Pyrazolylphenanthroimidazole heterocycles: Synthesis, biological and molecular docking studies

Article abstract of DOI:10.1039/d0nj02214d

The synthesis of a series of novel pyrazolylphenanthroimidazoles 6a-6j has been accomplished utilizing a multi-step synthetic protocol, and characterized through physical and spectral techniques. Among them, the molecules possessing para-bromo (6d), para-methyl (6f) and para-nitro (6j) phenyl substituents on the pyrazole scaffold displayed similar anti-inflammatory activity and the one with no substituents on the aryl unit (6a) exhibited the highest anti-inflammatory profile. While investigating the DPPH radical scavenging activity, the synthesized chemical entity with a para-methoxyphenyl group attached at the pyrazole structural motif (6i) revealed the highest activity when compared to the other synthesized molecules. Furthermore, the evaluation of cytotoxic activity of the synthesized molecule (6a) exerted significant activity against both the pancreatic cell lines such as AsPC1 and SW1990. Besides, while performing the molecular docking studies of 6a with B-cell lymphoma 2, an appreciable binding affinity (-9.04 kcal mol-1) has been observed. The results of the present examination imply that these chemical entities could be used as efficient intermediates for the construction of biopertinent molecules. This journal is

Full text of DOI:10.1039/d0nj02214d

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Pyrazolylphenanthroimidazole heterocycles: Synthesis, biological
and molecular docking studies
Received 00th January 20xx,
Accepted 00th January 20xx
Ramar Sivaramakarthikeyana, Shunmugam Iniyavala, Wei-Meng Limb, Ling-Wei Hiib, Chun-Wai
Maib,c, Chennan Ramalingana*
DOI: 10.1039/x0xx00000x
Synthesis of a series of novel pyrazolylphenanthroimidazoles 6a-6j has been accomplished utilizing multi-step synthetic
protocol and characterized through physical and spectral techniques. Among them, the molecules possessing para-bromo
(6d), para-methyl (6f) and para-nitro (6j) phenyl substituents on the pyrazole scaffold displayed similar anti-inflammatory
activity and the one with no substituents on the aryl unit (6a) exhibited the highest anti-inflammatory profile. While
investigating the DPPH radical scavenging activity, the synthesized chemical entity with para-methoxyphenyl group
attached at pyrazole structural motif (6i) revealed the highest activity when compared to the other synthesized molecules.
Furthermore, evaluation of cytotoxic activity of synthesized molecule 6a exerted significant activity against both the
pancreatic cell lines such as AsPC1 and SW1990. Besides, while performing the molecular docking studies of 6a with B-cell
lymphoma 2, appreciable binding affinity (-9.04 Kcal/mol) has been observed. The results of the present examination imply
that these chemical entities could be used as efficient intermediates for the construction of biopertinent molecules.
cancer, oral cancer, and colorectal cancer have high cure rates
when detected early and can be treated based on best
practices. When comparing rest of the cancer types, the
Introduction
In the 21st century, cancer is a static problem in non-
communicable diseases. Cancer is a comprehensive collection
of causes considered by the imprecise growth of irregular cells
that can spread to parts of the body while it is a significant
concern in health around the globe and a foremost cause of
death.1 Now-a-days, failing to decrease the burden of cancer
especially in low and middle income countries is due to poor
lifestyle, environmental inhabitants and economies, which are
the key features and, a few of the risk factors including
tobacco consumption, alcohol utilization, minimum physical
activity and unhealthy diets are burden of infectious origin.
The aforementioned ones are the reasons to believe that the
death rate is rising every year and entire prevention of cancer
is questionable.2 According to WHOs prediction, the rise in
global cancer is nearly 22.2 million by 2030. Several types of
cancer treatments available and the treatments type depend
on the type of cancer as well as its progression. Some of the
most common cancer types, such as breast cancer, cervical
pancreatic cancer has the least 5-year survival rate because of
its poor prognosis and, there is no noteworthy enhancement
in its survival since 1975. Extensive types of cancer treatment
include chemotherapy, surgical treatment, immunotherapy,
targeted therapy, radiation therapy, hormone treatment and
stem cell transplant etc. There are numerous drugs available
commercially in the dispensary to treat cancers, unfortunately,
the majority of them are associated with severe side effects
and subsequently, it is an inspiring and challenging duty to
save survives. Meanwhile, all the observations are related to
discover a potent drug molecule in this field.
Nitrogen possessing organic molecules, in general, play
vital role in drug discovery.4-8 Imidazole is a well-known
aromatic heterocyclic moiety containing two nitrogen atoms at
1 and 3 positions and its similarities are hopeful platforms in
medicinal chemistry as well as imidazole integrated structural
heterocyclic scaffolds in the development of an extensive
range of drugs through interest in the therapeutic areas
together with antifungal, antiviral, HIV inhibitor, anticancer,
antihelmintics, antihypertensive, antimicrobial, antihistamine,
antiulcer, antioxidant and anticoagulant activity.9-20 Various
ranges of biological influences of agents possessing imidazole
units are recognized to the unique fused benzene rings, which
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: 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.
can relate with
a diversity of targets of biological
importance.21,22 The imidazole ring is present in various
natural23-25 and synthetic26-28 bioactive compounds. Besides,
among the several targets, DNA chromosome is one of the
principal targets to investigate against the anticancer property
† 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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for the scientific community to recognize the interactions of enhanced activity. It has been reported that molecules
View Article Online
DOI: 10.1039/D0NJ02214D
small fragments by DNA to help new strategy and efficient possessing imidazole structural motif induces effective cell
therapeutic agents for monitoring gene expression.29 death through inhibition of PI3K mediated PI3K/Akt/mTOR
Curiously, imidazoles to interact the DNA through covalent or signalling pathway.45 On the other hand, molecules containing
non-covalent interactions (electrostatic, intercalation and pyrazole scaffold induces apoptosis through caspase-
groove binding).30 Numerous anticancer drugs possessing dependent pathways and inactivates protein kinase B/Akt
imidazole skeleton have been developed by researchers activity.46
Biological
activities
coupled
with
the
around the universe. Some of the imidazole tethered drug aforementioned imidazole and pyrazole scaffolds and our own
molecules which have been used in the health centres to treat expertise in the synthesis of novel heterocyclic molecules as
various types of cancers are dacarbazine, temozolomide, biological agents stimulated us to design and synthesize a
zoledronic acid, mercaptopurine, nilotinib, tipifarnib etc. (Fig. novel category of molecular hybrids tethered with imidazole
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1).
and pyrazole structural motifs as biological agents (Fig. 3).
Fig. 1. Anticancer drugs possessing imidazole structural motifs.
On the other hand, pyrazole, an aromatic five membered
heterocyclic molecule possessing double nitrogen atoms at the
adjacent places, and its similarities are gifted frameworks in
medicinal chemistry. Initially, Knorr created a pyrazole derived
compound in 1883, which directed to finding of antipyrine and
its derivatives 31
.
Subsequently the starter of antipyrine; the
first pyrazolone derivative was consumed in the treatment
32-37
therapy on pain, inflammation and fever in 1884
.
Pyrazole
and its by-products were broadly used as insect killer in
agrochemical division and studied for years their broad-
spectrum biological actions including insecticidal, acaricidal,
antibacterial, anticancer, herbicidal and antifungal activities.38-
44 Further, various pyrazole and its derived molecules also exist
in commercial for medicinal purpose as well as clinical trials.
The following are some of the drug molecules possessing
pyrazole structural motif (Fig. 2)
.
To synthesize biopertinent molecules, several strategies
can be adopted and a popular one is molecular hybridization
approach, which is nothing but hybridization of two or more
biopertinent scaffolds leading to a new chemical entity with
Fig. 2. Drug molecules possessing pyrazole scaffold.
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Reported herein are their synthesis, anti-inflammatory, Vilsmeier
-Haack reaction (i.e., utilizing POCl3 and DMF in
View Article Online
radical scavenging, and cytotoxic activities besides molecular dichloromethane) 47
.
A
multicomponenDtOI:o10n.e1039/D0NJ02214D
between pyrazole based carbaldehydes 4a 4j, aryl-9,10-dione
and NH4OAc in the ratio of 1:1:4, respectively in the presence
of ceric ammonium nitrate as a catalyst eventually produced
the desired titled target molecules 6a-6j in good yields The
structures of these novel chemical entities have unequivocally
been established based on physical and spectral methods
-pot reaction
docking studies of a representative molecule.
-
5
.
.
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The general plausible mechanistic route for the
construction of target chemical entities is provided in Scheme
. Lewis acid catalyzed nucleophilic reaction of the reactant
2
aldehyde produce the hydroxyl ammonium intermediate C,
A
wherein CAN acts a Lewis acid which abstracts electrons from
the carbonyl oxygen making the carbonyl carbon a facile
nucleophilic centre and ammonia, obtained from ammonium
acetate, as a neutral nucleophile. Intramolecular abstraction of
a proton from ammonium moiety by the hydroxyl functionality
Fig. 3. Imidazole and pyrazole integrated novel chemical entities.
in
intermediate
followed by elimination of a proton yields the diamine
intermediate . Lewis acid catalyzed double nucleophilic
reaction of diketone , wherein CAN acts as a Lewis acid and
the diamine intermediate acts as a neutral nucleophile,
provide dihydroxy diammonium intermediate . Intramolecular
proton abstraction by the hydroxyl units in followed by
elimination of water molecules affords imidazole 1,3-diium
intermediate which eventually loses a couple of protons to
furnish the target chemical entities.
C
followed by elimination of water provide the iminium
E. Further nucleophilic reaction with ammonia
Result and discussion
Chemistry
G
The schematic representation for the synthesis of imidazole
and pyrazole integrated chemical entities 6a-6j is represented
in scheme 1. Initially, pyrazole incorporated carbaldehydes 4a-
4j have been synthesized by the condensation of commercially
H
G
J
J
available arylhydrazine and appropriate aryl-alkyl ketones 1a
1j in glacial acetic acid followed by cyclization of the
arylhydrazone intermediates 3a 3j thus obtained using
-
L
-
Scheme 1. Synthesis of imidazole and pyrazole integrated hybrids 6a-6j
.
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potency of imidazole and pyrazole integrated target molecules
6a-6j towards inhibiting the denaturation of protein was
assessed. Diclofenac sodium was used as a standard one,
Biological Studies
Anti-inflammatory activity
View Article Online
DOI: 10.1039/D0NJ02214D
In vitro anti-inflammatory activity of the all synthesized target
compounds 6a-6j was assessed by the method of protein
denaturation.48,49 In this protein denaturation method, all the
secondary structure and tertiary structure of protein would be
which exhibited approximately 90
% inhibition of protein
denaturation The percentage of inhibitions of protein
.
denaturation upon the consumption of the chemical entities
6a-6j are calculated with respect to the inhibition of protein
denaturation by the standard diclofenac and the outcomes are
lost when external stress or a molecule is applied
recognized that familiar source of inflammation is
denaturation of protein As a part of present examination,
. It is well
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a
displayed in Fig. 4.
.
Scheme 2. Plausible mechanism for the construction of 6a-6j
4 | J. Name., 2012, 00, 1-3
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reducing antioxidant power, and DPPH radicaVliewscAartvicelenOgnilninge
DOI: 10.1039/D0NJ02214D
assay are accessible
. One of the most familiar radical
scavenging performances in organic DPPH (2,2-diphenyl-1-
picrylhydrazine) assay was initially described in 1958 in Blois
and was later cited by larger scientific community with slight
modification because of their simplicity.51,52 Accordingly, all
the synthesized imidazole and pyrazole integrated chemical
entities 6a-6j were investigated for their radical scavenging
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inhibition against DPPH (2,2-diphenyl-1-picrylhydrazine), a
reactive oxygen species. The activity was evaluated in terms of
inhibition of DPPH. The experiments were conducted in
triplicate and the outcomes, on comparison with the activity of
a standard ascorbic acid, was showed in Fig. 5.
Fig. 4. Anti-inlammatory activity of target molecules 6a-6j
.
The imidazole and pyrazole integrated molecule possessing
no substituents on the aryl moieties attached to pyrazole
scaffold (6a) showed similar activity when compared to the
standard diclofenac sodium under the investigation condition
.
Incorporation of a chloro moiety on the para position of the
aryl group attached to the pyrazole unit ends up with ~25%
reduction in inhibition. Replacing the chloro moiety by a fluoro
group, removal of chloro moiety from the para position
besides incorporation of bromo moiety on the meta position,
and replacing the chloro moiety by methoxy unit in 6b (i.e., in
6c, 6e, and 6i, respectively) culminated with almost similar
activity with that of 6b. Integration of chloro group on the
ortho position of an aryl group attached to pyrazole ring in 6a
as well as chloro groups on the ortho and para positions of the
Fig. 5. Radical scavenging activity of target molecules 6a-6j
.
The target imidazole and pyrazole tethered chemical entity
same (i.e., in 6g and 6h, respectively) offered appreciable with no substituents 6a displayed ~65% inhibition of DPPH
activity (~90% and ~85%, inhibitions, respectively) when radical on relating to the standard ascorbic acid. Incorporaꢀon
compared with the standard. Further, incorporation of bromo, of bromo and methoxy subsꢀtuents on the para posiꢀon of an
methyl and nitro substituent on the para position of an aryl aryl group tethered to pyrazole ring in 6a (i.e., in 6d and 6i
group tethered with pyrazole unit in 6a (i.e., 6d, 6f and 6j
exhibited almost similar acꢀvity while introducꢀon of chloro,
respectively) culminated with excellent activity despite a bit fluoro and methyl subsꢀtuents on the para posiꢀon of an aryl
lower than 6a
group tethered to pyrazole ring in 6a (i.e., in 6b 6c and 6f)
)
,
.
,
slightly reduced the acꢀvity. Also, integraꢀon of bromo
subsꢀtuent on the meta posiꢀon of an aryl group and chloro
subsꢀtuent on the para posiꢀon of an aryl group aꢁached to
the pyrazole ring in 6a (i.e., in 6e and 6g, respecꢀvely)
culminated with similar results. Subsꢀtuꢀng an addiꢀonal
chlorine moiety on the para posiꢀon of the aryl group in
addiꢀon to ortho posiꢀon in 6g (i.e., in 6h) reduced the acꢀvity
with a magnitude of ~5% while eliminaꢀon of chloro group
from the ortho posiꢀon and integraꢀon of nitro group on the
para posiꢀon in 6g (i.e., in 6j) slightly reduced the acꢀvity.
Among all, the one possessing methoxy subsꢀtuent on the
para posiꢀon of an aryl group (6i) exerted the best acꢀvity.
Radical scavenging activity
Considerable cellular injury stands up from reactive oxygen
species (ROS), at least in part of anthropological diseases for
example neurodegenerative disorders which including
Alzheimer disease, Parkinson disease, cancer, destroys in
memory,
mutagenesis,
autoimmune
disorder
and
cardiovascular diseases by obstructing the function of several
cellular mechanisms. Antioxidants are nothing but
representatives that scavenge the unrestricted radical
formation in our biological system. In spite of their availability
in large numbers in nature, it is extremely playing a crucial role
to develop the medicines with improved antioxidant profiles to
serve the chance around the globe.50 In order to evaluate the
radical scavenging property, several scavenging in vitro
approaches are existing, including some ABTS radical
scavenging technique, hydroxyl radical scavenging assay, ferric
Cytotoxicity studies
All the synthesized imidazole and pyrazole tethered chemical
entities (6a-6j) were investigated for their cytotoxicity on
human pancreatic cancer cells (SW1990 and AsPC1) and the
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non-cancerous cells MRC5 through the well established Cell-
Titer Glo
Luminescent Cell Viability Assay method.53-58 The Fig. 6
cytotoxicity results are furnished in Table 1
4
.
0
.
The results gathered through docking are represented in
.
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DOI: 10.1039/D0NJ02214D
®
.
The imidazole and pyrazole integrated chemical entity 6a
result an appreciable binding energy of -9.04 Kcal mol-1 besides
its estimated IC50 values of 238.12 nM. The target chemical
entity 6a exerted Van der Waals interaction with polar
Table 1. Cytotoxicity studies of target chemical entities against cancerous and non-
cancerous cells.
IC50 μM
hydrophilic amino acids such as glutamine glutamate and non
polar hydrophobic amino acids such as phenyl alanine, leucine,
tryptophan, glycine and valine Additionally, carbon-
hydrogen conventional bond interaction between aspartic
acid, an electrically stimulating polar hydrophilic amino acid,
and a carbon adjacent to the substituent possessing nitrogen
-
S.No.
Compound
MRC5
55.5 ± 3.50
>100
>100
>100
SW1990
30.3 ± 0.45
>100
>100
>100
AsPC1
32.4 ± 0.65
>100
>100
>100
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1
2
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6a
6b
6c
6d
6e
6f
.
a
>100
>100
>100
>100
>100
>100
of the pyrazole unit was observed. Further, a π-π interaction
6
between a non-polar hydrophobic amino acid phenyl alanine
and two aromatic rings, the one tethered with pyrazole
structural motif while one among the others fused with the
imidazole structural component of the target molecule. The
7
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11
6g
6h
6i
6j
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Gemcitabibe
36.1 ± 0.55
39.7 ± 1.19
59.8 ± 0.90
hydrophobic methionine amino acid exhibited
a π-σ
interaction with one of the phenyl rings attached at the
nitrogen of the pyrazole moiety besides its π-alkyl interactions
with pyrazole core as well as one of the phenyl rings integrated
at the carbon-3 of the pyrazole unit. In addition, aliphatic
hydrophobic amino acid alanine and electrically charged
hydrophilic amino acid of arginine showed -alkyl interactions
with the target molecules: when the former exhibited this
interaction with one of the phenyl moieties tethered at
pyrazole ring as well as one of the other aromatic rings fused
with imidazole scaffold, the latter exerted interaction with the
same aryl moiety fused with imidazole scaffold.
The target chemical entity possessing no substituent on the
aryl moieties exhibited the most potent cytotoxicity (IC50 value
of ~30 μM) against the human pancreatic cancer cells such as
SW1990 and AsPC1. The IC50 exhibited by 6a was comparable
to IC50 exhibited by gemcitabine, the first line chemotherapy
agent prescribed for pancreatic cancer patients. These results
suggested 6a may be as potential as gemcitabine in treating
pancreatic cancer. Integration of chloro and nitro substituents
on the para position of an aryl group attached at pyrazole
structural motif in 6a (i.e., in 6b and 6j, respectively) and
substitution of fluoro and methoxy moieties on the para
positions, bromo moiety on the meta position, and chloro
moieties on the ortho and para positions of an aryl group
Conclusion
A series of novel imidazole and pyrazole integrated molecular
hybrids 6a-6j was synthesized through multi-step synthetic
methodology and all the synthesized chemical entities were
characterized through physical and spectroscopic methods. In
the anti-inflammatory evaluation using protein denaturation
method, the chemical entity possessing no substituent on an
aryl group (6a), bromo, methyl and nitro substituents on the
para-position of the aryl group tethered at pyrazole motif (6d,
6f and 6j, respectively) exhibited almost similar activity when
compared with the anti-inflammatory standard diclofenac
sodium. Further, the radical scavenging activity by DPPH
method implies that the molecule integrated with no
substituents (6a) and methoxy substituent on the para-
position (6i) of an aryl group integrated at pyrazole unit
showed similar activity. Besides, imidazole and pyrazole
integrated chemical entity with no substituents (6a) exerted
the highest cytotoxic activity against both the tested
pancreatic cancer cell lines such as SW1990 and AsPC1 by
tethered at pyrazole unit (i.e., 6c
exerted IC50 value of >100 μM. Akin to the same, the rest of
the molecules with various substituents such as 6d 6f and 6g
, 6i, 6e and 6h, respectively)
,
also showed IC50 value of >100 μM. These results suggested
the substitutions may reduce its cytotoxicity effects against
pancreatic cancer cells. As indicated in Table 3, the activity of
all the chemical entities against non-cancerous cell such as
MRC5 was also examined and found that the IC50 against the
most active molecule was much greater. This examination
clearly implies that the most active chemical entity, the one
integrated with no substituents (6a) exhibits selective
cytotoxicity towards cancer cells sparing the non-cancerous
cells. Structural diversification on 6a in order to obtain further
superior result is under the way.
Molecular docking studies
Molecular docking is an attempt to find the most stable
position of the ligand in the active region of a receptor and to
know the binding affinity with a particular attempted protein.
Binding affinity, obtained from protein data bank, and the
molecular sources of interactions amongst the active sites of
BCL-2 (B-cell Lymphoma 2) and the structure of imidazole and
pyrazole integrated chemical entity 6a were assessed through
molecular docking examinations by utilizing Auto dock version
operating the Cell-Titer Glo Luminescent Cell Viability Assay
methodology. On the other hand, molecular docking between
B-cell lymphoma 2 (BCL-2) and the imidazole and pyrazole
integrated chemical entity 6a reflects significant binding
affinity (9.04 Kcal/mol). The results of anti-inflammatory,
radical scavenging and anticancer evaluations exposed that the
synthesized most active molecules could serve as efficient
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intermediates for the construction of efficient biological visualized by long- and short-wavelength UV lamps. All FT-IR
View Article Online
agents. To enhance the activity further, structural spectra in KBr pellets were recorded on DaOSI:h1i0m.1a0d39zu/DI0RNJT0r2a2c1e4rD-
diversifications of the aforesaid chemical entities are presently 100 spectrophotometer in the range of 4000–400 cm-1. 1H and
in progress.
13C NMR spectra were recorded on an NMR spectrometer
(Bruker AVANCE II 400 and 100 MHz) at 25°C with the use of
tetramethylsilane (TMS) as an internal standard and CDCl3 as
solvent; chemical shifts are expressed in terms of parts per
million (ppm). The H NMR spin multiplicities s, d, t, q and m
stand for singlet, doublet, triplet, quartet and multiplet,
respectively.
Experimental Section
General:
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All Chemicals were purchased from commercial sources. The
chemicals used herein are reagent grade and were utilized as-
received. All solvents were distilled /dried by employing
standard procedures before their utilization. Analytical thin-
layer chromatography (TLC) was performed on the pre-coated
TLC sheets of silica gel 60, F254 (Merck, Germany) and
General method for the synthesis of pyrazole based aldehydes
(4a-4j)
A mixture of individual ketones (1a-1j) (83.3mmol) and phenyl
hydrazine (
2
) (99.8 mmol) with glacial acetic acid (20mL) were
Fig. 6. 2D & 3D interactions of BCL-2 with 6a
.
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heated on a water bath for 30 minutes. Filtered the cold (KBr, cm-1):
reaction mixture after cooling the same and washed the solid 1446.6, 1417.6, 1354.0, 1307.7, 127D3O.0I,: 101.1105339/.4D,0N1J00282914.7D,
with dilute HCl followed by cold rectified spirit. 1056.9, 1012.6, 950.9, 923.9, 840.9, 813.9, 785.0, 738.7,
Journal Name
3300.2, 3062.9, 2916.2, 1595.1, 15V3i3ew.4A,rti1cl5e0O8nl.i3ne,
1
2
3
4
5
6
7
8

1
Recrystallization of the same from ethanol provided a pure 680.87, 638.4, 607.5; H NMR (400 MHz, CDCl3): 9.26 (s, 1H),
respective arylhydrazone (3a-3j).
7.99-7.93 (m, 4H), 7.83 (s, 1H), 7.71-7.60 (m, 7H), 7.50-7.45 (m,
Synthesis of carbaldehydes (4a-4j) was carried out by the 2H), 7.24-7.22 (m, 3H); 13C NMR (100 MHz, CDCl3): 151.8,
application of two moles of cold solution of Vilsmeier-Haack 147.7, 139.1, 135.6, 135.4, 134.7, 134.4, 130.1, 129.6, 128.4,
(VH) reagent [DMF (100mL) - POCl3 (26 mL, 0.28 mol adduct) in 128.3, 128.2, 120.0, 117.3, 117.2, 101.7. Anal. Calcd. for
DMF] with substituted phenylhydrazones (3a-3j). The reaction C30H19ClN4 (%): C, 76.51; H, 4.07; N, 11.90; Found: C, 76.95; H,
mixture was stirred at 70-80 °C for 5-6 hours and the progress 3.98; N, 11.76.
9
10
11
12
13
14
15
16
17
18
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20
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24
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
was monitored by TLC. After completion of the reaction, the
Synthesis of 2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
phenanthro[9,10-d] imidazole (6c):
content of the flask was cooled to ambient temperature then
poured into cold water. Saturated solution of sodium
bicarbonate was then added to neutralize the mixture and the
solid obtained was filtered, washed with water and dried to
get the respective aldehydes (4a-4j).47
A mixture of carbaldehyde 4c (0.5 g, 1.88 mmol), aryl-9,10-
dione
5 (0.39 g, 1.88 mmol) and ammonium acetate (0.58 g,
7.52 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the
General method for the synthesis of hybrid heterocycles (6a-6j)
To a mixture of respective carbaldehydes (4a-4j) (1 equi.), 9, target product 6c. Yield, 0.76 g, 89%; MP: 285-287°C; FT-IR
10-phenanthrenequinone (1 equi.) and ammonium acetate (4 (KBr, cm-1):

3304.1, 3061.0, 2916.2, 1593.2, 2216.2, 1593.2,
equi.) in acetonitrile (7 ml) and water (3 ml), was added ceric 1531.5, 1502.6, 1444.7, 1419.6, 1361.7, 1305.8, 1274.9,
ammonium nitrate (10 mol%) and refluxed. The refluxing was 1242.2, 1018.4, 954.7, 921.9, 817.8, 777.3, 702.0, 678.9, 678.9,
continued for 12 h while the progress was monitored by TLC. 609.5, 582.5, 513.1; H NMR (400 MHz, CDCl3): 9.26 (s, 1H),
1
After completion of the reaction, the content of the flask was 8.16 (s, 1H), 7.99-7.95 (m, 3H)7.78-7.73 (m, 3H), 7.63-7.48 (m,
cooled to ambient temperature and the obtained crude was 8H), 7.25 (br.s, 2H); 13C NMR (100 MHz, CDCl3):
 152.7, 147.8,
poured into crushed-ice with stirring. The precipitate thus 139.1, 135.7, 133.9, 132.4, 131.7, 131.6, 130.4, 129.3, 128.6,
formed, after some time, was filtered by using vacuum suction 128.4, 122.8, 120.0, 117.1, 116.0, 102.5. Anal. Calcd. for
and dried. Recrystallisation of ethanolic solution of the same C30H19FN4 (%): C, 79.28; H, 4.21; N, 12.33; Found: C, 79.54; H,
eventually furnished the target molecules 6a-6j
.
4.12; N, 12.54.
Synthesis of 2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-phenanthro[9,10- Synthesis of 2-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
d]imidazole (6a): phenanthro[9,10-d] imidazole (6d):
A mixture of carbaldehyde 4a (0.5 g, 2.02 mmol), aryl-9,10- A mixture of carbaldehyde 4d (0.5 g, 1.53 mmol), aryl-9,10-
dione (0.42 g, 2.02 mmol) and ammonium acetate (0.63 g, dione (0.32 g, 1.53 mmol) and ammonium acetate (0.47 g,
5
5
8.08 mmol) in the presence of ceric ammonium nitrate (10 6.12 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the of the reaction by adopting the general method, provided the
target product 6a. Yield, 0.81 g, 92%; MP: 244-245°C; FT-IR target product 6d. Yield, 0.69 g, 87%; MP: 298-299°C; FT-IR
(KBr, cm-1):
  3305.9, 2918.1, 1595.1, 1521.8, 1423.4, 1348.2,
3323.3, 2912.3, 1589.3, 1525.6, 1498.6, 1415.7, (KBr, cm-1):
1355.9, 1305.8, 1271.0, 1240.2, 1078.2, 954.7, 918.1, 817.8, 1232.5, 1109.0, 1068.5, 960.5, 862.1, 817.8, 758.0, 684.7,
1
756.1, 736.8, 680.8, 650.0, 609.5, 609.5, 574.7, 513.0, 470.6, 636.5, 497.6; H NMR (400 MHz, CDCl3): 9.24 (s, 1H), 8.12 (s,
1
439.7; H NMR (400 MHz, CDCl3): 9.30 (s, 1H), 8.44 (d, J= 8.4 1H), 8.00-7.95 (m, 3H), 7.81-7.79 (m, 3H), 7.70-7.68 (m, 3H),
Hz, 2H), 8.14 (s, 1H), 8.05-8.00 (m, 5H), 7.66-7.61 (m, 5H), 7.64-7.60 (m, 4H), 7.49-7.45 (m, 1H), 7.25 (br.d, J=2.80Hz, 2H);
7.51-7.49 (m, 2H), 7.26 (br.s, 2H); 13C NMR (100 MHz, CDCl3):

13C NMR (100 MHz, CDCl3):
 153.2, 147.8, 139.2, 135.8, 132.5,
154.3, 147.8, 139.2, 136.2, 131.6, 130.4, 129.5, 129.4, 129.2, 131.4, 130.8, 130.4, 129.3, 128.3, 123.2, 119.9, 117.1, 115.9,
128.3, 119.9, 117.2, 115.8, 102.0. Anal. Calcd. for C30H20N4 (%): 102.3. Anal. Calcd. for C30H19BrN4 (%): C, 69.91; H, 3.72; N,
C, 82.55; H, 4.62; N, 12.84; Found: C, 82.23, H, 4.78; N, 12.72.
10.87; Found: C, 69.69; H, 3.86; N, 10.99.
Synthesis of 2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H- Synthesis of 2-(3-(3-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
phenanthro[9,10-d] imidazole (6b): phenanthro[9,10-d] imidazole (6e):
A mixture of carbaldehyde 4b (0.5 g, 1.77 mmol), aryl-9,10- A mixture of carbaldehyde 4e (0.5 g, 1.53 mmol), aryl-9,10-
dione (0.37 g, 1.77 mmol) and ammonium acetate (0.55 g, dione (0.32 g, 1.53 mmol) and ammonium acetate (0.47 g,
5
5
7.08 mmol) in the presence of ceric ammonium nitrate (10 6.12 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the of the reaction by adopting the general method, provided the
target product 6b. Yield, 0.73 g, 88%; MP: 266-268°C; FT-IR target product 6e. Yield, 0.66 g, 84%; MP: 288-290°C; FT-IR
8 | J. Name., 2012, 00, 1-3
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Journal Name
(KBr, cm-1):
ARTICLE
1
2
3
4
5
6
7
8

3311.7, 3049.4, 2981.6, 2816.2, 1593.2, 1525.6, 1382.9, 1355.9, 1328.9, 1290.3, 1215.1, 115V5ie.w3,Arti1cl1e2O4nl.in5e,
1500.6, 1419.6, 1311.5, 1232.5, 1072.4, 1006.8, 956.6, 925.8, 1072.4, 1018.4, 941.2, 860.2, 817.8, 756.D1O, 7I:3110..100,3698/D80.N5,J0621231.43D,
1
1
829.3, 756.1, 734.8, 684.7, 634.5, 503.4, 474.4; H NMR (400 588.2, 516.9; H NMR (400 MHz, CDCl3): 9.24 (s, 1H), 8.12 (s,
MHz, CDCl3): 9.24 (s, 1H), 8.13 (s, 1H), 7.97-7.95 (m, 3H), 7.77 1H), 7.96 (d, J=8.0Hz, 2H), 7.81-7.77 (m, 3H), 7.64-7.60 (m, 4H),
(d, J=8.4Hz, 2H), 7.67-7.60 (m, 7H), 7.49-7.45 (m, 2H), 7.25 7.49-7.42 (m, 4H), 7.25 (br.d, J=2.8Hz, 2H); 13C NMR (100 MHz,
(br.d, J=3.2Hz, 2H); 13C NMR (100 MHz, CDCl3):
 153.1, 147.8, CDCl3):  153.4, 147.8, 139.2, 136.0, 131.7, 131.6, 129.1, 128.3,
139.2, 135.8, 134.5, 131.2, 130.5, 130.4, 129.6, 129.3, 128.3, 128.1, 119.9, 117.2, 116.6, 116.4, 115.8, 102.1. Anal. Calcd. for
119.9, 117.2, 115.9, 102.3. Anal. Calcd. for C30H19BrN4 (%): C, C30H18Cl2N4 (%): C, 71.29; H, 3.59; N, 11.09; Found: C, 71.53; H,
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
69.91; H, 3.72; N, 10.87; Found: C, 69.74; H, 3.80; N, 10.71.
3.68; N, 10.99.
Synthesis
phenanthro[9,10-d]imidazole (6f):
A mixture of carbaldehyde 4f (0.5 g, 1.91 mmol), aryl-9,10- A mixture of carbaldehyde 4i (0.5 g, 1.80 mmol), aryl-9,10-
dione (0.40 g, 1.91 mmol) and ammonium acetate (0.59 g, dione (0.37 g, 1.80 mmol) and ammonium acetate (0.55 g,
of
2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H- Synthesis of 2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-
1H-phenanthro[9,10-d]imidazole (6i):
5
5
7.64 mmol) in the presence of ceric ammonium nitrate (10 7.20 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the of the reaction by adopting the general method, provided the
target product 6f. Yield, 0.77 g, 90%; MP: 290-292°C; FT-IR target product 6i. Yield, 0.74 g 88%; MP: 282-284°C; FT-IR (KBr,
(KBr, cm-1):

3313.7, 2998.4, 2881.6, 2823.7, 2823.7, 1595.1, cm-1):

3300.6, 2972.3, 2877.9, 1593.2, 1514.1, 1456.2,
1560.4, 1512.1, 1400.3, 1234.4, 1076.2, 1047.3, 883.4, 827.4, 1338.6, 1236.3, 1188.1, 1109.0, 1082.0, 1045.4, 964.4, 941.2,
1
1
790.8, 752.2, 725.2, 686.6, 665.4, 534.2, 505.3, 435.9; H NMR 858.3, 754.1, 721.3, 686.6, 536.2, 495.7, 430.1; H NMR (400
(400 MHz, CDCl3): 9.21 (s, 1H), 8.14 (s, 1H), 7-95 (d, J=8.0H, MHz, CDCl3): 9.22 (s, 1H),8.14 (s, 1H),7.95 (d, J=8.0Hz, 2H),
2H), 7.62-7.58 (m,7H), 7.47-7.24 (m, 5H), 7.24 (br.d, J=2.80Hz, 7.68-7.59 (m, 8H), 7.47-7.43 (m, 2H), 7.25-7.23 (m, 2H), 7.17-
2H), 2.41 (s, 3H); 13C NMR (100 MHz, CDCl3):
  154.3,
154.4, 147.8, 7.14 (m, 3H), 3.86(s, 3H); 13C NMR (100 MHz, CDCl3):
139.2, 139.1, 136.3, 130.3, 130.1, 129.3, 129.0, 128.8, 128.2, 147.9, 139.3, 136.4, 128.9, 128.1, 124.0, 123.8, 117.3, 115.6,
119.8, 117.3, 115.7, 101.7, 21.4. Anal. Calcd. for C31H22N4 (%): 101.60, 55.8. Anal. Calcd. for C31H22N4O (%): C, 79.81; H, 4.75;
C, 82.64; H, 4.92; N, 12.44; Found: C, 82.89; H, 4.84; N, 12.62.
N, 12.01; Found: C, 79.99; H, 4.64; N, 12.18.
Synthesis of 2-(3-(2-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
phenanthro[9,10-d] imidazole (6g):
Synthesis of 2-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
phenanthro[9,10-d] imidazole (6j):
A mixture of carbaldehyde 4g (0.5 g, 1.77 mmol), aryl-9,10-
To a mixture of carbaldehyde 4j (0.5 g, 1.71 mmol), aryl-9,10-
dione 5 (0.36 g, 1.71 mmol) and ammonium acetate (0.53 g,
6.84 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the
target product 6j. Yield, 0.75 g, 92%; MP: 308-309°C; FT-IR
dione
5 (0.37 g, 1.77 mmol) and ammonium acetate (0.55 g,
7.08 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the
target product 6g. Yield, 0.77 g, 93%; MP: 244-245°C; FT-IR
(KBr, cm-1):
 3271.2, 2968.4, 2873.9, 1593.2, 1510.2, 1485.1,
(KBr, cm-1):
 3593.3, 3435.2, 3049.4, 2881.6, 2816.0, 1597.0,
1454.3, 1435.0, 1381.0, 1234.4, 1188.1, 1120.6, 1076.2,
1045.4, 958.6, 827.4, 752.2, 727.1, 686.6, 663.5, 615.2, 534.2,
501.4, 430.1; 1H NMR (400 MHz, CDCl3): 9.26 (s, 1H), 7.95 (d,
J=7.60Hz, 2H), 7.85 (s, 1H), 7.72 (d, J=7.2Hz, 1H), 7.67-7.55 (m,
8H), 7.49-7.45 (m, 3H), 7.23 (br.d, J=6.0Hz, 2H); 13C NMR (100
1504.4, 1458.1, 1402.2, 1382.9, 1228.6, 1089.7, 1066.6,
1010.7, 958.6, 900.7, 835.1, 756.1, 731.0, 688.5, 580.5, 524.6;
1H NMR (400 MHz, CDCl3): δ 9.26 (s, 1H), 8.17 (s, 1H), 7.97 (d,
J=7.6Hz, 2H), 7.75-7.73 (m, 3H), 7.64-7.56 (m, 8H), 7.49-7.45
(m, 1H), 7.25 (br.s, 2H); 13C NMR (100 MHz, CDCl3): δ 154.3,
147.8, 139.2, 136.2, 131.6, 130.4, 129.5, 129.4, 129.2, 128.3,
119.9, 117.2, 115.8, 102.0. Anal. Calcd. for C30H19N5O2 (%): C,
74.83; H, 3.98; N, 14.54; Found: C, 75.03; H, 4.11; N, 14.42.
MHz, CDCl3):
 152.8, 147.6, 139.1, 135.8, 133.5, 133.2, 131.8,
130.5, 130.4, 128.4, 128.3, 128.0, 119.9, 117.2, 101.3. Anal.
Calcd. for C30H19ClN4 (%): C, 76.51; H, 4.07; N, 11.90; Found: C,
76.79; H, 4.22; N, 11.78.
Anti-inflammatory activity by Protein denaturation method
Synthesis of 2-(3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-
1H-phenanthro[9,10-d] imidazole (6h):
For the anti
Mazushima et al.46 and Sakat et al.47 with slight modification
was adopted The reaction mixture 5 mL) is egg albumin
1 mL), phosphate buffered saline PBS, 1 4 mL; pH 6 ), and
synthesized molecule 1 mL; 1 mM . The control used was
equivalent amount of water double distilled). The mixture
was then heated at 70 °C for 5 min after cultivation at 37 °C ± 2
for 15 min After cooling, their absorbance was measured at
660 nm operating vehicle as blank As a reference drug,
-inflammatory (in vitro) evaluation, the method of
A mixture of carbaldehyde 4h (0.5 g, 1.58 mmol), aryl-9,10-
.
(2.
dione
5 (0.33 g, 1.58 mmol) and ammonium acetate (0.49 g,
(
0
.
(
.
.4
6.32 mmol) in the presence of ceric ammonium nitrate (10
mol%) in acetonitrile (7 ml) and water (3 ml), after completion
of the reaction by adopting the general method, provided the
target product 6h. Yield, 0.73 g, 91%; MP: 278-279°C; FT-IR
(
)
(
.
(KBr, cm-1):
 3055.2, 2976.2, 1595.1, 1510.2, 1435.0, 1408.0,
.
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diclofenac sodium
the absorbance determination
performed in triplicate. The protein denaturation inhibition
was then calculated in terms of (%). The percentage inhibition
was calculated as per the equation provided below.
(
1 mM
)
was used and treated the same for
The experiments were
Conflicts of interest
The authors declare no conflict of interest.
View Article Online
DOI: 10.1039/D0NJ02214D
.
Acknowledgements
Financial assistance provided by the Indian Council of Medical
Research, New Delhi, India (No.58/16/2013BMS) is gratefully
acknowledged.
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Radical scavenging activity by DPPH method
References
The evaluation of radical scavenging (2,2-Diphenyl-1-
picrylhydrazyl) has been made as per the technique existing in
the literature with minor modification.48,49 DPPH (2,2-
Diphenyl-1-picrylhydrazyl) (1.6 mg) was dissolved in DMSO (50
mL). DPPH soluꢀon (1.5 mL) was added to individual molecule
synthesized (1.5 mL; 100 μg/mL) and kept aside for 45 min
incubation under dark condition at room temperature. The
variations of absorbance at 517 nm were then measured. The
blank DPPH solution absorbance at 517 nm was utilized as
control. The experiments were conducted in triplicate. The
DPPH free radical scavenging activity was evaluated by
employing the below mentioned equation:
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culture method
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mM , then further diluted to 0 1 mM in sterile PBS The cells
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viability assay well plates were seeded with
51,56 Primarily, 384
cells 1,500 cells well for 24 h and then the cells were
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Pyrazolylphenanthroimidazole heterocycles: Synthesis, biological and molecular docking
studies
Ramar Sivaramakarthikeyan, Shunmugam Iniyaval, Wei-Meng Lim, Ling-Wei Hii, Chun-Wai Mai, Chennan
Ramalingan
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