EP4698279A1

EP4698279A1 - Compounds with activity against sars-cov-2

Info

Publication number: EP4698279A1
Application number: EP24719547.2A
Authority: EP
Inventors: Theodora Calogeropoulou; Maria Koufaki; Demetris PAPAHATJIS; Vassilios GORGOULIS; Petros DARAS; Apostolos AXENOPOULOS; Stylianos MYLONAS; Sotirios KATSAMAKAS; Konstantinos STAMATOPOULOS; Spyridon PETRAKIS; Ioannis KARAKASILIOTIS
Original assignee: Centre For Research And Technology Hellas; National and Kapodistrian University of Athens; Democritus University of Thrace; National Hellenic Research Foundation
Current assignee: Centre For Research And Technology Hellas; National and Kapodistrian University of Athens; Democritus University of Thrace; National Hellenic Research Foundation
Priority date: 2023-04-18
Filing date: 2024-04-17
Publication date: 2026-02-25
Also published as: WO2024218171A1

Abstract

Compounds of Formula (I), pharmaceutical compositions containing them and their use in the treatment of a SARS-CoV-2 infection.

Description

COMPOUNDS WITH ACTIVITY AGAINST SARS-COV-2

TECHNICAL FIELD OF THE INVENTION

The invention relates to compounds exhibiting activity against severe-acute-respiratory syndrome coronavirus 2.

BACKGROUND OF THE INVENTION

In March 2020, the coronavirus disease 2019 (COVID-19) caused by a novel, highly pathogenic severe-acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) was declared a global pandemic by the World Health Organization (WHO). COVID-19 characterized as serious respiratory disease affects not only the respiratory tract but also the gastrointestinal system, the central nervous system, the heart and the kidneys. It is highly contagious and has affected over 610 million people to date. The accelerated development of effective COVID-19 vaccines and antibodies has offered transient solutions to resolve the pandemic.

The human infection by SARS-CoV-2 involves the binding of the transmembrane homotrimeric spike (S) glycoprotein [through the Receptor Binding Domain (RBD) of its S1 subunit] to the host protein angiotensin-converting enzyme 2 (ACE2). During the intracellular life cycle, the virus expresses and replicates its genome RNA. Then, the virion is assembled, and the newly synthesized SARS CoV-2 particles are released, surrounded by the membrane of the host cells.

The interaction between RBD and ACE2 is of outmost importance due to the regulation of cross-species and human-to-human viral transmission (Rodriguez Y. etal., ACS Omega, 2022, 7(33), 28779-28789). The specific interaction, of spike glycoprotein with ACE2 is the current pharmacological target of vaccines and monoclonal antibodies that block the viral entry into the cells. Since they are subgenus or strain specific, they promote the emergence of new mutated variants (Sun X. et al., Nat Microbiol, 2022, 7(7), 1063-1074). Nonetheless, recently published data indicate the waning of vaccines between three and six months post-vaccination (Zhuang C. etal., Front Microbiol, 2022, 13, 828806). Recently approved treatments include the use of the combination of small molecules nirmatrelvir and ritonavir (Paxlovid) (Lamb Y.N., Drugs, 2022, 1-7), remdesivir and molnupiravir (Cave J. A. & Phizackerley D., Drug and Therapeutics Bulletin, 2022, 60(1), 2) However, none of the approved small molecules targets the inhibition of the S(RBD)-ACE2 protein-protein interaction (PPI). It has been reported (Omotuy Olaposi et al., Microbial Pathogenesis, 2023, 176, 105994) that the known drug bazedoxifene inhibited S(RBD)-ACE2 interaction and the benzimidazole fragment was identified as high affinity binder at RBD/ACE2 interface. Furthermore, patent application W02022/006498 discloses that cannabidiol cannabidivarin and cannabigerol inhibit viral cell death.

Thus, the development of novel S(RBD)-ACE2 PPI inhibitors represents an innovative and promising therapeutic approach to prevent the infection and/or spreading of SARS-CoV-2.

SUMMARY OF THE INVENTION

The present invention provides compounds which exhibit activity against SARS-CoV-2. Specifically, the compounds of the present invention inhibit the interaction between the Receptor Binding Domain (RBD) of the S1 subunit of SARS-CoV-2 spike glycoprotein and ACE2.

The present invention provides also a pharmaceutical composition comprising a compound of the present invention.

Furthermore, the present invention provides the use of a compound of the present invention in the treatment of a SARS-CoV-2 infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the viability of HEK293T cells treated with compounds of the present invention. Cell viability was measured 48 hrs later using a MTT assay. Cells treated with DMSO served as control. Data are presented as a mean of triplicates ± standard deviation (SD).

FIG. 2 shows the effects of compounds of the present invention on Spike RBD/ACE2 (aa19-615) interaction using a cell-based quantification assay. Interaction of PA- mCitrine-Spike RBD and cmyc-NL-ACE2 (aa19-615) proteins was quantified in HEK293T cells in the presence of the tested compounds. The cBRET signal corresponding to Spike RBD/ACE2 (aa19-615) interaction was quantified 48h post treatment. Data are presented as a mean of triplicates ± SD.

FIG. 3 shows (A) the viability of Vero E6 cells treated for 48h with compounds of the present invention; (B) the cytopathic effect (CPE) of SARS-CoV-2 in Vero E6 cells treated with compounds of the present invention at non-cytotoxic concentrations; (C) the quantification of SARS-CoV-2 viral load in the culture medium of cells treated for 48h with compounds of the present invention; (D) the suppression of SARS-CoV-2 CPE by a compound of the present invention. Data are presented as a mean of triplicates ± SD

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt thereof wherein

Q is selected from N, N⁺(X')CH2R⁴,

X is selected from Cl, Br, when Q is N, R¹ is selected from OCH3, NO2, OH, NH2, NHC(O)CHs, and when Q is N⁺(X’)CH₂R⁴, R¹ is selected from H, OCH₃, NO₂, OH, NH₂, NHC(O)CH₃,

R² is selected from OCH₃, NO₂, OH, NH₂, NHC(O)CH₃, H,

R³ is selected from R⁵ is selected from H, F, Cl, Br, CH₃, CN, OCH₃, SCH₃, NO₂, OH, NH₂, N(CH₃)₂, NHC(O)CH₃, SO₂NH₂, SO₃H, B(OH)_2I

Z is selected from CH₂, NH, O, S.

Preferably, Q is N and R¹ is selected from OCH₃, NO₂, OH, NH₂, or Q is N⁺(X')CH₂R⁴ and R¹ is selected from H, OCH₃, NO₂, OH, NH₂. More preferably, Q is N and R¹ is selected from OCH₃, NO₂, NH₂, or Q is N+(X')CH₂R4 and R¹ is selected from H, OCH₃, NO₂, NH₂.

Preferably, R² is selected from OCH₃, NO₂, or H. More preferably, R² is H.

Preferably, R³ is selected from

M , Preferably,

More preferably, R⁴ is selected from

Preferably, R⁵ is selected from H, F, Cl, Br, OCH3, NO2. More preferably, R⁵ is selected from Cl, NO2.

Preferably, Z is selected from CH2, O. More preferably, Z is CH2.

The compounds of formula (I) contain at least one basic functional group and are capable therefore of forming pharmaceutically acceptable salts by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic and pharmaceutically acceptable organic acids. Examples of inorganic acid addition salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate. Examples of organic acid addition salts include acetate, lactate, citrate, tartrate, succinate, maleate and fumarate. The pharmaceutically acceptable salts can be prepared by methods well known in the art, e.g. by treatment of a compound of Formula (I) with an acid, in the presence of a solvent, such as water or acetone.

The present invention includes all possible stereoisomers of compounds of Formula (I) and pharmaceutically acceptable salts thereof.

The compounds of Formula (I) can be prepared according to the following general processes (Scheme 1):

III

Scheme 1 As shown in Scheme 1 , compounds of Formula (II) can be prepared from the appropriately aryl-substituted 1 ,2-diamines through an N-alkylation reaction with the desired alkyl halide or mesylate using a base (e.g. NaH, K2CO3) and a solvent (e.g. THF, DMF). The N-alkylated intermediates can then undergo cyclization in the presence of a Lewis acid (e.g. BFs Et2O) or organic acid (e.g. pTsOH) in a solvent (e.g. DCM, toluene). Alternatively, compounds of Formula (II) can be obtained by N-alkylation of the appropriately aryl-substituted 2-nitroanilines followed by reduction of the nitro group using, for example, SnCh in a solvent (e.g. MeOH, AcOH). Cyclization of the monoalkylated diamine under the same conditions as above can afford the desired compounds. Finally, compounds of Formula (II) can be obtained starting from the appropriately substituted benzimidazoles through an N-alkylation reaction as above.

Compounds of Formula (III) can be prepared starting from quaternization of compounds of Formula (II) using, e.g., an alkyl halide in dioxane under reflux.

The compounds of the present invention are typically administered to a subject as a pharmaceutical composition.

Thus, the present invention provides also a pharmaceutical composition comprising a compound of the present invention as active ingredient and a pharmaceutically acceptable carrier.

The composition may be formulated, for example, for oral, parenteral, intramuscular, intravenous, intraperitoneal, subcutaneous, transdermal/intradermal, or inhalation administration. The composition may have different forms, such as solid or liquid forms, for example it can have the form of tablet, capsule, powder, solution, suspension, emulsion, gel, cream, ointment, spray, transdermal patch, or lotion. The pharmaceutically acceptable carrier may comprise one or more excipients, such as solvents, buffering agents, emulsifying agents, preservatives, antioxidants, chelating agents, diluents, binders, disintegrants, lubricating agents and the like, which are well known in the art.

Examples of solvents, include water, saline, phosphate buffered saline, ethanol, isopropyl alcohol and mixtures thereof.

Examples of buffering agents include citric acid monohydrate, sodium citrate, sodium lactate, calcium lactate, acetic acid, sodium acetate, potassium acetate, dibasic potassium phosphate, monobasic potassium phosphate sodium hydrogen phosphate and sodium bicarbonate.

Examples of emulsifying agents include sodium lauryl sulfate, lecithin, polysorbate, sorbitan monooleate, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, poloxamer nonionic surfactants, acacia, agar, alginic acid and sodium alginate.

Examples of preservatives include sodium benzoate, benzyl alcohol, benzalkonium chloride, benzethonium chloride, bronopol, cetrimide, ethanol, and parahydroxy benzoic acids and their alkyl esters. Examples of antioxidants include sodium metabisulfite, butylated hydroxyanisole, butylated hydroxytoluene and ascorbic acid.

Examples of chelating agents include ethylenediaminetetraacetic acid and salts thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, and the like), citric acid and salts thereof, fumaric acid and salts thereof, phosphoric acid and salts thereof, and tartaric acid and salts thereof.

Examples of diluents include lactose, sucrose, dextrose, mannitol sorbitol, inositol, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, microcrystalline cellulose, kaolin, sodium chloride, dry starch and cornstarch.

Examples of binders include hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, dextrose, xylitol, polyvinylpyrrolidone, polyethylene glycol, alginates and gelatin.

Examples of lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt and sodium lauryl sulfate.

The compounds of the present invention inhibit the interaction between the Receptor Binding Domain (RBD) of the S1 subunit of SARS-CoV-2 spike glycoprotein and ACE2. Therefore, the present invention provides also a method of treatment of a SARS-CoV-2 infection by administering to a subject a compound of the present invention. The compound of the present invention is typically administered in the form of a pharmaceutical composition.

Preferably, the subject is a mammal. More preferably, the subject is a human.

The compounds of the present invention may be administered via a variety of routes, including oral, intravenous, intramuscular, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical mucosal, nasal, buccal and sublingual route. The route of administration may depend upon various factors including the nature of the active ingredient, the age and the condition of the subject. The compounds of the present invention are generally administered daily, or more frequently, such as twice a day, or less frequently, such as every second day, or every week. The frequency of administration and the administered dosage depend on various factors, such as the severity of the infection, the route of administration, the age of the subject, and the form of the administered composition, and can be determined by a person skilled in the art by using common general knowledge EXAMPLES

In the Examples below, the following abbreviations are used: THF for tetrahydrofuran, DMF for dimethylformamide, Boc for tert-butyloxycarbonyl, MeOH for methanol, AcOH for acetic acid, EtOAc for ethyl acetate, DCM dichloromethane, DMSO for dimethylsulfoxide, TFA for trifluoroacetic acid, pTsOH for p-Toluenesulfonic acid, MS for mass spectroscopy, ESI for electrospray ionization, HRMS for high-resolution mass spectrometry, NMR for nuclear magnetic resonance, FCC for flash column chromatography, RT for room temperature, ppm for parts per million, min for minute, and h for hour.

In the assays described in the Examples below, the following abbreviations are used: DMSO for dimethylsulfoxide, MTT for 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, RBD for receptor binding domain, ACE2 for angiotensinconverting enzyme 2, DMEM for Dulbecco's Modified Eagle Medium, PCR for polymerase chain reaction, RNA for Ribonucleic acid, rt for room temperature, min for minute, and h for hour.

The compounds of the Examples were synthesized by following the general procedures described below:

General procedures for N-alkylation (Step 1)

(a) To an ice-cold solution of the appropriate amine (1 equivalent) in dry THF (0.3M) NaH (oil dispersion 60% w/w, 1.2 equivalents) was added portionwise. The reaction was warmed to RT and stirred at the same temperature for 30min followed by the addition of the appropriate alkyl halide or alkyl mesylate (1.05 equivalents). The reaction mixture was stirred at RT for 17 h, diluted with water and extracted twice with EtOAc. The combined organic layers were washed with water, dried over Na2SO4 and evaporated to dryness. Pure compound was obtained after FCC.

(b) A suspension of the corresponding amine (1 equivalent) and K2CO3 (2 equivalents) in DMF (0.3M) was stirred at RT for 10min and subsequently the appropriate alkyl halide (1.0-1.3 equivalents) was added. The resulting mixture was heated at 90 °C for 2 h, was left to cool to RT, diluted with water and the aqueous phase was extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness. Purification by FCC afforded the desired compound. (c) To an ice-cold suspension of NaH (60% w/w dispersion in mineral oil, 1.2 equivalents) in dry DMF (0.3M) the corresponding amine (1 equivalent) was added. After stirring for 30 minutes at 0 °C, the appropriate alkyl halide (1.0-1.05 equivalents) was added and the mixture was stirred at RT for 24h. Then the reaction was cooled to 0 °C and quenched with NH4CI saturated solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SC>4, filtered and the solvent removed in vacuo. The residue was purified by FCC to obtain the desired compound.

General procedure for N-Boc protection (Step 2): The appropriate aniline (1 equivalent) was added to a suspension of NaH (60% w/w dispersion in mineral oil, 2.05 equivalents) in dry THF (0.2M). After the suspension was stirred for 10 minutes, a solution of di-‘butyl-dicarbonate (1.2 equivalents) in dry THF (0.9M) was added slowly and the mixture was stirred at RT for 24h. Then the solution was cooled at 0 °C and water was added and extracted with ethyl acetate. The organic layer was dried over Na2SC>4, filtered and the solvent removed in vacuo. The residue was purified by FCC to afford the terf-butyl carbamate derivative.

General procedure for N-Boc deprotection (Step 3): To a solution of terf-butyl carbamate from Step 2 (1 equivalent) in dry CH2CI2 (0.1M) was added TFA (20 equivalents) at 0 °C and the reaction mixture was stirred at RT for 1 h. The solvent was evaporated in vacuo and the residue was diluted with sat. aq. NaHCCh and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SC>4, filtered and evaporated in vacuo. The residue was purified by FCC to afford the desired compound.

General procedures for nitro group reduction (Step 4):

(a) SnCh (5 equivalents) was added to a mixture of the appropriate nitro compound (1 equivalent) in CH3OH (0.1 M) and con. HCI (70 equivalents) and stirred at 50 °C overnight. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and washed with sat. aq. NaHCOs, brine, dried over Na2SO4, filtered and evaporated in vacuo. The crude mixture was purified by FCC to give the desired compound.

(b) Concentrated HCI (56 equivalents) was added to a mixture of the appropriate nitro compound (1 equivalent), SnCh2H2O (5 equivalents) in dry CH3OH (0.18M) and the mixture was stirred for 18-20h. The solvent was removed under vacuo followed by addition of water and adjustment of pH to 7 with NaHCOs solution and the aqueous phase extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2SC>4, filtered and the solvent removed under reduced pressure. The residue was used to the next step without further purification.

(c) To a suspension of the appropriate nitro compound (1 equivalent) in gl. CH3COOH (0.3M), cone. HCI (48 equivalents) was added followed by the addition of SnCl2'2H2O (0.45 g, 2 mmol). The reaction mixture was stirred at RT for 30 min. Solvents were azeotropically removed with toluene and to the residue, aq. NaOH (10 M) was added followed by brine. The aqueous phase was extracted with CH2CI2 and the combined organic layers dried over Na2SO4 and evaporated to dryness to afford the desired compound.

General procedures for cyclization (Step 5):

(a) To an ice-cold solution of trimethyl orthoformate (3 equivalents) in CH2Cl2 (0.1M), BF3'Et2O (2 equivalents) was added dropwise, followed by the addition of the appropriate diamine (1 equivalent). The resulting mixture was stirred at RT for 5 h and quenched with sat. aqueous NaHCCh. The aqueous phase was extracted twice with CH2CI2. The combined organic layers were dried over Na2SO4 and evaporated to dryness. The residue was purified by FCC to obtain the desired compound.

(b) A mixture of the appropriate diamine (1 equivalent), trimethyl orthoformate (1.5 equivalents) and catalytic amount of pTsOH H₂O (3.25%) in toluene (0.07M) was stirred at 80 °C for 15h. After completion of the reaction the solvent was removed in vacuo. The residue was purified by FCC to afford the desired compound.

General procedure for methoxy group deprotection (Step 6): To an ice-cold solution of the appropriate methoxy substituted compound (1 equivalent) in CH2CI2 (0.05M), BF3.S(CH₃)2 (10 equivalents, per methoxy group) was added and the reaction mixture was stirred at RT for 2 days. After completion of the reaction, the solvent and excess reagent was evaporated under argon stream. To the residue sat. aq. NaHCCh was added and extracted with ethyl acetate. The organic layer was washed with sat. aq.NaCI, dried over Na2SO4 and the solvent was evaporated in vacuo. The residue was purified by FCC to obtain the desired compound.

General procedure for quaternization (Step 7): To a solution of the appropriate alkylated benzimidazole (1 equivalent) in 1 ,4-dioxane (0.03M) the appropriate alkylating agent (1 .3 equivalent) was added, and the resulting mixture was stirred at 100 °C for 24 h. Then, the reaction was cooled to RT. The precipitate was filtered, and the solid was washed with dioxane and diethyl ether. The resulting compound was pure as obtained or was recrystallized from acetone when necessary.

Synthesis of the compounds of Examples 1-6

EXAMPLE 1

1-(naphthalen-2-ylmethyl)-4-nitro-1/7-benzo[d]imidazole (CKP20)

/\/¹-(naphthalen-2-ylmethyl)-3-nitrobenzene-1 ,2-diamine was prepared according to the general procedure described in Step 1 b by the reaction of 1 (0.15 g) and 2- (bromomethyl)naphthalene (0.29 g). Purification of the compound with FCC (n- Hexane/EtOAc 9:1 v/v) gave a red solid, 0.22 g (75% yield); ¹H-NMR (600 MHz, acetone-d₆): 64.61 (d, 2H, J = 5.4 Hz), 5.20 (m, 1 H), 6.57 (t, 1 H, J = 7.8 Hz), 6.82 (d, 2H, J = 7.8 Hz), 7.47-7.51 (m, 3H), 7.59 (dd, = 1.2 Hz, J₂ = 8.4 Hz), 7.85-7.87 (m, 1 H), 7.88-7.91 (m, 2H), 7.93 (s, 1 H); ESI-HRMS (m/z): [M-H]⁺ calcd. for C17H14N3O2 292.1086; found 292.1083.

CKP20 was prepared according to the general procedure described in Step 5a using intermediate 2 (0.12 g). Purification of the compound with FCC (n-Hexane/EtOAc 1 :1 v/v) gave a white solid, 0.114 g (94% yield); ¹H-NMR (600 MHz, acetone-dg): 65.87 (s, 2H), 7.39 (t, 1 H, J = 8.1 Hz), 7.48 (dd, 1 H, = 1.9 Hz, J₂ = 8.5 Hz), 7.49-7.58 (m, 2H),

7.86-7.92 (m, 3H), 7.96 (dd, 1 H, = 1.2 Hz, J₂ = 8.4 Hz), 8.03 (dd, 2H, = 1.2 Hz, J₂

= 7.8 Hz), 8.64 (s, 1 H); ESI-HRMS (m/z): [M+H]⁺ calcd. for C18H14N3O2 304.1086; found 304.1076.

EXAMPLE 2

1-([1 ,1'-biphenyl]-4-ylmethyl)-4-nitro-1/7-benzo[d]imidazole (CKP21)

CKP21 was prepared according to the general procedure described above for CKP20, using 4-biphenylmethyl bromide (0.32 g) in corresponding Step 1 b. Pure compound was obtained accordingly as white solid, 0.121 g; ¹H-NMR (600 MHz, acetone-cfe): 55.75 (s, 2H), 7.33-7.37 (m, 1 H), 7.42-7.48 (m, 5H), 7.60-7.63 (m, 2H), 7.64-7.67 (m, 2H), 7.98 (d, 1 H, J = 8.4 Hz), 8.05 (d, 1 H, J = 8.4 Hz), 8.61 (s, 1 H); ESI-HRMS (m/z): [M+H]⁺ calcd. for C20H16N3O2 330.1243; found 330.1232.

EXAMPLE 3

7-((4-nitro-1/7-benzo[c(]imidazol-1-yl)methyl)quinolone (CKP48)

CKP48 was prepared according to the general procedure described above for CKP20, using 7-(bromomethyl)quinoline hydrobromide (0.100 g) in Step 1 b. Pure compound was obtained accordingly as off-white solid, 0.028 g; Mp: 190.5-192.0 °C; ¹H-NMR (600 MHz, acetone-cfe): 5 5.96 (s, 2H), 7.43 (dt, 1 H, = 3.0 Hz, J₂ = 8.4 Hz), 7.95-8.62 (m,

4H), 8.08 (s,1 H), 8.31 (d, 1 H, J = 7.8Hz), 8.68 (s, 1 H), 8.88 (bs, 1 H); ESI-HRMS (m/z): [M+H]⁺ calcd. for C17H13N4O2 305.1039; found 305.1032

EXAMPLE 4

1-([1 ,1'-biphenyl]-4-ylmethyl)-1/7-benzo[c(]imidazol-4-amine (CKP28)

CKP28 was prepared according to the general procedure described in Step 4c by using compound CKP21 (0.090 g). Purification of the compound with FCC (DCM/MeOH 97:3 v/v) gave a white solid, 0.082 g (91% yield); ¹H-NMR (600 MHz, acetone-cfe): 54.85 (bs, 2H), 5.39 (s, 2H), 6.34 (dd, = 1.2 Hz, J₂ = 8.4 Hz), 6.60 (dd, 1 H, = 1.2 Hz, J₂ = 8.4 Hz), 6.82 (t, 1 H, J = 7.8 Hz,), 7.21-7.29 (m, 3H), 7.30-7.35 (m, 2H), 7.50-7.53 (m, 4H), 7.97 (s, 1 H); ESI-HRMS (m/z): [M+H]⁺ calcd. for C20H18N3 300.1501 ; found 300.1489.

EXAMPLE 5

1-(naphthalen-2-ylmethyl)-1/7-benzo[c(]imidazol-4-amine (CKP29)

CKP29 was prepared according to the general procedure described in Step 4c, using CKP20 (0.082 g). Pure compound was obtained accordingly as white solid, 0.073 g (89% yield); ¹H-NMR (600 MHz, acetone-cfe): 5 5.60 (s, 2H), 6.44 (dd, 1 H, = 1.2 Hz,

J₂ = 7.8 Hz), 6.69 (dd, 1 H, = 1.2 Hz, J₂ = 8.4 Hz), 6.90 (t, 1 H, J = 7.8 Hz), 7.41 (dd,

1 H, Ji = 1.8 Hz, J₂ = 8.4 Hz), 7.47-7.51 (m, 3H), 7.81 (s, 1 H), 7.84-7.89 (m, 4H), 8.11 (s, 1 H); ESI-HRMS (m/z): [M+H]⁺ calcd. for CI₈HI₆N₃274.1344; found 274.1333.

EXAMPLE 6 1-(quinolin-7-ylmethyl)-1/7-benzo[d]imidazol-4-amine (CKP49)

CKP49 was prepared according to the general procedure described in Step 4c, using CKP48 (0.039g). Pure compound was obtained accordingly as off-white solid, 0.031 g (89% yield); Mp: 204.0-205.0 °C decomposition; ¹H-NMR (600 MHz, acetone-cfe): 65.72 (s, 2H), 6.45 (d, 1 H, J = 7.8Hz), 6.71 (d, 1 H, J = 8.4Hz), 6.91 (t, 1 H, J = 8.4Hz), 7.47- 7.51 (m, 2H), 7.91-7.93 (m, 2H), 8.23 (s,1 H), 8.29 (d, 1 H, J = 8.4Hz), 8.87 (bs, 1 H); ESI- HRMS (m/z): [M+H]⁺ calcd. for C17H15N4 275.1297; found 275.1287.

EXAMPLE 7

4-methoxy-1-(naphthalen-2-ylmethyl)-1/7-benzo[c(]imidazole (CKP25) terf-butyl (3-methoxy-2-nitrophenyl)(naphthalen-2-ylmethyl)carbamate (4) was prepared according to the Step 1c by the reaction of fert-butyl (3-methoxy-2- nitrophenyl)carbamate 3 (0.15 g) and 2-(bromomethyl)naphthalene (0.13 g). The compound was used in the next step without further purification.

3-Methoxy-N-(naphthalen-2-ylmethyl)-2-nitroaniline (5) was prepared according to Step 3 using fert-butyl (3-methoxy-2-nitrophenyl)(naphthalen-2-ylmethyl)carbamate 4 (0.23 g). Purification of the compound with FCC (n-Hexane/EtOAc 8:2 v/v) gave a yellow solid, 0.14 g (79% yield over 2 steps); Mp: 111.0-112.0 °C; ¹H-NMR (600 MHz, CDCh): 53.88 (s, 3H), 4.59 (s, 2H), 6.29 (d, 1 H, J = 8.3 Hz), 6.37 (d, 1 H, J = 8.6 Hz), 7.16 (t, 1 H, J = 8.4 Hz), 7.37-7.55 (m, 3H), 7.72-7.86 (m, 4H); MS m/z: 331.08 [M+Na]⁺, 638.69 [2M+Na]⁺.

3-Methoxy-N¹-(naphthalen-2-ylmethyl)benzene-1 ,2-diamine (6) was prepared according to Step 4a using 3-methoxy-N-(naphthalen-2-ylmethyl)-2-nitroaniline (5) (0.120 g). Purification of the compound with FCC (n-Hexane/EtOAc 8:2 v/v) gave a brown oil, 0.089 g (82% yield); ¹H-NMR (600 MHz, CDCI₃): 53.70 (bs, 3H), 3.87 (s, 3H), 4.51 (s, 2H), 6.45 (t, 2H, J = 8.1 Hz), 6.75-6.79 (m, 1 H), 7.35-7.57 (m, 3H), 7.74-7.89 (m, 4H); MS m/z: 279.22 [M+H]⁺.

CKP25 was prepared according to Step 5b using 3-methoxy-N¹-(naphthalen-2- ylmethyl)benzene-1 ,2-diamine 6 (0.088 g). Purification of the compound with FCC (DCM/Acetone 9:1 v/v) gave a white solid, 0.061 g (67% yield); Mp: 163.0-164.0 °C; ¹H- NMR (600 MHz, Acetone-cfe): 54.00 (s, 3H), 5.68 (s, 2H), 6.69 (d, 1 H, J = 7.5 Hz), 7.05- 7.11 (m, 2H), 7.42 (d, 1 H, J = 8.4 Hz), 7.49 (dd, 2H, = 2.9 Hz, J₂ = 6.5 Hz), 7.81-7.90

(m, 4H), 8.21 (s, 1 H); MS m/z: 289.10 [M+H]⁺, 576.49 [2M]⁺, 598.80 [2M+Na]⁺.

EXAMPLE 8

1 -([1 , 1 '-biphenyl]-4-ylmethyl)-4-methoxy-1 /7-benzo[c(]imidazole (CKP27)

CKP27 was prepared according to CKP25 using terf-butyl (3-methoxy-2- nitrophenyl)carbamate 3 (0.17 g) and 4-biphenylmethyl bromide (0.166 g) in Step 1c. Pure compound was obtained accordingly as white solid; Mp: 150.0-151.0 °C; ¹H-NMR (600 MHz, Acetone-cfe): 5 4.01 (s, 4H), 5.57 (s, 3H), 6.71 (d, 1 H, J = 7.8 Hz), 7.08 (d, 1 H, J = 7.8 Hz), 7.13 (t, 1 H, J = 7.9 Hz), 7.35 (t, 2H, J = 7.3 Hz), 7.39 (d, 2H, J = 7.9 Hz), 7.44 (t, 2H, J = 7.6 Hz), 7.63 (dd, 4H, = 4.1 Hz, J₂ = 7.9 Hz), 8.18 (s, 1 H); MS m/z: 315.09 [M+H]⁺, 337.16 [M+Na]⁺, 628.72 [2M]⁺, 650.84 [2M+Na]⁺.

EXAMPLE 9

7-((4-methoxy-1/7-benzo[c(]imidazol-1-yl)methyl)quinolone (CKP44)

CKP44 was prepared according to the general procedures described also for CKP25, by using 7-(bromomethyl)quinoline hydrobromide (0.100 g) in Step 1c. Pure compound was obtained accordingly as white solid; Mp: 161.0-162.0 °C; ¹H-NMR (600 MHz, acetone-cfe): 64.01 (s, 3H), 5.78 (s, 2H), 6.72 (dd, 1 H, = 1.3 Hz, J₂ = 7.5 Hz), 7.07-

7.16 (m, 2H), 7.50 (ddd, 2H, = 3.0 Hz, J₂ = 8.4 Hz, J₃ = 14.1 Hz), 7.91-7.98 (m, 2H),

8.23-8.32 (m, 2H), 8.88 (dd, 1 H, J₁ = 1.8 Hz, J₂ = 4.3 Hz); MS m/z: 290.11 [M+H]⁺, 578.66 [2M]⁺, 600.81 , [2M+Na]⁺.

EXAMPLE 10

1-(naphthalen-2-ylmethyl)-1/7-benzo[c(]imidazol-4-ol (CKP26)

CKP26 was prepared according to Step 6 using compound CKP25 (0.026 g). Purification of the compound with FCC (DCM/Acetone, 95:5) gave a white solid, 0.015 g (60% yield); Mp: 235.0-236.0 °C; ¹H-NMR (600 MHz, CD3OD): 65.61 (s, 2H), 6.63 (d, 1 H, J = 7.7 Hz), 6.91 (d, 1 H, J = 8.2 Hz), 7.04 (t, 1 H, J = 7.9 Hz), 7.35 (d, 1 H, J = 8.5 Hz), 7.47 (dd, 1 H, Ji = 3.3 Hz, J₂ = 5.9 Hz), 7.72 (s, 2H), 7.78-7.85 (m, 3H), 8.19 (s, 1 H); MS m/z: 275.07 [M+H]⁺, 570.73 [2M+Na]⁺.

EXAMPLE 11

1 -([1 , 1 b i ph eny l]-4-y I m ethy I)- 1 /7- benzo[c/] i m idazo l-4-ol (CKP30)

CKP30 was prepared according to Step 6 using CKP27 (0.039 g). Pure compound was obtained accordingly as off-white solid, 0.023 g (62% yield); Mp: 258.0-259.0 °C; ¹H- NMR (600 MHz, CDCI₃): 5 5.42 (s, 2H), 6.88 8.0 Hz, J₂ = 22.3 Hz), 7.19-

7.23 (m, 2H), 7.29 (d, 2H, J = 7.8 Hz), 7.32-7.36 (m, 1 H), 7.42 (t, 2H, J = 7.6 Hz), 7.55 (dd, 4H, Ji = 7.8 Hz, J₂ = 20.7 Hz), 8.31 (s, 1 H); MS m/z: 301.02 [M+H]⁺, 622.66 [2M+Na]⁺.

Synthesis of the compounds of Examples 12-22

EXAMPLE 12

(E)-1-(naphthalen-2-ylmethyl)-3-(3-(4-nitrophenyl)allyl)-1/7-benzo[c(]imidazol-3-ium bromide (CKP31)

CKP31 was prepared according to Step 7 using 1-(naphthalen-2-ylmethyl)-1/7- benzo[d]imidazole (0.022 g) and (E)-1-(3-bromoprop-1-en-1-yl)-4-nitrobenzene (0.026 g). Pure compound was obtained as off-white solid, 0.031 g (74.8% yield); Mp: 189.2- 190.6 °C; ¹H-NMR (600 MHz, acetone-d₆): 64.98 (d, 2H, J = 6.4 Hz), 5.52 (s, 2H), 6.43 (dd, 1 H, Ji = 7.7 Hz, J₂ = 14.6 Hz), 6.58 (d, 1 H, J = 16.0 Hz,), 7.01-7.13 (m, 2H), 7.21 (ddd, 3H, Ji = 8.0 Hz, J₂ = 15.1 J₃ = 30.1 Hz), 7.31 (d, 2H, J = 8.4 Hz), 7.48 (d, 2H, J = 11.1 Hz), 7.55 (dd, 2H, Ji = 8.3 Hz, J₂ = 22.0 Hz), 7.63-7.70 (m, 2H), 7.78 (d, 2H, J = 8.4 Hz), 9.55 (s, 1 H); ESI-HRMS (m/z): [M-Br]⁺ calcd. for C27H22N3O2 420.1706; found 420.1704.

EXAMPLE 13 (E)-3-(3-(4-chlorophenyl)allyl)-1-(naphthalen-2-ylmethyl)-1/7-benzo[d]imidazol-3-ium bromide (CKP32)

CKP32 was prepared according to Step 7 using 1-(naphthalen-2-ylmethyl)-1/7- benzo[d]imidazole (0.021 g) and (E)-1-(3-bromoprop-1-en-1-yl)-4-chlorobenzene (0.025 g). Pure compound was obtained as white solid, 0.03 g (74.4% yield); Mp: 165.7-167.3 °C; ¹H-NMR (600 MHz, CD₃OD): 6 5.34 (d, 2H, J = 6.6 Hz), 5.49 (s, 2H), 5.93 (s, 1 H), 6.57 (dt, 1 H, Ji = 6.5 Hz, J₂ = 14.1 Hz), 6.93 (d, 1 H, J = 15.9 Hz), 7.35 (d, 1 H, J = 8.6 Hz), 7.47 (d, 1 H, J = 8.1 Hz), 7.52-7.59 (m, 2H), 7.69 (dt, 1 H, = 7.7 Hz, J₂ = 24.2 Hz), 7.89 (t, 1 H, J = 4.9 Hz), 7.94 (t, 1 H, J = 7.0 Hz), 8.03 (d, 1 H, J = 6.1 Hz); ESI-HRMS (m/z): [M-Br]⁺ calcd. for C27H22CIN2 409.1466; found 409.1461.

EXAMPLE 14

(E)-1-([1 ,1'-biphenyl]-4-ylmethyl)-3-(3-(4-nitrophenyl)allyl)-1/7-benzo[c(]imidazol-3-ium bromide (CKP33)

CKP33 was prepared according to Step 7 using 1-([1 ,1'-biphenyl]-4-ylmethyl)-1/7- benzo[d]imidazole [prepared according to Step 1a, using 1/7-benzo[d]imidazole (0.071 g) and 4-biphenylmethyl bromide (0.155 g)] (0.020 g) and (E)-1-(3-bromoprop-1-en-1- yl)-4-nitrobenzene (0.022 g). Pure compound was obtained as white solid, 0.014 g (36.9% yield); Mp: 236.0-238.0 °C decomposition; ¹H-NMR (600 MHz, CD3OD): 65.42 (d, 2H, J = 6.5 Hz), 5.82 (s, 2H), 6.75-6.88 (m, 1 H), 7.04 (d, 1 H, J = 15.9 Hz), 7.37 (t, 1 H, J = 7.6 Hz), 7.44 (t, 2H, J = 7.6 Hz), 7.60 (dd, 4H, = 7.8 Hz, J₂ = 12.3 Hz), 7.67-

7.80 (m, 6H), 7.96 (d, 1 H, J = 8.0 Hz), 8.04 (d, 1 H, J = 7.8 Hz), 8.22 (d, 2H, J = 8.5 Hz); ESI-HRMS (m/z): [M-Br]⁺ calcd. for C29H24N3O2 446.1863; found 446.1859.

EXAMPLE 15

(E)-1-(3,4-dimethoxybenzyl)-3-(3-(4-nitrophenyl)allyl)-1/7-benzo[c(]imidazol-3-ium bromide (CKP34)

CKP34 was prepared according to Step 7 using 1-(3,4-dimethoxybenzyl)-1/7- benzo[d]imidazole [prepared according to Step 1a, using 1/7-benzo[d]imidazole (0.154 g) and 3,4-dimethoxybenzyl methanesulfonate (0.337 g)] (0.021 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-nitrobenzene (0.024 g). Pure product was obtained as off-white solid, 0.018 g (55.3% yield); Mp: 146.0-148.0 °C decomposition; ¹H-NMR (600 MHz, CD3OD): 63.83 (s, 6H), 5.40 (d, 2H, J = 6.4 Hz), 5.68 (s, 2H), 6.79 (dq, 1 H, = 9.0 Hz, J₂ = 15.6 Hz), 6.95-7.04 (m, 3H), 7.10 (d, 1 H, J = 7.9 Hz), 7.17 (s, 1 H), 7.71 (p, 5H, J = 10.2 Hz), 7.99 (dd, = 7.7 Hz, J₂ = 29.0 Hz), 8.21 (d, 2H, J = 8.4 Hz), 9.62 (s, 1 H);

ESI-HRMS (m/z): [M-Br]⁺ calcd. for C25H24N3O4 430.1761 ; found 430.1759.

EXAMPLE 16

(E)-1-([1 ,1'-biphenyl]-4-ylmethyl)-4-methoxy-3-(3-(4-nitrophenyl)allyl)-1/7- benzo[d]imidazol-3-ium bromide (CKP40)

CKP40 was prepared according to Step 7 using CKP27 (0.020 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-nitrobenzene (0.020 g). Pure compound was obtained as off- white solid, 0.033 g (92% yield); Mp: 203.0-204.0 °C; ¹H-NMR (600 MHz, CD3OD): 5 4.09 (s, 3H), 5.52 (d, 2H, J = 6.3 Hz), 5.75 (s, 2H), 6.81 (dt, 1 H, = 6.2 Hz, J₂ = 16.0 Hz), 6.90 (d, 1 H, J = 16.1 Hz), 7.24 (d, 1 H, J = 8.2 Hz), 7.34-7.39 (m, 1 H), 7.41-7.50 (m, 3H), 7.55 (d, 1 H, J = 7.9 Hz), 7.58-7.62 (m, 4H), 7.70 (d, 4H, J = 8.2 Hz), 8.21 (d, 2H, J = 8.4 Hz); MS m/z: 476.07 [M-Br]⁺.

EXAMPLE 17

(E)-1-([1 ,1'-biphenyl]-4-ylmethyl)-3-(3-(4-chlorophenyl)allyl)-4-methoxy-1/7- benzo[c(]imidazol-3-ium bromide (CKP41)

CKP41 was prepared according to Step 7 using CKP27 (0.014 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-chlorobenzene (0.015 g). Pure compound was obtained as white solid, 0.018 g (50% yield); Mp: 205.0-206.0 °C; ¹H-NMR (600 MHz, CD3OD): 5 4.10 (s, 3H), 5.46 (d, 2H, J = 5.6 Hz), 5.75 (s, 2H), 6.59 (dt, 1 H, = 6.6 Hz, J₂ = 15.8

Hz), 6.82 (d, 1 H, J = 15.7 Hz), 7.23 (d, 1 H, J = 8.2 Hz), 7.35 (dd, 1 H, = 8.0 Hz, J₂ =

11.3 Hz), 7.41-7.50 (m, 4H), 7.55 (d, 4H, J = 8.2 Hz), 7.58-7.64 (m, 5H), 7.69 (d, 2H, J = 8.2 Hz); MS m/z: 465.11 [M-Br]⁺.

EXAMPLE 18

(E)-4-methoxy-1-(naphthalen-2-ylmethyl)-3-(3-(4-nitrophenyl)allyl)-1/7- benzo[c(]imidazol-3-ium bromide (CKP42)

CKP42 was prepared according to Step 7 using CKP25 (0.017 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-nitrobenzene (0.019 g). Pure compound was obtained as yellowish solid, 0.026 g (84 % yield); Mp: 227.0-228.0 °C; ¹H-NMR (600 MHz, CD3OD): 64.09 (s, 3H), 5.52 (d, 2H, J = 6.2 Hz), 5.88 (s, 2H), 6.78-6.85 (m, 1 H), 6.90 (d, 1 H, J = 15.9 Hz), 7.22 (d, 1 H, J = 8.2 Hz), 7.45-7.60 (m, 5H), 7.69 (d, 2H, J = 8.7 Hz), 7.85-7.97 (m, 3H), 8.02 (s, 1 H), 8.21 (d, 2H, J = 8.7 Hz), 9.63 (s, 1 H); MS m/z: 450.16 [M-Br]⁺. EXAMPLE 19

(E)-3-(3-(4-chlorophenyl)allyl)-4-methoxy-1-(naphthalen-2-ylmethyl)-1/7- benzo[d]imidazol-3-ium bromide (CKP43)

CKP43 was prepared according to Step 7 using CKP25 (0.020 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-chlorobenzene (0.021 g). Pure compound was obtained as white solid, 0.013 g (36% yield); Mp: 228.0-229.0 °C; ¹H-NMR (600 MHz, CDCI₃): 54.04 (s, 3H), 5.55 (d, 2H, J = 6.8 Hz), 5.96 (s, 2H), 6.55 (dt, 1 H, = 6.8 Hz, J₂ = 15.1 Hz),

6.90-6.98 (m, 2H), 7.14 (d, 1 H, J = 8.4 Hz), 7.25 (d, 2H, J = 8.4 Hz), 7.34-7.40 (m, 3H), 7.48 (dd, 2H, Jv = 3.2 Hz, J₂ = 6.2 Hz), 7.54 (d, 1 H, J = 8.5 Hz), 7.75-7.91 (m, 3H), 7.97 (s, 1 H), 11.73 (s, 1 H); MS m/z: 439.17 [M-Br]⁺.

EXAMPLE 20

(E)-1-(2-(naphthalen-2-yl)ethyl)-3-(3-(4-nitrophenyl)allyl)-1/7-benzo[c(]imidazol-3-ium bromide (CKP47)

CKP47 was prepared according to Step 7 using 1-(2-(naphthalen-2-yl)ethyl)-1/7- benzo[d]imidazole (CKP46) [prepared according to Step 1a using 1/7-benzo[c(]imidazole (0.078 g) and 2-(2-bromoethyl)naphthalene (0.186 g)] (0.020 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-nitrobenzene (0.023 g). Pure compound was obtained as off- white solid, 0.021 g (54.5% yield); Mp: 176.0-177.0 °C decomposition; ¹H-NMR (600 MHz, DMSO-cfe): 54.90 (s, 2H), 5.34 (d, 2H, J = 6.1 Hz), 6.69 (d, 1 H, J = 15.7 Hz), 6.85 (d, 1 H, J = 16.0 Hz), 7.45 (s, 3H), 7.68 (dd, = 11 .4 Hz, J₂ = 30.4 Hz), 7.77 (s, 1 H), 7.86 (d, 2H, J = 8.1 Hz), 8.07 (s, 1 H), 8.13-8.27 (m, 3H), 9.73 (s, 1 H); ESI-HRMS (m/z): [M-Br]⁺ calcd. for C28H24N3O2 434.1863; found 434.1859.

EXAMPLE 21

(E)-3-(3-(4-chlorophenyl)allyl)-1-(3,4-dimethoxybenzyl)-1/7-benzo[c(]imidazol-3-ium bromide (CKP50)

CKP50 was prepared according to Step 7 using CKP24 (0.020 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-chlorobenzene (0.022 g). Pure compound was obtained as white solid, 0.025 g (70.2% yield); Mp: 172.1-173.9 °C; ¹H-NMR (300 MHz, DMSO-cfe): 63.75 (d, 6H, J = 6.1 Hz), 5.34 (d, 2H, J = 6.1 Hz), 5.67 (s, 2H), 6.61 (m, 1 H), 6.84-7.01 (m, 2H), 7.10 (d, 1 H, J = 8.3 Hz), 7.24 (s, 1 H), 7.37-7.59 (m, 4H), 7.67 (m, 2H), 8.05 (m, 2H), 9.92 (s, 1 H); ESI-HRMS (m/z): [M-Br]⁺ calcd. for C25H24CIN2O2 419.1521 ; found 4190.1517. EXAMPLE 22

(E)-1-(benzo[d][1 ,3]dioxol-5-ylmethyl)-3-(3-(4-chlorophenyl)allyl)-1/7-benzo[c(]imidazol-

3-ium bromide (CKP58)

CKP58 was prepared according to Step 7 using 1-(benzo[d][1 ,3]dioxol-5-ylmethyl)-1/7- benzo[d]imidazole [prepared according to Step 1b using 1/7-benzo[d]imidazole (0.030 g) and 5-(bromomethyl)benzo[c(][1 ,3]dioxole (0.053 g)] (0.035 g) and (E)-1-(3- bromoprop-1-en-1-yl)-4-chlorobenzene (0.039 g). Pure compound was obtained as white foam, 0.06 g (90% yield); Mp: 190.0-191.0°C decomposition; ¹H-NMR (300 MHz, acetone-cfe): <55.57 (d, 2H, J = 6.6Hz), 5.96 (s, 4H), 6.80-6.85 (m, 2H), 7.11-7.16 (d, 1 H, J = 15.6Hz), 7.29-7.35 (m, 4H), 7.53-7.65 (m, 4H), 8.03-8.15 (m, 2H), 11.20 (s, 1 H);

ESI-HRMS (m/z): [M-Br]⁺ calcd. for C24H20CIN2O2 403.1208; found 403.1202.

Table 1. Structures of the compounds of examples 1-22.

Tested compounds were dissolved and serially diluted in DMSO. Their cytotoxicity was assessed using an MTT assay. HEK293T, SH-SY5Y or Vero E6 cells were cultured in 96-well plates (4 x 104 cells/well) at 37°C in a 5% CO2 atmosphere. Cells were treated for 48hrs with varying concentrations of the compounds (100-0.1 uM) or solvent. The final concentration of DMSO in culture did not exceed 1 %. Treated cells were incubated for 4hrs with 0,5 pg/mL MTT (Applichem). After incubation, the MTT- containing medium was removed and formazan crystals were dissolved in DMSO. The optical density (OD) of formazan solution at 570 nm and 630 nm was measured in a SPARK plate reader (Tecan).

Results

Cell viability was measured after 48h using a MTT assay. Cells treated with DMSO served as control. Data are presented as a mean of triplicates ± SD. FIG. 1 shows the viability of HEK293T cells treated with the compounds of the present invention showcasing the non-toxic profile of the compounds.

EXAMPLE 24

Cell-based assay for the interaction between Spike RBD and ACE2 receptor (aa19-615)

The effect of compounds in the interaction of Spike RBD and ACE2 receptor (aa19-615) was assessed, as previously described (Trepte P. et al. Molecular systems biology 2018, 14, e8071). HEK293T cells were transfected with plasmids encoding PA- mCitrine-S RBD and c-myc-NL-ACE2 receptor (aa19-615). The next day, compounds were added in varying non-cytotoxic concentrations and BRET/cBRET signals corresponding to Spike RBD/ACE2 (aa 19-615) protein interaction was quantified 48h later. The non-specific/auto-fluorescence signal of each compound was calculated and subtracted from all relevant interactions. For dose-dependent assays, HEK293T were treated with decreasing concentrations of compounds for 8h. The % inhibitory effect of compounds was calculated compared to control cBRET signal in cells treated with the solvent.

Results

FIG. 2 shows the effect of the tested compounds on Spike RBD/ACE2 (aa19-615) interaction using a cell-based quantification assay. Interaction of PA-mCitrine-Spike RBD and cmyc-NL-ACE2 (aa19-615) proteins was quantified in HEK293T cells in the presence of the tested compounds used at non-cytotoxic concentrations. The cBRET signal corresponding to Spike RBD/ACE2 (aa19-615) interaction was quantified 48 h post treatment. Data are presented as a mean of triplicates ± SD.

EXAMPLE 25

SARS-CoV-2 viral assay

SARS-CoV-2 (isolate 30-287) was obtained through culture in Vero E6 cells (ATCC® CRL-1586) from a COVID-19 patient in Alexandroupolis, Greece. The virus was recovered from a nasopharyngeal swab, after rinsing with 1 ml saline and double filtering through a 0.22 nm filter. Viral stock was prepared by infecting fully confluent Vero E6 cells in DM EM, supplemented with 10% fetal bovine serum (FBS) and 1 % antibiotics (penicillin-streptomycin) at 37 °C and 5% CO2. Four days after inoculation, the supernatant was frozen at -80 °C until use.

Infections were carried out in 24-well plates, using SARS-CoV-2 (M.O.I. of 0.01) on Vero E6 cells. Before cell treatment, the virus was pre-incubated for 30 minutes at 37 °C and 5% CO2 with the chemical substances (final concentration of 25 pM in DMEM). After the initial incubation, the medium containing the corresponding chemical and the virus was used to treat Vero cells for 48h. As controls, cells were treated only with SARS-CoV-2 or were left untreated for 48h. Cell morphology was observed by phase contrast in an inverted microscope in order to record cytopathic effect (CPE) after treatment. To determine the SARS-CoV-2 viral load, RNA was extracted from 24-well supernatants (100 pl) using NucleoSpin Dx Virus kit (Macherey Nagel). Multi-target realtime PCR was performed using COVID-19 SARS-CoV-2 Real-TM kit according to the manufacturer’s instructions (Sacace Biotechnologies).

Results FIG. 3, shows (A) Viability of Vero E6 cells treated for 48h with selected compounds; (B) Cytopathic effect (CPE) of SARS-CoV-2 in Vero E6 cells treated with selected compounds at non-cytotoxic concentrations. All compounds suppressed the CPE of SARS-CoV-2; (C) Quantification of SARS-CoV-2 viral load in the culture medium of cells treated for 48h with selected compounds; (D) CKP-25 suppresses SARS-CoV-2 CPE in a concentration dependent manner compared to the solvent DMSO (IC5o=5 M). All data are presented as a mean of triplicates ± SD.

Claims

1. A compound according to Formula (I) or a pharmaceutically acceptable salt thereof, wherein

Q is selected from N, N⁺(X')CH2R⁴, X is selected from Cl, Br, when Q is N, R¹ is selected from OCH3, NO2, OH, NH2, NHC(O)CHs, and when Q is N⁺(X)CH₂R⁴, R¹ is selected from H, OCH₃, NO₂, OH, NH₂, NHC(O)CH₃,

R² is selected from OCH₃, NO₂, OH, NH₂, NHC(O)CH₃, H,

R³ is selected from

R⁴ is selected from

R⁵ is selected from H, F, Cl, Br, CH₃, CN, OCH₃, SCH₃, NO₂, OH, NH₂, N(CH₃)₂, NHC(O)CH₃, SO2NH₂, SO₃H, B(OH)_2I

Z is selected from CH₂, NH, O, S.

2. The compound according to claim 1 , wherein Q is N and R¹ is selected from OCH₃, NO₂, OH, NH₂, or Q is N⁺(X’)CH₂R⁴ and R¹ is selected from H, OCH₃, NO₂, OH, NH₂.

3. The compound according to claim 1 or 2, wherein Q is N and R¹ is selected from OCH₃, NO₂, NH₂, or Q is N⁺(X’)CH₂R⁴ and R¹ is selected from H, OCH₃, NO₂, NH₂.

4. The compound according to any one of the preceding claims, wherein R² is selected from OCH₃, NO₂, H.

5. The compound according to any one of the preceding claims, wherein R² is H.

6. The compound according to any one of the preceding claims, wherein R³ is selected from

7. The compound according to any one of the preceding claims, wherein R³ is

8. The compound according to any one of the preceding claims, wherein R4 is

9. The compound according to any one of the preceding claims, wherein Z is selected from CH2, O.

10. The compound according to any one of the preceding claims, wherein Z is CH2.

11. The compound according to any one of the preceding claims, wherein R⁴ is

12. The compound according to any one of the preceding claims, wherein R⁵ is selected from H, F, Cl, Br, OCH3, NO2.

13. The compound according to any one of the preceding claims, wherein R⁵ is selected from Cl, NO2.

14. The compound according to any one of the preceding claims for use in the treatment of a SARS-CoV-2 infection.

15. A pharmaceutical composition comprising as active ingredient a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.