Design, synthesis, and biological evaluation of crenatoside analogues as novel influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.12.031

Natural products, especially derived from TCMH, have been found to exert antiviral effects against influenza virus. Crenatoside, a phenylethanoid glycoside from Pogostemon cablin Benth, which has been shown as a novel effective NA inhibitor previously, is considered as the leading compound for our further SARs studies. This work presented design, synthesis of novel crenatoside analogues from readily available d-Glucose and l-rhamnose in a convergent manner. Furthermore, their biological activities and SARs were also investigated. Especially, compound 2 h showed impressive IC₅₀ = 27.77 μg/mL against NAs, which is 3 folds more potent than the leading compound crenatoside (IC₅₀ = 89.81 μg/mL). These results would promise their therapeutic potential for influenza disease.

Full text of DOI:10.1016/j.ejmech.2015.12.031

European Journal of Medicinal Chemistry 109 (2016) 199e205
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of crenatoside analogues
as novel influenza neuraminidase inhibitors
,
*
Bao-Long Chen a, Ya-Jing Wang b, Hong Guo a, Guang-Yao Zeng a
a School of Pharmaceutical Sciences, Central South University, 172 Tongzipo Road, Yuelu District, Changsha 410013, PR China
b Hunan University of Chinese Medicine, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2015
Received in revised form
15 December 2015
Accepted 15 December 2015
Available online 4 January 2016
Natural products, especially derived from TCMH, have been found to exert antiviral effects against
influenza virus. Crenatoside, a phenylethanoid glycoside from Pogostemon cablin Benth, which has been
shown as a novel effective NA inhibitor previously, is considered as the leading compound for our further
SARs studies. This work presented design, synthesis of novel crenatoside analogues from readily available
D
-Glucose and
L
-rhamnose in a convergent manner. Furthermore, their biological activities and SARs
g/mL against NAs,
g/mL). These results
were also investigated. Especially, compound 2 h showed impressive IC50 ¼ 27.77
m
which is 3 folds more potent than the leading compound crenatoside (IC50 ¼ 89.81
would promise their therapeutic potential for influenza disease.
m
Keywords:
Design
Synthesis
© 2016 Elsevier Masson SAS. All rights reserved.
Neuraminidases inhibitors
Crenatoside derivatives
Influenza virus
1. Introduction
discovered by Sokolova, which exhibits potent inhibitory activity
against influenza A viruses [6]. Especially, neuraminidase (NA)
cleaves the specific linkage of the sialic acid receptor, resulting in
the release of the newly formed virions from the infected cells.
Additionally, the neuraminidase may facilitate the early stage of
influenza virus infection towards lung epithelial cells [7]. Hence,
neuraminidase has been an attractive target for the development of
novel anti-influenza drugs.
At present, the first-line drugs (oseltamivir and zanamivir)
recommended for flu treatment are NA inhibitors. Both of them are
sialic acid (Neu5Ac) analogues (Fig. 1). Though with tremendous
success, the treatment doesn't seem to be optimistic due to the
spontaneously arising and spreading of oseltamivir resistance
among influenza virus [8]. Therefore, developing novel NA in-
hibitors to combat influenza virus is desirable.
Natural products, especially those derived from traditional
Chinese medicine herbs (TCMH), are still the major source of
innovative therapeutic agents for infectious diseases, cancer, lipid
disorders and immunomodulation [9]. However, the structural
complexity, small content and unfavourable pharmacokinetic
properties of bioactive natural products limited their use in ther-
apeutic field [10]. Thus it is desirable to initiate a SARs study to
identify the drug candidate for further investigations.
Influenza virus causes worldwide outbreaks in humans and
animals every year with high morbidity and mortality. In the past
century, severe influenza outbreaks took place, including the
catastrophic H1N1 Spanish influenza in 1918 (caused more than 50
million deaths globally), the H2N2 Asian flu in 1957 (caused more
than 1 million deaths globally), and the H3N2 Hong Kong flu in
1968 (caused ~0.5 million deaths globally) [1]. The battle between
human and influenza has begun since ancient time, and will last for
a long time.
The enveloped, negative-stranded influenza virus belongs to the
family Orthomyxoviridae, which can be classified by the antigenic
properties of glycopro-teins located at the surface of the virus,
haemagglutinin (H) and neuraminidase (N). Sixteen subtypes have
currently been defined with the haemagglutinin protein (H1eH16)
and nine with the neuraminidase protein (N1eN9) accordingly [2].
Both haemagglutinin and neuraminidase are crucial for the repli-
cation and infectivity of influenza virus [3]. Many compounds
against NP and HA targets were found [4,5]. For example, a new
class of compounds featuring
a camphor moiety has been
Pogostemon cablin Benth, also known as “Guang-Huo-Xiang” in
China, is an important TCMH that has been widely used for
* Corresponding author.
E-mail address: zenggy2013@csu.edu.cn (G.-Y. Zeng).
http://dx.doi.org/10.1016/j.ejmech.2015.12.031
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

200
B.-L. Chen et al. / European Journal of Medicinal Chemistry 109 (2016) 199e205
treatment on common cold, nausea, diarrhoea, headaches and fever
[11]. In previous study, we have identified crenatoside as a novel
selective NA inhibitor from a collection of phenylethanoid glyco-
sides isolated from P. cablin Benth. The study partially revealed a
molecular basis for the anti-influenza activity of this TCMH. It also
suggested that caffeoyl and 1, 4-dioxanyl moieties are critical for
their inhibition and selectivity of NA from influenza A virus (H1N1)
(Fig. 2). However, several defects of crenatoside, including its poor
pharmacokinetic properties and small content in plants, limited its
use as a drug candidate. In this paper, we presented the design,
synthesis and biological evaluation of crenatoside analogues as
novel NA inhibitors, which not only reveal the structure-activity
relationships in crenatoside, but also provide drug candidate for
further investigations.
to reach intermediate 15. Subsequently, we focused on the chal-
lenging glycosidation. After multiple attempts, we found that C-20
acetylate promoted stereoselective glycosidation of compound 15
with phenylethanol 16 under AgOTf/NIS/-40 ꢀC conditions. This
gave our target compound 17 as diastereoisomers in moderate yield
[13]. In this protocol, phenylethanol 16 was prepared by reaction of
styrene oxide with hydrobromide as racemic isomers, which would
help our investigation of C-7 configuration on the biological ac-
tivities. Finally, deacetylation with NaOMe followed by intra-
molecular O-alkylation in the presence of NaH yields compound 7
as diastereoisomers (Scheme 2).
We then performed synthesis of analogues. On one hand,
debenzylidenation of compound 7 in the presence of 80%HOAc
afforded isomers 1a and 1b, which was then separated by HPLC.
The stereochemistry of 1a and 1b was elucidated by the difference
of the coupling constant for H-7/H-8 (J7-8
uration) [14]. The stereochemistry of C-7 of analogues is also
assigned in the same way. On the other hand, -rhamnose conju-
gated analogues 1c and 1d were synthesized in a convergent
manner. Compound 20 was prepared through a three-step protocol
b
¼ 3.6 Hz for
a config-
2. Design of crenatoside analogues
L
Generally, the design of crenatoside analogues is based on the
bioisosteric replacement and functionality “knock-out” strategy. In
medicinal chemistry, bioisosteric replacement is a frequently used
strategy in optimization to obtain the desired biological or physical
properties of a compound. And functionality “knock out” is also a
common protocol to clearly state the contributions of the structural
motif to the biological activities. Specifically, we would like to
replace the caffeoyl group with privileged drug scaffold and elim-
inate the rhamnose moiety to investigate the impact of these
functional groups on the biological activities. Furthermore, MOE
simulation showed that the bulky glucose 30- rhamnose moiety
impedes its binding with the NA (Fig. 3). Additionally, the substi-
tution effect of C-30 on the biological activity is still unexplored.
Therefore, we would like to incorporate privileged drug scaffold,
amino acid, onto the C-30 position.
from L-rhamnose according to the routine procedure. Subsequent
glycosylation of compound 20 with the compound 7 in the pres-
ence of TMSOTf at ꢁ30 ꢀC gave compound 21 in good yield [15],
which was then debenzylidenated and deacetylated to provide
isomers 1c and 1d [16] (Scheme 3).
As mentioned above, a series of C-30 substituted analogues were
also synthesized. Specifically, we would like to attach cinnamoyl
group and amino acid at C-30 position. Compound 7 was esterified
with different cinnamic acid derivatives, followed by debenzyli-
denation and deacetylation to provide compounds 2ae2 h in
moderate yield [17]. Similarly, compounds 3ae3d synthesized by
esterification and simultaneous removal of benzylidenation and
Boc groups using TFA/CH2Cl2 (Scheme 4).
3. Chemistry
4. Experimental
With considerations mentioned above, we initiated our syn-
thesis of the crenatoside analogues. The compound 7 can be served
as the versatile intermediate for the synthesis of crenatoside ana-
logues. The retrosynthestic analysis of compound 7 is shown in
Scheme 1. Compound 7 can be synthesized by intermediate 8
through C-30 functionalization and reductive debenzylation. The
intermediate 8 can be prepared by base-mediated intramolecular
alkylation of compound 9, which is routinely synthesized by gly-
cosidation of compound 10 and phenylethanol. The activated pre-
cursor can be prepared by protection of C-50, C60 hydroxyl groups
with benzyl acetide and activation of the anomeric carbon with
4.1. General remarks
1H and 13C NMR spectra were recorded on a Brucker AV 400
spectrometer or 500 using tetramethylsilane (TMS) as an internal
standard; J-values are in Hz. Mass spectra were recorded by ESI-
HRMS analysis, which was provided by Agilent 6520 Q-TOF LC/
MS spectrometer (Agilent, Germany). Solvent for anhydrous reac-
tion should be processed before use. Commercially obtained re-
agents were used without further purification. All reactions were
monitored by thin-layer chromatography (TLC) with Huang hai
GF254 silica gel coated plates.
phenyl sulfide of readily available
With the retrosynthetic analysis in mind, we then started the
synthetic endeavours. Starting from commercially available
D-glucose.
D
-
4.2. Procedure for the preparation of the unknown compounds
glucose, we obtained compound 12 by fully acetylation of hydroxyl
group with Ac2O in the presence of sodium acetate in good yield.
Sequential BF3$Et2O catalysed theo glycosylation, and Zemplen
deacetylation, led to the glucosinolate 13, of which the C-40, C60
hydroxyl groups were selectively protected with O-benzylidene
group [12] and C20, C30 hydroxyl groups were protected as diacetate
4.2.1. 2-b-bromo-Benzeneethanol, 4, 6-O-Benzylidene-2, 3-O-
acetyl- -D- glucopyranose (17)
b
A suspension of compound 15 (88.8 mg, 0.2 mmol) and com-
pound 16 (60 mg, 0.3 mmol) in dry CH2Cl2 (5 mL) containing
activated 4 Å molecular sieves (80 mg) was stirred under an at-
mosphere of argon at RT for 30 min. After cooling to ꢁ40 ꢀC, NIS
(54 mg, 0.24 mmol) and a solution of AgOTf in toluene (18 mg, 2 ml)
was added and the resulting mixture was stirred from ꢁ40 ꢀC to RT
for 2.5 h. The reaction was quenched with 0.3 ml Et3N and diluted
with CH2Cl2 (10 mL). The solution was washed with brine and the
organic layer was dried over Na2S04 and filtered. The filtrate was
concentrated in vacuo and the residue was purified by flash silica
gel column chromatography to give compound 17 (58 mg, 55.0%) as
a white powder.
Fig. 1. The structure of Neu5Ac, oseltamivir and zanamivir.

B.-L. Chen et al. / European Journal of Medicinal Chemistry 109 (2016) 199e205
201
Fig. 2. Phenylethanoid glycosides isolated from P. cablin Benth.
Compound 7 (7a
): m.p. 185e187 ꢀC; 1H NMR (400 MHz, CDCl3):
d
7.35e7.62 (m, 10H), 5.49 (s, 1H), 4.89e4.90 (d, J ¼ 3.6 Hz, 1H),
4.67e4.68 (m, 2H), 4.36e4.40 (dd, J ¼ 4.8, 10.8 Hz), 4.27e4.31 (dd,
J ¼ 3.6, 12.4 Hz, 1H), 3.76e3.96 (m, 2H, 8-H), 3.61e3.67 (m, 1H),
3.37e3.54 (m, 2H). 13C-NMR (100 MHz, CDCl3):
d 137.32, 136.82,
129.35, 128.74, 128.38, 128.12, 127.99, 126.28, 102.18, 99.66, 81.34,
73.15, 72.75, 70.86, 68.41, 68.19, 67.09.
Compound 7 (7b
): m.p. 182e184 ꢀC; 1H NMR (500 MHz, CDCl3)
d
7.35e7.55 (m, 10H), 5.59 (s, 1H), 4.76e4.80 (dd, J ¼ 3.2, 10.8 Hz,
1H), 4.57e4.59 (d, J ¼ 7.6 Hz, 1H), 4.42e4.46 (dd, J ¼ 4.4, 10.4 Hz,
1H), 4.09e4.13 (dd, J ¼ 3.2, 12.4 Hz, 1H), 3.86e4.06 (m, 2H),
3.48e3.53, 3.73e3.79 (m, 2H), 3.66e3.76 (m, 2H). 13C-NMR
(125 MHz, CDCl3):
d 136.83, 136.24, 129.36, 128.65, 128.61, 128.38,
126.60, 126.32, 102.21, 98.67, 81.30, 80.22, 78.03, 72.09, 70.82,
68.50, 68.28.
4.2.3.
b-D-Glucopyranose 1, 2-O-[(2a/b)-2-benzene-1, 2-
Fig. 3. Docking result:crenatoside in the active site of NA(PDB ID:3TI6). The red part is
rhamnose molecules. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
ethanediyl] (1a and 1b)
A suspension of 7 (370 mg, 0.1 mmol) in 80% HOAc (20 mL) was
heated to 90 ꢀC with stirring till all starting material was consumed.
The solvent was removed under reduced pressure, the residue was
purified by flash column chromatography to give 220 mg of 1a and
1b, 78% yield. Then HPLC is used to separate, afforded the 1a and 1b
respectively.
Compound 1a (7
DMSO-d6): 7.54e7.29 (m, 5H), 5.22e5.23 (s, 1H), 5.14e5.15 (s, 1H),
4.82e4.83 (d, J ¼ 3.5 Hz, 1H), 4.61e4.63 (s, 2H), 4.52e4.55 (d,
J ¼ 12.0 Hz, 1H), 4.39e4.40 (d, J ¼ 7.5 Hz, 1H), 4.07e4.10 (dd, J ¼ 4.0,
13.0 Hz, 1H), 3.41e3.67 (m, 2H), 3.33e3.37 (m, 1H), 3.04e3.09 (m,
1H), 3.24e3.28 (m, 1H), 2.92e2.96 (m, 1H). 13C-NMR (125 MHz,
4.2.2.
b-D-Glucopyranose 4, 6-O-Benzylidene-1, 2-O-[(2a/b)-2-
benzene-1, 2-ethanediyl] (7)
A solution of compound 17 (267 mg, 0.5 mmol) in MeOH con-
taining NaOMe (1.0 mmol) was stirred at RT for 2 h. Then the
mixture was neutralized by Amberlite IR-120 (Hþ form), filtered,
and the filtrate was concentrated. The residue was dissolved in dry
THF (15 mL) and NaH (84 mg, 7 mmol). The mixture was stirred
under an atmosphere of argon at RT for 5 h, and then refluxed
overnight. The solution was neutralized by 1 M/L HCl, and then
extracted with dichloromethane and the organic layer was dried
over Na2S04 and filtered. The filtrate was concentrated in vacuo and
the residue was purified by flash silica gel column chromatography
to give compound 7 (83.6 mg, 45.2%) as a white powder.
a
): m.p. 217e219 ꢀC; 1H-NMR (500 MHz,
d
DMSO-d6): d 139.21, 128.77, 127.90, 127.71, 99.11, 79.07, 73.77, 73.38,
71.66, 70.74, 66.68, 61.11; ESI-HRMS: C14H18O6Na for þ, calculated
305.1001, found 305.0972.
Compound 1b (7
DMSO-d6): 7.31e7.43 (m, 5H) 5.19e5.22 (m, 2H), 4.69e4.71 (m,
b
): m.p. 269e271 ꢀC; 1H-NMR (500 MHz,
d

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B.-L. Chen et al. / European Journal of Medicinal Chemistry 109 (2016) 199e205
Scheme 1. Retrosynthetic analysis of compound 7.
Scheme 2. Synthesis of key intermediate 7 and 1a, 1b.
Scheme 3. Synthesis of target molecules 1ce1d.
2H), 4.63e4.66 (dd, J ¼ 2.5, 10.5 Hz, 1H), 4.37e4.38 (d, J ¼ 7.5 Hz,
1H), 3.98e4.01 (dd, J ¼ 4.0, 12.0 Hz, 1H), 3.71e3.74 (m, 1H),
3.48e3.55 (m,1H), 3.55e3.59 (m,1H), 3.40e3.44 (m,1H), 3.18e3.22
(m, 1H), 3.30e3.34 (m, 1H), 3.04e3.08 (m, 1H, 40-H). 13C-NMR
(125 MHz, DMSO-d6): d 137.85, 128.70, 128.53, 127.12, 98.27, 79.97,
79.16, 76.87, 73.75, 71.02, 70.87, 61.23; ESI-HRMS: C14H18O6Na
for þ, calculated 305.1001, found 305.0977.

B.-L. Chen et al. / European Journal of Medicinal Chemistry 109 (2016) 199e205
203
Scheme 4. Synthesis of target molecules 2aeh,3aed.
4.2.4. 1,2-O-[(2
3-O-(2,3,4- tri-O-acetyl-
glucopyranose (21)
a
/
b
)-2-benzene-1,2-ethanediyl] 4,6-0-benzylidene-
heated to 90 ꢀC with stirring till all starting material was consumed.
The solvent was removed under reduced pressure. A solution of the
residue in MeOH containing NaOMe (2.0 mmol) was stirred at RT
for 2 h. Then the mixture was neutralized by Amberlite IR-120 (Hþ
form), filtered, and the filtrate was concentrated. The residue was
purified by flash column chromatography to give 302 mg of 1c and
1d, 71% yield. Then HPLC is used to separate, afforded the 1c and 1d
respectively.
a-L-rhamno-pyranosyl)-b-D-
A suspension of compound 7 (111 mg, 0.3 mmol) and compound
20 (119.25 mg, 0.36 mmol) in dry CH2Cl2 (5 mL) containing acti-
vated 4 Å molecular sieves (80 mg) was stirred under an atmo-
sphere of argon at RT for 30 min. After cooling to ꢁ30 ꢀC, NIS
(90 mg, 0.4 mmol) and TMSOTf (88.8 mg, 0.4 mmol) was added and
the resulting mixture was stirred from ꢁ30 ꢀC to RT for 4 h. The
reaction was quenched with 0.3 ml Et3N and diluted with CH2Cl2
(10 mL). The solution was washed with brine and the organic layer
was dried over Na2S04 and filtered. The filtrate was concentrated in
vacuo and the residue was purified by flash silica gel column
chromatography to give compound 21 (109 mg, 56.7%) as a pale
yellow oily matter.
Compound 1c (7
DMSO-d6): 7.31e7.49 (m, 5H), 5.12 (s,1H), 4.81 (s,1H), 4.63 (s, 2H),
a
): m.p. 173e175 ꢀC; 1H-NMR (500 MHz,
d
4.53 (s, 1H), 5.08 (s, 1H), 4.85 (d, J ¼ 2.5 Hz, 1H), 4.56e4.59 (d,
J ¼ 12.5 Hz, 1H), 4.45e4.56 (d, J ¼ 8.0 Hz, 1H, 10-H), 4.09e4.12 (dd,
J ¼ 3.5, 12.5 Hz, 1H), 3.86e3.91 (m, 1H), 3.70 (s, 1H), 3.46e3.64 (m,
4H), 3.31e3.33 (m, 1H), 3.09e3.22 (m, 2H), 2.95e2.99 (m, 1H),
1.10e1.11 (d, J ¼ 6.5 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6):
Compound 21 (7
a
) 1H-NMR (500 MHz, CDCl3):
d
7.32e7.57 (m,
d 138.86, 128.85, 127.86, 127.78, 100.76, 98.33, 78.86, 77.28, 74.63,
10H), 5.51 (s, 1H), 5.31e5.32 (dd, J ¼ 2.0, 4.0 Hz, 1H), 5.25e5.27 (m,
2H), 5.01e5.05 (m, 1H), 4.86 (d, J ¼ 3.0 Hz, 1H), 4.68e4.70 (d,
J ¼ 13.0 Hz, 1H), 4.61e4.63 (d, J ¼ 8.0 Hz, 1H), 4.37e4.40, 4.25e4.29
(dd, J ¼ 4.0, 10.5 Hz), 4.31e4.34 (m, 1H), 4.15e4.21 (m, 1H),
3.77e4.01 (m, 2H), 3.58e3.65 (m, 2H), 3.37e3.41 (m, 1H), 2.21 (s,
3H), 2.02 (s, 3H), 1.99 (s, 3H), 0.94e0.95 (d, J ¼ 6.0 Hz, 3H).
72.50, 71.60, 71.17, 70.91, 68.86, 68.54, 66.13, 60.96, 18.32; ESI-
HRMS: C20H28O10Na for þ, calculated 451.1580, found 451.1550.
Compound 1d (7
DMSO-d6):
b
): m.p. 217e219 ꢀC; 1H-NMR (500 MHz,
d
7.31e7.38 (m, 5H), 5.23 (s,1H), 4.74 (s,1H), 4.63 (s, 2H),
4.48 (s, 1H), 5.02 (s, 1H), 4.70e4.72 (dd, J ¼ 2.5, 10.5 Hz, 1H),
4.42e4.43 (d, J ¼ 8.0 Hz, 1H), 4.03e4.06 (dd, J ¼ 2.5, 12.0 Hz, 1H),
3.89e3.95 (m, 1H), 3.16e3.75 (m, 9H), 1.11e1.12 (d, J ¼ 6.0 Hz, 3H).
Compound 21 (7b d 7.30e7.50 (m,
) 1H-NMR (500 MHz, CDCl3):
10H), 5.63 (s, 1H), 5.35e5.36 (dd, J ¼ 1.5, 3.0 Hz), 5.26e5.29 (dd,
J ¼ 3.5, 10.0 Hz, 1H), 5.01e5.05 (d, J ¼ 1.5 Hz,1H), 4.96e5.01 (m, 1H),
4.78e4.8 (dd, J ¼ 2.5, 10.5 Hz, 1H), 4.55e4.57 (d, J ¼ 8.0 Hz, 1H),
4.44e4.47, 4.26e4.29 (m, 2H), 4.31e4.34 (m, 1H), 4.13e4.16 (m,1H),
3.77e3.93 (m, 2H), 3.67e3.73 (m, 2H), 3.60e3.63 (m, 1H), 2.06 (s,
3H), 1.96 (s, 3H), 1.96 (s, 3H), 0.96e0.97 (d, J ¼ 6.0 Hz, 3H).
13C-NMR (125 MHz, DMSO-d6)
d 137.78, 128.81, 128.37, 126.39,
100.31, 97.64, 80.92, 79.09, 76.70, 76.34, 72.45, 71.12, 71.03, 70.85,
68.69, 68.58, 61.12, 18.35; ESI-HRMS: C20H28O10Na for þ, calculated
451.1580, found 451.1549.
4.2.6. The general procedure of synthesis of 2ae2h
To a soln of different cinnamic acid analogues (0.5 mmol) and
compound 7 (0.4 mmol) were added EDCI (0.5 mmol) and a cata-
lytic amount of DMAP at 0 ꢀC. The soln was stirred for 24 h. The
solution was washed with brine and the organic layer was dried
4.2.5. 1, 2-O-[(2
acetyl- -L- rhamno - pyranosyl)-
A suspension of 21 (624 mg, 1.0 mmol) in 80% HOAc (20 mL) was
a/
b
)-2-benzene-1, 2-ethanediyl] 3-O-(2, 3, 4-tri-O-
a
b
-D-glucopyranose (1c and 1d)

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B.-L. Chen et al. / European Journal of Medicinal Chemistry 109 (2016) 199e205
over Na2S04 and filtered. The organic layer was concentrated and
applied to a column of silica gel to give 26.
Compound 2 g (7
CD3OD):
6.32e6.35 (d, J ¼ 16.0 Hz, 1H), 5.17e5.21 (m, 1H), 4.80 (d, J ¼ 3.5 Hz,
1H), 4.63e4.66 (m, 2H), 4.23e4.27 (dd, J ¼ 3.5, 12.5 Hz, 1H),
3.70e3.88 (m, 2H), 3.22e3.55 (m, 3H). 13C-NMR (125 MHz, CD3OD):
a
): m.p. 81e83 ꢀC; 1H-NMR (500 MHz,
7.59e7.62 (d, J ¼ 16.0 Hz, 1H), 6.98e7.51 (m, 8H),
d
A suspension of 26 (0.5 mmol) in 80% HOAc (20 mL) was heated
to 55 ꢀC with stirring till all starting material was consumed. The
solvent was removed under reduced pressure, the residue was
purified by flash column chromatography. Then a solution of the
product in MeOH containing HCl (1.0 mmol) was stirred at 0 ꢀC till
all starting material was consumed. The solvent was removed un-
der reduced pressure, then use of silica gel or Lipophilic Sephadex
LH-20 to Separation of this epimers.
d
167.41,148.29,145.90,145.47,137.97, 128.12,127.40,126.35, 121.63,
115.17, 113.81, 113.65, 98.91, 78.24, 74.50, 72.13, 71.27, 68.34, 66.12,
60.70; ESI-HRMS: C23H25O9 for þ, calculated 459.1499, found
445.1469.
Compound 2 h (7
CD3OD):
b
): m.p. 80e82 ꢀC; 1H-NMR (500 MHz,
Compound 2a (7
a
): m.p. 145e147 ꢀC; 1H-NMR (400 MHz,
d
7.57e7.60 (d, J ¼ 16.0 Hz, 1H), 6.76e7.37 (m, 8H),
CD3OD):
d
7.72e7.76 (d, J ¼ 16.0 Hz, 1H), 7.29e7.65 (m, 10H),
6.30e6.33 (d, J ¼ 16.0 Hz, 1H), 5.30e5.34 (m, 1H), 4.69e4.72 (dd,
J ¼ 2.5, 10.5 Hz, 1H), 4.60e4.61 (d, J ¼ 7.5 Hz, 1H), 3.93e4.09 (dd,
J ¼ 3.0, 12.0 Hz, 2H), 3.78e3.81 (dd, J ¼ 5.5, 12.0 Hz, 1H), 3.72e3.76
6.58e6.62 (d, J ¼ 16.0 Hz, 1H), 5.21e5.25 (m, 1H), 4.79e4.80 (d,
J ¼ 3.2 Hz, 1H), 4.62e4.66 (m, 2H), 4.23e4.27 (dd, J ¼ 3.6, 12.8 Hz),
3.70e3.90 (m, 2H), 3.25e3.60 (m, 3H). 13C-NMR (100 MHz, CD3OD):
(m, 1H), 3.44e3.68 (m, 3H). 13C-NMR (125 MHz, CD3OD):
d 167.48,
d
166.76, 145.27, 137.98, 134.34, 130.26, 128.72, 128.14, 127.94,
148.16, 145.7 9, 145.37, 136.88, 128.00, 127.76, 126.38, 125.90, 121.59,
115.07, 113.77, 113.73, 98.11, 78.45, 77.67, 76.98, 74.42, 71.41, 68.46,
60.81; ESI-HRMS: C23H25O9 for þ, calculated 459.1499, found
445.1464.
127.46, 117.43, 98.91, 78.24, 74.74, 72.14, 71.20, 68.30, 66.17, 60.68;
ESI-HRMS: C23H25O7 for þ, calculated 413.4404, found 413.4693.
Compound 2b (7b
): m.p. 113e115 ꢀC; 1H-NMR (500 MHz,
CD3OD):
d
7.71e7.74 (d, J ¼ 16.0 Hz, 1H), 7.26e7.58 (m, 10H),
6.55e6.58 (d, J ¼ 16.0 Hz, 1H), 5.33e5.37 (m, 1H), 4.70e4.73 (dd,
J ¼ 3.0, 10.5 Hz, 1H), 4.61e4.63 (d, J ¼ 8.0 Hz, 1H), 3.94e4.06 (dd,
J ¼ 3.0, 12.0 Hz, 2H), 3.79e3.82 (dd, J ¼ 5.0, 12.0 Hz, 1H), 3.74e3.78
4.2.7. The general procedure of synthesis of 3ae3d
To a soln of different Boc protected amino acids (0.5 mmol) and
compound 7 (0.4 mmol) were added DCC (1.5 mmol) and a catalytic
amount of DMAP at 0 ꢀC. The soln was stirred for 36 h. The solution
was washed with brine and the organic layer was dried over Na2S04
and filtered. The organic layer was concentrated and applied to a
column of silica gel to give 24.
(m, 1H), 3.47e3.69 (m, 3H). 13C-NMR (125 MHz, CD3OD):
d 166.81,
145.13, 136.87, 134.38, 130.07, 128.58, 128.01, 127.85, 127.78, 125.91,
117.49, 98.10, 78.44, 77.64, 77.03, 74.66, 71.43, 68.42, 60.79; ESI-
HRMS: C23H25O7 for þ, calculated 413.4404, found 413.4567.
Compound 2c(7
CD3OD):
a
): m.p. 116e118 ꢀC; 1H-NMR (400 MHz,
Compound 24 (0.5 mmol) was treated with TFA/CH2Cl2 (15 mL)
for 1.5 h at room temperature. The mixture was concentrated in
vacuo, and the residue was separate by Lipophilic Sephadex LH-20,
provided single configuration of 3ae3d.
d
7.65e7.69 (d, J ¼ 15.6 Hz, 1H), 7.30e7.53 (m, 9H),
6.39e6.43 (d, J ¼ 15.6 Hz, 1H), 5.15e5.23 (m, 1H), 4.80e4.81 (d,
J ¼ 3.6 Hz, 1H), 4.63e4.67 (m, 2H), 4.24e4.28 (dd, J ¼ 4.0, 12.8 Hz,
1H), 3.69e3.89 (m, 2H), 3.22e3.58 (m, 3H). 13C-NMR (100 MHz,
Compound 3a (7
CD3OD): 7.34e7.57(m, 5H), 5.19e5.24 (m, 1H), 4.79e4.80 (d,
a
): m.p. 113e115 ꢀC; 1H-NMR (400 MHz,
CD3OD):
d 167.39, 159.99, 145.52, 137.98, 129.85, 128.11, 127.41,
d
125.77, 115.48, 113.469, 98.93, 78.26, 74.49, 72.13, 71.25, 68.32,
66.14, 60.67; ESI-HRMS: C23H25O8 for þ, calculated 429.1549, found
429.1530.
J ¼ 3.6 Hz, 1H), 4.63e4.65 (d, J ¼ 7.6 Hz, 1H), 4.56e4.59 (d,
J ¼ 12.8 Hz,1H), 3.28e4.28 (m, 7H), 2.25e2.33 (m, 1H), 1.03e1.12 (d,
J ¼ 6.8 Hz, 6H). 13C-NMR (100 MHz, CD3OD):
d 168.67, 137.72,
Compound 2d (7
CD3OD):
b
): m.p. 98e100 ꢀC; 1H-NMR(500 MHz,
128.40, 128.02, 98.86, 78.05, 76.56, 72.55, 70.21, 68.21, 66.67, 60.53,
58.05, 29.57, 17.07, 16.91; ESI-HRMS: C19H28NO7 for þ, calculated
382.1866, found 382.1839.
d
7.62e7.65 (d, J ¼ 16.0 Hz, 1H), 6.77e7.41 (m, 9H),
6.33e6.37 (d, J ¼ 16.0 Hz, 1H), 5.32e5.36 (m, 1H), 4.67e4.70 (dd,
J ¼ 3.0,10.5 Hz,1H), 4.60e4.61 (d, J ¼ 7.5 Hz,1H), 3.94e4.06 (m, 2H),
3.80e3.83 (dd, J ¼ 5.0, 12.0 Hz, 1H), 3.74e3.78 (m, 1H), 3.47e3.69
Compound 3b (7
CD3OD): 7.31e7.37 (m, 5H), 5.34e5.38 (m, 1H), 4.69e4.73 (dd,
b
): m.p. 85e87 ꢀC; 1H-NMR (400 MHz,
d
(m, 3H). 13C-NMR (125 MHz, CD3OD):
d 167.50, 159.79, 145.46,
J ¼ 2.4, 10.4 Hz, 1H), 4.61e4.63 (m, 1H), 3.44e4.09 (m, 8H),
2.21e2.28 (m, 1H), 0.92e0.95(m, 6H). 13C-NMR (400 MHz, CD3OD):
136.85, 129.86, 128.03, 127.79, 125.94, 125.81, 115.40, 113.81, 98.11,
78.43, 77.66, 76.98, 74.43, 71.42, 68.49, 60.85; ESI-HRMS: C23H25O8
for þ, calculated 429.1549, found 429.1521.
d
168.59, 136.52, 128.06, 126.07, 97.74, 78.23, 77.30, 77.17, 76.39,
71.31, 67.85, 60.58, 58.00, 29.70,16.79,16.37; ESI-HRMS: C19H28NO7
for þ, calculated 382.1866, found 382.1839.
Compound 2e (7
CD3OD):
6.42e6.45 (d, J ¼ 15.5 Hz, 1H), 5.18e5.22 (m, 1H), 4.79e4.80 (d,
J ¼ 3.5 Hz, 1H), 4.63e4.65 (m, 2H), 4.23e4.27 (dd, J ¼ 3.5, 12.5 Hz,
1H), 3.92 (s, 1H), 3.70e3.88 (m, 2H), 3.23e3.56 (m, 3H). 13C-NMR
a
): m.p. 135e137 ꢀC; 1H-NMR (500 MHz,
7.64e7.68 (d, J ¼ 15.5 Hz, 1H), 6.84e7.51 (m, 8H),
Compound 3c (7a d 7.30e7.54 (m,
) 1H-NMR (500 MHz, CD3OD):
d
5H), 5.36e5.39 (m, 1H), 4.71e4.74 (d, J ¼ 3.2 Hz, 1H), 3.45e4.63 (m,
9H), 1.82e2.90 (m, 7H). 13C-NMR (125 MHz, CD3OD):
d
168.91,
136.54, 128.45, 128.19, 128.07, 126.28, 126.04, 97.73, 78.21, 77.22,
77.22, 76.62, 71.36, 67.88, 60.56, 51.75, 29.80, 27.94, 13.43; ESI-
HRMS: C19H28NO7S for þ, calculated 414.1586, found 414.1592.
(125 MHz, CD3OD):
d 167.38, 149.32, 148.02, 145.80, 137.99, 128.11,
127.44, 127.38, 126.33, 122.82, 115.12, 114.01, 110.34, 98.94, 78.27,
74.25, 72.14, 71.25, 68.35, 66.17, 60.69, 55.08; ESI-HRMS: C24H27O9
for þ, calculated 459.1655, found 459.1624.
Compound 3d (7b) d 7.32e7.57
1H-NMR (400 MHz, CD3OD):
(m, 5H), 5.18e5.23 (m, 1H), 4.79e4.82 (dd, J ¼ 2.4, 10.4 Hz, 1H),
b
): m.p. 139e141 ꢀC; 1H-NMR (500 MHz,
7.61e7.64 (d, J ¼ 16.0 Hz, 1H), 6.77e7.35 (m, 8H),
3.23e4.70 (m, 9H), 2.04e3.07 (m, 7H). 13C-NMR (400 MHz,
Compound 2f (7
CD3OD):
6.37e6.40 (d, J ¼ 16.0 Hz, 1H), 5.32e5.36 (m, 1H), 4.68e4.71 (dd,
J ¼ 2.5, 10.0 Hz, 1H), 4.60e4.62 (d, J ¼ 7.5 Hz, 1H), 3.94e4.07 (dd,
J ¼ 3.0, 12.0 Hz, 2H), 3.84 (s, 3H), 3.79e3.83 (dd, J ¼ 5.0,12.0 Hz,1H),
3.74e3.78 (m, 1H), 3.46e3.67 (m, 3H). 13C-NMR (125 MHz, CD3OD):
d
CD3OD):
d 169.05, 137.74, 128.45, 128.29, 128.08, 127.90, 127.81,
98.75, 78.04, 76.77, 72.42, 70.40, 68.12, 66.44, 60.53, 51.49, 29.79,
28.80, 13.71; ESI-HRMS: C19H28NO7S for þ, calculated 414.1586,
found 414.1592.
d
167.43, 149.12, 147.86, 145.71, 136.85, 128.01, 127.78, 126.33,
5. Results and discussions
125.94, 122.73, 115.00, 114.09, 110.29, 98.11, 78.45, 77.69, 77.02,
74.44, 71.41, 68.51, 60.83, 55.00; ESI-HRMS: C24H27O9 for þ,
calculated 459.1655, found 459.1616.
With crenatoside analogues in hand, we then determined their
inhibitory activities by subjecting them onto Neuraminidase

B.-L. Chen et al. / European Journal of Medicinal Chemistry 109 (2016) 199e205
205
Table 1
development and promise their therapeutic potential for the rising
challenge of influenza diseases.
In vitro inhibitory effects of compounds against influenza A NAs.
Sample
1000ug/mLInhibitory rate
mg/ml
IC50mg/ml
Acknowledgements
1a
3.9
NT
1b
1c
16.82
8.28
NT
NT
The authors are thankful to the authorities of School of Phar-
maceutical Sciences of Central South University, for providing lab-
oratory facilities. Gratitude is expressed to Shaogang Liu and Zhen
Li, Modern Analysis and Testing Center of CSU, for 1H NMR, 13C NMR
spectrums and ESI-HRMS spectrum.
1d
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
11.72
17.55
29.82
91.24
95.89
64.98
66.69
94.08
97.42
16.58
25.32
88.76
90.33
89.24
86.33
NT
NT
NT
42.91
29.64
97.95
78.03
29.91
27.77
NT
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.12.031.
NT
89.43
78.12
89.81
115.84
3d
References
Crenatoside
Isocrenatoside
[1] R. Salomon, R.G. Webster, The influenza virus enigma, Cell 136 (3) (2009)
402e410.
All compounds were tested in DMSO wells per dilution. NT: not tested.
[2] J. An, D.C.W. Lee, A.H.Y. Law, C.L.H. Yang, L.L.M. Poon, A.S.Y. Lau, S.J.M. Jones,
A novel small-molecule inhibitor of the avian influenza H5N1 virus deter-
mined through computational screening against the neuraminidase, J. Med.
Chem. 52 (9) (2009) 2667e2672.
[3] W. Fiers, M. De Filette, A. Birkett, S. Neirynck, W. Min Jou, A “universal” human
influenza A vaccine, Virus Res. 103 (1e2) (2004) 173e176.
[4] M.I. Lin, B.H. Su, C.H. Lee, S.T. Wang, W.C. Wu, P. Dangate, S.Y. Wang,
W.I. Huang, T.J. Cheng, O.A. Lin, Y.S. Cheng, Y.J. Tseng, C.M. Sun, Synthesis and
inhibitory effects of novel pyrimido-pyrrolo-quinoxalinedione analogues
targeting nucleoproteins of influenza A virus H1N1, Eur. J. Med. Chem. 102
(2015) 477e486.
[5] S. Niu, L. Si, D. Liu, A. Zhou, Z. Zhang, Z. Shao, S. Wang, L. Zhang, D. Zhou,
W. Lin, Spiromastilactones: a new class of influenza virus inhibitors from
deep-sea fungus, Eur. J. Med. Chem. 108 (2016) 229e244.
[6] A.S. Sokolova, O.I. Yarovaya, A.V. Shernyukov, Y.V. Gatilov, Y.V. Razumova,
V.V. Zarubaev, T.S. Tretiak, A.G. Pokrovsky, O.I. Kiselev, N.F. Salakhutdinov,
Discovery of a new class of antiviral compounds: camphor imine derivatives,
Eur. J. Med. Chem. 105 (2015) 263e273.
[7] M.N. Matrosovich, T.Y. M, T. Gray, N.A. Roberts, H.D. Klenk, Neuraminidase is
important for the initiation of influenza virus infection in human airway
epithelium, J. Virol. 78 (2004) 12665e12667.
[8] A. Moscona, Global transmission of oseltamivir-resistant influenza, N. Engl. J.
Med. 360 (2009) 953e956.
[9] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the
30 years from 1981 to 2010, J. Nat. Prod. 75 (3) (2012) 311e335.
[10] J. Clardy, C. Walsh, Lessons from natural molecules, Nature 432 (2004)
829e837.
[11] Z. Zhao, J. Lu, K. Leung, C.L. Chan, Z.-H. Jiang, Determination of patchoulic
alcohol in herba pogostemonis by GC-MS-MS, Chem. Pharm. Bull. 53 (7)
(2005) 856e860.
Inhibitors Screen Kit, with crenatoside and is crenatoside as refer-
ences. Based on the biological results (Table 1), the structure-
activity relationships (SARs) of the leading compound, crenato-
side, were discussed.
As summarized in Table 1, there are some interesting SARs. It is
interesting that the analogues 1ae1d, with the core structure of
crenatoside, do not show the activity of neuraminidase inhibitors.
It's suggesting that the core structure of crenatoside does not
guarantee the inhibitory activity of neuraminidase. This may be
explained by the spatial effect of the molecule.
It is found that compounds 2ae2 h are generally more potent
than the leading compound crenatoside. Among them, compound
2 h was the most potent one, with IC50 ¼ 27.77
mg/ml against NAs.
Carefully inspection into the structure of compounds 2ae2 h
revealed that their activity increases along with the number of
phenolic hydroxyl groups on the benzene ring. When substituted
with methoxy group on the C-30 phenyl moiety, the activity
decreased significantly. It is therefore proposed that the hydrogen
donor substitution at the C-30 phenyl moiety is favourable for the
activity.
Generally, the NA inhibitory activities of compounds 3ae3b
were better than those of compounds 3ce3d. It seemed that
compounds with methionine groups were superior to those com-
pounds containing valine groups. The docking analysis performed
by MOE simulation indicated that the amino group of 3ce3d
showed hydrogen bonding interactions with residues Arg371 and
Arg118. To some extent, this can explain why compounds 3ce3d
were potent than compounds 3ae3b.
[12] X.-F. Luo, F. Lei, Y. He, S.-C. Pei, L. Hai, S. Qian, Y. Wu, The synthesis of pen-
nogenin 3-O-
b-D-glucopyranosyl-(1/3)-[a-L-rhamnopyranosyl-(1/2)]-b-
D-glucopyranoside, J. Asian Nat. Prod. Res. 14 (4) (2012) 314e321.
[13] S. Ng, M.R. Jafari, W.L. Matochko, R. Derda, Quantitative synthesis of geneti-
cally encoded glycopeptide libraries displayed on M13 phage, ACS Chem. Biol.
7 (9) (2012) 1482e1487.
[14] Y. Yang, J. Zhang, Y. Liu, Q. Tang, Z. Zhao, W. Xia, Structural elucidation of a 3-
O-methyl-D-galactose-containing neutral polysaccharide from the fruiting
bodies of phellinus igniarius, Carbohydr. Res. 342 (8) (2007) 1063e1070.
[15] K. Arumugam, B. Varghese, J.N. Brantley, S.S.M. Konda, V.M. Lynch,
C.W. Bielawski, 1,6-Enyne cyclizations catalyzed by N-heterocyclic carbene
supported gold complexes: deconvoluting sterics and electronics, Eur. J. Org.
Chem. 2014 (3) (2014) 493e497.
[16] C.-C. Zou, S.-J. Hou, Y. Shi, P.-S. Lei, X.-T. Liang, The synthesis of gracillin and
dioscin: two typical representatives of spirostanol glycosides, Carbohydr. Res.
338 (8) (2003) 721e727.
[17] F.-Y. Zhou, J. She, Y.-G. Wang, Synthesis of a benzyl-protected analog of are-
narioside, a trisaccharide phenylpropanoid glycoside, Carbohydr. Res. 341
(15) (2006) 2469e2477.
The effect of C-7 configuration on the activity was also exam-
ined. We found that C-7a analogues are less potent than C-7b ones,
which indicated that the C-7 configuration exerts a significant ef-
fect on the inhibitory activity of neuraminidase.
In sum, we presented design, synthesis and SARs studies of
crenatoside as novel NA inhibitors. This work disclosed some
interesting SARs of phenylethanoid glycosides derivatives, which
not only enrich the NA inhibitor reservoir, but also facilitate further