Structure-activity relationships of 3-O-β-chacotriosyl oleanane-type triterpenoids as potential H5N1 entry inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.04.061

A series of 3-O-β-chacotriosyl oleanolic acid analogs have been designed, synthesized and evaluated as H5N1 entry inhibitors based on a small molecule inhibitor saponin 1 previously discovered by us. Detailed structure-activity relationships (SARs) studies on the aglycone of compound 1 indicated that the subtle modification of oleanolic acid as an aglycon has key influences on the antiviral activity. These results suggested that either the introduction of a disubstituted amide structure at the 17-COOH of OA or alteration of the C-3 configuration of OA from 3β-to 3α-forms can significantly improve the selective index while maintaining their antiviral activities in vitro. Compound 8 was selected for further mechanistic study because of its distinguished inhibition activity and good selective index. Molecular simulation study and surface plasmon resonance analysis confirmed that compound 8 stabilized HA2 subunit of hemagglutinin (HA) by binding with amino acid residues LYS-26, ASN-53, ASN-27 and ASN-50, therefore may prevent HA from conformational rearranging, which is a critical step for viral entry.

Full text of DOI:10.1016/j.ejmech.2016.04.061

Accepted Manuscript
Structure-activity relationships of 3-O-β-chacotriosyl oleanane-type triterpenoids as
potential H5N1 entry inhibitors
Gaopeng Song, Xintian Shen, Sumei Li, Yibin Li, Hongzong Si, Jihong Fan, Junhua
Li, Erqiang Gao, Shuwen Liu
PII:
S0223-5234(16)30354-3
10.1016/j.ejmech.2016.04.061
EJMECH 8576
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 September 2015
Revised Date: 22 April 2016
Accepted Date: 23 April 2016
Please cite this article as: G. Song, X. Shen, S. Li, Y. Li, H. Si, J. Fan, J. Li, E. Gao, S. Liu, Structure-
activity relationships of 3-O-β-chacotriosyl oleanane-type triterpenoids as potential H5N1 entry inhibitors,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.061.
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ACCEPTED MANUSCRIPT
A series of 3-O-β-chacotriosyl oleanolic acid analogs have been designed, synthesized and
evaluated as H5N1 entry inhibitor, of which compound 8 showed the strongest inhibition activity
with IC50 of 2.4 ꢀM.

ACCEPTED MANUSCRIPT
Relationships of
Structure-activity
3-O-β-chacotriosyl
oleanane-type triterpenoids as Potential H5N1 Entry Inhibitors
Gaopeng Songa*,1, Xintian Shen b,c,1 Sumei Lid, Yibin Lia, Hongzong Sie, Jihong Fana,
Junhua Lia, Erqiang Gaoa, Shuwen Liub*
aCollege of Materials and Energy, South China Agricultural University, Guangzhou, 510642,
China
bDepartment of Pharmacy, Maternal and Child Health Hospital of Bao’an District, Shenzhen,
518133, China.
cSchool of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China
dDepartment of Human anatomy, School of Medicine, Jinan University, Guangzhou, 510632,
China
eInstitute for Computational Science and Engineering, Qingdao University, Qingdao, 266071,
China
Abstract:
A series of 3-O-β-chacotriosyl oleanolic acid analogs have been designed, synthesized and
evaluated as H5N1 entry inhibitors based on a small molecule inhibitor saponin 1 previously
discovered by us. Detailed structure-activity relationships (SARs) studies on the aglycone of
compound 1 indicated that the subtle modification of oleanolic acid as an aglycon has key
influences on the antiviral activity. These results suggested that either the introduction of a
disubstituted amide structure at the 17-COOH of OA or alteration of the C-3 configuration of OA
from 3β- to 3α-forms can significantly improve the selective index while maintaining their
antiviral activities in vitro. Compound 8 was selected for further mechanistic study because of its
distinguished inhibition activity and good selective index. Molecular simulation analysis
confirmed that compound 8 stabilized HA2 subunit of hemagglutinin (HA) by binding with amino
acid residues LYS-26, ASN-53, ASN-27 and ASN-50, therefore may prevent HA from
conformational rearranging, which is a critical step for viral entry.
Keywords: 3-O-β-chacotriosyl saponins, H5N1 entry inhibitors, structure-activity relationships
1. Introduction
H5N1 avian influenza A virus has caused a serious impact on global morbidity,
mortality, and economy since 2009 [1, 2]. To date, there are two types of
* Corresponding author. Tel.: +86- 20- 6164 8538; fax: +86 -20 -6164 8655
E-mail address: liusw@smu.edu.cn (S. Liu).
* Corresponding author. Tel.: + 86-20-85280293; fax: + 86-20-85280292
E-mail address: vinsin1021@126.com(G. Song).
1These authors contributed equally to this work
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anti-influenza drugs have been approved for interruption of specific processes in
influenza infection. The one is the inhibitors of M2 ion channel such as amantadine
and rimantadine, which can stop the release of viral RNA into the cytoplasm [3]. The
other is the neuraminidase inhibitors such as zanamivir, oseltamivir, and peramivir,
which inhibit the activity of the enzyme and cause the tethered progeny virus to be
unable to separate from its host cells [4, 5]. However, the drug-resistant influenza
viruses emerged rapidly, which limited the use of those drugs [6-8]. Therefore, there
is an urgent task to discover novel anti-influenza drugs with new mechanism of action
to combat H5N1 virus.
The earliest and most critical step 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]. Thus, to identify and develop
potent entry inhibitors against H5N1 virus represents a favorable target for drug
discovery like many viruses entry inhibitors applied by the similar entry way [10, 11].
Pentacyclic triterpenes found in different plant organs, have received considerable
attention due to the increased understanding of their wide spectrum of antiviral
activities against several viruses such as HIV, IAV, and HCV [12-14]. It has been
proved that the defense activities of these triterpenes stem from their ability to prevent
various pathogen and herbivore infections in the host [15]. For example, betulinic acid
(BA), and its derivative bevirimat have been confirmed to exhibit inhibitory activity
against HIV entry [16, 17]. Zhou reported that echinocystic acid (EA), and its
derivatives showed strong inhibitory activity against HCV entry [18, 19].
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Glycyrrhizin (GL) is well known for its broad activity against several viruses in vitro
and in vivo including influenza A virus (IAV) [20].
Oleanolic acid (OA), an oleanane-type triterpene, is probably the most well-known
member of pentacyclic triterpenes. It is noteworthy that certain oleanane-type
triterpenoids with conserved structural features displayed in vitro anti-influenza virus
activity that is comparable to or higher than that of oseltamivir [21]. Particularly,
compound Y3, an OA-acetyl galactose conjugate, has displayed strong inhibitory
activity against IAV entry in vitro, of which biological effects are attributed to the
presence of the aglycon as well as of the sugar moiety [21]. In our previous study,
we have used an efficient HIV-based pseudotyping system to screen a saponin library
generated from semisynthesis, and discovered dioscin derivative 1 (Fig. 1) exhibited
good inhibitory activity against H5N1 entry with the IC50 values being 7.2-8.0 µM
[22]. Further SARs studies indicates that the trisaccharide moiety of compound 1,
namely chacotrioside is essential for activity and the subtle modifications of aglycone
of compound 1 may be tolerated without losing antiviral activity [22-24].
Based on the above results, given compound Y3 exhibits good antiviral activity
against IAV entry, in present communications, we replace the 6α-OH-diosgenin
residue of active compound 1 with OA to attempt the synthesis of several
3-O-β-chacotriosyl OA analogs 2-14 with the aim to investigate the SARs around OA
as aglycone. In order to study the influence of esterification of the 17-COOH group of
OA on the inhibitory activity, compounds 3-7 (Fig. 1) were synthesized. Compounds
8 and 9 (Fig. 1), bioisosteric surrogates of oleanolic acid ester, were derived to study
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the effects of acylation at the 17-COOH position of OA with alkyl amines on the
activity. Moreover, we investigated the effect of carbonyl group at the 17-COOH of
OA on the inhibitory activity. Erythrodiol methyl ether and oleanoaldehyde were
selected as the aglycone moieties and the resulting two saponins 10 and 11 with the
same 3-O-β-chacotriosyl residue were derived (Fig. 1). Compound 12 bears an α-OH
at the C-3 position when compared to compound 3 with a β-OH at the same position,
which provides a means to judge the effects of the C-3 configuration on the activity.
Structurally, OA belongs to oleanane-type triterpenes as do GA with both the C-29
COOH group shifted to C-28 and the incorporation of a carbonyl group at the C-11
position. Thus, two saponins 13 and 14 (Fig. 1) were synthesized to study the effect of
different types of oleanane-type triterpenes as the aglycone residue on the inhibitory
activity. The results of our dock studies are also presented to disclose the molecular
basis of the 3-O-β-chacotriosyl oleanane-type triterpenoids binding to HA.
2. Results and discussion
2.1. Chemistry
As depicted in Scheme 1, treatment of 3α-oleanolic acid 15 [25] with iodomethane
in the presence of K2CO3 in DMF provided 16 in good yield. Methyl esterification of
erythrodiol-3-benzyl ether 17 [26] was done by the similar route as that for compound
16 and then was subjected to 10 % Pd/C to afford erythrodiol-28-methyl ether 18.
3β-O-11-oxo-methyl oleanolate 19 [27] was prepared following literature procedures.
The preparation of the known compounds 3 and 14 was performed according to our
previous procedure [28]. As shown in Scheme 2, glycosylation of benzyl oleanolate
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20 [29], erythrodiol methyl ether 18, [olean-28-al-12-en-3α-ol (oleanoaldehyde)] 21
[26], methyl 3α-oleanolate 16, and 3β-O-11-oxo-methyl oleanolate 19 with
2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl trichloroacetimidate 22 [28] respectively
under the action of TMSOTf afforded the 3-O-β-glucopyranosides 23 [29] and 24-27.
Removal of the benzoyl groups with NaOMe in MeOH afforded 28 [29] and 29-32,
which were subjected to PivCl in the presence of pyridine to protect selectively the
3,6-OHs of the β-glucopyranosyl residues, leading to intermediates 33-37. Subsequent
glycosylation of the 2, 4-OHs in 33-37 with 2,3,4-tri-O-acetyl-L-rhamnopyranosyl
trichloroacetimidate 38 [28] with TMSOTf as the promoter, followed by deprotection
of the acyl groups with NaOH, provided target saponins 7, and 10-13, respectively.
The synthetic route toward target saponins 2, 4-6 and 8-9 is depicted in Scheme 3.
Hydrogenolysis of compound 7 over palladium/carbon in CH2Cl2-MeOH furnished
saponin 2 in quantitative yield. Treatment of 2 with different halohydrocarbon in the
presence of K2CO3 in DMF provided the target compounds 4-6. Protection of all the
OHs in 2 by using Ac2O in the presence of DMAP provided compound 39. The key
intermediate 39 was treated with oxalyl chloride to yield 28-acyl chloride, which was
then condensed with appropriate amines and finally deprotected with MeONa to
afford the corresponding target saponins 8 and 9.
2.2. Inhibition of the infection of H5N1 pseudovirus
The single-cycle pseudovirus was used instead of live H5N1 avian influenza virus
to evaluate the inhibitory activities of compounds 1-14 against H5N1 entry due to the
safety concerns in studying viral H5N1 pathogens. An HA/HIV pseudotyped virus
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model was performed to test saponins on H5N1 viral entry inhibitory effects, while
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. Our preceding study has shown that dioscin
derivative (1) has a good inhibitory effect on H5N1, and could inhibit H5N1 virus
production most effectively at the virus entry step of cell infection [22]. It should be
noted that the inhibitory activity of compound 1 is specific to hemagglutinin, since it
has no inhibitory activity toward VSV-G pseudovirus [22].
Compounds 2-14 were evaluated for the inhibitory activity against the entry of
H5N1 influenza virus based on an efficient HIV-based pseudotyping system with
luciferase report element established by us [9]. This pseudovirus was enveloped with
influenza glycoprotein HA and NA, and packed with HIV-backbone [9]. The results,
expressed as IC50, CC50 and SI (selectivity index), are illustrated in Table 1,
respectively. As shown in Table 1, all the compounds except compound 10 exhibited
inhibitory activity against H5N1 pseudovirus with a potency ranging from moderate
(IC50 > 5 ꢀM) to potent (IC50 < 5 ꢀM, which is comparable to the IC50 of a positive
compound CL-385319 [9, 30]). Notably, Compound 8 is the most active compound in
this study (Fig. 2 and Table 1).
It should be noted that the active compounds in series 3-9 and 12-14 show stronger
inhibition activity but lower cytotoxicity against MDCK cells than compound 1.
Representative compounds 3, 8 and 12 had no effect on VSV-G enveloped
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pseudovirus (Fig. 3A), which was similar as both compound 1 and HA targeted
compound CL-385319 [9, 30]. These results demonstrate that these compounds did
not inhibit HIV-backbone and luciferase reporting activity. In addition, compounds 3,
8 and 12 at 20 ꢀM•L-1 did not inhibit neuraminidase (NA) protein (Fig. 3B),
indicating that neuraminidase protein might not be the primary target of
3-O-β-chacotriosyl oleanolic acid analogs. Taken together, we postulated that
compounds 2-14 might interfere with the entry of influenza virus by targeting
hemagglutinin, the only other glycoprotein enveloped in the pseudovirus system.
2.3 SAR analysis of compounds 2-14
SAR exploration first focused on the influence of the oleanane-type triterpenes OA
and derivatives instead of diosgenin as aglycone residue on inhibition activity. As
expected, all of the compounds 2-9 exhibited higher or similar inhibitory activity in
relation to the lead compound 1, with the exception of compound 2, suggesting that
replacement of the aglycone moiety of compound 1 to oleanane-type triterpenes OA
with conserved structural features is able to enhance inhibition activity.
Noticeably, 3-O-β-chacotriosyl OA ester (compounds 3-7) exhibited more potent
anti-H5N1 activity than 2. The results verified the hypothesis that esterification of
carboxylic acid could significantly enhance the anti-H5N1activity, maybe due to the
better pharmacokinetic properties of ester derivatives 3-7 as prodrugs of 2. Among
ester derivatives, it was observed that the increase of the chain length at 17-COOH of
OA from one carbon to five carbons resulted in slightly reduced activity against
H5N1, while increasing the bulk of the ester derivatives from methyl group to
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cyclopentyl group or benzyl group could keep inhibition activity against H5N1.
Interestingly, there was significant difference in selectivity index (SI) among these
compounds when compared to compound 2, indicating that introduction of fit bulk
groups in the 17-COOH could significantly enhance SI while keeping inhibition
activity. The introduction of an amine structure at the 17-COOH position seemed to
have some impact on potency and selectivity index. Compounds 8 and 9 profiled
relatively stronger anti-H5N1 activity and better selectivity index compared with
compounds 3-7, revealing the importance of this structural modification for antiviral
activity. However, reduction of 17-COOH resulted in significantly decreasing the
activity against H5N1, such as compounds 10 and 11. The results suggested the C-17
group of carboxylic acid might play an essential role in the anti-H5N1 activity of
3-O-β-chacotriosyl oleanane-type triterpenoids, whereas reduction of COOH group
should be avoided.
The effects of configuration of 3-hydroxyl group on inhibition were further
examined. Taking into account the selectivity, the 3α-form 12 stood out among all
compounds evaluated, displaying similar inhibitory activity against H5N1 and lower
toxicity to MDCK cells than the 3β-forms 3. These results confirmed that the
configuration at C-3 is an important factor for the SI, rather than for anti-H5N1
activity. It seems that the diversity of structural skeleton of pentacyclic triterpenes
does not have a significant impact on the anti-H5N1 activity, since all the three types
of tested triterpenes 3, 13 and 14 exhibited similar inhibitory activity against H5N1.
Compound 8 had better selectivity indexes and comparable potency in the
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corresponding derivatives, suggesting compound 8 is exploitable as a lead compound.
Thus, compound 8 was chosen to investigate its mechanism of action against H5N1.
2.4 Compound 8 does not block H5 HA adsorption to chicken RBCs
The HA is able to absorb to chicken RBCs resulting in hemagglutination through
the interaction of the receptor-binding domain in HA1 subunit with the sialic acid
receptor in the RBC membrane. We investigated whether compound 8 can inhibit
hemagglutination by interfering with the H5 HA adsorption to RBCs. It was found
that compound 8 could not inhibit the adsorption of H5 HA to chicken RBCs even at
the highest concentration of 20 ꢀM, while the positive control antisera against H5
hemagglutinin (Anti-H5) could effectively inhibit H5 HA adsorption to chicken RBCs
with a titer at 1:16 (Fig. 4). This result indicated that compound 8 may not target the
receptor binding domain of the HA1. Taken together, we postulated that
3-O-β-chacotriosyl oleanolic acid derivatives could target to the membranes fusion
step medicated by the more conserved hemagglutinin transmembrance subunit HA2,
but not the receptor-binding domain of HA1.
2.5 Compound 8 interacted with hemagglutinin and HA2 subunit
HA-mediated fusion is mainly mediated by rearrangement of HA2 subunit at low
pH, therefore we detected interaction of compound 8 with HA and also HA2 subunit
at low pH by surface plasmon resonance (SPR). HA proteins of influenza
A/Vietnam/1194/2004(H5N1) (bought from Sino Biological Inc, Beijng, China) were
used as representative HA of H5N1 influenza strains. The sequences of HA protein of
influenza strains A/Thailand/Kan353/2004(H5N1) and A/Vietnam/1194/2004(H5N1)
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are 99% identical (561/563), with only two amino acid disparity in subunit HA1, the
sequences of HA2 subunits are 100% identical. Compound 8 potently interacted with
HA in a dose-dependent manner, and the equilibrium dissociation constant (KD) of
the interaction is 4.46e - 9 M (Fig 5A). Further study revealed that compound 8
potently interacted with HA2 subunit in a dose-dependent manner, the KD of the
interaction is 1.12e - 8 M (Fig 5B). Compound 8 did not interact with negative control
protein FKBP12 (FK506 Binding Protein 1A, 12kDa), indicated that interactions of
HA2 with compound 8 were specific (data not shown).
2.6 Molecular modeling for compound 8 binding to H5 hemagglutinin
We found that the HA2 sequence of A/Thailand/Kan353/2004 was 100% identical
to that of A/Vietnam/1194/2004, of which the neutral-pH crystal structure of the HA
has been well defined recently [9, 28]. On the basis of the previous results, we used
the neutral-pH crystal structure of the HA of H5N1 (Influenza strain
A/Vietnam/1194/2004, PDB: 2IBX) [9, 31] as a reference for our modeling work.
Using Surflex-Dock program of SYBYL 7.3, the predicted binding modes of
compound 8 is shown in Figure 6. Docking analysis displays that compound 8 can fit
inside the same cavity in the stem region of the HA2 near the fusion peptide like a
positive compound CL-385319 through Van der Waals interactions, hydrophobic
contacts, and hydrogen bonding networks. In our previous work, it was found that
compound CL-385319 might bind to both the residues M241, E251 and R3221 on the
A chain and the residues D1092, and F1102 on the B chain (subscripts 1 and 2 refer to
HA1 and HA2 subunits, respectively). However, as shown in Figure 6, at the top of
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cavity, compound 8 occupied the binding site with the C4-OH and C2-OH of the
L-rhamnose moiety linked to C4-OH of D-glucose forming stable hydrogen bonds
with LYS-261, and ASN-531 on the C chain, respectively. C6-OH of D-glucose and
C1-OH of the L-rhamnose moiety linked to C2-OH of D-glucose also could form
stable hydrogen bonds with ASN-502 and ASN-272 on the B chain, respectively. As
expected, the analysis of the binding models also gave our preceding study support,
suggesting that the chacotriosyl residue might have a very important reffect on the
anti-H5N1 activity, whereas simplifing the chacotriosyl moiety into an
α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl
moiety
or
α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl moiety should be avoided [22].
It is noteworthy that although compound 8 is different from CL-385319 in the
binding sites with the cavity surrounded by the residues, both of them are able to
block the formation of homotrimer of disulfide-linked HA1-HA2.
Conclusion
In summary, a series of 3-O-β-chacotriosyl oleanane-type triterpenes were designed
and synthesized as H5N1 entry inhibitors based on our previously discovered small
molecule inhibitor 1. The results verified the hypothesis that oleanane-type triterpenes
with conserved structural features as aglycon could significantly enhance the
anti-H5N1 activity in vitro. On the other hand, the preliminary SAR studies showed
that the anti-H5N1 activity of 3-O-β-chacotriosyl oleanane-type triterpenes depended
on the availability of the17-COOH groups, and the carbon chain functional groups at
17-COOH of OA. The above results indicated that the attachment of a disubstituted
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amide structure to 17-COOH of OA was helpful in enhancing inhibitory activity and
decreasing the toxicity against MDCK cells. Conversion of 3β-OH of oleanolic acid
to 3α-OH group can maintain antiviral activity and greatly enhance selectivity index.
Some 3-O-β-chacotriosyl oleanolic acid derivatives impaired the virus entry step of
cell infection by inserting into the cavity in the stem region of the HA2 near the fusion
peptide but the detail mechanism of action against H5N1 influenza virus need further
study. Among these derivatives, compound 8 exhibits excellent activity against H5N1
and may serve as a lead compound or scaffold for the further development of H5N1
entry inhibitors.
4. Experimental protocols
4.1. General methods
Solvents were purified in a conventional manner. Thin layer chromatography (TLC)
was performed on precoated E. Merck silica gel 60 F254 plates. Flash column
1
chromatography was performed on silica gel (200-300 mesh, Qingdao, China). H
13
NMR and C NMR spectra were taken on a JEOL JNM-ECP 600 spectrometer with
tetramethylsilane as an internal standard, and chemical shifts are recorded in ppm
values. Mass spectra were recorded on a Q-TOF Global mass spectrometer.
4.2 Methyl-3α-hydroxy-olean-12-en-28-oic acid (16)
To a solution of 3α-oleanolic acid (2.00 g, 4.38 mmol) in dry DMF (30 mL) was
added potassium carbonate (1.20 g, 8.76 mmol). After 30 min of stirring at room
temperature, iodomethane (0.82 mL, 13.14 mmol) was added and the mixture was
stirred for an additional 8 h. The solvents were evaporated and the crude residue was
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dissolved in a mixture of CH2Cl2 (150 mL) and hydrochloric acid (30 mL, 1.0 M).
The aqueous layer was extracted with CH2Cl2 (60 mL × 3). The combined organic
layer was washed with brine (150 mL), dried (Na2SO4) and concentrated under
reduced pressure. Recrystallization from EtOH provided the compound 16 (1.98 g,
95%) as a colourless solid; 1H NMR (CDCl3): δ 5.29 (t, 1H, J = 3.6 Hz, H-12), 3.63 (s,
3H, OCH3), 3.41 (t, 1H, J = 2.8 Hz, H-3), 2.87 (dd, 1H, J = 13.9, 4.5 Hz, H-18), 1.15
13
0.96, 0.94, 0.93, 0.91, 0.85, 0.73 (each s, each 3H, CH3); C NMR (CDCl3): δ 178.3
(C-28), 143.8 (C-13), 122.4 (C-12), 76.1, 51.5, 49.0, 47.4, 46.7, 45.9, 41.7, 41.3, 39.4,
37.3, 37.1, 33.9, 33.1, 32.9, 32.5, 32.4, 30.7, 28.3, 27.6, 26.1, 25.2, 23.6, 23.3, 23.1,
22.3, 18.3, 16.8, 15.1; HRESIMS calcd for C31H50O3Na 493.3652; found 493.3659.
4.3 Erythrodiol-28- methyl ether (18)
The compound 17 (2.10 g, 4.46 mmol) was dissolved in DMF (40 mL) and cooled to
0°C. Sodium hydride (60%, 446.0 mg, 11.16 mmol) was added, followed by
iodomethane (0.42 mL, 6.69 mmol) and TBAI (1.65 g, 4.46 mmol). As shown by
TLC, the reaction was complete after 6 h at room temperature. The mixture was
diluted with water (100 mL) and extracted with CH2Cl2 (200 mL × 3). The combined
organic extracts were combined, dried over Na2SO4, and concentrated. A mixture of
the above residue and 10% Pd/C (0.6 g) in THF (50 mL) was stirred at room
temperature under H2 at atmospheric pressure for 12 h. The reaction mixture was
filtered through Celite, and the insoluble substance was washed with THF (15 mL ×
3). The filtrate was concentrated in vacuo, and then purified by chromatography on a
silica gel column eluted with n-hexane-acetone (30:1) to afford 18 as a white solid
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(1.6 g, 89%). 1H NMR (CDCl3): δ 5.19 (t, 1H, J = 3.6 Hz, H-12), 3.36 (d, 1H, J = 9.2
Hz, OCH3-CH2-1), 3.31 (s, 3H, OCH3), 3.24 (dd, 1H, J = 11.3, 4.4 Hz, H-3), 2.90 (d,
1H, J = 9.2 Hz, OCH3-CH2-2), 1.99 (dd, 1H, J = 13.7, 4.6 Hz), 1.17, 1.01, 0.98, 0.92,
13
0.88, 0.87, 0.81 (each s, each 3H, CH3); C NMR (CDCl3): δ 144.7 (C-13), 122.1
(C-12), 80.1, 78.9, 59.4, 55.2, 47.6, 46.4, 42.7, 41.7, 39.8, 38.8, 38.6, 36.9, 36.5, 34.2,
33.2, 32.6, 32.0, 30.9, 29.7, 28.1, 27.2, 25.9, 25.7, 23.7, 23.5, 22.2, 18.4, 16.7, 15.6,
15.5; HRESIMS calcd for C31H52O2Na 479.3865; found 479.3868.
4.3. General procedure for the preparation of 23-27
To
a
solution
of
compounds
16,
and
18-21
(1
eq),
2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl trichloroacetimidate 22 (1.2 eq) and 4 Å
molecular sieves in dry CH2Cl2 was added TMSOTf (0.15 eq) at 0 °C under argon.
The reaction mixture was stirred for 0.5 h and warmed to room temperature for 0.5 h.
The reaction was quenched by Et3N and concentrated in vacuo. The residue was
purified by silica gel column chromatography (petroleum ether-EtOAc, 8:1) to give
compounds 23-27, respectively.
4.3.1
3β-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-erythrodiol-28-methyl
ether (24)
1
Compound 24 was synthesized as a white solid in 88% yield; H NMR (CDCl3): δ
7.27-8.03 (m, 20H, Ar-H), 5.91 (t, 1H, J = 9.7 Hz, H-3΄), 5.59 (t, 1H, J = 9.6 Hz,
H-4΄), 5.57 (dd, 1H, J = 9.7, 7.9 Hz, H-2΄), 5.18 (t, 1H, J = 3.7 Hz, H-12), 4.87 (d, 1H,
J = 7.9 Hz, H-1΄), 4.59 (dd, 1H, J = 11.9, 3.5 Hz, H-6΄-1), 4.55 (dd, 1H, J = 11.9, 6.6
Hz, H-6΄-2), 4.13-4.17 (m, 1H, H-5΄), 3.32 (d, 1H, J = 9.1 Hz, CH2OMe-1), 3.28 (s,
14

ACCEPTED MANUSCRIPT
3H, OCH3), 3.11 (dd, 1H, J = 11.8, 4.5 Hz, H-3), 2.87 (d, 1H, J = 9.1 Hz, CH2OMe-2),
13
1.12, 0.91, 0.89, 0.88, 0.86, 0.70, 0.64 (each s, each 3H, CH3); C NMR (CDCl3): δ
166.0, 165.9, 165.3, 165.0, 144.6 (C-13), 133.5, 133.2, 133.1, 129.9, 129.8 (two),
129.7 (two), 129.4, 128.9, 128.8, 128.4 (two), 128.3, 122.1 (C-12), 103.3 (C-1΄), 90.7,
80.1, 73.0, 72.2, 70.3, 63.4, 59.4, 55.4, 47.5, 46.4, 42.7, 41.6, 39.7, 38.8, 38.4, 36.5,
34.2, 33.3, 32.4, 32.0, 31.0, 27.6, 25.9 (two), 25.6, 23.7, 23.5, 22.2, 18.1, 16.7, 16.2,
15.4; HRESIMS calcd for C65H78O11Na 1057.5442; found 1057.5446.
4.3.2 3β-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-oleanoaldehyde (25)
1
Compound 25 was synthesized as a white solid in 80% yield; H NMR (CDCl3): δ
9.40 (s, 1H, CHO), 7.28-8.04 (m, 20H, Ar-H), 5.92 (t, 1H, J = 9.7 Hz, H-3΄), 5.60 (t,
1H, J = 9.7 Hz, H-4΄), 5.58 (dd, 1H, J = 9.7, 7.9 Hz, H-2΄), 5.36 (t, 1H, J = 3.5 Hz,
H-12), 4.88 (d, 1H, J = 7.9 Hz, H-1΄), 4.61 (dd, 1H, J = 12.1, 3.5 Hz, H-6΄-1), 4.56
(dd, 1H, J = 12.1, 6.6 Hz, H-6΄-2), 4.14-4.18 (m, 1H, H-5΄), 3.12 (dd, 1H, J = 11.8,
4.6 Hz, H-3), 2.65 (dd, 1H, J = 13.6, 4.5 Hz, H-18), 1.11, 0.95, 0.94, 0.84, 0.70, 0.69,
0.64 (each s, each 3H, CH3); 13C NMR (CDCl3): δ 207.5 (CHO), 166.0, 165.9, 165.3,
165.0, 143.0 (C-13), 133.4, 133.2, 133.1, 129.8, 129.7, 129.6, 129.4, 128.8 (two),
128.4 (two), 128.3 (two), 123.2 (C-12), 103.2 (C-1΄), 90.7, 73.0, 72.1, 72.0, 70.3, 63.4,
55.4, 49.1, 47.5, 45.6, 41.6, 40.4, 39.5, 38.7, 38.3, 36.6, 33.2, 33.1, 32.6, 30.7, 27.7
(two), 26.7, 25.8, 25.7, 23.4 (two), 22.1, 19.6, 18.0, 17.0, 16.2, 15.2; HRESIMS calcd
for C64H74O11Na 1041.5129; found 1041.5135.
4.3.3 3α-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-olean-12-en-28-oic acid
methyl ester (26)
15

ACCEPTED MANUSCRIPT
1
Compound 26 was synthesized as a white solid in 82% yield; H NMR (CDCl3): δ
7.28-8.02 (m, 20H, Ar-H), 5.92 (t, 1H, J = 9.7 Hz, H-3΄), 5.66 (t, 1H, J = 9.7 Hz,
H-4΄), 5.59 (dd, 1H, J = 9.8, 7.9 Hz, H-2΄), 5.10 (t, 1H, J = 3.4 Hz, H-12), 4.83 (d, 1H,
J = 7.9 Hz, H-1΄), 4.66 (dd, 1H, J = 12.0, 3.6 Hz, H-6΄-1), 4.52 (dd, 1H, J = 11.9, 5.4
Hz, H-6΄-2), 4.09-4.12 (m, 1H, H-5΄), 3.61 (s, 3H, OCH3), 3.38 (t, 1H, J = 3.1 Hz,
H-3), 2.82 (dd, 1H, J = 13.7, 4.1 Hz, H-18), 1.27, 1.02, 0.96, 0.95, 0.94, 0.77, 0.62
(each s, each 3H, CH3); 13C NMR (CDCl3): δ 178.3 (C-28), 166.1, 165.8, 165.3, 165.0,
143.4 (C-13), 133.4, 133.2, 133.0, 130.1, 129.8 (two), 129.7, 129.6, 129.0, 128.9,
128.4, 128.3 (two), 122.3 (C-12), 98.4 (C-1΄), 82.3, 73.1, 72.0, 71.8, 70.4, 63.2, 51.5,
49.0, 47.1, 46.8, 46.2, 41.7, 41.4, 39.2, 36.7, 36.5, 33.9, 33.2, 32.9, 32.5, 32.4, 30.7,
29.7, 28.5, 27.7, 25.7, 25.6, 23.7, 23.2, 22.9, 22.1, 20.9, 18.0, 16.8, 15.2; HRESIMS
calcd for C65H76O12Na 1071.5229; found 1071.5233.
4.3.4 3β-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-11-oxo-olean-12-en-28-oic
acid methyl ester (27)
1
Compound 27 was synthesized as a white solid in 83% yield; H NMR (CDCl3): δ
8.03 (dd, 2H, J = 8.0, 0.9 Hz, Ar-H), 7.95 (dd, 2H, J = 8.5, 1.3 Hz, Ar-H), 7.93 (dd,
2H, J = 8.4, 1.3 Hz, Ar-H), 7.85 (dd, 2H, J = 8.5, 1.3 Hz, Ar-H), 7.56 (tt, 1H, J = 7.4,
1.1 Hz, Ar-H), 7.51 (td, 2H, J = 7.4, 1.2 Hz, Ar-H), 7.44 (td, 3H, J = 7.9, 2.0 Hz,
Ar-H), 7.35-7.39 (m, 4H), 7.30 (t, 2H, J = 7.9 Hz, Ar-H), 5.92 (t, 1H, J = 9.7 Hz,
H-3΄), 5.64 (s, 1H, H-12), 5.59 (t, 1H, J = 9.7 Hz, H-4΄), 5.58 (dd, 1H, J = 9.7, 7.9 Hz,
H-2΄), 4.87 (d, 1H, J = 7.9 Hz, H-1΄), 4.66 (dd, 1H, J = 11.9, 3.2 Hz, H-6-1΄), 4.48 (dd,
1H, J = 12.0, 7.0 Hz, H-6-2΄), 4.14-4.17 (m, 1H, H-5΄), 3.63 (s, 3H, OCH3), 3.14 (dd,
16

ACCEPTED MANUSCRIPT
1H, J = 11.6, 4.8 Hz, H-3), 3.02 (dd, 1H, J = 13.9,4.0 Hz, H-1a), 2.79 (dt, 1H, J =
13.7, 3.4 Hz, H-18), 2.22 (s, 1H, H-9), 1.32, 1.06, 0.96, 0.95, 0.88, 0.70, 0.68 (each s,
13
each 3H, each CH3); C NMR (CDCl3): δ 200.1 (C-11), 177.5 (C-28), 178.7, 177.5,
168.7 (C-13), 128.0 (C-12), 104.8 (C-1΄), 90.1, 78.0, 74.1, 72.7, 70.1, 63.7, 61.7, 55.3,
51.9, 46.2, 45.0, 44.3, 43.4, 41.6, 39.3, 39.0, 38.8, 33.7, 32.9, 32.8, 31.6, 30.6, 28.2,
27.7, 27.2, 27.1, 25.8, 23.5, 23.4, 23.0, 18.9, 17.3, 16.6, 16.1; HRESIMS calcd for
C65H74O13Na 1085.5022; found 1085.5030.
4.4. General procedure for the preparation of 28-32
Compounds 23-27 were dissolved in CH2Cl2 and CH3OH (V:V = 1:1) and then
NaOMe was added until pH = 10, respectively. After stirred at r.t. for 6 h, the solution
was neutralized with Dowex 50 × 8 (H+) resin until pH = 7, filtered and concentrated
in vacuo. Then the residue was purified by silica gel column chromatography
(CH2Cl2-MeOH, 10:1) to provide compounds 28-32, respectively.
4.4.1 3β-O-(β-D-Glucopyranosyl)-erythrodiol-28-methyl ether (29)
1
Compound 29 was synthesized as a white solid in 95% yield; H NMR (CDCl3): δ
5.26 (s, 1H, OH), 5.19 (t, 1H, J = 3.1 Hz, H-12), 5.01 (s, 1H, OH), 4.37 (d, 1H, J =
7.8 Hz, H-1΄), 3.80-3.86 (m, 2H, H-6΄), 3.62 (t, 1H, J = 9.5 Hz, H-3΄), 3.54 (t, 1H, J =
8.6 Hz, H-4΄), 3.43-3.47 (m, 1H, H-5΄), 3.36 (d, 1H, J = 8.9 Hz, CH2OMe-1), 3.31 (s,
3H, OCH3), 3.27-3.30 (m, 1H, H-2΄), 3.17 (d, 1H, J = 10.9 Hz, H-3), 2.90 (d, 1H, J =
9.1 Hz, CH2OMe-2), 2.57-2.58 (m, 1H, H-18), 1.17, 1.04, 0.98, 0.95, 0.89, 0.88, 0.85
(each s, each 3H, CH3); 13C NMR (CDCl3): δ 144.6 (C-13), 122.0 (C-12), 105.2
(C-1΄), 90.1, 80.1, 76.3, 75.3, 73.8, 69.6, 61.7, 59.4, 55.5, 47.6, 46.4, 42.7, 41.7, 39.8,
17

ACCEPTED MANUSCRIPT
39.1, 38.8, 36.6, 36.5, 34.2, 33.3, 32.6, 32.0, 30.9, 28.0, 26.2, 26.0, 25.7, 23.7, 23.6,
22.2, 18.2, 16.7, 15.6; HRESIMS calcd for C37H62O7Na 641.4393; found 641.4396.
4.4.2 3β-O-(β-D-Glucopyranosyl)-oleanoaldehyde (30)
1
Compound 30 was synthesized as a white solid in 92% yield; H NMR (CDCl3): δ
9.41 (s, 1H, CHO), 5.36 (brs, 1H, H-12), 4.34 (d, 1H, J = 7.3 Hz, H-1΄), 3.78-3.83 (m,
2H, H-6΄), 3.59 (t, 1H, J = 8.8 Hz, H-3΄), 3.48-3.55 (m, 1H, H-4΄), 3.38-3.44 (m, 1H,
H-2΄), 3.27-3.28 (m, 1H, H-5΄), 3.14 (d, 1H, J = 10.8 Hz, H-3), 2.65 (d, 1H, J = 12.1
13
Hz, H-18), 1.14, 1.01, 0.94, 0.94, 0.91, 0.82, 0.74 (each s, each 3H, CH3); C NMR
(CDCl3): δ 207.4 (CHO), 143.0 (C-13), 123.1 (C-12), 105.2 (C-1΄), 90.1, 76.3, 75.3,
73.8, 69.4, 61.6, 55.5, 49.1, 47.6, 45.6, 41.7, 40.4, 39.6, 39.1, 38.6, 36.7, 33.1, 32.7,
30.7, 29.7, 28.0, 25.6, 23.4, 22.1, 18.2, 17.0, 16.7, 15.4; HRESIMS calcd for
C36H58O7Na 625.4080; found 625.4084.
4.4.3 3α-O-(β-D-Glucopyranosyl)-olean-12-en-28-oic acid methyl ester (31)
1
Compound 31 was synthesized as a white solid in 96% yield; H NMR (CDCl3): δ
5.30 (t, 1H, J = 3.3 Hz, H-12), 4.29 (d, 1H, J = 7.7 Hz, H-1΄), 3.83-3.86 (m, 2H, H-6΄),
3.65 (t, 1H, J = 9.2 Hz, H-3΄), 3.64 (s, 3H, OCH3), 3.57 (t, 1H, J = 9.1 Hz, H-4΄), 3.41
(t-like, 1H, J = 8.8, 8.1 Hz, H-2΄), 3.36 (brs, 1H, H-3), 3.28-3.31 (m, 1H, H-5΄), 2.87
(dd, 1H, J = 13.6, 4.0 Hz), 1.17, 0.94, 0.94, 0.93, 0.91, 0.86, 0.74 (each s, each 3H,
13
CH3); C NMR (CDCl3): δ 178.2 (C-28), 143.7 (C-13), 122.6 (C-12), 100.1 (C-1΄),
82.4, 76.4, 75.1, 73.6, 70.3, 62.3, 51.5, 49.9, 47.5, 46.7, 46.0, 41.8, 41.3, 39.5, 37.0,
36.9, 33.9, 33.4, 33.1, 32.5, 32.4, 30.7, 28.8, 27.7, 26.2, 23.6, 23.3, 23.1, 22.6, 21.1,
20.9, 18.1, 16.8, 15.3; HRESIMS calcd for C37H60O8Na 655.4180; found 655.4184.
18

ACCEPTED MANUSCRIPT
4.4.4 3β-O-(β-D-Glucopyranosyl)-11-oxo-olean-12-en-28-oic acid methyl ester (32)
1
Compound 32 was synthesized as a white solid in 95% yield; H NMR (CDCl3): δ
5.63 (s, 1H, H-12), 4.35 (d, 1H, J = 7.8 Hz, H-1΄), 3.79-3.81 (m 1H, H-6΄-1),
3.58-3.61 (m, 1H, H-6΄-2), 3.62 (s, 3H, OCH3), 3.53 (t, 1H, J = 8.5 Hz, H-4΄), 3.43 (t,
1H, J = 8.0 Hz, H-3΄), 3.27-3.29 (m, 1H, H-5΄), 3.16 (t, 1H, J = 8.1 Hz, H-2΄), 3.01
(dd, 1H, J = 13.5, 3.5 Hz, H-3), 2.80 (d, 1H, J = 13.3 Hz, H-1a), 2.30 (s, 1H, H-9),
13
1.36, 1.10, 1.02, 0.94, 0.94, 0.91, 0.83 (each s, each 3H, CH3); C NMR (CDCl3): δ
200.3 (C-11), 177.5 (C-28), 168.5 (C-13), 166.1, 165.9, 165.3, 165.0, 133.4, 133.2,
133.0, 129.9, 129.8, 129.7 (two), 129.6 (C-12), 129.5, 128.9, 128.8, 128.4, 128.3
(two), 127.9, 103.1 (C-1΄), 90.6, 73.0, 72.2, 72.0, 70.3, 63.4, 61.7, 55.3, 51.9, 46.2,
45.0, 44.3, 43.4, 41.6, 39.1, 39.0, 37.0, 33.7, 32.9, 32.8, 31.6, 30.7, 27.7 (two), 26.9,
25.8, 23.6, 23.4, 22.9, 18.9, 17.2, 16.2, 16.1; HRESIMS calcd for C37H58O9Na
669.3973; found 669.3976.
4.5. General procedure for the preparation of 33-37
Compounds 28-32 (1 mmol) were dissolved in dry CH2Cl2 (100 mL) and pyridine
(V:V = 1:1) and then cooled to -10°C. To the cold solution, pivaloyl chloride (6 mmol)
was added dropwise over a period of 10 min and the reaction mixture was allowed to
stir for another 12 h. After the reaction was complete as judged by TLC, the mixture
was concentrated in vacuo. Then the residue was diluted with EtOAc (100 mL) and
washed with 1 M HCl (50 mL × 3), saturated aq NaHCO3 (50 mL × 3), and brine (50
mL). The organic layer was dried over Na2SO4, filtered, and concentrated under
diminished pressure. Purification of the crude reaction product by column
19

ACCEPTED MANUSCRIPT
chromatography on silica gel [petroleum ether-EtOAc (3:1)] furnished compounds
33-37, respectively.
4.5.1 3β-O-(3,6-Di-O-Pivaloyl-β-D-glucopyranosyl)-olean-12-en-28-oic acid benzyl
ester (33)
Compound 33 was synthesized as a white solid in 77% yield; 1H NMR (CD3COCD3):
δ 7.36-7.40 (m, 4H, Ar-H), 7.32 (tt, 1H, J = 7.9, 1.7 Hz, Ar-H), 5.26 (t, 1H, J = 3.6 Hz,
H-12), 5.10 (d, 1H, J = 12.5 Hz, Ar-CH2-1), 5.06 (d, 1H, J = 12.5 Hz, Ar-CH2-2),
4.94 (t, 1H, J = 9.4 Hz, H-3΄), 4.47 (dd, 1H, J = 11.6, 2.2 Hz, H-6-1΄), 4.46 (d, 1H, J =
7.8 Hz, H-1΄), 4.09 (dd, 1H, J = 11.6, 7.4 Hz, H-6-2΄), 3.60-3.63 (m, 1H, H-5΄), 3.45
(t, 1H, J = 9.4 Hz, H-4΄), 3.38 (t, 1H, J = 8.1 Hz, H-2΄), 3.17 (dd, 1H, J = 11.8, 4.7 Hz,
H-3), 2.95 (dd, 1H, J = 14.2, 4.6 Hz, H-18), 1.20, 1.19 (each s, each 9H, 2 × (CH3)3C),
1.16, 1.04, 0.93, 0.92, 0.92, 0.81, 0.64 (each s, each 3H, each CH3); 13C NMR
(CD3COCD3): δ 177.4, 177.3, 176.5, 143.8 (C-13), 136.8, 128.3 (two), 127.9 (two),
127.8, 122.4 (C-12), 105.5 (C-1΄), 89.2, 77.4, 73.8, 72.6, 69.3, 65.5, 63.7, 55.5, 47.6,
46.5, 45.7, 41.5 (two), 39.3, 38.9, 38.5, 38.4 (two), 36.6, 33.5, 32.6, 32.4, 32.3, 30.4,
27.4 (two), 26.6, 26.5, 25.8, 25.4, 23.2, 23.0, 22.9, 18.0, 16.6, 15.9, 14.9; HRESIMS
calcd for C53H80O10Na 899.5650; found 899.5653.
4.5.2 3β-O-(3,6-Di-O-Pivaloyl-β-D-glucopyranosyl)-erythrodiol-28-methyl ether (34)
1
Compound 34 was synthesized as a white solid in 75% yield; H NMR (CDCl3): δ
5.19 (t, 1H, J = 3.5 Hz, H-12), 4.87 (t, 1H, J = 9.2 Hz, H-3΄), 4.46 (dd, 1H, J = 11.8,
2.4 Hz, H-6΄-1), 4.42 (d, 1H, J = 7.7 Hz, H-1΄), 4.22 (dd, 1H, J = 11.8, 7.0 Hz,
H-6΄-2), 3.56-3.58 (m, 1H, H-5΄), 3.48 (t, 1H, J = 9.4 Hz, H-4΄), 3.35 (d, 1H, J = 9.1
20

ACCEPTED MANUSCRIPT
Hz, CH2OMe-1), 3.31 (s, 3H, OCH3), 3.29 (dd, 1H, J = 9.2, 7.2 Hz, H-2΄), 3.18 (dd,
1H, J = 11.6, 4.4 Hz, H-3), 2.99-3.04 (m, 1H, H-18), 2.90 (d, 1H, J = 9.3 Hz,
CH2OMe-2), 1.26, 1.25 (each s, each 9H, 2 × C(CH3)3), 1.16, 1.02, 0.97, 0.95, 0.89,
13
0.88, 0.84 (each s, each 3H, CH3); C NMR (CDCl3): δ 180.3, 178.5, 144.7 (C-13),
122.0 (C-12), 104.8 (C-1΄), 90.3, 80.1, 78.1, 74.2, 72.6, 70.2, 63.7, 59.4, 55.5, 47.6,
46.4, 43.2, 42.7, 41.7, 39.8, 39.2, 39.1, 39.0, 38.8, 38.5, 37.5, 36.6, 36.5, 34.2, 33.2,
32.5, 32.0, 30.9, 28.2, 27.2, 27.1, 25.9, 25.8, 25.6, 23.7, 23.5, 22.2, 18.2, 16.7 (two),
15.5; HRESIMS calcd for C47H78O9Na 809.5544; found 809.5546.
4.5.3 3β-O-(3,6-Di-O-Pivaloyl-β-D-glucopyranosyl)-oleanoaldehyde (35)
1
Compound 35 was synthesized as a white solid in 78% yield; H NMR (CDCl3): δ
9.41 (s, 1H, CHO), 5.36 (t, 1H, J = 3.7 Hz, H-12), 4.87 (t, 1H, J = 9.3 Hz, H-3΄), 4.46
(dd, 1H, J = 11.8, 2.3 Hz, H-6΄-1), 4.42 (d, 1H, J = 7.8 Hz, H-1΄), 4.22 (dd, 1H, J =
11.8, 7.0 Hz, H-6΄-2), 3.55-3.59 (m, 2H, H-4΄, H-5΄), 3.48 (t, 1H, J = 9.4 Hz, H-2΄),
3.17 (dd, 1H, J = 11.8, 4.6 Hz, H-3), 2.65 (dd, 1H, J = 11.3, 4.8 Hz, H-18), 1.26, 1.22
(each s, each 9H, 2 × C(CH3)3), 1.14, 1.07, 0.94, 0.93, 0.92, 0.82, 0.74 (each s, each
3H, CH3); 13C NMR (CDCl3): δ 207.5 (CHO), 180.3, 178.5, 143.0 (C-13), 123.2
(C-12), 104.8 (C-1΄), 90.2, 78.0, 74.1, 72.6, 70.1, 63.7, 55.5, 49.1, 47.5, 45.6, 41.7,
40.4, 39.5, 39.1, 39.0, 38.8, 38.4, 36.7, 33.1 (two), 32.7, 30.6, 28.3, 27.7, 27.2, 27.1,
27.0, 26.7, 25.8, 25.5, 23.4, 22.1, 18.1, 17.0, 16.7, 15.3; HRESIMS calcd for
C46H74O9Na 793.5231; found793.5235.
4.5.4 3α-O-(3,6-Di-O-Pivaloyl-β-D-glucopyranosyl)-olean-12-en-28-oic acid methyl
ester (36)
21

ACCEPTED MANUSCRIPT
1
Compound 36 was synthesized as a white solid in 76% yield; H NMR (CDCl3): δ
5.28 (t, 1H, J = 3.3 Hz, H-12), 4.88 (t, 1H, J = 7.9 Hz, H-3΄), 4.47 (dd, 1H, J = 12.0,
1.6 Hz, H-6΄-1), 4.32 (d, 1H, J = 7.7 Hz, H-1΄), 4.28 (dd, 1H, J = 11.8, 5.1 Hz,
H-6΄-2), 3.63 (s, 3H, OCH3), 3.57 (t, 1H, J = 8.8 Hz, H-4΄), 3.52-3.53 (m, 1H, H-2΄),
3.37 (brs, 1H, H-3), 3.00-3.02 (m, 1H, H-5΄), 2.87 (dd, 1H, J = 13.6, 4.0 Hz, H-18),
1.27 (s, 9H, (CH3)3C), 1.22 (s, 9H, (CH3)3C), 1.16, 0.94, 0.93, 0.92, 0.91, 0.84, 0.72
(each s, each 3H, CH3); 13C NMR (CDCl3): δ 180.3, 178.5, 178.3, 143.6 (C-13), 122.4
(C-12), 100.0 (C-1΄), 82.1, 72.8, 74.2, 71.8, 70.1, 63.1, 51.5, 50.0, 47.5, 46.7, 45.9,
41.7, 41.3, 39.4, 39.1, 38.8, 37.0, 36.8, 33.9, 33.5, 33.1, 32.5, 32.4, 30.7, 28.5, 27.7,
27.1, 26.1, 23.6, 23.3, 23.1, 22.4, 21.1, 18.1, 16.8, 15.2; HRESIMS calcd for
C47H76O10Na 823.5336; found 823.5342.
4.5.5 3β-O-(3,6-Di-O-Pivaloyl-β-D-glucopyranosyl)-11-oxo-olean-12-en-28-oic acid
methyl ester (37)
1
Compound 37 was synthesized as a white solid in 80% yield; H NMR (CDCl3): δ
5.63 (s, 1H, H-12), 4.87 (t, 1H, J = 9.2 Hz, H-3΄), 4.46 (dd, 1H, J = 11.9, 2.4 Hz,
H-6-1΄), 4.41 (d, 1H, J = 7.7 Hz, H-1΄), 4.19 (dd, 1H, J = 11.8, 7.0 Hz, H-6-2΄), 3.64
(s, 3H, OCH3), 3.55-3.59 (m, 2H, H-4΄, H-5΄), 3.46 (t-like, 1H, J = 9.3, 8.2 Hz, H-2΄),
3.17 (dd, 1H, J = 11.8, 4.7 Hz, H-3), 3.01 (dd, 1H, J = 14.1, 2.2 Hz, H-1a), 2.81 (dt,
1H, J = 13.6, 3.3 Hz, H-18), 2.29 (s, 1H, H-9), 1.25, 1.21 (each s, each 9H, 2 ×
13
(CH3)3C), 1.36, 1.11, 1.01, 0.94, 0.93, 0.91, 0.84 (each s, each 3H, each CH3); C
NMR (CDCl3): δ 200.3 (C-11), 180.3, 178.7, 177.5 (C-28), 168.7 (C-13), 128.0
(C-12), 104.8 (C-1΄), 90.1, 78.0, 74.1, 72.7, 70.1, 63.7, 61.7, 55.3, 51.9, 46.2, 45.0,
22

ACCEPTED MANUSCRIPT
44.3, 43.4, 41.6, 39.3, 39.0, 38.8, 33.7, 32.9, 32.8, 31.6, 30.6, 28.2, 27.7, 27.2, 27.1,
25.8, 23.5, 23.4, 23.0, 18.9, 17.3, 16.6, 16.1; HRESIMS calcd for C47H74O11Na
837.5129; found 837.5135.
4.6 General procedure for the preparation of 7, 10-13
To a mixture of 33-37 (1 eq) and 4 Å molecular sieves in dried CH2Cl2 (30 mL) at
0 °C under argon was added TMSOTf (0.20 eq), followed by a solution of imidate 38
(5 eq) in CH2Cl2 (5 mL). After stirring at 0 °C for 2 h and then at r.t. for 1 h, the
reaction was quenched with Et3N. The solid was filtered, and the filtrate was
concentrated under vacuum to give yellow oil. The oil was subjected to column
chromatography on silica gel (EtOAc-petroleum ether, 1:4) to provide the desired
crude trisaccharide. The above trisaccharide mixture was dissolved in
THF-CH3OH-H2O (V:V:V = 1:1:1) and then NaOH (14 eq) was added. After stirred at
50 °C for 12 h, the solution was neutralized with Dowex 50 × 8 (H+) resin until pH =
7, filtered and concentrated in vacuo. Then the residue was purified by silica gel
column chromatography (CH2Cl2-MeOH, 6:1) to afford compounds 7, and 10-13,
respectively.
4.6.1 3β-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl]-olean-12-en-28-
oic acid benzyl ester (7)
1
Compound 7 was synthesized as a white solid in 66% yield for two steps; H NMR
(DMSO-d6): δ 7.28-7.35 (m, 5H, Ar-H), 5.17 (d, 1H, J = 1.3 Hz, Rha-H-1), 5.14 (t,
1H, J = 3.3 Hz, H-12), 5.01 (d, 1H, J = 12.7 Hz, Ar-CH2-1), 4.97 (d, 1H, J = 12.7 Hz,
Ar-CH2-2), 4.66 (d, 1H, J = 1.1 Hz, Rha-H-1), 4.26 (d, 1H, J = 7.7 Hz, H-1΄), 3.72
23

ACCEPTED MANUSCRIPT
(dd, 1H, J = 3.2, 1.9 Hz, Rha-H-2), 3.60 (dd, 1H, J = 3.1, 1.7 Hz, Rha-H-2), 3.57 (d,
1H, J = 10.9 Hz, H-6-1΄), 3.47 (dd, 1H, J = 9.4, 3.3 Hz, Rha-H-3), 3.41-3.43 (m, 1H),
3.40 (dd, 1H, J = 9.5, 3.2 Hz, Rha-H-3), 3.13-3.14 (m, 1H), 3.00 (dd, 1H, J = 10.9,
4.1 Hz, H-3), 2.78 (dd, 1H, J = 13.3. 4.0 Hz, H-18), 1.09 (d, 3H, J = 6.2 Hz, Rha-H-6),
1.05 (d, 3H, J = 6.2 Hz, Rha-H-6), 1.04, 0.90, 0.84, 0.83, 0.78, 0.71, 0.48 (each s,
each 3H, CH3);13C NMR (DMSO-d6): δ 177.1 (C-28), 149.6, 143.8 (C-13), 136.6,
128.8 (two), 128.4, 128.1 (two), 122.4 (C-12), 104.1 (C-1΄), 100.9 (Rha-C-1), 100.5
(Rha-C-1), 88.5, 77.6, 77.5, 76.6, 75.5, 72.3, 72.2, 70.9, 70.7, 70.6, 70.5, 69.2, 68.6,
65.8, 60.4, 55.7, 47.5, 46.5, 45.8, 41.6, 41.4, 38.8, 36.6, 33.5, 33.1, 32.7, 32.4, 30.7,
27.8, 27.4, 26.1, 26.0, 23.7, 23.3, 23.0, 18.2, 18.1, 17.0, 16.6, 15.5; HRESIMS calcd
for C55H84O16Na 1023.5652; found 1023.5652.
4.6.2 3β-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl] -erythrodiol-28-
methyl ether (10)
1
Similarly, 10 was prepared as a white solid in 68% yield for two steps; H NMR
(CD3OD): δ 5.38 (d, 1H, J = 1.6 Hz, Rha-H-1), 5.20 (t, 1H, J = 3.6 Hz, H-12), 4.86 (d,
1H, J = 1.6 Hz, Rha-H-1), 4.45 (d, 1H, J = 7.8 Hz, H-1΄), 3.98-4.02 (m, 2H),
3.91-3.93 (m, 1H), 3.85 (dd, 1H, J = 3.2, 2.0 Hz, Rha-H-2), 3.81 (dd, 1H, J = 12.1,
1.9 Hz, H-6-1΄), 3.76 (dd, 1H, J = 9.4, 3.3 Hz, Rha-H-3), 3.68 (dd, 1H, J = 12.1, 4.1
Hz, H-6-2΄), 3.64 (dd, 1H, J = 9.4, 3.3 Hz, Rha-H-3), 3.60 (t, 1H, J = 8.7 Hz), 3.55 (t,
1H, J = 8.8 Hz), 3.46 (t, 1H, J = 8.0 Hz), 3.42 (t, 1H, J = 9.5 Hz), 3.40 (t, 1H, J = 9.6
Hz), 3.35 (d, 1H, J = 13.7 Hz, OCH3-CH2-1), 3.34 (d, 1H, J = 13.6 Hz, OCH3-CH2-2),
3.29 (s, 3H, OCH3), 3.19 (dd, 1H, J = 11.7, 4.3 Hz, H-3), 2.96 (d, 1H, J = 9.2 Hz),
24

ACCEPTED MANUSCRIPT
2.05 (dd, 1H, J = 13.0, 4.0 Hz), 1.28 (d, 3H, J = 6.2 Hz, Rha-H-6), 1.23 (d, 3H, J =
6.2 Hz, Rha-H-6), 1.19, 1.07, 1.01, 0.99, 0.90, 0.89, 0.88 (each s, each 3H, CH3); 13C
NMR (CD3OD): δ 144.3 (C-13), 122.2 (C-12), 104.1 (C-1΄), 101.7 (Rha-C-1), 100.6
(Rha-C-1), 89.0, 79.7, 79.0, 77.8, 76.7, 75.1, 72.5, 72.3, 71.0, 70.7, 70.6, 69.4, 68.6,
69.5, 60.6, 58.2, 55.9, 46.3, 42.5, 41.4, 40.6, 39.7, 38.9, 38.8, 36.4, 36.3, 33.9, 32.4
(two), 27.1, 25.8, 25.4, 25.1, 23.3, 22.7, 22.0, 17.9, 16.6, 16.5, 16.1, 15.8, 14.8;
HRESIMS calcd for C49H82O15Na 933.5546; found 933.5552.
4.6.3 3β-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl]-oleanoaldehyde
(11)
1
Similarly, 11 was prepared as a white solid in 65% yield for two steps; H NMR
(DMSO-d6): δ 9.35 (s, 1H, CHO), 5.26 (brs, 1H, H-12), 5.19 (s, 1H, Rha-H-1), 4.73
(s, 1H, Rha-H-1), 4.30 (d, 1H, J = 8.0 Hz, H-1΄), 3.82-3.86 (m, 3H), 3.69 (d, 1H, J =
3.0 Hz, Rha-H-2), 3.62-3.64 (m, 1H), 3.55 (dd, 1H, J = 9.9, 4.1 Hz, Rha-H-3),
3.51-3.52 (m, 1H), 3.49 (dd, 1H, J = 8.9, 3.3 Hz, Rha-H-3), 3.42-3.45 (m, 2H),
3.30-3.32 (m, 1H), 3.23-3.27 (m, 3H), 3.17-3.18 (m, 1H, H-3), 3.02 (d, 1H, J = 13.2
Hz, H-18), 1.15 (d, 3H, J = 6.0 Hz, Rha-H-6), 1.11 (d, 3H, J = 6.0 Hz, Rha-H-6), 1.10,
13
0.95, 0.89, 0.88, 0.86, 0.76, 0.64 (each s, each 3H, CH3); C NMR (DMSO-d6): δ
207.9 (CHO), 177.3 (C-28), 128.8 (C-12), 103.3 (C-1΄), 100.0 (Rha-C-1), 99.8
(Rha-C-1), 88.0, 77.1, 75.5, 74.5, 71.4, 71.3, 70.0, 69.7, 69.5, 68.4, 68.0, 59.6, 58.2,
48.2, 46.7, 40.8, 35.9, 35.8, 32.3, 29.8, 27.0, 24.9, 22.7, 20.5, 17.1 (two), 15.8, 14.7;
HRESIMS calcd for C48H78O15Na 917.5233; found 917.5233.
4.6.4 3α-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl]-olean-12-en-28-
25

ACCEPTED MANUSCRIPT
oic acid methyl ester (12)
1
Similarly, 12 was prepared as a white solid in 71% yield for two steps; H NMR
(CD3OD): δ 5.38 (d, 1H, J = 1.6 Hz, Rha-H-1), 5.20 (t, 1H, J = 3.6 Hz, H-12), 4.86 (d,
1H, J = 1.6 Hz, Rha-H-1), 4.45 (d, 1H, J = 7.8 Hz, H-1΄), 3.98-4.02 (m, 2H),
3.91-3.93 (m, 1H), 3.85 (dd, 1H, J = 3.2, 2.0 Hz, Rha-H-2), 3.81 (dd, 1H, J = 12.1,
1.9 Hz, H-6-1΄), 3.76 (dd, 1H, J = 9.4, 3.3 Hz, Rha-H-3), 3.68 (dd, 1H, J = 12.1, 4.1
Hz, H-6-2΄), 3.64 (dd, 1H, J = 9.4, 3.3 Hz, Rha-H-3), 3.60 (t, 1H, J = 8.7 Hz), 3.55 (t,
1H, J = 8.8 Hz), 3.46 (t, 1H, J = 8.0 Hz), 3.42 (t, 1H, J = 9.5 Hz), 3.40 (t, 1H, J = 9.6
Hz), 3.35 (d, 1H, J = 13.7 Hz, OCH3-CH2-1), 3.34 (d, 1H, J = 13.6 Hz, OCH3-CH2-2),
3.29 (s, 3H, OCH3), 3.19 (dd, 1H, J = 11.7, 4.3 Hz, H-3), 2.96 (d, 1H, J = 9.2 Hz),
2.05 (dd, 1H, J = 13.0, 4.0 Hz), 1.28 (d, 3H, J = 6.2 Hz, Rha-H-6), 1.23 (d, 3H, J =
6.2 Hz, Rha-H-6), 1.19, 1.07, 1.01, 0.99, 0.90, 0.89, 0.88 (each s, each 3H, CH3); 13C
NMR (CD3OD): δ 144.3 (C-13), 122.2 (C-12), 104.1 (C-1΄), 101.7 (Rha-C-1), 100.6
(Rha-C-1), 89.0, 79.7, 79.0, 77.8, 76.7, 75.1, 72.5, 72.3, 71.0, 70.7, 70.6, 69.4, 68.6,
69.5, 60.6, 58.2, 55.9, 46.3, 42.5, 41.4, 40.6, 39.7, 38.9, 38.8, 36.4, 36.3, 33.9, 32.4
(two), 27.1, 25.8, 25.4, 25.1, 23.3, 22.7, 22.0, 17.9, 16.6, 16.5, 16.1, 15.8, 14.8;
HRESIMS calcd for C49H82O15Na 933.5546; found 933.5552.
4.6.53β-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl]-11-oxo-olean-12-
en-28-oic acid methyl ester (13)
1
Similarly, 13 was prepared as a white solid in 70% yield for two steps; H NMR
(CD3OD): δ 5.55 (s, 1H, H-12), 5.37 (d, 1H, J = 1.5 Hz, Rha-H-1), 4.86 (d, 1H, J =
1.7 Hz, Rha-H-1), 4.44 (d, 1H, J = 7.8 Hz, H-1΄), 3.96-4.00 (m, 2H), 3.89-3.94 (m,
26

ACCEPTED MANUSCRIPT
1H), 3.85 (dd, 1H, J=3.2, 1.8 Hz, Rha-H-2), 3.81 (dd, 1H, J = 12.1, 1.9 Hz, H-6-1΄),
3.75 (dd, 1H, J = 9.5, 3.4 Hz, Rha-H-3), 3.67 (dd, 1H, J = 12.1, 4.1 Hz, H-6-2΄), 3.65
(s, 3H, OCH3), 3.63 (dd, 1H, J = 9.5, 3.3 Hz, Rha-H-3), 3.60 (t, 1H, J = 8.7 Hz), 3.55
(t, 1H, J = 8.8 Hz), 3.46 (t, 1H, J = 8.2 Hz), 3.38-3.44 (m, 2H), 3.30-3.32 (m, 1H),
3.20 (dd, 1H, J = 11.8, 4.3 Hz, H-3), 3.03 (dd, 1H, J = 14.0, 4.1 Hz, H-1a), 2.73 (dt,
1H, J = 13.6, 3.4 Hz, H-18), 2.43 (s, 1H, H-9), 1.28 (d, 3H, J = 6.2 Hz, Rha-H-6),
1.23 (d, 3H, J = 6.2 Hz, Rha-H-6), 1.42, 1.13, 1.07, 0.97, 0.96, 0.93, 0.88 (each s,
each 3H, CH3); 13C NMR (CD3OD): δ 201.4 (C-11), 177.6 (C-28), 170.5 (C-13),
127.0 (C-12), 104.1 (C-1΄), 101.6 (Rha-1-C), 100.7 (Rha-1-C), 88.7, 79.0, 77.8, 76.7,
75.1, 72.5, 72.3, 71.0, 70.7, 70.6, 69.4, 68.6, 61.7, 60.6, 55.4, 51.1, 46.2, 45.1, 44.0,
43.5, 41.9, 39.2, 39.1, 36.8, 33.2, 32.6, 31.8, 31.3, 30.2, 27.4, 27.1, 25.8, 22.6, 22.5,
22.4, 18.2, 17.0, 16.6, 16.5, 15.8, 15.5; HRESIMS calcd for C51H66O11Na 877.4497;
found 877.4501.
4.7 3β-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl]-olean-12-en-28-
oic acid (2)
A mixture of compound 7 (0.30 g, 0.33 mmol) and 10% Pd/C (50 mg) in MeOH
(10mL) was stirred at room temperature under H2 at atmospheric pressure for 8 h. The
reaction mixture was filtered through Celite, and the insoluble substance was washed
with MeOH (5 mL × 3). The filtrate was concentrated in vacuo to give 2 as a white
solid (0.26 g, 95%). 1H NMR (CD3OD): δ 5.38 (s, 1H, Rha-H-1), 5.25 (t, 1H, J = 3.0
Hz, H-12), 4.86 (s, 1H, Rha-H-1), 4.44 (d, 1H, J = 7.7 Hz, H-1΄), 3.97-4.01 (m, 1H),
3.98 (dd, 1H, J = 3.2, 1.8 Hz, Rha-H-2), 3.91-3.93 (m, 1H), 3.85 (dd, 1H, J = 3.2, 1.9
27

ACCEPTED MANUSCRIPT
Hz, Rha-H-2), 3.81 (d, 1H, J = 10.8 Hz, H-6-1΄), 3.76 (dd, 1H, J = 9.5, 3.3 Hz,
Rha-H-3), 3.68 (dd, 1H, J = 12.0, 4.0 Hz, H-6-2΄), 3.64 (dd, 1H, J = 9.4, 3.2 Hz,
Rha-H-3), 3.60 (t, 1H, J = 8.7 Hz), 3.55 (t, 1H, J = 8.9 Hz), 3.46 (t, 1H, J = 8.2 Hz),
3.42 (t, 1H, J = 9.4 Hz), 3.39 (t, 1H, J = 9.5 Hz), 3.18 (dd, 1H, J = 11.6, 4.1 Hz, H-3),
2.86 (dd, 1H, J = 13.9, 3.7 Hz), 1.28 (d, 3H, J = 6.2 Hz, Rha-H-6), 1.22 (d, 3H, J =
6.2 Hz, Rha-H-6), 1.17, 1.06, 0.96, 0.95, 0.92, 0.87, 0.83 (each s, each 3H, each CH3);
13C NMR (CD3OD): δ 180.8 (C-28), 143.8 (C-13), 122.2 (C-12), 104.0 (C-1΄), 101.6
(Rha-1-C), 100.6 (Rha-1-C), 89.0, 79.0, 77.8, 76.7, 75.0, 72.5, 72.3, 71.0, 70.7, 70.6,
69.4, 68.6, 60.6, 55.9, 46.3, 45.9, 41.5, 41.3, 39.2, 38.8, 38.7, 36.5, 33.5, 32.6, 32.4,
32.2, 30.2, 27.4, 27.1, 25.8, 25.0, 23.1, 22.7, 22.6, 17.9, 16.6, 16.5, 16.3, 15.8, 14.6;
HRESIMS calcd for C48H78O16Na 933.5182; found 933.5184.
4.8 General procedure for the preparation of 3-6
A mixture of compound 2 (1 mmol) and K2CO3 (2 mmol) in DMF (10 mL) was
stirred at room temperature for 4 h. The iodomethane (6 mmol) or bromoalkane (3
mmol) was then dripped slowly into the mixture. After being stirred for another 10 h,
the reaction mixture was poured into the 40 mL distilled water and partitioned with
butyl alcohol (80 × 3 mL). The organic layer was washed with saturated sodium
chloride, dried over Na2SO4, and purified via silica gel column chromatography
(MeOH/CH2Cl2, 1:5) to afford 3-6 as white solids, respectively.
4.8.1 3β-O-[2,4-Di-O-(α-L-Rhamnopyranosyl)-β-D-glucopyranosyl]-olean-12-en-28-
oic acid methyl ester (3)
Compound 3 was prepared as a white solid in 93% yield; 1H NMR (CD3OD): δ 5.36
28