The Development of Novel Compounds Against Malaria: Quinolines, Triazolpyridines, Pyrazolopyridines and Pyrazolopyrimidines

molecules

Review The Development of Novel Compounds Against Malaria: Quinolines, Triazolpyridines, Pyrazolopyridines and Pyrazolopyrimidines

Luiz C. S. Pinheiro 1 ,Lívia M. Feitosa 1,2, Marilia O. Gandi 1,2, Flávia F. Silveira 1,3 and Nubia Boechat 1,2,3,* 1 Departamento de Síntese de Fármacos, Instituto de Tecnologia em Fármacos, Farmanguinhos-FIOCRUZ, Fundação Oswaldo Cruz, Rua Sizenando Nabuco 100, Manguinhos, Rio de Janeiro 21041-250, Brazil; luiz.pinheiro@far.fiocruz.br (L.C.S.P.); [email protected] (L.M.F.); [email protected] (M.O.G.); fl[email protected] (F.F.S.) 2 Programa de Pós-Graduação em Farmacologia e Química Medicinal, PPGFQM, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21041-250, Brazil 3 Programa de Pós-Graduação em Química, PGQu Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21041-250, Brazil * Correspondence: nubia.boechat@far.fiocruz.br; Tel.: +55-21-3977-2464

Academic Editors: Valeria Patricia Sülsen and Albertina Moglioni Received: 16 October 2019; Accepted: 11 November 2019; Published: 13 November 2019

Abstract: Based on medicinal chemistry tools, new compounds for malaria treatment were designed. The scaffolds of the drugs used to treat malaria, such as chloroquine, primaquine, amodiaquine, mefloquine and sulfadoxine, were used as inspiration. We demonstrated the importance of quinoline and non-quinoline derivatives in vitro with activity against the W2 chloroquine-resistant (CQR) Plasmodium falciparum clone strain and in vivo against Plasmodium berghei-infected mouse model. Among the quinoline derivatives, new hybrids between chloroquine and sulfadoxine were designed, which gave rise to an important prototype that was more active than both chloroquine and sulfadoxine. Hybrids between chloroquine–atorvastatin and primaquine–atorvastatin were also synthesized and shown to be more potent than the parent drugs alone. Additionally, among the quinoline derivatives, new mefloquine derivatives were synthesized. Among the non-quinoline derivatives, we obtained excellent results with the triazolopyrimidine nucleus, which gave us prototype I that inspired the synthesis of new heterocycles. The pyrazolopyrimidine derivatives stood out as non-quinoline derivatives that are potent inhibitors of the P. falciparum dihydroorotate dehydrogenase (Pf DHODH) enzyme. We also examined the pyrazolopyridine and pyrazolopyrimidine nuclei.

Keywords: malaria; P. falciparum; Pf DHODH; quinoline; triazolopyrimidine; pyrazolopyridine; pyrazolopyrimidine; chloroquine; primaquine; meﬂoquine

1. Introduction Malaria is one of the world’s most serious public health problems. According to the latest World Health Organization (WHO) World Malaria Report, no signiﬁcant gains were achieved in reducing malaria cases in the period from 2015 to 2017. The estimated number of malaria deaths in 2017, at 435,000, remained broadly unchanged relative to the previous year, which was 445,000 deaths (WHO, 2019) [1]. Due to the high parasitic resistance exhibited by Plasmodium falciparum to most drugs available, monotherapy is no longer used to treat malaria. To prevent recurrence and delay the development of parasite resistance, the WHO recommends the use of artemisinin-combined therapies (ACTs), which are based on the simultaneous use of drugs with

Molecules 2019, 24, 4095; doi:10.3390/molecules24224095 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 20 Molecules 2019, 24, 4095 2 of 20 To prevent recurrence and delay the development of parasite resistance, the WHO recommends the use of artemisinin-combined therapies (ACTs), which are based on the simultaneous use of drugs diwithfferent different modes modes of action of action [2]. Artemisinin [2]. Artemisinin derivatives derivatives have have very very short shortin vivo in vivohalf-lives, half-lives, but theybut they are fastare actingfast acting against against the intraerythrocytic the intraerythrocytic asexual blood-stageasexual blood-stage malaria parasites. malaria Therefore,parasites. artemisininTherefore, derivativesartemisinin arederivatives coadministered are coadministered with drugs with with drugs longer with half-lives longer [2half-lives]. There [2]. is an There urgent is an need urgent for novelneed for antimalarials novel antimalarials with better with safety better profiles safety thanprofiles current than medicinescurrent medicines due to the due resistance to the resistance against antimalarialagainst antimalarial drugs and drugs for theand prevention for the prevention of transmission of transmission and relapse and ofrelapse the disease of the [disease3–6]. [3–6]. InIn thethe recent recent literature, literature, a number a number of new of antimalarialnew antimalarial compounds compounds in different in stagesdifferent of preclinicalstages of andpreclinical clinical and development clinical development have been have described been described [7–10]. Notably, [7–10]. Notably, quinoline quinoline derivatives derivatives are still theare predominantstill the predominant class of antimalarialclass of antimalarial drugs. [11 drugs.–13]. [11–13]. TheThe exchangeexchange of thethe quinolinequinoline ringring withwith anotheranother heterocyclicheterocyclic ring isis anan importantimportant strategystrategy inin drugdrug designdesign andand thethe chemicalchemical modificationmodification ofof availableavailable drugsdrugs toto developdevelop novel,novel, biologicallybiologically activeactive compounds.compounds. Many heterocyclic compounds have been developed in an attempt to findfind new drugs toto treattreat malariamalaria [[14].14]. RecentRecent studies by Boechat and coworkers present the design and synthesis of a broad classclass ofof quinolinequinoline andand non-quinoline non-quinoline compounds compounds with with anti- Panti- falciparumP falciparum. These. compoundsThese compounds had their structureshad their modifiedstructures using modified some medicinalusing some chemistry medicinal tools, chemistry such as molecular tools, such hybridization. as molecular The hybridization. rational sequence The ofrational modifications sequence made of modi fromfications the parent made drugs from and the/or parent prototypes drugs to an obtaind/or prototypes the novel hybridto obtain compounds the novel ishybrid summarized compounds in Figure is summarized1. in Figure 1.

Figure 1. Rational approach to the design quinolinequinoline and non-quinoline compounds.compounds.

Therefore,Therefore, twenty-six twenty-six new new deriva derivativestives of ofthethe [1,2,4]triazolo[1,5- [1,2,4]triazolo[1,5-a]pyrimidinea]pyrimidine system system (1–26 (),1 –with26), withdifferent diff erentsubstituents substituents at the at2, 5 the and 2, 7 5positions, and 7 positions, were designed weredesigned using standard using medicinal standard medicinalchemistry chemistryand modeling and principles, modeling such principles, as isosteric such replacement, as isosteric based replacement, on ring isosterism based on ringwith isosterismthe antimalarial with thedrugs antimalarial mefloquine, drugs chloroquine mefloquine, and chloroquineamodiaquine and (Figure amodiaquine 2) [15]. Additionally, (Figure2)[ 15the]. CF Additionally,3 group present the CFin mefloquine3 group present was added in mefloquine at the 2 position was added of the at triazolopyrimidine the 2 position of the ring, triazolopyrimidine and aromatic and ring, aliphatic and aromaticamine moieties and aliphatic were amineincorporated moieties at were the incorporated 7 position, attaking the 7 position,into consideration taking into the consideration amodiaquine the amodiaquinescaffold. The scatrifluoromethylffold. The trifluoromethyl moiety is one moiety of the is most one ofwidespread the most widespread fluorine-containing fluorine-containing functional functionalgroups in groupsbioactive in molecules. bioactive molecules.Due to its highly Due to electronegative its highly electronegative feature, it can feature, exert it significant can exert significantelectronic influences electronic on influences neighbor oning neighboring groups. Another groups. advantage Another is advantage the improvement is the improvement in lipophilicity, in lipophilicity,making this moiety making useful this moiety for targeting useful formolecules targeting to moleculesenzymatic to active enzymatic sites [16–18]. active sites [16–18]. The reaction of 3-amino-1,2,4-triazoles 27a–c with ethyl acetoacetate or ethyl 4,4,4-trifluoroacetoacetate in toluene under reflux with catalytic p-toluenesulfonic acid, for 24 h produced [1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-ones 28a–d in 50–90% yield [19]. Compounds 28a–d were treated

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with POCl3 under reﬂux for 6 h to obtain the respective 7-chloro[1,2,4]triazolo[1,5-a]pyrimidines 29a–d in 58–90% yield. The nucleophilic aromatic substitution reaction of intermediates 29a–d with the appropriate amines [20] aﬀorded target compounds [1,2,4]triazolo[1,5-a]pyrimidine derivatives 1–26 in 30–90% yield (Figure3).

Figure 2. Rational approach to the design of [1,2,4]triazolo[1,5-a]pyrimidine derivatives 1–26.

The reaction of 3-amino-1,2,4-triazoles 27a–c with ethyl acetoacetate or ethyl 4,4,4- trifluoroacetoacetate in toluene under reflux with catalytic p-toluenesulfonic acid, for 24 h produced [1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-ones 28a–d in 50–90% yield [19]. Compounds 28a–d were treated with POCl3 under reflux for 6 h to obtain the respective 7-chloro[1,2,4]triazolo[1,5- a]pyrimidines 29a–d in 58–90% yield. The nucleophilic aromatic substitution reaction of intermediates 29a–d with the appropriate amines [20] afforded target compounds [1,2,4]triazolo[1,5- a]pyrimidineFigure 2. derivativesRational approach 1–26 in to30–90% the design yield of (Figure [1,2,4]triazolo[1,5- 3). a]pyrimidine derivatives 1–26.

Figure 3. Synthesis of [1,2,4]triazolo[1,5-a]pyrimidines 1–26. Reagents and conditions: (i) ethyl

acetoacetate or ethyl 4,4,4-trifluoroacetoacetate, TsOH (cat.), toluene, reflux, 24 h; (ii) POCl3, reflux, 6 h; (iii) appropriate amine, EtOH, 25 ◦C, 16–18 h; (iv) appropriate amine/azol, DMF, 120 ◦C, 12 h; (v) appropriate amine, EtOH, 25 ◦C, 43 h.

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Figure 3. Synthesis of [1,2,4]triazolo[1,5-a]pyrimidines 1–26. Reagents and conditions: (i) ethyl acetoacetate or ethyl 4,4,4-trifluoroacetoacetate, TsOH (cat.), toluene, reflux, 24 h; (ii) POCl3, reflux, 6 Moleculesh; (iii)2019 appropriate, 24, 4095 amine, EtOH, 25 °C, 16–18 h; (iv) appropriate amine/azol, DMF, 120 °C, 12 h; (v)4 of 20 appropriate amine, EtOH, 25 °C, 43 h.

Synthesized compounds 11––2626 were evaluated inin vitrovitro againstagainst thethe W2W2 chloroquine-resistantchloroquine-resistant (CQR) P.P. falciparum falciparum cloneclone strain, strain, which which showed showed IC50 IC values50 values in the in range the range of 0.023 of to 0.023 20 μ toM and 20 µ didM andnot didpresent not toxicity present to toxicity HepG2 tocells. HepG2 The trifluoromethyl cells. The trifluoromethyl group, as a substituent group, as at a substituentthe 2 position at of the the 2 position[1,2,4]triazolo[1,5- of the [1,2,4]triazolo[1,5-a]pyrimidine ring,a]pyrimidine contributed ring, to contributed an increase to anin increaseanti-P. infalciparum anti-P. falciparum activity. activity.Compounds Compounds 2 (2-naphthyl;2 (2-naphthyl; IC50 = 0.023 IC μ50M),= 50.023 (3,4-diCl;µM), IC5 50(3,4-diCl; = 0.55 μM), IC50 8 (4-OCH= 0.55 µ3;M), IC508 =(4-OCH 0.4 μM),3; ICand50 13= 0.4(4-CFµM),3; IC and50 = 130.3 (4-CFμM) 3were; IC50 the= 0.3mostµ M)potent were of the the most series. potent However, of the the series. 5-methyl-7- However,N'-(N the,N- 5-methyl-7-diethylpentane-1,4-diamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-N’-(N,N-diethylpentane-1,4-diamine)-2-(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine derivativea]pyrimidine that derivativecontained a that trifluoromethyl contained a trifluoromethylgroup showed poor group antimalarial showed poor activity. antimalarial Derivative activity. 2, which Derivative contains 2a, whichβ-naphthylamine contains a βgroup-naphthylamine in its structure group at in the its 7 structure position, at and the the 7 position, trifluoromethyl and the trifluoromethylgroup at the 2 groupposition, at thehas 2an position, important has contribution an important to contribution anti-P. falciparum to anti- activityP. falciparum when comparedactivity when with compared the other withderivatives the other that derivativescontain aryl/alkylamine that contain groups aryl/alkylamine at the 7 position. groups atDerivative the 7 position. 2 was therefore Derivative used2 wasas a thereforeprototype compound used as a prototype(prototype compoundI) for future investigations (prototype I) in for the future search investigations for compounds in for the the search treatment for compoundsof malaria (Figure for the 4). treatment of malaria (Figure4).

Figure 4. Structure of prototype I.

The inhibition of of P.P. falciparum falciparum dihydroorotatedihydroorotate dehydrogenase dehydrogenase (Pf (PfDHODH)DHODH) has has been been shown shown to tobe an be attractive an attractive strategy strategy to search to search for new for substanc new substanceses with antiplasmodial with antiplasmodial activity [21,22]. activity Synthetic [21,22]. Syntheticcompounds compounds have been identified have been as identiﬁedinhibitors of as class inhibitors 2 DHODH of class enzymes. 2 DHODH Some [1,2,4]triazolo[1,5- enzymes. Some [1,2,4]triazolo[1,5-a]pyrimidin-7-amines a]pyrimidin-7-amines were discovered by were Phillips discovered and coworkers by Phillips as inhibitors and coworkers of PfDHODH as inhibitors [23–26]. of Pf DHODH [23–26]. Following these good results, new 7-arylaminopyrazolo[1,5-a]pyrimidines Following these good results, new 7-arylaminopyrazolo[1,5-a]pyrimidines were designed by ring wereisosterism designed from by prototype ring isosterism I [27]. from The prototype [1,2,4]triazolo[1,5-I [27]. The [1,2,4]triazolo[1,5-a]pyrimidine ringa ]pyrimidinewas modified ring to was a modiﬁedpyrazolo[1,5- to aa pyrazolo[1,5-]pyrimidine ringa]pyrimidine and changed ring by and molecular changed byhybridization molecular hybridization(Figure 5). (Figure5). Different arylamines were incorporated into the structure to investigate the importance of the substituent at the 7 position of the pyrazolopyrimidine nucleus, and the β-naphthylamine moiety was also incorporated. In a previous work, we observed that a trifluoromethyl group at the 2 position of the triazolopyrimidine ring plays an important role in antiplasmodial activity. This time, CF3 was also added as a substituent in the 2 or 5 position of the pyrazolo[1,5-a]pyrimidine scaffold to investigate the influence of this group on the anti-P. falciparum activity.

Figure 5. Rational approach to the design of pyrazolo[1,5-a]pyrimidine derivatives 30–44.

Different arylamines were incorporated into the structure to investigate the importance of the substituent at the 7 position of the pyrazolopyrimidine nucleus, and the β-naphthylamine moiety was also incorporated. In a previous work, we observed that a trifluoromethyl group at the 2 position of the triazolopyrimidine ring plays an important role in antiplasmodial activity. This time, CF3 was also added as a substituent in the 2 or 5 position of the pyrazolo[1,5-a]pyrimidine scaffold to investigate the influence ofFigure this 5. group Rational on theapproach anti-P. to falciparum the designactivity. of pyrazolo[1,5-a]pyrimidine derivatives 30–44. Fifteen compounds, 30–44, were synthesized in 44%–92% yield using the same synthetic methodologyFifteen compounds, described above 30–44 (Figure, were6 )[synthesized15]. in 44%–92% yield using the same synthetic methodology described above (Figure 6) [15].

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MoleculesThese 2019 compounds, 24, x FOR PEER were REVIEW evaluated in vitro against the P. falciparum W2 CQR clone, in5 vivoof 20 against P. berghei-infected mouse model, and in vitro as PfDHODH inhibitors. In addition, a These compounds were evaluated in vitro against the P. falciparum W2 CQR clone, in vivo molecular docking study was performed to evaluate the possible binding mode of the 7- against P. berghei-infected mouse model, and in vitro as PfDHODH inhibitors. In addition, a arylaminopyrazolo[1,5-a]pyrimidine compounds to PfDHODH. Moleculesmolecular2019 , docking24, 4095 study was performed to evaluate the possible binding mode of the5 of 207- arylaminopyrazolo[1,5-a]pyrimidine compounds to PfDHODH.

Figure 6. Synthesis of pyrazolo[1,5-a]pyrimidines 30–44. Reagents and conditions: (i) ethyl acetoacetate Figureor ethyl 6. Synthesis4,4,4-trifluoroacetoacetate, of pyrazolo[1,5-a]pyrimidines TsOH (cat.),30 toluene,–44. Reagents reflux, and 20 conditions h; (ii) :POCl (i) ethyl3, reflux, acetoacetate 4 h; (iii) or Figure 6. Synthesis of pyrazolo[1,5-a]pyrimidines 30–44. Reagents and conditions: (i) ethyl acetoacetate ethylappropriate 4,4,4-trifluoroacetoacetate, amine, EtOH, 25 °C, TsOH 16–18 (cat.), h. toluene, reflux, 20 h; (ii) POCl3, reflux, 4 h; (iii) appropriate or ethyl 4,4,4-trifluoroacetoacetate, TsOH (cat.), toluene, reflux, 20 h; (ii) POCl3, reflux, 4 h; (iii) amine, EtOH, 25 ◦C, 16–18 h. Amongappropriate the amine,15 pyrazolopy EtOH, 25 °C,rimidines 16–18 h. synthesized, 13 exhibited anti-P. falciparum activity, with IC50 valuesThese ranging compounds from were 1.2 to evaluated 92.4 μM. Compoundsin vitro against 33 (R the1 = CFP. falciparum3, R2 = CH3),W2 38 CQR(R1 = CH clone,3, R2in = CH vivo3) Among the 15 pyrazolopyrimidines synthesized, 13 exhibited anti-P. falciparum activity, with againstand 44 (RP.1 = berghei CH3, R-infected2 = CF3) with mouse β-naphthylamine model, and inat the vitro 7 positionas Pf DHODH were the inhibitors. most active. InCompounds addition, IC50 values ranging from 1.2 to 92.4 μM. Compounds 33 (R1 = CF3, R2 = CH3), 38 (R1 = CH3, R2 = CH3) a33 molecularand 38 exhibited docking low studytoxicitywas and low performed IC50 values to of evaluate 1.2 and 5.1 the μM, possible respectively, binding and modeconsequently, of the and 44 (R1 = CH3, R2 = CF3) with β-naphthylamine at the 7 position were the most active. Compounds 7-arylaminopyrazolo[1,5-the highest SI (selectivity ainde]pyrimidinex) values compounds of 467.8 and to 79.6,Pf DHODH. respectively. Therefore, compounds 33 and 33 and 38 exhibited low toxicity and low IC50 values of 1.2 and 5.1 μM, respectively, and consequently, 38 wereAmong selected the15 for pyrazolopyrimidines in vivo in P. berghei-infected synthesized, mouse13 exhibitedmodel. On anti- dayP. 5 falciparumupon treatmentactivity, at with 5 mg/kg, IC50 the highest SI (selectivity index) values of 467.8 and 79.6, respectively. Therefore, compounds 33 and valuesadministered ranging orally, from 1.2 both to 92.4compoundsµM. Compounds reduced 33parasitemia(R1 = CF3 ,Rby2 50%.= CH Compound3), 38 (R1 = CH 44 3showed,R2 = CH the3) best and 38 were selected for in vivo inβ P. berghei-infected mouse model. On day 5 upon treatment at 5 mg/kg, 44inhibition(R1 = CH of3 ,Rthe2 Pf= DHODHCF3) with enzyme-naphthylamine with an IC at50 the= 0.16 7 position μM, which were was the more most active.potent Compoundsthan prototype33 administered orally, both compounds reduced parasitemia by 50%.µ Compound 44 showed the best andI (IC3850 =exhibited 0.70 μM), low whereas toxicity 33 andand low38 had IC50 ICvalues50 values of 1.2of 6.0 and and 5.1 4.0M, μM, respectively, respectively. and consequently, inhibition of the PfDHODH enzyme with an IC50 = 0.16 μM, which was more potent than prototype the highestCompounds SI (selectivity that had index) better values activity of against 467.8 and P. 79.6,falciparum respectively. were also Therefore, the most compounds active against33 and the I (IC50 = 0.70 μM), whereas 33 and 38 had IC50 values of 6.0 and 4.0 μM, respectively. 38PfDHODHwere selected enzyme for (Figurein vivo in7), P.which berghei indicated-infected that mouse inhibition model. of On this day enzyme 5 upon was treatment one of the at potential 5 mg/kg, Compounds that had better activity against P. falciparum were also the most active against the administeredmechanisms of orally, action both of the compounds 7-arylaminepyrazolo[1,5- reduced parasitemiaa]pyrimidines. by 50%. Compound 44 showed the best PfDHODH enzyme (Figure 7), which indicated that inhibition of this enzyme was one of the potential inhibitionThe proposed of the Pf DHODH bioisosteric enzyme replac withement an ICof 50the= [1,2,4]triazolo[1,5-0.16 µM, which wasa]pyrimidine more potent ring than on prototype prototypeI mechanisms of action of the 7-arylaminepyrazolo[1,5-a]pyrimidines. (ICI by50 =the0.70 pyrazolo[1,5-µM), whereasa]pyrimidine33 and 38 hadring ICwas50 values shown of to 6.0 be and a positive 4.0 µM, respectively.proposition according to the The proposed bioisosteric replacement of the [1,2,4]triazolo[1,5-a]pyrimidine ring on prototype molecularCompounds docking that study. had better These activity results against demonstratedP. falciparum thewere potential also the mostof 7-arylpyrazolo[1,5- active against the I by the pyrazolo[1,5-a]pyrimidine ring was shown to be a positive proposition according to the Pfa]pyrimidineDHODH enzyme derivatives (Figure as7), inhibitors which indicated of PfDHODH that inhibition and may of thisrepresent enzyme new was leads one offor the developing potential molecular docking study. These results demonstrated the potential of 7-arylpyrazolo[1,5- mechanismsdrugs against of malaria. action of the 7-arylaminepyrazolo[1,5-a]pyrimidines. a]pyrimidine derivatives as inhibitors of PfDHODH and may represent new leads for developing drugs against malaria.

Figure 7. Structure of pyrazolo[1,5-a]pyrimidines 30–44. Figure 7. Structure of pyrazolo[1,5-a]pyrimidines 30–44.

TheAlthough proposed quinoline bioisosteric Figurederivatives replacement 7. Structure continue ofof thepyrazolo[1,5- [1,2,4]triazolo[1,5-to dominatea]pyrimidines the aantimalarial]pyrimidine 30–44. ringdrug, on new prototype syntheticI by thehybrid pyrazolo[1,5- compoundsa]pyrimidine have been ringdescribed was shown in recent to beliterature a positive [28–34]; proposition however, according due to resistance to the molecular to this Although quinoline derivatives continue to dominate the antimalarial drug, new synthetic dockingclass of drugs, study. Thesesearching results for demonstratedother analogous the compounds potential of 7-arylpyrazolo[1,5-is extremely importanta]pyrimidine [35]. derivatives hybrid compounds have been described in recent literature [28–34]; however, due to resistance to this as inhibitorsIn an effort of Pf toDHODH obtain novel and may quinoline represent derivati newves leads for for the developing treatment drugsof malaria, against new malaria. quinolinyl- class of drugs, searching for other analogous compounds is extremely important [35]. 1H-1,2,3-triazolesAlthough quinoline (48–58) derivatives were designed continue to find to dominatenew compounds the antimalarial with anti- drug,P. falciparum new synthetic activity hybrid [36]. In an effort to obtain novel quinoline derivatives for the treatment of malaria, new quinolinyl- compoundsThe literature have been describes described several in recent compounds literature containing [28–34]; however,a 1H-1,2,3-triazole due to resistance ring with to P. this falciparum class of 1H-1,2,3-triazoles (48–58) were designed to find new compounds with anti-P. falciparum activity [36]. drugs,activity searching [37]. Previous for other works analogous described compounds by our group is extremely demonstrated important that [35 ].1,2,3-triazoles are active The literature describes several compounds containing a 1H-1,2,3-triazole ring with P. falciparum againstIn Mycobacterium an eﬀort to obtaintuberculosis novel [38] quinoline and Leishmania derivatives amazonensis for the[39]. treatment Therefore, ofit was malaria, planned new to activity [37]. Previous works described by our group demonstrated that 1,2,3-triazoles are active quinolinyl-1connect this Hring-1,2,3-triazoles to the 7-chloroquinoline (48–58) were moiety designed present to ﬁnd in newCQ and compounds amodiaquine with and anti- incorporateP. falciparum a against Mycobacterium tuberculosis [38] and Leishmania amazonensis [39]. Therefore, it was planned to activityvariety of [36 substituents]. at the 4 position (Figure 8). connectThe this literature ring to describes the 7-chloroquinoline several compounds moiety containing present in a CQ 1H -1,2,3-triazoleand amodiaquine ring withand incorporateP. falciparum a activity variety of [37 substituents]. Previous at works the 4 describedposition (Figure by our 8). group demonstrated that 1,2,3-triazoles are active against Mycobacterium tuberculosis [38] and Leishmania amazonensis [39]. Therefore, it was planned to connect this ring to the 7-chloroquinoline moiety present in CQ and amodiaquine and incorporate a variety of substituents at the 4 position (Figure8). Molecules 2019, 24, x FOR PEER REVIEW 6 of 20

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Figure 8. Rational approach to the design of quinoline derivatives 48–58.

The synthetic route that was used to prepare the 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinolines 48– 58 is shown in Figure 9. The raw material 4,7-dichloroquinoline (59) was treated with sodium azide in MeOH under reflux for 24 h to obtain the 4-azido-7-chloroquinoline (60) derivative in 70% yield. The 1,3-dipolar cycloaddition reaction between azide derivative 60 and the respective alkyne was performed with sodium ascorbate and a Cu(I) catalyst, in H2O/t-BuOH/THF (1:1:1) at 25 °C to obtain the 1,4- regioisomer of 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinolines 48–58 in 40–77% yield [38]. Alcohol derivative 55 was used for the synthesis of compounds 56, 57 and 58. Aldehyde 56 was prepared in 55% Figureyield, by 8. Rational the Swern approach oxidation to the of design 55. The of quinoline reaction derivatives of 55 with48 dimethylaminosulfur–58. trifluoride (DAST) in CH2Cl2 at 25 °C for 24 h produced the monofluorinated derivative 57 in 70% yield.The The synthetic difluorinated route thatderivative was used 58 towas prepare obtained the with 7-chloro-4-(1 60% yieldH-1,2,3-triazol-1-yl)quinolines from the reaction of aldehyde48– 5856 iswith shown DAST. in Figure9.

Figure 9. Synthesis of 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinolines 48–58. Reagents and conditions: (i) NaN3, MeOH, reﬂux, 24 h; (ii) appropriate acetylene, L-ascorbic acid sodium salt, CuSO4.5H2O, H O/t-BuOH/THF (1:1:1), 25 C, 24 h; (iii) ClCOCOCl, CH Cl , DMSO, TEA, 78 C, 6 h; (iv) DAST, 2 ◦ 2 2 − ◦ CH2Cl2, 25 ◦C, 24 h.

The raw material 4,7-dichloroquinoline (59) was treated with sodium azide in MeOH under reﬂux for 24 h to obtain the 4-azido-7-chloroquinoline (60) derivative in 70% yield. The 1,3-dipolar cycloaddition reaction between azide derivative 60 and the respective alkyne was performed with sodium ascorbate and a Cu(I) catalyst, in H2O/t-BuOH/THF (1:1:1) at 25 ◦C to obtain the 1,4-regioisomer of 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinolines 48–2caonima58 in 40–77% yield [38]. Molecules 2019, 24, x FOR PEER REVIEW 7 of 20

Figure 9. Synthesis of 7-chloro-4-(1H-1,2,3-triazol-1-yl)quinolines 48–58. Reagents and conditions: (i) MoleculesNaN20193, MeOH,, 24, 4095 reflux, 24 h; (ii) appropriate acetylene, L-ascorbic acid sodium salt, CuSO4.5H2O, H2O/t-7 of 20 MoleculesBuOH/THF 2019, 24, x (1:1:1), FOR PEER 25 °C, REVIEW 24 h; (iii) ClCOCOCl, CH2Cl2, DMSO, TEA, −78 °C, 6 h; (iv) DAST, CH2Cl27, of 20 25 °C, 24 h. AlcoholFigure 9. derivativeSynthesis of55 7-chloro-4-(1was used forH-1,2,3-triazol-1-yl)quinolines the synthesis of compounds 48–5856. ,Reagents57 and 58and. Aldehydeconditions: 56(i) was preparedWeNaN 3synthesized in, MeOH, 55% yield, reflux, elevenby 24 h; the (ii) Swernnew appropriate hybrid oxidation acet 7-chloro-4-(1ylene, of 55 L-ascorbic. TheH reaction-1,2,3-triazol-1-yl)quinolines acid sodium of 55 withsalt, CuSO dimethylaminosulfur4.5H2O, 48H2–O/t-58. Six trifluoridecompounds,BuOH/THF (DAST) 48, (1:1:1),50– in52 CH,25 56 °C,2 andCl 242 at58h; 25,(iii) exhibited◦ ClCOCOCl,C for 24 in h vitro producedCH2 Clactivity2, DMSO, the against monofluorinated TEA, −the78 °C,P. falciparum6 h; (iv) derivative DAST, W2 CHCQR572Clin 2clone,, 70% yield.with 25IC The °C,50 values difluorinated24 h. ranging derivativefrom 1.4 to58 46was μM. obtained None of withthese 60% compounds yield from were the toxic reaction to HepG2 of aldehyde cells. 56 withThe DAST. most active compound 56 (Figure 10) contained an aldehyde group at the 4 position of 1H- 1,2,3-triazol-1-yl,We synthesizedsynthesized with elevenaneleven IC50 = new new1.4 μ hybridMhybrid and SI 7-chloro-4-(17-chloro-4-(1 = 351. HH-1,2,3-triazol-1-yl)quinolines-1,2,3-triazol-1-yl)quinolines 4848––5858.. Six compounds, 4848, ,5050––5252, ,5656 andand 58,58 exhibited, exhibited in vitroin vitro activityactivity against against the P. the falciparumP. falciparum W2 CQRW2 clone, CQR clone,with IC with50 values IC50 values ranging ranging from 1.4 from to 1.446 μ toM. 46 NoneµM. None of these of these compounds compounds were were toxic toxic to HepG2 to HepG2 cells. cells. The most active active compound compound 5656 (Figure(Figure 10) 10 contained) contained an an aldehyde aldehyde group group at the at the4 position 4 position of 1H of- 11,2,3-triazol-1-yl,H-1,2,3-triazol-1-yl, with with an IC an50 IC = 501.4= μ1.4M andµM andSI = SI351.= 351.

Figure 10. Structure of compound 56.

Hybrid antimalarial drugs have advantages over combined drugs since they decrease the risk of adverse drug-drug interactions and facilitate treatment adherence. Through a rational drug design approach, single hybrid moleculesFigure with dual 10. Structure functionality of compound and/or 56 targets. have been planned and may have either the same mechanism of action as the precursor drugs or a distinct mechanism of action [40,41].Hybrid antimalarial drugs have advantages over combined drugs since they decrease the risk of adverseA hybrid drug-drug salt of interactions artesunate and and mefloquine, facilitate treatment MEFAS adherence. (61) (Figure Through 11), was a active rational against drug both design the approach,CQS 3D7 and single single CQR hybrid hybrid W2 strainsmolecules molecules of P. with withfalciparum dual dual functionality, functionalitywith IC50 = and/or 0.001 and μ /targetsorM. targets This have hybrid have been beensalt planned was planned at and least may and 5- mayfoldhave more haveeither eitherpotent the same thethan samemechanism mefloquine mechanism of alone, action of action moreas the potent asprecursor the precursorthan drugs artesunate drugsor a distinct against or a distinct mechanism 3D7, mechanismas effective of action as of actionartesunate[40,41]. [ 40,41 against]. W2, and more potent than mixtures of the drugs. In vivo tests against P. berghei- infectedA hybrid mice, saltat a ofdose artesunateartesuna of 10 temg/kg, and mefloquine, mefloquine, led to cure MEFASwithout ( 61recrudescence)) (Figure 1111),), of was parasitemia. active against The both in vivo the CQScytotoxicity 3D73D7 andand of CQR CQRMEFAS W2 W2 strainshas strains demonstrated of ofP. P. falciparum falciparum that, with its, with toxi IC 50cityIC=50 0.001is= 0.0015-foldµM. μ lowerM. This This hybridthan hybrid that salt ofsalt was mefloquine. was at least at least 5-fold The 5- morecombinedfold more potent fixedpotent than dose mefloquinethan of mefloquine ASMQ alone, (artesunate morealone, potent more + mefloquine) thanpotent artesunate than was artesunate against3-fold more 3D7, against astoxic e ff3D7,ective than as asMEFAS effective artesunate [42]. as againstMEFASartesunate W2,has against andbeen more demonstrated W2, potent and more than to potentmixturesbe an active than of the mibloodxtures drugs. schizonticidal ofIn the vivo drugs.tests and In against canvivo also P.tests berghei block against -infectedthe infectivityP. berghei- mice, atofinfected aP. dose falciparum mice, of 10 at mggametocytes, a/ kg,dose led of to10 280- cure mg/kg, and without 15-foldled to recrudescence cure more without effectiv ofrecrudescenceely parasitemia. than mefloquine of The parasitemia.in and vivo artesunatecytotoxicity The in alone, vivo of MEFASrespectivelycytotoxicity has demonstrated of[43]. MEFAS These has results that demonstrated its make toxicity this isthat 5-foldcompou its toxi lowerndcity thanvery is 5-fold thatpromising oflower mefloquine. than to target that The of both mefloquine. combined the asexual fixed The doseparasitescombined of ASMQ and fixed gametocytes, (artesunate dose of ASMQ +improvingmefloquine) (artesunate the wasantimalarial + mefloquine) 3-fold more effects. toxicwas 3-fold than MEFASmore toxic [42]. than MEFAS MEFAS has been[42]. demonstratedMEFASPRIMAS has been ( to62 bedemonstrated) (Figure an active 11) blood tois bealso schizonticidal an aactive hybrid blood salt and schizonticidal between can also blockartesunate and the can infectivity alsoand blockprimaquine of theP. infectivity falciparum under gametocytes,developmentof P. falciparum 280-by gametocytes, Boechat and 15-fold and 280- morecoworkers and eff ectively15-fold [44]. moreThis than hy mefloquineeffectivbrid wasely than anddesigned artesunatemefloquine with the alone, and goal artesunate respectively of minimizing alone, [43]. Theseprimaquinerespectively results toxicity. make[43]. These this Indeed, compound results the PRIMASmake very promisingthis hybrid compou salt to targetnd is more very both activepromising the asexual in vivo to parasitesand target in vitro both and and gametocytes,the less asexual toxic improvingthanparasites primaquine. and the gametocytes, antimalarial eimprovingffects. the antimalarial effects. PRIMAS (62) (Figure 11) is also a hybrid salt between artesunate and primaquine under development by Boechat and coworkers [44]. This hybrid was designed with the goal of minimizing primaquine toxicity. Indeed, the PRIMAS hybrid salt is more active in vivo and in vitro and less toxic than primaquine.

Figure 11. Structures of MEFAS and PRIMAS.

PRIMAS (62) (Figure 11) is also a hybrid salt between artesunate and primaquine under development by Boechat and coworkers [44]. This hybrid was designed with the goal of minimizing primaquine toxicity. Indeed, the PRIMAS hybrid salt is more active in vivo and in vitro and less toxic Figure 11. Structures of MEFAS and PRIMAS. than primaquine.

Molecules 2019, 24, x FOR PEER REVIEW 8 of 20

Hybrid compounds that have been designed through the incorporation of different pharmacophoric groups into the quinoline ring have generated effective derivatives. Some are already in the clinical trial phase [45]. MoleculesIn 2019an effort, 24, 4095 to enhance the anti-P. falciparum activity of quinoline derivatives 63–77, a series8 of of 15 20 moleculesMolecules 2019 was, 24, xdesigned FOR PEER REVIEWfrom the precursor drugs chloroquine and sulfadoxine (Figure 12)8 of [46]. 20 These compounds contain the pharmacophore groups of a 7-chloroquinoline and arylsulfonamide, HybridHybrid compounds compounds that that have beenhave designedbeen designed through thethrough incorporation the incorporation of different pharmacophoric of different separated by a distinct linker that is not found in the individual drug molecular frameworks. The 7- groupspharmacophoric into the quinoline groups ringinto havethe quinoline generated ring effective have derivatives.generated effective Some are derivatives. already in theSome clinical are chloroquinoline moiety was included because it is present in CQ, which is used to treat malaria, while trialalready phase in [the45]. clinical trial phase [45]. the arylsulfonamide moiety is present in sulfadoxine. The pharmacophore groups were connected by InIn anan eeffortffort to enhance the the anti- anti-P.P. falciparum falciparum activityactivity of of quinoline quinoline derivatives derivatives 63–6377–, 77a series, a series of 15 of a linker group containing 2–4 CH2 units. 15molecules molecules was was designed designed from thethe precursor precursor drugs drugs chloroquine chloroquine and and sulfadoxine sulfadoxine (Figure (Figure 12)[ 4612)]. These[46]. compoundsThese compounds contain contain the pharmacophore the pharmacophore groups ofgroups a 7-chloroquinoline of a 7-chloroquinoline and arylsulfonamide, and arylsulfonamide, separated byseparated a distinct by linker a distinct that is linker not found that inis thenot individualfound in the drug individual molecular drug frameworks. molecular The frameworks. 7-chloroquinoline The 7- moietychloroquinoline was included moiety because was itincluded is present because in CQ, it which is present is used in toCQ, treat which malaria, is used while to treat the arylsulfonamide malaria, while moietythe arylsulfonamide is present in moiety sulfadoxine. is present The in pharmacophoresulfadoxine. The groupspharmacophore were connected groups were by a connected linker group by containinga linker group 2–4 CHcontaining2 units. 2–4 CH2 units.

Figure 12. Rational approach to the design of compounds 63–77.

The synthetic route to prepare the N-(2-((7-chloroquinolin-4- yl)amino)alkyl)benzenesulfonamides 63–77 is shown in Figure 13. The nucleophilic substitution reaction of 4,7-dichloroquinoline (59) with the corresponding 1 diamine was performedFigureFigure to 12.obtain12. Rational intermediates approach to N the -(7-chloroquinolin-4-yl)alkyldiamine design of of compounds compounds 6363––7777. . 78–80 in 85- 90% yield [47]. The addition-elimination reaction between the N1-(7-chloroquinolin-4-yl)alkyl- diaminesTheThe synthetic 78–80synthetic and route the to appropriate prepareroute the sulfonylN-(2-((7-chloroquinolin-4-yl)amino)alkyl)benzenesulfonamidesto chlorideprepare in MeOH theand TEAN at-(2-((7-chloroquinolin-4- 25 °C afforded the N-(2- 63((7-chloroquinolin-4-yl)amino)alkyl)benzenesulfonamidesyl)amino)alkyl)benzenesulfonamides–77 is shown in Figure 13. 63–77 is shown in Figure 63– 7713. in 50–77% yield [48]. The nucleophilic substitution reaction of 4,7-dichloroquinoline (59) with the corresponding diamine was performed to obtain intermediates N1-(7-chloroquinolin-4-yl)alkyldiamine 78–80 in 85- 90% yield [47]. The addition-elimination reaction between the N1-(7-chloroquinolin-4-yl)alkyldiamines 78–80 and the appropriate sulfonyl chloride in MeOH and TEA at 25 °C afforded the N-(2- ((7-chloroquinolin-4-yl)amino)alkyl)benzenesulfonamides 63–77 in 50–77% yield [48].

Figure 13. Synthetic route used to prepare compounds 63–77. Reagents and conditions: (i) appropriate Figure 13. Synthetic route used to prepare compounds 63–77. Reagents and conditions: (i) appropriate diamine, reflux, 4 h; (ii) appropriate sulfonyl chloride, MeOH, TEA, 25 C, 24 h. diamine, reflux, 4 h; (ii) appropriate sulfonyl chloride, MeOH, TEA, 25◦ °C, 24 h. The nucleophilic substitution reaction of 4,7-dichloroquinoline (59) with the corresponding The 15 synthesized compounds 63–77 with different substituents at the 4 position of the diamine was performed to obtain intermediates N1-(7-chloroquinolin-4-yl)alkyldiamine 78–80 in 85-90% arylsulfonamide group were tested against the W2 chloroquine and sulfadoxine-resistant P. yield [47]. The addition-elimination reaction between the N1-(7-chloroquinolin-4-yl)alkyl-diamines falciparumFigure clone 13. Synthetic and for route their used cytotoxicity. to prepare compounds 63–77. Reagents and conditions: (i) appropriate 78–80 and the appropriate sulfonyl chloride in MeOH and TEA at 25 ◦C afforded the Alldiamine, the compounds reflux, 4 h; (ii) presented appropriate activity, sulfonyl and chloride, 10 of MeOH,them showed TEA, 25 °C,IC50 24 values h. ranging from 0.05 to N-(2-((7-chloroquinolin-4-yl)amino)alkyl)benzenesulfonamides 63–77 in 50–77% yield [48]. 0.40 μM in the anti-HPR2 assay, which are lower than those of CQ and sulfadoxine, and none of them The 15 synthesized compounds 63–77 with different substituents at the 4 position of the were Thetoxic 15 to BGMsynthesized cells. compounds 63–77 with different substituents at the 4 position of the arylsulfonamide group were tested against the W2 chloroquine and sulfadoxine-resistant P. falciparum arylsulfonamideA direct relationship group were was observedtested against between the theW2 increase chloroquine in the andnumber sulfadoxine-resistant of methylene groups P. clone and for their cytotoxicity. (CHfalciparum2) used clone as the and linker for andtheir the cytotoxicity. increase in activity. Compounds with 4 and 3 methylene group (CH2) All the compounds presented activity, and 10 of them showed IC values ranging from 0.05 to linkersAll showed the compounds greater presentedactivity against activity, P. and falciparum 10 of them than showed CQ, whileIC50 values the seriesranging of from compounds 0.05 to 0.400.40µ μMM in in thethe anti-HPR2anti-HPR2 assay,assay, whichwhich areare lower than th thoseose of CQ CQ and and sulfadoxine, sulfadoxine, and and none none of of them them containing 2 methylene groups were less active than CQ, with IC50 values in the range of 0.48 to 1.63 werewere toxic toxic to to BGM BGM cells.cells. μM. Compounds 68–72, with three methylene groups as the linker, showed IC50 values ranging from A direct relationship was observed between the increase in the number of methylene groups 0.10 toA 0.35direct μ M.relationship Compounds was 74 observed–77 containing between four the methylene increase in groups the number as the of linker methylene were thegroups most (CH(CH22)) usedused as as the the linker linker and and the the increase increase in activi in activity.ty. Compounds Compounds with with4 and 43 andmethylene 3 methylene group (CH group2) (CH linkers2) linkers showed showed greater greater activity activity against against P. falciparumP. falciparum thanthan CQ, CQ, while while the the series series of of compounds compounds containingcontaining 2 2 methylene methylene groups groups were were less less active active than than CQ, CQ, with with IC 50ICvalues50 values in in the the range range of 0.48of 0.48 to 1.63to 1.63µM. CompoundsμM. Compounds68–72 68, with–72, threewith three methylene methylene groups groups as the as linker, the linker, showed showed IC50 ICvalues50 values ranging ranging from from 0.10 0.10 to 0.35 μM. Compounds 74–77 containing four methylene groups as the linker were the most

Molecules 2019, 24, 4095 9 of 20 Molecules 2019, 24, x FOR PEER REVIEW 9 of 20 activeto 0.35 ofµM. the Compounds series, with74 IC–5077 valuescontaining ranging four from methylene 0.05–0.15 groups μΜ. as Compound the linker were73 (R the = H) most (IC active50 = 0.40 of Μ) μthe series, was an with exception. IC50 values Compounds ranging from with 0.05–0.15 substituentsµM. at Compound the 4 position73 (R of= theH) (ICarylsulfonamide50 = 0.40 µM)was group an increasedexception. Compoundsthe P. falciparum with substituents activity, atwhile the 4the position derivatives of the arylsulfonamide without a substituent group increased on the P.arylsulfonamide falciparum activity, group while were the the derivatives least potent. without Compounds a substituent 72 (R = on F; theIC50 arylsulfonamide = 0.10 μΜ), 74 (R group = CH3 were; IC50 Μ Μ Μ =the 0.05 least μ potent.), 75 (R Compounds = Cl; IC50 = 0.0972 (R μ=)F; and IC50 77= (R0.10 = F;µ M),IC5074 = 0.15(R = μCH)3 had; IC50 the= highest0.05 µM), SI75 values:(R = Cl; 3386.0, IC50 2489.0,= 0.09 µ 1102.2M) and and77 (R1031.3,= F; ICrespectively,50 = 0.15 µM) so hadthey the were highest also SIevaluated values: 3386.0,for their 2489.0, antimalarial 1102.2 andactivity 1031.3, in P.respectively, berghei-infected so they mice. were also evaluated for their antimalarial activity in P. berghei-infected mice. Compounds 7474 andand77 77were were partially partially active active and and inhibited inhibitedP. bergheiP. berghei-parasitemia-parasitemia by 27% by 27% and 30%,and 30%,respectively respectively on day on 5 day upon 5 upon treatment treatment at 10 mgat 10/kg, mg/kg, administered administered orally, orally, whereas whereas compounds compounds72 and 7275 andshowed 75 showed parasitemia parasitemia inhibition inhibition at rates at of rates 47% andof 47% 49%, and respectively. 49%, respectively. Due to to the the advantages advantages of ofthe the hybrids hybrids with with the best the results best results exhibited exhibited herein, herein, compound compound 75 (Figure75 (Figure14) was 14used) was as an used antimalarial as an antimalarial prototype prototype (prototype (prototype II) to proceedII) to with proceed other with studies other to studiesovercome to theovercome burden the of burdenresistance of resistancein P. falciparum in P.. falciparum .

Cl H N HN S O O 75- prototype II IC50 =0.09µM in vivo 49% Cl N Figure 14. Structure of prototype II. Figure 14. Structure of prototype II. Continuing the search for new quinoline derivatives with antimalarial activity, activity, a new class of hybridscaonima 81 81––8484 withwith atorvastatin atorvastatin (AVA) (AVA) was was planned planned inspired inspired by byprototype prototype II [49]. II [ 49The]. design The design of these of moleculesthese molecules was based was based on the on theproven proven AVA AVA antimalarial antimalarial activity. activity. The The molecular molecular hybridization hybridization of chloroquine derivativesderivatives81 81–84–84included included the the aminoquinoline aminoquinoline moiety moie withty with the pyrrole the pyrrole of AVA. of ToAVA. connect To connectthese two these pharmacophoric two pharmacophoric groups, the groups, 7-chloroquinoline the 7-chloroquinoline moiety was bound moiety to thewas pentasubstituted bound to the pyrrolepentasubstituted by a linker pyrrole group containingby a linker 2–4group CH 2containingunits. Hybrid 2–4 84CHwas2 units. made Hybrid by the 84 direct was attachmentmade by the of primaquinedirect attachment to the of pentasubstituted primaquine to the pyrrole pentasubstituted (Figure 15). pyrrole (Figure 15).

Figure 15. Rational approach to the design of compounds 81–84.

3caonima Molecules 2019, 24, 4095 10 of 20 Molecules 2019, 24, x FOR PEER REVIEW 10 of 20 Molecules 2019, 24, x FOR PEER REVIEW 10 of 20 The Paal–Knorr [50] reaction between the key intermediate used in prototype II synthesis [47] TheThe Paal–Knorr Paal–Knorr [50] [50 reaction] reaction between between the key the inte keyrmediate intermediate used in prototype used in II prototypesynthesis [47] II N-(7-chloroquinolin-4-yl)alkyldiamines 78–80 or primaquine and 1,4-diketone 85, new pyrrole- synthesisN-(7-chloroquinolin-4-yl)alkyldiamines [47] N-(7-chloroquinolin-4-yl)alkyldiamines 78–80 or primaquine78–80 orand primaquine 1,4-diketone and 85 1,4-diketone, new pyrrole-85, chloroquine 81–83 and pyrrole-primaquine 84 derivatives were obtained, respectively, in 18-64% newchloroquine pyrrole-chloroquine 81–83 and pyrrole-primaquine81–83 and pyrrole-primaquine 84 derivatives84 derivativeswere obtained, were respectively, obtained, respectively, in 18-64% yield (Figure 16). The N-(7-chloroquinolin-4-yl)alkyldiamines 78–80 were obtained as previously inyield 18-64% (Figure yield 16). (Figure The N 16-(7-chloroquinolin-4-yl)alkyldiamines). The N-(7-chloroquinolin-4-yl)alkyldiamines 78–80 were 78obtained–80 were as obtained previously as described [46]. previouslydescribed [46]. described [46].

Figure 16. Synthetic route used to prepare compounds 81–84. Reagents and conditions : (i) (i) pivalic pivalic acid, acid, Figure 16. Synthetic route used to prepare compounds 81–84. Reagents and conditions: (i) pivalic acid, THF: cyclohexane, 80–100 °C,C, 18–64%. THF: cyclohexane, 80–100 ◦°C, 18–64%. All compounds synthesized showed activity against against the the P.P. falciparum W2 clone, and none of All compounds synthesized showed activity against the P. falciparum W2 clone, and none of them were significantly significantly toxic to the BGM cell line. Chloroquine-AVA Chloroquine-AVA hybrids 81, 82 and 83 were the them were significantly toxic to the BGM cell line. Chloroquine-AVA hybrids 81, 82 and 83 were the most active, with IC50 valuesvalues of of 0.99, 0.99, 0.65 0.65 and and 0.40 0.40 μµM,M, respectively, respectively, which were in a similar range to most active, with IC50 values of 0.99, 0.65 and 0.40 μM, respectively, which were in a similar range to chloroquine (IC50 == 0.590.59 μµM),M), and and all were better than primaquineprimaquine (IC(IC5050 = 1.891.89 μµM) and atorvastatin chloroquine (IC50 = 0.59 μM), and all were better than primaquine (IC50 = 1.89 μM) and atorvastatin (IC50 == 10.310.3 μµM),M), with with good good SI SI values. values. Herein, Herein, the the activity activity of of the the compounds compounds increased increased with the length (IC50 = 10.3 μM), with good SI values. Herein, the activity of the compounds increased with the length of the carbon chain, which was similarly shown for the quinoline-sulfonamide derivatives that have of the carbon chain, which was similarly shown for the quinoline-sulfonamide derivatives that have four methylene groups asas thethe linker. Compound 8383 ((n = 4)4) (Figure (Figure 17)17) exhibited exhibited better better activity activity despite despite four methylene groups as the linker. Compound 83 (n = 4) (Figure 17) exhibited better activity despite the exchange of the terminal amine for a polysubstitutedpolysubstituted pyrrole ring and was 26-fold more active the exchange of the terminal amine for a polysubstituted pyrrole ring and was 26-fold more active than AVA. PrimaquinePrimaquine derivativederivative 8484 (IC(IC5050 = 1.41) was the least potent of the series. However, it was than AVA. Primaquine derivative 84 (IC50 = 1.41) was the least potent of the series. However, it was significantlysignificantly less toxic and more active thanthan primaquineprimaquine andand 7-fold7-fold moremore activeactive thanthan AVAAVA (IC (IC5050 == 10.3 significantly less toxic and more active than primaquine and 7-fold more active than AVA (IC50 = 10.3 µμM). This indicates that compound 84 is promising and may be a prototype in the search for a drug μM). This indicates that compound 84 is promising and may be a prototype in the search for a drug that is able to replace primaquine,primaquine, becausebecause 84 was shownshown toto bebe safersafer (SI(SI >> 1107) than primaquine (SI that is able to replace primaquine, because 84 was shown to be safer (SI > 1107) than primaquine (SI of 239). Additionally, Additionally, to to evaluate evaluate the the importance importance of of the the pyrrolic pyrrolic moiety moiety in in the the hybrid hybrid compounds, compounds, a of 239). Additionally, to evaluate the importance of the pyrrolic moiety in the hybrid compounds, a apentasubstituted pentasubstituted pyrrole pyrrole without without the the aminoquinolinyl aminoquinolinyl moiety moiety was was synthesized. synthesized. This This compound compound had pentasubstituted pyrrole without the aminoquinolinyl moiety was synthesized. This compound had hadno toxicity no toxicity in vitroin vitro andand was was more more potent potent (IC (IC50 =50 6.39= 6.39 μM)µM) than than AVA. AVA. This This result result suggests suggests that that the no toxicity in vitro and was more potent (IC50 = 6.39 μM) than AVA. This result suggests that the pentasubstituted pyrrole might be the pharmacophor pharmacophoricic group group of of AVA AVA for antimalarial activity. New pentasubstituted pyrrole might be the pharmacophoric group of AVA for antimalarial activity. New compounds containing pentasubstitutedpentasubstituted pyrrole-quinolines will be synthesized with the expectation compounds containing pentasubstituted pyrrole-quinolines will be synthesized with the expectation of enhanced potency and solubility and will also be assayed assayed for cerebral cerebral antimalarial activity to clarify of enhanced potency and solubility and will also be assayed for cerebral antimalarial activity to clarify the importance of AVA inin thisthis scascaffold.ffold. the importance of AVA in this scaffold.

Figure 17. Structure of compound 83. Figure 17. Structure of compound 83. In an effort to enhance the anti-P. falciparum activity of prototype II, new non-quinoline In an effort to enhance the anti-P. falciparum activity of prototype II, new non-quinoline derivatives 86–95 were planned [51]. Therefore, the pyrazolopyridine system was selected as an derivatives 86–95 were planned [51]. Therefore, the pyrazolopyridine system was selected as an

caonima

Molecules 2019, 24, 4095 11 of 20

In an eﬀort to enhance the anti-P. falciparum activity of prototype II, new non-quinoline derivatives 86–95 were planned [51]. Therefore, the pyrazolopyridine system was selected as an isostere of quinoline, and the literature has demonstrated that this heterocycle possesses antimalarial activity [52]. The design of the non-quinoline derivatives consists of a ring isosterism in which the 7-chloroquinoline moiety is replaced by the 1-phenyl-1H-pyrazolo[3,4-b]pyridine system. An N-(4-aminobutyl)benzenesulfonamide group was attached to the 4 position of this heterocyclic ring. The 1H-pyrazolo[3,4-b]pyridine ring remains separated from the benzenesulfonamide moiety by the linker containing four methylene groups, which is similar to the linker found in the individual molecular framework of prototype II (Figure 18).

Figure 18. Rational approach to the design of compounds 86–95.

The synthetic route for preparing the N-(4-((1-phenyl-1H-pyrazolo[3,4-b]pyridin-4- yl)amino)butyl)benzenesulfonamidesMolecules 2019, 24, x FOR PEER REVIEW 86–95 is shown in Figure 19. 12 of 20

3caonima

98 R 1 = C O 2 Et

Figure 19. Synthetic route used to prepare compounds 86–95. Reagents and conditions : (i) (i) EtOH, EtOH, reflux, reﬂux,

1 h,h, 70%;70%; (ii)(ii) HH33PO4,, 170 170 °C,◦C, 6 6 h, 86%; (iii) EtOH, reflux, reﬂux, 2 h, 86–88%; (iv) POCl3,, reflux, reﬂux, 6 6 h, h, 84%; (v) butane-1,4-diamine, 1,4-dioxane, 25–80 ◦°C,C, 1–4 h, 66–79%; (vi) appropriate sulfonyl chloride, MeOH,

TEA, 25 ◦°C,C, 24 h, 59–70%; (vii) Dowtherm, 250 ◦°C,C, 40 min, 71%; (viii) POCl33,, reflux, reﬂux, 6 h, 90%.

FromTen derivatives the reaction of 1-phenyl-1 of phenylhydrazineH-pyrazolo[3,4- and ethylb]pyridine 2-cyano-3-ethoxyacrylate, 86–95 were synthesized in ethanolwith different under reflux,substituents the ethylat the 5-amino-1-phenyl-1 4 position of the benzenesulfoH-pyrazole-4-carboxylatenamide group and (96) tested was preparedin vitro against at 70% the yield. W2 Hydrolysischloroquine followed and sulfadoxine-resistant by decarboxylation P. of thisfalciparum compound clone a ffandorded for 5-amino-1-phenyl-1 their cytotoxicity.H -pyrazolAll the (compounds97) in 86% exhibited yield. low Michael toxicity addition to BGM of cells97 andwith IC50 ethyl values 2-cyano-3-ethoxyacrylate lower than that of the control or diethyl drug 2-(ethoxymethylene)malonatesulfadoxine (IC50 = 15.0 μM). However, in ethanol the under observed reflux activity gave derivatives was lower98 thanand that99 in of 86% chloroquine and 88% yield, (IC50 = 0.55 μM) and the quinoline-sulfonamide hybrid II. The IC50 values ranged from 3.46 to 9.30 μM in the anti-HPR2 assay. Derivative (R2 = CH3) 87 with IC50 = 3.46 μM, derivative 89 (R2 = Cl) and 92 (R2 = CH3), both with IC50 values of 3.60 μM, were the most active against P. falciparum (Figure 20). Compound 89 also showed the highest SI value: >277.77. It was observed that the 3 compounds with R1 = CO2Et at the 5 position of the 1H-pyrazolo[3,4- b]pyridine ring showed higher activity than those with CN at the same position.

Figure 20. Structure of compound 87.

Continuing the search to obtain new non-quinoline antimalarials, we used prototype II to design the new derivatives N-(4-((1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4- yl)amino)butyl)benzenesulfonamides 105–113 [55]. The 7-chloroquinoline moiety was replaced by the 1H-pyrazolo[3,4-d]pyrimidine system by ring isosterism, and at the 4 position of the heterocyclic ring, the N-(4-aminobutyl)benzenesulfonamide moiety was attached, designing the 1H-pyrazolo[3,4- b]pyridine derivatives (Figure 21).

Molecules 2019, 24, x FOR PEER REVIEW 12 of 20

Molecules 2019, 24, 4095 12 of 20 respectively. From the reaction of diethyl 2-(((1-phenyl-1H-pyrazol-5-yl)amino)methylene)malonate 98 R (98) with phosphorus oxychloride under reflux, the1 = intermediate ethyl C O 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (100) was prepared2 Et in 84% yield [53]. However, the derivative 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (101) could not be obtained using this methodology. To obtain 101, the cyclization of ethyl 2-cyano-3-((1-phenyl-1H-pyrazol-5-yl)amino)acrylate (99) was performed in refluxing Dowtherm for 40 min and 4-oxo-1-phenyl-4,7-dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (102) was isolated by precipitation from hexane with a yield of 84% [20]. Derivative 102 was treated with phosphorus oxychloride to produce 101 in 90% yield. From the nucleophilic substitution reaction between 4-chloro-1-phenyl-1Figure 19. SyntheticH-pyrazolo[3,4- route used tob prepare]pyridines compounds100 and 86101–95and. Reagents butane-1,4-diamine, and conditions: (i) theEtOH, intermediates reflux, ethyl1 h, 4-((4-aminobutyl)amino)-1-phenyl-170%; (ii) H3PO4, 170 °C, 6 h, 86%; (iii) EtOH,H-pyrazolo[3,4- reflux, 2 h, 86–88%;b]pyridine-5-carboxylate (iv) POCl3, reflux, 6 h, 84%; (103 )(v) and 4-((4-aminobutyl)amino)-1-phenyl-1butane-1,4-diamine, 1,4-dioxane, 25–80H-pyrazolo[3,4- °C, 1–4 h, 66–79%;b]pyridine-5-carbonitrile (vi) appropriate sulfonyl (104 chloride,) were synthesizedMeOH, in 79%TEA, and 25 66%°C, 24 yield, h, 59–70%; respectively (vii) Dowtherm, [54]. The 250 addition-elimination °C, 40 min, 71%; (viii) reactionPOCl3, reflux, between 6 h, 90%. the appropriate sulfonyl chloride and amines 103 and 104 was performed in MeOH and TEA (1.0 mmol) at 25 ◦C to obtain targetTen compounds derivativesN-(4-((1-phenyl-1 of 1-phenyl-1HH-pyrazolo[3,4--pyrazolo[3,4-bb]pyridine]pyridin-4-yl)amino)butyl)benzenesulfonamides 86–95 were synthesized with different substituents86–95 in 59–70% at the yield 4 position [46]. of the benzenesulfonamide group and tested in vitro against the W2 chloroquineTen derivatives and sulfadoxine-resistant of 1-phenyl-1H-pyrazolo[3,4- P. falciparumb]pyridine clone 86and–95 werefor their synthesized cytotoxicity. with diAllfferent the compoundssubstituents exhibited at the 4 position low toxicity of the to benzenesulfonamideBGM cells and IC50 values group lower and tested than thatin vitro of theagainst control the drug W2 sulfadoxinechloroquine (IC and50 sulfadoxine-resistant= 15.0 μM). However,P. the falciparum observedclone activity and forwas their lower cytotoxicity. than that of All chloroquine the compounds (IC50 =exhibited 0.55 μM) low and toxicity the quinoline-sulfonamide to BGM cells and IC hybrid50 values II. lower than that of the control drug sulfadoxine (IC50The= 15.0 IC50µ M).values However, ranged the from observed 3.46 toactivity 9.30 μM was in lowerthe anti-HPR2 than that assay. of chloroquine Derivative (IC (R502= = 0.55CH3µ) M)87 withand theIC50 quinoline-sulfonamide = 3.46 μM, derivative 89 hybrid (R2 =II Cl). and 92 (R2 = CH3), both with IC50 values of 3.60 μM, were the mostThe ICactive50 values against ranged P. falciparum from 3.46 (Figure to 9.30 20).µM Compound in the anti-HPR2 89 also assay. showed Derivative the highest (R2 = SICH value:3) 87 with>277.77. IC50 = 3.46 µM, derivative 89 (R2 = Cl) and 92 (R2 = CH3), both with IC50 values of 3.60 µM, wereIt the was most observed active againstthat theP. 3 falciparumcompounds(Figure with 20R1). = CompoundCO2Et at the89 5 alsoposition showed of the the 1 highestH-pyrazolo[3,4- SI value: >b]pyridine277.77. ring showed higher activity than those with CN at the same position.

Figure 20. StructureStructure of compound 87.

ContinuingIt was observed the search that to obtain the 3 new compounds non-quinoline with antimalarials, R1 = CO2Et weat used the prototype 5 position II to ofdesign the 1theH-pyrazolo[3,4- newb]pyridine ringderivatives showed higher activityN-(4-((1-phenyl-1 than those withH-pyrazolo[3,4- CN at the samed]pyrimidin-4- position. yl)amino)butyl)benzenesulfonamidesContinuing the search to obtain new 105 non-quinoline–113 [55]. The antimalarials, 7-chloroquinoline we used moiety prototype wasII toreplaced design theby thenew 1H derivatives-pyrazolo[3,4-N-(4-((1-phenyl-1d]pyrimidineH system-pyrazolo[3,4- by ringd isosterism,]pyrimidin-4-yl)amino)butyl)benzenesulfonamides and at the 4 position of the heterocyclic 105ring,–113 the N[55-(4-aminobutyl)benzenesul]. The 7-chloroquinolinefonamide moiety wasmoiety replaced was attached, by the designing 1H-pyrazolo[3,4- the 1H-pyrazolo[3,4-d]pyrimidine bsystem]pyridine by derivatives ring isosterism, (Figure 21). and at the 4 position of the heterocyclic ring, the N-(4-aminobutyl)benzenesulfonamide moiety was attached, designing the 1H-pyrazolo[3,4-b]pyridine derivatives (Figure 21).

caonima

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Figure 21. RationalRational approach to the design of compounds 105–113.

The synthetic route for preparingpreparing NN-(4-((1-phenyl-1-(4-((1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-]pyrimidin-4- yl)amino)butyl)benzenesulfonamides 105–113 isis shownshown inin FigureFigure 2222..

2caonima

Figure 22.22. SyntheticSynthetic route route used used to to prepare prepare compounds compounds105 –105113–.113Reagents. Reagents and conditionsand conditions: (i) EtOH,: (i) EtOH, reﬂux,

reflux,2 h, 61–80%; 2 h, 61–80%; (ii) HCOOH, (ii) HCOOH, reflux, 12 reflux, h, 73–89%; 12 h, (iii) 73–89%; POCl 3(iii), reflux, POCl 243, h,reflux, 78–97%; 24 h, (iv) 78–97%; butane-1,4-diamine, (iv) butane- 1,4-diamine,CH3CN, 25 ◦ CHC, 243CN, h, 23–36%; 25 °C, 24 (v) h, appropriate 23–36%; (v) sulfonylappropriate chloride, sulfonyl DMF, chloride, TEA, 90 DMF,◦C, 24 TEA, h, 25–79%. 90 °C, 24 h, 25–79%. The reaction of the appropriate phenylhydrazine and 2-(ethoxymethylene)malononitrile in ethanol underThe reflux reaction for 2 hof was the performed appropriate to obtain phenylhydraz the 5-amino-1-phenyl-1ine and 2-(ethoxymethylene)malononitrileH-pyrazole-4-carbonitrile 114a –inc ethanolcompounds under in 61–80%reflux yieldfor 2 [56h ].was Reaction perfor ofmed the suitableto obtain 5-aminepyrazoles the 5-amino-1-phenyl-1114a–c andH-pyrazole-4- formic acid carbonitrileunder reflux 114a for– 12c compounds h afforded in the 61–80% 1-phenyl-1 yieldH [56].-pyrazolo[3,4- Reaction ofd ]pyrimidin-4-olsthe suitable 5-aminepyrazoles(115a–c) in 73–89% 114a– cyield and [ 57formic]. Derivatives acid under115a reflux–c were for treated 12 hwith afforded phosphorus the 1-phenyl-1 oxychlorideH-pyrazolo[3,4- under reflux ford]pyrimidin-4-ols 24 h to produce (115a4-chloro-1-phenyl-1–c) in 73–89% yieldH-pyrazolo[3,4- [57]. Derivativesd]pyrimidines 115a–c were(116a treated–c) in 78–97%with phos yieldphorus [51 ,oxychloride54]. Nucleophilic under refluxsubstitution for 24 h between to produce116a 4-chloro-1-phenyl-1–c and butane-1,4-diamineH-pyrazolo[3,4- in CH3CNd]pyrimidines at 25 ◦C for (116a 24 h– wasc) in performed78–97% yield to 1 [51,54].obtain intermediates Nucleophilic Nsubstitution-(1-phenyl-1 betweenH-pyrazolo[3,4- 116a–c andd]pyrimidin-4-yl)butane-1,4-diamines butane-1,4-diamine in CH3CN at 25(117a °C for–c) 24in h23–36% was performed yield [46, 51to]. obtain The addition-elimination intermediates N1-(1-phenyl-1 reaction betweenH-pyrazolo[3,4- diaminesd]pyrimidin-4-yl)butane-1,4-117a–c and the appropriate diaminessulfonyl chloride(117a–c) in 23–36% DMF and yield triethylamine [46,51]. The addition-e (TEA) at 90limination◦C for24 reaction h afforded between target diamines compounds 117a– cN and-(4-((1-phenyl-1 the appropriateH-pyrazolo[3,4- sulfonyl chlorided]pyrimidin-4-yl)amino)butyl)benzenesulfonamides in DMF and triethylamine (TEA) at 90 °C for 24 (105 h afforded–113) in target compounds N-(4-((1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-

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25–79%yl)amino)butyl)benzenesulfonamides yield [46,51]. The nine derivatives (105–113 of) in 1-phenyl-1 25–79% yieldH-pyrazolo[3,4- [46,51]. Thed ]pyrimidinenine derivatives (105 –of113 1-) thatphenyl-1 wereH-pyrazolo[3,4- synthesized wered]pyrimidine tested in (105 vitro–113for) that their were eﬃ synthesizedcacy against were the tested W2 chloroquine- in vitro for their and sulfadoxine-resistantefficacy against the W2P. falciparum chloroquine-clone and and sulfadoxine-resistant for their cytotoxicity. P. Thefalciparum anti-P. falciparumclone and activitiesfor their ofcytotoxicity. the quinoline The andanti- 1P.H -pyrazolo[3,4-falciparum activitiesd]pyrimidine of the systemsquinoline were and then1H-pyrazolo[3,4- compared.d Compounds]pyrimidine µ 105systems–108, were111, andthen113 compared.showed Compounds IC50 values ranging105–108, from111, and 5.13 113 to 43.40showedM IC in50 thevalues anti-HPR2 ranging assayfrom and5.13 to low 43.40 toxicity μM in to the BGM anti-HPR2 cells. assay Among and the low compounds toxicity to BGM (105–113 cells.) synthesized,Among the compounds six compounds (105– exhibited113) synthesized, anti-P. falciparum six compoundsactivity exhibitedin vitro againstanti-P. falciparum chloroquine-resistant activity in vitro parasites, against and chloroquine- none were toxicresistant to BGMparasites, cells. and This none study were showed toxic to BGM that compound cells. This study107 (R showed1 = F / thatR2 =compoundCH3) presented 107 (R1 = an F µ IC/ R502 =value CH3) of presented 5.13 M, an which IC50 wasvalue lower of 5.13 than μM, that which of the was control lower drug than sulfadoxine that of the (ICcontrol50 = 15.00drug µsulfadoxineM) in the anti-HRPII(IC50 = 15.00assay. μM) in Inthe addition, anti-HRPII most assay. of theIn addition, compounds most in of this the seriescompounds have higherin this SIseries values have than higher sulfadoxine. SI values However, than sulfadoxine. the 1H-pyrazolo[3,4- However,d ]pyrimidinethe 1H-pyrazolo[3,4- derivativesd]pyrimidine were not morederivatives potent were than not the more control potent drug than chloroquine the control drug (IC50 chloroquine= 0.55 µM) (IC and50 = quinoline0.55 μM) and prototype quinolineII (ICprototype50 = 0.09 II µ(ICM).50 = It 0.09 is interesting μM). It is tointeresting note that, to similarnote that, to 1similarH-pyrazolo[3,4- to 1H-pyrazolo[3,4-d]pyridined]pyridine87, the most 87, activethe most derivatives active derivatives in this in series this series have have CH3 CHat the3 at the 4 position 4 position of of the thebenzenesulfonamide benzenesulfonamide moiety. moiety. Compounds 1H-pyrazolo[3,4-d]pyrimidine 107 and 1H-pyrazolo[3,4-b]pyridine]pyridine 87 were equipotent; however, prototype IIII waswas still still the the most most active active (Figure (Figure 23). 23 ).It is It possible is possible to conclude to conclude that thatthe 1 theH- 1pyrazolo[3,4-H-pyrazolo[3,4-d]pyrimidined]pyrimidine and and1H-pyrazolo[3,4- 1H-pyrazolo[3,4-b]pyridineb]pyridine systems systems are promising are promising for further for furtherstudies studiesof their ofanti- theirP. falciparum anti-P. falciparum activities.activities.

Figure 23. Comparison of prototype II with 87 and 107..

New mefloquinemefloquine derivatives 118–133 werewere designeddesigned usingusing molecularmolecular hybridizationhybridization and ringring bioisosterism. The pharmacophoric subunit 2,8-bis-(trifluoromethyl)quinoline,2,8-bis-(trifluoromethyl)quinoline, which which is present in mefloquine,mefloquine, andand the the aminoaryl aminoaryl moiety moiety of amodiaquineof amodiaquine were were linked linked to provide to provide potent potent antimalarial antimalarial drugs (Figuredrugs (Figure 24)[58 ].24) The [58]. importance The importance of the arylmethanol of the arylmethanol moiety inmoiety mefloquine, in mefl alsooquine, present also inpresent quinine, in isquinine, highly importantis highly important for the antimalarial for the antimalarial activity however, activity the however, arylmethanol the arylmethanol moiety was replacedmoiety was by phenylaminoreplaced by phenylamino group present group in amodiaquine. present in amodiaquine. A variety of aliphatic, A variety aromatic of aliphatic, and heteroaromatic aromatic and substituentsheteroaromatic were substituents added to provide were theadded electronic to provide and lipophilic the electronic properties and to researchlipophilic the properties contributions to ofresearch each fragment the contributions to the activity of each profile fragment of this to newthe activity class of profile compounds. of this new Compound class of 134compounds.was also preparedCompound to compare134 was the importancealso prepared of the to 2,8-bis-(trifluoromethyl)quinoline compare the importance moietyof the versus 2,8-bis- the 7-chloroquinoline(trifluoromethyl)quinoline moiety for moiety antiplasmodial versus the activity. 7-chloroquinoline moiety for antiplasmodial activity.

Moleculescaonima 2019 , 24, x FOR PEER REVIEW 15 of 20

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Figure 24. Rational approach to the design of compounds 118–134.

The synthetic route to prepare the N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amine derivatives 118–133 is shown in Figure 25. Reaction of 2-(trifluoromethyl)aniline with ethyl 4,4,4- trifluoroacetoacetateFigure in 24. Rationalpolyphosphoric approach to theacid design (PPA) of compounds for 1183 –134h. produced 2,8- bis(trifluoromethyl)quinolin-4-ol (135) in 91% yield [59], which was then treated with phosphorus oxychlorideThe synthetic at 80 °C route to obtain to prepare 4-chloro-2,8-bis(trifluoromethyl)quinoline the N-substituted-2,8-bis(trifluoromethyl)-quinolin-4-amine (136) in 98% yield [15]. derivatives 118–133 is shown in Figure 25. Reaction of 2-(trifluoromethyl)aniline The nucleophilic aromatic substitution reaction of intermediates 136 with the appropriate amine withafforded ethyl target 4,4,4-trifluoroacetoacetate compounds 118–133 in 42–88% in polyphosphoric yield (Figure 24) acid[60]. (PPA) for 3 h produced 2,8-bis(trifluoromethyl)quinolin-4-olCompound 7-chloro-N-(pyridin-4-yl)quinolin-4-amine (135) in 91% yield [59], which (134) was thenobtained treated from with the phosphorus reaction of oxychloridepyridin-4-amine at 80 with◦C to the obtain commercially 4-chloro-2,8-bis(trifluoromethyl)quinoline available 4,7-dichloroquinoline ( (59136) in) in 89% 98% yield yield [15]. [15 ].

Figure 25. Synthetic route used to prepare compounds 118–134. Reagents and conditions: (i) ethyl Figure 25. Synthetic route used to prepare compounds 118–134. Reagents and conditions: (i) ethyl 4,4,4- 4,4,4-trifluoroacetoacetate, PPA, 150 ◦C, 3 h; (ii) POCl3, 80 ◦C, 4 h; (iii) appropriate amine, NaH, DMSO, trifluoroacetoacetate, PPA, 150 °C, 3 h; (ii) POCl3, 80 °C, 4 h; (iii) appropriate amine, NaH, DMSO, 25 25 C, 1–24 h; (v) pyridin-4-amine, EtOH, 25 C, 24 h. °C,◦ 1–24 h; (v) pyridin-4-amine, EtOH, 25 °C,◦ 24 h. The nucleophilic aromatic substitution reaction of intermediates 136 with the appropriate amine First, a comparison was made between the importance of the 2,8-bis(trifluoromethyl)quinoline afforded target compounds 118–133 in 42–88% yield (Figure 24)[60]. versus the 7-chloroquinoline moieties. Compounds 119 (IC50 = 8.4 ± 1.7 μM) and 134 (IC50 = 11.7 ± 3 Compound 7-chloro-N-(pyridin-4-yl)quinolin-4-amine (134) was obtained from the reaction of μM) were equipotent, demonstrating that substitution of the quinoline core was not relevant for anti- pyridin-4-amine with the commercially available 4,7-dichloroquinoline (59) in 89% yield [15]. P. falciparum activity. Compounds 132 (IC50 = 31.5 μM) and 133 (IC50 > 143 μM), which are both tertiary First, a comparison was made between the importance4caonima of the 2,8-bis(trifluoromethyl)quinoline versus the 7-chloroquinoline moieties. Compounds 119 (IC = 8.4 1.7 µM) and 134 (IC = 11.7 3 50 ± 50 ± µM) were equipotent, demonstrating that substitution of the quinoline core was not relevant for anti-P. falciparum activity. Compounds 132 (IC50 = 31.5 µM) and 133 (IC50 > 143 µM), which are both tertiary Molecules 2019, 24, 4095 16 of 20 Molecules 2019, 24, x FOR PEER REVIEW 16 of 20 amines, were less active and inactive, respectively. This proves that the presence of the quinoline-NH group in this class of molecules is important. The reduction of anti- P.P. falciparum activity of 132 and 133 cancan bebe justifiedjustified throughthrough thethe moremore rigidrigid conformationalconformational structures of the cycliccyclic tertiary amines compared to the the arylamino arylamino derivatives. derivatives. This This rigidity rigidity interferes interferes with with the the ability ability of ofthe the compounds compounds to tointeract interact with with the the biomacromolecule biomacromolecule receptor, receptor, although although the the NH NH group group could could still still interact interact with the bioreceptor viavia hydrogenhydrogen bonding.bonding. TheThe mostmost activeactive compoundcompound129 129(IC (IC5050 = 0.0830.083 μµM) was 3-fold more potent than chloroquinechloroquine (IC5050 = 0.250.25 μµM)M) (Figure 26).26). However, However, it was less active than mefloquinemefloquine (IC5050 == 0.0190.019 μµM).M). Moreover, Moreover, as as an an advantage, advantage, its its chemical chemical structure structure is simpler simpler than that of mefloquine mefloquine because it does not contain a stereogenic center, andand consequently, its synthesissynthesis in the laboratory is easier and less expensive. When the CH3 substituentsubstituent was exchanged for H or CF 3 onon the the triazole triazole ring, ring, as in 128128 (IC(IC5050 = 2.92.9 μµM) and 130 (IC(IC5050 = 11.511.5 μµM),M), respectively, significantlysignificantly lower activities were observed. The isosteric replacement of the 5-methyltriazole unit in 129 with the 5-methylthiadiazole unit in 131 (IC(IC50 == 1.81.8 μµM)M) decreased decreased the the activity, activity, showing the importance of the triazole nucleus for anti-P. falciparumfalciparumactivity. activity.Derivative Derivative123 123(IC (IC5050 = 9.6 μµM), with the amine β-naphthyl-naphthyl group, group, was three times more activeactive thanthan 122122 (IC(IC5050 = 28.028.0 μµM),M), with the amine phenyl group, showing the importance of lipophilicity in this region on biological activity; however, 123 was not selective and showed high toxicity. Aminepyridin-4-ylAminepyridin-4-yl derivativederivative119 119 (IC(IC5050 = 8.48.4 μµM)M) was was 2.5-fold 2.5-fold more more active and selective than aminepyridin-2-yl derivative 118 (IC50 == 19.619.6 μµM).M). Moreover, Moreover, when when we we evaluated the effect effect of the aminepyrimidin-2-yl derivative 120 (IC50 == 8.48.4 μµM),M), it it showed showed equipotent equipotent anti- anti-P.P. falciparum activity and the same toxicity as 119..

Figure 26. Structure of compound 129.

2. Conclusions During our research on new drugs for malaria treatment,treatment, we sought out medicinal chemistry to make structural modificationsmodifications and hybridizations to obtain new compounds that are more selective and less toxic. In the search ofof non-quinolinicnon-quinolinic compounds,compounds, aa seriesseries ofof [1,2,4]triazolo[1,5-[1,2,4]triazolo[1,5-aa]pyrimidine]pyrimidine derivatives were were designed. designed. These These compounds compounds exhibited exhibited anti- anti-P. P.falciparum falciparum activities,activities, and and none none of the of the compounds were toxic to HepG2 cells. Compound 2, with the CF group at the 2 position and compounds were toxic to HepG2 cells. Compound 2, with the CF3 group3 at the 2 position and β- β naphthylamine-naphthylamine at atthe the 7 position, 7 position, was was the the most most active, active, and and it ithas has an an important important contribution contribution to to anti- anti-P.P. falciparum activity with IC = 0.023 µM. Derivative 2 was used as a prototype compound (prototype falciparum activity with IC50 = 0.023 μM. Derivative 2 was used as a prototype compound (prototype I) for Ifuture) for futureinvestigations investigations in the search in the for search compounds for compounds for the treatment for the of treatment malaria. Bioisosteric of malaria. replacements Bioisosteric replacementsof the triazolo[1,5- of thea]pyrimidine triazolo[1,5- aring]pyrimidine on prototype ring on I prototypeby the pyrazolo[1,5-I by the pyrazolo[1,5-a]pyrimidinea]pyrimidine ring were β ringshown were to shownbe effective. to be eDerivativesffective. Derivatives with β-naphthylamine with -naphthylamine at the 7 position at the 7 positionwere the were most the active. most active. Compounds 33 (R = CF ,R = CH ) and 38 (R = CH ,R = CH ) exhibited low toxicity and Compounds 33 (R1 = CF3,1 R2 = CH3 3)2 and 383 (R1 = CH3,1 R2 = CH3 3) 2exhibited3 low toxicity and the low the low IC values of 1.2 and 5.1 µM, respectively, and consequently the highest SI values of 467.8 IC50 values50 of 1.2 and 5.1 μM, respectively, and consequently the highest SI values of 467.8 and 79.6, andrespectively. 79.6, respectively. These compounds These compounds showed a showed 50% redu a 50%ction reduction of parasitemia of parasitemia in the in in vivo the anti-in vivoP. bergheianti-P. berghei malaria evaluation. Compound 44 (R = CH ,R = CF ) showed the best inhibition of the malaria evaluation. Compound 44 (R1 = CH3,1 R2 = CF3 3) 2showed3 the best inhibition of the enzyme enzyme Pf DHODH with an IC = 0.16 µM, which was more potent than prototype I (IC = 0.70 µM). PfDHODH with an IC50 = 0.16 50μM, which was more potent than prototype I (IC50 = 0.7050 μM). A hybrid salt of artesunate and mefloquine,mefloquine, MEFAS (61),), hashas beenbeen demonstrateddemonstrated to be 280- and 15-fold more eeffectiveffective than both mefloquinemefloquine and artesunate alone, respectively, against P. falciparum gametocytes. Against Against P.P. berghei- berghei-infectedinfected mice, mice, a a dose dose of of 10 10 mg/kg mg/kg led to cure, and the toxicity of 61 was 5-fold lower than that of mefloquine.mefloquine. PRIMAS ( 62) is also a hybrid salt between artesunate and primaquine. This hybrid salt is more active in vivo and in vitro andand lessless toxictoxic thanthan primaquine.primaquine.

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The 1,2,3-triazol-1-yl quinoline derivatives exhibited activity in vitro against the P. falciparum W2 CQR clone without toxicity to HepG2 cells. The most active compound 56 showed an IC50 = 1.4 µM, and SI = 351. Compound 75 (R = Cl; IC50 = 0.09 µM) had an SI value of 1102.2 and was evaluated for its antimalarial activity in P. berghei-infected mice, showing 49% inhibition of parasitemia. This compound was used as an antimalarial prototype (prototype II) to proceed with other studies to overcome the burden of resistance in P. falciparum. From prototype II, derivatives 1H-pyrazolo[3,4-d]pyrimidine and 1H-pyrazolo[3,4-b]pyridine were designed. These cores were active against P. falciparum with 87 giving an IC50 = 3.46 µM and 107 showing an IC50 = 5.13 µM, which were lower than that of the control drug sulfadoxine; however, prototype II was still the most active. Chloroquine/primaquine-atorvastatin (AVA) hybrids showed similar activity to chloroquine but were better than primaquine. The primaquine derivative was significantly less toxic and more active than primaquine, 7-fold more active than AVA and was shown to be safer (SI > 1107) than primaquine (SI of 239). The 2,8-bis-(trifluoromethyl)quinoline derivatives are chemical structures simpler than those of mefloquine. Compound 129, the most active compound, showed an IC50 = 0.083 µM and was 3-fold more potent than chloroquine (IC50 = 0.25 µM). We can conclude that these compounds are promising for further studies of antimalarial. However, the antimalarial activity of promising compounds to reverse artemisinin resistance can only be seen by establishing the ring-stage survival assay (RSA) [61].

Author Contributions: L.C.S.P., L.M.F., M.O.G. and F.F.S. wrote the manuscript; N.B. reviewed the manuscript. Funding: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001 and PROEP/CNPQ-FAR/FIOCRUZ 407853/2017-0. Acknowledgments: The authors thank the Coordination of Improvement of Higher Education (CAPES) and the National Council of R&D of Brazil (CNPq) for the fellowships granted to the authors. We also thank the Foundation for Research of the State of Rio de Janeiro (FAPERJ), Technological Development Program on Products for Health (PDTIS), for financial support. L.C.S.P. and N.B. are recipients of research productivity fellowships from the CNPq and N.B. from FAPERJ (“Cientista do Nosso Estado”). Conflicts of Interest: The authors declare no conflict of interest.

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