Synthesis and anti-trypanosoma cruzi activity of diaryldiazepines

Article abstract of DOI:10.3390/molecules20010043

Chagas disease is a so-called "neglected disease" and endemic to Latin America. Nifurtimox and benznidazole are drugs that have considerable efficacy in the treatment of the acute phase of the disease but cause many significant side effects. Furthermore, in the Chronic Phase its efficiency is reduced and their therapeutic effectiveness is dependent on the type of T. cruzi strain. For this reason, the present work aims to drive basic research towards the discovery of new chemical entities to treat Chagas disease. Differently substituted 5,7-diaryl-2,3-dihydro-1,4-diazepines were synthesized by cyclocondensation of substituted flavones with ethylenediamine and tested as anti-Trypanosoma cruzi candidates. Epimastigotes of the Y strain from T. cruzi were used in this study and the number of parasites was determined in a Neubauer chamber. The most potent diaryldiazepine that reduced epimastigote proliferation exhibited an IC50 value of 0.25 μM, which is significantly more active than benznidazole.

Full text of DOI:10.3390/molecules20010043

Molecules 2015, 20, 43-51; doi:10.3390/molecules20010043
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Anti-Trypanosoma cruzi Activity
of Diaryldiazepines
Júlio César L. Menezes 1, Luana Beatriz A. Vaz 2, Paula Melo de Abreu Vieira 2,
Kátia da Silva Fonseca 2, Cláudia Martins Carneiro 2 and Jason G. Taylor 1,*
1
Departamento de Química, ICEB, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro,
CEP, Ouro Preto, MG 35400-000, Brazil; E-Mail: jc_menezes2@hotmail.com
2
Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas, ICEB II,
Morro do Cruzeiro, Universidade Federal de Ouro Preto, Ouro Preto 35400-000, Brazil;
E-Mails: luanavazz@yahoo.com.br (L.B.A.V.); paulinhamav@gmail.com (P.M.A.V.);
katia.fonseca@gmail.com (K.S.F.); claudiamartinscarneiro@gmail.com (C.M.C.)
* Author to whom correspondence should be addressed; E-Mail: jason@iceb.ufop.br;
Tel.: +55-313-559-1707.
Academic Editor: Derek J. McPhee
Received: 14 October 2014 / Accepted: 18 November 2014 / Published: 23 December 2014
Abstract: Chagas disease is a so-called “neglected disease” and endemic to Latin America.
Nifurtimox and benznidazole are drugs that have considerable efficacy in the treatment of
the acute phase of the disease but cause many significant side effects. Furthermore, in
the Chronic Phase its efficiency is reduced and their therapeutic effectiveness is dependent
on the type of T. cruzi strain. For this reason, the present work aims to drive basic
research towards the discovery of new chemical entities to treat Chagas disease. Differently
substituted 5,7-diaryl-2,3-dihydro-1,4-diazepines were synthesized by cyclocondensation of
substituted flavones with ethylenediamine and tested as anti-Trypanosoma cruzi candidates.
Epimastigotes of the Y strain from T. cruzi were used in this study and the number
of parasites was determined in a Neubauer chamber. The most potent diaryldiazepine that
reduced epimastigote proliferation exhibited an IC50 value of 0.25 μM, which is significantly
more active than benznidazole.
Keywords: diaryldiazepines; Trypanosoma cruzi; Chagas disease; epimastigote

Molecules 2015, 20
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1. Introduction
Seven-membered heterocycles with two nitrogen’s in a 1,4-relationship are classified as diazepines.
Diazepines are well known for their unique pharmacological activity and in particular, the
1,4-benzodiazepines are especially recognized for their effects on the central nervous system [1]. In the
last 10 years, a variety of 1,4-diazepines have been reported for various biological activities, such as
anti-schistosomal activity [2], apoptosis inhibitors [3], Nox4/Nox1 inhibitors [4] and anticonvulsant
activity, [5] amongst others. In the specific case of diaryldiazepines, antiproliferative and cytotoxic
activities were demonstrated in vitro against several human leukemic cells, such as Jurkat, HL60,
MOLT3, NCEB-1 and K562 [6]. However, in general, diaryldiazepines have been underexplored for
other types of diseases and this is surprising given the possibility to modulate its activity by
incorporating different substituents on the phenyl rings (Figure 1).
R 2
B
R 1
A
N
H N
Figure 1. Chemical structure of diaryldiazepines.
The present work is part of our ongoing medicinal chemistry project funded by the Brazilian
government, which aims to drive basic research towards solving neglected tropical diseases (NTD).
American trypanosomiasis, also known as Chagas disease [7], is included in the NTD list and is
endemic to 21 Latin American countries [8,9]. The protozoan hemoflagellate Trypanosoma cruzi is
the etiologic agent responsible for Chagas disease. The disease is a so-called “neglected disease”
because health markets in affected countries are insufficient to attract pharmaceutical industry to invest
in research and development. Thus, the biggest obstacle to treating the disease has been the discovery
and development of new chemical entities able to meet this deficit in innovation. The two drugs currently
available for treating Chagas disease are nifurtimox and benznidazole, which are chemically related
nitro-heterocycles (Figure 2).
N
O
C H 3
N
O
N
N
N
O
N
O
H
N
O
O
S
O
O
N ifu rtim o x
B e n zn id a zo le
Figure 2. Chemical structures of benznidazole and nifurtimox.

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45
Unfortunately, they both result in many significant side effects such as oedema, fever, rash,
agranulocytosis and neurotoxicity. In comparison with benznidazole, not only are the side effects of
nifurtimox more severe, it has lower efficacy and so the commercialization of nifurtimox was suspended
in Brazil, Argentina, Chile and Uruguay in the 1980s [10]. Additionally, the therapeutic effectiveness
of nifurtimox and benznidazole is dependent on the type of T. cruzi strain, since certain strains are
more resistant than others [11]. Thus, due to the limitations of these treatments, they are not ideal
drugs and this drives the search for new more efficient compounds.
A number of studies focused on the synthesis and in vitro activity of biologically active compounds
against T. cruzi have been reported in the literature. Recent examples include thiazolidinones [12],
1,2,4-triazole-3-thiones [13], aryloxyindole-4,9-diones [14], 4-arylthiazolylhydrazones [15],
oxadiazoles [16] and Morita–Baylis–Hillman adducts [17]. Promising results are indicated by potencies
that are either equal to or better than benznidazole with varying degrees of mammalian cell toxicity
observed depending on the compound class (Figure 3). The aryloxyindoles stand out for their high potency
but in general, IC50 values in the range of 4–30 μM have been considered promising candidate leads.
B r
O
N H
N
S
O
O
O
C O 2 E t
N
S
N
O
H N
E t
O
O
T h ia z o lid in o n e s
T h ia z o lid in o n e s
A ry lo x y in d o le -4 ,9 -d io n e s
IC 5 0 [μ m ]: 3 .9 (e p im a s tig o te s )
IC 5 0 [μ m ]: 1 8 .3 (e p im a s tig o te s )
IC 5 0 [μ m ]: 0 .0 2 (e p im a s tig o te s )
H
O H
N
O
N
N
H
N
C N
N
C l
O
N
N
S
C l
O
O 2
N
O
o x a d ia z o le s
IC 5 0 [μ m ]: 7 .5 (e p im a s tig o te s )
3 -h y d ro x y -2 -m e th y le n e -3 -(4 -n itro p h e n y l)-
p ro p a n e n itrile
4 -a ry lth ia z o ly lh y d ra z o n e s
IC 5 0 [μ m ]: 4 .5 (e p im a s tig o te s )
IC 5 0 [μ m ]: 2 8 .5 (e p im a s tig o te s )
Figure 3. Compounds with anti-T. cruzi activity.
In order to meet this deficit in new drug candidates for Chagas disease, we have prepared and tested
against T. cruzi six diaryldiazepines analogous.
2. Results and Discussion
The synthesis of the target diazepine was commenced with the esterification of 2-hydroxyacetaphenone
with substituted benzoyl chlorides as illustrated in Scheme 1.
A Baker-Venkataraman rearrangement induced by KOH affords 1,3-diketones 2a–f, which undergo
condensation under refluxing acetic acid to provide flavones 3a–f. All ester, 1,3-diketone and flavone
intermediates were confirmed by comparison of their melting point and NMR spectral data with
literature values. All data for these intermediates were in complete accordance with literature values.
Flavones 3a–f reacted with ethylenediamine to afford the required diaryldiazepines in reasonable

Molecules 2015, 20
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yields and high purity. Their spectroscopic data were in accordance with those previously reported [6].
Only diazepine 4d is novel and was therefore fully characterized by FT-IR, NMR and Mass
Spectrometry. To evaluate the possible influence of substituent effects, we prepared diaryldiazepines
which either contained an electron donating or withdrawing group.
O
C H 3
O
O
O
O
O H
O
K O H
+
N
C H 3
N
2 5 o C ,1 5 m in
5 0 o C , 0 .5 h
C l
O H
R
2 a -f
O
R
R
1 a -f
O
N
N H
A c O H , 1 h
1 0 0 o C
N H 2
H 2
N
O
R
R
1 0 0 o C , 2 h
O H
4 a -f
3 a -f
R = a : H , b :4 -C l, c : 4 -C H 3 , d : 3 ,4 ,5 -(O C H 3
)
3 , e : 4 -F , f: F u ra n
In th e c a s e o f 1 f-4 f:
R
O
=
Scheme 1. Synthesis of Diazepines 4a–f.
Once the final products were purified and fully characterized, we carried out in vitro bioassays using
epimastigotes forms (i.e., the form present in the midgut vector) of Y-strain T. cruzi, which provided
the inhibitory concentration as summarized in Table 1.
Table 1. Trypanocidal activity of diazepines 4a–f against the epimastigotes forms of
Y-strain T. cruzi.
Trypanocidal Activity
Compound
IC50 (μM)
Benznidazole
10.8
10.6
4a
4b
4c
4d
4e
4f
0.25
34.2
26.8
4.2
Not Active
IC50: Represents the concentration required to give 50% inhibition.
In Brazil, benznidazole is the only therapy used in etiological treatment with direct action against
intracellular trypomastigotes of the Trypanosoma cruzi parasite. Precisely for this reason, it is the only
drug used as a standard control in our experiments. However, due to numerous side effects caused by
this drug and resistance build up by different strains of the parasite, several compounds have been

Molecules 2015, 20
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studied in the search for new treatments [12–17]. Herein, 6 Diazepines compounds were assayed
directly against the epimastigotes forms of T. cruzi.
Among the diazepines tested, 4a, 4b and 4e were found to be the most potent trypanosomicides
against epimastigotes and in particular, diazepine 4b was 40 times more potent than our positive
control, benznidazole. The furan bearing diazepine was essentially inactive and it was therefore not
possible to calculate an IC50 for 4f with any statistical confidence due its low activity. No cytotoxicity
was observed for this same compound against human leukemic cancer cell lines [6].
Diazepine 4b showed 78% inhibition at the lowest concentration tested and this result highlights the
remarkable effect that the chloro group has on the activity of the 5,7-diaryldiazepine. In contrast, the
presence of electron donating methoxy groups are slightly unfavourable towards anti-trypanocidal
activity. Our results contrast to those of Ramajayam and collaborators [6] in which cytotoxicity
activity of 5,7-diaryldiazepines was highest for the methyl group bearing diaryldiazepine. Similarly,
electron-donating groups i.e., the methoxy substituents, lowered cytotoxicity. In the present study,
these analogues were also less active against T.cruzi.
Ramajayam and collaborators found that antiproliferative activity for halogen group bearing
diaryldiazepines was moderate, but from the data obtained in the present study, this analogue was
significantly more active against T. cruzi with compound 4e being the next most active against
epimastigotes of T. cruzi. In contrast, the IC50 for cytotoxicity of 4b against human leukemic cancer
cell lines Jurkat (81 μM), HL60 (>100 μM), Molt3 (64 μM), NCEB-1 (>100 μM) and K562 (65 μM)
suggest only moderate antiproliferative activity. Interestingly, the same compound demonstrated higher
activity against epimastigotes of T. cruzi than the aforementioned compound classes (see introduction)
that have been studied in recent years. These initial promising results will direct us in investigating
how substituents on both rings of the molecule contribute to the overall activity against the parasite.
In conclusion, diaryldiazepine 4b is the most active against epimastigotes of T. cruzi and is 40 times
more potent than the current medicine, benznidazole that is used to treat Chagas disease in Brazil.
Studies are ongoing to investigate the possible biological mechanism of action.
3. Experimental Section
All commercial reagents were used as received. TLC analyses were performed using silica gel
plates, using ultraviolet light (254 nm) or vanillin solution for visualization. Melting points are
uncorrected. IR spectra were recorded using samples that were prepared between NaCl plates, or
pressed in to KBr discs. For NMR data, the chemical shifts are reported in δ (ppm) referenced to
residual protons and 13C signals in deuterated chloroform or deuterated DMSO. The coupling constants
(J) are expressed in Hertz (Hz). Mass spectra were recorded on a Shimadzu LCMS-IT-TOF mass
spectrometer. Flavones 3a–f were prepared according to literature methods [18].
3.1. Chemistry
General method for the preparation of 5,7-diaryl-2,3- dihydro-1,4-diazepine derivatives is based on
the protocol described by Ramajayam and co-workers [6] and involves refluxing a mixture of flavone
(5–15 mmol) in ethylenediamine (20 mL) for 2 h. The cooled mixture was poured into ice water and the
yellow precipitate was filtered and finally recrystallized from methanol to give analytically pure samples.

Molecules 2015, 20
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5-(2-Hydroxyphenyl)-7-phenyl-2,3-dihydro-1H-1,4-diazepine. Compound 4a was obtained as a yellow
solid in 57% yield. MP. 209–211 °C (Lit MP. 208–210 °C [6]); (300 MHz, DMSO-d6) δ: 8.0 (1H, brs),
7.38–7.58 (7H, m), 7.0 (1H, t, J 8.5), 6.74 (1H, d, J 8.0), 6.45 (1H, t, J 8.0), 5.6 (1H, s), 3.8 (2H, bs),
3.6 (2H, bs); δC (75 MHz, DMSO-d6): 170.2, 167.2, 158.9, 140.4, 136.2, 132.5, 129.6, 127.19, 127.8,
121.2, 117.1, 114.1, 86.1, 50.4, 49.3.
5-(2-Hydroxyphenyl)-7-(4-chlorophenyl)-2,3-dihydro-1H-1,4-diazepine. Compound 4b was obtained
as a yellow solid in almost quantitative yield. MP. 238–240 °C (Lit MP. 243–245 °C [6]); υmax (cm−1):
1570, 1529, 1490, 1439, 1318, 1289, 1260, 1143, 837, 751; δH (300 MHz, DMSO-d6): 7.66–7.45 (5H, m),
7.08 (1H, t, J 7.8), 6.56 (1H, d, J 8.2), 6.38 (1H, t, J 7.0), 5.61 (1H, s), 3.65 and 3.95 (4H, bs); δC (75 MHz,
DMSO-d6): 170.8, 168.2, 157.8, 137.7, 135.2, 132.7, 129.8, 129.3, 129.0, 128.7, 128.0, 121.7, 116.9,
113.2, 86.6, 49.9, 49.0, 31.1.
5-(2-Hydroxyphenyl)-7-(4-methylphenyl)-2,3-dihydro-1H-1,4-diazepine. Compound 4c was obtained
as a yellow solid in 47% yield. MP. 233–235 °C (Lit.MP. 235–237 °C [6]); υmax (cm−1): 3221, 2922,
1604: δH (300 MHz, DMSO-d6): 8.14 (1H, s), 7.59–7.51 (3H, m), 7.28 (2H, d, J 8.0), 7.07 (1H, t, J 7.0),
6.54 (1H, d, J 8.5), 6.37 (1H, t, J 8.0), 5.62 (1H, s), 3.81 (2H, s), 3.56 (2H, s), 2.35 (3H, s); δC (75 MHz,
DMSO-d6): 170.8, 168.2, 158.9, 140.4, 136.2, 132.5, 129.6, 127.9, 127.8, 121.6, 117.1, 113.1, 86.1,
50.0, 49.0, 21.3.
5-(2-Hydroxyphenyl)-7-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1H-1,4-diazepine. Compound 4d was
obtained as a yellow solid in 43% yield and is a novel compound. MP. 229–230 °C; υmax (cm−1): 1582,
1531, 1499, 1309, 1244, 1124, 1009, 756; δH (300 MHz, DMSO-d6): 8.12 (1H, brs), 7.64 (1H, d, J 8.5),
7.08 (1H, t, J 7.8), 6.56 (1H, d, J 8.3), 6.42 (1H, t, J 7.0), 5.67 (1H, s), 3.84 (6H, s), 3.69 (3H, s), 3.54
(4H, bs); δC (75 MHz, DMSO-d6): 172.5, 170.5, 158.7, 153.1, 134.5, 132.4, 128.2, 121.4, 117.3, 113.2,
105.5, 86.2, 60.5, 56.5, 50.2, 49.1; MS m/z (EI): calcd. for C20H22N2O4 354.1580, found 354.1571.
5-(2-Hydroxyphenyl)-7-(4-fluorophenyl)-2,3-dihydro-1H-1,4-diazepine. Compound 4e was obtained as
a yellow solid in 49%. MP. 235–236 °C (MP. 234–236 °C Lit. [6]); υmax (cm−1): 1603, 1531, 1499,
1440, 1318, 1259, 1224, 1168, 1144, 845, 783, 751; δH (300 MHz, DMSO-d6): 7.68 (2H, t, J 8.7), 7.59
(1H, d, J 8.4), 7.3 (2H, t, J 8.7), 7.08 (1H, t, J 6.9), 6.55 (1H, d, J 9.3), 6.38 (1H, t, J 8.1), 3.65 and
3.95 (4H, bs); δC (75 MHz, DMSO-d6): 171.3, 168.4, 165.5 and 162.2 (d, JCF 247.5), 158.4, 135.639
and 135.601 (d, JCF 2.9), 132.9, 130.6, 130.5, 130.5, 128.3, 122.0, 117.1, 116.3 and 116.0 (d, JCF 59.7),
113.3, 86.6, 50.0, 49.2.
5-(2-Hydroxyphenyl)-7-(2-furyl)-2,3-dihydro-1H-1,4-diazepine. Compound 4f was obtained as a yellow
solid in 77% yield. M.P. 175–178 °C (Lit MP. 168–170 °C [6]); υmax (cm−1): 1602, 1537, 1511, 1329,
1301, 1253, 1149, 1023. δH (300 MHz, DMSO-d6): 7.87 (1H, s), 7.66 (1H, d, J 8.4), 7.21 (1H, d, J 3.3),
7.1 (1H, t, J 8.1), 6.68–6.66 (1H, m), 6.56 (1H, d, J 9.3), 6.44 (1H, t, J 8.1), 6.03 (1H, s), 3.65 and 3.95
(4H, bs); δC (75 MHz, DMSO-d6): 171.1, 168.5, 150.1, 147.1, 145.7, 132.9, 128.1, 121.9, 117.2, 113.5,
113.2, 111.7, 83.0, 50.0, 48.8.

Molecules 2015, 20
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3.2. Pharmacology
The IC50 values of the drug concentration (μM) necessary to kill 50% of the parasites, was obtained
by linear regression analysis.
3.3. Anti-Trypanosoma cruzi Activity
In this study, epimastigotes of the Y strain from T. cruzi were used. Epimastigotes obtained in
exponential growth phase were washed with LIT medium 2 times in sterile PBS at pH 7.2 (1500 g) for
10 min at 4 °C. The number of parasites was determined in a Neubauer chamber. Next, the parasites
were suspended in LIT medium, which was further supplemented with 10% FBS (Fetal Bovine Serum
inactivated at 56 °C), and the concentration of epimastigotes adjusted to 5 × 106 epimastigotes/mL.
With the goal of screening samples with possible biological activity against T. cruzi, compounds
were incubated in 48 well Nunc plates containing 800 μL of the suspension of parasites and 200 μL of
the test compounds at different concentrations (40, 20, 10 and 1.6 μg/mL) diluted in sterile DMSO for
72 h in duplicate. As a negative control, the parasites were incubated in the absence of test compound
and in the presence of 1% DMSO. Benznidazole (10 μg/mL) was used as positive control in tests
against T. cruzi. The activity was determined by counting in a Neubauer chamber and subsequent
statistical evaluation. The results are expressed as IC50. The tests were repeated 2 times with the
objective of evaluating the maintenance of the activity of the compounds and reproducibility of results.
Acknowledgments
This work was supported by the Brazilian funding agency Fundação de Amparo à Pesquisa do Estado
de Minas Gerais (FAPEMIG) under research grant project code APQ-00307-12, APQ-00356-13 and
the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) under research grant
project code 473461/2013-7. Authors gratefully acknowledge the generous financial support from
the Universidade Federal de Ouro Preto, FAPEMIG and the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq).
Author Contributions
JCLM carried out the synthesis of the diaryldiazepines; LBAV, PMAV, KSF and CMC evaluated
the biological activity against T. cruzi; JGT, PMAV and CMC wrote the paper. All authors read and
approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4a–f are available from the authors.
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