Discovery of dihydropyrrolidones as novel inhibitors against influenza A virus

Article abstract of DOI:10.1016/j.ejmech.2020.112334

More effective prophylactic and therapeutic strategies to combat influenza viruses are urgently required worldwide because the conventional anti-influenza drugs are facing drug resistance. Here, dihydropyrrolidones (DHPs), the products of an efficient multi-components reaction, were found to possess good activities against influenza A virus (IAV). Primary structure-activity relationship indicated that the activities of DHPs were greatly influenced by substituents and four of them had IC₅₀ values lower than 10 μM (DHPs 5–2, 8, 14 and 19: IC₅₀ = 3.11–9.23 μM). The activities against multiple IAV strains and mechanism of DHPs were further investigated by using 5–2 (IC₅₀ = 3.11 μM). It was found that 5–2 possessed antiviral effects against all the investigated subtypes of IAVs with the IC₅₀ values from 3.11 to 7.13 μM. Moreover, 5–2 showed very low cytotoxicity with CC₅₀ > 400 μM. Results of mechanism study indicated that 5–2 could efficiently inhibit replication of IAV, up-regulate the expression of key antiviral cytokines IFN-β and antiviral protein MxA, and suppress the production of the NDAPH oxidase NOX1 in MDCK cells. These results indicated that 5–2 could be used as a potential inhibitor against wide subtypes of IAVs.

Full text of DOI:10.1016/j.ejmech.2020.112334

European Journal of Medicinal Chemistry 199 (2020) 112334
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of dihydropyrrolidones as novel inhibitors against influenza
A virus
Teng Liu 1, Chenshu Dai 1, Huiting Sang, Fangzhao Chen, Yingna Huang, Hui Liao,
Shuwen Liu, Qiuhua Zhu*, Jie Yang**
Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment,
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China
a r t i c l e i n f o
a b s t r a c t
Article history:
More effective prophylactic and therapeutic strategies to combat influenza viruses are urgently required
worldwide because the conventional anti-influenza drugs are facing drug resistance. Here, dihy-
dropyrrolidones (DHPs), the products of an efficient multi-components reaction, were found to possess
good activities against influenza A virus (IAV). Primary structure-activity relationship indicated that the
activities of DHPs were greatly influenced by substituents and four of them had IC50 values lower than
Received 16 February 2020
Received in revised form
24 March 2020
Accepted 13 April 2020
Available online 4 May 2020
10
mechanism of DHPs were further investigated by using 5e2 (IC50 ¼ 3.11
possessed antiviral effects against all the investigated subtypes of IAVs with the IC50 values from 3.11 to
7.13 M. Moreover, 5e2 showed very low cytotoxicity with CC50 > 400 M. Results of mechanism study
indicated that 5e2 could efficiently inhibit replication of IAV, up-regulate the expression of key antiviral
cytokines IFN- and antiviral protein MxA, and suppress the production of the NDAPH oxidase NOX1 in
m
M (DHPs 5e2, 8, 14 and 19: IC50 ¼ 3.11e9.23
m
M). The activities against multiple IAV strains and
m
M). It was found that 5e2
Keywords:
Dihydropyrrolidones
Design and synthesis
Influenza a virus
Immunity regulation
Structure-activity relationship
m
m
b
MDCK cells. These results indicated that 5e2 could be used as a potential inhibitor against wide subtypes
of IAVs.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
fatality rate of approximately 40% since the first case of the disease
was found in China [8,9] and it may have the potential risk of
Influenza is a contagious disease caused by influenza viruses,
especially influenza A virus, leading to seasonal epidemics and
occasional pandemics, which has a tremendous threat to human
health and social security [1e3]. The WHO estimate that about 3e5
million cases of serious illnesses and 290,000 to 650,000 deaths
annually are associated with respiratory diseases caused by sea-
sonal influenza [4]. Additionally, the highly pathogenic H5N1 avian
influenza virus causes acute respiratory distress syndrome (ARDS)
and multi-organ disorder with approximately 60% lethality, which
receives significant attention [5]. Recent studies demonstrate that
the H5N1 avian influenza virus has capability of airborne infection
between ferrets resulted in four amino acid mutations in the HA
gene of IAV (N1581D/N2241 K/Q2261L/T3181I) [6,7]. In recent
years, the new type of reassorted influenza A virus H7N9 caused a
pandemic outbreak [10]. Therefore, effective prophylactic and
therapeutic strategies against influenza virus infection are highly
desired.
Compared with vaccinations, owing to the limitations of their
efficacy and safety, antiviral drugs are more vital to contain the
threat of influenza pandemic, particularly in the time lag between
virus identification and vaccine distribution [11e14]. Two major
classes of antiviral drugs have been approved for clinical treat-
ment of influenza, including M2 ion channel inhibitors (amanta-
dine and rimantadine), neuraminidase (NA) inhibitors (zanamivir
and oseltamivir). However, the rapid emergence of drug-resistant
influenza virus strains in recent years has limited their clinical
application and highlights the need for developing new antiviral
drugs [15e18]. Although novel anti-influenza drugs, such as
xofluza and pimodivir, targeting other viral proteins have been
developed [19e21], the on-market anti-influenza drugs still
cannot satisfy the clinical requirements. In this regard, it needs to
explore new treatment options in addition to the currently
available antiviral drugs.
* Corresponding author.
** Corresponding author.
E-mail addresses: zhuqh@smu.edu.cn (Q. Zhu), yj528@smu.edu.cn (J. Yang).
These authors contributed equally.
1
https://doi.org/10.1016/j.ejmech.2020.112334
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
Multi-component reactions (MCRs) have played an important
2. Results and discussion
role in the discovery of new drugs [22,23], for their advantages of
atom economy, high efficiency and fast building structural diversity
and complexity of compound libraries [24,25]. Considering that
heterocycles have diverse bioactivities, we are interested in
development of efficient MCRs for the synthesis of novel hetero-
cycles [26e31] and in their activities [32,33]. Dihydropyrrole-2-
ones are important heterocycles with a wide range of pharmaco-
logical activities, such as inhibition of p53ꢀMDM2 proteinꢀprotein
interaction (I) [34], agitation of serotonin 5-HT1A receptor agonist
(II) [35], analgesic activity (III) [36] and inhibition of vascular
endothelial growth factor receptor (VEGF-R) (IV) [37] (Fig. 1). The
great influence of substituents on the bioactivities of dihy-
dropyrrole-2-ones can be observed in the molecular structures of
these dihydropyrrole-2-ones. We developed convenient MCRs for
the synthesis of diverse tetra- and pentasubstituted dihy-
dropyrrolidones (DHPs) [38e40] and found that DHPs shown
inhibitory activity against caspase-3 (V) [40] and activities against
four human cancer lines (VI) (Fig. 1) [41]. Recently, some of DHPs
were found to have potential activities against IAV by high
throughput screening. Since the activities against IAV of DHPs were
not reported and the synthesis method of DHPs was highly efficient
MCRs, we designed and synthesized 29 DHPs to study their primary
structure-activity relationship (SAR) as novel inhibitors against
Puerto Rico/8/34 (H1N1) (PR8) IAV strain. Among them, DHPs 5e2,
8, 14 and 19 exhibited the potent inhibitory activity and the most
potent 5e2 was selected to study the activities against multiple
influenza A viral strains and its activity mechanism. Herein, we
reported these results.
2.1. Chemistry
Diverse DHPs 5e1ꢀ29 were designed and synthesized by the
four-component reaction (4CR) of but-2-ynedioates 1, amines 2 and
3, aldehydes 4 at room temperature. The 4CR, developed by us
previously [39], is shown in Scheme 1. Since aromatic and aliphatic
reactants 4 have very different influences on the 4CR activity, the
synthesis of DHPs needs to be proceed under different conditions
(conditions A and B) [39]. DHP 5e1 was synthesized in 65% yield
under conditions A and DHPs 5e2ꢀ29 were synthesized under
conditions B in 35e90% yields (all were new compounds except
5e17, 5e22, 5e26 and 5e29). The 1H NMR spectra of the four
known compounds are in accordance with those reported in the
literatures [31,42], and the structural characteristics of the 25 new
DHPs were determined by their IR, 1H NMR, 13C NMR and HRMS
data. The groups R1- R4 of 5e1ꢀ29 were shown in Table 1.
2.2. Biological evaluation
2.2.1. In vitro evaluation for activity against IAV
The activities of DHPs 5e1ꢀ29 against H1N1 were shown in
Table 1. Firstly, we studied the influence of R4 (5e1ꢀ16). It could be
seen that the most optimal group R4 was Ph (5e2, IC50 ¼ 3.11
mM),
followed by 4-FPh (5e8, IC50 ¼ 7.63
mM), 3-MeOPh (5e14,
mM) and 3-CF3Ph
M). Others showed much lower and even no
IC50 ¼ 9.23
(5e10, IC50 ¼ 11.82
activity. The phenyl with electron-withdrawing substituent made
mM), 1-naphthyl (5e15, IC50 ¼ 11.81
m
Fig. 1. Molecular structures of bioactive compounds containing a dihydropyrrole-2-one moiety in reported references.

T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
3
Scheme 1. Synthesis of diverse DHPs 5-1ꢀ29.
Table 2
Table 1
Inhibition activities and selectivity indexes of 5e2 against different IAV strains.
Molecular structures of DHPs 5e1ꢀ29 and their IC50 values against H1N1 (A/Puerto
Rico/8/34).
Influenza A Virus Strains
IC50
(
m
M)а
SI
a
DHP
R1
R2/R3
R4
IC50
/m
M
(mean SD)
A/Aichi/2/68 (H3N2)
A/FM-1/1/47 (H1N1)
A/Puerto Rico/8/34 (H1N1)
A/PR/8/34 (H1N1) with NA-H274Y
The influenza A virus 690 (H3)
5.26 1.17
3.58 0.13
3.11 0.23
6.48 0.55
7.13 0.78
>76.0
>111.6
>128.7
>61.6
>56.1
5e1
5e2
5e3
5e4
5e5
5e6
5e7
5e8
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
Me
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
Ph
H
Ph
222.5 29.1
3.11 0.23
>400
>400
>400
4-CNPh
4-NO2Ph
3-NO2Ph
4-BrPh
4-ClPh
4-FPh
4-CF3Ph
3-CF3Ph
4-CF3OPh
4-MePh
4-t-ButPh
3-MeOPh
1-naphthyl
5-Brthiphen-2-yl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
>400
>400
a
The samples were measured in MDCK cells in triplicate, and the data were
shown in mean SD.
7.63 1.05
67.22 9.79
11.82 2.16
128.4 39.5
68.5 16.0
>400
9.23 1.37
11.81 0.54
>400
5e9
5e10
5e11
5e12
5e13
5e14
5e15
5e16
5e17
5e18
5e19
5e20
5e21
5e22
5e23
5e24
5e25
5e26
5e27
5e28
5e29
Ribavirinb
4-CF3Ph is better than 3-CF3Ph for R2/R3 (comparing 5e2 and
5e21), but the case is just the opposite for R4 (comparing 5e9 and
5e10). 4-CF3Ph (5e2, IC50 ¼ 3.11
m
M) is the most suitable group R2/
R3, followed by 3-ClPh (5e19, IC50 ¼ 6.91
mM) and 4-MePh (5e22,
IC50 ¼ 18.99
m
M). Aliphatic group as R2/R3 such as cyclohexyl pro-
duces no activity (5-22). The influence of R1 relates to R2/R3,
changing R1 from Et to Me decreases activity as R2/R3 ¼ 4-CF3Ph
(comparing 5e2 and 5e28) but increases activity as R2/R3 ¼ 3-
CF3Ph (comparing 5e21 and 5e29). The antiviral effect of com-
pound 5e2 is similar to that of the positive control medicine
ribavirin.
>400
>400
4-ClPh
3-ClPh
4-BrPh
6.91 0.90
>400
30.94 9.30
18.99 3.40
>400
>400
>400
>400
>400
3-CF3Ph
4-MePh
4-isopropylPh
4-t-ButPh
4-PhOPh
4-MeOPh
cyclohexyl
4-CF3Ph
3-CF3Ph
2.2.2. Antiviral activity of 5e2 against multiple IAV strains
Ph
Ph
Ph
Ph
MTT-based assay as described in experimental section was
performed to evaluate the antiviral effect of 5e2 against different
influenza virus strains. MDCK cells were infected with the different
subtypes of IAV for 1 h, including H1N1, H3N2, the IAV 690 (H3
subtype) and oseltamivir-resistant H1N1 virus strain with an NA-
H274Y mutation, and then were incubated with 5e2. At 48 h post
infection (p.i.), the antiviral activity of 5e2 was measured by MTT-
based method. As shown in Table 2, 5e2 could efficiently inhibit
11.90 3.45
16.15 0.71
2.84 0.06
a
IC50: 50% inhibitory concentration.
The compound was used as a positive control medicine.
b
the activity disappear (5e3ꢀ7) except substituent containing F
(5e8ꢀ11) (4-CF3O is an electron-withdrawing substituent which
makes the chemical shift of CeH on pyrrole ring move to lower
filed, comparing the 1HNMR spectra of 5e1 and 5e11), in which
hydrogen bond is expected to be the main effect. The phenyl with
electron-donating substituent shows good and certain activities
(5e12 and 5e14) except the phenyl with large volume 4-t-But
which leads to no activities. Larger rigid aromatic ring naphthyl
affords good activity (5-15) but no activity found for smaller
electron-rich aromatic ring thiophenyl.
infection of multiple strains of IAV with IC50 from 3.11 to 7.13 mM,
which indicated 5e2 possibly possessed broad-spectrum of anti-
influenza virus activity.
Additionally, we observed the cytopathic effect (CPE) of MDCK
cells caused by influenza PR8 virus, it was found that 5e2 had a
certain protective effect by microscopic and found that 5e2 dis-
played a protective effect (Fig. 2A), compared to untreated cells. By
plaque reduction assay, we also found that 5e2 significantly
blocked plaque formation induced by influenza virus in a dose-
dependent manner (Fig. 2B), confirming the results from anti-
influenza virus assay. MTT assay was also applied to evaluate
in vitro cytotoxicity of compound 5e2 in MDCK and A549 cells. 5e2
exhibited low cytotoxicity towards the tested cells with the CC50
The influence of R2/R3 (5e17ꢀ27) is very different from and even
opposite to that of influence of R4. For example, a) Ph produces no
activity for R2/R3 (5e17) but is the most suitable group for R4 (5e2);
b) although CF3Ph is a relative suitable group for R2/R3 and R4, the
position influence of CF3 on phenyl ring is opposite for R2/R3 and R4:
(50% cellular cytotoxicity concentration) values more than 400
mM
(Fig. 3). The selectivity indexes (SI ¼ CC50/IC50) of 5e2 against

4
T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
Fig. 2. Biological effect of 5e2 on the MDCK cells infected with IAV PR8 strain. (A) The infected MDCK cells without (left) and with 5e2 (40
48 h p.i. and the lower was ten times larger than the upper. (B) 5e2 decreased viral plaque formation in a dose-dependent manner.
mM) (right). The photos were taken at
Fig. 3. The effect of 5-2 on cell viability. (A) The effect of 5-2 on MDCK cells viability. (B) The effect of 5-2 on A549 cells viability.
different IAV strains were shown in Table 2.
conditions to verify which period of influenza virus life cycle was
intervened by 5e2 was carry out. Employing time-of-addition
assay, we confirmed that 5e2 could effectively inhibit influenza
virus PR8 strain infection when cells were treated with 5e2 as
“post-infection” described (Fig. 5A), suggesting that the compound
displayed inhibitory activity after the virus entry into host cells.
2.3. Mechanism studies
2.3.1. Effect of 5e2 on the viral replication in IAV-infected MDCK
cells
Subsequently, PR8-infected-MDCK cells were treated with 40 mM of
To confirm whether 5e2 had an influence on the replication of
IAV, we detected the expression level of influenza viral protein and
mRNA in infected-cells by employing indirect immunofluorescence
microscopy, western blotting and qRT-PCR assays. As demonstrated
in Fig. 4A, the result showed that 5e2 significantly decreased the
yields of influenza viral NP in a dose-dependent manner. Further-
more, NP protein was hardly observed when MDCK cells were
5e2 at the different time points during the whole replication cycle
of influenza virus as shown in Fig. 5B. At 10 h p.i., the expressions
level of viral NP protein was measured using western blotting. The
result exhibited that influenza NP protein was decreased mainly
happened in at 0e2, 2e5 and 5e8 h during the virus infection
(Fig. 5C). Furthermore, the same result was confirmed in A549 cells
using qRT-PCR (Fig. 5D), revealing that 5e2 strongly inhibited
replication and translation of IAV.
treated with 40 mM of 5e2. Compared with the virus control, the
expression level of NP protein could also be restrained by 5e2 in a
dose-dependent manner (Fig. 4B). Simultaneously, the remarkably
reduction of influenza virus HA mRNA level in 5-2-treated
A549 cells was also observed (Fig. 4C), which suggested that 5e2
exerted an inhibitory effect on influenza virus replication without
cell specificity.
In addition, the expression level of viral HA mRNA in the IAV-
infected MDCK cells treated with serially diluted 5e2 was detec-
ted at 24 h, 48 h, and 72 h p.i. respectively by qRT-PCR assay. The
data indicated that 5e2 obviously reduced influenza HA mRNA
expression at all three time points (Fig. 4D). The result indicated
that the antiviral activity of 5e2 was still effective up to 72 h,
implying that 5e2 effectively suppressed multiple rounds of
influenza virus replication.
2.3.2. Effect of 5e2 on the expression of IFN-
influenza-infected MDCK cells
b and MxA in
Amplification and propagation of IAV is strictly dependent on
the host cell, which means that many factors in the host cell
involved in influenza virus life cycle. Through the in-depth study, a
series of innate immune regulatory factors of host cell with the
antiviral function has been discovered and gradually become an
important source for screening potential antiviral drugs. To identify
whether 5e2 had an effect on the regulation pathway of host cell
defense response to the virus, the expression level of various cy-
tokines was measured using corresponding primers by qRT-PCR
after the infected-cells were treated with 40
mM 5e2 for 24 h. As
Then, further investigation under different experimental
a result, we discovered that 5e2 could significantly increase the

T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
5
Fig. 4. Effect of 5e2 on the viral replication in IAV-infected MDCK cells. The expression level of influenza viral proteins and mRNA in infected-cells by employing indirect
immunofluorescence microscopy (A), western blotting (B) and qRT-PCR (C). (A) The green foci stand for viral NP proteins. The blue foci stand for nucleus counter stained with 4,6-
diamidino-2-phenylindole (DAPI) in indirect immunofluorescence microscopy assay. The protein and nucleus images were merged. Original magnification, 10 ꢁ . (D) The expression
level of viral HA mRNA was measured by qRT-PCR at the indicated time. Ribavirin was applied as a positive control. Data are shown as the mean SD. (*p < 0.05, ***p < 0.001). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
expression of IFN-
b
and MxA mRNA in compared with control
vicious cycle.Therefore, finding a way to effectively control virus-
induced ROS production is essential for alleviating patients’
symptoms. We unexpectedly found that 5e2 significantly
decreased the production of NADPH oxidase NOX1 in MDCK cells
treated in the same way as above, compared with control group
(Fig. 7), which mainly involved in the regulatory pathway of host
cell generated ROS. The above results shown that 5e2 could
effectively inhibit the tissue damage caused by excessive ROS
production.
group after influenza virus infection (Fig. 6). Type I interferon, as
the most important antiviral cytokine, is generated by almost all
cells and plays an important role in the host cell defense response
to virus infection. As the most classic modes of type I interferon,
they activate the downstream JAK/STAT pathway, followed by
initiating the expression of antiviral genes containing the antiviral
protein MxA. It has been reported that MxA is a key antiviral pro-
tein in innate immune pathway to prevent the replication of
influenza virus, and the up-regulated expression of MxA could in-
crease the survival rate of influenza-infected mice [43]. The results
3. Conclusion
indicated that 5e2 could induce the expression of IFN-
b in the
virus-infected cells, followed by activating the production of the
downstream relevant antiviral factor MxA to block virus
replication.
We designed and synthesized 29 DHPs (25 of them are new
compounds) to study the primary SAR of DHPs as novel inhibitors
against H1N1. It was found that their inhibition effects depended on
their substituents R1-R4 and four of them (5e2, 8, 14 and 19) shown
good activities against H1N1. In addition, we further investigated
the activities against different influenza A subtypes and the inhi-
bition mechanism of the most potent 5e2. The experimental re-
sults indicated: a) DHP 5e2 possessed antiviral effects against a
broad variety of influenza A viral strains including oseltamivir-
resistant strains and clinically relevant isolates with low cytotox-
icity; b) Mechanism study indicated that 5e2 significantly inhibited
2.3.3. Effect of 5e2 on the expression of NOX1 gene in MDCK cells
infected with influenza virus
On the other hand, host cells generate a large amount of ROS
after influenza infection, result in apoptosis by stimulating oxida-
tive stress signaling pathways [44]. However, influenza virus
replication could not be restrained due to cell damage thus the
continuous viral replication and increasing ROS production form a

6
T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
Fig. 5. Compound 5e2 could inhibit of viral replication. (A) Three modes of treatment, namely pre-infection, post infection and entire infection, were executed as described in
experimental section. (B) 5e2 was added to MDCK cells at different intervals. The production of the viral NP protein and mRNA was detected by western blotting (C) and qRT-PCR
(D) at 10 h p.i..The results represent the average of three independent experiments. Results are presented as mean SD. (NS, not significant, *p < 0.015, **p < 0.01 and ***p < 0.001).
Fig. 6. Up-regulated effect of 5e2 on the expression of IFN-b and MxA in influenza-infected MDCK cells. The expression level of IFN-b (A) and MxA (B) was measured using
corresponding primers by qRT-PCR after the virus-infected cells were treated with 40 mM of 5e2 for 24 h.
the transcription and replication of viral genome. It was also able to
regulate several factors induced by IAV infection, including key
antiviral cytokines IFN-b, antiviral protein MxA and NDAPH oxidase
NOX1. To sum up, the above-mentioned results highlight 5e2 as a
promising anti-influenza agent because of its virus inhibitory ac-
tivity and immunity regulation profiles. Further investigations into
the structure-activity relationship and mechanism of DHPs are
under way in our laboratory.
resolution three-in-one mass spectrometer (Thermo). The re-
actions were monitored by thin-layer chromatography (TLC) using
100e400 mesh silica gel plates (GF254) and were visualized using
UV lamp (254 and 365 nm). All the starting materials were pur-
chased from commercial suppliers and used without further
purification.
4.1.1. General procedures for the synthesis of DHPs 5e1ꢀ29
The procedures are reported in our previous work [39]. DHP 5e1
was synthesized by the procedures for the synthesis of tetrasub-
stituteddihydropyrrolidones and DHPs 5e2ꢀ29 were synthesized
by the procedures for synthesis of the pentasubstituteddihy-
dropyrrolidones. Generally, DHP 5e1 was synthesized by proced-
ures: (a) but-2-ynedioates 1 (1 mmol) and aromatic primary
amines 2 (1 mmol) were added into 5 mL of MeOH, kept at room
temperature for 10e30 min; (b) aromatic primary amines 3
4. Experimental section
4.1. Chemistry
All melting points were measured on an X-5 micro melting point
apparatus and were uncorrected. 1H and 13C NMR spectra were
recorded on a BrukerAvance 400 MHz spectrometer with CDCl3or
DMSO‑d6 as the solvent and TMS as the internal standard. The IR
spectra were recorded on a Bruker Vector 22 infrared spectropho-
tometer (KBr dicks) and expressed in wave number (cmꢀ1). High
resolution mass spectra (HRMS) were recorded on a MAT 95XP
(Thermo) High resolution mass spectrometer or an Ultra high
(1.6 mmol), 38% formaldehyde 4 (112 mL, 1.4 mmol), benzaldehyde
(212 mg, 2 mmol), and Cu(OAc)2$H2O (80 mg, 0.4 mmol) were
dropwise added into the above mixture in sequence and stirred at
room temperature for desired time. DHPs 5e2ꢀ29 were synthe-
sized by the procedures: (a) but-2-ynedioates 1 (1 mmol) and

T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
7
7.62e7.58 (m, 4H), 7.55 (d, J ¼ 8.7 Hz, 2H), 7.44 (d, J ¼ 1.8 Hz, 1H),
7.43 (d, J ¼ 1.8 Hz, 1H), 7.29 (d, J ¼ 2.5 Hz, 1H), 7.27 (s, 1H), 5.95 (s,
1H), 4.10 (q, J ¼ 7.1 Hz, 2H), 1.09 (t, J ¼ 7.1 Hz, 3H).13C NMR
(101 MHz, CDCl3) d:163.69, 163.66, 141.52, 141.48, 141.16, 138.96,
132.53, 128.30, 127.70 (q, J ¼ 33 Hz), 126.63 (q, J ¼ 32 Hz), 126.29 (q,
J ¼ 4 Hz), 125.76 (q, J ¼ 4 Hz), 124.03 (q, J ¼ 270 Hz), 123.60 (q,
J ¼ 270 Hz), 122.29, 121.59, 118.07, 112.64, 110.99, 62.09, 60.90,
13.77.IR (KBr)
n
(cmꢀ1): 3451, 2230, 1708, 1643, 1613, 1523, 1373,
1325, 1167, 1118, 1066, 1022, 756.HRMS-ESI (m/z): calcd for
C
28H20F6N3O3 ([MþH]þ):560.1403; Found:560.1405.
4.1.2.4. Ethyl
2-(4-nitrophenyl)-5-oxo-1-(4-(trifluoromethyl)
phenyl)-4-((4-(trifluoro-methyl) phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate (5-4). 54% yield, yellow solid, m.p. ¼181e182 ꢂC.
1H NMR (400 MHz, CDCl3)
d:8.45 (s, 1H), 8.20e8.17 (m, 1H),
8.17e8.15 (m, 1H), 7.66 (d, J ¼ 8.5 Hz, 2H), 7.61 (d, J ¼ 8.4 Hz, 2H),
7.56 (d, J ¼ 8.6 Hz, 2H), 7.52e7.50 (m, 1H), 7.50e7.48 (m, 1H), 7.29
(d, J ¼ 7.6 Hz, 2H), 6.02 (s, 1H), 4.11 (m, 2H),1.10 (t, J ¼ 7.1 Hz, 3H).13
C
Fig. 7. Effect of 5e2 on the&ensp;ROS-generating NADPH oxidase&ensp;NOX1 in
influenza-infected MDCK cells.
NMR (101 MHz, CDCl3)
d:163.64, 147.95, 143.43, 141.59, 141.10,
138.90, 128.49, 127.93, 127.61, 126.88, 126.55, 126.39, 126.35,126.32,
126.28, 125.83,125.80,125.76,125.72,125.37, 124.92, 123.98, 122.67,
122.35, 122.22, 121.60, 110.79, 77.29, 61.81, 60.95, 13.79.IR (KBr)
n
(cmꢀ1):3443, 1708, 1644, 1612, 1524, 1325, 1267, 1167, 1117, 1067,
aromatic/aliphatic primary amines 2 (1 mmol) were added into test
tube A with 3 mL of MeOH and kept at room temperature for
30 min; (b) aromatic/aliphatic primary amines 3 (1.6 mmol), aro-
matic aldehydes 4 (3.5 mmol), salicylic acid (41 mg, 0.3 mmol), and
Cu(OAc)2$H2O (80 mg, 0.4 mmol) were added into test tube B with
3 mL of MeOH, then the mixture in tube B was added into the above
mixture in tube A, and stirred at room temperature for desired
time. After completion of the reactions, the product mixture was
purified by preparative TLC with petroleum ether/ethyl acetate
(15:1e3:1) as eluent to afford the desired products DHPs 5e1ꢀ29.
1020,
755.HRMS-ESI
(m/z):
calcd
for
C27H20F6N3O5
([MþH]þ):580.1302; Found:580.1303.
4.1.2.5. Ethyl
2-(3-nitrophenyl)-5-oxo-1-(4-(trifluoromethyl)
phenyl)-4-((4-(trifluoro-methyl) phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate (5-5). 38% yield, white solid, m.p. ¼ 198e199 ꢂC.
1H NMR (400 MHz, CDCl3)
d
: 8.49 (s, 1H), 8.24 (t, J ¼ 1.9 Hz,1H), 8.15
(m,1H), 7.68 (d, J ¼ 8.5 Hz, 2H), 7.61 (m, 3H), 7.57 (s,1H), 7.55 (s,1H),
7.50 (d, J ¼ 7.9 Hz, 1H), 7.30 (d, J ¼ 8.5 Hz, 2H), 6.02 (s, 1H), 4.11 (m,
2H), 1.17e0.95 (m, 3H).13C NMR (101 MHz, CDCl3)
d:163.73, 163.58,
148.34, 141.77, 141.02, 138.85, 138.48, 133.02, 129.85, 127.74 (q,
J ¼ 32.8 Hz), 126.75 (q, J ¼ 32.8 Hz), 126.36 (q, J ¼ 3.8 Hz), 125.78 (q,
J ¼ 3.7 Hz), 124.02 (q, J ¼ 270.3 Hz), 123.58 (q, J ¼ 270.3 Hz), 123.70,
4.1.2. Structural characteristics of DHPs 5e1ꢀ29
4.1.2.1. Ethyl
5-oxo-1-(4-(trifluoromethyl)phenyl)-4-((4-(tri-
122.97, 122.47, 121.66, 110.80, 61.74, 60.94, 13.76.IR (KBr)
n
(cmꢀ1):
fluoromethyl)phenyl)amino)-2,5-dihydro-1H-pyrrole-3-carboxylate
(5-1). 65% yield, white solid, m.p. ¼142e143 ꢂC. 1H NMR (400 MHz,
3444, 2920, 1704, 1643, 1614, 1530, 1324, 1267, 1166, 1115, 1066,
CDCl3)
d
: 8.21 (s, 1H), 7.96 (d, J ¼ 8.7 Hz, 2H), 7.68 (d, J ¼ 8.8 Hz, 2H),
1020,
755.HRMS-ESI
(m/z):
calcd
for
C27H20F6N3O5
([MþH]þ):580.1302; Found:580.1302.
7.59 (d, J ¼ 8.5 Hz, 2H), 7.22 (d, J ¼ 8.4 Hz, 2H), 4.61 (s, 2H), 4.30 (q,
J ¼ 7.1 Hz, 2H), 1.29 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d:
164.17, 163.86, 141.95, 141.62, 141.36, 126.84 (q, J ¼ 32.7 Hz), 126.39
(q, J ¼ 3.7 Hz), 126.00 (q, J ¼ 32.7 Hz), 125.67 (q, J ¼ 3.7 Hz), 124.12
(q, J ¼ 270.0 Hz), 123.87 (q, J ¼ 271.6 Hz), 121.64, 118.58, 106.75,
4.1.2.6. Ethyl
2-(4-bromophenyl)-5-oxo-1-(4-(trifluoromethyl)
phenyl)-4-((4-(trifluoro-methyl)phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate (5e6). 46% yield, white solid, m.p. ¼185e186 ꢂC.
60.86, 48.06, 14.10.IR (KBr)
n
(cmꢀ1):3292, 2987, 1700, 1652, 1609,
1H NMR (400 MHz, CDCl3)
d
: 8.40 (s, 1H), 7.68 (d, J ¼ 8.6 Hz, 2H),
1527, 1455, 1394, 1326,1198, 1111,1067, 841, 758, 593. HRMS-ESI (m/
7.58 (m, 4H), 7.48e7.44 (m, 1H), 7.44e7.41 (m, 1H), 7.28 (d,
J ¼ 1.8 Hz, 1H), 7.19 (d, J ¼ 1.9 Hz, 1H), 7.18e7.14 (m, 1H), 5.87 (s, 1H),
z): calcd for C21H16F6N2O3 ([MþH]þ): 459.1126; Found: 459.1126.
4.11 (m, 2H), 1.10 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d:
4.1.2.2. Ethyl 5-oxo-2-phenyl-1-(4-(trifluoromethyl)phenyl)-4-((4-
(trifluoromethyl)phenyl)amino)-2,5-dihydro-1H-pyrrole-3-
carboxylate (5-2). 55% yield, white solid, m.p. ¼ 148e149 ꢂC. 1H
163.86, 163.83, 141.44, 141.11, 139.20, 134.92, 131.92, 129.13, 127.44
(q, J ¼ 32 Hz), 126.35 (q, J ¼ 38 Hz), 126.16 (q, J ¼ 3 Hz), 125,73 (q,
J ¼ 4 Hz), 124.09 (q, J ¼ 270 Hz), 123.78 (q, J ¼ 270 Hz), 122.51,
n
(cmꢀ1): 3461,
NMR (400 MHz, CDCl3)
d
: 8.40 (s, 1H), 7.72 (d, J ¼ 8.6 Hz, 2H), 7.57
121.99, 121.66, 112.04, 62.16, 60.80, 13.76.IR (KBr)
(m, 4H), 7.29 (m, 7H), 5.90 (s, 1H), 4.10 (q, J ¼ 7.1 Hz, 2H), 1.09 (t,
1706, 1644, 1612, 1523, 1487, 1370,1325, 1246,1166, 1117, 1067, 1015,
757.HRMS-ESI (m/z): calcd for C27H20BrF6N2O3 ([MþH]þ):613.0556;
Found:613.0555.
J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d:164.02, 164.00, 141.61,
141.08, 139.51, 135.67, 128.68, 128.57, 127.48, 127.19 (q, J ¼ 32.6 Hz),
126.132 (q, J ¼ 32.8 Hz), 126.02 (q, J ¼ 3.7 Hz), 124.13 (q, J ¼ 269.9),
123.76 (q, J ¼ 270.3 Hz), 121.86, 121.64, 112.90,62.83, 60.68, 13.73.IR
4.1.2.7. Ethyl
2-(4-chlorophenyl)-5-oxo-1-(4-(trifluoromethyl)
(KBr)
n
(cmꢀ1):3783, 3355, 2919, 1703, 1610, 1323, 1242, 1167, 1117,
phenyl)-4-((4-(trifluoro-methyl)phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate (5e7). 73% yield, white solid, m.p. ¼181e182 ꢂC.
1066, 836, 756, 695. HRMS-ESI (m/z): calcd for C27H20F6N2O3
([MþH]þ): 535.1442; Found: 535.1442.
1H NMR (400 MHz, CDCl3)
d
: 8.40 (s,1H), 7.67 (t, J ¼ 8.2 Hz, 2H), 7.58
(m, 4H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.29e7.23 (m, 2H), 7.18 (d,
4.1.2.3. Ethyl
2-(4-cyanophenyl)-5-oxo-1-(4-(trifluoromethyl)
J ¼ 8.3 Hz, 2H), 5.87 (s,1H), 4.21e3.97 (m, 2H),1.19e0.96 (m, 3H).13
C
phenyl)-4-((4-(trifluoro-methyl) phenyl)amino)-2,5-dihydro-1H-pyr-
NMR (101 MHz, CDCl3) d:163.87, 163.82, 141.44, 141.11, 139.20,
role-3-carboxylate (5-3). 40% yield, white solid, m.p. ¼ 186e187 ꢂC.
134.91, 131.92, 131.80, 131.56, 129.13, 127.459 (q, J ¼ 32.6 Hz), 126.34
1H NMR (400 MHz, CDCl3)
d: 8.43 (s, 1H), 7.65 (d, J ¼ 8.6 Hz, 2H),
(q, J ¼ 32.5 Hz), 126.16 (q, J ¼ 3.7 Hz), 125.73 (q, J ¼ 3.7 Hz), 124.08

8
T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
(q, J ¼ 270.0 Hz), 123.70 (q, J ¼ 2704 Hz), 121.99, 121.67, 112.03,
62.17, 60.80, 13.76.IR (KBr)
(cmꢀ1): 3444, 1706, 1644, 1612, 1522,
NMR (400 MHz, CDCl3)
d
:8.37 (s,1H), 7.73 (d, J ¼ 8.6 Hz, 2H), 7.59 (d,
n
J ¼ 8.5 Hz, 2H), 7.54 (d, J ¼ 8.7 Hz, 2H), 7.26 (m, 2H), 7.19 (d,
1369, 1325, 1167, 1117, 1067, 1015, 755.HRMS-ESI (m/z): calcd for
J ¼ 8.0 Hz, 2H), 7.11 (d, J ¼ 8.0 Hz, 2H), 5.88 (s, 1H), 4.17e3.90 (m,
C
27H19ClF6N2NaO3 ([MþNa]þ):591.0881; Found:591.0906.
2H), 2.27 (s, 3H), 1.19e0.96 (m, 3H).13C NMR (101 MHz, CDCl3)
d:
164.10, 163.99, 141.76, 140.78, 139.57, 138.38, 132.49, 129.39, 127.34,
127.07 (q, J ¼ 32.7 Hz), 125.99 (q, J ¼ 32.5 Hz), 126.02 (q, J ¼ 3.7 Hz),
125.70 (q, J ¼ 3.8 Hz), 124.15 (q, J ¼ 269.8 Hz), 123.81 (q,
J ¼ 270.2 Hz), 121.71, 121.60, 113.21, 62.65, 60.67, 21.05, 13.72.IR
4.1.2.8. Ethyl
2-(4-fluorophenyl)-5-oxo-1-(4-(trifluoromethyl)
phenyl)-4-((4-(trifluoro-methyl)phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate (5e8). 79% yield, white solid, m.p. ¼144e145 ꢂC.
1H NMR (400 MHz, CDCl3)
d
:8.41 (s, 1H), 7.68 (d, J ¼ 8.4 Hz, 2H), 7.58
(KBr) n
(cmꢀ1): 3283, 1705, 1644, 1611, 1521, 1370, 1325, 1166, 1118,
(m, 4H), 7.29 (s, 1H), 7.26 (s, 2H), 7.00 (t, J ¼ 8.3 Hz, 2H), 5.90 (s, 1H),
1067, 1022, 840, 755.HRMS-ESI (m/z): calcd for C28H23F6N2O3
4.10 (q, J ¼ 7.1 Hz, 2H), 1.09 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz,
([MþH]þ):549.1607; Found:549.1604.
CDCl3)
d
:163.91,163.87,162.53 (d, J ¼ 246 Hz),141.52,141.03,139.26,
131.45 (d, J ¼ 4 Hz), 129.18 (d, J ¼ 8 Hz), 127.39 (q, J ¼ 32 Hz), 126.57
(q, J ¼ 33 Hz), 126.09 (q, J ¼ 4 Hz), 125.73 (q, J ¼ 3 Hz), 124.10 (q,
J ¼ 270 Hz), 123.72 (q, J ¼ 270 Hz), 121.93, 121.79, 115.77 (q,
4.1.2.13. Ethyl 2-(4-(tert-butyl)phenyl)-5-oxo-1-(4-(trifluoromethyl)
phenyl)-4-((4-(tri-fluoromethyl)phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate
(5-13). 76%
yield,
yellow
solid,
J ¼ 22 Hz), 112.42, 62.11, 60.73, 13.73.IR (KBr)
n
(cmꢀ1): 3283, 1707,
m.p. ¼ 170e171 ꢂC. 1H NMR (400 MHz, CDCl3)
d:8.37 (s, 1H), 7.75 (d,
1645, 1611, 1514,1370, 1326,1232, 1166,1118, 1067, 1021, 756.HRMS-
J ¼ 8.1 Hz, 2H), 7.60 (d, J ¼ 8.3 Hz, 2H), 7.55 (d, J ¼ 8.5 Hz, 2H),
7.34e7.30 (m, 2H), 7.29e7.25 (m, 2H), 7.23e7.19 (m, 2H), 5.88 (s,
1H), 4.11 (m, 2H), 1.29 (s, 9H), 1.07 (t, J ¼ 7.1 Hz, 3H).13C NMR
ESI (m/z): calcd for
Found:553.1356.
C
27H20F7N2O3 ([MþH]þ):553.1357;
(101 MHz, CDCl3) d:164.13, 164.04, 151.56, 141.74, 140.84, 139.69,
4.1.2.9. Ethyl
5-oxo-1,2-bis(4-(trifluoromethyl)phenyl)-4-((4-(tri-
132.37, 126.98 (q, J ¼ 32.7 Hz), 127.05, 125.99 (q, J ¼ 32.5 Hz), 126.00
(q, J ¼ 3.8 Hz), 125.71 (q, J ¼ 3.8 Hz), 125.59, 124.15 (q, J ¼ 269.9 Hz),
123.83 (q, J ¼ 270.2 Hz), 121.75, 121.45, 113.34, 62.50, 60.59, 34.50,
fluoromethyl)phenyl) amino)-2,5-dihydro-1H-pyrrole-3-carboxylate
(5e9). 52% yield, yellow solid, m.p. ¼ 110e111 ꢂC. 1H NMR
(400 MHz, CDCl3)
d:8.42 (s, 1H), 7.76e7.50 (m, 8H), 7.44 (d,
31.14, 13.70.IR (KBr) n
(cmꢀ1): 2966, 1707, 1644, 1612, 1521, 1368,
J ¼ 8.2 Hz, 2H), 7.34e7.24 (m, 2H), 5.96 (s, 1H), 4.11 (q, J ¼ 7.1 Hz,
1325, 124, 1167, 1120, 1067, 1021, 841, 757.HRMS-ESI (m/z): calcd for
C
2H), 1.10 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d:163.78,
31H29F6N2O3 ([MþH]þ):591.2077; Found:591.2073.
141.42, 127.88, 126.27, 125.80, 125.76, 125.72, 122.19, 121.55,
111.58,62.11, 60.84, 31.53, 22.59, 13.75.IR (KBr)
n
(cmꢀ1):3292, 2924,
4.1.2.14. Ethyl
2-(3-methoxyphenyl)-5-oxo-1-(4-(trifluoromethyl)
1706, 1614, 1503, 1324, 1243, 1121,1066, 841, 768, 600, 504. HRMS-
ESI (m/z): calcd for C28H19F9N2O3 ([MþH]þ): 603.1240; Found:
603.1239.
phenyl)-4-((4-(trifluoro-methyl)phenyl)amino)-2,5-dihydro-1H-pyr-
role-3-carboxylate
(5-14). 51%
yield,
white
d:8.42 (s, 1H), 7.73 (d,
solid,
m.p. ¼ 137e138 ꢂC.1H NMR (400 MHz, CDCl3)
J ¼ 8.4 Hz, 2H), 7.57 (m, 4H), 7.27 (d, J ¼ 9.0 Hz, 2H), 7.22 (d,
J ¼ 7.9 Hz, 1H), 6.91 (d, J ¼ 7.6 Hz, 1H), 6.85e6.77 (m, 2H), 5.87 (s,
4.1.2.10. Ethyl
5-oxo-2-(3-(trifluoromethyl)phenyl)-1-(4-(tri-
fluoromethyl)phenyl)-4-((4- (trifluoromethyl)phenyl)amino)-2,5-
dihydro-1H-pyrrole-3-carboxylate (5-10). 65% yield, white solid,
1H), 4.11 (q, J ¼ 7.1 Hz, 2H), 3.77 (s, 3H), 1.11 (t, J ¼ 7.1 Hz, 3H).13
C
NMR (101 MHz, CDCl3) d:164.03, 163.99, 159.76, 141.67, 140.99,
m.p. ¼148e149 ꢂC. 1H NMR (400 MHz, CDCl3)
d
:8.52 (s, 1H), 7.68 (d,
139.56, 137.28, 129.69, 127.17 (q, J ¼ 32.7 Hz), 126.05 (q, J ¼ 32.6 Hz),
126.02 (q, J ¼ 3.7 Hz), 125.70 (q, J ¼ 3.7 Hz), 125.323 (q, J ¼ 35.2 Hz),
121.82, 121.60, 119.90, 113.70, 113.26, 112.86, 62.73, 60.68, 55.15,
J ¼ 8.6 Hz, 2H), 7.63 (d, J ¼ 5.2 Hz, 2H), 7.60 (s,1H), 7.55 (m, 3H), 7.43
(d, J ¼ 6.5 Hz, 2H), 7.31 (s, 1H), 5.96 (s, 1H), 4.12 (q, J ¼ 7.1 Hz, 2H),
1.12 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d:163.92, 163.69,
13.74.IR (KBr) n
(cmꢀ1): 3620, 3359, 2920, 2851, 1706, 1643, 1610,
141.75, 141.16, 139.10, 137.17, 131.05(q, J ¼ 32 Hz), 130.09, 129.38,
127.58 (q, J ¼ 33 Hz), 126.55 (q, J ¼ 33 Hz), 126.23 (q, J ¼ 4 Hz),
125.76 (q, J ¼ 4 Hz), 123.79 (q, J ¼ 270 Hz), 125.01 (q, J ¼ 4 Hz),
123.70 (q, J ¼ 273 Hz), 122.71, 122.28, 123.59 (q, J ¼ 273 Hz), 121.67,
1520, 1369, 1324, 1268, 1166, 1117, 1066, 751.HRMS-ESI (m/z): calcd
for C28H23F6N2O4 ([MþH]þ):565.1557; Found:565.1556.
4.1.2.15. Ethyl
2-(naphthalen-1-yl)-5-oxo-1-(4-(trifluoromethyl)
111.57, 62.14, 60.86, 13.63.IR (KBr)
n
(cmꢀ1): 3678, 3295, 1710, 1645,
phenyl)-4-((4-(trifluoro-methyl).
1612, 1522, 1369, 1326, 1251, 1168, 1121, 1069, 1022, 749.HRMS-ESI
phenyl)amino)-2,5-dihydro-1H-pyrrole-3-carboxylate (5-15). 35%
(m/z):
calcd
for
C
28H20F9N2O3
([MþH]þ):603.1325;
yield, white solid, m.p. ¼ 149e150 ꢂC. 1H NMR (400 MHz, CDCl3)
d:
Found:603.1325.
7.87 (d, J ¼ 8.2 Hz, 1H), 7.84e7.61 (m, 5H), 7.57 (m, 4H), 7.45e7.32
(m, 4H), 7.24 (m, 2H), 6.85 (s, 1H), 3.82 (m, 2H), 0.50 (m, 3H).13
NMR (101 MHz, CDCl3) : 164.32, 164.28, 141.66, 141.58, 139.70,
C
4.1.2.11. Ethyl
5-oxo-2-(4-(trifluoromethoxy)phenyl)-1-(4-(tri-
d
fluoromethyl)phenyl)-4-((4-(.
133.80, 131.95, 131.78, 129.25, 129.07, 126.60, 126.28, 126.04, 126.00,
125.96, 125.83, 125.79, 125.76, 125.72,125.46, 125.04, 123.33,123.11,
trifluoromethyl)phenyl)amino)-2,5-dihydro-1H-pyrrole-3-
carboxylate (5-11). 82% yield, white solid, m.p. ¼ 130e131 ꢂC.1H
122.53, 121.89, 120.86, 114.24, 60.52, 56.47, 13.26.IR (KBr) n
(cmꢀ1):
NMR (400 MHz, CDCl3)
d
: 8.43 (s, 1H), 7.69 (d, J ¼ 8.6 Hz, 2H), 7.58
3740, 3299, 2990, 1706, 1644, 1611, 1522, 1367, 1325, 1276, 1167,
1118, 1068, 1023, 841, 755. HRMS-ESI (m/z): calcd for C31H23F6N2O3
([MþH]þ) Calcd: 585.1607; Found: 585.1606.
(m, 4H), 7.34 (d, J ¼ 8.5 Hz, 2H), 7.28 (d, J ¼ 8.2 Hz, 2H), 7.16 (d,
J ¼ 8.4 Hz, 2H), 5.92 (s, 1H), 4.25e3.87 (m, 2H), 1.08 (t, J ¼ 7.1 Hz,
3H).13C NMR (101 MHz, CDCl3)
d: 163.86, 163.82, 149.13, 141.24,
139.20, 134.44, 128.95, 127.32, 126.19, 126.15, 125.76, 125.72, 122.07,
121.66, 121.00, 112.02, 61.94, 60.76, 31.52, 22.58, 14.02, 13.68.IR
(KBr)
4.1.2.16. Ethyl
2-(5-bromothiophen-2-yl)-5-oxo-1-(4-(tri-
fluoromethyl)phenyl)-4-((4- (trifluoromethyl)phenyl)amino)-2,5-
dihydro-1H-pyrrole-3-carboxylate (5-16). 50% yield, white solid,
n
(cmꢀ1): 3296, 1707, 1645, 1611, 1517, 1370, 1326, 1261, 1222,
1166, 1118, 1067, 1021, 840, 755.HRMS-ESI (m/z): calcd for
m.p. ¼ 155e156 ꢂC. 1H NMR (400 MHz, CDCl3)
d:8.39 (s, 1H),
C
28H20F9N2O4 ([MþH]þ):619.1274; Found:619.1276.
7.70e7.57 (m, 6H), 7.28 (d, J ¼ 2.0 Hz, 1H), 6.89 (d, J ¼ 3.8 Hz, 1H),
6.85 (d, J ¼ 3.8 Hz, 1H), 6.14 (s, 1H), 4.18 (m, 2H), 1.15 (t, J ¼ 7.1 Hz,
4.1.2.12. Ethyl 5-oxo-2-(p-tolyl)-1-(4-(trifluoromethyl)phenyl)-4-((4-
(trifluoromethyl) phenyl)amino)-2,5-dihydro-1H-pyrrole-3-
carboxylate (5-12). 47% yield, white solid, m.p. ¼ 166e167 ꢂC. 1H
3H).13C NMR (101 MHz, CDCl3)
d:163.75, 163.05, 141.30, 141.12,
141.03, 138.84, 129.51, 128.29, 127.89 (q, J ¼ 32.8 Hz), 126.53 (q,
J ¼ 32.4 Hz), 126.22 (q, J ¼ 3.7), 125.73 (q, J ¼ 3.8 Hz), 124.06 (q,

T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
9
J ¼ 270.0 Hz), 123.73 (q, J ¼ 269.6 Hz), 122.38, 122.20, 112.82, 110.85,
60.88, 58.47, 13.82.IR (KBr)
(cmꢀ1): 3282, 2987, 1709, 1646, 1612,
HRMS-ESI (m/z): calcd for C27H20F6N2O3 ([MþH]þ): 535.1444;
n
Found: 535.1444.
1526, 1372, 1326, 1243, 1167, 1121, 1067, 1023, 842, 740.HRMS-ESI
(m/z): calcd for
Found: 619.0124.
C
25H18BrF6N2O3S ([MþH]þ) Calcd: 619.0120;
4.1.2.22. Ethyl 5-oxo-2-phenyl-1-(p-tolyl)-4-(p-tolylamino)-2,5-
dihydro-1H-pyrrole- 3-carboxylate (5-22). 90% yield, white solid,
m.p. ¼ 156e157 ꢂC. 1H NMR (400 MHz, CDCl3)
d:8.16 (s, 1H), 7.33 (d,
4.1.2.17. Ethyl 5-oxo-1,2-diphenyl-4-(phenylamino)-2,5-dihydro-1H-
J ¼ 8.0 Hz, 2H), 7.27e7.16 (m, 6H), 7.07 (m, 5H), 5.76 (s, 1H), 4.01 (q,
pyrrole-3-carboxylate
(5-17). 90%
yield,
white
solid,
J ¼ 7.1 Hz, 2H), 2.33 (s, 3H), 2.22 (s, 3H), 1.01 (t, J ¼ 7.1 Hz, 3H).13
C
m.p. ¼ 113e114 ꢂC; 1H NMR (400 MHz, CDCl3)
d
:8.21 (s, 1H), 7.50 (d,
NMR (101 MHz, CDCl3) d:164.47, 163.88, 137.07, 135.98, 135.30,
J ¼ 8.0 Hz, 2H), 7.42e7.04 (m, 12H), 5.84 (s, 1H), 4.04 (q, J ¼ 7.0 Hz,
134.40,134.00,132.52,129.30,128.96,128.25,127.93,127.66,122.98,
2H), 1.03 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
:164.34,
122.65, 108.70, 63.13, 59.99, 20.90, 20.81, 13.79.IR (KBr) n
164.05, 141.96, 138.67, 136.78, 136.55, 134.12, 128.76, 128.41, 128.33,
128.05, 127.64, 125.62, 124.60, 122.69, 109.79, 63.17, 60.13, 13.76. IR
(cmꢀ1):3032, 2983,2924, 1709, 1632, 1515, 1450, 1393, 1290, 1211,
1110, 1035, 834, 823. The structural characteristics of 5e17 are
consistent with those reported in our previous work [42].
(KBr) n
(cmꢀ1): 3132, 2985, 2927, 1792, 1631, 1398, 1228, 1036, 755.
The structural characteristics of 5e17 are consistent with those
reported in our previous work [31].
4.1.2.23. Ethyl 1-(4-isopropylphenyl)-4-((4-isopropylphenyl)amino)-
5-oxo-2-phenyl-2,5-
dihydro-1H-pyrrole-3-carboxylate
(5-23).
4.1.2.18. Ethyl 1-(4-chlorophenyl)-4-((4-chlorophenyl)amino)-5-oxo-
2-phenyl-2,5-dihydro- 1H-pyrrole-3-carboxylate (5-18). 61% yield,
72% yield, yellow solid, m.p. ¼ 137e138 ꢂC. 1H NMR (400 MHz,
CDCl3) d:8.16 (s, 1H), 7.44e7.28 (m, 3H), 7.25e7.04 (m, 9H), 5.77 (d,
whitesolid, m.p. ¼ 135e136 ꢂC;1H NMR (400 MHz, CDCl3)
d
:8.28 (s,
J ¼ 5.8 Hz,1H), 3.98 (q, J ¼ 7.1 Hz, 2H), 2.85 (m, 2H),1.24 (m, 6H),1.15
1H), 7.46 (d, J ¼ 8.9 Hz, 2H), 7.27 (m, 9H), 7.15 (d, J ¼ 8.6 Hz, 2H), 5.79
(d, J ¼ 6.9 Hz, 6H), 0.98 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
(s, 1H), 4.07 (q, J ¼ 7.1 Hz, 2H), 1.08 (t, J ¼ 7.1 Hz, 3H).13C NMR
d:164.49, 164.08, 146.23, 145.26, 142.29, 137.08, 136.30, 134.20,
(101 MHz, CDCl3)
d:164.28, 163.75, 141.94, 137.02, 136.19, 135.04,
128.28, 127.91, 127.66, 126.72, 126.35, 122.79, 122.73, 108.97, 63.21,
60.00, 33.56, 33.48, 23.97, 23.75, 13.73.IR (KBr)
(cmꢀ1): 3465,
3285, 3034, 2961, 1698,1637, 1516, 1459, 1372, 1290, 1242, 1116,
130.98, 130.04, 128.92, 128.51, 128.49, 128.34, 127.55, 124.11, 123.59,
n
110.42, 62.99, 60.37, 13.82.IR (KBr)
n
(cmꢀ1): 3356, 2921, 2852, 1703,
1642, 1456, 1329, 1239, 1125, 889, 795, 694, 531. HRMS-ESI (m/z):
calcd for C25H20Cl2N2O3 ([MþH]þ): 467.0914; Found: 467.0913.
1032, 832, 756.HRMS-ESI (m/z): calcd for
C31H35N2O3
([MþH]þ):483.2642; Found:483.2639.
4.1.2.19. Ethyl 1-(3-chlorophenyl)-4-((3-chlorophenyl)amino)-5-oxo-
4.1.2.24. Ethyl
1-(4-(tert-butyl)phenyl)-4-((4-(tert-butyl)phenyl)
2-phenyl-2,5-dihydro- 1H-pyrrole-3-carboxylate (5-19). 47% yield,
amino)-5-oxo-2-phenyl- 2,5-dihydro-1H-pyrrole-3-carboxylate (5-
white solid, m.p. ¼166e147 ꢂC. 1H NMR (400 MHz, CDCl3)
d: 8.28 (s,
24). 54% yield, white solid, m.p. ¼ 150e151 ꢂC. 1H NMR (400 MHz,
1H), 7.67 (d, J ¼ 7.1 Hz, 1H), 7.36 (d, J ¼ 8.2 Hz, 1H), 7.34e7.26 (m,
5H), 7.22 (d, J ¼ 10.9 Hz, 2H), 7.16 (t, J ¼ 8.2 Hz, 2H), 7.08 (t,
J ¼ 9.1 Hz, 2H), 5.82 (s, 1H), 4.07 (q, J ¼ 7.1 Hz, 2H), 1.07 (t, J ¼ 7.1 Hz,
CDCl3)
d
:8.21 (s, 1H), 7.41 (d, J ¼ 2.1 Hz, 1H), 7.40 (d, J ¼ 2.1 Hz, 2H),
7.36 (d, J ¼ 2.1 Hz, 1H), 7.34 (s, 1H), 7.30 (d, J ¼ 1.8 Hz, 2H), 7.29 (s,
1H), 7.28e7.25 (m, 2H), 7.24 (m, 1H), 7.16 (d, J ¼ 2.0 Hz, 1H), 7.15 (d,
J ¼ 2.0 Hz, 1H), 5.81 (s, 1H), 4.02 (q, J ¼ 7.1 Hz, 2H), 1.35 (s, 9H), 1.26
3H).13C NMR (101 MHz, CDCl3)
d: 164.07, 163.87, 141.32, 139.82,
137.64, 136.03, 134.50, 134.03, 129.73, 129.35, 128.55, 128.38, 127.57,
125.67, 124.63, 122.72, 122.50, 120.86, 120.19, 111.37, 63.01, 60.47,
(s, 9H), 1.00 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d:164.49,
164.11, 148.47, 147.45, 142.22, 137.11, 136.04, 133.94, 128.30, 127.92,
127.66, 125.66, 125.28, 122.38, 122.32, 109.17, 63.17, 60.01, 34.35,
13.80.IR (KBr)
n
(cmꢀ1): 3449, 1699, 1638, 1591, 1481, 1369, 1271,
1121, 1032, 755.HRMS-ESI (m/z): calcd for
C
25H21Cl2N2O3
34.32, 31.35, 31.16, 13.72.IR (KBr) n
(cmꢀ1): 3286, 2961, 2869, 1699,
([MþH]þ):467.0924; Found:467.0924.
1637, 1518, 1370, 1237, 1121, 1032, 833, 755.HRMS-ESI (m/z): calcd
for C33H39N2O3 ([MþH]þ):511.2955; Found:511.2952.
4.1.2.20. Ethyl 1-(4-bromophenyl)-4-((4-bromophenyl)amino)-5-
oxo-2-phenyl-2,5-dihydro-1H
-pyrrole-3-carboxylate
(5-20).
4.1.2.25. Ethyl
5-oxo-1-(4-phenoxyphenyl)-4-((4-phenoxyphenyl)
68% yield, yellow solid, m.p. ¼ 167e168 ꢂC. 1H NMR (400 MHz,
amino)-2-phenyl-2,5- dihydro-1H-pyrrole-3-carboxylate (5-25).
CDCl3)
d
: 8.28 (s, 1H), 7.40 (m, 6H), 7.27 (q, J ¼ 4.3 Hz, 5H), 7.08 (d,
78% yield, white solid, m.p. ¼ 133e134 ꢂC. 1H NMR (400 MHz,
J ¼ 8.6 Hz, 2H), 5.79 (s, 1H), 4.07 (q, J ¼ 7.1 Hz, 2H), 1.07 (t, J ¼ 7.1 Hz,
3H).13C NMR (101 MHz, CDCl3)
: 164.22, 163.77, 141.75, 137.59,
136.15, 135.58, 131.86, 131.42, 128.54, 128.36, 127.55, 124.36, 123.80,
118.82, 117.66, 110.66, 62.92, 60.40, 13.83.IR (KBr)
(cmꢀ1): 3288,
CDCl3) d:8.32 (s, 1H), 7.40e7.27 (m, 8H), 7.22 (m, 5H), 7.10 (m, 2H),
d
7.04 (d, J ¼ 8.0 Hz, 2H), 6.97 (m, 4H), 6.89 (d, J ¼ 8.8 Hz, 2H), 5.75 (s,
1H), 4.06 (q, J ¼ 7.1 Hz, 2H), 1.07 (t, J ¼ 7.1 Hz, 3H).13C NMR
n
(101 MHz, CDCl3) d:164.58, 163.86, 157.31, 156.76, 155.01, 154.28,
2982, 1701, 1636, 1587, 1530, 1490, 1369, 1277, 1242, 1072, 1031,
755.HRMS-ESI (m/z): calcd for C25H21Br2N2O3 ([MþH]þ):554.9913;
Found:554.9916.
142.83, 136.74, 133.83, 131.60, 129.69, 128.38, 128.12, 127.66, 124.73,
124.67, 123.43, 123.15, 118.96, 118.85, 118.82, 118.72, 108.97, 63.49,
60.11, 13.89.IR (KBr)
n
(cmꢀ1): 3282, 3061, 1701, 1635, 1591, 1505,
1372, 1288, 1234, 1168, 1106, 1031, 837, 755.HRMS-ESI (m/z): calcd
4.1.2.21. Ethyl 5-oxo-2-phenyl-1-(3-(trifluoromethyl)phenyl)-4-((3-
(trifluoromethyl) phenyl)amino)-2,5-dihydro-1H-pyrrole-3-
carboxylate (5-21). 82% yield, white solid,m.p. ¼ 184e185 ꢂC;1H
for C37H31N2O5 ([MþH]þ):583.2227; Found:583.2229.
4.1.2.26. Ethyl 1-(4-methoxyphenyl)-4-((4-methoxyphenyl)amino)-
NMR (400 MHz, CDCl3)
d
:8.37 (s, 1H), 7.83 (s, 1H), 7.73 (d, J ¼ 7.7 Hz,
5-oxo-2-phenyl-2,5-
dihydro-1H-pyrrole-3-carboxylate
(5-26).
1H), 7.53e7.34 (m, 6H), 7.34e7.16 (m, 5H), 5.88 (s, 1H), 4.08 (q,
56% yield, yellow solid, m.p. ¼ 139e140 ꢂC; 1H NMR (400 MHz,
J ¼ 7.1 Hz, 2H), 1.07 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
CDCl3)
d
:8.24 (s, 1H), 7.41e7.09 (m, 9H), 6.88 (d, J ¼ 8.8 Hz, 2H), 6.78
d:164.07, 163.87, 141.35, 138.96, 137.01, 135.74, 131.23 (q,
(d, J ¼ 9.0 Hz, 2H), 5.73 (s, 1H), 4.04 (q, J ¼ 7.1 Hz, 2H), 3.82 (s, 3H),
J ¼ 32.4 Hz), 131.00 (q, J ¼ 32.4 Hz), 129.38, 128.93, 128.62, 128.50,
127.54, 125.85, 125.31, 122.79 (q, 271.1 Hz), 123.57 (q,
J ¼ 271.1 Hz), 122.18, 122.14, 121.17, 119.25, 119.21, 119.00, 118.96,
111.68, 63.03, 60.55, 13.74.IR (KBr)
(cmꢀ1):3302, 2924, 2851, 2309,
1696, 1636, 1497, 1371, 1274, 1233, 1095, 1029, 826, 763, 653, 507.
3.73 (s, 3H), 1.05 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
J
¼
d:164.66, 163.77, 157.32, 157.08, 143.22, 137.12, 131.52, 129.49,
128.26, 127.95, 127.67, 124.93, 124.66, 113.97, 113.64, 107.71, 63.46,
59.93, 55.40, 55.25, 13.87. IR (KBr)
(cmꢀ1):3291, 2926, 2840, 1693,
1631,1512,1293,1240,1179, 1111,1034, 829, 755, 701, 527. HRMS-ESI
n
n

10
T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
(m/z): calcd for C27H26N2O5 ([MþH]þ): 459.1914; Found:
459.1912.The structural characteristics of 5e26 are consistent with
those reported in the literatures [48].
MTT-based assay as described above. In addition, we observed the
protection of 5e2 (40 M) against virus-induced CPE by micro-
scopy. Independent experiments were performed at least three
times.
m
4.1.2.27. Ethyl
1-cyclohexyl-4-(cyclohexylamino)-5-oxo-2-phenyl-
2,5-dihydro-1H-pyrrole- 3-carboxylate (5-27). 48% yield, white
4.2.3. Plaque reduction assay
Confluent monolayers of MDCK cells were infected with influ-
enza PR8 virus at 100TCID50 for 1 h. The infected-cells were
solid,m.p. ¼ 93e94 ꢂC, 1H NMR (400 MHz, CDCl3)
d
:7.41e7.08 (m,
5H), 6.69 (s, 1H), 5.09 (s, 1H), 4.66 (m, 1H), 4.16e3.79 (m, 2H), 3.65
(t, J ¼ 11.8 Hz, 1H), 2.05 (m, 2H), 1.90e1.58 (m, 7H), 1.58e1.39 (m,
4H), 1.35e1.06 (m, 6H), 1.06e0.84 (m, 4H).13C NMR (101 MHz,
cultured with 1.5 ml of serum-free MEM containing 1 mg/ml TPCK,
2% agar (Sigma-Aldrich, USA) and serially diluted 5e2 for another
48e72 h as previously described after removing the unabsorbed
virus. Subsequently, cells were fixed with 4% paraformaldehyde
and then stained with crystal violet (Sigma-Aldrich, USA). The in-
hibition effect of the compound on viral plaque formation was
measured by the amount of plaques.
CDCl3)
61.33, 59.06, 54.12, 50.37, 34.93, 34.73, 30.81, 30.47, 25.95, 25.81,
25.49, 25.19, 24.72, 13.99.IR (KBr)
(cmꢀ1):3324, 2930, 2856, 1686,
d:165.27, 165.24, 138.94, 127.92, 127.88, 127.65, 102.92, 99.92,
n
1622, 1448, 1367, 1270, 1212, 1088, 893, 825, 763, 700. HRMS-ESI
(m/z): calcd for C25H34N2O3 ([MþH]þ): 411.2642; Found: 411.2638.
4.1.2.28. Methyl
5-oxo-2-phenyl-1-(4-(trifluoromethyl)phenyl)-4-
4.2.4. Time-of-addition assay
((4-(trifluoromethyl) phenyl)amino)-2,5-dihydro-1H-pyrrole-3-
To investigate which stage of influenza replication was inter-
vened by the 5e2, we performed time-of-addition methods as
previously reported with minor modifications [45], named pre-
infection treatment, post-infection treatment and entire infection
respectively. After 48 h p.i., culture supernatants of each group
were collected to detect the antiviral effect of 5e2. In addition,
MDCK cells were infected with the influenza PR8 virus for 1 h. After
carboxylate (5-28). 63% yield, white solid, m.p. ¼ 159e160 ꢂC; 1H
NMR (400 MHz, CDCl3)
d
:8.35 (s,1H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.61 (d,
J ¼ 8.5 Hz, 2H), 7.54 (d, J ¼ 8.7 Hz, 2H), 7.36e7.21 (m, 7H), 5.89 (s,
1H), 3.64 (s, 3H).13C NMR (101 MHz, CDCl3)
:164.32, 163.89, 141.48,
141.21, 139.44, 135.63, 128.77, 128.58, 127.33, 126.05, 126.01, 125.75,
125.71, 122.04, 121.67, 112.45, 62.78, 51.44.IR (KBr)
(cmꢀ1):3291,
d
n
2923, 2308, 1705, 1613, 1530, 1323, 1243,1120, 1067, 837, 758, 695,
497. HRMS-ESI (m/z): calcd for C26H18F6N2O3 ([MþH]þ): 521.1286;
Found: 521.1286.
removing unabsorbed virus, the cells were treated with 40 mM
5e2 at indicated time intervals (0e2, 2e5, 5e8, 8e10 and 0e10 h),
which covered one cycle of influenza virus replication. At 10 h p.i.,
the expression level of viral NP protein and HA mRNA was deter-
mined by western blotting or qRT-PCR.
4.1.2.29. Methyl
5-oxo-2-phenyl-1-(3-(trifluoromethyl)phenyl)-4-
((3-(trifluoromethyl).
phenyl)amino)-2,5-dihydro-1H-pyrrole-3-carboxylate (5-29). 59%
4.2.5. Immunofluorescence microscopy
yield, white solid, m.p. ¼ 123e124 ꢂC;1H NMR (400 MHz, CDCl3)
d:
Immunofluorescence staining was conducted with minor al-
terations as described previously [46]. Briefly, MDCK cells in 48-
well plates infected with the influenza PR8 virus at 100TCID50
were treated with different concentrations of 5e2. At 24 h p.i., cells
were washed twice with cold PBS and fixed with 4% para-
formaldehyde for 10 min, followed by blocking for 1 h at room
temperature with 3% bovine serum albumin (BSA). Cells were
incubated with anti-IAV NP antibody (1:250 dilution, Santa Cruz,
CA, USA) at 4 ꢂC overnight and with a fluorescein isothiocyanate
(FITC)-labeled secondary antibody (1:250 dilution, Santa Cruz, CA,
USA) for another 1 h at room temperature. Cell nucleus was visu-
alized by counterstaining with DAPI. The samples were captured on
8.35 (d, J ¼ 7.5 Hz,1H), 7.84 (d, J ¼ 5.8 Hz,1H), 7.72 (d, J ¼ 7.4 Hz,1H),
7.43 (m, 6H), 7.30 (s, 4H), 7.26 (s, 1H), 5.89 (d, J ¼ 3.1 Hz, 1H), 3.62 (s,
3H).13C NMR (101 MHz, CDCl3)
d:164.36, 163.87, 141.35, 138.92,
136.96,135.70,131.22 (q, J ¼ 32.5 Hz),130.98 (q, J ¼ 32.5 Hz),129.40,
128.93, 128.72, 128.54, 127.42, 126.03, 125.34, 123.81 (q, J ¼ 271 Hz),
123.59 (q, J ¼ 271 Hz), 122.22 (q, J ¼ 3.8 Hz), 121.29 (q, J ¼ 3.6 Hz),
119.37 (q, J ¼ 3.7 Hz), 119.04 (q, J ¼ 3.9 Hz), 111.26, 63.03, 51.38. The
structural characteristics of 5e29 are consistent with those re-
ported in our previous work [31].
4.2. Biological assays
a
Ti-Eclipse inverted fluorescence microscope (Nikon, Tokyo,
4.2.1. Cytotoxicity assay
Japan).
The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium-
bromide (MTT; Sigma, USA) assay was performed to assess the
cytotoxicity of compound 5e2. Briefly, MDCK or A549 cells were
seeded into 96-well tissue culture plates at 2 ꢁ 104 cells per well
and incubated overnight at 37 ꢂC with 5% CO2. 5e2 (concentrations
4.2.6. Western blotting assay
Two-fold serial dilutions 5e2 was added to infected-MDCK cells
after the adsorption of the influenza virus as described above. The
cells were harvested at 24 h p.i. and then equal amounts of protein
range from 1.56 to 400
quently, cells were incubated with MTT solutions at 37 ꢂC for 4 h
and added with 150 L DMSO to dissolve the formed formazan
m
M) was added into cells for 48 h. Subse-
lysate (50 mg) were separated by 10% SDS-PAGE. Polyvinylidenedi-
fluoride (PVDF) membrane were incubated overnight at 4 ꢂC with
m
primary antibodies against NP (1:2000 dilution, Abcam, Cambridge,
crystals. The absorbance at 570 nm was measured by a microplate
reader (GENios Pro, TECAN, Bedford, MA, USA) to calculate the 50%
cytotoxic concentration (CC50) of the 5e2 using the CalcuSyn
software.
MA, USA) and b-actin (1:1000 dilution, Cell Signaling Technology,
Beverly, MA, USA) as control after transfer. Hereafter, the anti-
mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated
secondary antibodies (1:2000 dilution, Cell Signaling Technology,
Beverly, MA, USA) were incubated at room temperature for 1 h.
Chemiluminescent signals of protein bands were detected with
lumiglo reagent (Cell Signaling Technology, Beverly, MA, USA) using
a FluorChem E imaging system (ProteinSimple, Silicon Valley, CA,
USA).
4.2.2. In vitro anti-influenza virus assay
MDCK cells were infected with different IAV at 100TCID50 for
1 h, the cells were then washed twice with PBS after removing the
unabsorbed virus and cultured with DMEM containing two-fold
serial dilutions 5e2 and 1 mg/ml TPCK for 48 h. Ribavirin (Sigma,
purity of 99%) was used as a positive control. Subsequently, the IC50
4.2.7. Quantitative real-time PCR assay
value of 5e2 was computed to investigate antiviral activity through
The levels of different gene were detected by qRT-PCR as

T. Liu et al. / European Journal of Medicinal Chemistry 199 (2020) 112334
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primer sequences for each gene were presented in supplemen-
tary data.
4.2.8. Statistical analysis
Statistical analyses were performed using GraphPad Prism (6.0).
The data were presented as mean standard deviation (SD) from
experiments in triplicate. Comparison of different groups was
performed using the Student’s t-test, followed by one-way ANOVA
with or without Tukey-Kramer for multiple comparisons. A value of
p < 0.05 was considered significant and marked with asterisk (*).
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
This work was supported by the Natural Science Foundation of
Guangdong Province, China (No.2019A1515011550) and the Na-
tional Natural Science Foundation of China (61975074).
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