In vitro anti‐trypanosomal activity of synthetic nitrofurantoin‐triazole hybrids against Trypanosoma species causing human African trypanosomosis

Human African trypanosomosis (HAT) which is also known as sleeping sickness is caused by Trypanosoma brucei gambiense that is endemic in western and central Africa and T. b. rhodesiense that is endemic in eastern and southern Africa. Drugs used for treatment against HAT first stage have limited effectiveness, and the second stage drugs have been reported to be toxic, expensive, and have time‐consuming administration, and parasitic resistance has developed against these drugs. The aim of this study was to evaluate the anti‐trypanosomal activity of nitrofurantoin‐triazole hybrids against T. b. gambiense and T. b. rhodesiense parasites in vitro. This study screened 19 synthesized nitrofurantoin‐triazole (NFT) hybrids on two strains of human trypanosomes, and cytotoxicity was evaluated on Madin‐Darby bovine kidney (MDBK) cells. The findings in this study showed that an increase in the chain length and the number of carbon atoms in some n‐alkyl hybrids influenced the increase in anti‐trypanosomal activity against T. b. gambiense and T. b. rhodesiense. The short‐chain n‐alkyl hybrids showed decreased activity compared to the long‐chain n‐alkyl hybrids, with increased activity against both T. b. gambiense and T. b. rhodesiense. Incorporation of additional electron‐donating substituents in some NFT hybrids showed increased anti‐trypanosomal activity than to electron‐withdrawing substituents in NFT hybrids. All 19 NFT hybrids tested displayed better anti‐trypanosomal activity against T. b. gambiense than T. b. rhodesiense. The NFT hybrid no. 16 was among the best performing hybrids against both T. b. gambiense (0.08 ± 0.04 μM) and T. b.rhodesiense (0.11 ± 0.06 μM), and its activity might be influenced by the introduction of fluorine in the para‐position on the benzyl ring. Remarkably, the NFT hybrids in this study displayed weak to moderate cytotoxicity on MDBK cells. All of the NFT hybrids in this study had selectivity index values ranging from 18 to greater than 915, meaning that they were up to 10–100 times fold selective in their anti‐trypanosomal activity. The synthesized NFT hybrids showed strong selectivity >10 to T. b. gambiense and T. b. rhodesiense, which indicates that they qualify from the initial selection criteria for potential hit drugs.

Human African trypanosomosis (HAT), also known as sleeping sickness, is caused by Trypanosoma brucei sub-species parasites transmitted by flies of the genus Glossina in sub-Saharan Africa [1].Glossina fuscipes and G. palpalis are the main vectors for transmission of HAT [2].There are two forms of HAT: chronic western and central African HAT caused by T. b. gambiense, whereby symptoms develop from months to years before the first stage progresses to the second stage of the central nervous system (CNS), before death [3].The second type is the acute eastern and southern African HAT caused by T. b. rhodesiense infection that takes weeks to months from the first stage to the second stage of the disease prior to death [4].
Treatment of HAT following a positive diagnosis relies solely on chemotherapy [5].Clinical anti-HAT drugs have been associated with major challenges [6], disadvantages [7], and limitations [8], leading to therapeutic failures [9].Pentamidine and suramin, which are drugs used to treat the first stage of HAT, have limited effectiveness against the first stage of T. b. gambiense and T. b. rhodesiense [5].The second-stage drugs eflornithine and melarsoprol have been reported to have toxic effects and be ineffective against T. b. rhodesiense; additionally, they are expensive, involve time-consuming parenteral administration, and most importantly, increase parasitic resistance [10].Therefore, there is an urgent need for the development of new trypanocides.However, developing effective trypanocides is a daunting task as it depends on the Trypanosoma sub-species and the diagnostic stage of the disease [11].
Drugs containing nitrogen functional groups have long been used in medicinal chemistry and have been reported to be effective against diseases such as cancer, leishmaniasis, malaria, trypanosomosis, and tuberculosis [12].Nitroaromatic or nitroheteromatic analogs induce therapeutic effects through diverse mechanisms of the bioreduction activation process [13].There has been a modification of these analogs to enhance efficacy and reduce toxic effects.Nitrofurantoin is one of the most widely used nitrofurans that possess antimicrobial activity.It is commonly used to treat cystitis, kidney infections, and urinary tract infections [14].
Synthesis and modification of derivatives are crucial to obtaining drugs with favorable pharmacokinetic and pharmaceutical properties.Different types of candidate drugs with similar structure and functionality are used to investigate their mode of action and wide range of biological activity.There is a continuous need to develop efficient antitrypanosomal drugs for HAT that are cost effective, safe and can also cater for non-parenteral/oral administration.In this study, synthesized nitrofurantointriazole (NFT) hybrids [15] were tested against T. b. gambiense and T. b. rhodesiense parasites in vitro.

| Chemistry
A series of NFT hybrids containing nitrofuran and 1,2,3-triazole pharmacologically active scaffolds were synthesized in low to high yields (30%-80%), as described by Zuma et al. [15].The NFT hybrids used in this study are shown in Table 1 together with their chemical structures.

| In vitro cultivation of mammalian cells
Madin-Darby bovine kidney cells (MDBK; NBL-1) were cultured and maintained in GIT medium supplemented with Dulbecco's modified eagle's medium (DMEM; Sigma Aldrich, Tokyo, Japan), 20% heat-inactivated fetal bovine serum (HI-FBS), and 1% penicillin streptomycin (Thermo Fisher Scientific, Yokohama, Japan).The cells were incubated at 37 C in 5% CO2, and the medium was replaced with a fresh medium every 3 to 4 days.When cells were approximately 80% confluent, they were passaged by removing the medium from the cell culture flask and washed once with 5 mL phosphatebuffered saline (PBS); 250 μL of pre-heated trypsin was added to dislodge adherent cells from the cell culture flask with a 5 mL pipette, then incubated for 1 min at 37 C.The cell suspension was completely sucked out from the cell culture flask, and approximately 11 mL of fresh medium was added to the cells as previously described by Nefertiti et al. [16].

| In vitro anti-trypasomal activity
The assay was conducted by adding 50 μL of each nitrofurantoin hybrid in 2% dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Osaka, Japan) to HMI-9 medium at various concentrations (25 to 0.003 ng/mL).The nitrofurantoin hybrids and commercial reference drugs, suramin, pentamidine, nifurtimox, eflornithine, and diminazene aceturate, were added to the Nunc ® MicroWell 96-well optical bottom plate (Thermo Fisher Scientific, Yokohama, Japan) and incubated at 37 C in a humidified atmosphere.Parasite viability was observed under a light microscope, and parasites were counted manually after every 24 h using a counting chamber.Fifty microliters of T. b. gambiense and T. b. rhodesiense parasites were inoculated at 5 Â 10

| In vitro cytotoxicity evaluation
Cytotoxicity evaluation of nitrofurantoin hybrids was conducted using the MDBK cell line (Obihiro University of Agriculture and Veterinary Medicine, Japan) as described by Li et al. [21].Approximately 1 Â 10 5 cells/ mL were seeded in a 96-well microtiter plate (Thermo Fisher Scientific, Yokohama, Japan); 50 μL of the cells were exposed to nitrofurantoin hybrids at varying concentrations (100 μg/mL to 0.80 μg/mL) and incubated for 72 h at 37 C in a 5% CO 2 incubator.Cell viability was detected by an optical microscope and Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) by adding 10 μL of CCK-8 solution to each well.After the incubation period, the plates were read using a GloMax ® -Multi Detection System plate reader (Promega, Japan) at 450 nm.The 96-well plates were incubated again after the initial absorbance reading at 37 C for 4 h; subsequently, the plates were read for the second time, and the experiments were each repeated three times [18].Selectivity index which is expressed as the ratio of 50% cytotoxic concentration (IC 50 ) of the cells over the inhibitory concentration (IC 50 ) of the trypanosome parasites to determine the nitrofurantointriazole hybrids that are active against the parasites but nontoxic to the mammalian cells [22].

| Statistical analysis
The activity of each hybrid was expressed as IC 50 ± S. D., which is the concentration of each hybrid that induced 50% inhibition of the parasites for experiments Chemical structure Chemical structure performed in triplicate.Nonlinear regression (curve fit) graphs were performed using GraphPad PRISM version 5 software (GraphPad Software, Inc., CA, USA).Cytotoxicity of the NFT hybrids was analyzed by Cell Counting Kit-8 (CCK-8) and measured by 50% cytotoxic concentration (IC 50 ) on MDBK cells and SI, which is the ratio of trypanosome inhibition to host cell toxicity.
The SI was calculated to allow for the possible identification of compounds with high efficacy and low cytotoxicity.This parameter reflects the quantity of the compound that is active against the pathogen but is not toxic toward the host cells [22].ClogP was calculated using Chem Axon Marvin Beans Software (Version 5.2.3_1,May 26, 2009) as log (coctanol/water).SI, which is expressed as the ratio of the 50% cytotoxic concentration (IC 50 ) of the cells versus the IC 50 of the parasites, is used to determine the NFT hybrids that are active against the parasites but nontoxic to the cells.SI = minimum toxic concentration (μg/mL)/minimum inhibitory concentration (μg/mL).
T A B L E 3 Anti-trypanosomal activity of nitrofurantoin-triazole hybrids against T. b. rhodesiense and cytotoxocity on MDBK cells.

| In vitro anti-trypanosomal activity
In the current study, 19 synthesized NFT hybrids were screened against T. gambiense and T. rhodesiense.
The findings in this study showed that an increase in the chain length and the number of carbon atoms in the nalkyl hybrids 5-9 influenced the increase in antitrypanosomal activity against T. b. gambiense and hybrids 4-9 against T. b. rhodesiense.Experts implemented criteria for screening potential compounds for infectious diseases (leishmaniasis, malaria, TB, and trypanosomosis) and the in vitro activity criteria for a "hit" compound against T. b. rhodesiense is IC 50 < 0.2 μg/mL and for T. cruzi is IC 50 < 1.0 μg/mL [23][24][25].
The short-chain n-alkyl hybrids 2, 3, 4, 5, and 10 showed decreased activity compared to long-chain nalkyl hybrids 6, 7, 8, and 9 with increased activity against both T. b. gambiense and T. b. rhodesiense (Tables 2 and 3).The results of this study agree with previous studies regarding the structure-activity relationship, chain length, and the number of carbon atoms do influence trypanocidal [26,27].According to Zuma et al. [28], the addition of carbons to extend the alkyl chain allows for more flexibility and structural diversity.Electrophilic aromatic substitution with electron withdrawing groups; Br, F, CF 3 , NO 2 and electron donating groups, methyl, isopropyl and tert-butyl on the phenyl ring had an influence on trypanocidal activity.Incorporation of additional electron-donating substituents in NFT hybrids 11-14 showed increased antitrypanosomal activity compared to electronwithdrawing substituents in NFT hybrids 15, 16, and 18-20, as shown in Tables 1-3.Complementary to the results, electronegativity has been reported to correlate with trypanocidal activity [29,30].The position of the substituents on the phenyl ring appeared to influence anti-trypanocidal activity.Hybrid 15 incorporating a bromine substituent in the para-position was marginally more active than hybrid 19 with a bromine substituent in the meta-position against both T. b. gambiense and T. b. rhodesiense (Tables 1-3).However, hybrids with the fluorine substituent 17 were more active in the para-position than 18 in the ortho-position or 20 in the meta-position against both T. b. gambiense and T. b. rhodesiense (Tables 1-3).Zhou et al. [31] reported that the nitro group position of synthesized 5-nitrofurans containing nitroaromatic rings influenced activity as it was in meta-position and more potent than those in ortho-or para-position.The effects of ortho-, meta-, and para-substitution on the biological activity of (E)-N,-1-diphenylmethanimine derivatives were investigated by Mkpenie et al. [32], and the results displayed that meta-and para-substituents were more active against some fungal and bacterial microorganisms.
Hu et al. [33] reported that 4-nitrobenzyl phosphoramide mustard displayed high potency against T. brucei, T. cruzi, and Leishmania donovani with IC 50 values of 0.007, 0., and 1.29 μM, respectively.The activity can be explained by the type I nitroreductase mechanism and the benzylic C-O bond necessary for prodrug activation.Kaiser et al. [34] reported the findings of 5-nitrofuran repurposed drugs and nitrofurantoin as being exceptionally potent with good selectivity against T. b. rhodesiense with IC 50 of 0.5 μM; SI:181 but inactive against other antiprotozoal parasites L. donovani amastigotes IC 50 2.12 μM; SI: 59 and T. cruzi  2 and 3).This poor activity in comparison to hybrids 2, 3, 6-16, 18, and 19 may be due to poor parasitic cell permeability and the hybrids being unstable, and this could also have influenced the lack of activity in Bains et al. [35].Poor antitrypanocidal activity against T. b. rhodesiense was also observed compared to T. b. gambiense in a study by Kibona et al [36] stated that drug resistance was associated with reduced susceptibility of T. b. rhodesiense.
A compound containing dinitrophynyl and its analogs were screened against T. b. rhodesiense, and most were potent with IC 50 ranging from 0.0075 to 0.05 μg/ mL in vitro [24].
Pharmacokinetic properties such as absorption, distribution, excretion, metabolism, permeability, potency, selectivity, and toxicity are very important when developing new drugs [37].It is crucial for a potential drug to have good solubility, which enables the compound's bioavailability and absorption and its ability to permeate biological membranes [38].Nitrofurantoin has been reported to have low lipophilicity, which makes it poorly soluble in oils, and it influences its ability for intestinal permeability by passive diffusion for oral administration [39].There is a need for the development of drugs that display optimal physicochemical properties for oral administration, as there are a limited number of orally administered anti-trypanocidal drugs [18].
The measure between lipophilicity and hydrophilicity of a compound is calculated by partition coefficient cLogP value [40].Lipinski's rule of five for drug-likeness indicates that the logarithm of the partition coefficient logP value requirement is ≤5 with < being the ideal target [41].Most NFT hybrids in this study showed good drug-like properties; the cLogP values were within the target range of cLogP <5 (1.52-4.74).Hybrids with a large logP value indicate high lipophilicity and poor water solubility, whereas hybrids with a low logP value showed high hydrophilicity associated with poor permeability and low [42,43].Sub-series 1 n-alkyl hybrids 2, 4-9 chain length had an influence on lipophilicity; as the chain length increased, there was an increase in cLogP and anti-trypanosomal activity (Table 2).The two most active n-alkyl hybrids, 8 (C11) and 9 (C12), had high cLogP values of 4.29 and 4.74, respectively.An increase in the lipophilicity of compounds tends to increase potency; however, due to high binding affinity, highly lipophilic compounds can bind to off-target hydrophobic environments, increasing the risk of toxicity [44].In a study conducted by Cao et al. [45], it was observed that the antiviral activity of highly potent compounds was partially affected by their configuration and lipophilicity.
There was no effect on lipophilicity observed on the NFT benzyl hybrids, as the electronic effect of the substituents varied.However, hybrids 11 and 17 that showed the strongest activity were among the least lipophilic, with cLogP values of 1.58 and 1.62, respectively.Zuma et al. [28] reported the same outcomes with regards to the effect of lipophilicity on the antimycobacterial activity of n-alkyl analogs with increasing side chain length.The benzyl sub-series, however, displayed no relation between the electronic effect of the substituents and lipophilicity.There were NFT hybrids within the cLogP target range, but they were found to be inactive, confirming that biological activity is dependent on many parameters apart from physicochemical properties [46].The difference in the anti-trypanosomal activity of the NFT hybrids observed in this study may be because of many factors, such as the bioassay, parasite species, and taking into consideration the different bio-activation enzymes between Trypanosoma species [47].
Hybrid 16, which was among the best performing hybrids against both T. b. gambiense; 0.08 ± 0.04 μM and T. b. rhodesiense 0.11 ± 0.06 μM, the activity might be influenced by the introduction of a fluorine in the para-position on the benzyl ring.Fluorination of aromatic rings by the replacement of hydrogen with fluorine has been reported to have an effect on stability, solubility, lipophilicity in terms of distribution, and absorption, as well as biological activity [48].A study by Cuevas-Herna'ndez et al. [49] showed a trifluoromethyl compound to have better trypanocidal activity than benznidazole, which is a commercially used drug for the treatment of Chagas disease caused by T. cruzi.
The trypanocidal drugs suramin, pentamidine, and diminazine aceturate used in this study showed more potency than all the NFT hybrids.However, nifurtimox and eflornithine showed no anti-trypanosomal activity at IC 50 TBI900) have been reported to be sensitive to melarsoprol, pentamidine, and suramin [24].Diminazene aceturate is used for the treatment of animal trypanosomosis; however, it has also been used for the first-stage treatment of T. b. gambiense and T. b. rhodesiense [52].
Hybrid 18 containing a nitro group substituent displayed moderate cytotoxicity with IC 50 of 22.20 ± 12.11 μM, and a study conducted by Li et al. [58] showed that the nitrobenzene-containing group on the furan ring of nitrofurantoin played a role in toxicity.There was no correlation observed between chain length and cytotoxicity of the n-alkyl hybrids, as well as the chain length of the benzyl hybrids bearing electron-donating methyl substituents.However, the benzyl hybrids with electron-withdrawing substituents suggested that the position of the substituent on the phenyl ring played a role in cytotoxicity.The bromine substituent at paraposition on hybrid 15 was nontoxic with IC 50 of 111.00 ± 53.06 μM, whereas when the bromine substituent (hybrid 19) was at meta-position, it displayed weak cytotoxicity with LC 50 of 53.96 ± 32.58 μM, respectively.Similar results were observed with hybrid 20 with a fluorine substituent in the ortho-position, which showed no toxicity with IC 50 of 138.56 ± 44.20 μM, but the fluorine substituent (hybrid 17) in the para-position displayed moderate cytotoxicity with LC 50 of 23.67 ± 5.26 μM, respectively.
As reported by Luethi et al. [59], para-substituents to the nitro moiety may increase toxicity influenced by mitochondrial impairment.Nitrofurantoin toxicity can also be mediated by redox cycling, which results in oxidative stress, and mitochondrial toxicity is caused by the production of oxygen electron-withdrawing nature of the nitro moiety [60].Hall et al. [61] showed findings of type I nitroreductases, which reduce the nitro group and are responsible for generating toxic metabolites for anti-parasitic activity that may also inhibit mammalian cell growth.Interference of the substituents on the benzyl ring is considered to influence toxicity, and substituents in the meta-position decrease toxicity, whereas para-position substituents decrease toxicity [62].
Toxicity and poor pharmacokinetics account for 20%-40% of drug failure in drug development, especially for candidate drugs, and compounds with high lipophilicity are associated with toxicity [43,63].The use of nitrofurantoin has been associated with hepatotoxicity and pulmonary toxicity; however, the mechanism remains undetermined for reasons that are so far unexplained [64].Mechanisms in in vitro studies indicated that an increase in the production of oxidants results in toxic metabolic products of nitrofurantoin in the presence of oxygen with long-term use [65].
Lack of suitable cell lines for cytotoxicity tests may hinder reliable results for cytotoxicity; MDBK cells may be susceptible, but they have been used for their bovine origin, epithelial traits, and adherent growth characteristics [66].Cytotoxic assay can also influence the optical quantitative measurement of electrochemical properties, and in this study, cell viability was evaluated by the CCK-8 assay.According to Cai et al. [67], the CCK-8 assay shows detection sensitivity using a highly water-soluble tetrazolium salt measured by the dye of WST-8 reduced by dehydrogenase in cells to form a water-soluble orange-colored product called formazan.The amount of formazan dye produced by cellular dehydrogenases is correlated with the number of living cells.Therefore, cell viability can be simply estimated by recording the optical density (OD) of formazan at 450 nm using a microplate reader.
All of the nitrofurantoin hybrids in this study had SI values ranging from 18 to greater than 915, and these results were in agreement with previous studies by Katsuno et al. [25] and Finiuk et al. [56] that the hybrids were up to 10-100 times fold selective in their antitrypanosomal activity.The synthesized NFT hybrids showed strong selectivity >10 to T. b. gambiense and T. b. rhodesiense, which indicates the initial criteria for selecting potential hit drugs [68].

| CONCLUSION
The anti-trypanosomal activity of 19 NFT-triazole hybrids was evaluated against T. b. gambiense and T. b. rhodesiense.Hybrids 9, 11, 13, 16, and 19 exhibited high anti-trypanosomal activity; even so, these hybrids showed weak to moderate cytotoxicity on the MDBK cells.The findings in the current study add to the library potential compounds for the design and development of trypanocidal drugs.These NFT-triazole hybrids must be further evaluated in vivo for trypanocidal activity using experimental animals.

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E Y W O R D S anti-trypanosomal activity, nitrofurantoin-triazole hybrids, T. b. rhodesiense, Trypanosoma brucei gambiense 1 | INTRODUCTION Anti-trypanosomal activity of 19 synthesized NFTtriazole hybrids against T. b. gambiense (IL1922), and T. b. rhodesiense (IL1501) are shown in (Tables2 and 3), respectively.The activities were expressed as IC 50 ± S.D., which is the concentration of each hybrid that induced 50% inhibition of the parasites.The nonlinear regression curves were also calculated to show the IC50 of anti-trypanosomal activity of NFT hybrids against T. b. gambiense (FigureS1A-S) and T. b. rhodesiense (Figure S2A-S).The hybrids were moderately active in comparison to reference drugs against T. b. gambiense and T. b. rhodesiense (Tables