Diversity-oriented generation and biological evaluation of new chemical scaffolds bearing a 2,2-dimethyl-2H-chromene unit: Discovery of novel potent ANO1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104000

Chemical territory bearing a 2,2-dimethyl-2H-chromene motif was expanded by utilizing an o-hydroxy aldehyde group of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde as a synthetic handle to install distinctive morphology and functionality of each scaffold. Cell based assays and in silico docking analysis led us to discover that these new compounds exhibit inhibitory effect on anoctamin1 (ANO1). ANO1 is amplified and highly expressed in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic cancer. Biological assays revealed that (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (3n, Ani-FCC) is a novel, potent and selective ANO1 inhibitor with an IC₅₀ value of 1.23 μM. 3n showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride channel activity of CFTR and the intracellular calcium signaling. Notably, 3n strongly decreased cell viability of PC-3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, 3n significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC-3 and FaDu cells. This study shows that a novel ANO1 inhibitor, 3n, can be a potential candidate for the treatment of cancers overexpressing ANO1, such as prostate cancer and esophageal cancer.

Full text of DOI:10.1016/j.bioorg.2020.104000

Bioorganic Chemistry 101 (2020) 104000
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Diversity-oriented generation and biological evaluation of new chemical
scaffolds bearing a 2,2-dimethyl-2H-chromene unit: Discovery of novel
potent ANO1 inhibitors
a,1
a,1
a,1
b
c
b,d
Yohan Seo , Jiwon Choi , Jeong Hwa Lee , Tae Gun Kim , So-hyeon Park , Gyoonhee Han ,
a,d,⁎
a,⁎
Wan Namkung
, Ikyon Kim
a
College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983, Republic of Korea
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea
Graduate Program of Industrial Pharmaceutical Science, Yonsei University, Incheon 21983, Republic of Korea
b
c
d
Interdisciplinary Program of Integrated OMICS for Biomedical Science Graduate School, Yonsei University, Seoul 03722, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chemical territory bearing a 2,2-dimethyl-2H-chromene motif was expanded by utilizing an o-hydroxy aldehyde
group of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde as a synthetic handle to install distinctive
morphology and functionality of each scaffold. Cell based assays and in silico docking analysis led us to discover
that these new compounds exhibit inhibitory effect on anoctamin1 (ANO1). ANO1 is amplified and highly ex-
pressed in various carcinomas including prostate cancer, esophageal cancer, breast cancer, and pancreatic
cancer. Biological assays revealed that (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-
Diversity-oriented synthesis
2
H-Chromene
Chemical space
Anoctamin 1 (ANO1)
Anticancer agent
Apoptosis
2
-en-1-one (3n, Ani-FCC) is a novel, potent and selective ANO1 inhibitor with an IC50 value of 1.23 μM. 3n
showed 144 times stronger activity on ANO1 inhibition than ANO2 inhibition and did not alter the chloride
channel activity of CFTR and the intracellular calcium signaling. Notably, 3n strongly decreased cell viability of
PC-3 and FaDu cells expressing high levels of ANO1 with a decrease in ANO1 protein levels. In addition, 3n
significantly enhanced apoptosis via activation of caspase 3 and cleavage of PARP in PC-3 and FaDu cells. This
study shows that a novel ANO1 inhibitor, 3n, can be a potential candidate for the treatment of cancers over-
expressing ANO1, such as prostate cancer and esophageal cancer.
1
. Introduction
o-hydroxy benzaldehyde group of 5-hydroxy-2,2-dimethyl-2H-chro-
mene-6-carbaldehyde (1) as a synthetic handle to add structural and
2
H-Chromene, also known as 2H-benzopyran, is a substructure of
functional diversification of each scaffold (Scheme 1) [19–23]. As part
of our continued research interest on design, synthesis, and biological
evaluation of novel chemical space [24–31], here we wish to report
efficient construction and preliminary biological investigation of new
chemical libraries based on a 2,2-dimethyl-2H-chromene motif [32].
Through biological screening, we were able to discover that these new
chromene-based chemical scaffolds inhibit ANO1.
many natural products. More specifically, 2,2-dimethyl-2H-chromene
moiety is embedded in a number of bioactive natural substances (Fig. 1)
[
1–5]. Thus, this functional motif has been frequently used as a partial
structure of the small-molecule medicinal agents in drug development
programs [6–10]. For example, Lee and co-workers developed potent
antiangiogenic agents through design and synthesis of ring-truncated
deguelin derivatives [11].
ANO1 plays an important role in a wide range of biological pro-
cesses in various cell types such as epithelial cells, smooth muscle cells,
intestinal pacemaker cells and sensory neurons [33–36]. ANO1 is also
highly expressed and plays a pivotal role in cancer progression such as
proliferation of cancer cells and metastasis in various carcinomas
Although several systematic approaches to 2H-chromene-containing
chemical libraries have been demonstrated in the literature [12–14], we
envisioned that new chemical space [15–18] having this moiety with
distinctive substitution patterns could be further generated by using an
⁎
Corresponding authors at: College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon
1983, Republic of Korea (W. Namkung and I. Kim).
2
E-mail addresses: wnamkung@yonsei.ac.kr (W. Namkung), ikyonkim@yonsei.ac.kr (I. Kim).
1
These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2020.104000
Received 19 October 2019; Received in revised form 21 April 2020; Accepted 3 June 2020
Available online 08 June 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
chalcones 3 [60] as shown in Scheme 3. Both electron-rich and elec-
tron-poor arylaldehydes participated well in these condensations to
afford the corresponding enones 3 in good to excellent yields.
The Petasis reaction, also known as the Petasis borono-Mannich
reaction, is a three component reaction of an aldehyde, an amine, and a
vinyl- or arylboronic acid to form a polyfunctionalized amine [61–63].
Since use of salicylaldehyde as an aldehyde partner of this reaction
leads to highly substituted 1,3-amino alcohols [64,65], we applied this
reaction to 1 to get benzylamines 4 (Scheme 4). Various 1,3-amino
alcohols 4 were readily accessed in good yields under mild conditions.
As some biological activities of heterocycles having a 1,3-amino alcohol
moiety were known, we hoped that our scaffold 4 may have interesting
pharmacological functions.
In addition, reaction of 1 with 4-nitrobenzyl bromide in the pre-
sence of KOt-Bu at 160 °C provided the corresponding benzofurans 5a
(
Scheme 5) [66,67]. Similarly, benzofuran (5b) having a pyridine
moiety at C2 position was accessed from 1 and 2-(chloromethyl)pyr-
idine [68].
To evaluate the biological effect of the pyran ring embedded in this
series of compounds, compound 7 was synthesized by following the
similar procedures as those of 2f and 3 (Scheme 6).
Fig. 1. Some Bioactive Natural Products Having a 2,2-Dimethyl-2H-chromene
Unit.
Biological investigation of the synthesized compounds revealed that
the compounds bearing a 2,2-dimethyl-2H-chromene motif such as 3h,
including head and neck squamous cell carcinoma (HNSCC), gastro-
intestinal, breast, and prostate cancers [37–41]. ANO1 and ANO2 are
identified as CaCC (Calcium-activated chloride channel) and share the
highest sequence homology between ANO family members (ANO1-
ANO10) [42]. ANO2 is highly expressed in olfactory sensory neurons
and plays an important role in the regulation of olfactory transduction.
Interestingly, recent evidence suggests that ANO2 is involved in the
reduction of spike generation in the thalamic cortex and hippocampal
CA1 neurons and ANO2 knockout mice show a lack of executive
function and severe impairment of motors [43,44].
3
n, 3o, 3p and 4d potently inhibited ANO1 channel activity with
IC50 < 10.2 μM, and 3n most potently inhibited ANO1 channel ac-
tivity with an IC50 value of 1.23 μM (Table 1). However, compounds
having no pyran rings (7a, 7b, and 7c) did not show an inhibitory effect
on ANO1 channel function, indicating the importance of this additional
pyran ring on ANO1 inhibition. Based on these observations, further
biological study with 3n was pursued.
2
.2. Inhibitory effect of 3n on ANO1 channel activity
Previous studies reported that selective downregulation of ANO1
without alteration of ANO2 may provide a novel anticancer therapy
We further measured the inhibitory effect of 3n on ANO1, ANO2
[
45,46]. ANO1 inhibitors can reduce cell viability by inhibiting cal-
and cystic fibrosis transmembrane conductance regulator (CFTR) using
YFP fluorescence in FRT cells stably expressing ANO1, ANO2, and
CFTR, respectively. As shown in Fig. 2A and 2B, ANO1 and ANO2
calcium-activated chloride channels were activated by ATP, a P2Y re-
ceptor agonist. 3n potently inhibited YFP fluorescence reduction by
iodide influx via ANO1, but 3n weakly inhibited ANO2-mediated YFP
fluorescence reduction. 3n inhibited ANO1 activity ~ 144 times more
strongly than ANO2 activity (Fig. 2C). To investigate the effect of 3n on
CFTR channel activity, CFTR was activated by forskolin and inhibited
with a specific inhibitor, CFTRinh-172. 3n did not alter the CFTR-
mediated YFP reduction up to 30 μM (Fig. 2D). To observe the effect of
modulin-dependent protein kinase II (CAMKII) and epidermal growth
factor receptor (EGFR) in cancers highly expressing ANO1, such as
breast cancer, head and neck cancer, and esophageal squamous cancer
(
ESCC) [39,47]. ANO1 is overexpressed in prostate cancer and plays
pivotal roles in the regulation of chloride efflux and cell volume in
androgen-independent prostate cancer [48]. Thus, ANO1 is considered
as a potential drug target in various cancers including prostate cancer.
To date, several ANO1 inhibitors have been reported including
CaCCinh-A01, tannic acid, T16Ainh-A01, idebenone, Ani9, luteolin and
Ani9-5f [45,46,49–52]. However, all these inhibitors are in the early
stages of drug discovery: novel, potent, and selective ANO1 inhibitors
for the drug development are still required. In the present study, novel
chemotype ANO1 inhibitors were identified via screening of new
chromene-based compounds and their anticancer effects as ANO1 in-
hibitors were investigated.
3
n on calcium signaling, the intracellular calcium concentration was
increased by ATP in PC-3 cells, and 3n did not significantly reduce the
ATP-induced increase in intracellular calcium concentration up to a
concentration of 10 μM indicating complete inhibition of ANO1
(
Fig. 2E). These results show that 3n is an ANO1 inhibitor with higher
selectivity for ANO1 compared to ANO2 and CFTR with no effect on
intracellular calcium signaling.
2
. Results and discussion
2
.1. Syntheses of novel 2,2-dimethyl-2H-chromene scaffolds
2.3. Effect of 3n on the expression levels of ANO1 protein and cell viability
in PC-3 and FaDu cells
The starting material 1 was easily prepared in gram quantities by
following the known procedure [53,54]. As a means to make a new
Reduction of ANO1 protein is known to significantly inhibit the
growth of prostate cancer and hypopharyngeal cancer cells highly ex-
pressing ANO1 [69,70]. To investigate the effect of 3n on the protein
expression levels of ANO1, 3n was treated with different doses in PC-3
and FaDu cells expressing high levels of ANO1. As shown in Fig. 3A and
B, 3n strongly decreased protein expression levels of ANO1 in a dose
dependent manner.
chemical scaffold from 1, we first chose the Rap-Stoermer reaction
[
55,56], which is a condensation reaction of a salicylaldehyde with an
α-halocarbonyl compound to give a 2-acylbenzofuran [57–59], a key
pharmacophore in many drug discovery researches. Thus, reactions of 1
with several α-bromoketones in the presence of K
2
CO were first at-
3
tempted to synthesize various benzofurans 2 (Scheme 2). The desired 2-
acylbenzofurans were obtained in excellent yields.
To determine whether the reduction of ANO1 protein by 3n inhibit
the proliferation of cancer cells, MTS assay was performed in PC-3 and
FaDu cells expressing ANO1 or knockout ANO1. The ANO1 knockout
In addition, an acetyl moiety in 2f was used for further derivatiza-
tion: Claisen-Schmidt aldol reactions of 2f with arylaldehydes provided
2

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
O
O
R
if R = Me
O
O
O
O
2
3
Ar
Ar
CHO
OH
N
O
O
O
OH
4
5
1
X
Y
O
Scheme 1. Synthetic Plans.
cells were established by CRISPR/Cas9 system. While 3n significant
inhibited cell viability in ANO1 expressing PC-3 and FaDu cells, much
less inhibition of cell viability by 3n in ANO1-knockout PC-3 and FaDu
cells was observed (Fig. 3C and 3D). These results suggest that 3n ex-
hibits anticancer effect via inhibition and reduction of ANO1 protein.
Of note, 3n exhibited ANO1-dependent and ANO1-independent cyto-
toxicity at high concentrations in PC-3 and FaDu cells (Fig. 3D), in-
dicating that 3n can also induce apoptosis via ANO1-independent
pathways.
activity was measured. As shown in Fig. 4A and 4B, the caspase 3 ac-
tivity was significantly increased by 3n in a dose dependent manner,
but the caspase 3 activation by 3n was completely blocked by Ac-
DEVD-CHO, a potent inhibitor of caspase 3. We stained apoptotic cells
with a caspase 3-substrate (NucView® Caspase-3 Substrates), which is
cleaved by caspase 3 in apoptotic cells and stains DNA with bright
green fluorescence. 3n significantly increased caspase 3-mediated
apoptotic cells in PC3 and FaDu cells (Fig. 4C and 4D). To further in-
vestigate the apoptotic mechanism of 3n, we observed levels of cleaved
PARP, a hallmark of apoptosis. The levels of cleaved PARP were sig-
nificantly increased by 3n in a dose dependent manner in PC-3 and
FaDu cells (Fig. 4E and 4F).
Down regulation of ANO1 significantly induced apoptosis via acti-
vation of caspase 3 and cleavage of poly ADP-ribose polymerase (PARP)
[
71]. To determine whether 3n exerts its anti-cancer effects via indu-
cing apoptosis in PC-3 and FaDu cells, the effect of 3n on caspase 3
These results revealed that 3n can induce apoptotic cell death in PC-
a,b
a
(1.5 equiv) in acetonitrile (2 mL) was stirred at 120 °C. b
Scheme 2. Synthesis of Benzofurans 2
.
A mixture of 1 (0.15 mmol), α-haloketone (1.2 equiv), and K
2
CO
3
c
Isolated yield (%). Ethyl bromoacetate (1.2 equiv) was used.
3

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Scheme 3. Synthesis of Chalcones 3a,b
.
a
To a mixture of 2h (0.12 mmol) and ArCHO (1.2 equiv) in EtOH (1 mL) was added K
CO (1 equiv) at rt. The reaction
2
3
mixture was stirred at 60 °C. b Isolated yield (%). MeOH was used instead of EtOH.
c
3
and FaDu cells expressing high levels of ANO1 via inhibition of ANO1
alkyl-hydrophobic interaction with ILE551. The calculated binding
activity and reduction of ANO1 protein levels.
energy between 3n and ANO1 was −51.69 kcal/mol.
2.4. Molecular docking analysis
3. Conclusions
A molecular docking study of 3n to ANO1 was performed using
Several new chemical scaffolds having a 2,2-dimethyl-2H-chromene
moiety were established via selective manipulation of the salicylalde-
hyde group of the starting material and their inhibitory effect of ANO1
was investigated. SAR analysis revealed a novel potent ANO1 inhibitor,
3n (IC50 = 1.23 μM). Selectivity analysis has shown that 3n is highly
previously reported protein structure and binding sites of ANO1
[
72–73]. In Fig. 5A, the binding mode of 3n was selected by low
CDOCKER energy values (-17.22 kcal/mol). As shown in Fig. 5B, 3n has
pi-cation interaction with LYS741, hydrogen bond with LYS645, and
4

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Scheme 3. (continued)
selective for ANO1. 3n did not alter CFTR activity and calcium sig-
naling, and is 144 times more selective for ANO1 than ANO2. In vitro
studies showed that 3n selectively reduced cell viability in an ANO1
dependent manner in PC-3 and FaDu cells. In addition, apoptosis is
strongly induced by 3n via activation of caspase 3 and cleavage of
PARP in PC-3 and FaDu cells expressing ANO1. This study could help to
identify promising drug candidates for the treatment of cancers with
ANO1 upregulation, including prostate cancer and hypopharyngeal
cancer.
C
12
H
13
O 205.0859, found 205.0864.
3
General procedure for the synthesis of 2: To a vial charged with 1
(30 mg, 0.15 mmol) in acetonitrile (2 mL) were added 2-bromo-4′-
methoxyacetophenone (40.4 mg, 1.2 equiv) and potassium carbonate
(60.9 mg, 3.0 equiv) at room temperature. After being stirred at 120 ℃
for 4 h, the reaction mixture was concentrated under reduced pressure,
extracted with CH
aqueous layer was extracted with CH
organic layer was dried over MgSO
2
Cl
2
(2 mL), and washed with H
Cl
, filtered, and concentrated in
2
O (5 mL). The
2
2
(1 mL) two more times. The
4
vacuo. The residue was purified by silica gel column chromatography
(
(
n-hexane:ethyl acetate:dichloromethane = 30:1:2) to give 2a
4
. Experimental section
45.7 mg, 93%).
4.1. General methods
Unless specified, all reagents and starting materials were purchased
from commercial sources and used as received without purification.
“
Concentrated” refers to the removal of volatile solvents via distillation
using a rotary evaporator. “Dried” refers to pouring onto, or passing
through, anhydrous magnesium sulfate followed by filtration. Flash
chromatography was performed using silica gel (230–400 mesh) with
hexanes, ethyl acetate, and dichloromethane as the eluents. All reac-
tions were monitored by thin-layer chromatography on 0.25 mm silica
plates (F-254) visualized with UV light. Melting points were measured
(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)(4-methoxyphenyl)
1
methanone (2a). Brown oil; H NMR (400 MHz, CDCl
3
) δ 8.03 (d,
J = 8.8 Hz, 2H), 7.40–7.37 (m, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.86 (d,
J = 10.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 5.69 (d, J = 10.0 Hz, 1H),
1
13
using a capillary melting point apparatus. H and C NMR spectra were
recorded on a 400 MHz NMR spectrometer and were described as
chemical shifts, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet), coupling constant in hertz (Hz), and number of protons.
HRMS were measured with electrospray ionization (ESI) and Q-TOF
mass analyzer.
1
3
3.86 (s, 3H), 1.46 (s, 6H); C NMR (100 MHz, CDCl ) δ 182.5, 163.4,
3
153.7, 152.5, 152.5, 131.8, 130.5, 130.3, 122.6, 120.8, 116.8, 115.8,
114.8, 113.9, 106.7, 77.2, 55.6, 28.0; HRMS (ESI-QTOF) m/z [M
+
+Na] calcd for C21
H
18NaO 357.1097, found 357.1064.
4
Compound 1 was prepared by following the literature procedure
[
53,54].
(
7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)(4-methoxyphenyl)
1
methanone (2b). Yellow solid, mp: 138.9–140.3 °C;
H NMR
(
400 MHz, CDCl ) δ 7.59 (d, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.44 (s, 1H),
3
5
-Hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde (1). Ivory
7.42 (s, 1H), 7.40 (s, 1H), 7.15 (dd, J = 2.0, 8.4 Hz, 1H), 6.90 (d,
J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.72 (d, J = 9.6 Hz, 1H),
1
solid, mp: 69.6–70.4 °C; H NMR (400 MHz, CDCl
3
) δ 11.65 (s, 1H),
1
3
9
.66 (s, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 10.0 Hz, 1H), 6.43
3.89 (s, 3H), 1.49 (s, 6H); C NMR (100 MHz, CDCl ) δ 183.7, 159.8,
3
1
3
(
d, J = 8.4 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 1.46 (s, 6H); C NMR
154.1, 152.8, 152.0, 139.0, 130.6, 129.6, 122.8, 122.0, 120.7, 119.1,
(
100 MHz, CDCl
3
) δ 194.7, 160.7, 158.8, 134.8, 128.7, 115.4, 115.2,
118.1, 115.8, 115.0, 113.9, 106.7, 77.3, 55.6, 28.0; HRMS (ESI-QTOF)
+
m/z [M+Na]+ calcd for C21
1
09.6, 109.0, 78.3, 28.6; HRMS (ESI-QTOF) m/z [M+H] calcd for
H
18NaO 357.1097, found 357.1071.
4
5

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Scheme 4. Synthesis of Benzylamines 4a,b. a A mixture of 1 (0.15 mmol), amine (1.2 equiv), and ArB(OH)
(1.2 equiv) in CH
Cl
2
(1 mL) was stirred at 60 °C for 18 h. b
2
2
Isolated yield (%).
6

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
[
1,1′-Biphenyl]-4-yl(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)
1
methanone (2e). Ivory solid, mp: 102.9–104.1 °C; H NMR (400 MHz,
CDCl ) δ 8.11 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.67 (d,
3
J = 7.2 Hz, 2H), 7.52–7.48 (m, 3H), 7.44 (d, J = 8.4 Hz, 2H), 6.92 (d,
J = 10.0 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 5.73 (d, J = 10.0 Hz, 1H),
Scheme 5. Synthesis of 5.
1
3
1
1
1
.50 (s, 6H); C NMR (100 MHz, CDCl ) δ 183.5, 154.1, 152.8, 152.2,
3
45.5, 140.1, 136.5, 135.3, 130.6, 130.1, 129.1, 128.4, 127.5, 127.3,
22.8, 120.8, 117.7, 115.8, 115.1, 106.7, 77.4, 28.0; HRMS (ESI-QTOF)
+
m/z [M+H] calcd for C26
H
21
O 381.1485, found 381.1441.
3
(
4-Chlorophenyl)(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)me-
1
thanone (2c). Yellow solid, mp: 88.3–90.8 °C; H NMR (400 MHz,
CDCl ) δ 7.95 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.42–7.38
m, 2H), 6.85–6.80 (m, 2H), 5.70 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H);
) δ 182.6, 154.2, 151.9, 139.2, 136.0, 130.9,
1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)ethan-1-one
(2f).
) δ 7.42 (s,
3
1
3
1
(
C
Yellow solid, mp: 107.9–109.8 °C; H NMR (400 MHz, CDCl
3
NMR (100 MHz, CDCl
3
1H), 7.39 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 10.0 Hz, 1H), 6.81 (d,
1
2
3
30.7, 129.0, 125.8, 122.9, 120.7, 117.9, 115.7, 115.2, 106.7, 77.4,
J = 8.4 Hz, 1H), 5.71 (d, J = 10.0 Hz, 1H), 2.57 (s, 3H), 1.48 (s, 6H);
+
13
8.0; HRMS (ESI-QTOF) m/z [M+Na] calcd for C20
H15ClNaO
3
C NMR (100 MHz, CDCl ) δ 188.0, 153.9, 152.5, 152.4, 130.6, 122.7,
3
61.0602, found 361.0655.
120.7, 115.7, 114.9, 114.3, 106.7, 77.3, 28.0, 26.4; HRMS (ESI-QTOF)
m/z [M+Na]+ calcd for C15
H
14NaO 265.0835, found 265.0796.
3
Ethyl 7,7-dimethyl-7H-furo[2,3-f]chromene-2-carboxylate (2g).
(
7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)(4-nitrophenyl)metha-
1
1
White solid, mp: 54.6–56.8 °C; H NMR (400 MHz, CDCl ) δ 7.43 (s,
none (2d). Yellow solid, mp: 140.2–141.0 °C; H NMR (400 MHz,
3
1
H), 7.36 (d, J = 8.8 Hz, 1H), 6.88 (d, J = 10.0 Hz, 1H), 6.80 (d,
CDCl
3
) δ 8.39 (d, J = 8.8 Hz, 2H), 8.17 (d, J = 8.4 Hz, 2H), 7.50 (s,
J = 8.4 Hz, 1H), 5.70 (d, J = 9.6 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H),
1
5
1
1
H), 7.45 (d, J = 8.4 Hz, 1H), 6.87 (s, 1H), 6.85 (d, J = 4.4 Hz, 1H),
1
3
1
3
1.47 (s, 6H), 1.41 (t, J = 7.0 Hz, 3H); C NMR (100 MHz, CDCl ) δ
.74 (d, J = 10.0 Hz, 1H), 1.50 (s, 6H); C NMR (100 MHz, CDCl
3
) δ
3
1
6
59.6, 153.0. 152.1, 145.0, 130.3, 122.0, 120.5, 115.7, 114.4, 106.6,
81.8, 154.7, 153.1, 151.5, 150.1, 142.8, 130.8, 130.4, 123.8, 123.1,
+
1.2, 27.8, 14.3; HRMS (ESI-QTOF) m/z [M+Na]
calcd for
20.6, 118.8, 115.6, 115.4, 106.6, 77.6, 28.1; HRMS (ESI-QTOF) m/z
+
C
H
16 16
NaO 295.0941, found 295.0968.
[
M+H] calcd for C20
H
16NO
5
350.1023, found 350.0984.
4
General procedure for the synthesis of 3: To a vial charged with
2
f (30 mg, 0.12 mmol) and benzaldehyde (15.2 μl, 1.2 equiv) in ethanol
a,b
a
Scheme 6. Synthesis of 7
.
A mixture of 2-hy-
(1.97 mmol),
CO
droxy-4-methoxybenzaldehyde
chloroacetone (1.2 equiv), and K
2
3
(3.0 equiv)
in acetonitrile (6.6 mL) was stirred at 130 °C. To a
mixture of 6 (0.16 mmol) and ArCHO (1.2 equiv)
in EtOH (1 mL) was added K
2
CO
3
(1.0 equiv) at rt.
b
The reaction mixture was stirred at 60 °C.
Isolated yield (%).
7

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Table 1
Table 1 (continued)
Compound Structure
ANO1 inhibition by a 2,2-Dimethyl-2H-chromene derivativesa.
IC50 (μM) of ANO1
NA
Compound
Structure
IC50 (μM) of ANO1
NAb
3
e
2
a
2
b
c
NA
NA
3
f
NA
2
3
3
g
NA
2
2
d
e
NA
NA
h
4.33
2
2
3
f
NA
NA
NA
3
3
i
j
NA
NA
g
a
3
3
b
c
NA
NA
3
3
k
l
NA
NA
3
d
NA
(
continued on next page)
8

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Table 1 (continued)
Table 1 (continued)
Compound
Structure
IC50 (μM) of ANO1
NA
Compound
Structure
IC50 (μM) of ANO1
NA
3
m
4f
3
3
3
n
o
p
1.23
1.73
10.2
4g
NA
NA
4
h
4
4
i
j
NA
NA
4
4
a
b
NA
NA
4
k
NA
4
4
c
NA
4
4
l
NA
NA
d
5.26
m
4
e
NA
4
n
NA
(
continued on next page)
9

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Table 1 (continued)
NMR (100 MHz, CDCl
3
) δ 179.1, 154.0, 153.6, 152.6, 144.1, 134.9,
1
1
30.8, 130.5, 129.1, 128.7, 122.8, 121.5, 121.0, 115.8, 115.0, 114.5,
Compound
Structure
IC50 (μM) of ANO1
NA
06.7, 77.3, 28.0; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for
C
22
H
18NaO 353.1148, found 353.1122.
3
4
o
1
NA
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2-methox-
1
5
5
7
a
b
a
NA
NA
NA
yphenyl)prop-2-en-1-one (3b). Yellow solid, mp: 143.3–144.2 °C; H
NMR (400 MHz, CDCl
3
) δ 8.24 (d, J = 16.0 Hz, 1H), 7.68 (dd, J = 1.2,
7
7
1
.6 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.55 (s, 1H), 7.43–7.37 (m, 2H),
.00 (t, J = 7.6 Hz, 1H), 6.94 (t, J = 10.0 Hz, 2H), 6.82 (d, J = 8.4 Hz,
13
H), 5.73 (d, J = 10.0 Hz, 1H), 3.94 (s, 3H), 1.49 (s, 6H); C NMR
(
100 MHz, CDCl
3
) δ 179.7, 159.1, 153.9, 153.8, 152.5, 139.6, 132.0,
1
1
30.5, 129.4, 123.9, 122.7, 122.2, 121.1, 120.8, 115.9, 114.8, 114.2,
+
11.4, 106.7, 77.2, 55.7, 28.0; HRMS (ESI-QTOF) m/z [M+Na] calcd
for C23
H
20NaO 383.1254, found 383.1251.
4
7
7
b
c
NA
NA
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(4-methox-
1
yphenyl)prop-2-en-1-one (3c). Yellow solid, mp: 138.8–139.3 °C;
H
NMR (400 MHz, CDCl ) δ 7.89 (d, J = 15.6 Hz, 1H), 7.64 (d,
3
J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.44 (s, 1H), 7.41 (d, J = 5.2 Hz, 1H),
6
1
1
1
.94 (t, J = 9.6 Hz, 3H), 6.83 (d, J = 8.8 Hz, 1H), 5.73 (d, J = 9.6 Hz,
13
H), 3.87 (s, 3H), 1.49 (s, 6H); C NMR (100 MHz, CDCl ) δ 178.1,
3
60.9, 152.8, 151.5, 142.9, 129.5, 129.5, 126.6, 121.6, 120.0, 118.1,
14.8, 113.8, 113.5, 113.0, 105.7, 102.8, 76.2, 54.5, 27.0; HRMS (ESI-
QTOF) m/z [M+Na]+ calcd for
C
H
20NaO
383.1254, found
23
4
a
IC50 values were determined using YFP fluorescence quenching assay in
3
83.1213.
FRT cells expressing ANO1 (mean ± S.E., n = 3).
b
NA when the inhibition rate at 100 µM is < 20%.
(
1 mL) was added K
After being stirred at 60 ℃ for 3 h, the reaction mixture was con-
centrated under reduced pressure, extracted with CH Cl (2 mL), and
washed with H O (5 mL). The water layer was extracted with CH Cl
1 mL) two more times. The organic layer was dried over MgSO , fil-
tered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (n-hexane:ethyl acetate:di-
chloromethane = 50:1:2) to give 3a (38.1 mg, 96%).
2
CO
3
(17.1 mg, 1.0 equiv) at room temperature.
2
2
2
2
2
(
4
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(p-tolyl)prop-
1
2
-en-1-one (3d). Yellow solid, mp: 136.4–137.8 °C;
H NMR
(
(
400 MHz, CDCl
3
) δ 7.90 (d, J = 15.6 Hz, 1H), 7.59–7.57 (m, 3H), 7.49
d, J = 16.0 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.0 Hz,
1
H), 6.93 (d, J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d,
1
3
J = 9.6 Hz, 1H), 2.40 (s, 3H), 1.49 (s, 6H); C NMR (100 MHz, CDCl )
3
δ 179.2, 153.9, 153.8, 152.5, 144.2, 141.4, 132.2, 130.5, 129.9, 129.4,
1
2
3
28.8, 127.3, 122.7, 121.0, 120.4, 115.8, 114.9, 114.3, 106.7, 77.3,
+
8.0, 21.7; HRMS (ESI-QTOF) m/z [M+Na] calcd for C23
H
20NaO
3
67.1305, found 367.1350.
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-phenylprop-2-
1
en-1-one (3a). Yellow solid, mp: 98.1–99.6 °C; H NMR (400 MHz,
CDCl
3
) δ 7.92 (d, J = 15.6 Hz, 1H), 7.69–7.67 (m, 2H), 7.58 (s, 1H),
7
6
.53 (d, J = 15.6 Hz, 1H), 7.44–7.42 (m, 4H), 6.93 (d, J = 9.6 Hz, 1H),
.83 (d, J = 8.4 Hz, 1H), 5.73 (d, J = 10.0 Hz, 1H), 1.50 (s, 6H); 13
C
10

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Fig. 2. Identification and characterization of 3n, a novel ANO1 inhibitor. (A-B) Effect of 3n on ANO1 and ANO2 activity in FRT-YFP cells expressing ANO1 and
ANO2. The cells were pretreated with the indicated concentrations of 3n for 20 min and then ANO1 and ANO2 was activated by 100 μM ATP. (C) Summary of dose
responses (mean ± S.E., n = 3). (D) Effect of 3n on CFTR activity in FRT-YFP cells expressing human CFTR. CFTR was activated by 10 μM forskolin (FSK) and
blocked by 10 μM CFTRinh-172. (E) Intracellular calcium concentration was measured using Flou-4 in PC-3 cells. The cells were pretreated with 1, 3 and 10 μM of 3n
for 20 min, then 100 μM ATP was applied.
(
E)-3-(Benzo[d][1,3]dioxol-5-yl)-1-(7,7-dimethyl-7H-furo[2,3-f]
1H), 6.82 (d, J = 8.4 Hz, 1H), 6.04 (s, 2H), 5.73 (d, J = 10.0 Hz, 1H),
13
chromen-2-yl)prop-2-en-1-one (3e). Yellow solid, mp: 71.6–72.8 °C;
1.49 (s, 6H); C NMR (100 MHz, CDCl ) δ 179.1, 153.9, 152.5, 150.2,
3
1
H NMR (400 MHz, CDCl
3
) δ 7.84 (d, J = 15.6 Hz, 1H), 7.55 (s, 1H),
148.6, 143.9, 130.5, 129.4, 125.7, 122.7, 121.1, 119.5, 115.8, 114.9,
7
.42 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 15.6 Hz, 1H), 7.21 (s, 1H), 7.16
114.1, 108.9, 106.9, 106.7, 101.8, 77.3, 29.9, 28.0; HRMS (ESI-QTOF)
(
d, J = 8.0 Hz, 1H), 6.92 (d, J = 10.0 Hz, 1H), 6.86 (d, J = 8.0 Hz,
m/z [M+H]+ calcd for C23
H
19
O 375.1227, found 375.1264.
5
Fig. 3. Effect of 3n on protein expression
levels of ANO1 and cell viability in PC-3 and
FaDu cells. (A-B) Western blot analysis of
ANO1 in PC-3 and FaDu cells expressing
ANO1. Cells were cultured with indicated
concentration of 3n for 24 h. ANO1-
knockout (KO) PC-3 or FaDu cells were used
as control. (C-D) PC-3 and FaDu cells ex-
pressing ANO1 or ANO1- knockout were
treated with 3n at the indicated concentra-
tion for 48 h. Cell viability was measured by
MTS analysis (mean
±
S.E., n = 3).
*
P < 0.05 **P < 0.01, ***P < 0.001.
11

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Fig. 4. Effect of 3n on caspase 3 activity and cleavage of PARP in PC-3 and FaDu cells expressing ANO1. (A-B) Cells were cultured with indicated concentration of 3n
for 12 h, and then 1 μM caspase 3 substrate were treated for 30 min. Caspase 3 was inhibited by 10 μM Ac-DEVD-CHO (mean ± S.E., n = 3). (C-D) Cells were
incubated with 10 μM 3n for 12 h, and then 1 μM caspase 3 substrate and 1 μM Hoechst 33,342 were treated for 30 min. Apoptotic cells were stained with green
fluorescence, and nucleus were stained by Hoechst 33,342 (blue). (E-F) Cells were cultured with indicated concentration of 3n for 12 h, and PARP, cleaved PARP, and
β-actin were detected using western blot assay. *P < 0.05 **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 5. Molecular docking and binding prediction of 3n to ANO1. (A) The binding mode between 3n and the active site of ANO1 shown as a stick model. (B) The
chemical binding residues of 3n-ANO1 were shown in 2D diagram.
12

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
J = 10.0 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 5.74 (d, J = 10.0 Hz, 1H),
1
3
1
1
1
2
4
.51 (s, 6H); C NMR (100 MHz, CDCl ) δ 179.0, 154.0, 153.7, 152.6,
3
44.1, 134.6, 133.5, 132.4, 131.0, 130.5, 128.8, 128.8, 127.9, 127.6,
26.9, 123.8, 122.8, 121.5, 121.0, 115.8, 115.0, 114.5, 106.7, 77.3,
8.0; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for
C
20NaO
26
H
3
03.1305, found 403.1313.
(
E)-3-(2,4-Dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]
chromen-2-yl)prop-2-en-1-one
(3f).
Yellow
solid,
mp:
1
1
1
1
21.4–123.1 °C; H NMR (400 MHz, CDCl
3
) δ 8.17 (d, J = 16.0 Hz,
H), 7.61 (d, J = 8.4 Hz, 1H), 7.55–7.51 (m, 2H), 7.41 (d, J = 8.8 Hz,
H), 6.92 (d, J = 9.6 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.54 (dd,
J = 2.0, 10.0 Hz, 1H), 6.48–6.48 (m, 1H), 5.72 (d, J = 10.0 Hz, 1H),
1
3
3
1
1
5
.92 (s, 3H), 3.86 (s, 3H), 1.49 (s, 6H); C NMR (100 MHz, CDCl
3
) δ
(E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(4-fluor-
ophenyl)prop-2-en-1-one (3j). Yellow solid, mp: 139.4–139.7 °C;
1
79.8, 163.3, 160.7, 154.1, 153.6, 152.4, 139.7, 131.2, 130.4, 122.6,
H
21.1, 119.8, 117.2, 115.9, 114.7, 113.7, 106.7, 105.6, 98.5, 77.4,
NMR (400 MHz, CDCl
3
) δ 7.88 (d, J = 15.6 Hz, 1H), 7.67 (t,
+
5.7, 55.6, 28.0; HRMS (ESI-QTOF) m/z [M+Na]
calcd for
J = 6.8 Hz, 2H), 7.58 (s, 1H), 7.47–7.42 (m, 2H), 7.12 (t, J = 8.4 Hz,
C
24
H
22NaO 413.1359, found 413.1295.
5
2H), 6.92 (d, J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d,
1
3
J = 10.0 Hz, 1H), 1.48 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 178.9,
1
1
65.5, 163.0, 154.0, 153.6, 152.6, 142.8, 131.1, 130.7, 130.6, 122.8,
21.1, 121.0, 116.4, 116.2, 115.7, 115.1, 114.5, 106.7, 77.3, 28.0;
+
HRMS (ESI-QTOF) m/z [M+H] calcd for C22
H
18FO 349.1234, found
3
3
49.1188.
(
E)-3-(3,5-Dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]
chromen-2-yl)prop-2-en-1-one (3g). Yellow solid, mp: 74.5–75.5 °C;
1
H NMR (400 MHz, CDCl ) δ 7.82 (d, J = 16.0 Hz, 1H), 7.57 (s, 1H),
3
7
.48–7.40 (m, 2H), 6.91 (d, J = 10.0 Hz, 1H), 6.83–6.81 (m, 3H), 6.53
13
(
(
s, 1H), 5.72 (d, J = 10.0 Hz, 1H), 3.84 (s, 6H), 1.49 (s, 6H); C NMR
100 MHz, CDCl
3
) δ 178.9, 161.2, 154.0, 153.5, 152.6, 144.1, 136.7,
(E)-3-(4-Chlorophenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-
1
1
7
30.5, 122.8, 121.9, 121.0, 115.7, 115.0, 114.6, 106.6, 106.6, 102.9,
yl)prop-2-en-1-one (3k). Yellow solid, mp: 122.6–123.5 °C; H NMR
+
7.3, 55.6, 28.0; HRMS (ESI-QTOF) m/z [M+Na]
calcd for
(400 MHz, CDCl ) δ 7.85 (d, J = 16.0 Hz, 1H), 7.60 (d, J = 8.0 Hz,
3
C
24
H
22NaO
5
413.1359, found 413.1319.
2H), 7.58 (s, 1H), 7.49 (d, J = 16.0 Hz, 1H), 7.41 (t, J = 9.0 Hz, 3H),
.91 (d, J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d,
6
1
3
J = 10.0 Hz, 1H), 1.49 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 178.8,
1
1
54.1, 153.5, 152.6, 142.6, 136.7, 133.4, 130.6, 129.8, 129.4, 122.8,
21.9, 120.9, 115.7, 115.1, 114.7, 106.7, 77.3, 28.0; HRMS (ESI-QTOF)
+
m/z [M+H] calcd for C22
H
18ClO 365.0939, found 365.0889.
3
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(4-(dimethyla-
mino)-phenyl)prop-2-en-1-one
(3h).
Orange
solid,
mp:
1
1
1
7
72.7–173.8 °C; H NMR (400 MHz, CDCl
3
) δ 7.90 (d, J = 15.2 Hz,
H), 7.58 (d, J = 8.8 Hz, 2H), 7.52 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H),
.34 (d, J = 15.2 Hz, 1H), 6.94 (d, J = 9.6 Hz, 1H), 6.81 (d,
(
E)-3-(4-Bromophenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-
J = 8.8 Hz, 1H), 6.69 (d, J = 8.8 Hz, 2H), 5.72 (d, J = 10.0 Hz, 1H),
1
yl)prop-2-en-1-one (3l). Yellow solid, mp: 145.8–146.2 °C; H NMR
1
3
3
1
1
.05 (s, 6H), 1.49 (s, 6H); C NMR (100 MHz, CDCl ) δ 179.3, 154.3,
3
(
(
400 MHz, CDCl ) δ 7.84 (d, J = 15.6 Hz, 1H), 7.58–7.49 (m, 6H), 7.43
3
53.5, 152.3, 152.2, 145.0, 130.7, 130.4, 122.6, 122.5, 121.2, 116.2,
d, J = 8.8 Hz, 1H), 6.91 (d, J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz,
16.0, 114.6, 113.2, 111.9, 106.7, 77.4, 40.3, 28.0; HRMS (ESI-QTOF)
13
1
H), 5.73 (d, J = 10.0 Hz, 1H), 1.49 (s, 6H); C NMR (100 MHz,
+
m/z [M+Na] calcd for C24
H
23NNaO 396.1570, found 396.1516.
3
CDCl
3
) δ 178.8, 154.1, 153.5, 152.6, 142.6, 133.8, 132.4, 131.5, 130.6,
1
2
4
30.0, 125.1, 122.8, 122.1, 121.00, 115.7, 115.1, 114.7, 106.7, 77.4,
+
8.0; HRMS (ESI-QTOF) m/z [M+Na] calcd for C22
H
17BrNaO
3
31.0253, found 431.0267.
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(naphthalen-
1
2
-yl)prop-2-en-1-one (3i). Yellow solid, mp: 142.0–143.2 °C; H NMR
) δ 8.10–8.06 (m, 2H), 7.90–7.83 (m, 4H), 7.64–7.61
(
(
400 MHz, CDCl
3
m, 2H), 7.55–7.50 (m, 2H), 7.43 (d, J = 8.4 Hz, 1H) 6.95 (d,
13

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
Methyl (E)-4-(3-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-ox-
give 4a (52.1 mg, 91%).
1
oprop-1-en-1-yl)benzoate (3m). Yellow solid, mp: 170.2–172.9 °C; H
NMR (400 MHz, CDCl ) δ 8.10 (d, J = 8.4 Hz, 2H), 7.92 (d,
3
J = 15.6 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.61–7.57 (m, 2H), 7.44 (d,
J = 8.4 Hz, 1H), 6.92 (d, J = 10.0 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H),
1
3
5
.74 (d, J = 10.0 Hz, 1H), 3.95 (s, 3H), 1.50 (s, 6H); C NMR
(
100 MHz, CDCl ) δ 178.7, 166.6, 154.2, 153.5, 152.7, 142.5, 139.1,
3
1
1
3
31.7, 130.6, 130.3, 128.5, 123.6, 122.9, 121.0, 115.7, 115.2, 115.0,
+
06.7, 29.9, 28.1; HRMS (ESI-QTOF) m/z [M+H] calcd for C24
H
O
21 5
89.1384, found 389.1371.
6-((4-Methoxyphenyl)(morpholino)methyl)-2,2-dimethyl-2H-
1
chromen-5-ol (4a). Ivory solid, mp: 99.7–101.3 °C; H NMR (400 MHz,
CDCl ) δ 12.13 (s, 1H), 7.29 (d, J = 7.6 Hz, 2H), 6.83 (d, J = 8.4 Hz,
3
2
H), 6.74 (d, J = 9.6 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 6.20 (d,
J = 8.0 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 4.31 (s, 1H), 3.76 (s, 7H),
13
2
.58–2.42 (m, 4H), 1.42 (s, 3H), 1.39 (s, 3H); C NMR (100 MHz,
CDCl ) δ 159.3, 153.2, 152.2, 131.4, 129.9, 129.2, 128.5, 117.2, 117.1,
3
1
14.2, 110.0, 107.6, 75.8, 75.6, 67.0, 55.3, 52.0, 28.1; HRMS (ESI-
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-
+
QTOF) m/z [M+K] calcd for C23
H
27KNO 420.1572, found 420.1510.
4
1
yl)prop-2-en-1-one (3n). Brown solid, mp: 116.2–117.1 °C; H NMR
(
400 MHz, CDCl
3
) δ 9.49 (s, 1H), 7.88 (d, J = 15.6 Hz, 1H), 7.49 (s,
H), 7.30 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 15.6 Hz, 1H), 7.03 (s, 1H),
.81–6.76 (m, 3H), 6.34 (s, 1H), 5.66 (d, J = 9.6 Hz, 1H), 1.46 (s, 6H);
1
6
1
3
C NMR (100 MHz, CDCl ) δ 179.3, 153.9, 153.7, 152.4, 134.0, 130.5,
3
1
1
29.5, 123.8, 122.6, 121.1, 116.0, 115.7, 115.1, 114.8, 113.8, 111.7,
+
06.6, 77.2, 28.0; HRMS (ESI-QTOF) m/z [M+H]
calcd for
C
20
H
18NO 320.1281, found 320.1234.
3
6
-((4-Methoxyphenyl)(piperidin-1-yl)methyl)-2,2-dimethyl-2H-
1
chromen-5-ol (4b). Ivory solid, mp: 101.4–102.9 °C;
H
NMR
) δ 13.10 (s, 1H), 7.27 (s, 1H), 6.84 (d, J = 8.8 Hz, 2H),
.78 (d, J = 10.0 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 6.17 (d, J = 8.4 Hz,
H), 5.57 (d, J = 10.0 Hz, 1H), 4.43 (s, 1H), 3.78 (s, 3H), 2.39 (s, 4H),
(
400 MHz, CDCl
3
6
1
1
1
1
3
.63 (s, 6H), 1.43 (s, 3H), 1.41 (s, 3H); C NMR (100 MHz, CDCl
3
) δ
59.1, 153.6, 152.9, 131.1, 130.3, 129.0, 128.2, 117.8, 117.4, 113.9,
(
E)-1-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(furan-2-yl)
1
109.8, 106.8, 75.7, 75.1, 55.2, 28.0, 27.9, 26.2, 24.2; HRMS (ESI-QTOF)
prop-2-en-1-one (3o). Dark yellow solid, mp: 138.4–138.9 °C; H NMR
m/z [M+H]+ calcd for C24
H
30NO 380.2220, found 380.2132.
3
(
400 MHz, CDCl
3
) δ 7.65 (d, J = 15.6 Hz, 1H), 7.55–7.54 (m, 2H), 7.42
(
s, 1H), 7.39 (d, J = 7.2 Hz, 1H), 6.91 (d, J = 10.0 Hz, 1H), 6.81 (d,
J = 8.4 Hz, 1H), 6.73 (d, J = 3.2 Hz, 1H), 6.52–6.51 (m, 1H), 5.71 (d,
1
3
J = 9.6 Hz, 1H), 1.48 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 178.8,
1
1
53.9, 153.6, 152.5, 151.7, 145.2, 130.5, 130.0, 122.7, 121.0, 119.0,
16.7, 115.8, 114.9, 114.4, 112.9, 106.6, 77.3, 28.0; HRMS (ESI-QTOF)
+
m/z [M+Na] calcd for C20
H
16NaO 343.0941, found 343.0995.
4
6
-((4-Methoxyphenyl)(pyrrolidin-1-yl)methyl)-2,2-dimethyl-2H-
1
chromen-5-ol (4c). Brown solid, mp: 116.8–117.9 °C;
H NMR
(
400 MHz, CDCl ) δ 12.73 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 6.81 (d,
3
J = 8.8 Hz, 2H), 6.74 (d, J = 10.0 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H),
6
.17 (d, J = 8.4 Hz, 1H), 5.55 (d, J = 10.0 Hz, 1H), 4.28 (s, 1H), 3.76 (s,
3
H), 2.63 (s, 2H), 2.47 (s, 2H), 1.82 (s, 4H), 1.42 (s, 3H), 1.38 (s, 3H);
(
E)-3-(5-Bromofuran-2-yl)-1-(7,7-dimethyl-7H-furo[2,3-f]
13
C NMR (100 MHz, CDCl ) δ 159.0, 152.9, 152.9, 134.6, 129.1, 128.4,
3
chromen-2-yl)prop-2-en-1-one
(3p).
Brown
solid,
mp:
1
28.1, 119.3, 117.3, 114.0, 110.0, 107.1, 75.8, 74.5, 55.3, 28.1, 27.9,
1
1
7
6
12.7–113.7 °C; H NMR (400 MHz, CDCl
3
) δ 7.57–7.51 (m, 2H),
3.6; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C23
2
H
28NO 366.2064,
3
.42–7.34 (m, 2H), 6.92 (d, J = 10.0 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H),
found 366.2005.
.66 (s, 1H), 6.45 (d, J = 3.6 Hz, 1H), 5.72 (d, J = 10.0 Hz, 1H), 1.48
1
3
(
s, 6H); C NMR (100 MHz, CDCl
3
) δ 178.4, 154.0, 153.6, 153.5,
1
1
52.6, 130.5, 128.5, 126.1, 122.8, 121.0, 119.4, 118.6, 115.8, 115.0,
+
14.8, 114.7, 106.7, 77.3, 28.0; HRMS (ESI-QTOF) m/z [M+H] calcd
for C20
H
16BrO 399.0226, found 399.0161.
4
General procedure for the synthesis of 4: To a solution of 1
(
30 mg, 0.15 mmol) in anhydrous dichloromethane (1 mL) were added
morpholine (15.2 μl, 1.2 equiv), and 4-methoxyphenylboronic acid
(
26.8 mg, 1.2 equiv) at room temperature. After being stirred at 60 ℃ for
8 h, the reaction mixture was diluted with CH Cl (2 mL) and washed
with H O (5 mL). The aqueous layer was extracted with CH Cl (1 mL)
, filtered, and
6
-((3,4-Dimethoxyphenyl)(morpholino)methyl)-2,2-dimethyl-2H-
1
1
2
2
chromen-5-ol (4d). Ivory solid, mp: 161.2–162.5 °C;
H NMR
2
2
2
(
400 MHz, CDCl ) δ 12.08 (s, 1H), 6.94 (s, 1H), 6.91 (d, J = 9.2 Hz, 1H),
3
two more times. The organic layer was dried over MgSO
4
6
1
3
.77 (d, J = 8.0 Hz, 1H), 6.72 (d, J = 10.0 Hz, 1H), 6.65 (d, J = 8.4 Hz,
H), 6.19 (d, J = 8.4 Hz, 1H), 5.55 (d, J = 10.0 Hz, 1H), 4.26 (s, 1H),
.82 (s, 3H), 3.82 (s, 3H), 3.74 (s, 4H), 2.59 (s, 2H), 2.43 (s, 2H), 1.39 (d,
concentrated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane:ethyl acetate:dichloromethane = 30:1:2) to
14

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
1
3
J = 9.6 Hz, 6H); C NMR (100 MHz, CDCl
3
) δ 153.2, 152.2, 149.2,
(m, 3H), 7.07 (d, J = 7.2 Hz, 1H), 6.76 (d, J = 10.0 Hz, 1H), 6.59 (d,
1
48.8, 132.0, 129.1, 128.5, 121.1, 117.2, 117.1, 111.1, 110.0, 107.7,
J = 8.4 Hz, 1H), 5.56 (d, J = 10.0 Hz, 1H), 4.39 (s, 1H), 3.03 (s, 4H),
13
7
6.1, 75.8, 67.0, 56.0, 55.9, 52.0, 28.0, 27.9; HRMS (ESI-QTOF) m/z [M
2.33 (s, 3H), 1.63 (s, 6H), 1.42 (s, 3H), 1.40 (s, 3H); C NMR (100 MHz,
+
+
H] calcd for C24
H
30NO
5
412.2118, found 412.2105.
CDCl ) δ 153.6, 153.0, 139.2, 138.3, 129.1, 128.6, 128.6, 128.3, 117.7,
3
1
17.4, 109.9, 106.9, 76.1, 75.8, 29.9, 28.6, 28.1, 28.0, 26.2, 24.3, 21.7;
+
HRMS (ESI-QTOF) m/z [M+K] calcd for C24
H
29KNO 402.1830, found
2
4
02.1834.
6
-((3,4-Dimethoxyphenyl)(piperidin-1-yl)methyl)-2,2-dimethyl-2H-
1
chromen-5-ol (4e). Ivory gum; H NMR (400 MHz, CDCl
3
) δ 13.04 (s,
1
H), 6.90 (s, 2H), 6.80–6.73 (m, 2H), 6.60 (d, J = 8.4 Hz, 1H), 6.16 (d,
J = 8.0 Hz, 1H), 5.55 (d, J = 10.0 Hz, 1H), 4.38 (s, 1H), 3.85 (s, 3H),
13
3
.83 (s, 3H), 2.35 (s, 4H), 1.63 (s, 6H), 1.40 (d, J = 6.4 Hz, 6H);
C
2,2-Dimethyl-6-(pyrrolidin-1-yl(m-tolyl)methyl)-2H-chromen-5-ol
1
NMR (100 MHz, CDCl
3
) δ 153.5, 153.0, 149.0, 148.7, 131.8, 129.0,
(4i). Ivory gum; H NMR (400 MHz, CDCl ) δ 12.73 (s, 1H), 7.26–7.22
3
128.3, 121.5, 117.8, 117.4, 111.0, 109.9, 106.9, 75.8, 75.6, 56.0, 55.9,
(m, 2H), 7.26 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.74 (d,
J = 10.0 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 6.17 (d, J = 8.4 Hz, 1H),
5.55 (d, J = 10.0 Hz, 1H), 4.27 (s, 1H), 2.63 (s, 2H), 2.48 (s, 2H), 2.32 (s,
+
52.0, 28.0, 26.2, 24.3; HRMS (ESI-QTOF) m/z [M+Na] calcd for
31NNaO 432.2145, found 432.2135.
C
25
H
4
13
3
H), 1.82 (s, 4H), 1.42 (s, 3H), 1.38 (s, 3H); C NMR (100 MHz, CDCl )
3
δ 153.0, 152.9, 142.3, 138.3, 128.6, 128.6, 128.4, 128.3, 128.2, 125.0,
1
19.1, 117.5, 109.9, 107.1, 75.8, 75.4, 28.1, 27.9, 23.6, 21.7; HRMS
(
ESI-QTOF) m/z [M+H]+ calcd for C23
H28NO
2
350.2115, found
350.2042.
6
-((3,5-Dimethoxyphenyl)(morpholino)methyl)-2,2-dimethyl-2H-
1
chromen-5-ol (4f). White solid, mp: 59.4–62.1 °C; H NMR (400 MHz,
CDCl
3
) δ 11.92 (s, 1H), 6.72 (d, J = 10.0 Hz, 1H). 6.67 (d, J = 8.4 Hz,
H), 6.58 (s, 1H), 6.34 (t, J = 2.2 Hz, 1H), 6.20 (d, J = 8.0 Hz, 1H), 5.55
d, J = 10.0 Hz, 1H), 4.21 (s, 1H), 3.75 (s, 10H), 2.62 (s, 2H), 2.48 (s,
1
(
13
2H), 1.40 (d, J = 9.2 Hz, 6H); C NMR (100 MHz, CDCl ) δ 161.1,
1
9
3
53.3, 152.2, 142.0, 129.2, 128.5, 117.2, 116.8, 110.0, 107.7, 106.6,
9.3, 76.7, 75.9, 67.0, 55.4, 52.2, 28.1, 28.0; HRMS (ESI-QTOF) m/z [M
2,2-Dimethyl-6-(morpholino(phenyl)methyl)-2H-chromen-5-ol (4j).
+
1
+
Na] calcd for C24
H
29NNaO
4
434.1938, found 434.1920.
White solid, mp: 59.2–61.4 °C; H NMR (400 MHz, CDCl
3
) δ 12.06 (s,
1
H), 7.39 (s, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.27–7.23 (m, 1H), 6.75 (d,
J = 10.4 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.21 (d, J = 8.4 Hz, 1H),
5
2
1
1
.58 (d, J = 10.0 Hz, 1H), 4.34 (s, 1H), 3.75 (s, 4H), 2.62 (s, 2H), 2.44 (s,
13
H), 1.43 (s, 3H), 1.40 (s, 3H); C NMR (100 MHz, CDCl ) δ 153.3,
3
52.2, 139.4, 129.3, 128.9, 128.7, 128.6, 128.1, 117.1, 117.0, 110.0,
+
07.7, 76.5, 75.9, 67.0, 52.1, 28.1; HRMS (ESI-QTOF) m/z [M+Na]
calcd for C22
H
25NNaO 374.1727, found 374.1674.
3
2
,2-Dimethyl-6-(morpholino(m-tolyl)methyl)-2H-chromen-5-ol
1
(
4g). Ivory solid, mp: 58.6–59.5 °C; H NMR (400 MHz, CDCl
3
) δ 12.08
(
s, 1H), 7.21–7.19 (m, 3H), 7.07 (d, J = 7.2 Hz, 1H), 6.75 (d.
J = 10.0 Hz, 1H), 6.67 (d, J = 8.4 Hz, 1H), 6.21 (d, J = 8.0 Hz, 1H),
5
.58 (d, J = 10.0 Hz, 1H), 7.30 (s, 1H), 3.76 (s, 4H), 2.65 (s, 2H), 2.44 (s,
13
2
H), 2.33 (s, 3H), 1.43 (s, 3H), 1.40 (s, 3H); C NMR (100 MHz, CDCl )
3
δ 153.2, 152.3, 139.4, 138.5, 129.3, 128.9, 128.5, 125.7, 117.2, 117.0,
1
10.0, 107.7, 76.6, 75.9, 67.0, 52.2, 31.7, 28.1, 28.0, 21.6; HRMS (ESI-
+
QTOF) m/z [M+Na]
calcd for C23
H
27NNaO
3
388.1883, found
2
,2-Dimethyl-6-(phenyl(piperidin-1-yl)methyl)-2H-chromen-5-ol
1
388.1871.
(
4k). Ivory solid, mp: 46.7–48.1 °C; H NMR (400 MHz, CDCl ) δ 12.98
3
(
s, 1H), 7.35 (s, 1H), 7.32–7.24 (m, 4H), 6.76 (d, J = 10.0 Hz, 1H), 6.59
(
d, J = 8.0 Hz, 1H), 6.16 (d, J = 8.4 Hz, 1H), 5.56 (d, J = 10.0 Hz, 1H),
13
4
.43 (s, 1H), 2.43 (s, 4H), 1.63 (s, 6H), 1.42 (s, 3H), 1.40 (s, 3H);
C
NMR (100 MHz, CDCl
3
) δ 153.5, 153.0, 139.3, 129.1, 128.7, 128.3,
1
27.9, 117.6, 117.4, 109.9, 107.0, 76.0, 75.8, 29.9, 28.1, 28.0, 26.2,
2
4.3; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C23
H
28NO 350.2115,
2
found 350.2045.
2
,2-Dimethyl-6-(piperidin-1-yl(m-tolyl)methyl)-2H-chromen-5-ol
(
4h). Ivory gum; 1H NMR (400 MHz, CDCl
3
) δ 13.0 (s, 1H), 7.21–7.14
15

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
2
,2-Dimethyl-6-(naphthalen-2-yl(pyrrolidin-1-yl)methyl)-2H-
1
chromen-5-ol (4o). Ivory solid, mp: 148.1–149.4 °C;
H NMR
(
400 MHz, CDCl ) δ 12.88 (s, 1H), 7.81 (t, J = 7.4 Hz, 2H), 7.77 (d,
3
J = 6.8 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H), 7.48–7.42 (m, 2H), 6.79 (d,
J = 9.6 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.19 (d, J = 8.4 Hz, 1H), 5.58
(
d, J = 10.0 Hz, 1H), 4.50 (s, 1H), 2.70 (s, 2H), 2.52 (s, 2H), 1.84 (s, 4H),
13
1
1
1
2
3
.44 (s, 3H), 1.38 (s, 3H); C NMR (100 MHz, CDCl ) δ 153.1, 152.9,
3
40.0, 133.4, 133.0, 128.8, 128.4, 128.3, 128.1, 127.7, 126.6, 126.2,
26.0, 125.8, 118.8, 117.3, 110.0, 107.3, 75.8, 75.6, 53.3, 29.9, 28.1,
2
,2-Dimethyl-6-(phenyl(pyrrolidin-1-yl)methyl)-2H-chromen-5-ol
+
7.9, 23.6; HRMS (ESI-QTOF) m/z [M+H] calcd for C26
H
28NO
2
1
(
4l). Yellow gum; H NMR (400 MHz, CDCl
3
) δ 12.73 (s, 1H), 7.44 (d,
86.2115, found 386.2037.
J = 7.2 Hz, 2H), 7.30–7.20 (m, 3H), 6.75 (d, J = 10.0 Hz, 1H), 6.68 (d,
Synthesis of 5a: To a solution of 1 (60 mg, 0.15 mmol) in anhy-
J = 8.4 Hz, 1H), 6.18 (d, J = 8.4 Hz, 1H), 5.56 (d, J = 10.0 Hz, 1H),
drous N,N-dimethylformamide (1 mL) were added 4-nitrophenyl bro-
mide (69.8 mg, 1.1 equiv) and potassium tert-butoxide (49.4 mg, 1.5
equiv) at room temperature. After being stirred at 160 ℃ for 20 h, the
reaction mixture was quenched with water (5 mL) and extracted with
ethyl acetate (2 mL). The aqueous layer was extracted with ethyl
acetate (1 mL) five more times. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (n-hexane:ethyl acetate:di-
chloromethane = 50:1:2) to give 5a (81.1 mg, 87%).
4
1
1
2
3
.31 (s, 1H), 2.64 (s, 2H), 2.49 (s, 2H), 1.86–1.79 (m, 4H), 1.43 (s, 3H),
13
.38 (s, 3H); C NMR (100 MHz, CDCl ) δ 153.0, 152.8, 142.4, 128.8,
3
28.4, 128.1, 127.9, 127.7, 119.1, 110.0, 107.2, 75.8, 75.4, 53.2, 28.1,
+
7.9, 23.6; HRMS (ESI-QTOF) m/z [M+K] calcd for C22
H25KNO
2
74.1517, found 374.1675.
2
,2-Dimethyl-6-(morpholino(naphthalen-2-yl)methyl)-2H-chromen-
1
5
-ol (4m). White solid, mp: 146.7–147.4 °C; H NMR (400 MHz, CDCl
3
)
7,7-Dimethyl-2-(4-nitrophenyl)-7H-furo[2,3-f]chromene
(5a).
1
δ 12.20 (s, 1H), 7.84–7.78 (m, 4H), 7.62 (d, J = 6.4 Hz, 1H), 7.51–7.45
Yellow solid, mp: 141.8–142.9 °C; H NMR (400 MHz, CDCl
3
) δ 8.29 (d,
(
m, 2H), 8.61 (dd, J = 4.4, 9.6 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.24
J = 8.8 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.4 Hz, 1H),
(
dd, J = 3.6, 8.0 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 4.52 (s, 1H), 3.77 (s,
7.15 (s, 1H), 6.87 (d, J = 9.6 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 5.75 (d,
13
13
4
H), 2.68 (s, 2H), 2.48 (s, 2H), 1.46 (s, 3H), 1.41 (s, 1H); C NMR
J = 9.6 Hz, 1H), 1.50 (s, 6H); C NMR (100 MHz, CDCl
3
) δ 152.6,
(
100 MHz, CDCl
3
) δ 153.3, 152.2, 137.2, 133.4, 133.1, 129.4, 129.0,
151.9, 151.7, 146.9, 136.6, 130.9, 124.6, 124.5, 122.3, 121.0, 115.8,
+
1
28.6, 128.0, 127.7, 126.4, 126.3, 125.9, 117.1, 116.8, 110.1, 107.8,
113.9, 107.0, 106.5, 105.8, 27.9; HRMS (ESI-QTOF) m/z [M+Na]
+
7
6.7, 75.9, 67.0, 52.3, 28.1, 27.9; HRMS (ESI-QTOF) m/z [M+K] calcd
calcd for C19
H
15NNaO 344.0893, found 344.0893.
4
for C26
H27KNO
3
440.1623, found 440.1529.
Synthesis of 5b: To a solution of 1 (60 mg, 0.30 mmol) in anhy-
drous N,N-dimethylformamide (1.1 mL) were added 2-(chloromethyl)
pyridine hydrochloride (49.6 mg, 1 equiv) and potassium carbonate
(
125.3 mg, 3 equiv) at room temperature. After being stirred at 50 ℃
for 11 h, the reaction mixture was quenched with water (5 mL) and
extracted with ethyl acetate (2 mL). The aqueous layer was extracted
with ethyl acetate (1 mL) five more times. The organic layer was dried
over MgSO filtered, and concentrated in vacuo to give the crude re-
4
sidue. To a vial charged with the crude residue in anhydrous N,N-di-
methylformamide (1 mL) was added potassium tert-butoxide (43.3 mg,
1
.5 equiv) at room temperature. After being stirred at 80 ℃ for 16 h, the
2
,2-Dimethyl-6-(naphthalen-2-yl(piperidin-1-yl)methyl)-2H-
reaction mixture was quenched with water (5 mL) and extracted with
ethyl acetate (2 mL). The aqueous layer was extracted with ethyl
1
chromen-5-ol (4n). White solid, mp: 145.9–146.7 °C;
H NMR
(
400 MHz, CDCl ) δ 13.11 (s, 1H), 7.84–7.76 (m, 4H), 7.59 (s, 1H),
3
acetate (1 mL) five more times. The organic layer was dried over MgSO
4
7
6
6
.50–7.44 (m, 2H), 6.81 (d, J = 10.0 Hz, 1H), 6.66 (d, J = 8.4 Hz, 1H),
filtered, and concentrated in vacuo. The residue was purified by silica
gel column chromatography (n-hexane:ethyl acetate:di-
chloromethane = 10:1:2) to give 5b (44.8 mg, 55%).
.17 (d, J = 8.0 Hz, 1H), 5.59 (d, J = 10.0 Hz, 1H), 4.61 (s, 1H), 2.52 (s,
13
H), 1.65 (s, 4H), 1.44 (s, 3H), 1.41 (s, 3H); C NMR (100 MHz, CDCl )
3
δ 153.5, 153.1, 137.1, 133.4, 133.1, 129.2, 128.6, 128.3, 128.1, 128.0,
1
2
4
27.7, 126.3, 126.2, 117.5, 117.4, 110.0, 107.1, 76.3, 75.8, 29.9, 28.1,
+
8.0, 26.2, 24.2; HRMS (ESI-QTOF) m/z [M+H] calcd for C27
H30NO
2
00.2271, found 400.2183.
2
-(7,7-Dimethyl-7H-furo[2,3-f]chromen-2-yl)pyridine (5b). Ivory
1
solid, mp: 59.7–61.3 °C; H NMR (400 MHz, CDCl
3
) δ 8.65 (d,
J = 4.8 Hz, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H),
.36–7.33 (m, 2H), 7.21–7.18 (m, 1H), 6.91 (d, J = 9.6 Hz, 1H), 6.77
7
1
3
(
d, J = 8.4 Hz, 1H), 5.73 (d, J = 10.0 Hz, 1H), 1.49 (s, 6H); C NMR
16

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
(
100 MHz, CDCl
3
) 154.5, 151.5, 151.4, 150.0, 149.6, 136.8, 130.6,
(E)-1-(6-Methoxybenzofuran-2-yl)-3-(2-(trifluoromethoxy)phenyl)
1
1
22.5, 121.1, 119.4, 116.1, 113.5, 106.6, 105.3, 76.6, 27.9; HRMS
prop-2-en-1-one (7c). Yellow solid, mp: 75.0–76.0 °C; H NMR
+
(
ESI-QTOF) m/z [M+Na] calcd for C18
00.0924.
H
15NNaO
2
300.0995, found
(400 MHz, CDCl ) δ 8.12 (d, J = 16.0 Hz, 1H), 7.82 (d, J = 7.6 Hz,
3
3
1H), 7.55–7.64 (m, 3H), 7.46 (t, J = 7.6 Hz, 1H), 7.31–7.40 (m, 2H),
13
General procedure for the synthesis of 7: To a solution of 2-hy-
7.09 (s, 1H), 6.97 (dd, J = 2.0, 8.8 Hz, 1H), 3.90 (s, 3H); C NMR
droxy-4-methoxybenzaldehyde (300 mg, 1.97 mmol, 1.0 equiv) in
acetonitrile (6.6 mL) were added chloroacetone (190.36 μl, 1.2 equiv)
and potassium carbonate (817.54 mg, 3.0 equiv) at room temperature.
After being stirred at 130 ℃ for 16 h, the reaction mixture was con-
centrated in vacuo and the crude residue was extracted with ethyl
acetate (5 mL × 3) three times. The organic layer was dried over
(100 MHz, CDCl ) δ 198.7, 178.6, 161.3, 157.5, 153.2, 148.0, 136.7,
3
131.5 128.7, 128.1, 127.1, 124.3, 123.6, 121.4, 120.5, 114.5, 114.2,
+
95.6; HRMS (ESI-QTOF) m/z [M+H] calcd for C19
H
14
F
3
O 363.0839,
4
found 363.0922.
4.2. Bioassay
MgSO , filtered, and concentrated in vacuo to give the crude residue.
4
The residue was purified by silica gel column chromatography (n-hex-
ane:ethyl acetate:dichloromethane = 50:1:2) to give 6 (354.4 mg,
4
.2.1. Cell culture
Fisher rat thyroid (FRT) cells stably expressing ANO1, ANO2 and
9
5%). To a vial charged with 6 (30 mg, 0.16 mmol) and pyrrole-2-
CFTR were established as described previously [46]. FRT cells were
cultured in Coon‘s modified F12 medium supplemented with 10% FBS,
carboxaldehyde (18 mg, 1.2 equiv) in ethanol (1 mL) was added K
1.0 equiv) at room temperature. After being stirred at 60 ℃ for 3 h, the
reaction mixture was concentrated under reduced pressure, extracted
with CH Cl (2 mL), and washed with H O (5 mL). The water layer was
extracted with CH Cl (1 mL) two more times. The organic layer was
dried over MgSO
2
CO
3
(
2
mM L-glutamine, 100 units/mL penicillin and 100 μg/mL strepto-
mycin. Prostate cancer cells (PC-3) and hypopharyngeal cancer cells
FaDu) were cultured in RPMI1640 medium containing 10% FBS, 100
units/ml penicillin and 100 μg/ml streptomycin.
2
2
2
(
2
2
4
, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (n-hexane:ethyl acet-
4
.2.2. Construction of ANO1-knock out (KO) cells
ate:dichloromethane = 15:1:2) to give 7a (25.7 mg, 62%).
PLentiCRISPRv2 vector containing ANO1 CRISPR Guide RNA (
CCTGATGCCGAGTGCAAGTA) and Cas9 (Clone ID: X35909) was pur-
chased from Genescript (Piscataway, NJ, USA). The DNA composed of
1
50 ng CRISPR/Cas9 with ANO1 gRNA, 1200 ng packaging plasmid
(
psPAX2) and 400 ng envelope plasmid (pMD2.G) were co-transfected
in HEK293T cells on 6 well plate. After 48 h, supernatant containing
lentivirus particles were collected and filtered by 0.45 μm syringe filter.
The lentiviral particle with medium (1:1 mixture) were treated with
cells on 24 well plate for 72 h. ANO1-knockout cells were selected by
puromycin (Sigma-Aldrich).
(
E)-1-(6-Methoxybenzofuran-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-
1
one (7a). ellow solid, mp: 176.6–177.2 °C; H NMR (400 MHz, CDCl
3
)
4.2.3. YFP fluorescence quenching analysis
δ 9.02 (s, 1H), 7.85 (d, J = 15.6 Hz, 1H), 7.50–7.56 (m, 2H), 7.18 (d,
J = 15.6 Hz, 1H), 7.00–7.07 (m, 2H), 6.94 (dd, J = 2.2, 8.6 Hz, 1H),
FRT cells stably expressing YFP-H148Q/I152L/F46L and ANO1,
3
ANO2 or CFTR were plated in 96-well plates at 2 × 10 cells per well
1
3
6
.74–6.78 (m, 1H), 6.33–6.38 (m, 1H), 3.87 (s, 3H);
C NMR
and incubated for 48 h. The 96-well plates were washed twice with
300 μl PBS and 100 μl PBS was left, and test compounds were treated
for 20 min. The 96-well plates were transferred to a microplate reader
and YFP fluorescence measurements were performed. To measure the
(
100 MHz, CDCl
3
) δ 178.9, 160.8, 157.2, 153.7, 133.6, 129.3, 123.4,
1
23.4, 120.7, 115.8, 114.9, 114.1, 112.9, 111.6, 95.6, 55.7; HRMS
+
(
ESI-QTOF) m/z [M+H] calcd for C16
H14NO
3
268.0968, found
-
-
2
68.1017.
ANO1-, ANO2- or CFTR-mediated I influx, 100 μl of 70 mM I solution
containing 200 μM ATP or 10 μM forskolin was injected at 1 or 2 s,
fluorescence was measured once every 0.4 s and continuous for 5–6 s.
The inhibitory effect of the test compound was measured by calculating
-
the initial slope of YFP fluorescence reduction by I influx. Analysis
were performed using FLUOstar Omega microplate reader and MARS
Data Analysis Software (BMG Labtech).
4
.2.4. Western blot analysis
(
E)-1-(6-Methoxybenzofuran-2-yl)-3-(thiophen-2-yl)prop-2-en-1-
1
Western blotting proceeded as previously reported [46]. PC3 and
one (7b). Pale yellow solid, mp: 127.0–127.7 °C; H NMR (400 MHz,
CDCl ) δ 8.03 (d, J = 15.6 Hz, 1H), 7.55–7.60 (m, 2H), 7.44 (d,
FaDu cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4),
3
1
mM EDTA, 1 mM Na
3
VO , 150 mM NaCl, 1% Nonidet P-40, 0.25%
4
J = 5.2 Hz, 1H), 7.38 (d, J = 3.6 Hz, 1H), 7.30 (d, J = 15.6 Hz, 1H),
1
3
sodium deoxycholate protease inhibitor mixture). Total cell lysates
were centrifuged at 13,000 rpm for 15 min at 4 °C and protein con-
centration were determined with a Bradford assay kit (Thermo Fisher
Scientific) according to the manufacturer‘s instructions. The super-
natant protein was separated via 4–12% Tris-glycine precast gel (KOMA
BIOTECH), and transferred to a PVDF membrane and then blocked with
7
.06–7.12 (m, 2H), 6.95 (dd, J = 2.2, 8.6 Hz, 1H), 3.89 (s, 3H);
C
NMR (100 MHz, CDCl
3
) δ 178.5, 161.1, 157.4, 153.3, 140.3, 136.4,
1
32.3, 129.0, 128.4, 123.5, 120.6, 120.1, 114.4, 113.7, 95.6, 55.7;
+
HRMS (ESI-QTOF) m/z [M+H] calcd for C16
85.0605.
H
13
O S 285.0580, found
3
2
5
0
% Bovine Serum Albumin (BSA) in Tris-buffered saline containing
.1% Tween 20 (TBST) for 1 h. Membranes were then incubated with
anti-ANO1 antibody (Abcam), anti-cleaved PARP (BD Biosciences) and
beta-actin (Santa Cruz Biotechnology). Next, the membranes were in-
cubated with HRP-conjugated anti-secondary IgG antibody (Enzo life
science) and visualized using the ECL Plus western blotting detection
system (GE Healthcare).
17

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
4
.2.5. Cell viability assay
(1990) 877–882.
[
6] Y.Q. Guo, G.H. Tang, L.L. Lou, W. Li, B. Zhang, B. Liu, S. Yin, Prenylated flavonoids
as potent phosphodiesterase-4 inhibitors from Morus alba: Isolation, modification,
and structure-activity relationship study, Eur. J. Med. Chem. 144 (2018) 758–766.
MTS analysis was performed on CellTiter 96® AQueous One Solution
Cell Proliferation Assay kit (Promega). Briefly, PC3 and FaDu cells were
plated in 96-well plates and incubated with 3% FBS containing medium
for 24 h. Once the cells reached ~20% confluency, the cells were
treated with test compound for 48 h. Then, the medium and compound
were exchanged every 24 h. After removing the cell culture medium,
MTS solution was added to the 96-well plate and re-incubated for 1 h.
Absorbance of formazan was measured by Infinite M200 microplate
reader (Tecan) at 490 nm.
[7] M.A. Arai, F. Ochi, Y. Makita, T. Chiba, K. Higashi, A. Suganami, Y. Tamura,
T. Toida, A. Iwama, S.K. Sadhu, F. Ahmed, M. Ishibashi, GLI1 Inhibitors Identified
by Target Protein Oriented Natural Products Isolation (TPO-NAPI) with Hedgehog
Inhibition, ACS Chem. Biol. 13 (2018) 2551–2559.
[8] H. An, S. Lee, J. Lee, D.H. Jo, J. Kim, Y.-S. Jeong, M.J. Heo, C.S. Cho, H. Choi,
J.H. Seo, S. Hwang, J. Lim, T. Kim, H.O. Jun, J. Sim, C. Lim, J. Hur, J. Ahn,
H.S. Kim, S.-Y. Seo, Y. Na, S.-H. Kim, J. Lee, J. Lee, S.-J. Chung, Y.-M. Kim, K.-
W. Kim, S.G. Kim, J.H. Kim, Y.-G. Suh, Novel Hypoxia-Inducible Factor 1α (HIF-1α)
inhibitors for angiogenesis-related ocular diseases: discovery of a novel scaffold via
ring-truncation strategy, J. Med. Chem. 61 (2018) 9266–9286.
[
9] B. Lin, J.-F. Huang, X.-W. Liu, X.-T. Ma, X.-L. Liu, Y. Lu, Y. Zhou, F.M. Guo,
T.T. Feng, Rapid, microwave-accelerated synthesis and anti-osteoporosis activities
evaluation of morusin scaffolds and morusignin L scaffolds, Bioorg. Med. Chem.
Lett. 27 (2017) 2389–2396.
4.2.6. Caspase 3 activity assay
PC-3 and FaDu cells were cultured in 96-well cell culture plate
about 30% confluence, test compound and Ac-DEVD-CHO, a caspase-3
inhibitor, were treated for 24 h. To measure caspase-3 activity, the cell
culture medium was replaced with PBS containing 1 μM NucView 488
caspase-3 substrate, and the cells were incubated at room temperature
for 30 min and then stained with 1 μM Hoechst 33342. The fluorescence
intensity of NucView 488 and Hoechst 33,342 were measured using
FLUOstar Omega microplate reader (BMG Labtech) and fluorescence
microscopy images were obtained with Lionheart FX automated live
cell microscope (BioTek).
[
[
10] H.S. Kim, M. Hong, S.-C. Lee, H.-Y. Lee, Y.-G. Suh, D.-C. Oh, J.H. Seo, H. Choi,
J.Y. Kim, K.-W. Kim, J.H. Kim, J. Kim, Y.-M. Kim, S.-J. Park, H.-J. Park, J. Lee, Ring-
truncated deguelin derivatives as potent Hypoxia Inducible Factor-1α (HIF-1α)
inhibitors, Eur. J. Med. Chem. 104 (2015) 157–164.
11] H.S. Kim, V.-H. Hoang, M. Hong, K.C. Kim, J. Ann, C.-T. Nguyen, J.H. Seo, H. Choi,
J.Y. Kim, K.-W. Kim, W.S. Byun, S. Lee, S. Lee, Y.-G. Suh, J. Chen, H.-J. Park, T.-
M. Cho, J.Y. Kim, J.H. Seo, J. Lee, Investigation of B, C-ring truncated deguelin
derivatives as heat shock protein 90 (HSP90) inhibitors for use as anti-breast cancer
agents, Bioorg. Med. Chem. 27 (2019) 1370–1381.
[
12] S. Oh, H.J. Jang, S.K. Ko, Y. Ko, S.B. Park, Construction of a polyheterocyclic
benzopyran library with diverse core skeletons through diversity-oriented synthesis
pathway, J. Comb. Chem. 12 (2010) 548–558.
4.2.7. Molecular docking analysis
[13] K.C. Nicolaou, J.A. Pfefferkorn, A.J. Roecker, G.Q. Cao, S. Barluenga, H.J. Mitchell,
Natural product-like combinatorial libraries based on privileged structures. 1.
general principles and solid-phase synthesis of benzopyrans, J. Am. Chem. Soc. 122
The structure of ANO1 was obtained from the Protein data bank
(
PDB ID: 5OYB) [72]. 3n were docked using the CHARMM force field in
(
2000) 9939–9953.
Discovery Studio software on the binding site of ANO1 [74]. For the
docking analysis, the binding site was defined within 10.7 Å sphere
centered from the result of 3D-QSAR analysis [73]. Based on the
docking results, in situ minimization and ligand conformational entropy
were determined to calculate binding energy of the most predictive
binding mode.
[14] K.C. Nicolaou, J.A. Pfefferkorn, H.J. Mitchell, A.J. Roecker, S. Barluenga, G.Q. Cao,
R.L. Affleck, J.E. Lillig, Natural product-like combinatorial libraries based on pri-
vileged structures. 2. construction of a 10,000-membered benzopyran library by
directed split-and-pool chemistry using NanoKans and optical encoding, J. Am.
Chem. Soc. 122 (2000) 9954–9967.
[
[
15] K. Passador, S. Thorimbert, C. Botuha, Heteroaromatic rings of the Future: ex-
ploration of unconquered chemical space, Synthesis 51 (2019) 384–398.
16] J.-L. Reymond, M. Awale, Exploring chemical space for drug discovery using the
chemical universe database, ACS Chem. Neurosci. 3 (2012) 649–657.
[
[
17] C.M. Dobson, Chemical space and biology, Nature 432 (2004) 824–828.
18] R.S. Bohacek, C. McMartin, W.C. Guida, The art and practice of structure-based
drug design: a molecular modeling perspective, Med. Res. Rev. 16 (1996) 3–50.
4.3. Statistical analysis
The results of multiple experiments are presented as the
[19] M. Srinivas Lavanya Kumar, J. Singh, S.K. Manna, S. Maji, R. Konwar, G. Panda,
Diversity oriented synthesis of chromene-xanthene hybrids as anti-breast cancer
agents, Bioorg. Med. Chem. Lett. 28 (2018) 778–782.
means ± S.E. Statistical analysis was performed with Student's t-test or
by analysis of variance as appropriate. A value of P < 0.05 was con-
sidered statistically significant. Dose-response curve and IC50 values
were calculated using GraphPad Prism Software.
[
20] N. Luise, P.G. Wyatt, Generation of polar semi-saturated bicyclic pyrazoles for
fragment-based drug-discovery campaigns, Chemistry (Easton) 24 (2018)
10443–10451.
[
21] J. Halder, D. Das, S. Nanda, A distinctive transformation based diversity oriented
synthesis of small ring carbocycles and heterocycles from biocatalytically derived
enantiopure alpha-substituted-beta-hydroxyesters, Org. Biomol. Chem. 16 (2018)
Acknowledgements
2
549–2575.
[
22] M.L.N. Rao, V.N. Murty, S. Nand, Functional group manoeuvring for tuning stability
and reactivity: synthesis of cicerfuran, moracins (D, E, M) and chromene-fused
benzofuran-based natural products, Org. Biomol. Chem. 15 (2017) 9415–9423.
We thank the National Research Foundation of Korea (NRF-
2
2
017R1A2A2A05069364,
NRF-2018R1A6A1A03023718,
NRF-
019R1I1A1A01061117, and NRF-2020R1A2C2005961) for generous
[23] L. Herrera, P. Barrio, I. Ibáñez, R. Román, N. Mateu, S. Fustero, 8-Iodonaphthalene-
1
-carbaldehyde: A versatile building block for diversity-oriented synthesis, Org.
financial support. This research was supported (in part) by Yonsei
University Research Fund (Post Doc. Researcher Supporting Program)
of 2019 (project no.: 2019-12-0013).
Lett. 18 (2016) 4722–4725.
[
[
[
[
[
24] J. Kim, M. Park, J. Choi, D.K. Singh, H.J. Kwon, S.H. Kim, I. Kim, Design, synthesis,
and biological evaluation of novel pyrrolo[1,2-a]pyrazine derivatives, Bioorg. Med.
Chem. Lett. 29 (2019) 1350–1356.
25] A. Dagar, G.H. Bae, J.H. Lee, I. Kim, Tandem [4+1+1] annulation approach to 4-
acyl-3,4-dihydropyrrolo[1,2-a]pyrazines: diastereoselective construction of dihy-
dropyrazine units from Pyrroles, J. Org. Chem. 84 (2019) 6916–6927.
26] S. Park, E.H. Kim, J. Kim, S.H. Kim, I. Kim, Biological evaluation of indolizine-
chalcone hybrids as new anticancer agents, Eur. J. Med. Chem. 144 (2018)
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104000.
4
35–443.
27] M. Nayak, D.K. Singh, I. Kim, Regiospecific synthesis of 5- and 6-acylated naphtho
References
[1,2-b]benzofurans via intramolecular alkyne carbonyl metathesis, Synthesis 49
(
2017) 2063–2073.
28] S.H. Moon, Y. Jung, S.H. Kim, I. Kim, Synthesis, characterization and biological
evaluation of anti-cancer indolizine derivatives via inhibiting beta-catenin activity
and activating p53, Bioorg. Med. Chem. Lett. 26 (2016) 110–113.
[
[
[
[
1] M. Costa, T.A. Dias, A. Brito, F. Proença, Biological importance of structurally di-
versified chromenes, Eur. J. Med. Chem. 123 (2016) 487–507.
2] J. Boyd, A. Han, Deguelin and its role in chronic diseases, Adv. Exp. Med. Biol. 929
[
[
29] Y. Jung, I. Kim, Deformylative intramolecular hydroarylation: Synthesis of Benzo[e]
pyrido[1,2-a]indoles, Org. Lett. 17 (2015) 4600–4603.
(
2016) 363–375.
3] Z.Q. Xu, M.T. Flavin, T.R. Jenta, Calanolides, the naturally occurring anti-HIV
agents, Curr. Opin. Drug. Discov. Devel. 3 (2000) 155–166.
30] M. Kim, Y. Jung, I. Kim, Domino Knoevenagel condensation/intramolecular aldol
cyclization route to diverse indolizines with densely functionalized pyridine units,
J. Org. Chem. 78 (2013) 10395–10404.
4] A.D. Patil, A.J. Freyer, D.S. Eggleston, R.C. Haltiwanger, B. Tomcowicz, A. Breen,
R.K. Johnson, Daleformis, a new phytoalexin from the roots of Dalea filiciformis: An
inhibitor of endothelin converting enzyme, J. Nat. Prod. 60 (1997) 306–308.
5] Y. Hano, Y. Yamagami, M. Kobayashi, R. Isohata, T. Nomura, Artonins E and F, two
new prenylflavones from the bark of Artocarpus communis Forst, Heterocycles 31
[
[
31] I. Kim, K. Kim, Expeditious synthesis of highly substituted indolizinones via a
palladium-catalyzed domino sequence, Org. Lett. 12 (2010) 2500–2503.
32] T.T. Talele, Natural-products-inspired use of the gem-dimethyl group in medicinal
chemistry, J. Med. Chem. 61 (2018) 2166–2210.
[
18

Y. Seo, et al.
Bioorganic Chemistry 101 (2020) 104000
[
[
[
33] K. Kunzelmann, Y. Tian, J.R. Martins, D. Faria, P. Kongsuphol, J. Ousingsawat,
F. Thevenod, E. Roussa, J. Rock, R. Schreiber, Anoctamins, Pflügers Arch. 462
Synthesis and bioactivity evaluation of novel 2-salicyloylbenzofurans as anti-
bacterial agents, Molecules 22 (2017) 687.
(
2011) 195–208.
[56] Y.H. Seo, K. Damodar, J.K. Kim, J.G. Jun, Synthesis and biological evaluation of 2-
aroylbenzofurans, rugchalcones A, B and their derivatives as potent anti-in-
flammatory agents, Bioorg. Med. Chem. Lett. 26 (2016) 1521–1524.
[57] H. Gao, X. Zhang, X.J. Pu, X. Zheng, B. Liu, G.X. Rao, C.P. Wan, Z.W. Mao, 2-
Benzoylbenzofuran derivatives possessing piperazine linker as anticancer agents,
Bioorg. Med. Chem. Lett. 29 (2019) 806–810.
34] F. Huang, J.R. Rock, B.D. Harfe, T. Cheng, X. Huang, Y.N. Jan, L.Y. Jan, Studies on
expression and function of the TMEM16A calcium-activated chloride channel, Proc.
Natl. Acad. Sci. U. S. A. 106 (2009) 21413–21418.
35] M.R. Opp, P.L. Rady, T.K. Hughes Jr., P. Cadet, S.K. Tyring, E.M. Smith, Human
immunodeficiency virus envelope glycoprotein 120 alters sleep and induces cyto-
kine mRNA expression in rats, Am. J. Physiol. 270 (1996) 963–970.
[58] S. Boddupally, P. Jyothi, M.V.B. Rao, K.P. Rao, Design and synthesis of anti-
microbial active (E)-(3-(substituted-styryl)-7H-furo[2,3-f]chromen-2-yl)(phenyl)
methanone derivatives and their in silico molecular docking studies, J. Heterocycl.
Chem. 56 (2019) 73–80.
[
[
36] C.R. Bader, D. Bertrand, R. Schlichter, Calcium-activated chloride current in cul-
tured sensory and parasympathetic quail neurones, J. Physiol. 394 (1987) 125–148.
37] Y. Sui, M. Sun, F. Wu, L. Yang, W. Di, G. Zhang, L. Zhong, Z. Ma, J. Zheng, X. Fang,
T. Ma, Inhibition of TMEM16A expression suppresses growth and invasion in
human colorectal cancer cells, PLoS ONE 9 (2014) e115443.
[59] P. Kushwaha, A.K. Tripathi, S. Gupta, P. Kothari, A. Upadhyay, N. Ahmad,
T. Sharma, M. Siddigi, R. Trivedi, K.V. Sashidhara, Synthesis and study of benzo-
furan-pyran analogs as BMP-2 targeted osteogenic agents, Eur. J. Med. Chem. 156
(2018) 103–117.
[
[
[
[
38] Y. Li, J. Zhang, S. Hong, ANO1 as a marker of oral squamous cell carcinoma and
silencing ANO1 suppresses migration of human SCC-25 cells, Med. Oral Patol. Oral
Cir. Bucal. 19 (2014) e313–319.
[60] C. Zhuang, W. Zhang, C. Sheng, W. Zhang, C. Xing, Z. Miao, Chalcone: A privileged
structure in medicinal chemistry, Chem. Rev. 117 (2017) 7762–7810.
[61] P. Wu, M. Givskov, T.E. Nielsen, Reactivity and synthetic applications of multi-
component Petasis reactions, Chem. Rev. 11245–11290 (2019).
39] A. Britschgi, A. Bill, H. Brinkhaus, C. Rothwell, I. Clay, S. Duss, et al., Calcium-
activated chloride channel ANO1 promotes breast cancer progression by activating
EGFR and CAMK signaling, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) E1026–1034.
2
+
-
40] W. Liu, M. Lu, B. Liu, Y. Huang, K. Wang, Inhibition of Ca -activated Cl channel
ANO1/TMEM16A expression suppresses tumor growth and invasiveness in human
prostate carcinoma, Cancer Lett. 326 (2012) 41–51.
[62] N.R. Candeias, F. Montalbano, P.M. Cal, P.M. Gois, Boronic acids and esters in the
Petasis-borono Mannich multicomponent reaction, Chem. Rev. 110 (2010)
6169–6193.
41] U. Duvvuri, D.J. Shiwarski, D. Xiao, C. Bertrand, X. Huang, R.S. Edinger, J.R. Rock,
H.D. Harfe, B.J. Henson, K. Kunzelmann, R. Schreiber, R.S. Seethala, A.M. Egloff,
X. Chem, V.W. Lui, J.R. Grandis, S.M. Gollin, TMEM16A induces MAPK and con-
tributes directly to tumorigenesis and cancer progression, Cancer Res. 72 (2012)
[63] N.A. Petasis, I. Akritopoulou, The boronic acid mannich reaction: a new method for
the synthesis of geometrically pure allylamines, Tetrahedron Lett. 34 (1993)
583–586.
[64] V. Pandey, B. Wang, C.D. Mohan, A.R. Raquib, S. Rangappa, V. Srinivasa,
J.E. Fuchs, K.S. Girish, T. Zhu, A. Bender, L. Ma, Z. Yin, Basappa, K.S. Rangappa,
P.E. Lobie, Discovery of a small-molecule inhibitor of specific serine residue BAD
phosphorylation, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) E10505–10514.
[65] P. Doan, A. Karjalainen, J.G. Chandraseelan, O. Sandberg, O. Yli-Harja, T. Rosholm,
R. Franzen, N.R. Candeias, M. Kandhavelu, Synthesis and biological screening for
cytotoxic activity of N-substituted indolines and morpholines, Eur. J. Med. Chem.
120 (2016) 296–303.
3
270–3281.
[
[
[
42] A. Picollo, M. Malvezzi, A. Accardi, TMEM16 proteins: unknown structure and
confusing functions, J. Mol. Biol. 427 (2015) 94–105.
43] Y. Zhang, Z. Zhang, S. Xiao, J. Tien, S. Le, T. Le, L.Y. Jan, H. Yang, Inferior olivary
TMEM16B mediates cerebellar motor learning, Neuron 95 (2017) 1103–1111.
44] F. Neureither, K. Ziegler, C. Pitzer, S. Frings, F. Mohrlen, Impaired motor co-
ordination and learning in mice lacking anoctamin 2 calcium-gated chloride
channels, Cerebellum 16 (2017) 929–937.
[66] Y. Xu, N.Y. Wang, X.J. Song, Q. Lei, T.H. Ye, X.Y. You, W.Q. Zuo, Y. Xia, L.D. Zhang,
L.T. Yu, Discovery of novel N-(5-(tert-butyl)isoxazol-3-yl)-N'-phenylurea analogs as
potent FLT3 inhibitors and evaluation of their activity against acute myeloid leu-
kemia in vitro and in vivo, Bioorg. Med. Chem. 23 (2015) 4333–4343.
[67] X.D. Yang, W.C. Wan, X.Y. Deng, Y. Li, L.J. Yang, L. Li, H.B. Zhang, Design,
synthesis and cytotoxic activities of novel hybrid compounds between 2-phe-
nylbenzofuran and imidazole, Bioorg. Med. Chem. Lett. 22 (2012) 2726–2729.
[68] F. Baharloo, M.H. Moslemin, H. Nadri, A. Asadipour, M. Mahdavi, S. Emami,
L. Firoozpour, R. Mohebat, A. Shafiee, A. Foroumadi, Benzofuran-derived benzyl-
pyridinium bromides as potent acetylcholinesterase inhibitors, Eur. J. Med. Chem.
93 (2015) 196–201.
[
[
[
45] Y. Seo, K. Ryu, J. Park, D.K. Jeon, S. Jo, H.K. Lee, W. Namkung, Inhibition of ANO1
by luteolin and its cytotoxicity in human prostate cancer PC-3 cells, PLoS ONE 12
(
2017) e0174935.
46] Y. Seo, H.K. Lee, J. Park, D.K. Jeon, S. Jo, M. Jo, W. Namkung, Ani9, A novel potent
small-molecule ANO1 inhibitor with negligible effect on ANO2, PLoS ONE 11
(
2016) e0155771.
47] A. Bill, A. Gutierrez, S. Kulkarni, C. Kemp, D. Bonenfant, H. Voshol, U. Duvvuri,
L.A. Gaither, ANO1/TMEM16A interacts with EGFR and correlates with sensitivity
to EGFR-targeting therapy in head and neck cancer, Oncotarget 6 (2015)
9
173–9188.
[
[
48] H. Wang, L. Zou, K. Ma, J. Yu, H. Wu, M. Wei, Q. Xiao, Cell-specific mechanisms of
[69] S.B. Pattle, N. Utjesanovic, A. Togo, L. Wells, B. Conn, H. Monaghan, E. Junor,
I. Johannessen, K. Cuschieri, S. Talbot, Copy number gain of 11q13.3 genes as-
sociates with pathological stage in hypopharyngeal squamous cell carcinoma, Genes
Chromosomes Cancer 56 (2017) 185–198.
2
+
TMEM16A Ca -activated chloride channel in cancer, Mol. Cancer 16 (2017) 152.
49] Y. Seo, J. Kim, J. Chang, S.S. Kim, W. Namkung, I. Kim, Synthesis and biological
evaluation of novel Ani9 derivatives as potent and selective ANO1 inhibitors, Eur. J.
Med. Chem. 160 (2018) 245–255.
[70] C. Ruiz, J.R. Martins, F. Rudin, S. Schneider, T. Dietsche, C.A. Fischer, L. Tornillo,
L.M. Terracciano, R. Schreiber, L. Bubendorf, K. Kunzelmann, Enhanced expression
of ANO1 in head and neck squamous cell carcinoma causes cell migration and
correlates with poor prognosis, PLoS ONE 7 (2012) e43265.
[
[
[
[
50] Y. Seo, J. Park, M. Kim, H.K. Lee, J.H. Kim, J.H. Jeong, W. Namkung, Inhibition of
ANO1/TMEM16A chloride channel by idebenone and its cytotoxicity to cancer cell
lines, PLoS ONE 10 (2015) e0133656.
51] W. Namkung, P.W. Phuan, A.S. Verkman, TMEM16A inhibitors reveal TMEM16A as
a minor component of calcium-activated chloride channel conductance in airway
and intestinal epithelial cells, J. Biol. Chem. 286 (2011) 2365–2374.
[71] Y. Song, J. Gao, L. Guan, X. Chen, J. Gao, K. Wang, Inhibition of ANO1/TMEM16A
induces apoptosis in human prostate carcinoma cells by activating TNF-α signaling,
Cell Death Dis. 9 (2018) 703.
2
+
52] W. Namkung, J.R. Thiagarajah, P.W. Phuan, A.S. Verkman, Inhibition of Ca -ac-
tivated Cl- channels by gallotannins as a possible molecular basis for health benefits
of red wine and green tea, FASEB J. 24 (2010) 4178–4186.
[72] S. Dang, S. Feng, J. Tien, C.J. Peters, D. Bulkley, M. Lolicato, J. Zhao,
K. Zuberbühler, W. Ye, L. Qi, T. Chen, C.S. Craik, Y.N. Jan, D.L. Minor Jr, Y. Cheng,
L.Y. Jan, Cryo-EM structures of the TMEM16A calcium-activated chloride channel,
Nature 552 (2017) 426–429.
53] M. Nayak, I. Kim, Alkyne carbonyl metathesis as a means to make 4-acyl chro-
menes: syntheses of ( ± )-deguelin and ( ± )-munduserone, J. Org. Chem. 80 (2015)
[73] Y.H. Lee, Yi, GS, Prediction of Novel Anoctamin1 (ANO1) Inhibitors Using 3D-
QSAR Pharmacophore Modeling and Molecular Docking, Int. J. Mol. Sci. 19 (2018)
3204.
1
1460–11467.
[
[
54] G.E. Henry, H. Jacobs, A short synthesis of 5-methoxy-2,2-dimethyl-2H-1-benzo-
pyran-6-propanoic acid methyl ester, Tetrahedron 57 (2001) 5335–5338.
[74] L.G. Ferreira, R.N. Dos Santos, G. Oliva, A.D. Andricopulo, Molecular docking and
structure-based drug design strategies, Molecules. 20 (7) (2015) 13384–13421.
55] P.T. Phan, T.T. Nguyen, H.T. Nguyen, B.N. Le, T.T. Vu, D.C. Tran, T.N. Pham,
19