Design and Synthesis of Marine Phidianidine Derivatives as Potential Immunosuppressive Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b01430

A series of novel marine phidianidine derivatives were designed, synthesized, and evaluated for their immunosuppressive activities during our search of potential immunosuppressive agents with high efficacy and low toxicity from marine sources. These compounds were tested for their inhibitory activity on Con A-induced T cell and lipopolysaccharide-induced B cell proliferation. Compounds 14a and 18c, displaying the most promising inhibitory effects and low toxicities, were further found to possess immune-regulatory activities upon cross-linking of T cell receptor (TCR) and B cell receptor (BCR) on purified T and B cells, respectively.

Full text of DOI:10.1021/acs.jmedchem.8b01430

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Article
Design and Synthesis of Marine Phidianidine
Derivatives as Potential Immunosuppressive Agents
Jin Liu, Heng Li, Kaixian Chen, Jianping Zuo, Yue-Wei Guo, Wei Tang, and Xu-Wen Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01430 • Publication Date (Web): 30 Nov 2018
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Design and Synthesis of Marine Phidianidine
Derivatives as Potential Immunosuppressive Agents
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Jin Liu,†,‡,1 Heng Li, ‡,¶,1, Kai-Xian Chen,†,§ Jian-Ping Zuo,‡,¶ Yue-Wei Guo,*,†,‡,§ Wei Tang,*,‡,¶, §
and Xu-Wen Li ,*,†,‡,§
†
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203,
China
‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
¶ Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China
§ Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory
for Marine Science and Technology (Qingdao), 1 Wenhai Road, Aoshanwei, Jimo, Qingdao,
266237, China
ABSTRACT: A series of novel marine phidianidine derivatives were designed, synthesized, and
evaluated for their immunosuppressive activities during our search of potential
immunosuppressive agents with high efficacy and low toxicity from marine sources. These
compounds were tested for their inhibitory activity on Con A-induced T cells and LPS-induced B
cells proliferation. Compounds 14a and 18c, displaying the most promising inhibitory effects and
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low toxicities, were further found to possess immune-regulatory activities upon cross-linking of
T cell receptor (TCR) and B cell receptor (BCR) on purified T and B cells, respectively.
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◼ INTRODUCTION
Over the past decades, immune disorders have been affecting a large population worldwide.
Those patients are commonly subjected to abnormal immune responses and inflammation,
manifesting with disturbance of immune cells populations, immune cells activation, upregulation
of inflammatory mediators, and tissue damage.1 Therapeutic immunosuppressants, e.g.
cyclosporin A (CsA), mycophenolate mofetil (MMF), rapamycin, tacrolimus, are available for
treating diverse autoimmune diseases, inflammation, and organ transplant, including rheumatoid
arthritis, multiple sclerosis, systemic lupus erythematosus, acute and chronic inflammation.1,2
However, despite the undeniable clinical advantages, the aforementioned common
immunosuppressive drugs, exemplified by CsA and MMF, were found to cause considerably
serious side effects, such as renal or liver toxicity, infection, malignancy, and other adverse
effects.3-7 Thus, the discovery of novel immunosuppressants with high efficacy and low adverse
effects remains a challengeable task.
In our continuous search of new potential immunosuppressive agents from marine sources, a
screening of our compound library of marine natural products (MNPs) and derivatives was
conducted, leading to the discovery of a series of novel immunologically active MNPs and their
synthetic derivatives, such as xishacorenes A−C,8 sarinfacetamide A,9 and phidianidine analogs.
Phidianidines A and B (1 and 2, Figure 1) are two novel indole alkaloids, bearing an uncommon
1,2,4-oxadiazole ring and a guanidine side chain, isolated from the marine opisthobranch
mollusk Phidiana militaris in 2011.10 Interestingly, these metabolites and their derivatives were
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found to exhibit widespread biological activities, such as cytotoxic, neuroprotective, dopamine
transporter (DAT) inhibitory, and protein tyrosine phosphatase-1B (PTP1B) inhibitory
activities,10-13 which are probably due to the unique combination of structure features, including
the indole ring (A), the oxadiazole ring (B), and the guanidine side chain (C) (Figure 1). In fact,
the 1,2,4-oxadiazole ring was proved to be responsible for the biological properties of some
previously reported immunologically active compounds, such as 4a and 4b (Figure 1).14,15
Therefore, in this study, phidianidine derivatives containing the 1,2,4-oxadiazole ring have been
designed and synthesized for the evaluation of their immunosuppressive activity. Through the
initial screening, compound 3a (Figure 1), bearing a modification at the C-moiety of
phidianidine B, was found to exhibit considerable inhibitory activity on lipopolysaccharide
(LPS)-induced B cell proliferation with low toxicity. The intriguing activity of 3a stimulated us
for the further structure modification and biological study of related compounds toward
immunosuppressive agents. In order to obtain simplified structures with improved activities, a
structure-activity relationship (SAR)-based synthetic strategy was employed for the synthesis of
phidianidine analogs towards novel and potential MNP-derived immunosuppressive agents.
Herein, we report the design and synthesis of immunosuppressive phidianidine derivatives and
their immune-regulatory activities.
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Figure 1 Structures of phidianidines, immunosuppressive analog 3a, and structurally related
immunosuppressive compounds (4a and 4b).
◼ RESULTS AND DISCUSSION
Chemistry. Four round syntheses have been carried out. The first round concerned the
synthesis of the natural product and its analogs (Scheme 1) with different guanidine chain
lengths, as initially to test their influence on the targeted activity. The second round was
designed to simplify the synthesis by replacing the guanidine chain to various aryl rings (Scheme
2), to increase the hydrophobicity and to evaluate the importance of the guanidine moiety in the
biological activity. These two-round syntheses were achieved before the screening, resulting in
the discovery of 3a as the lead compound for further structure modification. The third round was
aiming to greatly shorten the synthetic steps for easily obtaining more analogs for biological
evaluation. The last round was to replace the indole A-ring by a 3,4,5-trimethoxyphenyl ring
since the latter was proved to be crucial in the activity during our biological activity evaluation.
The TFA salts of the natural product phidianidine B (2) and its analogs 2a and 2b with shorter
guanidine side chains, were initially synthesized according to Lindsley’s synthetic route (Scheme
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1).11 The only difference was the use of more easily accessible 1,3-di-Boc-2-(trifluoromethylsulf
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onyl)guanidine (7) for the final guanidation in the last two steps.
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Scheme 1 Synthetic route to phidianidine B and its analogs with different guanidine chain
lengths.
In the second round, other phidianidine derivatives were designed to simplify the C moiety
(Figure 1) by replacing the guanidine side chain to different aryl rings as shown in Scheme 2.
The synthesis was similar as our previous reported route.16 The aryl aldehyde 8 (8a−8f) first
reacted with hydroxylamine hydrochloride (NH2OH·HCl), in the presence of NaOH in
EtOH/H2O (1:1), to afford the oxime 9. It was then dehydrated by dichloro(p-
cymene)ruthenium(II) dimer in acetonitrile (CH3CN), yielding the nitrile 10,17 which was further
treated by NH2OH·HCl and NaHCO3 in EtOH, to afford amidoxime 11. Esterification of 11 with
3-indoleacetic acid (12) by using 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and diisopropylethylamine (DIPEA) in CH2Cl2 gave compound 13.
Finally, an intramolecular cyclization of 13 in the presence of sodium acetate (NaOAc) in 30%
EtOH (in H2O) under reflux led to the oxadiazole product 3.18
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Scheme 2 Reagent and conditions: (a) NH2OH·HCl, NaOH, 50% EtOH, rt., overnight; (b)
Dichloro(p-cymene)ruthenium (II) dimer, CH3CN, 80 °C, 4h; (c) NH2OH·HCl, NaHCO3, EtOH,
rt., 4h; (d) HATU, DIPEA, CH2Cl2, rt., 2h; (e) NaOAc, 30% EtOH, reflux, overnight.
As will be discussed in the biological part, compound 3a displayed the strongest inhibitory
activity against LPS-induced B cell proliferation, with no cytotoxicity over murine splenocytes.
In order to rapidly obtain more analogs of 3a, a simplified synthetic strategy was applied to flip
the 1,2,4-oxadiazole ring of compound 3a, placing the indole ring at the C-3 position in
compound 14a, as displayed in Scheme 3. Consequently, the synthetic sequence could be
shortened to three steps, avoiding the costly ruthenium catalyst, and the yield could be greatly
improved. Based on this new synthetic strategy, we initially synthesized compound 14a as the
isomer of 3a. The similar immunosuppressive effects on LPS-induced B cell proliferation of both
compounds encouraged us to design and synthesize a series of 14a analogs. As for the synthetic
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route, by using the similar method shown in Scheme 2, 3-indoleacetonitrile (15) was transformed
to the amidoxime 16, which then underwent an esterification with various carboxylic acids,
followed by an immediate 1,2,4-oxadiazole ring closing under reflux, yielding a number of 14a
analogs 14b–14l (Scheme 3).
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Scheme 3 Reagent and conditions: (a) NH2OH·HCl, NaHCO3, EtOH, 65 °C, 4h; (b) carboxylic
acid, HATU, DIPEA, CH2Cl2, r.t., 2h; (c) NaOAc, 30% EtOH, reflux, overnight.
The last round synthesis was designed on the basis of the biological results of the previous
synthesized compounds 14a–14l, of which the 3,4,5-trimethoxyphenyl ring was associated to the
best biological activity, and thus was used to replace the indole ring of phidianidine B for the A-
moiety, whereas structural variations were introduced on the C-moiety, through aryl,
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bicycloalkyl, adamantyl or spiroalkyl substituents. The synthetic steps (Scheme 4) towards 18a–
18l were similar as those shown in Scheme 3, with only the replacement of the starting material
from 3-indoleacetonitrile (15) to 3,4,5-trimethoxybenzonitrile (19).
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Scheme 4 Reagent and conditions: (a) NH2OH·HCl, NaHCO3, EtOH, 65 °C , 4h; (b) carboxylic
acid, HATU, DIPEA, CH2Cl2, r.t., h; (c) i, NaOAc, 30% EtOH, reflux, overnight. (c) ii, for 18c,
18d, 18h, TFA/CH2Cl2, (1:10 v/v), rt, 8h.
Biological Activity. The spleen functions like a large lymph node, which plays a critical role
in the immune system. The proliferation of murine splenocytes, predominantly including T and
B cells, could be triggered by concanavalin A (Con A) and LPS, respectively, which can be
applied to evaluate the immunosuppressive activity of compounds in vitro. Ten phidianidine
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derivatives, including phidianidine B (2), 2a, 2b, 3a−3f, and 13a, were initially assayed on the
proliferation of T and B cells. Among them, 2, 2a and 2b, the three compounds with guanidine
side chains, showed no inhibitory activity at a concentration of 50 μM. However, among those
phenyl substituted derivatives, compound 3a exhibited remarkable inhibitory activity against
LPS-induced B cells proliferation with IC50 value of 2.8 μM, but only low activity on Con A-
induced T cells proliferation, and low cytotoxicity on murine splenocytes, at a concentration of
50 μM (Table 1). The above results indicated that the guanidine is not the optimal group for the
activity. In addition, when comparing the activity of 3a and its precursor 13a, the results shown
in Table 1 suggested that the opening of the oxadiazole ring would increase the cytotoxicity of
the compounds, which is not compatible with the development of an immunosuppressive activity.
Therefore, from these preliminary results, we deduced that the oxadiazole ring is needed for the
immunosuppressive activity, while the guanidine group is not necessary.
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Table 1 Immunosuppressive activity of compounds 3a–3f, and 13a.
Con A
stimulation stimulation
LPS
No.
Cytotoxicity
R
CC50 (μM)
>50
IC50 (μM)
>50
IC50 (μM)
2.8
3a
3b
OMe
H
>50
>50
>50
3c
3d
3e
3f
F
Cl
NO2
C2H5
OMe
–
13.6
42.8
>50
24.4
37.3
1.2
5.4
22.6
>50
7.5
19.5
0.04
13.6
>50
>50
13.9
10.4
0.4
13a
CsAa
a positive control, with the problem of high toxicity.
As discussed in the chemistry part, the simplified structural modification of compound 3a led
to its isomer 14a, with only different connection patterns of the oxadiazole, while the synthetic
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steps were greatly decreased and the yield was increased. It is worth noting that 14a exhibited
the similar effect against LPS-induced B cells proliferation as 3a, with IC50 value of 3.1 μM
(Table 2), which indicated that the linkage position of the 1,2,4-oxadiazole has no obvious
influence on the activity. Moreover, 14a also exhibited moderate inhibitory activity on the Con
A-induced T cells proliferation with IC50 value of 11.0 μM. In addition, 17a, the oxadiazole ring
opening precursor of 14a, was found to exhibit high cytotoxicity, which futher confirmed that the
oxadiazole ring is an important group for the optimization of the biological profile (Table 2).
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Table 2 Immunosuppressive activity of compounds 14a–14l.
Con A
stimulation stimulation
LPS
Cytotoxicity
CC50 (μM)
No.
IC50 (μM)
11.0
16.7
18.5
>50
IC50 (μM)
3.1
14a
14b
14c
14d
14e
14f
14g
14h
14i
>50
42.2
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>50
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11.1
11.1
>50
4.7
8.6
>50
>50
8.2
>50
40.6
33.6
>50
10.1
>50
>50
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13.9
9.8
>50
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6.9
11.2
>50
15.7
13.3
>50
14j
14k
14l
>50
6.8
17a
11.9
Based on the above results, eleven 14a analogs 14b–14l were rapidly synthesized by replacing
the 3,4,5-trimethoxyphenyl ring by other aromatic rings with either electron-donating groups (e.g.
14b–14h) or electron withdrawing groups (e.g. 14i–14j), or the combination of both groups (e.g.
14k), or aliphatic group (e.g. 14l). However, as shown in Table 2, none of them showed better
activity than 14a. Therefore, 3,4,5-trimethoxyphenyl ring was suggested to be the optimal group,
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leading to the last round synthesis towards 18a–18l. Among them, compound 18c with a p-
aminophenyl group at the C-moiety, showed significant inhibitory activities on both Con A-
induced T cells proliferation and LPS-induced B cells proliferation, with IC50 values of 0.8 and
1.7 μM, respectively, and without significant cytotoxicity at a concentration of 50 μM. All the
other compounds either showed no immunosuppressive activity or high cytotoxicity.
Interestingly, for compound 18c, when protecting its amino group to be –NHBoc group towards
18b or changing the position of the amino group to be –meta towards 18d, the cytotoxicity
would be greatly increased (Table 3). It is also worth mentioning that, although the
bicyclo[2,2,2]octane, adamantane, and spiro[3.3]heptane were generally considered to be good
replacing candidates of aromatic rings in some bioactive molecules for increasing their
druggability,19,20 the molecules designed with these functionalities (e.g. 18f–18i) showed high
cytotoxicity in our case, which limited them for immunosuppressant applications.
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Table 3 Immunosuppressive activity of compounds 18a–18l.
Con A
stimulation stimulation
IC50 (μM)
>50
1.6
LPS
Cytotoxicity
CC50 (μM)
No.
IC50 (μM)
>50
2.2
18a
18b
18c
18d
18e
18f
18g
18h
18i
>50
9.2
>50
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0.8
7.0
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2.7
2.5
4.8
5.2
>50
>50
23.8
1.7
5.5
>50
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3.6
4.1
5.6
>50
36.2
22.9
>50
26.5
12.7
25.8
28.5
31.3
21.2
25.0
18j
18k
18l
The synthesis of phidianidine derivatives, on the basis of our SAR analysis, resulted into the
discovery of two easily synthesized immunosuppressive compounds 14a and 18c with low
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cytotoxicity. In order to deeply understand the mechanism of the immunosuppressive activity of
the two compounds, further investigation of 14a and 18c on T cell receptor (TCR) and B cell
receptor (BCR)-mediated immune responses were evaluated upon cross-linking of TCR and
BCR on purified murine CD4+ T cells and CD19+ B cells, stimulated by anti-CD3/CD28 Abs
and anti-IgM/CD40 Abs, respectively. Abnormal T cells and B cells activation have been
implicated in mediating multiple aspects of inflammatory and autoimmune diseases and
cytokines play a central role in the initiation and regulation of immune responses which may
involve one or more biological effect including cell division, proliferation, migration and further
effector functions. In vitro stimulation with anti-CD3/CD28 mAbs could mimic the physiologic
cross-linking of TCR and induce the proliferation and activation of T cells. Similarly, cross-
linking of BCR plus CD40L lead to proliferation and activation of B cells.21,22
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The biological results showed that both compounds 14a and 18c dramatically suppressed
primary CD4+ T cells proliferation induced by anti-CD3/CD28 (Figure 2A). Inflammatory
cytokines IL-2 and IFN-γ have been proven to promote inflammation and tissue injury in
multiple diseases. Therefore, we also determined IL-2 and IFN-γ production upon T cells
activation by ELISA. Both 14a and 18c showed remarkable suppression effects on IL-2 and IFN-
γ production stimulated with anti-CD3/CD28 for 24 h (Figure 2B and 2C). CD25, also known as
IL-2RA, is generally regarded as the activation surface marker of T cells. Interestingly,
compounds 14a and 18c significantly downregulated the percentage of CD25 expression (Figure
2D), suggesting that both compounds exhibited potent suppressive activity on T cell functions.
As demonstrated in Figure 2D, compound 18c exhibited better effects than compound 14a,
which is in agreement with in vitro results for immunosuppressive activity (IC50 11.0 μM and 0.8
μM for 14a and 18c, respectively).21
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Figure 2. The inhibitory effects of compounds 14a and 18c on anti-CD3/CD28-induced CD4+ T
cells proliferation, activation and cytokines production. A: Primary CD4+ T cells (4×105/well)
were cultured with plate-coated anti-CD3 (5 μg/ml) anti-CD28 mAb (2 μg/ml) and in the
absence or presence of compounds (10, 3, and 1 μM) in 96-well plates for 48 h to determine T
cells proliferation. B and C: After stimulation with anti-CD3/CD28 mAbs for 24 h, the culture
supernatants were collected and assayed for release of inflammatory cytokines. D: Flow
cytometry analysis was performed to determine the percentage of T cells activation marker
CD25.
Ligation of BCR induces signals that are critical for activation of the quiescent B cells to
proliferate and differentiate into plasma cells. For B cells function, compounds 14a and 18c
strikingly inhibited CD19+ B cells proliferation induced by anti-IgM/CD40 Abs (Figure 3A) and
also showed regulatory effects on cytokines production, manifesting decreasing IL-6 and
increasing IL-10 secretion, in which IL-6 serves as an inflammatory cytokine, while IL-10
bearing an anti-inflammatory property (Figure 3B and 3C). In B cells, membrane-bound IgD
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serves as a fundamental activation marker and when B cells were activated, the expression of
IgD would decrease or disappear. Consistently, we found that the level of IgD was
downregulated after stimulation with anti-IgM/CD40 mAbs, while 14a and 18c could reverse
this effect (Figure 3D).22 Therefore, the above biological investigation indicated that compounds
14a and 18c exhibited the potent immunosuppressive effects in vitro, including T and B cells
proliferation, activation and cytokines production.
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Figure 3. The regulatory effects of compounds 14a and 18c on the proliferation, activation and
cytokines production from anti-IgM/CD40-primed CD19+ B cells. A: Primary CD19+ B cells
were cultured with F(ab')2 anti-mouse IgM (10 μg/ml) and purified hamster anti-mouse CD40
mAb (1 μg/ml) in the absence or presence of compounds (10, 3, and 1 μM) in 96-well plates for
48 h to determine B cells proliferation. B and C: After 24 h incubation, the culture supernatants
were collected and assayed for release of IL-6 and IL-10. D: The cells were collected and
determined the expression of IgD and data were shown as the mean fluorescence intensity (MFI).
◼ CONCLUSION
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In summary, a series of novel phidianidine derivatives, together with some previously reported
ones, were designed and synthesized on the basis of several rounds of SAR analysis towards
potential immunosuppressive agents. The synthetic steps of the target compounds were greatly
reduced and the overall yields were improved to be 40%−50%, compared to the synthesis of the
original natural products. Their inhibitory activity on Con A-induced T cells and LPS-induced B
cells proliferation, as well as their cytotoxicity on murine splenocytes, were evaluated in
comparison with the positive control CsA in vitro. Among them, 14a and 18c exhibited the most
remarkable inhibitory activity on T and B cells proliferation, with very low cytotoxicity. The
SAR analysis indicated that the 1,2,4-oxadiazole ring and the 3,4,5-trimethoxyphenyl ring were
important for the activity. It is worth mentioning that, although CsA has been used as an
immunosuppressive drug, the high cytotoxicity (CC50 = 1.2 μM) limited its medicinal
application.3,4 Comparing to CsA, the toxicity of our compounds 14a and 18c are much lower
(CC50 > 50 μM), and their selectivity index (SI = CC50/IC50) are even higher than CsA,
especially on the LPS-induced B cells proliferation (SICsA = 3.0; SI14a > 16.1; SI18c > 29.4). The
SI of 18c on Con A-induced T cells proliferation is also higher than the positive control CsA
(SICsA = 30.0; SI18c > 62.5). All these data indicate the promising immunosuppressive activity of
both compounds. In the light of such intriguing activity of 14a and 18c, they were further
investigated for their regulatory effects on TCR and BCR-mediated immune responses, which
demonstrated that both 14a and 18c showed dramatic effects on proliferation, activation and
cytokines release of T and B cells (Figure 4). Since redundancy activation of immune system and
inflammation have been consistently regarded as the jokers in immune disorders, our findings
may provide an alternative strategy for the discovery of novel immunosuppressants in clinical
application. Compounds 14a and 18c, as two lead compounds for the treatment of immune
disorders, will be further studied in vivo for their targeted immunological diseases.
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Figure 4. Marine phidianidine derived 14a and 18c with promising regulatory effects on TCR
and BCR-mediated immune responses.
◼ EXPERIMENTAL SECTION
1. Chemistry. All reagents and solvents were purchased from common commercial suppliers
and were used without further purification. 1H NMR spectra were recorded at 400 MHz, and 13C
NMR were recorded at 125 MHz on a Bruker NMR spectrometer instrument (Avance 400 for 1H,
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Avance 500 for C, Bruker Biospin AG, Uster, Switzerland). All H and C NMR shifts are
reported in  units (ppm) relative to the signals for CHCl3 (H 7.26, C 77.16), and MeOH (H
3.31, C 49.00). All coupling constants (J values) are reported in hertz (Hz). NMR abbreviations
are as follows: bs, broadened singlet; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and
dd, doublet of doublets. Thin-layer chromatography (TLC) was performed on pre-coated silica-
gel plates (HSGF254, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). ESI-MS
spectra were recorded on a Q-TOF Micromass spectrometer (1290-6545 UHPLC-QTOF,
Micromass, Wythenshawe, UK). Column chromatography was performed using a 200−300 mesh
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silica gel (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). All final compounds were
determined to have purities of >95% by one of the aforementioned methods, in combination with
the high performance liquid chromatography (HPLC).
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1.1. General Procedure for the Synthesis of 2, 2a and 2b. Compounds 6a−6c were synthesized
from the diamines 5a−5c in 5 steps, according to Lindsley’s synthetic route.11 To a CH2Cl2
solution of compounds 6a−6c (1.0 mmol) was successively added triethylamine (2.0 mmol) and
1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine (7, 1.2 mmol). The reaction mixture was stirred
at room temperature overnight, which was then stopped and washed by sat. aq. NaHCO3 (10 mL
x 2) and water (10 mL x 1), dried over anhydrous MgSO4 and concentrated. The residue was
subjected to silica gel chromatography with petroleum ether (PE)/ CH2Cl2 (1:1) to give the Boc
protected guanidine compounds, which was then deprotected by dissolving in 10 mL of a 9:1
(v/v) solution of CH2Cl2 and trifluoroacetic acid (TFA). The solution was stirred at ambient
temperature for 8 h, and the reaction mixture was diluted with 8 mL of MeOH and concentrated
in vacuo to afford compound 2, 2a and 2b, respectively. Phidianidine B (2) has been previously
described by Guo and Gavagnin.10
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1-(2-((5-((1H-indol-3-yl)methyl)-1,2,4-oxadiazol-3-yl)amino)ethyl)guanidine (2a). Brown
solid; yield 21% (Start from compound 5a over 7 steps); 1H NMR (400 MHz, CD3OD): δ 7.53 (d,
J = 7.9 Hz, 1H), 7.36 (d, J = 8.2 Hz,1H), 7.22 (s, 1H), 7.11 (t, J = 7.6 Hz,1H), 7.02 (t, J = 7.5 Hz,
13
1H), 4.24 (s, 2H), 3.35 (m, 4H); C NMR (125 MHz, CD3OD): δ 179.8, 170.1, 158.9, 138.1,
128.1, 124.7, 122.7, 120.1, 119.2, 112.4, 108.1, 42.9, 41.4, 24.1; HR-ESI: [M+H]+ calcd for
C14H18N7O 300.1567, found: 300.1567.
1-(4-((5-((1H-indol-3-yl)methyl)-1,2,4-oxadiazol-3-yl)amino)butyl)guanidine (2b). Brown
solid; yield 22% (Start from compound 5b over 7 steps); 1H NMR (400 MHz, CD3OD): δ 7.53 (d,
J = 8.0 Hz, 1H), 7.36 (d, J = 7.9 Hz,1H), 7.22 (s, 1H), 7.11 (t, J = 7.3 Hz,1H), 7.01 (t, J = 7.4 Hz,
13
1H), 4.22 (s, 2H), 3.18 (m, 4H), 1.63 (m, 4H); C NMR (125 MHz, CD3OD): δ 179.4, 170.0,
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158.6, 138.1, 128.1, 124.7, 122.7, 120.1, 119.2, 112.4, 108.1, 43.4, 42.1, 27.3, 27.2, 24.1; HR-
ESI: [M+H]+ calcd for C16H22N7O 328.1880, found: 328.1871.
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1.2. General Procedure for the Synthesis of compounds 13a−13f. To a solution of aldehyde 8
(1.0 mmol) in EtOH (5 mL) was added hydroxylamine hydrochloride (1.2 mmol) and sodium
acetate (2.0 mmol). The mixture was stirred at room temperature overnight. EtOH was removed
in vacuo. The residue was added water (5 mL) and extracted with ethyl acetate (3×5ml), washed
with brine (3×5ml), dried over anhydrous MgSO4, filtered and concentrated. The residue was
subjected to silica gel chromatography with petroleum ether (PE)/CH2Cl2 (3:2) to give oxime 9.
To a solution of oxime 9 (1.0 mmol) in acetonitrile (5 mL) was added [Ru2(p-PriC6H4Me)2(μ-
Cl)Cl]2 (0.05 mmol) and refluxed for 4 h. The mixture was filtered and the filtrate was
concentrated in vacuo. The residue was subjected to silica gel chromatography with PE/CH2Cl2
(2:1) to give nitrile 10. To a solution of nitrile 10 (1.0 mmol) in EtOH (5 mL) was added
hydroxylamine hydrochloride (1.2 mmol) and NaHCO3 (2.0 mmol). The mixture was refluxed
for 4 h. The reaction mixture was diluted with EtOAc, filtered and concentrated in vacuo. The
residue was added water (5 mL) and extracted with ethyl acetate (3×5 mL), washed with brine
(1×5 mL), dried over anhydrous MgSO4 and concentrated. The residue was subjected to silica
gel chromatography with EtOAc/MeOH (9:1) to give amidoxime 11. To a solution of 3-
indoleacetic acid (1.0 mmol) in CH2Cl2 (5 mL) was added DIPEA (1.3 mmol) and HATU (1.0
mmol) and stirred for 30 min, then amidoxime 11 (1.0 mmol) in CH2Cl2 (2 mL) was added and
stirred for 2 h. The mixture was filtered and the residue was washed with CH2Cl2, the solution
was combined and concentrated. The residue was subjected to silica gel chromatography with
PE/EtOAc (2:1) to give 13. The spectroscopic data of 13b−13f has been previously reported.16
((2-(1H-indol-3-yl)acetoxy)amino)(3,4,5-trimethoxyphenyl)methanamine (13a). White
solid; yield 34% (Start from compound 8a over 4 steps);1H NMR (400 MHz, CD3OD): δ 7.65 (d,
J = 7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.25 (s, 2H), 7.12 (t, 1H), 7.05 (m, 3H), 3.96 (s, 2H),
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3.86 (s, 6H), 3.77 (s, 3H); C NMR (400 MHz, CD3OD): δ 172.2, 159.6, 154.5, 141.3, 138.1,
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128.6, 128.1, 124.8, 122.6, 120.0, 119.4, 112.4, 108.4, 105.7, 61.1, 56.7, 31.0; HR-ESI: [M+H]+
calcd for C20H22N3O5 384.1554, found: 384.1550.
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1.3. General Procedure for the Synthesis of compounds 3a−3f. A solution of 13 (1.0 mmol)
and sodium acetate (2.0 mmol) in 30% EtOH/H2O (5 mL) was refluxed overnight. The EtOH
was then removed in vacuo, the residue was added water (5 mL) and extracted with ethyl acetate
(3×5 mL), washed with brine (1×5 mL), dried over anhydrous MgSO4 and concentrated. The
residue was subjected to silica gel chromatography with PE/EtOAc (5:1) to give compound 3.
The spectroscopic data of 3b−3f has been previously reported.16
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5-((1H-indol-3-yl)methyl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole 3a. White solid;
1
yield 90% ; H NMR (400 MHz, CD3OD): δ 7.57 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H),
7.35 (s, 2H), 7.29 (s, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 4.47 (s, 2H), 3.89 (s,
6H), 3.81 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 181.2, 169.4, 155.0, 141.8, 138.1, 128.2,
124.8, 123.5, 122.8, 120.2, 119.1, 112.5, 108.1, 105.8, 61.2, 56.7, 24.1; HR-ESI: [M-H]- calcd
for C20H18N3O4 364.1303, found: 364.1294.
1.4. General Procedure for the Synthesis of compounds 14a−14l and 18a−18l. To a stirred
solution of the nitrile 15 or 19 (1.0 mmol) in EtOH (5 mL) were added hydroxylamine
hydrochloride (1.5 mmol) and NaHCO3 (3.0 mmol). The mixture was stirred for 4 h at 65℃. The
reaction mixture was diluted with EtOAc, filtered and concentrated in vacuo. The residue was
added water (5 mL) and extracted with ethyl acetate (3×5 mL), washed with brine (1×5 mL),
dried over anhydrous MgSO4 and concentrated. The residue was subjected to silica gel
chromatography with EtOAc /MeOH (9:1) to give amidoxime 16 or 20. To a solution of
carboxylic acid (1.0 mmol) in CH2Cl2 (5 mL), were added DIPEA (1.3 mmol) and HATU (1.0
mmol), the reaction mixture was stirred for 30 min, after which amidoxime 16 or 20 (1.0 mmol)
in CH2Cl2 (2 mL) was added and stirred for 2 h. The mixture was filtered and the residue was
washed with CH2Cl2, the solution was combined and concentrated. The residue was subjected to
silica gel chromatography with PE/EtOAc (2:1) to give compound 17 or 21. A solution of
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compound 17 or 21 (1.0 mmol) and sodium acetate (2.0 mmol) in 30% EtOH/H2O (5 mL), was
refluxed overnight. The EtOH was removed in vacuo, and the residue was added water (5 mL)
and extracted with ethyl acetate (3×5 mL), washed with brine (1×5 mL), dried over anhydrous
MgSO4 and concentrated. The residue was subjected to silica gel chromatographic with
PE/EtOAc (5:1) to give compound 14 or 18a, 18b, 18e−18g, 18i−18l. For 18c, 18d, and 18h:
Compound 18b, NBoc-18d, or 18g (1.0 mmol) was dissolved in 5 mL of a 9:1 (v/v) solution of
CH2Cl2 and trifluoroacetic acid, and the solution was stirred at ambient temperature for 8 h.
Afterwards, the black-colored reaction mixture was diluted with 8 mL of MeOH and
concentrated in vacuo to afford compound 18c, 18d, and 18h, respectively. The spectroscopic
data of 14d,16 14h,16 14i,16 14l,16 18e23 and 18j24 has been previously reported.
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3-((1H-indol-3-yl)methyl)-5-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (14a). White solid;
yield 41% (Start from compound 15a over 3 steps); 1H NMR (400 MHz, CD3OD): δ 7.57 (d, J =
8.0 Hz, 1H), 7.40 (s, 2H), 7.35 (d, J = 8.2 Hz,1H), 7.23 (s, 1H), 7.10 (td, J = 7.5, 1.0 Hz, 1H),
7.00 (td, J = 7.5, 1.0 Hz,1H), 4.25 (s, 2H), 3.90 (s, 6H), 3.83 (s, 3H); 13C NMR (125 MHz,
CD3OD): δ 176.9, 171.9, 155.1, 143.4, 138.2, 128.4, 124.6, 122.6, 120.4, 119.9, 119.3, 112.4,
109.7, 106.5, 61.2, 56.8, 23.4; HR-ESI: [M+H]+ calcd for C20H20N3O4 366.1376, found:
366.1449.
3-((1H-indol-3-yl)methyl)-5-(2-methoxyphenyl)-1,2,4-oxadiazole (14b). White solid; yield
40% (Start from compound 15b over 3 steps); 1H NMR (400 MHz, CD3OD): δ 7.95 (dd, J = 7.8,
1.8 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.58 (td, J = 7.8, 1.8 Hz, 1H), 7.34 (d, J = 8.1 Hz,1H), 7.21
(s, 1H), 7.20 (d, J = 9.6 Hz, 1H), 7.08 (m, 2H), 7.01 (td, J = 7.0, 1.0 Hz, 1H), 4.27 (s, 2H), 3.94
(s, 3H); 13C NMR (125 MHz, CD3OD): δ 176.4, 171.2, 160.0, 138.2, 135.6, 132.3, 128.4, 124.5,
122.6, 121.8, 119.9, 119.4, 114.2, 113.5, 112.3, 109.9, 56.4, 23.4; HR-ESI: [M+H]+ calcd for
C18H16N3O2 306.1164, found: 306.1241.
3-((1H-indol-3-yl)methyl)-5-(3-methoxyphenyl)-1,2,4-oxadiazole (14c). White solid; yield
1
45% (Start from compound 15c over 3 steps); H NMR (400 MHz, CD3OD): δ 7.65 (d, J = 7.7
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Hz, 1H), 7.59 (m, 2H), 7.42 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.1 Hz,1H), 7.22 (s, 1H), 7.15 (ddd,
J = 8.4, 2.6, 1.0 Hz, 1H), 7.10 (td, J = 7.6, 1.0 Hz, 1H), 7.01 (td, J = 7.5, 1.0 Hz, 1H), 4.25 (s,
2H), 3.83 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 177.0, 172.0, 161.6, 138.2, 131.5, 128.4,
126.4, 124.6, 122.6, 121.3, 120.2, 119.9, 119.4, 113.7, 112.3, 109.8, 56.0, 23.4; HR-ESI:
[M+H]+ calcd for C18H16N3O2 306.1164, found: 306.1241.
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3-((1H-indol-3-yl)methyl)-5-(2,4-dimethoxyphenyl)-1,2,4-oxadiazole (14e). White solid;
yield 45% (Start from compound 15e over 3 steps); 1H NMR (400 MHz, CD3OD): δ 7.90 (d, J =
8.7 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.20 (s, 1H), 7.09 (td, J = 7.0, 1.0
Hz, 1H), 7.00 (td, J = 7.5, 1.0 Hz, 1H), 6.63 (m, 2H), 4.22 (s, 2H), 3.91 (s, 3H), 3.85 (s, 3H); 13C
NMR (125 MHz, CD3OD): δ 176.3, 171.0, 166.5, 161.7, 138.1, 133.7, 128.4, 124.5, 122.6, 119.9,
119.4, 112.3, 110.0, 107.3, 106.8, 99.7, 56.4, 56.2, 23.4; HR-ESI: [M+H]+ calcd for C19H18N3O3
336.1270, found: 336.1350.
3-((1H-indol-3-yl)methyl)-5-(2,5-dimethoxyphenyl)-1,2,4-oxadiazole (14f). White solid;
1
yield 44% (Start from compound 15f over 3 steps); H NMR (400 MHz, CDCl3): δ 7.75 (d, J =
7.8 Hz, 1H), 7.54 (d, J = 3.1 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.20 (m, 2H), 7.13 (td, J = 7.4,
1.0 Hz, 1H), 7.08 (dd, J = 9.1, 3.2 Hz, 1H), 6.98 (d, J = 9.2 Hz, 1H), 4.32 (s, 2H), 3.90 (s, 3H),
13
3.81 (s, 3H); C NMR (125 MHz, CDCl3): δ 174.8, 168.9, 153.5, 153.0, 136.4, 127.4, 123.2,
122.4, 120.6, 119.8, 119.3, 115.4, 114.0, 111.3, 110.3, 56.9, 56.1, 23.0; HR-ESI: [M+H]+ calcd
for C19H18N3O3 336.1270, found: 336.1347.
3-((1H-indol-3-yl)methyl)-5-(3,5-dimethoxyphenyl)-1,2,4-oxadiazole (14g). White solid;
yield 45% (Start from compound 15g over 3 steps); 1H NMR (400 MHz, CD3OD): δ 7.58 (d, J =
7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.22 (m, 3H), 7.09 (td, J = 7.6, 1.0 Hz, 1H), 7.01 (td, J =
7.5, 1.0 Hz, 1H), 6.71 (t, J = 2.3 Hz, 1H), 4.25 (s, 2H), 3.82 (s, 6H); 13C NMR (125 MHz,
CD3OD): δ 177.0, 172.0, 162.9, 138.2, 128.4, 126.8, 124.6, 122.6, 119.9, 119.3, 112.3, 109.8,
106.7, 106.2, 56.1, 23.4; HR-ESI: [M+H]+ calcd for C19H18N3O3 336.1270, found: 336.1350.
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3-((1H-indol-3-yl)methyl)-5-(4-fluoro-2-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (14j).
White solid; yield 40% (Start from compound 15j over 3 steps); 1H NMR (400 MHz, CD3OD): δ
8.03(dd, J = 8.7, 5.3 Hz, 1H), 7.73 (dd, J = 9.0, 2.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.54 (td, J
= 8.3, 2.6 Hz, 1H), 7.34 (d, J = 8.1 Hz, 2H), 7.21 (s, 1H), 7.10 (td, J = 7.1, 1.0 Hz, 1H), 7.01 (td,
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J = 7.1, 1.0 Hz, 1H), 4.29 (s, 2H); C NMR (125 MHz, CD3OD): δ 174.8, 172.0, 165.7, 138.1,
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136.1, 132.7, 128.4, 124.9, 124.5, 122.6, 120.7, 119.9, 119.4, 116.3, 112.3, 109.7, 23.3; HR-ESI:
[M+H]+ calcd for C18H12F4N3O 362.0911, found: 362.0919.
3-((1H-indol-3-yl)methyl)-5-(2-bromo-5-methoxyphenyl)-1,2,4-oxadiazole (14k). White
solid; yield 45% (Start from compound 15k over 3 steps); 1H NMR (400 MHz, CDCl3): δ 7.76 (d,
J = 7.8 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 3.1 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.21
(m, 2H), 7.15 (td, J = 6.9, 1.0 Hz, 1H), 6.94 (dd, J = 3.1, 8.9 Hz, 1H), 4.34 (s, 2H), 3.82 (s, 3H);
13C NMR (125 MHz, CDCl3): δ 174.8, 170.2, 158.9, 136.4, 135.6, 127.3, 126.4, 123.1, 122.5,
120.0, 119.8, 119.2, 116.7, 112.5, 111.3, 110.0, 55.9, 22.9; HR-ESI: [M+H]+ calcd for
C18H15BrN3O2 384.0342, found: 384.0338.
5-Phenyl-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (18a). White solid; yield 52% (Start
from compound 19a over 3 steps); 1H NMR (400 MHz, CDCl3): δ 8.23 (d, J = 7.0 Hz, 2H), 7.62
13
(t, J = 7.3 Hz, 1H), 7.56 (t, J = 7.3 Hz, 2H), 7.43 (s, 2H), 3.98 (s, 6H), 3.93 (s, 3H); C NMR
(125 MHz, CDCl3): δ 175.8, 168.9, 153.7, 132.9, 129.3, 128.3, 124.4, 122.3, 104.7, 61.1, 56.5;
HR-ESI: [M+H]+ calcd for C17H17N2O4 313.1110, found: 313.1182.
Tert-butyl (4-(3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)phenyl)carbamate (18b).
White solid; yield 50% (Start from compound 19b over 3 steps); 1H NMR (400 MHz, CDCl3): δ
8.15 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.41 (s, 2H), 3.97 (s, 6H), 3.92 (s, 3H) , 1.54 (s,
9H); 13C NMR (125 MHz, CDCl3): δ 175.5, 168.8, 153.7, 152.3, 142.8, 140.6, 129.6, 122.5,
118.6, 118.2, 104.8, 81.6, 61.1, 56.5, 28.4; HR-ESI: [M+H]+ calcd for C22H26N3O6 428.1743,
found: 428.1820.
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4-(3-(3,4,5-Trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)aniline (18c). White solid; yield 40%
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(Start from compound 19b over 4 steps); H NMR (400 MHz, CD3OD): δ 7.91 (d, J = 8.7 Hz,
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2H), 7.41 (s, 2H), 6.79 (d, J = 8.8 Hz, 2H), 3.93 (s, 6H), 3.84 (s, 3H); C NMR (125 MHz,
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CD3OD): δ 177.9, 169.5, 155.0, 154.4, 141.7, 130.9, 123.9, 115.3, 112.8, 105.7, 61.2, 56.7; HR-
ESI: [M+H]+ calcd for C17H18N3O4 328.1219, found: 328.1301.
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3-(3-(3,4,5-Trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)aniline (18d). White solid; yield 42%
1
(Start from compound 19d over 4 steps); H NMR (400 MHz, CD3OD): δ 7.95 (d, J = 7.9 Hz,
1H), 7.92 (s, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.44 (s, 2H), 7.38 (d, J = 7.2 Hz, 1H), 3.94 (s, 6H),
13
3.84 (s, 3H); C NMR (125 MHz, CD3OD): δ 176.9, 170.0, 155.1, 142.8, 142.1, 131.7, 126.5,
124.1, 123.4, 123.1, 118.7, 105.9, 61.2, 56.8; HR-ESI: [M+H]+ calcd for C17H18N3O4 328.1219,
found: 328.1301.
5-(Adamantan-1-yl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (18f). White solid; yield
1
48% (Start from compound 19f over 3 steps); H NMR (400 MHz, CDCl3): δ 7.31 (s, 2H), 3.93
13
(s, 6H), 3.89 (s, 3H), 2.13 (m, 9H), 1.80 (m, 6H); C NMR (125 MHz, CDCl3): δ 185.6, 168.0,
153.5, 140.3, 122.6, 104.6, 61.0, 56.4, 40.1, 36.3, 35.7, 27.9; HR-ESI: [M+H]+ calcd for
C21H27N2O4 371.1893, found: 371.1970.
Tert-butyl
(4-(3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)bicyclo[2.2.2]octan-1-
1
yl)carbamate (18g). White solid; yield 47% (Start from compound 19g over 3 steps); H NMR
(400 MHz, CDCl3): δ 7.29 (s, 2H), 3.93 (s, 6H), 3.89 (s, 3H), 2.13 (m, 6H), 1.99 (m, 6H), 1.43 (s,
9H); 13C NMR (125 MHz, CDCl3): δ 184. 8, 168.1, 153.7, 140.6, 122.4, 104.8, 61.1, 56.4, 49.9,
33.3, 30.3, 30.1, 28.6; HR-ESI: [M+H]+ calcd for C24H34N3O6 460.2369, found: 460.2370.
4-(3-(3,4,5-Trimethoxyphenyl)-1,2,4-oxadiazol-5-yl)bicyclo[2.2.2]octan-1-amine
(18h).
1
White solid; yield 41% (Start from compound 19g over 4 steps); H NMR (400 MHz, CD3OD):
δ 7.31 (s, 2H), 3.89 (s, 6H), 3.81 (s, 3H), 2.22 (m, 6H), 1.97 (m, 6H); 13C NMR (125 MHz,
CD3OD): δ 185.1, 169.2, 154.9, 141.8, 123.4, 105.6, 61.2, 56.7, 52.2, 34.2, 30.2, 30.0, 29.9; HR-
ESI: [M+H]+ calcd for C19H26N3O4 360.1845, found: 360.1923.
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5-(6,6-Difluorospiro[3.3]heptan-2-yl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (18i).
White solid; yield 45% (Start from compound 19i over 3 steps); 1H NMR (400 MHz, CDCl3): δ
7.30 (s, 2H), 3.93 (s, 6H), 3.90 (s, 3H), 3.75 (m, 1H), 2.66 (m, 8H); 13C NMR (125 MHz, CDCl3):
δ 181.4, 168.4, 153.7, 140.7, 122.2, 119.2, 104.7, 61.1, 56.4, 47.6, 47.1, 38.8, 29.7, 26.9; HR-ESI:
[M+H]+ calcd for C18H21F2N2O4 367.1391, found: 367.1462.
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5-(4-Butylphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (18k). White solid; yield
50% (Start from compound 19k over 3 steps); 1H NMR (400 MHz, CDCl3): δ 8.11 (d, J = 8.3 Hz,
2H), 7.41 (s, 2H), 7.35 (d, J = 8.4 Hz, 2H), 3.96 (s, 6H), 3.92 (s, 3H), 2.69 (m, 2H), 1.63 (m, 2H),
1.37 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 175.9, 168.8, 153.6,
148.6, 140.5, 129.3, 128.3, 122.4, 121.7, 104.6, 61.2, 56.4, 35.9, 33.4, 22.4, 14.0; HR-ESI:
[M+H]+ calcd for C21H25N2O4 369.1736, found: 369.1814.
5-(4-Hexylphenyl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole (18l). White solid; yield
51% (Start from compound 19l over 3 steps); 1H NMR (400 MHz, CDCl3): δ 8.12 (d, J = 8.3 Hz,
2H), 7.42 (s, 2H), 7.35 (d, J = 8.3 Hz, 2H), 3.97 (s, 6H), 3.92 (s, 3H), 2.69 (m, 2H), 1.65 (m, 2H),
1.31 (m, 6H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 176.0, 168.8, 153.6,
148.7, 140.5, 129.3, 128.3, 122.4, 121.8, 104.7, 61.1, 56.4, 36.2, 31.8, 31.2, 29.0, 22.7, 14.2;
HR-ESI: [M+H]+ calcd for C23H29N2O4 397.2049, found: 397.2127.
2. Biological assay.
2.1. Materials. Compounds were dissolved with 100% dimethylsulfoxide (DMSO, Sinopharm,
China) and then diluted with RPMI 1640 medium (Hyclone, South Logan, UT, USA) containing
10% fetal bovine serum (FBS, Hyclone). Cell Counting Kit-8 (CCK-8) was purchased from
Dojindo (Kumamoto, Japan). Concanavalin A (Con A), lipopolysaccharide (LPS, Escherichia
coli 055:B5) were purchased from Sigma (St Louis, MO, USA). Anti-CD3/CD28 antibodies,
F(ab')2 anti-mouse IgM antibodies were purchased from Thermo Fisher Scientific (Waltham,
MA, USA). Purified hamster anti-mouse CD40 was purchased from BD Pharmingen (San Diego,
CA, USA).
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2.2. Animals. Inbred 6-8-week-old female BALB/c mice were purchased from Shanghai
Lingchang Biotechnology Co., Ltd. (Certificate No.2013-0018, China). The mice were housed
under specific pathogen-free conditions with a controlled environment (12 h of light/12 h of dark
cycle, 22 ± 1 °C, 55 ± 5% relative humidity). All mice were fed standard laboratory chow and
water ad libitum and allowed to acclimatize in our facility for one week before any experiments
started. All experiments were carried out according to the National Institutes of Health Guide for
Care and Use of Laboratory Animals and were approved by the Bioethics Committee of the
Shanghai Institute of Materia Medica.
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2.3. Splenocytes Preparation from Mice. Female BALB/c mice were sacrificed and the spleens
were removed aseptically. Mononuclear cell suspensions were prepared after cell debris, and
clumps were removed. Erythrocytes were depleted with ammonium chloride buffer solution.
Cells were washed and resuspended in RPMI 1640 media containing 10% FBS, penicillin (100
U/mL), and streptomycin (100 μg/mL). Cells were counted by trypan blue exclusion.
2.4. Cell Viability Assay. Cell viability was assessed with CCK-8 reagent. Briefly, fresh
splenocytes (1×106 cells) were cultured in 96-well plates in triplicate with 200 μL RPMI 1640
media containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a
humidified, 37 °C, 5% CO2-containing incubator for 48 h in the presence or absence of indicated
concentrations of compounds. Subsequently, a total of 20 μL CCK-8 was added to each well.
After 6-8 h incubation, the absorbance value at 450 nm (570 nm calibration) was collected by a
microplate reader (Molecular Devices, Sunnyvale, CA, USA) and the cell viability was
calculated. The values of CC50 were estimated using the log(inhibitor) vs. normalized response
non-linear fit (GraphPad Prism 6.0).
2.5. Con A and LPS-induced Proliferation Assay. Splenocytes (5×105 cells) were cultured in
triplicate for 48 h, in the presence or absence of indicated concentrations of compounds, were
stimulated with 5 μg/ml of Con A or 10 μg/ml of LPS to induce T cells or B cells proliferation,
respectively. The cell culture was incubated in a humidified, 37 °C, 5% CO2-containing
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incubator for 48 h. Cells were pulsed with 0.5 μCi/well of [3H] thymidine for 8 h and harvested
onto glass fiber filters. The incorporated radioactivity was counted using a Beta Scintillation
Counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA). The values of IC50 were
estimated using the log(inhibitor) vs. normalized response non-linear fit (GraphPad Prism 6.0).
2.6. Purification of CD4+ T cells and CD19+ B cells. After mononuclear cell suspensions
preparation, mouse splenic polyclonal CD4+ T cells and CD19+ B cells were isolated using
EasySep™ mouse CD4+ T Cell isolation kit (Stemcell, Vancouver, BC, Canada) and EasySep™
mouse CD19+ B Cell isolation kit (Stemcell) according to the manufacturer’s instructions,
respectively. To acquire CD4+ T cells, immunomagnetic negative selection were performed for
removal with biotinylated antibodies recognizing specific cell surface markers. Unwanted cells
(CD8+ cells, B220+ cells, CD11b+ cells, and I-A+ antigen presenting cells from splenocytes) were
separated with an EasySep™ magnet. The purity of the CD4+ T cells was consistently >98%
determined by flow cytometry. Similarly, to acquire CD19+ B cells, mAb cocktails were added
to deplete CD4+ cells, CD8+ cells and CD11b+ cells from splenocytes by negative selection. The
purity of the CD19+ B cells was consistently >98%.
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2.7. In vitro Stimulation of Splenic CD4+ T cells and CD19+ B cells. For in vitro study,
purified CD4+ T cells were cultured with medium alone or anti-CD3 antibodies (5 μg/ml) and
anti-CD28 antibodies (2 μg/ml). Purified CD19+ B cells were stimulated with medium alone or
F(ab')2 anti-mouse IgM (10 μg/ml) plus anti-CD40 (1 μg/ml). After incubation, for 96-well
plates, the cultures were pulsed with 0.5 μCi/well [3H] thymidine to determine CD4+ T and
CD19+ B cells proliferation activity. For 24-well plates, the supernatants were collected to
determine the cytokine levels, meanwhile, the activation of T cells and B cells were detected by
flow cytometry.
2.8. ELISA. Cytokines in culture supernatants were determined by using mouse IL-2, IL-6, IL-
10 and IFN-γ ELISA kits (BD Pharmingen) according to the manufacturer’s instructions.
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Statistical analyses were conducted using GraphPad Prism 6.0 software and all data are
presented as the mean  sem.
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2.9. Flow Cytometry. Cells were washed with phosphate buffered saline (PBS) and then stained
with fixable viability dye eFluor™ 780 (eBioscience, San Diego, CA, USA) for 30 min at 4 ℃
to identify viable cells from the dead cells. Following, cells were blocked with anti-CD16/CD32
mAb (eBioscience) and stained with fluorescein isothiocyanate (FITC)-conjugated CD19,
phycoerythrin (PE)-conjugated CD25, and allophycocyanin (APC)-conjugated IgD. All
immunofluorescent mAbs used in this research were obtained from BD Biosciences (Franklin
Lakes, NJ, USA). The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
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◼ ASSOCIATED CONTENT
Supporting Information
Molecular Formula Strings (MFS), figures of NMR spectra, MS spectra, and HPLC purity for
the biologically evaluated compounds. The Supporting Information is available free of charge on
the ACS Publications website.
◼ AUTHOR INFORMATION
Corresponding Authors
*E-mail: xwli@simm.ac.cn; tangwei@simm.ac.cn; ywguo@simm.ac.cn
Author Contributions
1 These two authors contributed equally.
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
◼ ACKNOWLEDGEMENTS
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This research work was financially supported by the National Key Research and Development
Program of China (No. 2018YFC0310903), the National Natural Science Foundation of China
(NSFC) (Nos. 41676073, 81520108028), the NSFC/CNRS joint project (No. 81811530284), and
the SKLDR/SIMM Project (No. SIMM1705ZZ-01). Xu-Wen Li acknowledges the financial
support of "Youth Innovation Promotion Association" (No. 2016258) from Chinese Academy of
Sciences and SA-SIBS Scholarship Program. We thank Prof. Bastien Nay from Ecole
Polytechnique, France, for the helpful discussion about the synthetic work.
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◼ ABBREVIATIONS USED
Con A, concanavalin A; LPS, lipopolysaccharide; TCR, T cell receptor; BCR, B cell receptor;
CsA, cyclosporin A; MMF, mycophenolate mofetil; MNP, marine natural product; DAT,
dopamine transporter; PTP1B, protein tyrosine phosphatase-1B; SAR, structure-activity
relationship; TFA, trifluoroacetic acid; HATU, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate; DIPEA, diisopropylethylamine; PE, petroleum ether;
FBS, fetal bovine serum.
◼ REFERENCES
(1) Kahan, B. D. Individuality: the barrier to optimal immunosuppression. Nat. Rev. Immunol.
2003, 3, 831−838.
(2) Meyer, K. C.; Decker, C.; Baughman, R. Toxicity and monitoring of immunosuppressive
therapy used in systemic autoimmune diseases. Clin. Chest Med. 2010, 31, 565−588.
(3) Ader, J. L.; Rostaing, L. Cyclosporin nephrotoxicity: pathophysiology and comparison with
FK-506. Curr. Opin. Nephrol. Hypertens. 1998, 7, 539−545.
(4) Wang, X. L.; Tang, F.; Li, H. M. Bad responses of common immunosuppressants in organ
transplantation. J. Chin. Phys. 2002, 4, 1291−1293 (Chinese).
(5) Mignat, C. Clinically significant drug interactions with new immunosuppressive agents.
Drug. Saf. 1997, 16, 267-278.
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Journal of Medicinal Chemistry
(6) Wang, Y. R. Recent advances in immunosuppressive drugs. Chin. J. New Drugs. 2002, 11,
512−515.
1
2
3
4
5
6
7
8
9
(7) Smith, J. M.; Nemeth, T. L.; McDonald, R. A. Current immunosuppressive agents: efficacy,
side effects, and utilization. Pediatr. Clin. North Am. 2003, 50, 1283−1300.
(8) Ye, F.; Zhu, Z. D.; Chen, J. S.; Li, J.; Gu, Y. C.; Zhu, W. L.; Li, X. W.; Guo, Y. W.
Xishacorenes A-C, diterpenes with bicyclo[3.3.1]nonane nucleus from the Xisha soft coral
Sinularia polydactyla. Org. Lett. 2017, 19, 4183−4186.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(9) Ye, F.; Li, J.; Wu, Y.; Zhu, Z. D.; Mollo, E.; Gavagnin, M.; Zhu, W. L.; Li, X. W.; Guo, Y. W.
Sarinfacetamides A and B, nitrogenous diterpenoids with tricyclo[6.3.1.01,5]dodecane scaffold
from the south China Sea soft coral Sarcophyton infundibuliforme. Org. Lett. 2018, 20,
2637−2640.
(10) Carbone, M.; Li, Y.; Irace, C.; Mollo, E.; Castelluccio, F.; Pascale, A. D.; Cimino, G.;
Santamaria, R.; Guo, Y. W.; Gavagnin, M. Structure and cytotoxicity of phidianidines A and B:
First finding of 1,2,4-oxadiazole system in a marine natural product. Org. Lett. 2011, 13, 2516–
2519.
(11) Brogan, J. T.; Stoops, S. L.; Lindsley, C. W. T. Total synthesis and biological evaluation of
phidianidines A and B uncovers unique pharmacological profiles at CNS targets. ACS Chem.
Neurosci. 2012, 3, 658–664.
(12) Jiang, C. S.; Fu, Y.; Zhang, L.; Gong, J. X.; Wang, Z. Z.; Xiao, W.; Zhang, H. Y.; Guo, Y. W.
Synthesis and biological evaluation of novel marine-derived indole-based 1,2,4-oxadiazoles
derivatives as multifunctional neuroprotective agents. Bioorg. Med. Chem. Lett. 2015, 25, 216–
220.
(13) Zhang, L.; Jiang, C. S.; Gao, L. X.; Gong, J. X.; Wang, Z. Z.; Li, J. Y.; Li, J.; Li, X. W.;
Guo, Y. W. Design, synthesis and in vitro activity of phidianidine B derivatives as novel PTP1B
inhibitors with specific selectivity. Bioorg. Med. Chem. Lett. 2016, 26, 778–781.
(14) Li, Z.; Chen, W.; Hale, J. J.; Lynch, C.; Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach,
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