Dihydropyrimidinones: Efficient one-pot green synthesis using Montmorillonite-KSF and evaluation of their cytotoxic activity

Article abstract of DOI:10.1039/d0ra09072g

A simple, efficient, cost-effective, recyclable and green approach has been developed for the synthesis of new dihydropyrimidinone analogs via the Biginelli reaction. The methodology involves a multicomponent reaction catalyzed by "HPA-Montmorillonite-KSF"as a reusable and heterogeneous catalyst. This method gives an efficient and much improved modification of the original Biginelli reaction, in terms of yield and short reaction times under solvent free conditions. All the derivatives were subjected to cytotoxicity screening against a panel of four different human cancer cell lines viz. colon (Colo-205), prostate (PC-3), leukemia (THP-1) and lung (A549) to check their effect on percentage growth. MTT [3-(4,5-dimethylthiazol-yl)-diphenyl tetrazoliumbromide] cytotoxicity assay was employed to check IC50 values. Of the synthesized analogs, 16a showed the best activity with IC50 of 7.1 ± 0.8, 13.1 ± 1.4, 13.8 ± 0.9 and 14.7 ± 1.1 μM against lung (A549), leukemia (THP-1), prostate (PC-3) and colon (Colo-205) cancer lines, respectively. The 16a analog was further checked for its effect on cancer cell properties through clonogenic (colony formation) and scratch motility (wound healing) assays and thereby was found that it reduced both the colony formation and migratory properties of the lung cancer cell line (A549). Further, molecular docking studies were performed with 16a to show its binding mode. This journal is

Full text of DOI:10.1039/d0ra09072g

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Dihydropyrimidinones: efficient one-pot green
synthesis using Montmorillonite-KSF and
evaluation of their cytotoxic activity†
Cite this: RSC Adv., 2020, 10, 42221
ab Fahad A. Alharthi,c Ali Alsalme,c Aashiq Hussain,d Bashir A. Dar,e
*
Saleem Farooq,
df
Abid Hamid and S. Koulb
*
A simple, efficient, cost-effective, recyclable and green approach has been developed for the synthesis of
new dihydropyrimidinone analogs via the Biginelli reaction. The methodology involves a multicomponent
reaction catalyzed by “HPA-Montmorillonite-KSF” as a reusable and heterogeneous catalyst. This method
gives an efficient and much improved modification of the original Biginelli reaction, in terms of yield and
short reaction times under solvent free conditions. All the derivatives were subjected to cytotoxicity
screening against a panel of four different human cancer cell lines viz. colon (Colo-205), prostate (PC-3),
leukemia (THP-1) and lung (A549) to check their effect on percentage growth. MTT [3-(4,5-
dimethylthiazol-yl)-diphenyl tetrazoliumbromide] cytotoxicity assay was employed to check IC50 values.
Of the synthesized analogs, 16a showed the best activity with IC50 of 7.1 ꢀ 0.8, 13.1 ꢀ 1.4, 13.8 ꢀ 0.9 and
14.7 ꢀ 1.1 mM against lung (A549), leukemia (THP-1), prostate (PC-3) and colon (Colo-205) cancer lines,
respectively. The 16a analog was further checked for its effect on cancer cell properties through
clonogenic (colony formation) and scratch motility (wound healing) assays and thereby was found that it
reduced both the colony formation and migratory properties of the lung cancer cell line (A549). Further,
molecular docking studies were performed with 16a to show its binding mode.
Received 24th October 2020
Accepted 13th November 2020
DOI: 10.1039/d0ra09072g
rsc.li/rsc-advances
improve the capability to synthesize molecules in more facile,
efficient and in economical ways.2 The paradigms of organic
1. Introduction
synthesis have shied from the traditional concept of efficiency
in terms of chemical yield to one that also considers economic
and ecological values. The signicance of a particular reaction
can be judged on its capability to form products with dimen-
sions of high yield, chemo-, regio-, stereo- or enantio-selec-
tivity.3 In last one decade, there has been an incredible increase
on the accessibility of Multi-Component Reactions (MCRs), and
much still remains to be accomplished.4–9 MCRs are important
cornerstones in the diversity-oriented construction of molecular
complexity due to their ability to incorporate, in a fast and
efficient manner, three or more components into a single
product.10–14 Reactions that build up carbon–carbon bonds and
at the same time introduce nitrogen-containing functionalities
into the structural framework are especially attractive for the
rapid construction of organic molecules. As MCRs are one-pot
processes with simpler experimental conditions that do not
require the isolation of intermediates, they are perfect candi-
dates for combinatorial, and generation of products in a single
synthetic operation15,16 and are of increasing importance in
organic and medicinal chemistry.17–19 Since rapidity and diver-
sity are considered as key factors in modern drug discovery,
MCR strategies offer signicant advantages over conventional
linear-type syntheses, owing to their exceptional synthetic effi-
ciency.20 MCRs contribute to the requirements of an
In modern methods of drug discovery processes, design and
synthesis of drugs based on biological targets is of the great
interest to medicinal chemists. Additionally, semi-synthetic
processes for new compounds, obtained by molecular modi-
cation of the functional groups of lead compounds, are able to
generate structural analogs with greater pharmacological
activity and with fewer side effects.1 A constant enrichment in
the science of organic synthesis through improvement of the
synthetic methodologies is observed, driven by the needs to
aDepartment of Higher Education, Department of Chemistry, Government Degree
College for Boys, Baramulla, 193101, J&K, India. E-mail: farooqprince100@yahoo.
in; Fax: +91-1952-234214; Tel: +91-1952-234214
bBioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal
Road Jammu, 180001, J&K, India
cDepartment of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh, 11451, Saudi Arabia
dCancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal
Road Jammu, 180001, J&K, India
eDepartment of Higher Education, Department of Chemistry, Govt. Degree College
Sopore, Baramulla, 193201, J&K, India
fDepartment of Biotechnology, Central University of Kashmir, Ganderbal, 191201, J&K,
India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra09072g
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environmentally benign process by reducing the number of epithelial cell line (fR-2) followed by clonogenic (colony
synthetic steps, energy consumption and waste production.21 formation) and scratch motility (wound healing) assays of most
Functionalized nitrogen-heterocycles play a prominent role in potent molecule to analyze any effect on certain cancer prop-
medicinal chemistry and therefore they have been intensively erties. Molecular docking studies were carried out to establish
used as scaffolds in the eld of drug development.22–26 Despite the binding capabilities of the same derivative.
several reports on fused heterocycles, there is a continuing
demand for development of new methods for synthesis of novel
fused heterocycles due to their plethora of medicinal
applications.27
2. Results and discussion
2.1. Chemistry
In this context pyrimidine derivatives are of particular
interest because of their interesting pharmacological
prole.28–30 Dihydropyrimidin-2(1H)-ones (DHPMs), also known
as Biginelli compounds are easily accessible via a multicompo-
nent condensation process, rst reported more than a century
ago.31 The synthesis of 3,4-dihydropyrimidin-2(1H)-ones (Bigi-
nelli compounds) and their derivatives is of great importance
due to the biological investigation of these molecules via
molecular manipulation which have shown several activities
such as calcium channel blockers,32–35 a1a-adrenergic receptor
antagonists,35 mitotic kinesin inhibitors35 along with anti-
bacterial and anti-tubercular activities.36,37 Monastrol, a struc-
turally simple DHPM has been found as a novel cell-permeable
molecule that blocks normal bipolar mitotic spindle assembly
in mammalian cells by specically inhibiting the motor activity
of the mitotic kinesin Eg5, a motor protein required for spindle
bipolarity, and therefore, causes cell cycle arrest.38 There are
only a limited number of cell-permeable molecules currently
known to specically inhibit mitotic kinesin Eg5 and such
molecules having DHPM scaffolds can therefore be considered
as leads for the development of new anticancer drugs.
Based on the above cited ndings and inspiration from the
potential anticancer activity of DHPMs,39–44 we directed this
work towards the synthesis of a diverse series of novel DHPM
derivatives of biological interest using a simple, efficient, cost-
effective, recyclable and green approach via Biginelli reaction
involving the one-pot multicomponent reaction catalyzed by
novel “HPA-Montmorillonite-KSF” as a reusable and heteroge-
neous catalyst. All the newly synthesized compounds were
subjected to MTT cytotoxicity screening against a panel of four
different human cancer cell lines viz. colon (Colo-205), prostate
(PC-3), leukemia (THP-1) and lung (A549) along with normal
From the literature scan it is evident that DHPM moiety is
pharmacologically important and many researchers are
engaged to prepare their libraries by applying various modi-
cations using different synthetic strategies to shorten the reac-
tion time and increase the yield using a variation of different
catalysts. Some of the reported catalysts used include Cu(OTf)2/
MWI,45 Yb(PFO)3,46 HCl/EtOH,47 H3PO3/Pd-Cat, TMSCl/CAN,
silica immobilized Ni(II), Ca(OCl)2, Mg(NO3)2, PPA-SiO2,
sulfated tungstate, melamine trisulfonic acid, Cu(NO3)2$3H2O,
acidic ionic liquids, organocatalysts, H3PMo12O40, H3PW12O40,
H6P2W18O62$24H2O, H4PMo11VO40 (ref. 48) and other catalysts.
These chemical methods in spite of their potential utility,
involve expensive/toxic reagents and adverse reaction condi-
tions like, strong acids, long reaction times, high temperature,
stoichiometric amount of catalysts, environmental concerns
and poor yields. Therefore, in order to overcome these limita-
tions, the discovery of a new and efficient catalyst with high
catalytic activity, short reaction time, recyclability and simple
work-up procedures for the preparation of 3,4-dihydropyr-
imidin-2(1H)-ones under neutral, mild and practical conditions
is of prime interest. Biginelli reaction catalysed by hetero-
polyacids (HPAs) can be one of the sources for the synthesis of
large number of new molecules. HPA catalysts are widely
exploited for the production of ne organic chemicals, health
care products, pharmaceuticals and agrochemical products.49
HPAs are more reactive catalysts than conventional inorganic
and organic acids for reactions in solution50 and have been used
in organic transformations, such as synthesis of acylals, tetra-
hydropyranilation of phenols, thioacetalization and trans-
acetalization reactions. They are also used as industrial
catalysts for several liquid-phase reactions, including alcohol
dehydration, alkylation and esterication.51 HPAs that contain
Table 1 H5PV2W10O40 catalysed synthesis of DHPM in different solvents and under solvent free conditionsa
S. no.
Solvent
Time (h)
Amount of catalyst (mol%)
Yield (%)
Temperature (ꢁC)
1
2
3
4
5
6
7
8
9
1,4-Dioxane
Acetonitrile
Toluene
6 h
5 h
7 h
2 h
1 h
1 h
5 minutes
30 minutes
18 h
10
10
10
10
10
2
2
2
2
2
65
77
71
87
92
96
70
74
40
52
Reux
Reux
Reux
Reux
Ethanol
Solvent free
Solvent free
Solvent free
Solvent free
Ethanol
Reux
Reux
Microwave at 50 ꢁC
Sonication at 35 ꢁC
rt (25)
rt
10
Solvent free
14 h
a
Para-Methoxy benzaldehyde : urea : ethylacetoacetate in the ratio of 1 : 1.5 : 1.5.
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Scheme 1 The model for preparation of DHPMs.
super acidic properties have found numerous applications and ethyl acetoacetate and 1 equivalent of aldehyde under
during last three decades, as useful and versatile acid catalysts reux conditions for 1 h, affording the desired product in good
for some acid-catalyzed reactions. They are usually solids that yields (Table 1).
are insoluble in non-polar solvents while as highly soluble in
2.1.1. Optimization of reaction. The model reaction was
polar ones. They can be used in bulk or in supported forms, in carried out by using para-methoxy benzaldehyde (1 eq.), urea
both homogeneous and heterogeneous system. Furthermore, (1.5 eq.) and ethylacetoacetate (1.5) in solvent ethanol and
HPAs have several advantages, including high exibility in catalyst (H5PV2W10O40) under reux conditions for the genera-
modication of the acid strength, ease of handling, environ- tion of corresponding DHPM in good yield (Scheme 1), which
mental compatibility, non-toxicity, and experimental was identied by spectral analysis in light of literature.53
simplicity.52
Encouraged by the initial success with regard to the formation
Inspired by above mentioned facts, a novel green method- of 4f, a comprehensive optimization study was performed with
ology has been developed which involves the synthesis of DHPM the objective of reducing the reaction time and space,
analogs by Biginelli reaction and their in vitro screening against enhancing the yields, minimizing the temperature range and
different human cancer cell lines. The methodology involves the exploration study towards the use of other solvents and catalysts
multicomponent reaction catalyzed by HPA-Montmorillonite- for better yields due to the biological and synthetic importance
KSF (H5PV2W10O40) as a reusable and heterogeneous catalyst. of the DHPMs as described earlier. In the rst set of optimiza-
The strategy involves a three-component one-pot Biginelli-type tion experiments, the reaction was studied in different solvents
reaction for the condensation of urea, ethylacetoacetate and that included acetonitrile, 1,4-dioxane, toluene, ethanol at
different aldehydes to corresponding pyrimidinones in pres- reux conditions. The investigation was also extended for
ence of a catalytic amount of HPA-Montmorillonite-KSF. The solvent free reaction. The use of solvent free conditions had
best conditions were achieved under solvent free conditions profound effect in terms of time saving as the reaction was
(neat) using 2 mol% of HPA, 1.5 equivalents of urea/thiourea complete in 1 h and the yields were excellent (Table 1). The
Table 2 Synthesis of 3,4-dihydropyrimidin-2(1H)-ones
Mp (ꢁC)
Entry
Product
R
X
Yield (%)
Found
Reported
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
C6H5
C6H5
4-(Cl)–C6H4
2-(Cl)–C6H4
O
S
97
95
93
89
93
96
94
95
97
79
83
87
94
81
78
93
94
201–203
206–208
209–211
213–215
211–214
201–204
176–177
157–160
202–204
205–207
210–212
214
213–215
201–203
178
158–160
—
209–211
—
—
—
218–220
227–228
98–100
—
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
4-(Br)–C6H4
4-(MeO)–C6H4
2,3-(OMe)2–C6H3
2,4-(OMe)2–C6H3
3,4,5-(OMe)3–C6H2
4-(NO2)–C6H4
4-(F)–C6H4
2-(Br)-5-(OMe)–C6H3
Piperanal
2-(NO2)–C6H4
4-(OH)–C6H4
3-(OH)-4-(OMe)–C6H3
5-(Br)-2-(OMe)–C6H3
9
10
11
12
13
14
15
16
17
207–208
217–219
226–228
98–99
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Table 3 Comparison the results of the synthesis of 4-aryl-3,4-dihy-
dropyrimidin-2(1H)-one using different catalysts
The next part of the optimization study was to assess the
recycled use of catalyst. It may be stated that due to the
constantly rising environmental concerns in the eld of
chemistry, it is sensible to use easily recovered and recycled
catalysts, especially expensive or toxic metallic ones for the
next use,54 as only few of them meet the criterion of green
chemistry. For example, the recovery of ytterbium triate from
water seems cumbersome since water must be removed
through heating and then drying under vacuum at 100 ꢁC for 2
hours,55 and in the case of polymer-supported Yb(III) resin, the
activity of recycled resin is much lower than that of the original
one thus limiting the recyclability.56 Therefore, there is still
room for further search for recyclable catalysts to be used in
the Biginelli reaction that can convert a variety of aldehydes to
pyrimidinones in high yields under mild reaction conditions.
In order to show the merit of the present work in comparison
with some reported protocols, we compared the results of the
synthesis of 5-ethoxycarbonyl-4-phenyl-6-methyl-3,4-dihy-
dropyrimidin-2(1H)-one in the presence of montmorillonite
KSF, sulfuric acid, zeolite, silica-sulfuric acid, BF3–OEt2/CuCl,
H3PMo12O40 with HPA-Montmorillonite-KSF with respect to
the reaction times (Table 3). The yield of product in presence
HPA-Montmorillonite-KSF is comparable with these catalysts.
However, the reaction in presence of these catalysts required
longer reaction times than HPA-Montmorillonite-KSF. The
catalyst could be reused several times (six times) and did not
show any signicant negative inuence on the overall yields of
the reaction (Fig. 1).
2.1.2. Synthesis of novel 3,4-dihydropyrimidin-2(1H)-one
derivatives. To explore the newly designed Biginelli reaction,
versatile aldehydes (both aromatic and aliphatic aldehydes)
were used, with some of them obtained by modication of
natural products or through synthetic route. Natural product
osthol isolated from plant Prangos pabularia was oxidized into
corresponding aldehyde 5 by using selenium dioxide (Scheme
2). Other synthetic aldehydes were prepared by performing
Grignard and Vilsmeier reactions by following the procedure
reported earlier57 (Scheme 3). The purpose to use these alde-
hydes was to extend the scope of Biginelli reaction besides
generation of new library of DHPM products not reported
hitherto in the literature and more importantly to study their
bioevaluation proles (Scheme 4 and Table 4). The structures of
the newly synthesized compounds have been established on the
basis of elemental analysis and spectroscopic techniques and
are presented in section 4.1.4 (Experimental).
Entry Catalyst
Time (h) Yield (%)
1
2
3
4
5
6
7
Montmorillonite KSF
Sulphuric acid
Zeolite
Silica sulphur acid
BF3$OEt2/CuCl
48
18
12
16
18
5
82
71
80
91
71
80
96
H3PMo12O40
HPA-Montmorilonite-KSF (present work)
1
Fig. 1 Recyclability of the catalyst.
reaction was also optimized with respect to the use of
appropriate/optimum amount of catalyst (H5PV2W10O40) and
the best yields at minimum time were obtained by using
2 mol% of HPA. In addition to this, the reaction was monitored
at different temperature conditions in microwave; moreover
sonication methods were also tried. Microwave assisted reac-
tion adsorbed on silica gel (mesh 60–120) for 5–6 minutes with
H5PV2W10O40 as catalyst furnished product 4 in low yield.
ꢁ
However, under ultrasonication at 35 C with reaction time of
30 minutes yields were low as compared to that at reux.
Reaction was also performed at room temperature for stirring, it
takes long time for the completion of the reaction and yields
were comparatively poor. From the above results, it may be
concluded that the temperature affects not only yield of the
products, but also the reaction time. The results of the opti-
mization of the reaction conditions are summarized in (Table
1). This methodology is effective with a variety of substituted
aliphatic and aromatic aldehydes independent of the nature of
substituents (electron donating or electron withdrawing) in the
aromatic ring, representing an improvement to the classical
Biginelli's methodology as shown in Table 2.
Scheme 2 Oxidation of osthol to aldehyde 5.
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Scheme 3 Preparation of aldehydes using Grignard and Vilsmeier reactions.
Scheme 4 General procedure for preparation of novel DHPM analogs.
these anti-cancer molecules have been used in in vitro studies in
different cancer cells.58–61 We have in our lab used 5-uoro-
uracil, paclitaxel and doxorubicin as experimental positive
controls for Colo-205 and fR-2; PC-3 and A549; and THP-1,
respectively in MTT assay. Most of the synthesized analogs
displayed broad spectrum cytotoxic effect in a dose dependent
manner. The cytotoxicity prole indicates that the osthol
derived DHPM derivative 5a demonstrated cytotoxic effect
against colon (Colo-205), prostate (PC-3), leukemia (THP-1) and
lung (A549) cancer cell lines with IC50 of 37.6 ꢀ 1.1, 40.8 ꢀ 1.7,
65.8 ꢀ 1.4 and 67.8 ꢀ 1.7 mM, respectively; however, it was also
toxic against normal cell line fR-2 with IC50 of 68.8 ꢀ 1.1 mM.
DHPM derivatives with varying carbon chain length of R
group showed interesting results wherein compound 8a bearing
n-pentyl chain showed improved cytotoxic effect against colon
(Colo-205), prostate (PC-3) leukemia (THP-1) and lung (A549)
cancer cell lines with IC50 of 18.6 ꢀ 1.0, 22.2 ꢀ 1.6, 43.8 ꢀ 1.4
and 63.4 ꢀ 1.1 mM, respectively; however, still toxic against
normal cell line (fR-2) being with IC50 of 67.2 ꢀ 1.5 mM. On the
other hand compounds 6a, 7a and 9a bearing n-propyl, n-butyl
and n-hexyl R groups lost their activity with IC50 values not in
2.2. Biology
2.2.1. In vitro screening of the novel analogs against
human cancer cell lines. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was used to screen
the newly synthesized analogs against a panel of four different
human cancer cell lines viz. colon (Colo-205), prostate (PC-3),
leukemia (THP-1) and lung (A549) along with normal epithe-
lial cell line (fR-2). The newly synthesized analogs were screened
for their effect on cell growth/viability at the concentrations of
0.07, 0.7, 7.0 and 70.0 mM and concentration dependent growth
curves were drawn to determine the IC50 values (ESI Fig. S6† and
Table 5). The values are the average of triplicate analysis. The
range of 0.07–70 mM concentration of analogs was chosen based
on our initial IC50 values determination experiment wherein we
found most potent analog has IC50 around 7 mM; so maximum
IC50 we seeked to determine in repeat experiment discussed
below was in 10-fold range viz 70 mM. Furthermore, 5-uoro-
uracil, paclitaxel and doxorubicin are known chemotherapy
agents used against several cancer types particularly colon and
breast; pancreatic and lung; and leukemia, respectively. Also,
several studies have been already done by other groups where
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Table 4 Novel 3,4-dihydropyrimidin-2(1H)-ones
Entry
Product
R
X
Yield (%)
1
5a
O
96
2
3
4
5
6a
7a
8a
9a
O
O
O
O
93
95
93
89
6
10a
O
93
7
8
11a
12a
O
O
96
94
9
13a
14a
15a
O
O
O
95
92
92
10
11
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Table 4 (Contd.)
Entry
Product
R
X
Yield (%)
92
12
13
16a
O
17a
18a
O
O
87
89
14
15
19a
O
88
16
17
20a
21a
O
O
90
93
the range of #70 mM, thereby demonstrating the optimum cell line with IC50 of 27.3 ꢀ 1.8 mM, thereby making provision
chain length responsible for cytotoxicity. Compound 10a having for synthesis of similar DHPM analogs having more activity and
hydroxyl-styryl R group showed activity against PC-3 and THP-1 less toxicity. This observation is further supported in 20a and
with IC50 of 29.4 ꢀ 1.2, and 42.1 ꢀ 1.7 mM, respectively. 21a wherein an extra ethylene group in 20a enhances the activity
Compound 11a demonstrated selective cytotoxicity against (23.4 ꢀ 1.4 mM) by two fold as compared to 21a (48.1 ꢀ 2.1 mM).
prostate (PC-3) cancer cell line with IC50 of 29.3 ꢀ 1.3 mM while
Among all the tested DHPM analogs, 16a bearing a p-
as its corresponding chloro-derivative (12a) totally lost its methoxy phenyl moiety displayed the most potent cytotoxic
activity with IC50 values not in the range of #70 mM. Compound effect with IC50 of 14.7 ꢀ 1.1, 13.8 ꢀ 0.9, 13.1 ꢀ 1.4 and 7.1 ꢀ 0.8
13a showed broad spectrum cytotoxic effect against colon (Colo- mM against colon (Colo-205), prostate (PC-3), leukemia (THP-1)
205), prostate (PC-3), leukemia (THP-1) and lung (A549) cancer and lung (A549) cancer lines respectively. 17a and 19a with
cell line with IC50 of 59.4 ꢀ 1.6, 35.4 ꢀ 2.2, 23.2 ꢀ 1.4 and 30.3 ꢀ dimethoxy phenyl moieties showed moderate cytotoxicity
2.9 mM, respectively while toxicity against normal cell line (fR-2) against lung (A549), prostate (PC-3) and colon (Colo-205) cancer
determined with IC50 of 67.6 ꢀ 2.5 mM. The role of an extra lines, while as, 18a with trimethoxy phenyl moiety lost its
ethylene moiety in 15a as compared 14a is highlighted in terms activity with IC50 values not in the range of #70 mM. These
of its selective cytotoxic activity against colon (Colo-205) cancer observations demonstrate that the activity of such DHPM
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Table 5 IC50 values of analogs for 24 h treatment against colon (Colo-205), prostate (PC-3), leukemia (THP-1) and lung (A549) cancer cell lines
using MTT assay. Normal epithelial cell line (fR-2) was used as control. n ¼ 3. mean ꢀ SDa
Colon (Colo-205)
Compound
IC50
Prostate (PC-3) IC50
Leukemia (THP-1) IC50
Lung (A549) IC50
Normal (fR-2) IC50
5a
6a
37.6 ꢀ 1.1
>70.0
40.8 ꢀ 1.7
>70.0
65.8 ꢀ 1.4
>70.0
67.8 ꢀ 1.7
>70.0
68.8 ꢀ 1.1
>70.0
7a
>70.0
>70.0
>70.0
>70.0
>70.0
8a
9a
18.6 ꢀ 1.0
22.2 ꢀ 1.6
43.8 ꢀ 1.4
63.4 ꢀ 1.1
67.2 ꢀ 1.5
>70.0
>70.0
>70.0
>70.0
>70.0
10a
11a
12a
>70.0
>70.0
>70.0
29.4 ꢀ 1.2
29.3 ꢀ 1.3
>70.0
42.1 ꢀ 1.7
>70.0
>70.0
>70.0
>70.0
>70.0
>70.0
>70.0
>70.0
13a
14a
59.4 ꢀ 1.6
35.4 ꢀ 2.2
23.2 ꢀ 1.4
30.3 ꢀ 2.9
67.6 ꢀ 2.5
>70.0
>70.0
>70.0
>70.0
>70.0
15a
16a
17a
18a
27.3 ꢀ 1.8
14.7 ꢀ 1.1
38.2 ꢀ 1.4
>70.0
>70.0
>70.0
>70.0
>70.0
>70.0
>70.0
>70.0
13.8 ꢀ 0.9
47.9 ꢀ 2.1
>70.0
13.1 ꢀ 1.4
7.1 ꢀ 0.8
26.4 ꢀ 1.1
>70.0
>70.0
>70.0
19a
20a
21a
33.7 ꢀ 1.8
23.4 ꢀ 1.4
48.1 ꢀ 2.1
33.9 ꢀ 1.9
—
>70.0
>70.0
>70.0
—
>70.0
>70.0
>70.0
—
47.8 ꢀ 2.4
>70.0
>70.0
68.7 ꢀ 1.1
>70.0
—
>70.0
5-Fluorouracil
Paclitaxel
Doxorubicin
334.7 ꢀ 3.8
—
—
0.048 ꢀ 0.008
—
5.1 ꢀ 0.4
—
—
0.018 ꢀ 0.004
—
a
IC50 values are expressed in mM concentration. 5-Fluorouracil (Colo-205, fR-2), paclitaxel (PC-3, A549) and doxorubicin (THP-1) were used as
positive controls.
analogs increases as the number of methoxy groups decreases compound 16a inhibits clonogenic property of A549 cells
in the phenyl moiety of R group. All these results suggest that signicantly at 7 and 15 mM when compared with vehicle
the structural features have profound inuence on the biolog- DMSO; staurosporine is already known to have effect on
ical prole of a compound. It was concluded that the compound mobility and invasiveness of A549 cells,58 hence was used as
16a is the most potent derivative with minimum IC50 of 7.1 ꢀ positive control (Fig. 2). To check the anti-metastatic activity,
0.8 mM against lung cancer line (A549). Additionally the deriv- 16a was further evaluated by scratch motility (wound healing)
ative had least toxicity against normal epithelial cell line (fR-2) assay against A549 cell line. Aer A549 cells got attached over-
with IC50 value in the range of >70 mM.
night incubation, the cell monolayers were wounded and PBS
One of the hallmark traits of cancer cells is their ability to washed. This was followed by treatment with 16a/DMSO in
undergo isolated clonal growth. Clonogenic assay was per- culture media. The vehicle DMSO treated cells had almost
formed to test the colony formation property of single cell to completely lled in the cleared area, whereas treatment with 7
grow into a colony. This assay conrmed that most potent and 15 mM of compound 16a signicantly abrogates motility
Fig. 2 (A) A549 cells (1 ꢂ 103 cells per well) were cultured and treated with various concentration of compound 16a for 24 h at 37 ꢁC and then
stained with crystal violet (for details see Result section), number of stained colonies were counted, photographed (magnification, 100ꢂ; scale
bar, 200 mm) and (B) data was calculated from three independent experiments. Columns mean; bars SD of three independent experiments. **P <
0.01 compared with untreated control.
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Fig. 3 (A) A549 cells (0.5 ꢂ 105 cells per well) were grown to confluence in six well tissue culture plate and scratched with sterile tip, compound
16a was added to cultures as indicated. Scratched areas were photographed (magnification, 100ꢂ; scale bar, 200 mm) at zero hour and then
subsequently again 24 h later to assess the degree of wound healing. (B) The scratched areas were quantified in three random fields in each
treatment, and data were calculated from three independent experiments. Data were calculated from three independent experiments. Columns
mean; bars SD of three independent experiments. **P < 0.01 compared with untreated control.
Fig. 4 Molecular docking studies showing probable binding mode of 16a.
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and invasion potential of A549 cells (Fig. 3). Again staur-
osporine was used as positive control.62
4. Experimental
4.1. Chemistry
All reagents for chemical synthesis were obtained from Sigma
Aldrich and the solvents used in reactions were distilled and
dried prior to use. All the chemical reactions were monitored by
TLC on 0.25 mm silica gel 60 F254 plates (E. Merck) using 2%
ceric ammonium sulphate solution for detection of the spots.
Purication of compounds was carried out by column chro-
2.3. Molecular docking studies
In order to rationalize the obtained biological data and under-
stand the probable mode of binding, the most potent
compound (16a) were docked into the crystal structure. On
analysis, 16a was found to bind with better affinity on CDK2 and
PI3Kg which supports the in vitro cytotoxic activity data. The
interaction gure analysis of compound 16a with CDK2 reveals
that the dihydropyrimidinone moiety of 16a is involved in
bidentate H-bonding with Leu83 (hydrophobic) amino acid
residue and methoxy-benzene moiety is forming a pi–pi stack-
ing with Phe80 (hydrophobic) which favors more affinity of the
molecules for CDK2. Additionally, the “O” of methoxy-benzene
moiety is for forming third H-bond with Asp-145 of CDK-2.
Further the interaction gure analysis of compound 16a with
PI3Kg shows that the dihydropyrimidinone moiety of 16a is
involved in bidentate H-bonding with Tyr867 (hydrophobic) and
Asp964 (acidic) amino acid residue and esteric carbonyl oxygen
is forming H-bonding with the hinge residue Val882 (hydro-
phobic) (Fig. 4). The three H-bondings enhance the affinity of
the 16a molecule for both CDK-2 and PI3Kg.
We concluded earlier from SAR analysis that phenyl moiety
as side chain of dihydropyrimidone increased the specicity
and cytotoxicity of 16a. So, we did docking studies with 6a too
which has one of the simplest aliphatic (propyl) as side chain
for comparison. The docking data revealed that only two H-
bonding were being formed between 6a and the enzymes,
with Leu83 in case of CDK-2 and with Val882 and Glu880 in case
of PI3Kg compared to three in case of 16a. Further the absence
of methoxy-benzene moiety in 6a leads to loss of pi–pi stacking.
So, this docking comparison explains partially why phenyl
moiety containing 16a molecule has increased cytotoxicity.
1
matography using silica gel 60–120 mesh stationary phase. H
NMR and 13C NMR spectra (with chemical shis expressed in
d and coupling constants in hertz) were recorded on Bruker DPX
200, 400 and DPX 500 instruments using CDCl3 or CD3OD as the
solvents with TMS as internal standard. High resolution mass
spectra (HRMS) were recorded on Agilent Technologies 6540
instrument and IR recorded on an FT-IR Bruker (270-30) spec-
trophotometer. Melting points of compounds were recorded on
Buchi melting point apparatus B-542.
4.1.1. Preparation of catalyst H5PV2W10O40$10H2O (HPA).
Disodium hydrogen phosphate (Na2HPO4, 1.775 g, 25 mmol)
was dissolved in 50 mL of water and mixed with vanadium
pentoxide (V2O5, 6.1 g, 100 mmol) dissolved in 20 mL of 1 M
Na2CO3 and the solution so formed was boiled for 30 min (green
colour) and then cooled to room temperature. To this solution,
sodium tungstate dihydrate (Na2WO4$2H2O, 41.25 g, 250 mmol)
dissolved in 20 mL of water (black colour) was added. This
solution was kept at 90 ꢁC for 30 min (bluish green colour),
cooled and to it was added 50% sulfuric acid (50 mL) drop wise,
a wine red solution (pH ¼ 2) was obtained. Extraction of the
solution with diethyl ether (100 mL) afforded the orange-red
H5PV2W10O40$10H2O product.
The HPA so formed was dissolved in 150 mL water and
added dropwise to 500 mL aqueous suspension of 10 g Mont-
morillonite KSF. This mixture was stirred for 5 h and the water
was evaporated over water bath to get a dry powder, which was
kept overnight in hot air oven at 110 ꢁC. A portion of this solid
was then calcined at 425 ꢁC for 3 h to get a heteropoly acid clay
nano hybrid and was named as HCNH. The catalyst was char-
acterized using X-ray powder diffraction (XRD) and scanning
electron microscopy as discussed in ESI.†
4.1.2. Preparation of aldehyde 5 (Scheme 2). Compound 5
was prepared by adding selenium dioxide (1.3 mmol) to a solu-
tion of osthol (1.3 mmol) dissolved in glacial acetic acid (5 mL)
and stirred for about 3 h. On completion of the reaction
(monitored by TLC), the contents were poured into crushed ice
and extracted with dichloromethane (3 ꢂ 50 mL), the organic
layer was washed with water (2 ꢂ 10 mL) dried over sodium
sulfate and concentrated under vacuo to give crude product,
which on silica gel column chromatography yielded pure alde-
hyde in 50% yield. 1H NMR (CDCl3, 400 MHz): d 9.4 (s, 1H), 7.6
(1H, d, J ¼ 9.5 Hz), 7.3 (1H, d, J ¼ 8.6 Hz), 6.8 (1H, d, J ¼ 8.6 Hz),
6.5 (1H, t, J ¼ 7.3 Hz), 6.2 (1H, d, J ¼ 9.4 Hz), 3.8 (2H, d, J ¼ 7.3
Hz), 3.9 (3H, s), 1.9 (3H, s). 13C NMR (CDCl3, 100 MHz): d 195.4,
160.79, 160.30, 153.0, 150.74, 143.64, 139.81, 127.38, 114.2,
113.3, 113.1, 107.4, 56.1, 22.8, 9.2. IR (KBr) nmax cmꢃ1: 2923,
3. Conclusions
We have reported a catalytic method for the synthesis of dihy-
dropyrimidinones using HPA-Montmorillonite-KSF as an effi-
cient and eco-friendly heterogeneous inorganic catalyst. It is
noteworthy to mention that the catalyst is reusable and even
aer ve runs; the catalytic activity of H5PV2W10O40$10H2O was
retained as that of the freshly used catalyst. The short reaction
times, simple work-up and isolation of the products in high
yields with high purity, and mild reaction conditions, are some
of the highlights of current procedure. A diverse library of
DHPM analogs have been synthesized by using this catalyst and
most of the derivatives displayed impressive anti-proliferative
effect at 50 mM, with best cytotoxic effect observed for 16a
exhibiting the inhibition against all the tested cancer cell lines
with most potency against lung (A549) cancer cells as 7.1 ꢀ 0.8
mM while the toxicity against the normal epithelial cells was
apparently more than 10 fold higher (>70 mM). However, in vivo
studies are warranted to investigate the mechanisms of action
responsible for the cytotoxic activity of the most active
derivatives.
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1728, 1608, 1497, 1280, 1117, 1093. HR-ESIMS m/z: 259.0793
(calculated for C15H14O4, 258.0892).
4.1.4.3. Ethyl
4-butyl-1,2,3,4-tetrahydro-6-methyl-2-
ꢁ
oxopyrimidine-5-carboxylate 7a. White solid, (mp: 146–149 C).
1H NMR (400 MHz, CDCl3): d 4.2 (1H, m), 4.1 (2H, q, J ¼ 7.1 Hz),
4.1.3. Preparation aldehydes 15–20 (Scheme 3). To the
ethereal solution of substituted aldehyde (110 mmol), freshly
prepared Gigrand's reagent (110 mmol) was added at ꢃ5 ꢁC and
the contents were stirred for 2 h at 0–5 ꢁC. Aer the completion
of reaction, saturated solution of ammonium chloride (10 mL)
was added followed by dilution with water. The product was
extracted with ether (2 ꢂ 50 mL) and the combined organic
layer was washed with water (2 ꢂ 20 mL), dried over anhydrous
sodium sulphate and concentrated under vacuum to give the
corresponding secondary alcohol as a gummy mass (95% yield).
To a chilled solution of Scheme 3 products in DMF, phosphoryl
chloride was added slowly at 0 ꢁC for 1 hour. The contents were
stirred further for 2 hours and then allowed to attain room
temperature followed by heating on a water bath for 3 hours.
The reaction mixture was cooled and a saturated solution of
sodium acetate (15 mL) was added followed by dilution with
water. The contents of the reaction mixture were extracted with
ethyl acetate (4 ꢂ 25 mL), the organic layer was washed with
water (3 ꢂ 20 mL) and dried over sodium sulfate, ltered and
concentrated under vacuo to give crude product, which on silica
gel column chromatography yielded the required aldehydes57
(Scheme 3).
2.2 (3H, s), 1.4 (2H, m), 1.2 (7H, m), 0.9 (3H, t, J ¼ 7.4 Hz). 13
C
NMR (100 MHz, CDCl3): d 167.4, 154.8, 146.8, 101.5, 59.9, 51.4,
36.8, 31.4, 24, 22.5, 14.3, 14. IR (KBr) nmax cmꢃ1: 1744, 1717,
1705, 1637, 1440, 1107. HR-ESIMS m/z: 241.1546 (calculated for
C
12H20N2O3, 240.1474).
4.1.4.4. Ethyl-methyl-2-oxo-4-pentyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate 8a. White solid, (mp: 150–
152 ꢁC). 1H NMR (400 MHz, CDCl3): d 4.8 (1H, s), 4.1 (1H, m), 4
(2H, q, J ¼ 7.3 Hz), 3.2 (1H, s), 2.1 (3H, s), 1.4 (2H, m), 1.9 (6H,
m), 0.8 (3H, t, J ¼ 7.6 Hz). 13C NMR (100 MHz, CDCl3): d 167.4,
154.8, 146.8, 101.5, 59.9, 51.4, 31.4, 37.4, 24, 22.5, 18.5, 14.3, 14.
IR (KBr) nmax cmꢃ1: 1744, 1705, 1637, 1617, 1440. HR-ESIMS m/z:
255.1703 (calculated for C13H22N2O3, 254.1630).
4.1.4.5. Ethyl-4-hexyl-1,2,3,4-tetrahydro-6-methyl-2-oxo-
1
ꢁ
pyrimidine-5-carboxylate 9a. White solid, (mp: 153–156 C). H
NMR (400 MHz, CDCl3): d 4.6 (1H, s), 4.1 (1H, m), 4 (2H, q, J ¼
7.2 Hz), 3 (1H, s), 2.1 (3H, s), 1.4 (2H, m), 1.2 (8H, m), 0.8 (3H, t, J
¼ 7.3 Hz). 13C NMR (100 MHz, CDCl3): d 166.7, 153.2, 145.8,
100.3, 59.5, 50.4, 32.2, 24.3, 23.1, 19.29, 15.3, 14.2, 13.9. IR (KBr)
nmax cmꢃ1: 1744, 1705, 1637, 1617, 1440. HR-ESIMS m/z:
269.1853 (calculated for C14H24N2O3, 268.1787).
4.1.4. General procedure for the synthesis of DHPM
derivatives (Scheme 4). A mixture of aldehyde (10 mmol), 1,3-
dicarbonyl compound (15 mmol), urea or thiourea (15 mmol)
and HPA-Montmorillonite-KSF (2 mol%) was reuxed for 1 h
using solvent free conditions and the progress of the reaction
was monitored by TLC. Upon completion of the reaction, the
mixture was cooled to room temperature and the catalyst was
subsequently removed by ltration and the solution was poured
on to ice-water (30 mL). The resulting solid product was ltered
and recrystalized from ethanol to give the pure products. Aer
removing the reaction products by ltration, the solid catalyst
was washed with ethanol and reused for the second run of the
Biginelli reaction.
4.1.4.6. Ethyl-4-(3-hydroxystyryl)-1,2,3,4-tetrahydro-6-methyl-
2-oxopyrimidine-5-carboxylate 10a. Yellowish powder solid, (mp:
135–137 ꢁC). 1H NMR (400 MHz, CDCl3): d 7.3 (1H, m), 7.3 (1H,
m), 7.2 (1H, m), 7.2 (1H, m), 6.5 (1H, d, J ¼ 16 Hz), 6.2 (1H, dd, J
¼ 3.9, 16 Hz), 4.2 (1H, m), 4.1 (2H, q, J ¼ 7.4 Hz), 2.3 (3H, s), 1.3
(3H, t, J ¼ 7.3 Hz). 13C NMR (100 MHz, CDCl3): d 167.6, 158.4,
154.5, 144.6, 130.6, 130.1, 129.7, 128.8, 127.6, 118.3, 109.7,
101.8, 59.7, 53.7, 18.4, 14.9. IR (KBr) nmax cmꢃ1: 3240, 3117,
2960, 1693, 1514, 1462. HR-ESIMS m/z: 303.1339 (calculated for
C
16H18N2O4, 302.1267).
4.1.4.7. Ethyl-4-(3,4-dihydronaphthalen-2-yl)-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate 11a. Pale yellow solid,
(mp: 181–183 ꢁC). 1H NMR (400 MHz, CDCl3): d 7.1–7.2 (4H, m),
6.3 (1H, s), 5.3 (1H, s), 5 (1H, s), 4.1 (2H, q, J ¼ 7.6 Hz), 2.8 (2H, t,
J ¼ 7.6 Hz), 2.3 (3H, s), 2.2 (2H, m), 1.2 (3H, t, J ¼ 7.5 Hz). 13C
NMR (100 MHz, CDCl3): d 167.7, 155.7, 149.5, 142.3, 136.1,
135.2, 128.2, 128.4, 127.5, 125, 99.8, 61.1, 29.2, 23.9, 18.3, 14.8.
IR (KBr) nmax cmꢃ1: 3240, 3117, 2960, 1693, 1728, 1681, 1608,
1514. HR-ESIMS m/z: 313.1546 (calculated for C18H20N2O3,
312.1474).
4.1.4.1. Ethyl 4-(4-(6-methoxy-2-oxo-2H-chromen-7-yl)but-2-
en-2-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate 5a. Yellowish solid, (mp: 144–146 ꢁC). 1H NMR (400
MHz, CDCl3): d 7.6 (1H, d, J ¼ 9.5 Hz), 7.3 (1H, d, J ¼ 8.6 Hz), 7.2
(1H, s), 6.8 (1H, d, J ¼ 8.6 Hz), 6.5 (1H, t, J ¼ 7.3 Hz), 6.2 (1H, d, J
¼ 9.4 Hz), 4.9 (1H, s), 4.1 (2H, q, J ¼ 8 Hz), 3.9 (3H, s), 3.8 (2H, d,
J ¼ 7.6 Hz), 2.3 (3H, s), 1.9 (3H, s), 1.2 (3H, t, J ¼ 8 Hz). 13C NMR
(100 MHz, CDCl3): d 169.4, 161.8, 155.7, 154.6, 151.5, 149.3,
142.8, 134.5, 133.2, 126.1, 119.8, 119.3, 114.7, 106.8, 92.8, 61.5,
56.7, 48.9, 30.3, 18.9, 16.2, 14.6. IR (KBr) nmax cmꢃ1: 3340, 3012,
1728, 1681, 1072, 832. HR-ESIMS m/z: 413.4424 (calculated for
4.1.4.8. Ethyl-4-(1-chloro-3,4-dihydronaphthalen-2-yl)-6-
methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
12a.
White solid, (mp: 162–164 ꢁC). 1H NMR (400 MHz, CDCl3): d 7.6
(1H, m), 7.2 (2H, m), 7.1 (1H, m), 5.8 (1H, s), 5.3 (1H, s), 5.2 (1H,
s), 4.1 (2H, q, J ¼ 6.7 Hz), 2.8 (2H, m), 2.3 (3H, s), 2.4 (2H, m), 1.1
(3H, t, J ¼ 7.3 Hz). 13C NMR (100 MHz, CDCl3): d 167.5, 154.8,
150.3, 137.7, 137.4, 134.14, 129.5, 128.3, 127.9, 127.4, 126.2,
98.6, 61.4, 29.2, 25.1, 18.9, 15.2. IR (KBr) nmax cmꢃ1: 3235, 2936,
1693, 1700, 1681, 1596. HR-ESIMS m/z: 347.1156 (calculated for
C
22H24N2O6, 412.4358).
4.1.4.2. Ethyl
1,2,3,4-tetrahydro-6-methyl-2-oxo-4-
propylpyrimidine-5-carboxylate 6a. White solid, (mp: 156–158
ꢁC). 1H NMR (400 MHz, CDCl3): d 4.2 (1H, m), 4.2 (2H, q, J ¼ 6.7
Hz), 2.2 (3H, s), 1.4 (2H, m), 1.28 (4H, m), 0.9 (3H, t, J ¼ 6.9 Hz).
13C NMR (100 MHz, CDCl3): d 167.8, 156, 149.2, 102.2, 61.1,
51.9, 40.3, 18.4, 18.1, 14.6, 14.2. IR (KBr) nmax cmꢃ1: 1744, 1717,
1637, 1440, 1215. HR-ESIMS m/z: 227.1393 (calculated for
C
18H19ClN2O3, 346.1084).
4.1.4.9. Ethyl-4-((Z)-3-(methoxycarbonyl)-1-chloro-1-(3,4-
dimethoxyphenyl)prop-1-en-2-yl)-1,2,3,4-tetrahydro-6-methyl-2-
C
11H18N2O3, 226.1317).
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150.2, 144.6, 130, 127.1, 122.5, (2C, 104.5), 101.9, 59.7, 56.3, (2C,
55.3), 36.4, 19.5, 14.34. IR (KBr) nmax cmꢃ1: 3240, 2960, 1693,
1514, 1462. HR-ESIMS m/z: 377.1822 (calculated for
oxopyrimidine-5-carboxylate 13a. Brown powder solid, (mp: 126–
128 ꢁC). 1H NMR (400 MHz, CDCl3): d 7.1 (1H, d, J ¼ 2.7 Hz), 7
(1H, dd, J ¼ 2.3 & 8.3 Hz), 6.9 (1H, d, J ¼ 8.3 Hz), 4.5 (1H, s), 4.2
(2H, q, J ¼ 7.2 Hz), 3.6 (6H, s), 3.6 (3H, s), 2.7 (2H, s), 2.2 (3H, s),
1.3 (3H, t, J ¼ 6.9 Hz). 13C NMR (100 MHz, CDCl3): d 174, 169.9,
155.7, 151.3, 150.1, 148.5, 143.4, 134.3, 123.1, 121.6, 113.8, 113,
C
19H24N2O6, 376.1634).
4.1.4.15. Ethyl-4-(2,4-dimethoxystyryl)-1,2,3,4-tetrahydro-6-
methyl-2-oxopyrimidine-5-carboxylate 19a. Yellowish liquid. 1H
NMR (400 MHz, CDCl3): d 7.2 (1H, s), 7 (1H, m), 6.8 (1H, d, J ¼ 16
Hz), 6.2 (2H, m), 5.96 (1H, dd, J ¼ 3.2, 16 Hz), 5.5 (1H, s), 4.5 (1H,
d, J ¼ 4 Hz), 4.1 (2H, q, J ¼ 7.3 Hz), 3.7 (6H, s), 2.4 (3H, s), 1.2
(3H, t, J ¼ 7.1 Hz). 13C NMR (100 MHz, CDCl3): d 167.7, 159.8,
156.7, 154.2, 144.6, 130, 127.1, 122.5, 119.9, 104.5, 101.9, 93.8,
59.7, (2C, 55.3), 36.4, 19.5, 14.3. IR (KBr) nmax cmꢃ1: 3240, 2960,
1693, 1514, 1097. HR-ESIMS m/z: 347.1611 (calculated for
95.1, 61.5, 57.6, 56.7, 51.8, 36.3, 16.3, 14.7. IR (KBr) nmax cmꢃ1
:
3195, 2860, 1693, 1514, 1462. HR-ESIMS m/z: 453.1423 (calcu-
lated for C21H25ClN2O7, 452.1350).
4.1.4.10. Ethyl-1,2,3,4-tetrahydro-6-methyl-4-(2,2-
dimethylchroman-6-yl)-2-oxo-pyrimidine-5-carboxylate
14a.
1
ꢁ
Yellowish solid, (mp: 134–137 C). H NMR (400 MHz, CDCl3):
d 7.5 (1H, s), 7 (2H, m), 6.6 (1H, d, J ¼ 2 Hz), 5.4 (1H, m), 5.3 (1H,
s), 4.1 (2H, q, J ¼ 7.6 Hz), 2.7 (2H, t, J ¼ 7.5 Hz), 2.3 (3H, s), 1.7
(2H, t, J ¼ 7.5 Hz), 1.3 (6H, s), 1.2 (3H, t, J ¼ 7.6 Hz). 13C NMR
(100 MHz, CDCl3): d 165.8, 153.7, 145.9, 135, 127.7, 125.7, 120.9,
117.2, 101.6, 74.3, 59.9, 55.2, 54, 32.6, 30.2, 26.8, 22.5, 18.6, 14.1.
IR (KBr) nmax cmꢃ1: 3335, 3156, 1693, 1700, 1608, 1576, 1488.
HR-ESIMS m/z: 345.1822 (calculated for C19H24N2O4, 344.1736).
4.1.4.11. Ethyl-1,2,3,4-tetrahydro-6-methyl-4-((Z)-2-(2,2-
C
18H22N2O5, 346.1529).
4.1.4.16. Ethyl-4-((E)-2-(benzo[d][1,3]dioxol-6-yl)vinyl)-
1,2,3,4-tetrahydro-6-methyl-2-oxo pyrimidine-5-carboxylate 20a.
White solid, (mp: 181–184 ꢁC). 1H NMR (200 MHz, CDCl3): d 7.8
(1H, s), 6.9 (2H, m), 6.7 (1H, m), 6.6 (1H, d, J ¼ 2.2 Hz), 6.3 (1H,
d, J ¼ 16.0 Hz), 6.1 (2H, s), 5.9 (1H, dd, J ¼ 3.4 & 16.0 Hz), 5.6
(1H, s), 4.61 (1H, d, J ¼ 3.4 Hz), 4.1 (2H, q, J ¼ 7.2 Hz), 2.3 (3H, s),
1.2 (3H, t, J ¼ 7.2 Hz). 13C NMR (100 MHz, CDCl3): d 165.7, 154.2,
148.8, 146.9, 145.3, 136.8, 129.2, 124.3, 120.8, 109.1, 107.2, 102,
101.8, 60.2, 55.6, 19.4, 14.5. IR (KBr) nmax cmꢃ1: 3230, 2930,
1710, 1655, 1520, 1107. HR-ESIMS m/z: 331.1309 (calculated for
dimethylchroman-6-yl)vinyl)-2-oxo-pyrimidine-5-carboxylate 15a.
1
ꢁ
Yellowish solid, (mp: 120–123 C) H NMR (400 MHz, CDCl3):
d 7.3 (1H, s), 7.2 (2H, m), 6.7 (1H, dd, J ¼ 8.5 & 2.4 Hz), 6.2 (1H, d,
J ¼ 16 Hz), 5.9 (1H, dd, J ¼ 3.2 & 16.0 Hz), 4.5 (1H, d, J ¼ 4.0 Hz),
4.1 (2H, q, J ¼ 7.3 Hz), 2.4 (3H, s), 2.7 (2H, t, J ¼ 7.0 Hz) 1.7 (2H,
t, J ¼ 7.0 Hz), 1.2 (3H, t, J ¼ 7.1 Hz), 1.2 (6H, s). 13C NMR (100
MHz, CDCl3): d 168.6, 155.9, 154.6, 152.4, 130.5, 129, 128, 125.8,
122.8, 115.1, 100.7, 78.3, 61.5, 34.4, 27.4, 25, 19.5, 14.4. IR (KBr)
nmax cmꢃ1: 3240, 3117, 2960, 1693, 1514, 1097. HR-ESIMS m/z:
371.11979 (calculated for C21H26N2O4, 370.1893).
C
18H22N2O5, 330.1216).
4.1.4.17. Ethyl-4-(benzo[d][1,3]dioxol-6-yl)-1,2,3,4-tetrahydro-
6-methyl-2-oxopyrimidine-5-carboxylate 21a. White solid, (mp:
176–178 ꢁC). 1H NMR (200 MHz, CDCl3): d 7.9 (1H, s), 6.8 (1H, d,
J ¼ 8.7 Hz), 6.7 (1H, m), 6.7 (1H, d, J ¼ 1.6 Hz), 5.9 (2H, s), 5.6
(1H, s), 5.3 (1H, s), 4 (2H, q, J ¼ 7 Hz), 2.3 (3H, s), 1.18 (3H, t, J ¼
7 Hz). 13C NMR (100 MHz, CDCl3): d 165.6, 153.6, (2C, 147.2),
146.3, 137.9, 120, 108.1, 107, (2C, 101.1), 60, 55.4, 18.5, 14.2. IR
(KBr) nmax cmꢃ1: 3225, 2928, 1710, 1651, 1514, 1097. HR-ESIMS
m/z: 305.1131 (calculated for C18H22N2O5, 304.1059).
4.1.4.12. Ethyl-4-(4-methoxystyryl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxyl-ate 16a. Colourless liquid. 1H
NMR (400 MHz, CDCl3): d 7.2 (1H, s), 7 (2H, d, J ¼ 7.9 Hz), 6.8
(2H, d, J ¼ 8 Hz), 6.2 (1H, d, J ¼ 16 Hz), 5.9 (1H, dd, J ¼ 3.2, 16
Hz), 5.5 (1H, s), 4.5 (1H, d, J ¼ 4 Hz), 4.1 (2H, q, J ¼ 7.3 Hz), 3.7
(3H, s), 2.4 (3H, s), 1.2 (3H, t, J ¼ 7.1 Hz). 13C NMR (100 MHz,
CDCl3): d 167.6, 154.5, 144.6, 132.9, 129.7, 129.3, 127.4, 127.3,
118.3, 109.7, 101.8, 85.3, 59.7, 55.2, 36.4, 19.5, 14.4. IR (KBr)
nmax cmꢃ1: 3240, 2960, 1693, 1514, 1462, 1097. HR-ESIMS m/z:
317.1495 (calculated for C17H20N2O4, 316.1423).
4.1.4.13. Ethyl 4-(2,3-dimethoxystyryl)-1,2,3,4-tetrahydro-6-
methyl-2-oxopyrimidine-5-carboxylate 17a. Yellowish liquid, 1H
NMR (400 MHz, CDCl3): d 7.2 (1H, s), 6.8 (1H, d, J ¼ 16 Hz), 6.5
(3H, m), 5.9 (1H, dd, J ¼ 3.2, 16 Hz), 5.5 (1H, s), 4.5 (1H, d, J ¼ 4
Hz), 4.1 (2H, q, J ¼ 7.4 Hz), 3.7 (6H, s), 2.4 (3H, s), 1.2 (3H, t, J ¼
7.1 Hz). 13C NMR (100 MHz, CDCl3): d 167.7, 156.8, 155.7, 151.2,
144.6, 130, 127.1, 122.5, 119.9, 104.5, 101.9, 93.8, 59.7, (2C,
55.3), 36.4, 19.5, 14.3. IR (KBr) nmax cmꢃ1: 3240, 2960, 1693,
1514. HR-ESIMS m/z: 347.1601 (calculated for C18H22N2O5,
346.1529).
4.2. Biology
RPMI-1640 medium, Dulbecco's modied eagle's medium,
penicillin, streptomycin, fetal calf serum, sodium bicarbonate,
phosphate buffer saline, trypsin, gentamycin sulphate, tryphan
blue, ethanol, DMSO, paraformaldehyde were purchased from
Sigma Chemicals Co. Glacial acetic acid from Fischer scientic,
PBS and trichloroacetic acid (TCA) from Merck specialties
private limited. All the human cancer cell lines (Colo-205, PC-3,
THP-1, and A549) as well as normal epithelial cell line (fR-2)
were obtained from National Center for Cell Science, Ganesh-
khind, Pune-4111007 (India) and National Cancer Institute,
Biological Testing Branch DTP/DCTD/NCI, Frederick Cancer
Research and Development Center, Fairview Center, Suite 205,
1003 West 7th Street, Frederick, MD 21701-8527 (USA).
4.2.1. MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra
zolium bromide] assay. To determine IC50 value, all the deriv-
atives were further evaluated against colon (Colo-205), prostate
(PC-3), leukemia (THP-1) and lung (A549) cancer cell lines
including normal epithelial cell line (fR-2) using MTT assay.
Briey, Colo-205, PC-3, THP-1, A549 and fR-2 cells (3 ꢂ 103 cells
per mL per 100 mL per well) were plated into a 96 well tissue
4.1.4.14. Ethyl 4-(3,4,5-trimethoxystyryl)-1,2,3,4-tetrahydro-6-
methyl-2-oxopyrimidine-5-carboxylate 18a. Greyish liquid. 1H
NMR (400 MHz, CDCl3): d 7.2 (1H, s), 6.5 (2H, d, J ¼ 16 Hz), 6.2
(2H, s), 5.9 (1H, dd, J ¼ 3.2, 16 Hz), 5.5 (1H, s), 4.5 (1H, d, J ¼ 3.4
Hz), 4.1 (2H, q, J ¼ 7.6 Hz), 3.7 (9H, s), 2.4 (3H, s), 1.2 (3H, t, J ¼
7.1 Hz). 13C NMR (100 MHz, CDCl3): d 167.8, 156.8, (2C, 155.7),
42232 | RSC Adv., 2020, 10, 42221–42234
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culture plate. Cells were incubated either in RPMI-1640 or
Dulbecco's modied eagle's media containing 10% fetal calf
serum, supplemented with 100 units per mL penicillin,
100 mg Lꢃ1 streptomycin in a humidied atmosphere in 5.0%
CO2 at 37 ꢁC. Aer overnight cell attachment, the cells were
treated with test materials at concentrations of 0.07, 0.7, 7.0 and
70.0 mM for 24 h and then further treated with MTT (250 mg
mLꢃ1) for 3–4 h. Inhibition of formation of colored MTT for-
mazan was taken as an index of cytotoxicity activity. The amount
of colored formazan derivative was determined by measuring
optical density (OD) using TECAN microplate reader (Innite
M200 PRO) at 570 nm and percentage cell growth was calculated
by the formula (test OD/non-treated OD) ꢂ 100. Further the
graphs and IC50 values of different analogs and controls in
cancer cells of different tissue origin used for screening were
determined on excel.63
4.3. Molecular docking studies
All the computational studies were carried out in the Schro-
dinger suite 2012 molecular modeling soware. The 2D struc-
ture of the ligands was built in the maestro window. The
structures were then converted to their respective 3D structures,
with various conformers, tautomers and ionization states using
the Ligprep and Confgen modules.64 The three dimensional
structure of CDK2 and PI3Kg i.e., PDB ID 2DUV and PDB ID
1E7U respectively were downloaded from Protein Data Bank.65
The receptor was prepared for docking using the Protein Prep-
aration wizard. Extra precision (XP) scoring function of Glide
was used for carrying out the docking studies.66
Conflicts of interest
There are no conicts to declare.
4.2.2. Clonogenic (colony formation) assay. To evaluate
whether compound 16a inhibits clonogenic property of cancer
cells, colony formation assay was performed in A549 cells.
Briey, A549 cells were plated at a seeding density of (1 ꢂ 103
cells per well) in 6 well tissue culture grade plates. Aer 24 h the
culture medium was changed and new medium was added and
cells were exposed to various concentrations of 16a (5, 7 and 15
mM) along with vehicle DMSO for 24 h at 37 ꢁC incubator in 5%
CO2. Aer the treatment, the plates were placed in an incubator
with fresh media only for a time equivalent to at least six
potential cell divisions (to give colonies of >50 cells). Later on,
the obtained colonies were xed with 4% paraformaldehyde
and were stained with 0.5% crystal violet solution. The colonies
from the plates were counted and averaged from the observed
elds randomly (n ¼ 3) and photographed with Olympus C-7070
wide 700M inverted microscope camera (Fig. 2). Staurosporine
(25 nM) was used as positive control while as DMSO was used as
vehicle.
Acknowledgements
The author (SF) is grateful to DST SERB, India for providing
research funding bearing the Project No: TAR/2018/000088. The
authors (FAA, AA) are also grateful to King Saud University,
Riyadh, Saudi Arabia for providing research funding bearing the
Researchers Supporting Project Number RSP-2020/160.
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