molecules

Review Quinoxaline Derivatives as Antiviral Agents: A Systematic Review

Marc Montana 1,2, Vincent Montero 1, Omar Khoumeri 1 and Patrice Vanelle 1,3,*

1 Laboratoire de Pharmaco-Chimie Radicalaire, Institut de Chimie Radicalaire ICR, Aix Marseille Univ, CNRS, UMR 7273, 13005 Marseille, France; [email protected] (M.M.); [email protected] (V.M.); [email protected] (O.K.) 2 Oncopharma, Hôpital Nord, Assistance Publique-Hôpitaux de Marseille (AP-HM), 13005 Marseille, France 3 Service central de la qualité et de l’information pharmaceutiques (SCQIP), Assistance Publique-Hôpitaux de Marseille (AP-HM), 13005 Marseille, France * Correspondence: [email protected]; Tel.: +33-4-91-83-55-80; Fax: +33-4-91-79-46-77



Received: 26 May 2020; Accepted: 9 June 2020; Published: 16 June 2020


Abstract: Background: In recent decades, several viruses have jumped from animals to humans, triggering sizable outbreaks. The current unprecedent outbreak SARS-COV-2 is prompting a search for new cost-effective therapies to combat this deadly pathogen. Suitably functionalized polysubstituted quinoxalines show very interesting biological properties (antiviral, anticancer, and antileishmanial), ensuring them a bright future in medicinal chemistry. Objectives: Focusing on the promising development of new quinoxaline derivatives as antiviral drugs, this review forms part of our program on the anti-infectious activity of quinoxaline derivatives. Methods: Study compiles and discusses recently published studies concerning the therapeutic potential of the antiviral activity of quinoxaline derivatives, covering the literature between 2010 and 2020. Results: A final total of 20 studies included in this review. Conclusions: This review points to a growing interest in the development of compounds bearing a quinoxaline moiety for antiviral treatment. This promising moiety with different molecular targets warrants further investigation, which may well yield even more encouraging results regarding this scaffold.

Keywords: quinoxaline; antiviral; SAR; biological applications; chemistry

1. Introduction Humans have a long history of viral infections. For some viral diseases, vaccines and antiviral drugs have made it possible to prevent infections or have helped those infected to recover. Recent antiviral drug development has led to the discovery of effective new treatments to control Human Immunodeficiency Virus (HIV) and Hepatitis C virus (HCV) infections [1]. However, several viruses have jumped from animals to humans triggering sizable outbreaks. One example is the 2014–2016 outbreak of Ebola in West Africa, which resulted in over 28,000 infected patients and was responsible for over 11,000 deaths [2], making it the most lethal member of the Ebola family. The SARS-COV-2 outbreak worldwide continues to pose a serious threat to public health, with no reliable treatment yet available. In addition, the last decade has seen the development of only a few novel antivirals as remdesivir and favipiravir, initially used respectively as Ebola and influenzae treatment and proposed for repurposing in the SARS-CoV-2 outbreak, or sofosbuvir and daclatasvir, which have dramatically changed the prognosis in HCV infection [3,4]. However, this unprecedented SARS-CoV-2 crisis underlines the urgency of developing new cost-effective therapies to combat the deadly pathogen.

Molecules 2020, 25, 2784; doi:10.3390/molecules25122784 www.mdpi.com/journal/molecules Molecules 2020, 25, 2784x FOR PEER REVIEW 2 of 20 19

Nitrogen-containing heterocycles are promising compounds for the development of new drugs or novelNitrogen-containing potential lead molecules heterocycles [5,6–8]. are promising The quinoxaline compounds scaffold, for the a development bioisoster of of quinoline new drugs and or naphthalene,novel potential is leadone moleculesof the heterocycl [5–8]. Thees currently quinoxaline attracting scaffold, attention. a bioisoster of quinoline and naphthalene, is oneQuinoxaline, of the heterocycles formed currentlyby the fusion attracting of benzene attention. and pyrazine rings, is a white crystalline powder whoseQuinoxaline, melting point formed is 29–30 by °C the and fusion whose of benzene molecular and formula pyrazine is C rings,8H6N2 is [9,10]. a white Its crystallinesynthesis has powder been whoseintensively melting studied point in is the 29–30 past.◦C The and classic whose method molecular of formulaquinoxaline is C 8preparationH6N2 [9,10]. is Its to synthesis condense has o- phenylenediaminebeen intensively studied with a in dicarbonyl the past. The compound. classic method This procedure of quinoxaline requires preparation high temperatures, is to condense a strongo-phenylenediamine acid catalyst, withand long a dicarbonyl reaction compound. times [11,12]. This Other procedure strategies requires described high temperatures, for the synthesis a strong of quinoxalineacid catalyst, derivatives and long reaction involve times condensation [11,12]. Other of 1,2-diamines strategies described with α-diketones for the synthesis [13], 1,4-addition of quinoxaline of 1,2-diaminesderivatives involve to diazenylbutenes condensation [14], of 1,2-diamines cyclization–oxidation with α-diketones of phenacyl [13], bromides 1,4-addition [15], of and 1,2-diamines oxidative couplingto diazenylbutenes of epoxides [ 14with], cyclization–oxidation ene-1,2-diamines [16]. of Th phenacylere are also bromides several green [15], and synthetic oxidative methods, coupling like usingof epoxides recyclable with catalysts ene-1,2-diamines [17], one-pot [16]. Theresynthesis are also[18], severalmicrowave-assisted green synthetic synthesis methods, [19,20], like using and reactionsrecyclable in catalysts aqueous [17 medium], one-pot [21]. synthesis [18], microwave-assisted synthesis [19,20], and reactions in aqueousSuitably medium functionalized [21]. polysubstituted quinoxalines show very interesting biological propertiesSuitably (antiviral functionalized [22], anticancer polysubstituted [23], and quinoxalines antileishmanial show [24]), very ensuring interesting them biological a bright properties future in (antiviralmedicinal [22chemistry], anticancer [11,25]. [23 ],Many and antileishmanialdrug candidates [24 bearing]), ensuring quinoxaline them a brightcore structures future in have medicinal been identified,chemistry [such11,25 as]. S-2720 Many (Figure drug candidates 1), found to bearing be a very quinoxaline potent inhibitor core structures of HIV-1 havereverse been transcriptase identified, [26].such as S-2720 (Figure1), found to be a very potent inhibitor of HIV-1 reverse transcriptase [26].

H N S

CH3 Cl N CH3 O O

H C CH 2 3 Figure 1. Chemical structure of S-2720.

This review investigates the new quinoxaline derivatives that are showing promise as antiviral drugs as part part of of our our program program focu focusedsed on on the the anti-infectious anti-infectious activity activity of ofquinoxaline quinoxaline derivatives. derivatives. It compilesIt compiles and and discusses discusses recently recently published published studies studies concerning concerning the the therapeutic therapeutic potential potential of of the antiviral activity of quinoxaline derivatives, covering the literature betweenbetween 20102010 andand 2020.2020.

2. Methods 2. Methods 2.1. Background Definition 2.1. Background Definition The search method employed in this systematic review was to select studies that evaluated the The search method employed in this systematic review was to select studies that evaluated the biological activity and mechanism of action of quinoxaline derivatives. biological activity and mechanism of action of quinoxaline derivatives. 2.2. Data Sources and Searches 2.2. Data Sources and Searches Three different databases were used to conduct a comprehensive survey: MEDLINE/PubMed (NationalThree Library different of Medicine—databases werewww.ncbi.nlm.nih.gov used to conduct a /comprehensivepubmed), Web ofsurvey: Science MEDLINE/PubMed (Thomson Reuters (NationalScientific— Librarywww.webofknowledge.com of Medicine—www.ncbi.nlm.nih.g/), and Scienceov/pubmed), Direct (Elsevier Web of Sciencewww.sciencedirect.com (Thomson Reuters). TheScientific—www.webofknowledge.com/), search terms “quinoxaline” and “antiviral” and Science were Direct chosen (Elsevier so as to www.sciencedirect.com). detect everything published The searchaboutquinoxaline terms “quinoxaline” before applying and “antiviral” exclusion were criteria. chosen Searches so as were to detect conducted everything using thepublished limit dates about of 1quinoxaline January 2010 before and 1applying May 2020. exclusion criteria. Searches were conducted using the limit dates of 1 January 2010 and 1 May 2020. 2.3. Study Selection 2.3. Study Selection The review was performed in three main stages by three independent reviewers. In the first stage,The articles’ review titles was and performed abstracts in were three assessed main stages according by three to the independent eligibility criteria reviewers. (Table In1 ).the In first the stage,second articles’ stage, duplicatedtitles and abstracts articles werewere deleted.assessed Finally,according the to authors the eligibility read each criteria selected (Table full 1). text In andthe second stage, duplicated articles were deleted. Finally, the authors read each selected full text and

Molecules 20202020,, 2525,, 2784x FOR PEER REVIEW 3 of 1920 eliminated articles fitting the exclusion criteria. During this final stage, articles found in the reference eliminated articles fitting the exclusion criteria. During this final stage, articles found in the reference lists of selected manuscripts, but which had not been listed under the search terms in the databases, lists of selected manuscripts, but which had not been listed under the search terms in the databases, were added. were added.

Table 1.1. InclusionInclusion andand exclusionexclusion criteria.criteria. Parameter Inclusion Exclusion Parameter Inclusion Exclusion 1 Language English, French Any other language 1 Language English, FrenchExclusively in Any silico, other articles language that focus only Biological activity, In vitro 2 Type of study Biological activity, In vitro Exclusivelyon synthesis in silico, or other articles purely that chemical focus only on 2 Type of study and/or in vivo studies and/or in vivo studies synthesis or otherparameters purely chemical parameters Reviews, book chapters, posters, table of Type of Reviews, book chapters, posters, table of 3 Original manuscripts contents, personal opinions, indexes, 3 Typepublication of publication Original manuscripts contents, personal opinions, indexes, conferenceconference abstracts, abstracts, letters letters 44 Search Search terms terms Merely Merely citing citing keywords keywords in in text text Articles that evaluate the Mechanism of Articles that evaluate the Articles that concern non-human or animal 5 Mechanism ofbiological activity of quinoxaline Articles that concern non-human or 5 action biological activity of viruses action derivatives animal viruses quinoxaline derivatives

2.4. Data Extraction Process 2.4. Data Extraction Process The following information was extracted from all the selected studies: type of study, biological The following information was extracted from all the selected studies: type of study, biological matrix used, compound structure and nomenclature, and main conclusions. matrix used, compound structure and nomenclature, and main conclusions.

3. Results The database search identified 216 records. After the first evaluation phase (title/abstract), 175 The database search identified 216 records. After the first evaluation phase (title/abstract), records were excluded. Eight repeated files were discarded, leaving 33 articles. 175 records were excluded. Eight repeated files were discarded, leaving 33 articles. No other paper was added from the reference lists of the identified studies (which had not been No other paper was added from the reference lists of the identified studies (which had not been found in the initial search). A second phase was therefore conducted with a total of 33 articles. found in the initial search). A second phase was therefore conducted with a total of 33 articles. After the full-text reading, 13 articles were excluded and one was included, leaving a final total After the full-text reading, 13 articles were excluded and one was included, leaving a final total of of 21 studies included in this review. This process is illustrated by a flow diagram in Figure 2. 21 studies included in this review. This process is illustrated by a flow diagram in Figure2.

Figure 2. Flow diagram of study selection adapted from PRISMA [27]. Figure 2. Flow diagram of study selection adapted from PRISMA [27].

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After critical reading, articles were divided into categories according to the activity assessed in each study (Table2). A manuscript could be assigned to more than one category if a single study evaluated more than one type of antiviral activity.

Table 2. Activities associated with quinoxaline derivatives based on virus classification.

Activity Reference DNA viruses Herpesviridae [28–32] Poxviridae [33] Hepadnaviridae [34] RNA viruses Picornaviridae [35] Orthomyxoviridae [36] Filoviridae [2] Flaviviridae [31,37–42] Rhabdoviridae [43,44] Retroviridae [45–47]

4. Discussion

4.1. Quinoxaline Derivatives Active against DNA Viruses DNA viruses have DNA genomes that are replicated by either host or virally encoded DNA polymerases. DNA viruses with a large genome, particularly the families of Herpesviridae and Poxviridae, encode a number of proteins that counter host defenses. [48] Double-stranded DNA viruses can be subdivided into three groups: (1) those with a small size DNA genome (<10 kb), such as polyomaviruses and papillomaviruses; (2) those with a medium-sized DNA genome (ca., 35 kb), such as adenoviruses; and (3) those with a large DNA genome (ca., 130–250 kb), such as herpesviruses and poxviruses [49].

4.1.1. Quinoxaline Derivatives Active against Herpesviridae The Herpesviridae are a ubiquitous worldwide family responsible for viral infections. Among its members frequently encountered are, herpes simplex viruses (HSV-1 and HSV-2), human cytomegalovirus (HCMV), and Epstein Barr virus (EBV) [28,50,51]. Usually, these infections remain latent and patients are often asymptomatic, particularly immunocompetent populations. However, in immunocompromised patients especially (e.g., AIDS, cancer, etc.), there can be clinical symptoms such as meningitidis or pneumoniae leading to death. Treatments against HSV or HCMV like ganciclovir, valganciclovir, foscarnet, or cidofovir, are currently available but they are limited by toxicity and/or poor oral bioavailability [28,52,53]. In addition, drug resistance is emerging. Hence there is a need to identify improved agents that circumvent one or more of these problems. In 2012, a series of new [1,2,4]triazolo[4,3-a]quinoxaline derivatives and their pyrimido-quinoxaline isosters were synthesized and evaluated as potential antiviral agents. Twenty-two novel compounds were obtained. Among them, 1-(4-chloro-8-methyl[1,2,4]triazolo[4,3a]quinoxaline-1-yl)-3-phenyl thiourea 1 showed the highest antiviral activity in a plaque-reduction assay against Herpes simplex virus grown on Vero African monkey kidney cells, reducing the number of plaques by 25% at 20 µg/mL (Figure3). Nine other compounds reduced the number of plaques by less than 25% at 80 µg/mL, leading the authors to the conclusion that the thiourea moiety may be responsible for antiviral activity and highlighting the importance of the moieties selected in developing antiviral activity [29]. However, antiviral activity remained disappointing compared to positive control aphidicolin, which reduced the number of plaques by 100% at 5 µg/mL, even though compound 1 showed lower cytotoxicity. Molecules 2020, 25, 2784 5 of 19 Molecules 2020, 25, x FOR PEER REVIEW 5 of 20

HN HN S 1 HN N Molecules 2020, 25, x FOR PEER REVIEW H C N N 6 of 20 H3C N

Table 3. Chemical structure, and EC50 values againstN Clhuman cytomegalovirus (HCMV). N Cl Compound Structure EC50 (µM) FigureFigure 3. 3.Chemical Chemical structure structure of of 1-(4-chloro-8-methyl[1,2,4]triazolo[4,3 1-(4-chloro-8-methyl[1,2,4]triazolo[4,3aa]quinoxaline-1-yl)-3-phenyl]quinoxaline-1-yl)-3-phenyl Figure 3. Chemical structure of 1-(4-chloro-8-methyl[1,2,4]triazolo[4,3H a]quinoxaline-1-yl)-3-phenyl thioureathiourea1. 1. H C N O 3 O 4 <0.05 TheThe synthesis synthesis of fourof four new new aldehydo-sugar-N-(3-phenylquinoxalin-2-yl)hydrazones aldehydo-sugar-N-(3-phenylquinoxalin-2-yl)hydrazones2a-d 2a-dand and their H3CCHN O 3 acyclictheir C-nucleosideacyclic C-nucleoside analogues, analogues, 1-(4-phenyl-[1,2,4]triazolo[4,3- 1-(4-phenyl-[1,2,4]triazolo[4,3-a]quinoxalin-1-yl)alditolsa]quinoxalin-1-yl)alditols 3a-d (Figure 3a-d 4) (Figure 4) indicated that these compounds exhibited very weak antiviral activity against HSV-1 in a indicated(Figure 4) that indicated these compounds that these compounds exhibitedH3C exhibited very weakN very antiviral weakO antiviral activityCH3 activity against against HSV-1 HSV-1 in a plaque in a reductionplaque reduction infectivity5 infectivity assay in comparisonassay in comparison to aphidicolin to aphidicolin taken as taken reference as reference [30]. [30].>30 O N O H H3C N NHNH2 N N 6 O >1.2 H3C N N O N NH N N H N N N n = 4; aO = D-gluco-;OH b = D-galacto n(HOHC) n = 3; a = D-arabino-; b = D-xylo H n(HOHC) (CHOH)n HOH C (CHOH)n HOH2C CH OH S 2a-d CH2OH H 72a-d H C 3a-d N N >30 3 N FigureFigure 4. Chemical4. Chemical structure structure of new seriesof new of aldehydo-sugar-N-(3-phenylquinoxalin-2-yl)hydrazonesseries of aldehydo-sugar-N-(3-phenylquinoxalin-2-O H C N O andyl)hydrazones their acyclic and C-nucleoside their acyclic analogues, C-nucleoside3 1-(4-phenyl-[1,2,4]triazolo[4,3- analogues, 1-(4-phenyl-[1,2,4]triazolo[4,3-a]quinoxalin-1-yl)alditols.a]quinoxalin- H 1-yl)alditols. Recently, nine novel quinoxaline derivatives were synthetized via different nucleophilic reactions using ethylRecently, (6,7-dimethyl-2-oxo-3,4-dihydroquinoxalin-3-yl)acetate nine novel quinoxaline derivatives were synthetized4Nand via ethyl different (6-methyl-2-oxo-3,4- nucleophilic reactions using ethyl (6,7-dimethyl-2-oxo-3,4-dihydroquinoxalin-3-yl)acetate 4 and ethyl (6-methyl- dihydroquinoxalin-3-yl)acetatereactions using ethyl8 (6,7-dimethyl-2-oxo-3,4-dihydroquinoxalin-3-yl)acetate 5, 3-methylquinoxalin-2(1H3C NH)-one, andN 1,4-dihydroquinoxaline-2,3-dione 4 and<0.05 ethyl (6-methyl- as2-oxo-3,4-dihydroquinoxalin-3-yl)acetate precursors [31] (Table3). When their antiviral5, activity3-methylquinoxalin-2(1 against HCMVH)-one, was comparedand to1,4- the dihydroquinoxaline-2,3-dione as precursors [31] (Table 3). WhenN N their antiviral activity against standarddihydroquinoxaline-2,3-dione drug ganciclovir (EC 50as= precursors0.059Hµ3M),C [31] two (Table derivativesN 3).O When demonstrated their antiviral higher activity activity, against each H HCMV was compared to the standard drug ganciclovir (EC50 = 0.059 µM), two derivatives withHCMV EC50 150 µM),µM, two respectively) derivatives was Ganciclovir - 0.059 demonstrated higher activity, each with EC50 < 0.05 µM. Notably, the toxicity of 4 and 8 (CC50 = 108.47 comparable to the reference drug (CC50 >150 µM) and compound 6 showed the poorest safety profile EC50: Compound concentration that reduces viral replication50 by 50%. and >150 µM, respectively) was comparable to the reference drug (CC50 >150 µM) and compound 6 with CC50 = 2.34 µM. showed the poorest safety profile with CC50 = 2.34 µM. FourFour other other compounds compounds showed showed promisingpromising antiviralantiviral activity. Antiviral Antiviral activity activity was was observed observed to to dependdepend on on varying varying chemical chemical characteristics, characteristics, likelike thethe presence of of a a dimethylquinoxalinyl dimethylquinoxalinyl methylene methylene nucleusnucleus as as a commona common structural structural feature feature and and thethe presencepresence of a lipophilic ester ester function function (Figure (Figure 5).5).

The presence of a second CH3 favors antiviral actvity

H A lipophilic side chain enhances H C N O the activity 3 O

H3CCHN O 3

The methylene bridge is necessary for activity

FigureFigure 5. 5.Structure–activity Structure–activity relationship relationship ofof novelnovel quinoxalinequinoxaline derivatives derivatives with with anti anti HCMV HCMV activity. activity.

In addition, several quinoxaline derivatives are of current interest as anti HCMV agents [28]. Some 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides were synthetized and tested (Figure 6).

Molecules 2020, 25, 2784 6 of 19 MoleculesMolecules 2020 2020, 25, 25, x, FORx FOR PEER PEER REVIEW REVIEW 6 of6 of 20 20 Molecules 2020, 25, x FOR PEER REVIEW 6 of 20 Molecules 2020, 25, x FOR PEER REVIEW 6 of 20 Molecules 2020, 25, xTable FOR PEER 3. Chemical REVIEW structure, and EC50 values against human cytomegalovirus (HCMV). 6 of 20 TableTable 3. 3. Chemical Chemical structure, structure, and and EC EC50 50values values against against human human cytomegalovirus cytomegalovirus (HCMV). (HCMV). Table 3. Chemical structure, and EC50 values against human cytomegalovirus (HCMV). Table 3. ChemicalCompound structure, and EC50 values Structureagainst human cytomegalovirus EC (HCMV).50 (µM) TableCompoundCompound 3. Chemical StructureStructure structure, and EC50 values against human cytomegalovirusEC EC(HCMV).50 50(µM) (µM) Compound Structure EC50 (µM) Compound Structure HH EC50 (µM) Compound Structure HH3C3C NHN OO EC50 (µM) H3C NH O OO 4 4 4 H C HN O O <0.05<<0.050.05 4 H 3C N O O <0.05 4 3 O <0.05 4 HH3CCH3CCHNN OO 3 3 <0.05 H3CCHN O 3 H CCHN O H 3CCHN O 3 HH3C33C NN OO CHCH333 H3C N O CH3 5 5 5 H3C N O CH3 >30>>3030 5 H3C N OOO CH3 >30 5 NN OOO >30 5 NH O O >30 H O HN O HHC3C NH NNO NHNHNHNH2 H3C H N NHNH2 H3C N NHNH2 6 6 H 3C N ONHNH 2 >1.2>1.2 6 6 3 O 2 >1.2>1.2 HHC3C NN OOO 6 H3C NH O O >1.2 6 3 H O >1.2 H3C HN O H3C NH O OO OHOH H O OH O OH O OH SS HH S 7 7 HHCC NN NHN S >30>30 7 H3C3 N NH NNS >30 7 7 H3C N HN >30>30 7 3 N >30 H3C N OON N HHCC NN OOO N H3C3 N O O 3 HH H3C HN O O H C N O 3 H H NN N N 8 8 HH3C3C NN NN N <0.05<0.05 8 H3C N N <0.05 8 H3C N N <0.05 8 8 H3C N NNNNN <0.05<0.05 HH3C3C NN OON N H C NH O N H3C HN O N N 3 H N GanciclovirGanciclovir H3C NH - - O 0.059 0.059 Ganciclovir H - 0.059 GanciclovirECEC50 :50 Compound: Compound concentration concentration that that re reducesduces - viral viral replication replication by by 50%. 50%. 0.059 GanciclovirEC50 : Compound concentration that reduces - viral replication by 50%. 0.059 ECGanciclovir50: Compound concentration that reduces - viral replication by 50%. 0.059 EC50: Compound concentration that reduces viral replication by 50%. FourFour other other compounds compoundsEC showed showed: Compound promising promising concentration antiviral antiviral that reducesactivity. activity. viral Antiviral replicationAntiviral activity byactivity 50%. was was observed observed to to Four other compounds showed50 promising antiviral activity. Antiviral activity was observed to dependdependFour on on other varying varying compounds chemical chemical showed characteristics, characteristics, promising like like antiviral the the presence presence activity. of of a Antiviral adimethylquinoxalinyl dimethylquinoxalinyl activity was observed methylene methylene to dependFour on other varying compounds chemical showed characteristics, promising like antiviral the presence activity. of aAntiviral dimethylquinoxalinyl activity was observed methylene to nucleusdependnucleus ason Inas a varying a addition,common common chemical structural several structural characteristics, quinoxaline feature feature and and derivatives the like the presence presencethe presence are of of of a alipophilic current oflipophilic a dimethylquinoxalinyl interest ester ester function asfunction anti HCMV(Figure (Figure methylene agents5). 5). [28]. dependnucleusSome onas a 2-aryl-2-hydroxyvarying common chemical structural ethylaminecharacteristics, feature and substituted thelike presence the presence 1H,7 ofH -pyrido[1,2,3-a lipophilicof a dimethylquinoxalinyl esterde]quinoxaline-6-carboxamides function (Figure methylene 5). nucleus as a common structural feature and the presence of a lipophilic ester function (Figure 5). ThewereTheMolecules presence presence synthetized 2020, 25of of, ax aFORsecond and second PEER tested CH REVIEWCH3 (Figure 3 6). 7 of 20 The presence of a second CH3 The favorspresencefavors antiviral antiviral of a second actvity actvity CH3 The presencefavors antiviral of a second actvity CH favors antiviral actvity 3 O O favors antiviral actvity HH AA lipophilic lipophilic side side chain chain enhances enhances HHCC R NHN OO N A lipophilic theside the activity activitychain enhances H3C3 2 NH O OO the activity H3C HN O H A lipophilic sidethe activity chain enhances 3 O H3C N O N Cl the activity HH3CCH3CCHNN O OO 3 3 H3CCHN N O 3 H3CCHN O 3 R1 H3CCHN O 3 TheThe methylene methyleneO bridge bridge is is Thenecessarynecessary methylene for for bridgeactivity activity is

FigureFigure 6.6. GeneralGeneral structure structure Theof ofnecessary 2-aryl-2-hydroxy methylene for bridgeactivity ethylamine is substituted 11HH,7,7HH-pyrido[1,2,3--pyrido[1,2,3-

FigureFigurede.de. ]quinoxaline-6-carboxamides5.]quinoxaline-6-carboxamides 5. Structure–activity Structure–activity relationship relationship synthetized.synthetized.necessary of of novel novel quin for quin activityoxalineoxaline derivatives derivatives with with anti anti HCMV HCMV activity. activity. Figure 5. Structure–activity relationship of novel quinoxaline derivatives with anti HCMV activity. Figure 5. Structure–activity relationship of novel quinoxaline derivatives with anti HCMV activity. InFigureIn addition, addition,InIn 5. thisthis Structure–activity study,severalstudy, several the thequinoxaline quinoxaline pyridoquinoxalinepyridoquinoxaline relationship derivatives derivatives of novel nucleusnucleus quinare are oxalineof of current provedproved current derivatives interest to tointerest bebe awitha as useful usefulas anti anti HCMV nucleus,HCMVnucleus, HCMV activity. agents asagentsas somesome [28]. [28]. ofof thethe In addition, several quinoxaline derivatives are of current interest as anti HCMV agents [28]. SomeSomesynthetizedInsynthetized 2-aryl-2-hydroxy addition,2-aryl-2-hydroxy compoundsseveralcompounds ethylamine quinoxalineethylamine showed showed substituted asubstitutedderivatives favorablea favorable profile1 Hare1H,7 ,7HprofileofH-pyrido[1,2,3- for-pyrido[1,2,3-current established for interestestablishedede]quinoxaline-6-carboxamides drugs]quinoxaline-6-carboxamides as danti likedrugs HCMV as ganciclovir,like agents as ganciclovir, acyclovir,[28]. SomeIn 2-aryl-2-hydroxyaddition, several ethylaminequinoxaline substituted derivatives 1areH,7 ofH-pyrido[1,2,3- current interestde]quinoxaline-6-carboxamides as anti HCMV agents [28]. wereSomewerefoscarnet, acyclovir,synthetized 2-aryl-2-hydroxysynthetized andfoscarnet, and and aphidicolin tested tested ethylamine and (Figure (Figureaphidicolin [28] (Table 6).substituted 6). 4[28]), (Table 1H,7 4),H-pyrido[1,2,3- de]quinoxaline-6-carboxamides Somewere synthetized2-aryl-2-hydroxy and tested ethylamine (Figure substituted6). 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides were synthetized and tested (Figure 6). Table 4. HCMV pol/pra IC50 for lead compounds.

HCMV pol HCMV pra Compound Structure Activity Activity IC50 (nM) IC50 (nM) O O N N H O 9 N Cl 620 100 N H3C O

O O

O N N H 10 OH CH 4 1 3 N Cl N H3C O Ganciclovir - 1300 Acyclovir - >20,000 Foscarnet 2500 - Aphidicolin - 487 - Pol: enzymatic activity; Pra: antiviral activity.

The morpholinomethyl side chain afforded good levels of both enzymatic and antiviral activity whereas the benzofuran moiety resulted in extremely potent enzymatic and antiviral activity. However, despite excellent biological activity, the calculated log P of compound 10 proves the need to continue pharmacomodulation efforts aimed at improving its hydrosolubility. Finally, EBV is a very common virus that can increase the risk of developing certain rare cancers. The malignant transformation of normal human epithelial cells results from exposure to EBV and that transformation is dependent on the presence of phorbol esters, which stimulate cell proliferation through rapid activation of protein kinase C [32,54]. Novel 3-amioquinoxalin-2(1H)-one derivatives and derivatives with pyrimidine ring linked to quinoxaline through sulfur (Figure 7) exhibited properties against EBV antigen activation.

Molecules 2020, 25, x FOR PEER REVIEW 7 of 20

O O Molecules 2020, 25, x FOR PEER REVIEW 7 of 20 R2 N O O H N Cl R2 N N H R1 N Cl O N R Figure 6. General structure of 2-aryl-2-hydroxy1 ethylamine substituted 1H,7H-pyrido[1,2,3- O de.]quinoxaline-6-carboxamides synthetized. Figure 6. General structure of 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3- In thisde.]quinoxaline-6-carboxamides study, the pyridoquinoxaline synthetized. nucleus proved to be a useful nucleus, as some of the synthetizedMolecules 2020 compounds, 25, 2784 showed a favorable profile for established drugs like as ganciclovir, 7 of 19 acyclovir,In foscarnet,this study, and the aphidicolinpyridoquinoxaline [28] (Table nucleus 4), proved to be a useful nucleus, as some of the synthetized compounds showed a favorable profile for established drugs like as ganciclovir, acyclovir, foscarnet, and aphidicolin [28] (Table 4), Table 4. HCMV pol/pra IC50 for lead compounds. Table 4. HCMV pol/pra IC50 for lead compounds.

Table 4. HCMV pol/pra IC50 for lead compounds. HCMV pol HCMV pra HCMV pol Activity HCMV pra Activity CompoundCompound StructureStructure Activity Activity HCMVIC 50pol(nM) HCMV pra IC50 (nM) Compound Structure ActivityIC50 (nM) ActivityIC50 (nM) O O IC50 (nM) IC50 (nM) O O N N H O N N 99 N H Cl 620620 100 100 9 O 620 100 N N Cl H3C N H3C O O O O O O O N N O N NH 10 H 4 1 1010 OHOH CHCH3 4 41 1 3 N ClCl N H C N H33C O O GanciclovirGanciclovir - - 13001300 Ganciclovir - 1300 AcyclovirAcyclovir - - >20,000>20,000 FoscarnetAcyclovirFoscarnet 2500 2500 - - - >20,000 Aphidicolin - 487 - AphidicolinFoscarnet - 487 2500 - - Pol: enzymatic activity; Pra: antiviral activity. AphidicolinPol: enzymatic - activity; Pra: antiviral activity. 487 - The morpholinomethyl side chainPol: afforded enzymatic good activity; levels Pra: of both antiviral enzymatic activity. and antiviral activity whereasThe morpholinomethyl the benzofuran moiety side chain resulted afforded in extr goodemely levels potent of both enzymatic enzymatic and andantiviral antiviral activity. activity whereas the benzofuran moiety resulted in extremely potent enzymatic and antiviral activity. However,The morpholinomethyl despite excellent biological side acti chainvity, a thefforded calculated good log levels P of compound of both enzymatic10 proves the and need antiviral activity However,to continue despite pharmacomodulation excellent biological efforts acti aimedvity, the at improvingcalculated its log hydrosolubility. P of compound 10 proves the need towhereas continueFinally, the pharmacomodulation benzofuranEBV is a very common moiety efforts resultedvirus aimed that incan at extremely increa improvingse the potent risk its hydrosolubility.of developing enzymatic certain and antiviralrare cancers. activity. However, despiteTheFinally, malignant excellent EBV istransformation a biological very common activity,of normal virus that the human calculatedcan increaepithelialse logthe cells Prisk results of of compound developing from exposure10 certainproves to rareEBV the cancers.and need to continue Thepharmacomodulationthat malignant transformation transformation is dependent efforts of aimednormalon the presence athuman improving of ep phorbolithelial its esters, cells hydrosolubility. resultswhich stimulate from exposure cell proliferation to EBV and thatthrough transformationFinally, rapid EBV activation is is dependent a very of protein common on thekinase presence virus C [32,54]. that of phorbol Novel can increase 3-amioquinoxalin-2(1 esters, which the risk stimulate ofH developing)-one cell derivatives proliferation certain rare cancers. and derivatives with pyrimidine ring linked to quinoxaline through sulfur (Figure 7) exhibited throughThe malignant rapid activation transformation of protein kinase of normal C [32,54]. human Novel epithelial 3-amioquinoxalin-2(1 cells resultsH)-one from derivatives exposure to EBV and properties against EBV antigen activation. andthat derivatives transformation with pyri is dependentmidine ring on linked the presenceto quinoxaline of phorbol through esters, sulfur which (Figure stimulate 7) exhibited cell proliferation properties against EBV antigen activation. through rapid activation of protein kinase C [32,54]. Novel 3-amioquinoxalin-2(1H)-one derivatives and derivatives with pyrimidine ring linked to quinoxaline through sulfur (Figure7) exhibited properties againstMolecules EBV 2020 antigen, 25, x FOR activation. PEER REVIEW 8 of 20

OCH3 O CN X N HN N N N R2 N S N O

R1 N Cl

Figure 7. General structures of 3-aminoquinoxalin-2(1H)-one derivatives and derivatives with Figure 7. General structures of 3-aminoquinoxalin-2(1H)-one derivatives and derivatives with pyrimidine ring linked to quinoxaline through sulfur exhibiting anti EBV antigen activation. pyrimidine ring linked to quinoxaline through sulfur exhibiting anti EBV antigen activation. On a series of 22 original compounds, six derivatives demonstrated stronger inhibitory effect on EBVOn than a series oleanolic of 22 acid original as reference, compounds, without six deriva showingtives any demonstrated cytotoxicity. stronger The structure–activity inhibitory effect on relationshipEBV than proved oleanolic that disubstitutionacid as reference, with alkyl without groups showing on both any nitrogen cytotoxicity. of hydrazine The and structure–activity quinoxaline wasrelationship crucial for activity proved especially that disubstitution for the allyl group. with Thisalkyl high groups activity on couldboth resultnitrogen from of a hydrophobichydrazine and interactionquinoxaline between was crucial the alkyl for group activity and especially the hydrophobic for the allyl region group. of This the binding high activity site of could the receptor. result from a hydrophobic interaction between the alkyl group and the hydrophobic region of the binding site of the receptor. The presence of a methoxy group on the phenyl group and substitution with a pyrimidine nucleus linked to quinoxaline through sulfur were also conducive to activity [32].

4.1.2. Quinoxaline Derivatives Active against Poxviridae The Poxviridae family include 38 viruses that can infect a wide range of hosts, including mammals, birds, reptiles, and insects [55]. The causative agent of Smallpox and Molluscum contagiosum, two human specific diseases, belongs to the poxviruses. Although variola was globally eradicated, Molluscum contagiosum results from a usually benign infection with mild skin disease characterized by lesions that may appear anywhere on the body. Within 6–12 months, Molluscum contagiosum typically resolves without scarring, but may take as long as many years in some people with weakened immune systems [56]. In a series of nine new halophenyl pyrrolo[2,3-b]quinoxaline derivatives, none of the compounds proved inhibitory at subtoxic concentration except ethyl 2-(4-chlorophenyl)-1-methyl-2,4-dihydro- 1H-pyrrolo-[2,3-b]quinoxaline 11 (Figure 8), which inhibited the vaccinia virus and was considered as a potential lead compound for poxvirus inhibition, with an EC50 value of 2 µM in HEL cell cultures and moderate antiproliferative activity (CC50 >20 µM) [33].

CH3 O O H N Cl N N H C 3 Figure 8. Structure of the lead compound 11 for poxvirus inhibition.

4.1.3. Quinoxaline Derivatives Active against Hepadnaviridae Hepatitis B virus (HBV) is a member of the Hepadnaviridae family. This virus can cause liver infections that lead to various hepatic diseases such as hepatitis, cirrhosis, and hepatic cancer. A new class of 3-(1’,2’-dihydroxyeth-1’-yl)-1-phenylpyrazolo[3,4-b]quinoxaline demonstrated encouraging anti-hepatitis B activity at 100 µM, but the five most potent compounds were associated with high cytotoxicity (cytotoxicity >30% at 100 µM) [34].

4.2. Quinoxaline Derivatives Active against ARN Viruses

Molecules 2020, 25, x FOR PEER REVIEW 8 of 20

OCH3 O CN X N HN N N N R2 N S N O

R1 N Cl

Figure 7. General structures of 3-aminoquinoxalin-2(1H)-one derivatives and derivatives with pyrimidine ring linked to quinoxaline through sulfur exhibiting anti EBV antigen activation.

On a series of 22 original compounds, six derivatives demonstrated stronger inhibitory effect on EBV than oleanolic acid as reference, without showing any cytotoxicity. The structure–activity relationship proved that disubstitution with alkyl groups on both nitrogen of hydrazine and Molecules 2020, 25, 2784 8 of 19 quinoxaline was crucial for activity especially for the allyl group. This high activity could result from a hydrophobic interaction between the alkyl group and the hydrophobic region of the binding site of Thethe presencereceptor. ofThe amethoxy presence group of a onmethoxy the phenyl group group on the and phenyl substitution group with and a pyrimidinesubstitution nucleuswith a linkedpyrimidine to quinoxaline nucleus linked through to quinoxaline sulfur were through also conducive sulfur were to activity also conducive [32]. to activity [32].

4.1.2.4.1.2. Quinoxaline Derivatives Active againstagainst PoxviridaePoxviridae TheThe PoxviridaePoxviridaefamily family include include 38 viruses38 viruses that canthat infect can ainfect wide a range wide of range hosts, includingof hosts, mammals,including birds,mammals, reptiles, birds, and reptiles, insects [55and]. The insects causative [55]. agentThe causative of Smallpox agent and Molluscumof Smallpox contagiosum, and Molluscum two humancontagiosum, specific two diseases, human specific belongs diseases, to the poxviruses. belongs to the Although poxviruses. variola Although was globallyvariola was eradicated, globally Molluscumeradicated, contagiosumMolluscum contagiosum results from aresults usually from benign a usually infection benign with infection mild skin with disease mild characterized skin disease bycharacterized lesions that by may lesions appear that anywhere may appear on theanywhere body. Withinon the 6–12body. months, Within Molluscum6–12 months, contagiosum Molluscum typicallycontagiosum resolves typically without resolves scarring, without but may scarring, take as but long may as manytake as years long in as some many people years within some weakened people immunewith weakened systems immune [56]. systems [56]. InIn aa series of nine new halophenyl pyrrolo[2,3- b]quinoxaline]quinoxaline derivatives,derivatives, nonenone ofof thethe compounds provedproved inhibitoryinhibitory atat subtoxicsubtoxic concentrationconcentration exceptexcept ethylethyl 2-(4-chlorophenyl)-1-methyl-2,4-dihydro-2-(4-chlorophenyl)-1-methyl-2,4-dihydro- 11HH-pyrrolo-[2,3--pyrrolo-[2,3-bb]quinoxaline]quinoxaline11 11(Figure (Figure8), 8) which, which inhibited inhibited the the vaccinia vaccinia virus virus and and was was considered considered as aas potential a potential lead lead compound compound for for poxvirus poxvirus inhibition, inhibition, with with an an EC EC5050value value of of 2 2µ µMM inin HELHEL cellcell culturescultures andand moderatemoderate antiproliferativeantiproliferative activityactivity (CC(CC5050 >>2020 µM)µM) [33]. [33].

CH3 O O H N Cl N N H C 3 FigureFigure 8.8. Structure of the leadlead compoundcompound 1111 forfor poxviruspoxvirus inhibition.inhibition.

4.1.3. Quinoxaline Derivatives Active against Hepadnaviridae 4.1.3. Quinoxaline Derivatives Active against Hepadnaviridae Hepatitis B virus (HBV) is a member of the Hepadnaviridae family. This virus can cause liver Hepatitis B virus (HBV) is a member of the Hepadnaviridae family. This virus can cause liver infections that lead to various hepatic diseases such as hepatitis, cirrhosis, and hepatic cancer. A new infections that lead to various hepatic diseases such as hepatitis, cirrhosis, and hepatic cancer. A new class of 3-(1 ,2 -dihydroxyeth-1 -yl)-1-phenylpyrazolo[3,4-b]quinoxaline demonstrated encouraging class of 3-(1’,2’-dihydroxyeth-1’-yl)-1-phenylpyrazolo[3,4-b]quinoxaline0 0 0 demonstrated encouraging anti-hepatitis B activity at 100 µM, but the five most potent compounds were associated with high anti-hepatitis B activity at 100 µM, but the five most potent compounds were associated with high cytotoxicity (cytotoxicity >30% at 100 µM) [34]. cytotoxicity (cytotoxicity >30% at 100 µM) [34]. 4.2. Quinoxaline Derivatives Active against ARN Viruses 4.2. Quinoxaline Derivatives Active against ARN Viruses Human disease-causing RNA viruses include Orthomyxoviruses, Hepatitis C Virus (HCV), Ebola disease, SARS, and retroviruses including adult human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus (HIV). RNA viruses have RNA as genetic material, either single-stranded RNA or double-stranded RNA. Viruses may exploit the presence of RNA-dependent RNA polymerases for replication of their genomes or, in retroviruses, reverse transcriptase produces viral DNA, which can be integrated into the host DNA under its integrase function [57].

4.2.1. Quinoxaline Derivatives Active against Picornaviridae Enteroviruses, which include coxsackievirus A and B, belong to Picornaviridae, a single-stranded RNA virus family. They are implicated in various diseases, with a wide range of symptoms; and exceptionally, coxsakieviruses can be responsible for more severe diseases, such as flaccid paralysis myocarditis, pericarditis, encephalitis, or systemic neonatal disease [35,58,59]. To date, there are conventional treatments or vaccines against coxsackieviruses, which cause acute or chronic disease in infants, children, and immunocompromised persons. Molecules 2020, 25, x FOR PEER REVIEW 9 of 20

Human disease-causing RNA viruses include Orthomyxoviruses, Hepatitis C Virus (HCV), Ebola disease, SARS, and retroviruses including adult human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus (HIV). RNA viruses have RNA as genetic material, either single-stranded RNA or double-stranded RNA. Viruses may exploit the presence of RNA- dependent RNA polymerases for replication of their genomes or, in retroviruses, reverse transcriptase produces viral DNA, which can be integrated into the host DNA under its integrase function [57].

4.2.1. Quinoxaline Derivatives Active against Picornaviridae Enteroviruses, which include coxsackievirus A and B, belong to Picornaviridae, a single-stranded RNA virus family. They are implicated in various diseases, with a wide range of symptoms; and exceptionally, coxsakieviruses can be responsible for more severe diseases, such as flaccid paralysis myocarditis, pericarditis, encephalitis, or systemic neonatal disease [35,58,59]. To date, there are Molecules 2020, 25, 2784 9 of 19 conventional treatments or vaccines against coxsackieviruses, which cause acute or chronic disease in infants, children, and immunocompromised persons. InIn orderorder toto developdevelop moremore eeffectiveffective antivirals,antivirals, 1414 newnew quinoxalinequinoxaline derivativesderivatives (Figure(Figure9 )9) were were synthetizedsynthetized andand testedtested againstagainst aa panelpanel ofof virusesviruses forfor whichwhich thethe eefficacyfficacy ofof therapeutictherapeutic agentsagents waswas unsatisfactoryunsatisfactory [[35].35].

O N R1 S R O N 2 X R 3 FigureFigure 9.9. ChemicalChemical structurestructure ofof quinoxalinequinoxaline derivatives.derivatives.

AmongAmong thesethese new new quinoxalines, quinoxalines, ethyl ethyl 4-(((2,3-dimethoxyquinoxalin-6-yl)methyl)thio)benzoate 4-(((2,3-dimethoxyquinoxalin-6-yl)methyl)thio)benzoate11, 4-(((2,3-dimethoxyquinoxalin-6-yl)methyl)thio)benzoic11, 4-(((2,3-dimethoxyquinoxalin-6-yl)methyl)thio)benzoic acid 12 and acid ethyl 6-(((2,3-dimethoxyquinoxalin-12 and ethyl 6-(((2,3- 6-yl)methyl)thio)nicotinatedimethoxyquinoxalin-6-yl)methyl)thio)nicotinate13 displayed remarkable activity13 displayed against coxsackievirus remarkable B5activity (CBV5), withagainst an ECcoxsackievirus50 = 0.09 µM, B5 0.06 (CBV5),µM, and with 0.3 anµM, EC respectively50 = 0.09 µM, (Table 0.06 µM,5). The and absence 0.3 µM, of respectively cytotoxicity (Table towards 5). theThe Vero-76absence cells of cytotoxicity of compound towards11 led the to further Vero-76 experimental cells of compound/in silico 11 investigation led to further aimed experimental/in at determining silico its mechanisminvestigation of aimed action. at These determining investigations its mechanism demonstrated of action. that compound These investigations11 inhibits demonstrated CBV5 by targeting that thecompound early events 11 inhibits of attachment, CBV5 by entry targeting or uncoating, the early as events it can of favorably attachment, insert entry into aor hydrophobic uncoating, as pocket it can onfavorably the VP1 insert chain into of thea hydrophobic capsid protomer pocket implicated on the VP1 in chain the proteinof the capsid conformational protomer implicated changes during in the infectionMolecules of2020 the, 25, hostx FOR cell.PEER REVIEW 10 of 20 proteinMolecules conformational 2020, 25, x FOR PEER changes REVIEW during infection of the host cell. 10 of 20 Molecules 2020, 25, x FOR PEER REVIEW 10 of 20 TableTable 5.5. Chemical structures, structures, acti activityvity against against coxsackievir coxsackievirusus B5 and B5 cytotoxicity. and cytotoxicity. Table 5. Chemical structures, activity against coxsackievirus B5 and cytotoxicity. Table 5. Chemical structures, activity against coxsackievirus B5 and cytotoxicity. EC50 CBV5 CC50 Vero-76 cells CompoundCompound StructureStructure ECEC5050 CBV5 (µM)CC50 CCVero-7650 Vero-76 cells cells (µM) Compound Structure EC(µM)50 CBV5 CC50 Vero-76(µM) cells Compound O N Structure (µM) (µM) O N (µM) (µM) O N S 11 11 O N S 0.090.09 ± 0.010.01 >100 >100 11 O N S 0.09 ± 0.01± >100 11 O N 0.09 ± 0.01 >100 COOEt COOEt O N COOEt O N O N S 12 O N S 0.06 ± 0.01 >65 12 12 O N S 0.060.06 ± 0.010.01 >65 >65 12 O N 0.06 ± 0.01± >65 COOH COOH O N COOH O N O N S 13 O N S 0.3 ± 0.05 >100 13 O N S 0.3 ± 0.05 >100 13 13 N 0.30.3 ± 0.050.05 >100 >100 O N ± N COOEt N COOEt EC50: Compound concentration that reduces the plaqueCOOEt number of CBV5 by 50%. CC50: Concentration EC50: Compound concentration that reduces the plaque number of CBV5 by 50%. CC50: Concentration ECof a50 drug: Compound that will concentration kill 50% of cells that in reduces uninfected the plaque cell culture. number of CBV5 by 50%. CC50: Concentration ECof50 a: Compounddrug that will concentration kill 50% of cells that reducesin uninfected the plaque cell culture. number of CBV5 by 50%. CC50: Concentration of a drug thatof willa drug kill that 50% will of cells kill in50% uninfected of cells in cell uninfected culture. cell culture. 4.2.2. Quinoxaline Derivatives Active against Orthomyxoviridae 4.2.2. Quinoxaline Derivatives Active against Orthomyxoviridae 4.2.2.4.2.2. QuinoxalineInfluenza Quinoxaline viruses DerivativesDerivatives can cause Active Active contag againstious against respiratory OrthomyxoviridaeOrthomyxoviridae disease in humans and are responsible every Influenza viruses can cause contagious respiratory disease in humans and are responsible every year Influenzafor flu pandemics. viruses can cause contagious respiratory disease in humans and are responsible every yearInfluenza for flu pandemics. viruses can cause contagious respiratory disease in humans and are responsible every year Basedfor flu pandemics.on the planar polyaromatic system (chromophore), quinoxaline derivatives are good Based on the planar polyaromatic system (chromophore), quinoxaline derivatives are good yearcandidates forBased flu pandemics. toon combat the planar influenza polyaromatic viruses because system of(chr theiromophore), potential quinoxalineto target the NS1derivatives protein, are a highly good candidates to combat influenza viruses because of their potential to target the NS1 protein, a highly candidatesconservedBased on influenzato combat the planar virus influenza encoded polyaromatic viruses protein. becausesystem Since of th the (chromophore), eirN-terminal potential domain to target quinoxaline of the the NS1 NS1A protein, derivatives protein a resultshighly are good conserved influenza virus encoded protein. Since the N-terminal domain of the NS1A protein results candidatesconservedin a six-helical toinfluenza combat chain virus influenzafold encoded with a viruses deepprotein. cavity because Since at th the ofe N-terminal theircenter potential of domainthe double-stranded to of target the NS1A the NS1 proteinRNA-binding protein, results a highly in a six-helical chain fold with a deep cavity at the center of the double-stranded RNA-binding conservedinsurface, a six-helical a influenza small chain molecule virus fold encodedwithcould a fitdeep protein.into cavity the Sincecavity at the the andcenter N-terminal block of thevirus double-stranded domain replication of the [36,60–62]. NS1ARNA-binding protein 2,3,6- results surface, a small molecule could fit into the cavity and block virus replication [36,60–62]. 2,3,6- surface,substitued a smallquinoxaline molecule has couldalso yielded fit into compounds the cavity idandentified block as virus having replication valuable antiviral[36,60–62]. activity, 2,3,6- insubstitued a six-helical quinoxaline chain fold has with also a yielded deep cavity compounds at the centeridentified of the as having double-stranded valuable antiviral RNA-binding activity, surface, substituedparticularly quinoxaline under bis-2-furyl has also substitution. yielded compounds Maintaining identified bis-2-furyl as having substitution, valuable antivirala novel seriesactivity, of a smallparticularly molecule under could bis-2-furyl fit into substitution. the cavity Maintaining and block virusbis-2-furyl replication substitution, [36,60 a –novel62]. 2,3,6-substituedseries of particularlyquinoxaline derivativesunder bis-2-furyl was synthetized substitution. to determine Maintaining the influencebis-2-furyl of substitution, substitution ata positionnovel series 6. Two of quinoxalinequinoxaline has derivatives also yielded was synthetized compounds to determine identified the as influence having valuable of substitution antiviral at position activity, 6. Two particularly quinoxalinederivatives, derivativesone with 3-methoxyphenyl was synthetized togroup determine and onethe influencewith 2-furyl of substitution at position at 6, position showed 6. goodTwo underderivatives, bis-2-furyl one with substitution. 3-methoxyphenyl Maintaining group bis-2-furyland one with substitution, 2-furyl at position a novel 6, seriesshowed of good quinoxaline derivatives,activity with onean IC with50 of 6.23-methoxyphenyl and 3.5 µM, respectively group and (Table one 6).with RNA 2-furyl intercalation at position experiments 6, showed showed good activity with an IC50 of 6.2 and 3.5 µM, respectively (Table 6). RNA intercalation experiments showed activitythat both with compounds an IC50 of 6.2could and bind 3.5 µM, to the respectively NS1A RNA-binding (Table 6). RNA domain, intercalation demonstrating experiments the antiviral showed that both compounds could bind to the NS1A RNA-binding domain, demonstrating the antiviral thatpotential both ofcompounds these quinoxaline could bind derivatives to the NS1A[36]. RNA-binding domain, demonstrating the antiviral potential of these quinoxaline derivatives [36]. potential of these quinoxaline derivatives [36]. Table 6. Structure and activity of 2,3,6-substitued quinoxaline. Table 6. Structure and activity of 2,3,6-substitued quinoxaline. Table 6. Structure and activity of 2,3,6-substitued% Binding quinoxaline. at 50 % Intercalation at 50 Compound Structure % Binding at 50 % Intercalation at 50 Compound Structure % BindingµM at 50 % IntercalationµM at 50 Compound StructureO µM µM O µM µM N O O N N 14 O N NH 74.0 4.5 14 O HN 74.0 4.5 N H 14 N 74.0 4.5 O N OCH O OCH3 O O OCH3 O 3 N O O O N N O 15 O N NH O 79.5 5.9 15 O HN 79.5 5.9 15 N H 79.5 5.9 N O N O O 4.2.3. Quinoxaline Derivatives Active against Filoviridae 4.2.3. Quinoxaline Derivatives Active against Filoviridae 4.2.3. Quinoxaline Derivatives Active against Filoviridae

Molecules 2020, 25, x FOR PEER REVIEW 10 of 20 Molecules 2020, 25, x FOR PEER REVIEW 10 of 20 Table 5. Chemical structures, activity against coxsackievirus B5 and cytotoxicity. Table 5. Chemical structures, activity against coxsackievirus B5 and cytotoxicity. EC50 CBV5 CC50 Vero-76 cells Compound Structure EC50 CBV5 CC50 Vero-76 cells Compound Structure (µM) (µM) O N (µM) (µM) O N S 11 O N S 0.09 ± 0.01 >100 11 O N 0.09 ± 0.01 >100 COOEt COOEt O N O N S 12 O N S 0.06 ± 0.01 >65 12 O N 0.06 ± 0.01 >65 COOH COOH O N O N S 13 O N S 0.3 ± 0.05 >100 13 O N 0.3 ± 0.05 >100 N N COOEt COOEt EC50: Compound concentration that reduces the plaque number of CBV5 by 50%. CC50: Concentration EC50: Compound concentration that reduces the plaque number of CBV5 by 50%. CC50: Concentration of a drug that will kill 50% of cells in uninfected cell culture. of a drug that will kill 50% of cells in uninfected cell culture. 4.2.2. Quinoxaline Derivatives Active against Orthomyxoviridae 4.2.2. Quinoxaline Derivatives Active against Orthomyxoviridae Influenza viruses can cause contagious respiratory disease in humans and are responsible every Influenza viruses can cause contagious respiratory disease in humans and are responsible every year for flu pandemics. year for flu pandemics. Based on the planar polyaromatic system (chromophore), quinoxaline derivatives are good Based on the planar polyaromatic system (chromophore), quinoxaline derivatives are good candidates to combat influenza viruses because of their potential to target the NS1 protein, a highly candidates to combat influenza viruses because of their potential to target the NS1 protein, a highly conserved influenza virus encoded protein. Since the N-terminal domain of the NS1A protein results conserved influenza virus encoded protein. Since the N-terminal domain of the NS1A protein results in a six-helical chain fold with a deep cavity at the center of the double-stranded RNA-binding Moleculesin a six-helical2020, 25, 2784chain fold with a deep cavity at the center of the double-stranded RNA-binding 10 of 19 surface, a small molecule could fit into the cavity and block virus replication [36,60–62]. 2,3,6- surface, a small molecule could fit into the cavity and block virus replication [36,60–62]. 2,3,6- substitued quinoxaline has also yielded compounds identified as having valuable antiviral activity, substitued quinoxaline has also yielded compounds identified as having valuable antiviral activity, particularly under bis-2-furyl substitution. Maintaining bis-2-furyl substitution, a novel series of derivativesparticularly was under synthetized bis-2-furyl tosubstitution. determine Maintaining the influence bis-2-furyl of substitution substitution, at position a novel 6. series Two of derivatives, quinoxaline derivatives was synthetized to determine the influence of substitution at position 6. Two onequinoxaline with 3-methoxyphenyl derivatives was synthetized group and to determine one with the 2-furyl influence at positionof substitution 6, showed at position good 6. Two activity with derivatives, one with 3-methoxyphenyl group and one with 2-furyl at position 6, showed good anderivatives, IC of 6.2 one and with 3.5 3-methoxyphenylµM, respectively group (Table and6). one RNA with intercalation 2-furyl at position experiments 6, showed showed good that both activity50 with an IC50 of 6.2 and 3.5 µM, respectively (Table 6). RNA intercalation experiments showed activity with an IC50 of 6.2 and 3.5 µM, respectively (Table 6). RNA intercalation experiments showed compoundsthat both compounds could bind could to the bind NS1A to the RNA-binding NS1A RNA-binding domain, domain, demonstrating demonstrating the the antiviral antiviral potential of that both compounds could bind to the NS1A RNA-binding domain, demonstrating the antiviral thesepotential quinoxaline of these quinoxaline derivatives derivatives [36]. [36]. potential of these quinoxaline derivatives [36]. TableTable 6. 6.StructureStructure andand activity activity of 2,3,6-substitued of 2,3,6-substitued quinoxaline. quinoxaline. Table 6. Structure and activity of 2,3,6-substitued quinoxaline. % Binding at 50 % Intercalation at 50 CompoundCompound StructureStructure % Binding Binding at at 50 50 µ%M Intercalation % Intercalation at 50 at 50 µM Compound Structure µM µM O µM µM O N O N N 14 14 O HN 74.074.0 4.5 4.5 H 14 N 74.0 4.5 N O OCH O OCH3 O 3 O N O O N N O 15 O HN 79.5 5.9 15 15 H 79.579.5 5.9 5.9 N N O O 4.2.3. Quinoxaline Derivatives Active against Filoviridae 4.2.3. Quinoxaline Derivatives Active against Filoviridae 4.2.3.Molecules Quinoxaline 2020, 25, x FOR Derivatives PEER REVIEW Active against Filoviridae 11 of 20

Ebola andand Marburg Marburg belong belong to theto theFiloviridae Filoviridaefamily family of single-stranded of single-stranded RNA viruses.RNA viruses. These viruses These wereviruses responsible were responsible for the 2014–2015 for the outbreak 2014–2015 of hemorrhagicoutbreak of fever hemorrhagic in Western fever Africa, in resulting Western in Africa, a total ofresulting 28,616 infectedin a total people, of 28,616 including infected 11,310people, deaths, including for a11,310 case-fatality deaths, rate for ofa case-fatality 40%. There rate is currently of 40%. noThere antiviral is currently drug licensed no antiviral by the drug U.S. licensed Food and by Drug the U.S. Administration Food and Drug (FDA) Administration to treat Ebola infection;(FDA) to however,treat Ebola four infection; drugs calledhowever, ZMapp four remdesivir,drugs called Mab114, ZMapp andremdesivir, REGN-EB3, Mab114, are underand REGN-EB3, investigation, are asunder each investigation, has reduced theas each risk ofhas death reduced from the Ebola risk [of63 death,64]. Actually, from Ebola the [63,64]. outbreak Actually, is not yet the over, outbreak with newis not cases yet over, identified with innew the cases Democratic identified Republic in the Democratic of Congo, andRepublic the need of Congo, for antiviral and the candidates need for remainsantiviral strong.candidates remains strong. A criticalcritical virus–host virus–host interaction interaction required required for virusfor virus egress egress and dissemination and dissemination involves involves late-budding late- domainsbudding containingdomains containing PPxY motifs, PPxY which motifs, are which highly are conserved highly conserved in the matrix in the protein matrix of aprotein large number of a large of RNAnumber viruses. of RNA Targeting viruses. this Targeting interaction, this a interaction, novel series a of novel quinoxaline-2-mercapto-acetyl-urea series of quinoxaline-2-mercapto-acetyl- analogues (Figureurea analogues 10) were (Figure synthetized 10) were and evaluatedsynthetized for and their evaluated ability to inhibitfor their viral ability egress to ofinhibit Marburg viral and egress Ebola of inMarburg VP40 VLP and budding Ebola in assayVP40 inVLP HEK293T budding cells assay [2]. in Among HEK293T them, cells four [2]. compounds Among them, demonstrated four compounds strong RNAdemonstrated viral egress strong inhibition RNA viral potential. egress inhibition potential.

Presence of a final arylalkyl group substituted at the ortho and para position by a halogen or a methyl group improved potency Substitution of sulfur resulted in greatly reduced activity R O O N S N N H H

N CH3 Methyl substituents on the imido and amide nitrogen atoms resulted in greatly No suitable replacement of the reduced activity methyl substituent on the quinoxaline moiety

Figure 10. Structure–activity relationship of novel series of quinoxaline-2-mercapto-acetyl-

urea analogues. Figure 10. Structure–activity relationship of novel series of quinoxaline-2-mercapto-acetyl-urea analogues.

4.2.4. Quinoxaline Derivatives Active against Flaviviridae HCV is responsible for both acute and chronic hepatitis, ranging in severity from a mild illness lasting a few weeks to a serious, lifelong illness. Globally, an estimated 71 million people have chronic HCV infection and in 2016, approximately 399,000 people died from HCV [65]. Antiviral drugs can cure more than 95% of HCV patients, but access to treatment is poor due to its high cost, which is why research is ongoing. Several quinoxaline derivatives were evaluated for their anti-HCV potential. Even though novel quinoxaline derivatives synthetized using ethyl (6,7-dimethyl-2-oxo-3,4-dihydroquinoxalin-3- yl)acetate 4 and ethyl (6-methyl-2-oxo-3,4-dihydroquinoxalin-3-yl)acetate 5, 3-methylquinoxalin- 2(1H)-one, and 1,4-dihydroquinoxaline-2,3-dione as precursors failed to demonstrate any activity against HCV [31], in pyrido[2,3-g]quinoxalinone series, 5-chloro-3-(thiophen-2-yl)pyrido[2,3- g]quinoxaline-2(1H)-one 16 (Figure 11) was able to inhibit HCV replication in a subgenomic replication assay with EC50 = 7.5 ± 0.5 µM. However, it was also cytotoxic for GS4.1 cells (CC50 = 21 ± 20 µM) [37,38,55].

H N O

N N Cl S Figure 11. 5-chloro-3-(thiophen-2-yl)pyrido[2,3-g]quinoxaline-2(1H)-one 16.

Molecules 2020, 25, x FOR PEER REVIEW 11 of 20

Ebola and Marburg belong to the Filoviridae family of single-stranded RNA viruses. These viruses were responsible for the 2014–2015 outbreak of hemorrhagic fever in Western Africa, resulting in a total of 28,616 infected people, including 11,310 deaths, for a case-fatality rate of 40%. There is currently no antiviral drug licensed by the U.S. Food and Drug Administration (FDA) to treat Ebola infection; however, four drugs called ZMapp remdesivir, Mab114, and REGN-EB3, are under investigation, as each has reduced the risk of death from Ebola [63,64]. Actually, the outbreak is not yet over, with new cases identified in the Democratic Republic of Congo, and the need for antiviral candidates remains strong. A critical virus–host interaction required for virus egress and dissemination involves late- budding domains containing PPxY motifs, which are highly conserved in the matrix protein of a large number of RNA viruses. Targeting this interaction, a novel series of quinoxaline-2-mercapto-acetyl- urea analogues (Figure 10) were synthetized and evaluated for their ability to inhibit viral egress of Marburg and Ebola in VP40 VLP budding assay in HEK293T cells [2]. Among them, four compounds demonstrated strong RNA viral egress inhibition potential.

Presence of a final arylalkyl group substituted at the ortho and para position by a halogen or a methyl group improved potency Substitution of sulfur resulted in greatly reduced activity R O O N S N N H H

N CH3 Methyl substituents on the imido and amide nitrogen atoms resulted in greatly No suitable replacement of the reduced activity methyl substituent on the quinoxaline moiety

Figure 10. Structure–activity relationship of novel series of quinoxaline-2-mercapto-acetyl-urea Moleculesanalogues.2020, 25, 2784 11 of 19

4.2.4. Quinoxaline Derivatives Active against Flaviviridae 4.2.4. Quinoxaline Derivatives Active against Flaviviridae HCV is responsible for both acute and chronic hepatitis, ranging in severity from a mild illness lastingHCV a few isresponsible weeks to a serious, for both lifelong acute and illness. chronic Globally, hepatitis, an estimated ranging in71 severitymillion people from a have mild chronic illness lastingHCV infection a few weeks and toin a2016, serious, approximately lifelong illness. 399,000 Globally, people an died estimated from 71HCV million [65]. peopleAntiviral have drugs chronic can HCVcure more infection than and 95% in of 2016, HCV approximately patients, but 399,000access to people treatment died is from poor HCV due [to65 ].its Antiviral high cost, drugs which can is curewhy moreresearch than is 95% ongoing. of HCV patients, but access to treatment is poor due to its high cost, which is why researchSeveral is ongoing. quinoxaline derivatives were evaluated for their anti-HCV potential. Even though novel quinoxalineSeveral quinoxalinederivatives derivatives synthetized were using evaluated ethyl for (6,7-dimethyl-2-oxo-3,4-dihydroquinoxalin-3- their anti-HCV potential. Even though novel quinoxalineyl)acetate 4 derivatives and ethyl synthetized (6-methyl-2-oxo-3,4- using ethyldihydroquinoxalin-3-yl)acetate (6,7-dimethyl-2-oxo-3,4-dihydroquinoxalin-3-yl)acetate 5, 3-methylquinoxalin- 42(1andH)-one, ethyl and (6-methyl-2-oxo-3,4-dihydroquinoxalin-3-yl)acetate 1,4-dihydroquinoxaline-2,3-dione as precursors failed5, 3-methylquinoxalin-2(1 to demonstrate any Hactivity)-one, andagainst 1,4-dihydroquinoxaline-2,3-dione HCV [31], in pyrido[2,3- asg]quinoxalinone precursors failed series, to demonstrate 5-chloro-3-(thiophen-2-yl)pyrido[2,3- any activity against HCV [31], ing]quinoxaline-2(1 pyrido[2,3-g]quinoxalinoneH)-one 16 (Figure series, 5-chloro-3-(thiophen-2-yl)pyrido[2,3-11) was able to inhibit HCV replicationg]quinoxaline-2(1 in a subgenomicH)-one 16replication(Figure assay11) was with able EC50to = 7.5 inhibit ± 0.5 µM. HCV However, replication it was in also a subgenomic cytotoxic for replication GS4.1 cells assay(CC50 = with 21 ± EC20 50µM)= 7.5 [37,38,55].0.5 µM. However, it was also cytotoxic for GS4.1 cells (CC50 = 21 20 µM) [37,38,55]. ± ± H N O

N N Cl S Molecules 2020, 25, xFigure FigureFOR PEER 11.11. REVIEW5-chloro-3-(thiophen-2-yl)pyrido[2,3-5-chloro-3-(thiophen-2-yl)pyrido[2,3- gg]quinoxaline-2(1]quinoxaline-2(1HH)-one)-one 16.16. 12 of 20

However,However, grazoprevirgrazoprevir 1717,, aa novelnovel P2-P4P2-P4 quinoxalinequinoxaline macrocyclicmacrocyclic NS3NS3/4a/4a proteaseprotease inhibitorinhibitor withwith broadbroad activityactivity acrossacross genotypesgenotypes andand resistantresistant variants,variants, is currentlycurrently approved for thethe treatmenttreatment forfor HCVHCV [[39]39] (Figure(Figure 1212).).

H N

O ON O H H O NH N NH O O O O HN S O Figure 12. Chemical structure of grazoprevir.

TheThe structure–activitystructure–activity relationshiprelationship showsshows thatthat grazoprevir’sgrazoprevir’s eefficacyfficacy derivesderives fromfrom lipophiliclipophilic interactioninteraction atat P2 P2 position position in additionin addition to a contributionto a contribution from the from P2-P4 the constraint P2-P4 constraint [66]. The P2 [66]. quinoxaline The P2 moietyquinoxaline largely moiety avoids largely direct avoids interaction direct with interaction residues with Arg-155 residues and Asp-168, Arg-155 theand two Asp-168, most common the two resistance-associatedmost common resistance-associated residues, but interacts residues, with but theinteracts catalytic with His-57 the catalytic and Asp-81, His-57 which andexplains Asp-81, itswhich activity explains against its activity most HCV against genotypes most HCV and geno resistanttypes variants and resistant [67]. Modelingvariants [67]. studies Modeling showed studies that inshowed patients that who in failedpatients to achievewho failed sustained to achiev virologice sustained response virologic with simeprevir, response with grazoprevir simeprevir, was egrazoprevirfficacious because was efficacious of a strong because direct of cation–quinoxaline a strong direct cation–quinoxaline interaction with theinteraction Lys-155 with side chainthe Lys- of double155 side substitution chain of double R155K substitution/D168A [40 R155K/D168A]. More recently, [40]. eliminating More recently, the P2-P4 eliminating moiety the was P2-P4 considered moiety aimingwas considered at conformational aiming at flexibility conformational and exploration flexibility of and diverse exploration quinoxalines of diverse at position quinoxalines P2 in order at toposition improve P2 potencyin order and to improve resistance potency profile. and The resist structure–activityance profile. The relationship structure–activity indicated relationship that a small hydrophobicindicated that substituent a small hydrophobic at position 3substituent of P2 quinoxaline at position effectively 3 of P2 maintains quinoxaline activity effectively against maintains resistant variants,activity against as derivatives resistant with variants, a larger as derivatives group at position with a 3larger of P2 group quinoxaline at position shift 3 out of P2 of thequinoxaline binding shift out of the binding site [41,42]. Further investigations involved replacement of the quinoxaline moiety by a quinoline scaffold, leading to interesting analogues [68].

4.2.5. Quinoxaline Derivatives Active against Rhabdoviridae Vesicular stomatis virus (VSV) is a virus in the family Rhabdoviridae. This virus is zoonotic and in infected humans leads to a flu-like illness characterized by fever, headache, myalgia, weakness, and occasionally vesicular lesions of the mouth [69]. As VSV infection results in a short 3–5-day illness, no specific treatment is available and VSV is commonly used as a laboratory virus to study the properties of viruses. Indoloquinoxaline derivatives and their benzoindoloquinoxalines were synthetized and assessed for their anti-VSV activity, interferon-inducing ability, and cytotoxicity (Figure 13). Anti-viral activity was significantly reduced with annulation of benzene ring in indoloquinoxaline derivatives [43,44].

N N

N N N N R2N R2N

Figure 13. General structure of synthetized indoloquinoxalines and benzoindoloquinoxalines.

As these indoloquinoxalines were more active antivirals when they were added immediately after virus infection, it was supposed that their antiviral action was first mediated by interferon.

Molecules 2020, 25, x FOR PEER REVIEW 12 of 20

However, grazoprevir 17, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor with broad activity across genotypes and resistant variants, is currently approved for the treatment for HCV [39] (Figure 12).

H N

O ON O H H O NH N NH O O O O HN S O Figure 12. Chemical structure of grazoprevir.

The structure–activity relationship shows that grazoprevir’s efficacy derives from lipophilic interaction at P2 position in addition to a contribution from the P2-P4 constraint [66]. The P2 quinoxaline moiety largely avoids direct interaction with residues Arg-155 and Asp-168, the two most common resistance-associated residues, but interacts with the catalytic His-57 and Asp-81, which explains its activity against most HCV genotypes and resistant variants [67]. Modeling studies showed that in patients who failed to achieve sustained virologic response with simeprevir, grazoprevir was efficacious because of a strong direct cation–quinoxaline interaction with the Lys- 155 side chain of double substitution R155K/D168A [40]. More recently, eliminating the P2-P4 moiety was considered aiming at conformational flexibility and exploration of diverse quinoxalines at position P2 in order to improve potency and resistance profile. The structure–activity relationship Molecules 2020, 25, 2784 12 of 19 indicated that a small hydrophobic substituent at position 3 of P2 quinoxaline effectively maintains activity against resistant variants, as derivatives with a larger group at position 3 of P2 quinoxaline siteshift [ 41out,42 of]. the Further binding investigations site [41,42]. involvedFurther inve replacementstigations ofinvolved the quinoxaline replacement moiety of the by quinoxaline a quinoline scamoietyffold, by leading a quinoline to interesting scaffold, analogues leading to [ 68interesting]. analogues [68].

4.2.5. Quinoxaline Derivatives Active against Rhabdoviridae Vesicular stomatisstomatis virus (VSV)(VSV) is aa virusvirus inin thethe familyfamily RhabdoviridaeRhabdoviridae.. This virus isis zoonoticzoonotic andand in infectedinfected humans leads to a flu-likeflu-like illness characterized by fever, headache,headache, myalgia, weakness, and occasionallyoccasionally vesicular vesicular lesions lesions of of the the mouth mouth [69 ].[69]. As VSVAs VSV infection infection results results in a short in a 3–5-dayshort 3–5-day illness, noillness, specific no treatmentspecific treatment is available is andavailable VSV is and commonly VSV is usedcommonly as a laboratory used as virusa laboratory to study virus the properties to study oftheviruses. properties Indoloquinoxaline of viruses. Indoloquinoxaline derivatives and derivatives their benzoindoloquinoxalines and their benzoindoloquinoxalines were synthetized were andsynthetized assessed and for assessed their anti-VSV for their activity, anti-VSV interferon-inducing activity, interferon-inducing ability, and ability, cytotoxicity and cytotoxicity (Figure 13). Anti-viral(Figure 13). activity Anti-viral was significantlyactivity was reduced significantl withy annulation reduced with of benzene annulation ring inof indoloquinoxaline benzene ring in derivativesindoloquinoxaline [43,44]. derivatives [43,44].

N N

N N N N R2N R2N

Figure 13. General structure of synthetized indoloquinoxalinesindoloquinoxalines and benzoindoloquinoxalines.

MoleculesAs 2020 thesethese, 25 indoloquinoxalines,indoloquinoxalines x FOR PEER REVIEW were were more more active active antivirals antivirals when when they they were were added added immediately immediately13 after of 20 virusafter virus infection, infection, it was it supposed was supposed that their that antiviraltheir antiviral action action was first was mediated first mediated by interferon. by interferon. 4.2.6. Quinoxaline Derivatives Active against Retroviridae 4.2.6. Quinoxaline Derivatives Active against Retroviridae While there is no cure for HIV, there are very effective treatments that enable most people with the virusWhile to there live a is long no cure and for healthy HIV, therelife. Combination are very effective antiretroviral treatments therapy that enable is required mostpeople for durable with thevirologic virus tosuppression. live a long Reverse and healthy transcriptase life. Combination is one of the antiretroviral most frequent therapy targets is required for the treatment for durable of virologicHIV infection, suppression. since the Reverse blockage transcriptase of this enzyme is one can of thestop most an essential frequent targetsstep in forviral the replication. treatment ofHowever, HIV infection, a growing since number the blockage of cases ofof thisresistant enzyme HIV can strains stop and an essentialserious adverse step in events viral replication. due to the However,antiretroviral a growing therapy number administered of cases have of resistant encouraged HIV strainsattempts and to serious develop adverse new HIV events agents, due tomore the antiretroviralactive, less toxic, therapy and administeredwith increased have tolerability encouraged to mutation attempts to[70]. develop Some newquinoxaline HIV agents, derivatives more active, like lessHBY, toxic, HBQ, and and with S-2720 increased have demonstrated tolerability to high mutation potency [70 ].as Some reverse quinoxaline transcriptase derivatives inhibitors like (Figure HBY, HBQ,14). and S-2720 have demonstrated high potency as reverse transcriptase inhibitors (Figure 14).

H H N S N O

CH3 H3CO N S F N

O O O O H C CH 3 3 H3C CH3

HBY HBQ

Figure 14. Chemical structure of HBY and HBQ.

For thesethese reasons,reasons, design,design, synthesis, and evaluationevaluation of new quinoxalinequinoxaline derivatives was investigated.investigated. UsingUsing aa computational computational approach, approach, 58 58 quinoxaline quinoxaline compounds compounds were were identified, identified, and 25and new 25 quinoxalinenew quinoxaline and quinoxaline-relatedand quinoxaline-related compounds compounds were were synthetized synthetized and and evaluated evaluated as inhibitorsas inhibitors of reverseof reverse transcriptase transcriptase (RT). (RT). Chemical Chemical features features identified identified as crucial as forcrucial reverse for transcriptase reverse transcriptase inhibition wereinhibition the presence were the ofpresence a five- of or a six-membered five- or six-membered aromatic aromatic ring and ring a hydrophilic and a hydrophilic center that center can that be can be nitrogen, oxygen, or sulfur. Six of these derivatives presented the highest inhibitory activity at 100 µM, ranging from 56% to 99% of reverse transcriptase inhibition, and were considered as hit compounds. One compound was a particularly interesting derivative, with values comparable to those of commercial compound nevirapine when used at 10 µM (both showing reverse transcriptase inhibition % = 91) [45] (Table 7).

Molecules 2020, 25, 2784 13 of 19 nitrogen, oxygen, or sulfur. Six of these derivatives presented the highest inhibitory activity at 100 µM, ranging from 56% to 99% of reverse transcriptase inhibition, and were considered as hit compounds. One compound was a particularly interesting derivative, with values comparable to those of commercial compound nevirapine when used at 10 µM (both showing reverse transcriptase inhibitionMolecules 2020 %, =25,91) x FOR [45 PEER] (Table REVIEW7). 14 of 20 Molecules 2020, 25, x FOR PEER REVIEW 14 of 20 Molecules 2020, 25, x FOR PEER REVIEW 14 of 20 Molecules 2020, 25, x FOR PEER REVIEW 14 of 20 MoleculesTableTable 2020, 7.257., ActivityActivityx FOR PEER of new REVIEW potential potential quinoxaline quinoxaline derivati derivativesves as asinhibitors inhibitors of reverse of reverse transcriptase. transcriptase. 14 of 20 Table 7. Activity of new potential quinoxaline derivatives as inhibitors of reverse transcriptase. Table 7. Activity of new potential quinoxaline derivatives as inhibitors of reverse transcriptase. Table 7. Activity of new potential quinoxaline%RT Inhibitionderivatives as inhibitors of reverse transcriptase. Table 7. Activity of new potential quinoxaline%RT%RT Inhibitionderivati Inhibitionves as inhibitorsMT2 IC50 of reverseSelectivity transcriptase. Index CompoundCompound StructureStructure %RT100 Inhibition10 MT2MT2 IC IC5050 (µM)SelectivitySelectivity Index Index (SI) Compound Structure %RT100100µ InhibitionM10 10 µ M MT2(µM) IC 50 Selectivity(SI) Index Compound Structure %RTµM100 InhibitionµM10 MT2(µM) IC 50 Selectivity(SI) Index Compound Structure µM100 µM10 MT2(µM) IC 50 Selectivity(SI) Index Compound StructureH µM100 µM10 (µM) (SI) HN O µM µM (µM) (SI) NH O µM µM NH O NH O 1818 5656 7 7 NE NE NE NE 18 N O 56 7 NE NE 18 N 56 7 NE NE 18 N 56 7 NE NE 18 O N CH 56 7 NE NE O N CH3 O CH3 O H CH3 O HN CHO3 NH O3 NH O NH O N O 0.63 (0.53– 19 N 99 91 0.63 (0.53– 31,798 1919 N 9999 91 910.63 0.630.76) (0.53– (0.53–0.76) 31,798 31,798 19 N 99 91 0.630.76) (0.53– 31,798 19 O N 99 91 0.630.76) (0.53– 31,798 19 O N 99 91 0.76) 31,798 O CH 0.76) O CH3 O CH3 HCH3 HNCH3 O NH 3O NH O NH O N CHO 20 N CH3 64 37 64 (38–103) NE 20 N CH3 64 37 64 (38–103) NE 2020 H3C N CH3 6464 37 3764 (38–103) 64 (38–103) NE NE 20 H3C O O 3 64 37 64 (38–103) NE 20 H3C O N O CH3 64 37 64 (38–103) NE H3C CH O O H3C CH3 O O CH3 O O CHH3 CHHN3 O NH3 O CH NH O CH3 NH O CH3 N O CH3 21 N CH3 3 51 22 67 (54–87) 74 21 N CHCH3 51 22 67 (54–87) 74 21 N CH3 51 22 67 (54–87) 74 N CH3 2121 O O CH3 5151 22 2267 (54–87) 67 (54–87) 74 74 21 O N O CHCH33 51 22 67 (54–87) 74 O O CHCH3 O O CHCH3 3 O O CH3 H CH3 HN O CH3 NH O CH 3 22 NH O CH3 73 15 23 (21–25) NE 22 NH O CH3 73 15 23 (21–25) NE 22 N O CH3 73 15 23 (21–25) NE 22 N CHCH3 3 73 15 23 (21–25) NE 2222 N CH3 3 7373 15 1523 (21–25) 23 (21–25) NE NE N CH3 0.13 (0.09– Nevirapine N - CH3 98 91 0.13 (0.09– 14,353 Nevirapine N - CH3 98 91 0.130.17) (0.09– 14,353 Nevirapine - 98 91 0.130.17) (0.09– 14,353 Nevirapine - NE: Not98 evaluated.91 0.130.17) (0.09– 14,353 NevirapineNevirapine -- NE: Not98 98 evaluated.91 91 0.130.17) (0.09–0.17) 14,353 14,353 NE: Not evaluated. 0.17) NE:NE: Not Not evaluated. evaluated. Compound 19 displayed similar inhibitoryNE: Not activity evaluated. with nevirapine, with an EC50 = 3.1 nM vs. Compound 19 displayed similar inhibitory activity with nevirapine, with an EC50 = 3.1 nM vs. EC50 Compound= 6.7 nM respectively 19 displayed and similar can be consideredinhibitory activity a promising with nevirapine,lead compound with [45]. an EC 50 = 3.1 nM vs. EC50Compound =Compound 6.7 nM respectively 1919 displayeddisplayed and similar similarcan be consideredinhibitory inhibitory activitya activity promising with with leadnevirapine, nevirapine, compound with with [45]. an anEC EC50 =50 3.1= nM3.1 nMvs. vs. EC50 =Moreover,Compound 6.7 nM respectively this 19 displayednew class and similarofcan integrase be consideredinhibitory inhibitors activitya promising proved with very leadnevirapine, compoundeffective with against [45]. an EC HIV,50 = showing3.1 nM vs. a ECEC5050 =Moreover,= 6.7 nM respectively respectively this new class and and ofcan can integrase be be considered considered inhibitors a promising a promisingproved very lead lead effectivecompound compound against [45]. [45 HIV,]. showing a highEC50 Moreover,=therapeutic 6.7 nM respectively this index. new Three class and representatives ofcan integrase be considered inhibitors of this a promising class, proved raltegravir, very lead effectivecompound elvitegravir, against [45]. and HIV, dolutegravir showing a highMoreover, Moreover,therapeutic this this index. new new Three class class ofrepresentatives of integrase integrase inhibitors inhibitors of this class, proved proved raltegravir, very very e effectiveffective elvitegravir, against against and HIV, HIV, dolutegravir showing showing a a high highare currently Moreover,therapeutic available. this index. new However,Three class representatives of integrasefor two of inhibitors them of this ha veclass, proved led raltegravir, to reportedvery effective elvitegravir, cases againstof resistance, and HIV, dolutegravir showingindicating a therapeuticarehigh currently therapeutic index. available. index. Three However,Three representatives representatives for two of of them thisof this ha class, veclass, led raltegravir, raltegravir,to reported elvitegravir, elvitegravir,cases of resistance, and and dolutegravir dolutegravir indicating are aretohigh an currently therapeuticurgent needavailable. index. to develop However, Three otherrepresentatives for new two effective of them of this haanti-HIVve class, led raltegravir, toagents. reported In a elvitegravir,cases structure-based of resistance, and dolutegravirdrug indicating design currentlytoare an currently urgent available. needavailable. to However, develop However, other for twofor new two of effective them of them have anti-HIVha ledve led to reportedtoagents. reported In cases a cases structure-based of of resistance, resistance, drug indicating indicating design to an toapproach,are an currently urgent the need available. quinoxaline to develop However, scaffold other for was new two identified effective of them asanti-HIVha ave core led moiety toagents. reported to In design a casesstructure-based potential of resistance, novel drug indicating anti-HIV design urgentapproach,to an urgent need the to need quinoxaline develop to develop other scaffold new other e wasff newective identified effective anti-HIV asanti-HIV a agents. core moiety agents. In a structure-based to In design a structure-based potential drug novel design drug anti-HIV design approach, approach,agents.to an urgent A seriesthe need quinoxaline of toseven develop new scaffold other6-chloro-7-fluoroquino was new identified effective asanti-HIV a xalinecore moiety agents.derivatives to In design a structure-basedwith potential various novelsubstituents drug anti-HIV design at theagents.approach, quinoxaline A seriesthe quinoxaline scaof sevenffold wasnew scaffold identified6-chloro-7-fluoroquino was identified as a core as moiety axaline core moiety toderivatives design to design potential with potential various novel novelsubstituents anti-HIV anti-HIV agents. at agents.positionapproach, A 2 series theand quinoxaline 3of wasseven also new scaffold synthetized. 6-chloro-7-fluoroquino was identified Among asthem, axaline core two moiety derivatives derivatives to design with 23 potential variousand 24 substituentsnovelbearing anti-HIV bulky at positionagents. A 2 series and 3of was seven also new synthetized. 6-chloro-7-fluoroquino Among them,xaline two derivatives derivatives with 23 variousand 24 substituentsbearing bulky at Apositionsubstitutionagents. series ofA 2 sevenseriesand at position 3of new was seven 6-chloro-7-fluoroquinoxaline also 2 newand synthetized. 6-chloro-7-fluoroquino3 exhibited Among better acthem,tivity derivativesxaline two compared derivatives derivatives with to various unsuwith 23 bstitutedandvarious substituents 24 bearingsubstituents or less at positionbulky at 2 substitutionposition 2 and at position3 was also 2 and synthetized. 3 exhibited Among better acthem,tivity two compared derivatives to unsu 23 bstitutedand 24 bearing or less bulky substitutionsubstitutionsposition 2 and at [46]. position3 wasIn addition, also2 and synthetized. these3 exhibited two compoundsAmong better acthem,tivity revealed two compared derivatives no cytotoxicity to unsu 23 bstitutedand on VERO24 bearingor cellsless (Tablebulkybulky substitutionssubstitution at[46]. position In addition, 2 and these3 exhibited two compounds better activity revealed compared no cytotoxicity to unsubstituted on VERO or cells less (Tablebulky substitutions8).substitution at[46]. position In addition, 2 and these3 exhibited two compounds better activity revealed compared no cytotoxicity to unsubstituted on VERO or cells less (Tablebulky 8).substitutions [46]. In addition, these two compounds revealed no cytotoxicity on VERO cells (Table 8).substitutions [46]. In addition, these two compounds revealed no cytotoxicity on VERO cells (Table 8). 8).

Molecules 2020, 25, 2784 14 of 19 and 3 was also synthetized. Among them, two derivatives 23 and 24 bearing bulky substitution at position 2 and 3 exhibited better activity compared to unsubstituted or less bulky substitutions [46]. In addition, these two compounds revealed no cytotoxicity on VERO cells (Table8). Molecules 2020, 25, x FOR PEER REVIEW 15 of 20 Molecules 2020, 25, x FOR PEER REVIEW 15 of 20 Table 8. HIV activity and cytotoxicity study of the two most potent new 6-chloro-7- Table 8. HIV activity and cytotoxicity study of the two most potent new 6-chloro-7-fluoroquinoxaline MoleculesfluoroquinoxalineTable 2020 8., 25 HIV, x FOR activity PEER derivatives. and REVIEW cytotoxicity study of the two most potent new 6-chloro-7-fluoroquinoxaline15 of 20 derivatives. derivatives. CompoundTable 8. HIV activity and cytotoxicity Structure study of the tw Straino most IIIBpotent IC 50new(µ g6-chloro-7-fluoroquinoxaline/mL) Vero IC50 (µg/mL) Compound Structure Strain IIIB IC50 (µg/mL) Vero IC50 (µg/mL) Compoundderivatives. Structure Strain IIIB IC50 (µg/mL) Vero IC50 (µg/mL)

Compound Cl StructureN Strain IIIB IC50 (µg/mL) Vero IC50 (µg/mL) Cl N 2323 >11.78>11.78 >100>100 23 >11.78 >100 ClF NN F N 23 >11.78 >100

F N F F Cl N Cl N F 24 >15.45 >100 24 >15.45>15.45 >100>100 ClF NN F N 24 >15.45 >100 F F N F Recently, an approach aimed at dysregulation of gelatinase and pathogenesis of HIV led to the Recently, an approach aimed at dysregulation of gelatinase and pathogenesis of HIV led to the synthesis of two new classes of gelatinase inhibitoF rs bearing a quinoxalinone motif, based on this synthesis of two new classes of gelatinase inhibito rs bearing a quinoxalinone motif, based on this coplanarRecently, scaffold anan approachapproach being able aimedaimed to penetrate atat dysregulationdysregulation into the relatively ofof gelatinasegelatinase broad andand S1 pathogenesispathogenesis binding domain of HIV of gelatinase. led toto thethe coplanar scaffold being able to penetrate into the relatively broad S1 binding domain of gelatinase. synthesissynthesisThe acylamide of twotwo (Series newnew classesclasses I) and ofof acylhydrazone gelatinasegelatinase inhibitorsinhibito (Seriesrs bearingbearingII) linkage aa quinoxalinonequinoxalinone can also act as motif,motif, potent basedbased H-bonding onon thisthis The acylamide (Series I) and acylhydrazone (Series II) linkage can also act as potent H-bonding coplanaracceptor/donor scascaffoldffold to being interact able with to penetratethe active intoamino the acid relatively of the broadenzyme S1 [47] binding (Figure domain 15). of gelatinase. acceptor/donor to interact with the active amino acid of the enzyme [47] (Figure 15). TheThe acylamideacylamide (Series(Series I)I) andand acylhydrazoneacylhydrazone (Series II) linkage can alsoalso actact asas potentpotent H-bondingH-bonding acceptoracceptor/donor/donor toto interactinteract withwith thetheO activeactive aminoamino acidacid ofof thethe enzymeenzyme [[47]47] (Figure(Figure 15 15).). O H O NH R O N N R1 O N 1 N R3 H H N N R3 N OH O O HN N O N OR1 N OH R N N O N R2R H N 2 3 NN OCH O H N CH3 N O R 3 N CH3 2 N CH3 N CH3 S1 N CH S1 3 S1 S1 Figure 15. Structure and modelS1 of the binding site of new gelatinase inhibitors. Figure 15. Structure and model of the binding site of new gelatinaseS1 inhibitors. DerivativesFigure in series 15. Structure 1 displayed and model moderate of the activi bindingty with site of gelatinase new gelatinase enzymatic inhibitors. inhibition ranging DerivativesFigure in series 15. Structure 1 displayed and model moderate of the activi bindingty withsite of gelatinase new gelatinase enzymatic inhibitors. inhibition ranging from 34.79 ± 6.3 µM to >500 µM against 5.64 ± 0.6 µM for LY52 taken as reference. The best activity from 34.79 ± 6.3 µM to >500 µM against 5.64 ± 0.6 µM for LY52 taken as reference. The best activity was Derivativesobserved for in series a para 1 displayed-chloro phenyl moderate substi activityactivituent.ty with In gelatinaseseries II, enzymatictwo derivatives inhibition 25 rangingand 26 fromwas 34.79observed6.3 µforM toa >para500-chloroµM against phenyl 5.64 substi0.6tuent.µM for In LY52 series taken II, as two reference. derivatives The best 25 activityand 26 fromdemonstrated 34.79 ±± 6.3 similar µM to activity >500 µM with against LY52, 5.64 probably ± 0.6 µM because for LY52 the substitakentuents as reference. introduced The havebest activityenough demonstrated similarpara activity with LY52, probably because the substituents introduced25 26 have enough waswasspace observedobserved and the for rightfor a aorientation -chloropara-chloro phenyl to phenylguide substituent. the substi compounds Intuent. series In II,to series twofit into derivatives II, the two binding derivativesand cavitydemonstrated (Table25 and 9). 26 In space and the right orientation to guide the compounds to fit into the binding cavity (Table 9). In similardemonstratedaddition, activity as compoundsimilar with LY52,activity 26 probably displayedwith LY52, because slightlyprobably the more substituentsbecause potent the substi introducedactivitytuents than haveintroduced compound enough have space25 ,enough it andwas addition, as compound 26 displayed slightly more potent activity than compound 25, it was thespaceconcluded right and orientation the that right the orientationphenolic to guide thehydroxyl to compoundsguide groupthe compounds toco fituld into provide theto fit binding intoa more the cavity effectivebinding (Table cavityhydrogen-bonding9). In(Table addition, 9). In concluded that the phenolic hydroxyl group could provide a more effective hydrogen-bonding asaddition,interaction, compound as resultingcompound26 displayed in 26increased slightly displayed more affinity. slightly potent Substi activity moretution potent than with compound activity an aliphatic than25, it wascompoundgroup concluded led 25to , thatinactiveit was the interaction, resulting in increased affinity. Substitution with an aliphatic group led to inactive phenolicconcludedcompounds. hydroxyl that the group phenolic could hydroxyl provide agroup more co effuldective provide hydrogen-bonding a more effective interaction, hydrogen-bonding resulting in compounds. increasedinteraction, affi resultingnity. Substitution in increased with anaffinity. aliphatic Substi grouptution led towith inactive an aliphatic compounds. group led to inactive compounds.

MoleculesMolecules 20202020, 25, x25 FOR, 2784 PEER REVIEW 16 of15 20 of 19 Molecules 2020, 25, x FOR PEER REVIEW 16 of 20

Table 9. Enzymatic inhibition and cytotoxicity study of tested compounds. TableTable 9. Enzymatic 9. Enzymatic inhibition inhibition and and cytotoxici cytotoxicityty study study of tested of tested compounds. compounds. C8166 CompoundCompound StructureStructure Gelatinase Gelatinase IC 50IC(50µ M)(µM) C8166C8166 CC50 (µM) Compound Structure Gelatinase IC50 (µM) CC50 (µM) CC50 (µM) O O N CH3 N N CH3 HN 2525 N OH 9.399.390.7 ± 0.7 >100>100 25 N O 9.39± ± 0.7 >100

N CH3 N CH3 O O N H N N H HN 26 N OH OH 7.17 ± 0.95 >100 2626 N O OH 7.177.170.95 ± 0.95 >100>100 ±

N CH3 OCH3 N CH3 OCH3 LY52 5.64 ± 0.6 >100 LY52 5.64 ± 0.6 >100 LY52 5.64 0.6 >100 ± 5. Conclusion 5. Conclusion 5. Conclusions Quinoxaline represent an important class of nitrogen-containing heterocycles with a wide range Quinoxaline represent an important class of nitrogen-containing heterocycles with a wide range of potentialQuinoxaline biological represent activities. an importantThis review class points of nitrogen-containing to a growing interest heterocycles in the development with a wide rangeof of potential biological activities. This review points to a growing interest in the development of compoundsof potential bearing biological a quinoxaline activities. moiety This reviewfor antivira pointsl treatment. to a growing Regarding interest the in antiviral the development activity of of compounds bearing a quinoxaline moiety for antiviral treatment. Regarding the antiviral activity of quinoxalinecompounds derivatives, bearing astudies quinoxaline showed moiety that these for antiviral derivatives treatment. represented Regarding very encouraging the antiviral agents activity quinoxaline derivatives, studies showed that these derivatives represented very encouraging agents forof investigators quinoxaline as derivatives, they exhibit studies some activity showed ag thatainst these a large derivatives number representedof different veryviruses. encouraging Future for investigators as they exhibit some activity against a large number of different viruses. Future investigationsagents for investigatorsof this moiety as requiring they exhibit analysis some of activity structure–activity against a large relationships, number of as di ffwellerent as viruses. the investigations of this moiety requiring analysis of structure–activity relationships, as well as the mechanismsFuture investigations of action of of these this moietycompounds requiring coul analysisd give some of structure–activity more encouraging relationships, results and as may well as mechanisms of action of these compounds could give some more encouraging results and may providethe mechanisms to new useful of actiontherapeutics. of these compounds could give some more encouraging results and may provide to new useful therapeutics. Authorprovide Contributions: to new useful Conceptualization: therapeutics. M.M.; methodology: M.M.; formal analysis: M.M., V.M., O.K.; data Author Contributions: Conceptualization: M.M.; methodology: M.M.; formal analysis: M.M., V.M., O.K.; data curation; M.M., V.M., O.K.; writing—original draft preparation: M.M.; writing—review and editing: P.V.; curation;Author M.M., Contributions: V.M., O.K.;Conceptualization: writing—original M.M.;draft pr methodology:eparation: M.M.; M.M.; writing—review formal analysis: and M.M., editing: V.M., P.V.; O.K.; supervision: P.V. All authors have read and agreed to the published version of the manuscript. supervision:data curation; P.V. All M.M., authors V.M., have O.K.; read writing—original and agreed to the draft published preparation: version M.M.; of the writing—review manuscript. and editing: P.V.; Funding:supervision: This research P.V. All received authors haveno external read and funding. agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. ConflictsConflicts of Interest: of Interest: TheThe authors authors declare declare no noconflict conflict of interest. of interest. References ReferencesReferences 1. Blair, W.; Cox, C. Current Landscape of Antiviral Drug Discovery. F1000Research 2016, 5, F1000. 1. Blair, W.; Cox, C. Current Landscape of Antiviral Drug Discovery. F1000Research 2016, 5, F1000. 2. 1. Loughran,Blair, W.; H.M.; Cox, Han, C. Current Z.; Wrobel, Landscape J.E.; Decker, of Antiviral S.E.; Drug Ruthel, Discovery. G.; Freedman,F1000Research B.D.; Harty,2016, 5R.N.;, F1000. Reitz, [CrossRef A.B. ] 2. Loughran, H.M.; Han, Z.; Wrobel, J.E.; Decker, S.E.; Ruthel, G.; Freedman, B.D.; Harty, R.N.; Reitz, A.B. Quinoxaline-based[PubMed] inhibitors of Ebola and Marburg VP40 egress. Bioorg. Med. Chem. Lett. 2016, 26, 3429–3435. Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress. Bioorg. Med. Chem. Lett. 2016, 26, 3429–3435. 3. 2. Elfiky,Loughran, A.A. Ribavirin, H.M.; Han, Remdesivir, Z.; Wrobel, Sofosbuvir, J.E.; Decker, Galidesivir, S.E.; Ruthel, and G.; Tenofovir Freedman, against B.D.; Harty,SARS-CoV-2 R.N.; Reitz, RNA A.B. 3. Elfiky, A.A. Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependentQuinoxaline-based RNA polymerase inhibitors (RdRp): of Ebola A molecular and Marburg docking VP40 study. egress. Life Sci.Bioorg. 2020, 253 Med., 117592. Chem. Lett. 2016, 26, dependent RNA polymerase (RdRp): A molecular docking study. Life Sci. 2020, 253, 117592. 4. Pol,3429–3435. S.; Corouge, [CrossRef M.; Vallet-Pichard,][PubMed] A. Daclatasvir-sofosbuvir combination therapy with or without 4. Pol, S.; Corouge, M.; Vallet-Pichard, A. Daclatasvir-sofosbuvir combination therapy with or without 3. ribavirinElfiky, for A.A. hepatitis Ribavirin, C virusRemdesivir, infection: From Sofosbuvir, the clinicalGalidesivir, trials to real life. and Hepatic Tenofovir Med. againstEvid. Res. SARS-CoV-2 2016, 8, 21–26. RNA ribavirin for hepatitis C virus infection: From the clinical trials to real life. Hepatic Med. Evid. Res. 2016, 8, 21–26. 5. Shagufta;dependent Ahmad, RNA I. polymeraseAn insight (RdRp):into the Atherapeutic molecular potential docking study.of quinazolineLife Sci. 2020derivatives, 253, 117592. as anticancer [CrossRef ] 5. Shagufta; Ahmad, I. An insight into the therapeutic potential of quinazoline derivatives as anticancer agents.[PubMed Med. ]Chem. Com. 2017, 8, 871–885. agents. Med. Chem. Com. 2017, 8, 871–885. 6. 4. Shagufta;Pol, S.; Ahmad, Corouge, I. M.; Recent Vallet-Pichard, insight into A. the Daclatasvir-sofosbuvir biological activities of combination the synthetic therapy xanthone with derivatives. or without ribavirin Eur. 6. Shagufta; Ahmad, I. Recent insight into the biological activities of the synthetic xanthone derivatives. Eur. J. Med.for hepatitisChem. 2016 C,virus 116, 267–280. infection: From the clinical trials to real life. Hepatic Med. Evid. Res. 2016, 8, 21–26. J. Med. Chem. 2016, 116, 267–280. 7. Ahmad,[CrossRef I.; Shagufta.] Recent developments in steroidal and nonsteroidal aromatase inhibitors for the 7. Ahmad, I.; Shagufta. Recent developments in steroidal and nonsteroidal aromatase inhibitors for the 5. chemopreventionShagufta; Ahmad, of oestrogen-dependent I. An insight into the therapeuticbreast cancer. potential Eur. J. ofMed. quinazoline Chem. 2015 derivatives, 102, 375–386. as anticancer agents. chemoprevention of oestrogen-dependent breast cancer. Eur. J. Med. Chem. 2015, 102, 375–386. 8. Ahmad,Med. Chem.I.; Shagufta, Com. 2017S. An, 8 important, 871–885. [classCrossRef of orga] nic compounds with diverse biological activities. Int. J. 8. Ahmad, I.; Shagufta, S. An important class of organic compounds with diverse biological activities. Int. J. 6. Pharm.Shagufta; Sci. 2015 Ahmad,, 7, 19–27. I. Recent insight into the biological activities of the synthetic xanthone derivatives. Eur. J. Pharm. Sci. 2015, 7, 19–27. 9. Deepika,Med. Chem.Y.; Nath,2016 P.S.;, 116 ,Sachin, 267–280. K.; [ CrossRefShewta, ]S. Biological activity of quinoxaline derivatives. Int. J. Curr. 9. Deepika, Y.; Nath, P.S.; Sachin, K.; Shewta, S. Biological activity of quinoxaline derivatives. Int. J. Curr. Pharm. Res. 2011, 2, 33–46. Pharm. Res. 2011, 2, 33–46.

Molecules 2020, 25, 2784 16 of 19

7. Ahmad, I.; Shagufta. Recent developments in steroidal and nonsteroidal aromatase inhibitors for the chemoprevention of oestrogen-dependent breast cancer. Eur. J. Med. Chem. 2015, 102, 375–386. [CrossRef] 8. Ahmad, I.; Shagufta, S. An important class of organic compounds with diverse biological activities. Int. J. Pharm. Sci. 2015, 7, 19–27. 9. Deepika, Y.; Nath, P.S.; Sachin, K.; Shewta, S. Biological activity of quinoxaline derivatives. Int. J. Curr. Pharm. Res. 2011, 2, 33–46. 10. Tariq, S.; Somakala, K.; Amir, M. Quinoxaline: An insight into the recent pharmacological advances. Eur. J. Med. Chem. 2018, 143, 542–557. [CrossRef] 11. Cheeseman, G.W.H.; Cookson, R.F. Condensed Pyrazines. In The Chemistry of Heterocyclic Compounds; Weissberger, A., Taylor, E.C., Eds.; John Wiley & Sons, Inc.: New York, NY, USA, 1979; Volume 35, pp. 78–111. 12. Pereira, J.A.; Pessoa, A.M.; Cordeiro, M.N.D.S.; Fernandes, R.; Prudencio, C.; Noronha, J.P.; Vieira, M. Quinoxaline, its derivative and applications: A state of the art review. Eur. J. Med. Chem. 2015, 97, 664–672. [CrossRef][PubMed] 13. Brown, D.J.; Ellman, J.A. The Chemistry of Heterocyclic Compounds; Wiley: Amsterdam, The Netherlands, 2004. 14. Aparicio, D.; Attanasi, O.A.; Filippone, P.; Ignacio, R.; Lillini, S.; Mantellini, F.; Palacios, F.; De Los Santos, J.M. Straightforward access to pyrazines, piperazinones, and quinoxalines by reactions of 1,2-diaza-1,3-butadienes with 1,2-diamines under solution, solvent-free, or solid-phase conditions. J. Org. Chem. 2006, 71, 5897–5905. [CrossRef][PubMed] 15. Kunkuma, V.; Bethala, L.A.P.D.; Bhongiri, Y.; Rachapudi, B.N.P.; Potharaju, S.S.P. An efficient synthesis of quinoxalines catalyzed by monoammonium salt of 12-tungstophosphoric acid. Eur. J. Med. Chem. 2011, 2, 495–498. [CrossRef] 16. Antoniotti, S.; Dunach, E. Direct and catalytic synthesis of quinoxaline derivatives from epoxides and ene-1,2-diamines. Tetrahedron Lett. 2002, 43, 3971–3973. [CrossRef] 17. Cai, J.J.; Zou, J.P.; Pan, X.Q.; Zhang, W. Gallium triflate catalyzed synthesis of quinoxaline derivatives. Tetrahedron Lett. 2008, 49, 7386–7390. [CrossRef] 18. Thakuria, H.; Das, G. One-pot efficient green synthesis of 1,4-dihydroquinoxaline-2,3-dione derivatives. J. Chem. Sci. 2006, 118, 425–428. [CrossRef] 19. Gris, J.; Glisoni, R.; Fabian, L.; Fernandez, B.; Moglioni, A.G. Synthesis of potential chemotherapic quinoxalinone derivatives by biocatalysis or microwave-assisted Hinserg reaction. Tetrahedron Lett. 2008, 49, 1053–1056. [CrossRef] 20. Rostamizadeh, S.; Jafari, S. The synthesis of quinoxalines under microwave irradiation Indian. J. Heterocycl. Chem. 2001, 10, 303–304. 21. Nageswar, Y.V.D.; Reddy, K.H.V.; Ramesh, K.; Murthy, S.N. Recent developments in the synthesis of quinoxaline derivatives by green synthetic approaches. Org. Prep. Proc. Int. 2013, 45, 1–27. [CrossRef] 22. Harmenberg, J.; Akesson-Johansson, A.; Graslund, A.; Malmfors, T.; Bergman, J.; Wahren, B.; Akerfeldt, S.; Lundblad, L.; Cox, S. The mechanism of action of the anti-herpes virus compound 2,3-dimethyl-6(2- dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline. Antiviral. Res. 1991, 15, 193–204. [CrossRef] 23. Montana, M.; Mathias, F.; Terme, T.; Vanelle, P. Antitumoral activity of quinoxaline derivatives: A systematic review. Eur. J. Med. Chem. 2019, 163, 136–147. [CrossRef][PubMed] 24. Cogo, J.; Cantizani, J.; Cotillo, I.; Pereira sangi, D.; Gonçalves Corrêa, A.; Ueda-Nakamura, T.; Prado Dias Filho, B.; Martin, J.J.; Vataru Nakamura, C. Quinoxaline derivatives as potential antitrypanosomal and antileishmanialagents. Bioorg. Med. Chem. 2018, 7, 4065–4072. [CrossRef] 25. González, M.; Cerecetto, H. Quinoxaline derivatives: A patent review (2006–present). Expert Opin. Ther. Pat. 2012, 22, 1289–1302. [CrossRef] 26. Kleim, J.P.; Bender, R.; Billhardt, U.M.; Meichsner, C.; Riess, G.; Rösner, M.; Winkler, I.; Paessens, A. Activity of a novel quinoxaline derivative against human immunodeficiency virus type 1 reverse transcriptase and viral replication. Antimicrob. Agents Chemother. 1993, 37, 1659–1664. [CrossRef] 27. Moher, D.; Liberati, A.; Tetzlaff, J.; Altman, D.G. The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. Open Med. 2009, 3, 123–130. 28. Tanis, S.P.; Strohbach, J.W.; Parker, T.T.; Moon, M.W.; Thaisrivongs, S.; Perrault, W.R.; Hopkins, T.A.; Knechtel, M.L.; Oien, N.L.; Wieber, J.L.; et al. The design and development of 2-aryl-2-hydroxy ethylamine substituted 1H,7H-pyrido[1,2,3-de]quinoxaline-6-carboxamides as inhibitors of human cytomegalovirus polymerase. Bioorg. Med. Chem. Lett. 2010, 20, 1994–2000. [CrossRef] Molecules 2020, 25, 2784 17 of 19

29. Henen, M.A.; El Bialy, S.A.A.; Goda, F.E.; Nasr, M.N.A.; Eisa, H.M. [1,2,4] Triazolo [4,3-a] quinoxaline: Synthesis, antiviral, and antimicrobial activities. Med. Chem. Res. 2012, 9, 2368–2378. [CrossRef] 30. El-Tombary, A.A.; El-Hawash, S.A. Synthesis, antioxidant, anticancer and antiviral activities of novel quinoxaline hydrazone derivatives and their acyclic C-nucleosides. Med. Chem. 2014, 10, 521–532. [CrossRef] [PubMed] 31. El-Zahabi, H.S.A. Synthesis, Characterization, and Biological Evaluation of Some Novel Quinoxaline Derivatives as Antiviral Agents. Arch. Pharm. (Weinheim) 2017, 350, e1700028. [CrossRef][PubMed] 32. Galal, S.A.; Abdelsamie, A.S.; Tokuda, H.; Suzuki, N.; Lida, A.; Elhefnawi, M.M.; Ramadan, R.A.; Atta, M.H.; El Diwani, H.I. Part I: Synthesis, cancer chemopreventive activity and molecular docking study of novel quinoxaline derivatives. Eur. J. Med. Chem. 2011, 46, 327–340. [CrossRef] 33. Manta, S.; Gkaragkouni, D.N.; Kaffesaki, E.; Gkizis, P.; Hadjipavlou-Litina, D.; Pontiki, E.; Balzarini, J.; Dehaen, W.; Komiotis, D. A novel and easy two-step, microwave-assisted method for the synthesis of halophenyl pyrrolo[2,3-b]quinoxalines via their pyrrolo precursors. Eval. Their Bioactivity Tetrahedron Lett. 2014, 55, 1873–1876. [CrossRef] 34. Fakhreldin, Y.; El-Kousy, S.M.; El Ashry, M. Synthesis and novel chemical reaction for a new class of

3-(10,20-dihydroxyeth-10-yl)-1-phenylpyrazolo [3,4-b] quinoxaline series “C-nucleosides” as antiviral agents. Life Sci. 2012, 9, 234–239. 35. Carta, A.; Sanna, G.; Briguglio, I.; Madeddu, S.; Vitale, G.; Piras, S.; Corona, P.; Peana, A.T.; Laurini, E.; Fermeglia, M.; et al. Quinoxaline derivatives as new inhibitors of coxsackievirus B5. Eur. J. Med. Chem. 2018, 145, 559–569. [CrossRef][PubMed] 36. You, L.; Cho, E.J.; Leavitt, J.; Ma, L.C.; Montelione, G.T.; Anslyn, E.V.; Krug, R.M.; Ellington, A.; Robertus, J.D. Synthesis and evaluation of quinoxaline derivatives as potential influenza NS1A protein inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 3007–3011. [CrossRef] 37. Carta, A.; Briguglio, I.; Piras, S.; Corona, P.; Boatto, G.; Nieddu, M.; Giunchedi, P.; Marongiu, M.E.; Giliberti, G.; Iuliano, F.; et al. Quinoline tricyclic derivatives. Design, synthesis and evaluation of the antiviral activity of three new classes of RNA-dependent RNA polymerase inhibitors. Bioorg. Med. Chem. 2011, 19, 7070–7084. [CrossRef][PubMed] 38. Briguglio, I.; Loddo, R.; Laurini, E.; Fermeglia, M.; Piras, S.; Corona, P.; Giunchedi, P.; Gavini, E.; Sanna, G.; Giliberti, G.; et al. Synthesis, cytotoxicity and antiviral evaluation of new series of imidazo[4,5-g]quinoline and pyrido[2,3-g]quinoxalinone derivatives. Eur. J. Med. Chem. 2015, 105, 63–79. [CrossRef][PubMed] 39. Summa, V.;Ludmerer, S.W.; McCauley,J.A.; Fandozzi, C.; Burlein, C.; Claudio, G.; Coleman, P.J.;Dimuzio, J.M.; Ferrara, M.; Di Filippo, M.; et al. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob. Agents Chemother. 2012, 56, 4161–4167. [CrossRef] 40. Guo, Z.; Black, S.; Hu, Y.; McMonagle, P.; Ingravallo, P.; Chase, R.; Curry, S.; Asante-Appiah, E. Unraveling the structural basis of grazoprevir potency against clinically relevant substitutions in hepatitis C virus NS3/4A protease from genotype 1a. J. Biol. Chem. 2017, 292, 6202–6212. [CrossRef] 41. Rusere, L.N.; Matthew, A.N.; Lockbaum, G.J.; Jahangir, M.; Newton, A.; Petropoulos, C.J.; Huang, W.; Kurt Yilmaz, N.; Schiffer, C.A.; Ali, A. Quinoxaline-Based Linear HCV NS3/4A Protease Inhibitors Exhibit Potent Activity against Drug Resistant Variants. ACS Med. Chem. Lett. 2018, 9, 691–696. [CrossRef] 42. Matthew, A.N.; Zephyr, J.; Hill, C.J.; Jahangir, M.; Newton, A.; Petropoulos, C.J.; Huang, W.; Kurt Yilmaz, N.; Schiffer, C.A.; Ali, A. Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants. J. Med. Chem. 2017, 60, 5699–5716. [CrossRef] 43. Shibinskaya, M.O.; Karpenko, A.S.; Lyakhov, S.A.; Andronati, S.A.; Zholobak, N.M.; Spivak, N.Y.; Samochina, N.A.; Shafran, L.M.; Zubritsky, M.J.; Galat, V.F. Synthesis and biological activity of 7H-benzo[4,5]indolo[2,3-b]-quinoxaline derivatives. Eur. J. Med. Chem. 2011, 46, 794–798. [CrossRef] [PubMed] 44. Shibinskaya, M.O.; Lyakhov, S.A.; Mazepa, A.V.; Andronati, S.A.; Turov, A.V.; Zholobak, N.M.; Spivak, N.Y. Synthesis, cytotoxicity, antiviral activity and interferon inducing ability of 6-(2-aminoethyl)-6H- indolo[2,3-b]quinoxalines. Eur. J. Med. Chem. 2010, 45, 1237–1243. [CrossRef] 45. Fabian, L.; Taverna Porro, M.; Gómez, N.; Salvatori, M.; Turk, G.; Estrin, D.; Moglioni, A. Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV agents targeting reverse transcriptase enzyme. Eur. J. Med. Chem. 2020, 188, 111987. [CrossRef][PubMed] Molecules 2020, 25, 2784 18 of 19

46. Patel, S.B.; Patel, B.D.; Pannecouque, C.; Bhatt, H.G. Design, synthesis and anti-HIV activity of novel quinoxaline derivatives. Eur. J. Med. Chem. 2016, 117, 230–240. [CrossRef][PubMed] 47. Yang, L.; Wang, P.; Wu, J.F.; Yang, L.M.; Wang, R.R.; Pang, W.; Li, Y.G.; Shen, Y.M.; Zheng, Y.T.; Li, X. Design, synthesis and anti-HIV-1 evaluation of hydrazide-based peptidomimetics as selective gelatinase inhibitors. Bioorg. Med. Chem. 2016, 24, 2125–2136. [CrossRef][PubMed] 48. Nathanson, N.; González-Scarano, F. Patterns of infection. Viral Pathogenesis 2016, 81–94. 49. Ryu, W.S. Discovery and Classification. Mol. Virol. Human Pathog. Viruses 2017, 3–20. 50. Villareal, E.C. Current and potential therapies for the treatment of herpes-virus infections. Prog. Drug Res. 2003, 60, 263–307. 51. Brideau, R.J.; Knechtel, M.L.; Huang, A.; Vaillancourt, V.A.; Vera, E.E.; Oien, N.L.; Hopkins, T.A.; Wieber, J.L.; Wilkinson, K.F.; Rush, B.D.; et al. Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses. Antiviral Res. 2002, 54, 19–28. [CrossRef] 52. Faulds, D.; Heel, R.C. Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections. Drugs 1990, 39, 597–638. [CrossRef] 53. Lalezari, J. Drug-resistant herpes: Cidofovir gel in limbo. Interview with Jay Lalezari, M.D. Interview by John S. James. AIDS Treat. News 1997, 283, 5–6. 54. Kolb, T.M.; Davis, M.A. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) provokes a prolonged morphologic response and ERK activation in Tsc2-null renal tumor cells. Toxicol. Sci. 2004, 81, 233–242. [CrossRef][PubMed] 55. Louten, J. Essential Human Virology, 1st ed.; Elsevier, Academic Press: Cambridge, MA, USA, 2016; p. 344. 56. Badri, T.; Gandhi, G.R. Molluscum Contagiosum. Treasure Island (FL): StatPearls Publishing. January 2020. Available online: https://www.ncbi.nlm.nih.gov/books/NBK441898/ (accessed on 5 May 2020). 57. Poltronieri, P.; Sun, B.; Mallardo, M. RNA Viruses: RNA Roles in Pathogenesis, Coreplication and Viral Load. Curr. Genomics 2015, 16, 327–335. [CrossRef][PubMed] 58. Gaaloul, I.; Riabi, S.; Harrath, R.; Hunter, T.; Hamda, K.B.; Ghzala, A.B.; Huber, S.; Aouni, M. Coxsackievirus B detection in cases of myocarditis, myopericarditis, pericarditis and dilated cardiomyopathy in hospitalized patients. Mol. Med. Rep. 2014, 10, 2811–2818. [CrossRef] 59. Lee, C.J.; Huang, Y.C.; Yang, S.; Tsao, K.C.; Chen, C.J.; Hsieh, Y.C.; Chiu, C.H.; Lin, T.Y. Clinical features of coxsackievirus A4, B3 and B4 infections in children. PLoS ONE 2014, 9, e87391. [CrossRef] 60. Hale, B.G.; Randall, R.E.; Ortín, J.; Jackson, D. The multifunctional NS1 protein of influenza A viruses. J. Gen. Virol. 2008, 89, 2359–2376. [CrossRef] 61. Yin, C.; Khan, J.A.; Swapna, G.V.; Ertekin, A.; Krug, R.M.; Tong, L.; Montelione, G.T. Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses. J. Biol. Chem. 2007, 282, 20584–20592. [CrossRef] 62. Min, J.Y.; Krug, R.M. The primary function of RNA binding by the influenza A virus NS1 protein in infected

cells: Inhibiting the 20-50 oligo (A) synthetase/RNase L pathway. Proc. Natl. Acad. Sci. USA 2006, 103, 7100–7105. [CrossRef] 63. Mulangu, S.; Dodd, L.E.; Davey, R.T., Jr.; Tshiani Mbaya, O.; Proschan, M.; Mukadi, D.; Lusakibanza Manzo, M.; Nzolo, D.; Tshomba Oloma, A.; Ibanda, A.; et al. Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. N. Engl. J. Med. 2019, 381, 2293–2303. [CrossRef] 64. Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of High-Consequence Pathogens and Pathology (DHCPP), Viral Special Pathogens Branch (VSPB). Available online: https://www.cdc.gov/vhf/ebola/treatment/index.html (accessed on 5 May 2020). 65. World Health Organisation. Hepatitis C. Available online: https://www.who.int/news-room/fact-sheets/ detail/hepatitis-c (accessed on 5 May 2020). 66. Liverton, N.J.; Holloway, M.K.; McCauley, J.A.; Rudd, M.T.; Butcher, J.W.; Carroll, S.S.; DiMuzio, J.; Fandozzi, C.; Gilbert, K.F.; Mao, S.S.; et al. Molecular modeling-based approach to potent P2-P4 macrocyclic inhibitors of hepatitis C NS3/4A protease. J. Am. Chem. Soc. 2008, 130, 4607–4609. [CrossRef] 67. Romano, K.P.; Ali, A.; Aydin, C.; Soumana, D.; Ozen, A.; Deveau, L.M.; Silver, C.; Cao, H.; Newton, A.; Petropoulos, C.J.; et al. The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors. PLoS Pathog. 2012, 8, e1002832. [CrossRef][PubMed] Molecules 2020, 25, 2784 19 of 19

68. Shah, U.; Jayne, C.; Chackalamannil, S.; Velázquez, F.; Guo, Z.; Buevich, A.; Howe, J.A.; Chase, R.; Soriano, A.; Agrawal, S.; et al. Novel Quinoline-Based P2-P4 Macrocyclic Derivatives As Pan-Genotypic HCV NS3/4a Protease Inhibitors. ACS Med. Chem. Lett. 2014, 5, 264–269. [CrossRef][PubMed] 69. Quiroz, E.; Moreno, N.; Peralta, P.H.; Tesh, R.B. A human case of encephalitis associated with vesicular stomatitis virus (Indiana serotype) infection. Am. J. Trop. Med. Hyg. 1988, 39, 312–314. [CrossRef][PubMed] 70. National Institute of Allergy and Infectious Diseases. Antiretroviral Drug Discovery and Development. Available online: https://www.niaid.nih.gov/diseases-conditions/antiretroviral-drug-development (accessed on 5 May 2020).

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