Synthesis and trypanocide activity of chloro-l-tyrosine and bromo-l-tyrosine derivatives

Article abstract of DOI:10.1007/s00044-018-2249-y

Twenty-two halogenated l-tyrosine derivatives were synthesized to examine new substances for the treatment of Chagas disease. The synthesis of these derivatives with different degree of substitution in the amino group with methyl iodide, giving primary, tertiary, and quaternary amino acids. All compounds were tested in vitro against intracellular amastigotes of Trypanosoma cruzi, and the cytotoxicity were evaluated over monocytic cell line U-937. Compound 25 was the most active against T. cruzi with a EC₅₀ of 75.52 μM compared with benznidazole with a EC₅₀ of 58.79 μM. Compounds 3, 4, 7, and 15 were the derivatives with the best selectivity index (SI) with values of 7.5, 8.3,12.1, and 8.6, respectively. Finally, compound 7 was the safer and the more promising derivative against T. cruzi.

Full text of DOI:10.1007/s00044-018-2249-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2249-y
ORIGINAL RESEARCH
Synthesis and trypanocide activity of chloro-L-tyrosine and bromo-L-
tyrosine derivatives
Manuel Pastrana Restrepo1 Elkin Galeano Jaramillo1 Alejandro Martínez Martínez1 Sara Robledo Restrepo2
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Received: 20 June 2018 / Accepted: 15 September 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Twenty-two halogenated L-tyrosine derivatives were synthesized to examine new substances for the treatment of Chagas
disease. The synthesis of these derivatives with different degree of substitution in the amino group with methyl iodide,
giving primary, tertiary, and quaternary amino acids. All compounds were tested in vitro against intracellular amastigotes of
Trypanosoma cruzi, and the cytotoxicity were evaluated over monocytic cell line U-937. Compound 25 was the most active
against T. cruzi with a EC50 of 75.52 µM compared with benznidazole with a EC50 of 58.79 µM. Compounds 3, 4, 7, and 15
were the derivatives with the best selectivity index (SI) with values of 7.5, 8.3,12.1, and 8.6, respectively. Finally, compound
7 was the safer and the more promising derivative against T. cruzi.
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●
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Keywords Chagas disease Bromo-tyrosine Chloro-tyrosine T. cruzi
Introduction
of infection, due to the conditions geographies that favor
vector replication, mother-to-child transmission, and con-
sumption of food contaminated by the parasite. The increase
in annual cases in the America region is ∼56,000, leading to
about 12,000 deaths annually (Cucunubá et al. 2016).
Chemotherapeutic options for the control and treatment of
these protozoa infections are limited to few drugs, which in
many cases are associated with severe toxicity and variable
efficiency.
Currently, there is no vaccine for the treatment of this
disease, in addition, efforts to eradicate the vectors that
transmit the parasite have not given the best results, with
chemotherapy being the best option so far for the treatment
of this disease (Quijano-Hernandez and Dumonteil 2011).
Nifurtimox (NFX) and benznidazole (BZN), two hetero-
cyclic nitro compounds, are the conventional treatment for
the Chagas disease. The side effects of both drugs are a
major drawback of their use. Anorexia, weight loss, psychic
disturbances, skin manifestations, and bone marrow
depression. Frequently, these side effects force the physi-
cian to stop treatment (Castro et al. 2006). The use of both is
widely accepted in the acute phase of the disease, but their
use in the chronic phase in controversial (Coura and Castro
2002).
Chagas disease is an infectious disease caused by the pro-
tozoa Trypanosoma cruzi. This disease is one of the major
health problems in the Americas, affecting people in almost
21 countries in the region and the World Health Organi-
zation (WHO) estimates that there are ∼6–7 million people
infected around the world, with the highest infection
occurring in the American continent (Pinheiro et al. 2017).
Chagas disease has been increasingly detected in the United
States of America, Canada, and many European and some
Western Pacific countries. This is due mainly to population
mobility between Latin America and the rest of the world
(Antinori et al. 2017). About 100 million people are at risk
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2249-y) contains supplementary
material, which is available to authorized users.
* Elkin Galeano Jaramillo
elkin.galeano@udea.edu.co
1
Departamento de Farmacia, Facultad de Ciencias Farmacéuticas y
Alimentarias, Productos Naturales Marinos, Universidad de
Antioquia, UdeA, Calle 70 # 52-21, laboratorio 2-131,
Medellín, Colombia
In the last 40 years, there are no new drug developments.
This fact together with the resistance of the parasite against
conventional treatment encourages the discovery and
2
Facultad de Medicina, Programa de Estudio y Control de
Enfermedades Tropicales (PECET), Universidad de Antioquia
UdeA, Calle 62 # 52-59, laboratorio 632, Medellín, Colombia

Medicinal Chemistry Research
Scheme 1 Intermediate
synthesis from L-tyrosine
development of new, safer, and more effective antiprotozoal
agents to treat new cases.
formed by the reaction of L-tyrosine with thionyl chloride
(SOCl2) in methanol (Cao et al. 1999), later the protection
of the amino group was done by the reaction with Boc2O/
TEA at room temperature (Rehman et al. 2010). The bro-
mination and chlorination of the aromatic ring to give mono
and di halogenated compounds, were done using N-bro-
mosuccinimide (NBS) and N-chlorosuccinimide (NCS)
(Bovonsombat et al. 2008), producing the intermediates
Natural products have been used as a source of new
molecules with pharmacological activities, including their
potential as antiparasitic agents. Bromotyrosines from
marine sponges of the order Verongida is between the most
active and selective antiparasitic molecules that have been
isolated (Galeano et al. 2011). Bromotyrosines derivatives,
such as aeroplysinin have shown relevant activities against
T. cruzi with a 26.6% of growth inhibition at 9.3 µΜ
(Galeano et al. 2012). Structure–activity analyses have led
to the determination of some key structural requirements for
the activity. The variation of the substitutions of amino
groups, besides halogenation degree, has shown correla-
tions in their antiparasitic potentials. Computational studies
between bromotyrosines derivatives and their chemical
properties were conducted to identify a potential pathway-
related trypanocidal activity. Therefore, in this work, we
explored the synthesis and structural correlation of L-tyr-
osine derivatives with different halogenation degrees and
methylation degree in the amino group as potential trypa-
nocide agents.
1
(Int1–Int4). The H NMR analysis of (Int1–Int4) shows
the presence of a signal at 3.80 ppm (s, 3H) that represents
the –RCOOCH3 group of the methyl ester. The signal at
1.40 ppm (s, 9H) represents the three methyl groups of the
tert-butyl in the carbamate moiety. The mono halogenated
compounds show in the aromatic region of the spectra, three
signals that corresponds to the protons at 2, 5, and 6 posi-
tions in the aromatic ring with J around 2.0 and 7.5 MHz
corresponding to ortho and meta couplings, while the di
halogenated compounds only show in the aromatic region
of the spectra one signal, due to the symmetric ring, for two
protons at 2 and 6 positions. These intermediates were
divided into two sets, one of them were treated with TFA/
DCM (Hulme and Rosser 2002), to cleavage the amino
group (1a–1d). The other set was O-Methylated with
methyl iodide in basic media (Liu et al. 2014) and later
treated with TFA/DCM (1e–1h) (Scheme 1).
Results and discussion
The syntheses of primary amines were done by the
hydrolysis of compounds (1a–1h) with K2CO3/MeOH (Ase-
mave et al. 2018), getting compounds (2–9) (Scheme 2).
The syntheses of methyl ester tertiary amines were done
by the reductive amination of compounds (1a–1h) with
CH2O(ac)/NaCNBH3 getting compounds (10a–10h) (Rai-
mer and Lindel 2013). These set of compounds were divi-
ded into two, one of them were treated with K2CO3/MeOH
to get the tertiary amines (11–18) (Scheme 3). The 1H NMR
Chemistry
Synthesis of bromo-N-substituted and chloro-N-substituted
derivatives were made using as starting material L-tyrosine.
For the synthesis of 22 products it is necessary to synthesize
initially four intermediates, which have their amino and
carboxylic acids protected with a carbamate and methyl
ester group, respectively. Methyl esters derivatives were

Medicinal Chemistry Research
analyses of tertiary amines shows the presence of a signal at
2.88 ppm (s, 6H) for the di-methylation of the amino group.
O-methyl-phenolic derivatives were confirmed by 1H NMR
by the signal at 3.80 ppm (s, 3H).
compounds, 51:100:49 (X:X + 2:X + 4) to di bromine
compounds, 100:32 (X:X + 2) to mono chlorine compounds
and 100:65:10 to di chlorine compounds (X:X + 2:X + 4),
all the mass spectra are shown in the supplementary mate-
rial. The optical activities of the primary amines, com-
pounds 2–9, shows negative optical rotatory power; tertiary
amines, compounds 11–18, shows positive rotatory power,
and quaternary amines, compounds 20–25 shows negative
rotatory power. The substitution over the amino group
changes the sense of the rotatory power, where the sub-
stitutions of the aromatic only affect the magnitude of the
rotatory angle.
The other set of compounds (11c–11h), were treated with
MeI/(CH3)2CO to get the methyl ester quaternary amines
(Tars et al. 2014), which were treated with K2CO3/MeOH to
1
get the quaternary amines (20–25), (Scheme 4). The H
NMR analyses of quaternary amines shows the presence of
a signal at 2.97 ppm (s, 9H) for the tri-methylation of the
amino group. O-methyl-phenolic derivatives were con-
1
firmed by H NMR by the signal at 3.80 ppm (s, 3H).
The HRMS analysis of the 22 L-tyrosine derivatives
determines the kind of halogen present in the aromatic ring
by the distribution of the isotopic pattern of the bromine and
chlorine derivatives. The relation between X, X + 2, X + 4,
where X represents the molecular ion peak (M+ or [M + H]
+), shows a relation of 100:98 (X:X + 2) to mono bromine
Computational study
The geometry of the synthesized compounds was optimized
with DFT/B3LYP as implemented in Guassian-09 (Gaussia,
Revision D.01 2009) quantum chemistry package. Using
the DFT/ B3LYP calculation method with 6-311++G (d,p)
basis set in the ground state, the optimized molecular geo-
metric parameters were performed. The stable conformers
of the most active compounds are shown in Fig. 1. In Table
1, the Mulliken population analysis of aromatic carbons and
the magnitude of the dipole moment of the 22 L-tyrosine
derivatives are shown. The analysis of the Mulliken index is
a parameter to determine the variation of atomic charges
over carbon atoms, influenced by the type and the degree of
the halogen in the aromatic ring.
The analysis of the Mulliken index revealed that the type
and degree of halogenation pattern affect the electronic
density in the aromatic ring. The presence of chlorine or
bromine atoms increases the electronic density at carbons 2,
4, and 6, while decreasing the electronic density at carbons
3 and 5. The effect is higher with aromatic rings di halo-
genated, where the bromine is the halogen with more
influence in the electronic distribution by the formation of
sigma (σ)-holes (Lim and Beer 2018). The σ-hole
Scheme 2 Primary amines derivatives syntheses
Scheme 3 Tertiary amines
derivatives syntheses

Medicinal Chemistry Research
Scheme 4 Quaternary amines
derivatives syntheses
interactions are electron-deficient regions that arise from the
anisotropic distribution of electron density on the molecules
with halogen atoms. These regions together π–π interactions
(Meyer et al. 2003) produce modification in the electron
donor substituents regions or electron attractants in aromatic
systems. Some author had shown that halogen atoms in
aromatic systems play an important role in the formation of
non-covalent interactions with anions, peptide bonds, and
nucleic acids (Mendez et al. 2017), where bromine atoms
generating the strongest non-covalent interactions (Riley
et al. 2011).
T. cruzi amastigotes with EC50 values from 189.85 to 335.9
µM. Di brominated derivatives were more active than their
structural di chlorinated analogs. There is possibly a direct
relationship between the electronegativity of the halogen
atom involved and the interaction with its biological target,
where lower electronegativity interactions could be the
answer. Some authors have found structural correlations
between molecules with the carbonyl group and aromatic
ring moiety, and π–π stacking interaction with key amino
acid residues: Tyr342, Trp312, Arg53, and Glu230, on the
trans-sialidase of Trypanosoma cruzi (TcTS), a non-
homologous enzyme in humans. This enzyme catalyzes
the transfer of sialic acid from the host glycoconjugate
surface to the T. cruzi mucin-like glycoprotein surface, and
its inhibition produce a selective inhibition of the parasite
(Kashif et al. 2018). Additionally, studies realized with the
functionalization of aromatic hydrazones derivatives with
halogen moieties (bromide and chloride), have shown that
halogenated derivatives influences to have the best inter-
action with the parasite enzymes: cysteine-protease and
trypanothione reductase. It is due to hydrophobic nature of
the active site of these parasitic enzymes (Romero and
López 2017); therefore, aromatic halogenated derivatives of
amino acid should have a wide selective range of inhibition
of parasitic enzymes. Which play pivotal roles in the biol-
ogy and pathogenesis of the Chagas disease. The synthesis
of new iodine derivatives could help us to evaluate this
hypothesis. Additionally, O-methyl derivatives were more
actives than their phenolic analogs. The highest SI was
shown by the compounds 3, 4, 7, and 15, where the com-
pound 7 was the most promissory against T. cruzi, an O-
methyl-di brominated L-tyrosine derivative.
Biological activity
Compounds (2–9, 11–18, 20–25) were tested against T.
cruzi (Tulahuen strain) using BZN as the positive control
drug. For every compound, the median effective con-
centration EC50 and the median lethal concentration LC50 in
µM were determined. In addition, the selectivity index (SI)
was calculated by dividing the LC50 by the EC50 (SI =
LC50/EC50). Table 2 shows the anti-trypanosomal activity of
the 22 L-tyrosine derivatives tested.
All the synthetic derivatives (except 16, 18, 20, 21, 23,
and 25) showed LC50 values higher than 300 µM, making
them potentially non-cytotoxic against mammalian cells U-
937. Compound 7 was the less cytotoxic LC50 of 1014.1
µM, while compound 21 was the most cytotoxic for U-937
macrophages. Chlorinated derivatives were more cytotoxic
than their structural brominated analogs and O-methyl
derivatives were more cytotoxic than their phenolic analogs.
All compounds evaluated, except compounds 5 and 14,
showed activity against T. cruzi amastigotes at concentra-
tions <200 µM. According to the proposed scale to grade
the antitrypanosomal activity, the compound 25 was the
most active, with an EC50 value of 75.52 µM, while com-
pounds 2, 3, 4, 7, 8, 9, 15, 16, 18, 11, 20, 21, 22, and 24
exhibited an activity that could be considered moderate with
EC50 values varying from 77.42 to 173.06 µM. The
remaining compounds 5, 14, and 23 showed low activity for
Conclusions
Antitrypanosomal and cytotoxicity activities of 22 chloro
and bromo N-substituted L-tyrosine derivatives were pre-
pared and evaluated. Bromotyrosine derivatives, the

Medicinal Chemistry Research
Fig. 1 Stable conformers of
most active compounds
compounds isolated firstly from marine sponges, were the
most active derivatives against T. cruzi parasites. In this
study, it was established that the type of halogenation, in
addition to the degree of methylation on the amino group,
have influenced the antiparasitic activity of all the com-
pounds. In general, O-methyl-di halogenated compounds
with tertiary and quaternary amines were the most cytotoxic
derivatives, and O-methyl di-halogenated compounds were
the most active, where the derivatives with quaternary
amines stood out as the most active synthesized com-
pounds. The biological activity of di-halogenated deriva-
tives could be explained by the highest electronic density
over the aromatic ring, making possible stronger hydro-
phobic interactions between these compounds and aromatic
amino acids residues in the active site of the cysteine-
protease and trypanothione reductase enzymes.
Compounds 3, 4, 7, and 15 showed the higher SI against
T. cruzi amastigotes, where the compounds 7 is a promising
candidate to lead the development of new drug candidates
against Chagas disease. This is the first synthetic report for
12 compounds: 11–15, 18, 20–25. Most of the tertiary and
quaternary amines were synthesized for the first time, also is
the first report of antitrypanosomal and cytotoxic activity
for 20 compounds: 2–9, 11–18, and 20, 21, 24, 25. The
structural simplicity of the compound, in addition to the
high yields of products, are advantages for their future
production for in vivo trypanocide evaluation.
Experimental
General
All the laboratory reagents were analytical grade (Sigma-
Aldrich). Column chromatography and HPLC solvents were
liquid chromatography grade (Merck). Compound pur-
ifications were made by column chromatography using
Merck silica gel 60 and mixtures of hexane and ethyl
acetate. Purifications of biologically tested compounds were
made by HPLC Agilent 1200 with DAD detector (254, 280,
and 366 nm) and Eclipse XDB-C18 column using a mixture
of MeOH and H2O with 0.1% formic acid as the mobile
1
phase. H NMR and 13C NMR were recorded on Bruker
ultrashield (300 and 75 MHz) and Ascend III HD (600 and
125 MHz) with 5 mm cryoprobe TCI, using CDCl3, D2O,
and DMSO-d6 as deuterated solvents. Signals were assigned
using two-dimensional heteronuclear correlations (COSY
and HSQC). HRMS were recorded using electrospray
ionization (ESI–MS) in a UPLC-Q-Tof, reference Xevo-
XS-Qtof, waters. The drying and cone gas was nitrogen set
to flow rates of 300–30 L/h, respectively. Methanol samples
solutions (1 × 10−5 M) were directly introduced into the ESI
spectrometer at a flow rate of 10 μL/min. A capillary voltage
3.5 kV was used in the positive scan mode, and the cone
voltage set to Uc = 10 V. All compounds decomposed over
300 °C.

Medicinal Chemistry Research
Table 1 Dipole moments and
Mulliken index of 22 L-tyrosine
derivatives
Compound
Dipole moment (Debye)
Mulliken index
C1
C2
C3
C4
C5
C6
2
2.65287
2.09822
2.14052
2.29668
2.56023
2.80198
2.4184
1.024
1.184
0.783
0.819
1.217
1.524
1.017
0.001
1.280
1.462
1.175
1.242
1.385
1.723
0.257
−0.924
−0.912
−0.993
−0.884
−1.195
−1.362
−1.239
−0.175
−1.108
−1.150
−1.317
−1.294
−1.294
−1.476
0.183
0.904
1.332
0.892
1.073
1.143
1.932
1.337
0.253
0.917
1.422
0.888
1.225
1.108
1.964
0.057
0.197
0.921
1.224
1.316
1.996
−0.581
−2.249
−0.295
−1.556
−0.963
−2.663
−0.817
−0.701
−0.589
−2.325
−0.256
−1.599
−0.979
−2.717
−0.545
−0.829
−0.183
−1.423
−1.020
−2.653
0.370
1.750
0.173
1.439
0.746
1.956
0.409
−0.178
0.306
1.736
0.257
1.494
0.684
1.935
0.035
0.228
0.245
1.338
0.639
1.858
.−0.062
0.041
−0.855
−1.092
−0.475
−0.987
−1.166
−1.511
−0.753
0.586
3
4
5
6
7
8
9
1.61863
1.72658
2.04629
1.7982
11
12
13
14
15
16
17
18
20
21
22
23
24
25
−0.668
−0.843
−0.559
−0.830
−0.959
−1.260
−0.539
−0.187
−0.527
−0.780
−0.916
−1.140
−0.386
−0.017
2.1168
1.40875
2.72373
1.43062
2.87559
12.07689
14.76151
14.15019
17.67397
12.48706
15.48015
−0.094 0.032
1.234
1.278
1.407
1.598
0.250
−1.434
−1.365
−1.421
−1.438
0.148
−0.003 −0.476
0.141 −0.661
−0.076 −0.118
L-tyrosine derivatives syntheses
11.22 mmol), and NCS (2535 mg, 19 mmol), respectively.
Previous mixtures was dissolved, each one in MeOH (15
mL) and Boc2O (2300 µL, 10 mmol), and TEA (2000 µL)
was added and was stirred for 24 h at room temperature.
The solvent was removed, the crude was adjusted to pH = 3
and later extracted with AcOEt. The organic phase was
dried with Na2SO4 and purified in column chromatography
using as mobile phase Hexane–AcOEt (3:1).
Synthesis of intermediates (Int1–Int4)
To MeOH (50 mL) at −5 °C, SOCl2 (2190 µL, 30 mmol)
was dropped slowly to maintain the temperature below 0 °
C. Then L-tyrosine (5000 mg, 27.6 mmol) was added. The
resulting mixture was stirred at 70 °C for 24 h. As the
mixture cooled, the product was precipitated by the addition
of diethyl ether (100 mL). The precipitate was collected,
washed with ethyl ether (20 mL) and dried to give L-tyr-
osine methyl ester hydrochloride. The product was tested by
TLC mobile phase nBuOH–AcOH–H2O (4:1:1) showing
quantitative conversion. This reaction was done twice. The
mono bromination of L-tyrosine methyl ester (2000 mg,
8.63 mmol) was done with NBS (2000 mg, 11.21 mmol) in
MeOH (20 mL) stirred at room temperature for 24 h. The
solvent was removed, and the crude was partitioned with
AcOEt–H2O. The aqueous phase was recovered and eva-
porated to get red oil. This crude was tested by TLC mobile
phase nBuOH–AcOH–H2O (4:1:1) showing the formation
of mono bromine compound. This crude was not purified to
the next reaction. Compounds di-brominated, mono
chlorinated and di-chlorinated were obtained in the same
way using NBS (3380 mg, 19 mmol), NCS (1500 mg,
(Int1),
methyl-(S)-3-(3-bromo-4-hydroxyphenyl)-2-((tert-
butoxycarbonyl)-amino) propanoate White solid, 1615
1
mg, two stages yield 50%. H NMR (300 MHz, CDCl3) δ
7.21 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 8.3 Hz, 2.0 Hz, 1H),
6.87 (d, J = 8.3 Hz, 1H), 5.07 (d, 1H), 4.51 (d, 1H), 3.71 (s,
3H), 2.97 (m, 2H), and 1.41 (s, 9H).
(Int2), methyl-(S)-3-(3,5-dibromo-4-hydroxyphenyl)-2-((tert-
butoxycarbonyl)-amino) propanoate White solid, 2740
1
mg, two stages yield 70%. H NMR (300 MHz, CDCl3) δ
7.02 (s, 2H), 5.07 (d, 1H), 4.51 (d, 1H), 3.73 (s, 3H), 3.13–
2.98 (m, 1H), 2.90 (m, 1H), 1.42 (s, 9H).
(Int3),
methyl-(S)-3-(3-chloro-4-hydroxyphenyl)-2-((tert-
butoxycarbonyl)-amino) propanoate White solid, 1565
1
mg, two stages yield 55%. H NMR (300 MHz, CDCl3) δ

Medicinal Chemistry Research
Table 2 In vitro cytotoxicity and antitrypanosomal activities of L-
tyrosine derivatives
Synthesis of compounds (1e–1h)
Compound
Cytotoxicity on
Antitrypanosomal
Selectivity
indexc
The O-methylation of (Int1–Int4) were done by dissolving
1500 mg of each intermediate in acetone with 800 mg
K2CO3 and 750 µL MeI. The mixture was stirred for 12 h at
room temperature. The solvent was removed and purified in
column chromatography using as mobile phase Hexane–
AcOEt (3:1). The products obtained in the last step were
dissolved in DCM (3 mL) at 0 °C. After 5 min TFA (1000
µL) was added to the mixture and left to react for 24 h at
room temperature. Later the solvents and reagents were
evaporated, and the crude of the reaction was adjusted to
pH = 7. The product was tested by TLC mobile phase
Hexane–AcOEt (3:1) showing quantitative conversion for
compounds (1e–1h). (1e) 920 mg, two steps yield 80%. (1f)
972 mg, two steps yield 80%. (1g) 885 mg, two steps yield
80%. (1h) 916 mg, two steps yield 80%.
U-937 cells LC50 activity on intracellular
(µM)a
amastigotes EC50 (µM)b
2
>768.9
158.41 19.99
104.43 8.55
134.95 10.20
335.9 36.78
146.66 16.05
84.13 3.68
149.79 5.22
130.25 8.32
99.26 6.59
131.32 10.35
>82.07
>2.7
7.5
3
783.2 22.12
1123.2 38.49
>799.7
4
8.3
5
>2.4
4.1
6
598.3 44.50
1014.1 15.86
874.8 43.54
>757.2
7
12.1
5.9
8
9
>5.8
<7.0
3.1
11
>694.1
12
404.3 36.77
725.5 109.97
514.9 57.53
894.2 24.48
73.2 4.72
>776.05
13
<8.8
0.7
14
213.93 39.19
103.92 17.20
77.42 10.49
173.06 24.83
101.31 5.81
149.58 17.39
95.85 5.28
117.28 4.09
189.85 5.30
114.39 23.09
75.52 6.18
58.79 2.68
15
8.6
16
1
17
>4.5
1.7
18
168.4 13.25
148.8 10.43
69.9 3.40
>630.52
Primary amines synthesis
20
1
21
0.7
Synthesis of (2–9) compounds
22
>5.4
0.5
23
99.0 8.58
>733.2
150 mg of compounds (1a–1h) were dissolved in a mixture
of MeOH–H2O–THF (1:1:1) (5 mL) and then K2CO3 was
added (100 mg), and the mixture was stirred for 24 h at
room temperature. The crude of reaction was adjusted to
pH = 6. The compounds were tested by TLC mobile phase
nBuOH–AcOH–H2O (4:1:1) showing quantitative conver-
sion. The final products (2–9) were purified with RP-HPLC
using as a mobile phase a mixture of MeOH (0.01% F.A.)
and H2O (0.01% F.A.).
24
>6.4
2.2
25
167.0 7.56
>768.5
Benznidazole
>12.7
Data represent, mean value of three replicates standard deviation
aLC50: lethal concentration 50 in µM
bEC50: effective concentration 50 in µM
cSI: selectivity index = LC50/EC50
7.07 (s, 1H), 6.90 (s, 2H), 5.03 (s, 1H), 4.51 (s, 1H), 3.71 (s,
3H), 2.96 (s, 2H), and 1.41 (s, 9H).
(2)
(S)-2-amino-3-(3-bromo-4-hydroxyphenyl)-propanoic
(Int4), methyl-(S)-3-(3,5-dichloro-4-hydroxyphenyl)-2-((tert-
acid White solid, 140 mg, yield 100%. 1H NMR (300
MHz, D2O, DMSO-d6) δ 7.32 (d, J = 1.9 Hz, 1H, H-2),
7.00 (dd, J = 8.4, 1.9 Hz, 1H, H-6), 6.83 (d, J = 8.3 Hz, 1H,
H-5), 4.05 (m, 1H, H-8), 3.00 (m, 2H, H-7). 13C NMR (75
MHz, D2O, DMSO-d6) δ 172.62 (COOH), 153.82 (C-4),
135.45 (C-2), 131.59 (C-6), 128.96 (C-1), 118.34 (C-5),
111.24 (C-3), 55.50 (C-8), 35.94 (C-7). ½αŠ2D5=−12.2°. ESI-
Qtof-HRMS, m/z: calcd. for C9H11NO3Br [M + H]+ was
259.9922. Found 259.9915.
butoxycarbonyl)-amino) propanoate White solid, 1930
1
mg, two stages yield 65%. H NMR (300 MHz, CDCl3) δ
7.02 (s, 2H), 5.07 (d, 1H), 4.51 (m, 1H), 3.73 (s, 3H), 3.13–
2.98 (m, 1H), 2.90 (dd, 1H), and 1.42 (s, 9H).
Synthesis of compounds (1a–1d)
Intermediates NHBoc cleavage. (Int 1) (1615 mg, 4.31
mmol), (Int 2) (2740 mg, 6.04 mmol), (Int 3) (1565 mg,
4.74 mmol), and (Int 4) (1930 mg, 5.29 mmol) in separate
reactions were dissolved in DCM (3 mL) at 0 °C. After 5
min TFA (1000 µL) was added to the mixture and left to
react for 24 h at room temperature. Later the solvents and
reagents were evaporated, and the crude of the reaction was
adjusted to pH = 7. The product was tested by TLC mobile
phase Hexane–AcOEt (3:1) showing quantitative conver-
sion for (Int1–Int4) producing 1180 mg (1a), 2130 mg
(1b), 1080 mg (1c), and 1385 mg (1d).
(3)
(S)-2-amino-3-(3,5-dibromo-4-hydroxyphenyl)-propa-
noic acid White solid, 144 mg, yield 100%. 1H NMR
(300 MHz, D2O, DMSO-d6) δ 7.32 (s, 2H, H-2, H-6), 3.71–
3.68 (m, 1H, H-8), 2.99 (dd, 1H, H-7), and 2.83 (dd, 1H, H-
7). 13C NMR (75 MHz, D2O, DMSO-d6) δ 174.18 (COOH),
150.75 (C-4), 134.56 (C-2, C-6), 131.54 (C-1), 112.56 (C-3,
C-5), 56.74 (C-8), and 36.12 (C-7). ½αŠ2D5=−15.5°. ESI-
Qtof-HRMS, m/z: calcd. for C9H10NO3Br2 [M + H]+ was
337.9043. Found 337.9027.

Medicinal Chemistry Research
(4)
(S)-2-amino-3-(3-chloro-4-hydroxyphenyl)-propanoic
(9) (S)-2-amino-3-(3,5-dichloro-4-methoxyphenyl)-propa-
noic acid White solid, 142 mg. 1H NMR (300 MHz,
D2O, DMSO-d6) δ 7.20 (s, 2H, H-2, H-6), 3.74 (s, 4H, H-8,
OCH3), 3.02 (dd, 1H, H-7), 2.88 (dd, 1H, H-7). 13C NMR
(75 MHz, D2O, DMSO-d6) δ 174.36 (COOH), 152.09 (C-
4), 135.25 (C-1), 131.48 (C-2, C-6), 130.39 (C-3, C-5),
acid White solid, 140 mg, yield 100%. 1H NMR (300
MHz, D2O, DMSO-d6) δ 7.16 (d, J = 2.0 Hz, 1H, H-2),
6.95 (dd, J = 8.4, 2.1 Hz, 1H, H-6), 6.83 (d, J = 8.3 Hz, 1H,
H-5), 3.70 (dd, 1H, H-8), 3.00 (dd, 1H, H-7), 2.85 (dd, 1H,
H-7). 13C NMR (75 MHz, D2O, DMSO-d6) δ 174.51
(COOH), 152.46 (C-4), 132.33 (C-2), 130.78 (C-6), 129.64
(C-1), 121.77 (C-3), 118.57 (C-5), 57.21 (C-8), and 36.66
(C-7). ½αŠ2D5=−10.1°. ESI-Qtof-HRMS, m/z: calcd. for
C9H11NO3Cl [M + H]+ was 216.0427. Found 216.0438.
62.64 (C-8), 56.98 (OCH3), and 36.82 (C-7). ½αŠ25=−12.2°.
D
ESI-Qtof-HRMS, m/z: calcd. for C10H12NO3Cl2 [M + H]+
was 264.0194. Found 264.0199.
Synthesis of compounds (10a–10h)
(5) (S)-2-amino-3-(3,5-dichloro-4-hydroxyphenyl)-propanoic
acid White solid, 142 mg, yield 100%. 1H NMR (300
MHz, D2O, DMSO-d6) δ 7.12 (s, 2H, H-2, H-6), 3.70 (m,
1H, H-8), 3.01 (dd, 1H, H-7), and 2.84 (dd, 1H, H-7). 13C
NMR (75 MHz, D2O, DMSO-d6) δ 174.48 (COOH), 148.81
(C-4), 131.00 (C-2, C-6), 129.87 (C-1), 123.73 (C-3, C-5),
57.01 (C-8), 36.54 (C-7). ½αŠ2D5=−13.6°. ESI-Qtof-HRMS,
m/z: calcd. for C9H10NO3Cl2 [M + H]+ was 250.0038.
Found 250.0065.
The N,N-dimethylation was done by a reductive amination
using aqueous formaldehyde at 37% and NaCNBH3. 1
equivalent of compounds (1a–1h) were dissolved in MeOH
(15 mL), 2.2 equivalents of formaldehyde, and 1.5 equiva-
lents of NaCNBH3. The reactants were stirred for 18 h at
room temperature. Later the solvents were evaporated, the
crude of reaction was partitioned with AcOEt–H2O and
purified by column chromatography using as a mobile phase
AcOEt–Hexane (2:1). (10a): (1a) (500 mg, 1.82 mmol),
formaldehyde (300 µL, 4 mmol) and NaCNBH3 (172 mg,
2.73 mmol). White solid, 383 mg, yield 70%. (10b): (1b)
(500 mg, 1.41 mmol), formaldehyde (231 µL, 3.1 mmol)
and NaCNBH3 (133 mg, 2.11 mmol). White solid, 376 mg,
yield 70%. (10c): (1c) (500 mg, 2.17 mmol), formaldehyde
(355 µL, 4.8 mmol) and NaCNBH3 (133 mg, 2.04 mmol).
White solid, 390 mg, yield 70%. (10d): (1d) (500 mg, 1.89
mmol), formaldehyde (309 µL, 4.15 mmol) and NaCNBH3
(178 mg, 2.83 mmol). White solid, 386 mg, yield 70%.
(10e): (1e) (500 mg, 1.82 mmol), formaldehyde (300 µL, 4
mmol) and NaCNBH3 (172 mg, 2.73 mmol). White solid,
383 mg, yield 70%. (10f): (1f) (500 mg, 1.41 mmol), for-
maldehyde (231 µL, 3.1 mmol) and NaCNBH3 (133 mg,
2.11 mmol). White solid, 376 mg, yield 70%. (10g): (1g)
(500 mg, 2.17 mmol), formaldehyde (355 µL, 4.8 mmol)
and NaCNBH3 (133 mg, 2.04 mmol). White solid, 390 mg,
yield 70%. (10h): (1h) (500 mg, 1.89 mmol), formaldehyde
(309 µL, 4.15 mmol) and NaCNBH3 (178 mg, 2.83 mmol).
White solid, 386 mg, yield 70%.
(6) (S)-2-amino-3-(3-bromo-4-methoxyphenyl)-propanoic
acid White solid, 142 mg. 1H NMR (300 MHz, D2O,
DMSO-d6) δ 7.56 (d, J = 2.1 Hz, 1H, H-2), 7.29 (dd, J =
8.5 Hz, 2.1 Hz, 1H, H-6), 7.11 (d, J = 8.5 Hz, 1H, H-5),
4.06–3.84 (m, 4H, H-8, OCH3), 3.23 (dd, 1H, H-7), 3.07
(dd, 1H, H-7). 13C NMR (75 MHz, D2O, DMSO-d6) δ
164.63 (COOH), 146.03 (C-4), 125.30 (C-2), 121.43 (C-6),
120.57 (C-1), 104.57 (C-5), 102.60 (C-3), 47.52 (OCH3),
47.35 (C-8), and 26.64 (C-7). ½αŠ25 = −8.4°. ESI-Qtof-
D
HRMS, m/z: calcd. for C10H13NO3Br [M + H]+ was
274.0079. Found 274.0096.
(7) (S)-2-amino-3-(3,5-dibromo-4-methoxyphenyl)-propa-
noic acid White solid, 144 mg. 1H NMR (300 MHz,
D2O, DMSO-d6) δ 7.59 (s, 2H, H-2, H-6), 3.97–3.80 (m,
4H, H-8, OCH3), 3.23 (dd, 1H, H-7), 3.07 (dd, 1H, H-7).
13C NMR (75 MHz, D2O, DMSO-d6) δ 164.20 (COOH),
144.24 (C-4), 126.41 (C-2, C-6), 125.17 (C-1), 109.32 (C-3,
25
D
C-5), 52.11 (C-8), 47.14 (OCH3), and 26.65 (C-7). ½αŠ
=
−11.1°. ESI-Qtof-HRMS, m/z: calcd. for C10H12NO3Br2
[M + H]+ was 351.9184. Found 351.9227.
Tertiary amines synthesis
(8)
(S)-2-amino-3-(3-chloro-4-methoxyphenyl)-propanoic
Synthesis of (11–18) compounds
acid White solid, 141 mg. 1H NMR (300 MHz, D2O,
DMSO-d6) δ 7.20 (s, 1H, H-2), 7.06 (d, J = 8.5 Hz, 1H, H-
6), 6.96 (d, J = 8.4 Hz, 1H, H-5), 3.73 (s, 4H, H-8, OCH3),
3.02 (dd, 1H, H-7), 2.87 (dd, 1H, H-7). 13C NMR (75 MHz,
D2O, DMSO-d6) δ 174.76 (COOH), 155.19 (C-4), 132.35
(C-2), 130.89 (C-6), 130.23 (C-1), 123.11 (C-3), 114.77 (C-
150 mg of compounds (10a–10h) were dissolved in a
mixture of MeOH–H2O–THF (1:1:1) (5 mL) and then
K2CO3 (100 mg) was added and the mixture was stirred for
24 h at room temperature. The crude of reaction was
adjusted to pH = 6. The compounds were tested by TLC
mobile phase nBuOH–AcOH–H2O (4:1:1) showing quan-
titative conversion. The final products (12–18) were purified
5), 57.69 (OCH3), 57.28 (C-8), 36.71 (C-7). ½αŠ25=−9.4°.
D
ESI-Qtof-HRMS, m/z: calcd. for C10H13NO3Cl [M + H]+
was 230.0584. Found 230.0594.

Medicinal Chemistry Research
with RP-HPLC using as a mobile phase a mixture of MeOH
(0.01% F.A.) and H2O (0.01% F.A.).
= 8.5 Hz, 1H, H-5), 3.90 (s, 3H, OCH3), 3.78 (dd, 1H, H-8),
3.25 (dd, 1H, H-7), 3.10 (dd, 1H, H-7), 2.93 (s, 6H, N(CH3)
2). 13C NMR (75 MHz, D2O, DMSO-d6) δ 163.05 (COOH),
145.86 (C-4), 125.11 (C-2), 121.23 (C-6), 120.89 (C-1),
104.37 (C-5), 102.46 (C-3), 63.38 (C-8), 47.57 (OCH3),
(11) (S)-3-(3-bromo-4-hydroxyphenyl)-2-(dimethylamino)-
propanoic acid White solid, 108 mg, yield 75%. 1H
NMR (300 MHz, D2O, DMSO-d6) δ 7.34 (d, J = 1.8 Hz,
1H, H-2), 7.01 (dd, J = 8.4, 1.8 Hz, 1H, H-6), 6.81 (d, J =
8.3 Hz, 1H, H-5), 3.91 (m, 1H, H-8), 3.22-3.07 (m, 1H, H-
7), 2.92 (dd, 1H, H-7), 2.80 (s, 6H, N(CH3)2). 13C NMR
(75 MHz, D2O, DMSO-d6) δ 171.41 (COOH), 153.65 (C-
4), 135.30 (C-2), 131.45 (C-6), 129.14 (C-1), 118.20 (C-5),
111.05 (C-3), 71.00 (C-8), 42.91 (N(CH3)2), and 33.52 (C-
32.11 (N(CH3)2), and 24.00 (C-7). ½αŠ25=53.8°. ESI-Qtof-
D
HRMS, m/z: calcd. for C12H17NO3Br [M + H]+ was
302.0392. Found 302.0398.
(16)
(S)-3-(3,5-dibromo-4-methoxyphenyl)-2-(dimethyla-
mino)-propanoic acid White solid, 123 mg, yield 85%.
1H NMR (300 MHz, D2O, DMSO-d6) δ 7.57 (s, 2H, H-2,
H-7), 3.90 (s, 3H, OCH3), 3.82 (dd, 1H, H-8), 3.28 (d, 1H,
H-7), 3.06 (dd, 1H, H-7), 2.95 (s, 6H, N(CH3)2). 13C NMR
(75 MHz, D2O, DMSO-d6) δ 163.23 (COOH), 143.01 (C-
4), 125.80 (C-2, C-6), 124.46 (C-1), 108.54 (C-3, C-5),
63.16 (C-8), 51.93 (OCH3), 33.03 (N(CH3)2), and 23.48 (C-
7). ½αŠ2D5=61.0°. ESI-Qtof-HRMS, m/z: calcd. for
C12H16NO3Br2 [M + H]+ was 379.9497. Found 379.9490.
7). ½αŠ25 = 60.3°. ESI-Qtof-HRMS, m/z: calcd. for
D
C11H15NO3Br [M + H]+ was 288.0235. Found 288.0243.
(12)
(S)-3-(3,5-dibromo-4-hydroxyphenyl)-2-(dimethyla-
mino)-propanoic acid White solid, 110 mg, yield 75%.
1H NMR (300 MHz, D2O, DMSO-d6) δ 7.38 (s, 2H, H-2,
H-6), 4.09–3.93 (m, 1H, H-8), 3.21 (dd, 1H, H-7), 2.92 (dd,
1H, H-7), 2.82 (s, 6H, N(CH3)2). 13C NMR (75 MHz, D2O,
DMSO-d6) δ 170.79 (COOH), 150.63 (C-4), 134.73 (C-2,
C-6), 130.60 (C-1), 112.70 (C-3, C-5), 70.01 (C-8), 42.91
(17) (S)-3-(3-chloro-4-methoxyphenyl)-2-(dimethylamino)-
propanoic acid White solid, 120 mg, yield 85%. 1H
NMR (300 MHz, D2O, DMSO-d6) δ 7.20 (s, 1H, H-2), 7.05
(s, 1H, H-6), 6.94 (s, 1H, H-5), 3.72 (s, 3H, OCH3), 3.64-
3.48 (m, 1H, H-8), 3.07 (dd, 1H, H-7), 2.94–2.80 (m, 1H,
H-7), and 2.73 (s, 6H, N(CH3)2). 13C NMR (75 MHz, D2O,
DMSO-d6) δ 173.04 (COOH), 154.61 (C-4), 132.43 (C-2),
130.40 (C-1), 130.32 (C-6), 122.66 (C-3), 114.58 (C-5),
73.75 (C-8), 57.65 (OCH3), 40.25 (N(CH3)2), and 34.56 (C-
7). ½αŠ2D5=49.8°. ESI-Qtof-HRMS, m/z: calcd. for
C12H17NO3Cl [M + H]+ was 258.0897. Found 258.0898.
(N(CH3)2), 32.82 (C-7). ½αŠ25 = 64.8°. ESI-Qtof-HRMS, m/
D
z: calcd. for C11H14NO3Br2 [M + H]+ was 365.9340. Found
365.9350.
(13) (S)-3-(3-chloro-4-hydroxyphenyl)-2-(dimethylamino)-
propanoic acid White solid, 106 mg, yield 75%. 1H
NMR (300 MHz, D2O, DMSO-d6) δ 7.16 (s, 1H, H-2), 6.90
(dd, 1H, H-6), 6.81 (d, 1H, H-5), 3.81 (m, 1H, H-8), 3.11
(dd, 1H, H-7), 2.89 (dd, 1H, H-7), 2.78 (s, 6H, N(CH3)2).
13C NMR (75 MHz, D2O, DMSO-d6) δ 171.60 (COOH),
152.45 (C-4), 131.90 (C-2), 130.68 (C-6), 128.70 (C-1),
121.51 (C-3), 118.30 (C-5), 71.60 (C-8), 43.56 (N(CH3)2),
(18)
(S)-3-(3,5-dichloro-4-methoxyphenyl)-2-(dimethyla-
mino)-propanoic acid White solid, 121 mg, yield 85%.
1H NMR (300 MHz, D2O, DMSO-d6) δ 7.20 (s, 2H, H-2,
H-6), 3.73 (s, 3H, OCH3), 3.59 (dd, 1H, H-8), 3.09 (dd, 1H,
H-7), 2.86 (dd, 1H, H-7), 2.74 (s, 6H, N(CH3)2). 13C NMR
(75 MHz, D2O, DMSO-d6) δ 172.59 (COOH), 151.90 (C-
4), 135.35 (C-1), 131.32 (C-2, C-6), 130.21 (C-3, C-5),
73.03 (C-8), 62.46 (OCH3), 44.15 (N(CH3)2), and 34.21 (C-
and 33.76 (C-7). ½αŠ25 = 54.9°. ESI-Qtof-HRMS, m/z: calcd
D
for C11H15NO3Cl [M + H]+ was 244.0740. Found
244.0760.
(14)
(S)-3-(3,5-dichloro-4-hydroxyphenyl)-2-(dimethyla-
mino)-propanoic acid White solid, 106 mg, yield 75%.
1H NMR (300 MHz, D2O, DMSO-d6) δ 7.14 (s,2H, H-2, H-
6), 4.02 (m,1H, H-8), 3.17 (dd,1H, H-7), 2.94 (dd,1H, H-7),
2.82 (s,6H, N(CH3)2). 13C NMR (75 MHz, D2O, DMSO-d6)
δ 170.64 (CCOH), 149.23 (C-4), 131.31 (C-2, C-6), 129.16
(C-1), 123.18 (C-3, C-5), 69.83 (C-8), 43.59 (N(CH3)2), and
7). ½αŠ25 = 53.5°. EM-ESI m/z 292.6 [M]+.
D
Quaternary amines synthesis
Synthesis of (20–25) compounds
33.52 (C-7). ½αŠ25 = 59.1°. ESI-Qtof-HRMS, m/z: calcd. for
D
C11H14NO3Cl2 [M + H]+ was 278.0351. Found 278.0352.
The quaternization of amine group was done using 150 mg
of (3c–3h), methyl iodide (350 µL), and acetone (3 mL) at
room temperature by 18 h. The solid were filtered, washed
with acetone and dried. Later the solids were dissolved in a
mixture of MeOH–H2O–THF (1:1:1) (5 mL) and then
K2CO3 was added (100 mg) and the mixture was stirred for
(15) (S)-3-(3-bromo-4-methoxyphenyl)-2-(dimethylamino)-
propanoic acid White solid, 121 mg, yield 85%. 1H
NMR (300 MHz, D2O, DMSO-d6) δ 7.56 (d, J = 2.0 Hz,
1H, H-2), 7.30 (dd, J = 8.5 Hz, 2.1 Hz, 1H, H-6), 7.08 (d, J

Medicinal Chemistry Research
24 h at room temperature. The crude of reaction was
adjusted to pH = 6. The compounds were tested by TLC
mobile phase nBuOH–AcOH–H2O (4:1:1) showing quan-
titative conversion. The final products were purified with
RP-HPLC using as a mobile phase a mixture of MeOH
(0.01% F.A.) and H2O (0.01% F.A.).
(24) (S)-1-carboxy-2-(3-chloro-4-methoxyphenyl)-N,N,N-tri-
methylethan-1-aminium White solid, 173 mg, yield
100%. 1H NMR (300 MHz, D2O, DMSO-d6) δ 7.21 (s, 1H,
H-2), 7.06 (d, J = 8.5 Hz, 1H, H-6), 6.95 (d, J = 8.5 Hz, 1H,
H-5), 3.71 (s, 4H, OCH3, H-8), 3.13 (s, 10H, N(CH3)3, H-
7), 2.93 (dd, 1H, H-7). 13C NMR (75 MHz, D2O, DMSO-
d6) δ 171.51 (COOH), 155.23 (C-5), 132.44 (C-2), 131.00
(C-6), 129.58 (C-1), 123.06 (C-3), 114.79 (C-5), 80.81 (C-
(20) (S)-1-carboxy-2-(3-chloro-4-hydroxyphenyl)-N,N,N-tri-
methylethan-1-aminium White solid, 170 mg, yield
100%. 1H NMR (300 MHz, D2O, DMSO-d6) δ 7.17 (d, J =
1.9 Hz, 1H, H-2), 6.97 (dd, J = 8.4, 1.9 Hz, 1H, H-6), 6.84
(d, J = 8.4 Hz, 1H, H-5), 4.04 (m, 1H, H-8), 3.23 (m, 1H,
H-7), 3.18 (s, 9H, N(CH3)3), 2.94 (m, 1H, H-7). 13C NMR
(75 MHz, D2O, DMSO-d6) δ 169.90 (COOH), 152.52 (C-
4), 132.35 (C-2), 130.90 (C-6), 127.40 (C-1), 121.51 (C-3),
118.44 (C-5), 77.63 (C-8), 53.77 (N(CH3)3), and 32.48 (C-
7). ½αŠ2D5= −47°2. ESI-Qtof-HRMS, m/z: calcd. for
C12H17NO3Cl [M]+ was 258.0901. Found 258.0897.
8), 57.8 (OCH3), 53.73 (N(CH3)3), and 33.06 (C-7). ½αŠ25=
D
−51.9°. ESI-Qtof-HRMS, m/z: calcd. for C13H19NO3Cl [M]
+
was 272.1053. Found 272.1057.
(25) (S)-1-carboxy-2-(3,5-dichloro-4-methoxyphenyl)-N,N,N-
trimethylethan-1-aminium White solid, 170 mg, yield
100%. 1H NMR (300 MHz, D2O, DMSO-d6) δ 7.15 (s, 2H,
H-2, H-6), 3.70 (s, 3H, OCH3), 3.61 (m, 1H, H-8), 3.09 (s,
10H, N(CH3)3, H-7), 2.90 (dd, 1H, H-7). 13C NMR (75
MHz, D2O, DMSO-d6) δ 171.08 (COOH), 151.96 (C-4),
134.53 (C-1), 131.38 (C-2, C-6), 130.18 (C-3, C-5), 80.74
(C-8), 62.3 (OCH3), 53.53 (N(CH3)3), and 33.08 (C-7).
½αŠ2D5=−54.4°. ESI-Qtof-HRMS, m/z: calcd. for
C13H19NO3Cl2 [M]+ was 292.0507. Found 292.0519.
Compounds were stored at room temperature and prior to
the biological evaluations; they were solubilized in 0.5%
DMSO and then, diluted to the appropriate concentration in
culture media.
(21) (S)-1-carboxy-2-(3,5-dichloro-4-hydroxyphenyl)-N,N,N-
trimethylethan-1-aminium White solid, 172 mg, yield
100%. 1H NMR (300 MHz, D2O, DMSO-d6) δ 7.15 (s, 2H,
H-2, H-6), 3.97 (m, 1H, H-8), 3.23 (dd, 1H, H-7), 3.14 (s,
9H, N(CH3)3), 2.95 (dd, 1H, H-7). 13C NMR (75 MHz,
D2O, DMSO-d6) δ 169.9, 140.05 (C-4), 131.09 (C-2, C-6),
128.48 (C-1), 123.95 (C-3, C-5), 77.70 (C-8), 53.9 (N(CH3)
3), and 32.46 (C-7). ½αŠ25=−53°1. ESI-Qtof-HRMS, m/z:
D
calcd. for C12H16NO3Cl2 [M]+ was 292.0508. Found
292.0507.
Biological activity assays
In vitro culture of human promonocytic cell line U-937
(ATCC CRL-1593.2)
(22) (S) 1-carboxy-2-(3-bromo-4-methoxyphenyl)-N,N,N-tri-
methylethan-1-aminium White solid, 173 mg, yield
100%. 1H NMR (300 MHz, D2O, DMSO-d6) δ 7.51 (d, J =
2.0 Hz, 1H, H-2), 7.26 (dd, J = 8.5, 2.1 Hz, 1 H, H-6), 7.04
(d, J = 8.5 Hz, 1H, H-5), 3.87 (s, 3H, OCH3), 3.80 (m, 1H,
H-8), 3.28 (s, 9H, N(CH3)3), 3.24 (dd, 1H, H-7), 3.08 (dd,
1H, H-7). 13C NMR (75 MHz, D2O, DMSO-d6) δ 161.8
(COOH), 146.45 (C-4), 125.51 (C-2), 121.67 (C-6), 120.28
(C-1), 104.63 (C-5), 102.62 (C-3), 71.58 (C-8), 47.89
Cells were cultured under standard conditions in RPMI
1640 medium enriched with 10% fetal bovine serum (FBS)
and 1% penicillin–streptomycin (compkete medium) and
incubation at 37 °C with 5% CO2.
In vitro culture of T. cruzi
(OCH3), 43.77 (N(CH3)3), and 23.29 (C-7). ½αŠ25=−53.7°.
Epimastigotes of T. cruzi (Tulauen strain), transfected with
β-galactosidase gene, were maintained in vitro in biphasic
NNN medium and incubation at 26 °C.
D
ESI-Qtof-HRMS, m/z: calcd for C13H19NO3Br [M]+ was
316.0547. Found 316.0458.
(23) (S)-1-carboxy-2-(3,5-dibromo-4-methoxyphenyl)-N,N,N-
trimethylethan-1-aminium White solid, 168 mg, yield
100%. 1H NMR (300 MHz, D2O, DMSO-d6) δ 7.59 (s, 2H,
H-2, H-6), 3.93 (s, 3H, OCH3), 3.85 (m, 1H, H-8), 3.33 (s,
10H, N(CH3)3, H-7), 3.11 (dd, 1H, H-7). 13C NMR (75
MHz, D2O, DMSO-d6) δ 170.98 (COOH), 153.89 (C-4),
135.65 (C-2, C-6), 135.10 (C-1), 119.06 (C-3, C-5), 80.76
(C-8), 62.3 (OCH3), 53.54 (N(CH3)3), and 32.84 (C-7).
Antitrypanosomal activity assay
The in vitro antitrypanosomal activity was evaluated against
the T. cruzi (Tulauen strain) transfected with the β-galac-
tosidase gene as described elsewhere (Buckner et al. 1996).
The effectiveness of each compound was determined
according to the reduction of the amastigotes of T. cruzi in
the infected cells. Briefly, 100 µL of U-937 human cells at a
concentration of 2.5 × 105 cells/mL in RPMI-1640, 10%
SFB were adhered to the bottom of each well of 96 wells
½αŠ2D5=−59.3°.
ESI-Qtof-HRMS,
m/z:
calcd
for
C13H19NO3Br2 [M]+ was 393.9653. Found 393.9701.

Medicinal Chemistry Research
tissue culture microplate using 0.1 µg/mL of PMA. After
2 days in culture, adhered cells were infected with epi-
mastigotes (3-day growth) in 5:1 parasites:cell proportion.
Twenty-four hours later, after infection, each compound
was added to infected cells at various concentrations (100–
25–6.25–1.56 µg/mL). BZN was used as a control of the
trypanocidal drug. Cells were exposed to compounds during
72 h under incubation at 37 °C, 5% CO2. The effect of
compounds in the viability of intracellular parasites was
determined measuring the β-galactosidase activity by col-
orimetric assay using 100 µM CPRG substrate and 0.1%
nonidet P40. After 3 h of incubation, the amount of obtained
product was measured after reading of each well at 570 nm
in a spectrophotometer (Varioskan, Thermo). The absor-
bance was registered as optical densities (OD). Infected
cells incubated in the complete compound-free RPMI-1640
medium were used as a control of infection (negative con-
trol). Non-specific absorbance was corrected by subtracting
absorbance (OD) of the blank. Determinations were done in
triplicate in at least two independent experiments (Ramírez–
Prada et al. 2017).
to 100% of infection
%Infection ¼ ðA=BÞx100
ð1Þ
ð2Þ
%Reduction ¼ 100 À %Infection
Percentages of reduction of parasites data were used to
calculate the EC50 using the linear regression model Probit.
The activity of each compound was established accord-
ing to EC50 values, using the following scale, active: EC50
< 50 µM, moderately active: EC50 in the 50–90 µM range
and low activity: EC50 > 90 µM.
In turn, cytotoxicity was determined according to cell
growth reduction obtained for each compound and each
concentration. First, viability percentages were calculated
using Eq. (3) and formula (4). The optical density of
exposed cells C, the optical density of non-exposed cells D.
The optical density of control corresponds to 100% of
viability.
%Viability ¼ ðC=DÞx100
ð3Þ
ð4Þ
Cytotoxic activity in mammalian cells
%Reduction ¼ 100 À %Viability
Cytotoxic activity of compounds was assessed based on the
viability of U-937 cells determined by the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)
method. Shortly, 100,000 cells/mL in complete RPMI-1640
and the corresponding concentration of each compound
(200–50–12.5–3.125 µg/mL) were incubated at 37 °C, 5%
CO2 during 72 h. The effect of concentration of each
compound was determined by adding 10 µL/well of MTT
solution (0.5 mg/mL) and incubated at 37 °C for 3 h. The
reaction was stopped with 100 µL of 50% isopropanol
solution with 10% sodium dodecyl sulfate (SDS) and
incubated for 30 min. Cell viability was determined based
on the quantity of formazan produced, which was measured
with a Bio-Rad ELISA reader set at 570 nm. As a viability
control, cultured cells in the absence of extracts were used.
Doxorubicn was used as a cytotoxicity control. Nonspecific
absorbance was corrected by subtracting absorbance (OD)
of the blank. Determinations were done in triplicate in at
least two independent experiments (Mesa et al. 2017).
Percentages of reduction of viability data were used to
calculate the LC50 using the linear regression model Probit
(Finney 1971).
The toxicity was defined according to LC50 values, using
the following scale, high cytotoxicity: LC50 < 200 µM,
moderate cytotoxicity: LC50 in the 200–300 µM range and
potentially non-cytotoxicity: LC50 > 300 µM.
Acknowledgements The authors would like to thank the Colombian
Department of Science, Technology and Innovation—Colciencias
(Grant CT-695-2014) and to University of Antioquia (CODI CIQF-
176, CIQF-155 and estrategia de sostenibilidad 2014-2015) for their
financial support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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