Synthesis, molecular properties prediction and anticancer, antioxidant evaluation of new edaravone derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.03.024

A series of new edaravone derivatives 3-7 have been synthesized, characterised using various spectroscopic techniques and screened for their in vitro anti-cancer, antioxidant activities. Structure of 5d was further substantiated through single crystal X-ray diffraction. Among the tested, 5l exhibited pronounced activity against PC3 cancer cells. Compounds 5i, 5l, 7c showed potent activity against A549 cancer cells. Products 5k, 6, 7c demonstrated good antioxidant activity with MIC values of 18.60, 16.27, 16.07 μg/mL respectively. Further the reported analogues were also tested on normal HEK293T cells and displayed low to good safer profiles. Derivatives 5l and 7c have come out to be safer potent anticancer, antioxidant agents. Additionally, the target products were subjected to their molecular properties prediction and drug likeness by employing Molinspiration and Osiris property explorer toolkits. None of them violated Lipinski's boundaries classifying the title compounds as potential anticancer and antioxidant agents.

Full text of DOI:10.1016/j.bmcl.2016.03.024

Accepted Manuscript
Synthesis, molecular properties prediction and anticancer, antioxidant evalua-
tion of new edaravone derivatives
Naveen Polkam, Venkat Ragavan Ramaswamy, Parsharamulu Rayam,
Tejeswara Rao Allaka, Hasitha Shilpa Anantaraju, Sriram Dharmarajan,
Yogeeswari Perumal, Durgaiah Gandamalla, Narsimha Reddy Yellu, Sridhar
Balasubramanian, Jaya Shree Anireddy
PII:
S0960-894X(16)30244-X
http://dx.doi.org/10.1016/j.bmcl.2016.03.024
BMCL 23667
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 January 2016
3 March 2016
8 March 2016
Please cite this article as: Polkam, N., Ramaswamy, V.R., Rayam, P., Allaka, T.R., Anantaraju, H.S., Dharmarajan,
S., Perumal, Y., Gandamalla, D., Yellu, N.R., Balasubramanian, S., Anireddy, J.S., Synthesis, molecular properties
prediction and anticancer, antioxidant evaluation of new edaravone derivatives, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
Synthesis, molecular properties prediction
and anticancer, antioxidant evaluation of
new edaravone derivatives
Naveen Polkama, Venkat Ragavan Ramaswamyb, Parsharamulu Rayama, Tejeswara Rao Allakaa, Hasitha Shilpa
Anantarajuc, Sriram Dharmarajanc, Yogeeswari Perumalc, Durgaiah Gandamallad, Narsimha Reddy Yellud,
Sridhar Balasubramaniane, Jaya Shree Anireddya,*.

Bioorganic & Medicinal Chemistry Letters
Synthesis, molecular properties prediction and anticancer, antioxidant
evaluation of new edaravone derivatives
Naveen Polkama, Venkat Ragavan Ramaswamyb, Parsharamulu Rayama, Tejeswara Rao Allakaa, Hasitha
Shilpa Anantarajuc, Sriram Dharmarajanc, Yogeeswari Perumalc, Durgaiah Gandamallad, Narsimha Reddy
Yellud, Sridhar Balasubramaniane, Jaya Shree Anireddya,
aCentre for Chemical Sciences and Technology, IST, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500085, Telangana State,
India
bNuray Chemicals Private Limited, Plot. No. 111, SIDCO Industrial estate, Kakkalur 602001, Tamil Nadu, India
cDepartment of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad Campus, Jawahar Nagar 500078, Telangana State, India
dDepartment of Pharmacology and Toxicology, UCPSc, Kakatiya University, Warangal 506009, Telangana State, India
eX-ray Crystallography Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of new edaravone derivatives 3-7 have been synthesized, characterized using various
spectroscopic techniques and screened for their in vitro anti-cancer, antioxidant activities.
Structure of 5d was further substantiated through single crystal X-ray diffraction. Among the
tested, 5l exhibited pronounced activity against PC3 cancer cells. Compounds 5i, 5l, 7c showed
potent activity against A549 cancer cells. Products 5k, 6, 7c demonstrated good antioxidant
activity with MIC values of 18.60, 16.27, 16.07 µg/mL respectively. Further the reported
analogues were also tested on normal HEK293T cells and displayed low to good safer profiles.
Derivatives 5l and 7c have come out to be safer potent anticancer, antioxidant agents.
Additionally, the target products were subjected to their molecular properties prediction and
drug likeness by employing Molinspiration and Osiris property explorer toolkits. None of them
violated Lipinski’s boundaries classifying the title compounds as potential anticancer and
antioxidant agents.
Keywords:
Anticancer activity
Antioxidant activity
Crystallography
Edaravone
Lipinski Rule of Five
The putative reason for the oxidative stress in a healthy organism
is due to the delicate imbalance between the consumption and
production of free radicals.1,2 This disparity blends in the
development of major health problems such as cancer,
Alzheimers disease, atherosclerosis, DNA damage.2,3 Cancer, in
which a group of cells play relentless growth and spread to
distant sites in the body, is the challenging disorder in the present
scenario leading to morbidity and mortality across the globe.4
According to WHO estimates, 8.2 million cancer deaths were
registered in 2012 and is expected to rise to 13.1 million by
2030.5 Existing chemotherapeutic agents failed to cross the
cancer boundaries and deliver the expected profiles due to the
resistance developed to them.6 Obviously, there is a need for the
identification and generation of novel chemical entities targeting
molecular pathways, efficient drug diffusion to cancer tissues for
winning the hurdles in cancer treatment. Pyrazole represents a
privileged scaffold of heterocycles and its derivatives are
endowed with significant biological applications in agro and
pharmaceutical industries7 as antifungal, antimycobacterial,8
antimicrobial, antioxidant,9 anticancer10 agents. Ludwig Knorr
1883.11 The well-known pharmaceuticals in comprising the
pyrazole template includes celecoxib, fipronil, dipyrone, and
many more are already in the market (Fig. 1). Unfortunately the
clinical usage of pyrazole analogues is limited in several
countries due to severe side effects such as bone marrow
depression,
carcinogenesis,
cutaneous
reactions
and
agranulocytosis.12,13 This limitation encourages the generation of
newer candidates with improved potency and safer profiles and
thus becomes the focal point of current research.
Recently a new pyrazolin compound namely Edaravone
(EDA, 3-methyl-1-phenyl-2-pyrazolin-5-one) also known as
MCI-186 (Scheme 1), is the first novel neuroprotective,
antioxidant medical drug that was clinically approved by Japan in
2001 for the treatment of acute stroke due to cerebral thrombosis
and embolism,14 brain ischemia,14,15 myocardial infarction,16
oxidative damage to various extracerebral organs in both animal
and human models.14 Based on series of experiments, the
preponderant anti-oxidant mechanism of EDA is proposed to be
due to single electron transfer to free radicals14,17 resulting in the
formation of stable 2-oxo-3-(phenylhydrazonono) butanoic acid
(OPB) as final product (Scheme 1). Further the scavenging
first synthesized the pyrazole compound antipyrine (Fig. 1) in
———
 Corresponding author. Tel.: +91 8008103809; fax: +91 040 2305 8729; e-mail: jayashreeanireddy@gmail.com (J.S. Anireddy)

activity also depends of the electron density on pyrazolone ring,18
lipophilicity of the molecule19 and thus ameliorates postischemic
inflammation responses generated by the highly reactive oxygen
species (ROS)20 namely hydroxyl (OH•)21 and peroxyl (LOO•)
radicals.22 Previously Markovic et al. have synthesized 4-
aminomethylidene derivatives of EDA and reported remarkable
antitumor activity against human breast cancer MDA-MB 361
and human cancer MDA-MB 453 cell lines by in vitro assays.23
Scheme 2. Reagents and conditions: (I) phenyl hydrazine, ethanol, reflux, 3
h, 90%; (II) TPP, DIAD, MeOH, DCM, 0 °C-rt, 8.5 h, 80%; (III) NBS, DMF,
rt, 10 min, 60%; (iv) R1-B(OH)2, Cs2CO3, Pd(PPh3)4, DMF, 90 °C, 9 h, 60%-
70%.
Scheme 3. Reagents and conditions: (I) n-BuLi, CO2, THF, -78 °C-rt, 2.5 h,
60%; (II) R2-NH2, EDCI, HOBt, DIPEA, DMAP, DCM, 0 °C-rt, 8 h, 73%-
78%.
Figure 1. Some of the drugs based on pyrazole template.
Finally the Suzuki coupling of 4 with different hetero aromatic or
aromatic boronic acids catalyzed by the loading of Pd(0) complex
in the presence of Cs2CO3 afforded 5a-l (Fig. 3) in moderate
yields.24 Initially, we performed the reaction of 4 with phenyl
boronic acid in the presence of various bases and solvents as
summarized in Table 1. Among them Cs2CO3 in DMF afforded
5a in 60% yield. The plausible reason for the increment in yield
might be due to more basic nature of Cs2CO3 than K2CO3 which
activates the boronic acid. Having optimised the protocol (Table
1, entry 5), a library of 12 compounds 5a-l were synthesized in
good yield varying from 60-70%. A variety of boronic acids
possessing electron-rich and electron-deficient as well as catalyst
sensitive (5b presence of -OH) substituents underwent smooth
coupling with 4 to produce 5a-l thus validating the scope of the
present protocol. We decided to investigate further by
introducing an additional point of diversity by incorporating
carboxylic acid into EDA nucleus. Lithiation of 4 with n-BuLi
followed by purging with carbon dioxide gas resulted in the
formation of 6 (Scheme 3) in moderate yield.27 Effective peptide
Scheme 1. Mechanistic pathway of EDA towards ROS adopted from T.
Watanabe et al.14
From the published data, EDA analogues synthesized by
functionalization of pyrazol-5-one ring are of at most interest and
can be decisive for the biological activity.22 Literature of Suzuki
couplings inspired us to append aromatic/hetero aromatic
moieties and also an amide group at C-4 position of EDA frame
with a desire to increase the lipophilic factor which promotes
permeation of the resulting derivatives in the lipid phase thus
reaching the biological membranes.24 In the light of the above
stimulating properties, we have made an attempt to design the
title compounds as depicted in Figure 2 and to ascertain their
anticancer and antioxidant profiles. It is proposed to modify the
carbonyl of EDA to methoxy since under physiological
conditions in presence of demethylase, a methoxy group is
converted to hydroxyl which might make better interactions with
the receptor. Further, the designed molecules were subjected to
computational sensitivity and structural analysis for predicting
their molecular properties and drug likeness by using
Molinspiration (2014) and Osiris property explorer (2014)
softwares.
coupling
reagents
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI), 1-hydroxy benzotriazole
(HOBt) and N,N-diisopropylethylamine (DIPEA) (Hunig’s base)
assisted the conversion of 6 and respective amine to produce 7a-
d (Fig. 3) in excellent yields.28 Analytical and spectroscopic data
along with HRMS are consistent with the proposed structures.
1
Disappearance of the H NMR signal of 3 at δ 5.54 ppm and
appearance of a 13C NMR peak at δ 80.87 ppm confirmed 4,
while its IR spectrum band at 814 cm-1 further proved its
formation. Additionally, the molecular ion peaks in the ratio 1:1
due to (M+H)+ and (M+2+H)+ in the mass spectra supported its
formation. Loss of 13C NMR characteristic peak of 4 at δ 80.87
ppm validated the formation of 5a-l. Appearance of a 13C NMR
resonance signal at δ 163.74 ppm established the formation of 6.
Manifestation of IR values at 3423 cm-1 (O-Hacid) and 1678 cm-1
(C=Oacid) supported the formation of 6. Occurrence of broad
singlet at δ 6.97 ppm (NH) on NMR time scale asserted 7a.
Display of IR signal at 3310-3314 cm-1 (N-Hamide) and typical
value for amide moieties at 1649-1651 cm-1 (amide-I band due to
C=O stretch) and 1596 cm-1 (amide-II band due to the
combination of C-N+N-H stretch) affirmed the products 7a-d.
The single crystal X-ray diffraction studies of compound 5d
unambiguously confirmed its structure (Fig. 4).29
Figure 2. Structure based design of edaravone analogues.
The syntheses of target compounds were accomplished by the
pathways as outlined in Scheme 2 and Scheme 3. Briefly,
condensation between ethyl acetoacetate and phenyl hydrazine
furnished the parent core EDA in excellent yield.23 Subsequent
Mitsunobu reaction afforded the methoxy functionalized pyrazole
scaffold 3 in 80% yield.25 Further treatment of 3 with NBS
generated the key intermediate bromo compound 4 in moderate
yield.26
In silico study of molecular properties and drug likeness: Nearly
30% of drugs fail before reaching the clinics due to poor
pharmacokinetics.30 Good oral availability can be achieved by

right balance between partitioning and solubility properties. In
order to achieve better analogues of edaravone, we have
performed computational investigations of all the synthesized
compounds 3-7 for the prediction of their adsorption,
distribution, metabolism and excretion (ADME) properties
(Table 2), Lipinski’s rule of five (Table 2), toxicity liabilities
(Table 4) and drug likeness (Table 4).
Table 2
Theoretical predictiona of ADME properties of compounds 3-7
TPSA
Compd.
EDA
3
miLogP
1.78
2.06
2.80
3.79
3.31
4.95
4.68
3.54
3.83
3.37
3.84
3.69
3.95
4.24
4.21
1.56
2.58
3.14
3.26
2.64
%Abs
97.72
99.66
99.66
99.66
92.68
99.66
99.66
91.45
93.29
99.66
96.47
96.47
99.66
99.66
99.66
86.79
89.62
89.62
89.62
86.44
(Å2)
32.67
27.06
27.06
27.06
47.29
27.06
27.06
50.85
45.53
27.06
36.29
36.29
27.06
27.06
27.06
64.36
56.16
56.16
56.16
65.39
MW
nON
3
3
3
3
4
3
3
4
5
3
4
4
3
3
3
5
nOHNH
nviolations
Volume (Å3)
161.59
178.77
196.66
250.18
258.20
294.17
284.88
267.04
301.27
240.89
275.72
292.53
255.11
266.74
266.74
205.77
298.37
314.95
311.90
323.91
Nrotb
174.20
188.23
267.13
264.33
280.33
314.39
320.42
289.34
324.38
270.36
294.35
308.38
282.32
278.36
278.36
232.24
321.38
335.41
355.82
351.41
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
3
3
3
3
3
5
3
4
5
3
3
3
3
5
5
5
6
4
5a
5b
5c
5d
5e
5f
5g
5h
5i
Table 1
Optimisation of reaction conditions of 5aa
Entry
Solvent
Base
Temperature Time Yieldb
5j
5k
5l
(°C)
90
(%)
1
Acetonitrile –water
K2CO3
15 h
NRc
6
5
5
5
6
7a
7b
7c
7d
(8:2)
2
3
4
5
Ethanol-water (8:2)
KOH
TEA
90
90
90
90
15 h
10 h
9 h
NRc
30
amiLogP: logarithm of compound partition coefficient between n-octanol and
water; %Abs: percentage of absorption; TPSA: topological polar surface area;
MW: molecular weight; nON: number of hydrogen bond acceptors; nOHNH:
number of hydrogen bond donors; Nrotb: number of rotable bonds.
DMF
DMF
DMF
K2CO3
Cs2CO3
52
9 h
60
aAll the reactions were carried out with 4 (100 mg, 0.37 mmol), PhB(OH)2
(45 mg, 0.37 mmol), Pd(PPh3)4 (17 mg, 4 mmol%) and base (482 mg, 1.4
mmol) in solvent (1 mL).
Molinspiration calculations: The methodology used by
Molinspiration31 as a sum of fragment-based contributions and
correction factors is very robust and is able to process practically
all organic and most organometallic molecules. Lipinski’s rule of
five32 acts as a filter for drug like properties and states that a
potential molecule is orally active if it’s a) molecular weight is
≤500 da b) logP ≤5 c) number of hydrogen bond acceptors ≤10
d) number of hydrogen bond donors ≤5. Compounds 3-7
presented milogP (P is the partition coefficient of the molecule in
the water/octanol system) which accounts for molecular
hydrophobicity varied from 1.56 to 4.95 (<5) suggesting them for
good bioavailability (Table 2). It is generally considered that
hydrogen bonding is an important parameter for describing
permeability of drugs.33 All the derivatives 3-7 under study have
numerous hydrogen bond acceptors (≤6) and numerous hydrogen
bond donors (≤1) as shown in Table 2. Number of rotable bonds
(nrotb) is an important indicator for molecular flexibility and
conformational change for binding to the receptor or channels. It
is revealed that the criteria for nrotb should be ≤10.34 The
compounds under study exhibits a high number of nrotb (2-6)
exhibiting their conformational flexibility. Volume, percentage of
absorption (%ABS), molecular polar surface area (PSA) for the
compounds 3-7 are presented in Table 2. PSA has been shown to
be a very good descriptor characterizing drug absorption and
transportation properties apart from intestinal absorption,
bioavailability, human intestinal epithelial adenocarcinoma
(Caco-2) cells permeability and blood-brain barrier (BBB)
penetration. Magnitude of absorption is expressed by percentage
of absorption and is calculated by the expression: %ABS = 109-
0.345*PSA. PSA, a sum of fragment contributions of polar atoms
such as oxygen, nitrogen and attached hydrogens is calculated by
Ertl et al. methodology.35 PSA and volume are inversely related
to %ABS. Compounds 3, 4, 5a, 5c, 5d, 5g, 5j, 5k and 5l have
maximum absorption (99.66%) due to their least corresponding
polar surface area and volume. PSA and logP are considered to
be the two most important parameters for predicting oral
bioavailability of a drug though not sufficient criteria.36 TPSA is
associated with the transport properties of drug across the
membranes, prediction in the BBB and intestinal crossing.
Molecules with TPSA/PSA in the range ≤160 Å2 have good
intestinal absorption and ≤60 Å2 has BBB penetration.37 Among
the EDA analogues except 6, 7d remaining all displayed TPSA
≤60 Å2 suggesting them as better BBB agents. 6, 7d have come
out to be better intestinal absorbers. The bioactivity scores of all
the new derivatives were presented in the Table 3 by means of
numerical assignment. High score implies higher the probability
bisolated yield after column chromatography.
cNR = no reaction.
Figure 3. Structures and yields of products 5-7.
Figure 4. ORTEP view for the compound 5d. Displacement ellipsoids are
drawn at the 30% probability level and H atoms are represented by circles of
arbitrary radii.

○
○
○
○
○
○
○
○
○
○
○
○
○
○
○
○
○
●
5a
5b
5c
5d
5e
5f
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
○
●
●
●
●
●
●
●
●
●
●
□
□
□
□
□
●
3.17
2.82
4.36
4.01
3.00
3.03
2.95
3.10
3.00
3.27
3.51
3.51
0.99
2.36
2.65
2.97
2.29
-0.95
-4.35
-4.05
-5.95
-5.59
-5.12
-4.38
-4.25
-4.37
-4.36
-4.66
-4.69
-4.69
-2.28
-3.59
-3.66
-4.33
-3.61
-3.72
4.05
4.24
3.29
4.31
-4.63
2.49
4.12
4.00
3.12
3.51
2.83
3.38
4.41
6.26
6.96
7.67
7.06
7.38
0.45
0.28
0.30
0.33
0.20
0.42
0.46
0.44
0.44
0.42
0.40
0.41
0.36
0.32
0.31
0.28
0.31
0.61
for a molecule to be active. In this connection molecules with
score >0.00 indicates more active, value between -0.50 to 0.00
are moderately active and <-0.50 are inactive.
Table 3
Bioactivity scoresa of the synthesized compounds 3-7
Compd.
EDA
3
GPCRL
-1.40
-0.47
-0.77
-0.12
-0.03
-0.01
-0.06
-0.03
-0.04
-0.17
-0.08
0.09
ICM
-1.09
-0.34
-0.34
-0.15
-0.10
-0.11
-0.39
-0.15
-0.18
-0.14
-0.19
0.00
KI
NRL
-1.73
-1.06
-1.09
-0.42
-0.20
-0.19
-0.29
-0.22
-0.19
-0.50
-0.31
-0.17
-0.34
-0.40
-0.35
-0.72
-0.53
-0.65
-0.53
-0.48
PI
EI
-2.14
-0.78
-0.74
-0.10
-0.02
0.07
-1.42
-0.86
-1.06
-0.35
-0.28
-0.19
-0.34
-0.23
-0.25
-0.41
-0.28
-0.08
-0.33
-0.34
-0.32
-0.60
-0.17
-0.20
-0.20
-0.17
-0.66
-0.45
-0.56
-0.13
-0.06
-0.03
-0.10
-0.07
-0.12
-0.15
-0.14
0.02
5g
5h
5i
4
5a
5b
5c
5j
5k
5l
5d
5e
0.10
e
6
□
□
□
□
□
●
0.06
7a
7b
7c
7d
Dox
5f
-0.06
-0.03
-0.06
0.02
5g
5h
5i
5j
-0.07
-0.12
-0.09
-0.46
-0.18
-0.23
-0.17
-0.19
-0.16
-0.21
-0.20
-0.24
-0.23
-0.29
-0.23
-0.25
-0.03
-0.11
-0.07
-0.59
-0.22
-0.33
-0.23
-0.23
-0.14
-0.17
-0.15
-0.21
-0.19
-0.31
-0.22
-0.20
aMUT: mutagenic; TUM: tumorigenic; IRRIT: irritant; RE: reproductive
effect.
5k
5l
bCLP: c(logP); S: Solubility; DL: Drug likeness; DS: drug score.
6
c○: red
7a
7b
7c
d●: green
e□: yellow
The drug score (DS) is the combination of druglikeness, c(logP),
logS, molecular weight and toxicity risks in one handy value than
may be used to judge the compound's overall potential to qualify
for a drug. This value is calculated by multiplying contributions
of the individual properties with the first equation:
7d
aGPCRL: GPCR ligand; ICM: Ion channel modulator; KI: Kinase inhibitor;
NRL: Nuclear receptor ligand; PI: Protease inhibitor; EI: Enzyme inhibitor.
Osiris calculations: Drug likeness may be defined as a complex
balance of various molecular properties and structural features
which determine whether a particular molecule is similar to the
known drugs. The Osiris38 calculations for 3-7 are presented in
Table 4. The results of toxicity risk predictor are colour coded
indicating potential toxicity regarding the category specified.
Green colour indicates drug conform behaviour, red colour being
high risk of undesired effects; yellow colour indicates medium
risk fragments. From Table 4, it is found that reported
compounds 3-7 have come out to be only tumorigenic except 5b
which has an effect on the reproductive system. Further 6,
peptide analogues 7a-d were irritants and showed medium risk
fragments against the reproductive system. Compounds 3, 4
conformed drug like behavior. Drug solubility is an important
factor which aids in the movement of drug from the site of
administration into the blood stream. Poor solubility leads to poor
absorption.36 More than 80% of marketed drugs has logS >-4.
Osiris estimated logS values are measured in mol/liter. In case of
3-7, values of logS are around -4. Drug likeness of compounds 3-
7 is in the comparable zone of standard drug doxorubicin (Dox).
DS = π*[(1/2)+(1/2)Si]*πti .... (1)
Where S = 1/(1+expap+b
)
Si is the contributions calculated directly from c(logP), logS,
molecular weight and drug likeness (pi) via the second equation
which describes a spline curve. Parameters ‘a’ and ‘b’ are (1, -5),
(1, 5), (0.012, -6) and (1, 0) for c(logP), logS, molecular weight
and drug likeness respectively. The ti is the contributions taken
from the 4 toxicity risk types and its values are 1.0, 0.8 and 0.6
for no risk, medium risk and high risk respectively. The reported
compounds 3-7 exhibited moderate to good scores compared
with a standard drug (see Table 4).
Having prepared a range of novel edaravone derivatives, all of
them were screened for in vitro anticancer activity against a panel
of human cancer cell lines namely prostate adenocarcinoma
(PC3), lung carcinoma (A549) by MTT colorimetric assay as per
ATCC protocol39 and the results are reported in Table 5 as
percentage of inhibition. From the results, it is interesting to note
that Suzuki product 5l and peptide product 7c have demonstrated
highest activity among the tested series 3-7 against PC3 and
A549 cell lines respectively. Close inspection of the screening
data reveals the fact that compounds 5i, 5l, 7c demonstrated
superior activity >80% of inhibition, 5c, 5d, 5f, 5h exhibited
promising activity >70% of inhibition and 3, 5a, 5b, 5e, 7b
presented mediocre activity >52% of inhibition towards A549
cells and 5l displayed moderate activity of 59% of inhibition
against PC3 cells indicating the selectivity of these class of
compounds towards a particular cell line. Introduction of hetero
Table 4
Osiris calculations of the target compounds EDA, 3-7
Compd.
Toxicity Risksa
PhysicochemicalPropertiesb
MUT
TUM
IRRIT
RE
○
CLP
1.82
1.51
2.23
S
DL
3.72
3.38
-2.14
DS
c
EDA
○
○
●
●
○
●
●
-2.04
-2.26
-3.10
0.12
0.93
0.49
d
3
4
●
●
●
●

aromatic residue as in 5d, which also was shown to be a potent
agent against the above mentioned lung cancer cell line. The
activity trend of regio isomers 5l (m-CH3) and 5k (p-CH3) against
the cell lines indicate that presence of methyl group at meta
position is preferred over para region. Increment in the number of
inhibition) > 5a (65% inhibition) > 5k (43% inhibition)
indicating their selectiveness. From the activity data the
following structure-activity relationships (SARs) can be inferred
(Fig. 5). For the structures 4, 5a, 5b, 6 introduction of hydroxyl
group (5a vs. 5b) led to the decrease in activity against PC3 cells
among the series 5 and introduction of a carboxylic acid group (4
vs. 6) resulted in loss of activity against both the cell lines among
the series 5-7, hence these structures are considered to be not
beneficial for the anticancer activity. For 5l [4-(3-
methylphenyl)], a remarkable increase of anticancer activity
against PC3 is observed compared with compound 5a (4-phenyl),
and this change is thought to be beneficial for both the cell lines,
similar is the increase of activity against A549 for 5i [4-(2-
methoxymethyl)phenyl] vs. the cytotoxicity of 5a. For the
peptide series 7, the higher the electron withdrawing nature of the
substituent, the higher is the activity within the following four 4-
carboxamides: 7c (N-[(4-chlorophenyl)methyl]) > 7d (N-[4-
methoxyphenyl]methyl]) > 7b [N-(2-phenylethyl)] > 7a [N-4-
(phenyl)methyl)] > 6 (4-carboxylic acid) against all the cells (see
Table 5). Notably for 7c, however a generalization that amide
linker is always beneficial for the activity against lung cancer
cells (A549) is inconclusive as remaining compounds (7a, 7b,
7d) did not show the expected activity and depends on the nature
of the substituents present (7d) or absent (7a, 7b) on the N-
phenylmethyl group attached to amide bond. The highest activity
of 7c might be due to enhanced lipophilicity, pharmacokinetic
properties, physicochemical properties, electronegativity and
endurance for metabolic distruction due to presence of a halogen
bond.40 As anticipated, all the designed compounds presented
remarkable anti proliferative activity than the parent core EDA,
which might be due to the electronic effects of various
substituents and higher lipophilicities (Table 2) of the new
derivatives, albeit lesser activity than the standard drug. Further
assay was also performed on human normal cell lines HEK293T
to study their safety profiles. Compounds 5c, 5d, 5e, 5i, 5l
showed better safer profiles with percentage of growth higher
than 53.00. 5f, 5h, 7c, 7d displayed moderate safer profiles.
Sequential increment of methoxy groups for the series 5a, 5h and
5f also led to the increment of safety profiles. In a dose response
study compounds 5c and 5l showed consistent growth inhibition
when tested against prostate and lung cancers (Fig. 6).
Table 5
In vitro anticancer, antioxidant activity of the designed
edaravone compounds 3-7
Compound
Cytotoxicitya
IC50
(µg/mL)
% of inhibition at 25M
PC3
1.43
A549
19.22
55.79
30.80
65.70
55.87
71.00
73.46
54.74
74.68
40.05
70.70
80.53
63.87
43.99
85.87
22.06
46.70
52.67
87.02
60.91
--c
HEK293T
EDA
2.40
22.98
5.96
54.40 ± 4.59
49.18 ± 4.01
36.96 ± 3.96
32.20 ± 3.05
37.03 ± 2.62
53.56 ± 5.17
50.67 ± 5.91
41.98 ± 3.34
38.99 ± 2.83
30.08 ± 3.40
24.98 ± 2.81
20.05 ± 3.91
35.97 ± 3.80
18.60 ± 1.83
26.40 ± 3.63
16.27 ± 1.97
23.63 ± 3.03
26.98 ± 3.94
16.07 ± 3.81
28.42 ± 2.40
--e
3
4
5.91
6.38
5a
16.23
4.90
28.19
5.58
5b
5c
27.60
32.41
30.19
33.03
36.58
24.34
36.05
46.33
18.51
59.26
3.19
53.64
54.96
57.84
48.11
22.17
46.33
56.54
40.88
19.72
70.45
12.06
22.25
31.75
49.37
44.28
--d
5d
5e
5f
5g
5h
5i
5j
5k
5l
6
7a
8.71
7b
7c
29.74
45.80
34.64
--b
7d
Standard
aCytotoxic activity against two human cancer cell lines (prostate, lung) and
human normal cell line (HEK293T).
bDoxorubicin IC50 is 2.1 µM.
cDoxorubicin IC50 is 3.3 µM.
dDoxorubicin IC50 is 1.2 µM.
eAscorbic acid IC50 is 8.09 ± 1.04 µg/mL.
methoxy groups increased the activity particularly with respect to
5f (o,p-OCH3) > 5h (p-OCH3) > 5a (-Ph) > 5b (p-OH) against all
the panel of cell lines consumed in the present study which could
be explained by the augmented electron density and lipophilicity
of the methoxy group in the molecules 5f and 5h, the effect of
increased electron density is counter balanced by the
hydrophilicity of the hydroxyl group in 5b. Further enhancement
can be observed if the methoxy group is separated by a carbon
residue at the ortho position i.e., 5i (o-CH2OCH3) which is due to
the increased lipophilicity as well as reduced steric effects. For
compounds 5h (p-OCH3), 5k (p-CH3), 5a (Ph) the activity profile
is 5h (24% inhibition) and thus larger than for 5k (18%
inhibition), which in turn is larger than for 5a (16% inhibition)
against PC3 cell line following the trend of electronic effects (+I-
inductive effect) i.e., analogue featuring substituent with more +I
effect demonstrated higher activity. The exact pattern, however,
is not observed for A549 cell line, the trend being 5h (70%
Figure 5. SAR summary of anti-cancer activities for compounds 5-7.
Figure 6. Dose response curves of the most active compounds on cancer cell
lines.

15.
16.
Gao, M.; Shi, L.; Zhang, H.; Chen, B.; Wang, Q.; Xuan, Z.; Du,
G. ZhongguoYaoxueZazhi (Beijing, China) 2011, 46, 932.
Walker, J. R.; Fairfull-Smith, K. E.; Anzai, K.; Lau, S.; White, P.
J.; Scammells, P. J.; Bottle, S. E. Med. Chem. Commun. 2011, 2,
436.
(a) Kawai, H.; Nakai, H.; Suga, M.; Yuki, S.; Watanabe, T.; Saito,
KI. J. Pharmacol. Exp. Ther. 1997, 281, 921; (b) Yamamoto, T.;
Yuki, S.; Watanabe, T.; Mitsuka, M.; Saito, KI.; Kogure, K. Brain
Res. 1997, 762, 240; (c) Perez-Gonzalez, A.; Galano, A. J. Phys.
Chem. B 2012, 116, 1180.
Nakagawa, H.; Ohyama, R.; Kimata, A.; Suzuki, T.; Miyata, N.
Bioorg. Med. Chem. Lett. 2006, 16, 5939.
Chegaev, K.; Cena, C.; Giorgis, M.; Rolando, B.; Tosco, P.;
Bertinaria, M.; Fruttero, R.; Carrupt, P. A.; Gasco, A. J. Med.
Chem. 2009, 52, 574.
(a) Zheng, Z.; Yenari, M. A. Neurol. Res. 2004, 26, 884; (b)
Barone, F. C.; Feuerstein, G. Z. J. Cereb Blood Flow Metab. 1999,
19, 819.
Borges, R. S.; Queiroz, A. N.; Silva, J. R.; Mendes, A. P. S.;
Herculano, A. M.; Chaves Neto, A. M. J.; Da Silva, A. B. F.
Struct. Chem. 2013, 24, 349.
Perez-Gonzalez, A.; Galano, A. J. Phys. Chem. B 2011, 115,
10375.
Markovic, V.; Eric, S.; Juranic, Z. D.; Stanojkovic, T.; Joksovic,
L.; Rankovic, B.; Kosanic, M.; Joksovic, M. D. Bioorg. Chem.
2011, 39, 18.
Jose, G.; Suresha Kumara, T. H.; Nagendrappa, G.; Sowmya, H.
B. V.; Sriram, D.; Yogeeswari, P.; Sridevi, J. P.; Guru Row, T. N.;
Hosamani, A. A.; SujanGanapathy, P. S.; Chandrika, N.;
Narendra, L. V. Eur. J. Med. Chem. 2015, 89, 616.
Holzer, W.; Plagens, B.; Lorenz, K. Heterocycles 1997, 45, 309.
Pelleter, J.; Renaud, F. Org. Process Res. Dev. 2009, 13, 698.
Nagaki, A.; Takahashi, Y.; Yoshida, J. Chem. - Eur. J. 2014, 20,
7931.
Md-Saleh, S. R.; Chilvers, E. C.; Kerr, K. G.; Milner, S. J.;
Snelling, A. M.; Weber, J. P.; Thomas, G. H.; Duhme-Klair, A.
K.; Routledge, A. Bioorg. Med. Chem. Lett. 2009, 19, 1496.
Crystallographic data CCDC 1426671 contains supplementary
crystallographic data for the structure 5d. These data can be
Additionally the series of compounds were tested to ascertain
their in vitro antioxidant activities by the DPPH method41 and the
results are summarized in Table 5. From the assay, it was evident
that all the designed compounds exhibited potent antioxidant
profiles probably due to the additional contribution of these
analogues due to extended conjugation and delocalization to
stabilise the resulting pyrazolyl radical cation species42 formed
during the reaction with DPPH radical. Dominant radical
scavenging activity was observed for 6 (IC50 = 16.27 µg/mL), 7c
(IC50 = 16.07 µg/mL) which may be due to H-atom transfer from
OH and NH groups and also presence of large number of
scavenging sites.9 Compounds 5i, 5k displayed better activity
with IC50 values at 20.05 µg/mL and 18.60 µg/mL respectively.
Analogues 5h, 5l, 7a, 7b and 7d demonstrated moderate activity
with IC50 values at 24.98 µg/mL, 26.40 µg/mL, 23.63 µg/mL,
26.98 µg/mL, 28.42 µg/mL respectively.
In summary, we have designed and synthesized a library of
edaravone derivatives in good to excellent yields and tested their
anticancer and antioxidant profiles. All the synthesized
compounds demonstrated better biological responses than the
parent nucleus EDA. In particular 5i, 5l and 7c have showed
superior anticancer activity. The reported analogues showed
mediocre to good safer profiles against human normal cell lines
HEK293T. Products 5k, 6 and 7c exhibited best antioxidant
activity. The designed analogues showed suitable drug like
properties and are expected to present good bioavailability
profile. Further mechanistic studies and structural modification of
identified hits are on-going for the generation of newer analogues
with enhanced efficacy as anticancer and antioxidant agents.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Acknowledgments
One of the author (N.P.) is thankful to University Grants
Commission (UGC), New Delhi, Govt. of India for financial
assistance in the form of a research fellowship.
obtained
free
of
charge
[or
at
the
www.ccdc.cam.ac.uk/conts/retrieving.html
from
Cambridge Crystallographic Data Centre (CCDC), 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033;
email: deposit@ccdc.cam.ac.uk].
Bakht, M. A.; Yar, M. S.; Abdel-Hamid, S. G.; Qasoumi, S. I. A.;
Samad, A. Eur. J. Med. Chem. 2010, 45, 5862.
Supplementary Material
30.
Supplementary data comprising experimental procedures,
spectral data for all new compounds, assay of biological study
related to this article can be found at
31.
32.
http://www.molinspiration.com. v2014.11, 20-08-2015.
Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv.
Drug Deliv. Rev. 1997, 23, 3.
Rajanarendar, E.; Rama Krishna, S.; Nagaraju, D.; Govardhan
Reddy, K.; Kishore, B.; Reddy, Y. N. Bioorg. Med. Chem. Lett.
2015, 25, 1630.
Ahsan, M. J.; Samy, J. G.; Khalilullah, H.; Nomani, M. S.;
Saraswat, P.; Gaur, R.; Singh, A. Bioorg. Med. Chem. Lett. 2011,
21, 7246.
Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
Alafeefy, A. M.; Alqasoumi, S. I.; Ashour, A. E.; Masand, V.; Al-
Jaber, N. A.; Ben Hadda, T.; Mohamed, M. A. Eur. J. Med. Chem.
2012, 53, 133.
33.
34.
References
1.
Yasuda, D.; Takahashi, K.; Kakinoki, T.; Tanaka, Y.; Ohe, T.;
Nakamura, S.; Mashino, T. Med. Chem. Commun. 2013, 4, 527.
Perez-Gonzalez, A.; Galano, A. J. Phys. Chem. B 2011, 115, 1306.
Hata, K.; Lin, M.; Katsumura, Y.; Muroya, Y.; Fu, H.; Yamashita,
S.; Nakagawa, H. J. Radiat. Res. 2011, 52, 15.
2.
3.
35.
36.
4.
5.
Detchokul, S.; Williams, E. D.; Parker, M. W.; Frauman, A. G. Br.
J. Pharmacol. 2014, 171, 5462.
37.
38.
Da Silva, M. M.; Comin, M.; Duarte, T. S.; Foglio, M. A.; de
Carvalho, J. E.; do Vieira, M C.; Formagio, A. S. Molecules 2015,
20, 5360.
http://www.organic-chemistry.org/prog/peo/ 26/03/2014: version
2, 20-08-2015.
World
Cancer
Report
2014,
http://www.iarc.fr/en/publications/books/wcr/wcr-order.php
6.
Puthiyapurayil, P.; Poojary, B.; Chikkanna, C.; Buridipad, S. K.
Eur. J. Med. Chem. 2012, 53, 203.
7.
Patel, M. V.; Bell, R.; Majest, S.; Henry, R.; Kolasa, T. J. Org.
Chem. 2004, 69, 7058.
Horrocks, P.; Pickard, M. R.; Parekh, H. H.; Patel, S. P.; Pathak,
R. B. Org. Biomol. Chem. 2013, 11, 4891.
Rangaswamy, J.; Kumar, H. V.; Harini, S. T.; Naik, N. Bioorg.
Med. Chem. Lett. 2012, 22, 4773.
Mubeen, M.; Kini, S. G.; Pai, K. S. R. Der Pharma Chemica
2015, 7, 215.
39.
40.
Gerlier, D.; Thomasset, N. J. Immunol. Methods 1986, 94, 57.
Polkam, N.; Rayam, P.; Anireddy, J. S.; Yennam, S.; Anantaraju,
H. S.; Dharmarajan, S.; Perumal, Y.; Kotapalli, S. S.; Ummanni,
R.; Balasubramanian, S. Bioorg. Med. Chem. Lett. 2015, 25, 1398.
Blois, M.S. Nature 1958, 181, 1199.
Borges, R. S.; Queiroz, A. N.; Mendes, A. P. S.; Araujo, S. C.;
Franca, L. C. S.; Franco, E. C. S.; Leal, W. G.; Da Silva, A. B. F.
Int. J. Mol. Sci. 2012, 13, 7594.
8.
9.
41.
42.
10.
11.
12.
Knorr, L. Chem. Ber. 1883, 17, 546.
Tewari, A. K.; Srivastava, P.; Singh, V. P.; Singh, A.; Goel, R. K.;
Mohan, C. G. Chem. Pharm. Bull. 2010, 58, 634.
Uramaru, N.; Shigematsu, H.; Toda, A.; Eyanagi, R.; Kitamura,
S.; Ohta, S. J. Med. Chem. 2010, 53, 8727.
Watanabe, T.; Tahara, M.; Todo, S. Cardiovasc. Ther. 2008, 26,
101.
13.
14.