Further discovery of caffeic acid derivatives as novel influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.10.020

Eight series of compounds, each series containing two to five compounds were prepared by structural modifications of a lead, which was previously discovered as a mild influenza neuraminidase (NA) inhibitor. On the basis of the biological result, a detailed structure-activity relationship (SAR) was derived and discussed. Several caffeic acid derivatives that acted as non-competitive inhibitors were close or superior to the lead and also presented good antiviral activities in cells. Besides, it was interesting to find that modifications of the lead with different strategies could result in selective inhibition against N1 or N2. The preliminary docking analysis indicated that the 150-cavity of the enzymes played an important role in the selective inhibition.

Full text of DOI:10.1016/j.bmc.2013.10.020

Accepted Manuscript
Further Discovery of caffeic acid derivatives as Novel Influenza Neuraminidase
inhibitors
Yuanchao Xie, Bing Huang, Kexiang Yu, Wenfang Xu
PII:
S0968-0896(13)00885-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.10.020
BMC 11171
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 September 2013
30 September 2013
17 October 2013
Please cite this article as: Xie, Y., Huang, B., Yu, K., Xu, W., Further Discovery of caffeic acid derivatives as Novel
Influenza Neuraminidase inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/
j.bmc.2013.10.020
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Further Discovery of caffeic acid derivatives as Novel Influenza
Neuraminidase inhibitors
Yuanchao Xie a, Bing Huang b, Kexiang Yu b, Wenfang Xu a,*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44, West Culture
Road, Jinan, Shandong 250012, P. R. China
b Institute of poultry science, Shandong Academy of Agricultural Sciences, 1, Jiaoxiao Road, Jinan, Shandong
250023, P. R. China
ABSTRACT
Eight series of compounds, each series containing two to five compounds were prepared by
structural modifications of a lead, which was previously discovered as a mild influenza
neuraminidase (NA) inhibitor. On the basis of the biological result, a detailed structure-activity
relationship (SAR) was derived and discussed. Several caffeic acid derivatives that acted as
non-competitive inhibitors were close or superior to the lead and also presented good antiviral
activities in cells. Besides, it was interesting to find that modifications of the lead with different
strategies could result in selective inhibition against N1 or N2. The preliminary docking analysis
indicated that the 150-cavity of the enzymes played an important role in the selective inhibition.
Keywords: Caffeic acid; Influenza; Inhibitor; Neuraminidase.
* Corresponding author: Wenfang Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University 44,
West Culture Road, Jinan 250012, P. R. China
Tel/Fax: +86-531-88382264; E-mail address: wenfxu@gmail.com, yuanchaox623@aliyun.com.
1. Introduction
Influenza viruses that have a high mutation rate pose a great threat to human. In the 20th
century, there were three influenza pandemics (H1H1, H2N2 and H3N2), killing tens of millions
of people.1 The first outbreak of the highly pathogenic avian influenza A (H5N1) virus occurred in
1997, and since then this virus has been continuing to evolve and causing global concern. More
recently, some novel emerging viral strains, such as the 2009 swine-origin H1N1 virus2 and the
2013 Chinese H7N9 virus3 also give rise to seriously public panic.

Flu vaccine is an effective method for preventing influenza virus infection, but it needs
reformulating each year to match antigenic variations and is inadequate to cope with an
unpredictable pandemic. The influenza neuraminidase (NA) plays an important role in the life
cycle of influenza virus and has been discovered as an important target for anti-influenza drugs
design.4 Till now, three NA inhibitors, zanamivir, oseltamivir and peramivir have been approved
for the treatment and prophylaxis of influenza. Among them, oseltamivir administered orally is the
first line defense drug against influenza. However, in recent years more and more influenza virus
strains are resistant to it, such as the seasonal H1N1 viruses5 and avian H5N1 strains.6 The other
two drugs are mainly administered via inhaled nebulized or intravenous routes, which is very
inconvenient for patients. Therefore, it is still necessary to find novel effective drugs with good
properties to cope with influenza viruses.
In recent three years, only a few influenza NA inhibitors have been reported and most of
them are mainly limited to the derivatives of zanamivir or oseltamivir.7-12 Because zanamivir and
oseltamivir have perfectly occupied the active site of NA, the structural modification of them is
hard to discover better compounds. It seems that the development of NA inhibitors has been in a
difficult position. However, it is worth noting that many natural products, especially some small
plant-derived polyphenols have been found to exert good antiviral effects against influenza virus
and show moderate inhibitory activity against NA. These compounds contain various structures,
such as flavonoids13, xanthones14,15 and diarylheptanoids,16 which are suitable to be leads for the
design of novel influenza NA inhibitors.
In our previous studies, we built a compound library containing a variety of structures based
on the reported flavonoid NA inhibitors.17,18 Among them, we discovered that the caffeic acid
derivatives present good anti-influenza virus activity and NA inhibitory activity in vitro, such as
compound 1 and 2 (Fig. 1). Besides, compound 2 was found to be a non-competitive inhibitor
with Ki = 11.5 ± 0.25 µM. In the present study, we took compound 2 as the lead to make further
modifications. Eight series of compounds, each series containing two to five compounds were
prepared by different synthetic methods. The enzyme inhibition assay indicated that the
compounds containing caffeic acid fragment displayed much better activities. Several compounds
were closed or superior to the lead and also showed good antiviral activities in cells. Besides, it
was interesting to find that modifications of the lead with different strategies could result in

selective inhibition against N1 or N2.
Fig. 1. Two caffeic acid derivatives discovered in our previous studies
2. Chemistry
In order to make sure if the caffeic acid fragment was necessary for the activity and explore
the structure-activity relationship (SAR), four series of compounds without the caffeic acid
fragment were designed and synthesized in Scheme 1. Compound 5 was a key intermediate that
can be synthesized form the starting material, 2-amino-4-nitrophenol (compound 3) through three
serial steps: acylation with 2-ethylbutanoic acid, Williamson ether reaction with ethyl
chloroacetate and reduction of the nitro group by hydrogen. The target compounds 6a-6c were
prepared by reaction of compound 5 with acyl chloride or sulfuryl chloride and hydrolysis with
NaOH.
Because the basic group, such as amino group or guanidine group was important for the
activity of classical influenza NA inhibitors,19 compound 7, 9 and 10 were designed. For
synthesizing compound 7a, the amino group of compound 5 was first protected with Boc, then
hydrolyzed to give the Boc-protected acid. Deprotection of the intermediate with TFA afforded
the target compound. For synthesizing compound 7b, compound 5 was first guanylated with
N,N’-bis(tert-butoxycarbonyl)thiourea (SC(NHBoc)2)/HgCl2. The following hydrolysis and Boc
deprotection would afford compound 7b. Compounds 9 and 10 can also be synthesized with the
same methods from compounds 8a and 8b, which were obtained by two step form compound 5,
acylation with acyl chlorides and nitration with HNO3/H2SO4. The position of the NO2 on the
benzene ring of compounds 8 can be determined by the spectra of 1H-NMR.
To synthesize compounds 11a-11c, compound 5 was first converted the isocyanate and then
reacted with different amines. After hydrolysis with NaOH, the urea compounds were obtained.

Scheme 1. Reagents and conditions: (a) 2-ethylbutanoyl chloride, NaHCO3, THF, H2O, rt; (b) ethyl chloroacetate,
NaI, K2CO3, acetone, reflux; (c) H2, Pd/C, CH3CH2OH, rt; (d) acyl chloride or sulfuryl chloride, NaHCO3,
THF,H2O, rt; (e) NaOH, CH3OH, H2O, rt; (f) for 7a: 1. (Boc)2O, Et3N, THF, rt; 2. NaOH, CH3OH, H2O, rt; 3.
CH2Cl2, CF3COOH; for 7b: 1. SC(NHBoc)2, HgCl2, Et3N, DMF; 2. NaOH, CH3OH, H2O, rt; 3. CH2Cl2,
CF3COOH; (g)HNO3, H2SO4; (h) 1. triphosgene, dioxane, 40 oC; 2. THF, Et3N, R5NH2, rt.
In Scheme 2, three series of compounds containing the caffeic acid fragment were designed
and
synthesized.
For
improving
the
yields,
the
acetylated
caffeic
acid,
(E)-3-(3,4-diacetoxyphenyl)acrylic acid that had a relatively better solubility in common organic
solvents was used as the material. This compound was converted into the corresponding acyl
chloride by using oxalyl chloride and reacted with the reduced aniline products of compounds 8.
After hydrolysis with NaOH, compounds 12 were obtained. Compounds 13 were prepared by
reaction of compound 5 and different aldehydes in the present of NaBH3CN. A little amount of
acetic acid could be favorable for this reaction. Condensation of compound 5 with various Boc
protected amino acid afforded compounds 15a-15f, which were subsequently treated with 3M
HCl/EtOAc to remove the Boc group. Compounds 14a-14c and 16a-16f were prepared with the
same methods of synthesizing compounds 12.

Scheme 2. Reagents and conditions: (a) R6COCl, NaHCO3, THF, H2O, rt; (b) H2SO4, HNO3, 0 oC; (c) H2, Pd/C,
CH3CH2OH, rt; (d) 1. (E)-3-(3,4-diacetoxyphenyl)acrylic acid, oxalyl chloride, DMF, anhydrous THF, rt; 2.
NaHCO3, THF, H2O, rt; 3. NaOH, CH3OH, H2O, rt; (e) R7CHO, NaBH3CN, CH3COOH; (f) Boc-L-amino acid,
TBTU, Et3N,rt; (f) 3M HCl/EtOAc, rt.
In order to explore whether the double bond of the caffeic acid fragment was important for
the NA inhibitory activity. In Scheme 3, compounds 17a and 17b were prepared by reduction of
the double bond of compounds 12a and 12b.
Scheme 3. Reagents and conditions: H2, Pd/C, CH3CH2OH, rt.
3. Results and discussion
3.1. In vitro inhibitory activities on NAs
Generally, influenza NAs were divided into two groups: group-1 NAs containing N1, N4, N5,
N8 and group-2 NAs containing N2, N3, N6, N7, N9. It had been found that there were some
structural differences between the two group NAs in the region adjacent to the enzyme’s catalytic
center.20 Therefore, we chose NAs from two kinds of influenza viruses ( H9N2 and H5N1) to

determine the inhibitory effects of the designed compounds.
Table 1 listed the in vitro inhibitory effects of compounds 6, 7, 9, 10 and 11 against
influenza A NAs. Compared with the lead (compound 2), the twelve compounds with diverse
structures exhibited decreased activities, suggesting that the caffeic acid fragment was important.
Among compounds 6a-6a and 11a-11c, which had amides and ureas in their structures,
Compound 11a was the best. It seemed that the urea structures with unsaturated groups, especially
the aromatic groups favored the activity. Compounds 7a and 7b that had basic groups (the amino
group and the guanidine group) at C-4 position only presented weak inhibition at the concentration
of 100 µM, while compounds 9 and 10 with the basic groups at C-5 position exhibited improved
activities. Besides, compounds 9a and 10a with the amino group were better than compounds 9b
and 10b with the guanidine group. According to the pharmacophore model of classical influenza
NA inhibitors, the COOH group and basic groups were essential for the activity.21 However,
compounds 9 and 10 did not show potent activities against NA, which was probably because the
necessary groups could not bind with the corresponding pockets in the enzymatic active site.
Table 1
In vitro inhibitory effects of compounds 6, 7, 9, 10 and 11 against influenza A NAs
compound
IC50 (µM, H9N2
and H5N1)
compound
IC50 (µM, H9N2
and H5N1)
6a
> 100
9b
54.3/61.2
6b
6c
7a
7b
9a
78.6/86.7
> 100
10a
10b
11a
11b
11c
69.1/75.8
83.2/NT
49.7/55.6
89.8/NT
> 100
> 100
> 100
46.4/58.5
Table 2 listed the in vitro inhibitory effects of compounds 12, 14 and 16 against influenza A
NAs. All the ten compounds containing the caffeic acid fragments showed good NA inhibitory

activities, and several compounds, such as 12a, 12b, 16b and 16d were superior or closed to the
lead. The general order of potency for NA inhibition was compounds 12 > compounds 16 >
compounds 14, which indicated that the position of the caffeic acid fragment had a certain effect
on the activity. This should be carefully considered in the further studies. Besides, It was
noteworthy that compounds 12 and 16 selectively inhibited N2 and compounds 14 selectively
inhibited N1. The lead compound also presented slight selective inhibition against N2. Structurally,
compounds 14 had branched substituted groups at the amide nitrogen atoms, while compounds 12,
16 and the lead did not. According to the crystal structures of some group-1 NAs, there were at
least two cavities, such as the 150-cavity and 430-cavity adjacent to the active site.20,22 The
branched side chains of compounds 14 may contribute to multiple interactions with N1 and lead to
the selective inhibition. This strategy of structural modification could be used to look for highly
potent and selective group-1 NA inhibitors. Compound 9a (Table 1) was a mild NA inhibitor with
IC50 values of 46.4/58.5 against N2 and N1, respectively, while compound 12a obtained by
condensation of compound 9a and caffeic acid was about 15-fold more potent against N1.
Moreover, modifications on the NH2 at C-4 position would also affect the activity, since
compound 12b with isobutyryl group at this site was less potent than compound 12a. Generally,
compounds 16a-16e with different amino acids as linkers connecting the caffeic acid fragment
exhibited similar activities to the lead. The side chains of the amino acids had little influence on
the activity.
Table 2
In vitro inhibitory effects of compounds 12a,12b, 14a-14c and 16a-16e against influenza A NAs
compound
IC50 (µM, H9N2 and
H5N1)
compound
IC50 (µM, H9N2 and
H5N1)
12a
12b
14a
14b
3.2/10.7
5.8/14.2
25.2/11.3
24.1/16.4
16a
16b
16c
16d
9.2/17.6
7.9/16.1
9.4/16.6
8.5/14.0

14c
16.0/11.4
16e
9.8/16.2
All compounds were tested in triplicate wells per dilution. Oseltamivir carboxylate was used as the positive control
with IC50 values = 0.0028 µM (H9N2) and 0.016 µM (H5N1). NT: not tested.
In Table 3, it could be found that the activities of compounds 17 were five-fold less potent
than those of compounds 12, which proved that the double bond of caffeic acid fragment
contributing to structural constraints played an important role in the activity. Besides, the two
compounds also showed obvious selective inhibition against N2 like compounds 12.
Table 3
In vitro inhibitory effects of compounds 17a and 17b against influenza A NAs
compound IC50 (µM, H9N2
and H5N1)
17a
17b
15.8/42.7
14.2/38.2
3.2. Enzyme inhibition kinetics
In the kinetic studies, compounds 12a, 14c and 16b were found to be non-competitive
inhibitors with Ki = 2.70 ± 0.25 µM (N2), 32.5 ± 1.2 µM (N1) and 9.50 ± 0.50 µM (N1),
respectively by the means of Lineweaver-Burk and Dixon plots analysis (Fig. 2). In Recent years,
many natural products, especially polyphenol compounds have also been discovered as
non-competitive NA inhibitors. However, the exact binding site of this kind of inhibitors in the
enzyme is still unclear.

Fig. 2. Graphical determination of the type of inhibition for compound 12a, 14c and 16c. (A, B, C):
Lineweaver–Burk plot for the inhibition of compound 12a, 14c and 16c on NA from influenza virus for the
hydrolysis of substrate in the presence of different concentrations. (D, E, F): Dixon plot for inhibition of compound
15d on NA from influenza virus for the hydrolysis of substrate in the presence of different concentrations of
substrate
3.3. Docking analysis
Except for the NA active site and the adjacent several cavities, we did not find other sites that
were suitable for the docking analysis in the enzyme surface by using SYBYL-X. Therefore, we
made a preliminary binding analysis by docking compound 12a in the region around the active
site of N1 and N2. According to the result, it could be found that there was a relative large cavity,
named 150-cavity adjacent to the active site of N1 (Fig. 3, A), and the caffeic acid fragment of
compound 12a bound to this pocket. In the N2 enzyme, though the size of the 150-cavity was
small and constricted by the closed 150-loop (Fig. 3, B), the caffeic acid fragment of compound
12a could penetrate into the cavity under the closed 150-loop. This kind of interactions would to a
great extent increase the binding affinity of compound 12a with N2 relative to N1. The selective
inhibition of this compound against N2 was thus explicable.

Fig. 3. The docking result of compound 12 with N1 and N2
3.4 In vitro anti-influenza virus activity
Some compounds were further evaluated for their anti-influenza virus activities as well as the
cellular toxicity by the CPE reduction assay (Table 4).23 The assay results showed that most of
them possessed in vitro anti-influenza virus (H9N2) activity. Among them, compounds 12a, 12b
and 16d exhibited good antiviral activities with IC50 values of 13.8 µM, 14.5 µM and 13.5 µM,
respectively, and were about 2-fold more potent than the lead compound.
Table 4
In vitro anti-influenza virus (A/Chicken/Shandong/LY/08) activities in Chicken embryo fibroblast cells using the
CPE reduction assay
Compound
12a
EC50 (µM)a
13.8
CC50 (µM)b
> 100
> 100
> 100
87.6
Compound
16b
EC50 (µM)a
25.4
CC50 (µM)b
> 100
70.4
12b
14.5
16c
22.9
14a
56.4
16d
13.5
89.7
14b
NT
16e
NT
68.3
14c
NT
78.2
17a
NT
47.2
16a
33.6
> 100
OSc
0.24
> 20.0
a: EC50: concentration required to achieve 50% protection against virus-induced cytopathic effect
b: CC50: concentration of 50% cellular toxicity
c: OS: oseltamivir carboxylate, the positive control
NT: not tested.
4. Conclusion

In summary, we designed and synthesized eight series of compounds by structural
modifications of compound 2, a mild influenza NA inhibitor discovered in our previous work.
Among them, compounds 12, 14 and 16 that contained the caffeic acid fragment were much more
potent than compounds 6, 7, 9, 10 and 11. several compounds were closed or superior to the lead,
such as compounds 12a, 12b and 16c. Compounds 17 with a saturated bond instead of the double
in the caffeic acid fragment exhibited decreased activities. The present study demonstrated that the
caffeic acid fragment played an important role in the NA inhibitory activity. Compounds 12a was
the best compound with IC50 values of 3.2/10.7 µM and also exhibits good antiviral activity in
cells. The docking analysis indicated that the 150-cavity that was different between the two
enzymes mainly contributed to the selective inhibition of N2.
H5N1 influenza virus is a great threat to human and many isolated strains of the virus have
been resistant to oseltamivir. In this study, we happened to find that compounds 14 could
selectively inhibit N1. Though they are not potent, the strategy of structural modifications may be
useful to look for more potent and selective NA inhibitors. Caffeic acid itself has a variety of
potential pharmacological effects, such as anti-inflammatory, anti-cancer and antiviral activities.
In our studies, we have proved that caffeic acid derivatives have good anti-influenza virus
activities and NA activities. We hope that they could be new candidates for developing new
anti-influenza agents.
5. Experiments
5.1. chemistry
Caffeic acid, amino acid methyl esters, L-ornithine, 2-amino-4-nitro phenol (compound 3),
N,N’-bis(tert-butoxycarbonyl)thiourea and other compounds not specifically mentioned in the
synthetic routes were commercially available. Solvent for anhydrous reaction should be processed
before use. 1H-NMR spectra were determined on a Brucker Avance 300 spectrometer or 600 using
TMS as an internal standard. The solvents for NMR were DMSO-d6 (δ 2.5 for 1H), CD3OD (δ 3.3
1
for H). ESI-MS was determined on API 4000 LC/MS spectrometer (Applied Biosystems, USA)
and HRMS analysis was provided by Agilent 6520 Q-TOF LC/MS spectrometer (Agilent,
Germany). All reactions were monitored by thin-layer chromatography (TLC) on 25.4×76.2 mm
silica gel plates (GF-254). Silica gel used for column chromatography was 200-300 mesh. Melting

points were determined on an electrothermal melting point apparatus and were uncorrected.
5.1.1. The general synthetic procedure for compounds 6a-6c
Compound 5 was prepared according to the reported method from 2-amio-4-nitro phenol,
compound 3.18 To a mixture of compound 5 (0.46 g, 1.5 mmol) and NaHCO3 (0.50 g, 6 mmol) in
20 mL THF/H2O (5:1) was added acyl chloride or sulfuryl chloride (1.5 mmol) slowly. The
reaction mixture was stirred for 1h at room temperature, then concentrated and extracted with
EtOAc. The combined organic extracts were washed with saturated NaHCO3, 1 M aqueous HCl,
dried and concentrated.
Without further purification, the obtained crude was dissolved in 15 mL CH3OH and 5 mL
H2O followed by the addition of NaOH (0.6 g, 15 mmol). The mixture was allowed to be stirred
for 15 min at room temperature and then concentrated. The remaining solution was acidated with
HCl to a pH of 3 and extracted with EtOAc. The combined extracts were dried over anhydrous
MgSO4, and concentrated. Chromatographic purification of the crude product with EtOAc and
petroleum afforded the target compounds.
2-(2,4-bis(2-ethylbutanamido)phenoxy)acetic acid (6a)
White solid, yield: 37%. mp = 124-125 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.09 (br, 1H), 9.10
(s, 1H), 8.08 (s, 1H), 8.02 (d, 1H, J = 2.4 Hz), 7.20 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 6.87 (d, 1H, J = 9.0
Hz), 4.66 (s, 2H), 3.29-3.33 (m, 5H), 2.28-2.33 (m, 1H), 1.52-1.59 (m, 2H), 1.41-1.49 (m, 2H), 1.07 (t,
6H, J = 7.2 Hz), 0.87 (t, 6H, J = 7.2 Hz). HRMS calcd for C20H31N2O5+ [M+H] + 379.2233; found:
379.2230.
2-(2-(2-ethylbutanamido)-4-(4-methylphenylsulfonamido)phenoxy)acetic acid (6b)
White solid, yield: 42%. mp = 158-160 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.10 (br, 1H), 9.93
(s, 1H), 9.05 (s, 1H), 7.83 (s, 1H), 7.62 (d, 2H, J = 7.8 Hz), 7.32 (d, 2H, J = 7.8 Hz), 6.84 (d, 1H, J =
8.4 Hz), 6.73 (d, 1H, J = 8.4 Hz), 4.65 (s, 2H), 2.33 (s, 3H), 2.28-2.32 (m, 1H), 1.49-1.55 (m, 2H),
1.40-1.45 (m, 2H), 0.84 (t, 6H, J = 7.2 Hz). HRMS calcd for C21H27N2O6S+ [M+H] + 435.1590; found:
435.1584.
2-(2-(2-ethylbutanamido)-4-(furan-2-carboxamido)phenoxy)acetic acid (6c)
1
White solid, yield: 46%. mp = 192-193 ºC. H NMR (600 MHz, DMSO-d6): δ 13.13 (br, 1H),
10.11 (s, 1H), 9.18 (s, 1H), 8.29 (s, 1H), 7.91 (s, 1H), 7.51 (d, 1H, J = 8.4 Hz), 7.32 (d, 1H, J = 3.0 Hz),
6.97 (d, 1H, J = 8.4 Hz), 6.68 (d, 1H, J = 1.2 Hz), 4.71 (s, 2H), 2.34-2.37 (m, 1H), 1.53-1.60 (m, 2H),

1.44-1.49 (m, 2H), 0.88 (t, 6H, J = 7.2 Hz). HRMS calcd for C19H23N2O6+ [M+H] + 375.1556; found:
375.1551.
5.1.2. The synthetic procedure for 2-(4-amino-2-(2-ethylbutanamido)phenoxy)acetic acid
trifluoroacetic acid salt (7a)
To a solution of compound 5 (0.31g, 1.0 mmol) and Et3N (0.29 ml, 2 eq) in 20 ml THF was
added (Boc)2O ( 0.22g, 1.0 mmol). The reaction mixture was allowed to be stirred for 6h at room
temperature, and then concentrated. The obtain residue was hydrolyzed with NaOH by the method
above to give Boc-7a. The intermediate was dissolved in CH2Cl2 (1.0 mL), followed by the
addition of TFA (1.0 mL, 13.0 mmol). The reaction mixture was stirred at room temperature for 24
h. The solvent was evaporated and dried under vacuum. The residue was triturated with ether to
1
give a solid product that was collected by filtration. White solid, yield: 47%. mp = 179-181 ºC. H
NMR (300 MHz, DMSO-d6): δ 10.39 (br, 2H), 9.27 (s, 1H), 8.12 (d, 1H, J = 2.4 Hz), 7.04 (d, 1H, J =
9.0 Hz), 6.93 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 4.77 (s, 2H), 2.39-2.47 (m, 1H), 1.39-1.64 (m, 4H), 0.87 (t,
+
6H, J = 7.2 Hz). HRMS calcd for C14H21N2O4 [M+H] + 281.1501; found: 281.1498.
5.1.3. The synthetic procedure for 2-(2-(2-ethylbutanamido)-4-guanidinophenoxy)acetic acid
trifluoroacetic acid salt (7b)
Compound 5 (0.31g, 1.0 mmol), Et3N (0.29 ml, 2 eq) and SC(NHBoc)2 (0.33 g, 1.2 mmol,
1.2 eq) were dissolved in 15 ml DMF. To the mixture, HgCl2 (0.33 g, 1.2 mmol, 1.2 eq) was
slowly added and the reaction mixture was allowed to be stirred at room temperature over night.
The suspended reaction solution was diluted with EtOAc and filtered through a pad of Celite. The
filtrate was concentrated to give an oil product which was hydrolyzed with NaOH by the method
above to give N, N’-diBoc-7b. The following deprotection with TFA afforded compound 7b.
1
White solid, yield: 38%. mp = 190-192 ºC. H NMR (600 MHz, DMSO-d6): δ 9.70 (s, 1H), 9.23 (s,
1H), 8.05 (d, 1H, J = 3.0 Hz), 7.41 (br, 4H), 7.04 (d, 1H, J = 8.4 Hz), 6.91 (dd, 1H, J = 8.4 Hz, 3.0 Hz),
4.72 (s, 2H), 2.41-2.43 (m, 1H), 1.53-1.58 (m, 2H), 1.42-1.48 (m, 2H), 0.87 (t, 6H, J = 7.2 Hz). HRMS
+
calcd for C15H23N4O4 [M+H] + 323.1719; found: 323.1714.
5.1.4. The general synthetic procedure for compounds 9a, 9b, 10a and 10b
Compound 5 (0.92 g, 3.0 mmol) was acylated with acetyl chloride or isobutyryl chloride by
the reported method. The dried product was dissolved in 5 mL concentrated H2SO4 under ice bath.
Then, 0.85 mL 65% HNO3 was slowly added, and the mixture continued to be stirred for less than

10 minutes. The solution was poured into 50 mL water and the suspension was filtrated to give
compound 8a and 8b. ethyl 2-(4-acetamido-2-(2-ethylbutanamido)-5-nitrophenoxy)acetate (8a):
1
yellow solid, yield: 51%. mp = 132-134 ºC. H NMR (600 MHz, DMSO-d6): δ 10.17 (s, 1H), 9.43 (s,
1H), 8.45 (s, 1H), 7.61 (s, 1H), 5.00 (s, 2H), 4.19 (q, 2H, J = 7.2 Hz), 2.52-2.57 (m, 1H), 2.03 (s, 3H),
1.53-1.61 (m, 2H), 1.43-1.51 (m, 2H), 1.22 (t, 3H, J = 7.2 Hz), 0.86 (t, 6H, J = 7.2 Hz). ESI-MS m/z:
396.3 [M+H]+.
Compound 8a or 8b (2.0 mmol) was dissolved in 20 ml dried CH3CH2OH and hydrogenated
at room temperature with hydrogen balloon in the presence of 0.15 g of 10% Pd/C for 5h. The
mixture was then filtered followed by rotary evaporation of the solvent to give the anilines.
According to the synthetic procedure of compounds 7a and 7b, compounds 9a, 9b, 10a and 10b
were obtained.
2-(4-acetamido-5-amino-2-(2-ethylbutanamido)phenoxy)acetic acid trifluoroacetic acid salt
(9a)
White solid, yield: 35%. mp = 138-140 ºC. 1H NMR (600 MHz, DMSO-d6): δ 9.59 (s, 1H), 9.07 (s,
1H), 7.71 (s, 1H), 6.62 (s, 1H), 4.91 (s, 2H), 2.31-2.35 (m, 1H), 2.03 (s, 3H), 1.49-1.56 (m, 2H),
1.40-1.46 (m, 2H), 0.86 (t, 6H, J = 7.2 Hz). HRMS calcd for C16H24N3O5+ [M+H] + 338.1716; found:
338.1714.
2-(5-amino-2-(2-ethylbutanamido)-4-isobutyramidophenoxy)acetic acid trifluoroacetic acid
salt (9b)
White solid, yield: 37%. mp = 151-152 ºC. 1H NMR (600 MHz, DMSO-d6): δ 9.61 (s, 1H), 9.10 (s,
1H), 7.79 (s, 1H), 6.71 (s, 1H), 4.68 (s, 2H), 2.60-2.65 (m, 1H), 2.33-2.37 (m, 1H), 1.50-1.57 (m, 2H),
+
1.40-1.47 (m, 2H), 1.12 (d, 6H, J = 6.6 Hz), 0.86 (t, 6H, J = 7.2 Hz). HRMS calcd for C18H28N3O5
[M+H] + 366.2029; found: 366.2024.
2-(4-acetamido-2-(2-ethylbutanamido)-5-guanidinophenoxy)acetic acid trifluoroacetic acid
salt (10a)
White solid, yield: 30%. mp = 96-98 ºC. 1H NMR (600 MHz, CD3OD-d6): δ 8.22 (s, 1H), 7.05 (s,
1H), 4.80 (s, 2H), 2.36-2.47 (m, 1H), 2.17 (s, 2H), 1.50-1.77 (m, 2H), 0.96 (t, 6H, J = 7.5 Hz). HRMS
+
calcd for C17H26N5O5 [M+H] + 380.1934; found: 380.1929.
2-(2-(2-ethylbutanamido)-5-guanidino-4-isobutyramidophenoxy)acetic acid trifluoroacetic
acid salt (10b)

White solid, yield: 37%. mp = 191-193 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.25 (br, 1H), 9.73
(s, 1H), 9.28 (s, 1H), 8.49 (s, 1H), 8.16 (s, 1H), 7.35 (br, 4H), 6.99 (s, 1H), 4.77 (s, 2H), 2.61-2.67 (m,
1H), 2.37-2.41 (m, 1H), 1.52-1.59 (m, 2H), 1.43-1.49 (m, 2H), 1.11 (d, 6H, J = 7.2 Hz), 0.86 (t, 6H, J =
+
7.2 Hz). HRMS calcd for C19H30N5O5 [M+H] + 408.2247; found: 408.2244.
5.1.5. The general synthetic procedure for compounds 11a-11c
To a solution of compound 5 (0.62 g, 2.0 mmol) and Et3N (0.84 mL, 6 mmol) in 20 mL
dioxane was added triphosgene (0.20 g, 0.67 mmol) in one portion. The whole mixture was stirred
at 40 oC for 15 min, then added into a solution of amine (R5NH2, 2.0 mmol) and Et3N (0.57 mL, 4
mmol) in 15 mL THF. The reaction mixture was stirred for 4h at room temperature, then
concentrated and extracted with EtOAc. The combined organic extracts were washed with
saturated NaHCO3, 1 M aqueous HCl, dried and concentrated. After hydrolysis and
chromatographic purification according to the procedure above, compounds 11a-11c were
obtained.
2-(4-(3-benzylureido)-2-(2-ethylbutanamido)phenoxy)acetic acid (11a)
White solid, yield: 62%. mp = 195-197 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.10 (br, 1H),
9.12 (s, 1H), 8.50 (s, 1H), 7.97 (s, 1H), 7.28-7.34 (m, 4H), 7.20-7.25 (m, 2H), 6.89 (d, 1H, J = 9.0
Hz), 6.44 (t, 1H, J = 6.0 Hz), 4.65 (s, 2H), 4.28 (d, 2H, J = 6.0 Hz), 2.29-2.33 (m, 1H), 1.52-1.59
(m, 2H), 1.42-1.49 (m, 2H), 0.87(t, 6H, J = 7.2 Hz). HRMS calcd for C22H28N3O5+ [M+H] +
414.2029; found: 414.2025.
2.2-(2-(2-ethylbutanamido)-4-(3-(prop-2-yn-1-yl)ureido)phenoxy)acetic acid (11b)
White solid, yield: 67%. mp = 186-187 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.12 (br, 1H),
9.12 (s, 1H), 8.52 (s, 1H), 7.96 (s, 1H), 7.20 (d, 1H, J = 9.0 Hz), 6.90 (d, 1H, J = 9.0 Hz), 6.27 (t,
1H, J = 6.0 Hz), 4.66 (s, 2H), 3.86 (d, 2H, J = 6.0 Hz), 3.09 (s, 1H), 2.29-2.34 (m, 1H), 1.52-1.59
(m, 2H), 1.42-1.49 (m, 2H), 0.87(t, 6H, J = 7.2 Hz). HRMS calcd for C18H24N3O5+ [M+H] +
362.1716; found: 362.1711.
(R)-2-(2-(2-ethylbutanamido)-4-(3-(1-hydroxybutan-2-yl)ureido)phenoxy)acetic acid (11c)
White solid, yield: 52%. mp = 117-119 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.12 (br, 1H),
9.12 (s, 1H), 8.38 (s, 1H), 7.94 (d, 1H, J = 2.4 Hz), 7.17 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 6.88 (d, 1H,
J = 9.0 Hz), 5.82 (d, 1H, J = 7.8 Hz), 4.65 (s, 2H), 3.46-3.52 (m, 1H), 3.42 (dd, 1H, J = 10.8 Hz,
3.6 Hz), 3.32 (dd, 1H, J = 10.8 Hz, 5.4 Hz), 3.86 (d, 2H, J = 6.0 Hz), 2.28-2.34 (m, 1H), 1.50-1.62

+
(m, 3H), 1.40-1.50 (m, 2H), 1.32-1.38 (m, 1H), 0.80-0.89 (m, 9H). HRMS calcd for C19H30N3O6
[M+H] + 396.2135; found: 396.2132.
5.1.6. The general synthetic procedure for compounds 12a and 12b
Compounds 8a and 8b (2.0 mmol) were reduced with hydrogen in the presence of Pd/C. The
acylated caffeic acid, (E)-3-(3,4-diacetoxyphenyl)acrylic acid was converted to the acyl chloride 18,
and then reacted with the reduction products of compounds 8a and 8b. The subsequent hydrolysis
and chromatographic isolation afforded compounds 12a and 12b.
(E)-2-(4-acetamido-5-(3-(3,4-dihydroxyphenyl)acrylamido)-2-(2-ethylbutanamido)phenoxy)a
cetic acid (12a)
Off-White solid, yield: 47%. mp = 264-266 ºC. 1H NMR (300 MHz, DMSO-d6): δ 13.16 (br,
1H), 9.47 (s, 1H), 9.39 (s, 1H), 9.28 (s, 1H), 9.19 (s, 1H), 9.15 (s, 1H), 7.99 (s, 1H), 7.59 (s, 1H),
7.39 (d, 1H, J = 15.6 Hz), 7.03 (d, 1H, J = 1.8 Hz), 6.91 (dd, 1H, J = 8.4 Hz, 1.8 Hz), 6.77 (d, 1H,
J = 8.4 Hz), 6.64 (d, 1H, J = 15.6 Hz), 4.68 (s, 2H), 2.31-2.41 (m, 1H), 2.05 (s, 3H), 1.39-1.61 (m,
+
4H), 0.87 (d, 6H, J = 7.2 Hz). HRMS calcd for C25H30N3O8 [M+H] + 500.2033; found: 500.2029.
(E)-2-(5-(3-(3,4-dihydroxyphenyl)acrylamido)-2-(2-ethylbutanamido)-4-isobutyramidopheno
xy)acetic acid (12b)
Off-White solid, yield: 35%. mp = 246-248 ºC. 1H NMR (300 MHz, DMSO-d6): δ 13.16 (br,
1H), 9.47 (s, 1H), 9.40 (s, 1H), 9.28 (s, 1H), 9.20 (s, 1H), 9.15 (s, 1H), 8.06 (s, 1H), 7.37-7.43 (m,
3H), 7.02 (d, 1H, J = 1.8 Hz), 6.91 (dd, 1H, J = 8.1 Hz, 1.8 Hz), 6.77 (d, 1H, J = 8.1 Hz), 6.55 (d,
1H, J = 15.6 Hz), 4.70 (s, 2H), 2.55-2.65 (m, 1H), 2.32-2.42 (m, 1H), 1.35-1.63 (m, 4H), 1.11 (d,
+
6H, J = 6.9 Hz), 0.87 (d, 6H, J = 7.2 Hz). HRMS calcd for C27H34N3O8 [M+H]+ 528.2346; found:
528.2342.
5.1.7. The general synthetic procedure for compounds 14a-14c
To a solution of compound 5 (0.62 g, 2.0 mmol), aldehyde (4.0 mmol, 2eq) and 0.5 ml acetic
acid in 30 mL ethanol was added NaBH3CN (0.50 g, 4eq) slowly. The reaction was stirred at 56 oC
for 6h, and the concentrated. Chromatographic purification gave compounds 13a-13c. Reaction of
compounds 13 and (E)-3-(3,4-diacetoxyphenyl)acrylic acid, followed by hydrolysis afforded
compounds 14a-14c based on the methods above.
(E)-2-(4-(3-(3,4-dihydroxyphenyl)-N-(2-ethylbutyl)acrylamido)-2-(2-ethylbutanamido)pheno
xy)acetic acid (14a)

1
Light yellow solid, yield: 27%. mp = 108-110 ºC. H NMR (300 MHz, DMSO-d6): δ 13.10
(br, 1H), 10.02 (br, 1H), 9.39 (br, 1H), 7.99 (d, 1H, J = 2.1 Hz), 7.30 (d, 1H, J = 15.6 Hz), 7.07 (d,
1H, J = 8.4 Hz), 6.92 (dd, 1H, J = 8.4 Hz, 1.8 Hz), 6.65-6.71 (m, 3H), 6.03 (d, 1H, J = 15.6 Hz),
4.59 (s, 2H), 3.66 (d, 2H, J = 6.9 Hz), 2.34-2.42 (m, 1H), 1.31-1.60 (m, 5H), 1.19-1.30 (m, 4H),
+
0.84 (t, 6H, J = 7.2 Hz), 0.79 (t, 6H, J = 7.2 Hz). HRMS calcd for C29H39N2O7 [M+H]+ 527.2757;
found: 527.2755.
(E)-2-(4-(3-(3,4-dihydroxyphenyl)-N-(thiophen-2-ylmethyl)acrylamido)-2-(2-ethylbutanamid
o)phenoxy)acetic acid (14b)
Light yellow solid, yield: 35%. mp = 92-94 ºC. 1H NMR (300 MHz, DMSO-d6): δ 13.13 (br,
1H), 9.37 (s, 1H), 9.18 (br, 1H), 9.07 (br, 1H), 7.89 (d, 1H, J = 2.1 Hz), 7.42 (d, 1H, J = 15.6 Hz),
7.40 (d, 1H, J = 3.3 Hz), 7.00 (d, 1H, J = 8.7 Hz), 6.86-7.02 (m, 2H), 6.66-6.82 (m, 4H), 6.09 (d,
1H, J = 15.6 Hz), 5.03 (s, 2H), 4.62 (s, 2H), 2.33-2.38 (m, 1H), 1.38-1.59 (m, 4H), 0.84 (t, 6H, J =
7.2 Hz). HRMS calcd for C28H31N2O7S+ [M+H] + 539.1852; found: 539.1846.
(E)-2-(4-(N-benzyl-3-(3,4-dihydroxyphenyl)acrylamido)-2-(2-ethylbutanamido)phenoxy)acet
ic acid (14c)
Light yellow solid, yield: 39%. mp = 90-92 ºC. 1H NMR (300 MHz, DMSO-d6): δ 13.15 (br,
1H), 9.36 (s, 1H), 9.15 (s, 1H), 9.07 (br, 1H), 7.89 (d, 1H, J = 1.8 Hz), 7.42 (d, 1H, J = 15.6 Hz), 7.
20-7.31 (m, 5H), 6.97 (d, 1H, J = 8.7 Hz), 6.82 (dd, 1H, J = 8.7 Hz, 1.8 Hz), 6.66-6.73 (m, 3H),
6.16 (d, 1H, J = 15.6 Hz), 4.93 (s, 2H), 4.76 (s, 2H), 2.31-2.42 (m, 1H), 1.35-1.58 (m, 4H), 0.83 (t,
+
6H, J = 7.2 Hz). HRMS calcd for C30H33N2O7 [M+H] + 533.2282; found: 533.2278.
5.1.8. The general synthetic procedure for compounds 16a-16e
Boc-L-amino acid (3.0 mmol) and Et3N (0.86 ml, 6.0 mmol) were dissolved in 20 mLCH2Cl2,
and TBTU (1.06 g, 3.3 mmol, 1.1eq) was added in one portion at room temperature. After stirring
for 15 min, compound 5 (3.0 mmol) dissolved in 20 mL CH2Cl2 was added to the reaction mixture
with additional Et3N (0.43 ml, 3.0 mmol). The reaction was allowed to stir for 4h, then
concentrated and extracted with EtOAc. The combined extracts were washed with saturated
NaHCO3, 1 M aqueous HCl, dried and concentrated to give intermediates 15a-15e.
Compounds 15 were dissolved in 20 mL 3M EtOAc/HCl and stirred overnight. The solvent
was removed under reduced pressure to give the amine products. According to the synthetic
method for compounds 12, compounds 16a-16e were prepared.

(E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)acetamido)-2-(2-ethylbutanamido)phenoxy)
acetic acid (16a)
White solid, yield: 24%. mp = 123-125 ºC. 1H NMR (600 MHz, DMSO-d6): δ 13.16 (br, 1H),
9.95 (s, 1H), 9.37 (br, 1H), 9.14-9.15 (br, 2H), 8.30 (t, 1H, J = 6.0 Hz), 8.14 (d, 1H, J = 2.4 Hz),
7.40 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.26 (d, 1H, J = 15.6 Hz), 6.97 (d, 1H, J = 1.8 Hz), 6.94 (d, 1H,
J = 9.0 Hz), 6.86 (dd, 1H, J = 8.4 Hz, 1.8 Hz), 6.75 (d, 1H, J = 8.4 Hz), 6.46 (d, 1H, J = 15.6 Hz),
4.69 (s, 2H), 3.96 (d, 2H, J = 6.0 Hz) 2.31-2.36 (m, 1H), 1.43-1.59 (m, 4H), 1.11 (d, 6H, J = 6.9
+
Hz), 0.87 (d, 6H, J = 7.2 Hz). HRMS calcd for C25H30N3O8 [M+H] + 500.2033; found: 500.2025.
(S,E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)propanamido)-2-(2-ethylbutanamido)phe
noxy)acetic acid (16b)
White solid, yield: 28%. mp = 134-136 ºC. [α]2D5 = +21.4 (c, 1.35, CH3OH). H NMR (300
1
MHz, DMSO-d6): δ 13.16 (br, 1H), 9.99 (s, 1H), 9.37 (br, 1H), 9.14-9.15 (br, 2H), 8.26 (d, 1H, J =
7.2 Hz), 8.18 (d, 1H, J = 2.4 Hz), 7.42 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.25 (d, 1H, J = 15.6 Hz),
6.93-6.96 (m, 2H), 6.84 (dd, 1H, J = 8.4 Hz, 1.8 Hz), 6.75 (d, 1H, J = 8.4 Hz), 6.47 (d, 1H, J =
15.6 Hz), 4.69 (s, 2H), 4.49-4.54 (m, 1H), 2.26-2.39 (m, 1H), 1.38-1.64 (m, 4H), 1.32 (d, 3H, J =
+
+
6.9 Hz), 0.87 (d, 6H, J = 7.2 Hz). HRMS calcd for C26H32N3O8 [M+H] 514.2189; found:
514.2184.
(S,E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)-3-methylbutanamido)-2-(2-ethylbutanam
ido)phenoxy)acetic acid (16c)
Light yellow solid, yield: 39%. mp = 138-140 ºC. [α]2D5 = +60.4 (c, 0.5, CH3OH). H NMR
1
(300 MHz, DMSO-d6): δ 13.06 (br, 1H), 10.061 (s, 1H), 9.34 (s, 1H), 9.12 (s, 1H), 9.11 (s, 1H),
8.19 (d, 1H, J = 2.4 Hz), 8.11 (d, 1H, J = 8.7 Hz), 7.43 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.25 (d, 1H, J
= 15.6 Hz), 6.92-6.96 (m, 2H), 6.84 (dd, 1H, J = 8.1 Hz, 1.8 Hz), 6.74 (d, 1H, J = 8.1 Hz), 6.59 (d,
1H, J = 15.6 Hz), 4.69 (s, 2H), 4.36-4.42 (m, 1H), 2.26-2.39 (m, 1H), 1.97-2.10 (m, 1H),
1.38-1.65 (m, 4H), 0.92 (d, 3H, J = 6.6 Hz), 0.87 (d, 6H, J = 7.2 Hz). HRMS calcd for
+
C28H36N3O8 [M+H] + 542.2502; found: 542.2490.
(S,E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)-4-methylpentanamido)-2-(2-ethylbutana
mido)phenoxy)acetic acid (16d)
Light yellow, yield: 33%. mp = 139-142 ºC. [α]2D5 = +36.2 (c, 0.77, CH3OH). H NMR (300
1
MHz, DMSO-d6): δ 13.09 (br, 1H), 10.06 (s, 1H), 9.35 (br, 1H), 9.12 (br, 2H), 8.19 (d, 1H, J = 8.1

Hz), 8.17 (d, 1H, J = 2.4 Hz), 7.45 (dd, 1H, J = 8.7 Hz, 2.4 Hz), 7.25 (d, 1H, J = 15.6 Hz),
6.92-6.96 (m, 2H), 6.84 (dd, 1H, J = 8.4 Hz, 1.8 Hz), 6.75 (d, 1H, J = 8.4 Hz), 6.48 (d, 1H, J =
15.6 Hz), 4.79 (s, 2H), 4.53-4.61 (m, 1H), 2.29-2.39 (m, 1H), 1.38-1.67 (m, 7H), 0.85-0.95 (m,
+
12H). HRMS calcd for C29H38N3O8 [M+H] + 556.2659; found: 556.2652.
(S,E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)-3-phenylpropanamido)-2-(2-ethylbutana
mido)phenoxy)acetic acid (16e)
Light yellow solid, yield: 28%. mp = 154-156 ºC. [α]2D5 = +24.8 (c, 1.15, CH3OH). 1H NMR
(300 MHz, DMSO-d6): δ 10.12 (s, 1H), 9.54 (br, 1H), 9.35 (br, 1H), 9.15 (br, 1H), 8.35 (d, 1H, J =
8.1 Hz), 8.16 (d, 1H, J = 2.4 Hz), 7.43 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 7.15-7.33 (m, 6H), 6.93-6.96
(m, 2H), 6.82 (dd, 1H, J = 8.4 Hz, 1.8 Hz), 6.73 (d, 1H, J = 8.4 Hz), 6.42 (d, 1H, J = 15.6 Hz),
4.72-4.81 (m, 1H), 4.61 (s, 2H), 3.07 (dd, 1H, J = 13.5 Hz, 4.8 Hz), 2.88 (dd, 1H, J = 13.5 Hz, 9.6
Hz), 2.29-2.39 (m, 1H), 1.41-1.63 (m, 4H), 0.88 (d, 6H, J = 7.2 Hz). HRMS calcd for
+
C32H36N3O8 [M+H] + 590.2502; found: 590.2501.
5.1.9. The general synthetic procedure for compounds 17a and 17b
Compounds 12a or 12b (0.2 mmol) was dissolved in 5 ml dried CH3CH2OH and
hydrogenated at room temperature with hydrogen balloon in the presence of 50 mg of 10% Pd/C
over night. The reaction mixture was filtered through a pad of Celite. The filtrate was concentrated,
and then dried to give compounds 17a and 17b.
2-(4-acetamido-5-(3-(3,4-dihydroxyphenyl)propanamido)-2-(2-ethylbutanamido)phenoxy)ac
etic acid (17a)
Off-White solid, yield: 89%. mp = 198-200 ºC. 1H NMR (300 MHz, DMSO-d6): δ 13.14 (br,
1H), 9.24 (s, 1H), 9.18 (s, 1H), 9.16 (s, 1H), 8.62 (br, 1H), 7.99 (s, 1H) 7.29 (s, 1H), 6.61-6.64 (m,
2H), 6.47 (dd, 1H, J = 8.1 Hz, 1.8 Hz), 4.64 (s, 2H), 2.69-2.74 (m, 2H), 2.50-2.56 (m, 2H),
2.29-2.37 (m, 1H), 2.00 (s, 3H), 1.35-1.63 (m, 4H), 0.86 (d, 6H, J = 7.2 Hz). HRMS calcd for
+
C25H32N3O8 [M+H] + 502.2189; found: 502.2181.
2-(5-(3-(3,4-dihydroxyphenyl)propanamido)-2-(2-ethylbutanamido)-4-isobutyramidophenox
y)acetic acid (17b)
Off-White solid, yield: 91%. mp = 218-220 ºC. 1H NMR (300 MHz, DMSO-d6): δ 13.16 (br,
1H), 9.16-9.20 (m, 3H), 8.62 (br, 1H), 8.05 (s, 1H) 7.17 (s, 1H), 6.60-6.64 (m, 2H), 6.46 (dd, 1H,
J = 8.1 Hz, 1.8 Hz), 4.65 (s, 2H), 2.71 (t, 2H, J = 7.5 Hz ), 2.51-2.58 (m, 3H, merged in DMSO),

2.29-2.39 (m, 1H), 1.37-1.63 (m, 4H), 1.09 (d, 6H, J = 6.9 Hz), 0.86 (d, 6H, J = 7.2 Hz). HRMS
+
calcd for C27H36N3O8 [M+H] + 530.2502; found: 530.2500.
5.2. Influenza A (H9N2 and H5N1) Neuraminidase Inhibition Assay
The NA inhibition assay was performed according to a standard method13. Influenza virus
suspensions (H9N2 virus: A/Chicken/Shandong/LY/08 and H5N1 virus: A/duck/China/QJ/01)
obtained from the allantoic fluid of embryonated chicken eggs were inactivated and used as the
enzyme on the basis that NA was present on the viral surface. The substrate,
2’-(4-methylumbelliferyl)-α-D-acetylneuraminic acid sodium salt hydrate (4-MU-NANA) (Sigma,
M8639) was cleaved by NA to yield a fluorescent product which can be quantified. Compounds
tested were dissolved in DMSO and diluted to the corresponding concentrations in MES buffer
(32.5 mM 2-(N-morpholino)-ethanesulfonic acid, 4 mM CaCl2, pH 6.5) if not dissolved in MES
directly. In a 96-well plate, 10 µL of the diluted virus supernatant, 70 µL of MES buffer and 10 µL
of compounds at different concentration were added successively, and then incubated for 5 min at
37 °C. The reaction was started by the addition of the substrate. After incubation for 30 to 60 min,
the reaction was terminated by adding 150 µL 0.2 M glycine-NaOH (pH 10.2) or 0.034 M NaOH
in water. Fluorescence was recorded (excitation at 360 nm and emission at 450 nm), and substrate
blanks were subtracted from the sample readings. The 50% inhibitory concentration (IC50) was
calculated by plotting percent inhibition of neuraminidase activity versus the inhibitor
concentration.
5.3. In vitro anti-influenza virus assay and cytotoxicity assay
In vitro anti-influenza virus assay and cytotoxicity assay were determined by MTS assay with
slight modification23. Chicken embryo fibroblast cells were grown in 96-well plates for 24h at 37
oC. The compounds dissolved in DMSO were serially diluted by two-fold in DMEM (containing 1%
serum). 50 µL of compound at different concentrations was mixed with an equal volume of 1
TCID50 (50% tissue culture infective dose) of influenza virus (H9N2 virus:
A/Chicken/Shandong/LY/08). After 1 hour incubation at 37 °C, the compound-virus preparation
was added to the cells. At 2 days post infection, 20 µL MTS and PMS mixture solution was added
per well and the plate was incubated for 3h away from light. The optical density was measured at
OD490nm to determine the antiviral activity, expressed as the compound concentration producing
50% inhibition of virus-induced CPE (EC50), as well as the cytotoxicity of the compounds,

expressed as the compound concentration causing 50% cellular toxicity (CC50).
Acknowledgment
This work was supported by the 12th Five Years National Science and Technology Major
Project (2011ZX09401-015) and Doctoral Fund of Ministry of Education of China (No
20110131110037). We greatly thank Bing Huang and Kexiang Yu for their work in the biological
activity assays.
References
1. Laver, G.; Garman, E. Microbes Infect. 2002, 4, 1309.
2. Smith, G. J.; Vijaykrishna, D.; Bahl, J.; Lycett, S. J.; Worobey, M.; Pybus, O. G.; Ma, S. K.;
Cheung, C. L.; Raghwani, J.; Bhatt, S.; Peiris, J. S.; Guan, Y.; Rambaut, A. Nature 2009, 459,
1122.
3. Wu, S.; Wu, F.; He, J. Lancet 2013, 381, 1539.
4. Xie, Y.; Gong, J.; Li, M.; Fang, H.; Xu, W. Curr. Med. Chem. 2011, 18, 1050.
5. Wu, W. L.; Lau, S. Y.; Chen, Y.; Wang, G.; Mok, B. W.; Wen, X.; Wang, P.; Song, W.; Lin, T.;
Chan, K. H.; Yuen, K. Y.; Chen, H. Antiviral Res. 2012, 93, 144.
6. Takano, R.; Kiso, M.; Igarashi, M.; Le, Q. M.; Sekijima, M.; Ito, K.; Takada, A.; Kawaoka, Y. J.
Infect. Dis. 2013, 207, 89.
7. Wen, W. H.; Wang, S. Y.; Tsai, K. C.; Cheng, Y. S.; Yang, A. S.; Fang, J. M.; Wong, C. H.
Bioorg. Med. Chem. 2010, 18, 4074.
8. Ye, D.; Shin, W. J.; Li, N.; Tang, W.; Feng, E.; Li, J.; He, P. L.; Zuo, J. P.; Kim, H.; Nam, K. Y.;
Zhu, W.; Seong, B. L.; No, K. T.; Jiang, H.; Liu, H. Eur. J. Med. Chem. 2012, 54, 764.
9. Feng, E.; Shin, W. J.; Zhu, X.; Li, J.; Ye, D.; Wang, J.; Zheng, M.; Zuo, J. P.; No, K. T.; Liu, X.;
Zhu, W.; Tang, W.; Seong, B. L.; Jiang, H.; Liu, H. J. Med. Chem. 2013, 56, 671.
10. Lin, C. H.; Chang, T. C.; Das, A.; Fang, M. Y.; Hung, H. C.; Hsu, K. C.; Yang, J. M.; von Itzstein,
M.; Mong, K. K.; Hsu, T. A.; Lin, C. C. Org. Biomol. Chem. 2013, 11, 3943.
11. Rungrotmongkol, T.; Frecer, V.; De-Eknamkul, W.; Hannongbua, S.; Miertus, S. Antiviral Res.
2009, 82, 51.
12. Mohan, S.; McAtamney, S.; Haselhorst, T.; von Itzstein, M.; Pinto, B. M. J. Med. Chem. 2010, 53,
7377.
13. Liu, A. L.; Wang, H. D.; Lee, S. M.; Wang, Y. T.; Du, G. H. Bioorg. Med. Chem. 2008, 16, 7141.

14. Ryu, Y. B.; Curtis-Long, M. J.; Lee, J. W.; Kim, J. H.; Kim, J. Y.; Kang, K. Y.; Lee, W. S.; Park,
K. H. Bioorg. Med. Chem. 2009, 17, 2744.
15. Dao, T. T.; Dang, T. T.; Nguyen, P. H.; Kim, E.; Thuong, P. T.; Oh, W. K. Bioorg. Med. Chem.
Lett. 2012, 22, 3688.
16. Grienke, U.; Schmidtke, M.; Kirchmair, J.; Pfarr, K.; Wutzler, P.; Durrwald, R.; Wolber, G.; Liedl,
K. R.; Stuppner, H.; Rollinger, J. M. J. Med. Chem. 2010, 53, 778.
17. Xie, Y.; Huang, B.; Yu, K.; Shi, F.; Xu, W. Medicinal Chemistry Research 2013, 22, 3485.
18. Xie, Y.; Huang, B.; Yu, K.; Shi, F.; Liu, T.; Xu, W. Bioorg. Med. Chem. Lett. 2013, 23, 3556.
19. von Itzstein, M. Nat. Rev. Drug Discov. 2007, 6, 967.
20. Russell, R. J.; Haire, L. F.; Stevens, D. J.; Collins, P. J.; Lin, Y. P.; Blackburn, G. M.; Hay, A. J.;
Gamblin, S. J.; Skehel, J. J. Nature 2006, 443, 45.
21. Liu, Y.; Zhang, J.; Xu, W. Curr. Med. Chem. 2007, 14, 2872.
22. Cheng, L. S.; Amaro, R. E.; Xu, D.; Li, W. W.; Arzberger, P. W.; McCammon, J. A. J. Med.
Chem. 2008, 51, 3878.
23. Yu, M.; Liu, A.; Du, G.; Naesens, L.; Vanderlinden, E.; De Clercq, E.; Liu, X. Chem. Biol. Drug
Des. 2011, 78, 596.

Graphical abstract
Compounds containing the caffeic acid fragment exhibited better NA inhibitory activities and selectively
inhibited N1 or N2.
1