Synthesis of Lanostane-Type Triterpenoid N-Glycosides and Their Cytotoxicity against Human Cancer Cell Lines

Article abstract of DOI:10.1002/cbdv.201800113

Seventeen lanostane-type triterpenoid derivatives (2 – 18), including 11N-glycosides (8 – 18), were synthesized from the natural triterpenoid, lanosterol (1), and were evaluated for their cytotoxicity against the human cancer cell lines, HL-60, A549, and MKN45, as well as the normal human lung cells, WI-38. Among them, N-β-d-2-acetamido-2-deoxyglucoside (10) showed cytotoxicity against HL-60, A549, MKN45, and WI-38 cells (IC₅₀ 0.0078 – 2.8 μm). However, N-β-d-galactoside (12) showed cytotoxicity against HL-60 and MKN45 cells (IC₅₀ 0.0021 – 4.0 μm), but not the normal WI-38 cells. Furthermore, Western blot analysis suggested that 12 induces apoptosis by activation of caspases-3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or steroid N-glycosides to increase their cytotoxicity and apoptosis-inducing activities.

Full text of DOI:10.1002/cbdv.201800113

Article Type: Full Paper
Synthesis of Lanostane-Type Triterpenoid N-Glycosides and Their
Cytotoxicity against Human Cancer Cell Lines
Motohiko Ukiyaa*, Teppei Hayakawaa, Kouta Okazakia, Maiko Hikawaa, Hiroyuki Akazawaa, Wei
Lib, Kazuo Koikeb, and Makoto Fukatsua
a College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo
101-8308, JAPAN, ukiya.motohiko@nihon-u.ac.jp
b Faculty of Pharmaceutical Science, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-
8510, JAPAN
Seventeen lanostane-type triterpenoid derivatives (2–18), including 11 N-glycosides (8–18), were
synthesized from the natural triterpenoid, lanosterol (1), and were evaluated for their cytotoxicity
against the human cancer cell lines, HL-60, A549, and MKN45, as well as the normal human lung
cells, WI-38. Among them, N-β-D-2-acetamido-2-deoxyglucoside (10) showed cytotoxicity against
HL-60, A549, MKN45, and WI-38 cells (IC50 0.0078–2.8 μM). However, N-β-D-galactoside (12)
showed cytotoxicity against HL-60 and MKN45 cells (IC50 0.0021–4.0 μM), but not the normal WI-38
cells. Furthermore, western blot analysis suggested that 12 induces apoptosis by activation of
caspases-3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or
steroid N-glycosides to increase their cytotoxicity and apoptosis-inducing activities.
Keywords: terpenoid • glycosylation • N-glycoside • cytotoxicity • apoptosis
Introduction
Triterpenoids are naturally occurring lipids and some of them are known to be precursors of steroids.
Triterpenoid glycosides have been reported to possess various biological activities, including anti-
inflammatory, antitumor promoter, antiviral, and cytotoxic activities.[1-4] Naturally occurring
triterpenoid glycosides usually have an O-bond between the aglycone (triterpenoids) and sugar
moieties. Recently, N-glycosides were synthesized from steroids and reported to have cytotoxicity
against human cancer cell lines. On the basis of this information, we aimed to synthesize lanostane-
type triterpenoid N-glycosides and evaluate their cytotoxicity against human normal and cancer cell
lines.[5-8] Lanostane-type triterpenoids are considered to be promising agents for the development of
anticancer drugs[9] and their derivatives have been shown to have cytotoxic and apoptosis-inducing
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effects.[10] To the best of our knowledge, this is the first report on the synthesis of lanostane-type
triterpenoid N-glycosides, and the evaluation of their cytotoxicity and proapoptotic effects.
Results and Discussion
Synthesis
In this study, 17 lanostane-type triterpenoid derivatives (2–18) were synthesized from the natural
triterpenoid, lanosterol (1). As shown in Scheme 1, lanostane-type triterpenoid amine (7) was
synthesized from 1, and the amine (7) was glycosylated with six D-type monosaccharides, D-glucose
(D-Glc), D-2-deoxyglucose (2-deoxy-D-Glc), D-N-acetylglucosamine (D-GlcNAc), D-galactose (D-
Gal), D-fucose (D-Fuc; 6-deoxy-D-Gal), and D-talose (D-Tal; 2’-epimer of D-Gal), and two L-type
monosaccharides, L-glucose (L-Glc) and L-arabinose (L-Ara; 6’-nor-D-Gal). Furthermore, in order to
evaluate the influence of the acetoxy group at C-3, 3β-hydroxy-25,26,27-trinorlanost-8-en-24-al
methoxy amine N-β-D-galactoside (18) was synthesized.
Starting from commercially available lanosterol (a mixture of lanosterol (1) and dihydrolanosterol
(1’)), the hydroxyl group was protected by acetylation to yield a mixture of lanosterol 3-O-acetate (2)
and dihydrolanosterol 3-O-acetate (2’). This mixture was oxidized with meta-chloroperoxybenzoic
acid (m-CPBA) and separated from unreacted 2’ to give a mixture of (24S)- and (24R)-lanosterol
epoxide 3-O-acetate (3). The mixture was treated with perchloric acid to afford a mixture of (24S)-
and (24R)-3β-acetoxylanost-8-ene-24,25-diol (4), which was then treated with sodium periodate to
yield 3β-acetoxy-25,26,27-trinorlanost-8-en-24-al (5). The aldehyde (5) was treated with
methoxylamine hydrochloride to give 3β-acetoxy-24-methoxyimino-25,26,27-trinorlanost-8-ene (6)
as a mixture of (24E) and (24Z) isomers. Compound 6 was reduced with borane tert-butylamine
complex to give 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene (7). The glycosylation of
7 with D-Glc, 2-deoxy-D-Glc, D-GlcNAc, D-Fuc, D-Tal, and L-Glc produced the corresponding one N-
glycosides (8–10, 13, 14, and 15, respectively). In contrast, glycosylation of 7 with D-Gal and L-Ara
gave the two corresponding N-glycoside anomers (11 and 12 for D-Gal, 16 and 17 for L-Ara).
Compound 18, which is the C-3 deacetoxy derivative of 12 (β-D-Gal), was also synthesized from 4
after hydrolysis.
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O
N
R
24
21
22
26
O
20
vii)
18
25
27
23
17
7
12
11
i)
19
13 16
14 15
ii)
O
9
8
1
O
2
3
10
O
O
5
7
30
H
4
6
O
HO
H
H
O
28
29
H
OH
OH
HO
OH
6'
OH
2 (24-ene form)
+
2' (24-dihydro form)
1
(24-ene form)
+
4'
3 (24R/S mixture)
O
O
O
O
5'
HO
HO
HO
2'
HO
(
R =
1'
OH
1' (24-dihydro form)
3'
HO
HO
HO
OH
NHAc
8
9
10
11
OH
O
(
-D-Glucose)
-D-2-Deoxyglucose)
(
-D-N-Acetylglucosamine)
(
-D-Galactose)
H
OH
OH
HO
HO
OH
HO
iv)
iii)
OH
HO
HO
O
O
OH
O
O
HO
OH
HO
HO
O
O
OH
HO
15
13
( -D-Fucose)
14
12
O
O
(
-D-Talose)
(
-L-Glucose)
(
-D-Galactose)
H
H
5
4 (24R/S mixture)
HO
HO
O
H
N
O
O
O
HO
HO
N
H
OH
OH
v)
vi)
O
17
-L-Arabinose)
16
-L-Arabinose)
(
(
O
O
O
6 (24E/Z mixture)
7
H
O
H
N
viii), iv), v), vi), vii)
O
4
OH
HO
HO
HO
H
HO
18
-D-Galactose)
(
i) Ac2O, Pyridine ii) m-CPBA, CHCl3 iii) HClO4, 1,2-Dimethoxyethane iv) NaIO4, THF v) Methoxyamine hydrochloride, Methanol, Pyridine
vi) Borane tert-butylamine, 1,4-Dioxane, Ethanol, HCl vii) Monosaccharide, AcOH, DMF viii) KOH, MeOH
Scheme 1. Synthetic schemes of 2–18.
The stereochemistry at anomeric position (C(1’)) of 8–17 was assigned as follows: the anomeric
proton of 8 (D-Glc), 9 (2-deoxy-D-Glc), 10 (D-GlcNAc), and 13 (D-Fuc) appeared at δ(H) 4.71 (d,
3JHH = 8.2), 4.68 (dd, 3JHH = 1.4, 10.5), 4.91 (d, 3JHH = 9.6), and 4.60 (d, 3JHH = 8.7), respectively.
Their coupling constants were in good agreement with the reference data for β-glycosides[11-14] and the
anomeric configurations of 8, 9, 10, and 13 were assigned as β. The anomeric proton signals of the L-
arabinoside isomers (16 and 17) appeared at δ(H) 4.52 (d, 3JHH = 8.7) and 5.20 (d, 3JHH = 5.5),
respectively. Comparing these data with reference data[14, 15], we determined that 16 and 17 were α-
and β-L-arabinosides, respectively. However, it was difficult to assign the anomeric stereochemistry
of compound 12 (D-Gal) by its anomeric proton signal in 1H-NMR because the signal overlapped with
that of three other protons. Thus, 1H-, and 13C-NMR were measured at 40 °C and 2D-NMR
experiments (heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond
correlation (HMBC), 1H-1H correlation spectroscopy (COSY), and nuclear Overhauser effect
spectroscopy (NOESY)) were performed. As a result, the anomeric proton was assigned at δ(H) 4.61
(d, 3JHH = 8.9), which indicated that 12 was β-D-galactoside. In addition, the H-C(1’) signal showed
correlation peaks with H-C(3’) and H-C(5’) in the NOESY experiment, which indicated that they
were oriented on the same face as the cyclohexane ring (Figure 1). Based on these data, the anomeric
configuration of 12 was determined to be β. Another galactoside (11) was determined to be α-D-
galactoside by comparison of the anomeric proton signal at δ(H) 5.23 (d, 3JHH = 5.5) with reference
data.[14] Because the assignment of the anomeric configuration of 14 by 3JHH coupling constant was
difficult, the 13C-1H coupling constant (1JCH) of the 13C-NMR signal at C(1’) was measured and
compared with that of α- and β-D-talose. Specifically, the coupling constant 1JCH at δ(C) 100.0 (C(1’))
of 14 was 155.3 Hz, as measured by the non-decoupling 13C-NMR experiment. The 1JCH of α- and β-
D-talose were 168.7 Hz (δ(C) 96.0) and 159.1 Hz (δ(C) 96.5) in non-decoupling 13C-NMR,
respectively. Since the 1JCH of 14 at C-1’ was in good agreement with that of β-D-talose, 14 was
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determined to be β-D-taloside. To determine the anomeric configuration of 15, 1H-, and 13C-NMR,
and 2D-NMR experiments were performed. The anomeric proton signal (δ(H) 4.71–4.73) showed
correlation peaks with H-C(3’) and H-C(5’) in the NOESY experiment, which indicated that they
were oriented on the same face as the cyclohexane ring (Figure 1). Based on these results, 15 was
determined to be β-L-glucoside. The C(3) deacetoxy D-galactoside (18) was determined to be β-D-
galactoside by comparing its 1H-NMR data with that of 12.
aglycone
aglycone
12
15
Figure 1. Sugar moiety of 12 and 15 and representative nuclear Overhauser effect correlation (
)
Cytotoxic Activity of Compounds 1–18
Among the glycosides (8–18), β-D-2-deoxyglucoside (9), β-D-2-acetamido-2-deoxyglucoside (10),
and β-D-galactoside (12) showed cytotoxicity at IC50 values of 9.4, 2.8, and 0.0021 μM, respectively,
against leukemia cell lines (HL-60), which were higher than that of amine (7; >100 μM). In contrast
with the reference compound, cisplatin (IC50 = 1.3 μM), 12 (IC50 = 0.0021 μM) showed potent activity.
In addition, 10 showed cytotoxicity against lung cancer (A549; IC50 = 0.67 μM) and stomach cancer
cell lines (MKN45; IC50 = 0.0078 μM), which were more potent than that of cisplatin (IC50 of 18.4 and
7.0 μM, respectively) (p < 0.01). With respect to structure-activity relationships, β-D-2-
deoxyglucoside (9) and β-D-2-acetamido-2-deoxyglucoside (10) showed more potent cytotoxicity
than did β-D-glucoside (8), which indicated that the hydroxyl group at C(2’) may decrease the
activity. In comparison with β-D-galactoside (12), α-D-galactoside (11; C(1’) epimer of 12), β-D-
glucoside (8; C(4’) epimer of 12), β-D-taloside (14; C(2’) epimer of 12), β-D-fucoside (13; 6’-deoxy
derivative of 12), and α-L-arabinoside (16; 6’ nor derivative of 12) showed no cytotoxicity against
HL-60. These results showed that, in the case of the lanostane side chain N-glycosides, the
stereochemistry at C(1’), C(2’), and C(4’) and the presence of the 6’-hydroxymethylene group were
essential for cytotoxicity against HL-60 cells.
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Table 1. Cytotoxic Activities (IC50 S.D. [μM]) of Lanostane-type Triterpenoid Derivatives 1–18 against One Human Normal and Three
Human Cancer Cell Linesa
Compounds
HL-60
A549
MKN45
WI-38
(Leukemia)
(Lung)
(Stomach)
(Normal
Lung)
1
2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3b
4b
>100
30.8 4.8
5
38.7 6.9
46.7 4.4
98.9 1.8
>100
6c
>100
>100
>100
>100
7
8
9
>100
>100
9. 4 0.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
11
2.8 0.8
0.67 0.07
0.0078 0.0033
0.62 0.03
>100
>100
>100
>100
12
13
0.0021 0.0004
>100
4.0 2.1
>100
>100
>100
>100
>100
14
>100
>100
>100
>100
>100
1.3 0.1
>100
>100
>100
>100
>100
18.4 1.9
>100
>100
>100
>100
>100
7.0 0.1
>100
>100
>100
>100
>100
20.1 2.4
15
16
17
18
Cisplatind
a Cells were treated with compounds (1 × 10-4 –1 × 10-6 M) for 48 h, and cell viability
was analyzed by the MTT assay. IC50 Values were calculated from five point
triplicates. b (24R) and (24S) mixture. c (24E) and (24Z) mixture. d Reference
compound.
Western Blot Analysis for Apoptosis-related Proteins.
In this study, β-D-galactoside (12), which showed the most potent cytotoxicity against HL-60 cells,
was selected and its proapoptotic mode of action was evaluated by western blot for the apoptosis-
related proteins, caspases-3, 8, and 9. After treatment of HL-60 cells with 12 (1 μM), the expression
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levels of procaspases-3, 8, and 9 were decreased, and cleaved caspases-3, 8, and 9 were detecteed after
48 h (Figure 2). These results suggestedthat compound 12-induced cell death occurs through thhe
activation of caspases-3, 8, and 9.
0 24 48 [h]
-Actin
Procaspase-3
Cleaved Caspase-3
Procaspase-8
Cleaved Caspase-8
Procaspase-9
Cleaved Caspase-9
Figure 2. Western Blot Analysis for Apoptosis-related Proteins
HL-60 cells were treated with 12 (1 M) for 0–48 h. The expression levels of the apoptosis-related proteins were assessed by western
blotting; -actin was used as the loading control. The results are shown from one representative experiment among three independent runs.
Conclusions
In this study, we synthesized 17 lanostane-type triterpenoid derivatives including 11 N-glycosiddes and
evaluated their cytotoxicity against one normal human lung and three human cancer cell lines. Among
them, β-D-2-acetamido-2-deoxyglucosidee (10) not only showed potent cytotoxicity against the three
cancer cell lines but also showed cytotoxiicity against the normal cells. In contrast, β-
D
-galactosside
(12) showed potent cytotoxicity only agaiinst HL-60 cells and its mode of action was suggested to be
apoptosis by western blotting. To the bestt of our knowledge, this is the first report of the evaluation of
lanostane-type N-glycosides and their proapoptotic effects. We believe that these results will be useful
for the synthesis of other tetracyclic triterrpenoids or steroid N-glycosides to increase their cytotoxicity
and apoptosis-inducing activities.
Experimental Section
General
Silica gel 60 (SiO2, 230–400 mesh; Merck KGaA, Darmstadt, Germany) was used for open coluumn
chromatography. Reversed-phase (RP) prreparative high-performance liquid chromatography (HHPLC)
was conducted using a refractive index detector with an octadecyl silica (ODS) column (Superiorex
ODS column, 25 cm × 4.6 cm i.d.; Shiseiddo Co., Ltd., Tokyo, Japan) with a mobile phase of MeeOH (1
ml/min; HPLC system I), or with an ODS column (Pegasil ODS SP100 column, 25 cm × 10 cm i.d.;
Senshu Scientific Co., Ltd., Tokyo, Japann) with a mobile phase of MeOH (4 ml/min; HPLC system II)
or EtOH/H2O 75:25 (2 ml/min; HPLC system III) at 25 ºC. The infrared (IR) spectra were recorded
using a Perkin-Elmer Spectrum One Fourrier-transform IR (FTIR) spectrophotometer and potassium
bromide (KBr) disks, with ν in cm-1. The nuclear magnetic resonance (NMR) spectra were recoorded
using a JEOL ECX-400 spectrometer (1H, 400 MHz; 13C, 100 MHz) or JEOL ECA-500 (1H, 50
0
MHz; 13C, 125 MHz) at room temperaturre (r.t.; 20 °C) or 40 °C in deuterated chloroform (CDCl3),
CDCl3/deuterated methanol (CD3OD), orr deuterated pyridine (C5D5N) with tetramethylsilane as an

internal standard. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was
performed using an Agilent 1100 LC/MSD time-of-flight (TOF) system (ionization mode: positive or
negative, capillary voltage: 3000 V, and fragmentor voltage: 225 V); HR-atmospheric-pressure
chemical ionization (APCI)-MS was performed using the Agilent 1100 LC/MSD TOF system
(ionization mode: positive, capillary voltage: 3000 V, fragmentor voltage: 40 V, and corona current:
2.0 μA).
Chemicals and Materials
The chemicals used were purchased from the following sources: methoxyamine hydrochloride, borane
tert-butylamine complex, D-galactose, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT), and DMSO from Sigma-Aldrich Japan Co. (Tokyo, Japan); lanosterol (63 : 37
mixture of lanosterol and dihydrolanosterol based on HPLC analysis), N-acetyl-D-glucosamine, D-
fucose, D-talose, L-glucose, and L-arabinose from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan);
Roswell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), and penicillin-
streptomycin from Invitrogen Co. (Auckland, NZ); anti-procaspase-3, anti-cleaved caspase-3, anti-
procaspase-8, anti-cleaved caspase-8, anti-procaspase-9, anti-cleaved caspase-9, and anti-β-actin
primary antibodies from Cell Signaling Technology (Beverly, MA, U.S.A.); enhanced
chemiluminescence (ECL) Plus Western Blotting Detection System from GE Healthcare
(Buckinghamshire, U.K.); sodium periodate, 2-deoxy-D-glucose, and cisplatin from Wako Pure
Chemical Industries Ltd. (Osaka, Japan); and perchloric acid and D-glucose from Kanto Chemical Co.
Inc. (Tokyo, Japan). All other chemicals and reagents were of analytical grade.
Preparation of Compounds
Purification of lanosterol (1). For the evaluation of cytotoxicity, a mixture of lanosterol (1) and
dihydrolanosterol (1’) (20.2 mg) was purified by HPLC (System I) to give purified 1 (tR = 19.3 min,
8.0 mg), which was identified by spectral comparison with literature data.[16]
Synthesis of lanosterol 3-O-acetate (2). (i) Starting from purified 1: To a solution of purified
lanosterol (1) (5.1 mg, 0.01 mmol) in dried pyridine (1 ml), acetic anhydride (1 ml, 9.9 mmol) was
added and stirred at r.t. overnight. The reaction mixture was diluted with H2O and extracted with
diethyl ether. The organic layer was washed sequentially with 1 M HCl, saturated NaHCO3 aq, and
H2O, and was then dried over Na2SO4. The dried organic layer was evaporated under reduced pressure
to yield a mixture of lanosterol 3-O-acetate (2) (5.2 mg, 100%), which was identified by spectral
comparison with literature data.[16] (ii) Starting from crude 1: To a solution of a crude lanosterol (1 : 1’
= 63 : 37) (2.1 g, 4.9 mmol) in dried pyridine (30 ml), acetic anhydride (30 ml, 32 mmol) was added
and stirred at r.t. overnight. The reaction mixture was diluted with H2O and extracted with diethyl
ether. The organic layer was washed sequentially with 1 M HCl, saturated NaHCO3 aq, and H2O, and
was then dried over Na2SO4. The dried organic layer was evaporated under reduced pressure to yield a
mixture of lanosterol 3-O-acetate (2) and dihydrolanosterol 3-O-acetate (2’) (2.3 g, 99%). The
products were used for the next reaction without purification.
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Synthesis of (24R)- and (24S)-lanosterol epoxide 3-O-acetate (3). To a solution of a mixture of 2
and 2’ (2.3 g, 4.9 mmol) in dried CHCl3 (35 ml), m-CPBA (0.88 g, 5.1 mmol) was added at 0 °C and
stirred for 10 min. The mixture was warmed to r.t. and stirred for 12 h. Afterward, the reaction
mixture was diluted with CHCl3, washed sequentially with 1 M NaOH aq. and H2O, and was then
dried over Na2SO4. The dried solution was evaporated under reduced pressure to yield crude products
(3.3 g). The products were purified with Si-gel column chromatography (hexane/AcOEt = 1 : 0 to 9 :
1) to give 3 (0.86 g, 36%) as a 24R/24S mixture, which was identified by spectral comparison with
literature data.[10]
Synthesis of (24R)- and (24S)- 3β-acetoxylanost-8-ene-24,25-diol (4). To a solution of 3 (0.86 g,
1.8 mmol) in 1,2-dimethoxyethane (85 ml), a solution (21 ml) formed by adding perchloric acid (0.2
ml) to H2O (21 ml) was added and the reaction mixture was stirred at r.t. overnight. Afterward, the
reaction mixture was diluted with H2O and extracted with diethyl ether. The organic layer was washed
sequentially with 2 M Na2CO3 aq. and H2O, and was then dried over Na2SO4. The dried solution was
evaporated under reduced pressure to yield 4 (0.73 g, 81%) as a mixture of (24R)- and (24S)-
stereoisomers, which was identified by spectral comparison with literature data.[17]
Synthesis of 3β-acetoxy-25,26,27-trinorlanost-8-en-24-al (5). To a solution of 4 (0.60 g, 1.2
mmol) in THF (22 ml) and H2O (7 ml), sodium periodate (1.1 g, 5.2 mmol) was added and stirred at
r.t. for 1.5 h. Afterward, 2 M HCl was added and the mixture was extracted with CHCl3. The organic
layer was washed with H2O, dried over Na2SO4, and then evaporated under reduced pressure to yield
5 (0.51 g, 96%), which was identified by spectral comparison with literature data.[10]
Synthesis of (E)- and (Z)-3β-acetoxy-24-methoxyimino-25,26,27-trinorlanost-8-ene (6) To a
solution of 5 (0.49 g, 1.1 mmol) in pyridine (12 ml) and methanol (0.5 ml), methoxyamine
hydrochloride (0.15 g, 1.8 mmol) was added and stirred at r.t. for 1 h. Afterward, the reaction mixture
was diluted with H2O and extracted with CHCl3. The organic layer was washed sequentially with 1 M
HCl and H2O, and was then dried over Na2SO4. The solution was evaporated under reduced pressure
to yield 6 (0.51 g, 98%) as a mixture of (24E)- and (24Z)-stereoisomers. Amorphous solid. IR (KBr):
873, 1033, 1054, 1079, 1251, 1371, 1455, 1632, 1727, 2951, 3434. 1H-NMR (400 MHz, CDCl3): 0.69
(s, 3H, Me); 0.88 (s, 9H, Me); 0.94 (d, J = 6.4, 3H, H-C(21)); 1.00 (s, 3H, Me); 2.05 (s, 3H, MeCO);
3.81 (s, 1.5H, OMe); 3.88 (s, 1.0H, OMe); 4.50 (dd, J = 4.6, 11.5, 1H, H-C(3)); 6.63 (t, J = 5.3, 0.3H,
H-C(24)); 7.37 (t, J = 6.2, 0.5H, H-C(24)). HR-ESI-MS (pos.): 472.3782 ([M + H]+, C30H49NO3 ;
+
calc. 472.3791).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene (7). To a solution of 6
(0.50 g, 1.1 mmol) in 1,4-dioxane (12 ml) and ethanol (3 ml), borane tert-butylamine complex (0.30
g, 3.4 mmol) was added. The reaction mixture was cooled to 0 °C, 3 M HCl (1 ml) was added
dropwise, and the mixture was stirred at 0 °C for 2.5 h. Afterward, solid Na2CO3 and saturated
NaHCO3 aq. were added until gas evolution ceased, and the reaction mixture was extracted with
CHCl3. The organic layer was dried over Na2SO4 and evaporated under reduced pressure to yield
crude product (0.63 g). A portion of the product (14 mg) was purified with HPLC (System II) to give
purified 7 (tR 16.4 min, 6.4 mg). The other portion was used for synthesis of N-glycosides (8–17)
without purification. Amorphous solid. IR (KBr): 983, 1010, 1032, 1250, 1371, 1466, 1736, 2950,
3439. 1H-NMR (400 MHz, CDCl3): 0.69 (s, 3H, Me); 0.87 (s, 3H, Me); 0.88 (s, 6H, Me); 0.92 (d, J =
6.0, 3H, H-C(21)); 1.00 (s, 3H, Me); 2.05 (s, 3H, MeCO); 2.85–2.92 (m, 2H, H-C(24)); 3.54 (s, 3H,
OMe); 4.50 (dd, J = 4.4, 11.7, 1H, H-C(3)). HR-ESI-MS (pos.): 474.3970 ([M + H]+, C30H51NO3 ;
+
calc. 474.3947).
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Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-glucoside (8).
The amine 7 (77.0 mg, 0.16 mmol) and D-glucose (97.2 mg, 0.54 mmol) were dissolved in DMF (1.5
ml) and acetic acid (0.5 ml) and stirred at 40 °C for 28 h. The reaction mixture was concentrated
under reduced pressure to give crude product (182.2 mg). The product was subjected to Si-gel column
chromatography (CHCl3 : acetone, 5 : 5 to 4 : 6) to give crude 8 (34.8 mg), which was further purified
by HPLC (system III) to give purified 8 (tR 19.0 min, 20.8 mg, 20%). Amorphous solid. IR (KBr):
900, 1031, 1083, 1247, 1372, 1451, 1734, 2948, 3401. 1H-NMR (400 MHz, C5D5N): 0.74 (s, 3H, H-
C(18)); 0.94 (s, 3H, H-C(28)); 0.96 (s, 6H, H-C(29), H-C(30)); 0.98 (d, J = 6.4, 3H, H-C(21)); 1.02 (s,
3H, H-C(19)); 2.09 (s, 3H, MeCO); 3.11–3.14 (m, 1H, H-C(24)); 3.37–3.41 (m, 1H, H-C(24)); 3.90 (s,
3H, OMe); 3.96–3.98 (m, 1H, H-C(Glc)); 4.22-4.37 (m, 3H, H-C(Glc)); 4.42 (dd, J = 5.5, 11.9, 1H,
H-C(6’)); 4.60 (d, J = 11.4, 1H, H-C(6’)); 4.71 (d, J = 8.2, 1H, H-C(1’)); 4.70–4.73 (m, 1H, H-C(3)).
13C-NMR (100 MHz , C5D5N): 16.4 (C(18)); 17.1 (C(29)); 18.7 (C(6)); 19.3 (C(21)); 19.6 (C(19));
21.5 (MeCO); 21.6 (C(11)); 24.7 (C(30)); 24.9 (C(2)); 25.5 (C(23)); 26.9 (C(7)); 28.3 (C(28)); 28.8
(C(16)); 31.4 (C(15)); 31.6 (C(12)); 34.7 (C(22)); 35.7 (C(1)); 37.2 (C(20)); 37.4 (C(10)); 38.4 (C(4));
45.0 (C(13)); 50.4 (C(14)); 51.0 (C(5)); 51.1 (C(17)); 54.3 (C(24)); 62.9 (NOMe); 63.4 (C(6’)); 72.1,
72.2 (C(2’) or C(4’)); 80.5, 80.7 (C(3’) or C(5’)); 81.0 (C(3)); 95.4 (C(1’)); 134.9 (C(8), C(9)); 171.0
(MeCO). HR-ESI-MS (pos.): 658.4324 ([M + Na]+, C36H61NO8Na+; calc. 658.4295).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-2-
deoxyglycoside (9). The amine 7 (85.1 mg, 0.18 mmol) and D-2-deoxyglucose (88.6 mg, 0.54 mmol)
were reacted by the same method as used for 8 to give crude product (214.7 mg). The product was
subjected to Si-gel column chromatography (CHCl3 : acetone, 8 : 2 to 6 : 4) to give crude 9 (34.8 mg),
which was further purified by HPLC (system III) to give purified 9 (tR 39.5 min, 3.6 mg, 3%).
Amorphous solid. IR (KBr): 824, 875, 1050, 1075, 1266, 1384, 1451, 1494, 1714, 2924, 3435. 1H-
NMR (400 MHz , C5D5N): 0.76 (s, 3H, Me); 0.94 (s, 3H, Me); 0.96 (s, 3H, Me); 0.97 (s, 3H, Me);
1.00 (d, J = 6.9, 3H, Me); 1.02 (s, 3H, Me); 2.09 (s, 3H, MeCO); 2.37–2.46 (m, 1H, H-C(2’)); 2.59–
2.63 (m, 1H, H-C(2’)); 2.99–3.06 (m, 1H, H-C(24)); 3.25–3.32 (m, 1H, H-C(24)); 3.73 (s, 3H, OMe);
3.85–3.88 (m, 1H, H-C(2-DeoxyGlc)); 4.10 (t, J = 9.1, 1H, H-C(2-DeoxyGlc)); 4.26–4.32 (m, 1H, H-
C(2-DeoxyGlc)); 4.43 (dd, J = 5.1, 11.5, 1H, H-C(2-DeoxyGlc)); 4.59 (dd, J = 2.3, 11.9, 1H, H-C(2-
DeoxyGlc)); 4.68 (dd, J = 1.4, 10.5, 1H, H-C(1’)); 4.72 (dd, J = 4.1, 11.5, 1H, H-C(3)). HR-APCI-MS
+
(pos.): 620.4499 ([M + H]+, C36H62NO7 ; calc. 620.4526).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-2-acetamido-2-
deoxyglucoside (10). The amine 7 (101.1 mg, 0.21 mmol) and D-N-acetylglucosamine (139.4 mg,
0.63 mmol) were reacted by the same method as used for 8 to give crude product (286.5 mg). The
product was subjected to Si-gel column chromatography (CHCl3 : acetone, 9 : 1 to 5 : 5) to give crude
10 (17.6 mg), which was further purified by HPLC (system III) to give purified 10 (tR 33.0 min, 4.6
mg, 3%). Amorphous solid. IR (KBr): 1030, 1082, 1246, 1372, 1455, 1558, 1735, 2948, 3399 cm-1.
1H-NMR (400 MHz , C5D5N): 0.75 (s, 3H, Me); 0.94 (s, 3H, Me); 0.96 (s, 6H, Me); 1.01 (d, J = 5.0
Hz, 3H, H-C(21)); 1.02 (s, 3H, Me); 2.09 (s, 3H, MeCO); 2.17 (s, 3H, MeCO); 3.14–3.20 (m, 1H, H-
C(24)); 3.38–3.45 (m, 1H, H-C(24)); 3.80 (s, 3H, OMe); 3.93–3.98 (m, 1H, H-C(GlcNAc)); 4.23 (t, J
= 9.2, 1H, H-C(GlcNAc)); 4.34–4.41 (m, 1H, H-C(GlcNAc)); 4.37 (t, J = 9.6, 1H, H-C(GlcNAc));
4.60 (br.d, J = 11.5, 1H, H-C(GlcNAc)); 4.72 (dd, J = 4.1, 11.5, 1H, H-C(3)); 4.76 (t, J = 9.6, 1H, H-
C(GlcNAc)); 4.91 (d, J = 9.6, 1H, H-C(1’)); 8.91 (d, J = 9.2, 1H, NH). HR-APCI-MS (pos.):
+
677.4738 ([M + H]+, C38H65N2O8 ; calc. 677.4741).
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Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-α-D-galactoside (11)
and 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-galactoside (12). The amine
7 (78.3 mg, 0.17 mmol) and D-galactose (100 mg, 0.56 mmol) were reacted by the same method as
used for 8 to give crude product (243.2 mg). The product was diluted with H2O and extracted with
ethyl acetate to give a mixture of 11 and 12 (39.2 mg), which was further purified by HPLC (system
III) to give purified 11 (tR 35.1 min, 3.6 mg, 3%) and 12 (tR 32.9 min, 7.1 mg, 7%).
Data for 11: Amorphous solid. IR (KBr): 1035, 1247, 1371, 1733, 2950, 3500. 1H-NMR (400 MHz ,
C5D5N): 0.74 (s, 3H, Me); 0.94 (s, 3H, Me); 0.96 (s, 6H, Me); 0.97 (d, J = 8.7, 3H, H-C(21)); 1.01 (s,
3H, Me); 2.08 (s, 3H, MeCO); 3.09–3.16 (m, 1H, H-C(24)); 3.24–3.31 (m, 1H, H-C(24)); 3.79 (s, 3H,
OMe); 4.39–4.44 (m, 2H, H-C(Gal)); 4.54–4.60 (m, 1H, H-C(Gal)); 4.71 (dd, J = 4.5, 11.4, 1H, H-
C(3)); 4.86 (dd, J = 2.8, 8.3, 1H, H-C(Gal)); 5.04–5.09 (m, 1H, H-C(Gal)); 5.17–5.22 (m, 1H, H-
C(Gal)); 5.23 (d, J = 5.5, 1H, H-C(Gal)). HR-ESI-MS (pos.) 658.4293 ([M + Na]+, C36H61NO8Na+;
calc. 658.4295).
Data for 12: Amorphous solid. IR (KBr) 1035, 1248, 1372, 1458, 1736, 2927, 3431. 1H-NMR (400
MHz, C5D5N): 0.73 (s, 3H, H-C(18)); 0.94 (s, 3H, H-C(28)); 0.96 (s, 6H, H-C(29), H-C(30)); 0.96 (d,
J = 6.4, 3H, H-C(21)); 1.02 (s, 3H, H-C(19)); 2.09 (s, 3H, MeCO); 3.18–3.24 (m, 1H, H-C(24)); 3.33–
3.40 (m, 1H, H-C(24)); 3.83 (s, 3H, OMe); 4.11 (t, J = 5.9, 1H, H-C(5’)); 4.24 (dd, J = 2.8, 8.3, 1H,
H-C(3’)); 4.50 (d, J = 6.0, 2H, H-C(6’)); 4.64–4.68 (m, 3H, H-C(Gal)); 4.72 (dd, J = 4.1, 11.5, 1H, H-
C(3)). 1H-NMR (500 MHz , C5D5N, 40 °C): 0.74 (s, 3H, H-C(18)); 0.94 (s, 3H, H-C(28)); 0.96 (s, 6H,
H-C(29), H-C(30)); 0.97 (d, J = 6.6, 3H, H-C(21)); 1.02 (s, 3H, H-C(19)); 2.06 (s, 3H, MeCO); 3.17–
3.23 (m, 1H, H-C(24)); 3.31–3.36 (m, 1H , H-C(24)); 3.80 (s, 3H, OMe); 4.03 (t, J = 5.7, 1H, H-
C(5’)); 4.17 (dd, J = 3.0, 8.5, 1H, H-C(3’)); 4.43 (br.d, J = 5.7, 2H, H-C(6’)); 4.55–4.59 (m, 2H, H-
C(4’), H-C(2’)); 4.61 (d, J = 8.9, 1H, H-C(1’)); 4.69–4.71 (m, overlapped with H2O, 1H, H-C(3)). 13C-
NMR (125 MHz , C5D5N, 40 °C): 16.5 (C(18)); 17.2 (C(29)); 18.8 (C(6)); 19.4 (C(21)); 19.7 (C(19));
21.5 (MeCO); 21.7 (C(11)); 24.8 (C(30)); 25.0 (C(2)); 25.7 (C(23)); 27.1 (C(7)); 28.4 (C(28)); 28.8
(C(16)); 31.5 (C(15)); 31.7 (C(12)); 34.8 (C(22)); 35.9 (C(1)); 37.2 (C(20)); 37.6 (C(10)); 38.5 (C(4));
45.2 (C(13)); 50.5 (C(14)); 51.2 (C(5), C(17)); 53.8 (C(24)); 62.6 (NOMe); 63.0 (C(6’)); 69.8 (C(2’));
70.8 (C(4’)); 77.1 (C(3’)); 79.0 (C(5’)); 81.2 (C(3)); 95.9 (C(1’)); 135.1 (C(8), C(9)); 171.0 (MeCO).
+
HR-APCI-MS (pos.): 636. 4517 ([M + H]+, C36H62NO8 ; calc. 636.4475).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-fucoside (13).
The amine 7 (62.5 mg, 0.13 mmol) and D-fucose (64.0 mg, 0.39 mmol) were reacted by the same
method as used for 8 to give crude product (109.0 mg). The product was subjected to Si-gel column
chromatography (CHCl3 : acetone, 8 : 2 to 5 : 5) to give crude 13 (39.9 mg), which was further
purified by HPLC (system III) to give purified 13 (tR 30.9 min, 3.9 mg, 5%). Amorphous solid. IR
(KBr): 1030, 1247, 1371, 1453, 1735, 2948, 3418. 1H-NMR (400 MHz , C5D5N): 0.74 (s, 3H, Me);
0.95 (s, 3H, Me); 0.96 (s, 6H, Me); 0.97 (d, J = 5.0, 3H, H-C(21)); 1.02 (s, 3H, Me); 1.60 (d, J = 6.4,
3H, H-C(6’)); 2.09 (s, 3H, MeCO); 3.19–3.26 (m, 1H, H-C(24)); 3.31–3.38 (m, 1H, H-C(24)); 3.82 (s,
3H, OMe); 3.80–3.88 (m, 1H, H-C(Fuc)); 4.10 (d, J = 2.8, 1H, H-C(Fuc)); 4.17 (dd, J = 3.6, 8.7, 1H,
H-C(Fuc)); 4.54 (t, J = 9.4, 1H, H-C(Fuc)); 4.60 (d, J = 8.7, 1H, H-C(1’)); 4.72 (dd, J = 4.1, 11.4, 1H,
+
H-C(3)). HR-APCI-MS (pos.): 620.4474 ([M + H]+, C36H62NO7 ; calc. 620.4526).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-taloside (14).
The amine 7 (81.8 mg, 0.17 mmol) and D-talose (89.5 mg, 0.50 mmol) were reacted by the same
method as used for 8 to give crude product (174.6 mg). The product was purified by HPLC (system
II) to give purified 14 (tR 7.5 min, 15.5 mg, 14%). Amorphous solid. IR (KBr): 1033, 1267, 1371,
1712, 2916, 3381. 1H-NMR (400 MHz , CD3OD-CDCl3 = 5 : 3): 0.72 (s, 3H, H-C(18)); 0.88 (s, 3H,
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H-C(28)); 0.90 (s, 6H, H(29), H-C(30)); 0.94 (d, J = 5.9, 3H, H-C(21)); 1.03 (s, 3H, H-C(19)); 2.04
(s, 3H, MeCO); 2.75–2.82 (m, 1H, H-C(24)); 2.87–2.94 (m, 1H, H-C(24)); 3.59 (s, 3H, OMe); 3.59–
3.63 (m, 2H, H-C(6’)); 3.66–3.69 (m, 1H, H-C(5’)); 3.89 (dd, J = 3.7, 5.5, 1H, H-C(4’)); 4.14 (t, J =
5.0, 1H, H-C(3’)); 4.18 (dd, J = 3.7, 5.5, 1H, H-C(2’)); 4.46 (dd, J = 7.3, 9.6, 1H, H-C(3)); 4.55 (d, J
= 3.2, 1H, H-C(1’)). 13C-NMR(100 MHz, CD3OD-CDCl3 = 5 : 3): 16.3 (C(18)); 17.0 (C(29)); 18.9
(C(6)); 19.2 (C(21)); 19.7 (C(19)); 21.6 (MeCO); 21.8 (C(11)); 24.7 (C(30)); 24.8 (C(2)); 24.9
(C(23)); 27.1 (C(7)); 28.4 (C(28)); 29.0 (C(16)); 31.6 (C(15)); 31.8 (C(12)); 34.6 (C(22)); 36.1 (C(1));
37.2 (C(20)); 37.7 (C(10)); 38.5 (C(4)); 45.3 (C(13)); 50.6 (C(14)); 51.3 (C(5)); 51.4 (C(17)); 55.5
(C(24)); 63.1 (NOMe); 64.5 (C(6’)); 72.2, 72.9, 73.3 (C(2’), C(3’), or C(5’)); 82.3 (C(3)); 83.5
(C(4’)); 100.0 (C(1’)); 135.1, 135.4 (C(8) or C(9)); 172.7 (MeCO). HR-ESI-MS (pos.): 658.4283 ([M
+ Na]+, C36H61NO8Na+; calc. 658.4295).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-L-glucoside (15).
The amine 7 (52.5 mg, 0.11 mmol) and L-glucose (59.4 mg, 0.33 mmol) were reacted by the same
method as used for 8 to give crude product (124.9 mg). The product was purified by HPLC (system
III) to give purified 15 (tR 39.0 min, 8.8 mg, 12%). Amorphous solid. IR (KBr) 1031, 1078, 1265,
1371, 1710, 2910, 3076. 1H-NMR (400 MHz , C5D5N): 0.74 (s, 3H, H-C(18)); 0.94 (s, 3H, H-C(28));
0.95 (s, 3H, H-C(30)); 0.96 (s, 3H, H-C(29)); 0.98 (d, J = 6.5, 3H, H-C(21)); 1.01 (s, 3H, H-C(19));
2.09 (s, 3H, MeCO); 3.19–3.26 (m, 1H, H-C(24)); 3.29–3.35 (m, 1H, H-C(24)); 3.88 (s, 3H, OMe);
3.95–3.99 (m, 1H, H-C(5’)); 4.24–4.29 (m, 1H, H-C(4’)); 4.31–4.33 (m, 2H, H-C(2’), H-C(3’)); 4.42
(dd, J = 5.5, 11.9, 1H, H-C(6’)); 4.60 (dd, J = 2.3, 11.9, 1H, H-C(6’)); 4.69–4.71 (m, 1H, H-C(3));
4.71–4.73 (m, 1H, H-C(1’)). 13C-NMR (100 MHz , C5D5N): 16.4 (C(18)); 17.1 (C(29)); 18.7 (C(6));
19.3 (C(21)); 19.6 (C(19)); 21.5 (MeCO); 21.6 (C(11)); 24.7 (C(30)); 24.9 (C(2)); 25.4 (C(23)); 26.9
(C(7)); 28.3 (C(28)); 28.8 (C(16)); 31.4 (C(15)); 31.6 (C(12)); 34.7 (C(22)); 35.7 (C(1)); 37.1 (C(20));
37.4 (C(10)); 38.4 (C(4)); 45.0 (C(13)); 50.4 (C(14)); 51.0 (C(5)); 51.2 (C(17)); 53.7 (C(24)); 62.7
(NOMe); 63.3 (C(6’)); 72.1, 72.2 (C(2’) or C(4’)), 80.5 (C(3’)); 80.7 (C(5’)); 81.0 (C(3)); 95.1
(C(1’)); 134.9 (C(8), C(9)); 171.0 (MeCO). HR-ESI-MS (pos.): 658.4311 ([M + Na]+, C36H61NO8Na+;
calc. 658.4295).
Synthesis of 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-α-L-arabinoside
(16) and 3β-acetoxy-24-methoxyamino-25,26,27-trinorlanost-8-en-24-methoxy amine N-β-L-
arabinoside (17). The amine 7 (69.2 mg, 0.15 mmol) and L-arabinose (82.1 mg, 0.54 mmol) were
reacted by the same method as used for 8 to give crude product (161.5 mg). The product was purified
by HPLC (system II) to give crude 16 (tR 8.5 min, 33.8 mg) and 17 (tR 7.8 min, 11.2 mg), which was
further purified with HPLC (system III) to give purified 16 (tR 42.4 min, 7.1 mg, 8%) and 17 (tR 41.7
min, 6.3 mg, 7%).
Data for 16: Amorphous solid. IR (KBr): 1035, 1267, 1714, 2985, 3521. 1H-NMR (400 MHz ,
C5D5N): 0.74 (s, 3H, Me); 0.94 (s, 3H, Me); 0.95 (s, 3H, Me); 0.96 (s, 3H, Me); 0.96 (d, J = 6.0, 3H,
H-C(21)); 1.01 (s, 3H, Me); 2.08 (s, 3H, MeCO); 3.18–3.25 (m, 1H, H-C(24)); 3.32–3.39 (m, 1H, H-
C(24)); 3.75–3.76 (m, 1H, H-C(Ara)); 3.83 (s, 3H, OMe); 4.17–4.19 (m, 1H, H-C(Ara)); 4.29–4.31
(m, 1H, H-C(Ara)); 4.36 (dd, J = 1.4, 11.9, 1H, H-C(Ara)); 4.52 (d, J = 8.7, 1H, H-C(1’)); 4.61 (dt, J
= 3.2, 9.2, 1H, H-C(Ara)); 4.71 (dd, J = 4.1, 11.4, 1H, H-C(3)). HR-ESI-MS (pos.): 628.4196 ([M +
Na]+, C35H59NO7Na+; calc. 628.4189).
Data for 17: Amorphous solid. IR (KBr) 1033, 1267, 1712, 2923, 3527. 1H-NMR (400 MHz ,
C5D5N): 0.75 (s, 3H, Me); 0.94 (s, 3H, Me); 0.96 (s, 6H, Me); 0.98 (d, J = 6.4, 3H, H-C(21)); 1.01 (s,
3H, Me); 2.09 (s, 3H, MeCO); 3.12–3.18 (m, 1H, H-C(24)); 3.28–3.35 (m, 1H, H-C(24)); 3.79 (s, 3H,
OMe); 4.22–4.27 (m, 1H, H-C(Ara)); 4.40–4.43 (m, 1H, H-C(Ara)); 4.67–4.70 (m, 1H, H-C(Ara));
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4.71 (dd, J = 4.6, 11.5, 1H, H-C(3)); 4.95–5.00 (m, 1H, H-C(Ara)); 5.07 (dd, J = 5.5, 12.0, 1H, H-
C(Ara)); 5.20 (d, J = 5.5, 1H, H-C(1’)). HR-ESI-MS (pos.): 628.4187 ([M + Na]+, C35H59NO7Na+;
calc. 628.4189).
Synthesis of 3β-hydroxy-24-methoxyamino-25,26,27-trinorlanost-8-ene N-β-D-galactoside
(18): Compound 4 (107.4 mg, 0.21 mmol) was dissolved in 5% KOH-MeOH (10 mL) and extracted
with diethyl ether to give lanost-8-ene-3β,24,25-triol (84.6 mg, 0.18 mmol), which was used under the
same reaction conditions as those used to synthesize 5, 6, and 7 to give 3β-hydroxy-25,26,27-
trinorlanost-8-ene-24-methoxyamine (89.6 mg). The amine (89.6 mg, 0.21 mmol) and D-galactose
(100.0 mg, 0.56 mmol) were then reacted by the same method as used for 8 to give crude product
(29.1 mg). The product was purified by HPLC (system III) to give purified 18 (tR 12.1 min, 3.8 mg,
3%). Amorphous solid. IR (KBr): 1031, 2945, 3197, 3677. 1H-NMR (400 MHz , C5D5N): 0.77 (s, 3H,
Me); 0.97 (s, 3H, Me); 0.97 (d, J = 6.0, 3H, H-C(21)); 1.09 (s, 3H, Me); 1.11 (s, 3H, Me); 1.28 (s, 3H,
Me); 3.17–3.24 (m, 1H, H-C(24)); 3.33–3.40 (m, 1H, H-C(24)); 3.46–3.52 (m, 1H, H-C(3)); 3.83 (s,
3H, OMe); 4.10 (t, J = 6.0, 1H, H-C(Gal)); 4.24 (dd, J = 2.7, 7.8, 1H, H-C(Gal)); 4.50 (d, J = 5.5, 2H,
H-C(6’)); 4.63–4.64 (m, 2H, H-C(Gal)); 4.68 (d, J = 8.7, 1H, H-C(1’)). HR-ESI-MS (pos.): 616.4177
([M + Na]+, C34H59NO7Na+; calc. 616.4189).
Cytotoxicity Assay
Cytotoxicity assays were performed as previously described.[18] Briefly, 3 × 103 cells/well of HL-60
(leukemia), A549 (lung), MKN45 (stomach), or WI-38 (normal lung) cell lines were treated with the
indicated compounds for 48 h prior to adding MTT solution to each well. After incubation for 3 h, the
generated blue formazan was solubilized using 0.04 M HCl in isopropanol. The absorbance at 570 nm
(top) and 630 nm (bottom) were measured by a microplate reader.
Western Blot Analysis
Western blot analysis was performed according to a previously reported method.[18] Briefly, HL-60
cell (from 1 × 106 cells/well) lysates containing 20 μg of total protein were separated by 15% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene
difluoride (PVDF) membranes. After blocking, the membranes were incubated with anti-caspase-3,
anti-cleaved caspase-3, anti-caspase-8, anti-cleaved caspase-8, anti-caspase-9, anti-cleaved caspase-9,
or anti-β-actin primary antibodies at 4 °C overnight. The signals were detected with the ECL Plus
Western Blotting Detection System and imaged using the cooled CCD camera system Atto Light-
Capture II.
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-
number.
This article is protected by copyright. All rights reserved.

Acknowledgements
LCMS and NMR were performed in the Center for Creative Materials Research, College of Science
and Technology (CST), Nihon University. This work was partly supported by JSPS KAKENHI Grant
Number 15K08004 and by the fund of CST, Nihon University. We would like to thank Editage
(www. editage.jp) for English language editing.
Author Contribution Statement
T. H., K. O., M. H., H. A., W. L., and K. K. participated in the acquisition and interpretation of the
data. M. F. contributed to the polishing of the manuscript. M. U. participated in the conception and
design of the research, in the acquisition and interpretation of the data, and in the drafting of the
manuscript.
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