Synthesis, and the antioxidant, neuroprotective and P-glycoprotein induction activity of 4-arylquinoline-2-carboxylates

Article abstract of DOI:10.1039/c4ob00488d

An efficient formic acid catalyzed one-pot synthesis of 4-arylquinoline 2-carboxylates in water via three-component coupling of arylamines, glyoxylates and phenylacetylenes has been described. 4-Arylquinoline 2-carboxylates 1o and 1q displayed significant

Full text of DOI:10.1039/c4ob00488d

Organic &
Biomolecular Chemistry
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Synthesis, and the antioxidant, neuroprotective
and P-glycoprotein induction activity of
4-arylquinoline-2-carboxylates†‡
Cite this: Org. Biomol. Chem., 2014,
12, 6267
Jaideep B. Bharate,a,b Abubakar Wani,c Sadhana Sharma,c Shahi Imam Reja,d
Manoj Kumar,d Ram A. Vishwakarma,*a,b Ajay Kumar*c and Sandip B. Bharate*a,b
An efficient formic acid catalyzed one-pot synthesis of 4-arylquinoline 2-carboxylates in water via three-
component coupling of arylamines, glyoxylates and phenylacetylenes has been described. 4-Arylquino-
line 2-carboxylates 1o and 1q displayed significant antioxidant activity as indicated by their Fe-reducing
power in the ferric reducing ability of plasma (FRAP) assay. The compounds were found to react directly
with hydrogen peroxide, which might be one of the mechanisms of their antioxidant effect. Compounds
1o and 1q effectively quenched H2O2 and amyloid-β-generated reactive oxygen species (ROS) and also
displayed significant protection against H2O2-induced neurotoxicity in human neuroblastoma SH-SY5Y
cells. Additionally, all compounds exhibited promising P-glycoprotein induction activity in human adeno-
carcinoma LS-180 cells, indicating their potential to enhance amyloid-β clearance from Alzheimer’s
brains. Furthermore, all compounds were relatively non-toxic to SH-SY5Y and LS-180 cells (IC50 > 50 µM).
The promising antioxidant, ROS quenching, neuroprotective and Pgp-induction activity of these com-
pounds strongly indicate their potential as anti-Alzheimer’s agents.
Received 4th March 2014,
Accepted 24th June 2014
DOI: 10.1039/c4ob00488d
www.rsc.org/obc
enhanced accumulation of amyloid-β protein.4 Thus, ROS does
damage to the nerve cells by this feed forward loop mechan-
Introduction
The brains of Alzheimer’s patients are characterized by ism. The use of antioxidants has been shown to reduce the
accumulation of amyloid-β plaques, neurofibrillary tangles of accumulation of amyloid-β protein in the mouse Alzheimer’s
hyperphosphorylated tau protein and loss of neurons. The disease (AD) model and slowed functional decline in clinical
neurons loaded with amyloid-β plaques and neurofibrillary studies in mild to moderate AD.5 Therefore, discovery of new
tangles are stressed by unusually high levels of reactive oxygen antioxidants for the effective treatment of Alzheimer’s disease
species (ROS).1 Amyloid-β induced oxidative damage plays an is one of the current areas in the development of Alzheimer’s
important role in the pathogenesis of Alzheimer’s disease. disease therapeutics. Furthermore, the amyloid hypothesis
Numerous reports suggest that deposition of amyloid-β protein proposes that Alzheimer’s disease is caused by an imbalance
can lead to accumulation of hydrogen peroxide and thus result between amyloid-β production and clearance, resulting in
in oxidative damage to the neurons.2 There is considerable evi- increased amounts of amyloid-β in various forms such as
dence for involvement of ROS in amyloid-β induced oxidative monomers, oligomers, insoluble fibrils and plaques in the
damage.3 Evidence also exists for the role of ROS in the CNS.6 The Aβ efflux is regulated through a P-glycoprotein (Pgp)
efflux pump, and thus the Pgp deficiency at the blood–brain
barrier has been reported to increase amyloid-β deposition.7
aMedicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu 180001, India. E-mail: sbharate@iiim.res.in,
ajaykmahajan@hotmail.com, ram@iiim.res.in; Fax: +91-191-2569333;
Tel: +91-191-2569111
bAcademy of Scientific & Innovative Research (AcSIR), Indian Institute of Integrative
Medicine (CSIR), Canal Road, Jammu 180001, India
cCancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR),
In vivo studies indicated that Pgp inducers are able to increase
amyloid-β clearance.8 With the aim of discovering effective
anti-Alzheimer’s agents, herein we screened a series of 4-aryl-
quinoline carboxylate esters for their antioxidant and Pgp-
induction activity.
The quinoline ring system occurs widely among alkaloids9
and is a key structural component of several pharmaceuticals,
agrochemicals, dyestuffs, and materials. The quinoline
scaffold has been reported to possess diverse range of pharma-
cological activities.9,10 The well known antimalarial
natural products quinine and quinidine alkaloids isolated
Canal Road, Jammu 180001, India
dDepartment of Chemistry, Guru Nanak Dev University (GNDU), Amritsar 143005,
India
†IIIM Publication number IIIM/1669/2014
‡Electronic supplementary information (ESI) available: Experimental details and
NMR spectra of all new compounds. See DOI: 10.1039/c4ob00488d
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Table 1 Solvent and catalyst optimization studiesa
Reaction medium
(catalyst and solvent)
Yieldb
(%)
Entry
Temp.
Time
1
2
3
4
5
6
7
8
50 mol% Cu–Mn B, CH2Cl2
50 mol% Cu–Mn B, water
50 mol% Cu–Mn B, CH2Cl2
20 mol% Fe–PILC, CH2Cl2
20 mol% Fe–PILC, water
20 mol% Cu-turnings, CH2Cl2
20 mol% Fe2O3, CH2Cl2
10 mol% Pd(OAC)2, CH2Cl2
50 mol%, PdCl2, CH2Cl2
10 mol%, Pd (TFA)2, CH2Cl2
10 mol% In(OTf)2, CH2Cl2
10 mol% TFA, water
rt
rt
70 °C
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
12 h
12 h
12 h
12 h
12 h
12 h
12 h
12 h
0
0
0
0
0
0
0
0
0
Fig. 1 Methods for one-pot synthesis of 4-arylquinoline-2-carboxy-
lates from arylamines, glyoxylates and phenylacetylenes.
from Cinchona bark comprise the quinoline scaffold.11
Because of the promising biological activities of the scaffold,
methods to construct structurally diverse quinoline derivatives
have been extensively studied.12 Most of the well known classi-
cal methods such as the Skraup synthesis (involves sulphuric
acid),12c the Doebner–Miller reaction (SnCl4),12d,e and the
Friedlander synthesis (Lewis acids)12f,g,i involve harsh reaction
conditions. All of these are two-component reactions, and thus
have limitations for diversity-oriented synthesis. Diversity-
oriented one-pot protocols such as the Povarov reaction (3CC
of aromatic amine, aldehyde and alkyne),13 and 3CC of two
molecules of ortho-haloacetophenones with urea or primary
amines14 have also been reported. Amongst these multicompo-
nent protocols, the Povarov reaction15 is one of the most
9
12 h
12 h
10
11
12
13
14
15
16c
0
45 min
45 min
45 min
30 min
30 min
45 min
75
50
70
95
92
88
10 mol% formic acid, water
100 mol% formic acid, water
50% formic acid in water
10% formic acid in water
rt, room temperature. a Reagents and conditions: 2a (1 mmol), 3a
(1 mmol), 4a (1.2 mmol) and reaction medium (catalyst and
solvent). b Isolated yields. c Optimized reaction conditions.
elegant and direct one-pot approach for synthesis of functiona- these catalysts (entries 1–5). Several other commercial catalysts
lized quinolines. In the literature, the reagents used for the such as Cu-turnings, Fe2O3, Pd(OAc)2, PdCl2, Pd(TFA)2 were
Povarov reaction include HClO4-montmorillonite,13 Cu- also unsuccessful (entries 6–10). The use of 10 mol% In(OTf)2
(OTf)2,16 I2/MeNO2,17 CuI/La(OTf)2,18 AuCl3/CuBr,19 FeCl3,20 produced product 1a in good yield (entry 11). Trifluoroacetic
and AgOTf.21 Most of the protocols involve the use of a metal acid and formic acid produced product 1a in good yields
catalyst, organic solvents and also require heating or longer (50–95%; entries 12–16). A brief examination of formic acid
reaction times. Thus, development of a metal-free eco-friendly loading showed that 10% formic acid in water is an optimal
green protocol for synthesis of this class of compounds will be condition to produce the desired product in good yield
of great use. Herein, we report metal-free formic acid-catalyzed (entry 16), which was selected for further studies.
multicomponent synthesis of functionalized quinolines 1
Next, the scope of the optimized protocol was investigated
through 3CC of arylamines 2, glyoxylates 3, and phenylacety- for a variety of arylamines and phenylacetylenes (Table 2). A
lenes 4 in water (Fig. 1). 4-Arylquinolines displayed promising variety of aromatic amines such as 4-methoxy-substituted, 3,4-
antioxidant, neuroprotective and Pgp-induction activity, indi- methylene-dioxy, and 4-benzyloxy substituted anilines partici-
cating their potential as anti-Alzheimer’s agents.
pated well in this reaction. The phenylacetylenes substituted
with various electron-donating (e.g. methyl, t-butyl, OMe, acety-
lene, OCF3) as well as electron-withdrawing (e.g. F, Cl, Br, CF3)
groups produced the corresponding 4-arylquinoline-2-carboxy-
lates in excellent yields (Table 2).
Results and discussion
Chemistry
A possible mechanism for formic acid-catalyzed synthesis
Initially, experimental exploration of reaction parameters, of 4-arylquinoline 2-carboxylate 1a from aniline 2a, glyoxylate
including catalyst, solvent, temperature and reaction time, was 3a and phenylacetylene 4a is depicted in Fig. 2. The reaction
conducted using the model multicomponent reaction (MCR) mechanism involves a cascade of reactions involving initial
between aniline 2a, ethyl glyoxylate 3a and phenyl acetylene 4a condensation of aniline 2a and ethyl glyoxylate 3a to
(Table 1). Catalyst-free MCR was first attempted by varying form imine intermediate I. Next, there is a protonation of the
solvent, reaction temperature and time; however, no product nitrogen of the imine which facilitates the attack by phenyl-
was formed. To start with, first we investigated the catalytic acetylene 4a, leading to cyclization to produce dihydroquino-
effect of heterogeneous metal catalysts Cu–Mn spinel oxide,22 line V. The dihydroquinoline
and Fe–PILC;23 however, the reaction does not proceed with 4-arylquinoline 2-carboxylate 1a.
V on oxidation produces
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Table 2 Scope of the reactiona
Entry
Product
Yieldb (%)
88
Entry
Product
Yieldb (%)
85
1a
1k
1b
1c
1d
88
82
92
1l
82
78
78
1m
1n
1e
1f
92
85
1o
1p
88
80
1g
1h
80
82
1q
1r
78
86
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Table 2 (Contd.)
Entry
Product
Yieldb (%)
92
Entry
Product
Yieldb (%)
88
1i
1s
1j
92
1t
92
a Reagents and conditions: 2 (1.0 mmol), 3 (1.0 mmol), 4 (1.2 mmol) in 10% formic acid in water, stirred at rt for 45 min. b Isolated yields.
optical density of hydrogen peroxide at 240 nm.25 The reaction
of hydrogen peroxide started immediately after the addition of
compounds 1o and 1q. The OD was taken 10 seconds after the
addition of compounds. Compounds 1o and 1q displayed
instantaneous rate of reaction 3.0 × 10−3 and 2.0 × 10−3 moles
s−1, respectively. Thus, the ability of these compounds to
directly interact with hydrogen peroxide should be one of the
mechanisms of their observed antioxidant activity.
Next, the electrochemical behaviour of compound 1o (1.0 ×
10−3 M) was investigated by cyclic voltammetry. One anodic
peak at potential 0.4 V vs. Ag/Ag+ was observed, which may be
Fig. 2 Proposed mechanism.
due to the presence of a conjugated imine group of quinoline
which possesses lone pair of electrons. The cyclic voltammetry
measurement indicates that the peak at 0.4 V vs. Ag/Ag+ is the
Antioxidant and neuroprotection activity
oxidation peak of the imino nitrogen atom.
Quinoline class of compounds are known to possess antioxi-
dant activity due to their metal chelation ability.24 Therefore,
herein all synthesized compounds were screened for antioxi-
dant potential via checking their Fe-reducing power and free-
radical scavenging activity. Several analogs showed significant
antioxidant activity at 100 µM in the ferric reducing ability of
plasma (FRAP) assay, where compounds 1o and 1q were found
to be better than others. None of the compound displayed sig-
nificant DPPH free radical scavenging activity. Further experi-
ments showed that all analogs were relatively non-toxic
in human neuroblastoma SH-SY5Y cells with an IC50 value
of >50 µM (Table 3).
With a view that compounds 1o and 1q may interact directly
with hydrogen peroxide to reduce its damaging effect on cells,
the direct reaction of hydrogen peroxide with these com-
pounds was investigated. The rate of disappearance of hydro-
gen peroxide was calculated by measuring the reduction of
Further, the ROS-quenching activity of compounds 1o and
1q was investigated using the in vitro assay in human neuro-
blastoma SH-SY5Y cells. Cells were differentiated for seven
days by exposure to 10 µM of retinoic acid. The ROS was gener-
ated by treatment of differentiated SH-SY5Y cells with 500 µM
of hydrogen peroxide for 30 min. The DCFH-DA fluorescence
for H2O2 treated cells was considered to be 100%. Compounds
1o and 1q at 100 µM effectively quenched the acute ROS as
indicated by the decreased level of DCF fluorescence to 53%
and 76% by these compounds, respectively (Fig. 3).
Next, the effect of compounds on amyloid-β generated ROS
was investigated. The SH-SY5Y cells treated with aggregated
amyloid-β 1–42 peptide (10 µM) displayed 15% rise in the ROS
level in comparison with control samples (100%). The
SH-SY5Y cells co-treated with compounds 1o and 1q showed
significantly reduced level of ROS to 96% and 99% respectively
(Fig. 4).
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Table 3 Antioxidant activity and effect of compounds on cell viability
of SH-SY5Y cells
Antioxidant activity
FRAP assaya,c
DPPH assayb,c
% viability of
% Fe reducing % free-radical
power
SH-SY5Y cells at
Entry
scavenging activity 50 µMd
Control
Ascorbic acid
—
—
100
nd
100 0.0
5.8 0.9
8.4 1.2
32.8 2.5
12.3 1.4
15.7 0.8
7.0 1.5
20.5 1.9
16.9 0.7
13.9 1.6
7.4 1.1
10.0 1.7
5.4 1.1
3.4 0.17
10.8 1.6
41.5 3.2
5.4 0.9
48.3 0.1
8.7 0.5
6.7 0.06
2.9 0.3
100 0.0
5.0 0.32
1.1 0.35
2.2 0.41
4.3 0.19
7.6 1.5
7.8 0.64
5.4 1.4
6.8 1.02
5.0 0.06
7.2 0.82
6.4 0.36
5.7 0.79
7.2 1.52
6.1 0.27
5.3 1.28
6.4 0.57
8.5 0.39
5.8 0.88
5.1 0.80
7.9 0.72
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
87.7 7.3
99.8 4.7
115.7 11.7
107.8 7.9
99.1 2.2
69.3 1.1
68.7 2.4
96.6 3.2
98.41 4.3
71.1 2.5
90.7 1.9
92.0 2.2
99.6 10.8
56.0 10.4
96.4 9.4
88.1 6.6
82.3 11.4
111.9 9.2
92.5 6.4
89.6 10.3
Fig. 4 Quenching of amyloid-β induced ROS by compounds 1o and 1q
in SH-SY5Y cells. Cells were treated with 1o and 1q (at 50 µM), 30 min
before the treatment of aggregated amyloid-β 1–42 peptide (10 µM).
Data are mean of three independent experiments. Statistical compari-
sons were made using the Bonferroni test. The p value <0.05 was con-
sidered to be significant. p value *<0.05, **<0.01, ***<0.001. The symbol
@ represents comparison between control and samples treated with
amyloid-β, whereas the symbol
* represents comparisons between
samples treated with amyloid-β and those of treated with compounds
1o and 1q along with amyloid-β.
1s
1t
nd, not determined. a Ferric reducing ability of ascorbic acid (100 µM)
was considered to be 100%, whereas FRAP value for other compounds
was calculated in comparison with ascorbic acid. b DPPH free radical
scavenging activity of ascorbic acid was considered to be 100%. The
free radical scavenging activity of test compounds was calculated in
comparison with ascorbic acid. c Data in the FRAP and DPPH assays
are mean SD of three independent experiments. d Data for the cell
viability assay are mean SD of three similar experiments.
Fig. 5 Neuroprotective effect of compounds 1o and 1q against H2O2-
induced toxicity in SH-SY5Y cells. Cells were treated with 1o and 1q at
indicated concentrations 30 minutes prior to the treatment with H2O2
(100 µM) for 24 h. Data are mean of three independent experiments.
Statistical comparisons were made using the Bonferroni test. The p
value <0.05 was considered to be significant. p value *<0.05, **<0.01,
***<0.001. The symbol @ represents comparison between control and
samples treated with H2O2, whereas the symbol * represents compari-
sons between samples treated with H2O2 and those of treated with 1o
and 1q along with H2O2.
compounds 1o and 1q strongly reversed the H2O2-induced
neurotoxicity. The viability of hydrogen peroxide treated cells
(59%) was restored to 93% and 97% by pre-treatments with 1o
(50 µM) and 1q (50 µM) respectively (Fig. 5). However, the treat-
ment with Aβ 1–42 (10 µM) did not induce any significant
damage to SH-SY5Y cells in 24 h (data not shown), therefore,
the neuroprotective effect of compounds 1o and 1q against Aβ
induced neurotoxicity could not be analysed in this study.
Fig. 3 ROS quenching by compounds 1o and 1q in SH-SY5Y cells. Cells
were treated with 100 µM of 1o and 1q, 30 minutes before the treatment
of H2O2 (500 µM). Data are mean of three independent experiments.
Statistical comparisons were made using the Bonferroni test. The
p value <0.05 was considered to be significant. p value *<0.05, **<0.01,
***<0.001.
Pgp-induction activity
The recent two independent clinical studies6,26 observed that
AD patients have decreased clearance of CNS amyloid-β com-
pared to healthy volunteers. The Aβ-clearance occurs primarily
Encouraged by these results, we hypothesized that these
compounds may effectively provide neuroprotection to differ-
entiated SH-SY5Y cells against oxidative stress. Interestingly,
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oxidant activity of these compounds appears to be due to their
ability to directly interact with ROS. Furthermore, the signifi-
cant Pgp-induction activity of 4-arylquinolines indicates their
potential application in promoting efflux of toxins from the
body and enhancing amyloid-β clearance from the AD brain.
These findings clearly indicate their potential as a new lead for
anti-Alzheimer’s therapeutics.
Table 4 Pgp-induction activity and effect of 4-arylquinoline-2-
carboxylates on cell viability in LS-180 cells
% Rh123 accumulation in
LS-180 cells after 48 ha,b
% viability of LS-180
cells at 50 µMb
Entry
Control
Rifampicin
100
100
nd
70.6 7.9**
69.8 3.2**
78.3 3.8**
60.9 8.4***
69.6 9.1**
76.7 5.0**
93.0 6.9
72.0 7.1**
71.9 9.9**
81.5 2.5*
79.0 3.8**
71.0 6.4**
65.1 11.4**
61.0 2.3***
70.9 3.3**
59.6 2.1***
69.9 7.9**
60.2 4.7***
57.8 8.2***
71.4 5.9**
66.5 6.9***
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
106.1 9.3
100 6.5
115.6 6.2
114.8 6.0
134.8 1.1
98.1 6.6
127.1 5.2
135.5 4.8
61.9 2.3
65.5 4.9
98.6 0.9
99.2 6.4
111.9 7.4
45.6 5.0
86.2 4.2
80.9 4.8
64.2 3.7
95.6 9.2
117.0 12.2
117.0 6.4
Experimental section
General
All chemicals were obtained from Sigma-Aldrich Company and
were used as received. 1H, 13C and DEPT NMR spectra were
recorded on Bruker-Avance DPX FT-NMR 500 and 400 MHz
instruments. Chemical data for protons are reported in parts
per million (ppm) downfield from tetramethylsilane and are
referenced to the residual proton in the NMR solvent (CDCl3,
7.26 ppm). Carbon nuclear magnetic resonance spectra (13C
NMR) were recorded at 125 MHz or 100 MHz: chemical data
for carbons are reported in parts per million (ppm, δ scale)
downfield from tetramethylsilane and referenced to the
carbon resonance of the solvent (CDCl3, 77 ppm). ESI-MS and
HRMS spectra were recorded on an Agilent 1100 LC-Q-TOF and
HRMS-6540-UHD machines. IR spectra were recorded on a
Perkin-Elmer IR spectrophotometer. Melting points were
recorded on digital melting point apparatus.
1s
1t
nd, not determined. a Pgp induction activity of compounds was
checked at 5 µM and was measured in terms of the % intracellular
accumulation of rhodamine 123/total protein (μg) inside LS-180 cells.
The decrease in % intracellular accumulation (compared to control) of
Rh123 indicates induction of Pgp. Rifampicin (10 μM) was used as a
reference Pgp inducer. The statistical comparisons were made between
control vs. compounds. The p value <0.5 was considered to be
significant. p value *<0.05, **<0.01, ***<0.001. b Values are shown as
average of three experiments SD.
General procedure for preparation of 4-arylquinoline-
2-carboxylates
A
mixture of arylamine (1.0 mmol), ethyl glyoxylate
(1.0 mmol), and phenylacetylene (1 mmol) in 10% formic
acid–water was stirred at room temperature for 45 min. The
reaction mixture was then extracted with ethyl acetate and
combined organic layers were evaporated on a vacuum rota-
vapor. The crude product was purified by silica-gel column
chromatography (mesh 100–200) to obtain desired quinoline-
2-carboxylates 1a–t in 78–92% yield.
via a Pgp efflux pump, thus drugs with the ability to induce
Pgp have a great potential to emerge as novel AD therapeutics.
The compounds prepared herein are structurally similar to
nifedipine, a 4-(phenyl)-1,4-dihydropyridine-3,5-dicarboxylate
methyl ester, which is known to induce Pgp-expression.27
Therefore, we decided to screen all the compounds for Pgp
induction activity. The Pgp induction activity was determined
in colorectal adenocarcinoma LS-180 cells, which are known to
have constitutively high expression of Pgp. Cells treated with
5 µM of each compound for 48 h displayed significant induction
of Pgp activity, as displayed by the increased efflux of rhoda-
mine-123 (Table 4). The compounds 1o and 1q showed %
intracellular rhodamine-123 level of 57% and 60% respectively
in comparison with untreated control samples. Furthermore,
these compounds were found to be non-toxic to LS-180 cells
(IC50 > 50 μM).
Ethyl 6-methoxy-4-phenylquinoline-2-carboxylate (1a).28
Yellow solid; m.p. 155–156 °C; 1H NMR (CDCl3, 400 MHz): δ
8.29 (d, J = 12.0 Hz, 1H), 8.10 (s, 1H), 7.56–7.50 (m, 5H), 7.45
(d, J = 8.0 Hz, 1H), 7.22 (s, 1H), 4.58–4.53 (q, J = 8.0, 12.0 Hz,
2H), 3.81 (s, 3H), 1.49 (t, J = 4.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz): δ 165.6, 159.5, 148.0, 145.4, 144.3, 137.9, 132.7,
129.3, 129.2, 128.8, 128.7, 122.8, 121.8, 103.2, 62.1, 55.5, 14.4;
IR (CHCl3): νmax 3400, 3055, 2925, 1729, 1715, 1620, 1492,
1413, 1366, 1224, 1107 cm−1; ESI-MS: m/z 308.00 [M + H]+;
HRMS: m/z 308.1279 (ESI) calcd for C19H17NO3
+
H+
(308.1281).
Ethyl 6-methoxy-4-p-tolylquinoline-2-carboxylate (1b). White
solid; m.p. 141–142 °C; H NMR (CDCl3, 400 MHz): δ 8.28 (d,
Conclusion
1
In summary, we have developed a simple and efficient, econo- J = 12.0 Hz, 1H), 8.08 (s, 1H), 7.46–7.42 (m, 4H), 7.37 (d, J =
mically viable formic acid-catalyzed one-pot multicomponent 8.0 Hz, 2H), 4.58–4.52 (q, J = 8.0, 16.0 Hz, 2H), 3.82 (s, 3H),
protocol for preparation of structurally diverse 4-arylquinoline 2.48 (s, 3H), 1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3,
2-carboxylates. Compounds displayed significant antioxidant 100 MHz): δ 165.7, 159.5, 148.1, 145.3, 144.3, 138.6, 134.8,
and Pgp-induction activity and relatively low toxicity. The anti- 132.7, 129.5, 129.3, 122.8, 121.8, 103.3, 62.1, 55.6, 21.4, 14.3;
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IR (CHCl3): νmax 3400, 2919, 1716, 1620, 1585, 1497, 1472, 1714, 1619, 1553, 1463, 1249, 1028 cm−1; ESI-MS: m/z 338.10
1253, 1105 cm−1; ESI-MS: m/z 322.00 [M + H]+; HRMS: m/z [M + H]+; HRMS: m/z 338.1391 (ESI) calcd for C20H19NO4 + H+
322.1445 (ESI) calcd for C20H19NO3 + H+ (322.1438).
Ethyl 6-methoxy-4-(thiophen-3-yl)quinoline-2-carboxylate
(338.1387).
Ethyl 6-methoxy-4-m-tolylquinoline-2-carboxylate (1h).28
(1c). Brown solid; m.p. 153–154 °C; 1H NMR (CDCl3, Pale yellow solid; m.p. 139–140 °C; 1H NMR (CDCl3, 400 MHz):
400 MHz): δ 8.26 (d, J = 8.0 Hz, 1H), 8.14 (s, 1H), 7.60 (s, 1H), δ 8.28 (d, J = 8.0 Hz, 1H), 8.09 (s, 1H), 7.45–7.42 (m, 2H),
7.55–7.53 (m, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.40–7.37 (m, 2H), 7.36–7.31 (m, 3H), 7.24 (s, 1H), 4.58–4.53 (m, 2H), 3.81 (s, 3H),
4.58–4.53 (m, 2H), 3.86 (s, 3H), 1.49 (t, J = 4.0 Hz, 3H); 13C 2.47 (s, 3H), 1.49 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3,
NMR (CDCl3, 100 MHz): δ 165.6, 159.6, 145.4, 144.4, 142.8, 100 MHz): δ 165.7, 159.5, 148.2, 145.4, 144.3, 138.6, 137.9,
138.4, 132.8, 129.2, 128.5, 126.7, 125.1, 122.8, 121.6, 103.2, 132.7, 130.1, 129.3, 129.2, 128.6, 126.4, 122.7, 121.7, 103.4,
62.1, 55.6, 14.4; IR (CHCl3): νmax 3415, 2960, 1732, 1714, 1620, 62.1, 55.5, 21.5, 14.4; IR (CHCl3): νmax 2978, 2932, 1738, 1713,
1555, 1475, 1268, 1226 cm−1; ESI-MS: m/z 314.00; [M + H]+; 1620, 1474, 1406, 1367, 1257, 1224, 1106 cm−1; ESI-MS: m/z
HRMS: m/z 314.0844 (ESI) calcd for C17H15NO3S
(314.0845).
+ +
H+ 322.10 [M H]+; HRMS: m/z 322.1410 (ESI) calcd for
C20H19NO3 + H+ (322.1438).
Ethyl
4-(4-(trifluoromethyl)phenyl)-6-methoxyquinoline-2-
Ethyl 8-phenyl-[1,3]dioxolo[4,5-g]quinoline-6-carboxylate (1i).
carboxylate (1d). Brown solid; m.p. 161–162 °C; 1H NMR Brown solid; m.p. 237–238 °C; 1H NMR (CDCl3, 400 MHz):
(CDCl3, 400 MHz): δ 8.31 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.84 δ 8.00 (s, 1H), 7.64 (s, 1H), 7.56–7.47 (m, 5H), 7.19 (s, 1H), 6.13
(d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, (s, 2H), 4.56–4.51 (m, 2H), 1.47 (t, J = 4.0 Hz, 3H); 13C NMR
1H), 7.10 (s, 1H), 4.59–4.54 (q, J = 8.0, 16.0 Hz, 2H), 3.83 (s, (CDCl3, 100 MHz): δ 165.6, 151.1, 149.9, 148.2, 146.8, 145.6,
3H), 1.49 (t, J = 4.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): 138.1, 129.4, 128.8, 128.6, 125.6, 120.4, 107.0, 102.2, 100.9,
δ 165.4, 159.8, 146.4, 145.4, 144.3, 141.6, 133.0, 129.8, 128.8, 62.1, 14.4; IR (CHCl3): νmax 3435, 2918, 1696, 1462, 1276, 1239,
1
125.8, (t, JCF = 3.77 Hz), 123.1, 121.8, 102.8, 62.3, 55.7, 14.4; 1111, 1036 cm−1; ESI-MS: m/z 322.00 [M + H]+; HRMS: m/z
19F NMR (376.50 MHz, CDCl3): δ −62.81 (s, 3F); IR (CHCl3): 322.1077 (ESI) calcd for C19H15NO4 + H+ (322.1074).
νmax 3434, 2927, 2851, 1620, 1512, 1476, 1324, 1228,
1066 cm−1; ESI-MS: m/z 376.00 [M + H]+; HRMS: m/z 376.1162 Yellow solid; m.p. 220–221 °C; 1H NMR (CDCl3, 400 MHz):
(ESI) calcd for C20H16F3NO3 + H+ (376.1155).
δ 7.98 (s, 1H), 7.63 (s, 1H), 7.39–7.33 (m, 4H), 7.22 (s, 1H),
Ethyl 8-p-tolyl-[1,3]dioxolo[4,5-g]quinoline-6-carboxylate (1j).
Ethyl 4-(4-tert-butylphenyl)-6-methoxyquinoline-2-carboxy- 6.12 (s, 2H), 4.57–4.51 (m, 2H), 2.47 (s, 3H), 1.47 (t, J = 8.0 Hz,
late (1e).28 White solid; m.p. 174–175 °C; 1H NMR (CDCl3, 3H); 13C NMR (CDCl3, 100 MHz): δ 165.7, 151.1, 149.8, 148.4,
400 MHz): δ 8.28 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 7.58 (d, J = 146.8, 145.6, 138.6, 135.1, 129.4, 129.3, 125.7, 120.4, 106.1,
8.0 Hz, 2H), 7.52 (d, J = 12.0 Hz, 2H), 7.45–7.43 (dd, J = 4.0, 102.1, 101.0, 62.1, 21.3, 14.4; IR (CHCl3): νmax 3401, 2921,
4.0 Hz, 1H), 7.31 (s, 1H), 4.57–4.52 (m, 2H), 3.84 (s, 3H), 1.48 1703, 1617, 1504, 1463, 1376, 1237, 1110 cm−1; ESI-MS: m/z
(t, J = 8.0 Hz, 3H), 1.42 (s, 9H); 13C NMR (CDCl3, 100 MHz): 336.00 [M
+
H]+; HRMS: m/z 336.1236 (ESI) calcd for
δ 165.7, 159.4, 151.8, 148.1, 145.4, 144.4, 134.9, 132.7, 129.2, C20H17NO4 + H+ (336.1230).
129.1, 125.8, 122.6, 121.9, 103.6, 62.1, 55.6, 34.8, 31.4, 14.4; IR
Ethyl 8-(4-(trifluoromethyl)phenyl)-[1,3]dioxolo[4,5-g]quino-
(CHCl3): νmax 3401, 2961, 2866, 1739, 1715, 1620, 1498, 1474, line-6-carboxylate (1k). Brown solid; m.p. 161–162 °C; 1H NMR
1251, 1224 cm−1; ESI-MS: m/z 364.10 [M + H]+; HRMS: m/z (CDCl3, 400 MHz): δ 8.02 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.74
364.1903 (ESI) calcd for C23H25NO3 + H+ (364.1907).
(s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.09 (s, 1H), 6.18 (s, 2H),
Ethyl 4-(4-bromophenyl)-6-methoxyquinoline-2-carboxylate 4.59–4.54 (m, 2H), 1.49 (t, J = 4.0 Hz, 3H); 13C NMR (CDCl3,
(1f). Pale yellow solid; m.p. 171–172 °C; 1H NMR (CDCl3, 100 MHz): δ 165.0, 152.2, 150.7, 147.9, 145.7, 144.4, 141.2,
1
400 MHz): δ 8.28 (d, J = 8.0 Hz, 1H), 8.06 (s, 1H), 7.70 (d, J = 129.8, 125.9 (t, JCF = 3.77 Hz), 125.6, 131.3, 131.0, 120.3,
8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 3H), 7.13 (s, 1H), 4.58–4.53 (m, 106.1, 102.7, 100.5, 62.7, 14.4; 19F NMR (376.50 MHz, CDCl3)
2H), 3.82 (s, 3H), 1.49 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, δ −62.95 (s, 3F); IR (CHCl3): νmax 3400, 2919, 2850, 1617, 1419,
100 MHz): δ 165.5, 159.7, 146.7, 145.4, 144.3, 136.8, 132.9, 1090 cm−1; ESI-MS: m/z 390.00 [M + H]+; HRMS: m/z 390.0953
132.1, 131.0, 130.9, 128.9, 123.1, 123.0, 121.7, 102.9, 102.8, (ESI) calcd for C20H14F3NO4 + H+ (390.0948).
62.2, 55.6, 14.4; IR (CHCl3): νmax 3400, 2923, 2850, 1730, 1715,
1619, 1488, 1473, 1434, 1367, 1274, 1226 cm−1; ESI-MS: m/z Pale yellow solid; m.p. 126–127 °C; 1H NMR (CDCl3, 400 MHz):
387.90 [M
H]+; HRMS: m/z 388.0374 (ESI) calcd for δ 7.98 (s, 1H), 7.63 (s, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.32–7.28
C19H16BrNO3 + H+ (386.0386).
(m, 3H), 7.20 (s, 1H), 6.13 (s, 2H), 4.56–4.51 (m, 2H), 2.46 (s,
Ethyl 8-m-tolyl-[1,3]dioxolo[4,5-g]quinoline-6-carboxylate (1l).
+
Ethyl 6-methoxy-4-(4-methoxyphenyl)quinoline-2-carboxylate 3H), 1.47 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz):
(1g).28 Pale yellow solid; m.p. 119–120 °C; 1H NMR (CDCl3, δ 165.7, 151.1, 149.8, 148.5, 146.8, 145.6, 138.5, 138.1, 130.0,
400 MHz): 8.27 (d, J = 8.0 Hz, 1H), 8.07 (s, 1H), 7.51 (d, J = 129.3, 128.4, 126.4, 125.8, 120.4, 107.0, 102.1, 101.0, 62.1, 21.5,
8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.10 (s, 2H), 14.4; IR (CHCl3): νmax 3400, 2922, 1617, 1462, 1384, 1237,
4.58–4.53 (m, 2H), 3.92 (s, 3H), 3.83 (s, 3H), 1.49 (t, J = 8.0 Hz, 1037 cm−1; ESI-MS: m/z 336.3602 [M + H]+; HRMS: m/z
3H); 13C NMR (CDCl3, 100 MHz): δ 165.8, 160.0, 159.4, 147.8, 336.3600 (ESI) calcd for C20H17NO4 + H+ (336.3607).
145.4, 144.4, 132.7, 130.6, 130.2, 129.4, 122.8, 121.8, 114.2,
Ethyl 8-(3-chlorophenyl)-[1,3]dioxolo[4,5-g]quinoline-6-car-
103.3, 62.1, 55.6, 55.4, 14.4; IR (CHCl3): νmax 3400, 2019, 2850, boxylate (1m). Yellow solid; m.p. 133–134 °C; 1H NMR (CDCl3,
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400 MHz): δ 7.97 (s, 1H), 7.64 (s, 1H), 7.48 (d, J = 4.0 Hz, 3H), (m, 2H), 1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ
7.37–7.35 (m, 1H), 7.12 (s, 1H), 6.14 (s, 2H), 4.57–4.52 (m, 2H), 165.6, 158.4, 148.0, 145.5, 144.3, 137.8, 136.1, 132.8, 129.4,
1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 165.5, 129.0, 128.8, 128.7, 128.6, 128.2, 127.6, 123.2, 121.8, 104.9,
151.3, 150.1, 146.8, 145.6, 139.8, 134.8, 130.1, 129.4, 128.7, 70.2, 62.1, 14.4; IR (CHCl3): νmax 3400, 2923, 1732, 1601, 1462,
127.5, 125.3, 120.3, 107.1, 102.3, 100.5, 62.1, 29.6, 14.1; IR 1384, 1237, 1035 cm−1; ESI-MS: m/z 384.10 [M + H]+; HRMS:
(CHCl3): νmax 3434, 2923, 1742, 1620, 1464, 1241, 1082 cm−1
;
m/z 384.1595 (ESI) calcd for C25H21NO3 + H+ (384.1594).
Ethyl 6-(benzyloxy)-4-(3-chlorophenyl)quinoline-2-carboxy-
late (1s). Yellow solid; m.p. 153–154 °C; 1H NMR (CDCl3,
ESI-MS: m/z 356.00 [M + H]+; HRMS: m/z 356.0656 (ESI) calcd
for C19H14ClNO4 + H+ (356.0684).
Ethyl
8-(4-ethynylphenyl)-[1,3]dioxolo[4,5-g]quinoline-6- 400 MHz): δ 8.30 (d, J = 12.0 Hz, 1H), 8.05 (s, 1H), 7.53 (m,
1
carboxylate (1n). Pale yellow solid; m.p. 142–143 °C; H NMR 1H), 7.49 (s, 2H), 7.46–7.42 (m, 1H), 7.38–7.32 (m, 5H), 7.30 (s,
(CDCl3, 400 MHz): δ 7.97 (s, 1H), 7.67 (d, J = 8.0 Hz, 3H), 7.46 1H), 7.18 (d, J = 4.0 Hz, 1H), 5.08 (s, 2H), 4.47–4.52 (m, 2H),
(d, J = 8.0 Hz, 2H), 7.13 (s, 1H), 6.14 (s, 2H), 4.57–4.51 (m, 2H), 1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 165.4,
3.19 (s, 1H), 1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 158.7, 146.5, 145.4, 144.3, 139.6, 136.1, 134.8, 132.6, 130.1,
100 MHz): δ 165.5, 151.2, 150.1, 147.4, 146.8, 145.6, 138.5, 128.7, 128.3, 129.3, 128.8, 127.6, 127.6, 123.5, 121.7, 104.7,
132.5, 129.4, 125.3, 122.6, 120.3, 107.1, 102.2, 100.6, 83.0, 78.5, 70.3, 62.2, 14.4; IR (CHCl3): νmax 3399, 2921, 1556, 1348, 1042,
62.1, 14.1; IR (CHCl3): νmax 3400, 2924, 1714, 1620, 1497, 1483, 772 cm−1; ESI-MS: m/z 418 [M + H]+; HRMS: m/z 418.1205 (ESI)
1369, 1212, 1239, 1114 cm−1; ESI-MS: m/z 346.00 [M + H]+; calcd for C25H20ClNO3 + H+ (418.1204).
HRMS: m/z 346.1082 (ESI) calcd for C21H15NO4
(346.1074).
+
H+
Ethyl
6-(benzyloxy)-4-(4-fluorophenyl)quinoline-2-carboxy-
1
late (1t). Pale yellow solid; m.p. 134–135 °C; H NMR (CDCl3,
Ethyl 8-(4-(trifluoromethoxy)phenyl)-[1,3]dioxolo[4,5-g]qui- 400 MHz): δ 8.30 (d, J = 12.0 Hz, 1H), 8.05 (s, 1H), 8.54–8.51
noline-6-carboxylate (1o). Brown solid; m.p. 117–118 °C; 1H (dd, J = 4.0, 12.0 Hz, 1H), 7.41–7.34 (m, 6H), 7.23–7.17 (m, 4H),
NMR (CDCl3, 400 MHz): δ 7.97 (s, 1H), 7.64 (s, 1H), 7.53 (d, J = 5.09 (s, 2H), 4.58–4.52 (m, 2H), 1.48 (t, J = 8.0 Hz, 3H); 13C
1
8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.11 (s, 1H), 6.14 (s, 2H), NMR (CDCl3, 125 MHz): δ 165.6, 163.9 (d, JCF = 251.5 Hz),
4.57–4.51 (m, 2H), 1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 158.5, 146.9, 145.4, 144.3, 136.1, 132.9, 131.14, 131.08, 129.0,
2
100 MHz): δ 165.5, 151.2, 150.1, 149.5, 146.8, 146.7, 145.7, 128.7, 128.2, 127.5, 123.3, 128.8, 116.0 (d, JCF = 21.8 Hz),
1
136.7, 130.9, 125.3, 121.8, 120.8 (d, JCF = 100.60 Hz), 119.2, 104.7, 70.2, 62.2, 14.4; 19F NMR (376.50 MHz, CDCl3):
107.2, 102.3, 100.5, 62.1, 14.4; 19F NMR (376.50 MHz, CDCl3) δ δ −112.96 (s, 1F); IR (CHCl3): νmax 3400, 2925, 1715, 1618,
−57.72 (s, 3F); IR (CHCl3): νmax 3400, 2921, 2851, 1742, 1590, 1605, 1497, 1378, 1224 cm−1; ESI-MS: m/z 402.00 [M + H]+;
1503, 1463, 1384, 1161, 1035 cm−1; ESI-MS: m/z 406.00 HRMS: m/z 402.1499 (ESI) calcd for C25H20FNO3
[M + H]+; HRMS: m/z 406.0936 (ESI) calcd for C20H14F3NO5 + (402.1500).
H+ (406.0897).
+
H+
Ethyl 6,7-dimethoxy-4-phenylquinoline-2-carboxylate (1p).
Yellow solid; m.p. 140–141 °C; 1H NMR (CDCl3, 400 MHz):
FRAP assay
δ 8.02 (s, 1H), 7.69 (s, 1H), 7.56–7.53 (m, 5H), 7.20 (s, 1H), The FRAP assay was performed in a 96-well microplate using a
4.58–4.53 (m, 2H), 4.06 (s, 3H), 3.87 (s, 3H), 1.48 (t, J = 8.0 Hz, slightly modified protocol of Benzie and Strain (1996).29
3H); 13C NMR (CDCl3, 100 MHz): δ 165.7, 152.8, 151.5, 147.6, Briefly, the FRAP reagent was prepared by mixing 10 ml of
145.6, 145.5, 138.1, 129.3, 128.8, 128.6, 123.8, 120.3, 109.4, 300 mM acetate buffer with 1 ml of 10 mM 2,4,6-tripyridyl-
102.9, 62.1, 56.4, 56.1, 14.5; IR (CHCl3): νmax 3400, 2924, 1735, S-triazine (TPTZ) in 40 mM of hydrochloric acid and 1 ml of
1714, 1497, 1483, 1239, 1212, 1113, 1027 cm−1; ESI-MS: m/z 20 mM FeCl3·6H2O. The freshly prepared FRAP reagent
338.10 [M
+
H]+; HRMS: m/z 338.1387 (ESI) calcd for (195 µl) was added to all wells of the 96-well plate. Test com-
pounds dissolved in methanol (5 µl) were added to the final
C20H19NO4 + H+ (338.1387).
Ethyl 4-(3-chlorophenyl)-6,7-dimethoxyquinoline-2-carboxy- concentration of 100 µM. Ascorbic acid (100 µM) was used as a
late (1q). Brown solid; m.p. 141–142 °C; 1H NMR (CDCl3, positive control. The absorbance was read at 593 nm after
400 MHz): δ 8.00 (s, 1H), 7.71 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H), 30 min incubation in the dark.
7.42 (d, J = 8.0 Hz, 2H), 7.11 (s, 1H), 4.57–4.55 (m, 2H), 4.07 (s,
3H), 3.89 (s, 3H), 1.48 (t, J = 8.0 Hz, 3H); 13C NMR (CDCl3,
100 MHz): δ 165.6, 153.0, 151.8, 149.9, 146.0, 145.6, 145.6,
DPPH assay
134.7, 130.1, 130.8, 128.8, 123.7, 121.2, 120.3, 109.5, 102.5, The DPPH assay was carried out in a 96-well microplate.
62.1, 56.4, 56.1, 14.4; IR (CHCl3): νmax 3391, 2924, 1738, 1619, Briefly, 190 µl of each test compound dissolved in methanol
1492, 1370, 1240, 771 cm−1; ESI-MS: m/z 372.00 [M + H]+; was added into each well of the 96-well plate with a final con-
HRMS: m/z 372.1000 (ESI) calcd for C20H18ClNO4 + H+ centration of 100 µM. Ascorbic acid (100 µM) was used as a
(372.0997).
positive control. The reaction was started by adding 10 µl
Ethyl 6-(benzyloxy)-4-phenylquinoline-2-carboxylate (1r). methanolic solution of DPPH (100 µM) to all the samples.
Pale yellow solid; m.p. 109–110 °C; 1H NMR (CDCl3, 400 MHz): After 30 min incubation while shaking in the dark, absorbance
δ 8.30 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.53–7.50 (m, 5H), was recorded at 517 nm. The DPPH free-radical scavenging
7.45–7.43 (m, 2H), 7.38–7.36 (m, 5H), 5.07 (s, 2H), 4.58–4.52 activity was calculated as percentage inhibition using the
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following formula:
Measurement of redox potential
The electrochemical behaviour of compound 1o (1.0 × 10−3 M)
was investigated by cyclic voltammetry at the platinum elec-
trode in 0.01 M TBAP (tetrabutylammonium perchlorate)/
acetonitrile solution with a scan rate of 50 mV s−1. The cyclic
voltammogram of compound 1o was scanned between the
potential range of 1.2 V to −1.2 V versus Ag/Ag+.31
Inhibition ð%Þ ¼ ðA0 ꢀ A1=A0Þ ꢁ 100
where A0 is the absorbance of the control and A1 is the absor-
bance of the test sample.
Cell culture and treatments
Human colorectal adenocarcinoma LS-180 and human neuro-
blastoma SH-SY5Y cells were purchased from ECACC, England.
LS-180 cells were grown in MEM growth medium and SH-SY5Y
cells were grown in MEM : F12 nutrient medium mixed in the
ratio of 1 : 1. The media for both the cell lines were sup-
plemented with 1% MEM non-essential amino acids along
with 10% FCS, 100 U penicillin G and 100 µg ml−1 of strepto-
mycin. Cells were grown in 5% CO2 at 37 °C with 95% humid-
ity. All the test compounds were dissolved in DMSO for
treatment of either SH-SY5Y or LS-180 cells, while the
untreated control cultures received only the vehicle (DMSO <
0.2%).
Preparation of Aβ 1–42 peptide
Aβ 1–42 was dissolved in water containing 0.1% NH3 to
prepare 1 mM stock. The Aβ peptide solution was further
diluted with the same volume of PBS and the aggregation was
induced by incubating the peptide at 37 °C for 3 days before use.
ROS generation analysis
The ROS scavenging activity of compounds against acute oxi-
dative stress produced by hydrogen peroxide was checked in
differentiated SH-SY5Y cells. Cells were treated with the test
compounds 1o and 1q at 50 or 100 µM 30 minutes prior to the
treatment with 10 µM of Aβ 1–42 for 24 h or 500 µM of H2O2
for 30 min. The cell permeable ROS probe DCFH-DA (10 µM)
was added to each well at the time of treatment with com-
pounds 1o and 1q. The cells were washed once with PBS, tryp-
sinized and resuspended in PBS for the measurement of
fluorescence on a fluorimeter (BioTek SYNERGYMx) at 504/
529 nm. The total level of ROS was calculated by dividing fluo-
rescence with the total protein present in each sample.
Differentiation of SH-SY5Y cells
The SH-SY5Y cells were differentiated into neurons by treat-
ment with 10 µM of retinoic acid for seven days before treat-
ment with test compounds. During differentiation the retinoic
acid concentration was maintained at 10 µM by replacing the
media every 48 h.30
Cell viability assay
The cell proliferation assay was performed in human colorectal
adenocarcinoma LS-180 and human neuroblastoma SH-SY5Y
cells (differentiated with 10 µM of retinoic acid for seven days).
Cells (1 × 104) were seeded into each well of the 96-well micro-
plate for 24 h. Cells were treated with 50 µM of each com-
pound for 24 h. The MTT dye was then added to each well 4 h
prior to the termination of experiment. Formazan crystals were
dissolved in DMSO before recording absorbance at 570 nm.
Cell viability of the untreated control sample was considered
to be 100%, while viability of test samples was calculated
using the following formula:
Neuroprotection assay against hydrogen peroxide
The neuroprotection assay was performed in differentiated
human neuroblastoma SH-SY5Y cells. Briefly, cells (1 × 104)
were seeded into each well of 96-well plate for 24 h. Compounds
1o and 1q were added 30 min prior to the treatment with
100 µM of H2O2 for 24 h. At the end of treatments, cell viability
was analysed by the MTT assay as described under the section
heading Cell viability assay.
Pgp-induction assay
All synthesized compounds were screened for their ability to
induce Pgp using the rhodamine 123 (Rh123) cell exclusion
method. In this method, the Pgp function was evaluated in
terms of rhodamine 123 (Rh123) accumulation and efflux.32
Briefly, the protocol used is as follows: colorectal LS-180 cells
were seeded at a density of 2 × 104 per well of a 96-well
plate and were allowed to grow for the next 24 h. Cells
were further incubated with the test compounds to a final con-
centration of 5 µM and rifampicin (positive control) to a final
concentration of 10 µM in complete media for 48 h. The final
concentration of DMSO was kept at 0.1%. The drugs were
removed and cells were incubated with HANKS buffer for
40 minutes before further incubation with HANKS buffer (con-
taining 10 µM of Rh123 as a Pgp substrate) for 90 minutes. At
the end of Rh123 treatment cells were washed four times with
OD ðtestÞ
% cell viability ¼
ꢁ 100
OD ðcontrolÞ
Direct interaction of compounds with hydrogen peroxide
This assay was performed using the protocol of Beers and
Sizer (1952).25 A standard curve for hydrogen peroxide was gen-
erated using different concentrations (0.0125 to 0.1 M). The
rate of disappearance of hydrogen peroxide in the presence of
compounds 1o and 1q was calculated by measuring optical
density at different time points. The rate of reaction was calcu-
lated using the following formula:
Δ½Aꢂ
Rate of disappearance of hydrogen peroxide ¼ ꢀ
Δt
where Δ[A] is the change in concentration and Δt is the corres- cold PBS followed by cell lysis for 1 h using 200 µl of lysis
ponding change in time.
buffer (0.1% Triton X-100 and 0.2 N NaOH). A total of 100 µl of
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Organic & Biomolecular Chemistry
lysate was used for reading fluorescence of Rh123 at 485/
529 nm. Samples were normalized by dividing fluorescence of
each sample with total protein present in the lysate.
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Data are expressed as mean SD of three independent experi-
ments unless otherwise indicated. The comparisons were
made between control and treated groups or the entire
intragroup using the Bonferroni test through the Instat-2
software. p values *<0.05 were considered significant. p value
*<0.05, **<0.01, ***<0.001.
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Acknowledgements
The authors are thankful to analytical department, IIIM for
NMR, MS and IR analysis of our compounds. AK is thankful to
CSIR for the award of Senior Research Associateship. This
work was supported by CSIR 12th FYP grant # BSC-0205.
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