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In Vitro and In Vivo Trypanocidal Efficacy of Synthesized Nitrofurantoin Analogs

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 management depending mainly on chemotherapy. Moreover, only few drugs are available and these have adverse effects on patients, are costly, show poor accessibility, and parasites develop drug resistance to them. Therefore, novel trypanocidal drugs are urgently needed. Here, the effects of synthesized nitrofurantoin analogs were evaluated against six species/strains of animal and human trypanosomes, and the treatment efficacy of the selected compounds was assessed in vivo. Analogs 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 relationship analysis suggested that the trypanocidal activity and cytotoxicity were related to the 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 treatment efficacy. These results indicate that nitrofurantoin analogs with high hydrophilicity are required for in vivo assessment to determine if they are promising leads for developing trypanocidal drugs.


Introduction
African trypanosomes, transmitted by tsetse fly, cause several diseases in both humans and livestock on the Sub-Saharan African continent. Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei gambiense and T. b. rhodesiense. The disease has devastating socio-economic impacts, such as reduced income generation, negative effects on the education of children, increased healthcare costs, and long-term health consequences [1,2]. In contrast, animal African trypanosomosis, which is caused by T. congolense, T. vivax, and T. brucei brucei, represents a major health concern in animal development, and its deleterious impacts can be manifested in terms of reduced livestock productivity, costly disease control, and trade implications, as well as negative impacts on agriculture and ment, and its deleterious impacts can be manifested in terms of reduced livestock productivity, 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 infection. It is mechanically transmitted by hematophagous biting flies such as Tabanus spp., Stomoxys spp., and species of tsetse flies [5]. Animal trypanosomoses (AT), including animal African trypanosomosis and surra, are economically important diseases in animals. They cause high mortality, low milk and meat production, poor carcass quality, reduced reproductive performance, and decreased draught animal power and manure production, as well as immunosuppression in livestock [3,5]. HAT/AT control requires an integrated approach involving the use of trypanocidal drugs [3,6], reduction in the number of vectors, and low contact between hosts and vectors [7]. Owing to the challenges in vector control programs and vaccine development, HAT/AT control primarily depends on chemotherapy using anti-parasitic agents. Currently, the nifurtimox-eflornithine combination therapy (NECT) and fexinidazole are approved for treating HAT caused by T. b. gambiense [8]. Particularly, fexinidazole was the first oral drug approved for HAT treatment, which shows satisfactory treatment efficacy against second-stage HAT, as compared to the nifurtimox-eflornithine combination therapy [9]. In contrast, no orally administered and safe drugs are available against HAT caused by T. b. rhodesiense and AT. Therefore, the screening and development of safe and effective compounds for treating HAT/AT are urgently required.
Nitrofurantoin is a hypoxic agent with activity against a myriad of anaerobic pathogens. This nitrofuran compound contains a Schiff base derived from 5-nitrofuraldehyde, which is known to be effective against a wide spectrum of gram-positive and gram-negative bacteria and various pathogens, including trypanosomes [10,11]. Nitrofurantoin analogs show good safety profiles, enhanced anti-mycobacterial potency, improved lipophilicity, and a reduced protein-binding affinity. Nitrofurans are broad-spectrum redoxactive antibiotics, with dose-dependent bacteriostatic or bactericidal activity [10,12], and have been used in animal feeds, pharmaceuticals, and other applications [11,13].
The clinical drug furazolidone belongs to the group of nitrofuran antibiotics and has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish [11,14]. Nifurtimox, another nitrofuran derivative, which was developed in the 1960s, has been used to treat Chagas disease caused by T. cruzi. More recently, the combination of nifurtimox and eflornithine was evaluated for the treatment of late-stage T. b. gambiense sleeping sickness [15][16][17]. Furthermore, a study revealed that chemically synthesized analogs of nitrofurantoin had significantly enhanced antimycobacterial activity [11]. Overall, these reports suggest that some nitrofurantoin analogs can be used against African trypanosomes. Therefore, the aim of the study was to evaluate the in vitro trypanocidal activity of these chemically synthesized nitrofurantoin analogs (Table 1) and in vivo treatment efficacy of three promising analogs against human and animal trypanosomes. ment, and its deleterious impacts can be manifested in terms of reduced livestock productivity, 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 infection. It is mechanically transmitted by hematophagous biting flies such as Tabanus spp., Stomoxys spp., and species of tsetse flies [5]. Animal trypanosomoses (AT), including animal African trypanosomosis and surra, are economically important diseases in animals. They cause high mortality, low milk and meat production, poor carcass quality, reduced reproductive performance, and decreased draught animal power and manure production, as well as immunosuppression in livestock [3,5]. HAT/AT control requires an integrated approach involving the use of trypanocidal drugs [3,6], reduction in the number of vectors, and low contact between hosts and vectors [7]. Owing to the challenges in vector control programs and vaccine development, HAT/AT control primarily depends on chemotherapy using anti-parasitic agents. Currently, the nifurtimox-eflornithine combination therapy (NECT) and fexinidazole are approved for treating HAT caused by T. b. gambiense [8]. Particularly, fexinidazole was the first oral drug approved for HAT treatment, which shows satisfactory treatment efficacy against second-stage HAT, as compared to the nifurtimox-eflornithine combination therapy [9]. In contrast, no orally administered and safe drugs are available against HAT caused by T. b. rhodesiense and AT. Therefore, the screening and development of safe and effective compounds for treating HAT/AT are urgently required.
Nitrofurantoin is a hypoxic agent with activity against a myriad of anaerobic pathogens. This nitrofuran compound contains a Schiff base derived from 5-nitrofuraldehyde, which is known to be effective against a wide spectrum of gram-positive and gram-negative bacteria and various pathogens, including trypanosomes [10,11]. Nitrofurantoin analogs show good safety profiles, enhanced anti-mycobacterial potency, improved lipophilicity, and a reduced protein-binding affinity. Nitrofurans are broad-spectrum redoxactive antibiotics, with dose-dependent bacteriostatic or bactericidal activity [10,12], and have been used in animal feeds, pharmaceuticals, and other applications [11,13].
The clinical drug furazolidone belongs to the group of nitrofuran antibiotics and has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish [11,14]. Nifurtimox, another nitrofuran derivative, which was developed in the 1960s, has been used to treat Chagas disease caused by T. cruzi. More recently, the combination of nifurtimox and eflornithine was evaluated for the treatment of late-stage T. b. gambiense sleeping sickness [15][16][17]. Furthermore, a study revealed that chemically synthesized analogs of nitrofurantoin had significantly enhanced antimycobacterial activity [11]. Overall, these reports suggest that some nitrofurantoin analogs can be used against African trypanosomes. Therefore, the aim of the study was to evaluate the in vitro trypanocidal activity of these chemically synthesized nitrofurantoin analogs (Table 1) and in vivo treatment efficacy of three promising analogs against human and animal trypanosomes.  ment, and its deleterious impacts can be manifested in terms of reduced livestock productivity, 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 infection. It is mechanically transmitted by hematophagous biting flies such as Tabanus spp., Stomoxys spp., and species of tsetse flies [5]. Animal trypanosomoses (AT), including animal African trypanosomosis and surra, are economically important diseases in animals. They cause high mortality, low milk and meat production, poor carcass quality, reduced reproductive performance, and decreased draught animal power and manure production, as well as immunosuppression in livestock [3,5]. HAT/AT control requires an integrated approach involving the use of trypanocidal drugs [3,6], reduction in the number of vectors, and low contact between hosts and vectors [7]. Owing to the challenges in vector control programs and vaccine development, HAT/AT control primarily depends on chemotherapy using anti-parasitic agents. Currently, the nifurtimox-eflornithine combination therapy (NECT) and fexinidazole are approved for treating HAT caused by T. b. gambiense [8]. Particularly, fexinidazole was the first oral drug approved for HAT treatment, which shows satisfactory treatment efficacy against second-stage HAT, as compared to the nifurtimox-eflornithine combination therapy [9]. In contrast, no orally administered and safe drugs are available against HAT caused by T. b. rhodesiense and AT. Therefore, the screening and development of safe and effective compounds for treating HAT/AT are urgently required.
Nitrofurantoin is a hypoxic agent with activity against a myriad of anaerobic pathogens. This nitrofuran compound contains a Schiff base derived from 5-nitrofuraldehyde, which is known to be effective against a wide spectrum of gram-positive and gram-negative bacteria and various pathogens, including trypanosomes [10,11]. Nitrofurantoin analogs show good safety profiles, enhanced anti-mycobacterial potency, improved lipophilicity, and a reduced protein-binding affinity. Nitrofurans are broad-spectrum redoxactive antibiotics, with dose-dependent bacteriostatic or bactericidal activity [10,12], and have been used in animal feeds, pharmaceuticals, and other applications [11,13].
The clinical drug furazolidone belongs to the group of nitrofuran antibiotics and has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle, and farmed fish [11,14]. Nifurtimox, another nitrofuran derivative, which was developed in the 1960s, has been used to treat Chagas disease caused by T. cruzi. More recently, the combination of nifurtimox and eflornithine was evaluated for the treatment of late-stage T. b. gambiense sleeping sickness [15][16][17]. Furthermore, a study revealed that chemically synthesized analogs of nitrofurantoin had significantly enhanced antimycobacterial activity [11]. Overall, these reports suggest that some nitrofurantoin analogs can be used against African trypanosomes. Therefore, the aim of the study was to evaluate the in vitro trypanocidal activity of these chemically synthesized nitrofurantoin analogs (Table 1) and in vivo treatment efficacy of three promising analogs against human and animal trypanosomes.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.

In Vitro Experiment
A summary of the trypanocidal activity and cytotoxicity data of the nitrofurantoin analogs is shown in Table 2.
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.
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.

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 (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). 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 (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).

Discussion
In the present study, the trypanocidal activity of 19 nitrofurantoin analogs was evaluated against six human and animal trypanosomes in vitro. Investigation of the structureactivity relationship within the series revealed that the trypanocidal activity of short alkyl analogs (1-4) in sub-series 1 decreased with increasing chain lengths up to four carbons (n = 4, 4) (Figure 2a). The trypanocidal activity in long alkyl chain analogs (5-12) increased with increasing chain lengths up to 12 carbons (n = 12, 12) (Figure 2a). The cytotoxicity within the analogs of sub-series 1 mostly increased with increasing chain lengths ( Figure  2a). The strongest trypanocidal activity of these long alkyl chains (9)(10)(11) in sub-series 1 may be related to their higher lipophilicity, which increased the cLogP value. This feature is typically associated with better permeation of a molecule through biological tissues/membranes, as well as interactions with transporter proteins and enzymes [18]. Similar results were previously observed against the T. cruzi parasite [19,20]. Various studies have indicated a relationship between trypanocidal activity and the number of carbon atoms in the alkyl chain of the compounds [21,22]. Group IV (11) Group V (12) Group VI (NF) Figure 1. In vivo analysis of selected nitrofurantoin analogs and nitrofurantoin (NF). Evaluation of parasitemia in mice infected with T. congolense and intraperitoneal or orally treated with nitrofurantoin analogs (9, 11, and 12) and nitrofurantoin. The Y axis shows log 10 scale, and the data are shown as mean ± standard deviation. Parasitemia was not observed in group II (diminazene aceturate treatment). (a) Treatment of mice by intraperitoneal injection at 0.1 mg/kg. (b) Treatment of mice by oral administration at 10 mg/kg.

Discussion
In the present study, the trypanocidal activity of 19 nitrofurantoin analogs was evaluated against six human and animal trypanosomes in vitro. Investigation of the structureactivity relationship within the series revealed that the trypanocidal activity of short alkyl analogs (1-4) in sub-series 1 decreased with increasing chain lengths up to four carbons (n = 4, 4) (Figure 2a). The trypanocidal activity in long alkyl chain analogs (5-12) increased with increasing chain lengths up to 12 carbons (n = 12, 12) (Figure 2a). The cytotoxicity within the analogs of sub-series 1 mostly increased with increasing chain lengths (Figure 2a). The strongest trypanocidal activity of these long alkyl chains (9)(10)(11) in sub-series 1 may be related to their higher lipophilicity, which increased the cLogP value. This feature is typically associated with better permeation of a molecule through biological tissues/membranes, as well as interactions with transporter proteins and enzymes [18]. Similar results were previously observed against the T. cruzi parasite [19,20]. Various studies have indicated a relationship between trypanocidal activity and the number of carbon atoms in the alkyl chain of the compounds [21,22].
Additionally, we showed that the trypanocidal activity of analogs bearing electronwithdrawing 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 ( Figure 2b). Electronegativity was also reported to affect the trypanocidal activity [23][24][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 electronwithdrawing group in the orthoor 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 ( Table 2), 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 IC 50 <10 µM and selectivity index >10 [26]. Despite being mild to weakly toxic towards Madin-