepatitis B virus (HBV), human immunodeficiency virus (HIV-1) and the newly detected severe acute respiratory syndrome (SARS-CoV-2).

Chemistry
First, we synthetized the bis(POC)allylphosphonate 7 from dimethylallylphosphonate according our previously reported method [14]. Then, optimized olefin cross-metathesis reaction between 7 and 2-methylprop-2-en-1-ol (8) was performed in dry dichloromethane with a Hoveyda-Grubbs catalyst (15 mol%) under ultrasonic irradiation at 55 • C during 24 h [17], (Scheme 1). The desired compound 9 was obtained in an excellent 94% yield with only E-isomer; no trace of Z-isomer was detected (NOESY NMR, see Supplementary Materials). Compound 9 was converted to the corresponding mesylate and the obtained sulfone was used directly in the next step without further purification. The introduction of the azido group on the mesylated compound was realized with sodium azide in DMF at room temperature for 5 h to afford compound 10 in excellent 93% yield. Among them, the compound 5 exhibits significant potency against human hepatitis C virus (HCV) infections at 10 μM (95% of inhibition) meanwhile our synthesized bis(POC)-(E)-2-methyl-but-2-enylguanine 6 (unpublished data) showed antiviral activity against hepatitis B virus (HBV) with an EC50 of 8.5 μM without significant cytotoxicity ( Figure 1). For nucleosides, it appears that chemical alterations (such as a methyl group at 2′ position) at the sugar (or acyclic side-chain) moiety or at the heterocycle such as 1,2,3triazoles [16] often lead to marked differences in antiviral activity. Based on these findings and on our data for compounds 5 and 6, we were interested in the synthesis of hitherto unknown (1,4-disubstituted-1,2,3-triazol)-(E)-2-methyl-but-2-enyl nucleosides phosphonate prodrugs and we wish to compare the impact of bis(POC) with the mixed HDP/POC biolabile moiety on the antiviral activity. All compounds were evaluated against hepatitis B virus (HBV), human immunodeficiency virus (HIV-1) and the newly detected severe acute respiratory syndrome (SARS-CoV-2).
It is worth noting that the substituent on triazole ring has a dramatic effect on HBV activity. In fact, compounds with hydrophobic aryl groups (electron-donating 11a-e and electron-withdrawing 11l) exhibited good activity (except for 11b), whereas triazolyl-ANPs with alkyl (11f,g) or polar substituents such as alcohol (11h), aldehyde (11i), formamide (11j) and amide (11k) and carboxylic derivatives (11m-q) have only low activity. We can speculate that the hydrophobic pocket (formed by A87, F88, P177, L180, and M2) found in the rear of the RT dNTP binding site of HBV could interact with hydrophobic aryl residues through donor atom-π interaction [21].
Compound 15j, a HDP/POC prodrug, a ribavirin analog, is the most active drug against HBV in this serial, with 62% inhibition at 10 µM. However, generally speaking, the bis(POC) ester prodrugs are more active than their HDP/POC counterparts. The cytotoxicity of test compounds was performed in different cell systems. Positive control cycloheximide exhibited expected toxicity in the peripheral blood mononuclear (PBM) and HepG2 cells, with an IC 50 (µM) of 1.0 and 1.5 µM, respectively (data not shown).

Chemistry General Section
Commercially available chemicals were provided as reagent grade and used as received. Some reactions requiring anhydrous conditions were carried out using oven-dried glassware and under an atmosphere of dry argon. All anhydrous solvents were provided from commercial sources as very dry reagents. The reactions were monitored by thin layer chromatography (TLC) analysis using silica gel precoated plates (Kieselgel 60F254, E. Merck). Compounds were visualized by UV irradiation and/or spraying with sulfuric acid (H 2 SO 4 5% in ethanol) stain followed by charring at average 150 • C. Flash column chromatography was performed on silica gel 60 M (0.040-0.063 mm, E. Merck). The infrared spectra were measured with the Perkin-Elmer Spectrometer. The 1 H and 13 C NMR spectra were recorded on the BrukerAvance DPX 250 or BrukerAvance 400 Spectrometer (Bruker, Champs sur Marne, France). Chemical shifts are given in ppm and are referenced to the deuterated solvent signal or to TMS as internal standard and multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Carbon multiplicities were assigned by distortionless enhancement by polarization transfer (DEPT) experiments. 1 H and 13 C signals were attributed on the basis of H-H and H-C correlations. High resolution mass spectra were performed on a Bruker Q-TOF MaXis spectrometer (Bruker Daltonics, Bremen, Germany) by the "Fédération de Recherche" ICOA/CBM (FR2708) platform. LC-MS data were acquired on a Thermo-Fisher UHPLC-MSQ system equipped with an electron spray ionization source (ESI). The temperature of the source was maintained at 350 • C. Initially, the cone voltage was set at 35 V and after 5 min was increased to 75 V. In full scan mode, data were acquired between 100 and 1000 m/z in the positive mode with a 1.00 s scan time. In addition, a UV detection was performed with a diode array detector at three wavelengths 273, 254 and 290 nm, respectively. A water/methanol (70%/30%) solution mixture with 0.1% formic acid was used as the mobile phase. The composition of the mobile phase was increased to 100% methanol with 0.1% formic acid with a 7% ramp. The flow rate was set at 0.300 mL min −1 . Samples diluted in the mobile phase were injected (3 µL) on a C18 column (X-terra, Waters), with a 2.1 mm internal diameter, and 100 mm length, and placed into an oven at 40 • C. The electronic extraction of ions was performed and the subsequent areas under the corresponding chromatographic peaks determined. The compounds' name follows the IUPAC recommendations.

HBV Assay
In Huh7 cells: antiviral activity against HBV in cell culture was measured as previously described [22]. Briefly, Huh7 cells were transfected with plasmid pCI_HBVpg1820 containing 1.1 unit of HBV genome under the control of a human cytomegalovirus (CMV) promoter. The drugs were stored at 1000× in DMSO at −20 • C. For each experiment, the drugs were diluted in DMEM cell culture medium (Thermo Fisher Scientific, Artenay, France) to a final concentration of 10 µM. All drugs were tested in triplicate in two independent experiments. After 4 days of culture, intracapsid viral DNA was quantified by duplex real-time PCR, using primers described elsewhere, on a LightCycler 480 II apparatus (Roche Diagnostics France, Meylan, France) using TaqMan Universal PCR Mastermix II without UNG (Thermo Fisher Scientific, Artenay, France). Cells were quantified using primers by real-time PCR, using primers HPRT1_F (5 -TGCAGACTTTGCTTTCCTTGGTC) and HPRT1_R (5 -CAAGCTTGCGACCTTGACCATC), on a LightCycler 480 II apparatus (Roche) using a LightCycler ® 480 SYBR Green I Master (Roche Diagnostics France, Meylan, France). Cycling reactions were performed with the following parameters: 5 min at 95 • C, followed by 45 cycles of 10 s at 95 • C, 15 s at 60 • C, 10 s at 72 • C. The inhibition of HBV replication (%) and cell culture viability (%) were quantified using the following formulas. In HepAD38 cells [23] were seeded at 50,000 cells/well in collagen-coated 96-well plates. Test compounds or 2 ,3 -didéoxy-3 -thiacytidin (3TC) (control) were added to HepAD38 cells to a final concentration of 10 µM.
Real-Time PCR for HBV DNA. The experiment lasted 7 days. On day 7, total DNA was purified from the supernatant using a commercially available kit (DNeasy 96 Blood & Tissue kit, Qiagen). The HBV DNA was amplified in a real-time PCR assay using a LightCycler 480 (Roche) as described elsewhere [24]. All samples were tested in duplicate in two to three independent experiments. Analysis: The concentration of compound that inhibited HBV DNA replication by 50% (EC 50 ) was determined by linear regression.

HIV Assay
The assay was performed as described by Schinazi et al. [25,26] with minor modifications [26]. Briefly, human PBM cells were stimulated with PHA/IL-2 prior to infection with HIV-1 LAI (MOI 0.1) for 5 days in the presence of test compounds with a final concentration of 10 µM (LA-338-366) or AZT (control). The supernatants were harvested, and the HIV-1 RT was quantified. The median effective concentrations (EC 50/90 ) were determined using the method of Belen'kii and Schinazi [27].

SARS-CoV-2 Assay
Vero E6 (ATCC CRL-1586) cells were grown in a minimal essential medium (MEM) (Life Technologies, Carlsbad, CA, USA) with 7.5% heat-inactivated fetal calf serum (FCS), at 37 • C with 5% CO 2 with 1% penicillin/streptomycin (PS, 5000 U.mL −1 and 5000 µg.mL −1 , respectively; Life Technologies) and supplemented with 1% non-essential amino acids (Life Technologies). SARS-CoV-2 strain BavPat1 was obtained from Pr Drosten through EVA GLOBAL (https://www.european-virus-archive.com, accessed on 23 February 2021). To prepare the virus working stock, a 25 cm 2 culture flask of confluent Vero E6 cells growing with MEM medium with 2.5% FBS (Life Technologies) was inoculated at MOI 0.001. The cell supernatant medium was harvested at the peak of infection and supplemented with 25 mM HEPES (Sigma, St. Louis, MO, USA) before being stored frozen in small aliquots at −80 • C. All experiments were conducted in a BSL3 laboratory. One day prior to infection, for the antiviral screening 5 × 10 4 Vero E6 cells were seeded in 100 of µL the assay medium (containing 2.5% FCS) in 96 well plates. The next day, four 2-fold serial dilutions of compounds (20 µM to 2.5 µM, in duplicate) were added to the cells (25 µL/well, in the assay medium) as well as two internal well controls of viral inhibition corresponding to the addition of 10 µM of Remdesivir (BLDpharm, Hyderabad, India). Three virus control wells were supplemented with 25 µL of assay medium (positive controls hereafter named vc) and three cell control wells were supplemented with 50 µL of the medium (negative controls, hereafter named nc). After 15 min, 25 µL of a virus mix diluted in 2.5% FCS-containing medium was added to the wells at MOI 0.002. Three days after infection, the cell supernatant media was discarded and CellTiter-Blue ® reagent (Promega, Madison, WI, USA) was added following the manufacturer's instructions. Plates were incubated for 2 h prior to recording the fluorescence (560/590 nm) with a Tecan Infinite 200Pro machine. From the measured OD 590nm , the inhibition percentage was calculated as follows: ((OD 590nm value-mean OD 590nm value of vc)/(mean OD 590nm of nc-mean OD 590nm value of vc))*100. All data obtained were analyzed using GraphPad Prism 7 software (Graph pad software).

Cytotoxicity Assays
Assays were performed in human peripheral blood mononuclear (PBM) and human liver (HepG2) cells via MTS assay using the CellTiter 96 ® Non-Radioactive Cell Proliferation (Promega) kit as previously described 3 . Cytotoxicity was expressed as the concentration of test compounds that inhibited cell proliferation by 50% (IC 50 ) and calculated using the Chou and Talalay method [28].