Synthesis of novel pentacyclic triterpene-Neu5Ac2en derivatives and investigation of their: In vitro anti-influenza entry activity

Article abstract of DOI:10.1039/c7md00245a

Sialic acid derivatives, analogs, and their conjugates are important pharmacophores. Modification of the C-4 hydroxyl group of sialic acid can lead to derivatives, such as zanamivir, with potent anti-influenza activities. Herein, we described the synthesis of C-4-modified sialic acid derivatives via conjugation with naturally derived pentacyclic triterpenes, which are active ingredients of traditional Chinese medicine, and the evaluation of their in vitro anti-influenza virus activity in MDCK cells. Interestingly, a set of configurational isomers was obtained during the de-O-acetylation reaction of two pentacyclic triterpene-sialic acid conjugates under Zemplén conditions, and a mechanism was proposed. Owing to the attachment of the Neu5Ac2en moiety, all synthesized conjugates displayed lower hydrophobicity than their parent compounds. In comparison with ursane- and lupane-type triterpenes, oleanane-type triterpene-functionalized Neu5Ac2en conjugates were most promising. The insertion of a (1,2,3-triazol-4-yl)-methyl between the amide bond and Neu5Ac2en caused a substantial decrease in activity. Compound 15a exhibited the highest inhibitory activity (IC₅₀ = 8.3 μM) and selectivity index (SI = 22.7). Further studies involving hemagglutination inhibition and neuraminidase inhibition suggested that compound 15a inhibited virus-induced hemagglutination with no effect on the enzymatic activity of neuraminidase, indicating that the antiviral activity appeared to be mediated via interaction with hemagglutinin at the initial stage of viral infection.

Full text of DOI:10.1039/c7md00245a

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ARTICLE
Synthesis of novel pentacyclic triterpene-Neu5Ac2en derivatives
and investigation of their in vitro anti-influenza entry activity
Yongying Shi,a,† Longlong Si,a,† Xu Han,a,† Zibo Fan,a Shouxin Wang,a,c Man Li,a Jiaqi Sun,a Yongmin
Zhang,b Demin Zhoua and Sulong Xiaoa,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Dedicated to Professor Lihe Zhang on the Occasion of His 80th Birthday.
www.rsc.org/
Sialic acid derivatives, analogs and their conjugates are important pharmacophores. Modification of C-4 of sialic acid can
lead to derivatives with potent anti-influenza activities, such as Zanamivir. Herein, we described the synthesis of C-4
modified sialic acid derivatives by conjugation with naturally derived pentacyclic triterpenes, an active ingredient of
traditional Chinese medicine, and the evaluation of their in vitro anti-influenza virus activity in the MDCK cells.
Interestingly, a set of configurational isomers was obtained during the de-O-acetylation reaction of two pentacyclic
triterpene-sialic acid conjugates under Zemplén conditions and a mechanism was proposed. Due to the attachment of
Neu5Ac2en moiety, all synthesized conjugates showed lower hydrophobicity than their parent compounds. Compared
with ursane- and lupane-type triterpenes, oleanane-type triterpene functionalized Neu5Ac2en conjugates were the most
promising. The insertion of a (1,2,3-triazol-4-yl) methyl between the amide bond and Neu5Ac2en caused a substantial
decrease in activity. Compound 15a showed the highest inhibitory activity (IC50 = 8.3 μM) and selectivity index (SI = 22.7).
Further studies involving hemagglutination inhibition and neuraminidase inhibition suggested that compound 15a
inhibited virus-induced hemagglutination with no effect on the neuraminidase enzymatic activity, indicating that the
antiviral activity appears to be mediated by interaction with hemagglutinin at the initial stage of viral infection.
for interruption of specific processes in influenza infection.[5] However,
frequent clinical applications of the marketed anti-influenza drugs
combined with the high mutation rate of the RNA genome of the
1. Introduction
Influenza A virus, which causes a substantial number of deaths during
annual epidemics and occasional pandemics, belongs to the family of
single-stranded negative sense RNA viruses or Orthomyxoviridae.[1] The
most well-known influenza pandemics in the 20th century, “Spanish Flu
(1918/H1N1)”, “Asian Flu (1957/H2N2)” and “Hong Kong Flu
(1968/H3N2)”, resulted in the deaths of more than 50 million people
globally.[2] Up to now influenza viruses still infect 3 to 5 million people
worldwide every year.[3] Although anti-influenza vaccines are available,
their efficacy is limited due to the rapid mutation in the two surface
antigens (hemagglutinin (HA) and neuraminidase (NA)), leaving the
immune system unable to cope with the essentially new antigens.[4]
Currently, only two classes of anti-influenza drugs, including M2 ion-
channel inhibitors (amantadine and rimantadine) and NA inhibitors
(oseltamivir, zanamivir and peramivir), have been approved by the FDA
influenza virus lead to the rapid emergence of drug-resistant influenza
virus strains.[6] Therefore, there is a continuous and urgent need for
discovering new drugs to overcome resistance and looming threats of
sporadic outbreaks of pandemic influenza A strains.[7]
The first step in influenza A virus infection involves attachment to
cells through binding of viral HA to cell-surface receptors containing
sialic acid (also called α-5-N-acetylneuraminic acid, Neu5Ac), leading to
internalization of viral particles into endosome.[8] One approach to
combating cellular infection is the inhibition of the recognition process
by using sialic acid analogues that compete with the cell surface sialyl
oligosaccharides, for viral HA. Unfortunately, sialic acid and its
derivatives, such as α-methyl sialoside, bind only weakly to HA with
dissociation constants in the millimolar range.[9] Since the crystal
structure of the influenza virus HA complexed with its receptor was first
published by Weis et al. in 1988,[10] there have been many efforts to
search for sialic acid derivatives with high affinity for HA. Knowles’
group reported that the binding affinity of sialic acid can be increased
by introducing hydrophobic aglycons at C-2 position, with dissociation
constants in the micromolar range.[11] Rudrawar et al also reported that
the introduction of hydrophobic group at C-3 of Neu5Ac could lock
open the 150-loop of an influenza
A
virus group-1 sialidase.[12]
Therefore, four kinds of hydrophobic pentacyclic triterpene, which are
widely distributed in plant kingdom and generally believed to enhance
the immunity of host plants and increase plant resistance to
pathogens,[13] were selected to conjugate with sialic acid via C-2 in our
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previous study.[14] However, those derivatives only showed weak anti-
influenza entry activity with IC50 ranging from 41.2 to 95.2 μM, possibly
due to the fact that when the sialic acid groups bind to the HA polymer
backbone, the linking groups are brought too close to the HA surface.
The structures of influenza HA complexed with several different 2-
substituted sialic acid derivatives, such as Neu5Ac2N4 and Neu5Ac2N6,
have been determined by X-ray crystallography.[15] In all cases the 4-OH
of sialic acid points away from the binding site into solution and does
not have any interactions with the HA protein, indicating that 4-
modified sialosides would not interfere with the binding to HA. For
example, Wu et al.[16] reported that a polymeric 4-N-linked sialoside
strongly inhibited influenza virus HA activity with Ki of 10-6 M. Moreover,
the addition of certain functionalities at the C-4 position of unsaturated
sialic acid analog, such as 4-amino Neu5Ac2en and 4-guanidino
Neu5Ac2en, should enhance its binding with the influenza virus
neuraminidase (NA).[17] There have been many efforts to develop anti-
influenza inhibitors based on the Neu5Ac2en.[18] However, the report of
C-4 triazole modified analogs of Neu5Ac2en is rare. In 2006, Li et al.
reported the synthesis of C-4 triazole analogs of Zanamivir and found
one derived from 3-hydroxy pentyne showing 61% protective activity
(50 μM) against infection by avian influenza virus.[19] One year later, Lu
et al. reported the synthesis of C-4 triazole analogs of Neu5Ac2en
derivatives as multivalent sialic acid containing scaffolds.[20]
Ac2O, pyridine, DMAP; (d) TMSOTf, EtOAc; (e) Me3SiN3, t-BuOH; (f)
CuSO4, sodium L-ascorbate, DCM/H2O (1:1, v/v).
Neu5Ac2en 7 was synthesized from commercially available sialic acid 4
in 48% yield through a three-step process described by Chandler and
co-workers.[23] The structures of compounds 6 and 7 were confirmed by
1H and 13C NMR (Supporting Information). Subsequent conjugation of
3a-c with 7 via copper catalyzed azide-alkyne 1,3-dipolar cycloaddition
reaction (CuAAC) provided the 5,7,8,9-tetra-O-acetyl protected
intermediates 8a-c with good yield. In a similar way, conjugation of 3d
with 7 afforded intermediate 8d in 87% yield.
As an initial experiment, de-O-acetylation of 8a was carried out under
Zemplén conditions.[24] However, two compounds, 9a and its isomer
10a, were separated by standard silica gel chromatography in 52% and
31% yield, respectively (Scheme 2). We observed that 9a was more
polar than its isomer 10a (based on silica gel migratory ability). The
structures of the two isomers have been fully ascertained by ESI-HRMS
and NMR spectroscopy. Figure 1 (A) represents the characteristic parts
(δ = 3.5-6.5 ppm) of the 1H NMR spectra (in CD3OD) of conjugates 9a
and 10a. The chemical shifts of the H3 and H5 protons of 10a were
significantly shifted to the lower field compared to its isomer 9a. In
addition, the coupling constant between H4 and H5 of 9a was 9.8 Hz
when these two protons are trans, while this coupling constant was
close to 5.4 Hz when they are cis. To further demonstrate the
configurations of the two isomers 9a and 10a, 2D NOE spectra were
also acquired (Figure 1 (B)). The NOE experiment of compound 9a
showed a NOE effect between the H4 proton and the H6 protons of the
carbohydrate ring, while the absence of this effect in its isomer 10a. On
the contrary, an NOE effect of 10a between H4 proton and H5 protons of
the carbohydrate ring was observed obviously, while the absence of this
effect in its isomers 9a. Therefore, we concluded that the H4 of 9a and
10a are β-orientation and α-orientation, respectively. Similar results
were observed for the de-O-acetylation of 8b under Zemplén conditions
and two isomers 9b and 10b were also obtained. To the best of our
knowledge, there are no other reports about this reaction. We wonder
whether the triazolyl group facilitates the formation of these isomers.
Therefore, de-O-acetylation of 7 was carried out under the same
condition (Scheme 3). We found that the reaction finished quickly as
shown by TLC analysis, yielding azido compound 11 exclusively in 88%
yield. Subsequent direct conjugation with alkynyl-functionalized
pentacyclic triterpene derivatives 3c-d in a similar manner as described
in Scheme 1 afforded conjugates 9c-d with high yields.
Our recent works on the anti-influenza virus activities of pentacyclic
triterpene,[14,21] along with published reports,[15,17,19] lead us to further
explore novel sialic acid derivatives in which petacyclic triterpenes are
attached at the 4-position of sialic acid. To ensure a stable linkage to
the 4-hydroxy group under assay conditions, a triazolyl or amide bond
was introduced as linkage between the two moieties. Interestingly, a
set of configurational isomers was obtained during the de-O-acetylation
reaction of 8a and 8b under Zemplén conditions. In addition, a five-fold
more potent anti-influenza activity was found for compound 15 with
better selectivity index (SI
= 22.7) when conjugating pentacyclic
triterpene to sialic acid via C-4 position. Herein we describe the
synthesis and in vitro anti-influenza virus activity of those novel
pentacyclic triterpene-Neu5Ac2en conjugates
2. Results and discussion
2.1 Chemistry
The target pentacyclic triterpene-Neu5Ac2en conjugates were
synthesized as described in Scheme 1-5. The pentacyclic triterpenes
used to conjugate with Neu5Ac2en included oleanolic acid (OA, 1a),
ursolic acid (UA, 1b), echinocystic acid (EA, 1c) and betulinic acid (BA,
1d). Scheme 1 depicts the synthesis of conjugates 8a-d. Firstly, alkynyl-
functionalized pentacyclic triterpene derivatives 3a-d were prepared
according to published procedures.[22] The key intermediate 4-azido-
Scheme 2. Reagents and conditions: (a) MeONa/MeOH.
R1
R1
R1
OAc
OAc
R3
R3
R3
AcO
H
R1
O
N
N
AcHN
N
R3
O
COOH
R2
N
N
N
a
b
O
O
N
COOMe
R2
R2
H
N
HO
HO
HO
f
O
2a: R1
2b: R1 = H, R2 = H, R3
2c: R1
=
CH3
,
R2
=
H, R3
=
=
CH3
H
3a
1a: R1
1b: R1
1c: R1
=
=
=
CH3
,
R2
=
H, R3
=
H
: R1
3b
=
=
CH3
, R2 = H, R3 = H
R2
H, R2 = H, R3 = CH3
: R1
H, R2 = H, R3 = CH3
HO
= CH3, R2 = OH, R3 = H
3c: R1 = CH3, R2 = OH, R3
= H
CH3
O
,
R2 = OH, R3 = H
8a: R1
8b: R1
=
=
CH3
, R2 = H, R3 = H
H, R2
=
R2
H, R3 = CH3
OH
OAc
OAc
AcO
HO
AcO
8c: R1 = CH3
,
=
OH, R3
= H
AcO
AcO
COOMe
OAc
COOH
c
O
COOMe
AcO
O
e
COOMe
O
d
N
HO
AcHN
AcO
OAc
AcO
AcHN
O
OH
AcHN
OAc
N3
HO
AcO
AcO
AcHN
N
6
7
4
5
O
f
N
N
COOMe
H
N
H
N
O
N
N
b
a
N
COOH
O
O
O
8d
HO
HO
HO
HO
3d
2d
1d
Scheme 1. Reagents and conditions: (a) TBTU, DIPEA, THF; (b)
propargylamine, K2CO3, DMF; (c) H+-exchange resin, RT, MeOH; then
2 | J. Name., 2012, 00, 1-3
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Figure 1. (A) Characteristic portions of the 400-MHz proton spectra of
conjugates 9a (red) and 10a (blue). (B) Section of NOESY spectra of
Scheme 5. Reagents and conditions: (a) PPh3, THF, H2O; (b) EDC,
Na2CO3, DMF, 2a, 2b or 2c, 60oC; (c) EDC, Na2CO3, DMF, 2d, 60oC;
(d) MeONa/MeOH.
Table 1. Calculated AlogP values of pentacyclic triterpene-Neu5Ac2en
conjugates.[a]
compounds 9a and 10a in CD3OD at 25oC.
Compound
AlogP
Compound
AlogP
Compound
AlogP
OA
UA
EA
BA
8a
8b
8c
6.447
6.492
5.345
6.546
4.756
4.802
3.654
4.856
9a
9b
9c
3.619
3.664
2.517
3.718
3.619
3.664
4.775
4.821
15c
15d
16a
16b
16c
16d
3.673
4.874
3.638
3.683
2.536
3.737
Scheme 3. Reagents and conditions: (a) MeONa/MeOH; (b) CuSO4,
sodium L-ascorbate, 3c, THF/H2O (1:1, v/v); (c) CuSO4, sodium L-
ascorbate, 3d, THF/H2O (1:1, v/v).
9d
10a
10b
15a
15b
Based on these results, we propose a plausible mechanism for the
formation of the two isomers (Scheme 4). The first step is the removal
of the acetate protecting groups by sodium methoxide in methanol to
afford triol 9. Removal of H4 hydrogen by methoxide forms an allylic
carbanion, which is further stabilized by resonance with an extended
enolate to form the intermediate 13. Racemization occurs when a
proton attacks from upper or down side of the planar carbanion to
generate 9 or 10, respectively.
8d
[a] AlogP values (Ghose and Crippen octanol-water partition coefficient
at 25°C) were calculated using Pipeline Pilot software, version 7.5
(Accelrys Corp., San Diego, CA, USA).
Neu5Ac2en derivatives were calculated using Pipeline Pilot software
version 7.5 (Accelrys Corp., San Diego, CA, USA) and the results are
presented in Table 1. All the studied conjugates showed decreased
hydrophobicity compared with their parent compounds, with AlogP
values within the range of 2.52-4.86. Benefiting from the hydrophilic
Neu5Ac2en moiety, the AlogP value decreased about 2.8 for each series
(for example, OA vs 9a and 16a), which means that their solubility in
water are about 630–fold higher than their parent compounds.
2.3 Cytotoxic activity
Alternatively, reduction of the azide group in 7 was carried out
through treatment with Ph3P in THF/H2O (1:1) to yield intermediate 14,
which was condensed with intermediate 2a-d in the presence of EDC to
yield 15a-d in 62-75% yields. Followed by de-O-acetylation under
Zemplén conditions, 16a-d were afforded quantitatively (Scheme 5).
2.2 Calculated AlogP
Lipophilicity governs the interaction of a given molecule with the
intestinal membrane. A compound must possess hydrophobic
properties for passive transport across intestinal epithelia. It is
known that pentacyclic triterpenes are hydrophobic molecules with
relative high lipophilicity. The different substituents such as the
sugar part at C3-OH and/or C28-COOH contribute to different
degrees of lipophilicity. In this study, the AlogP of pentacyclic
triterpene-
After completion of the synthesis, all synthesized conjugates were
evaluated for their in vitro anti-influenza activity against
A/WSN/33 (H1N1) virus in the MDCK cell line. To exclude the
possibility that the observed anti-influenza virus activity is due to
non-specific cytotoxic activity, preliminary screening of the
synthesized conjugates 8a-d, 9a-d, 10a-b, 15a-d and 16a-d was
performed to evaluate their cytotoxic activity in MDCK cells based
on CellTiter-Glo assay.[21] The commercial antitumor agent
paclitaxel (PTX) was used as a positive control. The results of the
cytotoxicity test at two different concentrations (10 μM and 100
μM) are shown in Figure 2. No significant cytotoxicity was
observed at concentration below 10 μM. However, compounds
8a-b
8c
,
8d and 16a-b showed strong cytotoxicity while compounds
9b and 10b showed moderate cytotoxicity at high
,
concentration (100 μM). Except compound 15b, most of the UA-
Neu5Ac2en conjugates showed strong (8b and 16b) to middle (9b
Scheme 4. Proposed mechanism for the formation of the two
isomers during de-O-acetylation reaction under Zemplén
conditions.
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and 10b) cytotoxicity at the same concentration. Two
Figure 3. Compound 15a inhibited virus-induced CPE in MDCK cells. The
antiviral efficacy of 15a was observed in terms of cellular morphology at
40 h post-infection.
Figure 2. The cytotoxicity screening of pentacyclic triterpene-
Neu5Ac2en conjugates 8a-d, 9a-d, 10a-b, 15a-d and 16a-d using
CellTiter-Glo® Assay. DMSO and paclitaxel were used as negative and
positive control, respectively. Error bars indicate standard deviations of
In addition, CPE in the virus-infected cells were microscopically
observed. Morphologically, the influenza virus-infected cells showed a
rounded-up appearance and detached from the dish in the absence of
15a at 40 h post-infection. Compound 15a inhibited the virus-induced
CPE significantly and no cytotoxicity was morphologically observed at a
concentration up to 100 μM (Figure 3).
triplicate experiments
.
BA-Neu5Ac2en conjugates
(
8a and 16a) also showed strong
cytotoxicity to MDCK cells at high concentration (100 μM).
2.4 In vitro anti-influenza virus activity in MDCK cells
2.5 Identifying HA as the potential target of compound 15a
Except for compounds 8a-b, 8d and 16a-b with strong cytotoxic
As described in our previous study,[14,21] we found that the molecular
basis of the anti-influenza activity of pentacyclic triterpenes is likely due
to their high affinity to HA protein, which is a surface glycoprotein of
influenza virus and essential for viral attachment to host cells. In this
study, a hemagglutination inhibition (HI) assay was performed to
evaluate the potential interaction of the envelope HA protein with 15a.
A three-fold serial dilution of compound 15a from 40 to 1.5 μM was
activity as described above, the other 13 pentacyclic triterpene-
Neu5Ac2en conjugates were evaluated against the influenza virus
strain A/WSN/33 (H1N1) that was propagated in MDCK cells by the
cytopathic effect (CPE) reduction assay.[25] Curcumin, a small-
molecule entry inhibitor targeting the HA1 domain,[25-26] was
utilized as positive controls. The concentrations required to inhibit
viral replication by 50% (IC50) are summarized in Table 2. We also added. As expected, no hemagglutination was elicited by compound
15a alone (lower row, Figure 4) and the virus caused hemagglutination
(lane 1, upper row, Figure 4). However, like anti-HA antibody,
compound 15a inhibited the hemagglutination of red blood cells (RBCs)
caused by the influenza virus at the concentration from 40 to 1.5 μM
(lane 2-5, upper row, Figure 4), which implied that 15a and anti-HA
antibody had the same target, HA, and the inhibition of viral infection
might be resulted from the inhibition of the hemagglutinin-sialic acid
receptor interaction.
determined the cytotoxic effects by CellTiter-Glo assay and the
cytotoxicity of each compound was expressed as the
concentration required to induce 50% cell death (CC50) of the
MDCK cells. As shown in Table 2, we found that: 1) Compounds
15a and 15c exhibited appreciated effect against influenza virus
A/WSN/33
with IC50 at 8.3 and 15.5 μM (SI > 22.7), respectively, which were more
potent or comparable to that of the diarylheptanoid anti-influenza
entry inhibitor curcumin. 2) Conjugation of Neu5Ac2en with oleanane-
type pentacyclic triterpene was more effective than with ursane- or
lupine-type pentacyclic triterpene. Most of the conjugates with ursolic
acid and betulinic acid scaffold showed no anti-influenza activity but
cytotoxicity except compound 15b, which showed potent activity
On the other hand, Neu5Ac2en was found to be an influenza NA
inhibitor with a Ki of 4 μM in earlier studies.[27] To examine
whether the designed pentacyclic triterpene-Neu5Ac2en
derivatives inhibit NA enzymatic activity, untreated or compound
15a-treated influenza A/WSN/33 virus was tested for enzymatic
against A/WSN/33 virus with an IC50 of 19.2 μM. 3) The insertion of a activity by 4-methylumbelliferyl-α-D-N-acetylneuraminic acid
(1,2,3-triazol-4-yl) methyl between the amide bond and Neu5Ac2en
caused a substantial decrease in activity (eg. 15c vs 8c, 16c vs 9c),
indicating the linker between pentacyclic triterpene and Neu5Ac2en
played an important role in their antiviral activity. 4) Compared with our
previous study, the anti-influenza activities of the conjugates of sialic
acid to pentacyclic triterpene via C-4 position were almost 5-fold more
potent than those linked via C-2 position (IC50: 8.3 μM vs 41.2 μM[14]).
sodium salt hydrate solution (MUNANA). Zanamivir, a derivative of
Neu5Ac2en by introduction of a guanidine group linked to C-4,
was used as
a positive control. Compared with Zanamivir,
compound 15a showed no inhibition of the enzymatic activity of
influenza A/WSN/33 virus (SI Figure 1), implying that NA is not a
target of compound 15a in its inhibition of viral infection
3. Experimental
Table 2. In vitro anti-influenza virus activity and cytotoxicity of the active
compounds
3.1 Chemistry
Compd
8c
9a
IC50 (µM)a CC50(µM)b
SIc
Compd
15a
15b
15c
15d
16c
16d
curcumin
IC50 (µM)
CC50 (µM)
SI
All chemicals were used as supplied without further purification. The
synthesis of compounds 2a-d, 3a-d, 4-7 and 14 have been reported
previously.[22b,28] High-resolution mass spectra (HRMS) were obtained
with an APEX IV FT_MS (7.0T) spectrometer (Bruker) in positive ESI
mode. NMR spectra were recorded on a Bruker DRX 400 spectrometer
>100
28.3±2.6
>100
ND
>200
ND
>200
ND
ND
>7.1
ND
>3.6
ND
8.3±1.3
19.2±3.5
15.5±0.5
>100
40.5±3.9
>100
188.1±10.1 22.7
135.9±6.3
>200
ND
>200
7.1
>32.3
ND
>4.9
ND
9b
9c
9d
55.0±4.1
>100
10a
10b
>100
>100
ND
ND
ND
ND
ND
48.3±5.7
1
6.7±1.2
7.2
at ambient temperature. H NMR chemical shifts are referenced to the
internal standard TMS (δH = 0.00) or the solvent signal (δH = 3.31 for the
central line of CD3OD). 13C NMR chemical shifts are referenced to the
[a] Concentration inhibiting viral replication by 50%. The values are means of at
least three independent determinations; the corresponding standard deviations
are noted. [b] 50% cytotoxicity concentration. [c] Selectivity index, defined by
CC50/IC50
.
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0.78, 0.61 (s, 3H each, 2 × CH3), 0.71 (d, 1H, J = 11.6 Hz); 13C NMR (100
MHz, CDCl3): δ 178.50, 170.70, 170.44, 170.28, 161.28, 146.14, 145.40,
139.27, 126.10, 121.96, 106.85, 78.99, 77.02, 71.38, 67.92, 62.26, 58.35,
55.17, 53.50, 52.77, 48.39, 47.69, 47.56, 42.40, 39.76, 39.58, 39.02,
38.82, 38.66, 37.21, 36.97, 35.12, 32.78, 30.92, 28.22, 27.86, 27.25,
25.02, 23.39, 22.94, 21.28, 21.04, 20.86, 20.79, 18.31, 17.26, 16.75,
15.75, 15.57; ESI-HRMS calcd for C51H76N5O12 [M+H]+: 950.5485; found
950.5493.
Figure 4. Comparison of the behaviors of 15a vs. anti-HA antibody in
inhibition of influenza virus-induced aggregation of chicken
erythrocytes. 15a exerted identical capability as anti-HA antibody in
hemagglutination inhibition in a dose-dependent manner.
solvent signal (δC= 77.00 for the central line of CDCl3, δC= 49.00 for the
central line of CD3OD). Reactions were monitored by thin-layer
chromatography (TLC) on a precoated silica gel 60 F254 plate (layer
thickness 0.2 mm; E. Merck, Darmstadt, Germany) and detected by
staining with a yellow solution containing Ce(NH4)2(NO3)6 (0.5 g) and
(NH4)6Mo7O24.4H2O (24.0 g) in 6% H2SO4 (500 mL), followed by heating.
Flash column chromatography was performed on silica gel 60 (200-300
mesh, Qingdao Haiyang Chemical Co. Ltd.). The calculated AlogP and
water solubility values were determined using Pipeline Pilot software,
Vers. 7.5 (Accelrys Corporation, San Diego, CA, USA)
3.1.5 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β,16α-dihydroxy-
olean-12-en-28-oyl)amino] methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-
(1,2,3-triacetoxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic
acid
methyl ester (8c). Prepared from 3c and 7 according to general
procedure A, and the residue was purified by chromatography (eluent:
PE/Act = 3:2) over silica gel to afford compound 8c as a white solid in
1
91% yield. Rf = 0.30 (PE/Act = 3:2); H NMR (400 MHz, CDCl3): δ 7.74 (s,
1H), 7.49 (d, 1H, J = 8.2 Hz), 7.09 (br s, 1H), 6.04 (s, 1H), 5.68 (br s, 1H),
5.57 (d, 1H, J = 2.3 Hz), 5.54 (br t, 1H), 5.36-5.35 (m, 1H), 4.81 (d, 1H, J =
10.8 Hz), 4.66 (d, 1H, J = 10.5 Hz), 4.57 (dd, 1H, J = 9.5, 4.3 Hz), 4.46-
4.39 (m, 1H), 4.29 (d, 1H, J = 12.6 Hz), 4.25-4.20 (m, 2H), 3.82 (s, 3H),
3.24 (br s, 2H), 2.88 (d, 1H, J = 12.4 Hz), 2.14 (t, 1H, J = 13.1 Hz), 2.09 (s,
3H, CH3CO), 2.07 (s, 6H, 2 × CH3CO), 1.91-1.90 (m, 3H), 1.68-0.98 (m,
other aliphatic ring protons), 1.72 (s, 3H, CH3CO), 1.33, 0.99, 0.92, 0.90,
0.89, 0.79, 0.70 (s, 3H each, 7 × CH3), 0.74 (d, 1H, J = 11.6 Hz); 13C NMR
(100 MHz, CDCl3): δ 178.31, 170.76, 170.64, 170.60, 170.05, 161.06,
146.00, 145.57, 142.99, 123.25, 121.51, 106.75, 78.77, 77.32, 74.34,
71.75, 67.85, 62.09, 58.69, 55.18, 52.66, 49.27, 47.70, 46.77, 46.37,
41.84, 41.34, 39.69, 38.69, 38.50, 36.85, 35.55, 34.98, 34.65, 32.43,
29.97, 27.99, 27.11, 26.90, 25.86, 23.27, 22.60, 20.91, 20.73, 20.68,
18.11, 17.00, 15.61, 15.56; ESI-HRMS calcd for C51H76N5O13 [M+H]+:
966.5434; found 966.5440.
3.1.1 General procedure A for the CuAAC reaction. To a solution of
alkyne (0.45 mmol) and azide (0.30 mmol) in DCM/H2O (1:1 v/v, 12 mL)
was added CuSO4 (48 mg, 0.30 mmol) and sodium ascorbate (119 mg,
0.60 mmol). The resulting solution was stirred vigorously for 12 hours at
room temperature. The reaction mixture was extracted with DCM (3 ×
10 mL). The combined organic layer was dried over Na2SO4, filtered and
concentrated. The residue was purified by column chromatography
over silica gel.
3.1.2 General procedure B for the de-O-acetylation reaction. The
per-O-acetylated Neu5Ac2en pentacyclic triterpene conjugate was
dissolved in MeOH (~5 mL per 100 mg compound) and a solution of
MeONa (30% in MeOH, 0.1 equiv [mol acetate]-1) was added. The
solution was stirred at room temperature for 3 hours. After completion
(TLC), the reaction mixture was neutralized with Amberlite IR-120 (H+)
ion-exchange resin, filtered and concentrated. The crude product was
3.1.6 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β-hydroxy-lup-20(29)-
en-28-oyl)amino]methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-(1,2,3-
triacetoxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl
ester (8d). Prepared from 3d and 7 according to general procedure A,
and the residue was purified by chromatography (eluent: PE/Act = 3:2)
over silica gel to afford compound 8d as a white solid in 87% yield. Rf =
0.35 (PE/Act = 3:2); 1H NMR (400 MHz, CDCl3): δ 7.73 (s, 1H), 6.90 (s,
1H), 6.69 (s, 1H), 6.00 (s, 1H), 5.79 (s, 1H), 5.54 (d, 1H, J = 4.2 Hz), 5.38
(br t, 1H), 4.78 (d, 1H, J = 9.4 Hz), 4.71-4.74 (m, 2H), 4.59 (s, 1H), 4.45
(br s, 2H), 4.24-4.19 (m, 2H), 3.81 (s, 3H), 3.19-3.17 (m, 1H), 3.08 (t, 1H,
J = 8.3 Hz), 2.45 (t, 1H, J = 10.5 Hz), 2.09 (s, 3H, CH3CO), 2.06 (s, 6H, 2 ×
CH3CO), 2.00 (d, 1H, J = 12.8 Hz), 1.80 (s, 3H, CH3CO), 1.79-0.88 (m,
other aliphatic ring protons), 1.67, 0.96, 0.95, 0.87, 0.80, 0.76 (s, 3H
each, 6 × CH3); 13C NMR (100 MHz, CDCl3): δ 176.89, 170.75, 170.66,
170.50, 170.27, 161.18, 150.78, 146.26, 121.95, 109.60, 106.77, 79.03,
76.84, 71.25, 67.88, 62.15, 58.30, 55.72, 55.44, 52.83, 50.66, 50.11,
48.73, 46.77, 42.52, 40.85, 38.93, 38.80, 38.34, 37.80, 37.25, 34.88,
34.49, 33.49, 30.88, 29.52, 28.10, 27.48, 25.68, 22.99, 21.05 (2C), 20.87,
20.80, 19.49, 18.36, 16.28, 16.22, 15.49, 14.72; ESI-HRMS calcd for
purified by column chromatography over silica gel.
3.1.3 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β-hydroxy-olean-12-
en-28-oyl)amino]methyl-[1,2,3]triazol-1-yl)]-6(S)-((1R,2R)-(1,2,3-
triacetoxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl
ester (8a). Prepared from 3a and 7 according to general procedure A,
and the residue was purified by chromatography (eluent: PE/Act = 3:2)
over silica gel to afford compound 8a as a white solid in 93% yield. Rf =
1
0.33 (PE/Act = 3:2); H NMR (400 MHz, CDCl3): δ 7.79 (s, 1H), 7.31-7.33
(m, 1H, overlap with CDCl3), 6.79 (br s, 1H), 6.03 (s, 1H), 5.69 (d, 1H, J =
9.6 Hz), 5.54 (d, 1H, J = 4.4 Hz), 5.37-5.36 (m, 2H), 4.71 (td, 2H, J = 12.4,
1.9 Hz), 4.46 (dd, 1H, J = 15.1, 5.2 Hz), 4.38-4.31 (m, 2H), 4.21 (dd, 1H, J
= 12.4, 7.3 Hz), 3.82 (s, 3H), 3.23-3.18 (m, 1H), 2.56 (d, 1H, J = 10.5 Hz),
2.09 (s, 3H, CH3CO), 2.06 (2 × s, 6H, 2 × CH3CO), 2.02-0.92 (m, other
aliphatic ring protons), 1.77 (s, 3H, CH3CO), 1.15, 0.98, 0.90, 0.88, 0.87,
0.78 (s, 3H each, 6 × CH3), 0.72 (d, 1H, J = 11.2 Hz), 0.57 (s, 3H, CH3); 13
C
NMR (100 MHz, CDCl3): δ 178.61, 170.65, 170.41, 170.31, 170.23,
161.24, 146.11, 145.39, 144.35, 123.15, 122.21, 106.82, 78.91, 77.08,
71.38, 67.85, 62.24, 58.44, 55.12, 52.72, 48.21, 47.53, 46.67, 46.22,
41.95, 41.92, 39.36, 38.79, 38.48, 36.96, 35.13, 34.14, 33.03, 32.64,
32.37, 30.74, 28.16, 27.27, 27.18, 25.85, 23.94, 23.63, 23.48, 22.83,
21.00, 20.83, 20.75, 18.29, 16.68, 15.70, 15.41; ESI-HRMS calcd for
C51H75N5NaO12 [M+Na]+: 972.5304, found 972.5309.
C
51H75N5NaO12 [M+Na]+: 972.5304, found 972.5298.
3.1.7 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β-hydroxy-olean-12-
en-28-oyl)amino]methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-(1,2,3-
trihydroxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl
ester (9a) and 5-acetylamino-4(R)-[4-[(3β-hydroxy-olean-12-en-28-
oyl)amino]methyl-[1,2,3]triazol-1-yl]]6(S)-((1R,2R)6-(1,2,3-trihydroxy-
propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl ester (10a).
Prepared from 8a according to general procedure B, and the residue
was purified by chromatography (eluent: DCM/MeOH = 10:1) over silica
gel to afford compound 9a and 10a as white solids in 52% and 31% yield,
respectively. Compound 9a: Rf = 0.18 (DCM/MeOH = 10:1); 1H NMR
(400 MHz, CD3OD): δ 7.82 (s, 1H), 6.01 (d, 1H, J = 2.4 Hz), 5.61 (dd, 1H, J
= 9.8, 2.2 Hz), 5.33 (t, 1H, J = 3.4 Hz), 4.56 (d, 1H, J = 10.9 Hz), 4.46-4.33
(m, 3H), 3.95-3.90 (m, 1H), 3.85-3.81 (m, 4H), 3.68 (dd, 1H, J = 11.5, 6.2
Hz), 3.65 (d, 1H, J = 8.7 Hz), 3.13 (dd, 1H, J = 11.4, 4.5 Hz), 2.80 (dd, 1H, J
= 13.0, 3.6 Hz), 2.06 (dt, 1H, J = 13.6, 3.9 Hz), 1.89-1.85 (m,3H), 1.87 (s,
3H, CH3CO), 1.77 (t, 1H, J = 13.7 Hz), 1.62-0.95 (m, other aliphatic ring
protons), 1.15, 0.96, 0.94 (s, 3H each, 3 × CH3), 0.91 (s, 6H, 2 × CH3),
0.77 (s, 3H, CH3), 0.73 (d, 1H, J = 11.1 Hz), 0.53 (s, 3H, CH3); 13C NMR
3.1.4 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β-hydroxy-urs-12-en-
28-oyl)amino]methyl-[1,2,3]triazol-1-yl)]-6(S)-((1R,2R)-(1,2,3-
triacetoxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl
ester (8b). Prepared from 3b and 7 according to general procedure A,
and the residue was purified by chromatography (eluent: PE/Act = 3:2)
over silica gel to afford compound 8b as a white solid in 93% yield. Rf =
0.35 (PE/Act = 3:2); 1H NMR (400 MHz, CDCl3): δ 7.74 (s, 1H), 7.18 (d, 1H,
J = 8.9 Hz), 6.76 (brs, 1H), 6.03 (d, 1H, J = 1.6 Hz), 5.70 (d, 1H, J = 9.5 Hz),
5.53 (dd, 1H, J = 4.7, 1.3 Hz), 5.33-5.38 (m, 2H), 4.70-4.74 (m, 2H), 4.45
(dd, 1H, J = 14.6, 5.2 Hz), 4.27-4.34 (m, 2H), 4. 20 (dd, 1H, J = 12.5, 7.2
Hz), 3.82 (s, 3H), 3.20-3.22 (m, 1H), 2.09, 2.07, 2.06 (s, 3H each, 3 ×
CH3CO), 1.92-0.97 (m, other aliphatic ring protons), 1.78 (s, 3H, CH3CO),
1.09, 0.99, 0.94, 0.89 (s, 3H each, 4 × CH3), 0.86 (d, 3H, CH3, J = 6.3 Hz),
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(100 MHz, CD3OD): δ 180.36, 173.76, 163.51, 147.58, 146.68, 145.07, (eluent: DCM/MeOH = 10:1) over silica gel to afford compound 9c as a
124.13, 123.29, 107.29, 79.67, 78.44, 71.16, 69.71, 64.78, 59.95, 56.70, white solid in 85% yield. Rf = 0.15 (DCM/MeOH = 10:1); 1H NMR (400
53.07, 50.09, 48.96, 47.63, 47.51, 42.86, 42.52, 40.63, 39.84, 38.10, MHz, CD3OD): δ 7.82 (s, 1H), 7.57 (t, 1H, J = 5.2 Hz), 5.99 (d, 1H, J = 2.4
35.86, 35.08, 34.21, 33.79, 33.54, 31.61, 28.74, 28.46, 27.85, 26.39, Hz), 5.63 (dd, 1H, J = 9.8, 2.2 Hz), 5.45 (br t, 1H), 4.56 (d, 1H, J = 10.9 Hz),
24.51, 24.03, 22.55, 19.45, 17.75, 16.34, 15.97; ESI-HRMS calcd for 4.46-4.30 (m, 4H), 3.92 (ddd, 1H, J = 8.4, 5.1, 2.8 Hz), 3.85-3.81 (m, 4H),
C
45H70N5O9 [M+H]+: 824.5168; found 824.5174. Compound 10a: Rf
=
3.68 (dd, 1H, J = 11.6, 5.3 Hz), 3.66 (d, 1H, J = 9.4 Hz), 3.14 (dd, 1H, J =
1
0.21 (DCM/MeOH = 10:1); H NMR (400 MHz, CD3OD): δ 7.80 (t, 1H, J = 11.0, 4.9 Hz), 2.88 (dd, 1H, J = 13.6, 3.3 Hz), 2.35 (t, 1H, J = 13.2 Hz),
5.6 Hz), 7.77 (s, 1H), 6.11 (d, 1H, J = 5.4 Hz), 5.58 (t, 1H, J = 5.4 Hz), 5.33 1.98-1.89 (m, 4H), 1.87 (s, 3H, CH3CO), 1.64-1.01 (m, other aliphatic ring
(br t, 1H), 4.64 (dd, 1H, J = 11.1, 5.4 Hz), 4.51 (dd, 1H, J = 11.1, 1.0 Hz), protons), 1.36, 0.97, 0.96, 0.92, 0.88, 0.77 (s, 3H each, 6 × CH3), 0.74 (d,
4.37 (dq, 2H, J = 15.0, 5.4 Hz), 3.94 (ddd, 1H, J = 8.8, 5.3, 2.8 Hz), 3.85- 1H, J = 11.2 Hz), 0.56 (s, 3H, CH3); 13C NMR (100 MHz, CD3OD): δ 180.12,
3.81 (m, 4H), 3.66 (dd, 1H, J = 11.4, 5.4 Hz), 3.61 (dd, 1H, J = 9.4, 0.8 Hz), 173.73, 163.49, 147.55, 146.56, 144.92, 124.17, 122.96, 107.31, 79.64,
3.13 (dd, 1H, J = 11.4, 4.5 Hz), 2.79 (dd, 1H, J = 13.3, 3.4 Hz), 2.07 (dt, 1H, 78.45, 75.56, 71.15, 69.69, 64.77, 59.96, 56.79, 53.08, 50.01, 49.96,
J = 13.6, 3.4 Hz), 1.88 (dd, 1H, J = 8.8, 3.2 Hz), 1.85 (s, 3H, CH3CO), 1.77 48.11, 48.05, 42.77, 42.26, 40.75, 39.92, 39.83, 38.07, 36.38, 36.10,
(t, 1H, J = 13.6 Hz), 1.64-0.98 (m, other aliphatic ring protons), 1.15, 35.97, 33.93, 33.29, 31.92, 31.23, 28.73, 27.88, 27.29, 25.38, 24.46,
0.96, 0.93, 0.92, 0.90, 0.78, (s, 3H each, 6 × CH3), 0.73 (d, 1H, J = 11.2 22.57, 19.43, 17.77, 16.35, 16.20; ESI-HRMS calcd for C45H70N5O10
Hz), 0.49 (s, 3H, CH3); 13C NMR (100 MHz, CD3OD): δ 180.46, 173.54, [M+H]+: 840.5117; found 840.5125.
163.79, 148.09, 145.86, 145.03, 125.51, 124.16, 104.61, 79.66, 73.83,
3.1.10 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β-hydroxy-lup-
71.45, 69.77, 64.79, 56.69, 54.48, 53.10, 48.97, 48.36, 47.56, 47.50, 20(29)-en-28-oyl)amino]methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-
42.85, 42.60, 40.60, 39.83, 38.10, 35.93, 35.05, 34.26, 33.80, 33.53, (1,2,3-trihydroxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic
acid
31.61, 28.74, 28.45, 27.85, 26.43, 24.51, 24.02, 22.42, 19.45, 17.48, methyl ester (9d). To a solution of 3d (222 mg, 0.45 mmol) and 11 (99
16.36, 16.00; ESI-HRMS calcd for C45H69N5NaO9 [M+Na]+: 846.4987, mg, 0.30 mmol) in THF/H2O (1:1 v/v, 12 mL) was added CuSO4 (48 mg,
found 846.4993.
3.1.8 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β-hydroxy-urs-12-en- solution was stirred vigorously for 12 hours at room temperature. After
28-oyl)amino]methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-(1,2,3- removal of THF and water, the residue was purified by chromatography
0.30 mmol) and sodium ascorbate (119 mg, 0.60 mmol). The resulting
trihydroxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl (eluent: DCM/MeOH = 10:1) over silica gel to afford compound 9d as a
ester (9b) and 5-acetylamino-4(R)-[4-[(3β-hydroxy-urs-12-en-28- white solid in 83% yield. Rf = 0.19 (DCM/MeOH = 10:1); 1H NMR (400
oyl)amino]methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-(1,2,3-trihydroxy-
MHz, CD3OD): δ 7.82 (s, 1H), 6.02 (d, 1H, J = 2.4 Hz), 5.61 (dd, 1H, J = 9.6,
propyl))-5,6-dihydro-4H-pyran-2-carboxylic acid methyl ester (10b). 2.0 Hz), 4.70 (s, 1H), 4.58-4.53 (m, 2H), 4.49-4.42 (m, 2H), 4.29 (d, 1H, J
Prepared from 8b according to general procedure B, and the residue = 15.1 Hz), 3.94 (ddd, 1H, J = 8.9, 5.1, 2.9 Hz), 3.78-3.85 (m, 4H), 3.69
was purified by chromatography (eluent: DCM/MeOH = 10:1) over silica (dd, 1H, J = 11.4, 5.2 Hz), 3.66 (d, , 1H, J = 9.4 Hz), 3.07-3.13 (m, 2H),
gel to afford compound 9b and 10b as white solids in 49% and 28% 2.51 (dt, 1H, J = 12.7, 3.0 Hz), 2.12 (td, 1H, J = 13.6, 3.0 Hz), 1.88 (s, 3H,
yield, respectively. Compound 9b: Rf = 0.19 (DCM/MeOH = 10:1); 1H CH3CO), 1.86-1.78 (m, 2H), 1.72-0.90 (m, other aliphatic ring protons),
NMR (400 MHz, CD3OD): δ 7.83 (s, 1H), 6.02 (d, 1H, J = 2.3 Hz), 5.62 (d, 1.68, 0.98, 0.94, 0.87, 0.84, 0.76 (s, 3H each, 6 × CH3), 0.68 (dd, 1H, J =
1H, J = 9.2 Hz), 5.32 (br t, 1H), 4.56 (d, 1H, J = 10.9 Hz), 4.46-4.36 (m, 8.4, 2.7 Hz); 13C NMR (100 MHz, CD3OD): δ 179.27, 173.87, 163.51,
3H), 3.92 (ddd, 1H, J = 8.6, 5.1, 2.8 Hz), 3.83 (dd, 1H, J = 11.5, 2.8 Hz), 152.28, 147.58, 123.35, 110.02, 107.34, 79.66, 78.52, 71.15, 69.71,
3.81 (s, 3H), 3.64-3.70 (m, 2H), 3.14 (dd, 1H, J = 11.0, 5.0 Hz), 2.14 (d, 1H, 64.78, 59.95, 56.93, 53.10, 52.07, 51.40, 50.10, 48.13, 43.46, 42.08,
J = 5.6 Hz), 2.03-2.10 (m, 1H), 1.87 (s, 3H, CH3CO), 1.68-0.95 (m, other 40.11, 39.96, 39.20, 39.01, 38.32, 35.65, 35.54, 33.92, 31.95, 30.58,
aliphatic ring protons), 1.10 (s, 3H, CH3), 0.96 (s, 6H, 2 × CH3), 0.92 (s, 3H, 28.66, 28.05, 27.00, 22.58, 22.16, 19.65, 19.46, 16.99, 16.85, 16.18,
CH3), 0.89 (d, 3H, J = 6.4 Hz, CH3), 0.77 (s, 3H, CH3), 0.72 (d, 1H, J = 11.0 15.12; ESI-HRMS calcd for
Hz), 0.56 (s, 3H, CH3); 13C NMR (100 MHz, CD3OD): δ 180.17, 173.72, 824.5173.
C
45H70N5O9 [M+H]+: 824.5168; found
163.49, 147.54, 146.63, 139.71, 127.28, 123.19, 107.30, 79.62, 78.40,
3.1.11 Synthesis of 5(S)-acetylamino-4(S)-[(3β-hydroxy-olean-12-en-
71.15, 69.67, 64.76, 59.96, 56.67, 54.11, 53.09, 50.04, 49.85, 48.91, 28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-triacetoxy-propyl))-5,6-dihydro-4H-
43.22, 40.82, 40.26, 39.94, 39.82, 38.55, 38.04, 35.80, 34.11, 31.88, pyran-2-carboxylic acid methyl ester (15a). To a solution of 14 (188.5
28.92, 28.77, 27.87, 25.25, 24.33, 24.04, 22.58, 21.59, 19.41, 17.74, mg, 0.44 mmol) and 2a (321 mg, 0.56 mmol) in DMF (20 mL), Na2CO3
17.73, 16.41, 16.13; ESI-HRMS calcd for C45H70N5O9 [M+H]+: 824.5168; (201 mg, 1.9 mmol) was added. The resulting mixture was stirred
found 824.5163. Compound 10b: Rf = 0.22 (DCM/MeOH = 10:1); 1H vigorously for 24 hours at 60 oC. After removal of DMF under vacuum,
NMR (400 MHz, CD3OD): δ 7.75 (s, 1H), 6.12 (d, 1H, J = 5.4 Hz), 5.57 (t, the residue was purified by chromatography (eluent: PE/Act = 2:1) over
1H, J = 5.4 Hz), 5.31 (br t, 1H), 4.63 (dd, 1H, J = 11.0, 5.4 Hz), 4.51 (d, 1H, silica gel to afford compound 15a as a white solid in 66% yield. Rf = 0.33
1
J = 11.1 Hz), 4.30-4.39 (m, 2H), 3.95-3.92 (m, 1H), 3.83-3.81 (m, 4H), (PE/Act = 2:1); H NMR (400 MHz, CDCl3): δ 6.15-6.16 (m, 1H), 6.04 (d,
3.66 (dd, 1H, J = 11.3, 5.4 Hz), 3.60 (d, 1H, J = 9.3 Hz), 3.14 (dd, 1H, J = 1H, J = 7.3 Hz), 5.91 (d, 1H, J = 1.9 Hz), 5.49 (d, 1H, J = 4.5 Hz), 5.31-5.30
10.8, 4.70 Hz), 2.14-2.04 (m, 2H), 1.85 (s, 3H, CH3CO), 1.72-0.97 (m, (m, 2H), 4.70-4.72 (m, 2H), 4.26-4.22 (m, 2H), 4.18 (dd, 1H, J = 12.4, 7.4
other aliphatic ring protons), 1.10 (s, 3H, CH3), 0.96 (s, 6H, 2 × CH3), 0.93 Hz), 3.79 (s, 3H), 3.21 (dd, 1H, J = 9.8, 3.3 Hz), 2.86 (dd, 1H, J = 10.4, 3.4
(s, 3H, CH3), 0.89 (d, 3H, J = 6.3 Hz, CH3), 0.78 (s, 3H, CH3), 0.72 (d, 1H, J Hz), 2.10, 2.07, 2.06, 1.89 (s, 3H each, 4 ×CH3CO), 1.87-0.97 (m, other
= 11.1 Hz), 0.51 (s, 3H, CH3); 13C NMR (100 MHz, CD3OD): δ 180.29, aliphatic ring protons), 1.13, 0.99, 0.90, 0.89, 0.88, 0.78, 0.76 (s, 3H
173.56, 163.81, 148.10, 145.77, 139.71, 127.35, 125.51, 104.61, 79.67, each, 7 × CH3), 0.73 (d, 1H, J = 11.2 Hz); 13C NMR (100 MHz, CDCl3): δ
73.85, 71.46, 69.78, 64.80, 56.68, 54.49, 54.22, 53.10, 48.95, 48.88, 178.54, 171.44, 170.55, 170.40, 170.03, 161.82, 144.30, 143.48, 122.62,
48.39, 43.27, 40.83, 40.80, 40.35, 39.98, 39.84, 38.66, 38.06, 35.78, 110.78, 78.96, 77.32, 71.51, 67.86, 62.21, 55.20, 52.37, 49.19, 47.58,
34.13, 31.87, 28.91, 28.75, 27.89, 25.27, 24.34, 24.00, 22.40, 21.54, 46.74, 46.14, 45.98, 41.81, 40.97, 39.36, 38.73, 38.40, 37.04, 34.05,
19.42, 17.69, 17.49, 16.42, 16.15; ESI-HRMS calcd for C45H69N5NaO9 33.37, 32.97, 32.81, 30.61, 28.11, 27.34, 27.15, 25.80, 23.53, 23.41,
[M+Na]+: 846.4987, found 846.4988.
3.1.9 Synthesis of 5(S)-acetylamino-4(S)-[4-[(3β,16α-dihydroxy- for C48H72N2NaO12 [M+Na]+: 891.4977, found 891.4984.
23.15, 20.89, 20.75, 20.65, 18.30, 17.23, 15.61, 15.30; ESI-HRMS calcd
olean-12-en-28-oyl)amino]methyl-[1,2,3]triazol-1-yl]]-6(S)-((1R,2R)-
(1,2,3-trihydroxy-propyl))-5,6-dihydro-4H-pyran-2-carboxylic
3.1.12 Synthesis of 5(S)-acetylamino-4(S)-[(3β-hydroxy-urs-12-en-
acid 28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-triacetoxy-propyl))-5,6-dihydro-4H-
methyl ester (9c). To a solution of 3c (229 mg, 0.45 mmol) and 11 (99 pyran-2-carboxylic acid methyl ester (15b). To a solution of 14 (188.5
mg, 0.30 mmol) in THF/H2O (1:1 v/v, 12 mL) was added CuSO4 (48 mg, mg, 0.44 mmol) and 2b (321 mg, 0.56 mmol) in DMF (20 mL), Na2CO3
0.30 mmol) and sodium ascorbate (119 mg, 0.60 mmol). The resulting (201 mg, 1.9 mmol) was added. The resulting mixture was stirred
solution was stirred vigorously for 12 hours at room temperature. After vigorously for 24 hours at 60 oC. After removal of DMF under vacuum,
removal of THF and water, the residue was purified by chromatography the residue was purified by chromatography (eluent: PE/Act = 2:1) over
6 | J. Name., 2012, 00, 1-3
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silica gel to afford compound 15b as a white solid in 75% yield. Rf = 0.35 J = 11.1, 4.9 Hz), 2.91 (dd, 1H, J = 9.9, 3.5 Hz), 2.11 (dt, 1H, J = 14.5, 4.0
(PE/Act = 2:1); 1H NMR (400 MHz, CDCl3): δ 6.60 (d, 1H, J = 8.8 Hz), 6.11 Hz), 1.96 (s, 3H, CH3CO), 1.93-1.89 (m, 2H), 1.73-0.97 (m, other aliphatic
(d, 1H, J = 7.2 Hz), 5.88 (s, 1H), 5.50 (d, 1H, J = 4.2 Hz), 5.29 (br t, 1H), ring protons), 1.17, 0.98, 0.96, 0.93, 0.90, 0.84, 0.78 (s, 3H each, 7 ×
5.25 (s, 1H), 4.72-4.70 (m, 2H), 4.30-4.32 (m, 2H), 4.18 (dd, 1H, J = 12.5, CH3); 13C NMR (100 MHz, CD3OD): δ 180.69, 174.46, 164.28, 145.66,
7.6 Hz), 3.78 (s, 3H), 3.22-3.20 (m, 1H), 2.31 (s, 1H), 2.19-2.11 (m, 2H), 145.03, 123.83, 111.77, 79.71, 78.54, 71.17, 70.02, 64.88, 56.77, 52.82,
2.10, 2.07, 2.06, 1.90 (s, 3H each, 4 × CH3CO), 1.94-0.91 (m, other 49.64, 49.00, 48.36 47.65, 47.33, 42.96, 42.35, 40.71, 39.84, 38.21,
aliphatic ring protons), 1.08, 0.99, 0.93, 0.91 (s, 3H each, 4 × CH3), 0.85 35.11, 34.63, 34.17, 33.54, 31.57, 28.76, 28.60, 27.87, 26.40, 24.56,
(d, 3H, J = 6.1 Hz, CH3), 0.79, 0.78 (s, 3H each, 2 × CH3), 0.72 (d, 1H, J = 23.98, 23.48, 22.82, 19.51, 18.29, 16.33, 15.96; ESI-HRMS calcd for
11.5 Hz); 13C NMR (100 MHz, CDCl3): δ 178.49, 171.42, 170.53, 170.43,
C
42H66N2NaO9 [M+Na]+: 765.4661, found 765.4669.
170.01, 161.78, 144.07, 137.67, 125.77, 110.91, 78.89, 77.25, 71.49,
3.1.16 Synthesis of 5(S)-acetylamino-4(S)-[(3β-hydroxy-urs-12-en-
67.80, 62.22, 55.10, 52.54, 52.31, 49.15, 47.62, 47.42, 46.11, 42.10, 28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-trihydroxy-propyl))-5,6-dihydro-4H-
39.55, 39.15, 38.66, 38.51, 37.60, 36.91, 33.09, 30.73, 28.12, 27.72, pyran-2-carboxylic acid methyl ester (16b). Prepared from 15b
27.10, 24.26, 23.36, 23.27, 23.17, 21.10, 20.85, 20.72, 20.64, 18.23, according to general procedure B, and the residue was purified by
17.41, 16.98, 15.68, 15.38; ESI-HRMS calcd for C48H73N2O12 [M+H]+: chromatography (eluent: DCM/MeOH = 10:1) over silica gel to afford
869.5158; found 869.5150.
3.1.13 Synthesis of 5(S)-acetylamino-4(S)-[(3β,16α-dihydroxy-olean- 10:1); 1H NMR (400 MHz, CD3OD): δ 7.49 (d, 1H, J = 7.9 Hz), 5.81 (d, 1H,
12-en-28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-triacetoxy-propyl))-5,6- J = 2.5 Hz), 5.26 (t, 1H, J = 3.2 Hz), 4.73 (dt, 1H, J = 9.8, 2.4 Hz), 4.28 (t,
compound 16b as a white solid in 87% yield. Rf = 0.32 (DCM/MeOH =
dihydro-4H-pyran-2-carboxylic acid methyl ester (15c). To a solution of 1H, J = 10.5 Hz), 4.23 (t, 1H, J = 10.7 Hz), 3.89 (ddd, 1H, J = 8.5, 5.2, 2.8
14 (188.5 mg, 0.44 mmol) and 2c (330 mg, 0.56 mmol) in DMF (20 mL), Hz), 3.82 (dd, 1H, J = 11.4, 2.9 Hz), 3.77 (s, 3H), 3.66 (dd, 1H, J = 11.4, 5.4
Na2CO3 (201 mg, 1.9 mmol) was added. The resulting mixture was Hz), 3.59 (d, 1H, J = 10.0 Hz), 3.15 (dd, 1H, J = 11.0, 4.8 Hz), 2.26 (d, 1H, J
stirred vigorously for 24 hours at 60 oC. After removal of DMF under = 11.0 Hz), 2.12 (dt, 1H, J = 14.0, 3.6 Hz), 1.97 (s, 3H, CH3CO), 1.96-0.98
vacuum, the residue was purified by chromatography (eluent: PE/Act = (m, other aliphatic ring protons), 1.12, 0.98, 0.97, 0.96 (s, 3H each, 4 ×
2:1) over silica gel to afford compound 15c as a white solid in 68% yield. CH3), 0.89 (d, 3H, J = 6.4 Hz, CH3), 0.87, 0.78 (s, 3H each, 2 × CH3); 13C
Rf = 0.30 (PE/Act = 2:1); 1H NMR (400 MHz, CDCl3): δ 6.80 (d, 1H, J = 6.9 NMR (100 MHz, CD3OD): δ 180.64, 174.36, 164.23, 145.58, 139.38,
Hz), 6.59 (d, 1H, J = 8.6 Hz), 5.86 (s, 1H), 5.47 (s, 2H), 5.30 (br s, 1H), 127.02, 111.83, 79.61, 78.52, 71.09, 69.90, 64.82, 56.70, 53.83, 52.82,
4.82-4.75 (m, 2H), 4.29-4.08 (m, 4H), 3.79 (s, 3H), 3.23 (br s, 1H), 3.11 (d, 49.05, 48.18, 43.28, 40.92, 40.55, 40.17, 39.98, 39.82, 38.93, 38.11,
1H, J = 14.3 Hz), 2.45 (s, 1H), 2.10, 2.06, 2.05, 1.89 (s, 3H each, 4 × 34.47, 31.93, 28.97, 28.82, 27.87, 24.65, 24.43, 24.06, 22.95, 21.62,
CH3CO), 1.95-0.96 (m, other aliphatic ring protons), 1.25, 1.00, 0.95, 19.47, 18.47, 17.73, 16.44, 16.15; ESI-HRMS calcd for C42H67N2O9
0.92, 0.89, 0.84, 0.79 (s, 3H each, 7 × CH3), 0.74 (d, 1H, J = 10.9 Hz); 13C [M+H]+: 743.4841; found 743.4846.
NMR (100 MHz, CDCl3): δ 178.79, 171.19, 170.68, 170.47, 170.05,
3.1.17 Synthesis of 5(S)-acetylamino-4(S)-[(3β,16α-dihydroxy-olean-
161.90, 144.30, 141.77, 123.08, 111.16, 78.86, 77.66, 73.62, 71.77, 12-en-28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-trihydroxy-propyl))-5,6-
68.03, 62.33, 55.32, 52.34, 49.94, 48.79, 47.12, 46.49, 45.67, 42.08, dihydro-4H-pyran-2-carboxylic acid methyl ester (16c). Prepared from
41.03, 40.00, 38.74, 38.61, 36.94, 36.15, 33.84, 32.65, 32.39, 29.63, 15c according to general procedure B, and the residue was purified by
28.04, 27.23, 27.08, 26.61, 23.29, 23.21, 20.84, 20.76, 20.73, 18.21, chromatography (eluent: DCM/MeOH = 10:1) over silica gel to afford
17.64, 15.76, 15.66; ESI-HRMS calcd for C48H73N2O13 [M+H]+: 885.5107; compound 16c as a white solid in 84% yield. Rf = 0.29 (DCM/MeOH =
found 885.5108.
3.1.14 Synthesis of 5(S)-acetylamino-4(S)-[(3β-hydroxy-lup-20(29)- J = 2.4 Hz), 5.40 (br t, 1H), 4.68-4.70 (m, 1H), 4.23-4.24 (m, 3H), 3.89
en-28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-triacetoxy-propyl))-5,6-dihydro- (ddd, 1H, J = 8.6, 5.2, 2.8 Hz), 3.82 (dd, 1H, J = 11.3, 2.9 Hz), 3.76 (s, 3H),
10:1); 1H NMR (400 MHz, CD3OD): δ 7.39 (d, 1H, J = 7.8 Hz), 5.78 (d, 1H,
4H-pyran-2-carboxylic acid methyl ester (15d). To a solution of 14 3.66 (dd, 1H, J = 11.5, 5.4 Hz), 3.59 (d, 1H, J = 9.4 Hz), 3.16 (dd, 1H, J =
(188.5 mg, 0.44 mmol) and 2d (321 mg, 0.56 mmol) in DMF (20 mL), 11.0, 5.0 Hz), 3.09 (dd, 1H, J = 14.1, 3.4 Hz), 2.20 (t, 1H, J = 13.5 Hz), 1.97
Na2CO3 (201 mg, 1.9 mmol) was added. The resulting mixture was (s, 3H, CH3CO), 1.92 (dd, 1H, J = 5.9, 2.4 Hz), 1.84-1.01 (m, other
stirred vigorously for 24 hours at 60 oC. After removal of DMF under aliphatic ring protons), 1.34, 0.98, 0.97, 0.95, 0.89, 0.86, 0.78 (s, 3H
vacuum, the residue was purified by chromatography (eluent: PE/Act = each, 7 × CH3); 13C NMR (100 MHz, CD3OD): δ 180.43, 174.29, 164.18,
2:1) over silica gel to afford compound 15d as a white solid in 62% yield. 145.59, 144.34, 123.68, 111.61, 79.62, 78.47, 74.78, 71.10, 69.95, 64.83,
1
Rf = 0.35 (PE/Act = 2:1); H NMR (400 MHz, CDCl3): δ 6.35 (d, 1H, J = 7.5 56.86, 52.82, 50.48, 49.77, 48.43, 48.33, 47.58, 42.91, 41.89, 40.98,
Hz), 6.03 (d, 1H, J = 8.4 Hz), 5.86 (d, 1H, J = 2.0 Hz), 5.50 (d, 1H, J = 3.2 39.97, 39.83, 38.14, 36.42, 36.03, 34.17, 33.26, 31.00, 30.69, 28.76,
Hz), 5.32-5.28 (m, 1H), 4.83 (t, 1H, J = 8.2 Hz), 4.70-4.74 (m, 2H), 4.58 (s, 27.89, 27.59, 26.26, 24.49, 22.90, 19.47, 18.24, 16.37, 16.27; ESI-HRMS
1H), 4. 30-4.23 (m, 2H), 4.17 (dd, 1H, J = 12.5, 7.5 Hz), 3.80 (s, 3H), 3.22- calcd for C42H67N2O10 [M+H]+: 759.4790; found 759.4787.
3.18 (m, 1H), 3.04 (dt, 1H, J = 11.2, 4.1 Hz), 2.40 (dt, 1H, J = 9.5, 3.1 Hz),
3.1.18 Synthesis of 5(S)-acetylamino-4(S)-[(3β-hydroxy-lup-20(29)-
2.15 (s, 1H), 2.09, 2.08, 2.06, 1.90, (s, 3H each, 4 × CH3CO), 1.93-0.85 (m, en-28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-trihydroxy-propyl))-5,6-dihydro-
other aliphatic ring protons), 1.66, 0.97, 0.96, 0.93, 0.83, 0.76 (s, 3H 4H-pyran-2-carboxylic acid methyl ester (16d). Prepared from 15d
each, 6 × CH3), 0.68 (d, 1H, J = 9.0 Hz); 13C NMR (100 MHz, CDCl3): δ according to general procedure B, and the residue was purified by
177.06, 171.29, 170.58, 170.43, 170.01, 161.82, 150.58, 144.47, 111.21, chromatography (eluent: DCM/MeOH = 10:1) over silica gel to afford
109.46, 78.95, 77.53, 71.54, 67.82, 62.18, 55.73, 55.33, 52.43, 50.57, compound 16d as a white solid in 81% yield. Rf = 0.32 (DCM/MeOH =
50.14, 48.12, 46.74, 46.45, 42.38, 40.71, 38.83, 38.69, 37.89, 37.69, 10:1); 1H NMR (400 MHz, CD3OD): δ 7.69 (d, 1H, J = 8.5 Hz), 5.76 (d, 1H,
37.17, 34.31, 33.37, 30.79, 29.37, 27.97, 27.33, 25.52, 23.06, 20.88, J = 2.4 Hz), 4.83-4.86 (m, 1H, overlap with water), 4.69 (br s, 1H), 4.59
20.75, 20.62, 19.31, 18.25, 16.10, 16.00, 15.43, 14.60; ESI-HRMS calcd (br s, 1H), 4.25 (t, 1H, J = 10.6 Hz), 14.22 (t, 1H, J = 10.6 Hz), 3.90 (ddd,
for C48H73N2O12 [M+H]+: 869.5158; found 869.5160.
3.1.15 Synthesis of 5(S)-acetylamino-4(S)-[(3β-hydroxy-olean-12-en- (dd, 1H, J = 11.4, 5.4 Hz), 3.60 (d, 1H, J = 9.4 Hz), 3.13 (dd, 1H, J = 10.9,
28-oyl)amino]-6(S)-((1R,2R)-(1,2,3-trihydroxy-propyl))-5,6-dihydro-4H- 5.1 Hz), 3.07 (dt, 1H, J = 11.1, 4.3 Hz), 2.59 (ddd, 1H, J = 12.8, 3.2 Hz),
1H, J = 8.6, 5.2, 2.8 Hz), 3.82 (dd, 1H, J = 11.4, 2.9 Hz), 3.78 (s, 3H), 3.66
pyran-2-carboxylic acid methyl ester (16a). Prepared from 15a 2.14 (d, 1H, J = 10.9 Hz), 1.96 (s, 3H, CH3CO), 1.87-0.92 (m, other
according to general procedure B, and the residue was purified by aliphatic ring protons), 1.68 (s, 3H, CH3), 1.00 (2 × s, 6H, 2 × CH3), 0.96,
chromatography (eluent: DCM/MeOH = 10:1) over silica gel to afford 0.87, 0.76 (s, 3H each, 3 × CH3), 0.71 (d, 1H, J = 8.7 Hz); 13C NMR (100
compound 16a as a white solid in 86% yield. Rf = 0.31 (DCM/MeOH = MHz, CD3OD): δ 179.32, 174.31, 164.19, 152.18, 145.80, 112.11, 110.05,
10:1); 1H NMR (400 MHz, CD3OD): δ 5.83 (d, 1H, J = 2.5 Hz), 5.28 (t, 1H, J 79.63, 78.60, 71.10, 69.90, 64.84, 57.10, 56.91, 52.85, 52.11, 51.45,
= 3.3 Hz), 4.74 (dd, 1H, J = 9.4, 2.5 Hz), 4.55 (s, 1H), 4.31-4.22 (m, 2H), 48.69, 48.66, 48.11, 43.49, 42.02, 40.13, 39.95, 39.16, 38.91, 38.35,
3.89 (ddd, 1H, J = 8.6, 5.3, 2.9 Hz), 3.82 (dd, 1H, J = 11.4, 2.9 Hz), 3.78 (s, 35.64, 33.75, 31.98, 30.56, 28.66, 28.05, 26.98, 22.78, 22.20, 19.68,
3H), 3.66 (dd, 1H, J = 11.4, 5.4 Hz), 3.60 (d, 1H, J = 10.0 Hz), 3.15 (dd, 1H,
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19.47, 16.84, 16.79, 16.16, 15.16; ESI-HRMS calcd for C42H66N2NaO9
[M+Na]+: 765.4661, found 765.4667.
3.2 Bioassays
influenza entry inhibitor, although more studies are necessary to
investigate its anti-influenza effect in vivo.
3.2.1 Cytotoxicity assay. The assay was performed as previously
described with some modifications.[28] Cells were seeded in 96-well
plates in DMEM supplemented with 10% FBS and cultured overnight at
37 oC in 5% CO2. Then the test compounds were added and the cells
were further incubated at 37 oC in 5% CO2 for 40 hours. Cell viability
was assessed using the CellTiter-Glo assay kit as recommended by the
supplier, and the plates were read using a plate reader (Tecan Infinite
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (Grants Nos. 81373271, 81361168002,
81573269 and 21572015). The authors gratefully acknowledge Dr.
M2000 PRO; Tecan Group Ltd., Mannedorf, Switzerland) Viability was Hongwei Jin (State Key Laboratory of Natural and Biomimetic
calculated using the background-corrected absorbance as follows:
Drugs, Peking University) for carrying out the AlogP
Viability (%) = A of experiment well/A of control well × 100%.
3.2.2 CPE reduction assay. MDCK cells (ATCC CCL-34, Manassas, VA,
determinations.
USA) were seeded into 96-well plates in DMEM supplemented with 10%
FBS and incubated overnight at 37 oC under 5% CO2. The culture
medium was replaced by test compound and influenza virus (MOI =
0.1)-DMEM supplemented with 1% FBS and 2 μg/mL TPCK-treated
trypsin. The final concentration of DMSO was 1%. After 40 hours of
incubation, CellTiter-Glo reagent (Promega Corp., Madison, WI, USA)
was added, and assessed by the CellTiter-Glo assay as described above.
3.2.3 The hemagglutination inhibition (HI) assay. HI assay was
performed as described previously.[30] Briefly, compound from a 3-fold
serial dilution in saline was mixed with an equal volume of influenza
virus (2 HA units) in the V-bottomed 96-well microplates. Subsequently,
50 μL of freshly prepared chicken red blood cells (cRBCs) (1% v/v in
saline) (Beijing Yuabio Biotechnology Co., Ltd, China) was added to each
well. The mixture was incubated for 30 min at room temperature
before observing cRBC aggregation on the plate.
Conflict of Interest
The authors declare no competing interests.
References
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A series of pentacyclic triterpene-Neu5Ac2en derivatives were
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7.
Zemplén conditions,
a pair of configurational isomers was
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separated and their configurations were characterized by detailed
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AlogP values within the range of 2.52-4.86. The most potent
inhibitory activity against influenza A/WSN/33 (H1N1) virus was
displayed by compounds 15a and 15c with IC50 at 8.3 and 15.5 μM,
respectively, which were comparable to the known anti-influenza
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Graphical abstract
The C-4 of sialic acid is not important for its binding with hemagglutinin and could be replaced with hydrophobic
moieties.