In Vitro and in Vivo Trypanocidal Efficacy of Synthesized Nitrofurantoin Analogs

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Article In Vitro and In Vivo Trypanocidal Efﬁcacy of Synthesized Nitrofurantoin Analogs

Linous Munsimbwe 1, Anna Seetsi 2 , Boniface Namangala 3, David D. N’Da 4, Noboru Inoue 5 and Keisuke Suganuma 6,*

1 Ministry of Fisheries and Livestock, Department of Veterinary Services, Mulungushi House, P.O. Box 50600, Ridgeway, Lusaka 15100, Zambia; [email protected] 2 Unit for Environmental Science and Management, Faculty of Natural and Agricultural Sciences, North-West University, Potchefstroom 2531, South Africa; [email protected] 3 Department of Paraclinical Studies, School of Veterinary Medicine, University of Zambia, P.O. Box 32379, Lusaka 10101, Zambia; [email protected] 4 Centre of Excellence for Pharmaceutical Sciences (PHARMACEN), North-West University, Potchefstroom 2520, South Africa; [email protected] 5 OIE Reference Laboratory for Surra, National Research Centre for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; [email protected] 6 Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan * Correspondence: [email protected]; Tel.: +81-155-49-5697

Abstract: African trypanosomes cause diseases in humans and livestock. Human African trypanosomiasis is caused by Trypanosoma brucei rhodesiense and T. b. gambiense. Animal trypanosomoses have major effects on livestock production and the economy in developing countries, with disease Citation: Munsimbwe, L.; Seetsi, A.; management depending mainly on chemotherapy. Moreover, only few drugs are available and these Namangala, B.; N’Da, D.D.; Inoue, N.; have adverse effects on patients, are costly, show poor accessibility, and parasites develop drug Suganuma, K. In Vitro and In Vivo resistance to them. Therefore, novel trypanocidal drugs are urgently needed. Here, the effects of Trypanocidal Efficacy of Synthesized synthesized nitrofurantoin analogs were evaluated against six species/strains of animal and human Nitrofurantoin Analogs. Molecules 2021, 26, 3372. https://doi.org/ trypanosomes, and the treatment efficacy of the selected compounds was assessed in vivo. Analogs 10.3390/molecules26113372 11 and 12, containing 11- and 12-carbon aliphatic chains, respectively, showed the highest trypanocidal activity (IC50 < 0.34 µM) and the lowest cytotoxicity (IC50 > 246.02 µM) in vitro. Structure-activity Academic Editors: Paula A. C. Gomes relationship analysis suggested that the trypanocidal activity and cytotoxicity were related to the and Diego Muñoz-Torrero number of carbons in the aliphatic chain and electronegativity. In vivo experiments, involving oral treatment with nitrofurantoin, showed partial efficacy, whereas the selected analogs showed no treat- Received: 26 April 2021 ment efficacy. These results indicate that nitrofurantoin analogs with high hydrophilicity are required Accepted: 25 May 2021 for in vivo assessment to determine if they are promising leads for developing trypanocidal drugs. Published: 2 June 2021 Keywords: animal trypanosomosis; human African trypanosomiasis; nitrofurantoin analog; try- Publisher’s Note: MDPI stays neutral panocidal drug with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction African trypanosomes, transmitted by tsetse fly, cause several diseases in both humans and livestock on the Sub-Saharan African continent. Human African trypanosomiasis Copyright: © 2021 by the authors. (HAT) is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. The disease has Licensee MDPI, Basel, Switzerland. devastating socio-economic impacts, such as reduced income generation, negative effects on This article is an open access article distributed under the terms and the education of children, increased healthcare costs, and long-term health consequences [1, conditions of the Creative Commons 2]. In contrast, animal African trypanosomosis, which is caused by T. congolense, T. vivax, Attribution (CC BY) license (https:// and T. brucei brucei, represents a major health concern in animal development, and its creativecommons.org/licenses/by/ deleterious impacts can be manifested in terms of reduced livestock productivity, costly 4.0/). disease control, and trade implications, as well as negative impacts on agriculture and

Molecules 2021, 26, 3372. https://doi.org/10.3390/molecules26113372 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 11 Molecules 2021,, 26,, xx FORFOR PEER REVIEW 2 of 11

ment, and its deleterious impacts can be manifested in terms of reduced livestock produc- ment, and its deleterious impacts can be manifested in terms of reduced livestock produc- Molecules 2021, 26, 3372 tivity, costly disease control, and trade implications, as well as negative impacts on2 of agri- 11 tivity, costly disease control, and trade implications, as well as negative impacts on agriculture and food security [3,4]. Surra is an animal trypanosomosis caused by T. evansi in- culture and food security [3,4]. Surra is an animal trypanosomosis caused by T. evansi infection. It is mechanically transmitted by hematophagous biting flies such as Tabanus spp., fection. It is mechanically transmitted by hematophagous biting flies such as Tabanus spp., Stomoxys spp., and species of tsetse flies [5]. Animal trypanosomoses (AT), including ani- foodStomoxys security spp., [3,4 and]. Surra species is an of animal tsetse flies trypanosomosis [5]. Animal trypanosomoses caused by T. evansi (AT),infection. including It isani- mal African trypanosomosis and surra, are economically important diseases in animals. mechanicallymal African transmittedtrypanosomosis by hematophagous and surra, are economically biting flies such important as Tabanus diseasesspp., Stomoxysin animals. They cause high mortality, low milk and meat production, poor carcass quality, reduced spp.,They and cause species high of mortality, tsetse flies low [5]. milk Animal and trypanosomoses meat production, (AT), poor including carcass animalquality, African reduced reproductive performance, and decreased draught animal power and manure production, trypanosomosisreproductive performance, and surra, are and economically decreased draught important animal diseases power in and animals. manure They production, cause as well as immunosuppression in livestock [3,5]. highas well mortality, as immunosuppression low milk and meat in production, livestock [3,5]. poor carcass quality, reduced reproductive HAT/AT control requires an integrated approach involving the use of trypanocidal performance,HAT/AT and control decreased requires draught an integrated animal power approach and involving manure production, the use of trypanocidal as well as drugs [3,6], reduction in the number of vectors, and low contact between hosts and vectors immunosuppressiondrugs [3,6], reduction in livestockin the number [3,5]. of vectors, and low contact between hosts and vectors [7]. Owing to the challenges in vector control programs and vaccine development, [7].HAT/AT Owing to control the challenges requires an in integrated vector control approach programs involving and the vaccine use of development, trypanoci- HAT/AT control primarily depends on chemotherapy using anti-parasitic agents. Cur- dalHAT/AT drugs [ 3control,6], reduction primarily in the depends number on of chemotherapy vectors, and lowusing contact anti-parasitic between agents. hosts and Cur- rently, the nifurtimox–eflornithine combination therapy (NECT) and fexinidazole are ap- vectorsrently, [ 7the]. Owingnifurtimox to the–eflornithine challenges combination in vector control therapy programs (NECT) and fexinidazole vaccine develop- are approved for treating HAT caused by T. b. gambiense [8]. Particularly, fexinidazole was the ment,proved HAT/AT for treating control HAT primarily caused depends by T. b. gambiense on chemotherapy [8]. Particularly, using anti-parasitic fexinidazole agents. was the first oral drug approved for HAT treatment, which shows satisfactory treatment efficacy Currently,first oral thedrug nifurtimox–eflornithine approved for HAT treatment, combination which therapy shows (NECT) satisfactory and fexinidazoletreatment efficacy are against second-stage HAT, as compared to the nifurtimox–eflornithine combination ther- approvedagainst second for treating-stageHAT HAT caused, as compared by T. b. to gambiense the nifurtimox[8]. Particularly,–eflornithine fexinidazole combination was therapy [9]. In contrast, no orally administered and safe drugs are available against HAT theapy first [9] oral. In drug contrast, approved no orally for HAT administered treatment, and which safe shows drugs satisfactory are available treatment against effi- HAT caused by T. b. rhodesiense and AT. Therefore, the screening and development of safe and cacycaused against by T. second-stage b. rhodesiense HAT, and asAT. compared Therefore, to the screening nifurtimox–eflornithine and development combination of safe and effective compounds for treating HAT/AT are urgently required. therapyeffective [9]. compounds In contrast, for no treating orally administered HAT/AT are and urgently safe drugs required. are available against HAT Nitrofurantoin is a hypoxic agent with activity against a myriad of anaerobic patho- causedNitrofurantoin by T. b. rhodesiense is a hypoxicand AT. Therefore,agent with theactivity screening against and a myriad development of anaerobic of safe patho- and gens. This nitrofuran compound contains a Schiff base derived from 5-nitrofuraldehyde, effectivegens. This compounds nitrofuran for compound treating HAT/AT contains are a Schiff urgently base required. derived from 5-nitrofuraldehyde, which is known to be effective against a wide spectrum of gram-positive and gram-nega- whichNitrofurantoin is known to is be a hypoxic effective agent against with a activitywide spectrum against aof myriad gram- ofpositive anaerobic and pathogens. gram-negative bacteria and various pathogens, including trypanosomes [10,11]. Nitrofurantoin ana- Thistive nitrofuran bacteria and compound various contains pathogens, a Schiff including base derived trypanosomes from 5-nitrofuraldehyde, [10,11]. Nitrofurantoin which ana- is logs show good safety profiles, enhanced anti-mycobacterial potency, improved lipo- knownlogs showto beeffective good safety against profiles, a wide enhancedspectrum of anti gram-positive-mycobacterial and potency, gram-negative improved bacteria lipo- andphilicity, various and pathogens, a reduced including protein-binding trypanosomes affinity. [ 10Nitrofurans,11]. Nitrofurantoin are broad-spectrum analogs show redox- philicity, and a reduced protein-binding affinity. Nitrofurans are broad-spectrum redox- goodactive safety antibiotics, profiles, with enhanced dose-dependent anti-mycobacterial bacteriostatic potency, or bactericidal improved lipophilicity, activity [10,12], and and a active antibiotics, with dose-dependent bacteriostatic or bactericidal activity [10,12], and reducedhave been protein-binding used in animal affinity. feeds, Nitrofurans pharmaceuticals, are broad-spectrum and other applications redox-active [11,13]. antibiotics, have been used in animal feeds, pharmaceuticals, and other applications [11,13]. with dose-dependentThe clinical drug bacteriostatic furazolidone or belongs bactericidal to the activity group [of10 ,nitrofuran12], and have antibiotics been used and in has The clinical drug furazolidone belongs to the group of nitrofuran antibiotics and has animalbeen feeds,widely pharmaceuticals, used as an antibacterial and other and applications antiprotozoal [11,13 feed]. additive for poultry, cattle, been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, andThe farmed clinical fish drug [11,14]. furazolidone Nifurtimox, belongs another to thenitrofuran group of derivative, nitrofuran which antibiotics was developed and has and farmed fish [11,14]. Nifurtimox, another nitrofuran derivative, which was developed beenin the widely 1960s used, has as been an antibacterial used to treat and Chagas antiprotozoal disease feedcaused additive by T. cruzi for poultry,. More cattle,recently, and the in the 1960s, has been used to treat Chagas disease caused by T. cruzi. More recently, the farmedcombination fish [11 ,of14 nifurtimox]. Nifurtimox, and another eflornithine nitrofuran was evaluated derivative, for which the treatment was developed of late-stage in combination of nifurtimox and eflornithine was evaluated for the treatment of late-stage theT. 1960s, b. gambiense has been sleeping used to sickness treat Chagas [15–17 disease]. Furthermore, caused by T.a study cruzi. Morerevealed recently, that chemically the com- T. b. gambiense sleeping sickness [15–17]. Furthermore, a study revealed that chemically binationsynthesized of nifurtimox analogs of and nitrofurantoin eflornithine washad evaluatedsignificantly for enhanced the treatment antimycobacterial of late-stage T. activ- b. synthesized analogs of nitrofurantoin had significantly enhanced antimycobacterial activ- gambiense sleeping sickness [15–17]. Furthermore, a study revealed that chemically synthe- ity [11]. Overall, these reports suggest that some nitrofurantoin analogs can be used sizedity analogs[11]. Overall, of nitrofurantoin these reports had suggest significantly that some enhanced nitrofurantoin antimycobacterial analogs activity can be [ 11 used]. against African trypanosomes. Therefore, the aim of the study was to evaluate the in vitro Overall,against these African reports trypanosomes. suggest that Therefore, some nitrofurantoin the aim of analogsthe study can was be to used evaluate against the African in vitro trypanocidal activity of these chemically synthesized nitrofurantoin analogs (Table 1) and trypanosomes.trypanocidal activity Therefore, of these the chemically aim of the synthesized study was to nitrofurantoin evaluate the analogsin vitro (Tabletrypanoci- 1) and in vivo treatment efficacy of three promising analogs against human and animal trypano- dalin activity vivo treatment of these efficacy chemically of three synthesized promising nitrofurantoin analogs against analogs human (Table and1 animal) and in trypano- vivo somes. treatmentsomes. efficacy of three promising analogs against human and animal trypanosomes. Table 1. Structures of synthesized nitrofurantoin analogues 1–19. TableTable 1. Structures1. Structures of synthesizedof synthesized nitrofurantoin nitrofurantoin analogues analogues1– 191–.19. MW Cpd. MW Name Structure Cpd. (g/mol) MW (g/mol) Name Name Structure Structure (g/mol) (E)-3-Methyl(E)-3-Methyl-1-([(5-nitrofuran-2-yl)-1-([(5-nitrofuran-2-yl) 11 252.18252.18 (E)-3-Methyl-1-([(5-nitrofuran-2-yl) 1 252.18 methylene]amino)imidazolidinemethylene]amino)imidazolidine-2,4-dione-2,4-dione methylene]amino)imidazolidine-2,4-dione

(E)-3-Ethyl(E)-3-Ethyl-1-([(5-nitrofuran-2-yl)-1-([(5-nitrofuran-2-yl) 22 266.21266.21 (E)-3-Ethyl-1-([(5-nitrofuran-2-yl) 2 266.21 methylene]amino)imidazolidinemethylene]amino)imidazolidine-2,4-dione-2,4-dione methylene]amino)imidazolidine-2,4-dione

(E)-1-([(5-Nitrofuran-2-yl) 3 280.24 (E)-1-([(5(E)-1-([(5-Nitrofuran-2-yl)-Nitrofuran-2-yl) 3 280.24280.24 methylene]amino)-3-propylimidazolidine-2,4-dione methylene]amino)methylene]amino)-3-propylimidazolidine-2,4-dione-3-propylimidazolidine-2,4-dione

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Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW Table 1. Cont. 3 of 11 Molecules 2021,, 26,, xx FORFOR PEER REVIEW 3 of 11 Molecules Molecules 20212021,, 2626,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 1111 Molecules 20212021,, 2626,, xx FORFOR PEER REVIEW 33 ofof 1111 Molecules Cpd.Molecules 2021 2021 MW, ,26 26 (g/mol), ,x x FOR FOR PEER PEER REVIEW REVIEW Name Structure 33 ofof 1111 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 Molecules 2021, 26, x FOR PEER REVIEW 3 of 11 (E)-3-Butyl(E)-3-Butyl-1-([(5-nitrofuran-2-yl)-1-([(5-nitrofuran-2-yl) 4 294.26294.26 (E)-3-Butyl-1-([(5-nitrofuran-2-yl) 4 294.26 methylene]amino)imidazolidine(E)methylene]amino)imidazolidine-2,4-dione-3-Butyl-1-([(5-nitrofuran-22,4-yl)-dione 4 294.26 methylene]amino)imidazolidine(E)-3-Butyl-1-([(5-nitrofuran--2,42-yl)-dione 44 294.26294.26 methylene]amino)imidazolidine(E(E)-)3-3-Butyl-Butyl-1-1-([(5-([(5-nitrofuran-nitrofuran--2,42-2-yl)--yl)dione 44 294.26294.26 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E)-3-Butyl-1-([(5-nitrofuran--2,42,42-yl)--dionedione 4 294.26 methylene]amino)imidazolidine(E)-3-Butyl-1-([(5-nitrofuran---2,42-yl)--dione 4 294.26 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E()E-1)-([(53-Butyl-Nitrofuran-1-([(5-nitrofuran-2-yl)methylene]--2,4-22,4-yl)-dione-dione 45 294.2630,829 methylene]amino)imidazolidine(E)-1-([(5(E)-1-([(5-Nitrofuran-2-yl)methylene]-Nitrofuran-2-yl)methylene]-2,4-dione 5 30,82930,829 methylene]amino)imidazolidineamino)(E)-1-([(5-3-pentylimidazolidine-Nitrofuran-2-yl)methylene]-2,4-2,4-dione-dione 5 30,829 methylene]amino)imidazolidineamino)(E)-1-amino)-3-pentylimidazolidine-2,4-dione([(5-3-pentylimidazolidine-Nitrofuran-2-yl)methylene]-2,4-2,4-dione-dione 55 30,82930,829 amino)(E(E)-)1-1--([(5-3([(5-pentylimidazolidine-Nitrofuran-Nitrofuran-2-2-yl)methylene]-yl)methylene]-2,4-dione 55 30,82930,829 amino)amino)(E)-1--([(5-33--pentylimidazolidinepentylimidazolidine-Nitrofuran-2-yl)methylene]--2,42,4--dionedione 5 30,829 amino)(E)-1--([(5-3--pentylimidazolidine-Nitrofuran-2-yl)methylene]--2,4--dione 5 30,829 amino)amino)(E()E-1)--3([(5-3-3Hexyl-pentylimidazolidine--pentylimidazolidineNitrofuran-1-([(5-nitrofuran-2-yl)methylene]-2,4-2,4-2-dioneyl)-dione 56 30,829322.32 amino)(E)-3--3Hexyl-pentylimidazolidine(E)-3-Hexyl-1-([(5-nitrofuran-2-yl)-1-([(5-nitrofuran-2,4-2--yl)dione 6 322.32322.32 methylene]amino)imidazolidineamino)(E)-3--3Hexyl-pentylimidazolidine-1-([(5-nitrofuran-2,4--2,42--yl)dione-dione 6 322.32 methylene]amino)imidazolidineamino)(E)methylene]amino)imidazolidine-2,4-dione-3-3-Hexyl-pentylimidazolidine-1-([(5-nitrofuran-2,4--2,42-yl)dione-dione 66 322.32322.32 methylene]amino)imidazolidine(E(E)-)3-3-Hexyl-Hexyl-1-1-([(5-([(5-nitrofuran-nitrofuran--2,42-2-yl)--yl)dione 66 322.32322.32 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E)-3-Hexyl-1-([(5-nitrofuran---2,42,42-yl)--dionedione 6 322.32 methylene]amino)imidazolidine(E)-3-Hexyl-1-([(5-nitrofuran---2,42-yl)--dione 6 322.32 methylene]amino)imidazolidinemethylene]amino)imidazolidine((EE))--33--HeptylHexyl--11--([(5([(5--nitrofurannitrofuran--2,4--22,42--yl)-yl)dione-dione 67 322.32336.34 methylene]amino)imidazolidine(E)-3-Heptyl-1-([(5-nitrofuran-2,4-2-yl)-dione 7 336.34 methylene]amino)imidazolidine(E)-3-Heptyl(E)-3-Heptyl-1-([(5-nitrofuran-2-yl)-1-([(5-nitrofuran--2,42-yl)-dione 7 336.34336.34 methylene]amino)imidazolidine(E)-3-Heptyl-1-([(5-nitrofuran-2,4-2--yl)dione 77 336.34336.34 methylene]amino)imidazolidine(E(E)-)methylene]amino)imidazolidine-2,4-dione3-3-Heptyl-Heptyl-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-2-2--yl)-dioneyl) 77 336.34336.34 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E)-3-Heptyl-1-([(5-nitrofuran--2,42,4-2--yl)-dionedione 7 336.34 methylene]amino)imidazolidine(E)-3-Heptyl-1-([(5-nitrofuran--2,4-2--yl)-dione 7 336.34 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E(E)-)1-3-([(5-Heptyl-Nitrofuran-1-([(5-nitrofuran-2-yl)methylene]-2,4--2,42--yl)dione-dione 78 336.34350.37 methylene]amino)imidazolidine(E)-1-([(5-Nitrofuran-2-yl)methylene]-2,4-dione 8 350.37 methylene]amino)imidazolidineamino)(E)-1-([(5-3--octylimidazolidineNitrofuran-2-yl)methylene]-2,4-2,4-dione-dione 8 350.37 methylene]amino)imidazolidineamino)(E)-1-([(5-(3E-)-1-([(5-Nitrofuran-2-yl)methylene]-octylimidazolidineNitrofuran-2-yl)methylene]-2,4-2,4-dione-dione 88 350.37350.37350.37 amino)(E(E)-)1-1-([(5--([(53-octylimidazolidine-Nitrofuran-Nitrofuran-2-2-yl)methylene]-yl)methylene]-2,4-dione 88 350.37350.37 amino)amino)(E)-1-([(5amino)-3-octylimidazolidine-2,4-dione--33--octylimidazolidine-octylimidazolidineNitrofuran-2-yl)methylene]--2,42,4--dionedione 8 350.37 amino)(E)-1-([(5--3--octylimidazolidine-Nitrofuran-2-yl)methylene]--2,4--dione 8 350.37 amino)(amino)E)-1-([(5-3-3--octylimidazolidineNitrofuranoctylimidazolidine-2-yl)methylene]-2,4-2,4-dione-dione 89 350.37364.4 amino)(E)-1-([(5-3--octylimidazolidineNitrofuran-2-yl)methylene]-2,4-dione 9 364.4 amino)amino)(E)-1-([(5--33--nonylimidazolidine-octylimidazolidineNitrofuran-2-yl)methylene]--2,42,4--dionedione 9 364.4 amino)amino)(E)-1-([(5--3(3E--nonylimidazolidine)-1-([(5-Nitrofuran-2-yl)methylene]-octylimidazolidineNitrofuran-2-yl)methylene]--2,42,4--dionedione 99 364.4364.4364.4 amino)(E(E)-)1-1-([(5--([(53-nonylimidazolidine-Nitrofuran-Nitrofuran-2-2-yl)methylene]-yl)methylene]-2,4-dione 99 364.4364.4 amino)amino)(E)-1-([(5amino)-3-nonylimidazolidine-2,4-dione--33--nonylimidazolidinenonylimidazolidine-Nitrofuran-2-yl)methylene]--2,42,4--dionedione 9 364.4 amino)(E)-1-([(5--3--nonylimidazolidine-Nitrofuran-2-yl)methylene]--2,4--dione 9 364.4 (E)amino)-amino)3(E-Decyl)-1-([(5-3--3-1nonylimidazolidine---nonylimidazolidineNitrofuran([(5-nitrofuran-2-yl)methylene]-2-yl)methylene]-2,4-2,4-dione-dione 109 378.42364.4 (E)amino)-3-Decyl-3--1nonylimidazolidine-([(5-nitrofuran-2-yl)methylene]-2,4-dione 10 378.42 (E)-amino)3-Decylamino)imidazolidine-3--1nonylimidazolidine-([(5-nitrofuran-2,42-yl)methylene]--dione2,4-dione 10 378.42 (E)amino)-3-Decylamino)imidazolidine-3--1nonylimidazolidine-([(5-nitrofuran--2,42-yl)methylene]--dione2,4-dione 1010 378.42378.42 (E(E)-)3-3-Decyl-Decylamino)imidazolidine(E)-3-Decyl-1-([(5-nitrofuran-2-yl)methylene]-1-1-([(5-([(5-nitrofuran-nitrofuran--2,42-2-yl)methylene]--yl)methylene]dione 1010 378.42378.42378.42 (E)-3-Decylamino)imidazolidineamino)imidazolidine-1-([(5-nitrofuran---2,42,42-yl)methylene]--dionedione 10 378.42 (E)-3-Decylamino)imidazolidine-1-amino)imidazolidine-2,4-dione([(5-nitrofuran---2,42-yl)methylene]--dione 10 378.42 (E)-3(E-Decyl)-amino)imidazolidine1amino)imidazolidine-([(5-1--Nitrofuran([(5-nitrofuran-2-yl)methylene]--2,4-22,4-yl)methylene]-dione-dione 1011 378.42392.45 (E)-amino)imidazolidine1-([(5-Nitrofuran-2-yl)methylene]-2,4-dione 11 392.45 amino)(E)-amino)imidazolidine1--([(53-undecylimidazolidine-Nitrofuran-2-yl)methylene]-2,4-dione-2,4-dione 11 392.45 amino)(E)-amino)imidazolidine1-3([(5-undecylimidazolidine-Nitrofuran-2-yl)methylene]-2,4-dione-2,4-dione 1111 392.45392.45 amino)(E(E)-)1-1-3([(5-([(5-(undecylimidazolidineE)-1-([(5-Nitrofuran-2-yl)methylene]-Nitrofuran-Nitrofuran-2-2-yl)methylene]-yl)methylene]-2,4-dione 1111 392.45392.45392.45 amino)amino)(E)-1--3([(53--undecylimidazolidineundecylimidazolidine-Nitrofuran-2-yl)methylene]--2,42,4--dionedione 11 392.45 amino)(E)-1-amino)-3-undecylimidazolidine-2,4-dione-3([(5--undecylimidazolidine-Nitrofuran-2-yl)methylene]--2,4--dione 11 392.45 amino)amino)((EE))--13--3([(5-Dodecyl3-undecylimidazolidine-undecylimidazolidine-Nitrofuran-1-([(5-nitrofuran2-yl)methylene]-2,4-2,4-2--dione-yl)dione 1112 392.45406.48 amino)(E)-3--3Dodecyl-undecylimidazolidine-1-([(5-nitrofuran-2,4-2--dioneyl) 12 406.48 methylene]amino)imidazolidineamino)(E)-3--3Dodecyl-undecylimidazolidine-1-([(5-nitrofuran--2,42,4-2--dioneyl)dione 12 406.48 methylene]amino)imidazolidineamino)(E)-3--3Dodecyl-undecylimidazolidine-1-([(5-nitrofuran--2,42,4-2--dionedioneyl) 1212 406.48406.48 methylene]amino)imidazolidine(E(E)-)3-3-Dodecyl-Dodecyl(E)-3-Dodecyl-1-([(5-nitrofuran-2-yl)-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-2-2-dioneyl)-yl) 1212 406.48406.48406.48 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E)-3-Dodecyl-1-([(5-nitrofuran--2,42,4-2--dioneyl)dione 12 406.48 methylene]amino)imidazolidine(E)-3methylene]amino)imidazolidine-2,4-dione-Dodecyl-1-([(5-nitrofuran--2,4-2--dioneyl) 12 406.48 methylene]amino)imidazolidinemethylene]amino)imidazolidine((EE))--13--([(5Dodecyl-Nitrofuran-1-([(5--nitrofuran2-yl)methylene]-2,4-2,4-2-dioneyl)-dione 1213 406.48356.33 methylene]amino)imidazolidine(E)-1-([(5-Nitrofuran-2-yl)methylene]-2,4-dione 13 356.33 amino)methylene]amino)imidazolidine(-E3)--(31--([(5phenylpropyl)imidazolidine-Nitrofuran-2-yl)methylene]-2,4--dione2,4 -dione 13 356.33 amino)methylene]amino)imidazolidine(-E3-)(3-1-phenylpropyl)imidazolidine([(5-Nitrofuran-2-yl)methylene]-2,4--dione2,4 -dione 1313 356.33356.33 amino)(-E3(E-)(3-)1-1-phenylpropyl)imidazolidine([(5-([(5-Nitrofuran-Nitrofuran-2-2-yl)methylene]-yl)methylene]-2,4 - dione 1313 356.33356.33 amino)amino)(--E33-)-(3-(31--phenylpropyl)imidazolidine([(5phenylpropyl)imidazolidine(E)-1-([(5-Nitrofuran-2-yl)methylene]-Nitrofuran-2-yl)methylene]--2,42,4 --dionedione 13 356.33356.33 amino)(--E3-)-(3-(31--phenylpropyl)imidazolidine([(5-Nitrofuran-2-yl)methylene]--2,4 --dione 13 356.33 amino)amino)(-amino)-3-(3-phenylpropyl)imidazolidine-2,4-dioneE3-(3E-)(3-)1(3--3phenylpropyl)imidazolidine([(5--phenylpropyl)imidazolidineBenzyl-Nitrofuran-1-([(5-nitrofuran-2-yl)methylene]-2-yl)-2,4-2,4 -dione-dione 1314 356.33328.28 amino)-3(E-(3)--3phenylpropyl)imidazolidine-Benzyl-1-([(5-nitrofuran-2-yl)-2,4 -dione 14 328.28 amino)methylene]amino)imidazolidine-(3E-(3)-3-phenylpropyl)imidazolidine-Benzyl-1-([(5-nitrofuran--2,42-yl)--dione2,4 -dione 14 328.28 amino)methylene]amino)imidazolidine-3(E-(3)--3phenylpropyl)imidazolidine-Benzyl-1-([(5-nitrofuran-2,4-2-yl)--dione2,4 -dione 1414 328.28328.28 methylene]amino)imidazolidine(E(E)-)3-3-Benzyl-Benzyl-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-2-2--yl)-dioneyl) 1414 328.28328.28 methylene]amino)imidazolidinemethylene]amino)imidazolidine(E)-3-Benzyl(E)-3-Benzyl-1-([(5-nitrofuran-2-yl)-1-([(5-nitrofuran--2,4-2,42--yl)-dionedione 14 328.28328.28 methylene]amino)imidazolidine(E)-3-Benzyl-1-([(5-nitrofuran--2,4-2--yl)-dione 14 328.28 (Emethylene]amino)imidazolidine)methylene]amino)imidazolidine-3-((Ep)--Methylbenzyl)3-Benzyl-1-([(5-1-nitrofuran-([(5-nitrofuran-2,4-2,42-yl)-dione-dione- 2-yl) 1415 328.28342.31 (Emethylene]amino)imidazolidine)-3-(p-Methylbenzyl)methylene]amino)imidazolidine-2,4-dione-1-([(5-nitrofuran-2,4-dione-2-yl) 15 342.31 (Emethylene]amino)imidazolidine)-3-(p-Methylbenzyl)-1-([(5-nitrofuran-2,4-dione-2-yl) 15 342.31 (Emethylene]amino)imidazolidine)-3-(p-Methylbenzyl)-1-([(5-nitrofuran-2,4-dione-2-yl) 1515 342.31342.31 (E(methylene]amino)imidazolidineE)-)3-3-(-p(p-Methylbenzyl)-Methylbenzyl)-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-dione-2-2-yl)-yl) 1515 342.31342.31 (Emethylene]amino)imidazolidinemethylene]amino)imidazolidine)-3-(p-Methylbenzyl)-1-([(5-nitrofuran--2,42,4--dionedione-2-yl) 15 342.31 (Emethylene]amino)imidazolidine)-3(-E()p-(-1EMethylbenzyl)-)-3-(([(5p-Nitrofuran-Methylbenzyl)-1-([(5-nitrofuran-2-yl)-1--2([(5-yl)methylene]-nitrofuran--2,4--dione-2 -yl) 15 342.31342.31 (Emethylene]amino)imidazolidine)methylene]amino)imidazolidine-3(E-()p--1Methylbenzyl)-([(5-Nitrofuran-1--2([(5-yl)methylene]-nitrofuran-2,4-2,4-dione-dione-2 -yl) 1516 342.31396.28 amino)methylene]amino)imidazolidine-3-((Ep)--(trifluoromethyl)benzyl)1methylene]amino)imidazolidine-2,4-dione-([(5-Nitrofuran-2-yl)methylene] imidazolidine-2,4-dione -2,4- 16 396.28 amino)methylene]amino)imidazolidine-3-((Ep)--(trifluoromethyl)benzyl)1-([(5-Nitrofuran-2-yl)methylene] imidazolidine-2,4-dione -2,4- 16 396.28 amino)methylene]amino)imidazolidine-3-((E(pE)--(trifluoromethyl)benzyl))1-1-([(5-([(5-Nitrofuran-Nitrofuran-2-2-yl)methylene]-yl)methylene] imidazolidineimidazolidine-2,4-dione -2,4- 1616 396.28396.28 amino)amino)--33--(((Epp)---(trifluoromethyl)benzyl)(trifluoromethyl)benzyl)1-([(5-Nitrofurandione-2 -yl)methylene] imidazolidineimidazolidine --2,42,4-- 16 396.28 amino)--3--(((Ep)---(trifluoromethyl)benzyl)(trifluoromethyl)benzyl)1-([(5(E)-1-([(5-Nitrofuran-2-yl)methylene]-Nitrofurandione-2 -yl)methylene] imidazolidineimidazolidine --2,4-- 1616 396.28396.28 amino)amino)-3-3-((-Ep(p)--(trifluoromethyl)benzyl)-1(trifluoromethyl)benzyl)-([(5-Nitrofurandione- 2-yl)methylene] imidazolidine imidazolidine -2,4-2,4-- 16 396.28396.28 amino)(E-3)-(3p--((trifluoromethyl)benzyl)p-Nitrobenzyl)amino)-3-(dionep-(triﬂuoromethyl)benzyl)-1-([(5 -nitrofuran imidazolidine-2-yl) -2,4- 16 396.28 amino)(E-3)-(3p--((trifluoromethyl)benzyl)p-Nitrobenzyl)dionedione-1-([(5 -nitrofuran imidazolidine-2-yl) -2,4- 1617 396.28373.28 amino)(E-3)--3(p-(-p(trifluoromethyl)benzyl)-Nitrobenzyl)dione-1-([(5 -nitrofuran imidazolidine-2-yl) -2,4- 17 373.28 (methylene]amino)imidazolidineE)-3-(p-Nitrobenzyl)imidazolidine-2,4-dionedione-1-([(5 -nitrofuran-2,4-dione-2-yl) 17 373.28 ((methylene]amino)imidazolidineE))--3--((p--Nitrobenzyl)dione--1--([(5([(5 --nitrofuran-2,4-dione--2--yl) 17 373.28 (methylene]amino)imidazolidineE(E)-)3-3-(-p(p-Nitrobenzyl)-Nitrobenzyl)-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-dione-2-2-yl)-yl) 1717 373.28373.28 (methylene]amino)imidazolidinemethylene]amino)imidazolidineE)-3-(p-Nitrobenzyl)-1-([(5-nitrofuran--2,42,4--dionedione-2-yl) 17 373.28 (E()methylene]amino)imidazolidineE-methylene]amino)imidazolidine3)--3(p-(-pMethoxybenzyl)(-ENitrobenzyl))-3-(p-Nitrobenzyl)-1-([(5-nitrofuran-2-yl)-1-1([(5-([(5-nitrofuran-nitrofuran--2,4-2,4--dione-dione-2--2yl)-yl) 17 373.28373.28 (E()methylene]amino)imidazolidineE-3)--3(p-(-pMethoxybenzyl)-Nitrobenzyl)-1-1([(5-([(5-nitrofuran-nitrofuran-2,4-dione-2-2yl)-yl) 1718 373.28358.31 (E)methylene]amino)imidazolidine-3-(p-Methoxybenzyl)methylene]amino)imidazolidine-2,4-dione-1-([(5-nitrofuran-2,4-dione-2-yl) 18 358.31 ((EE))methylene]amino)imidazolidine--33--((pp--Methoxybenzyl)Methoxybenzyl)--11--([(5([(5--nitrofurannitrofuran-2,4-dione--22--yl)yl) 18 358.31 ((E)methylene]amino)imidazolidine)--3--((p--Methoxybenzyl)--1--([(5([(5--nitrofuran-2,4-dione--2--yl) 18 358.31 (E(E)methylene]amino)imidazolidine-)3-3-(-p(p-Methoxybenzyl)-Methoxybenzyl)-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-dione-2-2-yl)- yl) 1818 358.31358.31 (E)methylene]amino)imidazolidine-3-(p-Methoxybenzyl)-1-([(5-nitrofuran-2,4-dione-2-yl) 18 358.31 (E(E)methylene]amino)imidazolidine-methylene]amino)imidazolidine)3--3(-p((-pEMethoxybenzyl)-)-3-(Bromobenzyl)p-Methoxybenzyl)-1-([(5-nitrofuran-2-yl)-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-2,4-dione-dione-2-2-yl)-yl) 1819 358.31407.18358.31 (Emethylene]amino)imidazolidine)-3-(p-Bromobenzyl)-1-([(5-nitrofuran-2,4-dione-2-yl) 1819 358.31407.18 (Emethylene]amino)imidazolidine)-3-(p-Bromobenzyl)methylene]amino)imidazolidine-2,4-dione-1-([(5-nitrofuran-2,4-dione-2-yl) 19 407.18 ((EEmethylene]amino)imidazolidine))--33--((pp--Bromobenzyl)Bromobenzyl)--11--([(5([(5--nitrofurannitrofuran-2,4-dione--22--yl)yl) 1919 407.18407.18 ((Emethylene]amino)imidazolidine))--3--((p--Bromobenzyl)--1--([(5([(5--nitrofuran-2,4-dione--2--yl) 19 407.18 (E(methylene]amino)imidazolidineE)-)3-3-(-p(p-Bromobenzyl)-Bromobenzyl)-1-1-([(5-([(5-nitrofuran-nitrofuran-2,4-dione-2-2-yl)-yl) 1919 407.18407.18 (Emethylene]amino)imidazolidine)-3-(p-Bromobenzyl)-1-([(5-nitrofuran--2,4--dione-2-yl) 19 407.18 (Emethylene]amino)imidazolidinemethylene]amino)imidazolidine)-3-(p-Bromobenzyl)-1-([(5-nitrofuran-2,4-2,4-dione-dione-2-yl) 19 407.18 methylene]amino)imidazolidine2.(E Results)-3-(p-Bromobenzyl)-1-([(5-nitrofuran-2-yl) -2,4-dione 19 407.18407.18 methylene]amino)imidazolidine2. Results -2,4-dione methylene]amino)imidazolidine2. methylene]amino)imidazolidine-2,4-dioneResults -2,4-dione 2. Results 2.2.1 Results. In Vitro Experiment 2.12.2. Results ..Results In Vitro Experiment 2.12. Results. InA Vitro summary Experiment of the trypanocidal activity and cytotoxicity data of the nitrofurantoin 2.12. Results.. InInA Vitro summary Experiment of the trypanocidal activity and cytotoxicity data of the nitrofurantoin 2.12.2.1 Results. .In AIn Vitro summaryVitro Experiment Experiment of the trypanocidal activity and cytotoxicity data of the nitrofurantoin 2.1analogs. InAA Vitro summarysummary is shown Experiment ofofin theTablethe trypanocidaltrypanocidal 2. activityactivity andand cytotoxicitycytotoxicity datadata ofof thethe nitrofurantoinnitrofurantoin analogs2.1. InA Vitrosummary is shown Experiment ofin Tablethe trypanocidal 2. activity and cytotoxicity data of the nitrofurantoin analogsAA summary issummary shown inof of Tablethe the trypanocidal trypanocidal 2. activity activity and and cytotoxicity cytotoxicity data data of of the the nitrofurantoin nitrofurantoin analogsA summary is shown inof Tablethe trypanocidal 2.. activity and cytotoxicity data of the nitrofurantoin analogsanalogsA summary is is shown shown ofin in Tablethe Table trypanocidal 2 2. . activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2. analogs is shown in Table 2.

Molecules 2021, 26, 3372 4 of 11

2. Results 2.1. In Vitro Experiment A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table2. Trypanocidal activities differed among the trypanosome species. However, T. congolense IL3000 and T. b. rhodesiense Chirundu were more sensitive to these nitrofurantoin analogs than the other trypanosome strains/species. The strongest trypanocidal activity (IC50 < 0.031 µM) against T. congolense IL3000 and T. b. rhodesiense Chirundu was observed with the long chain analogs 9–12, bearing side chains of 9–11 carbons, respectively (n = 9–12), in sub-series 1. These analogs showed relatively moderate (0.16–0.65 µM) activity against T. evansi Tansui, T. b. brucei GUTat3.1, T. b. gambiense IL1922, and T. b. rhodesiense IL1501. Furthermore, variation in the trypanocidal activity for analogs in sub-series 2 was found based on the electronegativity of para-substituents on the phenyl ring. The trypanocidal activity of analogs bearing electron-withdrawing groups (16, 17, and 19) was stronger than that of analog 14, featuring a neutral group and those of analogs 15 and 18 possessing electron-donating groups. In contrast, electronegativity had no effect on cytotoxicity within sub-series 2.

2.2. In Vivo Experiment The in vivo trypanocidal activity of three selected nitrofurantoin analogs (9, 11, and 12) showed strong trypanocidal activity and were used to conﬁrm the results of in vitro assay and nitrofurantoin. These three analogs were evaluated. All mice treated with nitrofurantoin and its analogs by intraperitoneal injection at 0.1 mg/kg, died within 9 dpi, because of high parasitemia (Figure1a). All mice orally treated with the three analogs at 10 mg/kg also succumbed to high parasite levels within 9 dpi. In contrast, two of three mice orally treated at 10 mg/kg with nitrofurantoin, survived until end of the experiment period (Figure1b). Molecules 2021, 26, 3372 5 of 11

Table 2. In vitro biological activities of synthesized nitrofurantoin analogs.

Cytotoxicity Trypanocidal Activity Selectivity Index IC ± SD IC ± SD (µM) c 50 (IC of Trypanosomes/IC of MDBK) d 50 (µM) c 50 50 clogP Tbb Tbr Tbb Tbg Tbr Tbr Tc Tev ID N a Tbg IL1922 Tbr IL1501 Tc IL3000 Tev Tansui MDBK b GUTat3.1 Chirundu GUTat3.1 IL1922 IL1501 Chirundu IL3000 Tansui 1 1 −0.04 3.61 ± 0.40 3.60 ± 0.26 7.02 ± 0.094 0.73 ± 0.17 0.19 ± 0.076 1.61 ± 0.098 65.21 ± 12.78 18 18 9 89 342 41 2 2 0.36 10.81 ± 4.84 11.31 ± 4.80 11.40 ± 5.24 0.51 ± 0.25 0.42 ± 0.16 5.39 ± 0.53 44.79 ± 1.69 4 4 4 87 108 8 3 3 0.88 14.13 ± 0.34 14.73 ± 1.32 14.02 ± 1.42 0.81 ± 0.24 0.33 ± 0.18 6.53 ± 0.61 39.29 ± 1.09 3 3 3 49 120 6 4 4 1.33 33.29 ± 24.29 17.25 ± 2.23 32.74 ± 0.82 1.38 ± 0.34 0.31 ± 0.20 7.89 ± 0.53 35.78 ± 3.49 1 2 1 26 117 5 5 5 1.77 4.24 ± 1.99 3.42 ± 1.47 5.69 ± 1.66 NA 0.35 ± 0.15 5.44 ± 0.17 39.07 ± 5.86 9 11 7 NA 112 7 6 6 2.22 5.76 ± 4.01 2.59 ± 0.28 2.61 ± 0.12 0.17 ± 0.11 0.08 ± 0.047 3.76 ± 0.47 25.18 ± 3.52 4 10 10 146 321 7 7 7 2.66 2.06 ± 0.75 1.99 ± 0.75 1.45 ± 0.86 0.056 ± 0.026 0.040 ± 0.011 1.02 ± 0.10 92.28 ± 6.52 45 46 64 1654 2323 90 0.039 ± 0.030 ± 8 8 3.10 1.89 ± 0.69 1.40 ± 0.73 1.38 ± 0.82 0.81 ± 0.082 84.74 ± 8.99 45 61 62 2195 2809 105 0.0084 0.0050 0.017 ± 0.010 ± 9 9 3.55 0.57 ± 0.22 0.44 ± 0.17 0.60 ± 0.54 0.27 ± 0.025 251.18 ± 58.27 443 567 420 14,645 25,639 918 0.0067 0.0035 0.017 ± 0.011 ± 10 10 3.99 0.65 ± 0.56 0.62 ± 0.70 0.61 ± 0.06 0.28 ± 0.025 110.45 ± 14.38 170 177 180 6645 10,502 402 0.0044 0.0044 0.021 ± 0.011 ± 11 11 4.44 0.33 ± 0.12 0.33 ± 0.12 0.24 ± 0.13 0.16 ± 0.014 >254.81 764 771 1052 11,933 21,978 1602 0.0094 0.0035 0.012 ± 12 12 4.88 0.34 ± 0.12 0.23 ± 0.13 0.26 ± 0.11 0.031 ± 0.011 0.20 ± 0.017 >246.02 715 1095 934 7937 21,322 1253 0.0045 13 2.46 7.29 ± 3.16 4.83 ± 3.22 5.90 ± 3.75 0.14 ± 0.067 0.14 ± 0.030 3.11 ± 0.37 >280.64 39 58 48 2,041 2058 90 14 1.73 2.88 ± 0.26 2.34 ± 0.64 2.83 ± 0.61 0.27 ± 0.12 0.18 ± 0.017 1.26 ± 0.10 >304.62 106 130 108 1142 1702 241 15 2.24 8.24 ± 3.11 2.89 ± 0.51 6.10 ± 0.047 0.15 ± 0.068 0.13 ± 0.044 4.23 ± 0.40 34.28 ± 11.5 4 12 6 231 271 8 16 2.60 0.52 ± 0.081 0.61 ± 0.26 0.96 ± 0.0087 0.02 ± 0.012 0.02 ± 0.073 0.44 ± 0.046 56.62 ± 40.76 108 93 59 3121 3802 128 17 1.67 1.07 ± 0.74 1.05 ± 0.70 0.50 ± 0.012 0.04 ± 0.094 0.04 ± 0.017 0.61 ± 0.050 20.10 ± 1.02 19 19 45 497 469 33 18 1.57 9.91 ± 0.092 9.78 ± 0.31 10.14 ± 0.62 0.33 ± 0.22 0.14 ± 0.030 2.40 ± 0.19 80.09 ± 36.89 8 8 8 244 585 33 19 2.50 2.13 ± 0.23 2.04 ± 0.091 2.47 ± 0.69 0.041 ± 0.025 0.049 ± 0.033 1.15 ± 0.11 28.74 ± 5.69 14 14 12 700 587 25 Nitrofurantoin −0.22 1.42 ± 0.15 1.62 ± 0.28 1.42 ± 0.25 NA 0.36 ± 0.043 1.96 ± 0.50 Nifurtimox 4.66 ± 19.7 4.58 ± 2.38 4.35 ± 1.59 1.46 ± 0.35 1.06 ± 0.22 2.62 ± 1.40 Eﬂornithine 38.56 ± 9.88 36.66 ± 12.87 45.99 ± 17.07 35.65 ± 10.16 16.13 ± 2.93 57.21 ± 17.56 0.041 ± 0.014 ± 0.029 ± 0.00097 ± Pentamidine 0.050 ± 0.012 0.33 ± 0.054 0.0023 0.0031 0.0062 0.00019 0.066 ± 0.064 ± Suramin 0.076 ± 0.011 0.066 ± 0.071 7.17 ± 0.87 0.38 ± 0.058 0.0052 0.0018 a number of carbon atoms in alkyl side chain. b clogP of compounds D of compounds were referred to Zuma et al. [11]. c represents the mean ± the standard deviation (SD) from triplicate biological experiments. d value as rounded off to the nearest digit. NA—not analyzed. Molecules 2021, 26, x FOR PEER REVIEW 4 of 11

Furthermore, variation in the trypanocidal activity for analogs in sub-series 2 was found based on the electronegativity of para-substituents on the phenyl ring. The trypanocidal activity of analogs bearing electron-withdrawing groups (16, 17, and 19) was stronger than that of analog 14, featuring a neutral group and those of analogs 15 and 18 possessing electron-donating groups. In contrast, electronegativity had no effect on cytotoxicity within sub-series 2.

2.2. In Vivo Experiment The in vivo trypanocidal activity of three selected nitrofurantoin analogs (9, 11, and 12) showed strong trypanocidal activity and were used to confirm the results of in vitro assay and nitrofurantoin. These three analogs were evaluated. All mice treated with nitrofurantoin and its analogs by intraperitoneal injection at 0.1 mg/kg, died within 9 dpi, Molecules 2021, 26, 3372 6 of 11 because of high parasitemia (Figure 1a). All mice orally treated with the three analogs at 10 mg/kg also succumbed to high parasite levels within 9 dpi. In contrast, two of three mice orally treated at 10 mg/kg with nitrofurantoin, survived until end of the experiment period (Figure 1b). 1010 1010

) Group I ) Group I L 109 L 109

/ Group III (9) / Group III (9)

l l

l l 108 108

e e Group IV (11) Group IV (11)

c c

(

7 7

a a Group V (12) Group V (12)

i i 10 10

e e 6 Group VI (NF) 6 Group VI (NF)

i i 10 10

r r 5 5

a a 10 10

P 104 104 0 5 10 0 5 10 Days post infection Days post infection

(a) (b)

Figure 1. In vivo analysisanalysis of selectedselected nitrofurantoin analogs and nitrofurantoin (NF).(NF). Evaluation of parasitemia in mice infected withwith T.T. congolense congolenseand and intraperitoneal intraperitoneal or or orally orally treated treated with with nitrofurantoin nitrofurantoin analogs analogs (9, 11 (,9 and, 11,12 and) and 12 nitrofurantoin.) and nitrofurantoin.The Y axis The shows Y axis logshows 10scale, log 10 and scale, the and data the are data shown are shown as mean as mean± standard ± standard deviation. deviation. Parasitemia Parasitemia was was not not observed observed in in group II (diminazene aceturate treatment). (a) Treatment of mice by intraperitoneal injection at 0.1 mg/kg. (b) Treatment group II (diminazene aceturate treatment). (a) Treatment of mice by intraperitoneal injection at 0.1 mg/kg. (b) Treatment of of mice by oral administration at 10 mg/kg. mice by oral administration at 10 mg/kg. 3. Discussion 3. DiscussionIn the present study, the trypanocidal activity of 19 nitrofurantoin analogs was eval- uatedIn against the present six human study, and the animal trypanocidal trypanosomes activity ofin 19vitro. nitrofurantoin Investigation analogs of the wasstructure evalu-– activityated against relationship six human within and the animal series trypanosomes revealed that inthe vitro trypanocidal. Investigation activity of theof short structure– alkyl analogsactivity relationship(1–4) in sub- withinseries 1 the decreased series revealed with increasing that the trypanocidal chain lengths activity up to offour short carbons alkyl (analogsn = 4, 4) ((Figure1–4) in sub-series2a). The trypanocidal 1 decreased activity with increasing in long alkyl chain chain lengths analogs up to four(5–12 carbons) increased (n = with4, 4) (Figureincreasing2a). Thechain trypanocidal lengths up activityto 12 carbons in long ( alkyln = 12, chain 12) analogs(Figure (2a).5–12 The) increased cytotoxicity with withinincreasing the analogs chain lengths of sub up-series to 12 1 carbonsmostly increased (n = 12, 12 with) (Figure increasing2a). The chain cytotoxicity lengths (Fig withinure 2a).the analogsThe strongest of sub-series trypanocidal 1 mostly activity increased of these with increasinglong alkyl chainchains lengths (9–11) (Figure in sub2-seriesa). The 1 maystrongest be related trypanocidal to their higher activity lip ofophilicity, these long which alkyl increased chains (9– the11) cLogP in sub-series value. T 1his may feature be re- islated typically to their associated higher lipophilicity, with better which permeation increased the of acLogP molecule value. through This feature biological is typically tissues/membranes,associated with better as well permeation as interactions of a moleculewith transporter through proteins biological and tissues/membranes, enzymes [18]. Sim- ilaras well results as interactionswere previously with observed transporter against proteins the T. and cruzi enzymes parasite [18 [19].,20 Similar]. Various results studies were havepreviously indicated observed a relationship against the betweenT. cruzi trypanocidalparasite [19,20 activity]. Various and studies the number have indicated of carbon a atomsrelationship in the alkyl between chain trypanocidal of the compounds activity [21 and,22 the]. number of carbon atoms in the alkyl chain of the compounds [21,22]. Additionally, we showed that the trypanocidal activity of analogs bearing electron- withdrawing groups (16, 17, and 19) was stronger than that of the compounds containing a neutral group (14) and those bearing electron-donating groups (15 and 18) in sub-series 2 (Figure2b). Electronegativity was also reported to affect the trypanocidal activity [ 23–25]. Our results are similar to the previous findings, except for the differences in the values. The trypanocidal activity of the nitrofurantoin analogs increased with increasing chain length, up to a certain number of carbon atoms and electronegativity. Our results confirm previous findings and extend the range of promising compounds that are optimized by a combination of chemical modifications with long alkyl chain and high electronegativity. This can be strategized using a fragment-based, drug-discovery approach to enhance the trypanocidal activity of nitrofurantoin. The presence of an electronegative electron- withdrawing group in the ortho- or meta-position on the phenyl ring may also influence activity. However, these structure–activity relationships must be confirmed in further experiments by investigating derivatives bearing such substituents. Furthermore, the selectivity index, which indicates the selectivity of the antipathogenic action of a compound in the presence of normal/healthy mammalian cells (Table2), showed that most analogs had a selectivity index >10, which is a minimum criterion value for selecting a synthetic drug hit [26]. Therefore, most analogs were hits, with activity IC50 <10 µM and selectivity index >10 [26]. Despite being mild to weakly toxic towards Madin– Molecules 2021, 26, 3372 7 of 11

Darby bovine kidney cells [27,28], these analogs were up to 10–100-fold selective in their trypanocidal action. Hall et al. suggested that trypanosomal type I nitroreductase reduces nitrofuran compounds, and that the toxic intermediate metabolites kill the parasites [29]. They also revealed that an increased expression level of this enzyme makes T. b. brucei more sensitive to nifurtimox and its related compounds [12]. The current study revealed differences in Fig. 2-a & b the sensitivities of the evaluated trypanosome species and strains to the 19 nitrofurantoin analogs. This may explain the difference in the expression level of trypanosomal type I nitroreductase or enzymatic activity in each trypanosome, leading to differences in the a sensitivity of the tested compounds.b

Trypanocidal Trypanocidal Chemicals Cytotoxicity Chemicals Electronegativity activity activity High High High High High High

16 1

Short alkyl chain Lowest 17

15 Long alkyl chain

Highest Low Low Low 12

(a) (b)

Figure 2. Summary of structure–activity relationship. The summary of structure–activity relationship between trypanocidal activity and alkyl chain length (a) or electronegativity (b).

Nitrofurantoin (Macrobid®) is an antibiotic that is orally used to treat bladder infections. Nitrofurantoin shows extremely low lipophilicity (clogP −0.22), which may explain its poor trypanocidal activity, as it cannot efficiently permeate parasitic biomembranes. In contrast, analogs 1 and 2 have low cLogP values of −0.04 and 0.36, respectively [11], which are relatively higher than those of nitrofurantoin, suggesting their moderately lipophilic nature. They were found to possess moderate (0.19–0.73 µM) activity against the T. b. rhodesiense Chirundu and T. congolense IL3000 strains. HAT and AT are endemic diseases, mainly occurring in rural areas of developing countries, and persist because of the lack of healthcare and veterinary medicine infrastructure. Therefore, oral drugs are highly convenient for treating these diseases. Although they showed strong trypanocidal activities in vitro, no treatment efficacy was observed with nitrofurantoin analogs 9, 11, and 12 against T. congolense infection in vivo, after both oral and intraperitoneal administration. Oral treatment with nitrofurantoin, however, demonstrated partial efficacy. nitrofurantoin possessed weak trypanocidal activity in vitro, but dissolved in water with a lower clogP than that of the tested analogs, which were more lipophilic and thus had higher clogP values. This result supports that high-hydrosolubility nitrofurantoin analogs should be developed and evaluated for their in vivo trypanocidal activity, in further studies. Analogs 1 and 2 showing moderate in vitro trypanocidal activity and higher solubility (lower cLogP) than the current selectees may be viable starting points for developing oral treatments for HAT and AT in endemic areas [30]. Molecules 2021, 26, 3372 8 of 11

4. Materials and Methods 4.1. Chemicals The nitrofurantoin analogs (Table1) were synthesized in moderate yields (50–65%) in a single-step N-alkylation process through nucleophilic substitution (SN2) [11]. The series of analogs was divided into two sub-series based on the nature of the substituent at the N-3 position. Thus, sub-series 1 comprised N-alkyl analogs 1–12, whereas sub-series 2 comprised benzyl-substituted analogs 13–19. The latter sub-series featured analogs 15 and 18, which bore the electron-donating groups CH3 and OCH3, respectively, whereas analogs 16, 17, and 19 possessed the electron-withdrawing groups CF3, NO2, and Br, respectively. Analog 14 bore a neutral group H, and analog 13 possessed an ethyl bridge between the nitrofurantoin scaffold and the benzyl group. The analogs possessed lipophilic properties, with lipophilicity coefﬁcients (clogP) in the targeted range of 1–5 [31,32]. They showed moderate to no toxicity in human embryonic kidney (HEK-293) and Chinese hamster ovary cells and exhibited anti-mycobacterial activity with 90% minimum inhibitory concentrations of 0.5–35 µM. The most potent antimycobacterial nitrofurantoin derivative was analog 8, which was 30-fold more potent than nitrofurantoin [11]. These analogs were dissolved at 10 mg/mL in dimethyl sulfoxide and stored at −30 ◦C until use.

4.2. In Vitro Cultivation of Trypanosomes and Madin–Darby Bovine Kidney Cells The bloodstream forms of T. b. brucei GUTat3.1, T. b. rhodesiense IL1501, T. b. rhodesiense Chirundu, T. b. gambiense IL1922, and T. evansi Tansui strains were propagated in culture at 37 ◦C, whereas the T. congolense IL3000 strain was propagated at 33 ◦C in Hirumi’s modiﬁed Iscove’s medium-9, supplemented with 20% heat-inactivated fetal bovine serum [33]. The bloodstream form cultures were maintained by replacing the entire culture supernatant with fresh medium, every other day.

4.3. Evaluation of Trypanocidal Activity and Cytotoxicity of Nitrofurantoin Analogs In Vitro The trypanocidal activity and cytotoxicity of nitrofurantoin analogs were evaluated according to previous reports [34,35], with some modifications. In brief, bloodstream forms adjusted at 2.5 × 103 cells/mL (T. b. brucei GUTat3.1, T. b. gambiense IL1922, and T. b. rhodesiense IL1501), 1 × 104 cells/mL (T. evansi Tansui and T. b. rhodesiense Chirundu), and 1 × 105 cells/mL (T. congolense IL3000), were cultivated in Nunc® MicroWell 96-well optical bottom plates (Thermo Fisher Scientific, Waltham, MA, USA) and exposed to various analogs at seven different concentrations for 3 days, in an incubator at 37 ◦C (33 ◦C for T. congolense IL3000). After 3 days of incubation, the condition of the bloodstream forms was observed with phase-contrast microscopy. Twenty-five microliters of the Cell-TiterGlo® (Promega, Madison, WI, USA) reagent was added into each well, and bioluminescence was measured using a GloMax®-Multi+Detection System plate reader (Promega). Additionally, Madin–Darby bovine kidney cells were cultivated (density, 1 × 104 cells/mL) in a 96-well cell culture plate (Thermo Fisher Scientific) with the analogs, at seven different concentrations for 3 days at 37 ◦C. Next, 10 µl of the Cell Counting Kit- 8 solution (Dojindo, Kumamoto, Japan) was added into each well before and after 4 h of incubation. Absorbance was measured at 450 nm using a GloMax®-Multi+Detection System plate reader. The half maximum inhibitory concentration (IC50) of each analog in trypanosomes and the Madin–Darby bovine kidney cells was calculated using the GraphPad Prism version 8 software (GraphPad, Inc., San Diego, CA, USA).

4.4. In Vivo Evaluation of the Treatment Efﬁcacy of Selected Nitrofurantoin Analogs Healthy female 8-week-old BALB/c mice (CLEA Japan, Inc., Tokyo, Japan) were used in this study. All animals had ad libitum access to normal chow and water. Molecules 2021, 26, 3372 9 of 11

The virulent T. congolense IL3000 strain was propagated in a mouse and used for infection. The parasites were passaged once in a mouse before the experiment. The experimental mice were intraperitoneally infected with 100 µL of T. congolense in phosphate-buffered saline, containing 10% glucose at 1 × 103 cells/mouse. First, the mice were randomly divided into six groups of three mice, each as follows. Group I (no treatment), mice were not infected and were only treated with solvent (10% dimethyl sulfoxide–phosphate–buffered saline). Group II received 3.5 mg/kg of diminazene aceturate treatment. Groups III (9), IV (11), V (12), and VI were treated with nitrofurantoin and the mice were infected and intraperitoneally treated with 0.1 mg/kg of the compound. In the second experiment, the mice were randomly divided into six groups of three mice, each as follows—group I received no treatment; group II received 3.5 mg/kg of diminazene aceturate treatment; and groups III (9), IV (11), V (12), and VI received nitrofurantoin—the mice were infected and orally treated with 10 mg/kg of the compound. Treatment was initiated at 2 days post-infection (dpi) and continued for seven con- secutive days. The treatments were freshly prepared each day. Parasitemia was observed using a cell counting chamber until 14 dpi.

5. Conclusions In conclusion, our results indicate that nitrofurantoin may be suitable as a lead compound for developing novel trypanocidal drugs, provided that compounds with good oral bioavailability and in vivo activity can be designed, particularly against animal African trypanosomosis caused by T. congolense and some conﬁned endemic acute HAT infections caused by the T. b. rhodesiense Chirundu strain. Our preliminary in vivo experiments also showed that NF and its analogs with high water solubility should be evaluated to determine their treatment efﬁcacy in mouse models, in further studies.

Author Contributions: Conceptualization, K.S. and D.D.N.; Investigation, K.S., L.M. and A.S.; Bio resources, B.N. and D.D.N.; Writing draft preparation, K.S., L.M. and D.D.N.; Review and editing, K.S., L.M., A.S., B.N., D.D.N., and N.I.; Funding, K.S. All authors have read and agreed to the published version of the manuscript. Funding: The study was supported by the Japan Society for the Promotion of Science (JSPS), KAK- ENHI Grant Number 16K18793 [Grants-in-Aid for Young Scientists (B)] and Ohyama Health Founda- tion, Inc. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Institutional Review Board Statement: The experiment was approved by the animal ethics commit- tee of Obihiro University of Agriculture and Veterinary Medicine (Approval No. 20-9). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank the Japan Society for the Promotion of Science (JSPS) and Ohyama Health Foundation, Inc. for providing financial assitance. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Sample Availability: Samples of the compounds are available from D.D.N.

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