Simple and convenient preparation of novel 6,8-disubstituted quinoline derivatives and their promising anticancer activities

Article abstract of DOI:10.3906/kim-1301-30

A short and easy route is described for 6,8-disubstituted derivatives of quinoline and 1,2,3,4-tetrahydroquinoline from 6,8-dibromoquinolines 2 and 7 by various substitution reactions. While copper-promoted substitution of 6,8- dibromide 2 produced monomethoxides 3 and 4, a prolonged reaction time mainly afforded dimethoxide 6 instead of 5, whose aromatization with DDQ and substitution reaction of dibromide 7 with NaOMe in the presence of CuI also gave rise to dimethoxide 6. Several 6,8-disubstituted quinolines were obtained by treatment of 6,8-dibromoquinoline (7) with n-BuLi followed by trapping with an electrophile [Si(Me)₃ Cl, S₂ (Me)₂; and DMF]. Furthermore, 7 was also converted to mono and dicyano derivatives. The anticancer activities of compounds 2, 7, 6, 12, 13, 15, and 16 against HeLa, HT29, and C6 tumor cell lines were tested, and 6,8-dibromo-1,2,3,4- tetrahydroquinoline (2) and 6,8-dimethoxyquinoline (6) showed significant anticancer activities against the tumor cell lines. TUeBITAK.

Full text of DOI:10.3906/kim-1301-30

Turk J Chem
Turkish Journal of Chemistry
(2013) 37: 896 – 908
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1301-30
Research Article
Simple and convenient preparation of novel 6,8-disubstituted quinoline
derivatives and their promising anticancer activities
1
4
4
Salih OKTEN, Osman C¸ AKMAK,2,∗ Ramazan ERENLER,3 Onem YUCE, S¸aban TEKIN
¨
¨
¨
˙
1Department of Primary Education, Division of Science Education, Faculty of Education, Kırıkkale University,
Kırıkkale, Turkey
2
˙
Department of Chemistry, Faculty of Arts and Science, Yıldız Technical University, Davutpa¸sa, Istanbul, Turkey
3Department of Chemistry, Faculty of Arts and Science, Gaziosmanpa¸sa University, Tokat, Turkey
4Department of Biology, Faculty of Arts and Science, Gaziosmanpa¸sa University, Tokat, Turkey
Received: 10.01.2013
•
Accepted: 13.05.2013
•
Published Online: 04.11.2013
•
Printed: 29.11.2013
Abstract: A short and easy route is described for 6,8-disubstituted derivatives of quinoline and 1,2,3,4-tetrahydroquinoli-
ne from 6,8-dibromoquinolines 2 and 7 by various substitution reactions. While copper-promoted substitution of 6,8-
dibromide 2 produced monomethoxides 3 and 4, a prolonged reaction time mainly afforded dimethoxide 6 instead of 5,
whose aromatization with DDQ and substitution reaction of dibromide 7 with NaOMe in the presence of CuI also gave
rise to dimethoxide 6. Several 6,8-disubstituted quinolines were obtained by treatment of 6,8-dibromoquinoline (7) with
n-BuLi followed by trapping with an electrophile [Si(Me)3 Cl, S2 (Me)2, and DMF]. Furthermore, 7 was also converted
to mono and dicyano derivatives. The anticancer activities of compounds 2, 7, 6, 12, 13, 15, and 16 against HeLa,
HT29, and C6 tumor cell lines were tested, and 6,8-dibromo-1,2,3,4-tetrahydroquinoline (2) and 6,8-dimethoxyquinoline
(6) showed significant anticancer activities against the tumor cell lines.
Key words: Anticancer effect, bromoquinoline, cyanoquinoline, lithium–bromine exchange, methoxyquinoline, quinoline
derivatives
1. Introduction
The quinoline and 1,2,3,4-tetrahydroquinoline skeletons are often used in the designs of many synthetic com-
pounds with diverse pharmacological properties. Quinoline bromides specifically contain a key structural com-
ponent of numerous compounds, which can undergo metal–halogen exchanges1 and couplings;2 therefore, they
are useful fine chemicals. However, their prices tend to be quite high because of the complex and difficult syn-
thetic methods involved in the production of these quinoline bromides.3 Therefore, the development of simple
and cheap synthetic methods for the syntheses of quinoline derivatives is important. Although different methods
have been described in the literature, efficient, large-scale, cheap technologies are still needed. Current strategies
for the synthesis of quinoline derivatives include cyclization reactions starting from benzene (or cyclohexane)
derivatives with N-functionalities, but these compounds have also been prepared by various conventionally
named reactions, such as Skraup,4 Friedl¨ander,5 Doebner-von Miller,6 Pfitzinger,7 Conrad-Limpach,8 and
Combes.9 Unfortunately, these methods for quinoline synthesis often do not allow for adequate diversity and
substitution on the quinoline ring system.
∗Correspondence: cakmak.osman@gmail.com
Dedicated to Professor Metin Balcı on the occasion of his 65th birthday
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For instance, a bromoquinoline-based derivative synthesis is often restricted due to the difficulty in
the preparation of bromoquinolines. However, recently, we developed an efficient synthesis method for 6,8-
dibromoquinolines 2 and 7 based on the bromination of 1,2,3,4-tetrahydroquinoline (1),10 and we synthesized
a series of trisubstituted quinoline derivative from a corresponding tribromoquinoline. As an extension of
that study, we report here a convenient synthesis for 6,8-disubstituted quinoline derivatives from brominated
quinolines 2 and 7 via a metal–bromine exchange, and the values for the brominated products 2 and 7, as
precursors to the corresponding disubstituted quinolines, are presented. In addition, because many quinoline
derivatives demonstrate impressive anticancer activities,11−14 we studied the anticancer activities of the 6,8-
disubstitued quinoline derivatives against several tumor cell lines; overall, 6,8-dibromide 2 and 6,8-dimethoxide
6 showed promising anticancer activities.
2. Results and discussion
2.1. Synthesis and structural assignment
Dibromides 2 and 7 were synthesized according to previously reported procedures starting from 1,2,3,4-
tetrahydroquinoline (1) (Schemes 1 and 2).10
In the first step, dibromide 2 was treated with MeONa in the presence of CuI in boiling DMF to give a
mixture of methoxyquinolines 3 and 4, and the proportion of the products depended on the reaction time. In
the 20-h reaction period (entry 1), monomethoxides 3 and 4 were obtained in a ratio of 1:1, whereas prolonged
reaction times up to 120 h raised the amount of monomethoxide 4 and dimethoxide 5 formed to a ratio of
1:3 (entry 3). Surprisingly, the longer reaction times (entries 4 and 5) also mainly resulted in the formation
of aromatized product 6. We assume that the high temperature conditions in the presence of CuI with longer
reaction times induced aromatization of the dimethoxy compound 5 to give 6.
Br
Br2
Entry Time
Ratio of the products
N
N
H
CHCl3, 2h, rt
3
4
5
6
H
Br
2, 90%
1
2
3
4
5
20 h
48 h
120 h
144 h
192 h
50: 50:
0:
0
0
0
1
15: 50: 35:
0: 25: 75:
0: 17: 68: 15
NaOMe
DMF/CuI, 150 oC
0:
8: 14: 78
MeO
MeO
MeO
Br
+
+
+
N
OMe
N
H
N
H
N
H
OMe
Br
OMe
3
4
6
5
Scheme 1. Copper-assisted methoxylation of 2.
The reaction mixture in entry 2 was separated by column chromatography using SiO2 with hex-
ane/AcOEt. The solvent polarity was gradually increased from 9:1 to 9:3, and the first fraction was found
to be a mixture of 6-methoxide 3 and 8-methoxide 4. Methoxide 4 was isolated as a pure compound in a
yield of 14% in the second fraction, and the following fraction contained 4 and 6,8-dimethoxide 5 as a mixture.
Lastly, 5 was isolated in pure form (18% yield) in the final fraction.
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DDQ aromatization of compound 5 in benzene at reflux for 40 h provided 6,8-dimethoxyquinoline (6).
The reaction mixture of 3 and 4 (entry 1) was also aromatized, and products 8 and 9 were easily isolated by
column chromatography (Scheme 2).
MeO
Br
MeO
DDQ
NaOMe
Benzene, 44 h,
80 oC
N
DMF, CuI,
150 oC, 20 h
N
N
H
OMe
OMe
Br
7, 83%
5
6, 78% from 5
84% from 7
Benzene, 44 h,
80 oC
DDQ
Br
MeO
NaOMe
DDQ
+
3
4
Benzene,
80 oC, 40 h
DMF, CuI,
150 oC, 20 h
N
N
H
Br
+
8, 42%
Br
Ratio = 50:50 (1H– NMR)
2
Br
N
OMe
9, 44%
Scheme 2. Synthesis of 6, 8, and 9.
To obtain pure dimethoxide 6, the reaction with 2 was repeated for 144 h (entry 4) and 192 h (entry 5).
However, the aromatized product 6 along with products 4 and 5 were produced instead of pure dimethoxide 5
(Scheme 2). We achieved pure dimethoxide 6 by direct treatment of 6,8-dibromoquinoline 7 with NaOCH3 in
the presence of CuI. 6,8-Dimethoxyquinoline 6 was obtained as the sole product in a yield of 84% (Scheme 2).
It is reported that Alfonsi et al. carried out many reactions for synthesis of 5,8-dimethoxy quinoline (5).
One of the best yields of the sequential alkylation/gold-catalyzed annulation reactions of anilines with propar-
gylic bromide was only 28%.15 Therefore, we developed an alternative, more efficient and simple preparation
method for compound 5.
The 1 H NMR spectra of dimethoxide 6 are quite similar to those of the starting material, dibromide
3, and consist of the same signal systems except for the higher shifted aryl protons present due to the 2 MeO
donor groups in 6.
Structural characterizations of all of the methoxide compounds were further confirmed by mass spec-
2
troscopy and other 2-dimensional NMR spectra. Compound 4 provided JCH couplings through an HMBC
correlation, and clear evidence for the positions of the MeO group (8- or 6-methoxide) and the aromatic protons
H-5 and H-7 was observed. The fact that C-8 (146.8 ppm) correlated with H-70 (6.72 ppm) but not with H-5
(6.77 ppm) confirmed that the MeO group was attached to C-8. Furthermore, H-5 (6.77 ppm) and H-7 (6.72
ppm) correlate with C-6 (133.6 ppm), which is in agreement with the suggested structure for 4.
To demonstrate the value of 6,8-dibromide 7 as a precursor for other useful compounds, we investi-
gated the metal–halogen exchange reaction of 7. As a result, 6,8-bis(methylsilyl)quinoline 11 (76%) and 6,8-
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bis(methylthio)quinoline 12 (78%) were obtained in moderate yields. In the NMR spectra, the 2 silyl signals
in 11 and 2 methylthiol signals in 12 were clearly observed, and these results confirmed the formation of the
compounds. Moreover, the similarities in the signal systems for compounds 11 and 12 with that of 7 were
quite helpful for the identification of the structures (Scheme 3).
Br
N
Br
7
n-BuLi/THF (2 eq)
-78 oC
Me3Si
Si(CH3)3Cl
OHC
Li
DMF
N
N
N
SiMe3
CHO
11, 76%
Li
10
13, 31%
(CH3)2S2
MeS
N
SMe
12, 78%
Scheme 3. Preparations of 6,8-disubstituted quinoline derivatives 11–13.
Lejejs et al. obtained methylthioquinoline 12 with a long sequential reaction procedure (10 steps)
starting from 6-nitro-8-aminoquinoline, in which synthesis and physicochemical properties of 6-methylthio-
8-mercaptoquinoline (precursor of 12) were reported.16 The authors claimed that the earlier method17 was
more complicated and their new method reduced the number of reaction stages and increased the yield (overall
yield: 20%). Thus, we developed an effective and simple method for the synthesis of valuable compound 12
starting from 1,2,3,4-tetrahydroquinoline using just 3 sequential, precisely selective and simple reactions that
all proceed in high yields.
Preparation of quinoline-6,8-dicarbaldehyde 13 led to a lower yield (31%) than expected, as the dilithiated
quinoline in THF at –78 ◦ C was trapped by DMF. The 1 H NMR spectra of 13, which consisted of 2 aldehyde
singlet signals (δ 11.48 and 10.30) and 5 signals of aromatic H-atoms, (δ 9.21, dd, H-2), 7.67 (dd, H-3), 8.46
(d, H-4), 8.67 (d, H-5), and 8.80 (d, H-7), matched fairly well with the proposed structure. In addition, the
13 C NMR spectrum of 13, consisting of 11 signals with 2 C = O signals (δ 191.8 and 190.7), was also in good
agreement with the suggested structure (Scheme 3).
Finally, dibromide 7 was treated with CuCN in boiling DMF. The nucleophilic substitutions of 7 resulted
in the formation of a mixture of cyanoquinolines 14, 15, and 16 (Scheme 4). According to the 1 H NMR and
mass spectral data, 3 cyanoquinolines were formed. The conversion and product ratio were 83% and 47:12:22
for 14–16, respectively, as assigned by 1 H NMR. Furthermore, the products were easily isolated by silica
gel column (SiO2 , AcOEt/hexane); this process yielded 9%, 43%, and 20% 8-bromo-6-cyanoquinoline 14, 6-
bromo-8-cyanoquinoline 15, and 6,8-dicyanoquinoline 16, respectively. However, when the reaction time was
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prolonged, polymerization occurred instead of the formation of dicyanide 16 as the sole product. In the case of
the lower reaction temperature and longer reaction time (100 ◦ C and 2 days), no conversion occurred.
NC
Br
NC
Br
CuCN
+
+
DMF, 150 oC
N
N
N
N
Br
CN
CN
Br
7
14, 9%
15, 43%
16, 20%
Scheme 4. Synthesis of the cyanoquinoline derivatives 14–16.
In the 1 H NMR spectrum, 6,8-dicyanide 16 displays the same signal system as dibromide 7, except with
higher field shifting. Furthermore, the 2-dimensional NMR spectra provided information about the position of
the cyanide group. For example, in the HBMC spectra of 14, the characteristic cyanide C-atom (119.0 ppm)
correlated with H-5 and H-7 (δ 8.22 and 8.25, respectively), which confirmed that the cyanide group was bonded
to C-6 in compound 14.
2.2. Antiproliferative activities of quinoline compounds against HeLa cells
In the present study, the antiproliferative activities of 2, 6, 7, 12, 13, 15, and 16 were tested against HeLa
cells in vitro at 5, 10, 20, 30 40, 50, 75, and 100 µg/mL concentrations.18 The results showed that only
6,8-dibromo-1,2,3,4-tetrahydroquinoline (2) significantly inhibited proliferation of HeLa cells at 10 µg/mL and
higher concentrations (P ≤ 0.05) tested (Figure 1). It was the most potent antiproliferative compound against
HeLa cells among the compounds tested in this study.
120.00
100.00
5–FU
2
6
80.00
60.00
7
40.00
20.00
12
13
15
16
0.00
0
5
10
20
30
40
50
75
–20.00
–40.00
–60.00
Concentration (µg/mL)
Figure 1. Antiproliferative activities of quinoline derivatives 2, 6, 7, 12, 13, 15, and 16 on the proliferation of HeLa
cells in vitro. All compounds were tested at 5, 10, 20, 30 40, 50, 75, and 100 µg/mL concentrations. Compound 2
significantly inhibited proliferation of HeLa cells at 10 µg/mL and higher concentrations tested (P ≤ 0.05). The data
show the averages of 2 individual experiments. The DMSO and 5-FU were used as the negative and positive control
respectively in all experiments.
2.3. Antiproliferative activities of quinoline compounds against HT-29 cells
Quinoline compounds 2, 6, and 12 were tested against proliferation of HT-29 cells at 5, 10, 20, 30, 40, 50, 75, and
100 µg/mL concentrations. The results showed that compound 2 more significantly inhibited the proliferation
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of HT-29 cells than control compound 5-FU at 30 µg/mL and higher concentrations (P ≤ 0.05) (Figure 2).
Compound 6 was more inhibitory against HT-29 cells than 5-FU at 70 µg/mL and higher concentrations (P ≤
0.05) (Figure 2).
90.00
80.00
70.00
60.00
50.00
40.00
30.00
5-FU
2
20.00
10.00
0.00
6
12
5
10
20
30
40
50
Concentration (µg/mL)
75 100
Figure 2. Antiproliferative activities of quinoline derivatives 2, 6, and 12 on the proliferation of HT-29 cells in
vitro.All compounds were tested at 5, 10, 20, 30 40, 50, 75, and 100 µg/mL concentrations. Compounds 2 and 6
significantly inhibited (P ≤ 0.05) the proliferation of HT-29 cells at 30 µg/mL and 70 µg/mL concentrations and higher
concentrations, respectively. The data show the averages of 2 individual experiments. The DMSO and 5-FU were used
as the negative and positive control respectively in all experiments.
2.4. Antiproliferative activities of quinoline compounds against C6 cells
The antiproliferative activities of 2, 6, 12, and 13 on C6 cells were also tested at 5, 10, 20, 30 40, 50, 75, and
100 µg/mL concentrations. Compound 2 was the most antiproliferative compound tested at 30 µg/mL and
higher concentrations (P ≤ 0.05) (Figure 3).
100.00
80.00
60.00
5-FU
2
40.00
20.00
6
12
13
0.00
5
10
20
30
40
50
75 100
–20.00
–40.00
Concentration (µg/mL)
Figure 3. The antiproliferative activities of 2, 6, 12, and 13 on C6 cells in vitro. All compounds were tested at 5, 10,
20, 30 40, 50, 75, and 100 µg/mL concentrations. Compound 2 was the most antiproliferative compound tested at 30
µg/mL and higher concentrations (P ≤ 0.05). The data show the averages of 2 individual experiments. The DMSO and
5-FU were used as the negative and positive control respectively in all experiments.
3. Conclusion
We developed a simple and convenient route to a variety of 6,8-disubstituted quinolines, which are difficult
to prepare by traditional methodologies, by starting with a commercially available, cheap starting material,
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tetrahydroquinoline 1. Our suggested methods are more convenient and efficient for known compounds 6,15 8,19
and 9,20 which valuable fine chemicals, and 12,16 and they are fully characterized. The prepared compounds
are often the starting points for other polyfunctionalized quinoline derivatives. For instance, the products can
be easily brominated at the 3-position of the quinoline ring, which could give 3,6,8-trisubstituted quinoline
derivatives, in the presence of Br2 and pyridine according to the Eisch procedure.21 On the other hand, the
bromomethoxide compounds 3, 4, 8, and 9 and the bromocyanides 14 and 15 can also be converted to other
disubstituted quinolines due to their bromine groups. Compounds 322 and 423 are known but they are protected
by patents. We are currently working on bromination of the methoxytetrahydroquinolines 3–5 to further study
their functionalization and to investigate their anticancer activities.
In addition, 6,8-dibromo-1,2,3,4-tetrahydroquinoline (2) significantly inhibited the proliferation of HeLa,
HT-29, and C6 cells in vitro at concentrations of 10 µg/mL, 30 µg/mL, and 30 µg/mL and higher concentra-
tions, respectively, as compared to a control cancer drug, 5-Fluorouracil (Figures 1–3). However, the compound
6,8-dimethoxyquinoline (6) selectively inhibited the proliferation of HT-29 cells only (Figure 2) at concentra-
tions of 70 µg/mL and higher. In contrast, compound 6 and the others tested did not exert any antiproliferative
activity against the cell lines used (Figures 1–3). Therefore, compound 2 was the most potent antiprolifera-
tive compound tested in this study. The results suggest that this compound may be a novel anticancer drug
candidate.
The 6,8-dibromide of the 1,2,3,4-tetrahydroquinoline 2 structure (but not the 6,8-dibromide functionality
of quinoline 7) and the 6,8-dimethoxy groups of quinoline 6 could be responsible for the antiproliferative
potentials of the compounds because of the possible reactivities of both groups. Therefore, we propose that
compounds with both groups might exert stronger anticancer activities. Although the results show anticancer
potentials for 6,8-dibromide 2 and 6,8-dimethoxide 6, the anticancer potentials of other proposed, substituted
quinolines and tetrahydroquinolines and their mechanisms of action need to be determined. The selective
and potent anticancer activities of compounds 2 and 6 need to be tested in further pharmacological studies.
Furthermore, variations in these substituents on the lead compounds, along with their mechanisms of action for
their anticancer activities, are being studied and will be reported in due course.
4. Experimental
4.1. General
Thin layer chromatography was performed on silica F254 0.255 mm plates, and spots were visualized by UV at
254 nm. Classic column chromatography was performed using (70–230 mesh) silica gel. Melting points were
determined on a capillary melting point apparatus. Solvents were concentrated at reduced pressure. IR spectra
were recorded on an IR instrument. Mass spectra were recorded on a spectrometer under electron-impact (EI)
and chemical ionization conditions. NMR analyses were recorded on a NMR instrument for the 1 H NMR (400
MHz) and for the 13 C NMR (100 MHz) spectra.
4.2. Synthesis of the methoxy derivatives of 1,2,3,4-tetrahydroquinoline (Scheme 2) (entry 2)
Freshly cut Na (1.5 g, 65.2 mmol) was added to dry MeOH (40 mL) under an Ar atmosphere. After complete
dissolution, the warm solution was diluted with dry DMF, and vacuum-dried CuI (1.78 g, 3.44 mmol) was
added. Next, 6,8-dibromo-1,2,3,4-tetrahyroquinoline (2) (1 g, 3.44 mmol) in dry DMF (25 mL) was added to
the mixture, which was stirred magnetically under an Ar atmosphere at reflux (ca. 150 ◦ C) for 48 h. The
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reaction progress was monitored by TLC. After cooling to rt, H2 O (50 mL) was added to the mixture, and the
aq layer was extracted with CHCl3 (3 × 50 mL). The organic layers were combined, washed with H2 O (3 × 25
mL), and dried (Na2 SO4). After filtration and removing the solvent, the residue was filtered through a short
silica gel column (5.0 g). A mixture (400 mg) of 8-bromo-6-methoxyquinoline (3), 6-bromo-8-methoxyquinoline
(4), and 6,8-dimethoxyquinoline (5) was obtained in a ratio of 15:50:35, respectively, as assigned by 1 H NMR.
The mixture was purified by silica gel (SiO2 , 60 g) column chromatography, eluting with AcOEt/hexane, (600
mL, 1:9). Compounds 5 and 6 were collected as a mixture in the first eluent (320 mL). However, after the
solvent polarity was increased to 2:9 AcOEt/hexane, compound 4 (112 mg, 14%) was isolated in pure form
(second eluent 150 mL). After the solvent polarity was increased to 3:9 (AcOEt/hexane), the third eluent (100
mL) was a mixture of products 4 and 5. Lastly, 6,8-dimethoxide 5 (120 mg, 18%) was isolated as a pure
substance, as the fourth eluent (240 mL). The reaction was repeated for 20 (entry 1) and 120 h (entry 3) under
the same reaction conditions. In the 20-h reaction, the product ratio of 6-bromo-8-methoxide 4 and 8-bromo-
6-methoxide 3 was 50:50, respectively. In the 120-h reaction, 6-bromo-8-methoxide 4 and 6,8-dimethoxide 5
were isolated in a ratio of 25:75, respectively (entry 3). The reactions were also repeated for 144 h and 192 h
using the same reaction conditions. The ratios of the products are shown in Scheme 1 (entries 4 and 5). The
reaction was also repeated under the same reaction conditions with CuI but not MeONa for 3 days. However,
no conversion (aromatization) was observed.
8-Bromo-6-methoxy-1,2,3,4-tetrahydroquinoline (3). l H NMR (400 MHz, CDCl3): δ 6.90 (d,
1H, J75 = 2.4 Hz, 1H, H-7), 6.57 (d, J57 = 2.4 Hz, 1H, H-5), 4.1 (s, 1H, NH), 3.73 (s, 3H, -OMe), 3.32 (t, J23
= 5.6 Hz, 2H), 2.74 (t, J43 = 6.4 Hz, 2H), 1.94 (m, 2H).
6-Bromo-8-methoxy-1,2,3,4-tetrahydroquinoline (4). Yellow oil. IR (KBr, cm−1): 3424 (NH),
l
2931, 2836, 1579, 1500, 1463, 1413, 1359, 1328, 1247, 1191, 1108, 1012, 867, 829, 732, 567; H NMR (400 MHz,
CDCl3): δ 6.77 (d, J57 = 1.6 Hz, 1H, H-5), 6.72 (d, J75 = 1.6 Hz, 1H, H-7), 4.1 (s, 1H, NH), 3.82 (s, 3H,
-OCH3), 3.32 (t, J23 = 5.5 Hz, 2H), 2.74 (t, J43 = 6.4 Hz, 2H), 1.94 (m, J32 = 5.5 Hz, J34 = 6.3 Hz, 2H);
13 C NMR (100 MHz, CDCl3): δ 146.8, 133.6, 125.3, 124.1, 110.8, 107.1, 55.6 (-OMe), 41.3, 26.5, 22.0; MS
m/z (rel. int.%): 240 (50, M+), 242 (48), 225 (52), 226 (50), 145 (100), 146 (34), 129 (14), 117 (18), 101 (9),
90 (14), 76 (9), 62 (10); Anal. Calcd for C10 H12 BrNO (242.11): C, 49.61%; H, 5.00%; found: C 49.57%; H
4.95%.
6,8-Dimethoxy-1,2,3,4-tetrahydroquinoline (5). Pale brown oil. IR (KBr, cm−1): 3411 (NH),
2931, 2836, 2358, 2331, 1602, 1504, 1461, 1374, 1274, 1251, 1215, 1197, 1149, 1112, 1053, 1016, 935, 829; l H
NMR (400 MHz, CDCl3): δ 6.2 (d, J57 = 2.1 Hz, 1H, H5), 6.3 (d, J75 = 2.1 Hz, 1H, H-7), 3.29 (t, J23
=
5.1 Hz, 2H, H-2), 1.97 (m, J32 = 5.4 Hz, J34 = 6.3 Hz, 2H, H-3), 2.76 (t, J43 = 6.4 Hz, 2H, H-4), 3.8 (s, 3H,
OMe), 3.75 (s, 3H, OCH3) 4.1 (s, NH); 13 C NMR (100 MHz, CDCl3): δ 151.6, 147.7, 128.5, 122.0, 104.8, 96.8,
55.8, 55.4, 41.8, 27.0, 22.5; MS m/z (rel. int.%): 192 (50, M+), 177 (100), 134 (24), 117 (6), 106 (7), 90 (6),
76 (5), 64 (4); Anal. Calcd for C11 H15 NO2 (193.24): C, 68.37%; H, 7.82%; found: C 68.32%; H 7.78%.
4.3. Synthesis of 6,8-dimethoxyquinoline (6)
DDQ (504 mg, 1.76 mmol) in dry benzene (20 mL) was added to a solution of 6,8-dimethoxide 5 (165 mg, 0.84
mmol) in dry benzene (20 mL). The reaction mixture was stirred at 80 ◦ C for 40 h under an Ar atmosphere.
Reaction progress was monitored by TLC. After the mixture had cooled, the dark green solid was filtered,
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and the solvent was removed under reduced pressure. The residue was purified by a short silica column (1:9,
AcOEt/hexane). 6,8-Dimethoxyquinoline (6) was obtained in a yield of 78% (145 mg), as a yellowish oil: (6).
IR (KBr, cm−1): 2958, 2937, 1620, 1596, 1575, 1504, 1450, 1423, 1380, 1332, 1261, 1216, 1190, 1167, 1116,
l
1051, 1130, 995, 941, 835, 788, 770, 665, 620; H NMR (400 MHz, CDCl3): δ 8.57 (d, J23 = 4.0 Hz, 1H, H-2),
7.75 (d, J43 = 8.2 Hz, 1H, H-4), 7.14 (dd, J32 = 4.1 Hz, J34 = 8.2 Hz, 1H, H-3), 6.5 (d, 1H), 6.4 (d, 1H), 3.85
(s, 3H, OMe), 3.68 (s, 3H, OMe); 13 C NMR (100 MHz, CDCl3): δ 158.1, 156.1, 146.5, 136.7, 134.5, 129.8,
121.9, 101.0, 96.7, 55.8 (OCH3), 55.3 (OCH3); MS m/z (rel. int.%): 187 (100, M+), 188 (70), 189 (10), 159
(59), 158 (42), 157 (44), 144 (21), 143 (22), 129 (35), 115 (46), 116 (27), 102 (22), 88 (29), 75 (22), 62 (22).
Anal. Calcd for C11 H11 NO2 (189.21): C, 69.83%; H, 5.86%; found: C 69.74%; H 5.81%.
4.4. Synthesis of 8-bromo-6-methoxyquinoline (8) and 6-bromo-8-methoxyquinoline (9)
A solution of DDQ (504 mg, 1.76 mmol, 2.1 eq) in dry benzene (20 mL) was added to a mixture of 8-bromo-
6-methoxide 3 and 6-bromo-8-methoxide 4 mixture (50:50, 200 mg, 0.83 mmol) in dry benzene (20 mL). The
mixture was refluxed at 80 ◦ C for 2 days under an Ar atmosphere. Reaction progress was monitored by TLC.
After completing the reaction, filtering off the a dark green solid and removing the solvent under reduced
pressure, the reaction mixture (200 mg) was chromatographed (silica gel, 25 g) by eluting with AcOEt/hexane
(600 mL, 1:9). 6-Bromo-8-methoxyquinoline 9 (78 mg, 44%) and 8-bromo-6-methoxyquinoline 8 (76 mg, 42%)
were isolated in their pure forms and their Rf values were 0.65 and 0.40, respectively (AcOEt/hexane, 1:9).
l
8-Bromo-6-methoxyquinoline (8). White solid; mp: 78-79 ◦ C. H NMR (400 MHz, CDCl3): δ 8.94
(d, 1H, H-2), 8.05 (d, J43 = 4.0 Hz, 1H), 7.43 (dd, J32 = 4 Hz, 1H, H-3), 7.15 (d, J57 = 1.6 Hz, 1H, H-5),
7.58 (d, J75 = 1.6 Hz, 1H, H-7), 4.10 (s, 1H, OMe); 13 C NMR (100 MHz, CDCl3): δ156.0, 149.4, 139.0, 135.0,
130.0, 125.3, 122.6, 121.6, 120.5, 56.4; Anal. Calcd for C10 H8 BrNO (238.08): C, 50.45%; H, 3.39%; found: C
50.40%; H 3.33%.
6-Bromo-8-methoxyquinoline (9). White solid; mp: 75–76 ◦ C. IR (KBr, cm−1): 3027, 2358, 2341,
1585, 1546, 1467, 1440, 1348, 1301, 1232, 1181, 1151, 1081, 1021, 962, 862, 808, 775, 603, 593; l H NMR (400
MHz, CDCl3): δ 8.91 (dd, J24 = 2.8 Hz, J23 = 4.0 Hz, 1H, H-2), 8.07 (dd, J43 = 8.0 Hz, 1H), 7.47 (dd, J32
= 4.0 Hz, J34 = 8.0 Hz, 1H, H-3), 7.10 (d, J57 = 2.8 Hz, 1H, H5), 7.78 (d, J75 = 2.8 Hz, 1H, H-7), 3.94 (s,
1H, -OMe); 13 C NMR (100 MHz, CDCl3): δ157.5, 148.6, 141.3, 135.6, 130.9, 130.0, 125.9, 122.2, 105.4, 55.8;
MS m/z (rel. int.%): 236 (6, M+), 238 (6), 205 (35), 208 (35), 126 (100), 127 (14), 98 (34), 97 (19), 73 (44),
74 (25), 63 (39), 49 (50); Anal. Calcd for C10 H8 BrNO (238.08): C, 50.45%; H, 3.39%; found: C 50.41%; H
3.32%.
4.5. Synthesis of 6,8-dimethoxyquinoline (6)
Freshly cut sodium (0.7 g, 30 mmol) was added to dry methanol (25 mL) under nitrogen gas atmosphere. When
dissolution was complete, the warm solution was diluted with dry dimethylformamide by addition of vacuum
dried cuprous iodide (0.49 g, 1.72 mmol). After dissolution, 6,8-dibromoquinoline (3) (0.5 g, 1.72 mmol) was
added to dry DMF (15 mL). The reaction mixture was stirred magnetically under a nitrogen gas atmosphere
at reflux (ca. 150 ◦ C) for 20 h. The reaction’s progress was monitored by TLC until the starting material was
all consumed. After cooling to room temperature, H2 O (50 mL) and chloroform (70 mL) were added to the
reaction mixture. The organic layers were separated, washed with H2 O (2 × 20 mL), and dried over sodium
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sulfate. The solvent was removed and the crude product was passed through a short column packed with silica
gel (5 g). After filtration and purification, the resultant product was 6,8-dimethoxyquinoline (6) in a yield of
84% (0.28 g), as a pure yellowish oil substance.
4.6. Synthesis of 6,8-bis(trimethylsilyl)quinoline (11)
n-Butyl lithium (2.65 mL, 1.6 M, 4.26 mmol, 4.1 eq) was added to a vacuum-dried flask containing dibromide
7 (0.3 g, 1.04 mmol) in THF (20 mL) at –78 ◦ C and stirred for 90 min. Next, chlorotrimethylsilane (Me3 SiCl,
0.24 g, 2.2 mmol) was added to the mixture at –78 ◦ C, and the mixture was stirred at –78 ◦ C for 90 min and
then at room temperature for 1 h. After quenching with water (30 mL), the aqueous layer was extracted with
diethyl ether (3 × 25 mL). The combined organic layers were washed sequentially with water (3 × 25 mL) and
dried over sodium sulfate. The solvent was removed by reduced pressure, and the crude product was purified
using short silica gel column chromatography (hexane, Rf = 0.5). Recrystallization from CH2 Cl2 /hexane (2:1,
6 mL) gave product 11 (240 mg, 76%). Pale yellow solid; mp: 70–72 ◦ C. IR (KBr, cm−1): 3048, 3015, 2952,
2897, 1600, 1558, 1475, 1401, 1338, 1257, 1233, 1139, 1094, 998, 871, 833, 808, 772, 745, 688, 616; l H NMR
(400 MHz, CDCl3): δ 8.91 (dd, J23 = 4 Hz, J24 = 1.6 Hz, 1H, H-2), 7.37 (dd, J32 = 4 Hz, J34 = 8 Hz, 1H,
H-3), 8.12 (dd, J42 = 1.6 Hz, J43 = 8 Hz, 1H, H-4), 8.0–7.98 (d, H5 , H7), 0.48 (s, 9H, Si(CH3)3), 0.38 (s, 9H,
Si(CH3)3); 13 C NMR (100 MHz, CDCl3): δ152.9, 149.4, 140.0, 139.5, 138.0, 136.0, 135.1, 126.9, 120.7, –0.15,
–1.06; MS m/z(rel. int.%): 271 (21, M+), 272 (14), 273 (6), 257 (100), 258 (22), 259 (10), 241 (7), 227 (8), 198
(10), 169 (7), 155 (8), 121 (20), 107 (7), 72 (13), 68 (13); Anal. Calcd for C15 H23 NSi2 (273.52): C, 65.87%;
H, 8.48%. Found: C, 65.78%; H, 8.41%.
4.7. Synthesis of 6,8-bis(methylthio)quinoline (12)
n-Butyl lithium (2.65 mL, 1.6 M, 4.26 mmol, 4.1 eq) was added to a vacuum-dried flask containing dibromide
7 (0.3 g, 1.04 mmol) in THF (20 mL) at –78 ◦ C. After stirring for 90 min, 1,2-dimethyldisulfate ((CH3 S)2 ,
1.96 mL, 2.08 mmol) was added to the mixture at –78 ◦ C. The mixture was stirred at –78 ◦ C for 90 min and
then at room temperature for 1 h. After quenching the reaction with water (30 mL), the aqueous layer was
extracted with diethyl ether (3 × 25 mL). The combined organic layers were washed sequentially with water
(2 × 25 mL) and dried over sodium sulfate. The solvent was removed by reduced pressure, and the crude
product was purified using short silica gel column chromatography (hexane, Rf = 0.55). Recrystallization from
CH2 Cl2 /hexane (2:1, 5 mL) gave compound 12 (180 mg, 78%). Pale green sphere; mp: 92–94 ◦ C. IR (KBr,
l
cm−1): 3060, 3022, 2982, 2359, 2338, 1582, 1555, 1474, 1420, 1365, 1185, 995, 968, 865, 830, 787, 769, 607; H
NMR (400 MHz, CDCl3): δ 8.83 (dd, J23 = 4.4 Hz, J24 = 1.6 Hz, 1H, H-2), 8.00 (dd, J42 = 1.6 Hz, J43
= 8.4 Hz, 1H, H-4), 7.4 (dd, J32 = 4.4 Hz, J34 = 8.4 Hz, 1H, H-3), 7.24 (d, 1H), 7.22 (d, 1H), 2.59 (s, 3H,
SCH3), 2.57 (s, 3H, SCH3); 13 C NMR (100 MHz, CDCl3): δ 148.2, 143.8, 140.6, 137.8, 135.0, 128.4, 122.2,
122.0, 118.0, 15.6, 14.3; MS m/z (rel. int.%): 220 (100, M+), 221 (15), 222 (10), 187 (64), 188 (8), 175 (15),
174 (29), 173 (35), 159 (18), 128 (15), 129 (15), 115 (20), 102 (18), 86 (15), 80 (5); Anal. Calcd for C11 H11 NS2
(221.34): C, 59.69%; H, 5.01%; found: C 59.60%; H 4.92%.
4.8. Synthesis of quinoline-6,8-dicarbaldehyde (13)
n-Butyl lithium (2.65 mL, 1.6 M, 4.26 mmol, 4.1 eq) was added to a solution of dibromide 7 (0.3 g, 1.04
mmol) in THF (20 mL) in a vacuum-dried flask at –78 ◦ C. The mixture was stirred for 90 min, and then
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dimethylformamide (DMF, 0.15 g, 2.08 mmol) was added to the mixture at –78 ◦ C. The mixture was stirred
at –78 ◦ C for 90 min and then at room temperature for 1 h. After quenching with water (30 mL), the aqueous
layer was extracted with diethyl ether (3 × 25 mL). The combined organic layers were washed sequentially
with water (3 × 25 mL) and dried over sodium sulfate. The solvent was removed by reduced pressure, and
the crude product was purified by short silica gel column chromatography (AcOEt/hexane, 2:1, Rf = 0.23).
Recrystallization of the product from CH2 Cl2 /hexane (2:1, 5 mL) gave pure 13 (57 mg, 31%). White sphere;
mp: 193–195 ◦ C. IR (KBr, cm−1): 3056, 3037, 2865, 1697, 1677, 1608, 1572, 1494, 1460, 1425, 1257, 1157, 977,
l
900, 786, 745, 640, 574; H NMR (400 MHz, CDCl3): δ 9.21 (dd, J23 = 4.1 Hz,J24 = 1.8 Hz, 1H, H-2), 8.80
(d, J75 = 1.6 Hz, 1H, H-7), 8.67 (d, J57 = 1.6 Hz, 1H, H-5), 8.46 (d, J43 = 8.36 Hz, 1H, H-4), 7.67 (dd, J32
= 4.3 Hz, J34 = 8.2 Hz, 1H, H-3), 11.48 (s, 1H, -CHO), 10.30 (s, 1H, -CHO); 13 C NMR (100 MHz, CDCl3):
δ 191.8, 190.7, 153.8, 149.7, 137.8, 136.8, 133.7, 132.7, 128.2, 127.8, 122.9; MS m/z (rel. int.%): 185 (100,
M+), 186 (9.2), 156 (11.5), 127 (13.7), 118 (7.5), 101 (7.4), 86 (5), 76 (10), 56 (9); Anal. Calcd for C11 H7 NO2
(185.18): C, 71.35%; H, 3.81%; found: C 71.30%; H 3.74%.
4.9. Synthesis of the cyanoquinolines
6,8-Dibromoquinoline (7) (582 mg, 2 mmol) was dissolved in freshly distilled DMF (50 mL) and mixed with
cuprous cyanide (7.16 g, 8 mmol, 4 eq). The reaction mixture was stirred magnetically at reflux (ca. 150
◦ C) under Ar for 6 h. The hot resulting brown mixture was poured into a solution of hydrated ferric chloride
(4 g) and concentrated hydrochloric acid (1 mL) in water (10 mL) (any remaining residues were conveniently
transferred with hot dimethylformamide). The reaction mixture was maintained at 60–70 ◦ C for 20 min to
decompose the complex.
The separatory funnel was lit with light to observe the layers because the interface was obscured by dark
colors. The aqueous layer was extracted with warm toluene (4 × 50 mL), and the extracts were combined with
the organic layer, washed with dilute hydrochloric acid (1:1, 25 mL), water, and 10% aqueous sodium hydroxide.
The remaining organic layer was filtrated to remove the dark insoluble matter and dried over Na2 SO4 . After
evaporation of the solvent, NMR analysis of the residue (0.38 g) indicated the formation of 14, 15, and 16 in
a ratio of 12:47:22, respectively, and the reaction conversion was 83%.
The reaction mixture (380 mg) was applied to a short silica gel (60 g) chromatography column eluting with
AcOEt/hexane (600 mL, 1:9). 6-Bromo-8-cyanoquinoline 15 was collected as the first eluent (190 mL, 46 mg,
9%), and then the solvent polarity was increased to a ratio of 2:9 (AcOEt/hexane). 8-Bromo-6-cyanoquinoline
14 (240 mL, 200 mg, 43%) was next obtained, and then the polarity of the solvent was increased to the ratios
of 3:9 and 4:9 (AcOEt/hexane). 6,8-Dicyanoquinoline 16 (240 mL, 70 mg, 20%) was isolated in pure form.
The reaction was repeated for 16 h using the same reaction conditions. After the reaction, a complex
reaction mixture was obtained; polymerization likely occurred.
The reaction was repeated at 100 ◦ C for 2 days under the same reaction conditions. However, the reaction
did not run at the reaction temperature.
8-Bromo-6-cyanoquinoline (14). White needle crystalline; mp: 176–178 ◦ C. IR (KBr, cm−1): 3076,
l
3046, 2229, 1581, 1567, 1483, 1361, 1307, 1238, 1200, 1135, 1093, 1032, 887, 840, 785, 723; H NMR (400 MHz,
CDCl3): δ 9.13 (dd, J23 = 4 Hz, J24 = 2 Hz, 1H, H-2), 7.60 (dd, J32 = 4 Hz, J34 = 8 Hz, 1H, H-3), 8.19
(dd, J43 = 8 Hz, J42 = 1.6 Hz, 1H, H-4), 8.22 (d, J57 = 2.0 Hz, 1H, H-5), 8.25 (d, 1H, H7); 13 C NMR (100
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MHz, CDCl3): δ 152.8, 146.2, 138.3, 135.4, 134.9, 129.2, 123.6, 119.0, 115.7, 115.0; MS m/z (rel. int.%): 231
(100, M+), 233 (98), 152 (64), 125 (46), 98 (32), 75 (29), 78 (28), 62 (21), 50 (18), 49 (22); Anal. Calcd for
C10 H5 BrN2 (233.06): C, 51.53%; H, 2.16%. Found: C, 51.48%; H, 2.13%.
6-Bromo-8-cyanoquinoline (15). White needle crystalline; mp: 180–182 ◦ C. IR (KBr, cm−1): 3073,
l
3046, 2231, 1583, 1563, 1483, 1363, 1311, 1236, 1098, 1141, 1095, 1033, 883, 842, 784, 725; H NMR (400 MHz,
CDCl3): δ 9.08 (d, J23 = 4 Hz, 1H, H-2), 7.58 (dd, J32 = 4 Hz, J34 = 8 Hz, 1H, H-3), 8.17–8.22 (m, 3H, H-4,
H-5, H-7); 13 C NMR (100 MHz, CDCl3): δ 152.8, 146.1, 138.2, 135.5, 134.9, 129.2, 123.6, 119.0, 115.8, 114.8;
MS m/z (rel. int.%): 231 (100, M+), 233 (97), 232 (11), 152 (65), 125 (48), 98 (34), 75 (30), 74 (30), 62 (22),
49 (23), 50 (20); Anal. Calcd for C10 H5 BrN2 (233.06): C, 51.53%; H, 2.16%. Found: C, 51.49%; H, 2.11%.
6,8-Dicyanoquinoline (16). Yellow sphere; decomposed at melting point (260 ◦ C). IR (KBr, cm−1):
3062, 2362, 2229, 1590, 1569, 1488, 1452, 1375, 1321, 1163, 1031, 930, 902, 881, 785; l H NMR (400 MHz,
CDCl3): δ 9.27 (dd, J23 = 4 Hz, J24 = 1.6 Hz, 1H, H-2), 7.74 (dd, J32 = 4 Hz, J34 = 8 Hz, 1H, H-3), 8.36
(dd, J43 = 8.4 Hz, J42 = 1.6 Hz, 1H, H-4), 8.5 (d, J57 = 1.6 Hz, 1H, H5), 8.3 (d, J75 = 1.6 Hz, 1H, H-7); 13 C
NMR (100 MHz, CDCl3): δ 155.3, 148.2, 138.2, 136.8, 135.8, 127.8, 124.4, 116.5, 115.5, 115.2, 110.5; MS m/z
(rel. int.%): 178 (100, M+), 179 (13), 151 (35), 124 (10), 98 (9), 74 (17), 61 (6); Anal. Calcd for C11 H5 N3
(179.18): C, 73.74%; H, 2.81%. Found: C, 73.70%; H, 2.77%.
4.10. Cell culture and cell proliferation assay
HeLa cells (human cervix carcinoma), HT29 (human colon carcinoma) cells, and C6 (rat brain tumor cells)
were grown in Dulbecco’s modified eagle’s medium (DMEM, Sigma) and supplemented with 10% (v/v) fetal
bovine serum (Sigma, Germany) and PenStrep solution (Sigma, Germany) at 37 ◦ C in a 5% CO2 humidified
atmosphere. For the proliferation assay, cells were plated in 96-well culture plates (COSTAR, Corning, USA) at
a density of 30,000 cells per well. Vehicle (DMSO), 5-Florouracil (5-FU), and various quinoline compounds at
the concentrations of (5, 10, 20 30, 40, 50, 75, and 100 µg/mL) were added to each well. The concentration of the
DMSO was less than 0.5% in all assays. The antiproliferative activities of the compounds were tested by using
a BrdU Cell Proliferation ELISA kit (Roche, Germany) according to the manufacturer’s procedure. Briefly, the
cells were incubated with the compounds overnight before applying the BrdU Cell Proliferation ELISA reagent.
Then the cells were pulsed with a BrdU labeling reagent for 4 h, followed by fixation in a FixDenat solution
for 30 min at room temperature. Thereafter, cells were incubated with a 1:100 dilution of anti-BrdU-POD
for 90 min at rt. The amount of cell proliferation was assessed by determining the A450 nm of the culture
media after the addition of the substrate solution using a microplate reader (Ryto, China). The results were
reported as percentages based on the inhibition of cell proliferation, where the optical density measured for the
vehicle-treated cells was considered to be 100% proliferated. The experiments were repeated at least twice. The
percent inhibition of cell proliferation was calculated as follows: [1 – (Atreatments/Avehicle control)] × 100.
4.11. Statistical analysis
The data are presented as the means ꢀ SEM of the 6 measurements for each cell type. The differences between
the treatment groups were tested using ANOVA and P values, where values of <0.05 were considered significant.
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5. Acknowledgments
¨
This study was supported by grants from the Scientific and Technological Research Council of Turkey (TUBITAK,
˙
Project number: 112T394).
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