Design, Synthesis and Molecular Docking Studies of Some Tetrahydropyrimidine Derivatives as Possible Fascin Inhibitors

Article abstract of DOI:10.1002/cbdv.201800339

Eight derivatives of tetrahydropyrimidine scaffold were designed and prepared as hybrid compounds possessing the structural features of both monastrol as an anticancer drug and nifedipine as a fascin blocking agent. All of the compounds were evaluated for

Full text of DOI:10.1002/cbdv.201800339

Title: Design, Synthesis and Molecular Docking Studies of some
Tetrahydropyrimidine Derivatives as Possible Fascin Inhibitors
Authors: Narges Riahi, Amirhosein Kefayat, Ahmad Ghasemi,
Mohammadhosein Asgarshamsi, Mojtaba Panjehpour, and
Afshin Fassihi
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201800339
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10.1002/cbdv.201800339
Chemistry & Biodiversity
Chem. Biodiversity
Design, Synthesis and Molecular Docking Studies of some
Tetrahydropyrimidine Derivatives as Possible Fascin Inhibitors
Narges Riahi,a Amirhosein Kefayat,b Ahmad Ghasemi,c Mohammadhosein Asgarshamsi,a
Mojtaba Panjehpoor,d Afshin Fassihi a,*
aDepartment of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences,
81746-73461, Isfahan, Iran
E-mail: fassihi@pharm.mui.ac.ir (A. Fassihi)
bDepartment of Oncology, Cancer Prevention Research Center, Isfahan University of Medical science,81746-73461, Isfahan, Iran.
cDepartment of Basic Medical Sciences, Neyshabur University of Medical Sciences, Neyshabur, Iran.
dDepartment of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, 81746-
73461, Isfahan, Iran
Eight derivatives of tetrahydropyrimidine scaffold were designed and prepared as hybrid compounds possessing the structural
features of both Monastrol as an anticancer drug and Nifedipine as a Fascin blocking agent. All of the compounds were evaluated
for their cytotoxic potency and the ability to inhibit 4T1 breast cancer cells migration. Then they were investigated in silico for their
ability to inhibit the Fascin protein using molecular docking simulation. The most potent compound was 4d and the weakest one was
4a according to the in vitro cytotoxicity assay. The corresponding IC50 values were 193.70 and 248.75 µmol, respectively. The least
cytotoxic compound (4a) was one of the strongest ones in binding to the Fascin binding site according to the molecular docking
results. 4a and 4e inhibited the 4T1 cells migration better than other compounds. They were more potent than Nifedipine in inhibiting
the migration process. In silico studies proved 4h to be the most potent Fascin inhibitor in terms of ΔGbind although it was not
inhibiting migration. The controversy between the in vitro and in silico results may cancel the theory of the involvement of the Fascin
inhibition in the migration inhibition. However, the considerable anti-migratory effects of some of the synthesized compounds
encourage performing further in vivo experiments to introduce novel tumor metastasis inhibitors.
Keywords: Tetrahydroprimidines • Fascin • Cytotoxicity • Migration • Molecular Docking
Introduction
Tumor metastasis has been recognized as the dissemination of malignant cells from the primary tissue to distant sites via organ-
specific patterns.[1] Progress in the knowledge of tumor invasion has yielded a list of drug targets like enzymes, receptors, and
multiple signaling pathways.[2] Now it is known that the process of metastasis consists of sequential and interlinked steps, all of
which depend on the hemostasis factors that promote tumor cell proliferation, angiogenesis, migration, invasion and extravasation
into organ parenchyma.[3] It seems that local invasion and metastasis of malignant cells are the main features of solid cancer cells.
Furthermore, resistance to cytotoxic drugs is attributed to mutations of the target or cell migration and/or invasion which are the
main causes of treatment failure. The metastatic process is a promising therapeutic target and many efforts have been invested in
the identification of cellular structures involved in the cell migration process.
Filopodia are thin finger-like and highly dynamic actin-rich membrane protrusions that are essential for migration.[4] Filopodia
enable migrating cells to navigate their surroundings and determine the direction of migration.[5] Filopodia extend beyond the leading
edge of migrating cells and are supported by rigid tightly bonded actin filaments.[6] Fascin is an evolutionarily conserved actin binding
protein that is localized in filopodia. This protein with its uniquely folded 4-β-3 foil structure is up-regulated as the part of the program
of epithelial to mesenchymal cancer cell progression.[7] Fascin gives special motility and invasion properties to the cancer cells.
Based on in vivo and in vitro conducted model studies, it has been demonstrated that Fascin as a primary molecular biomarker
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plays a significant role in tumor progression. Depletion or inhibition of this protein results in reduced cell migration. In contrast,
Fascin is up-regulated by the invasion capacity of the cancer cells. Additionally, these alterations provide a target for designing
novel agents to inhibit cancer cell migration.[8]
Two major binding sites were recognized for binding activity of Fascin. Each binding site is essential for filopodia formation
inside the cells. Some macrocyclic ketones have been recognized that occupy Fascin binding sites and specifically inhibit its
biochemical function of filopodia formation induction. These Fascin inhibitors block tumor cell migration and inhibit invasion and
metastasis. Migrastatin and its structural analogs are potent Fascin inhibitors of metastatic tumor cell migrations.[9] Some small
molecules have also been identified according to a cell based Fascin bioassay as potent Fascin inhibitors. Nifedipine, Baclofen and
Amantadine are three examples of these inhibitors.[10] The chemical structures of these compounds are illustrated in Figure 1.
O
O
NH
O
O
NO2
O
O
H3COOC
H3C
COOCH3
N
CH3
H
OH
Migrastatine
Nifedipine
O
H2N
OH
NH2
Amantadine
Baclofen
Cl
Figure 1. Structures of Migrastatin, Nifedipine, Baclofen and Amantadin
Monastrol is a prototype anticancer drug that selectively inhibits dimeric kinesin Eg5 regulation (Figure 2). As a
tetrahydropyrimidine derivative, Monastrol has a structural similarity to Nifedipine, a dihydropyridine derivative which inhibits Fascine.
From the chemical structure point of view, tetrahydropyrimidine scaffold can be regarded as the aza analog of dihydropyridine.
Tetrahydropyrimidine and dihydropyridine derivatives have also been identified to have similar pharmacological properties. Calcium
channel blocking, anti-tubercular, anti-microbial and anti-cancer activities are some of these shared pharmacological properties.[11]
Effective treatment of solid cancer can be achieved by cytotoxic compounds that are also able to inhibit the ability of cancer
cells to invade through the extracellular matrix and establish secondary tumors. Thus, a specific design of hybrid cytotoxic and
anti-invasive/antimetastatic compounds for the treatment of solid cancer must not be ignored. Medicinal chemistry efforts based on
cell biology and structure-activity relationship have well-defined pharmacophores of promising cytotoxic anti-migrastatic agents in
both synthetic and natural sources.[12-14]
In the present study, we aimed at the synthesis of some hybrid tetrahydropyrimidine derivatives possessing the structural
features of both Monastrol and Nifedipine. This strategy which is illustrated in Figure 2 may provide the designed molecules
biological properties of cytotoxicity and the ability to inhibit tumor cell migration. The anti-migratory activity would be a good additive
feature for cytotoxic agents. In fact, this feature would change nothing in the process of the solid cancer treatment with a strong
cytotoxic compound; but, in the case of moderate cytotoxic agents those cells which were not destroyed by the compound would not
have any chance to migrate which leads to invasion. The designed compounds were prepared in the lab and the cytotoxic effects as
well as the migration inhibitory ability of the synthesized compounds were evaluated. All of the compounds were also investigated
in silico for their abilities to inhibit the Fascin protein using molecular docking simulation.
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OH
O
NO2
O
H3C
COOCH3
O
O
NH
H3C
N
CH3
H3C
N
S
H
H
Nifedipine
Monastrol
O
NO2
NH
R
O
H3C
N
S
H
Figure 2. Design of tetrahydropyrimidines with cytotoxic and migration inhibitory activity based on Nifedipin and Monastrol
structures
Results and Discussion
Chemistry
Eight 1,2,3,4-tetrahydropyrimidine derivatives (4a-h) were synthesized as described in Scheme 1. General structure and the
structural details of the designed compounds are provided in Table 1.
Table 1. General structure and the structural details of the designed compounds
NO2
O
R
O
NH
H3C
N
X
H
(4a-h)
Compound
Nitro position
R
X
4a
4b
4c
4d
4e
4f
ortho
ortho
meta
meta
ortho
ortho
meta
meta
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
O
O
O
O
S
S
S
S
4g
4h
3
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The core 1,2,3,4-tetrahydropyridine ring was constructed according to the Biginelli multicomponent cyclocondensation reaction.[15] In
brief, a mixture of the proper aldehyde (1,1'), alkyl acetoacetate (2,2') and urea/thiourea (3,3') was refluxed in a suitable solvent
using the proper Lewis acid catalyst to provide the 4a-h products. For compounds 4a-d, absolute ethanol and cobalt hydrogen
sulfate were the solvent and catalyst, respectively. Tetrahydrofuran as the solvent and polyphosphate ester as the Lewis acid
catalyst were used for the preparation of 4e-h. The structures of the title compounds were confirmed by IR and 1H-NMR
spectroscopy. Some of the characterization data of the prepared compounds are summarized as follows. All compounds showed in
the IR spectra an absorption band at 1662–1716 cm-1, typical of the stretch vibrations belonging to the carboxylate C=O group. The
1
ureide carbonyl or thiocarbonyl groups appeared at 1626–1645 cm-1 and 1566-1670 cm-1, respectively. The H-NMR spectra of all
final compounds contained a singlet in the 2.27–2.30 ppm region due to the CH3 protons at the C6 position. The characteristic peak
for the C4-H of the 1,2,3,4-tetrahydropyrimidine ring appeared at 5.29–5.94 ppm confirming the formation of this heterocycle. It was
a singlet peak for 4a-d, but a doublet (J=2.8-3.6 Hz) for the 2-thiocarbonyl analogues of them, compounds 4e-h. This doublet
appeared as a result of the coupling of C4 proton with the hydrogen attached to N3. It can be explained in this way that in 4a-d, N3
does not bear the proton all the times as a consequence of the tautomerism between N3-H and the ureide carbonyl group. In 4e-h
derivatives which contain a thiocarbonyl group, for the weaker electronegativity of the sulfur atom, this tautomerism is not as
extensive as in the carbonyl analogs. Thus, the proton on the N3 atom spends most of its time on this atom which is vicinal to the
C4 atom and able to couple with its hydrogen. C6-CH3 and C4-H peaks are indicatives for the presence of the
1,2,3,4-tetrahydropyrimidine ring. The spectral features of 4c, 4e, 4f and 4g are provided in detail as follows. The characterization
data of 4a, 4b, 4d and 4h have previously been reported. [16,17]
NO2
NO2
O
H
O
O
R
(1,1')
Abs. EtOH or THF
Co(HSO4)2 or PPE
O
NH
R
O
NH2
X
H3C
N
X
H
H3C
O
H2N
(4a-h)
(2,2')
(3,3')
1= 2-Nitro 1'= 3-Nitro
2= R: CH3 2'= R: C2H5
3= X:O
3'= X:S
Scheme 1. Synthesis of alkyl-6-methyl-4-[2(3)-nitrophenyl]-2-(thi)oxo-1,2,3,4-tetrahydroprimidine-5-carboxylate (4a-h)
Methyl 6-methyl-4-(3-nitrophenyl)-2-oxo-1,2,3,4-tetrahydroprimidine-5-carboxylate (4c)
Mol. Formula: C13H13N3O5, Yield: 40%, MW: 291.26 g.mol-1, MP: 280-282 °C, FT-IR (KBr): ϒ (cm-1) 3356,3219 (N-H ureide), 3098
(C-H aromatic), 2959 (C-H aliphatic), 1698 (C=O ester), 1640 (C=O ureide), 1534 (C=C alkene), 1458 (C=C aromatic), 1H-NMR
(DMSO-d6): δ (ppm) 2.28 (s, CH3-C(6), 3H), 3.54 (s, CH3COO, 3H), 5.29 (s, H-C(4), 1H), 7.67-8.13 (m, H-C(2),H-C(4),H-C(5),
H-C(6) phenyl, H-N(1), 5H), 9.38 (s, H-N(3) , 1H), Mass m/z (%): 290.0 (M-1); Anal. Calcd. (%) for C13H13N3O5: C, 53.61; H, 4.50; N,
14.43. Found: C, 53.72; H, 4.32; N, 14.71.
Methyl 6-methyl-4-(2-nitrophenyl)-2-thioxo-1,2,3,4-tetrahydroprimidine-5-carboxylate (4e)
Mol. Formula: C13H13N3O4S, Yield: 41%, MW: 307.33 g.mol-1, MP: 213.8-215 °C, FT-IR (KBr): ϒ (cm-1) 3243 (N-H ureide), 3176 (C-H
aromatic), 3009(C-H aliphatic), 1716 (C=O ester), 1670 (C=S ureide), 1571 (C=C aromatic), 1523 (C=C alkene), 1464 (C=C
aromatic), 1H-NMR (DMSO-d6): δ (ppm) 2.28 (s, CH3-C(6), 3H), 3.39 (s, CH3COO, 3H), 5.89 (d, J = 2.8, H-C(4), 1H), 7.48-7.55
(m, H-C(4),H-C(6) phenyl, 2H), 7.42 (t, J = 7.6 Hz, H-C(5) phenyl, 1H), 7.89 (d, J = 8.0 Hz, H-C(3) phenyl, 1H), 9.67 (s, H-N(1) , 1H),
10.48 (s, H-N(3) , 1H), Mass m/z (%): 306.3 (M-1); Anal. Calcd. (%) for C13H13N3O4S: C, 50.81; H, 4.26; N, 13.67. Found: C, 50.62;
H, 4.19; N, 13.81.
4
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Ethyl 6-methyl-4-(2-nitrophenyl)-2-thioxo-1,2,3,4-tetrahydroprimidine-5-carboxylate (4f)
Mol. Formula: C14H15N3O4S, Yield: 46%, MW: 321.35 g.mol-1, MP: 219-221 °C, FT-IR (KBr): ϒ (cm-1) 3229 (N-H ureide), 3169 (C-H
aromatic), 2987 (C-H aliphatic), 1714 (C=O ester), 1655 (C=S ureide), 1572 (C=C aromatic), 1524 (C=C alkene), 1474 (C=C
aromatic), 1H-NMR (DMSO-d6): δ (ppm) 0.92 (t, J= 7.2 Hz, CH3CH2COO, 3H), 2.29 (s, CH3-C(6), 3H), 3.84-3.87 (q, J = 6 Hz,
CH3CH2COO, 2H), 5.94 (d, J = 2.8 Hz, H-C(4), 1H), 7.49-7.55 (m, H-C(4),H-C(6) phenyl, 2H), 7.75 (t, J = 8.0 Hz, H-C(5) phenyl, 1H),
7.92 (d, J = 8.0 Hz, H-(C3) phenyl, 1H), 9.59 (s, N(1) , 1H), 10.45 (s, N(3), 1H), Mass m/z (%): 320.35 (M-1); Anal. Calcd. (%) for
C14H15N3O4S: C, 52.33; H, 4.70; N, 13.08. Found: C, 52.52; H, 4.39; N, 12.87.
Methyl 6-methyl-4-(3-nitrophenyl)-2-thioxo-1,2,3,4-tetrahydroprimidine-5-carboxylate (4g)
Mol. Formula: C13H13N3O4S, Yield: 57%, MW: 307.33 g.mol-1, MP: 251-252 °C FT-IR (KBr): ϒ (cm-1) 3308 (N-H ureide), 3175 (C-H
1
aromatic), 2955 (C-H aliphatic), 1662 (C=O ester), 1566 (C=S ureide), 1531 (C=C alkene), 1459 (C=C aromatic), H-NMR (DMSO-
d6): δ (ppm) 2.30 (s, CH3-C(6), 3H), 3.55 (s, CH3COO, 3H), 5.32 (d, J = 3.6 Hz, H-C(4), 1H), 7.64-7.69 (m, H-C(5),H-C(6) phenyl,
2H), 8.06 (bs, H-C(2) phenyl, 1H), 8.13-8.16 (m, H-C(4) phenyl, 1H) 9.78 (s, H-N(1), 1H), 10.52 (s, H-N(3), 1H), Mass m/z (%):
306.3 (M-1); Anal. Calcd. (%) for C13H13N3O4S: C, 50.81; H, 4.26; N, 13.67. Found: C, 50.55; H, 4.05; N, 13.43.
Biology
Cytotoxicity
The cytotoxicities of all compounds were determined by MTT assay. All compounds were tested for cytotoxic properties against
HeLa cells. Data are given in Table 2 and Figure 3 in terms of IC50 values and cell viability at different concentrations, respectively.
According to Table 2, the most potent compound in terms of cytotoxicity was 4d with an IC50 value of 193.70 µmol and the
weakest one was 4a. The studied compounds can be ordered as 4d>4f>4b>4g>4h>4e>4c>4a according to their cytotoxic
potencies. From this small set of compounds, no accurate structure-activity relationships but the fact of the higher cytotoxic potency
of ethyl ester compounds over the methyl ester ones can be concluded.
Table 2. The cytotoxicity results of the studied compounds against HeLa cells in terms of IC50 (µmol)
Compd.
IC50
4a
4b
4c
4d
4e
4f
4g
4h
248.75
208.45
245.26
193.70
217.29
204.02
211.71
216.37
Migration assay
The ability of the less cytotoxic compounds (4a, 4c, 4e, and 4h) to inhibit cancer cell migration was investigated on 4T1 breast
cancer cell line by inexpensive in vitro scratch assay. Cells were treated with the compounds at the concentration of 50 µg/mL. The
results of this assay are provided in Figure 4. As shown in this figure, the PBS treated cells exhibited 50 percent wound closure
activity. The cells treated with compounds showed inhibition of wound closure in contrast to Nifedipine-treated cells (Nifedipine as
the positive control). The anti-migratory effects were more apparent for 4a and 4e which were more potent than Nifedipine. However,
other compounds did not exhibit any inhibitory effects on the cancer cells migration.
The obtained results demonstrate that some of the prepared compounds which were not cytotoxic could inhibit migration of
the breast cancer cells and this fact was more apparent for 4a and 4e. Compound 4e was one of the compounds with high in silico
affinity, compared to other molecules, to the Fascin binding site.
Docking studies
Molecular docking simulation was performed to investigate the binding mode of the prepared compounds to the Fascin protein.
Crystallographic 3D structure of 3LLP was used in docking studies. A blind ligand-protein docking approach was utilized to find the
ligand binding site inside the protein. Since the target molecule was too big to be covered by one grid box, four separate grid boxes
with different centers were applied for each compound then autodock calculations were run. Based on the results obtained from
docking in each grid box, binding site was determined. Nifedipine was docked as the known inhibitor of the Fascin protein.
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The results of this part of the study including the estimated free binding energy values (ΔGbind) of the docked positions, expressed in
kcal/mol, and the favorable interactions with the key amino acid residues at the active site of Fascin 3D structure are provided in
Table 3. Among the studied compounds 4c, 4d, 4e and 4h were recognized potent in docking studies in terms of the estimated
binding free energy changes and interactions within the Fascin active site. ΔGbind values of the docking poses of these three
compounds were within the range of -8.21 to -8.72 kcal/mol, not very different from the binding free energy change for Nifedipine
(-9.8 kcal/mol). Molecular interactions of the best docked conformation of Nifedipine are provided in Figure 5. Figure 6 demonstrates
the interactions of 4h, the most potent compound in terms of in silico inhibition of the Fascin protein with the lowest ΔGbind
(-8.72 kcal/mol), with the active site residues of Fascin.
The obtained results show that all the docked compounds except 4b were located in the same biding site as Nifedipine. They
formed hydrophobic interactions with almost the same residues inside the binding site as Nifedipine did. The corresponding ΔGbind
values were not as high as the ΔGbind of Nifedipine, but can be regarded acceptable for the in silico inhibitory potential.
Figure 3. Cytotoxicity results of the studied compounds in terms of cell viability
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Figure 4. Analysis of the 4a, 4c, 4e, and 4h ability to inhibit 4T1 cancer cell migration by in vitro scratch assay. A) The images of the
scratched area are acquired at 0 and 24 hours. The orange lines define the areas lacking cells. B) Percentage of the scratch area
closure after 24 h. (*:P ≤0. 05, **:P ≤0.005 , ***:P ≤0.001 , ns : not significant)
As can be seen in Figures 5, in the docking simulation of Nifedipine the two oxygen atoms of NO2 group formed hydrogen bonds
with Lys220. The NO2 oxygens were also involved in electrostatic interactions with this residue. 4h as the most potent in silico
inhibitor of Fascin established the same hydrogen bonds and electrostatic interactions in its best docked form inside the binding site.
A π–cationic interaction between the nitrophenyl ring and Lys220 was also observed for 4h which has an important role in fixing the
molecule inside the binding site (Figure 6). These hydrogen bonds and π–cationic interaction were common in all the four most
potent compounds. The OCH3 moieties of 3,5-dicarboxylate esters in Nifedipine formed extra hydrogen bonds with Lys220 and
Leu256. The studied compounds lacked the C5 ester group but, the OC2H5 moiety of 3-carboxylate ester in 4h was involved in
hydrogen bondng with Lys220. Such an interaction was not observed in other potent compounds; instead, N1-H/N3-H and/or the
carbonyl group in C2 position of tetrahydroprimidine ring were involved in some hydrogen bonding with the binding site residues.
The dihydropyridine NH hydrogen donated hydrogen to Val10 for a hydrogen bond and N1-H of the tetrahydroprimidine ring of 4h
did the same. None of these interactions were observed for the least potent compound, 4b with ΔGbind of -5.91 kcal/mol (Table 3). It
is noteworthy that the π–cationic interaction between the nitrophenyl ring and Lys220 compensated the lack of hydrogen bonding.
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Figure 5. Nifedipine docked inside the Fascin active site. The 3D ligand interaction diagram shows hydrogen bonding (green
dashed line) and the 2D diagram demonstrates electrostatic interactions (blue dashed line) of the nitro group with the positively
charged residue Lys220.
Figure 6. Compound 4h inside the Fascin binding site. The 3D ligand interaction diagram shows hydrogen bonding (green dashed
line) and the 2D diagram demonstrates electrostatic interactions (blue dashed line) of the nitro group with the positively charged
residue Lys220.
The most potent compound in terms of migration inhibition, 4e, had the same interactions with the Fascin binding site as the
weakest in vitro inhibitor, 4h. As can be seen in Figure 7 the ortho nitro group established electrostatic and hydrogen bonds with
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Lys220. Here again, a π–cationic interaction between the nitrophenyl ring and Lys220 was also observed. Other amino acids
involved in interactions were almost the same as what was observed for compound 4h and Nifedipine.
Figure 7. Compound 4e inside the Fascin binding site. The 3D ligand interaction diagram shows hydrogen bonding (green dashed
line) and the 2D diagram demonstrates electrostatic interactions (blue dashed line) of the nitro group with the positively charged
residue Lys220. π–cationic interaction is shown with orange lines in both diagrams.
It should be noted that all the studied compounds as well as Nifedipine were docked into the same pocket inside the Fascin
protein and the docking results had a high degree of reproducibility according to the clustering histograms in the docking output files.
Thus, all the studied compounds were docked into their actual binding site inside the Fascin protein. The reason behind the
controversy between the in vitro and in silico results might be that the scratch assay is not a representative of the inhibition of Fascin
and other cellular targets. Theses targets may also be inhibited by some of the studied compounds.
Conclusions
In the present study, it was aimed at the synthesis of
a series of alkyl-6-methyl-4-[2(3)-nitrophenyl]-2-(thi)oxo-1,2,3,4-
tetrahydroprimidine-5-carboxylates which possess structural features of both Monastrol as an anti-cancer drug and Nifedipine as a
Fascin blocker compound. All of the compounds were investigated in silico for their ability to inhibit the Fascin protein using
molecular docking simulation. It should be considered that in the case of abnormal proliferation of a tissue, if the proliferation is
completely inhibited, then there would not be any need to inhibit cell migration. Thus, it seemed to us that the anti-migratory activity
would be a good additive feature for moderately active cytotoxic agents. Those cells which were not destroyed by the cytotoxic
activity of the compound would not have any chance for migration which leads to invasion. In fact, this feature would change nothing
in the process of the solid cancer treatment with a strong cytotoxic agent.
The studied compounds were ordered as 4d>4f>4b>4g>4h>4e>4c>4a according to their cytotoxic potency. The most potent
one as a cytotoxic compound was 4d with an IC50 of 193.70 µmol and the weakest one was 4a (IC50=248.75 µmol). The least
cytotoxic compound was one of the strongest compounds in binding to the Fascine binding site according to molecular docking
simulation results (ΔGbind= -7.81 kcal/mol). The controversy between the in vitro and in silico results may cancel the postulation of
the involvement of the Fascin inhibition in the migration inhibition. However, the considerable anti-migratory effects of some of the
synthesized compounds proved it to be reasonable to perform further in vivo experiments to introduce novel inhibitors of tumor
metastasis.
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Table 3. Interactions between the docked 1,2,3,4-tehtrhydropyridine derivatives and fascin binding site residues
Amino acid in
Hydrogen bond
Amino acids in
Van der Waals and electrostatic
contacts
Amino acid in
π-cation
ΔGbind
hydrogen bond
electrostatic interaction
with NO2
interaction with Lys2220
(kcal/mol)
Compd.
4a
Distance (Å)
θ Angle (°)
Lys220
Lys220
Lys220
Ala7
1.85
1.72
1.89
1.69
164
158
120
160
Ala9,Ile12,Leu48, Lys220,Val221,
Phe254,Ala255,Leu256, Phe216
Lys220
-C6H4-NO2
-7.81
4b
4c
Ala7,Arg308,Gln295,Trp171,Val169,Pro170,
Tyr152
-
-
-5.91
-8.34
Lys220
Ile12
1.96
1.75
2.04
Ala9,Val10,Gln11,Ile12,Leu48,
Lys220,Leu256
Lys220
-C6H4-NO2
141
123
138
Val10
4d
4e
Lys220
Lys220
Ile12
1.73
1.74
2.17
1.59
1.68
1.90
2.13
158
160
168
161
148
163
135
136
Val10,Ile12,Leu48,Arg217 ,Lys220,
Val221,Phe254,Ala255,Leu256
Lys220
Lys220
-C6H4-NO2
-C6H4-NO2
-8.43
-8.21
Lys220
Lys220
Lys220
Val10
Ala9,Val10, Leu48,Glu49
Phe216,Lys220,Leu256
4f
Lys220
Ala9,Val10,Ile12,Leu48,Phe216,Lys220,
Val221,Ala255,Leu256
Lys220
Lys220
Lys220
Lys220
-
-6.51
-7.49
-8.72
-9.8
2.11
4g
Lys220
Lys220
Lys220
Val10
1.80
1.82
1.77
1.94
1.90
2.13
2.21
1.80
1.99
130
142
159
122
131
161
154
144
148
Ala9,Val10,Gln11,Ile12,Leu48,Glu49,
Lys220
-
4h
Val10,Ile12,Leu48,Phe216 ,Arg217,Lys220,
Val221,Leu256
-C6H4-NO2
-
Nifedipine
Val10
Ala9,Val10, Ile12, Leu48, Phe216,Lys220,
Val221,Ala255,Leu256
Leu256
Lys220
Lys220
Lys220
10
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10.1002/cbdv.201800339
Chemistry & Biodiversity
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Experimental Section
Chemistry
All chemicals, reagent and solvents were obtained from commercial suppliers and were freshly used after purification by standard
procedures. Melting points were determined on an Electrothermal 9200 Melting Point apparatus and were uncorrected. 1H-NMR
spectra were recorded in DMSO-d6 on a Bruker-Ultrashield 400MHz spectrometer (Germany). All the chemical shifts were reported
as (δ) values (ppm) against tetramethylsilane as an internal standard. IR spectra were recorded with a Perkin Elmer IR
spectrophotometer. Electrospray mass spectra (ESI-MS) were obtained in negative and positive ion mode on a SHIMADZU LCMS-
2010 EV spectrometer using methanol as solvent, a capillary voltage of 4500 V and a cone voltage of 10 V. All experiments were
monitored by analytical thin layer chromatography (TLC) on pre-coated silica gel 60 F254 aluminum plates (Merck, Germany).
Suitable names for the compounds were given with the aid of ChemOffice 2014 software. The final products were prepared in 16-
61%. Their purity was determined by thin layer chromatography using several solvent systems of different polarities and data
obtained from elemental analysis, to an accuracy of within ±0.4%. General procedure for the preparation of the compounds is
provided in Scheme 1.
Synthesis of the Lewis acid catalysts
Cobalt hydrogen sulfate Co(HSO4)2 was prepared according to the method of Memarian et al.[18] Poly phosphate ester was
synthesized using the procedure reported by Kappe and his colleagues.[19]
General procedure for the preparation of Biginelli compounds using cobalt hydrogen sulfate as the catalyst
A mixture of the proper aldehyde (1,1'; 3.0 mmol), methyl (ethyl) aceto acetate ester (2,2'; 3.0 mmol), urea (3, 3.9 mmol) and
Co(HSO4)2 as the Lewis acid catalyst (0.9 mmol) in EtOH (4 mL) was heated at 85 ºC for 24 hours. After the completion of the
reaction indicated by thin layer chromatography (TLC) (chloroform:methanol, 10:1), the mixture was cooled down to room
temperature and water was added. Stirring was continued for several minutes for dissolving the catalyst and the excess of urea.
Solid products (4a-d) were filtered and washed with water. Pure products were obtained by recrystallization from ethanol. The
chemical structures of the products were characterized by spectroscopic data of IR and 1H-NMR.
General procedure for the synthesis of Biginelli compounds using poly phosphate ester as the catalyst
A mixture of the proper aldehyde (1,1'; 1 mmol), methyl (ethyl) aceto acetate ester (2,2'; 1.0 mmol) and thiourea (3', 1.5 mmol) was
added to a solution of polyphosphate ester (150 mg) in tetrahydrofurane (4 mL) then refluxed for 17 hours. The reaction progress
was monitored using TLC until the reactants disappeared in the reaction mixture. Then it was cooled down to room temperature and
some crushed ice was added. After stirring for some more hours the resulted precipitate was filtered off, washed with cold water and
dried in a vacuum oven for 24 hours to give dry 4e-h.
Biology
Cell Culture
HeLa and 4T1 cell lines were purchased from Pasteur Institute, Iran. Cells were maintained in RPMI-1640 afforded with 10 % (v/v)
fetal bovine serum (FBS) and Penicillin/Streptomycin (50 IU/ml, 50 mg/ml) at 37 °C in a humidified atmosphere containing 5% CO2.
In vitro cytotoxicity assay
The cytotoxicity assay of the selected compounds was performed against HeLa cell line according to a previously reported
method.[20] HeLa cells were sub-cultured regularly via Trypsin/ethylene diamine tetra-acetic acid (EDTA). To evaluate the cell
viability, a test with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed. MTT is reduced to
11
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10.1002/cbdv.201800339
Chemistry & Biodiversity
Chem. Biodiversity
formazan crystals in the mitochondria of viable cells, and when the crystals are dissolved in DMSO they produce a dark-purple color.
Briefly, HeLa cells were plated in 96-well plates and grown overnight. Then cells were exposed to serial concentrations of selected
compounds (7.8–500 µg/mL) and incubated at 37°C for 48 h. At the end of the incubation time, 20 µL of MTT solution (5 mg/mL)
was added and incubated for further 3 hours. The medium containing unreacted MTT was removed. Subsequently, 15 mL of DMSO
was added to each well to dissolve the formazan crystals. Finally, the absorbance of the dissolved formazan was measured at
540 nm in an Enzyme-Linked Immuno Sorbent Assay (ELISA) reader. All the experiments were performed in duplicate. Statistical
analyses were achieved using the SPSS software package (v.17). One-way ANOVA tests and TukeyPost Hoc test were used with a
confidence level of 0.05 to assess the statistically significant homogeneous subsets. IC50 values were calculated using GraphPad
Prism software.
Migration assay
Convenient and inexpensive in vitro scratch assay reported by Liang and his colleagues was employed in this research for analysis
of cell migration.[21] The 4T1 cancer cells were seeded into 12-well culture plates at a 5 × 105 cells/mL concentration and cultured
overnight prior to serum starvation for 24 hours. The cell monolayers were then incubated with 10 mg/mL of Mytomycin C to stop
cell proliferation. The monolayers were carefully wounded using a pipette tip and washed three times with PBS to remove detached
cells. The wounded monolayers were then incubated for 24 hours with Nifedipine, 4c, 4a, 4e, and 4h at the same concentrations
(50 µg/mL). The wounds photographs were taken at the onset of the test, when the scratch was made and after 24 hours. Cell
migration was exhibited as the percentage of scratch closure according to borders (orange lines) closing.
Molecular docking studies
In order to gain some insight into the binding mode of the newly designed compounds with Fascin protein, it was decided to focus
on the inhibition of this protein by carrying out molecular docking studies. Crystallographic structure of Fascin was retrieved from the
RCSB protein Data Bank, PDB code: 3LLP. The docking studies were performed using AUTODOCK 4.2 software package, with the
implemented empirical free energy function and the Lamarckian genetic algorithm (LGA).[22]
Ligand preparation
For ligand preparation, three-dimensional structures of the compounds were constructed and optimized using the Polak-Ribiere
conjugate gradient algorithm and PM3 semi-empirical method implemented in Hyperchem software. These optimized structures
were used as inputs of the Auto Dock tools. Then the partial charges of atoms were calculated using the Gasteiger-Marsili
procedure implemented in the Auto Dock tools package.[23] Non-polar hydrogens of the compounds were merged and then rotatable
bonds were assigned. PDB format of this ligand was converted to PDBQT file to generate atomic coordinates.
Protein preparation
All irrelevant hetero atoms including water molecules were removed from the crystal structure using notepad software. All missing
hydrogens were added, and after determining the Kolman united atom charges, non-polar hydrogens were merged into their
corresponding carbons using Auto dock tools. As the final part of the process of the protein preparation, desolvation parameters
were assigned to protein atom.
Molecular docking simulation
Using Auto grid implemented in the Auto dock program, the grid maps were calculated. Since the target molecule was too big to be
covered by one grid box, four separate grid boxes with different centers were applied for each compound then autodock calculations
were run. Based on the results obtained from docking in each grid box, binding site was determined. Then a grid box was chosen to
be large enough to contain the determined binding site of the protein and the significant regions of the surrounding surface. The grid
map of 126×126×126 points and a grid point spacing of 0.375 Ǻ (roughly a quarter of the length of a carbon-carbon single bond)
were applied using the Autogrid. The Lamarckian Genetic Algorithm (LGA) approach was selected as the search algorithm for the
12
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10.1002/cbdv.201800339
Chemistry & Biodiversity
Chem. Biodiversity
global optimum binding position search provided by Autodock 4.2. For the LGA method, 100 independent docking runs were carried
out for each atom. An initial population of random individuals with a population size of 150 individuals was considered. For
investigation of hydrogen bonding and π–π stacking interactions which are established through the docking procedure
AutoDockTools and DS Visualizer 3.5 software were utilized.
Acknowledgment
The authors would like to thank the School of pharmacy, Isfahan University of medical science, Isfahan, Iran for financing this
project.
Author Contribution Statement
This article was extracted from the PharmD research project of Narges Riahi. She performed all the synthesis procedures by herself
and the biological assays with the help of Amirhosein Kefayat and Ahmad Ghasemi. Mohammadhosein Asgarshamsi wrote some
parts of the article and prepared the solutions for migration inhibition assay. Afshin Fassihi was the supervisor of the chemistry part
of the research and Mojtaba Panjehpoor was the supervisor of the biological part of the project.
Conflicts of interest
There are no conflicts of interest in this report.
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10.1002/cbdv.201800339
Chemistry & Biodiversity
Chem. Biodiversity
Graphical Abstract
15
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