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Review Quinoxaline Moiety: A Potential Scaffold against Mycobacterium tuberculosis

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

1 Aix Marseille Univ, CNRS, ICR, Equipe Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 13005 Marseille, France; [email protected] (M.M.); [email protected] (V.M.); [email protected] (O.K.) 2 Assistance Publique-Hôpitaux de Marseille (AP-HM), Oncopharma, 13015 Marseille, France 3 Assistance Publique-Hôpitaux de Marseille (AP-HM), Service Central de la Qualité et de l’Information Pharmaceutiques (SCQIP), 13005 Marseille, France * Correspondence: [email protected]; Tel.: +33-4-91-83-55-80; Fax: +33-4-91-79-46-77

Abstract: Background. The past decades have seen numerous efforts to develop new antitubercular agents. Currently, the available regimens are lengthy, only partially effective, and associated with high rates of adverse events. The challenge is therefore to develop new agents with faster and more efficient action. The versatile quinoxaline ring possesses a broad spectrum of pharmacological activities, ensuring considerable attention to it in the field of medicinal chemistry. Objectives. In continuation of our program on the pharmacological activity of quinoxaline derivatives, this review focuses on potential antimycobacterial activity of recent quinoxaline derivatives and discusses their structure—activity relationship for designing new analogs with improved activity. Methods. The review compiles recent studies published between January 2011 and April 2021. Results. The final total of 23 studies were examined. Conclusions. Data from studies of quinoxaline and quinoxaline Citation: Montana, M.; Montero, V.; 1,4-di-N-oxide derivatives highlight that specific derivatives show encouraging perspectives in the Khoumeri, O.; Vanelle, P. Quinoxaline Moiety: A Potential Scaffold against treatment of Mycobacterium tuberculosis and the recent growing interest for these scaffolds. These in- Mycobacterium tuberculosis. Molecules teresting results warrant further investigation, which may allow identification of novel antitubercular 2021, 26, 4742. https://doi.org/ candidates based on this scaffold. 10.3390/molecules26164742 Keywords: quinoxaline; Mycobacterium; tuberculosis; SAR; biological applications; chemistry Academic Editors: Jonathan Sellars, Alistair Brown and Jean-Marc Sabatier 1. Introduction Received: 11 June 2021 In 2020, the World Health Organization estimated that about 10 million people (range: Accepted: 3 August 2021 8.9–11.0 million) contracted tuberculosis (TB) in 2019, which was responsible for 1.4 million Published: 5 August 2021 deaths [1]. Treatment of drug-susceptible active tuberculosis consists of a standard 6-month regimen of four antimicrobials (usually isoniazid, rifampin, pyrazinamide, and ethamb- Publisher’s Note: MDPI stays neutral utol) [2,3]. However, these regimens are lengthy, only partially effective, and associated with regard to jurisdictional claims in with high rates of adverse events. The challenge is therefore to develop new agents with published maps and institutional affil- iations. faster and more efficient action. Nitrogen-containing heterocycles are of particular interest for the development of new drugs or novel potential lead molecules [4–8]. Quinoxaline, formed by the fusion of two aromatic rings, benzene and pyrazine, is one of the heterocycles receiving the most attention. Copyright: © 2021 by the authors. The versatile quinoxaline ring possesses a broad spectrum of pharmacological activ- Licensee MDPI, Basel, Switzerland. ities (antiviral [9], anticancer [10], antileishmanial [11]), ensuring considerable attention This article is an open access article to it in the field of medicinal chemistry [12]. In addition, as quinoxaline is also a part of distributed under the terms and well-known wide-spectrum antibiotics echinomycin, levomycin, and actinoleutin, quinoxa- conditions of the Creative Commons Attribution (CC BY) license (https:// line derivatives are expected to have antimycobacterial activity. One of them, clofazimine, creativecommons.org/licenses/by/ initially known as B663, is currently under intensive clinical investigation for the treatment 4.0/).

Molecules 2021, 26, 4742. https://doi.org/10.3390/molecules26164742 https://www.mdpi.com/journal/molecules MoleculesMolecules 20212021,, 2626,, 4742x FOR PEER REVIEW 22 ofof 2323

ofthe drug-resistant treatment of TBdrug-resistant to assess the compound’sTB to assess treatmentthe compound’s outcomes treatment with multidrug-resistant outcomes with andmultidrug-resistant extensively drug-resistant and extensively TB (Figure drug-resistant1). TB (Figure 1).

NNH CH3

N N CH3

1 Cl

Figure 1. Chemical structure of clofazimine.

Some quinoxaline-1,4-di-quinoxaline-1,4-di-NN-oxide-oxide derivatives derivatives have have also also shown shown excellent excellent antimicrobial antimicro- Mycobacterium tuberculosis activitiesbial activities against against Mycobacterium tuberculosis[13–15 [13–15],], indicating indicating the great the great interest interest of these of types of structure for the development of new structural classes of anti-TB drugs. The these types of structure for the development of new structural classes of anti-TB drugs. oxidation of nitrogen in a quinoxaline ring has a pronounced effect on antimycobacterial The oxidation of nitrogen in a quinoxaline ring has a pronounced effect on antimycobac- activity [16]. In addition, quinoxaline 1,4-di-N-oxides are species that suffer a bioreductive terial activity [16]. In addition, quinoxaline 1,4-di-N-oxides are species that suffer a biore- process under the hypoxic conditions [17] found in tuberculous granulomas, where non- ductive process under the hypoxic conditions [17] found in tuberculous granulomas, replicating persistent forms of Mycobacterium tuberculosis bacilli can survive, leading to the where nonreplicating persistent forms of Mycobacterium tuberculosis bacilli can survive, need for long treatments and the risk of treatment resistance [13]. In continuation of our leading to the need for long treatments and the risk of treatment resistance [13]. In contin- program on the pharmacological activity of quinoxaline derivatives, we decided to focus uation of our program on the pharmacological activity of quinoxaline derivatives, we de- on the development of new quinoxalines and quinoxaline 1,4-di-N-oxide derivatives as cided to focus on the development of new quinoxalines and quinoxaline 1,4-di-N-oxide antitubercular drugs. This review compiles studies published between 2011 and 2021 and derivatives as antitubercular drugs. This review compiles studies published between 2011 discusses the potential antimycobacterial activity of recent quinoxaline derivatives. and 2021 and discusses the potential antimycobacterial activity of recent quinoxaline de- 2.rivatives. Methods 2.1. Data Sources and Searches 2. Methods The research was conducted using three databases: MEDLINE/PubMed, Web of Science,2.1. Data andSources Science and DirectSearches Elsevier (Table1). The research was conducted using three databases: MEDLINE/PubMed, Web of Sci- ence, and ScienceTable Direct 1. Data Elsevier sources (Table and searches. 1). Boolean SiteTable Keyword 1. Data 1 sources and searches. Keyword 2 Date Filter Operator Boolean MEDLINE/PubMed Site Keyword 1 Keyword 2 Date Filter (National Library of Operator 01 January Document type: Medicine—www.ncbi.nlm. MEDLINE/PubMedQuinoxaline (National AND Tuberculosis 2011–01 April 01 Januaryjournal Document articles nih.gov/pubmed Library of Medicine— 2021 Quinoxaline AND Tuberculosis 2011–01 type: journal (accessed on 13 April 2021) www.ncbi.nlm.nih.gov/pubmed April 2021 articles Web of Science (Thomson (accessed on 13 April 2021) Reuters Scientiﬁc—www. Quinoxaline Tuberculosis Document type: Web of Science (ThomsonAND Reu- 2011–2021 webofknowledge.com (topic) (Topic) onlyDocument articles ters Scientific— Quinoxaline Tuberculosis (accessed on 13 April 2021) AND 2011–2021 type: only ar- www.webofknowledge.com (ac- (topic) (Topic) Science Direct Elsevier Documentticles type: (www.sciencedirect.com Quinoxalinecessed on 13 April 2021) AND Tuberculosis 2011–2021 only research (accessed on 13 April 2021) articlesDocument Science Direct Elsevier type: only re- (www.sciencedirect.com (ac- Quinoxaline AND Tuberculosis 2011–2021 search arti- cessed on 13 April 2021) cles

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2.2. Study Selection The review was performed by two independent reviewers as described by PRISMA [18]. Eligibility criteria were predetermined by the authors (Table2).

Table 2. Inclusion and exclusion criteria.

Parameter Inclusion Exclusion 1 Language French, English Any other language In vitro and/or in vivo studies, Articles that focus only on synthesis or other 2 Type of study biological activity purely chemical parameters 3 Type of publication Original manuscripts Book chapters, posters, reviews 4 Search terms Merely citing keywords in the text

In the ﬁrst step, duplicates were eliminated. Then, articles’ titles and abstracts were evaluated according to the inclusion criteria. The authors read each selected full text and eliminated articles ﬁtting the exclusion criteria. During this stage, the references of the relevant articles were examined to identify additional studies not retrieved in computerized databases.

3. Results The database search identified 148 records; 27 repeated files were discarded, leaving 121 articles. After the evaluation phase (title/abstract) and full-text reading, 98 records were excluded. The final total of 23 studies were included in this review. No other paper was added from the reference lists of the identified studies. During the 2011–2015 and the 2016–2021 periods, six articles (two concerning 1,4-di-N- oxide-quinoxaline derivatives and four concerning quinoxaline derivatives) and 17 articles (six concerning 1,4-di-N-oxide-quinoxaline derivatives and 11 concerning quinoxaline derivatives) were published, respectively.

4. Discussion 4.1. Quinoxaline Derivatives as Antimycobacterial Agents A wide variety of methods for the synthesis of functionalized quinoxalines have already been reported in the literature. In general, quinoxaline derivatives are obtained either by condensation of o-phenylenediamine and its derivatives with a dicarbonyl compound under conventional conditions or reaction of 1,2-diaza-1,3-butadienes with 1,2-diamines [19–22]. Eco-friendly approaches using recyclable catalysts [23,24], oxidative cyclisation of 1,2-diamines and phenacyl bromides [25], microwave-assisted synthesis [26,27], reactions in aqueous medium [28], or one-pot synthesis [29] have also been described.

4.1.1. Tricyclic Quinoxaline Derivatives Between 2017 and 2020, three series of original benzo[g]quinoxaline-5,10-dione derivatives were synthesized using a multistep synthetic route from 2,3-dihydroxynaphtalene and evaluated as potential antimycobacterial agents [30–32] (Figure2). Thirteen novel compounds belonging to the pyridine series, 11 new pyrazoline derivatives, and 11 7-[3-(substituted) phenylprop-2-enoyl]quinoxaline derivatives were obtained; they displayed moderate antimycobacterial activity [30–32] (Table3). MoleculesMolecules 2021, 262021, x, FOR26, 4742 PEER REVIEW 4 of 234 of 23

R R

N O O N HN N H2N N N N N O O

O O N

R N O

FigureFigure 2. Chemical 2. Chemical structures structures of of new new series series benzo[ gg]quinoxaline-5,10-dione]quinoxaline-5,10-dione derivatives. derivatives.

Pyrroloquinoxaline moiety represents a very attractive scaffold in medicinal chem- Thirteen novel compounds belonging to the pyridine series, 11 new pyrazoline de- istry for its biological anti-infectious properties, including antimycobacterial activity [33]. rivatives,This, pyrrolo[1,2- and 11 7-[3-(substituted)a]quinoxaline was phenylpr chosen asop-2-enoyl]quinoxaline scaffold and led to the synthesis derivatives of 23 newwere obtained;pyrrolo[1,2- they displayeda]quinoxaline moderate derivatives antimycobacterial (Figure3) that displayed activity [30–32] very encouraging (Table 3). antimycobacterial activity [34]. Table 3.Ten Chemical compounds structure, showed minimal both inhibitory interesting concentration antimycobacterial (MIC) values activity against and moderate Mycobacterium tuberculosiscytotoxicity. [30–32]. Antimycobacterial activity was observed to depend on varying chemical characteristics, like the replacement of tertiary nitrogen by an NH group, leading to Compoundcompounds 7 –10 with improved solubilityStructure and the lowest minimal inhibitory concentrationMIC (µg/mL) H C CH (MIC) value (5 µg/mL) (Table3 4).N 3 Modification of the linker between R2 and pyrrolo[1,2-a]quinoxaline moiety led to a minor variation in antibacterial activity, although substitutions at the R2 position with an aromatic group led to the most active compounds (Figure4). Derivative N-((7-chloro-1-(2- fluorophenyl)pyrrolo[1,2-a]quinoxalin-3-yl)methyl)-2-(3,4-dimethoxyphenyl)ethanamine (9) was identified as offeringN the best oral bioavailability. Determination of enzyme inhibition 2 50 and molecular docking studies revealed that theseO original derivatives could act by targeting the classic anti-TB drug target InhA by forming H bondsN as well as Pi–Pi interactions. In addition, severalH2N novelN 4,5-dihydropyrrolo[1,2-a]quinoxalines and pyrrolo[1,2- a]quinoxaline-2-ones were synthesized from the reaction between 2-(1H-pyrrol-1-yl)anilines and imidazo[1,2-a]pyridine-3-carbaldehyde or isatin andN are of current interest as antimycobacterial agents [35] (Figure5). O

Five 4-substitued 4,5-dihydropyrrolo[1,2-a]quinoxalines and only one pyrrolo[1,2- H3C a]quinoxaline-2-one derivative displayed potent antimycobacterial activity, with MIC ranging from 6.25 to 12.5 µg/mL. However, no clear structure–activity relationship could be demonstrated from the small number of synthesized compounds [35].

O 3 12.5 HN N N

N O

Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW 4 of 23

R R

N O O N HN N H N N N N N H2N N N N N O O

O O N

N R N O

Figure 2. Chemical structures of new series benzo[g]quinoxaline-5,10-dione derivatives. Molecules 2021, 26, 4742 5 of 23 Thirteen novel compounds belonging to the pyridine series, 11 new pyrazoline derivatives, and 11 7-[3-(substituted) phenylprop-2-enoyl]quinoxaline derivatives were obtained; they displayed moderate antimycobacterial activity [30–32] (Table 3).

Table 3. Chemical structure,Table 3. Chemical minimal structure, inhibitory minimal concentration inhibitory concentrationconcentration (MIC) values (MIC)(MIC) valuesvalues against againstagainstMycobacterium Mycobacterium tuberculosis [30–32]. tuberculosis [30–32].[30–32]. Compound Structure MIC (µg/mL) Compound Structure MIC (µg/mL) H3C CH3 3 N 3

N 2 2 50 50 2 O 50 N H N N N H2N N N O

H C H3C

O 3 3 HN 12.5 12.5 HN N N Molecules 2021, 26, x FOR PEER REVIEW 5 of 23 MoleculesMolecules 2021 2021, ,26 26, ,x x FOR FOR PEER PEER REVIEW REVIEW N 55 of of 23 23

O O OO OO N NN 4 4 25 25 44 2525 Cl N ClCl NN O OO

O O OO OO N NN 5 12.5 5 55 12.512.5 12.5 H CO N HH33COCO NN 3 O OO

O O OO OO N NN 6 25 6 66 2525 25 H CO N HH33COCO NN 3 OH O OHOH OO

RifampicinRifampicin –– 0.25 0.25 RifampicinRifampicin – – 0.25 0.25 IsoniazidIsoniazid –– 0.20 0.20 IsoniazidIsoniazid – – 0.20 0.20 Pyrroloquinoxaline moiety represents a very attractive scaffold in medicinal chemis- PyrroloquinoxalinePyrroloquinoxaline moiety moiety represents represents a a very very attractive attractive scaffold scaffold in in medicinal medicinal chemis- chemis- try for its biological anti-infectious properties, including antimycobacterial activity [33]. trytry forfor its4.1.2.its biologicalbiological Hybrids anti-infectiousanti-infectious and Conjugates propertiproperties,es, includingincluding antimycobacterialantimycobacterial activityactivity [33].[33]. This, pyrrolo[1,2-a]quinoxaline was chosen as scaffold and led to the synthesis of 23 new This,This, pyrrolo[1,2-pyrrolo[1,2-aa]quinoxaline]quinoxaline waswas chosenchosen asas scaffoldscaffold andand ledled toto thethe synthesissynthesis ofof 2323 newnew pyrrolo[1,2-a]quinoxalineMany classes derivatives of organic (Figure compounds 3) that displayed have very previously encouraging antimy- been evaluated for their anti- pyrrolo[1,2-pyrrolo[1,2-aa]quinoxaline]quinoxaline derivatives derivatives (Figure (Figure 3) 3) that that displayed displayed very very encouraging encouraging antimy- antimy- cobacterial activity [34]. cobacterialcobacterialmycobacterial activity activity [34]. [34]. activity. A rational design strategy based on combining the biological R properties of different bioactive structures into a single compound could lead to new RR22 X 2 compounds with increased activity, combined modes of action, or improved tolerance XX R N R N R2 R N R2 R 1 N proﬁlesR 1 comparedN to parentRR2 structures.R 1 N RR2 R11 N R11 N 2 R11 N 2 N Then, the quinoxalineN ring fused with azetidinoneN andnn thiazolidinone, two struc- NN NN NN n N N N NN tural scaffolds possessingNN antitubercular activityNN [36–38], showed in vitro activity against Mycobacterium tuberculosis. Quinoxaline derivatives with 2-chloro, dimethylamino and nitro substitution showed comparable activity to that of isoniazid (MIC = 0.67–0.97 µg/mL F F F FF versus 0.46 µg/mL)FF [39] (Figure6). FF

XX = = N, N, O O RR1 = = H, H, Cl Cl nn = = 1, 1, 2, 2, 3 3 X = N, O R11 = H, Cl n = 1, 2, 3 RR1 = = H, H, Cl Cl RR2 = = H, H, RR1 = = H, H, Cl, Cl, F, F, Br Br R11 = H, Cl R22 = H, R11 = H, Cl, F, Br RR2 = = H, H, 3-CF 3-CF3-Ph,-Ph, RR2 = = CH CH3, , R22 = H, 3-CF33-Ph, R22 = CH33, 3-CF 3-CF3-Ph,-Ph, CH CH3, , C C6HH5,, 3-CF33-Ph, CH33, C66H55, CH CH3, , C C6HH5 (CH (CH3))2 CH33, C66H55 (CH33)22 C C6HH5 2-Cl-6-F-Ph 2-Cl-6-F-Ph C66H55 2-Cl-6-F-Ph 4-Br-Ph 4-Br-Ph4-Br-Ph 3,4-OCH -Ph 3,4-OCH3,4-OCH33-Ph-Ph 3 Figure 3. Chemical structures of new series of pyrrolo[1,2-a]quinoxaline derivatives. FigureFigure 3. 3. Chemical Chemical structures structures of of new new series series of of pyrrolo[1,2- pyrrolo[1,2-aa]quinoxaline]quinoxaline derivatives. derivatives. Ten compounds showed both interesting antimycobacterial activity and moderate TenTen compoundscompounds showedshowed bothboth interestinginteresting anantimycobacterialtimycobacterial activityactivity andand moderatemoderate cytotoxicity. Antimycobacterial activity was observed to depend on varying chemical cytotoxicity.cytotoxicity. AntimycobacterialAntimycobacterial activityactivity waswas observedobserved toto dependdepend onon varyingvarying chemicalchemical characteristics, like the replacement of tertiary nitrogen by an NH group, leading to com- characteristics,characteristics, like like the the replacement replacement of of tertiary tertiary nitrogen nitrogen by by an an NH NH group, group, leading leading to to com- com- pounds 7–10 with improved solubility and the lowest minimal inhibitory concentration poundspounds 77––1010 withwith improvedimproved solubilitysolubility andand thethe lowestlowest minimalminimal inhibitoryinhibitory concentrationconcentration (MIC) value (5 µg/mL) (Table 4). (MIC)(MIC) value value (5 (5 µg/mL) µg/mL) (Table (Table 4). 4).

Molecules 2021, 26, x FOR PEER REVIEW 5 of 23

O O N 4 25 Cl N O

O O N 5 12.5

H3CO N O

O O N 6 25

H3CO N OH O

Rifampicin – 0.25 Isoniazid – 0.20

Pyrroloquinoxaline moiety represents a very attractive scaffold in medicinal chemistry for its biological anti-infectious properties, including antimycobacterial activity [33]. Molecules 2021, 26, 4742 This, pyrrolo[1,2-a]quinoxaline was chosen as scaffold and led to the synthesis of 236 new of 23 pyrrolo[1,2-a]quinoxaline derivatives (Figure 3) that displayed very encouraging antimycobacterial activity [34].

R2 X R R R1 N R1 N 2 R1 N 2 N N N n N N N

F F F

X = N, O R1 = H, Cl n = 1, 2, 3 Molecules 2021, 26, x FOR PEER REVIEW 7 of 23 R1 = H, Cl R2 = H, R1 = H, Cl, F, Br

R2 = H, 3-CF3-Ph, R2 = CH3,

3-CF3-Ph, CH3, C6H5,

CH3, C6H5 (CH3)2

C6H5 2-Cl-6-F-Ph 4-Br-Ph 3,4-OCH -Ph Molecules 2021, 26, x FOR PEER REVIEW 3 7 of 23

Figure 3. Chemical structures of new series of pyrrolo[1,2-a]quinoxaline]quinoxaline derivatives.derivatives.

Ten compounds showed both interesting antimycobacterial activity and moderate cytotoxicity. Antimycobacterial activity was observed to depend on varying chemical characteristics, like the replacement of tertiary nitrogen by an NH group, leading to com- Figure 4. Structure–activitypounds 7relationship–10 with improved of novel quinoxalinesolubility and derivatives the lowest with minimal antimycobacterial inhibitory concentration activity. (MIC) value (5 µg/mL) (Table 4). In addition, several novel 4,5-dihydropyrrolo[1,2-a]quinoxalines and pyrrolo[1,2- a]quinoxaline-2-ones were synthesized from the reaction between 2-(1H-pyrrol-1-yl)anilines and imidazo[1,2-a]pyridine-3-carbaldehyde or isatin and are of current interest as

FigureFigure 4. Structure–activity 4. Structure–activityantimycobacterial relationship relationship of ofagents novel novel quinoxalinequinoxaline [35] (Figure derivatives derivatives 5). with with antimycobacterial antimycobacterial activity. activity.

In addition, several novel 4,5-dihydropyrrolo[1,2-a]quinoxalines and pyrrolo[1,2- R2 a]quinoxaline-2-ones were synthesized from the reaction betweenR 2-(1H-pyrrol-1-yl)anilines andN imidazo[1,2-a]pyridine-3-carbaldehyde or isatin and2 are of current interest as antimycobacterial agents [35] (Figure 5).R N 3 N NH R2 NH R R 1 N O 2 R N N 3 N N NH NH R 1 O R1 N N R1 = H, Cl, CH3 R1 = H, F R R2 = 2-Br, 4-Cl, 4-CH3 R2 = H, Cl, F,1 I, CH3, OCF3 R1 = H, Cl, CH3 R1 = RH, F= H, CH , CH -Ph 3 3 2 R2 = 2-Br, 4-Cl, 4-CH3 R2 = H, Cl, F, I, CH3, OCF3 Figure 5.5. GeneralGeneral structurestructure of of novel novelR 4,5-dihydropyrrolo[1,2- = H,4,5-dihydropyrrolo[1,2- CH , CH -Ph a]quinoxalinesa]quinoxalines and pyrrolo[1,2- and pyrrolo[1,2- 3 3 2 a]quinoxaline-2-ones synthesized. synthesized. Figure 5. General structure of novel 4,5-dihydropyrrolo[1,2-a]quinoxalines and pyrrolo[1,2- a]quinoxaline-2-ones synthesized. Five 4-substitued 4,5-dihydropyrrolo[1,2-a]quinoxalines and only one pyrrolo[1,2- a]quinoxaline-2-oneFive 4-substitued derivative 4,5-dihydropyrrolo[1,2- displayeda]quinoxalines potent antimycobacterial and only one pyrrolo[1,2- activity, with MIC ranginga]quinoxaline-2-one from 6.25 to derivative 12.5 µg/mL. displayed However, potent noantimycobacterial clear structure–activity activity, with relationship MIC could ranging from 6.25 to 12.5 µg/mL. However, no clear structure–activity relationship could be bedemonstrated demonstrated fromfrom the the small small numb number of ersynthesize of synthesized compoundsd compounds [35]. [35].

4.1.2.4.1.2. Hybrids Hybrids andand ConjugatesConjugates ManyMany classes classes ofof organic organic compounds compounds have prhaveeviously previously been evaluated been forevaluated their anti- for their anti- mycobacterialmycobacterial activity.activity. A A rational rational design design strategy stra basedtegy on based combining on combining the biological the prop- biological properties of different bioactive structures into a single compound could lead to new com- ertiespounds of different with increased bioactive activity, structurescombined mode intos of a action, single or compoundimproved tolerance could profiles lead to new com- poundscompared with to increased parent structures. activity, combined modes of action, or improved tolerance profiles comparedThen, to the parent quinoxaline structures. ring fused with azetidinone and thiazolidinone, two structural scaffoldsThen, possessingthe quinoxaline antitubercular ring fused activity with [36–38], azetidinone showed in vitroand thiazolidinone,activity against My- two structural cobacterium tuberculosis. Quinoxaline derivatives with 2-chloro, dimethylamino and nitro scaffoldssubstitution possessing showed comparable antitubercular activity activity to that of [36–38], isoniazid showed(MIC = 0.67–0.97 in vitro µg/mL activity ver- against My- cobacteriumsus 0.46 µg/mL) tuberculosis [39] (Figure. Quinoxaline 6). derivatives with 2-chloro, dimethylamino and nitro substitution showed comparable activity to that of isoniazid (MIC = 0.67–0.97 µg/mL versus 0.46 µg/mL) [39] (Figure 6).

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Table 4. Chemical structure and MIC values against Mycobacterium tuberculosis of the best candidates from pyrrolo[1,2-a]quinoxalineTable series.4. Chemical structure and MIC values against Mycobacterium tuberculosis of the best candi- TableTable 4.4. ChemicalChemical structurestructure andand MICMIC valuesvalues againstagainst MycobacteriumMycobacterium tuberculosistuberculosis ofof thethe bestbest candi-candi- Tabledates from4. Chemical pyrrolo[1,2- structurea]quinoxaline and MIC series. values against Mycobacterium tuberculosis of the best candi- datesdates fromfrom pyrrolo[1,2-pyrrolo[1,2-aa]quinoxaline]quinoxaline series.series. Compounddates from pyrrolo[1,2-a]quinoxaline series. Structure MIC (µg/mL) Compound Structure MIC (µg/mL) Compound Structure MIC (µg/mL)

Cl N ClCl NN Cl N HN 7 7 HNHN 5 77 HN 55 5 7 N 5 N

F FF F Br Br

Cl N ClCl NN Cl N HN 8 8 HNHN 5 8 HN 5 5 N N

F FF F OCH3 OCH33 OCH3OCH3 OCHOCH3 OCH33

Cl N 9 Cl N 5 9 99 HN 55 9 HN 5 5 N N

F FF F Br Br

H N HH NN H N HN 10 HNHN 5 10 10 HN 5 5 N N

F FF F

Modification of the linker between R2 and pyrrolo[1,2-a]quinoxaline moiety led to a ModificationModification ofof thethe linkerlinker betweenbetween RR22 andand pyrrolo[1,2-pyrrolo[1,2-aa]quinoxaline]quinoxaline moietymoiety ledled toto aa minorModification variation in of antibacterial the linker between activity, Ralthough2 and pyrrolo[1,2- substitutionsa]quinoxaline at the R2 position moiety withled to an a minorminor variationvariation inin antibacterialantibacterial activity,activity, althoughalthough substitutionssubstitutions atat thethe RR22 positionposition withwith anan minor variationAs in vitaminantibacterial B6 activity, is an important although substitutions cofactor inat athe large R2 position number with of animportant enzymic reac- aromatic group led to the most active compounds (Figure 4). Derivative N-((7-chloro-1--((7-chloro-1- aromatic group led to the most active compounds (Figure 4). Derivative N-((7-chloro-1--((7-chloro-1- (2-fluorophenyl)pyrrolo[1,2-(2-fluorophenyl)pyrrolo[1,2-tions and is also usedaa]quinoxalin-3-yl)methyl)-2-(3,4-dimethoxyphenyl)ethana-]quinoxalin-3-yl)methyl)-2-(3,4-dimethoxyphenyl)ethana- in bioinorganic chemistry as a ligand, a new series of 13 quinoxalines (2-fluorophenyl)pyrrolo[1,2-(2-fluorophenyl)pyrrolo[1,2-aa]quinoxalin-3-yl)methyl)-2-(3,4-dimethoxyphenyl)ethana-]quinoxalin-3-yl)methyl)-2-(3,4-dimethoxyphenyl)ethanamine (9))bearing waswas identifiedidentified the pyridoxal asas offeringoffering the moietythe bestbest oraloral was bioavailability.bioavailability. synthesized DeterminationDetermination and tested against ofof enzymeenzymeMycobacterium tuberculosis mine (9)) waswas identifiedidentified asas offeringoffering thethe bestbest oraloral bioavailability.bioavailability. DeterminationDetermination ofof enzymeenzyme inhibitioninhibition andand molecularmolecular dockingdocking studiesstudies revealedrevealed thatthat thesethese originaloriginal derivativesderivatives couldcould inhibitioninhibition(Figure andand molecularmolecular7). In a dockingdocking hydrazone studiesstudies series, revealedrevealed the thatthat 7-chloroquinoxaline thesethese originaloriginal derivativesderivatives derivative couldcould ( 11) showed the best act by targeting the classic anti-TB drug target InhA by forming H bonds as well as Pi–Pi act by targetingactivity, the with classic the anti-TB MIC drug of target 72.72 InhAµM. by The forming number H bonds of as nitrogen well as Pi–Pi and chlorine atoms in the interactions.interactions. interactions.interactions.radical moiety plays an important role in antimycobacterial activity [40].

Molecules 2021, 26, x FOR PEER REVIEW 8 of 23

H H MoleculesMolecules 20212021,, 2626,, 4742x FOR PEER REVIEW NN NN 88 ofof 2323

R R N N Cl N N S H H N N O O H H NN NN R R : 4-CH3-Ph 4-OH-3-OCH3-Ph R N N Cl N N S H 4-OH-Ph 2-OH-Ph H N N 4-OCH3-Ph 3-NO2-Ph O 4-Cl-Ph 2-Cl-Ph O 4-(CH ) N-Ph furanyl 3 2 Figure 6. General structure of original quinoxaline derivatives containing azetidinone and thiazoli- R : 4-CH3-Ph 4-OH-3-OCH3-Ph dinone moieties. 4-OH-Ph 2-OH-Ph

As vitamin B6 is 4-OCHan important3-Ph cofactor3-NO in2-Ph a large number of important enzymic reactions and is also used 4-Cl-Phin bioinorganic chemistr2-Cl-Phy as a ligand, a new series of 13 quinoxalines bearing the pyridoxal moiety was synthesized and tested against Mycobacterium tubercu- 4-(CH3)2N-Ph furanyl losis (Figure 7). In a hydrazone series, the 7-chloroquinoxaline derivative ( 11) showed the bestFigure activity, 6. General with structure the MIC ofof of originaloriginal 72.72 quinoxalineµM.quinoxaline The number derivatives derivatives of nitrogen containing containing and azetidinone azetidinone chlorine andatoms and thiazolidi- thiazoli- in the radicalnonedinone moieties. moieties.moiety plays an important role in antimycobacterial activity [40].

As vitamin B6 is an important cofactor in a large number of important enzymic reac- HO CH3 tions and is also used in bioinorganic chemistry as a ligand, a new series of 13 quinoxalines bearing the pyridoxal moiety was Nsynthesized and tested against Mycobacterium tuberculosis (Figure 7).N In aNH hydrazoneN series, the 7-chloroquinoxaline derivative (11) showed the best activity, with the MIC of 72.72 µM. The number of nitrogen and chlorine atoms in the radical moiety plays an11 importantOH role in antimycobacterial activity [40]. Cl N

FigureFigure 7. 7. LeadLead compound compound of of hydrazones hydrazonesHO CH and and3 NN-acylhydrazones-acylhydrazones containing containing vitamin vitamin B6 and and different different heteroaromaticheteroaromatic nuclei. N InIn order order Nto prevent NH N excessive toxicity du duee to high lipophilicity and considering the biosafetybiosafety profile proﬁle of sugar conjugates [41–43] [41–43],, some new sugar conjugates of quinoxaline were synthesized andand evaluated evaluated11 OH for for their their antitubercular antitubercular activity. activity. Of Of a series a series of six of sugarsix sugar con- Molecules 2021, 26, x FOR PEER REVIEWCl N 9 of 23 conjugatesjugates of quinoxaline of quinoxaline compounds, compounds, all the all synthesized the synthesized derivatives derivatives demonstrated demonstrated interesting in- terestingantimycobacterialFigure 7. Lead antimycobacterial compound activity, of hydrazones withactivity, the MICwith and N rangingthe-acylhydrazones MIC fromranging 0.65 containingfrom to 9.6 0.65µM[ vitaminto 439.6] µM (Figure B6 [43]and8 different(Figure). 8).heteroaromatic nuclei. H RCHO H N O In order to preventH excessive toxicity due to high lipophilicity and considering the NH NH N O aldoses N O biosafety2 2 profile of sugar conjugates [41–43], some new sugar conjugates of quinoxaline N O were synthesized and evaluatedNH for their antitubercular activity. Of a seriesN Rof six sugar N N 2 CH3COOH/ N N H conjugates of quinoxaline compounds, all the synthesized derivatives demonstrated in- H CH3CH2OH H 12teresting antimycobacterial 13 activity, with the MIC ranging from 0.65 to 9.6 µM [43] (Figure 8). Ose = glucose: 13a mannose: 13b maltose: 13c lactose: 13d ribose: 13e xylose: 13f

Figure 8. SchematicSchematic route for the synthesis of sugar conjugates of quinoxaline.

somerism of sugar may influence antimycobacterial activity; this was supported by the fact that the ribose derivative was seven times more potent than the xylose derivative. Chalcones are compounds with a wide range of biological activities, including antitubercular activity [44,45]. Of a series of 14 original quinoxalinyl chalcones, two derivatives demonstrated equivalent activity to pyrazinamide and ciprofloxacin taken as reference with the MIC = 3.12 µg/mL. The structure–activity relationship showed that the presence of a hydroxyl substituent at position 3 on the phenyl ring increases antimycobacterial activity. Replacing phenyl with naphthyl resulted in reduced antimycobacterial activity, indicating that the hydrophobicity/lipophilicity balance plays an important role in these derivatives’ activity [46] (Table 5).

Table 5. Chemical structure of some quinoxalinyl chalcones and their MIC values against Mycobac- terium tuberculosis.

Compound Structure MIC (µg/mL) O N 14 3.12

N OH O N OH 15 3.12

N O N 16 50

N Br O N Br 17 25

Molecules 2021, 26, 4742 9 of 23

The ribose conjugate (13e) exhibited the best antimycobacterial activity, with the MIC = 0.65 µM. All the sugar conjugates were more active than the initial quinoxaline- 2,3-(1H,4H)-dione and the intermediate 3-hydrazono-3,4-dihydroquinoxalin-2(1H)-one at the MIC > 59.2 µM and the MIC = 27.2 µM, respectively. The structure–activity relationship showed that monosaccharides had better activity than disaccharides and that the aldopentoses were more potent than the aldohexoses. As in silico docking analysis revealed that these compounds could interact with DNA gyrase, it was concluded that the stereoisomerism of sugar may influence antimycobacterial activity; this was supported by the fact that the ribose derivative was seven times more potent than the xylose derivative. Chalcones are compounds with a wide range of biological activities, including antitubercular activity [44,45]. Of a series of 14 original quinoxalinyl chalcones, two derivatives demonstrated equivalent activity to pyrazinamide and ciprofloxacin taken as reference with the MIC = 3.12 µg/mL. The structure–activity relationship showed that the presence of a hydroxyl substituent at position 3 on the phenyl ring increases antimycobacterial activity. Replacing phenyl with naphthyl resulted in reduced antimycobacterial activity, indicating that the hydrophobicity/lipophilicity balance plays an important role in these derivatives’ activity [46] (Table5). The efficacy of quinoxaline-derived chalcones has recently been confirmed by the preclinical evaluation of a new series of derivatives. Of the synthesized compounds, six molecules inhibited Mycobacterium tuberculosis growth, with the MIC ranging from 3.13 to 12.5 µg/mL [47]. Further investigations on the lead compound (20) also demonstrated that this derivative exhibited synergistic effect with moxifloxacin and did not cause mutagenic- ity or genotoxicity (Table6). In addition, the modification of nalidixic acid, a representative of the quinolone and fluoroquinolone antibiotics which are an essential component of treatment strategies for drug-resistant TB [1], on its –COOH group led to the formation of original quinoxaline conjugates which showed encouraging antimycobacterial activity. Among the synthesized derivatives, quinoxalines with azide as side chain (25 and 26) exhibited the two best Molecules 2021, 26,activities, x FOR PEER withREVIEW the percentage of inhibition of Mycobacterium tuberculosis at 6.25 µg/mL 11 of 23

of 93% and 91%, respectively, compared to 100% for ciproﬂoxacin taken as reference [48] (Figure9). As Schiff basesconjugates possess which antimicrobial showed encouraging and antitubercular antimycobacterial properties activity. [49], synthesis Among of the synthe- some quinoxaline-incorporatedsized derivatives, quinoxalines Schiff bases with was performed.azide as side Among chain (25 an and original 26) exhibited series of the two best ten different Schiffactivities, bases with resulting the percentage from the of reaction inhibition between of Mycobacterium 2-((3-methylquinoxalin-2- tuberculosis at 6.25 µg/mL of yl)oxy)acetohydrazide93% and (3191%,) and respectively, various heterocyclic/aromatic compared to 100% for aldehydes, ciprofloxacin ﬁve compoundstaken as reference [48] exhibited potent(Figure antitubercular 9). activity [49] (Figure 10).

O O H S N O O N H H H N N N N N NNCH3 H N O NNCH N O H3C 3 H H 25 H3C 26 Figure 9. ChemicalFigure structures9. Chemical of structures lead compounds of lead compounds in nalidixic in acid nalidixic series. acid series.

As Schiff bases possess antimicrobial and antitubercular properties [49], synthesis of some quinoxaline-incorporated Schiff bases was performed. Among an original series of ten different Schiff bases resulting from the reaction between 2-((3-methylquinoxalin-2- yl)oxy)acetohydrazide (31) and various heterocyclic/aromatic aldehydes, five compounds exhibited potent antitubercular activity [49] (Figure 10).

O O N O N O N Ar NH ArCHO NH NH2 NCH3 NCH3 27a-j 27 27a: Ar = C6H5 % inhibition at MIC 6.25 µg/mL = 23 27b: Ar = 4-Cl-Ph C % inhibition at MlC 6.25 µg/mL = 15 27c: Ar = 3-NO2-Ph % inhibition at MIC 6.25 µg/mL = 6 27d: Ar = 4-OH-Ph . % inhibition at MIC 6.25 µg/mL = 58 - 27e: Ar = 3,4 (OCH3)2-Ph % inhibition at MIC 6.25 µg/mL = 25 27f: Ar = 4-OCH3-Ph % inhibition at MIC 6.25 µg/mL = 10 27g: Ar = 4-N(CH)2-Ph 2 % inhibition at MIC 6.25 µg/mL= 32 27h: Ar = 3-indoyl % inhibition at MIC 6.25 µg/mL = 7 27i: Ar = 2-furyl % inhibition at MIC 6.25 µg/mL = 56 27j: 5-(4-nitrophenyl)-2-furfuryl % inhibition at MIC 6.25 µg/mL = 50 Rifampicin % inhibition at MIC 6.25 µg/mL = 95

Figure 10. Percentage of inhibition of quinoxaline-incorporated Schiff bases against Mycobacterium tuberculosis.

Finally, spiroheterocyclic structures are well known for their antimycobacterial properties [50,51]. Spiropyrrolidine tethered indenoquinoxaline heterocyclic hybrids were synthesized and evaluated against Mycobacterium tuberculosis. Of the 11 synthesized compounds, bearing m-nitro, p-bromo and o-chloro substituents on the aryl ring showed interesting antimycobacterial activity, with MIC values ranging from 1.56 to 6.25 µg/mL [52] (Table 7).

Molecules 2021, 26, x FOR PEER REVIEW 9 of 23 MoleculesMolecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 99 ofof 2323 Molecules 2021, 26, x FOR PEER REVIEW 9 of 23

HH RCHO H NH O H RCHORCHO HH NN OO NH NH HNH O aldoses NH O NH2NH2 NN OO aldosesaldoses NN OO NHNH22NHNH22 N O aldoses N O NH N R NN OO NHNH2 CH3COOH/ N N NN RR HN O N N NH22 CHCH33COOH/COOH/ NN NN HH NHN NN CH3CH OH HN N H HH CHCH3CHCH2OHOH HH 12 13 H CH33CH22OH H 1212 13 13 OseOse == glucose:glucose: 13a13a Ose = mannose:glucose: 13a 13b mannose:mannose:mannose: 13b13b maltose:maltose: 13c13c lactose:maltose: 13d 13c lactose:lactose:lactose: 13d13d ribose:ribose: 13e13e xylose:ribose: 13e13f xylose:xylose:xylose: 13f13f

Figure 8. Schematic route for the synthesis of sugar conjugates of quinoxaline. FigureFigure 8.8. SchematicSchematic routeroute forfor thethe synthesissynthesis ofof sugarsugar conjugatesconjugates ofof quinoxaline.quinoxaline. The ribose conjugate (13e) exhibited the best antimycobacterial activity, with the MIC TheThe riboseribose conjugateconjugate ((13e13e))) exhibitedexhibitedexhibited thethethe bestbestbest antimycobacterialantimycobacterialantimycobacterial activity,activity,activity, withwithwith thethethe MICMICMIC = 0.65 µM. All the sugar conjugates were more active than the initial quinoxaline-2,3- == 0.650.65 µM.µM. AllAll thethe sugarsugar conjugatesconjugates werewere moremore activeactive thanthan thethe initialinitial quinoxaline-2,3-quinoxaline-2,3- (1H,4H)-dione and the intermediate 3-hydrazono-3,4-dihydroquinoxalin-2(1H)-one at the (1(1(1HH,4,4,4HH)-dione)-dione)-dione andandand thethethe intermediateintermediateintermediate 3-hy3-hy3-hydrazono-3,4-dihydroquinoxalin-2(1drazono-3,4-dihydroquinoxalin-2(1HH)-one)-one)-one atatat thethethe MIC > 59.2 µM and the MIC = 27.2 µM, respectively. The structure–activity relationship MICMIC >> 59.259.2 µMµM andand thethe MICMIC == 27.227.2 µM,µM, respectively.respectively. TheThe structure–activitystructure–activity relationshiprelationship showed that monosaccharides had better activity than disaccharides and that the aldo- showedshowed thatthat monosaccharidesmonosaccharides hadhad betterbetter activiactivitytyty thanthanthan disaccharidesdisaccharidesdisaccharides andandand thatthatthat thethethe aldo-aldo-aldopentoses were more potent than the aldohexoses. As in silico docking analysis revealed pentosespentoses werewere moremore potentpotent thanthan thethe aldohexoses.aldohexoses. AsAs inin silicosilico dockingdocking analysisanalysis revealedrevealed that these compounds could interact with DNA gyrase, it was concluded that the stereoi- thatthatthat thesethesethese compoundscompoundscompounds couldcouldcould interactinteractinteract withwithwith DNDNDNAA gyrase,gyrase, itit waswas concludedconcluded thatthat thethe stereoi-stereoisomerism of sugar may influence antimycobacterial activity; this was supported by the somerismsomerism ofof sugarsugar maymay influenceinfluence antimycobactantimycobacterialerial activity;activity; thisthis waswas supportedsupported byby thethe fact that the ribose derivative was seven times more potent than the xylose derivative. factfactfact thatthatthat thethethe riboseriboseribose derivativederivativederivative waswaswas sevensevenseven timestimestimes moremoremore potentpotentpotent thanthanthan thethethe xylosexylosexylose derivative.derivative.derivative. Chalcones are compounds with a wide range of biological activities, including an- ChalconesChalcones areare compoundscompounds withwith aa widewide rangerange ofof biologicalbiological activities,activities, includingincluding an-antitubercular activity [44,45]. Of a series of 14 original quinoxalinyl chalcones, two deriva- tituberculartituberculartitubercular activityactivityactivity [44,45].[44,45].[44,45]. OfOfOf aaa seriesseriesseries ofofof 141414 originaloriginaloriginal quinoxalinylquinoxalinylquinoxalinyl chalcones,chalcones,chalcones, twotwotwo deriva-deriva-derivatives demonstrated equivalent activity to pyrazinamide and ciprofloxacin taken as refer- tivestivestives demonstrateddemonstrateddemonstrated equivalentequivalentequivalent activityactivityactivity tototo pypypyrazinamiderazinamiderazinamide andandand ciprofloxacinciprofloxacinciprofloxacin takentakentaken asasas refer-refer-reference with the MIC = 3.12 µg/mL. The structure–activity relationship showed that the pres- enceence withwith thethe MICMIC == 3.123.12 µg/mL.µg/mL. TheThe structure–structure–activityactivity relationshiprelationship showedshowed thatthat thethe pres-presence of a hydroxyl substituent at position 3 on the phenyl ring increases antimycobacterial enceence ofof aa hydroxylhydroxyl substituentsubstituent atat positionposition 33 onon thethe phenylphenyl ringring increasesincreases antimycobacterialantimycobacterial activity. Replacing phenyl with naphthyl resulted in reduced antimycobacterial activity, activity.activity. ReplacingReplacing phenylphenyl withwith naphthylnaphthyl resuresultedltedlted ininin reducedreducedreduced antimycobacterialantimycobacterialantimycobacterial activity,activity,activity, Molecules 2021, 26,indicating 4742 that the hydrophobicity/lipophilicity balance plays an important role in these 10 of 23 indicatingindicatingindicating thatthatthat thethethe hydrophobicity/lipophilicityhydrophobicity/lipophilicityhydrophobicity/lipophilicity balancebalancebalance playsplaysplays ananan importantimportantimportant rolerolerole ininin thesethesethese derivatives’ activity [46] (Table 5). derivatives’derivatives’ activityactivity [46][46] (Table(Table 5).5). Table 5. Chemical structure of some quinoxalinyl chalcones and their MIC values against Mycobac- TableTable 5.5. ChemicalChemical structurestructure ofof somesome quinoxalinylquinoxalinyl chalconeschalconeschalcones andandand theirtheirtheir MICMICMIC valuesvaluesvalues againstagainstagainst Mycobac-Mycobac- Table 5.teriumChemical tuberculosis. structure of some quinoxalinyl chalcones and their MIC values against Mycobacterium tuberculosis. teriumteriumterium tuberculosis.tuberculosis.tuberculosis. Molecules 2021, 26, x FORCompound PEERCompound REVIEW Structure Structure MIC (µg/mL) MIC (µg/mL) 11 of 23 CompoundCompound StructureStructure MICMIC (µg/mL)(µg/mL) O OO N 14 14 NN 3.12 3.12 1414 conjugates which showed encouraging antimycobacterial 3.12activity.3.12 Among the synthesized derivatives, quinoxalinesN with azide OHas side chain (25 and 26) exhibited the two best NN OHOH O activities, with the percentage ofOO inhibition of Mycobacterium tuberculosis at 6.25 µg/mL of 93% and 91%, respectively,NN compared to OHOH100% for ciprofloxacin taken as reference [48] 15 N OH 3.12 15 1515 (Figure 9). 3.123.12 3.12 N NN O O O H OO N S16 N NN O O 50 16 1616 N H 5050 50 H H N N N N N Br NNNN CH BrBr N 3 O H OO N O NNCH N O H C NN BrBr 3 Molecules 2021, 26, x FOR PEER REVIEW 17 3 N H Br 25 10 of 23 17H 1717 2525 25 25 H3C N 26 NN Figure 9. Chemical structures of lead compoundsO in nalidixic acid series. N 18 18 As Schiff bases possess antimicrobial and antitubercular 25properties 25 [49], synthesis of some quinoxaline-incorporatedN Schiff bases was performed. Among an original series of

StreptomycinStreptomycinten different Schiff bases resulting from the reaction between 6.25 2-((3-methylquinoxalin-2- 6.25 CiproﬂoxacinCiprofloxacinyl)oxy)acetohydrazide (31) and various heterocyclic/aromatic 3.12 aldehydes, 3.12 five compounds PyrazinamidePyrazinamideexhibited potent antitubercular activity [49] (Figure 10). 3.12 3.12

TheO efficacy of quinoxaline-derived chalcones has recentlyO been confirmed by the preclinical evaluation of a new series of derivatives. Of the synthesized compounds, six N O N O N Ar molecules inhibitedNH MycobacteriumArCHO tuberculosis growth, with the MICNH ranging from 3.13 to 12.5 µg/mL NH[47].2 Further investigations on the lead compound (20) also demonstrated that NCH3 this derivative exhibited synergistic effect with moxifloxacinNCH3 and did not cause27a-j mutagen- icity or genotoxicity (Table 6). 27 27a: Ar = C H Table 6. MIC values against Mycobacterium6 tuberculosis5 of quinoxaline-derived% inhibition chalcones. at MIC 6.25 µg/mL = 23 27b: Ar = 4-Cl-Ph C % inhibition at MlC 6.25 µg/mL = 15 Compound 27c: Ar = 3-NOStructure2-Ph % inhibitionMIC at MIC(µg/mL) 6.25 µg/mL = 6 27d: Ar = 4-OH-PhO . % inhibition at MIC 6.25 µg/mL = 58 27e: Ar = 3,4-(OCH ) -Ph 3 2 N % inhibition at MIC 6.25 µg/mL = 25 19 27f: Ar = 4-OCH3-Ph % inhibition at 12.5MIC 6.25 µg/mL = 10 27g: Ar = 4-N(CH)2-Ph 2 % inhibition at MIC 6.25 µg/mL= 32 27h: Ar = 3-indoyl N % inhibition at MIC 6.25 µg/mL = 7 27i: ArOCH = 2-furyl3O % inhibition at MIC 6.25 µg/mL = 56 27j: 5-(4-nitrophenyl)-2-furfurylN % inhibition at MIC 6.25 µg/mL = 50 Rifampicin % inhibition at MIC 6.25 µg/mL = 95 20 3.13 N FigureFigure 10. Percentage 10. Percentage of inhibition of inhibition of ofquinoxa quinoxaline-incorporatedline-incorporated SchiffSchiff bases bases against againstMycobacterium Mycobacterium tuberculosis tuberculosis.. OCH 3 Finally, spiroheterocyclicO structures are well known for their antimycobacterial properties [50,51].H Spiropyrrolidine3CO tethered indenoquinoxalineN heterocyclic hybrids were syn- 21 12.5 thesized and evaluated against Mycobacterium tuberculosis. Of the 11 synthesized com- H CO N pounds, bearing3 m-nitro, p-bromo and o-chloro substituents on the aryl ring showed interesting antimycobacterialO activity, with MIC values ranging from 1.56 to 6.25 µg/mL [52] (Table 7). H3CO N 22 6.25 H3CO N OCH 3 O

H3CO N 23 12.5

HO N O H CO N 24 3 5

Rifampicin <0.2 Moxifloxacin <0.2 Isoniazid 0.39

In addition, the modification of nalidixic acid, a representative of the quinolone and fluoroquinolone antibiotics which are an essential component of treatment strategies for drug-resistant TB [1], on its –COOH group led to the formation of original quinoxaline

Molecules 2021, 26, x FOR PEER REVIEW 10 of 23 Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW 10 of 23 Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW 10 of 23

O O N 18 N 25 18 25 N N Streptomycin 6.25 Streptomycin 6.25 Ciprofloxacin 3.12 Ciprofloxacin 3.12 Pyrazinamide 3.12 Pyrazinamide 3.12 The efficacy of quinoxaline-derived chalcones has recently been confirmed by the The efficacy of quinoxaline-derived chalcones has recently been confirmed by the preclinical evaluation of a new series of derivatives. Of the synthesized compounds, six preclinical evaluation of a new series of derivatives. Of the synthesized compounds, six molecules inhibited Mycobacterium tuberculosis growth,growth, withwith thethe MICMIC rangingranging fromfrom 3.133.13 toto molecules inhibited Mycobacterium tuberculosis growth,growth, withwith thethe MICMIC rangingranging fromfrom 3.133.13 toto Molecules 2021, 26,12.5 4742 µg/mL [47]. Further investigations on the lead compound (20)) alsoalso demonstrateddemonstrated thatthat 11 of 23 12.5 µg/mL [47]. Further investigations on the lead compound (20) also demonstrated that thisthis derivativederivative exhibitedexhibited synergisticsynergistic effecteffect with moxifloxacin and did not cause mutagen- this derivative exhibited synergistic effect with moxifloxacin and did not cause mutagen- icityicity oror genotoxicitygenotoxicity (Table(Table 6).6). icityicity oror genotoxicitygenotoxicity (Table(Table 6).6). Table 6. MIC values against Mycobacterium tuberculosis of quinoxaline-derived chalcones. Table 6. MIC values against Mycobacterium tuberculosis ofof quinoxaline-derivedquinoxaline-derived chalcones.chalcones. Table 6. MIC values against Mycobacterium tuberculosis ofof quinoxaline-derivedquinoxaline-derived chalcones.chalcones. Compound Structure MIC (µg/mL) Compound Structure MIC (µg//mL) Compound Structure MIC (µg//mL) O O N 19 19 N 12.5 12.5 19 12.5 N NN OCH3O N OCH3O OCH3O 3 N N 20 20 3.13 3.13 20 3.13 N N OCH3 OCHOCH3 OCH3 O O H3CO N 21 HH3COCO N 12.5 21 H3CO N 12.5 21 21 12.5 12.5 H3CO N HH3COCO NN H3CO N O O H3CO N H3CO N H3CO N 22 3 6.25 22 2222 6.256.25 6.25 22 H3CO N 6.25 H3CO N H3CO N 3 OCH3 OCHOCH3 OCH3 O O H3CO N 23 HH3COCO NN 12.5 23 H3CO N 12.5 23 23 12.5 12.5 HO N HO N O O H3CO N H3CO N 24 H3CO N 5 2424 H3CO 55 24 24 5 5 N N Rifampicin <0.2 Rifampicin <0.2 RifampicinMoxifloxacin <0.2 <0.2 MoxiﬂoxacinMoxifloxacin <0.2 <0.2 IsoniazidIsoniazid 0.39 0.39 IsoniazidIsoniazid 0.39 0.39 InIn addition,addition, thethe modificationmodification ofof nalidixicnalidixic acid,acid, aa representativerepresentative ofof thethe quinolonequinolone andand In addition, theFinally, modification spiroheterocyclic of nalidixic structures acid, a representative are well known of forthetheir quinolone antimycobacterial and proper- fluoroquinolonefluoroquinolone antibioticsantibiotics whichwhich areare anan esseessential component of treatment strategies for fluoroquinoloneties antibiotics [50,51]. Spiropyrrolidine which are an esse tetheredntial component indenoquinoxaline of treatment heterocyclic strategies hybrids for were synthe- drug-resistant TB [1], on its –COOH group led to the formation of original quinoxaline drug-resistant TBsized [1], and on evaluatedits –COOH against groupMycobacterium led to the formation tuberculosis of original. Of the 11quinoxaline synthesized compounds, bearing m-nitro, p-bromo and o-chloro substituents on the aryl ring showed interesting antimycobacterial activity, with MIC values ranging from 1.56 to 6.25 µg/mL [52] (Table7).

4.1.3. Other Structures Several derivatives of 2-substituted quinoxalines have also shown interesting antitubercular activities. Thus, quinoxaline alkynyl derivatives demonstrated antimycobacterial activity. Of the 19 original quinoxalines bearing diverse substituents on the alkynyl group synthesized from quinoxaline-2-ol derivatives, seven compounds had MIC90 < 10 µM and ﬁve compounds had MIC90 ranging from 10 to 20 µM[53] (Table8). Molecules 2021, 26, 4742 12 of 23

Molecules 2021, 26, x FOR PEER REVIEW 12 of 23 Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW 12 of 23

Table 7. Chemical structure of some spiropyrrolidine tethered indenoquinoxaline heterocyclic hybrids and their MIC values Table 7. Chemical structure of some spiropyrrolidine tethered indenoquinoxaline heterocyclic hy- against MycobacteriumTable 7. Chemical tuberculosis structure. of some spiropyrrolidine tethered indenoquinoxaline heterocyclic hy- Tablebrids and7. Chemical their MIC structure values against of some Mycobacterium spiropyrrolidine tuberculosis. tethered indenoquinoxaline heterocyclic hybrids and their MIC values against Mycobacterium tuberculosis. Compound Structure MIC (µg/mL) Compound Structure MIC (µg/mL) Compound Structure MIC (µg/mL) N N

28 28 N NH 3.125 3.125 28 N NH 3.125

O N Br O2N Br O2N Br O2N N N

N NH 29 29 N NH 6.25 6.25 29 N NH 6.25

O N Cl O2N Cl O2N Cl N N

30 N NH 12.5 30 30 N NH 12.5 12.5

O N Cl O2N Cl O2N Cl O2N N N

N NH 31 N NH 1.56 31 31 1.56 1.56 O N O2N O2N O2N O2N O2N Ethambutol 1.56 Ethambutol 1.56 EthambutolRifampicin 0.1 1.56 RifampicinRifampicin 0.1 0.1 Isoniazid 0.05 IsoniazidIsoniazid 0.05 0.05 4.1.3. Other Structures 4.1.3. Other StructuresThe presence of an electron-withdrawing group at the C6 position of the quinoxaline Several derivativesmoiety plays of 2-substituted an important quinox rolealines in antimycobacterial have also shown activity: interesting four an- of the most active Several derivatives of 2-substituted quinoxalines have also shown interesting antitubercular activities.compounds Thus, arequinoxaline derivatives alkynyl bearing derivatives NO at this demonstrated position with antimycobac- MIC < 3 µM. titubercular activities. Thus, quinoxaline alkynyl derivatives2 demonstrated antimycobac-90 terial activity. Of the Moreover,19 original screening quinoxalin of aes library bearing of compounds diverse substituents identified three on originalthe alkynyl 2-carboxyquinoxaline terial activity. Of the 19 original quinoxalines bearing diverse substituents on the alkynyl group synthesized from quinoxaline-2-ol derivatives, seven compounds had MIC90 < 10 compounds showing activity against Mycobacterium tuberculosis with90 MIC99 values ranging group synthesized from quinoxaline-2-ol derivatives, sevenseven compoundscompounds hadhad MICMIC90 << 1010 µM and five compoundsfrom 3.1 tohad 12.5 MICµM[90 ranging54]. Among from them, 10 to 20 the µM lead [53] compound (Table 8). ( 39) which exhibited a MIC 90 99 µM and five compounds had MIC90 rangingranging fromfrom 1010 toto 2020 µMµM [53][53] (Table(Table 8).8). of 3.1 µM was shown to noncovalently and noncompetitively inhibit DprE1, an enzyme Table 8. Chemicalessential structure for and bacterial MIC values wall against synthesis Mycobacterium by forming tuberculosis hydrophobic of the interactions best candi- and hydrogen Table 8. Chemical structure and MIC values against Mycobacterium tuberculosis ofof thethe bestbest candi-candidates from the quinoxalinebonds (Figure alkynyl 11 series.). dates from the quinoxaline alkynyl series. Compound Structure MIC90 (µM) 90 Compound Structure MIC90 (µM)(µM) OH N OH N OH 32 N 4.26 32 4.26 O N N O2N N O2N N 2

Molecules 2021, 26, x FOR PEER REVIEW 12 of 23

Table 7. Chemical structure of some spiropyrrolidine tethered indenoquinoxaline heterocyclic hybrids and their MIC values against Mycobacterium tuberculosis.

Compound Structure MIC (µg/mL)

28 N NH 3.125

Br O2N N

29 N NH 6.25

O2N Cl N

30 N NH 12.5

Cl O2N N

N NH 31 1.56

O2N O N 2 Ethambutol 1.56 Rifampicin 0.1 Isoniazid 0.05

4.1.3. Other Structures Several derivatives of 2-substituted quinoxalines have also shown interesting antitubercular activities. Thus, quinoxaline alkynyl derivatives demonstrated antimycobac- Molecules 2021, 26,terial 4742 activity. Of the 19 original quinoxalines bearing diverse substituents on the alkynyl 13 of 23 Molecules 2021, 26, x FOR PEER REVIEW 13 of 23 group synthesized from quinoxaline-2-ol derivatives, seven compounds had MIC90 < 10 µM and five compounds had MIC90 ranging from 10 to 20 µM [53] (Table 8). Table 8. Chemical structure and MIC values against Mycobacterium tuberculosis of the best candidates from the quinoxaline Table 8. Chemical structure and MIC values against Mycobacterium tuberculosis of the best candi- alkynyl series. O dates from the quinoxaline alkynyl series. N 33 4.55 Compound Structure MIC (µM) Compound Structure MIC90 (µM)90 N OH N O 32 N 4.26 MoleculesMolecules 20212021,, , 26 26,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 32 34 13134.26 ofof 2323 1.13 MoleculesMolecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 1313 ofof 2323 Molecules 2021, 26, x FOR PEER REVIEW 13 of 23 O2N N Cl N O OO NN O O S CH3 N N OO 33 3333 35 NN O 4.554.55 4.55 6.47 3333 4.554.55 NN N N N OO O NN OO 34 N O O S CH31.13 34 3434 N N 1.131.13 1.13 3434 36 O 1.131.13 1.59 Cl N ClCl NN ClCl NN O O2N OON OO OO SS CHCH3 NN O S CH33 O 3535 N O SO CH33 O 6.476.47 35 35 NN OO 3 6.47 35 N OON 6.476.47 37 O CH3 1.80 NN N N O N NOO 2 O O OS CH OO SS CHCH33 O NN OO SS CHCH3 3636 N O OSO CH33 O 1.591.59 36 36 N NO 1.59 1.59 36 38 OO S1.59 2.74 OO22NN NN O 2N N O22N NO N N 2 2 OO OO OO Rifampicin N O O 0.01 NN OO CH 3737 NN CHCH33 1.801.80 37 N CHCH3 1.80 37 37 The presence of an electron-withdrawingCH gr33 oup at the C6 position1.80 1.80 of the quinoxaline OO NN NN moiety playsO2 2anN importantN role in antimycobacterial activity: four of the most active com- OO2NN NN O22N N OO pounds are derivatives bearing NO2 at this positionO with MIC90 < 3 µM. OO OO Moreover, screeningNN of a library OofO compounds identified three original 2-carbox- 3838 NN O SS 2.742.74 yquinoxaline compoundsN showing activity againstS Mycobacterium tuberculosis with MIC99 3838 S 2.742.74 38 values ranging from 3.1 to 12.5 µM [54]. Among them, the lead compound2.74 (39) which OO2NN NN O22N N exhibited aO MIC22N 99 of 3.1 NµM was shown to noncoval ently and noncompetitively inhibit RifampicinRifampicin 2 0.01 0.01 Rifampicin DprE1, an enzyme essential for bacterial wall synthesis by formin 0.01g hydrophobic interac- RifampicinRifampicin 0.01 0.01 tions and hydrogen bonds (Figure 11). TheThe presencepresence ofof anan electron-withdrawingelectron-withdrawing grgroupoup atat thethe C6C6 positionposition ofof thethe quinoxalinequinoxaline TheThe presencepresence ofof anan electron-withdrawingelectron-withdrawing grgroupoup atat thethe C6C6 positionposition ofof thethe quinoxalinequinoxaline moietymoietyThe playsplays presence anan importantimportant of an electron-withdrawing rolerole inin antimycobacterialantimycobacterial group activity: activity:at the C6 fourfour position ofof thethe of mostmost the quinoxaline activeactive com-com- moietymoiety playsplays anan importantimportant rolerole inin antimycobacterialantimycobacterialO activity:activity: fourfour ofof thethe mostmost activeactive com-com- poundsmoietypounds plays areare derivativesderivatives an important bearingbearing role inNONO antimycobacterial22 atat thisthis positionposition withwithactivity: MICMIC four9090 << 33of µM.µM. the most active com- poundspounds areare derivativesderivatives bearingbearingN NONO22 atat thisthis positionposition withwith MICMIC9090 << 33 µM.µM. poundsMoreover,Moreover, are derivatives screeningscreening bearing ofof aa library libraryNO2 atOH ofthisof compoundscompounds position with identifiedidentified MIC90 < threethree3 µM. originaloriginal 2-carbox-2-carbox- Moreover,Moreover, screeningscreening ofof aa librarylibrary ofof compoundscompounds identifiedidentified threethree originaloriginal 2-carbox-2-carbox- yquinoxalineyquinoxalineMoreover, compoundscompounds screening shshofowing owinga library activityactivity of compounds againstagainst MycobacteriumMycobacterium identified three tuberculosistuberculosis original withwith 2-carbox- MICMIC9999 yquinoxalineyquinoxaline compoundscompoundsF C shshowingowingN N activityactivity againstagainst MycobacteriumMycobacterium tuberculosistuberculosis withwith MICMIC9999 valuesyquinoxalinevalues rangingranging compounds fromfrom3 3.13.1 toto sh 12.512.5owing µMµM activity [54].[54]. AmongAmong against them,them,Mycobacterium thethe leadlead tuberculosiscompoundcompound with((3939)) whichwhichMIC99 values ranging from 3.1 to 12.5 µMH [54]. Among them, the lead compound (39) which valuesexhibited ranging a MIC from9999 of 3.1 39toµM 12.5 was µM shown [54]. toAmong noncoval them,CHently3 the and lead noncompetitively compound (39)) inhibitwhichwhich exhibitedexhibited aa MICMIC99 ofof 3.13.1 µMµM waswas shownshown toto noncovalnoncovalO entlyently andand noncompetitivelynoncompetitively inhibitinhibit exhibitedexhibited aa MICMIC9999 ofof 3.13.1 µMµM waswas shownshown toto noncovalnoncovalentlyently andand noncompetitivelynoncompetitively inhibitinhibit DprE1,exhibitedDprE1, anan a enzymeenzyme MIC99 of essentialessential 3.1 µM for forwas bacterialbacterial shown wawato llnoncovalll synthesissynthesisently byby forminforminand noncompetitivelygg hydrophobichydrophobic interac- interac-inhibit DprE1,DprE1, anan enzymeenzymeFigure essentialessential 11. Structure forfor bacterialbacterial of the wawa leadllll synthesissynthesis compound byby formin 3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)formingg hydrophobichydrophobic interac-interac- tionsDprE1,tions andand an hydrogenhydrogen enzymeFigure essential bondsbonds 11. Structure (Figure(Figure for bacterial of 11).11). the lead wa compoundll synthesis 3-((4-met by forminhoxybenzyl)amino)-6-(trifluoromethyl)quinox-g hydrophobic interac- tionstions andand hydrogenhydrogenaline-2-carboxylicquinoxaline-2-carboxylic bondsbonds (Figure(Figure acid, 11).11). a new acid, DprE1 a new inhibitor. DprE1 inhibitor. OO OO NN4.2. Quinoxaline-1,4-di-N-oxideO Derivatives Active against Mycobacterium tuberculosis N OH NN Quinoxaline-1,4-di-OHOH N-oxide derivatives are a class of compounds with a variety of OHOH F C NbiologicalN properties, including antitumor, anti-inflammatory, and anti-infectious activity FF33CC NN NN FF33CC NN[2]. TheHNNH classic method of quinoxaline-1,4-di-N-oxide preparation uses benzofuroxane N- F3C 39 N HN CHCH3 3939 oxideH as the reagentO [55].CH33 3939 OO CHCH33 39 OO CH3 O FigureFigure 11.11. StructureStructure ofof thethe leadlead compoundcompound 3-((4-met3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinox-hoxybenzyl)amino)-6-(trifluoromethyl)quinox- Figure 11. Structure of the lead compound 3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylicFigurealine-2-carboxylic 11. Structure acid,acid, of the aa newnew lead DprE1DprE1 compound inhibitor.inhibitor. 3-((4-met hoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid, a new DprE1 inhibitor. aline-2-carboxylic acid, a new DprE1 inhibitor. 4.2.4.2. Quinoxaline-1,4-di-N-oxideQuinoxaline-1,4-di-N-oxide DerivativesDerivatives ActiveActive againstagainst MycobacteriumMycobacterium tuberculosistuberculosis 4.2.4.2. Quinoxaline-1,4-di-N-oxideQuinoxaline-1,4-di-N-oxide DerivativesDerivatives ActiveActive againstagainst MycobacteriumMycobacterium tuberculosistuberculosis Quinoxaline-1,4-di-Quinoxaline-1,4-di-NN-oxide-oxide derivativesderivatives areare aa classclass ofof compoundscompounds withwith aa varietyvariety ofof Quinoxaline-1,4-di-Quinoxaline-1,4-di-NN-oxide-oxide derivativesderivatives areare aa classclass ofof compoundscompounds withwith aa varietyvariety ofof biologicalbiologicalQuinoxaline-1,4-di- properties,properties, includingincludingN-oxide antitumor,antitumor, derivatives anan areti-inflammatory,ti-inflammatory, a class of compounds andand anti-infectiousanti-infectious with a variety activityactivity of biologicalbiological properties,properties, includingincluding antitumor,antitumor, ananti-inflammatory,ti-inflammatory, andand anti-infectiousanti-infectious activityactivity [2].biological[2]. TheThe classicclassic properties, methodmethod including ofof quinoxaline-1,4-di-quinoxaline-1,4-di- antitumor, anNNti-inflammatory,-oxide-oxide preparationpreparation and uses usesanti-infectious benzofuroxanebenzofuroxane activity NN-- [2].[2]. TheThe classicclassic methodmethod ofof quinoxaline-1,4-di-quinoxaline-1,4-di-NN-oxide-oxide preparationpreparation usesuses benzofuroxanebenzofuroxane NN-- oxide[2].oxide The asas classic thethe reagentreagent method [55].[55]. of quinoxaline-1,4-di-N-oxide preparation uses benzofuroxane N- oxideoxide asas thethe reagentreagent [55].[55].

Molecules 2021, 26, 4742 14 of 23

4.2. Quinoxaline-1,4-di-N-oxide Derivatives Active against Mycobacterium tuberculosis Quinoxaline-1,4-di-N-oxide derivatives are a class of compounds with a variety of bio- Molecules 2021, 26, x FOR PEER REVIEWlogical properties, including antitumor, anti-inﬂammatory, and anti-infectious activity14 of [2 23]. The classic method of quinoxaline-1,4-di-N-oxide preparation uses benzofuroxane N-oxide as the reagent [55].

4.2.1. Hybrids and Conjugates 1,2,3-triazole and and their their derivatives derivatives are are known known for for their their various various biological biological activities activi- tiesincluding including antifungal antifungal and antibacterial and antibacterial activity activity [56]. In [ 56order]. Into orderstudy the to studyinfluence the of inﬂu- the encesubstitution of the substitutionat the C2 position at the in C2 quinoxaline-1,4-di- position in quinoxaline-1,4-di-N-oxide series,N -oxidethirty-one series, 1,2,3-tria- thirty- onezole 1,2,3-triazoleanalogs of quinoxaline-1,4-di-analogs of quinoxaline-1,4-di-N-oxide wereN synthesized-oxide were synthesizedand evaluated and against evaluated My- againstcobacteriumMycobacterium tuberculosis, tuberculosis, yielding 16yielding compounds 16 compounds with the MIC with ranging the MIC from ranging 12.5 fromto 25 12.5µg/mL to 25[56]µg/mL (Figure [56 12).] (Figure 12).

O X N O N R N N Y N CH3

X = H, Cl R : 4-CH2CH3 2-F 3-NO2 Y = H, Cl 4-F 2-Cl 3-CF3

4-Cl 2-NO2 3-Cl-5Cl 4-Br 4-NO 2 Figure 12. General structure of 1,2,3-triazole analogs of quinoxaline-1,4-di-NN-oxide.-oxide.

More specifically,specifically, in in 2-(((1-(substituted 2-(((1-(substituted phenyl)-1 phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-H-1,2,3-triazol-4-yl)methoxy)car- 3-methylquinoxaline-1,4-dioxidebonyl)-3-methylquinoxaline-1,4-dioxide series, only series, 2-chloro only and 2-chloro 2- or 3-nitroand 2- or compounds 3-nitro compounds exhibited highexhibited activity. high Inactivity. 3-(((1-(substituted In 3-(((1-(substituted phenyl)-1 phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6-H-1,2,3-triazol-4-yl)methoxy)car- chloro-2-methylquinoxaline-1,4-dioxidebonyl)-6-chloro-2-methylquinoxaline-1,4-dioxide series, unsubstituted, series, unsubstituted,ortho-, and orthopara-substituted-, and para- derivativessubstituted exhibitedderivatives high exhibited activity. high The mostactivi activety. The compound most active ofthe compound three series, of the 6-chloro- three 2-methyl-3-(((1-phenyl-1series, 6-chloro-2-methyl-3-(((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxaline-1,4-dioxide,H-1,2,3-triazol-4-yl)methoxy)carbonyl)quinoxa- ex- µ hibitedline-1,4-dioxide, good antimycobacterial exhibited good activity,antimycobact with theerial MIC activity, of 12.5 withg/mL the MIC against of 12.5 the µg/mL tested µ strainsagainst versus the tested 3.1 strainsg/mL forversus reference 3.1 µg/mL drug for isoniazid. reference In thedrug 2-(((1-(substituted isoniazid. In the phenyl)-12-(((1-(sub-H- 1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3-methylquinoxaline-1,4-dioxidestituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3-methylquinoxa- series, para onlyline-1,4-dioxide-substituted series, halogen only para compounds-substituted exhibited halogen compounds activity. These exhibited structure–activity activity. These relationships also demonstrated that the introduction of an electron-withdrawing group structure–activity relationships also demonstrated that the introduction of an electron- resulted in less active compounds. withdrawing group resulted in less active compounds. In addition, hybridization of quinoxaline 1,4-di-N-oxide with chalcone, fluoroquinolone, In addition, hybridization of quinoxaline 1,4-di-N-oxide with chalcone, fluoroquino- and thiazolidinone scaffolds was investigated [57,58]. Of the 10 original chalcones newly lone, and thiazolidinone scaffolds was investigated [57,58]. Of the 10 original chalcones synthesized by Claisen–Schmidt condensation, only one displayed potency, with the newly synthesized by Claisen–Schmidt condensation, only one displayed potency, with MIC = 3.1 µM compared to 6.2 µM, 0.3 µM, and 0.04 µM, respectively, for ethambutol, the MIC = 3.1 µM compared to 6.2 µM, 0.3 µM, and 0.04 µM, respectively, for ethambutol, isoniazid, and rifampicin taken as reference. In this same study, four of the five most potent isoniazid, and rifampicin taken as reference. In this same study, four of the five most po- derivatives were fluoroquinolone analogs, with MIC values ranging from 1.6 to 3.1 µM tent derivatives were fluoroquinolone analogs, with MIC values ranging from 1.6 to 3.1 (Table9). The structure–activity relationship showed that the presence of an electron- µM (Table 9). The structure–activity relationship showed that the presence of an electron- withdrawing substituent like a halogen atom at position C6 or C7 of the quinoxaline withdrawing substituent like a halogen atom at position C6 or C7 of the quinoxaline moi- moiety enhances the activity. In addition, the presence of CF3 at position C3 promotes antimycobacterialety enhances the activity. activity, In while addition, the nature the presence of the lateral of CF side3 at position chain substituent C3 promotes at the anti- C2 positionmycobacterial of the activity, quinoxaline while group the nature seems of to the have lateral less influence side chain on substituent biological activityat the C2 [57 po-]. sition of the quinoxaline group seems to have less influence on biological activity [57].

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Table 9. Antimycobacterial activity of quinoxaline derivatives based on molecular hybridization of quinoxaline 1,4-di-N- Table 9. Antimycobacterial activity of quinoxaline derivatives based on molecular hybridization of oxide withTableTable the chalcone 9.9. AntimycobacterialAntimycobacterial and ﬂuoroquinolone activityactivity of scaffolds.of quinoxalinequinoxaline derivativesderivatives basedbased onon molecularmolecular hybridizationhybridization ofof quinoxaline 1,4-di-N-oxide with the chalcone and fluoroquinolone scaffolds. quinoxalinequinoxaline 1,4-di-1,4-di-NN-oxide-oxide withwith thethe chalconechalcone anandd fluoroquinolonefluoroquinolone scaffolds.scaffolds. CompoundCompound Structure Structure MIC (µM) MIC (µM) CompoundCompound StructureStructure MICMIC (µM)(µM) O OO O O OO Cl N ClCl NN 40 40 3.1 3.1 4040 3.13.1 N CH N CH3 NN CHCH33 O OO O OO O O O OO OO N N O NN NN OO 41 41 3.1 3.1 4141 3.13.1 Cl N CF3 ClCl NN CFCF33 O OO O OO O O OO N N NN NN CH CHCH3 42 42 CH33 1.6 1.6 4242 1.61.6 F N CF F N CF3 FF NN CFCF33 O OO F FF O OO N O O NN OO N N NN NN 43 43 OCH3 1.6 1.6 4343 OCHOCH33 1.61.6 N CF N CF3 NN CFCF33 O OO O OO S O O SS OO N N NN NN CH3 44 CHCH33 3.1 44 4444 3.13.1 3.1 F N CF F N CF3 FF NN CFCF33

O OO Ethambutol 6.2 EthambutolEthambutolEthambutol 6.2 6.2 6.2 Rifampicin 0.3 RifampicinRifampicinRifampicin 0.3 0.3 0.3 IsoniazidIsoniazid 0.04 0.04 IsoniazidIsoniazid 0.04 0.04 In thiazolidinone series, out of 26 novel derivatives, four compounds displayed po- InIn thiazolidinonethiazolidinoneIn series,series, thiazolidinone outout ofof 2626 novelnovel series, derivatives,derivatives, out of 26 four novelfour compoundscompounds derivatives, displayeddisplayed four compounds po-po- displayed tent antimycobacterial activity, four compounds—moderate activity [58] (Table 10). The tenttent antimycobacterialantimycobacterialpotent activity,activity, antimycobacterial fourfour compoundcompound activity,s—moderates—moderate four compounds—moderate activityactivity [58][58] (Table(Table activity 10).10). TheThe [58 ] (Table 10). The presence of an electron-withdrawing group at the para position of the phenyl group is presencepresence ofof anan electron-withdrawingelectron-withdrawingpresence of an electron-withdrawing groupgroup atat thethe parapara group positionpositionposition atthe ofofof theparathethe phenylphenylphenylposition groupgroupgroup of the isisis phenyl group is essential for higher activity and the presence of a halogen atom at position C7 of the essentialessential forfor higherhigheressential activityactivity for andand higher thethe activitypresencepresence and ofof a thea halogenhalogen presence atomatom of aatat halogen positionposition atom C7C7 ofof at thethe position C7 of the quinoxaline nucleus increases antimycobacterial activity. Replacement of halogen atoms quinoxalinequinoxaline nucleusnucleusquinoxaline increasesincreases nucleus antimycobacterialantimycobacterial increases antimycobacterial activity.activity. ReplacementReplacement activity. ofof halogen Replacementhalogen atomsatoms of halogen atoms with a methyl or methoxy resulted in decreased antimycobacterial activity. withwith aa methylmethyl oror with methoxymethoxy a methyl resultedresulted or methoxy inin decreaseddecreased resulted antimycobacterialantimycobacterial in decreased antimycobacterial activity.activity. activity.

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Table 10. Antimycobacterial activity of quinoxaline derivatives based on molecular hybridization of quinoxaline 1,4-di-N- Table 10. Antimycobacterial activity of quinoxaline derivatives based on molecular hybridization oxide withTable the thiazolidinone 10. Antimycobacterial scaffold. activity of quinoxaline derivatives based on molecular hybridization Tableof quinoxaline 10. Antimycobacterial 1,4-di-N-oxide activity with the of thiazolidinone quinoxaline derivatives scaffold. based on molecular hybridization of quinoxaline 1,4-di-N-oxide with the thiazolidinone scaffold. of quinoxaline 1,4-di-N-oxide-oxide withwith thethe thiazolidinonethiazolidinone scaffold.scaffold. Compound Structure MIC (µg/mL) Compound Structure MIC (µg/mL) Compound StructureO MIC (µg/mL) OO S S O F N O F N N O 45 45 N 45 1.56 1.56 45 N 1.56 NN

O O F O F OO S S O F N O F N N O 46 N 1.56 46 46 1.56 1.56 46 N 1.56 NN

O O Cl O Cl O O S S S O Cl N O ClCl NN N O NN 47 1.56 47 47 N 1.561.56 1.56 NN O OO F FF O F OO S S O Cl N O Cl N N O 48 N 48 1.56 48 48 N 1.56 1.56 NN

O O Cl Cl Rifampicin 0.125 RifampicinRifampicin 0.125 0.125 As many isonicotinic acid hydrazide derivatives such as isoniazid demonstrated in- AsAs manymany isonicotinicisonicotinic acidacid hydrazidehydrazide derivativesderivatives suchsuch asas isoniazidisoniazid demonstrateddemonstrated in-in- terestingAs many anti-TB isonicotinic activities,As many acid an original hydrazide isonicotinic series derivatives acidof hybrids hydrazide such resulting as derivatives isoniazid from the demonstrated such fusion as between isoniazid in- demonstrated terestingteresting anti-TB anti-TB activities, activities, an an original original series series of of hybrids hybrids resulting resulting from from the the fusion fusion between between terestingquinoxaline anti-TB 1,4-di- interestingactivities,N-oxide anand anti-TB original isoniazid activities, series was of an synthetized; hybrids original resulting series it showed of hybridsfrom high the resulting fusionactivity between fromagainst the fusion between quinoxalinequinoxaline 1,4-di- 1,4-di-NN-oxide-oxide and and isoniazid isoniazid was was synthetized; synthetized; it it showed showed high high activity activity against against quinoxalineMycobacterium 1,4-di- tuberculosisquinoxalineN-oxide, andwith 1,4-di- isoniazid IC90N -oxideranging was and fromsynthetized; isoniazid < 1.16 to was it 50.60showed synthetized; µM high versus activity it showed0.21 againstµM high for activity against Mycobacterium tuberculosis, with IC90 ranging from < 1.16 to 50.60 µM versus 0.21 µM for Mycobacterium tuberculosis,, with IC9090 rangingranging fromfrom << 1.161.16 toto 50.6050.60 µMµM versusversus 0.210.21 µMµM forfor isoniazid taken asMycobacterium reference (Figure tuberculosis 13). As, isonicotinic with IC90 ranging hydrazide from derivatives <1.16 to 50.60 can beµM con- versus 0.21 µM for isoniazidisoniazidisoniazid takentakentaken asasas referencereferencereference (Figure(Figure(Figure 13).13).13). AsAsAs isonicotinicisonicotinicisonicotinic hydrazidehydrazidehydrazide derivativesderivativesderivatives cancancan bebebe con-con-considered as a prodrug,isoniazid these taken new hybrids as reference could (Figure act according 13). As to isonicotinicthe original mechanism hydrazide derivatives of can be sidered as a prodrug,considered these new as a hybrids prodrug, could these act new according hybrids couldto the actoriginal according mechanism to the original of mechanism action where the hydrolysis of thethe compoundscompounds wouldwould leadlead toto thethe formationformation ofof isoniazidisoniazid action where theof hydrolysis action where of thethe the compoundscompounds hydrolysis of wouldwould the compounds leadlead toto thethewould formationformation lead ofof to isoniazidisoniazid the formation of isoniazid which would act according to its own mechanism of action and of quinoxaline derivatives which would act whichaccording would to its act own according mechanism to its ownof action mechanism and of quinoxaline of action and derivatives of quinoxaline derivatives which would act synergistically with isoniazid. PositionPosition 77 unsubstitutedunsubstituted oror substitutedsubstituted byby which would act whichsynergistically would act with synergistically isoniazid. PositionPosition with isoniazid. 77 unsubstitutedunsubstituted Position oror 7 unsubstitutedsubstitutedsubstituted bybyor substituted by an electron-withdrawing or electron-releasing groupgroup doesdoes notnot differdiffer inin activityactivity [59].[59]. an electron-withdrawingan electron-withdrawing or electron-releasing or electron-releasing groupgroup doesdoes notnot differdiffer group inin does activityactivity not differ[59].[59]. in activity [59].

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O O O O N R7 N NH2 H NH N R7 N N + H N N R6 N CH3 O R6 N CH3 56 : Isoniazid O O

49: R6: H; R7: H 49a: R6: H; R7: H 50: R6: H; R7: Cl 50a: R6: H; R7: Cl 51: R6: H; R7: OCH3 51a: R6: H; R7: OCH3 52: R6: H; R7: CH3 52a: R6: H; R7: CH3 53: R6: CH3; R7: CH3 53a: R6: CH3; R7: CH3 54: R6: H; R7: F 54a: R6: H; R7: F 55: R : H; R : CF 55a: R : H; R : CF 6 7 3 6 7 3 FigureFigure 13. 13.Design Design of of 1,4-di- 1,4-di-NN-oxide-oxide quinoxaline-2-ylmethylene quinoxaline-2-ylmethylene isonicotinic isonicotinic acid acid hydrazide hydrazide derivatives. derivatives.

TheThe quinoxaline-2-carboxamidequinoxaline-2-carboxamide 1,4-di- N-oxide-oxide derivatives derivatives used used as as precursors precursors for for the thesynthesis synthesis of ofthese these new new series series were were also also evaluated evaluated against against MycobacteriumMycobacterium tuberculosis. tuberculosis. In Inthese these series, series, molecules molecules substituted substituted with with an electron-w an electron-withdrawingithdrawing group group at position at position 7 such 7 such as CF3, Cl, or F on the quinoxaline ring were the most active compounds, with as CF3, Cl, or F on the quinoxaline ring were the most active compounds, with the respec- the respective IC90 of 1.07, 1.25, and 4.65 µM. In order to explore the structural require- tive IC90 of 1.07, 1.25, and 4.65 µM. In order to explore the structural requirements for anti- ments for anti-TB activity, molecular modelling studies were performed on a series of TB activity, molecular modelling studies were performed on a series of 23 original quinox- 23 original quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives, with IC90 values rang- aline-2-carboxamide 1,4-di-N-oxide derivatives, with IC90 values ranging from 3.86 µg to ing from 3.86 µg to 100 µg [60]. The reliable pharmacophore generated was composed 100 µg [60]. The reliable pharmacophore generated was composed of one aromatic ring, of one aromatic ring, four hydrophobic substituents, three hydrogen acceptors, and one four hydrophobic substituents, three hydrogen acceptors, and one hydrogen donor. The hydrogen donor. The binding mode of these derivatives was similar to that of coumarin in binding mode of these derivatives was similar to that of coumarin in novobiocin, which novobiocin, which is known to be an inhibitor of Mycobacterium tuberculosis DNA gyrase is known to be an inhibitor of Mycobacterium tuberculosis DNA gyrase active site. active site.

4.2.2.4.2.2. Other Other Structures Structures InIn order order to to develop develop new new antitubercular antitubercular agents, agents, a a series series of of 33 33 original original quinoxaline-1,4- quinoxaline-1,4- di-di-NN-oxide-oxide derivatives derivatives variously variously substituted substituted at at the the C2 C2 position position were were synthesized synthesized and and eval- eval- uateduated [ 61[61].]. Of Of these, these, 17 17 showed showed signiﬁcant significant activity activity against againstMycobacterium Mycobacterium tuberculosis tuberculosis, with, with thethe MIC MIC ranging ranging from from 0.39 0.39 to 6.25to 6.25µg/mL µg/mL and and no no cytotoxic cytotoxic effects effects onVERO on VERO cells cells (Table (Table 11). Compounds11). Compounds bearing bearing a thioether a thioether linkage linkage and and a sterically a sterically bulky bulky aromatic aromatic group group at at the the terminalterminal side side chain chain on on the the C2 C2 position position are are the the best best representatives representatives of of these these original original series. series. Additionally, the inhibitory effect of methyl, ethyl, isopropyl, and n-propyl esters ofTable quinoxaline 11. Chemical 1,4-di- structureN-oxide of so onmeMycobacterium quinoxaline-1,4-di- tuberculosisN-oxide derivativeswas assessed variously [62 ].substituted Of the 18at originalthe C2 positionesters ofand the their 1,4-di- MICN values-oxide against synthesized, Mycobacterium eight derivatives tuberculosis. showed similar activity toCompound that of isoniazid taken as reference (TableStructure 12). MIC (µg/mL) The steric effect of the ester groupO at position 7 plays a crucial role in enhancing biological effects. The (CH3)2CH substituentO at position 7 enhances antimycobacterial activity. COOCH3 or COOCH2CH3 attachedN at positionS 2 on the quinoxaline ring and CF3 substituent at position 3 also result in increased antimycobacterial activity. 57 1.56 Inﬂuence of substitution at positions 2, 3, and 7 was also assessed in a new series of 22 new N-oxide-containing compoundsN leadingCH3 to the synthesis of nine quinoxaline 1,4-di-N-oxide derivatives [63] (Figure 14). O

Molecules 2021, 26, x FOR PEER REVIEW 17 of 23

O O O O N R7 N NH2 H NH N R7 N N + H N N R6 N CH3 O R6 N CH3 56 : Isoniazid O O

49: R6: H; R7: H 49a: R6: H; R7: H 50: R6: H; R7: Cl 50a: R6: H; R7: Cl 51: R6: H; R7: OCH3 51a: R6: H; R7: OCH3 52: R6: H; R7: CH3 52a: R6: H; R7: CH3 53: R6: CH3; R7: CH3 53a: R6: CH3; R7: CH3 54: R6: H; R7: F 54a: R6: H; R7: F 55: R : H; R : CF 55a: R : H; R : CF 6 7 3 6 7 3 Figure 13. Design of 1,4-di-N-oxide quinoxaline-2-ylmethylene isonicotinic acid hydrazide derivatives.

The quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives used as precursors for the synthesis of these new series were also evaluated against Mycobacterium tuberculosis. In these series, molecules substituted with an electron-withdrawing group at position 7 such as CF3, Cl, or F on the quinoxaline ring were the most active compounds, with the respective IC90 of 1.07, 1.25, and 4.65 µM. In order to explore the structural requirements for anti- TB activity, molecular modelling studies were performed on a series of 23 original quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives, with IC90 values ranging from 3.86 µg to 100 µg [60]. The reliable pharmacophore generated was composed of one aromatic ring, four hydrophobic substituents, three hydrogen acceptors, and one hydrogen donor. The binding mode of these derivatives was similar to that of coumarin in novobiocin, which is known to be an inhibitor of Mycobacterium tuberculosis DNA gyrase active site.

4.2.2. Other Structures In order to develop new antitubercular agents, a series of 33 original quinoxaline-1,4- di-N-oxide derivatives variously substituted at the C2 position were synthesized and evaluated [61]. Of these, 17 showed significant activity against Mycobacterium tuberculosis, with Molecules 2021, 26the, 4742 MIC ranging from 0.39 to 6.25 µg/mL and no cytotoxic effects on VERO cells (Table 18 of 23 11). Compounds bearing a thioether linkage and a sterically bulky aromatic group at the terminal side chain on the C2 position are the best representatives of these original series.

Table 11. Chemical structure of some quinoxaline-1,4-di-N-oxide derivatives variously substituted at the C2 position and Table 11. Chemical structure of some quinoxaline-1,4-di-N-oxide derivatives variously substituted their MICat values the C2 against positionMycobacterium and their MIC tuberculosis values against. Mycobacterium tuberculosis.

CompoundCompound Structure Structure MIC (µg/mL) MIC (µg/mL) O O N S 57 57 1.56 1.56 MoleculesMolecules 202120212021,,,, 262626,,,, xxxx FORFORFORFOR PEERPEERPEERPEER REVIEWREVIEWREVIEWREVIEW N CH3 181818 ofofof 232323 Molecules 2021, 26, x FOR PEER REVIEW 18 of 23

O OO OO OO OO N S CH N S CH333 N S CH3 58 5858 0.780.78 0.78 5858 0.780.78 N CH N CH333 N CH3 OO OO OO OO OO OO NN SS FF NN SS FF 59 5959 0.390.39 0.39 5959 0.390.39 N CH NN CHCH333 N CH3 OO OO OO OO OO OO N S OCH NN SS OCHOCH333 N S OCH3 60 6060 0.780.78 0.78 6060 0.780.78 N CH NN CHCH333 N CH3 OO OO OO OO OO NN O NN NHNH NN SS NH NN SS 6161 NN 0.390.39 61 6161 N 0.390.39 0.39 N CH N CH333 N CH3 OO OO OO OO OO SS O SS NN SS NN SS 6262 NN 1.561.56 62 6262 N 1.561.56 1.56 N CH NN CHCH333 N CH3 OO OO IsoniazidIsoniazid 0.2 0.2 IsoniazidIsoniazidIsoniazid 0.2 0.2 0.2 Additionally,Additionally, thethe inhibitoryinhibitory effecteffect ofof methyl,methyl, ethyl,ethyl, isopropyl,isopropyl, andand nn-propyl-propyl estersesters ofof Additionally,Additionally, thethe inhibitoryinhibitory effecteffect ofof methyl,methyl, ethyl,ethyl, isopropyl,isopropyl, andand nn-propyl-propyl estersesters ofof quinoxalinequinoxaline 1,4-di-1,4-di-NN-oxide-oxide onon MycobacteriumMycobacterium tuberculosistuberculosis waswaswas assessedassessedassessed [62].[62].[62]. OfOfOf thethethe 181818 orig-orig-orig- quinoxalinequinoxaline 1,4-di- 1,4-di-NN-oxide-oxide on on Mycobacterium Mycobacterium tuberculosis tuberculosis was was assessed assessed [62]. [62]. Of Of the the 18 18 orig- orig- inalinalinal estersestersesters ofofof thethethe 1,4-di-1,4-di-1,4-di-NN-oxide-oxide synthesized,synthesized, eighteight derivativderivativeses showedshowed similarsimilar activityactivity toto inalinal estersesters ofof thethe 1,4-di-1,4-di-NN-oxide-oxide synthesized,synthesized, eighteight derivativderivativeses showedshowed similarsimilar activityactivity toto thatthat ofof isoniazidisoniazid takentaken asas referencereference (Table(Table 12).12). thatthat ofof isoniazidisoniazid takentaken asas referencereference (Table(Table 12).12).

Molecules 2021, 26, 4742 19 of 23

Molecules 2021, 26, x FOR PEER REVIEW 19 of 23 Molecules Molecules 2021 2021, ,26 26, ,x x FOR FOR PEER PEER REVIEW REVIEW 1919 of of 23 23 Molecules 2021, 26, x FOR PEER REVIEW 19 of 23 Table 12.Molecules Chemical 2021,, 26 structure,, xx FORFOR PEERPEER REVIEWREVIEW on methyl, ethyl, isopropyl, and n-propyl esters of quinoxaline 1,4-di-19 Nof -oxide23 series and their

MIC values against Mycobacterium tuberculosis. Table 12. Chemical structure on methyl, ethyl, isopropyl, and n-propyl esters of quinoxaline 1,4-di- TableTable 12.12. ChemicalChemical structurestructure onon methyl,methyl, ethyl,ethyl, isopropyl,isopropyl, andand nn-propyl-propyl estersesters ofof quinoxalinequinoxaline 1,4-di-1,4-di- NTable-oxide 12. series Chemical and their structure MIC valueson methyl, against ethyl, Mycobacterium isopropyl, and tuberculosis. n-propyl esters of quinoxaline 1,4-di- NTableN-oxide-oxide 12. seriesseries Chemical andand theirtheirstructure MICMIC on valuesvalues methyl, againstagainst ethyl, MycobacteriumMycobacterium isopropyl, and tuberculosis.tuberculosis. n-propyl esters of quinoxaline 1,4-di- N-oxide series and their MIC values against Mycobacterium tuberculosis. Microplate Alamar Blue Assay CompoundN-oxide series and their MIC values Structure against Mycobacterium tuberculosis. MicroplateMicroplate AlamarAlamar blueblue µ CompoundCompound StructureStructure Microplate Alamar blue MIC ( g/mL) Compound Structure Microplateassayassay MICMIC Alamar (µg/mL)(µg/mL) blue Compound Structure assay MIC (µg/mL) OO assay MIC (µg/mL) O O O OO O OO O O O NN O HH3CC OO 63 H33C O N 0.14 63 6363 H3C O N 0.140.14 0.14 63 H3C O 0.14 63 N CF3 0.14 63 NN CFCF33 0.14 N CF 3 N CF3 OO O O OO O O O OO O OO O O O NN O SS HH3CC OO S 64 H33C O N S 0.10 64 6464 H 3C O N S 0.100.10 0.10 64 H3C O 0.10 64 N CF3 0.10 64 NN CFCF33 0.10 N CF 3 N CF3 OO O OOO O O O OO O OO O O HH3CC O NN O 33 OO 65 H3C O N 0.15 6565 H3C O N 0.150.15 65 65 O 0.15 0.15 65 N CF3 0.15 65 NN CFCF33 0.15 N CF 3 N CF3 OO O OO OOO CH3 O O O CHCH33 OO O OO CH 3 O N O CH3 O NN O SS HH3CC OO S 66 H33C O N S 0.08 6666 H3C O N S 0.080.08 66 H3C O 0.08 66 66 N CF3 0.08 0.08 66 NN CFCF33 0.08 N CF 3 N CF3 OO O OOO OO CH3 O O O CHCH33 OO O OO CH 3 O N O CH3 O NN O HH3CC OO CHCH3 67 H33C O N CH33 0.14 6767 H3C O N CH 3 0.140.14 H3C O CH3 6767 N CF3 0.140.14 67 67 NN CFCF33 0.14 0.14 N CF 3 N CF3 OO O O O OO O CH3 O O O CHCH33 OO O OO CH 3 O N O CH3 O NN O HH3CC OO 68 H33C O N 0.15 6868 H3C O N 0.150.15 68 H3C O 0.15 68 NN CFCF3 0.15 68 68 N CF33 0.15 0.15 N CF 3 N CF3 Molecules 2021, 26, x FOR PEER REVIEW OO 20 of 23 Molecules 2021, 26, x FOR PEER REVIEW O 20 of 23 O O O O CH3 O O CH3 O O N CH3 H3C O N CH3 69 69 H3C O 0.13 0.13 69 CH3 0.13 N CFCH3 3 N CF3

O O O O O O O O H3C N H3C O N OCH3 70 O OCH3 0.14 70 0.14 70 N CH3 0.14 N CH3 O O Isoniazid 0.12 Isoniazid 0.12 Isoniazid 0.12 The steric effect of the ester group at position 7 plays a crucial role in enhancing bio- The steric effect of the ester group at position 7 plays a crucial role in enhancing biological effects. The (CH3)2CH substituent at position 7 enhances antimycobacterial activ- logical effects. The (CH3)2CH substituent at position 7 enhances antimycobacterial activity. COOCH3 or COOCH2CH3 attached at position 2 on the quinoxaline ring and CF3 sub- ity. COOCH3 or COOCH2CH3 attached at position 2 on the quinoxaline ring and CF3 substituent at position 3 also result in increased antimycobacterial activity. stituent at position 3 also result in increased antimycobacterial activity. Influence of substitution at positions 2, 3, and 7 was also assessed in a new series of Influence of substitution at positions 2, 3, and 7 was also assessed in a new series of 22 new N-oxide-containing compounds leading to the synthesis of nine quinoxaline 1,4- 22 new N-oxide-containing compounds leading to the synthesis of nine quinoxaline 1,4- di-N-oxide derivatives [63] (Figure 14). di-N-oxide derivatives [63] (Figure 14). O O O O N CN O N CN R = H, CH Cl, F, O R = 1 3, R = ROCH1 = H, OCF CH3, Cl, F, OCH3, OCF3 N R R1 3, 3 N R R1 X X O O X = O,S X = O,S Figure 14. Design of the quinoxaline 1,4-di-N-oxide derivatives. Figure 14. Design of the quinoxaline 1,4-di-N-oxide derivatives.

For quinoxaline phenyl derivatives, MIC90 values ranged from 12 to 30.8 µM versus For quinoxaline phenyl derivatives, MIC90 values ranged from 12 to 30.8 µM versus 0.1 µM for the standard drugs isoniazid and rifampicin taken as reference. Antimycobac- 0.1 µM for the standard drugs isoniazid and rifampicin taken as reference. Antimycobac- terial activity was affected by the presence of a para substituent on the phenyl ring: absence terial activity was affected by the presence of a para substituent on the phenyl ring: absence of substitution led to the derivative with lowest activity while the derivatives substituted of substitution led to the derivative with lowest activity while the derivatives substituted by a methoxy group displayed the highest activity. However, no clear evidence of impact by a methoxy group displayed the highest activity. However, no clear evidence of impact from electron-withdrawing and electron-donating groups was observed. In addition, iso- from electron-withdrawing and electron-donating groups was observed. In addition, isosteric substitution of the phenyl ring by furan led to a more active compound, with MIC90 steric substitution of the phenyl ring by furan led to a more active compound, with MIC90 of 5.2 µM. of 5.2 µM. 5. Conclusions 5. Conclusions This review highlights the growing interest in the development of compounds bear- This review highlights the growing interest in the development of compounds bearing a quinoxaline moiety for antimycobacterial treatment, some of these compounds having a quinoxaline moiety for antimycobacterial treatment, some of these compounds having reached the preclinical evaluation phase. From the published studies, both quinoxa- ing reached the preclinical evaluation phase. From the published studies, both quinoxaline and quinoxaline-1,4-di-N-oxides with a variety of substituents in positions 2, 3, 6, and line and quinoxaline-1,4-di-N-oxides with a variety of substituents in positions 2, 3, 6, and 7 showed anti-TB activity. From these data, it appears that the quinoxaline moiety repre- 7 showed anti-TB activity. From these data, it appears that the quinoxaline moiety represents an interesting scaffold against Mycobacterium tuberculosis. Some structure–activity sents an interesting scaffold against Mycobacterium tuberculosis. Some structure–activity relationships can be proposed for further investigations on this scaffold. Firstly, it appears relationships can be proposed for further investigations on this scaffold. Firstly, it appears that quinoxaline derivatives with the most potent antimycobacterial activity are unsubsti- that quinoxaline derivatives with the most potent antimycobacterial activity are unsubstituted at positions 1, 4, 5, and 8, although the presence of the pyrrolo substituent at position tuted at positions 1, 4, 5, and 8, although the presence of the pyrrolo substituent at position

Molecules 2021, 26, x FOR PEER REVIEW 20 of 23

O CH3 O O N CH3 H3C O 69 0.13 CH3 N CF3

O O O O

H3C N O OCH3 70 0.14 N CH3

O Isoniazid 0.12

The steric effect of the ester group at position 7 plays a crucial role in enhancing biological effects. The (CH3)2CH substituent at position 7 enhances antimycobacterial activity. COOCH3 or COOCH2CH3 attached at position 2 on the quinoxaline ring and CF3 substituent at position 3 also result in increased antimycobacterial activity.

Molecules 2021, 26, 4742 Influence of substitution at positions 2, 3, and 7 was also assessed in a new series20 of of 23 22 new N-oxide-containing compounds leading to the synthesis of nine quinoxaline 1,4- di-N-oxide derivatives [63] (Figure 14).

O O N CN O R = R1 = H, CH3, Cl, F, OCH3, OCF3 N R R1 X O X = O,S Figure 14. Design of the quinoxaline 1,4-di- N-oxide derivatives.

For quinoxaline phenyl derivatives, MIC90 valuesvalues ranged ranged from 12 to 30.8 µµMM versus 0.1 µMµM for the standard drugsdrugs isoniazidisoniazid andand rifampicinrifampicin taken taken as as reference. reference. Antimycobacte- Antimycobac- terialrial activity activity was was affected affected by by the the presence presence of of a para a parasubstituent substituent on on the the phenyl phenyl ring: ring: absence absence of ofsubstitution substitution led led to theto the derivative derivative with with lowest lowest activity activity while while the derivativesthe derivatives substituted substituted by a bymethoxy a methoxy group group displayed displayed the highest the highest activity. activity. However, However, no clear no evidenceclear evidence of impact of impact from fromelectron-withdrawing electron-withdrawing and electron-donating and electron-donat groupsing groups was observed.was observed. In addition, In addition, isosteric iso- stericsubstitution substitution of the of phenyl the phenyl ring byring furan by fu ledran to led a to more a more active active compound, compound, with with MIC MIC90 of90 of5.2 5.2µM. µM. 5. Conclusions 5. Conclusions This review highlights the growing interest in the development of compounds bearing This review highlights the growing interest in the development of compounds bear- a quinoxaline moiety for antimycobacterial treatment, some of these compounds having ing a quinoxaline moiety for antimycobacterial treatment, some of these compounds hav- reached the preclinical evaluation phase. From the published studies, both quinoxaline and ing reached the preclinical evaluation phase. From the published studies, both quinoxa- quinoxaline-1,4-di-N-oxides with a variety of substituents in positions 2, 3, 6, and 7 showed line and quinoxaline-1,4-di-N-oxides with a variety of substituents in positions 2, 3, 6, and anti-TB activity. From these data, it appears that the quinoxaline moiety represents an 7 showed anti-TB activity. From these data, it appears that the quinoxaline moiety repre- interesting scaffold against Mycobacterium tuberculosis. Some structure–activity relation- sents an interesting scaffold against Mycobacterium tuberculosis. Some structure–activity ships can be proposed for further investigations on this scaffold. Firstly, it appears that relationships can be proposed for further investigations on this scaffold. Firstly, it appears quinoxaline derivatives with the most potent antimycobacterial activity are unsubstituted thatat positions quinoxaline 1, 4,5, derivatives and 8, although with the the most presence potent of theantimycobacterial pyrrolo substituent activity at position are unsubsti- 1 does tutednot result at positions in a loss 1, of 4, biological 5, and 8, although activity. Substituents the presence in of positions the pyrrolo 2 and substituent 3 appear at to position play an important role in anti-TB activity as the presence of alkynyl derivatives, azide, hydrazone or acylhydrazone, chalcone, azetidinone, thiazolidinone, carboxylic acid substituent leads to significant activity. Various substituents such as thioether linkage, ester, carboxamide, chalcone, ketone, thiazolidinone, or hydrazide at position 2 lead to derivatives with potent antimycobacterial activity in quinoxaline 1,4-di-N-oxide series. The presence of a trifluoromethyl or methoxy group at position 3 of the quinoxaline moiety increases the activity. Substitution at positions 6 and 7 is not crucial, but derivatives substituted with ester, halogen, nitro, trifluoromethyl group, chalcone, or heterocycle at these positions exhibited excellent antimycobacterial activity. Hybridization of different pharmacophoric moieties with a quinoxaline nucleus might help to develop an effective molecule for TB treatment. These results warrant further investigations, which may allow identification of novel antitubercular candidates based on this scaffold.

Author Contributions: Conceptualization and methodology, M.M.; formal analysis, M.M. and V.M.; data curation, V.M. and M.M.; writing—original draft preparation, M.M. and V.M.; writing—review and editing, O.K. and P.V.; supervision, P.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2021, 26, 4742 21 of 23

References 1. WHO. Global Tuberculosis Report 2020. Available online: https://www.who.int/publications/i/item/9789240013131 (accessed on 16 April 2021). 2. Keri, R.S.; Pandule, S.S.; Budagumpi, S.; Nagaraja, B.M. Quinoxaline and quinoxaline-1,4-di-N-oxides: An emerging class of antimycobacterials. Arch. Pharm. (Weinh.) 2018, 351, e1700325. [CrossRef] 3. Barthod, L.; Lopez, J.G.; Curti, C.; Bornet, C.; Roche, M.; Montana, M.; Vanelle, P. News on therapeutic management of MDR-tuberculosis: A literature review. J. Chemother. 2018, 30, 1–15. [CrossRef] 4. Shagufta; Ahmad, I. An insight into the therapeutic potential of quinazoline derivatives as anticancer agents. MedChemCom 2017, 8, 871–885. [CrossRef] 5. Kalaria, P.N.; Karad, S.C.; Raval, D.K. A review on diverse heterocyclic compounds as the privileged scaffolds in antimalarial drug discovery. Eur. J. Med. Chem. 2018, 158, 917–936. [CrossRef] 6. Kerru, N.; Gummidi, L.; Maddila, S.; Gangu, K.K.; Jonnalagadda, S.B. A Review on Recent Advances in Nitrogen-Containing Molecules and Their Biological Applications. Molecules 2020, 25, 1909. [CrossRef][PubMed] 7. Heravi, M.M.; Zadsirjan, V. Prescribed drugs containing nitrogen heterocycles: An overview. RCS Adv. 2020, 10, 44247–44311. 8. Deepika, Y.; Nath, P.S.; Sachin, K.; Shewta, S. Biological activity of quinoxaline derivatives. Int. J. Curr. Pharm. Res. 2011, 2, 33–46. 9. Montana, M.; Montero, V.; Khoumeri, O.; Vanelle, P. Quinoxaline Derivatives as Antiviral Agents: A Systematic Review. Molecules 2020, 25, 2784. [CrossRef][PubMed] 10. 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] 11. Barea, C.; Pabón, A.; Pérez-Silanes, S.; Galiano, S.; Gonzalez, G.; Monge, A.; Deharo, E.; Aldana, I. New Amide Derivatives of Quinoxaline 1,4-di-N-Oxide with Leishmanicidal and Antiplasmodial Activities. Molecules 2013, 18, 4718–4727. [CrossRef] 12. González, M.; Cerecetto, H. Quinoxaline derivatives: A patent review (2006–present). Expert Opin. Ther. Pat. 2012, 22, 1289–1302. [CrossRef][PubMed] 13. Zanetti, S.; Sechi, L.A.; Molicotti, P.; Cannas, S.; Bua, A.; Deriu, A.; Carta, A.; Paglietti, G. In vitro activity of new quinoxalin 1,4-dioxide derivatives against strains of Mycobacterium tuberculosis and other mycobacteria. Int. J. Antimicrob. Agents 2005, 25, 179–181. [CrossRef] 14. Ortega, M.A.; Montoya, M.E.; Jaso, A.; Zarranz, B.; Tirapu, I.; Aldana, I.; Monge, A. Antimycobacterial activity of new quinoxaline- 2-carbonitrile and quinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives. Pharmazie 2001, 56, 205–207. [CrossRef] 15. Villar, R.; Vicente, E.; Solano, B.; Perez-Silanes, S.; Aldana, I.; Maddry, J.A.; Lenaerts, A.J.; Franzblau, S.G.; Cho, S.H.; Monge, A.; et al. In vitro and in vivo antimycobacterial activities of ketone and amide derivatives of quinoxaline 1,4-di-N-oxide. J. Antimicrob. Chemother. 2008, 62, 547–554. [CrossRef][PubMed] 16. Crawford, P.W.; Scamehorn, R.G.; Hollstein, U.; Ryan, M.D.; Kovacic, P. Cyclic voltammetry of phenazines and quinoxalines including mono- and di-N-oxides. Relation to structure and antimicrobial activity. Chem. Biol. Interact. 1986, 60, 67. [CrossRef] 17. Chowdhury, G.; Kotandeniya, D.; Daniels, J.S.; Barnes, C.L.; Gates, K.S. Enzyme-activated, hypoxia-selective DNA damage by 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide. Chem. Res. Toxicol. 2004, 17, 1399–1405. [CrossRef][PubMed] 18. Moher, D.; Liberati, A.; Tetzlaff, J.; Altman, D.G. The PRISMA Group Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med. 2009, 3, 123–130. 19. 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. 20. 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] 21. Brown, D.J.; Ellman, J.A. The Chemistry of Heterocyclic Compounds; Wiley: New York, NY, USA, 2004. 22. 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] 23. 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] 24. 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] 25. Kumar, K.; Mudshinge, S.R.; Goyal, S.; Gangar, M.; Nair, V.A. A catalyst free, one pot approach for the synthesis of quinoxaline derivatives via oxidative cyclisation of 1,2-diamines and phenacyl bromides. Tetrahedron Lett. 2015, 56, 1266–1271. [CrossRef] 26. 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] 27. Rostamizadeh, S.; Jafari, S. The synthesis of quinoxalines under microwave irradiation. Indian J. Heterocycl. Chem. 2001, 10, 303–304. 28. Shaabani, A.; Maleki, A. Green and efﬁcient synthesis of quinoxaline derivatives via ceric ammonium nitrate promoted and in situ aerobic oxidation of alpha-hydroxy ketones and alpha-keto oximes in aqueous media. Chem. Pharm. Bull. 2008, 56, 79–81. [CrossRef] Molecules 2021, 26, 4742 22 of 23

29. 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] 30. Kumar, S.; Kumar, N.; Drabu, S. Synthesis of benzo[g]quinoxaline-5,10-dione based pyridine derivatives and their antimycobacterial activity. Orient. J. Chem. 2017, 33, 821–828. [CrossRef] 31. Kumar, S.; Kumar, N.; Drabu, S. Synthesis of benzo[g]quinoxaline-5,10-dione based pyrazoline derivatives and their antimycobacterial activity. Int. J. Pharm. Sci. Res. 2018, 9, 498–508. 32. Kumar, S.; Kumar, N.; Drabu, S. Synthesis and in vitro antimycobacterial activity of 7-[3-(substituted) phenylprop-2-enoyl]-2,3- diphenyl-5H,10H-benzo[g]quinoxaline-5,10-diones. Indian J. Heterocycl. Chem. 2017, 27, 25–31. 33. Guillon, J.; Reynolds, R.C.; Leger, J.M.; Guie, M.A.; Massip, S.; Dallemagne, P.; Jarry, C. Synthesis and preliminary in vitro evaluation of antimycobacterial activity of new pyrrolo[1,2-a]quinoxaline-carboxylic acid hydrazide derivatives. J. Enzym. Inhib. Med. Chem. 2004, 19, 489–495. [CrossRef] 34. Wang, T.; Tang, Y.; Yang, Y.; An, Q.; Sang, Z.; Yang, T.; Liu, P.; Zhang, T.; Deng, Y.; Luo, Y. Discovery of novel tuberculosis agents with pyrrolo[1,2-a]quinoxaline based scaffold. Bioorg. Med. Chem. Lett. 2018, 28, 2084–2090. [CrossRef] 35. Makane, V.B.; Vamshi Krishna, E.; Karale, U.B.; Babar, D.A.; Kalari, S.; Rekha, E.M.; Shukla, M.; Kaul, G.; Sriram, D.; Chopra, S.; et al. Synthesis of novel 4,5-dihydropyrrolo[1,2-a]quinoxalines, pyrrolo[1,2-a]quinoxalin]-2-ones and their antituberculosis and anticancer activity. Arch. Pharm. (Weinh.) 2020, 353, e2000192. [CrossRef][PubMed] 36. Vijaykumar, M.M.; Nagaraja, T.S.; Shameer, H.; Jayachandran, E.; Sreenivasa, G.M. N-substituted-3-chloro-2-azetidinones: Synthesis and characterization of new novel anti-inflammatory agents. J. Pharm. Sci. Res. 2009, 1, 83–92. 37. Sandip, J.; Mahesh, P.; Veeresh, M.; Pradeepkumar, R.; Shivalingarao, M.; Satyanarayana, D.; Mangala, G. Synthesis and antimycobacterial activity of a novel series of isonicotinyl hydrazide derivatives. Arch. Pharm. Chem. Life Sci. 2009, 342, 723–731. 38. Amit, V.; Shailendra, K.S. 4-Thiazolidinone—A biologically active scaffold. Eur. J. Med. Chem. 2008, 43, 897–905. 39. Puratchikody, A.; Natarajan, R.; Jayapal, M.; Doble, M. Synthesis, in vitro antitubercular activity and 3D-QSAR of novel quinoxaline derivatives. Chem. Biol. Drug Des. 2011, 78, 988–998. [CrossRef][PubMed] 40. Nogueira, T.C.M.; dos Santos Cruz, L.; Lourenço, M.C.; de Souza, M.V.N. Design, synthesis and anti-tuberculosis activity of hydrazones and n-acylhydrazones containing vitamin B6 and different heteroaromatic nucleus. Lett. Drug Des. Discov. 2019, 16, 792–798. [CrossRef] 41. Calvaresi, E.C.; Hergenrother, P.J. Glucose conjugation for the specific targeting and treatment of cancer. Chem. Sci. 2013, 4, 2319–2333. [CrossRef] 42. Xavier, N.M.; Goulart, M.; Neves, A.; Justino, J.; Chambert, S.; Rauter, A.P.; Queneau, Y. Synthesis of sugars embodying conjugated carbonyl systems and related triazole derivatives from carboxymethyl glycoside lactones. Evaluation of their antimicrobial activity and toxicity. Bioorg. Chem. 2011, 19, 926–938. [CrossRef] 43. Peraman, R.; Kuppusamy, R.; Killi, S.K.; Reddy, Y.P. New Conjugates of Quinoxaline as Potent Antitubercular and Antibacterial Agents. Int. J. Med. Chem. 2016, 2016, 6471352. [CrossRef][PubMed] 44. Gomes, M.N.; Braga, R.C.; Grzelak, E.M.; Neves, B.J.; Muratov, E.N.; Ma, R.; Klein, L.L.; Cho, S.; Oliveira, G.R.; Franzblau, S.G.; et al. QSAR-driven design, synthesis and discovery of potent and selective chalcone derivatives with antitubercular activity. Eur. J. Med. Chem. 2017, 137, 126–138. [CrossRef][PubMed] 45. Lin, Y.M.; Zhou, Y.; Flavin, M.; Zhou, L.M.; Nie, W.; Chen, F.C. Chalcones and flavonoids as anti-tuberculosis agents. Bioorg. Med. Chem. 2002, 10, 2795–2802. [CrossRef] 46. Desai, V.G.; Desai, S.R.; Gaonkar, S.N.; Palyekar, U.; Joshi, S.; Dixit, S. Novel quinoxalinyl chalcone hybrid scaffolds as enoyl ACP reductase inhibitors: Synthesis, molecular docking and biological evaluation. Bioorg. Med. Chem. Lett. 2017, 27, 2174–2180. [CrossRef][PubMed] 47. Muradás, T.C.; Abbadi, B.L.; Villela, A.D.; Macchi, F.S.; Bergo, P.F.; de Freitas, T.F.; Sperotto, N.; Timmers, L.; Norberto de Souza, O.; Picada, J.N.; et al. Pre-clinical evaluation of quinoxaline-derived chalcones in tuberculosis. PLoS ONE 2018, 13, e0202568. 48. Peraman, R.; Varma, R.V.; Reddy, Y.P. Re-engineering nalidixic acid’s chemical scaffold: A step towards the development of novel anti-tubercular and anti-bacterial leads for resistant pathogens. Bioorg. Med. Chem. Lett. 2015, 25, 4314–4319. [CrossRef][PubMed] 49. Achutha, L.; Parameshwar, R.; Madhava Reddy, B.; Harinadha Babu, V. Microwave-Assisted Synthesis of Some Quinoxaline- Incorporated Schiff Bases and Their Biological Evaluation. J. Chem. 2013, 2013, 578438. [CrossRef] 50. Kumar, R.R.; Perumal, S.; Senthilkumar, P.; Yogeeswari, P.; Sriram, D. Discovery of antimycobacterial spiro-piperidin-4-ones: An atom economic, stereoselective synthesis, and biological intervention. J. Med. Chem. 2008, 51, 5731–5735. [CrossRef] 51. Prasanna, P.; Balamurugan, K.; Perumal, S.; Yogeeswari, P.; Sriram, D. A regio- and stereoselective 1,3-dipolar cycloaddition for the synthesis of novel spiro-pyrrolothiazolyloxindoles and their antitubercular evaluation. Eur. J. Med. Chem. 2010, 45, 5653–5661. [CrossRef] 52. Arumugam, N.; Almansour, A.I.; Suresh Kumar, R.; Ibrahim Alaqeel, S.; Siva Krishna, V.; Sriram, D. Anti-tubercular activity of novel class of spiropyrrolidine tethered indenoquinoxaline heterocyclic hybrids. Bioorg. Chem. 2020, 99, 103799. [CrossRef] 53. Raphoko, L.A.; Lekgau, K.; Lebepe, C.M.; Leboho, T.C.; Matsebatlela, T.M.; Nxumalo, W. Synthesis of novel quinoxaline-alkynyl derivatives and their anti-Mycobacterium tuberculosis activity. Bioorg. Med. Chem. Lett. 2021, 35, 127784. [CrossRef] 54. Neres, J.; Hartkoorn, R.C.; Chiarelli, L.R.; Gadupudi, R.; Pasca, M.R.; Mori, G.; Venturelli, A.; Savina, S.; Makarov, V.; Kolly, G.S.; et al. 2-Carboxyquinoxalines kill mycobacterium tuberculosis through noncovalent inhibition of DprE1. ACS Chem. Biol. 2015, 10, 705–714. [CrossRef] Molecules 2021, 26, 4742 23 of 23

55. Xu, Y.; Wu, F.; Yao, Z.; Zhang, M.; Jiang, S. Synthesis of quinoxaline 1,4-di-N-oxide analogues and crystal structure of 2- carbomethoxy-3-hydroxyquinoxaline-di-N-oxide. Molecules 2011, 16, 6894–6901. [CrossRef] 56. Srinivasarao, S.; Nandikolla, A.; Suresh, A.; Ewa, A.K.; Głogowska, A.; Ghosh, B.; Kumar, B.K.; Murugesan, S.; Pulya, S.; Aggarwal, H.; et al. Discovery of 1,2,3-triazole based quinoxaline-1,4-di-N-oxide derivatives as potential anti-tubercular agents. Bioorg. Med. Chem. 2020, 100, 103955. [CrossRef] 57. Santivañez-Veliz, M.; Pérez-Silanes, S.; Torres, E.; Moreno-Viguri, E. Design and synthesis of novel quinoxaline derivatives as potential candidates for treatment of multidrug-resistant and latent tuberculosis. Bioorg. Med. Chem. Lett. 2016, 26, 2188–2193. [CrossRef] 58. Zhang, H.; Zhang, J.; Qu, W.; Xie, S.; Huang, L.; Chen, D.; Tao, Y.; Liu, Z.; Pan, Y.; Yuan, Z. Design, Synthesis, and Biological Evaluation of Novel Thiazolidinone-Containing Quinoxaline-1,4-di-N-oxides as Antimycobacterial and Antifungal Agents. Front. Chem. 2020, 8, 598. [CrossRef] 59. Torres, E.; Moreno, E.; Ancizu, S.; Barea, C.; Galiano, S.; Aldana, I.; Monge, A.; Pérez-Silanes, S. New 1,4-di-N-oxide-quinoxaline- 2-ylmethylene isonicotinic acid hydrazide derivatives as anti-Mycobacterium tuberculosis agents. Bioorg. Med. Chem. Lett. 2011, 21, 3699–3703. [CrossRef] 60. Radwan, A.A.; Abdel-Mageed, W.M. In Silico Studies of Quinoxaline-2-Carboxamide 1,4-di-N-Oxide Derivatives as Antimy- cobacterial Agents. Molecules 2014, 19, 2247–2260. [CrossRef] 61. Pan, Y.; Li, P.; Xie, S.; Tao, Y.; Chen, D.; Dai, M.; Hao, H.; Huang, L.; Wang, Y.; Wang, L.; et al. Synthesis, 3D-QSAR analysis and biological evaluation of quinoxaline 1,4-di-N-oxide derivatives as antituberculosis agents. Bioorg. Med. Chem. Lett. 2016, 26, 4146–4153. [CrossRef] 62. Palos, I.; Luna-Herrera, J.; Lara-Ramírez, E.E.; Loera-Piedra, A.; Fernández-Ramírez, E.; Aguilera-Arreola, M.G.; Paz-González, A.D.; Monge, A.; Wan, B.; Franzblau, S.; et al. Anti-Mycobacterium tuberculosis Activity of Esters of Quinoxaline 1,4-Di-N-Oxide. Molecules 2018, 23, 1453. [CrossRef] 63. Dos Santos Fernandes, G.F.; de Souza, P.C.; Moreno-Viguri, E.; Santivañez-Veliz, M.; Paucar, R.; Pérez-Silanes, S.; Chegaev, K.; Guglielmo, S.; Lazzarato, L.; Fruttero, R.; et al. Design, Synthesis, and Characterization of N-Oxide-Containing Heterocycles with in Vivo Sterilizing Antitubercular Activity. J. Med. Chem. 2017, 60, 8647–8660. [CrossRef]