Cyclic Depsipeptide BE-43547A2: Synthesis and Activity against Pancreatic Cancer Stem Cells

Article abstract of DOI:10.1002/anie.201709744

Asymmetric total synthesis of the cyclic depsipeptide BE-43547A₂ was achieved in 15 linear steps on a 350 mg scale in one batch. The synthesis features the highly diastereoselective construction of an α-hydroxy-β-ketoamide through α-hydroxylation with a d.r. of up to 86:1. BE-43547A₂ significantly reduces the percentage of pancreatic cancer stem cells (PCSCs) in Panc-1 cell cultures, and dramatically reduces the tumorsphere-forming capability of Panc-1 cells. An in vivo tumor-initiation assay, a gold standard for cancer stem cell assays, confirmed that BE-43547A₂ can abolish the tumorigenesis of Panc-1 cells. The anti-PCSC activity of BE-43547A₂ could make this depsipeptide scaffold a promising starting point for discovering new PCSC-targeting drugs.

Full text of DOI:10.1002/anie.201709744

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Synthesis and anti-pancreatic cancer stem cell activity of
BE-43547A₂
Authors: Yuanjun Sun, Yahui Ding, Dongmei Li, Ruifei Zhou, Xiuwen
Su, Juan Yang, Xiaoqian Guo, Chuanke Chong, Jinghan
Wang, Weicheng Zhang, Cuigai Bai, Liang Wang, and Yue
Chen
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201709744
Angew. Chem. 10.1002/ange.201709744
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http://dx.doi.org/10.1002/ange.201709744

10.1002/anie.201709744
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COMMUNICATION
Synthesis and anti-pancreatic cancer stem cell activity of BE-
43547A2
Yuanjun Sun,# Yahui Ding,# Dongmei Li, Ruifei Zhou, Xiuwen Su, Juan Yang, Xiaoqian Guo, Chuanke
Chong, Jinghan Wang, Weicheng Zhang, Cuigai Bai, Liang Wang,* and Yue Chen*
Abstract: The asymmetric total synthesis of cyclic depsipeptide BE-
43547A2 was achieved in 15 linear steps on a 350 mg scale in one
batch. Our synthesis is featured with highly diastereoselective
construction of α-hydroxy-β-ketoamide via α-hydroxylation with a d.r.
up to 86:1. BE-43547A2 can significantly reduce the percentage of
pancreatic cancer stem cells in Panc-1 cells, and dramatically ablate
the tumorsphere forming capability of Panc-1 cells. The tumor-
initiating assay in-vivo, a gold standard for cancer stem cell assays,
confirmed BE-43547A2 could abolish the tumorigenesis of Panc-1
cells. The anti-PCSC activity of BE-43547A2 will make this
depsipeptide scaffold a start for discovering new PCSC-targeting
drug.
BE-43547 family indicated its high cytotoxicity against various
cancer cell lines.[5] Recently, Poulsen and co-workers made a
break-through in designing the first synthetic route to the BE-
43547 macrocyclic scaffold.[6] With their chemical synthesis of
ent-BE-43547A1 and the biosynthesis of the BE-43547 family,
they determined the absolute configuration of BE-43547A1~A2
1
and proposed that the originally reported H NMR data for BE-
43547A1 and A2 were incorrect.[6] Meanwhile they demonstrated
that BE-43457A1 and A2 possess significant hypoxia-selective
growth inhibitory activity against human pancreatic cancer Panc-
1 cells. We also confirmed hypoxia selectivity of BE-43547A2
against MCF-7 and K562 cell lines (see the supporting
information, Table S3). Since intermittent[7] and sustained[8]
hypoxia have been reported to induce CSC-like properties
resulting in the invasiveness and metastasis of pancreatic
cancer cells, we speculated that BE-43457s may target PCSC.
For validation of BE-43457s' selectivity towards PCSC, a highly
efficient synthetic route to provide sufficient BE-43457s is
important. Herein, we report our total synthesis of BE-43547A2
along with its biological activity of selectively ablating PCSC.
Pancreatic cancer, one of the most refractory cancers, has an
overall 5-year survival rate of only 1-4%.[1] Pancreatic cancer
stem cells (PCSC) may play a key role in the carcinogenesis,
metastasis and drug resistance of pancreatic cancer.[2] For
example, CD24+CD44+ESA+ pancreatic cancer cells, the
commonly recognized PCSC, were able to generate tumors in
half of the tested mice with 100 cells injected, which
demonstrated 100-fold higher tumorigenic potential than regular
pancreatic cancer cells.[3] However, compounds targeting
pancreatic cancer stem cells are still very rare. Triptolide,
AZD7762 and LDE225 with modest PCSC selectivity, have
entered clinical trial against pancreatic cancer.[4] Therefore, new
type of compounds that can effectively target PCSC is in urgent
need.
Scheme 1. Structure of the BE-43547 family.
On the other hand, the BE-43547 family, isolated from
Streptomyces sp., is composed of cyclic depsipeptides
structurally characterized by 4-amido-2,4-pentadienoate (APD)
and α-hydroxy-β-ketoamide. Preliminary biological assays of the
Scheme 2. Retrosynthetic analysis of BE-43547A2.
The retrosynthetic analysis is shown in Scheme 1. Mesylation of
the primary alcohol 2 followed by elimination was used to
introduce APD.[9] Macrolactamization between C11 and N10
could be employed to form 2. Disconnection of 3 through the
C1−O35 bond resulted in the alcohol 4 and acid 5. The alcohol 4
could be prepared by the hydroxylation of compound 6 at C15.
[*]
Y. Sun, # Y. Ding, # D. Li, R. Zhou, X. Su, J. Yang, X. Guo, C. Chong,
J. Wang, W. Zhang, L. Wang, * Prof. Dr. Y. Chen*
The State Key Laboratory of Medicinal Chemical Biology, College of
Pharmacy and Tianjin Key Laboratory of Molecular Drug Research,
Nankai University
Tianjin 300071 (China)
E-mail: lwang@nankai.edu.cn
yuechen@nankai.edu.cn
C. Bai
High-throughput Molecular Drug Discovery Center,Tianjin
International Joint Academy of Biomedicine
Tianjin 300457 (China)
[#]
These authors contributed equally to this work.
Supportinginformation for this article can be found under:
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10.1002/anie.201709744
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COMMUNICATION
Scheme 3. Total synthesis of BE-43547A2. DIBAL-H = diisobutylaluminum hydride, EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, HOBt =
1-hydroxybenzotriazole, DMP = Dess-Martin periodinane, CSO = camphorsulfonyloxaziridine, TBAF = tetra-n-butylammonium fluoride, DIC = diisopropyl
carbodiimide, HATU = O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene.
Table 1. Optimization of the conditions for the α-hydroxylation of 6[a]
Entry
base
eq. (base)
oxidant
d.r.[b]
Yield[c]
1
2
LiHMDS
NaHMDS
1.0
1.0
17a
17a
5:1
9:1
5%
35%
Figure 1. Optimized structures for the intramolecular H-bond involving
potassium enolate 1 (A) and potassium enolate 2 (B) of the model compound;
proposed potassium enolate with a six-membered ring (C).
3
4
5
6
7
KHMDS
KHMDS
KHMDS
KHMDS
KHMDS
1.0
0.2
0.1
0.2
0.2
17a
17a
17a
17b
17c
19~40:1
68~86:1
62:1
70~88%
71~81%
39%
39:1
28%
Reactions reported for direct α-hydroxylation to construct the
acyclic α-hydroxy-β-ketoamide are rare,[11] and we planned to
stereoselectively install the OH group at C15 based on the facial
bias of the C15-C18-C19 allylic system of enolated 6. Different
conditions were tested (Table 1), and 0.2 eq. of KHMDS as the
base and D-CSO[12] as the oxidant were applied as the
optimized conditions to provide 16 in a d.r. of 68~86:1 (for other
conditions, see the Supporting Information, Table S1). Density
functional theory calculations indicated that the optimized
structure for the intramolecular hydrogen bond involving
potassium enolate (Figure 1A) was 2.8 kcal/mol lower in energy
than the linear potassium enolate (Figure 1B). These results
suggested that the N13-H may be involved in an intramolecular
hydrogen bond with O36 to form a six-membered ring, which
helped to enhance the facial selectivity in the hydroxylation
reaction (Figure 1C).
14:1
13%
[a] All reactions were carried out with 6 (0.20 mmol) and CSO (0.21 mmol). [b]
d.r. was determined by high performance liquid chromatography. [c] Yield was
determined by 1H NMR using 1,3,5-trimethoxybenzene as an internal
standard.
Synthesis began with the construction of compound 12 (for the
synthesis of 12, see the Supporting Information, Scheme S1).
Reduction of 12 with DIBAL-H provided aldehyde 13, which was
then subjected to the aldol reaction[10] with 8 to give 7 in d.r. >
20:1. Hydrolysis of 7 followed by amide coupling with 14
provided compound 15. Oxidation of the alcohol 15 with DMP
provided ketone 6 in a 93% yield with d.r. = 2:1.
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10.1002/anie.201709744
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Deprotection of TBS for 16 by TBAF provided alcohol fragment 4. compound compared to the other derivatives. Xenograft
With the key fragments 4 and 5 (see the Supporting Information,
Scheme S2) in hand, we completed the total synthesis of BE-
43547A2. First, fragment 4 was coupled with fragment 5 under
DIC and DMAP conditions to give 3 in a 70% yield. All three
protective groups in compound 3 were removed in the presence
of TFA, and the resulting mixture was subjected to the key ring
closing reaction under HATU and DIPEA conditions.[13]
Macrolactamization proceeded smoothly to produce 630 mg of
compound 2. Finally, mesylation of 2 followed by elimination
afforded 350 mg of BE-43547A2. The spectroscopic data
obtained for synthetic 1 were different from those of BE-43547A2
reported in the patent[5], but they were in accordance with the
data of biosynthetic BE-43547A2 reported by Poulsen.
zebrafish model experiments using Panc-1 cells further
demonstrated that BE-43547A2 has higher anti-pancreatic
cancer activities than the compounds 2a and TBDPS-ent-
vinylamycin[14] (see the Supporting Information, Figure S4 and
S5). The significantly reduced inhibitory activities of 1c, 2, 2a
and 2b implied that both the double bond between C8-C9 and
the S configuration at C15 are essential to maintain the inhibitory
activity against the Panc-1 cell line.
To evaluate the effect of BE-43547A2 on PCSC, we first
analyzed the activity of BE-43547A2 against the pancreatic stem
cell (with CD24+CD44+ESA+ biomaker) in Panc-1 cells. Some
anti-cancer drugs and reported anti-PCSC compounds[4, 15] were
used as comparison (Figure 3A). Based on the results at their
respective concentration about 2×IC50 (Figure 3B), the
percentage of stem cells increased by 8.8-fold after Dox
treatment, consistent with CSC being resistant to conventional
chemotherapy. While both resveratrol[15b] and triptolide (clinical
trial, phase II)[4b] resulted in minimal change to the percentage
of stem cell. Upon treatment with two reported PCSC-targeting
compounds, AZD-7762 (clinical trial, phase Ib)[4c] and LDE-225
(clinical trial, phase I)[4a], 3.4-old and 1.5-fold decrease in the
proportion of CD24+CD44+ESA+ stem cells were observed. The
same trend was also found in the sulforaphane[15a] treated group
(3.0-fold). To our delight, the percentage of CD24+CD44+ESA+
stem cells significantly declined to 0.075% after BE-43547A2
treatment, which was over 21-fold less than that in the untreated
control (1.6%), indicative of the PCSC selectivity of BE-43547A2
is significantly superior to that of other reported PCSC-targeting
compounds.[4, 15]
Figure 2. The IC50 values of BE-43547A2 and its derivatives against Panc-1
cells.
Based on our synthetic route, 4 derivatives (1c, 2, 2a and 2b)
were easily prepared (see the Supporting Information, Scheme
S4) to evaluate the influence of the double bond between C8-C9
and the absolute configuration at C15 (Figure 2). From the
results of the MTT assay, BE-43547A2 was the most effective
Figure 3. BE-43547A2 decreased the percentage of pancreatic cancer stem cell in Panc-1 cells, inhibited tumorsphere formation in vitro and reduced the tumor
initiation frequency. (A) The IC50 value of investigated compounds against Panc-1 cells. (B) Percentage of CD24+CD44+ESA+ cells in Panc-1 cells after treatment
with compounds at the concentration about 2×IC50. Dox: 4 µM; triptolide: 0.4 µM; resveratrol: 350 µM; LDE225: 33 µM; sulforaphane: 26 µM; AZD7762: 14 µM;
BE-43547A2: 2 µM. (C) Quantitative analysis of tumorsphere formation. Dox, sulforaphane and AZD7762 was used at the concentration about 1/2×IC50 (1 µM, 6.5
µM and 3.5 µM respectively). Paclitaxel was used at the concentration of 1.5×IC50 (10 nM). (D) Representative images of Panc-1 spheres treated with BE-
43547A2, Dox and paclitaxel for 7 days. (E) BE-43547A2 reduced the tumor initiation frequency (TIF) in the tumor initiation assay. (TIF refers to the average
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10.1002/anie.201709744
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COMMUNICATION
number of cells determined to be required to cause tumor growth in the recipient cohort. The results shown represent the mean ± SD of three independent
experiments. * p < 0.05 vs control, ** p < 0.01 vs control, *** p < 0.001 vs control).
PCSC could be enriched in non-adherent spherical clusters of
cells which was called tumorsphere. In order to further examine
the effect of BE-43547A2 on PCSC, we conducted tumorsphere
formation assay (Figure 3C and 3D). With good performance in
the CD24+CD44+ESA+ biomarker assay, sulforaphane, AZD7762
as well as Dox were chosen as the controls.The results (Figure
3C) indicated that treatment with BE-43547A2 at the
This work was supported by the National Natural Science
Foundation of China (NSFC) (NO. 81573282 to Y.C.; NO.
81703350 to L.W.), the National Science Fund for Distinguished
Young Scholars (NO. 81625021) to Y.C.
Keywords: BE-43547 • cyclic depsipeptide • cancer stem cells •
hydroxylation • pancreatic cancer
concentration of 1/2×IC50 induced
a
significant 29-fold
tumorspheres forming reduction compared to the untreated
control, which is superior to sulforaphane and AZD7762 (1.1-fold
and 2.1-fold of reduction, respectively).
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The most convincing test for cancer stem cells is the limiting
dilution tumor-initiating assay (Figure 3E). According to the
result, the Dox-treated group (at a concentration of 1 µM)
displayed no reduction of the tumor-initiating frequency. In
contrast, the BE-43547A2-treated group (at a concentration of
0.25 µM) displayed a 6.8-fold reduction of the tumor-initiating
frequency, and no tumor was observed after treatment with 1 µM
BE-43547A2. These results confirmed that BE-43547A2 could
abolish the capability of tumorigenesis of Panc-1 pancreatic cells
in vivo.
[3]
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In conclusion, we have accomplished the asymmetric total
synthesis of BE-43547A2 on a 350 mg scale in 15 linear steps.
Due to the rare report of the direct α-hydroxylation of acyclic β-
ketoamide,
our
synthesis
is
featured
with
highly
diastereoselective construction of α-hydroxy-β-ketoamide (d.r.
up to 86:1). This efficient synthetic sequence can not only
provide abundant BE-43547A2, but also resulted in several
derivatives for further biological assays. The preliminary
structure-activity-relationship study included in-vitro assay and
in-vivo assay with xenograft zebrafish model (see the supporting
information, Figure S4 and S5), both experiments indicated that
the C8-C9 double bond and 15S configuration are essential to
maintain the inhibitory activity against the Panc-1 cell line. More
importantly, we discovered that BE-43547A2 exhibited superior
PCSC selectivity compared to other compounds targeting PCSC.
The percentage of CD24+CD44+ESA+ stem cells decreased by
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tumorsphere forming assay, the number of tumorspheres formed
in the BE-43547A2-treated group was around 46-fold less than
that in the Dox-treated group. The tumor-initiating assay further
confirmed that BE-43547A2 could abolish the capability of
tumorigenesis of Panc-1 pancreatic cells. All of the biological
assays indicate that BE-43547A2 can selectively ablate
pancreatic cancer stem cells. These properties are highly
desirable for selecting PCSC drug candidates in preclinical study.
The highly productive total synthesis and exciting anti-PCSC
activities, along with the unknown mechanism of action, warrant
BE-43547A2 as a promising beginning for the discovery of novel
PCSC-targeting drugs.
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Acknowledgements
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COMMUNICATION
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COMMUNICATION
Yuanjun Sun, # Yahui Ding, # Dongmei Li,
Ruifei Zhou, Xiuwen Su, Juan Yang,
Xiaoqian Guo, Chuanke Chong,
Weicheng Zhang, Cuigai Bai, Liang
Wang,* and Yue Chen*
Page No. – Page No.
Synthesis and anti-pancreatic cancer
stem cell activity of BE-43547A2
The total synthesis of BE-43547A2 was achieved in 15 linear steps on a 350 mg scale. Our synthesis is
featured with highly diastereoselective construction of α-hydroxy-β-ketoamide. We discovered that BE-
43547A2 can reduce the percentage of pancreatic cancer stem cells by 21-fold. In the tumorsphere
forming assay, the number of tumorspheres formed in the BE-43547A2-treated group was about 46-fold
less than that in the Dox-treated group. The tumor-initiating assay in murine model further confirmed
that BE-43547A2 could abolish the capability of tumorigenesis of Panc-1 cells.
This article is protected by copyright. All rights reserved.