Discovery of 4-anilinoquinolinylchalcone derivatives as potential NRF2 activators

Article abstract of DOI:10.3390/molecules25143133

Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be an effective means to prevent the development of cancer, and natural curcumin stands out as a potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them, (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC50) value of 1.95 μM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level. Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction, which contributed to the enhancement of affinity and stability between the ligand and receptor. Compound 13b has been identified as the lead compound for further structural optimization.

Full text of DOI:10.3390/molecules25143133

molecules
Article
Discovery of 4-Anilinoquinolinylchalcone
Derivatives as Potential NRF2 Activators
Yu-Tse Kao , Yi-Siao Chen , Kai-Wei Tang , Jin-Ching Lee 3,5,6, Chih-Hua Tseng 4
1
2,3
4
,
1
3,6,
and Yeh-Long Chen 1,6,7,
*
Cherng-Chyi Tzeng , Chia-Hung Yen
*
1
Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University,
Kaohsiung 807, Taiwan; u104850010@Kmu.edu.tw (Y.-T.K.); tzengch@kmu.edu.tw (C.-C.T.)
Program in Environmental and Occupational Medicine, College of Medicine, Kaohsiung Medical University,
Kaohsiung 807, Taiwan; wl01420323@gmail.com
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 807, Taiwan; jclee@kmu.edu.tw
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan;
dadaking1107@gmail.com (K.-W.T.); chihhua@kmu.edu.tw (C.-H.T.)
Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Drug Development and Value Creation Research Center, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
2
3
4
5
6
7
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
*
Correspondence: chyen@kmu.edu.tw (C.-H.Y.); yeloch@kmu.edu.tw (Y.-L.C.);
Tel.: +886-7-3121101 (ext. 2684) (C.-H.Y.); Fax: +886-7-3125339 (C.-H.Y.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 12 June 2020; Accepted: 6 July 2020; Published: 8 July 2020
Abstract: Activation of nuclear factor erythroid-2-related factor 2 (NRF2) has been proven to be
an effective means to prevent the development of cancer, and natural curcumin stands out as a
potent NRF2 activator and cancer chemopreventive agent. In this study, we have synthesized a
series of 4-anilinoquinolinylchalcone derivatives, and used a NRF2 promoter-driven firefly luciferase
reporter stable cell line, the HaCaT/ARE cells, to screen a panel of these compounds. Among them,
(E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one (13b) significantly
increased NRF2 activity in the HaCaT cell with a half maximal effective concentration (EC ) value of
50
1
.95 µM. Treatment of compound 13b upregulated HaCaT cell NRF2 expression at the protein level.
Moreover, the mRNA level of NRF2 target genes, heme oxygenase-1 (HO-1), glutamate-cysteine
ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD) were significantly
increased in HaCaT cells upon the compound 13b treatment. The molecular docking results exhibited
that the small molecule 13b is well accommodated by the bound region of Kelch-like ECH-associated
protein 1 (Keap1)-Kelch and NRF2 through stable hydrogen bonds and hydrophobic interaction,
which contributed to the enhancement of affinity and stability between the ligand and receptor.
Compound 13b has been identified as the lead compound for further structural optimization.
Keywords: 4-anilinoquinolinylchalcone derivatives; nuclear factor erythroid-2-related factor 2 (NRF2)
activators; cancer chemopreventive agent
1
. Introduction
The nuclear factor erythroid-2-related factor (NRF2) is a transcription factor that is sensitive to
oxidative stress. In normal conditions, NRF2 activity is suppressed by Kelch-like ECH-associated
protein 1 (Keap1), which is a substrate adaptor for Cullin-3 (CUL3) [ ]. NRF2 will then be
ubiquitinated by CLU3 and be degraded by proteasome. Under oxidative stress conditions, reactive
1–3
Molecules 2020, 25, 3133; doi:10.3390/molecules25143133
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3133
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oxygen species (ROS) or electrophiles oxidize key sensor cysteine residues in Keap1 and change its
conformation, which disrupts the interaction between Keap1 and NRF2 [ ]. NRF2 then can enter the
4
nucleus and forms dimer with sMaf. This dimer binds to the antioxidant response element (ARE)
and activated a variety of antioxidant proteins and detoxification enzymes, which play important
roles in antioxidant response and cancer prevention [5,6]. Therefore, activation of NRF2 has long been
considered as a potential strategy to prevent oxidative damage caused by UV irradiation or carcinogens
exposure [7].
In addition to Keap1, the activation of different protein kinases has been shown to activate
NRF2 [
genes such as heme oxygenase-1 (HO-1), NAD(P)H/quinone oxidoreductase 1 (NQO1), glutamate
cysteine ligase modifier subunit (GCLM), -glutamyl cysteine synthase, glutathione peroxidase
GPx), and several members of the glutathione S-transferase family that express an ARE in their
8]. The NRF2-regulated genes include almost all of the relevant antioxidants and cytoprotective
γ
(
promoter [9]. Small molecules that activate NRF2 signaling are being investigated as potential anticancer
or anti-inflammatory agents [10]. A wide variety of dietary and synthetic compounds that function
as potent inducers of ARE-regulated gene expression have been shown to exert chemopreventive
activities, e.g., sulforaphane [11], dithiolethione [12], curcumin [13], and caffeic acid phenethyl ester
(CAPE) [14]. It is notable that both curcumin and CAPE, which bear a α,β-unsaturated ketone moiety,
can function as Michael acceptors, and therefore modify cysteine thiols present in Keap1. Chalcones
are Michael acceptors and constitute an important group of natural products belonging to the flavonoid
family [15].
Certain chalcone derivatives have been reported as potent NRF2 activators (Figure 1).
For examples, (E)-1-(2-methoxyphenyl)-3-[2-(trifluoromethyl)phenyl]prop-2-en-1-one (Compound
1
) [16] was found to activate the expression of GCLM and NQO1 in the small intestine with 3-fold and
5
4
-fold higher respectively compared to sulforaphane. Wu et al. have revealed that (E)-2-methoxy-
-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl)phenyl acrylate (compound ) [17] increased the
2
expression of NRF2-dependent antioxidant genes, such as glutamate-cysteine ligase complex (GCLC)
and HO-1, and their corresponding proteins, together with counteracting neuronal death induced
by H O in PC-12 cells. Lee et al. demonstrated that (E)-1-(2,5-dihydroxy-4-methoxyphenyl)-3-(4-
2
2
hydroxyphenyl)prop-2-en-1-one (compound
via the Keap1/NRF2 pathway. Lee et al. found that (E)-1-phenyl-3-(2,4,6-tris(methoxymethoxy)
phenyl)prop-2-en-1-one (compound ) [19] prevented TNF- -induced inflammation in HT-29 cells
through the nuclear translocation of NRF2 and a consequent increase of HO-1 expression. Kachadourian
et al. revealed that (E)-1-(2,5-dihydroxyphenyl)- 3-phenylprop-2-en-1-one (compound ) [20] was
3) [18] could increase the antioxidant endogenous defense
4
α
5
capable of increasing the NRF2–ARE activity and glutathione (GSH) levels. Pinner et al. discovered
that flavokawains A [21], a methoxylated chalcone from Kava (Piper methysticum), activated NRF2,
increasing the expression of antioxidant and heat shock (Hsp) response genes, in HepG2 cells.
The quinoline ring can be recognized in various biologically active compounds with clinical
applications. The most noteworthy example is neratinib, an irreversible tyrosine kinase inhibitor,
which is used as an adjuvant therapy in people with early stage breast cancer in which HER2 is
overexpressed. Ciprofloxacin is another example of quinoline derivative, which inhibits DNA gyrase
activity and clinically used for the treatment of bacteria infection. Other examples of quinoline
derivatives such as quinine and primaquine were found to possess antimalaria activity. Therefore,
preparation and extensive biological evaluations on quinoline derivatives have continuously attracted
our attention.

Molecules 2020, 25, 3133
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Figure1. Some of NRF2 activators and our target compounds containing chalcone moiety (1) (E)-1-(2-
methoxyphenyl)-3-[2-(trifluoromethyl)phenyl]prop-2-en-1-one, ( ) (E)-2-methoxy-4-(3-(4-methoxyphenyl)-
-oxoprop-1-en-1-yl)phenyl acrylate, (3) (E)-1-(2,5-dihydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)
2
3
prop-2-en-1-one, (4) (E)-1-phenyl-3-(2,4,6-tris(methoxymethoxy)phenyl)prop-2-en-1-one, (5) (E)-1-(2,5-
dihydroxyphenyl)-3-phenylprop-2-en-1-one.
Recently, we have synthesized certain 2-substituted 3-arylquinoline derivatives and the evaluation
of their anti-inflammatory effects in LPS-activated murine macrophage cell line J774A.1 cells.
2-(4-Fluoro-benzoyl)-3-(3,4,5-trimethoxyphenyl)quinoline significantly decreases the secretion of
pro-inflammatory cytokines (TNF- and IL-6) in the LPS-activated macrophages [22]. The present
α
report describes the synthesis of novel hybrid molecules, which bears both quinoline and chalcone
moieties and evaluate for their NRF2 activating activities.
2
. Chemistry
-Methylquinolin-4-ol (
methylquinolin ( ), which was then oxidized with selenium oxide to afford 4-chloroquinoline-2-
carbaldehyde ( ) [23
2
6) was treated with POCl3 to give the intermediate 4-chloro-2-
7
8
,
24], as described in Scheme 1. Aldol condensation of compound
8
with
◦
appropriately substituted acetophenones under a basic condition at 0 C gave quinolinylchalcone
derivatives, compounds 9–11, in a yield of 50–60%. These quinolinylchalcones were obtained as
geometrically pure and with trans-configuration (J Ha–Hb = 15.2–16.0 Hz) [25] of the sole (E)-form
isomers. The desired 4-anilinoquinolinylchalcone derivatives 12–14 were synthesized by the reaction of
compounds 9–11 respectively with various 4-substituted anilines under acidic condition. The structures
of 12–14 were determined by NMR spectrometer (spectra data can be found in Supplementary Materials)
and compound purity is determined by high performance liquid chromatography (HPLC), and all
final test compounds were >95% purity.

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Scheme 1. Synthesis of 4-anilinoquinolinylchalcone derivatives 12–14.
. Biological Results and Discussion
.1. Novel 4-Anilinoquinolinylchalcone Derivatives Induced ARE-Driven Luciferase Activity in HaCaT/
3
3
ARE Cells
In this study, we used a stable ARE-driven reporter system to screen the potential NRF2
activators [26]. Luciferase activities were determined after cells treated with test compounds
at a concentration of 10 M for 24 h. tert-Butyl hydroquinone (t-BHQ), a well-known NRF2
µ
activator in ARE-controlled gene transcription, showed 1880% induction of ARE-driven luciferase
activity, was used as the positive control. In addition, flavokawain A, which was identified
as a potential chemopreventative or chemotherapeutic agent by Pinner et al. [21], was
adapted as a second positive control, for the 1-phenylprop-2-en-1-one derivatives (R1 = H),
(
E)-1-phenyl-3-[4-(phenylamino)quinolin-2-yl]prop-2-en-1-one (12a, R = H, 7% of relative NRF2
2
activity) showed no induction of the luciferase activity while its 4-acetyl derivative (12b, R = COMe,
2
3
94% of relative NRF2 activity) was weakly active as shown in Table 1. Oximination of 12b decreased
NRF2 inductive activity in which the potency decreased in an order 12b 12d (287%) > 12e (95%) >
2f (26%). The 4-carboxylic acid derivative (12c, R = COOH, 75%) was less active than its 4-acetyl
>
1
2
counterpart (12b, R = COMe, 394%) but more active than the parent (12a, R = H, 7%) while amide
2
2
(12g, 9%) and ester (12h, 32%) derivatives were inactive. The same structure–activity relationships
were observed for the 1-(4-fluorophenyl)prop-2-en-1- one derivatives (R = F) in which the NRF2
1
inductive activity decreased in an order 13b (R = COMe, 2205%) > 13c (R = COOH, 147%) > 13a
2
2
(
R = H, 6%). Compound 13b was more active than both positive controls, t-BHQ (1880%) and
2
flavokawain A (2082%). Oximination of 13b gave 13d–13f, which exhibited less NRF2 inductive
effects, compounds 13d (1697%) and 13f (1850%) were comparable to the positive t-BHQ while
compound 13e (2145%) was more active. The ester derivative 13h (1767%) was approximately equal
active to t-BHQ in the NRF induction while the amide counterpart 13g (5%) was inactive. For the
1
-(4-methoxyphenyl)prop-2-en-1-one derivatives (R = OMe), the same structure–activity relationships
1
have also been obtained in which the NRF2 inductive activity decreased in an order 14b (937%) >
4c (212%) > 14a (7%). Oximination of 14b decreased NRF2 inductive activity in which the potency
1

Molecules 2020, 25, 3133
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decreased in an order 14b
>
14d (229%) > 14e (193%) > 14f (6%). The ester derivative 14h (127%) was
weakly active in the NRF induction while the amide counterpart 14g (5%) was inactive. Our results
indicated the best substituent for R is the acetyl group in all the 4-anilinoquinolinylchalcone derivatives
2
tested and (E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en- 1-one (13b
,
R = F, R = COMe) was the most potent NRF2 activator, which enhanced ARE promoter activity
1
2
to 2205%.
Table 1. Antioxidant response element (ARE)-luciferase gene expression and cell viability a.
Compounds a
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
4g
4h
% of Relative NRF2 Activity in HaCaT Cell
Cell Viability
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6.9 ± 1.2
394.4 ± 168.3
75.3 ± 32.9
287.1 ± 182.5
94.5 ± 17.4
25.5 ± 5.4
67.65 ± 0.6
71.2 ± 1.8
81.4 ± 0.8
68.4 ± 1.3
69.5 ± 0.9
78.8 ± 0.1
74.4 ± 1.8
71.1 ± 1.2
71.8 ± 0.7
95.7 ± 1.8
74.5 ± 1.1
87.5 ± 1.7
94.8 ± 0.7
88.7 ± 1.9
78.7 ± 0.5
82.7 ± 1.7
68.0 ± 1.5
92.0 ± 1.1
70.8 ± 1.9
75.6 ± 0.4
76.7 ± 0.2
66.3 ± 2.6
77.6 ± 1.5
76.8 ± 0.2
97.7 ± 1.5
95.9 ± 1.6
8.5 ± 1.9
32.4 ± 19.4
5.5 ± 0.17
2205.2 ± 144.9
147.3 ± 51.7
1697.3 ± 39.8
2144.7 ± 168.6
1850.4 ± 17.0
5.1 ± 0.42
1767.3 ± 103.6
7.1 ± 1.3
936.5 ± 39.8
212.6 ± 93.6
229.4 ± 68.4
193.3 ± 25.7
6.0 ± 0.3
5.1 ± 0.18
127.1 ± 36.5
1880.4 ± 97.2
2082.6 ± 117.6
t-BHQ
Flavokawain A
a
Concentration of each compound is 10 µM.
The cell viability of 4-anilinoquinolinylchalcone derivatives is also summarized in Table 1.
Most of them exhibited moderate cytotoxicity with the survival rate of less than 90% at 10
µM
concentration. However, compound 13b was relatively non-cytotoxic, with the survival rate of 96%
at the same concentration. Therefore, compound 13b and its analogs, 12b and 14b, were selected for
further evaluations. Results from Figure 2 indicated compounds 12b
,
13b, and 14b induced NRF2
0.24 M/1095% 48%,
24%, respectively. We have also compared
the cytotoxicity of these three compounds. A slight cytotoxicity of compounds 13b (CC50 > 100 M)
promoter activity in a dose-dependent manner with EC /Emax at 1.95
±
µ
±
50
0.85
±
0.04
µM/364%
±
15%, and 1.58
±
0.15
µM/393%
±
µ
was observed, while compound 14b and 12b are more toxic to keratinocyte, and the CC50 of these two
compounds are 28.4 µM and 9.3 µM, respectively as shown in Figure 3.

Molecules 2020, 25, 3133
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Figure 2. The effect of 12b, 13b, and 14b on NRF2 activity in the HaCaT/ARE cell. HaCaT/ARE cells
were treated with indicated concentrations of these three compounds for 18 h. Relative luciferase
activity was calculated as described in the Materials and Methods section. Data presented as mean
from three independent experiments.
±
SD
Figure 3. The cytotoxicity of 12b, 13b, and 14b in HaCaT cells. HaCaT cells were treated with
indicated concentrations of these three compounds for 72 h. Cell viability was determined by a
resazurin assay as described in the Materials and Methods section. Data presented as mean
three independent experiments.
±
SD from
3
.2. The Effect of Compound 13b on the NRF2 Pathway
To further evaluate the effect of compound 13b on NRF2 activation, the mRNA level of NRF2
target genes were determined by real-time PCR. Treatment of compound 13b significantly increased
the expression of HO1, G6PD, and GCLC (Figure 4). This suggested that compound 13b functionally
activates NRF2 signaling. Moreover, we found that treatment of compound 13b can effectively enhance
the expression of NRF2 protein without altering the mRNA of NRF2 (Figure 5). This finding indicated
that the possible mechanism of compound 13b is the activation of NRF2 via the accumulation of NRF2
protein, rather than the promotion of the NRF2 transcription.

Molecules 2020, 25, 3133
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Figure 4. Compound 13b increased the expression of NRF2 target genes in the HaCaT cell. HaCaT
cells were treated with indicated concentrations of 13b for 18 h. Total RNA was isolated and submitted
to reverse transcription and real-time PCR for the detection of the NRF2 target genes including HO1
(A
), GLCL (B), G6PD (C), and NQO1 (D). Data are expressed as normalized mRNA level and presented
as mean SD from three independent experiments. The asterisk (*) indicates a significant difference
±
between 13b treated groups and control group (* p< 0.05, *** p< 0.001, one-way ANOVA).
Figure 5. The compound 13b increased the NRF2 expression at the protein level in the HaCaT cell.
(A
) HaCaT cells were treated with indicated concentrations of compound 13b for 8 h. Protein expression
of NRF2 was determined by Western blot. ( ) HaCaT cells were treated with indicated concentrations
B
of 13b for 18 h. Total RNA was isolated and submitted to reverse transcription and real-time PCR for
the detection of the NRF2 mRNA level.
3
.3. Molecular Docking
The molecular docking study of compound 13b, the most potent NRF2 promoter, was carried
out by the Achilles Blind Docking Server. Compound 13b was docked with the Keap1-Kelch domain

Molecules 2020, 25, 3133
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(PDB code 5FNQ) and the docking pose with the lowest binding energy was shown in Figure 6A.
A channel-like shape in the middle of the structure of the Keap1-Kelch domain allowed compound 13b
to fit into the structure to form a stable complex. Once the channel was occupied by compound 13b
the construction of the Keap1-Kelch domain would be distorted and would no longer interact with
NRF2 resulting in the accumulation of the NRF2 protein. According to the docking results in Figure 6B,
compound 13b formed hydrophobic interactions with Val418, Val420, and Val561 and hydrogen bond
with Gly367, Val 512, and Val616. The lowest binding energy score between compound 13b and keap1
could reach to -9.60 kcal/mol. We speculated that the binding energy could reach the lowest score if the
Michael addition interaction between the chalcone of compound 13b and the cysteine of keap1 could
be calculated.
,
Figure 6.
FNQ). (
represents a hydrogen bond.
(
A) The proposed docking mode of compound 13b in the binding site of Keap1 (PDB code
5
B) The interaction illustrated by green lines represents the hydrophobic interaction, blue lines
4
. Conclusions
We have synthesized certain 4-anilinoquinolinylchalcone derivatives and evaluated for their
NRF2 activity. Among them, (E)-3-{4-[(4-acetylphenyl)- amino]quinolin-2-yl}-1-(4-methoxyphenyl)
prop-2-en-1-one (13b) was found to significantly induce the NRF2 activity in HaCaT cells with a higher
potency than that of t-BHQ and possessed no significant cell cytotoxicity (CC50 > 100 µM). The possible
mechanism of compound 13b is the activation of NRF2 via the accumulation of the NRF2 protein,
rather than the promotion of the NRF2 transcription. Our results of this work might be helpful for
discovering a novel class of potent NRF2 activators.

Molecules 2020, 25, 3133
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5
. Experimental
5
.1. General
All the chemical solvents and reagents used in this study were analytically pure without further
purification and commercially available. Melting points were determined on an Electrothermal
1
13
IA9100 melting point apparatus and are uncorrected. Nuclear magnetic resonance ( H and C)
spectra were recorded on a Varian Gemini 200 (Palo Alto, CA, USA) spectrometer or Varian-Unity-400
spectrometer. Chemical shifts were expressed in parts per million (δ) with tetramethylsilane (TMS)
as an internal standard. Thin-layer chromatography was performed on silica gel 60 F-254 plates
purchased from E. Merck (Darmstadt, Germany). Compound purity is determined by high performance
liquid chromatography (HPLC), and all final test compounds were > 95% purity. HPLC methods
used the following: HITACHI Chromaster 5110 (Hitachi High-Technologies, Tokyo, Japan) with UV
detection at 254 nm and auto sampler; column, Mightysil RP-18GP (250 mm
×
4.6 mm, 5 µm, Kento
chemical, Tokyo, Japan); mobile phase, method A: MeOH/0.01 M KH PO (50:50), pH = 2.64; method B.
2
4
MeOH/0.01M KH PO (50:50), pH = 2.94; method C. MeOH/0.01M KH PO (50:50), pH = 3.47; method
2
4
2
4
D. MeOH/0.01M KH PO (60:40), pH = 2.88; method E. MeOH/0.01M KH PO (60:40), pH = 2.94;
2
4
2
4
method F. MeOH/0.01M KH PO (60:40), pH = 3.47; and method G. MeOH/0.01M KH PO (80:20),
2
4
2
4
pH = 3.47; and the flow rate was 1 mL/min; sample injection, 100 µM (1.0 mg dissolved in 0.5 mL
DMSO, diluted with MeOH to 1 mL, then 0.5 mL of the compound was diluted with MeOH to 1 mL).
The elemental analyses were performed in the Instrument Center of the National Science Council
at National Cheng-Kung University and National Taiwan University using Heraeus CHN-O Rapid
EA(Waltham, MA, USA), and all values were within ± 0.4% of the theoretical compositions.
5
.2. General Procedure for the Preparation of 4-Chloroquinoline-2-Carbaldehyde 8
A mixture of 4-hydroxy-2-methylquinoline (3.18 g, 20 mmol) and POCl (10 mL) was heated at
6
3
◦
40 C for 2 h (TLC monitoring). After cooling, the reaction mixture was poured into ice-H O (200 mL)
2
1
and the concentrated NaOH solution was added until a pH of 10 was reached, and extracted with DCM
50 mL 3). The organic layer was washed with brine, dried over MgSO , and evaporated in vacuo to
give crude product
(
×
4
7
, which was used without further purification in the next step. A mixture
7
(2.62 g,
◦
1
5 mmol) and selenium dioxide (1.61 g, 15 mmol) in 1,4-dioxane (100 mL) was heated at 110 C for
2
h (TLC monitoring). After cooling, the mixture was treated with 5% NaHCO aqueous (160 mL),
3
extracted with DCM (100 mL
The crude product was purified by flash chromatography on silica gel (DCM) and recrystallized by
×
3), the organic layer was collected, dried over MgSO , and evaporated.
4
◦
◦
1
using EtOH to give
8. Orange solid; yield: 70%; Mp. 139.8-140.2 C ([22], lit: 138.1-139.2 C); H-NMR
(
CDCl ) δ 10.18 (s, 1H, CHO), 8.32-8.26 (m, 2H), 8.09 (s, 1H, 3-H), 7.91-7.87 (m, 1H), 7.82-7.77 (m, 1H);
3
1
3
C-NMR (CDCl ) δ 192.56, 152.34, 148.67, 144.20, 131.30, 130.85, 130.19, 128.05, 124.41, 117.57.
3
5
.3. General Procedure for the Aldol Condensation between Acetophenone and 8
To a suspension of appropriate 4-substituted acetophenone (10 mmol) with 3 M of NaOH (10 mL)
◦
in EtOH (30 mL) at 0 C, was added to
0
8 (10 mmol, 1.91 g) carefully, and the mixture was stirred at
◦
C for 2 h (TLC monitoring). The mixture was neutralized with 6 N HCl until pH 7, the resulted
precipitate thus obtained was collected, and the crude solid was recrystallized by using EtOH to give
compounds 9–11.
◦
(
E)-3-(4-chloroquinolin-2-yl)-1-phenylprop-2-en-1-one (
9
), white solid; yield: 70%; Mp. 137.6-138.3 C;
1
H-NMR (CDCl3)
δ
8.24-8.21 (m, 1H), 8.16-8.10 (m, 4H), 7.87 (d, 1H, J = 15.2 Hz, CH=C
H
C=O), 7.81 (m,
190.25, 153.41, 149.06,
H), 7.75 (s, 1H, 3-H), 7.68-7.60 (m, 2H), 7.55-7.51 (m, 2H); 1 C-NMR (CDCl3)
3
δ
1
1
1
43.21, 142.37, 137.64, 133.21, 130.96, 130.23, 128.78 (2C), 128.73 (2C), 128.29, 127.77, 126.30, 124.07,
21.37; Anal. Calcd for C H ClNO: C 73.60, H 4.12, N 4.77; found: C 73.44, H 4.13, N 4.75.
18
12
(
E)-3-(4-chloroquinolin-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (10), white solid; yield: 78%; Mp.
◦
66.9-167.3 C; H-NMR (CDCl ) δ 8.24-8.11 (m, 5H), 7.86 (d, 1H, J = 15.2 Hz, CH=CHC=O), 7.81 (m,
3
1
1

Molecules 2020, 25, 3133
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1
1
1
4
H), 7.74 (s, 1H, 3-H), 7.66 (m, 1H), 7.03-6.99 (m, 2H), 3.90 (s, 3H, OCH ); 13C-NMR (CDCl3)
δ
188.36,
3
63.79, 153.63, 149.07, 143.16, 141.51, 131.19 (2C), 130.91, 130.65, 130.19, 128.18, 127.72, 126.26, 124.07,
21.44, 113.97 (2C), 55.52; Anal. Calcd for C H ClNO : C 70.48, H 4.36, N 4.33; found: C 70.15, H
19
14
2
.48, N 4.37.
E)-3-(4-chloroquinolin-2-yl)-1-(4-fluorophenyl)prop-2-en-1-one (11), white solid; yield: 80%; Mp.
(
◦
1
1
73.1-173.9 C; H-NMR (CDCl )
δ
8.41 (d, 1H, J = 16.0 Hz, C
m, 2H), 7.96 (d, 1H, J = 16.0 Hz, CH=C C=O), 7.86 (m, 1H), 7.80-7.74 (m, 2H), 7.17-7.11 (m, 2H);
188.34, 164.22 (JCF = 250.1 Hz), 153.60, 147.98, 143.91, 143.83, 131.40 (JCF = 3.0
H=CHC=O), 8.35 (s, 1H, 3-H), 8.31-8.28
3
(
H
1
3
C-NMR (CDCl3)
δ
Hz), 130.93, 130.88, 130.83 (2C, JCF = 8.4 Hz), 129.59, 127.62, 124.25, 120.21, 120.19, 119.28, 115.65 (2C,
JCF = 21.2 Hz); Anal. Calcd for C H ClFNO: C 69.35, H 3.56, N 4.49; found: C 69.00, H 3.65, N 4.54.
18
11
5
.4. General Procedure for the Preparation for 4-Substituted-Quinolinyl Chalcones 12a–12g, 13a–13g, and
1
4a–14g
Compound 9, 10, or 11 (1.0 mmol) and appropriate para-substituted anilines (1.0 mmol) in
EtOH/HCl (30/1 mL) was refluxed for 2 h (TLC monitoring). The mixture was then cooled and
evaporated in vacuo to yield a residue, treated with ethyl acetate (50 mL), and the resulting precipitate
was filtered and washed with ethyl acetate. The crude product was purified by flash chromatography
on the silica gel (DCM/MeOH = 10/1), and recrystallized from EtOH to give 4-substituted-quinolinyl
chalcones 12a–12g.
(
E)-1-phenyl-3-[4-(phenylamino)quinolin-2-yl]prop-2-en-1-one hydrochloride (12a), yellow solid; yield:
◦
6%; Mp. 238.5-239.2 C; HPLC (method D), t = 13.467 min; purity: 99.2%; H-NMR (DMSO-d )
R 6
1
4
δ
7
1
1
11.10 (br s, 1H, NH), 8.89-8.80 (m, 2H), 8.58 (m, 1H), 8.27 (m, 2H), 8.05 (m, 1H), 7.81-7.70 (m, 3H),
.63-7.55 (m, 6H), 7.47-7.43 (m, 1H), 7.34 (s. 1H, 3-H); 13C-NMR (DMSO-d6)
188.53, 154.84, 147.47,
39.18, 137.03, 136.50, 134.65, 134.24, 134.06, 131.86, 129.91 (2C), 129.06 (2C), 128.99 (2C), 127.39, 127.12,
25.19 (2C), 123.45, 120.59, 116.91, 102.25; Anal. Calcd for C H N O 1.2HCl: C 73.13, H 4.91, N 7.11;
δ
·
24
18
2
found: C 73.03, H 4.97, N 7.02.
(
E)-3-{4-[(4-acetylphenyl)amino]quinolin-2-yl}-1-phenylprop-2-en-1-one hydrochloride (12b), yellow
◦
1
solid; yield: 47%; Mp. 248.7-249.2 C; HPLC (method F), t = 10.467 min; purity: 98.9%; H-NMR
R
(
1
1
DMSO-d6) δ11.17 (br s, 1H, NH), 8.90-8.83 (m, 2H), 8.58 (m, 1H), 8.27 (m, 2H), 8.13 (m, 2H), 8.07 (m,
1
3
H), 7.84-7.59 (m, 8H), 2.64 (s, 3H, CH3); C-NMR (DMSO-d6)
δ 196.87, 188.60, 148.40, 148.03, 142.90,
41.91, 136.51, 134.69, 134.60, 134.33, 134.09, 132.09, 129.99 (2C), 129.07 (2C), 129.02 (2C), 127.38, 124.00
(
2C), 123.62, 120.91, 117.61, 103.14, 26.72; Anal. calcd for C H N O2·1.1HCl: C 72.19, H 4.92, N 6.48;
26
20
2
found: C 72.37, H 4.63, N 6.45.
(
E)-4-{[2-(3-oxo-3-phenylprop-1-en-1-yl)quinolin-4-yl]amino}benzoic acid hydrochloride (12c), yellow
◦
1
solid; yield: 51%; Mp.298.3-298.9 C; HPLC (method F), t = 8.467 min; purity: 98.1%; H-NMR
R
(
7
DMSO-d6)
δ
11.16 (br s, 1H, NH), 8.85-8.80 (m, 2H), 8.53 (m, 1H), 8.27-8.25 (m, 2H), 8.13-8.06 (m, 3H),
188.65, 166.76, 154.31, 147.94, 141.48, 139.23, 136.52, 134.55,
.84-7.60 (m, 8H); 13C-NMR (DMSO-d6)
δ
1
34.45, 134.15, 132.20, 131.08 (2C), 129.11 (2C), 129.07 (2C), 128.80, 127.46, 124.32 (2C), 123.63, 120.70,
1
17.49, 102.85; Anal. calcd for C H N O3·1.1HCl: C 69.10, H 4.43, N 6.45; found: C 68.81, H 4.18,
25
18
2
N 6.48.
(
E)-3-{4-{{4-{(E)-1-[(2-aminoethoxy)imino]ethyl}phenyl}amino}quinolin-2-yl)}-1-phenylprop-2-en-1-one
◦
hydrochloride (12d), yellow solid; yield: 38%; Mp. 200.2-201.5 C; HPLC (method B), t = 8.613 min;
purity: 98.4%; H-NMR (DMSO-d )
R
1
δ 11.29 (br s, 1H, NH), 9.00-8.90 (m, 2H), 8.67 (m, 1H), 8.31-8.29 (m,
6
4
3
H), 8.06 (m, 1H), 7.89-7.59 (m, 9H), 7.46 (s, 1H, 3-H), 4.37 (t, 2H, J = 5.2 Hz), 3.18-3.16 (m, 2H), 2.32 (s,
H, CH3); 1 C-NMR (DMSO-d6)
δ 188.61, 155.12, 154.47, 147.63, 139.37, 138.30, 136.53, 134.72, 134.24,
3
134.10 (2C), 132.04, 129.13 (2C), 129.02 (2C), 127.43 (2C), 127.16, 124.73 (2C), 123.76, 120.66, 117.22,
1
02.89, 69.91, 38.23, 12.71; Anal. calcd for C H N O2·2.6HCl: C 61.67, H 5.29, N 10.27; found: C 61.32,
28
26
4
H 4.95, N 10.27.
(
E)-3-{4-{{4-{(E)-1-{[3-(dimethylamino)propoxy]imino}ethyl}phenyl}-amino}quinolin-2-yl}-1-phenylprop-2-
◦
en-1-one hydrochloride (12e), yellow solid; yield: 52%; Mp. 155.6-156.6 C; HPLC (method F), t = 7.647
R

Molecules 2020, 25, 3133
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1
min; purity: 96.1%; H-NMR (DMSO-d )
δ
9.26 (br s, 1H, NH), 8.41 (m, 1H), 8.13-8.07 (m, 3H), 8.00-7.98
6
(
2
m, 1H), 7.79-7.67 (m, 5H), 7.62-7.56 (m, 4H), 7.47 (m, 2H), 4.17 (t, 2H, J = 6.4 Hz), 2.52-2.49 (m,
13
H), 2.29 (s, 6H), 2.21 (s, 3H, CH ), 1.91-1.84 (m, 2H); C-NMR (DMSO-d6) δ 189.94, 153.52, 153.21,
3
1
1
48.92, 147.74, 144.47, 144.68, 137.38, 133.30, 130.60, 130.12, 129.65, 128.93 (C), 128.52 (2C), 126.97 (2C),
26.08, 125.63, 122.25, 120.83 (2C), 120.03, 103.68, 71.41, 55.41, 44.49 (2C), 26.34, 12.18; Anal. calcd for
C H N O ·0.3HCl: C 73.94, H 6.47, N 11.12; found: C 74.04, H 6.57, N 10.72.
31
32
4
2
(
E)-N-[2-(diethylamino)ethyl]-4-{[2-(3-oxo-3-phenylprop-1-en-1-yl)quino-lin-4-yl]amino}benzamide
◦
hydrochloride (12f), yellow solid; yield: 51%; Mp. 190.9-191.6 C; HPLC (method E), t = 4.667 min;
purity: 97.9%; H-NMR (DMSO-d )
R
1
δ 11.23 (br s, 1H, NH), 10.53 (br s, 1H, NH), 9.16 (m, 1H), 8.93-8.86
6
(
(
1
1
m, 2H), 8.62 (m, 1H), 8.28 (m, 2H), 8.14 (m, 2H), 8.07 (m, 1H), 7.83-7.59 (m, 6H), 7.53 (s, 1H, 3-H), 3.71
13
q, 2H, J = 6.0 Hz), 3.29-3.18 (m, 6H), 1.27 (t, 6H, J = 7.2 Hz); C-NMR (DMSO-d6)
δ
188.58, 165.76,
54.27, 147.82, 140.27, 139.38, 136.51, 134.72, 134.28, 134.08, 132.03, 131.76, 129.09 (2C), 129.02 (4C),
27.27, 124.25 (2C), 123.68, 120.75, 117.38, 102.86, 49.95, 46.66 (2C), 34.26, 8.39 (2C); Anal. calcd for
C H N O ·4.5HCl: C 56.70, H 5.60, N 8.53; found: C 56.69, H 5.61, N 8.54.
31
32
4
2
2
-(Diethylamino)ethyl (E)-4-{[2-(3-oxo-3-phenylprop-1-en-1-yl)quinolin-4-yl]amino}benzoate hydrochloride
◦
(
12g), yellow solid; yield: 52%; Mp. 158.7-159.3 C; HPLC (method A), t = 6.640 min; purity: 98.9%;
R
1
H-NMR (DMSO-d6)
δ
11.31 (br s, 1H, NH), 10.71 (br s, 1H, NH), 8.93-8.89 (m, 2H), 8.62 (m, 1H),
.29-8.19 (m, 4H), 8.07 (m, 1H), 7.84-7.71 (m, 5H), 7.63-7.59 (m, 3H), 4.68 (m, 2H), 3.56 (m, 2H), 3.25 (m,
H), 1.30 (t, 6H, J = 7.2 Hz); 13C-NMR (DMSO-d6)
188.64, 164.86, 154.05, 148.04, 142.40, 139.50, 136.53,
8
4
δ
1
1
5
34.67, 134.37, 134.16, 132.18, 131.23 (2C), 129.13 (2C), 129.06 (2C), 127.44, 126.76, 124.13 (2C), 123.86,
20.87, 117.71, 103.25, 59.48, 49.36, 47.03 (2C), 8.53 (2C); Anal. calcd for C H N O3·3.6HCl: C 59.58, H
31
31
3
.58, N 6.72; found: C 59.43, H 5.60, N 6.65.
(
E)-1-(4-Methoxyphenyl)-3-[4-(phenylamino)quinolin-2-yl]prop-2-en-1-one hydrochloride (13a), yellow
◦
1
solid; yield 48%; Mp. 220.6-221.1 C; HPLC (method G), t = 4.467 min; purity: 99.1%; H-NMR
R
(
8
7
DMSO-d6)
δ
11.05 (br s, 1H, NH), 8.85 (d, 1H, J = 15.6 Hz, C
.27-8.24 (m, 2H), 8.04 (m, 1H), 7.78 (m, 1H), 7.68 (d, 1H, J = 15.6 Hz, CH=C
.44 (m, 1H), 7.31 (s, 1H, 3-H), 7.12-7.08 (m, 2H), 3.88 (s, 3H, OCH3); 13C-NMR (DMSO-d6)
H
=CHC=O), 8.78 (m, 1H), 8.56 (m, 1H),
C=O), 7.62-7.54 (m, 4H),
186.53,
H
δ
163.93, 154.80, 147.66, 139.17, 137.05, 134.20, 133.91, 132.06, 131.60 (2C), 129.93 (2C), 129.53, 127.39,
1
27.08, 125.18 (2C) 123.41, 120.59, 116.89, 114.29 (2C), 102.12, 55.70; Anal. calcd for C H N O2·1.1HCl:
25
20
2
C 71.40, H 5.06, N 6.66; found: C 71.42, H 4.94, N 6.42.
(
E)-3-{4-[(4-Acetylphenyl)amino]quinolin-2-yl}-1-(4-methoxyphenyl)prop-2-en-1-one hydrochloride (13b),
◦
1
yellow solid; yield: 50%; Mp. 248.7-249.2 C; HPLC (method G), t = 6.087 min; purity: 99.0%; H-NMR
R
(
7
DMSO-d6)
δ 11.09 (br s, 1H, NH), 8.85-8.78 (m, 2H), 8.53 (m, 1H), 8.26 (m, 2H), 8.15-8.06 (m, 3H),
13
.84-7.71 (m, 4H), 7.62 (s, 1H, 3-H), 7.12 (m, 2H), 3.88 (s, 3H, OCH ), 2.64 (s, 3H, CH ); C-NMR
3
3
(
DMSO-d6)
δ 196.94, 186.61, 163.98, 153.94, 148.29, 141.96, 139.49, 134.57, 134.31, 134.04, 132.25, 131.64
(2C), 130.03 (2C), 129.54, 127.39, 123.95 (2C), 123.57, 120.94, 117.63, 114.33 (2C), 103.03, 55.74, 26.75;
Anal. Calcd for C H N O ·1.5HCl: C 67.96, H 4.96, N 5.87; found: C 67.67, H 5.08, N 5.79.
27
22
2
3
(
E)-4-{{2-[3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl]quinolin-4-yl}-amino}benzoic acid hydrochloride
◦
(
13c), orange solid; yield: 49%; Mp. 298.3-298.9 C; HPLC (method D), t = 9.900 min; purity: 96.7%;
R
1
H-NMR (DMSO-d6)
m, 1H), 8.27 (m, 2H), 8.12-8.04 (m, 3H), 7.82-7.68 (m, 4H), 7.59 (s, 1H, 3-H), 7.11 (m, 2H), 3.87 (s, 3H,
OCH3); 13C-NMR (DMSO-d6)
186.53, 166.71, 163.94, 154.20, 148.05, 141.45, 139.20, 134.31, 133.75,
32.37, 131.62 (2C), 131.00 (2C), 129.50, 128.71, 127.32, 124.23 (2C), 123.57, 120.64, 117.41, 114.28 (2C),
δ 11.14 (br s, 1H, NH), 8.87 (d, 1H, J = 16.0 Hz, CH=CHC=O), 8.80 (m, 1H), 8.57
(
δ
1
1
02.77, 55.70; Anal. Calcd for C H N O4·1.6HCl: C 64.68, H 4.51, N 5.80; found: C 64.61, H 4.62,
26
20
2
N 5.53.
(
E)-3-{4-{{4-{(E)-1-[(2-Aminoethoxy)imino]ethyl}phenyl}amino}quinolin-2-yl}-1-(4-methoxyphenyl)prop-2-
◦
en-1-one hydrochloride (13d), orange solid; yield: 39%; Mp. 177.9-181.7 C; HPLC (method B), t = 9.713
min; purity: 98.6%; H-NMR (DMSO-d )
R
1
δ 11.21 (br s, 1H, NH), 8.97 (d, 1H, J = 16.0 Hz, CH=CHC=O),
6
8.87 (m, 1H), 8.66 (m, 1H), 8.32-8.23 (m, 4H), 8.05 (m, 1H), 7.89-7.87 (m, 2H), 7.79 (m, 1H), 7.71 (d,
1
H, J = 16.0 Hz, CH=C C=O), 7.65-7.62 (m, 2H), 7.44 (s, 1H, 3-H), 7.13-7.10 (m, 2H), 4.37 (t, 2H,
H

Molecules 2020, 25, 3133
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J = 5.6 Hz), 3.89 (s, 3H, OCH ), 3.18 (m, 2H), 2.32 (s, 3H, CH ); 3C-NMR (DMSO-d6)
1
δ
186.60, 163.95,
3
3
1
1
55.12, 154.42, 147.82, 139.32, 138.30, 134.21, 134.06, 132.23, 131.67 (2C), 129.56, 127.44 (2C), 127.12,
24.70 (2C), 123.65, 120.66, 117.18, 114.30 (2C), 112.50, 102.79, 69.90, 55.73, 38.23, 12.70; Anal. calcd for
C H N O ·3.7HCl: C 56.59, H 5.19, N 9.10; Found: C 56.10, H 5.17, N 8.95.
29
28
4
3
(
E)-3-{4-{{4-{(E)-1-{[3-(Dimethylamino)propoxy]imino}ethyl}phenyl}-amino}quinolin-2-yl-1-(4-metho
◦
xyphenyl)prop-2-en-1-one hydrochloride (13e), orange solid; yield: 48%; Mp. 165.4-166.9 C; HPLC
1
(
8
4
method C), t = 15.673 min; purity: 99.1%; H-NMR (DMSO-d )
δ
9.41 (br s, 1H, NH), 8.48 (m, 1H),
C=O), 7.60-7.48 (m,
H), 7.11 (m, 2H), 4.21 (t, 2H, J = 6.0 Hz), 3.88 (s, 3H, OCH ), 3.11 (m, 2H), 2.72 (s, 6H), 2.24 (s, 3H,
R
6
.16-8.09 (m, 3H), 8.00 (m, 1H), 7.79-7.73 (m, 3H), 7.78 (d, 1H, J = 15.6 Hz, CH=CH
3
13
CH ), 2.15-2.08 (m, 2H); C-NMR (DMSO-d6)
δ
187.90, 163.39, 154.15, 153.27, 148.75, 147.85, 143.46,
3
1
41.87, 130.99 (2C), 130.38, 130.21, 129.41, 127.06 (2C), 126.12, 125.56, 122.50, 120.80 (2C), 120.03, 114.21
(
2C), 103.71, 70.56, 55.63, 54.03, 42.09 (2C), 24.16, 12.32; Anal. Calcd for C H N O3·4.8HCl: C 55.09,
32
34
4
H 5.60, N 8.03; Found: C 54.75, H 5.56, N 7.91.
(
E)-N-[2-(Diethylamino)ethyl]-4-{{2-[3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl]quinolin-4-yl}amino}ben
◦
zamide hydrochloride (13f), yellow solid; yield: 50%; Mp. 190.9-191.6 C; HPLC (method B), t = 5.473
R
1
min; purity: 95.4%; H-NMR (DMSO-d )
δ
11.20 (br s, 1H, NH), 10.52 (br s, 1H, NH), 9.14 (m, 1H),
=CHC=O), 8.86 (m, 1H), 8.63 (m, 1H), 8.29 (m, 2H), 8.14 (m, 2H), 8.06
m, 1H), 7.80 (m, 1H), 7.75-7.68 (m, 3H), 7.51 (s, 1H, 3-H), 7.12 (m, 2H), 3.88 (s, 3H, OCH ), 3.71 (m,
6
8
(
2
1
.92 (d, 1H, J = 16.0 Hz, CH
3
H), 3.29-3.18 (m, 6H), 1.27 (t, 6H, J = 7.2 Hz); 13C-NMR (DMSO-d6)
47.97, 140.26, 139.31, 134.24, 133.92, 132.26, 131.75, 131.64 (2C), 129.53, 129.01 (2C), 127.22, 124.23
2C), 123.64, 120.73, 117.33, 114.30 (2C), 102.76, 55.72, 49.95, 46.66 (2C), 34.27, 8.40 (2C) Anal. calcd for
δ 186.54, 165.76, 163.94, 154.25,
(
;
C H N O ·3.0HCl: C 60.81, H 5.90, N 8.86; found: C 60.81, H 6.05, N 8.86.
32
34
4
3
2
-(Diethylamino)ethyl (E)-4-{{2-[3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl]-quinolin-4-yl}amino}benzoate
◦
hydrochloride (13g), yellow solid; yield: 53%; Mp. 158.7-159.3 C; HPLC (method A), t = 7.553 min;
purity: 99.3%; H-NMR (DMSO-d )
R
1
δ 10.75 (br s, 1H, NH), 8.92-8.84 (m, 2H), 8.61 (m, 1H), 8.29 (m, 2H),
6
8
.19 (m, 2H), 8.05 (m, 1H), 7.82-7.73 (m, 4H), 7.62 (s, 1H, 3-H), 7.12 (m, 2H), 4.68 (m, 2H), 3.89 (s, 3H,
13
OCH ), 3.55 (m, 2H), 3.26-3.24 (m, 4H), 1.29 (t, 6H, J = 7.2 Hz); C-NMR (DMSO-d6)
δ
186.65, 164.83,
63.92, 153.48, 148.55, 142.70, 134.61, 133.99, 131.95, 131.62 (2C), 131.19 (2C), 129.59, 127.23, 126.33,
23.69 (2C), 121.48, 117.88, 116.03, 115.81, 114.30 (2C), 103.46, 59.39, 55.72, 49.30, 46.95 (2C), 8.48 (2C);
3
1
1
Anal. calcd for C H N O ·3.3HCl: C 59.68, H 5.68, N 6.52; found: C 59.34, H 5.81, N 6.33.
32
33
3
4
(
E)-1-(4-Fluorophenyl)-3-[4-(phenylamino)quinolin-2-yl]prop-2-en-1-one hydrochloride (14a), yellow
◦
1
solid; yield: 54%; Mp. 231.7-232.1 C; HPLC (method E), t = 11.213 min; purity: 98.8%; H-NMR
R
(
8
7
DMSO-d6)
δ
11.09 (br s, 1H, NH), 8.87 (d, 1H, J = 15.6 Hz, C
.39-8.34 (m, 2H), 8.05 (m, 1H), 7.78 (m, 1H), 7.74 (d, 1H, J = 15.6 Hz, CH=C
.46-7.40 (m, 3H), 7.33 (s, 1H, 3-H); 13C-NMR (DMSO-d6)
187.08, 165.55 (JCF = 251.6 Hz), 154.90,
H
=CHCO), 8.79 (m, 1H), 8.57 (m, 1H),
H
CO), 7.61-7.54 (m, 4H),
δ
1
1
47.39, 139.12, 137.01, 134.72, 134.31, 133.28 (JCF = 2.7 Hz), 132.20 (2C, JCF = 9.9 Hz), 131.72, 129.94 (2C),
27.45, 127.17, 125.22 (2C), 123.46, 120.52, 116.90, 116.17 (2C, JCF = 21.8 Hz), 102.29; Anal. calcd for
C H FN O·1.6HCl: C 67.55, H 4.39, N 6.56; found: C 67.40, H 4.43, N 6.68.
24
17
2
(
E)-3-{4-[(4-Acetylphenyl)amino]quinolin-2-yl}-1-(4-fluorophenyl)prop-2-en-1-one hydrochloride (14b),
◦
yellow solid; yield: 52%; Mp. 268.8-269.5 C; HPLC (method G), t = 6.087 min; purity: 99.1%;
R
1
H-NMR (TFA-d)
δ
8.36 (m, 1H), 8.24 (m, 2H), 8.06-7.97 (m, 5H), 7.83-7.76 (m, 2H), 7.62 (m, 2H), 7.42 (s,
H, 3-H), 7.17 (m, 2H), 2.75 (s, 3H, CH3); 13C-NMR (TFA-d)
202.37, 189.83, 165.99 (JCF = 259.3 Hz),
53.70, 145.54, 139.72, 136.71, 133.83, 133.49, 133.21, 130.55 (2C, JCF = 9.9 Hz), 129.97, 129.66, 129.30
1
1
δ
(
2C), 127.10, 122.80 (2C), 119.63, 118.37, 115.39, 114.49 (2C, JCF = 22.8 Hz), 98.74, 22.75; Anal. calcd for
C H FN O ·1.5HCl: C 67.14, H, 4.44, N 6.02; found: C 66.77, H 4.67, N 5.98.
26
19
2
2
(
E)-4-({2-[3-(4-Fluorophenyl)-3-oxoprop-1-en-1-yl]quinolin-4-yl}amino)-benzoic acid hydrochloride (14c),
◦
yellow solid; yield: 49%; Mp. 278.9-280.2 C; HPLC (method F), t = 10.780 min; purity: 99.3%;
H-NMR (DMSO-d6)
R
1
δ 13.02 (br s, 1H, OH), 11.07 (br s, 1H, NH), 8.83-8.77 (m, 2H), 8.51 (m, 1H),
8
.36-8.33 (m, 2H), 8.12-8.04 (m, 3H), 7.83-7.78 (m, 2H), 7.68 (m, 2H), 7.62 (s, 1H, 3-H), 7.44 (m, 2H);
1
3
C-NMR (DMSO-d6)
δ 187.17, 166.82, 165.52 (JCF = 242.8 Hz), 154.02, 148.04, 141.56, 139.44, 134.94,

Molecules 2020, 25, 3133
13 of 16
134.31, 133.29 (JCF = 2.6 Hz), 132.18 (2C, JCF = 9.6 Hz), 131.79, 131.06 (2C), 128.64, 127.40, 124.11 (2C),
1
23.54, 120.97, 117.56, 116.14 (2C, JCF = 21.8 Hz), 102.90; Anal. calcd for C H FN O3·1.7HCl: C 63.29,
25
17
2
H 3.97, N 5.90; found: C 63.02, H 4.12, N 5.83.
(
E)-3-{4-{{4-{(E)-1-[(2-Aminoethoxy)imino]ethyl}phenyl}amino}quinolin-2-yl}-1-(4-fluorophenyl)prop-2-
◦
en-1-one hydrochloride (14d), yellow solid; yield: 40%; Mp.180.1-180.8 C; HPLC (method A), t = 8.660
min; purity: 99.2%; H-NMR (DMSO-d )
R
1
δ 11.25 (br s, 1H, NH), 9.00-8.88 (m, 2H), 8.66 (m, 1H),
6
8
2
.41-8.28 (m, 5H), 8.04 (m, 1H), 7.87 (m, 2H), 7.78-7.74 (m, 2H), 7.63 (m, 2H), 7.45-7.41 (m, 3H), 4.37 (m,
13
H), 3.17 (m, 2H), 2.32 (s, 3H, CH3); C-NMR (DMSO-d6)
δ 187.10, 165.54 (JCF = 251.7 Hz), 155.10,
1
1
54.34, 147.63, 139.48, 138.32, 134.94, 134.15, 134.01, 133.29 (JCF = 2.3 Hz), 132.22 (2C, JCF = 9.1 Hz),
31.70, 127.40 (2C), 127.11, 124.64 (2C), 123.69, 120.78, 117.21, 116.09 (2C, JCF = 21.9 Hz), 102.95, 69.88,
3
8.21, 12.70; Anal. calcd for C H FN O2·1.9HCl: C 62.53, H 5.04, N 10.42, found: C 62.71, H 5.02,
28
25
4
N 10.53.
(
E)-3-{4-{{4-{(E)-1-{[2-(Dimethylamino)ethoxy]imino}ethyl}phenyl}-amino}quinolin-2-yl}-1-(4-fluoroph
◦
enyl)prop-2-en-1-one hydrochloride (14e), yellow solid; yield: 51%; Mp.131.2-132.7 C; HPLC (method E),
t = 4.793 min; purity: 97.7%; H-NMR (DMSO-d )
1
δ
9.26 (br s, 1H, NH), 8.41 (m, 1H), 8.17 (m, 2H),
R
6
8
4
.09 (d, 1H, J = 15.6 Hz, C
H=CHCO), 7.98 (m, 1H), 7.78-7.69 (m, 4H), 7.60-7.56 (m, 2H), 7.47-7.38 (m,
13
H), 4.17 (t, 2H, J = 6.0 Hz), 2.66 (m, 2H), 2.40 (s, 6H), 2.21 (s, 3H, CH ), 1.95-1.89 (m, 2H); C-NMR
3
(
(
(
DMSO-d6) δ 188.45, 165.09 (JCF = 251.0 Hz), 153.68, 153.17, 148.91, 148.71, 144.60, 141.73, 134.04
JCF = 3.1 Hz), 131.56 (2C, JCF = 9.8 Hz), 130.50, 130.12, 129.62, 126.99 (2C), 125.84, 125.62, 122.27, 120.76
2C), 120.04, 115.94 (2C, JCF = 22.0 Hz), 103.72, 71.16, 55.05, 43.86 (2C), 25.76, 12.20; Anal. calcd for
C H FN O ·1.5HCl: C 65.86, H 5.79, N 9.91. Found: C 65.48, H 5.93, N 9.86.
31
31
4
2
(
E)-N-[2-(Diethylamino)ethyl]-4-{{2-[3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl]quinolin-4-yl}amino}ben
◦
zamide hydrochloride (14f), yellow solid; yield: 50%; Mp. 175.6-176.1 C; HPLC (method A), t = 5.847
min; purity: 99.1%; H-NMR (DMSO-d )
R
1
δ 11.31 (br s, 1H, NH), 10.66 (br s, 1H, NH), 9.19 (m, 1H),
6
9
7
.01-8.91 (m, 2H), 8.68 (m, 1H), 8.40 (m, 2H), 8.16-8.05 (m, 3H), 7.82-7.68 (m, 4H), 7.53 (s, 1H, 3-H),
.47-7.43 (m, 2H), 3.71 (m, 2H), 3.29-3.17 (m, 6H), 1.27 (t, 6H, J = 7.2 Hz); 13C-NMR (DMSO-d6)
δ
187.12,
1
(
65.74, 165.55 (JCF = 251.7 Hz), 154.29, 147.76, 140.30, 139.47, 134.85, 134.23, 133.31 (JCF = 3.0 Hz), 132.24
2C, JCF = 9.0 Hz), 131.83, 131.72, 129.00 (2C), 127.23, 124.22 (2C), 123.80, 120.75, 117.41, 116.13 (2C,
JCF = 22.0 Hz), 103.05, 49.94, 46.65 (2C), 34.24, 8.40 (2C); Anal. Calcd for C H FN O2·3.6HCl: C 58.01,
31
31
4
H 5.43, N 8.73; found: C 57.84, H 5.44, N 8.93.
2
-(Diethylamino)ethyl (E)-4-{{2-[3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl]-quinolin-4-yl}amino}benzoate
◦
hydrochloride (14g), yellow solid; yield: 50%; Mp. 135.4-135.8 C; HPLC (method A), t = 8.073 min;
purity: 98.8%; H-NMR (DMSO-d )
R
1
δ 9.60 (br s, 1H, NH), 8.41 (m, 1H), 8.23-8.14 (m, 3H), 8.04-8.01 (m,
6
3
(
H), 7.81-7.76 (m, 3H), 7.62 (m, 1H), 7.54 (m, 2H), 7.45-7.40 (m, 2H), 4.53 (br s, 2H), 3.04 (br s, 4H), 1.20
m, 6H); 13C-NMR (DMSO-d6)
δ
188.35, 165.15, 165.14 (JCF = 250.2 Hz), 153.37, 148.99, 146.60, 146.47,
1
1
44.35, 134.01 (JCF = 3.0 Hz), 131.64 (2C, JCF = 9.1 Hz), 131.12 (2C), 130.34, 129.65, 126.11, 122.60, 122.21,
20.84, 118.65 (2C), 115.97 (2C, JCF = 22.0 Hz), 106.09, 60.19, 49.78, 47.00 (2C), 9.46 (2C); Anal. calcd for
C H FN O ·4.1HCl: C 56.32, H 5.20, N 6.36; found: C 56.21, H 5.49, N 6.29.
31
30
3
3
5
.5. Cytotoxicity and Antiviral Activity Assays
5
.5.1. Compounds
Compounds were dissolved in DMSO at 10 mM and then diluted in the culture medium.
5
.5.2. Cell Culture
Immortalized human keratinocytes HaCaT were cultured in the Dulbecco’s modified Eagle’s
medium (DMEM; Gibco BRL, Grand Island, NY, USA) with 10% heat-inactivated fetal bovine serum
HyClone, Logan, UT, USA), penicillin (100 U/mL), and streptomycin (100 g/mL) in a humidified
(
µ
incubator with 5% CO . HaCaT/ARE stable cells were grown in DMEM supplemented with 100
µg/mL
2
of hygromycin.

Molecules 2020, 25, 3133
14 of 16
5
.5.3. Luciferase Reporter Assay
4
HaCaT/ARE stable cells were seeded (1
×
10 cells/well) in a 96 well plate, then were treated with
indicated concentrations of compounds for 18 h. At the assay time point, resazurin (Cayman Chemical,
Ann Arbor, MI, USA) was added to a final concentration of 0.1 mg/mL and further incubated for 4 h at
◦
3
7 C. Fluorescence of the reduced resazurin (ex/em: 530 nm/590 nm) was measured from the culture
supernatant by using a Synergy HT Multi-Mode Reader (BioTek, Winooski, VT, USA) to determine
cell viability. The cells were then harvested for luciferase activity measurements according to the
manufacturer’s protocol (Promega Corporation, Madison, WI, USA). Relative luciferase activity was
calculated by normalizing luciferase activity to cell viability, and DMSO solvent control was used as
1
00% activity.
5
.5.4. Cell Viability Assay
Cells were seeded in a 96-well plate contained 100 µL culture medium in triplicate and treated
with several compounds for 72 h. At the assay time point, resazurin (Cayman Chemical) was added
◦
and further incubated for 4 h at 37 C. Fluorescence of the reduced resazurin was measured as
described previously.
5
.5.5. Quantitative Real-Time PCR (qRT-PCR)
RNA was prepared by using the TRIzol Reagent (Thermo Scientific, Madison, WI, USA) and was
reverse transcribed into cDNA using a TOOLs Easy Fast RT Kit (TOOLs Biotechnology, New Taipei City,
Taiwan). PCR was performed on an ABI StepOne Plus System (Applied Biosystems, Foster City, CA
USA) using the KAPA SYBR FAST qPCR Master Mix (2X) Kit (Sigma-Aldrich, St. Louis, MO, USA).
The mRNA level was normalized using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
,
®
0
mRNA level as the standard. The following primers were used: NRF2 forward: 5 -AGACGGTATGC
0
0
AACAGGACA and reverse: 5 -ACCATGGTAGTCTCAACCAGC; HO1 forward: 5 -GCCAGC
AACAAAGTGCAAG and reverse: 5 -GAGTGTAAGGACCCATCGGA; NQO1 forward: 5 -TGCAGC
GGCTTTGAAGAAGAAAGG and reverse: 5 -TCGGCAGGATACTGAAAGTTCGCA; GCLC forward:
-CTGGGGAGTGATTTCTGCAT and reverse: 5 -AGGAGGGGGCTTAAATCTCA; G6PD forward:
-CAACATCGCCTGCGTTA and reverse: 5 -CTTGACCTTCTCATCACGG; and GAPDH forward:
-GCAAATTCCATGGCACCGTCA and reverse: 5 -TCCTGGAAGATGGTGATGGGA.
0
0
0
0
0
0
0
5
5
5
0
0
5
.5.6. Western Blot
Cells were washed with PBS and harvested with RIPA lysis buffer (50 mM Tris (pH 7.5), 150 mM
NaCl, 1% Tx-100) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 10
leupeptin, 50 g/mL TLCK, 50 g/mL TPCK, 1 g/mL aprotinin, 1 mM NaF, 5 mM NaPPi, and 10 mM
Na VO ). The cell lysates were cleared via 10 min spins at 13,000 rpm, 4 C. Protein concentrations
µg/mL
µ
µ
µ
◦
3
4
were determined by a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Samples corresponded to
g of protein were used for Western blot according to our previous publication [ ]. The following
2
0
µ
7
antibodies were used: anti-NRF2 (GeneTex, Irvine, CA, USA) and anti-GAPDH (Abcam, Cambridge,
MA, USA).
5
.5.7. Molecular Docking Study
The crystal structure of the Keap1 Kelch domain (PDB code 5FNQ) [27] was acquired from
the RCSB Protein Date Bank. The 3D conformation of target compound 13b was produced by
ChemBio 3D Ultra 14.0. The molecular docking was performed by the Achilles Blind Docking Server
(http://bio-hpc.ucam.edu/achilles/). The “blind docking” approach was used for the docking of the
small molecule to the targets, which was done without a priori knowledge of the location of the binding
site by the system [28]. Visual representation of molecules was created with 3Dmol by Nicholas Rego
and David Koes [29].

Molecules 2020, 25, 3133
15 of 16
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/14/3133/s1.
Author Contributions: Y.-T.K. participated in synthesis, purification, and characterization of the chemical
compounds; Y.-S.C. participated in the biological activity, the interpretation of the results and in manuscript
writing; K.-W.T. participated in molecular modeling; J.-C.L. participated in the biological activity; C.-H.T. and C.-C.T.
participated in synthesis; C.-H.Y. and Y.-L.C. suggested the research idea, participated in the interpretation of the
results and in manuscript writing. All authors have read and agreed to the published version of the manuscript.
Funding: Financial support of this work by the Minister of Science and Technology of the Republic
of China (MOST 107-2320-B-037-015, MOST 108-2320-B-037-026-MY2, MOST 108-2320-B-037-025, MOST
1
07-2311-B-037-005-MY3, MOST 108-2319-B-037-001), the Livestock Research Institute, Council of Agriculture,
Taiwan [grant number 109AS-17.1.2-LI-L2],) and the Kaohsiung Medical University Research Center
KMU-TC108A03-2, KMU-DK109002-1) are gratefully acknowledged.
(
Acknowledgments: Authors were thankful to the Center for Research Resources and Development at Kaohsiung
Medical University for the instrumentation and equipment support.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds reported herein are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(