Novel Imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides as antiproliferative trypanosoma cruzi drugs: Computational screening from neural network, synthesis and in?vivo biological properties

Article abstract of DOI:10.1016/j.ejmech.2017.04.075

A new family of imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide with antiproliferative Trypanosoma cruzi properties was identified from a neural network model published by our group. The synthesis and evaluation of this new class of trypanocidal agents are d

Full text of DOI:10.1016/j.ejmech.2017.04.075

Accepted Manuscript
Novel Imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides as antiproliferative trypanosoma
cruzi drugs: Computational screening from neural network, synthesis and in vivo
biological properties
Angela Guerra, Pedro Gonzalez-Naranjo, Nuria E. Campillo, Javier Varela, María L.
Lavaggi, Alicia Merlino, Hugo Cerecetto, Mercedes González, Alicia Gomez-Barrio,
José A. Escario, Cristina Fonseca-Berzal, Gloria Yaluf, Jorge Paniagua, Juan A. Páez
PII:
S0223-5234(17)30357-4
10.1016/j.ejmech.2017.04.075
EJMECH 9426
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2017
Revised Date: 25 April 2017
Accepted Date: 29 April 2017
Please cite this article as: A. Guerra, P. Gonzalez-Naranjo, N.E. Campillo, J. Varela, Marí.L. Lavaggi,
A. Merlino, H. Cerecetto, M. González, A. Gomez-Barrio, José.A. Escario, C. Fonseca-Berzal, G.
Yaluf, J. Paniagua, J.A. Páez, Novel Imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides as antiproliferative
trypanosoma cruzi drugs: Computational screening from neural network, synthesis and in vivo biological
properties, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.075.
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.

ACCEPTED MANUSCRIPT

1
ACCEPTED MANUSCRIPT
Novel Imidazo[4,5-c][1,2,6]thiadiazine 2,2-Dioxides as Antiproliferative Trypanosoma Cruzi Drugs:
Computational Screening from Neural Network, Synthesis and in vivo Biological Properties
Angela Guerraa, Pedro Gonzalez-Naranjoa, Nuria E. Campillob, Javier Varelac, María L. Lavaggic, Alicia Merlinoc,
Hugo Cerecettoc, Mercedes Gonzálezc, Alicia Gomez-Barriod, José A. Escariod, Cristina Fonseca-Berzald, Gloria
Yalufe, Jorge Paniaguaf, and Juan A. Páeza*
a
Instituto de Química Médica. Consejo Superior de Investigaciones Científicas (CSIC), Juan de la Cierva 3,
28006 Madrid, Spain.
b
Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas (CSIC). Ramiro de
Maeztu 9, 28040 Madrid, Spain.
c
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química,
Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.
d
Departamento de Parasitología. Facultad de Farmacia. Universidad Complutense de Madrid, Pza. Ramón y
Cajal s/n, 28040, Madrid, Spain.
eInstituto de Investigaciones en Ciencias de la Salud (iics), Universidad Nacional de Asunción, Asunción-
Paraguay
f Laboratorios Silanes IDF, S.L. Calle Santiago Grisolia, Nº 2- PTM 148 Parque Tecnologico de Madrid 28760-
Tres Cantos-Madrid.
*Corresponding author. Dr. Juan A. Páez. Instituto de Química Médica (CSIC). Juan de la Cierva, 3, 28006-
Madrid. Spain. Phone: +34 915622900; Fax: +34 915644853; E-mail: jpaez@iqm.csic.es

2
ACCEPTED MANUSCRIPT
Abstract
A new family of imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide with antiproliferative Trypanosoma cruzi properties
was identified from a neural network model published by our group. The synthesis and evaluation of this new
class of trypanocidal agents are described. These compounds inhibit the growth of Trypanosoma cruzi,
comparable with benznidazole or nifurtimox. In vitro assays were performed to study their effects on the growth of
the epimastigote form of the Tulahuen 2 strain, as well as the epimastigote and amastigote forms of CL clone B5
of Trypanosoma cruzi. To verify selectivity towards parasite cells, the non-specific cytotoxicity of the most
relevant compounds was studied in mammalian cells, i.e. J774 murine macrophages and NCTC clone 929
fibroblasts. Furthermore, these compounds were assayed regarding the inhibition of cruzipain. In vivo studies
revealed that one of the compounds, 19, showed interesting trypanocidal activity, and could be a very promising
candidate for the treatment of Chagas disease.
Keywords: CODES®, Chagas disease, imidazo[4,5-c][1,2,6]thiadiazine, neural network, trypanocidal,
Trypanosoma cruzi

3
ACCEPTED MANUSCRIPT
1. INTRODUCTION
Chagas disease, also known as American trypanosomiasis, is the result of infection with the protozoan parasite
Trypanosoma cruzi. This zoonosis can be transmitted to humans by insect vectors that are found only in the
American continent, mainly in rural areas with unhealthy housing conditions where poverty is a general concern.
The World Health Organization (WHO) estimates that 6-7 million people are currently infected with Trypanosoma
cruzi in the 21 endemic countries of the American continent [1]. In recent years, migration and travel have
extended the distribution of Chagas disease to non-endemic areas of other continents, including Europe [2,3]
parts of the Western Pacific [4] and North America [5, 6]. Most people with Trypanosoma cruzi acquire their
infections in endemic countries [5].
Despite the efforts and initiatives taken by the WHO in terms of transmission control of Chagas disease, it is
estimated that over 25 million people are at risk of infection. The economic cost of Chagas disease was
estimated in 2013 at more than US $7 billion per year, with more than 7000 deaths per year caused by this
disease [7].
Currently, only two drugs are used for the treatment of Chagas disease. These are two nitro heterocyclic
compounds, i.e. a nitrofuran (nifurtimox) and a nitroimidazole (benznidazole) whose anti-Trypanosoma cruzi
activities were discovered empirically over four decades ago [8]. These drugs are not approved by the FDA and
are available only from the Centers for Disease Control and Prevention (CDC) under investigational drug
protocols [9]. The production of nifurtimox has been discontinued; thus, benznidazole is currently the only
commercially available treatment in most endemic countries. Two alternative drugs for Chagas disease treatment
are allopurinol and itraconazole, which have been used in some selected cases and in certain patients [10,11]
(Figure 1). Several clinical trials for the treatment of chronic Chagas disease with posaconazole [12, 13] and
E1224 [14, 15] have been completed in the last year. Unfortunately, these trials did not show significant
advantages in relation to benznidazole [16-18].

4
ACCEPTED MANUSCRIPT
Figure 1. Chemical structures of drugs introduced to the market and in clinical trials for Chagas disease.
The current treatments are unsatisfactory because of their low effectiveness in the chronic phase and their
adverse side effects, in addition to the fact that they do not address the serious consequences of this illness [8].
The side effects of these drugs include dermatological reactions [19] agranulocytosis and polyneuropathy [20].
Benznidazole and nifurtimox should not be taken by pregnant women [21] or by people with kidney or liver failure
[21- 23]. It has been found that benznidazole can provoke lymphoma in a rabbit model [24]. However, in humans
and at the doses used for Chagas treatment, lymphoma has not been detected. In addition, a mechanism for
cross-resistance to nifurtimox and benznidazole has been described [25].
It is, therefore, necessary and urgent to develop new and effective trypanocidal drugs for the suitable treatment of

5
ACCEPTED MANUSCRIPT
this disease. Motivated by our interest in the development of effective drugs for Chagas disease, a virtual
screening approach was applied to discover new hits against Trypanosoma cruzi. In this study, we report the
identification, synthesis and pharmacological assessment of a new family of heterocyclic compounds derived
from imidazothiadiazines with interesting trypanocidal properties.
2. RESULTS AND DISCUSSION
2.1 Rational Design
One of the key steps in drug design is the hit identification process. In line with our ongoing efforts on the
development of trypanocidal drugs, we developed a neural network model with a three-layered 4-4-1 architecture
to predict anti-T. cruzi activity against the epimastigote form of the Tulahuen strain with 78% accuracy in the
external prediction [26]. The codification of the structures was achieved with the CODES® program [27]. In this
context, we employed the model to predict the potential activity of a diverse set of compounds developed in our
laboratory. Among these compounds, we identified several imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide
derivatives showing interesting anti-T. cruzi activity.
Based on these hits, we proposed a virtual library of derivatives, taking into account that the structure of the
imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide system can have variations on the substituents at three different
positions. Thus, at position 6, a variety of groups such as alkyl, cycloalkyl, benzyl, aryl or heteroaryl were tested.
N1-substituted groups included hydrogen, methyl, benzyl, or 4-chlorobenzyl. At position 5 of the heterocycle, only
hydrogen and benzyl were proposed (Figure 2). Finally, we predicted the values of the percentage of growth
inhibition (PGIe) against epimastigotes of T. cruzi at a fixed dose for a set of 20 imidazo[4,5-c][1,2,6]thiadiazine
2,2-dioxides that include N-1H, N-1 substituted and N-1 and N-5 substituted imidazothiadiazines (Figure 2).

6
ACCEPTED MANUSCRIPT
Figure 2. Chemical structures of the imidazo[4,5-c][1,2,6]thiadiazine derivative family.
Thus, using the methodology developed previously, we achieved the codification of the molecules from a non-
supervised neural network using the CODES® program [27]. This program codifies each molecule into a set of
numerical parameters, exclusively taking into account the information on its chemical structure. A subsequent
dimension reduction procedure by means of supervised back-propagation neural network (NN) provided a set of
four parameters for each structure [28, 29] (Figure 3). Thus, the biological activity expressed as PGIe was
calculated and is presented in Table 1.
Figure 3. Protocol used to develop the neural network model.

7
ACCEPTED MANUSCRIPT
The calculated values indicate an interesting predicted activity (> 98% at 25 µM) for four derivatives that include
the N-1H 4, N-1 substituted 15, 17 and N-1,N-5 substituted 19 imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides. On
the basis of these findings, all compounds were tested in vitro as trypanomicidal agents against the Tulahuen 2
strain. The growth inhibition of the epimastigote form of T. cruzi by imidazothiadiazine derivatives 1-20 was
determined and the results are shown in Table 1.
Table 1. Predicted and experimental values of the PGIe and IC50 of imidazothiadiazine derivatives 1-20.
PGIe
PGIe
Compd
R2
R3
R1
IC50 (µM)
(calcd)a
(exp)b
Nifurtimox
7.7 ± 0.5
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
40
13
57
100
53
28
6
38.7± 5 > 25
2
CH2-CH3
43.0± 2 28.5 ± 0.8
42.2± 3 28.9 ± 0.4
72.3± 1 12.7± 0.6
39.6± 4 > 25
3
cyclopentyl
cyclohexyl
4
5
Ph
6
2-fluorophenyl
4-dimethylaminophenyl
3-thienyl
57.8 ± 2 20.2 ± 0.6
42.8± 1 28.5 ± 0.5
51.3 ± 5 22.2 ± 0.7
37.5 ± 5 > 25
7
8
59
59
6
9
3-pyridinyl
10
11
12
13
5-nitro-2-furyl
3-tetrahydrofuryl
CH2-Ph
31.3± 3 > 25
59
59
7
56.4± 4 20.3 ± 0.4
59.7± 2 16.2± 0.2
73.6± 1 11.5± 0.5
CH2-CH2-Ph

8
ACCEPTED MANUSCRIPT
14
15
16
17
18
19
20
CH3
H
H
H
H
H
3-thienyl
51
99
5
46.3± 3 26.2 ± 0.8
58.0± 1 16.7± 0.4
41.6± 1 29.5 ± 0.5
86.3± 2 7.0 ± 0.5
CH3
Ph
CH2-Ph
CH2-4-ClPh
CH2-4-ClPh
CH2-Ph
CH2-Ph
Ph
2-fluorophenyl
cyclohexyl
100
51
99
5
0± 5
> 25
CH2-Ph Ph
100.0± 1 4.5 ± 0.3
45.7± 3 28.7 ± 0.3
CH2-Ph cyclohexyl
aPGIe(calcd): Predicted value of the % of growth inhibition (PI) of the parasite by our NN model;
bPGIe(exp): Experimental value of the % of growth inhibition (PI) of the parasite at 25 µM.
2.2 Chemistry
The structures of the imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide system show variations on the substituents at
three different positions. At position 6, a variety of groups such as alkyl, cycloalkyl, benzyl, aryl and heteroaryl
were considered. N1-substituted groups such as hydrogen, methyl, benzyl, or 4-chlorobenzyl were considered. At
position 5 of the heterocycle, only hydrogen and benzyl were assessed.
The compounds prepared for this study are presented in Figure 2. Their general synthetic route (outlined in
Scheme I) involves formation of the parent 4-amino-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide [30]
bearing different substituents at C-5, and subsequent introduction of the substituents at N-1 or at N-1 and N-5.
The first step, preparation of the imidazo[4,5-c][1,2,6]thiadiazine derivatives, consists of the reaction of the 3,4,5-
triamino-2H-1,2,6-thiadiazine 1,1-dioxide 21 [31] with suitably functionalized aldehydes. Compound 21 was
prepared, according to a process described in three reaction steps, from sulfamide and sulfuryl chloride [31, 32].
So, the reaction of 21 with aliphatic aldehydes such as acetaldehyde, propionaldehyde,
cyclopentanecarbaldehyde, or cyclohexanecarbaldehyde afforded the corresponding 5-methyl, 5-ethyl, 5-
cyclopentyl or 5-cyclohexyl compounds 1-4, respectively (Figure 2). The preparation of 5-phenyl [30], 5-(2-
fluorophenyl), and 5-(4-dimethylaminophenyl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide 5-7 were carried

9
ACCEPTED MANUSCRIPT
out starting from 21 and the corresponding aromatic aldehydes. In a similar way, the reaction of 21 with
heterocyclic aldehydes such as 3-thienyl, 3-pyridyl, 5-nitro-2-furyl, or 3-tetrahydrofuryl afforded the corresponding
imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides 8-11, respectively. The 5-benzyl and 5-phenethyl-1H-[4,5-
c][1,2,6]thiadiazine 2,2-dioxides 12 and 13 were prepared from 21 and benzyl and phenethyl aldehydes,
respectively.
Scheme 1. General synthetic route for the preparation of imidazothiadiazine derivatives. Reagents and conditions:
a) H2O/AcOH, rt; b) MeI/K2CO3, acetone, reflux for 14, Me2SO4 /K2CO3/H2O for 15; R2-Br, Et3N, acetone, reflux for 16-18 ; c)
benzyl bromide, sodium hydroxide, H2O, reflux for 19; benzyl bromide, K2CO3, acetone, reflux for 20.
The second step of the route shown in Scheme 1 is the functionalization of the N-1 and N-5 positions. Different
alkylation procedures were tested to selectively obtain the final N-1-substituted derivatives. Substitution at
position 1 of the corresponding N-1H imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides was performed with alkyl
halides in acetone using either potassium carbonate or triethylamine as the base. When the alkylation reaction
was carried out with dimethyl sulfate or methyl iodide in potassium carbonate, it was possible to obtain only the
N-1 methyl derivatives 14 and 15, respectively (Scheme 1). The benzylation of 6-phenylimidazothiadiazine 5 in
potassium carbonate afforded a mixture of the monobenzyl derivative 16 and the dibenzyl derivative 19 at the N-1

10
ACCEPTED MANUSCRIPT
and N-5 position. The benzyl N-1 selectively mono-substituted derivatives were obtained by the reaction of the
corresponding halide in the presence of triethylamine. Thus, the reaction of N-1H-imidazo[4,5-c][1,2,6]thiadiazine
derivatives 4-6 with the corresponding bromides afforded the corresponding benzyl 16 and 4-chlorobenzyl
derivatives 17 and 18.
Finally, when the imidazo[4,5-c][1,2,6]thiadiazine had the same substituent at both the N-1 and N-5 positions, the
reaction was carried out in acetone and potassium carbonate or in an aqueous solution of sodium hydroxide.
Thus, the preparation of the substituted 1,5-dibenzylimidazothiadiazine 2,2-dioxides 19 and 20 were carried out
starting from imidazothiadiazine 5 or 4 and benzyl bromide, respectively (Scheme 1).
2.3. Pharmacology
2.3.1. Trypanocidal activity against the epimastigote form of T. cruzi strain Tulahuen 2
First, the percentage of growth inhibition of the epimastigote form of the Tulahuen 2 strain was determined at 25
µM of all compounds 1-20 using nifurtimox as the positive control. The compounds that showed a PGIe cut-off
higher than 70% (Table 1) were considered as good anti-T. cruzi agents [26, 33]. In general, as can be seen in
Table 1, the experimental PGIe values for derivatives 1-20 are in good agreement with those predicted by our
neural network model, showing an prediction accuracy greater than 80%.
According to PGIe(exp) values (Table 1), three N1-H derivatives 4, 12, 13, two N-1 substituted derivatives 15, 17
and N-1, N-5-substituted compound 19 showed interesting anti-T. cruzi properties in the epimastigote form of the
Tulahuen 2 strain, of all imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide derivatives considered. The IC50 values of
these imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide derivatives were calculated and the values are presented in
Table 2. The results show that derivatives 4, 12, 13, 15, 17 and 19 have excellent trypanocidal activity with
potency, in cases such as 17 and 19, equal to or greater than nifurtimox, used as the reference drug.

11
ACCEPTED MANUSCRIPT
Table 2. Trypanocidal activities against the epimastigote form of strain Tulahuen 2 presented as IC50 and non-
specific cytotoxicities (IC50) in murine macrophages (J774) of selected imidazothiadiazine derivatives.
SIb
IC50 (µM)a
IC50 (µM)a
Compd
Tulahuen 2 strain
J774
Nifurtimox
7.7 ± 0.5
16.2 ± 0.2
12.7 ± 0.6
11.5 ± 0.5
7.0 ± 0.5
16.7 ± 0.4
4.5 ± 0.3
316 ± 5
>400
41
> 25
>31
>34.8
<14.2
7.7
4
12
13
17
15
19
>400
>400
<100
128 ± 2
100 ± 5
22
a
b.
IC50 : Concentration that inhibit the 50% of parasite growth;
SI: Selectivity index defined as
IC50(J774)/IC50(Tulahuen2).
2.3.2. Evaluation of non-specific cytotoxicity in mammalian cell lines.
The non-specific cytotoxicity against J774 murine macrophages was assayed using the imidazo[4,5-
c][1,2,6]thiadiazine derivatives that showed an IC50 < 20 µM for the epimastigote form of T. cruzi strain Tulahuen
2. The results are presented in Table 2, including nifurtimox as the reference drug.
It was clearly observed that derivatives 4, 12, 13, 15, 17, and 19 showed selectivity for parasite cells; derivatives
4, 12, and 13 showed similar or better selectivity compared to the reference compound.
In view of these results, all derivatives were taken into account for additional studies. Thus, the cytotoxicity and
trypanocidal activity against another strain were evaluated.
2.3.3. Trypanocidal activity against the epimastigote form of T. cruzi strain CL-clone B5
The activity of the imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide derivatives 4, 12, 13, 15, 17, and 19, selected to
test against the epimastigote forms of T. cruzi strain CL-clone B5, is expressed as the PGIe. The IC50 values are
presented in Table 3, including nifurtimox and benznidazole as the reference drugs.

12
ACCEPTED MANUSCRIPT
Regarding the trypanocidal activity against T. cruzi strain CL-clone B5, the results indicate that all derivatives
showed higher IC50 values in relation to the Tulahuen strain, except for derivative 4 that showed the highest anti-
T. cruzi activity. Of note, this level of trypanocidal activity for compound 4 is ten times higher that of benznidazole,
the only available treatment for Chagas disease.
Table 3. Trypanocidal activity of imidazothiadiazine derivatives against the epimastigote form of the CL-clone B5
strain (PGIe, IC50e) and non-specific cytotoxicity (IC50) in murine fibroblasts (NCTC clone 929).
Anti-epimastigote activity
IC50 (µM)
NCTC-929
> 256
> 256
> 256
NDa
Compd
SI
PGIe (25 µM) IC50e (µM)
Nifurtimox
82.2 ± 2.8
49.0 ± 1.2
54.8 ± 3.3
17.7 ± 5.1
51.1 ± 0.5
11.3 ± 2.8
42.1 ± 7.4
60.8 ± 2.1
3.6
27.1
2.5
> 71.1
> 9.4
> 102.4
ND
Benznidazol
4
12
13
15
17
19
> 25
17.5
> 25
> 25
3.6
> 256
ND
> 14.6
ND
ND
ND
< 64
< 17.8
a ND: Not determined
2.3.4. Evaluation of non-specific cytotoxicity in murine fibroblasts (NCTC clone 929)
The non-specific cytotoxicity in murine fibroblasts was determined for the most interesting derivatives 4, 12, 13,
and 19 that showed trypanocidal activities against the Tulahuen 2 and CL-clone B5 strains. Selectivity indexes
were calculated in order to confirm the specific action of these drugs against the parasite (Table 3), showing that
derivatives 4, 12, 13, and 19 exhibited selectivity indexes on the same order or higher than that of benznidazole.

13
ACCEPTED MANUSCRIPT
2.3.5. Trypanocidal activity against intracellular amastigotes of T. cruzi (CL-clone B5 strain)
According to these preliminary results, the most interesting compounds, 4 and 19, were chosen to study their
activities against T. cruzi intracellular forms. The percentage of growth inhibition of the amastigote stage (PGIa)
are shown in Table 4, together with their IC50 values (IC50a). Nifurtimox and benznidazole were also tested as
reference drugs.
The results obtained from this study show that the N-1H derivative 4 did not have anti-amastigote activity at 25
µM. However, derivative 19 show interesting trypanocidal activity according to its capacity to inhibit the
amastigote form of T. cruzi.
Table 4. Trypanocidal activities of 19 and 4 against the intracellular amastigote form of the CL-clone B5 strain
(PGIa, IC50a).
Anti-amastigote activity
Compd
PGIa (25 µM) IC50a (µM)
Nifurtimox
100 ± 2.6
100 ± 0.0
90.2 ± 13.8
0.0 ± 9.0
< 0.5
0.8
Benznidazol
19
16.9
NDa
4
a ND: Not determined
2.3.6. Cruzipain inhibitory activity
Cruzipain, the most abundant endoproteinase of T. cruzi, has been described as one of the most promising
candidate targets for Chagas disease drug development [34,35]. Its biological relevance has been demonstrated
using different cruzipain inhibitors that block parasite development in vitro, regardless of the chemical structure
[14, 36]. Most cruzipain inhibitors described up to now belong to the peptidomimetic class, particularly 6-((3,5-
difluorophenyl)amino)-9-ethyl-9H-purine-2-carbonitrile, an imidazopyrimidine [37]. Taking into account that this

14
ACCEPTED MANUSCRIPT
compound shares structural motifs with our imidazothiadiazine family, the capacity to inhibit the cruzipain enzyme
was assessed in order to study the mechanism of action of the most active compounds 4, 12, 13, 15, 17, and 19
(Table 5). These six imidazothiadiazine derivatives presented variable degrees of cruzipain inhibition. The results
indicate that some imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides showed significant cruzipain inhibition, particularly
the N-1H derivatives 4 and 12. Therefore, trypanocidal activity may be mediated partly through cruzipain
inhibition. However, it is clearly that other mechanism of action should be involved since the most interesting
compound shows the highest value of IC50.
Table 5. Cruzipain inhibitory activity.
Compd
IC50 (µM)
12 ± 2
4
12
23 ± 3
13
39.0 ± 0.5a
27.4 ± 0.5a
86 ± 4
15
17
19
81 ± 4
mbth b
0.10 ± 0.01
b
a Percentage of inhibition of cruzipain at 100 µM. (m-bromophenyl)ethylketone thiosemicarbazone (reference
compound)
2.3.7. Mutagenicity study (Ames test)
An important issue in drug development is to establish the mutagenicity of the molecule. Regarding nifurtimox
and benznidazole, besides the side effects described above, these compounds show mutagenicity [38, 39].
Therefore, driven by our interest in determining the druggability of our compounds, we assessed the mutagenic
capacity of a representative set of derivatives including N-1H (12), N-1 substituted (15), and N-1, N-5-substituted
(19) compounds in the Ames test.

15
ACCEPTED MANUSCRIPT
For this, Salmonella typhimurium strain TA98 in the absence and presence of S9 was used in order to simulate
the metabolic process [40] (Table 6). Unlike the anti-Chagas drug nifurtimox, none of the studied compounds
were mutagenic according to the criterion that a compound is mutagenic when the number of revertant colonies is
at least two-fold higher than the spontaneous revertant frequencies for at least two consecutive dose levels [41].
This allowed us to confirm the druggability of these compounds.
Table 6. Ames test on selected imidazotiadiazine derivatives.
19
15
12
Nifurtimoxa
Db NRc,d
Db NRc,d Me Db NRc,d Me Db NRc,d Me
Me
500 18±5
167 15±3
56 10±1
19 9±3
500 10±2
167 11±2
56 10±1
19 10±1
500 12±3
167 14±4
56 12±2
19 9±2
10.0 144±11
3.0
1.0
0.5
0.0
62±2
43±17
29±6
21±4
(+)
-S9
6
0
7±3
6
0
5±3
6
0
9±2
10±3
10±3
10±3
500 13±3
167 15±1
56 5±2
19 6±1
500 8±1
167 9±1
56 7±2
19 14±5
500 10±0
167 26±9
56 8±1
19 4±2
0.0
0.5
1.0
3.0
31± 10
37± 5
39± 18
53± 9
+S9
6
0
9±2
6
0
15±8
11±1
6
0
9±6
10.0 64± 6
11±1
11±1
4-NPDf
Db
AFg
Db
NRc,d
NRc,d
-S9 20.0
1460±228
+S9 10.0
856±55
a The data for Nifurtimox were taken from reference [33]. b D: Doses in µg/plate. c NR: Number of revertants. d The
f
results are the mean of two independent experiments ±SD. e M: Mutagenicity, according to reference [41]. NPD:
4-nitro-o-phenylendiamine. g AF: 2-aminofluorene.

16
ACCEPTED MANUSCRIPT
2.3.8. In vivo trypanocidal assay
The compounds designated 19 and 4 were also chosen to evaluate their efficacy as trypanocidal agents in a
murine model of acute infection caused by T. cruzi strain Y. Benznidazole was also included as a reference drug,
and was administered to the animals in the same therapeutic scheme (i.e. 100 mg/kg/day p.o. for five
consecutive days). As shown in Figure 4, 19 reduced the number of blood trypomastigotes from the fourth day of
treatment. However, mice treated with 4 maintained high levels of parasitemia throughout the experiment.
Regarding survival, only one death was registered in the experimental group treated with 19, providing a survival
fraction of 87.5% (Figure 5).
Figure 4. Parasitemia levels in mice experimentally infected with T. cruzi (Y strain) and treated orally for five
consecutive days with 100 mg/kg/day of 19, 4 and benznidazole.
Figure 5. Survival rates of 19 and 4 treated mice expressed as the percentage of living animals during the acute
phase of the infection (30 dpi).

17
ACCEPTED MANUSCRIPT
3. CONCLUSIONS
This new family of imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide with anti-T. cruzi properties has been patented by
us in different countries [42-44] and in the USA [45], where the patent has been recently approved.
On the basis of this investigation, artificial neural network techniques (ANN) were used effectively to find new
trypanocidal agents with an innovative heterocyclic system containing an imidazothiadiazine 2,2-dioxide
structure. Six derivatives (4, 12, 13, 15, 17, and 19) of the evaluated compounds showed the capacity to inhibit
the epimastigote form of the Tulahuen 2 strain. The cruzipain studies provided insight into a possible mode of
action of these compounds.
In summary, the anti-epimastigote and anti-amastigote effects in the Tulahuen 2 and CL-clone B5 strains of
Trypanosoma cruzi, along with the absence of mutagenicity, suggest that these compounds can be regarded as
trypanocidal agents. Among them, the 1,5 substituted imidazothiadiazine 19 showed in vivo trypanocidal activity
and thus can be considered a potential agent for the treatment of Chagas disease.

18
ACCEPTED MANUSCRIPT
4. EXPERIMENTAL
4.1. Chemistry
1
Melting points were determined using a Reichert-Jung Kofler apparatus. H-NMR and 13C-NMR spectra were
recorded on Varian INNOVA-300 (300 MHz), Varian MERCURY-400 (400 MHz) and Varian UNITY-500 (500
MHz) spectrometers. Mass spectrometry (ES+) was performed using a Hewlett-Packard 1100SD apparatus.
Flash chromatography was performed using silica gel 60 (230-400 mesh) from Merck. High performance liquid
chromatography (HPLC) was performed using a Waters 2695 apparatus with a diode array UV/Vis detector and
coupled to a micromass ZQ using silica based columns X-BridgeTM C18 (2.1x100 mm, 3.5 µm). The sample
injection volume was set to 5 µL of a solution of 1 mg/mL methanol and experiments were carried out with a flow
rate of 0.25 µL and a different gradient elution specified in each case. Both solvents contained a 0.1% v/v formic
acid. Elemental analyses were performed on a Heraeus CHN-O Rapid Analysis apparatus. The purity of all
compounds was >95% prior to biological testing (Supporting Information).
4.1.1. General procedure for the preparation of 4-amino-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-
dioxides 1-13. To a suspension of 21 in water and acetic acid, the aldehyde was added in small fractions and
stirred at room temperature until the complete elimination of 21. The resulting precipitate was separated by
vacuum filtration and recrystallized in each case. Amounts of reagents, time reaction, conditions and specific
procedures are particularly specified in each case.
4.1.2. 4-Amino-6-methyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (1). From 21 (300 mg, 1.69
mmol), acetaldehyde (0.29 mL, 5.13 mmol), 12 mL water and 2 mL glacial acetic acid. Reaction time 24 h. Yield 1
(213 mg, 62%). Mp 303-5 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 11.91, 11.05 (bs, 2H, NH); 7.38 (bs, 2H, NH2);
2.30 (s, 3H, CH3). 13C-NMR (100 MHz, DMSO-d6): δ = 153.1 (C-4); 150.3 (C-7a); 148.0 (C-6); 101.8 (C-4a); 14.3
(CH3). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 1.70 min, [M+H]+ = 202.

19
ACCEPTED MANUSCRIPT
4.1.3. 4-Amino-6-ethyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (2). From 21 (1.5 g, 8.50 mmol),
propionaldehyde (0.90 mL, 12.47 mmol) in 40 mL water and 4 mL glacial acetic acid. Reaction time 24 h. Yield 2
1
(1.4 g, 78%). Mp 304-7 °C. H-NMR (400 MHz, DMSO-d6): δ = 11.93, 11.02 (bs, 2H, NH); 7.58 (bs, 2H, NH2);
13
2.68 (c, 2H, CH2CH3); 1.20 (t, 3H, CH2CH3, J = 7.5 Hz). C-NMR (125 MHz, DMSO-d6): δ = 153.7 (C-4); 152.0
(C-7a); 148.8 (C-6); 103.2 (C-4a); 21 .8 (CH2CH3); 12.0 (CH2CH3). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG:
18.00 min, tR: 1.53 min, [M+H]+ = 216.
4.1.4. 4-Amino-6-cyclopentyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (3). From 21 (300 mg, 1.69
mmol) and cyclopentanecarboxaldehyde (0.28 mL, 2.61 mmol) in 30 mL water and 4 mL glacial acetic acid.
1
Reaction time 96 h. Yield 3 (195 mg, 45%). Mp dec. H-NMR (400 MHz, DMSO-d6): δ = 11.86, 11.04 (bs, 2H,
NH); 7.43 (bs, 2H, NH2); 3.11 (bs, 1H, 61-H); 2.01 – 1.62 (m, 8H, 62-H / 63-H). 13C-NMR (100 MHz, DMSO-d6): δ
= 155.7 (C-4); 152.8 (C-7a); 150.5 (C-6); 101.4 (C-4a); 38.7 (C-61); 31.6 (2C, C-62); 25.0 (2C, C-63). HPLC-MS
(ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 3.93 min, [M+H]+ = 256.
4.1.5. 4-Amino-6-cyclohexyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (4). From 21 (1.5 g, 8.50
mmol), cyclohexanecarboxaldehyde (3.2 mL, 25.5 mmol) in 30 mL water and 4 mL glacial acetic acid. Reaction
time 24 h. Yield 4 (1.0 g, 46%). Mp 263-5°C (recrystallized from EtOH:H2O (1:1)). 1H-NMR (400 MHz, DMSO-d6):
δ = 11.82, 11.00 (bs, 2H, NH); 7.42 (bs, 2H, NH2); 2.67 (bs, 1H, 61-H); 1.94 – 1.18 (m, 10H, 62-H / 63-H / 64-H).
13C-NMR (100 MHz, DMSO-d6): δ = 155.8 (C-4); 152.9 (C-7a); 150.4 (C-6); 101.2 (C-4a); 37.2 (C-61); 30.9 (2C,
C-62); 25.4, 25.2 (3C, C-63 / C-64). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 5.80 min, [M+H]+
= 270.
4.1.6. 4-Amino-6-phenyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (5). From 21 (300 mg, 1.69
mmol), benzaldehyde (0.26 mL, 2.54 mmol) in 12 mL water and 2 mL glacial acetic acid. Reaction time 24 h.
1
Yield 5 (329 mg, 74%). Mp 296-8°C. H-NMR (500 MHz, DMSO-d6): δ = 12.72, 11.22 (bs, 2H, NH); 7.92 - 7.47
(bs, 2H, NH2); 7.91 (d, 2H, 62-H, J = 7.2 Hz); 7.55 - 7.47 (m, 3H, 63-H / 64-H). 13C-NMR (125 MHz, DMSO-d6): δ

20
ACCEPTED MANUSCRIPT
= 152.9 (C-4); 150.9 (C-7a); 147.7 (C-6); 130.1 (C-64); 129.1 (2C, C-63); 128.6 (C-61); 125.6 (2C, C-62); 103.1
(C-4a). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 6.03 min, [M+H]+ = 264.
4.1.7. 4-Amino-6-(2-fluorophenyl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (6). From 21 (300 mg,
1.69 mmol) and 2-fluorobenzaldehyde (0.28 mL, 2.54 mmol) in 12 mL water and 2 mL glacial acetic acid.
1
Reaction time 24 h. Yield 6 (253 mg, 53%). Mp 274-8°C (recrystallized from water). H-NMR (300 MHz;d6): δ =
12.57, 11.30 (bs, 2H, NH); 7.97, 7.79 (bs, 2H, NH2); 8.09 (t, 66-H, J = 7.5 Hz); 7.58 – 7.37 (m, 3H, 64-H, 65-H,
63-H). 13C-NMR(100 MHz;d6): δ = 159.8 (d, C-62, J = 247.1 Hz); 152.7 (C-4); 150.3 (C-7a); 142.8 (C-6); 133.2 (d,
C-64, J = 5.9 Hz); 130.2 (d, C-66, J = 1.5 Hz); 126.1 (d, C-65, J = 4.1 Hz); 117.2 (d, C-63, J = 21 .2 Hz); 116.9 (d,
C-61, J = 13.7 Hz); 103.3 (C-4a). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 6.87 min, [M+H]+ =
282.
4.1.8. 4-Amino-6-(4-dimethylaminephenyl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (7). From 21
(300 mg, 1.69 mmol), 4-dimethylaminebenzaldehyde (379 mg, 2.54 mmol) in 12 mL water and 2 mL glacial acetic
1
acid. Reaction time 26 h. Yield 7 (219 mg, 42%). Mp > 330 ºC. H-NMR (400 MHz, DMSO-d6): δ = 12.24, 11.12
(bs, 2H, NH); 7.50 (bs, 2H, NH2); 7.71 (d, 2H, 62-H, J = 8.6 Hz); 6.80 (d, 2H, 63-H, J = 8.6 Hz); 2.96 (s, 6H, N
(CH3)2). 13C-NMR (75 MHz, DMSO-d6): δ = 153.0 (C-4); 151.8 (C-64); 150.8 (C-7a); 148.9 (C-6); 127.3 (C-62);
115.6 (C-61); 112.2 (C-63) 102.8 (C-4a); 40.0 (2C, N (CH3)2). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00
min, tR: 7.20 min, [M+H]+ = 307.
4.1.9. 4-Amino-6-(3-thienyl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (8). From 21 (300 mg, 1.69
mmol) and thiophen-3-carboxaldehyde (0.23 mL, 2.54 mmol) in 12 mL water and 2 mL glacial acetic acid.
1
Reaction time 24 h. Yield 8 (240 mg, 53%). Mp 314-6°C (recrystallized from water). H-NMR (400 MHz;d6): δ =
12.70, 11.14 (bs, 2H, NH); 8.00 – 7.20 (bs, 2H, NH2); 8.04 (dd, 62-H, J62-H, 65-H = 1.2 Hz / J62-H, 64-H = 2.9 Hz); 7.70
(dd, 64-H, J62-H, 64-H = 2.9 Hz / J64-H, 65-H = 5.0 Hz); 7.55 (dd, 65-H, J62-H, 65-H = 1.2 Hz / J64-H, 65-H = 5.0 Hz). 13C-NMR
(100 MHz;d6): δ = 153.7 (C-4); 149.4 (C-7a); 143.9 (C-6); 131.0 (C-61); 128.6 (C-64); 125.7 (C-65); 125.5 (C-62);
104.2 (C-4a). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 4.98 min, [M+H]+ = 270.

21
ACCEPTED MANUSCRIPT
4.1.10. 4-Amino-6-(3-pyridyl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (9). From 21 (300 mg, 1.69
mmol) and 3-pyridincarboxaldehyde (0.25 mL, 2.54 mmol) in 12 mL water and 2 mL glacial acetic acid. Reaction
1
time 24 h. Yield 9 (154 mg, 35%). Mp 309-11°C (recrystallized from water). H-NMR (300 MHz, DMSO-d6): δ =
13.29, 12.90, 11.32 (bs, 2H, NH); 7.92, 7.15 (bs, 2H, NH2); 9.05 (s, 62-H); 8.60 (d, 64-H, J = 10.6 Hz); 8.22 (d,
66-H, J = 4.4 Hz); 7.55 (dd, 65-H, J64-H, 65-H = 10.6 Hz / J65-H, 66-H = 4.4 Hz). 13C-NMR (75 MHz, DMSO-d6): δ =
153.0 (C-4); 150.7 (C-64); 150.5 (C-7a); 146.8 ( C-62); 145.9 (C-6); 133.1 (C-66); 125.0 (C-61); 124.1 (C-65);
103.7 (C-4a). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 1.77 min, [M+H]+ = 265.
4.1.11. 4-Amino-6-(5-nitro-2-furyl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (10). From 21 (200 mg,
1.12 mmol) and 5-nitro-2-furaldehyde (240 mg, 1.72 mmol) in 12 mL water and 2 mL glacial acetic acid. Reaction
1
time 48 h. Yield 10 (153 mg, 46%). Mp dec. H-NMR (500 MHz, DMSO-d6): δ = 12.16, 11.29 (bs, 2H, NH); 8.15
(bs, 2H, NH2); 7.89 (d, 2H, 64-H, J = 3.9 Hz); 7.38 (d, 2H, 65-H, J = 3.9 Hz). 13C-NMR (125 MHz, DMSO-d6): δ =
153.9 (C-4); 148.3 (C-7a); 135.6 (C-6); 151.6 (C-63); 146.4 (C-62);115.3 (C-65); 112.6 (C-64); 107.0 (C-4a).
HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 6.90 min, [M+H]+ = 264.
4.1.12. 4-amino-6-(3-tetrahydrofuryl)-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (11). From 21 (300
mg, 1.69 mmol) and tetrahydrofuryl-3-carboxaldehyde (0.49 mL, 2.54 mmol) in 30 mL water and 2 mL glacial
1
acetic acid. Reaction time 48 h. Yield 11 (181 mg, 41%). Mp 238–40°C (recrystallized from ethanol). H-NMR
(500 MHz, DMSO-d6): δ = 11.97, 11.00 (bs, 2H, NH); 7.63 – 7.22 (bs, 2H, NH2); 3.98 – 3.74 (m, 4H, 62-H / 64-H);
3.54 – 3.45 (bs, 1H, 61-H); 2.30 – 2.23 (m, 1H, 65a-H); 2.12 – 2.05 (m, 1H, 65b-H). 13C-NMR (125 MHz, DMSO-
d6): δ = 152.9 (2C, C-4 / C-7a); 150.3 (C-6); 101.9 (C-4a); 71.2 (C-62); 67.4 (C-64); 38.3 (C-61); 31.3 (C-65).
HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 2.42 min, [M+H]+ = 258.
4.1.13. 4-Amino-6-benzyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (12). From 21 (300 mg, 1.69
mmol), phenylacetaldehyde (475 mg, 3.75 mmol) in 2 mL acetone, 30 mL water and 2 mL glacial acetic acid.
Reaction time 75 h. Yield 12 (190 mg, 40%). Mp 210-2°C (recrystallized from acetone). 1H-NMR (400 MHz;d6): δ

22
ACCEPTED MANUSCRIPT
= 12.9 – 10.5 (bs, 2H, NH); 7.45 (bs, 2H, NH2); 7.37 – 7.27 (m, 5H, Ar); 4.04 (s, 2H, 61-H). 13C-NMR (100
MHz;d6): δ = 153.8 (C-4); 149.2 (C-7a, C-6); 136.9 (C-62); 128.8 (2C, C-64); 128.7 (2C, C-63); 126.8 (C-65);
104.0 (C-4a); 34.5 ( C-61). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 5.88 min, [M+H]+ = 278.
4.1.14. 4-Amino-6-phenethyl-1H,5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (13). From 21 (300 mg, 1.69
mmol) and 3-phenylpropionaldehyde (0.34 mL, 2.54 mmol), 12 mL water and 2 mL glacial acetic acid. Reaction
time 24 h. Yied 13 (369 mg, 75%). Mp 190-2°C (recrystallized from EtOH:H2O (1:1)). 1H-NMR (300 MHz, DMSO-
d6): δ = 12.09, 11.96, 11.03 (bs, 2H, NH); 7.81, 7.66 (bs, 2H, NH2); 7.27 – 7.17 (m, 5H, Ar); 2.99 – 2.96 (m, 4H,
61-H / 62-H). 13C-NMR (125 MHz, DMSO-d6): δ = 154.2 (C-7a); 152.7 (C-4); 150.5 (C-6); 140.5 (C-63); 128.5
(2C, C-65); 128.3 (2C, C-64); 126.2 (C-66); 101.5 (C-4a); 32.9 (C-61); 29.9 (C-62). HPLC-MS (ES+): CH3CN/H2O
5:95-100:0, tG: 18.00 min, tR: 6.72 min, [M+H]+ = 292.
4.1.15. General procedure for N-1 substituted 5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides
(compounds 14, 16-18). A mixture of 4-amino-5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide derivative and the
appropriate base in acetone was boiled and stirred. Once reflux was achieved, the corresponding halide and a
catalytic amount of potassium iodide were added and the mixture was allowed to react until the complete
elimination of the N(H) derivative. The remaining solvent was eliminated under vacuum. The resulting crude
product was processed in each case. Work-up, amounts, the time of reaction, conditions, and specific procedures
are specified in each case.
4.1.16. 4-Amino-1-methyl-6-(3-thienyl)-5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (14). From 8 (300 mg,
1.11 mmol), K2CO3 (150 mg, 1.11 mmol), methyl iodide (150 mg, 11.1 mmol) in 60 mL acetone. Reaction time 4
days. To the residue aqueous HCl was added and the precipitate was purified by flash chromatography
(CH2Cl2:MeOH, 100:1) to afford 14 (197 mg, 63%). Mp 305-8°C. 1H-NMR (400 MHz;d6): δ = 13.04 (bs, 1H, NH);
8.00 – 7.20 (bs, 2H, NH2); 8.04 (dd, 62-H, J62-H, 65-H = 1.2 / J62-H, 64-H = 2.9); 7.70 (dd, 64-H, J62-H, 64-H = 2.9 / J64-H, 65-
H = 5.0); 7.55 (dd, 65-H, J62-H, 65-H = 1.2 / J64-H, 65-H = 5.0); 3.29 (CH3). 13C-NMR (100 MHz;d6) δ: 153.7 (C-4); 149.4
(C-7a); 143.9 (C-6); 131.0 (C-61); 128.6 (C-64); 125.7 (C-65); 125.5 (C-62); 104.2 (C-4a): 34.4 (CH3). HPLC-MS
(ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 9.42 min, [M+H]+ = 284.

23
ACCEPTED MANUSCRIPT
4.1.17. 4-Amino-1-benzyl-6-phenyl-5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (16). From 5 (300 mg,
1.15 mmol), Et3N (0.64 mL, 4.60 mmol), benzyl bromide (1.12 mL, 5.82 mmol) in 70 mL acetone. Reaction time 3
days. To the residue aqueous HCl was added and the precipitate was recrystallized from EtOH:H2O (1:1) to yield
16 (167 mg, 36%). Mp 304-6°C. 1H-NMR (400 MHz, DMSO-d6): δ = 13.01 (bs, 1H, NH); 8.12 (bs, 2H, NH2); 7.92
(d, 2H, 62-H, J = 7.0 Hz); 7.57 – 7.48 (m, 3H, 63-H / 64-H); 7.43 (d, 2H, 13-H, J = 7.3 Hz); 7.34 – 7.22 (m, 3H,
14-H / 15-H); 5.03 (s, 2H, 11-H). 13C-NMR (125 MHz, DMSO-d6): δ = 152.4 (C-4); 151.4 (C-7a); 148.2 (C-6);
137.3 (C-12); 130.1 (C-61); 128.9 (2C, C-63); 128.4 (C-64); 128.0 (2C, C-14); 127.7 (C-15); 127.0 (2C, C-13);
125.7 (2C, C-62); 103.3 (C-4a); 47.2 (C-11). ). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 12.95,
[M+H]+ = 354.
4.1.18. 4-Amino-1-(4-chlorobenzyl)-6-(2-fluorophenyl)-5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (17).
From 6 (500 mg, 1.78 mmol), Et3N (1.00 mL, 7.12 mmol), 4-chlorobenzyl bromide (740 mg, 3.56 mmol) in 100 mL
acetone. Reaction time 2 days. To the residue aqueous HCl was added and the precipitate was recrystallized
from acetone:H2O (1:2) affording 17 (158 mg, 39%). Mp 304-5°C. 1H-NMR (500 MHz, DMSO-d6): δ = 12.47 (bs,
1H, NH); 8.21 , 7.96 (bs, 2H, NH2); 8.10 (t, 1H, 66-H, J = 7.0 Hz); 7.58 – 7.37 (m, 7H, Ar); 5.02 (s, 2H, 11-H). 13C-
NMR (125 MHz, DMSO-d6): δ = 159.1 (d, C-62, J = 250.0 Hz); 152.4 (C-4); 150.7 (C-7a); 143.4 (C-6); 136.5 (C-
12); 132.4 (d, C-64, J = 8.2 Hz); 132.0 (C-15); 129.8 (2C, C-13 or C-14) 129.8 (d, C-66, J = 6.4 Hz); 128.2 (2C,
C-13 or C-14); 125.4 (C-65); 116.5 (d, C-63, J = 22.0 Hz); 116.2 (d, C-61, J = 11.9 Hz); 103.7 (C-4a); 47.0 (1C,
C-11). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 13.90, [M+H]+ = 406.
4.1.19. 4-Amino-6-cyclohexyl-1-(4-chlorobenzyl)-5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (18). From
4 (300 mg, 1.11 mmol), Et3N (1.00 mL, 7.12 mmol), chlorobenzyl bromide (465 mg, 2.27 mmol) in 40 mL acetone.
Reaction time 3 days. To the residue aqueous HCl was added and the precipitate was purified by flash
1
chromatography starting from 200:1 to 10:1 (CH2Cl2:MeOH) to afford 18 (125 mg, 29%). Mp 220-2°C. H-NMR
(500 MHz, DMSO-d6): δ = 11.97 (bs, 1H, NH); 7.89 (bs, 2H, NH2); 7.37 (d, 4H, Ar); 4.89 (s, 2H, CH2); 2.70 (m,
1H, 61-H); 1.95 – 1.20 (m, 10H, 62-H / 63-H / 64-H). 13C-NMR (125 MHz, DMSO-d6): δ = 157.2 (C-4); 153.0 (C-

24
ACCEPTED MANUSCRIPT
7a); 151.4 (C-6); 137.1 (C-12); 132.3 (C-15); 130.2 (2C, C-13 or C-14); 128.6 (2C, C-13 or C-14); 102.1 (C-4a);
46.6 (C, C-11); 37.7 (C-61); 31.2 (2C, C-62); 25.8, 25.5 (3C, C-63 / C-64). HPLC-MS (ES+): CH3CN/H2O 5:95-
100:0, tG: 18.00 min, tR: 11.67, [M+H]+ = 394.
4.1.20. 4-Amino-1-methyl-6-phenyl-5H-imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (15).To a suspension of 3
(300 mg, 1.11 mmol), K2CO3 (2,685 g, 19.10 mmol) in 30 mLof water, dimethyl sulphate (1.12 mL, 5.82 mmol)
was added. After 2 days, the reaction mixture was acidulated with aqueus HCl and the precipitate is filtered and
purified by flash chromatography (CH2Cl2:MeOH, 100:1) to afford 15 (111 mg, 36%). Mp 278-80°C. 1H-NMR (400
MHz, DMSO-d6): δ = 11.61 (bs, H, NH); 7.92 - 7.32 (bs, 2H, NH2); 7.92 (d, 2H, 62-H, J = 7.5 Hz); 7.51 - 7.43 (m,
13
3H, 63-H / 64-H); 3.28 (s, 3H, CH3). C-NMR (100 MHz, DMSO-d6): δ = 153.4 (C-4); 151.5 (C-7a); 148.4 (C-6);
129.7 (C-64); 129.0 (2C, C-63); 128.4 (C-61); 125.8 (2C, C-62); 105.3 (C-4a); 34.4 (CH3). HPLC-MS (ES+):
CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 9.23, [M+H]+ = 292.
4.1.21. 4-Amino-1,5-dibenzyl-6-phenylimidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (19). To a suspension of 6
(533 mg, 2.01 mmol), NaOH (200 mg, 5 mmol) in 100 mL water, benzylbromide was added dropwise (0.7 mL, 8.0
mmol) at reflux temperature. After 24 h, the reaction mixture was cooled, filtered and the residue was purified by
1
flash chromatography (CH2Cl2:MeOH, 100:1) to afford 19 (260 mg, 29%). Mp 238-40°C. H-NMR (300 MHz,
DMSO-d6): δ = 8.0 – 7.0 (bs, 2H, NH2); 7.63 – 7.60 (m, 2H, Ph); 7.54 – 7.50 (m, 3H, Ar); 7.39 – 7.20 (m, 10H, Ar);
13
5.53 (s, 2H, 51-H); 5.02 (s, 2H, 11-H). C-NMR (75 MHz, DMSO- CDCl3): δ = 153.7 (C-4); 152.8 (C-7a); 152.1
(C-6); 137.4 (C-12 or C-52); 136.2 (C-12 or C-52); 130.9 (C-61); 129.6, 129.2, 129.1, 128.5, 128.4, 128.2, 127.9,
127.5 (11C, Ar); 125.9 (C-62); 105.0 (C-4a); 49.2 (C51); 46.9 (C11). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG:
18.00 min, tR: 12.63, [M+H]+ = 444.
4.1.22. 4-Amino-1,5-dibenzyl-6-cyclohexylimidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide (20). To a suspension
of 14 (300 mg, 1.11 mmol) and K2CO3 (310 mg, 2.24 mmol) in 40 mL acetone, benzylbromide was added (0.27
mL, 2.27 mmol) at reflux temperature. After 3 days, the rection mixture was cooled, filtered and the residue was
purified by flash chromatography starting from 200:1 to 10:1 (CH2Cl2:MeOH) to afford 20 (59 mg, 12%). Mp 202-

25
ACCEPTED MANUSCRIPT
3°C. 1H-NMR (500 MHz, DMSO- CDCl3): δ = 7.05 – 7.04 (d, 2H, NH2); 7.62 – 7.60 (m, 2H, , Ar); 7.45 – 7.37 (m,
4H, , Ar); 7.33 – 7.24 (m, 4H, , Ar); 5.15 (s, 2H, 51-H); 4.80 (s, 2H, 11-H); 2.61- 2.56 (m, 1H, 61-H); 1.86 – 1.25
13
(m, 10H, 62-H / 63-H / 64-H). C-NMR (125 MHz, DMSO-d6): δ = 159.6 (C-4); 153.2 (C-7a); 152.0 (C-6); 136.8
(1C, C-12 or C-52); 134.5 (1C, C-12 or C-52); 129.2 (1C, C-15 or C-55); 127.6 (1C, 15 or C-55); 130.0; 129.3,
128.2, 125.3 (8C, C13 / C14 / C53 / C54); 103.7 (C-4a); 47.7 ( C51); 47.6 ( C11); 36.0 (C-61); 31.5 (2C, C-62);
25.8, 25.4 (3C, C-63 / C-64). HPLC-MS (ES+): CH3CN/H2O 5:95-100:0, tG: 18.00 min, tR: 14.08, [M+H]+ = 450.
4.2. Pharmacology
4.2.1. In vitro Tulahuen 2 antiepimastigote form activity. The parasites were cultured at 28ºC in axenic
medium (BHI-tryptose), supplemented with 5% fetal bovine serum. Cells cultured for 5-7 days (exponential
phase) were inoculated with 50 mL of fresh culture medium, giving an initial concentration of 1 x 106 cells/mL.
The growth of the parasite was followed for 11 days by culture absorbance measurements at 600 nm,
proportional to the number of cells present.
Before inoculation, a pre-established quantity of each test compound was incorporated into the medium,
dissolved in DMSO (dimethylsulfoxide). The final concentration of DMSO in the culture medium never exceeded
0.4%, using a blank (absence of product) with 0.4% DMSO. The compounds were incorporated into the culture
medium at a final concentration of 25 µM and, for those that were most active, the dose was progressively
decreased to 1 nM. The percentage of inhibition of growth (PGIe) of the parasite was evaluated in comparison
with the blank, using nifurtimox as the trypanosomicide reference drug. The PGIe was calculated as follows:
PGIe={1-[(Ap-A0p)/(Ac-A0c)]}x100, where Ap=A600nm of the culture containing the product on day 5; A0p=A600nm
of the culture containing the product after addition of the culture (day 0); Ac=A600nm of the blank on day 5;
A0c=A600nm of the blank on day
culture growth curve.
O. The A600nm taken on day 5 corresponds to the late exponential phase in the

26
ACCEPTED MANUSCRIPT
To determine IC50 values, parasite growth was followed in the absence (control) and presence of increasing
concentrations of the corresponding compound. The IC50 value was determined as the compound concentration
required to reduce the absorbance by half compared to that of the untreated controls.
4.2.2. In vitro CL antiepimastigote assay. The trypanocidal activity was evaluated by modifying a previously
described colorimetric method [46] against T. cruzi (CL strain, clone B5) parasites stably transfected with
Escherichia coli ß-galactosidase gene (lacZ) [47]. Epimastigote forms were axenically cultured at 28ºC in liver
infusion tryptose medium (LIT), supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100
µg/mL streptomicyn and 100 U/mL penicillin). Stock solutions of the compounds were prepared in
dimethylsulfoxide (DMSO) and added to the cultures in a final DMSO concentration lower than 0.2%.
Epimastigotes in the exponential phase of growth were seeded in 96-well plates at an initial concentration of 2.5
× 105 parasites/mL and incubated with the compounds for 72 h at 28ºC. The activity of the tested compounds was
evaluated by measuring absorbance at 595 nm after the addition to each well of 50 µL of a chlorophenol red ß -
D-galactopyranoside (CPRG) solution in 0.9% Triton X-100 (200 µM, pH 7.4), followed by incubation for 3 h at
37ºC. Nifurtimox and benznidazole were included in these assays as reference drugs. Antiepimastigote activity
was expressed as the percentage of growth inhibition (PGIe), as follows: PGIe=100-{[(APc-APm)/(Ac-Am)]*100},
where APc represents the absorbance of treated cultures, APm the absorbance of the compound in medium, Ac
the absorbance of cultures without compound (growth control) and Am the absorbance of the medium (blank).
The PGIe obtained were used to calculate the IC50 value for epimastigotes (IC50e) of each compound.
4.2.3. In vitro antiamastigote assay. This in vitro assay was performed in 48-well tissue culture plates and the
activity of the selected compounds was evaluated in the presence of the chromogenic substrate CPRG. NCTC-
929 fibroblasts were seeded at a density of 10,000 cells/well with 120 µL of MEM medium. The cells were
allowed to attach for 2-3 hours at 37ºC with 5% CO2 and, afterwards, NCTC-929 derived trypomastigotes (TCT)
of the CL-B5 lacZ strain were added in a 1:6 ratio (cells:TCT) and incubated for 24 h at a 33ºC with 5% CO2.

27
ACCEPTED MANUSCRIPT
Afterwards, infected cells were washed with PBS to remove non-penetrated trypomastigotes and then drugs in
DMSO were diluted in fresh medium (2 µL/mL) to give a final volume of 450 µL/well. The plates were incubated
for 7 days at 33ºC with 5% CO2, then 50 µL of a CPRG solution in 3% Triton X-100 (400 µM, pH 7.4) was added.
Finally, the plates were incubated at 37ºC for 3 h and the absorbance was read at 595 nm. Antiamastigote activity
was expressed as the percentage of growth inhibition (PGIa), calculated as: PGIa=100-{[(APc-APm)/(Aci-
Am)]*100}, were APc represents the absorbance of the treated group, APm the absorbance of the compound in
medium, Aci the absorbance of infected cells without compound (control of infection) and Am the absorbance of
the medium (blank). the absorbance of non-infected wells (only NCTC-929 cells) was subtracted from both
groups (experimental and control of infection) as the background. The PGIa values were used to calculate the
IC50 value of each compound against amastigotes (IC50a).
4.2.4. In vivo trypanocidal assay. Four groups of NMRI mice with an average weight of 20 g were
intraperitoneally infected by the injection of 104 blood trypomastigotes of the T. cruzi Y strain, previously obtained
by intracardiac puncture from anesthetized donor mice. The groups were distributed as follows: animals treated
only with the solvent (control of infection), animals treated with benznidazole (reference drug), animals treated
with 9 (experimental group 1) and animals treated with 4 (experimental group 2). This assay was carried out in
order to assess the efficacy of these compounds to reduce parasitemia levels in acute disease, following
previously established guidelines [48]. Accordingly, 100 mg/kg/day doses of the tested drugs were administered
orally for five consecutive days, with the initiated on the treatment the fifth day post-infection (5 dpi). A solution of
2% carboxymethylcellulose sodium salt (CMC) was used as the drug vehicle. Only animals with positive
parasitemia were included in the experiment. Parasitemia levels were estimated by counting trypomastigotes
from tail blood samples as previously described [49] at 5, 8, and 10 dpi. Survival of the animals was assessed
during acute infection (30 dpi).
All experiments were carried out according to directive 86/609/CEE and controlled in Spain by the then-current
Royal Decree 1201/2005 of 10 October on the protection of animals used for research and other scientific
purposes.

28
ACCEPTED MANUSCRIPT
4.2.5. Cytotoxicity assay in murine macrophages. The non-specific cytotoxicity against J774 murine
macrophages was assayed for the products that presented a percentage of growth inhibition of the epimastigote
form of T. cruzi of the Tulahuen 2 strain greater than 50% at 25 µM on day 5 of the study. For this, murine
macrophages were cultured in an atmosphere of 5% CO2 and 95% air at 37ºC for 48 h with the products
dissolved in DMSO at three concentrations: 100, 200 and 400 µM. Cell viability was determined based on the
conservation of mitochondrial activity by the MTT/formazan method. This assay included nifurtimox, as an anti-T.
cruzi reference drug.
4.2.6. Cytotoxicity assay in murine fibroblasts. NCTC-929 fibroblasts were seeded at a density of 15,000
cells/well in 96-well plates with 100 µL of MEM medium, and maintained for 3 h at 37ºC with 5% CO2. Following
cell attachment, the culture medium was replaced with 200 µL of compounds diluted in fresh medium and the
plates were incubated for 48 h at 37ºC with 5% CO2. Growth and medium controls were included in each plate.
Benznidazole was also assayed as the reference drug. Cell viability was evaluated by applying the resazurin
method [50], 20 µL of this substrate in PBS solution (2 mM, pH 7.0) was added before incubating the plates for
another 3 h (37ºC with 5% CO2). Finally, fluorescence intensity, with excitation at 535 nm and emission at 590
nm, was measured to assess cytotoxicity.
4.2.7. Cruzipain inhibitory activity [51]. The capacity to inhibit the cruzipain enzyme was assessed according to
the following protocol: cruzipain (10 µL) was incubated with a reaction mixture containing a final concentration of
50 mM of Tris-HCl buffer solution, pH 7.6, 10 mM of mercaptoethanol and 25, 50 or 100 µM of the inhibitor for 10
min at ambient temperature. Later, the chromogenic substrate Bz-Pro-Phe-Arg-p-NA was aggregated at a final
concentration of 150 µM, and the increase in absorbance at 410 nm was followed for 5 min at ambient