A convenient synthesis of N-linked diglycose derivatives based on one-pot tandem Staudinger/aza-Wittig/reduction and biological evaluation

Article abstract of DOI:10.1016/j.carres.2013.02.002

A series of novel N-linked diglycose derivatives 9 and 10 were conveniently and directly synthesized based on the key step of one-pot tandem Staudinger/aza-Wittig/reduction reaction from the azido sugar and sugar-derived aldehyde followed by deprotection.

Full text of DOI:10.1016/j.carres.2013.02.002

Carbohydrate Research 372 (2013) 15–22
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
A convenient synthesis of N-linked diglycose derivatives based
on one-pot tandem Staudinger/aza-Wittig/reduction and
biological evaluation
Pingzhu Zhang a, Yinbo Li a, Ming Liu a, Yanfei Wang a, Cuicui Li a, Donglai Ma a, Hua Chen a, Kerang Wang a,
Xiaoliu Li a,b, , Jinchao Zhang a
⇑
a Key Laboratory of Chemical Biology of Hebei Province, School of Chemistry and Environmental Science, Hebei University, Baoding, Hebei 071002, China
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-linked diglycose derivatives 9 and 10 were conveniently and directly synthesized
Received 13 November 2012
Received in revised form 5 February 2013
Accepted 13 February 2013
Available online 24 February 2013
based on the key step of one-pot tandem Staudinger/aza-Wittig/reduction reaction from the azido sugar
and sugar-derived aldehyde followed by deprotection. The biological activities against glycosidases (
amylase, -glucosidase, and b-glucosidase) and HIV-RT and antitumor activity of these compounds were
preliminarily evaluated.
a-
a
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
N-linked diglycoses
Staudinger/aza-Wittig/reduction
One pot reaction
Glycosidase inhibition
Antitumor
1. Introduction
glycosidase inhibition agents. Some synthetic strategies have been
exploited by using, for instance, epoxide opening,22 reductive ami-
nation,12,13 and Mitsunobu coupling reaction.23 However, the
development of a more convenient and efficient synthesis of the
amine-linked diglycose derivatives for the molecular diversity
and their biological activity investigation still remains a challenge.
Aza-Wittig reaction, as one of the powerful tools for construct-
ing C@N (or C–N) bonds, has been widely used in the synthesis of
nitrogenous compounds because of its mild reaction conditions
and high yields, and the convenient generation of the intermediate
of phosphinimines from the corresponding azido compounds by
the Staudinger reaction.24,25 And this methodology has also been
successfully applied in the synthesis of glycoside derivatives, such
as mono- and disaccharide glycosyl carbodiimides,26–28 five to
higher-membered aza-sugar derivatives,29–31 N-glycoside neogly-
cotrimers32 and sugar-aza-crown ethers.33 More recently, we have
developed a convenient and effective method for synthesizing
thiazolidin-4-one and thiazinan-4-one derivatives containing
glycoside moieties34–37 using the one pot tandem Staudinger/aza-
Wittig reaction strategy which generated the key intermediate
imine-linked diglycoside from the azido sugar and sugar-derived
aldehyde as shown in Scheme 1 route a. As the extensive applica-
tion of this strategy of Staudinger/aza-Wittig/reduction tandem
reaction, and in view of the promising bioactivity of nitrogen-con-
taining glycoside derivatives, we would like to report a convenient
Diglycose derivatives (Neodisacharides) are hydrolytically sta-
ble glycomimics in which the two sugars were linked through the
nonanomeric center. Since Pérez et al. isolated a hypoglycemic ac-
tive compound which was originally assigned as a 6,60-ether linked
diallose and named Coyolosa (Fig. 1)1 from Mexican traditional
medicine2 of the thorny palm Acrocomia mexicana in 1997, although
this assigned structure was later certified to be incorrect according
to the reported NMR data and structure activity studies of synthetic
compounds,3,4 increasing interest has been evoked in the synthesis
of diglycose derivatives for developing novel potential glycosidase
inhibition agents, and a variety of diglycose derivatives linked with
ether,3–7 thioether,8–10 amine11–14 and selenoether15–17 have been
recently discovered and synthesized. It is well known that the nitro-
gen-containing glycoside derivatives such as iminosugars, alkoids
and amine-linked pseudo saccharide analogs (for instance Vali-
doxylamine series) exhibit effective glycosidase inhibitory activity
due to the strong interaction between the amine group in the mol-
ecule and the enzyme.18–21 These promising bioactivities have at-
tracted considerable attention to the design and synthesis of
novel amine-linked diglycose derivatives for developing potential
⇑
Corresponding author. Tel./fax: +86 312 5971116.
E-mail address: lixl@hbu.cn (X. Li).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.02.002

16
P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
HO
HO
O
O
OH
OH
showed that by TLC checking 4aa accompanied with 5 was formed,
O
followed by MW at 80 °C for 5 min, 4aa disappeared and was trans-
formed into the eliminated intermediate 5. The above results
showed that the sugar-derived aldehyde (3a) and the intermediate
imine 4aa were more unstable in microwave irradiation than in the
OH HO
OH
OH
Figure 1. The originally reported structure of Coyolosa.
normal conditions and easily generated the eliminated a,b-unsatu-
rated imine 5. Thus, the novel amine-linked diglycose derivatives
were conveniently synthesized in the following procedure (Table 1,
entry 5): the mixture of azido sugar derivative 1a (1 mmol) and tri-
phenylphosphine (Ph3P, 1.2 mmol) in THF was stirred 0.5 h at room
temperature under N2 atmosphere; the sugar-derived aldehyde 3a
(1.0 mmol) was added, and stirred for another 2 h. Then the reac-
tion mixture was cooled to 0 °C, the mixture of glacial acetic acid
(2.0 mmol) and NaBH3CN (2.0 mmol) was added, and stirred for
0.5 h at 0 °C to complete the reaction. Saturated aqueous solution
of ammonium chloride was added to quench the reaction, after
work up and chromatographic purification, the protected amine-
linked diglycose 6aa was afforded in a yield of 51%. With the same
procedure, the compounds 6ab–6cc were synthesized in good
yields of 46–53% from the corresponding azido sugar derivative
(1a–1c) and sugar-derived aldehyde (3a–3c), respectively (Table 2
and Scheme 3).
Subsequently, the deacetylation of the intermediates 6 was car-
ried out in a methanolic solution of sodium methoxide at room
temperature. However, it was found that some of the deproteced
products were unstable, and an unseparated and unidentifiable
mixture without the main component was obtained in each case
of 6aa, 6ab, 6ba and 6bb. Only in the cases of 6ac, 6ca, 6cb, and
6cc, the deprotected diglycose products 9ac, 9ca, 9cb, and 9cc
were obtained in 43–67% yields by deacetylation and followed by
removing the isopropylidene under the treatment with 60% AcOH
aqueous solution at 45 °C (Table 2). Furthermore, the amines 9ac,
9ca, 9cb, and 9cc were also found to be not so stable at the ambient
conditions for a long time storage.48 Accordingly, we envisaged to
introduce an acetyl to the amino group to stabilize the product by
forming the amide derivative. Thus, 6aa–6cc was firstly treated
with AcCl to form the corresponding amide-linked diglycose inter-
mediates, followed by removing the O-acetyl and isopropylidene
protective groups to afford the corresponding amide-linked digly-
cose derivatives 10aa–10cc in good yields of 54–79%, respectively,
(Scheme 3 and Table 2).
and direct synthesis of the amine-linked diglycose derivatives 9
and 10 from azido sugars 1a–1c and sugar-derived aldehydes
3a–3c, as shown in Scheme 1 route b (see Scheme 2).
2. Results and discussion
2.1. The synthesis of amine-linked diglycose derivatives
Azido sugar derivatives (1a,38 1b,39 1c40) and sugar-derived
aldehydes (3a,41 3b,42 3c43) were synthesized from the correspond-
ing sugars according to the literatures.
The one pot tandem Staudinger/aza-Wittig/reduction reaction
was explored with azido sugar derivative (1a) and sugar-derived
aldehyde (3a) under the conditions in three steps as shown in
Scheme 2 and Table 1. Based on the previous studies34,35,37 the
reactions in the first two steps were first carried out at room tem-
perature, followed by the reduction of C@N with NaBH4, but in-
stead of the desired product (6aa), the acetamido derivative (8)
was obtained (entry 1), which possibly comes from the migration
of the Ac group from 3-O to the newly generated NH; when the
reduction took place in 0 °C, a small amount of the eliminated
product (7) was also obtained because of the deacetoxyation (entry
2). Both the side reactions may result from the weak basic feature
of NaBH4. To reduce the acetyl migration and the elimination in the
reduction step, the slightly acidic reagents of NaBH4–AcOH and
NaBH3CN–AcOH were examined, respectively (entries 3 and 4).
As a result, NaBH3CN–AcOH provided the desired product (6aa)
in a reasonable yield at lower reaction temperature (entry 5).
In order to improve the yield, the first two step reactions were
performed in higher temperature, but the eliminated product (7)
was increased without the total yield improvement with the tem-
perature going up (entries 6 and 7). Considering the successful
applications of microwave assisted organic synthesis in sugar
chemistry,44–46 we tried the second step with the microwave as-
sisted method. As shown in Table 1, under microwave irradiation
the reaction afforded only 7 at 80 °C (entry 8), which indicated that
the microwave irradiation benefited the formation of the
eliminated product 5. In order to examine the microwave effect,
the microwave irradiated reaction was performed at 25 °C,47 the
desired product (6aa) and a certain amount of the eliminated prod-
uct 7 were produced (entry 9), although the reaction at 25 °C affor-
ded the sole product (6aa) in non-microwave condition. In addition,
the imine 4aa was also unstable and eliminated AcOH to form the
eliminated intermediate 5 completely at 80 °C, which could be
clearly observed in the experiment of entry10: under the conditions
of microwave irradiation (MW) at 25 °C for 20 min, the reaction
2.2. Biological activities
The inhibitions against HIV-1 reverse transcriptase (HIV-RT)
and glycosidases, and the antitumor activity of some of compounds
9 and 10 were preliminarily evaluated. The cytotoxicity of the
compounds 9 and 10 against HeLa cell lines and human lung
adenocarcinoma epithelial cell line (A-549) was examined by the
modified Mosmann’s protocol.49 As shown in Table 3, compounds
9cb, 10ab and 10bb exhibited good cytotoxicity to A-549 with
the IC50 values of 23.32, 27.89 and 24.77 lM, respectively, similar
to that of the positive control Cisplatin, and most compounds have
Staudinger
H2
Aza-Wittig
R2 CHO
R1
Ph3P
H
N
R1
Phospinimine
R1
N
PPh3
N
C
H
R2
Reduction
R1
C
R2
N3
+
b
Amine
Imine
O
n
S
a
Condensation
N
R2
HS(CH2)nCO2H
( n=0,1)
R1
thiazolidin-4-one and
thiazinan-4-one derivatives
R1, R2 = sugar moieties
Scheme 1. Diagram of one-pot synthesis of amine-linked diglycose derivatives by tandem Staudinger/aza-Wittig /reduction.

P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
17
O
O
O
O
O
MeO
AcO
O
MeO
AcO
O
OMe
OAc
Ph3P
O
O
OMe
OAc
O
PPh3
step1
AcO
N
N
N3
AcO
N
3a
O
O
+
step2
AcO
AcO
OAc
OAc
O
O
Staudinger
aza-Wittig
OAc
OAc
1a
OAc
OAc
5
4aa
2a
5
6'
O
O
O
MeO
O
MeO
O
MeO
O
N
H
N
Ac
OAc
N
H
4'
1
2
1'
5
O
O
O
3' 2'
+
+
4
step3
reduction
3
OAc AcO
AcO
OAc
AcO
AcO
HO
O
O
O
OAc
OAc
OAc
8
6aa
7
Scheme 2. Synthesis of compound 6aa by one-pot tandem Staudinger/aza-Wittig reaction/reduction of 1a with 3a.
Table 1
Conditional optimization of the one-pot Staudinger/aza-Wittig/reduction of the azido sugar derivative 1a with the sugar-derived aldehyde 3a
Entry
Condition of step 2a
Temperature and timeb
Condition of step 3
Yield (%)
Aldehyde (3a) (equiv)
Reduction reagent (equiv)
Temperature and time
6aa
7
8
1
2
3
4
5
6
7
8
9
10
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
rt, 2 h
rt, 2 h
rt, 2 h
rt, 2 h
rt, 2 h
40 °C, 1.2 h
50 °C, 1 h
MW, 80 °C, 5 min
MW, 25 °C, 20 min
MW, 25 °C, 20 min then 80 °C, 5 min
NaBH4 (3.0)
NaBH4 (2.0)
NaBH3CN (2.0)
NaBH4–AcOH (2.0)
NaBH3CN–AcOH (2.0)
NaBH3CN–AcOH (2.0)
NaBH3CN–AcOH (2.0)
NaBH3CN–AcOH (2.0)
NaBH3CN–AcOH (2.0)
NaBH3CN–AcOH (2.0)
rt, 5 min
0
0
16
0
0
0
0
38
50
3
10
0
0
0
0
0
0 °C, 5 min
0 °C, 30 min
0 °C, 5 min
0 °C, 30 min
0 °C, 30 min
0 °C, 30 min
0 °C, 30 min
0 °C, 30 min
0 °C, 30 min
47
35
51
45
25
0
4
28
60
20
50
30
0
0
a
Condition of step 1: 1a, THF, Ph3P (1.2 equiv), rt 0.5 h, then step 2.
Reaction completion time (checked by TLC).
b
Table 2
inhibition, antitumor, and antiviral against HIV-RT were prelimi-
narily evaluated. The further study of the synthesis and biological
activities of such N-linked diglycose derivatives using this method-
ology is underway.
The yields of compounds 6, 9 and 10
Compounds 6, 9
and 10
Azido
sugar 1
Aldehyde
3
Yields
of 6
Yields
of 9
Yields of
10
a
aa
ab
ac
ba
bb
ca
cb
cc
a
a
a
b
b
c
c
c
a
b
c
a
b
a
b
c
51
51
53
47
52
50
49
46
—
—
79d
57d
61d
70d
63d
61e
70e
54e
a
3. Experimental
43b
a
—
—
a
3.1. General methods
65c
65c
67c
Melting points were measured on an SGWÒX-4 micro melting
point apparatus and are uncorrected. Optical rotations were deter-
mined on an SGWÒ-1 automatic polarimeter. 1H NMR, 13C NMR
a
b
c
Not obtained.
Condition: NaOMe (1 equiv), MeOH, rt.
Condition: NaOMe (1 equiv), MeOH, rt, then 60% AcOH, 45 °C.
Condition: (i) AcCl, Et3N (5 equiv), CH2Cl2; (ii) NaOMe (1 equiv), MeOH, rt.
spectra were measured on
a RT-NMR Bruker AVANCE 400
d
e
(400 MHz) and Bruker AVANCE 600 (600 MHz) spectrometer using
tetramethylsilane (Me4Si) as the internal standard. High-resolution
mass spectra (HRMS) were carried out on a FTICR-MS (Ionspec
7.0T) mass spectrometer in the electrospray-ionization (ESI) mode.
The microwave assisted reactions were performed on a DISCOVER
S-Class Auto Focused Microwave Synthesis System (CEM Corpora-
tion, USA), the microwave reactions at 25 °C were performed using
this microwave reactor equipped with a fiber-optic probe for direct
monitoring of the internal reaction temperature in a 10 mL sealed
reaction vessel, the reaction vessel is cooled from the outside by
compressed air (0.3–0.5 MPa) (air compressor WSC 21070A,
Shanghai Wispump Ind. Co., Ltd) which was precooled to ca. 0 °C
by passing through a coil immersed in a cooling bath (cooling li-
quid circulating pump, ꢀ20 to ꢀ15 °C, DLSB-10/20, Zheng Zhou
Great Wall Scientific Industrial and Trade Co. Ltd) before reaching
the reaction vessel while being irradiated by microwaves.
Condition: (i) AcCl, Et3N (5 equiv), CH2Cl2; (ii) NaOMe (1 equiv), MeOH, rt then
60% AcOH.
selectivity to A-549 than HeLa. The glycosidase inhibitory activities
of compounds 9 and 10 were determined with hydrolytic reactions
of a-amylase, a-glucosidase, and b-glucosidase using acarbose as a
control, respectively; and the HIV-RT inhibitory activities of the
compounds were measured using colorimetric reverse transcrip-
tase assay by comparison with AZT.50 However, the compounds
have not shown obvious activities with both of them.
In summary, we have synthesized a series of primary-primary
and primary-secondary N-linked diglycose derivatives conve-
niently by one-pot tandem Staudinger/aza-Wittig/reduction
process from azido sugar derivatives and sugar-derived aldehydes
at room temperature followed by deprotection, providing
a
The optical densities for examining the inhibitory activities
against glycosidase and HIV-RT and anti-tumor activity were
measured on a TU-1901 UV–vis spectrophotometer and a Bio-
Rad Model 3550 microplate spectrophotometer, respectively.
convenient and direct method for constructing such amine-linked
diglycose derivatives in good yields under very mild conditions.
The biological activities of compounds 9 and 10 for glycosidases

18
P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
H
N
R4
R3
iii
9ac, 9ca, 9cb, 9cc
H
N
R2
i
R1 N3
Ac
R1
R2 CHO
3a~3c
N
R4
1a~1c
6aa~6cc
ii,iii
R3
10aa~10cc
O
HO
OMe
O
OMe
OH
OMe
O
O
O
O
O
OMe
OAc
R3
HO
R1=
AcO
a
O
O
O
a
HO
=
b
c
b
c
OAc
O
O
HO
OH
AcO
OH
O
OAc
O
O
OMe
OAc
O
OMe
OH
O
O
OMe
OMe
O
O
O
R2
c
b
=
c
AcO
a
R4
=
a
b
HO
O
AcO
OAc
HO
HO
OH
AcO
OAc
OH
Scheme 3. Synthesis of compounds 9 and 10 by one-pot tandem Staudinger/aza-Wittig /reduction of azido sugar derivative 1 with the sugar-derived aldehyde 3 followed by
deprotection. Reagents and conditions: (i) Ph3P (1.2 equiv), THF, rt, 0.5 h, then R2-CHO (3a–3c) (1 equiv), rt, 2 h, and then NaBH3CN–AcOH (2 equiv), 0 °C, 0.5 h; (ii) AcCl
(5 equiv), Et3N (5 equiv), CH2Cl2, 0 °C; (iii) NaOMe (1 equiv), MeOH, rt, then 60% AcOH, 45 °C.
Table 3
Antitumor activities
Compounds
IC50 M)
9ac
148.52 3.69 116.01 4.34 161.51 3.39 346.99 2.88 222.65 3.43 133.89 1.39 118.78 3.46
A-549 129.71 7.12 23.32 1.16 43.81 2.15 204.85 3.89 27.89 1.23 60.23 1.19 128.4 47.28 24.77 0.63 19.41 5.67
9cb
9cc
10aa
10ab
10ac
10bb
10bb
Cisplatin
(
l
HeLa
44.10 0.82 20.01 1.36
Thin-layer chromatography (TLC) was performed on precoated
plates (Qingdao GF254) with detection by UV light or with phospho-
molybdic acid in EtOH/H2O followed by heating. Column chroma-
tography was performed using a SiO2 (Qingdao 300–400 mesh).
J = 10.2 Hz, CH, H-3), 5.93 (d, 1H, J = 3.6 Hz, CH, H-10); 13C NMR
(150 MHz, CDCl3) d: 20.58, 20.62, 20.67, 20.70, 26.36, 26.75
(CH3), 43.64 (CH2), 54.21 (OCH3), 56.74 (CH), 64.32 (CH2), 69.16
(CH), 70.43 (CH), 70.85 (CH), 73.01 (CH), 76.34 (CH), 83.65 (CH),
97.26 (C-1), 104.54 (C-10), 112.90 (C), 169.53, 169.73, 170.07,
3.2. General procedure for the synthesis of compounds 6
170.24 (O@C). HRMS (ESI): Calcd C
25H37NO14 for (M+H+):
575.7156. Found: 575.7141.
Azido sugar derivative 1 (1.0 mmol) was dissolved in 2 mL
anhydrous THF. Ph3P (1.2 mmol) was then added to the solution
with stirring under nitrogen atmosphere, the mixture was stirred
at room temperature for 0.5 h and the sugar-derived aldehyde 3
(1 mmol) was added to the solution, stirred for another 2.0 h at
rt. Then, it was cooled to 0 °C, a mixture of NaBH3CN (126 mg,
3.2.2. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-glucopyranosid-
6-yl)-N-(1,2-O-isopropylidene-3,5-dideoxy-3-enyl-a-xylos-5-yl)
amine (7)
Colorless oil; ½a D22
ꢁ
+72.6° (c 0.1, CHCl3); 1H NMR (400 MHz,
CDCl3) d: 1.43 (s, 3H, CH3), 1.45 (s, 3H, CH3), 2.00 (s, 3H, CH3),
2.03 (s, 3H, CH3), 2.07 (s, 3H, CH3), 2.70–2.73 (m, 2H, H-6), 3.33
(s, 2H, H-50), 3.40 (s, 3H, OCH3), 3.87–3.92 (m, 1H, H-5), 4.85 (dd,
1H, J = 3.6 Hz, J = 10.2 Hz, H-2), 4.91 (d, 1H, J = 3.6 Hz, H-1), 4.97
(t, 1H, J = 19.4 Hz, H-4), 5.09 (s, 1H, H-30), 5.27–5.29 (m, 1H, H-
20), 5.47 (t, 1H, J = 19.5 Hz, H-3), 6.05 (d, 1H, J = 5.2 Hz, H-10); 13C
NMR (100 MHz, CDCl3) d:21.08 (CH3), 21.29 (CH3), 21.65 (CH3),
28.20 (CH3), 28.55 (CH3), 47.05 (C-50), 49.77 (C-6), 55.77 (OCH3),
68.85 (C-5), 70.53 (C-3), 70.72 (C-4), 71.43 (C-2), 84.03 (C-20),
96.95 (C-1), 98.99 (C-30), 106.56 (C-10), 112.40, 160.73 (C-40),
170.23, 170.45, 170.57 (O@C). HRMS (ESI): Calcd C21H31NO11 for
(M+H+): 473.6020. Found: 473.6031.
2 mmol) and AcOH (115 lL, 2.0 mmol) was added. After continu-
ously stirring for 0.5 h at 0 °C, the reaction was quenched with
the addition of saturated aqueous ammonium chloride solution,
and the mixture was extracted with ethyl acetate. The combined
extracts were washed with water, dried over MgSO4 and evapo-
rated, and the residue was purified by a column chromatography
using petroleum ether/ethyl acetate (v/v = 1:1) as the eluent to ob-
tain the product 6aa–6cc.
3.2.1. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-glucopyranosid-
6-yl)-N-(1,2-O-isopropylidene-3-O-acetyl-5-dexoy-a-D-xylos-5-
yl) amine (6aa)
Colorless oil; ½a D20
ꢁ
+31.6° (c 0.1, CHCl3); 1H NMR (600 MHz,
3.2.3. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-
a-
D-glucopyranosid-
CDCl3) d: 1.30 (s, 3H, CH3), 1.32 (s, 3H, CH3), 2.00 (s, 3H, CH3),
2.02 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.14 (s, 3H, CH3), 2.90 (td, 1H,
J = 2.4 Hz, J = 12.0 Hz, CH2), 2.96–3.04 (m, 2H, CH2, CH), 3.25 (dd,
1H, J = 10.2 Hz, J = 13.2 Hz, CH2), 3.32 (dd, 1H, J = 8.4 Hz,
J = 14.4 Hz, CH2), 3.48 (s, 3H, OCH3), 4.16 (td, 1H, J = 2.4 Hz,
J = 5.4 Hz, CH), 4.37 (td, 1H, J = 2.4 Hz, J = 5.4 Hz, CH), 4.57 (dd,
1H, J = 4.2 Hz, J = 10.2 Hz, CH), 4.87 (t, 1H, J = 9.6 Hz, CH, H-4),
4.97 (d, 1H, J = 4.2 Hz, CH), 5.20 (s, 1H, CH, H-1), 5.50 (t, 1H,
6-yl)-N-(1,2-O-isopropylidene-5-dexoy-a-D-xylos-5-yl)
acetamide (8)
Colorless oil; ½a D20
ꢁ
+38.6° (c 0.1, MeOH); 1H NMR (600 MHz,
CDCl3) d:1.30 (s, 3H, CH3), 1.51 (s, 3H, CH3), 2.00 (s, 3H, CH3),
2.07 (s, 6H, CH3), 2.13 (s, 3H, CH3), 3.35 (s, 3H, OCH3), 3.44–3.48
(m, 2H, H-6), 3.91 (s, 1H, H-4), 3.95–3.97 (m, 1H, H-5), 4.09–4.16
(m, 2H, H-50), 4.58 (d, 1H, J = 3.5 Hz, H-2), 4.80–4.86 (m, 2H, H-1,
H-4), 4.92 (d, J = 3.6 Hz, 1H, H-30), 5.03 (s, 1H, H-20), 5.44 (t, 1H,

P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
19
J = 19.4 Hz, H-3), 5.88 (d, 1H, J = 3.6 Hz, H-1); 13C NMR (150 MHz,
3.2.8. N-(1,2:5,6-Di-O-isopropylidene-3-deoxy-
a
-D
-allos-3-yl)-N-
CDCl3) d: 20.60, 20.63, 21.47, 25.94, 26.84 (CH3), 45.41 (CH2),
51.28 (OCH3), 55.99 (CH), 69.65 (CH2), 69.89 (CH), 70.56 (CH),
70.63 (CH), 73.66 (CH), 78.44 (CH), 84.83 (CH), 96.72 (C-1),
104.58 (C-10), 111.51 (C), 169.79, 169.94, 170.05, 172.77 (O@C).
HRMS (ESI): Calcd C25H37NO14 for (M+H+): 575.7156. Found:
575.7143.
(1,2-O-isopropy-lidene-3-O-acetyl-5-deoxy-a-D-xylos-5-yl)
amine (6ca)
Colorless oil; ½a D20
ꢁ
+69.0° (c 0.1, CHCl3); 1H NMR (600 MHz,
CDCl3) d: 1.31 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.37 (s, 3H, CH3),
1.44 (s, 3H, CH3), 1.52 (s, 6H, CH3), 2.26 (s, 3H, CH3), 2.78 (dd,
1H, J = 3.6 Hz, J = 12.6 Hz, CH2), 3.00 (dd, 1H, J = 7.2 Hz,
J = 12.6 Hz, CH2), 3.05 (dd, 1H, J = 4.2 Hz, J = 10.2 Hz, CH2), 3.77
(dd, 1H, J = 3.6 Hz, J = 9.6 Hz, CH2), 3.96 (t, 1H, J = 7.2 Hz, CH),
4.18 (t, 1H, J = 7.2 Hz, CH), 4.33–4.38 (m, 2H, CH), 4.51 (d, 1H,
J = 3.6 Hz, CH), 4.65 (t, 1H, J = 4.2 Hz, CH), 5.22 (d, 1H, J = 3.0 Hz,
CH), 5.81 (d, 1H, J = 3.6 Hz, H-10), 5.92 (d, 1H, J = 3.6 Hz, H-1); 13C
NMR (150 MHz, CDCl3) d: 20.77, 25.25, 26.29, 26.48, 26.64, 26.75
(CH3), 48.66 (CH2), 60.13 (OCH3), 62.19 (CH), 64.89 (CH2), 75.52
(CH), 76.72 (CH), 78.24 (CH), 78.99 (CH), 79.33 (CH), 83.48 (CH),
99.79 (C-1), 103.77 (C-10), 109.46, 111.97, 112.03 (C), 169.81
(O@C). HRMS (ESI): Calcd C22H35NO10 for (M+H+): 473.6512.
Found: 473.6522.
3.2.4. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-
a
-D
-glucopyranosid-
6-yl)-N-(methyl 2,3-di-O-acetyl-5-dexoy-b-D-ribofuranosid-5-
yl) amine (6ab)
Colorless oil; ½a D22
ꢁ
+67.6° (c 0.1, CHCl3); 1H NMR (400 MHz,
CDCl3) d: 1.98–2.06 (m, 15H, CH3), 2.91–3.22 (m, 4H, H-6), 3.45
(s, 6H, OCH3), 3.52–4.01 (m, 8H, CH, CH2), 4.11–4.14 (m, 1H, CH),
4.51–4.53 (m, 1H, CH), 4.79–4.99 (m, 5H, CH), 5.27 (s, 1H, CH),
5.51–5.52 (m, 1H, CH); 13C NMR (100 MHz, CDCl3) d: 20.86 (CH3),
53.53 (CH2), 56.36 (CH2), 57.04, 59.33 (OCH3), 69.59 (CH), 70.24
(CH), 70.91 (CH), 71.30 (CH), 74.60 (CH), 75.20 (CH), 76.25 (CH),
97.75 (C-1), 107.53 (C-10), 169.80, 169.92, 170.29, 170.58, 170.66
(O@C). HRMS (ESI): Calcd C25H37NO15 for (M+H+): 591.7150.
Found: 591.7136.
3.2.9. N-(1,2:5,6-Di-O-isopropylidene-3-deoxy-
a
-D
-allos-3-yl)-N-
(methyl 2,3-di-O-acetyl-5-deoxy-b-D-ribofuranosid-5-yl) amine
(6cb)
3.2.5. N,N-Bis(methyl 2,3,4-tri-O-acetyl-6-deoxy-
a-
D
-
Colorless oil; ½a D20
ꢁ
+20.5° (c 0.1, CHCl3); 1H NMR (600 MHz,
glucopyranosid-6-yl) amine (6ac)
CDCl3) d: 1.34 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.46 (s, 3H, CH3),
1.48 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.51 (s, 6H, CH3), 2.58 (dd,
1H, J = 7.2 Hz, J = 11.4 Hz, CH2), 2.96 (dd, 1H, J = 8.4 Hz,
J = 11.4 Hz, CH2), 3.10 (dd, 1H, J = 4.8 Hz, J = 9.6 Hz, CH2), 3.38 (s,
3H, OCH3), 3.80 (dd, 1H, J = 3.0 Hz, J = 9.6 Hz, CH2), 3.99 (t, 1H,
J = 7.8 Hz, CH), 4.23–4.26 (m, 2H, CH), 4.40 (td, 1H, J = 3.0 Hz,
J = 7.2 Hz, CH), 4.58 (d, 1H, J = 6.0 Hz, CH), 4.63 (t, 1H, J = 4.2 Hz,
CH), 4.65 (d, 1H, J = 6.0 Hz, CH), 4.97 (s, 1H, H-1), 5.81 (d, 1H,
J = 3.6 Hz, H-10);13C NMR (150 MHz, CDCl3) d:25.06 (CH3), 25.37
(CH3), 26.27 (CH3), 26.49 (CH3), 26.50 (CH3), 26.75 (CH3), 51.84
(CH2), 55.21 (OCH3), 61.73 (CH), 65.06 (CH2), 75.74 (CH), 77.94
(CH), 78.96 (CH), 82.68 (CH), 85.33 (CH), 86.83 (CH), 104.33 (C-
1), 109.55 (C), 109.66 (C-10), 111.97, 112.33 (C), 169.81 (O@C).
HRMS (ESI): Calcd C22H35NO11 for (M+H+): 489.6506. Found:
489.6522.
Colorless oil; ½a D22
ꢁ
+45.8° (c 0.1, CHCl3,); 1H NMR (600 MHz,
CDCl3) d: 2.02 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.08 (s, 3H, CH3),
2.09 (s, 9H, CH3), 2.86 (t, 1H, J = 15.0 Hz, CH2), 3.02 (t, 1H,
J = 17.4 Hz, CH2), 3.10 (t, 1H, J = 17.4 Hz, CH2), 3.24 (dd, 1H,
J = 21.6 Hz, J = 13.2 Hz, CH2), 3.47 (s, 3H, OCH3), 3.53 (s, 3H,
OCH3), 4.14 (t, 1H, J = 15.6 Hz, CH), 4.46 (td, 1H, J = 2.4 Hz,
J = 15.0 Hz, CH), 4.74–4.91 (m, 4H, CH), 4.95 (d, J = 5.4 Hz, 1H, H-
10), 5.02 (d, 1H, J = 5.4 Hz, H-1), 5.50–5.53 (td, 2H, J = 2.4 Hz,
J = 8.4 Hz, CH); 13C NMR (150 MHz, CDCl3) d: 20.64, 20.67 (CH3),
52.75, 55.01 (C-6), 56.44, 56.88 (OCH3), 64.26 (C-4), 69.01, 69.10
(C-2), 69.88, 70.50 (C-5), 70.76 (C-3), 97.04, 97.21 (C-1), 169.69,
169.84, 170.06, 170.26 (O@C). HRMS (ESI): Calcd C28H41NO17 for
(M+H+): 663.7964. Found: 663.7954.
3.2.6. N-(Methyl 2,3-di-O-acetyl-5-deoxy-b-D-ribofuranosid-5-
yl)-N-(1,2-O-isopropylidene-3-O-acetyl-5-dexoy-
a-
D-xylos-5-yl)
3.2.10. N-(1,2:5,6-Di-O-isopropylidene-3-deoxy-
a
-D
-allos-3-yl)-
amine (6ba)
N-(methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-glucopyranosid-6-yl)
Colorless oil; ½a D20
ꢁ
ꢀ24.8° (c 0.1, CHCl3); 1H NMR (600 MHz,
amine (6cc)
CDCl3) d: 1.31 (s, 3H, CH3), 1.52 (s, 3H, CH3), 2.09 (s, 3H, CH3),
2.14 (s, 3H, CH3), 2.16 (s, 3H, CH3), 3.04–3.13 (m, 3H, CH2), 3.37–
3.39 (m, 1H, CH2), 3.43 (s, 3H, OCH3), 4.58 (d, 1H, J = 3.6 Hz, CH),
4.62–4.65 (m, 2H, CH), 4.92 (s, 1H, H-10), 5.17 (t, 1H, J = 5.4 Hz,
CH), 5.22 (m, 1H, CH), 5.24 (d, 1H, J = 4.8 Hz, CH, H-10), 5.93 (d,
1H, J = 3.6 Hz, H-1); 13C NMR (150 MHz, CDCl3) d: 20.46, 20.54,
20.61, 26.07, 26.74 (CH3), 53.47 (CH2), 56.27 (OCH3), 56.75 (CH),
72.67 (CH2), 72.97 (CH), 74.57 (CH), 75.59 (CH), 76.56 (CH), 83.52
(CH), 104.54 (C-10), 106.70 (C-1),112.90 (C), 169.53, 169.73,
170.07, 170.24 (O@C). HRMS (ESI): Calcd C22H33NO12 for (M+H+):
503.6342. Found: 503.6357.
Colorless oil; ½a D20
ꢁ
+67.2° (c 0.1, CHCl3); 1H NMR (600 MHz,
CDCl3) d: 1.34 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.44 (s, 3H, CH3),
1.53 (s, 3H, CH3), 2.00 (s, 3H, CH3), 2.02 (s, 3H, CH3), 2.07 (s, 3H,
CH3), 2.65 (dd, 1H, J = 2.4 Hz, J = 12.6 Hz, CH2), 2.90 (dd, 1H,
J = 7.8 Hz, J = 12.6 Hz, CH2), 3.06 (dd, 1H, J = 4.8 Hz, J = 9.0 Hz,
CH2), 3.41 (s, 3H, OCH3), 3.75–3.78 (dd, 1H, J = 3.0 Hz, J = 9.0 Hz,
CH2), 3.86 (td, 1H, J = 2.4 Hz, J = 10.2 Hz, CH), 3.40 (t, 1H,
J = 7.8 Hz, CH), 4.16 (t, 1H, J = 7.8 Hz, CH), 4.35 (td, 1H, J = 3.6 Hz,
J = 7.2 Hz, CH), 4.61 (t, 1H, J = 4.2 Hz, CH), 4.86 (dd, 1H, J = 4.2 Hz,
J = 10.8 Hz, CH), 4.93 (d, 1H, J = 3.6 Hz, H-10), 4.99 (t, 1H,
J = 9.6 Hz, CH), 5.47 (t, 1H, J = 9.6 Hz, CH), 5.79 (d, 1H, J = 4.2 Hz,
H-1); 13C NMR (150 MHz, CDCl3) d: 20.66, 20.66, 20.66, 25.34,
26.20, 26.49, 26.68 (CH3), 48.65 (CH2), 55.15 (OCH3), 62.56 (CH),
65.19 (CH2), 69.24 (CH), 70.19 (CH), 70.36 (CH), 71.06 (CH), 75.78
(CH), 78.05 (CH), 79.22 (CH), 96.47 (C-1), 104.32 (C-10), 109.54
(C), 112.09 (C), 169.77, 170.07, 170.18 (O@C). HRMS (ESI): Calcd
3.2.7. N,N-Bis(methyl 2,3-di-O-acetyl-5-deoxy-b-D-
ribofuranosid-5-yl) amine (6bb)
Colorless oil; ½a D20
ꢁ
ꢀ11.3° (c 0.1, CHCl3); 1H NMR (400 MHz,
CDCl3) d: 2.10 (s, 3H, CH3), 2.12 (s, 3H, CH3), 2.14 (s, 3H, CH3),
2.15 (s, 3H, CH3), 2.95–2.97 (m, 1H, H-5), 3.21–3.28 (m, 3H, H-5,
H-50), 3.43 (s, 3H, OMe), 3.45 (s, 3H, OMe), 4.63–4.65 (m, 2H, H-
4, H-40), 4.95 (s, 2H, H-1, H-10), 5.18–5.22 (m, 2H, H-3, H-30), 5.30
(d, 2H, J = 4.8 Hz, J = 15.2 Hz, CH, H-20, H-2); 13C NMR (100 MHz,
CDCl3) d: 20.81, 21.33 (CH3), 55.30, 55.88 (C-5), 59.78, 60.70
(OCH3), 73.30, 73.57 (C-3), 75.17 (C-4), 75.32, 76.21 (C-2),
107.17, 107.36 (C-1), 169.68, 169.99, 170.38, 170.41 (O@C). HRMS
(ESI): Calcd C22H33NO13 for (M+H+): 519.6336. Found: 519.6341.
C
25H39NO13 for (M+H+): 561.7320. Found: 561.7334.
3.3. General procedure for the synthesis of compounds 9
Compound 6 was dissolved in dry methanol, sodium methoxide
(1.0 equiv) was added, then the reaction mixture was stirred at
room temperature till the completion of reaction. Ion exchange re-
sin was added to adjust the pH of the solution to 7. The resin was

20
P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
filtered off and methanol was evaporated under reduced pressure.
The crude product was purified by silica gel column chromatogra-
phy using acetoacetate/methanol (v/v = 10:1) as the eluent to ob-
tain a colorless oil product. The product was dissolved in 60%
AcOH and the reaction mixture was stirred at 45 °C, until the reac-
tion completed (checked by TLC). The solvent was evaporated un-
der reduced pressure and residue was purified by flash column
chromatography (ethyl acetate/methanol (v/v = 8:1)) to afford the
compound 9.
HRMS (ESI): Calcd C16H29NO10 for (M+H+): 395.5018. Found:
395.5007.
3.4. General procedure for the synthesis of compounds 10aa–
10bb
The compounds 6aa–6bb were dissolved in dry CH2Cl2 under an
ice-water bath, Et3N (5.0 equiv) and AcCl (5.0 equiv) were added,
the reaction mixture was stirred at 0 °C till the completion of reac-
tion (checked by TLC). Excess reagent was decomposed with satu-
rated aqueous NaHCO3 solution and the mixture was extracted
with ethyl acetate three times. The combined extracts were
washed with water, brine, dried over MgSO4 and evaporated, and
the resulting product was dissolved in dry methanol, sodium
methoxide (1.0 equiv) was added, then the reaction mixture was
stirred at room temperature, ion exchange resin was added to ad-
just the pH of the solution to 7 after the completion of reaction
(checked by TLC). The resin was filtered off and methanol was
evaporated under reduced pressure. The crude product was puri-
fied by silica gel column chromatography (ethyl acetate/methanol
(v/v = 10:1)) to afford the compounds 10aa–10bb.
3.3.1. N,N-Bis(methyl 6-deoxy-a-D-glucopyranosid-6-yl) amine
(9ac)
White solid; ½a 2D1
ꢁ
+86.7° (c 0.1, MeOH); 1H NMR (400 MHz,
CD3OD) d: 2.97–3.43 (m, 8H, CH2, CH), 3.48 (s, 6H, OCH3), 3.62 (t,
J = 3.6 Hz, 2H, CH), 3.96–3.97 (m, 1H, CH), 4.10–4.11 (m, 1H, CH),
4.67 (d, J = 3.6 Hz, 1H, H-1), 4.72 (d, J = 3.6 Hz, 1H, H-1); 13C NMR
(100 MHz, CD3OD) d: 48.46, 52.54 (CH2), 55.46, 55.51 (OCH3),
71.71 (CH), 72.12 (CH), 73.43 (CH), 73.62, 73.62 (CH), 73.76 (CH),
74.57 (CH), 75.07 (CH), 101.11 (C-1), 101.14 (C-10). HRMS (ESI):
Calcd C14H27NO10 for (M+H+): 369.4520. Found: 369.4527.
3.3.2. N-(1,2-O-Isopropylidene-3-deoxy-
a
-
D
-allos-3-yl)-N-(1,2-
-xylos-5-yl) amine (9ca)
+89.2° (c 0.1, MeOH); 1H NMR (600 MHz,
3.4.1. N-(Methyl 6-deoxy-
a
-
D
-glucopyranosid-6-yl)-N-(1,2-O-
-xylos-5-yl) acetamide (10aa)
+37.1° (c 0.1, MeOH); 1H NMR (600 MHz,
O-isopropylidene-5-deoxy-a-D
Colorless oil; ½a D22
ꢁ
isopropylidene-5-deoxy-a-D
Colorless oil; ½a D22
ꢁ
CD3OD); d: 1.31 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.46 (s, 3H, CH3),
1.53 (s, 3H, CH3), 2.92–2.95 (m, 2H, CH2), 3.11–3.14 (m, 1H, CH2),
3.25–3.27 (m, 1H, CH), 3.69–3.71 (m, 2H, CH2), 3.82 (dd, 1H,
J = 3.0 Hz, J = 9.6 Hz, CH2), 3.90 (d, 1H, J = 3.6 Hz, CH), 4.11–4.13
(m, 1H, CH), 4.21 (d, 1H, J = 4.2 Hz, CH2), 4.48 (d, 1H, J = 3.6 Hz,
CH), 4.81 (t, 1H, J = 4.2 Hz, CH), 5.83 (d, 1H, J = 3.6 Hz, H-1), 5.90
(d, 1H, J = 3.6 Hz, H-10); 13C NMR (150 MHz, CD3OD) d: 25.34,
25.39, 25.65, 25.77 (CH3), 45.99 (CH2), 63.27 (CH2), 65.27 (CH),
75.72 (CH), 76.36 (CH), 78.36 (CH), 79.19 (CH), 79.25 (CH), 85.52
(CH), 104.75 (C-1), 104.85 (C-10), 111.25 (C), 112.11 (C). HRMS
(ESI): Calcd C17H29NO9 for (M+H+): 391.5194. Found: 391.5175.
CD3OD) d: 1.24 (s, 3H, CH3), 1.38 (s, 3H, CH3), 2.14 (s, 3H, CH3),
3.07 (t, 1H, J = 9.6 Hz, CH2), 3.31 (s, 3H, OCH3), 3.37 (dd, 1H,
J = 4.2 Hz, J = 9.6 Hz, CH2), 3.48 (dd, 1H, J = 4.2 Hz, J = 9.6 Hz, CH2),
3.27(m, 1H, CH), 3.55 (t, 1H, J = 9.6 Hz, CH2), 3.69 (d, 1H, J
=9.6 Hz, CH), 3.74 (m, 1H, CH), 3.97 (m, 2H, CH), 4.25 (m, 1H,
CH), 4.44 (t, 1H, J = 3.6 Hz, CH), 4.63 (t, 1H, J = 3.6 Hz, CH), 5. 83
(d, 1H, J = 3.6 Hz, H-10);13C NMR (150 MHz, CD3OD) d: 20.44,
24.95, 25.63 (CH3), 46.34, 51.29 (CH2), 54.36 (OCH3), 71.69 (CH),
71.99 (CH), 72.16 (CH), 73.61 (CH), 74.30 (CH), 78.72 (CH), 85.17
(CH), 99.73 (C-10), 104.75 (C-1), 173.50 (O@C). HRMS (ESI): Calcd
C
17H29NO10 for (M+H+): 407.5188. Found: 407.5176.
3.3.3. N-(1,2-O-Isopropylidene-3-deoxy-a-D-allos-3-yl)-N-
3.4.2. N-(Methyl 6-deoxy- -glucopyranosid-6-yl)-N-(methyl
a
-D
(methyl 5-deoxy-b-
D
-ribofuranosid-5-yl) amine (9cb)
5-deoxy-b-D-ribofuranosid-5-yl) acetamide (10ab)
White solid; ½a 2D2
ꢁ
ꢀ16.8° (c 0.1, MeOH); 1H NMR (600 MHz,
White solid; ½a 2D2
ꢁ
ꢀ13.8° (c 0.5, MeOH); 1H NMR (400 MHz,
CD3OD) d: 1.36 (s, 3H, CH3), 1.53 (s, 3H, CH3), 2.84–2.87 (m, 2H,
CH2), 3.27–3.29 (m, 2H, CH2), 3.71 (d, 1H, J = 3.6 Hz, CH), 3.81
(dd, 1H, J = 3.0 Hz, J = 9.6 Hz, CH), 3.90 (d, J = 4.2 Hz, 1H, CH),
3.91–3.93 (m, 1H, CH); 3.96–4.02 (m, 2H, CH), 4.77 (s, 1H, H-10),
4.83 (t, 1H, J = 4.2 Hz, CH), 5.83 (d, 1H, J = 3.6 Hz, H-1); 13C NMR
(150 MHz, CD3OD) d: 25.29, 25.54 (CH3), 51.96 (CH2), 53.79
(OCH3), 59.30 (CH), 62.93 (CH2), 70.70 (CH), 73.48 (CH), 74.83
(CH), 77.15 (CH), 81.38 (CH), 81.97 (CH), 104.77 (C-1), 108.53 (C-
10), 111.88 (C). HRMS (ESI): Calcd C15H27NO9 for (M+H+):
365.4696. Found: 365.4686.
CD3OD) d: 2.20 (s, 3H, CH3), 3.05–3.14 (m, 1H, CH2), 3.35 (s, 3H,
OCH3), 3.36 (s, 3H, OCH3), 3.41–3.43 (m, 1H, CH2), 3.52–4.01 (m,
8H, CH, CH2), 4.11–4.14 (m, 1H, CH), 4.66 (d, J = 3.6 Hz, 1H, H-1),
4.74 (s, 1H, H-10); 13C NMR (100 MHz, CD3OD) d: 21.90 (CH3),
51.60, 52.08 (CH2), 55.57, 55.76 (OCH3), 72.69 (CH), 73.45 (CH),
73.63 (CH), 74.70 (CH), 75.07 (CH), 76.14 (CH), 81.97 (CH),
101.11 (C-1), 109.99 (C-10), 174.42 (O@C). HRMS (ESI): Calcd
C
15H27NO10 for (M+H+): 381.4690. Found: 381.4679.
3.4.3. N,N-Bis(methyl 6-deoxy-a-D-glucopyranosid-6-yl)
acetamide (10ac)
White solid; ½a 2D2
ꢁ
+63.2° (c 0.1, MeOH); 1H NMR (400 MHz,
3.3.4. N-(1,2-O-Isopropylidene-3-deoxy-a-D-allos-3-yl)-N-
(methyl 6-deoxy-
a
ꢁ
-
D
-glucopyranosid-6-yl) amine (9cc)
CD3OD), d: 2.22 (s, 3H, CH3), 3.05–3.15 (m, 2H, CH2), 3.36 (s, 3H,
OCH3), 3.37 (s, 3H, OCH3), 3.41–3.44 (m, 2H, H-6, CH2), 3.58–3.79
(m, 6H, CH, CH2), 3.84–3.87 (m, 1H, CH), 3.97–4.01 (m, 1H, CH),
4.66 (d, 1H, J = 3.6 Hz, H-10), 4.69 (d, 1H, J = 3.6 Hz, H-1); 13C
NMR (100 MHz, CD3OD) d: 21.83 (CH3), 48.46, 52.54 (CH2), 55.46,
55.51 (OCH3), 71.71 (CH), 72.12 (CH), 73.43 (CH), 73.62, 73.62
(CH), 73.76 (CH), 74.57 (CH), 75.07 (CH), 101.11 (C-1), 101.14 (C-
10), 174.99 (O@C). HRMS (ESI): Calcd C16H29NO11 for (M+H+):
411.5012. Found: 411.5021.
White solid; ½a 2D2
+86.7° (c 0.1, MeOH); 1H NMR (600 MHz,
CD3OD) d: 1.37 (s, 3H, CH3), 1.54 (s, 3H, CH3), 2.94 (dd, 1H,
J = 9.0 Hz, J = 12.0 Hz, CH2), 3.02 (dd, 1H, J = 3.0 Hz, J = 12.0 Hz,
CH2), 3.14 (t, 1H, J = 9.6 Hz, CH), 3.30 (dd, 1H, J = 4.2 Hz,
J = 9.6 Hz, CH2), 3.32 (m, 1H, CH2), 3.39 (dd, 1H, J = 3.6 Hz,
J = 9.6 Hz, CH2), 3.41 (s, 3H, OCH3), 3.59 (d, 1H, J = 9.0 Hz, CH),
3.63 (dd, 1H, J = 2.4 Hz, J = 9.0 Hz, CH), 3.68 (dd, 1H, J = 3.6 Hz,
J = 6.0 Hz, CH), 3.81 (dd, 1H, J = 3.0 Hz, J = 9.6 Hz, CH), 3.90 (q, 1H,
J = 3.0 Hz, CH), 4.67 (d, 1H, J = 3.6 Hz, H-10), 4.78 (t, 1H, J = 3.6 Hz,
CH), 5.81 (d, 1H, J = 4.2 Hz, H-1); 13C NMR (150 MHz, CD3OD)
d:25.25, 25.50 (CH3), 48.48 (CH2), 54.35 (OCH3), 59.76 (CH), 62.91
(CH2), 70.48 (CH), 70.78 (CH), 72.18 (CH), 72.57 (CH), 73.55 (CH),
77.27 (CH), 82.13 (CH), 99.87 (C-1), 104.709 (C-10), 112.08 (C).
3.4.4. N-(Methyl 5-deoxy-b-D-ribofuranosid-5-yl)-N-(1,2-O-
isopropylidene-5-deoxy-a-D-xylos-5-yl) acetamide (10ba)
Colorless oil; ½a D22
ꢁ
ꢀ16.8° (c 0.1, MeOH); 1H NMR (600 MHz,
CD3OD) d: 1.31 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.20 (s, 3H, CH3),

P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
21
3.39 (s 3H CH3), 3.52 (dd, 1H, J = 9.0 Hz, J = 15.6 Hz, CH2), 3.57 (dd,
1H, J = 7.2 Hz, J = 14.4 Hz, CH2), 3.84–3.87 (m, 1H, CH), 3.88–4.11
(m, 5H, CH, CH2), 4.31–4.33 (m, 1H, CH), 4.49 (d, 1H, J = 3.6 Hz,
CH), 4.78 (s, 1H, H-10), 5.89 (d, 1H, J = 3.6 Hz, H-1); 13C NMR
(150 MHz, CD3OD) d: 20.40, 24.96, 25.62 (CH3), 45.72 (CH2),
53.45 (OCH3), 54.61 (CH), 72.75 (CH2), 74.29 (CH), 74.29 (CH),
78.82 (CH), 81.41 (CH), 85.16 (CH), 104.78 (C-10), 108.98 (C-1),
111.28 (C), 173.00 (O@C). HRMS (ESI): Calcd C16H27NO9 for
(M+H+): 377.4866. Found: 377.4881.
1H, J = 5.4 Hz, CH), 3.90 (m, 2H, CH, CH2), 3.95 (m, 1H, CH), 4.20
(m, 1H, CH), 4.45 (dd, 1H, J = 4.2 Hz, J = 8.4 Hz, CH), 4.66 (dd, 1H,
J = 5.4 Hz, J = 10.2 Hz, CH), 4.75 (t, 1H, J = 4.2 Hz, CH), 4.80 (s, 1H,
CH, H-10), 5.80 (d, 1H, J = 3.0 Hz, H-1); 13C NMR (150 MHz, CD3OD)
d: 21.27, 25.26, 25.73 (CH3), 48.88 (CH2), 54.75 (OCH3), 60.13 (CH),
62.91 (CH2), 70.85 (CH), 72.11 (CH), 72.80 (CH), 73.39 (CH), 73.68
(CH), 79.01 (CH), 99.79 (C-1), 103.77 (C-10), 112.65 (C), 170.18
(O@C). HRMS (ESI): Calcd C18H31NO11 for (M+H+): 437.5510.
Found: 437.5523.
3.4.5. N,N-Bis(methyl 5-deoxy-b-
D
-ribofuranosid-5-yl)
3.5.3. N-(1,2-O-Isopropylidene-3-deoxy-a-D-allos-3-yl)-N-
acetamide (10bb)
(methyl 6-deoxy-
a
ꢁ
-
D
-glucopyranosid-6-yl) acetamide (10cc)
White solid; ½a 2D2
ꢁ
+22.8° (c 0.1, MeOH); 1H NMR (400 MHz,
White solid; ½a 2D3
+70.0° (c 0.1, MeOH); 1H NMR (600 MHz,
CD3OD) d: 2.20 (s, 3H, CH3), 3.45 (s, 6H, OCH3), 3.49 (dd, 1H,
J = 12.0 Hz, J = 21.0 Hz, CH2), 3.63 (dd, 1H, J = 12.6 Hz, J = 23.4 Hz,
CH2), 3.88 (m, 3H, CH, CH2), 3.93–4.01 (m, 3H, CH), 4.09–4.17 (m,
2 H, CH), 4.75 (s, 1H, H-1), 4.77 (s, 1H, H-10);13C NMR (100 MHz,
CD3OD) d: 21.90 (CH3), 51.63 (C-5), 54.67 (C-5), 55.95, 56.15
(OCH3), 74.04 (CH), 74.72 (CH), 75.79 (CH), 76.23 (CH), 81.90
(CH), 82.51 (CH), 110.14, 110.44 (C-1), 173.91 (O@C). HRMS
(ESI): Calcd C14H25NO9 for (M+H+): 351.4368. Found: 351.4371.
CD3OD) d: 1.31 (s, 3H, CH3), 1.57 (s, 3H, CH3), 2.26 (s, 3H, CH3),
3.10 (t, 1H, J = 8.4 Hz, CH2), 3.37 (s, 3H, OCH3), 3.38 (m, 2H, CH2),
3.51 (m, 4H, CH, CH2), 3.64 (m, 2H, CH), 4.28 (t, 1H, J = 9.0 Hz,
CH), 4.54 (m, 1H, CH), 4.67 (dd, 1H, J = 3.6 Hz, J = 10.8 Hz, CH),
4.79 (t, 1H, J = 4.2 Hz, CH), 5.77 (d, 1H, J = 4.2 Hz, H-1); 13C NMR
(150 MHz, CD3OD) d: 21.33, 25.04, 25.18, 25.66, 25.69 (CH3),
46.10 (CH2), 60.14 (OCH3), 62.84 (CH), 74.08 (CH2), 74.72 (CH),
76.47 (CH), 79.15 (CH), 80.78 (CH), 85.17 (CH), 104.40 (C-1),
104.49 (C-10), 173.57 (O@C). HRMS (ESI): Calcd C19H31NO10 for
(M+H+): 433.5686. Found: 433.5671.
3.5. General procedure for the synthesis of compounds 10ca–
10cc
3.6. Biological activity assays
The compounds 6ca–6cc were dissolved in dry CH2Cl2, Et3N
(5.0 equiv) and AcCl (5.0 equiv) were added, the reaction mixture
was stirred at 0 °C till the completion of reaction (checked by
TLC). Excess reagent was decomposed with saturated aqueous
NaHCO3 solution and the mixture was extracted with ethyl acetate.
The combined extracts were washed with water, dried over MgSO4
and evaporated, and the residue was dissolved in dry methanol, so-
dium methoxide (1.0 equiv) was added, then the reaction mixture
was stirred at room temperature; Ion exchange resin was added to
adjust the pH of the solution to 7 after the completion of reaction
(checked by TLC). The resin was filtered off and methanol was
evaporated under reduced pressure. The crude product was puri-
fied by column chromatography (ethyl acetate/methanol (v/
v = 10:1)) to afford a colorless oil compound, then dissolved in
60% AcOH, the mixture was stirred at 45 °C, till the completion of
reaction (checked by TLC), The solvent was evaporated under re-
duced pressure to afford a crude product then purified by silic
gel flash column chromatography (ethyl acetate/methanol (v/
v = 8:1)) to afford compounds 10ca–10cc.
3.6.1. Inhibition of glycosidases
The inhibitory activity of the synthesized compounds against
amylase, -glucosidase and b-glucosidase: -glucosidase (yeast)
and b-glucosidase (almonds) were obtained from Fluka; -amylase
(Bacillaceae, 4000 U/mg) from Sanland-chen International Inc, Xia-
men, China. Two substrates, p-nitrophenyl -glucopyranoside
-salicin, were purchased from Sigma Chemical
a-
a
a
a
a
(PNPG) and (ꢀ)-
D
Co. All other commercial reagents were used as received. Each en-
zyme assay was measured as follows.
The
a-amylase assay was performed using 1% starch as sub-
strate in phosphate buffer, pH 6.0, at 50 °C. The enzyme solution
(0.1 mL, 5 mg of solid enzyme in 50 mL of pH 6.0 phosphate buf-
fer), 0.1 mL of inhibitor (1 mg/mL) and 1 mL of buffer were incu-
bated for 10 min, and then 1 mL of substrate was added. After
10 min, 2 mL of 3,5-dinitrosalicylic acid was added, and then the
reaction was heated in boiling water for 5 min. The solution was fi-
nally diluted to 20 mL after cooling down. Absorbance readings
were taken on a TU-1901 UV–vis spectrophotometer at 540 nm
using distilled deionized water as a blank control and acarbose
as a positive control.
3.5.1. N-(1,2-O-Isopropylidene-3-deoxy-
a
-
D
-allos-3-yl)-N-(1,2-
-xylos-5-yl) acetamide (10ca)
+50.4° (c 0.5, MeOH); 1H NMR (600 MHz,
O-isopropylidene-5-deoxy-
a
-D
The b-glucosidase assay was performed using (ꢀ)-
D-salicin
Colorless oil; ½a D22
ꢁ
(2 mg/mL) as the substrate in phosphate buffer, pH 4.8, at 35 °C.
The enzyme solution (0.1 mL, 10 mg of solid enzyme in 10 mL of
pH 4.8 acetate buffer), 0.1 mL of inhibitor (1 mg/mL), and 0.9 mL
of buffer were incubated for 10 min, and then 0.8 mL of substrate
was added. After 10 min, 2 mL of 3,5-dinitrosalicylic acid was
added, and then the reaction mixture was heated in boiling water
for 5 min. The solution was finally diluted to 20 mL after cooling
down. Absorbance readings were taken on a TU-1901 UV–vis spec-
trophotometer at 540 nm using distilled deionized water as a blank
control.
CD3OD) d: 1.33 (s, 3H, CH3), 1.58 (s, 3H, CH3), 2.26 (s, 3H, CH3),
3.43 (s, 3H, CH3), 3.55–3.59 (m, 1H, CH2), 3.74 (m, 1H, CH2), 3.85
(m, 1H, CH2), 3.93 (dd, J = 3.0 Hz, J = 15.6 Hz, 1H, CH2), 4.09 (dd,
1H, J = 2.4 Hz, J = 9.6 Hz, CH), 4.39 (m, 1H, CH), 4.45 (dd, 1H,
J = 5.4 Hz, J = 9.0 Hz, CH), 4.48 (m, 2H, CH), 4.56 (dd, J = 5.4 Hz,
J = 9.0 Hz, 1H, CH), 4.78 (t, 1H, J = 4.8 Hz, CH), 5.83 (d, 1H,
J = 3.6 Hz, H-1), 5.92 (d, 1H, J = 3.6 Hz, H-10); 13C NMR (150 MHz,
CD3OD) d: 21.36, 25.07, 25.71 (CH3), 51.97 (CH2), 55.13 (OCH3),
60.14 (CH), 62.67 (CH2), 73.23 (CH), 73.59 (CH), 74.76 (CH), 79.83
(CH), 80.86 (CH), 81.82 (CH), 103.92 (C-1), 108.90 (C-10), 112.73
(C). HRMS (ESI): Calcd C17H29NO10 for (M+H+): 407.5188. Found:
407.5175.
The a-glucosidase assay was performed using PNPG (1 mg/mL)
as substrates in phosphate buffer, pH 6.8, at 37 °C. The enzyme
solution (0.1 mL, 10 mg of solid enzyme in 10 mL of pH 6.8 phos-
phate buffer), 0.1 mL of inhibitor (1 mg/mL), 1.9 mL of buffer, and
0.05 mL glutathione (reduced, 1 mg/mL) were incubated for
10 min, and then 0.15 mL of substrate was added. The reaction
was quenched with 10 mL of sodium carbonate (0.1 mol/L) after
10 min, and the solution was finally diluted to 20 mL after cooling
down. Absorbance readings were taken on a TU-1901 UV–vis
3.5.2. N-(1,2-O-Isopropylidene-3-deoxy-a-D-allos-3-yl)-N-
(methyl 5-deoxy-b- -ribofuranosid-5-yl) acetamide (10cb)
White solid; ½aꢁ2DD2 +51.4° (c 0.1, MeOH); 1H NMR (600 MHz,
CD3OD) d: 1.33 (s, 3H, CH3), 1.58 (s, 3H, CH3), 2.26 (s, 3H, CH3),
3.43 (s, 3H, OCH3), 3.57 (m, 1H, CH2), 3.70 (m, 2H, CH2), 3.79 (q,

22
P. Zhang et al. / Carbohydrate Research 372 (2013) 15–22
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The cytotoxicity of the compounds against HeLa cell lines (hu-
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thelial cell line (A-549) was examined by the modified
Mosmann’s protocol as follows: Briefly, cells (104 cells per well)
were plated in 96-well culture plates and cultured overnight at
37 °C in a 5% CO2 humidified incubator. Compounds were added
to the wells at final concentrations of 1, 10 and 100 lmol/L. Control
wells were prepared by the addition of DMEM. Wells containing
DMEM without cells were used as blanks. The plates were incu-
bated at 37 °C in a 5% CO2 incubator for 48 h. Upon completion
of the incubation, stock MTT dye solution (10
added to each well. After 4 h of incubation, the supernatant was re-
moved and dimethyl sulfoxide (DMSO) (100 L) was added to dis-
lL, 5 mg/mL) was
l
solve the MTT. The optical density of each well was measured on a
microplate spectrophotometer at a wavelength of 570 nm. The
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Acknowledgments
The financial supports from the 973 Program (2010CB534913),
the National Natural Science Foundation of China (20972039,
21172051), the Natural Science Foundations of the Department
of Education of Hebei (12966417D, Y2011119, and ZH2011110),
the Natural Science Foundations of Hebei University (2009-175)
and open research fund of the State Key Laboratory of Natural
and Biomimetic Drugs, Peking University (20080205) are gratefully
acknowledged.
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