Synthetic iminosugar derivatives as new potential immunosuppressive agents

Article abstract of DOI:10.1021/jm050169t

Several iminosugar derivatives were synthesized, and their effects on the secretion of IL-4 and IFN-γ from the mouse splenocytes were examined. The effects on membrane expression of other T cell-associated molecules (CD3, CD4, CD8) and B cell-associated m

Full text of DOI:10.1021/jm050169t

3688
J. Med. Chem. 2005, 48, 3688-3691
Synthetic Iminosugar Derivatives as New
Potential Immunosuppressive Agents
Xin-Shan Ye,†,* Fang Sun,†,‡ Min Liu,§ Qin Li,†
Yuhang Wang,† Guisheng Zhang,‡ Li-He Zhang,† and
Xiao-Lian Zhang§,*
The State Key Laboratory of Natural and Biomimetic
Drugs, School of Pharmaceutical Science, Peking University,
Xue Yuan Road #38, Beijing 100083, China, College of
Chemistry and Environmental Science, Henan Normal
University, Xinxiang 453002, China, and Department of
Immunology, College of Medicine, Wuhan University,
Wuhan 430071, China
Figure 1. Structures of the iminosugar derivatives 1-5.
Scheme 1a
Received February 22, 2005
Abstract: Several iminosugar derivatives were synthesized,
and their effects on the secretion of IL-4 and IFN-γ from the
mouse splenocytes were examined. The effects on membrane
expression of other T cell-associated molecules (CD3, CD4,
CD8) and B cell-associated molecules (CD19) were also inves-
tigated. The experimental data demonstrated that synthetic
iminosugars hold potential as immunosuppressive agents.
The search to find better immunosuppressants in
transplantation has been stimulated by the need to
improve transplant survival and by the reduction of the
toxicity of current agents. The main immunosuppressive
agents currently used in organ transplantation, such as
cyclosporin A (CyA), tacrolimus, mycophenolate mofetil,
and sirolimus, have significant side-effects including
nephrotoxicity, neurotoxicity, infection, cancer, new-
onset posttransplant diabetes mellitus, hyperlipidemia,
and hypertension.1-5 At present there is no antidote for
the toxicity of these transplant drugs. Although many
treatment options for organ transplant patients have
been developed,6 there remains a great need for effective
and safe immunosuppressive agents.
Iminosugars, also called the “sugar-shaped alkaloids”,
are carbohydrate analogues in which the ring oxygen
has been replaced by nitrogen and are found to be
widespread in plants and microorganisms.7 They are
frequently found to be potent inhibitors of many carbo-
hydrate-processing enzymes involved in important bio-
logical systems.8-10 These unique molecules promise a
new generation of iminosugar-based medicines in a wide
range of diseases such as diabetes,11 viral infections,12
tumor metastasis,13 and lysosomal storage disorders.14
However, these iminosugar derivatives as immunosup-
pressive agents are less explored, and little is known
about their inhibition effects on immune system re-
sponses. So far only castanospermine (CAST), an in-
dolizidine alkaloid derived from the Australian rainfor-
est plant Castanaospermum australe,15 was found to
exhibit some immunosuppressive activity.16-18 We re-
port herein the synthesis of several iminosugar deriva-
tives 1-5 (Figure 1) and their effects on the secretion
of IL-4 and IFN-γ from the mouse splenocytes. Further-
a Reagents and conditions: (a) NBS, acetone-water, 75 °C, 97%;
(b) Ph3PdCH2, THF, 80 °C, 79%; (c) PCC, CH2Cl2, 80%; (d)
NH2OH‚HCl, KHCO3, 85 °C, 96%; (e) LiAlH4, Et2O, r.t.; then
CbzCl, K2CO3, THF, 93%; (f) Hg(OAc)2, THF, 85 °C; then KCl/
H2O/CHCl3, r.t.; (g) NaBH4, O2, DMF, r.t., 65%; (h) H2/Pd/C, HOAc:
H2O:THF ) 4:2:1, r.t., 92-97%.
more, the effects on membrane expression of other T
cell-associated molecules (CD3, CD4, CD8) and B cell-
associated molecules (CD19) were investigated.
The synthesis of iminosugars 1, 2, and 3 was achieved
in six steps starting from a D-galactose derivative 719
by way of a chain extension-amination-cyclization
sequence (Scheme 1). The key synthetic step involved
an intramolecular amidomercuration reaction.
As shown in Scheme 1, tetra-O-benzyl-D-galactopy-
ranose (7), easily prepared from the thiogalactoside 6,20
underwent Wittig methylenation by modified Martin’s
procedure21 to give the corresponding heptenitol 8 in
high yield. Oxidation of 8 with PCC produced the ketone
* To whom correspondence should be addressed. Fax: (+86)10-
62014949. E-mail: xinshan@bjmu.edu.cn or zhangxl65@hotmail.com.
† Peking University.
‡ Henan Normal University.
§ Wuhan University.
10.1021/jm050169t CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/04/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3689
Scheme 2a
Figure 2. The inhibition effects on the secretion of IL-4 in
mouse by the five compounds at 25 µM. Data are means (
SEM of at least three independent experiments, p < 0.05.
a Reagents and conditions: (a) PCC, 4 Å MS, CH2Cl2, r.t., 86%;
(b) NaClO2, NaH2PO4, CH3CN-H2O, H2O2, r.t., 60%; (c) H2/Pd-
C, HOAc:H2O:THF) 4:2:1, r.t., 92% for 4, 98% for 5.
9 which was readily converted to the oxime 10 by
treatment with hydroxylamine hydrochloride in the
presence of potassium bicarbonate. Although it was
reported22 that the reduction of the oxime in the glucose
derivative gave preponderantly the corresponding D-
gluco aminoheptenitol, we found in the galactose case
it was different. Reduction of the oxime 10 followed by
protection for the amino group afforded 11 as a chro-
Figure 3. The inhibition effects on the secretion of IFN-γ in
mouse by the five compounds at 25 µM. Data are means (
SEM of at least three independent experiments, p < 0.05.
D-galacto epimers in excellent yield (ratio ∼L1-.a5l:1tr)o. Wainthd
matographically inseparable mixture of
the mixture in hand, the mercury-mediated cyclization
of unsaturated amidoalditols was attempted. Upon
treatment with mercuric acetate, after ligand exchange
with the saturated KCl and reductive oxygenation using
NaBH4-O2, we fortunately obtained the three separable
cyclized isomeric alcohols 12, 13, and 14 in 26%, 29%,
and 10% isolated yield, respectively. Theoretically, in
this step four possible stereoisomers could be formed.
But we isolated only three isomers presumably due to
the stereoselectivity of aminomercuration for the D-
galacto amidoalditol derivative. It can be also explained
by the outcome of the cyclized products (the ratio of 13
+ 14 to 12 is 39% to 26%, that is 1.5 to 1 in accord with
the ratio of the starting material 11). Finally, catalytic
hydrogenolysis of 12, 13, and 14 over Pd-C in the mixed
solvent of acetic acid, water, and THF gave the corre-
sponding iminosugar 1, 2, and 3 in the form of acetate.
The structure identification of iminosugars 1-3 and
their precursors 12-14 was based on the analysis of
their 1D (1H, 13C) and 2D NMR spectroscopy (COSY,
HSQC, gHMBC, NOESY) (see the Supporting Informa-
tion), and structures of 1 and 2 were further confirmed
by comparison with the published data.21,23 Although 1
and 2 are known compounds used as R-galactosidase
inhibitors, by use of the modified procedure we prepared
iminosugars 1-3 at the same time in an efficient way
which involved in less synthetic steps.
aldehyde 15 in 86% isolated yield. Oxidation of 15 with
sodium chlorite provided the acid 16. Full deprotection
of 15 over Pd-C in acetic acid led to the iminosugar
derivative 4 in the form of acetate. Likewise, the L-altro
epimer 5 was prepared from the alcohol 13 in the same
manner.
To assay the effects of the five synthetic iminosugars
1-5 (in the form of acetate) on the secretion of cytokines
from the splenocytes in mouse, the spleen cells were
induced by Concanavalin A with 25 µM concentration
of the compounds at 37 °C, 5% CO2 for 72 h. The
secretion of IL-4 was detected from the supernatant of
spleen cells by the use of mice ELISA kit. Compared to
the control, the levels of IL-4 secretion were reduced
69.2%, 92.3%, 84.6%, 30.8%, and 92.3% when including
25 µM of 1, 2, 3, 4, and 5, respectively (Figure 2). It
was found that among the five synthetic iminosugar
derivatives, compounds 5 and 2 displayed the strongest
inhibition ability to the IL-4 secretion.
The assay of the secretion of IFN-γ from splenocytes
was similar to the assay of IL-4. The supernatant of
spleen cells were detected by mice IFN-γ ELISA kit. The
level of IFN-γ secretion were reduced 75.9%, 86.2%,
51.6%, 31.2%, and 24.2% when including 25 µM of
compound 1, 2, 3, 4, and 5 respectively (Figure 3). The
reduction efficiency of iminosugar 2 is the strongest
among the five compounds.
The corresponding carboxylic acid derivatives 4 and
5 of iminosugars 1 and 2 were also synthesized (Scheme
2). The alcohol 12 was treated with PCC to give the
T cells and B cells are two major types of lymphocytes.
CD3 and CD19 molecules are hallmarks on the T cells
and B cells, respectively. T cells contain two classes of

3690 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Letters
Figure 4. Effects of the compound 2 and 5 on the expression of CD3 (A), CD4 (B), CD8 (D), and CD19 (C) in mouse splencytes.
CyA was used as 30 µM concentration in the test; a, b, c represented as three different concentrations respectively: 10 µM, 30
µM, and 90 µM. Data are means ( SEM of at least three independent experiments.
T lymphocytes, T helper cells, which contain CD4
markers, and T cytotoxicity cells, which contain CD8
markers. The effects of these iminosugars on the
expression of CD3, CD4, CD8, and CD19 were measured
by the flow cytometer (FCM).24 We chose compound 2
and 5, due to their strongest inhibition effects on the
secretion of cytokines in mice, to further measure the
effects on the expression of major immune molecules on
the lymphocytes. It was first found that both compounds
2 and 5 had inhibition effects on CD3, hallmarks on the
surfaces of T cells, CD4, markers of CD4+T cells, and
CD19 molecules which were hallmarks on the surfaces
of B cells (Figure 4 A, B, C); however, they had
stimulating effects on CD8 (Figure 4 D). Compound 2
had even higher effects on all of these molecules than
compound 5 did. These results suggested that the
synthetic compound 2 and 5 had specific inhibition
effects on the CD4+T cells and B cells, while CyA had
the strongest inhibition effects on CD3 and CD4 mol-
ecules, and had no effects on CD19 and CD8 molecules.
The 50% inhibitory concentrations (IC50) was deter-
mined by the tetrazolium chlorimetric reduction assay
(MTT assay), which measures the mitochondrial dehy-
drogenase activity of surviving cells. IC50 of these five
compounds (1, 2, 3, 4, and 5) (in the form of acetate)
and CyA to mouse splenocytes proliferation by 72 h MTT
assay were 519 µM, 332 µM, 272 µM, 340 µM, 346 µM,
and 19.4 µM, respectively. These results showed that
these five synthetic compounds were much less toxic
than CyA to mouse PBMCs.
enormous therapeutic potential in many diseases. How-
ever, the effects of iminosugars on immune system and
the application as immunosuppressants have been less
investigated. To explore the potential application of this
class of compounds as immunosuppressive agents, D-
galacto- and L-altro- iminosugar derivatives were de-
signed and synthesized in an efficient way. Further-
more, the effects of the synthetic compounds 1-5 on the
secretion of cytokines and immune molecules surfaces
expression were examined.
CD4+T helper (Th) cells usually control proliferation
and differentiation, whereas CD8+T cytotoxic (Tc) cells
usually kill aberrant cells. CD4+T cells recognize the
antigen peptide and MHC II complex and are activated
as Th1 and Th2 cells. Th1 cells secrete IFN-γ and IL-2,
while Th2 cells secrete IL-4, IL-5, IL-6, and IL-10. IFN-γ
mediate Th1 type of cellular immune response, while
IL-4 and IL-5 stimulate B cells to secrete antibodies.25
The ratio of the two Th cell types, Th1 and Th2, is
closely correlated with the outcome of many diseases.
Th1 responses predominate in organ-specific autoim-
mune disorders, acute allograft rejection, and in some
chronic inflammatory disorders. In contrast, Th2 re-
sponses predominate in Omann’s syndrome, transplan-
tation tolerance, chronic graft-versus-host disease, sys-
temic sclerosis, and allergic diseases.
These iminosugars may have different mechanisms
to inhibit cytokine secretion and CD markers expression
from CyA (form complex with cyclophilin). CyA selec-
tively inhibits the transcription and expression of IL-2
gene and mainly inhibits CD4 Th cells, while imino-
sugars are carbohydrate mimetics and are potent in-
hibitors of many carbohydrate-processing enzymes. All
cytokines and CD molecules are N-glycoproteins. The
It is well-known iminosugars mimicking the struc-
tures of monosaccharides inhibit glycosylation process-
ing enzymes because of a structural resemblance to the
sugar moiety of the natural substrate and might have

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3691
(6) Stock, P. G. The year in review - ATC 2002. Am. J. Transplant.
2003, 3, 373-380.
(7) Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J. Sugar-
mimic glycosidase inhibitors: natural occurrence, biological
activity and prospects for therapeutic application. Tetrahedron:
Asymmetry 2000, 11, 1645-1680.
(8) Dwek, R. A. Glycobiology: Toward understanding the function
of sugars. Chem. Rev. 1996, 96, 683-720.
(9) Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Recent
developments of transition-state analogue glycosidase inhibitors
of non-natural product origin. Chem. Rev. 2002, 102, 515-553.
(10) Stu¨tz, A. E. Iminosugars as Glycosidase Inhibitors: Nojirimycin
and Beyond; Wiley-VCH: Weinheim, 1999.
(11) Andersen, B.; Rassov, A.; Westergaard, N.; Lundgren, K. Inhibi-
tion of glycogenolysis in primary rat hepatocytes by 1,4-dideoxy-
1,4-imino-d-arabinitol. Biochem. J. 1999, 342, 545-550.
(12) Durantel, D.; Branza-Nichita, N.; Carrouee-Durantel, S.; But-
ters, T. D.; Dwek, R. A.; Zitzmann, N. Study of the mechanism
of antiviral action of iminosugar derivatives against bovine viral
diarrhea virus. J. Virol. 2001, 75, 8987-8998.
(13) Goss, P. E.; Baker, M. A.; Carver, J. P.; Dennis, J. W. Inhibitors
of carbohydrate processing: A new class of anticancer agents.
Clin. Cancer Res. 1995, 1, 935-944.
(14) Sawkar, A. R.; Cheng, W.-C.; Beutler, E.; Wong, C.-H.; Balch,
W. E.; Kelly, J. W. Chemical chaperones increase the cellular
activity of N370S beta-glucosidase: A therapeutic strategy for
Gaucher disease. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15428-
15433.
(15) Hohenschutz, L. D.; Bell, E. A.; Jeweess, P. J. Castanospermine,
a 1,6,7,8-tetrahydroxyoctahydroindolizine alkaloid, from seeds
of Castanospermum Australe. Phytochemistry 1981, 20, 811-
814.
(16) Grochowicz, P. M.; Hibberd, A. D.; Smart, Y. C.; Bowen, K. M.;
Clark, D. A.; Cowden, W. B.; Willenborg, D. O. Castanospermine,
an oligosaccharide processing inhibitor, reduces membrane
expression of adhesion molecules and prolongs heart allograft
survival in rats. Transplant Immunol. 1996, 4, 275-285.
(17) Walter, S.; Fassbender, K.; Gulbins, E.; Liu, Y.; Rieschel, M.;
Herten, M.; Bertsch, T.; Engelhardt, B. Glycosylation processing
inhibition by castanospermine prevents experimental autoim-
mune encephalomyelitis by interference with IL-2 receptor signal
transduction. J. Neuroimmunol. 2002, 132, 1-10.
(18) Wall, K. A.; Pierce, J. D.; Elbein, A. D. Inhibitors of glycoprotein
processing alter T-Cell proliferative responses to antigen and
to interleukin 2. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5644-
5648.
(19) Koto, S.; Morishima, N.; Miyata, Y.; Zen, S. Preparation of
2,3,4,6-tetra-O-benzyl-D-mannose. Bull. Chem. Soc. Jpn. 1976,
49, 2639-2640.
immunosuppressive effects by these iminosugars may
hold possibly the N-glycoprotein-processing inhibition
effects on these glycoproteins. The data present here
demonstrated that compared with the synthetic D-
galacto type of iminosugars, the L-altro type of imino-
sugars had stronger reduction effects on the immune
system. The cytokines (e.g. IFN-γ) secreted by the Th1
subset act primarily in cell-mediated response, whereas
those (e.g. IL-4) secreted by the Th2 subset function
mostly in B-cell activation and humoral response. The
compound 2 had the strongest inhibition effects on both
secretion of IFN-γ and IL-4 among these compounds and
therefore might hold reduction efficiency to both hu-
moral response and cell-mediated immune system. On
the other hand, compound 5 had specifically the strong-
est effects on the secretion of IL-4 and only minor
inhibition effects on the secretion of IFN-γ and therefore
might have inhibition efficiency to the Th2-mediated
humoral immune reaction. Both compound 2 and 5
inhibited the expression of CD3, CD4, and CD19, so that
they may specifically inhibit the function of CD4+T cells
and B cells. However, compounds 2 and 5 had some
stimulating effects on the expression of CD8 molecules.
The mechanisms of these differences are not well-
known.
Our study first found that L-altro iminosugars 2 and
5 are much less toxic than CyA, which is a well-known
immunosuppressive drug. Our results demonstrated
that synthetic iminosugars, especially the L-altro type
of iminosugars, hold the potential as immunosuppres-
sive agents. In summary, the described findings may
open a new avenue in the development of a new class
of drugs possessing immunosuppressive activity.
Acknowledgment. This work was financially sup-
ported by the National Natural Science Foundation of
China (No. 20172005 and 20372004 for X.Y., No.
30370310 and 30270076 for X.Z.), and the Fund for
Oversea Returnee of National Educational Ministry of
China, the Scientific Research Grant (No: 2002AA301-
B15) from Hubei Province (X.Z.), and Peking University
(X.Y.).
(20) Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.;
Wong, C.-H. Programmable one-pot oligosaccharide synthesis.
J. Am. Chem. Soc. 1999, 121, 734-753.
(21) Martin, O. R.; Saavedra, O. M.; Xie, F.; Liu, L.; Picasso, S.; Vogel,
P.; Kizu, H.; Asano, N. alpha- and beta-homogalactonojirimycins
(alpha- and beta-homogalactostatins): Synthesis and further
biological evaluation. Bioorg. Med. Chem. 2001, 9, 1269-1278.
(22) Liu, P. S. Total synthesis of 2,6-dideoxy-2,6-imino-7-O-(.beta.-
Supporting Information Available: Full experimental
procedures and characterization data for all compounds. This
material is available free of charge via Internet at http://
pubs.acs.org.
D-glucopyranosyl)-D-glycero-L-gulo-heptitol hydrochloride.
potent inhibitor of.alpha.-glucosidases. J. Org. Chem. 1987, 52,
A
References
(1) Myers, B. D.; Sibley, R.; Newton, L.; Tomlanovich, S. J.; Boshkos,
C.; Stinson, E.; Luetscher, J. A.; Whitney, D. J.; Krasny, D.;
Coplon, N. S. et al. The long-term course of cyclosporine-
associated chronic nephropathy. Kidney Int. 1988, 33, 590-600.
(2) Brayman, K. L.; Stephanian, E.; Matas, A. J.; Schmidt, W.;
Payne, W. D. Sutherland, D. E. R. Analysis of infectious
complications occurring after solid organ transplantation. Arch.
Surg. 1992, 127, 38-48.
4717-4721.
(23) Shilvock, J. P.; Nash, R. J.; Watson, A. A.; Winters, A. L.;
Butters, T. D.; Dwek, R. A.; Winkler, D. A.; Fleet, G. W. J.
Piperidine analogues of D-galactose as potent inhibitors of
R-galactosidase: Synthesis by stannane-mediated hydroxym-
ethylation of 5-azido-1,4-lactose. Structural relationships be-
tween D-galactosidase and L-rhamnosidase inhibitors. J. Chem.
Soc., Perkin Trans. 1 1999, 2747-2754.
(24) Zhang, X.-L.; Liu, M.; Xie, P.; Wan, S.; Ye, J. T.; Zhou, X.; Wu,
J. Specific inhibition effects of N-pentafluorobenzyl-1-deoxy-
nojirimycin on human CD4+ T cells. Bioorg. Med. Chem. Lett.
2004, 14, 3789-3792.
(3) Sheil, A. G. R. Cancer in organ transplant recipients: part of
an induced immune deficiency syndrome. Br. Med. J. 1984, 288,
659-670.
(4) Ganadian Multicentre Transplant Study Group, A randomized
clinical trial of cyclosporine in cadaveric renal transplantation.
N. Engl. J. Med. 1983, 309, 809-815.
(5) 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. et al. Prevention of organ allograft
rejection by a specific Janus kinase 3 inhibiter. Science 2003,
302, 875-878.
(25) Sallustro, F.; Lanzavecchia, A.; Mackay, C. R. Chemokines and
chemokine receptors in T-cell priming and Th1/Th2-mediated
responses. Immunol Today 1998, 19, 568-574.
JM050169T