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Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and evaluation of antibacterial and
antioxidant activity of novel 2-phenyl-quinoline
analogs derivatized at position 4 with aromatically
substituted 4H-1,2,4-triazoles
Donatella Verbanac, Ritu Malik, Mahesh Chand, Khushbu Kushwaha, Monika
Vashist, Mario Matijašić, Višnja Stepanić, Mihaela Perić, Hana Čipčić Paljetak,
Luciano Saso & Subhash C. Jain
To cite this article: Donatella Verbanac, Ritu Malik, Mahesh Chand, Khushbu Kushwaha,
Monika Vashist, Mario Matijašić, Višnja Stepanić, Mihaela Perić, Hana Čipčić Paljetak, Luciano
Saso & Subhash C. Jain (2016): Synthesis and evaluation of antibacterial and antioxidant
activity of novel 2-phenyl-quinoline analogs derivatized at position 4 with aromatically
substituted 4H-1,2,4-triazoles, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.1080/14756366.2016.1190714
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1190714
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
!2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1190714
RESEARCH ARTICLE
Synthesis and evaluation of antibacterial and antioxidant activity of
novel 2-phenyl-quinoline analogs derivatized at position 4 with
aromatically substituted 4H-1,2,4-triazoles
Donatella Verbanac
1
, Ritu Malik
2
, Mahesh Chand
2
, Khushbu Kushwaha
2
, Monika Vashist
2
, Mario Matijas
ˇic
´
1
,
Vis
ˇnja Stepanic
´
3
, Mihaela Peric
´
1
, Hana C
ˇipc
ˇic
´Paljetak
1
, Luciano Saso
4
, and Subhash C. Jain
2
1
University of Zagreb School of Medicine, Center for Translational and Clinical Research, Zagreb, Croatia,
2
Department of Chemistry, University of
Delhi, Delhi, India,
3
Laboratory for Epigenomics, Division of Molecular Medicine, Rud
er Bos
ˇkovic
´Institute, Zagreb, Croatia, and
4
Department of
Physiology and Pharmacology ‘Vittorio Ersparmer’, Sapienza University of Rome, Rome, Italy
Abstract
A set of novel quinolone–triazole conjugates (12–31) were synthesized in three steps in good
yields starting from 2-phenylquinoline-4-carboxylic acid. All the intermediates, as well as the
final 1,2,4-triazolyl quinolines were fully characterized by their detailed spectral analysis utilizing
different techniques such as IR,
1
H NMR,
13
C NMR, and finally mass spectrometry. All the
synthesized compounds were evaluated in vitro for their potential antibacterial activity and
their preliminary safety profile was assessed through cytotoxicity assay. Additionally, six
selected conjugates were evaluated for their antioxidative properties on the basis of density
functional theory calculations, using radical scavenging assay (DPPH) and cellular antioxidant
assay. The reported results encourage further investigation of selected compounds and are
shading light on their potential pharmacological use.
Keywords
Cytotoxicity assays, DFT calculations using
DPPH and CAA, 1,2,4-triazole–quinoline
conjugates
History
Received 17 April 2016
Revised 7 May 2016
Accepted 8 May 2016
Published online 9 June 2016
Introduction
The development of resistance to currently used antibacterial
therapy has obliged the scientists, chemists, and biologists, to
further search for more effective agents with less or no side
effects. This is even more relevant to the present day scenario as
the primary and opportunistic bacterial and/or fungal infections
still continue to escalate with the increased number of immune
compromised patients (mostly due to the diseases such as AIDS,
cancer, and also as a consequence of long-term therapy after
transplants)
1
. This is the reason why extensive research is going
on around the world, to discover novel molecules to fight such
infections.
Despite plenty of research on different heterocyclic molecules,
the azole ring still remains interesting chemical fragment for the
development of novel molecules especially in the antibacterial
and antifungal therapeutic area. This is due to the fact that most of
the existing azole derivatives possessed both bacterial and fungal
static action and can be orally applied owing to favorable
bioavailability. They possess broad spectrum of activities against
most of the yeasts and filamentous fungi
2
.
Among azoles, 1H-1,2,4-triazole derivatives are considered to
be more interesting as they possess important pharmacological
activities such as antifungal
3,4
, antiviral
5
, antioxidant
6–8
, anti-
asthmatic
9
, anticonvulsant
10
, antidepressant
11
, antithyroid
12
, anti-
HIV
13
, anti-inflammatory
14
and anticancer
15–17
.
On the other hand, quinoline derivatives are known to possess
diverse pharmacological properties such as antioxidant
18,19
,
antibiotic
20,21
, cardiovascular
22
, anti-TB
23
, antiplatelet
24
, antic-
ancer
25,26
, receptor antagonists
27
, NK3 receptor antagonists-II
28
,
anti-inflammatory
29
, antimicrobial
30,31
, selective estrogen recep-
tor modulators (SERMs)
32
and protein kinase inhibitor
33
.
Keeping above in mind, we decided to synthesize conjugates of
the above-mentioned moieties to study the biological profile of the
resulting product. Genesis of our chemistry is derived from the fact
that few earlier conjugates of 1,2,4-triazole and quinoline, where
these molecules are linked via oxygen- or amide-containing linkers
in a single frame, are known to possess biological activities like
antimalarial
34
, antimicrobial
35
, anti tubercular
36
, antitumor
37,38
and antiviral
39
. However, the literature data about molecules that
contain both of these bioactive ligands directly linked/bonded to
each other in a single molecular frame are unknown.
As a result of our research programs involving the synthesis of
new bioactive molecules
40–47
, we report herein for the first time
synthesis of a new set of directly coupled quinolone–triazole
conjugates. We also assessed biological activity profiles of these
conjugates, with particular emphasis on their antioxidative
properties.
Methods
Chemistry
All chemicals were purchased from commercial suppliers (Merck,
SD Fine, and Spectrochem) and used as such. Solvents used in
extraction and purification were distilled prior to use. Products
Address for correspondence: Subhash C. Jain. E-mail: jainsc48@
hotmail.com
Downloaded by [Zagreb University] at 00:29 20 June 2016
were purified by column chromatography using silica gel as an
adsorbent. Melting points were determined on an electronic
apparatus and are uncorrected.
1
H and
13
C NMR spectra were
recorded at 400 MHz on a JEOL spectrometer (Tokyo, Japan) and
at 300 MHz on a Bruker spectrometer (Billerica, MA) using
CDCl
3
/DMSO-d
6
as solvent and tetramethylsilane (TMS) as
internal standard. TMS was used as a reference for both
1
H and
13
C NMR spectra. In
1
H NMR abbreviations s, d, dd, t, q, and m
represents singlet, doublet, double doublet, triplet, quartet, and
multiplet respectively. Coupling constants Jvalues are given in
hertz and the chemical shifts are given in . Elemental analyses
were performed on a PerkinElmer Series II, CHNS/O Analyzer
2400 (Waltham, MA). Mass spectra were recorded on JEOL-JMS-
DX303 mass spectrometer (Tokyo, Japan).
General procedure for the synthesis of compounds 3–5
Indol-2,3-dione/5-fluoro-indol-2,3-dione/5-methyl-indol-2,3-dione
(10 g) and NaOH (8.16 g) were stirred together in water (80 mL) in
a round bottom flask. To the reaction mixture, acetophenone
(8.16g) was added and contents refluxed. Reaction was monitored
on thin layer chromatography (TLC) and after its completion the
reaction mixture was cooled and acidified with conc. HCl solution.
The precipitate obtained was collected, washed, and dried to
afford 3–5.
2-Phenylquinoline-4-carboxylic acid (3). White solid. Yield:
14.72 g, 87%, m.p. 208–210 C. IR (KBr)
max
: 3442, 2465, 1953,
1705, 1601, 1550, 1448, 1354, 1259, 1204, 1082, 894, 781, 760,
732, 699 cm
1
.
1
H NMR (, DMSO-d
6
, 400 MHz): 11.38 (brs,
1H, –COOH), 8.79 (d, 1H, J¼8.80 Hz), 8.47 (s, 1H), 8.29–8.27
(m, 2H), 8.19 (d, 1H, J¼8.80 Hz), 7.85–7.66 (m, 2H), 7.59–7.52
(m, 3H).
13
C NMR (, DMSO-d
6
, 100 MHz): 167.50, 155.73,
148.50, 138.01, 137.03, 129.64, 129.56, 128.64, 127.28, 126.97,
125.39, 123.61, 119.28. Mass Spectral data, TOF MS ES+ m/z
(%): 250 (M
+
+1). Anal. Calcd for C
16
H
11
NO
2
: C, 77.10; H, 4.45;
N, 5.62. Found: C, 77.14; H, 4.42; N, 5.60.
General procedure for the synthesis of compounds 6–8
Prepared compound 3/4/5 (10 g) was added to abs. ethanol
(150 mL) in a flask, followed by conc. H
2
SO
4
(5 mL). The
resulting reaction mixture was refluxed and completion of
reaction was monitored by TLC. The reaction mixture was
cooled and poured over crushed ice in a beaker. The resulting
contents were rendered alkaline by adding sufficient amount of
ammonia solution. The mixture was then extracted thrice with
diethyl ether. The combined ethereal solution was dried over
anhydrous sodium sulfate and the solvent was removed by
distillation to get the desired compounds 6/7/8.
Ethyl 2-phenylquinoline-4-carboxylate (6). Yellow color oil.
Yield: 9.90 g, 89%. IR (film)
max
: 2983, 1724, 1623, 1594, 1513,
1338, 1248, 1230, 1193, 1143, 1119, 1037, 827, 749 cm
1
.
1
H
NMR (, CDCl
3
, 400 MHz): 8.73 (d, 1H, J¼8.72 Hz), 8.38 (s,
1H), 8.23–8.19 (m, 3H), 7.76 (t, 1H, J¼7.32 Hz), 7.62 (t, 1H,
J¼7.32 Hz), 7.56–7.52 (m, 3H), 4.53 (q, 2H, –COOCH
2
CH
3
),
1.50 (t, 3H, –COOCH
2
CH
3
).
13
C NMR (, CDCl
3
, 100 MHz):
166.45, 156.72, 149.22, 138.84, 136.07, 130.28, 129.85, 129.68,
128.91, 127.71, 127.46, 125.38, 123.98, 120.19, 61.90, 14.32.
Mass Spectral data, TOF MS ES+ m/z (%): 278 (M
+
+1). Anal.
Calcd for C
18
H
15
NO
2
: C, 77.96; H, 5.45; N, 5.05. Found: C,
78.02; H, 5.48; N, 5.02.
General procedure for the synthesis of compounds 9–11
A mixture of compounds 6–8(5 g) and hydrazine hydrate
(1.31 mL) was heated at 50–60 C temperature with constant
stirring. The solid that separated out on cooling was filtered and
crystallized to give their corresponding carbohydrazide 9–11.
2-Phenylquinoline-4-carbohydrazide (9). White crystalline
solid. Yield: 4.46 g, 94%, m.p. 214–216 C. IR (KBr)
max
:
3385, 2917, 2366, 1701, 1606, 1483, 1449, 1360, 1287, 1246,
1229, 1149, 1039, 932, 889, 830, 754 cm
1
.
1
H NMR (, DMSO-
d
6
, 400 MHz): 10.03 (s, 1H, –NH), 8.31 (d, 1H, J¼7.8 Hz), 8.25–
8.23 (m, 2H), 8.14 (m, 1H), 8.03 (m, 1H), 7.78–7.74 (m, 2H),
7.60–7.48 (m, 3H), 4.52 (brs, 2H, –NH
2
).
13
C NMR (, DMSO-
d
6
, 100 MHz): 166.35, 155.71, 147.92, 140.76, 138.24, 129.55,
129.47, 129.43, 128.36, 127.02, 126.69, 125.05, 123.36, 116.84.
Mass Spectral data, TOF MS ES+ m/z (%): 264 (M
+
+1). Anal.
Calcd for C
16
H
13
N
3
O: C, 72.99; H, 4.98; N, 15.96. Found: C,
73.04; H, 5.01; N, 15.97.
General procedure for the synthesis of compounds 12–26
The synthesized carbohydrazides 9–11 (200 mg) and substituted
benzaldehyde (92 mg) were dissolved in 10 mL of glacial acetic
acid in a round bottom flask. To the mixture, ammonium acetate
(84 mg) was added. The reaction mixture was stirred for a period
of 6–8 h at room temperature. The progress of the reaction was
monitored by TLC. After completion, the reaction mixture was
poured into ice-cold water and neutralized with ammonia. The
precipitated product was filtered, washed with water, and
crystallized from chloroform/methanol to give the desired
product.
2-Phenyl-4-[5-(4-hydroxyphenyl)-4H-[1,2,4]-triazol-3-
yl]quinoline (13). Cream-colored solid. Yield: 254 mg, 92%, m.p.
270–272 C. IR (KBr)
max
: 3257, 2925, 1654, 1606, 1585, 1545,
1513, 1365, 1267, 1235, 1166, 845, 766 cm
1
.
1
H NMR
(, DMSO-d
6
, 400 MHz): 12.08 (s, 1H, 4NH,D
2
O exchangeable),
10.06 (s, 1H, –OH,D
2
O exchangeable), 8.36–8.33 (m, 2H), 8.27–
8.25 (m, 2H), 8.20–8.16 (m, 3H), 7.85 (m, 1H), 7.67 (d, 2H,
J¼7.1 Hz), 7.57 (d, 2H, J¼7.5 Hz), 6.88 (d, 2H, J¼7.8 Hz).
13
C
NMR (, DMSO-d
6
, 100 MHz): 162.56, 159.71, 155.77, 149.14,
147.89, 145.17, 143.53, 141.46, 138.05, 130.34, 129.96, 129.60,
128.91, 127.15, 125.13, 123.47, 117.14, 115.79. Mass spectral
data, TOF MS ES+ m/z (%): 365 (M
+
+1). Anal. Calcd for
C
23
H
16
N
4
O: C, 75.81; H, 4.43; N, 15.38. Found: C, 75.94; H,
4.48; N, 15.39.
General procedure for the synthesis of compounds 27–31
The synthesized carbohydrazides 9–11 (200 mg) and heterocyclic
aldehyde (78 mg) were dissolved in 10 mL of glacial acetic acid in
a round bottom flask. To the mixture, ammonium acetate (84 mg)
was added. The reaction mixture was stirred for a period of 6–8 h
at room temperature. The progress of the reaction was monitored
by TLC. After completion of the reaction, the reaction mixture
was poured into ice-cold water and neutralized with ammonia.
The precipitated product was filtered, washed with water, and
crystallized from chloroform/methanol to give the desired
product.
2-Phenyl-4-[5-(furan-2-yl)-4H-[1,2,4]-triazol-3-yl]quinoline
(27). White solid. Yield: 230 mg, 88%, m.p. 198–200 C. IR
(KBr)
max
: 3182, 3052, 2925, 1655, 1623, 1591, 1544, 1349,
1284, 1270, 1158, 938, 765 cm
1
.
1
H NMR (, DMSO-d
6
,
400 MHz): 12.16 (s, 1H, 4NH,D
2
O exchangeable), 8.34–8.30
(m, 2H), 8.25–8.22 (m, 2H), 8.15 (m, 1H), 7.85–7.82 (m, 2H),
7.67 (m, 1H), 7.56–7.52 (m, 3H), 6.97 (m, 1H), 6.56 (m, 1H).
13
C NMR (, DMSO-d
6
, 100 MHz): 162.94, 155.65, 148.96,
147.98, 144.48, 140.65, 138.37, 129.77, 129.45, 128.48, 126.88,
126.77, 123.67, 123.26, 124.87, 119.21, 116.87. Mass spectral
data, TOF MS ES+ m/z (%): 339 (M
+
+1). Anal. Calcd for
C
21
H
14
N
4
O: C, 74.54; H, 4.17; N, 16.56. Found: C, 74.60; H,
4.20; N, 16.58.
2D. Verbanac et al. J Enzyme Inhib Med Chem, Early Online: 1–7
Downloaded by [Zagreb University] at 00:29 20 June 2016
In vitro biological screening
Antibacterial activity assay
Bacterial strains, Staphylococcus aureus (American Type Culture
Collection (Manassas, VA; ATCC), 29213), Streptococcus
pneumoniae (ATCC, 49619), Streptococcus pyogenes (ATCC,
700294), Haemophilus influenzae (ATCC, 49247), and
Escherichia coli (ATCC, 25922), were purchased from ATCC
and utilized to evaluate antibacterial activity of compounds.
Antibacterial activity was determined by the standard broth
microdilution method with azithromycin as comparator.
Minimum inhibitory concentrations (MICs) were established
according to guidelines of the Clinical Laboratory Standards
Institute
48
with the exception that lysed blood was substituted by
5% horse serum for Streptococcus medium. Double dilutions of
tested compounds were prepared in 128–0.5 mg/mL concentration
range within microplate wells. Bacteria were grown on appropri-
ate agar plates (Becton Dickinson, Franklin Lakes, NJ). Inocula
were prepared by direct colony suspension method and micro-
plates were inoculated with 5 104 CFU/well. Results were
determined by visual inspection after 20-h incubation at 37 C.
Cytotoxicity assays
A549 human lung adenocarcinoma cell line (ATCC, CCL-185),
HepG2 human hepatocellular carcinoma cell line (ATCC, HB-
8065), MDA-MB-231 human breast adenocarcinoma cell line
(ATCC, HTB-26), PC-3 human prostate adenocarcinoma cell line
(ATCC, CRL-1435), and THP-1 human acute monocytic leuke-
mia cell line (ATCC, TIB-202) were purchased from ATCC. Cell
lines were maintained in complete DMEM/F12 medium (Sigma-
Aldrich Chemie GmbH, Taufkirchen, Germany; D8437), or
complete RPMI1640 (Sigma, R7388) for THP-1 cells, supple-
mented with 10% Fetal Bovine Serum (Sigma, F7524) at 37 Cin
5% CO
2
atmosphere.
Cytotoxicity assay was performed using MTS Cell Titer 96
AQueous One Solution Cell Proliferation Assay (Promega
Corporation, Madison, WI; G3580)
49
. Double dilutions of tested
compounds were prepared in 100–0.2 mM concentration range
within microplate wells. 5 104 cells were added per well and
incubated overnight at 37 Cin5%CO
2
atmosphere. 15 mL of MTS
reagent was dispensed per well and plates incubated for 1–4 h at
37 Cin5%CO
2
atmosphere. The absorbance was recorded at
490 nm using Wallac Victor2 microplate reader (PerkinElmer Life
and Analytical Sciences, Turku, Finland). Results were analyzed in
GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).
DPPH-free radical scavenging assay
The DPPH (1,1-diphenyl-1,2-picryl hydrazyl) (Sigma-Aldrich
Chemie GmbH, Taufkirchen, Germany) method was used to
determine the free radical scavenging activity of compounds
50
.
Dilutions of tested compounds and ascorbic acid as a standard
antioxidant comparator were prepared in 1000–1 mg/mL con-
centration range. 1 mL of compound solution was added to
1 mL of freshly prepared DPPH solution (3.9 mg/50 mL
ethanol) and the reaction mixture incubated in the dark at
room temperature for 20 min. Absorbance (A) was measured at
517 nm using Analytik Jena UV Winaspect Specrod PC
250 spectrophotometer (Analytic Jena AG, Jena, Germany).
Inhibition of the DPPH radical by the compounds was
calculated according to the following formula:
DPPH scavenging activity %ðÞ¼A0 A1ðÞ=A0½100
Where A0 is the absorbance of the control and A1 is the
absorbance of the sample. The results are averages of three
measurements. The EC
50
value, compound concentration to reduce
50% of the DPPH, was calculated using GraphPad Prism software.
Cellular antioxidant activity (CAA) assay
OxiSelect Cellular Antioxidant Assay Kit (Cell Biolabs Inc., San
Diego, CA; STA-349) was used to assess antioxidant activity of
the compounds within a cell in a standard cell culture environ-
ment. The assay employs cell-permeable fluorogenic probe 20,70-
dichlorodihydrofluorescein diacetate (DCFH-DA), which is
diffused into cells, deacetylated by cellular esterases and oxidized
by free radicals to fluorescent 20,70-dichloro-dihydrofluorescein
(DCF), with the fluorescence intensity being proportional to the
reactive oxygen species levels within the cell cytosol
51
. The assay
was performed according to manufacturer’s instructions. In brief,
HepG2 cells were seeded at 5 104 per well in 96-well black
microplates and incubated overnight at 37 Cin5%CO
2
atmosphere. The medium was removed and wells washed with
sterile phosphate-buffered saline (PBS). Double dilutions of
compounds were prepared in 500–8 mM concentration range in
cell medium (50 mL) and DCFH-DA probe was added to wells
(50 mL). The cells were incubated 1 h at 37 Cin5%CO
2
atmosphere. After the medium was removed and cells washed
with sterile PBS, free radical initiator solution was added to all
wells (100 mL) and fluorescence read using a fluorescence
microplate reader (Victor 2 Wallac, PerkinElmer Life and
Analytical Sciences, Turku, Finland) (excitation 480 nm/emission
530 nm). The readouts were saved in increments of 15 min for a
total of 180 min. Results were analyzed in GraphPad Prism
software and quantified as IC
50
values.
Density functional theory (DFT) calculations
The reaction enthalpies BDE, IP, PA, and ETE and corresponding
free energies (Table 2 in Supplementary Material) were calculated
by a known method
52
. These reaction parameters were calculated
by applying the DFT model (U)B3LYP/6-31 + G(d, p) as
implemented in software Gaussian 03 (Gaussian, Inc.,
Wallingford, CT)
53
. Calculations were performed for the gas
and aqueous phases. Equilibrium geometries in unionized and
anionic closed-shell ground electronic states as well as of
corresponding radical and radical cation open-shell doublet
ground electronic states were fully optimized in the gas phase.
The minima were confirmed by no imaginary vibrational
frequencies at temperature of 298.15 K and pressure of 1 atm.
The free energies of solvation DG*hyd for all studied molecular
species at 1 M standard state in water were determined at the gas
phase equilibrium geometries by using integral equation formal-
ism of polarizable continuum model (IEFPCM) of solvation with
Bondi radii and tight SCF convergence criterion
54
. The free
energies (Table 2 in Supplementary Material) were calculated by
employing corresponding thermodynamic cycles
55
. Lipophilicity
values logP were calculated by OpenBabel
56
.
Results and discussion
Chemistry
The synthetic route employed for the preparation of a set of novel
quinoline-triazole conjugates (12–31) is shown in Scheme 1.
2-Phenylquinoline-4-carboxylic acid (3) was converted into its
ethyl ester 6by using absolute ethanol in the presence of
concentrated sulfuric acid. The ester was purified and characterized
by its IR spectrum which showed characteristic absorption band at
1724 cm
1
for ester group. Its
1
H NMR spectrum displayed signals
at 1.50 (t, 3H) for –COOCH
2
CH
3
and at 4.53 (q, 2H) for
–COOCH
2
CH
3
. Compound 6on treatment with hydrazine hydrate
gave 2-phenylquinoline-4-carbohydrazide (9), which exhibited
DOI: 10.1080/14756366.2016.1190714 Antibacterial and antioxidant activity of novel 2-phenyl-quinoline analogs 3
Downloaded by [Zagreb University] at 00:29 20 June 2016
characteristic absor ption band at 3385 cm
1
for NHNH
2
stretching
in its IR spectrum. Further its
1
H NMR did not show any signal
corresponding to the ester group indicating thereby complete
conversion. Final confirmation of structure of compound 9came
from its mass spectrum, which showed M
+
+1 signal at m/z 264. The
hydrazide obtained was cyclocondensed with 4-hydroxybenzalde-
hyde in the presence of ammonium acetate in glacial acetic acid at
room temperature to finally give a cream-colored solid compound
characterized as 13 (Scheme 1). Its
1
H NMR spectrum showed two
characteristic D
2
O exchangeable broad signals at 12.08 and
10.06 integrating for one proton each, indicating the presence4NH
and –OHprotons, respectively. In IR spectrum, absorption band at
1654 cm
1
indicated the presence of imido bond (C¼N) which was
further supported by the absence of –CH¼N-proton in
1
H NMR
spectrum thus confirming that cyclization has taken place. The other
protons of the phenyl group and quinoline skeleton were observed at
8.36–8.33 (m, 2H), 8.27–8.25 (m, 2H), 8.20–8.16 (m, 3H), 7.85
(m, 1H), 7.67 (d, 2H, J¼7.1 Hz), 7.57 (d, 2H, J¼7.5 Hz), 6.88 (d,
2H, J¼7.8 Hz). The
13
C NMR spectrum showed all expected
characteristic signals. On the basis of above analysis, and mass
spectrum compound 13 was characterized as 2-phenyl-4-[5-(4-
hydroxyphenyl)-4H-[1,2,4]-triazol-3-yl]quinoline. Similar set
of the above-described reactions were repeated with substituted
aryl and hetero aromatic ring aldehydes to obtain corresponding
desired compounds.
Biological activity
All final products (12–31) have been profiled in vitro, in terms of
their antibacterial activity and cytotoxicity. The cytotoxicity of a
compound is closely associated with potential adverse effects on
particular cells, tissues, or organs of drugs intended for human use.
The antibacterial activity was determined against five different
bacterial species: two gram-negative (E. coli,H. influenzae) and
three gram-positive (S. aureus,S. pneumoniae,S. pyogenes).
However, neither of the compounds showed substantial and
significant antibacterial activity (Supplementary Table S1).
Although molecules containing quinoline and 4H-1,2,4-triazole
fragments are often recognized in vitro as potent antibacterials
57
this has not to be general case
58
. In our case for 2-phenyl 4H-
1,2,4-triazole substituted quinolines, no antibacterial activity has
been detected. This may be either due to the specific unfavorable
structural factors such as site of substitution
57
or unfavorable
physicochemical properties
59
.
N
H
O
NN
N
COOH
COCH3COOC2H5
Ph
CONHNH2
Ph
Ph
+
RRR
R
O
ab
c
3 R = H
4 R = F
5 R = CH3
6 R = H
7 R = F
8 R = CH3
9 R = H
10 R = F
11 R = CH3
12
NPh
R
12 R = H; R1 = H; R2 = OH; R3 = OCH3
13 R = H; R1 = R3 = H; R2 = OH
14 R = H; R1 = R3 = H; R2 = F
15 R = R1 = R2 = R3 = H
16 R = F; R1 = H; R2 = OH; R3 = OCH3
17 R = F; R1 = R3 = H; R2 = OH
18 R = F; R1 = R3 = H; R2 = F
19 R = F; R1 = R2 = R3 = H
20 R = CH3; R1 = H; R2 = OH; R3 = OCH3
21 R = CH3; R1 = R3 = H; R2 = OH
22 R = CH3; R1 = R3 = H; R2 = F
23 R = CH3; R1 = R2 = R3 = H
24 R = H; R1 = H; R2 = R3 = OH
25 R = F; R1 = H; R2 = R3 = OH
26 R = CH3; R1 = H; R2 = R3 = OH
12-26
NH
N
N
R1R2
R3
dd
NPh
R
27 R = H; X = O
28 R = H; X = S
29 R = F; X = O
30 R = F; X = S
31 R = CH3; X = O
NH
N
N
X
27-31
Scheme 1. Synthesis of quinolone–triazole conjugates (12nju). Reagents and conditions: ai ¼NaOH, H
2
O, ref lux, 4–6 h; b–¼abs. C
2
H
5
OH, conc.
H
2
SO
4
, ref lux, 18–20 h; c8 ¼N
2
H
4
H
2
O, heat, 50–60 C, 4–6 h; d–¼aldehyde, glc. CH
3
COOH, CH
3
COONH
4
, 6–8 h at room temperature.
4D. Verbanac et al. J Enzyme Inhib Med Chem, Early Online: 1–7
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The antibacterials are generally hydrophilic molecules, while
these derivatives are very lipophilic with clogP values around 5 or
higher. Absence or very weak of antibacterial activity generally
indicates the potential of specific compounds for their long-term
use, for example as anti-inflammatory agents in the treatment of
chronic disease, without potential risks to induce resistance.
The cytotoxic effect of compounds was evaluated on five
different human cell lines (A549, HepG2, MDA-MB-231, PC-3,
and THP-1) using MTS test (Table 1). The test quantifies
metabolic activity of cells by measuring their metabolism through
the released NADH levels, thus indicating whether a compound
impairs any of the key cellular metabolic pathways.
The cytotoxicity of a compound is closely associated with its
potential adverse drug effects. In addition, MTS test is very often
used for the estimation of antiproliferative capacity of a
compound when followed through longer period of time. The
results of compound cytotoxicity screening, here performed
during 24 h, are shown in Table 1. 5-Aryl 4H-1,2,4-triazolyl
compounds 13,17,21, and 26 displayed significant cytotoxic
effect on THP-1 cell line. Derivatives 25 as well as 27–30 showed
weak cytotoxic effect on THP-1 cells. Only compounds 17 and 26
were additionally cytotoxic for HepG2 cell line. With respect to
these data, all the other compounds could be considered suitable
for further in vitro profiling in cellular assays.
The cytotoxicity of the active compounds toward the most
sensitive THP-1 cell-line is determined by substituents at position
5 of the triazole ring. The observed cytotoxic activity may be
attributed to the presence of hydrogen-bond accepting centers at
specific positions in these substituents, the chemical feature that is
absent in the inactive analogs.
In addition, 5-aryl 4H-1,2,4-triazolyl derivatives which mod-
erately inhibited metabolic activity in the most sensitive cell line
(THP-1) are (poly)phenolic compounds. It is also well known that
many polyphenols possess antioxidant features since they can
directly scavenge-free radicals by donating H-atom and/or
modulate activities of various proteins included directly or
indirectly in free radical production
60
. Therefore, some of the 5-
aryl 4H-1,2,4-triazolyl derivatives have been tested for their
antioxidant activity.
At first, assuming that free hydroxyl (OH) group of the active
compounds (Table 2) can donate hydrogen atom to a free radical,
the radical scavenging activity of these derivatives was estimated
in silico by using common approach
52
. Derivatives 12,16, and 20
containing guaiacyl-like group were also included in computa-
tions. Values of the calculated parameters gas-phase bond
dissociation enthalpy (BDEg) and aqueous bond dissociation
free energy (BDFEaq) were used for comparing radical scaven-
ging capacities of our polyphenolic derivatives mutually as well
as with corresponding natural polyphenols (Table 2). As expected,
compounds 24,25, and 26 with catechol fragment have stronger
radical scavenging ability than compounds 13,17, and 21 with
para-phenyl substituent and compounds 12,16, and 20 with
guaiacyl-like moiety
52
. Compounds within each of these three
subgroups have mutually similar radical scavenging capacities.
Additionally, the order of radical scavenger capacities of the three
subgroups corresponds to the order of the natural polyphenols
quercetin, apigenin, and tamarixetin containing analogous poly-
phenolic fragments (Table 2). The obtained density functional
theory (DFT) results indicate that for our compounds, the radical
scavenging activities of (poly)phenolic fragments are quite
independent on the rest of their structures.
The DPPH assay provides an easy and rapid in vitro method
commonly used to evaluate antioxidant activity of natural plant
Table 2. Comparison of calculated gas-phase (g) bond dissociation enthalpies (BDE) and corresponding aqueous
(aq) free energies (all given in kJ/mol) as well as estimated lipophilicity values logP of the 5-aryl-4H-1,2,4-triazolyl
derivatives and natural polyphenols. Experimentally determined radical scavenging (DPPH assay) and cellular
antioxidant (CAA) activities are also presented.
Compounds BDE
g
BDFE
aq
logP IC
50
(DPPH) (mg/mL) IC
50
(CAA) (mmol/mL)
12 342.7 335.8 5.07 198.1 4500
16 (F) 343.6 334.8 5.21 77.68 4500
20 (Me) 340.6 341.0 5.38 9.35 4500
13 342.2 324.0 5.06 NA NA
17 (F) 343.2 323.7 5.20 NA NA
21 (Me) 341.6 322.9 5.37 NA NA
24
a
305.9 306.9 4.77 5.61 114.4
25 (F) 306.8 307.8 4.90 3.57 100.9
26 (Me) 305.4 306.6 5.07 6.00 405.4
Tamarixetin 346.9 332.8 2.29
Apigenin 349.3 327.7 2.58
Quercetin 310.3 294.4 1.99 31
Ascorbic acid 9.67
a
For compounds with the catechol moiety, the calculated data for more active para-OH group are listed.
Table 1. The results of compound cytotoxicity screening expressed as
IC
50
values in mM.
Compounds HepG2 THP-1 A549 MDA-MB-231 PC-3
12 4100 4100 4100 4100 4100
13 4100 37 / / /
14 4100 4100 / / /
15 4100 4100 / / /
16 4100 4100 4100 4100 4100
17 95 37 / / /
18 4100 4100 / / /
19 4100 4100 / / /
20 4100 4100 4100 4100 4100
21 4100 45 / / /
22 4100 4100 / / /
23 4100 4100 / / /
24 4100 4100 4100 4100 4100
25 4100 78 4100 4100 4100
26 55 39 4100 4100 4100
27 4100 87 / / /
28 4100 95 / / /
29 4100 76 / / /
30 4100 70 / / /
31 4100 4100 / / /
Staurosporine 3,18 0,2 / / /
DOI: 10.1080/14756366.2016.1190714 Antibacterial and antioxidant activity of novel 2-phenyl-quinoline analogs 5
Downloaded by [Zagreb University] at 00:29 20 June 2016
extracts and chemical compounds which act as free radical
scavengers. The activity of six selected compounds assessed using
the DPPH assay demonstrates considerable radical scavenging
activity (Table 2). Ascorbic acid was used as a comparator in this
assay, yielding IC
50
value of 9.67 mg/mL. Four compounds
displayed radical scavenging capabilities comparable to ascorbic
acid, with the highest DPPH-scavenging activity shown by
compound 25 (IC
50
value of 3.57 mg/mL).
There are many chemical assays used to quantify radical
scavenging activity of compounds. However, their efficacy to
predict in vivo antioxidant activity is modest since they do not
address critical issues such as uptake, distribution, and metabol-
ism of antioxidants which may significantly impact their
bioavailability, stability, and tissue retention. In addition, in
such a way, it is not possible to assess indirect antioxidant
activities of compounds attainable in the cell
60
. With respect to all
this, cellular antioxidant assay (CAA) serves as a more biologic-
ally relevant method for assessing the antioxidant activity of
compounds in vivo. CAA was employed to test antioxidant
activity of 6 selected compounds in HepG2 cell line (Table 2).
Catecholic compounds 24 and 25 displayed considerable antioxi-
dant activity in cellular system with IC
50
values of 114 mM and
101 mM, respectively. Quercetin was used as a comparator in this
assay exhibiting IC
50
value of 31 mM.
Conclusions
The novel quinolone–triazole conjugates were synthesized in
three steps starting from 2-phenylquinoline-4-carboxylic acid.
The acid was converted into carbohydrazide derivative via its
ethyl ester. Carbohyrazide on cyclocondensation using ammo-
nium acetate in acetic acid in the presence of aryl/hetero aryl
aldehyde gave finally the target molecules. These novel
compounds were evaluated in vitro for their potential antibac-
terial activity. No significant antibacterial activity has been
observed, qualifying these compounds for additional develop-
ment as promising leads in other therapeutic areas where
chronic, long-term application is required. Moreover, when
their preliminary safety profile was assessed through cytotox-
icity assays on five different cell lines, we have seen potential
for further cell-based assays profiling since only moderate
inhibition of cell-metabolism activity has been observed on the
most sensitive THP-1 cell line. And finally, based on DFT
calculations, six selected conjugates were evaluated for their
antioxidative properties on radical scavenging assay (DPPH)
and CAA. A plausible way to increase antibacterial activity
would be to decrease lipophilicity. For example, by synthetizing
derivatives without phenyl ring at position 2 (which will reduce
clogP by 2 or 3 units). However, his approach needs to be
further investigated.
Nevertheless, the obtained results in all these assays are
advocating in terms that additional synthesis of new derivatives
and further investigations in this therapeutic area might provide
interesting and potentially promising results that can finally be
applied for enriching our knowledge and experience in the
development of new chemical leads with this specific biological
activity.
Acknowledgements
M.C. and M.V. are thankful to UGC and K.K. to CSIR New Delhi (India)
for research fellowships. Additionally, V.S. is grateful to the University of
Zagreb Computing Centre SRCE for supporting the computer cluster
where computations were done.
Declaration of interest
The authors declare no conflict of interest.
This work was supported by the research grants received from
the University of Delhi, India (number DRCH/R&D/2010-13) and
from the Croatian Science Foundation, Croatia (number 5467).
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Supplementary material available online
DOI: 10.1080/14756366.2016.1190714 Antibacterial and antioxidant activity of novel 2-phenyl-quinoline analogs 7
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