Design, synthesis, and biological evaluation of novel 6h-benzo[c]chromen-6-one derivatives as potential phosphodiesterase ii inhibitors

Article abstract of DOI:10.3390/ijms22115680

Urolithins (hydroxylated 6H-benzo[c]chromen-6-ones) are the main bioavailable metabolites of ellagic acid (EA), which was shown to be a cognitive enhancer in the treatment of neurodegenerative diseases. As part of this research, a series of alkoxylated 6H-benzo[c]chromen-6-one derivatives were designed and synthesized. Furthermore, their biological activities were evaluated as potential PDE2 inhibitors, and the alkoxylated 6H-benzo[c]chromen-6-one derivative 1f was found to have the optimal inhibitory potential (IC₅₀: 3.67 ± 0.47 μM). It also exhibited comparable activity in comparison to that of BAY 60-7550 in vitro cell level studies.

Full text of DOI:10.3390/ijms22115680

International Journal of
Molecular Sciences
Article
Design, Synthesis, and Biological Evaluation of Novel
6H-Benzo[c]chromen-6-one Derivatives as Potential
Phosphodiesterase II Inhibitors
Long Tang 1,2,3 , Jianchun Jiang 1,2,*, Guoqiang Song 3, Yajing Wang 3, Ziheng Zhuang 3, Ying Tan 3, Yan Xia 3,
Xianfeng Huang 3 and Xiaoqing Feng 3,
*
1
Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry, Nanjing 210042, China;
00009205@cczu.edu.cn
2
3
School of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou 213164, China;
sgq@cczu.edu.cn (G.S.); cathywang@cczu.edu.cn (Y.W.); zzh2010@cczu.edu.cn (Z.Z.);
15435108@smail.cczu.edu.cn (Y.T.); xy19105500550xy@163.com (Y.X.); huangxf@cczu.edu.cn (X.H.)
Correspondence: jiangjc@icifp.cn (J.J.); fxqfw@cczu.edu.cn (X.F.)
*
Abstract: Urolithins (hydroxylated 6H-benzo[c]chromen-6-ones) are the main bioavailable metabo-
lites of ellagic acid (EA), which was shown to be a cognitive enhancer in the treatment of neurode-
generative diseases. As part of this research, a series of alkoxylated 6H-benzo[c]chromen-6-one
derivatives were designed and synthesized. Furthermore, their biological activities were evaluated
as potential PDE2 inhibitors, and the alkoxylated 6H-benzo[c]chromen-6-one derivative 1f was found
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
to have the optimal inhibitory potential (IC50: 3.67
in comparison to that of BAY 60-7550 in vitro cell level studies.
±
0.47 µM). It also exhibited comparable activity
Citation: Tang, L.; Jiang, J.; Song, G.;
Wang, Y.; Zhuang, Z.; Tan, Y.; Xia, Y.;
Huang, X.; Feng, X. Design, Synthesis,
and Biological Evaluation of Novel
6H-Benzo[c]chromen-6-one
Keywords: alkoxylated 6H-benzo[c]chromen-6-one; phosphodiesterase II; inhibitors; biological activities
Derivatives as Potential
Phosphodiesterase II Inhibitors. Int. J.
Mol. Sci. 2021, 22, 5680. https://
doi.org/10.3390/ijms22115680
1. Introduction
Ellagic acid (EA) is an important part of the pharmacological activity of mellow
compounds, which are widely distributed in the bark, root, leaf, and fruit of plants. It has a
wide range of pharmacological activities, such as antioxidation, antitumor, antimutation,
Academic Editor: Oxana
V. Galzitskaya
antibacterial, anti-inflammatory, antiviral, and myocardial protection [1]. In recent years,
EA showed great therapeutic potential in the treatment of neurodegenerative diseases
such as typical Alzheimer’s disease (AD) and Parkinson’s disease (PD) because of its
neuroprotective effects and ability to delay neurodegeneration of aging in the mouse
Received: 15 April 2021
Accepted: 25 May 2021
Published: 26 May 2021
model of AD [2–8]. However, the applications of EA were limited in the field of health
care products and cosmetics due to poor oral bioavailability. There are no EA derivatives
available on the market in the pharmaceutical field.
Urolithins are main intestinal metabolites, which are derived from the metabolism
of EA through tannolactone ring and dihydroxylation [9–12]. Urolithins have more ad-
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published maps and institutional affil-
iations.
vantages in absorption than EA, which may be the final material basis for the biological
activities of EA [13]. Urolithins are widely distributed in the urine, feces, and bile of
mammals such as humans, rats, mice, cattle, and pigs [13–15]. The reports on the biological
activity of urolithins mainly focus on the antioxidation, anti-inflammatory, and antitumor
properties, as well as its regulation of intestinal flora and inhibition of end products of
protein glycosylation [9,10,13,16,17]. Some studies reported that both Urolithin A and
Urolithin B are involved in neuroprotection because they can significantly inhibit the for-
mation of protein glycosylation end products [18]. Urolithin A can activate neuroprotective
mechanisms, such as activating SIRT-1 in differentiated ReNcell VM human nerve cells.
SIRT-1 is a deacetylase that regulates DNA expression, cell apoptosis, and aging, and
participates in the physiological or pathological processes of organisms by deacetylating
Copyright:
© 2021 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 (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2021, 22, 5680. https://doi.org/10.3390/ijms22115680
https://www.mdpi.com/journal/ijms

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substrate proteins. The activation of SIRT-1 is related to the neuroprotective effect of
Urolithin A in nerve cells [19]. Urolithin B can cross the blood-brain barrier and has few
side effects on the body, so it may provide therapeutic benefits for neurodegenerative dis-
eases associated with oxidative stress and microglial activation [20]. Therefore, urolithins
show great therapeutic potential in the treatment of neurodegenerative diseases.
Phosphodiesterase inhibition received much attention for the potential treatment of
central nervous system (CNS) disorders. The role of PDEs in the augmentation of cyclic
nucleotide signaling makes these enzymes as attractive targets for the enhancing neuronal
communication. PDE2A is the only subtype of PDE2 in mammalian species, which is mainly
expressed in brain and heart, but remains low in peripheral tissues [21–24]. Compared with
normal people, the level of PDE2A in the brain of AD patients is increased. Laurent Gomez
et al. summarized the development of PDE2 inhibitors for the treatment of cognitive
disorders [25,26]. In 2013, Zhu et al. reported the composite crystal structure of PDE2 and
the highly selective inhibitor BAY 60-7550 (protein number: 4HTX, http://www.rcsb.org,
accessed on 20 March 2021), which provided the basis for the rational design of small
molecule ligands targeted to PDE2 [27]. However, there are no PDE2 drugs in the markets
so far. Therefore, further studies are urgently needed to develop PDE2 inhibitors to meet
clinical needs.
In the present study, urolithins were selected as leading compounds, and a series of
novel urolithin(6H-benzo[c]chromen-6-one)-derivatives were designed and synthesized
to screen PDE2 inhibitors. The potential neuroprotective effects of resultant compounds
were evaluated through PDE2 inhibition effects firstly. The compounds with higher PDE2
inhibition activity were further evaluated by corticosterone-induced cytotoxicity in the
hippocampal HT-22 cells.
2. Results and Discussion
2.1. Synthetic Pathways
Our initial exploration focused on introducing different substituents into the hydroxy
scaffold at the 3-position of urolithin while keeping the lactone ring fixed. Thus, various
kinds of novel 6H-benzo[c]chromen-6-one derivatives were designed and synthesized.
The general routes for synthesizing 6H-benzo[c]chromen-6-one derivatives 1 and 2 are
shown in Scheme 1 [28]. First, the reaction of 2-bromobenzoic acids and resorcinol in the
presence of CuSO4 and NaOH provides the intermediate I and II. Next, intermediate I and
II were reacted with various kinds of halides including iodomethane, alkyl bromides, and
heterocyclic bromides to generate the desired 6H-benzo[c]chromen-6-one derivatives 1 and
2 in moderate to good yields.
Scheme 1. Synthesis of 6H-benzo[c]chromen-6-one derivatives 1 and 2. Reagents and conditions: (
H2O, reflux, 1.2 h; (b) K2CO3, DMF, 80~120 C, 3.5~30 h.
a
) 5% CuSO4, NaOH,
◦
2.2. Enzymatic Assays
Our previous work reported [29] that a propylphenyl group in the classic PDE2
inhibitor BAY 60-7550 was well-accommodated through a hydrophobic-induced binding
pocket located under L770 of the protein. Therefore, for urolithin-derivatives in this work,
only by bearing certain length and lipophilicity can the R1 group interact properly within

Int. J. Mol. Sci. 2021, 22, 5680
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the pocket. Based on this rule, the 6H-benzo[c]chromen-6-one derivatives 1 and 2 were
evaluated based on their effects on PDE2 inhibitory activities. As shown in Figures 1 and 2
,
most of the derivatives gave the appropriate values of ClogP (2.0–5.0), indicating the good
blood-brain barrier penetration. Moreover, compounds 1
in vitro for PDE2 inhibitory activities, showing that R1 group remarkably affected the PDE2
inhibitory activities (as illustrated in Table 1). For compounds 1
, when R1 group alkane
a–u and 2a–w were also tested
a–u
substituted groups with less than five carbons, the PDE2 inhibitory activity is relatively
good. To our delight, compound 1f significantly affected the PDE2 inhibitory activities
(IC50 = 3.67
±
0.47 µM). For compounds 2a–w, although compound 2i had the best PDE2
inhibitory activity, it also needed to be reformed.
Figure 1. CLoP values of 6H-benzo[c]chromen-6-one derivatives 1.

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Figure 2. CLoP values of 6H-benzo[c]chromen-6-one derivatives 2.
2.3. Cell Assays
Corticosterone can induce PDE2A, PDE3B, PDE4A, PDE4D, PDE10, and PDE11 ex-
pression in HT-22 cells, which results in significant cell lesion [30 31]. Thus, corticosterone-
,
induced HT-22 neurotoxicity was generally employed as classical cell model to screen
PDE-inhibitors for curing neurodegenerative disease, such as AD and PD.
To determine whether compound 1
HT-22 cells against corticosterone-induced neurotoxicity by PDE2 inhibition, the viability
of HT-22 cells was measured in the presence of 100 M corticosterone (positive control),
corticosterone absence (control), and different concentrations of 1f. As shown in Figure 3
the cell viability was significantly decreased when HT-22 cells were exposed to 100
corticosterone (p < 0.001), which indicated that HT-22 neurotoxicity model was successfully
built. Compound 1 significantly increased cell viability of HT-22 cells in a dose-dependent
manner at concentration of 6.25~25 M, and the best concentration was 12.5 M (p < 0.01).
In addition, the effect was similar with the BAY 60-7550 at a concentration of 1.0 M. Our
results showed compound 1 possessed good protective effects on HT-22 cells, which was
associated with PDE2 inhibition.
f could promote neuron proliferation and protect
µ
,
µM
f
µ
µ
µ
f

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Table 1. In vitro PDE2 inhibitory activity of 6H-benzo[c]chromen-6-one derivatives 1 and 2.
a
a,b
Compound
PDE2 IC50 (µM)
Compound
PDE2 IC50
>50
(µM)
1a
1b
1c
1d
1e
1f
28.82 ± 7.8
25.40 ± 5.13
25.80 ± 5.45
>50
2a
2b
2c
>50
>50
>50
2d
2e
2f
37.65 ± 4.9
3.67 ± 0.47
>50
17.77 ± 4.07
>50
1g
1h
1i
2g
2h
2i
30.29 ± 7.38
>50
18.69 ± 2.72
17.88 ± 1.87
43.22 ± 3.67
>50
14.88 ± 1.78
>50
1j
2j
1k
1l
2k
2l
>50
19.27 ± 2.38
>50
>50
1m
1n
1o
1p
1q
1r
2m
2n
2o
2p
2q
2r
>50
27.51 ± 1.49
>50
>50
>50
>50
25.16 ± 6.91
23.31 ± 4.28
>50
>50
27.56 ± 2.14
8.14 ± 0.54
>50
1s
1t
2s
>50
2t
Insoluble
29.39 ± 5.01
23.88 ± 545
>50
1u
>50
2u
2v
2w
a
BAY 60-7550 was used as the reference compound with an IC50 of 8.4 nM. b Results are expressed as the mean of
at least three experiments.
Figure 3. Compound 1f protected cells against corticosterone (CORT)-induced cytotoxicity in a
concentration-dependent manner. HT-22 cells were treated with 100 M CORT for 30 min and
compound 1f was added for 24 h. Cell viability was measured by MTS assay. Results are expressed as
the mean
±
standard error of the mean (SEM) of six independent experiments performed in triplicates.
*** p < 0.001, compared to control group; # p < 0.05 and ## p < 0.01, compared to vehicle-treated
CORT group.

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3. Experiments
3.1. Synthesis
All chemicals, reagents, and solvents were analytical grade, purchased from commer-
cial suppliers (i.e., Aladdin, Adamas, and Acros).
The reactions were monitored via thin-layer chromatography (TLC) performed on
HSGF254 plates and visualized under UV light. Melting points were measured with an
1
X4-A microscopic melting point apparatus. HNMR spectra were recorded on a Bruker-
BioSpin GmbH spectrometer at 300 and 400 MHz; 13CNMR spectra were recorded on
a BrukerBioSpin GmbH spectrometer at 75 and 100 MHz; the coupling constants are
given in Hz. Mass (HRMS) analysis was obtained using Agilent 6200 Accurate-Mass TOF
LC/MS system with Electrospray Ionization (ESI). HPLC analysis was obtained using
SHIMADZULC-20AB.
3.1.1. 3-Hydroxy-6H-benzo[c]chromen-6-one (I)
2-Bromobenzoic acid (30 mmol, 6.03 g), resorcinol (60 mmol, 6.60 g), sodium hydroxide
(60 mmol, 0.8 g), and water (30 mL) were added in the 100 mL sealed tub, and the reaction
was stirred 20 min at 100 ◦C. Then, 5% copper sulfate solution (9.6 mL) was added to the
system and heated to reflux for 1 h. The progress of reaction was monitored through TLC
(PE:EA = 5:1). At the end of the reaction, the reaction solution was added dropwise to
the ice-water mixture, and the filtered solid continued to be washed with water. The filter
cake was recrystallized twice with methanol and glacial acetic acid to obtain compound I
◦
(
3.50 g, 55.0%) [26]. M.p. 250–251 C. 1H NMR (300 MHz, DMSO-d6)
(dt, J = 8.0, 3.0 Hz, 1H), 8.21–8.15 (m, 2H), 7.89 (m, 1H), 7.57 (m, 1H), 6.85 (dd, J = 9.0, 3.0
Hz, 1H), 6.76 (d, J = 3.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
= 161.06, 160.32, 152.57,
δ = 10.36 (s, 1H), 8.27
δ
135.65, 135.52, 130.11, 128.04, 125.21, 122.02, 119.39, 113.60, 109.82, 103.40; HRMS (ESI) m/z
calcd for C13H9O3+[M + H]+: 213.0473; found: 213.0470. Chromatographic purity: 98.0%
(HPLC).
3.1.2. General Procedures for Synthesis of Compounds 1a–u [28]
The anhydrous DMF (30 mL) was added to a 250 mL round bottom flask, compound I
(9.4 mmol, 2.0 g), anhydrous K2CO3 (12.2 mmol, 1.7 g), and halide (12.2 mmol) were added,
and the temperature was controlled at 80~120 ◦C; the reaction was stirred for 24 h. The
reaction was monitored by TLC (PE:EA = 3:1). When the reaction was over, the reaction
solution was added to the ice-water mixture to obtain brown crude solid. After suction
filtration and drying, the brown crude solid was purified by column chromatography to
obtain compounds 1a–u.
3-Methoxy-6H-benzo[c]chromen-6-one (1a): Using iodomethane as the star◦ting material,
1
the desired white solid 1a was isolated (1.30 g, 61%). M.p. 131.5–132.8 C. H NMR
(400 MHz, DMSO-d6)
δ = 8.35 (d, J = 8.0 Hz, 1H), 8.29 (d, J = 12.0 Hz, 1H), 8.22 (dd,
J = 8.0 Hz, 1H), 7.95–7.90 (m, 1H), 7.62 (t, J = 6.0 Hz, 1H), 7.06–6.99 (m, 2H), 3.88 (s, 3H); 13
C
NMR (100 MHz, DMSO-d6) δ = 161.67, 160.96, 152.54, 135.78, 135.19, 130.14, 128.53, 125.17,
122.40, 119.68, 112.78, 111.09, 101.91, 56.23; HRMS (ESI) m/z calcd for C14H11O3+[M + H]+:
227.0630; found: 227.0626. Chromatographic purity: 98.2% (HPLC).
Ethoxy-6H-benzo[c]chromen-6-one (1b): Using bromoethane as th◦e starting material, the
desired yellow solid 1b was isolated (1.70 g, 75%). M.p. 115.2–117.7 C. 1H NMR (300 MHz,
DMSO-d6)
δ = 8.35–8.22 (m, 3H), 7.93 (t, J = 6.0 Hz, 1H), 7.62 (t, J = 6.0 Hz, 1H), 6.98
(d, J = 6.0 Hz, 2H), 4.13 (q, J = 3.0 Hz, 2H), 1.39 (t, J = 6.0 Hz, 3H); 13C NMR (75 MHz,
DMSO-d6) δ = 160.97, 152.55, 135.81, 135.25, 130.15, 128.52, 125.20, 122.41, 119.68, 113.10,
110.99, 102.31, 64.28, 14.94; HRMS (ESI) m/z calcd for C15H13O3+[M + H]+: 241.0786; found:
241.0782. Chromatographic purity: 97.6% (HPLC).
3-Isopropoxy-6H-benzo[c]chromen-6-one (1c): Using 2-bromopropane as the starting ma-
terial, the desired yellow solid 1c was isolated (1.70 g, 71%). M.p. 58.7–59.1 ◦C. 1H NMR
(300 MHz, DMSO-d6) δ = 8.68–7.33 (m, 5H), 6.96 (s, 2H), 4.77 (m, 1H), 1.32 (d, J = 6.0 Hz,

Int. J. Mol. Sci. 2021, 22, 5680
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6H); 13C NMR (75 MHz, DMSO-d6)
δ
= 160.98, 159.92, 152.59, 135.76, 135.23, 130.13,
128.45, 125.22, 122.34, 119.65, 113.83, 110.83, 103.23, 70.42, 22.13; HRMS (ESI) m/z calcd for
C16H15O3+[M + H]+: 255.0943; found: 255.0940. Chromatographic purity: 97.8% (HPLC).
3-Propoxy-6H-benzo[c]chromen-6-one (1d): Using 1-bromopropane as the starting mate-
◦
rial, the desired white solid 1d was isolated (1.99 g, 83%). M.p. 67.6–69.2 C. 1H NMR
(300 MHz, DMSO-d6)
7.61–7.55 (m, 1H), 6.99–6.93 (m, 2H), 4.04–3.99 (m,2H), 1.83–1.71 (m, 2H), 1.01 (t, J = 6.0 Hz
3H); 13C NMR (75 MHz, DMSO-d6)
= 161.07, 160.95, 152.50, 135.74, 135.21, 130.11,
δ = 8.28 (t, J = 6.0 Hz, 1H), 8.23–8.17 (m, 2H), 7.92–7.86 (m, 1H),
,
δ
128.46,125.12, 122.35,119.64, 113.06, 110.95,102.29, 70.00, 22.35, 10.80; HRMS (ESI) m/z
calcd for C16H15O3+[M + H]+: 255.0943; found: 255.0940. Chromatographic purity: 98.9%
(HPLC).
3-(Sec-butoxy)-6H-benzo[c]chromen-6-one (1e): Using 2-bromobutane as the starting ma-
terial, the desired yellow solid 1e was isolated (1.79 g, 71%). M.p. 73.2–75.8 ◦C. 1H NMR
(400 MHz, DMSO-d6)
(m, 2H), 4.60–4.53 (m, 1H), 1.74–1.58 (m, 2H), 1.28 (d, J = 8.0 Hz, 3H), 0.95 (t, J = 4.0 Hz, 3H);
13C NMR (100 MHz, DMSO-d6)
= 160.97, 160.25, 152.58, 135.73, 135.24, 130.12, 128.41,
δ = 8.36–8.19 (m, 3H), 7.93–7.88 (m, 1H), 7.62–7.57 (m, 1H), 7.00–6.97
δ
125.19, 122.31, 119.64, 113.83, 110.83, 103.21, 75.23, 28.89, 19.35, 9.92; HRMS (ESI) m/z
calcd for C17H17O3+[M + H]+: 269.1099; found: 269.1095. Chromatographic purity: 99.2%
(HPLC).
3-Butoxy-6H-benzo[c]chromen-6-one (1f): Using 1-bromobutane as the starting material,
◦
the desired yellow solid 1f was isolated (1.41 g, 56%). M.p. 53.6–55.5 C. 1H NMR (300 MHz,
DMSO-d6)
(d, J = 6.0 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 1.74(t, J = 9.0 Hz, 2H), 1.50–1.42 (m, 2H), 0.95
(t, J = 9.0 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
= 161.13, 160.98, 152.54, 135.78, 135.25,
δ = 8.34–8.20 (m, 3H), 7.91 (t, J = 9.0 Hz, 1H), 7.60 (t, J = 9.0 Hz, 1H), 7.01–6.99
δ
130.14, 128.49, 125.16, 122.39, 119.67, 113.13, 110.98, 102.34, 68.30, 31.03, 19.17, 14.15; HRMS
(ESI) m/z calcd for C17H17O3+[M + H]+: 269.1099; found: 269.1095. Chromatographic
purity: 98.7% (HPLC).
3-(Pentyloxy)-6H-benzo[c]chromen-6-one (1g): Using 1-bromopentane as the starting ma-
terial, the desired yellow solid 1g was isolated (1.49 g, 56%). M.p. 69.4–71.8 ◦C. 1H NMR
(400 MHz, DMSO-d6)
7.01–6.98 (m, 2H), 4.08 (t, J = 6.0 Hz, 2H), 1.79–1.72 (m, 2H), 1.46–1.32 (m, 4H), 0.91 (t,
J = 8.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
= 161.10, 160.97, 153.65, 135.77, 135.23,
δ = 8.34–8.20 (m, 3H), 7.91 (t, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H),
δ
130.13, 128.47, 125.14, 122.37, 119.66, 113.10, 110.95, 103.11, 68.56, 29.42, 28.12, 22.36, 17.18;
HRMS (ESI) m/z calcd for C18H19O3+[M + H]+: 283.1256; found: 283.1252. Chromato-
graphic purity: 99.2% (HPLC).
Ethyl 2-((6-oxo-6H-benzo[c]chromen-3-yl)oxy)acetate (1h): Using ethyl 2-bromoacetate
as the starting material, the desired yellow solid 1h was isolated (1.40 g, 50%). M.p.
1
124.7–125.8 ◦C. H NMR (300 MHz, DMSO-d6)
δ
= 8.36–8.21 (m, 3H), 7.93 (t, J = 7.5 Hz, 1H),
7.62 (t, J = 7.5 Hz, 1H), 7.04 (dt, J = 9.0, 3.0 Hz, 2H), 4.94 (s, 2H), 4.20 (q, J = 7.0 Hz, 2H), 1.24
(t, J = 7.5Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
δ
= 168.79, 160.88, 159.94, 152.37, 136.32,
135.03, 130.17, 128.74, 125.29, 122.52, 120.20, 113.06, 111.79, 103.34, 66.14, 62.31, 15.96; HRMS
(ESI) m/z calcd for C17H15O5+[M + H]+: 299.0841; found: 299.0837. Chromatographic
purity: 98.3% (HPLC).
3-((Tetrahydro-2H-pyran-4-yl)methoxy)-6H-benzo[c]chromen-6-one
(bromomethyl)tetrahydro-2H-pyran as the starting material, the desired yellow solid 1i
was isolated (1.60 g, 55%). M.p. 133.0–134.8 ◦C. 1H NMR (300 MHz, CDCl3)
= 8.34 (d,
(1i):
Using 4-
δ
J = 9.0 Hz, 1H), 7.96 (dd, J = 18.0, 9.0 Hz, 2H), 7.78 (t, J = 7.5 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H),
6.91–6.82 (m, 2H), 4.04 (dd, J = 12.0, 9.0 Hz, 2H), 3.87 (t, J = 6.0Hz, 2H), 3.46 (t, J = 12.0 Hz,
2H), 2.17–2.06 (m, 1H), 1.80 (d, J = 3.0 Hz, 2H), 1.57–4.13 (m, 2H); 13C NMR (75 MHz, DMSO-
d6) δ = 161.09, 160.97, 152.53, 135.79, 135.22, 130.15, 128.53, 125.19, 122.41, 119.69, 113.13,
111.08, 102.44, 73.00, 67.06, 34.77, 29.60; HRMS (ESI) m/z calcd for C19H19O4+[M + H]+
:
311.1205; found: 311.1201. Chromatographic purity: 97.3% (HPLC).

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3-(Benzyloxy)-6H-benzo[c]chromen-6-one (1j): Using (bromomethyl)benzene as the start-
ing material, the desired yellow solid 1j was isolated (2.81 g, 99%). M.p. 113.2–115.3 ◦C.
1H NMR (300 MHz, DMSO-d6)
δ
= 8.31 (dd, J = 15.0, 9.0 Hz, 2H), 8.21 (dd, J = 9.0, 1.2 Hz,
1H), 7.94–7.89 (m, 1H), 7.61–7.58 (m, 1H), 7.52–7.48 (m, 2H), 7.45–7.35 (m, 3H), 7.12–7.07
(m, 2H), 5.24 (s, 2H); 13C NMR (75 MHz, DMSO-d6)
= 160.95, 160.70, 152.48, 136.92,
δ
135.81, 135.17, 130.16, 128.99, 128.61, 128.53, 128.37, 125.26, 122.47, 119.76, 113.44, 111.34,
102.91, 70.25; HRMS (ESI) m/z calcd for C20H15O3+ [M + H]+: 303.0943; found: 303.0940.
Chromatographic purity: 96.8% (HPLC).
3-(2-(1H-pyrazol-1-yl)ethoxy)-6H-benzo[c]chromen-6-one (1k): Using 1-(2-bromoethyl)-1H-
pyrazole as the starting material, the desired white solid 1k was isolated (1.24 g, 43%).
M.p. 164.3–166.6 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ
= 8.32 (d, J = 9.0 Hz, 1H), 8.27–8.19
(m, 2H), 7.93–7.88 (m, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.63–7.57 (m, 1H), 7.48 (d, J = 3.0 Hz
1H), 7.01–6.95 (m, 2H), 6.27 (t, J = 3.0 Hz, 1H), 4.55 (m, 2H), 4.46 (m, 2H); 13C NMR
75 MHz, DMSO-d6) = 160.94, 160.43, 152.48, 139.39, 135.84, 135.12, 131.08, 130.17, 128.67,
,
(
δ
125.29, 122.49, 119.77, 113.16, 111.46, 105.72, 102.63, 67.60, 50.93; HRMS (ESI) m/z calcd
for C18H15N2O3+[M + H]+: 307.1004; found: 307.1001. Chromatographic purity: 96.5%
(HPLC).
3-(Pyrimidin-2-yloxy)-6H-benzo[c]chromen-6-one (1l): Using 2-bromopyrimidine as the
starting material, the desired yellow solid 1l was isolated (1.83 g, 67%). M.p. 174.0–176.6 ◦C
.
1H NMR (400 MHz, DMSO-d6)
(d, J = 12.0 Hz, 1H), 7.98 (t, J = 10.0 Hz, 1H), 7.70 (t, J = 10.0 Hz, 1H), 7.41–7.29 (m,
3H); 13C NMR (100 MHz, DMSO-d6)
= 164.80, 160.66, 154.69, 151.92, 135.92, 134.55,
δ = 8.70 (d, J = 8.0 Hz, 2H), 8.45(d, J = 8.0 Hz, 2H), 8.28
δ
130.26, 129.58, 125.31, 123.09, 120.57, 119.02, 117.93, 115.50, 111.00; HRMS (ESI) m/z calcd
+
for C17H11N2O3
[
M + H]+: 291.0691; found: 291.0686. Chromatographic purity: 98.2%
(HPLC).
3-(2-Hydroxyethoxy)-6H-benzo[c]chromen-6-one (1m): Using 2-bromoethanol as the start-
ing material, the desired white solid 1m was isolated (2.34 g, 98%). M.p. 125.7–126.4 ◦C
1H NMR (400 MHz, DMSO-d6)
= 8.32 (d, J = 8.0 Hz, 1H), 8.27–8.24 (m, 1H), 8.21 (dd,
.
δ
J = 8.0, 4.0 Hz, 1H), 7.94–7.90 (m, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.03–7.00 (m, 2H), 5.01
(t, J = 6.0 Hz, 1H), 4.11 (t, J = 4.0 Hz, 2H), 3.76 (q, J = 4.0 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) δ = 161.16, 161.07, 152.52, 135.90, 135.23, 130.19, 128.61, 125.23, 122.42, 119.67,
113.25, 111.05, 102.43, 70.65, 59.87; HRMS (ESI) m/z calcd for C15H13O4+[M + H]+: 257.0736;
found: 257.0732. Chromatographic purity: 98.3% (HPLC).
3-((Tetrahydrofuran-2-yl)methoxy)-6H-benzo[c]chromen-6-one
(
1n):
Using 2-
(bromomethyl)tetrahydrofuran as the starting material, the desired yellow solid 1n was
◦
isolated (1.31 g, 47%). M.p. 82.8–84.1 C. 1H NMR (400 MHz, CDCl3)
δ = 8.36 (d, J = 8.0 Hz,
1H), 8.00 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.51 (t, J = 8.0 Hz
,
1H), 6.96 (dd, J = 8.0, 4.0 Hz, 1H), 6.89 (d, J = 4.0 Hz, 1H), 4.37–4.30 (m, 1H), 4.08–3.95
(m, 3H), 3.91–3.85 (q, J = 8.0 Hz, 1H), 2.18–2.09 (m, 1H), 2.06–1.94 (m, 2H), 1.85–1.76 (m,
1H); 13C NMR (100 MHz, CDCl3)
δ = 161.55, 160.74, 152.49, 135.15, 134.88, 130.54, 127.76,
123.77, 121.11, 119.97, 112.90, 111.28, 102.27, 76.85, 70.86, 68.72, 28.19, 25.76; HRMS (ESI)
m/z calcd for C18H17O4+[M + H]+: 297.1049; found: 297.1045. Chromatographic purity:
99.1% (HPLC).
3-((1,3-Dioxolan-2-yl)methoxy)-6H-benzo[c]chromen-6-one (1o): Using 2-(bromomethyl)-1,3-
dioxolane as the s◦tarting material, the desired white solid 1o was isolated 1.93 g, 69%).
1
M.p. 135.6–137.8 C. H NMR (400 MHz, CDCl3)
δ = 8.35 (d, J = 8.0 Hz, 1H), 8.00 (d,
J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.81–7.77 (m, 1H), 7.51 (t, J = 8.0 Hz, 1H), 6.96
(dd, J = 12.0, 4.0 Hz, 1H), 6.90 (d, J = 4.0 Hz, 1H), 5.34 (t, J = 4.0 Hz, 1H), 4.14–3.96 (m,
6H); 13C NMR (100 MHz, CDCl3)
δ = 161.45, 160.29, 152.43, 135.02, 134.90, 130.54, 127.86,
123.84, 121.14, 120.01, 112.74, 111.61, 102.49, 101.66, 69.01, 65.41; HRMS (ESI) m/z calcd for
C17H15O5+[M + H]+: 299.0841; found: 299.0839. Chromatographic purity: 98.6% (HPLC).

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3-((3,5-Dimethoxybenzyl)oxy)-6H-benzo[c]chromen-6-one (1p): Using 1-(bromomethyl)-3,5-
dimethoxybenzene as the starting material, the desired white solid 1p was isolated (3.37 g,
99%). M.p. 133.8–135.0 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ
= 8.23–8.11 (m, 3H), 7.82 (t,
J = 9.0 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.00– 6.97 (m, 2H), 6.58 (d, J = 3.0 Hz, 2H), 6.40 (t,
J = 3.0 Hz, 1H), 5.09 (s, 2H), 3.68 (s, 6H); 13C NMR (75 MHz, DMSO-d6)
= 161.03, 160.94,
δ
160.56, 152.43, 139.24, 135.76, 135.13, 130.13, 128.58, 125.22, 122.43, 119.73, 113.39, 111.34,
106.01, 102.87, 99.98, 70.04, 55.65; HRMS (ESI) m/z calcd for C22H19O5+[M + H]+: 363.1154;
found: 363.1151. Chromatographic purity: 98.4% (HPLC).
3-((1,3-Dimethyl-1H-pyrazol-5-yl)methoxy)-6H-benzo[c]chromen-6-one (1q): Using 5-
(bromomethyl)-1,3-dimethyl-1H-pyrazole as the starting material, the desired whitesolid
◦
1q was isolated (2.92 g, 97%). M.p. 165.1–167.6 C. 1H NMR (300 MHz, CDCl3)
δ
= 8.34 (dd,
J = 9.0, 1.5 Hz, 1H), 8.00–7.93 (m, 2H), 7.82–7.76 (m, 1H), 7.54–7.49 (m, 1H), 6.95 (m, 2H),
6.16 (s, 1H), 5.07 (s, 2H), 3.87 (s, 3H), 2.26 (s, 3H); 13C NMR (75 MHz, CDCl3)
= 161.39,
δ
159.70, 152.41, 147.46, 136.99, 134.98, 134.89, 130.57, 128.02, 124.00, 121.17, 120.01, 112.96,
111.84, 107.14, 102.50, 60.81, 36.53, 13.44; HRMS (ESI) m/z calcd for C19H17N2O3+[M + H]+:
321.1161; found: 321.1158. Chromatographic purity: 97.1% (HPLC).
3-(Pyridin-3-ylmethoxy)-6H-benzo[c]chromen-6-one (1r): Using 3-(bromomethyl)pyridine
as the starting material, the desired yellow solid 1r was isolated (1.42 g, 50%). M.p.
1
143.6–145.3 ◦C. H NMR (300 MHz, CDCl3)
δ
= 8.69 (d, J = 30.0 Hz, 2H), 8.35 (dd, J = 9.0,
3.0 Hz, 1H), 7.98 (dd, J = 12.0,6.0 Hz, 2H), 7.853–7.76 (m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.37
(dd, J = 9.0, 3.0 Hz, 1H), 6.98 (dd, J = 9.0, 3.0 Hz, 1H), 6.97 (d, J = 3.0 Hz,1H), 5.15 (s, 2H);
13C NMR (75 MHz, CDCl3)
δ = 161.39, 160.06, 152.50, 149.79, 149.07, 135.42, 134.95, 134.10,
131.74, 130.58, 127.97, 124.00, 123.65, 121.17, 120.03, 112.92, 111.75, 102.64, 67.94; HRMS
(ESI) m/z calcd for C19H14NO3+[M + H]+: 304.0895; found: 304.0891. Chromatographic
purity: 96.2% (HPLC).
3-(Cyclopentylmethoxy)-6H-benzo[c]chromen-6-one (1s): Using (bromomethyl)cyclopentane
as the starting material, the desired yellow solid 1s was isolated (2.13 g, 77%). M.p.
1
90.6–91.2 ◦C. H NMR (300 MHz, CDCl3)
δ
= 8.34 (dd, J = 9.0, 1.5 Hz, 1H), 7.98 (dd, J = 6.0
,
1.2 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.79–7.74 (m, 1H), 7.51–7.45 (m, 1H), 6.90 (dd, J = 9.0,
3.0 Hz, 1H), 6.83 (d, J = 3.0 Hz, 1H), 3.89 (d, J = 6.0 Hz, 2H), 2.47 (m, 1H), 1.96–1.79 (m, 2H),
1.752–1.55 (m, 4H), 1.44–1.32 (m, 2H); 13C NMR (75 MHz, CDCl3)
δ = 161.61, 161.19, 152.56,
135.25, 134.85, 130.52, 127.61, 123.67, 121.05, 119.89, 112.87, 110.87, 102.12, 72.67, 38.90,
29.48, 25.45; HRMS (ESI) m/z calcd for C19H19O3+[M + H]+: 295.1256; found: 295.1251.
Chromatographic purity: 98.4% (HPLC).
3-((4-[Hydroxymethyl]benzyl)oxy)-6H-benzo[c]chromen-6-one
(4-(bromomethyl)phenyl)methanol as the starting material, the desired white solid 1t
was isolated (2.40 g, 77%). M.p. 147.0–149.2 ◦C. 1H NMR (300 MHz, DMSO-d6)
= 8.33 (d,
(
1t):
Using
δ
J = 9.0 Hz, 1H), 8.29–8.21 (m, 2H), 7.96–7.90 (m, 1H), 7.65–7.60 (m, 1H), 7.49 (d, J = 9.0 Hz,
2H), 7.40 (d, J = 9.0 Hz, 2H), 7.12–7.07 (m, 2H), 5.29 (t, J = 9.0 Hz, 1H), 5.24 (s, 2H), 4.55
(d, J = 6.0 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.95, 160.68, 152.45, 142.94, 135.79,
135.17, 130.15, 128.58, 128.24, 127.02, 125.21, 122.44, 119.72, 113.45, 111.27, 102.87, 70.14,
63.10; HRMS (ESI) m/z calcd for C21H17O4+[M + H]+: 333.1049; found: 333.1044.Chromato-
graphic purity: 97.2% (HPLC).
3-(2-Morpholinoethoxy)-6H-benzo[c]chromen-6-one (1u): Using 4-(2-bromoethyl)morpholine
as the starting material, the desired yellow solid 1u was isolated (1.22 g, 40%). M.p.
100.8–102.8 ◦C 1H NMR (300 MHz, CDCl3)
. δ = 8.35 (dd, J = 9.0, 1.2 Hz, 1H), 7.96 (dd,
J = 18.0, 6.0 Hz, 2H), 7.78 (m, 1H), 7.50 (t, J = 7.5 Hz, 1H), 6.94–6.90 (dd, J = 9.0, 3.0 Hz, 2H),
4.18 (t, J = 6.0 Hz, 2H), 3.76 (t, J = 7.5 Hz, 4H), 2.85 (t, J = 4.5 Hz, 2H), 2.61 (t, J = 4.5 Hz, 4H);
13C NMR (75 MHz, CDCl3)
δ = 161.50, 160.55, 152.52, 135.09, 134.91, 130.55, 127.80, 123.80,
121.10, 119.96, 112.88, 111.29, 102.25, 66.91, 66.25, 57.43, 54.13; HRMS (ESI) m/z calcd for
C19H20NO4+[M + H]+: 326.1314; found: 326.1310. Chromatographic purity: 99.2% (HPLC).

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3.1.3. General Procedure for the Synthesis of 3-Hydroxy-6H-benzo[c]chromen-6-one (II)
Using 2-bromo-5-methoxybenzoic acid (60 mmol, 13.79 g) acid as the starting material,
the synthesis method was similar to that of compound , the desired yellow solid II was
isolated (6.82 g, 47.0%). M.p. 258.4–261.7 C. H NMR (300 MHz, DMSO-d6) = 10.22
(s, 1H), 8.21 (d, J = 9.0 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 3.0 Hz, 1H), 7.49 (dd,
J = 9.0, 3.0 Hz, 1H), 6.83 (dd, J = 9.0, 3.0 Hz, 1H), 6.75 (d, J = 3.0 Hz, 1H), 3.89 (s, 3H); 13
I
◦
1
δ
C
NMR (75 MHz, DMSO-d6) δ = 160.97, 159.41, 158.94, 151.62, 128.97, 124.57, 124.36, 123.99,
120.48, 113.55, 111.27, 109.96, 103.30, 56.01; HRMS (ESI) m/z calcd for C14H10O4+[M+H]+:
242.0579; found: 242.0575. Chromatographic purity: 97.2% (HPLC).
3.1.4. General Procedures for Synthesis of Compounds 2a–w
The anhydrous DMF (30 mL) was added to a 250 mL round bottom flask, compound
II (9.4 mmol, 2.28 g), anhydrous K2CO3 (12.2 mmol, 1.7 g) and halides (12.2 mmol) were
added, and the temperature was controlled at 80~120 ◦C, the reaction was stirred for 24 h.
The reaction was monitored by TLC (PE:EA = 3:1). When the reaction was over, the reaction
solution was to the ice-water mixture to obtain brown crude solid. After suction filtration
and drying, the brown crude solid was purified by column chromatography to obtain
compounds 2a–w.
3,8-Dimethoxy-6H-benzo[c]chromen-6-one (2a): Using iodomethane as the starting mate-
rial, the desired white solid 2a was isolated (1.23 g, 51%). M.p. 146.2–148.8 ◦C. 1H NMR
(400 MHz, DMSO-d6)
1H), 7.47 (dd, J = 9.0, 3.0 Hz, 1H), 6.96 (s, 1H), 6.94 (t, J = 4.0 Hz, 1H), 3.89 (s, 3H), 3.84
(s, 3H); 13C NMR (100 MHz, DMSO-d6)
= 160.85, 160.83, 159.23, 151.55, 128.55, 12.50,
δ = 8.22 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 3.0 Hz,
δ
124.34, 124.31, 120.79, 112.69, 111.30, 111.21, 101.79, 56.15, 56.04; HRMS (ESI) m/z calcd for
C15H13O4+[M + H]+: 257.0736; found: 257.0731. Chromatographic purity: 99.2% (HPLC).
3-Ethoxy-8-methoxy-6H-benzo[c]chromen-6-one (2b): Using bromoethane as the starting
material, the desired white solid 2b was isolated(1.14 g, 45%). M.p. 141.1–143.8 ◦C. 1H
NMR (400 MHz, DMSO-d6)
δ = 8.26 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.62 (d,
J = 4.0 Hz, 1H), 7.51 (dd, J = 8.0, 4.0 Hz, 1H), 6.98 (s, 1H), 6.96 (t, J = 4.0 Hz, 1H), 4.13
(q, J = 8.0 Hz, 2H), 3.90 (s, 3H), 1.37 (t, J = 8.0 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.89, 160.10, 159.23, 151.55, 128.61, 124.52, 124.39, 124.32, 120.78, 113.03, 111.32, 111.10,
102.20, 64.19, 56.05, 14.95; HRMS (ESI) m/z calcd for C16H15O4+[M + H]+: 271.0892; found:
271.0890. Chromatographic purity: 98.1% (HPLC).
8-Methoxy-3-propoxy-6H-benzo[c]chromen-6-one (2c): Using 1-bromopropane as the start-
1
ing material, the desired white solid 2c was isolated (1.39 g, 52%). M.p. 109.3–111.6 ◦C. H
NMR (400 MHz, DMSO-d6)
J = 4.0 Hz, 1H), 7.45 (dd, J = 8.0, 4.0 Hz, 1H), 6.94–6.90 (m, 2H), 3.99 (t, J = 6.0 Hz, 2H), 3.88
(s, 3H), 1.75 (m, 2H), 1.00 (t, J = 8.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
= 160.86,
δ = 8.18 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.56 (d,
δ
160.23, 159.20, 151.52, 128.58, 124.46, 124.33, 124.27, 120.75, 113.00, 111.29, 111.08, 102.20,
69.95, 56.02, 22.37, 10.81; HRMS (ESI) m/z calcd for C17H17O4+[M + H]+: 285.1049; found:
285.1045. Chromatographic purity: 99.3% (HPLC).
3-(Sec-butoxy)-8-methoxy-6H-benzo[c]chromen-6-one (2d): Using 2-bromobutane as the
starting material, the desired yellow solid 2d was isolated (0.95 g, 34%). M.p. 64.5–66.7 ◦C
.
1H NMR (400 MHz, DMSO-d6)
δ = 8.21 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.59
(d, J = 4.0 Hz, 1H), 7.47 (dd, J = 8.0, 4.0 Hz, 1H), 6.95 (s, 1H), 6.92 (d, J = 4.0 Hz, 1H),
4.56–4.49 (m, 1H), 3.89 (s, 3H), 1.73–1.57 (m, 2H), 1.27 (d, J = 6.0 Hz, 3H), 0.94 (t, J = 8.0 Hz,
3H); 13C NMR (100 MHz, DMSO-d6)
δ = 160.89, 159.40, 159.20, 151.62, 128.62, 124.57, 124.35,
124.27, 120.77, 113.84, 111.32, 111.01, 103.23, 75.18, 56.03, 28.90, 19.37, 9.94; HRMS (ESI) m/z
calcd for C18H19O4+[M + H]+: 299.1205; found: 299.1201. Chromatographic purity: 98.7%
(HPLC).
3-Butoxy-8-methoxy-6H-benzo[c]chromen-6-one (2e): Using 1-bromobutane as the starting
1
material, the desired white solid 2e was isolated (1.82 g, 65%). M.p. 102.5–104.4 ◦C. H

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NMR (300 MHz, DMSO-d6)
1H), 7.51 (dd, J = 9.0, 3.0 Hz, 1H), 6.98 (s, 1H), 6.96 (t, J = 4.5 Hz, 1H), 4.07 (t, J = 6.0 Hz
2H), 3.90 (s, 3H), 1.78–1.68 (m, 2H), 1.52–1.3840 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H); 13C NMR
(75 MHz, DMSO-d6) = 160.84, 160.25, 159.20, 151.52, 128.58, 124.43, 124.29, 124.24, 120.75,
δ = 8.26 (d, J = 9.0 Hz, 1H), 8.19–8.15 (m, 1H), 7.62 (d, J = 3.0 Hz,
,
δ
112.99, 111.31, 111.08, 102.21, 68.21, 56.02, 31.06, 19.17, 14.14; HRMS (ESI) m/z calcd for
C18H19O4+[M + H]+: 299.1205; found: 299.1201. Chromatographic purity: 99.1% (HPLC).
8-Methoxy-3-(pentyloxy)-6H-benzo[c]chromen-6-one (2f): Using 1-bromopentane as the
starting material, the desired white solid 2f was isolated (1.34 g, 46%). M.p. 73.1–75.5 ◦C
.
1H NMR (300 MHz, DMSO-d6)
δ = 8.23 (d, J = 9.0 Hz, 1H), 8.15–8.12 (m, 1H), 7.59 (d,
J = 3.0 Hz, 1H), 7.49 (dd, J = 8.0, 3.0 Hz, 1H), 6.97–6.93 (m, 2H), 4.03 (t, J = 7.5 Hz, 2H),
3.89 (s, 3H), 1.78–1.69 (m, 2H), 1.44–1.34 (m, 4H), 0.93–0.88 (m, 3H); 13C NMR (75 MHz,
DMSO-d6) δ = 160.89, 160.26, 159.22, 151.54, 128.62, 124.50, 124.39, 124.32, 120.77, 113.05,
111.29, 111.09, 102.21, 68.49, 56.04, 28.70, 28.13, 22.37, 14.40; HRMS (ESI) m/z calcd for
C19H21O4+[M + H]+: 313.1362; found: 313.1359. Chromatographic purity: 98.5% (HPLC).
3-Isopropoxy-8-methoxy-6H-benzo[c]chromen-6-one (2g): Using 2-bromopropane as the
starting material, the desired white solid 2g was isolated (0.97 g, 36%). M.p. 88.3–90.7 ◦C
.
1H NMR (300 MHz, DMSO-d6)
J = 3.0 Hz, 1H), 7.48 (dd, J = 9.0, 3.0 Hz, 1H), 6.95–6.91 (m, 2H), 4.75 (m, 1H), 3.89 (s, 3H),
1.32 (d, J = 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-d6)
= 160.89, 159.19, 159.05, 151.60,
δ = 8.21 (d, J = 9.0 Hz, 1H), 8.14–8.11 (m, 1H), 7.58 (d,
δ
128.60, 124.55, 124.34, 124.26, 120.76, 113.81, 111.30, 110.98, 103.20, 70.33, 56.03, 22.14; HRMS
(ESI) m/z calcd for C17H17O4+[M + H]+: 285.1049; found: 285.1045. Chromatographic
purity: 98.6% (HPLC).
3-(2-Hydroxyethoxy)-8-methoxy-6H-benzo[c]chromen-6-one (2h): Using 2-bromoethanol
as the starting material, the desired yellow solid 2h was isolated (1.51 g, 56%). M.p.
166.8–168.0 ◦C 1H NMR (300 MHz, DMSO-d6)
. δ = 8.21 (d, J = 9.0 Hz, 1H), 8.15–8.11
(m, 1H), 7.57 (d, J = 3.0 Hz, 1H), 7.47 (dd, J = 9.0, 3.0 Hz, 1H), 6.98–6.94 (m, 2H), 4.98 (t,
J = 6.0 Hz, 1H), 4.08 (dd, J = 6.0, 3.0 Hz, 2H), 3.89 (s, 3H), 3.77 (q, J = 6.0 Hz, 2H); 13C NMR
(75 MHz, DMSO-d6) δ = 160.86, 160.25, 159.19, 151.50, 128.55, 124.48, 124.33, 124.26, 120.76,
113.05, 111.26, 111.14, 102.28, 70.56, 59.89, 56.02; HRMS (ESI) m/z calcd for C16H15O5+[M +
H]+: 287.0841; found: 287.0837. Chromatographic purity: 98.8% (HPLC).
8-methoxy-3-(2-(Methylthio)ethoxy)-6H-benzo[c]chromen-6-one
(
2i):
Using
(2-bromoethyl)(methyl)sulfane as the starting material, the desired white solid 2i was
◦
isolated (1.18 g, 40%). M.p. 110.1–111.8 C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.27 (d,
J = 9 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.52 (dd, J = 9.0, 3.0 Hz,
1H), 7.03–6.98 (m, 2H), 4.26 (t, J = 6.0 Hz, 2H), 3.90 (s, 3H), 2.89 (t, J = 7.5 Hz, 2H), 2.18 (s,
3H); 13C NMR (75 MHz, CDCl3)
δ = 165.60, 164.53, 164.05, 156.28, 133.29, 129.33, 129.13,
125.61, 117.84, 116.16, 116.12, 107.21, 72.70, 60.82, 37.28, 20.45; HRMS (ESI) m/z calcd for
C17H17O4S+[M + H]+: 317.0769; found: 317.0765. Chromatographic purity: 97.8% (HPLC).
Ethyl 2-((8-Methoxy-6-oxo-6H-benzo[c]chromen-3-yl)oxy)acetate (2j): Using ethyl 2-bromoacetate
as the starting material, the desired white solid 2j was isolated (1.58 g, 51%). M.p.
1
151.5–152.8 ◦C. H NMR (300 MHz, DMSO-d6)
δ
= 8.28 (d, J = 9.0 Hz, 1H), 8.21 (t, J = 6.0 Hz
,
1H), 7.62 (d, J = 3.0 Hz, 1H), 7.52 (dd, J = 9.0, 3.0 Hz, 1H), 7.03– 6.99 (m, 2H), 4.92 (s, 2H),
4.20 (q, J = 6.0 Hz, 2H), 3.90 (s, 3H), 1.23 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz, DMSO-d6)
δ
= 168.85, 160.80, 159.42, 159.11, 151.39, 128.39, 124.63, 124.48, 124.40, 120.98, 113.01, 111.93,
111.42, 102.75, 65.41, 61.25, 56.09, 14.52; HRMS (ESI) m/z calcd for C18H17O6+[M + H]+:
329.0947; found: 329.0943. Chromatographic purity: 97.4% (HPLC).
3-(Cyclopentylmethoxy)-8-methoxy-6H-benzo[c]chromen-6-one
(bromomethyl)cyclopentane as the starting material, the desired white solid 2k was isolated
(0.90 g, 30%). M.p. 146.1–148.2 ◦C. 1H NMR (300 MHz, DMSO-d6)
= 8.26 (d, J = 9.0 Hz
(
2k):
Using
δ
,
1H), 8.17 (t, J = 4.5 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.51 (dd, J = 9.0, 3.0 Hz, 1H), 6.99–6.95
(m, 2H), 3.94 (d, J = 6.0 Hz, 2H), 3.90 (s, 3H), 2.38–2.28 (m, 1H), 1.82–1.74 (m, 2H), 1.65–1.51
(m, 4H), 1.38–1.27 (m, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.92, 160.44, 159.28, 151.59,

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128.66, 124.55, 124.44, 124.39, 120.83, 113.15, 111.39, 111.15, 102.35, 72.61, 56.09, 38.88,
29.44, 25.41; HRMS (ESI) m/z calcd for C20H21O4+[M + H]+: 325.1362; found: 325.1360.
Chromatographic purity: 98.1% (HPLC).
8-Methoxy-3-((tetrahydrofuran-2-yl)methoxy)-6H-benzo[c]chromen-6-one (2l): Using 2-
(bromomethyl)tetrahydrofuran as the starting material, the desired white solid 2l was
◦
isolated (1.71 g, 56%). M.p. 113.1–115.5 C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.27 (d,
J = 9.0 Hz, 1H), 8.18 (dd, J = 9.0, 3.0 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.51 (dd, J = 9.0, 3.0 Hz,
1H), 7.03–6.94 (m, 2H), 4.23–4.15 (m, 1H), 4.11–3.98 (m, 2H), 3.90 (s, 3H), 3.84–3.77 (m,
1H), 3.73–3.66 (m, 1H), 2.08–1.99 (m, 1H), 1.94–1.77 (m, 2H), 1.72–1.65 (m, 1H); 13C NMR
(75 MHz, DMSO-d6) δ = 160.86, 160.14, 159.27, 151.51, 128.57, 124.52, 124.36, 120.83, 113.09,
111.35, 111.29, 102.39, 76.81, 71.08, 67.97, 56.06, 28.06, 25.67; HRMS (ESI) m/z calcd for
C19H19O5+[M + H]+: 327.1154; found: 327.1150. Chromatographic purity: 99.2% (HPLC).
3-((1,3-Dioxolan-2-yl)methoxy)-8-methoxy-6H-benzo[c]chromen-6-one (2m): Using 2-
(bromomethyl)-1,3-dioxolane as the star◦ting material, the desired white solid 2m was
isolated (0.93 g, 30%). M.p. 147.9–150.1 C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.28 (d,
J = 9.0 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.52 (dd, J = 9.0, 3.0 Hz,
1H), 7.04–6.99 (m, 2H), 5.24 (t, J = 3.0 Hz, 1H), 4.12 (d, J = 6.0 Hz, 2H), 4.01–3.96 (m, 2H),
3.90 (s, 3H), 3.89– 3.85 (m, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.87, 159.76, 159.38,
151.49, 128.52, 124.63, 124.48, 124.45, 120.94, 113.12, 111.62, 111.41, 102.62, 101.57, 69.02,
65.03, 56.10; HRMS (ESI) m/z calcd for C18H17O6+[M + H]+: 329.0947; found:329.0943.
Chromatographic purity: 97.6% (HPLC).
3-(Cyclohexylmethoxy)-8-methoxy-6H-benzo[c]chromen-6-one
(bromomethyl)cyclohexane as the starting material, the desired white solid 2n was isolated
(1.85 g, 38%). M.p. 139.8–140.1 ◦C. 1H NMR (300 MHz, DMSO-d6)
= 8.25 (d, J = 9.0 Hz,
(
2n):
Using
δ
1H), 8.18–8.14 (m, 1H), 7.61 (d, J = 3.0 Hz, 1H), 7.50 (dd, J = 9.0, 3.0 Hz, 1H), 6.99–6.95
(m, 2H), 3.90 (s, 3H), 3.87 (d, J = 6.3 Hz, 2H), 1.84–1.65 (m, 6H), 1.33–1.04 (m, 5H); 13C
NMR (75 MHz, DMSO-d6) δ = 160.91, 160.43, 159.26, 151.58, 128.66, 124.54, 124.42, 124.37,
120.81, 113.10, 111.37, 111.12, 102.33, 73.67, 56.08, 37.43, 29.65, 26.49, 25.70; HRMS (ESI) m/z
calcd for C21H23O4+[M + H]+: 339.1518; found: 339.1514. Chromatographic purity: 97.5%
(HPLC).
3-(2-(1,3-Dioxolan-2-yl)ethoxy)-8-methoxy-6H-benzo[c]chromen-6-one (2o): Using 2-(2-
bromoethyl)-1,3-dioxolane as the starting material, the desired white solid 2o was isolated
(1.22 g, 38%). M.p. 151.6–153.2 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.27 (d, J = 9.0 Hz,
1H), 8.20–8.17 (m, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.51 (dd, J = 9.0, 3.0 Hz, 1H), 7.00–6.96
(m, 2H), 5.02 (t, J = 4.5 Hz, 1H), 4.18 (t, J = 6.0 Hz, 2H), 3.95–3.91 (m, 2H), 3.90 (s, 3H),
3.83–3.79 (m, 2H), 2.07 (m, 2H); 13C NMR (75 MHz, CDCl3)
δ = 161.64, 159.88, 159.13,
151.57, 128.66, 124.48, 123.11, 122.82, 120.96, 112.70, 111.33, 110.92, 102.20, 101.86, 65.02,
64.08, 55.76, 33.59, 29.73; HRMS (ESI) m/z calcd for C19H19O6+[M + H]+: 343.1103; found:
343.1101. Chromatographic purity: 96.9% (HPLC).
8-Methoxy-3-((tetrahydro-2H-pyran-4-yl)methoxy)-6H-benzo[c]chromen-6-one (2p): Using
4-(bromomethyl)tetrahydro-2H-pyran as the starting material, the desired yellow solid 2p
was isolated (1.79 g, 36%). M.p. 168.1–168.5 ◦C. 1H NMR (400 MHz, CDCl3)
δ = 7.94 (d,
J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 4.0 Hz, 1H), 7.39 (dd, J = 8.0, 4.0 Hz, 1H),
6.91 (dd, J = 8.0, 4.0 Hz, 1H), 6.86 (d, J = 4.0 Hz, 1H), 4.06 (dd, J = 12.0, 4.0 Hz, 2H), 3.94 (s,
3H), 3.88 (d, J = 8.0 Hz, 2H), 3.51–3.45 (m, 2H), 2.19–2.0 (m, 1H), 1.82–1.78 (m, 2H), 1.56–1.46
(m, 2H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.91, 160.29, 159.31, 151.59, 128.64, 124.60,
124.45, 124.41, 120.86, 113.14, 111.41, 111.25, 102.42, 72.99, 67.06, 56.10, 34.79, 29.62; HRMS
(ESI) m/z calcd for C20H21O5+[M + H]+: 341.1311; found: 341.1307. Chromatographic
purity: 97.4% (HPLC).
3-(Benzyloxy)-8-methoxy-6H-benzo[c]chromen-6-one (2q): Using (bromomethyl)benzene
as the starting material, the desired white solid 2q was isolated (1.04 g, 33%). M.p.
141.7–142.5 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.27 (d, J = 9.0 Hz, 1H), 8.20 (d, J = 9.0 Hz,

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1H), 7.62 (d, J = 3.0 Hz, 1H), 7.51–7.48 (m, 3H), 7.45–7.33 (m, 3H), 7.10–7.04 (m, 2H), 5.22 (s,
2H), 3.90 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
δ = 162.53, 159.77, 158.99, 151.43, 136.98,
128.96, 128.33, 124.30, 121.58, 120.60, 113.25, 111.25, 102.70, 70.15, 55.98; HRMS (ESI) m/z
calcd for C21H17O4+[M + H]+: 333.1049; found: 333.1045. Chromatographic purity: 98.7%
(HPLC).
3-((4-[Hydroxymethyl]benzyl)oxy)-8-methoxy-6H-benzo[c]chromen-6-one (2r): Using (4-
(bromomethyl)phenyl)methanol as the starting material, the desired white solid 2r was
◦
isolated (1.26 g, 37%). M.p. 192.4–194.6 C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.27 (d,
J = 9.0 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 3.0 Hz, 1H), 7.51 (dd, J = 9.0, 3.0 Hz, 1H),
7.44 (dd, J = 6.0, 3.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.08–7.03 (m, 2H), 5.20 (t, J = 6.0 Hz,
3H), 4.51 (d, J = 3.0 Hz, 2H), 3.90 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.86, 159.87,
159.32, 151.49, 142.91, 135.27, 128.55, 128.18, 127.01, 124.57, 124.40, 120.88, 113.43, 111.44,
111.39, 102.84, 70.11, 63.11, 56.08; HRMS (ESI) m/z calcd for C22H19O5+[M + H]+: 363.1154;
found: 363.1150. Chromatographic purity: 98.6% (HPLC).
3-((3,5-Dimethoxybenzyl)oxy)-8-methoxy-6H-benzo[c]chromen-6-one (2s): Using 1-
(bromomethyl)-3,5-dimethoxybenzene as the starting material, the desired white solid
2s was isolated (1.59 g, 43%). M.p. 153.6–155.3 ◦C. 1H NMR (300 MHz, CDCl3)
δ = 7.89
(d, J = 9.0 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.73 (d, J = 3.0 Hz, 1H), 7.35 (dd, J = 9.0, 3.0 Hz,
1H), 6.96 (dd, J = 9.0, 3.0 Hz, 1H), 6.89 (d, J = 3.0 Hz, 1H), 6.59 (d, J = 3.0 Hz, 2H), 6.42
(t, J = 3.0 Hz, 1H), 5.05 (s, 2H), 3.91 (s, 3H), 3.80 (s, 6H); 13C NMR (75 MHz, DMSO-d6)
δ = 161.04, 160.88, 160.82, 159.76, 159.33, 151.48, 139.36, 128.54, 124.62, 124.44, 120.90, 113.41,
111.51, 111.35, 106.65, 105.99, 102.85, 99.96, 70.00, 56.08, 55.67, 55.49; HRMS (ESI) m/z
calcd for C23H21O6+[M + H]+: 393.1260; found: 393.1257. Chromatographic purity: 98.3%
(HPLC).
3-((2-Fluorobenzyl)oxy)-8-methoxy-6H-benzo[c]chromen-6-one (2t):Using 1-(bromomethyl)-2-
fluorobenzene as the starting material, the desired white solid 2t was isolated (2.47 g, 75%).
M.p. 203.6–204.9 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ = 8.30 (d, J = 9.0 Hz, 1H), 8.23 (d,
J = 9.0 Hz, 1H), 7.64–7.58 (m, 2H), 7.52 (dd, J = 9.0, 3.0 Hz, 1H), 7.49–7.42 (m, 1H), 7.33–7.24
(m, 2H), 7.15 (d, J = 3.0 Hz, 1H), 7.07 (dd, J = 9.0, 3.0 Hz, 1H), 5.25 (s, 2H), 3.91 (s, 3H);
13C NMR (75 MHz, DMSO-d6)
δ = 159.49, 159.27, 151.59, 130.17, 130.06, 129.80, 128.58,
124.55, 124.42, 124.37, 123.26, 122.89, 121.09, 115.70, 115.42, 112.90, 111.78, 111.00, 102.74,
64.17, 55.79; HRMS (ESI) m/z calcd for C21H16FO4+[M + H]+: 351.0954; found: 351.0950.
Chromatographic purity: 97.6% (HPLC).
3-((1,3-Dimethyl-1H-pyrazol-5-yl)methoxy)-8-methoxy-6H-benzo[c]chromen-6-one (2u): Us-
ing 5-(bromomethyl)-1,3-dimethyl-1H-pyrazole as the starting material, the desired white
solid 2u was isolated (1.71 g, 52%). M.p. 184.6–186.6 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ
= 8.29 (d, J = 9.0 Hz, 1H), 8.21 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 3.0 Hz, 1H), 7.52 (dd, J = 9.0,
3.0 Hz, 1H), 7.15 (d, J = 3.0 Hz, 1H), 7.07 (dd, J = 9.0, 3.0 Hz, 1H), 6.19 (s, 1H), 5.23 (s, 2H),
3.90 (s, 3H), 3.76 (s, 3H), 2.12 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ = 165.59, 164.15, 164.09,
156.20, 150.86, 142.74, 133.24, 129.37, 129.23, 129.16, 125.71, 118.13, 116.53, 116.17, 111.87,
107.73, 65.66, 60.85, 18.37; HRMS (ESI) m/z calcd for C21H19N2O4+[M + H]+: 351.1267;
found: 351.1263. Chromatographic purity: 98.7% (HPLC).
8-Methoxy-3-(pyrimidin-2-yloxy)-6H-benzo[c]chromen-6-one (2v): Using 2-bromopyrimidine
as the starting material, the desired white solid 2v was isolated (1.29 g, 23%). M.p.
212.3–214.0 ◦C
.
1H NMR (300 MHz, DMSO-d6)
δ
= 8.69 (d, J = 6.0 Hz, 2H), 8.38 (dd,
J = 9.0, 6.0 Hz, 2H), 7.68 (d, J = 3.0 Hz, 1H), 7.57 (dd, J = 9.0, 3.0 Hz, 1H), 7.38 (d, J = 3.0 Hz
1H), 7.33 (t, J = 4.5 Hz, 1H), 7.27 (dd, J = 9.0, 3.0 Hz, 1H), 3.93 (s, 3H); 13C NMR (75 MHz,
,
CDCl3) δ = 160.11, 158.86, 152.24, 149.99, 126.84, 123.51, 122.38, 122.22, 120.83, 117.40, 115.74,
114.80, 110.18, 109.98, 54.79; HRMS (ESI) m/z calcd for C18H13N2O4+[M + H]+: 321.0797;
found: 321.0794. Chromatographic purity: 96.8% (HPLC).
3-(2-(1H-pyrazol-1-yl)ethoxy)-8-methoxy-6H-benzo[c]chromen-6-one (2w): Using 1-(2-
bromoethyl)-1H-pyrazole as the starting material, the desired white solid 2w was isolated

Int. J. Mol. Sci. 2021, 22, 5680
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(1.93 g, 61%). M.p. 159.0–161.1 ◦C. 1H NMR (300 MHz, DMSO-d6)
δ
= 8.32 (d, J = 9.0 Hz,
1H), 8.22 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 3.0 Hz, 1H), 7.67 (d, J = 3.0 Hz, 1H), 7.58–7.53 (m,
2H), 7.05–6.98 (m, 2H), 6.32 (t, J = 3.0 Hz, 1H), 4.59 (t, J = 6.0 Hz, 2H), 4.50 (t, J = 4.5 Hz, 2H),
3.95 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
δ = 160.83, 159.59, 159.35, 151.49, 139.37, 131.06,
128.47, 124.61, 124.41, 120.90, 113.10, 111.60, 111.39, 105.70, 102.55, 67.55, 56.08, 50.95; HRMS
(ESI) m/z calcd for C19H17N2O4+[M + H]+: 337.1110; found: 337.1106. Chromatographic
purity: 98.9% (HPLC).
3.2. Enzymatic Assays
The in vitro inhibitory activity of compounds 1a–u and 2a–w against PDE2, which
used Bio-cAMP as substrate, are listed in Table 1. The specific experimental method was
reported below [32].
Preparation of 1*Rection Buffer: we took 1 mL 10*Rection Buffer, added 9 mL ultra-
pure water to swirl and mix well, and put it on ice; preparation of PDE2 solution: 210
µL of
1*Rection Buffer were added into a centrifuge tube, then 30 L PDE2 were added, mixed
µ
well, equal to 8-times dilution, and placed on ice; preparation of compound solution with
gradient concentration: DMSO was used as the solvent to prepare 10 mM compound
solution, and then diluted with 1*Rection Buffer. The solution with final concentrations of
40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM was prepared, respectively,
and the solution was vortex mixed.
We took 2
croplate, added 4
and 4 L PDE2 into A10, added 2
added 6 L 1*Rection Buffer to A13, and ensured the three groups are parallel to each other.
Centrifugation was carried out at 1000 R and 25 C for about 2 min. The reaction time was
µ
L of the diluted compound solution of each concentration into the mi-
L PDE2 solution, numbered them A1–A9, added 2 L 1*Rection Buffer
L compound solution and 4 L 1*Rection Buffer to A11,
µ
µ
µ
µ
µ
µ
◦
0.5 h in the microporous plate thermostatic oscillator. After the reaction, 4 µL Bio-cAMP
was added to A1–A13, and centrifugation was carried out at 1000 R and 25 ◦C for about
2 min and reacted in a microporous plate thermoelectric oscillator for 1 h.
Anti-Camp Alohascreen Acceptor and Streptavidin Coated Donor Bead were pur-
chased from Perkin Elmer Company according to response volume: 1* Immunoassay
Buffer 549.32
µL and 3.70
µL anti-cAMP AlohaScreen Acceptor were collected under dark
conditions. We took 1.98
µL Streptavidin coated Donor Bead, mixed well, added 15 µL
mixture to each well after 1 h reaction, and reacted it in the dark for 1 h. After the reaction,
we tested it with multilabel microplate detector. According to the detection results, the
inhibition rate was calculated according to the dilution ratio of the experimental system
and the formula, and the IC50 of compound was calculated by GraphPad Prism 5.
3.3. Cell Assays
3.3.1. Solution Preparation
We added 943
20 mM stock solution, froze it at
medium before use. The test compound was dissolved in DMSO to obtain a 20 mM stock
solution and stored at
µ
L of DMSO solution to 8.2 mg of dexamethasone acetate to obtain a
−
20 ◦C, and diluted it to 2 mM with serum-containing
◦
−
20 C. Diluted with serum-containing medium to a concentration of
2
×
100, 2 50, 2 25, 2
×
×
×
12.5, 2
×
6.25, 2
×
3.125 µM before use. The control sample Bay
60-7550 stock solution with a concentration of 1 mM was diluted with serum-containing
medium to a concentration of 2 × 0.1, 2 × 1, 2 × 2, and 2 × 4 µM before use.
3.3.2. Cell Culture and Dexamethasone Intervention
After◦resuscitation, HT-22 cells were cultured in DMEM medium (containing 10%
FBS) at 37 C with 5% CO2. After the cells grew to more than 80% cell fusion, we passaged
and seeded the plate, and the density of each well should be 10,000 cells/100
µL/well and
cultured for 24 h. After the cells adhered to the wall, drug intervention was given.

Int. J. Mol. Sci. 2021, 22, 5680
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3.3.3. Drug Intervention
10 L solution of 2 mM dexamethasone acetate was cultured at 37 C for 30 min.
L drug solution of 2 100, 2 50, 2 25, 2 12.5, 2 6.25, and 2 3.125 M were
added to make the final concentration of dexamethasone acetate 100 M, and the final
concentrations of the drug were 100, 50, 25, 12.5, 6.25, 3.125 M. After preparation, it took
◦
µ
100
µ
×
×
×
×
×
×
µ
µ
µ
another 24 h to continue the culture. There were 5 replicate wells for each sample, and the
normal control group, dexamethasone acetate-affected group, and positive control group
were set at the same time (final concentration 0.1, 1, 2, and 4 µM).
3.3.4. MTT Color Experimental and Data Processing
After the incubation, 50
to incubate. Discarded the upper matrix and the 150
the blue-violet precipitate completely, the shaker was used to shake for 10 min. Finally,
the wavelength A 490 was measured. The data were expressed as mean sd (n = 5), and
µL of MTT was added to each well, and it needed another 4 h
µL DMSO was added. To dissolve
±
one-way anova analysis of variance was used for data statistics; p < 0.05 is defined as a
significant difference.
4. Conclusions
In this paper, a series of new 6H-benzo[c] chromen-6-one derivatives were designed
and synthesized with PDE2 as the potential therapeutic target, and the PDE2 inhibitory
activity was tested at the enzyme level. The results showed that part of compounds
exhibited good PDE2 inhibitory activity, such as 1f, 1h, 1i, 1l, 1s, 2e, and 2i. Among
them, 1f has the highest PDE2 inhibitory activity with IC50 value of 3.67
protective effects of compound 1 against corticosterone-induced HT-22 neurotoxicity
were further verified. Compound 1 can protect HT-22 cells from corticosterone-induced
±
0.47 µM. The
f
f
death and rescue corticosterone-induced toxicity in a dose-dependent manner, which is
probably associated with PDE2 inhibition. In the future, we will design and synthesize
more active PDE2-inhibitors based on an analysis of the structure-activity relationship
between compound 1f and 4HTX for the potential treatment of AD. In addition, the in vivo
test would be performed in mouse models of AD to obtain more convincing and potentially
therapeutic molecules.
Author Contributions: Synthesis, materials preparation and biological studies, L.T., Y.T., Y.X.; Synthe-
sis and characterization, L.T., Y.X.; Biological activity studies and Validation, Y.W., Z.Z.; Supervision,
J.J., G.S.; Writing-original draft, L.T., X.F.; Writing—review and editing, L.T., X.H. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China, grant
number 81803471.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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