COMPOUNDS AND METHODS FOR PREVENTION AND TREATMENT OF

CORONAVIRUS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of and priority to U.S. Provisional

Application Ser. Nos. 63/018,690, filed May 1, 2020, which is incorporated herein by reference in entirety.

FIELD OF THE INVENTION [0002] The present invention provides some anti-coronaviral compounds and the method and composition/pharmaceutical composition for prevention and treatment of coronavirus infections, particularly the disease caused by SARS-COV-2.

BACKGROUND OF THE INVENTION [0003] severe acute respiratory syndrome (SARS) outbreak in November 1 ^st , 2002 to June 18 ^th , 2003 led to 801 deaths in over 29 countries and 8465 probable cases around the world according to the World Health Organization (WHO) (Chen et al, 2005). SARS, an enveloped b coronavirus containing positive-sense, single-stranded RNA, has a genome size of about 30 kb, in which open reading frame (ORF) la and lb encode for two respective polyproteins (pps), ppla and pplab (Hegyi etal, 2002; Needle et cil, 2015). To complete its lifecycle, successful replication and proteolytic processing are imperative (Herold et al,

1998). Indeed, the consensus functions of these virus-encoded proteolytic proteins are found in all coronaviruses, specifically papline-like protease (PLpro) and chymotrypsin-like protease (3CLpro) (Herold et al. , 1998). In proteolytic processing of ppla and pplab, PLpro and 3CLpro cleave the first three sites and the remaining 11 locations, respectively, yielding a total of 16 nonstructural proteins (nspl-16) (Hegyi et al. , 2002; Needle et al. , 2015). Thus, 3CLpro inhibition has been regarded as a molecular approach in anti-SARS drug discovery and developments (Chen et al, 2005; Jo et al, 2020). [0004] SARS-COV-2 is a novel coronavirus that spreads rapidly since its identification in patients with severe pneumonia in Wuhan, China (named as COVID-19), has been reported in 25 countries, with nearly 72000 laboratory-confirmed cases and a death toll of 1775worldwideasofFebruary17 ^th ,2020(Coronavirusdisease2019(Covid-19)Situation Report 28,2020;Li& DeClercq,2020).Devastatingly,nodrugorvaccinehasyetbeen approvedtotreathumancoronaviruses(Li& DeClercq,2020).Concerningthecurrent outbreakofSARS-CoV-2andthetherapeuticexperienceofSARSandMERS (anotherb coronavirus),manystudiesextensivelyinvestigatethepossibility ofusingtheexisting antiviralagentsusedforHIV,hepatitisB virus,hepatitisC virusandinfluenzainfectionsfor thetreatmentorinterventionofSARS-COV-2(DeClercq& Li,2016;Li& DeClercq,

2020).Inthemeantime,SARS-CoV-2hasbeencharacterizedasanenv eloped,positive- sense,single-strandedRNA b coronavirus,similartoSARSandMERS(Li& DeClercq, 2020).Consistentwiththecharacteristicsofcoronaviruses,SARS-C oV-2genomeencodes structuralproteins(e.g.,spikeglycoproteins),nonstructuralpro teins(e.g.,3CLpro,PLpro, helicase,RNA-dependentRNA polymerase),andaccessoryproteins.Regardingtheavailable genomicsequenceofSARS-COV-2,SARSandMERS,ahigh-levelconservat ionofthe proteolyticsitesandproteolyticenzymeswasfound,whencerepurpos ingSARSandMERS proteaseinhibitorsfortreatmentofSARS-COV-2isworthconsidering (Liuetal.,2020).As 3CLproplaysapivotalroleinSARS,itisreasonabletoapproachprotea seinhibitionby targetingthe3CLproofSARS-COV-2insteadofitsPLprotointerceptit slifecycle(Chenet al,2005;Joetal,2020;Liuetal,2020). [0005] Currently,disulfiram,anapproveddrugtotreatalcoholdependence, hasbeen reportedtoinhibitthePLproofMERSandSARSincellculturesbuthasye tbeenevaluated clinically(Li& DeClercq,2020).Inaddition,clinicaltrialsofHIV proteaseinhibitors (lopinavirandritonavir)inSARS-CoV-2patientshavealsocommenced ,yetitisuncertainif itcaneffectuallyinhibitthoseofSARS-CoV-2,asHIV andbcoronavirusproteasesbelong totheasparticproteasefamilyandthecysteineproteasefamily,resp ectively(Li& DeClercq, 2020;Zumlaetal,2016).Ontheotherhand,remdesivir,anucleotidean alogofRNA dependentRNA polymeraseinhibitorapprovedforHIV treatment,iscurrentlyunderclinical trialsinSARS-CoV-2patientswithestimatedcompletiondatesinApri l,2020;galidesivir, anothernucleotideanalogofRNA dependentRNA polymeraseinhibitorinearly-stage clinicalstudiesforHCV treatment,hasshownbroad-spectrum antiviralactivitiesagainst severeacuterespiratorysyndrome(SARS),MiddleEastrespiratorysy ndrome(MERS)in preclinicalstudies(Wangetal,2020;Zumlaetal,2016).However,one mightexpectthata nucleosideanalogcanelicittoxicitythatarestillbeyondourknowle dge(Feng,2018). [0006] Thereareyettofindvaccinesorantiviraldrugstopreventortreathum an coronavirusinfections.Thereisanurgentneedforexploringanddeve lopingasafeanti- coronavirustherapy,particularlyagainstSARS-COV-2.

BRIEFSUMMARY OFTHEINVENTION [0007] Itisunexpectedlyfoundinthepresentinventionthatsometriterpene sare effectiveininhibitionofcoronavirusinfections,especiallySARS- COV-2. [0008] Inoneobject,thepresentinventionprovidesamethodforpreventinga nd/or treatingacoronavirusinfection,comprisingadministeringtoasubj ectinneedthereofa compoundorapharmaceuticallyacceptablesaltthereof,inwhichthec ompoundisselected from thegroupconsistingof:

(Ib),

(Ie), whereinRiisO,a-OH orb-H;R2isH orOH;R ₃ isO,a-H,b-OH,b-OAcorH2;R4isH or OH; R5isH,OH orORx;R ₆ isCOORxorCOO(CH2)n-CH3;nisanintegerfrom 0-3;R7is H,Oh,ORxorOAc;R ₈ isCH3orCOORx;R21isCH3,COORx,orCOO(CH2)n-CH3;nisan integerfrom 0-3;thedottedlinerepresentsasinglebondoradoublebond,RxisH oraCi-8 alkyl;and

whereineachofR21,R22,R23,R24,R25,R26,R27,R28,R29,R30,XiandX2 isH,OH,Ci-8alkyl, NRx,SRx,ORx,pyrazoline,cysteine,glutathione,halogen,COORx,or COO(CH2)n-CH3;n isanintegerfrom 0-3;eachofYi,Y2,Zi,Z2,WiandW21SH,OH,Ci-8alkyl,orXiandX2 togetherform -0-,orY1andY2,ZiandZ2,orWiandW2togetherform anepoxy. [0009] Insomeparticularexamplesofthepresentinvention,thecompoundiss elected from thegroupconsistingof:

(sulphurenicacid), [0010] Inonefurtheraspect,thepresentinventionprovidesa composition/pharmaceuticalcompositionforpreventingand/ortrea tingacoronavirus infection,particularlySARS-COV-2comprisingatherapeuticallyef fectiveamountofanyof thecompoundsdisclosedhereinorpharmaceuticallyacceptablethere of,oritsmixture,in combinationofapharmaceuticallyacceptablecarrier. [0011] Optionally,thecomposition/pharmaceuticalcompositionaccording tothe inventionmaycompriseatleastoneadditionalanti-viraltherapeuti cagent. [0012] Inoneyetaspect,thepresentinventionprovidesauseofanyofthecomp ounds disclosedhereinorpharmaceuticallyacceptablesalts,oritsmixtur eformanufacturinga medicamentforpreventingand/ortreatingacoronavirusinfection,p articularlySARS-COV- 2 [0013] Itistobeunderstoodthatboththeforegoinggeneraldescriptionandt he followingdetaileddescriptionareexemplaryandexplanatoryonlyan darenotrestrictiveof theinvention.

BRIEFDESCRIPTION OFTHE SEVERALVIEWSOFTHE DRAWINGS [0014] Theforegoingsummary,aswellasthefollowingdetaileddescriptiono fthe invention,willbebetterunderstoodwhenreadinconjunctionwiththe appendeddrawings. Forthepurposeofillustratingtheinvention,thereareshowninthedr awingsembodiments whicharepresentlypreferred. [0015] Inthedrawings: [0016] Figure1showstheinhibitoryprofilesofAR100-DS1,AR101DS1,AR101D S2, ARIOIDS3andAR101DS4attheconcentrationof20mM.*,P<0.05;**,P <.001;***, P<.001. [0017] Figure2A showstherelative3CLproactivity(%)ofAR101DS2(0.5p/5FP),and IC50=39pM. [0018] Figure2B showstherelative3CLproactivity(%)ofAR101DS2 (0.125p/1.25FP),andIC50=15.82pM. [0019] Figure2C showstherelative3CLproactivity(%)ofAR101DS3(0.5p/5FP),and IC50=14.61pM. [0020] Figure2D showstherelative3CLproactivity(%)ofARIOIDS3(0.5p/5FP),and IC50=11.06pM. [0021] Figure2Eshowstherelative3CLproactivity(%)ofAR101DS3 (0.125p/1.25FP),andIC50=11.7pM. [0022] Figure3showstherelative3CLproactivity(%)ofAR100DS1(0.125p/1. 25FP), andIC50=21.31mM.

DETAILED DESCRIPTION OFTHEINVENTION [0023] Theabovesummaryofthepresentinventionwillbefurtherdescribedwi th referencetotheembodimentsofthefollowingexamples.However,itsh ouldnotbe understoodthatthecontentofthepresentinventionisonlylimitedto thefollowing embodiments,andalltheinventionsbasedontheabove-mentionedcont entsofthepresent inventionbelongtothescopeofthepresentinvention. [0024] Unlessdefinedotherwise,alltechnicalandscientifictermsusedher einhavethe samemeaningascommonlyunderstoodbyapersonskilledinthearttowhi chthisinvention belongs. [0025] Asusedherein,thesingularforms “a”, “an”,and “the”includepluralreferents unlessthecontextclearlydictatesotherwise.Thus,forexample,ref erenceto “asample” includesapluralityofsuchsamplesandequivalentsthereofknowntot hoseskilledintheart. [0026] Inthepresentinvention,toevaluatetheeffectofprospectingdrugso nproteolytic processinginhibitioninhigh-throughput,thesyntheticpeptidesla belledfluorescence resonanceenergytransfer(FRET)pairswereemployedasthoseusedint hepreviousstudies, inwhichthequenchedfluorophoreisreleaseduponcleavageoftheFRET -labelledpeptides, generatingfluorescentsignalsthatcanbemonitoredinreal-time(Ch enetal,2005;Jeanet al.,1995;Joetak,2020).Itisconfirmedinthepresentinventionthat anyoritsmixtureof thecompoundsdisclosedhereiniseffectiveininhibitionofacystein eprotease,particularly 3CLproofSARS-CoV-2. [0027] Thepresentinventionprovidesamethodforpreventingand/ortreatin ga coronavirusinfection,particularlySARS-COV-2comprisingadminis teringtoasubjectin needthereofacompoundorpharmaceuticallyacceptablesaltthereof, oritsmixture,inwhich thecompoundisselectedfrom thegroupconsistingof:

(1)

(Ie), whereinRiisO,a-OH orb-H;R.2isH orOH;R ₃ isO,a-H,b-OH,b-OAcorH2;R4isH or OH; R5isH,OH orORx;R ₆ isCOORxorCOO(CH2)n-CH3;nisanintegerfrom 0-3;R7is H,Oh,ORxorOAc;R ₈ isCH3orCOORx;R21isCH3,COORx,orCOO(CH2)n-CH3;nisan integerfrom 0-3;,thedottedlinerepresentsasinglebondoradoublebond,RxisH oraCi-8 alkyl;and (2) whereineachofR21,R22,R23,R24,R25,R26,R27,R28,R29,R30,XiandX2 isH,OH,Ci-8alkyl, NRx,SRx,ORx,pyrazoline,cysteine,glutathione,halogen,COORx,or COO(CH2)n-CH3;n isanintegerfrom 0-3;eachofYi,Y2,Zi,Z2,WiandW21SH,OH,Ci-8alkyl,orXiandX2 togetherform -O-,orY1andY2,ZiandZ2,orWiandW2togetherform anepoxy. [0028] Thepresentinventionalsoprovidesacomposition/pharmaceuticalco mposition forpreventingand/ortreatingacoronavirusinfection,particularl ySARS-COV-2infection, whichcomprisesatherapeuticallyeffectiveamountofacompoundasdi sclosedhereinor mixturethereofandapharmaceuticallyacceptablecarrier. [0029] Inoneembodiment,thecompoundis

(1)

(Ie); wherein Ri is O, a-OH or b-H; R2 is H or OH; R3 is O, a-H, b-OH, b-OAc or H2; R4 is H or OH; R5 is H, OH or ORx; R ₆ is COORx or COO(CH2)n-CH3; n is an integer from 0-3; R7 is H, Oh, ORx or OAc; R ₈ is CH3 or COORx; R21 is CH3, COORx, or COO(CH2)n-CH3; n is an integer from 0-3; the dotted line represents a single bond or a double bond, Rx is H or a Ci-8 alkyl; or (2) wherein each of R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, Xi and X2 is H, OH, Ci-8 alkyl , NRx, SRx, ORx, pyrazoline, cysteine, glutathione, halogen, COORx, or COO(CH2)n-CH3; n is an integer from 0-3, Rx is H or a Ci-8 alkyl; each of Y 1, Y2, Zi, Z2, Wi and W2 is H, OH, Ci-8 alkyl , Xi and X2 together form -0-, or Y 1 and Y2, Zi and Z2, or Wi and W2 together form an epoxy.

[ 0030 ] In the example of the invention, the compound of formula (I) may be:

[0031] Inanotherexampleoftheinvention,thecompoundofformula(I)maybe:

R ₇ =H;R ₈ =CH ₃ [0032] Inoneyetexampleoftheinvention,thecompoundofformula(I)maybe:

,, [0033] Infurtherexampleoftheinvention,thecompoundofformula(I)maybe: [0034] Inoneparticularexampleoftheinvention,thecompoundofformula(I) maybe lanostane: 【0035】 Ina

oritsderivatives,suchas [0036] Accordingly,thepreferredcompoundisselectedfrom thegroupconsistingof:

((20x)-3b,15a,16a-trihydroxy-24-methyllanosta-7,9(l1),24( 24l)-trien-21-oicacid;15a- hydroxydehydrotumulosicacid),

(methyl25-hydroxy-3-epidehydrotumulosate(methyl)), (15α-acetyldehydrosulphurenicacid), (versisponicacidD), HOOC )-16,29-dihydroxy-24- methylidenelanosta-7,9(11)-dien-21-oicacid), HOOC ( _{dehydroeburicoic acid),}

[0037] Accordingtotheinvention,themostpreferredcompoundisselectedfr om the groupconsistingof:

[0038] Inoneparticularexampleoftheinvention,thecompoundofformula(II )is [0039] Theterm “coronavirus”asusedhereinreferstoaCoronaviruseinthesubfa mily Orthocoronavirinae,thefamilyCoronaviridae,orderNidovirales,a ndrealm Riboviria, whichisenvelopedviruseswithapositive-sensesingle-strandedRNA genomeanda nucleocapsideofhelicalsymmetry.Theyhavecharacteristicclub-sh apedspikesthatproject from theirsurface,whichinelectronmicrographscreateanimagereminisc entofthesolar coronafrom whichtheirnamederives.Coronavirusescausediseasesinmammalsand birds, includinghumans.Inhumans,coronavirusescauserespiratorytracti nfections,including commoncold,severeacuterespiratorysyndrome(SARS),MiddleEastre spiratorysyndrome (MERS),andSARS-COV-2. [0040] Theterm “cysteineprotease”asusedhereinreferstothiolproteases,are enzymes thatdegradeproteins,sharingacommoncatalyticmechanism thatinvolvesanucleophilic cysteinethiolinacatalytictriadorduad.Oneexampleofcysteinepro teaseinavirusis 3CLproinSARS-COV-2. [0041] Theterm “treat,” “treating”or “treatment”asusedhereinreferstotheapplication oradministrationofacompositionincludingoneormoreactiveagents toasubjectafflicted withadisease,asymptom orconditionsofthedisease,oraprogressionofthedisease,with thepurposetocure,heal,alleviate,relieve,alter,remedy,amelior ate,improve,oraffectthe disease,thesymptomsorconditionsofthedisease,thedisabilitiesi nducedbythedisease,or theprogressionofthedisease. [0042] Theterm “prevent,” “prevention”or “preventing”asusedhereinreferstothe preventionoftherecurrence,onset,ordevelopmentofavirusinfecti on,oneormore symptomsthereof,orarespiratoryconditionassociatedwith,potent iatedby,orpotentiatinga coronavirusinfectioninasubject. [0043] Theterm “subject”asusedhereinincludeshumanornon-humananimals,suc has companionanimals(e.g.dogs,cats,etc.),farm animals(e.g.cattle,sheep,pigs,horses,etc.), orexperimentalanimals(e.g.rats,mice,guineapigs,etc.). [0044] Theterm “therapeuticallyeffectiveamount”asusedhereinreferstoanam ountof apharmaceuticalagentwhich,ascomparedtoacorrespondingsubjectw hohasnotreceived suchamount,resultsinaneffectintreatment,healing,prevention,o rameliorationofa disease,disorder,orsideeffect,oradecreaseintherateofadvancem entofadiseaseor disorder.Theterm alsoincludeswithinitsscopeamountseffectivetoenhancenormal physiologicalfunction. [0045] Foruseintherapy,thetherapeuticallyeffectiveamountofthecompou ndis formulatedasapharmaceuticalcompositionforadministration.Acco rdingly,theinvention furtherprovidesapharmaceuticalcompositioncomprisingatherapeu ticallyeffectiveamount ofanyoritsmixtureofthesecompoundsdisclosedherein,andoneormor epharmaceutically acceptablecarriers. [0046] Forthepurposeofdeliveryandabsorption,atherapeuticallyeffecti veamountof theactiveingredientaccordingtothepresentinventionmaybeformul atedintoa pharmaceuticalcompositioninasuitableform withapharmaceuticallyacceptablecarrier. Basedontheroutesofadministration,thepharmaceuticalcompositio nofthepresent inventioncomprisespreferablyfrom 0.1% to100% inweightofthetotalweightoftheactive ingredient. [0047] Theterm "pharmaceuticallyacceptablecarrier"usedhereinreferstoacarrie r(s), diluent(s)orexcipient(s)thatisacceptable,inthesenseofbeingco mpatiblewiththeother ingredientsoftheformulationandnotdeleterioustothesubjecttobe administeredwiththe pharmaceuticalcomposition.Anycarrier,diluentorexcipientcommo nlyknownorusedin thefieldmaybeusedintheinvention,dependingtotherequirementsof thepharmaceutical formulation.Saidcarriermaybeadiluent,vehicle,excipient,ormat rixtotheactive ingredient.Someexamplesofappropriateexcipientsincludelactose ,dextrose,sucrose, sorbose,mannose,starch,Arabicgum,calcium phosphate,alginates,tragacanthgum,gelatin, calcium silicate,microcrystallinecellulose,polyvinylpyrrolidone,cell ulose,sterilizedwater, syrup,andmethylcellulose.Thecompositionmayadditionallycompri selubricants,suchas talc,magnesium stearate,andmineraloil;wettingagents;emulsifyingandsuspendin gagents; preservatives,suchasmethylandpropylhydroxybenzoates;sweetene rs;andflavoring agents. [0048] Thecompositionofthepresentinventioncanprovidetheeffectofrapi d, continued,ordelayedreleaseoftheactiveingredientafteradminist rationtothepatient. Accordingtotheinvention,thepharmaceuticalcompositionmaybeada ptedfor administrationbyanyappropriateroute,includingbutnotlimitedto oral,rectal,nasal, topical,vaginal,orparenteralroute(suchasintramuscular,intrav enous,subcutaneous,and intraperitoneal),transdermal,suppository,andintranasalmethod s. [0049] Regardingparenteraladministration,itispreferablyusedinthefor m ofasterile watersolution,whichmaycompriseothersubstances,suchassaltsorg lucosesufficientto makethesolutionisotonictoblood.Thewatersolutionmaybeappropri atelybuffered (preferablywithapH valueof3to9)asneeded.Preparationofanappropriateparenteral compositionundersterileconditionsmaybeaccomplishedwithstanda rdpharmacological techniqueswellknowntopersonsskilledintheart. [0050] Inoneparticularexampleoftheinvention,thepharmaceuticalcompos itionis formulatedfororaladministration.Suchformulationsmaybeprepare dbyanymethodknown intheartofpharmacy.Accordingtothepresentinvention,theform ofsaidcompositionmay betablets,pills,powder,lozenges,packets,troches,elixers,susp ensions,lotions,solutions, syrups,softandhardgelatincapsules,suppositories,sterilizedin jectionfluid,andpackaged powder. [0051] Intheinvention,themethodandcomposition/pharmaceuticalcomposi tionare effectiveintreatingavirusinfectionthroughaninhibitionofacyst eineproteaseinavirus, particularlyanRNA-dependentvirus.Accordingly,theinventionals oprovidesamethodand composition/pharmaceuticalcompositionfortreatmentand/orpreve ntionofavirusinfection throughinhibitionofacysteineproteaseinavirus,comprisingusing thecompounds disclosedhereinorpharmaceuticallyacceptablesaltthereof. [0052] Exemplifiedviruseswhichareresponsiveinclude,withoutlimitatio n,a coronavirus,andaHIV.Preferably,theviralinfectionisacoronavir us.Morepreferably,the viralinfectionisaSARS,MERSandSARS-COV-2. [0053] Inanotheraspect,thepresentinventionprovidesamethodfortreatin gor preventinganRNA-dependentvirusinfectionthroughinhibitingacys teineproteaseina virus.OneexampleofthevirusisanRNA-dependentvirus,suchasSARS, MERSand SARS-COV-2;particularlySARS-COV-2. [0054] Inonefurtheraspect,thepresentinventionprovidesa composition/pharmaceuticalcompositionfortreatingand/orpreven tingavirusinfection throughinhibitingacysteineproteaseinavirus,whichcomprisesany ofthecompounds disclosedherein,pharmaceuticallyacceptablesaltthereof,oritsm ixture.Optionally,the composition/pharmaceuticalcompositionmaycompriseatleastonead ditionalanti-viral therapeuticagent. [0055] Inonefurtheraspect,thepresentinventionprovidesauseofanyofthe compoundsdisclosedhereinformanufacturingamedicamentfortreati ngorpreventinga virusinfectionthroughinhibitingacysteineproteaseinavirus. [0056] Thepresentinventionisfurtherillustratedbythefollowingexample s,whichare providedforthepurposeofdemonstrationratherthanlimitation. [0057] Examples [0058] MaterialsandMethods [0059] I.FRET proteaseassayswiththeSARS-CoV-23CLpro [0060] TheestablishmentofanED-FRETplatform followstheprotocolgivenbyJoet al.(2020).Briefly,acustom proteolytic,fluorogenicpeptidewithDABCYL andEDANSon ends,DABCYL-TSAVLQSGFRKMG-EDANS(Genomics,Taiwan),containsthe consensus nsp4/nsp5cleavagesequencethatcanberecognizedby3CLproofSARS-C oV-2.The peptideisdissolvedindistilledwaterandincubatedwith3CLproofSA RS-CoV-2. Measurementsofthespectral-basedfluorescencearedeterminedbyaS PARK® multimode microplatereaderprovidedbyTECAN.Theproteolyticactivityisdete rminedat37°C by fluorescentintensityofEDANSuponpeptidehydrolysisasafunctiono ftime,inwhich λexcitation=340nm,λemission=490nm,bandwidths=9,15nm,respec tively.Priortotheassay, theemissionwavelengthofthetestingdrugsat340nm excitationisexaminedtoensurethat itdoesnotoverlapwiththeemissionspectrum ofEDANS. [0061] Assaysareconductedintriplicateinblack96-wellmicroplates(Grei ner)in100 μL assaybufferscontaining3CLproofSARS-CoV-2andthecustomizedpept ide.InSARS 3CLproassay,1pM SARS-CoV-23CLprocontaining50mM TrispH 6.5isincubatedwith 5pM fluorescentsubstrateat37°C for3hbeforemeasuringRelativeFluorescenceUnit (RFU). [0062] II.Inhibitionassaysinthepresenceofthecompoundsaccordingtothe invention [0063] Therearefiveexamplecompounds(AR100-DS1,AR101-DS1,AR101-DS2, AR101-DS3,andAR101-DS4)inthefirstscreeningprocessat20pM (n=3).Thefive compoundsaregiveninthetablebelow. [0064] Atfirst,theSARS-CoV-23CLproandArjildrugswillbemixedand pre-incubateat37°C for1h.Thosemanifestinginhibitoryactivityagainst3CLpro ofSARS-CoV-2willbeinvestigatedfurtheratdifferentconcentratio nsto characterizetheirIC50values,usingGraphPadPrism 7.03(GraphPadSoftware, SanDiego,CA,USA). [0065] Basedtheknowledgeandsequence-basedSARS-CoV-23CLpro,the efficacyof3CLproinhibitingthecompoundsaccordingtotheinventio nwere assessedinvitrotodeterminetheirtherapeuticpotentialinSARS-Co V-2treatment. ConcerningthatnodrugorvaccinehasyetbeenapprovedtotreathumanS ARS- CoV-2infection,developingabroad-spectrum antiviralagenttocombatagainst SARS-CoV-2isofutmostimportanceandurgency.EnactmentofED-FRET technologyanditsworkflow providesarobust,high-throughputdrugdiscoveryin thelab.Meanwhile,identificationofSARS-CoV-23CLproinhibitinga gentsfrom the5examplecompoundsaccordingtotheinventionactsasguidelineso fprobable therapeuticdosesinclinicalassessmentandpromptspatentapplicat ioninthe future,contributingtoantivirallibraryconstruction. [0066] III.Results [0067] 1.IdentificationofSARS-CoV-23CLproinhibitors [0068] Todeterminetheefficacyofthefiveexamplecompoundsaccordingto theinvention,SARS-CoV-23CLpro,IQFpeptidesubstrate,andtheexam ple compoundswereincubatedintheconstructedED-FRETplatform at0.5mM,5pM and20pM,respectively.AsshowninFigure1,allfiveoftheexamplecom pounds substantiallyinhibitedthepeptide-cleavingactivityofSARS-CoV- 23CLpro. Amongthesefiveexamplecompounds,AR101DS2andAR101DS3manifested theutmostinhibitoryeffectuponat20pM.Inparticular,theSARS-CoV -2 3CLproinhibitionatthepercentageofabout50% wasfoundafterthree-hour treatmentofAR101DS3,whileAR101DS2demonstratedaninhibitionby4 0% in thesamecondition,markingtheirpotentialsincombatingthepolypro tein processingcycleofSARS-CoV-2. [0069] 2.Characterizationofthehalfmaximalinhibitoryconcentrationof inhibitors [0070] TheinhibitoryprofilesofAR100-DS1,AR101DS1,AR101DS2, AR101DS3andAR101DS4attheconcentrationof20pM weredeterminedand giveninFigure1.AsgiveninFigure1,thehalfmaximalinhibitoryconc entration (IC)towardSARS-CoV-23CLprowascharacterizedbytreatingthecompo undsat theindicatedconcentrationsrangingfrom 0pM to200pM.TheIC50valuesof AR101DS2andAR101DS3weregiveninFigures2A-2E.AsshowninFigure 2A,AR101DS2hadanIC50valueof39pM inthepresenceof0.5pM SARS- CoV-23CLproand5pM IQFpeptidesubstrate(FP).Meanwhile,theinhibitionof 0.125pM AR101DS2onSARS-CoV-23CLproand1.25pM IQFpeptide substratewasdetermined(seeFigure2B),theIC50valueofAR101DS2ag ainst SARS-CoV-2situatedat15.82pM.AsfortheIC50valueofAR101DS3,50% SARS-CoV-23CLproinhibitionwasreachedat14.61pM in0.5pM SARS-CoV-2 3CLproand5pM IQFpeptidesubstrate(seeFigure2C),at11.06pM in0.25pM SARS-CoV-23CLproand2.5pM IQFpeptidesubstrate(seeFigure2D),orat 11.7pM in0.125pM SARS-CoV-23CLproand1.25pM IQFpeptidesubstrate (seeFigure2E). [0071] TheIC50valuesofAR100DS1weregiveninFigure3.AR100DS1 hadanIC50valueof21.31mM inthepresenceof0.125pM SARS-CoV-23CLpro and1.25pM IQFpeptidesubstrate. [0072] Giventheabove,alltheexamplecompoundsincludingAR100DS1, AR101DS1,AR101DS2,AR101DS3andAR101DS4wereconfirmedtohave theinhibitoryeffectonSARS-CoV-23CLprohighlightstheirtherapeu tic potentialsagainstSARS-CoV-2,particularlyAR100DS1,AR101DS2and AR101 DS3. [0073] Allpublications,patents,andpatentdocumentscitedhereinabovear e incorporatedbyreferenceherein,asthoughindividuallyincorporat edbyreference. [0074] Theinventionhasbeendescribedwithreferencetovariousspecifican d preferredembodimentsandtechniques.However,oneskilledintheart will understandthatmanyvariationsandmodificationsmaybemadewhilere maining withinthespiritandscopeoftheinvention.

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