{New 4-aminoquinolines as moderate inhibitors of P. falciparum malaria} J. Serb. Chem. Soc. 86 (2) 115–123 (2021) Original scientific paper JSCS–5408 115 New 4-aminoquinolines as moderate inhibitors of P. falciparum malaria IGOR OPSENICA1#, MILICA SELAKOVIĆ2#, MIKLOŠ TOT1, TATJANA VERBIĆ1#, JELENA SRBLJANOVIĆ3, TIJANA ŠTAJNER3, OLGICA DJURKOVIĆ DJAKOVIĆ3 and BOGDAN ŠOLAJA1,4*# 1University of Belgrade – Faculty of Chemistry, Studentski trg 16, P.O. Box 51, 11158 Belgrade, Serbia, 2Innovative Centre, Faculty of Chemistry, Ltd. Studentski Trg 12–16, 11158 Belgrade, Serbia, 3Institute for Medical Research, University of Belgrade, Dr. Subotića 4, P.O. Box 39, 11129 Belgrade, Serbia and 4Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade, Serbiа (Received 25 December 2020, revised 17 January, accepted 18 January 2021) Abstract: Synthesis of novel aminoquinoline derivatives has been accom- plished and their activity against malaria strains has been examined. The com- pounds showed moderate in vitro antimalarial activity against two P. falci- parum strains, 3D7 (CQ susceptible clone) and Dd2 (CQ resistant clone). Three aminoquinolines were further examined for antimalarial efficacy in a mouse model using a modified Thompson test. In this model, mice were inf- ected with P. berghei-infected red blood cells, and drugs were administered orally. Antimalarial 3 was found toxic at a dose of 320 (mg/kg)/day in 3/6 mice, however, 2/6 mice of the same group survived through day 31, and one of them was cured. Keywords: quinoline; bromobenzyl derivatives; antimalarials; β-hematin inhi- bitory activity. INTRODUCTION Climate change and global warming are closely associated with growing threat from infectious diseases. Increase in global temperatures favors the dev- elopment and spread of tropical diseases and the associated vector organisms.1 Warming will contribute to area expansion populated with malaria-transmitting mosquitoes. With 300–500 million clinical cases and nearly one million deaths every year, malaria is a major global public health problem.2 Among the five known species of the Plasmodium genus that cause human malaria (Plasmodium species: P. falciparum, P. ovale, P. vivax, P. malarie and P. knowlesi), P. falci- * Corresponding author. E-mail: bogdan.solaja@sanu.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC201225005O ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 116 OPSENICA et al. parum is th-e major cause of mortality. Malaria parasites use hemoglobin (Hb) as a source of nutrients, and after digestion of host Hb in the acidic food vacuole (FV) of P. falciparum amino acids and heme are released. The parasites use Hb amino acids as building blocks for their own protein synthesis. The heme moiety ferriprotoporphyrin-IX is toxic to the host, and the parasite has developed several mechanisms of detoxification: sequestration of heme into insoluble hemozoin,3 and degradation of heme with hydrogen peroxide4,5 and glutathione-mediated mechanisms6 in the FV and cytosol, respectively. The clinical symptoms of malaria appear during the asexual intraerythrocytic stage; consequently, efforts to develop an effective drug have mainly focused on this stage of infection.7 Mala- ria has been known for centuries, and there is a lot of information regarding transmission, prevention and treatment of the disease. Despite this, it is still one of the most widespread infectious diseases in the world.8 One of the main causes is development of widespread drug resistance9,10 especially to chloroquine (CQ), the most widely used antimalarial (Fig. 1).11,12 Most of 4-amino-7-chloroquino- line based compounds (ACQs) act as inhibitors of hemozoin formation,8 but it is also reported that they may act as inhibitors of oxidative13 and gluthatione- -mediated14 heme degradation. Resistance to ACQs is closely related to reduced accumulation of drugs caused by mutations in drug transporters (PfCRT, Pgh1, and PfMRP).15,16 The synthesis and development of novel ACQ-based derivat- ives has been the subject of extensive research. Efforts have focused mostly on three subclasses: bisquinolines, sidechain modified 4-aminoquinolines, and hyb- rid 4-aminoquinolines.17,18 Fig. 1. Structures of CQ and potent antimalarials. During our studies a variety of ACQ-based compounds were identified as potential antimalarial agents.19–22 Recently, we synthesized a series of simplified analogues with the ACQ component attached via an ethylene or propylene linker to different benzyl or pyridyl-groups (Fig. 1).23 The synthesized compounds showed promising antimalarial activities, and exhibited better potencies against CQ susceptible (CQS) and CQ resistant (CQR) strains as compared to CQ and mefloquine. The structure–activity data indicated that a substitution on the ben- zene ring had a significant influence on the antimalarial activity. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 4-AMINOQUINOLINES AS INHIBITORS OF MALARIA 117 In order to better understand the possible influence of bromine substituents on the antimalarial activity, we synthesized ten new aminoquinoline derivatives and here we analyze their activity (Scheme 1). Scheme 1. Synthesis of aminoquinoline derivatives 4–13. In addition, we have synthesized four hybrid derivatives (Fig. 2) that include previously established antimalarial pharmacophores-aminoquinoline24 and tetra- hydroimidazo[1,2-a]pyrazine.12 Fig. 2. Structures of hybrid derivatives. EXPERIMENTAL For the sake of journal space the full experimental details for the synthesis of tested compounds are given in the Supplementary material to this paper. In vitro antimalarial activity In vitro cultures of P. falciparum, chloroquine-sensitive 3D7 and chloroquine-resistant Dd2 strains were maintained as described previously.25 For drug assays, parasites were syn- chronized with 5 % sorbitol and ring-stage parasites were seeded in 96-well plates at a 2 % parasitemia and 0.75 % hematocrit. The compounds were initially dissolved in DMSO at a concentration of 50 mM and further dilutions were made in complete culture medium (final DMSO concentration was ≤0.2 %). After an initial screen at 0.5 µM concentration, com- pounds that inhibited parasite growth for at least 50 % were further titrated to obtain IC50 and IC90 values at eight two-fold dilutions (3 independent experiments were performed for each compound, each with 3 replicates per condition). Control experiments using chloroquine and/or artemisinin were performed in parallel with the tested compounds. Parasite inhibition was assayed after 48 h of incubation in the presence of a drug by the colorimetric LDH assay. The test is based on the evaluation of plasmodial lactate dehydrogenase (pLDH) activity and was performed according to the previously described method.26 IC50 and IC90 values were obtained using a sigmoidal dose–response model with the variable slope fitted to results using GraphPad Prism. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 118 OPSENICA et al. In vivo antimalarial activity Antimalarial activity of novel compounds was tested in mice infected with Plasmodium berghei ANKA strain using a modified version of the Thompson test.27 Prior to antimalarial efficacy experiments, all compounds were tested for toxicity. Female C57BL/6 mice aged 12 to 14 weeks and weighing 19–21 g were used. Mice were housed at 5 to 6 animals per cage at the Institute for Medical Research Animal Facility under a natural photo period and were offered drinking water and standard feed ad libitum. Mice were infected intraperitoneally (i.p.) with 250 μL of a PBS suspension containing 106 parasitized erythrocytes from the peripheral blood of a donor mouse. Compounds were suspended in 0.5 % hydroxyethylcellulose–0.1 % Tween 80 and administered orally at des- ignated doses in a volume of 200 μL once a day (on day 3, 4 and 5 post-infection (p.i.)). Mice were monitored daily and any clinical symptoms (e.g., ruffled fur, decreased loco- motion, lethargy, loss of appetite, lacrimation, salivation, diarrhoea, convulsions and weight loss) were noted. Parasitaemia was monitored by microscopic examination of Giemsa-stained peripheral blood smears using mouse tail blood twice a week, starting immediately before the initiation of treatment. The first time point served to check the efficacy of experimental inf- ections, whereas all later points served to monitor compound efficacy. Mice were observed for 30 days from the day of infection. Cure was defined as the survival of treated mice with para- site clearance at 31 days p.i. For toxicity experiments, naive mice were treated according to the described protocol of compound administration. Mice were observed daily during a period of 30 days after the first day of drug administration. All animal studies were approved by a local (Institute for Medical Research) ethics committee. In vivo experiments were approved by the Veterinary Directorate of the Ministry of Agriculture and Environmental Protection of Serbia (decision no. 323-07-02444/2014- -05/1). β-Hematin inhibitory activity assay. The inhibition of β-hematin formation is expressed as the molar equivalent of compound, relative to hemin, that inhibits β-hematin formation by 50 % and determined by slightly modified BHIA assay introduced by Parapini et al.28 Briefly, as described previously,29 50.0 µL of 16 mM solution of hemin in DMSO was distributed to 1.5 mL centrifuge tubes; compound dissolved in DMSO (Ccomp≈80 mM) was added to hemin in doses ranging from 0.125 to 5 mole equivalents (pure DMSO was added to control samples). 100.0 µL of ultrapure water was added to each tube, and β-hematin formation initiated by the addition of 200.0 µL of 8 M acetate buffer (pH 5.2). The final concentration of DMSO per tube was kept constant at 25 %. Tubes were incubated at 37.0±0.1 °C for 18 h and then centrifuged. The remaining pellet was resuspended in 0.500 mL DMSO to remove unreacted hematin. Tubes were then centrifuged again, DMSO-soluble fraction removed and the pellet, consisting of a pure precipitate of β-hematin, dissolved in 1.000 mL 0.1 M NaOH. 10.0 µL aliquots were transferred to 96-well microplate and diluted with 190.0 µl 0.1 M NaOH (200.0 µl of 0.1 M NaOH was used as a blank). The absorbance was measured at 405 nm, with correction at 670 nm (LKB 5060-006 Micro Plate Reader, Vienna, Austria). Expe- riments were performed in duplicate. A calibration curve of hemin dissolved in 0.1 M NaOH was made in the cHE range of 0.4×10-5–4.0×10-5 M, samples were diluted, if needed, to fit the calibration curve range. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 4-AMINOQUINOLINES AS INHIBITORS OF MALARIA 119 RESULTS AND DISCUSSION Synthesis All compounds were synthesized in high yield by coupling commercially available aryl aldehydes with aminoquinolines via reductive amination using sodium borohydride and acetic acid (Scheme 1). Each of the final compounds was then purified by dry-flash chromatography, and tested as > 95 % pure based on m.a. and/or HPLC analysis. Antimalarial activity The in vitro antimalarial activity of the synthesized compounds was evalu- ated against the CQS 3D7 and the CQR Dd2 P. falciparum strains, using CQ and artemisinin (ART) as positive controls (Table I). Eight compounds (1–3, 5, 6, 8, 11 and 12) were quite potent, with IC50 values within 19–48 nM range and appeared more active against the CQR Dd2 strain than CQ. Among them, deri- vatives 3 and 6 were 5-fold more active than CQ.30 In addition, 2 and 6 were as active as CQ and ART against the CQS 3D7 strain (2: IC50 = 22.05 nM; 6: IC50 = 14.02 nM). The results indicate that the length of the methylene chain affects the antiplasmodial activity. The potency of our compounds against the CQS 3D7 strain was increased up to C4 spacer, and further chain lengthening resulted in a decrease of the activity (7–9 vs. 10). On the other hand, the sub- stitution of chlorine atom with hydrogen at the 7-position of the quinoline ring did not significantly affect the in vitro antimalarial activity against the CQS strain (9: IC50 = 30.04 nM; 12: IC50 = 25.45 nM). TABLE I. In vitro antimalarial activity, IC50 / nM (geometric mean) Compound Strain 3D7a Dd2b 1 32.20c 40.82c 2 22.05c 37.97c 3 30.11c 18.81c 4 N.D. (>500)d N.D.d 5 43.50 34.04 6 14.02 26.07 7 41.08 N.D.e 8 45.75 35.22 9 30.04 N.D.d 10 61.66 N.D.d 11 291.10 48 12 25.45 30.72 13 76.22 216.71 CQ 16.17(5)e 129.18(5)e ART 13.09(3)e 15.93(2)e aP. falciparum CQ susceptible clone; bP. falciparum CQ resistant clone; ctaken from reference25; dN.D. – not determined; enumber of replicates ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 120 OPSENICA et al. In order to get an initial insight into the compound’s antimalarial mechanism of action, antimalarials 2, 3 and 8 were submitted to BHIA.28 The results showed that 3 and 8 interfered with hematin polymerization with the same potency as CQ (3: IC50 = 1.40; 8: IC50 = 0.81 vs. CQ: IC50 = 1.23), indicating the same mech- anism of action. On the other hand, compound 2 (without bromine attached to benzene ring) showed significantly lower BHIA (2: IC50 = 3.46). This difference in blocking the hemozoin formation requires further research, and we will pay full attention to this issue in the near future. Reported results of in vitro antimalarial activity for 2 and 3 showed that these compounds are good candidates for in vivo testing. Additionally, low tox- icities to human liver carcinoma cell line HepG2 and high metabolic stability in mouse and human liver microsomes of antimalarial 2 further supported antimal- arial efficacy studies in a mouse model.23 The bromo derivatives 3 and 8 were examined for antimalarial efficacy. A modified Thompson test model of malaria was used to determine the blood schizonticidal efficacy of the test compounds. Mice were infected with P. berghei-infected red blood cells. The test compounds were administered orally at doses of 80, 160 and 320 (mg/kg)/day on days 3, 4, and 5 after parasite inoculation; infection was confirmed by positive blood smear results for all the mice on study day 3 (prior to drug administration). At a dose of 320 (mg/kg)/day, compound 3 appeared active, since it afforded survival of 2/6 mice, with parasite clearance in one and parasite recrudescence in the other mouse. However, these results should be taken with caution since 3 experimental animals died on day 7 (D7), indicating no activity. Of the two mice that survived to D31, one had a positive blood smear and was therefore not cured, but the other one (1/6 tested) was cured based on a negative blood smear as well as absence of detection of P. berghei DNA by PCR (performed as previously described31) of blood, liver and spleen. At concentrations of 160 and 80 (mg/kg)/ /day only suppression activity was observed. No acute toxicity of compound 3 was detected, the mice that died or were euthanized prior to day 31 showed typi- cal gross lesions such as gray swollen liver, dark spleen, and pale emaciated car- cass (like the control mice group), which indicated fatal malaria infection. Anti- malarial 8 was tested at doses of 160 and 80 (mg/kg)/day. At 160 mg/kg/day it was found toxic, and at the lower concentration only suppression activity was observed, Table II. The hybrid derivatives 14–17 did not meet our expectations as they exhibited extremely poor in vitro activity, of IC50 > 1000 nM, against the CQR strain. However, their BHIA (IC50 ≈ 1) indicated that they may interfere with hematin polymerization to the same extent as CQ (CQ: IC50 = 1.23; 14: IC50 = 0.95; 15: IC50 = 1.19; 16: IC50 = 0.72; 17: IC50 = 0.96). The inconsistency between in vitro activity and BHIA could indicate the inability of hybrid derivatives to accumulate in the food vacuole of the parasite. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 4-AMINOQUINOLINES AS INHIBITORS OF MALARIA 121 TABLE II. In vivo antimalarial activity; groups of four, five, or six, P. berghei (ANKA strain) infected mice were treated p.o. once per day on days 3–5 post infection with aminoquinolines suspended in 0.5 % hydroxyethylcellulose-0.1 % Tween 80. Mice alive on day 31 with no parasites as detected by PCR are considered cured Compd. Dose, (mg/kg)/day Mice dead/day died Mice alive on day 31/total Mean survival time, day 2b 160c 1/13, 1/14, 1/15, 1/17 1/5 18 80 1/12, 2/13, 1/14, 1/28 0/5 16 3 320 3/7, 1/22 2/6 18 160c 1/15, 1/16, 2/17, 1/21, 1/24 0/6 18 80 1/12, 1/13, 2/14 0/4 13 8 80 2/14, 2/15, 1/19, 1/26 0/6 17 Infected controls 0 All mice died on day 6–8 aTaken from reference22 CONCLUSIONS In this manuscript we reported our findings on the antimalarial activity of simple aminoquinoline tethered benzyl derivatives with introduced bromines in positions C3 and C4. Although the synthesized compounds showed acceptably low in vitro activities, the antimalarial 3 was found toxic in vivo at a dose of 320 (mg/kg)/day in 3/6 mice, however, 2/6 mice of the same group survived through day 31, and one of them was cured. The detected toxicity of compound 3 indi- cates that the basic structure of presented aminoquinolines has yet to be improved in the near future. SUPPLEMENTARY MATERIAL Additional data are available electronically at the pages of journal website:.https:// //www.shd-pub.org.rs/index.php/JSCS/index, or from the corresponding author on request. Acknowledgments. This research was financially supported by the Ministry of Education, Science and Technological Development of Serbia (Contract no: 451-03-68/2020-14/200168 and 451-03-68/2020-14/200288), and Serbian Academy of Sciences and Arts, project F-80. И З В О Д НОВИ ДЕРИВАТИ 4-АМИНОХИНОЛИНА КАО УМЕРЕНИ ИНХИБИТОРИ ПАРАЗИТА Plasmodium falciparum ИГОР ОПСЕНИЦА1, МИЛИЦА СЕЛАКОВИЋ2, МИКЛОШ ТОТ1, ТАТЈАНА ВЕРБИЋ1, ЈЕЛЕНА СРБЉАНОВИЋ3, ТИЈАНА ШТАЈНЕР3, ОЛГИЦА ЂУРКОВИЋ ЂАКОВИЋ3 и БОГДАН ШОЛАЈА1,4 1Универзитет у Београду-Хемијски факултет, Студентски трг 16, 11158 Београд, 2Иновациони центар Хемијског факултета, Студентски трг 12–16, 11158 Београд, 3Институт за медицинска истраживања Универзитета у Београду, Др. Суботића 4, 11129 Београд и 4Српска академија наука и уметности, Кнез Михаилова 35, 11000 Београд Синтетисани су нови деривати аминохинолина и испитана је антималаријска активност једињења на два соја P. falciparum, хлорокин осетљивом клону 3D7 и хлорокин резистентом клону Dd2. У наставку истраживања испитана је in vivo активност деривата који су у in vitro условима испољили највећу активност. Применом аминохинолина 3 у дози 320 (mg/kg)/дан преживела су два од шест мишева, при чему је један миш излечен. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 122 OPSENICA et al. Међутим, при истој дози у 3/6 тестиране животиње исказана је токсичност аминохино- лина 3, што указује да је потребно даље истраживање на побољшању структуре овог типа једињења. (Примљено 25. децембра 2020, ревидирано 17. јануара, прихваћено 18. јануара 2021) REFERENCES 1. S. Altizer, R. S. Ostfeld, P. T. J. Johnson, S. Kutz, C. D. Harvell, Science 341 (2013) 514 (https://doi.org/10.1126/science.1239401) 2. WHO_TDR (2019), http://www.who.int/tdr/diseases-topics/malaria/en/index.html (accessed December 22, 2020) 3. D. Rathore, D. Jani, R. Nagarkatti, S. Kumar, Drug Discov. Today Ther. Strategy 3 (2006) 153 (https://doi.org/10.1016/j.ddstr.2006.06.003) 4. P. Loria, S. Miller, M. Foley, L. Tilley, Biochem. J. 339 (1999) 363 (https://doi.org/10.1042/bj3390363) 5. V. Papalexis, M. A. Siomos, N. Campanale, X. guo Guo, G. Kocak, M. Foley, L. Tilley, Mol. Biochem. Parasitol. 115 (2001) 77 (https://doi.org/10.1016/S0166-6851(01)00271- 7) 6. H. Ginsburg, O. Famin, J. Zhang, M. Krugliak, Biochem. Pharmacol. 56 (1998) 1305 (https://doi.org/10.1016/s0006-2952(98)00184-1) 7. M. Delves, D. Plouffe, C. Scheurer, S. Meister, S. Wittlin, E. A. Winzeler, R. E. Sinden, D. Leroy, PLoS Med. 9 (2012) e1001169 (htps://doi.org/10.1371/journal.pmed.1001169) 8. Treatment and Prevention of Malaria: Antimalarial Drug Chemistry, Action and Use, H. M. Staines, S. Krishna (Eds.), Series: Milestones in Drug Therapy; M. J. Parnham, J. Bruinvels (Series Eds.), Springer, Basel, 2012 (https://www.springer.com/series/4991) 9. N. J. White, J. Clin. Invest. 2113 (2004) 1084 (https://doi.org/10.1172/JCI21682) 10. J. K. Baird, N. Engl. J. Med. 352 (2005) 1565 (https://doi.org/10.1056/NEJMra043207) 11. J. Wiesner, R. Ortmann, H. Jomaa, M. Schlitzer, Angew. Chem. Int. Ed. 42 (2003) 5274 (https://doi.org/10.1002/anie.200200569) 12. M. A. Phillips, J. N. Burrows, C. Manyando, R. H. Van Huijsduijnen, W. C. Van Voorhis, T. N. C. Wells, Nat. Rev. Dis. Prim. 3 (2017) 17050 (https://doi.org/10.1038/nrdp.2017.50) 13. M. Foley, L. Tilley, Pharmacol. Ther. 79 (1998) 55 (https://doi.org/10.1016/s0163- 7258(98)00012-6) 14. O. Famin, M. Krugliak, H. Ginsburg, Biochem. Pharmacol. 58 (1999) 59 (https://doi.org/10.1016/S0006-2952(99)00059-3) 15. S. A. W. I. M. Hastings, P. G. Bray, Science 298 (2002) 74 (https://doi.org/10.1126/science.1077573) 16. M. Henry, S. Alibert, C. Rogier, J. Barbe, B. Pradines, Curr. Top. Med. Chem. 8 (2008) 563 (https://doi.org/10.2174/156802608783955593) 17. V. V. Kouznetsov, A. Gómez-Barrio, Eur. J. Med. Chem. 44 (2009) 3091 (https://doi.org/10.1016/j.ejmech.2009.02.024) 18. K. Kaur, M. Jain, R. P. Reddy, R. Jain, Eur. J. Med. Chem. 45 (2010) 3245 (https://doi.org/10.1016/j.ejmech.2010.04.011) 19. J. C. Burnett, D. Opsenica, K. Sriraghavan, R. G. Panchal, G. Ruthel, A. R. Hermone, T. L. Nguyen, T. A. Kenny, D. J. Lane, C. F. McGrath, J. J. Schmidt, J. L. Vennerstrom, R. Gussio, B. A. Šolaja, S. Bavari, J. Med. Chem. 50 (2007) 2127 (https://doi.org/10.1021/jm061446e) ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 4-AMINOQUINOLINES AS INHIBITORS OF MALARIA 123 20. B. A. Šolaja, D. Opsenica, K. S. Smith, W. K. Milhous, N. Terzić, I. Opsenica, J. C. Burnett, J. Nuss, R. Gussio, S. Bavari, J. Med. Chem. 51 (2008) 4388 (https://doi.org/10.1021/jm800737y) 21. M. Videnović, D. M. Opsenica, J. C. Burnett, L. Gomba, J. E. Nuss, Z. Selaković, J. Konstantinović, M. Krstić, S. Šegan, M. Zlatović, R. J. Sciotti, S. Bavari, B. A. Šolaja, J. Med. Chem. 57 (2014) 4134 (https://doi.org/10.1021/jm500033r) 22. J. Konstantinović, M. Videnović, J. Srbljanović, O. Djurković-Djaković, K. Bogojević, R. Sciotti, B. Šolaja, Molecules 22 (2017) 343 (https://doi.org/10.3390/molecules22030343) 23. I. M. Opsenica, M. Tot, L. Gomba, J. E. Nuss, R. J. Sciotti, S. Bavari, J. C. Burnett, B. A. Šolaja, J. Med. Chem. 56 (2013) 5860 (https://doi.org/10.1021/jm4006077) 24. N. Terzić, J. Konstantinović, M. Tot, J. Burojević, O. Djurković-Djaković, J. Srbljanović, T. Štajner, T. Verbić, M. Zlatović, M. Machado, I. S. Albuquerque, M. Prudêncio, R. J. Sciotti, S. Pecic, S. D’Alessandro, D. Taramelli, B. A. Šolaja, J. Med. Chem. 59 (2016) 264 (https://doi.org/10.1021/acs.jmedchem.5b01374) 25. W. Trager, J. B. Jensen, Science 193 (1976) 673 (https://doi.org/10.1126/science.781840) 26. M. T. Makler, J. M. Ries, J. A. Williams, J. E. Bancroft, R. C. Piper, B. L. Gibbins, D. J. Hinrichs, Am. J. Trop. Med. Hyg. 48 (1993) 739 (https://doi.org/10.4269/ajtmh.1993.48.739) 27. I. Opsenica, J. C. Burnett, R. Gussio, D. Opsenica, N. Todorović, C. A. Lanteri, R. J. Sciotti, M. Gettayacamin, N. Basilico, D. Taramelli, J. E. Nuss, L. Wanner, R. G. Panchal, B. A. Šolaja, S. Bavari, J. Med. Chem. 54 (2011) 1157 (https://doi.org/10.1021/jm100938u) 28. S. Parapini, N. Basilico, E. Pasini, T. J. Egan, P. Olliaro, D. Taramelli, D. Monti, Exp. Parasitol. 96 (2000) 249 (https://doi.org/10.1006/expr.2000.4583) 29. I. M. Opsenica, T. Verbić, M. Tot, R. J. Sciotti, B. S. Pybus, O. Djurković-Djaković, K. Slavić, B. A. Šolaja, Bioorg. Med. Chem. 23 (2015) 2176 (https://doi.org/10.1016/j.bmc.2015.02.061) 30. J. Srbljanović, B. Bobić, T. Štajner, A. Uzelac, I. Opsenica, N. Terzić-Jovanović, N. Bauman, B. A. Šolaja, O. Djurković-Djaković, J. Glob. Antimicrob. Resist. 23 (2020) 20 (http://dx.doi.org/10.1016/j.jgar.2020.07.027) 31. J. Srbljanović, T. Štajner, J. Konstantinović, N. Terzić-Jovanović, A. Uzelac, B. Bobić, B. A. Šolaja, O. Djurković-Djaković, Int. J. Antimicrob. Agents 50 (2017) 461 (https://doi.org/10.1016/j.ijantimicag.2017.06.002). ________________________________________________________________________________________________________________________ (CC) 2021 SCS. 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