Design and synthesis of substituted dihydropyrimidinone derivatives as cytotoxic and tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103317

An operationally simple Biginelli protocol was employed for the synthesis of new C6-carbon based aryl α-haloacrylamide-linked dihydropyrimidinone derivatives. The synthesized compounds were appraised for their in vitro antiproliferative potential against a selected panel of human cancer cell lines especially MCF-7 (human breast cancer), MDA-MB-231 (human breast cancer), HCT-116 (human colon cancer), HCT-15 (human colorectal adenocarcinoma), HT-29 (human colon adenocarcinoma) and DU145 (human prostate cancer) along with normal lung fibroblasts (HFL-1). Preferably, compounds containing α-haloacrylamide (10a-g) functionality were found to exhibit most significant cytotoxicity (IC₅₀ value 0.54 ± 0.12 to 8.35 ± 0.82 μM) against the listed cancer cell lines, particularly towards breast cancer cell lines MCF-7 and MDA-MB-231 (IC₅₀ value 0.54 ± 0.12 to 3.70 ± 0.24 μM). In the seam of synthesized compounds, compound 10f exhibited potent antiproliferative activity against breast cancer cell lines namely MCF-7 (IC₅₀ value 0.54 ± 0.12 μM) and MDA-MB-231 (IC₅₀ value 1.18 ± 0.32 μM). Further to understand the underlying apoptosis mechanisms, different staining techniques such as AO/EB, DCFDA, and DAPI staining were performed. To know the extent of apoptosis and loss of mitochondrial membrane potential in MCF-7 cell lines, annexin V-FITC/PI and JC-1 were performed. Cell cycle analysis revealed that compound 10f arrested the cells at G2/M phase in a dose-dependent manner. The compound 10f also found to exhibit significant inhibition of tubulin polymerization (IC₅₀ of 6.91 ± 0.43 μM) with microtubule destabilizing properties. Molecular docking studies also revealed that compound 10f efficiently interacted with critical catalytically active residues Ser178, Val238, and Val318 of the α/β-tubulin by a hydrogen bond.

Full text of DOI:10.1016/j.bioorg.2019.103317

BioorganicChemistry93(2019)103317
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of substituted dihydropyrimidinone derivatives as
cytotoxic and tubulin polymerization inhibitors
Sravani Sanaa, Ramya Tokalaa, Deepti Madanlal Bajajb, Narayana Nageshd, Kiran Kumar Bokarad,
Gaddam Kiranmaid, Uppu Jaya Lakshmia, Swapna Vadlamanic, Venu Tallab,
⁎
Nagula Shankaraiaha,
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
b Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
c Department of Pharmaceutical Technology in Process Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
d CSIR-Centre for Cellular and Molecular Biology, Medical Biotechnology Complex, ANNEXE II, Uppal Road, Uppal, Hyderabad 500007, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
An operationally simple Biginelli protocol was employed for the synthesis of new C6-carbon based aryl α-ha-
loacrylamide-linked dihydropyrimidinone derivatives. The synthesized compounds were appraised for their in
vitro antiproliferative potential against a selected panel of human cancer cell lines especially MCF-7 (human
breast cancer), MDA-MB-231 (human breast cancer), HCT-116 (human colon cancer), HCT-15 (human colorectal
adenocarcinoma), HT-29 (human colon adenocarcinoma) and DU145 (human prostate cancer) along with
normal lung fibroblasts (HFL-1). Preferably, compounds containing α-haloacrylamide (10a-g) functionality were
Tubulin polymerization
Biginelli reaction
α-bromoacrylamide dihydropyrimidinones
Cytotoxicity
Apoptosis
found to exhibit most significant cytotoxicity (IC50 value 0.54
0.12 to 8.35
0.82 µM) against the listed
0.12
cancer cell lines, particularly towards breast cancer cell lines MCF-7 and MDA-MB-231 (IC50 value 0.54
to 3.70
0.24 µM). In the seam of synthesized compounds, compound 10f exhibited potent antiproliferative
activity against breast cancer cell lines namely MCF-7 (IC50 value 0.54
0.12 µM) and MDA-MB-231 (IC50
value 1.18
0.32 µM). Further to understand the underlying apoptosis mechanisms, different staining tech-
niques such as AO/EB, DCFDA, and DAPI staining were performed. To know the extent of apoptosis and loss of
mitochondrial membrane potential in MCF-7 cell lines, annexin V-FITC/PI and JC-1 were performed. Cell cycle
analysis revealed that compound 10f arrested the cells at G2/M phase in a dose-dependent manner. The com-
pound 10f also found to exhibit significant inhibition of tubulin polymerization (IC50 of 6.91
0.43 μM) with
microtubule destabilizing properties. Molecular docking studies also revealed that compound 10f efficiently
interacted with critical catalytically active residues Ser178, Val238, and Val318 of the α/β-tubulin by a hy-
drogen bond.
1. Introduction
division and placed as an important target for anticancer therapy [4]. A
large number of natural as well as semi-synthetic derivatives [5] have
Cancer is a pernicious disease connecting to a bundle of cellular
processes, in which a group of cells become abnormal, remarked as a
major public health problem worldwide [1]. Universal cancer epidemic
is set to promote and estimated 12 million deaths per year by 2030 [2].
Despite the heroic developments, cancer still remained as a focus of
interest due to the emergence of multi-drug resistance, toxicity and
clearly drives the need for the development of new anticancer agents
for better clinical benefits [3].
been found to inhibit tubulin polymerization. Nonetheless, no ideal
member of this class has been succeeded clinically because of toxicity,
flat water solubility leading to poor patient compliance and multi-drug
resistance [6]. Therefore, it created an urgent need to develop novel
tubulin inhibitors, hoping to find a way to solve the unsolved problems
to date and to come-up with new binding sites of tubulin expanding the
scope for future cancer treatment [7].
Pyrimidine acts as a potential framework for DNA and RNA, eluci-
dating its importance in drug discovery and development [8]. Small
molecule inhibitors containing dihydropyrimidinone as a core have
Microtubule is the cage of tubulin dimers, involves in the dynamics
of chromosomes specifically, mitotic spindle formation during cell
⁎ Corresponding author.
E-mail address: shankar@niperhyd.ac.in (N. Shankaraiah).
https://doi.org/10.1016/j.bioorg.2019.103317
Received 16 May 2019; Received in revised form 17 September 2019; Accepted 26 September 2019
Availableonline26September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

S. Sana, et al.
BioorganicChemistry93(2019)103317
Fig. 1. Structures of minor groove binder, mitotic Egs and tubulin inhibitors.
been observed to possess distinct pharmacological activities such as
anticancer (B and F, Fig. 1) [9], antihypertensive [10], analgesic [11],
antimicrobial [12], anti-inflammatory [13], antitubercular [14], anti-
HIV, A2B adenosine receptor antagonists and antibacterial activity
[15]. Besides, dihydropyrimidinone was found to exert their cytotoxi-
city through varied mechanisms, includes inhibition of Eg5 kinesin
[16], α-amino-β-carboxymuconate-ε-semi aldehyde decarboxylase
[17], and tubulin polymerization. Monastrol (A, Fig. 1) is the first di-
hydropyrimidinone based small molecule identified as an inhibitor of
mitotic spindle protein, Eg5 that helps in spindle formation during
mitosis [18]. Continually, CYT-997, a highly cytotoxic pyrimidine de-
rivative was identified against a range of cancer cell lines through mi-
crotubule destabilization and also acts as vascular disrupting agent
[19].
synthesized derivatives were also evaluated for their in vitro anti-
proliferative activities on selected cancer cell lines and tubulin poly-
merization inhibition.
2. Results and discussion
2.1. Chemistry
The synthesis of desired derivatives 6a–s and 10a–g were depicted
in Scheme 1 and 2. 6-Chloro methyl dihydropyrimidinones 4a–g were
synthesized by Biginelli reaction between substituted aromatic alde-
hydes 2a–g, 4-chloro ethyl acetoacetate and urea in presence of conc.
HCl in ethanol as solvent. Similarly, this reaction was also performed
with heterocyclic aldehydes 2h and 2i, 4-chloro ethyl acetoacetate and
urea in the presence of zinc chloride in THF [28]. These derivatives
were subsequently undergoes nucleophillic substitution with different
monocyclic and bicyclic phenolic alcohols using K2CO3 in acetonitrile
to provide ether bridged dihydropyrimidinones 6a–s. The nitro group
was reduced to its corresponding amine in the presence of Fe-powder
and anhydrous CaCl2 in 10:1 (v/v) of ethanol: H2O, followed by cou-
pling with bromoacrylic acid using EDC in DMF to afford the desired
bromoacrylamide-linked dihydropyrimidinone derivatives 10a–g
(Scheme 2).
In addition, the diversely targeted covalent strategy has widened the
drug discovery since the 1990s with clear insights of hetero Michael
addition reaction with thiols of a cys-residue of a particular protein,
expands the ligand binding affinity and selectivity towards specific
proteins [20]. α-Haloacryloyl is one such functional group animated
from cytotoxic natural products like brostallicin (C, Fig. 1) [21], ver-
ongiaquinol, bromovulone, punaglandins and discorhabdin C, alkylates
DNA through a reactive electrophile generated by thiol interaction
[22]. Besides, pironetin (D, Fig. 1) [23] is a naturally derived α/β
unsaturated lactone known till date as cell cycle inhibitor at M-phase by
covalently binding to cysteine-316 of α-tubulin. Ottelione A (E, Fig. 1)
is the naturally derived, most cytotoxic of sulfhydryl reactive drugs,
also inhibits colchicine binding to tubulin [24]. Some of the other
earlier examples, such as 2,4-dichloro benzyl thiocyanate interact
covalently with tubulin at multiple cysteine residues, T138067 is also
found to interact with β-tubulin, especially with cysteine-241 thiol
group. Colchicine analogues with chloro acetyl groups attached to A-
ring alkylates both cys-239 and cys-354 of β-tubulin [25].
All the synthesized derivatives were carefully analyzed by using
HRMS, 1H, 13C NMR and FT-IR. The most potent compound 10f was
characterized by 1H NMR and showed a sharp singlet at δ 10.16 ppm
which details the proton after amidation reaction. The two signals at δ
6.73 and 6.28 ppm correspond to the olefinic protons of bromoacrylic
acid. Protons of eCH2e which is adjacent to phenoxy group resonated
as a multiplet at δ 5.21 and 5.17 ppm. The triplet and quartet of five
protons each at δ 4.00 and 1.06 ppm belong to the eCH3e and eCH2e
of ester protons respectively. The two singlets of each signal at δ 9.18
and 7.80 ppm account for the NeH protons of cyclic urea of pyr-
imidinone ring. Characteristic methyl protons appeared as a singlet at δ
2.27 ppm and rest of the protons lie in the aromatic region at δ
7.57–6.96 ppm. Similarly, 13C NMR interpretation of compound 10f
details the presence of carbonyl carbon at δ 165.1 ppm. The methyl and
methylene carbons of the ester group were resonated at δ 14.3 and
54.2 ppm respectively. The signal such as eCH2e adjacent to the phe-
noxy ring was appeared at δ 60.2 ppm. Tertiary carbon of pyrimidinone
ring showed a characteristic peak at δ 64.5 ppm and all the remaining
carbons resonates from δ 161.1–102.1 ppm. The spectroscopic data (1H
and 13C NMR) of all other newly synthesized derivatives are in agree-
ment with the respective structures as depicted in the experimental
Next, the rapid association of molecular diversity is an important
achievement in modern drug discovery where addressing the challenges
like intrinsic atom economy, time and energy savings etc., Similarly,
multi-component reactions (MCR) also address these challenges with
simpler procedures utilizing three or more reactants in a single reaction
flask to generate a product with most of the atoms in the starting ma-
terials [26]. In the way of our commitment towards the development of
bioactive molecules [27] and in view of above-mentioned prominence
of alkylating groups and synthetic feasibility, herein we proposed aryl
α-haloacrylamide-linked dihydropyrimidinone derivatives with the in-
troduction of the alkylating group at C6 position to increase the inter-
actions with cysteine residues (Fig. 2). Furthermore, the newly
2

S. Sana, et al.
BioorganicChemistry93(2019)103317
Fig. 2. Design of aryl α-haloacrylamide-linked dihydropyrimidinone derivatives.
section. The HRMS (ESI) of all the compounds showed an [M + H]+ or
[M+2]+ peaks corresponding to their respective molecular formula.
colorectal adenocarcinoma), HT-29 (human colon adenocarcinoma)
and DU145 (human prostate cancer) along with normal lung fibroblasts
(HFL-1) purchased from American Type Culture Collection (ATCC)
USA, by utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The IC50 (μM) values from the in vitro cytotoxic
evaluation of the synthesized derivatives 6a–s and 10a–g as well as
standard drugs were implied in Table 1. Remarkably, the tested deri-
vatives showed good to moderate cytotoxicity on the selected cancer
cell lines. Note worthily, preliminary results indicated that among the
acrylamide series, all the derivatives 10a–g, showed significant
2.2. Biological evaluation
2.2.1. Evaluation of in vitro cytotoxic effects
The newly synthesized dihydropyrimidinone derivatives 6a–s and
10a–g were screened for their cytotoxicity against various human
cancer cell lines MCF-7 (human breast cancer), MDA-MB-231 (human
breast cancer), HCT-116 (human colon cancer), HCT-15 (human
Scheme 1. Synthesis of ethyl 2-oxo-6-(phenoxymethyl)-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 6a–p.
3

S. Sana, et al.
BioorganicChemistry93(2019)103317
Scheme 2. Synthesis of five-membered and α-haloacrylamide based 1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate derivatives 6q–s & 10a–g.
antiproliferative activities against the tested cancer cell lines and the
bearing five-membered heterocyclic rings thiophenyl (10a), furanyl (10b)
ether bridged series 6a–s of dihydropyrimidinone showed moderate
showed potent to moderate cytotoxicity ranging from 1.72
0.12 μM to
antiproliferative activities with the IC50 values 7.21
1.46 µM to
8.17
1.02 μM against all the tested cancer cell lines. Substitution of a
40.0 µM and above. Among the ether bridged series, derivatives 6i
bulky group such as phenyl (10g) also displayed significant cytotoxicity
possessing heterocyclic coumarin ring was active with an IC50 value of
against breast (MCF-7, MDA-MB-231) and prostate cancer cell lines
7.21
1.46 µM and 7.52
0.28 µM against HCT15 and DU145 re-
(DU145) with an IC50 value of 1.57
0.28 μM, 1.06
0.14 μM and
spectively. Derivative 6j with substitution of 4-chloro 3-methyl phenyl
was found to be active on prostate cancer cell line (DU145). Further-
more, derivative 6p derived from monastrol was found to be active
against HCT-116 and MDA-MB-231 cell lines with an IC50 value of
1.50
0.25 μM respectively. It is evident from IC50 values that the po-
sition of acrylamide on dihyropyrimidinone ring has not greatly influ-
enced the cytotoxicity but ether bridge containing acrylamide at the para
position has displayed remarkable cytotoxicity on all the tested cancer
cell lines. Notably, in MDA-MB-231 and MCF-7 breast cancer cell lines,
compounds 10a–g exhibited potent antiproliferative activities with the
13.85
0.25 μM and 15.21
1.49 μM, respectively.
Interestingly, the synthesized compounds 10a–g of acrylamide deri-
vatives showed potent in vitro antiproliferative activities against all the
IC50 value ranging from 0.54
0.12 to 3.70
0.24 µM, which pre-
tested cancer cell lines with the IC50 value ranging from 0.54
0.12 to
sumes that these results may help in identification of new anti-breast
cancer agents with enhanced activity. Based on the in vitro cytotoxicity,
the Structure Activity Relationship (SAR) of the entitled compounds was
constructed and represented in Fig. 3 and the most potent compound 10f
was selected for further studies towards the mechanism of cancer cell
growth inhibition and tubulin polymerization inhibition.
8.35
0.82 µM. Although compound 10e has shown an exceptional IC50
of 24.60
1.52 μM on HCT-15 colon cancer cell line. From the precise
investigation of the IC50 values, it was inferred that the derivative 10f was
more potent to bring about cytotoxicity on all the tested cancer cell lines.
It was found to be most significant against MCF-7 breast cancer cell line
with an IC50 value 0.54
0.12 μM in as much as in MDA-MB-231 breast
cancer cell line 50% inhibition was observed at 1.18
derivative also found to be active in prostate (DU145) and colon cancer
(HCT-116, HCT-15, HT-29) with IC50 value of 2.50 0.19 μM,
2.66 0.11 μM, 3.78 0.40 μM and 6.36 0.20 μM respectively. It
could be observed from the in vitro cytotoxicity studies that compounds
0.32 μM. This
2.3. Effect of 10f on tubulin polymerization inhibition
To investigate the inhibitory activity of 10f by its interactions with
microtubule system [29], the tubulin polymerization inhibitory poten-
tial was evaluated for 10f at different concentrations (10, 5, 2.5, 1.25,
4

S. Sana, et al.
BioorganicChemistry93(2019)103317
Table 1
IC50 (μM) values[a] against human cancer cell lines by derivatives 6a–s and 10a–g by MTT assay.
Compound
MCF7b
MDA-MB-231c
HCT -116d
HCT-15e
HT 29f
DU145g
HFL-1h
6a
35.40
25.26
26.72
> 50
28.60
34.56
29.43
27.48
29.62
38.14
39.26
> 50
33.83
> 50
28.41
40.84
25.62
29.12
> 50
2.18
0.42
0.16
0.22
29.14
36.83
40.15
28.95
40.87
29.63
> 50
38.73
43.19
35.92
> 50
38.82
35.44
48.82
25.49
15.21
38.84
43.47
25.12
1.72
1.26
0.16
1.22
1.05
0.83
0.83
23.62
33.89
35.47
29.12
29.45
28.89
39.46
34.53
40.54
28.11
40.22
36.87
25.23
36.80
> 50
13.85
40.21
36.33
28.94
3.56
0.89
30.12
40.21
29.83
28.46
36.85
31.60
26.75
34.42
7.21
1.36
0.24
0.58
0.63
0.17
0.87
0.24
1.85
> 50
39.12
30.71
> 50
28.95
30.71
26.72
25.19
29.84
28.62
> 50
25.23
28.47
39.18
35.47
38.11
29.46
> 50
38.8
39.34
47.74
43.81
40.01
38.48
33.58
28.89
29.48
7.52
0.74
0.43
1.31
0.61
0.72
0.62
1.93
0.39
34.06
33.69
41.28
38.23
37.23
31.32
48.69
53.03
52.85
47.86
34.94
48.01
41.39
29.42
47.17
29.25
39.54
47.41
28.98
47.63
35.36
43.23
40.63
29.47
50.04
19.28
ND
6.41
6.95
5.86
9.87
8.12
9.76
9.57
3.15
8.16
14.23
6.95
4.20
9.41
6.28
3.63
0.60
2.46
6.29
8.62
8.27
6.84
6.75
2.26
4.50
11.21
0.65
6b
0.25
0.44
1.65
0.32
1.54
0.68
0.76
1.15
0.22
0.23
0.15
0.17
0.19
1.43
0.84
6c
6d
6e
1.45
0.78
0.36
0.34
1.83
1.24
1.21
1.37
0.85
0.28
0.90
0.56
1.44
6f
6g
6h
0.83
1.17
1.20
6i
1.46
0.28
6j
28.61
39.13
25.25
33.89
40.22
26.77
35.46
29.64
29.88
> 50
4.70
1.43
1.41
0.12
0.27
0.29
0.25
0.66
1.83
1.64
11.85
23.01
30.17
27.44
30.75
29.80
29.17
30.34
> 50
29.12
2.90
1.29
1.61
0.58
0.73
0.68
0.51
1.69
0.92
6k
6l
0.27
0.45
1.66
0.43
1.49
1.21
0.16
0.93
0.16
0.69
1.42
0.43
1.20
0.34
6m
0.26
6n
6o
0.68
1.61
0.32
1.66
6p
0.25
0.22
0.63
1.04
6q
6r
6s
0.2
1.27
10a
0.25
0.12
0.17
0.26
8.17
1.02
0.64
0.42
0.82
0.12
0.26
0.25
0.14
10b
3.15
0.72
0.26
0.34
0.24
0.12
0.28
2.08
0.56
0.28
0.12
0.16
0.32
0.14
4.43
0.16
0.18
0.20
0.31
0.11
0.19
0.062
0.005
5.90
0.13
0.55
0.61
7.25
3.80
0.23
0.25
0.47
0.14
0.19
0.25
10c
2.64
1.83
5.70
5.80
6.60
1.71
10d
2.65
1.13
6.40
6.17
8.35
2.60
10e
3.70
1.25
5.70
24.60
3.78
1.52
3.50
3.30
10f
0.54
1.18
2.66
0.40
0.13
6.36
2.50
10g
1.57
1.06
5.30
4.30
7.10
1.50
5-FUi
3.2
0.53
0.6
0.32
1.16
ND
3.9
1.1
0.98
ND
Podoph yllotoxini
Monastroli
ND
0.014
ND
1.21
0.06
0.08
0.24
0.02
0.07
0.003
ND
12.9
0.02
ND
ND
> 100
ND
ND
[a]
[b,c]
[d,e,f]
[g]
[h]
50% Inhibitory concentration after 72 h of drug treatment,
Human breast cancer,
Human colon cancer,
Human prostate cancer,
Human normal
[i]
lung fibroblasts,
Reference compound. All the values are expressed as Mean
SEM in which each treatment was performed in triplicate wells.
0.625 µM) with podophyllotoxin 5 µM and paclitaxel 3 µM) serving as a
positive control. The cell cycle arrest in G2/M phase in most of the
cancer cells is frequently associates with the tubulin polymerization
inhibition. The compound 10f also displayed G2/M cell cycle arrest,
hence in order to understand the concealed mechanism of the active
compound, we performed cell-free in vitro tubulin polymerization assay
by monitoring the increase in fluorescence emission at 440 nm (ex-
citation wavelength is 360 nm) for 1 h at 37 °C (Fig. 4a). The experi-
ment was performed in duplicates and from the Fig. 4b, it was inferred
that 22.4%, 30.5%, 75.3% of tubulin polymerization were observed in
the treatment group of 10f at the concentrations of 2.5, 5.0, and
10.0 µM, respectively, compared to the vehicle group (DMSO). To our
delight, the compound 10f displayed potent tubulin polymerization
cells was inhibited by 10f in a dose-dependent manner through the
tubulin polymerization inhibition.
3. Molecular docking
To elucidate the binding mode and type of interactions with tubulin
(PDB ID: 1SA0), we have performed molecular docking studies with the
most active compound 10f using the GLIDE docking module of
Schrödinger suite 2017-1 [30]. From the docking study, it was observed
that the top-ranked conformation of compound 10f was well accom-
modated inside the colchicine binding site of the tubulin. As shown in
Fig. 5a, docking studies suggest that the compound 10f binds well in the
colchicine-binding domain at the α/β-tubulin interface. More detailed
analysis of the inhibitor-tubulin complex (Fig. 5b) exposed various
hydrogen-bonding interactions that appear to play a key role in the
inhibition with an IC50 value of 6.91
0.43 µM and correlated well
with the anti-proliferative potency, affirming that the growth of cancer
Fig. 3. Structure Activity Relationship (SAR) of aryl α-haloacrylamide and ether-linked dihydropyrimidinone derivatives.
5

S. Sana, et al.
BioorganicChemistry93(2019)103317
Fig. 4. (a) Effect of compound 10f on the tubulin polymerization: tubulin polymerization was monitored by the increase in fluorescence at 360 nm (excitation) and
440 nm (emission) for 1 h at 37 °C. (b) % Dose-response inhibition of tubulin polymerization by compound 10f at final concentrations of 10, 5, 2.5, 1.25, 0.625 µM.
Podophyllotoxin and paclitaxel, were used as reference standards. Data expressed as mean
SEM (n = 3).
binding mode. Compound 10f showed three hydrogen bonding inter-
actions with the catalytically active residues Ser178, Val238 and
Val318. The nitrogen atom at N-1 position of dihydropyrimidinone ring
acts as hydrogen bond acceptor and involved in the hydrogen bond
interaction with back bone NH2 of Val318 (d ¼ 3.7 Å), and the nitrogen
atom at N-3 position of dihydropyrimidinone ring acts as hydrogen
bond acceptor and involved in the hydrogen bond interaction with
main chain NH2 of Ser178 (d ¼ 3.5 Å). Similarly, the oxygen atom of
ethyl ester showed a hydrogen-bonding interaction with the side chain
NH2 of Val318 (d ¼ 3.6 Å). Additionally, several hydrophobic inter-
actions were observed between the compound 10f and the active site
residues, e.g., Val74, Tyr202, Val238, Cys241, Leu242, Leu248,
Ala250, Leu252, Leu255, Met259, Val315, Pro348 and Val351 which
stabilizes the binding of the compound 10f in the colchicine-binding
domain of α/β-tubulin interface. To deepen and understand our find-
ings, we tried the superimposition of colchicine with the ligands and the
resultant superimposition poses suggest that ether bridged phenyl ring
of ligands under investigation occupied identical position with respect
to tri-methoxy phenyl ring of colchicine and the carbonyl group of ester
in 10f superimposes with the amide carbonyl of the colchicine as no-
ticed in Fig. 5c.
4. Analysis of cell cycle effect
Most of the chemotherapeutic agents exhibit their growth inhibitory
potential by the obstruction of cell cycle sequence at a specific check
point [31]. From the in vitro cytotoxicity results, it was clear that the
compound 10f displayed noticeable cytotoxicity against MCF-7 breast
cancer cells. Cell cycle analysis study was performed, wherein HEK-293
(Human embryonic kidney cells) considered as control and MCF-7 cells
were treated with the compound 10f for 48 h at 0.5 µM. The data ob-
tained clearly indicated that these compounds show cell cycle arrest at
the G2/M phase in comparison with the untreated control cells. The 10f
compound showed 22.39% and 31.41% of cell accumulation in G2/M
phase at 0.5 µM concentration in MCF-7 cells, whereas they exhibited
17.27% and 18.97% of cell accumulation at 0.5 µM concentration with
HEK-293 cells. The cell cycle assay data indicate the accumulation of
Fig. 5. (a) Docking model of most potent compound 10f and (b) its ligand interactions in the colchicine binding site of α/β-tubulin (PDB ID: 1SA0). The yellow
dashed lines represent hydrogen bonds. (c) Poses represent the superimposition of potential ligands (10f) and colchicine at the colchicine binding domain: The
potential ligands were shown as ball and stick model, while the interacting aminoacids were denoted as thin tubes and the colchicine was shown as green ball and
stick model in the black background.
6

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BioorganicChemistry93(2019)103317
Fig. 6. (a) Cell cycle analysis showing the effect of 10f on HEK-293 (control cells) and MCF-7 cells 48 h. A. Untreated HEK-293 cells B. HEK-293 cells after treatment
with 0.5 µM 10f C. Untreated MCF-7 cells D. MCF-7 cells after treatment with 0.5 µM 10f. The cell cycle assay was performed by Propidium Iodide (PI) staining
method. (b) Effect of % Dose-response inhibition by compound 10f at a concentration of 0.5 µM in different phases of cell cycle. Data expressed as mean
SEM
(n = 3).
MCF-7 cells at G2/M phase on the treatment of cells with 0.5 µM of
compound 10f (Fig. 6a and b). Further, there is no change in the per-
centage of HEK-293 cells at G2/M phase even after the treatment with
0.5 µM of compound 10f.
simplifies the identification of necrotic cells (Q1-UL; AV-/PI+), late
apoptotic cells (Q1- UR; AV+/PI+), early apoptotic cells (Q3-LR; AV
+/PI-) and live cells (Q2-LL; AV-/PI-) [34]. MCF-7 breast cancer cells
were treated with compound 10f at different concentrations of 0.25,
0.5, and 1 μM for 72 h. It can be observed from Fig. 9a that the per-
centage of late and early apoptotic cells significantly enhanced with an
increase in the concentration of compound 10f, which represents the
dose-dependent apoptosis induction Fig. 9b on MCF-7 breast cancer
cells.
5. Apoptosis detection studies
5.1. Identification of apoptotic cells
DAPI (4ꞌ,6-diamidino-2-phenylindole) is a dominant chromosome
counter stain, binds immutably to the AT clusters in the minor groove of
DNA, by reason of which it reveals the nuclear contraction through
chromatin condensation. DAPI penetrates less efficiently into the
flawless membrane in comparison to the disintegrated membrane and
thereby figure out the apoptotic cells from the live ones. Hence,
apoptotic cells appear brighter rather than live cells. Therefore, it is of
high value to study the nuclear damage induced by compound 10f in
MCF-7 by staining with DAPI [32]. As shown in Fig. 7 in the control (a),
the living cells show normal morphology and the cell nuclei preserve
intact. However, MCF-7 cells were treated with 0.5 µM (b), 1 µM (c),
2.0 µM (d) and 4.0 µM (e) of 10f for 72 h, the cells exhibit the re-
presentative apoptotic features including nuclear shrinkage, pyknotic
or fragmented bright nuclei, and chromatin condensation.
5.4. Effect on mitochondrial membrane potential (DΨm)
Disruption of active mitochondria is the unique peculiarity of the
initial stage of programmed cell death. Alterations in the oxidation-
reduction potential of the mitochondrial membrane lead to the en-
hanced generation of reactive oxygen species (ROS) and thereby loss of
mitochondrial membrane potential (DΨm) [35]. Therefore, it is a de-
cisive task to study the effect of compound 10f on mitochondrial
membrane potential (DΨm). The membrane-permeate JC-1 dye was
used for determining DΨm, in which J-aggregates in healthy mi-
tochondria, therefore, stains red in color whereas depolarized mi-
tochondria consist of J-monomers so they will stain in green. MCF-7
cells were treated with compound 10f at different concentrations for
72 h and then subjected to JC-1 staining. Flow cytometric analysis re-
vealed that the compound 10f caused a noteworthy enhancement in the
depolarized cell population i.e., J-monomers from control 1.82 to
91.38% at 2.0 μM in a dose-dependent manner as shown in Fig. 10.
5.2. Detection of morphology during apoptosis induction
Dual AO/EB staining is a simple method to identify apoptosis-as-
sociated changes of cell membranes during the process of apoptosis
thereby helpful in tumor drug sensitivity test. AO can efficiently in-
filtrate the immaculate membrane and smears the nuclei green, while
EB can only infuse disrupted cell membrane and stain the nucleus red
[33]. This study is generally used to differentiate the apoptotic, necrotic
and live cells. From Fig. 8, it can be inferred that the cell population in
control exhibited the regular morphology and appeared green in color.
However, 10f treated MCF-7 cells clearly disclosed the distorted mor-
phological characteristics such as cell contraction, membrane blebbing,
chromatin condensation and apoptotic body formation implying that
the compound had induced apoptosis in a dose-dependent manner.
5.5. Effect of 10f on intracellular ROS generation
Enhanced free radical generation is believed to contribute to the
damage of mitochondrial potential due to oxidative stress and thereby
leads to apoptosis [36]. Hence, to examine the degree of ROS genera-
tion by compound 10f, the intracellular ROS generation was driven
using the DCFDA staining. MCF-7 cells were treated with increased
doses of compound 10f, where increased green fluorescence pro-
portionality is observed with increase in the concentration, in which
DCFDA is oxidized to 2′,7′-dichlorofluorescein (DCF) in the presence of
ROS generation. From Fig. 11, it surely indicates the potential of 10f in
inducing ROS generation as compared to the control and thereby it
could be one of the mechanisms considered in prompting apoptosis in
MCF-7 breast cancer cells.
5.3. Quantification of apoptosis induction
Annexin V-FITC/Propidium iodide dual staining assay expedites the
quantification of apoptotic cell death using flow cytometry. This assay
7

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BioorganicChemistry93(2019)103317
Fig. 7. Effect of 10f on nuclear morphology of cancer cells after DAPI staining. Breast cancer (MCF-7) cells treated with and without compound 10f at various
concentrations 0.5, 1, 2 and 4 μM for 72 h and compared with control. The images were captured with fluorescence microscope at 200X. Red scale Bar in the image
indicates 50 μm.
6. Conclusion
panel of human cancer cell lines. From the preliminary screening, it was
inferred that compound 10f displayed a substantial biological response
against all the tested cancer cell lines specifically on MCF-7 breast
In conclusion, a new series of dihydropyrimidinone derivatives were
designed and synthesized by employing multi-component Biginelli re-
action with the introduction of α-haloacrylamide at the 6th position
through ether linked mono and bicyclic frameworks. Later, in vitro cy-
totoxic potential for these derivatives were screened against a selected
cancer cell line with IC50 of 0.54
0.1 μM. Induction of apoptosis was
evident by AO/EB staining, DAPI nuclear staining, and Annexin V/
Propidium Iodide assay. Compound 10f caused the collapse of mi-
tochondrial membrane potential (ΔΨM), elevated generation of ROS
Fig. 8. AO/EB staining in breast cancer cells (MCF-7), cells treated with and without compound 10f at various concentrations 0.5, 1, 2 and 4 μM for 72 h and
compared with control. The images were captured with fluorescence microscope at 200X. Red scale Bar indicates 50 μm.
8

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BioorganicChemistry93(2019)103317
Fig. 9. (a) Effect of compound 10f on cell apoptosis induction of MCF-7 cells was measured by Annexin V-Alexa Flour 488/propidium iodide staining assay. MCF-7
cells were treated with compound 10f ranging from 0.25 to 1.0 μM concentration and control (untreated cells) for 72 h. Then 10,000 cells from each sample were
analyzed by flow cytometry. The percentage of cells positive for Annexin V-Alexa Flour 488 and/or Propidium iodide is represented inside the quadrants. Cells in the
upper left quadrant (Q1-UL; AV-/PI+): necrotic cells; lower left quadrant (Q2-LL; AV−/PI−): live cells; lower right quadrant (Q3-LR; AV+/PI−): early apoptotic
cells and upper right quadrant (Q4- UR; AV+/PI+): late apoptotic cells. (b) % Dose-response inhibition by compound 10f at final concentrations of 0.25, 0.5, 1 µM.
Data expressed as mean
SEM (n = 3).
and cell cycle arrest at G2/M phase in MCF-7 cancer cell line in a dose
dependent manner. Molecular modelling studies supported the possible
binding mode of action by 10f to the α/β-tubulin. In addition, com-
pound 10f inhibited tubulin polymerization by 50% at a concentration
7. Experimental section
7.1. Chemistry
of 6.91
0.43 μM in an in vitro assay with microtubule-destabilizing
General Methods. All the reagents and solvents were obtained from
commercial suppliers and were used without further purification.
Analytical thin layer chromatography (TLC) was performed on MERCK
pre-coated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization
of the spots on TLC plates was achieved by UV light. 1H and 13C NMR
spectra were recorded on Bruker 500 MHz by making a solution of
properties. Overall, the current studies demonstrate that the new α-
haloacrylamide-linked dihydropyrimidinone derivatives can be devel-
oped as potent tubulin inhibitors and their further structural mod-
ifications may generate promising new anti-breast cancer leads in
cancer therapy.
Fig. 10. Effect on mitochondrial membrane potential. MCF-7 cells were treated with various concentrations (0.25, 0.5, 2 μM) of compound 10f for 72 h and JC-1
staining was performed. The control represents the cells without compound 10f treatment.
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BioorganicChemistry93(2019)103317
(m, 2H), 7.32–7.31 (m, 1H), 7.30 (d, J = 6.2 Hz, 3H), 7.28–7.26 (m,
2H), 5.33 (d, J = 11.9 Hz, 1H), 5.24 (d, J = 3.4 Hz, 1H), 5.16 (d,
J = 12.0 Hz, 1H), 4.03–3.99 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6): δ 165.2, 156.3, 152.4, 146.1, 144.5, 134.5,
129.7, 129.2, 128.9, 128.0, 128.0, 127.2, 126.9, 126.8, 124.3, 119.2,
107.5, 102.0, 64.3, 60.3, 54.5, 14.3; HRMS (ESI): m/z calc. for
C24H22N2O4 403.1658; found 403.1674 [M+H]+
.
7.1.1.3. Ethyl
6-((3-hydroxyphenoxy)methyl)-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6b). White solid; yield 82%;
mp:183–186 °C; FT-IR (cm−1): 2922, 2876, 1834, 1715, 1693, 1487,
754; 1H NMR (500 MHz, DMSO-d6): δ 9.23 (s, 1H), 7.85 (s, 1H),
7.43–7.31 (m, 4H), 7.31–7.20 (m, 4H), 6.67–6.61 (m, 2H), 5.23–5.20
(m, 2H), 4.97 (d, J = 12.0 Hz, 1H), 4.00–3.97 (m, 2H), 1.04 (t,
J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 165.1, 159.6,
152.3, 146.0, 144.5, 130.4, 128.9, 127.9, 126.7, 107.9, 102.3, 101.9,
64.3, 60.2, 54.5, 14.3; HRMS (ESI): m/z calculated for C20H20N2O5
Fig. 11. ROS generated before and after the treatment of 10f to HEK-293 and
MCF-7 cells. Data expressed as mean
SEM (n = 3).
samples in the DMSO-d6 as solvent using tetramethyl silane (TMS) as
the internal standard. Chemical shifts for 1H and 13C are reported in
parts per million (ppm) downfield from tetra methyl silane. Spin mul-
tiplicities are described as s (singlet), brs (broad singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). Coupling constant (J) values
are reported in hertz (Hz). HRMS were determined with Agilent QTOF
mass spectrometer 6540 series instrument. Wherever required, column
chromatography was performed using silica gel (60–120). The reactions
wherever anhydrous conditions required are carried under nitrogen
positive pressure using freshly distilled solvents. All evaporation of
solvents was carried out under reduced pressure using rotary eva-
porator below 45 °C. Melting points were determined with an electro
thermal digital melting point apparatus IA9100 and are uncorrected.
The names of all the compounds given in the experimental section were
taken from ChemBioDraw Ultra, Version 12.0.
369.1450 found 369.1483 [M+H]+
.
7.1.1.4. Ethyl
2-oxo-4-phenyl-6-((pyridin-4-yloxy)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6c). Yellow solid; yield 88%;
mp:178–182 °C; FT-IR (cm−1): 2927, 2868, 1839, 1788, 1367, 758;
1H NMR (500 MHz, DMSO-d6): δ 7.68 (d, J = 6.9 Hz, 2H), 7.41–7.13
(m, 6H), 7.03 (s, 1H), 6.06 (d, J = 6.9 Hz, 2H), 5.14 (s, 1H), 5.06 (d,
J = 13.7 Hz, 1H), 4.92 (d, J = 13.7 Hz, 1H), 4.06–3.88 (m, 2H), 1.08 (t,
J = 6.8 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 177.7, 165.7, 146.2,
141.4, 128.6, 127.5, 126.7, 117.6, 59.6, 55.1, 54.8, 14.5; HRMS (ESI):
m/z calculated for C19H19N3O4 354.1454 found 354.1450 [M+H]+
.
7.1.1.5. Ethyl
2-oxo-4-phenyl-6-((quinolin-8-yloxy)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6d). White solid; yield 79%;
mp:183–185 °C; FT-IR (cm−1): 2927, 2856, 1839, 1788, 1367, 748;
1H NMR (500 MHz, DMSO-d6): δ 9.43 (s, 1H), 9.28 (s, 1H), 7.83 (d,
J = 8.7 Hz, 2H), 7.75 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 6.9 Hz, 1H),
7.40–7.34 (m, 2H), 7.29 (d, J = 8.9 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H),
6.76–6.70 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 5.34 (d, J = 11.9 Hz, 1H),
5.17 (d, J = 3.4 Hz, 1H), 5.09 (d, J = 11.9 Hz, 1H), 4.06–4.00 (m, 2H),
1.07 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 165.2, 156.3,
152.4, 146.1, 144.5, 134.5, 129.7, 129.2, 128.9, 128.0, 128.0, 127.2,
126.9, 126.8, 124.3, 119.2, 107.5, 102.0, 64.3, 60.3, 54.5, 14.3; HRMS
(ESI): m/z calculated for C23H21N3O4 404.1610 found 404.1632 [M
7.1.1. Ethyl
2-oxo-6-(aryloxymethyl)-4-aryl/heteroaryl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6a–s)
To a mixture of chlorinated derivatives of dihydropyrimidinone
derivatives 4a–i (1 equiv.), potassium carbonate (2.5 equiv.) in acet-
onitrile, under 80 °C was added different aryl/ heteroaryl alcohols
(5a–k, 0.75 equiv.) and stirred under reflux till complete consumption
of the starting materials as determined by TLC. The solvent was then
removed using rotary evaporator and extracted using ethyl acetate
(25 mL × 3) and water. The organic layer was concentrated under in
vacuo and the residue obtained was chromatographed on silica gel
(elution with hexane/EtOAc = 7:3–5:5) to provide the 2-oxo-6-(ar-
yloxymethyl)-4-aryl/heteroaryl-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate derivatives 6a–s in moderate to good yields.
+H]+
.
7.1.1.6. Ethyl
6-((2-amino-4-chlorophenoxy)methyl)-2-oxo-4-phenyl-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (6e). White solid; yield
82%; mp:179–184 °C; FT-IR (cm−1): 2949, 2832, 1837, 1768, 1347,
774; 1H NMR (500 MHz, DMSO-d6): δ 9.29 (s, 1H), 7.84 (s, 1H), 7.28 (d,
J = 7.1 Hz, 3H), 7.21 (d, J = 7.2 Hz, 2H), 6.78 (d, J = 8.6 Hz, 1H), 6.66
(d, J = 2.4 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H), 5.36 (s, 2H), 5.19 (s, 3H),
4.07–4.00 (m, 2H), 1.08 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz,
DMSO-d6): δ 165.2, 152.3, 146.6, 144.5, 143.6, 140.8, 128.9, 127.9,
126.8, 126.2, 115.1, 114.3, 113.7, 101.2, 64.6, 60.3, 54.5, 14.3; HRMS
(ESI): m/z calculated for C20H20ClN3O4 402.1221 found 402.1218 [M
7.1.1.1. Ethyl 6-((2-(2-bromoacrylamido)-phenoxy)methyl)-2-oxo-4-aryl-
1,2,3,4-tetrahydro pyrimidine-5-carboxylate (10a–g). To a mixture of 2-
bromoacrylic acid (9, 1 equiv.), ethyl 6-(phenoxy methyl)-2-oxo-4-aryl-
1,2,3,4-tetrahydropyrimidine-5-carboxylate amines (8a–g, 1 equiv.), in
DMF, EDC (2 equiv.) was added and stirred at 25 °C till complete
consumption of the starting materials as determined by TLC. The
reaction mixture was then quenched with ice-cold water and
extracted using ethyl acetate (25 mL × 3). The organic layer was
concentrated under in vacuo and the residue obtained was
+H]+
.
7.1.1.7. Ethyl 6-((4-chloro-3-methylphenoxy)methyl)-2-oxo-4-(p-tolyl)-
1,2,3,4-tetrahydropyrimi dine-5-carboxylate (6f). White solid; yield
92%; mp:189–192 °C; FT-IR (cm−1): 2949, 2822, 1827, 1799, 1370,
747; 1H NMR (500 MHz, DMSO-d6): δ 9.20 (s, 1H), 7.80 (s, 1H), 7.32 (d,
J = 8.8 Hz, 1H), 7.13 (s, 4H), 7.02 (d, J = 2.5 Hz, 1H), 6.86 (d,
J = 8.7 Hz, 1H), 5.17 (d, J = 4.2 Hz, 2H), 5.01 (d, J = 12.0 Hz, 1H),
4.02–3.98 (m, 2H), 2.28 (d, J = 7.9 Hz, 6H), 1.06 (t, J = 7.1 Hz, 3H);
13C NMR (125 MHz, DMSO-d6): δ 165.1, 157.1, 152.4, 145.6, 141.6,
137.1, 136.9, 129.9, 129.4, 126.6, 125.6, 118.0, 114.4, 102.2, 64.5,
60.2, 54.2, 21.1, 20.2, 14.3; HRMS (ESI): m/z calculated for
chromatographed
on
silica
gel
(elution
with
hexane/
EtOAc = 6:4–4:6) to provide the bromoacrylamido-4-phenoxymethyl-
2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives
in moderate to good yields.
7.1.1.2. Ethyl
6-((naphthalen-2-yloxy)methyl)-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6a). White solid; yield 89%;
mp:185–189 °C; FT-IR (cm−1): 2922, 2862, 1897, 1710, 1370, 738;
1H NMR (500 MHz, DMSO-d6): δ 9.32 (s, 1H), 7.87 (d, J = 5.1 Hz, 2H),
7.85 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.50–7.46 (m, 1H), 7.40–7.35
C
22H23ClN2O4 415.1425 found 415.1436 [M+H]+
.
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BioorganicChemistry93(2019)103317
7.1.1.8. Ethyl
6-((3-chloro-4-fluorophenoxy)methyl)-2-oxo-4-(p-tolyl)-
5.21 (s, 1H), 5.11 (d, J = 14.1 Hz, 1H), 4.94 (d, J = 13.9 Hz, 1H),
4.06–4.02 (m, 2H), 1.09 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ
177.7, 165.2, 152.6, 145.4, 144.5, 141.1, 128.9, 128.0, 126.8, 117.9,
116.1, 103.1, 60.5, 54.6, 53.0, 14.3; HRMS (ESI): m/z calculated for
1,2,3,4-tetrahydropyrimi dine-5-carboxylate (6g). White solid; yield
89%; mp:187–190 °C; FT-IR (cm−1): 2922, 2853, 1822, 1733, 1398,
762; 1H NMR (500 MHz, DMSO-d6): δ 9.26 (s, 1H), 7.81 (s, 1H), 7.37 (t,
J = 9.1 Hz, 1H), 7.27 (d, J = 6.1 Hz, 1H), 7.13 (s, 4H), 7.05–6.98 (m,
1H), 5.18 (t, J = 7.0 Hz, 2H), 5.03 (d, J = 12.0 Hz, 1H), 4.04–3.98 (m,
2H), 2.27 (s, 3H), 1.07 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-
d6): δ 165.1, 155.0, 155.0, 153.6, 152.4, 151.7, 145.3, 141.5, 137.1,
129.4, 126.6, 120.0, 117.7, 117.5, 117.1, 115.8, 115.7, 102.4, 65.2,
60.3, 54.1, 21.1, 14.3; HRMS (ESI): m/z calculated for C21H20ClFN2O4
C
19H18ClN3O4 388.1064 found 388.1079 [M+H]+
.
7.1.1.14. Ethyl
4-([1,1′-biphenyl]-4-yl)-2-oxo-6-((pyridin-4-yloxy)
methyl)-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6m). Yellow solid;
yield 87%; mp: 193–197 °C; FT-IR (cm−1): 2976, 2876, 1862, 1712,
1609, 1395, 755; 1H NMR (500 MHz, DMSO-d6): δ 7.70 (d, J = 7.6 Hz,
3H), 7.67–7.61 (m, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.45 (t, J = 7.7 Hz,
2H), 7.35 (t, J = 6.8 Hz, 1H), 7.32 (d, J = 8.2 Hz, 2H), 7.22 (s, 1H),
6.08 (d, J = 7.7 Hz, 2H), 5.99 (d, J = 6.5 Hz, 1H), 5.20 (d, J = 2.3 Hz,
1H), 5.11 (d, J = 13.9 Hz, 1H), 4.91 (d, J = 13.9 Hz, 1H), 4.07–3.95
(m, 2H), 1.09 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ
177.7, 165.5, 146.0, 144.4, 141.3, 140.2, 139.7, 129.3, 127.4, 127.1,
127.0, 117.8, 116.0, 60.2, 54.4, 54.0, 14.5; HRMS (ESI): m/z calculated
419.1174 found 421.1087 [M+2]+
.
7.1.1.9. Ethyl
2-oxo-6-((pyridin-4-yloxy)methyl)-4-(p-tolyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (6h). Yellow solid; yield 87%;
mp:190–196 °C; FT-IR (cm−1): 2982, 2843, 1838, 1892, 1489, 742;
1H NMR (500 MHz, DMSO-d6): δ 7.66 (d, J = 7.5 Hz, 2H), 7.12 (d,
J = 7.7 Hz, 3H), 7.01 (d, J = 7.8 Hz, 2H), 6.32 (s, 1H), 6.02 (d,
J = 7.5 Hz, 2H), 5.03 (s, 1H), 4.98 (d, J = 13.5 Hz, 1H), 4.88 (d,
J = 13.5 Hz, 1H), 4.00–3.90 (m, 2H), 2.24 (s, 3H), 1.07 (t, J = 7.0 Hz,
3H); 13C NMR (125 MHz, DMSO-d6): δ 177.7, 166.3, 159.5, 145.2,
141.8, 135.8, 128.8, 126.6, 117.2, 58.5, 57.6, 54.8, 21.1, 14.8; HRMS
(ESI): m/z calculated for C20H21N3O4 368.1610 found 368.1622 [M
for C25H23N3O4 430.1767 found 430.1782 [M+H]+
.
7.1.1.15. Ethyl
4-(3-(benzyloxy)phenyl)-2-oxo-6-((pyridin-4-yloxy)
methyl)-1,2,3,4-tetrahydro pyrimi dine-5-carboxylate (6n). Light brown
solid; yield 76%; mp: 195–198 °C; FT-IR (cm−1): 2967, 2886, 1878,
1705, 1619, 1385, 765; 1H NMR (500 MHz, DMSO-d6): δ 9.58 (s, 1H),
7.90 (s, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.41 (d, J = 7.3 Hz, 4H), 7.33 (t,
J = 6.9 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H),
6.88–6.77 (m, 2H), 6.12 (d, J = 7.5 Hz, 2H), 5.20 (d, J = 2.9 Hz, 1H),
5.09 (d, J = 14.2 Hz, 1H), 5.05 (s, 2H), 4.94 (d, J = 14.2 Hz, 1H), 4.06
(t, J = 6.8 Hz, 2H), 1.10 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz,
DMSO-d6): δ 177.6, 165.2, 158.9, 152.3, 145.9, 144.7, 141.0, 137.4,
130.1, 128.9, 128.3, 128.1, 119.1, 118.0, 114.1, 113.6, 103.4, 69.6,
60.7, 54.4, 52.8, 14.3; HRMS (ESI): m/z calculated for C26H25N3O5
+H]+
.
7.1.1.10. Ethyl
2-oxo-6-(((2-oxo-2H-chromen-4-yl)oxy)methyl)-4-(p-
tolyl)-1,2,3,4-tetrahydropyrimi dine-5-carboxylate (6i). White solid;
yield 83%; mp:198–202 °C; FT-IR (cm−1): 2922, 2853, 1832, 1793,
1389, 752; 1H NMR (500 MHz, DMSO-d6): δ 9.57 (s, 1H), 7.87 (d,
J = 3.0 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.72–7.66 (m, 1H), 7.42 (d,
J = 7.6 Hz, 2H), 7.18 (d, J = 7.9 Hz, 4H), 5.98 (s, 1H), 5.37 (d,
J = 11.5 Hz, 1H), 5.25 (d, J = 11.6 Hz, 1H), 5.21 (d, J = 3.2 Hz, 1H),
4.05–3.97 (m, 2H), 2.28 (s, 3H), 1.05 (t, J = 7.1 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6): δ 165.1, 155.0, 155.0, 153.6, 152.4, 151.7,
145.3, 141.5, 137.1, 129.4, 126.6, 120.0, 117.7, 117.5, 117.1, 115.8,
115.7, 102.4, 65.2, 60.3, 54.1, 21.1, 14.3; HRMS (ESI): m/z calculated
460.1872 found 460.1869 [M+H]+
.
7.1.1.16. Ethyl 4-(3-hydroxyphenyl)-6-((naphthalen-2-yloxy)methyl)-2-
oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6o). White solid; yield
79%; mp: 188–189 °C; FT-IR (cm−1): 2967, 2881, 1896, 1702, 1609,
1395, 755; 1H NMR (500 MHz, DMSO-d6): δ 9.43 (s, 1H), 9.28 (s, 1H),
7.83 (d, J = 8.7 Hz, 4H), 7.48 (d, J = 6.9 Hz, 1H), 7.40–7.34 (m, 2H),
7.29 (d, J = 8.9 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 6.76–6.70 (m, 2H),
6.66 (d, J = 8.1 Hz, 1H), 5.34 (d, J = 11.9 Hz, 1H), 5.17 (d, J = 3.4 Hz,
1H), 5.09 (d, J = 11.9 Hz, 1H), 4.06–4.00 (m, 2H), 1.07 (t, J = 7.1 Hz,
3H); 13C NMR (125 MHz, DMSO-d6): δ 165.2, 157.9, 156.4, 152.5,
145.9, 145.7, 134.5, 129.8, 129.7, 129.2, 128.0, 127.2, 126.9, 124.2,
119.2, 117.3, 114.9, 113.7, 107.5, 102.2, 64.4, 60.3, 54.4, 14.3; HRMS
(ESI): m/z calculated for C24H22N2O5 419.1607 found 419.1610 [M
for C24H22N2O6 435.1556 found 435.1568 [M+H]+
.
7.1.1.11. Ethyl 6-((4-chloro-3-methylphenoxy)methyl)-4-(4-chlorophenyl)-2-
oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6j). White solid; yield
90%; mp:172–176 °C; FT-IR (cm−1): 2983, 2868, 1849, 1765, 1368,
748; 1H NMR (500 MHz, DMSO-d6): δ 9.31 (s, 1H), 7.88 (s, 1H), 7.42
(d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.7 Hz, 3H), 7.27 (d, J = 8.3 Hz, 2H),
7.02 (s, 1H), 6.87–6.85 (m, 1H), 5.22 (d, J = 3.0 Hz, 1H), 5.18 (d,
J = 12.0 HZ, 1H), 5.03 (d, J = 11.9 HZ, 2H), 4.03–3.98 (m, 2H), 2.29 (s,
3H), 1.07 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 165.0,
157.1, 152.1, 146.2, 143.4, 136.9, 132.5, 129.9, 128.9, 128.6, 125.7,
118.0, 114.4, 101.6, 64.5, 60.3, 53.9, 20.2, 14.3; HRMS (ESI): m/z
+H]+
.
calculated for C21H20Cl2N2O4 435.0878 found 437.0872 [M+2]+
.
7.1.1.17. Ethyl 6-((3,5-dimethoxyphenoxy)methyl)-4-(3-hydroxyphenyl)-
2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6p). Off white solid;
yield 73%; mp:187–191 °C; FT-IR (cm−1): 2931, 2611, 1869, 1739,
1738, 1675, 1385, 795; 1H NMR (500 MHz, DMSO-d6): δ 9.41 (s, 1H),
9.16 (s, 1H), 7.82–7.77 (m, 1H), 7.10 (t, J = 7.8 Hz, 1H), 6.71–6.64 (m,
3H), 6.18 (d, J = 2.1 Hz, 2H), 6.13 (t, J = 2.1 Hz, 1H), 5.19 (d,
J = 12.0 Hz, 1H), 5.13 (d, J = 3.3 Hz, 1H), 4.92 (d, J = 12.1 Hz, 1H),
4.05–3.99 (m, 2H), 3.70 (s, 6H), 1.09 (t, J = 7.1 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6): δ 165.2, 161.6, 160.2, 157.9, 152.4, 145.9,
145.7, 129.8, 117.2, 114.9, 113.7, 102.1, 94.0, 64.3, 60.3, 55.6, 54.3,
14.3; .HRMS (ESI): m/z calculated for C22H24N2O7 429.1662 found
7.1.1.12. Ethyl 6-((3-chloro-4-fluorophenoxy)methyl)-4-(4-chlorophenyl)-
2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6k). White solid;
yield 90%; mp:170–175 °C; FT-IR (cm−1): 2973, 2853, 1839, 1714,
1398, 748; 1H NMR (500 MHz, DMSO-d6): δ 9.38 (d, J = 1.2 Hz, 1H),
7.91 (s, 1H), 7.41 (dd, J = 11.3, 4.5 Hz, 2H), 7.39–7.32 (m, 1H),
7.31–7.24 (m, 3H), 7.02 (d, J = 9.1, 1H), 5.21 (d, J = 7.7 Hz, 2H), 5.05
(d, J = 12.0 Hz, 1H), 4.05–3.97 (m, 2H), 1.06 (t, J = 7.1 Hz, 3H); 13C
NMR (125 MHz, DMSO-d6): δ 164.9, 154.9, 153.6, 152.2, 151.7, 145.9,
143.4, 132.5, 128.9, 128.6, 120.0, 117.7, 117.5, 117.1, 115.7, 115.7,
101.7, 65.1, 60.4, 53.9, 14.3; HRMS (ESI): m/z calculated for
431.1678 [M+2]+
.
C
20H17Cl2FN2O4 439.0628 found 441.0647 [M+2]+
.
7.1.1.18. Ethyl 6-((2-amino-4-chlorophenoxy)methyl)-2-oxo-4-(thiophen-
2-yl)-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6q). Off white solid;
yield 73%; mp:189–193 °C; FT-IR (cm−1): 2986, 2611, 1879, 1563,
1384, 1175, 785; 1H NMR (500 MHz, DMSO-d6): δ 9.44 (s, 1H), 8.02 (s,
1H), 7.39 (d, J = 5.0 Hz, 1H), 6.97 –6.92 (m, 1H), 6.90 (d, J = 3.0 Hz,
1H), 6.76 (d, J = 8.6 Hz, 1H), 6.65 (d, J = 2.2 Hz, 1H), 6.48 (dd,
7.1.1.13. Ethyl
4-(4-chlorophenyl)-2-oxo-6-((pyridin-4-yloxy)methyl)-
1,2,3,4-tetrahydropyrim idine-5-carboxylate (6l). Off white solid, yield
85%; mp:185–189 °C; FT-IR (cm−1): 2972, 2853, 1864, 1710, 1388,
753; 1H NMR (500 MHz, DMSO-d6): δ 7.82 (s, 1H), 7.70 (d, J = 6.2 Hz,
2H), 7.31 (d, J = 6.4 Hz, 2H), 7.24 (s, 3H), 6.10 (d, J = 6.1 Hz, 2H),
11

S. Sana, et al.
BioorganicChemistry93(2019)103317
J = 8.5, 2.2 Hz, 1H), 5.47 (d, J = 3.4 Hz, 1H), 5.34 (s, 2H), 5.24 (d,
J = 13.6 Hz, 1H), 5.05 (d, J = 13.6 Hz, 1H), 4.13–4.09 (m, 2H), 1.16 (t,
J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.9, 152.4, 148.3,
146.8, 143.7, 140.7, 127.2, 126.2, 125.4, 124.3, 115.1, 114.1, 113.7,
101.5, 64.5, 60.4, 49.8, 14.4; HRMS (ESI): m/z calculated for
6.88 (d, J = 8.2 Hz, 1H), 6.35 (d, J = 7.2 Hz, 1H), 5.32 (d, J = 7.8 Hz,
2H), 5.23 (d, J = 8.4 Hz, 1H), 4.07–3.99 (m, 2H), 1.08 (t, J = 14.1,
7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 165.1, 160.3, 152.3,
147.6, 145.6, 144.5, 128.9, 128.4, 128.3, 128.0, 126.8, 125.4, 121.8,
114.1, 102.0, 65.2, 60.4, 54.6, 14.3; HRMS (ESI): m/z calculated for
C
18H18ClN3O4S 408.0785 found 408.0768 [M+H]+
.
C
23H21BrClN3O5 534.0431 found 536.0420 [M+2]+
.
7.1.1.19. Ethyl 6-((3,5-dimethoxyphenoxy)methyl)-2-oxo-4-(thiophen-2-
yl)-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (6r). Light brown solid;
yield 78%; mp:188–191 °C; FT-IR (cm−1): 2898, 2609, 1859, 1563,
1394, 1275, 785; 1H NMR (500 MHz, DMSO-d6): δ 9.34 (s, 1H), 8.02 (s,
1H), 7.40 (d, J = 5.0 Hz, 1H), 6.96 (d, J = 4.9 Hz, 1H), 6.93 (d,
J = 3.3 Hz, 1H), 6.18 (d, J = 2.1 Hz, 2H), 6.13 (d, J = 2.0 Hz, 1H),
5.49 (d, J = 3.5 Hz, 1H), 5.21 (d, J = 12.2 Hz, 1H), 4.90 (d,
J = 12.2 Hz, 1H), 4.10–4.08 (m, 2H), 3.70 (s, 6H), 1.14 (t,
J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.8, 161.5,
160.2, 152.5, 148.2, 146.2, 127.2, 125.4, 124.3, 102.2, 93.9, 93.9,
64.1, 60.5, 55.6, 49.9, 14.4; HRMS (ESI): m/z calculated for
7.1.1.24. Ethyl 6-((2-(2-bromoacrylamido)-4-chlorophenoxy)methyl)-4-
(4-chlorophenyl)-2-oxo-1,2,
3,
4-tetrahydropyrimidine-5-carboxylate
(10d). White solid; yield 89%; mp: 196–199 °C; FT-IR (cm−1): 3368,
3112, 2889, 1698, 1675, 1638, 1412, 890; 1H NMR (500 MHz, DMSO-
d6): δ 9.61 (s, 1H), 9.40 (s, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.91 (s, 1H),
7.40 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 5.4 Hz, 3H), 7.08 (d, J = 8.9 Hz,
1H), 6.87 (d, J = 2.5 Hz, 1H), 6.35 (d, J = 2.5 Hz, 1H), 5.31 (s, 2H),
5.23 (d, J = 3.1 Hz, 1H), 4.04–3.99 (m, 2H), 1.08 (t, J = 7.1 Hz, 3H);
13C NMR (125 MHz, DMSO-d6): δ 165.0, 160.3, 152.1, 147.6, 146.0,
143.4, 132.5, 128.9, 128.7, 128.3, 125.5, 123.7, 121.8, 114.2, 101.5,
65.2, 60.5, 54.0, 14.3; HRMS (ESI): m/z calculated for
C
20H22N2O6S 419.1277 found 419.1261 [M+H]+
.
C
23H20BrCl2N3O5 568.0042 found 570.0033 [M+2]+
.
7.1.1.20. Ethyl 6-((3,5-dimethoxyphenoxy)methyl)-4-(furan-2-yl)-2-oxo-
1,2,3,4-tetrahydropyrimid ine-5-carboxylate (6s). Light brown solid;
yield 69%; mp:183–187 °C; FT-IR (cm−1): 2920, 2651, 1851, 1556,
1389, 1285, 760; 1H NMR (500 MHz, DMSO-d6): δ 9.25 (s, 1H), 7.88 (s,
1H), 7.58 (d, J = 1.0 Hz, 1H), 6.38 (d J = 3.1 Hz, 1H), 6.19 (d,
J = 2.1 Hz, 2H), 6.16–6.10 (m, 2H), 5.29 (d, J = 3.4 Hz, 1H), 5.22 (d,
J = 12.2 Hz, 1H), 4.93 (d, J = 12.2 Hz, 1H), 4.08–4.04 (m, 2H), 3.71 (s,
6H), 1.12 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.8,
161.5, 160.2, 155.6, 152.6, 147.0, 142.8, 110.8, 106.1, 99.4, 94.0,
64.1, 60.3, 55.6, 48.2, 14.4; HRMS (ESI): m/z calculated for
7.1.1.25. Ethyl 6-((2-(2-bromoacrylamido)-4-chlorophenoxy)methyl)-4-
(4-bromophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(10e). White solid; yield 82%; mp: 198–205 °C; FT-IR (cm−1): 3366,
3207, 2926, 1696, 1685, 1587, 1253, 875; 1H NMR (500 MHz, DMSO-
d6): δ 9.59 (s, 1H), 9.38 (s, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.91 (s, 1H),
7.38 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 6.4 Hz, 3H), 7.08 (d, J = 7.9 Hz,
1H), 6.86 (d, J = 3.5 Hz, 1H), 6.33 (d, J = 3.5 Hz, 1H), 5.29 (s, 2H),
5.23 (d, J = 3.3 Hz, 1H), 4.04–3.99 (m, 2H), 1.08 (t, J = 7.1 Hz, 3H);
13C NMR (125 MHz, DMSO-d6): δ 164.8, 160.3, 155.6, 152.6, 147.7,
146.7, 142.9, 128.3, 128.2, 125.5, 125.3, 123.7, 121.9, 114.1, 110.9,
106.2, 99.2, 65.0, 60.4, 48.2, 14.4; HRMS (ESI): m/z calculated for
C
20H22N2O7 403.1505 found 403.1520 [M+H]+
.
C
23H20Br2ClN3O5 611.9536 found 613.9519 [M+2]+
.
7.1.1.21. Ethyl 6-((2-(2-bromoacrylamido)-4-chlorophenoxy)methyl)-2-
oxo-4-(thiophen-2-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate
7.1.1.26. Ethyl 6-((4-(2-bromoacrylamido)phenoxy)methyl)-2-oxo-4-(p-
tolyl)-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (10f). White solid;
yield 95%; mp: 199–202 °C; FT-IR (cm−1): 3274, 2967, 2881, 1732,
1702, 1609, 1495, 755; 1H NMR (500 MHz, DMSO-d6): δ 10.16 (s, 1H),
9.18 (d, J = 1.4 Hz, 1H), 7.80 (s, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.14 (s,
4H), 7.02–6.96 (m, 2H), 6.73 (d, J = 3.0 Hz, 1H), 6.28 (d, J = 3.0 Hz,
1H), 5.18 (d, J = 9.7 Hz, 2H), 4.99 (d, J = 12.0 Hz, 1H), 4.01–3.99 (m,
2H), 2.27 (s, 3H), 1.06 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-
d6): δ 165.1, 161.1, 155.0, 152.4, 145.9, 141.6, 137.1, 132.3, 129.4,
126.6, 125.9, 125.6, 122.4, 115.3, 102.1, 64.5, 60.2, 54.2, 21.1, 14.3;
(10a). White solid; yield 88%; mp: 192–196 °C; FT-IR (cm−1): 3346,
3119, 2986, 1689, 1654, 1527, 1203, 875; 1H NMR (500 MHz, DMSO-
d6): δ 9.61 (s, 1H), 9.49 (s, 1H), 8.14–7.95 (m, 2H), 7.41 (d, J = 4.9 Hz,
1H), 7.23 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.9 Hz, 1H), 7.03–6.91 (m,
2H), 6.87 (d, J = 2.6 Hz, 1H), 6.35 (d, J = 2.6 Hz, 1H), 5.51 (d,
J = 3.4 Hz, 1H), 5.29 (d, J = 13.3 Hz, 2H), 4.11–4.07 (m, 2H), 1.16
(t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.8, 160.3,
152.4, 148.1, 147.6, 145.8, 128.3, 128.2, 127.2, 125.5, 125.4, 124.4,
123.7, 121.8, 114.0, 102.2, 65.0, 60.6, 49.9, 14.4; HRMS (ESI): m/z
calculated for C21H19BrClN3O5S 539.9996 found 541.9971 [M+2]+
.
HRMS (ESI): m/z calculated for C24H24BrN3O5 514.0978 found
516.0968 [M+2]+
.
7.1.1.22. Ethyl 6-((2-(2-bromoacrylamido)-4-chlorophenoxy)methyl)-4-
(furan-2-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
7.1.1.27. Ethyl
4-([1,1′-biphenyl]-4-yl)-6-((4-(2-bromoacrylamido)
(10b). White solid; yield 95%; mp: 190–195 °C; FT-IR (cm−1): 3369,
3285, 2981, 1695, 1656, 1635, 1253, 831; 1H NMR (500 MHz, DMSO-
d6): δ 9.63 (s, 1H), 9.41 (s, 1H), 8.02 (d, J = 2.6 Hz, 1H), 7.92 (s, 1H),
7.58 (d, J = 5.9 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 8.9 Hz,
1H), 6.87 (d, J = 2.6 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 6.35 (d,
J = 2.6 Hz, 1H), 6.17 (d, J = 3.1 Hz, 1H), 5.36 (d, J = 13.3 Hz, 1H),
5.29 (d, J = 3.4 Hz, 1H), 5.24 (d, J = 13.3 Hz, 1H), 4.09–4.06 (m, 2H),
1.13 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.8, 160.3,
155.6, 152.6, 147.7, 146.7, 142.9, 128.3, 128.2, 125.5, 125.3, 123.7,
121.9, 114.1, 110.9, 106.2, 99.2, 65.0, 60.4, 48.2, 14.4; HRMS (ESI):
m/z calculated for C21H19BrClN3O6 524.0224 found 526.0223 [M
phenoxy)methyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(10g). White solid; yield 78%; mp: 200–206 °C; FT-IR (cm−1): 3364,
3151, 2881, 1685, 1646, 1527, 1418, 813; 1H NMR (500 MHz, DMSO-
d6): δ 9.61 (s, 1H), 9.40 (s, 1H), 8.02 (d, J = 2.5 Hz, 1H), 7.91 (s, 1H),
7.40 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 13.6 Hz, 3H), 7.08 (d, J = 8.9 Hz,
1H), 6.87 (d, J = 2.5 Hz, 1H), 6.35 (d, J = 2.5 Hz, 1H), 5.31 (s, 2H),
5.23 (d, J = 3.1 Hz, 1H), 4.11 (d, J = 7.0 Hz, 2H), 1.08 (t, J = 7.1 Hz,
3H); 13C NMR (125 MHz, DMSO-d6): δ 165.2, 161.2, 154.9, 152.4,
146.1, 143.5, 140.1, 139.9, 129.4, 127.4, 127.2, 127.0, 126.1, 122.5,
115.4, 102.0, 64.5, 60.4, 54.1, 14.3; HRMS (ESI): m/z calculated for
C
29H26BrN3O5 576.1134 found 578.1136 [M+2]+
.
+2]+
.
7.2. Pharmacology
7.2.1. Cell culture
7.1.1.23. Ethyl 6-((2-(2-bromoacrylamido)-4-chlorophenoxy)methyl)-2-
oxo-4-phenyl-1,2,3,4-tetra- hydropyrimidine-5-carboxylate (10c). White
solid; yield 95%; mp: 198–202 °C; FT-IR (cm−1): 3367, 3151, 2891,
1692, 1675, 1643, 1425, 955; 1H NMR (500 MHz, DMSO-d6): δ 9.63 (d,
J = 9.4 Hz, 1H), 9.38 (d, J = 9.4 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.88
(d, J = 7.2 Hz, 1H), 7.29 (d, J = 6.9 Hz, 6H), 7.09 (d, J = 8.7 Hz, 1H),
Cancer cell lines such as colon (HCT-116 and HCT-15, HT-29),
Prostate (DU 145) breast (MCF-7, MDA MB-231) were maintained in
Roswell Park Memorial Institute (RPMI) media while normal lung fi-
broblasts cell line (HFL-1) were maintained in F12k medium
12

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BioorganicChemistry93(2019)103317
supplemented with 10% fetal bovine serum (FBS) stabilized with 1%
antibiotic-antimycotic solution (Sigma) cells were maintained in 5%
CO2 and 98% relative humidity at 37 °C in incubator. When the cells
reached up to 80–90% of confluency, they were sub-cultured using
0.25% trypsin/1 mM EDTA solution for further passage. The com-
pounds were dissolved in DMSO to prepare the stock solution of 10 mM.
Further dilutions were made accordingly with respective media to get
required concentration (0–50 µM).
effect by arresting the specific checkpoint in cell cycle. Here MCF-7
cancer cells were incubated with compound 10f at 0.5 μM for 48 h.
Untreated and treated cells were harvested, washed and fixed overnight
in 70% ethanol in PBS at −20 °C. Fixed cells were pelleted and stained
with cell cycle analysis reagent propidium iodide (50 μg/ml) with
RNase A for 20 min. at 37 °C in dark according to the manufacturer
instructions and about 10,000 events were acquired and analyzed on a
flow cytometer BD FACSVerseTM (BD Biosciences, USA).
7.2.2. Evaluation of in vitro cytotoxic effects
7.2.6. Identification of apoptotic cells
It is a colorimetric assay that measures the reduction of MTT (3-
(4,5-dimethyl thiazol- 2-yl)-2,5-diphenyl tetrazolium bromide) to in-
soluble formazon by mitochondrial succinate dehydrogenase enzyme.
Since reduction of MTT can only happen in metabolically active cells,
the level of activity is the measure of the viability of the cells [37].
Briefly, cells were seeded in 96-well plates at a density of 1000 to 4000
cells per well in 100 μL of complete medium and allowed to grow
overnight for attachment onto the wells. Then the cells were treated
with various concentrations (0–50 µM) of the compounds for a period of
72 h. For cell growth inhibition analysis after 24 h of cell seeding, be-
fore treatment (day 0), absorbance was measured by adding MTT in few
wells. After 72 h incubation, 100 μL of MTT (0.5 mg/ml) was added and
incubated at 37 °C for 4 h. Then MTT reagent was aspirated and the
formazan crystals formed were dissolved by the addition of 200 μL of
DMSO for 20 min. at 37 °C. The formazon product quantity was mea-
sured by using a spectrophotometric microtiter plate reader (Spectra
Max, M4 Molecular devices, USA) at 570 nm wavelength. The day 0
absorbance was subtracted from the 72 h plates for determining growth
inhibition and data were plotted as a percentage of untreated control.
Morphological changes in nucleus were observed through DAPI
staining. After treatment with compound 10f for 72 h, breast cancer cell
line MCF-7 cells were washed with PBS and permeabilized with 0.1%
Tween 20 for 10 min followed by staining with 1 μM DAPI. Control and
treated cells were observed with fluorescence microscope with excita-
tion at 359 nm and emission at 461 nm using DAPI filter at 200x
magnification.
7.2.7. Detection of morphology during apoptosis induction
MCF-7 cells were plated at a concentration of 1 × 106 cells/ml and
treated with different concentrations of compound 10f and the plates
were incubated for 72 h. 10 μL of fluorescent dyes containing Acridine
Orange (AO) and Ethidium Bromide (EB) were added into each well in
equal volumes (10 μg/ml) respectively then the cells were visualized
immediately under fluorescence microscope (Nikon, Inc. Japan) with
excitation (488 nm) and emission (550 nm) at 200X magnification.
7.2.8. Quantification of apoptotic cells
It was performed by the method given by Rieger et al [38] with
slight modifications. Briefly, 1x 105 cells were seeded in a 12-well plate
and treated with different concentration of compound 10f for 72 h.
Then Untreated and treated cells were harvested and the cells were
processed with Annexin V-FITC and Propidium Iodide (PI) staining
(BioLegend) according to the manufacturer’s instructions. Further, flow
cytometric analysis was performed using a flow cytometer (BD FACS-
VerseTM, USA). Apoptosis and necrosis were analyzed with quadrant
statistics on propidium iodide-negative cells, fluorescein positive cells
and propidium iodide (PI)-positive cells, respectively.
7.2.3. Effect of 10f on tubulin polymerization inhibition
Tubulin polymerization kit was procured from Cytoskeleton, Inc.
(BK011). To study the effect of compound 10f, fluorescence based in
vitro tubulin polymerization assay was performed following the man-
ufacturer’s protocol. The reaction mixture having porcine brain tissue
(2 mg/mL) in 80 mM PIPES at pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA,
1.0 mM GTP and glycerol in the presence and absence of test compound
10f (final concentration of 10 mM) was prepared and added to each
well of 96-well plate. Tubulin polymerization was followed by a time
dependent increase in fluorescence due to the insertion of a fluores-
cence reporter into microtubules as polymerization takes place.
Spectramax M4 Multi mode Micro plate Detection System was used to
measure Fluorescence emission at 440 nm (excitation wavelength is
360 nm). podophyllotoxin was used as positive control in the assay at
5 µM final concentration. The IC50 value was calculated from the drug
concentration required for inhibiting 50% of tubulin assembly com-
pared to control. Another compound namely, Paclitaxel will stabilize
the microtubule. The IC50 value was calculated from the drug con-
centration required for inhibiting 50% of tubulin assembly compared to
control.
7.2.9. Effect on mitochondrial membrane potential
Measurement of mitochondrial membrane potential, MCF-7 cells
(1x106 cells/ml) were seeded in 12 well plates and allowed to adhere
for overnight. The cells were incubated with compound 10f at 0.25, 0.5,
and 2 μM concentrations for 72 h. Cells were collected and washed with
PBS and resuspended in solution of JC-1 (1 μM) and incubated for
30 min in incubator at 37 °C. The cells were washed twice with PBS and
analysed by flow cytometer (BD FACSVerse™, USA).
7.2.10. Effect of 10f on intracellular ROS generation
7.2.4. Molecular docking
DCFDA staining was performed to determine the reactive oxygen
species (ROS) levels as per reported method [39] with slight mod-
ifications. For this experiment, MCF-7 cells were plated at cell density
of 3.5x105 cells/well into 12-well plates in Roswell Park Memorial In-
stitute (RPMI) supplemented with 10% FBS. Then the cells were treated
with the compound 10f at various concentrations for 72 h. Then the
DCFDA reagent was added at 10 μM concentration for 15 min and the
fluorescent intensity was obtained using fluorescent spectrophotometer
(Hitachi F7000). The fluorescence readings were taken thrice to elim-
inate errors.
The crystal co-ordinates of α,β-tubulin subunits were retrieved from
the protein data bank (PDB ID: 1SA0). The 3D structure of compound
10f was drawn on Maestro Molecule Builder of Schrödinger. The mo-
lecule was optimized using OPLS_2005 force field in LigPrep module of
Schrödinger. Docking procedure was performed according to the stan-
dard protocol implemented in maestro software, version 9.9 and the
compound 10f was docked into the α,β -tubulin interphase. The ligand-
protein complex was analyzed for interactions and 3D pose of most
active compound 10f was imaged using Schrödinger.
7.2.5. Analysis of cell cycle effect
Flow cytometric analysis (FACS) was performed to calculate the
distribution of the cell population in various cell cycle phases. In gen-
eral the novel compounds exert their cytotoxic or growth inhibitory
Declaration of Competing Interest
The authors declare no conflicts of interest.
13

S. Sana, et al.
BioorganicChemistry93(2019)103317
Acknowledgements
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