Synthesis and antitumoral activity of novel analogues monastrol-fatty acids against glioma cells

Article abstract of DOI:10.1039/c8md00169c

Monastrol is a small cell-permeable heterocyclic molecule that is recognized as an inhibitor of mitotic kinesin Eg5. Heterocyclic-fatty acid derivatives are a new class of compounds with a broad range of biological activities. This work describes a comparative study of the in vitro antitumoral activity of a series of new long-chain monastrol analogues against rat glioblastoma cells. The novel analogues C6-substituted monastrol and oxo-monastrol were synthesized via Biginelli multicomponent condensation of fatty β-ketoester in good yields using a simple approach catalyzed by nontoxic and free-metal sulfamic acid. Following synthesis, their in vitro antitumoral activities were investigated. Notably, all analogues tested were active against rat glioblastoma cells. Superior activity was observed by analogues derived from palmitic and stearic fatty acid chains; these compounds were the most potent molecules, showing 13-fold higher potency than monastrol with IC₅₀ values of 5.11 and 6.85 μM, respectively. These compounds could provide promising new lead derivatives for more potent antitumor drugs.

Full text of DOI:10.1039/c8md00169c

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Marinho, M. A. Hort, A. Horn and D. Russowsky, Med. Chem. Commun., 2018, DOI:
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RESEARCH ARTICLE
Synthesis and antitumoral activity of novel hybrid monastrol–
fatty acids against glioma cells†
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
b
b
Franciele S. De Oliveira, Patrick M. De Oliveira, Luana M. Farias, Rafael C. Brinkerhoff, Rui
b
b
b
Carlos M. A. Sobrinho, Tamara M. Treptow, Caroline R. Montes D’Oca, Marcelo A. G. Marinho,
DOI: 10.1039/x0xx00000x
a
c
a
d
b*
Mariana A. Hort, Ana P. Horn, Dennis Russowsky, and Marcelo G. Montes D’Oca
www.rsc.org/
Monastrol is a small cell-permeable heterocyclic molecule that is recognized as an inhibitor of mitotic kinesin Eg5.
Heterocyclic–fatty acid hybrid derivatives are a new class of compounds with a broad range of biological activities. This
work describes a comparative study of the in vitro antitumoral activity of a series of new long-chain monastrol hybrid
analogs against rat glioblastoma cells. The novel hybrids C6-substituted monastrol and oxo-monastrol were synthesized via
Biginelli multicomponent condensation of fatty β-ketoester in good yields using a simple approach catalyzed by nontoxic
and free-metal sulfamic acid. Following synthesis, their in vitro antitumoral activities were investigated. Notably, all
hybrids tested were active against rat glioblastoma cells. Superior activity was observed by hybrids derived from palmitic
and stearic fatty acid chains; these compounds were the most potent molecules, showing 13-fold higher potency than
monastrol with IC50 values of 5.11 and 6.85 ꢀM, respectively. These compounds could provide promising new lead
derivatives for more potent antitumor drugs.
1
1-13
as calcium channel blockers.
potential antitumor agents, involved in blocking mitosis by
However, they are also
Introduction
1
4,15
Multicomponent reactions (MCRs) are chemical reactions inhibiting kinesin Eg5, a motor protein.
involving three or more chemical compounds in one step, molecule libraries (>500 gmol ) have demonstrated this ability
generating final heterocyclic compounds that contain majority of DHPMs, mainly monastrol (Figure 1).
parts of precursors. Due to their atom economy and typically capable of disrupting cell mitosis (ordered series of events
simple and low-energy requirements, they are powerful tools primarily mechanical in that identical copies of the genome are
for organic synthesis and medicinal chemistry in the synthesis moved within the cell) have shown promise for the treatment
Studies using small-
-
1
1
5-19
Compounds
1
,2
3
,4
14
of diverse and complex structures organics.
of various tumor cell lines.
The most important MCRs are the Biginelli and Hantzsch
reactions. Biginelli reactions involve simple condensation of
aldehydes, urea, or thiourea and acetoacetates, generating
compounds
known
as
5Biginelli
compounds
or
dihydropyrimidinones (DHPMs). A wide range of biological
processes are directly affected by DHPMs, which have high
pharmacological
potential
(with
anti-inflammatory,
bactericidal, antiviral, antifungal, and antioxidant activities)
and have important applications as biological modulators in
6
-10
Figure 1. Nifedipine and monastrol calcium channel blockers
medicinal chemistry.
Due to structural similarities with
dihydropyridines (DHPs) including Hantzsch adducts such as
nifedipine (Figure 1), a modulator of the calcium channel
obtained from Hantzsch MCRs, DHPMs have been investigated
There is a great need for novel synthesized drugs with
possible applications as antitumor agents, due to the large
mortality rate associated with cancer and wide variety of
cancer types. Cancer can be conceived as a generic term for a
a.
Programa de Pós-graduação em Ciências Fisiológicas, Instituto de Ciências large group of diseases that generally involve uncontrolled cell
Biológicas, Universidade Federal do Rio Grande - FURG, Rio Grande, RS, Brazil
division and subsequent tumor formation. Such tumors can
b.
Laboratório Kolbe de Síntese Orgânica, Universidade Federal do Rio Grande -
FURG, Rio Grande, RS, Brazil
Laboratório de Ensaios Farmacológicos e Toxicológicos, Instituto de Ciências the body or affect other vital functions. Brain tumors are one
progress rapidly and generate metastases in various parts of
2
0
c.
Biológicas, Universidade Federal do Rio Grande - FURG, Rio Grande, RS, Brazil
Laboratório de Sínteses Orgânicas, Instituto de Química, Universidade Federal do
of the most diverse and interesting categories of tumors, being
challenging in their biology and pathology. Glioblastoma is
the main and most malignant type of central nervous system
(CNS) tumor, and it is difficult to treat, which limits the
prognosis in such patients. Patients with this type of cancer
d
21
Rio Grande do Sul, Av. Bento Gonçalves 9500, 91501-970, Porto Alegre-RS, Brazil
*
Corresponding author: E-mail: dqmdoca@furg.br. Phone/Fax: +55 53 32336960
The authors declare no competing interests.
Electronic Supplementary Information (ESI) available: [ H and C NMR spectra of
the compounds]. See DOI: 10.1039/x0xx00000x
†
1
13
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have about 14 months of survival after diagnosis even with found to possess the most potent biological response,
treatment, and this treatment presents several side effects reducing cell viability by 40% at 5 μM.
beyond the comorbidities associated with this pathology.
Hence, it is necessary to search for new treatments with
greater effectiveness that can prolong the life expectancy of MG human). These results indicate that the hydroxyl group in
In these studies, compared to monastrol, compounds
1 and
showed better potency against glioma cells (C6 rat and U-138-
2
2
2
such individuals.
the aromatic ring of different heterocyclic cores and the
Russowsky et al. synthesized a series of monastrol analogs presence of a long fatty chain are essential for the antitumoral
and investigated antitumoral activity against several human activities of the new heterocyclic–fatty acid hybrids. This also
cancer cell lines. In that work, piperastrol was considered a suggests that the increased lipophilicity of fatty acid DHPM
potent antitumor agent against breast (MCF 7), kidney (786-0), and PHQs may be a promising approach for the development
colon (HT-29), melanoma (UACC62), and ovary (OVCAR03) of new antitumor compounds.
2
3
tumor cell lines. Excellent results were obtained with
piperastrol and monastrol derivatives, which were more active
than oxo-derivatives, suggesting the importance of the
presence of sulfur for anti-proliferative activity.
According to literature, is well known that the molecular
combinations derived from two biologically active molecules
can result in novel hybrid molecules with enhanced biological
2
4
activities. Biginelli and other heterocyclic compounds are
interesting core structures for the development of new
bioactive compounds. Fatty acids are derived from renewable
raw materials and exhibit various biological activities.
In this context, several researchers are synthesizing these two
bioactive components to yield bioactive hybrid molecules with
specific desirable features. Heterocyclic–fatty acid hybrid
derivatives are a new class of heterocyclic compounds with a
broad range of biological activities and significance in the field
of medicinal chemistry. Over the last few years, many studies
Figure 2. Hybrids monastrol–palmitic acid (1) and PHQ–stearic acid
(
2) investigated against glioma cell lines.
Based on previous studies, in the present study a series of
novel hybrid monastrol and oxo-monastrol fatty alkyl C6-
substituted were synthesized and their in vitro antitumoral
activities were investigated against rat glioblastoma cells.
To examine their biological responses to structural
variations, a positive-control monastrol (see Figure 1) was
synthesized using the Biginelli multicomponent protocol
2
5
have emphasized the significance of such derivatives.
Building on our previous antitumoral studies using fatty
2
6-29
acid derivatives,
this work investigated the antitumoral
activity of novel monastrol and oxo-monastrol fatty alkyl C6-
substituted hybrids against malignant rat GBM cells (C6); the
hybrids were synthesized via Biginelli condensation MCRs
using a simple approach catalyzed by nontoxic and free-metal
sulfamic acid. In this context, we selected palmitic, stearic, and
oleic fatty acid derivatives to evaluate the effects of these
slightly different molecular structures.
between ethyl acetoacetate, 3-hydroxy benzaldehyde (3), and
thiourea in methanol using InCl as a catalyst. The presented
3
23
spectroscopic data are in agreement with the literature. In
addition, a fatty positive control, hybrid oxo-monastrol C5-
substituted (1, Figure 2) derived from palmitic chain, was
synthesized at good yields from palmitic acetoacetate in
accordance with previous work.
2
6
Results and Discussion
Afterward, the novel hybrid fatty alkyls C6-substituted
were synthesized using Biginelli multicomponent protocol.
Previously, we described the synthesis of fatty analogs of
dihydropyrimidinones C5-substituted using the Biginelli
multicomponent protocol. After synthesis, their antitumoral
activities against glioma cell lines (C6 rat and U-138-MG
Initially, the fatty β-ketoesters 4a-c derived from palmitic
C16:0), stearic (C18:0), or oleic (cis-C18:1) acids were obtained
(
by acylation of Meldrum acid with DCC, DMAP, and pyridine,
followed by methanolysis, in accordance with literature.
29
2
6
human) were investigated. The compounds derived from
palmitic and oleic chains obtained from 3-
hydroxybenzaldehyde and urea or thiourea reduced cell
viability the most at 50 M (ca. 70–80%) and the hybrid oxo-
monastrol–palmitic acid ( , Figure 2) was the most potent,
reducing cell viability by ca. 50% at 10 M. Moreover, the
Next, the 6-substituted oxo-monastrol- and monastrol–fatty
acid 5a-c and 6a-c were synthesized from urea or thiourea,
respectively, β-ketoesters 4a-c and 3-hydroxybenzaldehyde
(3), under metal-free conditions using sulfamic acid as a
catalyst (Scheme 1).
ꢀ
1
ꢀ
DHPM–fatty acids did not increase the mortality rate of
nontumor cells by at least 20-fold and 4-fold the active glioma
concentration (25 mM). In addition, the hybrids showed a
large safety range for neural cells demonstrating non-toxicity
to organotypic hippocampal culture. In another study, the
antiproliferative activities of new fatty acid analogs of
polyhydroquinoline (hybrid PHQ–fatty acids) against the
2
8
glioma cell line were investigated. The fatty compounds were
synthesized via the Hantzsch multicomponent protocol from
several aldehydes and dimedone. The stearic fatty alkyl PHQ
Scheme 1. Multicomponent synthesis of new DHPMs–fatty acid
hybrids 5a-c and 6a-c under metal-free catalysis conditions.
hybrid (2, Figure 2) derived from 3-hydroxybenzaldehyde was
2
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The yields of 5a-c and 6a-c are showed in Table 1. These and C5-substituted derivative
ARTICLE
1
(fatty positive control). The
products were purified by column chromatography and MTT assay was performed 48 h after exposing rat glioma cells
characterized by usual spectroscopic methods. to each compound at selected concentrations (5, 10, 25, and
0 µM). In the control group, we used the same amount of
Table 1. Yields of new hybrids oxo-monastrol- and monastrol– DMSO (vehicle) in which the new molecules were solubilized.
5
fatty acids
5
,
6a-c
.
As shown in Figure 3, compounds 5a and 5b (palmitic and
stearic oxo-monastrol derivate, respectively) reduced cell
C6- substituted fatty
alkyl DHPMs
Yield
(%)
Entry Fatty β−ketoesters
viability at concentrations as low as 5
(
only 10
ꢀ
oleic acid oxo-monastrol derivate) was effective starting at
M while compound 5c
ꢀM. Compounds 6a and 6b (palmitic and stearic
monastrol derivate, respectively) also significantly reduced cell
viability in a concentration-dependent manner, showing a
1
78
85
78
81
74
80
reduction of about 50% at a concentration of 5 ꢀM.
These results demonstrate that these compounds were
more effective than the other compounds, providing evidence
that the C6 substituent influenced the biological activity of the
molecule while the size of the chain did not show interference.
Similar results were found in the cell-counting assay (Figure 4).
2
3
According to Russowsky et al., compounds with a thio- group
are more effective at reducing tumor cell proliferation than
compounds with an oxo-group. Our results support this notion
in that monastrol showed antitumoral activity while oxo-
monastrol did not (in addition, UACC.62 cells treated with
monastrol showed significant changes in morphology, which
were not observed with oxo-monastrol).
2
3
4
5
When we compared IC50 values (Table 2) in the MTT assay,
the results indicated that adding a fatty chain to molecules
increased their antitumoral activity. This may suggest that
increased lipophilicity could contribute to a better Eg5
inhibition improving the transport of molecules across
biological barriers by active processes mediated by fatty acid
3
0, 31
binding proteins (FABP).
As shown in Table 2, compounds 6a and 6b were the most
potent molecules, with IC50 values of 5.11 (4.13–6.32) and 6.85
OH
O
(6.18–7.60) ꢀM, respectively, making them 13 times more
potent than monastrol. These molecules were also 2.5 times
more potent than hybrid C5-substituted fatty DHPM
positive control).
In conclusion, this work described the synthesis of novel
C6-substituted monastrol and oxo-monastrol fatty acids from
fatty acid families at good yields (60–85%). We tested the
antitumoral activities of each compound against C6 rat glioma
cells using the MTT assay. All of the molecules tested exhibited
antitumoral effects, and the molecules of saturated fat chains
derived from thiourea (monastrol–fatty acids derivatives),
MeO
NH
1 (fatty
(
)14
N
S
H
6
a
induced
a
significant drop in cell viability at low
concentrations. The effect followed a concentration-response
curve, qualifying the molecules 6a and 6b as the most potent.
Cell-counting tests demonstrated the action of monastrol–
fatty acids in that all such compounds decreased the number
of tumor cells after treatment. In addition, these results
suggest that all C6-substituted monastrol– and oxo-monastrol–
fatty acid hybrids may have higher antitumoral activities than
monastrol itself, except for molecule 5c derived from oleic acid
and urea.
6
Finally, our results indicate that compounds 6a and 6b
,
derived from palmitic (C16:0) and stearic acid (C18:0), were
the most potent molecules with IC50 values of 5.11 and
Antitumoral activity
6.85 ꢀM, respectively, making them promising compounds for
future experiments in vivo. The actions of these compounds
are better than monastrol itself, a kinesin-inhibiting agent.
After synthesis, the antitumoral activities of hybrids 5,6a-c
were evaluated and compared to monastrol (positive control)
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Figure 4. Counts of rat glioma (C6) cells after 48 h of treatment
Figure 3. MTT assay results for rat glioma (C6) cells after 48 h
of treatment with concentrations of 5, 10, 25, and 50 μM. C =
at concentrations of 5, 10, 25, and 50 μM. C = DMSO-treated
DMSO-treated control cells. ANOVA followed by Tukey’s post- control group for each compound. ANOVA followed by Tukey’s
test, **p < 0.01 and ***p < 0.001 with respect to the control.
post-test, **p < 0.01 and ***p < 0.001 with respect to
controls.
4
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Table 2. IC50 and log P values from monastrol, oxo-monastrol- and monastrol–fatty acid hybrids.
ARTICLE
Molecule
Molecule
IC50 (µM)
Log P
IC50 (µM)
Log P
oxo-
thio-
1
6.68
6.06
87.83
1.57
(
(
(
14.47-19.23)
(±0.47)
(58.34-132.2)
(±0.47)
7
9.37
5.72
5.11
7.15
49.76-126.6)
(±0.47)
(4.13-6.32)
(±0.47)
2
1.77
5.47
6.85
7.98
18.65-25.41)
(±0.47)
(6.18-7.60)
(±0.47)
3
8.36
25.87-
6.90)
1
66.4
6.23
7.66
(
(
78.40-353.00)
(±0.47)
(±0.47)
5
1
With their respective confidence limits (CL 95%).
(
TMS), which was used as an internal standard. The coupling
3
Experimental Section
constants ( J) are reported in Hz and refer to apparent peak
multiplicities.
Apparatus and Chemistry
Synthesis
Sulfamic acid (98 wt.%), urea and thiourea were purchased from
Sigma-Aldrich Chemical and 3-hydroxybenzaldehyde was bought
from Alfa Aesar. All organic solvents used for synthesis were of
analytical grade. Solvents were dried and freshly distilled. Column
chromatography was performed on a silica gel 60 Å (ACROS
Organics, 0.035–0.070 mesh). Reactions were monitored using thin-
layer chromatography (TLC) on silica gel plates (Merck 60GF245)
with hexane:ethyl acetate as eluent. Spots were visualized using
iodine. Yields refer to chromatographically and spectroscopically
homogeneous materials. Melting points were obtained on a
Fisatom 430D. NMR spectra were recorded using a Brucker AVANCE
General procedure for the synthesis of fatty acid oxo-monastrol
and monastrol derivatives 5,6a-c
β-ketoesters (1 mmol) 4a-c, 3-hydroxybenzaldehyde (3,1 mmol),
urea or thiourea (1.3 mmol), and sulfamic acid (0.6 mmol) were
added to a round-bottom flask of 25 mL, and the reaction contents
were dissolved in methanol (3 mL). The reaction was refluxed for 24
h and monitored by TLC. After this interval, the methanol was
removed and the crude reaction was purified by column
chromatography (7:3 hexane; ethyl acetate).
1
13
III 400 spectrometer ( H at 400 MHz and C at 100 MHz) in
deuterochloroform (CDCl ) as the solvent. The chemical shift data
3
are reported in units of δ (ppm) downfield from tetramethylsilane
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Methyl
4-(3-hydroxyphenyl)-2-oxo-6-pentadecyl-1,2,3,4- Biological assays
tetrahydropyrimidine-5-carboxylate (5a):
weight 458.63: g.mol ; Yield: 78%; H NMR (400 MHz, CDCl ) δ 7.93 General cell culture procedures
C
27
H
42
N
2
O
4
; molecular
-1
1
3
(br. s., 1H), 6.99 – 7.19 (m, 1H), 6.80 (br. s., 1H), 6.71 (d, J = 7.82 Hz,
1
–
6
1
3
H), 6.75 (d, J = 7.83 Hz, 1H), 5.27 (br. s., 1H), 3.62 (br. s., 3H), 2.52 The experiments were performed using C6 rat cells obtained from
2.75 (m, 2H), 1.45 – 1.64 (m, 2H), 1.16 – 1.38 (m, 24H), 0.89 (t, J = American Type Culture Collection (atcc®CCL-107™). The cells were
1
3
.85 Hz, 3H). C NMR (100 MHz, CDCl ) δ 166.0, 156.8, 154.1, cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
3
50.8, 144.9, 129.9, 118.0, 115.3, 113.3, 100.9, 55.1, 51.3, 50.6, supplemented with 10% fetal bovine serum (FBS) and containing
1.9, 29.7, 29.7, 29.6, 29.6, 29.4, 28.3, 22.7, 14.1.
penicillin/streptomycin (1%) and fungizone® (1%). They were kept
in a humidified atmosphere with 5% CO at a temperature of 37 °C.
2
Methyl
6-heptadecyl-4-(3-hydroxyphenyl)-2-oxo-1,2,3,4-
molecular Treatment and experimental groups
weight 486.69 g.mol ; Yield: 85%; H NMR (400 MHz, CDCl ) δ 8.03
tetrahydropyrimidine-5-carboxylate (5b):
29 46 2 4
C H N O ;
-1
1
3
(br. s., 1H), 7.09 (t, J = 7.95 Hz, 1H), 6.83 (s, 1H), 6.75 (d, J = 7.82 Hz, The compounds were prepared as described above and dissolved in
1
1
1
1
5
H), 6.71 (d, J = 8.07 Hz, 1H), 6.49 (br. s., 1H), 5.27 (d, J = 2.69 Hz, cell culture-grade DMSO. Glioma cells were seeded at a density of
4
H), 3.63 (s, 3H), 2.49 – 2.71 (m, 2H), 1.44 – 1.61 (m, 2H), 1.19 – 1×10 cell per well in DMEM/10% FBS in 96-well plates. Cell cultures
1
3
.35 (m, 24H), 0.90 (t, J = 7.10 Hz, 3H). NMR C (100 MHz, CDCl
3
) δ were exposed to concentrations of 5, 10, 25, and 50 μM of the
66.0, 156.7, 154.4, 150.7, 144.9, 129.9, 118.1, 115.4, 113.2, 100.9, molecules for 48 h, while the control group was treated with vehicle
5.1, 51.3, 31.9, 29.8, 29.7, 29.7, 29.6, 29.4, 29.4, 28.4, 22.7, 14.1.
(1% of DMSO).
(Z)-Methyl
6-(heptadec-8-en-1-yl)-4-(3-hydroxyphenyl)-2-oxo- Cell viability assay
1
,2,3,4-tetrahydropyrimidine-5-carboxylate
(5c):
29 44 2 4
C H N O ;
-1
1
molecular weight: 484.67 g.mol ; Yield: 68%; H NMR (400 MHz, After 48 h of treatment, cell viability was evaluated using an MTT
CDCl ) δ 8.21 (br. s., 1H), 7.25 – 7.35 (m, 6H), 5.99 (br. s., 1H), 5.40 assay. MTT solution (sterile stock solution of 5 mg/mL) was added
d, J = 2.93 Hz, 1H), 5.31 – 5.39 (m, 2H), 3.63 (s, 3H), 2.66 – 2.78 (m, to the incubation medium at a final concentration of 0.5 mg/mL.
3
(
2
0
1
3
2
H), 1.98 – 2.09 (m, 4H), 1.57 – 1.67 (m,.2H), 1.24 – 1.42 (m, 21H), The cells were left for 60 min at 37°C in a humidified 5% CO2
.90 (t, J = 6.80 Hz, 3H). NMR C (100 MHz, CDCl ) δ 165.8, 153.5, atmosphere. Then the medium was removed and the plates were
3
51.1, 143.7, 130.0, 129.8, 128.8, 127.9, 126.5, 100.7, 55.6, 51.1, shaken for 30 min using DMSO as a solvent. The optical density of
1.9, 31.9, 31.9, 29.8, 29.7, 29.5, 29.4, 29.4, 29.3, 29.3, 29.3, 29.2, each well was measured at 492 nm. The results of the MTT assay
13
8.3, 27.3, 27.2, 22.7, 14.1.
are expressed as percentages of control group values.
Methyl
4-(3-hydroxyphenyl)-6-pentadecyl-2-thioxo-1,2,3,4- Cell proliferation assay
S; molecular
weight: 474,70 g.mol ; Yield: 81%; H NMR (400 MHz, CD
tetrahydropyrimidine-5-carboxylate (6a): C27
42 2 3
H N O
-1
1
3
3
OD) δ C6 cells were cultured at 5×10 cells/well in DMEM containing 10%
7
6
1
.14 (t, J = 7.83 Hz, 1H), 6.76 – 6.79 (m, 1H), 6.75 – 6.76 (m, 1H), FBS, in 96-well plates for 48 h. Then the cells were trypsinized, and
.69 – 6.72 (m, 1H), 5.28 (s, 1H), 3.66 (s, 3H), 2.69 – 2,84 (m, 2H), 100 µL aliquots were taken from each well for counting in a
13
.56 – 1.69 (m, 2H), 1.31 (s, 24H), 0.92 (t, J = 7.10 Hz, 3H). C NMR Neubauer chamber, using a microscope stereoscope.
OD) δ 175.2, 166.1, 157.4, 148.9, 144.6, 129.3, 117.4,
(100 MHz, CD
3
114.5, 113.1, 101.3, 54.8, 50.3, 31.7, 30.3, 29.4, 29.4, 29.3, 29.3, Lipophilicity calculations
29.3, 29.1, 29.1, 28.2, 22.3, 13.0.
The physicochemical parameter Clog P (the logarithm of n-
6-heptadecyl-4-(3-hydroxyphenyl)-2-thioxo-1,2,3,4- octanol/water partition coefficient P), based on established
tetrahydropyrimidine-5-carboxylate (6b): C29 S; molecular chemical interactions, was calculated using ChemDraw, Level: Ultra,
weight: 502.75 g.mol ; Yield: 74%; H NMR (400 MHz, CDCl ) δ 7.81 Version: 12.0.2.1076 (CambridgeSoft, Cambridge, MA, USA).
br. s., 1H), 7.34 (br. s., 1H), 7.19 (t, J = 7.95 Hz, 1H), 6.83 (d, J = 7.58
Hz, 1H), 6.78 (s, 1H), 5.36 (d, J = 3.42 Hz, 1H), 3.67 (s, 3H), 2.66 – Calculating the half maximal inhibitory concentration (IC50
Methyl
46 2 3
H N O
-1
1
3
(
)
2
3
1
2
.77 (m, 2H), 1.54 – 1.67 (m, 2H), 1.28 (s, 24H), 0.90 (t, J = 6.85 Hz,
13
H). C NMR (100 MHz, CDCl
3
) δ 174.8, 165.6, 156.3, 147.4, 143.8, The half maximal inhibitory concentration (IC ) was calculated
5
0
30.2, 118.7, 115.6, 113.6, 102.2, 55.8, 51.5, 31.9, 31.6, 29.7, 29.6, using GraphPad PRISM ® Prism 5 software for Windows Version 5.3.
9.6, 29.5, 29.5, 29.3, 29.3, 28.2, 22.7, 14.1.
IC50 was determined from a linear regression, which was related to
the inhibition percentage as a function of the logarithm of the
(Z)-Methyl
6-(heptadec-8-en-1-yl)-4-(3-hydroxyphenyl)-2-thioxo- concentrations tested, assuming a confidence interval of 95% (p
1
,2,3,4-tetrahydropyrimidine-5-carboxylate
(6c):
C
29
H
44
N
2
O
3
S; <0.05).
molecular weight: 500.74 g.mol ; Yield: 60%; H NMR (400 MHz,
CDCl ) δ 8.43 (s, 1H), 7.90 (br. s., 1H), 7.25 – 7.36 (m, 5H), 5.39 (d, Statistical analysis
J = 3.42 Hz, 1H), 5.34 – 5.39 (m, 2H), 3.64 (s, 3H), 2.76 – 2.85 (m,
-1
1
3
1
H), 2.62 – 2.74 (m, 1H), 1.96 – 2.11 (m, 4H), 1.56 – 1.67 (m, 2H), Data were analyzed for statistical significance by one-way analysis
1
3
1
CDCl
1
2
.22 – 1.44 (m, 20H), 0.90 (t, J = 6.80 Hz, 3H). C NMR (100 MHz, of variance (ANOVA) followed by a post-hoc test for multiple
) δ 174.8, 165.6, 156.3, 147.4, 143.8, 130.2, 118.7, 115.6, comparisons (Tukey test) using GraphPad Prism®software. Data are
13.6, 102.2, 55.8, 51.5, 31.9, 31.6, 29.7, 29.6, 29.6, 29.5, 29.5, expressed as the mean ± standard deviation. Differences were
3
9.3, 29.3, 28.2, 22.7, 14.1.
considered significant at p < 0.05.
6
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C8MD00169C
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22. O. Van Tellingen, B. Yetkin-Arik, M. De Gooijer, P. Wesseling,
T. Wurdinger and H. De Vries, Drug Resistance Updates, 2015,
Conflict of Interest
1
9, 1-12.
3. D. Russowsky, R. F. S. Canto, S. A. A. Sanches, M. G. M.
The authors declare no competing interests.
2
D'Oca, A. de Fatima, R. A. Pilli, L. K. Kohn, M. A. Antonio and J. E.
de Carvalho, Bioorganic Chem., 2006, 34, 173-182.
2
2
1
2
Battastini, C. G. Salbego, J. B. Hoppe, P. S. Taborda, S. B. Rosa, L.
A. Piovesan, C. D. Montes D'Oca, D. Russowsky and M. G.
Montes D'Oca, Eur. J. Med. Chem., 2015, 95, 552-562.
27. D. S. dos Santos, L. A. Piovesan, C. R. M. D’Oca, C. R. L. Hack,
T. G. Treptow, M. O. Rodrigues, D. B. Vendramini-Costa, A. L. T.
Ruiz, J. E. de Carvalho and M. G. M. D’Oca, Bioorg. Med. Chem.,
Acknowledgments
4. B. Meunier, Acc. Chem. Res., 2008, 41, 69–77.
5. V. Venepally, R. C. R. Jala, Eur. J. Med. Chem., 2017, 141
13–137.
,
The authors are thankful for financial support from Coordenação de
Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação
6. T. G. M. Treptow, F. Figueiro, E. H. F. Jandrey, A. M. O.
de Amparo
a Pesquisa do Estado do Rio Grande do Sul
(FAPERGS/PRONEM), and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq). Fellowships from CAPES are also
acknowledged.
2
2
015, 23, 340-347.
8. D. da Costa Cabrera, S. B. Rosa, F. S. de Oliveira, M. A.
References
Marinho, C. R. M. D'Oca, D. Russowsky, A. P. Horn and M. G. M.
D'Oca, MedChemComm, 2016, , 2167-2176.
9. R. C. Brinkerhoff, H. F. Tarazona, P. M. de Oliveira, D. C.
Flores, C. D. R. M. D'Oca, D. Russowsky and M. G. M. D'Oca, RSC
Advances, 2014, , 49556-49559.
0. X. L. Liu, B. Testa and A. Fahr, Pharm. Res., 2011, 28, 962-
1
1
2
. I. Ugi, A. Domling and W. Horl, Endeavour, 1994, 18, 115-
22.
. T. Zarganes-Tzitzikas, A. L. Chandgude and A. Domling, Chem.
7
2
Rec., 2015, 15, 981-996.
4
3
3
4
2
5
. A. Domling, W. Wang and K. Wang, Chem. Rev., 2012, 112,
083–3135.
. R. C. Cioc, E. Ruijter and R. V. A. Orru, Green Chem., 2014, 16
958-2975.
. H. G. O. Alvim, T. B. Lima, A. L. de Oliveira, H. C. B. de
3
9
3
77.
1. A. A. R. Mota, P. H. P. R. Carvalho, B. C. Guido, H. C. B. de
,
Oliveira, T. A. Soares, J. R. Corrêa and B. A. D. Neto, Chem. Sci.,
014, 5, 3995-4003.
2
Oliveira, F. M. Silva, F. C. Gozzo, R. Y. Souza, W. A. da Silva and B.
A. D. Neto. J. Org. Chem. 2014, 79, 3383−3397.
6. J. Kim, C. Park, T. Ok, W. So, M. Jo, M. Seo, Y. Kim, J. H. Sohn,
Y. Park, M. K. Ju, J. Kim, S. J. Han, T. H. Kim, J. Cechetto, J. Nam,
P. Sommer and Z. No, Bioorg. Med. Chem. Lett., 2012, 22, 2119-
2
7
124.
. S. N. Mokale, S. S. Shinde, R. D. Elgire, J. N. Sangshetti and D.
B. Shinde, Bioorg. Med. Chem. Lett., 2010, 20, 4424-4426.
. A. R. Trivedi, V. R. Bhuva, B. H. Dholariya, D. K. Dodiya, V. B.
Kataria and V. H. Shah, Bioorg. Med. Chem. Lett., 2010, 20, 6100-
8
6
9
102.
. P. Sharma, N. Rane and V. K. Gurram, Bioorg. Med. Chem.
Lett., 2004, 14, 4185-4190.
0. H. A. Stefani, C. B. Oliveira, R. B. Almeida, C. M. P. Pereira, R.
C. Braga, R. Cella, V. C. Borges, L. Savegnago and C. W. Nogueira,
1
Eur. J. Med. Chem., 2006, 41, 513-518.
1
1. K. S. Atwal, G. C. Rovnyak, J. Schwartz, S. Moreland, A.
Hedberg, J. Z. Gougoutas, M. F. Malley and D. M. Floyd, J. Med.
Chem., 1990, 33, 1510-1515.
1
Moreland, A. Hedberg and B. C. Oreilly, J. Med. Chem., 1991, 34
2. K. S. Atwal, B. N. Swanson, S. E. Unger, D. M. Floyd, S.
,
8
1
06-811.
3. G. C. Rovnyak, K. S. Atwal, A. Hedberg, S. D. Kimball, S.
Moreland, J. Z. Gougoutas, B. C. Oreilly, J. Schwartz and M. F.
Malley, J. Med. Chem., 1992, 35, 3254-3263.
1
4. T. M. Kapoor, T. U. Mayer, M. L. Coughlin and T. J. Mitchison,
J. Cell Biol., 2000, 150, 975-988.
5. R. Sakowicz, J. T. Finer, C. Beraud, A. Crompton, E. Lewis, A.
1
Fritsch, Y. Lee, J. Mak, R. Moody, R. Turincio, J. C. Chabala, P.
Gonzales, S. Roth, S. Weitman and K. W. Wood, Cancer Res.,
2
1
1
004, 64, 3276-3280.
6. R. Heald, Cell, 2000, 102, 399-402.
7. S. Brier, D. Lemaire, S. DeBonis, E. Forest and F. Kozielski,
Biochemistry, 2004, 43, 13072-13082.
8. J. C. Cochran, J. E. Gatial, T. M. Kapoor and S. P. Gilbert, J.
Biol. Chem., 2005, 280, 12658-12667.
9. J. C. Cochran, J. E. Gatial, Z. Maliga, T. M. Kapoor, T. J.
Mitchison and S. P. Gilbert, Biophys. J., 2005, 88, 648A-648A.
1
1
2
2
0. A. Levitzki and S. Klein, Mol. Asp. Med., 2010, 31, 287-329.
1. K. L. Ligon, K. Wilkinson and C. D. Stiles, in Pathology and
Epidemiology of Cancer, Springer, 2017, pp. 291-311.
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Synthesis and antitumoral activity of novel hybrid
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