Design, synthesis and biological evaluation of honokiol derivatives as influenza neuraminidase inhibitors

Article abstract of DOI:10.1080/10286020.2018.1509854

Honokiol, a natural polyphenol, which was reported to have satisfactory influenza neuraminidase (NA) inhibitory activity, was structurally modified. Twenty-three compounds were synthesized and the ortho-effects in the epoxidation and hydrolyzation reactions were studied. The derivatives were evaluated for NA inhibitory activity and the benzoylhydrazone derivatives showed much better anti-NA activity than honokiol. Structure-activity relationship analysis suggested that the polyphenols exhibited better anti-NA activity than monophenols and biphenols. Furthermore, probable binding mode of drug with target revealed that the most active compound had much stronger interactions with the active site of NA than honokiol suggesting the potent anti-influenza virus activity.

Full text of DOI:10.1080/10286020.2018.1509854

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Design, synthesis and biological evaluation of
honokiol derivatives as influenza neuraminidase
inhibitors
Ding Lin, Yang-Jie Yi, Meng-Wu Xiao, Jia Chen, Jiao Ye, Ai-Xi Hu, Wen-Wen
Lian, Ai-Lin Liu & Guan-Hua Du
To cite this article: Ding Lin, Yang-Jie Yi, Meng-Wu Xiao, Jia Chen, Jiao Ye, Ai-Xi Hu, Wen-Wen
Lian, Ai-Lin Liu & Guan-Hua Du (2018): Design, synthesis and biological evaluation of honokiol
derivatives as influenza neuraminidase inhibitors, Journal of Asian Natural Products Research,
DOI: 10.1080/10286020.2018.1509854
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1509854
Design, synthesis and biological evaluation of honokiol
derivatives as influenza neuraminidase inhibitors
Ding Lina, Yang-Jie Yia, Meng-Wu Xiaoa, Jia Chena, Jiao Yea, Ai-Xi Hua, Wen-Wen
Lianb, Ai-Lin Liub and Guan-Hua Dub
aCollege of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China;
bInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China
ABSTRACT
ARTICLE HISTORY
Received 20 May 2018
Revised 6 August 2018
Accepted 6 August 2018
Honokiol, a natural polyphenol, which was reported to have satis-
factory influenza neuraminidase (NA) inhibitory activity, was struc-
turally modified. Twenty-three compounds were synthesized and
the ortho-effects in the epoxidation and hydrolyzation reactions
were studied. The derivatives were evaluated for NA inhibitory
activity and the benzoylhydrazone derivatives showed much bet-
ter anti-NA activity than honokiol. Structure-activity relationship
analysis suggested that the polyphenols exhibited better anti-NA
activity than monophenols and biphenols. Furthermore, probable
binding mode of drug with target revealed that the most active
compound had much stronger interactions with the active site of
NA than honokiol suggesting the potent anti-influenza
virus activity.
KEYWORDS
Influenza; neuraminidase
inhibitor; honokiol;
polyphenol; structure-
activity relationship
1. Introduction
Influenza is an acute viral infectious disease of the respiratory tract causing 0.25–0.5
million deaths worldwide annually [1]. Influenza virus neuraminidase (NA), an
enzyme located on the viral surface, which plays a key role in the release of virions
from infected host cells, has been established as a primary target for anti-influenza
drugs [2]. Several NA inhibitors (NAIs), such as oseltamivir [3] and zanamivir [4],
have been used in prophylaxis and clinical therapy for influenza. Nonetheless, the
CONTACT Ai-Xi Hu
axhu@hnu.edu.cn; Ai-Lin Liu
liuailin@imm.ac.cn
Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/
10286020.2018.1509854.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
D. LIN ET AL.
Figure 1. Natural polyphenolic NAIs.
drug resistance caused by the high mutability of influenza virus has become increas-
ingly severe [5–7]. There is an immediate need to develop new anti-influenza agents
with novel skeleton.
In recent years, a growing body of researches suggested NAIs from natural prod-
ucts were of great potential for the development of anti-influenza drugs. A variety of
natural products have been found possessing favorable anti-NA activity, especially
polyphenolic compounds (Figure 1) such as flavones [8,9], catechins [10,11], chal-
cones [12,13], stilbenoids [14,15], etc. However, the research of natural NAIs was
blocked by the limitations of natural structure.
We hope to improve the biological activity of natural NAIs by chemical modifica-
tion. Through analyzing the structure-activity relationship of natural NAIs, hydroxyl
and other polar groups were considered as the essential functional groups for good
NA inhibitory effects [16]. In this work, honokiol (Figure 2), one of the simplest nat-
ural polyphenol, which was reported to have satisfactory inhibitory activity against
the NA of historic influenza A strain PR/8/34 and the oseltamivir-resistant strain B/
55/08 [17], was chosen to be structurally modified. In order to enhance the anti-NA
activity, hydroxyl and other polar groups were introduced into the structure of hono-
kiol. The derivatives were evaluated for their anti-NA activity in vitro and the struc-
ture-activity relationship (SAR) was discussed.
2. Results and discussion
2.1. Chemistry
The epoxidation reaction was used to modify the allyl of honokiol. However, acciden-
tal phenomena were observed in which there were up to four new components in the

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. Structural modification of honokiol.
Figure 3. Reaction process of the epoxidation and hydrolyzation.
reaction mixture. By monitoring the reaction and characterization of every compo-
nents, the reaction process was speculated as shown in Figure 3. Influenced by ortho-
hydroxy, the epoxidation of allyl was much faster, and the hydrolyzation of epoxypro-
pane was much easier which could even happen by absorbing moisture from air.
The mechanisms of ortho-effect in the epoxidation and hydrolyzation were
assumed as described in Figure 4. The phenolic hydroxyl could form hydrogen bond
with peroxy acid to promote the epoxidation occurring in the ortho-position. And
the intramolecular hydrogen bond makes the epoxypropane in the ortho-position
more likely to be attacked by nucleophile.
According to the above research, compounds 1–3 were prepared from honokiol
via the tandem process of epoxidation and hydrolyzation by controlling the dosage of
m-chloroperoxybenzoic acid and the temperature of hydrolyzation (Scheme 1).
Compound 4, a new found natural product extracted from Magnolia officinalis in
2013 [18] was synthesized starting from honokiol by Wacker-type intramolecular cyc-
lization [19]. Compound 5 was obtained from 4 by Wacker oxidation [19]. And the
condensation reaction of 5 with hydrazide afforded acethydrazone (6) and substituted
benzoylhydrazones (7–23) containing E and Z configurations [20].

4
D. LIN ET AL.
Figure 4. The mechanism of ortho-effect.
Scheme 1. Synthetic route of compounds 1–23. (a) i: m-CPBA (2.0 eq), CH2Cl2, r.t.; ii: NaOH, THF/
H2O, r.t., 38%; (b) i: m-CPBA (3.5 eq), CH2Cl2, r.t.; ii: NaOH, THF/H2O, r.t., 71%; (c) i: m-CPBA (3.5
eq), CH2Cl2, r.t.; ii: NaOH, THF/H2O, reflux, 66%; (d) PdCl2, NaOAc, O2, DMA/H2O, 60 ꢀC, 86%; (e)
PdCl2, O2, DMA/H2O, 60 ꢀC, 64%; (f) acethydrazide, HOAc, EtOH, 50 ꢀC, 59%; (g) substituted benzoyl-
hydrazide, HOAc, EtOH, reflux, 53–96%.
2.2. Anti-NA activity and SAR
The anti-NA efficacy of compounds 1–23 was preliminarily evaluated at the concen-
tration of 40 lg/ml and the inhibition rates were presented in Figure 5. As a matter
of experience, the introduction of polar groups in appropriate position may improve
the anti-NA activity because it may enhance the interaction between drug and NA
[21–23]. Therefore, compounds 1–3 were designed and synthesized, but the introduc-
tion of hydroxyl on the side chain made little difference to anti-NA activity compared
with honokiol. In addition, to study the effect of phenolic hydroxyl, benzofuran deriv-
atives 4–6 were synthesized and evaluated. Among them, compound 5 showed obvi-
ously better anti-NA activity than honokiol, and acetylhydrazone (6) exhibited the
highest inhibition rate (40.87%). Acetylhydrazone (6) contain the structure of acylhy-
drazone which was reported to have potent anti-influenza and anti-NA efficiency
[24–27]. Therefore, for further improving the NA inhibitory activity, benzoylhydra-
zones (7–23) were synthesized.
All of the benzoylhydrazones (7–23) showed better anti-NA efficiency than hono-
kiol. Some of the benzoylhydrazones displayed more than 50% inhibitory rate against
NA and they were further tested for IC50 values which are presented in Table 1.
Among them, compound 16 exhibited the most potent anti-NA activity with an IC50
value of 14.87 lg/ml (35.88 lM). And it is shown that the anti-NA activity of benzoyl-
hydrazones (7–23) was greatly influenced by the substituents. In a whole, the

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Inhibiꢀon rate/%
99.6
100
90
80
70
60
50
40
81.0
73.9
70.9
72.7
61.5
55.4
46.3
49.2
40.9
43.7
39.0
39.0
33.3
29.3
24.8
25.2
24.1
22.2
23.9
30
20
8.2
7.7
4.6
10
0
4.4
2.1
Figure 5. The inhibition rates of honokiol and compounds 7–23 against influenza NA at the con-
centration of 40 lg/ml using Zanamivir as positive control.
Table 1. The IC50 of compounds 11, 12, and 14–17 against influenza NA.
Compd.
R
IC50 /lgꢂmlꢃ1
IC50 /lM
11
12
14
15
16
17
2-NO2
4-NO2
2-OH
3-OH
4-OH
32.97
39.39
22.56
17.74
14.87
31.28
73.35
88.82
54.43
42.80
35.88
72.67
2,4-diOH
improvement of anti-NA activity by substituents could be ranked as:
OH > NO2 > NH2 ꢁ NHAc > F ꢁ CF3.
Just like the phenomenon occurring in natural NAIs [16], the compounds with poly-
phenol (13–16) exhibited better anti-NA activity than those with monophenol (3–12
and 17–22). This suggested that we could introduce additional phenol unit into NAIs
to enhance the activity. Furthermore, it is worth noting that unlike the low-active poly-
phenols 1, 2 and honokiol, the phenols of high-active polyphenols 13–16 and those
natural NAIs [28] with excellent anti-NA activity are not directly linked. It is suggested
that the phenols of polyphenolic NAIs should be apart at a certain distance.
2.3. Molecular docking
To better understand the potency of active compounds and guide further structur-
e–activity relationship studies, honokiol, the natural product without modification,

6
D. LIN ET AL.
Figure 6. Comparison of the interaction models of honokiol and 16(E/Z) with A/H1N1-NA (3TI6). A.
Binding models of honokiol with NA. B–D. Binding models of 16(E/Z) with NA.
and the most potent compound 16 were docked into the active site of NA. The pre-
dicted binding mode showed honokiol could only be attached to the entrance of the
enzyme active site with a week interaction (Figure 6(A)). The possible cause for this
is that the structure of honokiol is too small to occupy the active cavity or form
hydrogen bonds with other amino acid residues. Compound 16 is a mixture of cis-
trans isomers. It is displayed that both E and Z configurations of compound 16 are
well accommodated in the catalytic cavity of NA (Figure 6(B)) and could be stabilized
via interacting with NA active site by forming hydrogen bonds with several key resi-
dues (Arg118, Arg371, Arg152, Asn294, Thr-225) (Figure 3(C, D)).
3. Conclusions
In conclusion, honokiol, a natural polyphenol NAI was structurally modified and
evaluated for NA inhibitory activity in vitro. As a result, compounds 1–23 were syn-
thesized and the ortho-effects in the epoxidation and hydrolyzation were studied in
detail. The derivatives containing benzoylhydrazone (7–23) showed much better anti-
NA activity than honokiol. The polyphenolic derivatives (14–17) showed better anti-
NA inhibitory activity than that of the monophenols (4–13 and 18–23) and biphenols
(1–3 and honokiol). The structure-activity relationship was discussed and the

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
amelioration of anti-NA activity by substituents could be ranked as: OH > NO2>
NH2 ꢁ NHAc > F ꢁ CF3. Furthermore, molecular docking indicated that the most
active compound 16 has much stronger interactions with the NA active site than that
of honokiol. All of the above results gave suggestion for the future study that poly-
phenols are a kind of active structure of NAIs. The phenols of polyphenolic NAIs
should be apart at a certain distance and we could introduce additional phenol unit
into NAIs to enhance the anti-NA activity.
4. Experimental
4.1. Chemistry
All the reagents were commercially available and used directly without further purifi-
cation. Melting points were determined on an X-4 binocular microscope melting
1
point apparatus (Beijing Taike Instruments Co., Ltd., Beijing, China). H and 13C
NMR spectra were recorded on a Bruker-400 (Bruker, Karlsruhe, Switzerland), using
tetramethylsilane (TMS) as the internal standard and chemical shifts (d) were
expressed in ppm. Mass spectra were obtained by an Agilent 1100 series LC-MS
(Agilent Technologies Inc., Santa Clara, CA). Elemental analyses were performed on
a Vario EL III (Elementar Analysensysteme GmbH, Langenselbold, Germany).
4.2. Synthesis of compound 1
To a solution of honokiol (5 mmol) in CH2Cl2 (10 ml) was added m-chloroperoxy-
benzoic acid (75%, 10 mmol) in five portions over 8 h and kept stirring for 2 h at
room temperature. Then the reaction was quenched with saturated Na2SO3 solution,
neutralized by NaHCO3 and extracted with CH2Cl2. The combined extract was dried
over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in
THF (10 ml) and NaOH solution (1 M, 10 ml) was added. After it was stirred for 1 h
at room temperature, the mixture was acidized by diluted hydrochloric acid and
extracted with ethyl acetate. The organic layer was dried, concentrated, and purified
by silica gel chromatography (petroleum ether/ethyl acetate ¼ 4/1, v/v) to afford 1 as
1
a brown solid. Yield 38%; m.p. 32–34 ꢀC. H NMR (400 MHz, DMSO-d6) d 9.20 (s,
1H, Ar-OH), 7.33 (s, 1H, Ar-H), 7.21 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.99 (d, J ¼ 1.3 Hz,
1H, Ar-H), 6.90 (dd, J ¼ 8.2, 1.3 Hz, 1H, Ar-H), 6.82 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.73
(d, J ¼ 8.2 Hz, 1H, Ar-H), 6.00–5.88 (m, 1H, CH2CH¼CH2), 5.06 (d, J ¼ 17.1 Hz, 1H,
CH2CH ¼ CH2), 5.01 (d, J ¼ 9.9 Hz, 1H, CH2CH ¼ CH2), 4.85–4.77 (m, 1H,
CH2CH(OH)CH2OH), 3.63–3.55 (m, 2H, CH2CH(OH)CH2OH), 3.27 (d, J ¼ 6.7 Hz,
2H, CH2CH ¼ CH2), 3.21 (dd, J ¼ 15.7, 8.3 Hz, 1H, CH2CH(OH)CH2OH), 3.00 (dd,
J ¼ 15.7, 8.3 Hz, 1H, CH2CH(OH)CH2OH); 13C NMR (101 MHz, CDCl3) d 158.9,
150.7, 137.7, 132.1, 130.2, 129.5, 129.0, 128.7, 127.9, 127.8, 125.9, 115.5, 115.5, 109.9,
83.4, 64.8, 39.3, 31.1. LC-MS m/z: 301.0 [M þ H]þ. Elemental analysis: Found: C,
71.92%, H, 6.66%; calcd for C18H20O4, C, 71.98%, H, 6.71%.

8
D. LIN ET AL.
4.3. Synthesis of compound 2
To a solution of honokiol (5 mmol) in CH2Cl2 (10 ml) was added m-chloroperoxy-
benzoic acid (75%, 17.5 mmol) in five portions over 8 h and kept stirring for 2 h at
room temperature. Then the reaction was quenched with saturated Na2SO3 solution,
neutralized by NaHCO3 and extracted with CH2Cl2. The combined extract was dried
over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in
THF (10 ml) and NaOH solution (1M, 10 ml) was added. After it was stirred at room
temperature for 1 h, the mixture was acidized by diluted hydrochloric acid and
extracted with ethyl acetate. The organic layer was dried, concentrated, and purified
by silica gel chromatography (petroleum ether/ethyl acetate ¼ 4/1-2/1, v/v) to afford 2
1
as a yellow solid. Yield 71%; m.p. 56–58 ꢀC. H NMR (400 MHz, DMSO-d6) d 9.29 (s,
1H, Ar-OH), 7.25 (s, 1H, Ar-H), 7.23 (d, J ¼ 8.2 Hz, 1H, Ar-H), 7.09 (d, J ¼ 1.8 Hz,
1H, Ar-H), 6.99 (dd, J ¼ 8.2, 1.8 Hz, 1H, Ar-H), 6.84 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.74
(d, J ¼ 8.2 Hz, 1H, Ar-H), 5.03 (br, 1H, OH), 4.85–4.78 (m, 1H,
CH2CH(OH)CH2OH), 3.65–3.53 (m, 2H, CH2CH(OH)CH2OH), 3.22 (dd, J ¼ 15.8,
8.4 Hz, 1H, CH2CH(OH)CH2OH), 3.12–3.05 (m, 1H, CH2CHOCH2), 3.01 (dd,
J ¼ 15.8, 8.4 Hz, 1H, CH2CH(OH)CH2OH), 2.74 (dd, J ¼ 10.4, 5.6 Hz, 1H,
CH2CHOCH2), 2.70 (d, J ¼ 5.0 Hz, 1H, CH2CHOCH2), 2.66 (dd, J ¼ 14.3, 5.6 Hz, 1H,
CH2CHOCH2), 2.54 (dd, J ¼ 5.0, 2.6 Hz, 1H, CH2CHOCH2); 13C NMR (101 MHz,
DMSO-d6) d 158.7, 153.0, 131.1, 130.9, 129.0, 128.5, 128.1, 127.0, 126.2, 116.2, 116.1,
108.6, 83.9, 63.5, 52.8, 46.6, 37.7, 31.4. LC-MS m/z: 317.1 [M þ H]þ. Elemental ana-
lysis: Found: C, 68.29%, H, 6.33%; calcd for C18H20O5, C, 68.34%, H, 6.37%.
4.4. Synthesis of compound 3
To a solution of honokiol (5 mmol) in CH2Cl2 (10 ml) was added m-chloroperoxy-
benzoic acid (75%, 17.5 mmol) in five portions over 8 h and kept stirring for 2 h at
room temperature. Then the reaction was quenched with saturated Na2SO3 solution,
neutralized by NaHCO3, and extracted with CH2Cl2. The combined extract was dried
over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in
THF (10 ml) and NaOH solution (1M, 10 ml) was added. After it was stirred and
refluxed for 1 h, the mixture was acidized by diluted hydrochloric acid and extracted
with ethyl acetate. The organic layer was dried, concentrated, and purified by silica
gel chromatography (petroleum ether/ethyl acetate ¼ 2/1-1/1, v/v) to afford 3 as a yel-
1
low solid. Yield 66%; m.p. 50–52 ꢀC. H NMR (400 MHz, DMSO-d6) d 9.23 (s, 1H,
Ar-OH), 7.35 (s, 1H, Ar-H), 7.23 (d, J ¼ 8.2 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 6.94
(dd, J ¼ 8.2, 1.9 Hz, 1H, Ar-H), 6.81 (d, J ¼ 8.2 Hz, 1H, Ar-H), 6.73 (d, J ¼ 8.2 Hz, 1H,
Ar-H), 5.14 (s, 2H, OH), 4.86–4.77 (m, 1H, CH2CH(OH)CH2OH), 3.88–3.78 (m, 1H,
CH2CH(OH)CH2OH), 3.61–3.58 (m, 2H, CH2CH(OH)CH2OH), 3.55 (dd, J ¼ 11.0,
5.0 Hz,
1H,
CH2CH(OH)CH2OH),
3.46
(dd,
J ¼ 11.0,
5.0 Hz,
1H,
CH2CH(OH)CH2OH), 3.22 (dd, J ¼ 15.8, 8.4 Hz, 1H, CH2CH(OH)CH2OH), 3.00 (dd,
J ¼ 15.8, 8.4 Hz, 1H, CH2CH(OH)CH2OH), 2.72 (dd, J ¼ 13.6, 6.3 Hz, 1H,
CH2CH(OH)CH2OH), 2.61 (dd, J ¼ 13.6, 6.3 Hz, 1H, CH2CH(OH)CH2OH); 13C
NMR (101 MHz, DMSO-d6) d 158.6, 152.7, 131.5, 131.1, 129.3, 128.9, 128.9, 127.9,
127.0, 126.2, 116.0, 108.6, 83.9, 71.7, 63.5, 49.8, 31.4. LC-MS m/z: 335.1 [M þ H]þ.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
Elemental analysis: Found: C, 64.71%, H, 6.61%; calcd for C18H22O6, C, 64.66%,
H, 6.63%.
4.5. Synthesis of compound 4
A mixture of honokiol (10 mmol), NaOAc (1 mmol) and PdCl2 (0.15 mmol) in the co-
solvent of DMA and H2O (35 ml, v/v¼ 6:1) was stirred under O2 (0.8 Mpa) at 60 ꢀC for
16h. After cooling, the mixture was diluted with water and extracted with ethyl acetate.
The combined extracts were dried over Na2SO4 and concentrated under reduced pres-
sure. The residue was then purified by silica gel chromatography (petroleum ether/ethyl
1
acetate ¼ 8/1, v/v) to afford 4 as light yellow oil. Yield 86%; H NMR (400 MHz, CDCl3)
d 7.53 (d, J¼ 1.5 Hz, 1H, benzofuran 4-H), 7.50 (d, J ¼ 8.4 Hz, 1H, benzofuran 7-H),
7.26 (dd, J¼ 8.4, 1.5 Hz, 1H, benzofuran 6-H), 7.10–7.07 (m, 2H, C6H3 3,5-H), 6.94 (d,
1H, J¼ 8.8 Hz, C6H3 6-H), 6.41 (s, 1H, benzofuran 3-H), 6.04–5.93 (m, 1H,
CH2CH¼CH2), 5.21 (s, 1H, OH), 5.12–5.04 (m, 2H, CH2CH ¼ CH2), 3.36 (d,
J ¼ 6.7 Hz, 2H, CH2CH ¼ CH2), 2.49 (s, 3H, CH3); 13C NMR (101 MHz, CDCl3) d
156.6, 154.4, 151.1, 138.1, 132.3, 131.8, 130.8, 130.2, 129.0, 128.8, 124.4, 120.9, 116.0,
115.8, 111.3, 102.9, 39.6, 14.2. LC-MS m/z: 265.1 [Mþ H]þ. Elemental analysis: Found:
C, 81.85%, H, 6.08%; calcd for C18H16O2, C, 81.79%, H, 6.10%.
4.6. Synthesis of compound 5
A mixture of 4 (10 mmol) and PdCl2 (0.15 mmol) in the co-solvent of DMA and
H2O (40 ml, v/v ¼ 4:1) was stirred under O2 (0.8 Mpa) at 60 ꢀC for 16 h. After cooling,
the mixture was diluted with water and extracted with ethyl acetate. The combined
extracts were dried over Na2SO4 and concentrated under reduced pressure. The resi-
due was then purified by silica gel chromatography (petroleum ether/ethyl
1
acetate ¼ 4/1, v/v) to afford 5 as a white powder. Yield 64%; m.p. 118–120 ꢀC. H
NMR (400 MHz, CDCl3) d 7.53–7.48 (m, 2H, benzofuran 4,7-H), 7.26–7.24 (m, 1H,
benzofuran 6-H), 7.10–7.07 (m, 2H, Ar-H), 6.97 (d, 1H, J ¼ 7.8 Hz, Ar-H), 6.41 (s,
1H, benzofuran 3-H), 5.35 (br, 1H, OH), 3.66 (s, 2H, CH2), 2.49 (s, 3H, CH3), 2.18
(s, 3H, COCH3); 13C NMR (101 MHz, CDCl3) d 191.0, 158.3, 157.1, 154.7, 132.8,
131.2, 130.5, 130.1, 129.5, 129.3, 123.9, 120.7, 116.3, 111.8, 102.7, 50.2, 29.2, 14.2. LC-
MS m/z: 280.9 [M þ H]þ. Elemental analysis: Found: C, 77.07%, H, 5.79%; calcd for
C18H16O3, C, 77.12%, H, 5.75%.
4.7. Synthesis of compound 6
To a solution of 5 (0.5 mmol) and acethydrazide (1.5 mmol) in EtOH (20 ml) acetic
acid (0.2 ml) was added. After stirred at 50 ꢀC for 5 h, the reaction was concentrated,
poured into saturated salt water, shocked, and placed until precipitation. Then the
precipitate was separated by filtration, washed with water, and dried to get 6 as a
1
light yellow solid. Yield 59%; m.p. 92–95 ꢀC. H NMR (400 MHz, CDCl3) d 7.58–7.51
(m, 3H, Ar-H), 7.10–6.94 (m, 3H, Ar-H), 6.43–6.41 (m, 1H, Ar-H), 5.70 (br, 1H,
OH), 5.38 (br, 1H, OH), 3.68–3.51 (m, 2H, CH2), 2.50 (s, 3H, CH3), 2.49 (s, 1H,

10
D. LIN ET AL.
CH3), 2.35 (s, 2H, CH3); LC-MS m/z: 337.0 [M þ H]þ. Elemental analysis: Found: C,
71.34%, H, 5.96%, N, 8.35%; calcd for C20H20N2O3, C, 71.41%, H, 5.99%, N, 8.33%.
4.8. Synthesis of substituted benzoylhydrazide
Substituted benzoylhydrazide was prepared according to the literature proto-
col [29–33].
4.9. General procedure for the synthesis of substituted
benzoylhydrazone (7–23)
To a solution of 5 (0.5 mmol) and substituted benzoylhydrazide (0.55 mmol) in EtOH
(20 ml) acetic acid (0.2 ml) was added. After stirred and refluxed for 5 h, the reaction
was concentrated, poured into saturated salt water, shocked, and placed until precipi-
tation. Then the precipitate was separated by filtration, washed with water, and dried
to afford substituted benzoylhydrazone (7–23).
4.9.1. Compound 7
1
White solid. Yield 90%; m.p. 99–101 ꢀC; H NMR (400 MHz, CDCl3) d 7.87–7.73 (m,
2H, Ar-H), 7.53–7.44 (m, 5H, Ar-H), 7.26–6.93 (m, 4H, Ar-H), 6.40 (s, 1H, Ar-H),
5.56 (br, 1H, OH), 3.78–3.62 (m, 2H, CH2), 2.48 (s, 3H, CH3), 2.18–1.88 (m, 3H,
CH3); 13C NMR (101 MHz, Acetone-d6) d 164.4, 156.8, 155.0, 154.1, 149.3, 135.2,
134.2, 133.6, 132.5, 131.4, 130.3, 130.0, 129.7, 129.4, 128.6, 127.7, 125.9, 122.0, 117.6,
117.4, 110.8, 103.8, 45.2, 15.9, 14.1. LC-MS m/z: 399.2 [M þ H]þ. Elemental analysis:
Found: C, 75.30%, H, 5.54%, N, 6.99%; calcd for C25H22N2O3, C, 75.36%, H, 5.57%,
N, 7.03%.
4.9.2. Compound 8
1
Light yellow solid. Yield 53%; m.p. 91–93 ꢀC; H NMR (400 MHz, DMSO-d6) d 9.37
(br, 1H), 7.70–7.60 (m, 1H, Ar-H), 7.52–7.26 (m, 4H, Ar-H), 7.17–6.82 (m, 5H, Ar-
H), 6.60–6.54 (m, 1H, Ar-H), 3.67–3.47 (m, 2H, CH2), 2.45 (s, 3H, CH3), 1.87–1.69
(m, 3H, CH3); 13C NMR (101 MHz, Acetone-d6) d 163.3, 162.2, 161.6, 156.9, 155.0,
153.9, 134.4, 134.3, 132.8, 132.7, 132.6, 132.5, 130.3, 130.2, 129.8, 129.7, 125.9, 122.1,
122.0, 117.3, 110.9, 103.9, 45.2, 16.5, 14.2. LC-MS m/z: 414.1 [M þ H]þ. Elemental
analysis: Found: C, 72.70%, H, 5.64%, N, 10.14%; calcd for C25H23N3O3, C, 72.62%,
H, 5.61%, N, 10.16%.
4.9.3. Compound 9
1
Light yellow solid. Yield 71%; m.p. 111–113 ꢀC; H NMR (400 MHz, DMSO-d6) d
9.99–9.97 (m, 1H, NH), 9.39 (br, 1H, OH), 7.67–7.56 (m, 3H, Ar-H), 7.50–7.46 (m,
1H, Ar-H), 7.41–7.36 (m, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.03 (d, J ¼ 7.5 Hz, 1H, Ar-
H), 6.90 (d, J ¼ 7.5 Hz, 1H, Ar-H), 6.62–6.52 (m, 3H, Ar-H), 5.71 (br, 2H, NH2),
3.72–3.50 (m, 2H, CH2), 2.45 (s, 3H, CH3), 2.11–1.86 (m, 3H, CH3); 13C NMR
(101 MHz, Acetone-d6) d 164.8, 162.8, 156.9, 155.0, 154.1, 133.1, 131.1, 130.8, 130.5,
130.3, 130.2, 129.8, 127.5, 127.3, 126.0, 125.9, 122.0, 117.6, 117.2, 114.1, 110.9, 103.8,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
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50.3, 16.7, 14.2. LC-MS m/z: 414.0 [M þ H]þ. Elemental analysis: Found: C, 72.59%,
H, 5.57%, N, 10.22%; calcd for C25H23N3O3, C, 72.62%, H, 5.61%, N, 10.16%.
4.9.4. Compound 10
1
Light yellow solid. Yield 84%; m.p. 127–129 ꢀC; H NMR (400 MHz, DMSO-d6) d
10.33 (s, 1H, NH), 10.21–10.17 (m, 1H, NH), 9.39 (s, 1H, OH), 7.88–7.78 (m, 2H,
Ar-H), 7.69–7.63 (m, 3H, Ar-H), 7.51–7.46 (m, 1H, Ar-H), 7.41–7.36 (m, 1H, Ar-H),
7.16 (s, 1H, Ar-H), 7.04 (d, J ¼ 8.0 Hz, 1H, Ar-H), 6.91 (d, J ¼ 8.0 Hz, 1H, Ar-H),
6.62–6.57 (m, 1H, Ar-H), 3.74–3.56 (m, 2H, CH2), 2.45 (s, 3H, CH3), 2.08 (s, 1H,
CH3), 2.07 (s, 3H, CH3), 1.88 (s, 2H, CH3). LC-MS m/z: 456.2 [M þ H]þ. Elemental
analysis: Found: C, 71.24%, H, 5.54%, N, 9.17%; calcd for C27H25N3O4, C, 71.19%, H,
5.53%, N, 9.22%.
4.9.5. Compound 11
1
Yellow solid. Yield 63%; m.p. 109–111 ꢀC; H NMR (400 MHz, CDCl3) d 8.12 (d,
J ¼ 8.0 Hz, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.72 (d, J ¼ 7.4 Hz, 1H, Ar-H), 7.67–7.64
(m, 1H, Ar-H), 7.63–7.58 (m, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.49 (d, J ¼ 6.5 Hz, 1H,
Ar-H), 7.28 (dd, J ¼ 6.5, 2.0 Hz, 1H, Ar-H), 7.21 (dd, J ¼ 8.3, 1.7 Hz, 1H, Ar-H), 6.93
(d, J ¼ 8.3 Hz, 1H, Ar-H), 6.42 (s, 1H, Ar-H), 5.55 (br, 1H), 5.30 (br, 1H), 3.68–3.27
(m, 2H, CH2), 2.50 (s, 3H, CH3), 2.18–1.81 (m, 3H, CH3); 13C NMR (101 MHz,
CDCl3) d 169.0, 156.9, 154.7, 154.5, 147.4, 145.5, 133.7, 130.9, 130.5, 130.3, 130.2,
129.7, 129.3, 128.8, 127.9, 126.0, 124.0, 123.5, 120.6, 116.2, 111.6, 102.7, 44.3, 14.8,
14.2. LC-MS m/z: 444.1 [M þ H]þ. Elemental analysis: Found: C, 67.65%, H, 4.73%,
N, 9.53%; calcd for C25H21N3O5, C, 67.71%, H, 4.77%, N, 9.48%.
4.9.6. Compound 12
1
Yellow solid. Yield 95%; m.p. 99–111 ꢀC; H NMR (400 MHz, CDCl3) d 8.31 (d,
J ¼ 8.0 Hz, 2H, Ar-H), 7.99–7.92 (m, 2H, Ar-H), 7.54–7.47 (m, 2H, Ar-H), 7.29–7.25
(m, 1H, Ar-H), 7.10 (s, 1H, Ar-H), 7.06 (m, 1H, Ar-H), 7.00–6.93 (m, 1H, Ar-H),
6.41 (s, 1H, Ar-H), 3.79–3.65 (m, 2H, CH2), 2.49 (s, 3H, CH3), 2.18–1.92 (m, 3H,
CH3); 13C NMR (101 MHz, CDCl3) d 168.2, 156.8, 154.4, 151.8, 149.8, 143.5, 131.4,
131.1, 130.9, 130.4, 130.2, 129.7, 129.4, 128.5, 128.2, 124.0, 123.9, 122.7, 120.7, 116.1,
111.4, 102.7, 44.4, 14.7, 14.2. LC-MS m/z: 444.2 [M þ H]þ. Elemental analysis: Found:
C, 67.63%, H, 4.77%, N, 9.55%; calcd for C25H21N3O5, C, 67.71%, H, 4.77%,
N, 9.48%.
4.9.7. Compound 13
Light yellow solid. Yield 96%; m.p. 105–107 ꢀC; 1H NMR (400 MHz, CDCl3) d
9.17–9.04 (m, 3H, Ar-H), 7.53–7.45 (m, 2H, Ar-H), 7.24–7.18 (m, 1H, Ar-H),
7.10–7.02 (m, 2H, Ar-H), 6.97–6.91 (m, 1H, Ar-H), 6.40 (s, 1H, Ar-H), 3.83–3.52 (m,
2H, CH2), 2.49 (s, 3H, CH3), 2.19–1.97 (m, 3H, CH3); 13C NMR (101 MHz, CDCl3) d
165.4, 156.8, 155.3, 154.4, 151.8, 147.8, 131.4, 131.1, 130.5, 130.2, 129.7, 129.4, 128.8,
124.0, 120.5, 116.0, 111.4, 102.6, 44.6, 15.0, 14.1. Elemental analysis: Found: C,
61.45%, H, 4.17%, N, 11.44%; calcd for C25H20N4O7, C, 61.47%, H, 4.13%, N, 11.47%.

12
D. LIN ET AL.
4.9.8. Compound 14
1
White solid. Yield 82%; m.p. 113–115 ꢀC; H NMR (400 MHz, CDCl3) d 7.55–7.51
(m, 2H, Ar-H), 7.50–7.42 (m, 2H, Ar-H), 7.28–7.26 (m, 1H, Ar-H), 7.10 (s, 1H, Ar-
H), 7.08–7.03 (m, 2H, Ar-H), 6.97 (d, J ¼ 8.1 Hz, 1H, Ar-H), 6.93–6.88 (m, 1H, Ar-
H), 6.44–6.40 (s, 1H, Ar-H), 5.63 (br, 1H), 5.27 (br, 1H), 3.76–3.63 (m, 2H, CH2),
3.51 (br, 1H, OH), 2.50 (s, 3H, CH3), 2.49–2.18 (m, 3H, CH3); LC-MS m/z: 415.0
[M þ H]þ. Elemental analysis: Found: C, 72.51%, H, 5.37%, N, 6.71%; calcd for
C25H22N2O4, C, 72.45%, H, 5.35%, N, 6.76%.
4.9.9. Compound 15
1
White solid. Yield 68%; m.p. 117–119 ꢀC; H NMR (400 MHz, CDCl3) d 7.55–7.48
(m, 3H, Ar-H), 7.40 (s, 1H, Ar-H), 7.33 (d, J ¼ 7.6 Hz, 1H, Ar-H), 7.26–7.24 (m, 1H,
Ar-H), 7.17–7.07 (m, 2H, Ar-H), 7.03 (d, J ¼ 8.5 Hz, 1H, Ar-H), 6.97 (d, J ¼ 8.5 Hz,
1H, Ar-H), 6.41 (s, 1H, Ar-H), 5.60 (br, 1H), 5.28 (br, 1H), 3.73–3.65 (m, 2H, CH2),
3.52 (br, 1H), 2.49 (s, 3H, CH3), 2.17–1.88 (m, 3H, CH3); 13C NMR (101 MHz,
Acetone-d6) d 164.6, 164.2, 157.0, 155.1, 148.9, 136.5, 134.7, 133.7, 131.3, 130.6, 130.4,
130.3, 130.2, 128.6, 127.9, 125.9, 122.1, 119.4, 115.6, 111.0, 110.9, 103.9, 50.3, 15.9,
14.2. LC-MS m/z: 415.1 [M þ H]þ. Elemental analysis: Found: C, 72.53%, H, 5.40%,
N, 6.72%; calcd for C25H22N2O4, C, 72.45%, H, 5.35%, N, 6.76%.
4.9.10. Compound 16
1
White solid. Yield 77%; m.p. 134–136 ꢀC; H NMR (400 MHz, CDCl3) d 7.87–7.68
(m, 2H, Ar-H), 7.55–7.47 (m, 3H, Ar-H), 7.32–7.28 (m, 1H, Ar-H), 7.15–7.07 (m,
2H, Ar-H), 7.01–6.89 (m, 2H, Ar-H), 6.42 (s, 1H, Ar-H), 5.28 (br, 1H), 3.72–3.66 (m,
2H, CH2), 3.53 (br, 1H, OH), 2.50 (s, 3H, CH3), 2.18 (s, 3H, CH3). LC-MS m/z: 415.1
[M þ H]þ. Elemental analysis: Found: C, 72.43%, H, 5.31%, N, 6.75%; calcd for
C25H22N2O4, C, 72.45%, H, 5.35%, N, 6.76%.
4.9.11. Compound 17
1
Yellow solid. Yield 59%; m.p. 116–118 ꢀC; H NMR (400 MHz, CDCl3) d 7.78 (d,
J ¼ 1.8 Hz, 1H, Ar-H), 7.74 (dd, J ¼ 8.4, 1.8 Hz, 1H, Ar-H), 7.56 (d, J ¼ 1.8 Hz, 1H,
Ar-H), 7.54–7.47 (m, 3H, Ar-H), 7.29 (dd, J ¼ 8.4, 1.8 Hz, 1H, Ar-H), 7.07 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 6.95–6.85 (m, 1H, Ar-H), 6.43 (s, 1H, Ar-H), 5.70 (br, 1H),
5.33 (br, 1H), 3.80–3.52 (m, 2H, CH2), 2.50 (s, 3H, CH3), 2.18–1.78 (m, 3H, CH3);
13C NMR (101 MHz, DMSO-d6) d 162.2, 160.7, 157.4, 156.6, 155.9, 155.7, 153.4,
133.8, 132.5, 131.3, 129.8, 128.8, 128.6, 128.2, 125.7, 124.8, 120.9, 116.6, 116.3, 115.1,
110.1, 103.0, 43.8, 16.5, 13.9. LC-MS m/z: 431.0 [M þ H]þ. Elemental analysis: Found:
C, 69.69%, H, 5.11%, N, 6.48%; calcd for C25H22N2O5, C, 69.76%, H, 5.15%,
N, 6.51%.
4.9.12. Compound 18
1
White solid. Yield 77%; m.p. 109–111 ꢀC; H NMR (400 MHz, CDCl3) d 7.79–7.72
(m, 1H, Ar-H), 7.56–7.47 (m, 4H, Ar-H), 7.24–7.21 (m, 2H, Ar-H), 7.10–6.93 (m,
3H, Ar-H), 6.41 (s, 1H, Ar-H), 5.74 (br, 1H), 5.42 (br, 1H), 3.88–3.46 (m, 2H, CH2),
2.49 (s, 3H, CH3), 2.18 (s, 1H, CH3), 1.89 (s, 2H, CH3). LC-MS m/z: 417.0 [M þ H]þ.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
Elemental analysis: Found: C, 72.03%, H, 5.12%, N, 6.68%; calcd for C25H21N2O3F, C,
72.10%, H, 5.08%, N, 6.73%.
4.9.13. Compound 19
1
White solid. Yield 87%; m.p. 91–93 ꢀC; H NMR (400 MHz, CDCl3) d 7.55–7.43 (m,
4H, Ar-H), 7.26 (dd, J ¼ 8.2, 1.6 Hz, 1H, Ar-H), 7.20–7.12 (m, 2H, Ar-H), 7.10 (s,
1H, Ar-H), 7.09–7.00 (m, 1H, Ar-H), 6.98–6.94 (m, 1H, Ar-H), 6.41 (s, 1H, Ar-H),
5.34 (br, 1H), 3.77–3.61 (m, 2H, CH2), 2.49 (s, 3H, CH3), 2.18–1.89 (m, 3H, CH3).
LC-MS m/z: 417.1 [M þ H]þ. Elemental analysis: Found: C, 72.16%, H, 5.04%, N,
6.77%; calcd for C25H21N2O3F, C, 72.10%, H, 5.08%, N, 6.73%.
4.9.14. Compound 20
1
White solid. Yield 67%; m.p. 109–111 ꢀC; H NMR (400 MHz, CDCl3) d 7.54–7.46
(m, 4H, Ar-H), 7.24 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.15–7.09 (m, 3H, Ar-H), 7.07–6.93
(m, 2H, Ar-H), 6.41 (s, 1H, Ar-H), 5.73 (br, 1H), 5.40 (br, 1H), 3.85–3.45 (m, 2H,
CH2), 2.49 (s, 3H, CH3), 2.18–1.88 (m, 3H, CH3). LC-MS m/z: 417.1 [M þ H]þ.
Elemental analysis: Found: C, 72.17%, H, 5.09%, N, 6.80%; calcd for C25H21N2O3F, C,
72.10%, H, 5.08%, N, 6.73%.
4.9.15. Compound 21
1
White solid. Yield 74%; m.p. 85–87 ꢀC; H NMR (400 MHz, CDCl3) d 8.31–8.16 (m,
1H, Ar-H), 7.54–7.46 (m, 2H, Ar-H), 7.25–7.19 (m, 1H, Ar-H), 7.16 (s, 1H, Ar-H),
7.10–6.94 (m, 3H, Ar-H), 6.93–6.84 (m, 1H, Ar-H), 6.40 (s, 1H, Ar-H), 5.46 (br, 1H),
3.80–3.58 (m, 2H, CH2), 2.48 (s, 3H, CH3), 2.17–1.88 (m, 3H, CH3). LC-MS m/z:
435.0 [M þ H]þ. Elemental analysis: Found: C, 69.19%, H, 4.59%, N, 6.48%; calcd for
C25H20N2O3F2, C, 69.12%, H, 4.64%, N, 6.45%.
4.9.16. Compound 22
Light yellow solid. Yield 86%; m.p. 85–87 ꢀC; 1H NMR (400 MHz, CDCl3) d
8.17–7.93 (m, 2H, Ar-H), 7.78–7.67 (m, 1H, Ar-H), 7.55–7.42 (m, 3H, Ar-H),
7.28–7.20 (m, 1H, Ar-H), 7.12–6.90 (m, 3H, Ar-H), 6.37 (s, 1H, Ar-H), 5.76 (br, 1H),
3.70–3.46 (m, 2H, CH2), 2.46 (s, 3H, CH3), 2.16–1.88 (m, 2H, CH3). LC-MS m/z:
466.1 [M þ H]þ. Elemental analysis: Found: C, 67.02%, H, 4.57%, N, 5.95%; calcd for
C26H21N2O3F3, C, 66.95%, H, 4.54%, N, 6.01%.
4.9.17. Compound 23
1
White solid. Yield 90%; m.p. 95–97 ꢀC; H NMR (400 MHz, CDCl3) d 7.98–7.85 (m,
2H, Ar-H), 7.72 (d, J ¼ 7.5 Hz, 2H, Ar-H), 7.53–7.48 (m, 2H, Ar-H), 7.27–7.23 (m,
1H, Ar-H), 7.18–7.13 (m, 1H, Ar-H), 7.11–7.06 (m, 1H, Ar-H), 6.99–6.93 (m, 1H,
Ar-H), 6.41 (s, 1H, Ar-H), 5.46 (br, 1H), 3.78–3.64 (m, 2H, CH2), 2.49 (s, 3H, CH3),
2.18–1.90 (m, 3H, CH3). LC-MS m/z: 466.0 [M þ H]þ. Elemental analysis: Found: C,
67.00%, H, 4.48%, N, 6.06%; calcd for C26H21N2O3F3, C, 66.95%, H, 4.54%, N, 6.01%.

14
D. LIN ET AL.
4.10. Anti-NA activity assay
All the target compounds and natural compound honokiol were tested for their
anti-NA activity in vitro. The NA inhibition assay was performed according to the
standard method [14]. 2’-(4-Methylumbelliferyl)-a-D-acetyl neuraminic acid
(MUNANA) was used as a substrate of NA. The cleavage of MUNANA by NA
could produce a fluorescent product, which would emit a fluorescence of 450 nm
under the irradiation of excitation wavelength of 360 nm. The degree of fluores-
cence can reflect the activity of NA sensitively. In the enzyme reaction system, a
certain concentration of the target compounds with the NA enzyme was suspended
in MES buffer (pH ¼ 6.5). The reaction was started by adding the substrate
MUNANA. After the reaction was incubated for 40 min at 37 ꢀC, it was terminated
by adding sodium hydroxide solution. The plate was read within 20 min under the
parameters of excitation wavelength at 360 nm and emission wavelength at 450 nm.
The inhibition rates of the tested compounds were obtained according to the decre-
ment of the fluorescence intensity. The IC50 (the concentration required for 50%
inhibition) was calculated by plotting the percent of inhibition of NA activity versus
the inhibitor concentration.
4.11. Docking study
Molecular docking was performed by AutoDock (version 4.2 [34] with default
parameters, The Scripps Research Institute and Olson Laboratory, http://autodock.
scripps.edu/). The crystal structure data of H1N1 neuraminidase oseltamivir com-
plex (PDB Code: 3TI6) is downloaded from RSCB Protein Data Bank. The con-
formational energy of docked compound was minimized by MM2 force field. The
protein and ligand were prepared in the standard manner using AutoDock Tools
(version 1.5.6, The Scripps Research Institute and Olson Laboratory, http://auto-
dock.scripps.edu/) [34]. A grid was centered on the catalytic active site [–28.60, 14.
23, 21.11]. The dimension of binding box was set as 46 ꢄ 38 ꢄ 48 grid points with
ꢀ
the points separated by 0.375 Å. The results were analyzed and visualized by
PyMOL (http://www. pymol.org/).
Acknowledgments
The authors are thankful to the Institute of Materia Medica of Chinese Academy of
Medical Sciences and Peking Union Medical College for providing the biological activ-
ity assays.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the National Natural Science Fundation of China (No.
21442014) and the National Natural Science Foundation of China (No. 81673480).

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15
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