Synthesis and biological evaluation of celastrol derivatives as potential immunosuppressive agents

Article abstract of DOI:10.1021/acs.jnatprod.0c00067

Celastrol, a friedelane-type triterpenoid isolated from the genus Triperygium, possesses antitumor, anti-inflammatory, and immunosuppressive activities. A total of 42 celastrol derivatives (1a?1t, 2a?2l, and 3a?3j) were synthesized and evaluated for their immunosuppressive activities. Compounds 2a?2e showed immunosuppressive effects, with IC₅₀ values ranging from 25 to 83 nM, and weak cytotoxicity (CC₅₀ > 1 μM). Compound 2a, with a selectivity index value 31 times higher than that of celastrol, was selected as a lead compound. Further research showed that 2a exerted its immunosuppressive effects by inducing apoptosis and inhibiting cytokine secretion via Lck- and ZAP-70-mediated signaling pathways.

Full text of DOI:10.1021/acs.jnatprod.0c00067

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Article
Synthesis and Biological Evaluation of Celastrol Derivatives as
Potential Immunosuppressive Agents
Qi-Wei He,# Jia-Hao Feng,# Xiao-Long Hu, Huan Long, Xue-Feng Huang, Zhen-Zhou Jiang,
Xiao-Qi Zhang, Wen-Cai Ye, and Hao Wang*
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ABSTRACT: Celastrol, a friedelane-type triterpenoid isolated from the genus Triperygium, possesses antitumor, anti-inflammatory,
and immunosuppressive activities. A total of 42 celastrol derivatives (1a−1t, 2a−2l, and 3a−3j) were synthesized and evaluated for
their immunosuppressive activities. Compounds 2a−2e showed immunosuppressive effects, with IC50 values ranging from 25 to 83
nM, and weak cytotoxicity (CC50 > 1 μM). Compound 2a, with a selectivity index value 31 times higher than that of celastrol, was
selected as a lead compound. Further research showed that 2a exerted its immunosuppressive effects by inducing apoptosis and
inhibiting cytokine secretion via Lck- and ZAP-70-mediated signaling pathways.
utoimmune diseases are emerging noncommunicable
Adiseases, affecting 5−8% of the population of developed
countries and imposing a heavy burden of morbidity and
mortality on the population.1 Immunosuppressive agents are
the main clinical drugs for treatment of some autoimmune
diseases, including systemic lupus erythematosus, rheumatoid
arthritis, psoriasis, and glomerulonephritis, and organ trans-
plants.2 Most of the representative immunosuppressive drugs
are developed from microbial secondary metabolites such as
cyclosporin A, rapamycin, and tacrolimus. Although these
immunosuppressants exert satisfactory therapeutic effects on
organ transplantation and autoimmune diseases, their clinical
applications are considerably limited due to their side effects,
such as renal or liver toxicity, infections, malignancy, and other
adverse effects.3 Thus, there is an urgent need to explore new
potential immunosuppressants with high efficacy and low
toxicity.
Tripterygium wilfordii Hook f, known as Leigongteng
(Thunder God Vine) in Traditional Chinese Medicine, is
widely used to treat autoimmune and inflammatory diseases.
The single-herb preparation, “Leigongteng Duogan Tablets”,
has been used clinically as a medicine to treat autoimmune
diseases, such as systemic lupus erythematosus (SLE),
dermatomyositis, and rheumatoid arthritis, for half a century
in China.4,5 Celastrol, a friedelane-type triterpenoid isolated
from T. wilfordii, possesses a variety of biological activities,
including anti-inflammatory,6 anticancer,7−9 antiobesity,10,11
antifungal,12 and antiviral activities.13,14 Recently, celastrol was
reported to display immunosuppressive properties by inhibit-
ing T cell proliferation and Th17 cell induction.15 However,
the high toxicity and narrow safety margins of celastrol limit its
clinical applications.
In this work, a series of celastrol derivatives (1a−1t, 2a−2l,
and 3a−3j) were designed and synthesized. The immunosup-
pressive activities and toxicities of the celastrol derivatives were
evaluated in concanavalin A (Con A)-induced spleen cells in
vitro, and the structure−activity relationships (SARs) are also
discussed. Compound 2a, a methanamide analogue of celastrol
displaying the highest immunosuppressive effect (IC50 = 25
nM) with a selectivity index (SI) approximately 31 times
higher than that of celastrol, was selected as the lead
compound. Further investigations demonstrated that 2a
Received: January 19, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Scheme 1. Synthesis of Celastrol Derivatives 1a−1t
displayed an immunosuppressive effect by inducing apoptosis
and cytokine secretion via Lck- and ZAP-70-mediated signaling
pathways. These results establish a foundation for further
structural modifications of celastrol and the development of
new immunosuppressive agents with high efficiency and low
toxicity.
immunosuppressive activities of the indole substitution at the
quinone methide moiety. Owing to the instability of these
indole-substituted intermediate products, they were used
immediately in the next step without further purification.
Subsequently, the phenolic groups were acetylated in the
presence of DMAP and Ac2O. The stereochemistry of C-6
indole-substituted celastrol derivatives demonstrated that
nucleophilic addition to celastrol and related quinone methide
RESULTS AND DISCUSSION
■
structures was to the β-face (6R).17 With the aid of H−1H
1
The celastrol derivatives 1a−1t were synthesized by
nucleophilic reaction between celastrol and organic halides in
DMF at room temperature using NaHCO3 as the base
(Scheme 1). Next, amide derivatives 2a−2l were synthesized
via reaction of the activated carboxylic acid with the
corresponding amine (Scheme 2). Finally, a series of C-6
indole-substituted celastrol derivatives, 3a−3j, were synthe-
sized by a Friedel−Crafts reaction16 to verify the influence on
1
COSY, HSQC, and HMBC experiments, all the H and 13C
NMR data of 3b were totally assigned. The NOESY spectrum
showed the significant correlations between the H-2′ of the
indole moiety with the β-positioned H3-25, and no correlation
was observed between H-6 and H3-25, also indicating that the
relative configuration of H-6 was α-oriented (Scheme 3).
B
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Scheme 2. Synthesis of Celastrol Derivatives 2a−2l
The proliferation of murine splenocytes, predominantly
including T cells, could be triggered by Con A, which can be
applied to evaluate the immunosuppressive activities of
compounds in vitro.18 In this study, 42 celastrol derivatives
and cyclosporin A (CsA), the positive control, were tested in
vitro for their cytotoxic effects on splenocytes and inhibitory
effects on Con A-induced T cell proliferation. The values of
cytotoxicity index (CC50), immunosuppressive index (IC50),
and the SI of the 42 celastrol derivatives and CsA are shown in
Table 1. Among these compounds, the derivatives with amide
moieties 2a−2l (IC50 = 25−36.2 nM) displayed stronger
activities than ester derivatives 1a−1t (IC50 = 45.4−48.7 nM).
Furthermore, alkyl amide derivatives (2a−2e) showed potent
immunosuppressive activities, with IC50 values ranging from
approximately 25 to 83 nM; they also showed relatively low
cytotoxic effects on splenocytes, with CC50 values of >1.0 μM,
comparable to that of celastrol, with an IC50 value of 83 nM
and a CC50 value of 130 nM. However, other amide analogues
with heteroatom or heterocycle (2f−2l), ester analogue (1a−
1t), and C-6 indole-substituted celastrol derivatives (3a−3j)
exhibited no or little immunosuppressive activities. These
results indicated that the beneficial influence on immunosup-
pressive activities is associated with the amidation of a shorter
N-alkyl residue at the C-29 hydroxycarbonyl unit of celastrol
and that an intact quinone methide moiety is crucial for their
immunosuppressive and cytotoxic activities. Compound 2a, a
methanamide analogue of celastrol (IC50 = 25 nM), displayed
the best immunosuppressive activity compared to celastrol
(IC50 = 82 nM) and the positive control CsA (IC50 = 12.6
nM). To examine the cytotoxicity of 2a, activated and
unactivated mouse spleen cells were treated with different
concentrations of 2a and celastrol for 48 h. The results showed
that 2a possesses lower cytotoxicity than celastrol (Figure 1).
Collectively, compound 2a displayed the best immunosup-
pressive activity, exhibiting an SI value 31 times greater than
celastrol, and was selected as the lead compound for further
study.
Apoptosis is an essential mechanism in eradicating activated
T cells during the shutdown process of excess immune
responses, and it maintains an appropriate immune homeo-
stasis. However, the deficient apoptosis of activated T cells is
related to a wide variety of immune disorders.19 As a
representative of these celastrol derivatives, 2a has been
under investigation in in vitro experiments. Flow cytometry
(FCM) was employed to test whether 2a could lead to T cell
apoptosis. As shown in Figure 2A and B, 2a increased the
percentage of apoptosis by annexin V-FITC/PI staining in a
dose-dependent manner. The apoptosis rates of T cells were
36.3%, 52.6%, and 68.4%, respectively. The results indicated
that the mechanism of action of 2a is involved in the induction
of apoptosis of T cells. Furthermore, Bcl-2 and Bax are
apoptotic inhibitors or inducers. Cell survival in the initiation
phase of apoptosis mostly depends on the balance between the
Bcl-2 and Bax.20 Thus, the Bcl-2/Bax ratio could be used to
determine whether a cell has undergone apoptosis. Moreover,
Bcl-2 has been reported to inhibit Bax expression in
mitochondria and inhibit subsequent activation of caspase-3
and caspase-9, leading to cell death.21 The results showed that
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Scheme 3. Synthesis of Celastrol Derivatives 3a−3j
2a significantly increased the ratio of Bax/Bcl-2 (Figure 2C
and D) and induced the release of cytochrome c from the
mitochondria to the cytoplasm (Figure 2C and E).
Furthermore, Figure 2F and G demonstrate that 2a activated
caspase-3 and caspase-9. These results suggest that 2a induces
apoptosis of T cells through a mitochondrial apoptotic
pathway.
γ and interleukin-4 are the trait cytokines of Th1 and Th2 cells,
respectively. Our results showed that 2a (0.01, 0.1, and 1 μM)
decreased the level of IL-4 in the supernatant fluid of cultured
cells to 135.2
respectively, compared with the control group (167.8
13.8, 78.2
7.4, and 39.1
5.8 pg/mL,
9.4
pg/mL; Figure 3A). In addition, Figure 3B shows that 2a
(0.01, 0.1, and 1 μM) could also inhibit the secretion of INF-γ
T cells play an important role in maintaining the body’s
immune function, and the secretion of cytokine in T cells is
one of the important functions of its immune regulation. T-
helper cells are one of the important subpopulations of T cells;
they secrete a variety of cytokines, regulate the body’s immune
response, and play an important role in transplantation
immunity.22 According to the different patterns of cytokines,
the helper T cells are divided into distinct subsets of Th1 and
Th2 cells, known as subclasses of T-helper cells. Th1 cells
secrete pro-inflammatory cytokines, the responses of which
predominate in chronic inflammatory and autoimmune
diseases.23 Th2 cells generate cytokines, the responses of
which predominate in Omann’s syndrome, systemic sclerosis,
transplantation tolerance, chronic graft versus host disease, and
allergic diseases. The cytokines secreted by T cells play an
important role in regulating the body’s immune response. IFN-
to 264.5
37.5, 204.1
17.8, and 142.9
15.4 pg/mL,
respectively, compared with the control group (375.8 19.6
pg/mL). These results indicate that 2a can also exert
immunosuppressive activity by mediating the cytokine
secretion of T cells. Lymphocyte-specific kinase (Lck) is a
cytoplasmic tyrosine kinase of the Src family expressed in T
cells. The activation of Lck is critical for T cell activation, and
selective inhibition of Lck can produce immunosuppressive
effects. Indeed, an appropriate balance between active and
inactive Lck is essential for safe TCR (T cell receptor)
sensitivity, and abnormal T cell responsiveness occurs if the
balance is broken. A growing body of evidence has shown that
increased Lck phosphorylation is associated with T cell hyper-
responsiveness, which can induce an autoimmune disease
similar to that exhibited by SLE patients.24 Upon TCR
stimulation, ZAP-70 is then recruited to the phosphorylated
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Table 1. In Vitro Cytotoxicity on Lymph Node Cells and Inhibitory Effects of Compounds and Cyclosporin A (CsA) on
Splenocytes Co-stimulated by Concanavalin A
a
a
compound
CC50 (μM)
IC50 (nM)
SI
compound
CC50 (μM)
IC50 (nM)
SI
1a
1b
1c
1d
1e
1f
1g
1h
1i
2.4
1.4
1.2
0.9
1.5
1.3
1.7
1.3
1.4
1.1
1.1
1.3
1.1
1.9
1.7
1.8
1.8
6.7
2.0
15.1
1.2
1.1
1202
820
924
739
1088
454
2.0
1.3
1.3
1.2
1.4
2.8
1.7
1.1
1.3
1.6
1.4
1.0
1.0
1.2
1.6
1.1
1.7
1.4
1.4
8.1
47.7
34.6
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
3a
3b
3c
3d
3e
3f
3g
3h
3i
1.1
1.2
0.8
1.2
1.4
1.9
0.8
1.1
1.0
1.4
≥10
5.6
≥10
4.1
≥10
≥10
3.0
≥10
≥10
≥10
0.1
64
63
83
225
112
323
362
312
294
16.5
19.3
6.9
5.5
16.4
3.4
2.1
3.6
3.4
6.9
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1.5
994
1181
1121
721
1j
1k
1l
205
771
≥10 000
≥10 000
≥10 000
≥10 000
≥10 000
≥10 000
≥10 000
≥10 000
≥10 000
≥10 000
83
1255
1055
1602
1040
1548
1070
4872
1462
1875
25
1m
1n
1o
1p
1q
1r
1s
1t
3j
2a
2b
celastrol
CsA
b
31
9.8
26
77.8
a
Selectivity index [SI] is determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the
b
concentration of the compound needed to inhibit the proliferation to 50% (IC50) of the control value. Cyclosporin A was used as the positive
control.
Figure 1. Stimulated or nonstimulated spleen cells from 6- to 8-week-old BALB/c mice were incubated with different amounts of 2a and celastrol
for 48 h. Cell viability was measured by CCK-8 assay. (A) Effects of different concentrations of 2a on stimulated or nonstimulated spleen cells. (B)
Effects of different concentrations of celastrol on stimulated or nonstimulated spleen cells. (C) Effects of different concentrations of 2a and celastrol
on stimulated spleen cells, respectively. (D) Effects of different concentrations of 2a and celastrol on nonstimulated spleen cells, respectively. The
data are represented as mean SD for the three independent experiments. *p < 0.05 compared with the control group.
ITAMs and is subsequently activated by Lck. Activated ZAP-
70 phosphorylates LAT, resulting in the activation of multiple
pathways and eventually leading to IL-2 production and T cell
activation. Therefore, as key kinases responsible for regulating
the TCR signaling pathway, Lck and Zap-70 play key roles in
mediating T cell immune responses.25 Our results showed that
after stimulating with Con A the total and phosphorylated
levels of Lck and ZAP-70 were higher than those in the control
group. However, the total and phosphorylated levels of Lck
and ZAP-70 were significantly decreased in the 2a+ConA
group in a dose-dependent fashion (Figure 4). In addition, 2a
(0.01, 0.1, and 1 μM) could also significantly decrease the
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Figure 2. Effects of 2a on the apoptotic and mitochondrial apoptosis pathway in Con A-induced T cells. (A) Annexin V−PI double staining.
Distribution of viable (lower left, annexin V−PI−) necrotic (upper left, annexin V−PI+) late apoptotic (upper right, annexin V+PI+) and early
apoptotic (lower right, annexin V+PI−). (B) Bar graphs showing the quantitative results of AV−PI. (C) Original bands of Bax, Bcl-2, and
cytochrome c in the cytoplasm and mitochondria, which were normalized to β-actin. (D) Quantitative analysis of the ratio of Bax/Bcl-2. (E)
Quantitative analysis of the ratio of cytochrome c (cytoplasm/mitochondria). (F) Activity of active caspase-3. (G) Activity of active caspase-9. The
data are represented as mean SD for three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with a control group.
Figure 3. Effects of 2a on the secretion of IL-4, IFN-γ, and IL-2. (A) Level of IL-4 in the supernatant of cells. (B) Level of IFN-γ in the supernatant
of cells. (C) Level of IL-2 in the supernatant of cells. The data are represented as means SD for the three independent experiments. *p < 0.05,
**p < 0.01, and ***p < 0.001 compared with the control group.
secretion of IL-2 to 128.7 5.2, 79.1 4.5, and 51.1 15.1
pg/mL, respectively, compared to the control group (152.9
8.7 pg/mL) in Con A-stimulated mouse splenocytes (Figure
3C). These results were consistent with the mediation effect
on the cytokine secretion effect mentioned above, indicating
that 2a could inhibit the activation and function of T cells by
inhibiting p-Lck and p-ZAP-70 expression.
activities and toxicities were evaluated on ConA-induced
spleen cells in vitro for the first time. Among these compounds,
2a−2e, amide derivatives of celastrol, showed potent
immunosuppressive effects, with IC50 values ranging from 25
to 83 nM and weak cytotoxicity (CC50 > 1 μM). Compound
2a, the hit compound, displayed the highest immunosuppres-
sive effect (IC50, 25 nM), with an SI value of 47.7, 31-fold
higher than that of celastrol (SI = 1.5). Further research
demonstrated that 2a exhibited an immunosuppressive effect
In conclusion, a total of 42 celastrol derivatives were
designed and synthesized, and their immunosuppressive
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Figure 4. Effects of 2a on the expression of Lck and ZAP-70. (A) The cells were analyzed by Western blot to detect the expression of Lck, ZAP-70,
and phosphorylated Lck and ZAP-70, which were normalized to β-actin. (B−E) Relative densitometric analysis of the individual protein bands. The
data are shown as the mean SD for the three independent experiments. &&p < 0.01 compared with control and *p < 0.05; **p < 0.01; ***p <
0.001 compared with the 2a (0 μM) group.
dissolved in CH2Cl2 (2 mL) in a 10 mL sealed tube. After rigorous
stirring at room temperature, FeCl3·6H2O (0.65 mg, 0.0024 mmol)
was added to the solution, and the reaction mixture was stirred for 24
h. The reaction was quenched with water (10 mL), and the aqueous
phase was separated and extracted with EtOAc (3 × 20 mL). The
solvent was removed in vacuo, and the residue was used for the next
step without further purification. Ac2O (0.10 mL) and DMAP were
added to a solution of the intermediate residue in CH2Cl2 (2 mL) at
room temperature under nitrogen. After stirring for 24 h, the solvent
was removed under reduced pressure. The residue was purified by
flash chromatography (EtOAc/hexanes) to produce a gray solid.
Chemical names, yields, and physical data of 1a−1t, 2a−2k, and
3a−3j are described in the Supporting Information.
Biological Materials. Cell Counting Kit-8 (CCK-8) was obtained
from Dojindo Laboratories (Japan). Fetal bovine serum and RPMI
1640 medium were from Invitrogen (San Diego, CA, USA).
Cyclosporin A and concanavalin A were from Sigma Chemical Co.
(St. Louis, MO, USA). BCA protein determination kit, chemilumi-
nescence kit, caspase-3 and caspase-9 activity determination kits, red
blood cell lysis buffer, RIPA lysis buffer, and PMSF (protease
inhibitor) were purchased from Beyotime (Shanghai, People’s
Republic of China). ELISA kits for mice (IL-4, IL-2, and INF-γ)
were from EIAAB Science Co., Ltd. (Wuhan, People’s Republic of
China). Penicillin, streptomycin, annexin V−PI double staining kit,
mitochondrial protein extraction kit, and HRP-conjugated goat anti-
rabbit (mouse) IgG were obtained from KeyGEN BioTECH
(Nanjing, People’s Republic of China). Primary rabbit antibodies
for Bcl-2, Bax, cytochrome c, ZAP-70, and phospho-ZAP-70 (Tyr319)
were obtained from Cell Signaling Technology (Beverly, MA, USA).
Primary rabbit antibodies against Lck, phospho-Lck (Tyr394), were
purchased from Gene Tex (San Antonio, TX, USA).
by inducing apoptosis and inhibiting cytokine secretion via
Lck- and ZAP-70-mediated signaling pathways. These results
can provide some new insights into further structural
modification of celastrol to improve therapeutic activities and
reduce adverse effects.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Celastrol was purchased
from Dalian Meilun Pharm Co., Ltd. (Dalian, China), with over 99%
purity. The other chemicals and solvents were purchased from
commercial suppliers and were further purified or dried when
necessary. Column chromatography was performed with silica gel 60
(200−300 mesh). TLC analyses were performed on precoated silica
gel GF254 plates (Qingdao Haiyang Chemical, China). H and 13C
1
NMR spectra were recorded on a Bruker ARX-400 or AVANCE
NEO-500 NMR spectrometer in the indicated solvents (TMS as
internal standard), and the chemical shifts were expressed in δ (ppm)
and the coupling constants (J) in Hz. HRESIMS data were acquired
on an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies,
USA), equipped with an ESI ion source operating in positive mode.
Procedures for the Synthesis of Compounds 1a−1t.
Celastrol (22 mg, 0.048 mmol) in DMF (2 mL) was treated with
NaHCO3 (20 mg, 0.24 mmol) and the appropriate alkyl halide (0.05
mmol). After stirring for 24 h at room temperature, deionized water
(15 mL) was added and the mixture was extracted with EtOAc (2 ×
20 mL). The combined organic layers were washed with brine, dried
using anhydrous MgSO4, filtered, and evaporated in a vacuum until
dry. Each residue was further purified by flash column chromatog-
raphy on silica gel (EtOAc/hexanes) to produce a dark red solid.
Procedures for the Synthesis of Compounds 2a−2k.
Celastrol (22 mg, 0.048 mmol) in DMF (2 mL) was treated with
Hunig’s base (20 μL, 0.12 mmol), PyBOP (50 mg, 0.096 mmol), and
the appropriate amine (0.05 mmol). After stirring for 24 h at room
temperature, deionized water (15 mL) was added, and the mixture
was extracted by EtOAc (2 × 20 mL). The combined organic layers
were washed with brine, dried over MgSO4, and evaporated via rotary
evaporator to yield a dark oil. The crude product was purified by flash
chromatography (hexane/EtoAc), resulting in a dark orange to red
solid.
Animals. Male BALB/c mice (6−8 weeks old, 18−25 g) were
purchased from Nanjing University Experimental Animal Center
(Nanjing, People’s Republic of China). Mice were acclimatized for 1
week before use, and they were maintained with free access to pellet
food and water in plastic cages at 22−25 °C and kept on a 12 h light/
dark cycle. All experimental procedures were approved by the Ethical
Committee of the China Pharmaceutical University (No. SYXK2016-
0011).
Preparation of Spleen Cells from Mice. The assay was
performed similarly to our previous study with slight modifications.23
BALB/c mice were sacrificed to collect their spleens aseptically. Next,
Procedure for the Synthesis of Compounds 3a−3j. Celastrol
(0.048 mmol, 22 mg) and the indole derivative (0.72 mmol) were
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the spleens were ground by passing them through a sterile plastic
strainer with 200 mesh. A single-cell suspension was prepared after
cell debris and clumps were removed. Erythrocytes were lysed in red
blood cell lysis buffer. Then, cell pellets were washed two times with
culture medium. Cells were washed three times with culture medium
and were resuspended in RPMI 1640 medium containing 10% FBS at
the indicated concentration.
Immunosuppressive Activity and Cytotoxicity. Spleen cells
were seeded in 96-well plates at a density of 1 × 105 cells/well and
stimulated with ConA (5 μg/mL) in the presence of different
concentrations (0.78−10 μM) of test compounds or cyclosporin A for
48 h. For the proliferation assay, 20 μL of CCK-8 was added to all
wells 4 h before the end of the incubation. Finally, the absorbance
intensity at 540 nm was detected with a microplate reader (Tecan,
Austria). For the cytotoxicity assay, cells were incubated in a 96-well
plate at a density of 5 × 105 cells/well with various concentrations of
treatment. The absorbance intensity at 450 nm was detected on a
microplate reader (Tecan, Austria).
Apoptosis Assay. Annexin V−PI double staining was conducted
using a previous method.24 Spleen cells (3 × 106 cells/well in six-well
plates) were stimulated with ConA (5 μg/mL) with various
concentrations of 2a (0.01, 0.1, 1 μM) for 48 h. Cell lysates were
washed twice with cold PBS, and the density of cells was adjusted to 1
× 106 cells/mL using binding buffer. Finally, all samples were
performed according to the specifications of the commercial kit and
stained with both annexin V-FITC (fluorescein isothiocyanate) and
propidium iodide (PI). Then, samples were analyzed using a
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA,
USA).
University, Nanjing 210009, People’s Republic of China;
orcid.org/0000-0003-3994-9806; Email: wanghao@
cpu.edu.cn
Authors
Qi-Wei He − State Key Laboratory of Natural Medicines, School
of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Jia-Hao Feng − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Xiao-Long Hu − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Huan Long − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Xue-Feng Huang − State Key Laboratory of Natural Medicines,
School of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Zhen-Zhou Jiang − Jiangsu Key Laboratory of Drug Screening,
China Pharmaceutical University, Nanjing 210009, People’s
Republic of China
Xiao-Qi Zhang − Institute of Traditional Chinese Medicine and
Natural Products, Jinan University, Guangzhou 510632,
People’s Republic of China
Wen-Cai Ye − Institute of Traditional Chinese Medicine and
Natural Products, Jinan University, Guangzhou 510632,
People’s Republic of China
Active Caspase-3 and Caspase-9 Activity Assays. Spleen cells
at a density of 3 × 106 cells/well were seeded in 24-well plates and
stimulated with Con A (5 μg/mL) in the presence of various
concentrations of 2a (0.01, 0.1, 1 μM) for 48 h. After the cell
collection, the activities of caspase-3 and caspase-9 were determined
following the specifications of the commercial kit.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00067
Measurement of Cytokines. Spleen cells at a density of 3 × 106
cells/well were seeded in 24-well plates and were stimulated with
ConA (5 μg/mL) in the presence of different concentrations of 2a
(0.01, 0.1, 1 μM) for 48 h. After this, the culture medium was
collected to perform the ELISA assays for IL-4, IL-2, and IFN-γ. The
experimental procedure was conducted following the manufacturer’s
instructions. Finally, the absorbance intensity at 450 nm was detected
on a microplate reader (Tecan, Austria).
Author Contributions
#Q.-W. He and J.-H. Feng contributed equally and are joint
first authors.
Notes
The authors declare no competing financial interest.
Western Blot Analysis. Spleen cells at a density of 5 × 106 cells/
well were seeded in six-well plates and were treated with ConA (5 μg/
mL) in the presence of various concentrations of 2a (0.01, 0.1, 1 μM)
for 24 h. Then, cells were collected and washed twice with PBS.
Protein extraction and Western blotting were conducted as in our
previous study.23 The images were analyzed using the ImageJ
software.
ACKNOWLEDGMENTS
■
This study was funded by the National Natural Science
Foundation of China (Nos. 81573309, 81973206, 81773995),
the Funding of Double First-rate Discipline Innovation Team
(Nos. CPU2018PZQ17, CPU2018PZQ18, CPU2018GF05),
the Local Innovative and Research Teams Project of
Guangdong Pearl River Talents Program (No.
2017BT01Y036), Major National Science and Technology
Projects of the Chinese Thirteen Five-year Plan (No.
2017ZX09309024), the National Key R&D Program of
China (No. 2017YFC1703802), and China Postdoctoral
Science Foundation (No. 2019M662006, 2019TQ0357).
Statistics. The significance of intergroup differences was
determined using one-way analysis of variance. The results are
expressed as means SD for the indicated numbers of independent
experiments. Significance was determined for p values of <0.05.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00067.
REFERENCES
■
(1) Mika, A.; Stepnowski, P. J. Pharm. Biomed. Anal. 2016, 127,
Chemical name, synthetic yields, spectral data, as well as
NMR and HR-ESIMS spectra of the all synthetic
compounds (PDF)
207−231.
(2) Hackstein, H.; Thomson, A. W. Nat. Rev. Immunol. 2004, 4, 24−
35.
(3) Liu, Y.; Wang, Y. B.; Zhang, Q.; She, J. X. Chin. J. New Drugs.
2011, 20, 1981−1988.
AUTHOR INFORMATION
Corresponding Author
Hao Wang − State Key Laboratory of Natural Medicines, School
of Traditional Chinese Pharmacy, China Pharmaceutical
■
(4) Luo, D.; Zuo, Z.; Zhao, H.; Tan, Y.; Xiao, C. Front. Med. 2019,
13, 556−263.
(5) Tao, X. L.; Cush, J. J.; Garret, M.; Lipsky, P. E. J. Rheumatol.
2001, 28, 2160−2167.
H
https://dx.doi.org/10.1021/acs.jnatprod.0c00067
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(6) Allison, A. C.; Cacabelos, R.; Lombardi, V. R.; Alvarez, X. A.;
Vigo, C. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2001, 25,
1341−1357.
(7) Bufu, T.; Di, X.; Yilin, Z.; Gege, L.; Xi, C.; Ling, W. Anti-Cancer
Drugs 2018, 29, 530−538.
(8) Mou, H.; Zheng, Y.; Zhao, P.; Bao, H.; Fang, W.; Xu, N. Toxicol.
In Vitro 2011, 25, 1027−1032.
(9) Kannaiyan, R.; Shanmugam, M. K.; Sethi, G. Cancer Lett. 2011,
303, 9−20.
(10) Liu, J.; Lee, J.; Salazar Hernandez, M. A.; Mazitschek, R.;
Ozcan, U. Cell 2015, 161, 999−1011.
(11) Kyriakou, E.; Schmidt, S.; Dodd, G. T.; Pfuhlmann, K.;
Simonds, S. E.; Lenhart, D.; Geerlof, A.; Schriever, S. C.; De Angelis,
M.; Schramm, K. W. J. Med. Chem. 2018, 61, 11144−11157.
(12) Luo, D. Q.; Wang, H.; Tian, X.; Shao, H. J.; Liu, J. K. Pest
Manage. Sci. 2010, 61, 85−90.
(13) Tseng, C. K.; Hsu, S. P.; Lin, C. K.; Wu, Y. H.; Lee, J. C.;
Young, K. C. Antiviral Res. 2017, 146, 191−200.
(14) Yu, J. S.; Tseng, C. K.; Lin, C. K.; Hsu, Y. C.; Wu, Y. H.; Hsieh,
C. L.; Lee, J. C. Antiviral Res. 2017, 137, 49−57.
(15) Venkatesha, S. H.; Moudgil, K. D. Inflammation Res. 2019, 8,
285−296.
(16) Evans, D. A.; Scheidt, K. A.; Fandrick, K. R.; Lam, H. W.; Wu, J.
J. Am. Chem. Soc. 2003, 125, 10780−10781.
́
(17) Klaic, L.; Trippier, P. C.; Mishra, R. K.; Morimoto, R. I.;
Silverman, R. B. J. Am. Chem. Soc. 2011, 133 (49), 19634−19637.
(18) Tiegs, G.; Hentschel, J.; Wendel, A. J. Clin. Invest. 1992, 90,
196−203.
(19) Zhang, Z.; Zhang, X.; Zhao, Z.; Yan, R.; Xu, R.; Gong, H.; Zhu,
H. Bioorg. Med. Chem. 2012, 20, 3359−3367.
(20) Kim, S.; Park, S.; Sapkota, K.; Kim, M.; Kim, S. J. Pharm.
Pharmacol. 2011, 63, 1358−1367.
(21) Danial, N. N.; Korsmeyer, S. J. Cell 2004, 116, 205−219.
(22) Romagnani, S. Annu. Rev. Immunol. 1994, 12, 227−257.
(23) Singh, V. K.; Mehrotra, S.; Agarwal, S. S. Immunol. Res. 1999,
20, 147−161.
(24) Jury, E. C.; Kabouridis, P. S.; Abba, A.; Mageed, R. A.; Isenberg,
D. A. Arthritis Rheum. 2003, 48, 1343−1354.
(25) Pham, T. A.; Hu, X. L.; Huang, X. J.; Ma, M. X.; Feng, J. H.; Li,
J. Y.; Hou, J. Q.; Zhang, P. L.; Nguyen, V. H.; Nguyen, M. T.; Xiong,
F.; Fan, C. L.; Zhang, X. Q.; Ye, W. C.; Wang, H. J. Nat. Prod. 2019,
82, 859−869.
I
https://dx.doi.org/10.1021/acs.jnatprod.0c00067
J. Nat. Prod. XXXX, XXX, XXX−XXX