Flavonoid-triazolyl hybrids as potential anti-hepatitis C virus agents: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2021.113395

A series of flavonoid-triazolyl hybrids were synthesized and evaluated as novel inhibitors of hepatitis C virus (HCV). The results of anti-HCV activity assays showed that most of the synthesized derivatives at a concentration of 100 μg/mL inhibited the ge

Full text of DOI:10.1016/j.ejmech.2021.113395

European Journal of Medicinal Chemistry 218 (2021) 113395
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Flavonoid-triazolyl hybrids as potential anti-hepatitis C virus agents:
Synthesis and biological evaluation
,
,
c
,
,
Han Zhang a 1, Xin Zheng b 1, Jichong Li a, Qingbo Liu a **, Xiao-Xiao Huang a,
Huaiwei Ding d ***, Ryosuke Suzuki c, Masamichi Muramatsu c ****, Shao-Jiang Song a
,
,
, *
a Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China
b Department of Cardiology, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China
c Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
d Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of flavonoid-triazolyl hybrids were synthesized and evaluated as novel inhibitors of hepatitis C
virus (HCV). The results of anti-HCV activity assays showed that most of the synthesized derivatives at a
concentration of 100 mg/mL inhibited the generation of progeny virus. Among these derivatives, 10m and
10r exhibited the most potent anti-HCV activity and inhibited the production of HCV in a dose-
dependent manner. Interestingly, 10m and 10r had no significant inhibitory effect on viral translation
or replication. Additional action mechanism studies revealed that the most potent compounds, 10m and
Received 21 October 2020
Received in revised form
6 February 2021
Accepted 17 March 2021
Available online 31 March 2021
10r, significantly inhibited viral entry to 34.0% and 52.0%, respectively, at 10
further effective application of 10m and 10r as potential HCV preventive agents.
© 2021 Elsevier Masson SAS. All rights reserved.
mM. These results suggest
Keywords:
Flavonoid-triazolyl hybrids
Anti-HCV activity
HCV entry Inhibitors
1. Introduction
plays a very important role in virus replication and virus particle
formation. Because it is essential for the viral life cycle, NS3-4 A is
considered as a drug development target [8]. Although direct-
acting antiviral (DAA) treatment can cure 90% of HCV-infection
cases, this therapy is costly, especially in many upper-middle-
income, high-income countries. Therefore, the development of
more anti-HCV drugs is necessary.
Flavonoids represent a diverse group of natural polyphenols
that are widely found in many medicinal plants, fungi and vege-
tables and possess various biological and pharmacological activities
[9,10]. In particular, flavonoids have attracted increasing attention
due to their inhibitory activity against several viruses, including
HCV. Accumulating evidence has shown the ability of certain fla-
vonoids to inhibit HCV propagation at different stages (Fig. 1)
[11e13]. Additionally, several flavonoids, such as silymarin, have
been proven to provide a beneficial and protective role in liver
injury and fibrosis resulting from HCV infection [14,15]. Based on
these therapeutic viewpoints, flavonoids seem to be promising
scaffolds on which to build new derivatives with enhanced anti-
HCV potency. Many studies have reported that the introduction of
prenylated groups on a flavonoid skeleton can increase lipophilicity
and endow the molecule with a high affinity towards biological
membranes, leading to robust biological activity [16,17].
Hepatitis C virus (HCV) currently infects more than 180 million
people globally, approximately 3% of the world population [1].
Annually, approximately 3 million people worldwide are newly
infected with HCV, and more than 350,000 people die from HCV-
related liver disease [2,3]. HCV is an enveloped, positive-sense
single-stranded RNA virus belonging to the Flaviviridae family
[4,5]. After infection, the HCV genome encodes a single polyprotein
that can be cleaved by host and viral proteases into 10 kinds of viral
proteins, including structural proteins (core, E1, and E2) and
nonstructural proteins (NS2, NS3/4 A, NS4B, NS5A, and NS5B) [5].
NS3/4 A is a serine protease that is critical for cleaving four sites in
the HCV polyprotein [6], as well as host factors such as interferon
promoter stimulator-1 (IPS-1) [7]. The cleavage of viral proteins
* Corresponding author.
** Corresponding author.
*** Corresponding author.
**** Corresponding author.
E-mail addresses: liuqingbolily@163.com (Q. Liu), dinghuaiwei627@163.com
(H. Ding), muramatsu@niid.go.jp (M. Muramatsu), songsj99@163.com (S.-J. Song).
1
These authors made equal contribution to the article.
https://doi.org/10.1016/j.ejmech.2021.113395
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
Fig. 1. Chemical structures of reported anti-HCV flavone compounds and anti-HCV compounds containing triazole moiety.
Furthermore, cyclization involving the prenylated group and the
adjacent phenolic hydroxyl group of flavonoids to form pyrano-
flavonoids enhances bioactivities and decreases toxicity [18].
Inspired by these beneficial functions, a pyranoflavonoid was
designed and totally synthesized as a chemical scaffold [19].
Triazole, one of the most important nitrogen heterocycles, can
form various noncovalent interactions with different biological
targets via hydrogen bonds, van der Waals interactions, hydro-
phobic interactions, or dipole-dipole bonds, which has wide ap-
plications in the pharmaceutical field and possesses diverse
therapeutic properties [20e22]. Many studies have also shown that
triazole-containing compounds (Fig. 1) possess anti-HCV effects
[23e27], suggesting that the triazole moiety plays an intriguing
role in the development of new anti-HCV agents. Therefore,
different triazole groups were combined with the pyranoflavonoid
scaffold to design a series of flavonoid derivatives as potential anti-
HCV agents. All the designed compounds were evaluated for po-
tential anti-HCV activity by using mCherry-NLS-IPS visualization
system. In addition, the outstanding compound was selected for
further evaluation of its inhibition of HCV progeny virus production
and protein expression. Furthermore, assessments of their inhibi-
tory effects on HCV replication and entry were also carried out to
investigate the mode of action of the selected compounds against
HCV.
dimethylformamide (DMF) and used without further purification.
Finally, a 1,3-dipolar cycloaddition reaction of intermediate 9 with
these substituted azides in the presence of CuSO4_5H2O and sodium
L
-ascorbate resulted in the formation of 1,4-substituted triazolyl
derivatives 10a-10s in yields of 55.8%e80.3%.
2.2. Biological evaluation
2.2.1. In vitro cytotoxic activity
The cytotoxic activity of all the synthesized flavonoid derivatives
in initial screening was tested using Cell Proliferation Kit II (XTT).
The toxicity of triazole-modified isopentenyl flavonoids (10a-10s)
was generally lower than that of parent compound 8, indicating
that most of the derivatives were nontoxic to Huh7 cells (except
compounds 10g and 10j) at 100 mg/mL (Fig. 2).
2.2.2. Establishment of the mCherry-NLS-IPS visualization system
and initial screening
The HCV NS3/4 A protein cleaves the 508-cysteine of IPS-1 and
then prevents the inhibition of viral proliferation [30]. In this study,
based on the characteristics of the NS3/4 A protein, a visualization
system was prepared by overexpressing the recombinant protein
mCherry-NLS-IPS in Huh7 cells. In the absence of HCV infection,
mCherry-NLS-IPS is localized on mitochondria, and the cytoplasmic
region fluoresces red (Fig. 3). When Huh7 cells are infected, NS3/4 A
cleaves the IPS-1 protein, and the nuclear localization sequence
(NLS) sequence induces protein transfer from the cytoplasm into
the nucleus, and then, the nuclear area fluoresce red [31].
The system enables a quick determination of whether the cells
are infected with HCV. Therefore, it is suitable for screening anti-
viral drugs. Therefore, we used this system for the initial screening
of all the prepared derivatives, and the results indicated that
compounds 10d, 10e, 10m, 10o and 10r showed significant HCV
inhibitory activity (Fig. 4).
2. Results and discussion
2.1. Chemistry
The synthetic route for flavanone and flavanone triazole hybrid
synthesis is presented in Scheme 1. Compound 8 was prepared
from 2,4-dihydroxyacetophenone (1) and p-hydroxybenzaldehyde
(3). 2,4-Dihydroxyacetophenone was efficiently protected with
methoxymethyl (MOM) chloride using K2CO3 in acetone to obtain
2,4-bis(methoxymethoxy)acetophenone (2) [28]. Compound 3 was
alkylated with prenyl bromide to produce key intermediate 4 in a
yield of 29.3%. Compound 4 was cyclized by using 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) to produce bicyclic intermediate
5. The synthesis of chalcone 6 was based on a known methodology
[29], and it was then cyclized using sodium acetate and deprotected
with 3 N aqueous HCl to produce 8. Then, compound 8 was reacted
with bromopropyne in acetone in the presence of potassium car-
bonate to obtain intermediate 9. Substituted azides were prepared
from their corresponding halides with sodium azide in N,N-
2.2.3. Inhibition of HCV progeny virus production and protein
expression
The results of the preliminary screening showed that 10d, 10e,
10m, 10o and 10r may effectively inhibit HCV infection. Subse-
quently, the supernatant of the HCV sample culture was harvested
and used to reinfect Huh7 cells. After 48 h, HCV RNA in the rein-
fected cells was detected. The results showed that compounds 10d,
10e, 10m, 10o and 10r at a concentration of 100
mg/mL had a sig-
nificant inhibitory effect on the production of HCV progeny (Fig. 5).
2

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
Scheme 1. Synthesis route of 8 and its derivatives. Reagents and conditions: (a) CH3OCH2Cl, K2CO3, acetone, reflux (b) prenyl bromide, KOH, r. t. (c) DDQ, toluene, reflux (d)
KOHeH2OeCH3CH2OH, ice-bath (e) CH3CH2OH/CH3COONa, reflux (f) MeOH, dilute HCl (aq.) (g) K2CO3, acetone, reflux (h) CuSO4_5H2O, ReN3, Na-L-ascorbate, n-butanol/H2O (1:1).
Fig. 2. Cell toxicity of selected compounds 8 and 10a-10s were determined by Cell Proliferation Kit II (XTT). Huh7 cells were seeded per well overnight, treated with compounds
(100 mg/mL) for 72 h before assessing toxicity by Cell Proliferation Kit II (XTT).
Fig. 3. Structural representation of IPS-1 denoting the aa positions of the CARD, Proline-rich region (Pro), transmembrane domain (TM), and the NS3/4 A cleavage site at C508.
Interestingly, the study found that the length of fatty side chains
(10d and 10e) can significantly affect this anti-HCV activity, with
fatty acid chains that are too long or too short conferring the
compound with a weaker antiviral effect. In addition, derivatives
with aromatic side chains (10m, 10o and 10r) showed stronger
antiviral activity than derivatives with fatty acid chains (10d, 10e).
3

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
Fig. 4. The anti-HCV activity of parent compound 8 and all derivatives (10a-10s) was tested using the mCherry-NLS-IPS visualization system at a concentration of 100 mg/ml. Huh7-
mCherry-NLS-IPS cells were infected with the HCV (JFH1). After 72 h of treatment, the change of the fluorescence signal in the cells was observed by using BZ-X710 KEYENCE.
Fig. 5. Anti-HCV activity of synthetic derivatives in the supernatant. After treated Huh7-infected cells of each compound for 72 h, supernatant was harvested for reinfection to
detect viral infectivity. Total RNA was extracted from reinfected Huh7 cells and subjected to RT-PCR for HCV 50-UTR region to detect the level of viral RNA.
In summary, compared with parent compound 8, the introduction
of triazole fragments can reduce the toxicity of a compound and
improve its antiviral activity as well as prevent HCV infection to a
certain extent.
To confirm the anti-HCV effect of compounds 10m, 10o and 10r,
we treated Huh7 cells with compounds 10m, 10o and 10r at the
PCR, and IFNa (10U/mL) was used as a positive control (Fig. 6B).
Both 10m and 10r have no or little cytotoxic activity. The results
showed that compounds 10m and 10r exhibited potential anti-HCV
activity with IC50 values of 5.285
addition, compounds 10m and 10r inhibited HCV production in a
dose-dependent manner. At the concentration of 30 M, the
mM and 9.004 mM, respectively. In
m
indicated concentrations (1
mM, 10
mM, 100
mM). Western blot as-
inhibitory effects of compounds 10m and 10r are comparable to the
says were performed to determine the HCV protein expression
levels. As shown in Fig. 6, treatment with these compounds
resulted in a significant dose-dependent reduction in HCV protein
expression compared to that in the control group (Fig. 6A).
Furthermore, after treatment with compounds 10m and 10r for
3 days (1 mM, 3 mM, 10 mM, 30 mM), HCV production was measured
by the reinfection of Huh7 cells with the viral culture supernatant.
Forty-eight hours after reinfection, HCV RNA was detected by RT-
positive drug (10U/mL).
2.2.4. Selected compounds10m and 10r have no effect on HCV
replication
Antiviral compounds routinely target specific steps of the virus
lifecycle, including attachment, entry, replication or assembly
[32,33]. Our data showed that compounds 10m and 10r were active
against HCV protein expression and progeny virus production after
4

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
Fig. 6. Inhibitory effect on viral protein expression and infectivity. (A) NS3 and Core protein expression. Huh7 cells were treated with compounds at indicated concentrations, and
the cell lysate was subjected to Western blotting with anti-NS3, anti-Core and anti-GAPDH antibodies. (B) Supernatant was harvested to reinfect Huh7 cells and test viral infectivity.
Total RNA was extracted from reinfected cells and subjected to RT-PCR to detect HCV RNA. Data is presented as the mean SD of 3 independent experiments. *P < 0.05, **P < 0.01,
and ***P < 0.001 compared with the positive drug.
the HCV attachment step. Therefore, we preliminarily speculated
that these compounds have a high probability of inhibiting pro-
cesses after HCV attachment, including entry, translation, or
replication.
To further investigate the mechanism of action of compound
and evaluate their antiviral effect on HCV infection, a subgenomic
construct (SGR-Luc-JFH1) based on the HCV JFH1 backbone was
used to assess HCV replication (Fig. 7A) [34,35]. The results are
shown in Fig. 7B. Compared with the level of the control group,
there was no significant decrease in the luciferase signal after
Huh7 cells were first treated with compounds 10m and 10r for 24 h
and then infected with HCVpp. After 48 h, the cells were harvested
to detect luciferase activity (Fig. 8A). Interestingly, our data sug-
gested that compound pretreatment also inhibited the HCV entry
step (Fig. 8B). Similarly, wild-type HCV was used for the pretreat-
ment test. Huh7 cells were treated with 10m and 10r for 24 h,
followed by HCV infection. HCV RNA was detected 48 h later. Our
data suggested that 10m and 10r also somehow inhibited virus
attachment in the pretreatment assay of both the HCVpp and wild-
type infection systems (Fig. 8B and C), although 10m and 10r
inhibited HCV infection more significantly during the post infection
treatment (Fig. 6). All together, these results demonstrate that
compounds 10m and 10r inhibit HCV production by targeting the
HCV entry step, mainly HCV internalization.
10 mM treatment for 3 days, indicating that 10m and 10r did not
suppress viral translation or replication. These results led us to
speculate that the two compounds may target the HCV entry step,
such as inhibiting internalization.
The entry of HCV into cells represents the beginning of viral
infection, which is essential for initiating viral infection and spread.
In addition, viral entry is recognized as an important factor for graft
reinfection and establishment of persistent infection. Preventing
virus entry can effectively eradicate HCV infection before the viral
cycle begins, and it may prevent the cell-to-cell transmission
required for virus transmission [38]. Therefore, there is an urgent
need for new antiviral prevention and treatment strategies.
2.2.5. The inhibitory effect of selected compounds on HCV entry
Viral entry includes attachment and internalization that is
considered as another target for the development of antiviral drugs
[7]. If virus entry can be effectively blocked, it is possible to prevent
cells from viral infection [36]. To confirm their mechanism, com-
pounds 10m and 10r were investigated for their capacities to
inhibit HCV entry using a HCV pseudo-particle (HCVpp) assay [37].
Fig. 7. 10m and 10r do not inhibit HCV replication. (A) Subgenomic luciferase reporter gene JFH1 construct (SGR-Luc-JFH1) (not to scale). Luc, firefly luciferase gene; EMCV, EMCV
IRES. (B) SGR was transfected to Huh7 cells, and then treated with 10m or 10r. Luciferase activity in cell extracts was measured after 72 h of incubation.
5

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
Fig. 8. (A) Schematic diagram of compound pre-treatment following with HCVpp infection. (B) 10m and 10r inhibited HCVpp entry. (C) The effect of pre-treatment with 10m or 10r
during wildtype HCV infection.
Compounds 10m and 10r differ from other entry inhibitors re-
ported in the literature in terms of structural type and from other
marketed drugs in their mode of action (antiviral drugs currently
on the market or being evaluated in clinical trials are mainly
focused on targeting HCV nonstructural protein maturation or viral
RNA synthesis). Therefore, compounds 10m and 10r represent a
new class of anti-HCV entry inhibitors, and the results herein show
that they can interfere with the internalization of HCV from the cell
surface into the cell. Notably, the development of natural products
into inhibitors can reduce costs, which increases the prospects for
improving the approach to treatment, especially for countries or
patients with limited resources [39]. In addition, entry inhibitors
may have a synergistic effect with DAA, which may allow the use of
small-dose combination regimens to reduce the cost and potential
side effects of DAA [40,41]. More importantly, some reports sug-
gested that DAA-resistance mutations in the HCV replication region
were detected in HCV patients [42]. Current treatment options are
limited by drug resistance, therefore the development of entry in-
hibitors plays an important role in HCV treatment. In summary, as a
supplement to current treatment methods, HCV entry inhibitors
represent a promising new class of antiviral drugs that can target
the early steps of the viral life cycle.
of viruses attaching to the cell surface. We employed the HCVpp
model to confirm that whether HCV entry can be inhibited by these
compounds. As expected, our data showed that compounds 10m
and 10r inhibited virus entry by 34.0% and 52.0%, respectively, at
noncytotoxic concentrations. These results indicated that 10m and
10r may be potential HCV entry inhibitors that exhibit effective
antiviral activity and prevent HCV infections. In the future, they can
be used in combination with therapeutic drugs at different stages
or with different targets in the HCV infection cycle to establish new
therapeutic methods.
4. Experimental protocols
4.1. Chemistry
4.1.1. General
Solvents and reagents were purchased from commercial sources
and used without further purification. 1H NMR and 13C NMR spectra
were measured on a Bruker ARX-400 or Bruker AVIII-600 spec-
trometer in DMSO‑d6 or CDCl3. Mass spectra data were recorded
with a Bruker Micro-TOF-Q instrument. The thin-layer chroma-
tography (TLC) analysis was conducted on HSGF254 precoated sil-
ica gel plates (Yantai Zifu Chemical Group Co., China). Silica gel
(Qingdao city, China) was used for column chromatography. The
purity (%) of compounds was evaluated by high-performance liquid
chromatography (HPLC, Agilent 1260 pump system) equipped with
UV detector (Agilent 1260 detector) using Shodex ODS-A column
3. Conclusion
By using a visualization anti-HCV compounds screening system,
the potential anti-HCV effects of compounds 10m, 10o and 10r
were revealed. Furthermore, these compounds inhibited viral
protein expression and de novo virus production in a dose-
dependent manner. However, treatment with 10m or 10r did not
significantly decrease the HCV subgenomic replicon system. It is
suggested that these compounds did not contribute to the inhibi-
tion of HCV replication. Therefore, we speculated that by regulating
the intracellular environment, the compounds inhibited the entry
(250 mm ꢀ 4.6 mm, 5
mm).
4.1.2. 1-(2-hydroxy-4-(methoxymethox)phenyl) ethan-1-one (2)
To a solution of 2,4-dihydroxyacetophenone (15.0 g, 0.10 mol) in
dry acetone (60 mL) was added anhydrous K2CO3 (10.0 g, 0.07 mol)
and stirred under reflux for 15 min. Methoxymethyl (MOM) chlo-
ride (7.6 mL, 0.105 mol) was added, and stirring at room
6

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
temperature continued for 15 min. Then, the mixture was refluxed
for 2 h. The reaction was quenched with H2O (60 mL) and extracted
with CH2Cl2 (3 ꢀ 20 mL). The combined organic layers were washed
with brine (60 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo. The crude product obtained was column chromato-
graphed on silica gel eluting with petroleum ether/ethyl acetate
(35:1) to afford compound 2 (17.0 g, 92.1% yield, obtained as white
4.1.6. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-(methoxymethoxy)
chroman-4-one (7)
Compound 6 (13.4 g, 0.04 mol) and sodium acetate (1.5 g,
0.02 mol) were added to 30 mL EtOH. The solution was heated to
70 ꢁC for 12 h. After cooling to room temperature, the mixture was
filtered. The solid was washed with 25 mL EtOAc, and the combined
organic phases were concentrated under vacuum. The residue was
chromatographed on silica gel with petroleum ether/ethyl acetate
(40:1) to afford 7 (10.7 g, 80.1% yield, obtained as yellowish oil). 1H
oil). 1H NMR (600 MHz, CDCl3,
d): 12.58 (1H, s), 7.60 (1H, d,
J ¼ 8.8 Hz), 6.54 (1H, d, J ¼ 2.4 Hz), 6.50 (1H, dd, J ¼ 8.8, 2.4 Hz), 5.16
(2H, s), 3.43 (3H, s), 2.51 (3H, s). The 1H NMR data are consent with
reference [29].
NMR (600 MHz, CDCl3,
d
): 7.87 (1H, d, J ¼ 8.7 Hz), 7.19 (1H, dd,
J ¼ 8.3, 2.3 Hz), 7.09 (1H, d, J ¼ 2.2 Hz), 6.81 (1H, d, J ¼ 8.2 Hz), 6.70
(1H, dd, J ¼ 8.7, 2.3 Hz), 6.68 (1H, d, J ¼ 2.3 Hz), 6.33 (1H, d,
J ¼ 9.8 Hz), 5.65 (1H, d, J ¼ 9.8 Hz), 5.35 (1H, dd, J ¼ 13.5, 2.8 Hz),
5.20 (1H, s), 3.47 (3H, s), 3.04 (1H, dd, J ¼ 16.8,13.5 Hz), 2.79 (1H, dd,
J ¼ 16.8, 2.8 Hz), 1.44 (6H, s).
4.1.3. 4-Hydroxy-3-(3-methylbut-2-enyl) benzaldehyde (4)
4-hydroxybenzaldehyde (100.0 g, 0.82 mol) was dissolved in
150 mL aqueous potassium hydroxide (180.0 g, 3.21 mol) solution
at ice water bath. To the solution was added 3,3-dimethylallyl
bromide (170.0 g, 1.14 mol) and 150 mL aqueous potassium hy-
droxide (180.0 g, 3.21 mol) in parallel over 2 h with vigorous stir-
ring. After the complete addition, the temperature was lowered to
25 ꢁC, and the reaction was continued for 24 h in the dark. The
reaction mixture was adjusted to pH 2 by addition of 3 N aqueous
HCl. The reaction was extracted with EtOAc (3 ꢀ 40 mL). The
combined organic layers were washed with 100 mL brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue was
chromatographed on silica gel with petroleum ether/ethyl acetate
(4:1) to afford 4 (31.2 g, 20.5% yield, obtained as red-brown oil). 1H
NMR (600 MHz, CDCl3, d): 9.84 (1H, s, CHO), 7.67 (2H, overlap), 6.93
(1H, d, J ¼ 8.1 Hz), 5.33 (1H, t, J ¼ 7.3 Hz), 3.42 (2H, d, J ¼ 7.3 Hz),1.79
(3H, s), 1.78 (3H, s).
4.1.7. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-hydroxychroman-4-
one (8)
To a solution of 7 (10.7 g, 0.03 mol) in MeOH (100 mL), 3 N
aqueous HCl (8 mL) was added and the reaction mixture was stirred
at 50 ꢁC. After 12 h stirring, evaporated solvent under reduced
pressure and quenched reaction mixture in 100 mL water. Extrac-
ted the mixture with EtOAc (3 ꢀ 40 mL) and organic layer was dried
over anhydrous Na2SO4 and evaporated solvent to get the product.
The crude product obtained was subjected to silica gel column
chromatography and on elution with 10% ethyl acetate in hexane
afforded the pure product (8.5 g, 90.5% yield, obtained as yellowish
oil). 1H NMR (600 MHz, CDCl3, d): 7.82 (1H, d, J ¼ 8.6 Hz), 7.18 (1H,
dd, J ¼ 8.3, 2.3 Hz), 7.07 (1H, d, J ¼ 2.2 Hz), 6.80 (1H, d, J ¼ 8.2 Hz),
6.53 (1H, dd, J ¼ 8.6, 2.3 Hz), 6.42 (1H, d, J ¼ 2.3 Hz), 6.33 (1H, d,
J ¼ 9.8 Hz), 5.65 (1H, d, J ¼ 9.8 Hz), 5.33 (1H, dd, J ¼ 13.4, 3.0 Hz),
3.03 (1H, dd, J ¼ 16.8, 13.4 Hz), 2.77 (1H, dd, J ¼ 16.8, 2.9 Hz), 1.44
(6H, s).
4.1.4. 2,2-dimethyl-2H-chromene-6-carbaldehyde (5)
Compound 4 (31.2 g, 0.16 mol) and DDQ (37.2 g, 0.16 mol) were
dissolved in toluene heated at reflux for 12 h. After reaction was
completed, reaction mixture was cooled to room temperature. The
precipitate was filtered and washed thoroughly with toluene. The
aqueous layer was extracted with EtOAc (3 ꢀ 40 mL) and the
combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue was
purified on silica gel with petroleum ether/ethyl acetate (15:1) to
afford 5 (13.1 g, 42.2% yield, obtained as red-brown oil). 1H NMR
(600 MHz, CDCl3, d): 9.80 (1H, s, CHO), 7.61 (1H, dd, J ¼ 8.3, 2.1 Hz),
7.49 (1H, d, J ¼ 2.1 Hz), 6.83 (1H, d, J ¼ 8.3 Hz), 6.34 (1H, d,
J ¼ 9.9 Hz), 5.67 (1H, d, J ¼ 9.9 Hz), 1.44 (6H, s).
4.1.8. General procedure for the synthesis of intermediate 9
To a solution of 8 (8.5 g, 0.03 mol) in dry acetone (30 mL) were
added anhydrous K2CO3 (8.3 g, 0.06 mol) and bromopropyne (5.4 g,
0.045 mol). The solution was refluxed until TLC showed complete
disappearance of the starting material (12 h). After cooling, acetone
was removed under reduced pressure. The mixture was then
extracted with 30 mL dichloromethane. The organic layer was
washed with 30 mL H2O and 30 mL brine, dried over Na2SO4, and
concentrated in vacuo. The crude product obtained was subjected to
silica gel column chromatography and on elution with petroleum
ether/ethyl acetate (6:1) afforded pure product (8.2 g, 86.3% yield,
obtained as yellowish oil). 1H NMR (600 MHz, CDCl3, d): 7.88 (1H, d,
J ¼ 8.8 Hz), 7.20 (1H, dd, J ¼ 8.3, 2.2 Hz), 7.09 (1H, d, J ¼ 2.2 Hz), 6.81
(1H, d, J ¼ 8.3 Hz), 6.67 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.58 (1H, d,
J ¼ 2.4 Hz), 6.34 (1H, d, J ¼ 9.8 Hz), 5.65 (1H, d, J ¼ 9.8 Hz), 5.36 (1H,
dd, J ¼ 13.5, 2.8 Hz), 4.71 (2H, d, J ¼ 2.4 Hz), 3.05 (1H, dd, J ¼ 16.8,
13.5 Hz), 2.79 (1H, dd, J ¼ 16.8, 2.8 Hz), 2.56 (1H, t, J ¼ 2.4 Hz), 1.45
(6H, s).
4.1.5. (E)-3-(2,2-dimethyl-2H-chromen-6-yl)-1-(2-hydroxy-4-
(methoxymethoxy)phenyl)prop-2-en-1-one (6)
Compound 5 (13.1 g, 0.07 mol) was dissolved with compound 2
(13.6 g, 0.07 mol) in 50 mL ethanol. Under nitrogen and ice water
bath, KOH (7.8 g, 0.14 mol) - H2O (53 mL) - EtOH (80 mL) solution
was slowly added dropwise over 1.5 h. Then it returned to room
temperature and the reaction was continued for 24 h. Upon
completion of reaction as monitored by TLC, the reaction mixture
was washed with ethyl acetate three times (3 ꢀ 40 mL). The organic
extract was washed with 50 mL water followed by 50 mL brine and
dried over Na2SO4 and concentrated under vacuum. The residue
was chromatographed on silica gel with petroleum ether/ethyl
acetate (25:1) to afford 6 (13.4 g, 50.0% yield, obtained as yellowish
4.1.9. General procedure for the synthesis of conjugates 10a-10s
To a solution of compound 9 (100.8 mg, 0.28 mmol) and the
appropriate azide (0.72 mmol) in n-butanol (10 mL) and H2O
(10 mL) was added CuSO4 _5H2O (180.0 mg, 0.72 mmol) and sodium
L
-ascorbate (280.0 mg, 1.43 mmol). The reaction was stirred for 24 h
at room temperature, until TLC analysis revealed a total con-
sumption of compound 9. The mixture was diluted with water
(20 mL), extracted three times with CH2Cl2 (15 mL). The combined
organic extracts were washed with 20 mL brine, dried over Na2SO4,
filtered, concentrated, and purified via silica gel chromatography
(petroleum ether/ethyl acetate, 8:1) to yield the target compounds
10a-10s. The structures of the compounds were defined via HRMS
oil). 1H NMR (600 MHz, CDCl3,
d): 13.40 (1H, s, HO), 7.83 (2H,
overlap), 7.43 (2H, overlap), 7.28 (1H, d, J ¼ 2.1 Hz), 6.81 (1H, d,
J ¼ 8.3 Hz), 6.64 (1H, d, J ¼ 2.4 Hz), 6.58 (1H, dd, J ¼ 8.9, 2.4 Hz), 6.36
(1H, d, J ¼ 9.8 Hz), 5.68 (1H, d, J ¼ 9.8 Hz), 5.22(2H, s), 3.49 (3H, s),
1.46 (6H, s).
7

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
and NMR data.
13C NMR (150 MHz, DMSO‑d6,
d): 190.7, 164.8, 163.5, 153.1, 142.3,
131.9, 131.6, 128.5, 128.2, 125.5, 125.1, 122.0, 121.3, 116.2, 115.1, 110.8,
102.3, 79.5, 76.8, 49.9, 43.5, 31.0, 30.1, 28.4, 28.2, 25.9, 22.4, 14.3.
ESI-HRMS: m/z 488.2547 [MþH]þ (Calcd for C29H34N3O4,
488.2544). HPLC purity: 98.04%.
4.1.9.1. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-methyl-1H-1,2,3-
triazol-4-yl)methoxy)chroman-4-one (10a). Yield 70.8% as a yellow
oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.19 (1H, s), 7.72 (1H, d,
J ¼ 8.8 Hz), 7.27 (1H, dd, J ¼ 8.1, 2.3 Hz), 7.26 (1H, d, J ¼ 2.3 Hz), 6.78
(1H, d, J ¼ 8.1 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H, dd, J ¼ 8.8,
2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.51 (1H, dd,
J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 4.05 (3H, s), 3.18 (1H, dd, J ¼ 16.8,
13.2 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.9 Hz), 1.38 (6H, s). 13C NMR
(150 MHz, DMSO‑d6, d): 190.7, 164.8, 163.6, 153.1, 142.5, 132.0, 131.6,
128.5, 128.3, 126.1, 125.5, 122.0, 121.3, 116.2, 115.1, 110.9, 102.3, 79.5,
76.8, 62.0, 43.5, 36.8, 28.2 ꢀ 2. ESI-HRMS: m/z 418.1765 [MþH]þ
(Calcd for C24H24N3O4, 418.1761). HPLC purity: 95.76%.
4.1.9.6. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-nonyl-1H-1,2,3-
triazol-4-yl)methoxy)chroman-4-one (10f). Yield 67.9% as a yellow
oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.24 (1H, s), 7.72 (1H, d,
J ¼ 8.8 Hz), 7.27 (1H, dd, J ¼ 8.2, 2.3 Hz), 7.25 (1H, d, J ¼ 2.3 Hz), 6.78
(1H, d, J ¼ 8.2 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H, dd, J ¼ 8.8,
2.4 Hz), 6.42 (1H, d, J ¼ 9.8 Hz), 5.77 (1H, d, J ¼ 9.8 Hz), 5.50 (1H, dd,
J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 4.35 (2H, t, J ¼ 7.3 Hz), 3.16 (1H, dd,
J ¼ 16.8, 13.1 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.79 (2H, m), 1.38
(6H, s), 1.27e1.17 (12H, overlap), 0.83 (3H, t, J ¼ 7.3 Hz). 13C NMR
4.1.9.2. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-ethyl-1H-1,2,3-
(150 MHz, DMSO‑d6, d): 190.6, 164.8, 163.5, 153.1, 142.3, 131.9, 131.6,
triazol-4-yl)methoxy)chroman-4-one (10b). Yield 55.8% as a yellow
128.5, 128.2, 125.4, 125.1, 122.0, 121.3, 116.2, 115.1, 110.8, 102.3, 79.5,
76.8, 62.1, 49.9, 43.5, 31.7, 30.2, 29.3, 29.1, 28.9, 28.2 ꢀ 2, 26.3, 22.6,
14.4. ESI-HRMS: m/z 530.3019 [MþH]þ (Calcd for C32H40N3O4, 530.
3013). HPLC purity: 96.04%.
oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.27 (1H, s), 7.72 (1H, d,
J ¼ 8.8 Hz), 7.27 (1H, dd, J ¼ 8.2, 2.3 Hz), 7.26 (1H, d, J ¼ 2.3 Hz),
6.80e6.70 (2H, overlap), 6.72 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.42 (1H, d,
J ¼ 9.8 Hz), 5.78 (1H, d, J ¼ 9.8 Hz), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz),
5.21 (2H, s), 4.39 (2H, q, J ¼ 7.4 Hz), 3.18 (1H, dd, J ¼ 16.8, 13.1 Hz),
4.1.9.7. 7-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)-2-(2,2-
2.71 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.43 (3H, t, J ¼ 7.3 Hz), 1.38 (6H, s). 13
C
dimethyl-2H-chromen-6-yl)chroman-4-one (10g). Yield 80.3% as a
NMR (150 MHz, DMSO‑d6, d): 190.7, 164.8, 163.6, 153.1, 142.4, 131.9,
131.6, 128.5, 128.2, 125.5, 124.7, 122.0, 121.3, 116.2, 115.1, 110.8, 102.3,
79.5, 76.8, 62.1, 45.1, 43.5, 28.2 ꢀ 2, 15.8. ESI-HRMS: m/z 432.1920
[MþH]þ (Calcd for C25H26N3O4, 432.1918). HPLC purity: 99.63%.
yellow oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.31 (1H, s), 7.71 (1H, d,
J ¼ 8.8 Hz), 7.40e7.30 (5H, AreH, overlap), 7.27 (2H, overlap), 6.79
(1H, d, J ¼ 8.2 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H, dd, J ¼ 8.8,
2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.61 (2H, s),
5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 3.18 (1H, dd, J ¼ 16.8,
13.1 Hz), 2.69 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.38(6H, s). 13C NMR
4.1.9.3. 7-((1-butyl-1H-1,2,3-triazol-4-yl)methoxy)-2-(2,2-dimethyl-
2H-chromen-6-yl)chroman-4-one (10c). Yield 73.7% as a yellow oil.
(150 MHz, DMSO‑d6, d): 190.7, 164.8, 163.6, 153.1, 142.7, 136.4, 132.0,
1H NMR (400 MHz, CDCl3,
d
): 7.99 (1H, s), 7.85 (1H, d, J ¼ 8.8 Hz),
131.6, 129.2 ꢀ 2, 128.7, 128.5, 128.4 ꢀ 2, 128.3, 125.5, 125.4, 122.0,
121.3, 116.2, 115.1, 110.9, 102.3, 79.5, 76.8, 62.0, 53.3, 43.5, 28.2 ꢀ 2.
ESI-HRMS: m/z 494. 2104 [MþH]þ (Calcd for C30H28N3O4,
494.2094). HPLC purity: 90.76%.
7.17 (1H, dd, J ¼ 8.3, 2.3 Hz), 7.07 (1H, d, J ¼ 2.3 Hz), 6.79 (1H, d,
J ¼ 8.3 Hz), 6.66 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.58 (1H, d, J ¼ 2.4 Hz), 6.32
(1H, d, J ¼ 9.8 Hz), 5.64 (1H, d, J ¼ 9.8 Hz), 5.33 (1H, dd, J ¼ 13.1,
2.8 Hz), 5.20 (2H, s), 4.35 (2H, t, J ¼ 7.3 Hz), 3.02 (1H, dd, J ¼ 16.8,
13.1 Hz), 2.77 (1H, dd, J ¼ 16.8, 2.8 Hz),1.88 (2H, m),1.43 (6H, s),1.35
(2H, m), 0.94 (3H, t, J ¼ 7.4 Hz). 13C NMR (100 MHz, CDCl3, d): 190.8,
164.7,163.5,153.4,143.1,131.4, 130.8,128.8,127.2,124.4,122.7,121.9,
121.4, 116.5, 115.2, 110.5, 101.9, 79.8, 76.6, 62.3, 50.2, 44.0, 32.2,
28.1 ꢀ 2, 19.7, 13.4. ESI-HRMS: m/z 460.2227 [MþH]þ (Calcd for
4.1. 9. 8. 2-(2, 2-dimethyl-2H-chromen-6-yl)-7-((1-(3-
methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one
(10h). Yield 70.2% as a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d):
8.31 (1H, s), 7.71 (1H, d, J ¼ 8.8 Hz), 7.30e7.25 (3H, overlap),
6.92e6.85 (3H, overlap), 6.80e6.75 (2H, overlap), 6.71 (1H, dd,
J ¼ 8.8, 2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.57
(2H,s), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 3.72 (3H, s), 3.18
(1H, dd, J ¼ 16.8, 13.1 Hz), 2.69 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.38 (6H, s).
C
27H30N3O4, 460. 2231). HPLC purity: 90.82%.
4.1.9.4. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-pentyl-1H-1,2,3-
triazol-4-yl)methoxy)chroman-4-one (10d). Yield 63.5% as a yellow
13C NMR (100 MHz, DMSO‑d6,
d): 190.7, 164.8, 163.5, 159.9, 153.1,
oil. 1H NMR (600 MHz, DMSO‑d6,
d
): 8.25 (1H, s), 7.73 (1H, d,
142.7, 137.8, 132.0, 131.6, 130.4, 128.5, 128.3, 125.5, 125.4, 122.0,
121.3, 120.5, 116.2, 115.1, 114.2, 114.0, 110.9, 102.3, 79.5, 76.8, 62.0,
55.6, 53.2, 43.5, 28.2 ꢀ 2. ESI-HRMS: m/z 524.2194 [MþH]þ (Calcd
for C31H30N3O5, 524.2180). HPLC purity: 98.73%.
J ¼ 8.8 Hz), 7.29e7.25 (2H, overlap), 6.80e6.76 (2H, overlap), 6.72
(1H, dd, J ¼ 8.8, 2.4 Hz), 6.42 (1H, d, J ¼ 9.8 Hz), 5.77 (1H, d,
J ¼ 9.8 Hz), 5.50 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.22 (2H, s), 4.35 (2H, t,
J ¼ 7.3 Hz), 3.16 (1H, dd, J ¼ 16.8, 13.1 Hz), 2.71 (1H, dd, J ¼ 16.8,
2.8 Hz),1.81 (2H, m), 1.38 (6H, s),1.29 (2H, m),1.20 (2H, m), 0.84 (3H,
4.1.9.9. 7-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)
t, J ¼ 7.3 Hz). 13C NMR (150 MHz, DMSO‑d6,
d
): 190.6, 164.8, 163.5,
methoxy)-2-(2,2-dimethyl-2H-chromen-6-yl)chroman-4-one (10i).
153.1, 142.3,131.9, 131.6, 128.5, 128.2, 125.4, 125.1, 122.0, 121.3, 116.2,
115.1, 110.8, 102.3, 79.5, 76.8, 62.1, 49.9, 43.5, 29.9, 28.5, 28.2 ꢀ 2,
22.0, 14.2. ESI-HRMS: m/z 474.2390 [MþH]þ (Calcd for C28H32N3O4,
474.2387). HPLC purity: 98.72%.
Yield 76.5% as a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d): 8.31
(1H, s), 7.71 (1H, d, J ¼ 8.8 Hz), 7.26 (2H, overlap), 6.77 (2H, overlap),
6.71 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.46 (3H, overlap), 6.43 (1H, d,
J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.52 (2H, s), 5.50 (1H, dd, J ¼ 13.1,
2.8 Hz), 5.21 (2H, s), 3.70 (6H, s), 3.18 (1H, dd, J ¼ 16.8, 13.1 Hz), 2.69
(1H, dd, J ¼ 16.8, 2.8 Hz), 1.38 (6H, s). 13C NMR (100 MHz, DMSO‑d6,
4.1.9.5. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-hexyl-1H-1,2,3-
triazol-4-yl)methoxy)chroman-4-one (10e). Yield 57.6% as a yellow
d
): 190.7, 164.7, 163.5, 161.1 ꢀ 2, 153.1, 142.7, 138.5, 132.0, 131.6,
oil. 1H NMR (600 MHz, DMSO‑d6,
d
): 8.26 (1H, s), 7.72 (1H, d,
128.5, 128.3, 125.5, 125.4, 122.0, 121.3, 116.2, 115.1, 110.9, 106.6 ꢀ 2,
102.3, 100.1, 79.5, 76.8, 62.0, 55.7 ꢀ 2, 53.3, 43.5, 28.2 ꢀ 2. ESI-
HRMS: m/z 554. 2285 [MþH]þ (Calcd for C32H32N3O6, 554.2286).
HPLC purity: 95.38%.
J ¼ 8.8 Hz), 7.27 (1H, dd, J ¼ 8.2, 2.3 Hz), 7.25 (1H, d, J ¼ 2.3 Hz), 6.78
(1H, d, J ¼ 8.2 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H, dd, J ¼ 8.8,
2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.78 (1H, d, J ¼ 9.8 Hz), 5.51 (1H, dd,
J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 4.36 (2H, t, J ¼ 7.3 Hz), 3.17 (1H, dd,
J ¼ 16.8, 13.1 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.80 (2H, m), 1.41
(3H, s), 1.38 (3H, s), 1.25e1.15 (6H, overlap), 0.82 (3H, t, J ¼ 7.3 Hz).
4.1.9.10. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(pyridin-4-
ylmethyl)-1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one
(10j).
8

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
Yield 69.3% as a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d
): 8.56
102.3, 79.5, 76.8, 62.0, 49.1, 43.5, 28.2 ꢀ 2. ESI-HRMS: m/z 562.1297
[MþH]þ (Calcd for C30H26Cl2N3O4, 562.1295). HPLC purity: 91.25%.
(2H, overlap), 8.38 (1H, s), 7.72 (1H, d, J ¼ 8.8 Hz), 7.27 (1H, dd,
J ¼ 8.1, 2.3 Hz), 7.26 (1H, d, J ¼ 2.3 Hz), 7.20 (2H, overlap), 6.78 (2H,
overlap), 6.72 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.79
(1H, d, J ¼ 9.8 Hz), 5.71 (2H, s), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.24
(2H, s), 3.19 (1H, dd, J ¼ 16.8, 13.1 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.8 Hz),
4.1.9.15. 7-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-
(2,2-dimethyl-2H-chromen-6-yl)chroman-4-one (10o). Yield 57.6%
as a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d): 8.32 (1H, s), 7.71
1.38 (6H, s). 13C NMR (100 MHz, DMSO‑d6,
d
): 190.7, 164.7, 163.6,
(1H, d, J ¼ 8.8 Hz), 7.58 (2H, overlap), 7.28 (3H, overlap), 7.26 (1H, d,
J ¼ 2.2 Hz), 6.78 (1H, d, J ¼ 8.1 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H,
dd, J ¼ 8.8, 2.4 Hz), 6.42 (1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz),
5.60 (2H, s), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 3.18 (1H, dd,
J ¼ 16.8,13.1 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.38 (6H, s). 13C NMR
153.1, 150.5 ꢀ 2, 145.2, 142.9, 132.0, 131.6, 128.5, 128.3, 126.0, 125.5,
122.8 ꢀ 2,122.0,121.3,116.2,115.1,110.9,102.3, 79.5, 76.8, 62.0, 52.0,
43.5, 28.2
ꢀ
2. ESI-MS: m/z 495.3 [MþNa]þ (Calcd for
C
29H26N4O4Na, 495.2). HPLC purity: 99.41%.
(100 MHz, DMSO‑d6,
d): 190.7, 164.7, 163.5, 153.1, 142.8, 135.8,
4.1.9.11. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(4-nitrobenzyl)-
132.2 ꢀ 2,132.0,131.6,130.7 ꢀ 2,128.5,128.3,125.5 ꢀ 2,122.0,121.9,
121.3, 116.2, 115.1, 110.9, 102.3, 79.5, 76.8, 62.0, 52.6, 43.5, 28.2 ꢀ 2.
ESI-HRMS: m/z 574.1164 [MþH]þ (Calcd for C30H27BrN3O4,
574.1161). HPLC purity: 99.28%.
1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one (10k). Yield 59.1% as
a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d): 8.39 (1H, s), 8.24 (2H,
overlap), 7.72 (1H, d, J ¼ 8.8 Hz), 7.54 (2H, overlap), 7.27 (1H, dd,
J ¼ 8.1, 2.3 Hz), 7.25 (1H, d, J ¼ 2.3 Hz), 6.77 (2H, overlap), 6.72 (1H,
dd, J ¼ 8.8, 2.4 Hz), 6.42 (1H, d, J ¼ 9.8 Hz), 5.81 (2H, s), 5.79 (1H, d,
J ¼ 9.8 Hz), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.23 (2H, s), 3.19 (1H, dd,
J ¼ 16.8, 13.1 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.8 Hz),1.38 (6H, s). 13C NMR
4.1.9.16. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(2-fluorobenzyl)-
1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one (10p). Yield 57.3% as
a yellow oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.30 (1H, s), 7.72 (1H,
(100 MHz, DMSO‑d6,
d
): 190.7, 164.7, 163.6, 153.1, 147.7, 143.8, 142.9,
d, J ¼ 8.8 Hz), 7.41 (1H, m), 7.36 (1H, m), 7.28e7.20 (4H, overlap),
6.79 (1H, d, J ¼ 8.1 Hz), 6.77 (1H, d, J ¼ 2.4 Hz), 6.72 (1H, dd, J ¼ 8.8,
2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.78 (1H, d, J ¼ 9.8 Hz), 5.69 (2H, s),
5.51 (1H, dd, J ¼ 13.2, 2.8 Hz), 5.22 (2H, s), 3.18 (1H, dd, J ¼ 16.8,
13.2 Hz), 2.70 (1H, dd, J ¼ 16.8, 2.8 Hz), 1.38 (6H, s). 13C NMR
132.0, 131.6, 129.6 ꢀ 2, 128.5, 128.3, 125.9, 125.5, 124.4 ꢀ 2, 122.0,
121.3, 116.2, 115.1, 110.9, 102.3, 79.5, 76.8, 62.0, 52.4, 43.5, 28.2 ꢀ 2.
ESI-HRMS: m/z 539.1968 [MþH]þ (Calcd for C30H27N4O6, 539.1965).
HPLC purity: 99.45%.
(150 MHz, DMSO‑d6, d): 190.7, 164.8, 163.5, 160.6, 153.1, 142.7, 131.9,
4.1.9.12. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(4-methylbenzyl)-
131.6, 131.3, 131.2, 128.5, 128.2, 125.6, 125.5, 125.3, 123.2, 122.0,
121.3, 116.2, 116.0, 115.1, 110.8, 102.3, 79.5, 76.8, 62.0, 47.4, 43.5,
28.2 ꢀ 2. ESI-HRMS: m/z 512.1990 [MþH]þ (Calcd for C30H27FN3O4,
512.1980). HPLC purity: 99.39%.
1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one (10l). Yield 58.8% as
a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d): 8.27 (1H, s), 7.71 (1H,
d, J ¼ 8.7 Hz), 7.20 (2H, overlap 7.04 (2H, overlap), 6.84e6.80 (2H,
overlap), 6.78 (1H, d, J ¼ 8.2 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H,
dd, J ¼ 8.7, 2.4 Hz), 6.42 (1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz),
5.75 (2H, s), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.20 (2H, s), 3.16 (1H, dd,
J ¼ 16.8, 13.1 Hz), 2.69 (1H, dd, J ¼ 16.8, 2.8 Hz), 2.27 (3H, s), 1.38
4.1.9.17. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(3-fluorobenzyl)-
1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one (10q). Yield 62.3% as
a yellow oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.35 (1H, s), 7.72 (1H,
(6H, s). 13C NMR (100 MHz, DMSO‑d6,
d
): 190.7, 164.8, 163.5, 153.1,
d, J ¼ 8.8 Hz), 7.42 (1H, m), 7.27 (2H, m), 7.17 (3H, m), 6.78 (1H, d,
J ¼ 8.1 Hz), 6.77 (1H, d, J ¼ 2.4 Hz), 6.72 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.43
(1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.65 (2H, s), 5.51 (1H, dd,
J ¼ 13.1, 2.8 Hz), 5.23 (2H, s), 3.19 (1H, dd, J ¼ 16.8,13.1 Hz), 2.70 (1H,
142.7, 138.0, 133.4, 132.0, 131.6, 129.8, 129.7 ꢀ 2, 128.5 ꢀ 2, 128.2,
125.5, 125.3, 122.0, 121.3, 116.2, 115.1, 110.8, 102.3, 79.5, 76.8, 62.0,
53.1, 43.5, 28.2 ꢀ 2, 21.2. ESI-HRMS: m/z 508.2193 [MþH]þ (Calcd
for C31H30N3O4, 508.2201). HPLC purity: 98.75%.
dd, J ¼ 16.8, 2.8 Hz), 1.38 (6H, s). 13C NMR (150 MHz, DMSO‑d6,
d):
190.7, 164.7, 163.5, 162.8, 153.1, 142.1, 139.1, 132.0, 131.6, 131.3, 128.5,
128.3, 125.6, 125.5, 124.5, 122.0, 121.3, 116.2, 115.5, 115.3, 115.1, 110.8,
102.3, 79.5, 76.8, 62.0, 52.6, 43.5, 28.2 ꢀ 2. ESI-HRMS: m/z 534.1799
[MþNa]þ (Calcd for C30H26FN3O4Na, 534.1800). HPLC purity:
95.89%.
4.1.9.13. 7-((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-
(2,2-dimethyl-2H-chromen-6-yl)chroman-4-one (10m). Yield 57.2%
as a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d): 8.29 (1H, s), 7.71
(1H, d, J ¼ 8.7 Hz), 7.52 (1H, m), 7.38 (2H,overlap), 7.26 (3H, over-
lap), 6.78 (1H, d, J ¼ 8.2 Hz), 6.77 (1H, d, J ¼ 2.4 Hz), 6.71 (1H, dd,
J ¼ 8.7, 2.4 Hz), 6.43 (1H, d, J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.72
(2H, s), 5.51 (1H, dd, J ¼ 13.1, 2.8 Hz), 5.22 (2H, s), 3.19 (1H, dd,
J ¼ 16.8,13.1 Hz), 2.69 (1H, dd, J ¼ 16.8, 2.8 Hz),1.38 (6H, s). 13C NMR
4.1.9.18. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(4-fluorobenzyl)-
1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one (10r). Yield 63.3% as
a yellow oil. 1H NMR (600 MHz, DMSO‑d6,
d): 8.31 (1H, s), 7.71 (1H,
(100 MHz, DMSO‑d6,
d
): 190.7, 164.7, 163.6, 153.1, 142.5, 133.6, 133.1,
d, J ¼ 8.8 Hz), 7.45e7.37 (2H, overlap), 7.27 (1H, dd, J ¼ 8.1, 2.3 Hz),
7.25 (1H, d, J ¼ 2.3 Hz), 7.23e7.19 (2H, overlap), 6.78 (1H, d,
J ¼ 8.1 Hz), 6.76 (1H, d, J ¼ 2.4 Hz), 6.71 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.43
(1H, d, J ¼ 9.8 Hz), 5.78 (1H, d, J ¼ 9.8 Hz), 5.61 (2H, s), 5.51 (1H, dd,
J ¼ 13.1, 2.8 Hz), 5.21 (2H, s), 3.18 (1H, dd, J ¼ 16.8,13.1 Hz), 2.70 (1H,
132.0, 131.6, 131.0, 130.8, 130.1, 128.5, 128.3, 128.2, 125.9, 125.5,
122.0, 121.3, 116.2, 115.1, 110.9, 102.3, 79.5, 76.8, 62.0, 51.1, 43.5,
28.2 ꢀ 2. ESI-HRMS: m/z 528.1649 [MþH]þ (Calcd for C30H27ClN3O4,
528.1675). HPLC purity: 96.29%.
dd, J ¼ 16.8, 2.8 Hz), 1.38 (6H, s). 13C NMR (150 MHz, DMSO‑d6,
d):
4.1.9.14. 7-((1-(3,5-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-
190.7,164.8,163.6,162.4,153.1,142.7,132.7,132.0,131.6,130.9,130.8,
128.5, 128.3, 125.5, 125.4, 122.0, 121.3, 116.2 ꢀ 2, 116.0, 115.1, 110.8,
102.3, 79.5, 76.8, 62.0, 52.5, 43.5, 28.2 ꢀ 2. ESI-HRMS: m/z 512.1962
[MþH]þ (Calcd for C30H27FN3O4, 512.1980). HPLC purity: 99.28%.
2-(2,2-dimethyl-2H-chromen-6-yl)chroman-4-one
(10n). Yield
): 8.27 (1H, s),
58.6% as a yellow oil. 1H NMR (400 MHz, DMSO‑d6,
d
7.70 (1H, d, J ¼ 8.8 Hz), 7.57 (2H, overlap), 7.47 (1H,m), 7.27 (1H, dd,
J ¼ 8.2, 2.2 Hz), 7.25 (1H, d, J ¼ 2.2 Hz), 6.77 (1H, d, J ¼ 8.2 Hz), 6.75
(1H, d, J ¼ 2.4 Hz), 6.70 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.42 (1H, d,
J ¼ 9.8 Hz), 5.79 (2H, s), 5.78 (1H, d, J ¼ 9.8 Hz), 5.50 (1H, dd, J ¼ 13.1,
2.8 Hz), 5.19 (2H, s), 3.16 (1H, dd, J ¼ 16.8, 13.1 Hz), 2.69 (1H, dd,
4.1.9.19. 2-(2,2-dimethyl-2H-chromen-6-yl)-7-((1-(4-(tri-
fluoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)chroman-4-one
(10s). Yield 73.2% as a yellow oil. 1H NMR (600 MHz, DMSO‑d6,
d):
J ¼ 16.8, 2.8 Hz),1.38 (6H, s). 13C NMR (100 MHz, DMSO‑d6,
d
): 190.7,
8.37 (1H, s), 7.76 (2H,overlap), 7.72 (1H, d, J ¼ 8.8 Hz), 7.51
(2H,overlap), 7.27 (1H, dd, J ¼ 8.2, 2.3 Hz), 7.25 (1H, d, J ¼ 2.3 Hz),
6.77 (2H, overlap), 6.72 (1H, dd, J ¼ 8.8, 2.4 Hz), 6.43 (1H, d,
164.7, 163.5, 153.1, 142.2, 136.4, 132.2 ꢀ 2, 132.0, 131.6, 130.8,
129.5 ꢀ 2, 128.5, 128.3, 125.8, 125.5, 122.0, 121.3, 116.2, 115.0, 110.9,
9

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
J ¼ 9.8 Hz), 5.79 (1H, d, J ¼ 9.8 Hz), 5.75 (2H, s) 5.51 (1H, dd, J ¼ 13.1,
2.8 Hz), 5.22 (2H, s), 3.19 (1H, dd, J ¼ 16.8, 13.1 Hz), 2.70 (1H, dd,
to 16000 g. 50
m
L of elution buffer was added to the column and
allowed to stand at room temperature for 1 min. The column was
centrifuged at 10,000 g for 1 min, RNA was collected, and the RNA
concentration was measured with a Nanodrop (Thermo-Fisher)
J ¼ 16.8, 2.8 Hz), 1.38 (6H, s). 13C NMR (150 MHz, DMSO‑d6,
d): 190.7,
164.7, 163.6, 153.1, 142.8, 141.1, 132.0, 131.6, 129.2 ꢀ 2, 129.1, 128.5,
128.3, 126.2 ꢀ 2, 125.7, 125.5, 124.6, 122.0, 121.3, 116.2, 115.1, 110.9,
102.3, 79.5, 76.8, 62.0, 52.7, 43.5, 28.2 ꢀ 2. ESI-HRMS: m/z 561.1932
[MþH]þ (Calcd for C31H27F3N3O4, 561.1931). HPLC purity: 94.42%.
spectrophotometer.
A Thunderbird probe one-step RT-PCR
(TOYOBO) kit was used for RNA quantification. The sequence of the
front primer (R6-130-S17) used was 50-CGGGAGAGCCATAGTGG-30,
the sequence of the rear primer (R6-130-R19) was 50-AGTACCA-
CAAGGCCTTTCG-30, and the TaqMan probe (R6-148-S21FT) was
FAM-50-CTGCGGAACCGGTGAGTACAC-30 TAMRA. Use the StepOne-
Plus real-time PCR biosystems for quantitative analysis.
4.2. Biological assays
4.2.1. Cell culture
Huh7 and 293T cells were cultured in Dulbecco’s modified Ea-
gle’s medium (DMEM, Invitrogen) with 10% fetal bovine serum
(FBS, Cell Culture Bioscience), 10 units/mL of penicillin, 10 mg/mL of
streptomycin in a humidified 5% CO2 incubator at 37 ꢁC.
4.2.7. Western blot analysis
Cells were lysed with RIPA buffer (25 mM Tris-HCl [pH 7.6],
150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS).
Laemmli buffer (4% SDS, 20% glycerin, 10% 2-mercaptoethanol,
0.004% bromophenol blue and 0.125 M tris-HCl [pH 6.8]) was then
added to the sample and heated for 10 min at 95 ꢁC. It was left to
stand for 5 min at 4 ꢁC. The membranes were blocked with 1% skim
milk solution and probed with a primary antibody (GAPDH, sigma,
1/1000; NS3, 1/1000 and Core Ab, 1/3000, National Institute of In-
fectious Diseases) followed by the corresponding secondary anti-
body (Anti-mouse IgG-conjugated HRP, cell signal, 1/5000 and anti-
rabbit IgG-conjugated HRP, cell signal, 1/5000). The PVDF mem-
brane was treated with a developer (GE health-care) and detected
using a chemiluminescence detection instrument LAS-3000 (Fuji-
film). ImageQuant LAS 4000 was used for detection and analysis.
4.2.2. Cytotoxicity assay
1 ꢀ 104 of Huh7 cells were seeded per well overnight, treated
with compounds for 72 h. The Cell Proliferation Kit II (XTT, Roche
Diagnostics, Mannheim, Germany) (The test concentration of all
compounds is 100
Viability Assay (The concentration gradients of compounds 10m
and 10r are 1 M, 3 M, 10 M, 30 M, respectively and the test
mg/mL) and CellTiter-Glo® Luminescent Cell
m
m
m
m
concentration of the positive drug is 10 U/mL.) were used for in vitro
proliferation assays according to the manufacturer’s instructions.
4.2.3. mCherry-NLS-IPS visualization system
The VSV-G, Pol and mCherry-NLS-IPS1 plasmids were trans-
fected into 1 ꢀ 107 293T cells according to the method provided by
the Trans IT LT1 (Mirus) kit. After 72 h, the supernatant of the
culture medium rich in mCherry-NLS-IPS recombinant virus was
4.2.8. HCV pseudovirus entry assay
HCVpp entry assay was performed as described [42]. After
treated 5 ꢀ 103 Huh-7 cells/well in a 96-well plate with compounds
for 24 h, cells were infected with HCVpp. After 6 h infection, virus
and compounds were washed out. Bright-Glo reagent (Promega)
and Lumat LB 9507 was performed for luciferase activity 48 h later.
For wildtype entry assay, cells were pre-treated with com-
pounds for 24 h then compounds were washed out. Cells were
infected with the HCV (multiplicity of infection (MOI) of 0.1) for 6 h.
RT-PCR was performed to detect HCV wildtype RNA 48 h later.
collected and then filtered through a 0.22 mM filter. Huh7 cells were
infected with mCherry-NLS-IPS recombinant virus, and the
expression of red fluorescence was observed one week later to
determine the establishment of a visualization system [29].
4.2.4. Primary screening
Cells were seeded in 96-well plates at 5 ꢀ 104 and cultured
overnight. Huh7-mCherry-NLS-IPS cells (multiplicity of infection
(MOI) of 1) were infected with the HCV (JFH1). After 6 h, the cells
were washed with the medium and cultured by adding a medium
containing different compounds, and sobosbuvir was used as a
positive medicine. After 72 h of treatment, the change of the fluo-
rescence signal in the cells was observed by using BZ-X710
KEYENCE.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
4.2.5. Assay of HCV progeny virus production
Cells were seeded in 96-well plates at 1 ꢀ 104 and cultured
overnight. Huh7 cells were infected with the HCV (multiplicity of
infection (MOI) of 0.1). After 6 h, the virus was washout with me-
dium, following with compounds treatment for 72 h. Then the
supernatant was harvested, and used to infect another Huh7 cells.
After 48 h, total RNA was extracted and HCV mRNA was determined
by RT-PCR.
The authors are very grateful to the support of the China Natural
Science Foundation (81872766), Key R & D projects of Liaoning
Province, China (2020JH2/10300058), the Young and Middle-aged
Science and Technology Innovative Talent Support Program of She-
nyang City (RC180250). This study was also supported by a grant
from the Research Program on Hepatitis from the Japan Agency for
Medical Research and Development (20fk0210053j0002). We are
very much appreciated Dr. Fukuhara in Hokkaido University and Dr.
Matsuura in Osaka University for providing us the mCherry-NLS-IPS1
reporter system.
4.2.6. RNA inhibition (RT-PCR)
For RNA extraction, column purification was performed using
RNeasy kit (QIAGEN) according to the manufacturer’s protocol.
100 mL of lysis buffer was added to the 96-well plate to lyse the
cells. Mix 70% ethanol with the dissolved sample and transfer to the
chromatography column. Centrifuge at 10,000 g for 15 s, then
Appendix A. Supplementary data
discard the waste. The column was washed once with 700
RW1 solution and twice with RPE solution. The column was rotated
m
L of
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113395.
10

H. Zhang, X. Zheng, J. Li et al.
European Journal of Medicinal Chemistry 218 (2021) 113395
j.bmcl.2011.10.048.
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