Design, synthesis of oleanolic acid-saccharide conjugates using click chemistry methodology and study of their anti-influenza activity

Article abstract of DOI:10.1016/j.ejmech.2019.111622

The development of entry inhibitors is an emerging approach to the inhibition of influenza virus. In our previous research, oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor. Herein, as a further study, we report the preparation of a series of OA-saccharide conjugates via the CuAAC reaction, and the anti-influenza activity of these compounds was evaluated in vitro. Among them, compound 11b, an OA-glucose conjugate, showed a significantly increased anti-influenza activity with an IC₅₀ of 5.47 μM, and no obvious cytotoxic effect on MDCK cells was observed at 100 μM. Hemagglutination inhibition assay and docking experiment indicated that 11b might interfere with influenza virus infection by acting on HA protein. Broad-spectrum anti-influenza experiments showed 11b to be robustly potent against 5 different strains, including influenza A and B viruses, with IC₅₀ values at the low-micromole level. Overall, this finding further extends the utility of OA-saccharide conjugates in anti-influenza virus drug design.

Full text of DOI:10.1016/j.ejmech.2019.111622

European Journal of Medicinal Chemistry 182 (2019) 111622
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis of oleanolic acid-saccharide conjugates using click
chemistry methodology and study of their anti-influenza activity
,
,
Yangqing Su a 1, Lingkuan Meng a 1, Jiaqi Sun b, Weijia Li a, Liang Shao a, Kexuan Chen a,
Demin Zhou b, Fan Yang a **, Fei Yu a
,
, *
a Medical School of Kunming University of Science and Technology, Kunming, Yunnan, 650500, China
b State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of entry inhibitors is an emerging approach to the inhibition of influenza virus. In our
previous research, oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor.
Herein, as a further study, we report the preparation of a series of OA-saccharide conjugates via the
CuAAC reaction, and the anti-influenza activity of these compounds was evaluated in vitro. Among them,
compound 11b, an OA-glucose conjugate, showed a significantly increased anti-influenza activity with an
Received 16 July 2019
Received in revised form
12 August 2019
Accepted 12 August 2019
Available online 13 August 2019
IC50 of 5.47 mM, and no obvious cytotoxic effect on MDCK cells was observed at 100 mM. Hemaggluti-
nation inhibition assay and docking experiment indicated that 11b might interfere with influenza virus
infection by acting on HA protein. Broad-spectrum anti-influenza experiments showed 11b to be robustly
potent against 5 different strains, including influenza A and B viruses, with IC50 values at the low-
micromole level. Overall, this finding further extends the utility of OA-saccharide conjugates in anti-
influenza virus drug design.
Keywords:
Influenza virus
Entry inhibitor
Oleanolic acid
Hemagglutinin
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
continually mutating influenza viruses. Therefore, drug treatment
is the best means of controlling the spread of influenza virus. At
Influenza A (IAV) and B (IBV) viruses are important causes of
upper respiratory tract infections [1]. IAV are classified into
different subtypes based on the two large glycoproteins, hemag-
glutinin (HA) and neuraminidase (NA), which are on the surface of
the viral envelope [1]. HA binds to the sialic acid receptor on the
target cell membrane and is involved in the initial stage of viral
infection. NA hydrolyzes the sialic acid receptor on the cell surface
to release the progeny influenza virus from the infected cells. In the
past 100 years, influenza virus epidemics, including H1N1 in 1918,
H2N2 in 1957, H3N2 in 1968, H5N1 in 2009 and H7N9 in 2013,
resulted in serious impact on global morbidity, mortality, and huge
economic loss [2e8]. Currently, the main measures to deal with
influenza virus infection are vaccination and medical treatment.
However, vaccines have certain limitations on the scope of use, and
seasonal influenza vaccines are not effective enough to prevent
present, there are two main classes of anti-influenza drugs
approved by FDA. The first class is NA inhibitors, including Tamiflu
and Zanamivir, which can inhibit the activity of NA and thereby
disable tethered progeny virus from escaping from the host cell
[9,10]. The second class is M2 ion channel inhibitors, including
Amantadine and Rimantadine, which can inhibit the activity of
influenza virus M2 ion channel protein, thereby blocking the
exfoliation process of the influenza virus [11]. However, the high
mutation rate of the RNA genome of the influenza virus, combined
with assortment of its multiple genomic segments, promotes
antigenic diversity and new subtypes, allowing viruses to become
resistant to existing antiviral drugs [12]. Therefore, it is still urgent
to design and develop new anti-influenza drugs.
As the first step to prevent virus infection, entry inhibition
presents a favorable strategy for drug discovery which promises an
efficient blockage of virus propagation in theory [13,14]. Recently,
we found that plant-derived pentacyclic triterpenes and their de-
rivatives have displayed inhibitory activity against influenza A virus
entry in vitro, and compound Y3, an oleanolic acid (OA)-acetyl
galactose conjugate, exhibited robust anti-H1N1 entry activity with
* Corresponding author.
** Corresponding author.
E-mail addresses: yangfan@kmust.edu.cn (F. Yang), feiyuz8@kmust.edu.cn
(F. Yu).
an IC50 of 4.05 mM [1]. Further study indicated that these triterpene
1
These authors contributed equally to the work.
https://doi.org/10.1016/j.ejmech.2019.111622
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
derivatives could bind tightly to the pocket of viral envelope HA
protein, thus blocking the attachment of viruses to host cells.
Therefore, OA became an interesting scaffold for designing novel
influenza virus entry inhibitors. Previous reports also showed that
the anti-influenza activity can be significantly improved by
coupling 17-COOH or 3-OH of OA with oligosaccharides [1,15e17].
Nevertheless, due to the steric hindrance of the 17-COOH, it is
hard to obtain OA-di- or tri-saccharide conjugates by the same
synthetic strategy employed for Y3. So, it is still unclear whether
the antiviral activity can be further enhanced by coupling 17-COOH
of OA with a disaccharide or trisaccharide. In order to investigate
this possibility, we present herein our effort to conjugate different
saccharide moieties with 17-COOH of OA through a triazole linker
rather than an amide bond via a Cu(I) azide-alkyne cycloaddition
reaction (CuAAC). All the synthesized compounds were evaluated
for their in vitro anti-influenza A virus (IAV) activities.
than that of OA. However, combined with the data shown in Fig. 1A,
the high inhibition rate of compounds 12a, 15b and 16b might
reflect the cellular toxicity rather than potency enhancement.
These results indicated that the number and configuration of the
hydroxyl groups of OA-oligosaccharide conjugates have critical ef-
fects on cytotoxicity and anti-viral activity. Compared with DMSO-
treated MDCK cells, influenza A/WSN/33 virus causes a severe CPE
in MDCK-infected cells. As shown in Fig. 2A, compound 11b
significantly reduced the CPE, indicating that these compounds
were able to protect MDCK cells from influenza virus-induced CPE.
Three compounds, 11b 13a, and 14b, identified with high inhi-
bition rate (>75%) and low cytotoxicity (cell viability > 75%, Fig. 1),
were selected for dose response assays. The results, expressed as
IC50 (compound concentration required to achieve 50% inhibition of
replication of A/WSN/33), CC50 (compound concentration required
to cause 50% death of uninfected MDCK cells) and SI (selectivity
index), are summarized in Table 1, respectively. All the three
compounds showed much higher anti-influenza A/WSN/33 activ-
ities than OA with IC50 at low-micromole level. Compound 11b
showed the strongest inhibitory activity in this study with an IC50
2. Results and discussion
2.1. Chemistry
value of 5.47
mM. In addition, the CC50 of these compounds were all
Click chemistry has recently emerged as one of the most
powerful tools in drug discovery. The design and synthesis of
pharmacologically relevant heterocyclic molecules have proven to
be a promising strategy, and the Cu(I)-catalyzed version of the
Huisen 1,3-dipolar cycloaddition reaction between azides and ter-
minal alkynes (CuAAC) for the construction of trizoles is one of the
powerful reactions for making carbonꢀheteroatomꢀcarbon bonds
with a wide variety of chemical and biological applications.
Therefore, the CuAAC reaction was chosen as the linking method to
couple OA with different saccharide molecules. As outlined in
Scheme 1, the 17-COOH of OA was firstly activated by TBTU and
then reacted with propynylamine to construct the terminal alkyne
module. Azido saccharides were synthesized via reported methods
and their spectral data were consistent with the data reported
previously [18e25]. To perform the CuAAC reaction, OA-
propargylamine and an azido saccharide were dissolved in
higher than 100
m
M.
2.2.2. Conjugate 11b inhibits virus-induced hemagglutination
The viral envelope protein HA plays a crucial role in the early
stage of influenza virus infection. HA protein can bind to sialic acid
on the surface of red blood cells (RBCs), causing agglutination [27].
Antibodies of HA at a sufficient concentration will interfere with
the virus attachment to red blood cells and thus inhibit hemag-
glutination [28,29]. Therefore, hemagglutination inhibition (HI)
assay was applied to investigate whether compound 11b targets HA
protein. In this assay, A/WSN/33 virus was pretreated with com-
pound 11b before the addition of chicken erythrocytes, and a
further incubation was conducted at room temperature for 30 min.
In parallel, inhibition of hemagglutination by anti-HA antibody was
used as the positive control. As shown in Fig. 2B, compound 11b
inhibited hemagglutination with a similar capability as anti-HA
antibody. This result suggests that 11b may have the same target
as anti-HA antibody, HA, and thus block the interactions of viruses
with target cells.
CH2Cl2eH2O, followed by the addition of sodium L-ascorbate and
CuSO4$5H2O. The reaction proceeded smoothly at room tempera-
ture to afford the target compounds (11a-20a) in good yields. Then
the O-acetyl groups of the saccharide were cleaved with 2 N NaOH/
MeOH to give the corresponding deprotected compounds
11be20b. The 1H and 13C NMR spectra of the synthesized com-
pounds 11a-20b are shown in supplementary materials.
2.2.3. Molecular docking analysis
We designed molecular docking calculations of compound 11b
with influenza HA protein to further understand the molecular
basis of the inhibitory properties of OA-saccharide conjugates. The
docked conformations of HA-11b were determined based on the
minimum free energy analyses using AutoDock 4.2 program (The
Scripps research institute, La Jolla, CA, USA).
2.2. Biological assays
2.2.1. In vitro anti-influenza virus activity in MDCK cells
The CellTiter-Glo assay and the cytopathic effect (CPE) reduction
assay were employed to evaluate the antiviral activities of com-
pounds (11a-20b) against the influenza virus strain A/WSN/33,
which was propagated in Madin-Darby canine kidney (MDCK) cells
[26]. As shown in Fig. 1A, most compounds had no significant
As shown in Fig. 3, compound 11b was docked into the binding
site of the sialic acid receptor on the HA protein with a binding
energy of ꢀ10.70 kcal/mol and an inhibition constant (Ki) of
14.44 nM. The corresponding binding energy and inhibition con-
stant of OA were ꢀ6.14 kcal/mol and 31.5 nM, respectively, indi-
cating that 11b binds to HA protein more tightly. In addition, we
found that 11b almost occupied the full site of sialic acid receptor
binding pocket on HA, which might explain why the anti-viral ac-
tivity was significant reduced after coupling OA with di- or tri-
saccharide.
The modeled structure of HA-11b complex indicated that the
C3eOH of OA could form a hydrogen bond with Gly225, while the
20-OH and 30-OH of the glucose moiety also formed four hydrogen
bonds with Arg133, Lys156, and Gly158, respectively. In addition,
Ser193 is involved in hydrogen bond interaction with the 28-CO of
OA and 60-OH of the glucose. Moreover, the triazole moiety further
enhanced the binding affinity of 11b to HA by interacting with
cytotoxicity against MDCK cells at a concentration of 100 mM,
except for compounds 12a, 15b and 16b. Then, the compounds
against the influenza A/WSN/33 virus were evaluated by the
CellTiter-Glo assay (Fig. 1B). The inhibition rate was significantly
decreased when OA is conjugated with a disaccharide. In addition,
when OA is coupled with maltotriose (compounds 20a and 20b),
the potency was completely abolished. These data suggested that a
larger saccharide module at 17-COOH may hinder the effective
binding of OA pharmacophore to the target protein, resulting in
reduced antiviral activity.
Compounds 11b, 12a, 13a, 14b, 15b and 16b displayed significant
anti-A/WSN/33 activity with inhibition rates over 75%, far better

Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
3
Scheme 1. Reagents and conditions: (a) Ac2O, DMAP, pyridine, rt, overnight; (b) HBr$AcOH, CH2Cl2, 0 ꢁC to rt, overnight; (c) NaN3, DMF, rt, overnight; (d) TBTU, DIEA, THF, rt,
overnight; (e) 2-propynylamine, Na2CO3, DMF, rt, 20 min; (f) CuSO4$5H2O, Na-L-ascorbate, azido-saccharide, CH2Cl2/H2O, rt, overnight; (g) 2N NaOH, MeOH, rt, 1 h.
Val155 and Leu194 through
p
-alkyl stacking interaction (Fig. 3C).
and B viruses in MDCK cells. Three of these strains, which belong to
the IAV H3N2 subtype, the Yamagata lineage, and the Victoria
lineage of IVB, showed strong OSV-p resistance. Interestingly,
compound 11b exhibited strong inhibitory activity against OSV-p
resistant strains A/Victoria/361/2011 (H3N2), and the influenza B
virus B/Sichuan/531/2018 (BV) and B/Massachusetts/2/2012 (BV)
Therefore, occupancy of the binding pocket for sialic acid receptor
might account for the molecular basis by which 11b blocks the HA-
sialic acid receptor interaction and thus the attachment of viruses
to the host cells.
(with IC50 values of 10.42, 26.12 and 11.51
addition,11b exerted remarkable activity against B/Yamagata/16/88
(BY), which was 4-fold more active than OSV-p (IC50 ¼ 5.54
mM, respectively). In
2.2.4. Conjugate 11b exhibits broad-spectrum anti-influenza
activity
mM).
Given that 11b occupies the pocket for sialic acid receptor of HA,
we inquired whether 11b exerts broad antiviral spectrum. OSV-p is
a highly effective anti-influenza virus inhibitor that inhibits mul-
tiple influenza virus subtypes, such as H1N1, H5N1, H9N2. There-
fore, OSV-p was used as a positive control. As shown in Table 2,
OSV-p and 11b were evaluated against five strains of influenza A
3. Conclusion
In summary, we have designed and synthesized a series of OA-
saccharide conjugates by using a facile and efficient CuAAC process

4
Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
Fig. 1. Inhibitory effects of OA-saccharide conjugates against influenza A/WSN/33 (H1N1) virus. (A) The cytotoxic effect-based screen of OA-saccharide conjugates (100
mM) using
CellTiter-Glo® assay. (B) The cytopathic effect-based screen of OA-saccharide conjugates (100 M). MDCK was utilized as the host cell to test A/WSN/33 virus infection; 1% DMSO
m
(final concentration) was used as the negative control, and Oseltamivir phosphate (OSV-p) was used as the positive control. Error bars indicate standard deviations of triplicate
experiments.
Fig. 2. (A) Validation of the protection of MDCK cells from influenza A/WSN/33 virus by compound 11b. (B) Comparisons of 11b and anti-HA antibody for inhibiting influenza virus-
induced aggregation of chicken erythrocytes. Compound 11b exerted a similar capability to inhibit hemagglutination to that of anti-HA antibody.
which allows OA to be easily combined with various carbohydrate
moieties. The anti-influenza activity of these compounds was
evaluated in vitro, where some of them showed desirable potency.
Among them, compound 11b exerted the best inhibitory activity
Moreover, 11b exerted broad spectrum activity even against OSV-p
resistant viruses. HI assay and docking simulation indicate that the
anti-viral activity of 11b is potentially due to its high affinity to HA
protein, thus blocking the attachment of influenza viruses to host
cells, confirming the initial design idea.
against influenza A/WSN/33 virus, with an IC50 value of 5.47 mM.

Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
5
Table 1
4. Experimental
Inhibitory activities of compound 11b, 13a, and 14b against infection of MDCK cells
by influenza A/WSN/33 virus.
4.1. Chemistry-general
Compounds
CC50
(m
M)a
IC50
(
m
M)b
SIc
OSV-pd
OA
11b
13a
14b
>100
>100
>100
>100
>100
1.82
72.2
5.47
7.22
>54.94
>1.38
>18.28
>13.85
>7.73
High Resolution Mass Spectra (HRMS) were obtained with an
APEX IV FT_MS (7.0 T) spectrometer (Bruker) in positive ESI mode.
NMR spectra were recorded on a Bruker DRX 400 spectrometer at
ambient temperature. 1H NMR chemical shifts were reported
12.93
relative to the internal standard TMS (
signal (
¼ 7.26 for the central line of CDCl3,
central line of CD3OD). 13C NMR chemical shifts were referenced to
the solvent signal (
¼ 77.16 for the central line of CDCl3,
d
¼ 0.00) or the solvent
H
a
50% cytotoxicity concentration.
d
d
¼ 3.31 for the
H
H
b
Concentration inhibiting viral replication by 50%. The values are means of at
least three independent determinations.
c
d
C
SI: selectivity index as CC50/IC50
Positive control.
.
d
d
¼ 49.00 for the central line of CD3OD). Reactions were moni-
C
tored by thinꢀlayer chromatography (TLC) on a precoated silica gel
60 F254 plate (layer thickness 0.2 mm; E. Merck, Darmstadt, Ger-
many) and detected by staining with a yellow solution containing
Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O·244H2O (24.0 g) in 6%
Fig. 3. Structural representative of 11b binding within HA protein (Protein Data Bank: 1RVT) according to blind docking calculation. (A) Overview of HA protein. The inhibitor pocket
is highlighted in red square. (B) Closer view of the inhibitor pocket. Protein and compound 11b are shown as gray surface and orange sticks, respectively. (C) 2D interaction plot with
green dashes and pink dashes indicating hydrogen bonds and hydrophobic interactions correspondingly. (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
Table 2
Comparisons of the anti-influenza virus activity of compound 11b with that of OSV-p.
Compounds
CC50
(
m
M)a
IC50(m
M)b
A/WSN/33
A/Victoria/361/2011(H3N2)
B/Yamagata/16/88(BY)
B/Sichuan/531/2018(BV)
B/Massachusetts/2/2012(BV)
OSV-p
11b
>100
>100
1.82
5.47
>100
10.42
23.45
5.54
>100
26.12
>100
11.51
a
CC50, the concentration required to reduce the viability of normal, non-infected MDCK cells by 50%. Values represent the mean of duplicate samples from three inde-
pendent experiments.
b
IC50, the concentration required to inhibit viral infection-induced CPE by 50%. Values represent the mean of duplicate samples from three independent experiments.

6
Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
H2SO4 (500 mL) followed by heating. Flash column chromatog-
raphy was performed on silica gel 60 (200 ꢀ 300 mesh, Qingdao
Haiyang Chemical Co. Ltd).
procedure A. The residue was purified by column chromatography
(petroleum ether/AcOEt, 1/2 v/v) to give 11a as a white solid
(117 mg, 83%). Mp 129.8e131.7 ꢁC. 1H NMR (600 MHz, CDCl3):
d
0.67, 0.78, 0.88, 0.89, 0.90, 0.99,1.16 (7 ꢂ CH3), 0.67e1.80 (m, other
4.2. Synthesis
aliphatic ring protons), 1.88 (s, 3H), 2.04 (s, 3H), 2.07 (s, 3H), 2.10 (s,
3H), 2.55 (d, J ¼ 4.44 Hz, 1H), 3.21 (dd, J ¼ 4.08, 11.22 Hz, 1H),
3.98e4.01 (m, 1H), 4.15 (dd, J ¼ 2.04, 12.66 Hz, 1H), 4.24e4.32 (m,
2H), 4.62(dd, J ¼ 5.64,15.06 Hz,1H), 5.25 (t, J ¼ 9.9 Hz,1H), 5.4e5.46
(m, 3H), 5.83 (d, J ¼ 8.82 Hz,1H), 6.60 (brs,1H), 7.78 (s, 1H). 13C NMR
4.2.1. General procedure a for the CuAAC “click” reaction (11a-20a)
To a solution of alkyne (0.16 mmol) and azide (0.19 mmol) in
CH2Cl2 (3 mL) and H2O (3 mL) was added CuSO4$5H2O (48 mg,
0.19 mmol) and Na-
L-ascorbate (75 mg, 0.38 mmol). The resulting
(150 MHz, CDCl3): d 15.49, 15.67, 16.73, 18.38, 20.29, 20.65 (2C),
solution was stirred vigorously for 12 h at rt. The reaction mixture
was diluted with H2O (10 mL), then extracted with CH2Cl2
(10 mL ꢂ 3). The combined organic layer was dried over Na2SO4,
filtered, and concentrated. The residue was purified by column
chromatography.
20.81, 20.87, 23.59, 23.71, 24.00, 25.90, 27.23, 27.35, 28.18, 30.81,
32.39, 32.41, 33.09, 34.18, 35.06, 37.03, 38.56, 38.85, 39.45, 42.05,
42.08, 46.31, 46.74, 47.64, 55.17, 61.59, 62.09, 67.68, 70.43, 72.77,
75.22, 79.03, 85.85, 121.23, 123.35, 144.51, 168.82, 169.42, 170.11,
170.63, 178.57. ESI-HRMS (m/z) [MþNa]þ calcd for C47H70N4O11Na,
889.4933; found, 889.4939.
4.2.2. General procedure B for the O-Deacetylation of OA-
Saccharide conjugates (11be20b)
4.2.6. Compound 11b
To the O-pentacetylated compound stirred in methanol was
added 2N NaOH (cat.). The mixture was stirred at rt. After
completion (TLC), the reaction mixture was neutralized with HCl
(1 M). Water was added, and the resulting suspension was filtered.
The crude product was purified by column chromatography.
Prepared from 11a (78 mg, 0.09 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 11b as a white solid (61 mg,
95%). Mp 190.0e191.8 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.63, 0.77,
0.90, 0.93, 0.94, 0.96, 1.16 (7 ꢂ CH3), 0.63e2.10 (m, other aliphatic
ring protons), 2.79 (dd, J ¼ 3.72, 13.38 Hz, 1H), 3.14 (dd, J ¼ 4.62,
11.46 Hz, 1H), 3.47e3.58 (m, 3H), 3.71 (dd, J ¼ 5.52, 12.3 Hz, 1H),
3.83e3.88 (m, 2H), 4.34 (d, J ¼ 15.12 Hz, 1H), 4.44 (d, J ¼ 15.12 Hz,
4.2.3. Compound OA-OBt
To OA (912 mg, 2 mmol) and TBTU (963 mg, 3 mmol) stirred in
20 mL THF was added DIEA (0.5 mL, 3 mmol). The mixture was
stirred at room temperature overnight. After completion (TLC), the
reaction mixture was filtered, and the filtrate was concentrated.
The crude filtrate was purified by column chromatography (pe-
troleum ether/AcOEt, 4/1 v/v) to give OA-OBt as a white solid
1H), 5.35 (t, J ¼ 3.3 Hz, 1H), 5.57 (d, J ¼ 9.18 Hz, 1H), 7.99 (s, 1H). 13
C
NMR (150 MHz, CD3OD):
d 16.13, 16.49, 17.79, 19.63, 24.03, 24.18
(2C), 24.21, 24.69, 26.62, 27.99, 28.63, 28.89, 31.76, 33.69, 33.90,
34.26, 35.19, 35.98, 38.26, 39.98, 40.76, 42.68, 43.01, 47.66, 47.77,
56.84, 62.55, 71.04, 74.13, 78.59, 79.83, 81.27, 89.70, 123.91, 124.37,
145.24, 146.27, 180.66. ESI-HRMS (m/z) [MþNa]þ calcd for
(1.01 g, 86%). Mp 184.4e185.6 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.79,
0.85, 0.91, 0.97, 1.00, 1.01, 1.22 (7 ꢂ CH3), 0.79e2.30 (m, other
aliphatic ring protons), 2.98 (dd, J ¼ 4.08, 13.68 Hz, 1H), 3.23 (d,
J ¼ 7.92 Hz, 1H), 5.38 (t, J ¼ 3.48 Hz, 1H), 7.36e7.42 (m, 2H), 7.53 (t,
J ¼ 7.86 Hz, 1H), 8.05 (d, J ¼ 8.4 Hz, 1H); 13C NMR (150 MHz, CDCl3):
C39H62N4O7Na, 721.4511; found, 721.4515.
4.2.7. Compound 12a
Prepared from OA-propargylamine (93 mg, 0.19 mmol), acetyl-
galactosyl azide (82 mg, 0.22 mmol) according to general proced-
ure A. The residue was purified by column chromatography (pe-
troleum ether/AcOEt, 1/2 v/v) to give 12a as a white solid (160 mg,
d
15.38, 15.59, 17.27, 18.30, 23.10, 23.45, 23.52, 25.73, 27.14, 28.05,
28.11, 30.65, 32.41, 32.82, 32.93, 33.66, 37.00, 38.48, 38.75, 39.44,
41.57, 41.89, 45.45, 47.54, 47.61, 55.21, 78.98, 108.15, 120.50, 123.90,
124.64, 128.49, 128.74, 142.08, 143.54, 173.66. ESI-HRMS (m/z)
[MþH]þ calcd for C36H52N3O3, 574.3964; found, 574.4016.
98%). Mp 145.2e146.8 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.68, 0.78,
0.88, 0.89, 0.90, 0.99, 1.16 (7 ꢂ CH3), 0.68e1.80 (m, other aliphatic
ring protons), 1.88 (s, 3H), 2.01 (s, 3H), 2.05 (s, 3H), 2.24 (s, 3H), 2.56
(dd, J ¼ 3.54,13.62 Hz,1H), 3.21 (dd, J ¼ 3.84,11.16 Hz,1H), 4.11e4.15
(m, 1H), 4.19e4.25 (m, 3H), 4.64 (dd, J ¼ 6.12, 15.06 Hz, 1H), 5.24
(dd, J ¼ 3.36, 10.26 Hz, 1H), 5.41 (t, J ¼ 3.36 Hz, 1H), 5.54e5.57 (m,
4.2.4. Compound OA-propargylamine
To OA-OBt (589 mg, 1 mmol) and Na2CO3 (106 mg, 1 mmol)
stirred in 8 mL DMF was added 2-propynylamine (0.1 mL,
1.5 mmol). The mixture was stirred at room temperature for
20 min. After completion (TLC), the solvent was removed under
reduced pressure. The mixture was resolved in AcOEt and washed
with water and brine twice. The organic layer was dried over
MgSO4, then filtered and concentrated. The crude residue was
purified by column chromatography (petroleum ether/AcOEt, 2/1 v/
v) to give OA-propargylamine as a white solid (473 mg, 93%). Mp
2H), 5.80 (d, J ¼ 9.24 Hz,1H), 6.60 (t, J ¼ 5.34 Hz,1H), 7.84 (s, 1H). 13
C
NMR (150 MHz, CDCl3):
d 15.47, 15.67, 16.66, 18.38, 20.36, 20.62,
20.75, 20.80, 23.58, 23.69, 24.04, 25.90, 27.24, 27.34, 28.17, 30.80,
32.37, 32.39, 33.09, 34.18, 34.99, 37.03, 38.55, 38.84, 39.43, 42.03
(2C), 46.26, 46.74, 47.64, 55.16, 61.22, 66.86, 67.95, 70.93, 74.06,
78.99, 86.38, 121.32, 123.29, 144.56, 145.36, 168.91, 169.98, 170.16,
170.39, 178.48. ESI-HRMS (m/z) [MþNa]þ calcd for C47H70N4O11Na,
889.4933; found, 889.4935.
201.3e202.7 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.78, 0.79, 0.91, 0.92,
0.99, 1.17 (7 ꢂ CH3), 0.73e2.20 (m, other aliphatic ring protons),
2.21 (t, J ¼ 2.52 Hz, 1H), 2.53 (d, J ¼ 9.48 Hz, 1H), 3.22 (dd, J ¼ 4.26,
11.34 Hz, 1H), 3.88e3.92 (m, 1H), 4.04e4.08 (m, 1H), 5.42 (t,
J ¼ 3.36 Hz, 1H), 6.11 (t, J ¼ 4.62 Hz, 1H). 13C NMR (150 MHz, CDCl3):
4.2.8. Compound 12b
Prepared from 12a (70 mg, 0.08 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 12b as a white solid (53 mg,
d
15.51, 15.69, 17.06, 18.39, 23.65, 23.70, 24.01, 25.87, 27.26, 27.35,
28.20, 29.47, 30.85, 32.32, 32.40, 33.11, 34.17, 37.05, 38.60, 38.88,
39.49, 42.13, 42.24, 46.42, 46.76, 47.63, 55.18, 71.74, 79.05, 79.77,
123.31, 144.87, 178.22.
93%). Mp 189.6e192.0 ꢁC. 1H NMR (600 MHz, CD3OD)
d: 0.62, 0.77,
0.90, 0.92, 0.94, 0.96, 1.16 (7 ꢂ CH3), 0.62e2.13 (m, other aliphatic
ring protons), 2.78 (dd, J ¼ 3.9, 13.38 Hz, 1H), 3.13 (dd, J ¼ 4.56,
11.4 Hz, 1H), 3.68 (dd, J ¼ 3.3, 9.54 Hz, 1H), 3.74 (d, J ¼ 6.9 Hz, 1H),
3.81 (dd, J ¼ 6, 6.72 Hz, 1H), 3.98 (d, J ¼ 2.7 Hz, 1H), 4.13 (t,
J ¼ 9.36 Hz, 1H), 4.34 (d, J ¼ 15.12 Hz, 1H), 4.44 (d, J ¼ 15.18 Hz, 1H),
5.35 (t, J ¼ 1.62 Hz, 1H), 5.52 (d, J ¼ 9.18 Hz, 1H), 8.02 (m, 1H). 13C
4.2.5. Compound 11a
Prepared from OA-propargylamine (78.88 mg, 0.16 mmol),
acetyl-glucosyl azide (71 mg, 0.19 mmol) according to general

Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
7
NMR (150 MHz, CD3OD)
d: 16.11, 16.49, 17.71, 19.63, 24.20, 24.21,
4.2.12. Compound 14b
24.70, 26.63, 28.00, 28.63, 28.88, 31.76 (2C), 33.70, 33.89, 34.24,
35.19, 36.04, 38.26, 39.98 (2C), 40.76, 42.70, 43.00, 47.66, 47.77,
56.85, 62.38, 70.34, 71.47, 75.48, 79.84, 80.01, 90.35, 123.36, 124.39,
145.25, 146.40, 180.62. ESI-HRMS (m/z) [MþNa]þ calcd for
Prepared from 14a (77 mg, 0.9 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 14b as a white solid (64 mg,
99%). Mp 182.0e183.8 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.54, 0.77,
C
39H62N4O7Na, 721.4511; found, 721.4511.
0.90, 0.91, 0.94, 0.95, 1.14 (7 ꢂ CH3), 0.54e2.11 (m, other aliphatic
ring protons), 2.79 (dd, J ¼ 3.84, 13.26 Hz, 1H), 3.13 (dd, J ¼ 4.38,
11.22 Hz, 1H), 3.70 (dd, J ¼ 3.36, 9.42 Hz, 1H), 3.85 (dd, J ¼ 0.84,
12.6 Hz, 1H), 3.93e3.94 (m, 1H), 4.00 (dd, J ¼ 1.92, 12.66 Hz, 1H),
4.12 (t, J ¼ 9.24 Hz, 1H), 4.36 (d, J ¼ 15.12 Hz, 1H), 4.44 (d,
J ¼ 15.06 Hz, 1H), 5.33 (t, J ¼ 3.42 Hz, 1H), 5.45 (d, J ¼ 9.12 Hz, 1H),
4.2.9. Compound 13a
Prepared from OA-propargylamine (80 mg, 0.16 mmol), acetyl-
mannosyl azide (71 mg, 0.19 mmol) according to general proced-
ure A. The residue was purified by column chromatography (pe-
troleum ether/AcOEt, 1/2 v/v) to give 13a as a white solid (112 mg,
8.02 (s, 1H). 13C NMR (150 MHz, CD3OD):
d 16.07, 16.48, 17.71, 19.61,
24.14, 24.20, 24.66, 26.62, 28.00, 28.62, 28.88, 31.77 (2C), 33.71,
33.90, 34.31, 35.19, 35.88, 38.27, 39.96, 39.98, 40.72, 42.69, 42.97,
47.63, 47.73, 56.84, 70.36, 70.93, 71.55, 74.99, 79.84, 90.74, 123.45,
124.35, 145.22, 146.32, 180.55. ESI-HRMS (m/z) [MþNa]þ calcd for
80%). Mp 139.5e141.3 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.45, 0.78,
0.87, 0.88, 0.89, 0.98, 1.14 (7 ꢂ CH3), 1.99 (s, 3H), 2.09 (s, 3H), 2.11 (s,
3H), 2.17 (s, 3H), 0.45e2.40 (m, other aliphatic ring protons), 2.55
(d, J ¼ 9.48 Hz, 1H), 3.21 (dd, J ¼ 1.86, 7.32 Hz, 1H), 3.94e3.97 (m,
1H), 4.20 (d, J ¼ 12.54 Hz, 1H), 4.29 (dd, J ¼ 5.4, 12.54 Hz, 1H),
4.36e4.46 (m, 2H), 5.28 (dd, J ¼ 2.94, 10.08 Hz, 1H), 5.35e5.40 (m,
C38H60N4O6Na, 691.4405; found, 691.4405.
4.2.13. Compound 15a
2H), 5.56 (s, 1H), 6.17 (s, 1H), 6.68 (t, J ¼ 4.86 Hz, 1H), 7.85 (s, 1H). 13
C
Prepared from OA-propargylamine (77 mg, 0.15 mmol), acetyl-
rhamnosyl azide (60 mg, 0.19 mmol) according to general proced-
ure A. The residue was purified by column chromatography (pe-
troleum ether/AcOEt, 1/2 v/v) to give 15a as a white solid (109 mg,
NMR (150 MHz, CDCl3):
d 15.42, 15.62, 16.45, 18.25, 20.56, 20.70,
20.73, 20.80, 23.60, 23.96, 25.75, 27.06, 27.19, 27.27, 28.14, 30.74,
32.23, 32.44, 33.03, 34.13, 34.95, 36.95, 38.54, 38.80, 39.30, 42.00,
42.02, 46.27, 46.69, 47.54, 55.16, 62.14, 64.90, 68.83, 70.97, 75.63,
78.98, 84.67, 122.13, 123.35, 144.34, 144.83, 169.31, 169.62, 169.91,
170.64, 178.69. ESI-HRMS (m/z) [MþNa]þ calcd for C47H70N4O11Na,
889.4933; found, 889.4938.
89%). Mp 158.4e160.1 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.52, 0.77,
0.86, 0.89, 0.98, 1.14 (7 ꢂ CH3), 1.99, 2.10, 2.13 (3 ꢂ CH3CO),
0.52e1.90 (m, other aliphatic ring protons), 2.49 (d, J ¼ 9.42 Hz, 1H),
3.19e3.23 (m, 1H), 3.78e3.82 (m, 1H), 4.34 (dd, J ¼ 5.64, 14.94 Hz,
1H), 4.48 (dd, J ¼ 5.28, 15.00 Hz, 1H), 5.16e5.22 (m, 2H), 5.38 (t,
J ¼ 3.36 Hz, 1H), 5.65 (dd, J ¼ 1.26, 2.76 Hz, 1H), 6.08 (d, J ¼ 1.20 Hz,
1H), 6.58 (t, J ¼ 5.40 Hz,1H), 7.78 (s, 1H). 13C NMR (150 MHz, CDCl3):
4.2.10. Compound 13b
Prepared from 13a (70 mg, 0.08 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 13b as a white solid (53 mg,
d
15.45, 15.69, 16.60, 17.64, 18.37, 20.67, 20.77, 20.89, 23.55, 23.71,
23.94, 25.87 (2C), 27.27, 27.34, 28.19, 30.82, 32.42, 32.43, 33.09,
34.16, 34.99, 37.04, 38.55, 38.86, 39.41, 42.05, 42.22, 46.34, 46.76,
47.62, 55.17, 69.23, 69.68, 70.97, 74.13, 79.04, 84.83, 121.87, 123.42,
144.42, 169.45, 169.89, 170.06, 178.59. ESI-HRMS (m/z) [MþNa]þ
calcd for C45H68N4O9Na, 831.4879; found, 831.4873.
93%). Mp 179.7e181.2 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.56, 0.77,
0.90, 0.92, 0.94, 0.96, 1.15 (7 ꢂ CH3), 0.56e2.12 (m, other aliphatic
ring protons), 2.78 (dd, J ¼ 3.54, 13.2 Hz, 1H), 3.13 (dd, J ¼ 4.5,
11.34 Hz, 1H), 3.51e3.53 (m, 1H), 3.73e3.79 (m, 3H), 3.91 (d,
J ¼ 2.16 Hz, 1H), 3.93 (d, J ¼ 2.22 Hz, 1H), 4.10 (s, 1H), 4.34e4.46 (m,
2H), 5.34 (t, J ¼ 3.06 Hz, 1H), 5.9 (s, 1H), 8.16e8.17 (m, 1H). 13C NMR
4.2.14. Compound 15b
Prepared from 15a (70 mg, 0.08 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 15b as a white solid (56 mg,
(150 MHz, CD3OD):
d 16.12, 16.48, 17.62, 19.62, 24.18 (2C), 24.68,
26.62, 27.99, 28.61, 28.88, 31.76 (2C), 33.70, 33.90, 34.29, 35.18,
35.77, 38.24, 38.27, 39.95, 39.98, 40.73, 42.70, 42.99, 47.63, 47.73,
56.83, 62.77, 67.88, 72.42, 75.08, 79.82, 81.69, 88.66, 124.38, 124.93,
145.17, 180.61. ESI-HRMS (m/z) [MþNa]þ calcd for C39H62N4O7Na,
721.4511; found, 721.4511.
95%). Mp 185.2e186.9 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.49, 0.76,
0.90, 0.94, 0.96, 0.95, 1.14 (7 ꢂ CH3), 0.49e2.11 (m, other aliphatic
ring protons), 1.37 (d, J ¼ 5.94 Hz, 3H), 2.77 (dd, J ¼ 3.54, 13.14 Hz,
1H), 3.13 (dd, J ¼ 4.26, 11.1 Hz, 1H), 3.47e3.56 (m, 2H), 3.67 (dd,
J ¼ 3.18, 9.24 Hz, 1H), 4.06 (d, J ¼ 3.06 Hz, 1H), 4.37 (d, J ¼ 11.34 Hz,
1H), 4.44 (d, J ¼ 15.06 Hz, 1H), 5.33 (s, 1H), 5.95 (s, 1H), 8.09 (s, 1H).
4.2.11. Compound 14a
Prepared from OA-propargylamine (79 mg, 0.15 mmol), acetyl-
arabinosyl azide (60 mg, 0.2 mmol) according to general proced-
ure A. The residue was purified by column chromatography (pe-
troleum ether/AcOEt, 1/2 v/v) to give 14a as a white solid (111 mg,
13C NMR (150 MHz, CD3OD):
d 16.11, 16.48, 17.68, 18.32, 19.65, 24.16
(2C), 24.20, 24.64, 26.56, 27.98, 28.61, 28.87, 31.76 (2C), 33.68, 33.91,
34.29, 35.19, 35.79, 38.25, 39.94, 39.96, 40.71, 42.73, 43.00, 47.70,
47.75, 56.80, 72.45, 73.23, 74.85, 77.11, 79.79, 88.57, 124.37, 124.74,
145.13, 180.61. ESI-HRMS (m/z) [MþNa]þ calcd for C39H62N4O6Na,
705.4562; found, 705.4566.
92%). Mp 152.1e153.9 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.63, 0.77,
0.87, 0.88, 0.89, 0.98, 1.15 (7 ꢂ CH3), 0.68e1.80 (m, other aliphatic
ring protons), 1.89 (s, 3H), 2.04 (s, 3H), 2.23 (s, 3H), 2.58 (dd,
J ¼ 3.54, 13.08 Hz, 1H), 3.21 (dd, J ¼ 4.2, 11.22 Hz, 1H), 3.94 (d,
J ¼ 13.32 Hz, 1H), 4.16e4.24 (m, 1H), 4.66 (dd, J ¼ 6.18, 15.06 Hz, 1H),
5.24 (dd, J ¼ 3.42, 10.08 Hz, 1H), 5.39 (t, J ¼ 3.3 Hz, 1H), 5.43 (s, 1H),
5.58 (t, J ¼ 9.9 Hz, 1H), 5.71 (d, J ¼ 9.12 Hz, 1H), 6.60 (t, J ¼ 5.34 Hz,
4.2.15. Compound 16a
Prepared from OA-propargylamine (89 mg, 0.17 mmol), acetyl-
xylosyl azide (57 mg, 0.19 mmol) according to general procedure
A. The residue was purified by column chromatography (petroleum
ether/AcOEt, 1/2 v/v) to give 16a as a white solid (106 mg, 78%). Mp
1H), 7.85 (s, 1H) 13C NMR (150 MHz, CDCl3):
d 15.41, 15.68, 16.66,
18.37, 20.37, 20.70, 21.08, 23.54, 23.69, 23.94, 25.90, 27.22, 27.33,
28.17, 30.80, 32.38, 32.52, 33.10, 34.17, 34.92, 37.03, 38.53, 38.84,
39.40, 41.94, 41.98, 46.23, 46.67, 47.63, 55.16, 67.29, 67.76, 68.26,
70.63, 79.01, 86.82, 121.34, 123.21, 144.48, 145.25, 169.00, 170.05,
170.35, 178.40. ESI-HRMS (m/z) [MþNa]þ calcd for C44H66N4O9Na,
817.4722; found, 817.4720.
142.2e143.3 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.55, 0.71, 0.81, 0.82,
0.91, 1.08 (7 ꢂ CH3), 0.55e1.80 (m, other aliphatic ring protons),1.81
(s, 3H), 1.99 (s, 3H), 2.01 (s, 3H), 2.49 (d, J ¼ 8.88 Hz, 1H), 3.14 (dd,
J ¼ 4.14, 11.22 Hz, 1H), 3.51 (t, J ¼ 10.56 Hz, 1H), 4.17e4.23 (m, 2H),
4.55 (dd, J ¼ 5.94, 15.18 Hz, 1H), 5.05e5.09 (m, 1H), 5.32e5.35 (m,
3H), 5.67 (d, J ¼ 8.76 Hz, 1H), 6.49 (t, J ¼ 5.28 Hz, 1H), 7.21 (s, 1H),

8
Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
7.68 (s, 1H)$13C NMR (150 MHz, CD3OD):
d
14.31, 14.55, 15.58, 17.24,
4.2.19. Compound 18a
19.20, 19.61, 19.64, 22.43, 22.57, 22.80, 24.76, 26.11, 26.21, 27.05,
29.69, 31.26, 31.38, 31.98, 33.03, 33.90, 35.91, 37.41, 37.72, 38.29,
40.88, 40.90, 45.15, 45.57, 46.50, 54.04, 64.44, 67.34, 69.38, 71.01,
77.91, 85.26, 120.15, 122.15, 143.36, 144.26, 167.80, 168.71, 168.97,
177.35. ESI-HRMS (m/z) [MþNa]þ calcd for C44H66N4O9Na,
817.4722; found, 817.4724.
Prepared from OA-propargylamine (60 mg, 0.12 mmol), acetyl-
lactosyl azide (126 mg, 0.19 mmol) according to general procedure
A. The residue was purified by column chromatography (petroleum
ether/AcOEt, 1/2 v/v) to give 18a as a white solid (120 mg, 86%). Mp
148.0e159.5 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.66, 0.78, 0.87, 0.89,
0.99,1.16 (7 ꢂ CH3),1.87,1.98, 2.06, 2.07, 2.09, 2.12, 2.17 (7 ꢂ CH3CO),
0.66e2.30 (m, other aliphatic ring protons), 2.53 (dd, J ¼ 3.42,
12.90 Hz, 1H), 3.21 (dd, J ¼ 3.78, 11.10 Hz, 1H), 3.89e3.97 (m, 3H),
4.09e4.17 (m, 3H), 4.24 (dd, J ¼ 4.80, 15.12 Hz, 1H), 4.49 (d,
J ¼ 11.52 Hz, 1H), 4.53 (d, J ¼ 7.92 Hz, 1H), 4.60 (dd, J ¼ 6.00,
15.12 Hz, 1H), 4.98 (dd, J ¼ 3.42, 10.44 Hz, 1H), 5.12e5.15 (m, 1H),
5.37 (d, J ¼ 3.18 Hz, 1H), 5.40 (dd, J ¼ 2.88, 6.36 Hz, 2H), 5.78 (dd,
J ¼ 2.64, 6.42 Hz, 1H), 6.62 (t, J ¼ 5.46 Hz, 1H), 7.7 (s, 1H). 13C NMR
4.2.16. Compound 16b
Prepared from 16a (55 mg, 0.07 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 16b as a white solid (45 mg,
97%). Mp 185.3e187.0 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.40, 0.67,
0.81, 0.84, 0.86, 1.04 (7 ꢂ CH3), 0.40e2.00 (m, other aliphatic ring
protons), 2.70 (dd, J ¼ 4.68, 13.74 Hz, 1H), 3.03 (dd, J ¼ 4.38,
11.28 Hz, 1H), 3.35e3.41 (m, 2H), 3.54e3.58 (m, 1H), 3.75 (t,
J ¼ 9.18 Hz, 1H), 3.89 (dd, J ¼ 5.46, 11.28 Hz, 1H), 4.25e4.35 (m, 2H),
5.23 (t, J ¼ 3.42 Hz, 1H), 5.39 (d, J ¼ 9.18 Hz, 1H), 7.85 (s, 1H). 13C
(150 MHz, CDCl3):
d 15.48, 15.68, 16.68, 18.36, 20.34, 20.63, 20.75,
20.77, 20.80, 20.83, 20.92, 22.77, 22.79, 23.58, 23.69, 24.04, 25.88,
28.16, 30.80, 32.33, 33.08, 34.17, 35.01, 37.02, 38.54, 38.85, 39.41,
42.03, 42.07, 46.27, 46.73, 47.62, 55.14, 60.52, 60.92, 61.81, 66.66,
69.09, 70.61, 70.91, 71.00, 72.70, 75.65, 75.95, 78.99, 85.62, 101.23,
121.35, 123.37, 144.53, 145.32, 169.05, 169.22, 169.68, 170.19, 170.24,
170.31, 170.52, 178.64. ESI-HRMS (m/z) [MþNa]þ calcd for
NMR (150 MHz, CD3OD):
d 15.90, 15.92, 16.34, 17.66, 19.45, 23.98,
24.02, 24.50, 26.44, 27.84, 28.45, 28.73, 31.61, 33.56, 33.75, 34.20,
35.04, 35.70, 38.11, 39.79, 39.82, 40.56, 42.52, 42.81, 47.47, 47.57,
56.67, 69.81, 70.69, 73.87, 78.59, 79.67, 90.15, 123.69, 124.12, 145.11,
146.15, 180.36. ESI-HRMS (m/z) [MþNa]þ calcd for C38H60N4O6Na,
691.4405; found, 691.4406.
C59H86N4O19Na, 1177.5778; found, 1177.5778.
4.2.20. Compound 18b
Prepared from 18a (80 mg, 0.07 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 18b as a white solid (52 mg,
4.2.17. Compound 17a
Prepared from OA-propargylamine (75 mg, 0.15 mmol), acetyl-
maltosyl azide (126 mg, 0.19 mmol) according to general proced-
ure A. The residue was purified by column chromatography (pe-
troleum ether/AcOEt, 1/2 v/v) to give 17a as a white solid (163 mg,
91%). Mp 190.5e192.0 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.60, 0.77,
0.91, 0.93, 0.94, 0.96, 1.15 (7 ꢂ CH3), 0.60e2.11 (m, other aliphatic
ring protons), 2.79 (dd, J ¼ 3.72, 13.38 Hz, 1H), 3.14 (dd, J ¼ 4.5,
11.34 Hz, 1H), 3.50 (dd, J ¼ 3.3, 9.72 Hz, 1H), 3.56e3.63 (m, 2H),
3.70e3.75 (m, 3H), 3.77e3.83 (m, 3H), 3.90e3.94 (m, 3H),
4.36e4.46 (m, 3H), 5.35 (t, J ¼ 3.36 Hz, 1H), 5.64 (d, J ¼ 9.18 Hz, 1H),
93%). Mp 153.9e154.5 ꢁC. 1H NMR (600 MHz, CDCl3):
d 0.66, 0.78,
0.87, 0.89, 0.98, 1.16 (7 ꢂ CH3), 0.66e1.80 (m, other aliphatic ring
protons), 1.85 (s, 3H), 2.02 (s, 3H), 2.04 (s, 6H), 2.07 (s, 3H), 2.11 (s,
3H), 2.14 (s, 3H), 2.53 (d, J ¼ 9.72 Hz, 1H), 3.21 (dd, J ¼ 3.96, 11.16 Hz,
1H), 3.49 (s, 1H), 3.98 (dd, J ¼ 2.28, 9.48 Hz, 1H), 4.06 (dd, J ¼ 1.56,
12.36 Hz, 1H), 4.14 (t, J ¼ 6.54 Hz, 1H), 4.23e4.27 (m, 3H), 4.48 (dd,
J ¼ 1.92, 12.42 Hz, 1H), 4.60 (dd, J ¼ 6, 15.18 Hz, 1H), 4.89 (dd, J ¼ 3.9,
10.5 Hz, 1H), 5.08 (t, J ¼ 9.9 Hz, 1H), 5.31e5.40 (m, 4H), 5.44e5.47
(m, 2H), 5.84 (d, J ¼ 10.98 Hz, 1H), 6.60 (t, J ¼ 5.28 Hz, 1H), 7.72 (s,
8.07 (s, 1H). 13C NMR (150 MHz, CD3OD):
d 16.01, 16.39, 17.63, 19.48,
24.04 (3C), 24.54, 26.46, 27.84, 28.47, 28.74, 31.60, 33.54, 33.74,
34.13, 35.03, 35.66, 38.11, 39.80, 39.83, 40.60, 42.51, 42.85, 47.51,
47.60, 56.68, 61.50, 62.51, 70.28, 72.52, 73.64, 74.79, 76.75, 77.15,
79.55, 79.67 (2C), 89.46, 105.11, 124.09, 124.21, 145.06, 145.92,
180.54. ESI-HRMS (m/z) [MþNa]þ calcd for C45H72N4O12Na,
883.5039; found, 883.5036.
1H); 13C NMR (150 MHz, CDCl3):
d 15.51, 15.68, 16.73, 18.38, 20.32,
20.71 (5C), 20.83, 20.90, 20.96, 23.60, 23.70 (2C), 24.04, 25.89,
27.25, 27.35, 28.18, 30.81, 32.38, 33.09, 34.19, 35.05, 37.04, 38.57,
38.86, 39.45, 42.06, 42.11, 46.31, 46.75, 47.64, 55.17, 61.55, 62.62,
68.00, 68.85, 69.32, 70.11, 71.07, 72.48, 75.25, 75.42, 79.03, 85.38,
96.00, 121.34, 123.38, 144.54, 145.38, 169.14, 169.56, 170.07, 170.13,
170.44, 170.66, 170.69, 178.65. ESI-HRMS (m/z) [MþNa]þ calcd for
4.2.21. Compound 19a
Prepared from OA-propargylamine (82 mg, 0.16 mmol), acetyl-
cellobiosyl azide (126 mg, 0.19 mmol) according to general pro-
cedure A. The residue was purified by column chromatography
(petroleum ether/AcOEt, 1/2 v/v) to give 19a as a white solid
(163 mg, 87%). Mp 143.7e145.2 ꢁC. 1H NMR (600 MHz, CDCl3):
C
59H86N4O19Na, 1177.5778; found, 1177.5778.
d
0.66, 0.78, 0.87, 0.89, 0.98, 1.16 (7 ꢂ CH3), 0.66e2.20 (m, other
4.2.18. Compound 17b
aliphatic ring protons), 1.86 (s, 3H), 1.99 (s, 3H), 2.02 (s, 3H), 2.05 (d,
6H), 2.11 (s, 3H), 2.12 (s, 3H), 2.53 (dd, J ¼ 3.33, 12.84 Hz 1H), 3.21
(dd, J ¼ 4.14, 11.28 Hz, 1H), 3.69e3.71 (m, 1H), 3.88e3.96 (m, 2H),
4.07 (dd, J ¼ 1.92, 12.5 Hz, 1H), 4.12 (dd, J ¼ 5.04, 12.36 Hz, 1H), 4.23
(dd, J ¼ 4.8, 15.12 Hz, 1H), 4.40 (dd, J ¼ 4.38, 12.6 Hz, 1H), 4.51 (d,
J ¼ 10.92 Hz, 1H), 4.56 (d, J ¼ 7.92 Hz, 1H), 4.60 (dd, J ¼ 6.06,
15.12 Hz, 1H), 4.95 (t, J ¼ 8.22 Hz, 1H), 5.09 (t, J ¼ 9.78 Hz, 1H), 5.17
(t, J ¼ 9.36 Hz, 1H), 5.37e5.43 (m, 3H), 5.77 (d, J ¼ 8.7 Hz, 1H), 7.73
Prepared from 17a (100 mg, 0.09 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 17b as a white solid (64 mg,
86%). Mp > 250 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.60, 0.77, 0.91,
0.93, 0.94, 0.96, 1.15 (7 ꢂ CH3), 0.60e2.11 (m, other aliphatic ring
protons), 2.79 (dd, J ¼ 3.72, 13.38 Hz, 1H), 3.14 (dd, J ¼ 4.5, 11.34 Hz,
1H), 3.50 (dd, J ¼ 3.3, 9.72 Hz,1H), 3.56e3.63 (m, 2H), 3.70e3.75 (m,
3H), 3.77e3.83 (m, 3H), 3.90e3.94 (m, 3H), 4.36e4.46 (m, 3H), 5.35
(t, J ¼ 3.36 Hz, 1H), 5.64 (d, J ¼ 9.18 Hz, 1H), 8.07 (s, 1H) 13C NMR
(s, 1H). 13C NMR (150 MHz, CDCl3):
d 15.49, 15.68, 16.68, 18.37, 19.39,
20.33, 20.57, 20.66, 20.81, 20.92, 23.59, 23.69, 24.04, 25.89, 27.23,
27.32, 28.17, 30.80, 32.33, 32.35, 33.08, 34.18, 35.02, 37.03, 38.55,
38.85, 39.43, 42.04, 42.07, 46.28, 46.74, 47.63, 55.15, 61.62, 61.69,
67.76, 70.54, 71.20, 71.63, 72.21, 72.43, 72.94, 75.89, 75.98, 79.02,
85.68, 100.96, 121.40, 123.38, 144.53, 145.33, 169.03, 169.23, 169.46,
169.74, 170.28, 170.36, 170.64, 178.70. ESI-HRMS (m/z) [MþNa]þ
calcd for C59H86N4O19Na, 1177.5778; found, 1177.5776.
(150 MHz, CD3OD):
d 16.01, 16.39, 17.63, 19.48, 24.04 (3C), 24.54,
26.46, 27.84, 28.47, 28.74, 31.60, 33.54, 33.74, 34.13, 35.03, 35.66,
38.11, 39.80, 39.83, 40.60, 42.51, 42.85, 47.51, 47.60, 56.68, 61.50,
62.51, 70.28, 72.52, 73.64, 74.79, 76.75, 77.15, 79.55, 79.67 (2C),
89.46, 105.11, 124.09, 124.21, 145.06, 145.92, 180.54. ESI-HRMS (m/z)
[MþNa]þ calcd for C45H72N4O12Na, 883.5039; found, 883.5038.

Y. Su et al. / European Journal of Medicinal Chemistry 182 (2019) 111622
9
4.2.22. Compound 19b
suspended in DMEM supplemented with 1% FBS, containing test
compound and 2 m g/mL TPCK-treated trypsin, and a final DMSO
concentration of 1% was added in each well. After 40 h of incuba-
tion, CellTiter-Glo reagent (Promega Corp., Madison, WI, USA) was
added, and the plates were read using a plate reader (Tecan Infinite
M2000 PRO; Tecan Group Ltd., Mannedorf, Switzerland).
Prepared from 19a (100 mg, 0.09 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 19b as a white solid (69 mg,
92%). Mp 199.6e201.8 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.61, 0.78,
0.91, 0.93, 0.94, 0.97, 1.16 (7 ꢂ CH3), 0.61e2.18 (m, other aliphatic
ring protons), 2.79 (d, J ¼ 9.48 Hz, 1H), 3.14 (dd, J ¼ 4.56, 11.34 Hz,
1H), 3.25e3.28 (m, 1H), 3.32e3.40 (m, 6H), 3.67e3.74 (m, 3H), 3.79
(t, J ¼ 9.30 Hz, 1H), 3.88e3.94 (m, 4H), 4.36 (d, J ¼ 15.18 Hz, 1H),
4.43e4.48 (m, 2H), 5.35 (t, J ¼ 3.42 Hz, 1H), 5.62 (d, J ¼ 9.24 Hz, 1H),
4.3.2. Cytotoxicity test
Cells were grown in 96-well plates containing 1% FBS overnight
and then cultured with increasing amounts of the test compounds
for 40 h. Cytotoxicity was assessed with the CellTiter-Glo assay as
above described.
8.00 (s, 1H). 13C NMR (150 MHz, CD3OD):
d 16.01, 16.40, 17.63, 19.48,
24.03 (2C), 24.05, 24.54, 26.46, 27.84, 28.46, 28.74, 31.61, 33.55,
33.74, 34.13, 35.04, 35.82, 38.11, 39.80, 39.83, 40.60, 42.51, 42.85,
47.50, 47.61, 49.85, 56.68, 61.45, 62.43, 71.33, 73.70, 74.88, 76.77,
77.82, 78.16, 79.52, 79.67, 89.26, 104.60, 123.77, 124.20, 145.07,
146.14, 180.50. ESI-HRMS (m/z) [MþNa]þ calcd for C45H72N4O12Na,
883.5039; found, 883.5031.
4.3.3. HI assay
Compound from a 2-fold serial dilution in salinewas mixed with
an equal volume of influenza virus (The HA titers of A/WSN/33
(H1N1) virus is 1:2 x (x¼1)) in the V-bottomed 96-well microplates.
Subsequently, 50 mL of freshly prepared chicken red blood cells
4.2.23. Compound 20a
(cRBC) (1% v/v in saline) was added to each well. The mixture was
incubated for 30 min at room temperature before observing cRBC
aggregation on the plate.
Prepared from OA-propargylamine (68 mg, 0.13 mmol), acetyl-
maltotriosyl azide (180 mg, 0.19 mmol) according to general pro-
cedure A. The residue was purified by column chromatography
(petroleum ether/AcOEt, 1/2 v/v) to give 20a as a white solid
(163 mg, 87%). Mp 146.0e147.7 ꢁC. 1H NMR (600 MHz CDCl3):
4.3.4. Docking simulation
Compound 11b was docked into the HA protein using AutoDock
4.2 [30]. The structural template of influenza HA (Protein Data
Bank: 1RVT [31]) was obtained from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do). To carry out blind
docking experiments, grids of points covering the whole HA head
and partial stem were generated with ADT [32]. Box size:
126 ꢂ 126 ꢂ 126 points with a standard space of 0.375 Å. Docking
simulations of the compound were carried out using the La-
marckian genetic algorithm and through a protocol with a number
of 50 GA runs, an initial population of 300 randomly placed in-
dividuals, a maximum number of 25 million energy evaluations, a
mutation rate of 0.02, and a crossover rate of 0.80. The resulting
conformations that differed by less than 2.0 Å in positional root-
mean-square deviation (rmsd) were clustered together. Other pa-
rameters were set as default. All the relevant torsion angles were
treated as rotatable during the docking process, thus allowing a
search of the conformational space. After running autogrid and
autodock, the possible poses of compounds in HA were obtained.
d
0.59, 0.74, 0.83, 0.85, 0.94, 1.11 (7 ꢂ CH3), 1.97, 1.98, 1.99, 2.00, 2.03,
2.04, 2.07, 2.13 (10 ꢂ CH3CO), 0.59e2.30 (m, other aliphatic ring
protons), 2.50 (d, J ¼ 9.9 Hz, 1H), 3.16e3.20 (m, 2H), 3.90e4.30 (m,
13H), 4.44 (d, J ¼ 11.04 Hz, 2H), 4.55 (dd, J ¼ 5.94, 15.18 Hz, 1H), 4.73
(dd, J ¼ 3.96, 10.32 Hz, 1H), 4.82 (dd, J ¼ 3.96, 10.5 Hz, 1H), 5.04 (t,
J ¼ 9.84 Hz, 1H), 5.27e5.44 (m, 8H), 5.82 (d, J ¼ 9.12 Hz, 1H), 6.69 (t,
J ¼ 5.4 Hz,1H), 7.74 (s, 1H). 13C NMR 150 MHz, CDCl3):
d 15.44,15.65,
16.62, 18.31, 20.26, 20.66, 20.75, 20.83, 20.88, 20.97, 23.52, 23.63,
23.86, 25.03, 25.84, 27.07, 27.11, 27.26, 28.12, 30.74, 32.31, 32.32,
33.04, 34.10, 34.90, 36.96, 38.50, 38.79, 39.37, 41.96, 42.02, 46.25,
46.65, 47.56, 50.22, 55.11, 61.41, 62.31, 62.73, 67.89, 68.56, 69.22,
69.38, 70.14, 70.47, 70.54, 71.03, 71.66, 72.47, 73.47, 75.01, 75.25,
79.01, 85.31, 95.71, 96.15, 121.57, 123.34, 144.34, 145.17, 169.18,
169.59, 169.84, 170.01, 170.04, 170.51, 170.53, 170.71, 170.73, 175.06,
178.77. ESI-HRMS (m/z) [MþNa]þ calcd for C71H102N4O27Na,
1465.6624; found, 1465.6621.
4.2.24. Compound 20b
Prepared from 20a (110 mg, 0.07 mmol) according to general
procedure B. The crude product was washed with water three times
without further purification to afford 20b as a white solid (54 mg,
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (grant no. 81560560) and Open-Fund Program
of the State Key Laboratory of Natural and Biomimetic Drugs (No.
K20170209).
73%). Mp 203.5e205.8 ꢁC. 1H NMR (600 MHz, CD3OD):
d 0.63, 0.78,
0.91, 0.93, 0.94, 0.97, 1.16 (7 ꢂ CH3), 0.63e2.14 (m, other aliphatic
ring protons), 2.80 (d, J ¼ 9.54 Hz, 1H), 3.14 (dd, J ¼ 4.5, 11.34 Hz,
1H), 3.28 (t, J ¼ 9.78 Hz, 1H), 3.45 (dd, J ¼ 3.72, 9.72 Hz, 1H),
3.51e3.93 (m, 17H), 4.36 (d, J ¼ 15.18 Hz, 1H), 4.44 (d, J ¼ 15.24 Hz,
1H), 5.16 (d, J ¼ 3.84 Hz, 1H), 5.25 (d, J ¼ 3.78 Hz, 1H), 5.35 (brs, 1H),
5.63 (d, J ¼ 9.06 Hz, 1H), 8.04 (s, 1H). 13C NMR (150 MHz, CD3OD):
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111622.
d
16.01, 16.36, 17.68, 19.49, 24.03 (2C), 24.05, 24.54, 26.45, 27.84,
28.48, 28.73, 31.61, 33.53, 33.75, 34.14, 35.04, 35.77, 38.11, 39.80,
39.83, 40.61, 42.50, 42.85, 47.51, 47.61 (2C), 56.69, 61.83, 62.13,
62.72, 71.46, 73.43, 73.61, 73.78, 74.24, 74.78, 74.93, 75.07, 78.12,
79.61, 79.68, 80.27, 81.32, 89.45, 102.75, 102.92, 123.89, 124.21,
145.07, 146.06, 180.57. ESI-HRMS (m/z) [MþNa]þ calcd for
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