LU102310B1 - Inhibition of virus protease - Google Patents

Abstract

The present invention relates to a composition, comprising at least one compound according to formula (I) for use in the treatment of diseases caused by betacoronaviruses as well as to a composition comprising at least one compound according to formula (I) and least one compound according to formula (II) which are bonded together via one or two linker systems.

Description

New LU Patent application Applicants: Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Jagiellonian University Our ref.: HEL17245LU LU102310 1 Inhibition of virus protease

TECHNICAL FIELD OF THE INVENTION The present invention relates to a composition, comprising at least one compound according to formula {lI} for use in the treatment of diseases caused by betacoronaviruses as well as to a composition comprising at least one compound according to formula (!) and least one compound according to formula (Il) which are bonded together via one or two linker systems.

BACKGROUND ART

[001] Coronaviruses have been considered a potential threat since 2002, when the SARS- CoV virus emerged in southern China to spread through continents and disappear shortly thereafter'“ rapidly. Ten years later, the second coronavirus — MERS-CoV ~ gave a final warning to be prepared’. Still, the emergence of the SARS-CoV-2 and subsequent pandemic met the public unprepared and paralyzed the modern world in an unprecedented way. At present, 1.4 million fatalities, have already been crossed and the northern hemisphere is facing the long the winter season, which due to the appearance of other comorbidities (low/dry air, coinfections, pollution, dysregulated immune responses) may still increase these numbers drastically” ®.

[002] While the worldwide hunt for a vaccine is bringing optimistic news, a timeline to an effective one remains to be unkown, and the dark horses face issues with the safety’. At the same time, worldwide efforts are undertaken to design and develop new antivirals”. While the development of novel antivirals is essential, realistically, the drug development timeline is too long to hope for the completely new compounds to find their way to the clinic during this season or even this pandemic. The new compounds are developed to become the future drugs for pandemics that are yet to come. Pragmatically, the focus should be and has been given to the repurposing of existing drugs with known safety profiles. The initial hits did not fulfill the hopes, and several inhibitors were already abandoned, e.g., the HIV-1 protease inhibitors lopinavir and ritonavir. Some others are still in clinic, but no evident proof of effectiveness has been provided. While there are high hopes given to the remdesivir, results of the clinical trials show that its efficacy in monotherapy is low. On the other hand, it is already speculated that identification of other antivirals effective against SARS-CoV-2 may give a chance to use the strategy developed for the HIV-1, employing several compounds targeting different molecular targets. In such a way emergence of resistant mutants is limited and the synergy of action allows for effective limitation of virus replication.

[003] The coronaviral genome encodes for several structural and non-structural proteins. The most promising targets are the Spike (S) protein, as the target for neutralizing antibodies and entry inhibitors, the nsp12 polymerase, targeted by several compounds including the remdesivir, nsp14 and nsp16 methyltransferases essential for the capping of viral RNA, and two cysteine proteases M” and PL", responsible for the viral proteome maturation and indispensable for the infection. PLpro is aiso responsible for type | interferon response attenuation.

[004] There is a need for further therapeutics for treatment of a SARS-CoV-2 infection. A 102310 potential target is SARS-CoV-2 PLP™.

SUMMARY OF THE INVENTION

[005] The present invention is related to a composition comprising at least one compound according to formula (I) Ry R Sy “WE H x | H R, (1) R, is selected from the group consisting of (C,-Cs) alky!, preferably methyl, preferably H; R. is selected from the group consisting of H, (C,-C;) alkyl, preferably methyl; preferably H; R4 is selected from the group consisting of H, (C,-Cz) alkyl, preferably methyl; preferably H: X is an anion; for use in the treatment of diseases caused by betacoronaviruses.

[006] A composition in which at least one compound according to formula (1) and at least one compound according to formula (Il) are bonded together via one or two linker systems wherein optionally a) the linker system is (Cz-C1c)alkyl, preferably (C,-Ciy)alkenyl, preferably (C;-Cajalkyl, or (C3- s)alkenyl; wherein optionally at least one or at least two CHz-groups in these alkyl or alkenyl groups are substituted by O, S, S(O)h.2, NH or N(C,-C4)alkyl and/or b) connecting is performed preferably via position 5, 7, 8 or by substitution of the nitrogen in position 6 in formula (I) and position 5, 7, 8 or by substitution of the nitrogen in position 6 or 10 in formula (Il) wherein the underlying aromatic system in formula (J) and (ll) is numbered a 7 “ © LU 5 : Tee "N° 177 - according to formula (IV): + CE (IV) As shown in the examples, the inventive compounds black the replication of the selected betacoronaviruses, including with nanomolar ICs, showing high selectivity and good effectiveness {see Figures 8 to 14).

BRIEF DESCRIPTION OF THE FIGURES

[007] Fig. 1: Inhibiton of PL" in the presence of ACF. (A) AMC assay using RLRGG-AMC as substrate and PL” performed in technical triplicates. Vertical Y-axis shows fluorescent signal (rising, as the substrate is proteolytically cleaved). Horizontal X-axis indicate time. An inhibition profile is observable. (B) As in (A), only with ISG15-AMC instead of RLRGG-AMC (C) Time course analysis of tri-ubiquitin K48-linked (2 pM) hydrolysis using 100 nM PL"? in the presence of different ACF concentrations.

[008] Fig. 2: The inhibition of virus replication by selected compounds. Figure shows RT- gPCR analysis of cell culture supernatants infected with SARS-CoV-2; tissue culture infectious dose (TCIDs,) = 1600 /mL) with presence of compounds after 48 h of infection. Experiment was performed in duplicate. The results are presented as average values with standard deviations (error bars).

[009] Fig. 3: The crystal structure of SARS-Cov2-PL"" in complex with proflavine. SCoV2- PL" is represented as a solid surface, whereas proflavine is represented as a stick model. (A) a zoom-in of the two proflavine molecules in the S3-S5 pockets of the enzyme active site. (B) a zoom-in on a proflavine molecule between two crystal neighbors of PLP.

[0010] Fig. 4: Details of the molecular interactions between the SARS-CoV2-PL™® and proflavines. (A, B) Both proflavine molecules bind cooperatively in the substrate pocket of the enzyme forming a non-covalent inhibitor. Both polar (gray) and hydrophobic (red) interactions as well as 1-1 stacking (green) are involved. Main residues involved in the interactions are shown as a stick-model. (C, D) 2D plots of molecular interactions between proflavine-|, proflavine-Il and PL°°. Each proflavine molecule uses different properties for form interaction. The aromatic nitrogen of proflavine-l must be desolvated for binding. This explains the higher affinity of acriflavine than proflavine as it contains N-methylated components.

[0011] Fig. 5: Both proflavine molecules mimic native substrate interactions. (A) Comparison of SARS-CoV2-PL°° complexed with proflavine (in gray) and iSG15 host-cell substrate (in cyan; PDB ID:6YVA). The active site is marked by a dashed oval. (B) Substrate recognition cleft interaction of proflavines and the C-terminal tail of 1ISG15. Proflavine molecules overlap with the RLRGG motif of the substrate. The two arginines in P3 and PS5 turn their lipophilic carbons in the same directions as the aromatic carbons of the proflavine-ll molecule in the 53-55 pockets. The amide nitrogen H-bond donors of the peptide backbone correspond well with the proflavine-Il donors and the interactions are well preserved. The side chain of the leucin in position P4 points exactly of the proflavine ring in the S4 pocket.

[0012] Fig. 6: Overlay of 'H,°N TROSY NMR spectra of PL” with different amount of ACF added. The entire spectrum is shown in the middle, magnification of a few regions above and below. A number of peaks shift with the addition of ACF. The bulk of the spectra remains similar to the reference apo-PLP®. This indicated that the overall fold of the protein is intact and the compound binds in a distinct binding pocket.

{0013] Fig. 7: The cytotoxicity of ACF in vitro. LU102310

[0014] Fig. 8: The inhibition of virus replication by ACF in A549%°F?* (A) and Vero (B) cells. Figure shows RT-gPCR analysis of cell culture supernatants infected with SARS-CoV-2, tissue culture infectious dose (TCIDse) = 1600 /mL) with presence of ACF after 24 h of infection. All experiments were performed at least in 3 biological repetitions, each in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (p< 0.05) indicates values that are significantly different from the control.

[0015] Figure 9: The inhibition of virus replication by ACF in Vero cells. Cells were infected with the SARS-CoV-2 in presence of Acriflavine for 48 h. Images show the reduction of cytopathic effect. Scale bar = 100 um.

[0016] Figure 10: The inhibition of virus replication by ACF in A549*°°?* cells. Cells were infected with the SARS-CoV-2 in presence of 500 nM Acriflavine, 10 pM Remdesivir or vessel control for 24 h and 48 h. Cell nuclei are denoted in blue, actin is denoted in red and SARS- CoV-2 N-protein is denoted in green. Each image represents maximum projection of 5 pm section. Scale bar = 20 ym.

[0017] Figure 11: Antiviral activity of ACF against SARS-CoV-2 in human airway epithelium. (A) In-house HAE (B) MucilAir™. The figures show RT-gPCR analysis of HAE culture supernatants infected with SARS-CoV-2; tissue culture infectious dose (TCIDs0) = 5000 /mL). Remdesivir and PBS were used as controls. The assay was performed at least in duplicate and median values with range are presented.

[0018] Figure 12: The inhibition of virus replication by ACF in well differentiated HAE cultures. Cells were infected with the SARS-CoV-2 in presence of 500 nM Acriflavine, 10 uM Remdesivir or vessel control. At day 6 p.i. cells were fixed and immunostained. Cell nuclei are denoted in blue, actin in red and SARS-CoV-2 N-protein in green. Each image represents maximum projection of 3 um section. Scale bar = 10 um.

[0019] Figure 13: Time of addition study. The inhibition of virus replication in Vero cells by ACF added after post-infection (time denoted on the x-axis). Figure shows RT-qPCR analysis of cell culture supernatants infected with SARS-CoV-2; tissue culture infectious dose (TCIDsy) = 1600 /ml). All experiments were performed at least in 3 biological repetitions, each in triplicate. The results are presented as average values with standard deviations (error bars). An asterisk (p <

0.05) indicates values that are significantly different from the control.

[0020] Figure 14: ACF inhibits betacoronaviruses. Replication of (A) MERS-CoV, (B) HCoV- OC43, HCoV-NL63 (C) and (D) FIPV in vitro in the presence or absence of inhibitors, as assessed with RT-gPCR on cell culture supernatants. Single round of infection was recorded {24 h). All experiments were performed at least in 2 biologicai repetitions, each in triplicate. The results are presented as average values with standard deviations (error bars}. An asterisk {p <

0.05) indicates values that are significantly different from the non-treated control. ACF: acriflavin, rem: remdesivir.

[0021] Figure 15: Proflavine molecule at the interface between SARS-CoV2-PLP® asymmetric units. (A) Most probably due to a crystai packing molecule of proflavine was found on top of the catalytic triad (C111, H272, D286). Important residues are highlighted as stick model. Two water molecules (red spheres) mediate a hydrogen bond with D286. Hydrogen bonds are represented as yellow dashed lines. (B) 2D plot of the molecular interactions between proflavine and {1102310 residues of SARS-CoV2-PL°".

[0022] Figure 16: . Comparison of SARS-CoV2-PL°" in the proflavine-bound and apo-state. Bound PL" is colored in gray; whereas the unbound PL" (PDB ID: 7D47) is colored in yellow. The BL2 loop is involved in an induced fit rearrangement upon the binding mostly due to the movement of the Tyr268. The side-chain of Tyr268 participates in a TT-T7 stacking with proflavine molecules.

[0023] Figure 17: Electron density map showing the fractional presence of additional aromatic proflavine-like molecules -1r stacked one on top of the other between two copies of SARS- CoV2-PL°® present in the crystal lattice. 2F,-F. electron density map is contoured at 2CE. The electron density of the identified proflavine molecules is colored in blue; whereas additional electron density is colored in green. The densities are most likely caused by weak and transiently-bound proflavines. We did not model them in the crystal structure as their electron density was much weaker than active site-bound molecules.

[0024] Figure 18: SARS-CoV-2 MP” activity is not significantly inhibited by ACF. The digestion of fluorogenic substrate was recorded in the absence and presence of ACF (A). Only small decrease of MP” activity is observed at physiologically irrelevant ACF concentration of 100 uM (B). The vertical shift in signal levels is caused by ACF absorbance.

[0025] Figure 19: (A) Summary of the high-throughput screening campaign (16 x 384-well plates). RLRGG-AMC peptide was used as a substrate for His-PLPro. Each dot represents the data of one compound in one well (n = 1). Controls without protease are located in column 24 and ACF as representative hit is depicted in blue. (B) Two representative screening plates with ACF and Proflavine Hemisulfate as hits (blue) are depicted side by side. (C) Dose response curves of eleven hits. These hits were re-ordered and re-tested in ten-point titrations on the primary screening assay (mean + SD, n = 2). Shown are inhibition curves and IC50 values. (D) as in (C} Analogs of ACF were tested in ten-point titrations on the primary assay (mean + SD, n = 3). (E) Inhibition curve of the known PLPro inhibitor GLR-0617 (mean + SD, n = 3).

[0026] Figure 20: Kinetic assays of His-PLpro DUB activity in the presence of different concentrations of ACF. RLRGG-AMC in (A) and ISG15-AMC in (B) were used as subtrates. Reactions were performed in triplicates. (C) Time course analysis of tri-ubiquitin K48-linked (2 UM) hydrolysis using 100 nM His-PLP°.

[0027] Figure 21: Composition of commercial acriflavine (CACF) sold by Sigma Aldrich (A8251): 56% proflavine (PF), 17% acriflavine (ACF) and about 26% of their side methylated derivatives.

[0028] Figure 22: Map of pETM-5a.

Co PAR ER ETERS FE LIES TRE EEE IR LU102310

DETAILED DESCRIPTION OF THE INVENTION FER € Fine As Sie A «3 NS Foie =; he NF pes hs À ~ ù Any JEL IT a RE fiflgsart Live tiens (00381 The solution of the present invention is described in the following, exemples in he appended examples, Huataisd in he Figures and reflscied in the claims STE $ RY oh F003 Definitions EX REY See repaire x - card oie Eben evienens sieve Ba Cent Sayan sa Te fener artery vatsrent ISO is nolud that as used herein, he singuie forma "a", “an”, and Ihe”, includes plus tnd atti tte ts cersieueses Mises a ar Iscadinveutsess His sexes Tina For oe on fy N fe Ÿ pigrances unless Ihe context ceary Ingioatey ofherwise, Thus, for sxample, reference (6 a crore dT Reveals sets pS SY MRT § ave vie SE rn enh daa swiss NEE ENE ah ee Ne Ben Tee ayy ti SEE aa pended an regent’ includes one or more of such Cilleranl reagents and referenda Tha method includes ot; A evo ciao oF een = ¥ parent bent er Lrremases Fa coger PEE eNienenues are DEE Los Sem met Sy ae tstgrence to equivalent siens and methods known io those of ordinary SAIS in ha art hal COUR evs Ivrea a Leu Sra ong inary dened Saas EH se cuted stasis st Mare Le modified ar substitute for ihe methods described Harein.

SES SES 3 3 onde N N aa a An A Stand Lace TE ae pri ge NES ~ CAT ad = ae im Ten a {R033 Urdess othanndss rrfcated, the term “ai least” preceding à series of elements is Io be ened enero enn et Fan Tan des SCE RE nie ed ire FE dan ete hie wns qui i end Len Tic of {BR smpmoynires me het understood tp refer to every clement In the series, Those skilled in the art will rooognize, or be Fler Ts spose doin) NI TSATY RN a TH ma N AS BEY RY NÉ ang pay biens oy LE One ais een fon dla prier AXE is Sscerain using no more than roufine axpertnertation, many equivaiante ID he Space pv ES Fe oh ns o es mi N Ey mari witty taney dee oo Data è 3 on Bas émbodiments of the bpeantion deserihen herein, Such SquvSEms are menden I be STTUITIDESSEN by the prosant mvantian. 3 Er Then Tans Wan yom} Fri 4 Dette ROA ora rad Ha Lex Sees Ness TP SAN : “Ë 5 GN Warf arret Wendl sp TRI] The term "and/or" wheraver used herein includes the meaning of Tang’, or” and Tall or . tree Srey INE sen Aen NF PIN ond TE TREY SNE EY en Sr hen) espere any other combination of the elements connected by said term”. 7 PES CE Se re J A J oy ey aE ey cand Pine, fon Save on pis foliose + = es Eve Ÿ A Eyed savons Tree 19H34] Throughout this specification and the olabms which follow, unless Ihe conta guiness von N A Epa a Less Nr Wau et steer ent A NEN ces aN Ivete en ae ne Np SER ERY 5H Ba ciherwise, the wind "COTES, and vanialions SUCH RS comprises’ and "oompnang , wi SS con ofan gman te oF Fay Penryn an Ge $ iors ca ey a 8 laden - SPIT PRES NERD SNE tendon enamine DONNE Sv id NE unglersigns fo Impä the nclusien of à slated integer oF stag oF groups of lagers Or SIEDS DUT ne aan narod rey heure Sd = ANA TNE per te NE eg SN SE Sree et SN TS ES ya ar Buena then Fore the SRCISION oF any offer Infeger OF slap DT group of Integer or sien. Wha used Rare the term Sansa ad en an a = à : y toast NES zn AR I nh BE 2 na Trenand cat ca net ya rene Shey “porgrisine” can he subalfoted with the teem COHEN oF TInsluding oF sometimes whan x od SATE 3 dan Fewest Sara” AFR eat code yet Top Lextignet oof oowesdy adores mme aan gast used herein with he ler “having”. When used hers “consisling off axciudes any element, sites. of rrypeaient nat specified NIE, HIT RGHEN THA RSS, FREIES hen Toney Ponies ait Sn SoU on rcmanon een beret NE dy puytan Fron ter dng pers om reread Frartennnebe (86281 The form “alkyl” refers lo à monoradics! of à saturnaied sirsight or branches hydracarbon. N A SA EN ne Kanada TET N oy dens im » Sor SR N SOT RZ HN Fraferably the aikyt group comprises from Tie 10 carbon atome, Le. 1,2 3 4A 88 FT 88 8 SEL YY aaa x AS a > + Len IN sand era ne = A fn A re TFUE VE ES 3 ny carbon atoms, more preferably Tio 5 carbon atoms, such as 1 lo 4 ar Te 2 carbon ais Eflaenyavermisnnts miiresf pravraieees Saar sien aaa a abbey ed an de on 3 board Seven bag add bene® dg odes Exemplary alkyd groups include mistihnd, ei. propyl, iscepropyl, bul, isobutyl larkhall à yo fx ab Ie ry foal vaya eu EE EE ea soordral À D eo iawn dha N dan pny st fem on Peauvrst mar pani IRosPentyt, Seo-pentst, mao-peript 1,2-cimaihyi-proDyt, sound, A Hex, soda, sae ern ST oufresol_ races mi Fost doves mu a hi TT andra dl Fran ears ee aa Piven dre Boned, Sathiduegl, shop, schen mach! Seti hekvi not n-decyl ard Ihe de FINS 2 TV Ren Ae CW Hae an I sn Fins Tian od avenge band wads 24e cod avionics Fewest nm red À SR ENN SR Py 100381 Thy term "alkylans” relers Io à diradical of à Saluraied straight or branched hpdrocarban. trmtoarabhs fix eines sven seein ane Seavert À Ha SY net Sie 1x SOS 9% N N OS SP N N A TN Froforabie the aikyiene comprises fan 1 In 10 carbon aloms, La, LEE 8 8 T0 creer ey aire PRR arenes KEN Pen BF Nuits era avesendy se ct fey BY ove daS ur odes carbon atoms, wee praferaniy T fo 5 oardon RENTE, such as 1 D 8 or 1 10 4 carbon stom, Yan avs ex hase ayo Hy otis NEN dra deen ti code aR Eg any a en Ey aa FI » 4 4 ad TS ROY three eben en À EXAMEN SKVENS groups Noibde methine, silanes fa, 1. 1e0udens, LU Seihviane, Frees denses di x = + pare Hogptas N NN deans FONE SRE OISE x 5" sAmriteeies FETE INE LS seed progyviène Le, 1 -5ropyiens, La organ COMICHOOH], 2. propyièane GEHL and MN hen Shen reed Fy ot EE St Frere ÿ CAN DE RS = À brrrtcaie ” 8 PS Par His Ne ER NN Ldgrapviena) the Luiviene amare leu, 1, Tbuiyimne, 1 3butvleng, ZA S-Dutylarnıs, 1.3 Ara as CU OS Ear ah Sonia ones Beata NE on De deve Tia yt E A dns NN À A Yow octave 1 Dies Dityisne, à Sbutyisne {ris or Fans or a mikturs heread), Td butvieng, 1, {-sn-butyiene, 1,2-80- Set ade od anna OR ON tae 8 À en à 3 Se OLY ENR STONY LEN en SEN Fan ew RS prepare tieian arret - Nanna Ea olen w 3 Guiyiaris, and 1, 3-aedudviane) the penddone kamera fag, 1,1-pentyfons, 1. Spender, TS ana Saas % of spexiratenenas N anette el sana FN Veen A odors Rd pass En MW eran prada ad) tae pentyiens, 1,4-peniyièens, Hoorn, 17—c-ponirisne, 1,1-sec-mentiyt 1 assed), Bw hexylenisomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1-isohexylene), and the like. LU102310

[0037] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 4, ie, 1, 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 10 carbon atoms, i.e, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen-1,2-diyl, vinyliden, 1-propen-12-diyl, 1-propen-1,3-diyl, 1-propen-2,3-diyl, allyliden, 1-buten-1,2-diyl, 1-buten-1,3-diyl, 1-buten-1,4-diyl, 1-buten-2,3-diyl, 1-buten-2,4-diyl, 1-buten-3,4-diyl, 2-buten-1,2-diyl, 2-buten-1,3-diyl, 2-buten-1,4-diyl, 2-buten-2,3-diyl, 2-buten- 2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.

[0038] The term "cycloalkyl" represents cyclic non-aromatic versions of "alkyl" with preferably 3 to 6 carbon atoms, such as 3 to 6 carbon atoms, ie., 3, 4, 5, or 6, carbon atoms, more preferably 5 to 6 carbon atoms, even more preferably 6 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0039] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 4, ie, 1, 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 10 carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyi (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-hepteny!, 5- heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1- decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9- decenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. LU102310

[0040] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 4, ie, 1, 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 10 carbon atoms, ie, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 10 carbon atoms and 1, 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen-1,2-diyl, vinyliden, 1-propen-1,2-diyi, 1-propen-1,3-diyl, 1-propen-2,3-diyl, allyliden, 1-buten-1,2-diyl, 1-buten-1,3-diyl, 1-buten-1,4-diyl, 1-buten-2,3-diyl, 1-buten-2,4-diyt, 1-buten-3,4-diyl, 2-buten-1,2-diyl, 2-buten-1,3-diyl, 2-buten-1,4-diyl, 2-buten-2,3-diyl, 2-buten- 2,4-diyl, 2-buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.

[0041] The term "aryl" or "aromatic ring" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 6 to 10 (e.g., 6 to 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Wherein “-(C;-Cs)alkyl(Cs-Cio)aryl” means that an aryl group comprising an alkyl substituent is attached to the overall molecule via that alkyl substituent.

[0042] The term “heteroaryl” means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of © and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), tetrazolyl, thiazolyl, isothiazoly!, thiadiazolyl {1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), benzofuranyl (1- and 2-), indolyl, isoindolyl, benzothienyl (1- and 2-), 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl (1,2,3- and 1,2,4-benzotriazinyt), pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl (1,5-, 1,6-, 1,7-, 1,8, and 2,6-), cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-),

phenazinyl, oxazolopyridinyl, isoxazolopyridinyl, pyrroiooxazolyl, and pyrrolopyrrolyl. Exemplar 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadinadil 102310 (1,2,5- and 1,2,3-), pyrrolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3- and 1,2,4-), thiazolyl, isothiazolyl, thiadiazolyl (1,2,3- and 1,2,5-), pyridyl, pyrimidinyl, pyrazinyl, triazinyl (1,2,3-, 1,2,4-, and 1,3,5-), and pyridazinyl. Wherein for example “-(C,-Cg)alkyl(Cs-Cig)heteroaryl” means that a heteroaryl group comprising an alkylsubstituent group is attached to the overall molecule via that alkyl substituent.

[0043] The term "halogen" means fluoro, chloro, bromo, or iodo; preferably chloro, or fluoro, more preferably fluoro.

[0044] The term "complex of a compound" as used herein refers to a compound of higher order which is generated by association of the compound with other one or more other molecules. Exemplary complexes of a compound include, but are not limited to, solvates, clusters, and chelates of said compound.

[0045] The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non- stoichiometric. A "hydrate" is a solvate wherein the solvent is water.

[0046] In isotopically labeled compounds one or more atoms are replaced by a corresponding atom having the same number of protons but differing in the number of neutrons. For example, a hydrogen atom may be replaced by a deuterium atom. Exemplary isotopes which can be used in the compounds of the present invention include deuterium, "'C, "°C, "“C, PN, "PF, ¥s, CI, and ‘#1.

[0047] The present invention relates to a composition comprising at least one compound according to formula (}) Rz AE Ra Sy NF NT H X | H Ry (1) for use in the treatment of diseases caused by betacoronaviruses.

[0048] R, is selected from the group consisting of (C;-Ce)alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec- butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl {iso-pentyl oder iso-amyl), 3- methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methyipentyl, 3- methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl.

(00481 À, ls selected from tha group consisting of {GG prafanably saletiad from he group consisting of methyl ethyl, prog, tut, panty, iso-propyt sec-propyt, shy ses tall, tert-hütyl, 2-pentyt, S-centyt, =-Methyibuiyt, I-methybutyt (Ho pantyt oder seam), # methyltut-0-yt, ZemathyibubZad à 2 dimebag, had, Shard, han, Z-methylpentst 3 methyipertyt, 2 Secimelhyibutil, and 2. 3-dimathylbutyl, more praferably math (00507 Ry le selected from the group consiating of {0,-Ogikyl, preferably seleciad from the group consisting of methyl) athsi, propyl, bub, peniyt, iso-propyil, SSc-prpyt, fso-buly, sec buts, tert-butyl, Zopentyl, S-peniyt, Smattpdbabyl, 3-melhythriyt fsepenhd oder Bean, > Meihyioul- Zul, THE VBUÈEUE, à -dimethyipropyt, hel, Shay, Shexyl, 2-meithytpentyt, 3 methyipentst, 2,2-dimeihyibulyt and 2,3-dimelhylbulyl, more preferably mathe. {O81 Xan anion HORE] À 5 preferably seloclad Fam the group consisting of napeyials, aiycaliylarsaniate, nitrate, benzoate, Haxyhasormoinais, oleate, bierfrate, hycrpamaphihoate, pantothenate, dicartonais, hydratermine, pamoals, carmsyiate, sethionais, polygalacluronats, propionals, satoylaie, laciobioraie, stearate, decanozte, adetnie, ralsais, sucoinels, esipiate, mancetate, izoclaie, qlucoptais, acutate, giulamale, muscals, separate, ghyoolate, bermenesuffonaie, hexanoaie, solanoale, sulfonate, oidonde, iodide, bromide, phosphate, phosphonate, lantais, maiate, citraie, tartrate, fumarate, gluconate, suffais or hemisaïiaie, more preferably chorkie, txiide, bromide and sulfonate, most preferably chorkie. [G0831 In one embcotimant, the treatment is caused by human and veterinarg coronavirtises that belong fo subgeners hibacovirus, nnbecovrus, embecovrus, merbecovrus and SRIDOCOVTUS, praferably coronaviruses, SO) In another embodiment, the treaiment 5 caused by human coronavirus HU {HG HK) human coronavirus D043 (HOOV-QC43) Middle Bast respralory syndrome-ralstecd SNTDIAVTUS (NERS-COUT (00581 {In & further embodiment, he treatment à caused by severe auvle respratory syndromeasrataled coronaviruses, pretoratdy SARS-CoV, more preferably SARE-Cov5. 15688] In another embodinent, te treatment Is caused By 8 virus ha svoive or mutals from fre species described In the Ihres paragraphe above 198871 in anoîher eitodiment In tha composition here ars at least bro compounds accordine to formula J) present.

[0088] Preferably in the frst compound {fai | Reis (0 0getot, preferably selected from the group consisine of mein, sited, propyl, bubd, anil, SOTGEN, sec-propyl, lso-hulyt, sec-butyl, terb-butyl, Z-pentyt, 3-pentyt, E-methylbutel, 3- methytbutyt (ko-neniyl oder soamyll Iumethylbut-2.yi, Qmethylbutb lad, 22-dimeihyipropst, apd, R-haxyl, Zhexyl, 2-methylpenty!, 3-melhylpentyl, 2'2-dimelhylbuty! and 2,2-dimelhylbutyt, mors preferably methyl Rp and R, arc Band in

[0059] the second compound (lb) R; is (C,-Cc)alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tr 102310 butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (iso-pentyl oder iso-amyl), 3-methylbut-2- yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methyipentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl, and one of R; or R; in the second compound is {C4-Cg)alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2- methylbutyl, 3-methylbutyl (iso-pentyl oder iso-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2- dimethylpropyt, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl and the other one of R; or Ry is H.

[0060] The composition may further comprise at least one compound according to formula (Il) Ra Rs Re z Xx

AA Y N NHR;

[0061] (mn

[0062] Ra is H, halogen, (C,-Cg)alkyl, (C4-Cs)cycloaikyl, or -O(C,-Cg)alkyl; preferably H.

[0063] Rs is H, halogen, (C,-Cg)alky!, (Cs-Ce)cycloalkyl, or -O(C,-Cg)alkyl; preferably H.

[0064] Rs is H, halogen, (C--Cs)alkyl, (C,-C:)cycloalkyl, or -O(C,-Cg)alkyl; preferably H

[0065] R; is H, (C,-C;) alkyl, preferably methyl,

[0066] Z is H, halogen, (C;-Ce)alkyl, {Cs-Ce)cycloalkyl, -O(C+-Cg alkyl, -(C1-Ce)alkyl(Ce-C+o}aryl, - (C,-Cs)alkyl(Cs-C-0}heteroaryl: preferably H.

[0067] Y is -NH,, -NHRz, halogen, (C,-Cs)alkyl, (C,-Ce)cycloalkyl; preferably —NH,

[0068] Rg is H, (C,-C;) alkyl, preferably methyl;

[0069] In one embodiment, Rs is (C,-Cs})alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propy!, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (iso-pentyl oder iso-amyl), 3-methylbut-2-yl, 2-methyibut- 2-yi, 2 2-dimethylpropyl, hexyl, 2-hexy!, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2- dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl.

[0070] In one embodiment, Rs is (C4-Cs)alkyl, preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methyibutyl (iso-pentyl oder iso-amyl), 3-methylbut-2-yl, 2-methylbut- 2-yl, 2,2-dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2- dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl.

cinhing of GUD oonsming of add Ham the QUOUP COURSE vu CESR € ASS == o Sat el Farah aaipoisd Ham i Daran ty > Oclalkyl, preferably sales Stu, tert Duty, depend . © À, qui FARES es cape de WE, LES > ce 3 woe, Ry ie {DoCaltkeyt © wi, lac-buiyt, SER Dub, 1 2-mathytbut- ermibocirmeunt, SNS Ar à ara fi, RG Fs . Five at Dsl STH TH SY ; . GOT in one embodiment, LA, SOSE SOUTH 1, Fomeihdbub ZA = 3x TS ASF i fl vassal à CPAS penning) FE ERE A ct 5 5e 4 a od ARTS i 3 oo I IR RTA A ya gar Syd 3 + {reread TRIN, RTS ITS, _ ard a 20-21 + x oe tien YL À, x Aa BONS, DE 203 , SE figep rent FASO ; Severed SIREN math, ei. pray Wl A-mathylboi (so-panty L émelhyipenmst, 3m VI Is a N a ct FAVES : Fyn wed à ESS ESS yl S-ethythutyt, In L Sheol, Sex, Ze WI en en 3 NE ARES BN on «DS x da " STENT, À Iron, het Scheel, bby nen > 2 SAMSON, HER si mare preferatiy methyl $ Must BSR ; Shiu shyt THE FEAR ER indioney od F4 À, = d-cimetiy LERNER ES SE SOSSE 0 $ : NI OL mE = a Ha grads FNM SEN $ imaihyibuiyi, and 2.34 ty saisoled from the grag pent CHITIN Ih tare dye SEITEN ran tea Ay ch Rene AR pi + 1 resafars Ny SSH fret ae A sv SR fens prada vi, tert DE = A 8 (Ce kl, prefer hull ee ul ast 5 Sadie er LOR. de iE They FAT VE, à eich EN ahs aT. nde, Re 8 {Dole wrong, Schu, se ul 2a, Dornathndbut Frs FISH 4 SNES % + 721 In one ambodined, 1 ino-propyt, sec-propyt, Be -methibub OU € as 3 SEES aR ; aa in FQ SX SS co SIAR AP SEEN à VERS > . . REN 100727 in € soyt, Bul, pend, og fl oder seam) 3 TL erahnen. de ae ttn ruse KASSE, 8 het Sie adar Æ - a SPESEN misting, sil pray Jorhat Usenet L é-meaihyigenttt 3 oh THT, * tgyied SD ISRSSITS A aaa PIS RES SEE > Ps ; i. snsthvibudy, SR ; *% Bean STE, & Kerl Shatin frere KEN Hag Sep ve ; tyxdnreæret VERYI, = refers raethyl SE S-dhimelhvipromd, buiyi more prataraliy n dim nf Af den à 2 3<imeihylbuiyi, more . OUD consisting of ; ihyibuiyt and 3 0 -cimaiiy ed For the group cons A Si ever eV FX SIN FI, SEEN © ‘ sigepidgeet Te HS SN ’ = TATEN HIVE erably saipoted Fo bl, Spend, LAYER LY ‘aferaiiy SEE : SEEN SPL, ie Srafersiiy 8 at lért-Duivt, | $ a IS SU Heyl Fa fel = SE DU, IST ) Nya rar, Ryde {Ge Cl, 3 yi acedratyl, sec-butyl, 1 2-mettylbut- Ta > = N N EN ANSASTUM = Sun Sas oF od TIRE YR In one emboatiment, By 8 { pad, SED- Dry, edu 2 Sy GE pi, Done ; FEE NE SIT he ee se F Si S-melhçih > ; > ay 3 ang à YF, IRDSPPORYS, RST), emai > £148 EE" $ Es > Zst DEN 80 } ; ip fee STE, = Foginantisd hata 0 hvid Beaded FFF À owing RGN ES A beae SIE 3 rt, rand, bab, § ; ER oder Æ at SITET ; Ah Tran, O 3 : $e SII EMS | axed ed ATTRA : matind, ethyl, propyl, 3-methylbutyt (sepany Soetindpeniy, Sona TIFT, 3 > ; 3 Nel FTE HERS Wn SOTERERIYHSE > niv, 2-melhylbutpt, 3 L hey, 3 hexyl, = S-nenhi Sonal 3 FRE SS, fans Fla ER, cn al FIERY, x Sida avid 3 3 ya FRE iy FIVE, Lo dd dimeihvipramd, hs Bd, mos reload may ¥ > : LR DÉS IT THIRTY : SIT LEARNER bingy of Syl Zach FE Secret ya, money un consisting of * N EN TS . ¢ dha SQUE CONS > . Fy ates sève TR Hoan dust Toparix ths © FARES at dirsethyloutyt, a ferably setected fom the 9 butyl, Spent, $1353 ; ation, oraforably sade Bahl, herbbutst © FR . A ee kw pra LES = tt mies SSI, ETES “ x . si XY ix {hel Rk, | winced, see WARE DC Sieht.

embocdinent v ig 5, SEIT, ISG, Ey SE, Aie {OS741 In ong ambachm bel, iso-propyt, sen kata wit 3-moihyibute Fe Ga SR {RES S i oa ball nantyl SOS, Rr soar), domed “ Fired és Ÿ 3 . wi Ada PRY = beat mnt SSSR Sa rn at SEY, > i | A 3, _ Enh aia à RSR FIRE they, athyf, propyl, bul, ı Hreyfbutyi (open! ode Srndpentyl, 3-methyt metryt, othyl, dy, Sometndtudy (cs hexpi, Emethypentt, is dréibuitet Sun ; Jha, Smet NA. = -cnethwlbuitet, deena FENIX No vrenorir at DESSEN * eon ab SPRY $, x ein S-poriyt, em ned rvs hay, SEE, abi methyl | 22-dimethyipront. Suivi, more praforatiy matin * RY SA HT ~~. ue nibh mee # SISISÈINI of SV À, NN ETC EN ON COTSISIHTR | SF bl, and = d-cimeihyibuk Tae Group CONSHÈNS niet. ang 2.3 ad from he gro Se set im An = fornbly sateclad from me 9 hut, Spent Last me STE REY geval ay TUNEL NL - § po abo AAN ~ nad on SED SSP A : BET I 5 RNA FU, à Poisoned SE WERE = na te mbodiment, X Is {GG |, SEC-PTODFL, metab, ss thyfbut-2+f, 2-meityibut GSTSi NT ong smpodiment, tl, negro, seer, wl Imeihpibub ded, 2 ni 0m Et WEE M3 AN suivi, pend ise PEAR 2 P0 1 oder soa), dre hyipenip de hd, propyt, Dull, pend ac-peniyi oder Sosa] © S-maihylpentpt, Shan of aad Bical TI i ; 3 A CECA IN ; A N mein, stiyt, op, 1 S-mettyibuly! {io-penty Lo emeaihyipentst ZA EEE US, ; Ay REY TEEN FRSA 3 sarl ST REES SE Bl, Somethnibulyl Zen NE hawyl, Sheol, Sa a CITE IS * ; anon SEER, ) fy st S<peniyt ns ea het, Ks 3 ; ais Math. . adda € SI + ha aR "TN - at, 22-dimathyfprons bull, more preferathe m St, NASEN wy m4 Hrretryibutyt, RARE ÿ SF fog cnet an 2,3 clive = cimethyibubd, ang HY to 40 mors SHITTY 23 N ai 40 to SE, HIQTE ba 5 5 100, proferably 10 à ITT fen cud Ces ay he 5 in TD Tr ME fn FIL mrad ERR EN tar win TRY DED wands 153 ang PAR ‘81 The malar ratio of ig to formula (3) may d FH SOMPOUTRIS 113 ang Fe) Tha FRIAR 3 FOODNIPRI te FAT A at à of DANG DO ps TEER OTS DINGY OO TR {ment ma I QE PERSE A $ N 8 SONTOGUTNI 8 Si miotar mio east one COMP Ion he overaii mena: Ihe al lsat ang 1 moi based on he ave ANE ; SE MO RENAN N S ID AE TIME À GE beayogaed £1 STOTETEINF 10 85 mois Hate og PURER 2 et y 101085 mont À aie DRE erably Th in AE he comnnsidion ang AO iors Dreier [VY TUN WRT SONT pe Saye GREY tan {8 RS NS Sa & = WE WS WN 55 TERA TY nes De TR HSID SR chen it He CEHTISOSHOTT tit may be Din 55, pre is 1 and ti in the come Anca di RY NS € ’ TOON ET Hy SRY QUES oN LNG Be dg cL £ ass peat TE OCT PAR ton reer: site GE Ln ; far ratio of PUREE SOIT “wine in formule LH ; Sri moines radio of WNL Sooo EN x Sa SAT ITICHAT TE ; ; ii range Sonor = RN in de AN van ut SANT to CO 2 MOY im SIN, WYER Saition at THAW TVA (OT sr EOS NR, ; nr tha composition at da, Ro, and À are FE al Sackmernt, Fu the compos N Ra, Ra Fe Ry, and 8 Gs lalla, proforabiy : $ emtodrnent ut file ds PAG FE EN Ra ig ESC inked FE ; 10077] in one amb he first compound (Ha) Ra, INN and Ry IS (Kr CB ara MRSS OR SS x Zr iy + AS NN OO OY ie RES ES Hz x ad nm SCT, ; "aferatiy HY ESS 3 {OF sra BOY HIN, FEMS EE BRR A FIER] praterainy mn Rs Re, ang X are HY i Pall, pari, mo-proged, td acter ae reset . x s, Ne, ang £ ; cree ms od Tir, Da A. Ce Seria ORY NE N + ERY R, Ss, Ss ; thai sprint LIFE} bl Tate METRE Q ; SOUS (HR Phi, Ng ; Shed avid HIE Plön seeps ? COATING (HRY ; Torrie, alin, * Scnetyibuhe {3 ; sshand compo © sing of meh 3: F + Sy Jemmst FINALS Seat SSSSNS COUR] DY SCAN of om : sm voestfraibutyt. 3 ’ x I Jaa © SS HH STORE sonsist = = nly, Sans SRE ; | Shand VISE, acted fram fire STR avi, Japan = ? 1 hey HA Las 3 Aa from hs = © 1 San 3 FSSSS N ae TERN, bois vr sealed | Dili, eri-Butyi, Den, Beg, 2 Shmathyiprop, B aR proferabiy mets, bl, Seo-buiyt, teri-Buh Oh Rad 2 Dci © Hail, more prafaraby yet. nos but nethytihet?- Ha A UA more à acetyl, seo hha Sd, Somathyibut Rey PR SAME, me AY POST daa, oo tributisi, and 2034 ; I SITE, IIE + RW shimaihyibuty x} ITE, sivinentet LOTHIAN had mal TYR, rani SETHE SBI 5

TESTS A RRR 5

[0078] In one embodiment, in the composition at least two compounds according to formula (I and at least two compounds according to formula (Il) are present, preferably (la), (Ib), (lla) A 02310 (lib) as defined above present.

[0079] Preferably, in the embodiment wherein (la), (Ib), (lla) and (!lb) are present in the composition, the molar ratio based on the overall molar ratio of compounds (la), (Ib), (lla) and (lib) in the composition is for (la) 5 to 30, preferably 10 to 25, more preferably 12 to 20 mol-%; (Ib) 1 to 10, preferably 2 to 8, more preferably 2 to 5 mol-%; (lla) 5 to 60, preferably 30 to 60, more preferably 50 to 60 mol-%; and (IIb) 5 to 30, preferably 15 to 30, more preferably 20 to 25 mol-%

[0080] Compound (I) and/or (Il) may be a solvate, hydrate, salt, complex, or isotopically enriched form, preferably a salt.

[0081] Compound (I) may be a sait, wherein the salt comprises an anion selected preferably from the group consisting of napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, iodide, bromide, phosphate, phosphonate, lactate, malate, citrate, tartrate, fumarate, gluconate, sulfate or hemisulfate, more preferably a suifate, hemisulfate, or chloride, most preferably chloride.

[0082] Compound (Il) may be a salt, preferably the salt comprises an anion selected preferably from the group consisting of napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, iodide, bromide, phosphate, phosphonate, lactate, malate, citrate, tartrate, fumarate, gluconate, sulfate or hemisulfate, more preferably a sulfate, hemisulfate, chloride, most preferably chloride.

[0083] A sulfonate used in the present invention may be a sulfonate according to formula (Ill)

Q ve 0 (Il)

wherein Ra is selected from Ihe group consating of phenyt, d-nitophent, 4-methyphenpt 102810 iflzoromethyphernt, trifigoromeathyt, and {£,-Cahaikyt {GRAY In one erntodiment, Ry is {GG lake andlor {O-Gslaikyt ls selected from the group consisting of methyl sib propel, Dull, peatyl, ise propyl, sec-prooyl iso-buiyt secbubd lortab 2-pontyt Spent, 2-meihyibutst, = melhyibeiyt {io-pertisi oder Hoanmyh, É-mathyhul-wi amethylbul-P-4l, 2 2-dimethylorepd, prafarably rmetiyt [OBES] The Invention 8 further direcled fo à composition In which ai east one compound according fo formula { and at feast one compound according fo formula (I) are hooded logeiter via one dr weg nker gysiams, PROSE] The finkar system may Se G-Onlaivdene or (CC alkenylarıs, preferably {Ge Calalvleng, or {DoxaikEnÿiane, wherein optionally at least ons or at least two THpgroups in these ald or aikerty! groups arg substluiied By QU 5, MOha, NN oy NES RS andlor connecing 5 performed praferably via position § 7, 8 or by substitution of the nitrogen in sositon 5 in formule {1 and position 5, 7, 8 or by substilufion of the nitrogen In position 5 or 10 wn formula {Hi wherein the underlying sromatii system in formula {1 and Gt 15 numbered Ÿ J $

© A AN :

3 Se et” € according lo formula (VE «wy

{IV}

10087] The acridine compounds {Hi of he invention can be produced by applying suitable kaowey method of synthesizing scridine derivatives, 8.0. as described in Prager, RH, Willams, ©. M.

Ecience of Synthesis (2008) 16, 987 Gensicka-Kowsiewska M, Cholewinski $3 Dsterahicka, K RSC Adv (3017) 7, 15776 Matsjova, M.

Janovec, LInmich, J 'ARRIVOC 5018 {y}, 134 arui references cited haan HIRE] Fraquanty sppied methods wale dng closures, aromalization of internitienty obtained oi and tetrahydroserndines, sx weil as ring rearrangement reactions.

Furthermore, synthetic mules D 800888 guinolines may Le adapted.

For example, this may vole conversion of dipharndaminas and carbowstc acids, using the Remihsen resction (Bemiisen, À.

Justus Liebigs Ann, Chem, (1884) 234, 1), arvlationt of phenvlacetonitriies (awdonuk, M: Gzyzewski J: Makosza, M, J Cham Spe, Chem Commun (1973) 7981 aniline 8csiophenones, ooupded fa anyl bromides og. va Ullman COuDÉNG and subsequently perform 8 CYCHZATON LACET appropriate conditions eg said catoivss Ullmann, F; Torre, À L.

Bee Sisch, Chem, Ges (18041 37, 2838, Mayer, F; Freund, VV Ber Diech Chem, Gas, (1922) 55, SCHE or via Z-enifiroberaidehyde derbestives, ag. oblained by the MoFrodyen-Sievers texclient, foliesved Dy conversion fo acridine derivatives {Graboyes, BL Anderson, § L.

Levin, ©. M; Resnick, TM, J Hetsrooyel, Ober, (1975) 12; 13351 Furihermmore, scridim [iM bones are used precursors of acridine derivatives, which can be obfained by various mæthods {sy described in Prager, RS, Willams, CAL Science of Synthesis (5005), 10281 (008071 Acriîme derivatives modified in position 5 can for example can be obtained by reacting the corresponding acte 10hHi-cne derivatives, 9.5, with phosphorous cxyoloride or Ihlame chioride fo give SOF dervetives (Anuradha, 8, Poonam, FOhenm, Biol Drug Des, (2017), 86,

926; Nakajima, M., Nagasawa, S., Matsumoto, K., Kuribara, T., Muranaka, A., Uchiyama, M. Nemoto, T., Angew. Chem. Int. Ed. (2020), 59, 6847), with phosphorous tribromide to give 9-H102310 derivatives (Kishimoto, M., Kondo,K., Akita, M., Yoshizawa, M. Chem. Commun. 2017, 53,1425) or with P4S10 to give 9-thiol-acridines (Poulallion, P, Galy, J.-P, Vincent, E.-J., Galy, A.-M,, Barbe, J., Atassi, G. J. Heterocyclic Chem. (1986), 23, 1141). These derivatives can then be processed further e.g. by substituting the introduced chlorine or by transition metal catalyzed couplings of bromine.

[0090] Fused derivatives containing two acridine rings can be synthesized by adaption of methods described in the literature, e.g. starting from diphenyl anilines and reacting these with dicarboxylic acids, using the the Bernthsen condensation (Eldho, N. V; Saminathan, M.: Ramaiah, D., Synth. Commun., (1999) 29, 4007).

[0091] Alternatively, other linking methods to connect two heterocycles can be applied, e.g. using linker systems with two activated functionalities (e.g. malonyl chloride, succinic anhydride, glutaric acid, succinyl chloride, 1,3-dibromopropane, 1,4-dibromobutane, glutaroyl dischloride, 1,3-diiodopropane, 1,4-diiodobutane, glutaraldehyde, 4-chlorobutanolyl chloride or 5- chloropentanoyl chloride, e.g. Fröhlich, T.; Reiter, C.; Saeed, M. E. M; Hutterer, C.: Hahn, F; Leidenberger, M.; Friedrich, O.; Kappes, B.; Marschall, M.; Efferth, T.; Tsogoeva, S. B., ACS Med. Chem. Lett. (2017), 9, 534-539; Fröhlich, T.; Hahn, F.; Belmudes, L.; Leidenberger, M..

[0092] The composition comprising the linker system may be for use in the treatment of betacoronaviruses, optionally i) the treatment is caused by human and veterinary coronaviruses that belong to subgenera hibecovirus, nobecovirus, embecovirus, merbecovirus and sarbecovirus, preferably coronaviruses or ii) the treatment is caused by human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV) or iii) the treatment is caused by severe acute respiratory syndrome-related coronaviruses (SARS- CoV, SARS-CoV-2) or iv)the treatment is caused by a virus that evolve or mutate from the species described in i) to iii).

[0093] Further, the invention is directed to a composition comprising at least the composition as descibed above and at least one pharmaceutically acceptable carrier, thus to a pharmaceutical composition.

[0094] “Pharmaceutical composition” refers to one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound or composition of the present invention and a pharmaceutically acceptable carrier.

iG . : RCE a ; ss SANS harahautio ia ; finie cuits quisiaih the rare FERAL 3 €, RCHLVENt, GXCSNE oF VSTIOS WIN which the them 1U102310 N Re + amet MILER TE SANS IR, Las 0 * N as an ni HEIST Carrier” refers 0 à diluen!, aciusvan!, aay tore Nquiïs, such as water and oi REFERS ) cr rs oan be sterile BSD, Jad Ww x 2 eu ouai on DIS Lun DS NDTHS : 3 ; [ry Ten x een far fr Bred Such pharmaceutics! DRNTIEIE oan nin incloding Aut nat Emden In RCFNFISIETSS, Hush phan fable of syrihetc ailing, & HAASE ARS 1 ha © pefralaum, arimel, vageiable ar syrihet pe ; nreferred carrier when SACHBUCH hose of PARDISUTT, animal VE | Ihe fe, Water 5 à preferred carrier wie VEINE WH DR : AVES ret served GERS. VOS SUN Man ; #1, miners! of, sesame all and the Eke wir Sovboss are - ofl, SOVÈERN CH minal D Fp WW EN Ki SS doxirnse ars OS OH, NOYLENT CH, 8 Cs orally Saline and anyooux HUGE UR, SOS Hon à admrksiersd orally, Saline and ° es Qaïine ; RTSSLAiONE DONDOSIEES 5 adminisiors Fe, imiétistered! intavennuaiy Saline = SETTLE ABN Eason Lal cS ang as RIN ANAS TE LENS ES LA POA = the RÉRETTASTELER © Coe $1 al CRNTENINITTI RRC HE HR SHEN ax TRE ; HE . x od FITTAIOSI MIDIS Sey PPANSRBIANA ETS Ga x AN a Lens N | FIA Ashen San ad Q[IFRIGEUIGE CON ? acai amnirvest an PERIL prafarrad owners when he pharmacsut | ROIÉONS ars protorably smploysd as igual PERSIE RAR 4e x to Si af RES NO PATER N x SANS + sourire = = FRASER WALL N Aa EN scittione and aqueous dextrose and giyosrol Heal SXCDIENIS incisde sich, Qutose, SCRADNES ai JE Sisishie marine! SxNiDIENIE leds si ; A 4 ktions, Rosie nhartaneutosi ai } N NN ars for indeciabis solugliong, Ruilshis KARTEN N sachin stearate, ayvoann CNY EE ON $y i >. SARIS JAR x PES a sab 3 + RES SN GS) wR CRITIQIE TON FHRCISN * ice four chalk silos Qa BOWEN 8 rer a ala mal rie RAR, ERE, ’ “ s Sven. vuter aires inctèse, SUISSE, gelatin, Mal rte ; HR, oyceret, ropyiens, gheood, water, sthangd RAINS ° OT felts fiesi SKAT TK, ghost © PRESSE PR Siig me iparate, aie, scciem chioride, dried SKIN AR SS . SHOT amaunis oF walling al TDNONIRAraie, BHO, SOCLM pad, oan ala conan mung amounts © © N © Lu ED + route LEN DIN REE TRES © . F natiRresee i the fke. The composition § desired, can vn ar tube the form of solulions, ami ie HEEL THE comp is. These compositions can take the form HR US OUR sf bv meee Served FREES COIMTIDOSIIONIS CSI E aiment an amidaifying agers, or pri bufferng agents. 7 ; susiainechralegse formulations and NSS au HS capsules, powders, susiamsdrsigy ; ; ; a Ka digs 8 SEEN, NSS, Cin I las mnr sions, emulsions, tatiels, pills, CRESS, à Here with raditons! hinders ang SUSSENSIONHS, Susi, fated 85 8 suppository, with TACHONA STRAIT IRENA NT ve Ly Sa as ROSEN x ce rs ean x ORTHO can Le SORTE ZN 5 pue segppioren mes ss the ke. The composifon can Ge fon ts an duds standard ces such as WI SRG, TIR a es, Ord fomnulallen can noiuds sis : a ol SAN aw righecarides, Gral Roms x AR SCCNUNN Sacoharne, LETTS BUCH 29 VONT siarch, magnesium SIEHTS, sadn Boal arades of manniio! ncioss, sarah, n em has anal Sarnen as femal spre 85 in 028 LS STE > . EY ER PEYTON EE Tea AFTER pharmaceutical i RGSS DF au HER aif sttaine PNArTiaoeLtc LE ES FRS ES SSSR > is, et Exampies of slabs > ea add $ x En si X 221 ENS © AAN a afuings THAGNESHEN carbonais, at, CN S “pe FEW Martin. Such CONTIDOSHIONS wl SSHLNOND. JE RERT INI imal Onipnnnet hu AHL ITE RAILS TE 8 N URES . re Fharmaceutiond Sones MOINE Frost form frmeiher agora in Ramingion S AERO a Ay sisratiy i pried form AHIR QE ow TREE RES NS . ax Fin at nang 20 FORTRAN FT QUEER SAREE aE wy . x Aa SS a Baur EN SEN 2 ; ; © ie therapeutically SÉSOIVE amount of the theese, § © as administration in tha © NEE EN NY AED > x © nye x NT ACRES RRR N SONS à MGTEDAUIE FORE ; ttes the fon kr DEORE ROTES SAFE SSRI Ÿ _ . a. ad ÉN SHOW sie MOSAIK STE arg of carrier sO a io proviie WR © sufaïe amount of carrier sa as fo î 5 ES WAT 8 Sita amo . > nieiratiqns FRE SR HOUId suit the mode of adminisiration J Aa ee a Hovriirt ae IL AWRY INRAUIXS QE Ra patent The formulation should suit SARA NG 5 > : ê PAT EN ves use an desoribag above, is dirvoiai! Io 8 cornocathon for use as Jesoribad : ze + dus Ai Ada > Tadd TE RAS SANTE EREE € TRS 15008) Further, the MIVENION is dred DD 8 Compe TE 1 CLES, BRE R © Intnc as an inhalatios cru TONTLISie] as Art MIN = iateri an an à aint fey in formiiairme RS an tien fer uns as Ges(rined ations 8 ÉNTTHISIE as 2 Bodiment, the somposdion far USE as desc GETTY in à further embodimert, HD Sompos RE ii à further embpot rai dag. Cg d Tes +

XANPLES OF THE INVENTION

EXAMPLES QF THEW + gs * 3 x X RRATHERIA: & ANS ETHODS {O08 MATERIALS ANT METH PASSE = ; > frs Feeney RN Sin snd ad aa x Fan OO was order from 10035] Plasmids and proteins x once (Sars-covt nand - 746-1950) was on : ay the FH Bee emus MICE {BRN LOL NRG x ited ia ARE Gene encoding the FLFro sequence | mired for Bool aynransion ang cloned FOREN LEE 808 i haiti LISAT ontimizad far Bool ÉXETEHNNEIT : ? ’ ca & jo. Ë ST dors (556, IDE FRIAR TIAA A ’ ‘ iment OX Mixte Co dant ENKÈ NCEP IANNIS à ifs PRESSE SNS > * ; x 2m 34 N EN FR Mg: Integrated DNA Tachnologies | for, mandy, pei sel with à Clorminal G-histas IF ARERR a. OA animes 5 TEA DERN + FR . one Tid ing ha Slice mathod™ lo saver! VROÈNS, mainly, pall 5 Shed path] vector Th using he Shoe malhod™ Po: for named paoiMia, a modified potMiii veoh MAREN SES smn Lag TES SYS DONS, 8 ma ; [OVEN BETZ and & new expression veolor ng fon SOQUENDS alter he staring coda ATG, SEL SLES SCHE : . x Date anna Sent RINT RAT TS SAIN ; Pie ur NS an inserlign of oplimized codons SOQUEMCE € 5 nai &-Ristiitine laa detMSa vector contains an insertion | 5 and foßlawen by he N- terminal 8 Histidine lag, EAN Based an Varma of ai” and finest b 5 ; i Tha GISOAAGTATUARAAG, based on Verma ef al”, be found ai HMGL-FEPF wehsäe, The SEES SPAS LAPS | | Tha YScIDT map can be found al HMOL-PERI ; CS ; TRY nrafînnss aile. The Yo MSP LAN to I TT cuivres at

N VNA ES N N DETTE RS NER CHINE ang sy 8 Thy FARINE ON © 54 ES and Na ransomed Als Warn 0 SERS ASF {inde B50 ool BR 54 STIG ARG US an ’ AA reer evened 7 RSS were ransformad into £ ool QUE à fem arrivons, Âfler the ODE reached 3 ale x Ï SH N 3 Anan vagy à 3 TRE OK PASTTIOS Word TA . Baverey IY eran dd NANETTE ÂHET 1 N > fie trot (TRY medie containing 100 mal Rana FTE. The vertor nothdSa IN ha = anaes TRS Sy ARs “où "SE ARS È + TRS UNE ae ASS 37 "0 in toro broth (TRY medias co NT with DIS mid FTG. The vector pe x al he To SN ened ma HET Wath 100 mind 3 LE Cn HER was cooing to 18 70 and supplormiented ton. For abel DroRis, pre {he HAS was toced to 18 3 for ull FFOIART axprassion. For bal onen, | NHANES vante st HT as aa QF SH Gam AXPTRESR ; A EEN a EC folded monn SHUDIE protein ang À was used for al tation (CH. SOS into 10 of 20 NS YRISSS MO SOUDE PÈRES i a wa NRE De RON ER Sen LA SE RAR SS SS . ‘ ia Sa LN HISEUREON LAE S flea > x SO weint mientis EN NY BRS Conan steamed HS dem ; Sa by RAO STHSINTISN mais od = i. PAPA rs cheri VA, nw SULTS Was gown Hi AE minims . fend od a Aer GDS rmachec 5 CHARTS WES Wh ; Sa pénis ice HiT 3 AJL SMa KES SNS FES = - Aas ARES SEH OROTISS, ANS . . u Es Lo à mois sunpemerted with 15N-Ammieni 3 RTL After ovarnintd induction, the TRINITY TRANS Liane fod wii (108m ITS Alte SYSSTIQUE ENRON, FES RRL Bro © . x Say Sa FETA FAA * > A ve. hp {te TREY art sunrises SES VERTE LE SUITE A cie Maia O30 ture Was cocted io 15°C and suppiomenisy tes © rasuspendad In sls buffer (30 TUTTE WER Couey Le 6 srt the Qellais weary rasuspendad in N SAE x ow RE pon San. arg the PSERSS TEN TRE © harvested rough centucation, and 8 caîs were harvested frough centrifüg mM Tris-HCI, pH 85, 350 mM NaCl, 10% glycerol, 10mM imidazole, 5mM beta- mercaptoethanol) and sonicated at 4 °C. The insoluble material was removed throddh 02310 centrifugation at 24,000 rpm. The fusion protein was first purified by Ni-NTA affinity chromatography, the supernatant was applied to nickel resin and washed with 10 times the column volume with lysis buffer followed by a wash step of lysis buffer supplemented with 20mM imidazol. The protein was eluted with 3 times column volume by a buffer containing high imidazole concentration, 20 mM Tris-HCI, pH 8.5, 350 mM NaCl, 5% glycerol, 350mM imidazole, 5mM beta-mercaptoethanoi. 1mg of TEV protease was added and the solution was dialyzed overnight at 4°C against a buffer containing low imidazole concentration, 20 mM Tris- HCI, pH 8.5, 150 mM NaCl, 5% glycerol, 10mM imidazole, 1mM Beta mercaptoethanol. The next day, the protein was applied to a nickel column and the flow though was collected followed by concentration using a top centrifuge concentrator with a 30kDa cut off up to a volume of 2mL. For the final step, the protein was applied to a size exclusion chromatography column, High load S75 (GE- Helathcare, chigaco, USA), pre-equilibrated with the final buffer, 20mM Tris pH

8.0, 40mM NaCl and 2mM DTT. The purity of the protein was accessed by SDS page gel.

[00101] MP construct comprising aminçacids 3264-3569 of SARS-CoV-2 polyprotein 1ab, N-terminal 6xHis and TEV protease cleavage site optimized for expression in E. coli was ordered from GeneArt and subcloned into expression plasmid pETDuet-1. Protein expression was carried out in E. cofi strain BL21 in TB media. After reaching ODg=1.2 the bacteria were induced by adding 0.5 mM IPTG, cultured for 3 h at 37°C and harvested by centrifugation. Cells were then resuspended in a lysis buffer containing 50 mM Tris pH 8.5, 300 mM NaCl, 5% glycerol, 1% Triton X-100, 2 mM B-mercaptoethanol, and protease inhibitor coctail and disintegrated by sonication. The lysate was cleared by centrifugation, filtered through a 0.45 um filter and the protein was purified by nickel affinity (Ni-NTA Agarose, Jena Bioscience). TEV cleavage was carried out overnight in 50mM Tris pH 8.5, 250 mM NaCl, 5% Glycerol and 4 mM B-mercaptoethanol. TEV and uncut MP” were removed by a second NINTA purification step. The protein was further purified by size exclusion chromatography (Superdex s75, GE Healthcare) in 50mM Tris pH 7.4, 150mM NaCl, 2mM B-mercaptoethanol.

[00102] PLPF° activity assay

[00103] The assay was designed to measure PL”*° protease activity under screening conditions in white 384-well Optiplates. The assay buffer contained 50 mM Tris (pH 8.0), 0.01 % (w/v) BSA and 10 mM DTT. RLRGG-AMC was used as fluorogenic substrate for PLPro 40 pl of PLPro protein (end concentration 60 nM) was incubated with 10 pl RLRGG-AMC substrate (end concentration 400 nM). The assay (final volume 50 ul} was incubated for 30 min. The release of AMC (Ex. 360 nm / Em. 487 nm) was measured on an Envision plate reader (Perkin Elmer, Waltham, MA).

[00104] PL"RC activity assay

[00105] The assay was designed to measure PLC protease activity under screening conditions in white 384-well Optiplates. The assay buffer contained 50 mM Tris (pH 8.0), 0.01 % (w/v) BSA and 10 mM DTT. RLRGG-AMC was used as fluorogenic substrate for PLPro 40 pl of PLPro protein (end concentration 60 nM) was incubated with 10 pl RLRGG-AMC substrate (end concentration 400 nM). The assay (final volume 50 ul) was incubated for 30 min. The release of AMC (Ex. 360 nm / Em. 487 nm) was measured on an Envision plate reader (Perkin Elmer, Waltham, MA).

[00106] Compound Screening

N A FEO an RR The fended [HOT XD ui ef BLY protein fan concentration GONAU in assay butter (80 mM u 02310

8.04 GOT % (wiv) SSA ang 10 mM OTT was added to the screening pates using * © A = \ Ë Na 7 NEN vestes evra mene Hae iy x dhuponaing Sysiom (Sic Teak} Compounds and DMSQ {as cor von wer & pa sie Se soryrening plates lo achiayve a final concentration af 10 al using à Ben = ss RS workstation (PerkinEimer, Waltham, LISAL The dng FEDUTDOSING cotisction _ was used te identify PLS iniibitors. The controts In Ihe plates {first two and last two columms } included CRMRD control {negative control with no compound) and control weitaus protein fe ban the * 5 > : 7813 STN SN XT $ pets Senate Sneha RENTER Ra + SEY assay windy) After sdclion of IQ uf RLRGG-AME subatr ate {aud concentration 450 ni Al Ihe MuiiFin, AMD Runrescence (Ex, 500 man / Em, S87 AA) WAS measured us 9 à Cm plate reader immediately after subatrate action {time peat D} and after 30 mas {ime porn TL Clualiiy and robustness of he assay was salculates using À factor, HHH 08) FLY inhibitor iy determination _ | = - = a = 3 + wd an oy EN ee “Nh I a Cn N © Sa SEN SS ARE AROrOSCANTE Was massive! Em 360887 am) on an Emission plate Rage AG wl of 8 TH ri Plpre sodulion in assay buffer (50 od Tris {pH 8.0) 0.01% {wiv} HEA and 1 3 mM a Ï } CORCANTIRENTT was adden, The ANAÏLTS was Incubated for 1 hour at RT, Then the reaction NN i Rx » x =e x sut am STAN A KA (100 nh, Anall rrapoctrais India voicciies of AMC release sert normalized fe he DMSO KNIE Hn VER Was oeloninled using GraphPad Frgm, The oxperiiernt was repested Ihres OTH PLY inhibitor 104 determination Cee FE nl Sagen solution iy assay buffer (B80 mAt Trig {pt SU 2. O1 % {wiv BEA and 18 mid OT) SONSENPETLINI was added, The mili was hioubeied for 1 hour ai R . Than the sosie as irätlated. by adding 10 ul of 9 HAT RLROUD-AMC 1300 old final} or 10 gl of UG ui Sta teal times, NER Lois and viruses Co ai 2" ; ce x a es a bog NET 5 X 2e a BHR Vert {Demapithecus sathiops: kidney apitheial AI LE Lledo à M N SA bu CRL-HSSS) calls, dervatire of HRT-19 gocacat ooforecial adencrarcinome: ATOO CCL Aa GREK (Fait cafes, kidney cortex; ATUC® COL-04j were oulfred in Duibecors MEM (Thermo Fisher Sotentifie, Poland) supplemented with 54 fetal bovine serum (hestinactivater Pharm Fisher Scientific, Poland} and antibletice: poniaifin (100 Limi} sireptermpoir (100 pail, and | SSH Pa TR cu La = ans SIHFOÏORAENT {5 VOTE, ASHE cells with ACER OVETEXETEESENT {ASEM ware nulls in Ihe JENS MANNS with supplementation with G48 (5 matt SloShop, Canedai FHM LLO-NES cells (ATOC GOLLY, Mucscs mulatia kidney apitheligl ces wR maintained in minimal essential medium (MER: bec pars Hanks’ MEM and ans met Barle's "a 3 J 3 x Ca aad Bay any an MER [Lis Toctriologies, Poland) S% fed bovine serum (restiactiaisn: Thermo Fisher Susie, Foland), penton { 100 Wim), streplornpoin {100 Wim 1 and ciprofloxacin {8 GE

HOE HH Primary human shin Sbroblasis (HEF) were cultured in Duibeccos MEM (Thermo ; >. x x : UR nen - Fisher Scientific, Poland) supplemanted with 10% fetal bovine serum theatinactivated, Thermo Fisher Scientific, Poland), 1% nonessentat amine soils (Life Technologies! and sntibiatics: poral {100 Wm), shaptamyoin (100 parmi, and ciprmfissacin {5 pain

[00117] Human airway epithelial (HAE) cells were isolated from conductive airways resected from transplant patients. The study was approved by the Bioethical Committee of the Medical University of Silesia in Katowice, Poland (approval no: KNW/0022/KB1/17/10 dated

16.02.2010). Written consent was obtained from all patients. Cells were dislodged by protease treatment, and later mechanically detached from the connective tissue. Further, cells were trypsinized and transferred onto permeable Transwell insert supports ( =6.5mm). Cell differentiation was stimulated by the media additives and removal of media from the apical side after the cells reached confluence. Finally, cells were cultured for 4-6 weeks to form well-differentiated, pseudostratified mucociliary epithelium. Alt experiments were performed in accordance with relevant guidelines and regulations. Commercially available MucilAir™- Bronchial (Epithelix Sarl, Switzerland) HAE cultures were also used for the ex vivo analysis. MucilAir™ cultures were maintained as suggested by the provider in MucilAir™ culture medium.

[00118] All cells were maintained at 37°C under 5% CO».

[00119] SARS-CoV-2 strain used in the study was isolated in house and is designated PL_P07 [GISAID Clade G, Pangolin lineage B.1] (accession numbers for the GISAID database: hCoV-19/Poland/PL_P07/2020). Reference SARS-CoV-2 strain 026V-03883 was kindly granted by Christian Drosten, Charité — Universitätsmedizin Berlin, Germany by the European Virus Archive - Global (EVAg); https://www.european-virus-archive.com/).

[00120] All SARS-CoV-2 stocks were generated by infecting monolayers of Vero cells. The cells were incubated at 37 °C under 5% CO». The virus-containing liquid was collected at day 2 post-infection (p.i.), aliquoted and stored at -80°C. Control samples from mock-infected cells was prepared in the same manner.

[00121] MERS-CoV stock (isolate England 1, 1409231v, National Collection of Pathogenic Viruses, Public Health England, United Kingdom) was generated by infecting monolayers of Vero cells. The cells were incubated at 37°C under 5% CO,. The virus-containing liquid was collected at day 3 p.i., aliquoted and stored at -80°C. Control samples from mock- infected cells were prepared in the same manner.

[00122] FIPV stock (strain 79-1146) was generated by infecting CRFK cells in 90% confluency. The cells were incubated at 37 °C under 5% CO,. The virus-containing liquid was collected at day 3 pi, aliquoted and stored at -80°C. Control samples from mock-infected cells were prepared in the same manner.

[00123] The HCoV-NL63 stock (isolate Amsterdam 1) was generated by infecting monolayers of LLC-MK2 cells. The cells were incubated at 32 °C under 5% CO; and then lysed by two freeze-thaw cycles at 6 days p.i. The virus-containing liquid was aliquoted and stored at -80°C. A control LLC-MK2 cell lysate from mock-infected cells was prepared in the same manner.

[00124] The HCoV-OC43 stock (ATCC: VR-1558) was generated by infecting monolayers of HRT-18 cells. The cells were incubated at 32 °C under 5% CO; and then lysed by two freeze- thaw cycles at 5 days post-infection (p.i.). The virus-containing liquid was aliquoted and stored at -80°C. A control HRT-18G cell lysate from mock-infected cells was prepared in the same manner.

MAN] vi lebfs were assessed by tration on fully confluent calls in 96 -Ayell plates, 2310 [ORE] Virus viekls Were assessed by ¥ néon on fully ci vent rod M 10 according to Ihe method of Reed and Musnch, Plates were rotted aa or 37 Char tl indicated above, and the oylopathic effect {TPE} was scored by observation under an inverted 0012381 Cell viabdlity assay TY Celt Viability Assay kit (Bionic TON ISTI Cet viability was evaluaied using the XTT Gel Viability Aes x Nate Industries, Cronmaet, OT, USA} according to the manufac ers prot vers, ; We > Lu GREK, HRT-18, LLO-MKC and HEF ces were cultured on S6-wei pistes, Cells wers i abated with AGF for 24 hat 370 in an aimosnphers containing §% CO: After moubatan, ihe med ° was discarded and 100 pl of fresh medium was added ko dach well | hen, 28 ul pi sotivaten 2. 3-is-{2-methoxy-nitre-6-autshençti-aH} tra sant “Scarbo “ice i N ee solution was added and samples were Inoubaied for 2 hat “7 SG. (he SES bares | As ‘ SS rer Was measured using & Spectra MAK 259 spsctirophotometser (Molecular Devices, San dase, GA USA) The obigined resulls were normalized fo the contra! samples, where cef viability was set to 100%. (001381 Virus repfcation inhibition assay a tas (TPP Trasadincen led im oulure mediem on S6-veil pirates (TPP Trasadingen, TOUT 291 Varo calls were seuded in cullure adm Sn se well DS te aa sen Switreriand! at 9 days before infection.

Subsonfiuant cells wars iniecied with SA SA Pc viruses at 1600 50% tissue culture infectiqus dose {TOIL Infection was performed © A © presence of 100 nl, 1 ut and 10 uM concentration of compounds fisted be Fable 2. Aer € ° > $ ; x “ex = x cut Fans a À ose Line wate oF Share IL QUES inoubation at 370, coolly were ringed Autor with PRS and fresh medium wil Wt compounds w 5 adden, The Infection was carried oui far 48 h and the cylopaitie effect (CPE) was assessed Cuivre supsmaiants wars colleciec fom weils where CPE reduction was shared, ne 3 For the detailed determination of the antiviral properties of MT re Jo were seeded in oullure medium on S8- well plates at à days belo infection Shug! art on $ wars infecled with BARS-CaV-E, MERS-CoV and FIPV viruses ai TOI, = 1608 and Hiv: . - - SOUT bay Snead hea an nsrformad in tha NLS3 ai TOY = 4000 and HCOV-OC43 at TOM = 3500 infoction was performed in the présente of AOF Control calls vers inoculated with the same volume of mock as negative sontret and wath 10 UM remdesivir 85 à positive control of poronavirus repo inhibition : Ate had itt S and fresh madam without or with = h of incubation at 37°C, calls were rinsad hvice with PRX and fresh di without a with ACE or remdesivir was added.

The Infection was carried out for 24 h whan the CPE in virus CONtroI was observed, Culture supsmaiants were coflsotet. {081541 Viele replication inhibition ox vi was évaluation by necting HAE Teun with SARS-CoV-2 virus al 8000 Tori in the presence of ACFE ramen or F BS. te concerimiions of ACE (400 nl and S00 nl} and the controls were added to Ihe apical side of the insernts followed by the addition of the virus diluted in PRS infection fime was ot $ Fours at 3H Alter the infection, the apical side of the HAE were washed Ihres Ames with on aad ganh COMSOUNG was reapplied and inoubaier again for 30 minutes at 97° © After the last incubation wilh the ATK, the samples {Spl} were ooliscted and the HAE aes fait in ahi ; X ë 5 ng 2aïrset . € 3e = ss < {x tire NY siierphase, Every Sa hours the HAE were incubated for 30 minutes with the ACE ditution 5 9 contrats, and the samples were collected, After collecting fast samples cells wore fixed with 8% parsformaidehyde and siained as described alow.

FORA} Virus yield was measured using ihe RT-gPCR method desorbed below.

[00133] Isolation of nucleic acids, reverse transcription and quantitative PCR

[00134] A viral DNA/RNA kit (A&A Biotechnology, Poland) was used for nucleic acid 102810 isolation from cell culture supernatants. RNA was isolated according to the manufacturer's instructions.

[00135] Viral RNA was quantified using quantitative PCR coupled with reverse transcription (RT-PCR) (GoTaq Probe 1-Step RT-qPCR System, Promega, Poland) using CFX96 Touch real-time PCR detection system (Bio-Rad, Poland). The reaction was carried out in the presence of the probes and primers indicated in the Table 1. The heating scheme was as follows: 15 min at 45°C and 2 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 58°C or 60°C (specified in Table 1). In order to assess the copy number for the N gene, standards were prepared and serially diluted.

[00136] ACF and virus visualization

[00137] Vero cells were seeded on coverslips in 12-well plate (TPP, Trasadingen, Switzeriand) at 2 days before experiment. ACF at 1 uM or DMSO at 0,01% concentration were applied on cells. After 1h cells were fixed with 3.7% paraformaldehyde (PFA) for 15 min. Fluorescent images were acquired under EVOS XL Core Imaging System.

[00138] A549*<E2* cells were seeded on coverslips in 12 well plate (TPP, Trasadingen, Switzerland) at 2 days before infection. Subconfluent cells were infected with SARS-CoV---2 in the presence of ACF or remdesivir. After 2h infection unbound virions were washed off twice with PES and fresh medium supplemented with compounds was added. The infection was carried out for 24 h whereupon cells were fixed with 3.7% paraformaldehyde (PFA) for 1 h. Fixed cells were permeabilized using 0,5% Tween-20 (10 min, room temperature [RT]) and unspecific binding sites were blocked with 5% bovine serum albumin (BSA) in PBS (4°C, overnight) prior to staining. For visualization of viral particles anti-SARS-CoV-2 nucleocapsid protein antibody (Bioss, bsm-41412M) at 1:200 dilution (2 h, RT) followed by Alexa Fluor 546 conjugated secondary antibody (Invitrogen, A-11003, 1:400 1h, RT) was used. After incubations with antibodies cells were washed thrice with 0,5% Tween-20. After labeling virions actin cytoskeleton was visualized using Alexa-Fluor 647 conjugated Phalloidin (4 U/mL, 1 h, RT, Thermo Scientific) and nuclear DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, 0.1 ng/mL, Sigma-Aldrich). Stained coverslips were mounted on glass slides with Prolong Diamond antifade mountant (Thermo Scientific, Poland). Fixed HAE cultures were permeabilized using 0,5% Triton X-100 (7 min RT) and unspecific binding sites were blocked with 5% BSA in PBS (1 h, 37°C) prior to staining. For visualization of virions anti-SARS-CoV-2 nucleocapsid protein antibody (Bioss bsm-41412M, 1:200 2 h, RT) coupled with goat anti mouse Alexa Fluor 546 (Invitrogen, A-11003, 1:400 1h, RT) were used. Nuclear DNA was stained with DAPI (0.1 ug/mL, 20 min, RT, Sigma-Aldrich) and actin cytoskeleton with Alexa Fluor 647 conjugated phalloidin (4 U/mL, 1 h, RT, Thermo Scientific). Stained HAE were cut from inserts and mounted on glass slides with cells facing coverslips.

[00139] Fluorescent images were acquired under Zeiss LSM 880 confocal microscope.

[00140] Table 1. Primers and probes used for a RT-qPCR. 0 Co “ Target | Primer Sequence Concentration | i JE SN oo [nM] ji SARS- Forward | CACATT GGC ACC CGC AAT € (SEQ ID NO: 1) | 600 | | CoV-2 | Reverse | GAG GAA CGA GAA GAG GCT TG (SEQ ID NO: | 800

=» Tre ITTY 3 ba ; È : rene PSE ENN ES Rd PSU 3 i i ede ss US RARER A y SC AIFAR PL i : Prrgggere ey ACT TOC TCAAGG AAC BAC ATT G X ; î ; Frobe : A no « > RS UN sisson} : PEERAGE i N ER SEAL N TE S—— : : ; + BHOTLSEC IR TT ROH ; 3 : 1d TE era ne ep TRATED O&K TTY Emi, Toad ; : da ps SE TEST ACC TOT TRA TOO GRATTO (3S rr | Fowed | GEG TART AC cet ; i 10 NO: 43 DRS REA Fous ann i i ; + WG INNS rir SAAT SRY i NE 3 IRAN : 1 : SES ON NN BN * «= = 3 TELS i 3 ; rgd ERE AES OT À © i OOS DOA AT {SEQ $ . : | 3 © : Ra JEUNES 1 TR 5 22 Xe Re ’ { a ; SERS. : PEYSTRE : ee SERRES ER ; ; er ; 5 D EE n 8 à IEAM i 3 $k 3 ; M2 EN OT AU NN NAAR : AH: : : Gay "SR CUTREEE OF SOG CAR AAT GOT GU 16 Wid : : Ë : Tan he ALLL Late AR SIRENS ' à Li ; : SFTDDE 155 ann pn sy ox mtn na nues : 8 i Doan X {RES IY AI 3 5 eee Sem an es ! : ES ee AR DOI MST to AAG CGT etal ; : a ; Forward : ARALGT GOT TL ; PE N 1 AR : errr ; | ; DO SRE EU TEE TR Ee SAR : } ; PE AN RN RETR RE RRR RE SEEDY 157 EN 1 i à I Cry SAR AST TTT GEO ATS {SE Hd i ; Een en : Ravarse POTE TOE AA AI 3 ee : HGoV. | Rover in gees : Ë AN i 2 SE EEE 3 SR ; ; Ï : Er Ep RE Léa : ; ; 193 N BEA DEN 3 a 3 ES T'en A à N TOO : : ferred FEY TAT 5 CA £3 i {3 5 i i Fao Rd 8 3 ; i | Prose PR IRE LAK US i a i LPS : > Lu 4 2 oy xd TUTTO : } 3 DAS SANVE 3 LN LE EN AL LT ; ges SREY EN TORR : Ë TX TOTRET GOT OGG AMG ARTAAT Gea IE Po Ë Ë FON SES SES : AAA Este 3 È 3 : Ne SM TTR RR My § Ë i : EAE nn 3 sera i Hi i { : NEO D TON GAT ATA AASEDID CSSS | i Forge | SAX OAK GET OF DNS GRU AR ; i $ Ë | Reverse 1 GAA CAA GOT L Se FF \ ; NOM IH EEE ES USA : Ë "és RE AF SOO CTE GAA CAR BEA TOY ; Ë : ; Probe i Lule À FH me ir Sp 44 A € 3 : SWAB NES A erry a TR 3 £ = SAS 3 Ÿ EE N SOT SAT ATS A SE RA TER Pod § + EEE rrrraa ; Sr CE a 5 ES LMI AR Ee HS \ : § TTT Forward TASS RAS TAG GOT GAT © 0 ; N + : yon 3 +24 een ange tree 3 : 3 HN I “EEE ETES Ran à i © 1 TRS INAS YQ dresse PPT ESPN RAT FOIE OAS 3 i È vere TARE ANAT OT TOC VERS GOT TTC ART {SEQ PN ; 3 a = pre CS whee 3 A2 RES ! : Moy.

Reverse AGCAGACC gage i PS EN i 8 Hs NES * ne SEE TETE ET TETE 2 pi Rs aR 33 à : SC HET PGA RE TT ENSA PINS SGA SOS ANG TA Hh i £ 3 ï 3 A x ANS IAAT TAS : 3 i N Frohe POIANA LA A ~~ : et i È rer f TYRES | BAS. vi Cr ! | (PAMSHOH (SEC INE IS.

Es dad SS ; nis : chive fx Siren AEE Generation of resistance mula ined By serai nassanes of the virus in the FRE TR Yi ; I see À ven N hinined Ly NET TASSE = > . : x dF RESET 9 SE SDISITIST SF > ; een RATES Ürugresisiant SARS-LOUE was obls esi starting at à concentration = ÊTRE EN en ¢ event tn 23 0 x y . S fed LAT . af ADF or remoesivr aim ng ai o wang of noreasing conmentrations of AD ) à niste and infecied with SARS-Cov-? NTÉSSHRS oF NICNTGSSIE pd i 12 well piste ang infested with FEE - Yorn coolly were sooded in 12 wei FERN RS ; ee EEE . t tu thal Bee Yam oolls wars SES ; moles were solace. equivarient to tre lesa VHD CDHS one SO was observer samples ware chiectet, RUHR SRS.

When ORE was obser ‘ N 0 | cs of the drug or PER.

When OPE was ob Senfion Was renested with Taw NSE OS oF Ing OUR of pe infmntisun HAS renpeat 3 WY the PIESSTICS O5 = ; 3 er the nant GEESE.

SON WEE 5 _ 553 3 rat He Fp DS ASE JOTI 3 . æ Co Fe a wang usa tir minal CONS fre IIE IY BR = 78 nase N After es i HEE, TOZSN ANG USS WER ; ford rare TT RSS A ATX SiQquotst, frozen and i | dire, dose was double every = passages) SRRARSRS, . © GER NEN NEL TPN Penn REE WES LG A ° > 7 . ws ; PERS ; Sing CONCENTETONS of the campaural (le dos isolated and viral RNA was SENT ONE © Wacker REA was oolated ang vira TAREE AAS = $v no ax ae aay DoHacten ; Tex <> a rue HE Re EN USE, N ; passages, culture DUPETTIGIENiS wa sre carried oui i innticats, PRBS.

X AU axnorimants ware carnied oui int FRR : SON AN At, ea ia Sid ea Se: STU IRL IS WESTEN sora sequenced (INGE, Hing) Al experiment SSQUENTRS | IR fait validation ns re. » ; FREY wade REE Ya pe Fais HF NIE HH vali $ oo + have produced NOLEN pe A ACK fo PL” in soiution we have produced tas To prove the Wading of AGE to PL aad MS medium, The remaining fad + : da cuivres in minimal MO medium \ 2 . N anc than he Hœctoris ANETTE HY TIRÉS SUR > i RAGE teted Hrofsst using Dy growing Ihe bacteria ouifure bed above NME amerments ware lateled protein using By 9 fio (he one Joscribed above, NMR saps 1 of DrOISST purification was identioa! io he € or Ta TEQUETIOY SOU Met DEM OF peda PUTIICHET H SO MMe speohomaisy {4 frequer td ’ OA NN Pres che one ee 1 LAS RINE SRSTIIHTNY oe Sy . recorded st 295 K on à Bruker Avance HES ny a ere aonuired with 40 scans PRCOIGSU ST 2NS $ ES ing TROSY SOSDTS VS SAT À ; | wih à 5 mn TO! onvoorabe, HN TROSY = MS SRE? A ie SEINS Nee was supped with à § mm TG cryoprobe.

H, TH ON Jebelled FLFre, Sample ganosnfratian was PES ESS ; NE Put ATi + SE, INRNURRET TENTE a anon 3 ot THEA x B12 DOMBIEN porns dt uniformly HE h 10H DRO Anselm var processed ang STI VV ES ROUE VENTE 5 nented with 10% LRO, Speci ED on Da : H 7,4 supplemented with 10% IA Cre ee . Si in PRS buffer oN T3 sunolamantad à _ Ce Snuoher and Q. 17 mt in PES buffer pH T4 aunple 3 {iv 35, D 1553-2030. Wayne Boucher and #3 5 a : = creer fas S 8 id NEFITS, 3 ; . XSARA suite of Srtocrama IV SF, 8 analyse using He ASARA suite of ı mY 3, robert TH IRB FRA à = à sit rames AT à Seen rich urimuhiiah SSI à, ; à oi Hiophemiatre Linivarsite of AETTISRICHIS, x Department of Biochemistry, University © © Hisatlon and structure soistion GOTSS {rysiaitration and structure soi 190145] Crysiaitzation

[00146] PI? for crystallization was purified as described before. In the last step of purification, the protein was applied on a size exclusion chromatography S75 using as cartisr 02310 buffer 20mM Tris pH 8, 40mM NaCl and 2mM Dithiothreitol (DTT). Purified protein was concentrated to 5-10 mg/ml using 30 kD cutoff Amicon Ultra Filter. PLP®-proflavine complex was prepared adding 5-molar excess compound to the concentrated protein. The final concentration of DMSO was never higher than 5%. Crystallization samples were prepared using commercial kits and an automated crystallization workstation (Mosquito, TTP LabTech). Both ACF and pure proflavine were used in the experiments. Crystals of PL”“-proflavine complex grew at room temperature in 0.05 M Hepes sodium salt pH 7, 0.05M magnesium sulfate and 1.6 M lithium sulfate. Crystals suitable for testing were moved in cryo-protectant solution containing the harvesting solution supplemented with 25% (v/v) glycerol and snap frozen in liquid nitrogen.

[00147] PL”-proflavine crystals were measured at Swiss Light Source (SLS, Villigen, Switzerland), beamline PXIII. The best dataset was collected at 2.7 À resolution and it was indexed and integrated using XDS software; scaled and merged using STARANISO webserver”. Crystal belongs to space group P6522. Matthews coefficient analysis suggested the presence of two PLP -proflavine molecules in the asymmetric unit. Molecular replacement solution was found using Phaser? and the apo PL" structure (PDB code: 6W9C) as searching model. Model and restraints for proflavine was prepared using Lidia, the ligand builder in Coot®. The initial model was subjected to several iterations of manual and automated refinement cycles using COOT and REFMACS, respectively®®*®. Throughout the refinement, 5% of the reflections were used for cross-validation analysis’, and the behavior of Rfree was employed to monitor the refinement strategy.

[00148] Chemical characterization of the ACF components

[00149] The commercial ACF is a mixture of 3,6-diaminoadridin-10-ium (proflavine) and 3,6-diamino-10-methylacridin-10-ium (acriflavine). In order to perform the study using fully characterized material we have used NMR and HPLC techniques to analyze the commercial product.

[00150] HPLC-UV/MS and 'H-NMR analyses of commercial acriflavine hydrochloride (cACF) and proflavine hemisulfate (PF)

[00151] HPLC-UV/MS analyses of commercial acriflavine hydrochloride and proflavine hemisulfate were performed on a Dionex UitiMate 3000 HPLC system coupled with a Thermo Finnigan LCQ ultrafleet mass spectrometer, using the following method: Waters X-Bridge C18 (4.6 x 30 mm, 3.5 um) column; gradient: 5 to 10% of acetonitrile + 0.1% formic acid v/v in water + 0.1% formic acid v/v over 9.5 min perd; 10 to 20% of acetonitrile + 0.1% formic acid v/v in water + 0.1% formic acid v/v over 4.0 min period; 20 to 95% of acetonitrile + 0.1% formic acid viv in water + 0.1% formic acid v/v over 1.0 min period: flow rate: 0.6 mL/min; UV detection at 254 nm. "H-NMR spectra were recorded at room temperature on a Bruker AV-HD400 operating at 400 MHz.

[00152] All biological experiments were conducted using the same batch of commercial acriflavine hydrochloride (cACF) purchased from Sigma Aldrich, which is sold as a mixture of 3 6-diaminoadridin-10-ium (proflavine; PF) and 3,6-diaminc-10-methylacridin-10-ium (acriflavine, ACF). In order to determine the exact composition of the obtained commercial acriflavine (CACF) the mixture was analyzed by HPLC-MS and 'H-NMR spectroscopy. Both methods show that the used batch of commercial acriflavine contains three main components and that the composition is as follows: 56% proflavine (PF), 17% acriflavine (ACF) and 22-23%102310 side methylated PF. The remaining 4-5% are other 3,6-diaminoacridine derivatives like for example side methylated ACF (4%). The NMR analysis is based on the fact that the protons of the methyl group at position 10 of acriflavine (ACF) have a very distinct chemical shift of 3.94 ppm (4.04 ppm for side methylated ACF), whereas the protons of the methyl group connected to the amino group at position 3, like it is the case in side methylated PF and ACF, have a much lower chemical shift of 2.87 and 2.98 ppm, respectively."* The aromatic protons of the different 3,6-diaminoacridine derivatives almost exactly coincide with each other and consequently one of those peaks can be used for calibration of the integrals in the "H-NMR spectrum. Integrating the overlapping doublets at 7.86-7.78 ppm as 2 protons and dividing the integrals of the separate signals for the methyl groups (4.04, 3.94 and 2.98 ppm) by 3 gives the percentages of side methylated PF (0.69:3 = 0.23; 23%) and side methylated ACF (0.13:3 = 0.04; 4%) as well as actual acriflavine (ACF) (0.52:3 = 0.17; 17%) present in the measured sample. As there are virtually no other 3,6-dimainoacridine derivatives other than PF, ACF and their side methylated derivatives, the remaining 56% can be attributed to proflavine (PF). This analysis fits perfectly the result obtained via the HPLC-MS technique. Commercial proflavine hemisulfate was also acquired from Sigma Aldrich and its purity was analyzed via HPLC-MS and "H-NMR spectroscopy as well. The sample turned out to be 99% pure proflavine (PF) and was used as a reference for the analysis of commercial acriflavine (cACF). The signals found in the recorded "H-NMR spectrum and the retention time obtained via the HPLC-UV-chromatogram (RT:

7.96 min) were in accordance with those of proflavine present in commercial acriflavine hydrochioride (RT: 7.89 min).

[00153] Statistical analysis

[00154] All experiments were performed in triplicate, and results are presented as mean + SEM unless otherwise indicated. One-way ANOVA with Tukey HSD post-hoc test was used to assess statistical significance of acquired results. When the parametric test assumptions were violated, the nonparametric Kruskal-Wallis with Dunn's post-hoc test was used. P values of 0.05 and less were considered significant.

[00155] RESULTS

[00156] HTS screen to identify PL" inhibitors

[00157] In order to identify inhibitors for the PL", we established a fluorogenic protease assay using an AMC-labelled substrate and carried out a high-throughput screening with small molecule compounds at a final concentration of 10 uM (Figure 15 A, B). We used a repurposing drug library consisting of 5,632 small molecule compounds, which are either approved or are/have been under different stages of clinical testing”. Compounds that inhibited PL" activity were selected upon calculation of the slope of activity (velocity) from time point 0 to 1. The average Z' factor for all plates was above 0.8, showing excellent screening quality. As a cut-off value for active compounds, we choose 4x standard deviation below median of all compound- treated wells (calculated per plate). After removing compounds that generated high fluorescence signal at time point 0 (suggesting unspecific compound interference), 11 compounds were selected for further conformation screens, which are listed in Table 2.

[00158] Verification of primary screening hits

[00159] Eleven of these hits could be reordered and serial dilution experiments were carried out to determine ICs; values in the original screening assay (Figure 19C). Notably, not all of these hits were active in inhibiting PLP. Some of the hits from the primary screening MAY, 02310 have initially been identified as hits due to autofluorescence or quenching effects and thus displayed weaker dose-response effects in the follow-up analyses, since we determine the enzyme velocity and not just the endpoints of a reaction. Importantly, ACF showed a dose- dependent PL" inhibition with an ICs; of 1.66 uM and was the most active compound (Figure 19C). We then repeated this experiment with ACF and two derivatives of ACF, namely acridine orange base (Figure 19D) and acridine-3,6-diamine sulfate (Figure 19D) as well as the published positive control GRL-0167"%a (Figure 19E). All derivatives dose-dependently inhibited PL”® although to different degree. The effect of ACF on PL enzymatic activity using the ISG15-AMC as substrate has also ben tested. As previously shown, PLP® cleaves 1SG15- AMC significantly faster than RLRGG-AMC. Nonetheless, the ICs; of ACF with ISG15-AMC (1.46 uM) (Figure 20B and Figure 19B) was comparable to the ICs, that was determined with the RLRGG-AMC substrate. Since AMC assays are fluorescence-based assays that are susceptible to autofluorescence or quenching effects of the compounds, further conducted gel- based de-ubiquiniting assays have been conducted to confirm the results. For this purpose, the protease activity of PL” and the inhibitory potential of ACF on tri-ubiquitin K48 chains have been tested. First, K48 tri-ubigiutin has ben incubated with PL" and samples of different time points were taken to analyze the cleavage of these chains in Western Blot assays. It could be seen that PLP efficiently cleaved K48 tri-ubiquitin to di-ubiquitin (Figure 1C). Next, PL" was incubated with either DMSO or 5, 15, 25 uM of ACF, respectively, before adding K48 tri-ubiquitin chains to the reaction mixture. Also in this independent assay format, ACF reduced the protease activity in a dose-dependent manner (Figure 1C), thereby confirming that ACF is a specific PL" inhibitor.

[00160] Cellular validation of the screening hits

[00161] The hit compounds were tested in cell culture and cytotoxicity was verified on Vero cells at three concentrations (100 nM, 1 uM, 10 uM) using the XTT assay. At the same time, the cytopathic effect (CPE) reduction assay was carried out. Amongst 13 compounds, three hampered the development of the cytopathic effect, but the RT-gPCR analysis revealed that only ACF inhibited virus replication (Figure 2).

[00162] Table 2. CPE reduction assay. Initial screen of 13 proposed compounds in given concentrations. Table shows results of cytopathic effect (CPE) reduction assay obtained by microscopic observations. CPE - cytopathic effect; RED - CPE reduction; TOX - toxicity.

‘Compound [10pM !1uM [0.1uM | Nereporin | ope | Evans Blue CPE | Clofazimine 1" CPE 0 ] | Hypericin from | | Hypericus | CPE | perforatum ee Dithanol | CPE US | Chlorophyllin | sodium copper | RED CPE (salt LOL - Acriflavine Protoporphyrin IX 001 CPE 0 Tannic acid ……-- CPE PHA-665752

Proflavine | hemisulfate salt | RED | CPE | LU102310 hydrate __ PL 1 Co _

[00163] Verification of the ACF as selective PL" inhibitor

[00164] NMR ligand-based binding analysis was carried out to validate PL" -ligand interaction. The direct interaction of ACF with PL" was also observed by NMR ligand based assay (peaks of the ACF partially disappear in the presence of the protein) and the results are presented in Figure 3.

[00165] ACF is not a SARS-CoV-2 M°” inhibitor

[00166] To check the specificity of ACF we have performed an enzymatic digestion assay using fluorescent MP substrate. The experiment shown that ACF is a very weak MP” inhibitor with less than 50% inhibition at 100 uM of ACF concentration (Figure 17). We can, therefore, conclude that M°° inhibition has no significant contribution in SARS-CoV-2 inhibition at sub- micromolar concentrations used in the other assays.

[00167] Structural analysis of SARS-CoV-2 PL""° in complex with proflavine

[00168] To gain insight into the molecular basis of the SARS-CoV2-PLpro inhibition by acriflavine, we determined the X-ray crystal structure of the protein in complex with proflavine, one of its principal components. Crystallization of acriflavine complex did not yield diffracting crystals most likely doe to heterogenic nature of the mixture. The proflavine-complex structure was solved in P6;22 space group. There are two molecules of complex in the asymmetric unit. Intriguingly, it was found a disulfate bond which bridges two Cys270 of adjacent Plpro molecules from different asymmetric units. Analysis of the electron density map allowed to build unequivocally 3 molecules of proflavine for each protein molecule (Figure 3). One of these is located at the interface with the symmetric mate and it could be considered as a crystallographic artifact (Figure 3 inlet and Figure 15). More interestingly the other two molecules of proflavine are TT-TT stacked to each other and they accommodate the S3-S5 pockets of the PLpro substrate recognition cleft which is defined by the loop connecting the helices a3 and a4 and the so-called “blocking loop” BL2*. Comparison with the apo structure of PL”® (PDB code: 7D47) reveals that although the overall structure is well preserved the BL2 loop undergoes an induced fit upon the binding. In particular, the side chain of Tyr268 rotates by about 57° inward the substrate recognition cleft and the loop moves by 2A in the same direction narrowing the substrate binding cleft (Figure 16).

[00169] Looking closer at the intramolecular interactions, several Plpro residues are involved in the binding. One of the molecules termed proflavine-l is allocated in the S4 pocket (Figure 4 A, B). The side chain Tyr273 is involved in a hydrogen bond (2.9A) with the primary amine group at position 3 of proflavine-| which sits at the bottom of the substrate binding cleft. This latter forms a stacked-like CH/TT interaction with Pro247 (3.9A) and a L-shaped CHAT interaction with Pro248 (4. 6A). Moreover, the side chain of Tyr264 is engaged in a T-shaped TT- TT stacking interaction (3.5 A) with the same molecule. The second molecule, termen proflavine- Il is TT-TT stacked at 3.5A on top of the other and occupies the S3 and S5 pockets (Figure 4 C, D). Gly163 and Asp164 form a hydrogen bond with the primary amine group at position 3 (2.9 A) and the imine group of the acridine moiety (2.9 A), respectively. In addition, Tyr268 forms a T-shape m-m staked interaction (5.1 A) with this proflavine-Il. Overall, this provides unique binding model where two proflavines, tightly TT-17 stacked, cooperate in blocking the substrate pocket. It is clear that, both molecules are requested for the inhibition. The electron densit analysis shows weaker trace of at least two more proflavines that can be allocated on top of he 02310 other two at optimal distance for - staking forming a continuous, DNA-like stacking from one to another PLpro molecule in the same asymmetric unit. Their electron density is much weaker and does not allow to build these molecules unambiguously (Figure 17). However, they are not involved in any interaction with PLP.

[00170] Recently, Shin et al. solved the crystal structure of SARS-CoV2-PL"" in complex with 1SG15 (interferon-induced gene 15) bearing the RLRGG recognition motif at the C-terminus (1). Comparison with our structure shows that proflavine molecule occupying S3 and S5 pockets well overlaps with the backbone of Arg151 and Arg153 in position P3 and PS, respectively (Figure 5). Whereas, the side chain of Leut52 in position P4 points exactly towards the other molecule of proflavine in the 84 pocket. All the interactions with PLP residues are well preserved. This finding suggests that profiavine inhibits SARS-CoV2-PL°" by limiting access to the substrate. Moreover, since the imine group of the proflavine-l molecule in S4 pocket is not involved in any polar interactions, it can, with without being bond by theory, be assumed that the methylated (acriflavine) form is preferred at this position due to lack of desolvation penalty. The amino group at position 3 of proflavine-Il is located in similar position as amide nitrogen of the glycine P2. Side-methylated proflavine, also present in commercial acriflavine preparations (see Figure 21), would, therefore, mimic the P2 amino acid. These two, expected interactions of commercial acriflavine, explain why it is a significantly more potent inhibitor than pure proflavine.

[00171] NMR validation of the direct PL°"-ACF interaction

[00172] In order to confirm that ACF binds directly to PL" in solution we have recorded 2D ‘H,‘°N TROSY spectra of the PL? in the absence and presence of different concentrations of ACF. The spectra indicated a well-folded, monomeric protein in solution (Figure 6). The addition of ACF caused several resonances to shift. The remaining bulk of the resonances was not altered. This indicates that the ACF binds to a spatially limited, distinct pocket of PL”°. Upon binding the fold and monomeric state of the protein was not altered. This proves that the third proflavine moiety observed in crystal structure at the interface between neighboring PLP"° molecules is a crystallization artifact and that ACF does not cause dimerization of the PLP? in solution. The multiple -1 stacked electron densities observed in X-ray data between adjacent active sites also do not seem to cause oligomerization of the PL” as this would cause much more resonances to shift upon addition of high concentration of ACF.

[00173] ACF and SARS-CoV-2 in cell culture

[00174] For the studies on the SARS-CoV-2, two cell culture models were used: Vero cells, which are broadly used simian model and human A549*“F* cells overexpressing the ACEZ receptor®®. All experiments were carried out in parallel. First the cytotoxicity of the compounds was evaluated on three human cell lines (A549°°%%" Vero, HRT-18) and primary human fibroblasts (Figure 7). The CCso values were estimated in both systems to be 3.1 uM for A549", 3.4 uM for Vero, 2.1 uM for HRT-18, and 12 uM for primary human fibroblasts. Interestingly, ACF shows lower cytotoxicity in primary cells, compared to cell lines, what may be linked with previously described antineoplastic activity.

[00175] Second, the full range dose-response experiment was carried out and the results are presented in Figure 8. The ICs, value calculated based on the presented data was 64 nM for the Vero cells and 86 nM for A549**2* cells. Selectivity index (SI) values for Vero and A549"“F** models are 53 and 36, respectively. LU102310

[00176] Inhibition was also confirmed using inverted light microscopy and confocal microscopy. The reduction of cytopathic effect on Vero cells was observed under light microscopy at 48 hours post infection at different concentrations of ACF (Figure 9). After 24 h and 48 h of infection in the presence of 500 nM ACF, confocal microscopy images show nearly complete inhibition of SARS-CoV-2 replication was observed after 24 and 48 h, as determined with the amount of viral proteins and number of infected cells. While inhibition was also noted for remdesivir, it was inferior compared to ACF (Figure 10).

[00177] Ex vivo inhibition of SARS-CoV-2 infection in HAE cultures.

[00178] The antiviral activity of ACF was analysed on the HAE ex vivo model. Two different concentrations were evaluated (400 nM and 500 nM), PBS and remdesivir were used as controls. Each analysis was performed in duplicate and samples were collected for 144 hours. Figure 11 shows the inhibition of SARS-CoV2 replication in the presence of ACF and remdesivir.

[00179] The results show that ACF hampers SARS-CoV-2 replication in the HAE ex vivo model. A lower viral yield was detected in the cultures treated with ACF in comparison with the PBS control. ACF treated HAE showed an even higher inhibition of virus replication than the positive control with 10 uM remdesivir after 144 h of infection. At time points of 48 and 72 hours of infection, the virus was under the detection limits in the ACF 500 nM treatment. {00180] Mechanism of action

[00181] To fully delineate the mechanic of the drug activity we carried out the time-of- addition experiment, where the cells were first infected with the virus and the ACF was added after 0, 2, 4 or 6 h. The results clearly show that the virus inhibition was maintaind also when the compound was not present during early stages of the infection. Consequently, it is inhibited mainly during the replication phase (Figure 13).

[00182] Pan-coronavirus activity of ACF

[00183] To verify whether ACF may be used as a generic anticoronaviral drug, its activity against other betacoronaviruses (MERS-CoV, HCoV-0QC43) and alphacoronaviruses (HCoV- NL63 and feline infectious peritonitis virus (FIPV) was tested. ACF inhibited MERS-CoV even stronger than SARS-CoV-2 (IC5= 21 nM, SI = 162). Surprisingly, ACF exhibited weaker action on HCoV-OC43, only minor, not statistically significant inhibition was observed (< 1 log at 1 uM, ICs; = 105 nM, SI = 27). No effect on any of tested alphacoronaviruses replication in tested concentrations (Figure 14) was observed.

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Claims (15)

1. A composition comprising at least one compound according to formula (I) Ra = R3 Sn NF NT H X | H R4 (1) R, is selected from the group consisting of (C--C;) alkyl, preferably methyt; R: is selected from the group consisting of H, (C,-C;) alkyl, preferably H; Rj is selected from the group consisting of H, (C;-Ce) alkyl, preferably H; X is an anion; for use in the treatment of diseases caused by betacoronaviruses.

2. The composition of claim 1, wherein a) the treatment is caused by human and veterinary coronaviruses that belong to subgenera hibecovirus, nobecovirus, embecovirus, merbecovirus and sarbecovirus, preferably coronaviruses or b) the treatment is caused by human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-0C43), Middle East respiratory syndrome-related coronavirus (MERS-CoV) or c) the treatment is caused by severe acute respiratory syndrome-related coronaviruses (SARS- CoV, SARS-CoV-2) or d)the treatment is caused by a virus that evolve or mutate from the species described in a) to c).

3. The composition of claim 1 or 2, wherein X is selected from the group consisting of napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, tecclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexancate, octanoate, sulfonate, chloride, iodide, bromide, phosphate, phosphonate, lactate, malate, citrate, tartrate, fumarate, gluconate, sulfate or hemisulfate, preferably chloride, iodide, bromide and sulfonate, most preferably chloride.

4. The composition of any one of claims 1 to 3, wherein R,, R and/or R; are independently selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl,

iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbuty! (iso-pentyl PP L102810 iso-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethyipropyl, hexyl, 2-hexyl, 3-hexyl, Z- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl, preferably methyl.

5. The composition of any one of claims 1 to 4, further comprising at least one compound according to formula (Il) Ra Rs Re z Xx

PF Y N NHR; (Il) wherein R, is H, halogen, (C;-Ce)alkyl, (Cs-Cg)cycloalkyl, or -O(Cy-Cs)alkyl; preferably H, Rs is H, halogen, (C,-Cg)alky!, (C3-Cs)cycloalkyl, or -O(C4-Cg)alkyl, preferably H: Rs is H, halogen, (C,-Cglalkyl, (Ca-Cg)cycloalkyl, or -O{C,-Cg)alkyl; preferably H; R;is H, (C,-C;) alkyl, preferably methyl or H; Z is H, halogen, (C,-Cs)alkyl, (Cz-Ce)cycloalkyi, -O(C,-Cs)alkyl, -(C,-Ce)alkyl(Cs-Cyo)aryl, -(C1- Cç)alkyl(Cs-Cyc)heteroaryl; preferably H; Y is -NHz, -NHRg, halogen, (C,-Cs)alkyl, (C:-Cs)cycloalkyl; preferably —NHz Re is H, (C4-Cglalkyl, preferably methyl,

6. The compasition of claim 5 for use, wherein a) the molar ratio of the at least one compound according to formula (1) is 5 to 100, preferably 10 to 40, preferably to 30 mol-% based on the overall molar ratio of parent compounds (I) and (Il) of the composition and compound (Il) is 0 to 95, preferably 60 to 90, more preferably 70 to 85 mol%.-% based on the overall molar ratio of parent compounds (I) and (II) of the composition and/or b) Ri. Rs, Re, Rr, and/or Rs are (C,-C;)alkyl, preferably independently selected from the group of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3-methylbutyl (iso-pentyl oder /so-amyl), 3-methylbut-2-yl, 2-methylbut- 2-yl, 2 2-dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2- dimethylbutyl, and 2,3-dimethylbutyl, more preferably methyl and/or c) Y is (C,-Cç)alkyl, preferably independently selected from the group of methyl, ethyl, propyl, butyl, pentyl, iso-propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl 2-pentyl, 3-pentyl, Lh 02310 methylbutyl, 3-methylbuty! (iso-pentyl oder iso-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2 2- dimethylpropyl, hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethytbutyl, more preferably methyl.

7. The composition of any one of claims 1 to 6 for use, wherein compound (I) and/or (Il) is a solvate, hydrate, salt, complex, or isotopically enriched form, preferably a salt.

8. The composition of claim 7, wherein compound (I) is a salt, wherein the salt comprises an anion selected preferably from the group consisting of napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, iodide, bromide, phosphate, phosphonate, lactate, malate, citrate, tartrate, fumarate, gluconate, sulfate or hemisulfate, more preferably a sulfate, hemisulfate, or chloride, most preferably chloride.

9. The composition of claim 7 wherein compound (Il) is a sait, preferably the salt comprises an anion selected preferably from the group consisting of napsylate, glycollylarsanilate, nitrate, benzoate, hexylresorcinate, oleate, bitartrate, hydroxynaphthoate, pantothenate, bicarbonate, hydrabamine, pamoate, camsylate, isethionate, polygalacturonate, propionate, salicylate, lactobionate, stearate, decanoate, edetate, maleate, succinate, estolate, mandelate, teoclate, gluceptate, acetate, glutamate, muscate, aspartate, glycolate, benzenesulfonate, hexanoate, octanoate, sulfonate, chloride, iodide, bromide, phosphate, phosphonate, lactate, malate, citrate, tartrate, fumarate, gluconate, sulfate or hemisulfate, more preferably a sulfate, hemisulfate, chloride, most preferably chloride.

10. The composition of claims 3, 8 and 9 wherein the sulfonate is a sulfonate according to formula (II)

I

TC 0 (I) wherein Ra is selected from the group consisting of phenyl, 4-nitrophenyl, 4-methylphenyl, 4- trifluoromethyphenyl, trifluoromethyl, and (C:-Cs)alky!.

11. The composition of claim 10, wherein LU102310 a) Rs is (C,-Cs)alkyl and/or b) (C:-Cs)alkyl is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, iso- propyl, sec-propyl, iso-butyl, sec-butyl, tert-butyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 3- methylbutyl (iso-pentyl oder iso-amyl), 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2-dimethylpropyl, preferably methyl.

12. A composition in which at least one compound according to formula | and at least one compound according to formula Il are bonded together via one or two linker systems wherein optionally a) the linker system is (C>-Cyo)alkylene, or (C,-Cip)alkenyl, preferably (Cs;-Cglalkyl, or (C3 s)alkenyl; wherein optionally at least one or at least two CHz-groups in these alkyl or alkenyl groups are substituted by O, S, S(O), NH or N(C,-C,)alkyl and/or b) connecting is performed preferably via position 5, 7, 8 or by substitution of the nitrogen in position 6 in formula (I) and position 5, 7, 8 or by substitution of the nitrogen in position 6 or 10 in formula (ll) wherein the underlying aromatic system in formula (I) and (ll) is numbered 1120 according to formula (IV): PT: (IV) and/or c) the composition is for use in the treatment of betacoronaviruses, optionally i) the treatment is caused by human and veterinary coronaviruses that belong to subgenera hibecovirus, nobecovirus, embecovirus, merbecovirus and sarbecovirus, preferably coronaviruses or ii) the treatment is caused by human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV) or iii) the treatment is caused by severe acute respiratory syndrome-related coronaviruses (SARS- CoV, SARS-CoV-2) or ivithe treatment is caused by a virus that evolve or mutate from the species described in i) to iii).

13. The composition of any one of claims 1 to 12 further comprising at least one pharmaceutically acceptable carrier.

14. The composition for use of any one of claims 1 to 13 formulated as an inhalative drug.

15. The composition for use of any one of claims 1 to 11 formulated as an oral drug.

LU102310