Effect of N-1 arylation of monastrol on kinesin Eg5 inhibition in glioma cell lines

Article abstract of DOI:10.1039/c8md00095f

An original and focused library of two sets of dihydropyrimidin-2-thiones (DHPMs) substituted with N-1 aryl groups derived from monastrol was designed and synthesized in order to discover a more effective Eg5 ligand than the template. Based on molecular docking studies, four ligands were selected to perform pharmacological investigations against two glioma cell lines. The results led to the discovery of two original compounds, called 20h and 20e, with an anti-proliferative effects, achieving IC₅₀ values of about half that of the IC₅₀ of monastrol in both cell lines. As with monastrol, flow cytometry analyses showed that the 20e and 20h compounds induced cell cycle arrest in the G₂/M phase, and immunocytochemistry essays revealed the formation of monopolar spindles due to Eg5 inhibition without any toxicity to Caenorhabditis elegans.

Full text of DOI:10.1039/c8md00095f

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RESEARCH ARTICLE
Effect of N-1 arylation of monastrol on kinesin
Eg5 inhibition in glioma cell lines†
Cite this: DOI: 10.1039/c8md00095f
Itamar Luís Gonçalves, ‡a Liliana Rockenbach,‡a Gustavo Machado das Neves,a
Gabriela Göethel,b Fabiana Nascimento,a Luciano Porto Kagami,a Fabrício Figueiró,c
Gabriel Oliveira de Azambuja,a Amanda de Fraga Dias,c Andressa Amaro,c
Lauro Mera de Souza,d Ivan da Rocha Pitta,e Daiana Silva Avila,f
b
Daniel Fábio Kawano,g Solange Cristina Garcia,
Ana Maria Oliveira Battastinic and Vera Lucia Eifler-Lima
*
a
An original and focused library of two sets of dihydropyrimidin-2-thiones (DHPMs) substituted with N-1 aryl
groups derived from monastrol was designed and synthesized in order to discover a more effective Eg5 li-
gand than the template. Based on molecular docking studies, four ligands were selected to perform phar-
macological investigations against two glioma cell lines. The results led to the discovery of two original
compounds, called 20h and 20e, with an anti-proliferative effects, achieving IC50 values of about half that
of the IC50 of monastrol in both cell lines. As with monastrol, flow cytometry analyses showed that the 20e
and 20h compounds induced cell cycle arrest in the G2/M phase, and immunocytochemistry essays re-
vealed the formation of monopolar spindles due to Eg5 inhibition without any toxicity to Caenorhabditis
elegans.
Received 19th February 2018,
Accepted 14th April 2018
DOI: 10.1039/c8md00095f
rsc.li/medchemcomm
istered alkylating agent that reaches the central nervous sys-
tem in therapeutic concentrations.3
Introduction
Gliomas are the most common primary cancer in the central
nervous system.1 Glioblastoma multiform is considered the
most aggressive and lethal form of glioma, with a patient sur-
vival rate limited to a few months.2 This context has been ob-
served even through the adoption of current therapeutic pro-
tocols, which involve a combination of surgery, radiotherapy
and chemotherapy. Chemotherapy is usually performed
worldwide with the pro-drug temozolomide, an orally admin-
The search for new anti-glioblastoma agents still repre-
sents a great challenge, mainly due to the many malignant
characteristics of glioblastoma multiform,4 such as: a)
undefined cell infiltration boundaries to adjacent healthy tis-
sue, which hinders complete surgical removal; b) multiple
mechanisms of drug resistance; c) the presence of areas of
hypoxia with inactive mitotic cells; d) the cancer stem cell
populations; and e) the presence of a blood–brain barrier,
which limits the bioavailability of drugs to the brain.5 In ad-
dition to these factors, a major challenge for the discovery of
new drugs for the treatment of cancer is finding molecules
that have good levels of tolerability and pharmacokinetic pro-
files, and produce minimal adverse effects; they must also
show efficacy in curing the disease or significantly increase
the patients' survival rate.
One of the most explored targets for anticancer drug de-
sign is mitotic cell division, a complex process that is of cru-
cial importance for cellular proliferation and tumor growth.
Mitotic inhibitors currently used as antitumor drugs include
taxanes and vinca alkaloids, such as paclitaxel, vincristine,
vinblastine and colchicine. These compounds affect the poly-
merization of microtubules by binding to tubulin. When
exerting activity directly on tubulin, these drugs affect differ-
ent cellular functions that are dependent on microtubule ac-
tivity, such as cell mobility, the transport of organelles and
a Laboratório de Síntese Orgânica Medicinal/LaSOM, Faculdade de Farmácia,
Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752, Porto Alegre/
RS, Brazil. E-mail: veraeifler@ufrgs.br
b Laboratório de Toxicologia – LATOX, Faculdade de Farmácia, Universidade
Federal do Rio Grande do Sul, Porto Alegre/RS, Brazil
c Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul,
Porto Alegre/RS, Brazil. E-mail: abattastini@gmail.com
d Instituto de Pesquisa Pelé Pequeno Príncipe, Faculdades Pequeno Príncipe,
Curitiba-PR, Brazil
e Núcleo de Pesquisa em Inovação Terapêutica, Universidade Federal de
Pernambuco, Recife/PE, Brazil
f Grupo de Pesquisa em Bioquímica e Toxicologia em Caenorhabditis elegans
(GBToxCE), Universidade Federal do Pampa-UNIPAMPA, Uruguaiana, RS, Brazil
g Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas,
Campinas-SP, Brazil
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00095f
‡ These authors have contributed equally to this work.
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vesicles6 and neuro-transportation; consequently, they trigger
side effects, such as peripheral neuropathy.7 Another way to
affect microtubule dynamics in cell division is the inhibition
of kinesin Eg5, a mitosis motor protein that regulates spindle
elongation at prophase; this contributes to centrosome sepa-
ration, through which genetic material moves to the poles of
the cell during metaphase. Inhibition of Eg5 produces dis-
ruption of the cell cycle in the G2/M phase and consequent
cell death by apoptosis.8 Therapeutic use of Eg5 inhibitors
has the potential of overcoming the resistance of neoplastic
tissue against drugs targeting the polymerization of microtu-
bules.9 Furthermore, Eg5 expression in non-proliferating
adult tissue is generally negligible; meanwhile, in neoplastic
tissue, Eg5 is highly expressed and related to aggressiveness
and lack of response to chemotherapy.10 Kinesin Eg5 is
overexpressed in many kinds of tumors, such as breast carci-
nogenesis,11 laryngeal squamous cell carcinoma,12 astrocytic
neoplasm,13 prostate cancer,14 bladder cancer15 and renal cell
carcinoma.8 Thus, inhibition of Eg5 increases selectivity over
mitotically active cells16 and cancer cells,8,11–15 which results
in fewer side effects and greater effectiveness; therefore, Eg5
is a promising anticancer target.
Monastrol 1 (Fig. 1) is responsible for the discovery of Eg5
as a new target for cancer treatment; it was identified in 1999
as the first small molecule to present targeted Eg5 inhibition.
Monastrol 1 received this name because it induces the forma-
tion of a single mitotic spindle, replacing the bipolar spin-
dles that are characteristic of cell division.17 Evidence sup-
ports that monastrol 1 effects allosteric inhibition of Eg5,
leading to a conformational change that prevents the release
of ADP from the protein in the ADP-Eg5 complex. The active
site of Eg5, therefore, remains occupied, preventing the bind-
ing of a new molecule of ATP.18
Based on this fact, since the identification of the role of
monastrol 1 in Eg5 inhibition, a variety of dihydropyrimidin-
2-thiones (DHPMs) have been obtained with the aim of devel-
oping more potent compounds (Fig. 1). Since 2005, many
researchers have been dedicated to searching for new
dihydropyrimidin-2-thiones with antitumor activity; these are
currently considered to be privileged structures due to the di-
verse pharmacological activities found for this class of com-
pounds.19,20 Monastrol 1 produced moderate allosteric inhi-
bition in Eg5, while its analogue p-monastrol 2 presented
insignificant effects on Eg5.18 In 2005 it was reported that
enastrol 3, enastron 4 and dimethylenastron 5 present greater
inhibition activity on Eg5 than monastrol.21 Among the
monastrol derivatives with low inhibitory capacity, it is also
possible to highlight structures 6 and 7, identified as mon-97
and fluorastrol.22 Recently, various inhibitors of Eg5 have
reached the clinical stage (Fig. 2, structures 8–13); however,
there are no drugs on the market that act on Eg5.10
Previous results obtained in our laboratory have shown
that DHPMs can inhibit the growth of glioma cancer
cells.23–25 In this current work, molecular modeling was
employed to elucidate the possible interactions of DHPMs
with the active site of Eg5, such as a hydrophobic pocket near
the N-1 position. With the aim of verifying this possibility
and, consequently, the effects of arylation on these interac-
tions, a focused library of new dihydropyrimidin-2-thiones
(DHPMs) were synthesized and their activities were
evaluated.
Results and discussion
Chemistry
In recent years, many approaches to structural changes in
DHPMs have focused on C-5 ester side chain modifications,26
C-6 substitution changes27 and, mainly, C-4 aromatic ring
substitutions.26 Few structural variations have been described
in the literature related to the substitution of an aromatic
ring in the N-1 position of the dihydropyrimidine nucleus;
thus, most of the molecules obtained in the present work are
novel. Once the DHPMs are built, the regioselectivity is high
and the reactions occur on N-3 instead of the less reactive
N-1. In fact, a few publications can be found wherein DHPM
N-1 alkylation was achieved;28,29 however, no publications de-
scribing arylation at N-1 of the DHPM have been found. For
this purpose, it appears that the best strategy is to link the ar-
omatic moiety to nitrogen 1 before the Biginelli reaction. Few
studies in the literature using this strategy with the aim of
functionalizing N-1 with an aryl group have been
found,22,30–37 and only one reports some pharmacological ac-
tivity;32 many of these approaches employed phenylthiourea,
with limited substituents linked to the aromatic ring. There
is potential for exploration of the development of routes for
obtaining N-1 aryl-substituted dihydropyrimidin-2-thiones
and prospecting their anticancer activities.
The two series of N-1-aryl-substituted dihydropyrimidin-2-
thiones, 19a–k and 20a–k, were synthesized in three steps by
a convergent approach where the N-1 substitution was added
Fig. 1 Monastrol 1 and derivatives.
Fig. 2 Inhibitors of Eg5 in clinical stage investigations.
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at the thiourea moiety in two steps followed by the Biginelli
reaction in the last step (Scheme 1). In order to compare
their pharmacological effects with monastrol 1, the series
19a–k has no substitution at the benzene ring; meanwhile,
the other series, 20a–k, contains an OH at C-3, like monastrol
1. The eleven N-l arylthioureas 16a–k were obtained in two
steps with high purity (Scheme 1), starting with the reaction
of benzoyl chloride 14 with ammonium thiocyanate; this al-
lows the isothiocyanate intermediate to react with anilines
with different substituents to provide 15a–k. Hydrolysis of
these intermediates with NaOH led to the mono-substituted
thioureas 16a–k. With the exception of 16e and 16f, the inter-
mediates obtained were used in consecutive steps without
purification. The yield range was 40% to 82%.
With the thioureas 16a–k in hand, the Biginelli reaction was
accomplished using ethyl acetoacetate 18 and benzaldehyde
17a or 3-hydroxybenzaldehyde 17b as building blocks. The reac-
tions were carried out in the presence of trimethylsilane chlo-
ride (TMSCl) as a promoting agent at room temperature for 72
hours. In all cases, the product precipitated quantitatively when
the reactional medium was poured in the water. All the com-
pounds were obtained with high purity and good yields, in ac-
cordance with the analytical data reported here and in the ESI.†
This methodology enabled the rapid attainment of 22
dihydropyrimidin-2-thiones with various substitution pat-
terns at N-1 using mild reaction conditions. The different
substitution patterns at N-1 included aromatic rings
exhibiting electron donor and acceptor substituents with dif-
ferent electronic effects on the ring (Table 1). In fact, the N-1
position in the dihydrothiopyrimidine core has been poorly
explored; to the best of our knowledge, with the exception of
compound 19a, the chemical library generated here is origi-
nal. Thus, this research represents an important contribution
to the structural diversity of this class of molecules.
kinesin Eg5 offered important information for the develop-
ment of new ligands for this protein. The allosteric binding
site of Eg5 has been further studied through molecular
modeling techniques and has been found to contain three
hydrophobic subpockets (P1–P3) which interact with the in-
hibitors.38,39 Analysis of the crystallographic pose of
monastrol within Eg5 demonstrated that the insertion of an
aromatic ring with a different pattern of substitution in the
N-1 nitrogen of the dihydropyrimidine nucleus may lead to
improvement in the interaction of dihydropyrimidin-2-
thiones with Eg5. These modifications may promote better
interaction of the ligand with Eg5 by the site identified as the
hydrophobic subpocket 3 (P3 in Fig. 3). Considering these
findings, 2 series of compounds with aromatic substituents
at N-1 were designed, and a virtual screening at the kinesin
Eg5 crystallographic complex (PDB ID: 1X88) was performed.
The validation step was performed through both re-
docking and cross-docking procedures by using eight distinct
crystallographic ligands. The results show that these parame-
ters are suitable for the vast majority of ligands, including
other DHPM derivatives and a trityl-cysteine, containing at
least one pose with a RMSD value fulfilling the adopted
criteria. The docking parameters, however, did not achieve
the arrangement of large compounds, such as ispinesib,
showing poses with RMSD equal to or higher than 4.0 Å (Ta-
ble S1 in the ESI†).
The cross-docking procedure may be considered a more dif-
ficult task if compared to re-docking, showing a 20% decrease
in success rate.40 Moreover, the ability of docking programs to
fit a ligand into a non-native protein conformer is lower when
the ligand possesses a very large structural difference in com-
parison with the native protein.41 Although high RMSD values
were observed for the large ligands, the obtained results sug-
gest high accuracy of the established parameters for both the
re-docking and cross-docking procedures, especially for ligands
that are structurally similar to monastrol 1.
The virtual screening of the designed DHPMs revealed
that the two best compounds, which displayed acceptable
poses and had high GoldScore values, showed ranks better
than monastrol 1 (Table 2). These compounds, 20e and 19h,
and their counterpart analogues 19e and 20h were then cho-
sen for further analyses in order to predict their interactions
with the kinesin binding site compared to the superposed
crystallographic ligands, monastrol and fluorastrol. More-
over, the ligands were evaluated according to their capacity
for exploring the aimed hydrophobic subpocket P3.
Selection of compounds by molecular docking
The determination of the crystallographic structure of
monastrol 1 and of different complex compounds with
The compounds 20e and 19e displayed similar binding
modes with strong resemblance to the crystallographic pose
of monastrol 1, especially at their aromatic moieties,
4-(3-hydroxyphenyl) and 4-phenyl, respectively; these may
interact with the subpocket P2 through hydrophobic interac-
tions with Gly117, Glu118, Arg119, Ala133 and Pro137. The
4-(3-hydroxyphenyl) group from 20e may further interact with
Glu118 through hydrogen bonds, whereas this interaction
may not be observed in 19e due to the lack of the hydroxyl
function. The N1 substituent of both compounds, a 4-nitro-
Scheme 1 (i) NH4SCN, acetone, reflux, 15 min; (ii) PhNH2, acetone,
reflux, 30 min. (iii) NaOH 5% to 10%, 10 to 30 min, 90 °C; (iv) TMSCl,
DMF, 1 h ultrasound, r.t. 72 h.
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Table 1 Structures of the as-synthesized compounds
Compound
R1
R2
H
Compound
R1
R2
19a
20a
OH
19b
H
20b
OH
19c
19d
H
H
20c
20d
OH
OH
19e
H
20e
OH
19f
H
H
20f
OH
OH
19g
20g
19h
H
20h
OH
19i
19j
19k
H
H
H
20i
20j
20k
OH
OH
OH
phenyl group, may explore the subpocket P3 through hydro-
phobic interactions with Leu160, Leu214, Gly217, Ala218 and
Arg221, through hydrogen bonds with Glu116, and through
an anion–π interaction between the side chain of Glu116 and
the aromatic ring (Fig. 4 and Tables S2–S4 in the ESI†). The
most crucial difference observed between these two mole-
cules relies on the 3-hydroxyl group of the phenyl moiety,
which may interact through hydrogen bonds with the main
chain carbonyl group of Glu118 and which is absent in 19e.
The importance of the 3-hydroxyl feature in monastrol deriva-
tives was previously characterized through comparison of
both class I inhibitors (monastrol, enastron and dimethyl-
enastron) and class II inhibitors (mon-97 and fluorastrol),
which contain this group as a common moiety; therefore, it
may be concluded that the 3-hydroxyl feature is essential for
the activity of the molecule.42
The second pair of analogue compounds, 19h and 20h, as-
sumed binding modes similar to the crystallographic pose of
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Fig. 3 Crystallographic complex of monastrol 1 with the allosteric binding site of kinesin Eg5 (PDB ID: 1X88) showing the three hydrophobic
subpockets: P1 (red), P2 (blue) and P3 (black).
fluorastrol 13, with the thione moiety pointing out towards
the surface and the aromatic components, 4-(3-hydroxy-
phenyl) and 4-phenyl, occupying the P2 subpocket. They may
interact with the following common residues: Glu118,
Arg119, Ala133 and Tyr211. Additionally, 19h may further
interact with the main chain carboxyl oxygen from Gly117
and with the main chain amine group from Arg119. The ob-
served difference in the pose orientation of both compounds
led to a considerable change in the N-1 substituent, which
may interact with the P1 subpocket through Tyr211, Glu215,
Ala218 and Ala219, assuming the same position as the
N-methyl group from fluorastrol; however, the N-methyl
group does not appear to interact with the binding site. The
ester moiety of 19h and 20h may interact with both P2 and
P3 through hydrophobic contacts with Glu116, Ile136, Pro137
and Leu214 (Fig. 5 and Tables S5–S7 in the ESI†).
The two best compounds from both series (19e, 20e, 19h
and 20h) were selected by the docking procedure. It was
shown that these compounds may interact with the kinesin
Eg5 binding site in at least two poses, assuming the
monastrol configuration or the fluorastrol configuration. This
finding is in agreement with that related by Prokopcová and
co-workers, in which the authors found that the docked com-
pounds may only adopt two docking poses related to class I
and class II DHPM analogues.22 The four compounds were
also investigated according to their predicted binding affini-
ties using X-Score and were compared to monastrol 1. The
predicted values for all the four compounds were higher than
those predicted for monastrol 1 (Table 3). These results sug-
gest that the evaluated compounds may bind with greater af-
finity to the binding site than monastrol.
Table 2 Virtual screening ranking of the designed DHPMs and the crys-
tallographic ligands based on their best poses according to their
GoldScore values. The ligands, shown in bold, were selected with their
counterparts for further binding mode analyses. The ligands are repre-
sented according to their PDB ligand codes: monastrol (NAT); enastron
(KZ9); dimethylenastron (EGB); fluorastrol (X7E); S-trityl-L-cysteine ana-
logue (V02); triphenylbutanamine analogue (DQ6); Sb743921 (6LX);
ispinesib (G7X)
Structure
GoldScore
Rank
Structure
GoldScore
Rank
6LX
V02
DQ6
G7X
20e
81.8666
73.3863
69.8569
67.2378
66.5438
65.0821
63.5348
62.7548
62.2357
60.8637
60.6676
60.6326
60.5947
60.5311
59.7063
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
19c
20a
20h
20b
19i
59.3787
58.8010
58.6443
58.1574
56.9552
56.5571
54.9600
54.8859
53.9910
52.4179
52.3876
50.0315
49.6185
49.5652
47.4533
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
X7E
19e
20j
20i
Antitumor activity
20g
19h
NAT
19g
EGB
20c
20f
20k
19k
19f
20d
19j
Cell viability. With the aim of verifying the anticancer ac-
tivity of the monastrol derivatives selected by molecular
docking, cell viability was analyzed by MTS assay after treat-
ment with 5, 10, 25, 50 and 100 μM of monastrol 1 or its de-
rivatives 19e, 20e, 19h and 20h for 24 hours. As can be ob-
served in Fig. 6, 19h, 20e and 20h showed better profiles of
KZ9
19a
19b
19d
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Fig. 4 (Left) The allosteric binding site of kinesin Eg5 (PDB ID: 1X88) illustrating the interactions with the crystallographic pose of monastrol 1 (A)
and the best docked poses of 19e (B) and 20e (C). The interactions are represented as dashed lines in the following colors: hydrogen bonds (dark
blue), hydrophobic interactions (red) and anion–π (cyan). The binding pocket amino acids were labeled with different colors to represent their
associations with a specific subpocket: P1 (red); P2 (blue); P3 (black). (right) Solid surface representations of the kinesin Eg5 binding site and the
respective ligands, indicating the hydrophobicity of the binding pocket.
anti-proliferative activity than monastrol 1 in both glioma cell
lines. In the human cell line U138 (Fig. 6A), 100 μM of
(45.6 3.24%, 40.2 4.84% and 40.8 5.27%, respectively).
Similar results were obtained with the C6 rat glioma cell line,
where the three N-1-aryl DHPMs, 20e, 19h and 20h, were
shown to be more cytotoxic than monastrol 1 (Fig. 6B). How-
ever, in this case, 20e and 20h were more effective than 19h
monastrol 1 decreased 20.8
2.99% while, the derivatives
20e, 19h and 20h decreased approximately 2-fold in compari-
son with monastrol cell viability in the U138 glioma cells
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Fig. 5 (Left) The allosteric binding site of kinesin Eg5 (PDB ID: 1X88) illustrating the interactions with the superposed crystallographic fluorastrol
(A) and the best docked poses of 19h (B) and 20h (C). The interactions are represented as dashed lines in the following colors: hydrogen bonds
(dark blue); hydrophobic interactions (red); anion–π (cyan); amide–π stacking (pink); and halogen interactions (dark green). The binding pocket
amino acids were labeled with different colors to represent their associations with a specific subpocket: P1 (red); P2 (blue); P3 (black). (right) Solid
surface representations of the kinesin Eg5 binding site and the respective ligands, indicating the hydrophobicity of the binding pocket.
in decreasing the viability of C6 glioma cells (84.4 0.55%,
79.4 8.15% and 51.6 4.39%, respectively, at 100 μM).
The IC50 values of these five DHPMs in glioma cell lines
U138 and C6 were calculated and are depicted in Table 4. In
both cell lines, compounds 20e, 19h and 20h reached IC50
values lower than half the IC50 of monastrol 1. Derivatives
20e, 19h and 20h showed greater cytotoxic effects against the
C6 rat cell line than against the U138 human cell line; this
probably occurred because C6 cells have a higher prolifera-
tive rate than U138 cells.
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Table 3 Predicted binding affinity values for the four compounds com-
Table 4 The IC50 values of each compound for glioma cell lines U138
pared to monastrol (NAT) calculated by X-Score
and C6
Structure
GoldScore
X-Score
Binding energy
Molecule
U138 IC50 (μM)
C6 IC50 (μM)
20e
19e
19h
20h
NAT
66.5438
63.5348
62.2357
58.6443
60.8637
6.64
6.56
6.39
6.20
6.02
−9.06
−8.94
−8.72
−8.46
−8.22
Monastrol 1
19e
20e
19h
20h
>200
>200
114.1
131.1
142.7
>200
>200
54.7
78.3
57.1
This first result of N-1-aryl DHPMs against glioma cell
lines endorses the importance of the 3-hydroxyl group of the
phenyl moiety (R2), which may interact through hydrogen
bonds with the main chain carbonyl group of Glu118 of Eg5,
as this group is the only difference between 19e and 20e.
Cell cycle and cell death. Following the cell viability as-
says, where two compounds, 20e and 20h, displayed better
inhibition against both glioma cell lines (U138 and C6) than
the template 1, cell cycle and cell death analyses were
performed to confirm the profile and the possible mecha-
nism of cell viability decrease through Eg5 inhibition. The
cells were treated with the IC50 concentrations of the mole-
cules; U138 was treated with 200 μM monastrol 1 or 150 μM
of 20e or 20h, while C6 was treated with 100 μM monastrol 1
or 50 μM of 20e or 20h for 24 h to analyze the cell cycle and
for 48 h to analyze cell death. As expected, and confirming
the profile of Eg5 inhibitors, the treatments with 20e and 20h
induced cell cycle arrest in the G2/M phase (Fig. 7A).
Monastrol 1 increased G2/M cells at 27.4 6.99; 20e increased
to 8.7 2.68 and 20h to 10.4 1.68. The N-1-aryl DHPMs
showed a similar profile of monastrol 1 on cell cycle arrest in
the U138 human cell line but not in the C6 rat cell line,
where a lower extent of arrest in G2/M phase was induced by
the synthesized compounds 20e and 20h in relation to
monastrol 1. This may have occurred as a consequence of a
combination of more potent inhibition of Eg5 by 20e and
20h and the doubling of the C6 population within 24 h,
which culminated in faster cell death in C6 in comparison
with the U138 cells, leading to cell death following cell cycle
arrest before 24 h of analysis; it may also have occurred be-
cause in C6 cells, these compounds act by more than one
mechanism.
As can be observed in Fig. 7B, 20e induced higher levels of
apoptosis and necrosis than monastrol 1 in U138; mean-
while, in C6, monastrol 1 and 20h induced higher levels of
apoptosis. Observing the cell cycle and cell death analyses, it
is possible to note that 20h showed similar behavior in both
cell lines, with a similar profile to that of the Eg5 inhibitor
monastrol 1; however, this behavior was obtained with a
smaller IC50 concentration.
The above results identify the importance of the 3-hy-
droxyl group of the phenyl moiety and are in agreement with
the molecular docking studies. Moreover, the insertion of an
aromatic ring with a different pattern of substitution in the
N-1 nitrogen of the dihydropyrimidin-2-thiones nucleus may
lead to improvement in the activity against Eg5 by extending
the interactions of the ligand within the binding site, espe-
cially with the hydrophobic subpocket 3 (P3) in Fig. 3. The
potential of these two molecules is the first step for drug de-
sign for further in vivo tests.
Immunocytochemistry.
Immunocytochemistry
was
performed by immunofluorescence to analyze the Eg5 inhibi-
tion of 1, 20e and 20h. The formation of monopolar spindles
due to Eg5 inhibition was observed when the cells were
treated with monastrol 1. Fig. 8B and F show the formation
of monopolar spindles due to Eg5 inhibition; as expected,
the N-1 aryl derivatives, 20e and 20h, present the same out-
comes. Fig. 8C, D, G and H show the formation of monopolar
spindles triggered by 20e and 20h, respectively. These immu-
nofluorescence assay results are in agreement with those ob-
served in the cell cycle and cell death analyses, where inter-
ruption of mitosis was observed by cell cycle arrest in the G2/
M phase and apoptosis induction for both tested com-
pounds. Therefore, these results indicate that 20e and 20h
are Eg5 kinesin inhibitors and new anti-glioma drug candi-
dates with greater potential than monastrol.
Fig. 6 Cell viability: U138 (A) and C6 (B) cells were treated with
monastrol 1 or N-1-aryl DHPMs 19e, 20e, 19h and 20h at 5, 10, 25, 50
and 100 μM for 24 h prior to the MTS assay. The percentage of cell via-
bility was calculated in relation to the control, which was considered
as 100% viability. Data represented are means SD of three indepen-
dent experiments. Data were analyzed by one-way ANOVA followed by
post-hoc comparisons (Tukey).
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Fig. 7 (A) Cell-cycle analysis: the cells were treated with the IC50 concentrations of the molecules; U138 cells were treated with 200 μM
monastrol 1 or 150 μM of 20e or 20h. The C6 cells were treated with 100 μM monastrol 1 or 50 μM of 20e or 20h for 24 h. After treatment, the
cells were permeabilized and stained with propidium iodide for determination of cell-cycle distribution (sub-G1, G0/G1, S and G2/M) by flow cytom-
etry. (B) Cell death: the cells were treated with the same concentrations cited above for 48 hours. Cell death was measured by flow cytometry with
annexin V/propidium iodide staining. Data were analyzed by FLOWJO® software. Data are representative of cells in necrosis or total apoptosis
(early plus late apoptosis). The values presented are the mean SD and were analyzed by one-way ANOVA followed by post-hoc comparisons
(Tukey).
In vivo toxicological investigation. A representation of the
concentration–response curves for each molecule is depicted
and 20h, LC50 = 29.80 mM). This is an important finding be-
cause it is the first time that the in vivo toxicity of monastrol
and DHPMs in C. elegans has been revealed. Overall, the mol-
ecules presented high LC50 values, reiterating their safety for
use as potential drugs for an in vivo glioma model.
in Fig. 9. The results showed that monastrol 1 with LC50
=
80.11 mM is the safest compound; however, the other com-
pounds also presented safe profiles (20e, LC50 = 32.90 mM
Fig. 8 Immunocytochemistry: U138 cells (A–D) were treated with 200 μM monastrol 1 or 150 μM of 20e or 20h, while C6 cells (E–H) were treated
with 100 μM monastrol 1 or 50 μM of 20e or 20h for 24 hours. Fixed cells were stained for DNA (blue) and α-tubulin (green). The images corre-
spond to a magnification of 600×. Imaging was performed on an Olympus FluoView™ 1000 197 confocal microscope.
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Chemical shifts are reported as d values (ppm) relative to
TMS (0.0 ppm). The NMR multiplicities brs, s, d, t, q, and m
stand for broad singlet, singlet, doublet, triplet, quartet and
multiplet, respectively.
The samples were analyzed by a high-resolution mass
spectrometer (HR-MS), LTQ-Orbitrap XL (Thermo Scientific),
operating at atmospheric pressure ionization (API) with an
electrospray ionization (ESI) source. Positive ions [M + H]+
were obtained by applying 4.5 kV to the ion source, 30 V to
the capillary and 120 V to the tube lens. The ions were
fragmented by collision-induced dissociation (CID) with a
normalized collision energy of 20. Nitrogen was used to
desolvate the samples at flow rates of 30 and 5 arbitrary units
in the sheath and auxiliary gas, respectively. The samples
were dissolved in methanol (HPLC-grade, J.T. Baker) at 50 μg
mL−1 and placed in chromatographic vials. The infusion was
aided by ultra-high performance liquid chromatography
(UHPLC – Acquity-Waters) at 100 μL min−1 using methanol–
water (60 : 40, v/v) without any chromatographic column. The
mass resolution was set at 15 000 FWHM (full width at half
maximum) and, for accuracy, the HR-MS was externally
calibrated (m/z 100 to 2000) just before the analysis with a
Pierce™ LTQ ESI Positive Ion (caffeine, MRFA, Ultramark-
1621) calibration solution (Thermo-Fisher).
Fig. 9 Mortality percentages of monastrol and its derivatives. Data are
presented as mean SD, N = 3.
The LC50 values of these compounds are relatively high in
comparison to those of other organic molecules tested in
worms.43,44 The low toxicity of monastrol and its derivatives
is relevant for future studies of their biological effects in
worm models of hyperplasia, which will be further pursued
by the present group.
Conclusions
A novel library of 22 N-1 aryl dihydropyrimidin-2-thiones
(19a–k, 20a–k) was synthesized using a forward synthetic
route with the aim of generating monastrol derivatives with
anticancer activity against glioma cell lines. The molecular
modeling studies were conducted with modifications at the
N-1 position in order to increase interactions with the P3
pocket, resulting in excellent Eg5 inhibition. The binding
poses predicted by GOLD appear to be in accordance with
the inhibition studies. It has been demonstrated here that
the two new N-1 aryl substituted compounds, 20h and 20e,
have about twice the amount of activity of monastrol 1
against glioma cells, confirming the success of N-1 aromatic
ring insertion in DHPMs. The greater enzyme inhibition may
lead to cell cycle arrest and apoptosis induction without toxi-
cological effects. All these findings present two new
dihydropyrimidin-2-thiones, 20h and 20e, as Eg5 kinesin in-
hibitors and new anti-glioma drug candidates with higher po-
tential than monastrol. Further molecular studies have been
developed in order to predict the binding affinities of these
ligands in Eg5 as well as in vivo analysis using a rat glioma
model.
FT-IR spectra were recorded on a Perkin Elmer BXII
spectrometer using an ATR probe. TLC analyses were
performed on Merck silica plates (60 F254). Melting points
(mp) were determined on a System Kofler type WME appara-
tus and are uncorrected. The term “room temperature” indi-
cates 20 °C to 30 °C. All products were identified through
their spectroscopic data and their melting points.
General preparation of substituted arylthioureas (16a–k)
To a solution of ammonium thiocyanate (1.1 equiv.) in dry
acetone at room temperature, benzoyl chloride (1 equiv.) was
added, and the solution was heated at 70 °C for 15 minutes.
Then, different anilines (1 equiv.) in acetone were added
dropwise, and the solution was heated for a further 30 mi-
nutes. The reaction mixture was poured into water at room
temperature and the precipitate was filtered and submitted
to alkaline hydrolysis in NaOH 2.50 M, 90 °C for 20 minutes.
Following this, the pH of the system was adjusted to 2 with
HCl and 8 with NH4OH. The product was filtered to a high
level of purity.
General preparation of N-1 aryl substituted didydropirimidin-
2-thiones
Experimental
Chemical analysis
A mixture of ethyl acetoacetate (1 equiv.), substituted phenyl-
thiourea (1 equiv.) and benzaldehyde or 3-hydroxy-
benzaldehyde (1 equiv.) was solubilized in DMF under ultra-
sound for 1 hour. TMSCl (6 equiv.) was then added dropwise,
and the mixture was stirred at room temperature for 72 h. Af-
ter three volumes of water were poured into the mixture, it
was submitted to ultrasound for one hour; the obtained pre-
cipitate was filtered and washed with water. The crude isolate
All chemicals were research grade and were used as obtained.
Nuclear magnetic resonance spectra (1H NMR and 13C NMR)
were recorded on an Anasazi spectrometer with standard
pulse sequences operating at 60 MHz for 1H NMR and 15
MHz for 13C NMR and on a Bruker Ascend NMR with stan-
1
dard pulse sequences operating at 400 MHz for H NMR and
100 MHz for 13C NMR, using DMSO-d6 or CDCl3 as solvent.
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was recrystallized from ethanol or purified by column
chromatography.
yield 34%, mp: 130 °C to 135 °C. FT-IR (ATR cm−1): 3176
(NH); 1701 (CO); 1628 (CC); 1171 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.16 (t, 3J = 7.0 Hz, 3H, CH3); 2.09 (s, 3H,
Ethyl
6-methyl-1,4-diphenyl-2-thioxo-1,2,3,4-tetra-
3
3
hydropyrimidine-5-carboxylate (19a). Green solid, yield 97%,
mp: 106 °C to 108 °C. FT-IR (ATR cm−1): 3160 (NH); 1683
(CO); 1646 (CC); 1174 (C–O). RMN H1 (CDCl3, 60 MHz, δ
ppm): 1.18 (t, 3J = 7.0 Hz, 3H, CH3); 2.10 (s, 3H, CH3); 4.14
(q, 3J = 7.0 Hz, 2H, CH2); 5.46 (m, 1H, CH); 6.94–7.62 (m,
10Harom); 8.43 (bs, 1H, NH). RMN C13 (CDCl3, 15 MHz, δ
ppm): 13.9 (CH3); 18.4 (CH3); 53.8 (CH); 60.5 (CH2); 106.7
(C); 126.3 (C-arom); 127.9 (C-arom); 128.6 (C-arom); 128.8 (C-
arom); 140.2 (C-arom); 142.0 (C-arom); 146.2 (C); 165.6 (C);
178.0 (C). HRMS/MS (m/z): calcd. C20H20N2O2S [M + H]+:
353.13182, found 353.13185.
CH3); 4.12 (q, J = 7.0 Hz, 2H, CH2); 5.45 (d, J = 3.0 Hz, 1H,
CH); 6.97–7.51 (m, 9Harom); 8.75 (d, 3J = 3.0 Hz, 1H, NH).
RMN C13 (CDCl3, 15 MHz, δ ppm): 14.1 (CH3); 18.6 (CH3);
54.1 (CH); 60.7 (CH2); 107.2 (C); 126.4 (C-arom); 128.1IJC-
arom); 128.9 (C-arom); 129.0 (C-arom); 129.8 (C-arom); 134.4
(C-arom); 141.4 (C-arom); 142.0 (C-arom); 145.5 (C); 165.6
(C); 177.9 (C). HRMS/MS (m/z): calcd. C20H19ClN2O2S [M +
H]+: 387.09285, found 387.09296.
Ethyl
1-(3-chlorophenyl)-4-(3-hydroxyphenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (20c). Yel-
low solid, yield 40%, mp: 75 °C to 80 °C. FT-IR (ATR cm−1):
3280 (OH); 1684 (CO); 1631 (CC); 1169 (C–O). RMN H1
Ethyl-4-(3-hydroxyphenyl)-6-methyl-1-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (20a). Yellow solid,
yield 69%, mp: 151 °C to 154 °C. FT-IR (ATR cm−1): 3278
(OH); 1681 (CO); 1629 (CC); 1167 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.19 (t, 3J = 7.0 Hz, 3H, CH3); 2.08 (s, 3H,
3
(CDCl3, 60 MHz, δ ppm): 1.19 (t, J = 7.0 Hz, 3H, CH3); 2.08
3
3
(s, 3H, CH3); 4.14 (q, J = 7.0 Hz, 2H, CH2); 5.40 (d, J = 3.0
3
Hz, 1H, CH); 6.58–7.48 (m, 8Harom); 7.84 (d, J = 3.0 Hz, 1H,
NH). RMN C13 (CDCl3, 15 MHz, δ ppm): 14.0 (CH3); 18.7
(CH3); 54.2 (CH); 61.0 (CH2); 107.2 (C); 113.4 (C-arom); 115.5
(C-arom); 118.2 (C-arom); 129.2 (C-arom); 130.1 (C-arom);
134.5 (C-arom); 141.2 (C-arom); 143.4 (C-arom); 145.0 (C);
156.5 (C-arom); 165.9 (C); 177.8 (C). HRMS/MS (m/z): calcd.
C20H19ClN2O3S [M + H]+: 403.08776, found 403.08790.
3
CH3); 4.14 (q, J = 7.0 Hz, 2H, CH2); 5.40 (m, 1H, CH); 6.59–
7.56 (m, 9Harom); 7.96 (bs, 1H, NH). RMN C13 (CDCl3, 15
MHz, δ ppm): 14.0 (CH3); 18.6 (CH3); 54.1 (CH); 60.9 (CH2);
106.9 (C); 113.5 (C-arom); 115.4 (C-arom); 118.2 (C-arom);
128.8 (C-arom) 129.1 (C-arom); 130.0 (C-arom); 140.0 (C-
arom); 143.6 (C-arom); 146.2 (C); 156.5 (C-arom); 166.1 (C);
178.1 (C). HRMS/MS (m/z): calcd. C20H20N2O3S [M + H]+:
369.12674, found 369.12673.
Ethyl
1-(4-bromophenyl)-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19d). Yellow solid,
yield 83%, mp: 117 °C to 122 °C. FT-IR (ATR cm−1): 3169
(NH); 1699 (CO); 1631 (CC); 1166 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.19 (t, 3J = 7.0 Hz, 3H, CH3); 2.09 (s, 3H,
Ethyl
1-(4-chlorophenyl)-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19b). Yellow solid,
yield 59%, mp: 133 °C to 136 °C. FT-IR (ATR cm−1): 3172
(NH); 1705 (CO); 1630 (CC); 1167 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.17 (t, 3J = 7.0 Hz, 3H, CH3); 2.08 (s, 3H,
3
3
CH3); 4.14 (q, J = 7.0 Hz, 2H, CH2); 5.46 (d, J = 3.0 Hz, 1H,
CH); 6.95–7.66 (m, 9Harom); 8.03 (d, 3J = 3.0 Hz, 1H, NH).
RMN C13 (CDCl3, 15 MHz, δ ppm): 13.9 (CH3); 18.4 (CH3);
53.9 (CH); 60.5 (CH2); 107.1 (C); 122.6 (C-arom); 126.2 (C-
arom); 127.9 (C-arom); 128.7 (C-arom); 131.6 (C-arom); 132.1
(C-arom); 139.2 (C-arom); 141.8 (C-arom); 145.4 (C); 165.4
(C); 177.8 (C). HRMS/MS (m/z): calcd. C20H19BrN2O2S [M +
H]+: 431.04232, found 431.04236.
3
3
CH3); 4.11 (q, J = 7.0 Hz, 2H, CH2); 5.44 (d, J = 3.0 Hz, 1H,
CH); 6.92–7.51 (m, 9Harom); 8.63 (d, 3J = 3.0 Hz, 1H, NH).
RMN C13 (CDCl3, 15 MHz, δ ppm): 14.1 (CH3); 18.6 (CH3);
54.2 (CH); 60.8 (CH2); 107.3 (C); 126.4 (C-arom); 128.2 (C-
arom); 128.9 (C-arom); 129.3 (C-arom); 131.4 (C-arom); 138.9
(C-arom); 142.0 (C-arom); 145.7 (C); 165.6 (C); 178.2 (C).
HRMS/MS (m/z): calcd. C20H19ClN2O2S [M + H]+: 387.09285,
found 387.09260.
Ethyl
1-(4-bromophenyl)-4-(3-hydroxyphenyl)-6-methyl-2-
(20d).
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
White solid, yield 98%, mp: 144 °C to 149 °C. FT-IR (ATR
cm−1): 3380 (OH); 3175 (NH); 1692 (CO); 1641 (CC); 1169
Ethyl
1-(4-chlorophenyl)-4-(3-hydroxyphenyl)-6-methyl-2-
(20b).
3
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(C–O). RMN H1 (CDCl3, 60 MHz, δ ppm): 1.18 (t, J = 7.0 Hz,
White solid, yield 25%, mp: 163 °C to 167 °C. FT-IR (ATR
cm−1): 3379 (OH); 3165 (NH); 1691 (CO); 1639 (CC); 1174
3H, CH3); 2.08 (s, 3H, CH3); 4.14 (q, 3J = 7.0 Hz, 2H, CH2);
5.37 (m, 1H, CH); 6.71–7.06 (m, 4Harom); 7.06–7.35 (m,
2Harom); 7.35–7.72 (m, 2Harom); 8.14 (bs, 1H, NH). RMN C13
(CDCl3, 15 MHz, δ ppm): 13.9 (CH3); 18.7 (CH3); 53.6 (CH);
60.7 (CH2); 106.2 (C); 112.2 (C-arom); 114.4 (C-arom); 117.1
(C-arom); 121.6 (C-arom); 128.8 (C-arom); 130.5 (C-arom);
131.0 (C-arom); 137.8 (C-arom); 141.9 (C-arom); 144.1 (C);
154.8 (C-arom); 164.2 (C); 176.0 (C). HRMS/MS (m/z): calcd.
C20H19BrN2O3S [M + H]+: 447.03724, found 447.03760.
Ethyl 6-methyl-1-(4-nitrophenyl)-4-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (19e). Yellow solid, yield
50%, mp: 208 °C to 211 °C. FT-IR (ATR cm−1): 3162 (NH);
1702 (CO); 1649 (CC); 1518 (NO); 1346 (NO); 1181
(C–O). RMN H1 (DMSO-d6, 60 MHz, δ ppm): 1.13 (t, 3J = 7.0
3
(C–O). RMN H1 (CDCl3, 60 MHz, δ ppm): 1.18 (t, J = 7.0 Hz,
3H, CH3); 2.06 (s, 3H, CH3); 4.04 (q, 3J = 7.0 Hz, 2H, CH2);
3
5.38 (d, J = 3.0 Hz, 1H, CH); 6.20–7.45 (m, 8Harom); 7.964 (d,
3J = 3.0 Hz, 1H, NH). RMN C13 (DMSO-d6, 15 MHz, δ ppm):
14.0 (CH3); 18.2 (CH3); 53.3 (CH); 60.3 (CH2); 105.9 (C); 113.2
(C-arom); 115.0 (C-arom); 117.0 (C-arom); 128.8 (C-arom);
129.8 (C-arom); 132.3 (C-arom); 133.0 (C-arom); 139.3 (C-
arom); 144.1; 145.6; 157.9 (C-arom); 165.3 (C); 177.1 (C).
HRMS/MS (m/z): calcd. C20H19ClN2O3S [M + H]+: 403.08776,
found 403.08705.
Ethyl
1-(3-chlorophenyl)-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19c). White solid,
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Hz, 3H, CH3); 2.06 (s, 3H, CH3); 4.15 (q, 3J = 7.0 Hz, 2H,
CH2); 5.35 (d, 3J = 4.0 Hz, 1H, CH); 7.32–7.78; (m, 7Harom);
8.16–8.47 (m, 2Harom); 10.19 (d, 3J = 3.0 Hz, 1H, NH). RMN
C13 (DMSO-d6, 15 MHz, δ ppm): 13.9 (CH3); 18.2 (CH3); 53.5
(CH); 60.3 (CH2); 105.9 (C); 123.3 (C-arom); 126.5 (C-arom);
127.9 (C-arom); 128.8 (C-arom); 132.1 (C-arom); 142.5 (C-
arom); 145.1 (C-arom); 146.9 (C); 146.9 (C-arom); 165.1 (C);
176.5 (C). HRMS/MS (m/z): calcd. C20H19N3O4S [M + H]+:
398.11690, found 398.11704.
3.0 Hz, 1H, CH); 6.48–7.09 (m, 4Harom); 7.10–7.48 (m,
5Harom); 9.67 (s, 1H, OH); 9.92 (d, J = 3.0 Hz, 1H, NH). RMN
3
C13 (DMSO-d6, 15 MHz, δ ppm): 13.7 (CH3); 18.2 (CH3); 52.9
(CH); 60.1 (CH2); 105.5 (C); 115.2 (C-arom); 126.3 (C-arom);
127.8 (C-arom); 128.8 (C-arom); 131.6 (C-arom); 142.9 (C-
arom); 147.0 (C); 157.1 (C-arom); 165.3 (C); 177.8 (C). HRMS/
MS (m/z): calcd. C20H20N2O3S [M + H]+: 369.12673, found
369.12665.
Ethyl 4-(3-hydroxyphenyl)-1-(4-hydroxyphenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (20g). Yel-
low solid, yield 48%, mp: 198 °C to 201 °C. FT-IR (ATR cm−1):
3150 (OH); 1697 (CO); 1650 (CC); 1183 (C–O). RMN H1
(DMSO-d6, 60 MHz, δ ppm): 1.13 (t, 3J = 7.0 Hz, 3H, CH3);
Ethyl
4-(3-hydroxyphenyl)-6-methyl-1-(4-nitrophenyl)-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (20e). Yel-
low solid, yield 70%, mp: 200 °C to 202 °C. FT-IR (ATR cm−1):
3147 (NH); 1707 (CO); 1641 (CC); 1518 (NO); 1344
(NO); 1173 (C–O). RMN H1 (DMSO-d6, 60 MHz, δ ppm):
3
3
2.02 (s, 3H, CH3); 4.07 (q, J = 7.0 Hz, 2H, CH2); 5.17 (d, J =
3.0 Hz, 1H, CH); 6.52–7.35 (m, 8Harom); 9.49 (s, 1H, OH); 9.65
3
3
1.14 (t, J = 7.0 Hz, 3H, CH3); 2.03 (s, 3H, CH3); 4.09 (q, J =
3
3
7.0 Hz, 2H, CH2); 5.24 (d, J = 3.0 Hz, 1H, CH); 6.58–7.72 (m,
6Harom); 8.14–8.51 (m, 2Harom); 9.54 (s, 1H, OH); 10.12 (d, J
(s, 1H, OH); 9.84 (d, J = 3.0 Hz, 1H, NH). RMN C13 (DMSO-
3
d6, 15 MHz, δ ppm): 14.0 (CH3); 18.2 (CH3); 53.5 (CH); 60.2
(CH2); 105.6 (C); 113.2 (C-arom); 115.2 (C-arom); 117.0 (C-
arom); 129.7 (C-arom); 131.9 (C-arom); 144.5 (C-arom); 146.7
(C); 157.2 (C-arom); 157.7 (C-arom); 165.6 (C); 177.8 (C).
HRMS/MS (m/z): calcd. C20H20N2O4S [M + H]+: 385.12165,
found 385.12155.
= 3.0 Hz, 1H, NH). RMN C13 (DMSO-d6, 15 MHz, δ ppm): 13.9
(CH3); 18.2 (CH3); 53.4 (CH); 60.3 (CH2); 106.2 (C); 113.3 (C-
arom); 115.0 (C-arom); 117.0 (C-arom); 123.9 (C-arom); 129.8
(C-arom); 132.2 (C-arom); 143.9 (C-arom); 144.9 (C-arom);
146.2; 147.0; 157.8 (C-arom); 165.2 (C); 176.4 (C). HRMS/MS
(m/z): calcd. C20H19N3O5S [M
+
H]+: 414.11181, found
Ethyl
1-(4-methoxyphenyl)-6-methyl-4-phenyl-2-thioxo-
414.11198.
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19h). Green solid,
yield 77%, mp: 144 °C to 148 °C. FT-IR (ATR cm−1): 3170
(NH); 1703 (CO); 1630 (CC); 1157 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.19 (t, 3J = 7.0 Hz, 3H, CH3); 2.11 (s, 3H,
CH3); 3.83 (s, 3H, CH3); 4.14 (q, 3J = 7.0 Hz, 2H, CH2); 5.46
(d, J = 3.0 Hz, 1H, CH); 6.79–7.40 (m, 9Harom); 7.87 (d, J =
3.0 Hz, 1H, NH). RMN C13 (CDCl3, 15 MHz, δ ppm): 14.1
(CH3); 18.5 (CH3); 53.9 (CH); 55.4 (CH3); 60.6 (CH2); 106.8
(C); 114.1 (C-arom); 126.4 (C-arom); 128.0 (C-arom); 128.8 (C-
arom); 133.2 (C-arom); 142.3 (C-arom); 146.7 (C); 159.4 (C-
arom); 165.7 (C); 178.7 (C). HRMS/MS (m/z): calcd.
C21H22N2O3S [M + H]+: 383.14238, found 383.14242.
Ethyl
6-methyl-4-phenyl-2-thioxo-1-(3-(trifluoromethyl)-
(19f).
phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate
Green solid, yield 42%, mp: 103 °C to 109 °C. FT-IR (ATR
cm−1): 3166 (NH); 1700 (CO); 1649 (CC); 1163 (C–O).
RMN H1 (CDCl3, 400 MHz, δ ppm): 1.22 (t, 3J = 7.1 Hz, 3H,
CH3); 2.11 (s, 3H, CH3); 4.17 (m, 2H, CH2); 5.52 (d, 3J = 3.6
Hz, 1H, CH); 7.45–7.32 (m, 6Harom); 7.75–7.56 (m, 3H); 8.01
3
3
3
(d, J = 3.0 Hz, 2H). RMN C13 (CDCl3, 100 MHz, δ ppm): 14.1
(CH3); 18.2 (CH3); 54.6 (CH); 60.9 (CH2); 107.7 (C); 122.1;
124.8; 125.7; 126.4; 128.4; 129.1; 141.0; 141.9; 145.2; 165.5;
178.4. HRMS/MS (m/z): calcd. C21H19F3N2O2S [M + H]+:
421.11920, found 421.11918.
Ethyl 4-(3-hydroxyphenyl)-1-(4-methoxyphenyl)-6-methyl-2-
Ethyl
4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1-(3-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(20h).
(trifluoromethyl)phenyl)-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate (20f). Yellow solid, yield 27%, mp: 70 °C to 74 °C.
FT-IR (ATR cm−1): 3291 (NH); 1683 (CO); 1634 (CC); 1165
Green solid, yield 60%, mp: 174 °C to 177 °C. FT-IR (ATR
cm−1): 3147 (NH); 1652 (CO); 1622 (CC); 1171 (C–O).
3
RMN H1 (DMSO-d6, 60 MHz, δ ppm): 1.16 (t, J = 7.0 Hz, 3H,
3
3
(C–O). RMN H1 (CDCl3, 400 MHz, δ ppm): 1.19 (t, J = 7.1 Hz,
CH3); 2.04 (s, 3H, CH3); 3.79 (s, 3H, CH3); 4.10 (q, J = 7.0 Hz,
3H, CH3); 2.05 (s, 3H, CH3); 4.14 (q, 3J = 7.0 Hz, 2H, CH2);
5.41 (d, 3J = 3.3 Hz, 1H, CH); 6.74 (m, 1Harom); 6.85 (m,
1Harom); 7.15 (t, J = 7.8 Hz, 1H); 7.53 (m, 2Harom), 7.66 (m,
2H, CH2); 5.31 (d, J = 4.0 Hz, 1H, CH); 6.56–7.35 (m, 8Harom);
3
9.52 (s, 1H, OH); 9.81 (d, 3J = 4.0 Hz, 1H, NH). RMN C13
(DMSO-d6, 15 MHz, δ ppm): 14.0 (CH3); 18.2 (CH3); 53.1
(CH); 55.3 (CH3); 60.1 (CH2); 105.6 (C); 113.2 (C-arom); 113.8
(C-arom); 114.9 (C-arom); 116.9 (C-arom); 129.7 (C-arom);
133.1 (C-arom); 144.2 (C-arom); 146.5 (C); 157.7 (C-arom);
158.7 (C-arom); 165.4 (C); 177.7 (C). HRMS/MS (m/z): calcd.
C21H22N2O4S [M + H]+: 399.13730, found 399.13752.
3
1Harom); 8.09 (d, J = 3.2 Hz, 1H, NH). RMN C13 (CDCl3, 100
MHz, δ ppm): 14.2 (CH3); 19.0 (CH3); 54.5 (CH); 61.3 (CH2);
107.8 (C); 113.6; 115.8; 118.5; 122.3; 125.0; 125.9; 130.4;
141.0; 143.5; 145.3; 156.5; 165.9; 178.3. HRMS/MS (m/z):
calcd. for C21H19F3N2O3S [M
437.11395.
+
H]+: 437.11412, found
Ethyl
1-(2,6-dimethylphenyl)-6-methyl-4-phenyl-2-thioxo-
Ethyl
1-(4-hydroxyphenyl)-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19i). Yellow solid,
yield 62%, mp: 124 °C to 128 °C. FT-IR (ATR cm−1): 3176
(NH); 1712 (CO); 1652 (CC); 1176 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.12 (t, 3J = 7.0 Hz, 3H, CH3); 2.03 (s, 3H,
CH3); 2.16 (s, 3H, CH3); 2.24 (s, 3H, CH3); 4.10 (q, J = 7.0 Hz,
2H, CH2); 5.52 (m, 1H, CH); 7.06–7.22 (m, 3Harom); 7.22–7.49
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19g). Brown solid,
yield 52%, mp: 173 °C to 177 °C. FT-IR (ATR cm−1): 3212
(OH); 1697 (CO); 1649 (CC); 1173 (C–O). RMN H1
(DMSO-d6, 60 MHz, δ ppm): 1.14 (t, 3J = 7.0 Hz, 3H, CH3);
3
3
3
2.08 (s, 3H, CH3); 4.07 (q, J = 7.0 Hz, 2H, CH2); 5.27 (d, J =
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(m, 5Harom); 7.91 (bs, 1H, NH). RMN C13 (CDCl3, 15 MHz, δ
ppm): 13.6 (CH3); 16.6 (CH3); 17.6 (CH3); 53.9 (CH); 60.0
(CH2); 104.9 (C); 126.4 (C-arom); 127.5 (C-arom); 128.2 (C-
arom); 135.7 (C-arom); 138.0 (C-arom); 138.3 (C-arom); 142.4
(C-arom); 145.1 (C); 165.4 (C); 175.6 (C). HRMS/MS (m/z):
calcd. C22H24N2O2S [M + H]+: 381.16312, found 381.16324.
Ethyl 1-(2,6-dimethylphenyl)-4-(3-hydroxyphenyl)-6-methyl-
3.0 Hz, 1H, NH). RMN C13 (CDCl3, 15 MHz, δ ppm): 13.9
(CH3); 18.4 (CH3); 19.4 (CH3); 19.7 (CH3); 53.8 (CH); 60.4
(CH2); 106.5 (C); 126.3 (C-arom); 127.8 (C-arom); 128.6 (C-
arom); 129.9 (C-arom); 137.1 (C-arom); 137.9 (C-arom); 142.3
(C-arom); 146.5 (C); 165.5 (C); 178.2 (C). HRMS/MS (m/z):
calcd. C22H24N2O2S [M + H]+: 381.16312, found 381.16291.
Ethyl 1-(3,4-dimethylphenyl)-4-(3-hydroxyphenyl)-6-methyl-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(20i).
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(20k).
Brown solid, yield 69%, mp: 163 °C to 167 °C. FT-IR (ATR
cm−1): 3166 (OH); 1705 (CO); 1636 (CC); 1174 (C–O).
RMN H1 (CDCl3, 60 MHz, δ ppm): 1.14 (t, 3J = 7.0 Hz, 3H,
CH3); 2.00 (s, 3H, CH3); 2.12 (s, 3H, CH3); 2.24 (s, 3H, CH3);
4.11 (q, 3J = 7.0 Hz, 2H, CH2); 5.46 (m, 1H, CH); 6.55–7.31
(m, 7Harom); 7.92 (bs, 1H, NH). RMN C13 (CDCl3, 15 MHz, δ
ppm): 14.0 (CH3); 17.1 (CH3); 18.1 (CH3); 18.2 (CH3), 54.6
(CH); 60.8 (CH2); 105.6 (C); 114.0 (C-arom); 115.4 (C-arom);
118.4 (C-arom); 128.5 (C-arom); 128.6 (C-arom); 128.9 (C-
arom); 130.1 (C-arom); 136.3 (C-arom); 137.3 (C-arom); 138.7
(C-arom); 144.1 (C-arom); 145.3 (C); 156.5 (C-arom); 166.2
(C); 176.3 (C). HRMS/MS (m/z): calcd. C22H24N2O3S [M + H]+:
397.15803, found 397.15898.
White solid, yield 57%, mp: 163 °C to 167 °C. FT-IR (ATR
cm−1): 3364 (OH); 3169 (NH); 1657 (CO); 1627 (CC); 1150
3
(C–O). RMN H1 (CDCl3, 60 MHz, δ ppm): 1.17 (t, J = 7.0 Hz,
3
3H, CH3); 2.08 (s, 3H, CH3); 2.26 (s, 6H, 2CH3); 4.14 (q, J =
3
7.0 Hz, 2H, CH2); 5.38 (d, J = 3.0 Hz, 1H, CH); 6.25–6.54 (m,
3
1Harom); 6.54–7.33 (m, 6Harom); 7.92 (d, J = 3.0 Hz, 1H, NH).
RMN C13 (DMSO-d6, 15 MHz, δ ppm): 14.0 (CH3); 18.3 (CH3);
19.1 (CH3); 19.4 (CH3); 53.2 (CH); 60.2 (CH2); 105.6 (C); 113.3
(C-arom); 114.9 (C-arom); 117.1 (C-arom); 129.8 (C-arom);
136.5 (C-arom); 136.7 (C-arom); 138.1 (C-arom); 144.3 (C-
arom); 146.3 (C); 157.7 (C-arom); 165.4 (C); 177.4 (C). HRMS/
MS (m/z): calcd. C22H24N2O3S [M + H]+: 397.15803, found
397.15800.
Ethyl
1-(3,5-dimethylphenyl)-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19j). Green solid,
yield 64%, mp: 135 °C to 138 °C. FT-IR (ATR cm−1): 3167
(NH); 1705 (CO); 1623 (CC); 1158 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.16 (t, 3J = 7.0 Hz, 3H, CH3); 2.10 (s, 3H,
Molecular docking
The molecules 19a–k and 20a–k were first drawn and mini-
mized on a CORINA webserver45 and were then minimized
using the semi-empiric method PM6 in Spartan 14 V.1.1.4
(ref. 46) in order to obtain tridimensional structures with
bond lengths and chemically appropriate angles.
3
CH3); 2.32 (s, 6H, 2CH3); 4.12 (q, J = 7.0 Hz, 2H, CH2); 5.45
(d, 3J = 3.0 Hz, 1H, CH); 6.45–6.94 (m, 2Harom); 7.01 (m,
3
1Harom); 7.15–7.42 (m, 5Harom); 8.64 (d, J = 3.0 Hz, 1H, NH).
RMN C13 (CDCl3, 15 MHz): 14.1 (CH3); 18.6 (CH3); 21.2
(2CH3); 54.1 (CH); 60.5 (CH2); 106.5 (C); 126.5 (C-arom);
128.0 (C-arom) 128.8 (C-arom); 130.4 (C-arom); 138.6 (C-
arom); 140.2 (C-arom); 142.4 (C-arom); 146.5 (C); 165.8 (C);
178.2 (C). HRMS/MS (m/z): calcd. C22H24N2O2S [M + H]+:
381.16312, found 381.16310.
Molecular docking simulations were accomplished using
the crystallographic structure of kinesin Eg5 bound with
monastrol (PDBID: 1X88) using GOLD 5.0.3 from the Cam-
bridge Crystallographic Data Center (CCDC).47 The molecular
docking procedure began with protein preparation using the
protein preparation wizard from Schrödinger Maestro
suite.48,49 The protein preparation was performed in Maestro
via: (a) insertion of hydrogen atoms, (b) assignment of bond
orders, (c) filling of missing side chains using Prime, (d) de-
letion of water molecules, (e) deletion of the protein chain B,
(f) assignment of ligand protonation states and metal bind-
Ethyl 1-(3,5-dimethylphenyl)-4-(3-hydroxyphenyl)-6-methyl-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(20j).
Green solid, yield 55%, mp: 197 °C to 200 °C. FT-IR (ATR
cm−1): 3168 (OH); 1697 (CO); 1628 (CC); 1149 (C–O).
RMN H1 (CDCl3, 60 MHz, δ ppm): 1.18 (t, 3J = 7.0 Hz, 3H,
CH3); 2.08 (s, 3H, CH3); 2.31 (s, 6H, 2CH3); 4.12 (q, 3J = 7.0
ing states at pH 7.0
2.0 using Epik, (g) protonation of
3
Hz, 2H, CH2); 5.37 (d, J = 3.0 Hz, 1H, CH); 6.40 (m, 1Harom);
amino acid residues at pH 7.0 using PropKA, and (h) re-
strained minimization of all atoms of the crystal structure
using the OPLS2005 force field with a convergence of heavy
atoms of 0.30 Å.
3
6.54–7.36 (m, 6Harom); 7.89 (d, J = 3.0 Hz, 1H, NH). RMN C13
(DMSO-d6, 15 MHz, δ ppm): 14.0 (CH3); 18.3 (CH3); 20.8
(2CH3); 53.2 (CH); 60.2 (CH2); 105.6 (C); 113.3 (C-arom);
114.9 (C-arom); 117.1 (C-arom); 129.8 (C-arom); 137.9 (C-
arom); 140.2 (C-arom); 144.3 (C-arom); 146.1 (C); 157.7 (C-
arom); 165.64 (C); 177.2 (C). HRMS/MS (m/z): calcd.
C22H24N2O3S [M + H]+: 397.15803, found 397.15819.
Before starting the virtual screening procedure using the
designed molecules, the docking parameters (e.g. grid size
and position) were validated according to the re-docking and
cross-docking results. Re-docking and cross-docking runs
were evaluated through comparison of the docked poses with
the respective crystallographic ligands, in accordance with
criteria previously reported by Caroli and coworkers.50 The
cross-docking method consisted of docking several different
ligands extracted from other crystallographic structures of
kinesin Eg5 (Table S8 in the ESI†) into the binding site of
PDB ID 1X88. The superposition of the crystallographic
Ethyl
1-(3,4-dimethylphenyl)-6-methyl-4-phenyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (19k). White solid,
yield 55%, mp: 189 °C to 191 °C. FT-IR (ATR cm−1): 3181
(NH); 1702 (CO); 1622 (CC); 1153 (C–O). RMN H1 (CDCl3,
60 MHz, δ ppm): 1.18 (t, 3J = 7.0 Hz, 3H, CH3); 2.10 (s, 3H,
3
CH3); 2.28 (s, 6H, 2CH3); 4.12 (q, J = 7.0 Hz, 2H, CH2); 5.45
3
3
(d, J = 3.0 Hz, 1H, CH); 6.71–7.43 (m, 8Harom); 8.10 (d, J =
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structures was achieved by UCSF Chimera,51 and the RMSD
analyses were performed using Discovery Studio Visualizer
16.1.52
[Tris-HCl 0.5 mM (pH 7.6); 3.5 mM trisodium citrate; 0.1%
(v/v) NP40; 100 μg mL−1 RNAse; 50 μg ml−1 propidium iodide
(PI)] at a density of 106 cells per ml. After 15 min, data were
collected using a flow cytometer (FACS Calibur cytometric
system; BD Bioscience, Mountain View, CA, USA) and ana-
lyzed by FLOWJO® software.
The virtual screening procedure of GOLD involved extrac-
tion of the bound ligands and of the co-factors for later com-
parison. The crystallographic ligands were included with the
designed DHPMs for rank comparison. The algorithms were
applied with 100% efficiency and the search efficiency flexi-
bility was set to 100% (flexible – default). The grid region was
set over the ligand (monastrol) with a 6 Å radius sphere. The
genetic algorithm (GA) runs were set at 10 runs per ligand;
the best solutions were saved for each ligand, and all 10 solu-
tions were analyzed. The docking poses were then ranked
using the dimensionless scoring function GoldScore. The
score scale indicates how good a pose is; the higher the
score, the better the docking result is likely to be. The best
two compounds were selected through visual inspection of
their poses and their respective ranking positions. The best
molecules were analyzed with their counterpart analogues in
accordance with their interactions with the kinesin binding
site using DSV 16.1 (ref. 52) and the Ligand Explorer Java
module.53
Annexin V/PI assay. Apoptotic cells were quantified using
an annexinV-fluorescein isothiocyanate–PI (annexinV-FITC–
PI) double staining kit in accordance with the manufacturer's
instructions (BD Biosciences, San Diego, CA, USA). The cells
were plated in 6-well plates and treated with 50 μM 20e or
20h or 100 μM monastrol 1 for 48 h. At the end of the treat-
ment, the cells were washed twice with cold PBS (pH 7.4) and
counted. Following this, 105 cells were suspended in binding
buffer containing FITC-conjugated annexin V and PI. The
samples were then agitated and incubated for 15 minutes at
room temperature in the dark. Apoptotic and necrotic cells
were quantified using a dual-color flow cytometric technique
on a FACSCalibur cytometric system (FACSCalibur; BD Biosci-
ence, Mountain View, CA, USA). The obtained data were ana-
lyzed with LOWJO® software (Tree Star, INC Ashland, OR,
USA). The cells were classified as follows: live, annexin−/PI−;
early apoptotic, annexin+/PI−; late apoptotic, annexin+/PI+, and
necrotic, annexin−/PI+.
Immunocytochemistry. To access the formation of mono-
polar spindles resulting from Eg5 inhibition, U138 and C6
glioma cells were left to adhere to confocal 4-well plates for
48 hours before treatment. Following this, the U138 cells
were treated with 200 μM monastrol or 150 μM of 20e or 20h,
while the C6 cells were treated with 100 μM monastrol or 50
μM of 20e or 20h for 24 hours. After treatment, the cells were
washed with PBS, fixed, and then incubated with anti-alpha-
tubulin (ab52866, Abcam Ltd., Cambridge, UK) for 90 mi-
nutes, followed by incubation with fluorescein (FITC)-conju-
gated goat anti-rabbit (Invitrogen, Life Technologies Ltd,
Paisley, UK) secondary antibody for one hour. The cells were
washed with PBS and incubated with Hoechst, a nuclear
marker. Imaging was performed on an Olympus FluoView™
1000 197 confocal microscope equipped with solid state la-
sers of 405, 198 473, 559, and 635 nm (Centro de Microscopia
Eletrônica da Universidade Federal do Rio Grande do Sul).
Statistical analysis. Data were analyzed for statistical sig-
nificance by one-way analysis of variance (ANOVA) followed
by post-hoc tests for multiple comparisons (Tukey test) using
GraphPad Prism Software® (GraphPad Software, INC, La
Jolla, CA, USA). Data are expressed as the mean S.E.M.
The binding affinities were predicted using the X-Score.
The X-Score is an empirical score function that correlates the
binding affinity with the dissociate constant of a protein–li-
gand complex (−log Kd). The X-Score values are positive num-
bers ranging from 3.0 to 9.0, predicting a milli-molar to a
nano-molar affinity.54
In vitro activity against glioma cells
Maintenance of cell lines. The human U138 and rat C6 gli-
oma cell lines were obtained from the American Type Culture
Collection (ATCC) (Rockville, MD, USA). The cells were grown
and maintained in Dulbecco's modified Eagle's medium
(DMEM) containing antibiotics (0.5 U ml−1 penicillin/strepto-
mycin) and supplemented with 15% and 5% (v/v) fetal bovine
serum (FBS), respectively (all from Gibco BRL, Carlsbad, CA,
USA). The cells were maintained at a temperature of 37 °C, a
minimum relative humidity of 95%, and an atmosphere of
5% CO2 in the air.
Assessment of glioma cell viability. For the [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium] (MTS) assay, U138 and C6 glioma
cells were seeded in 96-well plates and allowed to grow to
semi-confluence. The cells were treated with 5, 10, 25, 50 and
100 μM of monastrol or 19e, 20e, 19h or 20h for 24 h. At the
end of the treatment, 20 μL of MTS (5 mg mL−1) reagent was
added to each well for 2 h. At the end of the incubation time,
the absorbance was read at 490 nm.
Toxicity assays on Caenorhabditis elegans
The nematode strain used was N2 (wild type), originally
obtained from the Caenorhabditis Genetics Center (Univer-
sity of Minnesota, Twin Cities, MN, USA); the worms were
maintained on nematode growth medium (NGM) plates
seeded with Escherichia coli OP50 at 20 °C. Synchronization
of C. elegans cultures at the first larval stage (L1) was
achieved by washing the gravid nematodes from the plates
Cell-cycle analysis. The cells were plated in 6-well plates;
after reaching semi-confluence, they were treated with 50 μM
20e or 20h or 100 μM monastrol 1 (positive control) for 24 h.
At the end of the treatment, the cell medium was removed
and the cells were washed twice with PBS (pH 7.4), harvested,
centrifuged, and suspended with 400 μL staining solution
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into centrifuge tubes, which were lysed with a bleaching mix-
ture (1% NaOCl; 0.25 M NaOH), followed by flotation on a
30% (m/v) sucrose solution to separate the eggs from the
dissolved worms and bacterial debris. The eggs were washed
with M9 buffer (0.02 M KH2PO4, 0.04 M Na2HPO4, 0.08 M
NaCl, and 0.001 M MgSO4) and allowed to hatch overnight in
NGM agar plates without bacteria. The toxicities of monastrol
1, 20e and 20h to C. elegans were then evaluated through the
following toxicological endpoint: lethal dose 50% (LC50). LC50
was determined after acute exposure of the C. elegans L1
larva to five doses of monastrol 1, 20e and 20h, ranging from
6.25 to 200 mM. Accordingly, synchronized L1 larvae (2.500
worms per dose) were treated at 20 °C for 30 minutes by con-
stant agitation in a rotator with each dose of the compounds.
The worms were then washed three times with M9 buffer and
placed on OP50-seeded NGM plates (60 × 10 mm) to deter-
mine the number of surviving organisms (24 hours post-expo-
sure). The dose–response curves were obtained by plotting
the numbers of surviving worms, and the LC50 values were
calculated from those curves.
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Acknowledgements
The authors are thankful to CNPq/MCT, INCT-if (Instituto
Nacional de Ciência
Farmacêutica), ICNT- DCEN (Instituto Nacional de Ciência e
Tecnologia: Doenças Cerebrais, Excitoxicidade
e
Tecnologia para Inovação
e
Neuroproteção), FINEP, IQ-UFRGS and to Programa de Pós-
graduação em Ciências-Bioquímica da Universidade Federal
do Paraná. The authors are grateful to CNPq and CAPES for
their scholarships.
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