Structure-activity relationships of saponin derivatives: A series of entry inhibitors for highly pathogenic H5N1 influenza virus

Article abstract of DOI:10.1016/j.ejmech.2012.04.022

The occurrence of highly pathogenic avian influenza virus H5N1 highlights the urgent need for new classes of antiviral drugs. Theoretically, each of steps in influenza viral life cycle can be a target of antiviral therapeutics. However, up to date, no licenced entry inhibitor drug is available for H5N1 or any other influenza viruses. Our strategy for developing new anti-influenza therapeutics is to target the interaction between HA and sialic acid which is influenza viral receptor presented on host cell surface. Here, based on our previously discovered small molecule inhibitor saponin 1, intensive SAR studies around the sugar chain and aglycone were conducted. The results showed that both the chacotriosyl residue and the chlorogenin moiety of active compound 1 are important for the antiviral activity, although several subtle modifications can be made on particular positions.

Full text of DOI:10.1016/j.ejmech.2012.04.022

European Journal of Medicinal Chemistry 53 (2012) 316e326
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships of saponin derivatives: A series of entry inhibitors
for highly pathogenic H5N1 influenza virus
,
,
,
a,
Ning Ding a 1, Qing Chen b 1, Wei Zhang a, Sumei Ren a, Ying Guo b , Yingxia Li
**
*
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The occurrence of highly pathogenic avian influenza virus H5N1 highlights the urgent need for new
classes of antiviral drugs. Theoretically, each of steps in influenza viral life cycle can be a target of
antiviral therapeutics. However, up to date, no licenced entry inhibitor drug is available for H5N1 or any
other influenza viruses. Our strategy for developing new anti-influenza therapeutics is to target the
interaction between HA and sialic acid which is influenza viral receptor presented on host cell surface.
Here, based on our previously discovered small molecule inhibitor saponin 1, intensive SAR studies
around the sugar chain and aglycone were conducted. The results showed that both the chacotriosyl
residue and the chlorogenin moiety of active compound 1 are important for the antiviral activity,
although several subtle modifications can be made on particular positions.
Received 2 February 2012
Received in revised form
12 April 2012
Accepted 12 April 2012
Available online 21 April 2012
Keywords:
Influenza
H5N1
Entry inhibitors
Structureeactivity relationships
Pseudovirions
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
urgent need for new classes of agents to combat avian H5N1 vari-
ants that are resistant to treatment by targeting at other potential
Highly pathogenic H5N1 virus can possibly be the most severe
pandemic threat in near future. Till 2010, 59% of the 508 confirmed
human cases infected by H5N1 have proved mortal [1]. Two classes
of antiviral drugsdion channel inhibitors and neuraminidase
inhibitorsdare at present licensed for use against influenza A
viruses [2]. Adamantanes block the ion channel formed by the M2
protein, which is critical in the release of viral ribonucleoprotein
complexes (vRNPs) into the cytoplasm. Although ion channel
inhibitors can be effective against influenza virus A infection, they
have been reported to cause CNS side effects [3,4], and have given
rise to the rapid emergence of drug-resistant viral strains [5]. Thus
they are not recommended for a general and uncontrolled use [6].
Two neuraminidase inhibitors, oseltamivir and zanamivir, were
both approved in 1999 for treatment and preventive use for acute
uncomplicated flu caused by influenza A and B [7]. Neuraminidase
inhibitors interfere with the enzymatic activity of the NA protein,
which is critical for the efficient release of newly synthesized
viruses from infected cells. However, resistance of H5N1 to osel-
tamivir has also been observed recently [8]. Therefore, there is an
viral factors.
The earliest stage of influenza virus infection is viral entry,
which is mediated by interaction of viral envelope protein
hemagglutinin (HA) and its receptor on host cell surface, sialic acid
sugars [9]. Therefore, to identify and develop potent entry inhibi-
tors against H5N1 virus can be a potential antiviral treatment. In
our previous work [10], we used an efficient HIV-based pseudo-
typing system [11e14] to screen a saponin library generated from
semisynthesis, and discovered the first three small molecule H5N1
viral entry inhibitors (Fig. 1). These inhibitory molecules contained
a 2,4-di-O-(
known as a chacotriose. Chlorogenin 3-O-
chlorogenin 6- -O-acetyl-3-O- -chacotrioside (2) and methyl
ursolate 3-O- -chacotrioside (3) displayed strong inhibitory
activity against H5N1 entry with IC50 of 6.00e9.25 M [10]. The
preliminary structureeactivity relations (SARs) study [10] showed
that the 3-O- -chacotriosyl residue was critical for the activity,
since substituted the -chacotriosyl moiety of compound 1 by
rhamnopyranosyl-(1 / 4)- -glucopyranosyl or -rhamnopyr-
anosyl-(1 / 2)- -glucopyranosyl moiety leaded inactive. In
a
-
L
-rhamnopyranosyl)-
b
-
D
-glucopyranose trisaccharide
b
-chacotrioside (1),
a
b
b
m
b
b
a-L-
b-
D
a-L
b-D
addition, compounds with replacement of the aglycone moiety of
compound 1 to dihydrochlorogenin, dehydroisoandrosterone or
stigmasterine were all found to be inactive except for methyl
ursolate (compound 3).
* Corresponding author. Tel.: +86 21 51980120; fax: +86 21 51980127.
** Corresponding author. Tel./fax: þ86 010 63165176.
E-mail addresses: yingguo6@yahoo.com (Y. Guo), liyx417@fudan.edu.cn (Y. Li).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.022

N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
317
Fig. 1. Three saponin inhibitors for H5N1 viral entry.
Based on the above results, in this report, compound 1 was
2. Chemistry
chosen as the lead compound to design and synthesize a series of
analogs to further investigate the SARs of saponins with different
sugar chains and aglycones.
For understanding the function of sugar residue, we designed
and synthesized compounds 4e11 (Fig. 2). Chacotriose (compound
4) was synthesized and tested to investigate the sugar chain of
compound 1 alone on the viral entry inhibitory activity. In order to
The preparation of target compounds 4 and 5 was shown in
Scheme 1. The known building block 15 [10] was subjected to 1-
BBTZ [1-(benzoyloxy)-benzotriazole] in the presence of Et3N to
protect selectively the 3,6-OHs, leading to intermediate 16 [10].
Glycosylation of the 2,4-OHs in 16 with 2,3,4-tri-O-acetyl-L-rham-
nopyranosyl trichloroacetimidate 17 [10] with TMSOTf as the
catalyst led to the trisaccharide 18. Removal of the benzoyl groups
with MeONa in MeOH gave desired compound 5. Treatment of 5
with NBS in water/acetone mixture provided compound 4.
mimic the
1-thio- -chacotrioside 5 was synthesized as well. Compound 6,
with two -arabinosyl residues instead of the two -rhamnosyl
b-chacotriosyl linkage in active compounds 1, ethyl
b
L
L
residues, and compounds 7e11, on which one or two sugar
hydroxyls were blocked by methyl groups, were designed and
synthesized in order to further investigate the effect of sugar
moiety on the activity.
The preliminary SAR study also showed that reductive ring
opening of spirostan saponin 1 led to the disappearance of inhibi-
The synthesis of target saponins 6e9 and 12e14 was done by
the similar route as that for compounds 1 and 2 reported previously
by us [10,15]. As shown in Scheme 2, glycosylation of saponin
derivatives 20aed with 3,6-di-O-benzoyl-2,4-di-O-levulinoyl-
glucopyranosyl trichloroacetimidate 19 [10], respectively, under
the action of TMSOTf afforded the 3-O- -glucopyranosides 21aed
D-
b
tory activity [10]. However, 6
similar inhibitory activity as compound 1, indicating the 6-position
a
-OH acetylated compound 2 showed
[10]. Removal of the Lev (levulinoyl) groups with AcOHeNH2NH2
gave 22aed. Subsequent glycosylation of the 2,4-OHs in 22aed
of the aglycone might be further optimized. Therefore, compound
with
L-arabinosyl trichloroacetimidate 23 [16] and L-rhamnopyr-
12 with a carbonyl group on C6, compound 13 with a
and compound 14 with a -rhamnosyl residue attached to 6-
were designed and synthesized (Fig. 3).
b
-OH on C6,
anosyl trichloroacetimidates 24 [17], 25 [18], 26 [19] and 17 under
the “inverse addition conditions” [20] with TMSOTf as the catalyst
led to the intermediates 27e30 and 31bed. Final treatment with
L
a
-OH
Fig. 2. Saponins designed for SAR studies on the sugar chain.

318
N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
Fig. 3. Saponins designed for SAR studies on the aglycone.
MeONa in MeOH afforded target saponins 6e9 and 12e14
smoothly.
The synthetic route toward target saponins 10 and 11 is depicted
in Scheme 4. The trichloroacetimidates 37 and 38 with methylated
3 or 6-OH were employed as donors to carry out the glycosylation
with acceptor 20a, giving the glycosides 39 and 40, respectively.
Removal of the benzoyl groups from 39 and 40 led to compounds 41
and 42. Selectively protection of the 3-OH in 42 by using 1-BBTz in
the presence of Et3N afforded 43. The important intermediates 41
The stereochemistry of the glycosidic bond formed, was
confirmed by NMR spectroscopy. In the 1H NMR spectrum of
compound 6, the presence of three anomeric protons (
and 4.31) as doublets with J ¼ 6.0, 7.8, 6.8 Hz, respectively, sup-
ported the presence of three -glycosidic linkages. All structures
d 4.62, 4.60
b
containing the chacotriosyl residues have alpha stereochemistry for
and 43 were subjected to glycosylation with perbenzoylated L-
rhamnopyranosyl trichloroacetimidate 17, giving 44 and 45. After
global deprotection the target saponins 10 and 11 were provided.
the rhamnose moieties. For instance, 1H NMR of 7 showed two
anomeric protons at
J ¼ 1.4 Hz, respectively, supported the presence of two
glycosidic bonds. And the signal of an anomeric proton at
d
5.30 and 5.03 as a singlet and a doublet with
-rhamnosyl
4.53 as
-configuration of
a
d
3. Pharmacology
a doublet with J ¼ 7.8 Hz is consistent with the
b
the glucosidic linkage.
Influenza viral entry is mediated by HA, which is responsible for
viral attachment and membrane fusion. An HA/HIV pseudotyped
virus model was performed to test saponins on their viral entry
inhibitory effects. Two HAs we used, with sequence alignments 97%
similarity, were from highly pathogenic H5N1 viruses which iso-
lated from human and avian. Vesicular stomatitis virus glycopro-
tein (VSV-G) had a broad host range, so that VSVG/HIV pseudoviral
transduction was used as a specificity control to exclude inhibitory
effect on post-entry for HIV infection. Only a compound exhibited
inhibitory activity on HA/HIV transduction, but not on VSVG/HIV,
can be considered as an inhibitor of influenza viral entry. All
compounds listed in Table 1 had no effects on VSVG/HIV viral
infection (data not shown). It should be noted that no cytotoxicity
was detected for all the designed compounds under the
The key building block 20a was synthesized according to our
previous work [10]. 20bed were synthesized as shown in Scheme
3. Oxidation of the compound 32 [10] with Jones reagent at 0 ꢀC
in 20 min provided compound 33 [21] smoothly. Removal of the
3-O-benzyl group by hydrogenation afforded the key building block
20b [21]. Stereoselective reduction of the carbonyl group at C6 of
compound 33 to a
b-OH was achieved by using NaBH4 as the
reagent (led to 34 [22]), on which a benzoyl protecting group was
introduced to give compound 35. Then removal of the 3-O-benzyl
group led to the key building block 20c. Glycosylation of the 6-OH
in 32 with
L-rhamnopyranosyl trichloroacetimidate 17 with
TMSOTf as the catalyst led to the intermediate 36, which was then
subjected to Pd/CeH2, affording 20d.
Conditions: (a) BBtz, Et3N, CH2Cl2, 60%; (b) TMSOTf, CH2Cl2, -40º C, 31%; (c) CH3Na/CH3OH, 45%; (d)
(i) NBS, acetone/H2O; (ii) Ac2O, pyridine; (iii) CH3ONa, CH3OH, 55% for 3 steps.
Scheme 1. Synthesis of compounds 4 and 5.

N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
319
Conditions: (a) TMSOTf, CH2Cl2; 40% for 21b, 49% for 21c, 67% for 21d; (b) AcOH-NH2NH2, 84% for
22b, 73% for 22c, 94% 22d; (c) TMSOTf, CH2Cl2, 75% for 28, 30% for 31b, 28% for 32c, 55% for 31d; (d)
CH3Na/CH3OH, 21% for 6 (2 steps), 80% for 7, 41% for 8 (2 steps), 66% for 9 (2 steps), 34% for 12, 65%
for 13, 40% for 14.
Scheme 2. Synthesis of compounds 6e9 and 12e14.
concentrations for the antiviral activity screening. The effects of
compounds on HA mediated viral entry were summarized in
Table 1.
to the total loss of activity, suggesting that the 2,4-L-rhamnosyls of
chacotriosyl residue is essential for the activity. Whereas, blocks
one or two hydroxyls by methyls in the chacotriosyl residue
(compounds 7e11) did not affect the activity significantly, and only
slightly reduced activity was observed. We suppose that during the
interaction of chacotriosyl residue with the receptor the hydroxyls
contributed as composition of forces. Thus methylations on one or
two hydroxyls were not able to induce the total loss but
only the slight drop of activity. The supposition can be further
supported by the results that the monomethylated compounds (10
and 11) both had a slightly better antiviral activity than those
dimethylated (7e9).
4. Result and discussion
The activity of compounds 1e3 was reported by us previously
[10]. First, chlorogenin, chacotriose 4 and ethyl 1-thio-b-chaco-
trioside 5 were tested but showed no H5N1 entry inhibitory
activity, which indicated that the active saponin 1 acted as an
integral structure but not the sugar chain or the aglycone alone.
To further understand the role of the chacotriosyl residue,
compound 6 in which the two
L-rhamnosyl residues were replaced
For the saponins with the same 3-O-b-chacotriose residue (1, 2
by two -arabinosyls was tested. It’s interesting that the changes led
L
and 12e14), all of them except for 14 exhibited excellent inhibitory

320
N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
Conditions. (a) Jones reagent, acetone, 0º C, 91%; (b) Pd/C-H2, EtOAc/CH3OH, 60º C, 83% for 20b, 95%
for 20c, 87% for 20d; (c) NaBH4, CH3OH/1,4-dioxane, 0º C, 92%; (d) BzCl, DMAP, CH2Cl2, 92%; (e) 4Å
MS, TMSOTf (0.2 eq), CH2Cl2, 96%.
Scheme 3. Synthesis of compounds 20bed.
activity in the IC50 range of 6.0e20
compounds 1 and 2 with the same 6
m
M. We also noticed that
residue might has a more affinity toward the target on HA(QH)/HIV-
luc strains than that on HA(viet)/HIV-luc strains, thus methylations
on chacotriosyl residue induced the drop of inhibitory activity more
rapidly on HA(QH)/HIV-luc strains. Similarly, subtle modifications
of aglycone on C6 could also induce the change of selectivity, since
both compounds 12 and 13 inhibited HA(viet)/HIV-luc strains more
effectively than HA(QH)/HIV-luc strains.
a-substitution both showed
slightly better activity than compounds 12 or 13, which has
a carbonyl group or a -OH on C6, respectively. However, when
a rhamnosyl residue was introduced to the 6 -OH of compound 1,
b
a
compound 14 lost the inhibitoryactivity. These resultssuggested that
subtlemodificationsofaglycone on C6hadnoimportanteffecton the
antiviral activity but introduction of bulk groups should be avoided.
In addition, compounds 1 and 2 showed a slight selectivity
toward HA(QH)/HIV-luc strains, because HA of each H5N1 virus has
its own special chemical structure, whereas all the methylated
compounds 7e11 inhibited HA(viet)/HIV-luc strains more
effectively. The reverse of selectivity suggested that the chacotriosyl
5. Conclusion
In conclusion, based on our previously discovered small mole-
cule inhibitor saponin 1, intensive SAR studies around the sugar
chain and aglycone were conducted. The results showed that both
Scheme 4. Synthesis of compounds 10 and 11.

N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
321
Table 1
identical with those provided in the literature: Morillo, M.; Lequart,
V.; Grand, E.; Goethals, G. Usubillaga, A.; Villa, P.; Martin, P. Car-
bohydr. Res. 2001, 334, 281e287. The above peracetylated chaco-
triose was dissolved in CH3OH/CH2Cl2 (20 mL, v/v ¼ 1:1), and then
NaOMe was added until pH ¼ 8.5. After stirred at 35 ꢀC for 4 h, the
solution was neutralized with ion-exchange resin (Hþ) and then
filtered and concentrated. The residue was purified by Sephadex
LH-20 column chromatography (65% methanol in H2O) to afford 4
Effect of compounds on HA mediated viral entry (IC50, mM).
Compounds
HA(QH)/HIV-luca
HA(viet)/HIV-lucb
1
2
3
7.2
7.6
6.0
>50
>50
>50
>50
31
28
N
25
12
7.8
9.3
8.5
>50
>50
>50
>50
15
16
24
13
9
Chlorogenin
4
5
6
7
8
9
10
11
12
13
14
(a/
b
mixture) as a white powder (20.5 mg, 55%): Rf ¼ 0.13 (CH2Cl2/
CH3OH, 3:1); 1H NMR (CD3OD, 400 MHz):
d
5.36 (s, 1H), 5.19 (s-like,
0.27H), 5.16 (d, 0.25H, J ¼ 3.1 Hz), 4.87 (s, 0.34H), 4.53 (d, 0.38H,
J ¼ 9.8 Hz), 4.14 (d,1H, J ¼ 9.8 Hz), 3.98e3.93 (m, 3H), 3.92e3.77 (m,
6H), 3.76e3.58 (m, 8H), 3.58e3.49 (m, 2H), 3.45e3.32 (m),
3.21e3.14 (m, 1.72H), 2.90e2.73 (m, 1.79H), 1.40e1.15 (m); 13C NMR
20
18
>50
12
11
>50
(CD3OD, 100 MHz):
d 103.2, 103.0, 88.6, 81.1, 78.6, 78.3, 74.9, 73.7,
72.5e72.1, 70.9, 70.7, 61.6, 41.9, 17.9; ESI-HRMS calcd for
a
C18H32NaO14 [M þ Na]þ 495.1684, found 495.1684.
HA(QH): HA, H5N1(Goose/Qinghai/59/05).
HA(Viet): HA, H5N1(A/Viet Nam/1203/2004).
b
6.3. Synthesis ethyl 1-thio-b-chacotrioside (5)
the chacotriosyl residue and the chlorogenin moiety of active
compound 1 are important for the antiviral activity, although
several subtle modifications can be made on particular positions.
Such efforts are of vital importance for the further development of
novel and effective small molecule inhibitors.
To a mixture of acceptor 16 (518.4 mg, 1.20 mmol), imidate 17
(990.5 mg, 1.60 mmol) and powdered 4 A molecular sieves in dried
ꢀ
CH2Cl2 (30 mL) at ꢁ40 ꢀC was added TMSOTf (53.3 mg, 0.2 mmol).
After stirred at ꢁ40 ꢀC for 0.5 h and then at r.t. for 2 h, the reaction
was quenched with Et3N. The solid was filtered off. The filtrate was
concentrated and purified by column chromatography (EtOAc/
petroleum ether, 1:3) to afford compound 18 as an amorphous solid
(506.2 mg, 31%).
6. Experimental protocols
6.1. General methods
Compound 18 (506.2 mg, 0.38 mmol) was dissolved in CH3OH/
CH2Cl2 (20 mL, v/v ¼ 1:1), and then NaOMe was added until
pH ¼ 8.5. After stirred at 35 ꢀC for 4 h, the solution was neutralized
with ion-exchange resin (Hþ) and then filtered and concentrated.
The residue was purified by silica gel column chromatography
(CH2Cl2/CH3OH, 3:1) to afford compound 5 as a yellow syrup
(87.2 mg, 45%): Rf ¼ 0.20 (CH2Cl2/CH3OH, 3:1); 1H NMR (CD3OD,
All chemical reagents were used as supplied unless indicated.
Solvents used in organic reactions were distilled under an inert
atmosphere. Unless otherwise noted, all reactions were carried out
at room temperature and were performed under a positive pressure
ꢀ
of argon. Crushed 4 A molecular sieves were activated by thorough
flame-drying and cooled in vacuo prior to use. Flash column chro-
matography was performed on silica gel (200e300 mesh, Qingdao,
China). Amberlite 15 (Hþ-form) was used where acidic ion-
exchange resin is indicated. Analytical thin layer chromatography
(TLC) was performed on glass plates pre-coated with a 0.25 mm
thickness of silica gel. 1H NMR and 13C NMR spectra were taken on
a Jeol JNM-ECP 600 or a Bruker Avance III 400 spectrometer at r.t.
Chemical shifts of the 1H NMR spectra are expressed in ppm relative
to the solvent residual signal 7.26 in CDCl3 or to tetramethylsilane
400 MHz):
d
5.27 (s, 1H, H-100), 4.92 (s, 1H), 4.42 (d, 1H, J ¼ 9.8 Hz),
4.18e4.16 (m,1H), 3.95e3.94 (m, 2H), 3.82e3.80 (m, 2H), 3.66e3.52
(m, 5H), 3.48e3.38 (m, 3H), 2.82e2.67 (m, 2H), 1.25e1.21 (m, 9H);
13C NMR (CD3OD,100 MHz):
d 103.1, 84.7, 80,8, 80.0, 79.9, 79.5, 73.9,
73.7, 72.5, 72.4, 72.3, 72.2, 70.7, 70.6, 62.1, 24.6, 17.9, 17.8, 15.1; ESI-
HRMS calcd for C20H36NaO13S [M þ Na]þ 539.1769, found 539.1769.
6.4. Synthesis of chlorogenin 3
b
-O-[2,4-di-O-(
a-L
-arabinopyranosyl)-
b-D
-glucopranoside] (6)
(
d
¼ 0.00). Chemical shifts of the 13C NMR spectra are expressed in
ppm relative to the solvent signal 77.00 in CDCl3 or to tetrame-
ꢀ
To a mixture of 22a (50.0 mg, 0.06 mmol) and 4 A molecular
thylsilane (
d
¼ 0.00) unless otherwise noted. COSY, HMQC, and
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (2.0
mL,
HMBC were routinely used to definitively assign the signals of 1H
NMR and 13C NMR spectra. Electrospray (ESI) mass spectra were
recorded on a Global Q-TOF mass spectrometer.
0.01 mmol). After stirred at ꢁ20 ꢀC for 5 min donor 23 (133.1 mg,
0.22 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for
20 min and then at r.t. for 8 h. The reaction was quenched with
Et3N. The solid was then filtered off.
6.2. Synthesis of 2,4-di-O-(a-
(chacotriose, 4)
L
-rhamnopyranosyl)-
a
/
b
-
D
-glucopyranose
The residue was dissolved in CH3OH/CH2Cl2 (20 mL, v:v ¼ 1:1),
and then NaOMe was added until pH ¼ 8.5. After stirred at 35 ꢀC for
4 h, the solution was neutralized with ion-exchange resin (Hþ) and
then filtered and concentrated. The residue was purified by silica
gel column chromatography (CH2Cl2/MeOH, 10:1e4:1) to afford 6
as a white syrup (10.1 mg, 21% for two steps): Rf ¼ 0.52 (CHCl3/
To a stirred solution of compound 5 (41.0 mg, 0.079 mmol) in
acetone and H2O (5 mL, v/v ¼ 9:1) at ꢁ20 ꢀC was added NBS
(28.1 mg, 0.16 mmol). After 20 min, the reaction was quenched with
saturated aqueous NaHCO3 and concentrated. The residue was
dissolved in pyridine (5 mL) and Ac2O (2 mL). After stirred over-
night, the reaction mixture was quenched by addition of CH3OH
(5 mL) and concentrated. The residue was diluted with CHCl3
(20 mL) and washed with 1 mmol/L HCl aqueous solution, saturated
aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatog-
raphy to afford peracetylated chacotriose. The physical data are
MeOH, 3:1); 1H NMR (CD3OD, 600 MHz):
d
4.62 (d, 1H, J ¼ 6.0 Hz,
Ara-H-1), 4.60 (d, 1H, J ¼ 7.8 Hz, H-10), 4.39 (q, 1H, J ¼ 7.8 Hz, H-16),
4.31 (d,1H, J ¼ 6.8 Hz, Ara-H-1), 3.98 (dd,1H, J ¼ 12.4, 4.1 Hz, Ara-H-
4), 3.93 (dd, 1H, J ¼ 12.8, 2.3 Hz, Ara-H-4), 3.85e3.83 (m, 2H, 2ꢂ
Ara-H-3), 3.71 (t,1H, J ¼ 9.2 Hz, H-30), 3.69e3.53 (m,10H, 2ꢂ Ara-H-
2, H-60, H-50, 2ꢂ Ara-H-5, H-40), 3.46e3.32 (m, 4H, H-20, H-3, H-26),
2.33 (brd, 1H, J ¼ 10.1 Hz), 0.95 (d, 3H, J ¼ 6.9 Hz, Me), 0.86 (s, 3H,
Me), 0.79 (d, 3H, J ¼ 6.0 Hz, Me), 0.79 (s, 3H, Me); 13C NMR (CD3OD,

322
N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
150 MHz):
d
110.7, 105.4, 101.0, 82.3, 81.1, 79.9, 76.3, 74.1, 73.9, 73.2,
3), 2.33 (d, 1H, J ¼ 12.8 Hz), 1.25 (d, 3H, J ¼ 6.4 Hz, Rha-6-Me), 0.96
(d, 3H, J ¼ 7.3 Hz, Me), 0.86 (s, 3H, Me), 0.79 (d, 3H, J ¼ 5.5 Hz, Me),
72.6, 70.1, 68.0, 67.9, 63.9, 61.9, 57.5, 55.4, 52.9, 43.1, 42.8, 41.9, 41.2,
38.7, 37.7, 35.4, 33.2, 32.8, 32.6, 31.6, 30.9e30.0, 29.5, 26.2, 23.9,
22.3, 17.7, 17.1, 15.0, 14.6, 14.0; ESI-HRMS calcd for C43H70NaO17
[M þ Na]þ 881.4505, found 881.4505.
0.79 (s, 3H, Me); 13C NMR (CD3OD, 150 MHz):
d 110.6, 102.8, 102.4,
100.3, 82.2e82.0, 79.6, 78.7, 76.7, 72.8, 72.6, 70.6, 69.9, 69.7, 68.4,
67.9, 63.9, 62.0, 57.4, 57.2, 55.4, 52.8, 48.0, 43.0, 42.7, 41.8, 41.1, 38.7,
37.7, 35.3, 32.7, 32.5, 31.5, 30.4, 29.9, 29.4, 22.1, 18.1, 17.9, 17.5, 17.0,
14.9, 13.9, 9.3; ESI-HRMS calcd for C47H79O17 [M þ H]þ 915.5311,
found 915.5311.
6.5. Synthesis of chlorogenin 3
rhamnopyranosyl)- -glucopyranoside] (7)
b-O-[2,4-di-O-(2-O-methyl-a-L-
b-D
ꢀ
To a mixture of 22a (50.0 mg, 0.10 mmol) and 4 A molecular
6.7. Synthesis of chlorogenin 3
b-O-[2,4-di-O-(4-O-methyl-
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (2.0
mL,
a-L
-rhamnopyranosyl)- -glucopyranoside] (9)
b-D
0.02 mmol). After stirred at ꢁ20 ꢀC for 5 min donor 24 (147.0 mg,
0.50 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for
20 min and then at r.t. for 8 h. The reaction was quenched with
Et3N. The solid was then filtered off. The filtrate was concentrated
and purified by silica gel column chromatography (EtOAc/petro-
leum ether, 1:3) to give 28 as an amorphous solid (57.4 mg, 75%).
Compound 28 (57.4 mg, 0.04 mmol) was dissolved in CH3OH/
CH2Cl2 (20 mL, v:v ¼ 1:1), and then NaOMe was added until
pH ¼ 8.5. After stirred at 35 ꢀC for 4 h, the solution was neutralized
with ion-exchange resin (Hþ) and then filtered and concentrated.
The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH, 10:1e4:1) to afford 7 as a white amorphous solid
(30.4 mg, 80%): Rf ¼ 0.54 (CHCl3/MeOH, 8:1); 1H NMR (CD3OD,
ꢀ
To a mixture of 22a (50.0 mg, 0.06 mmol) and 4 A molecular
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (2.0
mL,
0.01 mmol). After stirred at ꢁ20 ꢀC for 5 min and then donor 26
(147.0 mg, 0.50 mmol) was added. The mixture was stirred
at ꢁ20 ꢀC for 20 min and then at r.t. for 8 h. Then the reaction was
quenched with Et3N. The solid was then filtered off. The filtrate was
concentrated and purified by silica gel column chromatography
(EtOAc/petroleum ether, 1:4e1:2) to give 30 as an amorphous solid.
Compound 30 was dissolved in CH3OH/CH2Cl2 (10 mL,
v:v ¼ 1:1), and then NaOMe was added until pH ¼ 8.5. After stirred
at 35 ꢀC for 4 h, the solution was neutralized with ion-exchange
resin (Hþ) and then filtered and concentrated. The residue was
purified by silica gel column chromatography (CH2Cl2/MeOH,
10:1e4:1) to afford 9 as a white amorphous solid (54.2 mg, 66% for
two steps): Rf ¼ 0.55 (CHCl3/MeOH, 8:1); 1H NMR (CD3OD,
600 MHz):
d
5.30 (s, 1H, Rha-H-1), 5.03 (d, 1H, J ¼ 1.4 Hz, Rha-H-1),
4.53 (d, 1H, J ¼ 7.8 Hz, H-10), 4.39 (q, 1H, J ¼ 7.8 Hz, H-16), 4.13e4.08
(m, 1H, Rha-H-5), 3.95e3.90 (m, 1H, Rha-H-5), 3.82 (dd,1H, J ¼ 11.9,
1.9 Hz, H-6a0), 3.68e3.65 (m, 4H, H-6b0, Rha-H-3), 3.61e3.57 (m,
2H, Rha-H-3), 3.54 (dd, 1H, J ¼ 3.7, 1.9 Hz, Rha-H-2), 3.45 (s, 3H,
OMe), 3.44 (s, 3H, OMe), 3.46e3.44 (m,1H, Rha-H-2), 3.40e3.29 (m,
H-20, H-30, H-40, H-50, Rha-H-4, H-26), 2.32 (d, 1H, J ¼ 12.4 Hz), 1.25
(d, 3H, J ¼ 6.0 Hz, Rha-6-Me),1.22 (d, 3H, J ¼ 6.0 Hz, Rha-6-Me), 0.96
(d, 3H, J ¼ 7.3 Hz, Me), 0.86 (s, 3H, Me), 0.79 (d, 3H, J ¼ 6.4 Hz, Me),
0.78 (s, 3H, Me), 0.70 (td, 1H, J ¼ 11.0, 3.7 Hz); 13C NMR (CD3OD,
600 MHz):
d
5.9 (d, 1H, J ¼ 1.7 Hz, Rha-H-1), 4.81 (d, 1H, J ¼ 1.6 Hz,
Rha-H-1), 4.53 (d, 1H, J ¼ 8.2 Hz, H-10), 4.40 (q, 1H, J ¼ 7.7 Hz, H-16),
4.22e4.19 (m, 1H, Rha-H-5), 3.95e3.91 (m, 1H, Rha-H-5), 3.88 (dd,
1H, J ¼ 3.3, 1.6 Hz, Rha-H-2), 3.80 (dd, 1H, J ¼ 3.3, 2.2 Hz, Rha-H-2),
3.79 (m, 2H, Rha-H-3), 3.75 (dd, 1H, J ¼ 9.9, 3.3 Hz, Rha-H-3), 3.70
(dd, 1H, J ¼ 9.9, 3.3 Hz, Rha-H-3), 3.64 (dd, 1H, J ¼ 12.1, 4.4 Hz, H-
6b0), 3.68e3.66 (m, 1H, H-26a), 3.56 (t, 1H, J ¼ 8.8 Hz, H-30), 3.55 (s,
3H, OMe), 3.54 (s, 3H, OMe), 3.51 (t,1H, J ¼ 8.8 Hz, H-40), 3.39 (t-like,
1H, J ¼ 8.8, 8.2 Hz, H-20), 3.29e3.27 (m, 1H, H-5), 3.10 (t-like, J ¼ 9.9,
9.4 Hz, Rha-H-4), 3.06 (t-like, 1H, J ¼ 10.4, 9.4 Hz, Rha-H-4), 2.34 (d,
1H, J ¼ 12.6 Hz), 1.26 (d, 3H, J ¼ 6.1 Hz, Rha-Me), 1.24 (d, 3H,
J ¼ 6.6 Hz, Rha-Me), 0.96 (d, 3H, J ¼ 7.1 Hz, Me), 0.86 (s, 3H, Me),
0.79 (d, 3H, J ¼ 6.1 Hz, Me), 0.79 (s, 3H, Me), 0.70 (td, 1H, J ¼ 11.0,
150 MHz): d 110.5, 100.2, 99.2, 82.5, 82.1, 79.8, 79.3, 78.7, 77.9, 76.7,
74.3, 74.1, 72.1, 72.0, 70.5, 69.9, 69.7, 67.9, 63.8, 59.2, 57.3, 55.3, 52.8,
47.9, 42.9, 42.7, 41.7, 41.0, 38.7, 37.6, 35.3, 32.7, 32.4, 31.5, 30.4, 29.9,
29.3, 22.1, 18.0, 17.9, 17.5, 16.9, 14.9, 13.9, 9.2; ESI-HRMS calcd for
C47H78NaO17 [M þ Na]þ 937.5131, found 937.5134.
6.6. Synthesis of chlorogenin 3
b-O-[2,4-di-O-(3-O-methyl-
a-L
-
3.8 Hz); 13C NMR (CD3OD, 150 MHz):
d 110.6, 102.8, 99.8, 84.5, 84.2,
rhamnopyranosyl)- -glucopyranoside] (8)
b
-
D
82.2, 79.7, 78.7, 76.7, 72.5, 72.2, 69.9, 69.6, 68.7, 67.9, 63.8, 6.1, 57.3,
55.3, 52.6, 47.9, 42.9, 42.8, 41.7, 38.6, 37.7, 32.7, 32.4, 31.4, 30.8, 30.2,
29.1, 22.1, 18.2, 18.1, 17.5, 17.0, 14.9, 9.3; ESI-HRMS calcd for
C47H79O17 [M þ H]þ 915.5311, found 915.5311.
ꢀ
To a mixture of 22a (50.0 mg, 0.10 mmol) and 4 A molecular
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (4.0
mL,
0.02 mmol). After stirred at ꢁ20 ꢀC for 5 min donor 25 (147.0 mg,
0.50 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for
20 min and then at r.t. for 8 h. Then the reaction was quenched with
Et3N. The solid was then filtered off. The filtrate was concentrated
and purified by silica gel column chromatography (EtOAc/petro-
leum ether, 1:4e1:2) to give 29 as an amorphous solid.
6.8. Synthesis of chlorogenin 3b-O-[3-O-methyl-2,4-di-O-(a-L-
rhamnopyranosyl)- -glucopyranoside] (10)
b-D
ꢀ
To a mixture of 41 (25.0 mg, 0.03 mmol) and 4 A molecular
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (1.0
mL,
Compound 29 was dissolved in CH3OH/CH2Cl2 (20 mL,
v:v ¼ 1:1), and then NaOMe was added until pH ¼ 8.5. After stirred
at 35 ꢀC for 4 h, the solution was neutralized with ion-exchange
resin (Hþ) and then filtered and concentrated. The residue was
purified by silica gel column chromatography (CH2Cl2/MeOH,
10:1e4:1) to afford 8 as a white amorphous solid (24.5 mg, 49% for
two steps): Rf ¼ 0.45 (CH2Cl2/MeOH, 5:1); 1H NMR (CD3OD,
0.01 mmol). After stirred at ꢁ20 ꢀC for 5 min 17 (92.3 mg,
0.15 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for
20 min and at r.t. for 8 h. The reaction was quenched with Et3N and
the solid was filtered off.
The residue was dissolved in MeOH (5 mL) and 10%-Pd/C
(500.0 mg) was added. After stirred for 24 h at room temperature
under an atmosphere of hydrogen gas, the mixture was filtered
through Celite pad, and the filtrate was concentrated.
600 MHz): d 5.21 (s, 1H, Rha-1-H), 4.89 (s, 1H, Rha-H-1), 4.54 (d, 1H,
J ¼ 7.7 Hz, H-10), 4.39 (q,1H, J ¼ 7.4 Hz, H-16), 4.17e4.15 (m, 2H, Rha-
H-5, Rha-H-2), 4.04 (d, 1H, J ¼ 2.8 Hz, Rha-H-2), 3.98e3.96 (m, 1H,
Rha-H-5), 3.81e3.79 (m, 1H, H-6a0), 3.70e3.65 (m, 1H, H-6b0), 3.58
(t, 1H, J ¼ 9.2 Hz, H-40), 3.54 (t, 1H, J ¼ 9.2 Hz, H-30), 3.48e3.44 (m,
2H, Rha-H-4, Rha-H-4), 3.44 (s, 3H, OMe), 3.43 (s, 3H, OMe), 3.39 (t,
1H, J ¼ 8.3 Hz, H-20), 3.38e3.27 (m, 4H, H-20, H-50, Rha-H-3, Rha-H-
The residue was dissolved in CH3OH/CH2Cl2 (20 mL, v:v ¼ 1:1),
and NaOMe was added until pH ¼ 8.5. After stirred at 35 ꢀC for 4 h,
the solution was neutralized with ion-exchange resin (Hþ) and then
filtered and concentrated. The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 10:1e4:1) to afford 10 as
syrup (13.1 mg, 46% for three steps): Rf ¼ 0.65 (CHCl3/MeOH, 3:1);

N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
323
1H NMR (DMSO-d6, 600 MHz):
d
4.83 (s, 1H, Rha-H-1), 4.80 (t, 1H,
6.11. Synthesis of 6 -OH chlorogenin 3
b
b-O-[2,4-di-O-(a-L-
J ¼ 6.0 Hz, OH), 4.76e4.74 (m, 4H, Rha-H-1, H-10and 2ꢂ OH), 4.70
(d, 1H, J ¼ 4.6 Hz, OH), 4.55 (t, 1H, J ¼ 6.4 Hz, OH), 4.48 (d, 1H,
J ¼ 6.8 Hz, OH), 4.36 (d, 1H, J ¼ 5.5 Hz, OH), 4.26 (q,1H, J ¼ 7.3 Hz, H-
16), 3.98e3.95 (m, 1H, Rha-H-5), 3.69e3.67 (m, 2H, Rha-H-5 and H-
20), 3.60e3.47 (m, 6H), 3.40 (s, 3H, OMe), 3.27e3.13 (m, 6H), 1.10 (d,
3H, J ¼ 6.4 Hz, Rha-Me), 1.08 (d, 3H, J ¼ 6.0 Hz, Rha-Me), 0.90 (d, 3H,
J ¼ 6.9 Hz, Me), 0.74 (s, 3H, Me), 0.72e0.70 (m, 6H), 0.60 (td, 1H,
rhamnopyranosyl)-b-D-glucopyranoside] (13)
To a solution of 31c (35 mg, 0.019 mmol) in dry CH2Cl2/MeOH
(10 mL, v:v ¼ 1:1) was added NaOMe to make the pH value of the
mixture at 10. Afterstirred at r.t. for24h, thesolutionwasneutralized
with ion-exchange resin (Hþ) and then filtered and concentrated.
The residue was purified by flash chromatography (CH2Cl2/MeOH,
3:1) to give 13 as a white amorphous solid (11.0 mg, 65%). Rf ¼ 0.25
J ¼ 6.0, 4.0 Hz); 13C NMR (DMSO-d6, 150 MHz):
d 108.3, 100.3, 97.0,
85.5, 80.1, 75.8, 75.3, 74.9, 73.4, 71.8, 71.7, 70.5, 68.7, 68.3, 67.2, 65.8,
61.8, 61.5, 55.4, 53.3, 50.7, 45.4, 41.9, 41.0, 37.0, 35.9, 33.3, 31.4, 31.2,
30.8, 29.7, 28.9e28.4, 27.0, 22.0, 17.9, 17.8, 16.1, 14.6, 13.9, 13.0; ESI-
HRMS calcd for C46H76NaO17 [M þ Na]þ 923.4975, found 923.4975.
(CH2Cl2/MeOH, 4:1); 1H NMR (CD3OD, 400 MHz): 5.19 (s,1H, H-100),
d
4.83 (s, 1H, H-1000), 4.53e4.51 (d, 1H, J ¼ 7.4 Hz, H-10), 4.41e4.36 (m,
1H), 4.18e4.13 (m, 1H), 3.94e3.89 (m, 2H), 3.84e3.76 (m, 4H),
3.71e3.47 (m, 6H), 3.46e3.33 (m, 5H), 1.04 (s, 3H), 0.98e0.94 (d, 4H,
J ¼ 7.0 Hz), 0.83 (s, 3H), 0.81e0.78 (d, 3H, J ¼ 6.3 Hz), 2.02e0.68 (m);
6.9. Synthesis of chlorogenin 3
b
-O-[6-O-methyl-2,4-di-O-(
a-L
-
13C NMR (CD3OD, 100 MHz):
d 110.6, 103.0, 102.4, 82.2, 80.0, 79. 7,
rhamnopyranosyl)- -glucopyranoside] (11)
b
-
D
79.0, 78.1, 76.6, 74.1, 73.7, 72.5, 72.4, 72.2, 70.7, 69.7, 67.9, 63.9, 62.0,
57.3, 55.8, 42.9, 41.8, 41.1, 40.7, 39.9, 36.9, 32.7, 32.4, 31.4, 30.8, 30.6,
29.9, 22.0, 18.0, 17.9, 17.5, 17.0, 14.9; ESI-HRMS calcd for C45H74NaO17
[M þ Na]þ 909.4818, found 909.4818.
ꢀ
To a mixture of 43 (100.0 mg, 0.10 mmol) and 4 A molecular
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (4.0
mL,
0.02 mmol). After stirred at ꢁ20 ꢀC for 5 min and then 17 (340.0 mg,
0.50 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for
20 min and then at r.t. for 8 h. Then the reaction was quenched with
Et3N and the solid was filtered off. The filtrate was concentrated
and purified by silica gel column chromatography (EtOAc/petro-
leum ether, 1:3) to give an amorphous solid.
6.12. Synthesis of 6-O-
a-L
-rhamnopyranosyl-chlorogenin 3
b-O-
[2,4-di-O-( -rhamnopyranosyl)-b-D
a
-
L
-glucopyranoside] (14)
ꢀ
To a mixture of 22d (100.0 mg, 0.04 mmol) and 4 A molecular
sieves in dried CH2Cl2 (4 mL) at ꢁ20 ꢀC was added TMSOTf (2.0
mL,
The solid was dissolved in CH3OH/CH2Cl2 (20 mL, v:v ¼ 1:1), and
NaOMe was added until pH ¼ 8.5. After stirred at 35 ꢀC for 4 h, the
solution was neutralized with ion-exchange resin (Hþ) and then
filtered and concentrated. The residue was purified by silica gel
column chromatography (CH2Cl2/MeOH, 8:1) to afford 11 as a white
amorphous solid (48.7 mg, 44% for two steps): Rf ¼ 0.65 (CHCl3/
0.01 mmol). After stirred at ꢁ40 ꢀC for 5 min 17 (244.1 mg,
0.20 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for
20 min and then at r.t. for 8 h. The reaction was quenched with Et3N
and the solid was filtered off. The filtrate was concentrated and
purified by silica gel column chromatography (EtOAc/petroleum
ether, 8:1) to give 31d as a white solid (95.4 mg, 55%).
MeOH, 3:1); 1H NMR (CD3OD, 600 MHz):
d
5.19 (d, 1H, J ¼ 1.3 Hz,
Compound 31d (95.4 mg, 0.04 mmol) was dissolved in CH3OH/
CH2Cl2 (10 mL, v:v ¼ 1:1), and then NaOMewas added until pH ¼ 8.5.
After stirring at 35 ꢀC for 4 h, the solution was neutralized with ion-
exchange resin (Hþ) and then filtered and concentrated. The residue
was purified by silica gel column chromatography (CH2Cl2/MeOH,
10:1e4:1) to afford 14 as a white amorphous solid (18.1 mg, 40%):
Rha-H-1), 4.77 (d, 1H, J ¼ 1.9 Hz, Rha-H-1), 4.50 (d, 1H, J ¼ 7.7 Hz, H-
10), 4.40 (q, 1H, J ¼ 7.3 Hz, H-16), 4.16e4.11 (m, 1H, Rha-H-5),
3.97e3.92 (m, 1H, Rha-H-5), 3.92 (dd, 1H, J ¼ 3.2, 1.9 Hz, Rha-H-
2), 3.82 (dd, 1H, J ¼ 3.7, 1.8 Hz, Rha-H-2), 3.67 (dd, 1H, J ¼ 9.6,
3.7 Hz, Rha-H-3), 3.68e3.30 (m, 7H, H-60, H-50, H-40, H-30, H-20, H-
26), 2.31 (brd, 1H, J ¼ 12.4 Hz), 1.25 (d, 3H, J ¼ 6.4 Hz, Rha-Me), 1.24
(d, 3H, J ¼ 6.4 Hz, Rha-Me), 0.96 (d, 3H, J ¼ 6.9 Hz, Me), 0.86 (s, 3H,
Me), 0.80 (d, 3H, J ¼ 5.5 Hz, Me), 0.79 (s, 3H, Me), 0.70 (td, 1H,
Rf ¼ 0.29 (CH2Cl2/MeOH, 4:1); 1H NMR (CD3OD, 600 MHz):
d 5.26 (s,
1H, Rha-H-1), 4.85 (s,1H, Rha-H-1), 4.75 (s,1H, Rha-H-1), 4.50 (d,1H,
J ¼ 8.2 Hz, H-10), 4.43 (q,1H, J ¼ 6.8 Hz, H-16), 4.20e4.15 (m,1H, Rha-
H-5), 3.94e3.90 (m, 3H, 2ꢂ Rha-H-2, H-50), 3.84 (dd, 1H, J ¼ 3.2,
1.8 Hz, Rha-H-2), 3.79 (brd,1H, J ¼ 11.0 Hz, H-6a0), 3.70e3.52 (m, 6H,
2ꢂ Rha-H-5, 2ꢂ Rha-H-3, H-6b0, H-26, Rha-H-4), 3.50e3.46 (m, 1H,
H-3), 3.43e3.29 (m, 6H, 2ꢂ Rha-H-4, H-20, H-30, H-40, Rha-H-3), 2.16
(brd, 1H, J ¼ 11.9 Hz), 2.09 (dt, 1H, J ¼ 11.9, 4.1 Hz), 1.26 (d, 3H,
J ¼ 6.4 Hz, Rha-Me), 1.25 (d, 3H, J ¼ 6.0 Hz, Rha-Me), 1.24 (d, 3H,
J ¼ 6.0 Hz, Rha-Me), 0.96 (d, 3H, J ¼ 6.9 Hz, Me), 0.88 (s, 3H, Me), 0.81
(d, 3H, J ¼ 6.4 Hz, Me), 0.80 (s, 3H, Me), 0.72 (td, 1H, J ¼ 11.9, 4.1 Hz);
J ¼ 10.5, 4.4 Hz); 13C NMR (CD3OD, 150 MHz):
d 110.7, 102.5, 100.6,
82.3, 79.9, 79.5, 79.0, 78.1, 75.7, 74.1, 73.9, 72.6, 72.3, 70.7, 70.1, 69.8,
68.0, 64.0, 57.5, 55.5, 52.9, 43.1, 42.8, 41.9, 41.2, 38.8, 37.8, 35.4, 32.8,
32.6, 31.6, 30.5, 30.0, 29.5, 22.2, 18.1, 18.0, 17.7, 17.0, 16.0, 14.0; ESI-
HRMS calcd for C46H76NaO17 [M þ Na]þ 923.4975, found 923.4975.
6.10. Synthesis of laxogenin 3b-O-[2,4-di-O-(a-L-rhamnopyranosyl)-
b-D-glucopyranoside] (12)
13C NMR(CD3OD, 150 MHz):
d 110.7, 104.0, 103.1, 102.1, 82.1, 80.7,
To a solution of 31b (22.0 mg, 0.013 mmol) in dry CH2Cl2/MeOH
78.6, 78.5, 76.6, 74.1, 73.8, 72.6, 72.3, 70.8, 70.1, 69.8, 68.0, 63.9, 57.5,
55.2, 51.8, 43.1, 41.9, 41.5, 41.2, 38.7, 37.8, 35.4, 32.8, 32.5, 31.7, 30.8,
30.6, 30.0, 22.2, 18.0, 17.9, 17.6, 17.1, 15.0, 14.0; ESI-HRMS calcd for
C51H84NaO21 [M þ Na]þ 1055.5397, found 1055.5398.
(10 mL, v:v ¼ 1:1) was added NaOMe to make the pH ¼ 10. The
mixture was stirred at r.t. for 24 h at 35 ꢀC. The solution was
neutralized with ion-exchange resin (Hþ) and then filtered and
concentrated. The residue was purified by flash chromatography
(CH2Cl2/CH3OH, 3:1) to give 12 as a white amorphous solid (4.0 mg,
34%): Rf ¼ 0.30 (CH2Cl2/CH3OH, 3:1); 1H NMR (CD3OD, 400 MHz):
6.13. Synthesis of 6b-O-benzoyl chlorogenin (20c)
d
5.19 (s, 1H, H-100), 4.83 (s, 1H, H-1000), 4.53e4.51 (d, 1H, J ¼ 7.4 Hz,
Compound 34 (741.8 mg, 1.42 mmol) was dissolved in pyridine
H-10), 4.44e4.37 (m, 1H), 4.13e4.06 (m, 1H), 3.95e3.90 (m, 2H),
3.84e3.33 (m, 16H), 1.04 (s, 3H), 0.98e0.94 (d, 4H, J ¼ 6.7 Hz),
0.81e0.76 (d, 9H, J ¼ 11.7 Hz), 2.35e0.84 (m); 13C NMR (CD3OD,
(10 mL), and benzoyl chloride (0.33 mL) was added dropwise at
0
ꢀC. The solution was stirred for 1 h at 0 ꢀC. The reaction mixture
was diluted with EtOAc and washed consecutively with water,
1 mol/L HCl aqueous solution, saturated NaHCO3 solution and
brine. The organic layer was dried (Na2SO4), filtered and concen-
trated. The residue was purified by silica gel column chromatog-
raphy (EtOAc/petroleum ether, 1:20) to provide 35 as white solid
(818.4 mg, 92%): Rf ¼ 0.15 (EtOAc/petroleum ether, 1:20); 1H NMR
100 MHz):
d 213.4, 110.6, 103.0, 102.4, 99.8, 81.9, 80.0, 79.5, 78.0,
77.5, 76.6, 74.0, 72.5, 72.3, 72.2, 72.1, 70.7, 69.9, 67.9, 63.7, 62.0, 57.5,
57.4, 54.9, 47.5, 42.9, 42.2, 42.1, 40.6, 38.8, 37.7, 32.5, 32.4, 31.4, 30.7,
29.9, 27.0, 22.4, 18.0, 17.9, 17.5, 16.8, 14.8, 13.5; ESI-HRMS calcd for
C45H72NaO17 [M þ Na]þ 907.4662, found 907.4662.

324
N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
(CDCl3, 400 MHz):
d
8.03 (d, 2H, J ¼ 7.0 Hz), 7.56e7.54 (m, 1H),
36.4, 31.6, 31.3, 30.3e27.8, 26.2, 21.3, 17.1, 16.4, 14.5, 13.1; ESI-HRMS
calcd for C57H72NaO15 [M þ Na]þ 1019.4763, found 1019.4763.
7.46e7.44 (m, 2H), 7.32e7.30 (m, 4H), 7.26e7.24 (m, 1H), 5.21 (d,
1H, J ¼ 2.7 Hz), 4.53 (d, 1H, J ¼ 11.7 Hz), 4.51 (d, 1H, J ¼ 11.7 Hz), 4.38
(q, 1H, J ¼ 7.8 Hz), 3.48e3.35 (m, 3H), 1.21 (s, 3H), 0.98 (d, 3H,
J ¼ 6.04 Hz), 0.90e0.88 (m, 7H).
6.16. Synthesis of 6
b
-O-benzoyl chlorogenin 3b-O-(3,6-di-O-
benzoyl-2,4-di-O-levulinoyl-
b-D-glucopyranoside) (21c)
Compound 35 (751.7 mg, 1.20 mmol) was dissolved in CH2Cl2/
EtOH (20 mL, v:v ¼ 1:1) containing 2 drops of Et3N. 10%-Pd/C
(200.0 mg) was added. After stirred for 12 h at room temperature
under an atmosphere of hydrogen gas, the mixture was filtered
through Celite pad and the filtrate was concentrated. The residue
was purified by silica gel column chromatography (EtOAc/petro-
leum ether, 1:2) to give 20c as a white solid (610.1 mg, 95%):
Rf ¼ 0.28 (EtOAc/petroleum ether, 1:3); 1H NMR (CDCl3, 400 MHz):
To a mixture of 20c (75.1 mg, 0.14 mmol), imidate 19 (153.8 mg,
0.21 mmol) and powdered 4 A molecular sieves in dried CH2Cl2
ꢀ
(10 mL) at ꢁ20 ꢀC was added TMSOTf (6.8
mL, 0.04 mmol). After
stirred at ꢁ20 ꢀC for 0.5 h, the reaction was quenched with Et3N.
The solid was then filtered off. The filtrate was concentrated and
purified by silica gel column chromatography (EtOAc/petroleum
ether, 1:3) to give 21c as a white solid (75.6 mg, 49%): Rf ¼ 0.3
(EtOAc/petroleum ether, 1:3); 1H NMR (CDCl3, 400 MHz):
d
8.03 (d, 2H, J ¼ 7.0 Hz), 7.58e7.56 (m, 1H), 7.46e7.44 (m, 2H), 5.21
(d, 1H, J ¼ 2.7 Hz), 4.38 (q, 1H, J ¼ 7.4 Hz), 3.66e3.64 (m, 1H),
3.47e3.45 (m, 1H), 3.37 (t, 1H, J ¼ 11.0 Hz), 1.21 (s, 3H), 0.98 (d, 3H,
J ¼ 7.0 Hz), 0.90e0.88(m, 7H). ESIMS: calcd for [M þ H]þ m/z 537.4;
found: 537.4.
d 8.08e7.92 (m, 6H), 7.55e7.53 (m, 3H), 7.44e7.39 (m, 6H), 5.49 (t,
1H, J ¼ 9.8 Hz), 5.28 (t, 1H, J ¼ 9.4 Hz), 5.19e5.18 (m, 1H), 5.08 (t, 1H,
J ¼ 9.3 Hz), 4.70 (d,1H, J ¼ 7.8 Hz), 4.55e4.53 (m,1H), 4.46e4.35 (m,
2H), 3.95e3.93 (m, 1H), 3.61e3.59 (m, 1H), 3.46e3.40 (m, 1H),
3.37e3.35 (m, 1H), 2.78 (s, 6H), 1.98 (s, 3H), 1.89 (s, 3H), 0.96 (d, 3H,
6.14. Synthesis of chlorogenin 6-O-(2,3,4-tri-O-benzoyl-
a-L
-
J ¼ 6.7 Hz); 13C NMR (CDCl3, 150 MHz):
d 205.8, 205.4, 176.8e165.8,
rhamnopyranosyide) (20d)
133.3e128.4, 109.2, 99.8, 83.5, 80.7, 79.9, 74.1, 73.1, 71.9, 71.6, 69.1,
66.8, 62.8, 62.1, 55.9, 53.9, 46.4, 42.5, 41.6, 40.6, 39.9, 39.5, 38.1, 37.8,
36.5, 35.6, 31.7e27.8, 23.9, 20.8, 19.3, 17.1, 16.6, 15.9, 14.5; ESI-HRMS
calcd for C64H78NaO16 [M þ Na]þ 1125.5182, found 1125.5182.
To a mixture of 32 (100.0 mg, 0.20 mmol), imidate 17 (154.0 mg,
ꢀ
0.30 mmol) and powdered 4 A molecular sieves in dried CH2Cl2
(10 mL) at ꢁ20 ꢀC was added TMSOTf (6.8
mL, 0.04 mmol). After
stirred at ꢁ20 ꢀC for 0.5 h, the reaction was quenched with Et3N.
The solid was then filtered off. The filtrate was concentrated and
purified by silica gel column chromatography (EtOAc/petroleum
ether, 1:20e1:10) to give 36 as a white solid (180.1 mg, 96%).
Compound 36 (180.1 mg, 0.18 mol) was dissolved in EtOAc
(5 mL) and EtOH (5 mL) and then 10%-Pd/C (50 mg) was added.
After stirred for 24 h at room temperature under an atmosphere of
hydrogen gas, the mixture was filtered through Celite pad, and the
filtrate was concentrated. The residue was purified by silica gel
column chromatography (EtOAc/petroleum ether, 1:2) to give 20d
as a white solid (142.7 mg, 87%): Rf ¼ 0.45 (EtOAc/petroleum ether,
6.17. Synthesis of laxogenin 3b-O-(3,6-di-O-benzoyl-b-D-
glucopyranoside) (22b)
Compound 21b (76.1 mg, 0.076 mmol) was dissolved in dry
CH2Cl2/MeOH (5 mL, v:v ¼ 4:1), and AcOH$NH2NH2 (140.0 mg,
1.52 mmol) was added. The solution was stirred at r.t. for 3 h. The
mixture was diluted with CH2Cl2 and washed consecutively with
water, 1 mol/L HCl aqueous solution, saturated NaHCO3 solution
and brine. The organic layer was dried (Na2SO4), filtered and
concentrated. The residue was purified by silica gel column chro-
matography (EtOAc/petroleum ether, 2:3) to provide 22b as syrup
(51.1 mg, 84%): Rf ¼ 0.2 (EtOAc/petroleum ether, 2:3); 1H NMR
1:2); 1H NMR (CDCl3, 600 MHz):
d
8.09 (d, 2H, J ¼ 7.7 Hz, PhH), 7.98
(d, 2H, J ¼ 6.0 Hz, PhH), 7.82 (d, 2H, J ¼ 7.7 Hz, PhH), 7.61 (t, 1H,
J ¼ 7.1 Hz, PhH), 7.53e7.25 (m, 8H, PhH), 5.80 (d, 1H, J ¼ 7.1 Hz, H-
30), 5.66 (t, 1H, J ¼ 9.4 Hz, H-40), 5.62 (brs, 1H, H-20), 5.01 (s, 1H, H-
10), 4.41 (q, 1H, J ¼ 7.1 Hz, H-16), 4.29e4.23 (m, 1H, H-50), 3.69 (brs,
1H, OH), 3.47e3.41 (m, 2H, H-26a, H-3), 3.35 (t, 1H, J ¼ 11.0 Hz, H-
26b), 1.31 (d, 3H, J ¼ 6.1 Hz, 60-Me), 0.97 (d, 3H, J ¼ 7.1 Hz, Me), 0.86
(s, 3H, Me), 0.77 (d, 3H, J ¼ 5.0 Hz, Me), 0.71 (s, 3H, Me); ESIMS:
calcd for [M þ H]þ m/z 891.5; found: 891.9.
(CDCl3, 400 MHz): d 8.12e8.06 (m, 4H), 7.62e7.56 (m, 2H),
7.48e7.40 (m, 4H), 5.22e5.19 (t, 1H, J ¼ 3.0 Hz), 4.72e4.62 (m, 2H),
4.61e4.56 (d, 1H, J ¼ 7.8 Hz, H-10), 4.45e4.36 (m, 1H), 3.77e3.73 (s,
2H), 3.72e3.61 (m, 2H), 3.50e3.43 (m, 1H), 3.40e3.32 (m, 2H),
3.00e2.83 (d, 1H, J ¼ 13.7 Hz), 2.47 (s, 1H), 1.98 (s, 3H), 1.72 (s, 3H),
1.25 (s, 6H), 0.98 (s, 3H), 0.96 (s, 3H); 13C NMR (CDCl3, 150 MHz):
d
210.2, 167.9, 166.7, 133.6, 133.2, 130.1, 129.9, 128.6, 128.5, 128.4,
109.3, 101.3, 80.4, 74.4, 72.1, 69.9, 66.9, 63.8, 62.1, 55.6, 56.5, 53.8,
46.7, 41.6, 40.9, 39.5, 37.3, 36.5, 31.9e28.7, 26.4, 22.7, 21.3, 17.1, 16.4,
14.5, 14.1, 13.2; ESIMS: calcd for [M þ H]þ m/z 801.4; found: 801.2.
6.15. Synthesis of laxogenin 3b-O-(3,6-di-O-benzoyl-2,4-di-O-
levulinoyl- -glucopyranoside) (21b)
b-D
6.18. Synthesis of 6
b-O-benzoyl chlorogenin 3b-O-(3,6-di-O-
To a mixture of 20b (60.6 mg, 0.14 mmol), imidate 19 (153.8 mg,
0.21 mmol) and powdered 4 A molecular sieves in dried CH2Cl2
benzoyl- -glucopyranoside) (22c)
b-D
ꢀ
(10 mL) at ꢁ20 ꢀC was added TMSOTf (6.8
m
L, 0.04 mmol). After
Compound 21b (104.1.1 mg, 0.094 mmol) was dissolved in dry
CH2Cl2/MeOH (5 mL, v:v ¼ 4:1), and AcOH$NH2NH2 (176.7 mg,
1.92 mmol) was added. The solution was stirred at r.t. for 3 h. The
mixture was diluted with CH2Cl2 (100 mL) and washed consecu-
tively with water (10 mL), 1 mmol/L HCl aqueous (3 ꢂ 10 mL),
saturated NaHCO3 solution (10 mL) and brine (10 mL). The organic
layer was dried (Na2SO4), filtered and concentrated. The residue
was purified by silica gel column chromatography (EtOAc/petro-
leum ether, 1:2) to provide 22b as syrup (62.5 mg, 73%): Rf ¼ 0.2
(EtOAc/petroleum ether, 1:2); 1H NMR (CDCl3, 400 MHz):
stirred at ꢁ20 ꢀC for 0.5 h, the reaction was quenched with Et3N. The
solid was then filtered off. The filtrate was concentrated and purified
by silica gel column chromatography (EtOAc/petroleum ether, 2:3)
to give 21b as a white solid (56.1 mg, 40%): Rf ¼ 0.15 (EtOAc/petro-
leum ether, 2:3); 1H NMR (CDCl3, 400 MHz):
d
8.07 (d, 2H, J ¼ 7.4 Hz),
7.97 (d, 2H, J ¼ 7.0 Hz), 7.56 (m, 2H), 7.44 (m, 4H), 5.50 (t, 1H,
J ¼ 9.4 Hz), 5.31 (t,1H, J ¼ 10.2 Hz), 5.14 (t,1H, J ¼ 9.4 Hz), 4.73 (d,1H,
J ¼ 7.8 Hz), 4.60e4.55 (m,1H), 4.49e4.39 (m, 2H), 3.95e3.92 (m,1H),
3.59e3.50 (m, 2H), 3.36 (t, 1H, J ¼ 11.4 Hz), 2.78 (s, 1H), 2.04 (s, 3H),
1.98 (s, 3H), 0.97 (d, 3H, J ¼ 6.7 Hz), 0.80 (d, 3H, J ¼ 5.9 Hz), 0.78 (s,
d
8.10e8.03 (m, 4H), 8.03e7.98 (d, 2H, J ¼ 7.0 Hz), 7.61e7.53 (m, 3H),
3H), 0.73 (s, 3H); 13C NMR (CDCl3, 150 MHz):
d
210.1, 205.8,
7.48e7.40 (m, 6H), 5.19e5.13 (s, 2H), 4.72e4.62 (m, 2H), 4.55e4.50
(d, 1H, J ¼ 7.8 Hz, H-10), 4.42e4.36 (m, 1H), 3.78e3.67 (m, 2H),
3.67e3.59 (m, 2H), 3.48e3.43 (m, 1H), 3.40e3.37 (m, 2H), 2.47 (s,
175.5e165.8, 133.4e128.4, 109.3, 99.2, 80.4, 78.4, 73.2, 71.9, 71.6,
69.1, 66.9, 62.8, 62.0, 56.5, 53.8, 46.7, 41.6, 40.9, 39.5, 37.9, 37.8, 37.2,

N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
325
1H), 1.17 (s, 3H), 0.80 (s, 3H); 13C NMR (CDCl3, 150 MHz): 167.9,
166.7, 166.3, 133.5, 133.3, 133.2, 132.9, 130.5, 130.0, 129.8, 129.6,
129.3, 128.4, 109.2, 101.3, 80.6, 79.2, 78.7, 78.5, 74.3, 74.0, 72.1, 69.8,
66.8, 63.8, 62.1, 55.9, 53.9, 46.4, 41.6, 40.6, 39.9, 38.1, 36.5, 35.6, 31.6,
31.4, 31.4, 30.7, 30.3, 29.4, 28.7, 20.8, 17.1, 16.6, 15.9, 14.5. ESI-HRMS
calcd for C54H66NaO12 [M þ Na]þ 929.4447, found 929.4446.
J ¼ 7.7 Hz, Ph), 7.44 (t, 2H, J ¼ 7.7 Hz, Ph), 7.35e7.28 (m, 5H, Ph), 4.96
(td,1H, J ¼ 11.0, 5.0 Hz, H-3), 4.60 (d,1H, J ¼ 12.1 Hz, PhCHH), 4.56 (d,
1H, J ¼ 11.5 Hz, PhCHH), 4.37 (q, 1H, J ¼ 7.1 Hz, H-16), 4.31 (d, 1H,
J ¼ 7.7 Hz, H-10), 3.76 (dd, 1H, J ¼ 9.9, 3.8 Hz, H-6a0), 3.76 (dd, 1H,
J ¼ 10.4, 5.5 Hz, H-6b0), 3.60 (s, 3H, OMe), 3.51 (t,1H, J ¼ 9.4 Hz, H-40),
3.48e3.42 (m, 2H, H-26 and H-50), 3.36 (td, 1H, J ¼ 8.8, 2.8 Hz, H-20),
3.11 (t, 1H, J ¼ 8.8 Hz, H-30), 0.96 (d, 3H, J ¼ 7.1 Hz, 21-Me), 0.94 (s,
3H, 18-Me), 0.78 (s, 3H, 19-Me), 0.78 (d, 3H, J ¼ 6.0 Hz, 27-Me);
6.19. Synthesis of laxogenin 3b-O-[3,6-di-O-benzoyl-2,4-di-O-(2,3,4-
tri-O-benzoyl - -rhamnopyranosyl)-
a
-
L
b-D-glucopyranoside] (31b)
13C NMR (CDCl3, 150 MHz):
d 166.2, 137.8, 132.9, 130.5e127.7, 109.3,
109.2, 108.6, 101.3, 85.2, 80.6, 78.5, 74.0, 73.9, 73.6, 72.8, 71.5, 70.5,
66.8, 62.0, 60.5, 56.0, 53.7, 48.7, 41.5, 40.5, 39.9, 39.8, 39.5, 37.9, 37.1,
36.8, 33.7, 31.6, 31.3, 30.2, 29.2, 28.8, 20.9, 17.1, 16.4, 14.5, 13.4;
ESIMS: calcd for [M þ H]þ m/z 803.5; found: 803.4.
ꢀ
To a mixture of 22b (80.0 mg, 0.10 mmol) and 4 A molecular
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (4.0
mL,
0.04 mmol). After stirred at ꢁ20 ꢀC for 5 min donor 17 (309.5 mg,
0.5 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for 20 min
and then at r.t. for 8 h. The reaction was quenched with Et3N. The
solid was then filtered off. The filtrate was concentrated and puri-
fied by silica gel column chromatography (EtOAc/petroleum ether,
1:4) to give 31b as an amorphous solid (51.5 mg, 30%): Rf ¼ 0.45
(EtOAc/petroleum ether, 1:2); 1H NMR (CDCl3 þ D2O, 400 MHz):
6.22. Synthesis of 6
a-O-benzoyl chlorogenin 3b-O-(6-O-methyl-b-
D-glucopyranoside) (42)
To a mixture of 20a (573.6 mg, 1.0 mmol), imidate 39 (832.9 mg,
ꢀ
1.28 mmol) and powdered 4 A molecular sieves in dried CH2Cl2
d
8.16e7.12 (m, 40H), 5.93e5.75 (m, 2H), 5.72e5.60 (m, 2H),
(20 mL) at 0 ꢀC was added TMSOTf (20.5
mL, 0.21 mmol). After stir-
5.58e5.38 (m, 5H), 5.20 (m, 1H), 5.08 (m, 1H), 5.03e4.92 (m, 3H),
4.52e3.30 (m, 7H), 2.35e0.64 (m); ESI-HRMS calcd for
C101H104NaO25 [M þ Na]þ 1739.6759, found 1739.6759.
ring at 0 ꢀC for 0.5 h and then at r.t. for 0.5 h, the reaction was
quenched with Et3N. The solid was then filtered off. The filtrate was
concentrated. The residue was dissolved in CH3OH/CHCl3 (60 mL,
v:v ¼ 1:1), and then NaOMe was added until pH ¼ 8.5. After stirredat
r.t. for 4 h, the solutionwas neutralized with ion-exchange resin (Hþ)
and then filtered and concentrated. The residue was purified by
silica gel column chromatography (CHCl3/MeOH ¼ 30:1) to afford 42
as a white amorphous solid (529.8 mg, 70% for two steps): Rf ¼ 0.47
6.20. Synthesis of 6b-O-benzoyl chlorogenin 3b-O-[3,6-di-O-
benzoyl-2,4-di-O-(2,3,4-tri-O-benzoyl -
a-
L
-rhamnopyranosyl)-b-D-
glucopyranoside] (31c)
To a mixture of 22b (90.6 mg, 0.10 mmol) and 4 A molecular
(CH2Cl2/MeOH ¼ 15:1); 1H NMR (DMSO-d6, 600 MHz):
d 7.96 (d, 2H,
ꢀ
sieves in dried CH2Cl2 (10 mL) at ꢁ20 ꢀC was added TMSOTf (4.0
m
L,
J ¼ 7.3 Hz, Ph), 7.66 (t, 1H, J ¼ 6.9 Hz, Ph), 7.52 (t, 1H, J ¼ 7.3 Hz, Ph),
4.97e4.83 (m, 3H, OH ꢂ 3), 4.29e4.28 (m,1H), 4.15 (d,1H, J ¼ 7.3 Hz,
H-10), 3.55e3.32 (m, 6H), 3.23 (s, 3H, OMe), 3.02 (t,1H, J ¼ 8.8 Hz, H-
40), 2.95 (t,1H, J ¼ 9.2 Hz, H-30), 2.84 (t,1H, J ¼ 7.8 Hz, H-20),1.99 (t,1H,
J ¼ 6.4 Hz), 0.90 (d, 3H, J ¼ 6.9 Hz, 21-Me), 0.88 (s, 3H,18-Me), 0.74 (s,
3H, 19-Me), 0.70 (d, 3H, J ¼ 6.4 Hz, 27-Me); 13C NMR (DMSO-d6,
0.04 mmol). After stirred at ꢁ20 ꢀC for 5 min donor 17 (309.5 mg,
0.5 mmol) was added. The mixture was stirred at ꢁ20 ꢀC for 20 min
and then at r.t. for 8 h. The reaction was quenched with Et3N. The
solid was then filtered off. The filtrate was concentrated and puri-
fied by silica gel column chromatography (EtOAc/petroleum ether,
1:5) to give 31b as an amorphous solid (51.0 mg, 28%): Rf ¼ 0.55
(EtOAc/petroleum ether, 1:2); 1H NMR (CDCl3, 400 MHz):
150 MHz):
d 165.5, 133.3, 130.1, 129.3, 129.0, 108.6, 101.5, 80.3, 77.1,
76.8, 75.5, 73.5, 72.7, 72.1, 70.1, 66.1, 62.0, 58.7, 55.4, 53.1, 48.2, 41.3,
40.4, 37.6, 36.7, 36.6, 35.5, 31.4, 31.0, 30.0, 29.7, 28.8, 28.7, 20.7, 17.2,
16.4, 14.8, 13.2; ESIMS: calcd for [M þ Na]þ m/z 735.4; found: 735.4.
d
8.12e8.06 (m, 4H), 7.94e7.80 (m, 10H), 7.77e7.73 (m, 2H),
7.68e7.65 (m, 2H), 7.56e7.48 (m, 6H), 7.44e7.34 (m, 14H), 7.24e7.10
(m, 7H), 5.80e5.74 (m, 1H), 5.66 (t, 2H, J ¼ 11.4 Hz), 5.56e5.40 (m,
3H), 5.35 (s, 1H), 5.18 (s, 1H), 5.05 (d like, 2H, J ¼ 9.8 Hz), 4.99e4.95
(m, 1H), 4.82 (d, 1H, J ¼ 7.4 Hz), 4.72e4.69 (m, 1H), 4.49e4.43 (m,
1H), 4.43e4.35 (m, 1H), 4.16e4.10 (m, 1H), 4.07e4.01(m, 2H),
3.94e3.88 (m, 1H), 3.82e3.76 (m, 1H), 3.49e3.44 (m, 1H), 3.39 (d,
1H, J ¼ 11.7 Hz), 2.04e2.02 (m, 1H), 1.14 (s, 3H), 0.79 (s, 3H),
2.05e0.71 (m); ESI-HRMS calcd for C108H110NaO26 [M þ Na]þ
1845.7178, found 1845.7178.
6.23. Synthesis of 6
a-O-benzoyl chlorogenin 3b-O-(3-O-benzoyl-6-
O-methyl- -glucopyranoside) (43)
b-D
To a mixture of 42 (466.0 mg, 0.70 mmol) and 1-BBTZ (470.8 mg,
2.0 mmol) in dried CH2Cl2 (20 mL) was added Et3N (0.25 mL,
1.8 mmol). After stirred at r.t. overnight, the mixture was concen-
trated and purified by silica gel column chromatography (EtOAc/
petroleum ether/CHCl3, 1:8:2) to give 43 as a white solid (281.8 mg,
53%): Rf ¼ 0.42 (EtOAc/petroleum ether, 1:2); 1H NMR (CDCl3,
6.21. Synthesis of 6a-O-benzoyl chlorogenin 3b-O-(3-O-methyl-6-
O-benzyl- -glucopyranoside) (41)
b
-D
600 MHz)
d
8.06e7.55 (m, 10H, AreH), 5.11 (t, 1H, J ¼ 9.2 Hz, H-30),
4.96 (td, 1H, J ¼ 11.0, 4.6 Hz, H-6), 4.45 (d, 1H, J ¼ 7.7 Hz, H-10), 4.37
(d, 1H, J ¼ 6.8 Hz, H-16), 3.77 (t, 1H, J ¼ 9.2 Hz, H-40), 3.71e3.64 (m,
3H, H-26, H-6a0), 3.58 (dd, 1H, J ¼ 9.1, 7.3 Hz, H-20), 3.51e3.44 (m,
2H, H-50, H-6b0), 3.36 (t, 1H, J ¼ 10.5 Hz), 0.96 (d, 3H, J ¼ 6.2 Hz, Me),
0.94 (s, 3H, Me), 0.77 (s, 3H, Me), 0.78 (d, 3H, J ¼ 7.7 Hz, Me); 13C
To a mixture of 20a (100.0 mg, 0.18 mmol), imidate 38 (120.0 mg,
ꢀ
0.22 mmol) and powdered 4 A molecular sieves in dried CH2Cl2
(10 mL) at 0 ꢀC was added TMSOTf (5
mL, 0.02 mmol). After stirred at
0 ꢀC for 0.5 h and then at r.t. for 0.5 h, the reactionwas quenched with
Et3N. The solid was then filtered off. The filtrate was concentrated
and purified bysilica gel column chromatography (EtOAc/petroleum
ether, 1:10) to give 39 as a white solid (162.0 mg, 86%).
NMR (CDCl3, 150 MHz):
d 166.3, 133.5, 132.9, 130.4, 130.1, 129.6,
128.4, 109.3, 100.7, 80.6, 78.4, 76.0, 74.0, 73.3, 72.9, 72.3, 70.9, 66.8,
62.0, 59.6, 56.0, 53.7, 48.7, 41.6, 40.6, 39.8, 37.9, 36.8, 33.7, 31.6, 31.3,
30.3, 29.0, 28.7, 20.9, 17.1,16.4,14.5,13.4; ESIMS: calcd for [M þ Na]þ
m/z 839.4; found: 839.6.
Compound 39 was dissolved in CH3OH/CHCl3 (20 mL, v:v ¼ 1:1),
and then NaOMe was added until pH ¼ 8.5. After stirred at r.t. for
4 h, the solution was neutralized with ion-exchange resin (Hþ) and
filtered and concentrated. The residue was purified by silica gel
column chromatography (CHCl3/MeOH, 30:1) to afford 41 as a white
amorphous solid (51.0 mg, 40%): Rf ¼ 0.48 (CHCl3/MeOH, 10:1); 1H
6.24. Plasmids and cell lines
HA (QH) gene is from a high pathogenic H5N1 influenza virus in
goose (Goose/Qinghai/59/05). HA (QH) was cloned into pcDNA3 and
NMR (CDCl3, 600 MHz):
d
8.03 (d, 2H, J ¼ 8.3 Hz, Ph), 7.56 (t, 1H,

326
N. Ding et al. / European Journal of Medicinal Chemistry 53 (2012) 316e326
[4] L.H. Pinto, L.J. Holsinger, R.A. Lamb, Influenza virus M2 protein has ion channel
activity, Cell 69 (1992) 517e528.
[5] A.J. Hay, C.A. Thompson, A. Geraghty, S. Hayhurst, S. Grambas, M.S. Bennett,
The role of the M2 protein in influenza virus infection, in: C. Hannoun (Ed.),
Options for the Control of Influenza Virus II, Excerpta Medica, Amsterdam,
1993, pp. 281e288.
[6] R. Bright, M. Medina, X. Xu, G. Perez-Oronoz, T. Wallis, X. Davis, L. Povinelli,
N. Cox, A. Klimov, Incidence of adamantine resistance among influenza A
(H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern,
Lancet 366 (2005) 1175e1181.
[7] http://www.fda.gov/drugs/drugsafety/informationbydrugclass/
ucm100228.html.
sequences were confirmed.Acodon-optimizedHA geneis fromA/Viet
Nam/1203/2004 (H5N1) and was cloned into pcDNA3. NA A/PR/8/34
influenza virus (H1N1) in the vector pEF6/V5-His-TOPO was kindly
provided by John C Olsen (University of North Carolina, Chapel Hill).
A549 was purchased from ATCC, and 293T was from Institute of
Basic Medical Sciences, Chinese Academy of Medical Sciences and
Peking Union College. Both cell lines were maintained in medium
according to the protocols supplemented with 10% FBS and peni-
cillin/streptomycin (25 units/ml).
[8] M. Kiso, K. Mitamura, Y. Sakai-Tagawa, K. Shiraishi, C. Kawakami, K. Kimura,
F.G. Hayden, N. Sugaya, Y. Kawaoka, Resistant influenza A viruses in children
treated with oseltamivir: descriptive study, Lancet 364 (2004) 759e765.
[9] J.J. Skehel, D.C. Wiley, Receptor binding and membrane fusion in virus entry:
the influenza hemagglutinin, Annu. Rev. Biochem. 69 (2000) 531e569.
[10] G.P. Song, S. Yang, W. Zhang, Y.L. Cao, P. Wang, N. Ding, Z.H. Zhang, Y. Guo,
Y.X. Li, Discovery of the first series of small molecule H5N1 entry inhibitors,
J. Med. Chem. 52 (2009) 7368e7371.
[11] H. Hofmann, K. Hattermann, A. Marzi, T. Gramberg, M. Geier, M. Krumbiegel,
S. Kuate, K. Uberla, M. Niedrig, S. Pohlmann, S protein of severe acute respi-
ratory syndrome-associated coronavirus mediates entry into hepatoma cell
lines and is targeted by neutralizing antibodies infected patients, J. Virol. 78
(2004) 6134e6142.
6.25. Production of HA/HIV and VSVG/HIV pseudovirions
Human embryonic kidney 293T cells were transiently co-
transfected with 8
and 5 g NA or 3
m
g hemagglutinin envelope expression plasmid
m
m
g VSVG envelope expression plasmid with 10
mg
Env-deficient HIV vector (pNL4-3-Luc-RꢁEꢁ) in 100-mm plates by
a standard Ca3(PO4)2 protocol. Sixteen hours post-transfection, cells
werewashedby PBS w/o Ca2þ, Mg2þ, thenadded 10 mL freshmedium
into each plate. Forty-eight hours post-transfection, the supernatants
were collected and filtered through a 0.45-micron pore size filter
(Nalgene) and the pseudovirions were directly used for infection.
[12] L. Yi, Z. Li, K. Yuan, X. Qu, J. Chen, G. Wang, H. Zhang, H. Luo, L. Zhu, P. Jiang,
L. Chen, Y. Shen, M. Luo, G. Zuo, J. Hu, D. Duan, Y. Mie, X. Shi, W. Wang, Y. Han,
T. Li, Y. Liu, M. Ding, H. Deng, X. Xu, Small molecules blocking the entry of
severe acute respiratory syndrome coronavirus into host cells, J. Virol. 78
(2004) 11334e11339.
6.26. Infection assay of HIV pseudovirions
[13] Y. Guo, R.B. Emily, J.Z. Wang, H.X. Xiao, J. Yu, J.W. Wang, L. Guo,
F.G. George, Y.J. Cao, C. Michael, L.J. Rong, Analysis of hemagglutinin-
To investigate the effect of the test compounds, A549 cells were
seeded into 24-well plates at 5 ꢂ 104/well and tested compounds
were added to each well incubating for 15 min followed by adding
HA/HIV or VSVG/HIV pseudovirions to each well and incubating at
mediated entry tropism of H5N1 avian influenza, Virol. J.
6 (2009).
doi:10.1186/1743-422X-6-39.
[14] W. Wang, E. Butler, V. Veguilla, R. Vassell, J.T. Thomas, M. Moos, Z. Ye,
K. Hancock, C.D. Weiss, Establishment of retroviral pseudotypes with
influenza hemagglutinins from H1, H3, and H5 subtypes for sensitive and
specific detection of neutralizing antibodies, J. Virol. Methods 153 (2008)
111e119.
[15] C.H. Li, T.T. Guo, P. Wang, H.S. Guan, Y.X. Li, Semisynthesis of several
stigmasterol saponins, Chin. J. Chem. 24 (2006) 917e922.
[16] M.S. Cheng, M.C. Yan, Y. Liu, L.G. Zheng, J. Liu, Synthesis of
b-hederin and
37 ꢀC. Cells were lysed in 50
mL of cell culture lysis reagent (Prom-
ega) 48 h post-infection. The luciferase activity was measured with
a luciferase assay kit (Promega) and an FB15 luminometer (Berthold
detection system) according to the supplier’s protocols.
hederacolchiside A1: triterpenoid saponins bearing a unique cytotoxicity-
inducing disaccharide moiety, Carbohydr. Res. 341 (2006) 60e67.
[17] T. Heidelberg, O.R. Martin, Synthesis of the glycopeptidolipid of mycobacte-
rium avium serovar 4: first example of a fully synthetic C-mycoside GPL,
J. Org. Chem. 69 (2004) 2290e2301.
[18] B. Kryczka, J. Lewkowski, A. Zawisza, Product class 15: glycosyl oxygen
compounds (except di- and oligosaccharides), in: Science of Synthesis,
Houben-Weyl Methods of Molecular Transformations, Georg Thieme Verlag,
Stuttgart, New York, 2007, pp. 971e1055.
[19] G.O. Aspinall, A.M. Crane, D.W. Gammon, I.H. Ibrahim, N.K. Khare, Synthesis of
allyl glycosides for conversion into neoglycoproteins bearing epitopes of
mycobacterial glycolipid antigens, Carbohydr. Res. 216 (1992) 337e355.
[20] R.R. Schmidt, A. Toepfer, Glycosylation with highly reactive glycosyl
donors: efficiency of the inverse procedure, Tetrahedron Lett. 32 (1991)
3353e3356.
Acknowledgment
This work was supported by National Natural Science Founda-
tion of China (81072525 and 30701035), the Central level, Scientific
Research Institutes for Basic R & D Special Fund Business (2010ZD03
and 2011ZD06) and the Fundamental Research Funds for the
Central Universities (10FX061).
References
[1] http://www.who.int/csr/disease/avian_influenza/country/cases_table_2011_
06_22/en/index.html.
[2] G. Neumann1, T. Noda, Y. Kawaoka, Emergence and pandemic potential of
swine-origin H1N1 influenza virus, Nature 459 (2009) 931e939.
[3] S.M. Wintermeyer, M.C. Nahata, Rimantadine: a clinical perspective, Ann.
Pharmacother. 29 (1995) 299e310.
[21] B. Yu, H.C. Tao, Glycosyl trifluoroacetimidates. 2. Synthesis of dioscin and
xiebai saponin I, J. Org. Chem. 67 (2002) 9099e9102.
[22] I. Izzo, F.D. Riccardis, G. Sodano, Synthesis of (25R)-5
,6 ,15 ,16 ,26-pentol, a cytostatic starfish steroid, J. Org. Chem. 63 (1998)
4438e4443.
a-cholestane-
3
b
b
a
b