Synthesis and biological evaluation of a new series of 4-alkoxy-2-arylquinoline derivatives as potential antituberculosis agents

Article abstract of DOI:10.3906/kim-1501-112

Three new series of 33 quinolone compounds, 2-(2-, 3-, and 4-fluorophenyl)-4-O-alkyl(C_5-15)quinolines (7a-k, 8a-k, and 9a-k), were synthesized from 2-(2-, 3-, and 4-fluorophenyl)-2,3-dihydroquinolin-4(1H )-one (4, 5, and 6) by the reaction of a

Full text of DOI:10.3906/kim-1501-112

Turk J Chem
Turkish Journal of Chemistry
(2015) 39: 850 – 866
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1501-112
Research Article
Synthesis and biological evaluation of a new series of 4-alkoxy-2-arylquinoline
derivatives as potential antituberculosis agents
1
Gonca TOSUN , Tayfun ARSLAN , Zeynep ISKEFIYELI , Murat KUC¸ UK
2
1
1,3
˙
˙
˙
¨
¨
,
4
5,∗
˘
S¸engu¨l ALPAY KARAOGLU , Nurettin YAYLI
1Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey
2Department of Chemistry, Faculty of Science, Giresun University, Giresun, Turkey
3Faculty of Engineering and Natural Sciences, Gu¨mu¨¸shane University, Gu¨mu¨¸shane, Turkey
4Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdo˘gan University, Rize, Turkey
5Faculty of Pharmacy, Karadeniz Technical University, Trabzon, Turkey
Received: 26.01.2015
•
Accepted/Published Online: 09.05.2015
•
Printed: 28.08.2015
Abstract: Three new series of 33 quinolone compounds, 2-(2-, 3-, and 4-fluorophenyl)-4-O-alkyl(C5−15)quinolines (7a–
k, 8a–k, and 9a–k), were synthesized from 2-(2-, 3-, and 4-fluorophenyl)-2,3-dihydroquinolin-4(1H )-one (4, 5, and
6) by the reaction of alkyl halides under basic conditions in DMF. The new compounds 7a–k, 8a–k, and 9a–k were
synthesized from flavonones 4–6, which can be considered new precursors for quinoline synthesis through a one-step
reaction. All the target compounds (7a–k, 8a–k, and 9a–k) were evaluated for their in vitro antimicrobial activity
against nine test microorganisms. They showed the most activity against Mycobacterium smegmatis with minimum
inhibitory concentrations (MIC) of 62.5–500 µg/mL, indicating their potential uses as antituberculosis agents. Among
them 8a–k (m-fluoride) were the most active compounds against M. smegmatis (MIC, 62.5–125 µg/mL). The newly
synthesized title compounds were also evaluated for their in vitro antioxidant activities using DPPH• radical scavenging
and FRAP tests. They showed at a low concentration (mg/mL) a range of SC50 values of 0.03–12.48 mg/mL (DPPH•)
and 0–722 µM (FRAP), respectively. The antioxidant results of compounds 7a–k, 8a–k, and 9a–k revealed that the
length of the alkyl chain was negatively correlated with antioxidant capacity.
Key words: Quinoline derivatives, flavonones, air oxidation, antimicrobial activity, antituberculosis activity, antioxidant
activity
1. Introduction
Natural, synthetic, semisynthetic, or natural product-derived alkaloid compounds are important sources of
new drugs and have a variety of biological activities in clinical trials. Naturally occurring quinolines have
been identified and reported to possess a high degree of various biological activities.1,2 Graveoline3 and
chimanine4,5 are alkaloids isolated from Ruta graveolens L. and Galipea longiflora, respectively, and showed
comprehensive pharmacological activities such as antibacterial and antitumor activities.6−11 Tumor angiogenesis
is a promising target of cancer therapy. A series of graveoline derivatives has been synthesized and tested for
their antiangiogenesis activities.3
The quinoline core has been synthesized previously by various conventional strategies such as Skraup,12
∗Correspondence: yayli@ktu.edu.tr
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Friedlander,13,14 Pfitzinger,15 and Pavarov.16 These classical synthetic methods are still frequently used for
the preparation of quinolines. However, in this work, a new and practical method was used for the synthesis
of substituted quinoline derivatives. The reaction sequence consists of an initial Aldol condensation (1–3) and
then intramolecular Michael addition of amines to an α,β -unsaturated carbonyl group using K-10 clay under
solvent-free conditions using a microwave to give compounds 4–6, and finally air oxidation of compounds 4–6
with alkyl halide under basic conditions afforded the target compounds 7a–k, 8a–k, and 9a–k.
Heterocyclic systems with a quinoline are widely used in medicinal chemistry17 and display many different
biological activities such as antiparasitic,18 antibacterial,2 cytotoxic and antineoplastic,19 antimycobacterial,20
and antiinflammatory activities.21 The biological activities of quinolin-4(1H)-one moiety depend on the bicyclic
heteroaromatic pharmacophore as well as on the peripheral substituents and their spatial relationship.
A
number of 2-phenylquinolone derivatives with a phenyl group attached to the C-2 position of quinolin-4(1H)-
one have expressed antimitotic activity.22 In spite of their wide range of pharmacological activities, very few
activity studies have been reported against tuberculosis for these group of compounds in comparison with
other classes.23 Tuberculosis is one of the most important diseases worldwide, with approximately three million
deaths per year.24 Tuberculosis is a problematic disease especially with respect to the ease of the spread of HIV
infection and the increases in the prevalence of drug resistance as well as multidrug-resistant strains.25 New
synthetic compounds are certainly required for the long-term control of tuberculosis.
On the basis of these observations and as a part of our continued research for new antimicrobial and
antioxidant agents, we report an efficient and simple method for the synthesis of 33 new series of quinolines
derivatives 2-(2-, 3-, 4-fluorophenyl)-4-O-alkyl quinolines with an increasing number of carbons (C5 –C15) in
the side chain. Thus, we wanted to determine the influence of the length of the carbon chain in the O-alkyl
substituent of the synthetic compounds 7a–k, 8a–k, and 9a–k. The antimicrobial (antibacterial, antifungal,
and antituberculosis) and antioxidant activities were also evaluated for the synthetic compounds 4–9.
2. Results and discussion
2.1. Chemistry
Many synthetic methods for quinoline synthesis have been used in the literature.12−16,26,27 However, many
of these classical synthetic approaches suffer from a limited source of precursors, harsh reaction conditions,
and low yields and selectivity. We first synthesized substituted quinoline starting from flavonone and alkyl
halide at room temperature, which was a single step process and which was a practical method for the
synthesis of substituted quinoline derivatives. This method can be used for naturally occurring or syn-
thetically prepared substituted quinolines starting from flavonones. The reaction sequences used for the
synthesis of the target compounds (7a–k, 8a–k, 9a–k) are outlined in the Scheme. Flavonones of 2-(2-
fluorophenyl),2,3-dihydroquinolin-4(1H )-one (4), 2-(3-fluorophenyl),2,3-dihydroquinolin-4(1H )-one (5), and 2-
(4-fluorophenyl),2,3-dihydroquinolin-4(1H )-one (6) were synthesized through the cyclization of the correspond-
ing (2E)-1-(2-aminophenyl)-3-(2-fluorophenyl)prop-2-en-1-one (1), (2E)-1-(2-aminophenyl)-3-(3-fluorophenyl)prop-
2-en-1-one (2), and (2E)-1-(2-aminophenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3), respectively, using K-10 clay
under solvent-free conditions using a microwave at 85 ◦ C (Scheme). Then compounds 4, 5, and 6 were dis-
solved in DMF and treated with KOH and alkyl halides (C5 -C15 -Br). The reaction mixture was stirred at room
temperature overnight and then was treated with 20 mL of distilled water and extracted with CH2 Cl2 . The
crude residue was purified by silica gel column chromatography to afford compounds 7a–k, 8a–k, and 9a–k in
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moderate yields (27%–51%). The reaction progress was monitored using thin layer chromatography. In order
to improve the yield, the reactions were carried out at higher temperatures, but the flavonone ring was opened
and N-alkyl derivatives of compounds 1, 2, and 3 occurred.
Scheme. Synthesis of the 4-alkoxy-2-arylquinoline derivatives (7a–k, 8a–k, and 9a–k).
The structures of the newly synthesized compounds 7a–k, 8a–k, and 9a–k were identified by spectro-
scopic data such as 1 H NMR, 13 C/APT NMR, 1 H-1 H COSY, UV-Vis, FT-IR, LC-MS/MS, and elemental
analyses (Tables 1–9). The mass spectra of these compounds (7a–k, 8a–k, and 9a–k) showed molecular ion
peaks at the appropriate m/z values and the results are listed in Tables 1, 4, and 7, respectively. In the 1 H
and 13 C NMR spectra of compounds 7a–k, 8a–k, and 9a–k, in particular, H3 showed peaks at δH 7.2 (1H, s)
and C3 at δC 98.2–102.1 ppm, which are an indication of quinoline ring systems. Moreover, the alkoxy moiety
of products 7a–k, 8a–k, and 9a–k exhibited characteristic signals at δH 4.2 (2H, t, J = 6.6) and δC 68.4 ppm
for –OCH2 – in the 1 H (Tables 2, 5, and 8) and 13 C NMR (Tables 3, 6, and 9) data,28,29 respectively.
2.2. Biological activities
Quinolines, 4(1H)-quinolines, and their hydroderivatives are the most biologically active natural and syn-
thetic compounds. Quinine, cinchonine, graveoline, and chimanine alkaloid derivatives are well known bioac-
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854

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tive agents. Various antiparasitic,18 antibacterial,2 cytotoxic and antineoplastic,19 antimycobacterial,20 and
antiinflammatory21 biological activity studies of quinolone derivatives have been conducted and are still needed
for new quinolone derivatives.
2.2.1. In vitro antibacterial screening
The antimicrobial activities of three new series of 33 quinolone compounds 2-(2-, 3-, and 4-fluorophenyl)-
4-O-alkyl(C5−15)quinolines (7a–k, 8a–k, and 9a–k) were tested against gram-negative, gram-positive, and
antifungal bacteria. The experimental results showed that antimicrobial activity was more effective on the
gram-positive bacteria than the others that were used. Additionally, the length of the alkyl chain on the
quinolone ring increases and the antimicrobial activity decreases as its interaction with the membrane lowers.
In the literature, quinolines including fluorine substitution showed antituberculosis activity against Myosotis
tuberculosis bacteria type.30 The compounds 4–6, 7a–k, 8a–k, and 9a–k were prescreened with an agar well
diffusion assay at 500 µg/mL concentration, and those that showed activity were further tested to determine
their minimal inhibition concentration (MIC) values (Table 10). The MIC values of tested compounds 7a–k,
8a–k, and 9a–k decreased slightly with the number of carbon atoms in the alkyl chain as shown in Figure 1. It
was difficult to attribute decreasing MIC values with the length of the carbon chain in the O-alkyl substituents.
The antimicrobial activity of compounds 4, 5, and 6 showed that there was no adverse activity against gram-
positive and antifungal bacteria apart from compound 6 out of nine different bacteria types in total examined
as gram-positive, acido-resistant, and antifungal bacteria. The antimicrobial results revealed that compounds
9d, 9e, and 9f only have higher activities against the gram-positive bacterium Enterococcus faecalis at a high
concentration (500 µg/mL) among other bacteria used in this work.
Furthermore, it was observed that initial compounds 4, 5, and 6 and apart from 7b and 7c out of syn-
thesized compounds and all other compounds have activities against tuberculosis bacteria type Mycobacterium
smegmatis. The experimental results showed that the longer the alkyl chain gets, the lower activity is for the
tested compounds 7a–k, 8a–k, and 9a–k.31 Antimicrobial activities of these three series of title compounds
showed that 8a–k is the most active (MIC, 62.5–125 µg/mL), 9a–k is the second (MIC, 125–250 µg/mL, except
9h), and 7a–k is the least active (MIC, 125–500 µg/mL) against tuberculosis bacteria (M. smegmatis). When
the fluoride was substituted at the m-position of the target compounds 8a–k showed higher antituberculosis
activity (MIC, 62.5–125 µg/mL) than the other o-, and p-fluoride substituted compounds 7a–k (MIC, 250–500
µg/mL, except compound 7a) and 9a–k (MIC, 125–250 µg/mL, except compound 9h) (Table 10), respectively.
2.3. In vitro antioxidant activity
Two or more antioxidant test methods with different strategies were generally utilized in antioxidant activity
determinations. Antioxidant activity differences appear in many cases between the results of different assays
due to different reaction mechanisms with varying effects of solvents and temperature, existence of sterical
issues, pH, and the matrix components. In the current study, two widely used antioxidant test methods were
used for the determination of antioxidant capacities of the synthesized compounds 7a–k, 8a–k, and 9a–k.
The DPPH• radical scavenging test has been used extensively for various types of samples including synthetic
compounds (Figure 2).32 The ferric reducing/antioxidant power (FRAP) method has also been utilized in
many investigations with synthetic organics (Figure 3). To overcome solubility issues of the compounds when
the solutions are mixed with FRAP reagent, the original method33,34 has been modified to contain methanol
in 3:2 ratio in water instead of using water as solvent in the preparation of FRAP reagent.
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Table 10. Screening for antimicrobial activity of the compounds (4, 5, 6, 7a–k, 8a–k, 9a–k).
Microorganisms and minimum inhibition concentration (MIC, µg/mL)
Gram (–)
Ec Yp Pa
Gram (+)
Ef
Mycobacterium
Ms
Fungi
Ca
Compounda
Sa Bc
Sc
7
8
9
4
5
6
a
b
c
d
e
f
g
h
i
j
k
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
32
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
125
62.5
125
-
-
-
-
-
-
-
-
-
-
-
-
-
-
nt
nt
-
-
-
-
-
-
-
-
-
-
-
-
-
-
nt
nt
-
250
-
-
-
-
-
-
-
-
-
-
-
125 125 125
-
-
125 125
62.5 250
500b
500 62.5 250
500 62.5 125
500 125 250
500 62.5 250
500 125 500
250 125 250
500 125 250
250 125 250
500b
500b
-
-
-
-
-
Ampicillin
Streptomycin nt nt
Fluconazole nt nt
> 128
nt
nt
2
nt
nt
< 1 nt
4
nt nt
nt nt
nt
< 8 < 8
a The letters represent subscripts of compounds 7, 8, and 9.
b These values belong to only 9 series compounds (9d–f), and 7d–f and 8d–f were inactive. Ec: Escherichia coli, Yp:
Yersinia pseudotuberculosis, Pa: Pseudomonas aeruginosa, Ef: Enterococcus faecalis, Sa: Staphylococcus aureus, Bc:
Bacillus cereus 702 Roma, Ms: Mycobacterium smegmatis, Ca: Candida albicans, Sc: Saccharomyces cerevisiae, -: no
activity observed in the concentration range tested (0–500 µg/mL), nt: not tested.
4.50
4.30
4.10
3.90
3.70
3.50
3.30
3.10
2.90
2.70
2.50
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Number of C in Alkyl Chain
Figure 1. Trend in antimicrobial activity as the chain length of alkyl substituent increases. pMIC is –logaritm of MIC
(minimum inhibitory concentration) values; higher pMIC values represent higher antimicrobial potential.
In quinolines, antioxidant activity changes in accordance with the functional groups related to the
quinoline core. For example, when phenolic hydroxyl groups or imine groups are connected to the quinoline ring,
antioxidant activity increases.35 When activities of DPPH, ABTS with 2-oxoquinoline, and of superoxide anion
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14.0
12.0
10.0
8.0
8 a-k
7 a-k
9 a-k
6.0
4.0
2.0
0.0
5C
6C
7C
8C
9C
10C
11C
12C
13C
14C
15C
Number of C in Alkyl Chain
Figure 2. SC50 values in DPPH radical scavenging test of 7a-k (A), 8a-k (B), and 9a-k (C). 50 µM final concentration
of DPPH was used. Lower values represent higher activities. SC50 value of the reference antioxidant vitamin C was 0.005
mg/mL, and that of the precursor compounds 4, 5, and 6 were 0.143, 0.047 and 0.553, respectively. Linear regression
lines obtained in MS Excel program are shown on the graphs.
800
700
600
500
9 a-k
400
300
200
100
0
8 a-k
7 a-k
14C
5C
6C
7C
8C
9C
10C
11C
12C
13C
15C
Number of C in Alkyl Chain
Figure 3. FRAP values (µM) of 7a-k, 8a-k, and 9a-k as a measure of antioxidant capacity. Higher values represent
higher activities.
and hydroxyl radical are examined, it can be seen that they show a better performance than the commercial
antioxidant butylated hydroxytoluene (BHT).36 Moreover, when hydrogen sending groups are connected to
elements with a 2-phenylquinoline-4(1H)-one core, antioxidant activities of FRAP and TBARS increase.37−39
However, it is seen that the activity decreases when the length of the alkyl chain tied to the quinoline ring
increases.40
2.3.1. DPPH• scavenging tests
Radical scavenging activity against DPPH• was determined by UV at 517 nm by using vitamin C as antioxidant
standard. The values are expressed as SC50 (mg/mL), the concentration of the samples resulting in 50%
scavenging of DPPH• radical. The SC50 value of vitamin C (0.005 mg/mL) was determined to compare with
synthesized compounds in the range of 0.032–12.482 mg/mL. The highest and lowest activities were observed
from compounds 9b and 7j, respectively, as seen in Figure 2. In this serial synthesis of alkylated derivatives
of quinolone, DPPH• radical scavenging activity decreased with increasing number of alkyl carbons, evident
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from the increasing trend of SC50 values from compounds 7a, 8a, and 9a to 7k, 8k, and 9k. An obvious
irregularity was observed with some of the compounds in this sense, as for 9b, 9i, and 9j; the reason behind
this could not be explained with the current data. Similar results were also observed with some other types of
natural product analogues with alkyl substitutions.41
110
90
70
50
30
10
0
100
200
300
400
500
600
700
800
900
1000
1100
-10
FRAP value (μ M)
Figure 4. Correlation graph between antioxidant capacity (FRAP values) and DPPH radical scavenging activity
(%Scavenging values at 0.5 mg/mL compound concentration) assay results; R2 value is 0.85.
2.3.2. Ferric reducing/antioxidant power (FRAP) test
The antioxidant activities of the synthesized samples were also determined by FRAP assay (Figure 3).33,34
The method is based on measurement of the iron reducing capacities of the compounds. FRAP values were
expressed as µM obtained from the calibration curve prepared with vitamin C at 62.5–1000 µM concentrations
and multiplying the corresponding concentration by two because of 1:2 stoichiometry of vitamin C with FeSO4 .
In general, FRAP results were similar to those of DPPH• radical scavenging activities. As the length of
the alkyl chain was increased from 5 to 15 carbon atoms, the FRAP activities in most compounds decreased,
evident from the decreasing trend of FRAP values from compounds 7a, 8a, and 9a to 7k, 8k, and 9k. The
lowest antioxidant activities were determined with 8k and 9k, with the longest alkyl chain. The same irregularity
was observed with FRAP tests as with DPPH• scavenging tests in compounds 9b and 9h–j, showing unexpected
activity. Nevertheless, the highest activities were obtained from compound 9b in DPPH• and FRAP tests.
The FRAP assay is based on the ability of antioxidant to reduce Fe3+ to Fe2+ in the presence of 2,4,6-
tri(2-pyridyl)-s-triazine (TPTZ), forming an intense blue Fe2+ -TPTZ complex with an absorption maximum
around 595 nm. This antioxidant test method is generally used in comparison with other antioxidant test
methods. When two antioxidant activity method results are compared, a correlation can be expressed to show
total antioxidant capacity. A good correlation of the results of two antioxidant test methods was observed
(Figure 4), R2 = 0.85. The test results clearly show that antioxidant activity decreases as the length of alkyl
chain increases. FRAP tests revealed that compounds 7c, 8c, and 9b are the most active ones. Furthermore,
DPPH test results showed that compounds 7c, 8b, and 9b were the most active ones (Figures 3). Antioxidant
activity decreases with the length of the alkyl chain in all three series of compounds synthesized, in which the
alkyl group changes from 5 carbons to 15 carbons. In addition, it is clear that FRAP and DPPH effectiveness
levels are parallel to each other (Figure 4). When antioxidant activities of these three series are examined, it
can be seen that 9a–k is the most active, 8a–k is the second, and 7a–k is the least active.
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3. Experimental
3.1. Materials and equipment
All starting chemical reagents (2-aminoacetophenone, 2-, 3-, and 4-fluorobenzaldehyde, bromoalkanes (C5−15),
potassium hydroxide, K-10 clay, 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical, and 2,4,6-tri(2-pyridyl)-s-
triazine) used in the synthesis were high grade commercial products purchased from Aldrich, Fluka, or Sigma
and used without further purification. The solvents (n-hexane, chloroform, ethyl acetate, dimethyl formamide,
methanol, diethyl ether, and dimethyl sulfoxide) used were either analytical grade or bulk solvents distilled before
use. Thin-layer chromatography (TLC) and column chromatography were carried out on Merck precoated 60
Kieselgel F254 analytical aluminum acidic plates and silica gel 60 (0.040–0.063 mm), respectively. A Milestone
microwave oven was used for microwave reactions. 1 H and 13 C NMR spectra were recorded on a Varian Mercury
200 MHz NMR with tetramethylsilane (TMS) as an internal standard. The mass spectral analyses were carried
out on a Micromass Quattro LC-MS/MS spectrophotometer. The elemental analyses were performed on a
Costech ESC 4010 instrument. Infrared spectra were obtained with a PerkinElmer 1600 FT-IR (4000–400
cm−1) spectrometer. Melting points were determined using a Thermovar apparatus fitted with a microscope
and are uncorrected. UV-Vis absorbance measurements and spectral analyses were carried out on a Unicam
UV2-100 at 25 ◦ C.
3.2. Methods
The known compounds 1, 2, and 3 were synthesized according to the literature.42−46
3.2.1. General procedure for the preparation of compounds 4, 5, and 6
(2E)-1-(2-aminophenyl)-3-(2-fluorophenyl)prop-2-en-1-one (1), (2E)-1-(2-aminophenyl)-3-(3-fluorophenyl)prop-
2-en-1-one (2), and (2E)-1-(2-aminophenyl)-3-(4-fluorophenyl)prop-2-en-1-one (3) (0.02 mol each) were dis-
solved in chloroform and uniformly adsorbed on the surface of K-10 clay (15 g each) in a Pyrex round bottomed
flask. The solvent was evaporated under vacuum, and then the adsorbed material was transferred to a Pyrex
tube (2 cm diameter, 30 mL) and inserted inside the Milestone microwave oven. The mixture was heated using a
fixed power of 350 W for 5 min at 85 ◦ C. The reaction mixture was dissolved in AcOEt (2 × 15 mL) and filtered
off. The extract was evaporated to leave a crude mixture, which was purified by column chromatography over
silica (hexane–AcOEt, 5:1) to afford the pure corresponding products (4, 5, and 6) in 92%, 90%, and 96% yields,
respectively; the assigned structures for the known compounds 2-(2-fluorophenyl)-2,3-dihydroquinolin-4(1H)-one
(4), 2-(3-fluorophenyl)-2,3-dihydroquinolin-4(1H)-one (5), and 2-(4-fluorophenyl)-2,3-dihydroquinolin-4(1H)-
one (6) were confirmed by their spectral properties (1 H, 13 C/APT, 2D-COSY NMR, IR, and LC-MS/MS)
and by comparison with literature data.47−52
3.2.2. General procedure for the preparation of compounds 7a–k, 8a–k, and 9a–k
To a solution of 2-(2-fluorophenyl)-2,3-dihydroquinolin-4(1H)-one (4), 2-(3-fluorophenyl)-2,3-dihydroquinolin-
4(1H)-one (5), and 2-(4-fluorophenyl)-2,3-dihydroquinolin-4(1H)-one (6) (2.41 g, 0.01 mol for each) in DMF (10
mL each) was added KOH (1.12 g, 0.02 mol each) and the resulting mixture was stirred at room temperature for
1 h. Then alkyl bromide (0.03 mol each) was added dropwise, and the reaction mixture was stirred overnight
at room temperature. Then the reaction mixture was poured into 20 mL of water. The resulting mixture
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was extracted with CH2 Cl2 (2 × 20 mL). The combined organic layer was washed with brine, dried over
Na2 SO4 , filtered, and concentrated in a vacuum to give crude products 7a–k, 8a–k, and 9a–k, which were
purified by column chromatography using a mixture of n-hexane–chloroform (8.5:0.5) as eluent (yields are
in Tables 1, 4, and 7). The structures of compounds 7a–k, 8a–k, and 9a–k were assigned on the basis of
NMR, IR, and mass spectral analyses (Tables 1–9), respectively, and they were named 2-(2-fluorophenyl)-4-O-
alkyl(C 5−15)quinoline (7a–7k), 2-(3-fluorophenyl)-4-O-alkyl(C5−15)quinoline (8a–8k), and 2-(4-fluorophenyl)-
4-O-alkyl(C5−15)quinoline (9a–9k). The physicochemical and 1 H and 13 C NMR data of compounds 7a–k,
8a–k, and 9a–k are given in Tables 8–10.
3.3. In vitro antibacterial activity
The newly synthesized compounds were screened for their in vitro antibacterial activity against Escherichia
coli ATCC35218, Yersinia pseudotuberculosis ATCC911, Pseudomonas aeruginosa ATCC43288, Enterococcus
faecalis ATCC29212, Staphylococcus aureus ATCC25923, Bacillus cereus 709 Roma, Mycobacterium smegmatis
ATCC607, Candida albicans ATCC60193, and Saccharomyces cerevisiae RSKK 251, which were obtained
from Refik Saydam Hygiene Center (Ankara, Turkey). All tested compounds were dissolved in hexane and
diluted with dimethyl sulfoxide (DMSO) to prepare solutions of 10.0 mg/mL.
The antimicrobial effects of the compounds were tested quantitatively in respective broth medium by
using double dilution, and the minimal inhibition concentration (MIC) values (µg/mL) were determined.53
The antibacterial and antifungal assays were performed in Mueller–Hinton broth (MH) (Difco, Detroit, MI,
USA) at pH 7.3 and buffered Yeast Nitrogen Base (Difco) at pH 7.0, respectively. Brain Heart Infusion broth
(BHI) (Difco) was used for M. smegmatis.54 The MIC was defined as the lowest concentration that showed
no growth. Ampicillin (10.0 mg/mL), streptomycin (10.0 mg/mL), and fluconazole (5.0 mg/mL) were used as
standard antibacterial and antifungal drugs, respectively. Hexane with a dilution of 1:10 with DMSO was used
as solvent. The smallest concentration with which the growth of the test microorganism was totally inhibited
is reported as MIC (µg/mL) value (Table 10).
3.4. In vitro antioxidant activity tests
3.4.1. DPPH• scavenging test
DPPH• radical scavenging activity of the compounds was determined by UV spectra at 517 nm (Figure 2).25
The assay is based on the absorbance change of the DPPH• when deactivated by the antioxidants, which
is observed with the naked eye as a color change from purple to yellow. Briefly, 750 µL of the compound
solutions was mixed with 750 µL of a 100 µM DPPH• in methanol and vortexed. The compound solutions
were tested at five different concentrations prepared with a twofold serial dilution starting with the highest
concentration estimated with a pretest measurement. DPPH• radical scavenging activity was evaluated by
using ascorbic acid as antioxidant standard, and the values are expressed as SC50 (mg/mL), the concentration
of the sample resulting in 50% scavenging of the radicals. Low SC50 values represent high antioxidant activity.
In order to make a correlation with the results of the FRAP test, the results of the DPPH• scavenging test
were also expressed in terms of %scavenging, the percentage of the DPPH• radicals remaining after reacting
DPPH• with the sample at 0.5 mg/mL concentration in comparison to the initial value.
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3.4.2. Ferric reducing/antioxidant power (FRAP) test
The antioxidant activities of the compounds were determined by FRAP assay.36 The method is based on the
measurement of the iron reducing capacities of the compounds. Working FRAP reagent was prepared by mixing
150 mL of 0.3 M acetate buffer at pH 3.6 with 15 mL of 10 mM TPTZ solution in 40 mM HCl and 15 mL of
20 mM FeCl3 .6H2 O. The preparation of FRAP reagent was modified using a methanol–water mixture in 3:2
ratio to make the medium more compatible with the compounds of relatively nonpolar nature; 50 µL of the
sample was mixed with 1.5 mL of freshly prepared FRAP reagent. The absorbance was read after an incubation
period of 20 min at room temperature at 595 nm against distilled water (Figure 3). A calibration curve was
constructed using aqueous solutions of vitamin C in the concentration range of 62.5–1000 µM. The absorbance
of the reagent blank and sample blank was subtracted from that of the samples. The corresponding vitamin
C concentrations for the compound test results were determined from the calibration curve, and the results
are expressed as µM FRAP by multiplying the value obtained from the calibration graph by two, as two to
one stoichiometry is present between vitamin C and FeSO4 . High FRAP values mean high total antioxidant
activity (Figure 3).
4. Conclusion
A new synthetic method was used to synthesize 4-alkoxy-2-arylquinoline derivatives (7a–k, 8a–k, and 9a–
k) starting from flavonone [2-aryl-2,3-dihydroquinolin-4(1H )-ones]. All the newly synthesized compounds
were tested for their antibacterial, antifungal, and antituberculosis properties against a panel of standardized
microorganisms, using ampicillin, streptomycin, and fluconazole for comparison. The observed MIC values
of the compounds (7a–k, 8a–k, and 9a–k) against M. smegmatis were in the range of 62.5–500 µg/mL.
m−Fluoride substituted new compounds 8a–k gave high antituberculosis activity (MIC, 62.5–125 µg/mL).
The compounds showed relatively good antioxidant activities in DPPH• radical scavenging and FRAP tests.
This study indicated that the results of the DPPH and FRAP assays provide essentially identical information
in regard to the antioxidant capability of the compounds, and so it is difficult to establish what additional
information could be gained apart from alkyl chain length. When the alkyl chain length increased from five
carbons (7a, 8a, and 9a) to 15 carbons (7k, 8k, and 9k), the antioxidant capacity decreased in both methods.
The antimicrobial data observed against M. smegmatis suggest that the new chemotype is promising in the
search for an antituberculosis drug candidate lead. Synthetic or natural flavonones should be evaluated for their
potential use in the synthesis of new bioactive quinoline derivatives.
Acknowledgment
This study was supported by grants from Karadeniz Technical University Research Fund (KTU-BAP 9699) in
Turkey.
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