The invention relates to new tri-substituted tetrahydrofurans and their use as antiviral agents in the treatment and/or prevention of coronavirus infections, including COVID- 19.

BACKGROUND ART

Type-2 coronavirus causing severe acute respiratory syndrome (SARS-CoV-2) is the causative agent of COVID-19. The emergence of this virus at the end of 2019 has posed a huge biomedical challenge. Other related coronaviruses have previously emerged in the human population, such as SARS coronavirus and MERS coronavirus. Finding effective antivirals against SARS-CoV-2 and other coronaviruses is therefore a global priority.

To date, few drugs with demonstrable therapeutic effect in COVID- 19 patients have been described, and their effects have been modest at best. A compound with a slight ability to improve the prognosis of these patients is Remdesivir, a nucleoside analogue exhibiting broad-range antiviral activity, for which an IC50 against SARS- CoV-2 on the order of 1 μM was shown (Pizzorno, A. et al. In vitro evaluation of antiviral activity of single and combined repurposable drugs against SARS-CoV-2. Antiviral Res 181 , 104878 (2020)). In addition, there are numerous compounds with potential anti-SARS-CoV-2 effect in vitro, some of which are in the pre-clinical or clinical research phase. This includes a potent in vitro activity has been described for Aplidin (plitidepsin), a compound belonging to the didemnin family that was initially developed as an antitumoral (White, K. M. et al. Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Science 371 , 926- 931 (2021).

Therefore, due to the need for safe and effective antiviral agents with a wide-spectrum of anti-viral activity, especially against coronaviruses, the present invention provides tri-substituted tetrahydrofurans that could be useful as antiviral agents and, more particularly, against coronavirus infections. Substituted tetrahydrofurans have been described in the document:Adiwidjaja G. et al. "Eiectrophile-induced cyclizations of silyl-substituted 4-alken-1-ols", LIEBIGS ANNALEN: ORGANIC AND BIOORGANIC CHEMISTRY, 1995, 501-507, which discloses that silyl-substituted 4-alken-1-ols cyclize in the presence of a wide range of electrophilic reagents to give substitutes tetrahydrofurans.

In the document: Corriu R. "SYNTHESES A PARTIR DE CARBANIONS ALLYLIQUES SILICIES I. CARBANIONS DERIVES DE MONOALLYLSILANES", Journal of Organomeiallic Chemistry 93 (1975). 71-80, the synthesis of carbon functional organosilanes has been disclosed.

Document WO 98/11073 A1 discloses quinolinol and alkyl silyl derivatives as antivirals. However, the compound discloses in this document are very different from the ones provided in the present invention.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of tri-substituted five-membered ring oxacycles, in particular, tetrahydrofurans, which are active compounds against coronaviruses and other types of viruses.

The first aspect of the present invention relates to a compound of formula (I): a isomer or a solvate thereof, wherein

Ri is selected from CH ₃ and (CH2) _n X, being n and integer between 1 and 4 and X an halogen,

R ₂ is selected from H and phenyl,

R ₃ is selected from H, phenyl, benzyl and C ₁ -C ₄ alkyl, wherein one of R ₂ and R ₃ is H and the other one is different from H , with the proviso that when R ₃ is C ₁ -C ₄ alkyl, R ₁ is (CH ₂ ) _n X and wherein the compound is not the compound of formula: in its racemic form.

In a preferred embodiment, the compound of formula (I) is not the compound of formula: in any of its enantiopure forms (the drawn formula and its mirror image).

The above structure, as defined, comprises a phenyl group in addition to the phenyl groups of the SiPh ₃ substituent and/or an alkyl group substituted by an halogen ((CH ₂ ) _n X, group).

The term "halogen" refers to fluoride, chloride, bromide or iodine.

The term “benzyl” refers to a -CH ₂ Ph group, wherein Ph represents a phenyl group.

The term “C ₁ -C ₄ alkyl” in the present invention refers to linear or branched saturated hydrocarbon chain that have 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, tert-butyl.

In a preferred embodiment, R ₁ is CH ₃ .

In a preferred embodiment, R ₂ is H and R ₃ is selected from phenyl and benzyl, more preferably R ₃ is benzyl.

In a preferred embodiment, R ₃ is H and R ₂ is phenyl. In a preferred embodiment, R ₃ is C ₁ -C ₄ alkyl, more preferably, iso-butyl and R ₁ is (CH ₂ ) _n X, being n equals 3 and/or X is Cl.

Unless otherwise stated, the compound of formula (I) is intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the substitution of a hydrogen atom for a deuterium atom or a tritium atom, or the substitution of a carbon atom for a carbon atom enriched in ¹³ C or ¹⁴ C or a nitrogen atom enriched in ¹⁵ N fall within the scope of this invention.

The term “isomers” in the present invention refers to stereoisomers, including mixtures of stereoisomers (for example, racemic mixtures).

It must be understood that compound of formula (I) encompasses all the isomers of said compound, i.e. all the geometric and optical forms, and their mixtures (for example, racemic mixtures). The present invention includes, within its scope, all the possible diastereomers, including their mixtures. The different isomeric forms may be separated or resolved there between using conventional methods or any given isomer can be obtained using conventional synthetic methods or by means of stereospecific, stereoselective or asymmetric synthesis.

The term “racemic mixture,” “racemic compound” or “racemate” refers to an equimolecular mixture of the two enantiomers of one compound.

The term “enantiomer” is used to describe one of a pair of molecular isomers which are mirror images of each other and non-superimposable.

The term "diastereomers" or "diastereoisomers" as used herein refers to stereoisomers that are not enantiomers. The diastereomers have different configurations at one or more of the equivalent (related) stereocenters and are not mirror images of each other.

In certain embodiments, the compound of formula (I) is a mixture of stereoisomers, e.g., a mixture of diastereomers and/or enantiomers, e.g., a racemic mixture. In another such embodiment, the compound is a mixture of diastereomers. In another such embodiment, the compound is a mixture of enantiomers.

In a preferred embodiment, compound of formula (I) is a diastereomer selected from or combinations thereof (R ₂ is H in I’ and I”). Preferably, R ₁ is CH ₃ in formulas (I’) and (I”); and when R ₁ is CH ₃ , R ₃ is phenyl or benzyl, preferably benzyl.

In these diastereomers of formula (I’) and (I”), the substituents R ₁ and R ₃ are in relative position trans. The difference between these two diastereomers is in that they have different configurations at the carbon atom bonded to the Si atom.

In an embodiment, the compound of formula (I) is a racemic mixture of the diastereomer of formula (I’).

In another embodiment, the compound of formula (I) is a racemic mixture of the diastereomer of formula (I”).

In a preferred embodiment, the compound of formula (I) is a mixture of the diastereomers of formula (I’) and (I”) as represented above. More preferably, the compound of formula (I) is an equimolecular mixture of said diastereomers of formula (I’) and (I”).

In a preferred embodiment, compound of formula (I) is a mixture of said diastereomers, more preferably an equimolecular mixture, of formula (I’) and (I”) being each diasteromer a racemic mixture. Even more preferably, compound of formula (I) is an equimolecular mixture of diastereomers of formula (I’) and (I”), wherein R ₁ is CH3 and R ₃ is benzyl, being each diastereomer a racemic mixture. In a preferred embodiment, the compound of formula (I) is the isomer of formula (I ”) (R ₂ is H): wherein R ₃ is C ₁ -C ₄ alkyl.

In a preferred embodiment, the compound of formula (I) is the isomer of formula (I’”) (R ₃ is H in I’”):

Preferably, R ₁ is CH ₃ in the formula (I’”).

In a preferred embodiment, the isomer of formula (I’”) is a racemic mixture.

In a preferred embodiment, the compound of formula (I) is selected from the next ones: wherein Ph represents a phenyl group.

More preferably, the compound of formula (I) is selected from the next isomers: wherein Ph represents a phenyl group. Preferably, these compounds are in form of a racemic mixture.

The next formula: represents any of the two possible diastereomers depending on the stereochemistry of the carbon atom bonded to the SiPh ₃ group, or a mixture thereof.

The compound of formula (I) may be in crystalline form, either as free compound or as solvate (e.g.: hydrate), and it is understood that both forms fall within the scope of the present invention. Solvation methods are generally known in the state of the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.

A second aspect of the invention refers to a compound of formula (I), a isomer or a solvate thereof, wherein

R ₁ , R ₂ and R ₃ are as defined in the first aspect of the present invention wherein one of R ₂ and R ₃ is H and the other one is different from H and with the proviso that when R ₃ is C ₁ -C ₄ alkyl, R ₁ is (CH2) _n X for use as a medicament.

Another aspect of the invention refers to a compound of formula (I), as defined in the second aspect of the invention, for use as antiviral agent, more preferably, in the treatment and/or prevention of coronavirus infection.

Coronavirus infection preferably refers to SARS-CoV-2, human coronavirus OC43, transmissible gastroenteritis virus (TGEV), or murine hepatitis virus (MHV) infection. More preferably, it is a SARS-CoV-2 infection. The term "treatment or prevention" as used herein, unless otherwise indicated, relates to reversing, alleviating and inhibiting the progress of, or preventing the disorder or condition to which it applies in such terms, one or more symptoms of such disorder or condition.

In another aspect, the invention relates to a method for the prevention and/or treatment of coronavirus infection in a subject, preferably SARS-CoV-2 infection, comprising the administration to said subject of a therapeutically effective amount of a compound of formula (I) as defined in the second aspect of the present invention.

In another aspect, the invention relates to the use of a compound of formula (I) as defined in the second aspect of the present invention for the preparation of a medicament for prevention and/or treatment of coronavirus infection.

Another aspect of the invention refers to a pharmaceutical composition comprising the compound of formula (I), as described as described in the first aspect of the invention, isomers or a solvate thereof.The pharmaceutical composition preferably includes at least one excipient, adjuvant and/or a pharmaceutically acceptable carrier.

The term “carrier” relates to a dilute, adjuvant or excipient with which the main ingredient is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those derived from oil, animal, vegetable or synthetic, such as peanut oil, soybean oil, mineral oil, sesame seed oil and similar. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Preferably, the carriers of the invention are approved by the regulatory agency of a state government or a federal government, or are enumerated in the United States Pharmacopoeia, in the European Pharmacopoeia or other pharmacopoeia recognised in general for use in animals and, more particularly, in humans.

A last aspect of the present invention refers to the pharmaceutical composition comprising the compound of formula (I), as described in the second aspect of the invention, isomers or a solvate thereof, for use in the prevention and/or treatment of a virus infection, preferably coronavirus infection and, even more preferably, SARS- CoV-2, human coronavirus OC43, TGEV, or MHV infection.

The pharmaceutical composition comprises a therapeutically effective amount of the compound of formula (I), isomers or solvates thereof that must be administered (also referred to herein as therapeutically amount effective thereof).

The therapeutically amount effective of the compound of formula (I), in addition to their dosage for treating a pathological condition with said compound, will depend on a number of factors, including age, the condition of the patient, the severity of the disease, administration route and frequency, the modular compound to be used, etc.

The pharmaceutical compositions of this invention can be used on their own or jointly with other drugs to provide combined therapy. The other drugs can form part of the same pharmaceutical composition or be provided as a separate pharmaceutical composition, to be administered simultaneously or at different intervals. Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid composition (solutions, suspensions or emulsions) for oral, topical or parenteral administration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples, are provided by way of illustration and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Automated real-time microscopy quantitation of ST cell viability at different concentrations of SPL-0 in the presence (a) or absence (b) of TGEV infection. Dots indicate experimental data (average of two technical replicates). The line indicates the inferred dose-response function.

Fig. 2. Automated real-time microscopy quantitation of ST cell viability at different concentrations of SPL-4 in the presence (a) or absence (b) of TGEV infection. Dots indicate experimental data (average of two technical replicates). The line indicates the inferred dose-response function.

Fig. 3. Automated real-time microscopy quantitation of ST cell viability at different concentrations of SPL-6 in the presence (a) or absence (b) of TGEV infection. Dots indicate experimental data (average of two technical replicates). The line indicates the inferred dose-response function.

Fig. 4. Automated real-time microscopy quantitation of ST cell viability at different concentrations of SPL-1 in the presence (a) or absence (b) of TGEV infection. Dots indicate experimental data (average of two technical replicates). The line indicates the inferred dose-response function.

Fig. 5. Automated real-time microscopy quantitation of ST cell viability at different concentrations of SPL-5 in the presence (a) or absence (b) of TGEV infection. Dots indicate experimental data (average of two technical replicates). The line indicates the inferred dose-response function.

Fig. 6. TGEV titration in ST cell cultures treated with different concentrations of SPL-0. Dots indicate experimentally determined viral titers relative to an untreated control (average of three technical replicates). The line indicates the inferred dose- response function.

Fig. 7. Quantitation of TGEV RNA by RT-qPCR in ST cell cultures treated with different concentrations of SPL-0. Dots indicate experimentally determined RNA concentrations relative to an untreated control (average of three technical replicates).

The line indicates the inferred dose-response function.

Fig. 8. Effect of SPL-0 on SARS-CoV-2 infection in VeroE6 cells. Viral infection was quantified by immunofluorescence in cultures treated with different concentrations of SPL-0 (a). The percentage of viable cells in uninfected cultures was determined by the MTT assay (b). Dots indicate the experimentally determined percentage of infected cells. The line indicates the inferred dose-response function.

Fig. 9. Effect of SPL-0 on SARS-CoV-2 infection in A549 cells. Viral infection was quantified by immunofluorescence in cultures treated with different concentrations of SPL-0 (a). The percentage of viable cells in uninfected cultures was determined by the MTT assay (b). Dots indicate the experimentally determined percentage of infected cells. The line indicates the inferred dose-response function.

Fig. 10. CVB3 titration in HeLa cell cultures treated with different concentrations of SPL-0. Dots indicate experimentally determined viral titers relative to an untreated control (average of three technical replicates). The line indicates the inferred dose- response function.

Fig.11. Effect of SPL-0 on the infectivity of vesicular stomatitis virus pseudotyped with the SARS-CoV-2 spike protein. Dots indicate the GFP signal observed in A549 cells inoculated with the viral pseudotype and treated with different concentrations of SPL-0. Error bars correspond to the standard error of the mean obtained from three technical replicates, (a) A549 cells expressing the SARS-CoV-2 receptor ACE2. (b) A549 cells co-expressing ACE2 and the serine protease TMPRSS2.

Fig. 12. Effect of SPL-0 on the titer of TGEV populations passaged in ST cells the presence or absence of SPL-0. TGEV was serially passaged 10 times in the absence (control regime) or the presence of 50 μM SPL-0 (selective regime). The effect of SPL-0 on the resulting viral populations was evaluated by performing infections of ST cells at different SPL-0 concentrations and quantifying the viral titer obtained after 24 h by the plaque assay. Error bars indicate the standard error of the mean from three technical replicates.

Examples

Compounds SPL-0, SPL-4 and SPL-6 within the present invention have been synthetized. Also, compounds SPL-1 and SPL-5, which are not within the present invention, have been synthetized for comparative purposes. The chemical structure of all these compounds is represented in the examples below.

Example 1 : Synthesis of compound SPL-0

This compound (SPL-0) was obtained in two reaction steps, starting from the corresponding commercially available epoxide, and with an overall yield of 51%.

Scheme 1. Synthesis of tri-substituted five-membered ring oxacycle SPL-0 ((2,5-trans-5- benzyl-2-methyltetrahydrofuran-3-yl)triphenylsilane).

1 -phenyl-4-(triphenylsilyl)hex-5-en-2-ol (A)

To a well-stirred solution of allyltriphenylsilane (2.59 g, 8.46 mmol, 1.0 equiv.) in 42 mL of dry tetrahydrofuran (THF) (0.2 M vs allyltriphenylsilane), cooled to -78 °C and under N ₂ atmosphere, was added sec-butyllithium 1.4 M (6.0 mL, 8.46 mmol, 1.0 equiv.). Then the system was allowed to reach -50 °C and this temperature was kept along 2 hours. After that, the system was cooled again to -78 °C and 2-benzyloxirane (1.14 g, 8.46 mmol, 1.0 equiv.) was added dropwise, solved in a little amount of dry THF. The reaction was stirred at -78°C until analysis via Thin Layer Chromatography (TLC) showed complete formation of alcohol A. It was quenched by addition of a mixture of n-hexane: ethyl ether: saturated aqueous NH ₄ CI (2:2:1), being the volume of this mixture = 25 times the millimole of allyltriphenylsilane and extracted with ethyl ether. The combined organic layers were dried over anhydrous MgSCU, filtered, and the solvent was removed under reduced pressure. This crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 90:10 solvent system) to afford 2.10 g of alcohol A (4.82 mmol, 57% yield). Spectral data was consistent with the known product (Cruz, D. A.; Sinka, V.; Martin, V. S.; Padron, J. I. J. Org. Chem. 2018, 83, 12632-12647).

¹ H NMR (CDCI ₃ , 600 MHz) (mixture syn/anti (1 :0.7)) 6 7.60-7.53 (m, 12H), 7.41 (m, 6H), 7.36 (m, 12H), 7.28 (m, 4H), 7.21 (m, 2H), 7.15 (m, 4H), 5.89 (m, 1 H), 5.71 (m, 1 H), 5.06-4.88 (m, 4H), 3.91 (m, 2H), 2.99 (brt, J = 10.3 Hz, 1 H), 2.89 (dd, J = 4.0 & 13.7 Hz, 1 H), 2.72-2.64 (m, 2H), 2.64-2.58 (dd, J = 8.9 & 13.7 Hz, 1 H), 2.54 (dd, J = 8.5 & 13.6 Hz, 1 H), 1.95 (ddd, J = 2.6, 6.8 & 14.0 Hz, 1 H), 1.91-1.82 (m, 2H), 1.74 (m, 1 H), 1.69 (m, 1 H), 1.53 (m, 1 H).

¹³ C NMR (CDCh, 150 MHz) (mixture syn/anti (1 :0.7)) 6 139.3 (CH), 138.8 (C), 138.6 (C), 138.3 (CH), 136.2 (6xCH), 136.1 (6xCH), 133.8 (3xC), 133.4 (3xC), 129.5 (3xCH),

129.4 (3xCH), 129.3 (2xCH), 129.3 (2xCH), 128.5 (2xCH), 128.5 (2xCH), 127.8 (6xCH), 127.8 (6xCH), 126.4 (CH), 126.3 (CH), 115.3 (CH ₂ ), 115.1 (CH ₂ ), 73.6 (CH,,

70.4 (CH), 44.5 (CH ₂ ), 42.6 (CH ₂ ), 36.3 (CH ₂ ), 36.2 (CH ₂ ), 30.5 (CH), 237.8 (CH). HRMS (ESP): m/z [M+Na] ⁺ calcd. for C ₃₀ H ₃₀ OSi: 457.1964; found: 457.1959.

2,5-frans-5-benzyl-2-methyltetrahydrofuran-3-yl)triphenyl silane (SPL-0)

To a well-stirred solution of bi s-homoallylsilyl alcohol A (300 mg, 0.69 mmol, 1.0 equiv.) in 6.9 mL of dry dichloromethane (DCM) (0.1 M) at room temperature under N ₂ atmosphere, was added FeBr ₃ (20.4 mg, 0.069 mmol, 0.10 equiv.). Once the reaction was complete, the process was quenched by addition of water. The aqueous phase was extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4, filtered, and the solvent was removed under reduced pressure. The crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 95:5 solvent system) to give 270 mg of SPL-0 (0.62 mmol, 90% yield).

¹ H NMR (CDCI3, 500 MHz) (diastereoisomeric mixture (1 :1)) 6 7.53 (m, 11 H), 7.42 (m, 6H), 7.38-7.33 (m, 12H), 7.29-7.22 (m, 5H), 7.21-7.14 (m, 4H), 7.11 (m, 2H), 4.29 (m, 1 H), 4.22 (m, 1 H), 4.06 (m, 1 H), 3.87 (m, 1 H), 2.92 (dd, J = 13.7 & 5.9 Hz, 1 H), 2.86 (dd, J = 13.5 & 5.5 Hz, 1 H), 2.71 (dd, J = 13.6 & 6.7 Hz, 1 H), 2.59 (dd, J = 13.7 & 7.0 Hz, 1 H), 2.28 (ddd, J = 12.7, 7.6 & 5.6 Hz, 1 H), 2.06 (m, 2H), 1.95-1.88 (ddd, J = 12.1 8.9 & 7.9 Hz, 1 H), 1.88-1.81 (dd, J = 19.2 & 9.8 Hz, 1 H), 1.74 (dt, J = 12.2 & 9.0 Hz, 1 H), 1.11 (d, J = 5.9 Hz, 3H), 1.07 (d, J = 5.9 Hz, 3H). ¹³ C NMR (CDCI3, 150 MHz) (diastereoisomeric mixture (1 :1)) 6 138.7 (C), 138.6 (C), 136.0 (6 x CH), 135.9 (6 x CH), 133.9 (3 x C), 133.8 (3 x C), 129.6 (4 x CH), 129.3 (4 x CH), 129.2 (4 x CH), 128.2 (2 x CH), 128.1 (2 x CH), 127.9 (5 x CH), 127.9 (5 x CH), 126.1 (CH), 126.0 (CH), 78.8 (CH), 78.7 (CH), 78.2 (CH), 77.3 (CH), 42.3 (CH ₂ ), 41.5 (CH ₂ ), 36.3 (CH ₂ ), 35.1 (CH ₂ ), 32.9 (CH), 30.7 (CH), 22.6 (CH ₃ ), 22.0 (CH ₃ ).

HRMS (ESP): m/z [M+Na] ⁺ calcd. for C ₃ oH ₃₀ OSi: 457.1964; found: 457.1960.

Example 2: Synthesis of compound SPL-1

Following the general procedure applied to SPL-0, this compound was synthesized in two steps with an overall yield of 36%.

Scheme 2. Synthesis of tri-substituted five-membered ring oxacycle SPL-1 ((2,5-trans-5- decyl-2-methyltetrahydrofuran-3-yl)triphenylsilane).

3-(triphenylsilyl)pentadec-1 -en-5-ol (B)

To a well-stirred solution of allyltriphenylsilane (2.94 g, 9.77 mmol, 1.2 equiv.) in 49 mL of dry THF (0.2 M vs allyltriphenylsilane), cooled to -78 °C and under N ₂ atmosphere, was added sec-butyllithium 1.4 M (7.0 mL, 9.77 mmol, 1.2 equiv.). Then the system was allowed to reach -50 °C and this temperature was kept along 2 hours. After that, the system was cooled again to -78 °C and commercially available 1 ,2- epoxydodecane (1.78 mL, 8.14 mmol, 1.0 equiv.) was added dropwise, solved in a little amount of dry THF. The reaction was stirred at -78 °C until analysis via TLC showed complete formation of alcohol B. It was quenched by addition of a mixture of n-hexane: ethyl ether: saturated aqueous NH4CI (2:2:1), being the volume of this mixture = 25 times the millimole of allyltriphenylsilane and extracted with ethyl ether. The combined organic layers were dried over anhydrous MgSCU, filtered and the solvent was removed under reduced pressure. This crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 90:10 solvent system) to obtain 2.84 g of alcohol B (5.86 mmol, 72% yield). Spectral data was consistent with the known product. ¹ H NMR (CDCI3, 400 MHz) (mixture syn/anti (1 :1)) δ 7.60-7.55 (dt, J = 1.5 & 8.0 Hz, 12H), 7.44-7.38 (m, 6H), 7.38-7.32 (m, 12H), 5.94-5.83 (m, 1 H), 5.80-5.68 (m, 1 H), 5.04-4.93 (m, 4H), 3.76-3.61 (m, 2H), 2.96 (m, 1 H), 2.61 (m, 1 H), 1.87 (ddd, J = 2.5 & 6.1 & 14.0 Hz, 1 H), 1.79-1.69 (m, 2H), 1.67-1.63 (m, 1 H), 1.63-1.58 (m, 1 H), 1.41- 1.18 (m, 36H), 0.90-0.85 (m, 6H).

¹³ C NMR (CDCI ₃ , 150 MHz) (mixture syn/anti (1 :1)) δ 139.6 (CH), 138.6 (CH), 136.2 (6 x CH), 136.1 (6 x CH), 133.8 (3 x C), 133.6 (3 x C), 129.5 (3 x CH), 129.4 (3 x CH), 127.8 (6 x CH), 127.7 (6 x CH), 115.2 (CH ₂ ), 114.9 (CH ₂ ), 73.2 (CH), 69.7 (CH), 38.0 (CH ₂ ), 37.1 (CH ₂ ), 36.7 (CH ₂ ), 36.2 (CH ₂ ), 31.9 (2xCH ₂ ), 30.9 (CH), 29.6 (8 x CH ₂ ),

29.3 (2 x CH ₂ ), 27.9 (CH), 25.9 (CH ₂ ), 25.3 (CH ₂ ), 22.7 (2 x CH ₂ ), 14.1 (2 x CH ₃ ).

HRMS (ESP): m/z [M+Na] ⁺ calcd. for C ₃₃ H ₄₄ OSi: 507.3059; found: 507.3050.

2,5-frans-(5-decyl-2-methyltetrahydrofuran-3-yl)triphenyl silane (SPL-1)

To a well-stirred solution of bis-homoallylsilyl alcohol B (1.0 equiv.) in dry DCM (0.1 M) at room temperature under N ₂ atmosphere, was added FeBrs (0.10 equiv.). Once the reaction was complete, the process was quenched by addition of water. The aqueous phase was extracted with DCM. The combined organic layers were dried over anhydrous MgSO ₄ , filtered, and the solvent was removed under reduced pressure. The crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 95:5 solvent system) to afford SPL-1.

¹ H NMR (CDCI3, 500 MHz) (diastereoisomeric mixture (1 :1)) 6 7.56 (m, 12H), 7.45- 7.34 (m, 18H), 4.26 (dq, J = 6.0 & 8.9 Hz, 1 H), 4.06 (dq, J = 5.9 & 9.5 Hz, 1 H), 3.93 (m, 1 H), 3.53 (quint, J = Q.7 Hz, 1 H), 2.34 (ddd, J = 5.3, 7.7 & 12.7 Hz, 1 H), 2.11 (m, 1 H), 1.94 (m, 3H), 1.63 (dt, J = 9.4 & 12.2 Hz, 1 H), 1.50-1.36 (m, 2H), 1.31-1.21 (m, 34H), 1.96 (d, J = 6.0 Hz, 3H), 1.08 (d, J = 6.1 Hz, 3H), 0.88 (t, J = 7.0 Hz, 6H).

¹³ C NMR (CDCI3, 125 MHz) (diastereoisomeric mixture (1 :1)) 6 136.0 (6 x CH), 135.9 (6 x CH), 134.0 (3 x C), 133.9 (3 x C), 129.6 (6 x CH), 127.9 (12 x CH), 78.5 (CH),

78.4 (CH), 77.8 (CH), 77.2 (CH), 37.0 (CH ₂ ), 36.1 (CH ₂ ), 35.9 (CH ₂ ), 35.5 (CH ₂ ), 33.0 (CH), 31.9 (2 x CH ₂ ), 30.9 (CH), 29.8 (CH ₂ ), 29.7 (CH ₂ ), 29.6 (6 x CH ₂ ), 29.3 (2 x CH ₂ ), 26.2 (2 x CH ₂ ), 22.8 (CH ₃ ), 22.7 (2 x CH ₂ ), 22.0 (CH ₃ ), 14.1 (2 x CH ₃ )

HRMS (ESP): m/z [M+Na] ⁺ calcd. for C ₃₃ H ₄₄ OSi: 507.3059; found: 507.3051. Example 3: Synthesis of compound SPL-4

This compound (SPL-4) was synthesized in two chemical steps from the commercially available 2-phenyloxirane with an overall yield of 4% (Scheme 3).

Scheme 3. Synthesis of tri-substituted five-membered ring oxacycle SPL-4 ((2,3,4-c/s-2- methyl-4-phenyltetrahydrofuran-3-yl)triphenylsilane.

2-phenyl-3-(triphenylsilyl)pent-4-en-1-ol (F)

Following the procedure described by Corriu (a) Corriu, R.; Masse, J. J. Organomet. Chem. 1973, 57, C5-C8. b) Corriu, R. J. P.; Masse, J.; Samate, D. J. Organomet. Chem. 1975, 93, 71-80), to a solution of Mg (56 mg, 2.32 mmol, 1.4 equiv.) in 5.5 mL of dry Et20 (0.3 M) was added allyl bromide D (758 mg, 2.00 mmol, 1.2 equiv.) dissolved in 2.0 mL of dry Et ₂ 0 (1.0 M). The reaction mixture was heated to reflux for 2 h. Then, 2-phenyloxirane (200 mg, 1.66 mmol, 1.0 equiv.) was dissolved in 1.7 mL of Et20 (1.0 M) and added dropwise at the refluxing temperature. Once the reaction was complete (checked by TLC), it was allowed to cool to room temperature, filtered through a pad of Celite and quenched with saturated aqueous NH4CI. The mixture was extracted with Et20 and the organic phase was dried over anhydrous MgSO ₄ filtered and concentrated under reduced pressure. This crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 90:10 solvent system) to give 111 mg of bis-homoallylsilyl alcohol F (0.26 mmol, 16% yield).

2,3,4-cis-2-methyl-4-phenyltetrahydrofuran-3-yl)triphenyl silane (SPL-4)

To a well-stirred solution of bis-homoallylsilyl alcohol F (70 mg, 0.17 mmol, 1.0 equiv.) in 1.7 mL of dry DCM (0.1 M) at room temperature under N2 atmosphere, was added FeBrs (15 mg, 0.051 mmol, 0.30 equiv.). Once the reaction was complete, the process was quenched by addition of water. The aqueous phase was extracted with DCM. The combined organic layers were dried over anhydrous MgSO4, filtered and the solvent was removed under reduced pressure. The crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 95:5 solvent system) to afford 15 mg of SPL-4 (0.036 mmol, 22 % yield). ¹ H NMR (CDCh, 400 MHz) δ 7.49 (m, 6H), 7.39 (m, 3H), 7.31 (m, 6H), 7.19 (m, 3H), 7.09 (m, 2H), 4.35 (dq, J = 8.4 & 5.9 Hz, 1 H), 3.76 (m, 2H), 3.43 (m, 1 H), 2.05 (dd, J = 8.4 & 7.2 Hz, 1 H), 1.25 (d, J = 5.9 Hz, 3H).

¹³ C NMR (CDCh, 100 MHz) δ 145.7 (C), 136.1 (6 x CH), 133.6 (3 x C), 129.6 (3 x CH), 128.4 (3 x CH), 127.8 (6 x CH), 127.6 (CH), 126.2 (CH), 79.0 (CH), 75.3 (CH ₂ ), 50.0 (CH), 41.5 (CH), 22.2 (CH ₃ ).

HRMS (ESP): m/z [M+Na] ⁺ cald. for C ₂ 9H ₂ 8ONaSi: 443.1807; found: 443.1805.

Example 4: Synthesis of compound SPL-5

This compound (SPL-5) was synthesized in two chemical steps from the commercially available 2-benzyloxirane with an overall yield of 35% (Scheme 4).

Scheme 4. Synthesis of tri-substituted five-membered ring oxacycle SPL-5 ((2,5-cis-2- methyltetrahydrofuran-3-yl)tri methylsilane.

1 -phenyl-4-(trimethylsilyl)hex-5-en-2-ol (G)

To a well-stirred solution of allyltrimethylsilane (0.59 mL, 3.72 mmol, 2.5 equiv.) in 19 mL of dry THF (0.2 M vs allyltrimethylsilane), cooled to -78 °C and under N ₂ atmosphere, was added sec-butyllithium 1.4 M (2.7 mL, 3.72 mmol, 2.5 equiv.). Then the system was allowed to reach -50 °C and this temperature was kept along 2 hours. After that, the system was cooled again to -78 °C and 2-benzyloxirane (200 mg, 1.49 mmol, 1.0 equiv.) was added dropwise, solved in a little amount of dry THF. The reaction was stirred at -78 °C until analysis via TLC showed complete formation of alcohol G. It was quenched by addition of a mixture of n-hexane:ethyl ethersaturated aqueous NH4CI (2:2:1), being the volume of this mixture = 25 times the millimole of allyltriphenylsilane and extracted with ethyl ether. The combined organic layers were dried over anhydrous MgSO ₄ , filtered and the solvent was removed under reduced pressure. This crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 90:10 solvent system) to afford 275 mg of alcohol G (1.11 mmol, 74% yield). 2,5-trans-5-benzyl-2-methyltetrahydrofuran-3-yl)trimethylsil ane (SPL-5)

To a well-stirred solution of bis-homoallylsilyl alcohol G (100 mg, 0.40 mmol, 1.0 equiv.) in 4.0 mL of dry DCM (0.1 M) at 10 °C under N ₂ atmosphere, was added I nCI ₃ (8.8 mg, 0.040 mmol, 0.10 equiv.). Once the reaction was complete, the process was quenched by addition of water. The aqueous phase was extracted with DCM. The combined organic layers were dried over anhydrous MgSO ₄ , filtered and the solvent was removed under reduced pressure. The crude reaction mixture was purified by flash silica gel column chromatography (n-hexane/EtOAc 95:5 solvent system) to afford 47 mg of SPL-5 (0.19 mmol, 47 % yield).

¹ H-NMR (CDCh, 400 MHz) (diastereoisomeric mixture (1 :0.7)) δ 7.28 (m, 3H), 7.21 (m, 5H), 4.13 (m, 1 H), 4.05 (m, 1 H), 3.97 (dq, J = 10.0 & 6.0 Hz, 1 H), 3.79 (dq, J = 10.3 & 5.9 Hz, 1 H), 3.05-2.99 (dd, J = 13.4 & 5.5 Hz, 1 H), 2.99-2.92 (dd, J = 13.5 & 5.7 Hz, 1 H), 2.67 (ddd, J = 13.3, 7.5 & 3.5 Hz, 2H), 1.97 (ddd, J = 12.2, 6.8 & 5.3 Hz, 1 H), 1.79 (m, 2H), 1.43 (dt, J = 12.6 & 9.3 Hz, 1 H), 1.29 (d, J = 6.1 Hz, 3H), 1.25 (d, J = 6.1 Hz, 2H), 0.97 (m, 2H), 0.00 (s, 15H).)

¹³ C-NMR (CDCI ₃ , 100 MHz) (diastereoisomeric mixture (1 :0.7)) 6 139.0 (C), 138.9 (C), 129.3 (3 x CH), 129.2 (2 x CH), 128.2 (3 x CH), 126.1 (2 x CH), 79.4 (CH), 79.2 (CH), 78.2 (CH), 77.3 (CH), 42.8 (CH ₂ ), 42.1 (CH ₂ ), 36.1 (CH ₂ ), 35.7 (CH), 34.2 (CH ₂ ), 33.2 (CH), 22.3 (CH ₃ ), 21.9 (CH ₃ ), -2.6 (6 x CH ₃ ).

HRMS (ESP): m/z [M+Na] ⁺ cald. for C ₁₅ H ₂₄ ONaSi: 271.1494; found: 271.1491.

Example 5: Synthesis of compound SPL-6

This compound (SPL-6) was synthesized in one chemical steps from the unsaturated tris-homoallylsilyl alcohol H with a yield of 47% (Scheme 5).

Scheme 5. Synthesis of tri-substituted five-membered ring oxacycle SPL-6 (2,3,5-c/s-2-(3- choropropyl)-5-isobutyltetrahydrofuran-3-yl)triphenylsilane) .

2,3,5-c/s-2-(3-choropropyl)-5-isobutyltetrahydrofuran-3-y l)triphenylsilane (SPL-

6) To a well-stirred and open-air solution of tris-homoallylsilyl alcohol H (100 mg, 0.28 mmol, 1.0 equiv) in dry dichloromethane (DCM) (0.1 M) at room temperature were added isovaleraldehyde (2.0 equiv) and the FeBrs (8.7 mg, 0.030 mmol, 0.3 equiv.). The reaction was monitored by TLC, and once complete, the process was quenched by the addition of the same amount of water as DCM. The aqueous phases were separated and washed with DCM. The combined organic layers were dried over anhydrous magnesium sulfate and filtered, and the solvent was removed under reduced pressure. The crude reaction was purified by flash silica gel column chromatography (n-hexane/EtOAc 95:5 solvent system) to afford 52 mg of SPL-6 (0.112 mmol, 47 % yield).

¹ H-NMR (CDCI ₃ , 400 MHz) δ 7.60-7.50 (m, 6H), 7.5-7.3 (m, 9H), 4.2 (td, J = 8.6 & 2.8 Hz, 1 H), 4.00-3.95 (m, 1 H), 3.50-3.33 (m, 2H), 2.3 (ddd, J = 12.0, 8.1 & 5.1 Hz, 1 H), 1.9 (dt, J = 11.8 & 8.1 Hz, 1 H), 1.90-1.80 (m, 1 H), 1.80-1.60 (m, 3H), 1.50-1.30 (m, 3H), 1 .2 (ddd, J = 13.2, 7.3 & 5.6 Hz, 1 H), 0.9 (dd, J = 6.6 & 3.2 Hz, 6H).

¹³ C-NMR (CDCh, 100 MHz) δ 136.1 (6 x CH), 134.0 (3 x C), 129.8 (3 x CH), 128.1 (6 x CH), 80.3 (CH), 76.8 (CH), 45.2 (CH2), 44.4 (CH2), 37.0 (CH2), 34.4 (CH2), 31.3 (CH), 30.1 (CH2), 26.0 (CH), 23.1 (CH3), 22.9 (CH3).

HRMS (ESP): m/z [M+Na]+ calcd for C29H35ONaSiCI, 485.2043; found, 485.2047.

Example 6: Antiviral activity of SPL-0 against transmissible gastroenteritis virus (TGEV), as determined by quantitation of virus-induced cell death

TGEV is an alphacoronavirus that naturally infects pigs (Sus sp.) and is commonly used as a model coronavirus. The effect of SPL-0 on cellular viability in the presence or absence of TGEV infection was determined in swine testis (ST) cells inoculated with TGEV at a multiplicity of infection (MOI) of 0.01 infectious particles per cell. Real- time automated microscopy was used for determining cellular permeability to a commercial cyanine that fluorescently labels nucleic acids (Cytotox®). A range of SPL-0 concentrations was tested in order to obtain a dose-response curve. The percentage of dead cells at 48 h after viral inoculation decreased as the SPL-0 dose increased (Figure 1a), indicating that SPL-0 partially reverts TGEV-induced cell death. A dose-response model of the form was pt to the experimental data by the least-squares method, where c is the compound concentration (μM), D is the percentage of dead cells, D _max is the top plateau, D _min is the bottom plateau, ECso is the half-maximal effective concentration, and n is Hill slope. The estimated EC ₅₀ of SPL-0 on TGEV-induced cell death was 13.1 μM (coefficient of determination: r ² = 0.999; Hill slope: n = 2.87), with a bottom plateau of 16.1% dead cells. In cell cultures not inoculated with TGEV, the percentage of dead cells at 48 h remained <12% (range 2.5-12.0%), and showed no correlation with SPL-0 concentration (Figure 1b). Therefore, SPL-0 exhibited no detectable cytotoxic effects in the examined dose range (2-100 μM), showing that the half-maximal cytotoxic concentration (CC ₅₀ ) was higher than 100 μM.

Example 7: Antiviral activity of SPL-4 against TGEV, as determined by quantitation of virus-induced cell death

The effect of SPL-4 on cellular viability in the presence or absence of TGEV infection was determined in ST cells inoculated with TGEV at an MOI of 0.01 infectious particles per cell. Real-time automated microscopy was used for determining cellular permeability to Cytotox®. A range of SPL-4 concentrations was tested in order to obtain a dose-response curve. The percentage of dead cells at 48 h after viral inoculation decreased as the SPL-4 dose increased (Figure 2a), indicating that SPL- 4 partially reverts TGEV-induced cell death. The estimated EC ₅₀ of SPL-4 on TGEV- induced cell death was 15.8 μM (r ² = 0.999, n = 2.52), with a bottom plateau of 11.4% dead cells. Therefore, the activity of SPL-4 against TGEV was similar to that of SPL- 0. In cell cultures not inoculated with TGEV, the percentage of dead cells at 48 h remained <12 % (range 3.1-12.0 %), and showed no correlation with SPL-4 dose (Figure 2b), indicating that SPL-4 had no detectable cytotoxic effects in the examined dose range (2-100 μM).

Example 8: Antiviral activity of SPL-6 against TGEV, as determined by quantitation of virus-induced cell death

The effect of SPL-6 on cellular viability in the presence or absence of TGEV infection was determined in ST cells inoculated with TGEV at an MOI of 0.01 infectious particles per cell. Real-time automated microscopy was used for determining cellular permeability to Cytotox®. A range of SPL-6 concentrations was tested in order to obtain a dose-response curve. The percentage of dead cells at 48 h after viral inoculation decreased as the SPL-6 dose increased (Figure 3a), indicating that SPL- 6 partially reverts TGEV-induced cell death. The estimated EC ₅₀ of SPL-6 on TGEV- induced cell death was 30.5 μM (r ² = 0.999, n = 2.13), with a bottom plateau of 14.8% dead cells. Therefore, SPL-6 showed a less potent activity against TGEV than SPL- 0 or SPL-4. In cell cultures not inoculated with TGEV, the percentage of dead cells at 48 h remained <8 % (range 6.1-7.6 %), and showed no correlation with SPL-6 dose (Figure 3b), indicating that SPL-6 had no detectable cytotoxic effects in the examined dose range (2-100 μM).

Example 9: Lack of antiviral activity of SPL-1 against TGEV, as determined by quantitation of virus-induced cell death

The effect of SPL-1 on cellular viability in the presence or absence of TGEV infection was determined in ST cells inoculated with TGEV at an MOI of 0.01 infectious particles per cell. Real-time automated microscopy was used for determining cellular permeability to Cytotox®. A range of SPL-1 concentrations was tested in order to obtain a dose-response curve. SPL-1 did not revert TGEV-induced cell death (Figure 4a). In cell cultures not inoculated with TGEV, the percentage of dead cells at 48 h remained <10 % (range 3.3-9.6 %). However, the percentage of dead cells increased slightly at the highest assayed dose (100 μM: Figure 4b).

Example 10: Lack of antiviral activity of SPL-5 against TGEV, as determined by quantitation of virus-induced cell death

The effect of SPL-5 on cellular viability in the presence or absence of TGEV infection was determined in ST cells inoculated with TGEV at an MOI of 0.01 infectious particles per cell. Real-time automated microscopy was used for determining cellular permeability to Cytotox®. A range of SPL-5 concentrations was tested in order to obtain a dose-response curve. SPL-5 did not revert TGEV-induced cell death (Figure 5a). SPL-0 and SPL-5 differ in that the tri-phenylsilyl group of SPL-0 has been replaced by a tri-methylsilyl group in SPL-5. Therefore, the tri-phenylsilyl group shown in formula (I) is necessary for antiviral activity. In cell cultures not inoculated with TGEV, the percentage of dead cells at 48 h remained <12 % (range 3.5-11 .9 %), and showed no correlation with SPL-5 dose (Figure 5b), indicating that SPL-5 had no detectable cytotoxic effects in the examined dose range (2-100 μM).

Example 11 : Antiviral activity of SPL-0 against TGEV as determined by viral titration ST cells were inoculated with TGEV at a MOI of 0.01 infectious particles per cell and, after 24 h of incubation under standard culture conditions, the culture medium was subjected to the plaque assay, which determines the number of viral infectious particles present per volume unit (viral titer). A range of SPL-0 concentrations was tested in order to obtain a dose-response curve. The TGEV titer in the harvested medium decreased as the SPL-0 dose increased (Figure 6). The estimated EC ₅₀ of SPL-0 against TGEV was 15.6 μM (r ² = 0.949, n = 1 .60), with a bottom plateau of 0% for the estimated viral titer.

Example 12: Antiviral activity of SPL-0 against TGEV quantified by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR)

ST cells were inoculated with TGEV at a MOI of 0.01 infectious particles per cell and, after 24 h of incubation under standard culture conditions, total RNA was extracted and TGEV RNA was quantified by RT-qPCR. A range of SPL-0 concentrations was tested in order to obtain a dose-response curve. The amount of TGEV RNA decreased as the SPL-0 dose increased (Figure 7). The EC ₅₀ of SPL-0 against TGEV was 4.35 μM (r ² = 0.951 , n = 1.16), with a bottom plateau of 0 % viral RNA.

Example 13: Antiviral activity of SPL-0 against murine hepatitis virus (MHV) quantified by RT-qPCR

MHV is a betacoronavirus that naturally infects mice (Mus musculus) and is commonly used as a model coronavirus. It belongs to the same taxonomic genus as SARS-CoV- 2 (betacoronaviruses). Mouse liver CCL9.1 cells were inoculated with MHV at a MOI of 0.01 infectious particles per cell and, after 24 h of incubation under standard culture conditions, total RNA was extracted and MHV RNA was quantified by RT-qPCR. A single dose of SPL-0 (20 μM) was tested to check for antiviral effects that reproduce the results obtained with the TGEV model. The amount of MHV RNA in cells treated with 20 μM SPL-0 was 17.0 ± 2.4 % of the amount obtained in control cultures receiving no drug, indicating that SPL-0 is active against MHV with an EC ₅₀ < 20 μM.

Example 14: Antiviral activity of SPL-0 against human coronavirus OC43 quantified by RT-qPCR

OC43 betacoronavirus is a causative agent of human common cold. It belongs to the same taxonomic genus as SARS-CoV-2 (betacoronaviruses). OC43 conveniently infects BHK-21 baby hamster kidney cells under laboratory conditions. BHK-21 cells were inoculated with OC43 coronavirus at a MOI of 0.01 infectious particles per cell and, after 4 days of incubation under standard culture conditions, total RNA was extracted and OC43 RNA was quantified by RT-qPCR. A single dose of SPL-0 (20 μM) was tested to check for antiviral effects that reproduce the results obtained with other coronaviruses. The amount of OC43 RNA in cells treated with 20 μM SPL-0 was 4.16 ± 0.95 % of the amount obtained in control cultures receiving no drug, indicating that SPL-0 is active against OC43 with an EC ₅₀ < 20 μM.

Example 15: Antiviral activity of SPL-0 against SARS-CoV-2 quantified by immunofluorescence in African green monkey cells

To determine the activity of SPL-0 against SARS-CoV-2, a dose-response assay was performed in the range of 0.2 μM to 50 μM using VeroE6 kidney cells from African green monkey (Cercopithecus aethiops). Cell cultures were inoculated with SARS- CoV-2 at an MOI of 0.01 infectious particles per cell and analyzed after 48 h. The percentage of infected cells was obtained by flow cytometry using a monoclonal antibody against the virus spike protein S as well as a fluorescently labelled secondary antibody (immunofluorescence). The percentage of infected cells decreased as the SPL-0 increased (Figure 8a). The EC ₅₀ of SPL-0 against SARS-CoV-2 in VeroE6 cells was 27.5 μM (r ² = 0.993, n = 4.58), with a bottom plateau of 1.6 % infected cells. In addition, for each dose, cell viability assays were performed in the absence of virus. For this, the MTT assay, a standard procedure for determining cell metabolic activity, was used. SPL-0 showed no cytotoxic effects over the range of concentrations tested (Figure 8b). The percentage of viable cells at 48 h remained >85 % (range 85.0- 93.3%), and showed no correlation with SPL-0 dose.

Example 16: Antiviral activity of SPL-0 against SARS-CoV-2 quantified by immunofluorescence in human lung cells

The activity of SPL-0 against SARS-CoV-2 was determined in A549 human lung cells, which provide a biologically relevant model, since that lungs are a primary target of SARS-CoV-2. For this, a dose-response assay was performed in the range of 0.2 μM to 50 μM. Cell cultures were inoculated with SARS-CoV-2 at an MOI of 0.01 infectious particles per cell and analyzed after 72 h. The percentage of infected cells was obtained by flow cytometry using a monoclonal antibody against the virus spike protein S as well as a fluorescently labelled secondary antibody (immunofluorescence). The percentage of infected cells decreased as the SPL-0 increased (Figure 9a). The EC ₅₀ of SPL-0 against SARS-CoV-2 in A549 cells was 12.9 μM (r ² = 0.994, n = 3.48), with an estimated bottom plateau of -0.37 % (i.e. effectively no infection). In addition, for each dose, cell viability assays were performed in the absence of virus. For this, the MTT assay was used. SPL-0 showed no cytotoxic effects over the range of concentrations tested (Figure 9b). The percentage of viable cells at 48 h remained >89 % (range 89.0-100 %), and showed no correlation with SPL-0 dose.

Example 17: Antiviral activity of SPL-0 against coxsackievirus B3 (CVB3), as determined by viral titration

CVB3 belongs to the picornavirus family. Hence, CVB3 is not a coronavirus. Coronaviruses and picornaviruses share certain broad features. Both are plus-strand RNA viruses. However, they are otherwise largely unrelated virus groups. The effect of SPL-0 on CVB3 infection was determined in human HeLa cells. HeLa cells were inoculated with CVB3 at a MOI of 0.01 infectious particles per cell and, after 24 h of incubation under standard culture conditions, the medium was subjected to the plaque assay, which determines the number of viral infectious particles present per volume unit (viral titer). A range of SPL-0 concentrations was to obtain a dose-response curve. The CVB3 titer in the harvested medium decreased as the SPL-0 dose increased (Figure 10). The EC ₅₀ of SPL-0 against CVB3 was 2.95 μM (r ² = 0.980, n = 0.828), with a bottom plateau of 0 % for the estimated viral titer. Therefore, the antiviral action of SPL-0 extends beyond coronaviruses.

Example 18: Effect of SPL-0 on in the infectivity of vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein

Viral pseudotypes consist of a model virus carrying the surface protein of another virus of interest, and allow examining how the virus of interest binds to cellular receptors and enters the host. Vesicular stomatitis virus (VSV) pseudotyped with the spike protein of SARS-CoV-2 was used to inoculate A549 cells expressing the SARS- CoV-2 receptor ACE2 in the presence or absence of SPL-0. A reporter gene encoding green fluorescent protein (GFP), carried by the VSV genome, was used to quantify infection by automated fluorescence microscopy. SPL-0 at 20 μM or 50 μM had no effect on infection levels (Figure 11a), indicating that SPL-0 did not appreciably inhibit viral entry mediated by the SARS-CoV-2 spike protein. It can be concluded that the mode of action of SPL-0 against SARS-CoV-2 is not at the level of receptor-mediated viral entry. Furthermore, analogous assays were performed in A549 cells co- expressing the SARS-CoV-2 receptor ACE2 and transmembrane protease, serine 2 (TMPRSS2). In the absence of SPL-0, TMPRSS2 expression elevated pseudotype infectivity by 30-fold, consistent with the observation that TMPRSS2 is an enhancer of SARS-CoV-2 entry. It was also found that SPL-0 inhibited pseudotype infectivity in A549 cells co-expressing ACE2 and TMPRSS2 (Figure 11 b), suggesting an antiviral mode of action related to host serine protease activity.

Example 19: Assessment of the ability of TGEV to evolve resistances against SPL-0. To examine the emergence of resistances against SPL-0, TGEV was serially passaged 10 times in ST cells at an MOI of 0.01 infectious particles per cell in the presence of 50 μM SPL-0 (selective regime), or in the absence of SPL-0 (control regime). After completion of serial transfers, the antiviral effect of SPL-0 was re- evaluated using the viruses obtained from each of these two regimes. For this, ST cells were inoculated at an MOI of 0.01 infectious particles per cell, and the viral titer was determined after 24 h by the plaque assay. Titers were expressed relative to those achieved in the absence of SPL-0 by the virus obtained from the control regime transfers (control baseline). Viruses obtained in the control and selective regimes showed similar responses to SPL-0 (Figure 12), therefore providing no evidence for the emergence of resistances against SPL-0 after repeated exposure of TGEV to the compound.

Table 1 and Table 2 show a summary of the results:

Table 1. Summary of the activity of different compounds against TGEV as determined by quantitation of virus-induced cell death Table 2. Summary of the activity of SPL-0 against different viruses by different methods

ND: not determined