Discovery of potent nitrotriazole-based antitrypanosomal agents: In vitro and in vivo evaluation

Article abstract of DOI:10.1016/j.bmc.2015.08.014

3-Nitro-1H-1,2,4-triazole- and 2-nitro-1H-imidazole-based amides with an aryloxy-phenyl core were synthesized and evaluated as antitrypanosomal agents. All 3-nitrotriazole-based derivatives were extremely potent anti-Trypanosoma cruzi agents at sub nM con

Full text of DOI:10.1016/j.bmc.2015.08.014

Accepted Manuscript
Discovery of potent nitrotriazole-based antitrypanosomal agents: In vitro and
in vivo evaluation
Maria V. Papadopoulou, William D. Bloomer, Howard S. Rosenzweig, Ivan P.
O’Shea, Shane R. Wilkinson, Marcel Kaiser, Eric Chatelain, Jean-Robert Ioset
PII:
S0968-0896(15)00681-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.014
BMC 12518
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 May 2015
5 August 2015
13 August 2015
Please cite this article as: Papadopoulou, M.V., Bloomer, W.D., Rosenzweig, H.S., O’Shea, I.P., Wilkinson, S.R.,
Kaiser, M., Chatelain, E., Ioset, J-R., Discovery of potent nitrotriazole-based antitrypanosomal agents: In vitro and
in vivo evaluation, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.08.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Discovery of potent nitrotriazole-based antitrypanosomal agents:
In vitro and in vivo evaluation
Maria V. Papadopouloua*, William D. Bloomera, Howard S. Rosenzweigb, Ivan P.
O’Sheac, Shane R. Wilkinsonc, Marcel Kaiserd,e , Eric Chatelainf, Jean-Robert
Iosetf
b
aNorthShore University HealthSystem, Evanston, IL, US; Oakton Community College, Des Plaines, IL,
c
d
US; School of Biological & Chemical Sciences, Queen Mary University of London, London, UK; Swiss
e
Tropical and Public Health Institute, Parasite Chemotherapy, Basel, Switzerland; University of Basel,
Basel, Switzerland; fDrugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland.
*Correspondence to: Maria V. Papadopoulou, Ph.D., NorthShore University HealthSystem, Department
of Radiation Medicine, 2650 Ridge Ave., Evanston IL, 60201, USA.
Tel: (847)570-2262; Fax: (847)570-1878
e-mail: mpapadopoulou@northshore.org
Running Title: Potent nitrotriazole-based antitrypanosomal agents

2
Abstract: 3-Nitro-1H-1,2,4-triazole- and 2-nitro-1H-imidazole-based amides with an aryloxy-
phenyl core were synthesized and evaluated as antitrypanosomal agents. All 3-nitrotriazole-
based derivatives were extremely potent anti-T. cruzi agents at sub nM concentrations and
exhibited a high degree of selectivity for the parasite. The 2-nitroimidazole analogs were only
moderately active against T. cruzi amastigotes and exhibited low selectivity. Both types of
compound were active against L. donovani axenic amastigotes with excellent selectivity for the
parasite, whereas three 2-nitroimidazole-based analogs were also moderately active against
infected macrophages. However, no compound demonstrated selective activity against T.b.
rhodesiense. The most potent in vitro anti-T. cruzi compounds were tested in an acute murine
model and reduced the parasites to an undetectable level after five days of treatment at 13
mg/kg/day. Such compounds are potential inhibitors of T. cruzi CYP51 and, being excellent
substrates for the type I nitroreductase (NTR) which is specific to trypanosomatids, work as
prodrugs and constitute a new generation of effective and more affordable antitrypanosomal
agents.
Key words: nitrotriazoles; nitroimidazoles; type I nitroreductase; antitrypanosomal agents;
Chagas disease; L. donovani
1Abbreviations: NTD, Neglected tropical diseases; T. brucei, Trypanosoma brucei; HAT,
human African trypanosomiasis; T. cruzi, Trypanosoma cruzi; Bnz, benznidazole (N-benzyl-2-
(2-nitro-1H-imidazol-1-yl)acetamide); Nfx, nifurtimox (4-(5-nitrofurfurylindenamino)-3-
methylthio-morpholine-1,1-dioxide); NTR, type I nitroreductase; TcNTR, T. cruzi NTR;
TbNTR, T. brucei NTR; CYP51, sterol 14α-demethylase enzyme; TcCYP51, T. cruzi CYP51;
IC50, concentration for 50% growth inhibition; SI, selectivity index; SAR, structure-activity
relationships; TDR, Tropical Diseases Research (http://www.who.int/tdr/en/).

3
1. Introduction
Parasitic infections caused by trypanosomatids constitute a major health problem in mainly
resource poor countries around the world and diseases acquired by such infections are considered
‘neglected’ because they have received limited funding for discovery, development and delivery
of new treatments. Amongst neglected tropical diseases (NTD)1, human African trypanosomiasis
(HAT; caused by T. b. rhodesiense and T. b. gambiense) is endemic throughout sub-Saharan
Africa while American trypanosomiasis (Chagas disease; caused by T. cruzi) affects populations
in South and Central America. In contrast, leishmaniasis (caused by Leishmania species) is
prevalent in many sub-tropical and tropical regions of the World [1]. It is estimated that together
these three insect transmitted diseases are responsible for more than 110,000 deaths per year [2].
Although the incidence of T. cruzi infection has significantly declined recently due to
implementation of vector control initiatives, the number of cases in non-endemic sites (United
States, Australia, Europe and Japan) is rising, primarily due to international population migration
and contaminated blood transfusions [3,4].
The treatment of neglected diseases is based on pharmaceuticals with poor efficacy,
significant side effects, high cost, poor oral bioavailability requiring a parenteral administration,
long treatment course and emergence of resistance. Thus, severe toxicity and long treatment
requirements coupled with limited efficacy are associated with nifurtimox (Nfx) and
benznidazole (Bnz), the two currently used medications for Chagas disease [5, 6]. Similarly,
drugs used to treat HAT and leishmaniasis are highly toxic (e.g. melarsoprol, antimonials), or
require i.v. administration (e.g. melarsoprol, suramin, DFMO, antimonials) resulting in severe
side effects, or are of high cost (e.g. DFMO, liposomal amphotericin B, miltefosine, and

4
paromomycin) [7-9]. Therefore, new effective, safe and affordable drugs are urgently needed for
the treatment of these neglected diseases.
Inhibitors of the fungal sterol 14α-demethylase enzyme (CYP51) and currently inhibitors of
the orthologous enzyme T. cruzi CYP51 (TcCYP51) have demonstrated promising efficacy
against Chagas disease in preclinical studies [10-15]. However, in patients with chronic Chagas
disease, the antifungal agent posaconazole was shown to be inferior to Bnz based on qPCR
detection of T. cruzi DNA, measured 10 months after the end of treatment [16]. Moreover, in
newly designed in vitro assays, nitroheterocyclic compounds were more efficacious trypanocidal
agents compared to CYP51 inhibitors [17], and in an acute murine infection model with Bnz and
posaconazole administered concomitantly or in sequence, a positive interaction was observed
between the two drugs [18]. These data suggest that inhibitors of sterol biosynthesis may not be
as efficient as single chemotherapeutic agents against Chagas disease as they are against fungal
infections, but they may be effective in combination with known or new nitroheterocyclic
antichagasic agents. However, combining agents in vivo cannot always be effective due to
differential pharmacokinetic behavior of each drug, failure to obtain an optimal required
concentration and possible unexpected adverse effects. Combining CYP51 inhibition
characteristics with bioreduction properties of a nitro-group in one molecule may offer a better
solution in drug development against Chagas disease and other trypanosomatid-induced diseases.
This strategy has been successfully used in numerous occasions, including drugs for cancer,
asthma and chronic obstructive pulmonary disease as well as neurodegenerative diseases [19-22].
We have demonstrated that a variety of 3-nitro-1H-1,2,4-triazole-based compounds exhibit
excellent anti-T. cruzi activity both in vitro and in vivo, with several analogs also showing
appreciable anti-T. b. rhodesiense activity in vitro [23-27]. Interestingly, 3-nitrotriazole-based

5
compounds are significantly more potent and less toxic than their 2-nitroimidazole-based
counterparts [23-27], with part of the trypanocidal activity being dependent on the parasite’s
expression of an oxygen-insensitive type I nitroreductase (NTR), an enzyme absent from most
other eukaryotes [23, 24, 26, 27]. Type I NTR, via a series of 2 electron reduction reactions
which lead to the production of toxic metabolites, is responsible for the trypanocidal activity of
Nfx, Bnz and other nitroheterocyclic prodrugs in general [29-32]. More recently, we have
developed 3-nitrotriazole-based amides with a linear, rigid core, as well as 3-nitrotriazole-based
carbinols (fluconazole analogs) which, in addition to being substrates for type I NTR, are also
inhibitors of TcCYP51 [33]. These bifunctional agents exhibit promising anti-T. cruzi activity in
vitro and in vivo and have excellent ADMET characteristics [33].
In the current work we have expanded our search for more potent bifunctional antichagasic
agents by investigating the role of an aryloxy-group in the scaffold of 3-nitrotriazole-based linear
amides. Imidazole-based inhibitors of TcCYP51 with an aryloxy group in their scaffold have
been previously identified [15]. Therefore, we hypothesized that 3-nitrotriazole-based analogs
could potentially be inhibitors of this enzyme, in addition to their capacity of being substrates of
type I NTRs.
2. Results and Discussion
2.1. Chemistry
The structures of synthesized compounds are shown in Table 1. Their synthesis is
straightforward, based on well-established chemistry and outlined in Scheme 1. Thus, amides 2-
14 were obtained by nucleophilic substitution of the appropriate chloroacetamides 1a-h with the
potassium salt of 3-nitro-1,2,4-triazole, 2-nitroimidazole or 1,2,4-triazole under refluxing

6
conditions. Chloroacetamides 1a-h were prepared from appropriate, commercially available
aryl/alkylamines and chloroacetyl chloride in the presence of triethylamine at room temperature.
Table 1. In vitro antiparasitic activity, host toxicity and physical properties of tested compounds.

7
aT.b. rhodesiense, strain STIB 900 trypomastigotes; b T. cruzi, strain Tulahuen C4 amastigotes; c
L. donovani axenic, strain MHOM-ET-67/L82 amastigotes; dCytotoxicity in the host L6 cells. SI
is the ratio: IC50 in L6 cells/IC50 in each parasite. The highest concentration tested in L6 cells
was 100 μg/mL and no solubility problems were observed for any compound. Reference drugs:
Melarsoprol (Melar.), Benznidazole (Bnz), Miltefosine (Milt.). The IC50 value of each reference
drug is the mean from multiple measurements in parallel with the compounds of interest.
Bnz/comp is the ratio of IC50 values against T. cruzi obtained by Bnz and each compound. PSA:
polar surface area. All physical properties were predicted by using the Marvin Calculator
(www.chemaxon.com). IC50 values are means of 2 to 3 measurements. The SD was < 5%.
Scheme 1: Synthesis of compounds on Table 1.

8
2.2. Biological Evaluation
2.2.1. Anti-parasitic activity
Compounds on Table 1 were screened for anti-parasitic activity against three trypanosomatids: T.
cruzi, T. b. rhodesiense and Leishmania donovani. The concentration of compound that inhibits
parasite growth by 50 % (IC50) was calculated from dose response curves for each parasite
(Table 1). Compounds were also tested for toxicity in L6 rat skeletal myoblasts, the host cells for
T. cruzi amastigotes, in order to calculate a selectivity index for each parasite (SI =
IC50L6/IC50parasite) (Table 1). According to the TDR (Special Programme for Research and Training
in Tropical Diseases, World Health Organization) criteria for antiparasitic activity and
selectivity, an IC50 of <4.0 µM, between 4.0-60 µM or >60 µM, designates ‘active’, ‘moderately
active’ or ‘inactive’ compounds, respectively, against T. cruzi amastigotes, while a SI of ≥50 is
required; for blood stream form (BSF) T. b. rhodesiense, IC50 values of <0.5 µM, between 0.5-
6.0 µM or > 6.0 µM identify ‘active’, ‘moderately active’ or ‘inactive’ compounds, respectively,
while a SI value of ≥100 is desired; finally, for L. donovani amastigotes, IC50 of <1 µM, between 1.0-
6.0 µM or > 6.0 µM, provides ‘active’, ‘moderately active’ or ‘inactive’ compounds, respectively,
whereas a SI value of ≥20 is ideal [34].
According to the criteria described above, all 3-nitrotriazole-based analogs, except 11, were
selectively active against T. cruzi amastigotes, whereas the 2-nitroimidazole-based analogs 12-
14, although active against T. cruzi, exhibited unacceptable SI values (Table 1). Compound 5,
the des-nitro analog of 3, was moderately active against T. cruzi but was also not selective
against the parasite.

9
Several 3-nitrotriazole-based compounds (2-4, 6, 9, 10) and one 2-nitro-imidazole-based
analog (13) were moderately active against T. b. rhodesiense, however, only the chloro-
biphenylamide 6 demonstrated an acceptable selectivity for this parasite (Table 1).
Interestingly and, for the first time, both 3-nitrotriazole-based- and 2-nitroimidazole-based
analogs demonstrated a remarkable and selective activity against L. donovani amastigotes (Table
1). Thus, only the des-nitro analog of 3, namely compound 5, the phenylpyrimidinylacetamide 8
and the N-benzylpiperidine-amide 11 weren’t active against L. donovani.
2.2.2. SAR analysis of antichagasic activity
The unsubstituted 3-nitrotriazole-based aryloxy-phenylamide 2 was a very potent anti-T. cruzi
agent displaying an IC50 of 65 nM against this parasite, 31-fold lower than that observed using
Bnz, and having a SI of 1416. When a p-chloro group was introduced in the terminal phenyl of
2, the anti-T. cruzi potency increased by 8-fold and the SI by 2.6 in analog 3. Therefore,
compound 3 was 254-fold more active than Bnz, and represents the most potent analog in this
series. This increase in potency may be related to the increased lipophilicity of 3 compared to 2
(Table 1). However, the presence of the nitro-group was far more important for activity than
lipophilicity. Therefore, the des-nitro analog of 3, compound 5, albeit more lipophilic than 3, was
only moderately active against T. cruzi, while its increased lipophilicity resulted in an
unfavorable SI (<50). The fact that compound 5 demonstrated moderate activity against T. cruzi
also indicates an alternative mechanism of action, independent of nitroreduction.
The fluoro-analog of 3, compound 4, was also more potent against T. cruzi than the
unsubstituted compound 2, with an IC50 value of 36 nM and a SI of 1954. However, 4 was 4.5-

10
fold less potent than 3, presumably due to its lower lipophilicity, but still 56-fold more potent
than Bnz (Table 1).
To better understand the role that the ether link in 3 plays in mediating anti-T. cruzi activity,
we included some biphenyl/biaryl compounds in the study. The biphenyl compound 6 has been
described before by our group [33]. Compound 6, which shares a similar lipophilicity with its
aryloxy-analog 3, was also very potent and selective against T. cruzi, however its potency was
ca. 5.6- fold lower than that of 3, signifying the importance of the ether link in the core of these
compounds for anti-T. cruzi activity. Similarly, the biphenyl compound 7 was about 2.8-fold less
potent against T. cruzi compared to the unsubstituted aryloxy-compound 2 and 4-fold less potent
than 6. In the latter case the drop in activity of 7 may be related to its significantly lower
lipophilicity compared to 6 rather than the meta-position of the terminal phenyl group.
When the second phenyl ring in 7 was replaced with a pyrimidine ring in 8, the anti-T. cruzi
activity was reduced by a factor of 14, despite the fact that both 7 and 8 share a similar clogP
value. However, a greater PSA value in 8 may negatively affect permeability and thus activity
[35].
Replacing the phenoxy-group of 2 with a benzyl-group in 9 did not affect anti-T. cruzi
activity. However, the SI of 9 was reduced >2.5, due to higher toxicity in host cells and was
independent of lipophilicity since both compounds share a similar clogP value (Table 1).
Inserting an oxygen in 9 to obtain the benzyloxy-analog 10 increased anti-T. cruzi activity and
selectivity compared to both analog compounds 9 and 2, consistent with an increase in the
lipophilicity of 10. In addition, 10 was about 2-fold less toxic than 9, presumably due to its
slightly greater PSA value which may decrease permeability [35]. Furthermore, when the
anilino-ring of 9 was replaced with a 4-piperidine-amino ring in 11, disturbance in the planarity

11
decreased significantly the activity against T. cruzi, resulting in an IC50 of 8 μM and an
unacceptable SI of ca. 14 for compound 11.
For comparison purposes we have synthesized and evaluated the anti-T. cruzi activity of 2-
nitroimidazole-based aryloxy-phenyl amides 12-14, analogs of 2-4, respectively. Although
compounds 12-14 were more lipophilic than their 3-nitrotriazole-based analogs 2-4, they
demonstrated significantly less activity (65- to 324-fold) against T. cruzi. In addition, albeit
compounds 12-14 were active/moderately active according to the TDR criteria, they
demonstrated an unfavorable SI, confirming again the superiority of the 3-nitrotriazole- versus
the 2-nitroimidazole ring against T. cruzi. From the above discussion it is concluded that a 3-
nitrotriazole ring coupled with a aryloxy-phenyl core is important for potent antichagasic
activity and superior selectivity. With the exception of compound 8, there was also a good
correlation between anti-T. cruzi activity and lipophilicity among the 3-nitrotriazole-based
analogs. Thus, antichagasic activity increased with increasing clogP value (Fig.1s in
supplementary material). Good correlation also existed between lipophilicity and antichagasic
activity in the 2-nitroimidazole-based analogs, although the limited number of analogs does not
allow definitive conclusions.
2.2.3. SAR analysis of anti-T. brucei activity
Moderate activity was observed against BSF of T. b. rhodesiense with several analogs (2-4, 6, 9,
10, 13). Only the 3-nitrotriazole-based biphenylamide 6, however, demonstrated an acceptable
selectivity (>100). Interestingly, there was relatively good correlation between anti-T. cruzi and
anti-T. b. rhodesiense activity (R2 = 0.77) among nitrotriazoles active against both parasites (Fig.
2s in supplementary material), something that was not observed before with other 3-nitrotriazole-

12
based scaffolds [23-25, 33]. Thus, anti-T. b. rhodesiense activity (similarly to anti-T. cruzi
activity) increased as the clogP increased with chloro-substitution enhancing potency in
comparison to fluoro- or no-substitution (compare 2, 3 and 4 in the 3-nitrotriazole-based
subgroup, and compare 12, 13 and 14 in the 2-nitroimidazole-based subgroup). Although there
was relatively good correlation between T. cruzi and T. b. rhodesiense IC50 values for the diaryl
derivatives 6-8 (R2=0.87), the 3-biphenyamide 7 was inactive against T. b. rhodesiense, in
contrast to its 4-biphenyl analog 6, which displayed moderate activity and selectivity. Similarly,
the 4-phenyl-pyrimidine amide 8 was inactive against T. b. rhodesiense. Once again, the des-
nitro analog of 3, compound 5, was deemed “inactive” against T. b. rhodesiense based on the
TDR criteria, although the trypanocidal effect it did display made it more potent than the nitro-
compounds 7 and 8, suggesting that the latter two may not be such good substrates for TbNTR.
2.2.4. SAR analysis of anti-L. donovani activity
This is the first report of 3-nitrotriazole- and 2-nitroimidazole-based compounds demonstrating
selective anti-L. donovani activity. If we consider the active/moderately active 3-nitrotriazole-
based analogs with the exception of 10, there was a relatively good correlation between anti-T.
cruzi and anti-L. donovani activity (R2 = 0.82), whereas in the case of compounds with better
structural similarity we observe an even better correlation, although the number of analogs in
these cases is inadequate for correlations. In addition, increasing lipophilicity resulted in an
increased anti-L. donovani activity (Table 1). Chloro-substitution was more beneficial for
activity than fluoro- or no substitution (compare 2, 3 and 4, as well as 12-14). The ether link in
the core was more beneficial for activity than the methylene- or methylenoxy-link (2 vs 9 and 10)
or no link at all (3 vs 6). Disturbance in the planarity of the core in 11, or deletion of the nitro

13
group in 5, abolished anti-L. donovani activity. Similarly, replacement of the middle phenyl ring
in 7 with a pyrimidine ring in 8, resulted in abolishment of the anti-L. donovani activity (Table
1). Two 3-nitrotriazoles (4 and 6) and one 2-nitroimidazole (13) demonstrated similar activity to
the reference compound miltefosine, whereas the 3-nitrotriazole-based p-chlorophenyloxyphenyl
amide 3 was ca. 4.8-fold more potent than miltefosine, with a SI of 853. Compounds 2, 12 and
14 were selectively active against this parasite at nM concentrations, but slightly less potent than
miltefosine. Finally, compounds 7, 9 and 10 were moderately active and with acceptable
selectivity against the parasite (Table 1). If we compare the corresponding 3-nitrotriazole-based
and 2-nitroimidazole-based analogs, the former were 1.6 to 4-fold more active than the latter
against L. donovani parasites (2 vs 12, 3 vs 13 and 4 vs 14).
Table 2. Antileishmanial activity and toxicity in infected macrophages
Activity in
Toxicity in
L.don. axen.
ID No
SI
Macrophages Macrophages Milt./Comp.
µ
IC50
M
µ
µ
M
IC50
M
IC50
Milt. 0.162 ± 0.017
6.894
>30
2
3
4
0.27
0.034
0.18
339
853
384
89
0.60
4.8
>27
80
84
>28
0.89
6
9
0.17
1.2
738
31
>84
>30
16
>84
>89
>89
0.95
0.12
0.21
12
0.79
79
13
14
0.14
0.29
937
418
15
14
>81
>84
1.2
0.55
Milt./Comp. is the ratio of the coresponding IC50 values against L. don. axen. amastigotes.

14
Compounds with substantial selective activity against L. donovani axenic amastigotes were
also tested for activity and toxicity in infected macrophages, a model more suitable for drug
screening (36), since it takes into account host cell-mediated effects (Table 2). Despite activity
against L. donvani axenic amastigotes, the 3-nitrotriazole-based analogs (2-4, 6 and 9) were not
particularly active in infected macrophages, whereas the 2-nitroimidazole-based analogs (12-14)
demonstrated moderate activity (IC50 <20 μM) in this assay. Reduced intracellular activity might
be associated with metabolic stability of the compounds in the macrophages or permeability
issues, although permeability studies through Caco-2 cells with some analogs did not indicate
any permeability problems.
2.2.5. Involvement of type I Nitroreductase
Representative analogs from Table 1 were evaluated as substrates of purified, recombinant
trypanosomal NTRs and compared to benznidazole (Table 3). Enzyme specific activity was
measured as oxidized NADH per min per mg of protein. All tested compounds were excellent
substrates of both TcNTR and TbNTR and metabolized by the T. cruzi enzyme at rates
comparable or better than that of Bnz. However, the same analogs were metabolized by TbNTR
at rates 1.6 to 2.2-fold higher than that of Bnz (Table 3). When these studies were extended to
investigate the role of NTR within the parasite itself, BSF T. b. brucei was used and induced to
express elevated levels of TbNTR. T. b. brucei is the model organism for all three sub-species (T.
b. rhodesiense, T. brucei gambiense and T. b. brucei) and can be handled in a level 2
containment lab (with appropriate clearance from Ministry of Agriculture) whereas T. b.
rhodesiense can only be used in a level 3 containment lab. In addition, the T. b. brucei genome
has been sequenced and recombinant DNA technology is well advanced (gene replacement,

15
inducible/constitutive gene expression, RNAi etc) allowing investigation of potential drug mode
of action. Such technology cannot be done by using T. b. rhodesiense.
Table 3. Activity of recombinant TbNTR and TcNTR towards selected analogs of Table 1.
NTR-overexpressing T. b. brucei were ca. 2- to 5-fold more sensitive to most of the tested
compounds with 6 being the exception (Table 4). However, there was no good correlation
between TbNTR/TcNTR enzymatic activity and specific antitrypanosomal activity. This,
coupled with the modest fold difference in susceptibility observed between cells expressing
different levels of TbNTR in comparison to the larger changes noted when using other
nitroheterocycles such as Nfx, indicates that this oxidoreductase may not be the only activator of
the aryloxy-phenylamide prodrug within the parasite or that other factors (e.g. permeability into
mitochondrion, compound stability and pharmacokinetic factors in general) may play a role in
the antiparasitic activities.

16
Table 4. Antimicrobial activity of selected nitrotriazoles against BSF of T. b. brucei
2.2.6. TcCYP51 inhibition
Azole-based scaffolds are effective inhibitors of the T. cruzi enzyme sterol 14α-demethylase,
TcCYP51 [37]. In addition, aryloxybenzylimidazoles have been described as CYP51 inhibitors
[15]. Therefore, in silico docking studies were performed by using the ligand binding pocket of
the crystal structure of 4H6O sterole-14α demethylase and Glide docking protocols, to
investigate whether or not the present compounds bind to the protein as the reference compound
15 (Fig. 1) [15]. The docking scores, which were a function of –logKd, are shown in Table 5.

17
Table 5. Docking scores of compounds in Table 1
and reference 15, in the ligand binding pocket of
4H6O sterole-14α demethylase. The score is a
function of –logKd.
Docked Score in the 4H6O
Compound
Ligand Binding Pocket
2
3
9.63
9.82
9.91
5.48
8.93
7.86
6.65
7.54
7.23
6.13
6.83
6.52
6.98
8.96
4
5
6
7
8
9
10
11
12
13
14
15
The 3-nitrotriazole-based aryloxyphenylamides 2-4 showed a higher docking score than the
reference compound 15, whereas the more rigid 3-nitrotriazole-based biphenylamide 6 showed a
similar docking score with 15. The rest of the compounds demonstrated docking scores lower
than compound 15, with compound 5 (the des-nitro analog of 3) showing the lowest docking
score (Table 5). Examples of docked poses for compounds 3 and 15 are given in Fig. 1, which

18
show coordination between the heme iron of the enzyme and the N4 or N3 of the nitrotriazole
and imidazole rings, respectively.

19
Fig. 1. Docked poses of compound 3 (top) and reference compound 15 (bottom). The
distance (in Å) is shown between the iron atom of the heme and the N4 (top) or N3 (bottom)
of the nitrotriazole and imidazole rings, respectively.
A distance of 2.0 and 2.76 Å is shown between the iron atom and the corresponding nitrogen
atom for compounds 3 and 15, respectively. The docking score seems to be affected primarily by
the distance between the N4 of the nitrotriazole ring and the iron atom of the heme in the active
site of the enzyme, whereas the orientation of the core group of each molecule can change
significantly, making a SAR correlation much more complicated in this case.
There was good correlation between anti-T. cruzi activity (IC50 values against T. cruzi) and
docking score (Fig. 2), suggesting that at least for compounds 2-4 and 6 the antichagasic activity
may be also related to T. cruzi CYP51 inhibition, an assumption than needs to be verified.
2.5
y = 0.6671x - 4.739
2
R² = 0.8191
i
z
1.5
u
r
c
T.
1
0.5
0
0
2
4
6
8
10
12
-0.5
-1
-1.5
Docking Score

20
Fig. 2. Correlation between antichagasic activity and docking scores.
2.2.7. In vivo antichagasic activity
The most active compounds (3, 4 and 6) against T. cruzi in vitro were evaluated for in vivo anti-
chagasic activity by using an acute T. cruzi infected murine model. Bnz was used in parallel as a
positive control. Groups of 5 mice/group were treated i.p. for 10 consecutive days with each
compound, at 13 mg/kg/day. In our previous work with 3-nitrotriazole-based rigid amides we
have seen in vivo antichagasic activity at daily doses of 15 mg/kg, therefore we decided to use a
slightly lower dose for the present tested analogs which demonstrate in vitro anti-T. cruzi activity
at low nM concentrations (Table 1). The mean ratio of parasite levels was calculated after 5 and
10 days of treatment. The data are summarized in Fig. 3 and images are presented in Fig. 4.
10
8
Day 5/Day 1
6
Day 10/ Day 1
4
2
0
Control
BNZ
comp. 3 comp. 4 comp. 6
Fig. 3. In vivo evaluation of the antichagasic efficacy of compounds 3, 4, 6 and benznidazole in
the acute murine model. All compounds (including Bnz) were administered i.p. at 13 mg/kg/day

21
for 10 consecutive days. Parasite ratios were calculated after 5 and 10 days of treatment. The P
values between each treated group and control group were < 0.0001. Groups of 5 mice/group
were used.
Vehicle
Comp. 3
Comp. 4
Fig. 4. Images from mice treated with vehicle or compounds 3 and 4 after 1, 5 and 10 days of
treatment.
All tested compounds weren’t toxic at the given dose and time-frame and all, including Bnz,
reduced the parasite load to undetectable levels (Fig. 4) after 5 days of treatment with statistical
significance (p < 0.0001).
3.
Conclusions:
3-Nitrotriazole-based aryloxyphenylamides are very potent and selective anti-T. cruzi agents
both in vitro and in vivo, being able to reduce the parasite load to undetectable limits after just 5
days of treatment of infected mice, at the very small i.p. dose of 13 mg/kg/day (Figures 3 and 4).

22
In addition, limited ADMET studies demonstrated that these compounds are metabolically stable
and have a very good Caco-2 permeability, suggesting a favorable profile regarding oral
administration (an example is given in the Supplementary material). However, since activity in
the acute model is not predictive for efficacy in chronic indeterminate Chagas patients, future
experiments in a chronic murine model will be necessary to validate these agents for human use.
Additionally, 2-nitro-imidazole-based aryloxyphenylamides demonstrated promising and
selective antileishmanial properties in the macrophage model (which is more appropriate for
drug screening) exhibiting IC50 values similar or better than pentostam (36). Therefore, this
scaffold should be further explored for lead drug optimization.
Both types of compounds act as substrates for trypanosomal type I NTR, whereas the 3-
nitrotriazole-based analogs demonstrated slightly better docking scores for 4H6O sterol-14α
demethylase than the reference compound 15, suggesting that the antichagasic properties of these
compounds may be partially attributed to T. cruzi CYP51 inhibition, but this needs to be
confirmed with enzymatic studies.
Finally, although the present compounds should be evaluated for mutagenicity/genotoxicity
due to their nitro group, it is worth mentioning that previous studies have shown that 3-
nitrotriazole-based amides are not mutagenic compared to their 2-nitroimidazole-based analogs
[26], and do not cause developmental toxicity in zebrafish [28]. Therefore, 3-nitrotriazole-based
aryloxyphenylamides constitute a new generation of potentially effective and more affordable
antichagasic agents in humans.
4. Experimental
4.1. Chemistry

23
4.1.1. General
All starting materials and solvents were purchased from Sigma-Aldrich (Milwaukee, WI), were
of research-grade quality and used without further purification. Solvents used were anhydrous
and the reactions were carried out under a nitrogen atmosphere and exclusion of moisture.
Melting points were determined by using a Mel-Temp II Laboratory Devices apparatus
(Holliston, MA) and are uncorrected. Proton NMR spectra were obtained on a Varian Inova-500
or an Agilent Hg-400 spectrometer at 500 or 400 MHz, respectively, and are referenced to Me4Si
or to the corresponding solvent, if the solvent was not CDCl3. High-resolution electrospray
ionization (HRESIMS) mass spectra were obtained on a Agilent 6210 LC-TOF mass
spectrometer at 11000 resolution. Thin-layer chromatography was carried out on aluminum
oxide N/UV254 or polygram silica gel G/UV254 coated plates (0.2 mm, Analtech, Newark, DE).
Chromatography was carried out on preparative TLC alumina GF (1000 microns) or silica gel
GF (1500 microns) plates (Analtech). All compounds were purified by preparative TLC
chromatography on silica gel or alumina plates and also checked by HPLC (Agilent 1100)
coupled to mass spectrometry (MS) and ultraviolet (UV) absorbance detectors. Purity was
determined by comparing the area under the curve of the test compound peak to the combined
areas of all peaks in the chromatogram, excluding DMSO and any peaks appearing in blank runs.
Priority reporting was given to the UV traces (since mass spectrometer response can vary widely
depending on a given compound’s ease of ionization). Purity of compounds lacking UV activity
was reported using mass spectrometry detection. Purity was ≥ 95% purity for all analogs.
4.1.2. Synthesis of chlorides 1a-h:
Some of the chlorides 1a-h are known in the literature and others are unknown or known through

24
chemical libraries but without any reference. The synthesis and spectroscopic data of 1a-h are
provided in the supporting material.
4.1.3. General Synthetic Procedure of amides 2-14:
The potassium salt of 3-nitro-1,2,4-triazole or 2-nitroimidazole or 1,2,4-triazole (1 eq) was formed in
CH3CN (6-10 mL), by refluxing with KOH (1.2 eq) for 30 min. To this suspension, 1a-h (1.1 eq) was
added and the reaction mixture was refluxed under a nitrogen atmosphere for 9 h. In certain cases, 1a-h
was added in CH3CN solution. The reaction mixture was checked by TLC for completion of the reaction
and the solvent was evaporated. The residue was dissolved in ethyl acetate and the inorganic salts were
filtered away. Upon preparative TLC (silica gel or alumina, depending on the mobility of the major band;
ethyl acetate-petroleum ether), the desired product was obtained usually as a powder. Purity was checked
also by HPLC and it was > 95%.
4.1.3.1. 2-(3-nitro-1H-1,2,4-triazol-1-yl)-N-(4-phenoxyphenyl]acetamide (2): Off white
microcrystallic powder (89%): mp 156-158 oC ; 1H NMR (500 MHz, (CD3OD) : 8.70 (s, 1H), 7.56 (d, J =
9.5 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.09 (t, J = 8.0 Hz, 1H), 6.97 (d, J = 9.0 Hz, 4H), 5.28 (s, 2H).
HRESIMS calcd for C16H13N5NaO4 m/z [M+Na]+ 362.0860 found 362.0863.
4.1.3.2. N-[4-(4-chlorophenoxy)phenyl]-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (3): White
microcrystallic powder (52%): mp 167-169 oC; 1H NMR (500 MHz, (CD3OD) : 8.70 (s, 1H), 7.58 (d, J =
9.5 Hz, 2H), 7.32 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 5.29 (s, 2H).
HRESIMS calcd for C16H12ClN5NaO4 m/z [M+Na]+ 396.0475, 397.0527, 398.0512, 399.0514 found
396.0470, 397.0526, 398.0514, 399.0515.

25
4.1.3.3. N-[4-(4-fluorophenoxy)phenyl]-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (4): Off white
o
1
powder (80%): mp 179-181 C (dec); H NMR (500 MHz, (CD3COCD3) : 9.68 (br s, 1H), 8.73 (s, 1H),
7.63 (d, J = 9.5 Hz, 2H), 7.14 (t, J = 9.0 Hz, 2H), 7.04 (m, 2H), 6.98 (d, J = 9.0 Hz, 2H), 5.40 (s, 2H).
HRESIMS calcd for C16H12FN5NaO4 m/z [M+Na]+ 380.0766 , 381.0793, found 380.0767, 381.0791.
4.1.3.4. N-[4-(4-chlorophenoxy)phenyl]-2-(1H-1,2,4-triazol-1-yl)acetamide (5): Off white powder
(88%): mp 157-159 oC; 1H NMR (500 MHz, (CClD3) : 8.32 (br s, 1H), 8.26 (s, 1H), 8.14 (s, 1H), 7.44 (d,
J = 8.5 Hz, 2H), 7.28 (d, J = 9.5 Hz, 2H), 6.97 (d, J = 9.5 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.02 (s, 2H).
HRESIMS calcd for C16H14ClN4O2 and C16H13ClN4NaO2 m/z [M+H]+ and [M+Na]+ 329.0800, 331.0776 and
351.0619, 353.0595 found 329.0804, 331.0784 and 351.0629, 353.060.
4.1.3.5. N-([1,1'-biphenyl]-3-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (7): White powder (72%):
o
1
mp 121-123 C; H NMR (400 MHz, CD3COCD3) δ: 9.77 (br s, 1H), 8.75 (s, 1H), 7.99 (s, 1H),
7.64-7.35 (m, 8H), 5.45 (s, 2H). HRESIMS calcd for C16H14N5O3 and C16H13N5NaO3 m/z [M+H]+ and
[M+Na]+ 324.10912 and 346.0911, found 324.1099 and 346.0915.
4.1.3.6. 2-(3-nitro-1H-1,2,4-triazol-1-yl)-N-(4-phenylpyrimidin-2-yl)acetamide (8): Off white powder (55
%): mp 175-177 oC (dec); 1H NMR (400 MHz, (CD3COCD3) : 10.04 (br s, 1 H), 8.74 (d, J = 5.2 Hz, 1H),
8.73 (s, 1H), 8.26 (dd, J = 8.4, 1.2 Hz, 2H), 7.77 (d, J = 5.2 Hz, 1H), 7.58-7.53 (m, 3H), 6.03 (s, 2H).
HRESIMS calcd for C14H12N7O3 m/z [M+H]+ 326.0996, found 326.0995.
4.1.3.7. N-(4-benzylphenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (9): White microcrystallic
o
1
powder (71%): mp 166-168 C; H NMR (400 MHz, (CD3COCD3) : 8.72 (s, 1H), 7.55 (dd, J = 8.8, 0.8
Hz, 2H), 7.29-7.16 (m, 7H), 5.39 (s, 2H), 3.95 (s, 2H). HRESIMS calcd for C17H16N5O3 and C17H15N5NaO3
m/z [M+H]+ and [M+Na]+ 338.1248 and 360.1067, found 338.1250 and 360.1076.

26
4.1.3.8. N-(3-(benzyloxy)phenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (10): White powder (77 %):
1
o
mp 147-149 C; H NMR (400 MHz, (CD3COCD3) : 9.67 (br s, 1 H), 8.73 (s, 1H), 7.55-7.30 (m, 6H),
7.24 (t, J = 8.0 Hz, 1H), 7.14 (dd, J = 8.0, 1.6 Hz, 1H), 6.79 (dd, J = 8.0, 1.5 Hz, 1H), 5.40 (s, 2H), 5.10
(s, 2H). HRESIMS calcd for C17H16N5O4 m/z [M+H]+ 354.1197, found 354.1195.
4.1.3.9. N-(1-benzylpiperidin-4-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (11): White
o
1
microcrystallic powder (68%): mp 152-155 C (dec); H NMR (400 MHz, (CClD3) : 8.37 (s, 1H), 7.33-
7.24 (m, 5H), 5.83 (br d, J = 8.0 Hz, 1H), 4.92 (s, 2H), 3.83 (m, 1H), 3.49 (s, 2H), 2.81 (d, J = 11.6 Hz,
2H), 2.12 (t, J = 11.2 Hz, 2H), 1.91 (d, J = 9.6 Hz, 2H), 1.50 (m, 2H). HRESIMS calcd for C16H21N6O3
m/z [M+H]+ 345.1670 found 345.1677.
4.1.3.10. 2-(2-nitro-1H-imidazol-1-yl)-N-(4-phenoxyphenyl]acetamide (12): Off white powder
(72%): mp 167-169 oC; 1H NMR (500 MHz, (CClD3+ 1 drop of acetone -6d) : 8.97 (br s, 1H), 7.45 (d, J
= 9.5 Hz, 2H), 7.27 (t, J = 7.5 Hz, 2H), 7.19 (s, 1H), 7.16 (s, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.90 (dd, J =
9.0, 7.5 Hz, 4H), 5.22 (s, 2H). HRESIMS calcd for C17H15N4O4 and C17H14N4NaO4 m/z [M+H]+ and
[M+Na]+ 338.1015 and 361.0907, found 338.0974 and 361.0915.
4.1.3.11. N-[4-(4-chlorophenoxy)phenyl]-2-(2-nitro-1H-imidazol-1-yl)acetamide (13): White
microcrystallic powder (46 %): mp 185-186 oC; 1H NMR (500 MHz, (CD3OD) : 7.55 (d, J = 9.0 Hz, 2H),
7.50 (d, J = 1.0 Hz, 1H), 7.32 (d, J = 9.5 Hz, 2H), 7.20 (d, J = 0.5 Hz, 1H), 6.98 (d, J = 9.0 Hz, 2H), 6.94
(d, J = 9.0 Hz, 2H), 5.34 (s, 2H). HRESIMS calcd for C17H14ClN4O4 and C17H13ClN4NaO4 m/z [M+H]+ and
[M+Na]+ 373.0698, 375.0675 and 395.0518, found 373.0696, 375.0672 and 395.0519.

27
4.1.3.12. N-[4-(4-fluorophenoxy)phenyl]-2-(2-nitro-1H-imidazol-1-yl)acetamide (14): Off white powder
(75 %): mp 194-196 oC (dec); 1H NMR (500 MHz, (CD3OD) δ: 7.52 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 1.0
Hz, 1H), 7.20 (d, J = 1.5 Hz, 1H), 7.08 (t, J = 8.5 Hz, 2H), 6.99 (dd, J = 9.5, 5.0 Hz, 2H), 6.95 (d, J = 9.5
Hz, 2H), 5.34 (s, 2H). HRESIMS calcd for C17H13FN4NaO4 m/z [M+Na]+ 379.0813, found 379.0821.
4.2. Biological evaluation.
4.2.1. In vitro screening:
In vitro activity against T. cruzi, T. b. rhodesiense, L. donovani and cytotoxicity assessment
using L6 cells (rat skeletal myoblasts) was determined using a 96-well plate format as previously
described [38]. Data were analyzed with the graphic program Softmax Pro (Molecular Devices,
Sunnyvale, CA, USA), which calculated IC50 values by linear regression from the sigmoidal dose
inhibition curves.
4.2.2. Activity against Leishmania donovani intracellular amastigotes; macrophage assay:
Mouse peritoneal macrophages (4 x 104 in 100 µL RPMI 1640 medium with 10% heat-
inactivated FBS) were seeded into wells of Lab-tek 16-chamber slides. After 24 hrs 1.2 x 105
amastigote Leishmania donovani in 100 µL were added. The amastigotes were taken from an
axenic amastigote culture grown at pH 5.4. Four hrs later the medium containing free amastigote
forms was removed and replaced by fresh medium. Next day the medium was replaced by
medium containing different compound dilutions. Parasite growth in the presence of the drug
was compared to control wells. After 96 hours of incubation the medium was removed and the
slides fixed with methanol for 10 min followed by a staining with a 10% Giemsa solution.
Infected and non-infected macrophages were counted for the control cultures and the ones
exposed to the serial drug dilutions. The infection rates were determined. The results were

28
expressed as % reduction in parasite burden compared to control wells, and the IC50 calculated
by linear regression analysis [39].
4.2.3. In vitro T. brucei brucei antiproliferating assays and susceptibility studies.
The BSF of T. brucei brucei parasites were seeded at 1 x 103 ml-1 in 200 µL of growth medium containing
different concentrations of nitroheterocycle. Where appropriate, induction of the TbNTR was carried out
o
by adding tetracycline (1 µg/mL). After incubation for 3 days at 37 C, 20 µL of Alamar blue was added
to each well and the plates incubated for a further 16 h. The cell density of each culture was determined as
described before [29] and the IC50 established.
4.2.4. Enzymatic activity studies with Type I NTRs.
Recombinant TbNTR and TcNTR were prepared and assayed as previously described [40, 41]. The
specific activity of purified his-tagged TbNTR was assessed spectrophotometrically at 340 nm using
various nitrotriazole substrates (100 µM) and NADH (100 µM) and expressed as nmol NADH oxidized
min-1 mg-1 of enzyme.
4.2.5. Molecular Modeling studies: Docking into the active site of 4H6O sterole-14 alpha
demethylase.
The crystal structure of CYP51 (4H6O.pdb) which is available in protein database was imported
for the analysis and found to be suitable for the docking experiments as there are no missing
atoms found in the active site pocket of the enzyme. Moreover, the “b” values of the atoms are
well within the average value of the crystal structure. 4H6O.pdb was validated with MolProbity
[42] and the structure optimized by using Schrodinger software (Schrodinger LLC, Version 9.2 ,
2014, New York). A MolProbity score of 1.5 (90th percentile; N=5412, 2.70Å 0.25Å) was

29
obtained. MolProbity score combines the clashscore, rotamer, and Ramachandran evaluations
into a single score, normalized to be on the same scale as X-ray resolution.
Next, the Ligand Binding Site (LBS) of the protein was prepared. The 4H6O.pdb is a co-
crystal structure, therefore the bound ligand (reference compound) present in the active site of
the CYP51 structure was extracted out, to visualize the binding pocket. Using the LigPrep tools,
3-D low energetic conformer of each compound was generated at pH = 7.4 1, retaining the
absolute chirality of the compounds.
Docking of the reference compound was carried out in the Glide-XP (extra precision) module
of the Schrodinger software (Glide Docking Tools, Schrodinger Inc, 2014, NY) and the docked
and crystal poses were compared. The RMSD between the two conformers was found to be < 1.5
Å, which validates our docking protocols. Using the same protocols, all other compounds were
docked into the active site pocket of CYP51, the binding poses were visualized and the Glide
scores were collected. In order to cross check the binding poses, we used another docking
engine, the Surflex dock tool implemented in SYBYLx1.3 (Sybylx 1.3, Tripos International,
St.Louis, MO, 2013). Both the Glide and Surflex docking engines generated similar binding
poses.
4.2.6. In vivo antichagasic activity assessment of selected compounds:
For in vivo studies, a Brazilian strain trypomastigotes from transgenic T. cruzi parasites
expressing firefly luciferase were used as described before [26]. Briefly, parasites were injected
in Balb/c mice (105 trypomastigotes per mouse) and three days later mice were anesthesized by
inhalation of isofluorane, followed by an injection with 150 mg/kg of D-luciferin potassium-salt
in PBS. Mice were imaged 5 to 10 min after injection of luciferin with an IVIS 100 (Xenogen,