Anti-parasite and cytotoxic activities of chloro and bromo L-tyrosine derivatives

Article abstract of DOI:10.21577/0103-5053.20180136

A series of twenty-one L-tyrosine derivatives with modifications in the halogenation pattern of the aromatic ring and different degree of methylations on the amine and phenolic hydroxyl groups were synthesized. The structures of all the intermediates and target compounds were confirmed unambiguous by spectroscopy analysis. Additionally, all compounds were evaluated against Plasmodium falciparum and Leishmania panamensis parasites between 20-702 μg mL^-1. The cytotoxic evaluation was done to determine the selectivity index for each compound. Six compounds had the lower EC50 (effective concentration 50) against L. panamensis. One of these compounds was the most active with an EC50 at 24.13 μg mL^-1 (76.07 μM). All derivatives showed no significant activity against P. falciparum and no compound has in vitro antifungal activity at 500 μg mL^-1.

Full text of DOI:10.21577/0103-5053.20180136

http://dx.doi.org/10.21577/0103-5053.20180136
J. Braz. Chem. Soc., Vol. 29, No. 12, 2569-2579, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives
Manuel Pastrana Restrepo,a Elkin Galeano Jaramillo,*,a Alejandro Martínez Martínez,a
Ana Mesa Arangob and Sara Robledo Restrepoc
aProductos Naturales Marinos, Departamento de Farmacia,
Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia (UdeA),
Calle 70 No. 52-21, Laboratorio 2-131, 050010 Medellín, Colombia
bGrupo de Investigación Dermatológica, Facultad de Medicina, Universidad de Antioquia (UdeA),
050010 Medellín, Colombia
cPrograma de Estudio y Control de Enfermedades Tropicales (PECET),
Facultad de Medicina, Universidad de Antioquia (UdeA), Calle 62 No. 52-59,
Laboratorio 632, 050010 Medellín, Colombia
A series of twenty-one L-tyrosine derivatives with modifications in the halogenation pattern
of the aromatic ring and different degree of methylations on the amine and phenolic hydroxyl
groups were synthesized. The structures of all the intermediates and target compounds were
confirmed unambiguous by spectroscopy analysis. Additionally, all compounds were evaluated
against Plasmodium falciparum and Leishmania panamensis parasites between 20-702 µg mL-1.
The cytotoxic evaluation was done to determine the selectivity index for each compound. Six
compounds had the lower EC50 (effective concentration 50) against L. panamensis. One of these
compounds was the most active with an EC50 at 24.13 µg mL-1 (76.07 µM). All derivatives showed
no significant activity against P. falciparum and no compound has in vitro antifungal activity at
500 µg mL-1.
Keywords: L. panamensis, P. falciparum, cytotoxic, L-tyrosine
Introduction
implications and almost half of the world population is at
risk of infection, combating malaria becomes one of the
priorities of WHO programs.
Diseases caused by protozoan parasites affect about
one billion people, with a notable incidence in tropical
countries. Diseases such as malaria and leishmaniasis are
more frequent in developing countries of tropical and sub-
tropical regions. These diseases are endemic problems in
Colombia and other Latin American countries.
Currently, malaria is still a common cause of death with
approximately 445,000 cases reported in 2016 in tropical
countries of Africa, Asia and America; and tragically most
of the victims are children under the age of five.1 Despite
the decrease in fatal cases recorded by the World Health
Organization (WHO) in recent years, an increase in the
spread of mosquitoes resistant to common insecticides
is observed, and more importantly, the emergence
of resistance to current drugs by different strains of
Plasmodium.2 Given that malaria is a disease with global
On the other hand, leishmaniasis disease affect more than
10 million people worldwide, principally tropical countries.
Protozoan parasites of the genus Leishmania responsible
for the infection are: L. panamensis, L. braziliensis,
L. guyanensis, L. mexicana and L. amazonensis.3 Currently,
chemotherapies to treat cutaneous leishmaniasis are based
on old drugs; unfortunately, all of these drugs have severe
toxic effects on patients. Side effects are associated with high
therapeutic doses used and long term treatment schemes.4
Compounds isolated from marine organisms are considered
as rich sources for drug discovery. Marine sponges of
Verongida order has a great diversity of bromotyrosine
compounds, it has shown anti-parasite activities against
T. cruzi, P. falciparum, and L. panamensis protozoa.5-8 We
reported isolations and identifications of more than sixteen
bromotyrosine isolated from sponges Verongula rigida and
Aiolochroia crassa with antiparasitic activity, and we found
*e-mail: elkin.galeano@udea.edu.co

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Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives
J. Braz. Chem. Soc.
a close relationship between the degree of bromination in
the aromatic ring, the methylation in the amino group and
their influence in the antiparasitic activity.9
and N-chlorosuccinimide (NCS),12 producing intermediates
(Int1-Int4). These intermediates were divided into two sets,
one of them were treated with trifluoracetic acid (TFA)
in dichloromethane (DCM),13 to cleavage the amino
group (1a-1d). The other intermediates were O-methylated
with MeI in basic media,14 and later treated with TFA in
DCM (2a-2d) (Scheme 1).
Due to the emerging resistance against current drugs,
it is mandatory to develop new, safer and more effective
antiparasitic agents. For that reason, in the present study,
the potentials of marine natural products derivatives
compounds, based on bioactive L-bromotyrosines, were
evaluated as new antiparasitic compounds. The syntheses
of novel chloro and bromo L-tyrosine derivatives, with
different grade of methylation of the amino group, lead
us to realize a structure-activity relationship between the
halogenations patterns in the aromatic ring of the L-tyrosine
and the substitution of the amino group, looking for the
best structural parameters for the development of new
antiparasitic compounds.
The 1H nuclear magnetic resonance (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 carbamate moiety. The mono halogenated
compounds show in the aromatic region of the spectra three
signals that corresponds to the protons at 2, 5, 6 positions in
the aromatic ring, 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 position.
The syntheses of primary amines were done by
the hydrolysis of compounds 1a-1d, 2a-2d with
K2CO3/MeOH,15 getting compounds 4-11. The syntheses
of methyl ester tertiary amines were done by the reductive
amination of compounds 1a-1d, 2a-2d with formaldehyde/
NaCNBH3, getting compounds 3a-3h.16 These set of
compounds were divided in two, one of them were treated
with K2CO3/MeOH to get the tertiary amines 12-18. The
other set was treated with MeI/acetone to get the methyl ester
quaternary amines,17 which were treated with K2CO3/MeOH
to get the quaternary amines 19-24. The synthesis of the
primary, tertiary and quaternary amines are shown in
Scheme 2. The 1H NMR analyses of tertiary and quaternary
amines show the presence of a signal at 2.88 ppm (s, 6H) for
the di-methylation of the amino group in the tertiary amines,
and 2.97 ppm (s, 9H) for the tri-methylation of the amino
Results and Discussion
Chemistry
Synthesis of bromo and chloro N-substituted derivatives
was made using as starting material L-tyrosine. For
the synthesis of 21 products, it was 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 formed by the reaction of L-tyrosine with
SOCl2/MeOH,10 later the protection of the amino group
was done by the reaction with Boc2O/triethylamine (TEA)
at room temperature.11 The bromination and chlorination
of the aromatic ring, getting mono and di halogenated
compounds, were done using N-bromosuccinimide (NBS)
Scheme 1. Intermediate synthesis from L-tyrosine. (a) SOCl2, MEOH; (b) NBS or NCS, MeOH; (c) Boc2O, TEA, MeOH; (d) MeI, K2CO3, acetone;
(e) TFA, DCM.

Vol. 29, No. 12, 2018
Restrepo et al.
2571
group in the quaternary amines. O-Methyl derivatives were
Compounds 4, 5, 6, 10, 21 and 24 were the most active
against L. panamensis with values lower than 50 µg mL-1.
Derivative 21 were the most active with EC50 of 76.07 µM.
Compounds 5 and 6 have the higher SI with values of 7.43
and 7.86, respectively. The twenty-one derivatives have no
activity against P. falciparum.
In this study, it is possible to establish that the type of
halogenation, in addition to the degree of methylation on
the amino group, have influence in the anti-leishmanial
activity of the compounds. Brominated compounds
with O-methyl substitution in para and primary amine
(such as compounds 8 and 9) were less active than their
correlated structure with free hydroxyl and additional
amine substitutions, compounds 4 and 5. In general,
tertiary and quaternary amines derivatives are less cytotoxic
than primary analogs. Mono-chloro compound 6 with
O-methyl substitution in para and primary amine is the
most promising derivative against L. panamensis.
confirmed by 1H NMR by the signal at 3.80 ppm (s, 3H).
Biological activity
Compounds were tested against P. falciparum (NF54
chloroquine sensitive strain) and L. panamensis (MHOM/
CO/87/UA140epir GP strain) using chloroquine and
amphotericin B as positive control drugs, respectively. A
cytotoxic test was done over human promonocytic cells
(U-937 ATCC CRL1593.2). For every active compound,
effective concentration 50 (EC50) and lethal concentration
50 (LC50), in µM units, were tested and selectivity index
(SI) was represented as LC50/EC50. In Table 1, it is shown
the anti-leishmanial and antiplasmodial activity of the
21 derivatives.
Compounds 12, 16, 18, 19, 20 and 22 were the most
cytotoxic, while compounds 5, 6, 7, 8, 9, 10, 15, 13, 14, 17
and 23 showed LC50 values higher than 200 µM, making
them potentially safe against mammalian cells U-937.
Compounds were evaluated at a final concentration
of 500 µg mL-1 against nineteen fungal strains of medical
Scheme 2. Synthesis of chloro and bromo L-tyrosine derivatives. (a) CH2O, NaCNBH3, MeOH; (b) MeI, acetone; (c) K2CO3, MeOH, THF, H2O.

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Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives
J. Braz. Chem. Soc.
Table 1. Cytotoxicity, anti-leishmanial and antiplasmodial activities of the L-tyrosine derivatives
U-937
L. panamensis
LC50b / (µg mL-1) [µM]
P. falciparum
Compound
LC50a / (µg mL-1) [µM]
> 40 [153.79]
SIc
> 1.29
7.43
LC50b / (µg mL-1) [µM]
> 20 [76.89]
SIc
4
30.84 1.06 [118.57 4.07]
35.73 5.22 [105.40 15.39]
30.83 1.42 [142.97 6.58]
> 0.97
7.51
5
265.50 7.5 [783.23 22.12]
242.23 8.3 [1123.35 38.49]
> 200 [799.77]
> 20 [59.00]
6
7.86
> 20 [92.20]
8.32
7
281.01 46.20 [1123.72 184.74] > 0.71
270.53 5.71 [1081.81 22.83]
> 20 [69.41]
> 4.59
< 0.5
1.97
12
> 10 [34.70]
69.09 8.57 [239.78 29.74]
101.9 13.10 [418.17 53.75]
185.34 36.86 [666.37 132.52]
157.96 24.35 [610.54 94.11]
75.67 8.57 [258.11 29.23]
82.25 15.46 [300.06 56.40]
138.59 36.23 [392.59 102.63]
35.39 1.74 [154.09 7.57]
> 0.14
1.75
0.77
0.24
0.27
1.99
2.58
5.67
> 0.25
3.68
0.36
13
176.8 26.8 [725.54 109.98]
143.2 15.97 [514.86 57.41]
38.53 1.15 [148.92 4.44]
20.49 1.69 [69.89 5.76]
164.03 12.19 [598.40 44.47]
358.00 5.6 [1014.13 15.86]
200.85 10.0 [874.55 43.54]
> 40 [151.45]
89.35 9.70 [366.66 39.80]
> 20 [71.90]
14
< 7.16
< 1.92
> 1.01
4.08
19
> 20 [77.30]
20
> 20 [68.22]
8
> 20 [72.96]
9
> 20 [56.65]
12.07
5.85
10
> 20 [87.08]
11
158.42 34.99 [599.84 132.48]
73.40 11.20 [242.91 37.06]
77.39 10.61 [203.09 27.84]
> 20 [75.72]
0.73
15
270.23 7.40 [894.32 24.49]
27.90 1.69 [73.21 4.43]
> 200 [776.06]
> 20 [66.19]
8.60
16
702.01 140.4 [1842.25 368.44]
> 20 [77.60]
0.04
17
534.31 77.33 [2073.29 300.06] > 0.37
< 10
18
49.16 4.93 [168.26 16.87]
> 40 [126.10]
150.97 7.98 [516.73 27.31]
24.13 2.50 [76.07 7.88]
132.22 17.27 [333.81 43.06]
148.72 17.80 [545.26 65.26]
28.35 1.40 [92.29 57.95]
0.06 0.01 [0.0.6 0.01]
NA
0.32
> 1.65
0.29
> 20 [68.45]
< 2.45
> 1.07
< 1.95
< 10
21
> 20 [63.05]
22
39.16 1.48 [98.86 3.73]
> 200 [733.27]
> 20 [50.49]
23
> 1.34
> 7.0
625
> 20 [73.32]
24
51.28 6.85 [651.12 7.56]
37.5 0.9 [40.58 0.97]
152.2 5.2 [475.81 16.25]
> 20 [65.11]
< 10
Amphotericin B
NA
NA
Chloroquine
NA
0.84 0.13 [2.62 0.40]
181
aLC50: lethal concentration 50; bEC50: effective concentration 50; cSI: selectivity index = LC50 / EC50. Data represent mean value standard deviation.
interest. No compound showed anti-fungal activity at this
concentration.
23 and 24 against some fungal strains and P. falciparum
parasite (3D7 strain).
Conclusions
Experimental
The synthesis, cytotoxicity, and activity of twenty-one
L-tyrosine derivatives against L. panamnesis, P. falciparum
and 19 fungal strains of medical importance are reported.
Compounds 4, 5, 6, 10, 21 and 24 were the most active
against L. panamensis strain tested. Additionally,
compounds 5, 6 and 10 have the higher SI, they were 4
and 6-fold more active than cytotoxic.
Structural simplicity of the compounds, in addition to
the high yields in obtaining them, is an advantage for their
production to in vivo anti-leishmanial evaluation. This is
the first synthetic report for eleven compounds: 12 to 15
and 18 to 24. Most of the tertiary and quaternary amines
were synthesized for the first time. Although none of the
compounds showed anti-fungal or antiplasmodial activity,
this is the first biological evaluation for compounds 8 to 20,
General
All the laboratory reagents were analytical grade
(Sigma-Aldrich). Column chromatography and HPLC
solvents were liquid chromatography grade (Merck).
Compound purifications 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 diode array detector (DAD, 254, 280 and 366 nm)
and Eclipse XDB-C18 column using a mixture of MeOH
1
and H2O 0.1% formic acid as the mobile phase. H 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

Vol. 29, No. 12, 2018
Restrepo et al.
2573
as deuterated solvents. Signals were assigned using
two-dimensional heteronuclear correlations (COSY
(correlation spectroscopy) and HSQC (heteronuclear
single quantum correlation)). High-resolution mass
spectra (HRMS) were recorded using electrospray
ionization (ESI-MS) in a UPLC-Q-Tof (ultra-performance
liquid chromatography quadrupole time of flight)
(Xevo-XS-QTof, Waters). The drying and cone gas was
nitrogen set to flow rates of 300-30 L h-1, respectively.
Methanol samples solutions (1 × 10-5 M) were directly
introduced into the ESI spectrometer at a flow rate of
10 µL min-1. A capillary voltage of 3.5 kV was used in the
positive scan mode, and the cone voltage (Uc) set to 10 V.
(300 MHz, CDCl3) d 7.21 (d, J 2.0 Hz, 1H), 6.94 (dd, J 8.3,
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), 1.41 (s, 9H).
Methyl (S)-3-(3,5-dibromo-4-hydroxyphenyl)-2-((tert-butoxy-
carbonyl)-amino) propanoate (Int2)
1
White solid, 2740 mg, 2 stages yield 70%; H NMR
(300 MHz, CDCl3) d 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).
Methyl (S)-3-(3-chloro-4-hydroxyphenyl)-2-((tert-butoxy-
carbonyl)-amino) propanoate (Int3)
1
White solid, 1565 mg, 2 stages yield 55%; H NMR
(300 MHz, CDCl3) d 7.07 (s, 1H), 6.90 (s, 2H), 5.03 (s,
Derivatives synthesis
1H), 4.51 (s, 1H), 3.71 (s, 3H), 2.96 (s, 2H), 1.41 (s, 9H).
Synthesis of intermediates (Int1-Int4)
Methyl (S)-3-(3,5-dichloro-4-hydroxyphenyl)-2-((tert-butoxy-
carbonyl)-amino) propanoate (Int4)
To MeOH (50 mL) at –5 °C, thionyl chloride (2190 µL,
30 mmol) was dropped slowly to maintain the temperature
under 0 °C. Then L-tyrosine (5000 mg, 27.6 mmol) was
added. The resulting mixture was stirred at 80 °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-tyrosine methyl ester hydrochloride. The
product was tested by thin layer chromatography (TLC)
mobile phase n-BuOH:AcOH:H2O (4:1:1) showing
quantitative conversion. This reaction was done twice. The
monobromination 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 EtOAc:H2O. The aqueous phase was recovered and
evaporated to get red oil. This crude was tested by TLC
mobile phase n-BuOH:AcOH:H2O (4:1:1) showing the
formation of mono bromine compound. This crude was
not purified to the next reaction. Compounds dibrominated,
monochlorinated and dichlorinated were obtained in the
same way using NBS (3380 mg, 19 mmol), NCS (1500 mg,
11.22 mmol) and NCS (2535 mg, 19 mmol), respectively.
The protections of the halogenated compounds were done
by the reaction in MeOH (15 mL) with Boc2O (2300 µL,
10 mmol) and TEA (2000 µL) for 24 h at room temperature.
The solvent was removed, the crude was adjusted to pH = 3
and later extracted with EtOAc. The organic phase was
dried with Na2SO4 and purified in column chromatography
using as mobile phase hexane:EtOAc (3:1).
1
White solid, 1930 mg, 2 stages yield 65%; H NMR
(300 MHz, CDCl3) d 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), 1.42
(s, 9H).
Synthesis of compounds 1a-1d
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 reacting 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:EtOAc (3:1) showing
quantitative conversion for Int1-Int4 producing 1180 (1a),
2130 (1b), 1080 (1c) and 1385 mg (1d).
Synthesis of compounds 2a-2d
The O-methylations 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:EtOAc (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 reacting 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:EtOAc (3:1) showing quantitative conversion for
compounds 2a-2d: 2a: 920 mg, two steps yield 80%;
2b: 972 mg, two steps yield 80%; 2c: 885 mg, two steps
yield 80%; 2d: 916 mg, two steps yield 80%.
Methyl (S)-3-(3-bromo-4-hydroxyphenyl)-2-((tert-butoxy-
carbonyl)-amino) propanoate (Int1)
1
White solid, 1615 mg, 2 stages yield 50%; H NMR

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Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives
J. Braz. Chem. Soc.
Synthesis of compounds 3a-3h
135.45, 131.59, 128.96, 118.34, 111.24, 55.50, 35.94;
[α]D25 –12.2; ESI-Q-Tof-HRMS, m/z: calcd. for C9H11NO3Br
[M + H]+ 259.9922, found 259.9915.
The N,N-dimethylation was done by a reductive
amination using aqueous formaldehyde at 37% and
NaCNBH3. One equivalent of compounds 1a-1d, 2a-2d
were dissolved in MeOH (15 mL), 2.2 equivalents of
formaldehyde and 1.5 equivalents of NaCNBH3. The
reactions were stirred 18 h at room temperature. Later the
solvents were evaporated, the crude reaction partitioned
with EtOAc:H2O (50:50) and purified by column
chromatography using as a mobile phase EtOAc:hexane
(2:1). (3a) 1a (500 mg, 1.82 mmol), formaldehyde (300 µL,
4 mmol) and NaCNBH3 (172 mg, 2.73 mmol); white
solid, 383 mg, yield 70%; (3b) 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%;
(3c) 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%; (3d) 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%;
(3e) 2a (500 mg, 1.82 mmol), formaldehyde (300 µL,
4 mmol) and NaCNBH3 (172 mg, 2.73 mmol); white
solid, 383 mg, yield 70%; (3f) 2b (500 mg, 1.41 mmol),
formaldehyde (231 µL, 3.1 mmol) and NaCNBH3 (133 mg,
2.11 mmol); white solid, 376 mg, yield 70%; (3g) 2c
(500 mg, 2.17 mmol), formaldehyde (355 µL, 4.8 mmol)
and NaCNBH3 (133 mg, 2.04 mmol); white solid, 390 mg,
yield 70%; (3h) 2d (500 mg, 1.89 mmol), formaldehyde
(309 µL, 4.15 mmol) and NaCNBH3 (178 mg, 2.83 mmol);
white solid, 386 mg, yield 70%.
(S)-2-Amino-3-(3,5-dibromo-4-hydroxyphenyl)-propanoic
acid (5)
White solid, 144 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.32 (s, 2H), 3.71-3.60 (m, 1H), 2.99 (dd,
1H), 2.83 (dd, 1H); 13C NMR (75 MHz, D2O, DMSO-d6)
d 174.18, 150.75, 134.56, 131.54, 112.56, 56.74, 36.12;
[α]D25 –15.5; ESI-Q-Tof-HRMS, m/z: calcd. for C9H10NO3Br2
[M + H]+ 337.9043, found 337.9027.
(S)-2-Amino-3-(3-chloro-4-hydroxyphenyl)-propanoic
acid (6)
White solid, 140 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.16 (d, J 2.0 Hz, 1H), 6.95 (dd,
J 8.4, 2.1 Hz, 1H), 6.83 (d, J 8.3 Hz, 1H), 3.70 (dd, 1H),
3.00 (dd, 1H), 2.85 (dd, 1H); 13C NMR (75 MHz, D2O,
DMSO-d6) d 174.51, 152.46, 132.33, 130.78, 129.64,
121.77, 118.57, 57.21, 36.66; [α]D25 –10.1; ESI-Q-Tof-
HRMS, m/z: calcd. for C9H11NO3Cl [M + H]+ 216.0427,
found 216.0438.
(S)-2-Amino-3-(3,5-dichloro-4-hydroxyphenyl)-propanoic
acid (7)
White solid, 142 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.12 (s, 2H), 3.70 (m, 1H), 3.01 (dd,
1H), 2.84 (dd, 1H); 13C NMR (75 MHz, D2O, DMSO-d6)
d 174.48, 148.81, 131.00, 129.87, 123.73, 57.01, 36.54;
[α]D25 –13.6; ESI-Q-Tof-HRMS, m/z: calcd. for C9H10NO3Cl2
[M + H]+ 250.0038, found 250.0065.
Primary amines synthesis
Synthesis of compounds 4-11
(S)-2-Amino-3-(3-bromo-4-methoxyphenyl)-propanoic
acid (8)
150 mg of compounds 1a-1d, 2a-2d 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 24 h
at room temperature. The crude of reaction was adjusted
to pH = 6. The compounds were tested by TLC mobile
phase n-BuOH:AcOH:H2O (4:1:1) showing quantitative
conversion. The final products (4-11) were purified with
reversed phase (RP)-HPLC using as a mobile phase a
mixture of MeOH (0.01% formic acid (F.A.)) and H2O
(0.01% F.A.).
1
White solid, 142 mg; H NMR (300 MHz, D2O,
DMSO-d6) d 7.56 (d, J 2.1 Hz, 1H), 7.29 (dd, J 8.5, 2.1 Hz,
1H), 7.11 (d, J 8.5 Hz, 1H), 4.06-3.84 (m, 4H), 3.23 (dd,
1H), 3.07 (dd, 1H); 13C NMR (75 MHz, D2O, DMSO-d6)
d 164.63, 146.03, 125.30, 121.43, 120.57, 104.57, 102.60,
47.52, 26.64; [α]D25 –8.4; ESI-Q-Tof-HRMS, m/z: calcd. for
C10H13NO3Br [M + H]+ 274.0079, found 274.0096.
(S)-2-Amino-3-(3,5-dibromo-4-methoxyphenyl)-propanoic
acid (9)
1
(S)-2-Amino-3-(3-bromo-4-hydroxyphenyl)-propanoic acid
(4)
White solid, 140 mg, yield 100; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.32 (d, J 1.9 Hz, 1H), 7.00 (dd, J 8.4,
1.9 Hz, 1H), 6.83 (d, J 8.3 Hz, 1H), 4.05 (m, 1H), 3.00 (m,
2H); 13C NMR (75 MHz, D2O, DMSO-d6) d 172.62, 153.82,
White solid, 144 mg; H NMR (300 MHz, D2O,
DMSO-d6) d 7.59 (s, 2H), 3.97-3.80 (m, 4H), 3.23 (dd,
1H), 3.07 (dd, 1H); 13C NMR (75 MHz, D2O, DMSO-d6)
d 164.20, 144.24, 126.41, 125.17, 109.32, 52.11, 47.14,
26.65; [α]D25 –11.1; ESI-Q-Tof-HRMS, m/z: calcd. for
C10H12NO3Br2 [M + H]+ 351.9184, found 351.9227.

Vol. 29, No. 12, 2018
Restrepo et al.
2575
(S)-2-Amino-3-(3-chloro-4-methoxyphenyl)-propanoic
acid (10)
m/z: calcd. for C11H15NO3Cl [M + H]+ 244.0740, found
244.0760.
1
White solid, 141 mg; H NMR (300 MHz, D2O,
DMSO-d6) d 7.20 (s, 1H), 7.06 (d, J 8.5 Hz, 1H), 6.96 (d,
J 8.4 Hz, 1H), 3.73 (s, 4H), 3.02 (dd, 1H), 2.87 (dd, 1H);
13C NMR (75 MHz, D2O, DMSO-d6) d 174.76, 155.19,
132.35, 130.89, 130.23, 123.11, 114.77, 57.69, 57.28,
36.71; [α]D25 –9.4; ESI-Q-Tof-HRMS, m/z: calcd. for
C10H13NO3Cl [M + H]+ 230.0584, found 230.0594.
(S)-3-(3,5-Dichloro-4-hydroxyphenyl)-2-(dimethylamino)-
propanoic acid (14)
White solid, 106 mg, yield 75%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.14 (s, 2H), 4.02 (m, 1H), 3.17 (dd,
1H), 2.94 (dd, 1H), 2.82 (s, 6H); 13C NMR (75 MHz, D2O,
DMSO-d6) d 170.64, 149.23, 131.31, 129.16, 123.18, 69.83,
43.59, 33.52; [α]D25 59.1; ESI-Q-Tof-HRMS, m/z: calcd. for
C11H14NO3Cl2 [M + H]+ 278.0351, found 278.0352.
(S)-2-Amino-3-(3,5-dichloro-4-methoxyphenyl)-propanoic
acid (11)
1
White solid, 142 mg; H NMR (300 MHz, D2O,
(S)-3-(3-Bromo-4-methoxyphenyl)-2-(dimethylamino)-
propanoic acid (15)
DMSO-d6) d 7.20 (s, 2H), 3.74 (s, 4H), 3.02 (dd, 1H), 2.88
(dd, 1H); 13C NMR (75 MHz, D2O, DMSO-d6) d 174.36,
152.09, 135.25, 131.48, 130.39, 56.98, 36.82; [α]D25 –12.2;
ESI-Q-Tof-HRMS, m/z: calcd. for C10H12NO3Cl2 [M + H]+
264.0194, found 264.0199.
White solid, 121 mg, yield 85%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.56 (d, J 2.0 Hz, 1H), 7.30 (dd, J 8.5,
2.1 Hz, 1H), 7.08 (d, J 8.5 Hz, 1H), 3.90 (s, 3H), 3.78 (dd,
1H), 3.25 (dd, 1H), 3.10 (dd, 1H), 2.93 (s, 6H); 13C NMR
(75 MHz, D2O, DMSO-d6) d 163.05, 145.86, 125.11,
121.23, 120.89, 104.37, 102.46, 63.38, 47.57, 24.00;
[α]D25 53.8; ESI-Q-Tof-HRMS, m/z: calcd. for C12H17NO3Br
[M + H]+ 302.0392, found 302.0398.
Tertiary amines
Synthesis of compounds 12-18
150 mg of compounds 3a-3h (except 3b) 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 24 h
at room temperature. The crude of reaction was adjusted
to pH = 6. The compounds were tested by TLC mobile
phase n-BuOH:AcOH:H2O (4:1:1) showing quantitative
conversion. The final products (12-18) were purified with
RP-HPLC using as a mobile phase a mixture of MeOH
(0.01% F.A.) and H2O (0.01% F.A.).
(S)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(dimethylamino)-
propanoic acid (16)
White solid, 123 mg, yield 85%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.57 (s, 2H), 3.90 (s, 2H), 3.82
(dd, 1H), 3.28 (d, 1H), 3.06 (dd, 1H), 2.95 (s, 6H);
13C NMR (75 MHz, D2O, DMSO-d6) d 163.23, 157.07,
125.80, 124.46, 108.54, 63.16, 51.93, 23.48; [α]D25 61.0;
ESI-Q-Tof-HRMS, m/z: calcd. for C12H16NO3Br2 [M + H]+
379.9497, found 379.9490.
(S)-3-(3-Bromo-4-hydroxyphenyl)-2-(dimethylamino)-
propanoic acid (12)
(S)-3-(3-Chloro-4-methoxyphenyl)-2-(dimethylamino)-
propanoic acid (17)
White solid, 108 mg, yield 75%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.34 (d, J 1.8 Hz, 1H), 7.01 (dd, J 8.4,
1.8 Hz, 1H), 6.81 (d, J 8.3 Hz, 1H), 3.91 (m, 1H), 3.22-3.07
(m, 1H), 2.92 (dd, 1H), 2.80 (s, 6H); 13C NMR (75 MHz,
D2O, DMSO-d6) d 171.41, 153.65, 135.30, 131.45,
129.14, 118.20, 111.05, 71.00, 42.91, 33.52; [α]D25 60.3;
ESI-Q-Tof-HRMS, m/z: calcd. for C11H15NO3Br [M + H]+
288.0235, found 288.0243.
White solid, 120 mg, yield 85%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.20 (s, 1H), 7.05 (s, 1H), 6.94 (s, 1H),
3.72 (s, 3H), 3.64-3.48 (m, 1H), 3.07 (dd, 1H), 2.94-2.80
(m, 1H), 2.73 (s, 6H); 13C NMR (75 MHz, D2O, DMSO-d6)
d 173.04, 154.61, 132.43, 130.40, 130.32, 122.66, 114.58,
73.75, 57.65, 40.25, 34.56; [α]D25 49.8; ESI-Q-Tof-HRMS,
m/z: calcd. for C12H17NO3Cl [M + H]+ 258.0897, found
258.0898.
(S)-3-(3-Chloro-4-hydroxyphenyl)-2-(dimethylamino)-
propanoic acid (13)
(S)-3-(3,5-Dichloro-4-methoxyphenyl)-2-(dimethylamino)-
propanoic acid (18)
White solid, 106 mg, yield 75%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.16 (s, 1H), 7.03 (d, 1H), 6.90 (dd,
1H), 6.81 (d, 1H), 3.81 (m, 1H), 3.11 (dd, 1H), 2.89 (dd,
1H), 2.78 (s, 6H); 13C NMR (75 MHz, D2O, DMSO-d6) d
171.60, 152.45, 131.90, 130.68, 128.70, 121.51, 118.30,
71.60, 43.56, 33.76; [α]D25 54.9; ESI-Q-Tof-HRMS,
White solid, 121 mg, yield 85%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.20 (s, 2H), 3.73 (s, 3H), 3.59 (dd, 1H),
3.09 (dd, 1H), 2.86 (dd, 1H), 2.74 (s, 6H); 13C NMR (75 MHz,
D2O, DMSO-d6) d 172.59, 151.90, 135.35, 131.32, 130.21,
73.03, 62.46, 34.21; [α]D25 53.5; ESI-MS m/z 292.6 [M]+.

2576
Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives
J. Braz. Chem. Soc.
Quaternary amines
DMSO-d6) d 170.9, 153.8, 135.6, 135.1, 119.0, 80.7, 62.3,
53.5, 32.8; [α]D25 59.3; ESI-Q-Tof-HRMS, m/z: calcd. for
C13H19NO3Br2 [M]+ 393.9653, found 393.9701.
Synthesis of compounds 19-24
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 (100 mg) was added and the mixture was
stirred 24 h at room temperature. The crude of reaction
was adjusted to pH = 6. The compounds were tested by
TLC mobile phase n-BuOH:AcOH:H2O (4:1:1) showing
quantitative 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.).
(S)-1-Carboxy-2-(3-chloro-4-methoxyphenyl)-
N,N,N-trimethylethan-1-aminium (23)
White solid, 173 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.21 (s, 1H), 7.06 (d, J 8.5 Hz, 1H), 6.95
(d, J 8.5 Hz, 1H), 3.71 (s, 4H), 3.13 (s, 10H), 2.93 (dd,
1H); 13C NMR (75 MHz, D2O, DMSO-d6) d 171.5, 155.2,
132.4, 131.0, 129.5, 123.0, 114.7, 80.8, 57.8, 53.7, 33.0;
[α]D25 51.9; ESI-Q-Tof-HRMS, m/z: calcd. for C13H19NO3Cl
[M]+ 272.1053, found 272.1057.
(S)-1-Carboxy-2-(3,5-dichloro-4-methoxyphenyl)-
N,N,N-trimethylethan-1-aminium (24)
(S)-1-Carboxy-2-(3-chloro-4-hydroxyphenyl)-
N,N,N-trimethylethan-1-aminium (19)
White solid, 170 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.15 (s, 2H), 3.70 (s, 3H), 3.61 (m, 1H),
3.09 (s, 10H), 2.90 (dd, 1H); 13C NMR (75 MHz, D2O,
DMSO-d6) d 171.0, 151.9, 134.5, 131.3, 130.1, 80.7, 62.3,
53.5, 33.0; [α]D25 54.4; ESI-Q-Tof-HRMS, m/z: calcd. for
C13H19NO3Cl2 [M]+ 292.0507, found 292.0519.
White solid, 170 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.17 (d, J 1.9 Hz, 1H), 6.97 (dd, J 8.4,
1.9 Hz, 1H), 6.84 (d, J 8.4 Hz, 1H), 4.04 (m, 1H), 3.23 (m,
1H), 3.18 (s, 9H), 2.94 (m, 1H); 13C NMR (75 MHz, D2O,
DMSO-d6) d 169.9, 152.5, 132.3, 130.9, 127.4, 121.5,
118.4, 77.6, 53.7, 32.4; [α]D25 47.2; ESI-Q-Tof-HRMS, m/z:
calcd. for C12H17NO3Cl [M]+ 258.0901, found 258.0897.
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.
(S)-1-Carboxy-2-(3,5-dichloro-4-hydroxyphenyl)-
N,N,N-trimethylethan-1-aminium (20)
White solid, 172 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.15 (s, 2H), 3.97 (m, 1H), 3.23 (dd,
1H), 3.14 (s, 9H), 2.95 (dd, 1H); 13C NMR (75 MHz,
D2O, DMSO-d6) d 169.9, 140.0, 131.0, 128.4, 123.9, 77.7,
53.9, 32.4; [α]D25 53.1; ESI-Q-Tof-HRMS, m/z: calcd. for
C12H16NO3Cl2 [M]+ 292.0508, found 292.0507.
Biological activity assays
The compounds were subjected to in vitro evaluation
for their cytotoxicity, anti-leishmanial, antiplasmodial and
anti-fungal activity.
Anti-leishmanial activity assay
(S)-1-Carboxy-2-(3-bromo-4-methoxyphenyl)-
N,N,N-trimethylethan-1-aminium (21)
The anti-leishmanial activity of compounds was
determined according to the ability of the compound to
reduce the infection by L. panamensis parasites. For this,
the anti-leishmanial activity was tested on intracellular
amastigotes of L. panamensis transfected with the green
fluorescent protein gene (MHOM/CO/87/UA140-EpiR-
GFP strain). Briefly, U-937 human cells at a density of
3 × 105 cells mL-1 in Roswell Park Memorial Institute
medium (RPMI)-1640 and 0.1 µg mL-1 of PMA (phorbol
12-myristate 13-acetate) were dispensed on 24-wells
microplate and then infected with stationary phase growing
L. panamensis promastigotes in a 15:1 parasites per cell
ratio. Plates were incubated at 34 °C and 5% CO2 for 3 h
and then the cells were washed twice with phosphate buffer
solution (PBS) to eliminate not internalized parasites. Fresh
RPMI-1640 was added to each well (1 mL) and plates
White solid, 173 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.51 (d, J 2.0 Hz, 1H), 7.26 (dd, J 8.5,
2.1 Hz, 1H), 7.04 (d, J 8.5 Hz, 1H), 3.87 (s, 3H), 3.80 (m,
1H), 3.28 (s, 9H), 3.24 (dd, 1H), 3.08 (dd, 1H); 13C NMR
(75 MHz, D2O, DMSO-d6) d 161.8, 146.4, 125.5, 121.6,
120.2, 104.6, 102.6, 71.5, 47.8, 43.7, 23.2; [α]D25 53.7; ESI-
Q-Tof-HRMS, m/z: calcd. for C13H19NO3Br [M]+ 316.0547,
found 316.0458.
(S)-1-Carboxy-2-(3,5-dibromo-4-methoxyphenyl)-
N,N,N-trimethylethan-1-aminium (22)
White solid, 168 mg, yield 100%; 1H NMR (300 MHz,
D2O, DMSO-d6) d 7.59 (s, 1H), 3.93 (s, 3H), 3.85 (m, 1H),
3.33 (s, 10H), 3.11 (dd, 1H); 13C NMR (75 MHz, D2O,

Vol. 29, No. 12, 2018
Restrepo et al.
2577
were incubated again to complete infection. After 24 h of
infection, the RPMI-1640 medium was replaced by fresh
culture medium containing each compound at four serial
dilutions (50, 12.5, 3.125 and 0.78 µg mL-1) and plates were
then incubated at 37 °C and 5% CO2 during 72 h, then,
cells were removed from the bottom plate with a trypsin/
ethylenediaminetetraacetic acid disodium salt dihydrate
(EDTA) (250 mg) solution. The cells were centrifuged
at 1100 rpm during 10 min at 4 °C, the supernatant was
discarded, and cells were washed with 1 mL of cold PBS
and centrifuged at 1100 rpm for 10 min at 4 °C. Cells were
washed two times employing PBS, as previously, and after
the last wash, the supernatant was discarded, and cells
were suspended in 500 µL of PBS. Cells were analyzed by
flow cytometry employing a flow cytometer (Cytomics FC
500MPL) reading at 488 (exciting) and 525 nm (emitting)
over an argon laser and counting 10,000 events. Infected
cells were determined according to the events for green
fluorescence (parasites). Infected cells exposed to control
drugs (amphotericin B) were used as a control for anti-
leishmanial activity (positive control), while infected cells
incubated in absence of any compound or drug were used
as a control for infection (negative control). Nonspecific
fluorescence was corrected by subtracting fluorescence
of unstained cells. All determinations were performed in
triplicate in at least two independent experiments.18,19
deionized water, 4 g L-lactate, 1.32 g Tris buffer and 0.022 g
acetylpyridine adenine dinucleotide in 200 mL deionized
water; pH 9.0) and 25 µL of NBT/PES solution (0.16 g
nitroblue tetrazolium salt and 0.08 g phenazine ethosulfate
in 100 mL deionized water) were added to each well of
a second flat-bottomed 96-well microtiter plate. After
freeze-thaw cycles, the culture in each of the wells of the
first plate was resuspended by pipetting and 15 µL of each
well was taken and added to the corresponding well of the
second plate (containing Malstat and NBT/PES reagents).
After an hour of incubation in the dark, color development
of the LDH reaction was monitored colorimetrically in a
spectrofluorometer (Varioskan, Thermo) reading at 650 nm.
The intensity of color in each experimental condition
was registered as optical densities (O.D.). Non-specific
absorbance was corrected by subtracting O.D. of the
blank. Determinations were done in triplicate in at least
two independent experiments.20
Antifungal activity
Antifungalactivitywasevaluatedagainst19fungalstrains
of medical importance as Candida tropicalis (ATCC 750),
C. tropicalis (ATCC 200956), C. lusitaniae (ATCC 2819),
C. albicans (ATCC 10231), C. krusei (ATCC 6258),
C. glabrata (ATCC 90030), Cryptococcus neoformans
(ATCC 90112), and C. gattii (ATCC 10865). In addition,
the following clinical isolates and strains of filamentous
fungi were included: Sporothrix schenckii (UdeA 7027),
S. globosa(UdeA0002), Fusariumoxysporum(UdeA8038),
F. graminearum (UdeA 6003), F. proliferatum (Micoteca da
Universidade do Minho (MUM) 16144), F. verticillioides
(MUM 16143), Neoscytalidium dimidiatum (UdeA 8023),
Aspergillus flavus (ATCC 204304), A. fumigatus
(ATCC 204305), Trichophyton interdigitale (ATCC 24198)
and Fusarium oxysporum (ATCC 48112). In vitro anti-
fungal activity of derivatives was determined following
the Anti-Fungal Susceptibility Testing Subcommittee of
the European Committee on Antibiotic Susceptibility
Testing (AFST-EUCAST) reference procedure for
yeasts and for filamentous.21 The anti-fungal tests
were carried out in RPMI buffered at pH = 7 with
3-morpholinopropanesulfonic acid (MOPS). Compounds
were evaluated at a final concentration of 500 µg mL-1. One
hundred microliters of each concentration diluted twice
were dispensed into 96-well microtitration plates and the
same volume of each inoculum at 1.5 × 105 UFC mL-1
were added. Minimal inhibitory concentration (MIC) was
defined as the lowest dilution that resulted in total inhibition
of visible growth after 24 or 48 h incubation at 35 °C of
Candida spp. and Cryptococcus spp., respectively. The
MICs for filamentous fungi were determined after 24 h
Antiplasmodial activity assay
Antiplasmodial activity was evaluated in vitro on
asynchronic cultures of P. falciparum (3D7 strain),
maintained in standard culture conditions. The effect
of each compound over the growth of the parasites was
determined by quantification of parasite death, based
on the measurement of lactate dehydrogenase (LDH)
activity released from the cytosol of damaged cells into
the supernatant. Briefly, unsynchronized P. falciparum
cultures were adjusted to 0.5% parasitemia and 1%
hematocrit in RPMI medium enriched with 3% lipid-rich
bovine serum albumin (Albumax II). Then, in each well
of a 96-wells plate, 100 µL of parasite suspension were
dispensed and subsequently exposed against 100 µL of
four serial dilutions of compounds (100, 25, 6.25 and
1.56 µg mL-1). Dilutions were prepared from a stock
solution of 1000 µg mL-1. Chloroquine (CQ) was used as
positive antiplasmodial drug control. Parasites unexposed
to any compound were used as a control of both growth and
viability (negative control). Plates were incubated for 48 h
at 37 °C in N2 (90%), CO2 (5%) and O2 (5%) atmosphere.
After incubation, plates were harvested, and parasites were
subjected to three 20-min freeze-thaw cycles. Meanwhile,
100 µL of Malstat reagent (400 µL Triton X-100 in 80 mL

2578
Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives
J. Braz. Chem. Soc.
of incubation at 28 °C, except for Sporothrix spp. and
T. interdigitale (ATCC 24198), which were incubated for
five days. The anti-fungals itraconazole and amphotericin
B (Sigma-Aldrich Co.) were included as a positive control
at a concentration range of 16 to 1 µg mL-1 against C. krusei
(ATCC 6258), C. parapsilosis (ATCC 22019), A. flavus
(ATCC 204304) and A. fumigatus (ATCC 204305).
when LC50 < 100 µg mL-1; moderate cytotoxicity when
LC50 is in 100-200 µg mL-1 range and low cytotoxicity
when LC50 > 200 µg mL-1.
The antiplasmodial activity of each evaluated compound
was evidenced by the reduction of the absorbance (O.D.).
Indeed, the viability percentage was calculated by
equation 3:
(3)
Cytotoxic activity in human macrophages
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-diphenyltetrazolium
bromide) method. Shortly, 100,000 cells mL-1 in complete
RPMI-1640 and the corresponding concentration of each
compound (200, 50, 12.5, 3.125 µg mL-1) were incubated at
37 °C, 5% CO2 during 72 h. The effect of each concentration
of each compound was determined by adding 10 µL well-1 of
MTT solution (0.5 mg mL-1) and incubating at 37 °C for 3 h.
The reaction was stopped with 100 µL of 50% isopropanol
solution with 10% sodium dodecyl sulfate (SDS) and
incubation 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.
Doxorubicin was used as a cytotoxicity control. Nonspecific
absorbance was corrected by subtracting absorbance (O.D.)
of the blank. Determinations were done in triplicate in at
least two independent experiments.18
Then, the inhibition growing percentage was calculated
according to the following equation 4:
%Inhibition = 100 – %Viability
(4)
Anti-leishmanial activity was determined according to
reduction of the median fluorescence intensity (MFI) of
infected cells percentages obtained for each experimental
condition by the cytometer. The number of parasites for
each concentration of tested compound was calculated by
equation 5, where the MFI in infected cells in the control
well corresponds to 100% of infection.
(5)
Then, inhibition percentage was calculated with
equation 6:
%Inhibition = 100 – %Infection
(6)
Statistical analysis
Cytotoxicity was determined according to viability
and mortality percentages obtained for each experimental
condition (synthesized compounds, amphotericin B,
benznidazole, chloroquine and culture medium). Results
were expressed as LC50 that corresponds to the concentration
necessary to kill 50% of cells, calculated by Probit analysis
(parametric method of linear regression that permits
doses-response analysis).22 Initially, viability percentages
were calculated by equation 1, where the O.D. of control
well corresponds to 100% of viability. In turn, mortality
percentage corresponds to 100% – %viability.
The anti-leishmanial and antiplasmodial activity was
graded as high, moderate or low according to the EC50
values, as follows: high activity when EC50 < 25 µg mL-1;
moderate activity when EC50 is in the 25-50 µg mL-1 range
and low activity when EC50 > 50 µg mL-1.
The selectivity index (SI) was calculated by dividing
the cytotoxic activity and the antileishmanial or
antitrypanosomal activity (SI = LC50/EC50).
Supplementary Information
Supplementary data (1H NMR, 13C NMR and
ESI-Qtof-HRMS spectra) are available free of charge at
http://jbcs.sbq.org.br as PDF file.
(1)
Then, the percentage of cell growth inhibition was
calculated using the equation 2:
Acknowledgments
%Cell growth inhibition = 100% – %Viability
(2)
The authors would like to thank the Colombian
Department of Science, Technology and Innovation
(Colciencias, grant CT-695-2014) and the University of
Antioquia (CODI, CIQF-176) for their financial support.
The cytotoxicity was graded as high, moderate or low
according to LC50 values, as follows: high cytotoxicity

Vol. 29, No. 12, 2018
Restrepo et al.
2579
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