A novel Anti-Cancer Stem Cells compound optimized from the natural symplostatin 4 scaffold inhibits Wnt/β-catenin signaling pathway

Article abstract of DOI:10.1016/j.ejmech.2018.06.046

Cancer stem cells (CSCs) are responsible for carcinogenesis, cancer progression, relapse, metastasis and drug resistance. Therefore, the development of drug molecules targeting CSCs plays a vital role in medicinal researching field. However, there are extremely rare molecules that selectively ablate CSCs. The research and development of drugs targeting CSCs is limited due to a lack of anti-CSCs lead compounds. In this study, an anti-CSCs lead compound 35b was discovered, which was derived from the natural chemical scaffold of Symplostatin 4. This compound exhibited a significantly suppressive effect on tumor growth both in vitro and in vivo. Additionally, 35b could significantly reduce the number of melanoma tumor spheres and decrease the percentage of ALDH⁺ melanoma cells. Further mechanism study illustrated that compound 35b could eliminate the melanoma CSCs by efficiently blocking Wnt/β-catenin signaling pathway. Collectively, our findings would provide a novel chemical scaffold and alternative idea of molecular design for development of anti-CSCs drugs.

Full text of DOI:10.1016/j.ejmech.2018.06.046

Accepted Manuscript
A novel Anti-Cancer Stem Cells compound optimized from the natural symplostatin 4
scaffold inhibits Wnt/β-catenin signaling pathway
Shuangwei Liu, Xian Gao, Lisong Zhang, Shuanglin Qin, Mingming Wei, Ning Liu, Rui
Zhao, Benlong Li, Ye Meng, Gang Lin, Cheng Lu, Xinhua Liu, Maodun Xie, Tongtong
Liu, Honggang Zhou, Min Qi, Guang Yang, Cheng Yang
PII:
S0223-5234(18)30537-3
10.1016/j.ejmech.2018.06.046
EJMECH 10516
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 April 2018
Revised Date: 16 June 2018
Accepted Date: 18 June 2018
Please cite this article as: S. Liu, X. Gao, L. Zhang, S. Qin, M. Wei, N. Liu, R. Zhao, B. Li, Y. Meng, G.
Lin, C. Lu, X. Liu, M. Xie, T. Liu, H. Zhou, M. Qi, G. Yang, C. Yang, A novel Anti-Cancer Stem Cells
compound optimized from the natural symplostatin 4 scaffold inhibits Wnt/β-catenin signaling pathway,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.046.
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ACCEPTED MANUSCRIPT
A Novel Anti-Cancer Stem Cells Compound Optimized from the Natural Symplostatin 4 Scaffold
Inhibits Wnt/β-Catenin Signaling Pathway
Shuangwei Liu,1, § Xian Gao,1, § Lisong Zhang,1, § Shuanglin Qin,1,2, § Mingming Wei,1, § Ning Liu,1 Rui
Zhao,1 Benlong Li,1 Ye Meng,1 Gang Lin,1 Cheng Lu,1 Xinhua Liu,1 Maodun Xie,1 Tongtong Liu,1
Honggang Zhou,1 Min Qi,3,* Guang Yang,1,* and Cheng Yang1,*

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A Novel Anti-Cancer Stem Cells Compound Optimized from the Natural Symplostatin 4 Scaffold
Inhibits Wnt/β-Catenin Signaling Pathway
Shuangwei Liu,1, § Xian Gao,1, § Lisong Zhang,1, § Shuanglin Qin,1,2, § Mingming Wei,1, § Ning Liu,1 Rui
Zhao,1 Benlong Li,1 Ye Meng,1 Gang Lin,1 Cheng Lu,1 Xinhua Liu,1 Maodun Xie,1 Tongtong Liu,1
Honggang Zhou,1 Min Qi,3,* Guang Yang,1,* and Cheng Yang1,*
1The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University,
Tianjin 300071, People’s Republic of China
2
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, People’s
Republic of China
3Tianjin Key Laboratory of Molecular Drug Research, Tianjin International Joint Academy of
Biomedicine, Tianjin, 300457, People’s Republic of China
Hightlights:
Cancer Stem Cells
Total Synthesis
Symplostatin 4
Depsipeptides
Abstract
Cancer stem cells (CSCs) are responsible for carcinogenesis, cancer progression, relapse,
metastasis and drug resistance. Therefore, the development of drug molecules targeting CSCs
plays a vital role in medicinal researching field. However, there are extremely rare molecules that
selectively ablate CSCs. The research and development of drugs targeting CSCs is limited due to a
lack of anti-CSCs lead compounds. In this study, an anti-CSCs lead compound 35b was discovered,
which was derived from the natural chemical scaffold of Symplostatin 4. This compound
exhibited a significantly suppressive effect on tumor growth both in vitro and in vivo. Additionally,
35b could significantly reduce the number of melanoma tumor spheres and decrease the
percentage of ALDH+ melanoma cells. Further mechanism study illustrated that compound 35b
could eliminate the melanoma CSCs by efficiently blocking Wnt/β-catenin signaling pathway.
Collectively, our findings would provide a novel chemical scaffold and alternative idea of
molecular design for development of anti-CSCs drugs.
INTRODUCTION
Medicinal studies on natural products exert a crucial impact upon both chemical biology and
drug discovery. More than half of all clinical medicines have their origin from natural sources,
especially anti-cancer drugs. However, the miscellaneous chemical structures and moderate
activities of natural molecules have severely impeded the progress of novel drug development.
Synthesis and optimization of natural product scaffolds have not only brought an effective
solution to drug discovery, but also prepared novel chemical structures with improved efficacies.
Cancer stem cells (CSCs) are defined as a type of rare cancer cells with embryonic or somatic
stem cell-like properties, including the ability of self-renewal and multipotent differentiation

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potential [1]. Although the origin of CSCs has not been well understood, the presence of these
cells has been found in most malignancies, such as hematological malignancy, lung cancer, breast
cancer, gliomas, ovarian cancer and melanoma [2]. Of note, an avalanche of evidence has
demonstrated that CSCs are critically involved in tumorigenesis, cancer invasion, metastasis,
tumor recurrence and therapeutic resistance [3]. Hence, CSCs are hypothesized to be the
progenitor cells for tumor recurrence at primary or distant locations after appropriate and
standard cancer therapy. However, drugs targeting common cancer cells generally have no effects
on CSCs, due to the special characteristics of CSCs. [4] Thus, it is urgent to probe into novel
therapeutic strategies targeting CSCs.
Wnt/β-catenin signaling pathway, a canonical Wnt pathway, is considered to be an important
regulator for the maintenance of self-renewal of CSCs. Constitutive activation of Wnt/β-catenin
[5]
signaling pathway in CSCs strongly contributes to cancer proliferation, relapse and metastasis.
In brief, in activated Wnt signaling pathway, activated disheveled homologue (Dvl)
phosphoprotein, a downstream effector of the receptor, inhibits Axin-mediated phosphorylation
and degradation of β-catenin, resulting in its accumulation in cytoplasm. Then, β-catenin
translocates into nucleus and interacts with T-cell factor/lymphoid-enhancing factor (TCF/LEF) to
activate the transcription of downstream targeted genes, which subsequently regulate cell
[6]
proliferation and survival. In diverse types of malignancies, dysregulation of the expression of
Wnt, Frizzled receptors, Axin, and mutation of APC or β-catenin has been proved to induce the
[7]
persistent activation of Wnt/β-catenin signaling pathway.
Therefore, targeting these key
regulators to control the self-renewal and differentiation of CSCs is a novel strategy to overcome
CSCs-associated tumorigenesis for cancer treatment.
Symplostatin 4 (also known as Gallinamide A), is a natural depsipeptide independently
isolated from the Symploca genus in Key Largo, Florida and from Schizothrix species of
cyanobacteria from the Carribean Coast of Panama. [8] Firstly, Symplostatin 4 was found to be one
of the most potent anti-malaria depsipeptides, as it strongly exhibited the activity of cultured P.
falciparum in vitro, with an IC50 of 50 nM. More importantly, Symplostatin 4 did not exhibit any
hemolytic activity against red blood cells, nor did it inhibit the proteasome. These above
properties have made this natural product as an ideal leading compound for development of new
type anti-malaria drugs. The total synthesis of this molecule has been reported previously by
[9]
Payne’s group
and Kaiser’s group [10], and the medicinal chemistry and mechanism on
anti-malaria efficacy has been extensively studied. [11]
Symplostatin 4 has also been demonstrated to exert moderate activities against Hela and
HT-29 cell lines (with IC50 of 12 and 53 uM, respectively). [12] However, there have no researching
reports concerning its structure−acꢀvity relaꢀonships (SARs) and mechanisꢀc studies. Structurally,
Symplostatin 4 possesses a unique conjugated unsaturated amide system, which might be the key
pharmacophore. It is hypothesized that this molecule is a covalent, irreversible inhibitor, as a
Michael acceptor, via nucleophilic attack by the sulfhydryl side chain of the active site cysteine.
Additionally, Symplostatin 4 also possesses an unusual 4(S)-amino-2-(E)-pentenoyl moiety and
analiphatic depsipeptide chain. Based on these unique structural features, we envisaged that
preparation of Symplostatin 4 and its analogues via our optimized synthetic route. It was
proposed that the structural modification might provide important alternation on anti-cancer
activity and stability of these drug molecules.

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RESULTS AND DISCUSSION
Chemistry
Retrosynthetic analysis of Symplostatin 4
[9, 10,
Previously, there were three synthetic reports on the total synthesis of Symplostatin 4.
13]
Generally, all of the synthetic strategies were the same, which was disconnected from C18-N
amide bond (Scheme 1). The final amide coupling step yielded a messy mixture. The natural
molecule 1 could only be purified by preparation HPLC. These synthetic route could not easily
generate pure Symplostatin 4 and its analogues in a useful amount, which might greatly impede
the anti-cancer medicinal chemistry study on this Symplostatin 4 Scaffold.
Scheme 1. Previous Retrosynthetic Analysis
To this end, an alternative method was employed to efficiently prepare Symplostatin 4. Our
retrosynthetic analysis was straightforward, as shown in Scheme 2. Initial disconnection of the
conjugated olefin from C8-C9, gave rise to two fragments: the aldehyde 4 and the phosphonate
ester 5. According the modified method, further disconnection through C18-N amide bond led to
sulfite ester 6 and amine 7.
Scheme 2. Alternative Retrosynthetic Analysis
Total synthesis of Symplostatin 4
The synthesis of the required fragment 6 started from L-Leu-OH (shown in Scheme 3).
Treatment of L-Leu-OH with NaNO2 in acidic aqueous solution produced alcohol 9 in 95% yield.
Allyl bromide was employed to protect the carboxylic acid. The resulting intermediate 10 was
esterified on the hindered alcohol with N-Cbz-L-Leu-OH under our previous optimized conditions

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delivered ester 11, [14] without any epimerization observed. Cleavage of allyl ester in the presence
of Pd(0) catalyst was followed by further coupling with 4-Chlorothiophenol final yield the key
fragment 6 in 86% yield over 2 steps.
Scheme 3. Synthesis of the Fragment 6
The synthesis of another key fragment 7 was shown in Scheme 4. A weinreb amide 13 was
prepared from the easily available starting material N-Boc-L-Ala-OH. Removal of Boc group by
dissolving in TFA solution and coupling with N-Fmoc-L-Leu-OH obtained a dipeptide 14 in 78%
overall yield. Deprotection of Fmoc gave rise to compound 7.
Scheme 4. Synthesis of the Fragment 7
Following preparation of both of the amino skeletons, HWE component 5 was synthesized
from known compound 15 in 2 steps (shown in Scheme 5). [15] The compound 15 was pretreated
with NaHMDS and was then reacted with 2-bromoacetyl bromide. The resulting 16 heated in
neat P(OEt)3 furnished the last fragment 5.
O
Br
Br
O
O
O
P
O
NaHMDS, THF
P(OEt)3
89%
Br
EtO
EtO
Ref
Boc
OH
HN
N
N
N
O
O
O
3 steps
75%
H
O
O
O
12
15
16
5
Scheme 5. Synthesis of the Fragment 5
With all of the coupling precursors in hands, the depsipeptide chain was constructed in 82%
yield by simply mixing intermediate 6 and 7 together in polar solvent. Cbz protecting group was
then cleaved and simultaneously di-methylated on the deprotected N-terminal in one-pot
reaction under hydrogenation conditions. The weinreb amide was then reduced by DIBAl-H. To
our delight, the terminal weinreb amide was reduced without cleavage of the ester bond, even
using excess reductant. The resulting aldehyde 4 was connected with compound 5 by HWE

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reaction to finally furnish the natural product.
Scheme 6. Fragment Coupling and Total Synthesis of Natural Product
Of note, the final step could be easily purified by flash chromatography on silica gel to obtain
the pure Symplostatin 4. More importantly, this synthesis could be achieved in gram-scale to
obtain the useful amount of material for medicinal research. By contrast, previously synthetic
approaches could only yield several milligrams of pure compound using preparation-HPLC.
Preparation of Symplostatin 4 analogues
Following the success of efficient access to natural product Symplostatin 4, we next
investigated the essential anti-cancer pharmacophore, followed by establishment of the
preliminary SARs of this natural molecule.
The unusual 4-(S)-amino-2-(E)-pentenoyl moiety (C1-C6 ring fragment) was taken place by
some easier amide and ester from the synthetic aldehyde 4 (shown in Scheme 7 and Table 1).
Wittig reaction brought conjugated ester 19 in 95% yield. Methyl ester bond was hydrolysis
delivered carboxylic acid 20. Amide coupling using HATU generated the analogues 21a-21c.
Scheme 7. Replacement of the C1-C6 Fragment
Table 1. Structure of Analogues 21a-c
Compounds
R1
Yield
65%
21a
21b
21c
52%
44%

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In addition, Cbz functionalized Symplostatin 4 was also synthesized through the same
synthetic strategy (Scheme 8 and Table 2). Weinreb amide 17 was reduced by DIBAl-H to
generate aldehyde 22. HWE reaction connected aldehyde 22 with two phosphate ester 5 and 23
to furnish the other two analogues of Symplostatin 4, 24a and 24b, with 55% and 70% yield,
respectively.
Scheme 8. Synthesis of Symplostatin 4 Analogues
Table 2. Structure of Analogues 24a and 24b
Compounds
24a
R2
Yield
55%
70%
65%
Me
Bn
i-Pr
24b
24c
Analogues of this shorter peptide chain was also synthesized to investigate its effect on
activity (Scheme 9 and Table 3). The dipeptide 25 was reduced and di-methylated under
hydrogenation conditions. The resulting intermediate 26 was converted into aldehyde 27 by LAH
reaction, in high yield. HWE reaction connected the aldehyde 27 with different various phosphate
esters, which easily derived from amino acids [x]. Four analogues, including 28a-28c, were
produced using the same combinatory step.
Scheme 9. Synthesis of Symplostatin 4 Analogues
Table 3. Structure of Analogues 28a-c
Compounds
28a
R3
Yield
70%
85%
Me
28b
(S)-Bn

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28c
28d
(R)-Bn
82%
60%
i-Pr
The synthesis of the depsipeptide chain (C9-C31) was 6 linear steps, which was still a
rate-limited project. To simplify it, we synthesized the peptide 29 in gram-scale efficiently, instead
of the depsipeptide fragment, by employing the solid-phase synthesis (Scheme 10, Table 4 and
Table 5). Weinreb amide 30 was obtained from crude peptide 29 by using excess
N-methoxy-methylamine hydrochloride. Hydrogenation followed by LAH reduction gave rise to
aldehyde analogue 32 in high yield. The same HWE strategy was used to prepare the peptide
analogues 33a and 33b. Cbz protected compounds 30 was directly reduced by LAH to give
N-Cbz-aldehyde intermediate 34, which was then converted into analogues 35a-35e in the
subsequent HWE steps.
Scheme 10. Synthesis the Symplostatin 4 Analogues
Table 4. Structure of Analogues 33a-b
Compounds
33a
R4
Yield
45%
55%
Me
i-Pr
33b
Table 5. Structure of Analogues 35a-e
Compounds Yield
R5

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35a
35b
35c
Me
72%
43%
79%
i-Pr
35d
35e
82%
84%
(S)-Bn
Following the success of solid-phase synthesis to construct this peptide chain,
N-functionalized groups were replaced for the biological evaluation. As shown in Scheme 11
(Table 6), the carbamate group Cbz of common intermediate 30 could be removed under catalytic
hydrogenation conditions to obtain the terminal amine 36. Amine 36 reacted with isocyanate to
form a carbamide 37a with 89% yield. Simultaneously, this amine coupled with carboxylic acids to
furnish amides 37b and 37c with 83% and 86% yield, respectively. The next two steps were the
same as the previously described procedures. Reduction of compound 37a-c followed by HWE
reaction finally obtained Symplostatin 4 analogues 40a-c in moderate yield.
Scheme 11. Synthesis the N-functionalized Peptides Symplostatin 4 Analogues
Table 6. Structure of Analogues 40a-c
Compounds
R6
Step-2 Yield Step-3 Yield
Step-4 Yield
52%
40a
89%
64%
40b
40c
83%
59%
47%
42%
86%
68%
In Vitro Anti-proliferative Activity.
The growth inhibitory potency of synthesized Symplostatin 4 derivatives was evaluated in
three breast cancer cells, two melanoma cells, one pancreatic cancer cell, and one lung cancer
cell, using MTT assays as described in the in vitro screening protocol. The Symplostatin 4 was
included as a comparison (Table 7). The ability of these new analogues to inhibit the growth of

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cancer cells was summarized in Table 6. Specifically, the Symplostatin 4 (1) exhibited moderate
potency against the tested cell lines (IC50 was from 31.2 to 45.6 μM), while the compounds 21a-c
and 19 had no inhibitory activity on these cell lines (IC50 > 100 μM), indicating that the unusual
5-member ring moiety (4-amino-2-(E)-pentenoyl) was essential pharmacophore structure. N-Cbz
modified analogue 24a exerted a 2-14 fold more potent antitumor activity than Symplostatin 4
against all examined cell lines. Especially, compound 24a exhibited remarkable growth inhibition
against both A375 and B16F10 cells, with the IC50 value of 5.49 and 4.23 μM, respectively, which
were 8-fold and 14-fold more potent than Symplostatin 4. However, when Bn group took the
place of Me at C-5, the structural modification would tune the anti-proliferative activity of the
corresponding analogue 24b. The analogues 28a-c, with two amino acid moiety shorterpeptide
chain, were subsequently evaluated for anti-proliferative activities on these cell lines. As a result,
the compound 28b with isopropyl substitution at C-5 was found to harbor similar anticancer
activity as 23a. Analogue 28a with Me group at C-5 led to slightly decreased antitumor activity.
While the C-5 substitutions were changed into (R)-Bn (28b) or (S)-Bn (28c), the growth inhibition
effect was almost abolished. Analogue 33a, which was a peptide analogue of natural product,
exhibited increased anti-proliferative activity of cancer cell lines. (4 fold more potent against two
melanoma cells, and 2-fold against PANC-1). In comparison with 33a, peptide analogue 33b with
Isopropyl at C-5 exhibited similar activity as Symplostatin 4. Compounds 35a and 35b, which were
N-Cbz modified peptide analogues, were found to significantly increase anti-proliferation against
breast cancer cells, melanoma cells and pancreatic cancer cells, with IC50 values varying from the
low micromolar to submicromolar range. Especially, the compound 35b was the most potent one.
Compared with Symplostatin 4, the IC50 values of analogue 35b were 2.54 μM against MCF-7
(18-fold), 0.922 μM against MDA-MB-231 (38-fold), 0.946 μM against 4T1 (36-fold), 0.890 μM
against A375 (51-fold), 0.547 μM against B16F10 (109-fold), 0.787 μM against PANC-1 (39-fold)
and 15.1 μM against A549 (2-fold). Alternatively, the synthetic analogues with alternative
functional groups on N-terniaml of 35b, which were urea analogue 40a, amide analogue 40b and
40c, led to dramatically decreased anti-tumor activity against all tested cancer cell lines.
Therefore, the compound 35b was selected as the leading compound for further investigation.
Table 7. Effects of Symplostatin 4 Analogues on Proliferation of Cancer Cell Lines
IC50
(μ
M) a
Breast Cancer
melanoma
pancreatic cancer Lung cancer
Compounds
MCF-7
MDA-M
B-231
34.9
4T1
A375
B16F10
PANC-1
A549
1
45.5
>100b
>100
>100
>100
32.9
34.6
>100
>100
>100
>100
14.96
79.8
45.6
>100
>100
>100
>100
5.49
59.7
>100
>100
>100
>100
4.23
31.2
>100
38.3
>100
>100
14.0
59.3
19.59
82.1
67
40.4
>100
>100
>100
>100
13.8
19
>100
>100
>100
>100
15.6
21a
21b
21c
24a
24b
24c
28a
28b
>100
21.23
75.1
>100
12.79
79.7
45.9
30.1
41.4
27.89
19.2
13.71
11.4
24.92
29.2
27.52
30.0
>100
57.7
>100
>100
>100
>100

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28c
28d
33a
33b
35a
35b
35c
35d
35e
40a
40b
40c
>100
18.6
38.9
>100
4.66
2.54
>100
>100
>100
10.1
11.2
14.9
94.8
89.45
38.2
98.3
1.96
0.922
>100
>100
>100
25.1
32.8
22.2
>100
>100
16.5
27.6
1.92
0.946
>100
>100
>100
35.3
40.8
20.1
>100
7.561
11.2
>100
30.47
15.1
86.9
12.76
15.7
>100
>100
50.5
52.5
15.1
4.66
>100
>100
>100
20.8
14.6
36.2
35.7
61.6
36.0
1.350
0.801
>100
>100
>100
44.2
0.892
0.522
>100
>100
>100
60.5
0.787
0.435
>100
>100
>100
>100
11.6
15.2
15.2
9.89
7.07
5.21
a
b
All values are the mean of three independent experiments. If a specific compound is given a value >100, it
indicates that a specific IC50 cannot be calculated from the data points collected, meaning “no effect”.
Compound 35b Inhibited Colony Formation of Melanoma Cells.
In consideration of its potent anti-proliferative activities against two melanoma cancer cells
(A375 and B16F10), the leading compound 35b was selected for colony formation assay.
Consequently, compound 35b exerted an inhibitory effect on colony formation in two malignant
melanoma cancer cells A375 and B16F10, as shown in Figure 1, which was consistent with the
anti-proliferative activity.
Figure 1. Inhibitory effects of compound 35b on colony formation of malignant melanoma cancer
cells. Colony formation assay was performed to measure the capacity of A375 and B16F10 cells to
form colonies. (A) Representative image of long-term growth assay of A375 and B16F10 cells
following continuous compound 35b for 7 days. (B) Quantification of clonogenic growth shown in
A. Two way ANOVA; mean ± sd, n = 3. **p<0.01.
Compound 35b Induced Apoptosis of Melanoma Cells.
Based on its promising anti-proliferative effects and potent activities in the colony formation
assay, compound 35b was selected for further mechanistic studies to determine whether the
growth inhibition induced by them in human melanoma cells was due to apoptosis. Both A375

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and B16F10 cells were treated with vehicle alone as control and also with 35b at different
concentrations for 48 h, following by staining with FITC-Annexin V and propidium iodide (PI). The
percentages of apoptotic A375 and B16F10 cells were determined by flow cytometry. As shown
in Figure 2, compound 35b significantly induced apoptosis of A375 and B16F10 cells in a
dose-dependent manner. The findings supported that the apoptosis of melanoma cells mediated
by this compound contributed to their anti-proliferative effects.
Figure 2. Induction of apoptosis in malignant melanoma cancer cells by compound 35b. (A) Flow
cytometry analysis of apoptotic A375 and B16F10 cells induced by 35b at different
concentrations. (B) Quantitative analysis of apoptosis shown in A. Two-way ANOVA ; mean ± sd, n
= 3. * P < 0.05; **, P < 0.01.
Compound 35b Regulated Apoptotic Related Proteins.
To identify the potential mechanisms of apoptosis induced by compound 35b, several
proteins related to apoptosis (Such as PARP, Caspase 3, Bax and P53) were determined by
Western blot (Ref 7-8). As shown in Figure 3, compound 35b also increased the formation of
cleaved PARP and active Caspase-3, two important hallmarks of apoptosis. Consistently,
treatment with compound 35b on A375 and B16F10 cells at different concentrations (0.5-4 μM)
for 48 h led to the up-regulation of antiapoptotic protein levels, including Bax and P53 in a
dose-dependent manner, which played a vital role in apoptosis. Together, these results indicated
that compound 35b induced apoptosis in melanoma cells.

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Figure 3. Effect of compound 35b on apoptosis-related protein levels of malignant melanoma
cancer cells. (A) Western blot analysis of biological markers for apoptosis induction by compound
35b treatment in A375 cells at different concentrations (48 h). (B) Western blot analysis of
biological markers for apoptosis induction by compound 35b treatment in B16F10 cells at
different concentrations (48 h).
Compound 35b Inhibited Migration in Melanoma Cells.
To further investigate the motility potential, wound healing assays were performed on A375
and B16F10 cells. As a result, the wound gaps were wider after treatment with compound 35b on
A375 and B16F10 cells (0.25 μM). Meanwhile, Transwell migration assay demonstrated that the
number of cells migrating to the lower chamber was also reduced when A375 and B16F10 cells
were treated with compound 35b (0.25 μM) for 24h. Together, the above findings indicated that
compound 35b inhibited the migration ability of A375 and B16F10 cells (Figure 4).

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Figure 4. Effect of compound 35b on migration ability of malignant melanoma cancer cells.(A)
A375 and B16F10 cells treated with or without compound 35b (0.25μM) were subjected to
wound-healing assay, representative images were recorded at 0 h, 24 h and 48 h. (B) Quantitative
analysis of migration distances shown in A. (C) Cells were seeded into the upper chamber of
transwell and incubated with compound 35b (0.25μM) for 24 h, migration assay were conducted.
Representative images of migrated cells were shown (left), quantitative analysis of migrated cells
were shown (right). Two-way ANOVA; mean ± sd, n = 3. * P < 0.05;
Compound 35b Inhibited Invasion in Melanoma Cells.
Further, transwell invasion assay was used to determine the effect of compound 35b on cell
invasion. As shown in Figure 5, compound 35b reduced the number of invaded cell through the
Matrigel-precoated filter as compared to the control group, indicating that compound 35b could
markedly inhibit the invasion capability of melanoma cells.

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Figure 5. Effect of compound 35b on invasion ability of malignant melanoma cancer cells.Cells
were seeded into the upper chamber of transwell and incubated with compound 35b (0.25 μM)
for 24 h, invasion assay were conducted.Representative images of invaded cells were shown (left),
quantitative analysis of invaded cells were shown (right). Two-way ANOVA ; mean ± sd, n = 3. * P
< 0.05;
Compound 35b Inhibited the Tumorsphere Formation Ability of Melanoma Cells.
The tumor sphere assays were performed to evaluate whether 35b inhibited the activity of
melanoma CSCs (shown in Figure 6). A375 or B16F10 cells pretreated with or without compound
35b for 48 h were plated into 24-well ultra-low attachment plates, followed by analysis on the
number and size of the tumor spheres after seven days of culture. As a result, compound 35b
administration dramatically decreased the number of tumor spheres, suggesting that compound
35b administration led to significant inhibition of activity of melanoma CSCs.
Figure 6. Effect of compound 35b on tumor spheres formation ability of melanoma cancer cells.(A)
The cells were pretreated with or without compound 35b for 48 h, then collected and plated into
24-well ultra-lowattachment plates. After 1 week of culture, spherical colonies were collected
and evaluated. Representative histograms were shown. (B) Quantitative analysis of spherical
colonies showed in A. Two-way ANOVA ; mean ± sd, n = 3. * P < 0.05; **, P < 0.01.
Compound 35b Treatment Reduced the Percentage of ALDH+ Cells.

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To further investigate the effects of compound 35b on melanoma CSCs, we assessed
whether 35b decreased the percentage of the ALDH+ cells. A375 and B16F10 cells were exposed
to 35b for 48 h, followed by staining with ALDH and subsequent detection by flow cytometry. [16]
The results showed that treatment with 35b significantly reduced the percentage of ALDH+ cells
(Figure. 7), which provided additional evidence for the important role of 35b in elimination of
melanoma CSCs.
Figure 7. Effect of compound 35b on ALDH+ subpopulation of malignant melanoma cancer
cells.(A) A375 cells were treated with or without compound 35b for 48 h, the ALDH+ cells were
analyzed by flow cytometry. Representative histograms were shown (left), quantitative analysis of
ALDH+ cells were showed(right). (B) B16F10 cells were treated with or without compound 35b for
48 h, the ALDH+ cells were analyzed by flow cytometry. Representative histograms were shown
(left),quantitative analysis of ALDH+ cells were showed(right). Two-way ANOVA ; mean ± sd, n = 3.
* P < 0.05; **, P < 0.01.
Compound 35b Treatment Reduced the Percentage of CD133+ Cells.
Convincing evidence from numerous studies indicated that CD133 was an important

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biomarker of the CSCs for various cancers, including melanoma. Therefore, to further validate the
effects of compound 35b on melanoma CSCs, we also assessed whether 35b decreased the
percentage of the CD133+ cells. A375 and B16F10 cells were treated with different concentrations
of compound 35b for 48 h, followed by staining with CD133-APC antibody and subsequent
[17]
detection by flow cytometry.
The results showed 35b could significantly reduce the numbers
of CD133+cells (Figure 8), which further confirmed the important role of 35b in elimination of
melanoma CSC
Figure 8. Effect of compound 35b on CD133+ subpopulation of malignant melanoma cancer
cells.(A) A375 cells were treated with or without compound 35b for 48 h, the CD133+ cells were
analyzed by flow cytometry. Representative histograms were shown (left), quantitative analysis of
CD133+ cells were showed(right). (B) B16F10 cells were treated with or without compound 35b
for 48 h, the CD133+ cells were analyzed by flow cytometry. Representative histograms were
shown (left),quantitative analysis of CD133+ cells were showed(right). Two-way ANOVA ; mean ±
sd, n = 3. **, P < 0.01, *** P < 0.001;
Compound 35b Inhibited crosstalk pathway between STAT3 and Wnt/β-catenin Signaling
Pathway in Melanoma Cells.
Further mechanism studies on the anti-CSCs effects of compound 35b aimed to determine
the possible signaling pathway, which might be blocked following compound 35b administration.
Firstly, luciferase reporter assays indicated the compound 35b could efficiently inhibit the activity
of TCF/LEF and markedly block the Wnt signaling pathway (shown in Figure 9A). Additionally,
Western blot revealed that compound 35b decreased the phosphorylation level of STAT3, protein
level of β-catenin and its downstream targets c-Myc in dose-dependent manners (Figure. 9B and
C). Collectively, these data suggested that compound 35b effectively eliminated CSCs of
melanoma cells by blocking the crosstalk between STAT3 and Wnt/β-catenin signaling pathways.

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Figure 9. Comound 35b inhibited STAT3 and Wnt/β-catenin signaling pathway in malignant
melanoma cancer cells. (A) 293T cells transfected with TCF/LEF reporter plasmid were treated
with compound 35b at different concentrations (48 h). Cells were harvested for luciferase assay,
luciferase activity was normalized to Renilla luciferase activity.(B) Western blot analysis of STAT3,
p-STAT3, β-catenin and c-Myc in the A375 cells treated by compound 35b at different
concentrations (48 h).(C)Western blot analysis of STAT3, p-STAT3, β-catenin and c-Myc in the
B16F10 cells treated by compound 35b at different concentrations (48 h).
Compound 35b Suppressed B16F10 Tumor Growth in vivo.
The leading compound 35b was further evaluated for its anticancer activity in vivo for its
suppression of B16F10 tumor growth. Consequently, tumor sizes were significantly suppressed in
mice treated with compound 35b in comparison with that in control group (Figure 10). Moreover,
there was no obvious difference of body weight in two groups. These results indicated that
compound 35b could distinctly inhibit B16F10 tumor growth in vivo.

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Figure 10. Effect of compound 35b on B16F10 tumor growth delay and regression in
vivo.C57BL/6J mice were subcutaneously injected with B16F10 cells. (A) Body weights of animals.
(B) The representative images of tumors dissected from vehicle-and compound 35b-treated mice
were shown. (C) Tumors were weighted on day 24. (D) Changes in the tumor volume of B16F10
xenografts. Two-way ANOVA ; mean ± sd, n = 5.****, P < 0.0001.
CONCLUSIONS
In this report, natural product Symplostatin 4 was totally synthesized through an alternative
and efficient manner. The whole synthesis provided gram-scale natural product Symplostatin 4 in
8 linear steps with 6% overall yield. And the purification process could be more straightforward
than the previously reported synthetic route. Based on these successful achievement, 22
synthetic analogues from Symplostatin 4 scaffold were prepared and evaluated by anti-tumor
assays. Among them, the compound 35b was demonstrated as a leading compound with great
suppression effect of tumor growth both in vitro and in vivo. Furthermore, the compound 35b
could significantly reduce the number of the tumor spheres and decrease the percentage of
ALDH+ melanoma cells, indicating that this leading compound 35b possessed the potent activity
against CSCs. Further mechanism study illustrated that compound 35b could ablate the CSCs of
melanoma through blocking the crosstalk between STAT3 and Wnt/β-catenin signaling pathways.
In summary, the leading compound 35b was a promising anti-CSCs agent that merited further
investigation.

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Experimental section
(S)-2-Hydroxy-4-methylpentanoic acid (9). A three-neck flask was charged with L-Leu-OH (10.0 g,
76.2 mmol), H2O (50 mL) and concentrated sulfuric acid (23.3 g, 91.4 mmol). The resulting
solution was cooled to 0 °C. A solution of NaNO2 (7.89 g, 0.114 mol, in 50 mL H2O) was then
added dropwise. The reaction mixture was stirred at room temperature overnight. The mixture
was extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with
brine (100 mL), dried over anhydrous NaSO4 and filtered. The solvent was concentrated in vacuo
to afford acid 9 as a pale yellow oil, which could be used directly without any further purification
(9.50 g, 95%). [α]D20= ‒21, (c = 1.0, MeOH). νmax (KBr): 3424, 2960, 2873, 2629, 1726, 1650, 1432,
1389, 1370 cm−1. 1H NMR (400 MHz, CDCl3) δ 4.29 (dd, J = 8.1, 5.3 Hz, 1H), 1.91 (dp, J = 13.4, 6.7
Hz, 1H), 1.63 (ddd, J = 8.1, 5.4, 1.9 Hz, 2H), 0.97 (d, J = 6.6 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
180.7, 69.0, 43.4, 24.6, 23.3, 21.5. HRMS-ESI (m/z): [M+Na]+ calcd. for C6H12O3Na+, 155.0679;
found, 155.0677.
Allyl-(S)-2-hydroxy-4-methylpentanoate (10). A round-bottom flask was charged with the acid 9
(5.00 g, 37.8 mmol), K2CO3 (10.5 g, 75.7 mmol) and dry DMF (70 mL). Allyl bromide (9.15 g, 75.7
mmol) was added and the resulting suspension was stirred overnight in a nitrogen atmosphere.
After the substrate was completely consumed (monitored by TLCTLC analysis), the reaction
mixture waswashed with H2O (1000 mL), and extracted by ethyl acetate (3 × 100 mL). The
combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl
acetate = 20:1 to 5:1) to afford desired compound 10 as a pale yellow oil (5.81 g, 89%). [α]D20 = ‒
13.6, (c = 1.0, MeOH). νmax (KBr): 3474, 3087, 2958, 2872, 2377, 1737, 1649, 1469, 1386, 1368,
1270 cm−1. 1H NMR (400 MHz, CDCl3) δ 5.92 (ddt, J = 17.1, 10.4, 5.8 Hz, 1H), 5.40 – 5.22 (m, 2H),
4.72 – 4.61 (m, 2H), 4.22 (ddd, J = 8.3, 5.9, 4.9 Hz, 1H), 2.67 (d, J = 6.0 Hz, 1H), 1.90 (ddt, J = 14.5,
13.2, 6.7 Hz, 1H), 1.61 – 1.52 (m, 2H), 0.95 (dd, J = 6.7, 4.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
175.7, 131.6, 119.1, 69.2, 66.2, 43.6, 24.5, 23.3, 21.7. HRMS-ESI (m/z): [M+Na]+ calcd. for
C9H16O3Na+, 195.0992; found, 195.0991.
Allyl (R)-2-((((benzyloxy) carbonyl)-L-isoleucyl) oxy)-4-methylpentanoate (11). A round-bottom
flask was charged with N-Cbz-L-Ile-OH(13.0 g, 49.0 mmol), compound 10 (2.80 g, 16.3 mmol) ,
DIC (6.18g, 49.0 mmol), and dichloromethane (55 mL). The resulting solution was cooled to 0 °C.
The reaction mixture was stirred for 15 min. DMAP (599 mg, 4.90 mmol) was added and the
resulting solution was stirred for further 6 h. The reaction mixture was washed with 1 N
hydrochloric acid solution, saturated sodium carbonate solution and brine. The combined organic
layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (petroleum ether: ethyl acetate= 30: 1 to
20
20:1) to afford desired compound 11 as a pale yellow oil (6.50 g, 95%). [α]D = –25.4, (c = 1.0,
MeOH). νmax(KBr): 3383, 2963, 2935, 2875, 1711, 1574, 1514, 1477, 1389, 1338, 1231, 1154,
1088, 980, 855, 748, 697 cm−1. 1H NMR (400 MHz, CDCl3) δ 7.26 (d, J = 4.4 Hz, 5H), 5.85 – 5.72 (m,
1H), 5.27 – 5.10 (m, 3H), δ 5.04 – 4.95 (m, 3H), 4.52 (d, J = 5.7 Hz, 2H), 4.33 (dd, J = 9.0, 4.3 Hz,

ACCEPTED MANUSCRIPT
1H), 1.88 (dt, J = 10.9, 5.5 Hz, 1H), 1.70 (ddt, J = 23.9, 12.7, 5.6 Hz, 2H), 1.57 (td, J = 8.2, 4.1 Hz,
1H), 1.51 – 1.39 (m, 1H), 1.21 – 1.06 (m, 1H), 0.96 – 0.74 (m, 12H). 13C NMR (100 MHz, CDCl3) δ
171.8, 170.0, 156.2, 136.4, 131.6, 128.7, 128.3, 128.2, 119.1, 71.9, 67.1, 66.0, 58.3, 39.9, 38.1,
24.7, 24.6, 23.1, 21.7, 15.4, 11.8. HRMS-ESI (m/z): [M+Na]+ calcd. for C23H33NO6Na+, 442.2200;
found, 442.2199.
(S)-1-((4-Chlorophenyl) thio)-4-methyl-1-oxopentan-2-yl ((benzyloxy) carbonyl)-L-isoleucinate (6).
A round-bottom flask was charged with compound 11 (4.0 g, 9.4 mmol), Morpholine (1.6 g，19
mmol), Pd(Ph3)4 (0.6 g, 0.5 mmol ) and THF (10 mL). The resulting suspension was stirred for 7h.
After the substrate was completely consumed (monitored by TLC analysis), The reaction mixture
was washed with 1 N hydrochloric acid solution, and extracted by ethyl acetate (3 × 20 mL ). The
combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo
to afford an intermediate (3.6 g). To solution of the intermediate in DMF (20 mL) without any
further purification, followed by PyBOP (3.2 g, 6.1 mmol) and DIPEA (0.8 g, 6.1 mmol). The
resulting solution was stirred overnight. The reaction mixture was washed with 1 N hydrochloric
acid solution, and extracted by ethyl acetate (3 × 20 mL ). The combined organic layers were dried
over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (petroleum ether: ethyl acetate= 10:1) to afford fragment 6
20
as a pale yellow oil (2.6g, 86% for two steps). [α]D = – 62, (c = 1.0, MeOH). νmax(KBr) = 3474,
1
2958, 2872, 2377, 1737, 1649, 1469, 1368, 1205, 1141, 1088, 932, 851, 748 cm−1. H NMR (400
MHz, CDCl3) δ 7.34 (ddd, J = 16.0, 14.1, 8.5 Hz, 10H), 5.40 (dd, J = 9.4, 3.6 Hz, 1H), 5.33 (d, J = 9.2
Hz, 1H), 5.13 (s, 2H), 4.50 (dd, J = 9.2, 4.4 Hz, 1H), 2.11 (dd, J = 10.0, 6.5 Hz, 1H), 1.94 – 1.66 (m,
3H), 1.54 (ddd, J = 13.2, 7.4, 4.0 Hz, 1H), 1.27 (d, J = 2.9 Hz, 1H), 1.04 (d, J = 6.8 Hz, 3H), 1.01 –
0.91 (m, 10H). 13C NMR (100 MHz, CDCl3) δ 196.7, 171.33, 156.7, 136.8, 136.2, 129.7, 128.6,
128.3, 128.2, 124.6, 77.9, 67.2, 58.6, 40.9, 37.5, 24.5, 24.4, 23.1, 21.6, 15.7, 11.6. HRMS-ESI (m/z):
[M+Na]+ calcd. for C23H33NO6Na+, 528.1582; found, 528.1580.
Tert-butyl (S)-(1-(methoxy (methyl) amino)-1-oxopropan-2-yl) carbamate (13). A round-bottom
flask was charged with N-Boc-L-Ala-OH (5.0 g, 26 mmol), N,O-dimethylhydroxylamine
hydrochloride (3.1 g, 32 mmol), HOBT (4.3 g, 32 mmol), DIPEA (17 g, 0.13 mol), EDCI (6.1 g, 0.32
mol) and dichloromethane(100 mL). The resulting suspension was cooled to 0 °C and was stirred
overnight. The reaction mixture was washed with 1 N hydrochloric acid solution, saturated
sodium carbonate solution, and extracted by ethyl acetate. The combined organic phase were
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (petroleum ether: ethyl acetate= 5:1 to 3: 1) to afford
20
weinreb amide 13 as a white solid (5.5 g, 90%). [α]D = –21.5, (c = 1.0, MeOH). m. p.139 ℃.
νmax(KBr):3293, 3049, 3008, 2975, 2823, 1660, 1540, 1456, 1423 cm−1. 1H NMR (400 MHz, CDCl3)
δ 5.25 (d, J = 7.4 Hz, 1H), 4.66 (t, J = 7.6 Hz, 1H), 3.75 (s, 3H), 3.19 (s, 3H), 1.42 (s, 9H), 1.29 (d, J =
6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 173.8, 155.3, 79.6, 61.7, 46.6, 32.2, 28.4, 18.8. HRMS-ESI
(m/z): [M+Na]+ calcd. for C10H20N2O4Na+, 255.1315; found, 255.1314.
(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)amino)-4-methyl-

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1-oxopentan-2-yl) carbamate (14). A three-neck flask was charged with weinreb amide 13 (3.00 g,
11.2 mmol) and dichloromethane (9 mL). The resulting solution was cooled to 0 °C in a nitrogen
atmosphere. TFA (17.2 g, 112 mmol) was then added slowly. The reaction mixture was stirred 2 h.
The solvent was concentrated in vacuo to afford an intermediate. To solution of the intermediate
(1.40 g, 6.20 mmol) in dichloromethane (40 mL). N-Fmoc-L-Leu-OH (2.20 g, 6.20 mmol), HOBT
(1.00 g, 7.44 mmol), DIPEA (4.00 g, 31.0 mmol) and EDCI (1.40 g, 1.44 mmol) was added under
nitrogen atmosphere. The resulting solution was stirred overnight. The reaction mixture was
washed with brine and dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl
acetate= 3:1 to 1: 1) to afford weinreb amide 14 as a white solid (2.25 g, 78% for two steps).
[α]D20 = –37.6, (c = 1.0, MeOH). m. p. 92 °C. νmax (KBr)= 3301, 3069, 2957, 2871, 1717, 1642, 1536,
1468, 1450, 1392, 1369, 1323, 1238, 1181, 1042, 759, 740 cm−1. 1H NMR (400 MHz, CDCl3) δ 7.76
(d, J = 7.5 Hz, 2H), 7.61 – 7.57 (m, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.30 (t, J = 7.3 Hz, 2H), 6.83 (d, J =
7.1 Hz, 1H), 5.35 (d, J = 8.6 Hz, 1H), 4.92 (t, J = 6.7 Hz, 1H), 4.39 (dt, J = 17.6, 10.4 Hz, 2H), 4.29 –
4.19 (m, 2H), 3.77 (s, 3H), 3.21 (s, 3H), 1.69 – 1.51 (m, 3H), 1.35 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 5.7
Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 172.8, 171.8, 156.2, 144.0, 143.8, 141.3, 127.8, 127.1, 125.2,
120.1, 67.0, 61.7, 53.5, 47.2, 45.8, 42.1, 32.3, 24.7, 23.1, 21.9, 18.3. HRMS-ESI (m/z): [M+Na]+
calcd. for C26H33N3O5Na+,490.2312; found, 490.2311.
(S)-2-amino-N-((S)-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)-4-methylpentanamide (7).
A
round-bottom flask was charged with weinreb amide 14 (1.00 g, 2.14 mmol), dichloromethane
(30 mL). Diethylamine (15 mL) was added and the resulting solution was stirred for 2 h. The
reaction mixture was washed with brine and extracted by ethyl acetate (3 × 30 mL). The
combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl
acetate= 4:1 to diethylamine : methanol = 4: 1) to afford desired compound 7 as a white solid
20
(406 mg, 77%). [α]D = – 0.1, (c = 1.0, MeOH). m. p. 181 °C. νmax(KBr): 3197, 3086, 2958, 2872,
1687, 1447, 1346, 1151, 1110 cm−1. 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 7.7 Hz, 1H), 4.83 –
4.64 (m, 1H), 3.73 (s, 3H), 3.15 (dd, J = 9.2, 4.9 Hz, 1H), 3.11 (s, 3H), 1.73 (dtd, J = 15.0, 8.6, 8.0,
4.7 Hz, 3H), 1.39 (ddd, J = 13.5, 8.7, 4.9 Hz, 1H), 1.24 – 1.14 (m, 3H), 0.86 (dd, J = 14.5, 6.6 Hz, 6H).
13C NMR (100 MHz, DMSO-d6) δ 175.3, 61.2, 52.7, 44.3, 44.2, 24.0, 23.3, 21.7, 17.5. HRMS-ESI
(m/z): [M+Na]+ calcd. for C11H23N3O3Na+, 268.1632; found, 268.1632.
(S)-4-Methoxy-5-methyl-1,5-dihydro-2H-pyrrol-2-one (15). A round-bottom flask was charged
with Boc-15 (4.00 g, 17.6 mmol) and dichloromethane (13 mL). The resulting solution was cooled
to 0 °C. TFA (17.2 g, 112 mmol) was then added slowly. The reaction mixture was stirred 1 h.
After the substrate was completely consumed (monitored by TLC analysis), toluene was added.
The reaction mixture was concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (petroleum ether: ethyl acetate= 1:1 to diethylamine : methanol = 4:
1) to afford desired compound 15 as a yellow solid (2.20 g, 97%). [α]D20 = +13, (c = 1.0, MeOH). m.
p. 112 °C. νmax(KBr): 3256, 2942, 2881, 2605, 2310, 1680, 1620, 1456, 1379, 1361, 1233, 1207,
1178, 1049, 987, 811 cm−1. 1H NMR (400 MHz, CDCl3) δ 6.88 (brs, 1H), 4.97 (s, 1H), 4.06 (d, J = 6.6
Hz, 1H), 3.77 (s, 3H), 1.29 (d, J = 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 179.6, 174.6, 92.9, 58.4,
53.4, 17.8. HRMS-ESI (m/z): [M+Na]+ calcd. for C6H9NO2Na+, 150.0525; found, 150.0525.

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(S)-1-(2-Bromoacetyl)-4-methoxy-5-methyl-1,5-dihydro-2H-pyrrol-2-one (16). A three-neck flask
was charged with compound 15 (2.00 g, 15.7 mmol) and dry THF (60 mL). The resulting solution
was cooled to –78 °C in a nitrogen atmosphere. NaHMDS (7.90 mL, 15.7 mmol) was added
dropwise. The reaction mixture was stirred for 15 min. Bromoacetyl bromide (2.60 g, 13.0 mmol)
was then added dropwise over 10 min. The resulting solution was stirred for further 3 h. After the
substrate was completely consumed (monitored by TLC analysis), the reaction mixture was
quenched with saturated NH4Cl solution, and extracted by ethyl acetate (3 × 100 mL). The
combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl
acetate= 5:1 to 1: 1) to afford desired compound 16 as a yellow solid (2.90 g, 75%). [α]D20 = + 84,
(c = 1.0, MeOH). m. p. 75 °C. νmax(KBr): 3115, 3043, 2969, 2944, 1721, 1692, 1619, 1454, 1344,
1189, 1094, 956, 812 cm−1. 1H NMR (400 MHz, CDCl3) δ 4.95 (s, 1H), 4.51 – 4.42 (m, 2H), 4.37 (d, J
= 12.8 Hz, 1H), 3.77 (s, 3H), 1.37 (d, J = 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 181.1, 169.3,
165.1, 92.5, 59.0, 56.0, 30.7, 16.6. HRMS-ESI (m/z): [M+Na]+ calcd. for C8H10BrNO3Na+, 269.9736;
found, 269.9733.
Aiethyl (S)-(2-(3-methoxy-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxoethyl) phosphonate (5).
A round-bottom flask was charged with compound 16 (1.0 g, 4.0 mmol) and triethyl phosphite
(3.3 g, 20 mmol). The resulting solution was stirred overnight in 55 °C. After the substrate was
completely consumed (monitored by TLC analysis), the reaction mixture was concentrated in
vacuo. The residue was purified by flash column chromatography on silica gel (petroleum ether:
ethyl acetate= 2:1 to dichloromethane: methanol=10: 1) to afford phosphonate ester 5 as a pale
20
yellow oil (1.2 g, 89%). [α]D = +59.9, (c = 1.0, MeOH). νmax(KBr): 3099, 2985, 2941, 1726, 1682,
1
1626, 1455, 1382, 1331, 1251, 1170, 1025, 970 cm−1. H NMR (400 MHz, CDCl3) δ 5.02 (s, 1H),
4.57 (d, J = 6.3 Hz, 1H), 4.14 (t, J = 6.9 Hz, 4H), 3.93 (dd, J = 22.2, 14.2 Hz, 1H), 3.84 (s, 3H), 3.93
(dd, J = 22.2, 14.2 Hz, 1H), 1.45 (d, J = 6.3 Hz, 3H), 1.29 (t, J = 6.9 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 180.8, 168.5, 164.1, 164.0, 92.1, 62.5, 58.9, 55.9, 36.4, 35.1, 16.9, 16.4, 16.3. HRMS-ESI
(m/z): [M+Na]+ calcd. for C12H20NO6PNa+, 328.0920; found, 328.0918.
(S)-1-(((S)-1-(((S)-1-(Methoxy
(methyl)
amino)-1-oxopropan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl
((benzyloxy)carbonyl)-L-isoleucinate (17). A round-bottom flask was charged with fragment 6
(1.84 g, 7.50 mmol), compound 7 (4.50 g, 9.00 mmol) and DMF (140 mL). The resulting solution
was stirred overnight in room temperature. The reaction mixture was washed with water (200 mL)
and extracted by ethyl acetate (3 × 300 mL). The combined organic layers were dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (petroleum ether: ethyl acetate= 1:1) to afford desired compound
20
17 as a pale yellow oil (3.70 g, 82%). [α]D = – 55, (c = 1.0, MeOH). νmax(KBr): 3301, 2961, 2874,
2377, 2310, 1725, 1653, 1533, 1456, 1339, 1046 cm−1. 1H NMR (400 MHz, CDCl3) δ 7.39-7.30 (m,
5H), 6.95 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 7.1 Hz, 1H), 5.25-5.07 (m, 4H), 4.94-4.85 (m, 1H),
4.53-4.44 (m, 1H), 4.38-4.36 (m, 1H), 3.74 (s, 3H), 3.19 (s, 3H), 2.07-1.99 (m, 1H), 1.77-1.54 (m,
6H), 1.47-1.39 (m, 2H), 1.31 (d, J = 6.7 Hz, 3H), 1.27-1.22 (m, 1H), 1.00 (d, J = 6.4 Hz, 3H),
0.96-0.83 (m, 14H). 13C NMR (100 MHz, CDCl3) δ 172.7, 171.2, 171.1, 170.1, 157.1, 136.0, 128.8,

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128.5, 128.1, 74.1, 67.3, 61.7, 59.0, 51.5, 45.6, 41.0, 40.6, 37.2, 32.2, 25.1, 24.8, 24.7, 23.3, 21.7,
18.2, 16.0, 11.8. HRMS-ESI (m/z): [M+Na]+ calcd. for C31H50N4O8Na+, 629.3521; found, 629.3520.
(S)-1-(((S)-1-(((S)-1-(Methoxy(methyl)amino)-1-oxopropan-2-yl)
amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl
dimethyl-L-isoleucinate
(18). A three-neck flask was charged with compound 17 (1.30 g, 2.15 mmol), Pd/C (500 mg) and
methanol (60 mL). The resulting suspension was stirred for 2 h in hydrogen atmosphere. 37%
formaldehyde solution (8 mL) was injected and the reaction mixture was stirred for further 24 h.
The reaction mixture was filtered with diatomaceous earth. The combined organic layers were
dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (petroleum ether: ethyl acetate= 5:1 to 2: 1) to afford
desired compound 18 as a white solid (861 mg, 80%). [α]D20 = – 78, (c = 1.0, MeOH). m. p. 82 °C.
νmax (KBr): 3303, 3080, 2961, 2874, 2784, 2376, 2310, 1739, 1670,1648, 1550, 1389, 1321, 1285,
1251, 1127 cm−1. 1H NMR (400 MHz, CDCl3) δ 6.73 (d, J = 5.7 Hz, 1H), 6.51 (d, J = 6.8 Hz, 1H), 5.29
– 5.09 (m, 1H), 4.85 (s, 1H), 4.48 (s, 1H), 3.74 (s, 3H), 3.20 (s, 3H), 2.93 (d, J = 10.1 Hz, 1H), 2.30 (s,
6H), 1.77 (dd, J = 25.3, 13.0 Hz, 3H), 1.58 (d, J = 28.0 Hz, 4H), 1.30 (d, J = 6.2 Hz, 3H), 1.18 – 1.10
(m, 1H), 0.89 (dd, J = 17.8, 6.4 Hz, 19H). 13C NMR (100 MHz, CDCl3) δ 172.6, 170.9, 170.8, 170.1,
72.5, 72.4, 61.7, 51.3, 45.8, 41.8, 41.6, 41.0, 33.5, 32.3, 25.1, 24.7, 24.5, 23.3, 23.1, 22.1, 21.5,
18.3, 15.8, 10.5. HRMS-ESI (m/z): [M+Na]+ calcd. for C25H48N4O6Na+, 523.3466; found, 523.3464.
(S)-4-Methyl-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxopropan-2-yl)amino)pentan-2-yl)amino)-1-oxopen
tan-2-yl dimethyl-L-isoleucinate (4). A three-neck flask was charged with compound 18 （290 mg,
0.58 mmol）and dry THF (6 mL). The resulting solution cooled to – 78 °C in a nitrogen atmosphere.
DIBAL-H (1.00 mL, 1.50 mmol) was added dropwise over 5 min and the reaction mixture was
stirred for 4 h. the reaction mixture was quenched with MeOH (0.20 mL). The reaction mixture
was poured into a saturated solution of sodium potassium tartrate (200 mL), added with diethyl
ether (200 mL) and stirred for about 1 hour. The mixture was separated and the aqueous phase
was extracted with ethyl acetate (3 × 100 mL). The organic phase was combined and dried over
anhydrous Na2SO4, filtered and concentrated in vacuo to afford aldehyde 4, which could be used
directly without any further purification
(S)-1-(((S)-1-(((S,E)-5-((S)-3-Methoxy-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-oxopent-3-en-2
-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl dimethyl-L-isoleucinate
(1). A three-neck flask was charged with phosphonate ester 5 (0.19 g, 0.60 mmol) and dry THF (4
mL). The resulting solution cooled to – 78 °C in a nitrogen atmosphere. LiHMDS (0.60 mL, 0.60
mmol ) was added dropwise. The reaction mixture was stirred for 15 min. A solution of aldehyde
4 (180 mg, 0.41 mmol, in 2 mL THF) was then added dropwise over 5 min. The resulting solution
was stirred for further 3 h. After the substrate was completely consumed (monitored by TLC
analysis), the reaction mixture was quenched with H2O, and extracted by ethyl acetate (3 × 10
mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated
in vacuo. The residue was purified by flash column chromatography on silica gel (petroleum ether:
ethyl acetate= 5:1) to afford desired compound 1 as a white solid (146 mg, 60%). [α]D20 = – 57, (c

ACCEPTED MANUSCRIPT
= 1.0, MeOH). m. p. 64 °C. νmax(KBr): 3301, 3079, 2962, 2936, 2873, 2789, 2352, 1731, 1652, 1630,
1549, 1455, 1382, 1327, 1291, 1260, 1225, 1056, 807 cm−1. 1H NMR (400 MHz, CDCl3) δ 7.41 (dd,
J = 15.5, 1.4 Hz, 1H), 6.96 (dd, J = 15.5, 5.1 Hz, 1H), 6.41 (d, J = 8.2 Hz, 1H), 6.17 (d, J = 8.2 Hz, 1H),
5.15 (dd, J = 9.5, 4.0 Hz, 1H), 5.03 (s, 1H), 4.71 (dd, J = 12.8, 6.2 Hz, 1H), 4.61 (q, J = 6.5 Hz, 1H),
4.42 (td, J = 8.7, 5.5 Hz, 1H), 3.86 (s, 3H), 2.93 (d, J = 10.3 Hz, 1H), 2.31 (s, 6H), 1.74 (td, J = 13.2,
12.0, 4.6 Hz, 4H), 1.69 – 1.59 (m, 4H), 1.48 (d, J = 6.6 Hz, 3H), 1.29 (d, J = 7.0 Hz, 3H), 1.14 (dt, J =
22.2, 7.6 Hz, 1H), 1.03 – 0.79 (m, 18H). 13C NMR (100 MHz, CDCl3) δ 180.8, 171.2, 170.7, 170.6,
169.8, 164.3, 148.3, 122.5, 93.1, 72.6, 58.9, 55.8, 51.6, 46.3, 41.7, 41.0, 40.8, 33.5, 25.1, 24.8,
24.5, 23.3, 23.0, 22.0, 21.4, 20.0, 17.1, 15.7, 10.5. HRMS-ESI (m/z): [M+Na]+ calcd. for
C31H52N4O7Na+, 615.3728; found, 615.3726.
Methyl(S,E)-4-((S)-2-((S)-2-((dimethyl-L-isoleucyl)oxy)-4-methylpentanamido)-4-methylpentanami
do) pent-2-enoate (19). A round-bottom flask was charged with aldehyde 4 (100 mg, 0.23 mmol),
methyl (triphenylphosphoranylidene) acetate (151 mg, 0.45 mmol) and dichloromethane (2 mL).
The resulting solution was stirred overnight. The reaction mixture was concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel (petroleum ether: ethyl
20
acetate= 10:1 to 5: 1) to afford desired compound 19 as a white solid (109 mg, 95%) [α]D = –
57.2, (c = 1.0, MeOH). m. p.112 °C. νmax(KBr): 3314, 3295, 3064, 2958, 2873, 2377, 2310, 1737,
1
1728, 1652, 1546, 1455, 1347, 1279, 1253, 1226, 1145, 1059 cm−1. H NMR (400 MHz, CDCl3) δ
6.85 (dd, J = 15.7, 5.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 8.1 Hz, 1H), 5.88 (dd, J = 15.7,
1.6 Hz, 1H), 5.10 (dd, J = 9.5, 3.9 Hz, 1H), 4.63 (h, J = 6.8 Hz, 1H), 4.45 (td, J = 8.6, 5.6 Hz,1H), 3.71
(s, 3H), 2.93 (d, J = 10.3 Hz, 1H), 2.29 (s, 6H), 1.87 – 1.80 (m, 1H), 1.67 (ddt, J = 31.7, 12.8, 7.6 Hz,
6H), 1.23 (d, J = 7.0 Hz, 3H), 1.13 (dd, J = 14.2, 7.7 Hz, 1H), 0.96 – 0.82 (m, 19H). 13C NMR (100
MHz, CDCl3) δ 171.3, 170.8, 170.7, 166.9, 148.7, 120.2, 72.6, 72.5, 51.7, 51.5, 45.8, 41.7, 40.9,
40.8, 33.4, 25.1, 24.9, 24.5, 23.3, 23.1, 22.0, 21.4, 19.8, 15.9, 10.5. HRMS-ESI (m/z): [M+Na]+
calcd. for C26H47N3O6 Na+, 520.3357; found, 520.3356.
(3S,6S,9S,12S,E)-3-((S)-Sec-butyl)-6,9-diisobutyl-2,12-dimethyl-4,7,10-trioxo-5-oxa-2,8,11-triazape
ntadec-13-en-15-oic acid (20). A round-bottom flask was charged with compound 19 (1.00 g, 2.01
mmol), LiOH (253 mg, 6.03 mmol) and aqueous tetrahydrofuran solution (20 mL, H2O: THF= 1:1).
1 N hydrochloric acid solution was added to maintain pH of 4. The mixture was extracted with
ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4, filtered and
concentrated in vacuo to afford acid 20, which could be used directly without any further
purification.
(S)-1-(((S)-1-(((S,E)-5-(Tert-butylamino)-5-oxopent-3-en-2-yl)amino)-4-methyl-1-oxopentan-2-yl)a
mino)-4-methyl-1-oxopentan-2-yl dimethyl-L-isoleucinate (21a). A round-bottom flask was
charged with acid 20 (200 mg, 0.41mmol), tert-butylamine (36.6 mg, 0.50 mmol), HOBT (67.6 mg,
0.5 mmol), DIPEA (265 mg, 2.05 mmol), EDCI (95.9 mg, 0.50 mmol) and dichloromethane (5 mL).
The resulting suspension was stirred overnight. The reaction mixture was washed with 1 N
hydrochloric acid solution, saturated sodium carbonate solution, and extracted by ethyl acetate.
The combined organic phase were dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The residue was purified by flash column chromatography on silica gel (petroleum ether:

ACCEPTED MANUSCRIPT
ethyl acetate= 3:1 to 1: 1) to afford desired compound 21a as a pale yellow oil (143 mg, 65%).
[α]D20 = – 39, (c = 1.0, MeOH). νmax(KBr): 3309, 3077, 2964, 2934, 2873, 2790, 1734, 1658, 1553,
1
1455, 1392, 1365, 1259, 1028, 800 cm−1. H NMR (400 MHz, CDCl3) δ 6.65 (dd, J = 15.2, 5.1 Hz,
1H), 6.30 (dd, J = 20.3, 7.9 Hz, 1H), 5.78 (d, J = 15.0 Hz, 1H), 5.52 (s, 1H), 5.16 – 5.04 (m, 1H), 4.60
(d, J = 7.0 Hz, 1H), 4.45 – 4.30 (m, 1H), 2.95 (d, J = 10.3 Hz, 1H), 2.31 (s, 6H), 1.82 – 1.72 (m, 4H),
1.70 – 1.59 (m, 4H), 1.37 (s, 9H), 1.23 (d, J = 7.6 Hz, 5H), 0.98 – 0.84 (m, 18H). 13C NMR (100 MHz,
CDCl3) δ 170.5, 169.9, 163.7, 155.9, 141.8, 123.1, 71.8, 71.4, 52.4, 50.8, 50.3, 44.8, 41.1, 40.6,
39.7, 39.4, 32.4, 27.7, 24.0, 23.8, 23.5, 22.5, 22.5,22.2, 20.6, 20.3, 19.1, 17.2, 14.7, 9.3. HRMS-ESI
(m/z): [M+Na]+ calcd. for C29H54N4O5Na+, 561.3986; found, 561.3984.
(S)-1-(((S)-1-(((S,E)-5-(Diethylamino)-5-oxopent-3-en-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amin
o)-4-methyl-1-oxopentan-2-yl dimethyl-L-isoleucinate (21b). The titled compound 21b was
obtained following the procedure described for 21a. Flash column chromatography (petroleum
20
ether: ethyl acetate= 5:1 to 1: 1). 115 mg, 52%, a pale yellow solid. [α]D = – 48.5, (c = 1.0,
MeOH). m. p. 63 °C. νmax(KBr): 3301, 3073, 2961, 2934, 2873, 2792, 2373, 1736, 1650, 1551, 1456,
1
1385, 1262, 1171, 1148, 1126, 1058, 980, 913 cm−1. H NMR (400 MHz, MeOD) δ 6.69 (dd, J =
15.0, 5.1 Hz, 1H), 6.37 (d, J = 15.2 Hz, 1H), 5.07 (d, J = 6.0 Hz, 1H), 4.62 – 4.54 (m, 1H), 4.48 – 4.40
(m, 1H), 3.44 (dt, J = 14.0, 7.1 Hz, 4H), 2.97 (d, J = 9.5 Hz, 1H), 2.30 (s, 6H), 1.83 – 1.75 (m, 3H),
1.67 (d, J = 10.1 Hz, 2H), 1.54 (d, J = 13.0 Hz, 2H), 1.29 (s, 7H), 1.21 (t, J = 7.0 Hz, 3H), 1.14 (t, J =
7.1 Hz, 3H), 0.99 – 0.86 (m, 18H). 13C NMR (100 MHz, MeOD) δ 173.7, 172.8, 172.2, 167.5, 147.1,
120.7, 73.5, 73.4, 52.9, 47.4, 43.6, 42.4, 42.2, 42.0, 41.6, 34.7, 30.7, 26.2, 25.8, 25.6, 23.5, 21.9,
21.7, 20.0, 16.0, 15.1, 14.4, 13.2, 10.9. HRMS-ESI (m/z): [M+Na]+ calcd. for C29H54N4O5Na+,
561.3986; found, 561.3985.
(S)-4-methyl-1-(((S)-4-methyl-1-(((S,E)-5-morpholino-5-oxopent-3-en-2-yl)amino)-1-oxopentan-2-y
l)amino)-1-oxopentan-2-yl dimethyl-L-isoleucinate (21c). The titled compound 21c was obtained
following the procedure described for 21a. Flash column chromatography (petroleum ether: ethyl
acetate= 5:1 to 1: 1). 99.7 mg, 44%, as a white solid. [α]D20 = – 46.7, (c = 1.0, MeOH). m. p.121 °C.
νmax(KBr): 3335, 2959, 2871, 2787, 1732, 1645, 1532, 1436, 1385, 1366, 1263, 1173, 1117, 976,
916 cm−1. 1H NMR (400 MHz, MeOD) δ 6.67 (dd, J = 15.2, 5.6 Hz, 1H), 6.47 – 6.41 (d, 1H), 5.06 (dd,
J = 9.8, 4.0 Hz, 1H), 4.61 – 4.53 (m, 1H), 4.39 (dd, J = 10.1, 5.2 Hz, 1H), 3.65 (m, J = 11.0, 5.1 Hz,
8H), 2.97 (d, J = 9.7 Hz, 1H), 2.30 (s, 6H), 1.80 (m, J = 17.4, 7.5 Hz, 3H), 1.66 (m, J = 15.0, 10.3, 5.1
Hz, 2H), 1.55 (m, J = 8.5, 4.5 Hz, 2H), 1.31 – 1.26 (m, 5H), 1.20 – 1.14 (m, 1H), 0.99 – 0.86 (m, 19H).
13C NMR (100 MHz, MeOD) δ 173.7, 172.9, 172.3, 172.3, 147.5, 120.3, 73.5, 73.4, 67.8, 67.7, 53.1,
47.5, 43.6, 42.1, 42.0, 41.6, 34.7, 26.1, 25.8, 25.6, 23.6, 23.4, 21.9, 21.7, 19.9, 16.0, 10.9.
HRMS-ESI (m/z): [M+Na]+ calcd. for C29H52N4O6Na+, 575.3779; found, 575.3778.
(S)-4-Methyl-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxopropan-2-yl)amino)pentan-2-yl)amino)-1-oxopen
tan-2-yl ((benzyloxy)carbonyl)-L-isoleucinate (22). A three-neck flask was charged with compound
17 （390 mg, 0.64 mmol）and dry THF (6 mL). The resulting solution cooled to – 78 °C in a
nitrogen atmosphere. DIBAL-H (0.64 mL, 0.96 mmol) was added dropwise over 5 min and the
reaction mixture was stirred 4 h. the reaction mixture was quenched with MeOH (0.20 mL). The
reaction mixture was poured into a saturated solution of sodium potassium tartrate (200 mL),
added with diethyl ether (200 mL) and stirred for about 1 hour. The mixture was separated and

ACCEPTED MANUSCRIPT
the aqueous phase was extracted with ethyl acetate (3 × 100 mL). The organic phase was
combined and dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford
aldehyde 22, which could be used directly without any further purification.
Diethyl(S)-(2-(2-benzyl-3-methoxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxoethyl)phosphonate (23).
A
round-bottom
flask
was
charged
with
(S)-5-benzyl-1-(2-bromoacetyl)-4-methoxy-1,5-dihydro-2H-pyrrol-2-one (1.00 g, 3.10 mmol) and
triethyl phosphite (2.60 g, 15.5 mmol). The resulting solution was stirred overnight in 55 °C. After
the substrate was completely consumed (monitored by TLC analysis), the reaction mixture was
concentrated in vacuo. The residue was purified by flash column chromatography on silica gel
(petroleum ether: ethyl acetate= 2:1 to dichloromethane: methanol=10: 1) to afford
20
phosphonate ester 23 as a pale yellow oil (823 mg, 70%). [α]D = +211.9, (c = 1.0, MeOH).
νmax(KBr): 3030, 2985, 1727, 1680, 1627, 1496, 1385, 1330, 1252, 1057, 1025, 970, 743, 703 cm−1.
1H NMR (400 MHz, CDCl3) δ 7.13 (q, J = 6.9, 5.2 Hz, 3H), 6.97 – 6.90 (m, 2H), 4.81 – 4.78 (m, 1H),
4.76 (s, 1H), 4.12 (h, J = 7.8, 7.3 Hz, 4H), 3.83 – 3.76 (m, 1H), 3.74 (s, 3H), 3.70 – 3.62 (m, 1H),
3.46 (dd, J = 14.0, 4.9 Hz, 1H), 3.04 (dd, J = 14.0, 2.7 Hz, 1H), 1.26 (dt, J = 10.6, 7.1 Hz, 6H). 13C
NMR (100 MHz, CDCl3) δ 177.9, 169.4, 164.2, 133.9, 129.5, 128.0, 126.9, 94.4, 62.3 , 59.8, 58.3,
36.0, 34.7, 34.4, 16.3. HRMS-ESI (m/z): [M+Na]+ calcd. for C18H24NO6PNa+, 404.1233; found,
404.1230.
(S)-1-(((S)-1-(((S,E)-5-((S)-3-Methoxy-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-oxopent-3-en-2
-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl
((benzyloxy)carbonyl)-L-isoleucinate (24a). A three-neck flask was charged with phosphonate
ester 5 (150 mg, 0.49) and dry THF (4 mL). The resulting solution cooled to – 78 °C in a nitrogen
atmosphere. LiHMDS (0.50 mL,0.49 ) was added dropwise. The reaction mixture was stirred for
15 min. A solution of aldehyde 22 (180 mg, 0.33mmol, in 2 mL THF) was then added dropwise
over 5 min. The resulting solution was stirred for further 3 h. After the substrate was completely
consumed (monitored by TLC analysis), the reaction mixture was quenched with H2O, and
extracted by ethyl acetate (3 × 10 mL). The combined organic layers were dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (petroleum ether: ethyl acetate= 5:1 to 1: 1) to afford desired
20
compound 24a as a pale yellow oil (127 mg, 55%). [α]D = – 14.3, (c = 1.0, MeOH). νmax(KBr):
3292, 2963, 2873, 2377, 2310, 1732, 1680, 1456, 1338, 1291, 1094, 1025, 968, 807 cm−1. 1H NMR
(400 MHz, CDCl3) δ 7.40 – 7.29 (m, 8H), 6.97 (dd, J = 15.5, 5.4 Hz, 2H), 6.38 (d, J = 8.1 Hz, 1H),
5.26 (d, J = 6.2 Hz, 1H), 5.14 – 5.11 (m, 2H), 5.01 (s, 1H), 4.70 (q, J = 7.0 Hz, 1H), 4.60 (q, J = 6.5 Hz,
1H), 4.52 – 4.43 (m, 1H), 4.20 (dd, J = 6.5, 4.4 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 1H), 3.19 (s, 1H), 1.98
(q, J = 8.3, 6.9 Hz, 1H), 1.89 (s, 1H), 1.70 (d, J = 5.9 Hz, 2H), 1.63 – 1.57 (m, 2H), 1.47 (d, J = 6.6 Hz,
3H), 1.30 (t, J = 6.4 Hz, 3H), 0.99 (dd, J = 7.0, 2.8 Hz, 3H), 0.94 – 0.87 (m, 15H). 13C NMR (100 MHz,
CDCl3) δ 179.5, 170.3, 169.9, 169.1, 168.6, 163.3, 156.1, 147.5, 134.8, 127.6, 127.4, 126.8, 121.4,
92.0, 73.4, 72.9, 66.2, 58.2, 57.8, 57.6, 54.6, 50.4, 45.1, 44.4, 39.8, 39.2, 35.9, 24.1, 23.8, 22.2,
20.3, 18.8, 17.0, 16.0, 14.8, 10.5. HRMS-ESI (m/z): [M+Na]+ calcd. for C37H54N4O9Na+, 721.3783;
found, 721.3781.
(S)-1-(((S)-1-(((S,E)-5-((S)-2-Benzyl-3-methoxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-oxopent-3-en-2-

ACCEPTED MANUSCRIPT
yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl
((benzyloxy)carbonyl)-L-isoleucinate (24b). The titled compound 24b was obtained following the
procedure described for 24a. Flash column chromatography (petroleum ether: ethyl acetate= 5:1
to 1: 1). 168 mg, 70%, as a yellow oil. [α]D20 = + 64.5, (c = 1.0, MeOH). νmax(KBr): 3308, 3064, 2958,
1
2873, 2350, 1730, 1681, 1632, 1537, 1348, 1329, 1027, 969,838.735 cm−1. H NMR (400 MHz,
CDCl3) δ 7.38 – 7.29 (m, 6H), 7.27 – 7.14 (m, 4H), 7.02 (dd, J = 15.8, 6.5 Hz, 1H), 6.94 (d, J = 6.1 Hz,
2H), 6.37 (d, J = 7.3 Hz, 1H), 5.23 (d, J = 5.0 Hz, 1H), 5.10 (m, J = 8.6 Hz, 2H), 4.88 (s, 1H), 4.79 (s,
1H), 4.74 (s, 1H), 4.46 (s, 1H), 4.21 (s,1H), 3.92 (s, 1H), 3.79 (s, 3H), 3.53 (dd, J = 13.8, 4.5 Hz, 1H),
3.10 (d, J = 13.4 Hz, 1H), 1.97 (s, 1H), 1.75 – 1.56 (m, 5H), 1.43 (m, J = 6.0 Hz, 1H), 1.30 (d, J = 6.5
Hz, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.92 – 0.89 (m, J = 12.4 Hz, 17H). 13C NMR (100 MHz, CDCl3) δ
177.9, 171.4, 170.9, 170.3, 169.8, 164.7, 157.2, 149.0, 135.9, 134.4, 129.7, 128.7, 128.5, 128.3,
128.0, 127.1, 122.4, 95.0, 74.5, 67.3, 59.8, 59.3, 58.4, 51.6, 46.2, 40.4, 40.2, 37.1, 34.7, 25.2, 24.9,
24.7, 23.3, 23.3, 21.5, 20.0, 15.9, 11.7. HRMS-ESI (m/z): [M+Na]+ calcd. for C43H58N4O9Na+,
797.4096; found, 797.4097.
(S)-1-(((S)-1-(((S,E)-5-((S)-2-Isopropyl-3-methoxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)-5-oxopent-3-en
-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl
((benzyloxy)carbonyl)-L-isoleucinate (24c). The titled compound 24c was obtained following the
procedure described for 24a. Flash column chromatography (petroleum ether: ethyl acetate= 5:1
to 1: 1). 85 mg, 65%, as a yellow oil. [α]D20 = – 26.2, (c = 1.0, MeOH). νmax(KBr): 3293, 2961, 2869,
2357, 2307, 1692, 1671, 1454, 1329, 1290, 1086, 1022, 966, 811 cm−1. 1H NMR (400 MHz, CDCl3)
δ 7.40 – 7.31 (m, 5H), 7.03 – 6.90 (m, 2H), 6.37 (d, J = 8.2 Hz, 1H), 5.21 (d, J = 6.4 Hz, 1H), 5.15 –
5.08 (m, 3H), 5.05 (d, J = 2.3 Hz, 1H), 4.71 (q, J = 7.2 Hz, 1H), 4.59 (d, J = 3.1 Hz, 1H), 4.46 (s, 1H),
4.21 (d, J = 5.8 Hz, 1H), 3.82 (s, 3H), 2.63 – 2.51 (m, 1H), 1.98 (s, 1H), 1.82 (s, 1H), 1.70 (s, 6H),
1.61 (d, J = 10.7 Hz, 1H), 1.49 – 1.40 (m, 1H), 1.34 – 1.23 (m, 6H), 1.10 (dd, J = 7.1, 2.2 Hz, 3H),
0.99 (d, J = 6.9 Hz, 3H), 0.95 – 0.88 (m, 12H), 0.74 (dd, J = 6.8, 2.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 179.6, 171.4, 171.0, 170.5, 170.3, 164.6, 157.2, 148.6, 135.9, 128.8, 128.6, 128.0, 122.8,
94.8, 74.6, 67.4, 64.0, 59.4, 58.6, 51.7, 46.3, 40.5, 40.2, 37.1, 29.8, 28.8, 25.3, 25.0, 24.8, 23.4,
23.3, 21.6, 21.5, 20.0, 19.0, 15.9, 15.5, 11.7. HRMS-ESI (m/z): [M+Na]+ calcd. for C39H58N4O9Na+,
749.4096; found, 749.4099.
Benzyl
((S)-1-(((S)-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carba
mate (25). A round-bottom flask was charged with ((benzyloxy)carbonyl)-L-leucyl-L-alanine (329
mg, 0.98 mmol), N,O-dimethylhydroxylamine hydrochloride (287 mg, 2.94 mmol), HBTU (1.10 g,
2.94 mmol), DIPEA (633 mg, 4.90 mol) and THF(100 mL). The resulting suspension was stirred
overnight. The reaction mixture was washed with 1 N hydrochloric acid solution, saturated
sodium carbonate solution, and extracted by ethyl acetate. The combined organic phase were
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (petroleum ether: ethyl acetate= 3:1 to 1: 1) to afford
weinreb amide 25 as a pale yellow oil (370 mg, 99.5%). [α]D20 = – 19.8, (c = 1.0, MeOH). νmax (KBr):
1
3370, 3035, 2958, 1722, 1650, 1537, 1322, 1082, 987, 851, 779 cm−1. H NMR (400 MHz,
DMSO-d6) δ 8.12 (d, J = 7.2 Hz, 1H), 7.47 – 7.25 (m, 6H), 5.02 (s, 2H), 4.77 – 4.64 (m, 1H), 4.08 (q,
J = 8.7 Hz, 1H), 3.73 (s, 3H), 3.10 (s, 3H), 1.62 (dq, J = 13.1, 6.7 Hz, 1H), 1.41 (dd, J = 8.0, 4.7 Hz,