3-Nitrotriazole-based piperazides as potent antitrypanosomal agents

Article abstract of DOI:10.1016/j.ejmech.2015.08.042

Novel linear 3-nitro-1H-1,2,4-triazole-based piperazides were synthesized and evaluated as antitrypanosomal agents. In addition, some bisarylpiperazine-ethanones which were formed as by-products were also screened for antiparasitic activity. Most 3-nitrotriazole-based derivatives were potent and selective against Trypanosoma cruzi parasites, but only one displayed these desired properties against Trypanosoma brucei rhodesiense. Moreover, two 3-nitrotriazole-based chlorophenylpiperazides were moderately and selectively active against Leishmania donovani. Although the bisarylpiperazineethanones were active or moderately active against T. cruzi, none of them demonstrated an acceptable selectivity. In general, 3-nitrotriazole-based piperazides were less toxic to host L6 cells than the previously evaluated 3-nitrotriazole-based piperazines and seven of 13 were 1.54-to 31.2-fold more potent antichagasic agents than the reference drug benznidazole. Selected compounds showed good ADMET characteristics. One potent in vitro antichagasic compound (3) was tested in an acute murine model and demonstrated antichagasic activity after a 10-day treatment of 15 mg/kg/day. However, neither compound 3 nor benznidazole showed a statistically significant P value compared to control due to high variability in parasite burden among the untreated animals. Working as prodrugs, 3-nitrotriazole-based piperazides were excellent substrates of trypanosomal type I nitroreductases and constitute a novel class of potentially effective and more affordable antitrypanosomal agents.

Full text of DOI:10.1016/j.ejmech.2015.08.042

Accepted Manuscript
3-Nitrotriazole-based piperazides as potent antitrypanosomal agents
Maria V. Papadopoulou, William D. Bloomer, Howard S. Rosenzweig, Ivan P. O'Shea,
Shane R. Wilkinson, Marcel Kaiser
PII:
S0223-5234(15)30226-9
10.1016/j.ejmech.2015.08.042
EJMECH 8078
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 June 2015
Revised Date: 17 August 2015
Accepted Date: 22 August 2015
Please cite this article as: M.V. Papadopoulou, W.D. Bloomer, H.S. Rosenzweig, I.P. O’Shea, S.R.
Wilkinson, M. Kaiser, 3-Nitrotriazole-based piperazides as potent antitrypanosomal agents, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.08.042.
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1
3
-Nitrotriazole-based piperazides as potent antitrypanosomal agents
a
a
b
Maria V. Papadopoulou *, William D. Bloomer , Howard S. Rosenzweig , Ivan P.
c
c
d,e
O’Shea Shane R. Wilkinson , Marcel Kaiser
a
b
NorthShore University HealthSystem, Evanston, IL, US; Oakton Community College, Des
c
Plaines, IL, US; School of Biological & Chemical Sciences, Queen Mary University of London,
d
London, UK; Swiss Tropical and Public Health Institute, Parasite Chemotherapy, Basel,
e
Switzerland; University of Basel, Basel, 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: Nitrotriazole-based piperazides as antiparasitic agents
1
Abbreviations: 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/).

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Abstract: Novel linear 3-nitro-1H-1,2,4-triazole-based piperazides were synthesized and
evaluated as antitrypanosomal agents. In addition, some bisarylpiperazine-ethanones which were
formed as by-products were also screened for antiparasitic activity. Most 3-nitrotriazole- based
derivatives were potent and selective against T. cruzi parasites, but only one displayed these
desired properties against T. b. rhodesiense. Moreover, two 3-nitrotriazole-based
chlorophenylpiperazides were moderately and selectively active against L. donovani. Although
the bisarylpiperazine-ethanones were active or moderately active against T. cruzi, none of them
demonstrated an acceptable selectivity. In general, 3-nitrotriazole-based piperazides were less
toxic to host L6 cells than the previously evaluated 3-nitrotriazole-based piperazines and seven
of 13 were 1.54- to 31.2-fold more potent antichagasic agents than the reference drug
benznidazole. Selected compounds showed good ADMET characteristics. One potent in vitro
antichagasic compound (3) was tested in an acute murine model and demonstrated antichagasic
activity after a 10-day treatment of 15 mg/kg/day. However, neither compound 3 nor
benznidazole showed a statistically significant P value compared to control due to high
variability in parasite burden among the untreated animals. Working as prodrugs, 3-nitrotriazole-
based piperazides were excellent substrates of trypanosomal type I nitroreductases and constitute
a novel class of potentially effective and more affordable antitrypanosomal agents.
Key words: Nitrotriazoles; piperazides; trypanocidal; leishmanicidal; Chagas disease.

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Introduction
American trypanosomiasis (Chagas disease), human African trypanosomiasis (HAT) and
leishmaniasis are caused by parasitic infections and constitute major health problems in poor
countries worldwide. They are considered ‘neglected’ because they have received limited
funding for discovery, development and delivery of new therapies. African trypanosomiasis is
endemic in many sub-Saharan African countries and is caused by Trypanosoma brucei
rhodesiense and Trypanosoma brucei gambiense. Chagas disease affects populations in South
and Central America and is caused by Trypanosoma cruzi, whereas leishmaniasis, caused by
more than 20 Leishmania species, occurs throughout tropical and sub-tropical regions [1]. It is
estimated that together these three diseases infect approximately 20 million people and are
responsible for more than 110,000 deaths per year [2].
Currently available pharmaceuticals for the treatment of neglected diseases suffer from poor
efficacy, toxic side effects, high cost, the need for intravenous administration in certain cases,
long duration of treatment, and the emergence of resistance. For example, severe toxicity and
long treatment requirements are associated with nifurtimox (nfx) and benznidazole (bnz), the two
medications used against Chagas disease in its initial acute stage [3, 4]. Similarly, drugs used to
treat HAT disease and leishmaniasis are highly toxic and/or are based on i.v. administration (e.g.,
eflornithine, antimony- and arsenic-based compounds) resulting in severe side effects, or are of
high cost (e.g,. eflornithine, liposomal amphotericin B, miltefosine, and paromomycin) [5-7].
Therefore, new effective, safe and affordable drugs are urgently needed for the treatment of these
neglected tropical diseases.
Although inhibitors of the fungal sterol 14α-demethylase enzyme (CYP51) and of the
orthologous enzyme T. cruzi CYP51 (TcCYP51) demonstrated promising efficacy against

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Chagas disease in preclinical studies [8-13], data from clinical trials using posaconazole were
proved disappointing [14]. Recent evidence indicates that nitroheterocyclics might be more
efficacious trypanocidal agents than CYP51 inhibitors [15], and combination treatments of
nitroheterocyclics with CYP51 inhibitors might offer a better solution [16].
We have shown that several chemical classes of 3-nitro-1H-1,2,4-triazole-based compounds
exhibit excellent antichagasic activity both in vitro and in vivo; furthermore, appreciable anti-
HAT activity was observed in vitro with several such analogs [17-22]. Interestingly, 3-
nitrotriazole-based compounds are significantly more potent and less toxic than their 2-
nitroimidazole-based counterparts [17-23], with part of the trypanocidal activity being dependent
on the parasite’s expression of a mitochondrially targeted, oxygen-insensitive type I
nitroreductase (NTR), an enzyme absent from most other eukaryotes [17, 18, 20-22]. Type I
NTRs, via a series of 2 electron reduction reactions leading to the production of toxic
metabolites, is responsible for the trypanocidal activity of Nfx, Bnz and other nitroheterocyclic
prodrugs [24-27].
Among the chemical classes of 3-nitro-1H-1,2,4-triazole-based compounds studied as
antitrypanosomal agents, several piperazine- and amide-analogs were of exceptional antichagasic
activity [18-20]. However, 3-nitrotriazole-based piperazines tend to be more toxic than 3-
nitrotriazole-based amides. In addition, 3-nitrotriazole-based amides demonstrate excellent
ADMET characteristics [20, 22]. Therefore, we decided to investigate 3-nitrotriazole-based
piperazides as antitrypanosomatid agents with potentially improved characteristics. The synthesis
and biological evaluation of such compounds is described in the present work. In addition, some
bisarylpiperazine-ethanones, which were produced during the synthesis of the piperazides as by-
products, were also evaluated as antitrypanosomal agents.

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Results and Discussion
Chemistry
The structures of tested compounds are shown in Table 1. Piperazides 2-14 were prepared in
good yields by nucleophilic substitution of chloroacetylpiperazides 1a-l with the potassium salt
of 3-nitrotriazole (or 2-nitroimidazole in one case) under refluxing conditions.
Chloroacetylpiperazides 1a-l and bisarylpiperazine-ethanones 15-19 were formed in the same
reaction between chloroacetylchloride and an appropriate piperazine. When the reaction time
was short (2 h), only the chloroacetylpiperazides 1a-l were formed by nucleophilic substitution
of the acylchloride. At longer reaction times (usually 24 h) nucleophilic substitution of the
alkylchloride also took place, and the bisarylpiperazine-ethanones 15-19 were formed in addition
to chloroacetylpiperazides 1a-l (Scheme 1).
Biological Evaluation.
Anti-parasitic activity and toxicity. Compounds were tested for antiparasitic activity against T.
cruzi amastigotes, bloodstream form (BSF) of T. b. rhodesiense and L. donovani axenic
amastigotes and, for toxicity in L6 rat skeletal myoblasts, the host cells for T. cruzi amastigotes.
Dose response curves were constructed from which the concentration of compound that inhibits
parasite or mammalian cell growth by 50 % (IC ) was calculated (Table 1). Selectivity index
5
0
(SI) for each compound towards each parasite was calculated from the equation: 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, denotes ‘active’, ‘moderately

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active’ or ‘inactive’ compounds, respectively, against T. cruzi amastigotes, whereas a SI of ≥50
is required; for blood stream form (BSF) T. b. rhodesiense, IC values of <0.5 µM, between 0.5-
5
0
6
.0 µM or > 6.0 µM identify ‘active’, ‘moderately active’ or ‘inactive’ compounds, respectively,
whereas a SI value of ≥100 is desired; finally, for L. donovani amastigotes, IC50 of <1 µM, between
.0-6.0 µM or > 6.0 µM, provides ‘active’, ‘moderately active’ or ‘inactive’ compounds, respectively,
1
whereas a SI value of ≥20 is ideal [28]. Most (9 of 12) of the 3-nitrotriazole-based piperazides
tested were deemed to be ‘active’ antichagasic agents, one was ‘active’ anti-HAT agent and two
of them moderately active antileishmanial agents, all displaying an acceptable selectivity (Table
1
). Although the bisarylpiperazine-ethanones were active or moderately active against T. cruzi,
none demonstrated acceptable selectivity (Table 1).
2
.2.1. SAR analysis for antichagasic activity
The arylpiperazides (2-7) exhibited appreciative antichagasic activity with IC50 values ranging
between 169 nM to 2.85 µM. There was a direct correlation between activity and the
compound’s lipophilicity with most lipophilic dichlorophenylpiperazide 3 being the most potent
agent of this sub-group (Table 1). In addition, compound 3 demonstrated the greatest selectivity
(
SI = 641), despite the fact that it was the most toxic analog of the subgroup against L6 cells
Table 1).
(
The two 3-nitrotriazole-based heteroarylpiperazides (8 and 9) were moderately active,
according to the criteria set, but with unacceptable selectivity. Therefore, introducing
heteroatoms in the phenyl group of arylpiperazides reduced antichagasic activity, most likely due
to decreased lipophilicity, and, thus, selectivity (Table 1). When the 3-nitrotriazole ring in 9 was
replaced with a 2-nitroimidazole ring in 10, the activity was completely lost irrespective of
lipophilicity (Table 1).

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Antichagasic activity increased in benzylpiperazides (11-13) with the most lipophilic
compound 13 exhibiting an IC of 73 nM against T. cruzi. However, and despite a SI of 302, the
5
0
high lipophilicity of 13 (clogP = 3.102) resulted in an IC of ca. 22 µM in L6 cells, designating
5
0
it as the most toxic analog among all tested piperazides (Table 1). Finally, introducing a second
amide in 14, also abolished antichagasic activity (Table 1).
The above SAR clearly shows that independently of variations in structure among all 3-
nitrotriazole-based piperazides tested, there was very good correlation between lipophilicity
(clogP values) and antichagasic activity (IC50 against T. cruzi). The correlation is described by a
nd
2
2
degree polynomial equation and the R value was 0.97 (Fig. 1). Other physical characteristics
of each structure such as electronegativity of the substituent on the phenyl ring or polar surface
area (PSA) did not play a major role in the anti-chagasic activity although those compounds that
had the lowest PSA values exhibited the lower IC50 values against T. cruzi (Table 1). Several
analogs (2-5, 11-13) were from 1.5 to 31-fold more potent than benznidazole.
With regard to bisarylpiperazine-ethanones (15-19), the active derivatives 16 and 17 violate 2
of the Lipinski rules of 5 (MW and clogP), while compound 15 violates one rule (clogP). The
antichagasic activity of 15-17 is most likely related to high lipophilicity coupled with their low
PSA value; however, these properties also contribute to the high toxicity of 16 and 17 and the
unacceptable selectivity of 15-17 towards T. cruzi parasite (Table 1). The two remaining
bisarylpiperazine-ethanones 18 and 19 are moderately active against T. cruzi, but also had poor
SI values towards this parasite.
2
.2.2. Analysis of anti-HAT activity

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Displaying an IC50 value of 231 nM and a SI of 260, only the dichlorobenzylpiperazide 12 was
selectively active against T. b. rhodesiense (Table 1). Four additional 3-nitrotriazole-based
piperazides (3, 4, 11 and 13) exhibited moderate anti-HAT activity, but had unacceptable
selectivity (Table 1). As observed with antichagasic activity (with the exception of compound 2),
there was good correlation between clogP values and anti-HAT activity among 3-nitrotriazole-
based piperazides (data not shown). None of the bisarylpiperazine-ethanones (15-19) exhibited
anti-HAT activity according to TDRs set criteria (Table 1).
2
.2.3. Analysis of antileishmanial activity
Two arylpiperazides (3, 4) and one benzylpiperazide (13) exhibited moderate but appreciable
antileishmanial activity with IC50 values against L. donovani axenic amastigotes < 2 µM.
However, only compounds 3 and 4 demonstrated acceptable selectivity towards the parasite
(Table 1). Once again, lipophilicity seems to be the driving force for the leishmanicidal activity.
One bisarylpiperazine-ethanone (17) exhibited moderate antileishmanial activity, but with an
unacceptable SI value of 6. Since 17 does not demonstrate the highest clogP value among
ethanones, it is not clear whether or not lipophilicity is the only factor contributing to
antileishmanial activity.
2
.2.4. Involvement of type I Nitroreductase
Representative 3-nitrotriazole-based piperazides (2-5, 9, 11-13) were evaluated as substrates of
purified, recombinant trypanosomal NTRs and compared to benznidazole (Table 2). 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. When tested against the T. cruzi enzyme, the
selected compounds were metabolized at rates comparable or slightly better than that of Bnz

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while the T. b. brucei enzyme could reduce the same analogs at rates 1.3 to 2.5-fold higher than
the reference 2-nitroimidazole (Table 2).
To determine whether NTR plays a role in metabolizing the substrates within the parasite, the
above subset of 3-nitrotriazole-based piperazides were tested against BSF T. b. brucei. In initial
screens, 2, 5 and 9 did not affect the growth of wild type parasites at concentrations up to the 30
µM and these were not analyzed further (Table 3).
For the remaining compounds T. b. brucei engineered to express elevated levels of TbNTR
were shown to be ca. 4- to 17-fold more susceptible to the 3-nitrotriazole-based piperazides
under study than parasites expressing wild type levels of the enzyme, with 3, 4, 11 and 12
generating a ≥ 7-fold shift in parasite sensitivity: nfx tested in parallel generated a 10-fold
difference in IC values. For these four structures that demonstrate activity against T. cruzi and
5
0
T. rhodesiense (Table 1), this difference in sensitivity indicates that all function as prodrugs in
the parasite itself with NTR playing a key role in their activation. When TbNTR/TcNTR
enzymatic activity and anti-trypanosomal activity were compared no obvious correlation was
observed possibly due to existence of additional targets in the trypanosome, permeability issues
with regard to mitochondrion, compound stability and pharmacokinetic factors in general.
2
.2.5. ADMET studies
Limited ADMET studies were performed with compounds 11 and 12 to check metabolic
stability, Caco-2 permeability and cytochrome P450 inhibition. These two compounds were
shown to be relatively stable with half lives (T ) of >180 and 72 min, respectively, in the
1
/2
presence of NADPH and T1/2 of >180 and 174 min, respectively, in the absence of NADPH
Table 4). Neither compound inhibited CYP3A4, CYP2C9, CYP2D6, CYP2C19 and CYP2C8,
(

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demonstrating an IC50 > 20 µM for all enzymes (data not shown). Both compounds exhibited
very good Caco-2 permeability with efflux ratios (Re) of 0.97 and 0.91, respectively (Table 5).
2
.2.6. In vivo antichagasic activity
The 3-nitrotriazole-based dichlorophenyl piperazide 3 was selected for in vivo evaluation
because of its good in vitro antichagasic activity (IC50 of 169 nM against T. cruzi), excellent
selectivity (SI of 641, the highest in the series) and relatively low toxicity (IC50 in L6 cells of
1
1
08.4 µM). Groups of 5 mice each were treated i.p. with compound 3 or bnz, at 15 mg/kg/day x
0 days. The mean ratio of parasite levels was calculated after 10 days of treatment. The data are
summarized in Fig. 2. No apparent toxicity was observed at the given dose and time-frame for
compound 3, which reduced the parasite ratio by 77% after 10-day treatment; the corresponding
reduction by bnz was 99%. However, this reduction was not statistically significant for either
compound 3 or bnz due to high variability in parasite burden among the untreated animals.
3
.
Conclusions
Piperazide-based compounds demonstrate a broad range of biological activities including, but
not limited to, antibacterial, antiparasitic, antifungal, anthelmintic and antitubercular activity [29-
3
3]. In particular, some metronidazole-bearing piperazides exhibit appreciable antigiardial and/or
antitrichomonal activity [31]. In addition, aryloxyphenyl piperazides have been described as
histamine H antagonists and serotonin reuptake inhibitors for the treatment of depression [34],
3
while other piperazine amides are considered useful for the treatment of obesity or diabetes [35,
3
6].

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Our novel 3-nitrotriazole-based aryl- and benzylpiperazides were potent and selective
antichagasic agents in vitro. In addition, two such analogs (3, 4) exhibited moderate but selective
antileishmanial activity, while another analog (12) demonstrated remarkable anti-HAT activity in
vitro. The antitrypanosomal activities of tested compounds were attributed, at least in part, to
their high lipophilicity and their activation by the type I nitroreductases. 3-Nitrotriazole-based
heteroarylpiperazides, dipiperazides and bisarylpiperazine-ethanones were varyingly active, but
toxic antichagasic agents in vitro. Although in vivo antichagasic activity was demonstrated with
compound 3, the data were not of statistical significance due to high variability in parasite
burden among the untreated animals. However, favorable ADMET studies with two analogs (11,
1
2) and lack of mutagenicity or developmental toxicity in previously studied 3-nitrotriazole-
based amides [20, 23] suggest that 3-nitrotriazole-based aryl/benzylpiperazides could be of real
value in the treatment of Chagas disease, provided that further in vivo evaluation in the chronic
mouse model is successful.
4
4
4
.
Experimental
.1. Chemistry
.1.1. General
All starting materials and solvents 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
4
Hg-400 spectrometer at 500 or 400 MHz, respectively, and are referenced to Me Si or to the

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corresponding solvent, if the solvent was not CDCl
3
. 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 (≥ 95% purity).
4
.1.2. Synthesis of chloroacetylpiperazides 1a-l: Compounds 1a-l and compounds 15-19 were
synthesized in the same reaction: In a dichloromethane solution (2-3 mL) of chloroacetylchloride
1.1 eq), a dichloromethane solution (8-10 mL) of the appropriate piperazine (1 eq) and
(
triethylamine (2.5 eq) was added dropwise and the reaction mixture was stirred overnight at
room temperature under a nitrogen atmosphere. The reaction mixture was evaporated and the
residue was extracted with ethyl acetate-brine. The organic layer was dried over Na SO and
2
4
chromatographed on silica preparative TLC to give the desired products. When the reaction was
run for 2 h, compounds 1a-l were the main products (>90%). Compounds 1a-l are known in the
literature and their spectroscopic data are provided in the supplementary material.
4
.1.3. Synthesis of compounds 2-14: The potassium salt of 3-nitro-1,2,4-triazole or 2-
nitroimidazole (1 eq) was formed in CH CN (6-10 mL), by refluxing with KOH (1.2 eq) for 30
3
min. To this suspension 1a-l (1.1 eq) was added and the reaction mixture was refluxed under a
nitrogen atmosphere for 9 h. If chloride 1 was an oil, it was added in CH CN solution. The
3
reaction mixture was checked by TLC for completion of the reaction and the solvent was

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evaporated. The residue was redissolved in ethyl acetate or acetone and the inorganic salts were
filtered away. Upon preparative TLC (usually on silica gel; ethyl acetate-petroleum ether), the
desired product was obtained as a powder. Purity was checked also by HPLC and it was ≥ 95%.
4
.1.3.1. 1-(4-(4-Chlorophenyl)piperazin-1-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanone (2):
o
1
Yellow powder (77%): mp 226 C (dec); H NMR (400 MHz, (CD
3
COCD
d) δ: 8.66 (s, 1H), 7.26 (d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 5.59 (s, 2H), 3.80 (t, J =
.2 Hz, 2H), 3.73 (t, J = 5.2 Hz, 2H), 3.35 (t, J = 5.2 Hz, 2H), 3.23 (t, J = 5.2 Hz, 2H).
3
+ a drop of DMSO-
6
5
+
+
HRESIMS calcd for C H ClN O and C H ClN NaO m/z [M+H] and [M+Na] 351.0967
1
4
16
6
3
14 15
6
3
and 373.0786, 375.0762 found 351.0964 and 373.0786, 375.0752.
4
.1.3.2. 1-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanone (3):
o
1
Yellow crystals (78%): mp 130-132 C (dec); H NMR (400 MHz, (CD
3
3
COCD ) δ: 8.60 (s, 1H),
7
3
2
3
.39 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 3.2 Hz, 1H), 7.00 (dd, J = 8.8, 3.2 Hz, 1H), 5.59 (s, 2H),
.84 (t, J = 5.2 Hz, 2H), 3.75 (t, J = 5.2 Hz, 2H), 3.44 (t, J = 5.2 Hz, 2H), 3.31 (t, J = 5.2 Hz,
+
H). HRESIMS calcd for C H Cl N O m/z [M+H] 385.0577, 387.0550, found 385.0578,
1
4
15
2
6
3
87.0549.
4
.1.3.3.
2-(3-Nitro-1H-1,2,4-triazol-1-yl)-1-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)
o
1
ethanone (4): Off white powder (74 %): mp 224 C (dec); H NMR (400 MHz, (CD COCD ) δ:
3
3
8
.60 (s, 1H), 7.55 (d, J = 9.2 Hz, 2H), 7.14 (d, J = 9.2 Hz, 2H), 5.59 (s, 2H), 3.86 (t, J = 5.2 Hz,

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2
H), 3.76 (t, J = 5.2 Hz, 2H), 3.54 (t, J = 5.2 Hz, 2H), 3.42 (t, J = 5.2 Hz, 2H). HRESIMS calcd
+
for C H F N O m/z [M+H] 385.1230, found 385.1237.
1
5
16
3
6
3
4
.1.3.4. 2-(3-Nitro-1H-1,2,4-triazol-1-yl)-1-(4-(p-tolyl)piperazin-1-yl)ethanone (5): Yellow
o
1
powder (70 %): mp 172-174 C (dec); H NMR (400 MHz, (CD COCD ) δ: 8.60 (s, 1H), 7.07
3
3
(d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 5.57 (s, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.72 (t, J =
5
19 6 3
.2 Hz, 2H), 3.26 (t, J = 5.2 Hz, 2H), 3.15 (t, J = 5.2 Hz, 2H). HRESIMS calcd for C15H N O
+
m/z [M+H] 331.1513, found 331.1516.
4
.1.3.5. 1-(4-(4-Methoxyphenyl)piperazin-1-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanone (6).
This compound is listed in the Aurora Screening Library but no reference is available: Orange
o
1
powder (88 %): mp 181-183 C (dec); H NMR (400 MHz, (CD COCD ) δ: 8.60 (s, 1H), 6.97
3
3
(d, J = 8.8 Hz, 2H), 6.85 (d, J = 9.2 Hz, 2H), 5.57 (s, 2H), 3.79 (t, J = 5.0 Hz, 2H), 3.74 (s, 3H),
3
.72 (t, J = 5.2 Hz, 2H), 3.19 (t, J = 5.0 Hz, 2H), 3.08 (t, J = 5.2 Hz, 2H). HRESIMS calcd for
+
+
C
15 19 6 4 18 6 4
H N O and C15H N NaO m/z [M+H] and [M+Na] 347.1462 and 369.1282 found
3
47.1466 and 369.1283.
4
.1.3.6. 2-(4-(2-(3-Nitro-1H-1,2,4-triazol-1-yl)acetyl)piperazin-1-yl)benzonitrile (7): Off white
o
1
powder (79 %): mp 92-94 C (dec); H NMR (400 MHz, CD COCD ) δ: 8.61 (s, 1H), 7.70 (dd, J
3
3
=
8.0, 1.6 Hz, 1H), 7.63 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.17 (ddd, J =
8
4
.4, 7.2, 0.8 Hz, 1H), 5.60 (s, 2H), 3.88 (t, J = 5.2 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.38 (t, J =
+
.8 Hz, 2H), 3.25 (t, J = 4.8 Hz, 2H). HRESIMS calcd for C15
H
16
N
7
O
3
m/z [M+H] 342.1309,
found 342.1309.

ACCEPTED MANUSCRIPT
15
4
.1.3.7. 2-(3-Nitro-1H-1,2,4-triazol-1-yl)-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (8): White
o
1
powder (67 %): mp 108-109 C (dec); H NMR (400 MHz, (CD COCD ) δ: 8.61 (s, 1H), 8.16
3
3
(ddd, J = 4.8, 2.0, 0.8 Hz, 1H), 7.56 (ddd, J = 10.8, 7.2, 2.0 Hz, 1H), 6.85 (dd, J = 8.8, 0.8 Hz,
1
H), 6.68 (ddd, J = 7.2, 5.2, 0.8 Hz, 1H), 5.58 (s, 2H), 3.77 (m, 4H), 3.69 (m, 2H), 3.61 (m, 2H).
+
+
HRESIMS calcd for C H N O and C H N NaO m/z [M+H] and [M+Na] 318.1309 and
1
3
16
7
3
13 15
7
3
3
40.1129 found 318.1313 and 340.1129.
4
.1.3.8. 2-(3-Nitro-1H-1,2,4-triazol-1-yl)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)ethanone (9):
o
1
White crystals (79 %): mp 106-108 C (dec); H NMR (400 MHz, (CD COCD ) δ: 8.61 (s, 1H),
3
3
8
.38 (d, J = 4.8 Hz, 2H), 6.65 (t, J = 4.8 Hz, 1H), 5.59 (s, 2H), 3.99 (br t, J = 5.4 Hz, 2H), 3.86
(br t, J = 5.4 Hz, 2H), 3.75 (br t, J = 5.4 Hz, 2H), 3.67 (br t, J = 5.4 Hz, 2H). HRESIMS calcd for
+
+
C H N O and C H N NaO m/z [M+H] and [M+Na] 319.1262 and 341.1081 found
12
15
8
3
12 14
8
3
3
19.1268 and 341.1083.
4
.1.3.9. 2-(2-Nitro-1H-imidazol-1-yl)-1-(4-(pyrimidin-2-yl)piperazin-1-yl)ethanone (10): Light
o
1
yellow crystals (77 %): mp 148-150 C (dec); H NMR (400 MHz, (CD COCD ) δ: 8.38 (d, J =
3
3
4
2
.8 Hz, 2H), 7.44 (d, J = 1.0 Hz, 1H), 7.13 (d, J = 1.0 Hz, 1H), 6.64 (t, J = 4.8 Hz, 1H), 5.60 (s,
H), 3.97 (br t, J = 5.0 Hz, 2H), 3.84 (br t, J = 5.4 Hz, 2H), 3.74 (br t, J = 4.8 Hz, 2H), 3.64 (br t,
+
+
J = 4.8 Hz, 2H). HRESIMS calcd for C H N O and C H N NaO m/z [M+H] and [M+Na]
1
3
16
7
3
13 15
7
3
3
18.1309 and 340.1129 found 318.1313 and 340.1136.

ACCEPTED MANUSCRIPT
16
4
.1.3.10.
2-(3-Nitro-1H-1,2,4-triazol-1-yl)-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)
o
1
ethanone (11): White microcrystals (69 %): mp 162-164 C; H NMR (400 MHz, (CD Cl) δ:
3
8
2
.36 (s, 1H), 7.61 (d, J = 7.6 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 5.16 (s, 2H), 3.69 (t, J = 5.0 Hz,
H), 3.61 (s, 2H), 3.54 (t, J = 4.8, Hz, 2H), 2.54 (t, J = 5.2 Hz, 2H), 2.50 (t, J = 5.2 Hz, 2H).
+
HRESIMS calcd for C H F N O m/z [M+H] 399.1387, found 399.1395.
1
6
18
3
6
3
4
.1.3.11.
1-(4-(3,4-Dichlorobenzyl)piperazin-1-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanone
o
1
(
12): White microcrystals (88 %): mp 140-142 C; H NMR (400 MHz, (CD
3
Cl) δ: 8.36 (s, 1H),
7
3
.47 (d, J = 2.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.17 (dd, J = 8.4, 2.0 Hz, 1H), 5.16 (s, 2H),
.68 (t, J = 4.8 Hz, 2H), 3.53 (t, J = 4.8 Hz, 2H), 3.51 (s, 2H), 2.53 (t, J = 4.8 Hz, 2H), 2.48 (t, J
+
=
4.8 Hz, 2H). HRESIMS calcd for C H Cl N O m/z [M+H] 399.0734, 401.0707 found
1
5
17
2
6
3
3
99.0738, 401.0710.
4
.1.3.12. 1-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)
o
1
ethanone (13): Off white microcrystals (84 %): mp 94-96 C; H NMR (400 MHz, (CD
.34 (s, 1H), 7.37-7.23 (m, 9H), 5.13 (s, 2H), 4.27 (s, 1H), 3.65 (br t, J = 5.0 Hz, 2H), 3.50 (br t,
J = 5.0 Hz, 2H), 2.47 (br t, J = 5.0 Hz, 2H), 2.42 (t, J = 5.0 Hz, 2H). HRESIMS calcd for
3
Cl) δ:
8
+
+
C H ClN O and C H ClN NaO m/z [M+H] and [M+Na] 441.1436, 443.1416 and
21
22
6
3
21 21
6
3
4
63.1256, 465.1235 found 441.1436, 443.1425 and 463.1254, 465.1233.
4
.1.3.13. 1-(4-(Cyclopropanecarbonyl)piperazin-1-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanone
o
1
(
3 3
14): White crystals (84 %): mp 63-65 C ; H NMR (400 MHz, (CD COCD ) δ: 8.59 (s, 1H),

ACCEPTED MANUSCRIPT
17
5
.57 (s, 2H), 3.80-3.50 (br m, 8H), 1.96 (m, 1H), 0.85-0.81 (m, 2H), 0.76-0.71 (m, 2H).
+
HRESIMS calcd for C H N NaO m/z [M+Na] 331.1125, found 331.1126.
1
2
16
6
4
4
1
6
3
4
.1.3.14. 1,2-bis(4-(4-chlorophenyl)piperazin-1-yl)ethanone (15): Off white powder (44%): mp
o
1
65-167 C ; H NMR (400 MHz, (CD Cl) δ: 7.23 (d, J = 9.2 Hz, 2H), 7.21 (d, J = 9.2 Hz, 2H),
3
.85 (d, J = 9.2 Hz, 2H), 6.83 (d, J = 9.2 Hz, 2H), 3.79 (t, J = 4.8 Hz, 4H), 3.30 (s, 2H), 3.18-
+
2 4
,12 (m, 8H), 2.69 (t, J = 4.8 Hz, 4H). HRESIMS calcd for C22H27Cl N O m/z [M+H]
33.1556, 435.1531 found 433.1560, 435.1535.
4
.1.3.15. 1,2-bis(4-(3,4-dichlorophenyl)piperazin-1-yl)ethanone (16): Off white powder (33%):
o
1
mp 79-81 C; H NMR (400 MHz, (CD Cl) δ: 7.29 (d, J = 9.2 Hz, 1H), 7.28 (d, J = 9.2 Hz, 1H),
3
6
3
.96 (d, J = 2.8 Hz, 1H), 6.95 (d, J = 2.8 Hz, 1H), 6.76-6.72 (m, 2H), 3.77 (t, J = 5.2 Hz, 4H),
4 4
.29 (s, 2H), 3.18-3.14 (m, 8H), 2.67 (t, J = 5.2, 4H). HRESIMS calcd for C22H25Cl N O m/z
+
[
M+H] 501.0777, 503.0750 found 501.0788, 503.0761.
4
.1.3.16. 1,2-bis(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)ethanone (17): Off white powder
o
1
(
49%): mp 150-152 C; H NMR (400 MHz, (CD Cl) δ: 7.51 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.8
3
Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.80 (t, J = 4.8 Hz, 4H), 3.30 (m,
+
1
5
0H), 2.70 (t, J = 4.8 Hz, 4H). HRESIMS calcd for C24
H
27
F
6
N
4
O m/z [M+H] 501.2084,
02.2114 found 501.2094, 502.2123.

ACCEPTED MANUSCRIPT
18
4
.1.3.17. 1,2-bis(4-(p-tolyl)piperazin-1-yl)ethanone (18): Off white powder (37%): mp 149-151
o
1
C; H NMR (400 MHz, (CD Cl) δ: 7.09 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.84 (d, J
3
=
8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.78 (m, 4H), 3.28 (s, 2H), 3.14 (m, 8H), 2.69 (t, J = 4.8
+
Hz, 4H), 2.28 (s, 3H), 2.27 (s, 3H). HRESIMS calcd for C H N O m/z [M+H] 393.2649,
2
4
33
4
3
94.2680 found 393.2659, 394.2688.
4
1
4
.1.3.18. 1,2-bis(4-(2-cyanophenyl)piperazin-1-yl)ethanone (19): Off white powder (55%): mp
o
1
3
15-117 C; H NMR (400 MHz, (CD Cl) δ: 7.62-7.47 (m, 4H), 7.10-7.00 (m, 4H), 3.86 (t, J =
.8 Hz, 4H), 3.33 (s, 2H), 3.27-3.16 (m, 8H), 2.77 (t, J = 4.8 Hz, 4H). HRESIMS calcd for
+
C H N O m/z [M+H] 415.2241 found 415.2239.
24
27
6
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 [37]. Data were analyzed with the graphic program Softmax Pro (Molecular Devices,
Sunnyvale, CA, USA), which calculated IC values by linear regression from the sigmoidal dose
5
0
inhibition curves.
4
.2.2. In vitro T. brucei brucei antiproliferating assays and susceptibility studies.
3
-1
T. brucei brucei bloodstream form parasites were seeded at 1 x 10 mL in 200 µL of growth medium
containing different concentrations of a nitrotriazole or nifurtimox. Where appropriate, induction of the
o
TbNTR was carried out 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 [24] and the IC established.
50

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19
4
.2.3. Enzymatic activity studies with Type I NTRs.
Recombinant TbNTR and TcNTR were prepared and assayed as previously described [38, 39]. The
activity of purified his-tagged TbNTR was assessed spectrophotometrically at 340 nm using various
-1
-
nitrotriazole substrates (100 µM) and NADH (100 µM) and expressed as nmol NADH oxidized min mg
1
of enzyme.
4
4
.2.4. ADMET studies
.2.4.1. Caco-2 permeability assay was performed as described before [40]. For Apical to Basolateral (A-
B) permeability, the compound was added to the apical (A) side and amount of permeation was
>
determined on the basolateral (B) side; for Basolateral to Apical (B->A) permeability, the compound was
added to the B side and the amount of permeation was determined on the A side. Assays were run for 2 h
in duplicate and the amount of compound present in each compartment was quantified by LC-MS/MS.
Control compounds for low and high permeability were included in each experiment as well as the P-gp
efflux control, talinolol [41].
4
.2.4.2. Inhibition of CYP P450 isoforms was assessed by using CYP2B6, CYP2C8, CYP2C9, CYP2C19,
o
CYP2D6 and CYP3A4. For each assay, human liver microsomes were incubated at 37 C with a probe
substrate for each CYP isoform in the presence of a compound at various concentrations (up to 20 µM)
for 5 min (except CYP2C19 for 35 min). The formation of metabolites for each isoform was quantified by
LC-MS/MS as a measure of enzyme activity and an IC50 value (the compound concentration which
produces 50% inhibition) was generated [42].
4
.2.4.3. Microsomal stability. Compounds were tested for microsomal stability by using pooled human
o
liver S9 microsomes (0.3 mg/mL), which were incubated with test compound at 37 C in the presence or
absence of the co-factor NADPH. The reaction was terminated, the supernatant recovered and test

ACCEPTED MANUSCRIPT
20
compound quantified by LC-MS/MS. A fixed compound-concentration was tested in duplicate at 5 time-
points and compound stability expressed as a function of time [43].
4
.2.5. In vivo antichagasic activity assessment of compound 3:
The study was performed as described before [20, 22]. Briefly, trypomastigote forms from
5
transgenic T. cruzi Y strain expressing firefly luciferase were injected in Balb/c mice (10
trypomastigotes per mouse) and three days later mice were anesthetized 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, Alameda, CA)
and the data acquisition and analysis were performed with the software LivingImage (Xenogen)
as described before [44]. Treatment with test compound or bnz was started 4 days after infection
at 15 mg/kg/day x 10 days, given i.p. The vehicle control was 2% methylcellulose + 0.5% Tween
8
0 and groups of 5 mice/group were used. Mice were imaged after a 10-day treatment. The ratio
of parasite levels was calculated for each animal dividing the luciferase signal after treatment by
the luciferase signal on the first imaging (before treatment). Mean values of all animals in each
group ± SD were used for plotting.
Acknowledgements:
The authors thank M. Cal, M. Jud and S. Keller (Swiss TPH) for parasite assay results and Dr.
Ana Rodriguez (New York University School of Medicine) for obtaining the in vivo data. This
work was supported in part: a) by internal funds of the Radiation Medicine Department at
NorthShore University HealthSystem; b) the National Institutes of Health and the National
Institute of Allergy and Infectious Diseases, Contract No. HHSN272201100009I (ADMET
studies). In addition, the Drugs for Neglected Diseases initiative (DNDi) received financial

ACCEPTED MANUSCRIPT
21
support from the Bill & Melinda Gates Foundation (BMGF) to perform the in vitro screenings
against parasites. The donors had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
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Captions:
Fig. 1. Correlation graph between lipophilicity and antichagasic activity in the 3-nitrotriazole-
based arylpiperazides. Compounds 2-9 and 11-13 were used.
Fig. 2. In vivo evaluation of the antichagasic efficacy of compound 3 and benznidazole (Bnz) in
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Graphical Abstract