Antiproliferative effect, alteration of cancer cell cycle progression and potential MET kinase inhibition induced by 3,4-dihydropyrimidin-2(1H)-one C5 amide derivatives

Article abstract of DOI:10.1016/j.ejphar.2021.173850

Cancer continues to be the second leading cause of death worldwide. Discovery of novel therapeutic agents has crucial importance for improvement of our medical management capabilities. Dysregulation of the MET receptor tyrosine kinase pathway plays an important role in cancer progression, making this receptor an attractive molecular target for anticancer drug discovery. In this study, twenty-seven 3,4-dihydropyrimidin-2(1H)-one C5 amide derivatives were synthesized and their cancer cell growth inhibitory activity was examined against MCF-7, HT-29 and MOLT-4 cells and also NIH/3T3 non-cancer cells by MTT assay. The antiproliferative effect of the most potent derivatives were tested against MET-dependent EBC-1 and MKN-45, lung and gastric cancer cell lines, respectively. MET kinase inhibition was measured by a Homogenous Time Resolved Fluorescence (HTRF) Assay. The influence of the test compounds on cell cycle was examined by RNase/PI flow cytometric assay. A number of compounds exhibited considerable antiproliferative effects against breast and colon cancer and leukemia cell lines, relatively sparing non-cancer cells. Some derivatives bearing benzothiazolyl carboxamide moiety at C5 position (15, 21, 23, 31, and 37) showed the highest activities with IC₅₀ values as low as 10.9 μM. These compounds showed antiproliferative effects also against MET-amplified cells and dose-dependently inhibited MET kinase activity. They also induced G0/G1 cell cycle arrest at lower doses and apoptosis at higher doses. Molecular docking and dynamics simulation studies confirmed the interaction of compound 23 with the active site of the MET receptor. These findings demonstrate that 3,4-dihydropyrimidin-2(1H)-one analogues may represent promising targeted anticancer agents.

Full text of DOI:10.1016/j.ejphar.2021.173850

European Journal of Pharmacology 894 (2021) 173850
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Antiproliferative effect, alteration of cancer cell cycle progression and
potential MET kinase inhibition induced by 3,4-dihydropyrimidin-2
(1H)-one C5 amide derivatives
,
c
Fatemeh Moosavia, Ahmad Ebadib, Maryam Mohabbatia, Tahereh Damghania
,
,
*
Motahareh Mortazavia, Ramin Miria, Omidreza Firuzia
a Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
b Department of Medicinal Chemistry, School of Pharmacy, Medicinal Plants and Natural Products Research Center, Hamadan University of Medical Sciences, Hamadan,
Iran
c Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cancer continues to be the second leading cause of death worldwide. Discovery of novel therapeutic agents has
crucial importance for improvement of our medical management capabilities. Dysregulation of the MET receptor
tyrosine kinase pathway plays an important role in cancer progression, making this receptor an attractive mo-
lecular target for anticancer drug discovery. In this study, twenty-seven 3,4-dihydropyrimidin-2(1H)-one C5
amide derivatives were synthesized and their cancer cell growth inhibitory activity was examined against MCF-7,
HT-29 and MOLT-4 cells and also NIH/3T3 non-cancer cells by MTT assay. The antiproliferative effect of the
most potent derivatives were tested against MET-dependent EBC-1 and MKN-45, lung and gastric cancer cell
lines, respectively. MET kinase inhibition was measured by a Homogenous Time Resolved Fluorescence (HTRF)
Assay. The influence of the test compounds on cell cycle was examined by RNase/PI flow cytometric assay. A
number of compounds exhibited considerable antiproliferative effects against breast and colon cancer and leu-
kemia cell lines, relatively sparing non-cancer cells. Some derivatives bearing benzothiazolyl carboxamide
Anticancer drug
Kinase inhibitor
Scaffold hopping
Drug design
Molecular modeling
moiety at C5 position (15, 21, 23, 31, and 37) showed the highest activities with IC50 values as low as 10.9 μM.
These compounds showed antiproliferative effects also against MET-amplified cells and dose-dependently
inhibited MET kinase activity. They also induced G0/G1 cell cycle arrest at lower doses and apoptosis at
higher doses. Molecular docking and dynamics simulation studies confirmed the interaction of compound 23
with the active site of the MET receptor. These findings demonstrate that 3,4-dihydropyrimidin-2(1H)-one an-
alogues may represent promising targeted anticancer agents.
1. Introduction
approaches for cancer therapy (Moosavi et al., 2019; Recondo et al.,
2020). Consequently, over the past few years, several small molecule
Cancer imposes a major burden on healthcare systems in the world
and according to the last WHO update, it is the second leading cause of
mortality worldwide, accounting for the loss of more than 9 million
human lives in 2018 (WHO, 2020).
inhibitors of MET pathway have been developed or are under intense
investigation for the treatment of multiple solid tumors as well as he-
matologic malignancies (Firuzi et al., 2019; Huang et al., 2020; Par-
ikhGhate, 2018).
Hepatocyte growth factor receptor (HGFR) or MET, is a receptor
tyrosine kinase (RTK) involved in tumorigenesis and cancer progression
(Koch et al., 2020). Aberrant activation of MET signaling pathway has
been reported in several malignancies, prompting great interest in the
development of anti-MET therapeutic strategies as target-based
Benzothiazoles (BTAs) possess several interesting biological activ-
ities and have received a great deal of attention for their diverse prop-
erties (Keri et al., 2015) including potent and selective antitumor
properties (SinghSingh, 2014). As anticancer agents, BTAs may interact
with various targets (Kamal et al., 2012; Kamal et al., 2012).
* Corresponding author.
E-mail address: firuzio@sums.ac.ir (O. Firuzi).
https://doi.org/10.1016/j.ejphar.2021.173850
Received 31 July 2020; Received in revised form 12 December 2020; Accepted 5 January 2021
Available online 9 January 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
Benzothiazole ring is the structural feature of some of the most potent
MET inhibitors; For example, compound 1 (Scheme 1) inhibits MET with
an IC50 value of 17.6 nM, a higher potency compared to a known MET
inhibitor foretinib (Lei et al., 2016).
On the other hand, important pharmacological properties have been
reported for 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives
(GangwarKasana, 2012; Guido et al., 2015; Kim et al., 2012; Mishra
et al., 2010; Mokale et al., 2010; Narkhede Harsha, Nevagi Reshma,
Manoj and Prabhjeet, 2014). Mayer and colleagues reported the syn-
thesis and anticancer potential of monastrol (2, Scheme 1), a DHPM
flagship compound for the first time (Mayer et al., 1999). Monastrol has
´
inspired the discovery of new anticancer agents over decades (de Fatima
et al., 2015; Guido et al., 2015); For instance, one of the most potent
monastrol analogues, piperastrol (3, Scheme 1), has shown considerable
anticancer effect against MCF-7, HT-29 and OVCAR03 cancer cell lines
(Russowsky et al., 2006). Moreover, pyrimidinone-peptoid hybrid ana-
logues like compound 4 (Scheme 1) (Wright et al., 2008) and DHPMs
bearing C5 amide moieties (compounds 5 and 6, Scheme 1) have shown
significant antiproliferative effects against different cancer cells
including breast and colon cancer cell lines (Kumar et al., 2009). Other
DHPM derivatives have shown antiproliferative effects against breast
cancer cells through the inhibition KIF11 (Kinesin family member 11 or
Eg5) mortor protein (Guido et al., 2015).
Scheme 2. Scaffold-hopping approach used in the design of 3,4-dihydropyr-
imidin-2(1H)-one C5 amide derivatives bearing benzothiazole moiety as anti-
cancer agents with potential MET inhibitory activity.
without correction. FT-IR (KBr) spectra were recorded on a Nicolet FT-IR
Magna 550 spectrophotometer. 1H NMR and 13C NMR spectra were
determined by a Bruker FT-400 MHz spectrometer in DMSO-d6. All the
chemical shifts were reported as (δ) values (ppm) against tetrame-
thylsilane as an internal standard. The MS spectra were recorded using
Agilent 7890A spectrometer at 70 eV. The purity of all compounds was
measured using 1H NMR and was found to be more than 95%.
Furthermore, the purity was also confirmed by elemental analysis. CHN/
CHNS analysis was performed using CHNS-932 Leco analyzer and the
results were within ±0.4% of the theoretical values indicating very pure
compounds.
In the present contribution, we used the combination of privileged
scaffolds as a fruitful approach for discovery of new derivatives. In this
context, novel DHPM-C5 amide derivatives were designed by scaffold
hoping that uses different structures including DHPM-based antitumor
compounds (i.e. monastrol, compounds 5 and 6), as well as benzothia-
zole containing antitumor and MET inhibitor derivatives (NSC745689,
triflocas, PMX610 (SinghSingh, 2014) and compound 1) (Scheme 2).
Obtained results indicated that designed DHPM derivatives are indeed
promising anticancer agents with good selectivity against cancer cells
compared to non cancer cells. The most promising derivatives also
exhibited antiproliferative effect against MET-dependent cancer cell
lines EBC-1 and MKN-45 cells. Moreover, these compounds inhibited
MET kinase activity in in vitro enzymatic assay. The cell cycle analysis
showed accumulation of cells in G0/G1 phase of the cell cycle and the
molecular docking study and dynamics simulation revealed that the
most potent synthesized derivative effectively binds with the kinase
domain of the MET receptor.
The progress of the reactions and purity of the products were
checked by analytical thin-layer chromatography (TLC) on pre-coated
silica gel 60 F254 aluminum plates (Merck, Germany).
2.1.1. General procedure for the synthesis of N-(aryl)-3-oxobutanamides
(9a-f)
Acetoacetamide derivatives (9a-f) were prepared as shown in
Scheme 3. 2,2,6-Trimethyl-1,3-dioxine-4-one (7) (2eq) and appropriate
arylamine (8a-f) (1eq) were mixed and refluxed for about 2–4 h in
xylene. The progress of the reactions was checked by monitoring the
product by TLC to find the best reaction time. Consequently, 4 h was set
as the optimum time for reactions. Upon completion of the reaction, the
solid product was filtered. Further purification was performed by crys-
tallization of obtained products in ethanol. Characteristics data for
prepared N-(aryl)-3-oxobutanamides as described below.
2. Materials and methods
2.1. Chemistry
All the reagents used for synthesis were bought from Merck, Ger-
many and used without any further purifications. Melting points were
determined by a Stuart™ SMP3 melting point apparatus and reported
Scheme 1. Chemical structures of previously reported 3,4-dihydropyrimidin-2
(1H)-one and benzothiazole derivatives with antiproliferative activities against
different cancer cells.
Scheme 3. Synthesis pathway of compounds 12–38. Reagents and conditions:
(i) xylene, reflux, 2–4 h, (ii) ACN, FeCl3 (0.25 mol %), reflux, 24 h.
2

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
2.1.1.1. N-(2-Benzothiazolyl)-3-oxobutanamide (9a). Yield 65%; mp
214–216 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.65 (br s, 1H, NH-
amide), 7.86 (d, J = 7.6 Hz, 1H, C4′-benzothiazole), 7.68 (d, J = 8.0 Hz,
1H, C7′H-benzothiazole), 7.35 (t, J = 8.4 Hz, 1H, C5′H-benzothiazole),
7.20 (t, J = 8.0 Hz, 1H, C6′H-benzothiazole), 3.73 (s, 2H, COCH2CO),
monitoring the product by TLC. On completion of the reaction, the solid
product was filtered, washed with small portions of ethanol, and then
dried. Further purification by preparative TLC using ethylacetate/pe-
troleum ether as eluent gave pure compounds 12–38. The structure of
new DHPM-C5-amide derivatives were elucidated by IR, MS and 1H-
NMR spectra along with CHN/CHNS analysis. The first report of syn-
thesis and characterization of compounds 12–17, 20, 22–24, 27–28 and
36–38 was published previously (Ebadi et al., 2017). The character-
ization data of these compounds are reported in Supplemental file. NMR
spectral data of newly synthesized DHPM derivatives are as follows:
2.23 (s, 3H, CH3CO); IR (KBr)
ν
cmꢀ 1: 3178.7 (N–H, amide), 3067.5
–
(C–H, aromatic), 2917.3 and 2859.2 (C–H, aliphatic), 1719.4 (C O,
–
+
–
–
ketone), 1661.3 (C O, amide); MS m/z (%): 234 (25) [M ], 177 (6),
150 (100), 123 (11), 43 (32); Anal. Calcd (%) for C11H10N2O2S: C, 56.39;
H, 4.30; N, 11.96; S, 13.69. Found: C, 56.20; H, 4.42; N, 12.11; S, 13.52.
2.1.1.2. N-(6-Ethoxy-2-benzothiazolyl)-3-oxobutanamide
(9b). Yield
2.1.2.1. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-phenyl-3,4-dihy-
dropyrimidin-2(1H)-one (12). Yield 65%; mp 281–284 ◦C; 1H NMR (400
MHz, DMSO-d6) δ (ppm): 11.93 (br. s, 1H, NH-amide), 9.11 (br. s, 1H,
N1H-DHPM), 7.92 (d, 1H, J = 7.6 Hz, C4ʹH-benzothiazole), 7.81 (br. s,
1H, N3H-DHPM), 7.71 (d, 1H, J = 8.0 Hz, C7ʹH-benzothiazole), 7.41 (t,
1H, J = 7.7 Hz, C5ʹH-benzothiazole), 7.22–7.35 (comp, 6H, C6ʹH-ben-
zothiazole and CH-phenyl), 5.62 (d, 1H, J = 2.8 Hz, C4H-DHPM), 2.20
41%; mp 206–208 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.88 (br
s, 1H, NH-amide), 7.62 (d, J = 8.8 Hz, 1H, C4′H-benzothiazole), 7.54 (d,
J = 2.4 Hz, 1H, C7′H-benzothiazole), 7.01 (d, J = 8.8 Hz, 1H,
C5′Hbenzothiazole), 4.06 (q, J = 6.8 Hz, 2H, –OCH2CH3), 3.75 (s, 2H,
COCH2CO), 2.23 (s, 3H, CH3CO), 1.35 (t, J = 6.4 Hz, 3H, –OCH2CH3); IR
(KBr)
ν
cmꢀ 1: 3190.2 (N–H, amide), 3085.8 (C–H, aromatic), 2962.5 and
2889.6 (C–H, aliphatic), 1720.6 (C O, ketone), 1661.9 (C O, amide);
(s, 3H, CH3-DHPM); IR (KBr)
ν
cmꢀ 1: 3437, 3259.1, 3280 (N–H), 3057
–
–
–
–
MS m/z (%): 278 (58) [M+], 220 (18), 194 (100), 165 (86), 43 (41);
Anal. Calcd (%) for C13H14N2O3S: C, 56.10; H, 5.07; N, 10.06; S, 11.52.
Found: C, 56.23; H, 4.95; N, 10.18; S, 11.41.
(C–H, aromatic), 2898 (C–H, aliphatic), 1683 (C O, DHPM), 1636
–
–
+
–
–
–
–
(C O, amide), 1598 (C C, alkene); MS m/z (%): 364 (M , 79), 215
(100), 187 (36), 172 (75), 151 (62), 131 (56). Anal. Calcd (%) for
C
19H16N4O2S: C, 62.62; H, 4.43; N, 15.37; S, 8.80. Found: C, 62.40; H,
4.41; N, 15.41; S, 8.77.
2.1.1.3. N-(5-bromopyridin-2-yl)-3-oxobutanamide (9c). Yield 54%; mp
193–195 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.36 (br s, 1H, NH-
amide), 8.64 (d, 1H, J = 2.3 Hz, C6ʹH-bromopyridin), 8.03 (dd, 1H, J =
8.6, 2.3 Hz, C4ʹH-bromopyridin), 7.89 (d, 1H, J = 8.6, C3ʹH-bromo-
2.1.2.2. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-phenyl-3,4-dihy-
dropyrimidin-2(1H)-thione (13). Yield 46%; mp 289–294 ◦C; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 12.18 (br. s, 1H, NH-amide), 10.27 (br. s,
1H, N1H-DHPM), 9.68 (br. s, 1H, N3H-DHPM), 7.93 (d, 1H, J = 7.2 Hz,
C4ʹH-benzothiazole), 7.72 (d, 1H, J = 7.6 Hz, C7ʹH-benzothiazole), 7.41
(t, 1H, J = 7.2 Hz, C5ʹH-benzothiazole), 7.27–7.38 (comp, 6H, C6ʹH-
benzothiazole and CH-phenyl), 5.63 (s, 1H, C4H-DHPM), 2.21 (s, 3H,
pyridin), 3.34 (s, 2H, COCH2CO), 2.13 (s, 3H, CH3CO); IR (KBr)
ν
cmꢀ 1
:
3182.2 (N–H, amide), 3015.8 (C–H, aromatic), 2982.5 (C–H, aliphatic),
–
–
–
1726.6 (C O, ketone), 1670.9 (C O, amide); MS m/z (%): 258 (M+2,
–
43), 256 (43) [M+], 199 (52), 171 (100), 43(41); Anal. Calcd (%) for
C9H9BrN2O2: C, 42.05; H, 3.53; N, 10.90. Found: C, 42.22; H, 3.52; N,
10.86.
CH3-DHPM); IR (KBr)
ν
cmꢀ 1: 3297.4 (N–H), 3166.2 (C–H, aromatic),
–
–
–
2989.1 (C–H, aliphatic), 1670.7 (C S, DHPM), 1643.1 (C O, amide),
–
+
–
1613 (C C, alkene); MS m/z (%): 380 (M ,100), 231 (46), 172 (54),
–
2.1.1.4. N-(4-methyl-2-pyridinyl)-3-oxobutanamide (9d). Yield 48%; mp
186–188 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.58 (br s, 1H, NH-
amide), 8.24 (d, 1H, J = 4.6 Hz, C6H-pyridine), 7.87 (s, 1H, C3H-
pyridine), 6.75 (d, 1H, J = 4.6 Hz, C5H-pyridine), 3.41 (s, 2H,
151 (83), 131 (58). Anal. Calcd (%) for C19H16N4OS2: C, 59.98; H, 4.24;
N, 14.73; S, 16.85. Found: C, 59.87; H, 4.22; N, 14.75; S, 16.81.
COCH2CO), 2.16 (s, 3H, CH3CO), 2.06 (s, 3H, CH3-pyridine); IR (KBr)
ν
2.1.2.3. 6-Methyl-5-N-(6-ethoxy-2-benzothiazolyl)carboxamide-4-phenyl-
3,4-dihydropyrimidin-2(1H)-one (14). Yield 80%; mp 227–232 ◦C; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (br. s, 1H, NH-amide), 9.06
(br. s, 1H, N1H-DHPM), 7.77 (br. s, 1H, N3H-DHPM), 7.58 (d, 1H, J =
8.8 Hz, C4ʹH-benzothiazole), 7.48 (d, 1H, J = 2.8 Hz, C7ʹH-benzothia-
zole), 7.20–7.38 (comp, 5H, CH-phenyl), 6.99 (dd, 1H, J = 8.8, 2.8 Hz,
C5ʹH-benzothiazole), 5.59 (d, 1H, J = 2.8 Hz, C4H-DHPM), 4.05 (q, 2H,
J = 6.8 Hz, –OCH2CH3), 2.19 (s, 3H, CH3-DHPM), 1.34 (t, 3H, J = 6.8
cmꢀ 1: 3094.6 (N–H, amide), 3023.7 (C–H, aromatic), 2958.8 (C–H,
–
–
–
aliphatic), 1719.3 (C O, ketone), 1659.7 (C O, amide); MS: m/z (%):
–
192 (56) [M+], 135 (61), 108 (100), 43(57); Anal. Calcd (%) for
C
10H12N2O2: C, 62.49; H, 6.29; N, 14.57. Found: C, 62.74; H, 6.31; N,
14.62.
2.1.1.5. N-(5,6-dimethyl-2-benzothiazolyl)-3-oxobutanamide (9e). Yield
63%; mp 173–175 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.82
(1H, brs, NH-amide); 7.70 (1H, s, C4′H- benzothiazole), 7.53 (1H, s,
C7′H-benzothiazole), 3.75 (2H, s, COCH2CO), 2.32 (3H, s, CH3-benzo-
thiazole), 2.31 (3H, s, CH3-benzothiazole), 2.23 (3H, s, CH3CO); IR (KBr)
Hz, –OCH2CH3); IR (KBr)
ν
cmꢀ 1: 3221.9 (N–H), 3074.5 (C–H, aro-
–
–
–
matic), 2917.2 (C–H, aliphatic), 1681.6 (C O, DHPM), 1652.1 (C O,
–
+
–
amide), 1551.4 (C C, alkene); MS m/z (%): 408.2 (M , 33), 215 (17),
–
194 (100), 166(21). Anal. Calcd (%) for C21H20N4O3S: C, 61.75; H, 4.94;
cmꢀ 1: 3188.2 (N–H, amide), 3075.5 (C–H, aromatic), 2923.1 and
N, 13.72; S, 7.85. Found: C, 61.50; H, 4.93; N, 13.67; S, 7.83.
ν
–
–
–
2879.6 (C–H, aliphatic), 1717.7 (C O, ketone), 1660.8 (C O, amide);
–
MS: m/z (%) 262 (38) [M+], 205 (6), 178 (100), 163 (30), 91 (6), 43
(14); Anal. Calcd. for C13H14N2O2S: C, 56.39; H, 4.30; N, 11.96; S, 13.69;
Found: C, 56.55; H, 4.11; N, 11.86; S, 13.80.
2.1.2.4. 6-Methyl-5-N-(5,6-dimethyl-2-benzothiazolyl)carboxamide-4-
phenyl-3,4-dihydropyrimidin-2(1H)-one
(15). Yield
53%;
mp
292–294 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.81 (br. s, 1H, NH-
amide), 9.06 (br. s, 1H, N1H-DHPM), 7.77 (br. s, 1H, N3H-DHPM),
7.63 (s, 1H, C4ʹH-benzothiazole), 7.47 (s, 1H, C7ʹH-benzothiazole),
7.20–7.36 (comp, 5H, CH-phenyl), 5.61 (d, 1H, J = 2.8 Hz, C4H-DHPM),
2.31 (s, 3H, CH3-benzothiazole), 2.29 (s, 3H, CH3-benzothiazole), 2.20
2.1.2. General procedure for the synthesis of 6-methyl-5-N-(aryl)
carboxamide-4-aryl (aryl/heteroaryl)-3,4-dihydropyrimidin-2(1H)-one
(12–38)
The synthesis of 3,4-dihydropyrimidin-2(1H)-one(thione) C5 amide
derivatives (12–38) was carried out by one-pot multicomponent reac-
tion as depicted in Scheme 3. The mixture of proper acetoacetamide (9a-
f) (1eq), urea or thiourea (11) (1.5eq) and appropriate aldehyde (10a-h)
(1eq) with 0.25% eq of FeCl3 was mixed in 15 ml of acetonitrile and
refluxed for 24 h. The optimum time of reaction was found out by
(s, 3H, CH3-DHPM); IR (KBr)
ν
(cmꢀ 1): 3363.6, 3211.4 (N–H, DHPM),
–
3085.8(C–H, aromatic), 2962.2 (C–H, aliphatic), 1694.9 (C O, DHPM),
–
+
–
–
–
1634.7 (C O, amide), 1558.4 (C C, alkene); MS m/z (%): 392 (M ,
–
65), 178 (100), 215 (44). Anal. Calcd (%) for C21H20N4O3S: C, 64.27; H,
5.14; N, 14.28; S, 8.17. Found: C, 63.84; H, 5.20; N, 14.23; S, 8.10.
3

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
2.1.2.5. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(4-nitrophenyl)-
3,4-dihydropyrimidin-2(1H)-one (16). Yield 77%; mp 257–261 ◦C; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 12.01 (br. s, 1H, NH-amide), 9.29
(br. s, 1H, N1H-DHPM), 8.23 (d, 2H, J = 8.8 Hz, CH-phenyl), 8.00 (br. s,
1H, N3H-DHPM), 7.92 (d, 1H, J = 7.2 Hz, C4ʹH-benzothiazole), 7.71 (d,
1H, J = 7.6 Hz, C7ʹH-benzothiazole), 7.57 (d, 2H, J = 8.8 Hz, CH-
phenyl), 7.41 (t, 1H, J = 7.2 Hz, C5ʹH-benzothiazole), 7.28 (t, 1H, J
= 7.6 Hz, C6ʹH-benzothiazole), 5.72 (s, 1H, C4H-DHPM), 2.22 (s, 3H,
NH-amide), 8.93 (br. s, 1H, N1H-DHPM), 8.22 (d, 2H, J = 8.8 Hz, CH-
phenyl), 8.12 (d, 1H, J = 4.8 Hz, C6H-pyridine), 7.79 (s, 1H, C3H-
pyridine), 7.74 (br. s, 1H, N3H-DHPM), 7.56 (d, 2H, J = 8.8 Hz, CH-
phenyl), 6.90 (d, 1H, J = 4.8 Hz, C5H-pyridine), 5.54 (d, 1H, J = 2.0
Hz, C4H-DHPM), 2.26 (s, 3H, CH3-DHPM), 2.10 (s, 3H, CH3-pyridine);
IR (KBr)
ν
cmꢀ 1: 3237.4 (N–H), 3115.9 (CH-aromatic) 2946.3 (C–H,
–
–
–
aliphatic), 1674.3 (C O, DHPM), 1655.5 (C O, amide), 1616.1 1415.1
–
–
–
(C C, alkene), 1520.1, 1347.1 (C–NO aromatic); MS m/z (%): 367
2
CH3-DHPM); IR (KBr)
ν
cmꢀ 1: 3255.3, 3107.6 (N–H), 2948.99 (C–H,
(M+, 13), 350 (100), 320 (17), 109 (33). Anal. Calcd (%) for
–
–
–
–
aliphatic), 1687.9 (C O, DHPM), 1650.1 (C O, amide), 1598.4 (C C,
C18H17N5O4: C, 58.85; H, 4.66; N, 19.06. Found: C, 58.62; H, 4.64; N,
–
–
alkene), 1523.6, 1345.9 (C–NO2 aromatic); MS m/z (%): 409 (M+, 24),
19.00.
392 (87), 260 (22), 218 (62), 176 (71). Anal. Calcd (%) for
C
19H15N5O4S: C, 55.74; H, 3.69; N, 17.11; S, 7.83. Found: C, 55.56; H,
2.1.2.10. 6-Methyl-5-N-(5,6-dimethyl-2-benzothiazolyl)carboxamide-4-
(4-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one (21). Yield: 75%, mp:
246–250 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.89 (br. s, 1H, NH-
amide), 9.22 (br. s, 1H, N1H-DHPM), 8.22 (d, 2H, J = 8.8 Hz, CH-
phenyl), 7.95 (br. s, 1H, N3H-DHPM), 7.64 (s, 1H, C4ʹH-benzothiazol),
7.57 (d, 2H, J = 8.8 Hz, CH-phenyl), 7.48 (s, 1H, C7ʹH-benzothiazol),
5.70 (d, 1H, J = 2.8 Hz, C4H-DHPM), 2.31 (s, 3H, CH3-benzothiazol),
2.29 (s, 3H, CH3-benzothiazol), 2.23 (s, 3H, CH3-DHPM); 13C NMR
(300 MHz, DMSO-d6) δ 16.6, 18.3, 18.5, 52.5, 100.5, 109.9, 116.9,
120.3, 122.8, 126.6, 131.1, 133.6, 136.3, 143.9, 145.7, 150.1, 150.9,
3.67; N, 17.08; S, 7.80.
2.1.2.6. 6-Methyl-5-N-(5-bromopyridin-2-yl)carboxamide-4-(4-nitro-
phenyl)-3,4-dihydropyrimidin-2(1H)-one
(17). Yield
35%;
mp
283–286 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.36 (br. s, 1H,
NH-amide), 9.06 (br. s, 1H, N1H-DHPM), 8.46 (d, 1H, J = 2.0 Hz, C6ʹH-
bromopyridin), 8.27 (d, 2H, J = 8.8 Hz, CH-phenyl), 7.99 (dd, 1H, J =
8.8, 2 Hz, C4ʹH-bromopyridin), 7.96 (d, 1H, J = 8.8, C3ʹH-bromopyr-
idin), 7.85 (br. s, 1H, N3H-DHPM), 7.60 (d, 2H, J = 8.8 Hz, CH-phenyl),
5.60 (s, 1H, C4H-DHPM), 2.17 (s, 3H, CH3-DHPM); IR (KBr)
ν
cmꢀ 1
:
156.5, 164.0; IR (KBr)
ν (cm-1): 3294.6, 3205.5 (N–H, DHPM and
–
–
–
3219.8, 3100.7 (N–H), 2960.9 (C–H, aliphatic), 1698.2 (C O, DHPM),
amide), 3075.7 (C–H,aromatic) 2919.9 (C–H, aliphatic), 1683.1 (C O,
–
–
–
–
–
–
–
1673.7 (C O, amide), 1562.4 (C C, alkene), 1516.5, 1349.6 (C–NO
DHPM), 1668.5 (C O, amide), 1616.1 1454.1 (C C, alkene), 1519.8,
–
–
2
aromatic); MS m/z (%): 433 (M+2, 13), 431 (M+, 13), 414 (100), 384
(20), 260 (29), 218 (54). Anal. Calcd (%) for C17H14BrN5O4: C, 47.24; H,
3.26; N, 16.20. Found: C, 47.07; H, 3.27; N, 16.13.
1345.1 (C–NO2 aromatic); MS m/z (%): 437 (M+, 9), 420 (21), 178
(100), 163 (46). Anal. Calcd (%) for C21H19N5O4S: C, 57.66; H, 4.38; N,
16.01; S, 7.33. Found: C, 54.27; H, 3.63; N, 16.57; S, 6.23.
2.1.2.7. 6-
Methyl-5-N-(6-ethoxy-2-benzothiazolyl)carboxamide-4-(4-
2.1.2.11. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(3-nitro-
nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one (18). Yield: 78%, mp:
282–287 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.87 (br. s, 1H, NH-
amide), 9.24 (br. s, 1H, N1H-DHPM), 8.23 (d, 2H, J = 8.8 Hz, CH-
phenyl), 7.97 (br. s, 1H, N3H-DHPM), 7.57 (m, 3H, CH-phenyl and
C4ʹH-benzothiazole), 7.49 (d, 1H, J = 2.5, C7ʹH-benzothiazole), 6.99
(dd, 1H, J = 8.8, 2.5 Hz, C5ʹH-benzothiazole), 5.70 (d, 1H, J = 2.8 Hz,
C4H-DHPM), 4.04 (q, 2H, J = 6.9 Hz, -OCH2CH3), 2.12 (s, 3H, CH3-
DHPM), 1.33 (t, 3H, J = 6.9 Hz, -OCH2CH3); 13C NMR (300 MHz,
DMSO-d6) δ 15.1, 18.1, 54.2, 64.1, 101.9, 105.7, 115.7, 121.6, 124.4,
128.2, 133.7, 142.6, 145.4, 147.3, 151.6, 152.4, 155.7, 158.9, 162.8; IR
phenyl)-3,4-dihydropyrimidin-2(1H)-one
(22). Yield
53%;
mp
232–238 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.00 (br. s, 1H,
NH-amide), 9.29 (br. s, 1H, N1H-DHPM), 8.20 (m, 1H, C2H-phenyl),
8.14 (m, 1H, C4H-phenyl), 7.99 (br. s, 1H, N3H-DHPM), 7.91 (d, 1H,
J = 7.8 Hz, C4ʹH-benzothiazol), 7.75 (d, 1H, J = 7.6 Hz, C6H-phenyl),
7.66 (m, 2H, C5H-phenyl and C7ʹH-benzothiazol), 7.41 (t, 1H, J =
8.0 Hz, C5ʹH-benzothiazol), 7.28 (t, 1H, J = 8.0 Hz, C6ʹH-benzothiazol),
5.74 (s, 1H, C4H-DHPM), 2.27 (s, 3H, CH3-DHPM); IR (KBr)
ν
cmꢀ 1
:
3469.1, 3217.7 (DHPM and amide), 3118.7 (CH-aromatic) 2968.5 (C–H,
–
–
–
aliphatic), 1685.7 (C O, DHPM), 1662.5 (C O, amide), 1590.5,
–
–
(KBr)
ν
(cm-1): 3226.3, 3108.4 (N–H, DHPM and amide), 2927.1 (C–H,
1466.4 (C C, alkene), 1520.1, 1348.6 (C–NO aromatic); MS m/z (%):
–
2
aliphatic), 1695.0 (C O, DHPM), 1675.1 (C O, amide), 1551.4 (C C,
409 (M+, 8), 392 (42), 218 (33), 176 (54), 150 (100). Anal. Calcd (%) for
–
–
–
–
–
–
alkene), 1521.5, 1347.1 (C–NO2 aromatic); MS m/z (%): 453 (M+, 9),
436 (13), 218 (42), 194 (100), 165 (67), 111 (38). Anal. Calcd (%) for
C21H19N5O5S: C, 55.62; H, 4.22; N, 15.44; O, 17.64; S, 7.07. Found: C,
51.65; H, 4.07; N, 16.27; S, 6.87.
C19H15N5O4S: C, 55.74; H, 3.69; N, 17.11; S, 7.83. Found: C, 55.54; H,
3.68; N, 17.04; S, 7.85.
2.1.2.12. 6-Methyl-5-N-(5,6-dimethyl-2-benzothiazolyl)carboxamide-4-
(3-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one (23). Yield 32%; mp
300–304 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.89 (br. s, 1H,
NH-amide), 9.24 (br. s, 1H, N1H-DHPM), 8.19 (m, 1H, C2H-phenyl),
8.12 (m, 1H, J = 8 Hz, C4H-phenyl), 7.96 (br. s, 1H, N3H-DHPM),
7.75 (d, 1H, J = 7.6 Hz, C6H-phenyl), 7.65 (m, 2H, C5H-phenyl and
C4ʹH-benzothiazol), 7.47 (s, 1H, C7ʹH-benzothiazol), 5.72 (d, 1H, J =
2.8, C4H-DHPM), 2.29 (s, 3H, CH3-benzothiazol), 2.28 (s, 3H, CH3-
2.1.2.8. 6-Methyl-5-N-(6-ethoxy-2-benzothiazolyl)carboxamide-4-(4-
nitrophenyl)-3,4-dihydropyrimidin-2(1H)-thione (19). Yield: 35%, mp:
296–301 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 12.12 (br. s, 1H, NH-
amide), 10.39 (br. s, 1H, N1H-DHPM), 9.78 (br. s, 1H, N3H-DHPM),
8.25 (d, 2H, J = 8.8 Hz, CH-phenyl), 7.54 (m, 3H, CH-phenyl and
C4ʹH-benzothiazole), 7.48 (d, 1H, J = 2.4 Hz, C7ʹH-benzothiazole), 7.00
(dd, 1H, J = 8.8, 2.4 Hz, C5ʹH-benzothiazole), 5.72 (d, 1H, J = 2.8 Hz,
C4H-DHPM), 4.04 (q, 2H, J = 6.8 Hz, -OCH2CH3), 2.26 (s, 3H, CH3-
benzothiazol), 2.25 (s, 3H, CH3-DHPM); IR (KBr)
ν
cmꢀ 1: 3355.4, 3218.1
–
–
(N–H), 3089.1 (CH-aromatic) 2920.3 (C–H, aliphatic), 1697.3 (C O,
–
–
–
DHPM), 1.33 (t, 3H, J = 6.8 Hz, -OCH2CH3); IR (KBr)
ν
(cm-1):
DHPM), 1672.4 (C O, amide), 1629.7, 1457.4 (C C, alkene), 1534.3,
–
3186.2 (N–H, DHPM and amide), 2977.1 (C–H, aliphatic), 1659.6 (C S,
1349.3 (C–NO2 aromatic); MS m/z (%): 437 (M+, 17), 420 (62), 260 (8),
178 (100). Anal. Calcd (%) for C21H19N5O4S: C, 57.66; H, 4.38; N, 16.01;
S, 7.33. Found: C, 57.45; H, 4.36; N, 15.95; S, 7.30.
–
–
–
–
–
–
DHPM), 1605.3 (C O, amide), 1541.1 (C C, alkene), 1523.4, 1347.1
(C–NO2 aromatic); MS m/z (%): 469 (M+, 13), 452 (13), 249 (18), 220
(13), 194 (100), 165 (54). Anal. Calcd (%) for C21H19N5O4S2: C, 53.72;
H, 4.08; N, 14.92; S, 13.66. Found: C, 48.36; H, 4.11; N, 15.73; S, 12.75.
2.1.2.13. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(2-nitro-
phenyl)-3,4-dihydropyrimidin-2(1H)-one
(24). Yield
35%;
mp
2.1.2.9. 6-Methyl-5-N-(4-methyl-2-pyridinyl)carboxamide-4-(4-nitro-
264–270 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.00 (br. s, 1H,
NH-amide), 9.26 (br. s, 1H, N1H-DHPM), 7.89–7.93 (m, 2H, C4ʹH-
nenzothiazol and C3H-phenyl), 7.70 (t, 1H, J = 7.6 Hz, C5H-phenyl),
phenyl)-3,4-dihydropyrimidin-2(1H)-one
(20). Yield
41%;
mp
250–253 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.00 (br. s, 1H,
4

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
7.62–7.74 (m, 2H, C7ʹH-benzothiazol and C6H-phenyl),7.50–7.56 (m,
2H, N3H-DHPM and C4H-phenyl), 7.40 (t, 1H, J = 7.6 Hz, C5ʹH-ben-
zothiazol), 7.27 (t, 1H, J = 7.6 Hz, C6ʹH-benzothiazol), 6.10 (s, C4H-
NH-amide), 9.02 (br. s, 1H, N1H-DHPM), 7.69 (br. s, 1H, N3H-DHPM),
7.64 (s, 1H, C4ʹH-benzothiazol), 7.49 (s, 1H, C7ʹH-benzothiazol), 7.22
(d, 2H, J = 8.8 Hz, CH-phenyl), 6.88 (d, 2H, J = 8.8 Hz, CH-phenyl),
5.56 (d, 1H, J = 2.8 Hz, C4H-DHPM), 3.69 (s, 3H, –OCH3), 2.31 (s,
3H, CH3-benzothiazol), 2.29 (s, 3H, CH3-benzothiazol), 2.19 (s, 3H,
DHPM), 2.20 (s, 3H, CH3-DHPM); IR (KBr)
ν
cmꢀ 1: 3308.7, 3123.6
–
–
–
(N–H), 2926.7 (C–H, aliphatic), 1707.1 (C O, DHPM), 1678.6 (C O,
–
amide), 1614.1, 1440.6 (C C, alkene), 1543.7, 1343.9 (C–NO aro-
CH3-DHPM); IR (KBr) ν
cmꢀ 1: 3424.6, 3244.3 (N–H), 3070.1 (C–H, ar-
–
–
2
matic); MS m/z (%): 409 (M+, 8), 260 (8), 242 (10), 215 (67), 198 (29),
177 (100), 150 (50). Anal. Calcd (%) for C19H15N5O4S: C, 55.74; H, 3.69;
N, 17.11; S, 7.83. Found: C, 55.60; H, 3.67; N, 17.04; S, 7.80.
omatic), 2920.7 (C–H, aliphatic), 1686.0 (C O, DHPM), 1672.5 (C O,
–
–
–
–
–
–
amide), 1622.9, 1456.9 (C C, alkene), 1028.5, 1249.3 (C–O, aromatic);
MS m/z (%): 422 (M+, 54), 245 (46), 215 (33), 178 (96), 162 (100).
Anal. Calcd (%) for C22H22N4O3S: C, 62.54; H, 5.25; N, 13.26; S, 7.59.
Found: C, 62.79; H, 5.23; N, 13.23; S, 7.62.
2.1.2.14. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(4-methox-
yphenyl)-3,4-dihydropyrimidin-2(1H)-one
(25). Yield: 67%, mp:
295–296 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.86 (br. s, 1H, NH-
amide), 9.08 (br. s, 1H, N1H-DHPM), 7.92 (d, 1H, J = 7.6 Hz, C4ʹH-
benzothiazol), 7.74 (br. s, 1H, N3H-DHPM), 7.70 (d, 1H, J = 8.0 Hz,
C7ʹH-benzothiazol), 7.41 (t, 1H, J = 7.6 Hz, C5ʹH-benzothiazol), 7.28 (t,
1H, J = 7.6 Hz, C6ʹH-benzothiazol), 7.22 (d, 2H, J = 8.8 Hz, CH-phenyl),
6.88 (d, 2H, J = 8.8 Hz, CH-phenyl), 5.57 (s, C4H-DHPM), 3.69 (s, 3H,
-OCH3), 2.19 (s, 3H, CH3-DHPM); 13C NMR (300 MHz, DMSO-d6) δ
18.1, 53.8, 55.5, 102.5, 114.3, 120.7, 122.0, 123.8, 126.5, 128.1, 131.8,
2.1.2.18. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(2-naphthyl)-
3,4-dihydropyrimidin 2(1H)-one (29). Yield: 51%, Rf = 0.44 (EtOAc/PE
3:1), mp: 294–298 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.97 (br. s, 1H,
NH- amide), 9.16 (br. s, 1H, N1H-DHPM), 7.85–7.92 (m, 5H, C4ʹH-
benzothiazol and CH-naphthalenyl and N3H-DHPM), 7.75 (s, 1H, CH-
naphthalenyl), 7.69 (d, 1H, J = 8.0 Hz, C7ʹHbenzothiazol), 7.47–7.5
(m, 3H, CH-naphthalenyl), 7.40 (t, 1H, J = 7.6, C5ʹHbenzothiazol), 7.26
(t, 1H, J = 7.6, C6ʹH-benzothiazol), 5.78 (s, 1H, C4H-DHPM), 2.24 (s,
3H, CH3-DHPM); 13C NMR (300 MHz, DMSO-d6) δ 18.1, 56.0, 103.7,
110.8, 119.7, 122.0, 123.8, 125.1, 125.5, 126.5, 126.8, 127.9, 128.3,
128.9, 131.3, 132.9, 133.2, 141.8, 144.8, 148.7, 151.4, 159.2, 165.6; IR
136.6, 144.8, 148.9, 152.4, 158.2, 159.1, 165.8; IR (KBr)
ν (cm-1):
3444.9, 3252.2 (N–H, DHPM and amide), 3067.1 (C–H, aromatic),
–
–
–
2930.2 (C–H, aliphatic), 1682.5 (C O, DHPM), 1638.1 (C O, amide),
–
–
–
1598.5, 1444.2 (C C, alkene), 1030.5, 1238.1 (C–O, aromatic); MS m/z
(KBr)
ν (cm-1): 3438.4, 3242.5 (N–H, DHPM and amide), 3056.3 (C–H,
(%): 394 (M+, 50), 245 (50), 215 (46), 162 (100). Anal. Calcd (%) for
aromatic), 2962.3 (C–H, aliphatic), 1682.8 (C O, DHPM), 1634.9
–
–
+
–
–
–
C
20H18N4O3S: C, 60.90; H, 4.60; N, 14.20; S, 8.13. Found: C, 54.86; H,
(C O, amide), 1597.7, 1442.5 (C C, alkene); MS m/z (%): 414 (M ,
–
5.01; N, 13.85; S, 8.93.
29), 264 (100), 235 (50), 182 (83), 151 (29). Anal. Calcd (%) for
C23H18N4O2S: C, 66.65; H, 4.38; N, 13.52; S, 7.74. Found: C, 62.58; H,
4.07; N, 12.88; S, 8.16.
2.1.2.15. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(4-methox-
yphenyl)-3,4-dihydropyrimidin-2(1H)-thione (26). Yield: 74%, mp:
285–290 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 12.12 (br. s, 1H, NH-
amide), 10.23 (br. s, 1H, N1H-DHPM), 9.62 (br. s, 1H, N3H-DHPM),
7.93 (d, 1H, J = 7.6 Hz, C4ʹHbenzothiazol), 7.72 (d, 1H, J = 8.0 Hz,
C7ʹH-benzothiazol), 7.42 (t, 1H, J = 7.6 Hz, C5ʹH-benzothiazol), 7.29 (t,
1H, J = 7.6 Hz, C6ʹH-benzothiazol), 7.20 (d, 2H, J = 8.4 Hz, CH-phenyl),
6.91 (d, 2H, J = 8.4 Hz, CH-phenyl), 5.58 (s, C4H-DHPM), 3.70 (s, 3H,
-OCH3), 2.21 (s, 3H, CH3-DHPM); 13C NMR (300 MHz, DMSO-d6) δ
17.4, 54.1, 55.5, 104.5, 114.5, 120.6, 122.1, 123.9, 126.6, 128.3, 131.7,
2.1.2.19. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(2-naphthyl)-
3,4-dihydropyrimidin-2(1H)-thione (30). Yield: 45%, mp: 248–251 ◦C;
1H NMR (400 MHz, DMSO-d6) δ: 12.21 (br. s, 1H, NH-amide), 10.32 (br.
s, 1H, N1H-DHPM), 9.76 (br. s, 1H, N3H-DHPM), 7.86–7.95 (m, 4H,
C4ʹHbenzothiazol and CH-naphthalenyl), 7.70–7.74 (m, 2H, CH-
naphthalenyl and C7ʹH-benzothiazol), 7.47–7.51 (m, 3H, CH-
naphthalenyl), 7.40 (t, 1H, J = 7.6, C5ʹH-benzothiazol), 7.26 (t, 1H, J
= 7.6, C6ʹH-benzothiazol), 5.79 (s, 1H, C4HDHPM), 2.67 (s, 3H, CH3-
135.5, 141.0, 150.2, 157.1, 159.4, 162.8, 166.4; IR (KBr)
ν
(cm-1):
DHPM); IR (KBr)
ν (cm-1): 3388.1, 3226.5 (DHPM and amide), 3106.4
–
3447.7, 3151.4 (N–H, DHPM and amide), 3107.1 (C–H, aromatic),
(C–H, aromatic), 2949.1 (C–H, aliphatic), 1681.8 (C S, DHPM), 1646.4
–
+
–
–
–
–
–
2958.2 (C–H, aliphatic), 1681.5 (C S, DHPM), 1657.5 (C O, amide),
(C O, amide), 1575.5, 1455.6 (C C, alkene); MS m/z (%): 430 (M ,
–
–
–
–
–
1584.2, 1441.9 (C C, alkene), 1066.9, 1246.3 (C–O, aromatic); MS m/z
67), 280 (100), 215 (54), 222 (42), 181 (63), 151 (67), 127 (33). Anal.
Calcd (%) for C23H18N4OS2: C, 64.16; H, 4.21; N, 13.01; S, 14.90. Found:
C, 60.53; H, 4.12; N, 12.76; S, 15.19.
(%): 410 (M+, 42), 261 (21), 234 (100), 202 (17), 174 (71), 150 (33) 127
(25). Anal. Calcd (%) for C20H18N4O2S2: C, 58.52; H, 4.42; N, 13.65; S,
15.62. Found: C, 63.76; H, 3.95; N, 12.31; S, 16.10.
2.1.2.20. 6-Methyl-5-N-(6-ethoxy-2-benzothiazolyl)carboxamide-4-(2-
naphthyl)-3,4-dihydropyrimidin-2(1H)-one (31). Yield: 68%, mp:
242–244 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.83 (br. s, 1H, NH-
amide), 9.13 (br. s, 1H, N1H-DHPM), 7.85–7.92 (m, 4H, CH-
naphthalenyl and N3H-DHPM), 7.75 (s, 1H, CH-naphthalenyl), 7.56
(d, 1H, J = 8.4 Hz, C4ʹH-benzothiazol), 7.46–7.51 (m, 4H, C7ʹH-ben-
zothiazol and CH-naphthalenyl), 6.98 (dd, 1H, J = 8.4, 2.4, C5ʹH-ben-
zothiazol), 5.75 (d, 1H, J = 2.4 Hz, C4H-DHPM), 4.03 (q, 2H, J = 6.8 Hz,
-OCH2CH3), 2.23 (s, 3H, CH3-DHPM), 1.33 (t, 3H, J = 6.8 Hz,
-OCH2CH3); 13C NMR (300 MHz, DMSO-d6) δ 14.4, 18.1, 54.8, 63.6,
102.9, 106.1, 115.6, 121.2, 125.1, 125.5, 126.5, 126.8, 127.4, 127.9,
128.3, 128.9, 129.8, 132.9, 133.2, 141.8, 143.8, 153.3, 155.7, 163.5,
2.1.2.16. 6-Methyl-5-N-(6-ethoxy-2-benzothiazolyl)carboxamide-4-(4-
methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (27). Yield 58%; mp
250–255 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.73 (br. s, 1H,
NH-amide), 9.04 (br. s, 1H, N1H-DHPM), 7.71 (br. s, 1H, N3H-DHPM),
7.58 (d, 1H, J = 8.8 Hz, C4ʹH-benzothiazol), 7.48 (d, 1H, J = 2.2 Hz,
C7ʹH-benzothiazol), 7.21 (d, 2H, J = 8.8 Hz, CH-phenyl), 6.98 (dd, 1H, J
= 8.8, 2.2 Hz, C5ʹH-benzothiazol), 6.88 (d, 2H, J = 8.8 Hz, CH-phenyl),
5.55 (d, 1H, J = 2.4 Hz, C4H-DHPM), 4.05 (q, 2H, J = 6.8 Hz,
–OCH2CH3), 3.69 (s, 3H, –OCH3), 2.19 (s, 3H, CH3-DHPM), 1.34 (t, 3H,
J = 6.8 Hz, –OCH2CH3); IR (KBr)
ν
cmꢀ 1: 3445.1, 3246.7 (N–H), 3032.1
–
(C–H, aromatic), 2928.7 (C–H, aliphatic), 1681.9 (C O, DHPM),
–
–
–
–
–
1638.2 (C O, amide), 1607.3, 14601.2 (C C, alkene), 1058.5, 1255.9
167.1; IR (KBr)
ν (cm-1): 3435.2, 3211.5 (N–H, DHPM and amide),
(C–O, aromatic); MS m/z (%): 438 (M+, 42), 245 (25), 215 (17), 194
(100), 162 (58). Anal. Calcd (%) for C22H22N4O4S: C, 60.26; H, 5.06; N,
12.78; S, 7.31. Found: C, 60.06; H, 5.05; N, 12.73; S, 7.30.
3078.8 (C–H, aromatic), 2921.6 (C–H, aliphatic), 1677.1 (C O,
–
–
–
–
–
DHPM), 1636.2 (C O, amide), 1605.5, 1461.4 (C C, alkene), 1091.4,
–
1258.2 (C–O, aromatic); MS m/z (%): 458 (M+, 19), 264 (25), 194
(100), 165 (21). Anal. Calcd (%) for C25H22N4O3S: C, 65.48; H, 4.84; N,
12.22; S, 6.99. Found: C, 68.27; H, 4.66; N, 11.57; S, 7.24.
2.1.2.17. 6-Methyl-5-N-(5,6-dimethyl-2-benzothiazolyl)carboxamide-4-
(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (28). Yield 71%; mp
228–232 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (br. s, 1H,
5

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
2.1.2.21. 6-Methyl-5-N-(4-chlorophenyl)carboxamide-4-(2-naphthyl)-
3,4-dihydropyrimidin-2(1H)-one (32). Yield: 57%, mp: 269–272 ◦C; 1H
NMR (400 MHz, DMSO-d6) δ: 9.76 (br. s, 1H, NH-amide), 8.87 (br. s, 1H,
N1H-DHPM), 7.91–7.97 (m, 4H, CH-naphthalenyl and N3H-DHPM),
7.77 (s, 1H, CH-naphthalenyl), 7.62 (d, 2H, J = 8.8 Hz, CH-phenyl),
7.52–7.56 (m, 3H, CH-naphthalenyl), 7.34 (d, 2H, J = 8.8, CH-
phenyl), 5.63 (d, 1H, J = 2.0 Hz, C4H-DHPM), 2.13 (s, 3H, CH3-
DHPM); 13C NMR (300 MHz, DMSO-d6) δ 17.2, 56.4, 105.5, 121.5,
124.9, 125.4, 126.4, 126.8, 127.1, 127.9, 128.3, 128.9, 132.9, 133.2,
C17H14N4O2S2: C, 55.12; H, 3.81; N, 15.12; S, 17.31. Found: C, 55.34; H,
3.78; N, 15.06; S, 17.25.
2.1.2.25. 6-Methyl-5-N-(6-ethoxy-2-benzothiazolyl)carboxamide-4-(thio-
phen-2-yl)-3,4-dihydropyrimidin-2(1H)-one (36). Yield 66%; mp
244–248 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.79 (br. s, 1H,
NH-amide), 9.21 (br. s, 1H, N1H-DHPM), 7.98 (br. s, 1H, N3H-DHPM),
7.57 (d, 1H, J = 8.8 Hz, C4ʹH-benzothiazole), 7.49 (d, 1H, J = 2.5 Hz,
C7ʹH-benzothiazole), 7.38 (dd, 1H, J = 4.8, 1.2 Hz, C5H-thiophen), 7.00
(dd, 1H, J = 8.8, 2.5 Hz, C5ʹH-benzothiazole), 6.93–6.96 (m, 2H, C3H
and C4H-thiophen), 5.91 (d, 1H, J = 3.2 Hz, C4H-DHPM), 4.06 (q, 2H, J
= 6.8 Hz, –OCH2CH3), 2.22 (s, 3H, CH3-DHPM), 1.34 (t, 2H, J = 6.8 Hz,
138.6, 139.4, 139.8, 142.1, 152.9, 165.9; IR (KBr)
ν (cm-1): 3403.3,
3271.8 (N–H, DHPM and amide), 3114.9 (C–H, aromatic), 2925.7 (C–H,
–
–
–
aliphatic), 1700.4 (C O, DHPM), 1670.1 (C O, amide), 1628.3,
–
+
–OCH2CH3); IR (KBr)
ν
cmꢀ 1: 3216.5 (N–H), 3072.7 (C–H, aromatic),
–
–
1457.4 (C C, alkene); MS m/z (%): 391(M , 13), 265 (100), 222 (18),
–
–
–
182 (46), 127 (21). Anal. Calcd (%) for C22H18ClN3O2: C, 67.43; H, 4.63;
N, 10.72. Found: C, 69.66; H, 4.29; N, 11.65.
2924.4 (C–H, aliphatic), 1681.5 (C O, DHPM), 1652.9 (C O, amide),
–
+
–
1607.1, 1463.1 (C C, alkene); MS m/z (%): 414 (M , 33), 221 (17), 194
–
(100). Anal. Calcd (%) for C19H18N4O3S2: C, 55.05; H, 4.38; N, 13.52; S,
2.1.2.22. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(4-acetox-
yphenyl)-3,4 dihydropyrimidin-2(1H)-one (33). Yield: 41%, mp:
289–291 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.93 (br. s, 1H, NH-
amide), 9.13 (br. s, 1H, N1H-DHPM), 7.92 (d, 1H, J = 7.2 Hz, C4ʹH-
benzothiazole), 7.82 (br. s, 1H, N3HDHPM), 7.71 (d, 1H, J = 7.2 Hz,
C7ʹH-benzothiazole), 7.42 (t, 1H, J = 7.6 Hz, C5ʹH-benzothiazole), 7.32
(d, 2H, J = 8.8 Hz, CH-phenyl), 7.28 (t, 1H, J = 7.6 Hz, C6ʹH-benzo-
thiazole), 7.09 (d, 2H, J = 8.8 Hz, CH-phenyl), 5.62 (d, 1H, J = 2.8 Hz,
C4H-DHPM), 2.23 (s, 3H, CH3-acetoxyphenyl), 2.21 (s, 3H, CH3-
DHPM); 13C NMR (300 MHz, DMSO-d6) δ 16.3, 22.7, 52.2, 102.0,
120.3, 122.0, 122.4, 123.8, 126.5, 127.9, 130.7, 142.0, 145.1, 148.9,
15.47. Found: C, 54.83; H, 4.36; N, 13.47; S, 15.41.
2.1.2.26. 6-Methyl-5-N-(5,6-dimethyl-2-benzothiazolyl)carboxamide-4-
(thiophen-2-yl)-3,4-dihydropyrimidin-2(1H)-one (37). Yield 44%; mp
295–297 ◦C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.83 (br. s, 1H,
NH-amide), 9.22 (br. s, 1H, N1H-DHPM), 7.99 (br. s, 1H, N3H-DHPM),
7.94 (s, 1H, C4ʹH-benzothiazole), 7.46 (s, 1H, C7ʹH-benzothiazole), 7.35
(d, 1H, J = 4.8 Hz, C5H-thiophen), 6.93–6.97 (m, 2H, C3H and C4H-
thiophen), 5.92 (d, 1H, J = 2.8 Hz, C4H-DHPM), 3.31 (s, 3H, CH3-
benzothiazol), 2.29 (s, 3H, CH3-benzothiazol), 2.23 (s, 3H, CH3-DHPM);
IR (KBr)
ν
cmꢀ 1: 3351.3, 3213.1 (N–H), 3083.3 (C–H, aromatic), 2962.4
–
–
–
150.2, 152.1, 158.7, 164.9, 168.7; IR (KBr)
ν (cm-1): 3441.1, 3245.0
(C–H, aliphatic), 1696.3 (C O, DHPM), 1673.9 (C O, amide), 1632.9,
–
+
–
–
(N–H, DHPM and amide), 3061.0 (C–H, aromatic), 2927.5 (C–H,
1434.3 (C C, alkene); MS m/z (%): 398 (M ,42), 221 (25), 178 (100).
–
–
–
–
aliphatic), 1758.7 (C O, ester), 1681.9 (C O, DHPM), 1639.6 (C O,
–
–
Anal. Calcd (%) for C19H18N4O2S2: C, 57.27; H, 4.55; N, 14.06; S, 16.09.
–
–
amide), 1599.4, 1444.4 (C C, alkene), 1015.5, 1250.6 (CO, aromatic);
Found: C, 57.05; H, 4.53; N, 14.01; S, 16.02.
MS m/z (%): 422 (M+, 75), 273 (75), 231 (54), 201 (33), 148 (100), 43
(38). Anal. Calcd (%) for C21H18N4O4S: C, 59.70; H, 4.29; N, 13.26; S,
7.59. Found: C, 56.83; H, 4.16; N, 13.91; S, 7.34.
2.1.2.27. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(isopropyl)-3,4-
dihydropyrimidin-2(1H)-one (38). Yield 44%; mp 263–268 ◦C; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 11.96 (br. s, 1H, NH-amide), 8.77 (br. s,
1H, N1H-DHPM), 7.95 (d, 1H, J = 7.6 Hz, C4ʹH-benzothiazole), 7.71 (d,
1H, J = 7.6 Hz, C7ʹH-benzothiazole), 7.42 (t, 1H, J = 7.6 Hz, C5ʹH-
benzothiazole), 7.27–7.31 (m, 2H, C6ʹH-benzothiazole and N3H-
DHPM), 4.34 (t, 1H, J = 3.2 Hz, C4H-DHPM), 2.10 (s, 3H, CH3-
DHPM), 1.63 (m, 1H, CH-isopropyl), 0.85 (d, 3H, J = 6.8 Hz, CH3-iso-
2.1.2.23. 6-Methyl-5-N-(5,6-dimethyl-2-benzothiazolyl)carboxamide-4-
(4-acetoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (34). Yield: 63%,
mp: 278–282 ◦C; 1H NMR (400 MHz, DMSO-d6) δ: 11.82 (br. s, 1H, NH-
amide), 9.08 (br. s, 1H, N1H-DHPM), 7.79 (br. s, 1H, N3H-DHPM), 7.65
(s, 1H, C4ʹH-benzothiazole), 7.48 (s, 1H, C7ʹH-benzothiazole), 7.32 (d,
2H, J = 8.4 Hz, CH-phenyl), 7.09 (d, 2H, J = 8.4 Hz, CH-phenyl), 5.59
(d, 1H, J = 2.8 Hz, C4H-DHPM), 3.11 (s, 3H, CH3-benzothiazol), 2.29 (s,
3H, CH3-benzothiazol), 2.23 (s, 3H, CH3-acetoxyphenyl), 2.20 (s, 3H,
CH3-DHPM); 13C NMR (300 MHz, DMSO-d6) δ 18.1, 19.9, 20.2, 21.2,
53.9, 102.6, 120.8, 121.8, 122.3, 127.9, 129.5, 132.6, 135.1, 142.0,
propyl), 0.77 (d, 3H, J = 6.8 Hz, CH3-isopropyl); IR (KBr)
ν
cmꢀ 1
:
3446.7, 3209.1 (N–H), 3029.1 (C–H, aromatic), 2959.2 (C–H, aliphatic),
–
–
–
–
–
1679.1 (C O, DHPM), 1633.8 (C O, amide), 1598.4, 1444.8 (C C,
–
alkene); MS m/z (%): 330 (M+, 8), 287 (100), 181 (29), 151 (92). Anal.
Calcd (%) for C16H18N4O2S: C, 58.16; H, 5.49; N, 16.96; S, 9.70. Found:
C, 57.92; H, 5.46; N, 16.90; S, 9.66.
144.7, 147.4, 150.2, 152.6, 158.2, 165.9, 169.64; IR (KBr)
ν (cm-1):
3223.8 (DHPM and amide), 3061.0 (C–H, aromatic), 2919.9 (C–H,
–
–
–
aliphatic), 1756.2 (C O, ester), 1672.2 (C O, DHPM and amide),
–
–
–
2.2. Biological assays
1548.7, 1453.9 (C C, alkene), 1031.2, 1252.9 (C–O, aromatic); MS m/z
(%): 450 (42) [M+], 273 (21), 178 (100), 148 (33), 43 (13). Anal. Calcd
(%) for C23H22N4O4S: C, 61.32; H, 4.92; N, 12.44; S, 7.12. Found: C,
62.53; H, 4.80; N, 11.96; S, 7.33.
DMEM, RPMI 1640 media, FBS (fetal bovine serum), penicillin and
streptomycin were purchased from Biosera (Austria). MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], doxorubicin
and cisplatin were purchased from EBEWE Pharma (Unterach am
Attersee, Austria).
2.1.2.24. 6-Methyl-5-N-(2-benzothiazolyl)carboxamide-4-(thiophen-2-yl)-
3,4-dihydropyrimidin-2(1H)-one (35). Yield 49%; mp 259–263 ◦C; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.94 (br. s, 1H, NH-amide), 9.27
(br. s, 1H, N1H-DHPM), 8.02 (br. s, 1H, N3H-DHPM), 7.93 (d, 1H, J =
7.6 Hz, C4ʹH-benzothiazole), 7.71 (d, 1H, J = 7.9 Hz, C7ʹH-benzothia-
zole), 7.42 (t, 1H, J = 7.6 Hz, C5ʹH-benzothiazole), 7.38 (d, 1H, J = 4.4
Hz, C5H-thiophen), 7.29 (t, 1H, J = 7.8 Hz, C6ʹH-benzothiazole),
6.92–6.96 (m, 2H, C3H and C4H -thiophen), 5.93 (s, 1H, C4H-DHPM),
MCF-7 (breast adenocarcinoma), HT-29 (colorectal adenocarci-
noma), NIH/3T3 (mouse embryonic fibroblast) and MKN-45 cells
(gastric adenocarcinoma cells with MET gene amplification) were ob-
tained from Iranian Biological Resource center, Tehran, Iran. MOLT-4
(acute lymphoblastic leukemia) cells were purchased from National
Cell Bank of Iran, Pasteur Institute, Tehran, Iran. EBC-1 cells (lung
squamous cell carcinoma with MET gene amplification) were obtained
from Japanese Collection of Research Bio Resources Cell Bank. HT-29
and NIH/3T3 cells were grown in DMEM medium supplemented with
20% and 10% FBS, respectively. All other cell lines were cultured in
RPMI 1640 medium supplemented with 10% FBS, with the exception of
2.22 (s, 3H, CH3-DHPM); IR (KBr)
ν
cmꢀ 1: 3436.6, 3281.4 (N–H),
–
3061.5 (C–H, aromatic), 2953.8 (C–H, aliphatic), 1683.2 (C O,
–
–
–
–
DHPM), 1636.8 (C O, amide), 1612.4, 1444.8 (C C, alkene); MS m/z
–
(%): 370 (M+, 62), 221 (92), 192 (50), 138 (100). Anal. Calcd (%) for
6

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
MKN-45 cells, which needed 20% FBS for optimal growth. All cells were
cultured at 37 ◦C in humidified air containing 5% CO2 and their media
2.2.3. Flow cytometric analysis of cell cycle
To explore the distribution of cells in different phases of cell cycle
after treatment with the most potent antiproliferative compounds (15,
21, 23, 31 and 37), we performed flow cytometric analysis by RNase/
propidum iodide assay (Gholampour et al., 2019). MCF-7 cells were
seeded in 6-well microplates (2 × 105 cells per well) and incubated at
contained 100 U/ml Penicillin-G and 100 μg/ml streptomycin.
2.2.1. Assessment of antiproliferative effect by MTT reduction assay
Antiproliferative activities of the test compounds were evaluated by
MTT assay (Kayani, Firuzi and Bordbar, 2018; Shekari et al., 2015). In
this assay, reduction of yellow MTT by cellular mitochondrial dehy-
drogenase enzymes to the purple formazan is determined as an index of
cell viability. Cells were seeded into 96-well plates (3–5 × 103 cells/-
well) and incubated for 24 h at 37 ◦C. Afterwards, 3–4 different con-
centrations of DHPM derivatives, cisplatin and doxorubicin were added
to wells in duplicates or triplicates. The plates were incubated at 37 ◦C
for another 72 h. After the incubation period, the media was removed
from each well and MTT solution in phosphate-buffered saline at a
concentration of 0.5 mg/ml was added to all wells. Upon formation of
37 ◦C for 48 h. The cells were then treated with 10, 25 and 50
μM
concentrations of compounds 15, 21, 23, 31 and 37 for 48 h, after which
were trypsinized, and collected in 1.5 ml microtubes. The cells were then
washed with 1 ml PBS and fixed in 70% ethanol overnight at ꢀ 20 ◦C.
After at least 24 h, the ethanol-fixed cells were washed twice with PBS
and stained with DNA staining solution containing PI 20
μg/ml and
RNase 200
μg/ml for 30 min at room temperature in the dark. In the end,
20,000 events were analyzed by a FACSCalibur flow cytometer (BD
Biosciences) and the percentage of the cells in sub-G1, G0/G1, S, and
G2/M phases was calculated using CellQuest software (BD, USA). Each
experiment was repeated at least 3 times.
formazan crystals after 4 h incubation, crystals were dissolved in 200 μl
DMSO. Finally, absorbance was measured at a wavelength of 570 nm
with background correction at 650 nm using a microplate reader
(Bio-Rad model 680, Japan) and IC50 values for each compound was
calculated with CurveExpert version1.34 for windows. Each experiment
was repeated 3–5 times.
2.3. In silico studies
2.3.1. Molecular docking study
Molecular docking analysis was carried out by GOLD 2018 docking
software (Jones et al., 1997). PDB code of 3ZZE was obtained from
Brook Haven Protein databank (www.rcsb.org) (Cui et al., 2012). All
compounds were sketched using MarvinSketch 18.20.0 (http://www.
chemaxon.com) and energy of compounds were optimized by steepest
descent algorithm with Open Babel 2.4.0 (O’Boyle et al., 2011). The
preparation of protein structure was performed via Discovery studio
2.2.2. MET kinase enzymatic assay
The inhibitory activity of the most potent analogues against MET
kinase was measured by a Homogeneous Time Resolved Fluorescence
(HTRF®) based assay kit (KinEASE kit, Cisbio). This assay is based on
measuring the level of phosphorylation of a peptide substrate by MET
kinase (Chen et al., 2020; Wang et al., 2020). An anti-phosphotyrosine
antibody labeled with Europium (Eu3+)-Cryptate functions as a FRET
donor which transfers energy to an acceptor molecule (XL665) tagged to
the end of the peptide substrate, by biotin-streptavidin complex.
Acceptor and donor come to close proximity whenever the phosphory-
lation and consequently anti-phosphotyrosine antibody binding to sub-
strate occurs. Recombinant intracellular kinase domain of MET protein
(Met active) was purchased from Merck-Millipore (Cat number:
14–526). Optimal assay conditions were established for enzyme, sub-
strate, and ATP concentrations as well as the reaction times.
`
client (Dassault Systemes BIOVIA, 2017) and the addition of all hydro-
gens were done. In order to validate and find the most appropriate
scoring function for docking analyses, the ligand 6XP was redocked in-
side 3ZZE using available scoring functions in GOLD namely CHEMPLP.
The RMSD values for CHEMPLP was 0.33 Å.
2.3.2. Molecular dynamics simulation
Molecular dynamics simulations were carried out using GROMACS
package version 2019.1 on a GPU Linux server for the complex of MET
with compound 23. MD simulations was applied by Amber99sb force
filed at a mean temperature of 300 K and at neutral pH (7.0). Chimera
software was used to prepare the topology through adding the AM1
partial charges. Force filed parameters were created using the software
Acpype, which is a tool written in Python to use Antechamber to
generate topologies for chemical entities. A solute box distance of 1.0
nm was defined, in which there were a minimum distance of 2 nm be-
tween any two periodic images of a protein complex and the edge of the
box. Then the box was filled with TIP3P water molecules and the system
neutralization was carried out using 0.15 mol/l sodium chloride. In
order to optimize the system, the steepest descent algorithm was used
through a 100 ps run until a tolerance of 10 kJ molꢀ 1. Afterwards, the
atomic positions of the macromolecule and ligand were restrained with
the force constant of 1000 kJ molꢀ 1 nmꢀ 2. The solvent molecules and
counter-ions were further soaked into the macromolecule complex
during a 500 ps NVT run. During NVT stage, the temperature was
regulated via V-rescale thermostat to 300 K. Then, the pressure of the
system was stabilized under the NPT ensemble over the course of the
100-ps equilibration phase. Generally, the MD simulations need to be
adequately long to be able to draw logistic conclusions from the studies.
During 100 ns simulation time, the production MD run was completed
under a well equilibrated system with a desired temperature and pres-
sure. The particle-mesh Ewald (PME) algorithm was applied for the
calculation of long-ranged electrostatic contributions. The length of all
covalent bonds was restrained by applying the LINCS constraint algo-
rithm. It is known that this algorithm is four times faster than the SHAKE
algorithm. Upon the terminating of the MD run, the protein was
centered in the box and trajectory was corrected in the point of periodic
boundary condition. To resolve the equilibrium time range, the root-
The procedure consisted of a kinase reaction phase followed by a
detection step. In the kinase reaction step, 2 μl of MET kinase enzyme
(0.25 ng/
μ
l final concentration) were preincubated with 4
μ
l of the test
M final
derivatives at different concentrations (10, 25, 50 and 100
μ
concentrations) in a kinase reaction buffer containing 50 mM HEPES,
0.1 mM sodium orthovanadate, 0.01% BSA, 0.02% NaN3, 10 mM MgCl2,
5 mM MnCl2, 2 mM DTT at pH 7.0 in white 384-well plate (Cisbio Cat
Number: 6007299) for 10 min. The reaction was started by adding 2 μl
ATP solution diluted in kinase buffer at the final concentrations of 25
μМ
and 2 l of TK substrate at the final concentrations of 1 M. The plate
μ
μ
was then incubated at room temperature for 50 min. Phosphorylation
level of the peptide substrate was detected by adding 5
gated antibody and 5
μ
l Eu3+-conju-
μ
l Steptavidin-XL665 at 125 nM final concentra-
tion. Following 1 h incubation at room temperature, the plate was read
at excitation of 337 nm and dual emission of 665 and 620 nm with a
delay of 150 ms using a Bio-Tek multimode plate reader (model Cytation
3). The percent inhibition was determined using the following
equations:
Ratio665/620 = Em ission665 nm / Em ission620 nm
ΔR = (Ratio Sam ple665/620 - Ratio Background665/620)*100/ Ratio Back-
ground665/620
Inhibition (%) = (ΔRControl - ΔRSam ple)*100 / ΔRControl
Blanks (background) contained all reagents except for the enzyme.
Control wells contained DMSO in the same dose as in the samples. The
final concentration of DMSO did not exceed 2%.
7

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
mean-square deviation (RMSD) Å was calculated over the entire run for
the protein backbone atoms of each snapshot over the first frame as the
reference. Moreover, cluster analysis of the trajectory within the equi-
librium time range was applied by Gromos with a cut off value of 0.13.
profile were predicted by suing Lipinski’s rule of five (RO5) (Clark-
Pickett, 2000; Lipinski et al., 2001). As shown in Table 1, all DHPM
compounds had desirable drug-likeness properties and passed RO5
criteria by having ≤ H-bond donors (HBD) ≤ 5, H-bond acceptors (HDA)
≤ 10, molecular mass (MM) ≤ 500, and the calculated LogP (cLogP) ≤ 5.
2.4. Statistical analysis
3.2. Biological evaluations
The analysis of the difference between various treatments at different
doses was performed with one-way analysis of variance (ANOVA) with
SPSS software, version 13.0 for Windows. The concentration-response
curves were generated using the software program GraphPad Prism
and IC50 values were calculated with CurveExpert Software, version
1.34 for Windows.
3.2.1. Assessment of the antiproliferative effect
The antiproliferative activities of 27 synthesized DHPMs were eval-
uated against MCF-7, HT-29 and MOLT-4 cancer cell lines as well as
NIH/3T3 non-cancer cells. Doxorubicin and cisplatin were used as
positive controls (Table 2). The concentration-response curves are
shown in Supplementary Figs. Most of the test compounds equally
showed considerable antiproliferative effects against all 3 cell lines. The
selectivity index (SI) was calculated as the ratio of the IC50 value against
NIH/3T3 non-cancer cells and the same value obtained against cancer
cell lines MCF-7, HT-29 and MOLT-4. Among the synthesized DHPM
derivatives, compounds 15, 21, 23, and 37 bearing 5,6-dimethyl-2-ben-
zothiazolyl carboxamide and compounds 19 and 31 having 6-ethoxy-2-
benzothiazolyl carboxamide at C5 position were among the most potent
derivatives. Compound 20 that possesses 4-methyl-2-pyridinyl carbox-
amide at C5 position showed the lowest activity. Five compounds 15,
21, 23, 31, and 37 with the lowest IC50 values, especially against MCF-7
cells, were chosen to be further investigated in terms of their influence
on cell cycle alterations in these cells and also evaluated for their in-
teractions with the MET receptor.
3. Results
3.1. Chemistry
DHPM-C5-amide derivatives were synthesized according to the
procedure shown in Scheme 3. For the preparation of desired com-
pounds, appropriate building blocks were synthesized according to
modified method of Clemens via condensation of 2,2,6-trimethyl-1,3-
dioxine-4-one with the appropriate arylamine (ClemensHyatt, 1985).
Various substituted N-aryl-acetoacetamides were synthesized by react-
ing arylamines (8a-f) and 2,2,6-trimethyl-1,3-dioxine-4-one (7) in
xylene as solvent (Scheme 3). The reaction mixture was heated at 120 ◦C
for 2–4 h. Six different acetoacetamides were synthesized bearing
various hetero/aromatic rings (9a-f). Obtained acetoacetamides were
applied as CH-acidic carbonyl building blocks for the multi-component
Biginelli dihydropyrimidine synthesis (P. Biginelli, 1893). Desired
compounds (12–38) were synthesized on the FeCl3-catalyzed cyclo-
condensation reaction of acetoacetamides (9a-f), substituted aldehydes
(10a-h) and (thio)urea (11). The structures of the synthesized com-
pounds were confirmed by IR, MS and NMR spectral analysis. Physico-
chemical properties determining eventual favourable pharmacokinetic
Additionally, MTT assay was also performed to examine the potential
antiproliferative activity of the most effective compounds 15, 21, 23,
31, and 37 against two MET-addicted cell lines, EBC-1 lung cancer, and
MKN-45 gastric cancer cells. Both cell lines harboring MET gene
amplification rely on MET signaling for survival, while their growth is
inhibited by MET targeted agents (Ai et al., 2018; Inokuchi et al., 2015).
According to obtained results (Table 3), all tested compounds showed
inhibitory effects against both cell lines. The IC50 values were found to
be in the range of 14.6–18.8 μM and 15.0–18.7 μM against EBC-1 and
MKN-45 cells, respectively.
Table 1
Physicochemical properties of reported DHPMs.
3.2.2. MET kinase inhibitory effect
Compound
MW
HBA
HBD
cLogP
Based on the cellular assays, the most promising compounds were
further screened for their MET inhibitory activities using HTRF assay
and the results were outlined in Fig. 1. The compounds were tested at
12
364
380
408
392
409
432
453
469
367
437
409
437
409
394
410
438
422
414
430
458
391
422
450
370
414
398
330
501.5
450.3
853.9
543.5
6
5
7
6
9
9
10
9
9
9
9
9
9
7
6
8
7
6
5
7
5
8
8
6
7
6
6
5
5
10
12
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
4
6
3.02
3.47
3.32
3.29
3.13
2.63
3.57
4.10
2.08
3.54
3.09
3.53
2.99
3.09
3.58
3.48
3.36
3.98
4.60
4.35
3.86
3.21
3.68
3.08
3.44
3.46
2.51
4.01
3.8
13
14
four concentrations 10, 25, 50, and 100 μM and exhibited greater than
15
30% inhibition against the MET kinase. Compounds 15, 23, and 37 at
16
the concentration of 100
and 54.9%, respectively.
μM inhibited MET kinase activity by 52.4, 55.8
17
18
19
20
3.2.3. Flow cytometric analysis of cell cycle alterations
21
RNase/PI flow cytometric determination of cell cycle was performed
to examine the effect of compounds 15, 21, 23, 31 and 37 on the dis-
tribution of MCF-7 cells in different phases of cell cycle. The cells were
treated with selected DHPM derivatives at three different concentrations
22
23
24
25
26
of 10, 25 and 50 μM. Representative histograms are shown in Fig. 2 and
27
the final results are reported in Fig. 3.
28
29
We observed that DHPM derivatives increased the cells in G0/G1
30
phase at lower concentrations of 10 and 25 μM. Consequently, at the
31
higher dose of 50
μM, the sub-G1 phase cells were significantly
32
increased, which shows apoptosis induction at higher doses by test
compounds. The only exception was compound 15, for which the
number of cells in G0/G1 phase increased dose-dependently up to the
33
34
35
36
concentration of 50 μM.
37
38
3.3. In silico study
Cabozantinib
Crizotinib
Paclitaxel
Doxorubicin
3.2
3.3.1. Molecular docking
1.4
Molecular docking analysis was carried out to further investigate the
8

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
Table 2
Table 2 (continued)
Antiproliferative activity of synthesized DHPM-C5 amide derivatives against
cancer and non-cancer cells assessed by MTT reduction assay.
Compound
Substitutions
HT-29
cells
MOLT-4
cells
MCF-7
cells
NIH/
3T3
cells
IC50 expressed in
μ
M (Selectivity index1)
R: 5,6-diMe
R’: 4-AcO
X: O
35.0 ±
26.8 ±
29.2 ±
41.7 ±
1.7 (1.2)
1.7 (1.6)
2.1 (1.4)
2.8
35
36
37
38
R:H
>100 (-)
53.3 ±
53.4 ±
97.1 ±
C4: 2-Thio2
X: O
6.0 (1.8)
1.1 (1.8)
2.9
Compound
Substitutions
HT-29
cells
MOLT-4
cells
MCF-7
cells
NIH/
3T3
R: 6-EtO
C4: 2-Thio2
X: O
26.7 ±
37.7 ±
29.7 ±
66.3 ±
0.7 (2.5)
5.0 (1.8)
1.8 (2.2)
3.9
cells
R: 5,6-diMe
R’: 2-Thio2
X: O
20.8 ±
23.9 ±
15.9 ±
33.3 ±
IC50 expressed in
μ
M (Selectivity index1)
0.8 (1.6)
2.3 (1.4)
3.3 (2.1)
1.9
12
13
14
R, R’: H
X: O
>100 (-)
45.8 ±
5.0 (-)
38.9 ±
3.7 (-)
>100
R:H
>100 (-)
77.6 ±
86.6 ±
>100
R, R’: H
X: S
41.9 ±
40.1 ±
1.1 (1.4)
38.9 ±
3.9 (2.0)
45.8 ±
2.1 (1.2)
30.2 ±
1.9 (2.5)
55.3 ±
6.9
C4: 2-
3.1 (-)
5.5 (-)
propyl2
X: O
3.0 (1.3)
49.4 ±
R: 6-EtO
R’: H
76.4 ±
15.3
Doxorubicin3
Cisplatin
-
177.2 ±
20.3 (0.1)
13.0 ±
12.9 ±
0.5 (0.9)
3.2 ± 0.5
(1.5)
37.6 ±
12.0 ±
2.8
3.9 (1.6)
X: O
3.1 (0.3)
16.1 ±
15
16
R: 5,6-diMe
R’: H
19.4 ±
17.4 ±
17.2 ±
31.0 ±
-
4.7 ±
1.4
2.9 (1.6)
2.0 (1.8)
1.4 (1.8)
4.5
0.9 (0.4)
1.6 (0.3)
X: O
1
The values represent the mean ± S.E.M. of 3-5 independent experiments.
R:H
36.4 ±
31.2 ±
38.6 ±
55.1 ±
Selectivity index (SI) was calculated by dividing IC50 value against NIH/3T3
R’: 4-NO2
X: O
1.8 (1.5)
3.1 (1.8)
0.6 (1.4)
2.9
2
cells to the same value obtained against each cancer cell line. These sub-
17
18
-
72.6 ±
53.4 ±
56.0 ±
61.1 ±
10.1
stitutions are attached to the C4 atom of the DHPM ring; 2-naph: 2-naphthalenyl;
2-Thio: thiophen-2-yl. 3 The IC50 value of doxorubicin is reported in nM.
6.8 (0.8)
26.9 ±
4.3 (1.1)
21.1 ±
3.0 (1.1)
18.5 ±
R: 6-EtO
R’: 4-NO2
X: O
41.8 ±
2.0
2.3 (1.6)
1.3 (2.0)
0.5 (2.3)
Table 3
19
R: 6-EtO
R’: 4-NO2
X: S
17.7 ±
16.5 ±
19.9 ±
33.7 ±
Antiproliferative effects of select DHPM derivatives against MET-dependent cell
lines assessed by MTT assay.
1.7 (1.9)
1.1 (2.0)
1.3 (1.7)
4.4
20
21
-
>100
>100
>100
>100
31.4 ±
0.2
Compound
EBC-1 cells
MKN-45 cells
R: 5,6-diMe
R’: 4-NO2
X: O
16.7 ±
2.1 (1.9)
14.0 ±
1.7 (2.2)
12.1 ±
1.6 (2.6)
IC50 (μM)
15
21
23
31
37
18.8 ± 1.8
16.7 ± 2.1
14.6 ± 2.9
16 ± 1.2
14.9 ± 2.5
15.4 ± 2.4
15.1 ± 1.6
18.7 ± 3.3
15.1 ± 1.3
22
23
24
25
26
27
28
29
30
31
R:H
46.2 ±
33.7 ±
35.6 ±
63.8 ±
R’: 3-NO2
X: O
2.2 (1.4)
2.8 (1.9)
4.0 (1.8)
7.5
R: 5,6-diMe
R’: 3-NO2
X: O
15.5 ±
13.6 ±
13.6 ±
34.7 ±
18.2 ± 1.3
1.2 (2.2)
0.5 (2.6)
1.0 (2.6)
6.8
IC50 values represent the mean ± S.E.M. of 3–4 independent experiments.
R: 5,6-diMe
R’: 2-NO2
X: O
73.7 ±
35.7 ±
27.4 ±
94.1 ±
3.1 (1.3)
3.3 (2.6)
7.2 (3.4)
9.0
R:H
50.3 ±
46.5 ±
37.2 ±
>100
>100
R’: 4-MeO
X: O
4.7 (-)
1.5 (-)
7.7 (-)
R:H
>100 (-)
29.3 ±
33.4 ±
R’: 4-MeO
X: S
1.5 (-)
3.3 (-)
R: 6-EtO
R’: 4-MeO
X: O
29.6 ±
21.3 ±
24.1 ±
46.4 ±
3.5 (1.6)
1.0 (2.2)
2.2 (1.9)
2.4
R: 5,6-diMe
R’: 4-MeO
X: O
27.7 ±
16.8 ±
21.6 ±
30.7 ±
1.7 (1.1)
1.3 (1.8)
3.4 (1.4)
3.9
R:H
32.2 ±
38.5 ±
55.3 ±
>100
C4: 2-naph2
X: O
2.8 (-)
4.4 (-)
8.2 (-)
R:H
24.0 ±
17.4 ±
19.9 ±
35.0 ±
C4: 2-naph2
X: S
5.5 (1.5)
1.7 (2.0)
3.2 (1.8)
2.8
R: 6-EtO
C4: 2-naph2
X: O
17.0 ±
13.5 ±
10.9 ±
45.6 ±
1.5 (2.7)
0.3 (3.4)
1.7 (4.2)
5.8
32
33
-
44.2 ±
34.5 ±
36.1 ±
38.8 ±
9.0
3.1 (0.9)
>100 (-)
6.7 (1.1)
55.3 ±
3.6 (1.1)
52.2 ±
Fig. 1. Met kinase inhibitory effect of synthesized DHPM derivatives assessed
by HTRF assay. The synthetic compounds 15, 21, 23, 31 and 37 were tested for
their MET inhibitory activities in vitro by an HTRF assay at concentrations of 10,
R:H
81.0 ±
5.5
R’: 4-AcO
X: O
9.0 (1.5)
3.7 (1.6)
25, 50 and 100 μM. Values are the mean ± S.E.M. of 3–4 independent exper-
34
iments. The difference with control was significant (*P < 0.05).
9

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
Fig. 3. Analysis of cell cycle distribution of MCF-7 cells treated with different
doses of synthesized DHPM derivatives bearing (2-benzothiazolyl)carboxamide
substitutions at C5. Analysis of cell cycle distribution was performed as
described in Fig. 2 by RNase/propidum iodide (PI) flow cytometric assay.
Values represent the mean ± S.E.M. of 3–4 independent experiments. The dif-
ference with control untreated cells was significantly different (*P < 0.05, #P
< 0.005).
Fig. 2. Cell cycle distribution of MCF-7 cells after treatment with DHPM de-
rivatives bearing (2-benzothiazolyl)carboxamide substitutions at C5. Distribu-
tion of MCF-7 cells in different phases of cell cycle after treatment with the most
potent antiproliferative compounds (15, 21, 23, 31 and 37) was analyzed by
RNase/propidum iodide (PI) flow cytometric assay. MCF-7 cells were seeded in
6-well microplates and treated with 10, 25 and 50 μM concentrations of test
compounds for 48 h. Afterwards, the cells were trypsinized, washed with PBS,
fixed in 70% ethanol overnight at ꢀ 20 ◦C and then stained with PI 20
μg/ml
and RNase 200 μg/ml. In the end, 20,000 events were analyzed by a FACS-
Calibur flow cytometer (BD Biosciences) and the percentage of the cells in sub-
G1, G0/G1, S, and G2/M phases were calculated. Representative histograms are
shown for each test compound.
interactions of the most promising compounds (15, 23 and 37) with the
active site of MET kinase. The crystallographic structure of MET receptor
was extracted from the Protein Data Bank (PDB code: 3ZZE). 3D Inter-
action patterns of compounds 15, 23 and 37 are demonstrated in Fig. 4.
Backbone structure of compound 15 made one important hydrogen
bond interaction from carbonyl group of amide linker to Asp1164. Also,
it has one key pi-pi stacked interaction from the benzene of benzothia-
zole moiety with Tyr1230 (Fig. 4A). Docking study of compound 23
revealed that Tyr1159 residue formed a key hydrogen bond interaction
with the nitro group of nitrophenyl moiety. Other hydrogen bond in-
teractions were formed between the carbonyl group of amide linker and
nitro group of nitrophenyl with Asp1164 and His 1094, respectively.
Moreover, the phenyl and thiazole rings of benzothiazole moiety made
two pi-pi t-shaped interaction with Tyr1230 (Fig. 4B). As shown in
Fig. 4C, the compound 37 has two important hydrogen bonds interac-
tion from carbonyl and NH groups of amide linker to Asp1164 and
Fig. 4. Schematic representation of the interactions between selected synthe-
sized derivatives with MET receptor kinase domain. 3D Interaction patterns of
synthesized derivatives 15 (A), 23 (B) and 37 (C) inside the active site of MET
kinase are shown. The formed hydrogen bonds and pi-pi interactions are
marked with green and purple colors, respectively. (D) Compound 23 and 6XP
molecule (in yellow sticks) were superimposed to compare their binding modes
in the ATP pocket of the receptor.
10

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
Ile1084, respectively. Also, it has one important pi-pi stacked interaction
from the benzene of benzothiazole ring to Tyr1230. In order to further
compare the binding of compound 23 with a known MET inhibitor, the
superimposition of this compound with 6XP molecule (a reported ligand
of the receptor, shown in yellow sticks) in the ATP pocket was per-
formed, which showed a good overlap (Fig. 4D).
inside the active site for the complex with compound 23. The amino
acids that formed stable hydrogen bond interactions involved ASP1164
with 93.20% and Ile1084 with 50.67%. The stability of the hydrogen
bond interactions with ASP1164 and Ile1084 with compound 23 are
shown in Fig. 5B. The cluster analysis was performed for complex of
compound 23 with MET kinase. The percent of population in cluser1
was calculated (89.18%). Moreover, MD interaction pattern in cluser1
with the highest percent population value with MET was illustrated in
Fig. 5C. MD interaction demonstrated that compound 23 made one
hydrogen bond interaction from the carbonyl group of amide linker with
Asp 1164. Also, the benzene of benzothiazole moiety formed one pi-pi t-
shaped interaction with Tyr1230.
3.3.2. Molecular dynamics simulation
MD simulations of 100 ns were applied to investigate the stability of
interaction of active site residues with compound 23 which has shown
best docking results. In this study crystallographic structure of MET
receptor was extracted from the Protein Data Bank (PDB code: 3ZZE).
RMSD values of backbone atoms of each frame as a function of time (ns)
during the runs was calculated in order to evaluate the conformational
stability of the enzyme in respect with the initial structure. The RMSD
abruptly rised at the beginning of the simulation, after which a regular
profile was detected about 80 ns for the complex. So, the last 20 ns was
considered as the equilibrium time range for further analyses (Fig. 5A).
During the equilibrium time range, the lifetime of hydrogen bonds
interaction was monitored between the ligands and amino acid residues
4. Discussion
In this study, we synthesized 27 DHPM-C5 amide derivatives and
tested their antiproliferative effects against 3 human cancer cell lines
including breast and colon cancer as well as leukemia cells. The findings
showed that some of the compounds bearing benzothiazole moiety at C5
possessed promising antiproliferative effects against all 3 cells with IC50
values as low as 10.9
μM. Synthesized derivatives in general were
equally effective against all 3 cancer types. Interestingly, they showed
good selectivity for cancer cell lines in comparison with non-cancer
cells. Moreover, the five analogues with the highest potencies dis-
played a remarkable growth inhibitory effect in MET-dependent lung
and gastric cancer cell lines. In parallel with the cellular results, they
also showed MET kinase inhibitory activity in the HTRF enzymatic
assay. The effects of these derivatives on cell cycle distribution were
evaluated by RNase/PI flow cytometric assay and the results showed
that they increased cells in G0/G1 phase and consequently induced
apoptosis at higher doses. Molecular modeling studies conformed the
good interaction of compound 21 with the MET receptor.
The DHPM-C5 amide derivatives were synthesized with scaffold
hoping approach, in which we attempted to identify different molecular
scaffolds and assemble them to develop new candidate derivatives with
improved anticancer effects. One-pot three components reaction to
provide 3,4-dihydropyrimidin-2(1H)-ones/thiones was first described
by Petro Biginelli, in 1891, setting a milestone for the synthesis of het-
erocyclic compounds (P Biginelli, 1891). The original Biginelli reaction
involved three building blocks including aldehydes, 1,3-dicarbonyl
compounds and (thio)urea (Kappe, 2000). The advantage of Biginelli
reaction is the variety of the building blocks that could expand the di-
versity of synthesized DHPMs with a wide range of biological activities
´
(Ramos et al., 2013; ZhuBienayme, 2006).
The physicochemical properties that favor an acceptable pharma-
cokinetic profile including absorption were predicted by Lipinski’s rule
of five (RO5) and all synthesized derivatives passed the criteria (Clark-
Pickett, 2000; Lipinski et al., 2001). The cellular uptake of xenobiotics
often correlates with their lipophilicity, which could be estimated
through the octanol-water partition coefficient (LogP) (Yang et al.,
2017). In our study, no correlation was observed between the anti-
proliferative effect of compounds in terms of their IC50 values and the
measured lipophilicity (LogP values) in the five cancer cell lines, sug-
gesting that membrane permeability is not decisive for the observed
anticancer activities. It has been recently shown that 80% of the recently
approved kinase inhibitors including crizotinib and cabozantinib fol-
lowed the RO5 by having LogP values between 1 and 5 (OBrienF Mog-
haddam, 2017). In comparison to standard anticancer agents, the cLogP
values of the most potent agents in our study (3.29–4.60) were higher
than doxorubicin (LogP 1.27), indicating higher lipophilicity of DHPM
compounds. Moreover, their LogP profile was revealed close to the
predicted values of paclitaxel (3.0), crizotinib (3.82), and cabozantinib
(4.01), suggesting that these molecules have similar membrane perme-
ability properties (https://go.drugbank.com. Accessed on 20-10-2020)
(Wishart et al., 2018).
Fig. 5. Molecular dynamics simulation of the interaction of compound 23 with
MET receptor kinase domain. (A) To resolve the MD equilibrium time range, the
root mean square deviation (RMSD, nm) against time (ns) of the complex of
MET with compound 23 during 100 ns MD simulation is shown. The equilib-
rium time ranges of 80 ns was observed for the complex . (B) Formation of
hydrogen bond interactions between compound 23 and important residues in
the MET active site are shown. (C) 3D MD interaction patterns of compound 23
with the highest percent population in cluser1 inside the active site of MET
kinase is shown. The formed hydrogen bond and pi-pi interactions were marked
with green and purple colors, respectively.
One of the main considerations in this study was the selection
11

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
between DHPM derivatives with either 2-oxo or 2-thio moieties. Most of
potent anticancer DHPM derivatives like monastrol (2, Scheme 1) have
thiocarbonyl at C2 position and replacing S with O may affect lip-
ophilicity of the synthesized compound. However, comparing 2-oxo
compounds 12, 18, 25 and 29 with 2-thio derivatives 13, 19, 26 and
30, respectively, it can be observed that S atom has a small impact on
lipophilicity. Moreover, in comparison with monastrol with a CLogP of
2.11, all synthesized DHPM-C5-amide derivatives are more lipophilic,
even those with carbonyl at C2 position.
MKN-45 gastric cancer cells. Both cell lines harboring MET gene
amplification rely on MET signaling for survival, and their growth is
inhibited by MET targeted agents. These potent derivatives were further
evaluated for their inhibitory activities towards MET kinase enzyme in
an HTRF assay. All tested compounds displayed dose-dependent inhib-
itory activity. Based on these findings, it could be assumed that the in-
hibition of MET pathway is one of the important mechanisms underlying
the anticancer effects of these analogues.
Additionally, cell cycle alterations induced by the synthesized de-
rivatives were very similar to the alterations caused by MET inhibitors in
previous studies, which have shown an increase in G0/G1 phase in
different cancer cells (Fan et al., 2019; Gao et al., 2013; Hughes et al.,
2016a; H.-t. Zhang et al., 2014). Previous reports suggest that the in-
duction of the G0/G1 cell cycle arrest by inhibition of MET in cancer
cells is highly correlated with reduced levels of G1 phase-specific
cyclins, cyclin-dependent kinase, and increased levels of CDK inhibi-
tory proteins (Akl et al., 2014; Fan et al., 2019; Que et al., 2012). Cap-
matinib as a recently approved MET inhibitor induced a robust G1 cell
cycle arrest followed by apoptosis in MKN-45 cells associated with
reduced levels of cyclin D3 and cyclin-dependent kinase 4 (CDK4), and
increased expression of p27KIP1 (Kou et al., 2018). Similar alterations of
G0/G1 cells induced by MET inhibitors have been observed in meso-
thelioma (Kanteti et al., 2016) and lung cancer cells (Ma et al., 2015).
Furthermore, induction of apoptosis by various MET inhibitors
including tivantinib (Wei et al., 2019) and AMG 337 (Hughes et al.,
2016b) have also been observed previously.
The antiproliferative assay results against MCF-7 (human breast
adenocarcinoma), HT-29 (human colorectal adenocarcinoma) and
MOLT-4 (human lymphoblastic leukemia) cells revealed that the com-
pounds were equally effective against all cancer types. Among the top 10
cancer types, colorectal cancer, breast cancer, and leukemia account for
9.2%, 6.6%, and 3.2% for mortality worldwide respectively (Bray et al.,
2018).
Selectivity index (SI) was also calculated as the ratio of the IC50 value
of the test compound against NIH/3T3 fibroblast cells to the same value
obtained in each cancer cell line. Most of the DHPM derivatives showed
SIs of higher than 1 and even up to 4.1. It is interesting to note that
standard chemotherapeutic drugs, doxorubicin and cisplatin, exhibit
lower SIs in comparison to designed DHPM derivatives.
DHPM derivatives bearing (2-benzothiazolyl)carboxamide sub-
stitutions at C5 in general exhibited higher antiproliferative effects
compared to derivatives that did not have benzothiazole moiety at this
position (17, 20, and 32). The compounds bearing C5 benzothiazole
substitutions had also higher SIs compared to the compounds devoid of
this moiety. These observations confirmed our basic assumption that
designing DHPM compounds with benzothiazole substitution could
enhance the antiproliferative efficacy.
It was observed in our in silico studies that compounds 15, 23 and 37
made important hydrogen bond interactions with Asp1164 and critical
pi-pi interactions with Tyr1230. Most known MET inhibitors such as
crizotinib, capmatinib and 6XP in 2WGJ, 5EOB, and 3ZZE PDB codes,
receptively have shown common hydrogen bond interactions with
Met1160 and critical pi-pi interaction with Tyr1230 (Baltschukat et al.,
2019; D. Zhang et al., 2012). Our previous pharmacophore studies have
also illustrated that establishing pi-pi interaction with Tyr1230 is even
more important than hydrogen binding with Met1160 for certain MET
kinase inhibitors (Damghani et al., 2020).
The influence of the S atom in thiocarbonyl derivatives on the anti-
proliferative activity of synthesized compounds can be deduced from
comparison between pairs of compounds that differ only in this moiety
including 12 vs. 13, 18 vs. 19, 25 vs. 26, and 29 vs. 30. In most instances,
the 2-thio compounds seemed to have slightly lower IC50 values
compared to 2-oxo derivatives. The exceptions were compound 12 (2-
oxo) compared to 13 (2-thio) against MCF-7 cells and 25 (2-oxo)
compared to 26 (2-thio) against HT-29 cell lines. However, the SI values,
an important parameter that determines the selectivity against cancer
cells compared to NIH/3T3 fibroblast cells, was mostly higher in 2-oxo
compounds.
Moreover, compound 23, in comparison with compounds 15 and 37,
has made better hydrogen bond interactions with the three important
residues Tyr1159, Asp1164 and His1094 and also two critical pi-pi t-
shaped interactions with Tyr1230 (Fig. 4B). Hence, we performed an MD
simulation of the interaction of compound 23 with MET in order to take
the MET dynamics into account and we observed that MD simulation
could confirm the docking interaction analysis. According to obtained
results, we conclude that active conformer of compound 23 probably
forms stable interactions with important residues in the binding site of
MET receptor.
Moreover, considering the antiproliferative effects, additional
structure-activity relationships may also be established:
- The presence of an aliphatic substitutions at C4 (compound 38)
instead of aromatic moieties led to the loss of activity.
- C4 substitution did not seem to have a major influence on the anti-
proliferative effect since derivatives bearing different moieties
including phenyl (15), 4-nitrophenyl (21), 3-nitrophenyl (23), 2-
naphthalenyl (31) and thiophen-2-yl (37) were all among the most
promising derivatives.
In vitro and in silico studies suggest that the inhibition of MET might
be a main mechanism for the anticancer effects of these promising
agents. However, it is already very well-known that MET targeting
agents such as crizotinib, cabozantinib, foretinib, etc and also other
receptor tyrosine kinase inhibitors have cross reaction with other ki-
nases (Q. Zhang, Zheng and Zhu, 2020). Therefore, it is very likely that
these DHPM derivatives target other kinases as well and there is a need
for further research on these compounds to determine their potential
effect on other targets.
- Regarding the influence of different benzothiazole moieties at C5 on
the antiproliferative effect, the presence of 5,6-dimethylbenzothia-
zolyl (15, 21, 23 and 37) and 6-ehtoxybenzothiazolyl (31) moi-
eties seemed to confer the highest activities.
Overactivation of MET signaling occurs through MET gene over-
expression, gene amplification and also activating mutations, which can
promote cancer cell proliferation, invasion, and metastasis (Moosavi
et al., 2019). Several investigators are hence making great efforts for
discovery of novel agents targeting the MET pathway and many of these
inhibitors have been approved or have entered the last phases of clinical
trials (Q.-W. Zhang, Ye and Shi, 2019).
5. Conclusions
In this study, twenty-seven 3,4-dihydropyrimidin-2(1H)-one C5
amide derivatives are presented as antiproliferative agents with poten-
tial MET inhibitory activity. We utilized scaffold hopping approach to
modify DHPM core in order to improve the anticancer effect. It was
assumed that the addition of benzothiazole moiety to the 3,4-dihy-
dropyrimidin-2(1H)-one heterocyclic ring, as has been observed in
various MET inhibitors, could render the compounds efficient anti-
proliferative agents through the modulation of MET receptor kinase
In the current study, the agents with the highest anticancer activities
including 15, 21, 23, 31, and 37 showed notable anti-proliferative ac-
tivity against two MET-dependent cell lines, EBC-1 lung cancer, and
12

F. Moosavi et al.
European Journal of Pharmacology 894 (2021) 173850
Clemens, R.J., Hyatt, J.A., 1985. Acetoacetylation with 2,2,6-trimethyl-4H-1,3-dioxin-4-
one: a convenient alternative to diketene. J. Org. Chem. 50, 2431–2435. https://doi.
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pathway. Compounds bearing benzothiazolyl carboxamide moiety at C5
position showed indeed promising antiproliferative activities against all
tested cancer cell lines including MCF-7, HT-29 and MOLT-4 cells with
´
Cui, J.J., McTigue, M., Nambu, M., Tran-Dube, M., Pairish, M., Shen, H., et al., 2012.
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2-(4-(1-(Quinolin-6-ylmethyl)-1 H-[1, 2, 3] triazolo [4, 5-b] pyrazin-6-yl)-1 H-
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IC50 values as low as 10.9 μM, while non-benzithazolyl derivatives (17,
20 and 32) showed much lower effects. The antiproliferative derivatives
with the highest potencies and good selectivity for cancer cells
compared to non-cancer cells (15, 21, 23, 31, and 37) were further
screened for MET kinase inhibitory potential. They inhibited the pro-
liferation of MET-addicted EBC-1 lung and MKN-45 gastric cancer cells.
Moreover, an HTRF-based assay further confirmed that these com-
pounds are capable of blocking MET kinase enzymatic activity. The
select agents also induced G0/G1 cell arrest in MCF-7 cells, followed by
apoptosis at higher doses, features well compatible with MET inhibitory
potential. Molecular modeling of compound 23 demonstrated that this
new derivative could bind effectively to the active site of MET kinase.
We conclude that 3,4-dihydropyrimidin-2(1H)-one bearing benzothia-
zolyl carboxamide moieties at C5 position are potent and selective
anticancer agents with high potential for the treatment of MET-driven
cancers.
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CRediT authorship contribution statement
Gangwar, N., Kasana, V.K., 2012. 3, 4-Dihydropyrimidin-2 (1H)-one derivatives:
organocatalysed microwave assisted synthesis and evaluation of their antioxidant
activity. Med. Chem. Res. 21, 4506–4511.
Fatemeh Moosavi: Conceptualization, Methodology, Writing -
original draft, Writing - review & editing. Ahmad Ebadi: Conceptuali-
zation, Methodology, Writing - original draft, Software. Maryam
Mohabbati: Methodology. Tahereh Damghani: participation in revi-
sion, Writing - original draft, Methodology. Motahareh Mortazavi:
participation in revision, Methodology. Ramin Miri: Conceptualization,
Supervision. Omidreza Firuzi: Conceptualization, Writing - review &
editing, Supervision.
Gao, W., Bing, X., Li, M., Yang, Z., Li, Y., Chen, H., 2013. Study of critical role of c-Met
and its inhibitor SU11274 in colorectal carcinoma. Med. Oncol. 30, 546–554.
Gholampour, M., Ranjbar, S., Edraki, N., Mohabbati, M., Firuzi, O., Khoshneviszadeh, M.,
2019. Click chemistry-assisted synthesis of novel aminonaphthoquinone-1, 2, 3-tri-
azole hybrids and investigation of their cytotoxicity and cancer cell cycle alterations.
Bioorg. Chem. 88, 102967, 102967.
Guido, B.C., Ramos, L.M., Nolasco, D.O., Nobrega, C.C., Andrade, B.Y., Pic-Taylor, A.,
et al., 2015. Impact of kinesin Eg5 inhibition by 3,4-dihydropyrimidin-2(1H)-one
derivatives on various breast cancer cell features. BMC Canc. 15, 283–298. https://
doi.org/10.1186/s12885-015-1274-1. http://www.chemaxon.com.
Acknowledgments
Huang, X., Li, E., Shen, H., Wang, X., Tang, T., Zhang, X., et al., 2020. Targeting the HGF/
MET Axis in cancer therapy: challenges in resistance and opportunities for
improvement. Front. Cell Dev. Biol. 8, 152–167.
This study was supported by grants from Shiraz University of Med-
ical Sciences, Shiraz, Iran (Grant number: 8950) and National Institute
for Medical Research Development (NIMAD), Tehran, Iran (Grant
number: 957652). The authors also wish to thank Vice-chancellor for
Research, Shiraz University of Medical Sciences for the postdoctoral
grant to F. Moosavi (Grant number: 1396-01-106-15688).
Hughes, P.E., Rex, K., Caenepeel, S., Yang, Y., Zhang, Y., Broome, M.A., et al., 2016a. In
vitro and in vivo activity of AMG 337, a potent and selective MET kinase inhibitor, in
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Hughes, P.E., Rex, K., Caenepeel, S., Yang, Y., Zhang, Y., Broome, M.A., et al., 2016b. In
vitro and in vivo activity of AMG 337, a potent and selective MET kinase inhibitor, in
MET-dependent cancer models. Mol. Canc. Therapeut. 15, 1568–1579.
Inokuchi, M., Otsuki, S., Fujimori, Y., Sato, Y., Nakagawa, M., Kojima, K., 2015. Clinical
significance of MET in gastric cancer. World J. Gastrointest. Oncol. 7, 317–327.
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Synthesis of chalcone-amidobenzothiazole conjugates as antimitotic and apoptotic
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2021.173850.
Kamal, A., Sultana, F., Ramaiah, M.J., Srikanth, Y., Viswanath, A., Kishor, C., et al.,
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