Design, Synthesis and Anticancer Evaluation of New Substituted Thiophene-Quinoline Derivatives

Article abstract of DOI:10.1016/j.bmc.2019.07.042

A series of new isoxazolyl, triazolyl and phenyl based 3-thiophen-2-yl-quinoline derivatives were synthesized adopting click chemistry approach. In addition, the synthesis of new useful synthon, (2-chloroquinolin-3-yl) (thiophen-2-yl) methanol, is reported. The obtained compounds were characterized by spectral data analysis and evaluated for their anticancer activity. All the derivatives were subjected to in vitro MTT cytotoxicity screening assay against a panel of four different human cancer cell lines, liver (HepG-2), colon (HCT-116), human cervical cancer (HeLa) and breast (MCF-7). Out of a library of 17 compounds, two compounds have been identified as potent and selective cytotoxic agents against HeLa and MCF-7 cell lines. SAR studies for such hybridized analogues were investigated and phenyl derivatives were proved to be more potent than isoxazole and triazole derivatives. Furthermore, the promising compounds were selected for in vitro inhibition of EGFR-TK and Topo II enzymes. Also, they were subjected to cell cycle arrest analysis and apoptosis assay on MCF-7 cells. Our recent finding highlights these thiophene-quinoline analogues as a promising class of compounds for further studies concerning new anticancer therapies.

Full text of DOI:10.1016/j.bmc.2019.07.042

Journal Pre-Proof
Design, Synthesis and Anticancer Evaluation of New Substituted Thiophene-
Quinoline Derivatives
Dina I.A. Othman, Khalid B. Selim, Magda A. El-Sayed, Atif S. Tantawy, Yhiya
Amen, Kuniyoshi Shimizu, Tatsuo Okauchi, Mitsuru Kitamura
PII:
S0968-0896(19)31149-6
https://doi.org/10.1016/j.bmc.2019.07.042
BMC 15026
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 July 2019
25 July 2019
26 July 2019
Please cite this article as: Othman, D.I.A., Selim, K.B., El-Sayed, M.A., Tantawy, A.S., Amen, Y., Shimizu, K.,
Okauchi, T., Kitamura, M., Design, Synthesis and Anticancer Evaluation of New Substituted Thiophene-Quinoline
Derivatives, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.042
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JOURNAL PRE-PROOF
Design, Synthesis and Anticancer Evaluation of New Substituted
Thiophene-Quinoline Derivatives
Dina I. A. Othman1, 2, Khalid B. Selim2, Magda A. El-Sayed2, Atif S. Tantawy2, Yhiya Amen3, 4,
Kuniyoshi Shimizu3, Tatsuo Okauchi1, Mitsuru Kitamura1
1Department of Applied Chemistry, Kyushu Institute of Technology1-1 Sensuicho,
2
Tobata, Kitakyushu, 804-8550 Japan. Department of Pharmaceutical Organic
Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
3Division of Systematic Forest and Forest Products Sciences, Department of Agro-
Environmental Sciences, Graduate School of Bioresource and Bioenvironmental
Sciences, Kyushu University, Fukuoka, Japan. 4Department of Pharmacognosy, Faculty
of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
Highlights
 New series of thiophene-quinoline hybrids were synthesized and screened for
anticancer activity. Some new compounds displayed strong anticancer activity
against MCF-7 cancer cells.
 The promising compounds were selected for in vitro inhibition of Epidermal
Growth Factor Receptor tyrosine kinase (EGFR-TK) and topoisomerase II
(Topo II) enzymes.
 The most potent compounds 7d and 7e were able to cause G2/M cell cycle arrest
and demonstrated activity as caspase-3 and caspase-9 activator and apoptosis
inducer.
Abstract
A series of new isoxazolyl, triazolyl and phenyl based 3-thiophen-2-yl-quinoline
derivatives were synthesized adopting click chemistry approach. In addition, the
synthesis of new useful synthon, (2-chloroquinolin-3-yl) (thiophen-2-yl) methanol, is
reported. The obtained compounds were characterized by spectral data analysis and
evaluated for their anticancer activity. All the derivatives were subjected to in vitro
MTT cytotoxicity screening assay against a panel of four different human cancer cell
lines, liver (HepG-2), colon (HCT-116), human cervical cancer (HeLa) and breast
(MCF-7). Out of a library of 17 compounds, two compounds have been identified as
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potent and selective cytotoxic agents against HeLa and MCF-7 cell lines. SAR studies
for such hybridized analogues were investigated and phenyl derivatives were proved to
be more potent than isoxazole and triazole derivatives. Furthermore, the promising
compounds were selected for in vitro inhibition of EGFR-TK and Topo II enzymes.
Also, they were subjected to cell cycle arrest analysis and apoptosis assay on MCF-7
cells. Our recent finding highlights these thiophene-quinoline analogues as a promising
class of compounds for further studies concerning new anticancer therapies.
Key words Click Chemistry; Thiophene-Quinoline Hybrid; Isoxale; Triazole; Cytotoxic
Activity; Apoptosis.
1. Introduction
Cancer, which is the result of uncontrolled growth of the cells, remains one of the life-
threatening diseases causing death worldwide [1]. As a result, the development of new
anticancer drugs with high efficacy and minimal side effects remains an active field of
investigation in medicinal chemistry. The synthesis and functionalization of quinoline
nucleus has become a major area of focus for synthetic organic chemists because of
their several pharmacological functions, especially as anticancer agent [2-7]. It was
reported that, quinoline derivatives may act as anticancer agents through a variety of
mechanisms such as cell cycle arrest in the G2 phase [8], topoisomerase inhibition [9],
inhibition of tubulin polymerization [10], and inhibition of tyrosine kinases which is the
most common mechanism [11, 12]. In addition, increased attention has been devoted to
the thiophene nucleus due to its biological activity. It has been found in many promising
anticancer compounds [13], such as Raltitrexed (Tomudex®) (Figure 1), which causes
DNA fragmentation and subsequent cell death through inhibition of the thymidylate
synthase enzyme [14]. In the same time, the diphenylmethanol framework have also
퐈
been explored as potential antiproliferative agents [15]. Thus, based on a hybrid
pharmacophore design [16], we envisioned that applying the structural features of
diphenylmethanol I, by replacing the 2 phenyl moieties with the previously reported
antitumor active quinoline and thiophene rings, might provide a new synthon II for the
discovery of new anticancer agents (Figure 1). Moreover, it was reported that,
substituted isoxazole III [17], triazole IV [17, 18] and phenyl derivatives V [19] could
enhance the antitumor activity in different previous studies. Therefore, our new
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synthone II was further modified by incorporating substituted isoxazole, triazole and
phenyl moieties through hydroxyl group functionality to get more potent anticancer
molecules with multi-targeted molecular mechanisms (Figure 1).
Herein, we wish to report our initial findings on the synthesis and in vitro
pharmacological evaluation of a series of new 3-[(substituted methoxy)(thiophen-2-
yl)methyl]-2-chloroquinoline derivatives. To the best of our knowledge, the synthesis
and pharmacological properties of this new series have not been explored earlier.
O
N
N
N
H
OH
O
S
S
`R NH
N
O
O
Ar1
HO
S
H
O
Raltitrexed
OH
OH
anticancer drug
Ar2
N
Cl
O


Ph
N
New designed hybrid synthone
Diphenyl methanol framework
with reported antitumor activity
S
R
N
Cl
S
Thiophene and quinoline
derivatives with reported
anticancer agents
S
S
O
O
N
X
O
N
X
X
O
O
N
Y
N
N
Cl
Y
Y
S
X= O, N
Y= C, N
R
OH
R
R

X= O, Y= C
X= N, Y=N
N
Cl
S
V


OH
Several
active
moities
N
S
H
N
H
O
N
incorporated with OH group
N
R
N
Cl
O
R
n
O
V
R
Proposed designed target compounds
III, IV
V
and benzyloxy derivatives with
Aryloxy derivatives
reported potent antitumor activity
Figure 1: The designed framework of our target compounds compared to some previously
reported anticancer agents.
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2. Results and Discussion
2.1 Chemistry
S
2-bromothiophene (2 eq.)
CHO
Cl
DMF, POCl3
80 oC, 16 h
OH
Mg (2 eq.), LiCl (0.6 eq.)
THF, 0 oC, rt, 2 h
80%
N
O
N
N
3
Cl
H
70%
2
1
Scheme 1: The synthesis of new synthon, (2-chloroquinolin-3-yl)(thiophen-2-yl) methanol (3).
Initially, 2-chloroquinolin-3-carbaldehyde (2) was prepared with the Vilsmeier reagent
(DMF+POCl3) through traditional Vilsmeier–Haack cyclization of acetanilide 1 [20].
Then, the Grignard reagent freshly prepared from 2-bromothiophene was reacted with
compound 2 to afford (2-chloroquinolin-3-yl)(thiophen-2-yl)methanol (3) (Scheme 1)
according to previously reported procedure [21]. Propargylation of alcohol 3 with
propargyl
bromide
furnished
our
desired
alkyne,
2-chloro-3-[(prop-2-
ynyloxy)(thiophen-2-yl)methyl]quinoline (4) (Table 1). Table 1 resumes all
experiments, which were carried out to reach the optimal reaction conditions for the
synthesis of our target alkyne 4. First, the alcohol 3 was reacted with propargyl bromide
(1 eq.) in the presence of K2CO3 (1 eq.) in acetone at rt for 48 h to yield 26 % of the
alkyne 4 (Entry 1). While, using stronger base like NaH in anhydrous THF gave 18 %
yield (Entry 2). In both cases, we suffered from the low yield of the target compound in
addition to the recovery of the starting material which may indicate the instability of
both alkyl bromide and NaH. In addition, NaH is insoluble in THF and DMF so the
reaction is not homo phase media. Therefore, the use of excess amount of NaH is
suggested to be better for this reaction. As a result, we decided to increase the
equivalences of both alkylating agent and base. Using 3 eq. of both propargyl bromide
and NaH in THF or DMF were verified to improve the yield of our alkyne 4 to 46 %
and 66 %, respectively (Entries 3 and 4). The formation of alkyne 4 could be easily
confirmed by the appearance of two proton signals at
2.52 and 4.24 ppm, assigned to
훿
one terminal alkyne proton and two methylenic protons, respectively.
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Click chemistry inspired approach, involving union of this terminal alkyne 4 with
freshly prepared oximes or azides, has been accomplished for the synthesis of our new
isoxazole and triazole derivatives, as shown in Scheme 2.
Table 1: Optimization of reaction conditions for the synthesis of alkyne 4.
S
S
Proparagyl bromide
base
O
OH
rt
N
Cl
4
N
Cl
conditions
3
Conditions
Entry Proparagyl bromide (eq.)
Base (eq.) Solvent
Time (h)
Yield (%)
1
2
3
4
1.1
1.1
3
K2CO3 (1) acetone
48
24
24
6
26
18
46
66
NaH (1)
NaH (3)
NaH (3)
THF
THF
DMF
3
The one-pot reaction presenting the 1,3-dipolar cycloaddition reaction of terminal
alkyne and aromatic oximes was adopted and proved to provide an efficient,
straightforward and metal-free reaction for the synthesis of substituted isoxazoles under
mild condition. In the present work, the terminal alkyne 4 was subjected to [3+2]
cycloaddition with numerous freshly prepared oximes in THF in the presence of N-
chlorosuccinimide (NCS) [17] to yield the corresponding isoxazole derivatives 5a-f. It
was noted that, this cycloaddition is regioselective, in that only 3,5-disubstituted
isoxazoles were isolated and confirmed by spectroscopic data. The structure of the
1
obtained isoxazole derivatives were assigned depending on H NMR spectrum which
exhibited the appearance of H-4 proton singlet peak (around 6.56 ppm) with two
protons (-CH2) at 4.5-4.8 ppm region.
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R
S
Cpd. No.
R
Yield (%)
O
5a
5b
5c
5d
5e
5f
4-CH3
4-OCH3
3,4-(OCH3)2 49
4-OH 10
3,4,5-(OCH3)3 41
27
40
O
N
OH
N
a
N
Cl
5a-f
3,5-(CH3)2
33
R
4
S
Cpd. No.
R
Yield (%)
R
N3
N
O
N
6a
6b
6c
6d
6e
OCH3 48
N
b
Cl
6a-e
N
NO2
CH3
CF3
Cl
36
35
71
69
R
Scheme 2: Reagents and conditions: a) Oxime derivatives (1 eq.), N-chlorosuccinimide (1eq.),
K2CO3 (4 eq.), THF, rt, overnight, b) Azide derivatives (1eq.), Na.ascorbate (0.3 eq.),
CuSO4.5H2O (0.03 eq.), H2O:t.BuOH(1:1), rt, overnight.
Similarly, all the triazole derivatives 6a-e were accessed under click chemistry
condition using the same key template alkyne 4 (Scheme 2). The Cu(I)-catalyzed
Huisgen 1,3-dipolar azide-alkyne cycloaddition reaction, has become one of the most
common click reaction used to provide various five-membered heterocyclic compounds
[22]. The Cu-catalyzed version permits the specific preparation of the 1,4-regioisomers,
and it could be performed at rt under aquous conditions (Sharpless condition) [22].
In the present work, all triazole derivatives 6a-e were accessed under Sharpless click
chemistry condition, using alkyne 4 (Scheme 2). A combination of sodium ascorbate
with CuSO4.5H2O was used as a source of Cu(I) and the reactions were conducted in t-
BuOH:H2O (1:1) at rt. All aryl azides needed in this part were freshly synthesized
through standard one-pot diazotization reaction of the appropriate aromatic amines
following by SNAr displacement of the diazonium group with NaN3 [23]. The structures
of the synthesized triazoles could be easily proved by the downfield H-5 proton singlet
peak (~8.49 ppm) and methylene protons at ~ 4.94 ppm. The structures of all the
obtained derivatives were characterized based on the spectroscopic data (1H -NMR, 13C-
NMR, IR and HR-ESIMS).
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Cpd. No.
R
Yield (%)
S
7a
7b
7c
7d
7e
H
57
86
45
64
62
S
Br
4-Br
3-CF3
4-F
R
O
OH
a
N
Cl
R
4-Cl
N
Cl
7f
2,4,6-(CH3)3 58
7a-f
3
Scheme 3: Reagents and conditions: a) (un)Substituted benzyl bromides (3eq.), NaH (3 eq.),
DMF, 0 oC, rt, 1-2.5 h
The synthesis of other analogues was also attempted through nucleophilic substitution
reaction of our new alcohol 3 with various benzyl bromide derivatives (Scheme 3).
This reaction was carried out under the previously mentioned conditions (Entry 4,
1
Table 1) to yield a series of benzyloxy derivatives 7a-f with characteristic H NMR
signal (around 4.5 ppm) corresponding to the benzylic protons.
2.2 Biological activity
2.2.1 MTT Cell Culture Cytotoxic Assay
The new (2-chloropyridin-3-yl)(thiophen-2-yl) methanol (3), its propargylated product 4
and the isoxazolyl derivatives 5a-f, triazolyl derivatives 6a-e and phenyl derivatives 7a-
f were tested for cytotoxic activity against a panel of four different human cancer cell
lines; liver (HepG2), cervix (HeLa), colon (HCT-116) and breast (MCF-7) using
colorimetric MTT cytotoxicity screening assay [24]. To investigate the most promising
analogues, each compound was preliminary tested in three independent experiments in
each of the cell line and the results are summarized in table 2 and presented as %
growth inhibition values (GI). Unfortunately, the triazolyl derivatives 6a-e did not
exhibit GI% higher than 50%. The other promising analogues, exhibiting significant
cytotoxic effect, (~ 50% GI) at the preliminary screening concentration, were further
assayed at different concentrations to generate the IC50 values as illustrated in table 3.
The standard drug used in this assay was 5-Flurouracil (5-FU).
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Table 2: Percentage Growth inhibition (GI%) of the target compounds using MTT
colorimetric assay method.
S
S
S
N
O
O
O
O
N
N
R
N
Cl
6a-e
N
Cl
5a-f
N
N
Cl
7a-f
R
R
Cpd
GI%[a]
HepG2
HeLa
HCT-116
MCF-
7
No.
5a
R
10mM
43.91 ± 2.12 22.03 ± 4.26
4-CH3
4-OCH3
31.77±11.29
17.93 ± 7.38
NA
NA
NA
NA
NA
NA
NA
NA
NA
5b
5c
20.31 ± 3.04
NA
NA
NA
NA
NA
NA
NA
NA
3,4(OCH3)2
4-OH
5d
64.51±2.35
14.27 ± 2.21
NA
5e 3,4,5-(OCH3)3 14.57±5.31
5f
6a
6b
3,5-(CH3)2
4-OCH3
4-NO2
NA
NA
NA
NA
32.52 ± 2.96
NA
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6c
6d
6e
7a
7b
7c
7d
7e
4-CH3
4-CF3
4-Cl
H
18.78 ± 2.47
NA
NA
13.03 ± 4.38
NA
NA
NA
8.39 ± 7.35
NA
35.86 ± 0.77
23.13 ± 2.16
NA
NA
NA
NA
45.63 ± 3.4
NA
4-Br
3-CF3
4-F
NA
30.89 ± 1.2
42.42 ± 4.35
21.98 ± 0.14
23.22 ± 0.7
NA
NA
52.90 ± 1.46
64.97 ± 0.4
47.94 ± 0.67
30.53±4.25 66.21 ± 1.42
NA 30.41 ± 1.11
4-Cl
NA
7f
2,4,6-(CH3)3 24.76± 3.14 42.48 ± 4.33 26.37 ± 7.59 21.49 ± 0.45
[a]
Each experiment was carried out in triplicate so that the values are the average of triplicate
analysis. NA: not active, exhibit GI% lower than 10 %. Bold values show the most promising
compounds.
Table 3: Cytotoxicity of the promising quinoline derivatives 3, 4, 5 and 7 against,
HepG2, HeLa, HCT-116 and MCF-7 cancer cell lines.
S
S
S
S
O
OH
O
O
O
N
R
N
Cl
N
Cl
N
Cl
5a-f
N
Cl
3
4
7a-f
R
[a]
흁
In vitro Cytotoxicity IC50 ( M)
Cpd. No.
R
HepG-2
HeLa
HCT-116
MCF-7
>100
5-FU[b]
>100
20.4±2.5
63.9±3.8
3
4
-
80.8±5.21 17.5±4.62 69.3±2.8
80.6±3.47
>100
-
>100
NA
34.7±2.31
NA
>100
5a
4-CH3
114.6±2.12
NA
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5d
7a
7c
7d
7e
4-OH 178.5±2.35
NA
NA
NA
NA
NA
NA
NA
NA
H
NA
NA
NA
NA
108.4±3.4
100.6±1.46
38.4±7.01
28.3±10.21
3-CF3
4-F
NA
73.93±6.9
NA
4-Cl
[a]
The unmentioned derivatives did not pass preliminary screening; IC50 are the mean of three
휇
separate experiment ± SD and represent concentration of the compound ( M) producing 50% cell
growth inhibition after 72 h of drug exposure, as determined by the MTT assay. Bold values
represent the IC50 values of the target compounds with lower value than the positive control; NA =
Not active. [b] 5-FU was used as the positive control.
2.2.1.1 Results and structure activity relationship study
As depicted from table 2, among the tested isoxazoles, compounds 5a and 5d with
methyl or hydroxyl substitution showed good activity against HCT-116 and HePG2
cancer cell lines, respectively. Whereas; compounds 5c and 5f have shown no activity
against same cell lines. While, among the triazoles, compounds 6a, 6c, 6d and 6e
showed weak selective cytotoxic activity against HePG2 and HCT-116 cancer cell lines
and no activity against HeLa and MCF-7 cancer cell lines. Interestingly, among the
tested benzyloxy derivatives, most compounds showed activity against all tested cancer
cell lines, particularly more selective activity towards HeLa and MCF-7 cancer cell
lines with high % GI value.
As shown in table 3, compounds 5a, 5d and 7a, 7c, 7d, 7e showed more potent
anticancer activity and compounds 7d and 7e displayed higher efficacy against MCF-7
with an IC values of 38.41 and 28.36 M, respectively. Their IC values were more
휇
50
50
potent than that of the reference drug. SAR analyses revealed that introducing a
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benzyloxy group as a lipophilic group at position-3 of the quinoline nucleus improves
the in vitro cytotoxic activity compared to isoxazolyl and triazolyl derivatives.
Moreover, close inspection of the substituents attached to the benzene ring in 7a-f series
reveals that the presence of halogen (F, CF3 or Cl group) could apparently enhance
cytotoxic activity (as in 7c, 7d, 7e). The 4-chloro-substituted derivative 7e was the most
active compound compared to other substituted derivatives. The above outcomes
illustrated that the electronic effect of the benzyloxyl group may be a factor contributing
to cytotoxicity, and also halogen group at the 3 or 4-position of the benzyloxyl group
may have a powerful impact. It is worth noting that, these observed results are in good
agreement with the previously reported results which revealed that compounds having
phenyl ring substituted with halogen group at 4-position displayed a significant
antitumor effect [25]. Furthermore, the cytotoxicity profile of the parent starting
materials 3 and 4 indicates that they could be promising synthons for future
investigation with IC values of 17.5 and 34.7 M, respectively, against HeLa cell line.
휇
50
2.2.2 In vitro inhibition of Epidermal Growth Factor Receptor kinase (EGFR-TK)
and Topoisomerase II (Topo II) enzymes (IC50 determination)
Quinoline scaffolds were emerged as potent dual inhibitors of EGFR kinase and human
Topo IIα activity at a micromolar level against different cancer cell lines comparable to
erlotinib [9, 11, 12, 26]. As a result, we aimed to evaluate the inhibitory activity of the
most promising quinoline derivatives toward both enzymes. Quinoline derivatives 3, 7d
and 7e were evaluated for their antiproliferative effect against the EGFR-overexpressing
tumor cell lines, HeLa and MCF-7 and their IC50 values were determined. The tested
compounds showed prominent inhibitory activities with IC50 values in the micromolar
range. Erlotinib, with high selectivity and potency to EGFR-TK, was selected as a
positive drug in this kinase assay. After treatment with different concentration of
compounds 3, 7d and 7e, IC50 was calculated from concentration–response curve to be
6.3, 2.75 and 3.66 µM, respectively. They were slightly weaker comparable to the
positive control (Table 4) (Figure 2). We concluded that, the tested compounds could
inhibit the function of EGFR-TK with a dose-dependent manner. Therefore, the anti-
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proliferative activity might attribute partly due to the interaction between EGFR-TK and
the tested compounds.
IC
EGFR -
50
7
6
5
4
Ref. Erlotinib
µM
3
2
1
0
3/HeLa
7d/MCF77e/MCF7
Ref/MCF7
Ref/HeLa
Figure 2: EGFR inhibitory chart of compounds 3, 7d and 7e, compared to Erlotinib.
Table 4: Results of in vitro EGFR inhibition (IC50 Values) for compounds 3, 7d and
7e.[a]
Compound No.
3
7d
7e Erlotinib
IC50 µM (MCF-7)
-
2.75 3.66
1.88
2.72
IC50 µM (HeLa) 6.3
-
-
[a] The IC50 values are represented as the means of the data obtained from three separate experiments.
Variation is generally 5%.
Then, we aimed to investigate the behavior of the same compounds toward Topo-II
enzyme which is crucial for cell viability and normal cell cycle progression. [27]. To
determine IC50, different concentrations of compounds 3, 7d and 7e was used. The IC50
values and % inhibition at various concentrations were calculated and listed in table 5
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(Figure 3). It was observed that, compounds 7d and 7e exhibited more potent Topo-II
inhibitory activity, while compound 3 displayed weaker activity, compared to the
positive control Topotecan.
Topo II
6
5
4
µM3
Ref.Topotecan
2
1
0
3/HeLa
7d/MCF-7 7e/MCF-7
Ref/MCF-7
Ref/HeLa
Figure 3: Topo-II inhibitory chart of compounds 3, 7d and 7e, compared to Topotecan.
Table 5: Results of in vitro Topo-II inhibition for compounds 3 and 7d and 7e.
Compound No. Conc. µM % Inhibition IC50 Cell line
25 µM
5 µM
80.67064
44.96212
20.03869
3.852974
93.6537
4.95
HeLa
3
1 µM
0.2 µM
25 µM
5 µM
88.49733
70.78566
43.29519
93.14798
0.23 MCF-7
0.44 MCF-7
7d
1 µM
0.2 µM
25 µM
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7e
5 µM
1 µM
84.68345
63.34096
36.27002
92.84744
85.59115
66.49123
31.97559
0.2 µM
25 µM
5 µM
0.48 MCF-7
Topotecan
1 µM
0.73
HeLa
0.2 µM
2.2.3 Caspases activity (Caspase-3 and Caspase-9)
Caspases are proteolytic enzymes which play a crucial role in the regulation of
apoptosis. The activation of these proteases leads to protein substrates cleavage inside
the cell and consequently induces irreversible apoptotic cell death. The activation of
caspases is considered the best recognized biochemical hallmark of apoptosis [28].
Thus, the activation of caspase-3 in MCF-7 and HeLa cells treated with compounds 3,
7d and 7e was investigated.
The concentration of active caspase-3 was measured using ELISA technique. The
experimental results were expressed in term of Caspase-3 concentration Pg/mL (Table
6). In addition, the fluorescence density produced by tested and reference compounds
was illustrated in figure 4. It was observed that, compound 3 increased caspase-3
concentrations in HeLa by 13 fold, while compounds 7d and 7e induced activation of
caspase-3 in MCF-7 by 31 and 34 fold, respectively compared to its control. While all
compounds were weaker than reference drug sorafenib. Thus, these compounds may be
considered as promising apoptotic inducers through activation of caspase-3.
Table 6: Results of caspase-3 activation for compounds 3 and 7d, e.[a]
Compound No. Caspase-3 conc.(Pg/mL) FLD Cell line
Control
3
22.42
287.2
617.7
-
12.8
35.5
HeLa
Ref.
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Control
7d
17.38
536.4
594.4
17.38
586.2
594.4
-
30.8
34.2
-
Ref.
Control
7e
MCF-7
33.7
34.2
Ref.
[a] Reference drug used in this assay was sorafenib.
Casp -3
40
35
30
25
20
15
10
5
Ref. Sorafenib
fld
0
3 /HeLa
Ref/MCF7
7d /MCF77e /MCF7
Ref/HeLa
Figure 4: Fluorescence response graph in Casp-3 assay.
Then, the activation caspase-9 in MCF-7 and HeLa cells treated with compounds 3, 7d
and 7e was examined. The experimental results were shown in table 7 and recognized in
term of Caspase-9 concentration ng/mL. Furthermore, the fluorescence density chart
displayed by the tested and reference compounds was shown in figure 5. As observed,
the treatment with compounds 7d and 7e increased the expression of caspase-9 in MCF-
7 by 40 and 29 fold, respectively compared to the control. Moreover, they displayed
better expression than the standard reference drug (5-FU). Compound 3 induced
activation caspase-9 in HeLa cells by 72 fold comparable to the control and slightly
weaker than standard 5-FU.
Table 7: Results of caspase-9 activation for compounds 3, 7d and 7e.
FLD
Cell
line
Compound No. Caspase-9 conc (ng/mL)
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Control
3
0.25
18.16
23.6
-
72.6
94.4
-
HeLa
5-FU
Control
7d
0.816
33.16
21.33
40.6
26.1
-
5-FU
Control
7e
0.819
23.76
21.33
MCF-7
29
26
5-FU
Casp-9
100
90
80
70
60
50
40
30
20
10
0
Ref.5-FU
fld
Ref/MCF7
7e /MCF7
3 /HeLa
Ref/HeLa
7d /MCF7
Figure 9: Fluorescence response graph in Casp-9 assay.
Altogether, our results suggested that, both compounds 7d and 7e were able to induce
caspase-dependent intrinsic pathway of apoptosis in MCF-7 cells. Besides, our synthon
3 might be considered as promising apoptotic inducer in HeLa cells for future studies.
2.2.4 Cell cycle analysis and apoptosis detection assay
Cell cycle is the sequence of growth and development steps that lead to DNA
replication and cell division. It consists of four distinct phases: G1 phase, S phase
(synthesis), G2 phase and M phase. Our promising target compounds 7d and 7e were
subjected to cell cycle analysis and apoptotic assay aiming to investigate their ability to
play an active role in the cell cycle progression on MCF-7 cells.
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2.2.4.1 Cell cycle analysis on MCF-7 cells by flow cytometry
To evaluate the effect of our compounds 7d and 7e on cell cycle progression of MCF-7
cancer cells, this cell line was incubated with IC50 concentration of our compounds for
24 h. The cell lines were stained with propidium iodide (PI) and analyzed by flow
cytometry using BD FASCCallbur reader. Quantification of the results (Table 8, Figure
10) revealed that compounds 7d and 7e induced pre G1 apoptosis by 16.28- 17.05 %.
The tested compounds showed a high percent of cell accumulation at G2/M phase
indicating the arrest of cell cycle at this phase before entering mitosis. The flow
cytometry data (the cellular DNA content of the stained cells) is illustrated in figure 11.
The cell population in G2/M increases from 4.82 % in the untreated cells to 19.33,
23.14 % in the cells treated with compounds 7d and 7e, respectively. Thus, our tested
compounds could arrest cell cycle progression dramatically on MCF-7 cells, and finally
resulted in inhibition of tumor cell proliferation.
Cell cycle analysis
80
70
%G0-G1
%S
%G2-M
%Apoptosis
phase%
60
50
40
30
20
10
0
7e
7d
MCF-7
Cont/
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Figure 10: The effect of compounds 7d and 7e on different phases in cell cycle in MCF-7 cell.
Table 8: Cell cycle analysis results for compounds 7d and 7e on MCF-7.[a]
Compound
No.
%Apoptosis
(%Pre-G1)
%G0-G1 %S %G2-M
Comment
pre G1 Apoptosis & cell
cycle arrest at G2-M
7d
47.2
17.19 19.33
15.35 23.14
16.28
pre G1 Apoptosis & cell
cycle arrest at G2-M
7e
44.28
67.39
17.05
0.66
Control
27.13
4.82
[a] The data are presented as the average of at least three independent experiments.
control
7e
7d
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Figure 11: The flow cytometry analysis histogram of treated and untreated MCF-7 cells.
Each histogram indicates cell cycle distribution of MCF-7 cells; the percent of cells in G1 (red fraction), S
(colorless fraction), and G2/M (red fraction) phases of the cell cycle. The blue fraction represents %
apoptosis
2.2.4.2 Apoptosis assay
The detection of apoptosis inducers has adopted a great attraction toward the
identification of powerful anticancer drugs [29]. Annexin V Apoptosis Detection Kit is
based on the observation that cells, which undergo apoptosis, are able to carry
phosphatidylserine (PS) from the inner surface of the plasma membrane to the cell
surface. Consequently, the presence of PS on the cell surface is considered as a vital
tool to identify the apoptotic cells. PS can be easily detected by staining with a
fluorescent conjugate of Annexin V, a protein that possess a high affinity for PS,
followed by flow cytometry analysis. The kit can differentiate between apoptosis and
necrosis when performing both Annexin V and PI staining in parallel. PI are capable of
entering the cell only when the plasma membrane is entirely destroyed. This permits the
distinction of the early apoptotic cells (negative for PI, but positive for PS) from the late
apoptotic and necrotic cells (positive for both PI and PS) [30].
As depicted in cell cycle analysis of MCF-7, our tested compounds exhibited the
presence of pre-G1 peak which gave indication of apoptosis. To confirm their ability to
induce apoptosis, cells were evaluated using this AnnexinV and PI double staining flow
cytometry, which stains both PS residues and DNA, respectively. Then, they were
incubated for 24 h and analyzed. Analyses of early and late apoptosis showed that, the
tested compounds were able to induce significant levels of cell apoptosis in dose-
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dependent manner, (Table 9, Figure 12). Compared to the control, MCF-7 cells treated
with compounds 7d and 7e exhibited the accumulation of early and late apoptotic cells.
For example, treatment of MCF-7 cells with compounds 7d and 7e for 24 h resulted in
16.28, 17.05 % of apoptotic cells, respectively as compared to 0.66 % of apoptotic cells
in the untreated control. These results revealed that these compounds could inhibit cell
growth through cell apoptosis induction.
Table 9: Apoptosis induction analysis using AnnexinV/ PI for tested compounds.
Compound
No.
% Viable (healthy)
cells
% Early
apoptosis
% Late
%
apoptosis
Necrosis
7d
7e
83.96
82.72
99.34
7.03
6.69
0.29
8.42
8.67
0.22
0.83
1.6
Control
0.15
In addition, figure 12 showed the results of fluorescence-activated cell sorting analysis
performed after treatment of MCF-7 cells with our compounds. The histogram of each
sample was divided into four quadrants to illustrate viable cells (lower left quadrant),
necrotic cells (upper left quadrant), apoptotic cells (lower right quadrant), and
necrotic/late apoptotic cells (upper right quadrant) [30].
PI
Control
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7e
7d
Annexin V
Figure 12: Apoptosis induction analysis using Annexin V/PI of
compounds 7d and 7e on MCF-7.
3. Conclusion
A series of thiophene-quinoline hybrids possessing isooxazole, trizole or phenyl
moieties anchored through OH functionality were designed and efficiently synthesized.
The prepared compounds were evaluated for in vitro cytotoxic activity against four
human cancer cell lines (HepG2, HeLa, MCF-7 and HCT-116), using MTT assay. The
most promising compounds 7d and 7e exhibited the most potent anticancer activity
against MCF-7, with IC values 38.41 M and 28.36 M, respectively. The analysis of
휇
휇
50
SARs indicated that the replacement of the isoxazole and triazole moieties with phenyl
one enhances the potent cytotoxicity. In addition, compounds with halogen group at 4-
position on the phenyl ring was beneficial to anticancer activity. Also, it was verified
that our promising compounds displayed strong inhibitory activity against EGFR-TK
and Topo II enzymes. Furthermore, they were subjected to cell cycle analysis and
apoptosis assay. They were proved to arrest MCF-7 cells at G2/M phase before entering
mitosis. Moreover, apoptosis induction capability was confirmed by the effect on early
and late apoptosis and augmentation of caspase-3 and caspase-9 levels. Thus, our tested
compounds might be considered as promising apoptotic inducers and provide promising
new templates for future development of anticancer agents. Our promising derivatives
might open the door for future structure modification, investigation and docking studies.
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In the future, we hope to study the chirality of our promising compounds and its effect
on the binding modes with their target active sites from the virtual screening point of
view.
4. Experimental part
4.1 Chemistry
General Methods: All reactions were carried out under a nitrogen atmosphere. NMR
spectra were recorded in CDCl3 [TMS for 1H,
= 0] or CDCl3 (for C13,
= 77.0) was
훿
훿
used as an internal standard] or DMSO-d6 [TMS (for 1H,
= 0), DMSO (for 1H,
=
훿
훿
13
2.5), or DMSO (for C,
= 40.0) was used as an internal standard]. High-resolution
훿
mass spectra (HRMS) were obtained by an electron ionization (EI) method with a
double focusing sector detector. Column chromatography was performed on silica gel.
The key precursor, 2-chloroquinoline-3-carbaldehyde (2) could be easily prepared from
acetanilide (1) employing a Vilsmeier–Haack protocol according to the previously
described literature procedures [20].
4.1.1 Synthesis of (2-chloroquinolin-3-yl))thiophen-2-yl)methanol (3):
In a dry two necked round bottom flask under an anhydrous atmosphere, Mg metal
(0.346 g, 14.2 mmol) was taken in dry THF (30 mL) to which was added 2-
bromothiophene (2.36 g, 14.2 mmol). The mixture was refluxed until all the Mg metal
o
reacted. Then, this freshly prepared grignard reagent was cooled to 0-5 C and 2-
chloroquinoline-3-carbaldehyde (2) (1.0 g, 7.1 mmol) and LiCl (0.150 g, 3.55 mmol)
was added. The reaction mixture was stirred at 0 oC for 30 min and stirred at rt for 2 h.
After completion of the reaction (monitored by TLC), the mixture was quenched with
saturated NH4Cl solution and extracted with EtOAc (3 x 30 mL). The organic layers
were collected, dried over anhydrous Na2SO4, concentrated under vacuum and purified
by recrystallization (hexan/EtOAc) to give the title compound as a yellow solid, m.p =
1
131 ºC. Yield = 1.26 g, 80%). RF (hexane/EtOAc; 7:3) = 0.42. H NMR (500 MHz,
CDCl3): 8.53 (s, 1H), 8.02 (d, 1H, J = 8.4 Hz), 7.87 (d, 1H, J = 8.0 Hz), 7.70 (t, 1H, J =
8.4 Hz), 7.57 (t, 1H, J = 8.0 Hz), 7.30 (d, 1H, J = 5.1 Hz), 7.01 (d, 1H, J = 3.5 Hz), 6.96
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13
(dd, 1H, J = 3.5, 5.1 Hz), 6.50 (d, 1H, J = 3.8 Hz), 2.71 (d, 1H, OH, J = 3.8 Hz). C
NMR (125 MHz, CDCl ): = 148.8, 147.0, 145.5, 136.1, 134.9, 130.64, 128.1, 127.9,
훿
3
127.7, 127.30, 126.9, 126.0, 126.0, 68.4. IR (ATR, cm-1): 3274 (OH), 1659, 1615, 1489,
851. HRMS-FAB (M+H)+: 276.0258 (Calculated for C14H10ClNOS, 275.0172).
4.1.2 Synthesis of 2-Chloro-3-[(prop-2-yn-1-yloxy)(thiophen-2-yl)methyl]quinoline
(4):
Sodium hydride (60% oil dispersion, 45 mmol, 3 eq., pre-washed with petroleum ether)
was suspended in DMF (12 mL). To that, a solution alcohol 3 (14 mmol) in DMF(10
ml), was added portion wise and stirred for 30 min at 0 °C, until the evolution of H2
ceased. Then, propargyl bromide (42 mmol, 3 eq.) was added drop wise over a period of
30 min and the suspension was slowly brought to rt. Progress of reaction was monitored
using TLC at regular intervals. After the completion of reaction (6 h), it was quenched
with saturated NH4Cl and extracted with CH2Cl2 (3 x 30 mL). The organic layers were
collected, dried over anhydrous Na2SO4 and concentrated under low vacuum.
Subsequent purification over silica gel column resulted in the isolation of pure product 4
in 66% yield as a light yellow solid. RF (hexane/EtOAc; 7:3) = 0.71.1H NMR: (500
MHz, CDCl3): δ = 8.50 (s, 1H), 8.02 (d, 1H, J = 8.5 Hz), 7.89 (d, 1H, J = 8.0 Hz), 7.75
(t, 1H, J = 8.5 Hz), 7.59 (t, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 4.3 Hz), 7.10 (d, 1H, J =
3.1 Hz), 6.97-6.96 (m, 1H), 6.35 (s, 1H), 4.33 (dd, 2H, J = 2.4, 15.8 Hz), 2.52 (t, 1H, J
13
= 2.4 Hz). C NMR (125 MHz, CDCl )
149.1, 147.1, 142.2, 136.2, 133.0, 130.6,
:훿 =
3
128.2, 127.9, 127.7, 127.6, 127.2, 126.7, 124.3, 78.8, 75.5, 73.7, 56.1. HRMS-EI(M+):
313.0328 (Calculated for C17H12ClNOS, 313.0328). IR (ATR, cm-1): 3205 (CH-of
alkyne), 2125 (-C≡C-), 1616, 1560 (C=C), 1134, 1070, 761.
4.1.3 General procedure for the synthesis of isoxazole derivatives 5a-f
To a suspension of appropriate aldehyde (10 mmol, 1 eq.) in a mixture of iced
water/EtOH (1:1, 10 mL), NH2OH.HCl (10 mmol, 1 eq.) was added, followed by aq.
solution of NaOH (50%, 25 mmol, 2.5 eq.), while keeping the temperature below 30 °C.
After being stirred at rt for 1h, the solution was extracted with Et2O. The aq. phase was
acidified to pH 6 by adding concentrated HCl while keeping the temperature below
30 °C and extracted with Et2O. The organic phase was dried over MgSO4, and the
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solvent was evaporated to give the oxime products in 60–90% yield, which were used
directly in the next reaction without further purification [17].
To a solution of oxime (10 mmol) in THF (10 mL) containing NCS (10 mmol)
and K2CO3 (40 mmol), alkyne 4 (3.1g, 10 mmol) was added and the suspension was
stirred overnight at rt until the completion of the reaction. The crude reaction mixture
was extracted with EtOAc (3 x 30 mL) and the combined organic layer was dried over
anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified
using column chromatography (SiO2, hexane/EtOAc; 7:3) to afford the title isoxazoles
5a-f.
2-Chloro-3-{[(3-(4-methylphenyl)isoxazol-5-yl)methoxy](thiophen-2-yl)methyl}
quinoline (5a):
White solid, m.p = 145-147 ºC, Yield = 27 %. RF (hexane/EtOAc; 7:3) = 0.45.
1H NMR (500 MHz, CDCl3)
= 8.51 (s, 1H), 8.02 (d, 1H, J = 8.4 Hz), 7.90 (d, 1H, J
:훿
= 8.0 Hz), 7.76 (t, 1H, J = 8.4 Hz), 7.69 (d, 2H J = 8.0 Hz), 7.60 (t, 1H, J = 8.0 Hz),
7.35 (dd, 1H, J = 3.5, 6.2 Hz), 7.26 (d, 2H, J = 8.0 Hz), 7.11 (d, 1H, J = 6.2 Hz), 7.00
(dd, 1H, J = 3.5, 8.6 Hz), 6.59 (s, 1H), 6.23 (s, 1H), 4.82 (dd, 2H, J = 13.6, 13.6 Hz),
2.39 (s, 3H).
13C NMR (125 MHz, CDCl )
:훿 =
168.6, 162.0, 161.0, 149.0, 147.2, 142.1, 132.8,
3
136.3, 130.8, 129.6, 128.3, 128.2, 128.0, 127.5, 127.3, 127.2, 126.8, 126.7, 121.3, 114.3,
101.5, 75.6, 61.8, 21.4. HRMS-EI (M+): 446.0753 (calculated for C25H19ClN2O2S,
446.0856).
IR (ATR, cm-1): 3125, 2923, 1615 (C=N), 1589, 1565, 1432, 1296, 1141, 735.
2-Chloro-3-{[(3-(4-methoxyphenyl)isoxazol-5-yl)methoxy](thiophen-2-yl)methyl}
quinoline (5b):
White solid, m.p = 98-100 ºC, Yield = 40 %. RF (hexane/EtOAc; 7:3) = 0.42.
1H NMR (500 MHz, CDCl )
: 훿
= 8.51 (s, 1H), 8.02 (d, 1H, J = 8.5 Hz), 7.90 (d, 1H, J
3
= 8.5 Hz), 7.76 (t, 1H, J = 8.5 Hz), 7.74 (d, 2H, J = 8.9 Hz), 7.60 (t, 1H, J = 8.5 Hz),
24

JOURNAL PRE-PROOF
7.35 (dd, 1H, J = 3.5, 6.2 Hz ), 7.11 (d, 1H, J = 6.2 Hz), 7.00 (dd, 1H, J = 3.5, 8.6 Hz),
6.97 (d, 2H, J = 8.9 Hz), 6.56 (s, 1H), 6.23 (s, 1H), 4.74 (dd, 2H, J = 13.6, 13.6 Hz),
3.86 (s, 3H). 13C NMR (125 MHz, CDCl )
:훿 =
168.6, 162.0, 161.0, 149.0, 147.2,
3
142.1, 136.3, 132.8, 130.8, 128.3, 128.2, 128.0, 127.5, 127.3, 127.2, 126.8, 126.8, 121.3,
114.3, 101.3, 75.6, 61.8, 55.3. HRMS-EI (M+): 462.0702 (calculated for
C25H19ClN2O3S, 462.0856).
IR (ART, cm-1): 2960, 1608 (C=N), 1528, 1429, 1248, 1138, 753.
2-Chloro-3-{[(3-(3,4-dimethoxyphenyl)isoxazol-5-yl)methoxy]thiophen-2-yl)
methyl}quinoline (5c):
Yellow solid, m.p = 70-73 ºC, Yield = 49 %. RF (hexane/EtOAc; 7:3) = 0.28.
1H NMR (500 MHz, CDCl )
: 훿
= 8.51 (s, 1H), 8.03 (d, 1H, J = 8.4 Hz), 7.90 (d, 1H, J
3
= 7.6 Hz), 7.77 (t, 1H, J = 8.4 Hz), 7.61 (t, 1H, J = 7.6 Hz), 7.41 (d, 1H, J = 2.0 Hz),
7.36 (dd, 1H, J = 2.0, 6.0 Hz), 7.29 (dd, 1H, J = 2.0, 6.0 Hz), 7.11 (d, 1H, J = 3.5 Hz),
7.01 (dd, 1H, J = 3.5, 8.3 Hz), 6.93 (d, 1H, J = 8.3 Hz), 6.58 (s, 1H), 6.24 (s, 1H),
13
4.82 (dd, 2H, J = 13.6, 13.6 Hz), 3.95 (s, 3H), 3.93 (s, 3H). C NMR (125 MHz,
CDCl ) = 168.6, 162.2, 150.6, 149.3, 148.9, 147.2, 142.1, 136.3, 132.8, 130.8, 128.2,
:훿
3
128.0, 127.5, 127.3, 127.2, 127.0, 126.8, 121.4, 120.0, 111.0, 109.2, 101.4, 75.6, 60.4,
55.9.
HRMS-EI (M+): 492.0934 (calculated for C26H21ClN2O4S, 492.0911). IR (ATR, cm-1):
2946, 1614 (C=N), 1587, 1369, 1140, 754.
2-Chloro-3-{[(3-(4-hydroxyphenyl)isoxazol-5-yl)methoxy](thiophen-2-yl)
methyl}quinoline (5d):
Yellow solid, m.p = 220-223 ºC, Yield = 10 %. RF (hexane/EtOAc; 7:3) = 0.27.
1H NMR (500 MHz, CDCl )
: 훿
= 8.50 (s, 1H), 8.03 (d, 1H, J = 8.5 Hz), 7.89 (d, 1H, J
3
= 8.5 Hz), 7.76 (t, 1H, J = 8.5 Hz), 7.68 (d, 2H, J = 8.8 Hz), 7.60 (t, 1H, J = 8.5 Hz),
7.35 (dd, 1H, J = 3.5, 6.2 Hz ), 7.11 (d, 1H, J = 6.2 Hz), 7.00 (dd, 1H, J = 3.5, 8.8 Hz),
6.92 (d, 2H, J = 8.8 Hz), 6.55 (s, 1H), 6.23 (s, 1H), 5.83 (br.s, 1H, OH), 4.15 (dd, 2H, J
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= 13.6, 13.6 Hz). 13C NMR (125 MHz, CDCl )
:훿 =
168.6, 162.1, 157.5, 157.4, 150.0,
3
149.0, 147.1, 142.0, 136.5, 132.8, 130.8, 128.7, 128.5, 128.1, 128.0, 127.6, 127.4, 127.2,
121.1, 115.9, 115.8, 101.4, 75.6, 61.8. HRMS-EI (M+): 448.0758 (calculated for
C24H17ClN2O3S, 448.0648). IR (ATR, cm-1): 3263 (OH), 2360, 1606 (C=N), 1515, 1272,
1169, 754.
2-Chloro-3-{[(3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)methoxy]thiophen-2-yl)
methyl}quinoline (5e):
White solid, m.p = 123-126 ºC, Yield = 41 %. RF (hexane/EtOAc; 7:3) = 0.23.
1H NMR (500 MHz, CDCl )
: 훿
= 8.50 (s, 1H), 8.03 (d, 1H, J = 8.5 Hz), 7.90 (d, 1H, J
3
= 8.5 Hz), 7.77 (t, 1H, J = 8.5 Hz), 7.61 (t, 1H, J = 8.5 Hz), 7.36 ( dd, 1H, J = 3.5, 6.2
Hz ), 7.12 (d, 1H, J = 3.5 Hz), 7.01 (s, 2H) , 7.00 (dd, 1H, J = 3.5, 8.6 Hz), 6.58 (s, 1H),
6.24 (s, 1H), 4.83 (dd, 2H, J = 13.6, 13.6 Hz), 3.93 (s, 6H), 3.91 (s, 3H).
13C NMR (125 MHz, CDCl ) = 162.3, 153.6, 149.0, 147.2, 142.0, 139.7, 136.3,
:훿
3
132.7, 130.8, 128.2, 128.0, 127.5, 127.4, 127.2, 126.9, 124.1, 104.7, 104.1, 101.6, 75.7,
61.9, 60.9, 56.3, 31.5, 22.6, 14.13. HRMS-EI (M+): 522.1021 (calculated for
C27H23ClN2O5S, 522.1016). IR (ATR, cm-1): 2935, 1584 (C=N), 1465, 1419, 1231,
1124, 758.
2-Chloro-3-{[(3-(3,5-dimethylphenyl)isoxazol-5-yl)methoxy](thiophen-2-yl)
methyl}quinoline (5f):
White solid, m.p = 103-105 ºC, Yield = 33 %. RF (hexane/EtOAc; 7:3) = 0.6.
1H NMR (500 MHz, CDCl )
: 훿
= 8.51 (s, 1H), 8.03 (d, 1H, J = 8.5 Hz), 7.91 (d, 1H, J
3
= 8.5 Hz), 7.78 (t, 1H, J = 8.5 Hz), 7.628 (t, 1H, J = 8.5 Hz), 7.41-7.42 ( m, 2H), 7.37-
7.34 (m, 1H), 7.13-7.08 (m, 2H), 7.00-6.99 (dd, 1H, J = 3.5, 8.6 Hz), 6.59 (s, 1H), 6.21
(s, 1H), 4.75 (dd, 2H, J = 13.6, 13.6 Hz), 2.38 (s, 3H), 2.36 (s, 3H).
13C NMR (125 MHz, CDCl3)
168.6, 162.7, 153.6, 149.0, 147.2, 142.1, 138.5,
:훿 =
136.3, 132.8, 131.7, 130.8, 128.5, 128.3, 128.0, 127.598, 127.3, 127.2, 126.8, 124.6,
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104.1, 101.7, 75.6,61.9, 60.9, 22.6, 22.2. HRMS-EI (M+): 460.0993 (calculated for
C26H21ClN2O5S, 460.1012). IR (ATR, cm-1): 3075, 1593(C=N), 1490, 1298, 1158, 851.
4.1.4 General procedure for the synthesis of triazole derivatives 6a-e:
The appropriate aromatic amine (20.0 mmol) was dissolved in aq. HCl (12.6 mL, 6 M)
and the solution was cooled to 0 °C. An aq. solution of sodium nitrite (1.40 g, 20.3
mmol) in water (7 mL) was then added slowly. The reaction temperature was not
allowed to rise above 5 °C. Then, an aq. solution of NaN3 (1.31 g, 20.1 mmol) in water
(7 mL) was added, and stirring was continued for 15 min at rt. The reaction mixture was
neutralized with saturated aq. NaHCO3, and the product was extracted with Et2O (6 x 50
mL). Then, the combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuum to give aryl azides in quantitative yield. The obtained azides
were used directly in the next copper-catalyzed click chemistry reaction without further
purification [23].
Alkyne 4 (3 mmol) and different freshly prepared aryl azides (3 mmol) were
suspended in a mixture of water/t-BuOH (1:1, 10 mL). Sodium ascorbate (0.9 mmol,
300 L of freshly prepared 1 M solution in water) was added, followed by
휇
CuSO4.5H2O (0.09 mmol, in 100 L of water). Then, the heterogeneous mixture was
μ
stirred vigorously until the completion of reaction. The reaction mixture was extracted
with EtOAc (3 x 30 mL) and the combined organic layer was dried over Na2SO4,
filtered and concentrated under vacuum. The residue was purified using column
chromatography (SiO2, hexane/EtOAc; 7:3) to afford the title 1,2,3-triazoles 6a-e.
2-Chloro-3-{[(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy](thiophen-2-
yl)methyl}quinoline (6a):
Yellow crystals, m.p = 65-67 ºC, Yield = 48 %. RF (hexane/EtOAc; 7:3) = 0.22.
27

JOURNAL PRE-PROOF
1H NMR (500 MHz, CDCl )
: 훿
= 8.49 (s, 1H), 8.02 (d, 1H, J = 8.5 Hz), 7.93 (s, 1H),
3
7.88 (d, 1H, J = 8.5 Hz), 7.75 (t, 1H, J = 8.5 Hz), 7.62 (d, 2H, J = 9.0 Hz), 7.59 (t, 1H, J
= 8.5 Hz), 7.33 (dd, 1H, J = 3.5, 6.2 Hz), 7.11 (d, 1H, J = 6.2 Hz), 7.02 (d, 2H, J = 9.0
Hz), 6.99 (dd, 1H, J = 3.5, 8.6 Hz), 6.26 (s, 1H), 4.91 (dd, 2H, J = 13.6, 13.6 Hz), 3.84
13
(s, 3H). C NMR (125 MHz, CDCl ) = 159.8, 149.2, 147.1, 144.9, 142.8, 136.5,
:훿
3
133.2, 130.6, 130.4, 128.2, 127.5, 128.0, 127.2, 126.8, 126.4, 122.0, 121.3, 114.7, 75.2,
62.8, 60.4, 55.6. HRMS-EI (M+): 462.0814 (calculated for C24H19ClN4O2S, 462.0917).
IR (ART, cm-1): 2368, 1590, 1560, 1516, 1138, 752.
2-Chloro-3-{[(1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy](thiophen-2-yl)
methyl}quinoline (6b):
Shiny yellow crystals, m.p = 76-78 ºC, Yield = 36 %. RF (hexane/EtOAc; 7:3) = 0.29.
1H NMR (500 MHz, CDCl )
: 훿
= 8.46 (s,1H), 8.39 (d, 2H, J = 9.0 Hz), 8.16 (s, 1H),
3
7.98 (d, 1H, J = 8.5 Hz), 7.96 (d, 2H, J = 9.0 Hz), 7.86 (d, 1H, J = 8.0 Hz), 7.74 (t, 1H,
J = 8.4 Hz), 7.59 (t, 1H, J = 8.0 Hz), 7.34 (dd, 1H, J = 3.5, 6.2 Hz ), 7.12 (d, 1H, J = 6.2
Hz), 7.00 (dd, 1H, J = 3.5, 8.6 Hz), 6.27 (s, 1H), 4.93 (dd, 2H, J = 13.6, 13.6 Hz).
13C NMR (125 MHz, CDCl ) = 147.2, 147.1, 146.2, 142.5, 141.0, 136.4, 130.0,
:훿
3
130.8, 128.2, 127.9, 127.3, 127.2, 126.9, 126.6, 126.3, 125.5, 124.8, 120.9, 113.3, 75.5,
62.6, 60.4. HRMS-EI (M+): 477.0759 (calculated for C23H16ClN5O3S, 477.0662). IR
(ATR, cm-1): 3088, 1596, 1522, 1338, 1137, 852.
2-Chloro-3-{[(1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)methoxy](thiophen-2-
yl)methyl}quinoline (6c):
Faint yellow crystals, m.p = 164-166 ºC, Yield = 35 %. RF (hexane/EtOAc; 7:3) = 0.4.
1H NMR (500 MHz, CDCl )
: 훿 =
8.55 (s,1H), 8.04 (d, 1H, J = 8.4 Hz), 7.97 (s, 1H),
3
7.88 (d, 1H, J = 8.1 Hz), 7.75 (t, 1H, J = 8.4 Hz), 7.59 (d, 2H, J = 8.4 Hz), 7.55 (t, 1H, J
= 8.1 Hz), 7.33 (dd, 1H, J = 3.5, 6.2 Hz), 7.31 (d, 2H, J = 8.4 Hz), 7.11 (d, 1H, J = 6.2
Hz), 6.99 (dd, 1H, J = 3.5, 8.6 Hz), 6.26 (s, 1H), 4.92 (dd, 2H, J = 13.6, 13.6 Hz), 2.42
28

JOURNAL PRE-PROOF
13
(s, 3H). C NMR (125 MHz, CDCl )
:훿 =
149.2, 147.2, 145.0, 142.8, 139.0, 136.5,
3
134.7, 133.2, 130.6, 130.2, 128.2, 128.0, 127.2, 126.8, 126.4, 124.8, 121.0, 120.5, 113.3,
75.2, 62.8, 21.1. HRMS-EI (M+): 446.0865(calculated for C24H19ClN4OS, 446.0968).
IR (ATR, cm-1): 3122, 2361, 1560, 1519, 1139, 855.
2-Chloro-3-{[(1-(4-trifloromethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy] (thiophen-
2-yl) methyl}quinoline (6d):
Yellow solid, m.p = 123-126 ºC, Yield = 71 %. RF (hexane/EtOAc; 7:3) = 0.36.
1H NMR (500 MHz, CDCl )
: 훿 =
8.48 (s, 1H), 8.11 (s, 1H), 7.99 (d, 1H, J = 8.5 Hz),
3
7.87 (d, 2H, J = 8.6 Hz), 7.84 (d, 1H, J = 8.5 Hz), 7.77 (d, 2H, J = 8.6 Hz), 7.72 (t, 1H,
J = 8.5 Hz), 7.56 (t, 1H, J = 8.6 Hz), 7.32 (dd, 1H, J = 3.5, 6.2 Hz), 7.11 (d, 1H, J = 6.2
Hz), 6.98 (dd, 1H, J = 3.5, 8.6 Hz), 6.23 (s, 1H), 4.92 (dd, 2H, J = 13.6, 13.6 Hz).
13C NMR (125 MHz, CDCl ) = 149.1, 147.1, 145.7, 142.6, 139.3, 136.4, 133.1, 130.7,
:훿
3
128.2, 127.9, 127.3, 127.2, 127.1, 127.0, 126.9, 126.5, 120.9, 120.4, 120.2, 114.1, 75.4,
62.6. HRMS-EI (M+): 500.0583 (calculated for C24H16ClF3N4OS, 500.0685). IR (ATR,
cm-1): 3070, 1647, 1616, 1321, 1165, 842.
2-Chloro-3-[((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)(thiophen-2-
yl)methyl]quinoline (6e):
White solid, with m.p = 147-149 ºC, Yield = 69 %. RF (hexane: EtOAc, 7:3) = 0.3.
1H NMR (500 MHz, CDCl3)
= 8.47 (s, 1H), 8.01 (s, 1H), 8.00 (d, 1H, J = 8.5 Hz),
:훿
7.85 (d, 1H, J = 8.5 Hz), 7.56 (t, 1H, J = 8.5 Hz), 7.66 (d, 2H, J = 8.7 Hz), 7.59 (t, 1H, J
= 8.5 Hz), 7.46 (d, 2H, J = 8.7 Hz), 7.32 (dd, 1H, J = 3.5, 6.2 Hz), 7.11 (d, 1H, J = 3.5
Hz), 6.98 (dd, 1H, J = 3.5, 8.6 Hz), 6.25 (s, 1H), 4.90 (dd, 2H, J = 13.6, 13.6 Hz).
13C NMR (125 MHz, CDCl )
:훿 =
149.1, 147.1, 145.4, 142.7, 136.4, 135.4, 134.5,
3
133.1, 130.7, 129.9, 128.2, 128.0, 127.3, 127.2, 127.0, 126.8, 126.5, 121.7, 121.0, 114.7,
75.3, 62.7. HRMS-EI (M+): 466.0319 (calculated for C23H16Cl2N4OS, 466.0422).
IR (ART, cm-1): 3103, 1617, 1588, 1560, 1502, 1496, 1229, 1138, 820.
29