Anti-Parasite Activity of Novel 3,5-Diiodophenethyl-benzamides

Article abstract of DOI:10.21577/0103-5053.20180160

Novel iodotyramides with para-substituted benzoic acids were synthesized via electrophilic aromatic substitutions and amide coupling via N,N'-diisopropylcarbodiimide (DIC) in dimethylformamide (DMF). All derivatives were in vitro screened against U-937 ma

Full text of DOI:10.21577/0103-5053.20180160

http://dx.doi.org/10.21577/0103-5053.20180160
J. Braz. Chem. Soc., Vol. 30, No. 1, 116-123, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
Anti-Parasite Activity of Novel 3,5-Diiodophenethyl-benzamides
Manuel Pastrana Restrepo, a Verónica Surmay Surmay,a Elkin Galeano Jaramillo*,a
and Sara Robledo Restrepob
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
bPrograma 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
Novel iodotyramides with para-substituted benzoic acids were synthesized via
electrophilic aromatic substitutions and amide coupling via N,N’-diisopropylcarbodiimide
(DIC) in dimethylformamide (DMF). All derivatives were in vitro screened against U-937
macrophages and Plasmodium falciparum, Leishmania panamensis and Trypanosoma cruzi
protozoan parasites. The hemolytic activity was evaluated on human red blood cells (RBC).
Compounds N-(4-hydroxy-3,5-diiodophenethyl)-4-methylbenzamide and N-(3,5-diiodo-
4-methoxyphenethyl)-4-methoxybenzamide were the most active against L. panamensis with
an effective concentration 50 (EC50) of 17.9 and 17.5 μg mL-1, respectively; while compounds
N-(3,5-diiodo-4-methoxyphenethyl)-4-methylbenzamide and N-(3,5-diiodo-4-methoxyphenethyl)-
4-methoxybenzamide were the most active for T. cruzi with EC50 values of 23.75 and
6.19 μg mL-1, respectively. In contrast, all derivatives showed high activity against P. falciparum
with EC50 < 25 μg mL-1, except compound N-(4-hydroxy-3,5-diiodophenethyl)-benzamide. No
compound was hemolytic over RBC. This report represents the importance of novel iodotyramides
as anti-parasites agents.
Keywords: anti-protozoan activity, cytotoxicity, L. panamensis, P. falciparum, T. cruzi,
iodotyramides
Introduction
inhibitory concentration (IC50) 20 μg mL-1.3 Other active
diiodotyramines isolated from marine organisms are
turbotoxins A and B both isolated from gastropod Turbo
marmorata. These diiodotyramines exhibited an acute
toxicity against ddY mice, with an dosage required to kill
99% of the test population (LD99) of 1.0 and 4.0 mg kg-1,
respectively.4 However, their synthetic analogs were 100-
fold less toxic when the quaternary ammonium group was
changed by a tertiary ammonium group.5 Due to iodinated
alkaloid metabolites, usually isolated as tyrosine and
tyramine derivatives, diiodotyramides and diiodotyrosines
show broad biological activities, but are not very common
in nature, therefore, chemical synthesis of these compounds
emerge as an opportunity to potentiate their biological
activity through structural changes and develop new active
agents that may overcome the problem of parasite resistance
against current drugs. For this reason, the potential of
synthetic compounds based on diiodotyramides aromatic
esters, derivatives of marine natural products, was evaluated
Marine organisms represent a large source of primary
and secondary bioactive metabolites. Halogenated alkaloids,
especially those with iodine atoms, are a type of secondary
metabolites with diverse biological activities.While iodinated
halometabolites are hard to find in nature, chloro and bromo
derivatives alkaloids are more common.1 Nevertheless,
ten iodotyramine derivatives (3,5-diiodotyramines and
tyrosine derivatives) with antitumoral activity were
recently isolated from the aqueous extract of ascidian
Didemnum rubeum.2 Moreover, iodine derivative alkaloid
3,5-diiodo-4-methoxyphenetylamine and their respective
urea analogs were isolated from the tunicates Lissoclinum
patella and Didemnum ternatanum. Both derivatives were
active against C. albicans strains and mouse lymphocytic
leukemia L1210 cell line at concentrations of half maximal
*e-mail: elkin.galeano@udea.edu.co

Vol. 30, No. 1, 2019
Restrepo et al.
117
in this study for cytotoxicity, but also for anti-protozoan
and hemolysis activities.
carbodiimide (DIC) in dimethylformamide (DMF)
catalyzed with 4-dimethylaminopyridine (DMAP),10
in yields ranging 45-50%. H NMR analysis of the
1
Results and Discussion
amides shows the respective singlet for two protons
in the downfield area corresponding to hydrogens at
ortho position in the iodo-tyramine moiety. Besides,
the signals corresponding to the aromatic protons in the
aromatic acids scaffold appear in the 6-8 ppm region.
p-Substituted aromatic acids show these signals as two
doublets confirming the symmetry of the aromatic ring.
The presence of methyl and O-methyl groups in the
aromatic rings was confirmed by the singlets at 2.34 and
Chemistry
The aromatic iodo-tyramides syntheses were made
in two stages (Scheme 1). The first is the synthesis of
compounds C and D, and later coupling with different
aromatic acids to afford the respective amides.
The protection of the amino group in the tyramine was
done in basic media with Boc2C,6 and the diiodination of
the aromatic ring with N-iodosuccinimide (NIS),7 getting
a di-halogenated compound in positions 3 and 5, obtaining
1
3.70 ppm. The H-1H correlation spectroscopy (COSY)
analysis shows the respective couplings of protons in
the aromatic acid and two methylene group in the iodo-
tyramine chain. The molecular formula of the respective
amides was confirmed by high-resolution mass spectra
(HRMS) analysis. In this way it was possible to determine
unambiguously the chemical structure of the new
derivatives synthesized.
1
compound A with a 70% yield for two steps. H nuclear
magnetic resonance (NMR) analysis of compound A shows
a singlet at 7.50 ppm for two protons, which corresponds
to hydrogen at 2, which confirm the diiodination at the
ortho position of the phenyl ring. The singlet at 1.44 ppm
for nine protons corresponds to t-butyl group for the
carbamate moiety. Compound B was produced by the
reaction of A with methyl iodide in basic media,8 with 80%
yield. 1H NMR spectra of compound B shows a singlet at
3.84 ppm for three protons, corresponding to O-methyl
group in the aromatic ring.
Acid hydrolysis of compounds A and B9 produce the
primary amines in compounds C and D. The loss of the
signal at 1.44 ppm in both compounds shows the hydrolysis
of the carbamate moiety.
Biological activities
The effect of the eight synthesized amides on cell
growth and viability on human U-937 macrophages
was assessed. In addition, the anti-parasite activity on
L. panamensis and T. cruzi intracellular amastigotes and
total sexual forms of P. falciparum was tested, according to
the ability of compounds to reduce the amount of parasite
in culture. The hemolytic assay was done on human red
blood cells (RBC) obtained from a blood bank. Results are
summarized in Table 1.
The amide syntheses were done by the coupling of C
and D with different aromatic acids using N,N’-diisopropyl-
Scheme 1. Aromatic diiodotyramides synthesis.

118
Anti-Parasite Activity of Novel 3,5-Diiodophenethyl-benzamides
J. Braz. Chem. Soc.
Table 1. Biological activities of diiodotyramides derivatives
U-937
L. panamensis
T. cruzi
EC50 / (μg mL-1)
P. falciparum
EC50 / (μg mL-1)
RBC
LC50 / (μg mL-1)
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
NA
Compound
LC50 / (μg mL-1) EC50 / (μg mL-1)
SI
6.97
4.55
1.46
< 0.77
0.97
0.63
0.73
2.36
NA
SI
5.2
SI
3.31
1
323.2 51.68
81.4 15.4
75.22 15.4
38.67 7.12
32.73 5.17
24.12 3.9
12.86 0.46
88.49 21.5
1.37 0.6
46.33 17.5
17.9 8.77
51.54 69.8
> 50
62.09 10.36
28.88 1.74
67.29 6.77
111.37 246.97
30.34 2.46
23.75 2.85
6.19 0.18
26 2.28
97.69 10.12
27.08 1.96
20.45 1.14
21.15 1.81
20.41 1.51
18.49 0.89
22.6 1.72
19.07 1.88
NA
2
2.81
1.11
0.03
1.08
1.02
2.08
3.4
3.01
3
< 9.77
> 9.45
> 9.79
> 10.81
> 8.84
10.48
NA
4
5
33.77 22.53
38.21 14.87
17.5 17.77
37.43 32.83
NA
6
7
8
Doxorubicin
Amphotericin B
Benznidazole
Chloroquine
NA
NA
36.6
> 200
155.2 5.2
8
0.09 0.01
NA
406.66
NA
NA
NA
NA
NA
NA
14.78 0.28
NA
> 13.53
NA
NA
NA
NA
NA
NA
3.36 0.4
46.19
NA
RBC: red blood cells; EC50: effective concentration 50; LC50: lethal concentration 50; SI: selectivity index = LC50/EC50; NA: no apply.
The anti-parasite activities were measured by determining
the effective concentration 50 (EC50). The anti-leishmanial
assay showed that compounds 2 and 7 were the most active
with EC50 of 17.9 and 17.5 μg mL-1, respectively. Compounds
1, 3, 5, 6 and 8 had moderate activity with EC50 values
between 25 to 50 μg mL-1. The compound with the highest
selectivity index (SI) for L. panamnesis was compound 1
with a value of 6.97 μg mL-1. It is the safest of the iodo-
tyramides derivatives. Compound 1 was more than 8-fold less
cytotoxic than amphotericin B. Anti-trypanosomal assays
showed that compounds 6 and 7 were the most active with
EC50 of 23.75 and 6.19 μg mL-1, respectively. Moreover,
compound 7 was more active than benznidazole. In turn,
compounds 2, 5 and 8 were moderately active with values
ranging from 25 to 50 μg mL-1. Compound 1 was the most
selective against T. cruzi with an SI of 5.2.
Didemnun rubeum and seven correlated new tyramides with
different substitution in para-position of the aromatic acid
moiety.Antiparasitic activity against T. cruzi, L. panamensis
and P. falciparum and also cytotoxicity and hemolysis assays
were reported. All compounds had LC50 < 100 μg mL-1,
except compound 1, making them potentially cytotoxic
for U-937 macrophages. Compounds 2-8 had high activity
against P. falciparum, where compound 6 was the most
promissory compound. Anti-trypanosomal assays showed
that compounds 6 and 7 had the higher activity, where 7
was the most active against T. cruzi. In turn, anti-leishmanial
assays showed that compounds 2 and 7 were the most
active. No compound produced hemolysis of RBC. Based
on these in vitro biological activities, further in vivo studies
are necessary to validate anti-parasite activity observed of
compounds 2, 6 and 7.
Finally, antiplasmodial activity showed that all
derivatives had a high activity with EC50 values < 25 μg mL-1,
except compound 1. No compound was more active than
chloroquine. Compound 8 had the highest SI with a value
of 10.48. Compounds 4, 5, 6 and 7 were also selective
with SI values > 8. The p-O-Me compounds were most
active than their more polar analogs against L. panamnesis
and T. cruzi. The core A, 3,5-diiodo-p-methoxybenzyl, is
correlated with a higher antiparasitic activity against the
three parasites tested. Those derivatives showed the best
selectivity index against P. falciparum. No compound was
hemolytic on human RBC.
Experimental
General remarks
All laboratory reagents were analytical grade (Sigma-
Aldrich®). Chromatography column 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 DAD detector (254, 280
and 366 nm) and Eclipse XDB-C18 column using a mixture
of MeOH and H2O 0.1% formic acid as the mobile phase.
1H and 13C NMR were recorded on Bruker Ascend III HD
(600 and 125 MHz) with 5 mm cryoprobe TCI, using CDCl3
and dimethyl sulfoxide (DMSO-d6) as deuterated solvents.
Signals were assigned using two-dimensional heteronuclear
Conclusions
In this work it is reported the complete synthesis
of N-(3,5-diiodo-4-methoxyphenethyl)-benzamide, an
iodotyramide isolated for the first time from ascidian

Vol. 30, No. 1, 2019
Restrepo et al.
119
correlations (COSY (correlation spectroscopy) and HSQC
(heteronuclear single quantum correlation)). 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
and 30 L h-1, respectively. Methanol samples solutions
(1 × 10-5 mol L-1) were directly introduced into the ESI
spectrometer at a flow rate of 10 μL min-1.A capillary voltage
3.5 kV was used in the positive scan mode, and the cone
voltage set to Uc = 10 V.
(3 mL) were placed into a round bottom flask equipped
with a magnetic stirring bar. The mixture was stirred at
room temperature for 3 h. The crude reaction mixture
was evaporated under reduced pressure and the residue
was neutralized with 10% NaHCO3 and filtered affording
white solids in a quantitative cleavage. 1H NMR (600 MHz,
DMSO-d6) d 7.64 (s, 2H, H-2), 2.97 (t, 2H, H-6), 2.76 (t,
2H, H-5).
Synthetic procedure for O-methyl-3,5-diiodo-tyramine
(compound D)
Chemistry
Compound B (1000 mg), HCl 37% (1 mL) and EtOAc
(3 mL) were placed into a round bottom flask equipped
with a magnetic stirring bar. The mixture was stirred at
room temperature for 3 h. The crude reaction mixture
was evaporated under reduced pressure and the residue
was neutralized with 10% NaHCO3 and filtered affording
white solids in a quantitative cleavage. 1H NMR (600 MHz,
DMSO-d6) d 7.75 (s, 2H, H-2), 3.72 (s, 3H, OCH3), 3.01
(q, 2H, H-6), 2.80 (t, 2H, H-5).
Synthetic procedure for tert-butyl-(4-hydroxy-3,5-diiodo-
phenethyl)-carbamate (compound A)
Tyramine (1500 mg, 10.9 mmol), Boc2O (2756 μL,
12 mmol), triethylamine (TEA) (600 μL) and MeOH
(20 mL) were placed into a round bottom flask equipped
with a magnetic stirring bar. The mixture was stirred at
room temperature for 24 h. The crude reaction mixture
was evaporated under reduced pressure and the residue was
purified by column chromatography over silica gel eluting
with hexane-ethyl acetate (4:1 to 1:1) affording a pale
yellow oil, quantitative conversion. The previous product
was dissolved in MeOH (20 mL) and NIS (5400 mg,
24 mmol) and it was placed into a round bottom flask
equipped with a magnetic stirring bar. The mixture was
stirred at room temperature for 24 h. The crude reaction
mixture was evaporated under reduced pressure and the
residue was purified by column chromatography over silica
gel eluting with hexane-ethyl acetate (4:1 to 1:1) affording
a pale yellow oil.Yield 70%, 3820 mg; 1H NMR (600 MHz,
CDCl3) d 7.50 (s, 2H, H-2), 5.71 (s, 1H, NH), 3.28 (q, 2H,
H-6), 2.66 (t, 2H, H-5), 1.44 (s, 9H, C(CH3)3).
General procedure for the synthesis of aromatic tyramides
(1-8)
Compound C or D (0.15 mmol), DIC (1.28 mmol,
200 μL), DMAP (0.5 mmol, 61 mg), aromatic acid (benzoic
acid, 4-methyl benzoic acid, 4-methoxy benzoic acid and
4-nitrobenzoic acid) (0.5 mmol) and DMF (10 mL) were
placed into a round bottom flask equipped with a magnetic
stirring bar. The mixture was stirred at room temperature
for 36 h. The crude reaction mixture was evaporated under
reduced pressure and the residue was purified by column
chromatography over silica gel eluting with hexane-ethyl
acetate (4:1 to 1:1) to obtain the aromatic iodotyramides.
Synthetic procedure for tert-butyl-(3,5-diiodo-4-methoxy-
phenethyl)-carbamate (compound B)
N-(4-Hydroxy-3,5-diiodophenethyl)-benzamide (1)
Yield 48% (51.1 mg, 0.10 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 9.33 (s, 1H, OH), 8.48 (t, J 4.7 Hz,
1H, NH), 7.78 (d, J 7.6 Hz, 2H, H-9), 7.61 (s, 2H, H-2), 7.51
(t, J 7.2 Hz, 1H, H-11), 7.45 (t, J 7.3 Hz, 2H, H-10), 3.41 (q,
J 6.1 Hz, 1H, H-6), 2.71 (t, J 6.7 Hz, 1H, H-5); 13C NMR
(151 MHz, DMSO-d6) d 166.33 (C-7), 153.58 (C-4),
139.29 (C-2), 135.85 (C-1), 134.66 (C-8), 131.05 (C-11),
128.24 (C-10), 127.10 (C-9), 87.12 (C-3), 40.60 (C-6),
32.76 (C-5); ESI-Qtof-HRMS m/z, calcd. for C15H14I2NO2
[M + H]+: 493.9114, found: 493.9159.
Compound A (1900 mg, 3.8 mmol), K2CO3 (552 mg,
4 mmol), MeI (373 μL, 6 mmol) and acetone (15 mL) were
placed into a round bottom flask equipped with a magnetic
stirring bar. The mixture was stirred at room temperature
for 24 h. The crude reaction mixture was evaporated under
reduced pressure and the residue was purified by column
chromatography over silica gel eluting with hexane-ethyl
acetate (4:1 to 1:1) affording a pale yellow oil.Yield 80%,
1500 mg; 1H NMR (600 MHz, CDCl3) d 7.59 (s, 2H, H-2),
3.84 (s, 3H, OCH3), 3.31 (q, 2H, H-6), 2.68 (t, 2H, H-5),
1.45 (s, 9H, C(CH3)3).
N-(4-Hydroxy-3,5-diiodophenethyl)-4-methylbenzamide (2)
Yield 45% (49.2 mg, 0.09 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 9.33 (s, 1H, OH), 8.40 (t, J 4.7 Hz,
1H, NH), 7.69 (d, J 7.6 Hz, 2H, H-9), 7.60 (s, 2H, H-2),
Synthetic procedure for 3,5-diiodo-tyramine (compound C)
Compound A (1000 mg), HCl 37% (1 mL) and EtOAc

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Anti-Parasite Activity of Novel 3,5-Diiodophenethyl-benzamides
J. Braz. Chem. Soc.
7.25 (d, J 7.5 Hz, 2H, H-10), 3.39 (q, J 6.0 Hz, 2H, H-6),
2.70 (t, J 6.7 Hz, 2H, H-5), 2.34 (s, 3H, CH3); 13C NMR
(151 MHz, DMSO-d6) d 166.18 (C-7), 153.57 (C-4),
140.86 (C-11), 139.28 (C-2), 135.89 (C-1), 131.85 (C-8),
128.75 (C-10), 127.12 (C-9), 87.11 (C-3), 40.58 (C-6),
32.81 (C-5), 20.95 (CH3); ESI-Qtof-HRMS m/z, calcd. for
C16H16I2NO2 [M + H]+: 507.9270, found: 507.9267.
DMSO-d6) d 166.25 (C-7), 156.68 (C-4), 140.93 (C-11),
140.12 (C-2), 139.80 (C-1), 131.81 (C-8), 128.78 (C-10),
127.13 (C-9), 91.14 (C-3), 60.72 (OCH3), 40.36 (C-6),
32.97 (C-5), 20.95 (CH3); ESI-Qtof-HRMS m/z, calcd. for
C17H17I2NO2 [M + H]+: 520.9349, found: 521.8941.
N-(3,5-Diiodo-4-methoxyphenethyl)-4-methoxybenzamide
(7)
N-(4-Hydroxy-3,5-diiodophenethyl)-4-methoxybenzamide
(3)
Yield 45% (50 mg, 0.09 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 8.37 (t, J 5.5 Hz, 1H, NH), 7.77
(d, J 8.7 Hz, 2H, H-9), 7.69 (s, 2H, H-2), 6.98 (d, J 8.7 Hz,
2H, H-10), 3.80 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.41 (q,
J 6.7 Hz, 2H, H-6), 2.74 (t, J 7.0 Hz, 2H, H-5); 13C NMR
(151 MHz, DMSO-d6) d 166.80 (C-7), 161.46 (C-11),
156.66 (C-4), 140.14 (C-1), 139.77 (C-2), 128.91 (C-9),
126.77 (C-8), 113.44 (C-10), 91.14 (C-3), 60.20 (OCH3),
55.32 (OCH3), 40.36 (C-6), 33.03 (C-5); ESI-Qtof-HRMS
m/z, calcd. for C17H18I2NO3 [M + H]+: 537.9376, found:
537.8847.
Yield 46% (51.9 mg, 0.10 mmol); white solid;1H NMR
(600 MHz, DMSO-d6) d 9.32 (s, 1H, OH), 8.34 (t, J 4.6 Hz,
1H, NH), 7.77 (d, J 7.9 Hz, 2H, H-9), 7.59 (s, 2H, H-2),
6.98 (d, J 7.9 Hz, 2H, H-10), 3.80 (s, 3H, OCH3), 3.38 (q,
J 6.3 Hz, 2H, H-6), 2.69 (t, J 6.8 Hz, 2H, H-5); 13C NMR
(151 MHz, DMSO-d6) d 166.75 (C-7), 161.43 (C-11),
153.56 (C-4), 139.25 (C-2), 135.93 (C-9), 128.91 (C-9),
113.43 (C-10), 87.12 (C-2), 55.32 (OCH3), 40.58 (C-6),
32.86 (C-5); ESI-Qtof-HRMS m/z, calcd. for C16H16I2NO3
[M + H]+: 523.9220, found: 523.9233.
N-(3,5-Diiodo-4-methoxyphenethyl)-4-nitrobenzamide (8)
Yield 45% (55.3 mg, 0.10 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 8.85 (t, J 5.5 Hz, 1H, NH), 8.31
(d, J 8.7 Hz, 2H, H-10), 8.01 (d, J 8.7 Hz, 2H, H-9), 7.70
(s, 2H, H-2), 3.71 (s, 3H, OCH3), 3.47 (q, J 6.7 Hz, 2H,
H-6), 2.77 (t, J 7.0 Hz, 2H, H-5); 13C NMR (151 MHz,
DMSO-d6) d 164.67 (C-7), 156.74 (C-4), 148.95 (C-11),
140.14 (C-8), 139.79 (C-2), 128.61 (C-9), 123.52 (C-10),
91.17 (C-3), 60.21 (OCH3), 40.56 (C-6), 32.75 (C-5);
ESI-Qtof-HRMS m/z, calcd. for C16H15I2N2O4 [M + H]+:
552.9121, found: 552.9148.
N-(4-Hydroxy-3,5-diiodophenethyl)-4-nitrobenzamide (4)
Yield 50% (58.0 mg, 0.10 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 9.34 (s, 1H, OH), 8.83 (t, J 5.0 Hz,
1H, NH), 8.31 (d, J 7.8 Hz, 2H, H-10), 8.01 (d, J 7.8 Hz,
2H, H-9), 7.61 (s, 2H, H-2), 3.44 (q, J 6.1 Hz, 2H, H-6),
2.72 (t, J 6.8 Hz, 2H, H-5);13C NMR (151 MHz, DMSO-d6)
d 164.64 (C-7), 153.65 (C-11), 148.95 (C-4), 140.22 (C-8),
139.28 (C-2), 135.61 (C-1), 128.62 (C-9), 123.52 (C-10),
87.14 (C-3), 40.77 (C-6), 32.57 (C-5); ESI-Qtof-HRMS
m/z, calcd. for C15H13I2N2O4 [M + H]+: 538.8965, found:
538.8981.
In vitro cytotoxicity
N-(3,5-Diiodo-4-methoxyphenethyl)-benzamide (5)
Yield 50% (53 mg, 0.10 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 8.51 (t, J 5.4 Hz, 1H, NH), 7.78
(d, J 7.7 Hz, 2H, H-9), 7.70 (s, 2H, H-2), 7.51 (t, J 7.3 Hz,
1H, H-11), 7.45 (t, J 7.6 Hz, 2H, H-10), 3.71 (s, 3H,
OCH3), 3.43 (q, J 6.6 Hz, 2H, H-6), 2.76 (t, J 6.9 Hz, 2H,
H-5); 13C NMR (151 MHz, DMSO-d6) d 166.40 (C-7),
156.69 (C-4), 140.08 (C-1), 139.82 (C-2), 131.11 (C-11),
128.27 (C-10), 127.11 (C-9), 91.15 (C-3), 60.22 (OCH3),
40.39 (C-6), 32.93 (C-5); ESI-Qtof-HRMS m/z, calcd. for
C16H16I2NO2 [M + H]+: 507.9271, found: 507.8814.
Cytotoxicity of the compounds was evaluated on
human U-937 macrophages (ATCC CRL-1593.2) in the
exponential growth phase and adjusted at 1 × 105 cells mL-1
in RPMI-1640 enriched with 10% fetal bovine serum (FBS).
100 mL of cells were dispensed in each well of a 96-well
microplate and 100 mL of 200, 50, 12.5 or 3.125 μg mL-1
concentration of each compound were added. Cells exposed
to compounds or standard drugs were incubated for 72 h at
37 °C and 5% of CO2. Cytotoxic activity of each compound
was determined according to the effect on the cell viability
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) micro enzymatic method in which
MTT is reduced to a purple product named formazan by
mitochondrial enzyme succinate dehydrogenase. Thus,
10 μL well-1 were added to each well, and plates were
incubated at 37 °C, 5% CO2 for 3 h. The reaction was
stopped by adding 100 μL well-1 of isopropanol with 50 and
N-(3,5-Diiodo-4-methoxyphenethyl)-4-methylbenzamide (6)
Yield 45% (48.9 mg, 0.09 mmol); white solid; 1H NMR
(600 MHz, DMSO-d6) d 8.43 (t, J 5.5 Hz, 1H, NH), 7.69
(d, J 6.4 Hz, 4H, H-9, H-2), 7.25 (d, J 8.0 Hz, 2H, H-10),
3.71 (s, 3H, OCH3), 3.42 (q, J 6.7 Hz, 2H, H-6), 2.75 (t,
J 7.0 Hz, 2H, H-5), 2.34 (s, 3H, CH3); 13C NMR (151 MHz,

Vol. 30, No. 1, 2019
Restrepo et al.
121
10% of sodium dodecyl sulfate (SDS). The concentration
of formazan was spectrophotometrically determined at
570 nm (Varioskan, Thermo) and the intensity of the color
(absorbance) was registered as optical densities (O.D.).
Cells exposed to control drugs chloroquine, amphotericin
B, benznidazole, and doxorubicin were used as positive
control for toxicity, while cell incubated in RPMI-1640
medium alone were used as negative control. Non-specific
absorbance was corrected by subtracting absorbance (O.D.)
of the blank. Determinations were done by triplicate in at
least two independent experiments.11
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 employing a flow
cytometer (Cytomics FC 500MPL) reading at 488 nm
(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 amphotericin B (standard anti-
leishmanial drug) were used as control for anti-leishmanial
activity (positive control), while infected cells incubated in
absence of any compound or drug were used as control for
infection (negative control). Nonspecific fluorescence was
corrected by subtracting fluorescence of unstained cells.All
determinations were performed by triplicate in at least two
independent experiments.11,13
Hemolytic activity
The ability to induce hemolysis was specifically
evaluated to compounds which showed antiplasmodial
activity following the method of cytotoxicity by
spectrophotometry on 96-well plates. Briefly, 500 μL of
human red blood cells (huRBC), adjusted to 5% hematocrit
in RPMI-1640 medium, were placed into each well of a
24-well plate and subsequently exposed to 200 μg mL-1
of compounds 1-8. Detection of free hemoglobin, after
48 h of incubation at 37 °C, was the evidence that the
compound induced hemolysis. The concentration of
free hemoglobin was spectrophotometrically read at
542 nm (Varioskan, Thermo) and O.D. were registered.
Non-specific absorbance was corrected by subtracting
absorbance of the blank. Determinations were done by
triplicate in at least two independent experiments.12
In vitro anti-trypanosomal activity
To test the effectiveness of compounds, 25,000 U-937
human macrophages/100 µL RPMI-1640 enriched with
10% FBS and 10 ng PMA were placed in each well of
96-wells cell culture plate. Cells were then infected with
epimastigotes (24 h of growing) of T. cruzi Tulahuen strain
transfected with β-gal gene (donated by Dr F. S. Buckner,
University of Washington) in 5:1, parasites:cell ratio.
After infection, 100 µL of each compound at 50, 12.5,
3.125 or 0.78 μg mL-1 were added to each well and plates
were incubated during 72 h at 37 ºC, 5% CO2. Finally,
the effect of each compound and each concentration on
the viability of intracellular parasites was determined by
measuring β-gal activity by colorimetric method after
adding 100 µM CPRG and 0.1% nonidet P-40. After
3 h of incubation at room temperature plates were read
at 570 nm in a spectrophotometer (Varioskan, Thermo)
and absorbance was recorded as O.D. Anti-trypanosomal
activity of benznidazole was used as positive control,
while RPMI-1640 medium was used as negative control.
Non-specific absorbance was corrected by subtracting O.D.
of the blank. Determinations were done by triplicate in at
least two independent experiments.14
In vitro anti-leishmanial activity
Anti-leishmanial activity of iodotyramides was
determined according to the ability of the compound to
reduce infection by L. panamensis parasites.13 Briefly, U-937
human cells at a density of 3 × 105 cells mL-1 in RPMI 1640
and 0.1 μg mL-1 of phorbol-12-myristate-13-acetate (PMA)
were dispensed on 24-wells microplate and then infected
with stationary phase growing L. panamensis promastigotes
(MHOM/CO/87/UA140-E-GFP strain) in a 15:1 parasites
per cell ratio. Plates were incubated at 34 °C and 5% CO2
for 3 h and then 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 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 incubated at 37 °C and 5% CO2 for 72 h;
then, cells were removed from the bottom plate with a
100 μL trypsin/ethylenediaminetetracetic acid (EDTA)
In vitro anti-plasmodial activity
Asynchronized P. falciparum 3D7 strain cultures
were adjusted to 0.5% parasitemia and 1% hematocrit in
RPMI medium enriched with 3% lipid-rich bovine serum
albumin-Albumax II. Then, 100 μL of parasite suspension

122
Anti-Parasite Activity of Novel 3,5-Diiodophenethyl-benzamides
J. Braz. Chem. Soc.
was dispensed into each well of 96-wells cell culture plate
and subsequently exposed to 100 μL of each compound
at 100, 25, 6.25 or 1.56 μg mL-1. Plates were incubated
for 48 h at 37 °C in N2 (90%), CO2 (5%) and O2 (5%)
atmosphere.After incubation, parasites were harvested and
subjected to three 20 min freeze-thaw cycles. Meanwhile,
100 μL of Malstat reagent (400 μL Triton X-100 in 80 mL
deionized water, 4 g L-1 lactate, 1.32 g Tris buffer and
0.022 g acetylpyridine adenine dinucleotide in 200 mL
deionized water; pH 9.0) and 25 mL of NBT/PES solution
(0.16 g nitroblue tetrazolium salt and 0.08 g phenazine
ethosulphate in 100 mL deionized water) were added to
each well of a second 96-well plate. After freeze-thaw
cycles, 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 (lactate
dehydrogenase) reaction was read in a spectrofluorometer
(Varioskan, Thermo) at 650 nm. The intensity of color in
each experimental condition was registered as fluorescent
units (F.U.). Non-specific fluorescence was corrected by
subtracting F.U. of the blank. Determinations were done
by triplicate in at least two independent experiments.
Chloroquine (CQ) was used as positive control and culture
medium was used as negative control.15
Anti-plasmodial activity of each evaluated compound
was evidenced by the reduction of growing parasites
calculated according to equation 3:
(3)
Anti-trypanosomal activity was calculated by reduction
of infection (viable parasites) in each tested concentration.
For this, reduction of the number of parasites was calculated
with equation 4:
(4)
Percentages of reduction of parasites data were used
to calculate the EC50 using the linear regression model
Probit.16
Anti-leishmanial, anti-plasmodial and anti-
trypanosomal activities were graded as high, moderate
or low according to the EC50 values, as follow: high
activity when the EC50 < 25 μg mL-1; moderate activity
EC50 ranging between 25-50 μg mL-1 and low activity
when the EC50 > 50 μg mL-1. SI was calculated by
dividing the cytotoxic activity and the anti-leishmanial,
anti-trypanosomal and antiplasmodial activity using the
following formula: SI = LC50/EC50.
Supplementary Information
Statistical analysis
Supplementary data (1H, 13C and 2D NMR spectra,
and ESI-Qtof-HRMS) are available free of charge at
http://jbcs.sbq.org.br as a PDF file.
Cytotoxicity of iodotyramides was tested in comparison
to doxorubicin, amphotericin B, benznidazole, chloroquine
and RPMI-1640 culture medium. The results were expressed
as LC50 that corresponds to the concentration necessary to
eliminate 50% of cells and calculated by Probit analysis. Cell
growth inhibition was calculated by equation 1:
Acknowledgments
The authors would like to thank the Departamento
Administrativo de Ciencia, Tecnología e Innovación de
Colombia-Colciencias (grant CT-695-2014) and to Comité
para el Desarrollo de la Investigación, Universidad de
Antioquia (Proyect CODI CIQF-176) for their financial
support.
(1)
The degree of toxicity was graded according
to the LC50 value using the following scale: high
cytotoxicity: LC50 < 100 μg mL-1; moderate cytotoxicity:
LC50 100-200 μg mL-1 range and potentially non-cytotoxicity:
LC50 > 200 μg mL-1.
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well corresponds to 100% of infection.
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