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European Journal of Medicinal Chemistry 73 (2014) 295e309

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

7-Chloroquinolinotriazoles: Synthesis by the azideealkynecycloaddition click chemistry, antimalarial activity, cytotoxicity andSAR studies

Guilherme R. Pereira a, Geraldo Célio Brandão b, Lucas M. Arantes c,Háliton A. de Oliveira Jr. a, Renata Cristina de Paula a, Maria Fernanda A. do Nascimento a,Fábio M. dos Santos d, Ramon K. da Rocha c, Júlio César D. Lopes c,Alaíde Braga de Oliveira a,*

aDepartamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Campus Pampulha,CEP 31270-901 Belo Horizonte, MG, Brazilb Faculdade de Farmácia, UFOP, Rua Costa Sena 171, CEP 35400-000 Ouro Preto, MG, BrazilcDepartamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Campus Pampulha, CEP 31270-901 Belo Horizonte, MG, Brazild Programa de Pós-Graduação em Bioinformática, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CampusPampulha, CEP 31270-901 Belo Horizonte, MG, Brazil

a r t i c l e i n f o

Article history:Received 19 April 2013Received in revised form8 November 2013Accepted 20 November 2013Available online 1 December 2013

Keywords:7-ChloroquinolinotriazolesQuinolinesTriazolesClick chemistryAntimalarial activityPlasmodium falciparumCytotoxicity in HepG2

* Corresponding author. Tel.: þ55 31 3409 6950; faE-mail addresses: alaidebraga@terra.com.br, alaid

Oliveira).

0223-5234/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2013.11.022

a b s t r a c t

Twenty-seven 7-chloroquinolinotriazole derivatives with different substituents in the triazole moietywere synthesized via copper-catalyzed cycloaddition (CuAAC) click chemistry between 4-azido-7-chloroquinoline and several alkynes. All the synthetic compounds were evaluated for their in vitro ac-tivity against Plasmodium falciparum (W2) and cytotoxicity to Hep G2A16 cells. All the products disclosedlow cytotoxicity (CC50 > 100 mM) and five of them have shown moderate antimalarial activity (IC50 from9.6 to 40.9 mM). As chloroquine analogs it was expected that these compounds might inhibit the hemepolymerization and SAR studies were performed aiming to explain their antimalarial profile. Newstructural variations can be designed on the basis of the results obtained.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Malaria remains one of the most serious problems in publicheath around the world, despite being one of the oldest diseases. Itis estimated that around 500 million people are infected in sub-tropical and tropical countries and 2.5 million deaths occur annu-ally. In Brazil, it is restricted to the Amazonia area. Malaria is aninfectious disease, caused by protozoa parasites from the genusPlasmodium that is transmitted by mosquitoes of the genusAnopheles. Plasmodium falciparum is responsible for the most lethal

x: þ55 31 3441 5575.e.braga@pq.cnpq.br (A.B. de

son SAS. All rights reserved.

form of malaria [1]. Chloroquine (1) was the most widely andeffective antimalarial clinically used drug but parasite resistanceled to its substitution by artemisinin and its semi-synthetic de-rivatives (artemether, artesunate). However, emergence ofP. falciparum resistance to these drugs is a serious cause of concern.To prevent the selection of resistant parasites, WHO has recom-mended the combination of artemisinins with traditional antima-larial drugs such as lumefantrine, amodiaquine andmefloquine andACT (Artemisinin Combination Therapy) formulations are presentlyadopted in most endemic countries [2,3]. Therefore, new drugs totreat human malaria are urgently needed. Synthesis of molecularhybrids containing different moieties which are representatives ofknown or putative antimalarial compounds is presently beingextensively exploited. Recently, the synthesis of 1,2,3-triazoles by aprocess known as Cu-mediated click chemistry [4] has been

Scheme 1. From chloroquine (1) to 7-chloroquinolinotriazole derivatives of types A, B and C, via click reaction between 4-azido-7-chloroquinoline (3) and different alkynes.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309296

explored to combine different molecules affording new analogs ofchloroquine [5], chalcones [6], naphthoquinones [7] and severalother hybrid antimalarial molecules have been synthesized [8e14].The aim of the present work was to synthesize new compoundswith a 7-chloroquinolinotriazole basic structure supportingdifferent side chains linked to the triazole moiety, via clickchemistry.

Considering chloroquine (1) as the basic antimalarial moiety, wehave planned the substitution of its N-alkyl chain by a 1,2,3-triazolemoiety supporting different substituents at position C-4. The finalproducts could be represented by three general structures A, B andC, as shown in Scheme 1. The three types would be obtainedby a click reaction between 4-azido-7-chloroquinoline and analkyne via copper-catalyzed cycloaddition reactions (CuAAC) [4](Scheme 1).

2. Synthetic chemistry

4-Azido-7-chloroquinoline (3), a key intermediate, was pre-pared from 4,7-dichloroquinoline (2) that is commerciallyavailable. The click reactions were carried out according to themethodology described by Sharpless and co-workers [4] for theCu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC)that is presently one of most widely used for the preparation of1,2,3-triazole hybrids [7e11]. The reactions were carried out indichloromethane or acetonitrile and water, in the presence ofCuSO4$5H2O and sodium ascorbate, stirring at room tempera-ture, for 2e24 h. The crude reaction products were generallypurified by preparative TLC or column chromatography. Theyields for the several click reactions reported here were in therange of 49e81%, except in two cases that were of 28 and 31%(Table 1).

The simplest route, leading to derivatives of group A, was carriedout using commercially available alkynes (Scheme 2). Compounds4e11were prepared in high yield reactions (49e77%) (Table 1). It isinteresting to emphasize the applicability of this process to thesynthesis of 7-chloroquinolinotriazole derivatives with differentclasses of side chains such as alkyl, aryl and alcohol groups, asdescribed here.

Ester analogs, representing type B 7-chloroquinolinotriazolederivatives, were obtained by reaction of propargyl alcohol (12)

and 1-pentynol (13) with different benzoyl chlorides leading to atotal of 14 new compounds (14e27) (Scheme 3).

Based on chloroquine (1) structure, it seemed interesting tosynthesize a series of 7-chloroquinolinotriazoles containing amethylamine side chain with a C4 unity between the two nitrogenatoms, although in quite different situations, such as a C4 aliphaticchain, as in chloroquine (1), and an aromatic triazole ring. Themethylamine group in the designed molecules would favor theiraction in the acidic parasite vacuole by inhibition of the hemepolymerization similarly to known antimalarial 4-aminoquinolines, including chloroquine (1) itself [3] (Scheme 4).The necessary propargylamine moiety was easily formed frombromopropine (28) by simple nucleophilic displacement, and it wasfurther used without isolation in the click reaction with 4-azido-7-chloroquinoline (3) in a one- pot-process that afforded a total offive type C 7-chloroquinolinotriazoles with an amine side chain(29e33).

The structure of the quinolinotriazole hybrids were assigned onthe basis of spectrometric data including HRMS-ESI-IT-TOF, IR, 1Hand 13C NMR.

3. Biological assays

3.1. Continuous cultures of P. falciparum

P. falciparum W2 clone, which is chloroquine-resistant andmefloquine-sensitive [15], was kept in a continuous culture at 37 �Cin human erythrocytes using the candle jar method [15,16]. Theantimalarial effect of the compounds was measured by the HRP2assay [17,18]. Parasites were kept in complete culture medium(RPMI) containing sodium bicarbonate (21 mM), D-glucose(11 mM), HEPES (25 mM), hypoxanthine (300 mM) and gentamicin(40 mg/ml) that was supplemented with 10% human plasma onculture dishes, the culture medium being changed daily. All ex-periments were performed in duplicate; the compounds weretested in triplicate at each concentration. The cultures with pre-dominantly ring-stage parasites were concentrated by sorbitol-synchronization [19]. A suspension of red blood cells with 0.05%parasitemia and 1.5% hematocrit was distributed in a 96-well mi-crotiter plate (180 ml/well). The parasite growth was evaluated bythe ELISA immunoenzymatic anti-HRP2 test, as summarized below.

Table 1Yields of click reactions affording 7-chloroquinolinotriazoles 4e33, in vitro anti-malarial activity (IC50 mM) against P. falciparum (W2 clone), cytotoxicity (CC50 mM,Hep G2A16 cells) and selectivity index (SI).

Compound Yieldsa IC50b CC50c SI

4 76% 40.3 >2864 >71.1

5 58% >154.0 >3086 <20.0

6 77% >156.0 352 <2.2

7 59% >80.0 574 <7.2

8 75% 9.6 151 15.7

9 49% 90.3 442 4.9

10 64% >80.0 >3.204 <40.0

11 56% >129.0 >2590 <20.0

14 82% >127.5 1202 � 32 <9.4

15 81% >113.1 347 � 27 <3.0

16 60% >119.0 >2550 <21.4

17 75% >108.7 >2173 <20.0

18 67% >111.6 >2231 <20.0

19 68% >123.1 >2462 <20.0

20 67% >127.5 >2550 <20.0

21 70% >127.5 >2550 <20.0

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309 297

3.2. Evaluation of the in vitro antimalarial activity by the HRP2assay

The HRP2 test was performed as previously described [17,18].Briefly, cultures of P. falciparum (1.5% hematocrit; 0.05% para-sitemia) were placed in 96 well microplates with the test com-pounds and controls at different concentrations, and wereincubated for 48 h under the same culture conditions, as describedabove. After 24 h incubation, the content of six wells correspondingto medium and no test sample controls were harvested and frozenin microtubes, as to allow subtracting the average value obtainedfrom these wells from the other wells for excluding the backgroundvalue (production of HRP2 during the first 24 h of incubation). Aftera total of 48 h incubation, the plates were frozen and thawed twicefor total erythrocyte lyses and 100 ml/well of the material wasplaced in another plate for the ELISA test. This plate was pre-coatedovernight at 4 �C with 1 mg/ml of the primary antibody anti-HRP2(MPFM-55A ICLLABs) and then the content was discarded, replacedby the blocking solution (PBS/BSA 2% 200 ml/well), incubated for2 h, and finally the content was discarded. The hemolyzed cultureswere transferred to the ELISA pre-coated plate, incubated (1 h,room temperature), discarded, incubated for 1 hwith 0.05mg/ml of

Table 1 (continued )

Compound Yieldsa IC50b CC50

c SI

22 81% >119.0 >2380 <20.0

23 67% >137.3 >2747 <20.0

24 68% >111.6 >2232 <20.0

25 45% >115.7 1066 � 11 <9.2

26 77% >126.9 303 � 25 <2.4

27 28% 22.5 484 � 35 21.5

29 40% >166.0 1851 � 57 <11.2

30 53% 27.4 1849 � 58 67.5

32 62% >151.9 >3039 <20.0

31 55% >152.8 1106 � 14 <7.2

33 31% 40.9 930 � 33 22.7

A Chloroquine (1) 0.19 810 � 15 4263.0

SI: Selectivity Index ¼ CC50/IC50.a Yields of isolated compounds in the last step (click reaction).b IC50: concentration that inhibits 50% of the parasite growth in relation to control

cultures with no drugs.c CC50: concentration that kills 50% of HepG2cells 24 h after incubation with the

compounds determined by the MTT method.

Scheme 2. Synthesis of type A 7-chloroquinolinotriazole derivatives via click reaction.

OH

CuSO .H OsodiumascorbateDCM/H Or.t .

R

Cl O

x

X= 1 or 3

Ox

O

R

DCMTEAr.t .

NCl

N

C

NN

O R

O

R=

x

X=3R=

OMe Et

F FF

Me

14 15 16

17 18

X=1R=

CF

20 21 22 23

24

Et

Et

F

F OMeF OMe

25 2619

Cl

27

NCl

N

Scheme 3. Synthesis of type B 7-chloroquinolinotriazole derivatives via click reaction.

Scheme 4. Structural similarities with chloroquine (1) and the amine series (type C 7-chloroquinolinotriazole derivatives) synthesized by one-pot-click reaction of 4-azido-7-chloroquinoline (3) and different propargyl alkylamines which were generated in situ.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309298

the secondary antibody (MPFG55P-ICLLAB; 100 ml/well), thenincubated with 100 ml/well of TMB chromogen (15 min at roomtemperature) in the dark. The reaction was stopped with 50 ml/L of1 M sulfuric acid and the absorbance was read at 450 nm in aspectrophotometer (Infinite�200 PRO, Tecan). The results wereevaluated with the software MicrocalOrigin 8.5 for determinationof the doseeresponse curves plotted with sigmoidal fit. The 50%inhibitory concentration growth of the parasites (IC50) was deter-mined by comparison with controls with standard drug andwithout drugs.

3.3. Cytotoxicity evaluation in human hepatome cell cultures e HepG2A16

The hepatome cells Hep G2A16 were maintained at 37 �C, 5%CO2 in 75-cm2 sterile culture flasks (Corning�) with RPMI 1640

culture medium supplemented with 5% FBS, penicillin (10 U/ml),and streptomycin (100 g/ml), with changes of medium twice aweek. The cells were maintained in weekly passages (at 1:3 di-lutions in sterile culture flasks) and grown to 80% [20]. They wereused for experiments after being trypsinized (0.05% trypsin/0.5 mMEDTA) and plated on 96 well microplates [21]. When confluent, themonolayers were trypsinized, washed, counted, diluted in com-plete medium, distributed in 96-well microplates (4 � 103 cells/well), then incubated for another 24 h at 37 �C. The test samplesand controls were diluted to a final concentration of 0.02% DMSO inculture medium to yield four concentrations in serial dilutionsstarting at 1000 mg/ml. After a period of 24 h incubation at 37 �C,18 ml of MTT solution (5 mg/ml in PBS) were added to each well,followed by another 1 h 30 min incubation at 37 �C. The superna-tant was then removed, and 180 ml of DMSO were added to eachwell. The culture plates were read in a spectrophotometer with a

Table 2Values of experimental antimalarial activity (pIC50), calculated acid dissociationconstants (pKa), total charge for chloroquine (1) and the most active 7-chloroquinolinotriazole derivatives.

Compound pIC50 pKa

(QN)apKa

(RN)bTotalchargec e QN

Totalcharged e TR

Chloroquine (1) 6.72 7.29 10.32 0.785 e

8 5.00 5.84 e 0.957 �0.13527 4.64 5.70 e 0.972 �0.13830 4.57 5.67 6.86 1.115 �0.19433 4.39 5.62 6.88 1.106 �0.1964 4.40 5.81 3.87 0.900 �0.099

a Logarithmic acid dissociation constant of the quinoline nitrogen e QN e ascalculated by Chemaxon’s Calculator Plugins [30].

b Logarithmic acid dissociation constant of the side chain nitrogen e RN e ascalculated by Chemaxon’s Calculator Plugins [30].

c Total charge distributed over quinoline ring as calculated by AM1 method inelectron units [35].

d Total charge distributed over triazole ring e TR e as calculated by AM1 methodin electron units [35].

Fig. 1. Correlations between the calculated pKa of the quinoline ring nitrogen and theexperimentally determined IC50 values for chloroquine (CQ) and for the quinolino-triazoles 8, 30, 33, 27 and 4.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309 299

570 nm filter [22]. The minimum cytotoxicity concentration wasdetermined as described with slight modifications. Each test wasperformed in duplicate; the concentration that killed 50% of thecells (CC50) were determined [23]. The selectivity index (SI) for theantimalarial activity was then calculated based on the rate betweenCC50 and IC50 for the in vitro activity against P. falciparum asdescribed [24]. Most of the compounds showed SI > 10 and couldbe considered non-toxic [25].

4. Results and discussion

The twenty-seven 7-chloroquinolinotriazole synthetic com-pounds were evaluated for their in vitro antimalarial activityagainst P. falciparum W2 strain that is chloroquine resistant andmefloquine-sensitive. Table 1 shows the values of IC50 for thein vitro antimalarial assay (HRP2 method), the CC50 values for thecytotoxicity (Hep G2A16 cells) and the SI for these compounds. Allthe compounds disclosed low cytotoxicity with CC50 > 100 mM,some of them reaching values > 3000 mM. Five of them showedmoderate antimalarial activity with IC50 < 50 mM, in the range of9.6e40.9 mM. SI values varied from 2.2 to 71.1; about half of themwere close to 20 as consequence of high CC50 values (lowcytotoxicity).

The most active 7-chloroquinolinotriazole derivative was com-pound 8 (type A) with an IC50 of 9.6 mM and a reasonable SI (15.7).Interestingly, the other active compounds, 30 (IC50 27.4 mM), 4 (IC5040.3 mM) and 33 (IC50 40.5 mM) have a nitrogen or an oxygen atomin the end of the triazole side chain. However, compound 29 wasapproximately sixfold less potent (IC50> 166.0 mM) than the closelyrelated compound 30. The only structural differences between 29and 30 are the substituents on the amino group of the triazole sidechain, diethyl and dimethyl, respectively. It is surprising that such asmall structural difference could have a remarkable effect in theantimalarial potency of these compounds. Considering that thesequinolinotriazole derivatives are putative inhibitors of heme poly-merization, it sounds reasonable to suppose that the observed ef-fect is a consequence of side chain variation of these triazolederivatives. Indeed, such effect has been previously shown for asystematic variation of the branching and basicity of the side chainof chloroquine in a series of 7-chloro-4-aminoquinolines [26,27].On the other hand, in the case of the alcohol derivative 30, thedecreased activity of the corresponding ester 19 (IC50> 123.1 mM) isa clear demonstration of the importance of a free OeH group for theantimalarial activity. Besides, all the benzoyl ester derivatives,except 27, disclosed low activity with IC50 > 100 mM and, therefore,the expectation that they could act as pro-drugs affording thesupposed more active alcohols after hydrolysis was not a favorableone. The effect of a side chain hydroxyl group in the antimalarialactivity of this series of 7-chloroquinolinotriazole derivatives isevidenced when comparing the piperidylamine 31(IC50 > 152.8 mM) and the corresponding hydroxy derivative 33(IC50 40.9 mM) that is about four times more potent.

As pointed out, it was expected that the synthesized com-pounds, as chloroquine (1) analogs, could inhibit the heme poly-merization that is involved in the parasite-mediated hemoglobindigestion. This process, the heme detoxification, leads to hemozoinformation (malarial pigment) that takes place in the digestive orfood vacuole of intra-erythrocytic parasites and is an establisheddrug target. The food vacuole is an acidic compartment (pH 5.0e5.4) where chloroquine (1) is trapped and concentrated by pro-tonation following its active uptake by parasite transporter(s), andthen binding to a parasite specific receptor [28]. Many other anti-malarials target the food vacuole indicating the importance of thisorganelle and its various functions to the survival of the parasite[29].

The acid-base character of quinoline derivatives should be takeninto account for its putative action in the food vacuole and the pKavalues of the presently described 7-chloroquinolinotriazole de-rivatives were calculated by the Marvin 5.11 (2012) Chemaxon’sCalculator Plugins [30] (Table 2).

A significative decrease of pKa was shown for the quinoline ringnitrogen of these quinolinotriazoles in comparison with chloro-quine: from 7.3 for chloroquine (1) to 5.6 to 5.8 for the triazolederivatives. This difference can be explained by the low resonancecontribution of the triazole ring, due to its aromatic character, tothe stabilization of the protonated form of the quinoline nitrogen.As a consequence of the pKa decrease for the quinoline nitrogen ofthe quinolinotriazole derivatives, in comparison to chloroquine (1),it would be expected that its protonation would be largelydecreased in the digestive vacuolewith pH 5.0e5.4. It is known thatthe chloroquineehematin interaction is largely a function of itsquinoline moiety, but inhibition of hemozoin formation and para-site growth is also influenced by the 4-amino side chain [31].Therefore, taken together, the lower basicity (pKa) of these nitro-gens in the quinolinotriazoles, in comparison with those in thecorresponding positions in the chloroquine (1) structure, mightaffect the antimalarial effect of the quinolinotriazole derivativesdescribed here.

Linear correlation analyses between the calculated pKa andexperimentally determined IC50 values described here disclose anexcellent correlation (R2 ¼ 0.993) for the active quinolinotriazoles8, 27, 30 and 33 (IC50 < 50 mM) while, when chloroquine (1) isinserted in the series, a slight degradation is registered (R2¼ 0.985).These results are in agreement with the recognized relevance of the

Fig. 2. Model structures, potential pharmacophores and electrostatic surfaces of compound 4 (A), MB (B) and compound 8 (C). Molecular surface colored according Discover StudioVisualizer [39].

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309300

quinoline nitrogen protonation for the antimalarial activity ofchloroquine and 4-aminoquinolines [32]. Therefore it is clear thatwhen a given pH value is considered, compounds with lower pKawill have a smaller population of the acidic form (protonated

species) than its conjugated basic form resulting in lower antima-larial activity, as can be seen in Fig. 1 that depicts the correlation ofthe calculated pKa versus pIC50. The active quinolinotriazole 4(IC50 ¼ 41 mM) does not fit to the curve of Fig. 1 what can be

Fig. 3. Schematic representations for the putative pharmacophores according to the structures of compound 4 (A), MB (B) and compound 8 (C) as shown in Fig. 2: in both cases, anaromatic ring plane is in the same plane of the pharmacophores.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309 301

explained by its differentiated structure showing at C4 a strongelectron donor p-N(Me2)-phenyl group, instead of alkyl groups, asin the other active compounds of this series. In this situation,compound 4 shows a high pKa (5.81), close to that one of compound8, the most active (lowest IC50) of this series but with a markeddecrease of the antimalarial activity.

Aiming to understand how charge distribution could affect theantimalarial effect of the quinolinotriazoles, semi-empirical calcu-lations were also undertaken for generation of the most stableconformers of the most stable microspecies in pH 5. Such a pH isbeing considered to assuring protonation of the quinoline nitrogenin each of the compounds and proximity to the pH of the digestivevacuole. Initially, the major microspecies at pH 5 was calculated byChemaxon’s Calculator Plugins [30] and then submitted to theOMEGA2 program from OpenEye [33] to generate a set of stableconformers for each compound. All conformations of each com-pound were submitted to MOPAC2012 [34] for minimization andcharge distribution calculations with AM1method [35]. Vibrationalfrequencies were calculated to ensure that all conformations aretrue minima, and only the most stable one was considered tocharge distribution comparisons. The partial charge calculationsperformed at semi-empirical level with AM1method show that thetotal charge in the quinoline ring of the quinolinotriazoles becomesmore positive in relation to chloroquine (1) (Table 2). Therefore, thetriazole ring effect on the quinoline system can be considered justas an inductive electron attracting group when compared with theamino group of chloroquine (1). Again, the analyses of the partialcharges indicate that compound 4 shows a distinct behavior incomparison with other quinolinotriazole derivatives. The totalcharge in both quinoline (QN) and triazole rings are less pro-nounced, being the quinoline ring (QN) less positive and the tri-azole ring (TR) less negative what is coherent with the lowerantimalarial activity of compound 4 in the group of the five activequinolinotriazoles. These results would suggest that not only theprotonation of the quinoline ring is important to the antimalarialactivity, but that, for the quinolinotriazole derivatives, the negativecharge in the triazole ring could interfere in its interaction withheme. Considering that the main target for chloroquine analogs isthe ferroprotoporphyrin IX [36], a high electron density in the tri-azole ring (TR) could also contribute to the activity. These distincteffects of compound 4might even lead to speculate on an alterationin the mechanism of action, concerning the detailed mechanism ofthe chemical interaction of this compound with the ferroproto-porphyrin IX for inhibition of the heme polymerization.

It is interesting to note the remarkable resemblance of the po-tential pharmacophores of compounds 4 and 8 with methyleneblue (MB), a synthetic thiazine dye that shows good antimalarialactivity. Recently, its in vitro activity against resistant strains ofP. falciparumwas reported and disclosed that it is 20e50 fold moreactive than chloroquine (1) [37,38]. A close look to the potentialpharmacophores of compounds 4, 8 andMBwas undertaken by theDiscover Studio Visualizer [39]. The following reference groupswere focused: a) the quaternary nitrogen (positive center), theunprotonated nitrogen of the p-N(Me2)-phenyl moiety (negativecenter) and the endocyclic nitrogen as a hydrogen bond acceptor ofthe MB structure, at pH 5, b) the protonated quinoline nitrogen(positive center), the unprotonated nitrogen of the p-N(Me2)-phenyl group (negative center) and the N1-triazole nitrogen as ahydrogen bond acceptor of compound 4, and 8) the protonatedquinoline nitrogen (positive center), the oxygen of the triazole sidechain (negative center) and the N1-triazole nitrogen as a hydrogenbond acceptor of compound 8. The model structures (Fig. 2) showthat the putative pharmacophores disclose similar positions inthese three compounds, including distances and angles, as can beseen in their schematic representation in Fig. 3. It is interesting tonotice that, in both cases, the aromatic ring position is conserved aswell. Might we ascribe the higher potency of quinolinotriazoles 4and 8 to their closer structural resemblance to MB? It should benoticed that in this case, compound 4, showing a more extendedconjugation, planarity and rigidity, would be expected to be morepotent. However compound 8 is about four times more active thancompound 4. Therefore the combination of a 1,2,3-triazole moietywith the 7-chloroquinoline unity brings in a new hydrogenacceptor pharmacophore in which the distance between the posi-tive center and the hydrogen acceptor is 6.2 in these triazole de-rivatives and is comparable to that one of 5.6 A for MB.

Finally, the SAR analyses described in the present study hasshown that the click reaction to afford triazole derivatives shouldstill be exploited to produce a diverse structural series of 7-chloroquinolinotriazoles with stronger electron donating group atposition C4 and a spacer between the quinoline and the triazolegroup. Indeed, very recently, the synthesis of a series of 1H-1,2,3-triazole-tethered-7-chloroquinoline-isatin conjugates with orwithout an alkyl chain and its in vitro antimalarial evaluationagainst W2-chloroquine resistant strain of P. falciparum demon-strated the applicability of our SAR studies. The activity profilesshowed dependence on the presence or absence of an alkyl chainbetween the 7-chloroquinoline ring and the triazole moiety, as well

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309302

as the length of this alkyl chain and the substituent at the C-5position of the isatin ring. IC50 values <5 mM, in the range of 3.85 to1.21 mM, are reported for the most active compounds, all of themwith the spacer alkyl chain between the 7-chloroquinoline ring andthe triazole moiety, while compounds without this spacer are lessactive [40]. However, the IC50 have not been exactly determined(IC50> 5 mM) not allowing an appreciation of the structural effect inthis series.

5. Conclusion

The present work reports the synthesis of twenty-seven 7-chloroquinolinotriazole derivatives in which a 1,2,3-triazole ringsupporting different substituents in position C4 is directly linked bythe N1 to the C4 quinoline position. Evaluation of the antimalarialactivity and cytotoxicity of three different structural series dis-closed that the most promising compounds have some structuralsimilarities such as a heteroatom in the triazole side chain. Thefacility to obtain these compounds via click chemistry by thecopper-catalyzed cycloaddition (CuAAC) reaction can be an ad-vantageous strategy for SAR investigation. The moderate to lowactivity of the 7-chloroquinolinotriazoles we have synthesized wasexplained by the low resonance contribution of the triazole ring tostabilize the acidic form of the quinoline ring what decreases thepKa of the quinoline nitrogen that is known to be important for theheme detoxification in themalaria parasite. Good correlations wereobserved between pIC50 and pKa, and semi-empirical calculationsconfirmed the low resonance effect of the triazole ring on thequinoline ring that could be foreseen. However, the extension ofthis effect on the antimalarial activity of quinolines was not esti-mated. On the other hand, the analyses of pharmacophoresdescribed in the present study has shown that the click reaction toafford triazole derivatives should still be exploited to produce adiverse structural series of 7-chloroquinolinotriazoles witha stronger electron donating group at position C4 and a spacerbetween the quinoline and the triazole group. The significance ofthe triazole side chain for the antimalarial activity was alsoappreciated. Taking into account the results of the present studythe synthesis of promising new similar derivatives is on progress.

6. Experimental section

6.1. General

Chemicals and reagents were purchased from commercialsuppliers and used as received unless noted otherwise. Reactionswere monitored by thin layer chromatography (TLC) on pre-coated0.2 mm silica gel 60 F254 (Merck) plates and visualized in severalways with an ultraviolet light source at 254 nm, by spraying withHanissam reagent (ceric ammonium molibidate-CAM), anisalde-hyde acid, Dragendorff or iodine chamber. All reactions were per-formed in standard dry glassware without inert atmosphere.Evaporation and concentration were done at standard rotavaporBüchi using vacuum pump. Thin-layer chromatography (TLC) wascarried out with silica gel 60 with fluorescent indicator (e.g., SilicaGel. F-254 or IB-F, Merck) and activated previously activated withheating at 100 �C overnight and visualized by UV light or Dra-gendorff indicator. Melting points (mps) were measured with aFisher-Jones melting point apparatus and are uncorrected. 1H and13C NMR spectra were measured on the Bruker. Advance DPX 200with FT analysis. Chemical shifts d (ppm) are reported in SiMe4.Coupling constants (J) are given in hertz. Used deuterated solventwere CDCl3 or DMSO-d6. Splitting patterns have been designated asfollows: s ¼ singlet; bs ¼ broad singlet; d ¼ doublet; t ¼ triplet;q ¼ quartet; qt ¼ quintuplet; td ¼ triplet doublet; m ¼ multiplet.

Protons signals attribution was done for each structural moiety ofthe molecules and the following abbreviations were used:Cq¼ chloroquinoline, Tr¼ triazole, Ch¼ cyclohexane, Ph¼ phenyl,Np ¼ naphthalene, To ¼ tolyl, An ¼ aniline, iPr ¼ isopropyl,Pr ¼ propyl, Ea ¼ ethylamine, Ma ¼ methylamine,Mo¼Morpholine, Pm ¼ pyrimidine, Bz ¼ benzoate, Po¼ Pentynol.Infrared spectra were recorded on FT-IR, Spectrum One, PerkineElmer, with system ATR and are reported in wave number (cm�1).High-resolution EI mass spectra were recorded on a ShimadzuLCMS instrument with direct injection. Samples were dissolved inmethanol-formic acid 0.1% solution.

7. Materials

4,7-Dichloroquinoline, benzoyl chloride, 4-methylbenzoyl chlo-ride, 4-methoxylbenzoyl chloride, 4-(trifluoromethyl)benzoyl chlo-ride, 4-fluorobenzoyl chloride, 4-tert-butylbenzoyl chloride, 4-ethylbenzoyl chloride, 2-ethylbenzoyl chloride, 2,3,5-trifluoro-4-methoxybenzoyl chloride, 2-ethynyl-6-methoxynaphthalene; 4-ethynyltoluene; 1-ethynyl-4-fluorobenzene; 1-ethynyl-4-fluorobenzene; 4-ethynyl-N,N-dimethylaniline; cyclohexylacetylene; 2-methyl-3-butyn-2-ol; phenylacetylene, molecular sieves A4, trie-thylamine, piperidin-4-ol, pent-4-yn-1-ol, prop-2-yn-1-ol, 3-bromoprop-1-yne, RPMI 1640 medium, sodium bicarbonate, D-glucose, HEPES, hypoxanthine, gentamicin, D-sorbitol, PBS, BSA, TMB,FBS, penicillin, streptomycin, tripsin/EDTA, DMSO were obtainedfrom SigmaeAldrich� USA, Ltd. Copper sulfate pentahydrate wasobtained from Reagen�, ascorbic acid was obtained from Synth�,MPFG-55P and MPFM-55A antibodies were purchased fromICLLABS�, sulfuric acid, sodium bicarbonate and sodium sulfate wereobtained from FMaia�, MTT, dimethylamine, piperidine and dieth-ylamine from Merck�, and were used without further purification,glassware from Hialoquímica Ltda.

8. Synthesis and characterization

8.1. 4-Azido-7-chloro-quinoline (3) [41]

To a solution of 4,7-dichloroquinoline (2) (2.0 g, 10 mmol) in5 mL anhydrous DMF and molecular sieves A4, sodium azide(1.3 g, 20 mmol) was added in one portion at room temperature,and the resulting mixture stirred at 85 �C for 8 h, when TLCindicated reaction completion. The reaction mixture was thenallowed to cool to room temperature and then it was diluted with100 mL CH2Cl2, washed with water (3 � 40 mL), dried overanhydrous Na2SO4, and evaporated to dryness. The resultingproduct residue was purified by small column chromatographyeluted with CH2Cl2/Hexane mixture 1:1 to yield the final pureproduct as colorless, needle-like crystals (1.8 g, 91%); mp 115 �C(from CH2Cl2/Hex); Rf (EtOAc/Hex 3:7) 0.29; IR nmax (neat)/cm�1

3036, 2118, 1608. 1H NMR (200 MHz, CDCl3): d Cq 8.76 (1H, d,J ¼ 5.0 Hz, H2), 8.00 (1H, d, J ¼ 2.0 Hz, H8), 7.88 (1H, d, J ¼ 8.8 Hz,H5), 7.49 (1H, dd, J ¼ 2.0 Hz, J ¼ 8.8 Hz, H6), 7.13 (1H, d, J ¼ 5.0 Hz,H3). 13C NMR (50 MHz, CDCl3) d 151.33, 149.59, 146.30, 136.57,128.23, 127.52, 123.77, 119.91, 108.73.

General methodology for route A. 4-Azido-7-chloroquinoline(3) and commercial alkyne was dissolved in CH2Cl2 (2 mL), fol-lowed by addition of CuSO4$5H2O (0.3 equivalents) and anaqueous solution of sodium ascorbate (0.6 equivalents) (2 mL)freshly prepared. The reaction mixture was left overnight andwas stopped when it was completed as shown in TLC. Work up ofthe reaction mixture with CH2Cl2, water (3 � 10 mL), dried overNa2SO4 and final purification by preparative TLC or columnchromatography.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309 303

8.2. 4-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-N,N-dimethylaniline (4)

Yield: 76%; mp 202.5e205.3 �C; IR (nmax, cm�1): 3129, 3056,2995, 2970, 2938, 2842, 1632, 1613, 1593, 1557, 1504, 1477, 1450,1446, 1433, 1394, 1372, 1352, 1265, 1235, 1206, 1187, 1164, 1118,1080, 1024, 953, 900, 857, 815, 804, 770, 731, 716, 677. 1H NMR(200 MHz, CDCl3): Cq d 9.04 (d, 1H, J ¼ 4.6 Hz, H2); 8.23 (d, 1H,J ¼ 1.8 Hz, H8); 8.13 (d, 1H, J ¼ 9.2 Hz, H5); 7.57 (dd, 1H, J ¼ 2.0 Hz,J¼ 9.2 Hz, H6); 7.51 (d,1H, J¼ 4.6 Hz, H3); Tr 8.10 (s, 1H, H5); An 7.79(d, 2H, J¼ 8.6 Hz, H2, H6); 6.68 (d, 2H, J¼ 8.6 Hz, H3, H5); 3.01 (s, 6H,2� eCH3). HRMS-ESI-IT-TOF: m/z cal C19H17ClN5 (M þ H) 350.1172,found 350.1106;

13C NMR (50 MHz, CDCl3): d 151.59; 150.95; 150.41; 149.24;141.31; 136.97; 129.45; 129.08; 127.12; 125.12; 120.86; 119.63;117.50; 115.93; 112.58, 40.54. HRMS-ESI-IT-TOF: m/z calculatedC19H17ClN5 (M þ H) 350.1172, found 350.1106; HRMS-ESI-IT-TOF:m/z cal C19H16ClN5Na (M þ H þ 2) 352.1143, found 352.1108.HRMS-ESI-IT-TOF:m/z calculated C19H16ClN5Na (MþNa) 372.0992,found 372.0957.

8.3. 7-Chloro-4-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)quinoline (5)

Yield: 58%; mp 165.1e168.5 �C; IR (nmax, cm�1): 3140, 3056,2923, 2852, 1609, 1591, 1561, 1504, 1493, 1455, 1438, 1420, 1342,1301, 1232, 1218, 1159, 1030, 973, 952, 879, 840, 824, 795, 769, 677.1H NMR (200 MHz, DMSO-d6): d Cq 9.12 (d, 1H, J ¼ 4.2 Hz, H2); 8.21(d, 1H, J ¼ 1.6 Hz, H8); 8.14 (d, 1H, J ¼ 9.0 Hz, H5); 7.85 (d, 1H,J¼ 4.6 Hz, H3); 7.72 (dd, 1H, J¼ 1.6 Hz, J¼ 9.0 Hz, H6); Tr 9.22 (s, 1H,H5); Ph 7.82 (dd, 2H, J ¼ 5.6 Hz, J ¼ 8.2 Hz, H2, H6); 7.30 (t, 2H,J ¼ 8.6 Hz, H3, H5). 13C NMR (50 MHz, DMSO-d6): d 162.96; 150.54;147.84; 144.71; 138.69; 138.84; 127.22; 126.47; 126.07; 125.91;124.64; 123.90; 121.63; 118.41; 114.92; 114.48, 114.05 (t, 2C,J ¼ 21.5 Hz). HRMS-ESI-IT-TOF: m/z cal C17H9ClFN4 (M � H)323.0499, found 323.0521. HRMS-ESI-IT-TOF: m/z cal C17H9ClFN4(M þ H þ 2) 327.0627, found 327.0503.

8.4. 7-Chloro-4-(4-p-tolyl-1H-1,2,3-triazol-1-yl)quinoline (6)

Yield: 77%; mp 1163.9e165.7 �C; IR (nmax, cm�1): 3112, 3048,2919, 2853, 1611, 1594, 1559, 1497, 1448, 1430, 1375, 1343, 1300,1234, 1192, 1128, 1084, 1027, 1017, 955, 911, 875, 849, 812, 767, 722,678. 1H NMR (200MHz, CDCl3): d Cq 9.04 (d,1H, J¼ 4.2 Hz, H2); 8.21(d, 1H, J ¼ 3.8 Hz, H8); 8.05 (d, 1H, J ¼ 9.0 Hz, H5); 7.58 (d, 1H,J¼ 4.2 Hz, H3); 7.72 (dd, 1H, J¼ 3.8 Hz, J¼ 9.0 Hz, H6); Tr 9.29 (s, 1H,H5); To 7.82 (d, 2H, J¼ 7.6 Hz, H2, H6); 7.26 (d, 2H, J¼ 7.6 Hz, H3, H5);2.34 (s, 3H, eCH3). 13C NMR (50 MHz, DMSO-d6): d 151.59; 150.40;148.80; 141.17; 139.08; 139.00; 137.09; 129.95; 129.59; 129.17;126.86; 126.07; 124.92; 120.98; 120.87; 120.82; 116.07, 21.56.HRMS-ESI-IT-TOF: m/z calculated C17H10ClN4 (M � H) 305.0593,found 321.0912. HRMS-ESI-IT-TOF: m/z cal C17H10ClN4 (M þ H)323.0877, found 323.0821.

8.5. 7-Chloro-4-(4-phenyl-1H-1,2,3-triazol-1-yl)quinoline (7)

Yield: 59%; mp 160.3e162.9 �C; IR (nmax, cm�1): 3144, 3127,3053, 1608, 1593, 1560, 1502, 1484, 1453, 1433, 1316, 1301, 1234,1212, 1187, 1155, 1081, 1033, 1021, 954, 920, 877, 833, 820, 811, 770,691. 1H NMR (200 MHz, DMSO-d6):d Cq 9.15 (d, 1H, J ¼ 4.4 Hz, H2);8.21 (bs, 1H, H8); 8.06 (d, 1H, J ¼ 8.8 Hz, H5); 7.90 (d, 1H, J ¼ 4.4 Hz,H3); 7.75 (d, 1H, J ¼ 9.0, H6); Tr 9.29 (s, 1H, H5); Ph 7.99 (d, 2H,J ¼ 7.2 Hz, H2, H6); 7.54e7.40 (m, 3H, H3, H4, H5). 13C NMR (50 MHz,DMSO-d6): d 152.41; 149.51; 147.24; 140.45; 135.51; 129.86; 129.15;129.05; 128.63; 128.18; 125.66; 125.64; 123.67; 120.19; 116.86.

HRMS-ESI-IT-TOF: m/z calculated C17H10ClN4 (M � 1) 305.0593,found 305.0712. HRMS-ESI-IT-TOF: m/z cal C17H10ClN4 (M þ Na)329.0570, found 329.0368. HRMS-ESI-IT-TOF: m/z cal C17H10ClN4(M þ Na þ 2) 331.0540, found 331.0362.

8.6. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propan-1-ol (8)

Yield: 75%;mp 107.0e112.1 �C; IR (nmax, cm�1): 3321, 3079, 3037,1608, 1578, 1566, 1557, 1492, 1417, 1373, 1352, 1301, 1278, 1200,1206, 1146, 1071, 1012, 963, 896, 880, 840, 819, 777, 769, 671. 1HNMR (200 MHz, CDCl3): Cq d 8.98 (d, 1H, J ¼ 4.6 Hz, H2); 8.10 (s, 1H,H8); 7.46 (d,1H, J¼ 9.0 Hz, H5); 7.45 (d, 2H, J¼ 4.6 Hz, H3); Tr 8.05 (s,1H, H5); Pr 3.87 (d, 2H, J ¼ 6.0 Hz, H1); 3.05 (d, 2H, J ¼ 7.2 Hz, H3);7.78 (m, 2H, J ¼ 6.0 Hz, J ¼ 7.2 Hz, H2). 13C NMR (50 MHz, CDCl3):d 151.08; 149.45; 148.28; 140.67; 136.37; 128.87; 128.22; 124.51;125.12; 122.92; 120.03; 115.60; 60.98, 31.80, 21.73. HRMS-ESI-IT-TOF: m/z calculated C14H13ClN4ONa (M þ Na) 311.0675, found311.0687. HRMS-ESI-IT-TOF: m/z cal C14H13ClN4ONa (M þ Na þ 2)313.0646, found 313.0957.

8.7. 2-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propan-2-ol (9)

Yield: 49%; mp 143.5e145.6 �C; IR (nmax, cm�1): 3438, 3160,3037, 2972, 2925, 1613, 1592, 1559, 1505, 1449, 1435, 1362, 1314,1237, 1176, 1128, 1101, 1033, 962, 925, 875, 855, 826, 816, 808, 775,726, 684, 671. 1H NMR (200 MHz, CDCl3): d 8.94 (bs, 1H); 8.21 (bs,1H, J ¼ 3.8 Hz, H8); 8.00 (bs, 1H); 7.92 (d, 1H, J ¼ 9.2 Hz); 7.48e7.41(m, 2H); 1.75 (s, 6H). 13C NMR (50 MHz, CDCl3): d 156.78; 151.37;150.08; 141.08; 136.94; 129.40; 128.86; 124.79; 121.53; 120.55;115.99; 68.71; 30.68. HRMS-ESI-IT-TOF:m/z calculated C14H14ClN4O(M þ H) 289.07779, found 289.0855; HRMS-ESI-IT-TOF: m/zcalculated C14H13ClN4ONa (M þ Na) 311.0676, found 311.062.

8.8. 7-Chloro-4-(4-cyclohexyl-1H-1,2,3-triazol-1-yl)quinoline (10)

Yield: 64%; mp 115.9e120.3 �C; IR (nmax, cm�1): 3127, 3047,2924, 2849, 1610, 1593, 1560, 1504, 1449, 1438, 1348, 1310, 1244,1115, 1046, 1018, 922, 875, 834, 822, 813, 767, 672. 1H NMR(200 MHz, DMSO-d6): d Cq 9.09 (d, 1H, J ¼ 4.2 Hz, H2); 8.21 (d, 1H,J ¼ 1.4 Hz, H8); 8.06 (d, 1H, J ¼ 9.2 Hz, H5); 7.76 (d, 1H, J ¼ 4.2 Hz,H3); 7.75 (dd, 1H, J¼ 1.4 Hz, J¼ 9.2, H6); Tr 8.54 (s, 1H, H5), Ch 1.90e1.80 (m,1H, H1); 1.78e1.30 (m,10H, H14, H15, H16, H17, H18). 13C NMR(50 MHz, DMSO-d6): d 153.10; 152.31; 149.47; 140.62; 135.33;128.84; 128.10; 125.73; 122.94; 120.19; 116.58; 34.59; 32.39; 25.65;25.60. HRMS-ESI-IT-TOF: m/z calculated C17H17ClN4Na (M þ Na)335.1039, found 335.1236. HRMS-ESI-IT-TOF: m/z cal C17H17ClN4Na(M þ Na þ 2) 337.1010, found 337.1045.

8.9. 7-Chloro-4-(4-(6-methoxynaphthalen-2-yl)-1H-1,2,3-triazol-1-yl)quinoline (11)

Yield: 56%; mp 229.8e232.9 �C; IR (nmax, cm�1): 3129, 3056,2995, 2938, 1632, 1613, 1593, 1557, 1504, 1477, 1446, 1433, 1394,1265, 1235, 1206, 1164, 1118, 1080, 1024, 953, 900, 877, 857, 815,804, 770, 677 1H NMR (200 MHz, DMSO-d6): d 1H NMR (200 MHz,DMSO-d6): Cq 9.19 (d, 1H, J ¼ 4.6 Hz, H2); 8.49 (bs, 1H, H8); 8.19 (d,1H, J¼ 9.2 Hz, H5); 8.07 (d,1H, J¼ 9.2 Hz, H6); 7.98e791 (m,1H, H3);Tr 9.37 (s, 1H, H5); Np 8.20 (d, 1H, J ¼ 2.0 Hz, H2); 7.98e7.91 (m, 2H,H4, H7); 7.90 (dd, 1H, J ¼ 2.2 Hz, J ¼ 9.2 Hz, H8); 7.38 (d, 1H, J ¼ 2.4,H7); 7.38 (dd, 1H, J ¼ 2.4, J ¼ 9.2, H5); 3.90 (s, 3H, eOCH3). 13C NMR(50 MHz, DMSO-d6): d 157.77; 152.51; 149.54; 147.44; 140.97;140.53; 138.86; 135.51; 134.30; 129.10; 128.56; 128.21; 127.65;125.83; 125.79; 125.04; 124.25; 120.19; 119.41; 119.30; 55.34.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309304

HRMS-ESI-IT-TOF: m/z calculated C22H16ClN4O (M þ 1) 387.1012,found 387.0430. HRMS-ESI-IT-TOF: m/z cal C22H16ClN4O(M þ H þ 2) 389.0983, found 389.0929.

General methodology for route B. To a solution of amine (8 mmol)in 4 mL of acetonitrile, 3-bromoprop-1-yne (2 mmol) was added indrops at room temperature, followed by sodium bicarbonate(8 mmol) and the resulting mixture was stirred at room tempera-ture overnight. The next step was carried in the same reaction pot.First, 4-azido-7-chloro-quinoline (1 mmol) dissolved in enoughCH2Cl2 was added to the reaction, followed by a 2 mL of a freshlyprepared aqueous solution of sodium ascorbate, made usingascorbic acid (53 mg, 0.3 mmol), NaHCO3 (25 mg, 0.3 mmol) andCuSO4.5H2O (0.3 mmol). The reaction mixture was stirred for 24 h,then 30 mL CH2Cl2 was added, washed with water (3 � 40 mL), theorganic layer was dried over anhydrous Na2SO4, and evaporated todryness. The resulting residue was purified by preparative TLC toafford the final compound.

8.10. N-((1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-N-ethylethanamine (29)

Yield: 40%; yellow oil; IR (nmax, cm�1): 3053, 2969, 2933, 2873,2815, 1611, 1594, 1561, 1504, 1454, 1437, 1372, 1345, 1299, 1229,1188, 1159, 1112, 1070, 1034, 1001, 954, 877, 813, 769, 733, 665,671 m. 1H NMR (200 MHz, CDCl3): Cq d 9.04 (d, 1H, J ¼ 4.2 Hz, H2);8.22 (s, 1H, H8); 8.03 (d, 1H, J ¼ 9.2 Hz, H5); 7.58 (d, 1H, J ¼ 9.2 Hz,H6); 7.50 (d, 1H, J ¼ 4.2 Hz, H3); Tr 8.01 (s, 1H, H5); Ea 3.92 (s, 2H, eCH2); 2.63 (q, 4H, J ¼ 7.0 Hz, 2� eCH2); 1.14 (t, 6H, J ¼ 7.0 Hz, 2� e

CH3). 13C NMR (50 MHz, CDCl3): d 151.51; 150.31; 146.56; 141.21;136.94; 129.47; 129.05; 124.88; 124.46; 120.71; 116.00; 47.78;47.10; 11.96. HRMS-ESI-IT-TOF: m/z calculated C16H19ClN5 (M þ H)316.1328, found 316.1075; HRMS-ESI-IT-TOF: m/z cal C16H19ClN5(M þ H þ 2) 318.1299, found 318.1054.

8.11. 1-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-N,N-dimethylmethanamine (30)

Yield: 53%; yellow oil; IR (nmax, cm�1): 3398, 2947, 2869, 2828,2782, 2647, 1611, 1595, 1563, 1505, 1456, 1439, 1371, 1348, 1303,1235,1189,1174,1115,1045,1023,1000, 956, 878, 849, 818, 762, 733,672. 1H NMR (200MHz, CDCl3): Cq d 9.03 (d,1H, J¼ 4.4 Hz, H2); 8.28(s, 1H, H5); 8.10 (d, 1H, J ¼ 9.0 Hz, H8); 7.59 (d, 1H, J ¼ 4.4 Hz, H3);7.56 (d, 1H, J¼ 9.0 Hz, H6); Tr 8.15 (s, 1H, H5); Ma 3.76 (s, 2H,eCH2);2.63 (s, 6H, 2� eCH3). 13C NMR (50 MHz, CDCl3): d 150.81; 149.12;144.83; 140.08; 135.55; 128.26; 127.80; 124.21; 119.64; 115.32;53.16; 44.35. HRMS-ESI-IT-TOF: m/z calculated C14H14ClN5 (M þ H)288.1016, found 288.2605; HRMS-ESI-IT-TOF: m/z cal C14H14ClN5(M þ H) 288.1016, found 288.0975. HRMS-ESI-IT-TOF: m/z calC14H13ClN5Na (MþHþ 2) 290.0986, found 290.0972. HRMS-ESI-IT-TOF: m/z calculated C14H13ClN5Na (M þ Na) 310.0835, found310.0788.

8.12. 4-((1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)morpholine (31)

Yield: 62%; mp 157.0e161.2 �C; IR (nmax, cm�1): 3150, 3063,2991, 2857, 2804, 2608, 1594, 1562, 1504, 1453, 1437, 1339, 1319,1310,1285,1263,1240,1225,1201,1140,1112,1071,1035,1012,1004,956, 915, 879, 849, 867, 847, 824, 813, 772, 733, 698, 670. 1H NMR(200 MHz, CDCl3): d Cq 8.90 (d, 1H, J ¼ 4.4 Hz, H2); 8.06 (s, 1H, H8);7.87 (d, 1H, J ¼ 9.2 Hz, H5); 7.40 (d, 1H, J ¼ 9.2 Hz, H6); 7.37 (d, 1H,J¼ 4.4 Hz, H3); Tr 7.95 (s, 1H, H5); Mo 3.71 (s, 2H,eCH2); 3.63 (s, 4H,2� eCH2O); 2.51 (s, 4H, 2� eCH2N). 13C NMR (50 MHz, CDCl3):d 151.28; 149.28; 145.01; 140.78; 136.62; 129.19; 128.77; 124.58;120.35; 115.79; 66.72; 53.42; 53.42. HRMS-ESI-IT-TOF: m/z cal

C16H17ClN5O (M þ H) 330.1121, found 330.0890. HRMS-ESI-IT-TOF:m/z cal C16H17ClN5O (M þ H þ 2) 332.1092, found 332.0845.

8.13. 7-Chloro-4-((4-(piperidin-1-yl)methyl)-1H-1,2,3-triazol-1-yl)quinoline (32)

Yield: 55%; mp 105.4e108.7 �C; IR (nmax, cm�1): 3150, 3068,2941, 2855, 2823, 2780, 2609, 1596, 1560, 1504, 1449, 1435, 1373,1317, 1301, 1252, 1228, 1199, 1183, 1156, 1109, 1069, 1033, 1010, 956,884, 877, 858, 819, 809, 788, 768, 670. 1H NMR (200 MHz, CDCl3):d Cq 9.06 (bs, 1H, H2); 8.23 (s, 1H, H5); 8.03 (d, 1H, J ¼ 9.0 Hz, H8);7.58 (d, 1H, J¼ 9.0 Hz, H6); 7.37 (d, 1H, J¼ 2.8 Hz, H3); Tr 8.04 (s, 1H,H5); Pm 3.83 (s, 2H, eCH2); 2.56 (s, 4H, 2� eCH2O); 1.63 (s, 4H, 2�eCH2); 1.49 (s, 2H, eCH2N). 13C NMR (50 MHz, CDCl3): d 151.34;150.05; 145.69; 140.94; 136.63; 129.18; 128.79; 124.74; 124.51;120.45; 115.82; 66.72; 54.48; 25.79; 23.99. HRMS-ESI-IT-TOF: m/zcalculated C17H19ClN5 (M þ 1) 328.1328, found 328.1076. HRMS-ESI-IT-TOF: m/z cal C17H19ClN5 (M þ H þ 2) 330.1299, found330.1048.

8.14. 1-((1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)piperidin-4-ol (33)

Yield: 31%; mp 158.0e163.3 �C; IR (nmax, cm�1): 3151, 2991,2935, 2857, 2830, 2804, 2608, 1594, 1562, 1504, 1454, 1437, 1369,1339, 1319,1310,1264,1240,1225, 1201,1140, 1112,1071, 1035, 1013,1004, 956, 915, 880, 867, 847, 824, 813, 772, 738, 671, 654. 1H NMR(200MHz, CDCl3): d Cq 8.45 (d, J¼ 4.6 Hz,1H, H2); 7.56 (d, J¼ 1.6 Hz,1H, H8); 7.37 (d, 1H, J ¼ 9.0 Hz, H5); 7.16 (d, 1H, J ¼ 4.6 Hz, H3); 7.10(dd, 1H, J ¼ 1.6 Hz, J ¼ 9.0 Hz, H6); Tr 8.03 (s, 1H, H5); 3.04 (s, 2H, eCH2N); Pm 2.16e2.10 (m, 2H, 2� eCHN); 1.55e1.51 (m, 2H, 2� e

CHN); 1.09e1.04 (m, 2H, 2� eCH); 0.77e0.72 (m, 2H, 2� eCH).Note: signal for eCHOH in the piperidinyl cycle was probablysuperimposed to the solvent signal. 13C NMR (50 MHz, CDCl3):d 152.40; 149.46; 144.67; 140.54; 135.41; 128.98; 128.15; 126.07;125.69; 120.29; 116.91; 66.17; 52.42; 50.69; 34.27. HRMS-ESI-IT-TOF: m/z calculated C17H10ClN4 (M � H) 305.0593, found305.0712. HRMS-ESI-IT-TOF: m/z cal C17H10ClN4 (M þ H) 344.1278,found 344.1268. HRMS-ESI-IT-TOF: m/z cal C17H10ClN4 (M þ H þ 2)346.1249, found 346.1265.

General methodology for route C. To a solution of acid chloride(30.00 mmol) in 4 mL CH2Cl2, pent-4-yn-1-ol (1.3 g, 20 mmol) wasadded in one portion at room temperature, then triethylamine(19.40 mmol, 2.58 mL) was added in drops and the final mixturewas stirred overnight, where upon TLC indicated reactioncompletion. The crudewas dilutedwith 20mL CH2Cl2, washedwithwater (3 � 40 mL), HCl 0.1 M (1 � 10 mL) and sodium hydroxide0.1 M (1 � 10 mL) then dried over anhydrous Na2SO4, and evapo-rated to dryness. The resulting product residuewas used in the nextstep without further purification.

In the next step, 4-azido-7-chloroquinoline (102 mg, 0.5 mmol)and an alkyne ester (0.5 mmol) were dissolved in CH2Cl2 (2 mL),added CuSO4$5H2O (38 mg, 0.15 mmol) and a freshly preparedsolution of sodium ascorbate, made using ascorbic acid (53 mg,0.3 mmol) and NaHCO3 (25 mg, 0.3 mmol) in 2 mL of water. Theresulting mixture was stirred overnight, the reaction wascompleted by TLC and final compound was elaborated with CH2Cl2,water (3 � 10 mL); dried over Na2SO4 and purified by preparativeTLC or chromatography column.

8.15. 1-Pent-4-ynyl-benzoate (34)

IR (nmax, cm�1): 3298, 3064, 2916, 2119, 1790, 1714, 1601, 1584,1491, 1451, 1388, 1353, 1314, 1268, 1212, 1175, 1113, 1070, 1027, 997,707, 6861H NMR (200 MHz, CDCl3): d Bz 7.94 (d, J ¼ 7.4 Hz, 2H, H2,

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309 305

H6); 7.58e7.42 (m, 3H, H3, H4, H5); Po 4.31 (t, J ¼ 6.2 Hz, 2H, H1);2.75 (bs, 1H, H5); 2.31 (t, J ¼ 7.4 Hz, 2H, H3); 1.21 (pseudo q,J ¼ 6.2 Hz, J ¼ 7.4 Hz, 2H, H2). 13C NMR (50 MHz, CDCl3): d 165.70;133.11; 130.30; 129.16; 128.55; 83.30; 71.30; 63.34; 27.23; 14.73.

8.16. Pent-4-ynyl 4-methylbenzoate (35)

IR (nmax, cm�1): 3297, 2959, 2120, 1784, 1712, 1611, 1577, 1509,1446, 1408, 1387, 1269, 1221, 1176, 1108, 1032, 1020, 1004, 840, 751,690. 1H NMR (200 MHz, CDCl3): d Bz 7.92 (d, J ¼ 7.8 Hz, 2H, H2, H6);7.21 (d, J ¼ 7.8 Hz, 2H, H3, H5); 2.42e2.33 (m, 3H, eCH3); Po 4.40 (t,J ¼ 6.2 Hz, 2H, H1); 2.42e2.33 (m, 3H, H3, H5); 2.04e1.94 (m, 2H,H2). 13C NMR (50 MHz, CDCl3): d 166.55; 143.61; 129.61; 129.09;127.50; 83.10; 69.19; 63.30; 27.74; 21.64; 15.38.

8.17. Pent-4-ynyl 4-tert-butylbenzoate (36)

IR (nmax, cm�1): 3300, 2963, 2906, 2870, 2121, 1785, 1716, 1608,1572, 1506, 1463, 1409, 1389, 1364, 1314, 1271, 1224, 1187, 1178, 1117,1039, 997, 854, 775, 705. 1H NMR (200 MHz, CDCl3): d Bz 7.98 (d,J¼ 8.6 Hz, 2H, H2, H6); 7.44 (d, J¼ 8.6 Hz, 2H, H3, H5); 1.34 (s, 9H, 3�CH3); Po 4.41 (t, J ¼ 6.4 Hz, 2H, H1); 2.36 (td, J ¼ 2.6 Hz, J ¼ 7.0 Hz,2H, H3); 2.05e1.97 (m, 3H, H2, H5). 13C NMR (50 MHz, CDCl3):d 166.46; 156.57; 129.61; 127.51; 125.36; 83.09; 69.21; 63.25;35.30; 31.14; 27.78; 15.38.

8.18. Pent-4-ynyl 4-etyl-benzoate (37)

IR (nmax, cm�1): 3300, 2966, 2119, 1784, 1713, 1610, 1575, 1509,1460, 1415, 1387, 1269, 1219, 1177, 1108, 1032, 1020, 1000, 852, 760,701. 1H NMR (200 MHz, CDCl3): d Bz 7.81 (d, J ¼ 8.4 Hz, 2H, H2, H6);7.10 (d, J ¼ 8.4 Hz, 2H, H3, H5); 2.55 (q, J ¼ 7.8 Hz, 2H, CH2); 1.10 (t,J¼ 7.8 Hz, 3H, CH3); Po 4.33 (t, J¼ 6.0 Hz, 2H, H1); 2.97e2.90 (m, 2H,H3); 2.25e2.15 (m, 3H, H2, H5). 13C NMR (50 MHz, CDCl3): d 166.39;149.70; 130.68; 129.65; 128.35; 127.68; 83.01; 69.15; 63.21; 29.02;27.70; 15.21.

8.19. Pent-4-ynyl 4-methoxybenzoate (38)

IR (nmax, cm�1): 3295, 2961, 2120, 1778, 1707, 1605, 1580, 1510,1461, 1420, 1387, 1316, 1272, 1251, 1221, 1165, 1114, 1102, 1025, 995,846, 769, 695. 1H NMR (200MHz, CDCl3): d Bz 7.37 (d, J¼ 9.0 Hz, 2H,H2, H6); 6.73 (d, J¼ 9.0 Hz, 2H, H3, H5); 3.68 (s, 3H, eOCH3); Po 4.30(t, J ¼ 6.0 Hz, 2H, H1); 2.96e2.89 (m, 2H, H3); 2.20e2.15 (m, 3H, H2,H5). 13C NMR (50 MHz, CDCl3): d 166.27; 163.42; 131.64; 122.68;113.66; 83.21; 69.19; 63.23; 55.46; 27.82; 15.43.

8.20. Pent-4-ynyl 4-(trifluoromethyl)benzoate (39)

IR (nmax, cm�1): 3309, 2963, 1720, 1586, 1514, 1412, 1323, 1272,1166, 1100, 1064, 1017, 863, 820, 775, 752, 703, 689. 1H NMR(200 MHz, CDCl3): d Bz 8.16 (d, J ¼ 7.0 Hz, 2H, H2, H6); 7.74 (d,J ¼ 7.0 Hz, 2H, H3, H5); Po 4.47 (t, J ¼ 6.0 Hz, 2H, H1); 2.43e2.40 (m,3H, H5, H3); 2.05e1.99 (m, 2H, H2). 13C NMR (50 MHz, CDCl3):d 163.45; 134.54; 132.21; 132.05; 128.71; 123.88 (q, 1C, J¼ 11.5 Hz);81.66; 68.94; 62.69; 26.08; 13.74.

8.21. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl-benzoate (14)

Yield: 82%; mp 35.2e42.7 �C; IR (nmax, cm�1): 3132, 3059, 2933,2895, 1713, 1609, 1602, 1584, 1571, 1504, 1491, 1450, 1432, 1314,1272, 1251, 1238, 1173, 1112, 1069, 1038, 1025, 977, 960, 874, 824,795, 774, 685, 660. 1H NMR (200MHz, CDCl3): d Cq 9.03 (bs,1H, H2);8.21 (s, 1H, H8); 7.98 (d,1H, J¼ 9.2 Hz,1H, H5); 7.55e7.36 (m, 2H, H3,

H6); Tr 7.87 (s, 1H, H5); Pr 4.47 (t, 2H, J ¼ 6.2 Hz, H1); 3.07 (t, 2H,J¼ 7.2 Hz, H3); 2.37 (pseudo q, 2H, J¼ 6.0 Hz, J¼ 7.4 Hz, H2); Bz 8.00(d, J ¼ 7.0 Hz, 2H, H2, H6); 7.55e7.36 (m, 3H, H3, H4, H5). 13C NMR(50 MHz, CDCl3): d 166.49; 151.33; 150.01; 149.96; 147.70; 140.94;136.63; 131.77; 129.46; 129.18; 128.74; 128.36; 124.74; 122.93;120.46; 115.81, 63.93, 28.18, 22.24. HRMS-ESI-IT-TOF: m/z calC21H18ClN4O2 (M þ H) 393.1118, found 393.1330. HRMS-ESI-IT-TOF:m/z cal C21H18ClN4O2 (M þ H þ 2) 395.1293, found 393.1089.

8.22. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl-4-ethylbenzoate (15)

Yield: 60%; mp 94.7e97.2 �C; IR (nmax, cm�1): 3134, 3094, 3049,2969, 2918, 1708, 1611, 1590, 1561, 1505, 1458, 1437, 1342, 1325,1306, 1283, 1245, 1228, 1179, 1114, 1068, 1042, 1021, 956, 918, 873,852, 821, 810, 773, 747, 697, 683. 1H NMR (200 MHz, CDCl3): d Cq8.87 (bs,1H, H2); 7.84e7.79 (m,1H, H5, H8); 7.40 (d,1H, J¼ 9 Hz, H6);7.29 (bs, 1H, H3); Tr 8.05 (s, 1H, H5); Bz 7.81 (d, 2H, J ¼ 8.4 Hz, H3,H5); 7.10 (d, 2H, J¼ 8.4 Hz, H2, H6); 2.54 (q, 2H, J¼ 7.6 Hz, CH2); 1.10(t, 3H, J ¼ 7.6 Hz, CH3); Pr 4.33 (t, 2H, J ¼ 6.0 Hz, H1); 2.93 (t, 2H,J¼ 7.2 Hz, H3), 2.20 (m, 2H, H2). 13C NMR (50MHz, CDCl3): d 166.59;151.32; 150.06; 149.90; 147.79; 140.95; 136.65; 129.64; 129.19;128.82; 127.92; 127.54; 124.79; 122.95; 120.49; 115.83, 63.78,28.90, 28.25, 22.33, 15.22. HRMS-ESI-IT-TOF: m/z calculatedC23H21ClN4O2 (M þ H) 421.1431, found 421.1355. HRMS-ESI-IT-TOF:m/z cal C23H21ClN4O2 (M þ H þ 2) 423.1431, found 423.1371.

8.23. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl-4-tert-butyl-benzoate (16)

Yield: 67%; mp 91.3e94.2 �C; IR (nmax, cm�1): 3134, 3047, 2921,1704,1611,1591,1561,1505,1480,1451,1429,1354,1304,1275,1255,1233, 1177, 1121, 1110, 1034, 1016, 995, 956, 918, 880, 872, 818, 750,689, 670. 1H NMR (200 MHz, CDCl3): d Cq 8.91 (bs, 1H, H2); 8.10 (d,J¼ 2.0 Hz,1H, H8); 7.90e7.86 (m,1H, H5); 7.47e7.32 (m, 2H, H3, H6);Tr 7.90e7.86 (m, 1H, H5); Bz 7.88 (d, 2H, J ¼ 8.2, H3, H5); 7.34 (d,J ¼ 8.2, H2, H6); 1.24 (s, 9H, 3� CH3); Pr 4.39 (t, 2H, J ¼ 6.2 Hz, H1);2.99 (t, 2H, J ¼ 7.2 Hz, H3); 2.26 (pseudo q, 2H, J ¼ 6.2 Hz, J ¼ 7.2 Hz,H2). 13C NMR (50 MHz, CDCl3): d 166.47; 156.64; 151.29; 149.96;147.73; 140.90; 136.57; 129.35; 129.10; 128.72; 127.24; 125.32;124.75; 122.92; 120.39; 115.74, 63.72, 34.98, 31.02, 28.19, 22.27.HRMS-ESI-IT-TOF: m/z calculated C25H25ClN4O2Na (M þ Na)471.1563, found 471.1610. HRMS-ESI-IT-TOF: m/z calC25H25ClN4O2Na (M þ Na þ 2) 473.1534, found 473.1482.

8.24. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl-4-methoxy-benzoate (17)

Yield: 81%; mp 94.2e97.9 �C; IR (nmax, cm�1): 3131, 3069, 3009,2933, 1698, 1605, 1579, 1561, 1510, 1480, 1451, 1431, 1376, 1356,1299, 1276, 1256, 1233, 1198, 1165, 1120, 1100, 1029, 995, 968, 951,881, 874, 843, 818, 786, 766, 694, 671. 1H NMR (200 MHz, CDCl3):d Cq 8.85 (bs, 1H, H2); 8.02 (s, 1H, H8); 7.88e7.80 (m, 1H, H5); 7.30e7.27 (m, 2H, H3, H6). Tr 7.88e7.80 (m, 1H, H5); Bz 7.88e7.80 (m, 2H,H3, H5); 6.73 (d, 2H, J¼ 8.6 Hz, H2, H6); 3.68 (s, 3H, OCH3); Pr 4.33 (t,J ¼ 5.6 Hz, 2H, H1); 2.93 (t, 2H, J ¼ 6.8 Hz, H3); 2.18 (pseudo q, 2H,J ¼ 5.6 Hz, J ¼ 6.8 Hz, H2). 13C NMR (50 MHz, CDCl3): d 166.04;163.19; 151.19; 149.83; 147.63; 140.73; 136.36; 131.32; 128.91;128.58; 124.67; 122.83; 122.25; 120.22; 115.58, 113.44; 63.52,55.24; 28.11, 22.17. HRMS-ESI-IT-TOF: m/z calculated C22H19ClN4O3(M þ 1) 423.1223, found 423.1204. HRMS-ESI-IT-TOF: m/z calC22H19ClN4O3 (M þ H þ 2) 425.1194, found 425.1187.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309306

8.25. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl-4-trifluorometoxy-benzoate (18)

Yield: 75%; mp 112.5e116.4 �C; IR (nmax, cm�1): 3130, 3089,3051, 2963, 2931,1712,1611,1590,1561,1505,1480,1450,1437,1412,1376, 1325, 1310, 1285, 1250, 1165, 1135, 1124, 1101, 1064, 1039, 1017,996, 954, 917, 878, 862, 853, 819, 808, 773, 704, 682. 1H NMR(200MHz, CDCl3): d Cq 8.98 (bs,1H, H2); 8.15e8.09 (m,1H, H8); 7.95(d, J ¼ 9.0 Hz, 1H, H5); 7.51 (d, J ¼ 9.0 Hz, 1H, H6); 7.40 (d, 1H,J¼ 4.0 Hz, H3); Bz 7.96 (d, 2H, J¼ 8.2, H2, H6); 7.64 (d, J¼ 8.2 Hz, 2H,H3, H5); Tr 7.86 (s, 1H, H5); Pr; 4.47 (t, 2H, J¼ 6.2 Hz, H1); 3.03 (t, 2H,J ¼ 7.2 Hz, H3); 2.30 (pseudo q, 2H, J ¼ 6.2 Hz, J ¼ 7.2 Hz, H2). 13CNMR (50 MHz, CDCl3): d 165.39; 151.39; 150.14; 147.70; 141.06;136.87; 134.83; 133.83 (d, 1C, J ¼ 38.6 Hz); 130.03; 129.77; 129.36;128.90; 128.72; 124.77, 123.04 (d, 1C, J ¼ 245.1 Hz), 122.93, 115.90,64.62, 28.27, 22.30. HRMS-ESI-IT-TOF: m/z calculatedC22H16ClF3N4O2Na (M þ Na) 483.0811, found 483.0529. HRMS-ESI-IT-TOF: m/z cal C22H16ClF3N4O2Na (M þ Na þ 2) 485.0782, found485.0624.

8.26. 3-(1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl-4-trifluorometoxy-benzoate (19)

Yield: 68%; mp 81.0e83.9 �C; IR (nmax, cm�1): 3150, 3066, 2968,2952, 2905, 2870, 1700, 1609, 1596, 1560, 1507, 1478, 1451, 1429,1359, 1278, 1254, 1228, 1189, 1126, 1114, 1028, 1014, 993, 962, 950,882, 872, 852, 819, 812, 769, 733, 704, 671. 1H NMR (200 MHz,CDCl3): d Cq 8.87 (d, 1H, J ¼ 4.6 Hz, H2); 8.05 (d, 1H, J ¼ 2.0 Hz, H8);7.86 (d, 1H, J ¼ 2.0 Hz, H5); 7.41 (dd, 1H, J ¼ 2.0 Hz, J ¼ 8.8 Hz, H6);7.31 (d, 1H, J ¼ 4.6 Hz, H3); Tr 7.85 (s, 1H, H5); Bz 7.80 (d, 2H,J ¼ 8.2 Hz, H3, H5); 7.09 (d, J ¼ 8.2 Hz, 2H, H2, H6); 2.27 (s, 3H, CH3);Pr 4.35 (t, 2H, J ¼ 6.2 Hz, H1); 2.96 (t, 2H, J ¼ 7.4 Hz, H3); 2.21(pseudo q, 2H, J ¼ 6.2 Hz, J ¼ 7.4 Hz, H2). 13C NMR (50 MHz, CDCl3):d 165.45; 151.22; 149.89; 147.66; 143.59; 140.80; 136.47; 129.40;128.99; 128.98; 128.63; 127.21; 124.71; 122.87; 120.30; 115.65,63.70, 28.13, 22.22, 21.52. HRMS-ESI-IT-TOF: m/z cal C22H20ClN4O2(M þ H) 407.1275, found 407.1539. HRMS-ESI-IT-TOF: m/z calC22H20ClN4O2 (M þ H þ 2) 409.1245, found 409.0144.

Route C (small chain) e To a solution of acid chloride(12.95 mmol) in 4 mL CH2Cl2, prop-2-yn-1-ol (0.48 g, 8.63 mmol)was added in one portion at room temperature, then triethylamine(19.40 mmol, 2.58 mL) was added in drops and the final mixturewas stirred overnight, where upon TLC indicated reactioncompletion. The crudewas dilutedwith 20mL CH2Cl2, washedwithwater (3 � 40 mL), HCl 0.1 M (1 � 10 mL) and sodium hydroxide0.1 M (1 � 10 mL) then dried over anhydrous Na2SO4, and evapo-rated to dryness. The resulting product residuewas used in the nextstep without further purification.

In the next step, 4-azido-7-chloroquinoline (102 mg, 0.5 mmol)and ester alkyne (0.5 mmol) were dissolved in CH2Cl2 (2 mL), addedCuSO4$5H2O (38 mg, 0.15 mmol) and a freshly prepared solution ofsodium ascorbate, made using ascorbic acid (53 mg, 0.3 mmol) andNaHCO3 (25 mg, 0.3 mmol) in 2 mL of water. The resulting mixturewas stirred overnight, the reaction was completed by TLC and finalcompound was elaborated with CH2Cl2, water (3 � 10 mL); driedover Na2SO4 and purified by preparatory TLC.

8.27. Prop-2-ynyl benzoate (40)

IR (nmax, cm�1): 3295, 2946, 2129, 1786, 1719, 1600, 1584, 1492,1451, 1435, 1369, 1315, 1262, 1211, 1175, 1105, 1095, 1069, 1026, 979,925, 872, 804, 777, 706, 671. 1H NMR (200 MHz, CDCl3): d 1H NMR(200 MHz, CDCl3): d Pr 4.76 (d, J ¼ 1.4 Hz, 2H, H1); 2.43 (bs, 1H, H3);Bz 7.90 (d, J ¼ 7.6 Hz, 2H, H2, H6); 7.36e7.23 (m, 3H, H3, H4, H5). 13C

NMR (50 MHz, CDCl3): d 165.61; 133.26; 129.71; 129.34; 128.38;77.86; 75.17; 52.37.

8.28. Prop-2-ynyl 4-methylbenzoate (41)

IR (nmax, cm�1): 3250, 2979, 2947, 2739, 2603, 2497, 2124, 1774,1712, 1609, 1508, 1475, 1444, 1397, 1383, 1366, 1267, 1222, 1209,1171, 1120, 1099, 1035, 1015, 981, 924, 851, 840, 824, 807, 752, 730,682. 1H NMR (200MHz, CDCl3): d Pr 4.88 (d, J¼ 2.4 Hz, 2H, H3); 2.52(t, J ¼ 2.4 Hz, 1H, H1); Bz 7.94 (d, J ¼ 8.2 Hz, 2H, H2, H6); 7.20 (d,J¼ 8.2 Hz, 2H, H3, H5); 2.38 (s, 3H,eCH3). 13C NMR (50MHz, CDCl3):d 165.27; 144.11; 129.17; 128.99; 126.17; 77.88; 75.03; 52.28; 21.82.

8.29. Prop-2-ynyl 4-tert-butylbenzoate (42)

IR (nmax, cm�1): 3297, 2964, 2906, 2871, 2129, 1786, 1721, 1608,1571, 1505, 1463, 1434, 1409, 1366, 1314, 1299, 1266, 1224, 1187,1112, 1093, 1041, 1016, 983, 925, 853, 874, 773, 706, 673. 1H NMR(200 MHz, CDCl3): d 1H NMR (200 MHz, CDCl3): d Pr 4.91 (d,J ¼ 2.4 Hz, 2H, H3); 2.52 (t, J ¼ 2.4 Hz, 1H, H1); Bz 8.00 (d, J ¼ 8.6 Hz,2H, H2, H6); 7.45 (d, J¼ 8.6 Hz, 2H, H3, H5); 1.35 (s, 9H, 3�eCH3). 13CNMR (50 MHz, CDCl3): d 165.76; 157.09; 129.71; 126.67; 125.47;77.98; 74.92; 53.58; 52.27; 31.09.

8.30. Prop-2-ynyl 4-ethyllbenzoate (43)

IR (nmax, cm�1): 3296, 2968, 2129, 1720, 1689, 1610, 1574, 1509,1433, 1417, 1369, 1310, 1263, 1177, 1095, 1051, 1019, 981, 925, 852,701, 675. 1H NMR (200MHz, CDCl3): d Pr 4.88 (d, J¼ 2.4 Hz, 2H, H1);2.52 (t, J¼ 2.4 Hz, 1H, H3); Bz 7.94 (d, J¼ 8.0 Hz, 2H, H2, H6); 7.23 (d,J ¼ 8.0 Hz, 2H, H3, H5); 2.65 (q, J ¼ 7.6 Hz, 3H, eCH2); 1.22 (t,J ¼ 7.6 Hz, 2H, eCH3). 13C NMR (50 MHz, CDCl3): d 165.80; 150.27;129.94; 128.42; 127.98; 77.91; 74.92; 52.26; 28.96; 15.18.

8.31. Prop-2-ynyl 2-ethyllbenzoate (44)

IR (nmax, cm�1): 3295, 2972, 2130, 1785, 1720, 1601, 1575, 1487,1448, 1366, 1290, 1252, 1229, 1198, 1140, 1129, 1077, 1056, 977, 751,708, 661. 1H NMR (200MHz, CDCl3): d Pr 4.88 (d, J¼ 2.4 Hz, 2H, H1);2.52 (t, J¼ 2.4 Hz, 1H, H3); Bz 7.94 (dd, J¼ 0.8 Hz, J¼ 7.4 Hz, 1H, H6);7.45e7.38 (m, 2H, H5, H4); 7.25 (dd, J ¼ 2.0 Hz, J ¼ 5.6 Hz, 1H, H3);2.98 (q, J ¼ 7.6 Hz, 2H, CH2); 1.26 (t, J ¼ 7.6 Hz, 3H, CH3). 13C NMR(50 MHz, CDCl3): d 166.67; 146.50; 132.51; 131.44; 130.85; 130.35;125.82; 77.87; 75.01; 52.23; 27.62; 15.94.

8.32. Prop-2-ynyl 2,4,5-trifluoro-3-methoxybenzoate (45)

IR (nmax, cm�1): 3300, 3078, 2954, 2847, 2132, 1804, 1724, 1620,1508, 1473, 1435, 1377, 1351, 1276, 1213, 1185, 1100, 1054, 982, 956,932, 910, 874, 780, 763, 733, 684. 1H NMR (200 MHz, CDCl3): d Pr4.94 (d, J¼ 2.4 Hz, 2H, H1); 2.58 (t, J¼ 2.4 Hz,1H, H3); Bz 7.56e7.474(m,1H, H6); 4.10 (s, 3H,eOCH3). 13C NMR (50MHz, CDCl3): d 161.87;155.23; 149.53 (m, 1C); 144.66 (m, 1C); 139.00 (m, 1C); 113.80 (m,1C); 112.43 (d, J ¼ 20.9 Hz, 1C); 77.11; 75.72; 62.42; 53.20.

8.33. Prop-2-ynyl 4-(trifluoromethyl)benzoate (46)

IR (nmax, cm�1): 3308, 2950, 2131, 1728, 1619, 1587, 1514, 1437,1412, 1371, 1323, 1265, 1166, 1126, 1095, 1064, 1016, 979, 924, 861,820, 790, 773, 752, 703, 689. 1H NMR (200MHz, CDCl3): d Pr 4.97 (d,J ¼ 2.0 Hz, 2H, H1); 2.54 (bs, 1H, H3); Bz 8.16 (d, J ¼ 8.2 Hz, 2H, H2,H6); 7.69 (d, J ¼ 8.2 Hz, 2H, H3, H5). 13C NMR (50 MHz, CDCl3):d 164.62; 134.55 (q, J ¼ 32.5 Hz, 1C); 132.75; 130.28; 123.69 (d,J ¼ 271.0 Hz, 1C); 120.98; 744.40; 75.57; 53.01.

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309 307

8.34. Prop-2-ynyl 4-methoxybenzoate (47)

IR (nmax, cm�1): 3238, 2952, 2118, 1705, 1604, 1578, 1511, 1465,1428, 1369, 1254, 1167, 1095, 1026, 1008, 982, 922, 844, 768, 726,694. 1H NMR (200MHz, CDCl3): d Pr 4.89 (d, J¼ 2.4 Hz, 2H, H1); 2.53(t, J ¼ 2.4 Hz, 1H, H3); Bz 8.02 (d, J ¼ 9.0 Hz, 2H, H2, H6); 6.92 (d,J ¼ 9.0 Hz, 2H, H3, H5); 3.86 (s, 3H, eOCH3). 13C NMR (50 MHz,CDCl3): d 163.83; 132.48; 132.06; 121.91; 113.86; 77.88; 75.03;55.61; 52.34.

8.35. Prop-2-ynyl 4-chloro-benzoate (48)

1H NMR (200 MHz, CDCl3): d Pr 4.91 (d, J ¼ 2.4 Hz, 2H, H1); 2.54(t, J ¼ 2.4 Hz, 1H, H3); Bz 7.99 (d, J ¼ 8.6 Hz, 2H, H2, H6); 7.42 (d,J ¼ 8.6 Hz, 2H, H3, H5). 13C NMR (50 MHz, CDCl3): d 165.05; 139.98;131.33; 128.95; 127.97; 77.87; 75.43; 52.05.

8.36. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-4-ethylbenzoate (20)

Yield: 67%; mp 95.7e102.1 �C; IR (nmax, cm�1): 3155, 2964, 2930,1707, 1609, 1594, 1562, 1507, 1455, 1417, 1343, 1299, 1273, 1246,1182, 1112, 1040, 1022, 982, 955, 882, 875, 853, 825, 766, 701, 656.1H NMR (200 MHz, CDCl3): d Cq 9.04 (d, J ¼ 4.6 Hz, 1H, H2); 8.21 (d,J ¼ 1.8 Hz, 1H, H8); 7.98 (d, J ¼ 9.2 Hz, 1H, H5); 7.59 (dd, J ¼ 1.8 Hz,J ¼ 9.0 Hz, 1H, H6); 7.49 (d, 1H, J ¼ 4.6 Hz, H3); Tr 8.23 (s, 1H, H5);5.62 (s, 2H, CH2); Bz 7.98 (d, J ¼ 8.2 Hz, 2H, H2, H6); 7.26 (d, 2H,J¼ 8.0 Hz, H3, H5); 2.94 (q, 2H, J¼ 7.6 Hz, CH2); 1.24 (t, 3H, J¼ 7.6 Hz,CH3). 13C NMR (50 MHz, CDCl3): d 166.65; 151.48; 150.52; 150.26;144.09; 140.90; 137.02; 130.05; 129.59; 129.07; 128.14; 127.04;126.02; 124.69; 120.62; 116.17, 57.75, 29.09, 15.33. HRMS-ESI-IT-TOF: m/z calculated C21H18ClN4O2 (M þ H) 393.1118, found393.0648. HRMS-ESI-IT-TOF: m/z cal C21H18ClN4O2 (M þ H þ 2)395.1089, found 395.1322.

8.37. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-2-ethylbenzoate (21)

Yield: 70%; mp 84.1e87.3 �C; IR (nmax, cm�1): 3138, 3095, 2985,2972, 1718, 1608, 1591, 1560, 1505, 1484, 1451, 1439, 1320, 1293,1257,1245,1229,1142,1115,1088,1038, 954, 877, 872, 845, 820, 811,752, 709, 672, 653. 1H NMR (200 MHz, CDCl3): d Cq 9.05 (d,J¼ 4.6 Hz,1H, H2); 8.21 (d, J¼ 2.0 Hz,1H, H8); 7.94 (d, J¼ 9.2 Hz,1H,H5); 7.50 (dd, J ¼ 2.0 Hz, J ¼ 9.0 Hz, 1H, H6); 7.46 (d, J ¼ 4.6 Hz, 1H,H3); Tr 8.25 (s, 1H, H5); 5.61 (s, 2H, CH2); Bz 7.92 (t, J ¼ 7.2 Hz, 1H,H6); 7.26 (d, J ¼ 7.2 Hz, 2H, H4); 7.40e7.27 (m, 2H, H3, H5); 2.99 (q,J ¼ 7.4 Hz, 2H, CH2); 1.22 (t, 3H, J ¼ 7.4 Hz, CH3). 13C NMR (50 MHz,CDCl3): d 167.35; 151.43; 150.52; 146.48; 143.94; 140.82; 136.95;132.91; 130.88; 130.41; 129.51; 128.98; 128.50; 125.87; 125.86;124.56; 120.54; 116.13, 57.67, 27.64, 15.96. HRMS-ESI-IT-TOF: m/zcalculated C21H18ClN4O2 (M þ 1) 393.1118, found 393.0917. HRMS-ESI-IT-TOF: m/z cal C21H18ClN4O2 (M þ H þ 2) 395.1089, found395.1322.

8.38. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-4-tert-butyl-benzoate (22)

Yield: 81%; yellow oil; IR (nmax, cm�1): 3075, 3963, 2905, 2869,1713,1609,1561,1505,1456,1438,1409,1364,1268,1238,1187,1113,1095,1039,1015, 954, 908, 878, 853, 820, 813, 774, 730, 706, 672. 1HNMR (200 MHz, CDCl3): d Cq 9.05 (d, J ¼ 4.6 Hz, 1H, H2); 8.21 (d,J ¼ 2.0 Hz, 1H, H8); 7.58 (dd, J ¼ 2.0 Hz, J ¼ 9.2 Hz, 1H, H6); 7.46 (d,J¼ 7.0 Hz,1H, H3); Tr 8.23 (s,1H, H5); 5.63 (s, 2H, CH2); Bz 8.03e7.96(m, 3H, H2, H6); 7.26 (d, J ¼ 8.6 Hz, 2H, H3, H5); 1.33 (s, 9H, 3� CH3).13C NMR (50 MHz, CDCl3): d 166.65; 157.37; 151.48; 150.24; 144.15;

140.97; 137.11; 129.83; 129.95; 129.05; 126.81; 126.05; 125.62;124.73; 120.67, 116.21, 57.75, 35.28, 31.21. HRMS-ESI-IT-TOF: m/zcalculated C23H22ClN4O5 (M þ 1) 421.1431, found 421.0539. HRMS-ESI-IT-TOF: m/z cal C23H22ClN4O5 (M þ H þ 2) 423.1402, found423.0546.

8.39. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methylbenzoate (23)

Yield:67%; mp 107.0e110.5 �C; IR (nmax, cm�1): 3144, 3111, 3087,2959, 2924, 2852, 1705, 1613, 1597, 1560, 1506, 1449, 1368, 1339,1314, 1296, 1269, 1238, 1191, 1116, 1069, 1044, 1022, 999, 951, 886,876, 842, 833, 815, 770, 746, 705, 684, 674. 1H NMR (200 MHz,CDCl3): d Cq 9.02 (d, J ¼ 4.0 Hz, 1H, H2); 8.17 (s, 1H, H8); 7.94 (d,J ¼ 9.0 Hz, 1H, H5); 7.55e7.36 (m, 2H, H3, H6); Tr 8.22 (s, 1H, H5);5.60 (s, 2H, H5); Bz 7.98 (d, J¼ 7.6 Hz, 2H, H2, H6); 7.55e7.36 (m, 3H,H3, H6, H4, H5). 13C NMR (50 MHz, CDCl3): d 166.52; 151.43; 150.21;143.88; 140.83; 136.97; 133.50; 129.85; 129.55; 129.54; 129.02;128.56; 126.02; 124.64; 120.55,116.14, 57.87. HRMS-ESI-IT-TOF:m/zcalculated C19H13ClN4O2Na (M þ Na) 387.0624, found 387.0787.HRMS-ESI-IT-TOF: m/z cal C19H13ClN4O2Na (M þ Na þ 2) 389.0595,found 389.0424.

8.40. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-2,4,5-trifluoro-3-methoxybenzoate (24)

Yield: 68%; mp 145.2e149.8 �C; IR (nmax, cm�1): 3054, 3017,3087, 2977, 2933, 2838, 1719, 1685, 1604, 1579, 1561, 1510, 1452,1431, 1297, 1258, 1191, 1171, 1105, 1092, 1026, 1001, 956, 876, 844,821, 810, 767, 696, 6701H NMR (200 MHz, DMSO): d Cq 9.10 (d,J¼ 4.4 Hz,1H, H2); 8.19 (d, J¼ 2.2 Hz,1H, H8); 8.03 (d, J¼ 9.0 Hz,1H,H5); 7.49 (d, 1H, J ¼ 4.4 Hz, H3); 7.69 (dd, J ¼ 2.2 Hz, J ¼ 9.0 Hz, 1H,H6); Tr 8.89 (s, 1H, H5); 5.57 (s, 2H, CH2); Bz 7.67e7.54 (m, 1H, H6);3.99 (m, 3H, OCH3). 13C NMR (50 MHz, DMSO): d 159.85; 150.47;147.89; 147.68; 147.50; 140.78; 138.66; 133.63; 127.27; 126.57;125.39; 123.69; 118.59; 115.21; 112.54; 112.46; 110.52,110.10, 60.59,56.70. HRMS-ESI-IT-TOF: m/z calculated C20H12ClF3N4O3Na(M þ Na) 471.0447, found 471.0593. HRMS-ESI-IT-TOF: m/z calC20H12ClF3N4O3Na (M þ H) 449.0628, found 449.0811. HRMS-ESI-IT-TOF: m/z cal C20H12ClF3N4O3Na (M þ H þ 2) 451.0599, found451.0542.

8.41. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-4-(trifluoromethyl) benzoate (25)

Yield: 45%; mp 85.7e92.4 �C; IR (nmax, cm�1): 3162, 3109, 3038,2969,1736,1715,1610,1596,1586,1561,1508,1442,1412,1376,1319,1278, 1263, 1238, 1171, 1139, 1108, 1091, 1062, 1016, 997, 955, 945,871, 863, 832, 814, 777, 702, 689, 671. 1H NMR (200MHz, CDCl3): Cqd 9.08 (bs, 1H, H2); 8.27 (bs, 1H, H8); 8.01 (d, 1H, J¼ 9.0 Hz, H5); 7.62(dd,1H, J¼ 1.0 Hz, J¼ 9.0 Hz, H6); 7.53 (d,1H, J¼ 3.8 Hz, H3); Tr 8.24(s, 1H, H5); 5.67 (s, 2H, CH2); Bz 7.72 (d, 2H, J ¼ 8.2 Hz, H2, H6); 8.21(d, 2H, J ¼ 8.2 Hz, H3, H5). 13C NMR (50 MHz, DMSO): d 165.54;151.36; 150.16; 143.56; 141.09; 137.44; 135.43; 134.78; 132.87;130.44; 129.93; 129.07; 126.20 (q, 1C, J ¼ 15.2 Hz); 124.72; 121.00;120.71; 116.24; 58.35. HRMS-ESI-IT-TOF: m/z calculatedC20H12ClF3N4O2Na (M þ Na) 455.0498, found 454.9954. HRMS-ESI-IT-TOF: m/z cal C20H12ClF3N4O2Na (M þ Na þ 2) 457.0469, found457.0449.

8.42. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-4-(methoxy) benzoate (26)

Yield: 77%; mp 94.9e101.8 �C; IR (nmax, cm�1): 3162, 3109, 3038,2969,1736,1715,1610,1596,1586,1561,1508,1442,1412,1376,1319,

G.R. Pereira et al. / European Journal of Medicinal Chemistry 73 (2014) 295e309308

1278, 1263, 1238, 1171, 1139, 1108, 1091, 1062, 1016, 997, 955, 945,871, 863, 832, 814, 777, 702, 689, 671. 1H NMR (200 MHz, CDCl3):d Cq 9.07 (bs, 1H, H2); 8.21 (s, 1H, H8); 8.05e8.01 (m, 1H, H5); 7.51(m, 2H, H3, H6); Tr 8.28 (bs, 1H, H5); 5.61 (s, 2H, CH2); Bz 8.05e8.01(m, 2H, H2, H6); 6.93 (d, 2H, J ¼ 8.6 Hz, H3, H5); 3.86 (s, 3H, eOCH3).13C NMR (50MHz, DMSO): d 166.11; 163.66; 151.35; 150.06; 144.09;136.78; 131.81; 129.36; 128.88; 128.90; 125.93; 124.59; 120.40;116.01; 113.69; 57.52; 55.44. HRMS-ESI-IT-TOF: m/z calculatedC20H16ClN4O3 (M þ H) 395.0910, found 395.0414. HRMS-ESI-IT-TOF: m/z cal C20H16ClN4O3 (M þ H þ 2) 397.0881, found 397.1116.

8.43. (1-(7-Chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl-4-(chloro) benzoate (27)

Yield: 28%; mp 86.7e87.8 �C; IR (nmax, cm�1): 3059, 2922, 1708,1610, 1593, 1561, 1504, 1457, 1435,1400,1345, 1267, 1235, 1171, 1115,1092, 1039, 1013, 954, 879, 847, 812, 768, 755, 683. 1H NMR(200 MHz, CDCl3): d Cq 9.03 (d, 1H, J¼ 4.6 Hz, H2); 8.21 (bs, 1H, H8);8.01e7.98 (m, 1H, H5); 7.51 (dd, 1H, J ¼ 1.8 Hz, J ¼ 9.0 Hz, H6); 7.54(d, 1H, J ¼ 4.6 Hz, H3); Tr 8.21 (bs, 2H, H8, H11); 5.61 (s, 2H, CH2); Bz8.01e7.98 (m, 2H, H2, H6); 7.39 (d, 2H, J ¼ 8.4 Hz, H3, H5). 13C NMR(50 MHz, DMSO): d 165.77; 151.48; 150.27; 143.68; 140.86; 140.06;137.12; 131.31; 129.67; 129.12; 128.99; 128.03; 126.13; 124.63;120.59; 116.19; 58.05. HRMS-ESI-IT-TOF: m/z calculatedC19H13Cl2N4O2 (M þ 1) 399.0415, found 399.0863. HRMS-ESI-IT-TOF: m/z calculated C19H12Cl2N4O2Na (M þ Na) 421.0235, found421.0180. HRMS-ESI-IT-TOF:m/z cal C19H12Cl2N4O2Na (M þ Naþ 2)423.0206, found 423.0533.

Acknowledgments

This work was supported by Programa Nacional de Excelência ePRONEX, Conselho Nacional de Desenvolvimento Científico e Tec-nológico e CNPq (Process number 555655/2009-1) and Fundaçãode Amparo à Pesquisa do Estado de Minas Gerais e FAPEMIG(Process number CDS APQ 01129-10). Fellowships to GRP (PDJFAPEMIG), GCB (PDJ CNPq), HAOJ (BIC CNPq), RCP (D CNPq), MFAN(DTI2 FAPEMIG).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2013.11.022

Conflict of interest

Authors declare no conflict of interest.

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