Antitrypanosomal activity of 5-nitro-2-aminothiazole-based compounds

Article abstract of DOI:10.1128/AAC.02006-15

A small series of 5-nitro-2-aminothiazole-based amides containing arylpiperazine-, biphenyl- or aryloxyphenyl groups in their core were synthesized and evaluated as antitrypanosomatid agents. All tested compounds were active or moderately active against T

Full text of DOI:10.1128/AAC.02006-15

AAC Accepted Manuscript Posted Online 23 November 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.02006-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Evaluating 5-nitrothiazoles as trypanocidal agents
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Ivan P. O'Shea , Mohammed Shahed , Benjamín Aguilera-Venegas and Shane R.
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Wilkinson .
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School of Biological and Chemical Sciences, Queen Mary University of London, London,
UK.
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Laboratory of Antioxidants, Institute of Nutrition and Food Technology, University of Chile,
Av. Macul 5540, Santiago, P.O. Box 138-11, Chile
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corresponding author: s.r.wilkinson@qmul.ac.uk
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Running title: Anti-T. brucei activity of a nitrothiazole series

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Abstract
The growth inhibitory properties of a 5-nitrothiazole series was evaluated against
Trypanosoma brucei. A subset of related compounds displayed the greatest potency towards
the parasite while exhibiting little cytotoxic effect on mammalian cells, with this anti-
parasitic activity being dependent on expression of a type I nitroreductase by the
trypanosome. We conclude that the 5-nitrothiazole class of nitroheterocycle may represent
new leads in the treatment of human African trypanosomiasis.
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Spread via the blood feeding habits of tsetse flies, parasites belonging to the Trypanosoma
brucei complex are responsible for human African trypanosomiasis (HAT) (1). Drugs
represent the only option to combat this infection but their use is often problematic (2). One
treatment that targets the cerebral stage of this disease is a nifurtimox-eflornithine
combination therapy (3, 4). In this medication eflornithine acts as an inhibitor of ornithine
decarboxylase, blocking polyamine biosynthesis (5, 6) while nifurtimox is converted to a
toxic metabolite following activation by a type I nitroreductase (NTR) (7, 8). As type I NTRs
are expressed by some unicellular eukaryotes but not by metazoan organisms, the
bioreductive activity of this enzyme has been exploited to develop a series of novel anti-
parasitic agents that often exhibit little or no toxicity towards cultured mammalian cells (9 &
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The 5-nitrothiazoles represent a class heterocyclic compounds of which niridazole and
nitazoxanide display potent antimicrobial and antihelmintic activities (11, 12). The mode(s)
of action of these agents is unclear with both structures shown to inhibit of key enzymes
involved in energy metabolism (13, 14) and able to function as prodrugs, undergoing
reduction to form adduct forming metabolites (15-17). To date only niridazole and its
derivatives have been screened for trypanocidal activity against T. brucei with this in
combination with suramin able to cure mice of trypanosomiasis (18). However, concerns over
its carcinogenic properties resulted in trials using niridazole being suspended (19). Here, we
assessed a 2-amide 5-nitrothiazole series for growth inhibitory activity against bloodstream
form (BSF) T. brucei (Table 1). Out of the fifteen compounds tested, seven had no effect on
trypanosomal growth at a concentration of 30 µM. For the remaining chemicals, detailed
inhibition assays were conducted generating dose response curves from which IC50s were
determined (Table 1). For NT2, NT4, NT6, NT7 and NT11 an appreciable trypanocidal

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activity (IC50’s >10.0 µM) equivalent to the potency exhibited by nifurtimox was noted with
the other agents being less effective (IC50s ~17 µM). Screening against two mammalian lines
(Table 1) revealed that NT2, NT10, NT12 and NT15 displayed toxicity towards THP-1 or
SK-N-SH cells with NT10 and NT12 having growth inhibitory effects against both lines. For
the remaining agents no growth inhibitory effect at concentrations up to 100 µM was
observed.
Before mediating its trypanocidal effects nifurtimox must undergo activation in a reaction
catalysed by a type I NTR (7). Using purified HIS-tagged TbNTR (Fig. 1A) we evaluated
whether the 2-amide 5-nitrothiazoles series could serve as substrates for this enzyme (Fig.
1B). Five compounds were shown to be “good” NTR substrates, generating a specific activity
~3-fold greater than that noted for nifurtimox (Fig. 1B). Of these structures, NT2, NT4, NT6
and NT7 are related in that they contained a saturated unbranched hydrocarbon chain.
However, the number of carbon atoms in this sequence and the associated increase in
lipophilicity did not affecting the specific activity displayed by TbNTR towards a given
substrate. Of the remaining compounds, three yielded activities similar to that observed for
nifurtimox while the others were not metabolised by TbNTR at an appreciable rate under the
conditions used here (Fig. 1B).
To investigate whether NTR plays a role in prodrug activation within the parasite itself the
susceptibility of BSF T. brucei engineered to over express this enzyme was evaluated (Table
1; Fig. 2) (8). Cells having elevated levels of TbNTR were up to 10-fold more sensitive to
NT2, NT4, NT6 or NT7 than controls. This effect was NTR specific as recombinant and wild
type parasite lines displayed similar sensitivities to the non-nitroaromatic compound G418
(IC50 ~0.6 µM). When these studies were extended to test other trypanocidal nitrothiazoles, a

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lower (~2) fold or no difference in IC50 was observed (Table 1; Fig. 2). This implies that for
these less effective trypanocidal compounds, NTR plays little or no role in the metabolism of
these structures within the parasite itself.
By comparing the specific activity values and growth inhibitory effects of each compound, a
number of structure activity relationships (SARs) were identified. In contrast to their non-
substituent counterparts’ addition of a methyl or tert-butyl group at the 4-position on the
thiazole ring generated compounds that were not TbNTR substrates and did not exhibit
trypanocidal activities: compare NT2 with NT3 and NT4 with NT5. This lack of activity
could be due to steric hindrance with the 4-alkyl side chain blocking the trypanosomal
enzyme from gain access to the adjacent 5-nitro grouping or could reflect an inductive effect
with the alkyl substituent on the thiazole backbone rendering nitroreduction energetically
unfavourable. Extending the SAR studies to investigate grouping attached to the thiazole ring
via a 2-amide linker revealed that compounds containing an unbranched, saturated
hydrocarbon chain (NT2, NT4, NT6, NT7) were efficiently metabolised by TbNTR with this
translating to a trypanocidal effect equivalent to that of the reference nitrofuran.
Encouragingly, these structures displayed also little/no in vitro toxicity to mammalian cells
suggesting that they warrant in vivo analysis. Modification of this saturated linear
hydrocarbon chain (incorporation of an unsaturated bond (NT8) or an ether linkage (NT13),
inclusion of halogen substituents (NT10-12) or its replacement with a hydrogen atom (NT1)
or a benzyl-containing grouping (NT9, NT15)) generated structures that displayed lower
TbNTR activity and/or had reduced potency towards BSF trypanosomes. Presumably, such
alterations to the saturated alkyl chain alter the affinity these variants have for the parasite
oxidoreductase. As the broad spectrum ant-infective agent nitazoxanide is structurally related
to NT9 and NT15 (all contain a phenyl group attached to the amide linker) we predict that
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this particular antimicrobial agent is unlikely to function as an effective TbNTR substrate
and/or display activity against BSF T. brucei. Intriguingly, despite being screened against a
wide range of microbial infectious agents including Trypanosoma cruzi and Leishmania the
potency of this particular nitrothiazole against T. brucei has not been reported.
There has been renewed interest in the use of nitroheterocyclic prodrugs for the treatment of
trypanosomatid infections with nifurtimox in combination with eflornithine now being used
to treat the form of HAT prevalent in West and Central Africa while the nitroimidazole
fexinidazole is under clinical evaluation against HAT, Chagas disease and visceral
leishmaniasis. In both cases these nitroheterocycles are converted to toxic metabolites by a
type I NTR activity (7, 20). Here, we have identified several trypanocidal nitrothiazoles,
including some that are activated by the type I NTR, as being potent against BSF T. brucei as
nifurtimox. Promisingly the most effective structures exhibited little or no toxicity to cultured
mammalian cells with trypanosomal expression of the type I NTR underlying their
selectivity. As such, these compounds warrant further attention in terms of developing novel
therapies targeting HAT and could potentially represent one component of a new
combinatorial treatment against this disease.
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28 Funding Information
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BAV acknowledges financial support by FONDECYT Postdoctorado 3130364.
131 Acknowledgments
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We would like to thank Martin Taylor (London School of Hygiene and Tropical Medicine)
and James Sullivan (Queen Mary University of London) for valuable discussions and
comments on the manuscript.
136 Abbreviations.
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BSF, bloodstream form; HAT, human African trypanosomiasis; NECT, nifurtimox-
eflornithine combination therapy; NTR, nitroreductase; SAR, structure activity relationship;
TbNTR, Trypanosoma brucei type I nitroreductase
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142 References
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Table 1. Structure and growth inhibitory properties of nitrothiazole compounds. All compounds tested satisfy the Lipinski’s Rule of 5 (see
PubChem database (http://pubchem.ncbi.nlm.nih.gov/)). Susceptibility of parasites and mammalian cells to nitrothiazole compounds was
assessed as previously described (7). Average IC50 values ± standard deviations were calculated from dose response curves performed in
ox
triplicate. TbNTR represents the T. brucei cell line overexpressing the type I nitroreductase. The figures in parenthesis correspond to the fold
ox
difference in IC50 values of the TbNTR , SK-N-SH and THP-1 cell lines when compared against wild type.
compound
Structure
IC50 (μM)
T. brucei
Mammalian line
ox
R
H
1
R
H
H
2
wild type
>30.00
TbNTR
SK-N-SH
THP-1
NT1
NT2
NT3
NT4
NT5
NT6
NT7
NT8
CH
CH
CH
CH
CH
CH
CH
3
3
2
2
2
2
2
4.67 ± 0.34
>30.00
3.67 ± 0.50
>30.00
6.47 ± 0.06
4.32 ± 0.90
>30.00
0.58 ± 0.11 (8)
0.51 ± 0.09 (7)
>100.00 (>21)
>100.00 (>27)
86.97 ± 0.99 (19)
>100.00 (>27)
CH
H
C(CH
H
3
CH
CH
CH
CH
3
3
2
2
3 3
)
CH
CH
3
2
0.64 ± 0.13 (10)
0.57 ± 0.02 (8)
>100.00 (>15)
>100.00 (>23)
>100.00 (>15)
>100.00 (>23)
CH
3
H
CHCH
2
H
NT9
benzyl
CH Cl
C(F)
CH(Br)CH
CH OCH
C(CH CH
3,5 dichlorobenzyl
H
H
H
H
H
CH
H
>30.00
NT10
NT11
NT12
NT13
NT14
NT15
NFX
2
16.12 ± 0.97
8.87 ± 0.70
17.41 ± 1.08
>30.00
>30.00
16.26 ± 1.24
4.12 ± 0.13
13.53 ± 0.50 (1) 18.33 ± 0.68 (1)
11.24 ± 0.58 (1) >100.00 (>11)
8.37 ± 1.84 (2) 19.83 ± 0.44 (1)
7.68 ± 0.56 (<1)
>100.00 (>11)
21.53 ± 0.19 (1)
3
3
2
3
3
)
2
3
3
8.17 ± 0.61 (2)
0.31 ± 0.06 (13)
>100.00 (>6)
>100.00 (>24)
6.75 ± 1.08 (<1)
64.80 ± 1.50 (16)
206

Figure 1. Activity of TbNTR toward different nitrothiazoles. (A) Samples obtained during purification of recombinant TbNTR were
analysed on by SDS-PAGE (10 %) stained with Coomassie blue. E. coli crude extract (lane 1) was loaded onto a Ni-NTA column and the flow
through (lane 2) collected. The column was washed with 50 mM imidazole (lane 3) and 100 mM imidazole (lane 4) containing buffers.
Recombinant protein was eluted in a buffer containing 500 mM imidazole; 0.5 % Triton X-100 (lane 5). Markers (M) are in kiloDaltons. The
~30 kDa band corresponding to recombinant TbNTR is indicated. (B) Activity of purified recombinant TbNTR was assessed by using
nitrothiazoles (NT1-15) as substrate (100 M) at a fixed concentration of NADH (100 M). Enzyme activity, expressed in nmoles of NADH
oxidised per minute per mg TbNTR, was then calculated using an ε value of 6,220 M-1 cm . Nfx (nifurtimox) was used as control and enzyme
activity determined as previously described (7). The enzyme activity values are the means of data from 3 assays ± standard deviations.
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Figure 2. Susceptibility of bloodstream form T. brucei over expressing TbNTR to nitrothiazoles. Dose-response curves of T. brucei (solid
line) and parasites expressing an ectopic copy of Tbntr (dashed line) towards representative nitrothiazoles. The growth inhibitory effect
expressed as IC50 values was determined (see Table 2). All data points are mean values ± standard deviations from experiments performed in
quadruplicate. Nifurtimox was used as drug control.