Design, synthesis and cytotoxic activity of N-Modified oleanolic saponins bearing A glucosamine

Article abstract of DOI:10.1016/j.ejmech.2017.11.004

A series of N-acyl, N-alkoxycarbonyl, and N-alkylcarbamoyl derivatives of 2′-deoxy-glucosyl bearing oleanolic saponins were synthesized and evaluated against HL-60, PC-3, and HT29 tumor cancer cells. The SAR studies revealed that the activity increased in order of conjugation of 2′ -amino group with carbamate > amide > urea derivatives. Lengthening the alkyl chain increased the cytotoxicity, the peak activity was found to around heptyl to nonyl substitutions. 2′-N-heptoxycarbonyl derivative 56 was found to be the most cytotoxic (IC₅₀ = 0.76 μM) against HL-60 cells. Due to the interesting SARs of alkyl substitutions, we hypothesized that their location in the cell was different, and pursued a location study using 2′-(4″-pentynoylamino) 2′-deoxy-glucosyl OA, which suggested that these compounds distributed mainly in the cytosol.

Full text of DOI:10.1016/j.ejmech.2017.11.004

Accepted Manuscript
Design, synthesis and cytotoxic activity of N-Modified oleanolic saponins bearing A
glucosamine
You-Yu Lin, She-Hung Chan, Yu-Pu Juang, Hsin-Min Hsiao, Jih-Hwa Guh, Pi-Hui
Liang
PII:
S0223-5234(17)30890-5
10.1016/j.ejmech.2017.11.004
EJMECH 9879
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 August 2017
Revised Date: 1 November 2017
Accepted Date: 2 November 2017
Please cite this article as: Y.-Y. Lin, S.-H. Chan, Y.-P. Juang, H.-M. Hsiao, J.-H. Guh, P.-H. Liang,
Design, synthesis and cytotoxic activity of N-Modified oleanolic saponins bearing A glucosamine,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Design, Synthesis and Cytotoxic Activity of N-Modified Oleanolic Saponins
Bearing A Glucosamine
1
2
1
1
1
You-Yu Lin, She-Hung Chan, Yu-Pu Juang, Hsin-Min Hsiao , Jih-Hwa Guh, Pi-Hui
*,1, 3
Liang
Y.-Y. Lin,Y.-P. Juang, H.-M. Hsiao, Prof. Dr. J.H. Guh, Prof. Dr. P.-H. Liang
. School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
1
E-mail: phliang@ntu.edu.tw
Prof. Dr. S.-H. Chan
2
. Department of Cosmetic Science, Providence University, Taichuang 433, Taiwan
Prof. Dr. P.-H. Liang
. The Genomic Research Center, Academia Sinica, Taipei 128, Taiwan
3
AUTHOR INFORMATION
Corresponding Author
E-mail: phliang@ntu.edu.tw
Key words: Oleanolic acid; Glycosylation; Cytotoxicity; Glycoside
1

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Abstract
A series of N-acyl, N-alkoxycarbonyl, and N-alkylcarbamoyl derivatives of
2
ʹ-deoxy-glucosyl bearing oleanolic saponins were synthesized and evaluated against HL-60,
PC-3, and HT29 tumor cancer cells. The SAR studies revealed that the activity increased in
order of conjugation of 2ʹ -amino group with carbamate > amide > urea derivatives.
Lengthening the alkyl chain increased the cytotoxicity, the peak activity was found to around
heptyl to nonyl substitutions. 2ʹ-N-heptoxycarbonyl derivative 56 was found to be the most
cytotoxic (IC = 0.76 µM) againt HL-60 cells. Due to the interesting SARs of alkyl
5
0
substitutions, we hypothesized that their location in the cell was different, and pursued a
location study using 2ʹ-(4ʺ-pentynoylamino) 2ʹ-deoxy-glucosyl OA, which suggested that
these compounds distributed mainly in the cytosol.
1
. Introduction
Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA, Figure 1), a pentacyclic
triterpenoid, can be found in more than 1600 plant species as either a free acid or as a saponin
[
1]. It is particularly abundant in the Oleaceae family, and named for the olive (Olea europaea
L.) plant species that still serves as the main source of commercial OA. Due to its natural
abundance and ready availability, it is a good natural starting material for synthetic
modification and drug discovery[2].
Many biological activities have been reported for OA and its derivatives[3, 4]. The
hepatoprotective effect of OA is particularly well known, and OA formulations are widely
sold over-the-counter in China for this purpose [5]. Other studies found that OA imparts
anti-inflammatory and moderate anti-cancer activity [6-8]. Attempts to improve the
anti-cancer activity by the chemical synthesis of several OA derivatives have been described
2

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[
9], such as 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid 2 (CDDO, Figure 1)[6, 10,
11], C ring oxidized OA[12], and 28-urea containing OA [13].
OA is poorly water soluble, which results in low absolute oral bioavailability.
Modifications to the hydrophobic scaffold of OA alone failed to significantly improve its
solubility, thus limiting opportunities to evaluate its activity [14]. In fact, most naturally
occurring OA molecules are glycosylated, bearing a glycan at either C-3 or C-28 via an ether
or ester linkage respectively, and are termed OA saponins. OA derivatives bearing an N-acetyl
glucosamine (such as compound 3, Figure 1) moiety have attracted a great deal of attention
due to their remarkable cytotoxicity; however, few OA saponins containing
N-acetyl-glucosamine moieties have been found in nature [15-18]. Albiziatrioside A (4, Figure
1
), is one example of an N-acetyl glucosamine bearing natural product; it was isolated from
Acacia tenuifolia, and exhibited potent cytotoxic activity (IC = 0.9 µg/mL against A2780
5
0
ovarian cancer cells).[16, 17] Lotoidoside D (5) (Figure 1) was found to have
anti-proliferative activity against the Hela cell line with IC value of 2.7 µM [18].
5
0
Figure 1. OA and OA saponin derivatives
It was noticed that glycans attached with specific linkages of triterpenoid might have
3

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important roles in boosting the cytotoxic activity [14, 18-20]. Synthesis of non-natural
saponins bearing different monosaccharide substitutions is one way to probe the glycan
linkages for their biological function and SARs [19], Liʹs group attached additional N-acetyl
glucosamine moieties to compound 3, to give a series of mono- to tetra-(N-acetylglucosamine)
OA saponins [21]; however, neither of them exhibited superior cytotoxicity against HL-60
cell lines than 3. In previous work, we developed a concise method for synthesizing OA
saponins with (1→3)-linked, (1→4)-linked, (1→6)-linked N-acetylglucosamine
oligosaccharide residues, and found those bearing (
D/
L
)-xylose and -arabinose at positions 3ʹ
L
and 4ʹ were more cytotoxic to HL-60 and HT-29 cell lines than those bearing other sugars;
unfortunately, the magnitude of this improvement was not great, compared to compound 3
[
20]. Accordingly, in order to clarify and investigate the SARs of the N-acetyl glucosamine
moiety, we set out to synthesize a series of N-modified derivatives of glucosamine-bearing
OA saponins containing N-acyl, N-alkoxycarbonyl, N-alkylcarbamoyl groups. N-modified
derivatives of galactosamine-bearing OA saponins were also synthesized, to investigate the
effect of the epimer of the glucose moiety on cytotoxic activity. We also sought to vary the
length of the carbon chain on these derivatives, a common strategy to improve compound
activity since a chain length within a particular range may permit penetration into the cell
membranes, and interaction with the target organelle. However, compounds with excessively
long carbon chains may exhibit poor solubility, and be prevented from interacting with the
target receptor due to micelle formation [22]. OA saponins bearing a variety of different
length of N-alkyl glucosamine moieties were found to have superior cytotoxicity against
HL-60 than 3. The dependence of alkyl chain substitutions on cytotoxicity prompted us to
visualize their location using 2ʹ-(4ʺ-pentynoylamino) 2ʹ-deoxy-glucosyl OA as a model
compound.
2
. Results and discussion
4

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2
.1. Chemistry
Our synthesis of compounds 13–15 is depicted in Scheme 1. With compound 6 in hand,
removal of the 1-thiotoluene group using N-bromosuccinimide (NBS) followed by
trichloroimidate formation using CCl CN and DBU gave trichloroacetimidate glycosyl donor
3
7
. Glycosylation of donor 7 with 28-Bn-OA (8)[20] in the presence of TMSOTf afforded
compound 9, which underwent hydrolysis in 4.0 N NaOH and a variety of different alcohols
to give the carbamate derivatives 10–12. Deprotection of the benzyl group with hydrogen in
the presence of 10% Pd/C afforded the desired products 13–15.
Scheme 1. Synthesis of saponins. (a) i. NBS, acetone/H O, -20 ℃ , 54%; ii. CCl CN, DBU,
2
3
CH Cl , r.t.; (b) 28-Bn-OA (8), TMSOTf, 4 Å MS, CH Cl , 0 ℃ , 74% for 3 steps; (c) CH Cl ,
2
2
2
2
2
2
1
0: MeOH, 11: EtOH and 12: n-propanol, 4.0 N NaOH(aq), rt; (d) H , 10% Pd/C, MeOH, rt.
2
6
0% (13), 46% (14) and 39% (15) for two steps.
5

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Group
acyl
Compound
R
Group
acyl
Compound
R
21, 3
31, 50
acyl
acyl
acyl
acyl
acyl
acyl
22, 41
23, 42
24, 43
25, 44
26, 45
27, 46
acyl
32, 51
33, 52
34, 53
35, 54
36, 55
37, 56
acyl
alkoxycarbonyl
alkoxycarbonyl
alkoxycarbonyl
alkoxycarbonyl
acyl
acyl
acyl
28, 47
29, 48
30, 49
alkoxycarbonyl
alkoxycarbonyl
carbamoyl
38, 57
39, 58
40, 59
Scheme 2. Synthesis of saponin library. (a) i. NBS, acetone/H O, -20 ℃ , 64%; ii. CCl CN,
2
3
DBU, CH Cl , rt; (b) 28-Bn-OA (8), TMSOTf, 4 Å MS, CH Cl , 0 ℃ , 55% for 3 steps; (c)
2
2
2
2
6

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ethylenediamine, EtOH , 70–80 ℃ , 72%; (d) H , 20% Pd/C, MeOH; (e) 21–26: (RCO) O,
2
2
pyridine, CH Cl , rt; (f) 27–33: RCOOH, EDC, HOBt, DMF, rt; (g) 34–39: CDI, TEA, ROH,
2
2
rt; (h) 40: DPPA, n-nonanoic acid, TEA, toluene, 80–90 ℃ ; (i) H , 10% Pd/C, MeOH, rt.
2
Reductive removal of the N-Troc group of compound 9 using Zn/(RCO) O allowed the
2
one-pot conversion of the N-Troc group into an N-acyl group, but generated a mixture of side
products, including those arising from trans-esterification. Accordingly, the N-Troc group was
replaced with an N-phthaloyl group (Phth), which could be efficiently removed using
ethylenediamine [23]. The thiol group of compound 16 was removed by NBS, and then
trichloroimidate formation with CCl CN and DBU resulted in glycosyl donor 17, which was
3
reacted with 28-Bn-OA (8) in catalytic TMSOTf to give compound 18 (Scheme 2). All the
glycoside protecting groups were removed using ethylenediamine at 70–80 ℃ to give
compound 19. Hydrogenation of the benzyl ester group of 19 with 20% Pd/C under hydrogen
gave compound 20. The amide derivatives 21–33 were obtained by the reaction of compound
1
9 with various anhydrides in the presence of pyridine, or with various acids in the presence
of coupling reagents (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI),
hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIPEA)) [24]. The carbamate
bearing derivatives were obtained by reacting compound 19 with various alcohols,
1
ʹ-carbonyldiimidazole (CDI), and trietheylamine (TEA) in DCM.[25] Ureido derivative 40
was synthesized by a Curtius rearrangement of 19 using diphenylphosphoryl azide (DPPA),
n-nonanoic acid and TEA [26]. The final compounds 3 and 41–59 were obtained by
hydrogenation using hydrogen and 10% Pd/C in methanol.
The 4-epimer derivatives (66 and 67), N-acetyl and N-heptanoyl galactosamine-bearing
OA saponins, were also synthesized (Scheme 3). Hydrolysis of the anomeric acetate group of
compound 60 followed by trichloroimidate formation with CCl CN and DBU gave glycosyl
3
donor 61. Glycosylation of donor 61 and 28-Bn-OA (8) in the presence of catalytic TMSOTf
afforded 62 in a 46% yield over three steps. After treatment with ethylenediamine in EtOH at
7

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7
0–80 ℃ , compound 63 was obtained, which was subjected to amide formation using the
corresponding anhydrides to afford compounds 64-65. Hydrogenation of 64 and 65 in the
presence of hydrogen and 10% Pd/C in methanol afforded 66 and 67, respectively. Overall,
twenty-five novel oleanolic saponins bearing N-acyl, N-alkoxycarbonyl, and
N-alkylcarbamoyl modification on 2ʹ-deoxy-glucosyl were synthesized
Scheme 3. Synthesis of the saponin library. (a) i. ethylenediamine, acetic acid, THF, 0 ℃ , 61%;
ii. CCl CN, DBU, CH Cl , rt; (b) 28-Bn-OA, TMSOTf, 4 Å MS, CH Cl , 0 ℃ , 46% for 2
3
2
2
2
2
steps; (c) ethylenediamine, EtOH, 70–80 ℃ , 85%; (d) i. (RCO) O, imidazole, CH Cl , rt; ii.
2
2
2
NaOMe, MeOH; (e) H , 10% Pd/C, MeOH, rt.
2
2
.2 Cytotoxic activity
The cytotoxic activities of the oleanolic acid saponins 3, 13–15, 41–59, and 66–67 were
evaluated against four human tumor cell lines (promyelocytic leukemia cells (HL-60),
prostate cancer cells (PC-3), colon adenocarcinoma (HT29)) using the
methyl-thiazol-tetrazolium (MTT) reduction test (Table 1) [27].
8

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Amongst the three cancer cell lines tested, these compounds were more cytotoxic to
HL-60 than the other two. Some of the compounds bearing an amide moiety displayed
significant cytotoxicity against the HL-60 cell line at concentrations below 10 µM, such as
compound 46 (IC 1.68 µM), 47 (IC 1.46 µM), 48 (IC 1.40 µM), and 49 (IC 1.57 µM).
5
0
50
50
50
Likewise, some carbamate derivatives also exhibited significant cytotoxicity at concentrations
of less than 10 µM, such as compound 53 (IC 2.23 µM), 54 (IC 1.46 µM), 55 (IC 1.44
5
0
50
50
µM), 56 (IC 0.76 µM), and 58 (IC 1.23 µM). Derivatives bearing longer carbon chains
5
0
50
were more cytotoxic than compound 3 up until the chain length reached eleven carbons, after
which cytotoxicity significantly decreased; for example, compound 51 (2ʹ-N-hexadecanoyl
moiety, IC >30 µM), and 52 (2ʹ-N-octadecanoyl moiety, IC >30 µM). For the carbamate
5
0
50
derivatives, the trend in cytotoxicity was similar to that of the amide derivatives; overall, all
were more potent inhibitors Of the derivatives bearing carbon chains of the same length, the
ureido derivative 59 had approximately the same activity (IC 2.14 µM) as the amide (48,
.
5
0
IC 1.40 µM) and carbamate (57, IC 1.42 µM). Comparing galactosamine compounds 66
5
0
50
(
(
IC 11.6 µM) and 67 (IC 2.69 µM) with glucosamine compounds 3 (IC 10.3 µM) and 45
50 50 50
IC 2.85 µM) showed that the activities were not significantly different, suggesting the
5
0
C4-epimer of glucosamine moiety to be well tolerated.
Against prostate cancer cells PC-3, derivatives were found to less active to this cell lines,
only those bearing longer carbon chains were found to be more active; for example, in amide
derivatives 41, 46-47 (IC 11.9–19.4 µM), and carbamate derivatives 53, 55-58 (IC 13.9–
5
0
50
1
6.1 µM). Against colon adenocarcinoma HT29, increasing the carbon length increased the
potency, such as amide derivatives 41, 43, 46-47 (IC 16.4–22.8 µM) and carbamate
5
0
derivatives 53, 55-58 (IC 18.1–22.0 µM).
5
0
9

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Table 1. In vitro cytotoxicity of OA and a series of newly synthesized compounds
a
Compound
IC (µM)
50
HL-60
PC-3
>30
>30
>30
>30
>30
HT29
OA
3
>30
>30
10.3 ± 0.26
11.4 ± 0.04
12.5 ± 0.33
21.5 ± 0.06
2.65 ± 0.01
6.83 ± 0.02
2.48 ± 0.01
10.6 ± 0.07
5.82 ± 0.20
2.85 ± 0.01
1.68 ± 0.00
1.46 ± 0.01
1.40 ± 0.01
1.57 ± 0.00
10.7 ± 0.01
>30
>30
1
1
1
2
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
7
3
4
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
6
7
8
1
>30
>30
>30
25.0 ± 1.59
11.9 ± 0.21
>30
>30
16.4 ± 8.51
>30
24.0 ± 0.07
>30
21.7 ± 1.22
>30
>30
>30
19.4 ± 2.22
14.7 ± 1.84
>30
21.9±0.76
22.8 ± 1.82
>30
21.4 ± 0.67
>30
>30
>30
>30
>30
>30
>30
>30
2.23 ± 0.06
1.46 ± 0.01
1.44 ± 0.05
0.76 ± 0.01
1.42 ± 0.00
1.23 ± 0.06
2.14 ± 0.03
11.6 ± 0.74
2.69 ± 0.03
10.6 ± 0.06
9.13 ± 0.04
15.0 ± 1.58
>30
18.1 ± 5.12
>30
13.9 ± 1.15
15.4 ± 4.65
15.4 ± 3.20
16.1 ± 3.21
>30
21.4 ± 2.63
21.7 ± 6.09
20.3 ± 5.09
22.0 ± 1.82
>30
23.4 ± 0.57
>30
23.3 ± 2.57
>30
>30
>30
22.4 ± 0.55
23.1 ± 1.30
-
b
b
Vincristine
-FU
1.8 ± 0.19
>25
5
> 30
>30
8.3 ± 0.65
a
IC values from MTT assays after 48 h of treatment. The value are mean ± SD of at least
5
0
b
three independent experiments. Unit is nM
1
0

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2
.3 Determination of the location of the OA derivatives in the cell by fluorescent labeling
Our study showed that the cytotoxicity of the glucosamine-bearing OA saponins tested
depended in part on the length of the carbon chain at the glucosamine nitrogen. However,
compound bearing a simple long-chain carbamate conjugation with the 3-hydroxyl group of
OA was not active (data not shown), indicating the glucosamine moiety to be essential. One
possible explanation for this is that the distribution of small molecules bearing carbon chains
in cancer cells depends on the length of the carbon chain. For example,
3
,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) derivatives bearing a linker
chain longer than nine carbons were found to accumulate in mitochondria, while those
bearing linker chain fewer than nine carbons long remained in the nucleus [28]. Apparently,
the distribution of our OA derivatives is affected by the length of the carbon chains, but as
none of them incorporate a fluorophore, quantification of their uptake by individual organelles
is not possible. Further study of this effect was undertaken using compound 68 (synthesis of
6
8 is in the Supporting Information), the alkyne moiety of which was expected to be
amenable to conjugation with a fluorogenic probe (69) using click chemistry to give the
highly fluorescent triazole-compound 70 (Scheme 4) [29].
A cytotoxic assay of 68 against HL-60 determined it to have an IC of 10.6 µM,
5
0
suggesting its activity to be similar to that of compound 43 (IC50 = 10.6 µM). HL-60 cells
were treated with or without compound 68, incubated for 1 day, fixed by alcohol,
permeabilized, and mixed with fluorogenic probe (69) and the click reagents (CuSO , TBTA
4
and Na ascorbate, Scheme 4). Fluorescence images were detected using a confocal
microscope (Figure 2). Another way to introduce a fluorophore was conducted by using
azido-bearing OA 71 which can be labelled with DBCO-488 in a copper-free procedure
(Scheme 4, synthesis of 71 is in the Supporting Information) to treat with cells. For
comparison, samples were also stained with CellBrite™ Blue (cell membrane marker) and
RedDot™2 (nucleus marker). As shown in Figure 2, the control group of cells (no OA
1
1

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compound treatment) did not fluoresce. The fluorescent signals corresponding to 70 were
found evenly distributed in the HL-60 cells (green channel), while fluorescent signals of
compound 72 were found mainly in the cytosol of the HL-60 cells. These results implied that
alkynyl 68 and azido 71 were not solely located in the cell membrane, albeit they had subtle
difference in the cell distributions. Since some of nature saponins were previously reported to
cause hemolysis by interacting with sterols of erythrocyte membrane [30, 31], this effect
caused concerns in the further development of saponins as pharmacological agents.
Distribution of OA compounds in the intact cells suggested that their cytotoxic effects were
from the interactions of compounds with the mitochondrion or other organelles, but not cell
membrane.
Scheme 4. Synthesis of fluorogenic probe (a) TBTA, CuSO , Na ascorbate, rt, 6 h.
4
1
2

A
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Figure 2. Click reaction to locate cellular uptake of (A) with compound 68 in the presence of
+
fluorogenic probe 69, TBTA and Cu , (B) Compound 71 in the presence of fluorogenic probe,
DBCO-488. The confocal images of HL-60 cell fluorescence Green: [3 + 2] cycloaddition of
the compounds 68 and 71; Blue: CellBrite™ Blue (Cell membrane); Red: RedDot™2
(nucleus).
2
.4 Effect on mitochondria
Since mitochondria are involved in wide variety of cellular responses and functions,
including energy production, they are implicated in cell growth, survival, differentiation and
apoptosis [32, 33]. Recently, saponins such as reevesioside F [34], triphenylphosphonium
cation-containing betulin, and betulinic acid derivatives [35] were reported to cause the death
of cancer cells by mitochondria-stress. As we have observed the apoptosis of these OA
compounds to HL-60 cells, we then examined their effect on mitochondria using compound
4
5 as a represented compound. After compound 45 treatment, HL-60 cells were stained by
1
3

ACCEPTED MANUSCRIPT
JC-1 to determine the ∆Ψm (mitochondria membrane potential) of the mitochondrial membrane,
and analyzed by flow cytometry. As shown in Figure 3, compound 45 induced mitochondrial
membrane depolarization with significant increase of JC-1 monomer (green color) in a dose
dependent manner, indicating that compound 45 caused mitochondria damage.
The above mentioned locations of compounds and mitochondrial membrane stability
study suggested that glucosamine bearing OA compounds with appropriate proper length of
carbon chain conjugation might target mitochondria and cause cancer cell apoptosis. Further
mechanistic studies of these OA compounds are ongoing in this laboratories, and results will
be reported in due course.
Figure 3. Effect of compound on ∆Ψm and related protein expression. HL-60 cells were
incubated in the absence or dose-dependent concentration of compound 45 for the indicated
1
4

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times. Cells were incubated with JC-1 for the detection of △Ψm using FACScan flow
cytometric analysis and mitochondria membrane potential loss (%).
3
. Conclusion
In summary, a practical, concise and versatile synthesis of N-modified derivatives of
ʹ-amino-2ʹ-deoxysugar-bearing oleanolic saponins has been developed and used to
2
synthesize twenty-five novel derivatives (13-15, 20, 41-59, 66-67), the cytotoxicity of which
was evaluated in an MTT assay. Of the twenty-five novel compounds synthesized, certain
amide and carbamate-based derivatives (41-42, 44-49 and 53-59) exhibited more potent
growth inhibition than compound 3 (2ʹ-N-acetyl-2ʹ-deoxy-glucosyl OA), the first time the
cytotoxicity of 3 has been surpassed.
Amide, carbamate, and urea derivatives of the same carbon chain length exhibited
near-identical cytotoxicity against HL-60 cells. Increasing the carbon chain length increased
the cytotoxicity of the derivative up until the chain reached eleven carbons in length for amide
derivatives; and nine carbons in length for the carbamate derivatives. The different activities
of these compounds may reflect their different lipophilicities, which may affect their uptake
by cancer cells. Carbamate derivative 56 (2ʹ-N-heptoxycarbonyl moiety) was found to be the
most cytotoxic (IC = 0.76 µM). The cytotoxicities of compounds 66 and 67 bearing a
5
0
galactosamine moiety were almost equal to that of gluocosamine compounds with the same
carbon length modification, suggesting that the configuration of the C-4 in the sugar moiety
does not significantly influence cytotoxicity. These SAR relationships should help inform the
future development of anti-cancer drugs.
Preliminary studies into the mechanism of cytotoxicity imparted by the synthesized
compounds were also undertaken. Images obtained in a cellular uptake assay clearly show
that compounds 68 and 71 enter the cells and locate mainly the cytosol. Direct location
measurement of OA derivatives using fluorogenic probe allowed us to detect the distribution
1
5

ACCEPTED MANUSCRIPT
of compounds in the intact cells. A mitochondrial membrane potential assay found that
compound 45 induced mitochondria damage and this result coincided on compound
distribution on cytosol from cell image. These findings suggest that the apoptosis caused by
the compounds synthesized proceeds via perturbation of the mitochondria; more detailed
mechanistic studies, antiproliferative and apoptotic pathway analyses are ongoing.
4
. Experimental
4
.1 Chemistry part
All reagents and solvents were of reagent grade and used without further purification,
unless otherwise stated. Reaction progress was monitored by analytical TLC on 0.25 mm E.
Merck silica gel 60 F , stained with p-anisaldehyde, ninhydrin, and cerium ammonium
2
54
1
13
molybdate, and visualized by heating. H and C NMR spectra were recorded on either a
Bruker AV-400 or AVIII-600-Cry spectrometer. Chemical shifts are referenced to residual
1
13
1
13
solvent peaks (CDCl : H δ = 7.24, C δ = 77.0; CD OD: H δ = 3.30, C δ = 49.0). Splitting
3
3
patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), dd (double of doublets),
m (multiplet). Coupling constants (J) are given in Hertz. The purities of all of the biologically
tested compounds were confirmed to be higher than 95% using an analytical HPLC with a
dual pump Shimadzu LC-20AT system. Mass spectra were recorded on a Bruker bioTOF III.
Melting points were measured on a FARGO melting point apparatus MP-1D (Mandarin
Scientific Co., Ltd.).
4
.1.1. p-Methylphenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-(2ʹ,2ʹ,2ʹ-trichloroethyloxycarbonyl
amino)-β- -glucopyranoside (6)
Trichloroethoxycarbonyl chloride (38.4 mL, 279.0 mmol) was added dropwise to a
solution of -glucosamine hydrochloride (50 g, 231.9 mmol) and NaHCO (58.6 g, 698 mmol)
D
D
3
in water (500 mL), at 0 ℃ , with stirring. The mixture was stirred at room temperature for 4 h,
then filtered and washed with CHCl to give a white solid (82.3 g, 232.1 mmol), which was
3
1
6

ACCEPTED MANUSCRIPT
dissolved in pyridine (400 mL). The solution was cooled to 0 ℃ ; then, DMAP and Ac O were
2
added, with stirring. The cooling bath was removed, and the mixture stirred for 5 h. The
mixture was concentrated in vacuo; dissolved in CH Cl , and washed with 1.0 N HCl,
2
2
NaHCO solution, and brine. The organic layer was collected, dried with Na SO , filtered, and
3
2
4
concentrated in vacuo to give a white syrup (120 g). The white syrup (120 g, 230 mmol) and
o
p-toluenethiol (57 g, 459 mmol) were dissolved in dry CH Cl (800 mL), and cooled to 0 C.
2
2
BF ℃ OEt (58.4 mL, 461 mmol) was added dropwise, the ice bath removed, and the mixture
3
2
stirred for 8h. The mixture was washed with NaHCO solution and brine. The organic layer
3
was separated, dried with Na SO and concentrated in vacuo. The residue was recrystallized
2
4
1
from methanol to afford 7 (80 g, 59% over 3 steps) as a white solid; H NMR (400 MHz,
CDCl ) δ 7.39 (d, J = 8.0 Hz, 2H, Ar-H), 7.09 (d, J = 8.0 Hz, 2H, Ar-H), 5.31 (d, J = 9.1 Hz,
3
1
H, H-1), 5.25 (t, J = 9.8 Hz, 1H, H-3), 4.98 (t, J = 9.8 Hz, 1H, H-4), 4.80–4.74 (m, 2H,
Troc-CH , H-2), 4.70 (d, J = 12.0, 1H, Troc-CH ), 4.20 (dd, J = 12.2, 5.1 Hz, 1H, H-6a), 4.14
2
2
(
dd, J = 12.2, 2.2 Hz, 1H, H-6b), 3.72–3.57 (m, 2H, H-5, N-H), 2.32 (s, 3H, STol-CH ), 2.06
3
1
3
(
s, 3H, Ac-CH ), 1.97 ppm (s, 6H, Ac-CH ); C NMR (100 MHz, CDCl ) δ 170.6, 169.4,
3 3 3
1
53.8, 138.7, 133.6, 129.7, 95.4 (Troc-CCl ), 86.7 (C-1), 75.7 (C-5), 74.5 (C-3), 73.2
3
(
Troc-CH ), 68.5 (C-4), 62.3 (C-6), 55.0 (C-2), 21.2 (STol-CH ), 20.7 (Ac-CH ), 20.6
2
3
3
+
(
Ac-CH ), 20.6 ppm (Ac-CH ); HRMS (ESI) calcd. for C H Cl NO S+Na [M+Na] :
3
3
22 26
3
9
6
08.0286, found: 608.0300.
4
.1.2.
2
8-O-benzyl-3-O-[3ʹ,4ʹ,6ʹ-tri-O-acetyl-2ʹ-deoxy-2ʹ-(2ʺ,2ʺ,2ʺ-trichloroethyloxycarbonylamino)
-β-D-glucopyranosyl]oleanolic ester (9)
Compound 6 (7.63 g, 13 mmol) was dissolved in a mixture of acetone/H O = 10/1 (165
2
mL) and cooled to -20 ℃ . NBS (11.57 g, 65 mmol) was added slowly and the mixture stirred
at -20 ℃ for 2 h. The cooling bath was removed and the mixture diluted with Na S O solution
2
2
3
1
7

ACCEPTED MANUSCRIPT
and NaHCO solution and the acetone removed in vacuo. The residue was diluted with
3
CH Cl , washed with water, and concentrated in vacuo to give an off-white solid, which was
2
2
purified by flash column chromatography to give a white solid (3.4 g, 7.1 mmol, 54 %). DBU
31.08 µL, 0.208 mmol) was added to a solution of this white solid (500 mg, 1.04 mmol) and
trichloroacetonitrile (312.9 µL, 3.12 mmol) in CH Cl (5 mL). The mixture was stirred at
(
2
2
room temperature for 2 h, then concentrated in vacuo, and the residue purified by flash
column chromatography (silica gel; EtOAc/hexane/TEA = 1:1:0.1) to give trichloroimidate
intermediate 7. A mixture of trichloroimidate intermediate 7 (650 mg, 1.04 mmol), benzyl
oleanolate (682 mg, 1.25 mmol), and 4 Å MS in dry CH Cl (13 mL) was stirred at 0 ℃ for 30
2
2
minutes. TMSOTf (9.4 µL, 0.052 mmol) was added and the mixture was stirred for another 30
minutes at 0 ℃ . The reaction was quenched by the addition of TEA (1 drop); then the mixture
was filtered, concentrated in vacuo, and purified by flash column chromatography (silica gel;
1
EtOAc/hexane/toluene = 1/4/2) to give compound 9, as a white solid (780 mg, 74 %); H
NMR (400 MHz, CDCl ) δ 7.33–7.27 (m, 5H, Ar-H), 5.29–5.17 (m, 3H, H-1, H-3′, H-12),
3
5
.09–4.96 (m, 3H, H-4′, COCH Ph), 4.70 (d, J = 12.0 Hz, 1H, Troc-CH ), 4.64–4.58 (m, 2H,
2 2
H-2′, Troc-CH ), 4.23 (dd, J = 12.0, 5.4 Hz, 1H, H-6aʹ), 4.08 (d, J = 12.0 Hz, 1H, H-6bʹ),
2
3
.70–3.60 (m, 2H, H-5ʹ, N-H), 3.08 (dd, J = 11.4, 4.3 Hz, 1H, H-3), 2.87 (d, J = 10.1 Hz, 1H,
H-18), 2.05 (s, 3H, Ac-CH ), 2.00 (s, 6H, Ac-CH ), 1.96 – 1.90 (m, 1H), 1.84–1.77 (m, 2H),
3
3
1
.68 – 1.60 (m ,5H), 1.58–1.54 (m, 2H), 1.52 – 1.49 (m, 1H), 1.48–1.42 (m, 2H), 1.39–1.33
(m, 2H), 1.32–1.26 (m, 2H), 1.25–1.20 (m, 2H), 1.19–1.14 (m, 2H),1.08 (s, 3H), 1.0 –0.97
1
3
(
m,1H), 0.89 (s, 6H), 0.87 (s, 3H), 0.85 (s, 3H), 0.74 (s, 3H), 0.56 ppm (s, 3H); C NMR
(
100 MHz, CDCl ) δ 177.42 (CO CH Ph), 170.7 (Ac-C=O), 169.5 (Ac-C=O), 153.9
3
2
2
(Troc-C=O), 143.7 (C-13), 136.4 (Ar-C), 128.4 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 122.4
(
C-12), 103.1 (C-1ʹ), 95.2 (Troc-CCl ), 90.8 (C-3), 74.7 (C-5ʹ), 71.9 (C-3ʹ), 71.5 (Troc-CH ),
3
2
6
8.9 (C-4ʹ), 65.9 (CO CH Ph), 62.4 (C-6ʹ), 56.8 (C-2ʹ), 55.5, 47.6, 46.7, 45.9, 41.6, 41.3, 39.3,
2 2
3
8.9, 38.4, 36.7, 33.8, 33.1, 32.7, 32.3, 30.7, 28.0, 27.6, 26.3, 25.8, 25.7, 23.6, 23.4, 23.0, 20.8,
1
8

ACCEPTED MANUSCRIPT
+
2
1
0.6, 18.2, 16.83, 16.42, 15.2 ppm; HRMS (ESI) calcd. for C H Cl NO +Na [M+Na] :
52 72 3 12
030.4012, found: 1030.4051.
4
.1.3. General procedure A for the synthesis of carbamates 13–15
Compound 9 was dissolved in an alcohol (3 mL, MeOH for 10 or EtOH for 11 or
n-propanol/ CH Cl = 1/2 for 12) and the solution diluted with 4.0 N NaOH (0.5 mL) and
2
2
stirred for 5 h. The mixture was concentrated in vacuo, dissolved in CH Cl , washed with
2
2
NH Cl solution and brine, and purified by flash column chromatography (silica gel;
4
CH Cl /MeOH = 20:1) to give deacylated product 10–12. This was dissolved in MeOH and
2
2
Pd/C (10%) was added. The mixture was stirred under H2(g) at room temperature for 8h. The
residue was filtered, concentrated in vacuo, and purified by flash column chromatography
(
silica gel; CH Cl /MeOH = 14/1) to give final products 13–15.
2 2
4
.1.4. 3-O-(2ʹ-N-methoxycarbonyl-2ʹ-deoxy-β- -glucopyranosyl)oleanolic acid (13)
D
According to the general procedure A, compound 13 (20 mg, 60%) was obtained from
1
compound 9 (50 mg, 49.5 µmol) as a white solid; m.p. 250 ℃ ; H NMR (600 MHz,
CD OD/CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.41 (d, J = 7.9 Hz, 1H, H-1ʹ), 3.82
3
3
(
dd, J = 11.9, 2.7 Hz, 1H, H-6aʹ), 3.71 (dd, J = 11.9, 5.0 Hz, 1H, H-6bʹ), 3.61 (s, 3H, CO CH ),
2 3
3
.46 –3.33 (m, 3H, H-2ʹ, H-3ʹ, H-4ʹ), 3.23 (ddd, J = 9.3, 5.0, 2.7 Hz, 1H, H-5ʹ), 3.09 (dd, J =
11.7, 4.4 Hz, 1H, H-3), 2.81 (dd, J = 13.9, 3.9 Hz, 1H, H-18), 1.99–1.92 (m, 1H), 1.87–1.82
(m, 3H), 1.75–1.62 (m, 4H), 1.60–1.55 (m, 2H), 1.54–1.48 (m, 3H), 1.46–1.40 (m, 1H), 1.38–
1
1
3
.31 (m, 2H), 1.29–1.23 (m, 3H), 1.20–1.15 (m, 1H), 1.14–1.12 (m, 1H), 1.12 (s, 3H), 1.07–
.02 (m, 1H), 0.92 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H), 0.74 ppm (s,
1
3
H); C NMR (150 MHz, CD OD /CDCl = 4/1) δ 181.5 (COOH), 158.8 (NC=O), 144.5
3
3
(
C-13), 123.0 (C-12), 104.6 (C-1ʹ), 90.9 (C-3), 76.5 (C-5ʹ), 74.9 (C-3ʹ), 71.5 (C-4ʹ), 62.4
C-6ʹ), 58.8 (C-2ʹ), 52.3 (CO CH ), 48.3, 47.1, 46.6, 42.3, 42.0, 39.9, 39.4, 39.1, 37.3, 34.5,
(
2
3
1
9

ACCEPTED MANUSCRIPT
3
3.4, 33.2, 31.2, 30.2, 28.3, 28.1, 26.33, 26.28, 24.0, 23.9, 23.6, 23.5, 18.8, 17.3, 16.7, 15.7
+
ppm; HRMS (ESI) calcd. for C H NO +Na [M+Na] : 698.4239, found: 698.4253; HPLC
3
8
61
9
purity 95.7% (t : 10.5 min, Mightysil, RP-18, 250 × 4.6 mm, 5 µm, H O/ACN = 40/60, 1
R
2
mL/min, 30 min).
4
.1.5. 3-O-(2ʹ-N-ethoxycarbonyl-2ʹ-deoxy-β-D-glucopyranosyl)oleanolic acid (14)
According to general procedure A, compound 14 (15 mg, 46%) was obtained from
1
compound 9 (48 mg, 47.5 µmol) as a white solid; m.p. 245 ℃ ; H NMR (600 MHz, CD OD
3
/
CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.40 (d, J = 8.1 Hz, 1H, H-1ʹ), 4.11–4.00 (m,
3
2
H, H-1″), 3.83 (dd, J = 11.9, 2.5 Hz, 1H, H-6aʹ), 3.69 (dd, J = 11.9, 5.2 Hz, 1H, H-6bʹ),
.44–3.39 (m, 1H, H-3ʹ), 3.38–3.33 (m, 2H, H-2ʹ, H-4ʹ), 3.23 (ddd, J = 9.3, 5.2, 2.5 Hz, 1H,
3
H-5ʹ), 3.10 (dd, J = 11.7, 4.3 Hz, 1H, H-3), 2.82 (dd, J = 13.8, 4.0 Hz, 1H, H-18), 2.00–1.93
m, 1H), 1.92–1.85 (m, 3H), 1.77–1.69 (m, 2H), 1.68–1.61 (m, 2H), 1.61–1.55 (m, 2H), 1.55–
(
1
1
3
.49 (m, 3H), 1.48–1.43 (m, 1H), 1.41–1.35 (m, 2H), 1.34–1.32 (m, 1H), 1.30–1.27 (m, 3H),
.21 (t, J = 7.1 Hz, 3H, H-2″), 1.19–1.16 (m, 1H), 1.13 (s, 3H), 1.07–1.03 (m, 1H), 0.95(s,
1
3
H), 0.92 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.79 (s, 3H), 0.76 ppm (s, 3H); C NMR (150
MHz, CD OD /CDCl = 4/1) δ 181.7 (COOH), 158.7 (NC=O), 144.8 (C-13), 123.3 (C-12),
3
3
1
05.0 (C-1ʹ), 91.0 (C-3), 76.9 (C-5ʹ), 75.3 (C-3ʹ), 71.8 (C-4ʹ), 62.6 (C-6ʹ), 61.4 (C-1″), 58.9
(C-2ʹ), 46.9, 42.6, 42.3, 40.2, 39.8, 39.65, 39.4, 37.6, 34.7, 33.7, 33.51, 33.47, 31.4, 30.4, 28.5,
2
8.4, 26.6, 26.4, 24.2, 23.9, 23.8, 19.0, 17.5, 16.9, 15.8, 15.0, 14.4 ppm; HRMS (ESI) calcd.
+
for C H NO +Na [M+Na] : 712.4395, found: 712.4417; HPLC purity 96.2% (t : 12.8 min,
3
9
63
9
R
Mightysil, RP-18, 250 × 4.6 mm, 5 µm, H O/ACN = 40/60, 1 mL/min, 30 min).
2
4
.1.6. 3-O-(2ʹ-N-propoxycarbonyl-2ʹ-deoxy-β- -glucopyranosyl)oleanolic acid (15)
D
According to general procedure A, compound 15 (15 mg, 39 %) was obtained from
1
compound 9 (55 mg, 54.5 µmol) as a white solid; m.p. 226 ℃ ; H NMR (600 MHz, CD OD
3
2
0

ACCEPTED MANUSCRIPT
/
CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.41 (d, J = 8.1 Hz, 1H, H-1ʹ), 4.00–3.88 (m,
3
2
3
H, H-1″), 3.82 (dd, J = 11.9, 2.7 Hz, 1H, H-6aʹ), 3.71 (dd, J = 11.9, 4.9 Hz, 1H, H-6bʹ),
.45–3.33 (m, 3H, H-2ʹ, H-3ʹ, H-4ʹ), 3.23 (ddd, J = 9.3, 4.9, 2.7 Hz, 1H, H-5ʹ), 3.09 (dd, J =
11.7, 4.4 Hz, 1H, H-3), 2.81 (dd, J = 13.9, 3.9 Hz, 1H, H-18), 1.98–1.92 (m, 1H), 1.87–1.83
(m, 3H), 1.75–1.68 (m, 2H), 1.67–1.63 (m, 1H), 1.63–1.55 (m, 5H), 1.54–1.48 (m, 3H), 1.46–
1
1
3
.41 (m, 1H), 1.40–1.30 (m, 3H), 1.29–1.25 (m, 2H), 1.19–1.13 (m, 2H), 1.12 (s, 3H), 1.07–
.02 (m, 1H), 0.93 (s, 3H), 0.93–0.91 (m, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.77 (s,
13
H), 0.75 ppm (s, 3H); C NMR (150 MHz, CD OD /CDCl = 4/1) δ 181.5 (COOH), 158.4
3
3
(
NC=O), 144.5 (C-13), 122.9 (C-12), 104.7 (C-1ʹ), 90.8 (C-3), 76.4 (C-5ʹ), 75.0 (C-3ʹ), 71.5
C-4ʹ), 67.1 (C-1″), 62.4 (C-6ʹ), 58.6 (C-2ʹ), 48.3, 46.6, 42.3, 42.0, 39.9, 39.4, 39.1, 37.3, 34.5,
(
3
1
7
3.4, 33.2, 31.2, 30.2, 29.9, 28.3, 26.34, 26.27, 24.0, 23.9, 23.6, 23.2, 22.9, 18.8, 17.3, 16.8,
+
5.7, 14.3, 10.6 ppm; HRMS (ESI) calcd. for C H NO +Na [M+Na] : 726.4552, found:
4
0
65
9
26.4575; HPLC purity 96.4% (t : 16.0 min, Mightysil, RP-18, 250 × 4.6 mm, 5 µm,
R
H O/ACN = 40/60, 1 mL/min, 30 min).
2
4
.1.7. p-Methylphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside
(16)
Phthalic anhydride (6.88 g, 46.4 mmol) was added to a stirring solution of
-glucosamine hydrochloride (10g, 46.4 mmol) and NaHCO (7.8 g, 92.8 mmol) in
D
3
H O/acetone = 1/1 (200 mL) at room temperature. The mixture was stirred for 4h, and then
2
the mixture was concentrated under reduced pressure to give a light yellow solid. The solid
was suspended in pyridine (200 mL), Ac O (20g, 196 mmol) and DMAP (114 mg, 0.92
2
mmol), and stirred 8 h. The mixture was concentrated in vacuo, diluted with CH Cl , washed
2
2
with 1.0 N HCl, NaHCO solution, and brine. The organic layers were collected, filtered and
3
concentrated to get light yellow syrup. The syrup and p-toluenethiol (11.5 g, 92.8 mmol) were
dissolved in CH Cl (350 mL), and BF ．OEt (13.15 g, 92.8 mmol) was added dropwise at
2
2
3
2
2
1

ACCEPTED MANUSCRIPT
o
0
C. The mixture was stirred at room temperature for 24 h. The mixture was then washed with
NaHCO solution and brine. The organic layer was separated, dried with MgSO and
3
4
concentrated in vacuo. The residue was recrystallized from ethanol to afford compound 16 as
1
a white solid (13.2 g, 53 % for three steps); H NMR (400 MHz, CDCl ) δ 7.84 (dd, J = 5.1,
3
3
.1 Hz, 2H, Ar-H), 7.73 (dd, J = 5.1, 3.1 Hz, 2H, Ar-H), 7.27 (d, J = 7.9 Hz, 2H, Ar-H), 7.05
(d, J = 7.9 Hz, 2H, Ar-H), 5.75 (t, J = 9.7 Hz, 1H, H-3), 5.62 (d, J = 10.5 Hz, 1H, H-1), 5.09 (t,
J = 9.7 Hz, 1H, H-4), 4.33–4.22 (m, 2H, H-2, H-6a), 4.17 (d, J = 12.2 Hz, 1H, H-6b), 3.85 (dd,
J = 10.1, 2.5 Hz, 1H, H-5), 2.30 (s, 3H, STol-CH ), 2.08 (s, 3H, Ac-CH ), 1.99 (s, 3H,
3
3
1
3
Ac-CH ), 1.81 ppm (s, 3H, Ac-CH ); C NMR (100 MHz, CDCl ) δ 170.6, 170.1, 169.4,
3
3
3
1
38.7, 134.4, 133.9, 129.6, 126.9, 123.7, 83.1 (C-1), 75.8 (C-5), 71.6 (C-3), 68.7 (C-4), 62.2
(
C-6), 53.6 (C-2), 21.1 (STol-CH ), 20.7 (Ac-CH ), 20.6 (Ac-CH ), 20.4 ppm (Ac-CH );
3
3
3
3
+
HRMS (ESI) calcd. for C H NO S+Na [M+Na] : 564.1299, found: 564.1324.
2
7
27
9
4
.1.8. 28-O-benzyl 3-O-[3ʹ,4ʹ,6ʹ-tri-O-acetyl-2ʹ-deoxy-2ʹ-phthalimido-β-
D-glucopyranosyl]
oleanolic ester (18)
NBS (5.6 g, 31.5 mmol) was added slowly to a solution of compound 16 (4.09 g, 7.55
mmol) in acetone/H O = 10/1 (100 mL) at -20 ℃ . After 2 h, the mixture was diluted with
2
Na S O solution, NaHCO solution, then concentrated in vacuo. The residue was diluted with
2
2
3
3
CH Cl , washed with water, concentrated in vacuo, and purified by column chromatography
2
2
(
silicagel; hexane/EtOAc = 1.5/1) to give anomer-OH intermediate as a white solid (2.1 g,
.82 mmol, 64 %). DBU (12.9 µL, 0.086 mmol), was added into a solution of this
intermediate (200 mg, 0.459 mmol) and trichloroacetonitrile (260 µL, 2.59 mmol) in CH Cl2
4
2
(4 mL), and the mixture was stirred under room temperature for 2 h. The mixture was
concentrated in vauco, then subjected to column chromatography (silica gel;
EtOAc/hexane/TEA = 1: 1: 0.1) to give trichloroimidate 17 as an oil. A mixture of
trichloroimidate intermediate 17 (200 mg, 0.345 mmol), benzyl oleanolate (200 mg, 0.366
2
2

ACCEPTED MANUSCRIPT
mmol), and 4 Å MS in dry CH Cl (3 mL) was stirred at 0 ℃ for 30 minutes. TMSOTf (4.3
2
2
µL, 0.024 mmol) was then added, stirred for 30 minutes at 0 ℃ , and then TEA (1 drop) was
added. The mixture was filtered, concentrated in vacuo, and purified by column
chromatography (silica gel; EtOAc/hexane/toluene = 1/4/2) to give compound 18 (250 mg, 75
1
%
); H NMR (400 MHz, CDCl ) δ 7.82 (dd, J = 5.4, 3.1 Hz, 2H, Ar-H), 7.70 (dd, J = 5.4, 3.1
3
Hz, 2H, Ar-H), 7.31–7.26 (m, 5H, Ar-H), 5.80 (dd, J = 10.6, 9.2 Hz, 1H, H-3ʹ), 5.34 (d, J =
.4 Hz, 1H, H-1ʹ), 5.24 (s, 1H, H-12), 5.10 (dd, J = 10.0, 9.2 Hz, 1H, H-4ʹ), 5.02 (q, J = 12.6
Hz, 2H, CO CH Ph), 4.36–4.26 (m, 2H, H-2ʹ, H-6aʹ), 4.14–4.09 (m, 1H, H-6bʹ), 3.89–3.81 (m,
8
2
2
1
H, H-5ʹ), 3.02 (dd, J = 11.6, 4.4 Hz, 1H, H-3), 2.86 (d, J = 9.8 Hz, 1H, H-18), 2.07 (s, 3H,
Ac-CH ), 2.01 (s, 3H, Ac-CH ), 1.84 (s, 3H, Ac-CH ), 1.97–1.88 (m, 1H), 1.81– 1.75 (m, 2H),
3
3
3
1
1
0
.68–1.65 (m, 1H), 1.64–1.58 (m, 4H), 1.57–1.48 (m, 4H), 1.43–1.37 (m, 1H), 1.33–1.28 (m,
H), 1.26–1.21 (m, 3H), 1.18–1.15 (1H), 1.13–1.07 (m, 3H), 1.04 (s, 3H), 0.97 –0.92 (m, 1H),
1
3
.88 (s, 3H), 0.86 (s, 3H), 0.79 (s, 3H), 0.55 (s, 3H), 0.51 (s, 3H), 0.38 ppm (s, 3H); C NMR
(
100 MHz, CDCl ) δ 177.4(COCH Ph), 170.7 170.23, 169.5, 143.6 (C-13), 136.4, 134.3
3 2
1
28.4, 127.9, 127.9, 123.5 (C-12), 99.9 (C-1ʹ), 90.7 (C-3), 71.5 (C-5ʹ), 70.7 (C-3 ʹ), 69.3
(
C-4ʹ), 65.9 (COCH Ph), 62.4 (C-6ʹ), 55.1 (C-2ʹ), 54.8, 47.5, 46.7, 45.8, 41.6, 41.3, 39.2, 38.3,
2
3
8.2, 36.6, 33.8, 33.1, 32.6, 32.3, 30.7, 27.5, 27.4, 25.8, 23.6, 23.34, 22.98, 20.77, 20.6, 20.5,
+
1
8.0, 16.8, 16.3, 15.2 ppm; HRMS (ESI) calcd. for C H NO +Na [M+Na] : 986.5025,
5
7
73
12
found: 986.5039.
4
.1.9. 3-O-(2ʹ-amino-2ʹ-deoxy-β- -glucopyranosyl)oleanolic acid (20)
D
Starting material 18 (100 mg, 0.104 mmol) was dissolved in ethanol/ethylenediamine =
/1 (3 mL) and the mixture was stirred at 80 ℃ for 6 h. The mixture was concentrated in
2
vacuo and toluene was added twice to azeotropically remove ethanol and ethylenediamine.
The residue was purified by column chromatography (silica gel; CH Cl /MeOH/TEA =
2
2
9
/1/0.1) to afford amine containing intermediate 19 (69 mg, 93%). Intermediate 19 (15 mg,
2
3

ACCEPTED MANUSCRIPT
0
.021 mmol) was dissolved in MeOH and 20% Pd/C was added. The mixture was stirred
under H (balloon) at room temperature for 8h. The residue was filtered, concentrated in
2
vacuo, and purified by column chromatography (silica gel; CH Cl /MeOH = 12/1) to
2
2
1
compound 20 (10 mg, 77%) as a white solid. m.p. 234–237 ℃ ; H NMR (600 MHz,
CD OD/CDCl = 4/1) δ 5.24 (t, J = 3.2, 1H, H-12), 4.33 (d, J = 7.9 Hz, 1H, H-1), 3.84 (dd, J
3
3
=
12.0, 2.4 Hz, 1H, H-6aʹ), 3.69 (dd, J = 12.0, 5.2 Hz, 1H, H-6bʹ), 3.32 (t, J = 8.8 Hz, 1H,
H-4ʹ), 3.29 (t, J = 8.8 Hz, 1H, H-3ʹ), 3.24-3.28 (m, 1H, H-5ʹ), 3.22 (dd, J =11.7, 4.3 Hz, 1H,
H-3), 2.84 (dd, J = 13.6, 3.5 Hz, 1H, H-18 ), 2.68 (t, J = 8.9 Hz, 1H, H-2ʹ), 2.05–1.96 (m, 1H),
1
1
.96–1.93 (m, 1H), 1.93–1.91 (m, 1H), 1.91–1.83 (m, 2H), 1.81–1.77 (m, 1H), 1.75–1.70 (m,
H), 1.70–1.63 (m, 2H), 1.63–1.58 (m, 2H), 1.58–1.53 (m, 3H), 1.53–1.45 (m, 2H), 1.45–1.34
(
(
(
m, 3H), 1.34–1.24 (m, 3H), 1.21–1.16 (m, 1H), 1.14 (s, 3H), 1.12–1.09 (m, 1H), 1.09–1.04
m, 1H), 1.03 (s, 3H), 1.01–0.95 (m, 2H), 0.94 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.89–0.86
1
3
m, 1H), 0.84 (s, 3H), 0.81 (s, 3H), 0.80–0.77 ppm (m, 1H); C NMR(150MHz,
CD OD/CDCl = 4/1) δ 182.1 (COOH), 145.1 (C-13), 123.2 (C-12), 105.7 (C-1ʹ), 90.2 (C-3),
3
3
7
3
1
6
7.6 (C-5ʹ), 76.9 (C-3ʹ), 71.6 (C-4ʹ), 62.6 (C-6ʹ), 58.7 (C-2ʹ), 56.6, 47.5, 47.1, 42.7, 42.5, 40.3,
9.8, 39.5, 37.7, 34.8, 33.8, 33.6, 33.5, 31.5, 28.9, 28.7, 26.7, 26.4, 24.3, 24.0, 19.1, 17.6, 17.1,
+
+
5.9 ppm; HRMS (ESI TOF-MS) [M +H] calcd. for C H NO : 618.4364; found:
3
6
59
7
18.4380.
4
.1.10. General procedures B for amide bond coupling and deprotection to afford 41-45
Compound 19 (22 mg) was diluted with CH Cl (3 mL); then, pyridine (0.5 mL), and
2
2
anhydrides (0.1 mL) were added. The reaction was stirred for 2 h to a mixture of partially
acetylated products. The mixture was concentrated, then dissolved in CH Cl /MeOH = 1/1 (4
2
2
mL), diluted with 4.0 N NaOH (aq) (0.1 mL), and stirred for 6 h. The mixture was
concentrated in vacuo, dissolved in CH Cl , washed with NH Cl and brine, concentrated in
2
2
4
vacuo, and purified by flash column chromatography (silica gel; CH Cl /MeOH = 18/1) to get
2
2
2
4

ACCEPTED MANUSCRIPT
deacylated products 22-26. Then, the deacylated products were dissolved in MeOH and 10%
Pd/C was added. The mixture was stirred under H at room temperature for 8h. The residue
2
was filtered, concentrated in vacuo, and purified by flash column chromatography (silica gel;
CH Cl /MeOH = 14/1).
2
2
4
.1.11. 3-O-(2ʹ-propanoylamino-2ʹ-deoxy-β-
Compound 41 (50 mg, 60%), as a white solid, was prepared according to the general
procedure B for amide bond coupling using 19 (88 mg, 0.124 mmol), propinoic anhydride
0.1 mL), pyridine (0.5 mL) and purified by column chromatography (silica gel;
CH Cl /MeOH = 15/1) to give compound 22, which was further deprotected using 10% Pd/C
D
-glucopyranosyl)oleanolic acid (41)
(
2
2
(
6 mg) and purified by column chromatography (silica gel; CH Cl /MeOH = 15/1). m.p. 217
2 2
1
℃
; H NMR (600 MHz, CD OD /CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.46 (d, J =
3 3
8
.3 Hz, 1H, H-1ʹ), 3.83 (dd, J = 11.9, 2.6 Hz, 1H, H-6aʹ), 3.71 (dd, J = 11.9, 5.0 Hz, 1H,
H-6bʹ), 3.65 (dd, J = 10.3, 8.3 Hz, 1H, H-2ʹ), 3.46 (dd, J = 10.3, 9.0 Hz, 1H, H-3ʹ), 3.36 (t, J =
.0 Hz, 1H, H-4ʹ), 3.24 (ddd, J = 9.0, 5.0, 2.6 Hz, 1H, H-5ʹ), 3.08 (dd, J = 11.7, 4.4 Hz, 1H,
H-3), 2.81 (dd, J = 13.7, 4.2 Hz, 1H, H-18), 2.23–2.17 (m, 2H, H-2″), 2.00–1.92 (m, 1H),
9
1
2
.89–1.83 (m, 3H), 1.76–1.69 (m, 2H), 1.67–1.60 (m, 2H), 1.60–1.56 (m, 2H), 1.54–1.50 (m,
H), 1.49–1.46 (m, 1H), 1.45–1.41 (m, 2H), 1.37–1.34 (m, 2H), 1.31–1.29 (m, 2H), 1.20–1.16
(
(
(
m, 2H), 1.14–1.09 (m, 6H), 1.07–1.02 (m, 2H), 0.93 (s, 3H), 0.91 (s, 3H), 0.90 (s, 3H), 0.88
13
s, 3H), 0.77 (s, 3H), 0.74 ppm (s, 3H); C NMR (150MHz, CD OD/CDCl = 4/1) δ 181.6
3
3
COOH), 176.6 (NC=O), 144.6 (C-13), 123.1 (C-12), 104.3 (C-1ʹ), 90.6 (C-3), 76.6 (C-5ʹ),
7
3
1
6
5.3 (C-3ʹ), 71.7 (C-4ʹ), 62.4 (C-6ʹ), 57.1 (C-2ʹ), 47.1, 46.7, 42.4, 42.1, 40.0, 39.6, 39.2, 37.4,
4.5, 33.5, 33.3, 32.6, 31.1, 30.1, 29.6, 28.5, 28.4, 26.3, 24.1, 23.9, 23.7, 23.3, 18.9, 17.4, 16.8,
+
5.7, 14.4, 11.3 ppm; HRMS (ESI) calcd. for C H NO +Na [M+Na] : 696.4446, found:
3
9
63
8
96.4464; HPLC purity 96.4% (t : 10.4 min, Mightysil, RP-18, 250 × 4.6 mm, 5 µm,
R
H O/ACN = 40/60, 1 mL/min, 30 min).
2
2
5

ACCEPTED MANUSCRIPT
4
.1.12. 3-O-(2ʹ-butanoylamino-2ʹ-deoxy-β-D-glucopyranosyl)oleanolic acid (42)
Compound 42 (9.8 mg, 51%), as a white solid, was prepared according to the general
procedure B for amide bond coupling using 19 (20 mg, 0.028 mmol), butyric anhydride (0.1
mL), and pyridine (0.5 mL); and purified by column chromatography (silica gel;
CH Cl /MeOH = 15/1) to give compound 23 which was further deprotected using 10% Pd/C
2
2
(
3 mg) and purified by column chromatography (silica gel; CH Cl /MeOH = 15/1); m.p. 196
2 2
1
℃
; H NMR (600 MHz, CD OD/CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.46 (d, J =
3 3
8
.3 Hz, 1H, H-1ʹ), 3.82 (dd, J = 11.9, 2.6 Hz, 1H, H-6aʹ), 3.71 (dd, J = 11.9, 5.0 Hz, 1H,
H-6bʹ), 3.66–3.63 (m, 1H, H-2ʹ), 3.44 (dd, J = 10.2, 9.0 Hz, 1H, H-3ʹ), 3.39–3.34 (m, 1H,
H-4ʹ), 3.24 (ddd, J = 9.7, 5.0, 2.6 Hz, 1H, H-5ʹ), 3.08 (dd, J = 11.7, 4.4 Hz, 1H, H-3), 2.81 (dd,
J = 13.9, 3.7 Hz, 1H, H-18), 2.16 (dd, J = 8.2, 7.0 Hz, 2H, H-2″), 1.99–1.92 (m, 1H), 1.89–
1
1
1
0
.83 (m, 3H), 1.75–1.68 (m, 2H), 1.66–1.61 (m, 4H), 1.60–1.55 (m, 2H), 1.54–1.50 (m, 3H),
.45–1.40 (m, 2H), 1.36–1.34 (m, 2H), 1.30–1.29 (m, 2H), 1.19–1.15 (m, 2H), 1.12 (s, 3H),
.06–1.03 (m, 1H), 0.95–0.92 (m, 6H), 0.91 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H),
13
.74 ppm (s, 3H); C NMR (150 MHz, CD OD/CDCl = 4/1) δ 181.5 (COOH), 175.8
3
3
(
NC=O), 144.6 (C-13), 123.0 (C-12), 104.2 (C-1ʹ), 90.5 (C-3), 76.6 (C-5ʹ), 75.3 (C-3ʹ), 71.7
C-4ʹ), 62.4 (C-6ʹ), 57.1 (C-2ʹ), 48.4, 47.1, 46.7, 42.4, 42.1, 40.0, 39.5, 39.1, 37.4, 34.5, 33.4,
(
3
1
3.3, 32.6, 31.2, 30.0, 29.60, 28.47, 28.3, 26.3, 24.1, 23.9, 23.3, 19.6, 18.9, 17.4, 16.8, 15.7,
+
4.3, 14.2 ppm; HRMS (ESI) calcd. for C H NO +Na [M+Na] : 710.4602, found: 710.4620;
4
0
65
8
HPLC purity 95.8% (t : 11.7 min, Mightysil, RP-18, 250 × 4.6 mm, 5 µm, H O/ACN = 40/60,
R
2
1
mL/min, 30 min).
4
.1.13. 3-O-(2ʹ-pentanoylamino-2ʹ-deoxy-β-
D
-glucopyranosyl)oleanolic acid (43)
Compound 43 (16.0 mg, 65%), as a white solid, was prepared according to the general
procedure B for amide bond coupling using 19 (25 mg, 0.035 mmol), valeric anhydride (0.1
2
6

ACCEPTED MANUSCRIPT
mL), and pyridine (0.5 mL); and purified by column chromatography (silica gel;
CH Cl /MeOH = 15/1) to give compound 24 which was further deprotected using 10% Pd/C
2
2
(
3 mg) and purified by column chromatography (silica gel; CH Cl /MeOH = 15/1); m.p. 206
2 2
1
℃
; H NMR (600 MHz, CD OD/CDCl = 4/1) δ 5.24 (s, 1H, H-12), 4.45 (d, J = 8.3 Hz, 1H,
3 3
H-1ʹ), 3.84 (d, J = 11.8 Hz, 1H, H-6aʹ), 3.72-3.60 (m, 2H, H-6bʹ, H-2ʹ), 3.44 (t, J = 9.5 Hz, 1H,
H-3ʹ), 3.35–3.30 (m, 1H, H-4ʹ), 3.26–3.21 (m, 1H, H-5ʹ), 3.12–3.07 (m, 1H, H-3), 2.83 (d, J =
1
1
1
3
3.6 Hz, 1H, H-18), 2.24–2.17 (m, 2H, H-2″), 2.02–1.94 (m, 1H), 1.90–1.85 (m, 2H), 1.78–
.70 (m, 2H), 1.68–1.63 (m, 2H), 1.62–1.57 (m, 5H), 1.56–1.48 (m, 4H), 1.48–1.44 (m, 1H),
.39–1.32 (m, 6H), 1.21–1.16 (m, 2H), 1.14 (s, 3H), 1.08–1.04 (m, 1H), 0.95 (s, 3H), 0.93 (s,
1
3
H), 0.93–0.90 (m, 6H), 0.90 (s, 3H), 0.80 (s, 3H), 0.76 ppm (s, 3H); C NMR (150 MHz,
CD OD /CDCl = 4/1) δ 181.8 (COOH), 176.2 (NC=O), 144.9 (C-13), 123.4 (C-12), 104.6
3
3
(C-1ʹ), 90.7 (C-3), 77.2 (C-5ʹ), 75.7 (C-3ʹ), 72.0 (C-4ʹ), 62.6 (C-6ʹ), 57.4 (C-2ʹ), 49.9, 47.4,
4
7.0, 42.7, 42.4, 40.3, 39.8, 39.5, 37.7, 37.1, 34.8, 33.8, 33.6, 33.5, 31.5, 30.5, 28.7, 28.7, 28.6,
2
6.6, 26.4, 24.3, 24.0, 23.9, 23.4, 19.2, 17.6, 17.0, 15.9, 14.1 ppm; HRMS (ESI) calcd. for
+
C H NO +Na [M+Na] : 724.4759, found: 724.4784; HPLC purity 95.1% (t : 9.8 min,
41
67
8
R
Mightysil, RP-18, 250 × 4.6 mm, 5 µm, H O/ACN = 30/70, 1 mL/min, 30 min).
2
4
.1.14. 3-O-(2ʹ-hexanoylamino-2ʹ-deoxy-β-
Compound 44 (13.2 mg, 49%) was prepared as a white solid according to the general
procedure B for amide bond coupling using 19 (27.0 mg, 0.038 mmol), hexanoic anhydride
0.1 mL), and pyridine (0.5 mL); and purified by column chromatography (silica gel;
CH Cl /MeOH = 15/1) to give compound 25 which was further deprotected using 10% Pd/C
D
-glucopyranosyl)oleanolic acid (44)
(
2
2
(
3 mg), and purified by column chromatography (silica gel; CH Cl /MeOH = 15/1); m.p. 204
2 2
1
℃
; H NMR (600 MHz, CD OD/CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.46 (d, J =
3 3
8
.3 Hz, 1H, H-1ʹ), 3.82 (dd, J = 11.9, 2.6 Hz, 1H, H-6aʹ), 3.71 (dd, J = 11.9, 4.9 Hz, 1H,
H-6bʹ), 3.63 (dd, J = 10.3, 8.3 Hz, 1H, H-2ʹ), 3.44 (dd, J = 10.3, 9.0 Hz, 1H, H-3ʹ), 3.36 (t, J =
2
7

ACCEPTED MANUSCRIPT
9
.0 Hz, 1H, H-4ʹ), 3.23 (ddd, J = 9.0, 4.9, 2.6 Hz, 1H, H-5ʹ), 3.08 (dd, J = 11.7, 4.4 Hz, 1H,
H-3), 2.83–2.78 (m, 1H, H-18), 2.19–2.15 (m, 2H, H-2″), 1.99–1.91 (m, 1H), 1.88–1.82 (m,
H), 1.75–1.68 (m, 3H), 1.66–1.62 (m, 2H), 1.61–1.56 (m, 6H), 1.54–1.48 (m, 4H), 1.44–1.41
3
(m, 1H), 1.35–1.33 (m, 2H), 1.31–1.29 (m, 3H), 1.18–1.16 (m, 2H), 1.11 (s, 3H), 1.07–1.02
(m, 1H), 0.93 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.88–0.86 (m, 6H), 0.77 (s, 3H), 0.74 ppm (s,
1
3
3
H); C NMR (150 MHz, CD OD/CDCl = 4/1) δ 181.5 (COOH), 175.9 (NC=O), 144.5
3 3
(
C-13), 123.0 (C-12), 104.1 (C-1ʹ), 90.5 (C-3), 76.5 (C-5ʹ), 75.2 (C-3ʹ), 71.6 (C-4ʹ), 62.4
C-6ʹ), 56.3 (C-2ʹ), 48.3, 47.1, 46.6, 42.3, 42.0, 39.9, 39.5, 39.1, 37.3, 37.1, 34.5, 33.4, 33.2,
(
3
1
2.5, 32.2, 31.2, 30.2, 28.5, 28.3, 26.3, 26.3, 25.8, 24.0, 23.9, 23.6, 22.9, 18.8, 17.3, 16.8, 15.7,
+
4.2 ppm; HRMS (ESI) calcd. for C H NO +Na [M+Na] : 738.4915, found: 738.4940;
4
2
69
8
HPLC purity 95.7% (t : 11.4 min, Mightysil, RP-18, 250 × 4.6 mm, 5 µm, H O/ACN = 30/70,
R
2
1
mL/min, 30 min).
4
.1.15. 3-O-(2ʹ-heptanoylamino-2ʹ-deoxy-β-
D
-glucopyranosyl)oleanolic acid (45)
Compound 45 (16.2 mg, 58%) was prepared as a white solid according to the general
procedure B for amide bond coupling using 19 (27.0 mg, 0.038 mmol), hexanoic anhydride
0.1 mL), pyridine (0.5 mL); and purified by column chromatography (silica gel;
CH Cl /MeOH = 15/1) to give compound 26 which was further deprotected using 10% Pd/C
(
2
2
(
3 mg) and purified by column chromatography (silica gel; CH Cl /MeOH = 15/1); m.p. 204
2 2
1
℃
; H NMR (600 MHz, CD OD/CDCl = 4/1) δ 5.23 (t, J = 3.5 Hz, 1H, H-12), 4.46 (d, J =
3 3
8
.3 Hz, 1H, H-1ʹ), 3.82 (dd, J = 11.9, 2.7 Hz, 1H, H-6aʹ), 3.71 (dd, J = 11.9, 5.0 Hz, 1H,
H-6bʹ), 3.63 (dd, J = 10.3, 8.3 Hz, 1H, H-2ʹ), 3.44 (dd, J = 10.3, 9.2 Hz, 1H, H-3ʹ), 3.36 (t, J =
.2 Hz, 1H, H-4ʹ), 3.23 (ddd, J = 9.2, 5.0, 2.7 Hz, 1H, H-5ʹ), 3.08 (dd, J = 11.7, 4.4 Hz, 1H,
H-3), 2.81 (dd, J = 13.8, 4.0 Hz, 1H, H-18), 2.17 (td, J = 7.3, 2.0 Hz, 2H, H-2″), 1.99–1.92 (m,
H), 1.88–1.83 (m, 3H), 1.75–1.69 (m, 2H), 1.66–1.62 (m, 2H), 1.61–1.56 (m, 5H), 1.54–1.49
m, 3H), 1.45–1.41 (m, 1H), 1.37–1.35 (m, 1H), 1.34–1.32 (m, 2H), 1.32–1.31 (m, 2H), 1.30–
9
1
(
2
8