Phenothiazine and amide-ornamented dihydropyridines: Via a molecular hybridization approach: Design, synthesis, biological evaluation and molecular docking studies

Article abstract of DOI:10.1039/c9nj03394g

A series of novel phenothiazinyldihydropyridine dicarboxamides 7a-7j was synthesized by adopting a multi-step synthetic strategy and characterized through physical and spectral techniques. Among them, the chemical entities with para-fluoro (7d), ortho-bromo and-fluoro (7f and 7i), ortho- A nd para-methyl (7e) and meta- A nd para-methoxy (7h) substituents exhibited either similar or superior anti-inflammatory activities with respect to the standard drug diclofenac sodium. Besides, the chemical entities with ortho-bromo and-fluoro substituents as well as meta-nitro substituents (7f, 7g and 7i) showed enhanced radical scavenging activities when compared to standard ascorbic acid. Furthermore, anticancer studies revealed that the meta- A nd para-chloro-substituted molecule 7a exerted the best activity against all the pancreatic cancer cells tested. Also, appreciable binding affinity (-8.10 kcal mol^-1) was observed during molecular docking between B-cell lymphoma 2 and 7a. The structural diversifications of the potent chemical entities besides further exploration in connection with the biological profiles of the same are underway.

Full text of DOI:10.1039/c9nj03394g

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PAPER
Phenothiazine and amide-ornamented
dihydropyridines via a molecular hybridization
approach: design, synthesis, biological evaluation
and molecular docking studies†
Cite this: New J. Chem., 2019,
43, 17046
Ramar Sivaramakarthikeyan,a Shunmugam Iniyaval,a Krishnaraj Padmavathy,a
a
Hui-Shan Liew,b Chin-King Looi,b Chun-Wai Maicd and Chennan Ramalingan
*
A series of novel phenothiazinyldihydropyridine dicarboxamides 7a–7j was synthesized by adopting a
multi-step synthetic strategy and characterized through physical and spectral techniques. Among them,
the chemical entities with para-fluoro (7d), ortho-bromo and -fluoro (7f and 7i), ortho- and para-methyl
(7e) and meta- and para-methoxy (7h) substituents exhibited either similar or superior anti-inflammatory
activities with respect to the standard drug diclofenac sodium. Besides, the chemical entities with ortho-
bromo and -fluoro substituents as well as meta-nitro substituents (7f, 7g and 7i) showed enhanced
radical scavenging activities when compared to standard ascorbic acid. Furthermore, anticancer studies
revealed that the meta- and para-chloro-substituted molecule 7a exerted the best activity against all the
pancreatic cancer cells tested. Also, appreciable binding affinity (À8.10 kcal molÀ1) was observed
during molecular docking between B-cell lymphoma 2 and 7a. The structural diversifications of the
potent chemical entities besides further exploration in connection with the biological profiles of the
same are underway.
Received 30th June 2019,
Accepted 8th October 2019
DOI: 10.1039/c9nj03394g
rsc.li/njc
The rising death rate every year because of cancer has made it
obvious that there is an immediate need for the development of
Introduction
Cancer, a type of inflammation, is the second leading cause of newer anticancer agents that can abolish cancer cells with no
death in the world. There were 9.6 million fatalities around the harm to regular tissues.
globe in 2018 due to life-threatening diseases and one in six
On the other hand, one of the vital targets of organic
was due to cancer. It is anticipated that the global death toll synthesis is a rapid assembly of diversified molecules, which
from cancer is increasing (approx. 12 million deaths by 2030). is a key paradigm of the recent discovery of drugs; in this
Specifically, the rate of death because of pancreatic cancer is regard, multi-component reactions (MCRs) represent a useful
found to be plausibly higher due to its highly invasive nature approach to address this challenge. MCRs are nothing but
and chemoresistance.1 It has been reported that in the United chemical reactions between more than two reactants at the
States, pancreatic cancer is the fourth leading cause of deaths same time in one pot.3 Several green chemistry principles are
from cancer.2 Although various types of treatments are available satisfied in these reactions and most of the atoms in each
for cancer, the treatments can cause one or more side effects. reactant are integrated into the eventual target. Furthermore, a
Numerous drugs are commercially available to treat cancers. one-pot reaction decreases the utilization of reagents and also
Unfortunately, almost all of them are associated with serious solvents, especially those used for purification.4 In addition to the
side effects; consequently, it is a challenging task to save lives. aforementioned advantages, easy methodologies and equipment
as well as their energy and time saving features have led to a
a Department of Chemistry, School of Advanced Sciences, Kalasalingam Academy of
reasonable number of efforts to design and implement MCRs not
Research and Education, (Deemed to be University), Krishnankoil, 626 126,
only in industries but also in academia.5 The expediency of MCRs
Tamilnadu, India. E-mail: ramalinganc@gmail.com, c.ramalingan@klu.ac.in
b School of Postgraduate Studies, International Medical University, 57000,
is even larger if these reactions offer ‘‘privileged biological
structural motifs’’, which are structural components that are
able to provide ligands for various functionalities, as well as
structurally isolated receptors of biological importance, subsequently
providing a platform to develop agents for various applications
Kuala Lumpur, Malaysia
c Department of Pharmaceutical Chemistry, School of Pharmacy, International
Medical University, 57000, Kuala Lumpur, Malaysia
d Centre for Cancer and Stem Cell Research, Institute for Research, Development
and Innovation, International Medical University, 57000, Kuala Lumpur, Malaysia
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj03394g including pharmaceuticals.6
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Dihydropyridines, known as DHPs, embody a category of
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organic molecules of smaller size possessing a pyridine core,
which is a vital structural component with a widespread biological
profile.7 Although five isomeric DHPs can exist theoretically,
most of the documented ones possess either a 1,4-dihydro or
1,2-dihydro structure.8 In particular, 1,4-DHPs have attracted
much attention owing to their utility as calcium channel modulators
with nifedipine, having a nitro functionality, besides the sym-
metrical substituents on its DHP possessing a first-generation
derivative, as the first marketed calcium antagonist. The second-
generation DHP drugs, namely, nitrendipine, felodipine, amlo-
dipine, nilvadipine and nisoldipine, containing unsymmetrical
substituents have also become products of commercial utility.
While 1,4-DHPs have largely been produced as cardiovascular
agents, a diverse range of DHPs have effectively been designed
and used as various biological agents, including antitumor,
vasodilators, antimycobacterial, and antihypertensive activity.9
Phenothiazines, a category of molecules possessing a structural
motif fused with a thiazine and two-phenyl rings with a puckered
conformation, have been recognized since the nineteenth century.
In 1883, Bernthsen synthesized for the first time the parent
molecule 10H-dibenzo-1,4-thiazine using elemental sulphur
and diphenylamine.10 Because of their attractive chemical
characteristics as well as significant biological properties, their
syntheses, structures, plausible reactions, properties and appli-
cations, especially with biological perspectives, were meticulously
reviewed three decades ago and a retrosynthetic approach for the
construction of phenothiazines was also depicted.11,12
In recent years, a large number of articles in connection with
these molecules have been published and to date, more than
6000 derivatives of phenothiazine have been obtained. A minimum
of eight to ten dozens of phenothiazine derivatives are utilized in
treatment, largely as neuroleptics. One of the major advantages of
this structural core is its ability to provide diversified derivatives, as
substituents could effectively be introduced on the nitrogen at the
10-position, carbons at 1–4 as well as 6–9 positions and also
oxidation could be made on sulphur so as to obtain sulfoxides
and sulfones. In addition, the homoaryl rings could also be
replaced by heteroaryl motifs, including pyrimidine, pyridine,
quinoline, pyridazine, 1,2,4-triazine and pyrazine, to furnish the
Fig. 1 Dihydropyridine, phenothiazine and amide hybridized novel chemical
entities.
and antimalarial.32–35 In our ongoing research programme
searching for novel small organic molecules as potent biological
agents, recently we were involved in the synthesis of phenothiazine/
carbazole-integrated novel chemical entities as biological
agents.36–39 In particular, in one piece of our research, we
synthesized a phenothiazine-tethered dihydropyrimidine analogue,
which exhibited radical scavenging property of up to 69.34%
(IC50 = 148 mM).36 Further, we developed a few new series
of molecules, specifically, acrylonitrile/acrylamide-integrated
carbazoles/phenothiazines as well as hydrazide-integrated carba-
zoles and all the series exerted appreciable anticancer profiles.37–39
In continuation of our research on the development of pheno-
thiazine/carbazole-incorporated novel chemical entities as potent
biological agents and also having the biological importance of
the dihydropyridine and amide structural motifs in mind, a
series of dihydropyridine, phenothiazine and amide hybridized
novel chemical entities were designed and synthesized as bio-
pertinent chemical entities. Described herein are the synthesis,
characterization, anti-inflammatory, radical scavenging and
anticancer evaluations as well as molecular docking studies of
the novel hybrids 4-(10-alkyl-10H-phenothiazin-3-yl)-2,6-dimethyl-
N3,N5-diaryl-1,4-dihydropyridine-3,5 dicarboxamides, as furnished
in Fig. 1.
Results and discussion
Chemistry
corresponding azaphenothiazines. Moreover, phenothiazine is The synthetic approach for the assembly of hybrid phenothia-
reported to be non-toxic, which is unusual, and it is acceptable zinyldihydropyridine dicarboxamides 7a–7j is furnished in
up to doses of the multi-gram scale in humans.13 Chemical Scheme 1. Initially, the active intermediate phenothiazine
entities possessing the phenothiazine structural motif show a carbaldehyde 3a was synthesized from the commercial phenothia-
variety of pharmacological/biological properties, including anti- zine 1 by adopting alkylation utilising bromoalkane in the
fungal, antibacterial, antitubercular, antiplasmin, anticancer, presence of KOH in N,N-dimethyl formamide (DMF) followed
antioxidant, antiviral, anti-inflammatory, anticonvulsant, anti- by a Vilsmeier–Haack reaction using POCl3 and DMF in dichloro-
psychotic, antihistaminic, antitussive and anti-emetic. Their methane.40 On the other hand, the intermediates, 3-oxo-N-
noteworthy properties were reviewed recently in several research substituted phenylbutanamides 6a–6j were synthesized from
papers, monographs and book chapters.14–31
the reaction between the b-keto ester 4 and appropriately
Besides, the amide functionality is one of the most important substituted anilines 5a–5j in the presence of potassium hydro-
structural units and can serve as a potential ligand component xide in toluene.41 A three-component one-pot reaction between
in the interaction between a molecule and host proteins via the phenothiazine carbaldehyde 3, appropriate 3-oxo-N-substituted
hydrogen bonding and/or van der Waals forces of attractions. phenylbutanamides 6a–6j and ammonium acetate in the ratio
Appropriate molecules incorporated with the amide function- of 1 : 2 : 1.5, respectively, in the presence of p-toluenesulfonic
ality display various biological properties, including anticancer acid in ethanol eventually produced the desired target hybrid
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Scheme 1 Synthesis of hybrid phenothiazinyldihydropyridine dicarboxamides 7a–7j.
phenothiazinyldihydropyridine dicarboxamides 7a–7j in good the six protons integral, while the ones connected to the aromatic
to excellent yields. The structures of all the target molecules moiety in 7j give signals at nearly 2.3 ppm as singlets with each of
were unequivocally established based on physical and spectral the six protons integral. Eventually, the protons of the methoxy
methods.
groups attached to the aromatic moiety in 7h resonate as six
In the 1H NMR spectra of the target molecules 7a–7j, the protons each as singlets at nearly 3.8 ppm.
secondary amino proton of the dihydropyridine structural unit
In the 13C NMR spectra of the target chemical entities 7a–7j,
resonates between 5.0 and 6.0 ppm as a singlet with one proton signals for the methyl carbons attached at the 2- and 6-positions
integral, while the protons attached to the nitrogens of the of dihydropyridine structural motif could be observed in the
amide moieties were observed between 8.2 and 10.0 ppm as a region between 18.0 and 20.0 ppm. The methyl carbons of the
singlet with one proton integral. The protons of the methyl ethyl group as well as methylene carbons of the ethyl group
groups attached to the 2- and 6-positions of dihydropyridine connected to the nitrogen of phenothiazine moiety resonate in
core provide a singlet with six protons integral in the region the regions 12.0–13.0 ppm and 41.0–43.0 ppm, respectively. The
2.0 to 2.5 ppm, while the protons of the methyl group of ethyl methine carbons situated at the 4-position of dihydropyridine
moiety connected to phenothiazine resonate in the region exhibit signals in the region between 40.0 ppm and 42.0 ppm
1.2 to 1.5 ppm as a three protons signal. The methylene protons while the carbons located at the 3- and 5-positions of the
of the ethyl group integrated at the nitrogen of the phenothiazine dihydropyridine core resonate in the region 103.0–107.0 ppm.
were observed in the region between 3.7 and 4.0 ppm as either a Further, the carbons present at the 2- and 6-positions of
broad doublet or quartet with two protons integral. Further, the the dihydropyridine could be observed in the regions 144.0–
methine proton tethered at the 4-position of dihydropyridine 146.0 and 143.0–145.0 ppm. The carbonyl carbon of amide
resonates in the region 4.5 to 4.8 ppm as a one proton singlet, functionality exhibit signals in the extreme down-field region.
while the aromatic protons of the targets exhibit a multiplet In addition, the carbons of two methyl groups attached at the
signal in the area between 6.5 and 8.0 ppm. Besides, the protons 2- and 4-positions of the phenyl rings in 7e provide signals
of the methyl moieties connected to the aromatic ring in 7e between 17.0 and 21.0 ppm, while the same integrated at the
provide signals between 2.1 and 2.3 ppm as singlets with each of 3- and 4-positions of the phenyl rings 7j offer signals between
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Scheme 2 General mechanism for the synthesis of the phenothiazinyldihydropyridine dicarboxamides.
20.0 ppm and 21.0 ppm. In addition, the carbons of two methoxy furnished in Scheme 2. Here, the reactant amide A undergoes
groups located at the 3- and 4-positions of the phenyl rings in 7h keto–enol tautomerism (C) in the presence of acid. Nucleo-
resonate between 54.0 ppm and 55.0 ppm. Besides, the signals philic attack of the enolic bond on the carbonyl carbon of the
for all the aromatic carbons were observed in the region between aldehyde provides the secondary alcoholic intermediate E via D,
115.0 ppm and 142.0 ppm. All these characteristics in addition to which upon abstraction of a proton followed by the elimination
IR and microanalysis confirmed the target molecules.
of water gives the olefinic intermediate G. On the other hand,
The schematic representation of the plausible mechanistic ammonium acetate is in equilibrium with its dissociated
pathway for the synthesis of the target dihydropyridines is ammonium and acetate ions, which further is in equilibration
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with ammonia and acetic acid. Nucleophilic attack of ammonia
on the carbonyl carbon of the keto group of another molecule
of amide A furnishes the intermediate H, which upon the
addition and elimination of a proton yields the amino alcohol
intermediate I. The addition of a proton followed by dehydration
on I provides the cationic intermediate K through J, which upon
elimination of a proton yields M, equilibrated with L. The olefinic
bond of M then attacks one of the oleinic carbons of G during
electronic shifts followed by proton abstraction and elimination,
yielding a primary amino group possessing the intermediate O
through N. The lone pair of electrons on the primary amino
group (i.e. the nucleophile) then attack the carbonyl carbon of the
intermediate as shown in O followed by electron shifts and
proton transfer to provide the hydroxyl possessing intermediate
Q through P. Proton abstraction followed by dehydration and
deprotonation eventually furnish the target dihydropyridine T
through the ammonium intermediate S.
Fig. 2 Anti-inflammatory activity with respect to diclofenac sodium.
comparatively less electronegative bromo moieties in 7a (i.e.
7b) reduced the inhibition by 7%, while substituting a chloro
group at meta-position by an electron-withdrawing nitro group
and chloro group at the para-position by hydrogen in 7a
(i.e. in 7g) increased the inhibition by 7%. Substituting
electron-withdrawing chloro units on both the meta- and para-
positions by hydrogens in 7a (i.e. 7c) improved the inhibition by
18%, while the introduction of electron-releasing methyl sub-
stituents on the meta- and para-positions of the aryl groups in
place of the hydrogens in 7c (i.e. 7j) also exhibited an almost
similar inhibition to that of 7c. Further, the incorporation of a
more electronegative fluoro substituent on the para-positions
(7d), electron-releasing methyl substituents on the ortho- and
para-positions (7e), electron-withdrawing bromo substituents
on the ortho-positions (7f), electron-releasing methoxy substituents
on the meta- and para-positions (7h) and more electronegative
fluoro substituents on the ortho-positions (7i) of the aryl moieties
of the amide scaffold furnished superior inhibition profiles and
the results were equal and even slightly higher than that of the
standard diclofenac sodium. Among all the chemical entities
tested, the one possessing more electronegative fluoro function-
ality on the ortho-positions of the aryl units of the amide scaffolds
(7i) furnished the best inhibition activity. Since the electronic effect
of the substituents does not follow systematically with respect to
the inhibition activity, a combination of two or more effects,
including inductive, mesomeric and steric/field effects, might have
played a role in proving the superior anti-inflammatory activity.
Biological activities
Anti-inflammatory activity
The anti-inflammatory activity (in vitro) of all the synthesized
chemical entities 7a–7j was evaluated using the protein denatura-
tion technique.42,43 Protein denaturation is a methodology wherein
the secondary structure and tertiary structure of a protein would be
lost when an external stress or a molecule is applied. When
denatured, the biological function of most biological proteins would
be lost. A well-documented basis of inflammation is the denatura-
tion of proteins. As part of the present investigation, the ability of
the target molecules 7a–7j to inhibit protein denaturation was
evaluated. Diclofenac sodium was used as a standard drug, which
exhibited 90% inhibition of protein denaturation. The percentage
inhibitions of protein denaturation upon the utilization of the
chemical entities 7a–7j are provided with respect to the inhibition
of protein denaturation by the standard and the results are
furnished in Table 1 and Fig. 2.
Here, a phenothiazine and amide-ornamented dihydropyridine
possessing an electron-releasing ethyl moiety on the nitrogen of
the phenothiazine and electron-withdrawing chloro moieties on
the meta- and para-positions of the aryl groups of the amide units
(7a) was used, and it exhibited 78% inhibition when compared
to the inhibition from the standard drug. Replacing meta-chloro
substituents by hydrogens and para-chloro substituents by
Radical scavenging activity
Reactive oxygen species (ROS) cause diverse neurodegenerative
diseases, including Alzheimer’s, autoimmune disorder, ulcer,
mutagenesis and carcinogenesis, by hindering the function of
numerous cellular components.44,45 Antioxidants are nothing but
agents that scavenge the free radicals formed in our biological
system. Although a plethora of radical scavenging agents are
available in nature, it is highly essential to develop a drugs with
better antioxidant profiles to meet global needs. To determine
the radical scavenging activity, a wide variety of in vitro methods,
including a hydroxyl radical scavenging assay, ferric reducing
antioxidant power and organic radical scavenging assay, are
available. Of all the methods, organic radical scavenging by
Table 1 Anti-inflammatory activities of 7a–7j
S. no.
Compound
Inhibition (%)
1
2
3
4
5
6
7
8
9
10
7a
7b
7c
7d
7e
7f
7g
7h
7i
78.44 Æ 0.73
71.58 Æ 0.92
96.11 Æ 1.39
103.90 Æ 0.88
100.76 Æ 0.54
100.63 Æ 0.97
85.59 Æ 0.70
103.75 Æ 1.25
105.69 Æ 1.13
95.78 Æ 1.52
7j
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Table 2 Radical scavenging of the target molecules 7a–7j
by nearly 30%. Substituting electron-releasing methyl moieties
on the ortho- and para-positions of the aryl units of the amide
scaffold (7e) furnished no significant improvement in the inhibition
when compared with 7b containing an electron-withdrawing unit.
Further, integration of electron-releasing methoxy substituents on
the meta- and para-positions of the aryl units as well as electron-
releasing methyl substituents on the same (7h and 7j, respectively)
reduced the radical scavenging activity by nearly 50% when com-
pared with the standard. Besides, the incorporation of the more
electronegative fluoro functionality on the para-positions of the aryl
units (7d) provided comparable activity with that of 7a though
slightly lower. However, the chemical entity with no substituents
on the aryl moieties of the amide scaffold (7c) furnished similar
activity with that of the standard, while the molecules with
electron-withdrawing bromo-, nitro- and fluoro-substituents on
the ortho-, meta- and ortho-positions, respectively, of the aryl
part of the amide scaffold (7f, 7g and 7i, respectively) exerted
superior activity when compared with the standard ascorbic acid.
Since the activity variations do not depend on any particular
factors, such as electronic, mesomeric and steric/field effects,
the overall activity may be a result of two or more combinations
of the same.
Entry
Compound
% scavenging
IC50 (mM)
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
7g
7h
7i
90.32 Æ 0.80
61.18 Æ 1.17
100.12 Æ 0.51
85.70 Æ 1.25
63.42 Æ 0.49
110.62 Æ 0.99
110.45 Æ 1.36
51.87 Æ 1.20
108.78 Æ 0.91
43.56 Æ 0.84
230.6
—
75.93
16.02
32.45
7.07
202.3
28.33
57.06
162.6
10
7j
Anticancer activity
Cancer is one of the deadliest diseases in the world and there are
several types of cancers that affect humans. Although numerous
types of drugs are available in the market to treat cancers, the
Fig. 3 Radical scavenging activity with respect to ascorbic acid.
DPPH is being widely used by researchers as it is the simplest development of newer drugs in connection with the same is
one. Consequently, all the target phenothiazine and amide- essential as there are no specific drugs that can yet completely
tethered dihydropyridines 7a–7j were evaluated for their radical cure the cancers, and also the drugs, in general, tend to become
scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), resistant over a period of time due to biological/environmental
a reactive oxygen species.46 The activity was estimated in terms of changes. In the present study, cytotoxicity studies of all the
DPPH inhibition and the outcomes are furnished in Table 2 and synthesized phenothiazine and amide-tethered dihydropyridines
Fig. 3 with respect to the activity of ascorbic acid, a standard one. 7a–7j were evaluated against the pancreatic cancer cell lines
The phenothiazine and amide-tethered dihydropyridine SW1990, AsPC1, BxPC3, Panc1 and the non-cancerous cell line
possessing electron-withdrawing chloro moieties on the meta- MRC5 by the Cell-Titre Glo Luminescent Cell Viability Assay
and para-positions of the aryl groups of the amide structural method. Against the tested target molecules, the percentages of
motif (7a) exhibited 90% inhibition of DPPH radicals when pancreatic cancer cells survival are displayed in Table 3.
compared with the inhibition by the standard ascorbic acid.
Molecular docking
Integration of a hydrogen and comparatively less electronegative
bromine on the meta- and para-positions, respectively, in place The molecular origins of the interactions among the BCL-2
of chlorine present on the aryl groups (7b) reduced the activity (B-Cell Lymphoma 2) active sites and the binding affinity, taken
Table 3 Anticancer activity of the target chemical entities 7a–7j
IC50 (mM)
Entry
Compound
SW1990
AsPC1
BxPC3
Panc1
MRC5
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
7g
7h
7i
8.63 Æ 1.07
4100
7.47 Æ 0.73
4100
6.31 Æ 1.00
16.91 Æ 0.24
54.07 Æ 6.71
30.76 Æ 0.60
15.93 Æ 0.81
28.48 Æ 1.24
15.01 Æ 0.32
17.36 Æ 0.13
59.93 Æ 2.80
11.83 Æ 1.94
7.39 Æ 0.03
22.87 Æ 0.11
50.12 Æ 3.19
30.26 Æ 1.04
19.37 Æ 1.77
29.29 Æ 1.16
12.16 Æ 0.09
18.79 Æ 0.37
26.72 Æ 2.21
19.48 Æ 0.11
70.51
4100
4100
47.05 Æ 0.45
22.15 Æ 0.80
45.22 Æ 2.04
22.42 Æ 1.56
16.72 Æ 2.31
4100
92.80 Æ 6.26
30.99 Æ 0.17
20.83 Æ 2.24
26.43 Æ 4.89
18.00 Æ 0.35
24.48 Æ 9.90
4100
61.53 Æ 3.94
30.12 Æ 0.08
25.05 Æ 9.03
18.71 Æ 1.95
15.20 Æ 0.42
18.69 Æ 0.87
89.07 Æ 4.62
18.82 Æ 0.86
10
7j
18.83 Æ 1.92
21.35 Æ 0.41
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Fig. 4 2D and 3D interactions of BCL-2 with 7a.
from the protein data bank, and the structure of 7a, a con- charged basic amino acid arginine and one of the aryl rings of the
formationally stabilized one, were evaluated through molecular amide structural motif were also noticed.
docking studies by utilizing Auto dock version 4.0. The outputs
obtained through docking are provided in Fig. 4.
The phenothiazine and amide-tethered dihydropyridine 7a
Conclusion
showed a binding energy of À8.10 kcal molÀ1 along with a
predicted IC50 value of 1.16 mM. The molecule 7a possessed van A series of phenothiazine and amide-tethered dihydropyridines
der Waals interaction with two polar hydrophilic amino acids, 7a–7j were synthesized through a multi-step synthetic metho-
namely glutamine and asparagine, and one non-polar amino dology and characterized using physical and spectral methods.
acid, namely threonine, as well as two non-polar hydrophobic Anti-inflammatory studies revealed that the molecules incorporated
amino acids, namely tryptophan and glycine. Additionally, a with fluoro substituent on the para-position (7d), bromo and fluoro
conventional hydrogen bond interaction between aspartic acid, substituents on the ortho-positions (7f and 7i), methyl substituents
an electrically charged acidic amino acid, and a hydrogen of the on the ortho- and para-positions (7e) as well as methoxy substituents
secondary amino functionality of the amide unit was noted. A on the meta- and para-positions (7h) showed either a similar or
p-donor hydrogen bond interaction between tyrosine, a polar superior inhibition profile in comparison with the inhibition profile
hydrophilic amino acid, and one of the phenyl rings of the of the standard anti-inflammatory drug diclofenac sodium. Further,
phenothiazine structural unit was also observed. Besides, there the radical scavenging activity studies implied that the molecules
existed an alkyl–alkyl interaction between a non-polar hydrophobic possessing bromo and fluoro substituents on the ortho-position of
amino acid, namely glycine, and an alkyl group of phenothiazine. the aryl moieties of the amide scaffolds as well as the nitro
Further, there were three p-alkyl interactions between the alkyl substituent on the meta-position of the aryl moieties of the amide
group present in the phenothiazine structural unit and non-polar structural unit (7f, 7g and 7i) exhibited superior activity when
hydrophobic amino acids, such as phenyl alanine, valine and compared to the standard ascorbic acid. Besides, the study of
alanine, observed. In addition, a couple more p-halo interactions, their anticancer activity exposed that among the target molecules,
such as an interaction between a polar hydrophilic amino acid, the chemical entity incorporated with meta- and para-chloro
tyrosine and a chloro group connected to one of the aryl units of substituents on the aryl ring of the amide structural motif (7a)
the amide scaffold, as well as an interaction between an electrically exerted superior activity against all the human pancreatic cancer
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cell lines. In addition, molecular docking between B-cell lym-
General method for the synthesis of 3-oxo-N-substituted
phoma 2 (BCL-2) with 7a furnished a high binding affinity phenylbutanamides (6a–6j). To a solution of ethylacetoacetate
(À8.10 kcal molÀ1). Further exploration of the anti-inflammatory, (2 g, 15.4 mmol) in toluene (20 mL) was added powdered KOH
radical scavenging and anticancer activities of the existing (15 mol%). The respective substituted anilines (17.0 mmol)
respective potent molecules, besides structural diversification were then successively added to the solution. The reaction
of the selected molecules that offered better results in order to mixture was heated so as to reflux and this was continued for
develop novel drugs, are presently under progress.
8 h under continuous stirring. The mixture was extracted with
dichloromethane after attaining room temperature, and washed
with water. The organic layer thus obtained was dried over
anhydrous sodium sulphate and evaporated. The crude mass
thus resulted was subjected to silica-gel column chromatography
utilizing 10–20% ethyl acetate in hexane to eventually yield the
Experimental
General
All the chemicals were purchased from commercial sources as respective substituted phenylbutanamides 6a–6j.41
reagent grade and used as received. By employing standard General method for the synthesis of the target hybrid
procedures, all the solvents were dried/distilled prior to their molecules 7a–7j. To a mixture of carbaldehyde 3 (1 equiv.), the
utilization. Pre-coated (silica-gel) TLC sheets were utilized for TLC respective substituted phenyloxobutanamides 6a–6j (2 equiv.)
and visualized by long- and short-wavelength UV lamps. Column and ammonium acetate (1.5 equiv.) in ethanol (15 mL) under
chromatography was performed on silica gel (100–200 mesh, reflux conditions, was added p-toluenesulfonic acid (15 mol%).
spherical) slurry packed in glass columns. The eluent systems The refluxing was continued for 12–15 h while the progress was
used for individual separations are furnished in the respective monitored by TLC. After completion of the reaction, the content
experimental procedures. All the FT-IR spectra in KBr pellets were of the flask was cooled to ambient temperature and the solvent
recorded on a Shimadzu IR Tracer-100 spectrophotometer in was removed under reduced pressure. The crude obtained was
the range of 4000–400 cmÀ1 1H and 13C NMR spectra were then extracted with dichloromethane after diluting with water.
.
recorded on an NMR spectrometer (Bruker AVANCE II 400 and The organic portion was separated, dried on anhydrous sodium
100 MHz) at 25 1C with the use of tetramethylsilane (TMS) as sulphate and evaporated under reduced pressure. The solid thus
an internal standard and CDCl3/DMSO-d6 as the solvent obtained was subjected to column chromatography utilizing 30–
and the chemical shifts are expressed herein in terms of parts 50% ethyl acetate in hexane as the eluent to afford the respective
per million (d ppm). The spin multiplicities s, d, t, q and target hybrid molecules 7a–7j in pure form as solids.
m stand for singlet, doublet, triplet, quartet and multiplet,
respectively.
Hybrid phenothiazinyldihydropyridinedicarboxamide 7a. A
mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan-
Synthesis of ethyl phenothiazine 2. In a typical experiment, a amide 6a (0.97 g, 3.92 mmol) and ammonium acetate (0.23 g,
round-bottom flask (250 mL) was charged with 10H-phenothiazine 2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
(0.025 mol) and DMF (50 mL). Powdered KOH (0.03 mol) was then in ethanol (15 mL) by adopting the general method followed
added into the flask and it was stirred at ambient temperature for by column chromatography (30–50% ethyl acetate in hexane)
2 h. To this solution, bromoethane (0.04 mol) was added in a drop- furnished the target hybrid phenothiazinyldihydropyridinedi-
wise fashion through a pressure equalizer for 20 min and the carboxamide 7a. Yield, 1.18 g (85%); MP: 250–252 1C; FT-IR
reaction was continued for 24 h at the same temperature. The (KBr, cmÀ1): n 3645.4, 3230.7, 3091.8, 2922.1, 2850.7, 1699.2,
progress of the reaction was monitored through TLC. The reaction 1670.3, 1581.6, 1508.3, 1465.9, 1375.2, 1323.1, 1234.4, 1130.2,
mixture was then extracted with dichloromethane (3 Â 20 mL), 1109.0, 1085.9, 1026.1, 875.6, 813.9, 746.4, 675.0, 644.2, 572.8,
washed well with water and dried over anhydrous sodium 509.2, 484.1, 466.7, 429.7; 1H NMR (400 MHz, CDCl3): d 8.24
sulphate. Evaporation of the dichloromethane followed by (s, 2H), 7.59–6.77 (m, 13H), 5.74 (s, 1H), 4.83 (s, 1H), 3.86 (br.d,
purification through column chromatography (5% ethyl acetate J = 6.60 Hz, 2H), 2.29 (s, 6H), 1.36 (t, J = 6.0 Hz, 3H); 13C NMR
in hexane) eventually yielded the title compound 2.40
(100 MHz, CDCl3): d 166.6, 144.8, 144.5, 139.9, 138.7, 136.2,
Synthesis of carbaldehyde 3. In a two-neck round-bottom 132.3, 128.1, 127.4, 127.3, 126.7, 126.6, 125.6, 124.9, 124.0,
flask (100 mL), DMF (1 mL) was charged under an inert atmo- 122.6, 122.5, 122.4, 115.7, 115.0, 113.8, 106.4, 41.8, 41.4, 19.2,
sphere. POCl3 (1.2 mL) was then added in a drop-wise fashion 13.0; anal. calcd for C35H28Cl4N4O2S (%): C, 59.17; H, 3.97;
at 0 1C and stirred for 45 min. Phenothiazine 2 (11.0 mmol) in N, 7.89; S, 4.51; found: C, 59.38; H, 3.91; N, 7.95; S, 4.44.
chloroform (20 mL) was then added drop-wise into the reaction
Hybrid phenothiazinyldihydropyridinedicarboxamide 7b. A
mixture. After completion of the addition, the temperature was mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan-
raised to 60 1C and the stirring was continued for 6 h. The amide 6b (1.0 g, 3.92 mmol), and ammonium acetate (0.23 g,
completion of the reaction was monitored through TLC. It was 2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
then extracted with dichloromethane (3 Â 20 mL), washed well in ethanol (15 mL) by adopting the general method followed by
with water, dried over magnesium sulphate and the solvent column chromatography (30–50% ethyl acetate in hexane)
was evaporated under reduced pressure. The reaction mixture yielded the target hybrid phenothiazinyldihydropyridinedi-
thus obtained was subjected to column chromatography carboxamide 7b. Yield, 1.27 g (88%); MP: 146–148 1C; FT-IR
(15–20% ethyl acetate in hexane) to yield the pure product 3.40 (KBr, cmÀ1): n 3793.9, 3664.7, 3591.4, 3140.1, 3047.5, 2357.0,
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2322.2, 1651.0, 1597.0, 1504.4, 1354.0, 1330.8, 1228.6, 1195.8, 1483.2, 1450.4, 1400.3, 1377.1, 1232.5, 1166.9, 1122.5, 1076.2,
1010.7, 1066.6, 958.6, 835.1, 756.1, 731.0, 688.5, 615.2, 580.5, 1028.0, 999.1, 950.9, 827.4, 752.2, 723.3, 663.5, 534.2, 435.9;
1
524.6, 424.3; H NMR (400 MHz, CDCl3): d 8.26 (2s, 2H), 7.65– 1H NMR (400 MHz, CDCl3): d 7.94 (s, 2H), 7.46–6.68 (m, 15H),
6.77 (m, 15H), 5.77 (s, 1H), 4.63 (s, 1H), 3.87 (d, J = 6.80 Hz, 2H), 3.86 (d, J = 5.60 Hz, 2H), 2.64 (s, 6H), 2.52 (s, 6H), 2.43 (s, 6H),
2.16 (s, 6H), 1.37 (t, J = 7.00 Hz, 3H); 13C NMR (100 MHz, 1.23 (t, J = 7.10 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 165.6, 147.9,
CDCl3): d 166.9, 144.8, 144.4, 139.9, 138.8, 136.1, 132.3, 128.0, 145.4, 144.3, 143.8, 142.4, 129.5, 128.5, 127.7, 126.7, 124.8, 122.4,
127.4. 127.3, 126.6, 125.6, 124.9, 123.9, 123.6, 122.6, 122.4, 122.3, 121.5, 114.0, 113.8, 112.4, 110.0, 104.7, 40.3, 22.3, 21.6, 20.9,
115.7, 114.9, 113.9, 106.3, 41.8, 41.3, 19.6, 13.2; anal. calcd 12.4; anal. calcd for C39H40N4O2S (%): C, 74.49; H, 6.41; N, 8.91;
for C35H30Br2N4O2S (%): C, 57.55; H, 4.14; N, 7.67; S, 4.39; S, 5.10; found: C, 74.70; H, 6.34; N, 8.86; S, 5.17.
found: C, 57.76; H, 4.18; N, 7.58; S, 4.32.
Hybrid phenothiazinyldihydropyridinedicarboxamide 7f. A
Hybrid phenothiazinyldihydropyridinedicarboxamide 7c. A mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan-
mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutanamide amide 6f (1.0 g, 3.92 mmol) and ammonium acetate (0.23 g,
6c (0.69 g, 3.92 mmol) and ammonium acetate (0.23 g, 2.94 mmol) 2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
in the presence of p-toluenesulfonic acid (15 mol%) in ethanol in ethanol (15 mL) by adopting the general method followed by
(15 mL) by adopting the general method followed by column column chromatography (30–50% ethyl acetate in hexane)
chromatography (30–50% ethyl acetate in hexane) yielded the provided the target hybrid phenothiazinyldihydropyridinedi-
target hybrid phenothiazinyldihydropyridinedicarboxamide 7c. carboxamide 7f. Yield, 1.25 g (87%); MP: 206–208 1C; FT-IR
Yield, 1.02 g (91%); MP: 152–153 1C; FT-IR (KBr, cmÀ1): n (KBr, cmÀ1): n 3884.6, 3782.4, 3630.0, 3547.0, 3236.5, 3039.8,
3647.3, 3554.8, 3290.5, 2966.5, 2877.7, 2358.9, 1797.6, 1597.0, 2966.5, 2872.0, 2360.8, 1716.6, 1666.5, 1593.2, 1508.3, 1442.7,
1508.3, 1438.2, 1436.9, 1398.3, 1330.8, 1224.8, 1193.9, 1159.2, 1381.0, 1232.5, 1188.1, 1120.6, 1045.4, 952.8, 889.1, 827.4,
1
1018.4, 941.2, 842.8, 748.3, 686.6, 607.5, 528.5, 430.1; H NMR 746.4, 725.2, 686.6, 613.3, 528.5, 432.0; 1H NMR (400 MHz,
(400 MHz, CDCl3): d 8.29 (s, 2H), 8.18–6.82 (m, 17H), 5.90 (s, 1H), CDCl3): d 8.25 (2s, 2H), 7.58–6.77 (m, 15H), 5.52 (s, 1H), 4.83 (s,
4.76 (s, 1H), 3.89 (d, J = 6.80 Hz, 2H), 2.24 (s, 6H), 1.38 (t, J = 1H), 3.87 (q, J = 6.80 Hz, 2H), 2.31 (s, 6H), 1.37 (t, J = 6.80 Hz,
7.20 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 167.2, 148.3, 145.0, 3H); 13C NMR (100 MHz, CDCl3): d 166.6, 144.8, 144.5, 139.9,
144.3, 140.7, 139.3, 139.2, 129.6, 127.5, 127.4, 126.3, 125.6, 125.4, 138.7, 136.1, 132.3, 132.2, 128.1, 127.3, 126.1, 125.5, 124.8,
123.3, 122.6, 118.4, 115.4, 115.3, 114.4, 105.7, 41.9, 40.9, 18.6, 123.9, 115.7, 114.9, 113.8, 106.4, 41.8, 41.4, 19.1, 13.0; anal.
12.9; anal. calcd for C35H32N4O2S (%): C, 73.40; H, 5.63; N, 9.78; S, calcd for C35H30Br2N4O2S (%): C, 57.55; H, 4.14; N, 7.67; S, 4.39;
5.60; found: C, 73.57; H, 5.56; N, 9.67; S, 5.66.
found: C, 57.74; H, 4.08; N, 7.60; S, 4.45.
Hybrid phenothiazinyldihydropyridinedicarboxamide 7d. A
Hybrid phenothiazinyldihydropyridinedicarboxamide 7g. A
mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan- mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan-
amide 6d (0.77 g, 3.92 mmol) and ammonium acetate (0.23 g, amide 6g (0.87 g, 3.92 mmol) and ammonium acetate (0.23 g,
2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%) 2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
in ethanol (15 mL) by adopting the general method followed by in ethanol (15 mL) by adopting the general method followed by
column chromatography (30–50% ethyl acetate in hexane) column chromatography (30–50% ethyl acetate in hexane)
provided the target hybrid phenothiazinyldihydropyridinedi- yielded the target hybrid phenothiazinyldihydropyridinedicarbox-
carboxamide 7d. Yield, 0.97 g (82%); MP: 224–226 1C; FT-IR amide 7g. Yield, 1.14 g (88%); MP: 160–162 1C; FT-IR (KBr, cmÀ1):
(KBr, cmÀ1): n 3425.5, 3037.8, 2954.9, 2829.5, 2669.4, 2370.5, n 3664.7, 3437.1, 3055.2, 2754.3, 1595.1, 1537.2, 1496.7, 1448.5,
1917.2, 1805.3, 1622.1, 1593.2, 1498.6, 1450.4, 1398.3, 1346.3, 1408.0, 1382.9, 1355.9, 1328.9, 1290.3, 1215.3, 1215.1, 1155.3,
1224.3, 1165.0, 1124.5, 1033.8, 1006.8, 952.8, 833.2, 752.8, 1072.4, 1018.4, 964.4, 941.2, 817.8, 756.1, 731.0, 688.5, 516.9,
1
833.2, 750.3, 684.7, 615.2, 578.6, 424.3; 1H NMR (400 MHz, 466.7; H NMR (400 MHz, DMSO-d6): d 9.92 (s, 2H), 8.75 (s, 2H),
CDCl3): d 8.35 (s, 2H), 7.70–6.92 (m, 15H), 5.89 (s, 1H), 4.68 8.45 (s, 2H), 8.03–7.89 (m, 4H), 7.63–7.54 (m, 2H), 7.28–6.87 (m, 6H),
(s, 1H), 3.85 (br.d, J = 6.00 Hz, 2H), 2.30 (s, 6H), 1.39 (t, J = 5.13 (s, 1H), 3.87 (br.d, J = 6.40 Hz, 2H), 2.18 (s, 6H), 1.27 (t, J =
7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 167.2, 148.6, 145.5, 6.20 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 168.4, 144.8, 143.1,
144.4, 140.3, 139.6, 139.4, 129.3, 127.7, 127.4, 126.3, 125.6, 141.6, 141.1, 139.3, 130.4, 128.0, 127.4, 126.7, 126.2, 125.9, 123.1,
125.4, 123.3, 122.7, 118.5, 115.3, 115.2, 114.4, 105.7, 41.8, 123.0, 122.6, 117.7, 115.8, 115.5, 113.9, 105.9, 41.4, 40.9, 18.0, 13.1;
40.8, 18.7, 13.0; anal. calcd for C35H30F2N4O2S (%): C, 69.06; anal. calcd for C35H30N6O6S (%): C, 63.43; H, 4.56; N, 12.68; S, 4.84;
H, 4.97; N, 9.20; S, 5.27; found: C, 69.22; H, 4.90; N, 9.32; S, 5.21. found: C, 63.65; H, 4.48; N, 12.52; S, 4.91.
Hybrid phenothiazinyldihydropyridinedicarboxamide 7e. A
Hybrid phenothiazinyldihydropyridinedicarboxamide 7h. A
mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutanamide mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutana-
6e (0.80 g, 3.92 mmol) and ammonium acetate (0.23 g, 2.94 mmol) mide 6h (0.93 g, 3.92 mmol) and ammonium acetate (0.23 g,
in the presence of p-toluenesulfonic acid (15 mol%) in ethanol 2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
(15 mL) by adopting the general method followed by column in ethanol (15 mL) by adopting the general method followed by
chromatography (30–50% ethyl acetate in hexane) gave the target column chromatography (30–50% ethyl acetate in hexane) furnished
hybrid phenothiazinyldihydropyridinedicarboxamide 7e. Yield, the target hybrid phenothiazinyldihydropyridinedicarboxamide 7h.
84%; MP: 182–184 1C; FT-IR (KBr, cmÀ1): n 3664.7, 3307.9, Yield, 1.19 g (92%); MP: 242–244 1C; FT-IR (KBr, cmÀ1): n 3238.5,
2966.5, 2881.6, 1940.3, 1884.4, 1797.6, 1618.2, 1593.2, 1512.1, 3140.1, 3076, 2833.4, 1637.6, 1606.7, 1514.1, 1465.9, 1408.0, 1328.9,
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1257.6, 1238.3, 1138.0, 1028.1, 964.4, 923.9, 846.8, 800.5, 748.4, 660 nm utilizing the vehicle as a blank. As a reference drug,
1
613.4, 565.1; H NMR (400 MHz, CDCl3): d 8.90 (s, 2H), 7.49–6.66 diclofenac sodium (1 mM) was utilized and treated similarly for
(m, 15H), 3.85 (br.s, 8H), 3.63 (s, 6H), 2.45 (s, 6H), 1.23 (t, J = 6.40 Hz, determination of the absorbance. The inhibition (%) of protein
3H); 13C NMR (100 MHz, CDCl3): d 165.3, 154.6, 147.8, 145.4, 144.2, denaturation was then calculated.
142.9, 129.6, 128.5, 127.7, 126.7, 126.4, 126.2, 125.8, 123.4, 122.3,
Radical scavenging activity by the DPPH method
121.6, 114.0, 113.9, 112.4, 109.9, 104.6, 55.0, 54.6, 40.9, 21.6, 11.6;
anal. calcd for C39H40N4O6S (%): C, 67.61; H, 5.82; N, 8.09; S, 4.63;
found: C, 67.84; H, 5.90; N, 8.00; S, 4.57.
The radical scavenging assay (DPPH) was carried out according
to the literature44 with minor modifications. 2,2-Diphenyl-1-
picrylhydrazyl (DPPH) (1.6 mg) was dissolved in DMSO (50 mL).
To 1.5 mL of each compound prepared (100 mg mLÀ1) was
added 1.5 mL of DPPH solution and the mixture was then kept
for 45 min incubation under dark conditions at room temperature.
The changes in absorbance at 517 nm were then measured. The
absorbance at 517 nm of the blank DPPH solution was used as
the control. The DPPH free radical scavenging activity was then
calculated.
Hybrid phenothiazinyldihydropyridinedicarboxamide 7i. A
mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan-
amide 6i (0.77 g, 3.92 mmol) and ammonium acetate (0.23 g,
2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
in ethanol (15 mL) by adopting the general method followed by
column chromatography (30–50% ethyl acetate in hexane) gave
the target hybrid phenothiazinyldihydropyridinedicarboxamide
7i. Yield, 0.96 g (80%); MP: 208–210 1C; FT-IR (KBr, cmÀ1): n
3880.7, 3817.1, 3628.1, 3039.8, 2999.3, 2902.8, 2831.5, 2673.3,
2370.5, 1809.2, 1664.5, 1602.8, 1506.4, 1454.3, 1398.3, 1350.1,
1296.1, 1244.0, 1172.7, 1031.9, 954.7, 835.1, 750.3, 615.2, 513.0,
Anticancer evaluation
Human pancreatic cancer cells SW1990, AsPC1, BxPC3 and
Panc1 as well as non-cancerous lung fibroblast MRC5 were
procured from the American Type Culture Collection, ATCC,
USA. All the cells were cultured in RPMI-FBS medium supplemented
with 100 IU mLÀ1 of penicillin and 100 mg mLÀ1 of streptomycin
(Sigma-Aldrich, St. Louis, MO, USA). All the pancreatic cancer cells
were incubated at 37 1C in 5% CO2, using the standard in vitro cell
culture method established previously.47–51
In order to evaluate the antitumor effects of the synthesized
chemical entities, stock solutions of the synthesized compounds
in DMSO (100 mM) were initially prepared, then further diluted
to 0.1 mM in sterile PBS. The cells were treated based on the cell
viability assay reported in the literature.47,52 First, 384-well plates
were seeded with cells in such a way that each well contained
1500 cells per well. Plates were incubated for 24 h and then the
cells were treated with the synthesized compounds for 72 h. Cell
viability was measured using the Cell-Titre Glo Luminescent Cell
Viability Assay (Promega, USA). The luminescence readings were
recorded using a SpectraMax M3 microplate reader (Molecular
Devices Corporation, USA).
1
435.9; H NMR (400 MHz, CDCl3): d 8.21 (d, 2H), 7.65 (s, 2H),
7.52 (s, 2H), 7.35–6.73 (m, 11H), 5.67 (s, 1H), 4.83 (s, 1H), 3.87
(br.d, J = 6.80 Hz, 2H), 2.33 (s, 6H), 1.35 (t, J = 7.00 Hz, 3H);
13C NMR (100 MHz, CDCl3): d 166.6, 144.8, 144.2, 140.0, 138.7,
136.1, 134.9, 132.4, 132.2, 128.4, 127.1, 126.6, 125.6, 124.8,
124.0, 122.6, 115.7, 115.0, 114.6, 113.8, 106.3, 41.8, 41.4, 19.1,
12.6; anal. calcd for C35H30F2N4O2S (%): C, 69.06; H, 4.97;
N, 9.20; S, 5.27; found: C, 69.31; H, 4.88; N, 9.11; S, 5.35.
Hybrid phenothiazinyldihydropyridinedicarboxamide 7j. A
mixture of carbaldehyde 3 (0.5 g, 1.96 mmol), aryloxobutan-
amide 6j (0.81 g, 3.92 mmol) and ammonium acetate (0.23 g,
2.94 mmol) in the presence of p-toluenesulfonic acid (15 mol%)
in ethanol (15 mL) by adopting the general method followed by
column chromatography (30–50% ethyl acetate in hexane) gave
the target hybrid phenothiazinyldihydropyridinedicarboxamide
7j. Yield, 1.03 g (84%); MP: 232–234 1C; FT-IR (KBr, cmÀ1): n
3547.0, 3315, 3283, 3047.5, 2972.3, 2875.8, 1942.3, 1593.2,
1512.1, 1392.6, 1338.6, 1188.1, 1111.0, 1080.1, 1045.4, 966.3,
889.1, 858.3, 754.1, 721.3, 686. 6, 619.1, 536.2, 499.5, 432.0;
1H NMR (400 MHz, CDCl3): d 8.91 (s, 2H), 7.33–6.56 (m, 15H),
3.86 (br.d, J = 5.60 Hz, 2H), 2.62 (s, 6H), 2.53 (s, 6H), 2.42 (s, 6H),
2.30 (s, 6H), 2.23 (s, 6H), 1.22 (t, J = 7.08 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d 165.4, 154.6, 147.9, 145.3, 144.3, 142.8,
142.4, 129.5, 128.5, 127.7, 126.7, 126.4, 126.2, 125.8, 123.4,
122.2, 121.5, 114.0, 113.9, 112.4, 109.9, 104.7, 41.6, 22.2, 22.0,
21.3, 11.5; anal. calcd for C39H40N4O2S (%): C, 74.49; H, 6.41;
N, 8.91; S, 5.10; found: C, 74.78; H, 6.50; N, 8.82; S, 5.05.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
Financial assistance provided by the Indian Council of Medical
Research, New Delhi, India (No. 58/16/2013BMS) is gratefully
acknowledged.
Anti-inflammatory activity by the protein denaturation method
The methods of Mazushima et al.42 and Sakat et al.43 were used
with a slight modification for the in vitro anti-inflammatory
study. The reaction mixture (2.5 mL) consisted of synthetic
derivatives (1 mL; 1 mM), egg albumin (0.1 mL) and phosphate
buffered saline (PBS, 1.4 mL; pH 6.4). Double distilled water
was utilized as the control (equal volume). The mixture was
then heated at 70 1C for 5 min after incubation at 37 1C Æ 2 for
15 min. Their absorbance, after cooling, was measured at
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