# Synthesis and in vitro inhibitory activity of N-arylsulfonamide adenosine analogues designed to target SARS-CoV-2 nsp14 N7-methyltransferase

Marcel Hausdorff, Adrien Delpal, Hugo Machin, Amina Tahir, Jim Zoladek, Floriane Gucciardi, Jitendriya Swain, Nathalie Gros, Delphine Muriaux, Sébastien Nisole, et al.

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Marcel Hausdorff, Adrien Delpal, Hugo Machin, Amina Tahir, Jim Zoladek, et al. Synthesis and in vitro inhibitory activity of N-arylsulfonamide adenosine analogues designed to target SARS-CoV-2 nsp14 N7-methyltransferase. Bioorganic Chemistry, 2025, 166, pp.109104. 10.1016/j.bioorg.2025.109104. hal-05371098

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# Synthesis and *in vitro* inhibitory activity of N-arylsulfonamide adenosine analogues designed to target SARS-CoV-2 nsp14 N7-methyltransferase

Marcel Hausdorff,<fnref n="1" /> Adrien Delpal,<fnref n="1" />,² Hugo Machin,ª Amina Tahir,ª Jim Zoladek,ᶜ Floriane Gucciardi,ᵈ
Jitendriya Swain,ᵈ Nathalie Gros,ᵈ Delphine Muriaux,ᵈ Sébastien Nisolec, Bruno Canard,ᵇ Jean-Jacques
Vasseur,ª Etienne Decroly*b and Françoise Debart*a

**Keywords:** adenosine - arylsulfonamide - bisubstrate - C-nucleoside - nsp14 inhibitors - prodrug - RNA cap methyltransferase - SARS-CoV-2

## ABSTRACT

This study presents the design, synthesis, and biological evaluation of novel N-arylsulfonamide nucleosides as inhibitors of the SARS-CoV-2 nsp14 N7-methyltransferase. Several compounds demonstrate potent nanomolar inhibition in vitro, with structure-activity relationship analysis revealing the importance of aryl substituents on activity and physicochemical properties. Approaches aimed to improve cellular delivery using carbonate and isobutyryl prodrugs show partial conversion to the active form upon incubation in cell extracts, while orthoester prodrugs remain stable and inactive. Additionally, a fluorescent analogue was synthesized to track intracellular uptake and distribution, confirming cytoplasmic localization. These results highlight the N-arylsulfonamide nucleoside scaffold as a promising antiviral chemotype against SARS-CoV-2, and emphasize the critical role of chemical modifications for optimizing biological activity and pharmacokinetic profiles toward effective COVID-19 therapies.

## 1. Introduction

The highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of COVID-19. The former attacks mainly the upper respiratory tract leading to severe infection mainly in elderly population. Current prophylaxis consists in the use of vaccines. Vaccination is not sufficient to stop outbreaks due to social behaviors, virus evolution by mutation in the spike protein, and the inability of vaccination to block inter-human transmission [1, 2]. Since 2020, considerable effort has been devoted to finding effective antiviral drugs, a task that is both challenging and laborious [3]. Several critical steps in the viral cycle have been identified and could be targeted to block replication of the virus and thus limit its spread [4, 5]. Targets involved in either entry pathways (ACE2 receptor, endocytosis) or non-structural proteins (nsp) that orchestrate the

<footnote>ªIBMM, University of Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote>ᵇAFMB, University of Aix-Marseille, CNRS, Marseille, France</footnote>

<footnote>ᶜIRIM, University of Montpellier, CNRS, Montpellier, France</footnote>

<footnote>ᵈCEMIPAI, University of Montpellier, CNRS, Montpellier, France</footnote>

<footnote>*corresponding authors: francoise.debart@umontpellier.fr; etienne.decroly@univ-amu.fr.</footnote>

<footnote n="1">¹These two first authors contributed equally to this work.</footnote>
SARS-CoV-2 replication/transcription machinery, are being explored [6, 7]. Among the 16 nsps, key enzymes such as PL<sup>pro</sup> (nsp3) [8], the main protease 3CL<sup>pro</sup> (nsp5) [9], the RNA-dependent RNA polymerase (RdRP, nsp12) [10], and the N7- and 2′O-methyltransferases (MTases) (nsp14, nsp10/nsp16)[11-13] have been validated as drug targets for the development of antiviral drugs [14]. Even though remdesivir [15] and molnupiravir [16] for nsp12 and nirmatrelvir [17, 18] for nsp5 have been FDA-approved drugs, drug resistant mutations to the inhibitor remdesivir or 3CL<sup>pro</sup> inhibitor nirmatrelvir have been reported [19-21]. Therefore, the search for new antiviral strategies against coronaviruses continues, and viral MTases emerge as promising therapeutic targets, due to their essential role in viral RNA capping [11, 12, 22-25].

Previous work

<figure><img src="image_2.png" /></figure>

Present work

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Chemical structures of some previous potent inhibitors of SARS-CoV-2 N7-MTase nsp14 with an N-arylsulfonamide fragment inserting into the RNA cap binding site and structural modulations with various</figcaption></figure>
bicyclic cores, pyrrolotriazine as nucleobase and 2',3'-OH masked with various groups, explored in the present work.

Over the past five years, several groups including our own have discovered potent inhibitors of the SARS-CoV-2 N7-MTase nsp14 [26-40], which catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the N7-position of the cap guanosine of viral mRNA. Most inhibitors are SAM-mimetics compounds designed as bisubstrates of the N7-MTase nsp14 with a nucleoside fragment (adenosine or analogues) occupying the SAM methyl donor binding pocket linked to various moieties capable of mimicking the cap guanosine in the cap binding site of nsp14 [28-30, 32-36, 38, 39]. In our previous work and that of other groups, an N-arylsulfonamide core fitting in the cap binding site was demonstrated as a key component of the bisubstrate pharmacophore resulting in potent nanomolar to subnanomolar inhibition of nsp14 N7-MTase *in vitro* [33, 34, 36, 38]. This essential scaffold was directly linked to the C5' ribose atom of an adenosine, a C7-arylethynyl-7-deaza-adenosine or a C-nucleoside with a C-C glycosidic bond. Despite the potent and selective inhibition of these adenosine mimetics on coronavirus N7-MTases, they generally showed only moderate to poor antiviral activity in cell-based assays [34, 36, 38]. This result calls for further work to favor cell internalization and metabolic stability of new compounds in order to enhance their antiviral activity in infected cells.

In this work, we aimed to increase the lipophilicity of a previously discovered N7-MTase nsp14 inhibitor **MH44** [33] by replacing the phenyl ring with various bicyclic structures or by synthesizing diverse prodrugs of this potent inhibitor (IC₅₀ 19 nM) for improved cell permeability (Fig. 1). Moreover, we prepared three C-nucleosides with a metabolically stable C-C glycosidic bond to slow down compound breakdown. This work was inspired by promising results obtained by Chen's group with nanomolar bisubstrate inhibitors of SARS-CoV-2 nsp14 N7-MTase having both a remarkable antiviral activity (EC₅₀ 0.72 µM) and low cytotoxicity for a C-nucleoside [34]. We have also modelled the cellular internalization of nsp14 inhibitors using a fluorescent adenosine bearing a dansyl moiety as a surrogate, in order to follow the fate of the N-arylsulfonamide nucleosides in cells by fluorescence microscopy. Our synthesized compounds were screened for their ability to both inhibit SARS-CoV-2 N7-MTase activity by using enzymatic assays and measure their antiviral activity in cell-based assays.

## 2. Results and discussion

### 2.1. Chemistry

#### 2.1.1. Synthesis of N-arylsulfonamide adenosines with bicyclic cores

With the aim of optimizing the properties of the nsp14 inhibitor **MH44** (Fig. 1) [33], we studied the functionalization of the sulfonamide bond by replacing the phenyl ring with various bicyclic cores,
as described previously by Chen's group [34]. First, we selected 7 compounds with several original bicyclic cores whose ΔG values determined from molecular modeling experiments were close to that of compound 3* with a 2-naphthyl group (ΔG ≈ -10.9 kcal/mol), which was one of the best nsp14 inhibitors in Chen's study (Table 1). The difference in the positioning of the naphthalene attachment to the sulfonamide function between 3* and 4a does not seem to have any significant impact on the ΔG value. In addition, a quinazoline-2,4-dione heterocycle (5g, Scheme 1), which is a type of pharmacophore scaffold with a wide range of biological functions and has attracted much attention in medicinal chemistry [41], was introduced on the sulfonamide function because of its similarity to guanine, whose place it is intended to occupy in the RNA binding pocket.

<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Synthesis of *N*-arylsulfonamide adenosine analogues with bicyclic cores **4a–f**, **4h** and **5g–h**. **Reagents and conditions:** (a) arylsulfonylchloride, Et<sub>3</sub>N, DMF, -10°C, 2.5 h, 28–90%; (b) EtOTs, KI, K<sub>2</sub>CO<sub>3</sub>, DMF, 50°C, 15h, 22–89%; (c) TFA/H<sub>2</sub>O 8/2 v:v, 25°C, 30 min, 20–72%.</figcaption></figure>

Compounds **4a–f**, **4h**, and **5g–h** were obtained in three steps by coupling 5'-amino-2',3'-O-isopropylidene adenosine **1** with the commercially available arylsulfonylchloride reagents giving the
protected intermediates **2a–g** in 28–90% yield (Scheme 1).[33, 36] The sulfonamide of **2a–f** was functionalized with an ethyl group using ethyl *p*-toluenesulfonate (EtOTs), potassium carbonate and potassium iodide to afford the *N*-ethyl-*N*-arylsulfonamide adenosines **3a–f** in 22–89% yield. The functionalization of **2g**, bearing a quinazolinedione is a particular case, since when treated under the same conditions as its analogues (Scheme 1), it gave two distinct products **2h** and **3h**. NMR analysis of these compounds shows that they result from the attack of EtOTs by the free nitrogen atoms of the quinazolinedione (**2h**) and that one of the two products is substituted on the NH of the sulfonamide (**3h**). The use of only 1.5 eq of EtOTs explains the low yields obtained for both compounds (25% for **2h** and 10% for **3h**). The 2',3'-OH of **2g–h**, **3a–f**, **3h** were then deprotected by treatment with a TFA/water mixture for 30 min to give the final compounds **4a–f**, **4h** and **5g–h**.

<table><caption>**Table 1**</caption><caption>cLogP and IC<sub>50</sub> values of N-arylsulfonamide adenosine analogues with bicyclic cores 4a–f, 4h, 5g–h and of C-nucleosides 6–8</caption><thead><tr><th>Compounds</th><th>Nucleobase</th><th>Aryl cores R<sup>a</sup></th><th>cLogP<sup>b</sup></th><th>SARS-CoV-2<br>nsp14 N7-MTase<br>IC<sub>50</sub> (µM)<sup>c</sup></th></tr></thead><tbody><tr><td>4a</td><td>adenine</td><td></td><td>1.641</td><td>8.02 ± 0.74</td></tr><tr><td>4b</td><td>adenine</td><td></td><td>0.354</td><td>9.69 ± 0.92</td></tr><tr><td>4c</td><td>adenine</td><td></td><td>1.806</td><td>23.8 ± 0.9</td></tr><tr><td>4d</td><td>adenine</td><td></td><td>2.039</td><td>0.058 ± 0.002</td></tr><tr><td>4e</td><td>adenine</td><td></td><td>1.480</td><td>0.307 ± 0.01</td></tr><tr><td>4f</td><td>adenine</td><td></td><td>0.970</td><td>3.83 ± 0.7</td></tr><tr><td>4h</td><td>adenine</td><td></td><td>0.433</td><td>1.96 ± 0.28</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>4a–f, 4h</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>5g–h</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>6–8</figcaption></figure>
<table><tr><td>5g<sup>d</sup></td><td>adenine</td><td></td><td>-2.026</td><td>32.3 ± 3.96</td></tr><tr><td>5h<sup>d</sup></td><td>adenine</td><td></td><td>-0.457</td><td>5.50 ± 0.7</td></tr><tr><td>3*<sup>d,e</sup></td><td>adenine</td><td></td><td>1.641</td><td>0.061<sup>f</sup></td></tr><tr><td>10*<sup>d,e</sup></td><td>Pyrrolo-1,2,4-triazin-4-amine</td><td></td><td>2.153</td><td>0.093<sup>f</sup></td></tr><tr><td>6</td><td>Pyrrolo-1,2,4-triazin-4-amine</td><td></td><td>0.656</td><td>0.051 ± 0.003</td></tr><tr><td>7</td><td>C5-quinolin-ethynyl pyrrolo-1,2,4-triazin-4-amine</td><td></td><td>3.181</td><td>0.010 ± 0.001</td></tr><tr><td>8</td><td>C5-quinolin-ethynyl pyrrolo-1,2,4-triazin-4-amine</td><td></td><td>5.076</td><td>2.4 ± 0.1</td></tr><tr><td>MH44<sup>f</sup></td><td>adenine</td><td></td><td>0.144</td><td>0.019 ± 0.002</td></tr><tr><td>MH252<sup>f</sup></td><td>7-quinolin-ethynyl-7-deazaadenine</td><td></td><td>2.890</td><td>0.00072 ± 0.0004</td></tr><tr><td>MH344<sup>f</sup></td><td>7-phenyl-ethynyl-7-deazaadenine</td><td></td><td>3.003</td><td>0.0012 ± 0.00025</td></tr></table>

<sup>a</sup> various aryl cores bound to the N-ethylsulfonamide function at 5' position of adenosine for 4a–f, 4h, 5g–h, 6–8.

<sup>b</sup> cLogP were calculated using ChemDraw

<sup>c</sup> values are the mean of three independent experiments. The MTase activity was measured using a filter binding assay (see experimental section).

<sup>d</sup> in 5g–h, 3* and 10* no ethyl group on the NH of sulfonamide

<sup>e</sup> values from the literature for compound 3* described by Chen's group [34].

<sup>f</sup> values from the literature described by our group [36].

### 2.1.2. C-nucleosides: replacement of adenine by pyrrolo[2,1-f][1,2,4]triazin-4-amine

Chen's group reported the synthesis and inhibition of viral replication of nucleoside analogues whose nucleobase is pyrrolo[2,1-f][1,2,4]triazin-4-amine [34]. Pyrrolotriazines are isosteres of natural purine nucleobases used to synthesize C-nucleosides. A number of biologically active nucleoside analogues contain this adenine isostere, such as remdesivir [42]. When the analogue of MH44 possessing a pyrrolotriazine is docked with nsp14 of SARS-CoV-2 (PDB 7R2V), the adenine replacement does not impact affinity with the enzyme as attested by the docking scores which are in the same range for the two compounds. However, lipophilicity is increased compared with the adenine-bearing
nucleoside. We therefore synthesized three pyrrolo[2,1-f][1,2,4]triazine C-nucleosides **6–8** functionalized at 5' position with N-aryl-N-ethylsulfonamides found in the most active inhibitors **MH44** [33] or **4d**, linked to a pyrrolotriazine (**6**) or a C5-quinolin-substituted pyrrolotriazine (**7–8**) (Scheme 2). Indeed, in the previous series of nucleoside analogues inhibiting nsp14, we discovered the most potent nsp14 inhibitor to date, which is an analogue of **MH44** carrying a 7-quinolin-ethynyl-7-deazaadenine in place of adenine (**MH252**, Fig. 1) [36]. The same inhibitor was confirmed by another group a few months later [38]. By analogy, quinoline was introduced at C5 of the pyrrolotriazine via an ethynyl linker in compounds **7** and **8**. The C-nucleosides should be resistant to hydrolytic and enzymatic cleavage since the sugar and heterocyclic aglycon are connected by a C-C rather a C-N bond in canonical nucleosides.

The starting C-nucleoside **9** was prepared according to described procedures (Scheme 2) [34, 43, 44]. Treatment of **9** with N-iodosuccinimide gave the iodo precursor **10** of the C5-modified pyrrolotriazine nucleosides **7** and **8** in 84% yield [45]. C-nucleosides **9** and **10** were converted to their 5'-amino analogues **11** and **12** by the Mitsonobu reaction before reacting with either with 3-cyano-4-fluorophenylsulfonyl chloride to give **13** and **14** in 65% and 71% yield respectively, or with 5,6,7,8-tetrahydro-2-naphthalenesulfonyl chloride to afford **15** in 91% yield. After obtaining *p*-OMe derivatives **16** and **17** by aromatic nucleophilic substitution (76% and 83%), they were treated with ethyl tosylate to introduce an ethyl group on the sulfonamide nitrogen atom to give compounds **18** and **19** in 75% and 70% yield respectively. Similarly, NH of **15** was functionalized with an ethyl group to yield **20** (28%). The C5-iodo pyrrolotriazine intermediates **21** and **22** were then engaged in Sonogashira coupling with 3-ethynyl-quinoline to obtain nucleosides **21** and **22** in 91% and 53% respectively. The *isopropylidene* protection of **18**, **21** and **22** was removed by treatment with a TFA solution to yield the final C-nucleosides **6–8**.
<figure><img src="image_8.png" /></figure>

<footnote>21, R = isopropylidene
h → 7, R = H</footnote>
**Scheme 2.** Synthesis of N-arylsulfonamide C-nucleosides 6–8. **Reagents and conditions:** (a) N-iodosuccinimide, DMF, rt, 24 h, 84%; (b) phthalimide, DIAD, triphenylphosphine, THF, rt, 2.5 h, 62–71%; (c) hydrazine hydrate, EtOH, 55 °C, 15 h, 33–75%; (d) arylsulfonylchloride, Et<sub>3</sub>N, DMF, -10°C, 2.5 h, 65–90%; (e) sodium methoxide, MeOH, 50 °C, 15 h, 73–83%; (f) EtOTs, KI, K<sub>2</sub>CO<sub>3</sub>, DMF, 50 °C, 15 h, 28–77%; (g) 3-ethynyl-quinoline, Et<sub>3</sub>N, Cul, Pd(PPh<sub>3</sub>)<sub>4</sub>, THF, 60 °C, 2.5 h, 53–91%; (h) TFA/H<sub>2</sub>O 8/2 v:v, rt, 30 min, 39–69%.

2.1.3. Synthesis of MH44 prodrugs

The prodrug approach is suitable to modify or mask negative features of the parent active compound
linked to either chemical or *in vivo* instability, poor aqueous solubility, low penetration into cells,
toxicity [46, 47]. It has been widely explored for nucleosides and nucleotides over the last few
decades, and several examples, selected from the many reported structures, as mericitabine,
balapiravir, molnupiravir, all *isobutyryl* esters of cytidine derivatives, testify to the successful
application of this strategy [48, 49]. The covalent attachment of lipophilic moieties (esters, amides)
aims to increase the passive diffusion through cell membrane. Moreover, Chen's group
demonstrated that the use of acyl prodrugs resulted in N7-MTase nsp14 inhibitors with significant,
albeit moderate, antiviral activity [34]. Therefore, we designed different prodrugs of MH44, one of
the most potent nsp14 inhibitors that had shown no inhibition of viral replication in SARS-CoV-2
infected cells, to attenuate the high polarity of the nucleoside. Masking of 2'-OH and 3'-OH as well as
the N6 position of adenosine was achieved as *isobutyrate* prodrugs **23–25** [34, 50], carbonate
prodrug **26** [51, 52] and pH-sensitive *orthoester* prodrugs **27–30** (Scheme 3) [53].

<figure><img src="image_9.png" /></figure>

**Scheme 3.** Synthesis of MH44 prodrugs **23–30**. **Reagents and conditions:** (a) *isobutyric acid*, DMAP, 3-ethylcarbodiimide hydrochloride, DMF, 0 °C, 2.5 h, 10–76%; (b) 1,1'-carbonyldiimidazole, DMF, rt, 30 min, 52%; (c) for **27**: trimethylorthoformate, *p*-toluenesulfonic acid, rt, 1.5 h, 96%; for **28**: triethylorthoformate, *p*-
toluenesulfonic acid, 70 °C, 6 h, 60%; for **29**: trimethylorthoacetate, *p*-toluenesulfonic acid, 70 °C, 2.5 h, 55%; for **30**: triethylorthoacetate, *p*-toluenesulfonic acid, 70 °C, 72 h, 75%.

The *isobutyrate prodrugs 23–25* with one, two or three acyl groups respectively, were obtained by reaction of **MH44** with *isobutyric acid* in the presence of 3-ethylcarbodiimide hydrochloride and DMAP in DMF. For **23**, the 2'-*O* or 3'-*O*-protected isomer was not isolated, thus a mixture of regioisomers was tested in cell assays. The carbonate nucleoside **26** was readily prepared by treating **MH44** by 1,1'-carbonyldiimidazole in DMF [52]. Finally, to mask the 2'-OH and 3'-OH with an orthoester function, **MH44** was dissolved in various orthoester solvents in the presence of *p*-toluenesulfonic acid to give the corresponding prodrugs **27–30** in yields ranging from 55 to 95%. The inhibitory activity of MH44 prodrugs against nsp14 has not been assessed, as we have previously demonstrated that 2',3'-*O*-protected nucleosides do not inhibit nsp14, demonstrating the crucial role of ribose 2'-OH and 3'-OH for inhibition of nsp14 [33].

## 2.2. Biology

### 2.2.1. Structure-Activity Relationship (SAR)

Compounds **4a–f**, **4h**, **5g–h** and **6–8** were tested for their ability to inhibit SARS-CoV-2 N7-MTase nsp14 using a radioactive MTase assay that involves measuring the [³H]-radiolabeled methyl transferred from the SAM onto the N7-guanine of short cap RNA substrate (GpppAC₄) [54]. IC₅₀ values were determined in a dose-response assay using increasing compound concentration (Table 1). In parallel, we performed molecular modeling experiments on the SARS-CoV-2 N7-MTase nsp14 in the presence of SAH (PDB 7R2V) using Autodock Vina [55]. Calculated ΔG values and IC₅₀ values are compared in Table S1 (SI). In our previous work [36], the calculated ΔG values which refer to predicted free energy of binding (the lower the values, the less energy is required to forming the enzyme-ligand complex in a specific conformation) were consistent with the IC₅₀ values of the active compounds. This prompted us to adopt the same approach for a rational design. The reference compounds MH44, MH252, and MH344 exhibit similar binding poses after docking experiments: the arylsulfonamide interacts with the side chain of Phe426 to form a π–π stacking interaction. This residue is crucial for enzyme activity, as shown by the PDB 5C8S crystal structure: it is this residue that orients the guanine of the viral RNA cap into the enzyme pocket. In addition, the nitrile group forms a hydrogen bond with the hydroxyl group of Thr328 present in the pocket. On the other side, the nucleoside moiety interacts with Gln354, Ala353, and Tyr368 to form hydrogen bond from 2'OH, N3, and N1, respectively. These interactions of the nucleoside moiety are consistent with the binding pose of SAH in the pocket from PDB 7R2V.

Compounds **4a** with a naphthalene and **4b** with a quinoline showed similar activities with IC₅₀ = 8.02 and 9.69 µM, respectively, but much higher than the IC₅₀ of the compound **3*** described by L.
Chen (61 nM) [34]. This result is surprising given the very similar docking scores (ΔG ≈ -10.8 kcal/mol), even though the sulfonamide adenosine is not connected to the same position of naphthalene or quinoline in **4a** and **4b** compared with **3***. Furthermore, the docking experiment shows a similar pose for these three compounds with a π-π interaction formed between the aromatic bicyclic sulfonamide and Phe 426. These results show a limit in our docking experiment or in the comparison of the IC₅₀ from pur assay and that described by L. Chen. Compound **4c** with a dansyl group showed an IC₅₀ of 23.8 µM, the dimethylamino group in position 5 of the naphthalene is rather detrimental for its inhibitory activity (**4a**, IC₅₀ 8.02 µM). Compound **4f** bearing a dihydrobenzofuran was less active (IC₅₀ = 3.8 µM) than its carbon "indane" analog **4e** (IC₅₀ = 0.307 µM). Only **4d** and **4e** showed sub-micromolar IC₅₀ (IC₅₀ = 58 nM and 307 nM, respectively), both bearing a saturated ring fused to the phenyl attached to the sulfonamide, and **4d** is more active than the broad spectrum MTase inhibitor sinefungin (IC₅₀ = 278 nM). Compounds **5g-h** and **4h** with a quinazolinedione showed increasing activity as a function of the number of ethyl groups (0, 2 or 3) on nitrogen atoms. Indeed, compound **5g** has a much lower activity (IC₅₀ = 32.3 µM) than its analog **5h** substituted on the quinazolinedione nitrogen atoms (IC₅₀ = 5.5 µM). As with all 5'-*N*-arylsulfonamide derivatives previously studied [33, 36], the compound activity enhanced when the sulfonamide nitrogen atom was also functionalized with an ethyl group (**4h**, IC₅₀ = 1.96 µM). Overall, replacing the phenyl ring of the arylsulfonamide moiety with a bicyclic ring preserves pharmacophore activity while maintaining the same pose of the compounds in the enzyme pocket, as shown by molecular docking experiments.

C-nucleosides **6** and **7** displayed IC₅₀ of 51 and 10 nM respectively, which are in the same range as the IC₅₀ of **MH44** (19 nM) [33], even though the ΔG values of **6** and **7** (-9.6 and -10.8 kcal/mol, respectively) are higher than ΔG of MH44 (-8.7 kcal/mol). These data show that replacement of adenine by a pyrrolotriazine is not detrimental to inhibitory activity against *N*7-MTase nsp14. The results are consistent with Chen's study showing that compound 3* and C-nucleoside 10* have comparable IC₅₀ values (Table 1) [34]. This is evidenced by molecular docking experiments, which show that pyrrolotriazine interacts with the pocket in the same way as the nucleobase adenine. In contrast, C-nucleoside **8** with the *N*-tetralin-*N*-ethylsulfonamide moiety at 5' position has a much lower activity with IC₅₀ = 2.4 µM. This result is surprising compared to its adenine-containing analogue **4d**, which has an IC₅₀ = 58 nM, especially as the docking score of **8** (ΔG -12.3 kcal/mol) was more favorable for a better affinity for the nsp14 enzyme than that of **4d** (ΔG -9.6 kcal/mol). Moreover, the higher lipophilicity of **8** (cLogP 5.27) could lead to solubility issues in aqueous media, thus limiting its activity. The most unexpected results concern C-nucleoside **8** and **MH252**, which have similar ΔG values (-11.5 and -11.7 kcal/mol, respectively), but very different IC₅₀ values (2.4 µM
and 0.72 nM, respectively) whereas the experimental data for **MH44** (IC₅₀ =19 nM) and **MH252** (IC₅₀ = 0.72 nM) were consistent with the docking results (ΔG = -8.7 and -11.5 kcal/mol, respectively). Here, docking experiments do not allow us to explain accurately the effects of structural modifications on the inhibitory efficacy observed experimentally (SI, Fig. S1).

### 2.2.2. Stability of prodrugs 23–30 and potent nsp14 inhibitors 6, MH44, MH252

To study the stability of prodrugs **23–30** and nucleosides **6, MH44** and **MH252** with remarkable inhibitory activity on N7-MTase nsp14, we monitored their fate in the presence of different media using HPLC with an "on-line cleaning method" developed several decades ago [56, 57]. First, the chemical stability was studied in Dulbecco's Modified Eagle Medium (DMEM), then in DMEM enriched with 10% fetal calf serum (FCS) and a mixture of antibiotics (Streptomycin and Azithromycin at 100 µg/L each) in order to evaluate the stability of compounds in the extra-cellular medium used for antiviral evaluation in cell culture systems. The third medium was total cell extract from Vero E6 cells, mimicking the fate of compounds inside the cells.

The poor solubility of compound **MH252** in various aqueous media resulted in too much variability in HPLC analyses to obtain reliable stability data. On the other hand, we were able to study the fate of **MH44** and **6**. No degradation was observed for **MH44** in any medium after 120 h of incubation period. Slight degradation of C-nucleoside **6** began after 48 h in DMEM, reaching 14% degradation after 120 h, which is well above incubation times in infected cell assays.

Nucleoside **25**, bearing three *isobutyryl* groups, was too poorly soluble in aqueous media for reliable HPLC analyses, as observed for compound **MH252**. Compound **23** bearing a single *isobutyryl* group showed a t₁/₂ of 4.5 h in DMEM and 6.5 h in FCS-DMEM, which increased to 53 h in cell extracts, and a signal corresponding to the parent **MH44** was observed in the HPLC chromatograms. The di-substituted analogue **24** showed equivalent t₁/₂ in DMEM and cell extracts (4.8 and 4.5 h respectively). It proved more stable in FCS-DMEM (t₁/₂ 12 h). Upon degradation of **24**, signals corresponding to **23** appeared, showing unmasking of a single hydroxyl. Several signals were observed as degradation of **24** leads to the formation of a mixture of 2'- and 3'-*O*-isobutyryl regioisomers.

Carbonate prodrug **26** degrades rapidly in DMEM and FCS-DMEM with a t₁/₂ of 33 and 31 minutes respectively. However, in cell extracts, its t₁/₂ increased to 3.2 h, and the signal corresponding to **MH44** (RT=14.45 min) was observed during the degradation of **26** (RT=16.90 min) in the HPLC chromatograms (SI, Fig. S1).

The orthoformiate nucleosides **27** and **28** remained intact in all media tested for up to 48 h and do not release the active inhibitor **MH44**. In contrast, the orthoacetate prodrugs **29** and **30** were degraded slowly in all media except in cell extracts for **30**. Methyl orthoacetate **29** has half-lives (t₁/₂)
ranging from 14.8 h (in FCS-DMEM) to 23.8 h (in cell extracts, Table 2). Ethyl orthoacetate **30** has $t_{1/2}$ times ranging from 15.2 h (in enriched DMEM) to 23.1 h (in DMEM, Table 2). FCS-DMEM degrades orthoacetates more rapidly than other media, which could be due to action mediated by the FCS enzymatic content. Despite the transformation observed for compounds **29** and **30**, the signal corresponding to the parent nucleoside **MH44** was not observed in the HPLC chromatograms, indicating that these compounds degrade into a different chemical species and probably cannot act as prodrugs. This chemical species could be the product of partial hydrolysis of the orthoester.

<table><caption>**Table 2**</caption><thead><tr><th>Prodrugs of MH44</th><th>Half-lives in DMEM $t_{1/2}$ (h)</th><th>Half-lives in FCS-DMEM $t_{1/2}$ (h)</th><th>Half-lives in cell extracts $t_{1/2}$ (h)</th></tr></thead><tbody><tr><td><b>23</b></td><td>4.5</td><td>6.5</td><td>53.0</td></tr><tr><td><b>24</b></td><td>4.8</td><td>12.0</td><td>4.5</td></tr><tr><td><b>25</b></td><td>n.d<sup>a</sup></td><td>n.d<sup>a</sup></td><td>n.d<sup>a</sup></td></tr><tr><td><b>26</b></td><td>0.6</td><td>0.5</td><td>3.2</td></tr><tr><td><b>27</b></td><td>stable<sup>b</sup></td><td>stable<sup>b</sup></td><td>stable<sup>b</sup></td></tr><tr><td><b>28</b></td><td>stable<sup>b</sup></td><td>stable<sup>b</sup></td><td>stable<sup>b</sup></td></tr><tr><td><b>29</b></td><td>20.1.</td><td>14.8</td><td>23.8</td></tr><tr><td><b>30</b></td><td>23.1</td><td>15.2</td><td>stable</td></tr></tbody></table>

### 2.2.3. Delivery of dansyl-containing nucleoside **4c** in A549 cells

In order to check the cellular uptake of nsp14 nucleoside inhibitors, we monitored their internalization in cells by fluorescence microscopy. As the introduction of a bulky fluorescent label could affect the cell penetration properties of the nucleoside inhibitor, we chose to use compound **4c** bearing a dansyl group, as a fluorescent probe to study its cellular uptake. This nucleoside **4c** is similar in size and lipophilicity to most nsp14 inhibitors with bicycles on sulfonamide shown in Table 1. Compared to **4d** with a tetralin moiety, their molecular structure and cLogP are very close. **4c** displays an absorption band at 260 nm for adenosine and a broader band at 345 nm for the dansyl group. The fluorescence emission of **4c** shows a broad band between 490 and 670 nm and a maximum at 565 nm with a fluorescence quantum yield of 4% at 20°C in water (< 0.5% DMSO).

To investigate the cellular internalization of compound **4c**, A549 cells were incubated for 30 minutes in DMEM supplemented with increasing concentrations of **4c** (Fig. 2a). Following extensive washing to remove extracellular compound, fluorescence microscopy analysis revealed a concentration-dependent intracellular accumulation (Fig. 2b and c). The fluorescence signal was

<footnote><sup>a</sup> not determined because of poor solubility in aqueous media</footnote>

<footnote><sup>b</sup> stable after 48 h incubation in different media</footnote>
predominantly localized in the cytoplasm, with marked enrichment in the perinuclear region, whereas no nuclear localization was detected (Fig. 2b).

<figure><img src="image_10.png" /><figcaption>**Fig. 2.** Cellular uptake and subcellular distribution of compound 4c in A549 cells. (a-c) Subconfluent A549 cells were incubated for 30 min in DMEM containing increasing concentrations of compound 4c. Following incubation, cells were washed and analyzed by fluorescence microscopy. (a) Schematic overview of the experimental workflow. (b) Representative fluorescence microscopy image of cells treated with 250 µM compound 4c, illustrating cytoplasmic accumulation. (c) Quantification of intracellular fluorescence intensity in individual cells. Each data point represents the mean fluorescence of 10 cells in a given field of view. (d) Subconfluent A549 cells were incubated for 30 min in DMEM at 250 µM of compound 4c. Following incubation, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed, and analyzed by fluorescence microscopy. The cells were then kept for 16 hours at 4°C and analyzed again by fluorescence microscopy. (e) A549 cells were pre-incubated with Mitotracker (30 min) to label mitochondria, followed by incubation with 250 µM compound 4c for an additional 30 min. Fluorescent signals were acquired to assess subcellular co-localization. Line profile analysis (gray value plot) was performed along the yellow line in the zoomed-in region to compare the spatial distribution of compound 4c with the mitochondrial marker.</figcaption></figure>

To determine whether the perinuclear accumulation was maintained actively or resulted from passive diffusion, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes after 4c exposure. PFA solution was replaced with fresh PBS, and fluorescence was assessed both immediately and after an additional 16-hour incubation at 4 °C (Fig. 2d). Notably, the perinuclear localization was lost after prolonged incubation, with the fluorescence redistributing diffusely throughout the cytoplasm. This observation suggests that the initial perinuclear enrichment of 4c is not a passive phenomenon but likely depends on active cellular processes. Altogether, these findings demonstrate that 4c is efficiently internalized and accumulates intracellularly in a concentration-dependent manner, with its subcellular localization pattern suggesting involvement of active or facilitated transport mechanisms.

To further investigate whether 4c accumulates in specific subcellular compartments, we examined the perinuclear fluorescence pattern, which suggested localization to an organelle-rich region
potentially corresponding to either the endoplasmic reticulum or the mitochondrial network. To assess the latter possibility, cells were co-stained with MitoTracker Red CMXRos, a mitochondria-specific fluorescent probe (Ex/Em: 579/599 nm), enabling visualization of potential colocalization. The spectral separation from the dansyl emission allowed clear dual-channel imaging without signal overlap (SI, Fig. S2-S3). Fluorescence microscopy revealed substantial overlap between **4c** and MitoTracker signals (Fig. 2d). Cross-sectional fluorescence analyses across individual cells revealed overlapping fluorescence peaks, indicating a positive spatial correlation between **4c** accumulation and mitochondrial structures. These results suggest that a significant portion of intracellular **4c** accumulates in or around mitochondria.

In addition to mitochondrial localization, we observed discrete regions of intense **4c** fluorescence that did not overlap with MitoTracker staining. Based on their morphology and spatial distribution, these regions likely correspond to other membrane-rich organelles, such as the endoplasmic reticulum or nuclear envelope (Fig. 2e, arrowheads “e” and “n”). The observed pattern suggests that **4c** preferentially accumulates in membrane-dense regions of the cytoplasm, including mitochondria. This subcellular distribution is consistent with the hydrophobic nature of **4c** and its potential affinity for lipid-rich environments. LogD values were calculated for several compounds (SI, Table S2) and compound **4c** displays higher LogD values than Chen’s compound **10*** in the pH range of various cell compartments, meaning that it is more lipophilic than **10*** (EC₅₀ = 0.72 µM) and could be trapped by lipid bylayers. Indeed, just because our compounds could cross the cell membrane and end up in the cytoplasm does not mean that they would have access to the replication machinery which is in vesicles surrounded by another lipid bilayer to cross. Unfortunately, there are no tests today that can evaluate the crossing of all these membranes to be sure that the inhibitors can reach their enzymatic target.

### 2.2.4. SARS-CoV-2 replication inhibition assays for anti-nsp14 drugs in pulmonary A549-hACE2 cells

To assess whether the inhibitory activity measured in SARS-CoV-2 replication assays translates to viral replication inhibition, the antiviral activity of the best nsp14 inhibitors **4d-e**, **6-8** as well as MH44 prodrugs **23-30**, and three very potent nsp14 inhibitors from previous studies **MH44** [33], **MH252** and **MH344** [36] was evaluated using a SARS-CoV-2 infected pulmonary A549-hACE2 cell line. Compounds were tested at three concentrations (50, 25, 12.5 µM) on non-infected cells to measure cytotoxicity, and on SARS-CoV-2-infected-cells to assess their antiviral effect. Results were expressed as the percentage (%) of toxicity and the percentage of infection inhibition (Fig. 3). The molecule
Remdesivir was used as a control. Three compounds achieve around 50% inhibition: compound **4d** at 25 µM and compound **4e** at 50 µM. Compound **MH252** also shows 50% of inhibition at 50 µM, but with high toxicity. A stable inhibitory effect of about 70% at the three concentrations tested is detected for compound **MH344** but immunofluorescence imaging shows that cells treated with this compound harbors a strong disorganization of the cytoplasm with apparent membrane vesiculations (SI, Fig. S4).

<figure><img src="image_11.png" /><figcaption>**Fig. 3.** SARS-CoV-2 inhibition and cytotoxicity assays in A549-hACE2 cells. (a) SARS-CoV-2 inhibition assay; (b) Drug cytotoxic assays. See Materials and Methods section for details.</figcaption></figure>

**3. Conclusions**

This study reports the structural optimization of *N*-arylsulfonamide-based SARS-CoV-2 nsp14 *N*7-methyltransferase inhibitors through the introduction of bicyclic moieties and synthesis of *C*-nucleoside analogues. Several compounds showed potent nanomolar inhibition, with SAR analyses highlighting the role of aryl substitutions on both enzymatic activity inhibition and physicochemical properties. In addition, we also synthesized various MH44 prodrugs. Although prodrugs improved
stability in biological media, limited intracellular release of the parent active inhibitor remains a
challenge, indicating a limited suitability for prodrug strategies making use of the 2',3'-diol.

Fluorescence microscopy confirmed cellular uptake and cytoplasmic accumulation of N-
arylsulfonamide-based SARS-CoV-2 nsp14 N7-methyltransferase inhibitors. Antiviral assays revealed
moderate efficacy and some cytotoxicity for some nsp14 inhibitors, indicating further optimization is
needed. Overall, the N-arylsulfonamide adenosine scaffold shows promise, warranting continued
development toward effective, cell-permeable MTase inhibitors.

**4. Experimental section**

**4.1. *Synthetic procedures***

Detailed synthetic procedures and spectral characterization data for compounds **2a–h**, **3a–f**, **3h**, **4a–f**, **4h**, **5g–h**, **6–8**, **11–30** are included in the Supporting Information. Only general synthetic procedures are reported in this experimental section.

4.1.1. *General method A for the synthesis of 2a–g, 13–15*

To a solution of **1**, **11** or **12** (1.00 eq) in anhydrous DMF (C = 0.1 M) were added Et₃N (2.00 eq) and the corresponding arylsulfonyl chloride (1.25 eq) in three portions at 10 °C under argon. After stirring at -10°C for 2.5 h, the reaction mixture was diluted with AcOEt and brine. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with a 2 M LiCl solution and brine, dried over Na₂SO₄ and concentrated *in vacuo*. The residue was purified by flash column chromatography to give the desired compound.

4.1.2. *General method B for the synthesis of 16–17*

A solution of 0.5 M MeONa in MeOH (5.00 eq) was added to a solution of **13** or **14** (1.00 eq) in MeOH (C = 0.2 M) under argon. The reaction mixture was stirred and heated at 50 °C until completion of the reaction. The crude was diluted with AcOEt and neutralized with a saturated NH₄Cl solution. The 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 to give the desired compound.

4.1.3. *General method C for the synthesis of 2h, 3a–f, 18–20*

A suspension of **2a–g** or **15–17** (1.00 eq), ethyl *p*-toluenesulfonate (1.50 eq), KI (0.10 eq) and K₂CO₃ (3.00 eq) in anhydrous DMF (C =0.1 M) was stirred under argon at 50 °C for 16 h. After cooling to room temperature, the reaction mixture was diluted with AcOEt and water. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with a 2 M LiCl solution
then brine, dried over Na₂SO₄ and concentrated *in vacuo*. The residue was purified by column chromatography to give the desired compound.

#### 4.1.4. General method D for the synthesis of 21-22

To a solution under argon of **19** or **20** (1.00 eq) in THF (C = 0.066 M) were added Et₃N (3.00 eq) and 3-ethynylquinoline (2.00 eq). After stirring for 30 minutes under an argon flow, Cul (0.30 eq) and Pd(PPh₃)₄ (0.10 eq) were added. After stirring for 2 h at 60°C, silica was added to the mixture and all the volatiles were removed *in vacuo*. Residue was purified by flash column chromatography (dry sample, silica gel, linear gradient 0-100% AcOEt in cyclohexane) to give **22** or **23** as a colorless foam.

#### 4.1.5. General method E for the synthesis of final compounds 4a-f, 4h, 5g-h, 6-8.

Compounds **2g-h**, **3a-f**, **3h**, **18**, **21-22** were treated with a mixture of trifluoroacetic acid/water (8/2 v:v, C = 0.08 M). After stirring at 25 °C for 0.5 h, the solvents were removed *in vacuo* and the crudes were co-evaporated three times with water and three times with MeOH. The residues were dissolved in AcOEt and saturated NaHCO₃ solution, aqueous layer was extracted with AcOEt, combined organics extracts were washed with brine, dried over anhydrous Na₂SO₄ and concentrated *in vacuo*. The residue was purified by column chromatography. Fractions containing the pure products were concentrated and trituration in Et₂O or pentane afforded the desired compounds as solids.

#### 4.1.6. General method F for the synthesis of isobutyryl prodrugs 23-25

To a solution at 0 °C under argon of isobutyric acid (1.20 eq for **23** and 3.00 eq for **24-25**), EDC (1.80 eq for **23** and 4.00 eq for **24-25**) and DMAP (0.50 eq) in anhydrous DMF (C = 0.35M) was added **MH44** (1.00 eq). After stirring at 0 °C for 2 h, the reaction mixture was diluted with AcOEt and saturated NH₄Cl solution. The aqueous layer was extracted with AcOEt and the combined organic extracts were washed with 2M LiCl solution, brine, dried over Na₂SO₄ and concentrated *in vacuo*. The residue was purified by flash column chromatography (liquid sample, silica gel, linear gradient 0-5% MeOH in DCM) to give **23-25** as a white solid with 97-99% purity determined by HPLC analysis at 260 nm.

### 4.2. Molecular docking

All molecular docking experiments were performed using Autodock Vina [55]. The X-ray crystal structure of SARS-CoV-2 nsp14 (PDB 7R2V) was used as the receptor and prepared in Chimera [58]. The ligand structures were drawn and minimized using MarvinSketch (ChemAxon). The receptor and ligand structures were converted to PDBQT format using MGL tools [59]. The exhaustiveness was set to 16 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 20 x 20 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 = 11.8, Y = -9.4, Z = -23.0.
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. Representations of protein-
ligand complexes were visualized using PyMOL (The PyMOL Molecular Graphics System, Schrödinger,
LLC) and Ligplot+, respectively [60].

4.3. Enzymatic assays

4.3.1. Expression and purification of recombinant protein

SARS-CoV-2 nsp14 (N7-MTase) coding sequence was cloned in fusion with a N-terminus hexa-histidine tag in pET28 plasmids.[33] The protein was 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 protein was 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 chromatography (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 protein was subsequently concentrated up to 12.5 µM and conserved at -20 °C in a buffer containing 50% of glycerol.

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

The SARS-CoV-2 nsp14 MTase assay was carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl₂, 1.9 µM SAM, and 0.1 µM ³H-SAM (Perkin Elmer)] in the presence of 0.7 µM GpppAC₄ synthetic RNA[33] and the MTase (10 nM). Briefly, the enzyme was 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 60 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 ³H-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)⁵Hillslope)]). IC₅₀ is defined as the inhibitory compound concentration that causes 50% reduction in enzyme activity.

4.4. Fluorescence imaging of 4c uptake
A549 cells were cultured on sterile glass coverslips in high-glucose Dulbecco's Modified Eagle's Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FCS; Serana) and 1% Penicillin-Streptomycin (Gibco). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. Compound 4c was prepared as a 50 mM stock solution in DMSO and diluted into fresh DMEM immediately prior to treatment, ensuring a final DMSO concentration of 1% (v/v) across all experimental conditions. For mitochondrial labeling, MitoTracker Red CMXRos (Thermo Fisher), reconstituted in DMSO, was added to DMEM at a 1 ‰ (v/v) dilution, following the manufacturer's instructions. Both 4c and MitoTracker treatments were performed for 30 min at 37 °C. For dual-staining experiments, cells were first incubated with MitoTracker for 30 min, followed by a 2-hour recovery period in fresh DMEM before incubation with 4c. Live imaging of compound 4c uptake was performed using an EVOS M5000 fluorescence microscope (Thermo Fisher) equipped with a 40× air objective and a DAPI filter cube, which partially excites and detects dansyl fluorescence.

For fixed-cell imaging, cells were washed in phosphate-buffered saline (PBS) and fixed for 10 minutes at room temperature with 4% paraformaldehyde (Thermo Fisher) in PBS. Coverslips were mounted onto glass slides using a 1:1 (v/v) glycerol-PBS mounting medium and secured with adhesive tape. Imaging was performed within 30 min post-fixation to minimize potential diffusion of compound 4c. High-resolution fluorescence imaging and colocalization analysis were conducted using a Leica Thunder microscope (Leica Microsystems) equipped with a 100× oil immersion objective and a DAPI cube for dansyl detection. All fluorescence images were processed and analyzed using ImageJ. For quantifying intracellular accumulation, individual cells were manually segmented based on morphology and fluorescence boundaries. The mean fluorescence intensity of each cell was measured within the segmented area, and values were averaged over multiple cells per field of view. For colocalization analysis, fluorescence intensity profiles were extracted along linear regions intersecting more than three MitoTracker fluorescence maxima to assess the spatial correlation between compound 4c and MitoTracker signals.

### 4.5. Cell-based assays

#### 4.5.1. Cell lines, cell culture and reagents

The human pulmonary alveolar A549-hACE2 cells were engineered using a lentiviral vector from Flash Therapeutics (Toulouse, France) to express the human receptor ACE2. A549 cells obtained from ECACC (#86012804) were transduced and maintained in RPMI supplemented with 10% heat inactivated fetal bovine serum (FBS), 50U/mL of penicillin, 50µg/mL of streptomycin, 25mM of HEPES and 1mM of sodium pyruvate at 37°C with 5% CO₂. Cells were then sorted by FACS in order to obtain a A549-hACE2 population stably expressing hACE2.
### 4.5.2. SARS-CoV-2 virus stock and titration for antiviral screening

The strain hCoV-19/France/GE1973/2020 was supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). The human sample from which strain hCoV-19/France/GE1973/2020 was isolated has been provided by Dr. L. Andreoletti, Robert Debré Hospital, Reims, France and was supplied through the European Virus Archive goes Global (EVAg) platform, a project that has received funding from the European Union's Horizon 2020 research and innovation program under the grant agreement No 653316. Viruses were propagated in VeroE6 cells with DMEM containing 25 mM HEPES at 37°C in a 5% CO₂ atmosphere and were harvested 72 hours post inoculation. Virus stocks were stored at -80°C. Virus titration from stocks and infected cell culture supernatants were monitored using plaque assays on a monolayer of VeroE6 cells, using 100µL of virus solution. Samples were serially diluted and the plaque forming unit (PFU) values were determined using crystal violet coloration on cells and subsequent scoring the amounts of wells displaying cytopathic effects. Calculations allow to determine the titer as the number of PFU/mL.

### 4.5.3. Assessment of cytotoxicity and antiviral activity

All experiments with infectious SARS-CoV-2 virus were conducted in the biosafety level 3 facility in CNRS CEMIPAI UAR3725 Montpellier. Antiviral activity was assessed by a cytopathic effect (CPE) reduction assay on A549-hACE2 cells. Briefly, 25,000 cells per well were cultured in 96-well culture plates for 24 hours. Compounds were diluted in RPMI from 10mM stock to different concentrations tested. Cells were incubated with 100µL of the diluted compounds for 2h. Subsequently, cells either were mock infected (for analysis of cytotoxicity of the compound) or infected with 400 PFU of virus per well (MOI of 0.01) in a total volume of 110µL of medium with compound. Cell viability was assessed 3 days post-infection by MTS assay using a Cell Titer 96 aqueous cell proliferation kit (Promega) or Viral Tox Glo™ (Tox Glo assay). Absorption at 495 nm (MTS) or luminescence (Tox Glo) were measured with an EnVision multilabel plate reader (PerkinElmer). Data were treated with GraphPad Prism v10.4.2, based on the following calculations :

$$ \text{Cell cytotoxicity, \% Toxicity} = \left(1 - \frac{\text{drug}}{\text{cell DMSO}}\right) \times 100 $$

$$ \text{Percentage of viral replication inhibition, \%Inhibit} = \left( \frac{\text{drug-infected cells}}{\text{cell DMSO-infected cells}} \right) \times 100 $$

Data are mean ± sd of three biological replicates.

### 4.5.4. Immunofluorescence and microscopy cell imaging

A549-hACE2 cells seeded on 96-well cycloolefin cell culture treated microplate (Greiner) were treated with compounds and infected with SARS-CoV-2 at a MOI 0.01. At 24 h post-infection, cells were washed with PBS (Eurobio, France) and fixed in 4% paraformaldehyde in PBS for 15 min at room
temperature, followed by permeabilization with 0.2% Triton X-100 (Sigma, Lezennes, France) in PBS for 5 min and blocking in 2% BSA in PBS for 15 min. Incubation with primary rabbit antibody anti-SARS-CoV-2 membrane (M) protein (Rockland, MA, USA, 100-401-A55) was performed for 2 h at room temperature. After washing with PBS, cells were incubated with secondary antibody Alexa488-labeled donkey-anti-rabbit (Invitrogen, Paris, France, A21206) for 1 h at room temperature. DAPI staining was used for the visualizing the cell nuclei. Brightfield and confocal fluorescence images were acquired using a Cell-Discoverer 7 LMS900 confocal laser-scanning microscope (Zeiss, Germany).

### CRediT authorship contribution statement

M. H., H. M. and A. T.: synthesis of the compounds; M. H.: conceptualization, investigation, methodology, HPLC monitoring of compounds fate in various media; A. D. and E. D.: biochemical assays, investigation, methodology; J. Z. and S. N.: delivery of the dansyl compound in cells, investigation, methodology, validation, writing – review & editing; F. G., J. S., N. G. and D. M.: cell-based assays, investigation, methodology, validation, writing – review & editing; J.-J. V. and B. C.: resources, supervision, review & editing; E. D. and F. D.: supervision, validation, funding acquisition, project administration, writing – original draft and writing – review & editing.

### Acknowledgements

The research described here was part of the Virage project (M. H., H. M., J.-J. V., F.D., B.C., E.D.) supported by grant ANR-20-CE11-0024-02 from the Agence Nationale de la Recherche (ANR). M. H. and H. M. acknowledge the ANR for financial support for their PhD work and Master 2 internship respectively (VIRAGE: ANR-20-CE11-0024-02). A. D., B. C. and E. D. has received EU-funding from SCORE project H2020 SC1-PHE-Coronavirus-2020, grant agreement ID: 101003627. J.Z. was supported by a grant from the ANR (ANR-21-CE15-0041). We acknowledge the imaging facility of Montpellier (MRI), member of the national infrastructure France-BioImaging supported by the Agence Nationale de la Recherche (ANR-10-INBS-04, "Investissements d'avenir").

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://delp
# Data availability

No data was used for the research described in the article

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