Design and synthesis of plant cyclopeptide Astin C analogues and investigation of their immunosuppressive activity

Article abstract of DOI:10.1016/j.bmcl.2018.05.050

To further investigate on the structure-activity relationships of immunosuppressive Astin C, seventeen analogues 1–17 were designed and synthetized via amino acid substitution strategy by the solid-phase peptide synthesis method for the first time. In comparison with Astin C (IC₅₀ = 12.6 ± 3.3 μM), only compounds 2 (IC₅₀ = 38.4 ± 16.2 μM), 4 (IC₅₀ = 51.8 ± 12.7 μM), 5 (IC₅₀ = 65.2 ± 15.6 μM), and 8 (IC₅₀ = 61.8 ± 12.4 μM) exhibited immunosuppressive activity in the Lymph node cells of mice. These results showed that the Astin C analogues containing _D-amino acid residues, hydrophobic long-chain alkyl substituents, and aryl substituents performed better than those carrying hydrophilic amino acid residues and short-chain alkyl substituents. Moreover compounds 15, 16, and 17 had no immunosuppressive activity, which suggested that cis-3,4-dichlorinated proline played an important role in the immunosuppressive activity of Astin C.

Full text of DOI:10.1016/j.bmcl.2018.05.050

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of plant cyclopeptide Astin C analogues and
investigation of their immunosuppressive activity
⁎
Fei Lia,c,d, Xi-Xi Guob,d, Guang-Zhi Zenga, Wei-Wei Qinb, Bo Zhangb, Ning-Hua Tana,b,
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s
Republic of China
b
State Key Laboratory of Natural Medicines, Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing
211198, People’s Republic of China
c
University of Chinese Academy of Sciences, Beijing 10049, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To further investigate on the structure-activity relationships of immunosuppressive Astin C, seventeen analogues
Astin C analogues
Solid-phase peptide synthesis
Immunosuppressive activity
1–17 were designed and synthetized via amino acid substitution strategy by the solid-phase peptide synthesis
method for the first time. In comparison with Astin
C
(IC50 = 12.6
3.3 μM), only compounds
2
8
(IC50 = 38.4
(IC50 = 61.8
16.2 μM),
4
(IC50 = 51.8
12.7 μM),
5
(IC50 = 65.2
15.6 μM), and
12.4 μM) exhibited immunosuppressive activity in the Lymph node cells of mice. These results
showed that the Astin C analogues containing D-amino acid residues, hydrophobic long-chain alkyl substituents,
and aryl substituents performed better than those carrying hydrophilic amino acid residues and short-chain alkyl
substituents. Moreover compounds 15, 16, and 17 had no immunosuppressive activity, which suggested that cis-
3,4-dichlorinated proline played an important role in the immunosuppressive activity of Astin C.
In our project on unique structure and potential activity of cyclo-
peptides from traditional Chinese medicines (TCMs), we have found
that Astins, a family of homomonocyclopeptides, named as Compositae-
type cyclopeptides, were only isolated from the roots and rhizomes of
Aster tataricus L. (Compositae)1–4, which is one of TCMs used for re-
lieving cough and eliminating phlegm.5 It is noteworthy that Astins
share an unusual structural similarity, which are mainly characterized
by the presence of four unnatural amino acids (L-Abu, L-allo-Thr, β-Phe,
and mono- or di-chlorinated L-Pro residues) and one proteinogenic
amino acid (L-Ser).1 So far, sixteen cyclopentapeptides (Astins A–I1, K-
P2) together with two unique skeleton cyclopeptides, Tataricins A and
B3 (Fig.1), have been isolated from A. tataricus. Previous studies have
shown that Astin A, B, and C with the cis-3,4-dichlorinated proline re-
sidue played an important role in antitumor activity.6,7 Cozzolino et al.
have reported that synthetized antineoplastic cyclic astin analogues can
kill tumor cells via caspase-mediated induction of apoptosis.8 Recently
we have demonstrated that Astin C could induce mitochondria-depen-
Astins. The first is Astin G, which was totally synthetized in 1999.9 The
second is Tataricins A and B, which were synthetized by our group in
2013.3 However, study on the structure-immunosuppressive activity of
Astin C has not been carried out to date. Herein, several amino acid
substitution strategies have been conducted to modify Astin C and se-
venteen analogues were synthetized for the first time by the solid-phase
peptide synthesis (SPPS) method. Their immunosuppressive activities
were screened against Lymph node cells and the structure-activity re-
lationships (SARs) have been discussed.
Astin C contains a cyclic pentapeptide core comprised of two L-Abu,
one L-Ser, one β-Phe, and one strikingly cis-3,4-dichlorinated L-Pro. Its
structure was divided into two parts, i.e. substitution part and con-
served sequence part (Fig. 2). Firstly, we synthetized two analogues by
the SPPS method, which bear 3,4-dehydro L-Pro in compound 15 and
cis-3,4-dihydroxylated L-Pro in compound 16, to study the role of the
cis-3,4-dichlorinated L-Pro on the immunosuppressive activity against
lymph node cells. Secondly, we synthetized other fifteen Astin C ana-
logues 1–14 and 17 via amino acid substitution strategy by the SPPS
method to study their SARs.
dent apoptosis of activated
T cells, and exhibited potential im-
munosuppressive activity in vitro and in vivo.4 To the best of our
knowledge, Astin C and its derivatives with a chlorinated proline have
not been synthetized. There are only two reports about the synthesis of
Firstly, we prepared the intermediate cis-3,4-diol-L-Pro10 P-5a, and
the synthetic route is shown in Scheme 1. Compound P-1 was prepared
⁎
Corresponding author at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
People’s Republic of China.
E-mail address: nhtan@cpu.edu.cn (N.-H. Tan).
These authors contributed equally to this work.
d
https://doi.org/10.1016/j.bmcl.2018.05.050
Received 27 March 2018; Received in revised form 24 May 2018; Accepted 26 May 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Li,F.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.05.050

F. Li et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 1. Reagents and conditions: (a) Boc2O, NaHCO3, dioxane : H2O = 1 :
1, rt, 24 h, 86%; (b) BnBr, K2CO3, DMF, rt, overnight, 92%; (c) Ph3P, I2, imi-
dazole, DCM, N2, reflux, 12 h, 97%; (d) DBU, ACN, reflux, overnight, 78%; (e)
K2OsO2(OH)4, K3Fe(CN)6, K2CO3, t-BuOH : H2O = 1 : 1, rt, 24 h, 83%.
Next, solid-phase synthesis of other Astin C analogues were carried
out by substituting the cis-3,4-dichlorinated proline residue with var-
ious amino acids. Considering that Astins is all consisted of L-amino
acid, we firstly introduced D-Thr and D-allo-Thr to synthetize Astin C
analogues 2 and 4. Moreover, hydrophobic amino acid residues (L-Ada,
L-Val, L-Leu, β-Phe, L-Tyr) and hydrophilic amino acid residues (L-Thr, L-
allo-Thr, L-Lys, L-Arg, L-His, (2S, 3S)-3-O-(pyrrole-2-carbonyloxy)-allo-
Thr) were also introduced to prepare Astin C analogues 1, 3, and 5–13,
respectively. Because of the same sequence of linear tetrapeptide with L-
Abu5-β-Phe4-L-Ser3-L-Abu2 in 1–16, we designed to cyclize at the Abu5/
substituted amino acid1 site. As an example, the synthetic route of
compound 2 was shown in Scheme 3.
Fig. 1. Structures of Astins family.
Compound 2 was synthetized using the Fmoc-Abu-CTC Resin. Fmoc
group protecting amino acids were successively coupled by HBTU and
DIPEA in DMF. 20% piperidine/DMF was used for deblocking. The
reaction was monitored by ninhydrin color reaction. After the last step,
the peptide resin was washed with DMF (three times), and MeOH (three
times), then dried by N2 bubbling over night. TFA in DCM was added to
the flask containing the peptide resin to release the crude linear peptide
from the resin. Cyclization was accomplished by using HATU and
DIPEA. The progress of the cyclization was monitored by LC-MS. The
solvent was removed under reduced pressure and the crude peptide was
purified by Prep-HPLC to get the final product 2.12 Similarly, com-
pounds 1, 3–13 were prepared by the above synthetic method.
With Astin C and seventeen analogues 1–17 in hand (Fig. 3), we
have screened immunosuppressive activity against lymph node cells, in
which CsA was used as the positive control. As shown in Table 1, Astin
C shows potential immunosuppressive activity. Compared with Astin C
Fig. 2. Substitution of the structure of Astin C.
starting with commercially available trans-4-hydroxy-L-proline and
Boc2O in the presence of NaHCO3 in dioxane/H2O, which reacted with
BnBr in DMF to give compound P-2 in excellent yield. By using Appel-
Lee reaction, compound P-2 was successfully converted to compound P-
3 in 97% yield. Subsequently, elimination of compound P-3 with DBU
in ACN afforded compound P-4a as a major product, which is oxidized
by K2OsO2(OH)4 to the diol P-5a as the major product.
With 3,4-dehydro-L-Pro P-4a and cis-3,4-diol-L-Pro P-5a in hand, cis-
3,4-dichlorinated L-Pro in Astin C was then modified. As depicted in
Scheme 2, cis-3,4-dichlorinated L-Pro was substituted by Ala (Ala
scan11), P-4a, and P-5a, respectively, and compounds 14, 15, and 16
were obtained by the SPPS method similar to Scheme 3. In addition,
cyclotetrapeptide 17 was prepared by cutting the cis-3,4-dichlorinated
proline residue.
(IC50 = 12.6
D-Thr residue and compound 4 (IC50 = 51.8
Thr residue showed comparable immunomodulatory activity, which
opened doors to us that the introduction of D-amino acids is a moderate
3.3 μM), compound 2 (IC50 = 38.4
16.2 μM) with
12.7 μM) with D-allo-
strategy. On the other hand, compound 5 (IC50 = 65.2
with hydrophobic long-chain alkyl substituent and compound
(IC50 = 61.8 12.4 μM) with β-Phe residue (aryl substituent) also
exhibit comparable immunosuppressive activity. Unfortunately, we
15.6 μM)
8
2

F. Li et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 2. Solid-phase synthesis of compounds 14, 15, 16, and 17.
Scheme 3. Solid-phase synthesis of compound 2.
found that all of hydrophilic L-amino acid residues were inactive
(IC50 > 100), including compounds 1, 3, and 10–13. These results
show that the Astin C analogues containing D-amino acid residues,
hydrophobic long-chain alkyl substituents, and aryl substituents per-
formed better than those carrying hydrophilic amino acid residues and
short-chain alkyl substituents. To further explore the role of the cis-3,4-
dichlorinated L-Pro, we synthetized compounds 14–17, which resulted
in loss of activity against lymph node cells (IC50 > 100), suggesting
that the significance of cis-3,4-dichlorinated proline to the
immunosuppressive activity.
In summary, seventeen Astin C analogues 1–17 were designed and
synthesized by the SPPS method and their immunosuppressive activity
was evaluated against lymph node cells for the first time. The results
indicated that compounds 2, 4, 5, and 8 exhibited immunosuppressive
activity. Analysis of the structure-activity relationships indicated that D
-
amino acid residues, hydrophobic long-chain alkyl substituents, and
aryl substituents might act as a guideline for the further modification of
Astin C. Moreover, we found that cis-3,4-dichlorinated proline plays an
3

F. Li et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 3. Structures of Astin C analogues 1–17.
Acknowledgments
Table 1
Screening of immunosuppressive activity.
This work was supported by the National Natural Science
Foundation of China (31470428), the National New Drug Innovation
Major Project of China (2017ZX09309027), the Fund of Chinese
Academy of Sciences (XDA09030301-4, Hundred Talents Program), the
Program for Jiangsu Province Innovative Research Team, and the Fund
for Introduction of High-level Talents from China Pharmaceutical
University.
Compunds
IC50 (mean
SD, μM)
Astin C
1
2
3
4
5
6
7
12.6
3.3
> 100
38.4
16.2
> 100
51.8
65.2
12.7
15.6
> 100
> 100
61.8
A. Supplementary data
8
12.4
9–17
CsA
> 100
0.5
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2018.05.050.
0.04
CsA was used as the positive control.
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5