Studies on the stereoselective synthesis and immunosuppressive activity of dihydroartemisinin-O-glycoside derivatives

Article abstract of DOI:10.1016/j.bmcl.2020.127338

Eight new dihydroartemisinin-O-glycosides were synthesized with their relative configurations were determined based on NMR spectrum. In vitro immunosuppressive assay showed that 10α-dihydroartemisinin-β-O-D-mannoside (19a) demonstrate 88percent inhibition towards T cells proliferation and 98percent reduction in IFN-γ levels in cell media. These results suggest that dihydroartemisinin-O-glycoside as a potential lead for further in vivo evaluation.

Full text of DOI:10.1016/j.bmcl.2020.127338

Journal Pre-proofs
Studies on the Stereoselective Synthesis and Immunosuppressive Activity of
Dihydroartemisinin-O-Glycoside Derivatives
Yu Yu, Jingfeng Yu, Xiaosu Zou, Wei Xu, Jinghua Zhang, Hongzhu Bian,
Xun Dong, Zhengwu Shen
PII:
S0960-894X(20)30449-2
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127338
BMCL 127338
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
23 March 2020
2 June 2020
6 June 2020
Please cite this article as: Yu, Y., Yu, J., Zou, X., Xu, W., Zhang, J., Bian, H., Dong, X., Shen, Z., Studies on the
Stereoselective Synthesis and Immunosuppressive Activity of Dihydroartemisinin-O-Glycoside Derivatives,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127338
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Studies on the Stereoselective Synthesis and Immunosuppressive Activity of
Dihydroartemisinin-O-Glycoside Derivatives
Yu Yu#, b, Jingfeng Yu#, b, Xiaosu Zoub, Wei Xub, Jinghua Zhanga,
a, b
Hongzhu Bianc, Xun Dongc and Zhengwu Shen*,
a. Shanghai Jiao Tong University School of Medicine
b. Shanghai University of Traditional Chinese Medicine
c. Yunnan Baiyao Group Co., Ltd.
Abstract: Eight new dihydroartemisinin-O-glycosides were synthesized with their relative
configurations were determined based on NMR spectrum. In vitro immunosuppressive assay showed
that 10α-dihydroartemisinin-β-O-D-mannoside (19a) demonstrate 88% inhibition towards T cells
proliferation and 98% reduction in IFN-γ levels in cell media. These results suggest that
dihydroartemisinin-O-glycoside as a potential lead for further in vivo evaluation.
Keyword: Dihydroartemisinin; Glycoside; Immunosuppressive;
Correspondence: ZW Shen, Shanghai Jiao Tong University, School of Medicine, 128 South
Chongqing Road, Shanghai 200023, P.R. China. E-mail: Jeff_shen_1999@yahoo.com
Artemisinin is a natural sesquiterpene lactone isolated from Artemisia annua 1 with a unique
peroxobridge hemiacetal motif. Artemisinin and its derivatives are widely reported for its
anti-malarial and anti-tumor activities.2-5 In addition, artemisinin derivatives have potent
immunosuppressive properties and have been reported effective for treating of autoimmune diseases
6
7-8
including Systemic Lupus Erythematosus (SLE) , Atopic Dermatitis (AD)
and Rheumatoid
Arthritis (RA).9 Currently clinically available artemisinin derivatives drugs include anti-malarial
drugs such as artesunate, artemether, arteether and dihydroartemisinin (DHA).10 Among them, DHA
demonstrated therapeutic effect in SLE patients and is currently under Phase II clinical trial
(ClinicalTrials.gov Identifier: NCT03396393). The mode of action may involves an increased
SIGIRR expression and a blockage of TLR4/NF-κB signal pathway. 11-12 However, a main limitation
of DHA is its structural stability due to an active hemiacetal hydroxyl group. β-aminoarteether
maleate (SM934, 3，Fig 1) improved the stability of DHA by protecting the hemiacetal hydroxyl
group. In addition, the solubility, bioavailability and immunosuppressive activity of SM 934 were
also significantly improved. In vitro studies showed SM 934 had outstanding inhibitory effects on
lymphocyte proliferation, with an IC50 of 1.2 μM for T lymphocytes proliferation and 2.6 μM for B
lymphocytes proliferation.13 SM934 is currently under the Phase II clinical trial for the SLE
treatment in China (ClinicalTrials.gov Identifier: NCT03951259).
H
5 H
H
6
4
7
8
2
O
3
O
O
O
1
H
O
O
O
O
O
12
H
13
H
O
9
O
CO2H
CO2H
O
O
11
10
O
NH2
OH
Artemisinin (1)
Dihydroartemisinin (2)
SM934 (3)
1

Figure 1. The structures of Artemisinin (1), Dihydroartemisinin (2) and SM934 (3)
Based on the safety and activity profile of DHA and SM934, it is of interest to look for other
artemisinin derivatives with improved activities. From medicinal chemistry point of view, DHA
contains an active hemiacetal hydroxyl group, a formation of ether or ester on this hydroxyl group is
a direct choice for structural modification. For example, the well-known anti-malarial drugs,
artemether and artesunate, were derived from DHA through this strategy. Another approach for DHA
structure modification is to conjugate a saccharide to DHA, as it is expected to improve the water
14
solubility and bioavailability of artemisinin. In 1992, Lin A. J. et al
prepared a series of
dihydroartemisinin α-O-glycosides and evaluated their anti-malarial activities. (Figure 2.) However,
the anti-malarial activity of dihydroartemisinin α-O-glycosides was not significantly improved. This
result could be attributed to the difficulty for water-soluble dihydroartemisinin α-O-glycosides to
penetrate the pellicle of plasmodium.
O
CH2OH
CH2OH
CH2OH
OH
OH
O
O
O
O
OH
OH
OH
O
CH2OH
O
O
O O
O
O
O
OH
OH
O
OH
O
OH
O
O O
O
O
O
O
O
O
O
OH
O O
O
OH
OH
H
H
H
H
H
H
H
H
OH
4a
4b
4c
4d
O
O
O
O
O
O
O
O
O
H
H
O
H
O
H
O
O
O
O
O
CH2OH
CH2OH
OH
OH
CH2OH
O
O
O
O
O
O
O
H
H
O
H
OH
OH
OH
H
OH OH
OH
OH
OH
5a
OH
OH
5b
5c
5d
Figure 2. Dihydroartemisinin α-O-glycosides reported 14
The glycosylation reaction used involves the hemiacetal hydroxyl group of the sugar
nuclephilically attacking the oxonium ion 6 (Figure 3.）obtained from the elimination of the
hemiacetal hydroxyl of dihydroartemisinin. Due to steric hindrance of artemisinin’s peroxyl bridge
and the 6α-methyl, the nucleophiles attack predominantly form the β-orientated acetal bond.
H
CH2OAc
CH2OAc
O
O
O
OAc
OAc
O
O
OAc
Br
OAc
OAc
H
10
O
OAc
9
8
6
7
Figure 3. The key reactive intermediates of glycosylation reaction.
2

Figure 4. The illustration of α/β facial nuclephilic attack to 10-position of 6
Ren Y.R. et al.15 use a slightly different strategy to synthesize the dihydroartemisinin-β-O
glycosides. Dihydroartemisinin was used as a nucleophile to attack bromineated saccharide (7).
Since the dihydroartemisinin is a mixture of 10α-(OH) dihydroartemisinin and 10β-(OH)
dihydroartemisinin, theoretically the final product should be a mixture of diastereomers. However,
only 10β-dihydroartemisinin-O-glycosides were isolated according to the literature. It is suspected to
be attributed to the fact that 10α-dihydroartemisinin has higher tendency to eliminate and form
artemisitene.
H
H
O
O
O
O
O
H
H
TMSBr / (COCl)2
O
O
O
O
O
O
H
H
O
O
O
H
H
O
O
X
R
N
N
N
N
10: X=Br
11: X=Cl
9: R=α-OTMS
2: R=-OH, X=Cl
OH
OH
OH
O
O
O
H
O
HO
HO
HO
H
O
OH
O
O
O
H
O
O
O
OH
OH
H
Piperazine
O
Glucose
N
OH
N
N
OH
OH
13b
13c
O
N
H
HO
OH
13a
12
Scheme 1. Synthesis of N-glycosylated DHA piperazine derivatives
In order to obtain 10α-dihydroartemisinin-O-glycoside, Nicoletta Basilico et al.16 used
piperazine as a nucleophile to react with 10β-halogenated dihydroartemisinin, and obtained
10α-piperazine substituted dihydroartemisinin derivative (12). The subsequent aminoglycosidation
reaction yielded three novel 10α-N-dihydroartemisinin-N-glycoside piperazine derivatives (Scheme
1. 13a-13c). These three compounds showed good anti-malarial and anti-cancer activities. However,
due to lack of comparison data, it still is difficult to conclude which configuration of
dihydroartemisinin-O-glycoside has better biological activities.
3

As described above, a numbers of dihydroartemisinin-O-glycosides were synthesized with their
anti-malarial and anti-cancer activities reported. However, the immunosuppressive activity of
dihydroartemisinin-O-glycoside were not yet explored. Herein, we report the synthesis of eight
dihydroartemisinin-O-glycoside derivatives and their immunosuppressive activities.
CH2OH
CH2OAc
CH2OAc
O
O
O
c or d
OH
b
a
OAc
OAc
OH
OAc
OH
OH
OAc
OAc
OH
OAc
15
OAc
16
14
CH2OH
CH2OAc
O
O
OH
OH
OAc
CH OAc
HN
2
O
O O
O
O
O O
O
CCl3
OAc
O
OAc
O
O
f
e
OH
OAc
OAc
O
H
H
OAc
H
H
17
18
4a
(a) Ac2O/NaOAc, 130oC. (b) Hydrazine acetate, DMF, r.t. (c) CNCCl3, DBU, DCM, 0 oC
(d) CNCCl3, K2CO3, DCM, r.t. (e) BF3.Et2O, DHA, DCE, -20 oC. (f) K2CO3, MeOH, r.t.
CH2OH
CH2OH
OH
OH
CH2OH
O
O
O
O
O
OH
OH OH
H
O
O
O O
O
O O
O
OH
O
OH
O
OH
O
O
O
OH
O
O
CH2OH
O
O O
O
H
OH OH
H
H
H
H
H
H
OH
4a
4b
19a
19b
OH
OH
O
CH2OH
CH2OH
O
OH
O
OH
OH
O
O
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
OH
OH
O
O
OH OH
O
O
OH
O O
H
H
H
H
H
H
H
H
19c
19d
19e
19f
Scheme 2. Synthesis of DHA-glycosides
As illustrated in Scheme 2, pentacetyl D-glucose (15) was used as starting material and
Schmidt reaction17 was used as the key step in our synthesis. After selectively removing 1-acetyl at
room temperature, the crude product 16 reacted with trichloroacetonitrile to form an active
intermediate trichloroacetimidate ester 17. Intermediate 17 underwent glucosidation reaction with
.
dihydroartemisinin catalyzed by BF3 Et2O. The 10β-dihydroartemisinin-O-tetraacetyl glucoside 18
was obtained. Hydrolyzation of the acetyl groups on 18 with K2CO3 in methanol, yielded the
10β-dihydroartemisinin-O-glucoside (4a) in 6.4% overall yield in four steps. In the synthesis, 17 was
converted into an active intermediate oxonium ion 8, then the hemiacetal hydroxyl group of
dihydroartemisinin nucleophilic attacking oxonium ion 8 to form corresponding glycoside. Since the
hemiacetal hydroxyl group of 10α-dihydroartemisinin can easily undergo elimination reaction in
acidic condition, the stereochemistry of the product is dependent on the relative speed of
4

gluosidation and elimination reaction. If the glycosidation speed is faster than the elimination, the
main product will be the mixture of 10β-dihydroartemisinin-O-glycoside and
10α-dihydroartemisinin-O-glycoside. If the glycosidation speed is slower than the elimination, the
main product will be the 10β-dihydroartemisinin glycoside since the 10α-dihydroartemisinin was
consumed. Due to the planar nature of the oxonium ion, as illustrated in Figure 5, the nucleophilic
attack of 10β-dihydroartemisinin hemiacetal hydroxyl group to the oxonium ion 8 is in favor from
the less steric hindrance below the Harworth structure of the saccharide to form the
10β-dihydroartemisinin-O-glycoside , and the two biggest groups on the saccharide moiety of the
product, 6-hydroxymethlene group and the 1-dihydroartemisinin were in equatorial conformation.
(Figure 5), In contrary, the 10α-dihydroartemisinin hemiacetal hydroxyl group tend to attack the
oxonium ion 8 from above the Harworth structure of the saccharide to form the
10α-dihydroartemisinin-O-glycoside (Figure 6). Hence, total eight dihydroartemisinin-O-glycosides
were
synthesized,
namely,
10β-dihydroartemisinin-α-O-D-glucoside
(4a);
10β-dihydroartemisinin-α-O-D-galactoside (4b); 10α-dihydroartemisinin-β-O-D-mannoside (19a);
10β-dihydroartemisinin-α-D-mannoside (19b); 10β-dihydroartemisinin-α-D-xyloside (19c);
10β-dihydroartemisinin-β-L-arabinoside (19d)； 10α-dihydroartemisinin-α-L-rhamnoside （19e）；
10β-dihydroartemisinin-α-D-maltoside (19f).
Glucosidation of 10α-Dihydroartemisinin (left),
Glucosidation of 10β-Dihydroartemisinin(Right)
Figure 5. The illustration of stereoselectivity of glucosidation of dihydroartemisinin
Among them, 4a, 4b, 19c-19f were obtained as the major product. In mannose case, the ortho
effect of axial orientated 2-acetoxyl increased the reactivity of the oxonium ion, the speed of
glycosidation reaction was faster than the elimination reaction, hence
a mixture of
10α-dihydroartemisinin-β-O-D-mannoside and 10β-dihydroartemisinin-α-O-D-mannoside was
obtained. Since 10α-hydroxyl-dihydroartemisinin is in favor of attacking the oxonium ion from
above the Harworth structure while 10β-hydroxyl-dihydroartemisinin tend to approach the oxonium
ion from below the Harworth structure. Therefore, 19a and 19b were obtained at a ratio of
approximately 1:1.
5

The relative configuration of compound 19a and compound 19b were determined by NMR
spectroscopic method (Figure 6). The chemical shift of H-1 on glucose of 19a was δ 5.03 ppm
(J1-2=1.6 Hz) and H-10 on dihydroartemisinin was δ 4.76 ppm (J9-10=9.2 Hz), while the chemical
shift of H-1 of compound 19b was δ 5.17 ppm (J1-2=2.0Hz), H-10 on dihydroartemisinin of 19b was
δ 5.11 ppm (J9-10=3.2 Hz). The chemical shift and the characteristics coupling constants of these
protons were consistent with the literature.18-19 The configuration of glucoside bonds of 19c-19f were
also determined by NMR spectroscopic method.18 The configuration of 19c, 19d and 19f were
assigned to 1α-xyloside, 1β-arabinoside and 1α-simaltoside respectively. Although, it is difficult to
determine the configuration of glucoside bond of rhamnoside（19e）by simply comparing the
coupling constant, the conformation analysis of rhamnoside (Figure 7) indicated the chemical shift
of H-1 of 1α-rhamnoside should shift towards the lower field due to the induction effect of axial
3-OH. Hence, the chemical shift of H-1 and H-10 of 19e justified the 1β, 10α configuration (Table
1).
Table 1. Selected NMR data of dihydroartemisinin glycosides
4a
4b
19a
19b
19c
19d
19e
19f
5.22 (3.7)
5.09 (3.2)
1α,10β
5.25 (3.9)
5.09 (3.3)
1α, 10β
5.05 (1.3)
4.78 (9.4)
1β,10α
5.19 (1.6)
5.13 (3.4)
1α, 10β
5.16 (3.6)
5.01 (3.3)
1α, 10β
5.23 (3.1)
5.02 (3.3)
1β, 10β
5.14 (1.6)
4.82 (9.4)
1β,10α
5.22 (3.8)
5.16 (3.7)
1α, 10β
δH-1 (JHz)
δH-10 (JHz)
configuration
Figure 6. The characteristic 1H-NMR of 19a and 19b
6

1α-arhamnoside
1β- rhamnoside
Figure 7. Conformation analysis of rhamnoside
Table 2. Dihydroartemisinin glycosides inhibiting T cells proliferation in vitro
CD4+ T Cell
IC50 (μM)
17.85
10.41
3.49
CD8+ T Cell
IC50 (μM)
19.41
8.63
Compounds
4a
4b
19a
2.65
19b
19c
>32
>32
10.85
14.07
3.83
9.52
19d
19e
12.95
3.36
19f
9.09
4.00
DHA
SM934
4.03
5.53
1.05
1.36
The dihydroartemisinin glycosides synthesized were assessed for their proliferation inhibitory
activities 13 on human T cells and the results were depicted in Table 2.
In h ib itin g C D 4 + T C e ll P ro life ra tio n in V itro
In h ib itin g C D 8 + T C e ll P ro life ra tio n in V itro
19a
19e
100
80
60
40
20
0
100
80
60
40
20
0
D H A
S M 934
-20
-20
0.1
1
10
100
0.1
1
10
100
C o n c t.(μ M )
C o n c t.(μ M )
Figure 8. Anti-proliferation activity of 19a and 19e
*dose response curves fitted by 4 parameters non-linear regression(GraphPad Prism). Error bar
expressed as mean±SD (n=3).
As shown in Table 2, both 19a and 19e demonstrated enhanced in vitro activity in suppressing
T cell proliferation in a dose dependent manner (Figure 8). The IC50 of 19a and 19e inhibiting CD4+
T cells proliferation was 3.49、3.83 μM, and 2.65、3.36 μM inhibiting CD8+ T cells respectively.
(Table 2).
7

In h ib itio n % o f IF N -γ le v e l
IF N -γ le v e l
25000
20000
15000
10000
5000
0
100
80
32μM
8μM
2μM
***
60
***
***
***
***
***
40
***
***
***
***
***
***
20
a
9
e
9
4
3
d
A
e
H
t
1
1
9
a
e
9
4
a
D
A
M
9
3
9
e
H
r
S
1
1
t
D
n
M
S
U
Figure 9. 19a and 19e impaired IFN-γ level.
Error bar represent mean±SD(n=3). *** P < 0.0001
Immune cell dysfunction and imbalance of pro- and anti- inflammatory processes are key
features of autoimmune diseases. Interferon gamma (IFN-γ) is a pivotal pro-inflammatory cytokine
involved in immune response and inflammation. Artemisinin analogs have been reported to
ameliorate IFN-γ levels and alter Th1 cell response in RA, SLE and multiple sclerosis murine
models. 20-23 Thus, it is of interest to explore whether 19a and 19e demonstrate similar activity in T
cell activation process. Human primary T cells were isolated from PBMC and co-incubated with
above compounds for 120 hrs under the stimulation of CD3/CD28 T cell activator. IFN-γ levels in
the cell culture media was tested through ELISA. As depicted in Table 3, 19a and 19e decreased
IFN-γ recovery in the cell media dose-dependently, concurring with the inhibition effect of T cell
proliferation. Among compounds tested, 19a demonstrated the highest potentency and efficacy in
inhibiting T cell proliferation and reducing IFN-γ levels. (Figure 9)
In addition, there are two interesting observations: 1). 19a and 19b are diastereomers, however,
19a was more potent than 19b in decreasing cell media IFN-γ. 2). Both 19a and 19e have
10α-dihydroartemisinin moiety with different glycoside configurations and are more potent
compared to other derivatives. Therefore, we hypothesizes that the configuration of
dihydroartemisinin plays an important role in immunosuppressive activity, and dihydroartemisinin
glycosides with 10α -configuration exhibit an improved activity.
Several mechanisms were proposed for immunosuppressive activities of artemisinin
derivatives.24 In addition, Zuo J. P. et al demonstrated that artemether impaired T cells activation and
proliferation, decreased IFN-γ and IL-2 secretion through ERK1/2 inhibition.25 Wang H. et al
observed similar results on artesunate. They also proved that artesunate increases the percentage of
CD4+CD25+Foxp3+ regulatory T cells (Treg). 26 SM934 (NCT03951259) was found to ameliorate
the symptoms of RA, reduced percentage of Th1 and Th17 populations in collagen-induced arthritis
mice model.27,28 However, it is still unknow how the stereochemistry of dihydroartemisinin
glycosides influences their immunosuppressive activities. Since 19a has superior activity of
inhibiting IFN-γ secretion and T cell proliferation than SM934 and DHA, we believe 19a worth for
further in vivo studies on autoimmune diseases animal models.
In summary, we synthesized eight novel dihydroartemisinin-O-glycosides, of which 19a and
8

19e showed foremost inhibitory effect towards T cell proliferation and IFN-γ media recovery. Hence,
19a and 19e may have the potential to exhibit similar therapeutic effect in vivo.
Acknowledgments
This project was funded by the National Natural Science Foundation of China，fund approval
number 81673278.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may
be considered as potential competing interests:
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Inhibiting CD8+ T Cell Proli
19a
19e
100
80
60
40
20
0
DHA
SM934
Concentr
-20
0.1
1
10
100
IFN-γ le
25000
20000
15000
10000
5000
0
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