An expeditious one-pot synthesis of 1,6-dideoxy-N-alkylated nojirimycin derivatives and their inhibitory effects on the secretion of IFN-γ and IL-4

Article abstract of DOI:10.1016/j.bmc.2007.11.036

An efficient 'one-pot' approach to the synthesis of 1,6-dideoxy-N-alkylated nojirimycin derivatives in good yields and with high stereoselectivity was developed. It was found that the synthetic N-alkylated iminosugars showed inhibitory effects on the release of the cytokines IFN-γ and IL-4 from the mouse splenocytes. The preliminary structure-activity relationship was deduced from the activities of N-substituted iminosugars. Apart from the cytokine inhibitory activities, a series of glycosidase inhibitory activities were also examined. The present experimental data demonstrated that synthetic iminosugars might hold potential as immunosuppressive agents.

Full text of DOI:10.1016/j.bmc.2007.11.036

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1605–1612
An expeditious one-pot synthesis of 1,6-dideoxy-N-alkylated
nojirimycin derivatives and their inhibitory effects on
the secretion of IFN-c and IL-4
Jian Zhou,a Yongmin Zhang,b Xia Zhou,c Jing Zhou,c Li-He Zhang,a
Xin-Shan Yea,* and Xiao-Lian Zhangc,*
aThe State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Xue Yuan Road #38, Beijing 100083, PR China
bEcole Normale Supe´rieure, De´partement de Chimie, UMR 8642 CNRS, 24 rue Lhomond, 75231 Paris Cedex 05, France
cDepartment of Immunology, State Key Laboratory of Virology,
Hubei Province Key Laboratory of Allergy and Immune-Related Diseases,
Wuhan University College of Medicine, Wuhan 430071, PR China
Received 9 September 2007; revised 10 November 2007; accepted 12 November 2007
Available online 17 November 2007
Abstract—An efficient ‘one-pot’ approach to the synthesis of 1,6-dideoxy-N-alkylated nojirimycin derivatives in good yields and
with high stereoselectivity was developed. It was found that the synthetic N-alkylated iminosugars showed inhibitory effects on
the release of the cytokines IFN-c and IL-4 from the mouse splenocytes. The preliminary structure–activity relationship was
deduced from the activities of N-substituted iminosugars. Apart from the cytokine inhibitory activities, a series of glycosidase inhib-
itory activities were also examined. The present experimental data demonstrated that synthetic iminosugars might hold potential as
immunosuppressive agents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
three decades. These compounds are frequently found to
be potent inhibitors of many carbohydrate-processing
enzymes.6,7 Since these enzymes are involved in a num-
ber of important biological processes,8–11 iminosugars
have tremendous potential as therapeutic agents in a
wide range of diseases such as diabetes,12 viral infec-
tions,13 tumor metastasis,14 and lysosomal storage disor-
ders.15,16 The wide range of biological activities
displayed by this class of compounds has resulted in
many synthetic efforts.17–19
The main clinically used immunosuppressive agents,
such as cyclosporin A (CyA), tacrolimus, mycopheno-
late mofetil, and sirolimus, have significant side effects
including nephrotoxicity, neurotoxicity, infection, can-
cer, new onset post-transplant diabetes mellitus, hyper-
lipidemia, and hypertension.1–5 Moreover, so far there
is no antidote for the toxicity of these organ transplan-
tation drugs. Stimulated by the need to improve trans-
plant survival and by the reduction of the toxicity of
current agents, the search to find more effective and
safer immunosuppressants is in great demand.
Since the information of crystallographic data relating
to enzymes and their active sites is only slowly increasing
due to the inherent problems experienced with the crys-
tallization of these frequently membrane-bound and
sensitive enzymes, rational drug design is still difficult.
So it is highly desired to synthesize diversity-oriented
small molecules to improve the understanding of struc-
ture–activity relationship (SAR) between bio-targets
and small molecules. In order to gain rapid access to
new iminosugar derivatives of biological interest, a ser-
ies of N-alkylated iminosugar compounds were designed
and synthesized by an expeditious ‘one-pot’ method
Iminosugars are a family of polyhydroxylated heterocy-
cles containing an endocyclic nitrogen atom. They have
been a subject of enduring scientific interest over the last
Keywords: Iminosugar; One-pot synthesis; Cytokine inhibition; Immu-
nosuppressive agent; Glycosidase inhibitor.
*
Corresponding authors. Tel.: +86 10 82801570; fax: +86 10
62014949; e-mail addresses: xinshan@bjmu.edu.cn; zhangxl65@whu.
edu.cn
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.036

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J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
with high stereoselectivity. Since the effects of iminosug-
ars on immune system and their application as immuno-
suppressants have been less investigated, and recently we
disclosed the iminosugars displayed immunosuppressive
activity,20 inhibitory effects of the synthetic compounds
on the secretion of IFN-c and IL-4 as well as their gly-
cosidase inhibitory activities were evaluated.
(4) also exhibit potent glycosidase inhibitory proper-
ties.23 With regard to the development of new fucosyl-
transferase inhibitors, many efforts24 have been
devoted to the synthesis and modifications of fuconojir-
imycin (5), but only limited iminosugars displayed fuco-
sidase or fucosyltransferase inhibitory activities.25 The
biological importance has warranted further exploration
of N-alkylated iminosugars. Considering the high poten-
tial of iminosugars as carbohydrate mimetics, the design
of a general and efficient synthetic approach to N-alkyl-
ated iminosugars appears to be an important issue. So
far there are few methods available in this area.26–28
For this purpose, a series of characteristic N-alkylated
iminosugars, which not only share the basic skeleton
of deoxynojirimycin (1), but also have a very similar
appearance to fuconojirimycin (5), were designed and
synthesized by an efficient way.
2. Results and discussion
2.1. Design
The deoxynojirimycin (DNJ) (1) (Fig. 1) family com-
prises potent inhibitors of the a-glucosidase mediated
N-linked protein glycosylation. N-Alkylated deoxynojir-
imycin and deoxygalactonojirimycin are inhibitors of
the ceramide-specific glucosyltransferase, an enzyme in-
volved in the first step in the glycosylation of many
sphingolipids.21 For instance, N-butyl-1-deoxynojirimy-
cin (Zavesta) (2) has been approved as medicine for
Gaucher disease, a severe lysosomal storage disorder.
The iminosugar N-hydroxyethyl-DNJ (miglitol) (3) is a
drug for treatment of type II diabetes.22 In addition,
N-alkylated bicyclic compounds such as swainsonine
2.2. Chemistry
The preparation of compounds 8a–h is outlined in
Scheme 1. The key material alkene 6, a precursor for
the synthesis of N-alkylated iminosugars, was obtained
from methyl a-D-glucopyranoside through five steps in
high overall yield (77%) according to the procedure
HO
(CH2)2OH
R
HO
HO
HO
HO
N
N
N
NH
OH
HO
HO
OH
HO
HO
OH
OH
OH
OH
3 Miglitol (Glyset)
5 fuconojirimycin
4 swainsonine
(DNJ)
1 R=H
(Zavesta)
2 R=Bu
Figure 1.
R
R
N
O
one-pot
a)
N
BnO
BnO
[H]
b)
BnO
BnO
HO
HO
BnO
BnO
HO
OMe
7
8
6
7a R= -CH2Ph (77%)
7b R= -Ph (73%)
8a R = H (95%)
8b R = -cyclohexyl (97%)
8c R = -nBu (100%)
8d R = -nDec (99%)
7c R= -nBu (68%)
7d R = -nDec (80%)
7e R=-(CH2)2OH(70%)
8e R= -(CH2)2OH(92%)
8g R= -(CH2)2NH2(83%)
7g R=-(CH2)2NHCbz(72%)
(CH2)2OMe
(CH2)2OMe
(CH2)2OH
(CH2)2NH2
N
b)
N
c)
N
BnO
BnO
HO
HO
BnO
BnO
(92%)
(96%)
BnO
HO
BnO
8f
7e
7f
(CH2)2NHAc
N
N
d)
e)
HO
HO
HO
HO
95% for two setps
OH
OH
8g
8h
Scheme 1. Synthesis of iminosugar derivatives. Reagents: (a) CF3COOH, MeOH then RNH2, NaCNBH3, CH3COOH; (b) Pd–C, H2; (c) Mel, NaH,
THF; (d) Ac2O, Py; (e) MeONa, MeOH.

J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
1607
described previously.29 The unsaturated compound 6
120
100
80
60
40
20
0
readily lost the methoxyl aglycon in the presence of cat-
alytic amount of acid to form 1,5-dicarbonyl compound
9 presumedly via the rearrangement of the hydrated
intermediate (Scheme 2). It was found some organic
acids such as trifluoroacetic acid can greatly accelerate
this transformation but inorganic acids such as hydro-
chloric acid are generally invalid.30
With dicarbonyl compound 9 in hand, the iminosugar
derivative 7a was prepared by a well-established double
reductive amination approach.31,32 In order to further
simplify the operation procedure, we performed the
two reactions in a facile ‘one-pot’ manner. That is, al-
kene 6 was treated with trifluoroacetic acid, which was
followed by reductive amination with benzylamine and
NaCNBH3 without purification of intermediate 9, yield-
ing iminosugar 7a in 77% isolated yield. In the same
way, iminosugars 7b–e and 7g were constructed in good
yields (Scheme 1). It is noteworthy that unlike the re-
ported approach,33 in our experiments the reductive
amination proceeded in a very stereospecific manner;
thus in each operation only one stereoisomer was gained
under the reaction conditions we used. The configura-
tions of products were confirmed by their NMR spectra
analyses. The coupling constants (J = 9.0–10.0 Hz) and
the peak type (triplet) of H-4 NMR signals clearly veri-
fied the configurations of each product. The N-hydroxy-
ethyl substituted compound 7e (miglitol derivative) was
readily methylated in THF using NaH as a base to yield
the corresponding methylate 7f. Finally, catalytic
hydrogenolysis of compounds 7a–g over Pd–C in the
mixed solvent of acetic acid, water, and THF provided
the corresponding deprotected iminosugars 8a–g. Com-
pound 8h was obtained via acetylation of N-aminoethyl
iminosugar 8g. It is noteworthy that the N-phenyl deriv-
ative 7b afforded the product 8b with the aromatic ring
reduced over hydrogenolysis.
8a
8h
Control CyA
8b
8f
8c
8d
8g
8e
Figure 2. The inhibition effects on the secretion of IFN-c in mouse by
the eight compounds at 60 lM. Data are means SEM of at least
three independent experiments, p < 0.05.
ity to the IFN-c secretion, whereas compound 8d even
showed comparable inhibitory activity with CyA. As
shown in Figure 2, most of the synthetic compounds
showed IFN-c inhibition effects. Compounds 8c and
8d with non-polar N-side chains showed better inhibi-
tion than the polar chain, suggesting that hydrophobic
character of the N-side chain may be important to the
activity. But 8b with the non-polar substituent did not
display significant inhibition, probably due to the steric
hindrance of the cyclohexyl substituent. It seems that
the carbon atom number of the side chain can be varied
based on the fact that both N-butyl compound 8c and
N-decanyl compound 8d exhibited better inhibition.
Although 8c and 8f have similar side chain length, their
inhibitory effects are different. Actually, compound 8f
with N-polar substituted side chain showed less inhibi-
tory effect.
The assay of the secretion of IL-4 from splenocytes was
similar to the assay of IFN-c. The secretion of IL-4 was
detected from the supernatant of spleen cells by mice IL-
4 ELISA kit. The level of IL-4 secretion was reduced
58.0%, 57.0%, 44.0%, 90.0%, 57.0%, 58.8%, 64.0%,
and 59.7% when including 60 lM of compounds 8a–h,
respectively (Fig. 3). The reduction efficiency of imino-
sugar 8d is the strongest (better than CyA) among the
eight compounds. Since all the synthetic compounds
showed IL-4 inhibitory activity, it was clear that the
intrinsic character of iminosugar is critical to the activ-
ity. It was also found the secretion of IL-4 is relatively
insensitive to the variety of the N-substituents in imino-
sugars compared with that of IFN-c. The strongest
inhibitor 8d has a 10-carbon side chain, indicating the
2.3. The cytokine inhibitory activity and SAR
To evaluate the effects of the eight synthetic iminosugars
8a–h on the secretion of cytokines from the splenocytes
in mouse, the splenocytes were induced by 10 lg/mL of
concanavalin A with 60 lM concentration of the com-
pounds at 37 ꢁC, 5% CO2 for 72 h. The secretion of
IFN-c was detected from the supernatant of spleen cells
by the use of mice ELISA kit. Compared to the control,
the levels of IFN-c secretion were reduced 9.0%, 9.1%,
55.5%, 61.0%, 29.0%, 16.0%, 41.0%, and 9.0% when
including 60 lM of 8a–h, respectively (Fig. 2). It was
found that among the eight synthetic iminosugars, com-
pounds 8c and 8d displayed the strongest inhibitory abil-
H
O
O
O
O
BnO
BnO
BnO
BnO
O
BnO
BnO
H
BnO
BnO
BnO
H
OMe
+
OMe
H
9
6
Scheme 2.

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J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
100
80
60
40
20
0
Contorl CyA 8a
8b 8c
8d
8e
8f
8g
8h
Figure 3. The inhibition effects on the secretion of IL-4 in mouse by
the eight compounds at 60 lM. Data are means SEM of at least
three independent experiments, p < 0.05.
hydrophobicity of the side chain could be important to
the activity.
2.4. The glycosidase inhibition and SAR
The good IFN-c and IL-4 inhibitory activities showed
by N-alkylated derivatives 8c and 8d let us investigate
whether there are some correlations between the cyto-
kine inhibition and the glycosidase inhibition. So the
inhibitory activities of these iminosugars against a series
of glycosidases were evaluated.
As depicted in Table 1, the parent structure 1,6-dide-
oxynojirimycin (8a) showed no inhibition toward the
tested glycosidases.34 Compound 8d with the best
IFN-c and IL-4 inhibitory activities did not show signif-
icant glycosidase inhibition activity at the maximal
tested concentration. The N-butyl derivative 8c dis-
played extensive inhibitory activity against different gly-
cosidases compared to the other N-alkyl derivatives.
Although no selectivity was observed, it was displayed
that the N-alkylated iminosugars could enhance the gly-
cosidase inhibition to some extent. The N-cyclohexyl
derivative 8b, which had comparable side chain in length
to 8c, did not exhibit glycosidase inhibition. The miglitol
analogue 8e only showed b-glucosidase inhibition. It
was anticipated the N-alkylated iminosugars could show
selective inhibition against different glycosidases by the
variation of the N-substituted side chains. Compared
with 8e, the methylation derivative 8f totally lost the gly-
cosidase inhibitory activity, resulting from the sensitive
modification of iminosugars. The hydroxyethyl isostere
8g and its acetylated derivative 8h were assayed against
glycosidases. Neither of them showed significant inhibi-
tion. It was also noticeable that the inhibitory activity to
a-L-fucosidase did not meet our expectation. The results
again verified the conclusion that the minimum struc-
tural requirement for inhibition of a-L-fucosidase is
strict at C-2, C-3, C-4 positions corresponding to those
of L-fucose.35
Based on the results of glycosidase inhibition, it is
apparent that there is no dramatic correlation between

J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
1609
the cytokine inhibitory activity and glycosidase inhibi-
tory activity. The potential immunosuppressive mecha-
nisms by which iminosugars act still await
investigation.
residue was purified by column chromatography on sil-
ica gel (petroleum ether/EtOAc 12:1) to provide 7a
(105 mg, 77%) as white solids. 1H NMR (500 MHz,
CDCl3):
d
1.31 (d, J = 6.0 Hz, 3H), 1.95 (t,
J = 10.5 Hz, 1H), 2.32–2.38 (m, 1H), 2.98 (dd, J = 4.5,
11.0 Hz, 1H), 3.18 (t, J = 9.0 Hz, 1H), 3.24 (d,
J = 13.5 Hz, 1H), 3.51 (t, J = 9.0 Hz, 1H), 3.53–3.58
(m, 1H), 4.05 (d, J = 14.0 Hz, 1H), 4.50–4.58 (ABq,
J = 11.5 Hz, 2H), 4.62 (d, J = 11.0 Hz, 1H), 4.81 (d,
J = 11.0 Hz, 1H), 4.95 (d, J = 11.0 Hz, 1H), 4.96 (d,
J = 11.0 Hz, 1H), 7.20–7.33 (m, 20H). 13C NMR
(75 MHz, CDCl3): d 16.52, 54.28, 56.50, 60.83, 72.45,
75.27, 75.54, 78.52, 84.21, 87.02, 126.90, 127.40,
127.50, 127.58, 127.66, 127.83, 127.86, 128.25, 128.27,
128.35, 128.66, 138.40, 138.54, 138.63, 138.98. HRMS
(ESI, positive) for C34H37NO3 Calcd 508.2846
[(M+H)+]. Found: 508.2843.
3. Conclusions
We have developed an efficient ‘one-pot’ approach for
the synthesis of 1,6-dideoxy-N-alkyl iminosugars with
high stereoselectivity and in good yields. The synthetic
iminosugars showed cytokines IFN-c and IL-4 inhibi-
tion activity. Especially the compound 8d displayed
the strongest inhibitory effects on both the secretion of
IFN-c and IL-4 among eight synthetic compounds.
Compared with the parent 1-deoxynojirimycin (DNJ)
and our previously published iminosugars,20 compound
8d seemed to be more potent to the inhibition of conca-
navalin A induced mouse splenocyte proliferation.36
IFN-c is a representative of Th1 cytokines which can
activate macrophages, NK cells, and cell-mediated
immunity (CMI), plus the secretion of certain Ig iso-
types, while IL-4 is a representative of Th2 cytokines
which tend to favor isotype switching in the humoral im-
mune response. Since the cytokines (e.g., IFN-c) se-
creted by the Th1 subset act primarily in cell-mediated
immune response, whereas those (e.g., IL-4) secreted
by the Th2 subset function mostly in B-cell activation
and humoral response, our findings might open a new
avenue in the development of a new class of drugs pos-
sessing immunosuppressive activity. Using the acquired
SAR, future work will focus on the extension of this syn-
thetic strategy to the preparation of other iminosugar
derivatives bearing a diverse range of N-substituted
chains. Our results also showed that there seems to be
no direct correlation between the cytokines inhibition
and the glycosidases inhibition. The studies on detailed
immunosuppressive mechanism of iminosugars will be
under investigation.
4.1.2. 2,3,4-Tri-O-benzyl-N-phenyl-1,6-dideoxynojirimy-
cin (7b). Compound 7b was prepared from 6 as de-
scribed in the preparation of 7a, yielding 7b (73%
yield) as a colorless oil after column chromatography
(petroleum ether/EtOAc 15:1). 1H NMR (500 MHz,
CDCl3):
d
1.03 (d, J = 6.0 Hz, 3H), 2.84 (t,
J = 10.5 Hz, 1H), 3.01–3.06 (m, 1H), 3.30–3.37 (m,
2H), 3.67 (t, J = 8.5 Hz, 1H), 3.78–3.83 (m, 1H), 4.63
(d, J = 11.0 Hz, 1H), 4.66 (s, 2H), 4.84 (d, J = 11.0 Hz,
1H), 4.93 (d, J = 11.0 Hz, 1H), 4.96 (d, J = 11.0 Hz,
1H), 7.04–7.37 (m, 20H). 13C NMR (125 MHz, CDCl3):
d 17.24, 56.38, 59.21, 72.46, 75.08, 79.21, 84.26, 86.29,
123.81, 123.90, 127.49, 127.62, 127.72, 127.87, 128.37,
128.97, 138.38, 138.46, 138.89, 150.67. HRMS (ESI, po-
sitive) for C33H35NO3 Calcd 494.2690 [(M+H)+].
Found: 494.2690.
4.1.3. 2,3,4-Tri-O-benzyl-N-butyl-1,6-dideoxynojirimycin
(7c). Compound 7c was prepared from 6 as described in
the preparation of 7a, yielding 7c (68% yield) as a color-
less oil after column chromatography (petroleum ether/
1
EtOAc 12:1). H NMR (500 MHz, CDCl3): d 0.90 (t,
J = 7.5 Hz, 3H, CH3), 1.18 (d, J = 6.0 Hz, 3H), 1.21–
1.29 (m, 2H), 1.33–1.40 (m, 2H), 2.18 (t, J = 11.0 Hz,
1H), 2.28–2.31 (m, 1H), 2.45–2.50 (m, 1H), 2.62–2.68
(m, 1H), 3.04–3.10 (m, 2H), 3.47 (t, J = 9.0 Hz, 1H),
3.58–3.63 (m, 1H), 4.60 (d, J = 11.0 Hz, 1H), 4.65–4.72
(ABq, J = 11.5 Hz, 2H), 4.82 (d, J = 11.0 Hz, 1H), 4.94
(d, J = 11.0 Hz, 1H), 4.96 (d, J = 11.0 Hz, 1H), 7.23–
7.35 (m, 15H). 13C NMR (125 MHz, CDCl3): d 14.00,
15.89, 20.62, 26.27, 52.25, 54.56, 59.78, 72.73, 75.32,
75.47, 78.93, 84.21, 87.06, 127.41, 127.54, 127.59,
127.77, 127.86, 127.88, 128.28, 128.34, 138.56, 138.98.
HRMS (ESI, positive) for C31H39NO3 Calcd 474.3003
[(M+H)+]. Found: 474.2993.
4. Experimental
4.1. Chemistry
4.1.1. Typical ‘one-pot’ procedure for the synthesis of
2,3,4-tri-O-benzyl-N-benzyl-1,6-dideoxynojirimycin (7a).
Compound 627 (120 mg, 0.27 mmol) was dissolved in the
mixed solvent of CF3COOH and MeOH (V/V
CF3COOH/MeOH = 1:40, 6 mL). The mixture was
heated at 60 ꢁC for 10 min until TLC analysis showed
the complete disappearance of the starting material. A
slurry of benzylamine (40 lL, 0.38 mmol) in MeOH
(1 mL) was treated with acetic acid to adjust the pH va-
lue to 5–6, and this solution was added to the reaction
mixture over a period of 0.5 h by ice-water bath cooling.
A portion of NaCNBH3 (51 mg, 0.81 mmol) was then
added, and the mixture was stirred for 18 h at room tem-
perature. The mixture was quenched with 0.5 mL 2 N
HCl aqueous solution. After removal of the solvent,
the residue was dissolved in 10% Na2CO3 (4 mL), and
extracted with EtOAc. The EtOAc layers were com-
bined, dried (Na2SO4), filtered, and concentrated. The
4.1.4. 2,3,4-Tri-O-benzyl-N-decanyl-1,6-dideoxynojirimy-
cin (7d). Compound 7d was prepared from 6 as de-
scribed in the preparation of 7a, yielding 7d (80%
yield) as white solids after column chromatography
(petroleum ether/EtOAc 10:1). 1H NMR (500 MHz,
CDCl3): d 0.88 (t, J = 6.5 Hz, 3H), 1.18 (d, J = 9.0 Hz,
3H), 1.21–1.40 (m, 16H), 2.18 (t, J = 11.5 Hz, 1H),
2.28–2.31 (m, 1H), 2.45–2.50 (m, 1H), 2.61–2.67 (m,
1H), 3.04–3.10 (m, 2H), 3.47 (t, J = 9.0 Hz, 1H), 3.58–

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J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
3.62 (m, 1H), 4.60 (d, J = 11.5 Hz, 1H), 4.65–4.72 (ABq,
J = 11.5 Hz, 2H), 4.82 (d, J = 11.5 Hz, 1H), 4.93 (d,
J = 11.5 Hz, 1H), 4.95 (d, J = 11.5 Hz, 1H), 7.25–7.35
(m, 15H). 13C NMR (75 MHz, CDCl3): d 14.11, 15.87,
22.67, 24.00, 27.48, 29.31, 29.55, 29.60, 31.89, 52.60,
54.53, 59.76, 72.77, 75.35, 77.42, 78.90, 84.16, 87.05,
127.45, 127.62, 127.79, 127.90, 128.31, 128.37, 138.55,
138.98. ESI-MS: 558 [M+H+]. Anal. Calcd for
C37H51NO3: C, 79.67; H, 9.22; N, 2.51. Found: C,
79.50; H, 9.29; N, 2.42.
NMR (500 MHz, CDCl3): d 1.21 (d, J = 5.5 Hz, 3H),
2.13 (t, J = 10.5 Hz, 1H), 2.34–2.40 (m, 2H), 2.87–2.93
(m, 1H), 3.07–3.10 (m, 2H), 3.17–3.23 (m, 1H), 3.27–
3.35 (m, 1H), 3.52 (t, J = 9.0 Hz, 1H, H-4), 3.54–3.60
(m, 1H), 4.62 (d, J = 10.5 Hz, 1H), 4.67 (d, J = 11.5,
14.5 Hz, 1H), 4.72 (d, J = 11.5 Hz, 1H), 4.85 (d,
J = 11.0 Hz, 1H), 4.97 (d, J = 11.0 Hz, 1H), 4.99 (d,
J = 11.0 Hz, 1H), 5.05 (s, 1H), 5.12–5.17 (m, 2H),
7.24–7.37 (m, 20H). 13C NMR (75 MHz, CDCl3): d
16.15, 37.92, 50.76, 54.23, 60.77, 66.67, 72.83, 75.34,
75.51, 78.49, 83.92, 86.80, 127.56, 127.62, 127.70,
127.82, 128.13, 128.31, 128.36, 128.39, 128.51, 136.53,
138.36, 138.40, 138.87, 156.31. ESI-MS: 595 [M+H+].
Anal. Calcd for C37H42N2O5: C, 74.72; H, 7.12; N,
4.71. Found: C, 75.00; H, 7.32; N, 4.55.
4.1.5. 2,3,4-Tri-O-benzyl-N-hydroxyethyl-1,6-dideoxynoj-
irimycin (7e). Compound 7e was prepared from 6 as de-
scribed in the preparation of 7a, yielding 7e (70% yield)
as white solids after column chromatography (petro-
1
leum ether/EtOAc 3:1). H NMR (500 MHz, CDCl3):
d 1.21 (d, J = 6.0 Hz, 3H), 2.14 (dd, J = 10.0, 11.5 Hz,
1H), 2.32 (dt, J = 4.0, 13.5 Hz, 1H), 2.37–2.41 (m, 1H),
2.43 (br, 1H), 2.97–3.02 (m, 1H), 3.10 (t, J = 9.0 Hz,
1H), 3.13 (dd, J = 4.5, 11.5 Hz, 1H), 3.56–3.62 (m,
2H), 4.60 (d, J = 11.0 Hz, 1H), 4.64–4.72 (ABq,
J = 11.5 Hz, 2H), 4.83 (d, J = 11.5 Hz, 1H), 4.94 (d,
J = 11.5 Hz, 1H), 4.96 (d, J = 11.5 Hz, 1H), 7.24–7.34
(m, 15H). 13C NMR (125 MHz, CDCl3): d 16.33,
52.83, 54.36, 58.52, 61.02, 72.86, 75.36, 75.52, 78.47,
83.90, 86.78, 127.48, 127.63, 127.69, 127.73, 128.31,
128.35, 128.39, 138.33, 138.79. ESI-MS: 462 [M+H+].
Anal. Calcd for C19H35NO4: C, 75.46; H, 7.64; N,
3.03. Found: C, 75.68; H, 7.68; N, 2.96.
4.1.8. 1,6-Dideoxynojirimycin (8a). A mixture of 7a
(37.0 mg, 0.073 mmol) and 10% Pd–C (10.0 mg) in
HOAc (2.0 mL), H2O (1.0 mL), and THF (0.5 mL)
was stirred for 24 h under H2 atmosphere. The catalyst
was then removed by filtration through Celite, and the
filtrate was concentrated. The residue was subjected to
a C-18 reversed-phase column chromatography (H2O)
to give 8a (in the form of acetate) (13.0 mg, 95%) as sol-
ids after lyophilization. The data were in good agree-
ment with those reported by Dhavale et al.38 1H NMR
(500 MHz, D2O): d 1.38 (d, J = 7.0 Hz, 3H), 1.97 (s,
3H), 2.89 (t, J = 12.0 Hz, 1H), 3.08–3.14 (m, 1H), 3.35
(t, J = 10.0 Hz, 1H, H-4), 3.43–3.46 (m, 2H), 3.73–3.75
(m, 1H). 13C NMR (125 MHz, D2O): d 15.41, 46.76,
55.84, 68.04, 73.41, 76.77. HRMS (ESI, positive) for
C6H13NO3 Calcd 148.0968 [(M+H)+]. Found: 148.0964.
4.1.6. 2,3,4-Tri-O-benzyl-N-methoxyethyl-1,6-dideoxynoj-
irimycin (7f). A suspended mixture of compound 7e
(88 mg, 0.19 mmol) and NaH (9 mg, 0.22 mmol) in
THF (5 mL) was vigorously stirred under ice-water bath.
MeI (14 lL, 2.2 mmol) was then added to the mixture.
The mixture was stirred overnight, and the reaction was
quenched with 0.5 mL MeOH. The reaction mixture
was concentrated. The syrup was diluted with CH2Cl2,
washed successively with saturated NaHCO3 and brine.
The organic layer was dried and concentrated. The
residue was chromatographed on silica gel with petro-
leum ether–EtOAc (8:1) to give 7f (83 mg, 92%) as white
solids. 1H NMR (500 MHz, CDCl3): d 1.21 (d,
J = 6.0 Hz, 3H), 2.29 (t, J = 11.0 Hz, 1H), 2.38 (dd,
J = 6.5, 8.5 Hz, 1H), 2.63–2.68 (m, 1H), 2.89–2.95 (m,
1H), 3.10 (t, J = 9.0 Hz, 1H), 3.14 (dd, J = 4.5, 11.5 Hz,
1H), 3.32 (s, 3H), 3.39–3.48 (m, 3H), 3.58–3.63 (m, 1H),
4.60 (d, J = 11.0 Hz, 1H), 4.65–4.71 (ABq, J = 11.0 Hz,
2H), 4.82 (d, J = 11.0 Hz, 1H), 4.93 (d, J = 11.0 Hz,
1H), 4.96 (d, J = 11.0 Hz, 1H), 7.24–7.34 (m, 15H). 13C
NMR (125 MHz, CDCl3): d 16.11, 29.67, 51.55, 55.28,
58.88, 60.30, 69.71, 72.77, 75.35, 75.52, 78.64, 83.91,
86.98, 127.44, 127.60, 127.61, 127.78, 127.89, 128.30,
128.36, 138.49, 138.54, 138.96. HRMS (ESI, positive)
for C30H37NO4 Calcd 476.2795 [(M+H)+]. Found:
476.2791.
4.1.9. N-Cyclohexyl-1,6-dideoxynojirimycin (8b). Com-
pound 8b was prepared from 7b as described in the prep-
aration of 8a, yielding 8b (97% yield) as solids after
1
lyophilization. H NMR (500 MHz, D2O): d 1.03–1.35
(m, 7H), 1.44–1.52 (m, 1H), 1.57–1.63 (m, 2H), 1.73–
1.81 (m, 3H), 2.16 (t, J = 11.5 Hz, 1H), 2.46–2.51 (m,
1H), 2.86 (t, J = 11.5 Hz, 1H), 3.04–3.10 (m, 2H), 3.20
(t, J = 9.0 Hz, 1H, H-4), 3.46–3.51 (m, 1H). 13C NMR
(125 MHz, D2O): d 14.35, 23.20, 25.94, 26.55, 26.80,
31.44, 49.72, 57.54, 58.75, 70.50, 75.97, 78.92. HRMS
(ESI, positive) for C12H23NO3 Calcd 230.1751
[(M+H)+]. Found: 230.1744.
4.1.10. N-Butyl-1,6-dideoxynojirimycin (8c). Compound
8c was prepared from 7c as described in the preparation
of 8a, yielding 8c (100% yield) in the form of acetate, as
solids after lyophilization. 1H NMR (500 MHz, D2O): d
0.92 (t, J = 7.0 Hz, 3H), 1.30–1.34 (m, 2H), 1.36 (d,
J = 6.0 Hz, 3H), 1.54–1.67 (m, 2H), 1.90 (s, 3H), 2.78 (t,
J = 11.5 Hz, 1H), 2.90–3.00 (m, 2H), 3.14 (dt, J = 5.0,
11.5 Hz, 1H), 3.28 (t, J = 9.5 Hz, 1H, H-4), 3.38–3.42
(m, 2H), 3.68–3.73 (m, 1H). 13C NMR (125 MHz,
D2O): d 13.63, 13.90, 20.23, 25.46, 53.24, 54.36, 61.72,
67.80, 73.71, 77.03. HRMS (ESI, positive) for
C10H21NO3 Calcd 204.1594 [(M+H)+]. Found: 204.1587.
4.1.7. 2,3,4-Tri-O-benzyl-N-(N0-benzyloxycarbonyl)-ami-
noethyl-1,6-dideoxynojirimycin (7g). Compound 7g was
prepared from 6 as described in the preparation of 7a
using mono-Cbz protected ethyldiamine,37 yielding 7g
(72% yield) as white solids after column chromatogra-
phy (petroleum ether/EtOAc 12:1) separation. 1H
4.1.11. N-Decanyl-1,6-dideoxynojirimycin (8d). Com-
pound 8d was prepared from 7d as described in the prep-
aration of 8a, yielding 8d (99% yield) as solids after
lyophilization. 1H NMR (500 MHz, CD3OD): d 0.89

J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
1611
(t, J = 6.5 Hz, 3H), 1.19 (d, J = 6.0 Hz, 3H), 1.26–1.32
(m, 14H), 1.45–1.48 (m, 2H), 2.12–2.17 (m, 2H), 2.44–
2.49 (m, 1H), 2.68–2.74 (m, 1H), 2.93–2.99 (m, 2H),
3.09 (t, J = 9.0 Hz, 1H, H-4), 3.45–3.50 (m, 1H). 13C
NMR (125 MHz, CD3OD): d 5.45, 6.90, 14.72, 16.07,
19.58, 21.44, 21.62, 21.67, 21.72, 24.05, 44.87, 48.77,
52.77, 61.85, 67.97, 71.18. HRMS (ESI, positive) for
C16H33NO3 Calcd 288.2533 [(M+H)+]. Found:
288.2536.
3H), 1.97 (s, 3H, Ac), 2.41–2.47 (m, 2H), 2.75–2.81 (m,
1H), 2.88–2.94 (m, 1H), 3.08 (t, J = 9.0 Hz, 1H), 3.12
(dd, J = 4.5, 11.5 Hz, 1H), 3.25 (t, J = 9.0 Hz, 1H, H-
4), 3.35 (t, J = 7.0 Hz, 2H), 3.53–3.58 (m, 1H). 13C
NMR (125 MHz, D2O): d 15.12, 22.62, 35.51, 50.91,
56.03, 60.53, 69.37, 75.40, 78.33, 174.97. HRMS (ESI,
positive) for C10H20N2O4 Calcd 233.1496 [(M+H)+].
Found: 233.1495.
4.2. Biological assay
4.1.12. N-Hydroxyethyl-1,6-dideoxynojirimycin (8e).
Compound 8e was prepared from 7e as described in
the preparation of 8a, yielding 8e (92% yield) as solids
after lyophilization. 1H NMR (500 MHz, D2O): 1.36
(d, J = 6.5 Hz, 3H), 2.77 (t, J = 11.5 Hz, 1H), 2.90–
2.94 (m, 1H), 3.00–3.05 (m, 1H), 3.27–3.35 (m, 2H),
3.41 (t, J = 9.5 Hz, 1H, H-4), 3.46 (dd, J = 4.5,
12.0 Hz, 1H), 3.69–3.74 (m, 1H), 3.81–3.91 (m, 2H).
13C NMR (125 MHz, D2O): d 14.29, 54.48, 54.96,
56.80, 62.41, 67.81, 73.72, 77.17. HRMS (ESI, positive)
for C8H17NO4 Calcd 192.1230 [(M+H)+]. Found:
192.1228.
4.2.1. General procedure for cytokine inhibition experi-
ments
4.2.1.1. Preparation and cultivation of splenocytes.
The spleens from BALB/c mice were taken out in sterile
conditions and soaked in non-serum RPMI-1640 cell
culture medium. The spleens were grinded using a wire
mesh. The cell suspension was filtered with a 200-mesh
nylon net. The filtrate of the splenocytes was centrifuged
at 2000g for 10 min and then the supernatant was re-
moved. The precipitate was dissolved in 5 mL of pH
7.2 Tris–NH4Cl solution and the cells was incubated
at 37 ꢁC for 6–10 min in order to lyse the red cells. Then
the cells were centrifuged at 2000g for 7 min and the cell
pellets were dissolved in RPMI-1640 culture medium
with 10% Newborn Calf Serum. Counting cells and
adjusting the concentration of cells solution to 5 · 106/
mL was done, followed by addition of 5 · 105 cells into
each well of 96-well plates for cultivation and assay.
4.1.13. N-Methoxyethyl-1,6-dideoxynojirimycin (8f).
Compound 8f was prepared from 7f as described in
the preparation of 8a, yielding 8f (96% yield) as solids
after lyophilization. 1H NMR (500 MHz, D2O): 1.27
(d, J = 6.5 Hz, 3H), 2.54 (t, J = 11.0 Hz, 1H), 2.62–
2.65 (m, 1H), 2.86–2.90 (m, 1H), 3.15–3.22 (m, 2H),
3.27 (dd, J = 7.5, 12.0 Hz, 1H), 3.31 (t, J = 9.5 Hz, 1H,
H-4), 3.36 (s, 3H), 3.60–3.65 (m, 2H), 3.68–3.72 (m,
1H). 13C NMR (125 MHz, D2O): d 14.84, 51.78, 55.87,
58.83, 61.75, 68.27, 68.59, 74.60, 77.82. HRMS (ESI, po-
sitive) for C9H19NO4 Calcd 206.1387 [(M+H)+]. Found:
206.1380.
4.2.1.2. Measurement of the secretion of IFN-c and IL-
4 from splenocytes. Subsequently, cells pretreated with
10 lg/mL concanavalin A (type IV) for 4 h were incu-
bated with different concentration of each compound
into each well, and incubated at 37 ꢁC, 5% CO2 for
72 h. The supernatant was collected and centrifuged at
2000g for 5 min. The supernatant was collected and
stored at ꢀ20 ꢁC until the assay. Ninety-six-well plates
were coated with anti-mouse IFN-c and IL-4 MAb in
advance (commercial products). Different concentra-
tions of drugs and IL-4 or IFN-c standards (500, 250,
125, 62.5, 31.25, 15.63 pg/mL) were added into each
well, and incubated at 20–25 ꢁC for 120 min. The levels
of IFN-c and IL-4 secreted from immunized mice
splenocytes were detected using the cytokine-specific
ELISA Kits (R&D). Standard curves were determined
using known concentration of the IL-4 or IFN-c.
According to the standard curve, the amount of the
samples was then determined.
4.1.14. N-Aminoethyl-1,6-dideoxynojirimycin (8g). Com-
pound 8g was prepared from 7g as described in the prep-
aration of 8a, yielding 8g (83% yield) as solids after
lyophilization. 1H NMR (500 MHz, D2O): 1.40 (d,
J = 6.0 Hz, 3H), 2.85 (t, J = 11.5 Hz, 1H), 2.95–3.01
(m, 1H), 3.24–3.46 (m, 6H), 3.48–3.54 (m, 1H), 3.73
(dt, J = 5.0, 9.5 Hz, 1H). 13C NMR (125 MHz, D2O):
d 14.18, 34.85, 49.27, 55.05, 62.46, 67.69, 73.63, 76.83.
HRMS (ESI, positive) for C8H18N2O3 Calcd 191.1390
[(M+H)+]. Found: 191.1388.
4.1.15. N-(N0-Acetyl)aminoethyl-1,6-dideoxynojirimycin
(8h). To solution of 8g (25 mg, 0.14 mmol) in pyridine
(5 mL) was added acetic anhydride (100 lL, 1.12 mmol)
under ice-bath cooling. After the mixture was stirred for
5 h, MeOH was added to quench the reaction. The sol-
vent was evaporated under reduced pressure. The resi-
due was co-evaporated with toluene (3 mL) three
times, the residue was then dissolved again in MeOH
(5 mL). One percentage of catalytic amount of MeO-
Na/MeOH (30% w/v) was added, after 20 min, the mix-
ture was neutralized to pH 7 by Dowex-50 [H+] resin.
The resin was removed by filtration through Celite,
and the filtrate was concentrated. The residue was sub-
jected to a C-18 reversed-phase column chromatography
(H2O) to give 8h (29 mg, 95%) as solids after lyophiliza-
4.2.2. General procedure for glycoside inhibition experi-
ments.39,40 Each assay was performed in phosphate and
nitrate buffer (25 mM), with p-nitrophenyl glycoside
derivatives of the sugars except for b-galactosidase assay
in which o-nitrophenyl b-galactoside was used. The
reactions were initiated by the addition of enzyme to a
solution of the substrate (0.4–5 mM) in the presence or
absence of various concentrations of iminosugars in buf-
fer which had been thermodynamically equilibrated.
After the mixture was incubated for 20–25 min at the
optimized temperature, the reaction was quenched by
the addition of 200 mM Na2CO3 solution. The absor-
bance of the resulting mixture was recorded at 400 nm
1
tion. H NMR (500 MHz, D2O): 1.22 (d, J = 6.0 Hz,

1612
J. Zhou et al. / Bioorg. Med. Chem. 16 (2008) 1605–1612
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23. Watson, A. A.; Fleet, G. W. J.; Asano, N.; Molyneux, R.
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Cronin, L.; O’Brien, J. L.; Murphy, P. V. J. Org. Chem.
2004, 69, 3565–3568; (b) Takahata, H.; Banba, Y.; Ouchi,
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Sohoel, H.; Bulow, A.; Bols, M. J. Am. Chem. Soc. 2001,
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values were determined as a concentration of an imino-
sugar that inhibits 50% of the enzyme activity.
Acknowledgments
This work was financially supported by the National
Natural Science Foundation of China, ‘973’ and ‘863’
Grants from the Ministry of Science and Technology
of China.
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