Synthesis and antiviral activity of lycorine derivatives

Article abstract of DOI:10.1080/10286020.2020.1844674

There are no effective antiviral drugs to treat hand, foot, and mouth disease. In this study, a series of lycorine derivatives were synthesized and evaluated against enterovirus 71 and coxsackievirus A16 in?vitro. Derivatives 7c-m with the phenoxyacyl gro

Full text of DOI:10.1080/10286020.2020.1844674

Journal of Asian Natural Products Research
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Synthesis and antiviral activity of lycorine
derivatives
Ya-Jun Yang , Jiang-Ning Liu & Xian-Dao Pan
To cite this article: Ya-Jun Yang , Jiang-Ning Liu & Xian-Dao Pan (2020) Synthesis and antiviral
activity of lycorine derivatives, Journal of Asian Natural Products Research, 22:12, 1188-1196, DOI:
10.1080/10286020.2020.1844674
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
2020, VOL. 22, NO. 12, 1188–1196
https://doi.org/10.1080/10286020.2020.1844674
Synthesis and antiviral activity of lycorine derivatives
Ya-Jun Yanga, Jiang-Ning Liub and Xian-Dao Pana
aBeijing Key Laboratory of Active Substance Discovery and Drug ability Evaluation Institute of
Material Medical, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
b
100050, China; Key Laboratory of Human Disease Comparative Medicine, National Health
Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medical College,
Chinese Academy of Medical Sciences, Beijing 100050, China
ABSTRACT
ARTICLE HISTORY
Received 31 July 2020
Accepted 28 October 2020
There are no effective antiviral drugs to treat hand, foot, and
mouth disease. In this study, a series of lycorine derivatives were
synthesized and evaluated against enterovirus 71 and coxsackievi-
rus A16 in vitro. Derivatives 7c-m with the phenoxyacyl group at
the C-1 position showed higher efficacy and lower toxicity than
lycorine. In addition, derivative 7e enhanced the survival rate to
40% in the mouse model of the lethal EV71 infection.
KEYWORDS
Lycorine; hand; foot; and
mouth disease; enterovirus
71; coxsackievirus A16
1. Introduction
After severe outbreaks in the past two decades, hand, foot, and mouth disease
(HFMD) has become a highly infectious illness for global public health [1, 2]. This
disease was common in children, who are younger than five years old, and could be
diagnosed in adults occasionally [3]. HFMD were usually benign, mild, and self-limit-
ing. However, serious cardiovascular and neurological complications could also occur
in some cases, which might be fatal to children [4]. Coxsackievirus A16 (CAV16) and
enterovirus 71 (EV71) were two major causes of HFMD [5]. There was an evidence
that EV71 was associated with serious complications [6]. Since 2007, HFMD has been
prevalent in China. More than 20 million cases and 3000 deaths were reported
CONTACT Jiang-Ning Liu
National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medical College,
Chinese Academy of Medical Sciences, Beijing 100021, China; Xian-Dao Pan xdp@imm.ac.cn Beijing Key
ljn_zb03038@126.com
Key Laboratory of Human Disease Comparative Medicine,
Laboratory of Active Substance Discovery and Drug ability Evaluation Institute of Material Medical, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing 100050, China
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
1189
Figure 1. The structure of lycorine.
between 2008 and 2018 [7]. An EV71 vaccine was approved for outbreaks of HFMD
in 2016 [8]. It not only did not treated infected patients but also not offer cross-pro-
tection caused by other pathogens. Up to now, there are no effective antiviral drugs
to treat HFMD.
In order to discover novel lead compounds for the treatment of HFMD, we carried
out the screening study of natural products according to an in-house collection. Some
tannins and alkaloids were identified to possess anti-EV71 activity in both in vitro
and in vivo, including geraniin [9], punicalagin [10], chebulagic acid [11], deferox-
amine [12], matrine [13], oxysophocarpine [14], and lycorine [15]. Among them,
lycorine (Figure 1) displayed the most effective potency against EV71, and moderate
cytotoxicity was also observed. Furthermore, its mechanism was associated with the
inhibition of viral proteases and the down-regulation of autophagy [15–17].
Encouraged by these results, we attempted to investigate the structure-activity rela-
tionship (SAR) study of lycorine to discover more potent derivatives with less cyto-
toxicity. In this work, a series of lycorine derivatives were designed and synthesized,
and their inhibitory activities against EV71 and CAV16 were evaluated both in
in vitro and in vivo.
2. Results and discussion
2.1. Chemistry
Starting from a commercially available lycorine (1), the target compounds were syn-
thesized according to the reported procedures (Scheme 1). Briefly, the hydrogenation
of lycorine hydrochloride afforded dihydrolycorine (2) [18]. Considering the differ-
ence of the C1- and C2-hydroxyls, 2-acetyllycorine 3, and 2-benzoyllycorine 4 were
synthesized by 1 eq acetic anhydride or benzoyl chloride, respectively. By more than
2 eq acylation reagents, the diesters 5 and 6 were achieved [19]. 1-acetyllycorine 7a
was prepared by the selective removal of the acetyl group at the C-2 position [20].
The oxidation of 5a with PhI(OAc)2 led to lactam 8, which was further hydrolyzed to
yield 9 [19]. The phenanthridine derivative 10 was obtained by the methylation and
Hoffman degradation of 1 [21]. The hydrogenation of compound 10 in reflux MeOH
(Methanol) afforded 11. The selective oxidation of 5a using the Dess–Martin reagent
afforded 2-O-lycorine 12, which was subsequently acylated to provide 13 [19]. The
treatment of 1 with TBSCl (tert-Butyldimethylsilyl chloride) afforded 2-TBS-lycorine

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Y.-J. YANG ET AL.
Scheme 1. The synthesis of lycorine derivatives. Reagents and conditions: a, 10% Pd/C, H2, H2O, rt;
b, Ac2O or PhCOCl (1.0 eq), Py, rt; c, Ac2O or PhCOCl (2.5 eq), Py, rt; d, HCl, MeOH, 60 ꢀC; e, PhIO,
TBAI, CH3CN/H2O 9:1, rt; f, K2CO3, MeOH/H2O 9:1, 60 ꢀC; g, MeI, DMF, rt then t-BuOK, t-BuOH,
reflux; h, 10% Pd/C, H2, MeOH, reflux; i, DMP, DMF, rt; j, Ac2O, Py, rt; k, TBSCl, AgNO3, TEA, DMF,
rt; l, acids, EDCI, DMAP, DCM, rt then TBAF, THF, rt.
14 with regioselectivity [19], which could be easily transformed to the monosubsti-
tuted ethers 7b–o at the C-1 position.
2.2. The cytotoxicity and antiviral activity in vitro
The synthesized lycorine derivatives were evaluated against EV71 and CAV16 in
human rhabdomyosarcoma (RD) cells. The CC50 (50% cytotoxic concentration) val-
ues for cytotoxicity and EC50 (50% effective concentration) values for antiviral activity
were measured as described previously. The selectivity index (SI) was calculated using
the ratio of CC50 and EC50. As a positive control, lycorine (1) showed potent

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1191
Table 1. The antiviral activity of lycorine derivatives against EV71 and CAV16 in vitro.
EV71
CAV16
Comp.
CC50 (mM)
EC50 (mM)
SI
EC50 (mM)
SI
1
2
3
4
5
6
7a
7b
7c
8
52.26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.26
>100
6.08
51.15
28.30
>100
2.42
23.12
–
>16.45
>1.96
>3.53
–
>41.32
>7.82
>60.98
–
1.57
31.14
>100
>100
>100
>100
2.58
33.27
>3.21
–
–
–
–
>38.76
>7.82
>84.03
–
>1.58
–
12.79
1.64
12.79
1.19
>100
>100
>100
>100
70.18
30.58
>100
63.12
>100
>100
70.18
30.58
9
–
–
–
10
11
12
13
–
>1.42
>3.27
>1.42
>3.27
inhibitory activity against EV71 and CAV16 with EC50 values of 2.26 and 1.57 mM,
respectively. Meanwhile, its cytotoxicity was 52.26 mM, leading to moderate SIs. Except 7g,
significantly decreased cytotoxicity (> 100 mM) was observed for the synthesized lycorine
derivatives, suggesting that these derivatives might be less toxicity than lycorine. As shown
in Table 1, for inhibitory activity against EV71, dihydrolycorine (2) displayed a loss of
activity, indicating that the double bond at the C-3 position played an important role.
Unexpectedly, a moderate activity for 2 was observed against CAV16. In general, 2-esters
(3 and 4) and 1,2-diesters (5 and 6) resulted in a reduction or a loss of activity. The deriv-
atives (8 and 9) oxidized at the C-6 position were totally inactive. Unfortunately, the activ-
ity of phenanthridine derivatives (10 and 11) also decreased. The 2-ketone derivatives (12
and 13) were reported to increase in an antiviral activity against dengue virus [20], but
the poor activity was observed for them against EV71 and CAV16. Compared with lycor-
ine, 1-acetyllycorine 7a showed similar activity against EV71 and CAV16 with improved
SI, but derivative 7b showed weaker activity. It suggested that the C-1 position might be
sensitive to the antiviral activity. Notably, when the phenoxyacyl group (7c) was intro-
duced at the C-1 position, an increase in both antiviral activity and SI was observed with
EC50 values of 1.19 mM for CAV16 and 1.64 mM for EV71.
To further investigate the effects of substitutions in the phenoxyacyl group, deriva-
tives 7d–n were designed and synthesized. Moreover, both antiviral activity and SI
were improved again (Table 2). The introduction of electro-withdrawing groups (7d,
7e, 7j) was usually superior to electron-donating groups (7k, 7l, 7m). The para-sub-
stitution of benzene ring (7d) was more suitable than ortho- (7i) and meta-substitu-
tion (7h). The change of benzene ring (7c) to naphthalene ring (7n) led to a slight
decrease. Among them, derivatives 7d, 7e, and 7j displayed good inhibitory effects
against EV71 with EC50 values in the range between 1.23 and 1.28 mM.
On the basis of above results, a preliminary SAR could be described. Modifications
on the double bond at the C-3 position and the methylene group at the C-6 position
were unfavorable for the activity. The maintenance of molecular scaffold and C2-
hydroxyl was also important for activity. It was suggested that further modification
was focused on the C1-hydroxyl.

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Y.-J. YANG ET AL.
Table 2. The antiviral activity of lycorine derivatives against EV71 in vitro.
EV71
Comp.
CC50 (mM)
EC50 (mM)
SI
7d
7e
7f
7g
7h
7i
7j
7k
7l
131.19
153.84
103.43
99.64
151.56
145.24
105.38
161.12
134.22
101.25
163.41
1.27
1.28
1.47
1.84
1.73
1.63
1.23
1.65
1.53
1.67
1.97
103.30
139.85
70.36
54.15
87.61
89.10
85.67
97.65
87.73
60.63
82.94
7m
7n
2.3. The treatment effect of 7e in the mouse model
Considering the good inhibitory effect and high SI in vitro, derivative 7e was selected
to further investigate the effect in the mouse model of the lethal EV71 infection
(Figure 2). All mice died within 10-day post infection (dpi) in the saline-treated
group. Ribavirin and lycorine, as control drugs, prolonged the survival rate of mice to
10% and 30%, respectively. As expected, derivative 7e enhanced the survival time of
infected mice to 40% under the treatment of 1.0 mg/kg dose. Compared to the saline-
treated group, treatment with 7e reduced the clinical scores by delaying the appear-
ance of symptoms, including weakness and paralysis in hind limbs.
3. Conclusion
In this study, a series of lycorine derivatives were synthesized and evaluated against
EV71 and CAV16 in vitro. Compared to lycorine, derivatives 7c–m with the phenox-
yacyl group at the C-1 position showed higher efficacy and lower toxicity. In add-
ition, derivative 7e enhanced the survival rate to 40% in the mouse model of the
lethal EV71 infection. These results provided a potential lead for the treatment
of HFMD.
4. Experimental
4.1. Chemistry
All chemical reagents and solvents were commercially available without further purifi-
cation. With tetramethylsilane (TMS) as internal standard, 1H-NMR spectra were
recorded on Varian Mercury 400 MHz spectrometers (California, USA). The API-
TOFMS 10000 instrument was used to measure mass spectra with HR-API mode
(Guangzhou, China). Compounds 2–14 were synthesized according to reported meth-
ods [18–21].
A solution of 14 (1 mmol), acids (1.2 mmol), EDCI (1-Ethyl-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride, 1.2 mmol), and DMAP (Dimethylaminopyridine,
0.12 mmol) in DCM (Dichloromethane, 15 ml) was stirred at room temperature for
12 h. Then, 50 ml DCM was added. The organic phase was washed with saturated

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
1193
Figure 2. The treatment effect of 7e in the mouse model. (A) The survival rates. (B) The clin-
ical scores.
NaHCO3 (20 ml), H2O (20 ml), and brine (20 ml). Dried over Na2SO4, the solvent
was evaporated. The residue was dissolved in 15 ml THF (Tetrahydrofuran) and
2 mmol TBAF (Tetrabutylammonium fluoride) was added. After the reaction, 50 ml
DCM was added. The organic phase was washed with saturated NaHCO3 (20 ml),
H2O (20 ml), and brine (20 ml). After evaporation under reduced pressure, the resi-
due was purified using silica gel column chromatography to afford compounds 7c–n.
4.1.1. 1-(Phenoxyacyl)-lycorine (7c)
1
Yield: 53.4%. H NMR (CDCl3): d 7.16–7.12 (m, 2H), 6.93–6.89 (m, 1H), 6.68–6.66
(m, 3H), 6.53 (s, 1H), 5.93 (d, J ¼ 8.0 Hz, 2H), 5.75 (s, 1H), 5.51 (s, 1H), 4.51 (s, 2H),
4.21 (s, 1H), 4.08 (d, J ¼ 16 Hz, 1H), 3.35–3.30 (m, 2H), 2.85 (d, J ¼ 8.0 Hz, 1H), 2.59
(m, 2H), 2.52 (d, J ¼ 8.0 Hz, 1H), 2.27 (q, J ¼ 8.0 Hz, 1H). HR-API-MS: m/z 422.1606
[M þ H]þ (calcd for C24H24NO6, 422.1598).
4.1.2. 1-(4-F-Phenoxyacyl)-lycorine (7d)
1
Yield: 45.6%. H NMR (CDCl3): d 6.82–6.78 (m, 2H), 6.60–6.54 (m, 4H), 5.96 (s,
1H), 5.91 (s, 1H), 5.69 (s, 1H), 5.49 (s, 1H), 4.47 (s, 2H), 4.16 (s, 1H), 4.13–4.11 (m,
1H), 3.34–3.31 (m, 2H), 2.84 (d, J ¼ 8.0 Hz, 1H), 2.57 (m, 2H), 2.51 (d, J ¼ 12.0 Hz,
1H), 2.28 (q, J ¼ 8.0 Hz, 1H). HR-API-MS: m/z 440.1517 [M þ H]þ (calcd for
C24H23FNO6, 440.1504).
4.1.3. 1-(4-Cl-phenoxyacyl)-lycorine (7e)
1
Yield: 84.1%. H NMR (CDCl3): d 7.08–7.04 (m, 2H), 6.66 (s, 1H), 6.58–6.54 (m,
3H), 6.00 (d, J ¼ 4.0 Hz, 1H), 5.94 (d, J ¼ 4.0 Hz, 1H), 5.76 (s, 1H), 5.52 (s, 1H), 4.52
(s, 2H), 4.20 (s, 1H), 4.10 (d, J ¼ 12.0 Hz, 1H), 3.36–3.28 (m, 2H), 2.85 (d,
J ¼ 12.0 Hz, 1H), 2.59 (m, 2H), 2.41 (d, J ¼ 8.0 Hz, 1H), 2.27 (q, J ¼ 8.0 Hz, 1H). HR-
API-MS: m/z 456.1222 [M þ H]þ (calcd for C24H23ClNO6, 456.1214).

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Y.-J. YANG ET AL.
4.1.4. 1-(4-Br-phenoxyacyl)-lycorine (7f)
1
Yield: 48.6%. H NMR (CDCl3): d 7.17 (d, J ¼ 8.0 Hz, 2H), 6.60 (s, 1H), 6.57 (s, 1H),
6.49 (d, J ¼ 8.0 Hz, 2H), 5.99 (s, 1H), 5.93 (s, 1H), 5.72 (s, 1H), 5.50 (s, 1H), 4.50 (s,
2H), 4.17 (s, 1H), 4.08 (d, J ¼ 8.0 Hz, 1H), 3.32–3.26 (m, 2H), 2.83 (d, J ¼ 12.0 Hz,
1H), 2.58 (s, 2H), 2.39 (s, 1H), 2.25 (s, 1H). HR-API-MS: m/z 500.0691 [M þ H]þ
(calcd for C24H23BrNO6, 500.0703).
4.1.5. 1-(4-I-Phenoxyacyl)-lycorine (7g)
1
Yield: 48.4%. H NMR (CDCl3): d 7.34 (d, J ¼ 8.0 Hz, 2H), 6.59 (s, 2H), 6.37 (d,
J ¼ 8.0 Hz, 2H), 5.99 (s, 1H), 5.93 (s, 1H), 5.70 (s, 1H), 5.49 (s, 1H), 4.49 (s, 2H), 4.15
(s, 1H), 4.08 (d, J ¼ 12.0 Hz, 1H), 3.33–3.23 (m, 2H), 2.81 (d, J ¼ 8.0 Hz, 1H), 2.57 (s,
2H), 2.36 (m, 1H), 2.24 (m, 1H). HR-API-MS: m/z 548.0568 [M þ H]þ (calcd for
C24H23INO6, 548.0565).
4.1.6. 1-(3-Cl-phenoxyacyl)-lycorine (7h)
1
Yield: 56.4%. H NMR (CDCl3): d 7.04–7.01 (m, 1H), 6.89–6.88 (m, 1H), 6.73 (s,
1H), 6.60 (s, 1H), 6.53–6.51 (m, 2H), 5.94 (s, 1H), 5.92 (s, 1H), 5.70 (s, 1H), 5.50 (s,
1H), 4.51 (s, 2H), 4.18 (s, 1H), 4.09 (d, J ¼ 8.0 Hz, 1H), 3.35–3.32 (m, 2H), 2.85 (d,
J ¼ 8.0 Hz, 1H), 2.59 (s, 2H), 2.50 (s, 1H), 2.31 (s, 1H). HR-API-MS: m/z 456.1219
[M þ H]þ (calcd for C24H23ClNO6, 456.1214).
4.1.7. 1-(2-Cl-phenoxyacyl)-lycorine (7i)
1
Yield: 64.3%. H NMR (CDCl3): d 7.28 (m, 1H), 6.97–6.94 (m, 1H), 6.85–6.82 (m,
1H), 6.57 (s, 1H), 6.50–6.47 (m, 2H), 5.95 (s, 1H), 5.91 (s, 1H), 5.68 (s, 1H), 5.46 (s,
1H), 4.61 (d, 2H), 4.19 (s, 1H), 4.14–4.06 (m, 2H), 3.33–3.32 (m, 2H), 2.83 (d,
J ¼ 8.0 Hz, 1H), 2.57 (s, 4H), 2.31 (s, 1H). HR-API-MS: m/z 456.1224 [M þ H]þ
(calcd for C24H23ClNO6, 456.1214).
4.1.8. 1-(4-No2-phenoxyacyl)-lycorine (7j)
1
Yield: 43.5%. H NMR (CDCl3): d 7.02 (d, J ¼ 4.0 Hz, 2H), 6.67 (d, J ¼ 8.0 Hz, 2H),
6.55 (s, 1H), 6.51 (s, 1H), 6.02 (s, 1H), 5.92 (s, 1H), 5.71 (s, 1H), 5.51 (s, 1H), 4.63
(d, J ¼ 16.0, 12.0 Hz, 2H), 4.19 (s, 1H), 4.08 (d, J ¼ 12.0 Hz, 1H), 3.33–3.31 (m, 1H),
3.24 (d, J ¼ 12.0 Hz, 1H), 2.83 (d, J ¼ 8.0 Hz, 1H), 2.59 (s, 2H), 2.43 (s, 1H), 2.24 (s,
1H). HR-API-MS: m/z 467.1453 [M þ H]þ (calcd for C24H23N2O8, 467.1449).
4.1.9. 1-(4-Meo-phenoxyacyl)-lycorine (7k)
1
Yield: 84.5%. H NMR (CDCl3): d 6.67–6.58 (m, 5H), 6.53 (s, 1H), 5.94 (s, 1H), 5.91
(s, 1H), 5.71 (s, 1H), 5.50 (s, 1H), 4.46 (s, 2H), 4.18 (s, 1H), 4.09 (d, J ¼ 8.0 Hz, 1H),
3.73 (s, 3H), 3.36–3.31 (m, 2H), 2.84 (d, J ¼ 8.0 Hz, 1H), 2.59 (s, 2H), 2.55 (s, 1H),
2.29 (s, 1H). HR-API-MS: m/z 452.1706 [M þ H]þ (calcd for C25H26NO7, 452.1709).
4.1.10. 1-(4-Me-phenoxyacyl)-lycorine (7l)
1
Yield: 55.3%. H NMR (CDCl3): d 6.91 (d, J ¼ 8.0 Hz, 2H), 6.62 (s, 1H), 6.55–6.63
(m, 3H), 5.94 (s, 1H), 5.92 (s, 1H), 5.71 (s, 1H), 5.50 (s, 1H), 4.48 (s, 2H), 4.16 (s,
1H), 4.08 (d, J ¼ 12.0 Hz, 1H), 3.32–3.31 (m, 2H), 2.84 (d, J ¼ 8.0 Hz, 1H), 2.58 (s,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
1195
2H), 2.51 (s, 1H), 2.28–2.27 (m, 1H), 2.24 (s, 3H). HR-API-MS: m/z 436.1753
[M þ H]þ (calcd for C25H26NO6, 436.1760).
4.1.11. 1-(4-Iso-Piv-phenoxyacyl)-lycorine (7m)
1
Yield: 59.8%. H NMR (CDCl3): d 7.00 (d, J ¼ 8.0 Hz, 2H), 6.66 (s, 1H), 6.59 (d,
J ¼ 8.0 Hz, 2H), 6.55 (s, 1H), 5.94 (s, 1H), 5.93 (s, 1H), 5.73 (s, 1H), 5.50 (s, 1H), 4.46
(s, 2H), 4.19 (s, 1H), 4.10 (d, J ¼ 8.0 Hz, 1H), 3.36–3.32 (m, 2H), 2.95–2.80 (m, 2H),
2.59 (m, 2H), 2.29 (s, 1H), 2.04 (s, 1H), 2.20 (d, 6H). HR-API-MS: m/z 464.2053
[M þ H]þ (calcd for C27H30NO6, 464.2040).
4.1.12. 1-(2-Naphoxyacyl)-lycorine (7n)
1
Yield: 59.6%. H NMR (CDCl3): d 7.73 (d, J ¼ 4.0 Hz, 1H), 7.65 (d, J ¼ 4.0 Hz, 1H),
7.49–7.47 (m, 1H), 7.42–7.39 (m, 1H), 7.35–7.32 (m, 1H), 7.06–7.04 (m, 1H), 6.78 (s,
1H), 6.63 (s, 1H), 6.38 (s, 1H), 5.88 (s, 1H), 5.87 (s, 1H), 5.73 (s, 1H), 5.46 (s, 1H),
4.66 (d, 2H), 4.20 (s, 1H), 3.95 (d, J ¼ 8.0 Hz, 1H), 3.25–3.22 (m, 1H), 3.05–3.02 (m,
1H), 2.80 (d, J ¼ 8.0 Hz, 1H), 2.52–2.36 (m, 3H), 2.10 (s, 1H). HR-API-MS: m/z
472.1767 [M þ H]þ (calcd for C28H26NO6, 472.1760).
4.2. Antiviral and cytotoxicity assay in RD cells
An EV71 strain FY0805 (HQ882182) and a CAV16 strain Shzh05-1 (262658) were
used in RD cells. The CC50 (50% cytotoxic concentration) values for cytotoxicity and
EC50 (50% effective concentration) values for the antiviral activity were measured as
described previously [10].
4.3. Mouse protection assay
Each ICR 10-day-old mouse (n ¼ 40) was intraperitoneally (i.p.) inoculated with
1 ꢁ 107 TCID50 of MP10 (HQ712020). After 2 h, different concentrations of ribavirin,
lycorine, and 7e in saline were injected to the infected mice with once daily for
6 days. The survival rates and symptoms were monitored daily for 2 weeks. The clin-
ical scores of infected mice were graded according to previously described meth-
ods [10].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was funded by National Natural Science Foundation of China (81502918), the Drug
Innovation Major Project (2018ZX09711-001-005), the Fundamental Research Funds for the
Central Universities (2016ZX350015) and the National Science and Technology Major Project
of the Ministry of Science and Technology of China (2018ZX09721003-008-026).

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