Synthesis of N-substituted iminosugar derivatives and their immunosuppressive activities

Article abstract of DOI:10.1016/j.carres.2010.01.021

Several N-alkyl and hydroxyethyl-substituted iminosugar derivatives, including l-altro and d-galacto epimers, were synthesized and the effects of these synthetic iminosugars on proliferation of mouse splenocytes were evaluated in the MTT assay. The experi

Full text of DOI:10.1016/j.carres.2010.01.021

Carbohydrate Research 345 (2010) 780–786
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of N-substituted iminosugar derivatives and their
immunosuppressive activities
a,c,
Guo-Liang Zhang a,b,c, Changshui Chen b, Yulan Xiong c, Li-He Zhang a,c, Jia Ye c, , Xin-Shan Ye
*
*
a State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Xue Yuan Road #38, Beijing 100191, China
b College of Basic Sciences, Huazhong Agricultural University, Wuhan 430070, China
c School of Pharmaceutical Sciences, Peking University, Xue Yuan Road #38, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Several N-alkyl and hydroxyethyl-substituted iminosugar derivatives, including L-altro and D-galacto
Received 15 December 2009
Received in revised form 26 January 2010
Accepted 29 January 2010
Available online 4 February 2010
epimers, were synthesized and the effects of these synthetic iminosugars on proliferation of mouse
splenocytes were evaluated in the MTT assay. The experimental data demonstrated that the N-alkylated
D
-galacto iminosugars showed better inhibitory activities than
L-altro analogues, which might hold poten-
tial as immunosuppressive agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Iminosugar
Synthesis
Alkylation
Immunosuppressive agent
MTT assay
1. Introduction
OH
HO
OH
HO
OH
R
R
N
N
The currently clinically used small-molecule immunosuppres-
sive agents, such as cyclosporin A (CyA), sirolimus (Rapamycin),
tacrolimus (FK506), and mycophenolate mofetil (MMF), have seri-
ous side effects including nephrotoxicity, neurotoxicity, infection,
new onset post-transplant diabetes mellitus, hyperlipidemia, and
hypertension. Furthermore, so far there is no antidotefor the toxicity
of these organ transplantation drugs. To improve transplant survival
and reduce the toxicity of current agents, there is a great need to find
highly efficacious immunosuppressants with low toxicity.1–4
Iminosugars, also known as the ‘sugar-shaped alkaloids’, have
been found to be potent inhibitors of many carbohydrate-process-
ing enzymes (i.e., glycosidases and glycosyltransferases).5–8 Be-
cause these enzymes are frequently involved in essential
biological processes,9,10 iminosugars have great potential as thera-
peutic agents in many diseases such as cancer,11 diabetes,12 viral
infections,13 and lysosomal storage disorders.14,15 However, the
inhibition effects of iminosugars on immune system responses
and their application as immunosuppressants have been less ex-
plored. To date only castanospermine, a naturally occurring indo-
lizidine alkaloid, has been found to exhibit immunosuppressive
activity.16 Our preliminary results disclosed that some synthetic
iminosugars (e.g., 1 and 2, Fig. 1) displayed immunosuppressive
OH
OH
HO
OH
OH
1: R=H
2: R=H
3: R=(CH2)3CH3
4: R=(CH2)5CH3
5 :R=(CH2)8CH3
9: R=(CH2)2OH
6: R=(CH2)3CH3
7: R=(CH2)5CH3
8: R=(CH2)8CH3
10: R=(CH2)2OH
Figure 1. Structures of iminosugars 1 and 2, and their derivatives 3–10.
activity.17,18 Encouraged by these experiments, to further under-
stand the structure–activity relationships (SARs) of this type of
compounds and to find iminosugars with better activities, we re-
port herein the synthesis of several N-alkylated iminosugar deriv-
atives and the evaluation of their immunosuppressive activities by
the MTT assay.
2. Results and discussion
2.1. Design
Nojirimycin (NJ) and 1-deoxynojirimycin (DNJ), isolated from
microorganism cultures and/or plants, are competitive inhibitors
of N-glycan-processing enzymes (Fig. 2).19,20 N-Alkylated
* Corresponding authors. Tel.: +86 10 82801570; fax: +86 10 62014949.
E-mail addresses: yejia@bjmu.edu.cn (J. Ye), xinshan@bjmu.edu.cn (X.-S. Ye).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.01.021

G.-L. Zhang et al. / Carbohydrate Research 345 (2010) 780–786
781
OH
OH
OH
OH OH
R
H
R
CH2CH2OH
N
N
N
N
HO
HO
HO
HO
HO
HO
HO
OH
OH
OH
OH
(DGJ)
OH
R=H
(DNJ)
R=H
R=nNon (NN-DGJ)
Miglitol (Glyset)
NJ
R=nBn (NB-DNJ)
Figure 2. Some emblematic iminosugars.
deoxynojirimycin and deoxygalactonojirimycin (DGJ) span a wide
range of biological activities. For instance, N-butyl-1-deoxynojiri-
mycin (NB-DNJ) has been approved as medicine for type I Gaucher
disease, the most common lysosomal storage disorder. In addition,
NB-DNJ was evaluated in Phase II clinical trials as an anti-HIV
agent.21,22 N-Hydroxyethyl-DNJ (miglitol) served as an oral drug
for the treatment of type II diabetes taken either alone or in com-
bination with other anti-diabetic medicines.23–25 In addition, the
galactose analogue N-nonyl-1-deoxygalactonojirimycin (NN-DGJ)
was in clinical trials as an anti-HBV and an anti-HCV agent.26,27
Based on our previous report,17 to investigate whether the N-alkyl-
ated side chain influences the immunosuppressive activity or not,
we designed some N-alkylated iminosugar derivatives 3–10 with
side chains consisting of four-, six- or nine-carbons, or a hydroxy-
ethyl group (Fig. 1).
from 2,3,4,6-tetra-O-benzyl-D-galactopyranose according to a pro-
cedure described previously.17 The alcohol 11 was treated with so-
dium methoxide to provide 12 smoothly, which was further
treated with 50% aqueous potassium hydroxide to cleave the
protecting group on the amino functionality, affording 13.28 The
N-alkylated side chains were successfully introduced to the imino-
sugar moiety by reductive amination29,30 leading to compounds 14,
15, and 16 in 70%, 89%, and 65% yield, respectively, based on the
recovered starting materials. Debenzylation of 14–16 under a
hydrogen atmosphere over Pd–C provided iminosugars 3–5 in
quantitative yield. In a similar way, iminosugar derivatives 6–8
were prepared from the corresponding D-galacto alcohol 17. Inter-
estingly, when the above-mentioned reductive amination condi-
tions were applied to the preparation of compounds 20, 21, and
22 from 19, the reaction gave a low yield (less than 40%). However,
when using ethanol instead of methanol as the solvent, satisfactory
yields (80–86%) were obtained. Finally, it is noteworthy that it was
not easy to obtain pure samples of iminosugars 6 and 7. The crude
product must be successively subjected to silica gel column
chromatography (CH2Cl2–MeOH as the eluent), C-18 reverse-phase
2.2. Chemistry
The preparation of iminosugars 3–8 is shown in Scheme 1. The
key materials, cyclized isomeric alcohols 11 and 17, were obtained
OBn
OBn
OBn
O
Cbz
H
N
N
N
a
b
BnO
BnO
BnO
BnO
BnO
90%
BnO
BnO
BnO
13
90%
BnO
O
OH
OH
11
12
OBn
OH
CH2(CH2)nCH3
OH
CH2(CH2)nCH3
N
N
c
d
BnO
BnO
HO
HO
BnO
OH
OH
3
:
:
n=2 (100%)
n=4 (100%)
14 : n=2 (70%)
15 : n=4 (89%)
16 : n=7 (65%)
4
5 : n=7 (100%)
OBn
OBn
OBn
OBn
OBn
OBn
O
H
Cbz
N
N
N
b
a
BnO
BnO
BnO
85%
90%
BnO
BnO
19
BnO
O
OH
OH
17
18
OH OH
OBn
OBn
CH2(CH2)nCH3
OH
CH2(CH2)nCH3
OH
N
N
c
d
HO
BnO
OH
BnO
6 : n=2 (90%)
7 : n=4 (92%)
8 : n=7 (100%)
20 : n=2 (80%)
21 : n=4 (86%)
22 : n=7 (80%)
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, rt; (b) 50% KOH, MeOH, 80 °C; (c) CH3(CH2)nCHO, MeOH or EtOH, HOAc then NaBH3CN, 80 °C; (d) Pd/C, H2, H2O/THF/
HOAc = 4:2:1.

782
G.-L. Zhang et al. / Carbohydrate Research 345 (2010) 780–786
column chromatography (H2O as eluent), and ion-exchange resin
column chromatography.
Starting from compound 13 and its isomer 19, the miglitol
derivatives 9 and 10 were obtained (Scheme 2). Initially, com-
pound 13 was treated with BrCH2CH2OH and K2CO3 in DMF in
the hope of getting 24, but no product was obtained. Although
many other bases and solvent systems such as THF/K2CO3, THF/
NaOH/tetra-n-butylammonium iodide (TBAI), and THF/Et3N/TBAI
were evaluated, all these efforts failed to give the desired product.
Eventually, treatment of 13 with 40% glyoxal aqueous solution
afforded the lactone 23 in 74% isolated yield.31 The carbonyl group
of 23 was subsequently reduced by LiAlH4, providing tetra-O-ben-
zyl-protected iminosugar derivative 24. It was noteworthy that
galacto iminosugar intermediates 25 and 26 were less stable than
their corresponding -altro isomers. Final deprotection of 24 by cat-
D-
L
alytic hydrogenolysis yielded the target compound 9, which was
purified by silica gel column chromatography, C-18 reverse-phase
column chromatography, and ion-exchange resin. Iminosugar 10
was prepared in the same manner as described for the preparation
of 9.
Figure 3. Effects of compounds 1–10 on concanavalin A-induced mouse spleno-
cytes proliferation were assessed by the MTT assay. Values are mean SEM. ***p
<0.001 versus control.
2.3. Biology
With all the synthetic compounds in hand, the effect of the N-
substituted iminosugars 3–10 and the N-unsubstituted iminosug-
ars 1–217 on concanavalin A (Con A)-induced proliferation of
splenocytes in mice were assessed by the MTT assay.32 The spleno-
configuration of the hydroxymethyl group was important to im-
prove the inhibitory activity. However, the mechanisms of these
differences are not understood.
cytes were induced by 2.5 lg/mL of concanavalin A with 30 lM
concentration of the iminosugars at 37 °C, 5% CO2 for 48 h, using
the Con A-treated splenocytes as the experimental control. Com-
pared with the control, the levels of cell proliferation were reduced
25.4%, 16.5%, 21.6%, 17.9%, 26.6%, 35.8%, 38.3%, 27.7%, 18.4%, and
3. Conclusions
N-Alkylated iminosugars 3–8 and miglitol derivatives 9 and 10
were designed and synthesized, and their immunosuppressive
18.2% when including 30
As shown in Figure 3, iminosugars 6 and 7 displayed the stron-
gest inhibition effects. The N-alkylation of -galacto iminosugar 2
lM of compounds 1–10, respectively.
activities were evaluated. It was clear that N-alkylation of the
lacto iminosugar gave rise to better inhibitory activities than that
of the -altro iminosugar epimer, and the -galacto iminosugar
D-ga-
D
L
D
was important for the improvement of the inhibitory activity and
the immunosuppressive effects were increased by 1.7–2.3-fold
(compared compounds 6–8 to 2). The six-carbon side chain
showed the best inhibitory effect among the three N-substituted
derivative with N-substituted six-carbon side chain showed the
strongest inhibitory activity. With this information, our future
work will focus on the preparation and evaluation of more N-
substituted D-galacto iminosugar derivatives.
iminosugars. However, in the case of the L-altro iminosugar epi-
mers, N-alkylation did not improve the activity (compared com-
pounds 3–5 to 1). Comparing compounds 6–8 with 10, it seemed
that iminosugars with N-substituted non-polar side chains showed
better inhibitory activity than that with polar chains. This suggests
that the hydrophobic character may be important to the immuno-
4. Experimental
4.1. General methods
All chemicals were purchased and used without further
purification. Tetrahydrofuran (THF) was distilled over sodium/
suppressive activity. By comparison of the inhibition of the
D-epi-
mer 7 (38.3%) with -epimer 4 (17.9%), it was clear that the
L
OH
HO
OBn
BnO
OBn
BnO
OBn
(CH2)2OH
OH
(CH2)2OH
H
H
N
O
N
N
N
c
a
b
BnO
BnO
OH
74%
57%
BnO
90%
BnO
BnO
13
HO
BnO
BnO
OH
O
OH
9
23
24
OBn
OBn
OH
OBn
OBn
OBn
OH
OBn
O
(CH2)2OH
OH
(CH2)2OH
OH
N
N
N
N
c
a
b
BnO
BnO
HO
BnO
BnO
19
BnO
OH
10
55%, three steps
BnO
OH
O
26
25
Scheme 2. Reagents and conditions: (a) glyoxal, MeOH/HOAc (v/v) = 200:1, NaBH4, 80 °C; (b) LiAlH4, THF, rt; (c) Pd/C, H2, H2O/THF/HOAc = 4:2:1.

G.-L. Zhang et al. / Carbohydrate Research 345 (2010) 780–786
783
benzophenone and methylene chloride (CH2Cl2) over calcium hy-
dride. The reactions were monitored with analytical TLC on Silica
Gel 60-F254-precoated aluminum plates and visualized under UV
(254 nm) and/or by staining with acidic ceric ammonium molyb-
date. Column chromatography was performed on silica gel (35–
tion (0.2 mL). The mixture was extracted with EtOAc (3 ꢀ 30 mL)
and washed with aqueous NaHCO3 (10 mL) and aqueous NaCl
(10 mL). The organic phases were combined, dried (Na2SO4), filtered,
and concentrated. Theresidue was purified by column chromatogra-
phy on silica gel (petroleum ether–EtOAc 4:1) to provide 14 (59 mg,
38%) as a yellow oil. The material 13 (63 mg) was recovered and the
conversion was 70%. 1H NMR (500 MHz, CDCl3): d 0.85 (t, 3H,
J = 7.5 Hz, –CH3), 1.15–1.45 (m, 4H, –CH2–), 2.81–2.93 (m, 2H, –
NCH2–), 3.10 (q, 1H, J = 5.0, 11.0 Hz, H-2), 3.20 (q, 1H, J = 5.5,
10.5 Hz, H-6), 3.41–3.46 (m, 2H, H-1, H-7), 3.53 (dd, 1H, J = 4.5,
10.0 Hz, H-7), 3.65 (dd, 1H, J = 2.5, 7.0 Hz, H-4), 3.75 (dd, 1H,
J = 6.5, 11.5 Hz, 1H), 3.88 (dd, 1H, J = 2.5, 5.5 Hz, H-5), 3.98 (dd, 1H,
J = 4.5, 7.0 Hz, H-3), 4.41 (d, 1H, J = 12.0 Hz), 4.47–4.53 (m, 5H),
4.56 (d, 1H, J = 12.5 Hz), 4.61 (d, 1H, J = 12.0 Hz), 7.22–7.32 (m,
20H). 13C NMR (125 MHz, CDCl3): d 13.95 (–CH3), 20.21 (–CH2–),
28.05 (–CH2–), 53.08 (–NCH2–), 59.05 (C-2 and C-6), 61.26 (C-1),
68.94 (C-7), 71.92, 72.33, 72.91, 73.11 (4 ꢀ –CH2Ph), 74.09 (C-4),
75.30 (C-5), 77.00 (C-3), 127.46, 127.58, 127.63, 127.79, 127.86,
127.91, 128.03, 128.22, 128.25, 128.39, 137.98, 138.07, 138.48,
138.58. MS-ESI: 610 [M+H]+. Anal. Calcd for C39H47NO5: C, 76.82;
H, 7.77; N, 2.30. Found: C, 77.06; H, 7.56; N, 2.36.
75 lm). NMR spectra were recorded on a Varian VXR-300M or Var-
ian INOVA-500M spectrometer. Mass spectra were recorded using
a PE SCLEX QSTAR spectrometer. Elemental analysis data were re-
corded on PE-2400C elemental analyzer. Concanavalin A was pur-
chased from Sigma. Other reagents were from commercial sources.
4.2. 1,3,4,5-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-
altro-heptitol O (7), N-cyclic carbamate (12)
D-glycero-L-
To a solution of 1117 (210 mg, 0.31 mmol) in MeOH (15 mL) was
added sodium methoxide (30% solution in MeOH) (0.5 mL,
0.009 mmol). The reaction mixture was stirred for 1 h at room tem-
perature. MeOH was removed under reduced pressure, and the res-
idue was extracted with EtOAc (3 ꢀ 20 mL). The organic phases
were combined, dried (Na2SO4), filtered, and concentrated. The res-
idue was purified by column chromatography on silica gel (petro-
leum ether–EtOAc 4:1) to provide 12 (159 mg, 90%) as a yellow oil.
1H NMR (500 MHz, CDCl3): d 3.42 (d, 1H, J = 3.0 Hz), 3.80–3.81 (m,
1H), 3.87–3.91 (m, 1H), 3.98 (dd, 1H, J = 2.5, 9.5 Hz), 4.11–4.18 (m,
3H), 4.27 (dd, 1H, J = 3.5, 10.0 Hz), 4.33–4.37 (m, 2H), 4.41 (d, 1H,
J = 12.0 Hz,), 4.43 (d, 1H, J = 12.5 Hz), 4.47 (d, 1H, J = 12.0 Hz),
4.51 (d, 1H, J = 11.5 Hz), 4.66 (s, 2H), 4.74 (d, 1H, J = 12.0 Hz)
(8 ꢀ –CH2Ph), 7.19–7.43 (m, 20H). 13C NMR (75 MHz, CDCl3): d
54.53, 55.05, 62.06, 64.65, 72.14, 72.37, 73.00, 73.07, 73.15, 73.51
(4 ꢀ –CH2Ph), 74.58, 127.37, 127.75, 128.03, 128.09, 128.16,
128.36, 128.47, 137.30, 137.67, 137.92, 138.42 (Ph), 156.14 (CO).
HRMS (ESI, positive) Calcd for C36H38NO6: 580.2694 [M+H]+.
Found: 580.2705.
4.5. 3,4,5,7-Tetra-O-benzyl-N-hexyl-2,6-dideoxy-2,6-imino-
glycero- -altro–heptitol (15)
D-
L
Compound 15 was prepared from 13 (500 mg, 0.9 mmol) as de-
scribed in the preparation of 14, providing 15 (340 mg, 59%) as a
yellow oil. The material 13 (170 mg) was recovered and the con-
version was 89%. 1H NMR (500 MHz, CDCl3): d 0.86 (t, 3H,
J = 7.5 Hz, –CH3), 1.14–1.42 (m, 8H, –CH2–), 2.80–2.92 (m, 3H, –
OH, –NCH2–), 3.10 (q, 1H, J = 6.0, 10.5 Hz, H-2), 3.20 (q, 1H,
J = 5.0, 10.5 Hz, H-6), 3.43 (dd, 1H, J = 5.5, 9.5 Hz, H-7), 3.40–3.47
(m, 1H, H-1), 3.54 (dd, 1H, J = 5.0, 10.0 Hz, H-7), 3.64 (dd, 1H,
J = 2.5, 6.5 Hz, H-4), 3.74 (dd, 1H, J = 6.5, 11.0 Hz, 1H), 3.87 (dd,
1H, J = 3.0, 6.0 Hz, H-5), 3.97 (dd, 1H, J = 4.5, 7.0 Hz, H-3), 4.41 (d,
1H, J = 12.0 Hz), 4.47 (d, 1H, J = 11.5 Hz), 4.49–4.52 (m, 4H), 4.55
(d, 1H, J = 12.0 Hz), 4.59 (d, 1H, J = 11.5 Hz), 7.17–7.32 (m, 20H).
13C NMR (125 MHz, CDCl3): d 14.00 (–CH3), 22.62, 25.77, 26.75,
31.68 (4 ꢀ –CH2–), 53.27 (–NCH2–), 58.99 (C-6), 59.05 (C-2),
61.32 (C-1), 68.93 (C-7), 71.96, 72.38, 72.92, 73.15 (4 ꢀ –CH2Ph),
74.05 (C-4), 75.31 (C-5), 77.00 (C-3), 127.50, 127.62, 127.67,
127.84, 127.91, 127.94, 128.07, 128.26, 128.42, 138.00, 138.07,
138.49, 138.60 (Ph). HRMS (ESI, positive) Calcd for C41H52NO5:
638.3840 [M+H]+. Found: 638.3843.
4.3. 3,4,5,7-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-
altro-heptitol (13)
D-glycero-L-
To a solution of 12 (220 mg, 0.38 mmol) in MeOH (15 mL) was
added 50% aqueous KOH (15 mL). The reaction mixture was stirred
for 20 h at 80 °C. MeOH was removed under reduced pressure, and
the residue was extracted with EtOAc (3 ꢀ 50 mL). The organic
phases were combined, dried (Na2SO4), filtered, and concentrated.
The residue was purified by column chromatography on silica gel
(petroleum ether–EtOAc 1:1) to provide 13 (190 mg, 90%) as a
white solid. 1H NMR (500 MHz, CDCl3): d 2.03 (br s, 2H, –OH and
–NH), 3.15–3.21 (m, 2H, H-2, H-6), 3.50 (dd, 1H, J = 1.5, 3.5 Hz,
H-5), 3.53 (dd, 1H, J = 5.5, 11.0 Hz, H-7), 3.61–3.66 (m, 2H, H-7,
H-1), 3.77 (dd, 1H, J = 3.0, 10.0 Hz, H-3), 3.78–3.82 (m, 2H, H-1,
H-4), 4.30 (d, 1H, J = 12.0 Hz), 4.37 (d, 1H, J = 12.5 Hz), 4.39 (br s,
2H), 4.48 (d, 1H, J = 12.0 Hz), 4.51 (d, 1H, J = 12.0 Hz), 4.56 (d, 1H,
J = 12.0 Hz), 4.70 (d, 1H, J = 12.0 Hz), 7.16–7.35 (m, 20H). 13C
NMR (125 MHz, CDCl3): d 54.17 (C-2), 54.79 (C-6), 63.59 (C-7),
69.91 (C-1), 71.79 (CH2Ph), 71.90 (CH2Ph), 72.26 (C-4), 72.93
(CH2Ph), 73.26 (CH2Ph), 74.93 (C-3), 76.75 (C-5), 127.50, 127.69,
127.81, 127.93, 127.98, 128.12, 128.30, 128.35, 137.90, 138.37,
138.42, 138.53 (Ph). HRMS (ESI, positive) Calcd for C35H40NO5:
554.2901 [M+H]+. Found: 554.2900.
4.6. 3,4,5,7-Tetra-O-benzyl-N-nonyl-2,6-dideoxy-2,6-imino-
glycero- -altro–heptitol (16)
D-
L
Compound 16 was prepared from 13 (390 mg, 0.71 mmol) as
described in the preparation of 14, providing 16 (190 mg, 40%) as
a yellow oil. The material 13 (150 mg) was recovered and the con-
version was 65%. 1H NMR (300 MHz, CDCl3): d 0.88 (t, 3H,
J = 7.2 Hz, –CH3), 1.23–1.27 (m, 14H, –CH2–), 2.82–2.89 (m, 2H, –
NCH2–), 3.10 (q, 1H, J = 5.1 Hz, H-2), 3.19 (q, 1H, J = 5.4 Hz, H-6),
3.40–3.47 (m, 2H, H-7, H-1), 3.54 (dd, 1H, J = 4.8, 9.9 Hz, H-7),
3.65 (dd, 1H, J = 2.7, 6.9 Hz, H-4), 3.74 (dd, 1H, J = 6.6, 11.4 Hz, H-
1), 3.87 (dd, 1H, J = 2.7, 6.0 Hz, H-5), 3.96 (dd, 1H, J = 4.5, 6.9 Hz,
H-3), 4.42 (d, 1H, J = 12.0 Hz), 4.44–4.55 (m, 6H), 4.60 (d, 1H,
J = 11.4 Hz), 7.23–7.30 (m, 20H). 13C NMR (75 MHz, CDCl3): d
14.09 (–CH3), 22.64, 25.70, 27.11, 29.24, 29.52, 29.59, 31.85
(7 ꢀ –CH2–), 53.14 (–NCH2–), 58.91 (C-6), 58.98 (C-2), 61.35 (C-
1), 68.85 (C-7), 71.94, 72.38, 72.89, 73.14 (4 ꢀ –CH2Ph), 73.98 (C-
4), 75.29 (C-5), 77.09 (C-3), 127.51, 127.68, 127.92, 128.09,
128.26, 128.43, 138.00, 138.05, 138.48, 138.59 (Ph). MS-ESI: 680
[M+H]+. Anal Calcd for C44H57NO5: C, 77.72; H, 8.45; N, 2.06.
Found: C, 77.88; H, 8.67; N, 1.97.
4.4. 3,4,5,7-Tetra-O-benzyl-N-butyl-2,6-dideoxy-2,6-imino-
glycero- -altro-heptitol(14)
D-
L
To a solution of 13 (140 mg, 0.25 mmol) in MeOH and acetic acid
(v/v 200:1, 10 mL) was added CH3(CH2)2CHO (160 L, 1.75 mmol).
l
After stirring for 1 h at 60 °C, a portion of NaBH3CN (80 mg,
1.25 mmol) was added. The reaction mixture was stirred overnight
at 80 °C and the reaction was quenched with 1 N HCl aqueous solu-

784
G.-L. Zhang et al. / Carbohydrate Research 345 (2010) 780–786
4.7. N-Butyl-2,6-dideoxy-2,6-imino-
D
-glycero-
L
-altro–heptitol (3)
128.30, 128.44, 137.00, 137.67, 137.85, 138.29, 157.60 (CO). MS-
ESI: 580 (M+H+). Anal Calcd for C36H37NO6: C, 74.59; H, 6.43; N,
2.42. Found: C, 74.39; H, 6.58; N, 2.26.
A mixture of 14 (43 mg, 0.07 mmol) and 10% Pd–C (10.0 mg) in
acetic acid (1.0 mL), H2O (4.0 mL), and THF (2.0 mL) was stirred for
48 h under a H2 atmosphere. The solid was removed by filtration
through Celite and the filtrate was concentrated. The residue was
passed through a C-18 reverse-phase column chromatography
(H2O as eluent) and ion-exchange resin (Dowex 1 ꢀ 8, OHꢁ form)
to give 3 (17.5 mg, 100%) as white solids after lyophilization.
4.11. 3,4,5,7-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-
D-glycero-D-
galacto-heptitol (19)
Compound 19 was prepared from 18 (220 mg, 0.38 mmol) as
described in the preparation of 13, providing 19 (178 mg, 85%) as
a yellow oil. 1H NMR (500 MHz, CDCl3): d 2.07 (br s, 2H, –OH, –
NH–), 3.06 (br s, 1H, H-6), 3.27–3.31 (m, 1H, H-2), 3.45 (t,
J = 8.5 Hz, 1H, H-7), 3.57–3.61 (m, 2H, H-7, H-1), 3.64 (dd, 1H,
J = 2.5, 9.0 Hz, H-3), 3.75 (dd, 1H, J = 5.5, 11.0 Hz, H-1), 3.99–4.01
(m, 2H, H-5, H-4), 4.47 (s, 2H), 4.54 (d, 1H, J = 11.5 Hz), 4.58 (d,
1H, J = 12.0 Hz), 4.66 (d, 1H, J = 12.5 Hz), 4.68 (d, 1H, J = 12.5 Hz),
4.73 (d, 1H, J = 11.5 Hz), 4.84 (d, 1H, J = 11.0 Hz) (8 ꢀ –CH2Ph),
7.24–7.33 (m, 20H). 13C NMR (125 MHz, CDCl3): d 52.25 (C-6),
53.85 (C-2), 58.84 (C-1), 68.91 (C-7), 72.83, 73.12, 73.33, 73.78
(4 ꢀ –CH2Ph), 75.20 (C-5), 77.61 (C-4), 79.36 (C-3), 127.40,
127.46, 127.61, 127.73, 127.84, 127.86, 127.95, 128.27, 128.31,
128.38, 128.42, 138.06, 138.32, 138.71, 138.74. MS-ESI: 554
[M+H]+. Anal Calcd for C35H39NO5: C, 75.92; H, 7.10; N, 2.53.
Found: C, 75.67; H, 7.08; N, 2.45.
½
a 2D6
ꢂ
ꢁ21.3 (c 0.6, H2O). 1H NMR (500 MHz, D2O): d 0.89 (t, 3H,
J = 7.5 Hz, –CH3), 1.20–1.28 (m, 2H, –CH2–), 1.40–1.46 (m, 2H, –
CH2–), 2.64–2.70 (m, 1H, –NCH2–), 2.74 (td, 1H, J = 2.5, 9.0 Hz, H-
2), 2.78–2.85 (m, 1H, –NCH2–), 2.95–2.98 (m, 1H, H-6), 3.75 (dd,
1H, J = 7.0, 12.5 Hz, H-7), 3.80–3.85 (m, 3H, 2H-1, H-7), 3.87–3.90
(m, 1H, H-3), 3.92 (dd, 1H, J = 3.5, 6.5 Hz, H-4), 3.98 (dd, 1H,
J = 2.5, 4.5 Hz, H-5). 13C NMR (125 MHz, D2O): d 13.61 (–CH3),
19.96 (–CH2–), 24.02 (–CH2–), 48.82 (–NCH2–), 55.75 (C-6), 59.59
(C-1), 60.14 (C-2), 61.03 (C-7), 63.86 (C-3), 69.57 (C-5), 70.02 (C-
4). HRMS (ESI, positive) Calcd for C11H23NO5Na: 272.1468
[M+Na]+. Found: 272.1472.
4.8. N-Hexyl-2,6-dideoxy-2,6-imino-D-glycero-L-altro–heptitol (4)
Compound 4 was prepared from 15 (63 mg, 0.1 mmol) as de-
scribed in the preparation of 3, providing 4 (27 mg, 100%) as white
4.12. 3,4,5,7-Tetra-O-benzyl-N-butyl-2,6-dideoxy-2,6-imino-
D-
solids. ½a 2D6
ꢂ
ꢁ22.6 (c 0.8, H2O). 1H NMR (500 MHz, D2O): d 0.86 (t,
glycero- -galacto-heptitol (20)
D
3H, J = 7.0 Hz, –CH3), 1.22–1.30 (m, 6H, –CH2–), 1.43–1.48 (m, 2H, –
CH2–), 2.64–2.70 (m, 1H, –NCH2–), 2.74 (td, 1H, J = 3.0, 8.5 Hz, H-2),
2.79–2.85 (m, 1H, –NCH2–), 2.96–2.99 (m, 1H, H-6), 3.75 (dd, 1H,
J = 7.0, 11.5 Hz, H-7), 3.80–3.88 (m, 3H, 2H-1, H-7), 3.89 (dd, 1H,
J = 3.0, 8.5 Hz, H-3), 3.91–3.94 (m, 1H, H-4), 3.98 (dd, 1H, J = 2.0,
4.0 Hz, H-5). 13C NMR (125 MHz, D2O): d 14.04 (–CH2–), 21.68,
22.72, 26.86, 31.64 (4 ꢀ –CH2–), 48.21 (–NCH2–), 57.59 (C-6),
58.86 (C-1), 60.16 (C-2), 61.16 (C-7), 66.51 (C-3), 70.66 (C-5),
70.94 (C-4). HRMS (ESI, positive) Calcd for C13H27NO5Na:
300.1781 [M+Na]+. Found: 300.1786.
Compound 20 was prepared from 19 (100 mg, 0.18 mmol) as
described in the preparation of 14 with the exception that etha-
nol was used in the reaction mixture instead of MeOH, providing
20 (88 mg, 80%) as a yellow oil. 1H NMR (500 MHz, CDCl3): d
0.85 (t, 3H, J = 7.0 Hz, –CH3), 1.14–1.29 (m, 4H, –CH2–), 2.56–
2.67 (m, 2H, –NCH2–), 2.82 (br s, 1H, –OH), 2.93 (br s, 1H, H-
6), 3.22–3.26 (m, 1H, H-2), 3.52–3.56 (m, 2H, H-1, H-7), 3.59
(dd, 1H, J = 2.5, 9.5 Hz, H-4), 3.66 (t, 1H, J = 8.0 Hz, H-7), 3.72
(dd, 1H, J = 5.5, 10.5 Hz, H-1), 4.07 (br s, 1H, H-5), 4.18 (br s,
1H, H-3), 4.35 (d, 1H, J = 12.0 Hz), 4.41 (d, 1H, J = 11.5 Hz), 4.46
(d, 1H, J = 12.0 Hz), 4.62 (d, 1H, J = 11.5 Hz), 4.71–4.76 (m, 3H),
4.96 (d, 1H, J = 11.5 Hz) (8 ꢀ –CH2Ph), 7.22–7.36 (m, 20H). 13C
NMR (125 MHz, CDCl3): d 13.97 (–CH3), 20.19 (–CH2–), 32.55 (–
CH2–), 49.96 (–NCH2–), 54.97 (C-6), 56.27 (C-1), 59.43 (C-2),
68.64 (C-7), 72.78, 73.26, 73.38 (3 ꢀ –CH2Ph), 74.34 (C-3),
74.58 (–CH2Ph), 76.74 (C-5), 80.74 (C-4), 127.23, 127.30,
127.34, 127.40, 127.59, 127.75, 128.16, 128.30, 128.38, 137.96,
138.49, 138.79, 139.28. MS-ESI: 610 [M+H]+. Anal. Calcd for
C39H47NO5: C, 76.82; H, 7.77; N, 2.30. Found: C, 76.64; H, 7.91;
N, 2.20.
4.9. N-Nonyl-2,6-dideoxy-2,6-imino-D-glycero-L-altro–heptitol (5)
Compound 5 was prepared from 16 (77 mg, 0.11 mmol) as de-
scribed in the preparation of 3, providing 5 (36 mg, 100%) as white
solids. ½a 2D6
ꢂ
ꢁ22.8 (c 0.8, H2O). 1H NMR (500 MHz, D2O): d 0.86 (t,
3H, J = 7.0 Hz, –CH3), 1.27–1.37 (m, 12H, –CH2–), 1.71–1.80 (m, 2H,
–CH2–), 3.30–3.47 (m, 3H, 2 ꢀ –NCH2–, H-2), 3.72–3.74 (m, 1H, H-
6), 3.97 (dd, 1H, J = 4.5, 13.0 Hz, H-7), 4.02–4.09 (m, 4H, H-7, H-3,
2 ꢀ H-1), 4.19–4.23 (m, 2H, H-4, H-5). 13C NMR (125 MHz, D2O):
d 14.12 (–CH3), 20.69, 22.75, 26.14, 28.84, 28.99, 29.11, 31.82
(7 ꢀ –CH2–), 49.24 (–NCH2–), 55.09 (C-6), 59.94 (C-1), 60.08 (C-
2), 61.13 (C-7), 63.38 (C-3), 69.28 (C-5), 69.95 (C-4). HRMS (ESI, po-
sitive) Calcd for C16H34NO5: 320.2431 [M+H]+. Found: 320.2430.
4.13. 3,4,5,7-Tetra-O-benzyl-N-hexyl-2,6-dideoxy-2,6-imino-
glycero- -galacto-heptitol (21)
D-
D
4.10. 1,3,4,5-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-
galacto-heptitol O (7), N-cyclic carbamate (18)
D
-glycero-
D
-
Compound 21 was prepared from 19 (31 mg, 0.06 mmol) as de-
scribed in the preparation of 20, providing 21 (31 mg, 86%) as a yel-
low oil. 1H NMR (500 MHz, CDCl3): d 0.87 (t, 3H, J = 7.5 Hz, –CH3),
1.19–1.28 (m, 8H, –CH2–), 2.55–2.66 (m, 2H, –CH2–), 2.93 (br s, 1H,
H-6), 3.21–3.26 (m, 1H, H-2), 3.50–3.56 (m, 2H, H-1, H-7), 3.59 (dd,
1H, J = 3.0, 9.5 Hz, H-4), 3.65 (t, 1H, J = 8.5 Hz, H-7), 3.71 (dd, 1H,
J = 5.5, 11.0 Hz, H-1), 4.06–4.07 (m, 1H, H-5), 4.17 (br s, 1H, H-3),
4.36 (d, 1H, J = 11.5 Hz), 4.41 (d, 1H, J = 12.0 Hz), 4.46 (d, 1H,
J = 11.5 Hz), 4.62 (d, 1H, J = 11.5 Hz), 4.71–4.76 (m, 3H), 4.96 (d,
1H, J = 12.0 Hz) (8 ꢀ –CH2Ph), 7.22–7.36 (m, 20H). 13C NMR
(125 MHz, CDCl3): d 14.05 (–CH3), 22.59, 26.70, 30.32, 31.70
(4 ꢀ –CH2–), 50.24 (–NCH2–), 54.98 (C-6), 56.28 (C-1), 59.47 (C-
2), 68.63 (C-7), 72.77, 73.25, 73.36 (3 ꢀ –CH2Ph), 74.40 (C-3),
74.57 (–CH2Ph), 76.75 (C-5), 80.75 (C-4), 127.22, 127.28, 127.33,
127.38, 127.58, 127.73, 128.14, 128.28, 128.37, 137.95, 138.49,
Compound 18 was prepared from 17 (210 mg, 0.71 mmol) as
described in the preparation of 12, providing 18 (159 mg, 90%) as
a yellow oil. The data were in good agreement with those reported
by Martin et al.28 1H NMR (500 MHz, CDCl3): d 3.34 (dd, 1H, J = 2.5,
4.0 Hz), 3.82–3.83 (m, 1H), 3.88 (dd, 1H, J = 3.0, 10.5 Hz), 3.94 (dd,
2H, J = 2.5, 6.5 Hz), 4.02 (t, 1H, J = 10.5 Hz), 4.09 (dd, 1H, J = 4.0,
8.5 Hz), 4.13 (ABq, 1H, J = 7.5 Hz, H-1), 4.32 (d, 1H, J = 12.0 Hz),
4.40 (d, 1H, J = 13.0 Hz), 4.43–4.46 (m, 3H), 4.55–4.57 (m, 1H),
4.59 (d, 1H, J = 12.0 Hz), 4.62 (d, 1H, J = 12.0 Hz), 4.70 (d, 1H,
J = 12.0 Hz), 7.12–7.35 (m, 20H). 13C NMR (125 MHz, CDCl3): d
49.42, 51.51, 62.69, 65.41, 71.42, 72.15, 72.19, 73.11, 73.62,
75.09, 127.38, 127.52, 127.70, 127.81, 127.98, 128.13, 128.28,

G.-L. Zhang et al. / Carbohydrate Research 345 (2010) 780–786
785
138.78, 139.28. MS-ESI: 638 [M+H]+. Anal. Calcd for C41H51NO5: C,
77.20; H, 8.06; N, 2.20. Found: C, 76.97; H, 8.06; N, 2.10.
(125 MHz, D2O): d 14.38 (–CH3), 23.09, 27.16, 28.02, 29.62, 29.71,
29.89, 32.29 (7 ꢀ –CH2–), 51.01 (–NCH2–), 55.92 (C-6), 59.28 (C-
2), 60.18 (C-7), 66.79 (C-1), 68.86 (C-3), 67.00 (C-5), 70.96 (C-4).
HRMS (ESI, positive) Calcd for C16H34NO5: 320.2431 [M+H]+.
Found: 320.2427.
4.14. 3,4,5,7-Tetra-O-benzyl-N-nonyl-2,6-dideoxy-2,6-imino-
D-
glycero- -galacto-heptitol (22)
D
Compound 22 was prepared from 19 (80 mg, 0.14 mmol) as de-
scribed in the preparation of 20, providing 22 (78 mg, 80%) as a yel-
low oil. 1H NMR (500 MHz, CDCl3): d 0.89 (t, 3H, J = 7.0 Hz, –CH3),
1.13–1.31 (m, 14H, –CH2–), 2.55–2.66 (m, 2H, –NCH2–), 2.93 (br s,
1H, H-6), 3.21–3.26 (m, 1H, H-2), 3.52–3.56 (m, 2H, H-1, H-7), 3.59
(dd, 1H, J = 3.0, 10.0 Hz, H-4), 3.65 (t, 1H, J = 8.5 Hz, H-7), 3.71 (dd,
1H, J = 5.5, 11.0 Hz, H-1), 4.06 (br s, 1H, H-5), 4.18 (br s, 1H, H-3),
4.36 (d, 1H, J = 12.0 Hz), 4.41 (d, 1H, J = 11.5 Hz), 4.46 (d, 1H,
J = 12.0 Hz), 4.62 (d, 1H, J = 11.5 Hz), 4.72–4.76 (m, 3H), 4.96 (d,
1H, J = 11.5 Hz) (8 ꢀ –CH2Ph), 7.23–7.30 (m, 20H). 13C NMR
(125 MHz, CDCl3): d 14.09 (–CH3), 22.65, 27.07, 29.28, 29.54,
29.57, 30.38, 31.85 (7 ꢀ –CH2–), 50.25 (–NCH2–), 54.97 (C-6),
56.31 (C-1), 59.49 (C-2), 68.65 (C-7), 72.79, 73.25, 73.37 (3 ꢀ –
CH2Ph), 74.45 (C-3), 74.58 (–CH2Ph), 76.75 (C-5), 80.77 (C-4),
127.23, 127.30, 127.34, 127.40, 127.59, 127.75, 128.15, 128.30,
128.38, 137.96, 138.49, 138.79, 139.29. HRMS (ESI, positive) Calcd
for C44H58NO5: 680.4310 [M+H]+. Found: 680.4302.
4.18. 1,3,4,5-Tetra-O-benzyl-2,6-dideoxy-2,6-imino-
altro-heptitol- lactone (23)
D-glycero-L-
To a solution of 13 (278 mg, 0.50 mmol) in MeOH and acetic acid
(v/v 200:1, 10 mL) was added 40% glyoxal aqueous solution (86 L,
l
1.88 mmol). After stirring for 1 h at 60 °C, a portion of NaBH4
(38 mg, 1.0 mmol) was added. The reaction mixture was stirred
overnight at 80 °C and the reaction was quenched by the addition
of 1 N HCl aqueous solution (0.2 mL). The mixture was extracted
with EtOAc (3 ꢀ 50 mL) and washed with aqueous NaHCO3
(10 mL) and aqueous NaCl (10 mL). The organic phases were com-
bined, dried (Na2SO4), filtered, and concentrated. The residue was
purified by column chromatography on silica gel (petroleum
ether–EtOAc 4:1) to provide 23 (221 mg, 74%) as a yellow oil. 1H
NMR (500 MHz, CDCl3): d 2.69 (td, 1H, J = 2.5, 10.5 Hz), 2.91 (td,
1H, J = 2.5, 10.5 Hz), 3.07 (d, 1H, J = 18.0 Hz), 3.36 (dd, 1H, J = 2.5,
4.0 Hz), 3.63–3.71 (m, 3H), 3.84 (dd, 1H, J = 2.5, 10.0 Hz), 3.93 (dd,
1H, J = 3.0, 11.0 Hz), 4.05 (d, 1H, J = 17.5 Hz), 4.19 (d, 1H,
J = 12.5 Hz), 4.31 (d, 1H, J = 11.5 Hz), 4.33 (d, 1H, J = 12.5 Hz), 4.36
(d, 1H, J = 11.5 Hz), 4.44 (d, 1H, J = 12.5 Hz), 4.48 (d, 1H,
J = 10.5 Hz), 4.49 (d, 1H, J = 12.5 Hz), 4.57 (d, 1H, J = 12.0 Hz), 4.67
(d, 1H, J = 12.5 Hz), 7.10–7.33 (m, 20H). 13C NMR (125 MHz, CDCl3):
d 53.56, 53.65, 61.72, 65.98, 70.04, 70.82, 72.18, 72.40, 73.09, 73.23,
73.42, 73.63, 127.79, 127.84, 127.87, 128.20, 128.32, 128.40, 128.43,
128.46, 128.49, 128.57, 137.11, 137.83, 137.89, 138.16, 167.41 (CO).
MS-ESI: 594 [M+H]+. Anal. Calcd for C37H39NO6: C, 74.85; H, 6.62; N,
2.36. Found: C, 74.62; H, 6.55; N, 2.21.
4.15. N-Butyl-2,6-dideoxy-2,6-imino-D-glycero-D-galacto-
heptitol (6)
Compound 6 was prepared from 20 (73 mg, 0.12 mmol) as de-
scribed in the preparation of 3, providing 6 (26.8 mg, 90%) as a
white solid. ½a 2D6
ꢂ
+26.8 (c 0.8, H2O). 1H NMR (300 MHz, D2O): d
0.73 (t, 3H, J = 7.2 Hz, –CH3), 1.06–1.18 (m, 2H, –CH2–), 1.22–1.28
(m, 2H, –CH2–), 2.46–2.64 (m, 2H, –NCH2–), 2.73–2.77 (m, 1H, H-
2), 3.09–3.18 (m, 1H, H-6), 3.49 (dd, 1H, J = 3.6, 9.9 Hz, H-4),
3.62–3.74 (m, 4H, H-7, H-1), 3.86 (br s, 1H, H-5), 3.93 (dd, 1H,
J = 5.7, 9.9 Hz, H-3). 13C NMR (75 MHz, D2O): d 15.92 (–CH3),
22.58, 33.28 (2 ꢀ –CH2–), 51.51 (–NCH2–), 58.32 (C-6), 60.40 (C-
2), 62.71 (C-7), 63.24 (C-1), 69.60 (C-3), 72.75 (C-5), 73.73 (C-4).
HRMS (ESI, positive) Calcd for C11H23NO5Na: 272.1468 [M+Na]+.
Found: 272.1466.
4.19. 3,4,5,7-Tetra-O-benzyl-N-hydroxyethyl-2,6-dideoxy-2,6-
imino-D-glycero- L-altro–heptitol (24)
To a solution of 23 (133 mg, 0.22 mmol) in dry THF (8 mL) was
added LiAlH4 (70 mg, 1.76 mmol) at ꢁ15 °C under argon and the
mixture was stirred for 5 h at room temperature. The reaction mix-
ture was slowly added EtOAc (3 mL) and 1 N NaOH (1 mL) at 0 °C
and was extracted with EtOAc (3 ꢀ 50 mL) and washed with aque-
ous NaHCO3 (10 mL) and aqueous NaCl (10 mL). The organic
phases were combined, dried (Na2SO4), filtered, and concentrated.
The residue was purified by column chromatography on silica gel
(petroleum ether–EtOAc 2:1) to provide 24 (76 mg, 57%) as a yel-
low oil. 1H NMR (500 MHz, CDCl3) d: 2.96 (td, 1H, J = 5.5, 14.5 Hz,
–NCH2–), 3.09 (td, 1H, J = 4.0, 15.0 Hz, –NCH2–), 3.26 (q, 1H,
J = 6.5 Hz, H-6), 3.33–3.36 (m, 1H, H-2), 3.43 (dd, 1H, J = 5.0,
10.0 Hz, H-7), 3.47–3.56 (m, 4H, 2H-1, 2 ꢀ CH2OH), 3.68 (dd, 1H,
J = 2.5, 7.5 Hz, H-4), 3.72 (dd, 1H, J = 6.5, 11.5 Hz, H-7), 3.79 (dd,
1H, J = 3.0, 5.0 Hz, H-3), 4.03 (dd, 1H, J = 5.0, 7.5 Hz, H-5), 4.44 (d,
1H, J = 12.0 Hz), 4.48–4.60 (m, 6H), 4.62 (d, 1H, J = 11.5 Hz) (8 ꢀ –
CH2Ph), 7.24–7.39 (m, 20H). 13C NMR (125 MHz, CDCl3): d 55.77
(–NCH2–), 60.46 (–CH2OH), 60.98 (C-2), 61.07 (C-6), 61.96 (C-7),
69.83 (C-1), 72.26, 72.63, 73.22, 73.35 (4 ꢀ –CH2Ph), 74.56 (C-4),
74.92 (C-3), 77.37 (C-5), 127.60, 127.64, 127.69, 127.77, 127.82,
127.88, 127.94, 128.33, 128.34, 128.45, 137.43, 138.06, 138.11,
138.43. HRMS (ESI, positive) Calcd for C37H44NO6: 598.3163
[M+H]+. Found: 598.3170.
4.16. N-Hexyl-2,6-dideoxy-2,6-imino-D-glycero-D-galacto-
heptitol (7)
Compound 7 was prepared from 21 (30 mg, 0.05 mmol) as de-
scribed in the preparation of 3, providing 7 (12 mg, 92%) as white
solids. ½a 2D6
ꢂ
+30.5 (c 0.8, H2O). 1H NMR (300 MHz, D2O): d 0.70 (t,
1H, J = 6.3 Hz, –CH3), 1.13–1.14 (m, 6H, –CH2–), 1.39 (br s, 2H, –
CH2–), 2.76–2.78 (m, 2H, –NCH2–), 3.02 (br s, 1H, H-2), 3.31–3.33
(m, 1H, H-6), 3.56 (dd, 1H, J = 3.0, 9.6 Hz, H-4), 3.65–3.74 (m, 3H,
H-7, H-1), 3.78 (dd, 1H, J = 6.6, 11.7 Hz, H-1), 3.94 (br s, 1H, H-5),
3.98 (dd, 1H, J = 5.7, 9.9 Hz, H-3). 13C NMR (75 MHz, D2O): d
15.92 (–CH3), 24.54, 28.62, 30.14, 33.49 (4 ꢀ –CH2–), 52.20 (–
NCH2–), 58.05 (C-6), 61.18 (C-2), 62.01 (C-7), 63.56 (C-1), 69.25
(C-3), 72.25 (C-5), 73.26 (C-4). HRMS (ESI, positive) Calcd for
C13H28NO5: 278.1962 [M+H]+. Found: 278.1962.
4.17. N-Nonyl-2,6-dideoxy-2,6-imino-D-glycero-D-galacto-
heptitol (8)
Compound 8 was prepared from 22 (43 mg, 0.06 mmol) as de-
scribed in the preparation of 3, providing 8 (20 mg, 100%) as a
white solid. ½a 2D6
ꢂ
ꢁ10.2 (c 0.4, H2O). 1H NMR (500 MHz, D2O): d
4.20. N-Hydroxyethyl-2,6-dideoxy-2,6-imino-D-glycero-L-altro-
heptitol (9)
0.86 (t, 3H, J = 6.0 Hz, –CH3), 1.23–1.32 (m, 12H, –CH2–), 1.60 (br
s, 2H, –CH2–), 3.07 (d, 2H, J = 7.5 Hz, –NCH2–), 3.33–3.34 (m, 1H,
H-2), 3.56–3.57 (m, 1H, H-6), 3.78 (d, 1H, J = 7.0 Hz, H-4), 3.84–
3.94 (m, 4H, H-1, H-7), 4.16–4.20 (m, 2H, H-3, H-5). 13C NMR
Compound 9 was prepared from 24 (43 mg, 0.07 mmol) as de-
scribed in the preparation of 3, providing 9 (15.3 mg, 90%) as a

786
G.-L. Zhang et al. / Carbohydrate Research 345 (2010) 780–786
white solid. ½a 2D6
ꢂ
ꢁ9.0 (c 0.8, H2O). 1H NMR (500 MHz, D2O) d:
Supplementary data
2.84–2.87 (m, 1H, H-2), 2.98 (t, 2H, J = 6.5 Hz, –NCH2–), 3.06 (dt,
1H, J = 3.0, 6.5 Hz, H-6), 3.66 (t, 2H, J = 6.0 Hz, 2 ꢀ CH2OH), 3.74–
3.83 (m, 4H, 2H-7 and 2H-1), 3.90 (dd, 1H, J = 3.5, 6.0 Hz, H-3),
3.93 (dd, 1H, J = 3.0, 7.5 Hz, H-4), 4.01 (dd, 1H, J = 3.0, 5.5 Hz, H-
5). 13C NMR (75 MHz, D2O) d: 53.54 (–NCH2–), 61.38 (–CH2OH),
61.76 (C-6), 62.07 (C-1), 63.20 (C-2), 64.58 (C-7), 68.85 (C-3),
72.16 (C-5), 72.37 (C-4). HRMS (ESI, positive) Calcd for C9H19NO6-
Na: 260.1105 [M+Na]+. Found: 260.1102.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.01.021.
References
1. Rosen, H. R. Gastroenterology 2008, 134, 1789–1801.
2. Halloran, P. F. N. Engl. J. Med. 2004, 351, 2715–2729.
3. Ganadian Multicentre Transplant Study Group N. Engl. J. Med. 1983, 309, 809–
815.
4. Changelian, P. S.; Flanagan, M. E.; Ball, D. J.; Kent, C. R.; Magnuson, K. S.; Martin,
W. H.; Rizzuti, B. J.; Sawyer, P. S.; Perry, B. D.; Brissette, W. H. Science 2003, 302,
875–878.
4.21. N-Hydroxyethyl-2,6-dideoxy-2,6-imino-D-glycero-D-
galacto-heptitol (10)
5. Compain, P.; Martin, O. R. Iminosugars: From Synthesis to Therapeutic
Applications; John Wiley & Sons: New York, 2007.
6. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
7. Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102,
515–553.
8. Stütz, A. E. Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond; Wiley-
VCH: Weinheim, 1999.
Compound 10 was prepared from 19 (90 mg, 0.16 mmol) as de-
scribed in the preparation of 23, providing yellow crude product
(64 mg) after column chromatography. The product was treated
under the conditions as described in the preparation of 24, yielding
crude product (53 mg). Finally, catalytic hydrogenolysis of the
crude product using the method in the preparation of 9 provided
9. Ganem, B. Acc. Chem. Res. 1996, 29, 340–347.
10 (21 mg, 55%, three steps) as white solids. ½a D26
ꢂ
+33.8 (c 0.8,
10. Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed. 1999, 38, 2300–2324.
11. Tsuruoka, T.; Fukuyasu, H.; Ishii, M.; Usui, T.; Shibahara, S.; Inouye, S. J. Antibiot.
1996, 49, 155–161.
12. Somsák, L.; Nagy, V.; Hadady, Z.; Docsa, T.; Gergely, P. Curr. Pharm. Des. 2003, 9,
1177–1189.
13. Durantel, D.; Branza-Nichita, N.; Carrouee-Durantel, S.; Butters, T. D.; Dwek, R.
A.; Zitzmann, N. J. Virol. 2001, 75, 8987–8998.
14. (a) Fan, J.-Q.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112–115; (b)
Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O. R.; Fan, J.-
Q. Eur. J. Biochem. 2000, 267, 4179–4186.
H2O). 1H NMR (300 MHz, D2O): d 2.54–2.62 (m, 1H, –NCH2–),
2.73–2.89 (m, 2H, H-2, –NCH2–), 2.99–3.05 (m, 1H, H-6), 3.32–
3.44 (m, 3H, H-4, –CH2OH), 3.49–3.55 (m, 3H, H-7, H-1), 3.64
(dd, 1H, J = 7.8, 10.8 Hz, H-1), 3.77 (br s, 1H, H-5), 3.84 (dd, 1H,
J = 5.4, 9.3 Hz, H-3). 13C NMR (75 MHz, D2O): d 53.15 (–NCH2–),
58.82 (C-6), 59.74 (C-2), 62.17 (C-7), 62.77 (CH2OH), 64.05 (C-1),
69.56 (C-3), 73.10 (C-5), 74.12 (C-4). HRMS (ESI, positive) Calcd
for C9H19NO6Na: 260.1105 [M+Na]+. Found: 260.1101.
15. Yu, Z.; Sawkar, A. R.; Whalen, L. J.; Wong, C.-H.; Kelly, J. W. J. Med. Chem. 2007,
50, 94–100.
16. Grochowicz, P. M.; Hibberd, A. D.; Smart, Y. C.; Bowen, K. M.; Clark, D.
A.; Cowden, W. B.; Willenborg, D. O. Transpl. Immunol. 1996, 4, 275–
285.
4.22. Cell proliferation assay
17. Ye, X.-S.; Sun, F.; Liu, M.; Li, Q.; Wang, Y.; Zhang, G.; Zhang, L.-H.; Zhang, X.-L. J.
Med. Chem. 2005, 48, 3688–3691.
18. Zhou, J.; Zhang, Y.; Zhou, X.; Zhou, J.; Zhang, L.-H.; Ye, X.-S.; Zhang, X.-L. Bioorg.
Med. Chem. 2008, 16, 1605–1612.
19. Asano, N. Curr. Top. Med. Chem. 2003, 3, 471–484.
20. Compain, P.; Martin, O. R. Curr. Top. Med. Chem. 2003, 3, 541–560.
21. Butters, T. D.; Dwek, R. A.; Platt, F. M. Glycobiology 2005, 15, 43–52.
22. Butters, T. D.; Dwek, R. A.; Platt, F. M. Chem. Rev. 2000, 100, 4683–4696.
23. Scott, L. J.; Spencer, C. M. Drugs 2000, 59, 521–549.
24. Asano, N. Glycobiology 2003, 13, 93–104.
Male BALB/c mouse splenocytes (4 ꢀ 105 cells per well), re-
treated with Con A alone (2.5
l
g/mL concentration) or along with
30 lM concentration of synthetic iminosugar compounds, were
incubated at 37 °C for 48 h under 5% CO2 in a RPMI-1640 medium
containing 10% fetal bovine serum (FBS). The proliferation of the
mouse splenocytes was assayed using the MTT reduction method.
MTT (5 mg/mL, 20 lL) dissolved in phosphate buffer solution (PBS)
was added to each well and the plates were incubated for 4 h at
37 °C. The 96-well microplates were centrifuged at 1500g for
6 min and the media were aspirated. The resultant crystals were
25. Mitrakou, A.; Tountas, N.; Raptis, A. E.; Bauer, R. J.; Schulz, H.; Raptis, S. A.
Diabetes Med. 1998, 15, 657–660.
´
26. Pavlovic, D.; Neville, D. C. A.; Argaud, O.; Blumberg, B.; Dwek, R. A.; Fischer, W.
B.; Zitzmann, N. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6104–6108.
27. Metha, A.; Carrouée, S.; Conyers, B.; Jordan, R.; Butters, T. D.; Dewk, R. A.; Block,
T. M. Hepatology 2001, 33, 1488–1495.
28. Martin, O. R.; Saavedra, O. M.; Xie, F.; Liu, L.; Picasso, S.; Vogel, P.; Kizu, H.;
Asano, N. Bioorg. Med. Chem. 2001, 9, 1269–1278.
29. Mellor, H. R.; Nolan, J.; Pickering, L.; Wormald, M. R.; Platt, F. M.; Dwek, R. A.;
Fleet, G. W. J.; Butters, T. D. Biochem. J. 2002, 366, 225–233.
30. Zitzmann, N.; Mehta, A. S.; Carrouée, S.; Butters, T. D.; Platt, F. M.; McCauley, J.;
Blumberg, B. S.; Dwek, R. A.; Block, T. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
1878–1882.
dissolved in 150 lL of DMSO for 10 min. Optical density was mea-
sured using a Microplate Reader at 570 nm. All data were pre-
sented as mean SEM. One-way ANOVA followed by Bonferroni
was used to test the difference between groups. Statistical signifi-
cance was indicated by p <0.05.
Acknowledgment
31. Hisken, W.; Fed, E. U.S. Patent 5,071,990, 1991.
32. Vyavahare, V. P.; Chakraborty, C.; Maity, B.; Chattopadhyay, S.; Puranik, V. G.;
Dhavale, D. D. J. Med. Chem. 2007, 50, 5519–5523.
This work was financially supported by the National Natural
Science Foundation of China.