Synthesis, antitrypanosomal activity and molecular docking studies of pyrimidine derivatives

Article abstract of DOI:10.1007/s00044-018-2253-2

This publication describes the synthesis of 28 pyrimidine derivatives of which eight are new. Their structures have been verified by spectroscopic techniques (IR, H1, C13 NMR data). The mass spectral analysis gave correctly the elemental compositions of a

Full text of DOI:10.1007/s00044-018-2253-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2253-2
ORIGINAL RESEARCH
Synthesis, antitrypanosomal activity and molecular docking studies
of pyrimidine derivatives
Sebastião José de Melo1 Zenaide Severina do Monte1 Aline Caroline da Silva Santos2
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Ana Catarina Cristovão Silva2 Luiz Felipe Gomes Rebello Ferreira3 Marcelo Zaldini Hernandes3
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Ricardo Oliveira Silva4 Emerson Peter da Silva Falcão5 Maria Carolina Accioly Brelaz-de-Castro6
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Rajendra M. Srivastava4,7 Valeria Rêgo Alves Pereira3
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Received: 2 April 2018 / Accepted: 15 October 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
This publication describes the synthesis of 28 pyrimidine derivatives of which eight are new. Their structures have been
verified by spectroscopic techniques (IR, H1, C13 NMR data). The mass spectral analysis gave correctly the elemental
compositions of all compounds. All 28 compounds have been evaluated in vitro against two different stages of Trypanosoma
cruzi: 1. epimastigote and 2. trypomastigotes. Compounds 3a, 3b, 3j, 3k, 3o, 3s and 5d presented greater activity than
benznidazole(BZN) for trypomastigotes. Compound 3a selectivity was similar to that of BZN. Molecular modeling using the
Docking approach was performed to determine binding of the ligand to the enzyme. The M.O. calculations using the GOLD
5.2 program provided information on the pyrimidine binding sites with the enzyme.
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Keywords Pyrimidine analogs Trypanosoma cruzi Epimastigote Trypomastigote Cytotoxicity Docking
Introduction
Chagas disease is a tropical parasitic disease caused by the
protozoan parasite Trypanosoma cruzi and it is transmitted
to humans through the bite of the blood-sucking triatomine
or “kissing bugs”. The symptoms change over the course of
the infection. In the early stages of the disease, patients
generally do not show any sign, but with time they appear
and may include fever, swollen lymph nodes, headache, or
local swelling at the site of the bite (Guimarães et al. 1968).
Some 70% of the people, after 8–12 weeks, enter the
chronic phase of the disease (Hidron et al. 2010). The
others, about 30% of the people, develop further symptoms
10–30 years after the initial infection, which may include
enlargement of the ventricles of the heart in 20 to 30% of
the cases, leading to heart failure (Toloza et al. 2016;
Medical Research Institute 2003). An enlarged esophagus
or an enlarged colon may also occur in 10% of the people
(Medical Research Institute 2003). Approximately 6–7
million of the people suffer with this disease worldwide,
and is more prevalent in Latin American countries accord-
ing to World Health Organization (World Health Organi-
zation 2017). In Brazil, about 1.15 million people suffer
with this Chagas disease (World Health Organization 2015).
Chagas disease was discovered by a Brazilian physician
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2253-2) contains supplementary
material, which is available to authorized users.
* Sebastião José de Melo
melosebastiao@yahoo.com.br
1
Department of Antibiotics, Federal University of Pernambuco
CEP, Recife, PE 50740-560, Brazil
2
Aggeu Magalhães Research Center, Oswaldo Cruz Foundation,
CEP, Recife, PE 50670-420, Brazil
3
Department of Pharmaceutical Sciences and Health Sciences
Center, CEP, Recife, PE 50740-560, Brazil
4
Department of Fundamental Chemistry, Federal University of
Pernambuco, CEP, Recife, PE 50740-560, Brazil
5
Department of Nucleus of Nutrition, Federal University of
Pernambuco, Vitória Academic Center (CAV), CEP, Vitoria de
Santo Antão, PE 55608-680, Brazil
6
Parasitology Laboratory, Federal University of Pernambuco,
Vitória Academic Center (CAV), CEP, Vitoria de Santo Antão, PE
55608-680, Brazil
7
Department of Antibiotics, Visiting Research Scientist, Federal
University of Pernambuco, Recife, PE 50740-560, Brazil

Medicinal Chemistry Research
Carlos Chagas in 1909. It is caused by the parasite Trypa-
nosoma cruzi, and transmitted to animals and people by
insect vectors. It is more prevalent in Latin American
countries especially in the poverty-ridden areas. Although
more than a century passed since this disease was well
recognized, there is no complete cure for it. Only two drugs
are being used for Chagas disease chemotherapy, namely,
benznidazole and nifurtimox. These drugs, however are
mainly used in the acute phase of the disease since they are
not much effective in the chronic stage. Therefore, there is a
great need to discover new and effective drugs to combat
this disease.
epidemiological importance of Chaga’s disease and the
promising properties of pyrimidine, were synthesized and
tested against T. cruzi epimastigotes and trypomastigotes,
28 pyrimidine analogs. Eight compounds, namely 3d, 3h,
3o–q, 3s, 5b and 6i had excellent results against epimasti-
gotes. On the other hand, 3a, b, 3d, 3f, 3j, k, 3o, p, 3s, 5d
and 6i had remarkable activity agaist tripomastigotes. These
results demonstrate the potential of pyrimidine analogs as
anti-T. cruzi agents.
Materials and Methods
In our ongoing work on pyrimidines, we decide to
investigate the title compounds against Trypanosoma cruzi.
The reason for choosing the present compounds was that
nucleic acid structure contains purines and pyrimidines,
hence these bases are extremely important. In fact, cytosine,
thymine and uracil are pyrimidines and make up the
nucleotide base, and both purines and pyrimidines are
required in the cell in equal quantities (Nakajima-Shimada
et al. 1996). In 1996, Nakajima-Shimada et al., examined
purine and pyrimidine analogs and found that one of the
pyrimidines, 3′-azido-3′-deoxythymidine (zidovudine),
significantly reduced the growth of the parasite at con-
centrations as low as 1uM. Since then more work appeared
where pyrimidine analogs were investigated against Try-
panosoma cruzi with interesting results (Nakajima-Shimada
et al. 1996; Luque et al. 2000; Hashimoto et al. 2012; Braga
et al. 2017; Woods et al. 2006; Woodring et al. 2015). For
examples, the 6-substituted thienopyrimidines showed
excellent potency against T. cruzi with low values of IC50
0.60 µM (Hashimoto et al. 2012). The same authors
observed that the slow step is due to the enzyme carbamoyl-
phosphate synthetase-II (CPSII) in the de novo biosynthetic
pathway of pyrimidines. In fact, the increased activity of
CPSII causes fast proliferation of T. cruzi in the host cell
cytoplasm. It has also been established that disruption of the
T. cruzi CPSII gene significantly reduced parasitic growth.
Therefore, we conclude that pyrimidine analogs are pro-
mising for chemotherapy against Chagas disease (Hashi-
moto et al. 2012).
General experimental procedures
All melting points were obtained on a BUCHI B-540
apparatus and are not corrected. The IR spectra were
recorded on a BRUKER IFS66 using the KBr wafer
technique. 1H, 13 C NMR spectra were determined on a
VARIAN UNMRS 400 MHz instrument with DMSO-
d6as an internal standard; chemical shifts are as units δ
(ppm). The mass spectra were recorded on a Mass Spec-
trometer (LCMS-SHIMADZU, LCMS-IT-TOF). Mon-
itoring the reactions and checking the purity of the final
products were carried out by thin layer chromatography
(TLC) using silica gel pre-coated aluminum sheets (60
F254, Merck). NIR range emissions. All emission spectra
were corrected by spectral response of the mono-
chromators and using silicon photodiode reference
detector, to monitor and compensate for variation in the
xenon lamp output, using typical correction spectra pro-
vided by the manufacturer.
Typical experimental procedures
Preparation of 4-amino-2,6-diaryl-5-cyanopyrimidines, 3a–s
An appropriate bisnitrile 2a–s and arylamidine 1a–s (Melo
et al. 2002; Do Monte et al. 2016; Melo et al. 2017) in
equimolar quantity (3.46 mmol) were dissolved in methanol
(10 mL) and refluxed for 6h. The contents were cooled to
room temperature and solvent evaporated to give a solid
mass which was crystallized and recrystallized from an
appropriate solvent.
The pyrimidine moieties occupy a unique importance in
pharmaceutical chemistry, as they are components of
nucleic acids (Do Monte et al. 2016), soon derivative of
pyrimidines is the most attractive for a medicinal chemist.
These heterocyclic compounds bearing pyrimidine moieties
have received considerable attention of the pharmaceutical
industry because of their interesting multifaceted pharma-
cological profiles, such as anti-inflammatory, analgesic,
antimicrobial and antiviral activities (Samvel et al. 2016;
Marzano et al. 2010; Falcão et al. 2006; Natarajan et al.
2014; Andrews and Mansur 2014). In view of the
4-Amino-5-cyano-2-(p-diethylaminophenyl)-6-(p-
chlorophenyl)-pyrimidine (3g)
This compound was obtained as colorless crystals from
methanol 86.72% yield, m.p.221–223 °C, Rf = 0.45; n-
hexane-ethyl acetate 7:3); IR, KBr, γmax cm−1: 3435
(-NH2asymm.), 3238 (NH2symm.), 2221 (CN), 1647 (C=N);

Medicinal Chemistry Research
1H-NMR (DMSO-d6 400 MHz), δ: 8.46 (b, 2H, C2–NH2);
8.24 (d, 2H, J: 9.2 Hz, H2’ and H6′); 7.97 (d, 2H, J: 9.2 Hz,
H2” and H6ʺ); 7.65 (d, 2H, J = 9.2 Hz, H3ʺ and H5”); 6.78
(d, 2H, J = 9.2 Hz, H3’ and H5′) and δ 3.03 (s, 6H); 13C-
NMR (DMSO-d6 300 MHz), δ: 166.5 (1 C, C6); 164.3 (1
C, C1ʺʺ); 164.2 (1 C, C2); 152.6 (1 C, C4′); 135.7 (1 C,
C4ʺ); 135.5 (1 C,C1ʺ); 130.3 (2 C, C2ʺ and C6ʺ); 130.0 (2 C,
C2′ and C6′); 128.5 (2 C, C3ʺ and C5ʺ); 123.1 (1 C, C1′);
116.8 (1 C, C4); 111.1 (2 C, C3′ and C5′); 82.4 (1 C, C5);
and δ 39.6 (2 C,C1‴). LCMS-IT-TOF: m/z molecular mass
found = 350.1130; calculated for C19H16N6O3 [M + 1]+,
mass = 350.8168.
4-Amino-5-cyano-2-(p- nitrophenyl)-6-(p- anisyl)-pyrimidine
(3n)
This compound was obtained as yellow crystalsfrom ACN
fromin 67.2% yield, m.p.236–2385 °C, Rf = 0,4(n-hexane-
ethylacetate 7:3); IR, KBr, γmax cm−1: 3436 (NH2asymm.),
3239 (NH2symm.), 2221 (CN), 1648 (C=N); 1H-NMR
(DMSO-d6 400 MHz), 1H-NMR (DMSO-d6400 MHz), δ:
8.42 (d, 2H, J = 8.04 Hz, H2′ and H6’); 8.35 (d, 2H, J =
8.8 Hz, H2ʺ and H6ʺ); 8.01 (b, 2H, C2–NH2); 8.19 (d, 2H,
J = 8.8 Hz, H3ʺ and H5ʺ); 7.07 (d, 2H, J = 8.0 Hz, H3′ and
H5′) and δ 3.84 (s, 3H); 13C-NMR (DMSO-d6 300 MHz),
δ: 166.3 (1C, C6); 164.2 (1C, C1ʺʺ); 164.0(1C, C2); 162.2
(1C, C4ʺ); 148.7 (1C, C1′); 142.6 (1C, C4ʺ); 130.3 (2C, C2′
and C6′); 130.1 (2C, C3ʺ and C5ʺ); 128.6 (1C, C1′); 123.6
(2C, C2ʺ and C6ʺ); 116.0 (1C, C4); 114.0 (2C, C3′ and
C5′); 84.3 (1C, C5); and δ: 55.4 (2C, C1′); LCMS-IT-TOF:
m/z molecular mass found 346.0885; calculated for
C18H13N5O3 [M-1]-, mass = 346.3275.
4-Amino-5-cyano-2-(p-aminophenyl)-6-(p-anisyl)-
pyrimidine (3i)
This compound was obtained as yellow crystals from
acetonitrile, yield = 56.85% yield, m.p.236–2385 °C, Rf =
0,4(n-hexane-ethyl acetate 7:3); IR, KBr, γmax cm-1: 3436
(NH2asymm.), 3239 (NH2symm.), 2221 (CN), 1648
(C=N); 1H-NMR (DMSO-d6 400 MHz), 1H-NMR
(DMSO-d6 400 MHz), δ: 8.60 (b, 2H, C2–NH2); 8.42 (d,
2H, J = 8.4 Hz, H2ʺ and H6ʺ); 8.35 (d, 2H, J = 8.4 Hz, H2′
and H6′); 8.02 (b, 2H, C4′–NH2); 8.19 (d, 2H, J = 8.4 Hz,
H3ʺ and H5ʺ); 7.07 (d, 2H, J = 8.4 Hz, H3′ and H5′) and δ
3.84 (s, 3H); 13C-NMR (DMSO-d6300 MHz), δ: 166.3 (1
C, C6); 164.2 (1 C, C1ʺʺ); 164.0(1 C, C2); 162.2 (1C, C4ʺ);
148.7 (1 C, C1′); 142.6 (1C, C4ʺ); 130.3 (2C, C2′ and C6′);
130.1 (2 C, C3ʺ and C5ʺ); 128.6 (1C, C1’); 123.6 (2C, C2ʺ
and C6ʺ); 116.0 (1C, C4); 114.0 (2C, C3′ and C5′); 84.3
(1C, C5); and δ 55.4 (2C, C1′). LCMS-IT-TOF: m/z
molecular mass found 318.2652; calculated for C18H15N5O
[M + 1]+, mass = 318.1277.
4-Amino-5-cyano-2-(p-methylphenyl)-6-(p-chlorophenyl)-
pyrimidine (3q)
This compound was obtained as colorless crystals from
MeOH in 96% yield, m.p. 289–291, Rf = 0.65 (n-hexane-
ethyl acetate-methanol 6:3:1); IR, KBr, γmax cm−1: 3346
(NH2asymm.), 3029 (NH2symm.), 2221 (CN), 1604
(C=N); 1H-NMR (DMSO-d6 400 MHz), δ: 8.85 (b, 2H,
C2–NH2); 8.39 (d, 2H, J: 7.6 Hz, H2′ and H6′); 8.13 (d,
2H, J: 8.8 Hz, H2ʺ and H6ʺ); 7.53 (d, 2H, J: 7.6 Hz, H3′ and
H5′); 7.66 (d, 2H, J: 8.8 Hz, H3ʺ and H5ʺ) and δ: 2.68 (s,
3H); 13C-NMR (DMSO-d6 400 MHz), δ: 167.0 (1C, C6);
164.5 (1C, C1ʺʺ); 164.0 (1C, C2); 1354 (1C, C4′); 135.8
(1C, C4ʺ); 135.3 (1C, C1ʺ); 130.5 (2C, C2ʺ and C6ʺ); 130.4
(2C, C2′ and C6′); 128.5 (2C, C3ʺ and C5ʺ); 128.4 (2C, C3′
and C5′); 116.2 (1C, C4); 82.8 (1C, C5); and δ 22.1 (1C,
C1‴); LCMS-IT-TOF: m/z molecular mass found 320.7756;
calculated for C18H13ClN4 M+, mass = 320.2028
4-Amino-5-cyano-2-(p-aminophenyl)-6-(p-nitrophenyl)-
pyrimidine (3k)
This compound was obtained as yellow crystals from ACN
in 90.98% yield, m.p.221.6–222 °C, Rf = 0.45 (n-hexane-
ethyl acetate 7:3); IR, KBr, γmax cm−1: 3449 (NH2asymm.),
32948 (NH2symm.), 2192 (CN), 1627 (C=N); 1H-NMR
(DMSO-d6 400 MHz), δ: 8.42(d, 2H, J: 8.4 Hz, H2′ and
H6′); 8.36 (d, 2H, J: 8.4 Hz, H2ʺ and H6ʺ);8.02 (b, 2H, C2–
NH2); 7.90 (d, 2H, J = 8.4 Hz, H3′ and H5′); 7.09 (d, 2H, J
= 8.4 Hz, H3ʺ and H5ʺ); and 7.06 (s, 2H, C4′–NH2); 13C-
NMR (DMSO-d6 300 MHz), δ: 166.5 (1C, C6); 164.3 (1C,
C1‴); 164.1 (1C, C2); 148.7 (1C, C4ʺ); 142.4 (1C, C4′);
136.2 (1C, C1ʺ); 131.7 (1C, C1′); 130.1 (2C, C3ʺ and C5ʺ);
128.6 (2C, C2′ and C6′); 128.5 (2C, C2ʺ and C6ʺ); 123.61
(2C, C3′ and C5′); 115.8 (1C, C4) and 85.2 (1C, C5).
LCMS-IT-TOF: m/z molecular mass found 332.3284; cal-
culated for C17H13N5O2 M+, mass = 332.1022.
Preparation of derivatives of 4-Amino-5-cyano-2-(p-
diethylaminophenyl)-6-(p-pyrrol-2-onephenyl)-pyrimidine
(6g) or 4-Amino-5-cyano-2-(p-anisyl)-6-(p-pyrrol-2-
onephenyl)-pyrimidine (6h)
To a mixture of compound 4-Amino-5-cyano-2-(p-diethy-
laminophenyl)-6-(p-chlorophenyl)-pyrimidine (3g) or 4-
Amino-5-cyano-2-(p-anisyl)-6-(p-chlorophenyl)-pyrimidine
(3h) with pyrrol-2-one in equimolar quantity (3.46 mmol) in
methanol (10 ml), was added to a few drops of piperidine,
and refluxed for 2 h, and allowed to cool. The contents were
cooled to room temperature and solvent evaporated to give
a solid mass which was crystallized and recrystallized from

Medicinal Chemistry Research
an appropriate solvent. (Al-Issa 2012; Melo 1989; Comins
et al. 2008)
equimolar quantity (3.46 mmol) in acetic acid (10 ml), and
refluxed for 2 h, and allowed to cool. The contents were
cooled to room temperature and solvent evaporated to give
a solid mass which was crystallized and recrystallized from
an appropriate solvent. (Do Monte et al. 2016; Melo et al.
2017)
14-Amino-5-cyano-2-(p-diethylaminophenyl)-6-(p-pyrrol-2-
onephenyl)-pyrimidine (6g)
This compound was obtained as yellow crystals from
MeOH in 82.9% yield, m.p.234.7–235.5 °C, Rf = 0.56 (n-
hexane-ethyl acetate 7:3); IR, KBr, γmax cm-1: 3334
(NH2asymm.), 3270 (NH2symm.), 2219, (CN), 1587 (C=N);
1H-NMR (DMSO-d6 400 MHz), δ: 8.20 (d, 2H, J: 9.2 Hz,
H2′ and H6′); 8.02 (d, 2H, J: 9.2 Hz, H2ʺ and H6ʺ); 7.70 (b,
2H, C2–NH2); 7.63 (d, 2H, J = 9.2 Hz, H3ʺ and H5ʺ); 6.76
(d, 2H, J = 9.2 Hz, H3′ and H5′); 6.52 (m, 1H,J = 6.2 Hz,
H3ʺʺ′); 6.38 (m, 1H, J = 6.2 Hz, H4ʺʺ′); 3.30 (s, 2H, H5ʺʺ′);
and 2.99 (s, 6H); 13C-NMR (DMSO-d6 300 MHz), δ: 164.5
(1C, C6); 164.2 (1C, C1ʺʺ); 164.1 (1C, C2); 161.4 (1C, C2ʺʺ
′); 153.8 (1C, C4′); 152.6 (1C, C4ʺʺ′); 138.0 (1C, C4ʺ);
135.0 (1C,C1ʺ); 130.4 (2C, C2ʺ and C6ʺ); 130.3 (2C, C2′
and C6′); 128.6 (1C, C3ʺʺ′); 125.4 (1C, C1′); 123.3 (2C,
C3ʺ and C5ʺ); 116.2 (1C, C4); 114.0 (2C, C3′ and C5′);
84.1 (1C, C5); 52.8 (1C, C5ʺʺ′); and δ: 39.4 (2C, C1‴).
LCMS-IT-TOF: m/z molecular mass found 396.0357; cal-
culated for C23H20N6O3 M +, mass = 396.1699.
4-Amino-5-cyano-2-(p-maleimidephenyl)-6-(p-anisyl)-
pyrimidine (6i)
This compound was obtained as yellow crystals from
MeOH in 79.5% yield, m.p.236.4–236.9, Rf = 0.7(n-hex-
ane-ethyl acetate 6:4); IR, KBr, max cm−1: 3476 (NH2a-
symm.), 3212 (NH2symm.), 2213 (CN), 1621 (C=N); 1H-
NMR (DMSO-d6 400 MHz); δ: 8.40 (b, 2H, C2–NH2);
7.98 (d, 2H, J = 11.6 Hz, H2′ and H6′); 7.79 (s, 2H, H1‴
and H2‴); 7.37 (d, 2H, J = 7.6 Hz, H2ʺ and H6ʺ); 7.20 (d,
2H, J = 7.6 Hz, H3ʺ and H5ʺ); 7.11 (d, 2H, J = 11.6 Hz, H3′
and H5′) and δ: 3.88 (s, 3H””); 13C-NMR (DMSO-d6 300
MHz), δ: 166.9 (1C, C6); 164.6 (1C, C1ʺʺ); 164.2 (1C, C1ʺ);
164.1 (2C, C1‴ and C2‴); 164.0 (1C, C2); 134.9 (1C, C1′);
130.9 (1C, C1ʺ); 130.2 (2C, C2′ and C6′); 128.5 (2C, C2ʺ
and C6ʺ); 124.0 (2C, C3′ and C5′); 113.8 (1C, C4); 113.7
(2C, C3ʺ and C5ʺ); 84.4 (1C, C5); and δ: 55.4 (2C, C1ʺʺ′).
LCMS-IT-TOF: m/z molecular mass found 398.0965; cal-
culated for C22H17N5O2 [M + 1]+, mass = 398.3862.
4-Amino-5-cyano-2-(p-anisyl)-6-(p-pyrrol-2-onephenyl)-
pyrimidine (6h)
In vitro Assays
This compound was obtained as brown crystals from MeOH
in 87.45% yield, m.p. 344.3–345.1, Rf = 0.2 (ethyl acetate-
methanol 9.7:0.3); IR, KBr, γmax cm−1: 3487 (NH2asymm.),
3186 (NH2symm.), 2210 (CN), 1697 (C=N); 1H-NMR
(DMSO-d6 400 MHz), δ: 8.20 (d, 2H, J = 12 Hz, H2′ and
H6′); 8.02 (d, 2H, J = 11.6 Hz, H2‴ and H6”); 7.80 (b, 2H,
C2–NH2); 7.67 (d, 2H, J = 11.6 Hz, H3ʺ and H5ʺ); 7.08 (d,
2H, J = 12 Hz, H3′ and H5′); 6.80 (m, 1H, J = 6.6, H3ʺʺ′);
6.72 (m, 1H, J = 6.6,H4ʺʺ′); 3.84 (s, 3H) and3.30 (s, 3H,
H5ʺʺ′) ;13C-NMR (DMSO-d6 300 MHz), δ: 165.5 (1C, C6);
164.5 (1C, C1ʺʺ); 164.3 (1C, C6); 162.3 (1C, C2ʺʺ′); 153.8
(1C, C4ʺʺ′); 152.5 (1C, C1ʺ); 130.3 (1C, C4ʺ); 130.1 (1C,
C3ʺʺ′); 129.5 (2C, C2′ and C6′); 125.3 (2C, C2ʺ and C6ʺ);
123.2 (1C, C1′); 122.9 (2C, C3ʺ and C5ʺ); 116.5 (1C, C4);
112.1 (2C, C3′ and C5′); 82.8 (1C, C5); 55.9 (1C, C1‴) and
47.3 (1C, C5ʺʺ′). LCMS-IT-TOF: m/z molecular mass
found 384.9397; calculated for C22H17N5O2 [M + 1]+,
mass = 384.4027.
Evaluation of cytotoxic activity
RAW 264.7 macrophage cell line were cultured in DMEM
medium, at 37 °C in a humidified atmosphere of 5% CO2.
The mediums contained 10% heat-inactivated FBS, 100U/
ml penicillin G and 2mM l-glutamine. Culture medium was
changed after 2–3 days and subcultured when cell popula-
tion density reached to 70–80% confluence. Macrofages 105
cells/well were added in 96-well plates and incubated for
24 h (37 °C and 5% CO2). Compounds were then added in
different concentrations (1 to 100 mg/ml) and incubated for
48 h. Two independent experiments were performed in
duplicate. MTT (3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphe-
nyltetrazoliumbromide, 5 mg/ml in PBS) was added to each
well and incubated again for 2 h. Culture medium and MTT
not reduced was removed and 100 μl of DMSO were added.
The amount of formazan was determined by measuring the
absorbance at 570 nm. The negative control was obtained in
wells containing only culture medium and cells (without
treatment). The activity of the reference drug benzonidazole
was also evaluated.The CC50 was calculated by regression
analysis with GraphPad Prism Software (La Jolla, CA,
USA).
Preparation of derivative of 4-Amino-5-cyano-2-(p-
maleimidephenyl)-6-(p-anisyl)-pyrimidine (6i)
To a mixture of compound 4-amino-5-cyano-2-(p-amino-
phenyl)-6-(p-anisyl)-pyrimidine with maleic anhydride in

Medicinal Chemistry Research
Scheme 1 Synthesis of pyrimidine analogs 3a–s, 5a–f and 6g–i
Trypanocidal activity evaluation
well plates at 27 °C along with different concentrations of
the compounds (0.19, 1.56, 6.25, 25, 100 μg/ml) for 96 h.
Each compound was tested in duplicate. Negative control
were untreated wells and the reference drug (benzonidazole)
was used as a positive control. Parasitic viability was
determined by direct counting in Neubauer’s chamber and,
Against epimastigote forms
To determine the antiproliferative effect for epimastigote
forms of DM28c strain, 106 parasites/ml, maintained in LIT
medium + 1% antibiotic + 20% FBS, were seeded in 96-

Medicinal Chemistry Research
from these values, the IC50 (concentration that inhibits the
growth of 50% of parasites) was obtained.
enzyme (PDB code 3IUT, http://www.rcsb.org), in which it
has co-crystallized with tetrafluorophenoxymethyl Ketone
Inhibitor (KB2). The binding site was defined as all atoms
within a radius of 6.0 Å from the co-crystallized ligand
(KB2). The GOLD 5.2 program (Gold software, version
5.2, Cambridge Crystallographic Data Centre 2017) was
applied for docking calculations, using the ChemPLP score
function. In order to take into account the degrees of free-
dom for the receptor, to better simulate the induced fit, the
following amino acids residues were treated as flexible,
using conformation library: CYS25, TRP26, ASP60,
SER64, LEU67, MET68, ASN70, MET160, ASP161 and
LYS162. Then, the BINANA (Durrant and Mccammon
2011) program was used to analyze the molecular interac-
tions of docking results, using default settings. The figures
were generated using the Pymol (DeLano 2002) and Lig-
Plot + (Laskowski and Swindells 2011) programs.
Against Trypomastigote forms
Trypomastigote forms (Y strain) were obtained from the
in vitro infection (107 parasites) of the Vero cell line, after
they obtained confluence in culture. To determine the
antiproliferative effect for trypomastigote forms of the Y
strain, 4 × 106 parasites/ml, maintained in RPMI + 1%
antibiotic + 5% FBS medium, were seeded in 96-well plates
at 37 °C together with different concentrations of the com-
pounds (0.19, 1.56, 6.25, 25, 100 μg/ml) for 24 h at 5% CO2
atmosphere. After this time, aliquots from each well were
collected, and the number of viable parasites was counted in
a Neubauer chamber. The wells that did not receive the
compounds were assumed as 100% of the number of viable
parasites. The reference drug, benzonidazol, was used as a
positive control. Each compound was tested in duplicate.
The dose-response curves were determined and the inhibi-
tion concentration of 50% of parasite growth (IC50) were
calculated by linear regression (GraphPad Prism, version
5.0)
Results and Discussion
Chemistry
We have synthesized 28 pyrimidine derivatives, 3a–s, 5a–f
and 6g–i (Scheme 1), of which eight, viz., 3g, 3i, 3k, 3n, 3q
and 6g–i are new. The compounds were obtained in good
yields (ranging from 57 to 98%). The synthesis of com-
pounds m,p-substituents 3a–s occurred by cyclization of
arylamidines 1a–s and bisnitriles 2a–s in presence of
piperidine as base. The molecules 6g, h were obtained
through of the reaction between compounds 3g or 3h with
the pyrrol-2-one. As well, the synthesis of pyrimidine
functionalized 6i occurred between the compound 3i with
the maleic anhydride. The synthesis of pyrimidine deriva-
tives 3a–s and functionalized pyrimidines 6g–i occur in 6
and 2 h, respectively.
Selectivity index
To verify how much the compounds were seletive for
parasites, the selectivity index (SI) was calculates for the
trypomastigotes form. SI consists of the ration of CC50/
IC50.Higher SI values indicates greater specificity for
parasite. To obtain the SI, micromolar values were con-
sidered. SI values less than 1 were considered as not indi-
cative of parasitic selectivity.
Statistical analysis
Statistical analysis was performed using non-parametric
tests. A simple linear regression test was performed to
obtain CC50 and IC50. The analysis to measure the varia-
bility between the treated group was the Wilcoxon test and
between the treated and control groups was the Mann–
Whitney test. All conclusions were taken at the significance
level of 5%.
All compounds 3a–s, 5a–f and 6g–i were characterized
1
using IR, H, and 13C NMR spectroscopy. Mass spectro-
metry helped to determine the exact mass of the com-
pounds. The 1H and 13C NMR spectra of compound 3i (Fig.
1) helped to assign the steructure correctly. 1H NMR spectra
of compound 3i showed one singlet at δ 3.85 p.p.m., which
Molecular Modeling
The structures of all molecules were drawn using the
SPARTAN 08’ program (Spartan ‘08 Tutorial and User’s
Guide; Wavefunction 2017) and the optimization of each
structure was carried out using the RM1 (Rocha et al. 2006)
method, available as part of the SPARTAN 08’ program,
using internal default settings for convergence criteria.
Docking analysis was carried out on the T. cruzi Cruzain
2
Fig. 1 Compounds 4-Amino-5-cyano-2-(p-aminophenyl)-6-(p-anisyl)-
pyrimidine (3i)

Medicinal Chemistry Research
was attributed to methoxyl group, and four dublets in the
were performed based on the previous work from our
research work (Do Monte et al. 2016; Melo et al. 2017),
where the signal at δ: 55.4 p.p.m. was attributed to meth-
oxyl group. There are three aromatic rings, containing two
p-substituted systems and a 2,4,5,6-substituted pyrimidine
ring.
In the H NMR spectra of compounds 6h and 6g, when
the substituent in the position 4ʺ is a pyrrol-2-one ring, three
additional signals are present, which have been attributed to
1
aromatic region, at δ: 7.09, 8.19, 8.35 and 8.42 p.p.m. H-
1H COSY spectrum indicates that there are two p-
substituted systems, where the signals at δ: 7.09 and 8.35
p.p.m. are coupling, and the signals at δ 8.19 and 8.42 ppm
1
are in the other ring. H-1H COSY spectrum of this com-
1
pound is presented in supplementary material. There are
also two broad signals at δ: 8.02 and 8.60 p.p.m., which
were attributed to amino groups. 13C NMR assignments
Table 1 Anti T. cruzi activities
of pyrimidine derivatives 3a–s,
5a–f and 6g–i
Pyrimindine
derivatives
T. cruzi
Cytotoxicity HNC
CC50 [µM]c
SI
Trypomastigote (Y strain) Epimastigotes (DM28c
IC50 [µM]a
strain)IC50 [µM]b
3a
3b
3c
3d
3e
3f
10.28
15.09
ND
83.37
ND
112.1
124.55
88.56
16.37
33.29
203.56
31.07
57.06
82.69
188.06
264.78
49.81
131.47
46.89
175.85
30.02
17.39
87.8
10.90
8.25
ND
ND
6.66
4.19
84.66
89.77
91,36
4.25
39.4
76.43
72.26
32.92
81.6
18.56
3.4
2.46
0.50
14.58
0.32
0.73
ND
66.33
13.96
97.16
78.37
ND
3g
3h
3i
3j
29.23
34.99
75.37
ND
6.43
7.57
0.66
ND
3k
3l
3m
3n
3o
3p
3q
3r
3s
5a
5b
5c
5d
5e
5f
ND
ND
34.92
9.09
5.04
3.30
0.30
0.81
0.77
2.20
ND
3.61
3.68
14.04
2.39
ND
58.58
108.97
39.65
61.98
ND
30.55
136.1
52.36
17.32
128.85
99.3
0.022
59.22
59
ND
ND
33.49
68.56
ND
3.85
1.45
ND
67.61
120.31
21
84.4
6g
6h
6i
55.13
ND
60.94
56.03
37.2
1.11
ND
82.43
4.44
10.53
10.19
40.49
3.65
11.34
Benznidazole
(BZN)
459.2
The data were acquired but were not sufficient to calculate the IC50/SI
HNC highest non-cytotoxic concentration, BZN benznidazole, IC50 inhibitory concentration for 50%, ND not
determined
aDetermined 24 h after incubation of the compounds, using Y strain trypomastigotes
bDetermined 4 days after incubation with compounds, using Dm28c epimastigotes
cCell viability of RAW 264.7 macrophages determined 48 h after treatment
dSelectivity index (SI) is the ratio of macrophages viability (HNC) to the IC50 on trypomastigotes

Medicinal Chemistry Research
Table 2 Docking results and
molecular interaction of Cruzain
with compounds 3d, 3f, 5a–f
and 6g–i
Residues Compounds and ChemPLPa
(3d) (3f) (5a) (5b)
(5c)
56.90 60.62 60.49 58.51 58.02
(5d) 58.14 (5e)
(5f)
(6g) 55.42 (6h)
(6i)
55.35 63.22
58.84 60.35
GLN19
GLN21
GLY23
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
HC
HC
—
—
—
—
HB
—
HC
HC
HC
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CYS25 HC
HC
HC
HB
HC
HC
HC
—
—
—
—
HC
T-STACK
HB
—
HC
HC
HB
HC
HC
HC
HB
—
HC
HC
HB
—
HC
HC
HB
—
TRP26
SER29
GLY65
HC
HB
—
HC
—
HC
—
—
HC
—
—
—
—
—
HC
HC
HC
—
LEU67 HC
MET68 HC
ASN69 HB
VAL137 HC
ALA138 HC
HC
HC
—
HC
HC
—
HC
HC
—
HC
HC
HB
—
HC
HC
HB
—
HC
HC
—
—
—
—
—
—
—
HC
—
—
HC
HC
HC
HC
HC
HC
—
HC
—
HC
—
HC
—
HC
—
GLN159
—
—
LEU160 HC
HC
HC
HB
—
HC
—
HC
HC
(2 × )
ASP161
—
—
—
—
—
—
—
—
—
—
—
HC
—
—
—
—
—
HIS162 HC
GLY163 HC
HC
HC
—
HC
HC
—
HC
HC
—
—
HC
HC
HC
HC
HC
HC
HC
HC
—
PI-STACK HC
HC
HC
—
HC
—
—
—
—
—
—
HC
—
LEU164
LEU204
GLY207
GLU208
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
HC
HC
HC
HC
—
HC
HC
HC hydrophobic contacts, HB hydrogen bond, Pi-stack π–π interaction, T-stack T-shaped stacking
interaction, — not presented interaction with residues
aThe GOLD docking scores are dimensionless
H5ʺʺ of the methylene group and H3ʺʺ and H4ʺʺ of the
methine groups. The signal at δ 3.30 ppm (s, 2H) was
attributed to H5ʺʺ for compound 6h. (There was not
observed coupling with H4ʺʺ nucleus because the dihedral
angle between them is close to 90°, being observed one
singlet with relative integration area equal to two. The
signals at δ 6.80 ppm (d, 1H, J: 6.6 Hz) and 6.72 p.p.m. (d,
1H, J: 6.6 Hz) were attributed at H3ʺʺ and H4ʺʺ nuclei,
respectively.)
All 28 compounds were evaluated concerning their
ability to inhibit the epimastigote, and trypomastigote. The
anti-epimastigote activity of the compounds varied with an
IC50 of 0.022 μM (5b) to 120.31 μM (5f) and for trypo-
mastigote from 6.66 μM (3d) to 108.97 μM (3r). Com-
pounds 3d, 3h, 3o, 3p, 3q, 3s, 5b and 6i showed greater
activity than BZN against epimastigotes. Compounds 3a,
3b, 3d, 3f, 3j, 3k, 3o, 3p, 3s, 5d and 6i showed greater
activity than BZN against trypomastigotes. It is described in
the literature that the compounds which have lower IC50
values than BZN are considered active against T. cruzi
(Romanha et al. 2010).
Cytotoxic and Trypanocidal Activity
After structural characterization of pyrimidine compounds,
3a–s, 5a–f and 6g–i (Table 1), the anti-parasitic activity and
host cell cytotoxicity were determined.
Compounds were evaluated concerning their ability to
inhibit the T. cruzi epimastigote (Dm28 strain) and trypo-
mastigotes (Y strain). Results were expressed in terms of
IC50 (µM). The cytotoxicity has been determined in RAW
264.7 macrophages and results are expressed as CC50 values
(Table 1).
Most compounds proved to be more toxic than the
reference drug. The most selective compounds (≥5× SI)
tested were 3a, 3b, 3f, 3j, 3k and 3o, with emphasis on
compound 3f, whose selectivity index (SI:14.58) was better
than that of BZN.
Computational tools are used aiming to design target-
specific compounds (Ferreira et al. 2015). These com-
pounds are often investigated in silico in relation to their
affinity with the biological target which is being drawn

Medicinal Chemistry Research
Fig. 2 The 3D a and 2D b
representations of the
intermolecular interactions that
occurs between the SZ24 (6i)
compound and the Cruzain
binding site of the amino acid
residues
using molecular docking (Waszkowycz et al. 2011). In this
study, the ligands were the compounds and the target was
the T. cruzi enzyme, cruzain. In silico, the 3d, 3f, 5a–f and
6g–i demonstrated a greater affinity for cruzain.
performed more hydrophobic contacts at Cruzain’s binding
site with the extra carbons of this ring. Also, the additional
oxygens (H-bond acceptor) has improved the chances that
these compounds to form hydrogen bonds at Cruzain’s
binding site which, in fact, has granted an additional
hydrogen bond for the compounds 5b, 5d–f, 6h and 6i (Fig.
5b, 5d–f and 6h), see figures presented in supplementary
material.
By analyzing the experimental results (IC50), the com-
pounds SZ24 (6i) has presented an IC50 of 4.44 µg/ml
(DM28c strain), with the reference BZN of IC50 10.53 µg/
ml, and 10.19 µg/ml (Y strain), with the reference of IC50
40.49 µg/ml, CC50 37.2 µg/mL and 459.2 µg/mL, respec-
tively. Alongside the good experimental results (IC50), the
docking solution obtained for the complex formed by the 6i
compound and the Cruzain enzyme (Table 1, Fig. 2) indi-
cates that this compound may have a good potential of
becoming a novel drug against the T. cruzi parasite, as it has
obtained a high predicted affinity for the Cruzain enzyme
binding site (ChemPLP: 60.35), which is a key enzyme for
this parasite as mentioned earlier. Furthermore, by obser-
ving the experimental results (IC50), the 6i compound has
shown an activity up to 2.5 times higher than the Benzo-
nidazole compound, which is the reference anti T. cruzi
drug.
Compound 3f showed excellent IC50 and CC50 experi-
mental results (Table 1). The anchoring solution obtained
for the complex formed by compound 3f and the enzyme
Cruzain indicates that this compound may also have an
excellent potential to become a novel drug against T. cruzi
parasite, as it presented a high affinity predicted for the
binding site of the enzyme Cruzain (ChemPLP: 60.62),
which is a key enzyme for this parasite as mentioned before.
One of the key amino acids present at Cruzain binding
site that has been favorable to the compound’s stability was
the SER29, which performed hydrogen bonds with eleven
molecules (Table 2). Other key amino acids (Table 2 and
Table 3 in supplementary material) were TRP26, LEU67,
We noted that the compound 3f presented an excellent
result (anti- trypomastigote IC50 13.96 µM and CC50 203.56
µM), with a better selective index than BZN (SI:14.58), and
also was appointed as a hit by the molecular docking studies
against cruzain. Compound 3a also presented an interesting
result (anti-trypomastigote IC50 10.28 µM and CC50 112.1
µM) against trypomastigotes, and a better dosage than the
standard drug, BZN (anti-trypomastigote IC50 40.49 µM and
CC50 459.2 µM). Therefore, these drugs may act as a
potential antagonist to T. cruzi. It is important here to
emphasize that in silico and in vitro should be done in
parallel, since molecules that do not have a good compu-
tational score may be early discaderd for in vitro studies,
leading to the lost of potential hits [24]. These ideas could
be proved in our current study.
Docking studies
By analyzing the ChemPLP score values, it seems that the
substitution of the heterocyclic ring on any of the com-
pounds, viz., 3a–s, 5a–f and 6g–i substituent on the para
positions of the phenyl rings has improved the affinity of the
3a–f and 3g–i compound for the Cruzain binding site. All
the nine compounds which have at least one heterocyclic
ring addition (5a–f and 6g–i) can be found in the top 11 (3d,
3f, 5a–f and 6g–i) top ranking compounds in terms of
ChemPLP score values (Table 2). Although all of the
molecules have been calculated, only those with the best
enzyme affinity results will be presented here. The others
are presented in the Supplementary Material. By analyzing
the ChemPLP score values. The docking position at Cru-
zain’s binding site for these nine compounds can be seen in
Fig. 2. The reason behind this behavior seems to be that the
compounds that have this additional heterocyclic ring

Medicinal Chemistry Research
MET68, ALA138, LEU160, GLY163 and GLU208 which
have performed hydrophobic contacts with most of the
compounds. Nevertheless, a few other amino acids also
performed important interactions (Table 2 and Table 3 in
supplementary material), like the residues TRP26 (pi-
stacking T shaped with one compound), ASN69 (hydrogen
bonds with six compounds) and LEU160 (hydrogen bond
with six compound). Therefore, these intermolecular inter-
actions draw a map of the important contacts present in this
molecular system, and is useful for planning future mole-
cular modifications for potency improvement.
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Conclusion
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https://doi.org/10.3390/
molecules200713384
A series of 28 pyrimidine derivatives have been synthetized,
of these, eight are unpublished molecules. The structures
have been verified by employing the spectroscopic techni-
ques (IR, H1 and C13 NMR data). The mass spectral ana-
lysis gave correctly the elemental composition of all
compounds, which verified the molecular weight. We
evaluated their anti-T. cruzi, cytotoxicity and molecular
modeling using the Docking approach. The pharmacologi-
cal evaluation led to the identification of two potent and
selective pyrimidines: compounds 3a and 3f. Concomitantly
the molecular docking of compounds 3f and 6i showed the
affinity of ChemPLP 60.35 and 60.62, respectively.
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Acknowledgements We gratefully acknowledge the financial support
received from Foundation for Science and Technology (Facepe),
National Counsel of Technological and Scientific Development
(CNPq); and Coordination for the Improvement of Higher Education
Personnel (CAPES)
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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