Phenothiazine and amide-ornamented novel nitrogen heterocyclic hybrids: synthesis, biological and molecular docking studies

Article abstract of DOI:10.1039/c9nj05489h

The synthesis of novel hybrids, namely, phenothiazine and amide-ornamented nitrogen heterocycles (25-34) has been accomplished utilizing a multi-step synthetic protocol and the structures have been established based on physical and spectral techniques. Of these, the hybrids possessing meta-nitro (26), para-fluoro (28), meta- and para-methyl (31), ortho-bromo (33) and ortho- and para-dimethyl (34) phenyl carboxamide scaffolds exhibited superior anti-inflammatory profiles over the standard diclofenac sodium. A hybrid integrated with a para-fluorophenyl carboxamide moiety (28) showed the highest DPPH radical scavenging activity among the chemical entities synthesized. Furthermore, the results of anticancer evaluations implied that the hybrid tethered with a phenyl carboxamide structural unit (29) exerted superior activity when compared with other hybrids against the pancreatic cancer cells SW1990 and AsPC1. Molecular docking between the hybrid 29 and B-cell lymphoma 2 reflects its appreciable binding affinity (-8.84 kcal mol^-1). The results revealed that these chemical entities can serve as potent biological agents and/or efficient intermediates for the construction of potent biological agents.

Full text of DOI:10.1039/c9nj05489h

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Chechia Hu, Tzu-Jen Lin et al.
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DOI: 10.1039/C9NJ05489H
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Phenothiazine and amide ornamented novel nitrogen heterocyclic
hybrids: Synthesis and their biological and molecular docking
studies
Received 00th January 20xx,
Accepted 00th January 20xx
Ramar Sivaramakarthikeyana, Ayyanar Karuppasamya, Shunmugam Iniyavala, Krishnaraj
DOI: 10.1039/x0xx00000x
Padmavathya, Wei-Meng Limb, Chun-Wai Maib,c, Chennan Ramalingana*
Synthesis of novel hybrids, phenothiaze and amide ornamented nitrogen heterocycles 25-34 has been accomplished
utilizing multi-step synthetic protocol and the structures have been established based on physical and spectral techniques.
Of those, the hybrids possessing meta- nitro (26), para- fluoro (28), meta- and para- methyl (31), ortho- bromo (33) and
ortho- and para- dimethyl (34) phenyl carboxamide scaffold exhibited superior anti-inflammatory profile over the standard
diclofenac sodium. A hybrid integrated with para- fluorophenyl carboxamide moiety (28) showed the highest DPPH radical
scavenging activity among the chemical entities synthesized. Further, the results of anticancer evaluation implied that the
hybrid tethered with phenyl carboxamide structural unit (29) exerted superior activity when compared with other hybrids
against the pancreatic cancer cells SW1990 and AsPCl. Molecular docking between the hybrid 29 and B-cell lymphoma 2
reflects its appreciable binding affinity (-8.84 Kcal / mol). The results revealed that these chemical entities could serve as
potent biological agents and / or serve as efficient intermediates for the construction of potent biological agents.
same. It is expected that the world death toll from cancer is
continuing to increase (~12 million deaths by 2030).2
Introduction
There are several types of treatments for cancer available
and the type of treatment depends on the type of cancer as
well as its advancement. Broad types of cancer treatment
comprise chemotherapy, surgery, immunotherapy, radiation
therapy, targeted therapy, hormone therapy and stem cell
transplant etc. Cancer and cancer treatment can cause one or
more side effects including appetite loss, constipation, fertility
issues in women and men, hair loss, memory or concentration
problems, nerve problems, pain, sexual health issues in
women and men, skin and nail changes, urinary and bladder
problems etc. There are hundreds of drugs commercially
available in the market to treat cancers, regrettably, most of
them are allied with severe side effects and consequently, it is
a challenging task to save lives. The rising rate of death year by
year, as a reason of cancer, has made apparent that there is an
abrupt need for the development of anticancer agents with
more potential that can eradicate cancer cells without harming
regular tissues. In this track, diverse design approaches are
being confirmed to identify potent molecules.
One of the foremost threats to universal health is non-
communicable diseases (NCDs) that are the origin for
significant number of deaths each year. Cancer is the second
most important cause of death in the world and caused by
abnormalities in cells, might be either because of exposure to
radiation / chemicals / even infectious agents or inherited. The
speedy propagation of unusual cells that develop away from
the usual boundaries, and then attacks adjacent parts of the
body and extending to other organs is one of the typical
characters of cancer. Stomach, lungs, pancreas, liver, breast
and colon are the usually affected organs, which culminated
with death every year. This non-communicable disease is a
main health defy not only in developed nations but also in
developing and under developed nations, where the number
of occurrences of the same is rapidly growing.1 There are 9.6
million deaths around the globe in 2018 due to this life-
threatening disease and one in six deaths is because of the
Molecular hybridization is an influential tool for designing a
drug that engrosses the hybridization of two or more potent
pharmacophores of either dissimilar biopertinent chemical
entities or similar biopertinent chemical entities into one
molecular scaffold.3 It is being extensively used due to the fact
that numerous molecules of such crossbreed nature exhibited
enhanced efficiency besides safer toxicity report, while
comparing with their parent molecules. Based on this strategy,
several biologically potent structural motifs, mainly
heterocyclic units are being investigated to recognize novel
lead compounds.
a. Department of Chemistry, School of Advanced Sciences, Kalasalingam Academy of
Research and Education (Deemed to be University), Krishnankoil, 626 126,
Tamilnadu, India.
E-mail ID: ramalinganc@gmail.com
b. School of Pharmacy, International Medical University, 126 Jalan Jalil Perkasa 19,
57000, Bukit Jalil, Kuala Lumpur, Malaysia.
c. Center for Cancer and Stem Cell Research, Institute for Research, Development
and Innovation (IRDI), International Medical University, 126, Jalan Jalil Perkasa
19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Dihydropyrimidines, a class of molecules possessing a six
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With respect to anticancer aspects, examination on
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membered cyclic structure with nitrogens at 1 and 3 positions,
are an imperative category of heterocycles. Over the few
decades, the interest on the same among the researchers has
been increased because of a couple of independent reasons.
One is comparatively easiness of the construction of the ring
structure with wide variety of derivatization4 and the other is
their recognition as valuable pharmacophores exhibiting a
wide variety of pharmacological profile including anti-
inflammatory,5 adrenergic antagonists,6 antihypertension,7
antimicrobial8 and so on. Of the research on biological aspects,
the interest in the anticancer properties of pyrimidines has
increased rapidly since the discovery of Monastrol, a novel low
molecular weight, cell permeable chemical entity that also
cause mitotic arrest in mammalian cells in mitosis with
monopolar spindles, in 1999.9 Representative examples of
biopertinent chemical entities possessing dihydropyrimidine
scaffold are furnished in Figure 1.
DOI: 10.1039/C9NJ05489H
chlorpromazine (Figure 2), a phenothiazine pharmacore
possessing drug, revealed that it can augment effects of
cytotoxicity of tamoxifen on the cancer associated with
breast.32 Molecules possessing phenothiazine structural motif
can also improve sensitivity of cisplatin, a drug being used for
chemotherapy.33 Particularly, a couple of derivatives of
9
phenothiazine
such
as
(2,4-dimethoxyphenyl)(10H-
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phenothiazin-10-yl)methanone have been examined in order
to understand their ability to hamper antiproliferative activity
and tubulin polymerization against six dozens of cancer cell
lines.34 The chemical entities exhibited notable inhibition of
cell growth on COLO 205 (colorectal adenocarcinoma), A498
(human kidney adenocarcinoma) and Duke’s type D (human
colon) cell lines.34 All these noteworthy results recommended
that phenothiazine scaffold could be considered as a capable
anticancer pharmacophore in drug discovery.
Fig 2. Representative anticancer molecules possessing phenothiazine
structural motif
On the other hand, amide moiety is a vital structural motif
which serves as possible ligand part in the interaction of a
chemical entity with host proteins through H- bonding and / or
vander-Waals power of attractions. Chemical entities
integrated with amide scaffolds exhibit diverse biological
activities that include antimalarial and anticancer etc.35-38
Fig 1. Representative examples of dihydropyrimidines with biological importance
Fused thiazines, especially phenothiazines are documented
since nineteenth century when Bernthsen synthesized the first
parent molecule, 10H-dibenzo-1,4-thiazine in 1883.10 Because
of their striking chemical character besides noteworthy
pharmacological / biological potency, their construction,
plausible reactions, properties, and applications especially
pharmacological / biological angle were scrupulously reviewed
three decades ago and retrosynthetic approach for the
construction of phenothiazine was also depicted.11,12
In the recent years, numerous articles related to these
fused thiazines were appeared and until present, over 6000
derivatives of the same were attained. Further, a minimum ten
dozen of these derivatives were exploited in treatment. An
important aspect of this scaffold is its capability to offer a wide
range of analogues (substituents could be incorporated at
various positions besides oxidation of sulphur and
replacement of homoaryl by heteroaryl units viz., pyrimidine,
pyridine, quinoline, pyridazine, 1,2,4-triazine, and pyrazine and
so on). In addition, this fused thiazine is reported to be
harmless, an unusual one, and it is tolerable up to doses of
multi-gram scale in humans.13 A diverse range of molecules
incorporated with phenothiazine unit exhibit various
Fig 3. Dihydropyridine, phenothiazine and amide hybridized
novel chemical entities
Having the biological magnitude of the aforementioned
scaffolds in mind, a series of fused thiazine, dihydropyrimidine
and amide hybridized novel molecules has been designed and
synthesized as biopertinent ones. Since it is known that cancer
is a type of inflammation, and also it is known that the ROC
may influence the cellular components which cause cancer,
preliminary anti-inflammatory evaluation and plausible radical
pharmacological/biological
properties.
Their
striking
characteristics were reviewed recently.14-31
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scavenging activities of the novel molecules have been alkylation utilising bromoalkane in the presencVeiewoAfrtiKclOe OHnlinine
determined. Further, anticancer evaluation of all the novel DMF followed by Vilsmeier-Haack reactiDoOnI:w10it.h103th9/eC9uNtiJl0iz5a4t8io9Hn
chemical entities have been executed against pancreatic of POCl3 and DMF in dichloromethane.40 A multicomponent
cancer cells (human) SW1990 and AsPCl. Described herein are one-step reaction between appropriate 3-oxo-N-substituted
synthesis, characterization, anti-inflammatory, radical phenylbutanamides 12-21, phenothiazine carbaldehyde 24,
scavenging and anticancer evaluations as well as molecular and urea in the ratio of 1.1:1:1.5, respectively in the presence
docking
studies
of
novel
hybrids, of p-toluenesulfonic acid in ethanol eventually produced the
phenothiazenyldihydropyrimidine carboxamides as provided in desired target hybrid phenothiazinyltetrahydropyrimidine
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Figure 3.
carboxamides 25-34 in good yields. The structures of novel
phenothiazinyltetrahydropyrimidinecarboxamides have
unequivocally been established based on physical and spectral
methods.
Results and discussion
Chemistry
In the proton NMR spectra of the target
phenothiazinyltetrahydropyrimidine carboxamides 25-34, one
of the secondary amino protons of dihydropyrimidine
structural unit resonates between 5.40 and 6.38 ppm as a
singlet with one proton integral while signal for the other
secondary amino proton of dihydropyrimidine scaffold merged
with aromatic signals. The proton connected to the nitrogen of
the amide moiety resonate from 7.34 to 8.15 ppm as a singlet
with one proton integral except in 27-29, wherein the signal
for this proton is merged with aromatic ones. The protons of
The sketch of the synthetic scheme for the construction of
hybrid phenothiazinyltetrahydropyrimidinecarboxamides 25-
34 is furnished in Scheme 1. Firstly, the synthesis of
intermediates, 3-oxo-N-substituted phenylbutanamides 12-21
has been achieved from the reaction of β-keto ester 1 with
appropriately substituted anilines 2-11 in the presence of KOH
in toluene.39 On the other hand, synthesis of the active
intermediate phenothiazine carbaldehyde 24 has been
accomplished from commercial phenothiazine 22 by adopting
Scheme 1. Synthesis of novel hybrid phenothiazinyltetrahydropyrimidine carboxamides 25-34
This journal is © The Royal Society of Chemistry 20xx
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the methyl group attached to the 6-position of carbon present at 6- position of the dihydropyrimidine
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dihydropyrimidine core offer a singlet with three protons resonate in the region between 144.42DaOnId: 101.41053.96/6C9pNpJm05.4T8h9He
integral in the region 2.15 to 2.28 ppm while the protons of carbon of the carbonyl group of amide unit exhibits signal in
the methyl moiety of ethyl unit attached to phenothiazine the region from 163.36 to 165.75 ppm while the carbon of the
observed in the region 1.28 to 1.39 ppm as a three-proton carbonyl group located between two secondary amino
triplet. The methylene protons of the ethyl moiety tethered at moieties possesses signal between 152.63 and 153.46 ppm. In
nitrogen of the phenothiazine resonate in the region from 3.78 addition, the methyl carbons tethered at ortho- and para-
to 3.89 ppm as either broad doublet or quartet with two positions of the phenyl ring in 34 offered signals at 20.80 and
protons integral. In addition, the methine proton connected at 17.94 ppm while the same attached at meta- and para-
4-position of dihydropyrimidine resonates in the region positions of the phenyl rings 31 provided signals at 20.04 and
between 5.16 and 5.27 ppm as a one proton singlet while the 19.22 ppm. Besides, the carbons of two methoxy moieties
aromatic protons of the targets exert multiplet in the region attached at meta- and para- positions of phenyl unit in 27
between 6.59 and 7.93 ppm. Besides, the protons of methyl resonate at 56.28 and 56.11 ppm. Further, the signals for all
groups tethered at aromatic ring in 31 offer signals at 2.14 and the aromatic carbons observed in the region from 111.23 to
2.16 ppm as singlets with three protons integral while the ones 145.50 ppm. All these characteristics in addition to
attached to the aromatic scaffold in 34 provide signals at 2.17 microanalysis and IR corroborate the target molecules.
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and 2.23 ppm as singlets with three protons integral.
The plausible mechanism for the formation of target
Eventually, the protons of the methoxy units tethered to the molecules is furnished in Scheme 2. Initially, nucleophilic
aromatic scaffold in 27 resonate as three protons singlet at addition reaction between respective phenothiazine aldehyde
3.76 and 3.81 ppm.
and urea provides their corresponding hemiaminals, 1-
In the carbon NMR spectra of the target (hydroxy(10-alkyl-10H-phenothiazin-3-yl)methyl)ureas (I). The
phenothiazinyltetrahydropyrimidine carboxamides 25-34, the corresponding hemiaminals then abstracts proton from the p-
signal for the methyl carbon tethered at 6-positions of toluenesulfonic acid to form respective oxonium ions, [(10-
dihydropyrimidine scaffold resulted in the region from 16.82 alkyl-10H-phenothiazin-3-yl)(ureido)methyl]oxonium ions (II)
to 18.00 ppm. The signals for methylene carbon (attached to which subsequently loses water molecule to afford uronium
nitrogen of phenothiazine unit) and methyl carbon of ethyl ions, 1-[(10-alkyl-10H-phenothiazin-3-yl)methylene]uroniums
group observed in the region 40.64-41.91 ppm and 11.90- (III). Respective amides, synthesized previously from
13.18 ppm, respectively. The methine carbon located at 4- ethylacetoacetate and aromatic amines, eventually attacks the
position of dihydropyrimidine exert signal in the region from iminium carbon of the intermediates III to furnish open chain
54.63 to 55.89 ppm while the signal for carbon situated at 5- intermediates IV with elimination of proton which
position of the dihydropyrimidine core observed in the region subsequently loses water to provide the title molecules.
between 103.43 and 105.83 ppm. Further, the signal for
Scheme 2. Plausible mechanism for the synthesis of target molecules 25-34.
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substituent on the ortho- position of the aryl moiety of the
Biological activities
Anti-inflammatory activity
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amide unit (33) exhibited percentage inDhOibI:it1i0o.1n03s9u/pCe9rNiJo0r54th89aHn
that of the standard though marginal improvement. Among all
the molecules evaluated, 34 was found to be the most active
one.
Anti-inflammatory evaluation (in vitro) of all the
synthesized
target
phenothiazinyltetrahydropyrimidine
carboxamides 25-34 has been carried out by employing the
method of protein denaturation.41,42 Protein denaturation is a
technique in which the secondary and tertiary structure of
protein would be lost when an external stress or a molecule is
applied. The biological function of most biological proteins
would be lost when denatured. As it is known, denaturation of
proteins is a well-documented basis of inflammation. As a
piece of the present exploration, capability of target molecules
25-34 for the inhibition of protein denaturation was assessed.
The standard drug used in this investigation was diclofenac
sodium which displayed ~90% inhibition of protein
denaturation. The percentage inhibitions of protein
denaturation upon the use of target molecules 25-34 are
furnished with respect to the inhibition by standard diclofenac
sodium and the results are provided in Table 1 and Figure 4.
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Fig 4. Antiinflammatory activity of 25-34 with respect to diclofenac sodium.
Table 1. Anti-inflammatory activity of 25-34.
Radical scavenging activity
S.No.
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Compound
% Inhibition
87.45 ± 0.99
100.67 ± 1.73
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102.72 ± 0.69
75.25 ± 1.25
100.67 ± 1.45
104.74 ± 1.09
Reactive oxygen species (ROS) are basis for various
neurodegenerative illnesses which include ulcer, Alzheimer’s,
mutagenesis, autoimmune disorder, and carcinogenesis by
hampering the function of several cellular components.
Antioxidants are agents that scavenge the free radicals formed
in our biological system. Although a diverse range of such
agents are present in nature, it is vital to develop a drug with
superior antioxidant activity. For the determination of radical
scavenging activity, various methodologies which include
hydroxyl radical scavenging assay, ferric reducing antioxidant
power, and organic radical scavenging assay are available.
Among all of them, organic radical scavenging by 2,2-diphenyl-
1-picrylhydrazyl (DPPH) is being extensively utilized by the
researchers due to its simplicity. Accordingly, all the target
phenothiazinyltetrahydropyrimidine carboxamides 25-34 have
been examined for radical scavenging activity against DPPH, a
reactive oxygen species.43 The activity has been assessed in
terms of inhibition of DPPH and the results are given in Table 2
and Fig. 5 on comparison with the activity of a standard
ascorbic acid.
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10
When the amide and phenothiazine integrated
dihydropyrimidine having chloromoieties on the meta- and
para- positions of the aryl unit of the amide group and ethyl
moiety on the nitrogen of the phenothiazine (25) was used, it
exhibits 87% inhibition on comparing to the inhibition of
standard diclofenc sodium. Replacing the chloro moieties on
the meta- position by nitro group and para- positions by
hydrogen in 25 (i.e., 26) enhanced the inhibition (13%) which is
slightly higher than that of the standard drug. Incorporation of
methoxy groups on the meta- and para- positions of the aryl
ring in place of chloro moieties in 25 (i.e., in 27) did not
improve significantly the inhibition, it exhibits almost similar
inhibition while the incorporation of bromo on the para-
position of the aryl moiety instead of chloro unit and replacing
the chloro group on the meta- position of the aryl group by
hydrogen in 25 (i.e., in 32) reduced the percentage of
inhibition by 12%. The chemical entities with no substituents
on the aryl scaffold of the amide unit (29) as well as fluoro
substituent on the ortho- position of the aryl moiety of the
amide unit (30) showed excellent results (97% and 98%
inhibition, respectively) when compared to the standard drug.
Further, integration of methyl substituents on the meta- and
para- positions as well as ortho- and para- positions of the aryl
group of amide unit (31 and 34, respectively) and also bromo
Fig 5. Radical scavenging activity of 25-34
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Table 2. Radical scavenging of target molecules 25-34.
carboxamides, the survival percentages of pancreatic cancer
cells are furnished in Table 3.
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DOI: 10.1039/C9NJ05489H
S. No.
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Molecule
25
% Scavenging
75.03 ± 0.89
81.48 ± 1.28
76.71 ± 1.75
87.43 ± 0.80
83.48 ± 0.98
74.72 ± 1.38
75.23 ± 2.05
77.18 ± 1.73
80.19 ± 1.32
82.26 ± 1.46
IC50 (µM)
-
The carboxamides possessing ethyl substituent on the 10-
position of phenothiazine unit and chloro functionalities on
the meta- and para- positions of the aryl moieties of amide
scaffold (25), nitro group on the meta- position of amide
scaffold (26), methoxy substituents on the meta- and para-
positions of the amide moiety (27), fluoro substituent on the
para- position of the amide unit (28) and methyl substituent
on the meta- and para- positions of the amide scaffold (31)
exerted IC50 value of greater than 100 μM against both the
cells investigated. The molecules 32 and 33 showed better
activity against the cell SW1990 while the molecule 34
exhibited superior activity against both the cells ASPC1 and
SW1990. Among all the molecules tested, the one possessing
ethyl substituent on the nitrogen of the phenothiazine and no
substituents on the amide unit (29) showed the best activity
against both the cancer cells tested. The activity of all the
molecules against a normal cell MRC5 is also investigated and
found that the IC50 against the most active molecule was much
higher. This result explicitly implies that the most active
molecule, the one possessing ethyl substituent on the nitrogen
of the phenothiazine and no substituents on the amide unit
(29) is less toxic.
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47.63
48.93
103.80
22.81
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25.46
381.76
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The
target
phenothiazinyltetrahydropyrimidine
carboxamide 29 possessing no substituents on the aryl group
of amide unit exhibited 83% inhibition of DPPH radical when
compared with the inhibition by ascorbic acid, the standard.
Incorporation of chloro moieties on the meta- and para-
positions of the aryl moiety of amide unit in 29 (i.e., in 25)
reduced the inhibition percentage to a magnitude of 8%,
however, integration of nitro group on the meta- position of
the aryl moiety of amide unit in 29 (i.e., in 26) reduced the
inhibition percentage to a magnitude of only 2%. In addition,
the carboxamide containing methoxy substituents on the
meta- and para- positions (27), fluoro substituent on the
ortho- position (30), methyl substituents on the meta- and
para- positions (31) and bromo substituent on the para
position (32) of the aryl moiety of amide unit showed almost
similar DPPH inhibition when compared with the molecule
possessing chloro substituents on the meta- and para-
positions of the aryl moiety of amide unit (25). On the other
hand, the molecules possessing bromo substituent on the
ortho- position of the aryl unit (33) and methyl substituents on
the ortho- and para- positions of the aryl moiety (34) exhibited
almost similar DPPH inhibition when compared with the one
possessing nitro substituent on the meta- position of the aryl
unit of amide scaffold (26). Among all the
Table 3. Cell viability of pancreatic cancer cells.
IC50 μM
ASPC1
>100
>100
>100
>100
15.7 ± 2.29
>100
>100
>100
S.No. Compound
MRC5
>100
>100
>100
SW1990
>100
>100
>100
>100
22.8 ± 4.24
>100
>100
82.7 ± 5.67
51.1 ± 3.31
76.0 ± 3.19
1
2
25
26
27
28
29
30
31
32
33
34
3
4
5
6
>100
83.7 ± 1.80
84.7 ± 4.44
>100
>100
70.7 ± 3.11
92.6 ± 3.12
7
8
>100
57.0 ± 4.12
9
10
phenothiazinyltetrahydropyrimidine
carboxamides
synthesized, the one tethered with fluoro substituent on the
para- position of the aryl moiety of the amide scaffold (28)
exerted highest DPPH inhibition (87%).
Molecular docking
The molecular sources of communications among the active
sites of BCL-2 (B-Cell Lymphoma 2) and binding affinity,
obtained from the protein data bank, and the structure of
carboxamide 29, a three dimensionally stabilized one
(conformationally stabilized), were assessed via molecular
docking studies by employing Auto dock version 4.0. The
results harvested through docking are given in Figure 6.
The phenothiazinyltetrahydropyrimidine carboxamide 29
exhibited its binding energy -8.84 Kcal/mol in addition to
predicted IC50 value of 368.23 nM. The chemical entity 29
show van der Waals communication with phenyl alanine,
hydrophobic side chain possessing amino acid, glutamic acid,
electrically charged side chain containing amino acid and
glycine. Further, a conventional hydrogen bond interaction
between an amino acid with electrically charged side chain
aspartic acid and NH of the pyrimidine core was observed.
Anticancer activity
One of the deadliest maladies in the globe is cancer and,
there are numerous varieties of cancers which affect human.
While several categories of drugs have been developed to
combat cancers, the discovery of newer drugs for the same is
vital as there are no precise drugs which could entirely cure
the cancer is existing. Also, the drugs, in common, would
become resistant over a period of time because of the
environmental / biological variations. In this piece of research,
the studies on cytotoxicity of the entire target
phenothiazinyltetrahydropyrimidine carboxamides 25-34 have
been assessed against AsPC1 and SW1990, the pancreatic
cancer cell lines by the methodology of Cell-Titre Glo
Luminescent Cell Viability Assay.44-48 Against the tested
6 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C9NJ05489H
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25
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60
Fig 6. 2D & 3D interactions of BCL-2 with 29.
inflammatory studies employing protein denaturation
Besides, an interaction between an amino acid with technique reveal that the target hybrid molecules integrated
hydrophobic side chain phenyl alanine and sulphur present in with m-nitro, p-fluoro, m,p-dimethyl, o-bromo and o,p-
the central ring of phenothiazine structural motif was also dimethyl phenyl carboxamide structural motifs exhibited
noted. In addition, there exist π-π stacked interactions enhanced activity than that of the standard diclofenac sodium
between aromatic unit of tyrosine, an amino acid with under the experimental conditions. Radical scavenging activity
hydrophobic side chain and one of the aromatic rings of the through DPPH method reflects that p-fluorophenyl
phenothiazine structural core as well as pyrimidine core of the carboxamide tethered hybrid (28) showed the highest activity
molecule. It has also been observed various either alkyl-alkyl among the molecules investigated. Further, anticancer
interactions or π-alkyl interactions between valine and evaluation against pancreatic cancer cell lines SW1990 and
methionine, amino acids with hydrophobic side chains and one AsPCl utilizing cell-titre glo luminescent cell viability assay
of the aryl rings of the phenothiazine structural scaffold, methodology disclose that the hybrid integrated with phenyl
valine, leucine and methionine, amino acids with hydrophobic carboxamide scaffold (29) provided the highest activity while
side chains and methyl group of the ethyl moiety integrated at comparing the rest of the molecules. Molecular docking
nitrogen of the phenothiazine, and also alanine, an yet other revealed appreciable binding affinity between the most active
amino acid with hydrophobic side chain with another one aryl chemical entity 29 and B-cell lymphoma 2. The results expose
ring as well as central ring of the phenothiazine scaffold.
that these chemical entities could serve as potent biological
agents and / or serve as efficient intermediates for the
construction of potent biological agents. Further, anticancer
exploration of the potent molecule with varied cell lines,
besides development of structurally diversified significant
hybrids which exert noticeable outcomes are currently under
the way.
Conclusions
A
series
of
phenothiazinyltetrahydropyrimidine
carboxamides 25-34 has been synthesized through multi-step
synthetic strategy. All the chemical entities have been
characterized using physical and spectroscopic methods. Anti-
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mL), washed well with aqua, dried over MgSO4 and the solvent
was evaporated under reduced pressDuOreI:.10T.1h0e3V9i/ecCwr9uANrdtJice0le5O4t8hnl9uinHes
resulted was subjected to column chromatography to furnish
the pure product 24.40
Experimental Section:
General
All Chemicals were procured from commercial suppliers as
reagent grade and utilized as received. By adopting standard
methods, all solvents were distilled/dried before their
utilization. Pre-coated TLC sheets (silica-gel) were utilized for
TLC and visualized by short- and long-wavelength UV lamps.
Column chromatography was executed on silica-gel (spherical,
100–200 mesh) slurry packed in glass columns. The eluents
utilized for individual separations are provided in the
respective experimental protocols. All FT-IR spectra (KBr pellet
form) were recorded in the range of 4000–400 cm-1 on a
General method for the synthesis of hybrid heterocycles (25-34):
To an ethanolic mixture of carbaldehyde 24 (1 equi. in 15
mL),
were
added
respective
substituted
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
phenyloxobutanamides 12-21 (1.1 equi.), urea (1.5 equi.) and
catalytic amount of p-toluenesulfonic acid (15 mol %). The
mass in the flask was refluxed for 12 h and the solvent was
evaporated utilizing rotary evaporator after attaining ambient
temperature. The crude thus obtained was then extracted with
DCM after diluting with aqua. The organic layer was separated,
dried on Na2SO4, and evaporated using a rotary evaporator.
The solid thus resulted was subjected to column
chromatography using the eluent system ethyl acetate in
hexane (40~50 %) to provide respective pure target hybrid
molecules 25-34 as solids.
Shimadzu IR Tracer-100 spectrophotometer. H and 13C NMR
1
spectra were recorded on NMR spectrometer (Bruker AVANCE
II 400 and 100 MHz) at 25 °C with the use of TMS as an internal
standard and DMSO-d6 / CDCl3 as solvent and chemical shifts
are denoted in terms of ppm ( ppm). The spin multiplicities
m, q, t, d and s stand for multiplet, quartet, triplet, doublet,
and singlet, respectively.
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 25
To a mixture of carbaldehyde 24 (0.5 g, 1.96 mmol) in
ethanol (15 ml), was added aryloxobutanamide 12 (0.53 g,
General method for the synthesis of 3-oxo-N-substituted
phenylbutanamides (12-21)
To a solution of ethylacetoacetae (2 g, 15.4 mmol) in 2.15 mmol), Urea (0.18 g, 2.94 mmol) and catalytic amount of
toluene (20 mL), was added powdered KOH (15 mol %). p-toluenesulfonic acid (10 mol%). After completion of the
Respective substituted anilines 2-11 (17.0 mmol) were then reaction by adopting the general method, it gave the target
added to the solution. The reaction mass was heated so as to hybrid phenothiazinyltetrahydropyrimidinecarboxamide 25
reflux and it was continued (8 h) while stirring. Then it was (hexane-ethyl acetate 6:4). Yield, 0.92g (89%); MP: 124-126°C;
extracted with DCM after attaining ambient temperature, and FT-IR (KBr, cm-1): ν 3645.4, 3230.7, 3091.8, 2922.1, 2850.7,
washed with aqua. The organic layer thus obtained was dried 1699.2, 1670.3, 1581.6, 1508.3, 1465.9, 1375.2, 1323.1,
over Na2SO4 (anhyd.) and evaporated. The crude mass thus 1234.4, 1130.2, 1109.0, 1085.9, 1026.1, 875.6, 813.9, 746.4,
1
obtained was subjected to silica-gel column chromatography 675.0, 644.2, 572.8, 509.2, 484.1, 466.7, 439.7; H NMR (400
utilizing 10~20% ethyl acetate in hexane to eventually provide MHz, CDCl3): δ 8.13 (s, 1H), 7.56-6.71 (m, 11H), 6.38 (s, 1H),
the respective substituted arylbutanamides 12-21.39
5.19 (s, 1H), 3.78 (br.d, J=6.02 Hz, 2H), 2.15 (s, 3H), 1.29 (t,
J=6.80 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 164.9, 153.5,
145.7, 144.1, 140.2, 137.3, 136.1, 132.5, 130.3, 127.5, 125.3,
122.7, 121.6, 119.2, 115.4, 115.2, 105.3, 55.9, 41.9, 17.9, 13.1;
Anal. Calcd. for C26H22Cl2N4O2S (%): C, 59.43; H, 4.22; N, 10.66;
S, 6.10; Found: C, 59.63; H, 4.33; N, 10.49; S, 6.02.
Synthesis of alkylphenothiazine 23
In a characteristic experiment, an RB flask (250 mL) was
charged with N, N-dimethylformamide (50 mL) and 10H-
phenothiazine (0.025 mol). Powdered KOH (0.03 mol) was
then added into the reaction vessel and it was stirred at
ambient temperature for 2h. To this mixture, bromoethane
(0.04 mol) was added (drop-wise) using a pressure equalizer
for 20 min. and the reaction was continued at the same
temperature for 24 hrs. The reaction progress was examined
through TLC. The mixture was then extracted with DCM (3 x 20
mL), washed well with aqua and dried over Na2SO4 (anhyd.).
Removal of the DCM and subsequent purification through
column chromatography eventually offered the title
compound 23.40
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 26
A mixture of carbaldehyde 24 (0.5 g, 1.96 mmol),
aryloxobutanamide 13 (0.48 g, 2.15 mmol), urea (0.18 g, 2.94
mmol) and catalytic amount of p-toluenesulfonic acid (10
mol%)
in
ethanol
provided
the
target
hybrid
phenothiazinyltetrahydropyrimidinecarboxamide 26 (hexane-
ethyl acetate 6:4). Yield, 0.85g (86%); MP: 142-144°C; FT-IR
(KBr, cm-1): ν 3240.4, 2956.8, 2926.0, 1598.9, 1529.5, 1463.9,
1425.4, 1384.8,1350.1, 1327.0, 1236.0, 1134.1, 1107.1, 1087.8,
1043.4, 945.1, 881.4, 815.8, 796.6, 736.8, 702.0, 671.2, 596.0,
1
538.1, 453.2, 418.5; H NMR (400 MHz, CDCl3): δ 8.15 (s, 1H),
7.93-6.74 (m, 12H), 6.22 (s, 1H), 5.27 (s, 1H), 3.81 (br.d, J=6.00
Hz, 2H), 2.24 (s, 3H), 1.31 (t, J=6.60 Hz, 3H); 13C NMR (100
MHz, CDCl3): δ 165.3, 153.1, 145.7, 145.5, 143.5, 134.8, 132.6,
130.5, 130.0, 128.4, 127.9, 127.4, 120.3, 118.9, 118.8, 105.4,
55.9, 41.9, 17.8, 13.1; Anal. Calcd. for C26H23N5O4S (%): C,
62.26; H, 4.62; N, 13.96; S, 6.39; Found: C, 62.43; H, 4.73; N,
13.84; S, 6.30.
Synthesis of carbaldehyde 24
In a two neck RB flask (100 mL) under inert atmosphere,
was charged DMF (1 mL). POCl3 (1.2 mL) was then added in a
drop-wise fashion at 0 °C and stirred for 45 minutes. A
chloroform (20 mL) solution of phenothiazine 23 (11.0 mmol)
was then added drop wise into the reaction mixture. After the
addition is completed, the temperature was increased to 60 °C
and the stirring was continued for 6 hrs. The reaction progress
was monitored through TLC. The reaction mixture, after
completion of the reaction, was extracted with DCM (3 × 20
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 27
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To a mixture of carbaldehyde 24 (0.5 g, 1.96 mmol) in
ARTICLE
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ethanol (15 ml), was added aryloxobutanamide 14 (0.51 g,
2.15 mmol), urea (0.18 g, 2.94 mmol) and catalytic amount of
p-toluenesulfonic acid (10 mol%). After completion of the
reaction by adopting the general method, it gave the target
hybrid phenothiazinyltetrahydropyrimidinecarboxamide 27
(hexane-ethyl acetate 6:5). Yield, 0.83g (82%); MP: 152-153°C;
FT-IR (KBr, cm-1): ν 3230.7, 3115.0, 2960.7, 2908.6, 2835.3,
1699.2, 1670.3, 1624.0, 1602.8, 1512.1, 1467.8, 1442.7,
1411.8, 1382.9, 1363.6, 1330.8, 1244.0, 1201.6, 1166.9,
1134.1, 1022.2, 964.4, 881.4, 844.8, 800.4, 752.2, 713.6, 653.8,
DOI: 10.1039/C9NJ05489H
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 30
A mixture of carbaldehyde 24 (0.5 g, 1.96 mmol),
aryloxobutanamide 17 (0.42 g, 2.15 mmol), urea (0.18 g, 2.94
mmol) and catalytic amount of p-toluenesulfonic acid (10
mol%)
in
ethanol
provided
the
target
hybrid
phenothiazinyltetrahydropyrimidinecarboxamide 30 (hexane-
ethyl acetate 6:5). Yield, 0.85g (91%); MP: 168-170°C; FT-IR
(KBr, cm-1): ν 3340.7, 3209.5, 2960.7, 2924.0, 2854.6, 1697.3,
1662.6, 1627.9, 1523.7, 1500.6, 1458.1, 1319.3, 1261.4,
1022.2, 910.4, 800.4, 748.3, 702.0, 663.5, 605.6, 578.6, 553.5,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
597.9, 543.9, 513.0, 489.9; H NMR (400 MHz, CDCl3): δ 7.78-
1
6.66 (m, 12H), 5.60 (s, 1H), 5.25 (s, 1H), 3.88 (br.d, J=6.05 Hz,
2H), 3.81 (s, 3H), 3.76 (s, 3H), 2.23 (s, 3H), 1.28 (t, J=6.72 Hz,
3H); 13C NMR (100 MHz, CDCl3): δ 165.3, 153.6, 145.9, 138.8,
131.1, 127.5, 125.8, 125.4, 122.7, 115.5, 112.0, 111.2, 105.0,
56.3, 56.1, 55.8, 41.9, 18.0, 12.9; Anal. Calcd. for C28H28N4O4S
(%): C, 65.10; H, 5.46; N, 10.85; S, 6.21; Found: C, 64.93; H,
5.58; N, 10.70; S, 6.11.
532.3, 486.0, 451.3; H NMR (400 MHz, CDCl3): δ 7.42 (s, 1H),
7.40-6.71 (m, 12H), 5.47 (s, 1H), 5.16 (s, 1H), 3.83 (q, J=6.67 Hz,
2H), 2.24 (s, 3H), 1.32 (t, J = 6.60 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 163.4, 152.6, 144.4, 143.4, 140.4, 135.0, 126.3, 125.3,
124.9, 124.6, 124.4, 123.4, 123.1, 122.7, 121.5, 120.8, 114.5,
113.9, 103.5, 54.9, 40.8, 17.15, 11.92; Anal. Calcd. for
C26H23FN4O2S (%): C, 65.81; H, 4.89; N, 11.81; S, 6.76; Found: C,
65.99; H, 4.80; N, 11.69; S, 6.63.
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 28
A mixture of carbaldehyde 24 (0.5 g, 1.96 mmol),
aryloxobutanamide 15 (0.42 g, 2.15 mmol), urea (0.18 g, 2.94
mmol) and catalytic amount of p-toluenesulfonic acid (10
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 31
To a mixture of carbaldehyde 24 (0.5 g, 1.96 mmol) in
ethanol (15 ml), was added aryloxobutanamide 18 (0.44 g,
2.15 mmol), urea (0.18 g, 2.94 mmol) and catalytic amount of
p-toluenesulfonic acid (10 mol%). After completion of the
reaction by adopting the general method, it gave the target
hybrid phenothiazinyltetrahydropyrimidinecarboxamide 31
(hexane-ethyl acetate 6:4). Yield, 0.85g (89%); MP: 220-222°C;
FT-IR (KBr, cm-1): ν 3396.6, 3319.4, 3219.1, 3091.8, 1701.2,
1672.2, 1618.2, 1535.3, 1500.6, 1462.0, 1384.8, 1325.1,
1284.5, 1284.5, 1238.3, 1199.7, 1165.0, 1132.2, 1107.1, 954.7,
893.0, 864.1, 823.6, 744.5, 700.1, 675.0, 651.9, 592.1, 565.1,
mol%)
in
ethanol
provided
the
target
hybrid
phenothiazinyltetrahydropyrimidinecarboxamide 28 (hexane-
ethyl acetate 6:5). Yield, 0.81g (87%); MP: 188-189°C; FT-IR
(KBr, cm-1): ν 3435.2, 3373.5, 3215.3, 3089.9, 1710.8, 1653.0,
1624.0, 1510.2, 1463.9, 1404.1, 1328.9, 1236.3, 1201.6,
1159.2, 1132.2, 1078.2, 1039.6, 1012.6, 941.2, 831.3, 810.1,
794.6, 761.8, 746.4, 702.0, 677.0, 657.7, 609.5, 580.5, 549.7,
511.1, 493.7, 453.2; 1H NMR (400 MHz, CDCl3): δ 7.32-6.81 (m,
13H), 5.58 (s, 1H), 5.24 (s, 1H), 3.89 (br.d, J=6.00 Hz, 2H), 2.24
(s, 3H), 1.39 (t, J=6.80 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ
165.7, 153.0, 144.7, 144.0, 139.0, 138.9, 135.9, 128.0, 127.4,
125.9, 123.4, 122.7, 121.8, 115.5, 115.3, 105.5, 54.6, 41.5,
17.5, 13.1; Anal. Calcd. for C26H23FN4O2S (%): C, 65.81; H, 4.89;
N, 11.81; S, 6.76; Found: C, 65.98; H, 4.99; N, 11.69; S, 6.70.
1
536.2, 513.0, 457.1; H NMR (400 MHz, CDCl3): δ 7.34 (s, 1H),
7.26-6.80 (m, 11H), 5.53 (s, 1H), 5.24 (s, 1H), 3.89 (br.d, J=6.80
Hz, 2H), 2.21 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 1.38 (t, J=6.80
Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 165.5,153.0, 144.8,
144.0, 139.0, 137.3, 136.4, 131.2, 129.7, 128.0, 127.4, 126.0,
125.3, 123.8, 122.7, 121.3, 117.6, 115.8, 114.7, 105.8, 54.7,
41.5, 20.0, 19.2, 17.5, 13.1; Anal. Calcd. for C28H28N4O2S (%): C,
69.40; H, 5.82; N, 11.56; S, 6.62; Found: C, 69.57; H, 5.96; N,
11.41; S, 6.50.
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 29
To a mixture of carbaldehyde 24 (0.5 g, 1.96 mmol) in
ethanol (15 ml), was added aryloxobutanamide 16 (0.38 g,
2.15 mmol), urea (0.18 g, 2.94 mmol) and catalytic amount of
p-toluenesulfonic acid (10 mol%). After completion of the
reaction by adopting the general method, it gave the target
hybrid phenothiazinyltetrahydropyrimidinecarboxamide 29
(hexane-ethyl acetate 6:4). Yield, 0.79 (88%); MP: 138-140°C;
FT-IR (KBr, cm-1): ν 3224.9, 3103.4, 2933.7, 1697.3, 1597.0,
1525.9, 1496.7, 1463.9, 1438.9, 1384.8, 1367.5, 1325.1,
1238.3, 1195.8, 1134.1, 1111.0, 1091.7, 1074.3, 1039.6, 889.8,
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 32
A mixture of carbaldehyde 24 (0.5 g, 1.96 mmol),
aryloxobutanamide 19 (0.55 g, 2.15 mmol), urea (0.18 g, 2.94
mmol) and catalytic amount of p-toluenesulfonic acid (10
mol%)
in
ethanol
provided
the
target
hybrid
phenothiazinyltetrahydropyrimidinecarboxamide 32 (hexane-
ethyl acetate 6:4). Yield, 0.91g (87%); MP: 114-116°C; FT-IR
(KBr, cm-1): ν 3288.6, 3103.4, 2958.8, 2924.0, 2854.6, 1753.9,
1701.2, 1653.0, 1624.0, 1585.4, 1502.5, 1463.9, 1390.6,
1330.8, 1236.3, 1197.7, 1136.0, 1109.0, 1010.7, 902.6, 817.8,
748.3, 700.1, 675.0, 601.7, 565.1, 501.4, 464.8, 428.2; 1H NMR
(400 MHz, CDCl3): δ 7.51 (s, 1H), 7.32-6.79 (m, 12H), 5.71 (s,
1H), 5.21 (s, 1H), 3.87 (br.d, J=6.00 Hz, 2H), 2.22 (s, 3H), 1.37 (t,
J=6.80 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 163.8, 152.9,
144.8, 143.2, 135.7, 130.7, 126.4, 124.6, 122.3, 121.6, 120.5,
1
750.3, 692.4, 663.5, 599.8, 530.4, 455.2; H NMR (400 MHz,
CDCl3): δ 7.26-6.81 (m, 14H), 5.53 (s, 1H), 5.25 (s, 1H), 3.89
(br.d, J = 5.81 Hz, 2H), 2.24 (s, 3H), 1.39 (t, J = 6.60 Hz, 3H); 13
C
NMR (100 MHz, CDCl3): δ 165.8, 153.0, 144.7, 144.0, 139.6,
138.9, 129.1, 128.9, 127.4, 126.0, 125.3, 123.5, 122.8, 120.0,
115.8, 115.7, 105.7, 54.7, 41.5, 17.5, 13.1; Anal. Calcd. for
C26H24N4O2S (%): C, 68.40; H, 5.30; N, 12.27; S, 7.02; Found: C,
68.59; H, 5.47; N, 12.11; S, 6.90.
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115.7, 114.3, 104.1, 54.8, 40.8, 16.8, 13.1; Anal. Calcd. for
C26H23BrN4O2S (%): C, 58.32; H, 4.33; N, 10.46; S, 5.99; Found:
C, 58.51; H, 4.42; N, 10.33; S, 5.90.
View Article Online
DOI: 10.1039/C9NJ05489H
Radical scavenging activity by DPPH method
The assay of radical scavenging (DPPH) has been
performed using the method furnished in the literature43 with
small modifications. Firstly, 2,2-diphenyl-1-picrylhydrazyl
(DPPH) (1.6 mg) was dissolved in DMSO (50 mL). To each
compound prepared (1.5 mL, 100 μg/mL), was added DPPH
solution (1.5 mL) and set aside for 45 min incubation under
dark condition at ambient temperature. The variations in
absorbance were then noted (517 nm). The absorbance of the
blank DPPH solution was used as control (517 nm). The DPPH
free radical scavenging activity was then calculated.
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 33
To a mixture of carbaldehyde 24 (0.5 g, 1.96 mmol) in
ethanol (15 ml), was added aryloxobutanamide 20 (0.55 g,
2.15 mmol), urea (0.18 g, 2.94 mmol) and catalytic amount of
p-toluenesulfonic acid (10 mol%). After completion of the
reaction by adopting the general method, it gave the target
hybrid phenothiazinyltetrahydropyrimidinecarboxamide 33
(hexane-ethyl acetate 6:5). Yield, 0.86g (82%); MP: 184-186°C;
FT-IR (KBr, cm-1): ν 3402.4, 3344.5, 3213.4, 2962.6, 1695.4,
1668.4, 1602.8, 1514.1, 1465.9, 1367.5, 1261.4, 1220.9,
1091.7, 1020.3, 902.6, 802.3, 750.3, 705.9, 665.4, 549.7, 511.1,
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Anticancer evaluation
Human pancreatic cancer cells AsPC1 and SW1990 were
purchased from ATCC, USA. RPMI-FBS medium supplemented
with 100 μg/mL of streptomycin (Sigma-Aldrich, St. Louis, MO,
USA) and 100 IU/mL of penicillin was used to culture all cells.
All pancreatic cancer cells were incubated at 37 °C in 5% CO2,
utilizing the standard cell culture method (in vitro) established
previously.44-48 Gemcitabine was used as a positive control.
With a view to assess antitumor effects of the synthesized
molecules, stock solutions of the same in DMSO (100 mM)
were initially prepared, then further diluted to 0.1 mM in
sterile PBS. As reported in the literature,44,49 the cells were
treated on the basis of cell viability assay. Firstly, 384-well
plates were seeded with cells in such a way that each well
contained 1,500 cells/well. Plates were incubated for 24 hours
and then the cells were treated with the synthesized
molecules for 72 hours. 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
464.8, 441.7; H NMR (400 MHz, CDCl3): δ 8.13 (s, 1H), 7.39-
6.77 (m, 12H), 5.50 (s, 1H), 5.19 (s, 1H), 3.82 (br.d, J=6.40 Hz,
2H), 2.27 (s, 3H), 1.32 (t, J=6.80 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 164.0, 152.7, 144.7, 141.8, 137.1, 132.7, 131.9, 131.1,
130.2, 129.5, 128.5, 127.3, 120.3, 119.9, 119.8, 103.4, 54.8,
40.6, 17.0, 11.9; Anal. Calcd. for C26H23BrN4O2S (%): C, 58.32;
H, 4.33; N, 10.46; S, 5.99; Found: C, 58.47; H, 4.40; N, 10.36; S,
5.91.
Hybrid phenothiazinyltetrahydropyrimidinecarboxamide 34
A mixture of carbaldehyde 24 (0.5 g, 1.96 mmol),
aryloxobutanamide 21 (0.44 g, 2.15 mmol), urea (0.18 g, 2.94
mmol) and catalytic amount of p-toluenesulfonic acid (10
mol%)
in
ethanol
provided
the
target
hybrid
phenothiazinyltetrahydropyrimidinecarboxamide 34 (hexane-
ethyl acetate 6:4). Yield, 0.87g (92%); MP: 198-200°C; FT-IR
(KBr, cm-1): ν 3394.7, 3203.7, 3105.3, 2972.3, 2825.7, 1705.0,
1676.1, 1631.7, 1498.6, 1462.0, 1359.8, 1328.9, 1244.0,
1201.6, 1159.2, 1112.9, 1041.5, 945.1, 898.8, 806.2, 752.2,
653.8, 613.3, 574.7, 549.2, 509.2, 453.2, 422.4; 1H NMR (400
MHz, CDCl3): δ 7.48 (s, 1H), 7.26-6.59 (m, 11H), 5.40 (s, 1H),
5.25 (s, 1H), 3.89 (br.d, J=6.80 Hz, 2H), 2.28 (s, 3H), 2.23 (s, 3H),
2.17 (s, 3H), 1.39 (t, J=6.80 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 164.9, 152.9, 145.3, 144.5, 136.0, 134.8, 132.8,
131.3, 127.4, 126.0, 125.7, 123.5, 122.6, 115.1, 104.9, 56.1,
41.9, 17.3, 12.9; Anal. Calcd. for C28H28N4O2S (%): C, 69.40;
H, 5.82; N, 11.56; S, 6.62; Found: C, 69.58; H, 5.99; N, 11.41; S,
6.54.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
Financial assistance provided by the Indian Council of Medical
Research, New Delhi, India (No.58/16/2013-BMS) is gratefully
acknowledged.
Anti-inflammatory activity by Protein denaturation method
For the anti-inflammatory (in vitro) study, the method
adopted in the literature was used with slight modification.41,42
The reaction mixture (2.5 mL) consisted of egg albumin (0.1
mL), synthesized molecule (1 mL; 1 mM) and phosphate
buffered saline (PBS, 1.4 mL; pH 6.4). Equal volume of water
(double distilled) served as control. The mixture was then
heated at 70 °C for 5 min after incubation at 37 °C ±2 for 15
min. Their absorbance, after cooling, was measured at 660 nm
utilizing vehicle as blank. As a reference drug, diclofenac
sodium (1 mM) was utilized and treated likewise for the
absorbance determination. The inhibition (%) of protein
denaturation was then calculated.
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DOI: 10.1039/C9NJ05489H
Table of content
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Phenothiazine and amide ornamented novel nitrogen heterocyclic hybrids: Synthesis and
their biological and molecular docking studies
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Ramar Sivaramakarthikeyan, Ayyanar Karuppasamy, Shunmugam Iniyaval, Krishnaraj
Padmavathy, Wei-Meng Lim, Chun-Wai Mai, Chennan Ramalingan
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