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Review Recent Advances in the Discovery of Novel Agents

Seong-Min Lee †, Min-Sun Kim †, Faisal Hayat and Dongyun Shin * College of Pharmacy, Gachon University, 191 Hambakmoe-ro, Yeonsu-gu, Incheon 21936, Korea; [email protected] (S.-M.L.); [email protected] (M.-S.K.); [email protected] (F.H.) * Correspondence: [email protected] Authors contributed equally to this work. †  Received: 3 October 2019; Accepted: 23 October 2019; Published: 28 October 2019 

Abstract: Parasitic diseases have serious health, social, and economic impacts, especially in the tropical regions of the world. Diseases caused by protozoan parasites are responsible for considerable mortality and morbidity, affecting more than 500 million people worldwide. Globally, the burden of protozoan diseases is increasing and is been exacerbated because of a lack of effective due to the drug resistance and toxicity of current antiprotozoal agents. These limitations have prompted many researchers to search for new drugs against protozoan parasites. In this review, we have compiled the latest information (2012–2017) on the structures and pharmacological activities of newly developed organic compounds against five major protozoan diseases, , , malaria, , and , with the aim of showing recent advances in the discovery of new antiprotozoal drugs.

Keywords: protozoan diseases; parasitic; giardiasis; leishmaniasis; malaria; trichomoniasis; trypanosomiasis

1. Introduction It is well known that parasitic diseases are a serious health problem, that has a deep impact on the global human population [1]. Among parasites, protozoan parasites, such as , , Plasmodium falciparum, Giardia intestinalis, and , are the major disease-causing organisms. They are responsible for spreading worldwide, especially in undeveloped countries, where a tropical or temperate climate and poor sanitary and hygiene conditions are common [2–4]. Protozoan parasites are single-celled eukaryotes characterized as a diverse polyphyletic group. The infections caused by these parasites are responsible for 500 million deaths worldwide [5–8]. These infections are considered neglected because relatively little attention has been devoted to their surveillance, prevention, and treatment [9]. According to the global burden of disease analysis report by the World Health Organization in 2008, around 17% of deaths worldwide are caused by neglected tropical diseases [10]. These neglected protozoan diseases are a group of tropical infections that are particularly dominant in low-income majority populations, and affect millions of people and animals [6,11]. The major neglected protozoan diseases are , leishmaniasis, trichomoniasis, amebiasis, and giardiasis [9,12,13]. The modes of transmission of these protozoan parasites differ from each other. Some of them are transmitted by insects that are vectors of Plasmodium species (malaria), T. brucei (human , HAT), T. cruzi (Chagas disease), and Leishmania species (leishmaniasis). In addition, E. histolytica (amebiasis), Cryptosporidium parvum (), Cyclospora cayetanensis (cyclosporiasis), and Giardia lamblia (giardiasis) are transmitted through food and water contaminated with fecal matter [12]. The biochemistry of a large number of protozoan parasites has been studied

Molecules 2019, 24, 3886; doi:10.3390/molecules24213886 www.mdpi.com/journal/molecules Molecules 2019, 24, 3886 2 of 45 during previous studies. According to the mode of of the parasites, many organic molecules have been manufactured in the last 50 years for the development of new antiprotozoal agents. Some of these agents, e.g., and (for amebiasis, trichomoniasis, and giardiasis), (for leishmaniasis), (for malaria and trichomoniasis), and (for HAT), and (for American trypanosomiasis) are very effective and are currently used in medical practice. However, the available drugs are not the final solution because of their existing resistance and toxicity [14–30]. The above-mentioned shortcomings promote the ongoing drug research effort to identify mechanistically novel, nontoxic, and cost-effective chemotherapies for the treatment of these neglected protozoan problems [31]. As a continuation of our studies on antiprotozoal drug development, we present the latest information with respect to recent drug developments against five major protozoan diseases, giardiasis, leishmaniasis, malaria, trichomoniasis, and trypanosomiasis in the form of a review. Notably, during the literature review, we found many articles on antiprotozoal drug discovery published between 2012 and 2017 [32–47]. Therefore, keeping their findings in mind, we wrote this review in a different style; we have described the five major protozoan diseases and compiled their latest synthetic and pharmacological data individually from 2012 to 2017 in Tables1–5. This tabular form of data has not been reported in previously published reviews.

2. Recent Progress of Antiprotozoan Agents

2.1. Anti-Giardiasis Giardiasis is caused by the protozoan parasite G. intestinalis (also known as G. lamblia or G. duodenalis). It is also known by the common name “beaver fever,” because this infection was reported in campers who drank contaminated water that was inhabited by beavers. Giardiasis is the most common in human beings, and it occurs in both developing and industrialized countries [48]. Its global incidence is believed to range between 20%–60% [49], with 2%–7% incidence in industrialized nations [50]. Giardia intestinalis was first described in 1681 after the Dutch microscopist Antonie van Leeuwenhoek observed the protozoan in his own diarrheic stools. This disease is often prevalent in poor countries and communities that have untreated water, inadequate sanitation, and poor dietary status [51]. The infection is caused by fecal–oral transmission and initiated by the ingestion of infectious cysts from contaminated water or through person-to-person contact. After excystation, flagellated trophozoites colonize the upper small intestine, where they attach to the epithelial lining but do not invade the mucosa. The duration of Giardia infection is variable; however, chronic infection and reinfection commonly occur [52]. Approximately 50% of the symptoms classically associated with giardiasis are asymptomatic including diarrhea, abdominal pain, nausea, vomiting, and anorexia. However, infected individuals can also develop extraintestinal and postinfectious complications [53,54]. Chronic extraintestinal sequelae may affect the joints, skin, eyes, and central nervous system, but the underlying mechanisms are unknown [53,54]. According to research data, Giardia has eight distinct genetic assemblages labelled as assemblage “A” through “H” [55,56], and assemblages “A” and “B” are responsible for infection in humans. Three classes of drugs are currently used for the treatment of giardiasis: metronidazole, analogs, and nitrofurans, such as (Figure1). Metronidazole is the most widely used drug for the treatment of giardiasis globally and it is generally effective and well-tolerated. However, the United States Food and Drug Administration (FDA) has not yet approved this drug for the treatment of Giardia infection because of its toxicity and major side effects, such as seizures, ataxia, peripheral neuropathy, transient myopia, gastric mucosal irritation, sperm damage, and hematuria [14–18]. Tinidazole is an N1-position modified 5-nitro imidazole, which has been approved by the FDA for the treatment of giardiasis. (NTZ) belongs to an emerging class of 5-nitrothiazole compounds with potential antigiardial activity [57]. However, although NTZ is generally well tolerated, some adverse effects such as abdominal pain, diarrhea, and nausea limit the safe use for human beings. Molecules 2019, 24, 3886 3 of 45 Molecules 2019, 24, x FOR PEER REVIEW 3 of 45 Molecules 2019, 24, x FOR PEER REVIEW 3 of 45 Molecules 2019, 24, x FOR PEER REVIEW 3 of 45 Molecules 2019, 24, x FOR PEER REVIEW 3 of 45 MepacrinesideMolecules effects, 2019 is, no24such, longerx FOR as PEER gastrointe available REVIEW instinal the Uniteddisturbances, States, and hemolytic it is being anemia, replaced disulfiram-like in most of its applicationsreactions3 of to45 side effects, such as gastrointestinal disturbances, hemolytic anemia, disulfiram-like reactions to withalcohol,side safer effects, and and suchhypersensitivity more as specific gastrointe drugs. reactions,stinal Furazolidone disturbances, as well also as hashemolyticevidence serious ofanemia, side tumorigenicity effects, disulfiram-like such asin gastrointestinalrodent reactions studies. to alcohol,side effects, and suchhypersensitivity as gastrointe reactions,stinal disturbances, as well as hemolyticevidence ofanemia, tumorigenicity disulfiram-like in rodent reactions studies. to disturbances,Furthermore,sidealcohol, effects, and hemolytic such hypersensitivityG. lamblia as gastrointe anemia, resistance disulfiram-likereactions,stinal to thisdisturbances, asdrug well reactions has as alsohemolyticevidence to been alcohol, reportedofanemia, tumorigenicity and hypersensitivity [58–60].disulfiram-like Therefore, in rodent reactions,reactions research studies. asto Furthermore,alcohol, and hypersensitivityG. lamblia resistance reactions, to this as drug well has as alsoevidence been reportedof tumorigenicity [58–60]. Therefore, in rodent researchstudies. wellfocusingalcohol,Furthermore, as evidence andon the hypersensitivityG. of developmentlamblia tumorigenicity resistance reactions,of novel, in to rodent this alternativeas drug studies.well has as drugs Furthermore,alsoevidence been for ofreportedthe tumorigenicity G.treatment lamblia [58–60]. resistanceof giardiasis Therefore,in rodent to this is researchstudies. highly drug Furthermore,focusing on the G. developmentlamblia resistance of novel, to this alternative drug has alsodrugs been for reportedthe treatment [58–60]. of giardiasisTherefore, isresearch highly hasdesirable.Furthermore,focusing also been onIn reportedtheview G. lambliadevelopment of these [58 resistance–60 considerations,]. Therefore,of novel, to this researchalternative drug rese archershas focusing alsodrugs have been onfor designed the reportedthe development treatment and [58–60]. synthesized of of Therefore,giardiasis novel, some alternative isresearch highlynovel desirable.focusing on In theview development of these considerations, of novel, alternative researchers drugs have for designed the treatment and synthesized of giardiasis some is highly novel drugsmoleculesfocusingdesirable. for theon forIn treatment the viewthe development treatment of these of giardiasis considerations,of giardiasis,of novel, is highly alternativetheir rese desirable. resultsarchers drugs are Inhave summarized view for designed the of thesetreatment inand considerations, Table synthesized of 1. giardiasis researcherssome is highly novel moleculesdesirable. Infor view the treatment of these considerations,of giardiasis, their rese resultsarchers are have summarized designed inand Table synthesized 1. some novel desirable.molecules Infor view the treatment of these considerations, of giardiasis, their rese resultsarchers are have summarized designed inand Table synthesized 1. some novel havemolecules designed for andthe treatment synthesized of giardiasis, some novel their molecules resultsfor are the summarized treatment ofin giardiasis,Table 1. their results are molecules for the treatmentTable of giardiasis, 1. Selected theirdata of results reported are antigiardial summarized agents. in Table 1. summarized in Table1. Table 1. Selected data of reported antigiardial agents. Table 1. Selected data of reported antigiardial agents. CompoundTable 1. Selected data of reported antigiardial agents.Activity Ref. CompoundTableTable 1. 1.Selected Selected data data of of reported reported antigiardial antigiardial agents. agents.Activity Ref. Compound ActivityG. lamblia~~~ Ref. Compound ActivityG. lamblia~~~ Ref. CompoundCompound ActivityActivity[ICG. 50 lamblia (µg/mL)]~~~ Ref.Ref. [ICG. 50lamblia (µg/mL)]~~~ 7 G.[ICG. lamblia4.62 lamblia50 (µg/mL)] ± 0.12~~~ 7 [IC4.6250 (µg/mL)] ± 0.12 7 [IC [IC(4.62µ50g /(µg/mL)]mL)] ± 0.12 [61] 7 50 4.62 ± 0.12 [61] 7 7 4.624.620.12 ± 0.12 [61] MTZ 0.86± ± 0.03 [61][61] MTZ 0.86 ± 0.03 [61] MTZ 0.86 ± 0.03 MTZMTZ 0.86 0.860.03 ± 0.03 MTZ 0.86± ± 0.03 G. lamblia~~~ G. lamblia~~~ [ICG.50 lamblia (µg/mL)]~~~ G.[ICG. lamblia 50lamblia (µg/mL)]~~~ 8 [ICG. lamblia50 84.2(µg/mL)] ~~~ 8 [IC50[IC(µ50g /(µg/mL)]84.2mL)] 8 [IC50 (µg/mL)]84.2 8 8 84.284.2 [62] 8 84.2 [62] [62] MTZ 1.22 [62][62] MTZ 1.22 [62] MTZ 1.22 MTZMTZ 1.221.22 MTZ 1.22

G. intestinalis~~~ G. intestinalis~~~ G. [ICintestinalis50 (µM)]~~~ G.G. intestinalis [ICintestinalis50 (µM)]~~~ 9a [ICG. intestinalis[IC(µ0.12250M)] (µM)] ~~~ 9a 50[IC0.12250 (µM)] [63] 9b9a [IC0.151500.122 (µM)] [63] 9a9b9a 0.1220.1220.151 [63] [63] Aminitrozole9b9a9b 0.1510.4900.1220.151 [63] Aminitrozole9b 0.1510.490 [63] AminitrozoleAminitrozoleMTZ9b 0.4905.3600.1510.490 AminitrozoleMTZ 5.3600.490 AminitrozoleMTZMTZ G. 5.360 lamblia0.4905.360 ~~~ MTZ G. lamblia5.360 ~~~ MTZ G.G.[IC lamblia lamblia5.36050 (µM)] ~~~ [ICG.[IC (lambliaµ50M)] (µM)]~~~ 10 G.50[IC lamblia19.5850 (µM)] ~~~ 10 [IC19.5850 (µM)] 1010 19.58[IC5019.58 (µM)] [64] 10 19.58 [64][64] 10 19.58 [64] MTZ 62.48 [64] MTZMTZ 62.4862.48 [64] MTZ 62.48 MTZ 62.48

MTZ 62.48 G.G. lamblia lamblia~~~ G. lamblia~~~ [IC50[ICG.(µ50 lambliag /(µg/mL)]mL)] ~~~ [ICG. 50lamblia (µg/mL)]~~~ 1111 [ICG.30.6 lamblia50 30.6(µg/mL)] ~~~ [65] 11 [IC50 (µg/mL)]30.6 [65] 11 [IC50 (µg/mL)]30.6 [65] 11 30.6 [65] MTZMTZ11 0.210.2130.6 [65] MTZ 0.21 [65] MTZ 0.21

MTZ G. intestinalis0.21 MTZ G. intestinalis0.21 ~~~ [ICG. 50intestinalis(µM)] ~~~ G. [ICintestinalis50 (µM)]~~~ 12a 0.027G. [ICintestinalis0.00250 (µM)]~~~ 12a G.0.027 intestinalis[IC± 50 ±(µM)] 0.002~~~ [ 66] 12b12a 0.0220.027[IC0.00150 ±(µM)] 0.002 12b12a 0.0220.027[IC± 50 ±(µM)]± 0.0010.002 12c12a 0.0270.0270.002 ± 0.002 [66] 12b 0.022± ± 0.001 12d12a12c12b 0.0210.0270.0220.005 ±± 0.0020.001 [66] 12b12c 0.0220.027 ± 0.0010.002 [66] 12d12b12c 0.0210.0220.027± ±± 0.0050.0010.002 [66] 12d12c 0.0210.027 ± 0.0050.002 [66] Nitazoxamide12c12d 0.0150.0270.021 ±± 0.0020.005 Nitazoxamide12d 0.021 0.015 ± 0.0050.002 NitazoxamideMebendazole12d 0.0460.021 0.015 ±± 0.0090.0050.002 NitazoxamideMebendazole 0.0150.046 ± 0.0020.009 NitazoxamideMebendazole 0.015 0.046 ±± 0.0020.009 Mebendazole 0.046 ± 0.009 Mebendazole 0.046 ± 0.009

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Table 1. Cont. Molecules 2019, 24, x FOR PEER REVIEW MTZ 1.224 ± 0.021 4 of 45 MTZ 1.224 ± 0.021 CompoundMTZ Activity 1.224 ± 0.021 Ref. Nitazoxamide 0.015 0.002 ± Mebendazole 0.046 0.009 ± [66] MTZ 1.224 0.021 ± G. duodenalis~~~ MTZ G. 1.224 duodenalis ± 0.021~~~ G.G.[IC duodenalis duodenalis50 (µg/mL)]~~~ [IC50 (µg/mL)] 13a [IC50[IC(µ50g /14.3(µg/mL)]mL)] 13a 14.3 13a13b13a 14.314.38.2 13b 8.2 13b13b 8.2 8.2 [67] [67][67] [67] G. duodenalis~~~ MTZ 11.4 MTZMTZ [IC 11.450 11.4(µg/mL)] MTZ 11.4 13a 14.3

13b 8.2

G. intestinalis~~~ G.G. intestinalis intestinalis~~~ [67] G. [ICintestinalis50 (µM)]~~~ [IC50[IC(µ50M)] (µM)] 14a [IC500.87 (µM)] 14aMTZ14a 0.870.8711.4 14b14a 0.390.87 [68] 14b14b 0.390.39 [68][68] 14b 0.39 [68]

MTZMTZ 1.401.40 MTZ 1.40 MTZ G. intestinalis1.40 ~~~

[IC50 (µM)] G.G. lamblia lamblia~~~ 14a G. lamblia0.87 ~~~ [ICG.50[IC (lambliaµ50M)] (µM)]~~~ [IC50 (µM)] 14b [IC500.39 (µM)] [68] [69][69] [69] DisulfiramDisulfiram (15 ()15) 6.6 6.6 [69] DisulfiramMTZ (15) 1.406.6 Disulfiram (15) 6.6

G. lamblia~~~

ImportantImportant Highlights Highlights of of Table Table1 Compounds1 Compounds [IC50 (µM)] Important Highlights of Table 1 Compounds Important Highlights of Table 1 Compounds AllAll ofof thethe novel novel compoundscompounds designeddesigned forfor giardiasisgiardiasis treatmenttreatment areare listedlisted inin TableTable1 1and and their [69]their All of the novel compounds designed for giardiasis treatment are listed in Table 1 and their eefficaciesfficaciesAll of werewere the comparednovelcompared compounds withwith thosethose designed ofof thethe standardforstandard giardiasis drugsdrugsDisulfiram treatment metronidazolemetronidazole (15are) listed (MTZ),(MTZ), in 6.6Table formononetin, formononetin, 1 and their efficacies were compared with those of the standard drugs metronidazole (MTZ), formononetin, aminitrozole,aminitrozole,efficacies were nitazoxanide, nitazoxanide, compared tizoxanide,with tizoxanide, those nitazoxanideof nitazoxanide the standard and and drugs mebendazole. mebendazole. metronidazole In In brief, brief, (MTZ), Zhang Zhang formononetin, et et al. al. isolated isolated aminitrozole, nitazoxanide, tizoxanide, nitazoxanide and mebendazole. In brief, Zhang et al. isolated 3,5-dica3,5-dicaffeoylquinicaminitrozole,ffeoylquinic nitazoxanide, acid acid from from tizoxanide,Artemisia Artemisia argyinitazoxanide argyi,, and and from from and it, it, mebendazole. developed developed a a series Inseries brief, of of esterZhang ester derivatives derivatives et al. isolated as as 3,5-dicaffeoylquinic acid from Artemisia argyi, and from it, developed a series of ester derivatives as potentialpotential3,5-dicaffeoylquinic antigiardial antigiardial acid agents. agents. from AmongstAmongst Artemisia the theargyi synthesizedsynthesized, and fromcompounds, it,compounds, developed Compound Compounda series of ester7 7was was derivatives reported reported as as thepotentialImportant most potent antigiardial Highlights inhibitor ofagents. Table against Amongst 1 CompoundsG. lamblia the(IC synthesized = 4.62 µ compounds,g/mL) [61]. A Compound series of 3-tetrazolylmethyl- 7 was reported as thepotential most potentantigiardial inhibitor agents. against Amongst G. lamblia the synthesized(IC5050 = 4.62 µg/mL)compounds, [61]. ACompound series of 3-tetrazolylmethyl- 7 was reported as the most potent inhibitor against G. lamblia (IC50 = 4.62 µg/mL) [61]. A series of 3-tetrazolylmethyl- 4H-chromen-4-ones4H-chromen-4-onesthe mostAll of potent the novel inhibitor were were compounds synthesized syagainstnthesized G.designed lamblia by by Cano Cano (ICfor et 50etgiardiasis al. =al. 4.62 via via anµg/mL) an Ugi-azidetreatment Ugi-azide [61]. multicomponentareA multicomponent series listed of in 3-tetrazolylmethyl- Table reaction reaction 1 and andtheir and 4H-chromen-4-ones were synthesized by Cano et al. via an Ugi-azide multicomponent reaction and evaluatedevaluatedefficacies4H-chromen-4-ones for werefor antigiardial antigiardial compared were activity syactivitywithnthesized those and, and, compoundbyof compound theCano st andardet 8al. 8was wasvia drugs found anfound Ugi-azide metronidazole to to be be the the multicomponent most most (MTZ), potential potential formononetin, antigiardialreaction antigiardial and agentevaluated (IC for= 84.2antigiardialµg/mL) activity in the seriesand, compound [62]. Navarrete-V 8 was foundázquez to be et the al. most [63] synthesizedpotential antigiardial a series agentaminitrozole,evaluated (IC5050 for = 84.2 nitazoxanide,antigiardial µg/mL) in activity tizoxanide,the series and, [62]. compoundnitazoxanide Navarrete-Vázquez 8 wasand mebendazole.found et to al. be [63] the In synthesizedmost brief, potential Zhang a serieset antigiardial al. isolated of four agent (IC50 = 84.2 µg/mL) in the series [62]. Navarrete-Vázquez et al. [63] synthesized a series of four of5-nitrothiazole3,5-dicaffeoylquinicagent four (IC 5-nitrothiazole50 = 84.2 compounds µg/mL) acid compounds fromin theand Artemisia series andreported [62]. reportedargyi Navarrete-Vázquez ,them and them fromas as novelit, novel developed antigiardialet antigiardial al. [63] a series synthesized agents. agents. of ester Amonga derivatives series of them,them, four as compounds5-nitrothiazole9a (IC compounds= 0.122 µM) and and 9breported(IC = 0.151them µM)as exhibitednovel antigiardial potential inhibitory agents. activityAmong against them, compoundspotential5-nitrothiazole antigiardial 9a (ICcompounds50 50 = agents. 0.122 µM)andAmongst andreported 9bthe50 (IC synthesized them50 = 0.151 as novelµM)compounds, exhibited antigiardial Compound potential agents. inhibitory7 wasAmong reported activity them, as compounds 9a (IC50 = 0.122 µM) and 9b (IC50 = 0.151 µM) exhibited potential inhibitory activity G.againstthecompounds intestinalis. most G. potent intestinalis. 9aSingh (ICinhibitor50 et =Singh al. 0.122 [64against et] µM) designed al. [64] G.and lamblia designed 9b and (IC developed(IC50 50and= =0.151 4.62 developed a µg/mL)µM) series exhibited a of [61].series chalconyl A of seriespotential chalconyl blended of 3-tetrazolylmethyl-inhibitory blended triazole triazoleactivity allied against G. intestinalis. Singh et al. [64] designed and developed a series of chalconyl blended triazole silatranes;allied4H-chromen-4-onesagainst silatranes; G. these intestinalis. compounds these were Singh compounds sy areetnthesized al. hybrids [64] are designedby hybrids of Cano three etandof pharmacologic al. three developed via pharmacologican Ugi-azide a sca seriesffolds, multicomponent of scaffolds, namelychalconyl chalcone,namely blended reaction chalcone, triazole triazole and allied silatranes; these compounds are hybrids of three pharmacologic scaffolds, namely chalcone, andtriazoleevaluatedallied metal silatranes; and complex for metalantigiardial these (silatranes). complex compounds activity (silatranes). All theand, are derivatives compoundhybrids All the of were three8derivatives was evaluated pharmacologicfound were to for be the evaluatedthe antigiardialscaffolds, most potential for namely activity;the antigiardial chalcone, among themtriazole compound and metal10 showed complex excellent (silatranes). activity All against the derivativesG. lamblia (ICwere= evaluated19.58 µM). for Compound the antigiardial11 is a activity;agenttriazole (IC andamong50 = 84.2metal them µg/mL) complex compound in the (silatranes). series 10 showed [62]. AllNavarrete-Vázquez excellentthe derivatives activity etwereagainst 50al. [63]evaluated G. synthesized lamblia for (IC the 50a series= antigiardial19.58 of µM). four activity; among them compound 10 showed excellent activity against G. lamblia (IC50 = 19.58 µM). derivativeCompound5-nitrothiazoleactivity; among of naturally11 is compounds thema derivative occurring compound ofand sesquiterpene naturally 10reported showed occurring lactone,themexcellent assesquiterpene which activitynovel was antigiardialagainst isolated lactone, G. from lamblia whichagents.Decachaeta was(IC Among50 isolated= incompta 19.58 them, fromµM).by BautistaCompound et al. 11 It showedis a derivative greater of antigiardial naturally activityoccurring (IC sesquiterpene= 30.6 µg/mL) lactone, than its which parent was compound isolated [from65]. DecachaetacompoundsCompound incompta 119a is(IC a derivative50 by = Bautista0.122 µM)of et naturally al.and It showed9b occurring(IC50 greater= 0.151 sesquiterpene50 antigiardialµM) exhibited lactone, activity potential which(IC50 = inhibitory was30.6 isolatedµg/mL) activity fromthan Decachaeta incompta by Bautista et al. It showed greater antigiardial activity (IC50 = 30.6 µg/mL) than NovelagainstDecachaeta nitazoxanide– G. intestinalis.incomptaN by-methylbenzimidazole Singh Bautista et al. et [64] al. It designed showed hybrids andgreater weredeveloped antigiardial designed a series and activity synthesizedof chalconyl (IC50 = by30.6 blended Soria-Arteche µg/mL) triazole than et allied al. [66 silatranes;], and evaluated these compounds for their in are vitro hybridsbiological of three activity. pharmacologic Compounds scaffolds,12a–d namelyexpressed chalcone, good

triazole and metal complex (silatranes). All the derivatives were evaluated for the antigiardial activity; among them compound 10 showed excellent activity against G. lamblia (IC50 = 19.58 µM). Compound 11 is a derivative of naturally occurring sesquiterpene lactone, which was isolated from Decachaeta incompta by Bautista et al. It showed greater antigiardial activity (IC50 = 30.6 µg/mL) than

Molecules 2019, 24, x FOR PEER REVIEW 5 of 45 its parent compound [65]. Novel nitazoxanide–N-methylbenzimidazole hybrids were designed and Molecules 2019, 24, 3886 5 of 45 synthesized by Soria-Arteche et al. [66], and evaluated for their in vitro biological activity. Compounds 12a–d expressed good antigiardial activity (IC50 = 0.021–0.027 µM) by inhibiting an G. antigiardialintestinalis culture. activity Using (IC50 a= novel0.021–0.027 methodologyµM) by based inhibiting on a double an G. intestinalis Sonogashiraculture. coupling Using reaction a novel in methodology2-amino-3,5-diiodopyridine, based on a double Leboho Sonogashira et al. synthesized coupling reaction a series inof 2-amino-3,5-diiodopyridine,2,3,5-trisubstituted 7-azaindoles, Leboho as etwell al. synthesizedas 2,5-disubstituted a series of 7- 2,3,5-trisubstituted azaindoles. These 7-azaindoles, synthesized as wellseries as were 2,5-disubstituted evaluated 7-against azaindoles. a G. Theseduodenalis synthesized strain, and series the wereresults evaluated showed that against compounds a G. duodenalis 13a (ICstrain,50 = 14.3 and µg/mL) the results and 13b showed (IC50 = that 8.2 compoundsµg/mL) were13a the(IC most50 =potent14.3 µ agentsg/mL) [67]. and Another13b (IC50 series= 8.2 ofµg 2-amino-4-arylthiazole/mL) were the most potent derivatives agents were [67]. Anotherprepared series and evaluated of 2-amino-4-arylthiazole by Mocelo-Castell derivatives et al. [68] were as preparedpotential andanti-giardial evaluated agents. by Mocelo-Castell The results etrevealed al. [68] that as potential compounds anti-giardial 14a (IC50 = agents. 0.87 µM) The and results 14b (IC revealed50 = 0.39 that µM) compounds were the most14a potent(IC50 = inhibitors0.87 µM) andof G.14b intestinalis(IC50 =. 0.39DisulfiramµM) were (compound the most 15 potent) wasinhibitors proposed ofto G.inactivate intestinalis G. .lamblia Disulfiram kinase, (compound and Castillo-15) wasVillanueva proposed et al. to hypothesized inactivate G. lambliathat it actskinase, on enzymes and Castillo-Villanueva of G. lamblia. Accordingly, et al. hypothesized compound that 15 it (IC acts50 on= 6.6 enzymes µM) was of G. efficient lamblia .inactivator Accordingly, of compoundimmunorece15ptor(IC 50tyrosine-based= 6.6 µM) was inhibition efficient inactivatormotif (ITIM). of immunoreceptorTherefore, it is feasible tyrosine-based that compound inhibition 15 could motif lead (ITIM). to new Therefore, pharmacotherapies it is feasible that against compound G. lamblia15 could[69] lead to new pharmacotherapies against G. lamblia [69] In summary, thethe reportedreported antigiardialantigiardial agentsagents couldcould bebe categorizedcategorized as thethe naturalnatural compoundscompounds and their analogues analogues (e.g., (e.g., 3,5-dicaffeoylquinic 3,5-dicaffeoylquinic acid acid derivatives derivatives and and 8-acyl 8-acyl and and 8-alkyl 8-alkyl incomptine incomptine A Aderivatives) derivatives) and and hybrids hybrids compounds compounds with with the theknow knownn acitve acitve drug drug such such as nitazoxanide-based as nitazoxanide-based and andbenzimidazole-based -based hybrids. hybrids. It is notewort It is noteworthyhy that the that hybrid the hybrid of nitazoxanide of nitazoxanide and andN- Nalkylbenzimidazole-alkylbenzimidazole tethered tethered by by amide amide linker linker ex exhibitedhibited good good activity activity profiles profiles compared with nitazoxanide or , which suggests that hybridization of active compounds could provide good option for antigiardial drug discovery.discovery.

Figure 1. Currently available antigiardial drugs. 2.2. Anti-Leishmaniasis 2.2. Anti-Leishmaniasis Leishmaniasis, a spread by the bite of an infected female phlebotomine sand fly, Leishmaniasis, a parasitic disease spread by the bite of an infected female phlebotomine sand has been known for many years it was first clinically described in 1756 by Alexander Russell, who fly, has been known for many years it was first clinically described in 1756 by Alexander Russell, who named it Aleppo boil [70]. Many names correlate to this group of diseases such as kala-azar, Dum-dum named it Aleppo boil [70]. Many names correlate to this group of diseases such as kala-azar, Dum- fever, white leprosy, espundia, and pian bois. A vector borne disease, leishmaniasis is caused by an dum fever, white leprosy, espundia, and pian bois. A vector borne disease, leishmaniasis is caused obligate intramacrophage protozoan, and it is characterized by its diversity and complexity [71,72]. by an obligate intramacrophage protozoan, and it is characterized by its diversity and complexity Approximately 21 Leishmania species have been identified to be pathogenic to humans. Leishmania [71,72]. Approximately 21 Leishmania species have been identified to be pathogenic to humans. is one of several genera within the family Trypanosomatidae, and its species are characterized by Leishmania is one of several genera within the family Trypanosomatidae, and its species are the possession of a kinetoplast, a unique form of mitochondrial DNA. Based on species type host characterized by the possession of a kinetoplast, a unique form of mitochondrial DNA. Based on immune system responses, leishmaniasis has three basic clinical forms: Cutaneous (with skin ulcers), species type host immune system responses, leishmaniasis has three basic clinical forms: Cutaneous mucocutaneous (with skin, mouth, and nose ulcers), and visceral (with liver, and spleen enlargement, (with skin ulcers), mucocutaneous (with skin, mouth, and nose ulcers), and visceral (with liver, and as well as bone marrow dysfunctions) [73,74]. , the most common form of the spleen enlargement, as well as bone marrow dysfunctions) [73,74]. Cutaneous leishmaniasis, the most disease, is caused by L. braziliensis, L. major, L. mexicana, L. tropica, and several other species [75]. It can common form of the disease, is caused by L. braziliensis, L. major, L. mexicana, L. tropica, and several be eventually defeated by the immune system; however, in most cases it progresses and is converted other species [75]. It can be eventually defeated by the immune system; however, in most cases it in to the mucocutaneous form, in which the parasites metastasize to the mucosal tissues. Mucosal

Molecules 2019, 24, 3886 6 of 45 leishmaniasis is usually caused by L. braziliensis, and it is associated with damage to the palate, nasal septum, and mucous membranes [76,77]. This form is usually refractory to therapy and can be fatal. The most dangerous form of the disease is , commonly called kala-azar. It can become fatal if it is not rapidly diagnosed and treated, it is responsible for most leishmaniasis-related deaths [78,79]. In visceral leishmaniasis, which is caused by L. donovani and L. infantum (synonym of L. chagasi), the parasite mainly infects the liver, spleen, and bone marrow. The infected host shows symptoms such as fever, weight loss, and anemia [80]. This disease has been recognized as an increasing health problem worldwide by the World Health Organization (WHO) [81], with high morbidity and mortality rates in Africa, Asia, and America. Among all tropical diseases, leishmaniasis is ranked fourth in morbidity and second in mortality rates [82]. Leishmaniasis is widespread, having been reported in 88 countries across all continents, with the exception of Antarctica [71,83]. It has an annual Molecules 2019, 24, x FOR PEER REVIEW 7 of 45 deathMolecules rate of approximately2019, 24, x FOR PEER 80,000 REVIEW people [84], and there are two million new cases occurring annually,7 of 45 with 12 million people currently infected globally [83,85]. The life cycle of Leishmania sp. begins when the invertebrate host (sand fly) feeds on infected mammalian blood, thereby imbibing the amastigotes present within the macrophages. In the intestine of the insect vector, the amastigote transforms into procyclic promastigotes, and later into metacyclic promastigotes. When the insect bites a mammalian host again, the inoculated virulent promastigotes enter the blood stream and are internalized by the macrophages, where they differentiate again into amastigotes, completing the cycle [86]. No effective vaccine is available against leishmaniasis; chemotherapy is the only effective way to treat all forms of the disease [87–89]. However, some drugs are available for the treatment of this disease. The first-choice treatment for leishmaniasis involves the use of the derivatives, sodium stibogluconate, which is highly toxic with serious side effects, and requires a prolonged treatment regimen [90,91]. Alternatives include , pentamidine, and amphotericin-B (Figure2) However, these drugs have not found extensive use owing to their severe toxicities and difficulties associated with parenteral administration and drug resistance [19–22]. Generally, the drugs currently used for the treatment of human cutaneous and visceral leishmaniasis are toxic, and can cause severe adverse reactions such as pancreatitis, pancytopenia, reversible peripheral neuropathy, nephrotoxicity, cardiotoxicity, bone pain, and myalgia [92,93]. Therefore, the development of novel, effective, and safe antileishmanial agents with reduced side effects is a major priority for health researchers. In view of these considerations, some researchers have designed and synthesized novel molecules for the treatment of leishmaniasis; Figure 2. Currently available antileishmanial drugs. their results are summarized inFigure Table 22.. Currently available antileishmanial drugs.

TableTable 2. Selected 2. Selected data data of reported of reported antileishmanial antileishmanial agents. agents. Table 2. Selected data of reported antileishmanial agents. Compound Activity Ref. CompoundCompound ActivityActivity Ref. Ref. L. amazonensis~~~ L. amazonensis~~~ L.L. amazonensis ~~~L. amazonensisL. amazonensis~~~ (Promastigotes)~~~ (Amastigotes)~~~ (Promastigotes)~~~(Promastigotes) (Amastigotes) (Amastigotes)~~~ [IC[IC50 50(µ (µM)]M)] [IC50 ([ICµM)]50 (µM)] [IC50 (µM)] [IC50 (µM)] 21a21a 20.74 20.74 0.5± 0.5 22.0 22.00.1 ± 0.1 [94] [94] 21a 20.74± ± 0.5 ± 22.0 ± 0.1 [94] 22b22b 16.4 16.4 1.1± 1.1 17.0 17.01.1 ± 1.1 22b 16.4± ± 1.1 ± 17.0 ± 1.1 PentamidinePentamidine 4.8 4.8 0.1± 0.1 1.9 0.1 1.9 ± 0.1 Pentamidine 4.8± ± 0.1 ± 1.9 ± 0.1

L. major~~~ L. donovani~~~ L. major ~~~ L. donovaniL. donovani~~~ [IC[IC50 50(µ (µg/mL)]g/mL)] [IC50 ([ICµg/mL)]50 (µg/mL)] [IC50 (µg/mL)] [IC50 (µg/mL)] [95] 2222 0.47 0.47 0.02± 0.02 1.5 0.17 1.5 ± 0.17 22 0.47± ± 0.02 ± 1.5 ± 0.17 AmBAmB 0.56 0.56 0.01± 0.01 - - [95] AmB 0.56± ± 0.01 - [95] SSGSSG -- 2.98 2.98 SSG - 2.98

L. donovani [IC50 (µg/mL)] L. donovani [IC50 (µg/mL)] 23a 98.75 23a 98.75 23b 93.75 23b 93.75 SSG 490 SSG 490 [96] [96]

Pentamidine 5.5 Pentamidine 5.5

L. donovani~~~ L. donovani~~~ (Amastigotes) [IC50 (µM)] (Amastigotes) [IC50 (µM)] [97] 24a 2.8 [97] 24a 2.8 24b 2.0 24b 2.0

Molecules 2019, 24, x FOR PEER REVIEW 7 of 45

Figure 2. Currently available antileishmanial drugs.

Table 2. Selected data of reported antileishmanial agents.

Compound Activity Ref. L. amazonensis~~~ L. amazonensis~~~ (Promastigotes)~~~ (Amastigotes)~~~ [IC50 (µM)] [IC50 (µM)] 21a 20.74 ± 0.5 22.0 ± 0.1 [94] 22b 16.4 ± 1.1 17.0 ± 1.1

Pentamidine 4.8 ± 0.1 1.9 ± 0.1

L. major~~~ L. donovani~~~

[IC50 (µg/mL)] [IC50 (µg/mL)] Molecules 2019, 24, 3886 22 0.47 ± 0.02 1.5 ± 0.17 7 of 45 AmB 0.56 ± 0.01 - [95]

TableSSG 2. Cont. - 2.98

Compound Activity Ref. L. donovani [IC50 (µg/mL)] L. donovani µ 23a [IC98.7550 ( g /mL)] 23b23a 98.7593.75 23b 93.75 SSG 490 SSG 490 [96] [96]

Molecules 2019, 24, x FOR PEER REVIEW PentamidinePentamidine 5.5 5.5 8 of 45 Molecules 2019, 24, x FOR PEER REVIEW 8 of 45 Molecules 2019, 24, x FOR PEER REVIEW SSG 49.7 8 of 45 SSG 49.7 SSG 49.7 L.L. donovani donovani~~~ (Amastigotes) [IC50 (µM)] (Amastigotes) [IC50 (µM)] [97] 24a24a 2.8 2.8 24b 2.0 Miltefosine24bSSG 49.7 8.42.0 Miltefosine 8.4 [97] Miltefosine 8.4

Miltefosine 8.4

L. donovani [IC50 (µM)] L. donovani [IC50 (µM)] L. donovaniPromastigotes[IC (µM)] L. donovaniPromastigotes50 [IC50 (µM)] 25a 12.7 25a PromastigotesPromastigotes12.7 25b 9.1 25b25a25a 12.712.79.1 [98] Pentamidine 4.8 [98] Pentamidine25b25b 9.1 9.14.8 [98] [98] PentamidinePentamidine 4.8 4.8 [98] AmB 0.3 AmBAmB 0.3 0.3

AmB 0.3 L. amazonensis~~~ L. donovani~~~ L.L. amazonensis ~~~ L. donovaniL. donovani~~~ L. amazonensis50 ~~~ L. donovani50 ~~~ [IC[IC50 50(µ (µM)]M)] [IC50 ([ICµM)]50 (µM)] L. amazonensis[IC50 (µM)]~~~ L.[IC donovani50 (µM)]~~~ 26a26a 0.130.13 0.31 0.31 26a [IC500.13 (µM)] [IC500.31 (µM)] 26a 0.13 0.17 0.31 [99] [99] [99] 26b26b 0.140.14 0.17 [99] 26b 0.14 0.17 26b 0.14 0.17

L. major ~~~ L. donovaniL. donovani~~~

L. majorµ ~~~ L.µ donovani~~~ [IC[IC50 50( (µM)]M)] [IC50 ([ICM)]50 (µM)] L.[IC major50 (µM)]~~~ L.[IC donovani50 (µM)]~~~ 27a27a 1.21.2 0.7± 0.7 10.5 10.50.6 ± 0.6 [100] [IC±50 (µM)] ±[IC50 (µM)][ 100] 27a27b 1.51.2 1.1± 0.7 4.0 0.6 10.5 ± 0.6 [100] 27b 1.5± ± 1.1 ± 4.0 ± 0.6 27b27a 1.21.5 ± 0.71.1 10.5 4.0 ±± 0.60.6 [100] PentamidinePentamidine 4.34.3 0.8± 0.8 5.5 0.8 5.5 ± 0.8 Pentamidine27b 4.31.5± ± 0.81.1 ± 5.54.0 ± 0.80.6 L. aethiopica [IC50 (µg/mL)] Pentamidine L.4.3 aethiopicaL. aethiopica± 0.8 [IC50 [IC(µ50g/ mL)](µg/mL)] 5.5 ± 0.8 Promastigotes Amastigotes PromastigotesL. aethiopica [IC50 (µg/mL)]Amastigotes 28 Promastigotes0.0142 ± 0.004 Amastigotes 0.13 ± 0.02 Promastigotes Amastigotes 2828 0.01420.0142 0.004± 0.004 0.13 0.130.02 ± 0.02[ 101] Miltefosine 3.19± ± 14 ± 0.3 ± 0.04 [101] MiltefosineMiltefosine28 0.0142 3.193.19 14±± 0.00414 0.3 0.04 0.13 0.3 ± ± 0.04 0.02 [101] ± ± [101] Miltefosine 3.19 ± 14 0.3 ± 0.04 [101] AmBAmB 0.0472 0.0472 0.002± 0.002 0.2 0.02 0.2 ± 0.02 AmB 0.0472± ± 0.002 ± 0.2 ± 0.02

AmB 0.0472 ± 0.002 0.2 ± 0.02 L. infantum [IC50 (µM)] L. infantum [IC50 (µM)] 29 1.5 ± 0.1 29 L. infantum 1.5 ± [IC 0.150 (µM)] 29 1.5 ± 0.1 [102] [102] Miltefosine 3.4 ± 0.6 Miltefosine 3.4 ± 0.6 [102] Miltefosine 3.4 ± 0.6

L. major [IC50 (µM)] L. major [IC50 (µM)] Promastigotes Amastigotes [103] PromastigotesL. major [IC50 (µM)]Amastigotes [103] 30a 2.8 ± 0.4 0.2 ± 0.1 30a Promastigotes2.8 ± 0.4 Amastigotes 0.2 ± 0.1 [103] 30a 2.8 ± 0.4 0.2 ± 0.1

Molecules 2019, 24, x FOR PEER REVIEW 8 of 45

SSG 49.7

Miltefosine 8.4

L. donovani [IC50 (µM)] Promastigotes 25a 12.7 25b 9.1 Pentamidine 4.8 [98]

AmB 0.3

L. amazonensis~~~ L. donovani~~~

[IC50 (µM)] [IC50 (µM)] 26a 0.13 0.31

[99]

26b 0.14 0.17

L. major~~~ L. donovani~~~

[IC50 (µM)] [IC50 (µM)] 27a 1.2 ± 0.7 10.5 ± 0.6 [100] 27b 1.5 ± 1.1 4.0 ± 0.6 Pentamidine 4.3 ± 0.8 5.5 ± 0.8 L. aethiopica [IC50 (µg/mL)] Promastigotes Amastigotes 28 0.0142 ± 0.004 0.13 ± 0.02 Molecules 2019, 24, 3886 8 of 45 Miltefosine 3.19 ± 14 0.3 ± 0.04 [101]

TableAmB 2. Cont . 0.0472 ± 0.002 0.2 ± 0.02

Compound Activity Ref. L. infantum [IC50 (µM)] µ 29 L. infantum 1.5[IC 50± 0.1( M)] 29 1.5 0.1 Molecules 2019, 24, x FOR PEER REVIEW ± 9 of 45 Molecules 2019, 24, x FOR PEER REVIEW 9 of 45 Molecules 2019, 24, x FOR PEER REVIEW [1029] of[102] 45 Molecules 2019, 24, x FOR PEER REVIEW 30b 6.4 ± 0.2 0.8 9 of 45 MiltefosineMiltefosine 3.4 3.40.6 ± 0.6 Molecules 2019, 24, x FOR PEER REVIEW 30b 6.4 ± 0.2 ± 0.8 9 of 45 30b 6.4 ± 0.2 0.8 30b 6.4 ± 0.2 0.8

30b 6.4 ± 0.2 0.8 µ L. majorL. major[IC50 [IC( 50M)] (µM)] PromastigotesPromastigotes AmastigotesAmastigotes [103] 30a30a 2.82.8 0.4± 0.4 0.2 0.1 0.2 ± 0.1 ± ± Pentamidine30b 6.4 4.6 0.2± 1.1 0.8 3 ±1 Pentamidine 4.6± ± 1.1 3 ±1 Pentamidine 4.6 ± 1.1 3 ±1 Pentamidine 4.6 ± 1.1 3 ±1 [103] Pentamidine 4.6 ± 1.1 3 ±1

Pentamidine 4.6 1.1 3 1 ± ±

L. major [IC50 (µg/mL)]

31a L. major [IC95.0050 (µg/mL)] L. major [IC50 (µg/mL)] 31b31a L. major [IC99.0095.0050 (µg/mL)] 31a L. major [IC 95.00(µg/mL)] 31b31a L. major50 [IC95.0099.0050 (µg/mL)] SSG31b 490.0099.00 SSG31b31a31a 95.00490.0095.0099.00 SSG31b 99.00490.00 [104] SSG31b 490.0099.00 SSG 490.00 [104] SSG 490.00 [104] Pentamidine 5.50 [104] [104] Pentamidine 5.50 [104] PentamidinePentamidine 5.505.50 Pentamidine 5.50 Pentamidine 5.50 L. donovani~~~

(Promastigotes)L. donovani [IC~~~50 (µM)] L.L. donovani donovani~~~ 32 (Promastigotes)L. donovani 9.0 ± 2.80 [IC~~~ 50 (µM)] 50 (Promastigotes)(Promastigotes) [IC50 [IC(µM)] (µM)] 32 (Promastigotes)L. donovani 9.0 ± 2.80 [IC~~~ 50 (µM)] [105] 32 9.0 ± 2.80 32 (Promastigotes)9.0 2.80 [IC50 (µM)] [105] Miltefosine32 11.9± 9.0 ±± 2.802.70 [105] [105] 32 9.0 ± 2.80 [105] MiltefosineMiltefosine 11.9 11.92.70 ± 2.70 Miltefosine 11.9± ± 2.70 [105] Miltefosine 11.9 ± 2.70 L. donovani~~~ Miltefosine 11.9 ± 2.70 (Antiamastigotes)L. donovani [IC~~~50 (µM)] L.L. donovani donovani~~~ 33a (Antiamastigotes)(Antiamastigotes)L. donovani5.12 [IC 50 ~~~[IC(µM)]50 (µM)] (Antiamastigotes) [IC50 (µM)] 33a (Antiamastigotes)L. donovani5.12 ~~~[IC50 (µM)] 33b33a33a 5.123.565.12 33b33a33b (Antiamastigotes)3.565.123.56 [IC50 (µM)] [106] 33b 3.56 33a 5.12 [106] [106] 33b 3.56 [106] Miltefosine33b 12.503.56 [106] Miltefosine 12.50 Miltefosine 12.50 [106] Miltefosine 12.50 Miltefosine 12.50

Miltefosine L. major12.50 [IC50 (µM)] L. major [IC (µM)] 34a L. major1.8750 [IC ± 0.3150 (µM)] L. major [IC50 (µM)] 34a34a L.1.87 major1.870.31 [IC ± 0.3150 (µM)] 34b34a 4.371.87± ± 0.020.31 34b34a34b L.4.37 major1.874.370.02 [IC ± 0.310.0250 (µM)] 34b 4.37± ± 0.02 [107] [107] 34b34a 1.874.37 ± 0.310.02 [107] PentamidinePentamidine 5.09 5.090.09 ± 0.09 [107] 34b 4.37± ± 0.02 [107] Pentamidine 5.09 ± 0.09 Pentamidine 5.09 ± 0.09 [107] Pentamidine 5.09 ± 0.09

Pentamidine L. donovani 5.09 ± [IC 0.0950 (µM)]

35 L. donovani0.84 ± [IC 0.1250 (µM)] L. donovani [IC50 (µM)] 35 L. donovani0.84 ± [IC 0.1250 (µM)] 35 0.84 ± 0.12 35 L. donovani0.84 ± [IC 0.1250 (µM)] [108] 35 0.84 ± 0.12 [108] Miltefosine 8.10 ± 0.60 [108] Miltefosine 8.10 ± 0.60 [108] Miltefosine 8.10 ± 0.60 [108] Miltefosine 8.10 ± 0.60 Miltefosine 8.10 ± 0.60

Molecules 2019, 24, x FOR PEER REVIEW 9 of 45

30b 6.4 ± 0.2 0.8

Pentamidine 4.6 ± 1.1 3 ±1

L. major [IC50 (µg/mL)] 31a 95.00 31b 99.00 SSG 490.00 [104]

Pentamidine 5.50

L. donovani~~~

(Promastigotes) [IC50 (µM)] 32 9.0 ± 2.80 [105]

Miltefosine 11.9 ± 2.70

L. donovani~~~

(Antiamastigotes) [IC50 (µM)] 33a 5.12 33b 3.56 [106]

Miltefosine 12.50

L. major [IC50 (µM)] 34a 1.87 ± 0.31 34b 4.37 ± 0.02 Molecules 2019, 24, 3886 9 of 45 [107]

Pentamidine 5.09 ± 0.09 Table 2. Cont.

Compound Activity Ref. L. donovani [IC50 (µM)] L. donovani [IC (µM)] 35 0.84 50± 0.12 35 0.84 0.12 Molecules 2019, 24, x FOR PEER REVIEW ± 10 of 45 Molecules 2019, 24, x FOR PEER REVIEW 10 of 45 Molecules 2019, 24, x FOR PEER REVIEW [10810] of[108] 45 Molecules 2019, 24, x FOR PEER REVIEW Miltefosine L. infantum 8.10 0.60 [IC50 (µM)] 10 of 45 Miltefosine L. infantum 8.10± ± [IC 0.6050 (µM)] Molecules 2019, 24, x FOR PEER REVIEW 36a 1.40 ± 0.09 10 of 45 36a L. infantum1.40 ± [IC 0.0950 (µM)] 36b36a L. infantum1.401.47 ± [IC 0.090.0750 (µM)] 36b L. infantum1.47 ± [IC 0.0750 (µM)] 36b36a36c 1.470.831.40 ± 0.070.040.09µ 36c36a L. infantum0.831.40[IC 50± 0.040.09( M)] [109] Miltefosine36b 2.841.47 ± 0.100.07 [109] Miltefosine36c36a 1.40 2.840.830.09 ± 0.100.04 [109] 36b 1.47± ± 0.07 Miltefosine36c36b 1.47 2.840.830.07 ± 0.100.04 36c 0.83± ± 0.04 [109] MiltefosineEdelfosine36c 0.83 0.822.840.04 ± 0.130.10 [109] [109] Edelfosine 0.82± ± 0.13 MiltefosineMiltefosine 2.84 2.840.10 ± 0.10 Edelfosine 0.82± ± 0.13 Edelfosine L. infantum~~~ 0.82 ± 0.13L. amazonesis~~~ EdelfosineEdelfosine L. infantum 0.82~~~ 0.82 0.13 ± 0.13L. amazonesis~~~ L. [ICinfantum50(µM)]~~~ ± L. amazonesis[IC50(µM)]~~~ [IC50(µM)] [IC50(µM)] 37a L. [ICinfantum5019.5(µM)] ~~~ L. amazonesis[IC114.650(µM)] ~~~ 37a L. infantum19.5 ~~~ L. amazonesisL. amazonesis114.6 ~~~ 37b37a [IC[IC50(5019.538.0µ(µM)]M)] [IC50(µ[ICM)]inactive114.650(µM)] 37b [IC38.050(µM)] [ICinactive50(µM)] 37b37a 38.019.5 inactive114.6 [110] 37a37a 19.519.5 114.6 114.6 [110] 37b37b 38.038.0 inactiveinactive [110] 37b 38.0 inactive Miltefosine 23.7 20.9 [110] [110] Miltefosine 23.7 20.9 [110] Miltefosine 23.7 20.9 Miltefosine 23.7 20.9 Miltefosine 23.7 20.9 Miltefosine 23.7 20.9 L. donovani [IC50 (µM)] L. donovani [IC50 (µM)]

PromastogoteL. donovani [IC50 Amastogote(µM)] PromastogoteL. donovani [IC (µM)]Amastogote 38a Promastogote8.57L. ± 1.58donovani 50 [IC50Amastogote (µM)] 1.11 ± 0.19 38a 8.57L. ± 1.58donovani [IC50 (µM)] 1.11 ± 0.19 [111] 38b38a PromastogotePromastogote8.576.27 ± 1.580.65 AmastogoteAmastogote 1.110.36 ± 0.190.10 [111] 38b Promastogote6.27 ± 0.65 Amastogote 0.36 ± 0.10[ 111 ] [111] 38b38a38a 8.576.278.57 1.58± 0.651.58 1.11 0.361.110.19 ± 0.100.19 ± ± Miltefosine38a38b 1.108.57 ± 0.261.58 8.101.11 ± 1.410.19 [111] Miltefosine38b 6.271.106.27 0.65± 0.260.65 0.36 8.100.360.10 ± 1.410.10 [111] Miltefosine38b 1.106.27± ± 0.260.65 ± 8.100.36 ± 1.410.10 Miltefosine 1.10 L.0.26 donovani 8.10[IC50 (µM)]1.41 Miltefosine 1.10±L. ± donovani0.26 [IC50± (µM)] 8.10 ± 1.41 Miltefosine39a 1.10L. donovani ± 0.263.75 ± 0.31µ 8.10 ± 1.41 39a L. donovani3.75[IC 50± [IC 0.31( M)]50 (µM)] 39b39a39a L. donovani3.753.755.020.31 ± [IC 0.310.4950 (µM)] 39b L. donovani5.02± ± [IC 0.4950 (µM)] 39a39c39b 5.021.833.750.49 ± 0.210.31 39b39c 1.835.02± ± 0.210.49 39a39c 1.833.750.21 ± 0.31 [112] [112] Miltefosine39b39c 1.83 8.105.02± ± 0.210.510.49 [112] MiltefosineMiltefosine39b 8.10 8.105.020.51 ± 0.510.49 Miltefosine39c 8.101.83± ± 0.510.21 [112] 39c 1.83 ± 0.21 [112] MiltefosineSSGSSG 53.12 53.12 8.104.56 ±± 0.514.56 [112] MiltefosineSSG 53.12 8.10± ±± 0.514.56

SSG 53.12 ± 4.56 SSG L. 53.12 donovani ± 4.56~~~ SSG L.L. donovani 53.12 donovani ± 4.56~~~

AmastigoteAmastigoteL. donovani [IC50 [IC(µM)]~~~50 (µM)] Amastigote [IC50 (µM)] 4040 Amastigote0.6L. donovani0.60.2 ± [IC0.250~~~ (µM)] 40 L. donovani±0.6 ± 0.2~~~ MiltefosineMiltefosine40 Amastigote 8.4 0.6 8.41.2 ± [IC0.21.250 (µM)] [113] Miltefosine Amastigote± 8.4 ± [IC1.250 (µM)] [113] Miltefosine40 8.40.6 ± 1.20.2 [113] 40 0.6 ± 0.2 [113] MiltefosineSSG 56.1 8.43.2 ± 1.2 MiltefosineSSG 56.1±3.2 8.4± ± 1.2 [113] SSG 56.1±3.2 [113] SSG 56.1±3.2

SSG 56.1±3.2 SSG L. infantum~~~56.1±3.2 L. tropica~~~ L. infantum~~~ L. tropica~~~ L.[IC infantum50 (µM)]~~~ L.[IC tropica50 (µM)]~~~ [IC50 (µM)] [IC50 (µM)] 41 L.[IC 0.23infantum50 ±(µM)] 0.05~~~ L.[IC 0.12 tropica50 ±(µM)] 0.03~~~ 41 L. 0.23infantum ± 0.05~~~ L. 0.12 tropica ± 0.03~~~ Clofazimine41 [IC4.480.2350 ±(µM)] 1.060.05 [IC 2.960.1250 ±(µM)] 1.230.03 Clofazimine [IC4.4850 ±(µM)] 1.06 [IC 2.9650 ±(µM)] 1.23 [114] Clofazimine41 4.480.23 ± 1.060.05 2.960.12 ± 1.230.03 [114] 41 0.23 ± 0.05 0.12 ± 0.03 [114] Clofazimine 4.48 ± 1.06 2.96 ± 1.23 ClofazimineAmB 0.084.48 ± 0.021.06 0.092.96 ± 0.041.23 [114] AmB 0.08 ± 0.02 0.09 ± 0.04 [114] AmB 0.08 ± 0.02 0.09 ± 0.04

AmB 0.08 ± 0.02 0.09 ± 0.04 AmB 0.08L. ± infantum0.02 [IC50 (µM)] 0.09 ± 0.04 L. infantum [IC50 (µM)] 42a 12.7 [115] 42a L. infantum12.7 [IC 50 (µM)] [115] 42b42a L. infantum12.78.0 [IC 50 (µM)] [115] 42b L. infantum8.0 [IC 50 (µM)] 42b42a 12.78.0 [115] 42a 12.7 [115] 42b 8.0 42b 8.0

Molecules 2019, 24, x FOR PEER REVIEW 10 of 45

L. infantum [IC50 (µM)] 36a 1.40 ± 0.09 36b 1.47 ± 0.07 36c 0.83 ± 0.04 [109] Miltefosine 2.84 ± 0.10

Edelfosine 0.82 ± 0.13

L. infantum~~~ L. amazonesis~~~

[IC50(µM)] [IC50(µM)] 37a 19.5 114.6 37b 38.0 inactive [110]

Miltefosine 23.7 20.9

L. donovani [IC50 (µM)] Promastogote Amastogote 38a 8.57 ± 1.58 1.11 ± 0.19 [111] 38b 6.27 ± 0.65 0.36 ± 0.10

Miltefosine 1.10 ± 0.26 8.10 ± 1.41

L. donovani [IC50 (µM)] 39a 3.75 ± 0.31 39b 5.02 ± 0.49 39c 1.83 ± 0.21 Miltefosine 8.10 ± 0.51 [112]

SSG 53.12 ± 4.56

L. donovani~~~

Amastigote [IC50 (µM)] 40 0.6 ± 0.2 Molecules 2019, 24, 3886 Miltefosine 8.4 ± 1.2 10 of 45 [113]

TableSSG 2. Cont. 56.1±3.2

Compound Activity Ref. L. infantum~~~ L. tropica~~~ L. infantum L. tropica [IC[IC50 50(µ (µM)]M)] [IC50 ([ICµM)]50 (µM)] 4141 0.230.23 0.05± 0.05 0.12 0.120.03 ± 0.03 ± ± Molecules 2019, 24, x FOR PEER REVIEW ClofazimineClofazimine 4.484.48 1.06± 1.06 2.96 2.961.23 ± 1.23[ 11411] of 45 Molecules 2019, 24, x FOR PEER REVIEW ± ± 11 of[114] 45 0.08 0.02 Molecules 2019, 24, x FOR PEER REVIEW ± 11 of 45 AmB 0.09 0.04 Molecules 2019, 24, x FOR PEER REVIEW AmB 0.08 ± 0.02 ± 0.09 ± 0.04 11 of 45

L. infantum µ L. infantum[IC50 [IC( 50M)] (µM)] Miltefosine42a42a 12.712.710.4 [115] Miltefosine42b42b 8.0 10.48.0 Miltefosine 10.4 [115] Miltefosine 10.4

L. donovani [IC50 (µM)] L. donovani [IC50 (µM)]

43a L. donovani1.94 [IC 50 (µM)] L. donovani [IC (µM)] 43b43a L. donovani1.941.650 [IC 50 (µM)] 43b43a43c43a 1.941.861.941.6 43b 1.6 43d43b43c 1.271.861.6 43c 1.86 [116] 43d43e43c 1.391.861.27 43d 1.27 [116] [116] 43d43e43e 1.391.271.39 [116] 43e 1.39 Pentamidine 1.84 PentamidinePentamidine 1.841.84

Pentamidine 1.84 L. donovani [IC50 (µg/mL)] L. donovani [IC50 (µg/mL)] L. donovani [IC (µg/mL)] 44a L. donovani502.29 [IC50 (µg/mL)] 44a44a L. donovani2.292.29 [IC50 (µg/mL)] 44a 2.29

[117] [117] [117] 44b44b 2.482.48 [117] 44b 2.48 [117] 44b 2.48

L. donovani [IC50 (µg/mL)] L. donovaniL. donovani[IC50 [IC(µ50g /(µg/mL)]mL)]

45a45a L. donovani1.6 1.6[IC 50 (µg/mL)] 45b45a45b L. donovani2.0 2.01.6[IC 50 (µg/mL)] 45b45a 1.62.0 [118] [118] MiltefosineMiltefosine45b 0.1710.1712.0 [118] Miltefosine 0.171 [118] Miltefosine 0.171 Miltefosine L. donovani0.171 ~~~ L. donovani~~~ (Amastigotes) [IC50 (µM)] (Amastigotes)L. donovani [IC~~~50 (µM)]

46a (Amastigotes)L. donovani5.4 [IC~~~50 (µM)] 46a 5.4 46b46a (Amastigotes)1.15.4 [IC50 (µM)] 46b46a46c 2.45.41.1 [119] 46b46c 1.12.4 [119] 46c 2.4 [119] AmB 0.10 AmB 0.10

AmB 0.10 L. amazonensis [IC50 (µM)] L. amazonensis [IC50 (µM)]

47a L. amazonensis0.095 [IC 50 (µM)] [120] 47b47a L. amazonensis0.1230.095 [IC 50 (µM)] [120] 47b47a46c 0.0950.2110.123 46c 0.211 [120] 47b46c 0.1230.211 46c 0.211

Molecules 2019, 24, x FOR PEER REVIEW 11 of 45

Miltefosine 10.4

L. donovani [IC50 (µM)] 43a 1.94 43b 1.6 43c 1.86 43d 1.27 [116] 43e 1.39

Pentamidine 1.84

L. donovani [IC50 (µg/mL)] 44a 2.29

[117] 44b 2.48

L. donovani [IC50 (µg/mL)] 45a 1.6 Molecules 2019, 24, 3886 45b 2.0 11 of 45 [118]

TableMiltefosine 2. Cont . 0.171

Compound Activity Ref. L. donovani~~~ L. donovani (Amastigotes)(Amastigotes) [IC50 [IC(µM)]50 (µM)] 46a46a 5.4 5.4 46b46b 1.1 1.1 Molecules 2019, 24, x FOR PEER REVIEW [11912] of 45 46c 2.4 Molecules 2019, 24, x FOR PEER REVIEW 46c 2.4 12 of[119] 45 Molecules 2019, 24, x FOR PEER REVIEW 12 of 45 Molecules 2019, 24, x FOR PEER REVIEW 12 of 45 AmB 0.10 Molecules 2019, 24, x FOR PEER REVIEW AmB 0.10 12 of 45

L. amazonensis [IC (µM)] L. amazonensis50 [IC50 (µM)] 47a47a 0.0950.095 AmB 0.124 [120] 47b47b 0.1230.123 AmB46c 0.2110.124 AmB46c 0.1240.211 AmB 0.124 AmB 0.124 [120]

AmB 0.124

L. infantum [IC50 (µM)] 48 L. infantum 29.43 ±[IC 1.3450 (µM)] L. infantum [IC50 (µM)] 48 29.43 ± 1.34 48 L. infantum 29.43 ±[IC 1.3450 (µM)] L. infantum [IC50 (µM)] [121] 48 L. infantum 29.43 ±[IC 1.3450 (µM)] AmB48 29.43 0.521.34 ± 1.34 [121] 48 29.43± ± 1.34 [121] AmB 0.52 ± 1.34 [121] [121] AmB 0.52 ± 1.34 AmBAmB 0.52 0.521.34 ± 1.34 [121] L. donovani± [IC50 (µM)] AmB 0.52 ± 1.34 PromastigotesL. donovani [IC50Amastigotes (µM)] L. donovani [IC50 (µM)] PromastigotesL. donovani [IC (µM)]Amastigotes 49 Promastigotes26.9L. donovani 50 [IC50Amastigotes (µM)] 10.6 [122] 49 PromastigotesPromastigotes26.9L. donovani Amastigotes [IC50Amastigotes (µM)] 10.6 [122] Miltefosine49 26.96.1 10.6 8.2 [122] [122] 49 Promastigotes26.9 Amastigotes 10.6 Miltefosine49 26.96.1 10.6 8.2 [122] Miltefosine 6.1 8.2 49 26.9L. infantum [IC50 (µM)] 10.6 [122] MiltefosineMiltefosine 6.16.1 8.2 8.2 L. infantum [IC50 (µM)] 50 Miltefosine L. infantum6.1L. infantum [IC50 [IC(µM)] (µM)] 8.2 L. infantum [IC50 (µM)]

L. infantum [IC50 (µM)] [123] 50 5.6 ± 0.14 [123] [123] 5050 5.6 5.60.14 ± 0.14 [123] 50 ±5.6 ± 0.14 [123] 50 5.6 ± 0.14 [123] 50 5.6 ± 0.14

L. major [IC50 (µM)] L. major µ PromastigotesL. major[IC 50 [IC( 50M)] (µM)]Amastigotes L. major [IC50 (µM)] PromastigotesPromastigotes AmastigotesAmastigotes 51a Promastigotes9.35L. major [IC50 (µM)]Amastigotes2.7 51a 9.35 2.7 51a51a Promastigotes9.359.35L. major [IC50 2.7(µM)]Amastigotes2.7 51a Promastigotes9.35 Amastigotes2.7 [124 ] [124] 51a 9.35 2.7 [124] 51b51b 0.080.08 - - [124] 51b 0.08 - [124] 51b 0.08 - [124] 51b 0.08 - 51b 0.08 -

Important Highlights of Table 2 Compounds Important Highlights of Table 2 Compounds ImportantCompounds Highlights of different of Table heterocycles 2 Compounds classes are included in Table 2 as potent antileishmanial ImportantCompounds Highlights of different of Table heterocycles2 Compounds classes are included in Table 2 as potent antileishmanial agentsCompounds and the results of different of their analysisheterocycles were classescompar areed withincluded those in of Table the standard 2 as potent drugs antileishmanial pentamidine, Importantagents and Highlights the results of theirTable analysis 2 Compounds were compar ed with those of the standard drugs pentamidine, sodiumagentsCompounds and stibogluconate the results of different of (SSG), their analysismiltefosine,heterocycles were amphoter classes compar areedicin-B withincluded (AmB), those in of edelfosine Table the standard 2 as and potent drugsclofazimine antileishmanial pentamidine, (CFM). sodium stibogluconate (SSG), miltefosine, amphotericin-B (AmB), edelfosine and clofazimine (CFM). agentssodiumInCompounds and brief,stibogluconate the aresults series of different of (SSG),of their thiosemicarbazide analysismiltefosine,heterocycles were amphoter classescompar deri vativesareedicin-B withincluded (AmB),were those in preparedof edelfosine Table the standard 2 asand and potent evaluated drugsclofazimine antileishmanial pentamidine, for (CFM). thier In brief, a series of thiosemicarbazide derivatives were prepared and evaluated for thier sodiumantileishmanialagentsIn and stibogluconatebrief, the aresults activities.series of (SSG),of their Compoundsthiosemicarbazide analysismiltefosine, were 21a amphoter and compar deri 21bvatives wereedicin-B with reported (AmB),were those preparedof asedelfosine the the standard most and and active evaluated drugsclofazimine candidates, pentamidine, for (CFM). thierwith sodiumantileishmanialIn brief,stibogluconate a activities.series (SSG),of Compoundsthiosemicarbazide miltefosine, 21a amphoter and deri 21bvatives wereicin-B reported (AmB),were prepared asedelfosine the most and and active evaluated clofazimine candidates, for (CFM). thierwith antileishmanial activities. Compounds 21a and 21b were reported as the most active candidates, with antileishmanial activities. Compounds 21a and 21b were reported as the most active candidates, with In brief, a series of thiosemicarbazide derivatives were prepared and evaluated for thier antileishmanial activities. Compounds 21a and 21b were reported as the most active candidates, with

Molecules 2019, 24, 3886 12 of 45 Molecules 2019, 24, x FOR PEER REVIEW 7 of 45

Figure 2. Currently available antileishmanial drugs.

Important Highlights of Table 22. Compounds Selected data of reported antileishmanial agents.

CompoundsCompound of diff erent heterocycles classes are includedActivity in Table 2 as potent antileishmanialRef. agents and the results of their analysis were compared with those of the standard drugs pentamidine, L. amazonensis~~~ L. amazonensis~~~ sodium stibogluconate (SSG), miltefosine, amphotericin-B (AmB), edelfosine and clofazimine (CFM). (Promastigotes)~~~ (Amastigotes)~~~ In brief, a series of thiosemicarbazide derivatives were prepared and evaluated for thier [IC50 (µM)] [IC50 (µM)] antileishmanial activities. Compounds 21a and 21b were reported as the most active candidates, with 21a 20.74 ± 0.5 22.0 ± 0.1 [94] IC values in the range of 16.4 µM to 22.0 µM, after screening the whole series against L. amazonensis 50 22b 16.4 ± 1.1 17.0 ± 1.1 cultures using promastigotes and amastigote assays [94]. Among the compounds developed by Rashid et al., compound 22 was found to be a highlyPentamidine active antileishmanial 4.8 ± 0.1 agent. Basically, 1.9 compound ± 0.1 22 is a designed hybrid compound in which two biological scaffolds, pyrazoline and pyrimidine are linked L. major~~~ L. donovani~~~ with each other. It showed excellent biological activity the highest among all theh hybrids in its series against L. major and L. donovani with IC values of 0.47 0.02[IC50 and (µg/mL)] 1.5 0.17 µg/[ICmL,50 (µg/mL)] respectively [95]. 50 ± ± Sangshetti et al. [96] synthesized 4,5,6,7-tetrahydrothieno[3,2-c]pyridine-based22 0.47 ± 0.02 hydrazone 1.5 ± 0.17 derivatives and determined their antileishmanial inhibitoryAmB activities. 0.56 Among ± 0.01 the derivatives - in this series,[95] compounds 23a and 23b showed significant biological activities against L. donovani promastigotes, µ SSG - 2.98 IC50 values of 98.75 and 93.75 g/mL, respectively compared to that of the strandard drug SSG (IC50 = 490 µg/mL). A series of chalcones are also included in Table2, and their activity against L. donovani [IC50 (µg/mL)] L. donovani cultures clearly showed that among them, the compounds containing chromane, pyridine, and a substituted 4-hydroxy phenyl ring (compounds23a 24a and 24b) exhibited98.75 excellent antileishmanial 23b 93.75 activities (IC50 values of 2.8 and 2.0 µM, respectively) [97]. Among a series of carboline derivatives SSG 490 reported by Manda et al. [98], compounds 25a (IC50 = 12.7 µM) and 25b (IC50 = 9.1 µM), which are β derivatives of the commercially available tetrahydro- -carboline prepared in a single procedure were[96] the best candidates against L. donovani (promastigotes). Zhu et al. [99] reported compounds 26a and 26b, which are derived from arylamidamide using the reaction between amino diarylfurans and Pentamidine 5.5 2-pyridyl thioimidate analogs, as the most active antileishmanial agents against both intracellular L. donovani and L. amazonensis amastigotes, with IC50 values ranging from 0.13 to 0.31 µM. A series of 4-alkapolyenylpyrrolo[1,2-a]quinoxaline derivatives, including compounds 27a and 27b, which exhibited remarkable inhibitory potential against two Leishmania spp.L. donovani strains~~~ namely L. major and

L. donovani (IC50 values between 1.2 and 10.5 µM), were prepared(Amastigotes) and reported [IC by50 (µM)] Ronga et al. [100]. [97] 24a 2.8 24b 2.0

Molecules 2019, 24, 3886 13 of 45

Of all the compounds synthesized by Bekhit et al. [101], compound 28, whose hybrid analog baasically resulted from hybridization with five-membered heterocyclici moieties, including 1,3,4-thiadiazoles and pyrazolines, exhibited the greatest potential in terms of antileishmanial activity. It was the best candidate among the series against L. aethiopica promastigotes (IC50 = 0.0142 µM) and amastigotes (IC50 = 0.13 µM). Compound 29, an acetamide derivative of (+) -dehydroabietylamine derivative reported by Dae-ayuela et al [102], was found to be the most potent leishmanicidal agent among the series (IC50 = 1.5 µM). It was even more active than the reference compound, miltefosine (IC50 = 3.4 µM). A series of 2-phenyl-3-(pyridin-4-yl)imidazo[1,2-a]pyrazine derivatives were synthesized by Marchand et al. [103], and their antileishmanial activities were evaluated. Among the synthesized molecules, compounds 30a and 30b were found to be the most potent analogs against L major promastigotea and amastigotes with IC50 values between 0.2 and 6.4 µM. Sangshetti et al. [105] synthesized a series of indolyl–coumarin hybrids, and after evaluating their antileishmanial potential in vitro reported that compounds 31a and 31b were excellent antileishmanial agents (IC50 values between 95 and 99 µg/mL) compared to the standard drug SSG (IC50 = 490 µg/mL) [104]. The sntileishmanial potentials of 1,3,6-trisubstituted β-carboline derivatives, synthesized by Lunagariya et al. were evaluated, and compound 32 (IC50 = 9.0 µM) was found to show an comparable antileishmanial activity comparable to that of the standard drug, miltefosine (IC50 = 11.9 µM). Kumar et al. [106] reported the synthesis of a new series of aryl substituted ketene dithioacetals, which were evaluated in vitro for their activity against L. donovani. Based on their results, compounds 33a and 33b were reported as the most potent antileishmanial agents, with IC50 values of 5.12 and 3.56 µM respectively. Among the 4-arylamino-6-nitroquinazolines synthesized by Saad et al. [107], compounds 34a and 34b were found to be the most potent inhibitors of L. major promastigotes (IC50 values of 1.87 and 4.37 µM, respectively) compared to the standard drug, pentamidine (IC50 = 5.09 µM). Gopinath et al. [108] developed a series of substituted quinoline analogs and assessed their antileishmanial activities. They found compound 35 to be the most active (IC50 = 0.84 µM). Among the diselenide and derivatives developed by Baquedano et al. [109] compounds 36a–c were found to be potent antileishmanial agents, with IC50 values of 1.40, 1.47 and 0.83 µM, respectively, against L. infantum intracellular amastigotes. An assessment of the antileishmanial potential of triazolopyridyl pyridyl ketone derivatives developed by Adam et al. [110] revealed that compounds 37a and 37b elicited potent growth inhibition against cultured Leishmania spp. promastigotes and amastiogotes with IC50 values ranging between 19.5 and 114.6 µM. Sharma et al. [111] synthesized Triazino indole–quinoline hybrid as antileishmanial agents targeting L. donovani. Their results showed that compounds 38a and 38b significantly inhibited L. donovani extracellular promastigotes and intracellular amastigotes with IC50 values ranging between 0.36 and 8.57 µM. Among the heteroretinoid-bisbenzylidine ketone hybrids developed by Tiwari et al. [112], compounds 39a–c were identified as the most potent agents against L. donovani intramacrophagic amastigotes, with IC50 values between 1.83 and 5.02 µM. Pandey et al. [113] synthesized indole-2-carboxamide derivatives using utilizing the isocyanide based multicomponent reaction and evaluated them against L. donovani. Their results showed that among them, compound 40 (IC50 = 0.6 µM) exhibited a more promising antileishmanial activity than those of standard drugs, including SSG (IC50 = 56.1 µM) and miltefosine (IC50 = 8.4 µM). Several Clofazimine analogs were synthesized by Barteselli et al. [114] and their antileishmanial activities were screened using an in vitro evaluation, which demonstrated that compound 41 was the most potent antileishmanial agent against L. infantum, and L. tropica. A series of 8 imidazole derivatives were developed by Vita et al. [115], and an in vitro analysis of their antileishmanial activities showed that out of the 8 compounds, compounds 42a and 42b were the most potent antileishmanial molecules with IC50 values of 12.7 and 8.0 µM against L. infantum respectively. Reddy et al. [116] synthesized a large series of benzyl phenyl ether derivatives and evaluated their biological activities. Their results showed that compounds 43a–e were potent antileishmanial agents and compounds 43b (IC50 = 1.6 µM), 43d (IC50 = 1.27 µM) and 43e (IC50 = 1.39 µM) showed the greater antileishmanial activity against L. donovani compared to that of the standard drug, pentamidine (IC50 = 1.84 µM). Zhang et al. [117] prepared a novel series of oxyneolignans virolin, Molecules 2019, 24, 3886 14 of 45 surinamensin, and analogs using an asymmetric synthetic method. Thereafter, their ability to inhibit the growth of different protozoal strains was tested. Their results showed that compounds 44a and 44b exerted the maximum antileishmanial activities with IC50 of 2.29 µg/mL and 2.48 µg/mL, respectively. The antiprotozoal activities of novel oxadiazolyl pyrrolo triazole diones derivatives synthesized by Dürüst et al. [118] was investigated. The results showed that compounds 45a (IC50 = 1.6 µg/mL) and 45b (IC50 = 2.0 µg/mL) were the most active antileishmanial agents against L. donovani. Pierson et al. [119] synthesized a series of novel 4-arylcoumarin derivatives, and the determination of their antiprotozoal activities against various biological strains revealed that compounds 46a–c, with different substitutions on phenyl rings, displayed the most potent activity against L. donovani amastigotes (IC50 = 1.1–5.4 µM). Patric et al. [120] developed a series of bis-pyridylimidamide derivatives, and an assessment of their antileishmanial activities showed that among them, compound 47a was the most active, and inhibited the L. amazonensis strain with an IC50 value of 0.095 µM, while the others, including 47b (IC50 = 0.123 µM) and 47c (IC50 = 0.211 µM), exhibited slightly more potent activity compared with (IC50 = 0.124 µM). Diaryl sulfide inhibits L. infantum promastigotes, and an evaluation of the inhibitory activity of its deriatives by Saccoliti et al. [121] showed that compound 48 inhibited L. infantum promastigotes by a dose-dependent amount with an IC50 value of 29.43 µM. Preeti et al. [122] developed tellurium derivate, immunomodulatory, and demonstrated that compound 49 could eliminate L. donovani promastigotes, and an evaluation by in vitro assay showed that it had a significant growth inhibitory effect on L. donovani promastigotes with an IC50 value of 26.9 µM. Rodríguez-Hernandez et al. [123] converted hederagenin into 1,2,3-trizolyl derivatives aiming to obtain antileishmanial and cytotoxic compounds. Of the synthesized compounds, compound 50 was found to be the most potent antileishmanial molecule, with an IC50 value of 5.6 µM. The thiadazole scaffold is a prevalent heterocyclic ring with activity. Tahghighi et al. [124] developed its derivatives, among which compounds 51a and 51b (IC50 between 0.08=9.35 µM) were found to be the most potent antileishmanial agents inhibiting extracellular promastigotes and amastigotes. In summary, antileishmanial agents of diverse scaffolds such as chalcone, arylamidine, thiohydrazone, and polyheteroaromatics, were reported. Som of the compounds exhibited comparable activity with pentamidine or miltefosine. In particular, 1,5-diphenylpenta-1,4-dien-3-one derivatives (39) and Ether-tether phenylamidine (43) displayed good activity profiles.

2.3. Anti-Malaria Malaria is a deadly mosquito-borne disease that mainly affects humans. It is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Five different species of Plasmodium, P. falciparum, P. ovale, P. malariae, P. vivax, and P. knowlesi are responsible for the spread of malaria; P. falciparum is considered the most dangerous and virulent form. The disease is transmitted via the sucking of human blood by infected female Anopheles mosquitoes [125], and symptoms include fever, fatigue, vomiting, and headache; in severe cases, it can cause yellow skin, seizures, coma, and death [126]. The symptoms usually begin 10 to 15 days after infection, and disease recurrence may be observed months later if not properly treated. In those who have recently survived an infection, reinfection usually causes milder symptoms. This partial resistance disappears over months to years if the person has no continuing exposure to malaria [126]. According to the 2014 world malaria report by the WHO, a total of 198 million cases of malaria and nearly 584,000 malaria deaths occurred in 2013 [127]. In 2013, 3.4 billion people were at risk of malaria, of whom 1.2 billion were at a higher risk with more than one case per 1000 people, especially in over 97 countries in tropical areas with the ongoing transmission of malaria. Ninety percent of the above-mentioned deaths occurred in sub-Saharan Africa, of which 77% were children under the age of five. In 2010, an estimated 660,000 malaria deaths were reported worldwide [128]. Additional reports suggest that malarial infection and mortality are more widespread than previously estimated by the report of Murray et al., with up to 200 million clinical cases and 1.2 million deaths reported in 2010 alone [129]. Because of its devastating effects on the human population, the WHO rates malaria as one of the top three infectious diseases Molecules 2019, 24, 3886 15 of 45 worldwide [130]. In brief, malaria has become one of the major causes of illness in humans, with approximately 250 million clinical cases reported around the world annually, particularly in poor or developing countries [127]. P. falciparum causes most of the severe cases and the most deaths, and nearly 80% of all reported cases and as mentioned above 90% of malaria-attributed deaths occur in Africa [131]. Moreover, P. vivax is the predominant species, in Asia, the Middle East, Central and South America, and the Western Pacific [132]. Malarial parasites exhibit a complex life cycle involving an insect vector (mosquito) and a vertebrate host (human), and all species exhibit a similar life cycle with only minor variations. The infection is initiated when sporozoites are injected into the human body through the saliva of a feeding Anopheles mosquito. When sporozoites enter the human body and travel through the bloodstream to the liver they transform into exoerythrocytic forms (EEFs) [133]. Based on the Plasmodium species, these forms are converted into mature exoerythrocytic-stage schizonts, or enter a dormant phase in which they are called hypnozoites, which only two species of Plasmodiums, P. vivax and P. ovale make. These hypnozoites reactivate several weeks to months (or years) after the primary infection and are responsible for malaria relapses weeks, months, or even years after the initial infection [134]. Fully developed exoerythrocytic-stage merozoites eventually exit the liver and re-enter the bloodstream [133]. They enter the red blood cells (RBCs) and replicate asexually causing RBC destruction, which leads to the characteristic symptoms associated with malaria such as anemia, fever, and chills [135]. A small percentage of these asexual blood-stage parasites then differentiate into sexual erythrocytic stages (female and male gametocytes) whose transmission back to the mosquito vector during a subsequent blood meal completes the life cycle [136]. Important antimalarial agents are presented in Figure3. In the past, malaria was treated with the bark of cinchona (Cinchona rubra [Rubiaceae]); however, at this time, it was not known that cinchona bark contains , which was later isolated and shown to have antimalarial properties [137,138]. Some medicinal chemists developed simpler synthetic analogs of quinine such as chloroquine, , , and , which all had a quinine pharmacophore, but did not have multiple stereogenic centers. Among them, chloroquine was found to be the most efficient drug, and it has served humanity for over five decades [42,139]. However, the spread of chloroquine resistance prompted medicinal chemists to re-investigate the chemistry and pharmacology of alternative 4-aminoquinoline antimalarials such as amodiaquine [132], which has proven to be effective against chloroquine-resistant parasite strains [23–27]. Amodiaquine is effective against many chloroquine-resistant strains of P. falciparum [126]. However, its clinical use has been severely restricted because of its associations with hepatotoxicity and agranulocytosis [28,29]. Although in recent years a natural endoperoxide and its semisynthetic derivatives , , and have been employed for the treatment of malaria owing to chloroquine resistance in parasites, but the global deployment of artemisinin-based combination therapy is limited by its relatively high cost of treatment, safety concerins during pregnancy, and early signs of resistance in Southeast Asia [140–142]. Despite the market availability of large numbers of antimalarial drugs, no perfect drug is known because individual drugs and drug combinations have their own limitations including poor compliance, side effects, toxicity, and resistance. Therefore, owing to the aforementioned conditions, some researchers have designed and synthesized novel molecules for antimalarial purposes. In this review article, we have compiled the latest data on antimalarial agents designed from 2012–2017. The details of these antimalarial agents are summarized in Table3. Molecules 2019, 24, x FOR PEER REVIEW 16 of 45 Molecules 2019, 24, x FOR PEER REVIEW 16 of 45 Moleculesand agranulocytosis 2019, 24, x FOR PEER[28,29]. REVIEW Although in recent years a natural endoperoxide artemisinin and16 ofits 45 andsemisynthetic agranulocytosis derivatives [28,29]. artemether Although, artesunate, in recent years and dihydroartemisinina natural endoperoxide have artemisininbeen employed and forits and agranulocytosis [28,29]. Although in recent years a natural endoperoxide artemisinin and its semisyntheticthe treatment ofderivatives malaria owing artemether to chloroquine, artesunate, resist andance dihydroartemisinin in parasites, but thehave global been deployment employed for of semisynthetic derivatives artemether, artesunate, and dihydroartemisinin have been employed for theartemisinin-based treatment of malaria combination owing totherapy chloroquine is limited resist byance its in relatively parasites, high but thecost global of treatment, deployment safety of the treatment of malaria owing to chloroquine resistance in parasites, but the global deployment of artemisinin-basedconcerins during pregnancy, combination and therapy early signs is limited of resistance by its relativelyin Southeast high Asia cost [140–142]. of treatment, Despite safety the artemisinin-based combination therapy is limited by its relatively high cost of treatment, safety concerinsmarket availability during pregnancy, of large andnumbers early ofsigns antimala of resistancerial drugs, in Southeast no perfect Asia drug [140–142]. is known Despite because the concerins during pregnancy, and early signs of resistance in Southeast Asia [140–142]. Despite the marketindividual availability drugs and of drug large combinations numbers of have antimala their rialown drugs, limitations no perfect including drug poor is compliance,known because side marketindividualeffects, toxicity,availability drugs and and resistance.of drug large combinations numbers Therefore, of have owing antimala thei tor theownrial aforementioned limitationsdrugs, no includingperfect conditions, drug poor is somecompliance, known researchers because side individualeffects,have designed toxicity, drugs and and and synthesized resistance. drug combinations Therefore, novel molecules haveowing thei forto rthe antimalarialown aforementioned limitations purposes. including conditions, In this poor review some compliance, researchers article, weside effects,have designedcompiled toxicity, and theand latestsynthesized resistance. data on Therefore,nove antimalariall molecules owing ag forents to theantimalarial designed aforementioned from purposes. 2012–2017. conditions, In this The review somedetails article,researchers of these we havehaveantimalarial designedcompiled agents andthe latestsynthesizedare summarized data on nove antimalarial inl moleculesTable 3. ag ents for antimalarialdesigned from purposes. 2012–2017. In this The review details article, of these we Moleculeshaveantimalarial compiled2019, 24 agents, 3886 the latestare summarized data on antimalarial in Table 3. agents designed from 2012–2017. The details of16 ofthese 45 antimalarial agents are summarized in Table 3.

Figure 3. Currently available antimalarial drugs. Figure 3. Currently available antimalarial drugs. Table 3. Selected data of reported antimalarial agents. FigureFigure 3. 3.Currently Currently available available antimalarial antimalarial drugs. drugs. Table 3. Selected data of reported antimalarial agents. Table 3. Selected data of reported antimalarial agents. CompoundsTable 3. Selected data of reported antimalarialActivity agents. Ref. Compounds Activity Ref. Compounds ActivityP. falciparum [IC50 (µM)] Ref. 62 P. falciparum1.60 [IC 50 (µM)] Compounds ActivityP. falciparum [IC (µM)] Ref. 62 1.60 50 P. falciparum [IC50 (µM)] 62 1.60 [143] Dihydro- 62 0.00111.60 [143[143]] artemisininDihydro- 0.0011 Dihydro-artemisininartemisinin 0.0011 [143] Dihydro- 0.0011 artemisinin P. falciparum [IC50 (nM)] P. falciparum [IC (nM)] 63a P. falciparum11.7 [IC 50 (nM)]50 63b63a 63a 11.713.511.7 63b P. falciparum13.5 [IC50 (nM)] Chloroquine63b 36.3713.5 [144] Chloroquine63a 11.7 36.37 [144] Chloroquine 36.37 [144] Molecules 2019, 24, x FOR PEER REVIEW 63b 13.5 17 of 45 ArtemisininArtemisinin 4.37 4.37 Chloroquine 36.37 [144] Artemisinin P. falciparum4.37 [IC 50 (µM)] P. falciparum [IC50 (µM)] Artemisinin 3D7 strain 4.37 W2 strain 3D7 64a 3.76 W25.97 strain strain 64b 8.49 4.58 [145] 64a 3.76 5.97 [145] 64b 8.49 4.58 Chloroquine 0.021 0.49 Chloroquine 0.021 0.49

P. falciparum [IC50 (µM)] W2 strain D6 strain 65a 0.071 0.065 65b 0.079 0.069 [146] Artemisinin 0.007 0.009

Chloroquine 0.63 0.014

P. falciparum [IC50 (nM)] 66 27.4 Chloroquine 300

[147]

Mefloquine 92

P. falciparum [IC50 (µM)] D6 strain W2 strain 67 2.56 3.41 Artemisinin <0.09 <0.09 [148]

Chloroquine <0.08 0.72

P. falciparum [IC50 (nM)] 68a 0.3 68b 0.7 68c 1.5 [149]

Artemisinin 19

P. falciparum [IC50 (nM)] 69 2.6 ± 0.4 Chloroquine 9.8 ± 2.8 [150] Dihydro- 1.1 ± 0.5 Artemisinin

P. falciparum [IC50 (µM)] [151] 3D7 K1

Molecules 2019, 24, x FOR PEER REVIEW 17 of 45 Molecules 2019, 24, x FOR PEER REVIEW 17 of 45 Molecules 2019, 24, x FOR PEER REVIEW P. falciparum [IC50 (µM)] 17 of 45 Molecules 2019, 24, x FOR PEER REVIEW 3D7P. falciparum strain [ICW250 (µM)] strain 17 of 45 Molecules 2019, 24, x FOR PEER REVIEW 64a 3D7P. falciparum3.76 strain [ICW250 (µM)]5.97 strain 17 of 45 64b64a 3D7P. falciparum8.493.76 strain [ICW250 (µM)]4.585.97 strain 64b64a 3D7P. falciparum8.493.76 strain [ICW250 (µM)]4.585.97 strain [145] 64b64a 3D73.768.49 strain W25.974.58 strain [145] Chloroquine64b64a 0.0218.493.76 4.585.97 0.49 [145] Chloroquine64b 0.0218.49 4.58 0.49 [145]

Molecules 2019, 24, 3886 Chloroquine 0.021 0.49 [145]17 of 45

Chloroquine P. 0.021falciparum [IC50 (µM)] 0.49

Chloroquine W2P. 0.021falciparum strain [IC50D6 (µM)] 0.49 strain Table 3. Cont. 65a W2P. 0.071falciparum strain [IC50D6 (µM)]0.065 strain

Compounds65b65a ActivityW2P. 0.0790.071falciparum strain [IC50D6 (µM)]0.0690.065 strain Ref. P. falciparum [IC50 (µM)] [146] Artemisinin65b65a W20.0070.0790.071 strain D6 0.0090.0690.065 strain P. falciparum [IC50 (µM)] [146] Artemisinin65b65a W20.0710.0070.079 strain D60.065 0.0090.069 strain 65a 0.071W2 0.065 [146] ChloroquineArtemisinin65b 0.0790.0070.63 D60.069 0.0140.009 strain strain [146] ChloroquineArtemisinin65b 0.0790.0070.63 0.069 0.0090.014 65a 0.071 0.065 [146[146]] Artemisinin 0.007 0.009 Chloroquine 65b P. falciparum0.630.079 [IC50 0.069(nM)] 0.014

ChloroquineArtemisinin66 P. falciparum0.63 0.007 27.4 [IC 50 0.009(nM)]0.014 Chloroquine 0.63 0.014 ChloroquineChloroquine66 P. falciparum 0.6327.4300 [IC 50 0.014(nM)]

Chloroquine66 P. falciparum27.4300 [IC 50 (nM)] Chloroquine66 P. falciparumP. falciparum27.4300 [IC [IC50 (nM)]50 (nM)] [147] 66 66 27.427.4 [147] ChloroquineMefloquine 30092 Chloroquine 300 [147] ChloroquineMefloquine 30092 [147[147]] 92 [147] Mefloquine 92 Mefloquine P. falciparum92 [IC 50 (µM)] Mefloquine 92 P.D6 falciparum strain [ICW250 (µM)] strain

67 P.D6 falciparum2.56 strain [ICW250 (µM)]3.41 strain P. falciparum [IC50 (µM)] 67 P.D6 falciparum2.56 strain [ICW250 (µM)]3.41 strain Artemisinin <0.09 <0.09 [148] P. falciparumD6 strain [IC W250 (µM)] strain Artemisinin67 D6<0.092.56 strain W2 <0.093.41 strain [148] Artemisinin67 67 D6<0.092.56 strain2.56 W2<0.09 3.413.41 strain Chloroquine <0.08 0.72 [148[148]] Artemisinin67 2.56< 0.09 <3.410.09 ChloroquineArtemisinin <0.09<0.08 <0.09 0.72 [148] Artemisinin <0.09 <0.09 ChloroquineChloroquine <0.08<0.08 0.72 0.72 [148] P. falciparum [IC50 (nM)] Chloroquine <0.08 0.72 P. falciparum [IC50 (nM)] Chloroquine68a <0.08 0.3 0.72 P. falciparum [IC50 (nM)] 68b68a P. falciparum0.70.3 [IC 50 (nM)]

68b68c68a 68a P. falciparum1.50.70.3 [IC 0.350 (nM)] 68b 68b68a68c P. falciparum0.31.50.7 [IC 0.750 (nM)] 68c 1.5 [149] 68b68a68c 0.70.31.5 [149[149]] 68b68c 1.50.7 [149] ArtemisininArtemisinin 19 19 Artemisinin68c 1.519 [149] Artemisinin 19 [149]

Artemisinin P. falciparum19 [IC 50 (nM)] Artemisinin P. falciparum19 [IC50 (nM)] 69 P. falciparum2.6 ± 0.4[IC 50 (nM)] 69 2.6 0.4 Chloroquine69 P. falciparum 9.82.6 ± 2.80.4[IC± 50 (nM)] Chloroquine 9.8 2.8 Chloroquine69 P. falciparum 9.82.6 ± [IC2.80.4± 50 (nM)] [150[150]] ChloroquineDihydro-69 P. falciparum2.6 9.8 ± [IC0.42.8 50 (nM)] [150] Molecules 2019, 24, x FOR PEER REVIEW Dihydro-Artemisinin1.1 1.1± 0.5 0.5 18 of 45 ChloroquineArtemisininDihydro-69 2.6 9.8 ± 0.42.8± [150] 1.1 ± 0.5 ChloroquineArtemisininDihydro- 9.8 ± 2.8 [150] 70a 0.62 1.1 ± 0.5 1.78 [150] ArtemisininDihydro- P. falciparum 50 µ 70b P. falciparum0.29 [IC[IC (µM)]500.496( M)] Dihydro- 1.1 ± 0.5 [151] Artemisinin P. falciparum3D7 [IC50 (µM)]K1 3D1.17 ± 0.5 K1 [151] Artemisinin P. falciparum3D7 [IC50 (µM)]K1 70a 0.62 1.78 [151] P. falciparum3D7 [IC50 (µM)]K1 70b 0.29 0.496 [151[151]] P. falciparum [IC50 (µM)] 3D7 K1 [151] Chloroquine 0.0053D7 0.254K1 Chloroquine 0.005 0.254

% Dead asexual parasites~~~

(P. falciparum) 3D7 RKL-2 71a 25.7 5.0 [152]

71b 20.0 1.0

P. falciparum [IC50 (µM)]~~~

3D7 strain 72a 3.71 [153] 72b 3.95 Chloroquine 0.18

P. falciparum [IC50 (µM)] 73a 0.018 73b 0.015 [154]

Chloroquine 0.062

P. falciparum [IC50 (µM)] 3D7 K1 74a 0.87 2.04 74b 0.3 2.11 [155]

Chloroquine 0.011 1.2

P. falciparum [IC50 (µM)]~~~

3D7 strain 75a 0.24 75b 0.09 [156]

Chloroquine 0.028

P. falciparum [IC50 (µM)]~~~

3D7 strain [157] 76a 0.007

Molecules 2019, 24, x FOR PEER REVIEW 18 of 45 Molecules 2019, 24, x FOR PEER REVIEW 18 of 45 Molecules 2019, 24, x FOR PEER REVIEW 18 of 45 Molecules 2019, 24, x FOR PEER REVIEW 70a 0.62 1.78 18 of 45 Molecules 2019, 24, x FOR PEER REVIEW 70a 0.62 1.78 18 of 45 70b70a 0.290.62 0.4961.78 70b70a 0.620.29 0.4961.78 70b70a 0.290.62 0.4961.78 70b 0.29 0.496 70b 0.29 0.496

Chloroquine 0.005 0.254 Chloroquine 0.005 0.254 Molecules 2019, 24, 3886 Chloroquine 0.005 0.254 18 of 45 Chloroquine 0.005 0.254 Chloroquine 0.005 0.254

Table 3. Cont. % Dead asexual parasites~~~ % Dead asexual parasites~~~ Compounds % ActivityDead( P.asexual falciparum parasites~~~) Ref. % Dead( P.asexual falciparum parasites~~~) % Dead3D( 7P.asexual % falciparum Dead parasites~~~ asexualRKL-2) 3D(P.7 falciparumparasitesRKL-2) 71a 25.73D(P.7 falciparumRKL-2) 5.0 [152] 71a 25.73D7 (P. falciparumRKL-25.0) [152] 71a 25.73D7 RKL-25.0 [152] 71a 25.73D 7 RKL-25.0 [152] 71b71a 20.025.7 1.05.0 [152[152]] 71b71a 20.025.7 5.01.0 71b 20.0 1.0 71b 20.0 1.0 71b P. falciparum20.0 [IC50 (µM)]~~~1.0 71b P. falciparum20.0 [IC50 (µM)]~~~ 1.0 7 3D strain50 P. falciparum3D7 strain[IC (µM)]~~~ 72a P. falciparumP. falciparum3.71 [IC 50[IC (µM)]~~~(µM)] 72a P. falciparum3D3.717 strain[IC 50 (µM)]~~~ 50 [153] 3D73D strainstrain [153] 72b 3D3.957 strain7 72b72a 3.953.71 [153] 72a 72a 3.713.71 Chloroquine72b72a 0.183.953.71 [153[153]] 72b72b 3.953.95 [153] Chloroquine72b 0.183.95 Chloroquine P. falciparum0.18 [IC 50 (µM)] ChloroquineChloroquine P. falciparum0.18 [IC 0.18 50 (µM)] Chloroquine73a 0.0180.18 73a P. falciparum0.018 [IC 50 (µM)] P. falciparumP. falciparum [IC[IC50 (µM)](µM)] 73b73a P. falciparum0.0150.018 [IC 50 50(µM)] 73b73a 0.0180.015 73b73a73a 0.0150.0180.018 [154] 73b73b 0.0150.015 [154] 73b 0.015 [154[154]] Chloroquine 0.062 [154] Chloroquine 0.062 [154] ChloroquineChloroquine 0.062 0.062 Chloroquine 0.062 Chloroquine 0.062 P. falciparum [IC50 (µM)] P. falciparum [IC50 (µM)] 7 1 3DP. falciparum [IC50 50K(µ M)] P. falciparum7 [IC (µM)]1 P. falciparum3D [IC50 (µM)]K 74a 0.87 50 2.04 P. falciparum3D73D 7 [IC (µM)]KK11 74a 0.873D7 2.04K1 74b 3D0.37 2.11K1 74b74a74a 0.870.3 0.87 2.042.112.04 74a 0.87 2.04 [155] 74b74a74b 0.870.3 0.3 2.112.112.04 [155] 74b 0.3 2.11 [155] 74b 0.3 2.11 [155] [155] Chloroquine 0.011 1.2 [155] ChloroquineChloroquine 0.011 0.011 1.2 1.2 Chloroquine 0.011 1.2 Chloroquine 0.011 1.2 Chloroquine 0.011 1.2 P. falciparumP. falciparum [IC50[IC (µM)]~~~(µM)] P. falciparum [IC50 (µM)]~~~50 3D73D7 strain strain P. falciparum3D7 [ICstrain50 (µM)]~~~ P. falciparum [IC50 (µM)]~~~ 75a 75a P. falciparum3D70.24 [ICstrain0.24 50 (µM)]~~~ 75a 3D70.24 strain 75b75a75b 3D70.090.24 strain0.09 [156[156]] 75b75a 0.240.09 75a 0.24 [156] 75b 0.09 [156] Chloroquine75b 0.09 0.028 Molecules 2019, 24, x FOR PEER REVIEW Chloroquine75b 0.0280.09 19[156] of 45 Chloroquine 0.028 [156]

Chloroquine 0.028 Chloroquine P. falciparum0.028 [IC (µM)] Chloroquine P. falciparum0.028 [IC50 (µM)]~~~50 P. falciparum3D [IC7 strain50 (µM)]~~~ 3D7 strain [157] P. falciparum [IC50 (µM)]~~~ 3D7 strain [157] 76a 76a P. falciparum0.007 [IC0.00750 (µM)]~~~ P. falciparum3D7 strain[IC50 (µM)]~~~ [157] 76a 3D0.0077 strain [157] 76a 3D0.0077 strain [157[157]] 76b76a 0.0070.017 76a 0.007 76b 0.017

P. falciparum [IC50 (µM)]~~~

K1 strain 77a 0.28 77b 0.095 [158]

Chloroquine 0,12

P. falciparum [IC50 (µM)]~~~

K1 strain 78 0.6

[115]

Chloroquine 0.14

P. falciparum [(IC50 µM)]~~~

W2 strain 79a 0.33 ± 0.095 79b 0.2 ± 0.05

[159]

Mefloquine 0.04 ± 0.01

P. falciparum [IC50 (µM)] 80a 0.006 80b 0.004 80c 0.006 [116]

Chloroquine 0.201

P. falciparum [IC50 (µg/mL)] 81a 0.608 [117] 81b 2.50

Molecules 2019, 24, x FOR PEER REVIEW 19 of 45 Molecules 2019, 24, x FOR PEER REVIEW 19 of 45 Molecules 2019, 24, x FOR PEER REVIEW 19 of 45 Molecules 2019, 24, x FOR PEER REVIEW 19 of 45

76b 0.017 76b 0.017 76b 0.017 Molecules 2019, 24, 3886 76b 0.017 19 of 45

Table 3. Cont. P. falciparum [IC50 (µM)]~~~

CompoundsP. Activity falciparumK1 strain [IC50 (µM)]~~~ Ref. P. falciparum [IC50 (µM)]~~~ 77a P. falciparumK1 0.28strain [IC 50 (µM)]~~~µ P. falciparumK1 strain[IC 50 ( M)] 77b77a K10.0950.28 strainK strain 77a 0.281 [158] 77b77a 0.0950.28 [158] 77b 77a 0.0950.28 [158] Chloroquine77b77b 0.0950,120.095 [158[158]] Chloroquine 0,12 ChloroquineChloroquine 0,12 0.12 Chloroquine P. falciparum0,12 [IC 50 (µM)]~~~

P. falciparumK1 strain [IC50 (µM)]~~~ P. falciparumP. falciparum [IC50[IC (µM)]~~~(µM)] 78 P. falciparumK1 strain0.6 [IC 50 (µM)]~~~50 K1 strainK1 strain 78 K1 0.6strain 78 78 0.6 0.6 [115] 78 0.6 [115[115]] Chloroquine 0.14 [115] [115] Chloroquine 0.14 ChloroquineChloroquine 0.14 0.14 Chloroquine 0.14

P. falciparum [(IC50 µM)]~~~

P. falciparumP. falciparumW2 strain[(IC50[(IC µM)]~~~µM)] P. falciparum [(IC50 µM)]~~~50 79a P. falciparum0.33W2 W2 ±strain[(IC 0.095 strain50 µM)]~~~ W2 strain 79b79a 79a 0.33W20.20.33 ±strain 0.0950.050.095 79a 0.33 ± 0.095± 79b79a 79b 0.330.2 0.2± 0.050.0950.05 79b 0.2 ± 0.05± [159] 79b 0.2 ± 0.05 [159[159]] [159] Mefloquine 0.04 ± 0.01 [159] Mefloquine 0.04 0.01 Mefloquine 0.04 ± 0.01± Mefloquine 0.04 ± 0.01 Mefloquine 0.04 ± 0.01

P. falciparum [IC50 (µM)] P. falciparum [IC (µM)] 80a P. falciparum0.006 [IC 50 (µM)]50 P. falciparum [IC50 (µM)] 80b80a 80a P. falciparum0.0060.004 0.006[IC 50 (µM)] 80a 80b 0.0060.004 80b80a80c 0.0060.004 80b 80c 0.0040.006 [116[116]] 80b80c 0.0040.006 80c 0.006 [116] Molecules 2019, 24, x FOR PEER REVIEW 80c 0.006 20[116] of 45 Molecules 2019, 24, x FOR PEER REVIEW ChloroquineChloroquine 0.201 0.201 20[116] of 45 Chloroquine 0.201 Chloroquine 0.201 Chloroquine P. falciparum0.201 [IC P. falciparum [IC50 (µg/mL)]50 (µg/mL)] 81a P. falciparum0.608 [IC50 (µg/mL)] [117] 81a P. falciparum [IC0.60850 (µg/mL)] 81b81a P. falciparum0.6082.50 [IC 50 (µg/mL)] [117] 81a 81b 0.6082.50 [117] 81b81a 0.6082.50 [117] 81b 2.50 81b 2.50 81c 1.71 [117]

81c 1.71

P. falciparum [IC50 (µg/mL)] P. falciparumP. falciparum [IC50 (µg/mL)][IC50 82a 13.2(µg/ mL)] 82b 82a 14.713.2 82b 14.7 [118[118]] Chloroquine 0.073 Chloroquine 0.073

P. falciparum [IC[IC50 (µM)](µM)] 83a 1.1 ± 1.1 83b 1.9 ± 0.9 83c 1.5 ± 0.14

[160]

Mefloquine 0.004 ± 0.02

P. falciparum [IC50 (µM)](µM)] 84a 0.106 84b 0.149 [161]

Chloroquine 0.0039

P. falciparum [IC50 (µM)](µM)] 85a 0.002 85b 0.003 Chloroquine 0.125 [120]

Artemisinin 0.004

P. falciparum [IC50 (nM)](nM)] W2W2 D6D6 86a 45.78 26.38 86b 9.47 4.10 [162] 86c 5.93 3.95

Artemisinin 6.7 9.00

P. falciparum [IC50 (nM)](nM)] [163] [163]

Molecules 2019, 24, x FOR PEER REVIEW 20 of 45 Molecules 2019, 24, x FOR PEER REVIEW 20 of 45 Molecules 2019, 24, x FOR PEER REVIEW 20 of 45 Molecules 2019, 24, x FOR PEER REVIEW 20 of 45

81c 1.71 81c 1.71 81c 1.71 81c 1.71

P. falciparum [IC50 (µg/mL)]

82a P. falciparum13.2 [IC 50 (µg/mL)]

82b82a P. falciparum13.214.7 [IC 50 (µg/mL)] 82b82a P. falciparum13.214.7 [IC 50 (µg/mL)] [118] 82b82a 14.713.2 [118] Chloroquine 0.073 [118] Molecules 2019, 24, 3886 Chloroquine82b 0.07314.7 20 of 45 [118] Chloroquine 0.073 P. falciparum [IC50 (µM)] Chloroquine 0.073 Table 3.83a Cont . P. falciparum1.1 ± [IC1.1 50 (µM)]

83a P. falciparum1.1 ± [IC1.1 50 (µM)] Compounds83b Activity1.9 ± 0.9 Ref. 83b83a P. falciparum1.11.9 ± 1.10.9[IC 50 (µM)] 83c P. falciparum1.5 ± 0.14[IC (µM)] 83b83c83a 1.51.91.1 ± ±± 0.14 0.91.1 50 83a 1.1 1.1 [160] 83c83b 1.51.9 ± ± 0.14 0.9± 83b 1.9 0.9 [160] 83c 1.5 ± 0.14± 83c 1.5 0.14 Mefloquine 0.004 ± ±0.02 [160] Mefloquine 0.004 ± 0.02 [160[160]] Mefloquine 0.004 ± 0.02 Mefloquine 0.004 0.02 Mefloquine 0.004 ± 0.02 ± P. falciparum [IC50 (µM)]

84a P. falciparum0.106 [IC 50 (µM)]

84b84a P. falciparum0.1060.149 [IC 50 (µM)] P. falciparum [IC (µM)] 84b84a P. falciparum0.1060.149 [IC 50 50(µM)] [161] [161] 84b84a 84a 0.1490.1060.106 Chloroquine 0.0039 [161] Chloroquine84b84b 0.00390.1490.149 [161] [161] Chloroquine 0.0039 Chloroquine P. falciparum0.0039 [IC 50 (µM)]

Chloroquine85a P. falciparum0.00390.002 [IC 50 (µM)] P. falciparum [IC50 (µM)] 85b85a P. falciparum0.0020.003 [IC 50 (µM)] 85a 0.002 Chloroquine85b85a P. falciparum0.0020.1250.003 [IC 50 (µM)] 85b 0.003 [120] Chloroquine85b85a 0.0030.1250.002 Chloroquine 0.125 [120[120]] Chloroquine85b 0.1250.003 Artemisinin 0.004 [120] ChloroquineArtemisinin 0.125 0.004 Artemisinin 0.004 [120]

Artemisinin 0.004 P. falciparum [IC50 (nM)] Artemisinin P. falciparum0.004 [IC50 (nM)] P. falciparumW2 [IC50 (nM)]D6 W2 D6 86a P. 45.78falciparumW2 [IC50 (nM)]26.38D6 86b86a 86a P.45.78 9.47falciparumW245.78 [IC50 26.38(nM)]4.10D6 86b86a 86b 45.789.47W29.47 26.38 4.104.10D6 [162[162]] 86c 5.93 3.95 [162] Molecules 2019, 24, x FOR PEER REVIEW 86a 86c 45.785.93 26.38 3.95 21 of 45 Molecules 2019, 24, x FOR PEER REVIEW 86b86c 9.475.93 4.103.95 21 of 45 Artemisinin 6.7 9.00 [162] Artemisinin86c86b 5.939.476.7 3.95 9.004.10 D6 Dd2 [162] Artemisinin86c 5.93D66.7 Dd2 9.003.95 87a 6.5 7.4 P. falciparum [IC50 (nM)] Artemisinin87a P. falciparum6.76.5 [IC50 (nM)] 9.007.4 [163] Artemisinin P. falciparum6.7 D6 [IC50 (nM)] Dd2 9.00 [163]

P. falciparum [IC50 (nM)] [163] 87a 6.5 7.4 P. falciparum [IC50 (nM)] [163] 6.7 [163] 87b 6.7 3.5 87b 6.7 3.5 87b 3.5

P. falciparum [IC50 (µM)] P. falciparumP. falciparum [IC50 (µM)]µ 88 0.02 [IC50 ( M)] 88 88 0.020.02

[164[164]] [164] Chloroquine 15~25 ChloroquineChloroquine 15~25 15~25

P. falciparum [IC50 (µM)] P. falciparum [IC50 (µM)] 89a 0.17 ± 0.01 89a 0.17 ± 0.01

[165] [165] 89b 0.23 ± 0.01 89b 0.23 ± 0.01

P. falciparum [IC50 (µM)] P. falciparum [IC50 (µM)] 90a 0.43 ± 0.02 90a 0.43 ± 0.02 90b 0.53 ± 0.04 90b 0.53 ± 0.04 90c 0.37 ± 0.03 90c 0.37 ± 0.03 [166] [166]

Chloroquine 0.06 Chloroquine 0.06

Important Highlights of Table 3 Compounds Important Highlights of Table 3 Compounds Compounds of different heterocycle classes are included in Table 3 as potent antimalarial agents Compounds of different heterocycle classes are included in Table 3 as potent antimalarial agents and the results of their assessment were compared to those of the standard drugs and the results of their assessment were compared to those of the standard drugs dihydroartemisinine, chloroquine, artemisinin, mefloquine, puromycin, artesunate, artemether, dihydroartemisinine, chloroquine, artemisinin, mefloquine, puromycin, artesunate, artemether, artesunic acid, artemisinin derived alcohol, di-artemisinin, , and quinone. artesunic acid, artemisinin derived alcohol, di-artemisinin, proguanil, and quinone. Pingaew et al. [143] synthesized 11 chalcone coumarin hybrids linked by the 1,2,3-triazole ring. Pingaew et al. [143] synthesized 11 chalcone coumarin hybrids linked by the 1,2,3-triazole ring. All the derivatives were screened, and the evaluation of their potent antimalarial activity showed All the derivatives were screened, and the evaluation of their potent antimalarial activity showed that among them, compound 62 significantly inhibited a P. falciparum culture with an IC 50 value of that among them, compound 62 significantly inhibited a P. falciparum culture with an IC 50 value of 1.60 µM. Nisha et al. [144] developed β-amino-alcohol tethered 4-aminoquinoline-isatin conjugates, 1.60 µM. Nisha et al. [144] developed β-amino-alcohol tethered 4-aminoquinoline-isatin conjugates, and evaluation of their antimalarial activities revealed that compounds 63a and 63b were the most and evaluation of their antimalarial activities revealed that compounds 63a and 63b were the most potent antimalarial agents, with IC50 values of 11.7 and 13.5 nM respectively. Kumar et al. [145] potent antimalarial agents, with IC50 values of 11.7 and 13.5 nM respectively. Kumar et al. [145] synthesized various triazole tethered isatin-ferrocene derivatives and evaluated their antimalarial synthesized various triazole tethered isatin-ferrocene derivatives and evaluated their antimalarial activities against chloroquine-susceptible and chloroquine-resistant P. falciparum strains. The results activities against chloroquine-susceptible and chloroquine-resistant P. falciparum strains. The results

Molecules 2019, 24, x FOR PEER REVIEW 21 of 45 Molecules 2019, 24, x FOR PEER REVIEW 21 of 45 D6 Dd2 87a 6.5D6 Dd27.4 87a 6.5 7.4

87b 6.7 3.5 87b 6.7 3.5

P. falciparum [IC50 (µM)] 88 P. falciparum0.02 [IC 50 (µM)] 88 0.02

[164] Chloroquine 15~25 [164] Molecules 2019, 24, 3886 Chloroquine 15~25 21 of 45

Table 3. Cont.

P. falciparum [IC50 (µM)] Compounds Activity Ref. 89a P. falciparum0.17 ± 0.01[IC50 (µM)] µ 89a P. falciparum0.17 ± 0.01[IC 50 ( M)] 89a 0.17 0.01 ± [165[165]] 89b 0.23 ± 0.01 [165] 89b 0.23 0.01 89b 0.23 ± 0.01±

P. falciparum [IC50 (µM)] µ 90a P. falciparumP. falciparum0.43 ± 0.02[IC[IC50 (µM)]50 ( M)] 90a 90a 0.430.43 ± 0.020.02 90b 0.53 ± 0.04± 90b 0.53 0.04 90b 0.53 ± 0.04± 90c 90c 0.370.37 ± 0.030.03 90c 0.37 ± 0.03± [166[166]] [166] Chloroquine 0.06 Chloroquine 0.06 Chloroquine 0.06

ImportantImportant Highlights Highlights of of Table Table3 Compounds3 Compounds Important Highlights of Table 3 Compounds CompoundsCompounds of of di differentfferent heterocycle heterocycle classes classes areare includedincluded inin TableTable3 3 as as potentpotent antimalarialantimalarial agents andand the Compoundsthe results results of their ofof different assessmenttheir heterocycleassessment were compared classes were are toco thoseinclmpareduded of the into standardTable those 3 as drugs ofpotent the dihydroartemisinine, antimalarial standard agentsdrugs chloroquine,dihydroartemisinine,and the results artemisinin, of chloroquine,their mefloquine, assessment artemisinin, puromycin, were artesunate,mefloquine,compared artemether, topuro thosemycin, artesunicof artesunate, the standard acid, artemether, artemisinin drugs derivedartesunicdihydroartemisinine, alcohol, acid, artemisinin di-artemisinin, chloroquine, derived proguanil, alcohol,artemisinin, and di-artemisinin, quinone. mefloquine, proguanil, puromycin, and quinone. artesunate, artemether, artesunicPingaewPingaew acid, et et artemisinin al. al. [ 143[143]] synthesized synthesized derived alcohol, 1111 chalconechalcone di-artemisinin, coumarincoumarin proguanil, hybrids linked and quinone. by the the 1,2,3-triazole 1,2,3-triazole ring. ring. AllAll the thePingaew derivatives derivatives et al. were [143]were screened, synthesizedscreened, and and 11 the thechalcone evaluationevaluation coum ofarinof theirtheir hybrids potentpotent linked antimalarialantimalarial by the 1,2,3-triazole activity showed ring. All the derivatives were screened, and the evaluation of their potent antimalarial activity showed thatthat among among them, them, compound compound62 62significantly significantly inhibitedinhibited aa P.P. falciparumfalciparum cultureculture withwith an IC 5050 valuevalue of of 1.601.60thatµ M.µM.among Nisha Nisha them, et al.et al. [compound144 [144]] developed developed 62 significantlyβ-amino-alcohol β-amino-alcohol inhibited tethered tethered a P. falciparum 4-aminoquinoline-isatin 4-aminoquinoline-isatin culture with an conjugates, IC conjugates, 50 value and of evaluationand1.60 evaluationµM. Nisha of their ofet antimalarial theiral. [144] antimalarial developed activities activities β-amino-alcohol revealed revealed that compounds tetheredthat compounds 4-aminoquinoline-isatin63a and 63a63b andwere 63b the were mostconjugates, the potent most and evaluation of their antimalarial activities revealed that compounds 63a and 63b were the most antimalarialpotent antimalarial agents, with agents, IC50 withvalues IC of50 values 11.7 and of 13.511.7 nM and respectively. 13.5 nM respectively. Kumar et al. Kumar [145] synthesizedet al. [145] varioussynthesizedpotent triazoleantimalarial various tethered agents,triazole isatin-ferrocene with tethered IC50 isatin-ferrocevalues derivatives of 11.7 andne and derivatives evaluated 13.5 nM their andrespectively. antimalarialevaluated Kumar their activities antimalarial et al. against [145] chloroquine-susceptibleactivitiessynthesized against various chloroquine-susceptible triazole and chloroquine-resistanttethered isatin-ferroce and chloroquine-resistantP.ne falciparumderivativesstrains. and P. falciparum evalua Theted results strains. their showedantimalarial The results that compounds activities against64a and chloroquine-susceptible64b were potent antimalarial and chloroquine-resistant agents. Several aminoalkylated P. falciparum quercetin strains. The derivatives results were synthesized by Helgren et al. [146] by using the mannich reaction were screened for antimalarial activity using an in vitro assay. The results demonstrated THAT compounds 65a and 65b were the most potent antimalarial agents against three drug-resistant malarial strains (D6 and W2), with IC50 values between 0.065 and 0.079 µM. A series of fosmidomycin analogs were developed by Phillips et al. [147] and analyzed in vitro. The results showed that of all the synthesized compounds, compound 66 was the most potent antimalarial molecule with an IC50 values of 27.4 nM against chloroquine- and mefloquine-resistant Dd2 strains of P.falciparum. Yadavet al. [148] synthesized a series of marine-derived indole alkaloid derivatives, and an evaluation of their biological activities showed that compound 67 was the most potent antimalarial agent. Le et al. [149] designed a novel series of 11-aza-artemisinin analogus and tested them to determine their ability to inhibit the growth of FcB1 strains of P. falciparum. The results showed that compounds 68a–c exerted the greatest antimalarial activities with IC50 values of 0.3, 0.7, and 1.5 µM respectively. Two dimers and two trimers of artemisinin hybrids were synthesized by Reiter et al. [150] and their antimalarial activities were investigated using an antimalarial assay against P. falciparum 3D7 strains. Of all the compounds, compound 69 was reported to be the most active antimalarial agents, with an IC value of 2.6 0.4 nM. Parthiban et al. [151] synthesized a series 50 ± of chloroquinoline-4H-chromene conjugates with piperazine and azpane rings as tethers, and the Molecules 2019, 24, 3886 22 of 45 determination of their antimalarial activities against 3D7 and K1 strains of P. falciparum showed that compounds 70a and 70b displayed the most potent antimalarial activity with IC50 values between 0.29 and 1.78 µM. Bhat et al. [152] developed a series of hybrid 4-aminoquinoline 1,3,5-triazine derivatives, and an assessment their antimalarial activities showed that compounds 71a and 71b were the most active inhibiting chloroquine-sensitive (3D7) and chloroquine-resistant (RKL-2) P. falciparum strains with IC50 values between 1 and 25 µM. The results of the docking studies, revealed that the most active compounds bound well with P. falciparum dihydrofolate reductase thymidylate synthase (pf -DHFR-TS). Different oxirane compounds were synthesized by Carneiro et al. [153] and subjected to antimalarial screening against chloroquine-sensitive 3D7 strains of P.falciparum after evaluation, compounds 72a and 72b were reported to be the most active species among 18 synthesized compounds. Karad et al. [154] synthesized various analogs of morpholinoquinoline-based conjugates with an incorporated pyrazoline ring, and the evaluation of their antimalarial activities in vitro using the biological assay revealed that compounds 73a and 73b were the most active antimalarial agents with IC50 values between 0.015 and 0.018 µM. Devender et al. [155] synthesized a new series of triazoles and in vitro antimalarial activity evaluation found that among the different compounds synthesized, compounds 74a and 74b displayed excellent biological activity against 3D7 and K1 P. falciparum strains, with IC50 value between 0.3 and 2.11 µM. Svogie et al. [156] prepared a series of indolyl-3-ethanone α-thioethers, and an evaluation of their antimalarial activities revealed that compounds 75a (IC50 = 0.24 µM) and 75b (IC50 = 0.09 µM) were the most active species against 3D7 P. falciparum strains. The development of new antimalarial agents, led to the synthesis of a new series of imidazo[4,5-c]quinolin-2-one derivatives by Patel et al. [157] using a four-step synthetic route. The evaluation of an antimalarial activities of derivatives using an allamar-Blue gametocytocidal assay showed that compound 76a was the most potent candidate among the series. Seebacher et al. [158] prepared a series of several azabicyclic compounds, and an evaluation of their antimalarial activities showed that compounds 77a and 77b, with IC50 values of 0.28 and 0.095 µM, respectively, showed a remarkable antimalarial activity against 3D7 P. falciparum strains. Further analysis proved that compound 77b was the most potent P. falciparum inhibitor of the active species. A new imidazole-based series of substituted ester and carbamate derivatives were synthesized by Vita et al. [115], and an in vitro investigation revealed that compound 78 showed the highest antimalarial effect against K1 P. falciparum strains (IC50 = 0.6 µM). Inam et al. [159] designed and synthesized several acylhydrazine derivatives, attached to chloroqunoline nuclei with piperazine rings. An evaluation of their antiprotozoal activities revealed that among the compounds, compounds 79a (IC50 = 0.33) and 79b (IC50 = 0.2 µM) were moderately active against w2 strains of P. falciparum. Patrick et al. [116] developed a large series of cationic benzyl phenyl ether derivatives for application in antiprotozoal drug development. Their results showed that compounds 80a–c were extremely potent P. falciparum inhibitors, with IC50 values of 0.006, 0.004, and 0.006 µM, respectively. The screening and evaluation of the antiprotozoal activities of fifteen 8,4-oxyneolignans analogs prepared by Rye et al. [117], using asymmetric synthesis revealed that compounds 81a–c showed IC50 values between 0.608 and 2.50 µM against P. falciparum. Dürüst et al. [118] developed several derivatives of triazoles coupled with 1,2,4-oxadiazole moieties, and an investigation of their antiprotozoal activities revealed that compounds 82a and 82b were the most active P. falciparum inhibitors, with an IC50 values of 13.2 and 14.7 µg/mL, respectively. A new series of metal complexes designed and prepared by Juneja et al. [160] were evaluated for their antimalarial activity against w2 P. falciparum strains. The in vitro study revealed that compounds 83a–c were the best candidates with IC50 values between 1.1 and 1.9 µM. McKeever et al. [161] developed aminoalkyl derivatives form guanidine diaromatic minor groove binder, and evaluated their antiprotozoal activities. The results showed that compounds 84a (IC50 = 0.106 µM) and 84b (IC50 = 0.149 µM) elicited a significant P. falciparum culture inhibitory. Patrick et al. [120] synthesized thirty-six 4,4”-Diamidino-m-terphenyl analogs, which were tested for their antiprotozoal drug development. Among these 36 compounds, compounds 85a and 85b, which bear a dimethyltetrahydropyrimidinyl ring at the para-position, exhibited significant antimalarial activity with IC50 values of 0.002 and 0.003 µM, respectively, which were lower than that of their parent drug, Molecules 2019, 24, 3886 23 of 45 chloroquine. From the study, it was evident that a substituent attached at position 4 of a phenyl ring would lead to the exhibition of excellent inhibitory action. Opsenica et al. [162] synthesized a novel series of aminochloroquinoline derivatives, and assessed their antimalarial activities in vivo using different P. falciparum strains. The results revealed that compounds 86a–c were notable antimalarial agents with IC50 values between 3.95 and 45.78 nM. Hanessian et al. [163] designed a novel series of pactamycin analogs, and an evaluation of their biological activities found that compounds 87a (IC50 = 3.5 nM) and 87b (IC50 = 6.7 nM) were the most potent molecules against D6 and Dd2 strains of P. falciparum. Abada et al. [164] evaluated 24 porphyrin precursors and derivatives against different protozoal strains. Their results showed that compound 88 was the most active member of the series. Its IC50 value 0.02 µM was 100 to 200 times lower than that of standard drug chloroquine (15–25 µM). Yeo S J et al. [165] synthesized chloroquine derivatives with phenylmethyl groups and unsaturated amides, which have anti-malarial activity. Among them, compounds 89a and 89b showed greater antimalarial activity against 3D7 P. falciparum strains with IC50 values of 0.17 and 0.23 µM, respectively. Singh et al. [166] synthesized 4-aminoquinolin-ferrocenyl-chalcone derivatives, and the evaluation of their pharmacological properties revealed that compounds 90a–c were the most potent compounds against W2 strain of P. falciparum; their IC50 values ranged from 0.37 to 0.53 µM. In summary, various chemical scaffolds and its analogues such as triazole-tethered chalcone- coumarin hybrids, chloroquinoline-isatin hybrids, flavonones, indolesulfonamides, artemisinin derivatives, N,N0-diaryl substituted piperizines, porphyrin derivatives, and polyaryls present significant antimalarial activities. In particular, artemisinin derivatives (68) in which lactone was transformed to lactam, then various hydrophilic substituents were introduced, showed good inhibitor activity. As novel scaffolds, symmetric terphenyl cyclic amidines (85) exhibited slightly better antimalarial activity than chloroquine and artemisinin.

2.4. Anti-Trichomoniasis Trichomoniasis is a protozoan infection caused by the flagellate protozoan T. vaginalis. It is one of the most prevalent nonviral sexually transmitted diseases worldwide [167]. The protozoan T. vaginalis affects both men and women. In women, the symptoms of this infection worsen during menstruation whereas in men the infection is largely asymptomatic; these asymptomatic men are considered carriers [168]. Trichomoniasis in men and women is associated with birth outcomes [169], infertility [170], cervical and prostate cancers [171], and pelvic inflammatory disease [172]. In men, this disease is characterized by irritation inside the penis, mild discharge, or slight burning after urination or ejaculation and in women, it is associated with yellow-green vaginal discharge with a strong odor. The infection may also cause discomfort during sex and urination, as well as irritation and itching of the female genital area. In rare cases, lower abdominal pain can occur. The symptoms usually appear in women within 5–28 days of exposure. Generally, the prevalence of T. vaginalis infection is found to be higher among women than men. A recent report on trichomoniasis revealed that the prevalence of trichomoniasis in non-human immunodeficiency virus (HIV)-infected persons was 10.1% among women vs. 2.0% among men [173], when compared to HIV-infected women (10%–20% prevalence of trichomoniasis) [174]. Data regarding the prevalence of T. vaginalis in men who have sex with men (MSM) are scarce. T. vaginalis infection damages the vaginal epithelium, which increases the risk of women being infected by HIV, and thereby considerably increasing the chances of infected women transmitting HIV to her sexual partner(s) [175,176]. In short, T. vaginalis is a co-factor in HIV transmission and acquisition [177,178]. According to a WHO report, approximately 248 million new cases of trichomoniasis are reported worldwide annually. It is believed that two to three million symptomatic infections occur annually among sexually active women in the United States [179]. In the Republic of Korea, 10.4% of women complaining of vaginal symptoms and signs were found to be infected with T. vaginalis [180]. According to the literature the pathogenicity of T. vaginalis is due to cysteine peptidases (CP) enzymes [181,182] which may present on their cell surfaces as secretion products of the Molecules 2019, 24, x FOR PEER REVIEW 24 of 45 Molecules 2019, 24, x FOR PEER REVIEW 24 of 45 usuallyMolecules 2019appear, 24, x inFOR women PEER REVIEW within 5–28 days of exposure. Generally, the prevalence of T. vaginalis24 of 45 usuallyinfection appear is found in towomen be higher within among 5–28 women days of than exposure. men. A Generally,recent report the on prevalence trichomoniasis of T. revealedvaginalis infectionusuallythat the prevalenceappear is found in towomen of be trichomoniasis higher within among 5–28 in women non-hudays of thanman exposure. men. immunodeficiency A Generally,recent report the viru on prevalence strichomoniasis (HIV)-infected of T. revealed vaginalispersons thatinfectionwas the10.1% prevalence is among found towomen of be trichomoniasis higher vs. 2.0% among among in women non-hu men than man[173], men.immunodeficiency when A comparedrecent report to viru HIV-infectedon strichomoniasis (HIV)-infected women revealed persons (10%– wasthat20% 10.1%theprevalence prevalence among of women trichomoniasis)of trichomoniasis vs. 2.0% among [174] in non-hu. Datamen [173],manregarding immunodeficiency when the compared prevalence to viru HIV-infected of sT. (HIV)-infected vaginalis women in men persons (10%– who 20%havewas 10.1%prevalence sex with among men of women trichomoniasis) (MSM) vs. are 2.0% scarce. among[174] T.. vaginalisData men regarding[173], infection when the compareddamages prevalence theto HIV-infected ofvaginal T. vaginalis epithelium, women in men which(10%– who haveincreases20% prevalencesex withthe risk men of of trichomoniasis) (MSM)women are being scarce. infected [174] T.. vaginalisData by HIV, regarding infection and thereby the damages prevalence considerably the ofvaginal T. increasing vaginalis epithelium, in the men chances which who increasesofhave infected sex withthe women risk men of transmitting(MSM)women are being scarce. HIV infected to T. her vaginalis by sexual HIV, infection partner(s)and thereby damages [175,176]. considerably the In vaginal short, increasing T.epithelium, vaginalis the chances is which a co- offactorincreases infected in HIV the women risktransmission of transmitting women and being acquisition HIV infected to her [177,178].by sexual HIV, partner(s)and According thereby [175,176]. to considerably a WHO In report,short, increasing T.approximately vaginalis the chancesis a 248co- factormillionof infected in new HIV women cases transmission of transmitting trichomoniasis and acquisition HIV are to reported her [177,178]. sexual worldwide partner(s) According annually. [175,176]. to a WHO It Inis report,believedshort, T.approximately thatvaginalis two tois threea 248co- millionfactor in newsymptomatic HIV cases transmission of trichomoniasis infections and acquisition occur are annually reported [177,178]. among worldwide According sexually annually. to active a WHO It women is report,believed in approximately the that United two to States three 248 million[179]. In newsymptomatic the casesRepublic of trichomoniasis infectionsof Korea, 10.4%occur are annuallyof reported women among worldwide complaining sexually annually. of active vaginal It women is symptoms believed in the that and United two signs to States threewere [179].foundmillion In to symptomatic thebe infectedRepublic with infectionsof Korea, T. vaginalis 10.4%occur [180]. annuallyof women among complaining sexually of activevaginal women symptoms in the and United signs States were found[179].According Into thebe infected Republic to the with ofliterature Korea, T. vaginalis 10.4%the pathogenicity[180]. of women complaining of T. vaginalis of vaginalis due to symptoms cysteine peptidases and signs were(CP) foundenzymesAccording to be[181,182] infected to whichthe with literature mayT. vaginalis present the pathogenicity [180]. on their cell surf of T.aces vaginalis as secretion is due products to cysteine of the peptidases parasite [183–(CP) Molecules 2019, 24, 3886 24 of 45 enzymes187]. AccordingThese [181,182] enzymes to whichthe play literature may a critical present the role pathogenicity on in their pathogenicity, cell surf of T.aces vaginalisas as well secretion as is in due the products to biological cysteine of the processespeptidases parasite of [183– (CP) this 187].protozoan.enzymes These [181,182] Although,enzymes which play MTZ, may a critical which present roleis approved on in their pathogenicity, cell by surf theaces FDA as as well is secretion the as drug in the products of biological choice of for the processes trichomoniasis, parasite of [183– this protozoan.[188]187].parasite Theserecent[ 183 Although,enzymes studies–187]. haveplay TheseMTZ, ashown critical enzymeswhich that roleis approved play thisin pathogenicity, adrug critical by ha ths rolee severalFDA as in wellis pathogenicity, toxicthe as drug ineffects the of biological choice [14–18], as well for asprocesses andtrichomoniasis, in the the clinical biologicalof this [188]resistanceprotozoan.processes recent of Although, of studiesmany this protozoan.microbes have MTZ, shown which has Although, reduced that is approved this its MTZ, drug efficien by whichha thscye several FDA[189,190]. is approved is toxicthe In drug effectsview by of the of choice[14–18], FDAthese for ismajor and thetrichomoniasis, drugdrawbacks,the clinical of choice resistancethere[188]for trichomoniasis, recentis an of urgent studiesmany needmicrobes have [188 for] shown recent the has development reduced studiesthat this its have drug efficien of shownnew hacys andseveral [189,190]. that efficient this toxic drugIn scaffolds vieweffects has of several[14–18], theseagainst major toxic andtrichomoniasis. edrawbacks,theffects clinical [14–18 ], resistance of many microbes has reduced its efficiency [189,190]. In view of these major drawbacks, thereTherefore,and is the an clinical researchersurgent resistanceneed have for thedesigned of manydevelopment and microbes synt ofhesized hasnew reduced and some efficient novel its effi agentsscaffoldsciency as [ 189inhibitorsagainst,190]. trichomoniasis. Inof viewT. vaginalis of these there is an urgent need for the development of new and efficient scaffolds against trichomoniasis. Therefore,growth.major drawbacks, In researchersthis review there article,have is andesigned we urgent have and needcompiled synt forhesized the the developmentlatest some data novel (from of agents new2012–2017) andas inhibitors effi oncient the scaofdevelopment T.ffolds vaginalis against Therefore, researchers have designed and synthesized some novel agents as inhibitors of T. vaginalis growth.oftrichomoniasis. novel Inantitrichomonial this review Therefore, article, agents. researchers we have The resultscompiled have are designed the summarized latest and data synthesized in(from Table 2012–2017) 4. some novel on the agents development as inhibitors growth. In this review article, we have compiled the latest data (from 2012–2017) on the development ofof novelT. vaginalis antitrichomonialgrowth. In agents. this review The results article, are we summarized have compiled in Table the latest 4. data (from 2012–2017) on the of novel antitrichomonial agents. The results are summarized in Table 4. development of novelTable antitrichomonial 4. Selected data agents. of reported The results antitrichomonal are summarized agents. in Table4. Table 4. Selected data of reported antitrichomonal agents. CompoundTable 4. Selected data of reported antitrichomonalActivity agents. Ref. Table 4. Selected data of reported antitrichomonal agents. Compound ActivityT. vaginalis [IC50 (µM)] Ref. Compound Activity Ref. Compound91a ActivityT. vaginalis0.331 [IC 50 (µM)] Ref. 91a91b T. vaginalis0.3310.221 [IC 50 (µM)] T. vaginalis [IC (µM)] [63] 91b91a 0.2210.331 50 91a 0.331 [63] MTZ91b 0.2210.290 [63] 91b [63] MTZ 0.2900.221

MTZMTZ T. vaginalis 0.290 [IC 50 (µM)]

92 T. vaginalis0.09 [IC 50 (µM)] µ 92 T.T. vaginalis vaginalis0.09[IC [IC 5050( (µM)]M)] 9292 0.09 [191] MTZ 0.72 [191][191 ] MTZ 0.72 MTZ 0.72 [191] MTZ 0.72

T. vaginalis [IC50 (µM)] T. vaginalis [IC (µM)] 93 T. vaginalis7.06 [IC 5050 (µM)] 9393 T. vaginalis7.06 [IC 50 (µM)] 93 7.06 Molecules 2019, 24, x FOR PEER REVIEW 25[192] of[192 45 ] MTZ 0.72 Molecules 2019, 24, x FOR PEER REVIEW MTZ 0.72 25[192] of 45 Molecules 2019, 24, x FOR PEER REVIEW MTZ 0.72 25[192] of 45 MTZ 0.72

T. vaginalis [IC (µg/mL)] T. vaginalis [IC5050 (µg/mL)] MTZ 1.56 12.5 MTZ MTZ-susceptibleT. vaginalisMTZ-1.56 [IC50MTZ-resistant (µg/mL)]MTZ-12.5 50 [193] 94aMTZ T.susceptible vaginalisMTZ-1.56 [IC (µg/mL)]resistantMTZ-12.5 [193] 1.56 3.125 94b MTZ- MTZ- 94a susceptible1.561.56 resistant3.125 [193]

MTZ94a94b susceptible 1.561.56 resistant3.125 12.5 [193] T. vaginalis [IC50 (µM)] 94b94a T. 1.56vaginalis [IC50 (µM)]3.125 95 0.023± 0.005µ 94b95 T.T. 1.56vaginalis vaginalis0.023± [IC [IC0.0055050( (µM)]3.125 M)] Nitazoxanide 0.107 ± 0.009 [66] 9595 0.023±0.023 0.005 Nitazoxanide 0.107± ± 0.009 [66][66 ] Nitazoxanide 0.107 0.009 NitazoxanideMTZ 0.1070.213 ±± 0.0090.004 [66]

MTZMTZ 0.213 0.213 ± 0.004 MTZ 0.213 ±± 0.004 T. vaginalis [IC50 (µg/mL)] 96 T.T. vaginalis vaginalis 83.9[IC[IC5050 ((µg/mL)]µg/mL)] 9696 T. vaginalis83.9 [IC50 (µg/mL)] 96 83.9 [62][62 ] MTZMTZ 0.037 [62] MTZ 0.037 [62] MTZ 0.037

T.foetus [IC50 (µM)] 97a T.foetus 22.2[IC50 (µM)] T.foetus [IC50 (µM)] 97a97b 22.211.3 97a 22.2 97b97c 11.324.5 97b97c 24.511.3 [194] 97c 24.5 [194] [194] MTZ 0.72 MTZ 0.72 MTZ 0.72

T. vaginalis [IC50 (µM)] 98a T. vaginalis9.86 [IC 50 (µM)] T. vaginalis [IC50 (µM)] 98a98b 9.869.79 98b98a 9.799.86 [195] 98b 9.79 [195] MTZ 0.72 [195] MTZ 0.72 MTZ 0.72 T. vaginalis [IC50 (µM)] 99 T. vaginalis23 [IC 50 (µM)] 99 T. vaginalis23 [IC 50 (µM)] 99 23 [196] MTZ 0.72 [196] MTZ 0.72 [196] MTZ 0.72

T. vaginalis [IC50 (µM)] 100a T. vaginalis10.49 ±[IC 1.0550 (µM)] T. vaginalis [IC50 (µM)] 100a100b 10.4925.60 ± 1.051.08 100a 10.49 ± 1.05 100b 25.60 ± 1.08 [197] 100b 25.60 ± 1.08 [197] MTZ 0.72 [197] MTZ 0.72 MTZ 0.72

T. vaginalis [IC50 (µg/mL)] T. vaginalis [IC50 (µg/mL)] [198] 101 4.3 ± 1.2 [198] T. vaginalis [IC50 (µg/mL)] 101 4.3 ± 1.2 [198] 101 4.3 ± 1.2

Molecules 2019, 24, x FOR PEER REVIEW 25 of 45 Molecules 2019, 24, x FOR PEER REVIEW 25 of 45 Molecules 2019, 24, x FOR PEER REVIEW 25 of 45 Molecules 2019, 24, x FOR PEER REVIEW 25 of 45

MTZ 1.56 12.5 MTZ 1.56 12.5 MTZ 1.56 12.5 MTZ 1.56 12.5

T. vaginalis [IC50 (µM)]

95 T. vaginalis0.023± [IC 0.00550 (µM)]

Nitazoxanide95 T. vaginalis 0.1070.023± ± [IC 0.0050.00950 (µM)] [66] Nitazoxanide95 T. vaginalis 0.1070.023± ± [IC 0.0050.00950 (µM)] [66] MTZ95 0.2130.023± ± 0.0050.004 Nitazoxanide 0.107 ± 0.009 [66] MTZ 0.213 ± 0.004 Nitazoxanide 0.107 ± 0.009 [66] MTZ T. vaginalis 0.213 [IC ± 0.00450 (µg/mL)]

MTZ96 T. vaginalis 0.21383.9 [IC ± 0.00450 (µg/mL)]

96 T. vaginalis83.9 [IC50 (µg/mL)] 96 T. vaginalis83.9 [IC50 (µg/mL)] [62] 96 83.9 MTZ 0.037 [62] MTZ 0.037 [62] Molecules 2019, 24, 3886 25 of 45 MTZ 0.037 [62] MTZ 0.037 T.foetus [IC50 (µM)]

Table 4.97aCont . T.foetus 22.2[IC50 (µM)]

97a T.foetus 22.2[IC50 (µM)] Compound97b Activity11.3 Ref. 97b97a97c T.foetus11.3 22.224.5[IC50 (µM)] T.foetus [IC (µM)] [194] 97b97c97a 24.511.322.250 [194] 97a97b97c 22.224.511.3 97b 11.3 [194] MTZ97c 0.7224.5 97cMTZ 24.50.72 [194][194 ]

MTZ 0.72 MTZMTZ T. vaginalis 0.72 [IC 50 (µM)]

98a T. vaginalis9.86 [IC 50 (µM)]

98b98a T. vaginalis9.869.79 [IC 50 (µM)] T. vaginalis [IC (µM)] 98b98a T. vaginalis9.799.86 [IC 5050 (µM)] [195] 98a98a 9.86 [195] MTZ98b 9.790.72 98b 9.79 [195][195 ] MTZ98b 0.729.79 [195] MTZMTZ 0.72 T. vaginalis [IC50 (µM)] MTZ 0.72 99 T. vaginalis23 [IC 50 (µM)] T. vaginalis [IC (µM)] 99 T. vaginalis23 [IC 5050 (µM)] 9999 T. vaginalis23 [IC 50 (µM)] 99 23 [196] MTZ 0.72 [196][196 ] MTZMTZ 0.72 [196] MTZ 0.72 [196]

MTZ 0.72

T. vaginalis [IC50 (µM)] T. vaginalis [IC (µM)] 100a T. vaginalis10.49 ±[IC 1.055050 (µM)]

100a100a T. vaginalis10.49 ±[IC 1.0550 (µM)] 100b 25.60 ±± 1.08 Molecules 2019, 24, x FOR PEER REVIEW 100b 25.60 1.08 26 of 45 100b100a T. vaginalis25.6010.49 ±[IC 1.081.0550 (µM)] Molecules 2019, 24, x FOR PEER REVIEW ± 26[197] of[197 45 ] 100b100a 25.6010.49 ± 1.081.05 [197] Molecules 2019, 24, x FOR PEER REVIEW MTZ 0.72 26 of 45 MTZ100b 25.60 0.72 ± 1.08 [197] MTZ 0.72 [197] MTZ 0.72 µ MTZ T.T. vaginalis vaginalis 8.5 [IC[IC ± 500.950 ((µg/mL)] g/mL)] MTZ 0.72 [198] 101MTZ101 T. vaginalis4.3 8.5 [IC ± 1.20.950 (µg/mL)] ± [198] MTZ101 T. vaginalis4.3 8.5 [IC ± 1.20.950 (µg/mL)] [198] [198] MTZ101 T. vaginalis 8.54.3 [IC ± 0.91.250 (µg/mL)] ± [198] 101 T. vaginalis4.3 ± [IC 1.250 (µM)] 102a T. vaginalis5.47 [IC 50 (µM)] T. vaginalis [IC (µM)] 102b102a T. vaginalis5.477.56 [IC 5050 (µM)] 102a102b102a 5.477.56 [199] 102b102b 7.56 [199][199 ] MTZ 0.72 [199] MTZMTZ 0.72 MTZ 0.72

T. vaginalis [IC50 (µM)] T. vaginalis [IC (µM)] 103 T. vaginalis9.73 ± [IC 1.135050 (µM)] 103103 9.73 ± 1.13 T. vaginalis± [IC50 (µM)] 103 9.73 ± 1.13 [200] [200] MTZMTZ 0.72 [200] MTZ 0.72 [200] MTZ 0.72

T. vaginalis [IC50 (µM)] 104 T. vaginalis4.80 ± [IC 0.5450 (µM)] 104 T. vaginalis4.80 ± [IC 0.5450 (µM)] 104 4.80 ± 0.54 [201] MTZ 0.72 [201] MTZ 0.72 [201] MTZ 0.72

T. vaginalis [IC50 (µg/mL)] T. vaginalisMTZ- [IC50 (µg/mL)]MTZ-

T.susceptible vaginalisMTZ- [IC50 (µg/mL)]resistantMTZ-

105a susceptible0.0294MTZ- resistant0.2363MTZ-

105b105a susceptible0.02940.0140 resistant0.23630.1122 [202] 105b105a105c 0.01360.01400.0294 0.10910.11220.2363 [202] 105b105c 0.01360.0140 0.10910.1122 [202] MTZ105c 0.01820.0136 0.36550.1091 MTZ 0.0182 0.3655 MTZ 0.0182 0.3655 Important Highlights of Table 4 Compounds Important Highlights of Table 4 Compounds Compounds of different class heterocycles are included in Table 4 as potent agents against Important Highlights of Table 4 Compounds trichomoniasisCompounds and of the different results ofclass their heterocycles assessments are were included compared in Table with those4 as potentof the standardagents against drugs nitazoxanide,trichomoniasisCompounds tizoxanide, and of the different results MTZ, ofclass albendazole, their heterocycles assessments and nonoxynol.are were included compared in Table with those4 as potentof the standardagents against drugs nitazoxanide,trichomoniasisIn brief, Navarrete-Vázqueztizoxanide, and the results MTZ, of albendazole, their et al. assessments [63] andsynthesized nonoxynol. were compared a novel serieswith thoseeight of of the nitrothiazole standard drugs and benzothiazolenitazoxanide,In brief, Navarrete-Vázqueztizoxanide,derivatives MTZ, and among albendazole, et al. them, [63] andsynthesizedcompounds nonoxynol. 91aa novel and 91bseries showed eight ofeffective nitrothiazole T. vaginalis and cellbenzothiazole growthIn brief, inhibition, Navarrete-Vázquezderivatives with and IC50 among values et al. them,of [63] 0.331 compoundssynthesized and 0.221 µM, 91aa novel respectively.and 91bseries showed eight During ofeffective nitrothiazolestructure-activity T. vaginalis and cellrelationshipbenzothiazole growth inhibition, studies derivatives of withadenosine and IC50 among values and uridine them,of 0.331 compoundsanalogs and 0.221 against µM, 91a T.respectively.and vaginalis 91b showed, Shokar During effective et structure-activityal. [191] T. reportedvaginalis relationshipthatcell growthcompound inhibition, studies 92 (ICof adenosinewith50 = 0.09 IC50 valuesµM) and wasuridine of 0.331the analogsmost and 0.221potent against µM, cand T.respectively. vaginalisidate for, Shokartrichomoniasis During et structure-activityal. [191] treatment. reported that compound 92 (IC50 = 0.09 µM) was the most potent candidate for trichomoniasis treatment. relationship studies of adenosine and uridine analogs against T. vaginalis, Shokar et al. [191] reported that compound 92 (IC50 = 0.09 µM) was the most potent candidate for trichomoniasis treatment.

Molecules 2019, 24, x FOR PEER REVIEW 26 of 45 Molecules 2019, 24, x FOR PEER REVIEW 26 of 45

MTZ 8.5 ± 0.9 MTZ 8.5 ± 0.9

T. vaginalis [IC50 (µM)] 102a T. vaginalis5.47 [IC 50 (µM)] 102b102a 7.565.47 102b 7.56 [199] [199] MTZ 0.72 MTZ 0.72

T. vaginalis [IC50 (µM)] 103 T. vaginalis9.73 ± 1.13 [IC50 (µM)] 103 9.73 ± 1.13

[200] Molecules 2019, 24, 3886 MTZ 0.72 [200]26 of 45 MTZ 0.72

Table 4. Cont.

Compound ActivityT. vaginalis [IC50 (µM)] Ref. 104 T. vaginalis4.80 ± 0.54 [IC50 (µM)] µ 104 T. vaginalis4.80[IC ± 500.54( M)] 104 4.80 0.54 ± [201] MTZ 0.72 [201][201] MTZMTZ 0.720.72

T. vaginalis [IC50 (µg/mL)] T. vaginalis [IC (µg/mL)] T.MTZ- vaginalis [IC50 50 (µg/mL)]MTZ- MTZ-susceptibleMTZ- MTZ-resistantMTZ- susceptible resistant 105a105a 0.0294susceptible0.0294 0.2363resistant 105b 0.0140 0.1122 105b105a 0.01400.0294 0.11220.2363 [202][202 ] 105c 0.0136 0.1091 105c105b 0.01360.0140 0.10910.1122 [202] 105c 0.0136 0.1091 MTZ 0.0182 0.3655 MTZ 0.0182 0.3655 MTZ 0.0182 0.3655

ImportantImportant Highlights Highlights of of Table Table 44 Compounds Compounds Important Highlights of Table 4 Compounds CompoundsCompounds of of different different class class heterocycles heterocycles are are included included in inTable Table 4 4as as potent potent agents agents against against trichomoniasistrichomoniasisCompounds and and theof the different results results of class of their their heterocyclesassessments assessments wereare were in comparedcluded compared in withTable with those 4 those as of potent the of the standard agents standard drugsagainst drugs nitazoxanide,nitazoxanide,trichomoniasis tizoxanide, tizoxanide, and the results MTZ, MTZ, albendazole,of albendazole, their assessments and and nonoxynol. nonoxynol. were compared with those of the standard drugs nitazoxanide,InIn brief, brief, Navarrete-Vázquez Navarrete-Vtizoxanide, MTZ,ázquez albendazole, et etal. al. [63] [63 synthesized] and synthesized nonoxynol. a novel a novel series series eight eight of ofnitrothiazole nitrothiazole and and benzothiazolebenzothiazoleIn brief, deriNavarrete-Vázquez derivativesvatives and and among among et al.them, them,[63] compounds synthesized compounds 91a a91a novelandand 91b series91b showedshowed eight effective of e ffnitrothiazoleective T. vaginalisT. vaginalis and cellcellbenzothiazole growth growth inhibition, inhibition, derivatives with with ICand IC50 50 valuesamongvalues of them, of0.331 0.331 compoundsand and 0.221 0.221 µM, 91aµ M,respectively. and respectively. 91b showed During During effective structure-activity structure-activity T. vaginalis relationshiprelationshipcell growth studies inhibition, studies of of adenosine with adenosine IC50 andvalues and uridine uridine of 0.331 analogs analogs and 0.221 against against µM, T. respectively.vaginalisT. vaginalis, Shokar, Shokar During et al. et structure-activity al.[191] [191 reported] reported thatthatrelationship compound compound studies 9292 (IC (ICof50 adenosine 50= =0.090.09 µM) µandM) was uridine was the the most analogs most potentpotent against cand candidate T.idate vaginalis for for,trichomoniasis Shokar trichomoniasis et al. [191] treatment. reported treatment. that compound 92 (IC50 = 0.09 µM) was the most potent candidate for trichomoniasis treatment. Interestingly, it has also been approved by the US FDA as a potential drug candidate for trichomoniasis treatment. In a one-step reaction of N-substituted β-lactams with a free azide group and 5- substituted isatins containing a terminal alkyne, Raj et al. synthesized a series of β-lactam-isatin-triazole conjugates, and one of them, compounds 93, was found to be capable of selectively inhibiting T. vaginalis growth with an IC50 value of 7.06 µM[192]. A novel series of metronidazole-chalcone conjugates were designed and developed by Anthwal et al. [193], and their antitrichomonal activities against a T. vaginalis culture were evaluated for in vitro. The results revealed that two of the compounds in the series, compounds 94a and 94b were the most effective candidates against both MTZ-susceptible and MTZ-resistant parasite strains. Soria-Arteche et al. [66] synthesized a series of hybrid compounds bearing nitazoxanide and N-methylbenzimidazole moieties using different reagents, and the evaluation of their antiprotozoal activities revealed that among the 13 synthesized molecules, compound 95 (IC50 = 0.023 µM) displayed greater antitrichomonal activity against T. vaginalis compared to those of the other 12 compounds. Several hybrid analogs bearing tetrazole and chromane as bioactive scaffolds were synthesized by Cano et al. [62] using the one pot Ugi-azide multicomponent reaction in the presence of InCl3 as catalyst. Thereafter, they were screened, and their antiprotozoal activities were evaluated and among them, compound 96 was identified as the most potent antitrichomonal agent against T. vaginalis with an IC50 value of 83.9 µM. Nisha et al. [194] prepared a series of N-propargylated-isatin Mannich derivatives as potential antitrichomonal agents, using the one-pot CuCl-catalyzed Mannich-type reaction. An evaluation of their activities against T. foetus revealed that three of the synthesized compounds 97a–c elicited promising inhibitory activity on T. foteus culture growth, with IC50 values between 11.3 and 24.5 µM. In order to develop novel and effective antitrichomonal agents, a series of Molecules 2019, 24, 3886 27 of 45 mono- and bis-uracil-isatin conjugates were prepared using single-step synthetic procedure. After they were evaluated against a T. vaginalis culture. Kumar et.al [195] found that compounds 98a (IC50 = 9.86 µM) and 98b (IC50 = 9.79 µM) were the best candidate. Using a single-step CuCl-catalyzed Mannich-type reaction, Nisha et al. [196] synthesized a large series of Mannish-based compounds in which isatin and 4-aminoquinoline ring are linked to the piperazine nucleus. An evaluation of their antitrichomonal activities revealed that compound 99 was the most potent agent against T. vaginalis with an IC50 value of 23 µM. A series of hybrid conjugates with incorporated β-lactone, triazole, and isatin nuclei were designed and prepared by Raj et al. [197] as as novel T. vaginalis inhibitors. The evaluation of the antitrichomonal activities of these compounds in-vitro demonstrated that compounds 100a and 100b were the most active agents inhibiting the growth of a T. vaginalis culture. Saleh et al. [198] synthesized a new series of hybrid compounds bearing 5-nitrothiazole moiety, and an in vitro assessment of their antiprotozoal activities, and consequently compound 101 displayed the most promising biological activity against T. vaginalis strains, with an IC50 value of 4.3 µg/mL. In a study aimed at developing of antiparasitic agents, Adams et al. [199] prepared thiosemicarbazone-derived ruthenium metal complexes, and after evaluating their inhibitory properties against the in vitro growth of a G3 T. vaginalis strain, they found that the compounds 102a (IC50 = 5.47 µM) and 102b (IC50 = 7.56 µM) displayed the most potent antitrichomonal activities. A novel combinatorial library of β-amino alcohol-based β-lactam–isatin chimeras were designed and developed by Nisha et al. [201], and an evaluation of their potential against T. vaginalis in vitro demonstrated that among the synthesized compounds, compound 103 possessed significant antitrichomonal activity, with an IC50 value of 9.73 µM. Stringer et al. [201] prepared a series of rhodium metal complexes and analyzed them for their antiprotozoal activity. The results showed that compound 104 displayed an IC50 value of 4.80 µM against a T. vaginalis culture. A novel series of coumarin-glyoxal hybrid compounds were synthesized by Gupta et al. [202], and their inhibitory activities against T. vaginalis, as well as their spermicidal activities were evaluated. The results led to the conclusion that compounds 105a–c displayed the most potent trichomonacidal activity.

2.5. Anti-Trypanosomiasis Parasitic infections caused by trypanosomatids constitute a major health problem in countries where poor sanitary conditions are prevalent. Diseases acquired by such infections are considered ‘neglected’ because they receive limited funding for the research and development of new treatments. Amongst the neglected tropical diseases (NTD), human African trypanosomiasis (HAT) is endemic throughout sub-Saharan Africa, while American trypanosomiasis (Chagas disease) affects populations in South and Central America.

2.5.1. HAT/African Sleeping Sickness HAT, also known as African sleeping sickness, is one of the most neglected diseases in regions of sub-Saharan Africa; it affects 70 million people in 36 countries. HAT is a vector-borne disease caused by the protozoa parasite [203–205]. It is transmitted through the bite of an infected tsetse fly or passed from an infected mother to her child through the placenta. This disease has two stages: the first stage involves parasite-included seizures of the hemolymphatic system, and the second involves the transmission of the parasites into the central nervous system (CNS) across the blood–brain barrier [206]. Infection of the CNS leads to a number of symptoms including mental impairment, severe headaches, fever, chronic encephalopathy, and eventual death. The development of effective vaccines would be an option for preventing this deadly disease; however, trypanosomes can evade the host immune’s system because of the high degree of antigenic variation in glycoproteins forming their surface coat [207–212]. Therefore, chemotherapy remains the only viable strategy for the treatment and control of infection. The current chemotherapy for HAT comprises only four drugs. Three of these drugs, , pentamidine, and melarsoprol (Figure4), were developed over 60 years ago, and exhibit severe side effects. Melarsoprol is an arsenical derivative used for the treatment of HAT in the Molecules 2019, 24, x FOR PEER REVIEW 28 of 45

‘neglected’ because they receive limited funding for the research and development of new treatments. Amongst the neglected tropical diseases (NTD), human African trypanosomiasis (HAT) is endemic throughout sub-Saharan Africa, while American trypanosomiasis (Chagas disease) affects populations in South and Central America.

2.5.1. HAT/African Sleeping Sickness HAT, also known as African sleeping sickness, is one of the most neglected diseases in regions of sub-Saharan Africa; it affects 70 million people in 36 countries. HAT is a vector-borne disease caused by the protozoa parasite Trypanosoma brucei [203–205]. It is transmitted through the bite of an infected tsetse fly or passed from an infected mother to her child through the placenta. This disease has two stages: the first stage involves parasite-included seizures of the hemolymphatic system, and the second involves the transmission of the parasites into the central nervous system (CNS) across the blood–brain barrier [206]. Infection of the CNS leads to a number of symptoms including mental impairment, severe headaches, fever, chronic encephalopathy, and eventual death. The development of effective vaccines would be an option for preventing this deadly disease; however, trypanosomes can evade the host immune’s system because of the high degree of antigenic variation in glycoproteins forming their surface coat [207–212]. Therefore, chemotherapy remains the only viable strategy for the treatment and control of infection. The current chemotherapy for HAT comprises only four drugs. Three of these drugs, suramin, pentamidine, and melarsoprol (Figure 4), were Molecules 2019, 24, 3886 28 of 45 developed over 60 years ago, and exhibit severe side effects. Melarsoprol is an arsenical derivative used for the treatment of HAT in the neurological stage, and they have many undesirable or fatal neurological(3%–10%) side stage, effects and in theyaddition have to many the development undesirable of or drug fatal (3%–10%)resistance, sideexhibiting effects a in drug addition failure to rate the developmentof up to 30%. Pentamidine of drug resistance, and suramin exhibiting are a used drug for failure treatment rate of in up the to early 30%. stage Pentamidine of the disease, and suramin before arethe usedinvolvement for treatment of the in theCNS. early Nevertheless, stage of the disease,they have before many the severe involvement side effects of the such CNS. as Nevertheless, low blood theypressure, have manydecreased severe level side of eff consciousness,ects such as low kidney blood pressure,problems, decreased low blood level cell of consciousness,levels, and wheezing kidney problems,[30]. Moreover, low blood , cell levels, which and is wheezing less toxic, [ 30is ].only Moreover, effective eflornithine, against T. brucei which gambiense is less toxic, subspecies is only e[213].ffective Treatment against T.with brucei a combination gambiense subspecies of [213 an]. Treatmentd eflornithine with is aless combination toxic, but ineffective of nifurtimox against and eflornithinethe T. brucei isrhodesiense less toxic, subspecies. but ineffective against the T. brucei rhodesiense subspecies.

Figure 4. Currently available antitrypanosomal drugs (use in medical practice).

2.5.2. Chagas Disease American trypanosomiasis (Chagas disease) is caused by the flagellate parasitic protozoan T. cruzi. This infection was first discovered by the Brazilian physician Carlos Chagas (1879 1934) in 1909. − While Trypanosoma cruzi is transmitted to animals and humans via insect vectors of the Triatominae, a subfamily of the Reduviidae family of insects, commonly known as “kissing bugs” [214], outbreaks of chagas disease are usually caused by foodborne T. cruzi since it produces a very aggressive acute form which is often missed by clinicians. This infection is one of the most threatening diseases in Central and South America. A large number of people, approximately 18 million annually, are infected with this parasite. Among them, 50,000 died owing to heart failure. This disease has become a major public health problem in 22 developing countries in Latin America, where more than eight million people suffer from this infection annually [215]. As depicted in its life cycle, the infection is transmitted by two predominant modes: The first is vectorial, through the infected feces/urine of triatomine bugs, and the second is by blood transfusion: the metacyclic trypomastigotes are released in the feces of the insect vector as it takes a blood meal, and they enter the bloodstream via a bite wound or mucosal membrane. Once inside the host, the metacyclic trypomastigotes invade nearby cells and differentiate into their intracellular amastigote form, which multiplies by binary fission. The transformation into trypomastigotes occurs prior to the release from the cells back into the bloodstream, proliferating the infection cycle. Other forms of transmission include congenital, blood transfusion, and contaminated Molecules 2019, 24, x FOR PEER REVIEW 29 of 45 Molecules 2019, 24, x FOR PEER REVIEW 29 of 45 2.5.2. Chagas Disease 2.5.2. Chagas Disease American trypanosomiasis (Chagas disease) is caused by the flagellate parasitic protozoan T. American trypanosomiasis (Chagas disease) is caused by the flagellate parasitic protozoan T. cruzi. This infection was first discovered by the Brazilian physician Carlos Chagas (1879−1934) in cruzi. This infection was first discovered by the Brazilian physician Carlos Chagas (1879−1934) in 1909. While Trypanosoma cruzi is transmitted to animals and humans via insect vectors of the 1909. While Trypanosoma cruzi is transmitted to animals and humans via insect vectors of the Triatominae, a subfamily of the Reduviidae family of insects, commonly known as “kissing bugs” Triatominae, a subfamily of the Reduviidae family of insects, commonly known as “kissing bugs” [214], outbreaks of chagas disease are usually caused by foodborne T. cruzi since it produces a very [214], outbreaks of chagas disease are usually caused by foodborne T. cruzi since it produces a very aggressive acute form which is often missed by clinicians. This infection is one of the most threatening aggressive acute form which is often missed by clinicians. This infection is one of the most threatening diseases in Central and South America. A large number of people, approximately 18 million annually, diseases in Central and South America. A large number of people, approximately 18 million annually, are infected with this parasite. Among them, 50,000 died owing to heart failure. This disease has are infected with this parasite. Among them, 50,000 died owing to heart failure. This disease has become a major public health problem in 22 developing countries in Latin America, where more than become a major public health problem in 22 developing countries in Latin America, where more than eight million people suffer from this infection annually [215]. As depicted in its life cycle, the infection eight million people suffer from this infection annually [215]. As depicted in its life cycle, the infection is transmitted by two predominant modes: The first is vectorial, through the infected feces/urine of is transmitted by two predominant modes: The first is vectorial, through the infected feces/urine of triatomine bugs, and the second is by blood transfusion: the metacyclic trypomastigotes are released triatomine bugs, and the second is by blood transfusion: the metacyclic trypomastigotes are released in the feces of the insect vector as it takes a blood meal, and they enter the bloodstream via a bite in the feces of the insect vector as it takes a blood meal, and they enter the bloodstream via a bite wound or mucosal membrane. Once inside the host, the metacyclic trypomastigotes invade nearby wound or mucosal membrane. Once inside the host, the metacyclic trypomastigotes invade nearby cells and differentiate into their intracellular amastigote form, which multiplies by binary fission. The cells and differentiate into their intracellular amastigote form, which multiplies by binary fission. The transformation into trypomastigotes occurs prior to the release from the cells back into the transformation into trypomastigotes occurs prior to the release from the cells back into the Moleculesbloodstream,2019, 24 proliferating, 3886 the infection cycle. Other forms of transmission include congenital, blood29 of 45 bloodstream, proliferating the infection cycle. Other forms of transmission include congenital, blood transfusion, and contaminated food, to a lesser extent. This disease is endemic to Latin America, transfusion, and contaminated food, to a lesser extent. This disease is endemic to Latin America, affecting people from Mexico to Argentina [216]. The origin and development of this disease is food,affecting to a people lesser extent.from Mexico This disease to Argentina is endemic [216]. to The Latin origin America, and development affecting people of this from disease Mexico is to divided into three phases: The (i) acute (short), (ii) latent (long lasting), and (iii) chronic (very serious Argentinadivided into [216 three]. The phases: origin The and (i) acute development (short), (ii) of latent this disease(long lasting), is divided and (iii) into chronic three phases:(very serious The (i) condition) phases. In the chronic phase, symptoms such as cardiomyopathy and malformation of the acutecondition) (short), phases. (ii) latent In the (long chronic lasting), phase, and symptoms (iii) chronic such (very as cardiomyopathy serious condition) and phases.malformation In the of chronic the intestines (e.g., megaesophagus and megacolon) have been reported. The administration of the phase,intestines symptoms (e.g., megaesophagus such as cardiomyopathy and megacolon) and malformation have been reported. of the intestines The administration (e.g., megaesophagus of the antiparasitic drugs benznidazole and nifurtimox is 100% effective in the short acute phase, and both antiparasitic drugs benznidazole and nifurtimox is 100% effective in the short acute phase, and both anddrugs megacolon) act through have the beengeneration reported. of free The radicals administration that kill ofthe the parasite. antiparasitic If no treatment drugs benznidazole occurs at this and drugs act through the generation of free radicals that kill the parasite. If no treatment occurs at this nifurtimoxstage, then isthis 100% infection effective silently in the progresses short acute into phase, the chronic and both phase drugs in act which through internal the generationorgans such of as free stage, then this infection silently progresses into the chronic phase in which internal organs such as radicalsthe heart, that peripheral kill the parasite. nervous If system, no treatment esophagus, occurs and at thiscolon stage, are irreversibly then this infection affected. silently Benznidazole progresses the heart, peripheral nervous system, esophagus, and colon are irreversibly affected. Benznidazole intoand thenifurtimox chronic phaseare no inlonger which efficient internal in organs the chronic such asphase, the heart, and the peripheral health of nervous the patients system, deteriorates esophagus, and nifurtimox are no longer efficient in the chronic phase, and the health of the patients deteriorates andrapidly colon leading are irreversibly to death, usually affected. due Benznidazole to heart failur ande. Therefore, nifurtimox the are development no longer eofffi newcient antichagasic in the chronic rapidly leading to death, usually due to heart failure. Therefore, the development of new antichagasic phase,drugs andis of thethe healthutmost of importance the patients and deteriorates urgency [216]. rapidly In Table leading 5, we to death, compiled usually the latest due to data heart (2012– failure. drugs is of the utmost importance and urgency [216]. In Table 5, we compiled the latest data (2012– Therefore,2017) on drug the developmentdevelopment ofagainst new antichagasictrypanosomiasis. drugs is of the utmost importance and urgency [216]. In2017) Table on5 ,drug we compiled development the latestagainst data trypanosomiasis. (2012–2017) on drug development against trypanosomiasis. Table 5. Selected data of reported antitrypanosomal agents. Table 5. Selected data of reported antitrypanosomal agents. Table 5. Selected data of reported antitrypanosomal agents. Compound Activity Ref. Compound Activity Ref. Compound T. Activityb. rhodesiense [IC50 (µM)] Ref. T. b. rhodesiense [IC50 (µM)] 114a T. b. rhodesiense0.60 [IC (µM)] 114a 0.60 50 114b 0.53 114b114a 0.530.60 114b 0.53 [217] [217][217 ] Melarsoprol 0.051 MelarsoprolMelarsoprol 0.051 0.051

T. b. rhodesiense [IC50 (µM)]µ T.T. b. b. rhodesiense rhodesiense [IC5050 (µM)]( M)] Molecules 2019, 24, x FOR PEER REVIEW 115a 0.16 30 of 45 115a115a 0.160.16 Molecules 2019, 24, x FOR PEER REVIEW 30 of 45 115b115b 0.100.10 [218][218 ] Molecules 2019, 24, x FOR PEER REVIEW 115b 0.10 30 of 45 MelarsoprolMelarsoprol 0.009 0.009 [218] Melarsoprol 0.009 EflornithineEflornithine 3.80 3.80 Eflornithine 3.80 T. brucei [IC (µM)] T. brucei [IC50 (µM)] T. brucei [IC50 (µM)] Melarsoprol116a116a 0.0130.840.84 116a 0.84 [219] Melarsoprol116b116b 0.0130.600.60 [219] Melarsoprol116b116c 0.0130.600.51 [219] 116c 0.51 116c 0.51

Melarsoprol 0.013

T. brucei [IC50 (µM)]

T. brucei [IC50 (µM)] T.T. brucei brucei [IC[IC50 (µM)](µM)] [220] 117 1.5 ± 0.4 [220][220 ] 117117 1.51.5 ± 0.40.4 [220] 117 1.5 ±± 0.4

T. brucei [IC50 (µM)] T. brucei [IC50 (µM)] 118a T. brucei0.25 [IC ± 0.150 (µM)] 118a T. brucei0.25 [IC500.1 (µM)] 118a 0.25 ±± 0.1 [221][221 ] 118a 0.25 ± 0.1 118b118b 0.670.67 ± 10.110.1 [221] ± 118b 0.67 ± 10.1 [221] 118b 0.67 ± 10.1 T. b. rhodesiense [IC50 (µM)]

T. b. rhodesiense [IC50 (µM)] T. b. rhodesiense [IC50 (µM)]

[222] 119 0.091 [222] 119 0.091 [222] 119 0.091

T. b. brucei [IC50 (µM)]

120a T. b. brucei0.7 [IC 50 (µM)] T. b. brucei [IC50 (µM)] 120a 0.7 [223] 120a 0.7 120b 0.5 [223] 120b 0.5 [223] 120b 0.5 T. brucei [IC50 (µM)]

121a T. brucei0.07 [IC ± 0.0150 (µM)] 121a T. brucei0.07 [IC± 0.0150 (µM)] 121a 0.07 ± 0.01 [224] 121b 0.05 ± 0.01 [224] 121b 0.05 ± 0.01 [224] 121b 0.05 ± 0.01 T. b. T. cruzi~~~ rhodesienseT. b. ~~~ T.[IC cruzi50 (µM)]~~~ rhodesiense[IC50T. (µM)] b. ~~~ [ICT. cruzi50 (µM)]~~~ 122a rhodesiense[IC0.21850 (µM)] ~~~ 0.373 [IC50 (µM)] 122a [IC0.21850 (µM)] 0.373 [225] 122a 0.218 0.373 [225] 122b 0.187 0.239 [225] 122b 0.187 0.239 122b 0.187 0.239

T. cruzi [IC50 (µM)] [226] 123 T. cruzi0.008 [IC50 (µM)] [226] T. cruzi [IC50 (µM)] 123 0.008 [226] 123 0.008

Molecules 2019, 24, x FOR PEER REVIEW 30 of 45 Molecules 2019, 24, x FOR PEER REVIEW 30 of 45 Molecules 2019, 24, x FOR PEER REVIEW 30 of 45 Molecules 2019, 24, x FOR PEER REVIEW 30 of 45

Melarsoprol 0.013 Melarsoprol 0.013 Melarsoprol 0.013 Melarsoprol 0.013

T. brucei [IC50 (µM)] T. brucei [IC50 (µM)]

T. brucei [IC50 (µM)] T. brucei [IC50 (µM)] [220] 117 1.5 ± 0.4 [220] 117 1.5 ± 0.4 [220] 117 1.5 ± 0.4 [220] 117 1.5 ± 0.4

T. brucei [IC50 (µM)] T. brucei [IC50 (µM)] 118a 0.25 ± 0.1 118a T. brucei0.25 [IC ± 0.150 (µM)] 118a T. brucei0.25 [IC ± 0.150 (µM)] [221] [221] Molecules 2019, 24, 3886 118b118a 0.670.25 ± ± 10.1 0.1 30 of 45 118b 0.67 ± 10.1 [221]

118b 0.67 ± 10.1 [221] Table 5.118bCont . T. b. rhodesiense0.67 ± 10.1 [IC 50 (µM)] T. b. rhodesiense [IC50 (µM)]

T. b. rhodesiense [IC50 (µM)] Compound Activity Ref. T. b. rhodesiense [IC50 (µM)] T. b. rhodesiense [IC50 (µM)] [222] 119 0.091 [222] 119 0.091 [222] 119 0.091 [222][222 ] 119119 0.0910.091

T. b. brucei [IC50 (µM)] T. b. brucei [IC50 (µM)] 120a 0.7 120a T. b. brucei0.7 [IC 50 (µM)] T. b. brucei [IC (µM)] 120a T. b. brucei0.7 [IC 50 (µM)] [223] [223] 120b120a120a 0.70.50.7 120b 0.5 [223][223 ]

120b120b 0.50.5 [223] 120b T. brucei 0.5[IC 50 (µM)] T. brucei [IC50 (µM)] 121a T. brucei0.07 [IC± 0.01(µ M)] 121a T. brucei0.07 [IC ± 0.015050 (µM)] 121a121a T. brucei0.070.07 [IC± 0.010.0150 (µM)] ± [224] 121a 0.07 ± 0.01 [224][224 ] 121b 0.05 ± 0.01 121b121b 0.050.05 ± 0.010.01 [224] 121b 0.05 ±± 0.01 [224]

121b 0.05 ± 0.01 T. b. T. b. rhodesienseT. b. T. cruzi T. cruzi~~~ rhodesiense[IC50T.( µb.M)] ~~~ [ICT. cruzi50 (µM)]~~~ rhodesiense~~~ T.[IC cruzi50 (µM)]~~~ 122a rhodesiense[IC0.21850T. (µM)] b. ~~~ [IC 0.37350 (µM)] [IC50 (µM)] T. cruzi~~~ 122a 0.218 [IC0.37350 (µM)] 122a rhodesiense[IC0.21850 (µM)] ~~~ 0.373 Molecules 2019, 24, x FOR PEER REVIEW [IC50 (µM)] 31 [of225 45] [IC50 (µM)] [225] 122a 0.218 0.373 [225] Molecules 2019, 24, x FOR PEER REVIEW 31 of 45 122a122b 0.2180.187 0.373 0.239 [225] Molecules 2019, 24, x FOR PEER REVIEW 31 of 45 122b 0.187 0.239 Molecules 2019, 24, x FOR PEER REVIEW 122b 0.187 0.239 31[225] of 45 122b 0.187 0.239 Benznidazole 2.153 ± 0.176 122b 0.187 0.239 T. cruzi µ Benznidazole T. cruzi2.153 [IC [IC± 0.17650 (µM)]( M)] Benznidazole T. cruzi2.153 [IC ± 0.17650 (µM)] [226] 123123 0.0080.008 [226] Benznidazole123 T. cruzi2.1530.008 [IC ± 0.17650 (µM)] [226] [226] 123 T.T. b. cruzi brucei0.008 [IC [IC50 50(µM)] (µM)] Benznidazole 2.153 0.176 [226] 124 0.2624 ± 0.0139 123 T. b. brucei0.008 ±[IC 50 (µM)] T. b. brucei [IC50 (µM)] 124 T.0.2624 b. brucei ±[IC 0.0139(µM)] 124 T. b.0.2624 brucei ± [IC 0.013950 (µM)] 124124 0.26240.2624 ± 0.01390.0139 ± [227] pentamidine 0.0032 ± 0.0004 [227] pentamidine 0.0032 ± 0.0004 [227][227 ] [227] pentamidinepentamidine 0.0032 0.0032 ± 0.00040.0004 pentamidine 0.0032 ±± 0.0004

T. brucei [IC50 (µM)]

125 T. brucei1.01 [IC 50 (µM)] T. brucei [IC50 (µM)] T. brucei [IC50 (µM)] 125 1.01 [98] 125125 T. brucei1.01 1.01[IC50 (µM)] Pentamidine 0.0041 125 1.01 [98][98 ] PentamidinePentamidine 0.0041 0.0041 [98] Pentamidine 0.0041 [98] T. cruzi [IC50 (µM)] Pentamidine 0.0041 µ 126 T. cruzi8.5[IC 50 ( M)] T. cruzi [IC50 (µM)] 126126 T. cruzi [IC8.58.5 50 (µM)] [228][228 ] T. cruzi [IC50 (µM)] NifurtimoxNifurtimox126 7.78.5 7.7 [228] 126 8.5 Nifurtimox 7.7 [228] [228] Nifurtimox T. b. rhodesiense7.7 [IC50 (µM)] Nifurtimox 7.7 127a 0.061 T. b. rhodesiense [IC50 (µM)] 127b127a T. b. rhodesiense0.0610.065 [IC50 (µM)] T. b. rhodesiense [IC50 (µM)] Melarsoprol127b127a 0.00390.0650.061 127a 0.061 [158] Melarsoprol127b 0.00390.065 Melarsoprol127b 0.00390.065 [158] MelarsoprolSuramin 0.00390.0075 [158] [158] Suramin 0.0075 Suramin 0.0075 Suramin T. b. 0.0075 T. cruzi~~~ rhodesienseT. b. ~~~ [ICT. cruzi50 (µM)]~~~ [IC50T. (µM)] b. rhodesiense~~~ T. cruzi~~~ T. b. [IC50 (µM)] rhodesiense50 ~~~ [IC (µM)] T.[IC cruzi50 (µM)]~~~ [229] rhodesiense[IC50 (µM)]~~~ [IC50 (µM)] 128a [IC501.81 (µM)] 0.25 [229] [229] 128a 1.81 0.25 [229] 128a 1.81 0.25 128a T.1.81 cruzi [IC50 (µM)]0.25

129 0.04 T. cruzi [IC50 (µM)] 129 T. cruzi0.04 [IC50 (µM)] 129 T. cruzi 0.04[IC50 (µM)] 129 0.04 [115] Benznidazole 1.95 [115] Benznidazole 1.95 [115] Benznidazole 1.95 [115] Benznidazole 1.95 T. b. rhodesiense [IC50 (µM)] [230] 130 0.009 T. b. rhodesiense [IC50 (µM)] T. b. rhodesiense [IC50 (µM)] [230] 130 0.009 [230] T. b. rhodesiense [IC50 (µM)] 130 0.009 [230] 130 0.009

Molecules 2019, 24, x FOR PEER REVIEW 31 of 45 Molecules 2019, 24, x FOR PEER REVIEW 31 of 45 Molecules 2019, 24, x FOR PEER REVIEW 31 of 45

Benznidazole 2.153 ± 0.176 Benznidazole 2.153 ± 0.176 Benznidazole 2.153 ± 0.176

T. b. brucei [IC50 (µM)] T. b. brucei [IC50 (µM)] 124 T. b.0.2624 brucei ± [IC 0.013950 (µM)] 124 0.2624 ± 0.0139 124 0.2624 ± 0.0139

[227] [227] pentamidine 0.0032 ± 0.0004 [227] pentamidine 0.0032 ± 0.0004 pentamidine 0.0032 ± 0.0004

T. brucei [IC50 (µM)] T. brucei [IC50 (µM)] 125 T. brucei1.01 [IC 50 (µM)] 125 1.01 125 1.01 [98] [98] Pentamidine 0.0041 [98] Pentamidine 0.0041 Pentamidine 0.0041

T. cruzi [IC50 (µM)] T. cruzi [IC50 (µM)] 126 T. cruzi 8.5[IC 50 (µM)] Molecules 2019, 24, 3886 126 8.5 31 of 45 126 8.5 [228] [228] Nifurtimox 7.7 [228] Nifurtimox 7.7 TableNifurtimox 5. Cont. 7.7

T. b. rhodesiense [IC50 (µM)] Compound T. Activityb. rhodesiense [IC50 (µM)] Ref. 127a T. b. rhodesiense0.061 [IC50 (µM)] 127a T. b. rhodesiense0.061 µ 127b127a 0.0650.061 [IC50 ( M)] Melarsoprol127b127a 0.00390.0650.061 127b 0.065 [158] Melarsoprol127b 0.00390.065 Melarsoprol 0.0039 [158] Melarsoprol 0.0039 [158][158 ] Suramin 0.0075 Suramin 0.0075 SuraminSuramin 0.0075 0.0075

T. b. T. cruzi~~~ rhodesienseT. b. ~~~ T. b. rhodesienseT. b. T.T. cruzi cruzi~~~ [ICT. cruzi50 (µM)]~~~ rhodesiense[IC5050 (µM)](µM)]~~~ [IC50 (µM)] rhodesiense~~~ [IC50 (µM)] [IC50 (µM)] [IC50 (µM)] [IC50 (µM)] [229] [229][229 ] [229] 128a128a 1.81 0.250.25 128a 1.81 0.25 128a 1.81 0.25

T. cruzi [IC50 (µM)] µ T.T. cruzi cruzi [IC[IC50 (µM)]( M)] 129 T. cruzi0.04 [IC50 (µM)] 129129 0.040.04 129 0.04 Molecules 2019, 24, x FOR PEER REVIEW 32 of 45 [115][115 ] Molecules 2019, 24, x FOR PEER REVIEW 32 of 45 BenznidazoleBenznidazole 1.95 1.95 [115] Molecules 2019, 24, x FOR PEER REVIEW 32[115] of 45 Benznidazole 1.95 Molecules 2019, 24, x FOR PEER REVIEW Benznidazole 1.95 32 of 45

Melarsoprol T. b. rhodesiense0.008[IC (µM)] T. b. rhodesiense [IC5050 (µM)] Melarsoprol 0.008 [230] 130130 T. b. rhodesiense0.0090.009 [IC50 (µM)] Melarsoprol T. b. rhodesiense0.008 [IC50 (µM)] [230] 130 0.009 [230] Melarsoprol130 0.0080.009 [230] Melarsoprol 0.008 T. b. rhodesiense [IC50 (µM)]

131 T. b. rhodesiense0.003 [IC 50 (µM)] 50 Pentamidine131 T.T. b. b. rhodesiense rhodesiense0.003 [IC 50 (µM)](µM)] T. b. rhodesiense [IC50 (µM)] [116] Pentamidine131 0.003 131131 0.0030.003 [116] PentamidineMelarsoprolPentamidine 0.0030.006 0.003 Pentamidine 0.003 [116][116 ] Melarsoprol 0.0066 [116] Melarsoprol 0.00 Melarsoprol T. b. rhodesiense0.006 [IC 50(µg/mL)] Melarsoprol 0.006 132 T. b. rhodesiense7.0 [IC 50(µg/mL)] T. b. rhodesiense [IC (µg/mL)] 132 T. b. rhodesiense7.0 [IC 50(µg/mL)] T. b. rhodesiense [IC50(µg/mL)] [118] 132132 7.07.0 Melarsoprol132 0.0057.0 [118][118 ] MelarsoprolMelarsoprol 0.005 0.005 [118] [118] Melarsoprol 0.005 Melarsoprol T. b. rhodesiense0.005 [IC50(µM)] T. b. rhodesiense [IC (µM)] 133a T. b. rhodesiense13.1 [IC5050(µM)] 133b133a133a T. b. rhodesiense13.120.213.1 [IC50(µM)] [161][161 ] T. b. rhodesiense [IC50(µM)] 133b133a133b 13.120.220.2 [161] PentamidinePentamidine133a 0.00513.1 0.005 133b 20.2 [161] Pentamidine133b 0.00520.2 [161] T. b. Pentamidine 0.005 T. cruzi~~~ Pentamidine rhodesienseT. b. ~~~0.005 [ICT. cruzi50 (µM)]~~~ rhodesiense[IC50T. (µM)] b. ~~~ T. b. T.[IC cruzi50 (µM)]~~~ 134a rhodesiense[IC0.00450 (µM)] ~~~ T. cruzi71.6~~~ rhodesiense~~~ [IC50 (µM)] 134b134a [IC0.0040.01950 (µM)] [IC0.0535071.6 (µM)] [IC50 (µM)] [120] Pentamidine134b134a 0.0040.0190.003 0.05371.6 - 134a 0.004 71.6 [120] PentamidineMelarsoprol134b 0.0190.0040.003 0.053 - 134b 0.019 0.053 [120] PentamidineMelarsoprol 0.0030.004 - [120] Pentamidine 0.003 - Melarsoprol 0.004 - BenznidazoleMelarsoprol 0.004- 1.30 - Benznidazole - 1.30

Benznidazole T. -brucei [IC50 (µM)] 1.30 Benznidazole - 1.30 135a T. brucei 0.52 [IC± 0.0150 (µM)] 135a T. brucei 0.52 [IC± 0.0150 (µM)] T. brucei [IC50 (µM)] 135a 0.52 ± 0.01 135a 0.52 ± 0.01 [231] 135b 0.57 ± 0.02 [231] 135b 0.57 ± 0.02 [231] [231] 135b 0.57 ± 0.02 135b 0.57 ± 0.02

Important Highlights of Table 5 Compounds Important Highlights of Table 5 Compounds Compounds of different class heterocycles are included in Table 5 as potent agents against Important Highlights of Table 5 Compounds ImportantTrypanosomaCompounds Highlights species of and different of Tablethe results 5class Compounds ofheterocycles their assessments are included were compared in Table 5to as those potent of standardagents against drugs TrypanosomaTryparsamide,Compounds species melarsoprol, of and different the results classdifluoromethylornitithine, ofheterocycles their assessments are included were pentamidine, compared in Table 5to as thosediminazene, potent of standardagents suramin, against drugs Compounds of different class heterocycles are included in Table 5 as potent agents against TrypanosomaTryparsamide,benznidazole species and melarsoprol, nifurtimox. and the results Amdifluoromethylornitithine, ongof their the assessmentssubstituted werebenzothiophe pentamidine, comparedne toderivatives thosediminazene, of standard developed suramin, drugs by Trypanosoma species and the results of their assessments were compared to those of standard drugs Tryparsamide,benznidazole and melarsoprol, nifurtimox. Amdifluoromethylornitithine,ong the substituted benzothiophe pentamidine,ne derivatives diminazene, developed suramin, by Tryparsamide, melarsoprol, difluoromethylornitithine, pentamidine, diminazene, suramin, benznidazole and nifurtimox. Among the substituted benzothiophene derivatives developed by benznidazole and nifurtimox. Among the substituted benzothiophene derivatives developed by

Molecules 2019, 24, x FOR PEER REVIEW 32 of 45 Molecules 2019, 24, x FOR PEER REVIEW 32 of 45

Melarsoprol 0.008 Melarsoprol 0.008

T. b. rhodesiense [IC50 (µM)] 131 T. b. rhodesiense0.003 [IC 50 (µM)] Pentamidine131 0.003 Pentamidine 0.003 [116] [116] Melarsoprol 0.006 Melarsoprol 0.006

T. b. rhodesiense [IC50(µg/mL)] 132 T. b. rhodesiense7.0 [IC 50(µg/mL)] 132 7.0 [118] Melarsoprol 0.005 [118] Melarsoprol 0.005

T. b. rhodesiense [IC50(µM)] Molecules 2019, 24, 3886 133a T. b. rhodesiense13.1 [IC50(µM)] 32 of 45 133b133a 20.213.1 [161] 133b 20.2 [161] Table 5. Cont. Pentamidine 0.005 Pentamidine 0.005 Compound ActivityT. b. Ref. T. cruzi~~~ T.rhodesiense b. rhodesienseT. b. ~~~ T. cruzi [ICT. cruzi50 (µM)]~~~ rhodesiense[IC5050 (µM)](µM)]~~~ [IC50 (µM)] [IC50 (µM)] 134a134a [IC0.00450 (µM)] 71.6 134b134a134b 0.0190.004 0.053 71.6 Pentamidine 0.003 - [120] Pentamidine134b 0.0030.019 0.053 - [120] Melarsoprol 0.004 - [120] PentamidineMelarsoprol 0.0040.003 - Melarsoprol 0.004 - BenznidazoleBenznidazole - 1.30 Benznidazole - 1.30

T. brucei [IC50 (µM)] T. brucei [IC (µM)] 135a T. brucei 0.52 [IC± 0.0150 (µM)] 135a135a 0.520.52 ± 0.010.01 ±

[231][231 ] 135b 0.57 0.02 135b 0.57 ±± 0.02 [231] 135b 0.57 ± 0.02

ImportantImportant HighlightsHighlights of Table 55 CompoundsCompounds Important Highlights of Table 5 Compounds CompoundsCompounds of different different class class heterocycles heterocycles are are in includedcluded in in Table Table 55 asas potent potent agents agents against against TrypanosomaTrypanosomaCompounds species of and anddifferent the the results resultsclass ofheterocycles their of their assessments assessments are included were werecompared in Table compared 5to as those potent to of those standardagents of against standard drugs drugsTryparsamide,Trypanosoma Tryparsamide, species melarsoprol, and melarsoprol, the results difluoromethylornitithine, of difluoromethylornitithine, their assessments were pentamidine, compared pentamidine, to thosediminazene, diminazene, of standard suramin, suramin,drugs benznidazolebenznidazoleTryparsamide, and melarsoprol, nifurtimox. Amdifluoromethylornitithine, Amongong the the substituted substituted benzothiophe benzothiophene pentamidine,ne derivatives derivativesdiminazene, developed developed suramin, by by Bhambrabenznidazole et al. and [217 nifurtimox.], compound Am14aongand the114b substitutedwere found benzothiophe to be the mostne derivatives active candidates developed for HATby treatment with IC50 values of 0.60 and 0.53 µM, respectively. In another series of imidazole compounds synthesized by Trunz et al. [218], compounds 115a and 115b demonstrated good potency against T.b.rhodesiense and their reported IC50 values were 0.16 and 0.10 µM, respectively. Bouchikhi et al. [219] designed a series of compounds based on glycosyl-isoindigo conjugates, and an evaluation of their antitrypanosomal activities revealed that among the compounds synthesized, compounds 116a–c expressed significant biological activity against T. b. brucei strains with IC50 values between 0.51 and 0.84 µM. Among the several halonitrobenzamides derivatives synthesized by Hwang et al. [220], and evaluated against a T. b. brucei culture compounds, compound 117 was found to be the most potent inhibitor (IC50 = 1.5 µM). An assessment of the antiprotozoal activities (antitrypanosomal) of a series of analogs developed by Samant et al. [221] revealed that among the synthesized molecules, compound 118 was the most active antitrypanosomal agent (IC50 = 0.25 µM). Ferrins et al. [222] synthesized various anilides and the evaluation of their antitrypanosomal activity revealed that among the compounds synthesized, compound 119 (IC50 = 0.091 µM) significantly inhibited a T. b. rhodesiense culture. A large series of convolutamine analogs were prepared by Pham et al. [223]. Basically, convolutamine, which is a natural product, is a highly effective antitrypanosomal. All the synthesized convolutamine derivatives were screened against T. b. brucei culture, and compounds 120a and 120b were reported as the most potent inhibitors with IC50 values of 0.7 µM and 0.5 µM, respectively. Samant et al. [224] synthesized a big library of naphthoquinone derivatives, and the evaluation of their antiprypanosomal activities in vitro showed that compounds 121a (IC50 = 0.07 µM) and 121b (IC50 = 0.05 µM) were the most potent compounds in the series. Papadopoulou et al. [225] synthesized a series of azole-based compounds. An assessment of their antitrypanosomal potentials Molecules 2019, 24, 3886 33 of 45

revealed that compounds 122a and 122b were the most active agents with IC50 values against T. b. rhodesiense and T. cruzi vetween 0.187 and 0.373 µM. Papadopoulou et al. [226] also designed and developed nitrotriazole-based amide derivatives; an investigation of their antitrypanosomal activities against T. cruzi revealed that among them compound 123 was the most potent agent against T. cruzi (IC50 = 0.008 µM). A series of heterocyclic spiro compounds synthesized by Zelisko et al. [227] were evaluated for their antitrypanosomal activities. The results showed that compound 124 was the most active molecule (IC50 = 0.2624 µM) against T. b. rhodesiense. An in vitro assessment of the inhibitory effect of a novel series of β-carboline analogs prepared by Manda et al. [98] against T. b. brucei revealed that compound 125 was the most potent inhibitory agent among the compounds (IC50 = 1.01 µM). Alves et al. [228] developed several semicarbazone derivatives, and evaluated their antiprotozoal activities in vitro by testing their potential to inhibit biological strains. Compound 126 (IC50 = 8.5 µM) appeared to be the most potent T. cruzi culture inhibitor, with an IC50 values of 8.5 µM. Several azabicyclo nonane type derivatives synthesized by Seebacher et al. [158] were screened for their anti-protozoal activity. The results showed that among them, compounds 127a (IC50 = 0.061 µM) and 127b (IC50 = 0.065 µM) exhibited the greatest activity profiles. Upadhayaya et al. [229] developed a series of quinolone- and indenoquinoline-based heterocycles, and an evaluation of their antiprotozoal potentials revealed that compound 128 was a potential candidate against T. cruzi and T. b. rhodesiense with IC50 values of 0.25 and 1.81 µM, respectively. Vita et al. [115] synthesized series of potent and effective imidazole incorporated phenylethanol derivatives, of which compound 129 exhibited the highest antitrypanosomal activity (IC50 = 0.04 µM), and showed more potency against T. cruzi strains. Martínez et al. [230] reported the synthesis of several bisguanidine T. b. rhodesiense strain inhibitors among which compound 130 (IC50 = 0.009 µM) was identified as the most active agent for trypanosomiasis treatment. Patrick et al. [116] developed a new series of benzyl phenyl ether diamidine derivatives, and investigated their potential against a T. b. rhodesiense culture. Compound 131, which exhibited a good therapeutic potential (IC50 = 0.003 µM), was identified as the best candidate. Dürüst et al. [118] synthesized a new series of 1,2,4-oxadiazole-linked triazole derivatives using the 1,3-dipolar cycloaddition reaction, and their antiprotozoal activities were analyzed. Of these compounds, compound 132 was identified as the most potent antitrypanosomal agents (IC50 = 7.0 µM). As already reported, Mckeever et al. [161] developed a series ofguanidine diaromatic minor grove binder aminoalkyl derivatives. They did not only evaluate the antimalarial activities of these compounds, they also evaluated their antitrypnosomal properties and found that compounds 133a (IC50 = 13.1 µM) and 133b (IC50 = 20.2 µM) were the most potent candidates against T. b. rhodesiense strains. A series of 1,3-dipyridylbenzene derivatives were evaluated for their antitrypanosomal activity. The results showed that compounds 134a and 134b were the best candidates against T. b. rhodesiense and T. cruzi strains [120]. Sola et al. [231] synthesized a novel series of huprine Y dimer derivatives, and assessment of their antiprotozoal activities using a bioassay showed that compounds 135a (IC50 = 0.52 µM) and 135b (IC50 = 0.57 µM) were the most significant T. brucei culture inhibitors.

3. Conclusions Infections caused by protozoan parasites such as giardiasis, leishmaniasis, malaria, trichomoniasis, and trypanosomiasis are responsible for considerable morbidity and mortality worldwide, with devastating social and economic consequences. The currently available drugs for the treatment of and protection against protozoan parasites were discovered over 50 years ago, and a number of factors limit their utility, such as high cost, poor compliance, drug resistance, low efficacy, and safety concerns. Therefore, the development of new and more effective drugs with fewer side effects presents a crucial challenge. Currently, research focused on the developing new drugs to protect against and treat protozoans are increasing steadily. In this review article, we have presented some of the developments in this field, with the aim of showing the recent significant advances in the discovery of new antiprotozoal drugs. Molecules 2019, 24, 3886 34 of 45

Author Contributions: Conceptualization, F.H., D.S., Literature search, S.-M.L., M.-S.K., F.H., Writing-original draft preparation, S.-M.L., M.-S.K., F.H., Writing-Review & Editing, D.S. Funding: This work was supported by grants from the Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. NRF-2015R1D1A1A01056620, NRF-2014M3C1A3054139, and NRF-2018M3A9C8024360). Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

HAT Human African trypanosomiasis MTZ Metronidazole ML Mucosal leishmaniasis SSG Sodium gluconate AmB Amphotericin-B CFM Clofazimine EEF Exoerythrocytic form RBC Red blood cell pf -DHFR-TS P.falciparum dihydrofolate reductase thymidylate synthase CP Cysteine peptidase NTD Neglected tropical disease CNS Central nervous system

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