Caffeic acid derivatives: A new type of influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.04.033

Recently, many natural products, especially some plant-derived polyphenols have been found to exert antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to build a small compound library with various structures. The enzyme inhibition result indicated that some compounds exhibited moderate activities against NA and compound 15d was the best with IC₅₀ = 7.2 μM and 8.5 μM against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was important for the activity according to the docking analysis. Besides, compound 15d was found to be a non-competitive inhibitor with K_i = 11.5 ± 0.25 μM by the kinetic study and also presented anti-influenza virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhibitors from caffeic acid derivatives to cope with influenza virus.

Full text of DOI:10.1016/j.bmcl.2013.04.033

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3556–3560
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Caffeic acid derivatives: A new type of influenza neuraminidase
inhibitors
Yuanchao Xie a, Bing Huang b, Kexiang Yu b, Fangyuan Shi a, Tianqi Liu a, Wenfang Xu a,
⇑
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, Shandong 250012, PR China
b Institute of Poultry Science, Shandong Academy of Agricultural Sciences, 1 Jiaoxiao Road, Jinan, Shandong 250023, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 January 2013
Revised 9 April 2013
Accepted 13 April 2013
Available online 22 April 2013
Recently, many natural products, especially some plant-derived polyphenols have been found to exert
antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our
research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to
build a small compound library with various structures. The enzyme inhibition result indicated that some
compounds exhibited moderate activities against NA and compound 15d was the best with IC50 = 7.2
and 8.5 M against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was
important for the activity according to the docking analysis. Besides, compound 15d was found to be a
non-competitive inhibitor with Ki = 11.5 0.25 M by the kinetic study and also presented anti-influenza
lM
l
Keywords:
Influenza
Neuraminidase
Amino acid
Caffeic acid
l
virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhib-
itors from caffeic acid derivatives to cope with influenza virus.
Ó 2013 Elsevier Ltd. All rights reserved.
Influenza virus, especially the highly pathogenic avian influenza
A (H5N1) virus is a great threat to human. In recent years, the
worldwide spread of H5N1 avian influenza virus in birds and the
increasing cases of bird-to-human transmission get heightened
concern. The viral surface protein, neuraminidase (NA) plays an
important role in the life cycle of influenza virus and has been dis-
covered as an important target for anti-influenza drugs design.
Oseltamivir, one kind of NA inhibitors is currently the first line de-
fense drug against influenza. However, more and more influenza
virus strains are resistant to oseltamivir, such as the seasonal
H1N1 viruses1 and avian H5N1 strains.2 In 2009, peramivir was ur-
gently authorized in America to cope with the novel swine-origin
H1N1 (A) influenza virus that was also resistant to oseltamivir.
At present, this drug has been approved in many countries, but it
is mainly administered intravenously, which is very inconvenient
for patients.
the NA activity by binding on some other sites of the enzyme,
rather than the catalytic center.
Caffeic acid, which is abundant in nature has a variety of poten-
tial pharmacological effects in vitro and in vivo, such as anti-
inflammatory, anti-cancer and antiviral activities.5 Some natural
products containing the fragment of caffeic acid, like chlorogenic
acid and its analogue also show inhibitory activities against influ-
enza NAs.6 A simple compound, caffeic acid phenethyl ester (CAPE,
Fig. 2) is found to have anti-mitogenic, anti-carcinogenic, anti-
inflammatory and especially anti-influenza virus properties
in vitro.7 In 2012, Liu et al. reported some novel dual-targeted
bifunctional zanamivir anti-influenza drugs formed by conjugation
of zanamivir with caffeic acid for simultaneous inhibition of influ-
enza virus NA and suppression of pro-inflammatory cytokine.
These conjugates provided remarkable protection of cells and mice
against influenza infections.8
According to recent studies, it seems hard to discover novel NA
inhibitors which would be better than oseltamivir simply based on
the NA active site. However, many natural products, especially
some small plant-derived polyphenols have been found to exert
antiviral effects against influenza virus and show inhibitory activ-
ities on NAs (Fig. 1).3,4 Unlike the classical ones, the structures of
these compounds are various and interestingly, most of them act
as noncompetitive NA inhibitors, indicating that they likely inhibit
Till now, except some natural products, there are no synthetic
compounds designed to target influenza NA based on caffeic acid.
Therefore, in our study, we took caffeic acid as the basic fragment
to design and synthesize a small compound library of caffeic acid
derivatives. Through determining the inhibitory effects on the N1
and N2 NAs of influenza virus, we discovered a new kind of NA
inhibitors with better activities than the natural products.
Compounds were synthesized using methods presented in
Schemes 1–3. Scheme 1 presented the synthesis of compounds
3a–3f. In order to increase the yields of target compounds, caffeic
acid was first acetylated to give (E)-3-(3,4-diacetoxyphenyl)acrylic
acid (compound 1) that had a relatively better solubility in
⇑
Corresponding author. Tel./fax: +86 531 88382264.
E-mail addresses: yuanchaox623@yahoo.cn (Y. Xie), wfxu@yahoo.cn (W. Xu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.033

Y. Xie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3556–3560
3557
Figure 1. Plant-derived polyphenols with inhibitory activities against influenza NAs.
Figure 2. Caffeic acid derivatives with activities against influenza NA or influenza virus.
common organic solvents. This compound was then converted to
acyl chloride and reacted with different amino acid methyl esters
in THF. After hydrolysis with NaOH, compounds 3a–3f were ob-
tained with acceptable yields.
hydrogen atmosphere, the Cbz group was removed with Pd/C,
therefore exposing the other NH2 that was acylated with com-
pound 1. After hydrolysis with NaOH, compounds 7a–7f were
obtained.
L
-Ornithine that contained two amino groups, a carboxyl group
and a relative long carbon chain can be modified with caffeic acid
and some other structures. In Scheme 2, the
-NH2 of N0-Cbz-
ornithine methyl ester hydrochloride (4) was first acylated to
In Scheme 3, compounds 11a–11e bearing cinnamic acid group
were prepared for making a comparison with compounds 12a–12e
on their NA inhibitory activities. The starting material, 2-amino-4-
nitrophenol (compound 8) was acylated with different acyl
chloride followed by reduction with Pd/C in the presence of H2.
a
L-
introduce different fragments on the skeleton of
L
-ornithine. Under
O
O
O
O
O
a
HO
HO
b
AcO
AcO
AcO
N
H
OH
n
OH
N
H
O
n
R1
R1
AcO
1
2a~2f
3a~3f
3c R1 =
H, n = 0
3b R1
n = 0
=
3a R1 =
n = 0
n = 0
3f R1
=
H, n = 2
3e R1
=
H, n = 1
3d R1
=
N
H
Scheme 1. Reagents and conditions: (a) (1) (COCl)2, DMF, anhydrous THF, rt; (2) amino acid methyl esters, NaHCO3, THF, rt; and (b) NaOH, CH3OH, H2O, rt.
O
O
O
O
b
AcO
AcO
a
N
H
O
CbzHN
O
CbzHN
c
O
HN
HN
R2
NH2HCl
R2
6a~6f
4
5a~5f
O
O
HO
HO
N
OH
H
HN
R2
7a~7f
=
O
O
O
7c R2
7f R2
=
7b R2
7e R2
=
7a R2
7d R2
O
O
O
O
=
=
=
Scheme 2. Reagents and conditions: (a) RCOCl, NaHCO3, THF, H2O, rt; (b) (1) H2, Pd/C, CH3COOH, CH3OH, rt; (2) compound 1, (COCl)2, DMF, anhydrous THF; (3) NaHCO3, THF,
H2O, rt; and (c) NaOH, CH3OH, H2O, rt.

3558
Y. Xie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3556–3560
OH
O
O
N
H
N
H
R3
OH
OH
OH
c
H
H
N
R3
11a~11e
O
N
R3
NH2
a
b
d
OH
O
O
O
HO
HO
NH2
10a~10e
R3
NO2
8
NO2
N
H
N
H
9a~9e
12a~12e
e
OH
O
O
O
O
HO
HO
HN
O
O
O
g
HN
O
f
O2N
O
O
O
O
HN
AcO
AcO
O
HN
R3
HN
13a~13d
R3
R3
14a~14d
11c R3
15a~15d
11a R3
=
11b R3
=
H3C
CH3CH2
CH3CH2
=
=
11d R3
=
=
11e R3
12e R3
=
=
12a R3
15a R3
=
=
12c R3
12d R3
12b R3
15b R3
=
=
H3C
H3C
15c R3
=
15d R3
=
Scheme 3. Reagents and conditions: (a) R3COCl, NaHCO3, THF, H2O, rt; (b) H2, Pd/C, CH3OH, rt; (c) (1) cinnamic acid, oxalyl chloride, DMF, anhydrous THF, rt; (2) NaHCO3,
THF, H2O, rt; (d) (1) caffeic acid, oxalyl chloride, DMF, anhydrous THF, rt; (2) NaHCO3, THF, H2O, rt; (e) ethyl chloroacetate, NaI, K2CO3, acetone, reflux; (f) (1) H2, Pd/C, CH3OH,
rt; (2) compound 1, oxalyl chloride, DMF, anhydrous THF, rt; (3) NaHCO3, THF, H2O, rt; and (g) NaOH, CH3OH, H2O, rt.
Reaction of the intermediates 10a–10e with cinnamic acid or caf-
feic acid gave compounds 11a–11e and 12a–12e. There were four
steps involved in the conversion of compounds 9a, 9c–9e to the fi-
nal acids 15a–15d and intermediates did not need to be purified.
The first step was alkylation of the phenolic hydroxyl group with
ethyl chloroacetate to form the corresponding ether. In this step,
a catalytic amount of NaI was very beneficial to the reaction. The
second step was reduction of the nitro group of the esters to form
the anilines. The third step was condensation of the anilines with
compound 1 to provide intermediates 14a–14d. The forth step
was hydrolysis of intermediates 14a–14d by aqueous NaOH to af-
ford the final acids.
Though the residues within the active site of NA are high con-
served, there are still some structural differences between group-
1 (N1, N4, N5, N8) and group-2 (N2, N3, N6, N7, N9) NAs in the re-
gion adjacent to the enzyme’s catalytic center. Therefore, we chose
NAs from two kinds of influenza viruses (H9N2 and H1N1) to
determine the inhibitory activities of our designed compounds.
From the data of Tables 1–3, it could be found that these com-
pounds were superior to the reported caffeic acid derivatives or
Table 1
In vitro inhibitory effects of compounds 3a–3f against influenza A NAs
Compound H9N2 IC50 H1N1 IC50 Compound H9N2 IC50 H1N1 IC50
(
l
M)
(
l
M)
(
l
M)
(lM)
3a
3b
3c
43.3
37.3
39.6
49.5
38.3
37.4
3d
3e
3f
27.2
13.5
16.8
30.1
27.3
27.7
Table 2
In vitro inhibitory effects of compounds 7a–7f against influenza A NAs
Compound H9N2 IC50 H1N1 IC50 Compound H9N2 IC50 H1N1 IC50
(l
M) M) M) M)
(l
(
l
(l
7a
7b
7c
37.9
33.6
30.3
31.3
34.7
31.9
7d
7e
7f
44.2
37.2
28.1
39.2
34.3
30.2
Table 3
In vitro inhibitory effects of compounds 11a–11e, compounds 12a–12e and compounds 15a–15d against influenza A NAs
Compound
H9N2 IC50
(l
M)
H1N1 IC50
(lM)
Compound
H9N2 IC50
(
lM)
H1N1 IC50 (lM)
11a
11b
11c
11d
11e
12a
12b
NT
NT
NT
NT
NT
27.4
23.6
40.2% at 100
42.9% at 100
42.6% at 100
47.2% at 100
45.2% at 100
17.7
l
l
l
l
l
M
12c
12d
12e
15a
15b
15c
15d
24.7
20.4
20.7
15.4
16.5
13.0
7.2
18.2
19.0
22.8
12.2
13.8
11.6
8.5
M
M
M
M
17.6
All compounds were tested in triplicate wells per dilution. Oseltamivir carboxylate was used as the positive control with IC50 values = 0.0017
(H1N1). NT: not tested.
lM (H9N2) and 0.010 lM

Y. Xie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3556–3560
3559
Figure 3. Graphical determination of the type of inhibition for compound 15d. (A) Lineweaver–Burk plot for the inhibition of compound 15 on NA from H9N2 influenza virus
for the hydrolysis of substrate in the presence of different concentrations for lines from bottom to top: 25, 12.5, 6.25 and 0.0 M. (B) Dixon plot for inhibition of compound
15d on NA from H9N2 influenza virus for the hydrolysis of substrate in the presence of different concentrations of substrate for lines from bottom to top: 20, 10.0, and 5.0 M.
l
l
Figure 4. Docking analysis of compound 15d. (A) Oseltamivir (the smaller compound) and 15d binding to the active site of NA (PDB ID: 2HU0), and (B) residues that formed
hydrogen bonds with compound 15d.
caffeic acid (IC50 >100
l
M against N2 and N1, data not shown) on
at the
skeleton of
a
-NH2 had little influence on the activity and generally, the
-ornithine was unfavorable to NA inhibition.
the NA inhibitory activities. Besides, most of these compounds pre-
sented similar activities against N1 and N2 NAs, except 3e and 3f in
Table 1. The two compounds contained chain-like amino acids, 3-
aminopropanoic acid and 4-aminopropanoic acid, which may favor
selective inhibition of the N2 NA.
Since 3e and 3f showed better activities relative to compounds
3a–3d, we further designed and synthesized compounds 7a–7f
that had a relative long chain based on the skeleton of L-ornithine.
L
In Table 3, it could be clearly found that compounds 11a–11e
were less potent than compounds 12a–12f, which proved that caf-
feic acid was appropriate for being modified as NA inhibitors. Com-
pounds 15a–15e with the structural fragment of phenoxy acetic
acid instead of phenol exhibited improved activities relative to com-
pounds 12a–12f. The introduction of the carboxyl group obviously
increased the potency, which was consistent with the structure–
activity relationship of classical NA inhibitors. Among the four com-
pounds, 15d with 2-ethylbutanamide group was the most potent
As shown in Table 2, however, the activities of compounds 7a–7f
were lower than those of 3e and 3f. The result did not come up
to our expectations. It could be found that the size of substituents
against N2 and N1 NA with IC50 = 7.2 and 8.5
lM, respectively. In
Table 4
In vitro anti-influenza virus (A/Chicken/Shandong/LY/08) activities in Chicken embryo fibroblast cells using the CPE reduction assay
a
b
a
b
Compound
EC50
(lM)
CC50
89.5
(
lM)
Compound
EC50
(lM)
CC50
(l
M)
3d
3e
7c
7f
12a
12b
>100
>100
NT
12e
15a
15b
15c
15d
OS carboxylate
22.6
NT
NT
NT
31.2
0.5
>100
60.4
86.7
94.3
>100
>10.0
>100
65.0
87.7
78.2
>100
NT
NT
58.6
NT: not tested.
a
EC50: concentration required to achieve 50% protection against virus-induced cytopathic effect.
CC50: concentration of 50% cellular toxicity.
b

3560
Y. Xie et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3556–3560
the kinetic study, compound 15d was found to be a non-competitive
inhibitor with Ki = 11.5 0.25 M against the N2 NA (H9N2) by the
was the best inhibitor among the designed caffeic acid derivatives
and could be as a lead for further modifications.
l
means of Lineweaver–Burk and Dixon plots analysis (Fig. 3).
The docking analysis (Fig. 4A) performed by the means of SYB-
YL-X 1.1 indicated that the 3,4-dihydroxyphenyl group of 15d
could penetrate deeply into the active site of NA, and presented
hydrogen bonding interactions with residues Glu119 and Arg156
(Fig. 4B). To some extent, this can explain the reason why com-
pounds 12a–12f were potent than compounds 11a–11e. However,
the other parts of 15d were mainly on the surface of the active
sites, which was not favorable to the activity. Notably, the acetic
acid group had strong interactions with residue Arg292, Asn294
and Asn247, a little like that of oseltamivir, interacting with resi-
due Arg118, Arg292, and Arg371. These interactions likely contrib-
uted the higher activities of compounds 15a–15d compared with
compounds 12a–12f.
NA is still an important target for anti-influenza drugs design.
Considering the current anti-influenza situation, it is still necessary
to discover novel NA inhibitors. The caffeic derivatives seem prom-
ising to be a new choice for development of anti-influenza virus
agents.
Acknowledgments
This work was supported by the 12th Five Years National Sci-
ence and Technology Major Project (2011ZX09401-015) and Doc-
toral Fund of Ministry of Education of China (No.
20110131110037). We greatly thank B.H. and K.Y. for their work
in the biological activities assays.
Generally, the NA inhibitory activities of compounds 12a–12e
and 15a–15e were better than those of compounds 3a–3f and
7a–7f. It seemed that caffeic acid derivatives bearing rigid struc-
tural fragments, especially with carboxyl groups were superior to
those containing flexible long chains with branched groups. Com-
pound 15d could be a new lead for further modifications to dis-
cover more potent inhibitors.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
033.
References and notes
In addition, some compounds were further evaluated for their
anti-influenza virus activities as well as the cellular toxicity by
the CPE reduction assay (Table 4). Compound 15d with the best en-
zyme inhibitory activity showed moderate anti-influenza virus
1. 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.
2. Takano, R.; Kiso, M.; Igarashi, M.; Le, Q. M.; Sekijima, M.; Ito, K.; Takada, A.;
Kawaoka, Y. J. Infect. Dis. 2013, 207, 89.
3. Dao, T. T.; Tung, B. T.; Nguyen, P. H.; Thuong, P. T.; Yoo, S. S.; Kim, E. H.; Kim, S.
K.; Oh, W. K. J. Nat. Prod. 2010, 73, 1636.
4. Dao, T. T.; Nguyen, P. H.; Won, H. K.; Kim, E. H.; Park, J.; Won, B. Y.; Oh, W. K.
Food Chem. 2012, 134, 21.
5. Touaibia, M.; Jean-Francois, J.; Doiron, J. Mini Rev. Med. Chem. 2011, 11, 695.
6. Kashiwada, Y.; Ahmed, F. A.; Kurimoto, S.; Kim, S. Y.; Shibata, H.; Fujioka, T.;
Takaishi, Y. J. Nat. Med. 2012, 66, 217.
7. Kishimoto, N.; Kakino, Y.; Iwai, K.; Mochida, K.; Fujita, T. Biocontrol Sci. 2005, 10,
155.
activity with EC50 value of 31.2 lM against H9N2. Due to the obvi-
ous toxicity of compounds 7c, 7f, 12a and 15a–15c, their anti-
influenza virus activities were not determined. It was noteworthy
that compound 12e with low cytotoxicity displayed more potent
activity relative to compound 15d, which was not consistent with
the enzyme inhibition result. As mentioned above, many plant-de-
rived polyphenols have been found to exert antiviral effects against
influenza virus. It seemed that the phenolic hydroxyl group of
compound 12e was important for the antiviral activity.
8. Liu, K. C.; Fang, J. M.; Jan, J. T.; Cheng, T. J.; Wang, S. Y.; Yang, S. T.; Cheng, Y. S.;
Wong, C. H. J. Med. Chem. 2012, 55, 8493.
In summary, we synthesized some caffeic acid derivatives and
discovered a new type of influenza NA inhibitors. Compound 15d