Dual active 1, 4-dihydropyridine derivatives: Design, green synthesis and in vitro anti-cancer and anti-oxidant studies

Article abstract of DOI:10.1016/j.bioorg.2020.104379

The present work describes the design of 1,4-dihydropyridines (1,4-DHPs) with diverse variations in structural and functional groups. The physico-chemical properties and drug-like molecule nature evaluations were carried out using SWISSADME. A simple, economical, eco-friendly, water-mediated and Para-Toluene sulfonic acid catalysed multicomponent and one-pot synthetic method from nitroketene N, S- acetals (NMSM) and corresponding aldehydes has been developed. All compounds (6a-u and 13a-h) were subjected to in vitro assays against two important human cancer cell lines Viz. are Laryngeal carcinoma (Hep2) and Lung adenocarcinoma (A549) cells. The reduction level of DPPH (%) used to evaluate the anti-oxidant properties. The 1,4-DHP derivatives, 6o, 6u and 6l displayed the potent anti-cancer activity with IC₅₀ value of 10 μM, 14 μM and 10 μM against the Hep2 and 8 μM, 9 μM and 50 μM against the A549 cells. Similarly, the anti-oxidant properties of 6o, 6l and 6u at a standard concentration of 50 μg, are found to be 70.12%, 63.90% and 59.57% respectively favours the 1,4-DHP derivatives dual activity potentials. The compounds, 6o and 6l found to be equivalent with standard drug, Doxorubicin.

Full text of DOI:10.1016/j.bioorg.2020.104379

Journal Pre-proofs
Dual active 1, 4-Dihydropyridine derivatives: Design, green synthesis and in
vitro anti-cancer and anti-oxidant studies
Parthiban Anaikutti, Parameshwar Makam
PII:
S0045-2068(20)31677-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104379
YBIOO 104379
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
21 February 2020
5 September 2020
12 October 2020
Please cite this article as: P. Anaikutti, P. Makam, Dual active 1, 4-Dihydropyridine derivatives: Design, green
synthesis and in vitro anti-cancer and anti-oxidant studies, Bioorganic Chemistry (2020), doi: https://doi.org/
10.1016/j.bioorg.2020.104379
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Dual active 1, 4-Dihydropyridine derivatives: Design, green synthesis and in
vitro anti-cancer and anti-oxidant studies
Parthiban Anaikutti‡, Parameshwar Makam†§, *
‡ National Centre for Sustainable Coastal Management, Anna University Campus, Chennai
600025, India.
† Chemical Science Research Group, Division of Research and Development, Lovely
Professional University, Phagwara, Punjab, 144411. India.
§ Dr. Param Laboratories, Phase-1, IDA, B.N. Reddy Nagar, Cherlapally, Hyderabad,
Telangana-500062, India.
*Corresponding author: parammakam@gmail.com, makam.22764@lpu.co.in;

Abstract
The present work describes the design of 1,4-dihydropyridines (1,4-DHPs) with diverse
variations in structural and functional groups. The physico-chemical properties and drug-like
molecule nature evaluations were carried out using SWISSADME. A simple, economical, eco-
friendly, water-mediated and Para-Toluene sulfonic acid catalysed multicomponent and one-
pot synthetic method from nitroketene N, S- acetals (NMSM) and corresponding aldehydes has
been developed. All compounds (6a-u and 13a-h) were subjected to in vitro assays against two
important human cancer cell lines Viz. are Laryngeal carcinoma (Hep2) and Lung
adenocarcinoma (A549) cells. The reduction level of DPPH (%) used to evaluate the anti-
oxidant properties. The 1,4-DHP derivatives, 6o, 6u and 6l displayed the potent anti-cancer
activity with IC50 value of 10 µM, 14 µM and 10 µM against the Hep2 and 8 µM, 9 µM and
50 µM against the A549 cells. Similarly, the anti-oxidant properties of 6o, 6l and 6u at a
standard concentration of 50µg, are found to be 70.12%, 63.90% and 59.57% respectively
favours the 1,4-DHP derivatives dual activity potentials. The compounds, 6o and 6l found to
be equivalent with standard drug, Doxorubicin.
Keywords: Green Chemistry; Nitroketene N, S-Acetals; 1, 4-Dihydropyridine; anti-cancer,
anti-oxidant.

Graphical abstract
O
R1
S OH
R1
MCR:
CHO
O
O2N
MeS
NO2
1 new ring
2 C-C bond
1 C-N bond
NO2
NO2
(10 mol%)
N
R2
NHR2
Water, RT, 15 mins
70-98%
MeS
NHR2 MeS
NHR2
Potent Molecules
Green Chemistry
Water Solvent
O
O2N
S
NO2
PTSA Catalyst
No tedious workup
O2N
S
NO2
O2N
S
NO2
N
N
H
N
N
H
No column chromatrpohy
N
N
H
6o
6l
10
50
6u
14
09
Hep2 (IC50; µM ):
A549 (IC50; µM):
10
08
DPPH Reduction (%): 70.12
63.90
59.57

1. Introduction
Over a century, 1,4-dihydropyridines (1,4-DHPs) derivatives have been gaining
momentum and becoming the choice of synthetic chemists and medicinal chemists for
developing novel molecules with therapeutic importance. Owing to their diversity in structural
and medicinal applications, the 1,4-DHPs scaffold has been categorized as one of the privileged
structures.1 The reviewed biological importance of 1,4-DHPs include - calcium channel
blockers, cardiovascular, anti-hypertensive, antitumor, anti-inflammatory, analgesic,
neuroprotectant, platelet anti-aggregator, anti-ischemic, anti-Alzheimer, anti-microbial and
insecticidal etc.1-6 The noticeable drug candidates in this class are Nifedipine 1, Felodipine 2
and Amlodipine 3 etc.3, 4, 6
Cl
NO2
Cl
H3CO2C
Me
CO2CH3
H3CO2C
Me
COOEt
H3CO2C
Me
CO2CH3
O
N
H
Me
N
H
Me
N
H
NH2
Nifedipine (1)
Amlodipine (3)
Felodipine (2)
Figure 1. Biologically important 1, 4-DHPs.
In addition to the biological importance, 1, 4-DHPs are useful source of hydride for
reductive amination and synthetic intermediates for producing biologically important
molecules.7 Since from 1882, first classical report for the synthesis of 1, 4-DHPs through
multicomponent reactions (MCR) by Hantzsch,8 the advances towards improving and
development of new methods have been under progress with great interest to generate
therapautically important molecules.3, 6, 9 The MCRs are one of the simplest, eco-friendly and
widely used in organic synthesis. This is due to their ability of simultaneous and selective
formation of more than two or three bonds by domino mechanism within a single step with
high yields and purity. In recent years, MCR has been paying much attention to the generation

10-14
of highly diverse and functionalized molecules
Thus, developing an environmentally
benign MCR has been recognized as one of the most important methods for developing the
molecules with ease and advantages like stereo and regio-selective synthesis, biocatalysis and
green chemistry14-19
.
Cancer, broadly a collection of more than 100 diseases with the abnormal and uncontrolled
division of cells and affecting major parts of the body. 20, 21 Globally by causing 1 in 6 deaths,
cancer has become the second leading cause of deaths with the total accountability of 9.6
million deaths in 2018. The women and men populations have been effected with diverse types
of cancers.22 The accessible and existing anticancer drugs have distinctive mechanisms of
action which ultimately make variations in their effects on diverse types of cancer and normal
cells. In addition, there is very insufficient evidence of biochemical alterations between normal
cells and cancerous cells and hence, a particular cure for cancer has demonstrated indefinable.
The advancement of resistance to existing multi-drug chemotherapy and their slow cure rate,
dreadful and toxic effects on patients are serious and pragmatic concerns of cancer.23 Hence,
the impeccable efforts are required for the identification of newer, efficient and less toxic
chemotherapeutic agents for treating the cancers.24
The reactive oxygen species (ROS), are highly reactive and unstable species that are
produced during many metabolic activities in the body. The produced ROS disturbs the
function and structure of healthy cells by quickly pairing up their odd free electrons with cells
and creating an unstable redox equilibrium leading into the origin and growth of different
human cancers, in addition to various other ailments.25-28 Controlling the intracellular ROS
generation by antioxidants is one of the key strategy to overcome cancer.29 Hence, developing
chemotherapeutic agents with the dual action of anti-cancer and anti-oxidant properties are of
great importance.

Hence in continuation of our efforts in medicinal chemistry research30-38 and considering
the importance of the 1, 4-DHP derivatives, the present work aimed to design, synthesise and
validate the dual action properties of 1, 4-DHP derivatives by in vitro anti-cancer and anti-
oxidant studies. Considering the hurdles in the reported synthetic methods, the present work
demonstrates construction of the highly functionalized 1, 4-DHP derivatives by simple,
convenient and eco-friendly synthetic methodology by using simple starting materials ((E)-N-
Methyl-1-(methylthio)-2-nitroethenamine) (NMSM), 4a and corresponding aldehydes (5a-r)
in the presence of catalytic PTSA and in water medium.
2. Results and Discussion
2.1.
Design and evaluation of Physico-chemical and Drug like molecule (DLM)
properties of 1, 4-DHP derivatives
In the design of the molecules, the privileged 1,4-DHP scaffold with nitro groups at 3rd and 5th
positions have been kept intact and the structural and functional variations have been intended
at the 1st, 2nd, 4th and 6th positions of the 1,4-DHP scaffold as shown in figure 2. The designed 1,
4-DHP derivatives intended for the synthesis with diverse structural and functional variations
were shown in Figure 3. The designed molecules were subjected to evaluate for their DLM
nature, which is ultimately dependent on their physicochemical properties. These properties
further aid in designing a molecule with better Absorption, Distribution, Metabolism, Excretion
(ADME) and Toxicity.39, 40 One of the highly influential rules in guiding the small-molecule
drug design is the ‘rule of 5’ (Ro5). The Ro5 summarizes that the molecule with a Log P not
greater than 5, molecular weight (MW) less than 500, hydrogen bond acceptors (HAs) and
donors (HDs) less than 10 and 5 respectively could lead to good DLM character.41-43 The other
rules by Veber’s group revealed that the molecules containing total polar surface area (TPSA),
rotatable bonds (RBs) and HAs and HDs equal to or less than, 140 Å, 10 and 12 respectively
found to possess better oral bioavailability.44 The works of Leeson and co-workers have

identified that there was a 16 to 23% increase in the number of rings, hydrogen bonding groups,
a number of RBs and MWs in the molecules approved between 1983 and 2002 than the drugs
approved before 1983.45 Hence, a precise understanding and modulation of physicochemical
properties became an important component for a drug candidates to contribute overall quality at
different important drug discovery stages.
Substituted Phyenylsystems
Heteroaromatic: Five-membered
Six membered
Fused
Aliphatic systems
R
O2N
R'
NO2
R''
Diverse aryl/
Alkyl chain systems
SMe
N
N
Diverse Amine systems
H
R'''
Diverse Aryl/
Alkyl chain systems
Positions of Structural and functional diversity
Figure 2. Design of the 1,4-DHP derivatives
The physicochemical properties of 1, 4-DHP derivatives were evaluated using the SwissADME
online website 46, 47 and the key parameters are being summarized in table 1 (for the complete
profile can be found in the supporting information). The physicochemical properties data reveal
that the MWs of the intended compounds are in the range from 326.33 to 576.66 with 6o as the
lowest and 6t as the highest respectively. The molecule, 6t violated the Lipinski’s standard of
MW less than 500. The iLogP values varied between 1.58 to 4.47 with the 6q as lowest and 6t
as highest respectively. The HAs and HDs of the molecules have been found in the range from
4 to 6 and 1 to 2 respectively. The compounds have shown to have number of RBs between 5 to
13. The molecules 6t, 6u and 13d revealed to be the molecules with the highest number of RBs
with 13, 11 and 10 respectively. The molecules, 6e stand top with the highest PTSA value of
178.03 and the molecules, 13f, 13g with the the lowest TPSA value of 110.15. Hence, a blend

of molecules with wide variations in their physicochemical properties have been designed with
violations of Lipinski's rule with molecules 6e and 6t and rest all the other compounds are in
accordance with the Lipinski and Veber proposals.
F
Cl
Br
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
6a
O
6b
6c
O
6d
6e
6f
OH
OH
O
O
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
6g
6h
6i
6j
6k
6l
HN
N
N
N
S
O
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
O2N
S
NO2
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
N
N
H
6m
6n
6o
6q
6r
6p
O2N
NO2
NH
O2N
NO2
O
O2N
NO2
S
N
S
N
N
H
S
N
N
H
6u
6t
6s
O
O2N
NO2
O2N
NO2
O2N
HN
NO2
O2N
NO2
N
N
N
H
N
H
N
N
N
N
H
N
H
N
N
H
H
H
13b
13c
13d
13a
O
O2N
NO2
O2N
N
NO2
O2N
HN
NO2
O2N
NO2
N
N
N
H
N
N
N
N
H
N
N
N
H
H
O
13h
13f
13g
13e
Figure 3. Library 1,4-DHPs with diversity: C6-methylthio group (6a-u) and with amines
replacement of C6-SMe (13a -h).

Additionally, based on all the physicochemical properties, the pharmacokinetic
behaviours Viz. are Gastrointestinal absorption and brain access of the of 1, 4-DHP derivatives
have been predicted and depicted graphically by the Brain Or IntestinaL EstimateD permeation
method (BOILED‐Egg) by computing the lipophilicity (WLOGP) againt the TPSA. The
molecules falling in the region of the white ellipse, yellow ellipse and grey area predicted to
have high intestinal absorption (HIA), Brain-Blood barrier (BBB) crossing and non-permeant
either in HIA or BBB. The colour-coding by blue dot and red dots of the points indicates the
predicted active effluation of the molecules or not by the P-glycoprotein of the Central Nervous
System (CNS) respectively. It is predicted that none of the molecule is falling in the yellow
ellipse, hence the molecules are with poor BBB permeation. Majority of the molecules are in
the white ellipse and predicted to have HIA. Rest of the compounds are falling in the grey
region predicted to have poor GI and BBB. Except for molecules, 13h, 6r and 6j, all are
predicted to have non-efflux action from the CNS by the P-glycoprotein.
Figure 4. The BOILED-Egg depiction of the designed and synthesized molecules.

Table 1. Evalauation of physico-chemical properties and In vitro biological activities of 1, 4-DHP derivatives
Physico-chemical propertiesa
Lipinski rule of five
In vitro Biological Activities
Compound
Veber rule
TPSA
Anti-Cancer (IC50)
Anti-Oxidant
Reduction level
of DPPH (%)b
MW
iLOGP HAs HDs Violations RBs
Violations
Hep2
A549
(µM)
(µM)
6a
6b
6c
6d
6e
6f
336.37
354.36
370.81
415.26
381.36
350.39
366.39
352.37
396.42
382.39
386.42
362.4
2.01
2.21
2.37
2.48
1.77
2.38
2.51
1.7
4
5
4
4
6
4
5
5
6
6
4
4
4
4
5
5
1
1
1
1
1
1
1
2
1
2
1
1
1
1
1
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
5
5
5
5
6
5
6
5
7
6
5
6
8
5
5
7
132.21
132.21
132.21
132.21
178.03
132.21
141.44
152.44
150.67
161.67
132.21
132.21
132.21
160.45
145.35
150.03
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
16
25
10
51.31098
67.89634
46.52439
72.86585
75.67073
62.13415
59.72561
49.96951
69.39024
22.07317
63.35366
63.90244
73.71951
52.40854
70.12195
39.81707
>100
30
>100
25
>100
>100
50
>100
>100
45
6g
6h
6i
>100
20
>100
10
2.7
6j
1.84
2.53
2.62
2.75
2.23
1.82
3.02
40
30
6k
6l
>100
10
>100
50
6m
6n
6o
6p
330.4
45
50
342.39
326.33
478.52
>100
10
>100
8
15
25

6q
337.35
375.4
1.58
1.88
3.41
4.47
3.44
2.8
5
4
4
6
4
4
4
4
4
5
4
4
5
1
2
1
1
1
2
2
2
2
2
1
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
5
5
145.1
148
0
0
0
0
0
0
0
0
0
0
0
0
0
>100
>100
24
>100
>100
30
52.04268
53.81098
66.76829
66.82927
59.57317
22.07317
39.81707
65.36585
39.81707
71.40244
46.52439
56.58537
55.60976
-
6r
6s
6t
488.56
576.66
420.53
395.41
409.44
361.4
9
132.21
150.67
132.21
118.94
118.94
118.94
118.94
128.17
110.15
110.15
119.38
13
11
7
>100
14
>100
9
6u
13a
>100
>100
>100
>100
24
>100
>100
>100
>100
29
13b
3.1
8
13c
2.73
3.02
2.78
2.37
2.46
2.56
8
13d
389.45
391.42
333.34
361.4
10
9
13e
13f
5
>100
>100
>100
10±0.8
80
13g
7
>100
>100
0.65±0.04
13h
375.38
5
Doxorubicin
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Paclitaxel
-
-
1.8±0.12
--
0.175±0.01
--
-
Ascorbic Acid
100.9146
a calculated using SwissADME online; bResults are the average of three independent experiments. cEvaluated at standard concentration of 50µg

2.2.
Synthesis
Electron
withdrawing
group
O
S
Michael donor site
5a
OH
CHO
O
O2N
MeS
NO2
NO2
NHMe
NO2
NO2
(10 mol%)
Michael acceptor site
N
NHMe
Water, RT, 15 mins
MeS
MeS
NHMe MeS
NHMe
4a
Me
6a
Good leaving
group
4a
4a
Electron
donating groups
Green Chemistry
NMSM (4a)
MCR
Water Solvent
PTSA Catalyst
1 new ring
2 C-C bond
1 C-N bond
No tedious workup
(a)
No column chromatrpohy
(b)
Scheme 1: (a) Reactivity profile of NMSM; (b) General synthesis method of 1,4-DHPs with
6a as the example.
The NMSM is a versatile intermediate, with an ethylene motif containing three functional
groups - NO2, S-methyl and N-methyl. Due to the presence of strong electron-withdrawing
nitro group on ethylene carbon, the NMSM can act as an excellent Michael acceptor. The
methylsulfanyl group is a good leaving in nucleophilic substitutions and also an electron
donor.48 It is considered to be one of the key intermediate in industrial scale synthesis of
ranitidine and nizatidine, marketed anti-ulcer drugs.48
The development of new methods by using NMSM to synthesised polysubstituted 1,4-DHP
via an efficient and convenient procedures are of great interest. Previously, the synthesis of
1,4-DHP derivatives are reported from NMSM by various methods using the 2-aminopyridine
catalyst34 and microwave conditions.49 However, the above mentioned reaction suffers from
the following drawbacks i) high temperatures ii) there is no chance for molecular diversity for
various derivatives in the amine functional group and iii) toxic catalyst. To overcome these, in
the present work, a simple, greener and eco-friendly synthetic methodology has been developed
towards the synthesis of highly functionalized 1,4-dihydropyridine by using simple starting
materials ((E)-N-Methyl-1-(methylthio)-2-nitroethenamine) NMSM 4a and aldehydes 5a in
the presence of PTSA in water medium at ambient temperature. This method provides clean

reaction with shorter reaction time, easy work-up and purification of products by
nonchromatographic method with high yields. To the best of our knowledge, no research group
has reported the reaction of two moles of NMSM with wide spectrum of aldehydes in the
presence of PTSA in water to afford corresponding highly functionalized multi-substituted 1,4-
DHP derivatives in a single step and with excellent yield. The NMSM reactivity attributes and
synthetic scheme are depicted in scheme 1.
Table 2. Optimization of reaction conditions 6a.
CHO
NO2
Catalyst
O2N
MeS
NO2
solvent / RT
MeS
NHMe
N
NHMe
Me
5a
4a
6a
Entry
1
Catalyst in (equivalents)
PTSA (0.1)
PTSA (0.1)
PTSA (0.1)
PTSA (0.1)
PTSA (0.1)
PTSA (0.1)
DNSA
Solvent
Time
Yield (%)
Toluene
Acetone
MeCN
10 h
5 h
35
45
60
70
80
98
45
40
45
35
82
70
80
-
2
3
5 h
4
Ethanol
Water:Ethanol
Water
1h
5
30 min
10 min
1h
6
7
Water
8
CH3SO3H
CF3COOH
TfOH
Toluene
Toluene
Toluene
Ethanol
Ethanol
Ethanol
Water
5h
9
1h
10
11
12
13
14
1h
InCl3
2h
ZnCl2
5h
CuCl
1h
No catalyst
24h
a
General conditions; 5a (1 equi), 4a (2 equi) and PTSA (0.1 equi) under water at room
temperature conditions. b Isolated yield after washing water solvent.

PTSA=Para-Toluenesulfonic acid, DNSA=2,4-Dinitrobenzenesulfonic acid, CH3SO3H=
Methanesulfonic acid, CF3COOH= Trifluoroacetic acid, TfOH=trifluoromethanesulfonic acid
(Triflic acid), InCl3= Indium(III) chloride, ZnCl2 = Zinc chloride, CuCl = Cuprous chloride.
Our initial studies are focused towards the identification of the optimum reaction
conditions test reactions of benzaldehyde 5a with NMSM 4a afforded N, 1-dimethyl-6-
(methylthio)-3,5-dinitro-4-phenyl-1,4-dihydropyridin-2-amine 6a was investigated by using
various Bronsted acids (Table 2). To begin with, the reaction was performed with PTSA in
toluene medium afforded poor yield of the product (0.1 equiv; Table 2, entry 1) at room
temperature. The reation was conduted with diverse polar solvents such as acetone, acetonitrile,
ethanol and ethanol-water mixture (1:1) (0.1 equiv; Table 2, entries 2-5) furnished moderate
yields of products. To our delight, a breakthrough result was achieved when the reaction was
carried out in green solvent, water medium (0.1 equiv; Table 2, entry 6) 6a afforded good yield
98% and the reaction time was shortened to just 5-10 minutes. This test reaction was then
investigated using DNSA in water medium (0.1 equiv; Table 2, entry 7) which resulted in poor
yield. Furthermore this test reaction was carried out other Bronsted acids like CH3SO3H,
CF3COOH and TfOH in toluene (0.1 equiv; Table 2, entries 8-10) medium furnished the
desired products but yield of the products was not satisfactory. The reaction was performed by
using InCl3, ZnCl2 and CuCl in ethanol medium (0.1 equiv; Table 2, entris 11-13) gave
moderate yields. However, product has to isolate in conventional workup and column
chromatography. The reaction did not take place in the absence of any catalyst in water medium
(entry 14). Based on the optimization result, PTSA-water condition was found to be the ideal
for this MCR, which afforded good yield. Further, it is important to note that in the PTSA-
water condition furnished the product as precipitate in the reaction vessel which can easily
purified by simple filtration technique, hence no need of extra purification techniques is
required to get pure desired product 6a. Hence, The optimised reaction involves simple stirring
two reactants at the ambient temparature using water as a solvent and PTSA as a catalyst. The

reaction completes with in 15 mins and can be observed from the thin layer chromatography
(TLC) and precipitation formation in the in the reaction vessel. The crude products were further
purified by the simple recrystallization from dichloromethane and hexane mixture (9:1) to
obtain analytically pure products (6a-u). The desired product was characterized by spectral
analysis Viz. are IR, 1H NMR, 13C NMR and HRMS. The spectral data of the compounds are
found to be in accordance with the reported literature (See supporting information).34 For
1
instance, the H NMR spectrum of 6a displaye a singlet peak at 5.94 ppm, a characteristic
signal for C4H. The singlet peak observed at 3.42 ppm corresponds to NMe and doublet peaks
observed at 3.09 ppm corresponds to NHMe peaks. The characteristic peak of good leaving
group, SMe is observed at 2.53 ppm as a singlet. The singlet peak located at 10.05 ppm
confirms the presence of NH. Similarly, in the 13C NMR spectrum, the 6a compound has
displayed 12 distinctive signals corresponding the its crabons. Among these, four, three and
five were observed in the regions corresponding to the aliphatic, aromatic CH and quaternary
carbons respectively. The plausible mechanism for the formation of 1,4-DHPs has been
represented in Scheme 2.
Based on the optimization results mentioned above (Scheme 1, Table 2), a plausible
reaction mechanism was proposed as shown in scheme 2. First an aldehydes 5a reacts with
PTSA condensed to give protonated benzaldehyde 7 which reacts with NMSM 4a through
Michael addition to generate the intermediate 8 which rearrange to give intermediates 9 and 10
by through PTSA. The intermediate 10 undergoes a N-cyclization with another mole of NMSM
4a to give intermediate 11 with loss of water molecule. Finally an intramolecular elimination
of thiomethanol to furnish N, 1-dimethyl-6-(methylthio)-3,5 dinitro-4-phenyl-1,4-
dihydropyridin-2-amine 6a.

H
HO
O2N
O
O2N
MeS
SO3
HO
O2N
H
MeS
NH
Me
NH
Me
MeS
NH
Me
9
SO3H
PTSA
H
O
4a
7
8
5a
SO3H
H
H O
O2N2
O2N
MeS
NO2
O2N
MeS
NO2
-SMe
NO2
SO3
NHMe
SMe
-MeSH
N
NHMe
N
MeS
NH
S
NHMe
-H2O
Me
Me
11
Me
Me
4a
10
6a
Scheme 2: The plausible mechanism for the formation of 1,4-DHPs
Further, the reactivity of the good labile group, SMe has been tested by reacting the
obtained 6a with the diverse aliphatic amines as nucleophiles in refluxing conditions using
water and ethanol (1:1) as solvent mixture to afford the corresponding products with good
yields as shown in scheme 3. The library of synthesised 1,4-DHPs with primary and secondary
amines substitutions (13a-h) have been presented in figure 3.
R1
R1
H2O: EtOH (1:1)
O2N
HN
NO2
R2-NH2
12a-g
O2N
MeS
NO2
Reflux, 1-3 h
80-92%
N
NHMe
N
NHMe
R2 Me
Me
6a, 6g
13a-h
6a-h, 13a-h: R1 = H; 6g, 13e: R1= OMe
12a, 13a: R2 = Benzyl; 12b, 13b: R2 = Phenylethyl; 12c, 13c: R2 = n-Butyl; 12d, 13d: R2 =
n-Hexyl; 12e,13f: R2 = N,N-Dimethyl; 12f, 13g: R2 = N,N-Diethyl; 12g, 13h: R2 = Morpholine
Scheme 3: The nucleophilic substitution of C2 methylsulfanyl group of 1,4-DHPs with primary
and secondary amines.
2.3.
Single-crystal X-ray diffraction (SXRD) studies

In the activity of drug molecules, the isomers play a key role and hence a better
understanding of possible and stable isomers of the molecules would benefit in developing the
strategies in designing potent molecules.50 The single crystals of the compound, 13f has been
grown in dichloromethane: hexane (9:1) mixture by slow evaporation at 5 °C - 15 °C. The
SXRD analysis of 13f have been considered as the representative structures of 1,4-DHP
derivatives to explicit the spatial orientation of the stable conformer and its crystal packing. In
the present study of 1,4-DHPs derivatives, at the chiral carbon, planar phenyl ring is stabilized
with the above the plane orientation and has been arranged perpendicular to the 1,4-DHP
scaffold (A in figure 5).
The packing of the 13f in crystal lattice depicts that the one molecule of 13f has been
are arranged in a head to head and are being stabilized by one intramolecular and two
intermolecular hydrogen bonds. The intramolecular hydrogen bond is strongly held between
one of the Oxygen atom of NO2 group at 3rd position and the Hydrogen of the NH-CH3 present
at 2nd position with a distance of 2.01 Å and an angle of 128.40° between the O, N and H.
Similarly, the distance between Oxygen atom of NO2 group and Nitrogen of the NH-CH3 is
found to be 2.632 Å with an angle of 70.63° between the O, N and C. Whereas, the two
intermolecular hydrogen bonds have been observed Viz. are between the Hydrogen of the NH-
CH3 present at 2nd position with the Oxygen atom of NO2 group at 3rd position of the another
molecule with a distance of 2.953 Å and an angle of 45.27° between the O, N and H. Similarly,
the other intermolecular hydrogen bond of the same molecules has been observed with the
Hydrogen of the NH-CH3 of another molecule with a distance of 2.953 Å and with an angle of
45.27° between the O, N and H. That means one molecule is sandwiched between two other
molecules with two intermolecular hydrogen bonds (B in figure 5). The complete alignment of
the molecules with all the expanded contacts with hydrogen bonding interactions along the C-

axis has been shown in C in figure 5. The crystal data summary of the 13f is provided in
supporting information.
Figure 5. SXRD results of 13f. (A) The ORTEP diagram with 40% ellipsoid probability, (B)
Hydrogen bonding interactions along the C-axis and (C) Cubical packing along the C-axis.
2.4.
In vitro anti-cancer and anti-oxidant and SAR studies of 6a-u and 13a-h
The anti-cancer activities of synthesized 1,4-DHPs (6a-u and 13a-h) have been
evaluated by using in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) reduction assay against two human cancer cell lines, Laryngeal carcinoma (Hep2) and
Lung adenocarcinoma (A549) cell lines and the results are presented in Table 1. Among all the
screened compounds, 4-(furan-2-yl)-N,1-dimethyl-6-(methylthio)-3,5-dinitro-1,4-dihydro
pyridin-2-amine, 6o found to the most potent molecule with IC50 values of 10 µM and 8 µM
against Hep2 and A549 cells respectively. Similarly the styrene substituted molecule, 6l found
to be another potent molecule against Hep2 cell lines with an IC50 value of 10 µM but tolerated
the activity against A549 cells with an IC50 value of 50 µM. The inhibition potentials of 6o and
6l against Hep2 cell lines are found to equivalent to that of a standard drug, Doxorubicin’s
inhibition value of 10 µM. Another molecule, 6u containing phenyl substitution and n-butyl
group substitution on both the N atoms of the 1,4-DHP has found to be most potent molecule
against A549 cells with an inhibition potential IC50 value of 9 µM and also retained the activity
against the Hep2 cells with an IC50 value of 14 µM. These are the most potent molecules found

in this investigation. The other succeeding molecules, 6p, 6b, 6l and 6a have displayed a decent
activity against Hep2 cell with an inhibition potential IC50 values of 15 µM, 16 µM, 20 µM and
20 µM respectively. Similarly against A549 cells, the 6p, 6b, 6l and 6a have displayed better
inhibition with 25 µM, 10 µM, 10 µM, 25 µM respectively. The other molecules which could
retain the activity with lower IC50 values include 13e, 6s, 6d, 6j, 6m and 6g with 24 µM, 24
µM, 30 µM, 40 µM, 45 µM and 50 µM respectively against Hep2 cells. Similarly, 13e, 6s, 6d,
6j, 6m and 6g have retained the activity against the A549 cells with an IC50 inhibition potential
of 29 µM, 30 µM, 25 µM, 30 µM, 50 µM and 45 µM respectively.
From the interpretation of results, the structure-activity relationship has been drawn and
graphically represented in figure 6. It is evident that the central core, N,1-dimethyl-6-
(methylthio)-3,5-dinitro-1,4-dihydropyridin-2-amine is an essential requirement for exhibiting
the activity. The substitutions at the 4th position have shown to exert a great impact on the
activity. The 6a, the simple phenyl substituted N,1-dimethyl-6-(methylthio)-3,5-dinitro-1,4-
dihydropyridin-2-amine derivative has retained the activity with 20 µM and 25 µM against
Hep2 and A549 cells respectively. When the 4th position of Hydrogen replaced with the
bioisosteric Fluorine, the resulting molecule 6b has greatly gained the activity with the 16 µM
and 10 µM against Hep2 and A549 cells respectively. Instead of Fluorine, the halogen with
bigger in size, Bromine is replaced, the resulting molecule 6d has lost two folds it’s activity
with 30 µM and 25 µM against Hep2 and A549 cells respectively. Similarly in 6g molecule,
the 4th position Hydrogen atom of 6a is replaced with methoxy group, the activity lost to a
greater extent with the values of 50 µM and 45 µM against Hep2 and A549 cells respectively.
But upon introduction of another methoxy group at the 2nd position of 6g, the resulting
molecule gained the activity to the greater extent and displayed a very good activity with 20
µM and 10 µM against Hep2 and A549 cells respectively. It is interesting to notice that the

activity of the molecule 6j, which is a result of removing the methyl from the 4th position
methoxy group of 6i, has exhibited more than two folds lesser activity than the 6i.
Similar to the functional group variations, the molecules derived from the structural
variations on the 4th position of N,1-dimethyl-6-(methylthio)-3,5-dinitro-1,4-dihydropyridin-2-
amine core have also greatly impacted in varying the the activities. In the case of 6o, the phenyl
system of 6a is replaced with the furan, a five membered heterocyclic system has been found
to be the most potent molecule with the 10 µM and 8 µM against Hep2 and A549 cells
respectively. Similarly, when the furan is replaced with the 1,3-diphenyl-1H-pyrazole system,
the molecule 6p has lost the activity from two to three folds with 15 µM and 25 µM against
Hep2 and A549 cells respectively. Similar to this, the aliphatic structural variations have made
their contributions in tailoring the activities. The 6l and 6m are the more potent molecules in
this category with styrene and n-Butyl group substitutions respectively on the N,1-dimethyl-6-
(methylthio)-3,5-dinitro-1,4-dihydropyridin-2-amine core. Among the 6l and 6m, the styrene
in the 6l has contributed to exert better activity with 10 µM and 50 µM whereas, the n-Butyl
group in 6m has lowered the activity with 45 µM and 50 µM against Hep2 and A549 cells
respectively.
The variations of alkyl systems replacing the methyl group on the -NCH3 has also
extended its contributions in the exerting the activity. In the case of 6u, n-Butyl group are
replaced with the methyl groups of the 6a and found to have the molecule with great efficiency
with the 14 µM and 9 µM against Hep2 and A549 cells respectively. This is another high potent
molecule of this series. But replacing the n-Butyl groups with benzyl systems resulted in losing
the activity with 24 µM and 30 µM against Hep2 and A549 cells respectively. Unfortunately,
the replacement of –SCH3 with the amines could not help in elevating the activity except the
molecule 13e. The 13e is a structural modification of 6g with the butan-1-amine has increased

the activity from 50 µM and 45 µM to 24 µM and 30 µM against Hep2 and A549 cells
respectively.
Decreasing order of activity
O
F
N
N
O
Enhancers:
Tolerators:
O
Br
O
O
OH
O
R
O2N
R'
NO2
R''
Enhancers:
Tolerators:
NHCH3
H3CS
Enhancers:
N
N
R
R
H
HN
R'''
N
Decreasers:
H
N
Tolerators:
Enhancers:
CH3
HN
H
N
Figure 6. SAR studies
In anti-oxidant activities, the molecules, 6e, 6m, 6d, 13e and 6o have displayed better
anti-oxidant reduction levels against DPPH with 75.67%, 73.71%, 72.86%, 71.40% and
70.12% respectively. Interestingly, the hit molecule from the anti-cancer activities, 6o has
exhibited better antioxidant activity and validated the 1,4-DHPs dual action capabilities.
3. Conclusions
In summary, the physico-chemical properties of the designed and synthesized diverse
multi-substituted 1,4-DHP derivatives have found to be in accordance with Lipinski and Veber
proposals, except three molecules. It is also predicted that these molecules have poor BBB
accessibility and moderate HIA attributes. The simple, economical, water-mediated and PTSA
catalysed three-component one-pot synthesis procedure has been developed and validated its

wide use by applying it to synthesis molecules from 6a to 6u in good yields. The impact of the
various structural and functional variations on the biological activities has been evaluated and
designed the SAR. The results clearly indicate that furan, pyrrole and styrene substitutions are
key for the activity enhancement and the N,1-dimethyl-6-(methylthio)-3,5-dinitro-1,4-
dihydropyridin-2-amine core is an essential requirement. Interestingly, the hit molecule from
the anti-cancer activities, 6o has exhibited better antioxidant activity. This study established
dual action of 1,4-DHP derivatives as anti-cancer and oxidant agents and aids the researchers
in finding the potent molecules with the further tuning of the molecules with appropriate
structural and functional variations.
4. Materials and methods
All the chemicals, aldehydes, amines, NMSM, MTT, propidium iodide (PI), Dulbecco's
Modified Eagle’s Medium (DMEM), antibiotics and fetal bovine serum (FBS) were purchased
from Sigma–Aldrich, Bangalore, India. The TLC plates procured from Merck are used to
monitor the reactions. The melting points were analysed by using open-ended capillary tubes
on VEEGO VMP-DS instrument and are uncorrected. Infra-Red (IR) spectra was recorded
using Nicolet-6700 spectrometer with KBr pellets. The Bruker Avance 400 spectrometer has
been used to record the 1H NMR (400 MHz), 13C NMR (100 MHz) and DEPT-135 spectra of
the synthesised compounds using either (CDCl3) or (DMSO-d6 + CCl4, 1:1) with
tetramethylsilane (TMS) as internal standard. The J values are calculated in Hz. In reporting
the spectral chemical shift values in 1H NMR, multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet and m = multiplet), coupling constant, integration have been used. The Water’s Q-
TOF micro mass spectrometer has been used for recording the high-resolution mass spectra in
electron spray ionization mode. At 298 K, the SXRD measurements were performed on Oxford
CrysAlis CCD area detector system equipped with a graphite monochromator and a Mo-Kα
fine-focus sealed tube (λ = 0.71073 Å).

4.1.
Experimental section
4.1.1. General procedure for the synthesis of multi-substituted 1,4-DHPs (6a-u)
A mixture of solution containing aldehyde (1.0 equiv), NMSM (2.0 equiv) and PTSA
(0.1 equiv) in water (3 mL) was stirred at room temperature for about 15 minutes. The solid
formation observed. The TLC with mobile phase of hexane: EtOAc mixture has been used to
monitor the completion of the reaction. The resulted solid was cooled to 0-5 °C and filtered
under vaccum. The crude products were recrystallized from dichloromethane and hexane
mixture (9:1) to obtain analytically pure products (6a-u). The spectral characterisation data and
spectra are included in the supporting information.
4.1.2. General procedure for synthesis of nucleophilic substitution of 1,4-DHPs with
amines (13a-h).
A solution of N,1-dimethyl-6-(methylthio)-3,5-dinitro-4-phenyl-1,4-dihydropyridin-2-
amine (1 equiv) in water: ethanol mixture (5 mL), added the aliphatic amine (1 equiv) at
ambient temparatures and were mixed well. Gradually, the temperature of the reaction mixture
has been raised to 80 °C and stirred until the reaction got completed. The progress of the
reaction has been monitored by TLC. Upon completion, the reaction mixture was gradually
cooled to room temperature and the resulting solid was filtered off. The crude product was
purified by the recrystallization from dichloromethane and to obtain spectrally pure products
13a-h. The spectral characterisation data and spectra are included in the supporting
information.
4.2.
Biological activity
4.2.1. Cell lines and culture conditions
The cancer cell lines, Hep2 and A549 were obtained from NCCS, Pune, India. The cells
were supplemented with 10% fetal bovine serum (FBS) and maintained in 1× Dulbecco's

Modification of Eagle's Medium (DMEM) at 37 °C in a CO2 incubator in an atmosphere of
humidified 5% CO2 and 95% air. The cells were maintained by routine subculturing in tissue
culture flasks. The culture medium was changed at every 48 h and the cells were split was
observed when they reached confluence. The IC50 values of the tested compounds were
determined by incubating the cells with different concentrations for 48 h.
4.2.2. In vitro anticancer activity
The MTT reduction test was used to determine the cell survival rate. As per the
literature described protocol, the MTT colourimetric assay has been performed.51 The 6
replicates in 96-well flat bottomed culture plates were used to perform the experiments. The
phosphate-buffered saline (PBS) was used to dissolve MTT at a concentration of 5 mg/mL.
After 48 h of incubation of Hep2 and A549 cells, with different concentrations of tested
compounds, 20 μL of a 5 mg/mL MTT solution was added and the plate was further incubated
for about 4 h. The 100 μL of DMSO was used to dissolve the compounds. By the absorbance
at 570 nm was used to measure the conversion of MTT to formazan by metabolically viable
cells. The percentage conversion of compounds with treated cells was used to evaluate the
effect of chemicals on the growth of cells and to determine the concentration that inhibited
50% of growth (IC50).
4.2.3. In vitro anti-oxidant activity
As per the literature procedure, all synthesized compounds were tested for their ability
of free radical scavenging activity.52 Different compounds with various concentrations (1, 10,
50, 100, 150 and 200 µg/ml, respectively) were taken in clean and sterile test tubes. 3.9 ml of
2,2-diphenyl-1-picrylhydrazyl (DPPH) solution (0.1 Mm) was dissolved in methanol. This
mixture was then added to each test tube and followed by the vigorous shaking. Added DPPH
solution with all the test tubes was shaken gradually and permitted to standpoint at 27 ºC in

place of dark for 45 mins. Then blank and positive controls were equipped in a similar way
without any test compounds. With concentrations of 1 to 200 µg/ml, the Ascorbic acid was
used as a standard. The UV spectroscopy at a wavelength of 517 nm was used to measure the
absorbance of the prepared samples. The experiment was replicated three times for consistant
results. Radical scavenging activity of the all synthesized compounds was estimated as an
inhibition percentage and was calculated by using the following formula:
Measurement of radical scavenging activity (%): A control – A sample / A control*100
Acknowledgements
The authors thank central instrumentation facilities (CIF) for characterizing the compounds.
Conflict of Interest: The authors declare none
ORCID: Parameshwar Makam: https://orcid.org/0000-0001-6550-9404
Supporting information
Supporting information includes spectral analyses data and spectras of all the synthesised
compounds.
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