Studies on synthesis and structure-activity relationship (SAR) of derivatives of a new natural product from marine fungi as inhibitors of influenza virus neuraminidase

Article abstract of DOI:10.3390/md9101887

Based on the natural isoprenyl phenyl ether from a mangrove-derived fungus, 32 analogues were synthesized and evaluated for inhibitory activity against influenza H1N1 neuraminidase. Compound 15 (3-(allyloxy)-4-hydroxybenzaldehyde) exhibited the most potent inhibitory activity, with IC₅₀ values of 26.96 μM for A/GuangdongSB/01/2009 (H1N1), 27.73 μM for A/Guangdong/03/2009 (H1N1), and 25.13 μM for A/Guangdong/05/2009 (H1N1), respectively, which is stronger than the benzoic acid derivatives (～mM level). These are a new kind of non-nitrogenous aromatic ether Neuraminidase (NA) inhibitors. Their structures are simple and the synthesis routes are not complex. The structure-activity relationship (SAR) analysis revealed that the aryl aldehyde and unsubstituted hydroxyl were important to NA inhibitory activities. Molecular docking studies were carried out to explain the SAR of the compounds, and provided valuable information for further structure modification.

Full text of DOI:10.3390/md9101887

Mar. Drugs 2011, 9, 1887-1901; doi:10.3390/md9101887
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Studies on Synthesis and Structure-Activity Relationship (SAR)
of Derivatives of a New Natural Product from Marine Fungi as
Inhibitors of Influenza Virus Neuraminidase
5
Jing Li 1,2, Dingmei Zhang 2,3,4, Xun Zhu 2,3,4, Zhenjian He 2,3,4, Shu Liu , Mengfeng Li 2,3,4,*,
Jiyan Pang 1,2,* and Yongcheng Lin 1,2,
*
1
School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275,
China; E-Mail: zsulijing@163.com
2
Guangdong Province Key Laboratory of Functional Molecules in Oceanic Microorganism,
Bureau of Education, Sun Yat-sen University, Guangzhou 510080, China;
E-Mails: dingmeizhang@yahoo.com.cn (D.Z.); zhuxun8@mail.sysu.edu.cn (X.Z.);
hzj100@163.com (Z.H.)
3
4
5
Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-sen University,
74 Zhongshan Road II, Guangzhou 510080, China
Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University,
74 Zhongshan Road II, Guangzhou 510080, China
Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan
Road II, Guangzhou 510080, China; E-Mail: liushu@mail.sysu.edu.cn
* Authors to whom correspondence should be addressed; E-Mails: ceslyc@mail.sysu.edu.cn (Y.L.);
cespjy@mail.sysu.edu.cn (J.P.); limf@mail.sysu.edu.cn (M.L.); Tel./Fax: +86-20-8403-9623.
Received: 2 September 2011; in revised form: 26 September 2011 / Accepted: 30 September 2011 /
Published: 11 October 2011
Abstract: Based on the natural isoprenyl phenyl ether from a mangrove-derived fungus,
32 analogues were synthesized and evaluated for inhibitory activity against influenza
H1N1 neuraminidase. Compound 15 (3-(allyloxy)-4-hydroxybenzaldehyde) exhibited the
most potent inhibitory activity, with IC50 values of 26.96 μM for A/GuangdongSB/01/2009
(H1N1), 27.73 μM for A/Guangdong/03/2009 (H1N1), and 25.13 μM for A/Guangdong/
05/2009 (H1N1), respectively, which is stronger than the benzoic acid derivatives
(~mM level). These are a new kind of non-nitrogenous aromatic ether Neuraminidase (NA)
inhibitors. Their structures are simple and the synthesis routes are not complex. The
structure-activity relationship (SAR) analysis revealed that the aryl aldehyde and
unsubstituted hydroxyl were important to NA inhibitory activities. Molecular docking

Mar. Drugs 2011, 9
studies were carried out to explain the SAR of the compounds, and provided valuable
1888
information for further structure modification.
Keywords: aromatic ether; marine fungus; neuraminidase inhibitor
1. Introduction
Influenza virus is a major global threat that can result in periodic epidemics [1], causing high
morbidity and mortality [2] as flu infections. Neuraminidase (NA) is a surface glycoprotein of
influenza virus that catalyzes the cleavage of terminal sialic acid (Chart 1A) residues to facilitate the
release as well as spread and transmission of the progeny virus particles from host to host. A number
of classes of NA inhibitors have been developed in the past few years, which have shown to be
effective in controlling influenza infection, however, most of them are stereochemically complex or
orally ineffective, and their synthesis routes are lengthy. In an attempt to develop chemically simple
inhibitors, benzoic acid derivatives (Chart 1B) have been studied as important NA inhibitors [3–6].
Their aromatic benzene ring has been used to replace the pyranose of sialic acid in designing NA
inhibitors, however, this class of inhibitors have high affinity for influenza virus but high IC50 values
(~mM level) [7]. Hence, further search for novel NA inhibitors, with simple chemistry and strong
potency, has attracted extensive interest.
Chart 1.
COOH
OH
HOOC
O
HO
COOMe
OH
O2N
OH
OH
NHAc
NHCOCH3
O
OH
IC50= 750μM
HO
A
B
C
The metabolites of marine fungi have yielded a lot of novel and bioactive secondary metabolites.
Previously we reported the new isoprenyl phenyl ether (Chart 1C) from a mangrove fungus (NO. B60)
from the South China Sea with slight cytotoxicity on hepG2 cell line [8], which is similar to benzoic
acid NA inhibitors in structure. Therefore, we have keenly become interested in designing and
synthesizing novel NA inhibitors by using the isoprenyl phenyl ether as model compound. In this
paper, we described the synthesis, biological activities and structure-activities relationship (SAR)
analysis of aromatic ether analogues, 1–32 (Chart 2). The modification of compounds is focused on:
carboxylic acid, ester, aldehyde or amide substituted at 1-position of the aromatic ether, and a variety
of substituents at 3- or 4-hydroxyl group of aromatic ether. Compared to the benzoic acid derivatives,
our compounds, which do not have the N atom in the molecule, have much more simple structures and
better activities. They are also easily obtained with good yields from commercially available materials.
In addition, Wang et al. [9] reported the active site of NA: a negatively charged group is favored for

Mar. Drugs 2011, 9
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binding to the pocket formed by Arg118, Arg292 and Arg371, and a positively charged group is
needed in the Asp151 and Arg156 pocket. There are two hydrophobic regions in which hydrophobic
groups can be introduced to enhance binding intensities: one is formed by Trp178, Ile222 and Arg224,
and the other is created by rotating Glu276. We carried out a surflex-docking study of compounds to
further account for the experimental results by comparing the binding action of these inhibitors to the
active sites of NA.
Chart 2. Structures of derivatives.
COOMe
COOMe
COOMe
COOMe
HO
HO
HO
HO
O
O
O
O
1
2
3
4
COOMe
COOMe
COOMe
O
COOMe
O
HO
HO
HO
O
O
O
O
OH
5
6
7
8
CHO
CHO
O
CHO
CHO
HO
HO
HO
O
O
O
10
9
11
12
CHO
O
CHO
CHO
OH
CHO
OH
O
O
O
O
13
14
15
16

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Chart 2. Cont.
CHO
CHO
CHO
O
CHO
O
O
O
O
O
O
O
17
18
19
20
COOMe
COOH
COOH
COOH
O
HO
O
O
O
O
24
O
O
O
21
22
23
COOMe
COOMe
O
COOMe
O
HO
O
O
O
OH
OMe
OMe
25
26
28
27
COOMe
CONH2
COOMe
O
O
O
O
HO
HO
OH
O
OMe
OMe
32
29
30
31

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2. Results and Discussion
2.1. Chemistry
The general synthetic route of 1–32 is outlined in Scheme 1. We chose 3,4-dihydroxybenzoic acid
methyl ester, 3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, isovanillic acid, and caffeic acid
as the starting materials. A series of aromatic ethers were synthesized through the Williamson ether
synthesis, followed by selective mono-deallylation using 10% Pd/C or aminationa of the intermediate
to provide part of target compounds [10]. Most of the compounds were easily obtained with excellent
yields (60–95%). Compound 2, 3, 5, 8, 14, 19, 24, 25, 27–30 and 32 were new derivatives. All new
compounds were fully characterized by HRMS and NMR. The purities (>95%) of all target
compounds were checked by HPLC using a LC-2010c equipped with UV detector.
Scheme 1. (a) K2CO3/Acetone; (b) Pd/C.
R1
R1
R1
a
b
R2 Br
R2O
R2O
HO
OR2
OH
OH
2.2. H1N1 Virus Neuraminidase Inhibition
All compounds were evaluated for inhibitory activities on the pandemic influenza H1N1 virus
[(A/Guangdong/03/2009 (H1N1)] neuraminidase in vitro, the NA inhibitors oseltamivir (Tamiflu) was
used as positive control. The results (Figure 1) showed that most of the derivatives inhibited H1N1
virus neuraminidase in the range of 15–59% inhibition at the 50 μM concentration. Among them,
compound 15 and 26 exhibited 50% or higher activities. In this screen, the model compound (Chart 1C)
had no activity. The SAR analysis revealed that: (1) Compared with compounds 16–18, 20 and 25,
compounds 9, 11–13, and 24 exhibited higher activities, it meant that substitution of 3- and 4-OH
would reduce the activities; (2) compound 13 showed weaker activity than compound 15, and it
indicated that OH group at C-4 position could improve the activity; (3) for compounds 9–13, it seems
that allyl ether is better than isopentyl ether, propyl ether, propargyl ether and isopentene ether, which
indicated that maybe the smaller group substituted in this series of compounds is helpful for the
activities; (4) previous studies showed that carboxylic acid is an important group in maintaining
activities. However, acid changed to aldehyde in 1-position of aromatic compounds was more
favorable for the bioactivities in compounds 17, 21, 25 and 30 in our present study.
The effect of compound 15 was further investigated on the neuraminidase activity of three pandemic
influenza H1N1 2009 virus strains [A/GuangdongSB/01/2009 (H1N1), A/Guangdong/03/2009 (H1N1)
and A/Guangdong/05/2009 (H1N1)]. The results (Figure 2) revealed significant inhibitory effects of
compound 15 on all three tested viral NA, with IC50 values of 26.96 μM for A/GuangdongSB/01/2009
(H1N1), 27.73 μM for A/Guangdong/03/2009 (H1N1), and 25.13 μM for A/Guangdong/05/2009
(H1N1), respectively. At the same time, compound 15 showed low cytotoxity (>600 μM) to virus host
cell—Madin-Darby canine kidney (MDCK) cells—and showed good selectivity index.

Mar. Drugs 2011, 9
Figure 1. The inhibitory effect of 32 compounds on the NA activity of the pandemic
1892
influenza H1N1 2009 viruses. Data points are presented as means ± SD of
triplicated experiments.
Figure 2. Compound 15 inhibits NA enzymatic activity. Compound 15 inhibits enzymatic
activity of NA derived from A/GuangdongSB/01/2009 (H1N1), A/Guangdong/03/2009
(H1N1) and A/Guangdong/05/2009 (H1N1) viruses. Viruses were incubated with
increasing concentrations of compound 15 and NA enzymatic activity was determined by a
chemiluminescence assay. Results were presented as means ± SD of triplicated experiments.
2.3. Molecular Docking Study
Molecular docking was performed to further assess the SAR of different substituents of the
inhibitors by their interactions with the NA residues. The studies focused on the ether chains, the
position of OH and the groups at C-1 position.

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Compounds 9–13 with different ether chains in C-4 position produced little difference in activity.
Introducing the hydrophobic groups in the small hydrophobic pocket formed by Trp178 and Ile222 as
well as the side chain of Arg224 could enhance binding action. In Figure 3(a), all of the ether chains
were near the pocket, but the allyl ether chain terminal displayed the best activity compared with other
substituents. The triarginyl cluster of Arg118, Arg292, and Arg371 at the pocket is the predominant
factor for orienting and stabilizing inhibitors. Compounds 17, 21, 25, 30 had different groups in
C-4 position. Relative to 21, 25 and 30, compound 17 with an aldehyde group which made strong
charge–charge interactions with the triarginyl pocket was more favorable for binding action (Figure 3(b)).
From Figure 3(c), we found that the OH group of compound 15 was much closer to the nucleophilic
oxygen of Asp151 compared to compound 13. Asp151 also showed significant favorable electrostatic
interaction. The flexibility of the molecule during the docking calculation permitted turning the tail of
the ether chains, thus the steric hindrance of the structure did not have a strong effect in binding with
Arg156. Molecular docking studies further supported the aforementioned SAR analysis.
Figure 3. (a) Comparison of the positions of compounds 9 (pink), 10 (green), 11 (purple),
12 (yellow) and 13 (gray) in the NA; (b) Comparison of the positions of 17 (gray),
21 (yellow), 25 (pink) and 30 (purple); (c) Comparison of the positions of 13 (yellow) and
15 (green); (d) Comparison of compound 15 (green) and oseltamivir (yellow) in
neuraminidase; (e) The interaction between the Arg residues and compound 15 (green) in
comparison with oseltamivir (yellow); (f) Molecular surfaces of neuraminidase with bound
compound 15.
(a)
(b)
(c)
(d)

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Figure 3. Cont.
(e)
(f)
The most potent compound 15 was docked into the active sites of NA. Oseltamivir is an efficacious
and commonly used neuraminidase inhibitor. The binding of compound 15 with NA was shown in
Figure 3(d) and Figure 3(e). The activity of compound 15 could be explained by its interactions with
the active residues of NA. The aldehyde group made strong charge–charge interactions with Arg118,
Arg292, and Arg371. Asp151 exhibited favorable electrostatic interaction with the OH group at C-4
position. At the same time, the ether chains were suitable for the hydrophobic region formed by
Trp178 and Ile222, as well as the side chain of Arg224. Compound 15 provided a binding mode with
NA for this series of compounds from its geometric and electronic features.
3. Experimental Section
3.1. Chemistry
All reagents and solvents were of commercial quality. Melting points were determined on an X-4
1
13
micromelting point apparatus and were uncorrected. H and CNMR data were recorded on a Varian
1
13
Inova 400 MB NMR spectrometer operating at 400 and 125 MHz for H and C, respectively. All
chemical shifts are in ppm (δ) with respect to tetramethylsilane (TMS) as internal standard, and
coupling constants (J) are in Hz. Mass spectra were obtained on DSQ (low resolution mass
spectrometer) and MAT95XP (high resolution mass spectrometer) instruments.
3.2. Synthetic Methods of Compounds
3.2.1. General Procedure 1: Synthesis of Compounds 1–5, 7–13, 16–25
The starting materials were dissolved in acetone and 5 equiv K2CO3 were added. The mixture was
stirred for 24 h and then poured into water. The aqueous layer was extracted with ethyl acetate. The
combined organic extracts were washed with saturated brine, dried over anhydrous MgSO4 and
removed the solvent under reduced pressure. Purification by column-chromatography on silica gel:
methyl 3-hydroxy-4-((3-methylbut-2-en-1-yl)oxy)benzoate (1). White solid. Mp 82–84 °C;
1H NMR (400 MHz, CDCl3) δ 7.60–7.58 (m, 1H), 7.58 (d, J = 2.9 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H),

Mar. Drugs 2011, 9
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5.69 (s, 1H), 5.47 (t, J = 6.8 Hz, 1H), 4.63 (d, J = 6.8 Hz, 2H), 3.87 (s, 3H), 1.81 (s, 3H), 1.75 (s, 3H).
MS: m/z 236 (M+).
methyl 3-hydroxy-4-(isopentyloxy)benzoate (2). White solid. Mp 68–70 °C; 1H NMR (400 MHz,
CDCl3) δ 7.59 (s, 1H), 7.58 (d, J = 0.8 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 5.68 (s, 1H), 4.12 (t, J = 6.6 Hz,
13
2H), 0.97 (s, 3H). C NMR (101 MHz, CDCl3) δ 166.97, 149.96, 145.25, 123.36, 122.46, 115.53,
110.38, 67.46, 51.99, 37.94, 24.96, 22.72. MS: m/z 238 (M+); HRMS calcd for C13H18O4: 238.1200 (M+),
found: 238.1201.
methyl 3-hydroxy-4-propoxybenzoate (3). White solid. Mp 66–68 °C; 1H NMR (400 MHz, CDCl3)
δ 7.60–7.59 (m, 1H), 7.58–7.57 (m, 1H), 6.84 (d, J = 9.0 Hz, 1H), 5.73 (s, 1H), 4.05 (t, J = 6.6 Hz,
2H), 3.87 (s, 3H), 1.92–1.79 (m, 2H), 1.05 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 166.91,
149.92, 145.49, 123.30, 122.81, 115.68, 110.70, 70.66, 51.78, 22.55, 10.51. MS: m/z 210 (M+); HRMS
calcd for C11H14O4: 210.0887 (M+), found: 210.0886.
1
methyl 4-(allyloxy)-3-hydroxybenzoate (4). White solid. Mp 65–67 °C; H NMR (400 MHz,
CDCl3) δ 7.61–7.59 (m, 1H), 7.58 (d, J = 2.1 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 6.06 (dt, J = 16.0,
5.5 Hz, 1H), 5.67 (s, 1H), 5.38 (dd, J = 26.5, 14.5 Hz, 2H), 4.67 (d, J = 5.5 Hz, 2H), 3.88 (s, 3H).
MS: m/z 208 (M+).
1
methyl 3-hydroxy-4-(prop-2-yn-1-yloxy)benzoate (5). White solid. Mp 107–109 °C; H NMR
(400 MHz, CDCl3) δ 7.61 (s, 1H), 7.59 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 5.78 (s, 1H), 4.81
13
(s, 2H), 3.87 (s, 3H), 2.58 (s, 1H). C NMR (101 MHz, CDCl3) δ 166.79, 148.40, 145.62, 124.49,
122.49, 116.35, 111.73, 57.13, 51.82. MS: m/z 206 (M+); HRMS calcd for C11H10O4: 206.0574 (M+),
found: 206.0572.
1
methyl 3,4-bis(benzyloxy)benzoate (7). Pale yellow solid. Mp 55–57 °C; H NMR (400 MHz,
CDCl3) δ 7.67–7.61 (m, 2H), 7.50–7.28 (m, 10H), 6.94 (d, J = 8.3 Hz, 1H), 5.20 (d, J = 10.5 Hz, 4H),
3.87 (s, 3H). MS: m/z 348 (M+).
1
methyl 3,4-dipropoxybenzoate (8). White solid. Mp 39–41 °C; H NMR (400 MHz, CDCl3)
δ 7.63 (dd, J = 8.4, 2.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 4.01 (td, J = 6.6,
13
2.1 Hz, 4H), 3.88 (s, 3H), 1.92–1.78 (m, 4H), 1.05 (t, J = 7.4 Hz, 6H). C NMR (101 MHz, CDCl3)
δ 167.11, 153.42, 148.67, 123.67, 122.63, 114.71, 112.34, 71.01, 70.68, 51.99, 22.72, 22.64, 10.58,
10.54. MS: m/z 252 (M+); HRMS calcd for C14H20O4: 252.1356 (M+), found: 252.1354.
3-hydroxy-4-((3-methylbut-2-en-1-yl)oxy)benzaldehyde (9). Brown solid. Mp 65–67 °C; 1H NMR
(300 MHz, CDCl3) δ 9.82 (s, 1H), 7.42 (s, 2H), 6.97 (s, 1H), 5.80 (s, 1H), 5.48 (s, 1H), 4.67 (s, 2H),
1.80 (d, J = 15.3 Hz, 6H). MS: m/z 206 (M+).
1
3-hydroxy-4-(isopentyloxy)benzaldehyde (10). Brown oil. H NMR (300 MHz, CDCl3) δ 9.84
(s, 1H), 7.42 (d, J = 10.2 Hz, 2H), 6.96 (d, J = 8.0 Hz, 1H), 5.72 (s, 1H), 4.18 (t, J = 6.4 Hz, 2H), 1.80
(dt, J = 21.9, 6.7 Hz, 3H), 1.02 (s, 3H), 1.00 (s, 3H). MS: m/z 208 (M+).
1
3-hydroxy-4-propoxybenzaldehyde (11). White-like solid. Mp 71–73 °C; H NMR (300 MHz,
CDCl3) δ 9.83 (s, 1H), 7.43 (dd, J = 8.2, 5.0 Hz, 2H), 6.95 (d, J = 8.1 Hz, 1H), 5.75 (s, 1H), 4.12
(t, J = 6.6 Hz, 2H), 1.91 (dd, J = 14.0, 6.6 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H). MS: m/z 180 (M+).

Mar. Drugs 2011, 9
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1
3-hydroxy-4-(prop-2-yn-1-yloxy)benzaldehyde (12). Pale white solid. Mp 82–84 °C; H NMR
(300 MHz, CDCl3) δ 9.85 (s, 1H), 7.45 (dt, J = 8.6, 2.2 Hz, 2H), 7.10 (d, J = 8.2 Hz, 1H), 5.77 (s, 1H),
4.87 (d, J = 2.4 Hz, 2H), 2.63 (t, J = 2.2 Hz, 1H). MS: m/z 176 (M+).
1
4-(allyloxy)-3-hydroxybenzaldehyde (13). Pale white solid. Mp 55–57 °C; H NMR (400 MHz,
CDCl3) δ 9.84 (s, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 8.3, 2.0 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H),
6.07 (ddd, J = 22.8, 10.9, 5.5 Hz, 1H), 5.80 (s, 1H), 5.41 (dddd, J = 20.6, 10.5, 2.6, 1.3 Hz, 2H), 4.71
(dt, J = 5.5, 1.4 Hz, 2H). MS: m/z 178 (M+).
1
3,4-bis((3-methylbut-2-en-1-yl)oxy)benzaldehyde (16). Colorless oil. H NMR (300 MHz, CDCl3)
δ 9.82 (s, 1H), 7.49–7.35 (m, 2H), 6.96 (d, J = 8.7 Hz, 1H), 5.56–5.46 (m, 2H), 4.67 (dd, J = 13.0,
6.6 Hz, 4H), 1.80 (s, 6H), 1.77 (s, 6H). MS: m/z 274 (M+).
1
3,4-bis(prop-2-yn-1-yloxy)benzaldehyde (17). Pale yellow solid. Mp 100–102 °C; H NMR
(300 MHz, CDCl3) δ 9.88 (s, 1H), 7.63–7.44 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H), 4.85 (dd, J = 9.3,
2.4 Hz, 4H), 2.63–2.49 (m, 2H). MS: m/z 214 (M+).
3,4-bis(allyloxy)benzaldehyde (18). Red oil. 1H NMR (300 MHz, CDCl3) δ 9.83 (s, 1H), 7.46–7.39
(m, 2H), 6.98 (d, J = 8.4 Hz, 1H), 6.17–6.00 (m, 2H), 5.46 (d, J = 18.7 Hz, 2H), 5.33 (dd, J = 11.2,
6.9 Hz, 2H), 4.69 (dd, J = 10.1, 5.2 Hz, 4H). MS: m/z 218 (M+);
1
3,4-bis(isopentyloxy)benzaldehyde (19). Brown oil. H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H),
7.33 (d, J = 53.8 Hz, 2H), 6.95 (d, J = 8.0 Hz, 1H), 4.08 (dd, J = 12.7, 6.5 Hz, 4H), 1.84 (dd, J = 12.9,
6.4 Hz, 2H), 1.73 (dd, J = 13.3, 6.6 Hz, 4H), 0.97 (d, J = 6.5 Hz, 12H). 13C NMR (101 MHz, CDCl3)
δ 191.33, 154.59, 149.52, 130.01, 126.79, 111.84, 110.84, 67.64, 37.91, 37.78, 25.33, 22.75.
MS: m/z 278 (M+); HRMS calcd for C17H26O3: 278.1876 (M+), found: 278.1873.
3,4-dipropoxybenzaldehyde (20). Brown oil. 1H NMR (300 MHz, CDCl3) δ 9.82 (s, 1H), 7.45–7.38
(m, 2H), 6.96 (d, J = 7.9 Hz, 1H), 4.05 (dd, J = 14.0, 6.9 Hz, 4H), 1.98–1.81 (m, 4H), 1.07 (dd, J = 11.1,
6.8 Hz, 6H). MS: m/z 222 (M+).
1
3,4-bis(prop-2-yn-1-yloxy)benzoic acid (21). White solid. Mp 157–159 °C; H NMR (400 MHz,
CDCl3) δ 7.83–7.71 (m, 2H), 7.11 (d, J = 8.5 Hz, 1H), 4.83 (dd, J = 9.9, 2.4 Hz, 4H), 2.55 (q, J = 2.4 Hz,
2H). MS: m/z 230 (M+).
3,4-bis(isopentyloxy)benzoic acid (22). White solid. Yield, %. Mp 137–139 °C; 1H NMR (300 MHz,
CDCl3) δ 7.72 (d, J = 8.4 Hz, 1H), 7.59 (s, 1H), 6.90 (d, J = 8.5 Hz, 1H), 4.10 (q, J = 6.3 Hz, 4H), 1.88
(dt, J = 13.5, 6.7 Hz, 2H), 1.81–1.69 (m, 4H), 1.00 (d, J = 6.5 Hz, 12H). MS: m/z 294 (M+).
3,4-dipropoxybenzoic acid (23). White solid. Mp 133–135 °C; 1H NMR (300 MHz, CDCl3) δ 7.71
(d, J = 9.9 Hz, 1H), 7.58 (s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 4.04 (dd, J = 11.2, 6.6 Hz, 4H), 1.96–1.81
(m, 4H), 1.08 (t, J = 7.4 Hz, 6H). MS: m/z 238 (M+).
(E)-methyl 3-(3-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)acrylate (24). White solid. Mp 103–105 °C;
1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 15.9 Hz, 1H), 7.15 (d, J = 2.1 Hz, 1H), 7.03 (dd, J = 8.5,
1.9 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.30 (d, J = 15.9 Hz, 1H), 5.67 (s, 1H), 4.79 (d, J = 2.4 Hz, 2H),
3.79 (s, 3H), 2.58 (t, J = 2.4 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 167.75, 146.53, 146.35, 144.56,

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129.26, 123.99, 121.59, 116.61, 113.88, 112.59, 111.70, 57.03, 51.78. MS: m/z 232 (M+); HRMS calcd
for C13H12O4: 232.0730 (M+), found: 232.0732.
(E)-methyl 3-(3,4-bis(prop-2-yn-1-yloxy)phenyl)acrylate (25). White solid. Mp 96–98 °C;
1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 15.9 Hz, 1H), 7.25–7.11 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H),
13
6.31 (d, J = 15.9 Hz, 1H), 4.77 (dd, J = 2.4, 1.2 Hz, 4H), 3.79 (s, 3H), 2.57–2.48 (m, 2H). C NMR
(101 MHz, CDCl3) δ 167.59, 149.54, 147.63, 144.37, 128.62, 123.25, 116.47, 114.45, 113.93, 78.23,
78.11, 76.44, 76.40, 57.10, 56.85, 51.74. MS: m/z 270 (M+); HRMS calcd for C16H14O4: 270.0887 (M+),
found: 270.0888.
3.2.2. General Procedure 2: Synthesis of Compounds 6, 14–15
A mixture of di-substituted 3,4-dihydroxybenzoic acid methyl ester or 3,4-dihydroxybenzaldehyde
with 10% Pd/C in 10% K2CO3–MeOH was stirred at room temperature for an appropriate time. After
the catalyst was filtered, the filtrate was concentrated in vacuo. The mixture was extracted with AcoEt.
The organic extracts were washed with brine and dried. The solvent was evaporated under reduced
pressure to give a residue, which was purified by column-chromatography on silica gel.
1
methyl 3-(allyloxy)-4-hydroxybenzoate (6). White solid. Mp 46–48 °C; H NMR (300 MHz,
CDCl3) δ 7.64 (d, J = 10.3 Hz, 1H), 7.56 (s, 1H), 6.96 (d, J = 3.9 Hz, 1H), 6.15–6.04 (m, 1H), 6.03
(s, 1H), 5.40 (dd, J = 26.7, 13.7 Hz, 2H), 4.68 (d, J = 5.5 Hz, 2H), 3.89 (s, 3H). MS: m/z 208 (M+).
1
4-hydroxy-3-((3-methylbut-2-en-1-yl)oxy)benzaldehyde (14). Brown oil. H NMR (400 MHz,
CDCl3) δ 9.79 (s, 1H), 7.41 (s, 1H), 7.38 (s, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.53 (s, 1H), 5.46
13
(t, J = 6.3 Hz, 1H), 4.62 (d, J = 6.8 Hz, 2H), 1.78 (s, 3H), 1.74 (s, 3H). C NMR (101 MHz, CDCl3)
δ 191.02, 151.79, 146.44, 139.68, 129.52, 127.56, 118.24, 114.29, 109.75, 65.77, 25.38, 18.30.
MS: m/z 206 (M+); HRMS calcd for C12H14O3: 206.09387 (M+), found: 206.0938.
3-(allyloxy)-4-hydroxybenzaldehyde (15). Pale white solid. Mp 113–115 °C; 1H NMR (300 MHz,
CDCl3) δ 9.80 (s, 1H), 7.42 (s, 2H), 7.06 (d, J = 14.5 Hz, 1H), 6.22 (s, 1H), 6.18–5.98 (m, 1H), 5.41
(dd, J = 21.1, 14.9 Hz, 2H), 4.70 (d, J = 9.2 Hz, 2H). MS: m/z 178 (M+).
1
(E)-methyl 3-(3,4-dihydroxyphenyl)acrylate (26). Pale white solid. Mp 164–166 °C; H NMR
(400 MHz, CDCl3) δ 7.58 (d, J = 22.4 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.2, 2.0 Hz, 1H),
6.87 (d, J = 8.2 Hz, 1H), 6.27 (d, J = 15.9 Hz, 1H), 5.58 (s, 2H), 3.79 (s, 3H). MS: m/z 194 (M+).
1
methyl 3-(isopentyloxy)-4-methoxybenzoate (27). Pale yellow oil. H NMR (400 MHz, CDCl3)
δ 7.63 (dd, J = 8.4, 2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 4.06 (t, J = 6.9 Hz,
2H), 3.88 (s, 3H), 3.86 (s, 3H), 1.83 (dt, J = 13.0, 6.5 Hz, 1H), 1.74 (q, J = 6.9 Hz, 2H), 0.96 (d,
13
J = 6.5 Hz, 6H). C NMR (101 MHz, CDCl3) δ 167.02, 153.41, 148.21, 123.32, 122.69, 113.59,
110.49, 67.51, 56.02, 51.98, 37.96, 25.14, 22.67. MS: m/z 252 (M+); HRMS calcd for C14H20O4:
252.1356 (M+), found: 252.1354.
3-methylbut-2-en-1-yl 4-methoxy-3-((3-methylbut-2-en-1-yl)oxy)benzoate (28). Colorless oil.
1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 42.4 Hz, 2H), 6.83 (s, 1H), 5.45 (s, 2H), 4.68 (d, J = 61.2 Hz,
13
4H), 3.88 (s, 3H), 1.75 (s, 12H). C NMR (101 MHz, CDCl3) δ 166.61, 153.33, 147.82, 138.86,

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138.22, 123.53, 122.87, 119.56, 118.96, 115.70, 113.68, 110.27, 65.97, 61.78, 55.73, 25.90, 18.27,
18.14. MS: m/z 304 (M+); HRMS calcd for C18H24O4: 304.1669 (M+), found: 304.1667.
3-methylbut-2-en-1-yl 3-hydroxy-4-methoxybenzoate (29). White solid. Mp 154–156 °C; 1H NMR
(400 MHz, CDCl3) δ 7.59 (d, J = 6.0 Hz, 2H), 6.82 (d, J = 9.0 Hz, 1H), 5.96 (s, 1H), 5.43 (t, J = 7.8 Hz,
13
1H), 4.76 (d, J = 7.1 Hz, 2H), 3.88 (s, 3H), 1.74 (d, J = 8.1 Hz, 6H). C NMR (101 MHz, CDCl3)
δ 166.37, 150.57, 145.25, 138.83, 123.68, 122.64, 118.75, 115.78, 109.93, 61.78, 55.91, 25.58, 18.08.
MS: m/z 236 (M+); HRMS calcd for C13H16O4: 236.1043 (M+), found: 236.1042.
3.2.3. Synthesis of 3,4-bis(prop-2-yn-1-yloxy)benzamide (30)
The mixture of 3,4-dihydroxybenzoic acid methyl ester and propargyl bromide were dissolved in
acetone and 5 equiv K2CO3 were added. Through general procedure 1, di-substituted compound was
obtained. Then it was dissolved in MeOH, 5 equiv of the amine were added and the mixture was
stirred at 40 °C for 24 h. The mixture were poured into water and extracted with AcoEt. Removing the
water of the aqueous layer and the target compound was obtained as white solid. Mp 145–147 °C;
1H NMR (400 MHz, DMSO) δ 7.83 (s, 1H), 7.60–7.49 (m, 2H), 7.22 (s, 1H), 7.10 (d, J = 8.5 Hz, 1H),
4.85 (dd, J = 14.6, 2.4 Hz, 4H), 3.57 (dt, J = 7.6, 2.4 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 167.14,
149.27, 146.16, 127.43, 121.25, 113.57, 113.04, 79.03, 78.88, 78.56, 78.44, 56.09, 56.00. MS: m/z 230
(M+ + H); HRMS calcd for C13H11NO3: 229.0733 (M+), found: 229.0732.
1
methyl 3-hydroxy-4-methoxybenzoate (31). White solid. Mp 65–67 °C; H NMR (400 MHz,
CDCl3) δ 7.64–7.57 (m, 2H), 6.87 (d, J = 8.4 Hz, 1H), 5.62 (s, 1H), 3.95 (s, 3H), 3.88 (s, 3H).
MS: m/z 182 (M+).
(E)-methyl 3-(4-hydroxy-3-((3-methylbut-2-en-1-yl)oxy)phenyl)acrylate (32). Yellow oil. 1H NMR
(400 MHz, CDCl3) δ 7.61 (d, J = 15.8 Hz, 1H), 7.06 (d, J = 9.6 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.27
(d, J = 15.9 Hz, 1H), 5.96 (s, 1H), 5.48 (s, 1H), 4.61 (s, 2H), 3.79 (s, 3H), 1.79 (d, J = 19.9 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 167.90, 148.51, 146.09, 145.22, 139.49, 126.93, 123.01, 118.85,
115.21, 114.86, 110.84, 66.03, 51.71, 25.85, 18.39. MS: m/z 262 (M+); HRMS calcd for C15H18O4:
262.1200 (M+), found: 262.1199.
3.3. Virus and Cell Culture
The pandemic influenza H1N1 2009 virus Influenza A strains A/GuangdongSB/01/2009 (H1N1),
A/Guangdong/03/2009 (H1N1) and A/Guangdong/05/2009 (H1N1) were kindly provided by Dr. Li at
the Sun Yat-sen University Zhongshan School of Medicine (Guangzhou, Guangdong, China.) who
isolated it during the human swine flu outbreak of 2009. Madin-Darby canine kidney (MDCK) cells
were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum
(FBS) (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 100 μg/mL streptomycin and 100 units/mL
penicillin (Invitrogen, Carlsbad, CA, USA). The cultures were maintained at 37 °C in a humidified
atmosphere of 5% CO2. All viruses were amplified and titrated in MDCK cells and stored at −80 °C
until use. Confluent MDCK monolayers in 6-well plates were washed three times with PBS and
incubated with viruses at a multiplicity of infection (MOI) of 0.001 in DMEM for 2 h at 37 °C. After
viral adsorption, media was removed, cells were washed three times with PBS and incubated with

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post-adsorption medium [DMEM, supplemented with 1% fetal bovine serum (FBS) (Hyclone, Logan,
UT, USA), 2 mM L-glutamine, 100 μg/mL streptomycin and 100 units/mL penicillin (Invitrogen,
Carlsbad, CA, USA), 25 mM HEPES buffer (pH 7.2), 0.1 mM non-essential amino acids, 1.0 mM
sodium pyruvate, 2 mg/mL L-1-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin
(Invitrogen, Carlsbad, CA, USA)].
3.4. Neuraminidase Activity Inhibition Assay
Influenza virus neuraminidase (NA) sensitivity to the test compounds was evaluated by using
an NA activity inhibition assay, based on the method of Maki Kiso et al. [11]. Briefly,
4-Methylumbelliferyl-N-acetylneuraminic acid (4-MUNANA, Sigma-Aldrich, St. Louis, MO, USA), at
a final concentration of 0.15 mM, was used as the fluorescent substrate. The virus solution
[(A/Guangdong/03/2009 (H1N1), ranging from 800 to 1200 fluorescence units of MU-NANA] was
prepared in MES assay buffer [32.5 mM MES (pH 6.5), 4 mM CaCl2, 0.1% NP-40, 0.3 mg/mL BSA],
the test compounds were dissolved in MES assay buffer at various concentrations (1, 10, 20, 40, 50,
80 μM), and Tamiflu® (Roche Laboratories Inc., Basel, Switzerland) was used as a positive control.
Diluted virus was mixed with each test compound in 50 μL MES assay buffer, and the mixtures were
incubated at 37 °C for 1 h with gentle agitation, then mixed with 50 μL 4-MUNANA buffer and
incubated at 37 °C for another 1 h with gentle agitation. The reaction was terminated by adding 100 μL
of 0.1 M NaOH in 80% ethanol (pH 10.0). Then the fluorescence was measured at an excitation
wavelength of 360 nm and an emission wavelength of 465 nm, using a SpectraMax M5 multi-detection
reader (Molecular Devices Corporation, Sunnyvale, CA, USA). Under the experimental conditions, a
linear relationship between the enzyme amount and the fluorescence was confirmed. Each NA
inhibition assay was done in triplicate. NA activity inhibition was determined using the following
formula as described previously [11]: NA activity inhibition (%) = (1 − OD465 of treated group/OD465
of control group) × 100%. The half maximal inhibitory concentration (IC50) was calculated by Bliss
software [12] and the data, expressed as means ± SD, were analyzed by SPSS.
3.5. Modelling of Enzyme-Substrate Complexes by Molecular Docking
Surflex-Dock was applied to study molecular docking. Crystal structure of NA was retrieved from
RCSB Protein Data Bank (PDB entry code: 2ht7) [13]. This is a particular structure with oseltamivir.
The protein structure was utilized in subsequent docking experiments without energy minimization.
All ligands and water molecules have been removed at first and the polar hydrogen atoms were added.
Automatic docking was employed. Other parameters were established by default in software. All
molecules were minimized using default parameter.
4. Conclusions
This study provided a new kind of non-nitrogenous small molecule NA inhibitor, some examples of
which had stronger activities than the reported benzoic acid compounds that have high affinity for
influenza virus but high IC50 values (~mM level) [7]. It also proved that the carboxyl group is not
necessary and replacement with an aldehyde group enhanced the activity. The OH group was

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indispensable to the activity, but the ether chains were not crucial to the activity, regardless of their
degree of unsaturation. In addition, these compounds were easily obtained with excellent yields.
Further studies aimed at developing an efficient anti-influenza virus candidate based on the SAR
analysis and the docking results are underway in our laboratory.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 20802094;
81102370), Natural Science Foundation of Guang dong Province, China (Grant No. S2011020001231;
S2011040003876), and China’s Marine Commonweal Research Project (Grant No. 201005022-2),
High-Tech Research (863) projects (2011AA09070201); China Postdoctoral Science Foundation
(20100480825, 20110490954).
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Samples Availability: Available from the authors.
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