Novel 3-nitrotriazole-based amides and carbinols as bifunctional antichagasic agents

Article abstract of DOI:10.1021/jm5015742

3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogues were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed.

Full text of DOI:10.1021/jm5015742

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Article
Novel 3-nitrotriazole-based amides and
carbinols as bifunctional anti-Chagasic agents
Maria V Papadopoulou, William D Bloomer, Galina I Lepesheva, Howard S Rosenzweig, Marcel
Kaiser, Benjamín Aguilera-Venegas, Shane R. Wilkinson, eric chatelain, and Jean-Robert Ioset
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5015742 • Publication Date (Web): 12 Jan 2015
Downloaded from http://pubs.acs.org on January 19, 2015
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Novel 3-nitrotriazole-based amides and carbinols as bifunctional
anti-Chagasic agents
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Maria V. Papadopoulou#*, William D. Bloomer#, Galina I. Lepesheva§, Howard S.
Rosenzweig‡, Marcel Kaiser†,¶, Benjamín AguileraꢀVenegas¥,£, Shane R.
Wilkinson£, Eric Chatelain¢, JeanꢀRobert Ioset¢
#
§
NorthShore University HealthSystem, Evanston, IL, US; Vanderbilt University School of Medicine,
‡
Department of Biochemistry School of Medicine, Nashville, TN, US; Oakton Community College, Des
†
Plaines, IL, US; Swiss Tropical and Public Health Institute, Parasite Chemotherapy, Basel, Switzerland;
¶
¥
University of Basel, Basel, Switzerland; University of Chile, Laboratory of Antioxidants, Institute of
£
Nutrition and Food Technology, Santiago, Chile; School of Biological & Chemical Sciences, Queen
¢
Mary University of London, London, UK; Drugs for Neglected Diseases initiative (DNDi), Geneva,
Switzerland.
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Running Title: Novel nitrotriazole-based compounds as bifunctional anti-Chagasics
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Abstract: 3ꢀNitroꢀ1Hꢀ1,2,4ꢀtriazoleꢀbased amides with a linear, rigid core and 3ꢀnitrotriazoleꢀ
based fluconazole analogs were synthesized as dual functioning antitrypanosomal agents. Such
compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion
of trypanosomatids and, at the same time, act as inhibitors of the sterol 14αꢀdemethylase (T.
cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to
monotherapy, we believe that this emerging class of bifunctional compounds may introduce a
new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in
vivo evaluation of such compounds is discussed.
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Key words: Bifunctional nitrotriazoles, carbinols, fuconazole analogs, T. cruzi CYP51, type I
nitroreductase, antitrypanosomal agents, Chagas disease
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ꢀ
INTRODUCTION
About one sixth of the world’s population, most living in developing countries, suffer from a
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series of infections collective known as neglected tropical diseases (NTDs).1 In addition to
killing more than 500,000 people each year, these illnesses impair physical and cognitive
development of children, make it difficult to farm or earn a living, and limit productivity in the
workplace. Two NTDs are American trypanosomiasis (also known as Chagas disease), which is
causes by the protozoan parasite Trypanosoma cruzi and is endemic in 21 countries across Latin
America, and human African trypanosomiasis (HAT) (also known as African sleeping sickness),
which is prevalent across subꢀSaharan Africa and caused by T. brucei rhodesiense and T. brucei
gambiense Although the number of incidences of both infections has significantly declined in
the past 20 years due to implementation of vector control initiatives, the diseases still remain a
major global health issue, especially since the number of cases in nonꢀendemic sites (United
States, Australia, Europe and Japan) is rising, primarily due to international population migration
and transfusion of contaminated blood.2ꢀ4
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The therapeutic options for Chagas are currently limited to two drugs, both nitroheterocyclic
compounds, nifurtimox (Nfx; Lampit®) and benznidazole (Bnz; Rochagan®), while the success
of treatment depends on the phase of the disease. Thus, cure rates of 65ꢀ80% can be achieved in
the acute phase, whereas only 15ꢀ30% cure rates have been reported among adults in the chronic
stage.5, 6 Besides the low efficacy in patients with chronic disease, safety and tolerability issues
are also major drawbacks for these drugs. Therefore, the development of new, safer and more
effective therapeutic treatments remains a key challenge for Chagas disease control.
Currently, inhibitors of the fungal sterol 14αꢀdemethylase enzyme (CYP51) represent a new
form of treatment against Chagas disease and demonstrated promising efficacy in preclinical
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studies.7, 8 Additional pathogenꢀspecific drug discovery efforts have resulted in inhibitors for the
orthologous enzyme T. cruzi CYP51 (TcCYP51).9ꢀ12 One such compound, VNI, showed
parasitological cures in both the acute and chronic in vivo Chagas model.11 However, the
antifungal agent posaconazole revealed ∼80% treatment failure in clinical trials in human
patients with chronic Chagas disease while Bnz was proven more efficacious.13 In addition, in
newly designed in vitro assays, nitroheterocyclic compounds were more efficacious trypanocidal
agents than four different CYP51 inhibitors, including antifungal drugs posaconazole and
ravuconazole and two pyridineꢀbased compounds,14 suggesting that inhibitors of sterol
biosynthesis may not be as efficient as single chemotherapeutic agents against Chagas disease as
they are against fungal infections. Interestingly, a recent study with Bnz and posaconazole
administered concomitantly or in sequence suggested a positive interaction between the two
drugs, at least in an acute murine infection model.15
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We have recently demonstrated that another class of nitroheterocyclic compounds, 3ꢀnitroꢀ
1Hꢀ1,2,4ꢀtriazoles, are very potent antichagasic agents both in vitro and in vivo, whereas several
analogs showed very good in vitro efficacy against T. brucei rhodesiense as well.16ꢀ19 These
studies clearly demonstrated that 3ꢀnitrotriazoleꢀbased compounds are significantly more potent
and less toxic than their 2ꢀnitroimidazoleꢀbased counterparts,19, 20 with part of the trypanocidal
activity being dependent on the parasite’s expression of an oxygenꢀinsensitive type I
nitroreductase (NTR), an enzyme absent from most other eukaryotes.16, 17, 19 This same enzyme,
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.21ꢀ24
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In this work we examined the possibility of synthesizing dualꢀfunctioning 3ꢀnitrotriazoleꢀ
based compounds; such compounds could potentially act as prodrugs via their reducible nitroꢀ
group and, at the same time, be inhibitors of CYP51 enzyme via their triazoleꢀbased scaffold.25
The synthesis of bifunctional agents has the advantage of combining the beneficial effects of
nitroheterocyclics with the beneficial effects of ergosterol biosynthesis inhibitors in one
molecule. To accomplish this task, we synthesized and evaluated in vitro and in vivo linear, rigid
3ꢀnitrotriazoleꢀbased amides and 3ꢀnitrotriazoleꢀbased carbinols (analogs of fluconazole), as
potential antitrypanosomal/antichagasic agents, NTR substrates and TcCYP51 inhibitors.
Based on our previous work,16ꢀ19 we have realized that most 3ꢀnitrotriazoleꢀbased analogs are
very good substrates of NTR enzyme. Therefore, our goal was to obtain 3ꢀnitrotriazoleꢀbased
structures demonstrating affinity for CYP51. Early studies with 3ꢀnitrotriazoleꢀbased amides
having a flexible core showed lack of affinity for TcCYP51. Therefore, we assumed a more
rigid, linear core should potentially provide a better fit in the active site of this enzyme. Thus,
amides 2-8 were synthesized (Table 1). We chose the class of amides because they demonstrated
better ADME properties than other 3ꢀnitrotriazoleꢀbased compounds.19 Alternatively, the core of
the CYP51 inhibitor fluconazole was used as a good starting point, and compounds 18-21 were
synthesized (Table 1). Carbinols 14-17 were synthesized as structures that combine the linearity
and rigidity of the amides 2-8 and share some of the features of the fluconazole core.
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ꢀ
RESULTS AND DISCUSSION
Chemistry
The structures of synthesized compounds are shown in Table 1. Their synthesis is
straightforward and based on wellꢀestablished chemistry outlined in Scheme 1. Thus, amides 2-9
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were obtained by nucleophilic substitution of chloroacetamides 1a-g with the potassium salt of
3ꢀnitrotriazole or 2ꢀnitroimidazole under refluxing conditions. Similarly, nitro(triazole/
imidazole)ꢀketones 10-13 were synthesized by nucleophilic substitution of αꢀbromoketones.
Carbinols 14-19 were obtained in one step by using a modified CoreyꢀChaykovsky reaction;26 in
this oneꢀpot reaction, trimethylsulfoxonium iodide was used in a 2ꢀpropanol : aqueous KOH
(1:1) solution and the appropriate ketone was added to form in situ an oxirane; then 3ꢀ
nitrotriazole was added followed by 1ꢀh refluxing. Although the yields by using this method
were low, the method is attractive due to its simplicity and short reaction time. The obtained
carbinols 14-19 were used as racemic mixtures. When 2ꢀnitroimidazole was used in the modified
CoreyꢀChaykovsky reaction, the bicyclic compound 20 was formed as the final product,
presumably via an intramolecular aromatic nucleophilic substitution of the nitroꢀgroup by the
hydroxylꢀgroup. The sulfonamide 21 was formed by nucleophilic attack of the sodium salt of 3ꢀ
nitrotriazole with the appropriate aziridineꢀsulfonamide (Scheme 1).
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Biological Evaluation.
Anti-parasitic activity. Compounds were tested for antitrypanosomal activity against T. cruzi
amastigotes and bloodstream form (BSF) of T. b. rhodesiense. Dose response curves were
constructed from which the concentration of compound that inhibits parasite growth by 50 %
(IC50) was calculated for each parasite (Table 1). In addition, compounds were 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). The TDR (Special
Programme for Research and Training in Tropical Diseases, World Health Organization) criteria
for activity were adapted to interpret the data.27 Thus, for T. cruzi amastigotes, an IC50 of <4.0
µM, between 4.0ꢀ60 µM or >60 µM, designates ‘active’, ‘moderately active’ or ‘inactive’
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compounds, respectively, whereas for BSF T. b. rhodesiense, IC50 values of <0.5 µM, between
0.5ꢀ6.0 µM or > 6.0 µM identify ‘active’, ‘moderately active’ or ‘inactive’ compounds,
respectively. In addition, SI values should ideally be ≥50 and ≥100 for T. cruzi and T. b.
rhodesiense, respectively.27
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All the 3ꢀnitrotriazoleꢀbased rigid amides (2, 3, 5, 6, 8, 9) except 4, and all the 3ꢀ
nitrotriazoleꢀbased carbinols (14, 17-19), as well as the fluconazoleꢀlike 3ꢀnitrotriazoleꢀbased
sulfonamide 21, were active antichagasic agents with acceptable selectivity indices (Table 1).
Only 3 compounds (two 3ꢀnitrotriazoleꢀbased amides and one 3ꢀnitrotriazoleꢀbased carbinol)
were active (2) or moderately active (5, 14) antiꢀHAT agents with acceptable SI values. In
addition, ketone 11, the precursor of carbinol 19 showed a moderate antiꢀHAT activity with
acceptable selectivity. As observed before, compound 7, the 2ꢀnitroimidazoleꢀbased analog of 6,
was borderline active against T. cruzi amastigotes but with an unacceptable SI value (Table 1).
Analysis of antichagasic activity. Linear, rigid amides 2-9: Although a detailed SARs
evaluation cannot be conducted due to the limited number of compounds studied, we made the
following observations: A chloroꢀsubstituent in the para position of a diphenylꢀcore in the rigid
amide 5 increased the efficacy against T. cruzi by a factor of 3 compared to the cyanoꢀsubstituted
analog 2. Although 5 was slightly more toxic to L6 cells than compound 2, its SI for T. cruzi was
also increased about 2ꢀfold compared to 2 due to its better antiꢀchagasic efficacy; this cannot be
attributed to lipophilicity since both compounds share similar clogP values, but perhaps to its
lower PSA value (Table 1). Chloroꢀsubstitution in the phenylthiazoleꢀcore of 3 was also
beneficial for its antichagasic activity and selectivity compared to the difluoroꢀsubstitution in the
analog 6. However, in this case, increased antichagasic efficacy may be related to the increased
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lipophilicity of 3 compared to 6. Reversing the order between the thiazole and phenyl rings in
amide 8 slightly decreased antichagasic activity compared to 6 and once again, this reduction
may be related to slightly lower lipohilicity compared to 6. Furthermore, this reversal lead to a
slightly more toxic compound (compare 8 to 3 and 6). Replacing the terminal phenyl ring in the
biphenyl core of 2 or 5 with a piperidinic group in 9, resulted in reduced efficacy against T.
cruzi, which seems to be attributed to a decreased clogP value (Table 1). Compound 4 with a
thiodiazole ring in the rigid core failed to show either good antichagasic activity or acceptable
selectivity. However, compound 4 has significant solubility problems and may have not been
evaluated properly. Although compound 7, the 2ꢀnitroimidazole analog of 6, showed borderline
antichagasic activity according to the TDR criteria, it was the most toxic rigid amide in L6 cells
and failed to show an acceptable SI for T. cruzi. Compound 7 was about 8.7ꢀfold less active
against T. cruzi compared to its 3ꢀnitrotriazoleꢀanalog 6, even though the latter was 1.5 times less
lipophilic, suggesting that the reason for the reduced antichagasic activity of 7 was related to the
2ꢀnitroimidazole ring. All 3ꢀnitrotriazoleꢀbased rigid amides, except 4, were more potent
antichagasic agents than Bnz, with compound 5 being 45ꢀfold more potent than Bnz (Table 1).
Carbinols and their precursors. With regard to the carbinols in Table 1, which were racemic
mixtures, we observe the following: Lipophilicity and the presence of the nitroꢀgroup in the
triazoleꢀring played a significant role in the antichagasic activity. Thus, the 3ꢀnitrotriazoleꢀbased
carbinol 14, with a terminal imidazole ring in the core, was significantly less lipophilic than
carbinol 17, with a terminal phenyl ring in the core, and 32ꢀfold less potent against T. cruzi
amastigotes than 17 (Table 1). On the other hand, carbinol 15, the analog of 14 without the 3ꢀ
nitro group in the triazole ring, had a negative clogP value and was inactive against T. cruzi.
Interestingly, carbinol 16, the analog of 17 without the 3ꢀnitro group in the triazole ring, was still
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active against T. cruzi, most likely because of its relatively high lipophilicity. However, the lack
of the nitroꢀgroup resulted in a 7ꢀfold reduction in antichagasic potency compared to 17.
Carbinol 18 is the monoꢀnitrotriazole analog of fluconazole. This compound was 36ꢀfold less
potent than its more lipophilic analog 19, which has an additional phenyl ring in the side chain.
However, compound 18 was 8.5ꢀfold more potent than fluconazole, which is less lipophilic and
lacks the nitroꢀgroup (Table 1). Carbinol 19 was the most potent antichagasic compound tested
and demonstrated the highest SI value. In addition, 19 was about 68ꢀfold more potent than Bnz
(Table 1). The bicyclic compound 20 was formed in situ from the corresponding 2ꢀ
nitroimidazole analog of fluconazole presumably by intramolecular nucleophilic aromatic
substitution of the nitroꢀgroup by the hydroxyl group. This compound, although slightly more
lipophilic than 18, was about 5ꢀfold less potent against T. cruzi, presumably because it lacks the
nitro group. In addition, compound 20 was moderately active and marginally selective (SI >
49.4) for T. cruzi parasite. In compound 21 we replaced the hydroxylꢀgroup with a tosylaminoꢀ
group. Compound 21 was selectively active against T. cruzi, but its potency was about 3ꢀfold less
compared to carbinol 17 with a similar lipophilicity. An increased PSA value compared to 17
may have played a role for this reduction in this case.
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Some ketones, precursors of the carbinols in Table 1, were also evaluated for antichagasic
activity. Interestingly, the 3ꢀnitrotriazoleꢀbased ketones 10 and 11, precursors of carbinols 18 and
19, respectively, were found to be selectively active against T. cruzi (Table 1). The pattern of
lipophilicity and the presence of the nitroꢀgroup in the triazole ring were consistent with our
predictions for efficacy. Thus, the more lipophilic 11 was more potent than 10, whereas ketone
12, the analog of 11 without the nitroꢀgroup, was only moderately active against T. cruzi and did
not fulfill the TDR selectivity criteria. Once again, the 2ꢀnitroimidazole analog of 11, ketone 13,
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was only moderately active against T. cruzi parasites, but also the most toxic compound to the
host cells. From this evaluation, it appears that 3ꢀnitrotriazoleꢀbased ketones represent another
class of antichagasic agents. Finally, compound 22, the precursor of carbinols 14 and 15, was
inactive against T. cruzi (Table 1).
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Analysis of anti-HAT activity. Linear, rigid amides 2-9: With regard to the antiꢀHAT
activity of amides 2-9, biphenyl amides 2 and 5 (which were active and moderately active,
respectively) were more potent against T. b. rhodesiense than phenylthiazoleꢀamides 3, 6, 7 and
8. In fact, none of the latter demonstrated an acceptable selective antiꢀHAT activity (Table 1).
The phenylꢀthiodiazoleamide 4 did not show any antiꢀHAT activity, in part due to solubility
issues as mentioned earlier, whereas the piperidinophenylamide 9 was inactive, perhaps because
of planarity issues in the side chain. Interestingly, the 2ꢀnitroimidazoleꢀbased phenylthiazoleꢀ
amide 7 demonstrated a lower IC50 value against T. b. rhodesiense than its 3ꢀnitrotriazoleꢀbased
analog 6, possibly due to the higher lipophilicity of the first. Nonetheless, both were inactive
against this parasite.
Carbinols and their precursors. Only carbinol 14 was moderately active with acceptable
selectivity against T. b. rhodesiense. Carbinol 17 with increased lipophilicity was more active
than 14 but not selective enough for this parasite. Similarly, carbinol 19 was more active than the
less lipophilic analog 18, but also lacked an acceptable selectivity for T. b. rhodesiense. Linear,
rigid carbinols lacking the nitroꢀgroup (15, 16) were inactive against T. b. rhodesiense.
Interestingly, the precursor of carbinol 19, the lipophilic enough 3ꢀnitrotriazoleꢀbased ketone 11,
was moderately active and had an acceptable selectivity for T. b. rhodesiense.
Involvement of type I Nitroreductase. Representative 3ꢀnitrotriazoleꢀbased linear rigid
amides and carbinols, with IC50 values against T. cruzi ranging from 33 nM to 3.29 ꢁM, were
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evaluated as substrates of recombinant TbNTR and TcNTR enzymes (Table 2). Enzyme specific
activity was measured as oxidized NADH per min per mg of protein. All tested nitrotriazoles
were excellent substrates of Tb and TcNTR enzymes. Thus, tested nitrotriazoles were
metabolized by Tb and TcNTR at rates approximately 2ꢀfold higher than Bnz, whereas TbNTR
can metabolize nitrotriazoles (and Bnz) at rates 2 to 3ꢀfold greater than those of TcNTR. In
addition, blood stream form T. b. brucei parasites overexpressing TbNTR were 2 to 3ꢀfold more
susceptible to compound 2 compared to parasites not induced to express elevated levels of
TbNTR (IC50: 0.36 ± 0.01 vs 0.84 ± 0.16). However, no correlation existed between parasitic
IC50 values (Table 1) and activation rate by TcNTR or TbNTR, suggesting that antiparasitic
activity was not exclusively dependent on NTRꢀinduced activation.
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TcCYP51 inhibition. Azoleꢀbased scaffolds are effective inhibitors of the T. cruzi enzyme
sterol 14αꢀdemethylase (TcCYP51).25 By using eburicol as substrate, TcCYP51 is a central
enzyme in parasitic sterol biosynthesis, an essential pathway in all life stages of T. cruzi for the
formation of ergosterol and related structures from lanosterol.28, 29 Being azoles, our compounds
could be potential inhibitors of TcCYP51. Moreover, reduction of the nitro group by P450
reductase (which normally reduces the Fe3+ to Fe2+ in the heme cofactor of CYP51) could
potentially cause irreversible binding to the protein. Benznidazole was found to be an irreversible
inhibitor of TcCYP51, although with weak affinity for the enzyme.30 Binding of a hererocyclic
compound to CYP51 can be quantified spectroscopically by measuring a doseꢀresponse type 2
shift that occurs upon coordination of a Lewisꢀbase atom of the ligand to the heme iron of the
enzyme.9 The first 3ꢀnitrotriazoleꢀbased compound we tested as a potential TcCYP51 ligand was
chlorothiopheneꢀ2ꢀsulfonamide 23, with a flexible core (Table 1). The biological properties of 23
have been described before17 and are included in Table 1 for reference. This compound displayed
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a week affinity toward TcCYP51, providing a high Kd value of 65.2 ± 5.3 (Table 3). Therefore,
we assumed that compounds with a more rigid core could better fit in the active site of the
enzyme and thus act as stronger inhibitors. Indeed, compound 2 with a linear rigid core inhibited
the enzyme with an initial I/E2 ratio (molar ratio of inhibitor/enzyme which causes a 2ꢀfold
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decrease in enzyme activity) of 4 in a 5 min reaction. However, this ratio was increased to 36 in
the 1 h reaction, indicating that inhibition by 2 was reversible. The 3ꢀnitroꢀtriazoleꢀfluconazole
analog 18 binds to TcCYP51 and induces the same typical type 2 spectral response as
fluconazole (Fig. 1), indicating at least initial coordination of the triazole N4 to the P450 heme
iron.9 However, 18 was a weaker binder than fluconazole (Kd 0.606 versus 0.230 ꢁM) (Table 3).
To the contrary, carbinol 19, an analog of fluconazole with an elongated rigid side chain, elicited
the spectral response in TcCYP51 with an apparent Kd 10ꢀfold stronger than fluconazole. In the
reconstituted CYP51 reaction, 19 produced I/E2 ratios of <1 and 3, in 5 min and 1 h reactions,
respectively (Table 3 and Fig. 2). Furthermore, for carbinol 19 at high I/E ratios (≥ 20), the
inhibition seems to be irreversible, although further experiments are needed for verification (Fig.
2). Correlation exists between in vitro antichagasic potency and inhibition of TcCYP51 for the
nitroꢀcompounds 2, 19 and 23. However, compound 18 was more potent in killing T. cruzi
amastigotes than fluconazole (Tables 1), although the latter was a stronger TcCYP51 inhibitor
(Table 3). This might be related to the nitroꢀgroup of 18, making this compound bifunctional.
Crystallography studies could shed more light on the orientation of these bifunctional agents in
the TcCYP51 active site.
In vivo antichagasic activity. Compounds 2, 5, 18 and 19 were evaluated for in vivo antiꢀ
chagasic activity by using an acute T. cruzi infected murine model as described before.19 Bnz
was used in parallel as a positive control. Infected mice (5 mice/group) were treated i.p. for up to
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10 consecutive days with each compound, at 15 mg/kg/day, except for compound 5 which was
administered at 13 mg/kg/day. The Parasite Index (PI), which is an indicator for parasite
clearance, was calculated after 5 and 10 days of treatment.19 The data from these studies are
summarized in Fig. 3. All tested compounds reduced the PI to < 4% after 10ꢀday treatment with
statistical significance compared to the 10ꢀday untreated control. The linear rigid amides 2 and 5
were able to reduce parasites to undetectable levels even after 5 days of treatment. However,
only compound 5 yielded a statistically significant different PI value compared to untreated
control after 5 days of treatment (P<0.0001), whereas for Bnz and compound 2 (treated in the
same experiment) the corresponding P value was 0.080 and 0.0819, respectively. Carbinols 18
and 19 provided a PI value of 24.9 and 42.04, respectively, after 5ꢀday administration but
without statistical significance compared to the untreated control (P = 0.1680 and 0.2706,
respectively). Bnz in this latter experiment provided a PI value of 13.38 after 5ꢀday treatment,
also not statistically different from the untreated control (P = 0.2706). None of the tested
compounds was toxic to mice at the given doses and administration schedule.
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In vitro ADME studies. To understand better the in vivo activity of compounds 2 and 19 and
explain the discrepancy between in vitro and in vivo potency, we performed some in vitro
ADME studies (Table 4). Compound 2 represents the linear rigid amides and 19 the carbinols.
Both compounds demonstrate high Caco 2 permeability31 and similar metabolic stability in the
presence of murine or human microsomes. Both compounds were quite stable, explaining their
good in vivo activity after 10ꢀday dosing. The fact that compound 19 was acting slower than 2
may be related to its stronger binding to TcCYP51, a membrane protein, whereas type I NTR is
mitochondrial based. Thus, if compound metabolites formed through NTRꢀactivation are the
most toxic chemical species for T. cruzi, compound 2 would be expected to act faster than 19.
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Obviously, additional pharmacokinetic parameters (such as volume distribution and protein
binding) which can affect the in vivo behavior of these compounds cannot be excluded.
It is concluded from the above data that 3ꢀnitrotriazoleꢀbased linear rigid amides and
carbinols are potent antichagasic agents via their dual functioning properties as substrates for
trypanosomal type I NTR and as inhibitors of CYP51. In addition, these compounds demonstrate
excellent Caco 2 permeability and metabolic stability in the presence of human or murine
microsomes. Carbinols were slower in clearing the parasites in vivo compared to the linear rigid
amides, however, both type of compounds were able to clear T. cruzi after 10ꢀday treatment.
This is apparent in the images shown in Fig. 4. We have also shown previously that 3ꢀ
nitrotriazoleꢀbased compounds are not mutagenic compared to their 2ꢀnitroimidazoleꢀbased
analogs,19 and do not cause developmental toxicity in zebrafish.20
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In comparison with some monofunctional 3ꢀnitrotriazoleꢀbased analogs, there is no
substantial difference in antichagasic in vitro activity. Thus, we have seen in vitro IC50 values
against T. cruzi amastigotes at low nM concentrations with several 3ꢀnitrotriazoleꢀbased
amides/sulfonamides or piperazines which, having a more flexible core, did not demonstrate
affinity for TcCYP51.17ꢀ19 In the present work, the bifunctional compounds which have been
tested in vivo were able to completely clear the parasites after 10ꢀday treatment, something that
was not always achievable with monofunctional 3ꢀnitrotriazoleꢀbased derivatives.19 However,
since in vitro activity does not always translate to in vivo efficacy, due to pharmacokinetic/
pharmacodynamic factors,19 and since activity in the acute model does not guarantee cures,
future experiments in a chronic murine model are necessary to determine the validity of these
bifunctional compounds for the treatment of Chagas disease.
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ꢀ
EXPERIMENTAL
Chemistry. General: All starting materials and solvents were purchased from SigmaꢀAldrich
7
8
9
(Milwaukee, WI), were of researchꢀgrade 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 MelꢀTemp II Laboratory
Devices apparatus (Holliston, MA) and are uncorrected. Proton NMR spectra were obtained on a
Varian Inovaꢀ500 or an Agilent Hgꢀ400 spectrometer at 500 or 400 MHz, respectively, and were
referenced to Me4Si or to the corresponding solvent if the solvent was not CDCl3. Highꢀ
resolution electrospray ionization (HRESIMS) mass spectra were obtained on a Agilent 6210
LCꢀTOF mass spectrometer at 11000 resolution. Thinꢀlayer 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).
Synthesis of chlorides 1a-g: Some of the chlorides 1a-g are known in the literature and others
are unknown or known through libraries but without any reference. The synthesis and
spectroscopic data of 1a-g are provided in the supporting material.
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General Synthetic Procedure of amides 2-9: The potassium salt of 3ꢀnitroꢀ1,2,4ꢀtriazole or 2ꢀ
nitroimidazole (1 eq) was formed in CH3CN (6ꢀ10 mL), by refluxing with KOH (1.2 eq) for 30
min. To this suspension 1a-g (1.1 eq) was added and the reaction mixture was refluxed under a
nitrogen atmosphere for 9 h. In certain cases, 1a-g 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
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preparative TLC (silica gel or alumina, depending on the mobility of the major band; ethyl
acetateꢀpetroleum ether), the desired product was obtained usually as a powder. Purity was
checked also by HPLC and it was > 95%.
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N-[4-(4-cyanophenyl)phenyl]-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (2):
o
1
White microcrystallic powder (80%): mp 235 C (dec); H NMR (500 MHz, (CD3)2CO) δ: 9.84
(br s, 1H), 8.76 (s, 1H), 7.90ꢀ7,75 (m, 8H), 5.46 (s, 2H). HRESIMS calcd for C17H13N6O3 and
C17H12N6NaO3 m/z [M+H]+ and [M+Na]+ 349.1044 and 371.0863, found 349.1056 and
371.0873.
N-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide
(3):
o
White powder (64%): mp > 235 C; 1H NMR (500 MHz, (CD3)2CO) δ: 8.82 (s, 1H), 7.95 (d, J=8.5
Hz, 2H), 7.64 (s, 1H), 7.46 (d, J=8.5 Hz, 2H), 5.70 (s, 2H). HRESIMS calcd for C13H10ClN6O3S and
+
C13H9ClN6NaO3S m/z [M+H] and [M+Na]+ 365.0218, 367.0191 and 387.0038, 389.0011, found
365.0231, 367.0200 and 387.0038, 389.0012.
N-[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]-2-(3-nitro-1H-1,2,4-triazol-1-yl) acetamide (4):
o
1
White powder (63%): mp > 235 C; H NMR (500 MHz, DMSOꢀ6d) δ: 8.93 (s, 1H), 7.96 (d,
+
J=8.5 Hz, 2H), 7.60 (d, J=8.5 Hz, 2H), 5.57 (s, 2H). HRESIMS calcd for C14H11ClN4O4S m/z [M+H]
366.0184, 368.0148, found 366.0178, 368.0158.
N-(4'-chloro-[1,1'-biphenyl]-4-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (5): Off white
o
1
powder (77%): mp > 245 C; H NMR (500 MHz, CD3OD + a drop of DMSOꢀ6d) δ: 8.73 (s,
1H), 7.69 (d, J=9.0 Hz, 2H), 7.61 (dd, J=9.0, 4.0 Hz, 4H), 7.43 (d, J=8.0 Hz, 2H), 5.32 (s, 2H).
ꢀ
HRESIMS calcd for C16H11ClN5O3 m/z [MꢀH] 356.05556, found 356.0563.
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N-(4-(3,4-difluorophenyl)thiazol-2-yl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (6): White
5
6
o
powder (60%): mp 169ꢀ171 C; 1H NMR (500 MHz, (CD3)2CO) δ: 11.71 (br s, 1H), 8.80 (s, 1H),
7
8
9
7.83 (ddd, J=14, 7.5, 2 Hz, 1H), 7.76 (m, 1H), 7.64 (s, 1H), 7.37 (dd, J= 19, 8.5 Hz, 1H), 5.69 (s, 2H).
+
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HRESIMS calcd for C13H9F2N6O3S m/z [M+H] 367.0419, found 367.0426.
N-(4-(3,4-difluorophenyl)thiazol-2-yl)-2-(2-nitro-1H-imidazol-1-yl)acetamide (7): Off white
powder (55%): mp 180ꢀ183 oC (dec); 1H NMR (500 MHz, (CD3)2CO) δ: 11.70 (br s, 1H), 7.82 (ddd,
J=12, 7.5, 2.5 Hz, 1H), 7.77 (m, 1H), 7.63 (s, 1H), 7.61 (s, 1H), 7.36 (ddd, J= 16.5, 10.5, 8.5 Hz, 1H),
+
7.22 (s, 1H), 5.70 (s, 2H). HRESIMS calcd for C14H10F2N5O3S and C14H9F2N5NaO3S m/z [M+H] and
+
[M+Na] 366.0467 and 388.0286 found 366.0466 and 388.0281.
N-(3-(2-methylthiazol-4-yl)phenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)acetamide (8): White,
o
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sticky solid (73%): mp 114ꢀ115 C (dec); H NMR (400 MHz, CDCl3 + 2 drops DMSOꢀ6d) δ:
10.39 (s, 1H), 8.61 (s, 1H), 8.11 (s, 1H), 7.62 (d, J=7.6 Hz, 2H), 7.39 (s, 1H), 7.36 (t, J=8.0 Hz, 1H),
+
5.27 (s, 2H), 2.75 (s, 3H). HRESIMS calcd for C14H13N6O3S m/z [M+H] 345.0764, found 345.0776.
2-(3-nitro-1H-1,2,4-triazol-1-yl)-N-(4-(piperidin-1-yl)phenyl)acetamide
(9):
Orange
o
1
microcrystals (79%): mp 180ꢀ182 C (dec); H NMR (400 MHz, CDCl3 + 1 drop DMSOꢀ6d) δ:
9.19 (br s, 1H), 8.44 (s, 1H), 7.37 (d, J=9.0 Hz,2H), 6.87 (d, J=9.0 Hz, 2H), 5.10 (s, 2H), 3.10 (t, J=5.5
+
Hz, 4H), 1.68 (m, 4H), 1.57 (m, 2H). HRESIMS calcd for C15H19N6O3 m/z [M+H] 331.1513, found
331.1523.
General Synthetic Procedure of ketones 10-13: As in the case of amides 2-9, the potassium salt of 3ꢀ
nitroꢀ1,2,4ꢀtriazole or 2ꢀnitroimidazole or 1,2,4ꢀtriazole (1 eq) was formed in CH3CN (6ꢀ10 ml), by
refluxing with KOH (1.2 eq) for 30 min and the appropriate αꢀbromoketone (1 eq) was added to this
suspension. The reaction mixture was refluxed under nitrogen atmosphere for 5ꢀ6 h. The procedure for
separation of the desired ketones 10-13 was analogous to that used for amides 2-9.
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1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethan-1-one (10): White crystals
5
6
o
(85%): mp 102ꢀ104 C; 1H NMR (500 MHz, CDCl3) δ: 8.30 (s, 1H), 8.09 (dd, J=9.0, 7.0 Hz, 1H),
7
8
9
7.10 (td, J=9.5, 2.5 Hz, 1H), 7.02 (td, J=9.0, 2.5 Hz, 1H), 5.69 (d, J=3.0 Hz, 2H). HRESIMS calcd for
+
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C10H7F2N4O3 m/z [M+H] 269.0481, found 269.0487.
1-(4-phenylphenyl)-2-(1H-1,2,4-triazol-1-yl)ethan-1-one (11): White powder (95%): mp
o
1
175ꢀ177 C; H NMR [500 MHz, (CD3)2CO] δ: 8.67 (s, 1H), 8.16 (d, J=8.5, 2H), 7.84 (d, J=8.5,
2H), 7.70 (d, J=7.5, 2H), 7.48 (t, J=7.5, 2H), 7.41 (t, J=7.5, 1H), 6.15 (s, 2H). HRESIMS calcd for
+
+
C16H13N4O3 and C16H12N4NaO3 m/z [M+H] and [M+Na] 309.0982 and 331.0802 found 309.0983 and
331.0804.
1-(4-phenylphenyl)-2-(1H-1,2,4-triazol-1-yl)ethan-1-one (12): White microcrystals (65%;
higher yield based on recovered ketone): mp 159ꢀ161 oC; 1H NMR (400 MHz, CDCl3) δ: 8.28 (s,
1H), 8.07 (d, J=8.8 Hz, 2H), 8.03 (s, 1H), 7.76 (d, J=8.8 Hz, 2H), 7.64 (d, J=6.8 Hz, 2H), 7.52ꢀ7.44 (m,
+
3H), 5.71 (s, 2H). HRESIMS calcd for C16H14N3O m/z [M+H] 264.1131, found 264.1154.
2-(2-nitro-1H-imidazol-1-yl)-1-(4-phenylphenyl)ethan-1-one (13): White powder (60 %):
mp 163ꢀ164 oC; 1H NMR [500 MHz, (CD3)2CO] δ: 8.20 (d, J=9.0 Hz, 2H), 7.92 (d, J=9.0 Hz, 2H),
7.78 (d, J=7.0 Hz, 2H), 7.57 (d, J=1.0 Hz, 1H), 7.53 (t, J=7.5 Hz, 2H), 7.46 (t, J=7.5 Hz, 1H), 7.23 (d,
+
J=1.0 Hz, 1H), 6.26 (s, 2H). HRESIMS calcd for C17H14N3O3 m/z [M+H] 308.1030, found 308.1028.
General Synthetic Procedure of carbinols 14-19 and compound 20: The CoreyꢀChaykovsky reaction
was applied for this synthesis and a oneꢀpot reaction was adapted from an international patent26 because
of its short time, albeit with low yields. Briefly, in a 2ꢀnecked 25 mL round bottom flask, KOH (2.4 eq)
was dissolved in 3 mL distilled water and an equal volume of 2ꢀpropanol was added.
Trimethylsulfoxonium iodide (1.2 eq) was added and stirred for 15 min under a nitrogen atmosphere.
Then, the appropriate ketone (1 eq) was added and stirring was continued for 15 min. Finally 3ꢀnitroꢀ
1,2,4ꢀtriazole or 2ꢀnitroimidazole or 1,2,4ꢀtriazole (1.2 eq) was added and the reaction mixture was
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refluxed for 1 h. 2ꢀPropanol was evaporated and the aqueous solution was neutralized with 1% HCl. This
solution was then extracted with dichloromethane and the organic layer washed with NaHCO3 solution.
The organic layer was dried over Na2SO4 and chromatographed by preparative TLC (silica gel, ethyl
acetate plus 1% MeOH). When 2ꢀnitroimidazole was used in the reaction mixture, a nucleophilic aromatic
substitution of the nitroꢀgroup by the hydroxyl took place and the bicyclicꢀcompound 20 was obtained as
the final product.
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2-[4-(1H-imidazol-1-yl)phenyl]-1-(3-nitro-1H-1,2,4-triazol-1-yl)propan-2-ol (14): Off white
powder (15%): mp 133ꢀ134 oC; 1H NMR [500 MHz, (CD3)2CO] δ: 8.47 (s, 1H), 8.06 (s, 1H), 7.72
(d, J=8.5 Hz, 2H), 7.60 (d, J=8.5 Hz, 2H), 7.58 (s, 1H), 7.10 (s, 1H), 5.09 (s, 1H), 4.69 (d, J=4.5 Hz, 2H),
+
1.68 (s, 3H). HRESIMS calcd for C14H15N6O3 m/z [M+H] 315.1200, found 315.1207.
2-[4-(1H-imidazol-1-yl)phenyl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (15): White powder
(32%): mp 138ꢀ140 oC; 1H NMR [500 MHz, (CD3)2CO] δ: 8.20 (s, 1H), 8.05 (s, 1H), 7.80 (s, 1H),
7.66 (d, J=8.5 Hz, 2H), 7.56 (d, J=8.5 Hz, 2H), 7.10 (s, 1H), 4.89 (s, 1H), 4.54 (d, J=4.0 Hz, 2H), 1.57 (s,
+
3H).HRESIMS calcd for C14H16N5O m/z [M+H] 270.1349, found 270.1363.
2-(4-phenylphenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (16): White powder (37%; based on
o
1
recovered ketone the yield was 50%): mp 118ꢀ120 C; H NMR [500 MHz, (CD3)2CO] δ: 1H
8.20 (s, 1H), 7.81 (s, 1H), 7.67ꢀ7.61 (m, 6H), 7.45 (t, J=7.5 Hz, 2H), 7.35 (t, J=7.5 Hz, 1H), 4.79 (s, 1H),
+
4.43 (s, 2H), 1.55 (s, 3H). HRESIMS calcd for C17H18N3O m/z [M+H] 280.1444, found 280.1453.
1-(3-nitro-1H-1,2,4-triazol-1-yl)-2-(4-phenylphenyl)propan-2-ol (17): White powder (19%):
mp was not determined; 1H NMR (500 MHz, CDCl3) δ: 8.17 (s, 1H), 7.63ꢀ7.58 (m, 4H), 7.51ꢀ7.44
(m, 4H), 7.38 (t, J=7.0 Hz, 1H), 4.52 (d, J=3.5 Hz, 2H), 1.67 (s, 3H). HRESIMS calcd for C17H16N4NaO3
+
m/z [M+Na] 347.1115, found 347.1124.
2-(2,4-difluorophenyl)-1-(3-nitro-1H-1,2,4-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)-propan-2-
ol (18): White powder (25%): mp 154ꢀ156 oC; 1H NMR (400 MHz, CDCl3) δ: 8.32 (s, 1H), 7.94
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(s, 1H), 7.87 (s, 1H), 7.42 (ddd, J=8.8, 6.4, 2.4 Hz, 1H), 6.88ꢀ6.79 (m, 2H), 5.66 (s, 1H), 4.99 (d, J=14.4
Hz, 1H), 4.76 (d, J=14.4 Hz, 1H), 4.64 (d, J=14.0 Hz, 1H), 4.34 (d, J=14.0 Hz, 1H). HRESIMS calcd for
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C13H19F2N7O3 m/z [M+H] 352.09697, found 352.0980.
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1-(3-nitro-1H-1,2,4-triazol-1-yl)-2-(4-phenylphenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol
(19): White powder (33%): mp 90ꢀ93 oC (dec); 1H NMR [500 MHz, (CD3)2CO] δ: 8.46 (s, 1H),
8.23 (s, 1H), 7.86 (s, 1H), 7.65ꢀ7.60 (m, 6H), 7.45 (t, J=8.0, 2H), 7.36 (t, J=7.5, 1H), 5.57 (s, 1H), 4.98 (d,
J=14.0, 1H), 4.95 (d, J=14.0, 1H), 4.91 (d, J=14.0, 1H), 4.79 (d, J=14.0, 1H). HRESIMS calcd for
+
C19H18N7O3 m/z [M+H] 392.1466, found 392.1469.
1-{[2-(2,4-difluorophenyl)-2H,3H-imidazo[2,1-b][1,3]oxazol-2-yl]methyl}-1H-1,2,4-triazole
o
1
(20): White powder (29 %): mp 134ꢀ137 C (dec); H NMR [500 MHz, (CD3)2CO] δ: 8.35 (s,
1H), 7.72 (s, 1H), 7.49 (ddd, J=17.5, 9.0, 6.5 Hz, 1H), 7.21 (ddd, J=11.5, 9.0, 2.5 Hz, 1H), 7.05 (dddd,
J=11.5, 9.0, 3.0, 1.0 Hz, 1H), 6.66 (d, J=1.5 Hz, 1H), 6.51 (d, J=1.5 Hz, 1H), 5.04 (d, J=15.0 Hz, 1H),
4.95 (d, J=15.0 Hz, 1H), 4.88 (dd, J=11.0, 2.0 Hz, 1H), 4.46 (dd, J=11.0, 2.0 Hz, 1H). HRESIMS calcd
+
+
for C14H12F2N5O and C14H11F2N5NaO m/z [M+H] and [M+Na] 304.1004 and 326.0824, found 304.1006
and 326.0829.
4-Methyl-N-[1-(3-nitro-1H-1,2,4-triazol-1-yl)-3-phenylpropan-2-yl]benzene-1-sulfonamide
(21): 3ꢀNitroꢀ1,2,4ꢀtriazole (0.68 mmol) was dissolved in dry 1ꢀpropanol (5 mL) and then NaH
(60%, 1.05 eq) was added. Then the commercially available (Aldrich) Sꢀ(+)ꢀ2ꢀbenzylꢀ1ꢀ(pꢀ
tosylaziridine) (0.68 mmol) was added and the reaction mixture was refluxed for 6 h. The
reaction mixture was chromatographed on silica gel plates with ethyl acetateꢀpetroleum ether
(60:40) as eluent. White crystals (36 %): mp 147ꢀ148 oC; 1H NMR (400 MHz, CDCl3) δ: 8.18 (s,
1H), 7.43 (d, J=8.0 Hz, 2H), 7.26 (m, 3H), 7.18 (d, J=8.0 Hz, 2H), 7.00 (m, 2H), 4.64 (d, J=7.2 Hz, 1H),
4.54 (dd, J=14.0, 4.0 Hz, 1H), 4.20 (dd, J=14.8, 6.8 Hz, 1H), 3.83 (m, 1H), 2.96 (dd, J=14.8, 6.8 Hz, 1H),
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2.75 (dd, J=14.4, 7.2 Hz, 1H), 2.42 (s, 3H). HRESIMS calcd for C18H20N5O4S m/z [M+H] 402.1231,
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found 402.1235.
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1-(4-(2-methyloxiran-2-yl)phenyl)-1H-imidazole (22): This compound was isolated during
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the synthesis of compound 14. White crystals (17%): mp 79ꢀ81 C; H NMR [500 MHz,
(CD3)2CO] δ: 8.05 (s, 1H), 7.58 (d, J=8.5 Hz, 2H), 7.56 (s, 1H), 7.53 (d, J=8.5 Hz, 2H), 3.00 (d, J=5.5
+
Hz, 1H), 2.77 (d, J=5.5 Hz, 1H), 1.71 (s, 3H). HRESIMS calcd for C12H13N2O m/z [M+H] 201.1022,
found 201.1024.
Biological evaluation. In vitro screening: In vitro activity against T. cruzi, T. b. rhodesiense,
and cytotoxicity assessment using L6 cells (rat skeletal myoblasts) was determined using a 96ꢀ
well plate format as previously described.31 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.
Enzymatic activity studies with Type I NTR. Recombinant TbNTR was prepared and assayed
as previously described.33,34 The activity of purified hisꢀtagged TbNTR was assessed
spectrophotometrically at 340 nm using various nitrotriazole substrates (100 µM) and NADH
(100 µM) and expressed as nmol NADH oxidized minꢀ1 mgꢀ1 of enzyme.
Testing T. cruzi CYP51 as a potential inhibition target. Selected compounds (2, 18 and 19)
were evaluated as CYP51 ligand/inhibitors using purified fullꢀlength T. cruzi CYP51 protein
sample.28 Binding affinities of the compounds were estimated using spectral titration. The
experiments were performed on a dualbeam Shimadzu UVꢀ240IPC spectrophotometer in the
wavelength range 350−450 nm at 25 °C in 2 mL tandem cuvettes at 2 ꢁM P450 and ligand
concentrations ranging from 0.5 to 10 ꢁM. The apparent dissociation constants were calculated
by plotting the absorbance changes in the difference spectra (ꢂA425−390) upon titration against
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free ligand concentration and fitting the data to a rectangular hyperbola in Sigma Plot Statistics.
Inhibitory potencies of the compounds were compared in the reconstituted CYP51 reaction 5 min
and 1 h, at 50/2/1 molar ratio substrate/inhibitor/enzyme. P450 concentration in the reaction
mixture was 1 ꢁM. The reaction products were analyzed on a reverseꢀphase HPLC system
(Waters) equipped with a C18Nova Pak column and a βꢀRAM detector (INUS Systems, Inc.).9
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.19 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 Dꢀluciferin potassiumꢀsalt 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.35 Treatment with test compounds was started 4 days after infection at a
specific concentration (usually 15 mg/kg/day x 10 days) by i.p. injection. The vehicle control
was 2% methylcellulose + 0.5% Tween 80 and groups of 5 mice/group were used. In the vehicle
control group 10 mice were used. Mice were imaged on days 5 and 10 as above. Parasite index
was calculated as the ratio of parasite levels in treated mice compared to the control group and
multiplied by 100. The ratio of parasite levels is 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 then used to calculate the parasite index.35
In vitro ADME studies. ADME in vitro studies were performed by APREDICA (Watertown,
MA). Samples were analyzed by LC/MS/MS using an Agilent 6410 mass spectrometer coupled
with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter
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software (Agilent). After separation on a C18 reverse phase HPLC column (Agilent, Waters, or
equivalent) using an acetonitrileꢀwater gradient system, peaks were analyzed by mass
spectrometry (MS) using ESI ionization in MRM mode.
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Microsomal stability screen: Each test compound was dissolved in DMSO and incubated (37
ºC) at 1 ꢁM final concentration with 0.3 mg/mL of microsomal protein in 100 mM potassium
phosphate, 3 mM MgCl2, pH 7.4, in the presence or absence of 2 mM NADPH (to detect
NADPHꢀfree degradation) for up to 60 min as has been described before.19 Data were reported as
% remaining parent compound.
Caco-2 monolayer permeability studies: Cacoꢀ2 cells grown in tissue culture flasks were
trypsinized, suspended in medium, and the suspensions were applied to wells of a collagenꢀ
coated BioCoat Cell Environment in 96ꢀwell format. The cells were allowed to grow and
differentiate for three weeks, feeding at 2ꢀday intervals. For Apical to Basolateral (A→B)
permeability, the test agent was added to the apical (A) side at 10 ꢁM final concentration and
amount of permeation was determined on the basolateral (B) side after 2 h assay time as
described before.19 Data were expressed as permeability (Papp): Papp = (dQ/dt)/C0A, where
dQ/dt is the rate of permeation, C0 is the initial concentration of test agent, and A is the area of
the monolayer.31
ꢀ
ACKNOWLEDGEMENTS:
The authors thank M. Cal, S. Keller and M. Jud (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 by internal funds of the Radiation Medicine Department at
NorthShore University HealthSystem. Experiments on T. cruzi CYP51 were funded by NIH
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(GM067871, G.I.L.). In vitro screenings against parasites were funded by DNDi. For that
project, DNDi received funding from the following donors: Department for Internationl
Development (DFID), UK; Bill & Melinda Gates Foundation (BMGF), USA; Reconstruction
Credit InstitutionꢀFederal Ministry of Education and Research (KfWꢀBMBF), Germany; and
DirectorateꢀGeneral for International Cooperation (DGIS), the Netherlands. Benjamín Aguileraꢀ
Venegas acknowledges financial support by FONDECYT Postdoctorado 3130364. The donors
had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
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ꢀ
ETHICAL STATEMENT: Animal studies were approved by the Institutional Animal Care
and Use Committee of New York University School of Medicine (protocol #81213). This
protocol adheres to the guidelines of the Association for Assessment and Accreditation of
Laboratory Animal Care International (AAALAC).
ꢀ
SUPPORTING INFORMATION AVAILABLE: “Synthesis, spectroscopic data and
references of precursor chloroacetamides 1a-g”.
ꢀ
ꢀ
*Correspondence to: Maria V. Papadopoulou, Ph.D., NorthShore University HealthSystem,
Department of Radiation Medicine, 2650 Ridge Ave., Evanston, IL, 60201, USA.
Tel: (847)570ꢀ2262; Fax: (847)570ꢀ1878
eꢀmail: mpapadopoulou@northshore.org
ABBREVIATIONS USED
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NTD, Neglected tropical diseases; T. cruzi, Trypanosoma cruzi; T. brucei, Trypanosoma brucei;
HAT, human African trypanosomiasis; Nfx, nifurtimox (4ꢀ(5ꢀnitrofurfurylindenamino)ꢀ3ꢀ
methylthioꢀmorpholineꢀ1,1ꢀdioxide); Bnz, benznidazole (Nꢀbenzylꢀ2ꢀ(2ꢀnitroꢀ1Hꢀimidazolꢀ1ꢀ
yl)acetamide); NTR, type I nitroreductase; TcNTR, T. cruzi NTR; TbNTR, T. brucei NTR;
CYP51, sterol 14αꢀdemethylase enzyme; TcCYP51, T. cruzi CYP51; IC50, concentration for
50% growth inhibition; SI, selectivity index; SARs, structureꢀactivity relationships; TDR,
Tropical Diseases Research (http://www.who.int/tdr/en/).
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