Synthesis and in vitro evaluation of Ca2 + channel blockers 1,4-dihydropyridines analogues against Trypanosoma cruzi and Leishmania amazonensis: SAR analysis

Article abstract of DOI:10.1016/j.parint.2017.08.005

Drugs containing the1,4-dihydropyridine (DHP) core have recently attracted attention concerning their antiparasitic effect against various species of Leishmania and Trypanosoma. This approach named drugs repositioning led to interesting results, which have prompted us to prepare 21 DHP's analogues. The 1,4-DHP scaffold was decorated with different function groups at tree points including the nitrogen atom (NH and N-phenyl), the aryl group attached to C-4 (various substituted aryl residues) and the carbon atoms 2 and 6 (bearing Ph or Me groups). Moreover, the products were evaluated for their cytotoxicity on three cancer and a non-tumoral cell lines. Only 6 of them were antiproliferative and their weak effect (CC₅₀ comprised between 27 and 98 μM) suggested these DHPs as good candidates against the intracellular amastigote forms of L. amazonensis and T. cruzi. L. amazonensis was sensitive to DHPs 5, 11 and 15 (IC₅₀ values at 15.11, 45.70 and 53.13 μM, respectively) while 12 of them displayed significant to moderate trypanocidal activities against T. cruzi. The best trypanocidal activities were obtained with compounds 2, 18 and 21 showing IC₅₀ values at 4.95, 5.44, and 6.64 μM, respectively. A part of the N-phenylated DHPs showed a better selectivity than their NH analogues towards THP-1 cells. 4-Chlorophenyl, 4-nitrophenyl and 3-nitrophenyl residues attached to the carbon atom 4 turned to be important sub-structures for the antitrypanosomal activity.

Full text of DOI:10.1016/j.parint.2017.08.005

Accepted Manuscript
Synthesis and in vitro evaluation of Ca2+ channel blockers
1,4-dihydropyridines analogues against Trypanosoma cruzi and
Leishmania amazonensis: SAR analysis
Luiz A.E. Pollo, Milene H. de Moraes, Júlia Cisilotto, Tânia B.
Creczynski-Pasa, Maique W. Biavatti, Mario Steindel, Louis P.
Sandjo
PII:
DOI:
Reference:
S1383-5769(17)30005-3
doi: 10.1016/j.parint.2017.08.005
PARINT 1706
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Parasitology International
Received date:
Revised date:
Accepted date:
5 January 2017
7 August 2017
8 August 2017
Please cite this article as: Luiz A.E. Pollo, Milene H. de Moraes, Júlia Cisilotto, Tânia
B. Creczynski-Pasa, Maique W. Biavatti, Mario Steindel, Louis P. Sandjo , Synthesis
and in vitro evaluation of Ca2+ channel blockers 1,4-dihydropyridines analogues against
Trypanosoma cruzi and Leishmania amazonensis: SAR analysis. The address for the
corresponding author was captured as affiliation for all authors. Please check if
appropriate. Parint(2017), doi: 10.1016/j.parint.2017.08.005
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ACCEPTED MANUSCRIPT
Synthesis and in vitro evaluation of Ca2+ channel blockers 1,4-dihydropyridines analogues
against Trypanosoma cruzi and Leishmania amazonensis: SAR analysis.
Luiz A. E. Polloa, Milene H. de Moraesb, Júlia Cisilottoa, Tânia B. Creczynski-Pasaa, Maique W. Biavattia,
Mario Steindelb*, Louis P. Sandjoa**
aDepartment of Pharmaceutical Sciences, Universidade Federal de Santa Catarina, 88040-900,
Florianópolis, SC, Brazil
bDepartment of Microbiology, Immunology and Parasitology, Universidade Federal de Santa Catarina,
88040-900 Florianópolis, SC, Brazil
* Corresponding authors. Tel.:+55 4837213408, E-mail: p.l.sandjo@ufsc.br(L.P. Sandjo);
Tel: +55 48 37212958; fax: +55 4837212011,E-mail: mario.steindel@ufsc.br(M.Steindel).
1

ACCEPTED MANUSCRIPT
Abstract
Drugs containing the1,4-dihydropyridine (DHP) core have recently attracted attention concerning their
antiparasitic effect against various species of Leishmania and Trypanosoma. This approach named drugs
repositioning led to interesting results, which have prompted us to prepare 21 DHP’s analogues. The
1,4-DHP scaffold was decorated with different function groups at tree points including the nitrogen atom
(NH and N-phenyl), the aryl group attached to C-4 (various substituted aryl residues) and the carbon
atoms 2 and 6 (bearing Ph or Me groups). Moreover, the products were evaluated for their cytotoxicity on
three cancer and a non-tumoral cell lines. Only 6 of them were antiproliferative and their weak effect
(CC50 comprised between 27-98 μM) suggested these DHPs as good candidates against the intracellular
amastigote forms of L. amazonensis and T. cruzi. L. amazonensis wassensitive to DHPs 5, 11 and 15
(IC50 values at 15.11, 45.70 and 53.13 μM, respectively) while 12 of them displayed significant to
moderate trypanocidal activities against T. cruzi. The best trypanocidal activities were obtained with
compounds 2, 18 and 21 showing IC50 values at 4.95, 5.44, and 6.64 μM, respectively. A part of the
N-phenylated DHPs showed a better selectivity than their NH analogues towards THP-1 cells.
4-Chlorophenyl, 4-nitrophenyl and 3-nitrophenyl residues attached to the carbon atom 4 turned to be
important sub-structures for the antitrypanosomal activity.
Keywords
1,4-dihydropyridines, Leishmanicidal activity, Trypanocidal activity, SAR.
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1. Introduction
Data reported in 2016 by WHO revealed that 6 to 7 million people worldwide are suffering from Chagas
disease, which is endemic in 21 Latin America countries with an annual incidence of 28 thousand cases
[1]. In the particular case of Trypanosoma cruzi, the transmission to humans mainly occurs through
excreta deposition, after biting of contaminated triatomines, blood transfusion from infected donors, the
consumption of infected food, organ transplantation and childbirth from infected mother [1].
Leishmaniasis, another worldwide vector-borne disease is caused by protozoan parasites of the genus
Leishmania with an estimation of 1.5 to 2 million new cases annually. Human leishmaniasis ranges from
self-healing skin ulcers to mucosal lesions up to a life-threatening progressive visceral disease [2].
Moreover, about 90% of reported human visceral leishmaniasis occurs in Brazil, Ethiopia, India, Somalia,
South Sudan and Sudan [2]. Chagas disease and leishmaniasis represent a difficult healthcare problem in
Brazil, especially because of the lack of vaccines and the existence of pernicious side effects associated
with these diseases treatments [3, 4]. Moreover, the cure also requires a long-term therapy [4]. Therefore,
search for unmet efficient and inexpensive chemotherapy remain imperative. DHPs acting as Ca2+
channel blockers (CCB) such as amlodipine, lacidipine, nimodipine, lercanidipine, nicardipine, bepridil,
cilnidipine nefidipine, azelnidipine and nitrendipine have been largely studied for their antiparasitic effect
against various species of Leishmania and Trypanosoma [5-8]. It turned out that severalof these pyridine
analogues are endowed with promising antiprotozoal activity either as a single compound or in
combination with other drugs [6]. Moreover, synthetic N-aryl-4-glycosylDHPs derivatives showed
interesting inhibitory effect against L. donovani [9]. The theory put forward is based on the ability of
DHPs containing compounds to suppress the calcium channel action in cell membranes [10]. This
mechanism has been considered for the search of new antiparasitic compounds since Ca2+ ions in
eukaryotic organisms (trypanosomatids) act as a second messenger that regulates various cellular
processes [10]. Moreover, previous studies demonstrated that during the invasion of the host cells, there is
an increase in the cytolosic calcium concentration in T. cruzi trypomastigotes [11]. Likewise, the
virulence during the invasion in L. mexicana amazonensis amastigotes is associated to their intracellular
Ca2+ pool content [12]. Therefore, search for compounds that can compromise the Ca2+ channel action in
these parasites represents one of the approaches to be explored. The use of Ca2+ channel blockers
derivatives in this study to target parasitic diseases joins the drug discovery strategy named drugs
repositioning or repurposing. Drug repositioning consists in giving an additional pharmacological
potential to a drug by targeting illness other than that for which it was initially intended. Based on the
results obtained from the repurposing assay, the drug itself can undergo some hemisynthesis or can
inspire the preparation of analogues. A number of drugs repurposing has been successfully made in
neglected tropical diseases (NTDs) and has also inspired the preparation of biological promising entities;
for instance, eflornithine an anticancer compound, is being used for the treatment of the African sleeping
sickness (caused by T. brucei gambiense and T. brucei rhodesiense) whereas pentanidine and
amphotericin B initially antifungal drugs are prescribed today as leishmaniasis chemotherapies. Besides,
miltefosine developed for breast cancer and other tumors, is actually employed as topical formulation
against skin metastases and for the treatment of leishmaniasis [13, 14]. The hemisynthetic derivative of
artemisinin, dihydroartemisinin used for the treatment of malaria is also endowed with promising activity
against the human metastatic melanoma cells [15]. On the Basis of the aforementioned observation, this
study aims to report the synthesis of 21 DHPs including 13 NH analogues and eight N-phenyl derivatives.
These compounds have been evaluated for their cytotoxic activity against three cancer cells and a
non-tumoral cell line as well as for their inhibitory effect against the intracellular amastigotes form of L.
amazonensis and T. cruzi.
2. Experimental section
2.1. General methods
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Vacuum liquid chromatography (VLC), column chromatography (CC) and thin layer chromatography
(TLC) were performed on silica gel 60 (particle size 90 % <45 mm), 200–300 mesh silica gel, and silica
gel GF254 (Merck), respectively. The spots were visualized using ultraviolet light at 254 and 366 nm.
NMR spectra were recorded on a Bruker Avance operating at 300 MHz. The accurate mass spectra were
recorded on a Xevo G2-S QTOF machine equipped with an electrospray probe. xlogP and PSA values
were calculated by using SwissTargetPrediction website.
2.2. Preparation of DHPs 1-7.
An amount of 0.2 g of an aldehyde (purchased from Sigma Aldrich) was dissolved in ethanol to give a
0.25 M solution, then 2.4 equivalents of ethyl acetoacetate (purchased from Sigma Aldrich) was added
with 1.2 equivalents of NH4OAc (purchased from Sigma Aldrich) in the reaction flask, the reaction
mixture was stirred under reflux temperature between 16 to 68 hours. The reaction was stopped and the
solvent was removed under reduced pressure, the crude reaction mixture was then subjected to a
liquid/liquid extraction system with brine/ethyl acetate, the combined organics were dried with Na2SO4,
concentrated under reduced pressure, and submitted to a chromatographic purification.
2.2.1. Diethyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1).
The reaction ran for 48 h and the compound was purified by flash column chromatography (FCC) using a
gradient from 100% CH2Cl2 to 10% MeOH in CH2Cl2, yielding a pale green solid (0.2000 g, 43%). 1H
NMR (300 MHz, CDCl3) δ 6.88 (d, J = 1.85 Hz, 1H), 6.80 (dd, J = 1.85, 8.30 Hz, 1H), 6.73 (d, J = 8.30
Hz, 1H), 5.63 (s, 1H), 4.94 (s, 1H), 4.10 (dq, J = 2.48, 7.13 Hz, 4H), 3.84 (s, 3H), 3,82 (s, 3H), 2.34 (s,
6H), 1.24 (t, J = 7.13 Hz 6H). 13C NMR (75 MHz, CDCl3) δ 167.7, 148.1, 147.3, 146.6, 119.8, 111.7,
110.8, 104.3, 59.8, 55.8, 39.1, 19.7, 14.4; HRESIMS m/z 412.1728 ([C21H27NO6+Na]+ calcd for m/z
412.1731).
2.2.2. Diethyl 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (2).
The reaction last 68 h and the compound was purified by FCC, in a gradient from 100% CH2Cl2 to 10%
MeOH in CH2Cl2, yielding a pale green solid (0.0655 g) 13 %. 1H NMR (300 MHz, CDCl3) δ 8.09 (d, J =
8.36 Hz, 2H), 7.45 (d, J = 8.36 Hz, 2H), 5.78 (s, 1H), 5.10 (s, 1H), 4.10 (q, J = 7.12 Hz, 4H), 2.36 (s, 6H),
1.21 (t, J = 7.12 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 167.1, 155.1, 144.6, 128.9, 123.3, 103.2, 92.1,
60.0, 40.1, 19.7, 14.3; HRESIMS m/z 397.1367 ([C19H22N2O6+Na]+ calcd for m/z 397.1370).
2.2.3. Diethyl 2,6-dimethyl-4-(4-hydroxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (3).
The reaction ran for 48 h and the compound was purified by FCC, in a gradient from 100% CH2Cl2 to
1
10% MeOH in CH2Cl2, yielding a pale yellow solid (0.3217 g, 57%). H NMR (300 MHz, DMSO-d6) δ
9.10 (s, 1H), 8.72 (s, 1H), 6.93 (d, J = 8.43 Hz, 2H), 6.58 (d, J = 8.43, 2H), 4.74 (s, 1H), 3.98 (m, 4H),
13
2.23 (s, 6H), 1.13 (t, J = 7.06 Hz, 6H). C NMR (75 MHz, DMSO-d6) δ 167.6, 155.9, 145.2, 139.4, 128.8,
115.0, 102.7, 59.4, 38.3, 18.7, 14.7; HRESIMS m/z 368.1419 ([C19H23NO5+Na]+ calcd for m/z 368.1468).
2.2.4. Diethyl 4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5- dicarboxylate (4)
The reaction ran for 44 h and the compound was purified by FCC, in a gradient from 100% CH2Cl2 to 2%
MeOH in CH2Cl2, yielding a pale green solid (0.2093 g, 43 %). 1H NMR (300 MHz, CDCl3) δ 8.09 (d, J =
8.36 Hz, 2H), 7.45 (d, J = 8.36 Hz, 2H), 5.78 (s, 1H), 5.10 (s, 1H), 4.10 (q, J = 7.12 Hz, 4H), 2.36 (s, 6H),
1.21 (t, J = 7.12 Hz 6H). 13C NMR (75 MHz, CDCl3) δ 167.8, 145.8, 143.9, 143.6, 140.1, 120.5, 113.8,
110.9, 104.3, 59.8, 55.8, 39.1, 19.7, 14.4; HRESIMS m/z 398.1525 ([C20H25NO6+Na]+ calcd for
398.1574).
2.2.5. (E)-diethyl 2,6-dimethyl-4-styryl-1,4-dihydropyridine-3,5-dicarboxylate (5)
The reaction ran for 29 h and the compound was purified by flash column chromatography, in a gradient
from 100% CH2Cl2 to 2% MeOH in CH2Cl2 yielding a pale yellow solid (0.5088 g, 63 %). 1H NMR (300
MHz, CDCl3) δ 7.25 (m, 5H), 6.25 (d, J = 15.9 Hz 1H), 6.14 (dd, J = 15.9, 6.15 Hz, 1H), 5.89 (s, 1H),
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4.64 (d, J = 6.14 Hz, 1H), 4.2 (m, 4H), 2.33 (s, 6H), 1.30 (t, J = 7.10 Hz, 6H). 13C NMR (75 MHz, CDCl3)
δ 167.7, 145.0, 137.8, 131.8, 128.4, 128.1, 126.9, 126.2, 101.4, 59.8, 36.5, 19.5, 14.4; HRESIMS m/z
378.1633 ([C21H24NO4+Na]+ calcd for 378.1676).
2.2.6. Diethyl 2,6-dimethyl-4-(3,4,5-trimethoxyphenyl)-1,4-dihydropyridine-3,5- dicarboxylate (6).
The reaction ran for 16 h and the compound was purified by FCC, in a gradient from 100% CH2Cl2 to 2%
MeOH in CH2Cl2 yielding a bright yellow solid (0.3967 g, 93 %). 1H NMR (300 MHz, CDCl3) δ 6.52 (s,
2H), 5.68 (s, 1H), 4.98 (s, 1H), 4.14 (m, 4H), 3.80 (s, 9H), 2.36 (s, 6H), 1.26 (t, J = 7.1 Hz 6H). 13C NMR
(75 MHz, CDCl3) δ 167.7, 152.6, 143.8, 143.4, 136.4, 104.9, 104.0, 60.8, 59.8, 55.9, 39.7, 19.7, 14.4;
HRESIMS m/z 442.1837 ([C22H29NO7+Na]+ calcd for m/z 442.1836).
2.2.7. Diethyl 4-(4-hydroxy-3,5-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5- dicarboxylate
(7).
The reaction ran for 20 h and the compound was purified by FCC, in a gradient from 100% CH2Cl2 to 2%
MeOH in CH2Cl2 yielding a bright yellow solid (0.1991 g, 45 %). 1H NMR (300 MHz, CDCl3) data: δ
6.54 (s, 2H), 5.65 (s, 1H), 5.38 (s,1H), 4.96 (s, 1H), 4.13 (m, 4H), 3.84 (s, 6H), 2.35 (s, 6H), 1.26 (t, J =
7.12 Hz, 6H). 13C NMR (75 MHz, CDCl3) data: δ 167.7, 146.5, 143.6, 139.1, 133.2, 104.8, 104.2, 59.8,
56.2, 39.5, 19.7, 14.4; HRESIMS m/z 428.1654 ([C21H27NO7+Na]+ calcd for m/z 428.1680).
2.3. Preparation of N-phenyl DHPs (8-15).
An amount of 0.2 g of the aldehyde was dissolved in ethanol to reach a 0.25 M solution, then 2.4
equivalents of ethyl acetoacetate were added with 1.2 equivalents of aniline in the reaction flask, the
reaction mixture was stirred under reflux temperature between 16 to 30 hours. The reaction was stopped
and the solvent was dried under reduced pressure, the crude reaction mixture was then subjected to a
liquid/liquid extraction system with brine/ethyl acetate, the combined organics were dried with anhydrous
Na2SO4, concentrated under reduced pressure, and purified by a chromatographic column.
2.3.1. Diethyl-4-(4-chlorophenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-3,5-
dicarboxylate (8).
The reaction ran for 20 h and the compound was purified by FCC, in a gradient from 70% to 100%
CH2Cl2 in hexane yielding a yellow solid (0.2770 g, 44 %). 1H NMR (300 MHz, CDCl3) δ 7.46 (m, 3H),
7.35 (d, J 8.32 Hz 2H), 7.28 (d, J = 8.32 Hz, 2H), 7.14 (m, 2H), 5.12 (s, 1H), 4.16 (q, J = 7.08 Hz 4H),
2.06 (s, 6H), 1.26 (t, J = 7.08 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 168.0, 147.5, 145.7, 140.3, 131.8,
130.2, 129.5, 128.9, 128.8, 128.2, 105.5, 60.1, 38.4, 18.7, 14.3; HRESIMS m/z 440.1642
(C25H26ClNO4+H]+ calcd for m/z 440.1623).
2.3.2. Diethyl-(4-nitrophenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (9).
The reaction ran for 28 h and the compound was purified by FCC, in a gradient 100% CH2Cl2 to 2%
MeOH in CH2Cl2 and further separated by reverse phase chromatography with 20% water in MeOH,
yielding a yellow solid (0.0459 g, 7%). 1H NMR (300 MHz, CDCl3) δ 8.17 (m, 2H), 7.57 (m, 2H), 7.47
(m, 3H), 7.13 (m, 2H), 5.24 (s, 1H), 4.15 (q, J = 7.10 Hz, 4H), 2.07 (s, 6H), 1.25 (t, J = 7.10 Hz, 6H). 13C
NMR (75 MHz, CDCl3) δ 167.6, 154.6, 148.2, 146.4, 140.0, 130.1, 129.7, 129.0, 123.5, 104.7, 60.3, 39.2,
18.8, 14.3; HRESIMS m/z 451.1853 ([C25H26N2O6+H]+ calcd m/z 451.1864).
2.3.3. Diethyl 4-(4-bromophenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-3,5- dicarboxylate (10).
The reaction ran for 24 h, the crude mixture was left to crystallize over 48 h by slow solvent evaporation,
the solid were filtered and washed with ethanol, yielding a white solid (0.1561 g, 30%). 1H NMR (300
MHz, CDCl3) δ 7.45 (m, 5H), 7.28 (m, 2H), 7.13 (m, 2H), 5.10 (s, 1H), 4.15 (q, J = 7.08 Hz, 4H), 2.06 (s,
6H), 1.26 (t, J = 7.08 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 167.9, 147.5, 131.1, 130.2, 129.5, 129.3,
128.8, 105.4, 60.1, 38.5, 18.7, 14.2; HRESIMS m/z 484.1116 ([C25H26BrNO4+H]+ calcd for m/z
484.1118).
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2.3.4. Diethyl 4-(4-hydroxyphenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-3,5- dicarboxylate (11).
The reaction ran for 16 h, and the compound was purified byFCC, in a gradient from 100% CH2Cl2 to 2%
MeOH in CH2Cl2 yielding a yellow solid (0.1203 g, 17%). 1H NMR (300 MHz, CDCl3) data: δ 7.43 (m,
3H), 7.23 (d, J 8.45 Hz 2H), 7.14 (m, 2H), 6.74 (d, J = 8.45 Hz, 2H), 5.08 (s, 1H), 4.15 (q, J = 7.26 Hz,
4H), 2.05 (s, 6H), 1.25 (s, J = 7.12, 6H). 13C NMR (75 MHz, CDCl3) data: δ 168.8, 154.7, 147.2, 140.4,
138.9, 130.2, 129.5, 128.7, 128.6, 115.0, 106.1, 60.3, 38.0, 31.0, 18.8, 14.3; HRESIMS m/z 422.1924
([C25H27NO5+H]+ calcd for m/z 422.1962), m/z 376.1530 [M-EtOH]+.
2.3.5. Diethyl 2,6-dimethyl-1-phenyl-4-(3,4,5-trimethoxyphenyl)-1,4-dihydropyridine-3,5- dicarboxylate
(12).
The reaction last 30 h, and the compound was purified by flash column chromatography, in a gradient
from 50% CH2Cl2 in hexane to 100% CH2Cl2 and to 2% MeOH in CH2Cl2 yielding a yellow solid (0.1824
g, 36%). 1H NMR (300 MHz, CDCl3) δ 7.39 (m, 3H), 7.08 (m, 2H), 6.61 (s, 2H), 5.11 (s, 1H), 4.14 (q, J =
13
7.10 Hz 4H), 3.81 (s, 6H), 3.79 (s, 3H), 2.03 (s, 6H), 1.24 (t, J = 7.10 Hz, 6H). C NMR (75 MHz, CDCl3)
δ 168.1, 152.8, 147.2, 142.7, 140.3, 136.3, 130.1, 129.5, 128.7, 119.9, 105.6, 104.3, 60.7, 60.0, 55.9, 38.8,
18.7, 14.4; HRESIMS m/z 496.2360 ([C28H32NO7+H]+ calcd for m/z 496.2330), m/z 518.2161 [M+Na]+.
2.3.6. Diethyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-3,5- dicarboxylate
(13).
The reaction ran for 30 h, and the compound was purified by flash column chromatography, in a gradient
from 70% CH2Cl2 in hexane to 100% CH2Cl2, yielding a yellow solid (0.2275 g, 41%). 1H NMR (300
MHz, CDCl3) δ 7.37 (m, 3H), 7.07 (m, 2H), 6.96 (d, J = 1.62 Hz, 1H), 6.88 (dd, J = 1.62, 8.12 Hz 1H),
6.78 (d, J 8.12 Hz 1H), 5.06 (s, 1H), 4.11 (q, J = 7.10 Hz, 4H), 3.83 (s, 3H), 3.80 (s, 3H), 2.01 (s, 6H),
1.21 (t, J = 7.10 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 168.2, 148.3, 147.3, 147.1, 140.4, 140.0, 130.2,
129.4, 128.7, 119.1, 111.2, 110.9, 105.9, 60.0, 55.8, 55.7, 38.2, 30.8, 18.6, 14.3; HRESIMS m/z 466.2261
([C27H32NO6+H]+ calcd for m/z 466.2224), m/z 488.2027 [M+Na]+.
2.3.7. Diethyl 4-(4-hydroxy-3-methoxyphenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-
3,5-dicarboxylate (14).
The reaction ran for 16 h, and the compound was purified by flash column chromatography, in a gradient
from 70% CH2Cl2 in hexanes to 100% CH2Cl2, yielding a brown solid (0.1375 g, 23%). 1H NMR (300
MHz, CDCl3) δ 7.42 (m, 3H), 7.12 (m, 2H), 6.97 (s, 1H), 6.84 (s, 2H), 5.08 (s, 1H), 4.14 (q, J = 7.12 Hz,
4H), 3.84 (s, 3H), 2.04 (s, 6H), 1.25 (t, J = 7.12 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 168.3, 147.1,
146.2, 144.0, 140.4, 139.2, 130.2, 129.5, 128.7, 119.7, 114.1, 110.6, 105.9, 60.0, 55.7, 38.3, 18.7, 14.3;
HRESIMS m/z 452.2027 ([C26H30NO6+H]+ calcd for m/z 452.2068), m/z 474.1886 [M+Na]+.
2.3.8. Diethyl 4-(4-hydroxy-3,5-dimethoxyphenyl)-2,6-dimethyl-1-phenyl-1,4-dihydro pyridine-
3,5-dicarboxylate (15).
The reaction ran for 20 h, and the compound was purified by flash column chromatography, in a gradient
from 70% CH2Cl2 in hexane to 100% CH2Cl2, yielding a dark brown solid (0.0740 g, 15%). 1H NMR
(300 MHz, CDCl3) δ 7.42 (m, 3H), 7.11 (m, 2H), 6.64 (s, 2H), 5.09 (s, 1H), 4.16 (q, J = 7.12 Hz 4H), 3.85
(s, 6H), 2.05 (s, 6H), 1.26 (t, J = 7.12 Hz 6H). 13C NMR (75 MHz, CDCl3) δ 168.2, 147.1, 146.7, 140.4,
138.3, 133.2, 130.2, 129.5, 128.7, 105.8, 104.2, 60.0, 56.2, 38.6, 30.9, 18.7, 14.4; HRESIMS m/z
482.2185 ([C27H32NO7+H]+ calcd for m/z 482.2185), m/z 518.1970 [M+Na]+.
2.4. Cytotoxicity assay.
Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 medium, penicillin, streptomycin,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and resazurin were acquired from
Sigma-Aldrich (Missouri, EUA). Fetal bovine serum was acquired by Gibco® (Waltham, MA USA) and
Dimethylsulfoxide (DMSO) was purchased from Vetec (Rio de Janeiro, Brazil). All compounds were
dissolved in DMSO for the study of the viability in cancer cells, stock solution of 30 µM, and stored at
-20°C.
6

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2.4.1. Cell Culture
Cell lines were obtained from Rio de Janeiro Cell Bank (BCRJ, Brazil). The human breast cancer MCF-7
and mouse breast cancer 4-T1 cell lines were cultured in RPMI and human melanoma SK-MEL-28,
human breast cancer MDA-MD-231 and normal mouse fibroblast NIH-3T3 cell lines were cultured in
DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and
10 mM HEPES. Cells were maintained at 37 °C in a CO2 5% humidified atmosphere.
2.4.2. Evaluation of cell viability in cancer cell lines and selectivity index
The cell viability was evaluated by Alamar Blue (resazurin) assay. Cells (1×104/well) were plated in
96-well culture microplate and incubated for 24 h with increasing concentrations of the compounds,
ranging from 5 μM to 100 μM. The same final concentration of DMSO (1%) without compounds was
used as negative control. Resazurin (150 µM/mL) was incubated by 4 h and the plate was read in
fluorescence spectrometer – LS55 PerkinElmer (530 nm excitation, 590 nm emission). The concentration
required to reduce the cell number by 50% (CC50) was determined by nonlinear regression analysis of the
logarithm of concentration as a function of the normalized response using the software Prism 5.0
(GraphPad Software). To obtain the selectivity index (SI), the CC50 of non-tumoral cell was divided by
CC50 of tumoral cell.
2.5. Antiparasitic assay
2.5.1. Trypanocidal and Leishmanicidal screening against intracellular amastigotes.
T. cruzi trypomastigotes expressing β-galactosidase were raised from infected L929 cell line maintained
at 37°C, CO2 5% in RPMI-1640 medium supplemented with FBS 5%. L. amazonensis
MHOM/BR/77/LTB0016 promastigotes, expressing β-galactosidase, were grown at 26°C, in Schneider's
insect medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with heat inactivated FBS 5%
and human urine 2%.
THP-1 cell line (ATCC TIB202) was grown at 37°C, 5% CO2, in RPMI-1640 without phenol red
(Sigma-Aldrich, CO. St. Louis, MO, USA) supplemented with FBS 10%, HEPES 12.5 mM, penicillin
(100 U/mL), streptomycin (100 μg/mL) and Glutamax (2 mM).
For assays, cells were cultivated in 96 well microplates (4.0 × 104 cells/well) in supplemented RPMI-1640
and treated with phorbol 12-myristate 13-acetate (PMA) 100 ng/mL for 72 h (37°C, CO2 5%) to allow cell
differentiation into non-dividing macrophages.
Four days old culture promastigotes of L. amazonensis (4.0 × 106 parasites/mL) was washed with
phosphate buffered saline (PBS, pH 7.4) and incubated in RPMI-1640 supplemented with 10% of
heat-inactivated human B+ serum for 1 h (34°C) to parasite opsonization. THP-1 cells were then
incubated with a parasite/cell ratio of 10:1 for 3 h (34°C, CO2 5%) for L. amazonensis and with a
parasite/cell ratio of 2:1 overnight (37 °C, CO2 5%) for T. cruzi. Thereafter, non-adherent parasites were
removed by two washes with PBS and infected cells incubated with 180 μL of full supplemented
RPMI-1640 medium for another 24h (34°C or 37°C, CO2 5%) to allow the transformation of
promastigotes/trypomastigotes into intracellular amastigotes.
The infected cells were treated with 20 μL of each compound serially diluted in DMSO 50 μM to
1.56 μM in triplicate, followed by incubation for 48h (34°C or 37°C, CO2 5%). Subsequently, cells were
carefully washed with PBS and incubated for 16h (34°C, 5% CO2) with 250 μL of chlorophenol
red-ß-d-galactopyranoside (Sigma–Aldrich Co., St. Louis, MO, USA) (CPRG) at 100 μM and Nonidet
P-40 (Amresco Inc, Solon, Ohio, USA) (NP-40) 0.1%. Optical density was read at 570/630 nm in an
Infinite M200 TECAN, Austria. Amphotericin B (Bristol-Myers, Squibb) and benznidazol (Sigma
Aldrich) were used as positive control and DMSO 1% as negative control. The concentration of each
7

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sample that reduced parasite viability by 50% when compared to untreated control (IC50) was estimated
by non-linear regression of concentration-response curves using the GraphPad Prism program.
2.5.2. Cytotoxicity assay
THP-1 cells were cultivated in 96 well microplates (4.0 × 106 cells/well), and treated with the compounds
serially diluted in concentrations ranging from 500 μM to 15.6 μM and incubated for 72h (37°C, 5% CO2).
Cell viability was assessed by the MTT assay. DMSO 1% (Merck), Benznidazol (Sigma Aldrich) and
Amphotericin B (Squibb) were used as negative and positive controls, respectively. The optical density
was read at 540 nm in an Infinite M200 TECAN microplate reader immediately after the dissolution of
formazan crystals with DMSO [16, 17]. DMSO 1% (v/v) and DMSO 50% (v/v) were used as negative and
positive controls, respectively. The IC50 values were calculated by a non-linear regression using the
GraphPad Prism program.
3. Results and discussion
3.1. Chemistry
DHPs were prepared on a one-pot manner based on the catalyst-free Hantzsch reaction [18]. So, 2.4
equivalents of ethyl acetoacetate and 1.2 equivalents of ammonium acetate (compounds 1-7) or aniline
(compounds 8-15) were added to an ethanolic solution of the aromatic aldehyde. The reaction mixture
was stirred under reflux conditions and monitored by thin layer chromatography (TLC) (Fig. 1). The
desired products were purified by silica gel chromatographic column. Their structures were established by
the mean of NMR spectroscopy and HR-ESI-QTOFMS data. The yields of DHPs containing a NH group
ranged 14 to 93% while the N-phenyl products were obtained with quantities comprised between 14 and
44%.
The synthesis of compounds 16-21 was performed as previously reported by Sandjo et al. [19] and the
preparation process is illustrated in figure 2. The cytotoxic [19] and antiparasitic effects of DHPs 16-21
(tables 1 and 2) guided the structural design used for the preparation of compounds 1-15.
2.2. Biological assays.
2.2.1. Cytotoxicity
Compounds 1-21 were evaluated for their cytotoxicity on three cancer cell lines including the human
melanoma SK-MEL-28, the breast cancer MCF-7 and the mouse breast tumor 4-T1. Toxicity of the
compounds was also assayed on the murine fibroblast cells NIH-3T3, a non-tumoral cell line, to calculate
the selectivity index (Table 1). Among the tested compounds, only DHPs 2, 11, 15, 18, 19, and 21
showed antiproliferative activity with CC50 values ranged between 27-84 μM. Compound 18 was the most
potent against SK-MEL-28 with CC50 of 27 μM. Furthermore, MCF-7 and 4-T1 were more sensitive to
compound 19 with CC50 of 28 μM and 30 μM, respectively. Compound 14 showed cytotoxicity
exclusively on 4-T1 and was 2.4-fold less toxic for NIH-3T3. In general, the active 1,4-dihypyridines did
not present the expected selectivity for the non-tumoral cells lines. While 11, 18, 19, and 21 displayed SI
values greater than 1.3, compounds 2 and 15 strongly inhibited the growth of non-tumoral cells than
cancer cells. The weak antiproliferative activity of the prepared DHPs against tumoral cells made them
good candidates for the evaluation against the parasites. This strategy was opted because the antiparasitic
bioassay was carried out on intracellular amastigote forms. Thus, the cytotoxic activity and the selectivity
values obtained were used as an initial insight into theTHP-1 (host cells) sensitivity while they are
exposed to 1-21.
8

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3.2.2. Antiparasitic activity.
3.2.2.1. Trypanocidal activity
Compounds were further evaluated for antiprotozoal activity against the amastigote form of two
trypanosomatids namely T. cruzi and L. amazonensis. The inhibitory concentration of 50% of the parasite
growth (IC50) was calculated for each compound. Besides, their toxicity to THP-1 (human monocytic cell
line) used as host cells for both parasites were performed in order to evaluate the selectivity index (SI =
CC50THP-1/IC50parasite) for each compound.
The first antiparasitic assays performed with compounds 16-21 against the intracellular amastigotes of T.
cruzi revealed interesting inhibitory effect with IC50 (SI) values at 5.44 μM (20.91) and 6.64 μM (17.03)
for compounds 18 and 21, respectively (Table 2). Compounds 18 and 21 had the Me group attached to
C-2 and C-6 while those bearing phenyl group at the position was devoid of trypanocidal effect.
Assuming that bulky groups attached to C-2 and C-6 of the DHP core disadvantage the potency of 16, 17,
19, and 20, Me groups were used to replace to phenyl groups for the next series of compounds to be
prepared. Among the products (1-7), 1-5 showed interesting trypanocidal activity with IC50 (SI),
respectively at 15.74 μM (8.19), 4.95 μM (9.30), 42.54 μM (>11.75), 35.78 μM (1.87), and 20.30 μM
(17.1). Less bulky N-phenylDHP analogues 8-15 were prepared and assayed on the abovementioned
parasites. Except for 10, 12 and 14, DHPs 8, 9, 11, 13, and 15 showed trypanocidal activity with IC50 (SI)
at 10.91 μM (38.17), 30.23 μM (>16.54), 16.79 μM (5.32), 24.39 μM (11.14), and 26.14 μM (5.43),
respectively.
3.2.2.2. Leishmanicidal activity
All prepared DHPs were tested on L. amazonensis and among the 21 compounds, only 5, 11, and 15
showed leishmanicidal activity with IC50 (SI) values at 15.11 μM (22.98), 45.7 μM (1.95), and 53.13 μM
(2.67), respectively. Compound 5 was highly selective towards the TPH-1 cell line compared to other
DHPs and was the most potent chemical entity.
3.2.3. SAR study
3.2.3.1. Trypanocidal effect and DHP structures
A diagnostic analysis of the tested DHP structures regarding their trypanocidal activity revealed that
4-(para-nitrophenyl)-1,4-dihydropyridine (2) was more potent than
4-(meta-nitrophenyl)-1,4-dihydropyridine (21). However, the position of NO2 in meta reduces the toxicity
against THP-1 cell by almost 2-fold if comparing their selectivity indexes. Replacing the NO2 group in 2
by a Cl group reduces the toxicity of compound 18 by 2.25-fold even though its anti-T. cruzi effect is
slightly inferior (1.12-fold) to that of 2. A para H-donor group strongly decreases the anti-T. cruzi activity
as observed for the activity of 3 and by comparing to those of 2 and 18. Moreover, 4-(para-bromophenyl)
DHP (19) inhibited T. cruzi with IC50 higher than 55 μM. This loss of the activity might be caused by the
size of the bromine atom instead of its electronic property, which is similar to that of chlorine.
In general, polyoxygenated compounds were moderately to weakly trypanocidal agents or not active.
However, the presence of a OMe group at the ortho position of the phenol in DHP 3 structure increase the
anti-T. cruzi effect as noticed with 4 but increase also the toxicity of this eventually (Fig. 3). An
additional OMe group to compound 4 to afford 7 abolishes the antiprotozoal activity meanwhile, the
methoxylated form of 4 (compound 1) was more potent than 4 by 2.23-fold. Compound 1 showed also a
good tolerance to THP-1 cell compared to 4 whereas an additional MeO group in 1 to form DHP 6
completely abolishes the activity.
The presence of a phenyl moiety in compound 9 strongly decreases the antiparasitic effect by 6.12-fold
compared to its free NH-containing derivative 2. Nevertheless, this N-phenyl residue (DHP 9) also
contributes to reduce the toxicity. Similar biological feature was observed with compound 8 (the
N-phenyl derivative of 18), which showed a better selectivity than 18 even though its anti-T. cruzi effect
was diminished by almost 2-fold (Fig. 4). In contrast, the N-Ph moiety in 11 (the N-phenyl derivative of 3)
rather improved the trypanocidal activity and increased the toxicity comparing to the activity of 3.
1-Ph-4-(4-chlorophenyl) DHP (8) was more active than its congeners 1-Ph-4-(4-nitrophenyl) DHP (9) and
1-Ph-4-(4-hydroxyphenyl) DHP (11) with a better selectivity towards the THP-1 cell line. Additional
MeO group in compounds 14 and 15 led to respectively, a loss and a decrease of the trypanocidal activity
when compared their data to those of 11 (Fig. 4). DHP 14 initially inactive on T. cruzi, recovered the
antitrypanosomal activity in its methoxylated form (13). Compounds bearing OH and MeO groups were
9

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moderately to weakly trypanocidal agents suggested the substituents unfavorable for the activity.
Moreover, DHPs bearing these functional groups did no show good selectivity.
Calculated physical properties including the lipophilicity (xlogP) and the polar surface area (PSA)
revealed that compounds with the best trypanocidal IC50 have xlogP values ranging 3.81 to 4.03 and PSA
values of 110.45 Å2. Nevertheless, no real correlations were found between the activity (IC50) and these
two parameters. It can be noted in table 2 that some compounds have the same xlogP or PSA values and
revealed different antiparasitic activity. Moreover, compounds 2, 5, 18, and 21, the most potent active
1,4-dihydropyridines corroborate the Lipinski rule of 5 (H-bond donors <5, H-bond acceptors <10, the
molecular weight < 500, and ClogP < 5) [20]. Thus, 4-(3-nitrophenyl)DHP, 4-(3-nitrophenyl)DHP and
4-(Cl-nitrophenyl)DHP cores seem to be the pharmacophoric unit that can inspire preparation of hit
analogues. The 4-(3-nitrophenyl)DHP and 4-(Cl-nitrophenyl)DHP scaffolds can easily be framed within
the chemical structure of NO2 containing Ca2+ channel blockers (azelnidipine, cilnidipine, lercanidipine,
nicardipine, nifendipine, nimodipine, nitrendipine, and isradipine) and the chlorinated DHP amlodipine
previously reported as antiparasitic entities against the trypomastigote form of T. cruzi [5] with interesting
inhibitory values. However, compounds 2, 18 and 21 can still be decorated with different function groups
in order to improve to antichagasic activity and still meet the rule of 5.
3.2.3.2. Leishmanicidal effect and DHP structures
The styryl group in compound 5 turned to be favourable for the leishmanicidal activity and less
aggressive towards THP-1 cells. In contrast, 11 and 15 the weakest active compounds contain
hydroxylated and methoxylated aryl groups. No concrete structure-activity relationships were found
between the active compounds except for 11 and 15 that are N-phenylated and contain OH and/or MeO
groups. The lack of leishmanicidal effect from the remaining DHPs might be explained by the lack of any
correlations between the Ca2+ channel action and the survival of L. amazonensis in its host. Otherwise, the
inhibition of the Ca2+ channel action perhaps does occur, but does not affect the proliferation of the
parasite in its host.
A comparison of various former studies on antiparasitic effect of DHPs used as CCB drugs revealed
that nifedipine and verapamil inhibited the macrophage growth but did not show any antiparasitic effect
on L. donovani. Despite the observation, the report claimed a critical contribution of Ca2+ in the
interaction macrophage-Leishmania [21]. In contrast, another study reported the proliferation of L.
donovani in macrophage despite the presence of nifedipine [22]. Because of these controversial results,
Tempone and co-workers studied a different CCB (nimodipine) on L. chagasi. During the in vitro
investigation, no decrease in L. chagasi growth was observed. Despite their results, the authors correlated
the leishmanicidal effect of the compound with its ability to induce apoptosis by affecting the
mitochondrial function [7]. Moreover, another interesting result revealed that nifedipine and verapamil
inhibited the sphingosine-activated plasma membrane Ca2+ in L. mexicana promastigotes [23]. On the
basis of these previous data, we concluded that the leishmanicidal effect of DHPs 11 and 15 might
occurred by a similar mechanism pathway namely the inhibition of the sphingosine-activated plasma
membrane Ca2+. This deactivation of the Ca2+ channel might also affect the host cell supporting the low
selectivity of 11 and 15 in assays reported in tables 1 and 2.
4. Conclusion
The study aimed to prepare 1,4-dihydropyridines related to Ca2+ channel blockers drugs and to evaluate
their cytotoxic and antiparasitic effects on L. amazonensis and T. cruzi. The results showed that some of
the compounds had a significant trypanocidal activity with a good selectivity on THP-1 cells. Their
selectivity was increased when a phenyl group was attached to the nitrogen atom although a slight
decrease of the antiparasitic effect was observed. Moreover, Br atom, MeO and OH groups seemed not to
be suitable substituents for the trypanocidal activity because of the weak effect of the compound or the
low selectivity. The study also showed that except for compound 5, the leishmanicidal effect of some
DHPs might be associated to their capability of inhibiting the Ca2+ channel.
Supplementary Material
1H and 13C NMR data of compounds 1-15 are associated with this work can be found online.
10

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Acknowledgments
LAEP and LPS would like to thank CNPq/PIBIC for supporting the chemical study within the
scientific initiation program of PGFAR/UFSC, as well as CAPES for the fellowship of post-graduation
students. This study is part of the collaboration work within the Research Network NaturalProducts
against Neglected Diseases (ResNetNPND http://www.resnetnpnd.org/).
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as potent antileishmanial agents, Eur. J. Med. Chem. 45 (2010) 2381-2388.
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[12] H.-G. Lu, L. Zhong, K.-P. Chang, R. Docampo, Intracellular Ca21 Pool Content and Signaling and
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Figure 1: Synthesis route of compounds 1-15
13

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Figure 2: Synthesis route of compounds 16-2119.
14

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Figure 3Analysis of the structure-Activity relationship between DHPs bearing a di-substituted nitrogen atom
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Figure 4. Analysis of the structure-Activity relationship between DHPs bearing NH and
N-phenyl groups.
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Table 1: CC50 and SI values of compounds against different cancer cell lines
Compound
CC50 (µM)
Selectivity Index (SI)*
NIH-3T3/
SK-MEL-28
NIH-3T3/
MCF-7
NIH-3T3/
4-T1
SK-MEL-28 MCF-7
4-T1
NIH-3T3
>100
1
>100
>100
>100
-
-
-
2
3
41
68
57
59
1,5
0,9
1,0
>100
>100
>100
>100
>100
>100
>100
>100
41
>100
>100
>100
>100
>100
>100
>100
>100
67
>100
>100
>100
>100
>100
>100
>100
>100
49
>100
>100
>100
>100
>100
>100
>100
>100
44
-
-
-
4
-
-
-
-
5
-
-
6
-
-
-
-
-
7
-
8
-
-
-
9
-
-
-
10
11
12
13
14
15
16
17
18
19
20
21
-
-
-
1,1
-
0,6
-
0,9
-
>100
>100
>100
71
>100
>100
>100
43
>100
>100
84
>100
>100
200
-
-
-
-
-
2,4
0,6
-
81
44
0,6
-
1,0
-
>100
>100
27
>100
>100
37
>100
>100
31
>100
>100
58
-
-
-
2,2
1,3
-
1,6
1,5
-
1,9
1,4
-
32
28
30
41
>100
56
>100
55
>100
64
>100
98
1,7
1,8
1,5
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Table 2 Antiparasitic effect of compounds 1-21 against the intracellular T. cruzi and L. amazonensis amastigotes as well as their selectivity towards the
THP-1 cell line.
THP-1 cells
T. cruzi
L. amazonensis
xLogP
PSA (Å2)
Compound
IC50 (μM)
15.74 (±0.58)
4.95 (±0.40)
42.54 (±17.39)
35.78 (±2.08)
20.30 (±2.16)
NA
SI
8.19
9.30
>11.75
1.87
17.10
-
CC50 (μM)
128.90 (±19.10)
46.05 (±1.77)
>500
91.66 (±18.01)
347.20 (±33.03)
NA
IC50
NA
NA
NA
NA
SI
-
1
2
3
4
5
3.39
3.81
3.08
3.09
3.92
3.40
3.10
5.68
5.46
5.79
4.74
5.06
5.05
4.74
4.75
6.41
6.30
4.03
4.14
5.80
3.81
83.09
110.45
84.86
94.09
64.63
92.32
103.32
55.84
101.66
55.84
76.07
83.53
74.30
85.3
94.53
64.63
64.63
64.63
64.63
92.32
110.45
-
-
15.11 (±3.67)
22.98
6
NA
7
8
9
NA
-
NA
NA
NA
NA
NA
-
-
-
10.91 (±2.12)
30.23 (±5.49)
NA
16.79 (±0.29)
NA
24.39 (±2.06)
NA
26.14 (±1.21)
NA
NA
5.44 (±0.13)
NA
NA
6.64 (±0.68)
10.18 (±0.3)
38.17
>16.54
416.50 (±92.01)
>500
NA
89.34 (±4.79)
NA
271.70 (±41.50)
NA
142.00 (±10.00)
NA
10
11
12
13
14
15
16
17
18
19
20
21
ref1
ref2
-
5.32
-
11.14
-
5.43
-
-
20.91
-
45.70 (±11.70)
1.95
-
-
-
2.67
-
-
-
NA
NA
NA
53.13 (±4.30)
NA
NA
NA
NA
NA
NA
-
NA
113.8 (±36.03)
~203.6
-
-
-
-
NA
17.03
>49.11
113.1 (±13.10)
>500
27.86 (±0.98)
-
0.14 (±0.02)
199
ref1: Benznidazol, ref2: Amphotericin B, NA: not active
18

ACCEPTED MANUSCRIPT
Change of the NO2 position from para
to meta slightly decreases of the activity
and reduces by 2-fold of the toxicity
Replacement of NO2 by Cl
slightly decreases of the activity
and reduces 2.25-fold the toxicity
R1
R2
O
The N-phenyl substitution
decreases the activity and
reduces the toxicity
O
EtO
OEt
R1 = NO2, R2 = H, R3 = H: IC50 4.95 M/SI 9.3
R1 = H, R2 = NO2, R3 = H: IC50 6.64 M/SI 17.03
R1 = Cl, R2 = H, R3 = H: IC50 5.44 M/SI 20.91
R1 = Cl, R2 = H, R3 = Ph:IC50 10.91 M/SI 38.17
N
R3
Anti-T. cruzi agents
Graphical abstract
19

ACCEPTED MANUSCRIPT
Highlights
Synthesis of 21 Ca2+ channel blocker 1,4-dihydropyridine analogues.
Evaluation of their antiparasitic effects against T. cruzi and L. amazonensis.
4 of the 12 active compounds on T. cruzi revealed promising IC50 values.
L. amazonensis was sensitive to three of the prepared 1,4-dihydropyridines.
20