Marcel Hausdorff

Adrien Delpal

Sarah Barelier

Laura Nicollet

Bruno Canard

Franck Touret

Agathe Colmant

Bruno Coutard

Jean-Jacques Vasseur

Etienne Decroly
email: etienne.decroly@univ-amu.fr

Françoise Debart
email: francoise.debart@umontpellier.fr

Structure-guided optimization of adenosine mimetics as selective and potent inhibitors of coronavirus nsp14 N7-methyltransferases

Keywords: 7-deaza-adenine, arylsulfonamide, bisubstrate, SARS-CoV-2, RNA cap methyltransferase, structure-guided design SARS, severe acute respiratory syndrome, MERS, Middle East respiratory syndrome, CoV, coronavirus, COVID19, coronavirus disease 2019, DV3, dengue virus serotype 3, MTase, methyltransferase, nsp14, non-structural protein 14, RdRp, RNA dependent RNA polymerase, 3CLpro, 3-chymotrypsin like protease, Mpro, main protease, PLpro, papainlike protease, SAM, S-adenosylmethionine, SAH, S-adenosylhomocysteine, hRNMT, human RNA methyltransferase, FBA, filter binding assay, SAR, structure-activity relationships

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The emergence of new viruses poses a global threat to human health. The COVID-19 pandemic caused by the SARS-CoV-2 illustrates what the world faces with emerging viruses in the absence of appropriate treatments or prophylactic vaccines. After three years of pandemic, approved antiviral treatments for CoV-diseases are still limited to molecules targeting the RNA dependent RNA polymerase (RdRp) activity or the main protease (Mpro) activity [1][2][START_REF] Akinosoglou | Oral Antiviral Treatment for COVID-19: A Comprehensive Review on Nirmatrelvir/Ritonavir[END_REF][START_REF] Spurr | New small molecule inhibitors of histone methyl transferase DOT1L with a nitrile as a non-traditional replacement for heavy halogen atoms[END_REF]. The discovery of potent and selective antiviral inhibitors to fight SARS-CoV-2 infection and likely future CoV outbreaks is an urgent research priority. To this end, significant activity has been observed in drug repurposing [5] and many approved drugs have been reported to block viral replication. Among them, the most promising nucleoside analogues, remdesivir and molnupiravir targeting viral RdRp, and nirmatrelvir inhibiting viral Mpro have shown relative efficacy in clinical trials depending on the stage of infection [6]. Over the past two years, great efforts have been made in structure-based drug design, an effective alternative for developing new antiviral agents targeting viral enzymes [7]. Among the four structural and 16 non-structural proteins encoded by CoVs [8], the papain-like protease (PLpro) and Mpro that are essential for CoV replication have been extensively targeted with peptidomimetics and small molecule inhibitors [START_REF] Owen | An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19[END_REF]. Two methyltransferases (MTases), which are engaged in the capping pathway of viral mRNA, are also essential for viral replication, as reverse genetic studies demonstrated that these enzymes play a key role in mRNA translation and prevent viral restriction mediated by host innate immune system [START_REF] Nencka | Coronaviral RNAmethyltransferases: function, structure and inhibition[END_REF][START_REF] Ogando | Structure-function analysis of the nsp14 N7guanine methyltransferase reveals an essential role in Betacoronavirus replication[END_REF][START_REF] Decroly | Biochemical principles and inhibitors to interfere with viral capping pathways[END_REF]. These MTases are responsible for the enzymatic cascade of viral RNA cap methylations: first the N7-methylation of the guanosine by guanine N7-MTase nsp14 and subsequently the 2'-OH methylation of the ribose of the first RNA adenosine by the 2'-O-MTase complex nsp10/nsp16 [8,[START_REF] Bouvet | In vitro reconstitution of SARS-coronavirus mRNA cap methylation[END_REF]. N7-MTase nsp14 was identified as a critical enzyme for the replication of SARS-CoV, as two mutations in the N7-MTase catalytic pocket abrogate virus viability [START_REF] Ogando | Structure-function analysis of the nsp14 N7guanine methyltransferase reveals an essential role in Betacoronavirus replication[END_REF]. Prior to the emergence of SARS-CoV-2, besides the unselective MTase inhibitors sinefungin and S-adenosyl homocysteine (SAH), few inhibitors of SARS-CoV nsp14 had been described, most of them discovered by highthroughput screening [START_REF] Aouadi | Toward the identification of viral capmethyltransferase inhibitors by fluorescence screening assay[END_REF]. In this context, our group had developed a rational drug design approach to synthesize competitive bisubstrate inhibitors that occupy both the Sadenosylmethionine (SAM) methyl donor binding pocket and the cap-binding pocket of SARS-CoV nsp14 [START_REF] Ahmed-Belkacem | Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase[END_REF]. In this series of adenine dinucleoside SAM analogues, the best inhibitor displayed submicromolar activity, with a selective inhibition of N7-MTase nsp14 over the human N7-MTase. Then, with the emergence of Covid-19, many libraries were screened on SARS-CoV-2 nsp14 but the inhibition results were rather poor [START_REF] Basu | Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14 RNA cap methyltransferase[END_REF][START_REF] Bobrovs | Discovery of SARS-CoV-2 nsp14 and nsp16 methyltransferase inhibitors by high-throughput virtual screening[END_REF][START_REF] Devkota | Probing the SAM binding site of SARS-CoV-2 nsp14 in vitro using SAM competitive inhibitors guides developing selective bisubstrate inhibitors[END_REF][START_REF] Kasprzyk | Identification and evaluation of potential SARS-CoV-2 antiviral agents targeting mRNA cap guanine N7-methyltransferase[END_REF][START_REF] Pearson | Development of a high-throughput screening assay to identify inhibitors of the SARS-CoV-2 guanine-N7-methyltransferase using RapidFire mass spectrometry[END_REF]. In contrast, rational drug design approaches lead to the identification of much more potent inhibitors active at nanomolar [START_REF] Bobileva | Potent SARS-CoV-2 mRNA cap methyltransferase inhibitors by bioisosteric replacement of methionine in SAM cosubstrate[END_REF][START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF][START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF][START_REF] Jung | Bisubstrate inhibitors of severe acute respiratory syndrome coronavirus-2 nsp14 methyltransferase[END_REF][START_REF] Bobileva | 3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors[END_REF] or subnanomolar concentration [START_REF] Bobileva | 3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors[END_REF]. All these new inhibitors are mimics of SAM/SAH and most of them have been modeled in the crystal structure of SARS-CoV nsp14, solved in the presence of SAM (PDB 5C8T) [START_REF] Ma | Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex[END_REF]. Very recently, several crystal structures of full-length nsp14 from SARS-CoV-2 were solved, bound to SAH (PDB 7R2V, 7QGI) or to a cap-analogue (PDB 7QIF) [START_REF] Czarna | Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity[END_REF][START_REF] Imprachim | Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development[END_REF]. A high-resolution crystal structure of the SARS-CoV-2 N7-MTase domain -expressed in fusion with the TELSAM epitope -was also reported [START_REF] Kottur | High-resolution structures of the SARS-CoV-2 N7-methyltransferase inform therapeutic development[END_REF]. These new structures provide crucial information for understanding the dynamics of nsp14 methyltransferase activity and pave the way for the structure-based development of new antivirals against CoVs.

Our previous work identified potent bisubstrate inhibitors bearing N-ethyl arylsulfonamide moieties linked to the 5'-position of an adenosine mimicking that of SAM as a key component occupying the cap RNA substrate binding site. Indeed, this N-ethyl arylsulfonamide motif likely accounts for the compounds'high affinity for SARS-CoV-2 nsp14, which results in a doubledigit nanomolar inhibition [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. When the sulfonamide linker was replaced by an amide or an N-acylsulfonamide linker, we showed that the inhibitory activity of the nucleoside analogues was lost [START_REF] Ahmed-Belkacem | Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase[END_REF][START_REF] Amador | Facile access to 4'-(N-acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14[END_REF]. In addition, a recent report confirmed the importance of the sulfonamide linker between the adenosine and the aromatic moiety, a 2-naphtalene in this case, to reach nanomolar inhibition of nsp14 [START_REF] Jung | Bisubstrate inhibitors of severe acute respiratory syndrome coronavirus-2 nsp14 methyltransferase[END_REF].

In the present work, we developed a structure-guided drug design approach to design a new series of inhibitors targeting nsp14 N7-MTase activity. We used the N-arylsulfonamide moiety as a guanine mimic of the cap structure and modified the adenine moiety in order to increase the interaction with the SAM binding pocket. We replaced adenine with its analogue, hypoxanthine (HXN), or a C7-substituted-7-deaza-adenine, or we added a methyl group at the N 6 position of adenine (N 6m A) (Fig. 1). Thus, we synthesized 26 final compounds 1-26 that were tested for N7-MTase inhibitory activity in vitro. In the most potent compounds, the adenine is replaced with 7-deaza-adenine bearing either a phenyl (C 7Ph ) or a 3-quinoline group (C 7Q ) attached to the C7-position of the nucleobase via an ethynyl linker. One of these compounds displays subnanomolar IC50 against the N7-MTase activity of SARS-CoVs nsp14 and is barely active on hN7-MTase (hRNMT). Docking predictions support the selective inhibition of the viral nsp14 MTase, as the phenyl or 3-quinoline group on the nucleobase occupies a SAM entry tunnel controlled by a mobile loop forming the substrate entry gate, which is absent in the RNMT human ortholog. Finally, the inhibition of the viral replication for the best nsp14 inhibitors was tested in infected cell cultures, where some of the compounds show inhibitory effect at the micromolar range.

Results and discussion

Rational design of compounds

We based the design of our new inhibitors on the molecular structure of the previous series of bisubstrate inhibitors containing N-ethyl arylsulfonamide moieties, which were shown to be crucial for the affinity and inhibition of the SARS-CoV-2 nsp14 N7-MTase (Fig. 1). Molecular docking studies supported that the arylsulfonamide moiety occupies the cap-guanosine binding pocket of SARS-CoV nsp14 (PDB ID: 5C8T) [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. The impact of the nature and position of the substituents on the aryl moiety was thus demonstrated, as a meta electron-withdrawing substituent (NO2, CN, or Cl) and a para electron-donating substituent (Me or OMe) were found to be the best combination to yield nsp14 inhibitors with double-digit nanomolar IC50.

Therefore, in the present work, the 26 synthesized compounds were designed with a phenylsulfonyl moiety bearing one substituent in meta (NO2, CN, or Cl) and one substituent in para (Me, OMe or Br) positions following the same rationale. Moreover, as functionalization of the nitrogen atom of the sulfonamide link with an alkyl group was shown to improve inhibition, an ethyl group was used in compounds 4-6, 12-16 and a methyl group in nucleoside analogues 17-26. Here, we focused on improving the interactions between the nucleobase moiety and N7-MTase nsp14, according to docking performed in nsp14 structure. By mimicking adenine, hypoxanthine with O6 instead of NH2 at C6 position in the purine ring was chosen to design compounds 1-6. Furthermore, the addition of a methyl group on the exocyclic NH2 of adenine in nucleosides 7-16 was intended to increase the lipophilic properties of the inhibitors to favor cellular uptake.

In compounds 17-26, adenine was replaced by two different 7-deaza-adenines bearing a phenyl or a 3-quinoline group attached to the C7-position of the nucleobase through an ethynyl linker. This modified adenine design was inspired by the work of Nencka et al. where replacement of adenine with various 7-substituted-7-deaza-adenines in SAH nucleoside analogues resulted in nanomolar inhibitors of SARS-CoV-2 nsp14 [START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF]. These modified nucleobases have been shown to effectively target a side cavity of SARS-CoV nsp14 (PDB 5C8T), which is a substrate entry gate communicating with the SAM binding site [START_REF] Imprachim | Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development[END_REF].

Combined with the N-methyl arylsulfonamide scaffold at the 5' position of the adenosine analogue, we anticipated that C7-aryl-substituted 7-deaza-adenine would yield much more potent and selective inhibitors of the SARS-CoV-2 nsp14 N7-MTase (PDB 7R2V). Because of the high similarity between SARS-CoV and SARS-CoV-2 structures (> 95% sequence identity, structural root-mean-square deviation (RMSD) 0.49 Å over 585 backbone atoms) our compounds were tested against both CoVs nsp14 enzymes.

Chemical synthesis

The synthesis of inosine analogues 1-6 began with the preparation of the previously described 5'-N-arylsulfonamide adenosines 27-32 [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF] (Scheme 1). Treatment of 27-32 with NaNO2 under acidic conditions with a mixture of acetic acid and water (85:15) during 72 h afforded the inosine derivatives 33-38 in satisfactory yields (59-80%) [START_REF] Kotra | Synthesis, biotransformation, and pharmacokinetic studies of 9-(β-d-arabinofuranosyl)-6azidopurine: a prodrug for ara-A designed to utilize the azide reduction pathway[END_REF]. The addition of a second portion of sodium nitrite after 48 h of reaction is crucial to complete the conversion of adenosine to inosine. Interestingly, no cleavage of the isopropylidene protection was observed, nor was any depurination. The 2', 3'-hydroxyls of intermediates 33-38 were deprotected with a mixture of formic acid and water (1:1) during 24-48 h to obtain the final compounds 1-6 in good yields (63-72%). The 7-ethynylaryl-7-deaza-adenine nucleosides 17-26 were synthesized from the readily accessible 7-deaza-7-iodo-2',3'-O-isopropylideneadenosine 52 [START_REF] Seela | 7-Functionalized 7-deazapurine β-d and β-l-ribonucleosides related to tubercidin and 7-deazainosine: glycosylation of pyrrolo[2,3-d]pyrimidines with 1-O-acetyl-2,3,5-tri-O-benzoyl-βd or β-l-ribofuranose[END_REF][START_REF] Kunkel | A 7-deazaadenosylaziridine cofactor for sequence-specific labeling of DNA by the DNA cytosine-C5 methyltransferase M.HhaI[END_REF][START_REF] Nauš | Synthesis and biological profiling of 6-or 7-(het)aryl-7-deazapurine 4′-C-methylribonucleosides[END_REF] (Scheme 3). Sonogashira cross-coupling reaction of 52 [START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF][START_REF] Milisavljevic | Antiviral activity of 7substituted 7-deazapurine ribonucleosides, monophosphate prodrugs, and triphoshates against emerging RNA viruses[END_REF] with commercially available phenylacetylene or prepared 3-ethynylquinoline [START_REF] Rodríguez | Synthesis of n-chloroquinolines and nethynylquinolines (n=2, 4, 8): homo and heterocoupling reactions[END_REF][START_REF] Smeyanov | K3PO4-KOH mixture as efficient reagent for the deprotection of 4-aryl-2-methyl-3-butyn-2-ols to terminal acetylenes[END_REF]

N N N N NH 2 O O O N S O O R 1 R 2 NH N N N O O O O N S O O R 1 R 2 NH N N N O O OH OH N S O O R 1 R 2 R 3 R 3 R
N N N NH 2 O O O N S O O R 1 R 2 N N N NH 2 O OH OH N S O O R 1 R 2 e c

RNA Methyltransferase Activity Assays

Compounds 1-26 were tested for their ability to inhibit the SARS-CoV and SARS-CoV-2 N7-MTases which are involved in viral RNA cap structure methylation. We also assessed the selectivity of the synthesized compounds, by determining the inhibition of the cognate human N7-MTase (hRNMT) and of the Dengue virus MTase (NS5). Briefly, the different purified MTases were incubated together with compounds 1-26 at 5 µM and the transfer of [ 3 H]radiolabeled methyl on synthetic capped RNA (GpppAC4) was determined by filter binding assay (FBA) [START_REF] Peyrane | High-yield production of short GpppA-and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions[END_REF].

MH44 from our previous series of nsp14 inhibitors is used here as a control (Fig. 1) [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. The replacement of the exocyclic NH2 (N 6 ) with an oxygen atom in inosine is detrimental for the inhibitory activity of the compounds 1-6 at 5 µM against CoV N7-MTase nsp14 (Table 1). The addition of a methyl group to the exocyclic NH2 (N 6 ) of adenine in 7-11 impairs the inhibition of both CoV MTase activity which is less impacted for compounds 12-16 bearing an ethyl on the sulfonamide bond. The two C7-substituted-7-deaza-adenine nucleoside series bearing either a phenyl [START_REF] Bobrovs | Discovery of SARS-CoV-2 nsp14 and nsp16 methyltransferase inhibitors by high-throughput virtual screening[END_REF][START_REF] Devkota | Probing the SAM binding site of SARS-CoV-2 nsp14 in vitro using SAM competitive inhibitors guides developing selective bisubstrate inhibitors[END_REF][START_REF] Kasprzyk | Identification and evaluation of potential SARS-CoV-2 antiviral agents targeting mRNA cap guanine N7-methyltransferase[END_REF][START_REF] Pearson | Development of a high-throughput screening assay to identify inhibitors of the SARS-CoV-2 guanine-N7-methyltransferase using RapidFire mass spectrometry[END_REF][START_REF] Bobileva | Potent SARS-CoV-2 mRNA cap methyltransferase inhibitors by bioisosteric replacement of methionine in SAM cosubstrate[END_REF] or a 3-quinoline group [START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF][START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF][START_REF] Jung | Bisubstrate inhibitors of severe acute respiratory syndrome coronavirus-2 nsp14 methyltransferase[END_REF][START_REF] Bobileva | 3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors[END_REF][START_REF] Ma | Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex[END_REF] exhibit potent inhibitory effect at 5 µM on SARS-CoV and SARS-CoV-2 N7-MTases. In addition, the five series of compounds barely inhibit the dengue virus NS5-MTase or human N7-MTase that methylates the mRNA cap structure, suggesting selectivity toward both SARS-CoV N7-MTases. [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. n.i: no inhibition detected at 5 µM. B*: nucleobase with A = adenine; HXN = hypoxanthine; N 6m A = N 6 -methyladenine; C 7Ph A = C7-phenylethynyl 7-deaza-adenine; C 7Q A = C7-quinolinethynyl 7-deaza-adenine.

Dose-response Testing and Docking of Compounds 6-26 against SARS-CoV-2 nsp14

To further decipher the antiviral activity of our compounds, we determined the IC50 values of the best inosine-derived inhibitor 6, N 6 -methyl adenosine derivatives 7-16 and 7-deaza-adenine nucleosides 17-26, on SARS-CoV-2 N7-MTase (Table 2). In parallel, we performed docking experiments against the structure of SARS-CoV-2 nsp14, solved in presence of SAH (PDB 7R2V) using Autodock Vina [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF] and compared the calculated ∆G values with the IC50 values determined by FBA upon dose response titration (Table 2).

Briefly, SARS-CoV-2 nsp14 was pre-incubated with increasing concentrations of inhibitors a The docking was performed on the SARS-CoV-2 nsp14 (PDB 7R2V). The ΔG value corresponds to the best pose showing a good fitting between the SAH and the nucleobase of the compounds. b Concentration inhibiting MTase activity by 50%; mean value from three independent experiments. c value from the literature [START_REF] Aouadi | Toward the identification of viral capmethyltransferase inhibitors by fluorescence screening assay[END_REF]. The MTase activity for IC50 determinations was measured using a filter binding assay. d MH44 was the best inhibitor of SARS-CoV-2 N7-MTase in our previous work [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF].

We next determined the selectivity of the best inhibitors of each series on SARS-CoVs and MERS-CoV N7-MTases (Table 3). The IC50 values of compounds 11, 16, 21 and 26 on these MTases show a similar range of N7-MTase inhibition demonstrating that these inhibitors might have pan-CoV activity, whereas they are barely active on the cognate hRNMT and not active on NS5 of DV3, confirming their selectivity toward a large range of CoV N7-MTases.

Remarkably, compounds 21 and 26 inhibit SARS-CoV-2 nsp14 with 47 000-and 230 000-fold much higher selectivity over hN7-MTase, respectively, compared to the previous inhibitor MH44, which was 2800-fold more selective.

Moreover, a dose-response assay showed no inhibition of SARS-CoV-2 2'O-MTase (nsp10/nsp16 complex) by compounds 11, 21 and 26 and a moderate inhibition by N 6 -methyl adenine derivative 16 (IC50 76.9 µM) confirming that these four compounds are highly selective in targeting CoV N7-MTases. a Concentration inhibiting MTase activity by 50%; mean value from three independent experiments. The MTase activity for IC50 determinations was measured using a filter binding assay as described above. n.i: no inhibition.

Structural basis for compound selectivity

Molecular modeling experiments of the best inhibitors of each series (compounds 6, 11, 16, 21 and 26) were further analyzed in order to rationalize their mode of action. Fig. 2 shows that the C7-phenyl and C7-quinoline 7-deaza-adenine in compounds 21 (panel D) and 26 (panel E) perfectly overlay the adenine nucleobase of SAH present in the structure of SARS-CoV-2 nsp14 (PDB 7R2V). Similar results were obtained in our docking experiments based on the structure of the SARS-CoV nsp14-nsp10 complex solved in the presence of SAM (PDB 5C8T) [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF], which shares 95% sequence identity with SARS-CoV-2 nsp14. The amine moiety on the adenine base makes a key interaction with the backbone carbonyl of Tyr368 (Fig. 2D, 2E and 2F), which is lost in the N 6 -methyl adenosine derivatives 7-16 (Fig. 2B and2C) and in the inosine analogues 1-6 (Fig. 2A). These modifications of N 6 -NH2 in compounds 1-16 likely impact the orientation of the nucleobase that does not superpose to SAH anymore, and give lower docking scores (Table 2). On the 5' side of the nucleoside inhibitors, the phenyl moiety substituted in meta (NO2, CN, or Cl) and para (Me, OMe and Br) positions sits in the cap binding pocket and makes a π-π stacking interactions with Phe426 (d = 3.6 -4 Å), as previously reported (Fig. 2, blue dashes) [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. Additionally, the docking poses of the two best inhibitors 21 and 26 suggest potential interactions of the methoxy substituent with the side-chains of Gln388 and Thr428 (Fig. 2D,2E and 2F).

SARS-CoV-2 inhibition assay in Vero E6 TMPRSS2 cells

To assess whether the inhibitory activity observed in SARS-CoV-2 N7-MTase nsp14 inhibition assays translates to viral replication inhibition, the antiviral activity of the best nsp14 inhibitors 17-26 (0.72 nM < IC50 < 25.6 nM) was evaluated in cell culture in a viral RNA yield reduction assay using qRT-PCR for viral RNA quantification [START_REF] Touret | Phylogenetically based establishment of a dengue virus panel, representing all available genotypes, as a tool in dengue drug discovery[END_REF][START_REF] Touret | In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication[END_REF][START_REF] Kaptein | A pan-serotype dengue virus inhibitor targeting the NS3-NS4B interaction[END_REF][START_REF] Touret | Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2[END_REF]. A dose-response inhibition assay was performed with compounds concentrations from 0.4 µM to 10 µM in Vero E6 TMPRSS2 cells infected with SARS-CoV-2 (BavPat; lineage B.1) (Fig. 4A). The results of this study reveal that most of the compounds exhibited no cytotoxicity for the cells at concentrations up to 10 µM (Fig. 4B). Compounds 22-26 containing C7-quinolin-ethynyl 7deaza-adenine moieties showed a moderate activity reaching barely 50% of viral inhibition at 10 µM concentration in comparison with compounds 17-21 with a phenyl-ethynyl substituent at C7 of adenine that did not display any inhibitory activity (Fig. 4A). The antiviral effect at 10 µM concentration, albeit weak, suggests that compounds 22-26 were able to enter, at least partly, VeroE6 TMPRSS2 cells to mediate inhibition of SARS-CoV-2 replication. Remdesivir was used as a positive control with the same concentrations.

Our results and the various studies published in the last two years on SARS-CoV-2 nsp14 inhibitors did not reveal any significant antiviral activity, which was unexpected given the potent nanomolar to subnanomolar inhibitions obtained on the purified enzyme [START_REF] Bobileva | Potent SARS-CoV-2 mRNA cap methyltransferase inhibitors by bioisosteric replacement of methionine in SAM cosubstrate[END_REF][START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF][START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF][START_REF] Jung | Bisubstrate inhibitors of severe acute respiratory syndrome coronavirus-2 nsp14 methyltransferase[END_REF][START_REF] Bobileva | 3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors[END_REF]. Several hypotheses could explain the moderate inhibition observed. The poor activity could result either delivery issue to the viral replication site or from the early metabolism of the compounds in the cells before they reach their target. Nevertheless, a recent study on compounds of similar chemical structure showed that they were stable in plasma and in human liver S9 fraction containing microsomal and cytosolic enzymes [START_REF] Jung | Bisubstrate inhibitors of severe acute respiratory syndrome coronavirus-2 nsp14 methyltransferase[END_REF]. Finally, even several reverse genetic studies have demonstrated that SARS-CoV-2 replication is strongly impaired by mutagenesis of the N7-MTase catalytic site [START_REF] Ogando | Structure-function analysis of the nsp14 N7guanine methyltransferase reveals an essential role in Betacoronavirus replication[END_REF], it cannot be ruled out that the antiviral effect of these mutations results from an unidentified nsp14 function and that the nsp14 N7-MTase activity is not absolutely required for viral replication in our cell system. Further work is now needed to address these different possibilities.

Conclusion

Altogether were stored at -20 °C for several months without any degradation.

General method A for the synthesis of compounds 33-38. To a solution of compounds 27-32 [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF] in acetic acid/water (85/15 v:v, C = 0.09 M) was added NaNO2 (15.00 eq). After stirring at 25 °C for 48 h, another NaNO2 portion (10.00 eq) was added and the mixture was allowed to react at 25 °C for 24 h. After completion of the reaction, the volatiles were removed in vacuo, the residue was diluted with AcOEt and washed with water. Aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (dry sample, silica gel, linear gradient 0-5% MeOH in CH2Cl2) to give the desired compound as a solid.

5'-deoxy-5'-N-(3-chloro-4-methylbenzenesulfonyl)-2',3'-isopropylideneinosine (33).

Following method A with 27 (163 mg, 0.33 mmol, 1.00 eq), 33 was obtained as an off-white solid (121 mg, 74%

5'-deoxy-5'-N-(3-cyano-4-methoxybenzenesulfonyl)-2',3'-isopropylideneinosine (35).

Following method A with 29 (100 mg, 0.20 mmol, 1.00 eq), 35 was obtained as an off-white solid (65 mg, 65%). Rf 0.37 (1:9 MeOH/CH2Cl2).

5'-deoxy-5'-N-(ethyl)-(3-chloro-4-methylbenzenesulfonyl)-2',3'-isopropylideneinosine (36).

Following method A with 30 (135 mg, 0.26 mmol, 1.00 eq), 36 was obtained as an off-white solid (81 mg, 60% [START_REF] Milisavljevic | Antiviral activity of 7substituted 7-deazapurine ribonucleosides, monophosphate prodrugs, and triphoshates against emerging RNA viruses[END_REF].

Following method A with 31 (100 mg, 0.18 mmol, 1.00 eq), 37 was obtained as an off-white solid (59 mg, 59%

5'-deoxy-5'-N-(ethyl)-(3-cyano-4-methoxybenzenesulfonyl)-2',3'-isopropylideneinosine (38).

Following method A with 32 (75 mg, 0.14 mmol, 1.00 eq), 38 was obtained as an off-white solid (60 mg, 80% General method B for the synthesis of compounds 40-44, 57-61, 62-66. To a solution of 39 obtained according to described procedures [START_REF] Yao | Selective inhibitors of histone methyltransferase DOT1L: design, synthesis, and crystallographic studies[END_REF][START_REF] Bhattarai | α,β-methylene-ADP (AOPCP) derivatives and analogues: development of potent and selective ecto-5′-nucleotidase (CD73) inhibitors[END_REF], 55 or 56 (1.00 eq) in anhydrous DMF (C = 0.1 M) were added Et3N (2.00 eq) and the corresponding benzenesulfonyl chloride reactant (1.25 eq) in three portions at -10 °C under argon. After stirring at -10 °C for 1.5-3 h, the reaction mixture was diluted with AcOEt and washed with brine. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with a 2M LiCl solution and brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography to give the desired compound as a solid.

5'-deoxy-5'-N-(3-nitro-4-bromobenzenesulfonyl)-2',3'-isopropylidene-N6-methyladenosine (40).

Following method B with 39 (400 mg, 1.25 mmol, 1.00 eq) and

5'-deoxy-5'-N-(3-nitro-4-methylbenzenesulfonyl)-2',3'-isopropylidene-N6-methyladenosine (41).

Following method B with 39 (400 mg, 1.25 mmol, 1.00 eq) and 4-methyl-3nitrobenzenesulfonyl chloride (368 mg, 1.56 mmol, 1.25 eq), 41 was obtained as a white foam (522 mg, 80%). Rf 0.53 (1:1 acetone/CH2Cl2).

5'-deoxy-5'-N-(3-chloro-4-methylbenzenesulfonyl)-2',3'-isopropylidene-N6-methyladenosine (42).

Following method B with 39 (400 mg, 1.25 mmol, 1.00 eq) and 3-chloro-4methylbenzenesulfonyl chloride (351 mg, 1.56 mmol, 1.25 eq), 42 was obtained as a white foam (582 mg, 91%). Rf 0.66 (1:1 acetone/CH2Cl2).

5'-deoxy-5'-N-(3-nitro-4-chlorobenzenesulfonyl)-2',3'-isopropylidene-N6-methyladenosine (43).

Following method B with 39 (500 mg, 1.56 mmol, 1.00 eq) and 4-chloro-3nitrobenzenesulfonyl chloride (499 mg, 1.95 mmol, 1.25 eq), 43 was obtained as a white foam (524 mg, 62%). Rf 0.65 (1:1 acetone/CH2Cl2).

5'-deoxy-5'-N-(3-cyano-4-fluorobenzenesulfonyl)-2',3'-isopropylidene-N6-methyladenosine (44).

Following method B with 39 (500 mg, 1.56 mmol, 1.00 eq) and 3-cyano-4fluorobenzenesulfonyl chloride (428 mg, 1.95 mmol, 1.25 eq), 44 was obtained as a white foam (712 mg, 90%). Rf 0.57 (1:1 acetone/CH2Cl2).

5'-deoxy-5'-N-(methyl)-(3-cyano-4-fluorobenzenesulfonyl)-2',3'-isopropylidene-7phenylethynyl-7-deazaadenosine (61).

Following method B with 55 (100 mg, 0.24 mmol, 1.00 eq) and 3-cyano-4fluorobenzenesulfonyl chloride (65 mg, 0.30 mmol, 1.25 eq), 61 was obtained as an off-white solid (128 mg, 89%). Rf 0.54 (1:9 MeOH/CH2Cl2). [M+H] + : 603.1820, Found 603.1814.

5'-deoxy-5'-N-(methyl)-(3-nitro-4-bromobenzenesulfonyl)-2',3'-isopropylidene-7-(quinolin-3ethynyl)-7-deazaadenosine (62).

Following method A with 56 (55 mg, 0.12 mmol, 1.00 eq) and 3-nitro-4-bromobenzenesulfonyl chloride (44 mg, 0.15 mmol, 1.25 eq), 62 was obtained as an off-white solid (75 mg, 87%

5'-deoxy-5'-N-(methyl)-(3-nitro-4-methylbenzenesulfonyl)-2',3'-isopropylidene-7-(quinolin-3-ethynyl)-7-deazaadenosine (63).

Following method A with 56 (40 mg, 0.08 mmol, 1.00 eq) and 3-nitro-4-methylbenzenesulfonyl chloride (25 mg, 0.11 mmol, 1.25 eq), 63 was obtained as an off-white solid (40 mg, 70%). Rf

5'-deoxy-5'-N-(methyl)-3-chloro-4-methylbenzenesulfonyl)-2',3'-isopropylidene-7-(quinolin-3-ethynyl)-7-deazaadenosine (64).

Following method A with 56 (55 mg, 0.12 mmol, 1.00 eq) and 3-chloro-4methylbenzenesulfonyl chloride (33 mg, 0.15 mmol, 1.25 eq), 64 was obtained as an off-white solid (55 mg, 71%). Rf 0.70 (1:9 MeOH/CH2Cl2).

5'-deoxy-5'-N-(methyl)-(3-nitro-4-methoxybenzenesulfonyl)-2',3'-isopropylidene-7-(quinolin-3-ethynyl)-7-deazaadenosine (65).

Following method A with 56 (55 mg, 0.12 mmol, 1.00 eq) and 4-methoxy-3nitrobenzenesulfonyl chloride (35 mg, 0.14 mmol, 1.25 eq), 65 was obtained as an off-white solid (69 mg, 86%

5'-deoxy-5'-N-(methyl)-(3-cyano-4-fluorobenzenesulfonyl)-2',3'-isopropylidene-7-(quinolin-3-ethynyl)-7-deazaadenosine (66).

Following method A with 56 (103 mg, 0.22 mmol, 1.00 eq) and 3-cyano-4fluorobenzenesulfonyl chloride (60 mg, 0.27 mmol, 1.25 eq), 66 was obtained as an off-white solid (130 mg, 91%). Rf 0.62 (1:9 MeOH/CH2Cl2). and concentrated in vacuo. The residue was purified by column chromatography (liquid deposit, silica gel, linear gradient 1-3% MeOH in CH2Cl2) to give the desired compound as a foam.

5'-deoxy-5'-N-(ethyl)-(3-nitro-4-bromobenzenesulfonyl)-2',3'-isopropylidene-N6methyladenosine [START_REF] Aouadi | Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10[END_REF].

Following method D with 40 (200 mg, 0.34 mmol, 1.00 eq), 47 was obtained as a white foam (129 mg, 62%). Rf 0.71 (1:9 MeOH/CH2Cl2). General method E for the synthesis of final compounds 1-6. Compounds 33-38 were treated with a formic acid/water solution (1/1 v:v, C = 0.05 M). After stirring at 25 °C for 24-48 h until completion of the reaction, solvents were removed in vacuo and the crude was co-evaporated three times with absolute EtOH. The residues were purified by flash column chromatography (dry sample, silica gel, linear gradient 0-10% MeOH in CH2Cl2). Fractions containing the pure products were concentrated and trituration in Et2O afforded the desired compounds as solids.

5'-deoxy-5'-N-(3-chloro-4-methylbenzenesulfonyl)inosine (1).

Following method E with 33 (121 mg, 0.24 mmol, 1.00 eq), 1 was obtained as an off-white solid (80 mg, 72%). Rf 0.07 (1:9 MeOH/CH2Cl2).

5'-deoxy-5'-N-(4-methoxy-3-nitrobenzenesulfonyl)inosine (2).

Following method E with 34 (123 mg, 0.24 mmol, 1.00 eq), 2 was obtained as an off-white solid (72 mg, 63%). Rf 0.12 (1:9 MeOH/CH2Cl2).

5'-deoxy-5'-N-(3-cyano-4-methoxybenzenesulfonyl)inosine (3).

Following method E with 35 (65 mg, 0.13 mmol, 1.00 eq), 3 was obtained as an off-white solid (40 mg, 67%

5'-deoxy-5'-N-(ethyl)-(3-chloro-4-methylbenzenesulfonyl)inosine (4).

Following method E with 36 (174 mg, 0.33 mmol, 1.00 eq), 4 was obtained as an off-white solid (112 mg, 70%

5'-deoxy-5'-N-(ethyl)-(4-methoxy-3-nitrobenzenesulfonyl)inosine (5).

Following method E with 37 (32 mg, 0.06 mmol, 1.00 eq), 5 was obtained as an off-white solid (21 mg, 71%

5'-deoxy-5'-N-(ethyl)-(3-cyano-4-methoxybenzenesulfonyl)inosine (6).

Following method E with 38 (60 mg, 0.11 mmol, 1.00 eq), 6 was obtained as an off-white solid (40 mg, 67%). Rf 0.08 (1:9 MeOH/CH2Cl2).

5'-deoxy-5'-N-(methyl)-(3-cyano-4-methoxybenzenesulfonyl)-7-(quinolin-3-ethynyl)-7-

deazaadenosine [START_REF] Ma | Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex[END_REF].

Following method F with 68 (100 mg, 0.15 mmol, 1.00 eq), 26 was obtained as an off-white solid (50 mg, 53%). Rf 0.33 (1:9 MeOH/CH2Cl2).

Expression and purification of recombinant proteins

SARS-CoV and SARS-CoV-2 nsp14 (N7-MTase), SARS-CoV-2 nsp10 and nsp16 (2'O-MTase), the human RNA N7-methyltransferase (hRNMT) and the Dengue virus serotype 3 methyltransferase (NS5 MTase) coding sequences were cloned in fusion with a N-terminus hexa-histidine tag in pET28 plasmids [START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF] and in Gateway-compatible plasmids as described in [START_REF] Peyrane | High-yield production of short GpppA-and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions[END_REF][START_REF] Egloff | An RNA cap (nucleoside-2'-O-)methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization[END_REF]. The MERS-CoV nsp14 was produced and purified as previously described [START_REF] Aouadi | Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10[END_REF]. The other proteins were expressed in E.coli C2566 and purified in a two-step IMAC using cobalt beads. Briefly, cells were lysed by sonication in a buffer containing 50 mM Tris pH 6.8, 300 mM NaCl, 10 mM imidazole, 5 mM MgCl2, and 1 mM BME, supplemented with 0.25 mg/mL lysozyme, 10 μg/mL DNase, and 1 mM PMSF. The proteins were next purified through affinity chromatography with HisPur Cobalt resin 480 (Thermo Scientific), washing with an increased concentration of salt (1 M NaCl) and imidazole (20 mM), prior to elution in buffer supplemented with 250 mM imidazole. The second step of purification was performed by size exclusion chomatography (GE Superdex S200) in a final buffer of 50 mM Tris pH 6.8, 300 mM NaCl, 5 mM MgCl2, and 1 mM BME and the proteins were subsequently concentrated up to 12.5 µM and conserved at -20 °C in a buffer containing 50% of glycerol.

Determination of the MTase activity by filter binding assay (FBA)

The

Molecular docking

All molecular docking experiments were performed using Autodock Vina [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF]. The X-ray crystal structure of SARS-CoV-2 nsp14 (PDB 7R2V) was used as the receptor and prepared in Chimera [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF]. The ligand structures were drawn and minimized using MarvinSketch (ChemAxon). The receptor and ligand structures were converted to PDBQT format using MGL Tools [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF]. The exhaustiveness was set to 10 and default parameters were used, unless otherwise stated. The ligand was treated as flexible and the protein was kept rigid. The grid size was set to 14 x 16 x 20 (Å) and the grid box's center points were set to target the active site of the protein, with the center at X = 14.2, Y = -12.3, Z = -20.1. The docking poses were further filtered for superposition of the adenine nucleobase present in our compounds with the SAH molecule observed in the crystal structure. 3D and 2D representations of protein-ligand complexes were visualized using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC) and Ligplot+, respectively [START_REF] Laskowski | LigPlot+: multiple ligand-protein interaction diagrams for drug discovery[END_REF].

SARS-CoV-2 inhibition assay in Vero E6 TMPRSS2 cells

The experimental procedures for the antiviral activity assay against SARS-CoV-2 in cell culture were as previously described [START_REF] Touret | Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2[END_REF][START_REF] Weiss | Niclosamide shows strong antiviral activity in a human airway model of SARS-CoV-2 infection and a conserved potency against the Alpha (B.1.1.7), Beta (B.1.351) and Delta variant (B.1.617.2[END_REF]. Vero E6 TMPRSS2 cells (ID 100978) were obtained from CFAR and were grown in minimal essential medium (Life Technologies) with 10% heatinactivated fetal calf serum (FCS; Life Technologies), at 37°C with 5% CO2 with 1% penicillin/streptomycin (5000 U/mL1 and 5000 μg/mL respectively; Life Technologies) and supplemented with 1% non-essential amino acids (Life Technologies) and G-418 (Life Technologies). SARS-CoV-2 strain BavPat (lineage B.1) was obtained from Pr. C. Drosten through EVA GLOBAL (https://www.european-virus-archive.com/). All experiments with infectious virus were conducted in a biosafety level 3 laboratory. One day prior to infection, 5 × 10 4 Vero E6 TMPRSS2 cells were seeded per well in 100 μL medium in 96 well culture plates. The next day, four 3-fold dilutions of compounds (from 0.37 μM to 10 µM in duplicate were added to the cells using a Tecan d300e (Tecan) and were supplemented with 25μL/well of assay medium). Virus control wells were supplemented with 25 μL of assay medium, and 25 μL of a virus mix diluted in medium was added to the wells. The amount of virus working stock used was calibrated prior to the assay, based on a replication kinetics, so that the viral replication was still in the exponential growth phase for the readout as previously described. On each

Fig. 1 .

 1 Fig. 1. Design of novel bisubstrate adenosine mimetics 1-26 as potential inhibitors of SARS-CoV-2 nsp14 N7-MTase derived from a previous series of N-arylsulfonamide adenosine analogues with MH44 as the best inhibitor (IC50 = 19 nM) [23].

Scheme 1 .c 2 .

 12 Scheme 1. Synthesis of 5'-N-arylsulfonamide inosine derivatives 1-6. (a) NaNO2, AcOH/H2O 85:15 v/v, 25 °C, 72 h, 59 -80%; (b) HCO2H / H2O 1:1 v/v, 25 °C, 24-48 h, 63-72%.To prepare N 6 -methyladenosine compounds 7-16, we first synthesized 5'-amino-2',3'-Oisopropylidene-N 6 -methyladenosine 39 following known procedures[START_REF] Yao | Selective inhibitors of histone methyltransferase DOT1L: design, synthesis, and crystallographic studies[END_REF][START_REF] Bhattarai | α,β-methylene-ADP (AOPCP) derivatives and analogues: development of potent and selective ecto-5′-nucleotidase (CD73) inhibitors[END_REF] (Scheme 2). Coupling of 39 with the corresponding commercially available benzenesulfonyl chloride reagents afforded the intermediates 40-44 in 62-91% yields[START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. To obtain compounds 45 and 46 with a p-OMe group, aromatic nucleophilic substitution was performed on 43 with a p-Cl atom and on 44 with a p-F atom using sodium methoxide in methanol at 50 °C. The amino function of compounds 40-42, 45, 46 was derivatized with an ethyl group using ethyl ptoluenesulfonate, potassium carbonate and potassium iodide to obtain the N-ethyl sulfonamide derivatives 47-51 in yields ranging from 48 to 90%. It is noteworthy that a catalytic amount of potassium iodide is required to form ethyl iodide, which is sufficiently reactive to be substituted by the deprotonated sulfonamide while the tosylate reagent is not. In addition, ethyl iodide was used in stoichiometric default to avoid the formation of by-products due to the possible reaction with at least the N 6 -amino group. Finally, the isopropylidene groups were removed from 40-42 and 45-51 by treatment with a TFA/water mixture for 30 min to give the desired compounds 7-16.

  led to the formation of 7ethynylphenyl-7-deaza-adenosine 53 and 7-ethynylquinoline-7-deaza-adenosine 54, respectively, both in high yields (85-95%). The 5'-aminomethyl derivatives 55 and 56 were obtained by reaction of 53 and 54 with mesyl chloride in dichloromethane and then with methylamine. The amino functions of 55 and 56 were functionalized with different benzenesulfonyl chlorides to give 57-66. From 61 and 66 bearing a fluorine atom in the paraposition of the phenyl ring, an aromatic nucleophilic substitution with sodium methoxide afforded 67 and 68 with a p-OMe group, respectively. Intermediate compounds 57-60, 62-65, 67-68 were deprotected in a TFA/H2O mixture to obtain the final 7-ethynylaryl-7-deaza-

  and the MTase activity was measured by FBA. After normalization, the IC50 values were deduced by Hill slope (Y = 100/[1 + ((X/IC50)^Hillslope)]) curve-fitting. Interestingly, the C7substituted-7-deaza-adenine nucleosides 17-26 exhibited a potent inhibitory effect with IC50 values in the subnanomolar or nanomolar range. In average, the series of compounds with the 3-quinoline substitution[START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF][START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF][START_REF] Jung | Bisubstrate inhibitors of severe acute respiratory syndrome coronavirus-2 nsp14 methyltransferase[END_REF][START_REF] Bobileva | 3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors[END_REF][START_REF] Ma | Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex[END_REF] showed slightly more potent inhibition than the phenylsubstituted compounds 17-21 and the best compound 26 displays an IC50 value of 0.72 nM. The ΔG obtained from the docking on SARS-CoV-2 N7-MTase also suggests that the presence of a 3-quinoline (22-26, average score of -11.4 kcal/mol) is slightly more beneficial than a phenyl (17-21, average score -10.4 kcal/mol). Regarding the substitution on the N-alkyl arylsulfonamide moiety, the effect on the IC50 values is the same for these two series (3-CN, 4-OMe > 3-NO2, 4-OMe > 3-NO2, 4-Me > 3-NO2, 4-Br > 3-Cl, 4-Me) and is in agreement with our previous results[START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF]. Furthermore, if we compare the inhibitory activity of 21 and 26 to that of MH44, which all harbor the same N-arylsulfonamide moiety (with 3-CN, 4-OMe as phenyl substituents), we notice that replacing adenine with C7-phenyl or C7-quinoline 7-deazaadenine significantly increases nsp14 inhibition (16-fold for 21 and 26-fold for 26). On the other hand, the replacement of adenine by hypoxanthine in 6 and the methyl on the exocyclic NH2 (N 6 ) of adenine in 7-16 result in a lower inhibitory effect on the nsp14 N7-MTase, as evidenced by their overall higher IC50 values in the micromolar or submicromolar range. These compounds also show higher ΔG values in the docking experiments, as expected (average score of -9.1 kcal/mol).

Fig. 2 .

 2 Fig. 2. Overlay of the best inhibitors of each series and the SAH molecule present in the structure of SARS-CoV-2 nsp14. The protein is represented in light grey cartoon, the main residues interacting with the inhibitors are shown in light grey lines. SAH is shown in dark grey sticks. Hydrogen bonds between the inhibitors and the protein are highlighted in green, π-stacking interactions are shown in blue. A. Compound 6 (purple sticks). B. Compound 11 (yellow sticks). C. Compound 16 (light brown sticks). D. Compound 21 (orange sticks). E. Compound 26 (beige sticks). F. 2D representation of the predicted interactions between the best inhibitor 26 and SARS-CoV-2 nsp14.

Fig. 3 .

 3 Fig. 3. A. Model of 26 (beige sticks) binding to SARS-CoV-2 nsp14 (PDB 7R2V, grey, top panel) or to SARS-CoV nsp14 (PDB 5C8T, teal, middle panel), showing the 3-quinoline moiety protruding from the SAM entry tunnel, which is absent in the human ortholog enzyme N7-methyltransferase (PDB 3BGV, yellow, bottom panel). B. Comparison of the structure of SARS-CoV-2 nsp14 (PDB 7R2V, grey cartoon) bound to 26 (beige sticks) with the apo structure (PDB 7QGI, light pink cartoon), showing the presence of a flexible loop closing the SAM entry gate.

Fig. 4 .

 4 Fig. 4. Dose-response assay on compounds 17-26 on (A) SARS-CoV-2 (B.1) viral inhibition in % of untreated infected control and (B) Vero E6 TMPRSS2 cell viability in % of untreated control. These experiments were performed in duplicate at four concentrations: 10 µM, 3.3 µM, 1.1 µM and 0.4 µM (from left to right bars). Remdesivir was used as a positive control with the same concentrations.

  , the SAR presented in this work demonstrates the possibility to optimize the inhibitory activity of adenosine-derived compounds, based on the SAM-bound SARS nsp14 crystal structures. By combining the substitution of the nucleobase at the C7 position with modifications on the ribose mimicking the GTP moiety, we obtained a series of compounds inhibiting the N7-MTase activity of SARS-CoV-2 nsp14 at nanomolar concentrations and one compound with subnanomolar IC50. The nanomolar inhibitions obtained with 7-deaza adenosine analogues as SAH mimetics are consistent with the results of Nencka et al.[START_REF] Otava | The structure-based design of SARS-CoV-2 nsp14 methyltransferase ligands yields nanomolar inhibitors[END_REF]. It is noteworthy that for the first time, the subnanomolar range could be reached by our scaffold combining the 5'-deoxy 5'-(N-methyl N-arylsulfonamide) moiety with C7-quinolin-ethynyl 7deaza-adenine. Docking analyses suggest that C7-adenine nucleobase superimposes perfectly with the adenine of the SAM in the nsp14 structures, while the 3-quinoline or the phenyl substituent on the nucleobase protrudes into the SAM entry channel. These substituents may thus block the mobile loop that controls the SAM entry gate in its open position and prevent SAM access to the MTase substrate[START_REF] Imprachim | Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development[END_REF]. Because the nsp14 protein from SARS-CoV, SARS-CoV-2 and MERS-CoV share a similar structural organization, we hypothesized that our compounds might have pan-CoV inhibitory activity. This was confirmed by demonstrating that the best compounds inhibit SARS-CoV, SARS CoV-2 and MERS-CoV nsp14 N7-MTase activity. In the event of the emergence of a new coronavirus that would lead to a potential pandemic, the development of pan-CoV inhibitors appears to be essential and our original inhibitors fit into this class. Conversely, we did not observe any inhibition of dengue virus NS5 MTase activity and the human N7-MTase involved in RNA capping is barely inhibited by our compounds, thus demonstrating their selectivity. The lack of inhibition of human N7-MTase is probably related to its different structural organization, as the SAM entry tunnel and the mobile loop recently described for nsp14 are absent in the human ortholog. This SAR study thus led to a new series of compounds that inhibit N7-MTases of coronaviruses with subnanomolar to nanomolar activity, while sparing the cognate human N7-MTase. The potent and selective inhibition of these adenosine mimetics on SARS-CoVs and MERS-CoV N7-MTases argues for further work to enable their internalization into cells towards DMV, the stability of compounds upon intracellular penetration and demonstrate cellular antiviral activity against CoVs. 4. Experimental section 4.1. Chemistry 4.1.1. General procedures. All dry solvents and reagents were purchased from commercial suppliers and were used without further purification. Thin-layer chromatography (TLC) analyses were carried out on silica plate 60 F254. Purifications by column chromatography were performed using Biotage Isolera 1 system or Buchi C-815 system with Flash-Pure cartridges (Buchi). NMR experiments were recorded on Bruker 500 or 600 MHz spectrometer at 20 °C. HRMS analyses were obtained with electrospray ionization (ESI) in positive mode on a Q-TOF Micromass spectrometer. Analytical HPLC was performed on a UHPLC Thermoscientific Ultimate 3000 system equipped with a LPG-3400RS pump, a DAD 3000 detector and an WPS-3000TBRS Autosampler, Column Oven TCC-3000SD. Nucleosides 1 -26 were analyzed by RP-HPLC on a Column Nucleodur C18 ec 100-3, 4.6 x 75 mm (Macherey Nagel) at 30 °C. The following HPLC solvent systems were used: 1% CH3CN in 50 mM TEAAc (buffer A), 80% CH3CN in 50 mM TEEAc (buffer B). Flow rate was 1 mL/min. UV detection was performed at 260 nm. Finals compounds 1-26

0. 51 ( 1 : 9

 5119 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 9.04 (d, J = 2.1 Hz, 1H) ; 8.65 (dd, J = 2.2, 0.7 Hz, 1H) ; 8.23 (d, J = 2.0 Hz, 1H) ; 8.20 (s, 1H) ; 8.05 (dd, J = 8.3, 1.0 Hz, 1H) ; 8.01 (dd, J = 8.2, 1.4 Hz, 1H) ; 7.96 (s, 1H) ; 7.91 (dd, J = 8.1, 2.0 Hz, 1H) ; 7.81 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H) ; 7.71 -7.63 (m, 2H) ; 6.23 (d, J = 2.7 Hz, 1H) ; 5.32 (dd, J = 6.5, 2.7 Hz, 1H) ; 5.02 (dd, J = 6.5, 3.8 Hz, 1H) ; 4.25 (ddd, J = 7.4, 5.3, 3.7 Hz, 1H) ;

  1 H-NMR (600 MHz, DMSO-d6) δ 12.40 (s, 1H) ; 8.27 -8.23 (m, 2H) ; 8.11 (br. s, 1H) ; 8.03 -7.99 (m, 2H) ; 7.50 (d, J = 9.0 Hz, 1H) ; 5.81 (d, J = 6.0 Hz, 1H) ; 5.54 (d, J = 6.1 Hz, 1H) ; 5.32 (d, J = 4.9 Hz, 1H) ; 4.53 (q, J = 5.5 Hz, 1H) ; 4.09 -4.04 (m, 1H) ; 4.00 (s, 3H) ; 3.95 -3.89 (m, 1H) ; 3.16 (dd, J = 13.6, 4.5 Hz, 1H) ; 3.07 (dd, J = 13.6, 6.4 Hz, 1H).

  1 H-NMR (600 MHz, DMSO-d6) δ 9.04 (d, J = 2.1 Hz, 1H) ; 8.64 (d, J = 2.3 Hz, 1H) ; 8.21 (d, J = 2.4 Hz, 1H) ; 8.18 (s, 1H) ; 8.08-8.03 (m, 2H) ; 8.03-7.99 (m, 2H) ; 7.81 (ddd, J = 8.5, 6.8, 1.5 Hz, 1H) ; 7.68 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H) ; 7.41 (d, J = 9.0 Hz, 1H) ; 6.10 (d, J = 6.0 Hz, 1H) ; 5.49 (d, J = 6.3 Hz, 1H) ; 5.35 (d, J = 5.0 Hz, 1H) ; 4.51 (q, J = 5.9 Hz, 1H) ; 4.12 (td, J = 5.0, 3.6 Hz, 1H) ; 4.03 (ddd, J = 7.4, 4.9, 3.6 Hz, 1H) ; 4.00 (s, 3H) ; 3.50 (dd, J = 14.1, 5.0 Hz, 1H) ; 3.19 (dd, J = 14.2, 7.5 Hz, 1H) ; 2.72 (s, 1H).

  hRNMT, DV3 NS5, SARS-CoV, SARS-CoV-2 and MERS-CoV nsp14 MTase assays were carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl2, 2μM SAM, and 0.1 μM 3 H-SAM (Perkin Elmer)] in the presence of 0.7 μM GpppAC4 synthetic RNA[START_REF] Ahmed-Belkacem | Potent inhibition of SARS-CoV-2 nsp14 N7-methyltransferase by sulfonamide-based bisubstrate analogues[END_REF] and the corresponding MTases. The SARS-CoV-2 2'O-MTase assay was performed in the same experimental conditions except that 7m GpppAC4 synthetic RNA was used in the presence of 1mM MgCl2.The concentration of MTases was adjusted in each assay in order to get similar enzymatic activity in the FBA during the linear phase of the enzymatic reaction.Briefly, the enzymes were first mixed with the compound suspended in 50% DMSO (2.5% final DMSO) before the addition of RNA substrate and SAM and then incubated at 30 °C. Reactions mixtures were stopped after 30 min by their 10-fold dilution in ice-cold water. Samples were transferred to diethylaminoethyl (DEAE) filtermat (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol. They were soaked with scintillation fluid (Perkin Elmer), and 3 H-methyl transfer to the RNA substrates was determined using a Wallac MicroBeta TriLux liquid scintillation counter (Perkin Elmer). For IC50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y = 100/[1 + ((X/IC50)^Hillslope)]. IC50 is defined as the inhibitory compound concentration that causes 50% reduction in enzyme activity.

61, 66 67, 68 d 57 -66 17 -26 55

				R 4
			N
			R 4	R 4	R 4
	56	R 4 4 R	= phenyl = 3-quinoline
	Scheme 3. (a) appropriate substituted alkyne reagents, Et3N, CuI, Pd(PPh3)4, THF, 60 °C, 2 h, 85-95%; (b) (i)
	MsCl, Et3N, CH2Cl2, -10 °C, 30 min, 80-87%, (ii) MeNH2 2M/THF, 50 °C, 72 h, 31-55%; (c) appropriate
	substituted benzenesulfonyl chloride reagents, Et3N, DMF, -10 °C, 1.5-3 h, 57-95%; (d) MeONa 0.5 M/MeOH,
	50 °C, 24-48 h, 73-83%; (e) TFA/H2O 8:2 v/v, 25 °C, 30 min, 16-66%.

Table 1

 1

	Screening for inhibitory activity of compounds 1-26 at 5 µM on various viral and cellular N7-MTases
		Compounds
		R 2
	R 1	O	S	O N R 3	O	B*		Percentage of MTase activity at 5 µM (%) a
				OH	OH
	N°	B*		R1		R2	R3	SARS-CoV nsp14	SARS-CoV-2 nsp14	DV3 NS5	hRNMT
	MH44	A		OMe CN Et	4.2 ± 2.2	0.1 ± 0.4	n.i	69.5 ± 16.4
	1			Me		Cl		71.8 ± 4.8	87.6 ± 3.3	n.i	n.i
	2	HXN		OMe NO2	H	91.7 ± 4.8	93.6 ± 7.9	n.i	n.i
	3			OMe CN		98.4 ± 5.0	86.0 ± 0.2	n.i	n.i
	4			Me		Cl		91.0 ± 8.6	75.5 ± 4.5	n.i	n.i
	5	HXN		OMe NO2	Et	69.8 ± 8.7	56.4 ± 3.9 86.4 ± 16.9	n.i
	6			OMe CN		48.1 ± 4.8	30.7 ± 1.6 89.8 ± 5.9 88.9 ± 15.9
	7			Br		NO2		54.0 ± 7.3	33.8 ± 2.0 78.1 ± 4.4	57.0 ± 9.8
	8			Me NO2		66.4 ± 6.9	38.7 ± 3.6 76.6 ± 9.5 66.4 ± 11.3
	9	N 6m A		Me		Cl	H	53.5 ± 3.3	45.4 ± 2.8	n.i	92.0 ± 17.7
	10			OMe NO2		41.9 ± 2.6	31.5 ± 1.4	n.i	n.i
	11			OMe CN		45.1 ± 0.7	25.4 ± 2.6	n.i	n.i
	12			Br		NO2		29.5 ± 0.6	15.2 ± 1.8 98.0 ± 2.3	n.i
	13			Me NO2		20.1 ± 1.7	10.9 ± 0.7 97.1 ± 5.1	n.i
	14	N 6m A		Me		Cl	Et	10.9 ± 1.8	8.3 ± 0.2	n.i	78.0 ± 1.6
	15			OMe NO2		16.4 ± 0.1	10.3 ± 0.3 90.0 ± 2.8	73.3 ± 5.1
	16			OMe CN		4.3 ± 0.2	4.2 ± 0.2	80.2 ± 7.0	55.9 ± 2.7
	17			Br		NO2		3.3 ± 1.0	5.0 ± 1.1	96.9 ± 1.5	95.9 ± 0.3
	18			Me NO2		1.0 ± 0.4	1.0 ± 0.9	93.3 ± 5.7	n.i
	19	C 7Ph A		Me		Cl	Me	2.4 ± 0.3	7.0 ± 4.6	94.8 ± 3.4	n.i
	20			OMe NO2		0.5 ± 0.3	0.0 ± 0.2	91.6 ± 3.2	n.i
	21			OMe CN		1.0 ± 1.0	2.4 ± 0.3	n.i	n.i
	22 23	C 7Q A		Br Me NO2 NO2	Me	5.5 ± 0.9 1.9 ± 0.2	5.4 ± 1.1 4.2 ± 0.2	n.i 96.0 ± 1.1 86.7 ± 25.7 52.3 ± 20.3

Table 2

 2 Comparison of ∆G values calculated by Autodock Vina with the IC50 values of 21 most active compounds on SARS-CoV-2 N7-MTase activity.

		SARS-CoV-2 nsp14	SARS-CoV-2 nsp14
	Compounds	Docking ∆G a	N7-MTase IC50 b
		(kcal/mol)	(nM)
	Sinefungin	-7.9	360 c
	MH44 d	-8.7	19 ± 2
	6	-8.3	4000 ± 2800
	7	-9.4	3408 ± 1837
	8	-9.0	2850 ± 1467
	9	-9.8	2180 ± 120
	10	-9.0	573 ± 122
	11	-9.6	468 ± 150
	12	-9.1	383 ± 56
	13	-8.7	187 ± 46
	14	-9.1	130 ± 89
	15	-8.3	274 ± 165
	16	-8.9	62 ± 2
	17	-10.2	17.3 ± 0.6
	18	-10.9	9.5 ± 0.6
	19	-10.5	25.6 ± 10
	20	-10.1	2.8 ± 1.2
	21	-10.1	1.2 ± 0.25
	22	-12.0	3.5 ± 1.8
	23	-10.9	2.1 ± 1.3
	24	-11.1	2.07 ± 0.5
	25	-11.3	1.3 ± 1
	26	-11.5	0.72 ± 0.4

Table 3

 3 Comparison of IC50 values of the most active compounds of each series on SARS-CoV, SARS-CoV-2, MERS-CoV, DV3 and human N7-MTase activities

			IC50 a (nM)
	Compounds	SARS-CoV-2	SARS-CoV	MERS-CoV	Human	DV3
		nsp14	nsp14	nsp14	RNMT	NS5
	11	468 ± 150	3994 ± 700	9766 ± 5600	56166 ± 7100	n.i
	16	62 ± 2	51.7 ± 32	471.9 ± 147	16100 ± 3860	n.i
	21	1.2 ± 0.25	0.8 ± 0.5	1.25 ± 0.25	56500 ± 17930	n.i
	26	0.72 ± 0.4	0.141 ± 0.02	2.7 ± 0.8	165300 ± 30700	n.i

  Hz, 1H) ; 7.49 (d, J = 8.1 Hz, 1H) ; 6.09 (d, J = 2.8 Hz, 1H) ; 5.28 (dd, J = 6.4, 2.8 Hz, 1H) ; 4.88 (dd, J = 6.4, 3.3 Hz, 1H) ; 4.13 (ddd, J = 6.7, 5.5, 3.3 Hz, 1H) ; 3.10 (dd, J = 13.6, 5.6 Hz, 1H) ; 3.03 (dd, J = 13.7, 6.9 Hz, 1H) ; 2.37 (s, 3H) ; 1.50 (s, 3H) ; 1.28 (s, 3H).13 C-NMR (150 MHz, DMSO-d6) δ 156.5, 147.4, 145.9, 140.6, 139.5, 139.3, 133.8, 131.8, 126.6, 125.1, 124.8,

	113.6, 89.1, 84.5, 83.3, 81.4, 44.4, 26.9, 25.1, 19.7. HRMS (ESI+): m/z calcd for
	C20H23ClN5O6S [M+H] + : 496.1052, Found 496.1046.
	5'-deoxy-5'-N-(3-nitro-4-methoxybenzenesulfonyl)-2',3'-isopropylideneinosine (34).
	Following method A with 28 (156 mg, 0.30 mmol, 1.00 eq), 34 was obtained as an off-white
	solid (123 mg, 79%). Rf 0.54 (1:9 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 12.43 (d,
	J = 3.9 Hz, 1H) ; 8.25 -8.18 (m, 2H, H8) ; 8.10 (t, J = 6.0 Hz, 1H) ; 8.03 (d, J = 3.9 Hz, 1H) ;
	7.94 (dd, J = 8.9, 2.3 Hz, 1H) ; 7.46 (d, J = 8.9 Hz, 1H) ; 6.09 (d, J = 2.8 Hz, 1H) ; 5.28 (dd, J
	= 6.4, 2.9 Hz, 1H) ; 4.88 (dd, J = 6.3, 3.4 Hz, 1H) ; 4.16 -4.10 (m, 1H) ; 4.00 (s, 3H) ; 3.18 -
	3.03 (m, 2H) ; 1.50 (s, 3H) ; 1.28 (s, 3H).

). Rf 0.44 (1:9 MeOH/CH2Cl2).

1

H-NMR (600 MHz, DMSO-d6) δ 12.43 (s, 1H) ; 8.23 (s, 1H) ; 8.03 (br. s, 1H) ; 8.02 (s, 1H) ; 7.71 (d, J = 1.9 Hz, 1H) ; 7.57 (dd, J = 8.0, 1.9 13 C-NMR (150 MHz, DMSO-d6) δ 156.5, 154.7, 147.4, 146.0, 139.3, 138.4, 132.4, 132.1, 124.8, 123.9, 115.1, 113.7, 89.0, 84.5, 83.3, 81.4, 57.4, 44.4, 26.9, 25.1. HRMS (ESI+): m/z calcd for C20H23N6O9S [M+H] + : 523.1242, Found 523.1223.

  3H).13 C-NMR (150 MHz, DMSO-d6) δ 156.5, 147.5, 146.0, 141.0, 139.5, 138.6, 134.0, 132.0, 126.8, 125.5, 124.8, 113.5, 89.1, 84.9, 83.4, 81.8, 48.9, 43.3, 26.9, 25.2, 19.7, 13.4. HRMS (ESI+): m/z calcd for C22H27ClN5O6S [M+H] + : 524.1365, Found 524.1369.

	5'-deoxy-5'-N-(ethyl)-(3-nitro-4-methoxybenzenesulfonyl)-2',3'-isopropylideneinosine
	). Rf 0.45 (1:9 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 12.45 (s,
	1H) ; 8.28 (s, 1H) ; 8.09 (s, 1H) ; 7.70 (d, J = 1.9 Hz, 1H) ; 7.58 (dd, J = 8.0, 1.9 Hz, 1H) ; 7.50

(d, J = 8.2 Hz, 1H) ; 6.17 (d, J = 2.3 Hz, 1H) ; 5.37 (dd, J = 6.4, 2.4 Hz, 1H) ; 5.01 (dd, J = 6.4, 3.3 Hz, 1H) ; 4.28 (ddd, J = 8.4, 5.4, 3.4 Hz, 1H) ; 3.55 (dd, J = 14.7, 5.3 Hz, 1H) ; 3.33 -3.26 (m, 1H) ; 3.18 -3.06 (m, 2H) ; 2.37 (s, 3H) ; 1.52 (s, 3H) ; 1.32 (s, 3H) ; 0.87 (t, J = 7.1 Hz,

  The residue was purified by flash column chromatography

	1H) ; 5.20 -5.13 (m, 2H) ; 4.93 (dd, J = 6.2, 2.8 Hz, 1H) ; 4.16 (td, J = 4.6, 2.8 Hz, 1H) ; 3.62
	-3.52 (m, 2H) ; 1.55 (s, 3H) ; 1.32 (s, 3H). 13 C-NMR (150 MHz, DMSO-d6) δ 157.7, 153.0, (liquid sample, silica gel, linear gradient 0-12% MeOH in CH2Cl2 1% NEt3) to give 55 as a
	149.5, 131.1, 128.7, 128.5, 127.2, 127.2, 122.4, 113.1, 102.1, 95.1, 91.2, 89.0, 85.7, 83.7, 82.7, foam (416 mg, 55%). Rf = 0.01 (1:9 MeOH/CH2Cl2). 1 H-NMR (500 MHz, DMSO-d6) δ 8.18
	81.0, 61.6, 27.1, 25.2. HRMS (ESI+): m/z calcd for C22H23N4O4 [M+H] + : 407.1714, Found (s, 1H) ; 7.90 (s, 1H) ; 7.62 -7.56 (m, 2H) ; 7.48 -7.38 (m, 3H) ; 6.18 (d, J = 3.1 Hz, 1H) ;
	407.1707. 5.25 (dd, J = 6.5, 3.2 Hz, 1H) ; 4.91 (dd, J = 6.5, 3.3 Hz, 1H) ; 4.15 (dt, J = 6.2, 3.7 Hz, 1H) ;
	2.76 -2.64 (m, 2H) ; 2.29 (s, 3H) ; 1.53 (s, 3H) ; 1.31 (s, 3H). 13 C-NMR (125 MHz, DMSO-
	2',3'-isopropylidene-7-(quinolin-3-ethynyl)-7-deazaadenosine (54). d6) δ 157.7, 153.1, 149.6, 131.1, 128.7, 128.6, 127.4, 127.2, 122.4, 113.4, 102.1, 95.2, 91.3,
	To a solution of 52 (1.00 g, 2.31 mmol, 1.00 eq) in THF (C = 0.066 M) under argon, were added 88.6, 84.0, 83.3, 82.7, 81.9, 53.2, 36.1, 27.1, 25.2. HRMS (ESI+): m/z calcd for C23H26N5O3
	Et3N (968 µl, 6.94 mmol, 3.00 eq) and 3-ethynylquinoline (709 mg, 4.63 mmol, 2.00 eq). After [M+H] + : 420.2030, Found 420.2040.
	stirring for 30 min under an argon flow, CuI (132 mg, 0.69 mmol, 0.30 eq) and Pd(PPh3)4 (267
	mg, 0.23 mmol, 0.10 eq) were added. After stirring for 2 h at 60°C, silica was added to the 5'-deoxy-5'-methylamino-2',3'-isopropylidene-7-(quinolin-3-ethynyl)-7-deazaadenosine (56)
	Methanesulfonyl chloride (205 µL, 2.65 mmol, 1.20 eq) was added dropwise to a solution of
	54 (1.01 g, 2.21 mmol, 1.00 eq) and Et3N (679 µl, 4.87 mmol, 2.20 eq) in anhydrous CH2Cl2
	DMSO-d6) δ 157.6, 153.1, 151.6, 149.6, 146.2, 138.0, 130.3, 128.8, 128.0, 128.0, 127.5, 126.9,
	116.8, 113.1, 101.9, 94.7, 89.1, 88.8, 85.9, 85.8, 83.8, 81.1, 61.6, 27.1, 25.2. HRMS (ESI+):
	m/z calcd for C25H24N5O4 [M+H] Methanesulfonyl chloride (228 µL, 2.95 mmol, 1.20 eq) was added dropwise to a solution of
	53 (1.00 g, 2.46 mmol, 1.00 eq) and Et3N (755 µl, 5.41 mmol, 2.20 eq) in anhydrous CH2Cl2 (liquid sample, silica gel, linear gradient 0-12% MeOH in CH2Cl2 1% NEt3) to give 56 as a
	(35.1 mL, C = 0.07 M) at -10 °C under argon. After stirring at -10° C for 30 min, the reaction foam (280 mg, 31%). Rf = 0.02 (1:9 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 9.04 2',3'-isopropylidene-7-phenylethynyl-7-deazaadenosine (53). mixture was diluted with a saturated NH4Cl solution. The aqueous layer was extracted with (d, J = 2.1 Hz, 1H) ; 8.65 (d, J = 2.3 Hz, 1H) ; 8.20 (s, 1H) ; 8.05 (dd, J = 8.5, 1.0 Hz, 1H, HQuin) Et3N (968 µl, 6.94 mmol, 3.00 eq) and phenylacetylene (508 µl, 4.63 mmol, 2.00 eq) were CH2Cl2 and the combined organic extracts were washed with brine, dried over Na2SO4 and ; 8.03 -7.97 (m, 2H) ; 7.81 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H) ; 7.67 (ddd, J = 8.1, 6.7, 1.2 Hz, 1H) added to a solution of 52 (1.00 g, 2.31 mmol, 1.00 eq) in THF (C = 0.066 M) under argon. After concentrated in vacuo. The residue was purified by flash column chromatography (liquid ; 6.20 (d, J = 3.3 Hz, 1H) ; 5.27 (dd, J = 6.4, 3.3 Hz, 1H) ; 4.91 (dd, J = 6.4, 3.3 Hz, 1H) ; 4.16 stirring for 30 min, under an argon flow, CuI (132 mg, 0.69 mmol, 0.30 eq) and Pd(PPh3)4 (267 sample, silica gel, linear gradient 0-50% acetone in CH2Cl2) to give the mesylate compound as (td, J = 5.9, 3.3 Hz, 1H) ; 2.75 -2.63 (m, 2H) ; 2.28 (s, 3H) ; 1.54 (s, 3H) ; 1.32 (s, 3H). 13 C-mg, 0.23 mmol, 0.10 eq) were added. After stirring for 2 h at 60°C, silica was added to the pressure, the mesylate compound (870 mg, 1.80 mmol) was treated with a solution of 2M mixture and all the volatiles were removed in vacuo. The crude was purified by flash column a colorless foam (950 mg, 80%). Rf = 0.54 (1:1 acetone/CH2Cl2). After drying under reduce NMR (150 MHz, DMSO-d6) δ 157.6, 153.2, 151.7, 149.7, 146.2, 138.0, 130.4, 128.8, 128.2,

). Rf 0.43 (1:9 MeOH/CH2Cl2).

1

H-NMR (500 MHz, DMSO-d6) δ 8.27 (s, 1H) ; 8.13 (d, J = 2.3 Hz, 1H) ; 8.08 (s, 1H) ; 7.96 (dd, J = 9.0, 2.3 Hz, 1H) ; 7.31 (d, J = 9.0 Hz, 1H) ; 6.16 (d, J = 2.3 Hz, 1H) ; 5.36 (dd, J = 6.4, 2.3 Hz, 1H) ; 4.99 (dd, J = 6.4, 3.4 Hz, 1H) ; 4.28 (dt, J = 8.4, 4.3 Hz, 1H) ; 3.99 (s, 3H) ; 3.56 (dd, J = 14.8, 5.2 Hz, 1H) ; 3.34 -3.26 (m, 1H) ; 3.11 (dp, J = 25.0, 7.3 Hz, 2H) ; 1.53 (s, 3H) ; 1.32 (s, 3H) ; 0.86 (t, J = 7.0 Hz, 3H).

13 C-NMR (125 MHz, DMSO-d6) δ 163.

5, 156.8, 148.6, 146.4, 139.4, 133.8, 132.7, 131.9, 124.8, 115.0, 113.6, 112.9, 101.2, 89.0, 85.1, 83.4, 81.8, 57.2, 48.9, 43.4, 26.9, 25.2, 13.4. HRMS (ESI+): m/z calcd for C23H27N6O7S [M+H] + : 531.1656, Found 531.1667. chromatography (dry sample, silica gel, linear gradient 0-5% MeOH in AcOEt) to give 53 as a colorless foam (801 mg, 85%). Rf = 0.63 (1:9 MeOH/AcOEt). 1 H-NMR (600 MHz, DMSO-d6)

δ 8.20 (br. s, 1H) ; 7.91 (s, 1H) ; 7.63 -7.55 (m, 2H) ; 7.47 -7.38 (m, 3H) ; 6.24 (d, J = 3.3 Hz, mixture and all the volatiles were removed in vacuo. The residue was purified by flash column chromatography (dry sample, silica gel, linear gradient 0-5% MeOH in AcOEt) to give 54 as a colorless foam (1.01 g, 95%). Rf = 0.57 (1:9 MeOH/AcOEt). 1 H-NMR (600 MHz, DMSO-d6) δ 9.04 (d, J = 2.2 Hz, 1H) ; 8.65 (d, J = 2.1 Hz, 1H) ; 8.19 (s, 1H) ; 8.05 (dd, J = 8.4, 1.0 Hz, 1H) ; 8.06 -7.98 (m, 2H) ; 7.80 (ddd, J = 8.5, 6.9, 1.5 Hz, 1H) ; 7.67 (ddd, J = 8.0, 6.8, 1.2 Hz, 1H) ; 6.26 (d, J = 3.2 Hz, 1H) ; 5.23 -5.16 (m, 2H) ; 4.95 (dd, J = 6.2, 2.9 Hz, 1H) ; 4.18 (td, J = 4.6, 2.8 Hz, 1H) ; 3.64 -3.53 (m, 2H) ; 1.55 (s, 3H) ; 1.33 (s, 3H). 13 C-NMR (150 MHz, + : 458.1823, Found 458.1826.

5'-deoxy-5'-methylamino-2',3'-isopropylidene-7-phenylethynyl-7-deazaadenosine (55).

MeNH2 in THF (18.0 mL, C = 0.1M) in a sealed vessel at 50 °C for 72 h. The mixture was concentrated in vacuo, the residue was diluted with AcOEt and brine. Aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo.

(31.6 mL, C = 0.07 M) at -10 °C under argon. After stirring at -10° C for 30 min, the reaction mixture was diluted with a saturated NH4Cl solution. The aqueous layer was extracted with CH2Cl2 and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (liquid sample, silica gel, linear gradient 0-50% acetone in CH2Cl2) to give the mesylate intermediate as a colorless foam (1.03 g, 87%). Rf = 0.43 (1:1 acetone/CH2Cl2). After drying, the mesylate compound (1.03 g, 1.92 mmol) was treated with a 2M MeNH2 solution in THF (19.2 mL, C = 0.1M) in a sealed vessel at 50 °C for 72 h. After completion of the reaction, the mixture was concentrated in vacuo, the residue was diluted with AcOEt and brine. Aqueous layer was extracted with AcOEt and the combined organic extracts were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography 128.0, 127.5, 126.9, 116.9, 113.4, 101.9, 94.9, 88.8, 88.7, 85.9, 84.2, 83.3, 82.0, 53.4, 36.3, 27.1, 25.2. HRMS (ESI+): m/z calcd for C26H27N6O3 [M+H] + : 471.2139, Found 471.2134.

  ). Rf 0.51 (1:9 MeOH/CH2Cl2).

	5.5, 3.7 Hz, 1H) ; 3.99 (s, 3H) ; 3.46 (dd, J = 14.1, 5.6 Hz, 1H) ; 3.22 (dd, J = 14.1, 7.3 Hz, 1H)
	; 2.68 (s, 1H) ; 1.54 (s, 3H) ; 1.33 (s, 3H). 13 C-NMR (150 MHz, DMSO-d6) δ 157.4, 154.6,
	152.9, 151.4, 149.2, 145.9, 138.6, 137.7, 132.7, 130.1, 128.6, 128.4, 128.3, 127.7, 127.2, 126.6,
	124.0, 116.5, 114.9, 113.5, 101.7, 94.7, 88.7, 88.6, 85.5, 83.3, 83.2, 81.4, 57.1, 51.1, 35.7, 26.7,
	24.9. HRMS (ESI+): m/z calcd for C33H32N7O8S [M+H]

1 H-NMR (600 MHz, DMSO-d6) δ 9.04 (d, J = 2.1 Hz, 1H) ; 8.64 (dd, J = 2.1, 0.8 Hz, 1H) ; 8.22 (d, J = 2.4 Hz, 1H) ; 8.20 (s, 1H) ; 8.05 (dd, J = 8.3, 1.0 Hz, 1H) ; 8.01 (dd, J = 8.2, 1.4 Hz, 1H) ; 7.99 -7.93 (m, 2H) ; 7.81 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H) ; 7.67 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H) ; 7.48 (d, J = 9.1 Hz, 1H) ; 6.24 (d, J = 2.7 Hz, 1H) ; 5.34 (dd, J = 6.6, 2.7 Hz, 1H) ; 5.02 (dd, J = 6.5, 3.8 Hz, 1H) ; 4.25 (ddd, J = 7.3, + : 686.2028, Found 686.2031.

  ) ; 2.40 (s, 3H).13 C-NMR (150 MHz, DMSO-d6) δ 157.6, 153.0, 151.7, 150.2, 146.1, 141.2, 137.9, 136.3, 134.1, 132.1, 130.4, 128.8, 128.0, 127.5, 127.1, 126.9, 125.9, 116.9, 102.0, 94.8, 88.7, 87.1, 86.2, 82.2, 72.9, 71.3, 52.1, 36.1, 19.7. HRMS (ESI+): m/z calcd for C30H28ClN6O5S [M+H] + : 619.1525, Found 619.1529. .3, 128.8, 128.0, 127.5, 126.9, 124.3, 116.9, 115.3, 102.0, 94.8, 88.7, 87.1, 86.1, 82.1, 72.9, 71.3, 57.4, 52.0, 36.1. HRMS (ESI+): m/z calcd for C30H28N7O8S [M+H]

	5'-deoxy-5'-N-(methyl)-(3-chloro-4-methylbenzenesulfonyl)-7-phenylethynyl-7-
	deazaadenosine (19).
	Following method F with 59 (67 mg, 0.11 mmol, 1.00 eq), 19 was obtained as an off-white
	solid (10 mg, 16%). Rf 0.50 (1:9 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 8.16 (s,
	1H) ; 7.92 (s, 1H) ; 7.79 (d, J = 2.0 Hz, 1H) ; 7.67 (dd, J = 8.0, 1.9 Hz, 1H) ; 7.61 -7.56 (m,
	3H) ; 7.46 -7.38 (m, 3H) ; 6.07 (d, J = 6.1 Hz, 1H) ; 5.46 (d, J = 6.2 Hz, 1H) ; 5.33 (d, J = 5.0 5'-deoxy-5'-N-(methyl)-(4-methoxy-3-nitrobenzenesulfonyl)-7-(quinolin-3-ethynyl)-7-
	Hz, 1H) ; 4.49 (q, J = 5.9 Hz, 1H) ; 4.11 (td, J = 5.2, 3.8 Hz, 1H) ; 4.04-3.99 (m, 1H) ; 3.50 (dd, deazaadenosine (25).
	Following method F with 65 (68 mg, 0.10 mmol, 1.00 eq), 25 was obtained as an off-white J = 14.1, 4.9 Hz, 1H) ; 3.17 (dd, J = 14.2, 7.5 Hz, 1H) ; 2.72 (s, 3H) ; 2.40 (s, 1H). 13 C-NMR (150 MHz, DMSO-d6) δ 157.6, 153.0, 150.1, 141.2, 136.3, 134.1, 132.1, 131.1, 128.7, 128.5, solid (40 mg, 62%). Rf 0.33 (1:9 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 9.04 (d, J
	127.2, 125.9, 122.5, 102.1, 95.1, 91.2, 87.1, 83.0, 82.1, 72.9, 71.3, 52.1, 36.1, 19.7. HRMS = 2.1 Hz, 1H) ; 8.66 -8.62 (m, 1H) ; 8.29 (d, J = 2.3 Hz, 1H) ; 8.18 (s, 1H) ; 8.08-8.03 (m, 2H)
	(ESI+): m/z calcd for C27H27ClN5O5S [M+H] + : 568.1416, Found 568.1411. ; 8.03-7.99 (m, 2H) ; 7.81 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H) ; 7.68 (ddd, J = 8.2, 6.8, 1.2 Hz, 1H) ;
	7.53 (d, J = 9.0 Hz, 1H) ; 6.10 (d, J = 5.9 Hz, 1H) ; 5.48 (d, J = 6.2 Hz, 1H) ; 5.34 (d, J = 5.1
	5'-deoxy-5'-N-(methyl)-(3-nitro-4-methoxybenzenesulfonyl)-7-phenylethynyl-7-Hz, 1H) ; 4.51 (q, J = 5.8 Hz, 1H) ; 4.12 (td, J = 5.1, 3.9 Hz, 1H) ; 4.04 (dt, J = 7.4, 4.5 Hz, 1H)
	deazaadenosine (20). ; 4.01 (s, 3H) ; 3.51 (dd, J = 14.2, 4.8 Hz, 1H) ; 3.23 (dd, J = 14.2, 7.5 Hz, 1H) ; 2.75 (s, 1H).
	3 Hz,
	1H) ; 5.33 (d, J = 5.1 Hz, 1H) ; 4.48 (q, J = 5.9 Hz, 1H) ; 4.10 (td, J = 5.1, 3.7 Hz, 1H) ; 4.04-
	3.99 (m, 4H) ; 3.50 (dd, J = 14.1, 4.9 Hz, 1H) ; 3.20 (dd, J = 14.2, 7.5 Hz, 1H) ; 2.73 (s, 3H).
	13 C-NMR (150 MHz, DMSO-d6) δ 157.6, 154.9, 153.0, 150.1, 138.9, 133.1, 131.1, 128.7, 1 H-NMR (500 MHz, DMSO-d6) δ 12.36 (s, 1H) ; 128.5, 127.2, 124.3, 122.5, 115.3, 102.1, 95.1, 91.2, 87.0, 83.0, 82.1, 72.9, 71.3, 57.4, 52.0,
	8.30 (s, 1H) ; 8.15 (s, 1H) ; 8.07 -8.01 (m, 2H) ; 7.34 (dd, J = 9.1, 1.4 Hz, 1H) ; 5.86 (dd, J = 36.1. HRMS (ESI+): m/z calcd for C27H27N6O8S [M+H] + : 595.1606, Found 595.1611.
	5.7, 1.4 Hz, 1H) ; 5.54 (br. s, 1H) ; 5.34 (br. s, 1H) ; 4.59 (br. s, 1H) ; 4.13 (br. s, 1H) ; 4.08 -
	4.02 (m, 1H) ; 3.99 (s, 3H) ; 3.64 -3.55 (m, 1H) ; 3.40 -3.32 (m, 1H) ; 3.27 -3.08 (m, 2H); 5'-deoxy-5'-N-(methyl)-(3-cyano-4-methoxybenzenesulfonyl)-7-phenylethynyl-7-
	0.98 (t, J = 7.1 Hz, 3H). 13 C-NMR (125 MHz, DMSO-d6) δ 163.4, 156.5, 148.2, 145.8, 139.1, deazaadenosine (21).
	133.8, 132.7, 132.1, 124.5, 115.0, 112.9, 101.1, 87.6, 82.8, 72.9, 71.2, 57.1, 49.6, 43.3, 13.6. Following method F with 67 (95 mg, 0.15 mmol, 1.00 eq), 21 was obtained as an off-white
	HRMS (ESI+): m/z calcd for C20H23N6O7S [M+H] + : 491.1343, Found 491.1341. solid (42 mg, 47%). Rf 0.51 (1:9 MeOH/CH2Cl2). 1 H-NMR (600 MHz, DMSO-d6) δ 8.19 (d, J
	= 2.3 Hz, 1H) ; 8.16 (s, 1H) ; 8.05 (dd, J = 9.0, 2.4 Hz, 1H) ; 7.91 (s, 1H) ; 7.61 -7.55 (m, 2H)
	General method F for the synthesis of final compounds 7-26. Compounds 40-42, 45-51, ; 7.46 -7.38 (m, 4H) ; 6.08 (d, J = 6.1 Hz, 1H) ; 5.46 (s, 1H) ; 5.33 (s, 1H) ; 4.48 (t, J = 5.6 Hz,
	57-60, 62-65, 67, 68 were treated with a mixture of trifluoroacetic acid/water (8/2 v:v, C = 1H) ; 4.13 -4.08 (m, 1H) ; 4.05 -3.98 (m, 4H) ; 3.49 (dd, J = 14.3, 5.0 Hz, 1H) ; 3.17 (dd, J =
	0.08 M). After stirring at 25 °C for 0.5 h, the solvents were removed in vacuo and the crudes 14.3, 7.4 Hz, 1H) ; 2.72 (s, 3H). 13 C-NMR (150 MHz, DMSO-d6) δ 163.7, 157.6, 152.9, 150.1,

were co-evaporated three times with water and three times with MeOH. The residues were dissolved in CH2Cl2/MeOH, then 0.5 eq of NEt3 was added before adding silica to prepare the dry sample. Purification by column chromatography (silica gel, linear gradient 0-10% MeOH Following method F with 60 (59 mg, 0.09 mmol, 1.00 eq), 20 was obtained as an off-white solid (11 mg, 20%). Rf 0.52 (1:9 MeOH/CH2Cl2).

1

H-NMR (600 MHz, DMSO-d6) δ 8.28 (d, J = 2.3 Hz, 1H) ; 8.16 (s, 1H) ; 8.05 (dd, J = 8.9, 2.4 Hz, 1H) ; 7.91 (s, 1H) ; 7.61 -7.56 (m, 2H) ; 7.53 (d, J = 8.9 Hz, 1H) ; 7.47 -7.38 (m, 3H) ; 6.07 (d, J = 5.9 Hz, 1H) ; 5.47 (d, J = 6.134.2, 133.0, 131.1, 129.5, 128.7, 128.5, 127.1, 122.5, 115.0, 113.1, 102.1, 101.3, 95.1, 91.2, 87.0, 83.0, 82.1, 72.9, 71.3, 57.1, 52.1, 36.1. HRMS (ESI+): m/z calcd for C28H27N6O6S [M+H] + : 575.1707, Found 575.1702. 3H13 C-NMR (150 MHz, DMSO-d6) δ 157.6, 154.9, 153.0, 151.6, 150.2, 146.1, 138.9, 137.9, 133.1, 130+ : 646.1715, Found 646.1721.

  13 C-NMR (150 MHz, DMSO-d6) δ 163.7, 157.6, 153.0, 151.7, 150.3, 146.2, 138.0, 134.2, 133.1, 130.4, 129.5, 128.8, 128.0, 127.5, 126.9, 116.9, 115.1, 113.1, 102.0, 101.3, 94.8, 88.7, 87.0, 86.2, 82.2, 72.9, 71.3, 57.2, 52.1, 36.1. HRMS (ESI+): m/z calcd for C31H28N7O6S [M+H] + : 626.1816, Found 626.1825.

Acknowledgments

culture plate, a control compound (Remdesivir, BLDpharm) was tested similarly. Plates were incubated for 2 days at 37°C prior to quantification of the viral genome by real-time RT-PCR.

To do so, 100 μL of viral supernatant was collected in S-Block (Qiagen) previously loaded with VXL lysis buffer containing proteinase K and RNA carrier. Cell viability was also determined in order to determine if the absence of viral replication was due to genuine inhibition by the molecules or as an artifact of molecule cytotoxicity, and as another method to observe viral replication inhibition. This was achieved by removing all remaining cell culture supernatant and using CellTiter Blue® (Promega) following manufacturer's instructions. RNA extraction was performed on the collected supernatant using the Qiacube HT automat and the QIAamp 96 DNA kit HT following manufacturer instructions. Viral RNA was quantified by real-time RT-qPCR (GoTaq 1-step qRt-PCR, Promega) using 3.8 μL of extracted RNA and 6.2 μL of RT-qPCR mix and standard fast cycling parameters, i.e., 10 min at 50°C, 2 min at 95°C, and 40 amplification cycles (95°C for 3 s followed by 30 s at 60°C). Quantification was provided by four 2 log serial dilutions of an appropriate T7-generated synthetic RNA standard of known quantities (10 2 to 10 8 copies/reaction). RT-qPCR reactions were performed on QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) and analyzed using QuantStudio 12K Flex Applied Biosystems software v1.2.3. Primers and probe sequences, which target SARS-CoV-2 N gene, were: Fw: GGCCGCAAATTGCACAAT; Rev: CCAATGCGCGACATTCC; Probe: FAM-CCCCCAGCGCTTCAGCGTTCT-BHQ1. For the evaluation of compounds cytotoxicity, the same culture conditions without addition of the virus, and cell viability was measured using CellTiter Blue® (Promega) following manufacturer's instructions. All data obtained were analyzed using GraphPad Prism 9 software (Graphpad software).

Author contributions

Notes

The authors declare no competing financial interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at :


Edward Marc Cushing

Sara Touhami

Fabrice Hollender

Fernando Lopez-Caballero

Denis Moiriat

Cédric Guyonnet-Benaize

Nikolaos Theodoulidis

Edwige Pons-Branchu

Sophie Sepulcre

Pierre-Yves Bard

Cecile Cornou

Aline Dechamp

Armand Marsical

Zafeiria Roumelioti

Building a Three Dimensional Model of Plio-Quaternary Basin of Argostoli (Cephalonia Island, Greece) From an Integrated Geophysical and Geological Approach to Perform Numerical Simulations of Seismic Motion

SUMMARY

Particular shallow geological configurations introduced by significant lithological and/or geometrical variations may cause ground motion amplification such as for sedimentary basins and covers overlying rock basements, where a large impedance contrast between the superficial soft layers and the harder deeper rocks. For the safety assessment of critical facilities, site effects associated with such geological conditions have to be quantified. In the framework of the PIA SINAPS@ project of the French Research Agency (ANR), a narrow Plio-Quaternary sedimentary basin next to Argostoli capital of Cephalonia (Greece) was selected as a test site for studying site effects and the nonlinear behavior of soil. We present here the geological and geophysical characterization that has led to the definition of the 3D structure and the different stratigraphic units of the Argostoli basin. From the 3D model, a numerical code (i.e. SEM3D) has been developed. The seismic phenomenon is simulated in its entirety: from the seismic source to the site using a representative model of the complexity of the wave path. In order to validate the proposed numerical model, several recorded earthquakes issued from the accelerometers array database are compared with the numerical simulated ones. The first obtained results show the importance of the in-situ measures on the regional scale simulations.

REFERENCES:

Haslinger, F., Kissling, E., Ansorge, J., Hatzfeld, D., Papadimitriou, E., Karakostas, V., Makropoulos, K., Kahle, H.-G., and Peter, Y. (1999). "3d crustal structure from local earthquake tomography around the gulf of Arta (Ionian region, NW Greece)." Tectonophysics,304(3),201202203204205206207208209210211212213214215216217218 The Cephalonia Island (Greece) area is located at the north-western end of the Aegean subduction frontal thrust that is linked to the dextral Cephalonia Transform Fault (west of Cephalonia). As the mean slip rate and the length of the Cephalonia Transform Fault are large, the seismic hazard is high in terms of earthquake frequency and magnitude (1a). The Plio-Quaternary Koutavos-Argostoli basin site was selected to host a vertical accelerometer array (1b).

ANR-11-RSNR-0022

INTERPRETATION OF GEOPHYSICAL DATA

A comprehensive geophysical survey encompassing HVSR, SDWA (AVA) and MASW acquisition allowed to perform 1) H/V ratio distribution map (3a) and typology (flat, broad peak, double peak,…(3b, 2c)) and 2) dispersion curves over each identified unit and Vs vertical profiles (3c). The interpretation of this data led to characterize two embedded series that have been identified on field (Stiff and Soft units : Pliocene and Quaternary). From the fundamental frequencies determined on SDWA arrays and taking in account the vertical Vs profile, it was then possible to construct cross sections (3e) showing the base of the Pliocene and Quaternary units by extrapolating the fo measurements along these profiles (3d). Complementary structural and geological information was used to complete these cross sections. Taking into account the four profiles, outcrops of stratigraphic limits drawn on the geological map and some complementary fo measurements (from SDWA arrays and complementary points), a 3D geological model was interpolated (3 surfaces -3f) using Arcgis software (TopoToRaster function).

3D VELOCITY MODEL AND GROUND MOTION SIMULATION

Two different cases were studied and compared. The first case is a layered model using the velocity model proposed by Haslinger et al. (1999) (for upper layers) (4a). This model does not allow to see the local effects. In order to take into account the geological characteristics of the site, a more refined model (4b) was studied considering the 3D geological model (4c). The two models have the size of: 27 x 30 x 50 km. The size of the elements constituting the mesh is 130 x 130 m in the horizontal directions (EW and NS). For the vertical direction, the size of the elements is adapted according to the properties of each layer. The amplification is evaluated between two sensors : one inside the basin and the other one outside the basin (4d). In the case of the stratified model there is no peak in the NS directions and the appearance of very small peaks in the EW direction (4e). In the case of the basin model the peaks appear in both directions (4f). The simulated results are compared to the amplification function calculated from the Argostoli recorded database (4g). It is noted that for the Basin model the three founded frequency peaks are practically the same as in the case of experimental data. The magnitudes, though, are less important.



  . Perron V, Hollender F, Mariscal A, Theodoulidis N, Andreou C, Bard PY, Cornou C, Cottereau R, Cushing EM, Frau A, et al. (2018) Accelerometer, velocimeter dense-array, and rotation sensor datasets from the sinaps@ postseismic survey (Cephalonia 2014-2015 aftershock sequence). Seismological Research Letters 89(2A):678-687 Sorel, D. (1989). L'évolution structurale de la Grèce nord-occidentale depuis le miocène, dans le cadre géodynamique de l'arc égéen. Paris 11. Theodoulidis, N., Hollender, F., Mariscal, A., Moiriat, D., Bard, P.Y., Konidaris, A., Cushing, M., Konstantinidou, K., Roumelioti, Z., 2018b. The ARGONET (Greece) seismic observatory : an accelerometric vertical array and its data. Seismol. Res. Lett. 89, 1555-1565. Underhill, J.R. (1989). Late Cenozoic deformation of the Hellenide foreland, western Greece. Geol. Soc. Am. Bull. 101, 613-634. ACKNOWLEDGEMENTS : we thank colleagues who participated to acquisition campains : Elise Delavaud, Myrtille Kuperminc, Vincent Perron and Michael Darche; Olivier Dufaure and Serge Miska (GEOPS) for technical assistance for Xray diffraction sample preparation and analysis. Thanks to Agis Konidaris and Chrisostomos Andreou for all assistance provided. Local authorities of Argostoli warmly welcomed this project. This Project was funded by the French ANR in the framework of the program Investissements d'avenir (PIA): SINAPS@ (ANR-11-RSNR-0022; www.institut-seism.fr/en/) CONCLUSIONS  New geological data from field survey, geophysical interpretation of fo distribution and typology and from borehole data  Dating the base of the Quaternary basin at ~411 ka  A 3D geological model of the Argostoli basin was performed by interpolation of depth control points coming from geological survey and interpretation of geophysical data.  Construction of integrated 3D velocity model at # scales . First simulation of EQ spectral amplification is in good agreement with empirical evaluation of amplification frequencies  Future simulation taking in account fault models (2014 Cephalonia EQ) 1 IRSN, Fontenay-aux-Roses, France 2 SSMat,CNRS, CentraleSupélec, Gif-Sur-Yvette, France, 3 CEA Cadarache, St Paul-lez-Durance, France 4 Univ. Grenoble Alpes, ISTerre / IRD, Grenoble, France 5 Setec International, Vitrolles, France 6 ITSAK, Thessalonique, Grèce 7 LSCE/IPSL, CEA-CNRS-UVSQ, Université Paris-Saclay, France 8 GEOPS, Université Paris-Saclay, France 9 CEA, DIF, Bruyères le Chatel, France. 10 Aristotle University, Thessaloniki, Greece , SDWA and MASW) (2b,c), geological and geotechnical analysis of the ARGONET borehole (2d) allowed to performed a new upgraded geological map that details the Plio-quaternary formations in the Argostoli basin. Taking advantage of the ARGONET borehole, dating performed on corals allowed to assess the middle Pleistocene age of the base of the Quaternary unit of Koutavos that was no to date established (~411 ka).

  Giardini et al (2013) Seismic Hazard Harmonization in Europe (SHARE): Online Data Resource, doi: 10.12686/SED-00000001calculated from recorded data by Perron et al. (2018).