A low toxic CRM1 degrader: Synthesis and anti-proliferation on MGC803 and HGC27

Article abstract of DOI:10.1016/j.ejmech.2020.112708

Chromosome region maintenance 1 (CRM1) is the sole nuclear exporter of several tumor suppressor, a growth regulatory protein as an attractive cancer drug target. In the present work, a novel CRM1 degrader was discovered from newly synthesized α, β-unsaturated-δ-lactone based on a natural product Goniothalamin. It induces apoptosis of both MGC803 and HGC27 cell lines via degrading CRM1. Selective inhibition was observed for the proliferation of gastric cancer cell lines MGC803, HGC27 comparing to Human Gastric Mucosal Epithelial Cell Line (GES1). For the first time, CRM1 inhibitor or degrader inducing apoptosis in gastric carcinoma was investigated.

Full text of DOI:10.1016/j.ejmech.2020.112708

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A low toxic CRM1 degrader: synthesis and anti-proliferation on MGC803 and HGC27
Hai-Wei Xu, Shilong Jia, Mengbo Liu, Xiaobo Li, Xia Meng, Xinxin Wu, Lu Yu,
Menglin Wang, Chengyun Jin
PII:
S0223-5234(20)30680-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112708
EJMECH 112708
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 May 2020
Revised Date: 12 July 2020
Accepted Date: 28 July 2020
Please cite this article as: H.-W. Xu, S. Jia, M. Liu, X. Li, X. Meng, X. Wu, L. Yu, M. Wang, C. Jin, A low
toxic CRM1 degrader: synthesis and anti-proliferation on MGC803 and HGC27, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112708.
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O
O
O
1l
Compound

A low toxic CRM1 degrader: synthesis and anti-proliferation on MGC803
and HGC27
Hai-Wei Xu1,2*, Shilong Jia1‡, Mengbo Liu1‡, Xiaobo Li1, Xia Meng1, Xinxin Wu1, Lu Yu1, Menglin Wang1,
Chengyun Jin1,2*
1Key Laboratory of Advanced Technology for Drug Preparation，Ministry of Education, School of Pharmaceutical
Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China.
2Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou
University, No. 100, KeXueDaDao, Zhengzhou 450001, China
Corresponding author: E-mail address: xhwei01@126.com (H. Xu); cyjin@zzu.edu.cn(C.Jin)
‡These authors contributed equally (S. jia and M. Liu)
Abstract:
Chromosome region maintenance 1 (CRM1) is the sole nuclear exporter of several tumor suppressor, a growth
regulatory protein as an attractive cancer drug target. In the present work, a novel CRM1 degrader was discovered
from newly synthesized α, β-unsaturated-δ-lactone based on a natural product Goniothalamin. It induces apoptosis
of both MGC803 and HGC27 cell lines via degrading CRM1. Selective inhibition was observed for the
proliferation of gastric cancer cell lines MGC803, HGC27 comparing to Human Gastric Mucosal Epithelial Cell
Line (GES1). For the first time, CRM1 inhibitor or degrader inducing apoptosis in gastric carcinoma was
investigated.
Keywords: CRM1/XPO1; synthesis; gastric cancer; degrader; structure-activity relationship
1.Introduction
Chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1), is the protein transporter. It is
responsible for the nucleocytoplasmic shuttling of most of the tumor suppressor proteins (TSP) and growth
regulatory factors(GRP), which belongs to the karyopherin β super family of transport receptors[1, 2]. CRM1, the
sole nuclear exporter of TSP, GRP including p53, Rb1 and so on, is an attractive cancer drug target[3]. Currently, an
increasing number of drug-like compounds that target CRM1 have been isolated or synthesized such as Leptomycin
B (LMB)[4, 5], KPT-330 (selinexor)[6, 7], KPT-335(verdinexor)[8, 9], KPT-185[9, 10], KPT-276[11, 12],
KPT-251[13], S109[14] and CBS9106[15]. Some of them have been investigated in clinical trials as a single agent
or in combination with bortezomib, selinexor, or dexamethasone[16], while CRM1 inhibitors have not
demonstrated adequate potency in the clinical settings. Among them, CBS9106 is a novel reversible CRM1
inhibitor with unique CRM1 degrading activity [15] and it is the only reported CRM1 degrader. Therefore it is
needed to discover and research new CRM1 inhibitor or degrader.
Gastric cancer (GC) is a highly aggressive malignant tumor, especially in East Asia such as China, South Korea and
Japan. Its high mortality rate prompts the urgent need for novel therapeutic agents[17]. In our screening of
therapeutic drugs against GC, some synthesized compounds showed apoptosis activity in MGC803. Among them
compound ll led to the downregulation of CRM1 in a dose dependent manner in MGC803. The downregulation of
CRM1 led by compound 1l was for the degradation of CRM1 through proteasome pathway. It was approved to be a
novel CRM1 degrader with a different skeleton of the first degrader CBS9106[15], had selective anti-proliferation
and induced apoptosis activity on MGC803, HGC27 cell lines comparing with Human Gastric Mucosal Epithelial
Cell Line (GES1).
1

2. Results and Discussion
2.1. Chemistry
Natural products LMB, Goniothalamin and their analogues showed CRM1 inhibition and anti-proliferation
activity on cancer cell lines. They were reported to bind to CRM1 through the α, β-unsaturated lactone moiety[18,
19]. Based on the pharmacophore of α, β-unsaturated lactone, a series of compounds with various linkers and aryl
groups were designed and synthesized to investigate the SAR for their anti-proliferation on MGC803.
a: R = 2-OEt; b: R = 2-OMe; c: R = 2-Bn; d: R = 4-SMe; e: R = 4-Et ; f: R = 4-CH2(CH2)2CH3;
g: R = 4-Cyclohexyl; h: R = 4-CH3OCH2CH2; i: R = 4-CF3O ; j: R = 4-PhCH2O; k: R = 3-(CH3CH2)2N ;
l: R = 2-Isopropyl-5-Me ; m: R = [2,3]Ph; n: R = [3,4]dioxole .
Scheme 1. Synthesis of (R)-6-(phenyloxidemethyl)-5, 6-dihydro-2H-pyran-2-ones (1a-1n)
Compounds 1a~1n with phenoxymethyl groups were synthesized shown in Scheme 1. They were prepared
from key intermediates (6a~6n) via olefin metathesis reaction catalyzed by grubbs-2. Compounds 6a~6n were
obtained by the acylation of compounds 5a~5n with Vinylmagnesium chloride, which were synthesized by grignard
reaction of compounds 4a-4n. Compounds 4a-4n was prepared by the reaction of substituted phenols (2a-2n) and
S-epoxy chloropropane (3)[20].
Scheme 2. Synthesis of (R)-6-((1H-indol-1-yl)methyl)-5,6-dihydro-2H-pyran-2-ones (7a~7d)
Compounds 7a~7d were synthesized in a similar way with compounds 1a~1n，and the only difference was in
the first step where indol analogues instead of the phenyl derivatives were used as the start materials to react with
S-epoxy chloropropane (3) (Scheme 2). Compound 12 with phenyl sulfane was synthesized by the same strategy,
which was oxidized using mCPBA to yield compound 13 (Scheme 3).
2

Scheme 3 Synthesis of (R)-6-(((4-chlorophenyl)sulfinyl) methyl)-5,6-dihydro-2H- pyran-2- ones (12, 13)
Compounds 18a-18d and 19a~19d with a linker of 3 atoms length were synthesized from compound 20 which
can be obtained commercially (Scheme 4). Compound 20 was transfer to compounds 21a-d by the acylation.
Compounds 18a-18d and 19a~19d were prepared by an intramolecular esterification in toluene under refluxing in
the present of pTSA.
18, 19
Comp.
a
b
c
d
R
Scheme 4. Synthesis of compounds 18a-d and 19a-d
2.2. Biology
The anti-proliferation of all synthesized compounds were evaluated on MGC803 by MTT assay (Table 1).
Compounds (1a~1n) containing benzyl groups and a two atoms length linker showed moderate anti-proliferation
activity on MGC-803 with IC50 of 2.3 - 47.7 μM. The most potent compound 1l had an IC50 of 2.3μM. Compounds
7a~7e with aniline or indole groups and compounds 18a~18d with a linker of 3 atoms length linker led to
significantly decreasing of efficacy with IC50>50μM. Compounds 19a~19d did not inhibit the proliferation of
MGC803. The SAR data clearly suggested that the benzyl groups and the two atoms length linker were very
important for their anti-proliferation on MGC803. The steric hindrance at the terminal of the linker and the more
atoms length linkers (compounds 7a-7d and 18a-18d) were not beneficial for efficacy. Thus compound 1l was
chosen for further study based on all the above data.
Table 1 the anti-proliferation of synthesized compounds on MGC803
Comp. MGC803/IC50(µM) Comp. MGC803/IC50(µM)
1a
1b
1c
1d
1e
1f
10.353±1.015
24.159±1.383
22.978±1.36
>50
7a
>50µM
7b
7c
25.620±1.409
>50µM
7d
7e
>50µM
12.425±1.094
16.950±1.229
10.593±1.025
>50µM
>50µM
13
>50µM
1g
1h
18a
18b
>50µM
>50µM
3

1i
8.489±0.929
35.439±1.549
>50µM
18c
18d
19a
19b
19c
19d
>50µM
>50µM
>50µM
>50µM
>50µM
>50µM
1j
1k
1l
2.314±0.364
47.687±1.678
11.976±1.078
24.803±1.395
1m
1n
5-Fu
The anti-proliferation activity of compound 1l (Fig 1A) was then evaluated on human gastric cancer cell lines.
Two gastric cancer cell lines (MGC803 and HGC27) and human gastric epithelial cells lines(GES1) were incubated
with it (24 h) at different concentrations, and then the effects of compound 1l on reducing cell viabilities were
assayed by MTT. As shown in Fig.1B, following treatment with compound 1l, the viability of the gastric cancer cell
lines decreased in a dose dependent manner. MGC803 and HGC27 cells lines showed the sensitivity to compound
1l in related to control treatment, causing 40~60% viability reduction at 10 μM for 24 h. However, it is almost no
toxicity to human gastric epithelial cells (GES1) (Fig. 1B). Taken together, these results suggested that compound
1l has selective anti-proliferation on gastric cancer cell lines versus normal human gastric epithelial cell lines.
B
A
Figure 1. the structure of compound 1l and its anti-proliferation activity on MGC803 and HGC27
2.2.1 Compound 1l induced apoptosis by the activation of Bcl-2 family proteins in gastric cancer cell lines
Further experiments were conducted to determine whether the anti-proliferation of compound 1l on the gastric
cancer cell lines were the result of apoptotic cell death. The inducing apoptosis of compound 1l on MGC803 and
HGC27 were evaluated by Annexin V and PI (Propidium Iodide) staining. As shown in Fig.2A, the numbers of
Annexin V positive cells gradually increased in a dose dependent manner in these two cell lines. The results
exhibited that exposure to compound 1l for 24 h significantly upregulated the levels of p53 and apoptosis-related
proteins including Cleaved PARP and Cleaved caspase-3, whereas downregulated CRM1 in MGC803 and HGC27
cells were observed.
In order to confirm the function of mitochondrial in the apoptosis caused by compound 1l, JC-1 was used to
measure MMP (ΔΨ). After treatment with compound 1l in a dose dependent manner, MMP (ΔΨ) was decreased in
MGC803 and HGC27 cell lines comparing with controls, as shown in Fig. 2B. The pro-apoptotic and anti-apoptotic
members of the Bcl-2 super family play primary roles in regulating mitochondrial associated apoptosis. After
treatment with compound 1l, the levels of the pro-apoptotic protein Bax was increased and the levels of the
anti-apoptosis proteins such as Bcl-2 and Bcl-xL were decreased in MGC803 and HGC27 cell ines comparing with
controls (Fig. 2B). In addition, it was observed that compound 1l increased the expression of cleaved caspase-3 and
cleaved caspase-9 in MGC803 and HGC27 cells in comparison with controls. These results indicated that the
mitochondrial pathway is an important factor in compound 1l mediated apoptosis in gastric cancer cells.
4

B
A
Figure 2. Apoptosis induced by compound 1l involves Mitochondrial pathway in gastric cancer cell lines.
(A) Cell apoptosis of MGC803 and HGC27 was detected by flow cytometry after treatment by compound 1l for 24
h. The protein levels of CRM1, PARP, Cleaved PARP, Cleaved caspase-3 and p53 were detected at different
concentrations by Western blot in MGC803 cells. (B) MMP (ΔΨ) was decreased by compound 1l treatment.
Membrane potential was measured by JC-1 dye retention using Flow Cytometry. The protein levels of Bcl-2,
Bcl-xL, Bax and Cleaved caspase-9 were determined by Western blot after compound 1l treatment.
2.2.2 Compound 1l-mediated CRM1 depletion depends on ubiquitin-proteasome pathway
C
A
B
Figure 3. Mitochondrial p53 pathway is involved in the inducing apoptosis of compound 1l in MGC803 cells
and compound 1l induces CRM1 protein degradation via the ubiquitin-proteasome pathway.
(A)CRM1 bound with p53 detected by immunoprecipitation assay in MGC803 cell lines after treatment with
compound 1l at 10 µM at various time points. Accumulation of Ranbp1 or p53 in nucleus was observed in
MGC803 cells with treatment of compound 1l at 10 μM for 6 h. The treated and untreated MGC803 cells with
compound 1l at 10 µM for 0 and 6 h are stained with Ranbp1 antibody (Green) and DAPI (Blue) and analyzed by
confocal microscopy. Westernblot analysis for expression of Ranbp1, p53 and CRM1 in the nucleus and cytoplasm.
(B) MTT assay detected the effect of siRNA targeting to p53 on the survival rate of MGC803 cells, *P<0.05 vs
control group. (C) MGC803 cells were treated with compound 1l (10 μM) in the present or absent of MG132 (0.05
μM ), MG132 (0.1 μM ) and bortezomib (2.5nM) for 24 h. Westernblot was used to detect the expression levels of
CRM1.
5

The levels of CRM1 protein in MGC803 and HGC27 cell lines with treatment of compound 1l were analyzed.
The results showed that compound 11 decreased CRM1 protein level in a dose dependent manner and the levels of
the p53 were increased in MGC803 and HGC27 cell lines comparing with controls (Fig. 2A), which suggested that
compound 1l was possible a CRM1 degrader. Therefore, we detected whether compound 1l could regulate the
expression of p53 by acting on the CRM1 protein. Immuno precipitation indicated that CRM1 bound with p53
dramatically decreased at 6 h (Fig. 3A). As revealed by immunofluorescence staining, notable nuclear
accumulation of Ranbp1 and p53 occurred at 6 h after compound 1l treatment. After exposure to compound 1l at 6
h, the accumulation of p53 in the nucleus increased, whereas the cytoplasmic level of Ranbp1 and CRM1 decreased,
as shown in Fig. 3A. In addition, the utilization of p53 siRNA with MGC803 cell lines, the cell death led by
compound 1l were decreased evidently, as shown in Fig. 3B. The results showed that p53 is required for compound
1l mediated apoptosis.
The degradation of CRM1 proceeds by the ubiquitin-proteasome pathway. To investigate this, MG132, a
proteasome inhibitor, was used to block the CRM1 degradation led by compound 1l. MGC803 and HGC27 cells
were treated with compound 1l (5 μM) for 24h in the presence or absence of MG132(0.05 μM). Reduced CRM1
protein levels were observed in compound 1l-treated cell lines. In contrast, in the presence of MG132, depletion of
CRM1 protein by compound 1l was almost completely abolished (Fig. 3C). Similar results were obtained with the
use of bortezomib, another proteasome inhibitor. These data demonstrated that compound 1l mediated CRM1
depletion depends on ubiquitin-proteasome pathway.
2.2.3. Compound 1l synergize with MG132 to increase the apoptosis of MGC803 and HGC27 cell lines
Some of CRM1 inhibitors have been investigated in clinical trials, while they have not demonstrated adequate
potency in the clinical settings as a single agent and they are more potency in combination with proteasome
inhibitor such as bortezomib[16]. In Fig. 4A, pretreatment with MG132 significantly increased apoptosis in
MGC803 and HGC27 cells, whereas treatment with compound 1l or MG132 alone decreased the activity of gastric
cancer cells only slightly or not at all. In addition, the data showed that pretreatment with MG132 increased
expression of Cleaved PARP, Cleaved caspase-3, p53 and Bax in two gastric cancer cells (Fig.4A). To investigate
the role of p53 in MG132 induced enhancement of compound 1l mediated apoptosis. Fig. 4B revealed that p53
translocated in cytoplasm and bound with anti-apoptotic Bcl family proteins including Bcl-2, Bcl-xl. These data
indicated that, combination of compound 1l with MG132 increased apoptosis via accumulate p53 in the
cytoplasmic, p53 translocated in cytoplasm and bound with Bcl-2 and Bcl-xl, which increased Bax expression and
induced apoptosis, despite degradation of CRM1 protein by compound 1l was completely blocked.
A
B
Figure 4. Compound 1l work synergistically with MG132increased apoptosis
(A) MGC803 and HGC27 cells were pretreated with/without 0.05 μM of MG132 for 1 h before exposure to 10 μM
6

of compound 1l for 24 h. Cell viability was determined by MTT assays. ***P < 0.01 vs. untreated group.MGC803
and HGC27 cells were pretreated with/without 0.05 μM of MG132 for 1 h before exposure to 10 μM of compound
1lfor 24 h. Cells apoptosis of MGC803 and HGC27 was detected by flow cytometry. Western blot assay was used
to detect the expression levels of CRM1. Cleaved PARP, Cleaved caspase-3, p53 and Bax after pretreatment with
the MG132 (0.05 μM) in MGC803 and HGC27 cells for 1 hour, followed by 24 h of compound 1l(10 μM)
incubation. (B) MGC803 cells were pretreated with 0.05 μM of MG132 for 1 h before exposure to 10 μM of
compound 1lfor 24 h. The indicated proteins were detected by immunoprecipitation with an antibody for
p53.MGC803 cells were pretreated with/without 0.05 μM of MG132 for 1 h before exposure to 10 μM of
compound 1l for 6 h. p53 in the nucleus or cytoplasm was detected by Immunofluorescence analysis. The treated
and untreated samples are stained with p53antibody (Green) and DAPI (Blue) and analyzed by confocal
microscopy.
3. Discussion
Gastric cancer is one of highly aggressive malignant tumor, while impactful chemotherapies for it are limited
[21]. Therefore, developing novel therapeutic drugs is an attractive area of gastric cancer research. In this study, we
examined the cytotoxic effects of a novel compound 1l in gastric cancer cells. Compound 1l exhibited strong
cytotoxic effect against gastric cancer cell lines with low toxic in Human Gastric Mucosal Epithelial Cell Line.
Furthermore, we discovered that p53 is required for compound 1l mediated apoptosis.
In recent years, CRM1 over expression has been detected in gastric cancer and caused dysfunction of cell fate
controls[22]. A number of studies have shown that targeting CRM1 as promising therapeutic targets for cancer
research. Our results support this suppose because we found that compound 1l showed antitumor effects in gastric
cancer cell lines in vitro. It was reported that CRM1 inhibitor KPT-185 induced apoptosis in ovarian and breast
cancer cell lines [23]. However, the mechanism of anti-gastric cancer activity of CRM1 inhibitor remain unknown.
Our study showed that compound 1l suppresses proliferation and induces apoptosis in MGC803 and HGC27 cell
lines in a dose dependent manner through mitochondrial pathway.
Up to present, CRM1 mediates the transport of more than 230 proteins, many of which are tumor suppressors
[24]. Therefore, the inhibition of CRM1 function induced the accumulation of tumor suppressor proteins in the
nucleus and reduced cytoplasmic degradation of these proteins, has been thought to be as significant mechanism of
the anti-gastric cancer activity of CRM1 inhibitor. In our study, the data suggest that the antitumor effects of
compound 1l is associate with tumor suppressor proteins p53. We observed that the expression levels of CRM1 was
decreased and p53 accumulated within nuclear after compound 1l treatment. In addition, knockdown of p53 by
specific siRNA in MGC803 cells, compound 1l induced mitochondrial apoptotic pathway was decreased evidently.
The ubiquitin-proteasome system (UPS) and autophagy are major intracellular protein degradation
systems[25]. The UPS pathway is essential to the cell cycle, transcription and cell survival. Numerous studies have
revealed that blockage of proteasome mediated protein degradative pathway stabilized the tumor suppressor p53,
and the proapoptotic proteins Bax[16, 26]. MG132, a26S proteasome inhibitor, whereas inhibition of the 26S
proteasome lead to accumulation of non-degraded ubiquitinated proteins in the cytoplasmic. In our study, we found
that co-incubated with the proteasome inhibitor MG132 or bortezomib, degradation of CRM1 protein by compound
1l was almost completely blocked. In addition, we observed that compound 1l treatment in combination with
MG132 exhibited enhanced expression of p53, Cleaved PARP, Cleaved caspase-3 and Bax and induced higher
levels of apoptosis in two gastric cancer cells when compared to cells treated with compound 1l or inhibitors alone.
p53 transcription-independent apoptosis is initiated by interacts with anti-apoptotic Bcl family proteins including
Bcl-2, Bcl-xl, induced cell apoptosis[27]. In this study, we found that p53 translocated in cytoplasm and bound with
Bcl-2 and Bcl-xl. Pretreatment with MG132, these results indicated that combined treatment significantly increased
apoptosis via accumulate p53 in the cytoplasmic, despite degradation of CRM1 protein by compound 1l was
completely blocked.
A critical component of any degrader/drug is permeability. Passive diffusion permeability is the most
important permeability for small molecules. Cell permeability of a drug depends on its molecular weight, polar
surface area and clogP. The permeability of the lipophilic molecules is better than that of polar molecules. Neutral
molecules are much more permeable than their charged forms. Compound 1l is a neutral and lipophilic molecule
with clogP of 3.32 and polar surface area of 35.5. Its molecular weight is 260.4. The small molecule structure and
the reasonable clogP suggest that compound 1l has a good permeability possibly.
In summary, the results in this work show that compound 1l is a novel CRM1 degrader that has major
cytotoxicity in gastric cancer cells and have little toxicity in human gastric epithelial cells. It is a potential
pro-apoptotic agent for prevention and treatment of gastric cancer.
4. Experimental section
4.1.General information
Reagents and solvents were purchased from Bide Pharmatech Ltd, Aladdin, Sinopharm Chemical Reagent Co,
7

1
Ltd. with purities of at least 97%. H NMR (400 MHz) and 13C NMR (100 MHz) spectrawere recorded with a
Bruker spectrometer. All reactions were monitored by thin-layer chromatography (TLC) on 25.4 mm× 76.2 mm
silica gel plates (GF-254) and UPLC-Mass on Waters ACQUITY UPLC H-Class or Q-Tof Micro HRMS on waters.
Melting points were determined on a Beijing Keyi XT4A apparatus. The silica gel used for column
chromatography was 200～300 mesh or recrystallization with solvents specified in the corresponding experiments.
4.2. General synthetic procedure for the compounds
4.2.1. General Synthesis of (R)-6-((2-ethoxyphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1a) as a sample
Grubbs-2 (188 mg, 0.221 mmol) was dissolved in 20 mL anhydrous DCM in an oven dried flask. Subsequently,
compound 6a（612 mg, 2.21mmol） was dissolved in 2 mL DCM and added drop wise under an nitrogen
atmosphere. The reaction was stirred for 15 hours at 50℃~60℃. The crude product was purified by
chromatography on silica gel with petroleumether/ ethylacetate (10:1) to afford 1a as a white solid (173 mg,
31.46%).1H NMR (400 MHz, CDCl3)δ 7.06 – 6.84 (m, 5H), 6.14 – 6.04(m, 1H), 4.95 – 4.75(m, 1H), 4.30 (dd, J =
10.2, 4.5 Hz, 1H ), 4.21 (dd, J = 10.2, 6.2 Hz, 1H), 4.19 – 4.00 (m, 2H), 2.79 – 2.58 (m, 2H), 1.45(t, J = 7.0 Hz,
3H). 13C NMR (100 MHz, CDCl3)δ 163.48, 148.11, 144.86, 122.71, 121.34, 121.09, 116.01, 113.97, 75.69, 70.53,
64.49, 26.41, 14.96.
4.2.2. Synthesis of (R)-6-((2-methoxyphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1b) Compound 6b (520 mg,
1.98mmol) was converted to 1b(145 mg, 31.22%) as a white solid by the same procedure as described for
compound 1a. m.p. 90.2~91.6℃; 1H NMR (400 MHz, CDCl3) δ 7.06 – 6.84 (m, 5H), 6.10 – 6.04 (m, 1H), 4.88 –
4.80 (m, 1H), 4.28 (dd, J = 10.2, 4.6 Hz, 1H), 4.19 (dd, J = 10.2, 6.2 Hz, 1H), 3.85 (s, 3H), 2.75 – 2.53 (m, 2H).13C
NMR (100 MHz, CDCl3) δ 163.42, 149.95, 147.78, 144.81, 122.48, 121.36, 121.00, 115.03, 112.31, 75.62, 70.19,
55.93, 26.45.HRMS: calcd for C13H14NaO4 [M+Na]+m/z: 257.0790, found: 257.0792.
4.2.3.Synthesis of (R)-6-((2-benzylphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1c) Compound 6c (550 mg,
1.71mmol) was converted to 1c(220mg, 43.81%) as a white solid by the same procedure as described for
compound 1a. m.p. 86.5~87.2℃; 1H NMR (400 MHz, DMSO-d6) δ 7.27 – 7.21m, 4H), 7.21 – 7.13 (m, 3H), 7.12 –
7.04 (m, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.91 (t, J = 7.4 Hz, 1H), 6.02-5.97 (m, 1H), 4.85 – 4.77 (m, 1H), 4.25 – 4.14
(m, 2H), 3.91 (s, 2H), 2.50 – 2.44 (m, 2H).13C NMR (100 MHz, DMSO-d6) δ 163.26 , 155.68, 146.80, 140.85,
130.28, 129.45, 128.65, 128.16 , 127.58, 125.71, 120.82, 119.95, 111.85, 75.63, 68.57, 35.45, 25.10. HRMS: calcd
for C19H18NaO3 [M+Na]+ m/z: 317.1154, found: 317.1153.
4.2.4. Synthesis of (R)-6-((4-(methylthio)phenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1d) Compound 6d (500
mg, 1.80mmol) was converted to 1d(200mg, 44.32%) as a gray solid by the same procedure as described for
compound 1a. m.p. 100.5~101.7℃; 1H NMR (400 MHz, CDCl3) δ 7.26 (s, 2H), 7.02 – 6.91 (m, 1H), 6.86 (d, J =
7.6 Hz, 2H), 6.08 (d, J = 9.6 Hz, 1H), 4.84 – 4.74 (m, 1H), 4.22 – 4.08 (m, 2H), 2.77 – 2.50 (m, 2H), 2.45 (s,
3H).13C NMR (100 MHz, CDCl3) δ 163.27, 156.63, 144.61, 129.99,129.89, 121.38, 115.30, 75.47, 68.72, 26.24,
17.77.HRMS: calcd for C13H14NaO3S [M+Na]+m/z: 273.0561, found: 273.0561.
4.2.5. Synthesis of (R)-6-((4-ethylphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1e) Compound 6e (560 mg,
2.15mmol) was converted to 1e(213 mg, 42.63%) as a white solid by the same procedure as described for
compound 1a. m.p. 65.1~66.7℃; 1H NMR (400 MHz, CDCl3) δ 7.12 (d, J = 8.5 Hz, 2H), 6.98 – 6.90 (m, 1H), 6.84
(d, J = 8.5 Hz, 2H), 6.07 (dd, J = 9.8, 1.7 Hz, 1H), 4.83 – 4.73 (m, 1H), 4.19 (dd, J = 10.0, 4.5 Hz, 1H), 4.13 (dd, J
= 10.0, 5.6 Hz, 1H), 2.71 – 2.59 (m, 2H), 2.59 – 2.49 (m, 2H), 1.21 (t, J = 7.6 Hz, 3H).13C NMR (100 MHz, CDCl3)
δ 163.43 , 156.21, 144.81, 137.30, 128.85, 121.30, 114.47, 75.60, 68.62, 27.99, 26.29, 15.87.HRMS: calcd for
C14H16NaO3 [M+Na]+m/z: 255.0997, found :255.0998.
4.2.6. Synthesis of (R)-6-((4-pentylphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1f) Compound 6f (550 mg,
1.82mmol) was converted to 1f(246 mg, 49.30%) as a white solid by the same procedure as described for
compound 1a. m.p. 68.0~69.1℃; 1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 8.5 Hz, 2H), 6.98 – 6.92 (m, 1H), 6.83
8

(d, J = 8.6 Hz, 2H), 6.14 – 6.02 (m, 1H), 4.85 – 4.75 (m, 1H), 4.19 (dd, J = 10.0, 4.5 Hz, 1H), 4.13 (dd, J = 10.0,
5.7 Hz, 1H), 2.72 – 2.50 (m, 4H), 1.62 – 1.56 (m, 2H), 1.39 – 1.24 (m, 4H), 0.89 (t, J = 6.9 Hz, 3H).13C NMR (100
MHz, CDCl3) δ 156.15, 144.75, 135.97, 129.37, 121.32, 114.33, 77.33, 75.56, 68.55, 35.00, 31.43, 31.38, 26.31,
22.54, 14.05.HRMS: calcd for C17H22NaO3 [M+Na]+m/z: 297.1467, found: 297.1467.
4.2.7. Synthesis of (R)-6-((4-cyclohexylphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1g) Compound 6g(470 mg,
1.49mmol) was converted to 1g(196 mg, 45.79%) as a white solid by the same procedure as described for
compound 1a. m.p. 85.5~86.3℃;1H NMR (400 MHz, CDCl3) δ 7.13(d, 7.5Hz, 2H), 6.98 –6.89(m, 1H), 6.84 (d, J =
8.0 Hz, 2H), 6.07 (d, 9.7Hz, 1H), 4.85 – 4.75 (m, 1H), 4.25 – 4.07 (m, 2H), 4.13 (dd, J = 10.0, 5.7 Hz, 1H), 2.72 –
2.50 (m, 1H), 2.50 – 2.36 (m, 1H), 1.93 – 1.79 (m, 4H), 1.73 (d, J = 13.0 Hz, 1H), 1.46 – 1.30 (m, 4H), 1.30 – 1.15
(m, 1H). 13C NMR (100 MHz, CDCl3) δ 163.37, 156.22, 144.71, 141.28, 127.76, 121.34, 114.36, 75.55, 68.56,
43.70, 34.68, 26.91, 26.33, 26.14. HRMS: calcd for C18H22NaO3 [M+Na]+ m/z: 309.1467, found: 309.1466.
4.2.8. Synthesis of (R)-6-((4-(2-methoxyethyl)phenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1h) Compound
6h(556mg, 1.91 mmol) was converted to1h(223 mg, 44.40%) as a white solid by the same procedure as described
for compound 1a. m.p. 47.3~48.1℃; 1H NMR (400 MHz, CDCl3) δ 7.14 (d, J = 8.5 Hz, 2H), 7.03 – 6.90 (m, 1H),
6.84 (d, J = 8.6 Hz, 2H), 6.06 (dd, J = 9.8, 1.7 Hz, 1H), 4.84 – 4.74(m, 1H), 4.18 (dd, J = 10.0, 4.6 Hz, 1H), 4.13
(dd, J = 10.0, 5.6 Hz, 1H), 3.56 (t, J = 7.0 Hz, 2H), 3.35 (s, 3H), 2.82 (t, J = 7.0 Hz, 2H), 2.70 – 2.59 (m, 1H), 2.58
– 2.48 (m, 1H).13C NMR (100 MHz, CDCl3) δ 163.40, 156.62, 144.83, 131.91, 129.86, 121.22, 114.48, 75.56,
73.72, 68.53, 58.64, 35.26, 26.21.HRMS: calcd forC15H18NaO4 [M+Na]+m/z: 285.1103, found: 285.1104.
4.2.9. Synthesis of (R)-6-((4-(trifluoromethoxy)phenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1i) Compound
6i(570 mg, 1.80mmol) was converted to 1i(250 mg, 48.13%) as a white solid by the same procedure as described
for compound 1a. m.p. 71.1~72.7℃; 1H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 8.7 Hz, 2H), 7.01 – 6.95 (m, 1H),
6.95 – 6.90 (m, 2H), 6.11 (dd, J = 9.4, 2.1 Hz, 1H), 4.88 – 4.78 (m, 1H), 4.26 – 4.13 (m, 2H), 2.75 – 2.64 (m, 1H),
2.63 – 2.50 (m, 1H).13C NMR (101 MHz, CDCl3) δ 163.22, 156.63, 144.59, 143.26, 122.57, 121.37, 119.24, 115.41,
75.39, 68.96, 26.15.HRMS: calcd forC13H11F3NaO4 [M+Na]+m/z: 311.0507, found: 311.0506.
4.2.10. Synthesis of (R)-6-((4-(benzyloxy)phenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1j) Compound 6j (530
mg, 1.57mmol) was converted to1j(210 mg, 43.20%) as a white solid by the same procedure as described for
compound 1a. m.p.146.0~147.3℃; 1H NMR (400 MHz, CDCl3) δ 7.47 – 7.28 (m, 5H), 6.98 – 6.93 (m, 1H), 6.92 –
6.88 (m, 2H), 6.88 – 6.82 (m, 2H), 6.11 –6.04 (m, 1H), 5.02 (s, 2H), 4.83 – 4.73 (m, 1H), 4.16 (dd, J = 10.0, 4.5 Hz,
1H), 4.11 (dd, J = 10.0, 5.6 Hz, 1H), 2.71 – 2.59 (m, 1H), 2.58 – 2.48(m, 1H).13C NMR (100 MHz, CDCl3) δ
163.42, 153.53, 152.53, 144.74, 137.14, 128.57, 127.94, 127.47, 121.34, 115.91, 115.64, 75.62, 70.66, 69.27,
29.70.HRMS: calcd forC19H18NaO4 [M+Na]+ m/z: 333.1103, found: 333.1102.
4.2.11. Synthesis of (R)-6-((3-(diethylamino)phenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1k) Compound 6k
(360 mg, 1.19mmol) was converted to 1k(150 mg, 45.91%) as a white solid by the same procedure as described for
compound 1a. m.p. 50.2~51.6℃; 1H NMR (400 MHz, CDCl3) δ 7.11 (t, J = 8.2 Hz, 1H), 6.98 – 6.91 (m, 1H), 6.34
(dd, J = 8.4, 1.8 Hz, 1H), 6.26 – 6.15 (m, 2H), 6.11 – 6.04 (m, 1H), 4.85 – 4.75 (m, 1H), 4.21 (dd, J = 10.0, 4.5 Hz,
1H), 4.13 (dd, J = 10.0, 6.0 Hz, 1H), 3.33 (q, J = 7.1 Hz, 4H), 2.71 – 2.50 (m, 2H), 1.15 (t, J = 7.1 Hz, 6H).13C
NMR (100 MHz, CDCl3) δ 163.46, 159.56, 144.79, 130.01, 121.35, 105.72, 100.50, 98.87, 75.63, 68.30, 44.36,
29.70, 26.41, 12.58.
4.2.12. Synthesis of (R)-6-((2-isopropyl-5-methylphenoxy)methyl)-5,6-dihydro-2H-pyran-2-one (1l) Compound 6l
(460 mg, 1.60mmol) was converted to 1l(186 mg, 44.79%) as a colorless oily liquid by the same procedure as
described for compound 1a.1H NMR (400 MHz, CDCl3) δ 7.11 (d, J = 7.7 Hz, 1H), 7.05 – 6.91 (m, 1H), 6.79 (d, J
= 7.7 Hz, 1H), 6.65 (s, 1H), 6.09 (d, J = 11.4 Hz, 1H), 4.90 – 4.80 (m, 1H), 4.21 (dd, J = 9.9, 4.3 Hz, 1H), 4.16 (dd,
J = 9.9, 5.7 Hz, 1H), 3.30 – 3.20 (m, 1H), 2.76 – 2.50 (m, 2H), 2.32 (s, 3H), 1.19 (d, J = 6.9 Hz, 6H).13C NMR (100
MHz, CDCl3) δ 163.47, 155.02, 144.76, 136.49, 134.06, 126.06, 121.93, 121.33, 112.14, 75.63, 68.51, 26.54, 26.43,
9

22.76, 22.73, 21.31.HRMS: calcd forC16H20NaO3 [M+Na]+ m/z: 283.1310, found: 283.1311.
4.2.13. Synthesis of (R)-6-((naphthalen-1-yloxy)methyl)-5,6-dihydro-2H-pyran-2-one (1m) Compound 6m (480
mg, 1.70mmol) was converted to 1m(153 mg, 35.39%) as a white solid by the same procedure as described for
compound 1a. m.p. 87.0~88.1℃; 1H NMR (400 MHz, DMSO-d6) δ 8.33 – 8.10 (m, 1H), 7.93 –7.87 (m, 1H), 7.61
– 7.48 (m, 3H), 7.44 (t, J = 7.9 Hz, 1H), 7.20 – 7.13 (m, 1H), 7.03 (d, J = 7.5 Hz, 1H), 6.08 – 6.02 (m, 1H), 5.05 –
4.95 (m, 1H), 4.46 – 4.34 (m, 2H), 2.78 – 2.60 (m, 2H).13C NMR (100 MHz, DMSO-d6) δ 163.36, 153.53, 146.95,
133.99, 127.47, 126.53, 126.14, 125.43, 124.80, 121.48, 120.39, 120.03, 105.45, 75.68, 68.90, 25.30.HRMS: calcd
for C16H14NaO3 [M+Na]+ m/z: 277.0841, found: 277.0842.
4.2.14. Synthesis of (R)-6-((benzo[d][1,3]dioxol-5-yloxy)methyl)-5,6-dihydro-2H-pyran-2-one (1n) Compound 6n
(530 mg, 1.92mmol) was converted to 1n(198 mg, 41.58%) as a white solid by the same procedure as described for
compound 1a. m.p. 96.5~97.4℃; 1H NMR (400 MHz, CDCl3) δ 7.00 – 6.90 (m, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.50
(d, J = 2.5 Hz, 1H), 6.33 (dd, J = 8.5, 2.5 Hz, 1H), 6.10 – 6.06 (m, 1H), 5.93 (s, 2H), 4.83 – 4.72 (m, 1H),4.13 (dd,
J = 10.0, 4.6 Hz, 1H), 4.08 (dd, J = 10.0, 5.4 Hz, 1H), 2.70 – 2.46 (m, 2H).13C NMR (100 MHz, CDCl3) δ 163.36,
153.66, 148.35, 144.68, 142.25, 121.33, 107.95, 105.79, 101.28, 98.34, 75.53, 69.59, 26.22.HRMS: calcd
forC13H12NaO5 [M+Na]+m/z: 271.0582, found: 271.0582.
4.3.General Synthesis Procedure for analogues of (R)-2-(phenoxymethyl)oxirane (4a~4n) phenol derivatives
2a~2n (1 equvi) and NaOH (3 equvi) was dissolved in 5 mL H2O in a flask.Subsequently, The reaction mixture
was stirred at room temperature for 30min. The reaction system was added drop wise to a 50 mL round bottom
flask containing (s)-epichlorohydrin (3 equvi) and tetrabutylammonium bromide (0.05 equvi). The reaction mixture
was stirred at room temperature. After the reaction is completed, The resulting mixture was diluted with water (10
mL) and extracted with ethylacetate (20 mL × 3).The combined organic phases were dried with Na2SO4,
concentrated to afford a crude product. The crude product was purified by chromatography on silica gel with
petroleum ether andethylacetate to afford4a~4n.
4.4.
General
Synthesis
Procedure
for
analogues
of
(R)-1-phenoxypent-4-en-2-ol
(5a~5n)
(R)-2-(phenoxymethyl)oxirane derivatives 4a~4n (1 equvi) and CuI (0.2 equvi) was dissolved in 20 mL anhydrous
THF in a flask. The reaction mixture was stirred at-10~0℃ for 10 min and ethylene magnesium chloride (1.5 equvi)
was added drop wise under an argon atmosphere. After the reaction is completed, The resulting mixture was diluted
with saturated ammonium chloride (50 mL) and the system was suction filtered with celite, and then evaporated
and extracted with ethylacetate (15 mL × 3).The combined organic phases were dried with Na2SO4, concentrated to
afford a crude product.The crude product was purified by chromatography on silica gel with petroleum ether
andethylacetate to afford 5a~5n.
4.5. General Synthesis Procedure for analogues of (R)-1-phenoxypent-4-en-2-yl acrylate (6a~6n) A solution of
5a~5n(1 equvi), anhydrousMgSO4 (2 equvi), and TEA (1.5 equvi) in Anhydrous CH2Cl2 (20 mL) was stirred at
room temperature. Acryloylchloride (1.5 equvi) was added drop wise. The reactionmixture waswas stirred atrt for
2h. After the reaction is completed, The resulting mixture was diluted with water (20 mL) and extracted with
CH2Cl2 (20 mL × 3), dried (MgSO4), and concentrated to afford acrude product. The crude product was purified by
chromatography onsilica gel with petroleum ether/ethylacetate to afford 6a~6n.
4.6. General Synthesis of (R)-6-((1H-indol-1-yl)methyl)-5,6-dihydro-2H-pyran-2-one (7a)as a sample Grubbs-2
(126 mg, 0.219 mmol) was dissolved in 20 mL anhydrous DCM in an oven dried flask. Subsequently, compound
11a（380 mg, 1.49mmol）was dissolved in 2 mL DCM and added drop wise under an nitrogen atmosphere. The
reaction was stirred for 15 hours at 50℃~60℃. The crude product was purified by chromatography on silica gel
with petroleumether/ ethylacetate (10:1) to afford 7a as a colorless oily liquid(143 mg, 42.28%).1H NMR (400
MHz, CDCl3) δ 7.63 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.26 – 7.19 (m, 1H), 7.19 – 7.04 (m, 2H), 6.89 –
6.66 (m, 1H), 6.54 (d, J = 2.5 Hz, 1H), 5.99 (d, J = 9.7 Hz, 1H), 4.90 – 4.68 (m, 1H), 4.46 (dd, J = 15.1, 4.8 Hz,
10

1H), 4.39 (dd, J = 15.0, 5.5 Hz, 1H), 2.36 – 2.17 (m, 2H).13C NMR (100 MHz, CDCl3) δ 163.17 (s), 144.32, 136.28
128.66, 128.57, 122.02, 121.30, 121.22, 119.83, 109.06, 102.46, 76.35, 48.99, 26.88.
4.6.1.Synthesis of (R)-6-((5-methoxy-1H-indol-1-yl)methyl)-5,6-dihydro-2H-pyran-2-one (7b) Compound
11b(430 mg, 1.51mmol) was converted to 7b(176 mg, 45.39%) as a paleyellow solid by the same procedure as
described for compound 7a.1H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 3.1 Hz, 1H), 7.09
(d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.9, 2.4 Hz, 1H), 6.84 – 6.73 (m, 1H), 6.46 (d, J = 3.1 Hz, 1H), 6.05 – 5.95 (m,
1H), 4.85 – 4.70 (m, 1H), 4.43 (dd, J = 15.1, 4.9 Hz, 1H), 4.37 (dd, J = 15.1, 5.6 Hz, 1H), 3.85 (s, 3H), 2.30 – 2.19
(m, 2H).13C NMR (100 MHz, CDCl3) δ 163.16, 154.27, 144.28, 131.56, 129.09, 121.32, 112.33, 109.82, 102.83,
102.03, 76.37, 55.86, 49.20, 26.86.HRMS: calcd forC15H15NNaO3 [M+Na]+m/z: 280.0950, found: 280.0951.
4.6.2. Synthesis of (R)-6-(indolin-1-ylmethyl)-5,6-dihydro-2H-pyran-2-one (7c) Compound 11c (500 mg, 1.94
mmol) was converted to 7c(210 mg, 47.14%) as a colorless oily liquidby the same procedure as described for
compound 7a.1H NMR (400 MHz, CDCl3) δ 7.08 (dd, J = 13.3, 7.1 Hz, 2H), 6.98 – 6.85 (m, 1H), 6.68 (t, J = 7.3
Hz, 1H), 6.44 (d, J = 7.8 Hz, 1H), 6.05 (dd, J = 10.1, 2.0 Hz, 1H), 4.78 – 4.60 (m, 1H), 3.59 – 3.46 (m, 2H), 3.38 (d,
J = 5.2 Hz, 2H), 3.00 (t, J = 8.4 Hz, 2H), 2.64 – 2.39 (m, 2H).13C NMR (100 MHz, CDCl3) δ 163.84, 152.14,
144.95, 129.48, 127.35, 124.65, 121.29, 117.98, 106.27, 55.13, 53.38, 29.69, 28.77, 27.25.HRMS: calcd
forC14H15NNaO2 [M+Na]+m/z: 252.1000, found: 252.1003.
4.6.3. Synthesis of (R)-6-(((6-chloro-1H-benzo[d] [1,2,3]triazol-1-yl)oxy)methyl) -5,6-dihydro-2H-pyran-2- one
(7d) Compound 11d(450 mg, 1.46 mmol) was converted to 7d(55 mg, 13.45%) as a grey-green solid by the same
procedure as described for compound 7a.m.p. 109.5~110.1℃; 1H NMR (400 MHz, CDCl3) δ 7.94 (d, 8.8Hz, 1H),
7.72 (d, J = 1.3 Hz, 1H), 7.43 – 7.35 (m, 1H), 7.07 – 6.92 (m, 1H), 6.13 (dd, J = 9.8, 1.9 Hz, 1H), 4.95 – 4.84 (m,
1H), 4.81 – 4.68 (m, 2H), 2.80 – 2.65 (m, 1H), 2.65 – 2.50 (m, 1H).13C NMR (101 MHz, CDCl3) δ 162.47, 144.04,
141.94, 135.22, 127.76, 126.40, 121.54, 121.26, 108.64, 80.29, 73.96, 25.49 .HRMS: calcd forC12H10ClN3NaO3
[M+Na]+m/z: 302.0308, found: 302.0308.
4.6.4. Synthesis of (R)-6-((methyl(m-tolyl)amino)methyl)-5,6-dihydro-2H-pyran-2-one (7e) Compound 11e (500
mg, 1.93 mmol) was converted to 7e(210 mg, 47.09%) as a yellow oily liquidby the same procedure as described
for compound 7a.1H NMR (400 MHz, CDCl3) δ 7.19 – 7.06 (m, 1H), 6.97 – 6.80 (m, 1H), 6.57 (d, J = 7.4 Hz, 1H),
6.52 (d, J = 6.2 Hz, 2H), 6.11 – 5.90 (m, 1H), 4.76 – 4.60 (m, 1H), 3.68 (dd, J = 15.4, 5.8 Hz, 1H), 3.60 (dd, J =
15.4, 5.8 Hz, 1H), 3.04 (s, 3H), 2.46 – 2.37 (m, 2H), 2.32 (s, 3H).13C NMR (100 MHz, CDCl3) δ 163.79, 148.86,
144.68, 139.12, 129.21, 121.47, 117.96, 112.89, 109.36, 76.15, 55.96, 39.96, 27.45, 21.95.HRMS: calcd
forC14H17NNaO2 [M+Na]+m/z:254.1157, found: 254.1156.
4.7. General Synthesis Procedure for analogues of (S)-1-(oxiran-2-ylmethyl)-1H-indole (9) Indole analogues 8 (1
equvi) and NaH (3equvi) was dissolved in 20 mL MeCN at ice-bathin a flask.Subsequently, The reaction mixture
was stirred at room temperature for 30min. (S)-epichlorohydrin (3 equvi) was added drop wise. The reaction
mixture was stirred at room temperature. After the reaction is completed, The resulting mixture was diluted with
water (10 mL) and evaporated, and then extracted with ethylacetate (20 mL × 3).The combined organic phases
were dried with Na2SO4, concentrated to afford a crude product. The crude product was purified by chromatography
on silica gel with petroleum ether / acetone to afford 9.
4.8. General Synthesis Procedure for analogues of (R)-1-(1H-indol-1-yl)pent-4-en-2-ol (10) Compounds 9 (1
equvi) and CuI (0.2 equvi) was dissolved in 20 mL anhydrous THF in a flask. The reaction mixture was stirred
at-10~0℃ for 10 min and ethylene magnesium chloride (1.5 equvi) was added drop wise under an argon
atmosphere. After the reaction is completed, The resulting mixture was diluted with saturated ammonium chloride
(50 mL) and the system was suction filtered with celite, and then evaporated and extracted with ethylacetate (15
mL × 3).The combined organic phases were dried with Na2SO4, concentrated to afford a crude product.The crude
product was purified by chromatography on silica gel with petroleum ether andethylacetate to afford 10.
11

4.9. General Synthesis Procedure for analogues of (R)-1-(1H-indol-1-yl)pent-4-en-2-yl acrylate (11) A solution of
10(1 equvi), anhydrousMgSO4 (2 equvi), and TEA (1.5 equvi) in Anhydrous CH2Cl2 (20 mL) was stirred at room
temperature. Acryloylchloride (1.5 equvi) was added drop wise. The reactionmixture was stirred atrt for 2h. After
the reaction is completed, The resulting mixture was diluted with water (20 mL) and extracted with CH2Cl2 (20 mL
× 3), dried (MgSO4), and concentrated to afford acrude product. The crude product was purified by
chromatography onsilica gel with petroleum ether/ethylacetate to afford 11.
4.10. General Synthesis Procedure for(R)-6-(((4-chlorophenyl)thio)methyl)-5,6-dihydro-2H-pyran-2-one(12)
Grubbs-2 (270 mg, 0.318 mmol) was dissolved in 20 mL anhydrous DCM in an oven dried flask. Subsequently,
compound 17（900 mg, 3.18 mmol） was dissolved in 2 mL DCM and added drop wise under an nitrogen
atmosphere. The reaction was stirred for 15 hours at 50℃~60℃. The crude product was purified by
chromatography on silica gel with petroleumether/ ethylacetate to afford 12 as a white solid (305 mg, 37.62%).
4.11. Synthesis of (6R)-6-(((4-chlorophenyl)sulfinyl)methyl)-5,6-dihydro-2H-pyran-2-one (13) A solution of 12
(1.00g, 3.93 mmol) in CH2Cl2 (20 mL),subsequently, m-chloroperoxybenzoic acid (812mg, 4.71mmol) is added to
the system under ice bath conditions. After the reaction is complete, a mixture of saturated solution of Na2S2O3 (10
mL) and NaHCO3 (10 mL) was added. The organic phase was washed with water (3 × 10mL) and brine (10 mL),
dried (Na2SO4), and concentrated to afford a crude product. The crude product was purified by chromatography on
1
silica gel with petroleum ether/acetone (8:1) to afford 13 as a gray solid (145 mg, 13.64%). m.p. 80.5~81.6℃; H
NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 6.99 – 6.84 (m, 1H), 6.11 (d, J = 10.0
Hz, 1H), 5.17 – 5.01 (m, 1H), 3.11 (dd, J = 13.4, 9.4 Hz, 1H), 2.95 (dd, J = 13.4, 3.2 Hz, 1H), 2.62 – 2.28 (m,
2H).13C NMR (101 MHz, CDCl3) δ 162.74, 144.29, 142.20, 137.74, 129.89, 125.18, 121.62, 71.67, 63.20,
29.24 .HRMS: calcd forC12H11ClNaO3S [M+Na]+ m/z: 293.0015, found: 293.0016.
4.12. General Synthesis Procedure for (R)-2-(((4-chlorophenyl)thio)methyl)oxirane (15)4-chlorobenzenethiol14
(2.00 g, 13.83mmol) and NaOH (1.66 g, 41.49mmol) was dissolved in 5 mL H2O in a flask.Subsequently, The
reaction mixture was stirred at room temperature for 30min. The reaction system was added drop wise to a 50 mL
round bottom flask containing (s)-epichlorohydrin (3.84 g, 41.49mmol) and tetrabutylammonium bromide (223 mg,
0.69mmol). The reaction mixture was stirred at room temperature. After the reaction is completed, The resulting
mixture was diluted with water (10 mL) and extracted with ethylacetate (20 mL × 3).The combined organic phases
were dried with Na2SO4, concentrated to afford a crude product. The crude product was purified by chromatography
on silica gel with petroleum ether andethylacetate to afford colorless oil liquid15 (1.05 g, 37.83%).
4.13. General Synthesis Procedure for(R)-1-((4-chlorophenyl)thio)pent-4-en-2-ol (16) Compound 15 (1.00g, 4.98
mmol) and CuI (190 mg, 0.99 mmol) was dissolved in 20 mL anhydrous THF in a flask. The reaction mixture was
stirred at-10~0℃ for 10 min and ethylene magnesium chloride(648 mg, 7.47 mmol) was added drop wise under an
argon atmosphere. After the reaction is completed, The resulting mixture was diluted with saturated ammonium
chloride (50 mL) and the system was suction filtered with celite, and then evaporated and extracted with
ethylacetate (15 mL × 3).The combined organic phases were dried with Na2SO4, concentrated to afford a crude
product.The crude product was purified by chromatography on silica gel with petroleum ether andethylacetate to
afford colorless oil liquid16 (970 mg, 85.10%).
4.14. General Synthesis Procedure for (R)-1-((4-chlorophenyl)thio)pent-4-en-2-yl acrylate (17) A solution of
16(950mg, 4.15mmol), anhydrousMgSO4 (999.79mg, 8.31mmol), and TEA (630.43mg, 6.23mmol ) in anhydrous
CH2Cl2 (20 mL) was stirred at room temperature. Acryloylchloride (563.85mg ,6.23mmol) was added drop wise.
The reactionmixture was stirred at r.t. for 2h. After the reaction is completed, The resulting mixture was diluted
with water (20 mL) and extracted with CH2Cl2 (20 mL × 3), dried (MgSO4), and concentrated to afford acrude
product. The crude product was purified by chromatography onsilica gel with petroleum ether/ethylacetate to afford
17 (970mg, 83.44%).
12

4.15. General Synthesis of (R)-N-(2-(6-oxo-3,6-dihydro-2H-pyran-2-yl)ethyl) benzamide(18a) Compound 21a
(2.49g, 6.6mmol) was dissolved in 25 mL toluene in a flask. Subsequently, the p-TSA (56.79mg, 0.33mmol) was
added. The reaction was stirred at 110℃ for 6 hours, reflux. The reaction mixture was filtered and the toluene was
removed by rotaryevaporation. The resulting mixture was diluted with water (20 mL) and extracted with
ethylacetate (20 mL × 3). The combined organic phases were dried with Na2SO4, concentrated to afford a crude
product. The crude product was purified by chromatography on silica gel with petroleum ether/acetone (2:1) to
1
afford 18a as a white acicular crystal (650 mg, 40.17%).m.p. 79.1~80.7℃. H NMR (400 MHz, CDCl3) δ 7.86 –
7.76 (m, 2H),7.57 – 7.38 (m, 3H), 6.98 – 6.87 (m, 1H), 6.80 (s, 1H), 6.11 – 5.95 (m, 1H), 4.69 – 4.52 (m, 1H), 3.78
– 3.58 (m, 2H), 2.46 – 2.37 (m, 2H), 2.19 – 2.09 (m, 1H), 2.06 – 1.95 (m, 1H).13C NMR (100 MHz, CDCl3) δ
145.28, 131.54, 128.60, 126.91, 121.22, 76.67, 36.55, 34.35, 29.38.
4.15.1. Synthesis of (R)-4-fluoro-N-(2-(6-oxo-3,6-dihydro-2H-pyran-2-yl)ethyl)benzamide (18b) Compound 21b
(1.53 g, 3.87 mmol) was converted to18b(185 mg, 18.16%) as a white acicular crystalby the same procedure as
described for compound 18a.m.p. 128.2~129.3℃. 1H NMR (400 MHz, DMSO-d6) δ 8.57 (t, J = 5.6 Hz, 1H), 8.01
– 7.83 (m, 2H), 7.29 (t, J = 8.9 Hz, 2H), 7.12 – 6.97 (m, 1H), 5.95 (dd, J = 9.8, 1.8 Hz, 1H), 4.58 – 4.48(m, 1H),
3.54 – 3.35 (m, 2H), 2.48 – 2.26 (m, 2H), 1.99 – 1.81 (m, 2H).13C NMR (100 MHz, CDCl3) δ 166.60, 164.72(d, J =
250 Hz), 164.01, 145.45, 130.41 (d, J = 2.8 Hz ), 129.33 (d, J = 8.9 Hz), 121.13, 115.55 (d, J = 21.8Hz), 76.78 ,
36.62, 34.29, 29.37.HRMS: calcd forC14H15FNO3 [M+Na]+ m/z: 264.1036, found: 264.1036.
4.15.2. Synthesis of (R)-N-(2-(6-oxo-3,6-dihydro-2H-pyran-2-yl)ethyl) cinnamamide(18c) Compound 21c (1.79 g,
4.44 mmol) was converted to 18c(265 mg, 22.02%) as a white acicular crystalby the same procedure as described
for compound 18a.m.p. 113.1~114.2℃. 1H NMR (400 MHz, DMSO-d6) δ 8.22 (t, J = 5.6 Hz, 1H), 7.56 (d, J = 6.8
Hz, 2H), 7.50 – 7.29 (m, 4H), 7.15 – 6.97 (m, 1H), 6.60 (d, J = 15.8 Hz, 1H), 5.95 (dd, J = 9.8, 1.8 Hz, 1H), 4.61 –
4.40 (m, 1H), 3.46 – 3.34 (m, 1H), 3.30 – 3.17 (m, 1H), 2.48 – 2.42 (m, 1H), 2.40 – 2.24 (m, 1H), 1.97 – 1.75 (m,
2H).13C NMR (100 MHz, DMSO-d6) δ 164.96, 163.71, 147.09, 138.59, 134.83, 129.39, 128.88, 127.45, 122.06,
120.15, 75.45, 34.63, 34.18, 28.69.HRMS: calcd forC16H17NNaO3 [M+Na]+ m/z: 294.1106, found: 294.1105.
4.15.3. Synthesis of (R)-5-chloro-N-(2-(6-oxo-3,6-dihydro-2H-pyran-2-yl)ethyl)thiophene-2-carboxamide (18d)
Compound 21d (1.60 g, 3.83 mmol) was converted to 18d(305 mg, 27.88%) as a white acicular crystalby the same
procedure as described for compound 18a.m.p. 145.0~145.7℃. 1H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 4.0 Hz,
1H), 6.95 – 6.91 (m, 1H), 6.89 (d, J = 4.0 Hz, 1H), 6.70 (br, 1H), 6.08 – 5.99 (m, 1H), 4.66 – 4.52 (m, 1H), 3.75 –
3.64 (m, 1H), 3.63 – 3.50 (m, 1H), 2.47 – 2.36 (m, 2H), 2.15 – 1.94 (m, 2H).13C NMR (100 MHz, CDCl3) δ 163.80,
161.17, 145.31, 137.53, 135.35, 127.11, 127.04, 121.18, 76.86, 36.65, 34.19, 29.37.HRMS: calcd for
C12H12ClNNaO3S [M+Na]+ m/z: 308.0124, found: 308.0123.
4.16. General Synthesis of N-(2-((2S)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)benzamide (19a)
Compound 21a (2.49g, 6.6mmol) was dissolved in 25 mL toluene in a flask. Subsequently, the p-TSA(56.79mg,
0.33mmol) was added. The reaction was stirred at 110℃ for 6 hours, reflux. The reaction mixture was filtered to
1
afford 19a as a white acicular crystal (728 mg, 41.92%) directly.m.p. 178.8~179.9℃. H NMR (400 MHz,
DMSO-d6) δ 8.56 (t, J = 5.3 Hz, 1H), 7.85 (d, J = 7.1 Hz, 2H), 7.59 – 7.41 (m, 3H), 5.21 (br, 1H), 4.80 – 4.59 (m,
1H), 4.22 – 4.07 (m, 1H), 3.56 – 3.26 (m, 2H), 2.66 (dd, J = 17.3, 4.5 Hz, 1H), 2.41 (dd, J = 17.3, 1.7 Hz, 1H), 1.94
– 1.80 (m, 3H), 1.79 – 1.65 (m, 1H).13C NMR (101 MHz, DMSO-d6) δ 170.12, 166.17, 134.47, 131.04, 128.20,
127.09, 73.51, 61.23, 38.53, 35.44, 35.07, 34.98.HRMS: calcd for C14H17NNaO4 [M+Na]+ m/z: 286.1055, found
m/z: 286.1058.
4.16.1. Synthesis of 4-fluoro-N-(2-((2S)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)benzamide (19b)
Compound 21b (1.53 g, 3.87 mmol) was converted to 19b(810 mg, 74.43%) as a white acicular crystalby the same
procedure as described for compound 19a.m.p. 182.7~183.6℃. 1H NMR (400 MHz, DMSO-d6) δ 8.66 – 8.50 (m,
1H), 7.92 (dd, J = 8.7, 5.6 Hz, 2H), 7.29 (t, J = 8.8 Hz, 2H), 5.20 (br, 1H), 4.82 – 4.58 (m, 1H), 4.22 – 4.01 (m, 1H),
13

3.53 – 3.38 (m, 2H), 2.65 (dd, J = 17.3, 4.5 Hz, 1H), 2.40 (dd, J = 17.3, 1.7 Hz, 1H), 1.84 (dd, J = 13.5, 6.5 Hz, 3H),
1.77 – 1.64 (m, 1H).13C NMR (100 MHz, DMSO-d6) δ 170.11, 165.10, 163.75 (d, J = 248.2 Hz), 130.92 (d, J = 2.8
Hz), 129.72 (d, J = 9.0 Hz), 115.11 (d, J = 21.7 Hz), 73.47, 61.22, 38.52, 35.48, 35.06, 34.95.HRMS: calcd
forC14H16FNNaO4 [M+Na]+ m/z: 304.0961, found: 304.0961.
4.16.2. Synthesis of N-(2-((2S)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)ethyl)cinnamamide (19c) Compound
21c(1.79 g, 4.44 mmol) was converted to 19c(810 mg, 63.11%) as a white acicular crystalby the same procedure as
described for compound 19a.m.p. 171.6~172.3℃. 1H NMR (400 MHz, DMSO-d6) δ 8.23 (t, J = 5.3 Hz, 1H), 7.56
(d, J = 7.2 Hz, 2H), 7.49 – 7.30 (m, 4H), 6.62 (d, J = 15.8 Hz, 1H), 5.21 (br, 1H), 4.78 – 4.59 (m, 1H), 4.13 (s, 1H),
3.46 – 3.35 (m, 1H), 3.29 – 3.17 (m, 1H), 2.65 (dd, J = 17.3, 4.4 Hz, 1H), 2.40 (dd, J = 17.3, 1.2 Hz, 1H), 1.95 –
1.62 (m, 4H).13C NMR (100 MHz, DMSO-d6) δ 170.11, 164.93, 138.55, 134.84, 129.38, 128.89, 127.45, 122.10,
73.33, 61.23, 40.08, 38.52, 35.02, 34.84.HRMS: calcd forC16H19NNaO4 [M+Na]+ m/z: 312.1212, found: 312.1210.
4.16.3. Synthesis of 5-chloro-N-(2-((2S)-4-hydroxy-6- oxotetrahydro-2H-pyran-2-yl)ethyl)thiophene- 2-
carboxamide(19d) Compound 21d (1.60 g, 3.83 mmol) was converted to 19d(650 mg, 55.90%) as a white acicular
crystalby the same procedure as described for compound 19a.m.p. 172.4~173.1℃. 1H NMR (400 MHz, DMSO-d6)
δ 8.67 (t, J = 5.5 Hz, 1H), 7.62 (d, J = 4.1 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 5.19 (br, 1H), 4.79 – 4.55 (m, 1H),
4.20 – 4.05 (m, 1H), 3.48 – 3.37 (m, 1H), 3.33 – 3.19 (m, 1H), 2.65 (dd, J = 17.3, 4.5 Hz, 1H), 2.39 (dd, J = 16.5,
3.9 Hz, 1H), 1.91 – 1.77 (m, 3H), 1.75 – 1.61 (m, 1H).13C NMR (100 MHz, DMSO-d6) δ 170.03, 160.09, 139.15,
132.71, 127.95, 127.75, 73.33, 61.23, 38.52, 35.32, 35.05, 34.95.HRMS: calcd for C12H14ClNNaO4S [M+Na]+ m/z:
326.0230, found: 326.0231.
4.17. General Synthesis Procedure for analogues oftert-butyl 2-((4S,6S)-6-(2-benzamidoethyl)-2, 2-dimethyl-1,3-
dioxan-4-yl) acetate(21) A solution of compound 20(1 equvi), anhydrousMgSO4 (2 equvi), and TEA (1.5 equvi) in
anhydrous CH2Cl2 (20 mL) was stirred at room temperature. Acyl chloride analogue(1.5 equvi) was added drop
wise. The reactionmixture was stirred atrt for 2h. After the reaction is completed, The resulting mixture was diluted
with water (20 mL) and extracted with CH2Cl2 (20 mL × 3), dried (MgSO4), and concentrated to afford acrude
product. The crude product was purified by chromatography onsilica gel with petroleum ether/ethylacetate to afford
21.
5. Material and Methods
5.1. Reagent and antibodies
Fetal bovine serum (FBS), Roswell Park MemorialInstitute 1640 (RPMI-1640) medium, and
penicillin-streptomycin were purchased from HyClone (Victoria,Australia).DAPI,MTT,JC-1 was purchased from
Sigma-Aldrich (St.Louis,MO).MG132and bortezomibwere purchased fromMCE (MedChem Express, New Jersey,
USA). The FITC/Annexin V Apoptosis DetectionKit was purchased from BestBio (Shanghai, China). The Nuclear
and Cytoplasmic Protein Extraction Kit was purchased from Beyotime (Shanghai, China). The ECL Western
blotting kit was purchased from ThermoFisher(Waltham,MA).Antibodies against CRM1, Ranbp1, p53 andCleaved
PARP , were purchased form Cell Signaling Technology (Danvers, MA). Antibodies against Bcl-xl,
PARP,LaminB1, Tubulin, Actin, Cleaved caspase-3were purchased formSanta Cruz Biotechnology (Santa Cruz,
CA). Antibodies against Bax,Bcl-2, p53 siRNA,Cleaved caspase-9 were purchased form Abcam (Cambridge, MA).
5.2 Cell lines and culture
The human gastric cancer cell lines MGC803 and HGC27 and gastric epithelial cells (GES1) were purchased
from the American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI-1640 medium
supplemented with 10% heat-inactivated FBS, and were maintainedat 37°C in an incubator under an atmosphere
containing 5% CO2.
5.3. Cell viability assay
Cells were seeded into a 96-well plate at a density of 5×104/well,cultured overnight and then treated with
compound 1l. Then, 20μL of MTT solution (5 mg/mL) wasadded tothe cells in each well,and maintained at 37°C
14

for 4h.Finally, formazan crystals were solubilizedwith 150 μl of DMSO ,and the absorbance intensity wasanalyzed
with a96-well platereader at 490 nm. Every experiment wasduplicated three times .
5.4Immunofluorescence analysis
MGC803 cells were treated with compound 1l and fixed for 30 minutes with 4% paraformaldehyde. Next, cell
member were permeabilized by 0.1% Triton X-100 for 10 minutes. After blocked with 10% normal goat serum for
30 min, MGC803 cells incubation with relevant antibody.In order to locate the cell, DAPI was used for nuclei
labeling,slides were analyzedusing Nikon confocal microscopy.
5.5. Western blot
Cells were seeded ata 100-mm tissueculture plate and cultured for 24 h. After culturing, the cells treatedwith
different concentrations of compound 1l for 24 h. Cells then were collected and lysed withice-cold RIPA buffer
(Beyotime, P0013B), followed by centrifugation. Protein concentration was quantified and normalized using BCA
assay kit.Thetotal cellular protein extracts wereboiled with 5×loading buffer and resolved by 10% or 12%
SDS-PAGE and blotted onto PVDF membranes. The membranes were blockedwith5% skimmed milk in PBST for
2 hat 37 °C, they were wash and treated withovernight at 4°C with appropriate antibodies, followed by HRP
conjugated anti-mouse, anti-goat or anti-rabbitsecondary antibodies. followed by HRP conjugated anti-mouse,
anti-goat or anti-rabbitsecondary antibodiesfor 2 hat 37 °C. Finally, each protein were visualized by using an ECL
Western blotting kit.
5.6. Apoptosis analysis
Cells were seeded at 50%confluency in a 6-well plate and overnight incubation, followed by treatment
withdifferent concentration of compound 1l for 24 h. Cells then washedwith PBS twice,and collected by
centrifugation.After that, cells incubated with fluoresceinisothiocyanate (FITC) conjugated Annexin V and PI by
use of FITC Annexin V/PIapoptosis kit according to the manufacturer’s instructions .
5.7.Measurement of loss of mitochondrial membrane potential
Mitochondrial membrane potential was assed using 2.5 μMJC-1.In brief, cells were plated into a 6-well plate
at a density of 2×105 cells/well and cultured overnight. Cells exposed to compound 1lfor 1, 3, 6, 12 and 24 h.After
that,cells were collected and stained with 2.5 µM JC-1 at 37ºC for 10 min in darkness. The cells rinsed twice with
PBS, suspended, and measured using flow cytometer.
5.8.Statistical analysis
All data are presented as mean ± SD. Statistical evaluation of signifcant differences was performedusing
theunpaired Student’s t-test. *, ** and *** respectively represent p＜0.05, p<0.02 and p<0.001.
Acknowledgment
This study was supported by Program for Science and Henan Province Natural Science Foundation (No.
182300410367 for Haiwei Xu), the Scientific Innovation Talent Award from the Department of Education of Henan
Province (No. 15HASTIT036 for Cheng-Yun Jin).
Appendix A. Supplementary data
The Supporting Information, NMR for compounds, is available free of charge on the ACS Publications website.
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Highlight：
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ovel small molecular CRM1 degrader with different skeleton from CBS9106.
CRM1 inhibitor or degrader induce the apoptosis in gastric carcinoma
elective inhibition for the proliferation of gastric cancer cell line MGC803, HGC27 comparing to Human
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Gastric Mucosal Epithelial Cell Line (GES1).
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Combination treatment of compound 1l and MG132 resulted in synergistic effects.

A low toxic CRM1 degrader: synthesis and anti-proliferation on MGC803
and HGC27
Hai-Wei Xu1, 2*, Shilong Jia1‡, Mengbo Liu1‡, Xiaobo Li1, Xia Meng1, Xinxin Wu1, Lu Yu1, Menglin Wang1,
Chengyun Jin1, 2*
1Key Laboratory of Advanced Technology for Drug Preparation，Ministry of Education, School of Pharmaceutical
Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China.
2Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou
University, No. 100, KeXueDaDao, Zhengzhou 450001, China
Corresponding author: E-mail address: xhwei01@126.com (H. Xu); cyjin@zzu.edu.cn(C.Jin)
‡These authors contributed equally (S. jia and M. Liu)
Abstract:
Chromosome region maintenance 1 (CRM1) is the sole nuclear exporter of several tumor suppressor, a growth
regulatory protein as an attractive cancer drug target. In the present work, a novel CRM1 degrader was discovered
from newly synthesized α, β-unsaturated-δ-lactone based on a natural product Goniothalamin. It induces apoptosis
of both MGC803 and HGC27 cell lines via degrading CRM1. Selective inhibition was observed for the
proliferation of gastric cancer cell lines MGC803, HGC27 comparing to Human Gastric Mucosal Epithelial Cell
Line (GES1). For the first time, CRM1 inhibitor or degrader inducing apoptosis in gastric carcinoma was
investigated.
Keywords: CRM1/XPO1; synthesis; gastric cancer; degrader; structure-activity relationship
1. Introduction
Chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1), is the protein transporter. It is
responsible for the nucleocytoplasmic shuttling of most of the tumor suppressor proteins (TSP) and growth
regulatory factors(GRP), which belongs to the karyopherin β super family of transport receptors[1, 2]. CRM1, the
sole nuclear exporter of TSP, GRP including p53, Rb1 and so on, is an attractive cancer drug target[3]. Currently, an
increasing number of drug-like compounds that target CRM1 have been isolated or synthesized such as Leptomycin
B (LMB)[4, 5], KPT-330 (selinexor)[6, 7], KPT-335(verdinexor)[8, 9], KPT-185[9, 10], KPT-276[11, 12], KPT-251[13]
,
S109[14] and CBS9106[15]. Some of them have been investigated in clinical trials as a single agent or in combination
with bortezomib, selinexor, or dexamethasone[16], while CRM1 inhibitors have not demonstrated adequate potency
in the clinical settings. Among them, CBS9106 is a novel reversible CRM1 inhibitor with unique CRM1 degrading
activity[15] and it is the only reported CRM1 degrader. Therefore it is needed to discover and research new CRM1
inhibitor or degrader.
Gastric cancer (GC) is a highly aggressive malignant tumor, especially in East Asia such as China, South Korea and
Japan. Its high mortality rate prompts the urgent need for novel therapeutic agents[17]. In our screening of
therapeutic drugs against GC, some synthesized compounds showed apoptosis activity on MGC803. Among them
compound ll led to the downregulation of CRM1 in a dose dependent manner in MGC803. The downregulation of
CRM1 led by compound 1l was for the degradation of CRM1 through proteasome pathway. It was approved to be a
novel CRM1 degrader with a different skeleton of the first degrader CBS9106[15], had selective anti-proliferation
and induced apoptosis activity on MGC803, HGC27 cell lines comparing with Human Gastric Mucosal Epithelial
Cell Line (GES1).
1

2. Results and Discussion
2.1. Chemistry
Natural products LMB, Goniothalamin and their analogues showed CRM1 inhibition and anti-proliferation
activity on cancer cell lines. They were reported to bind to CRM1 through the α, β-unsaturated lactone moiety[18, 19]
.
Based on the pharmacophore of α, β-unsaturated lactone, a series of compounds with various linkers and aryl
groups were designed and synthesized to investigate the SAR for their anti-proliferation on MGC803.
a: R = 2-OEt; b: R = 2-OMe; c: R = 2-Bn; d: R = 4-SMe; e: R = 4-Et ; f: R = 4-CH2(CH2)2CH3;
g: R = 4-Cyclohexyl; h: R = 4-CH3OCH2CH2; i: R = 4-CF3O ; j: R = 4-PhCH2O; k: R = 3-(CH3CH2)2N ;
l: R = 2-Isopropyl-5-Me ; m: R = [2, 3]Ph; n: R = [3, 4]dioxole.
Scheme 1 Synthesis of (R)-6-(phenyloxidemethyl)-5, 6-dihydro-2H-pyran-2-ones (1a-1n)
Compounds 1a ~ 1n with phenoxymethyl groups were synthesized shown in Scheme 1. They were prepared
from key intermediates (6a ~ 6n) via olefin metathesis reaction catalyzed by grubbs-2. Compounds 6a ~ 6n were
obtained by the acylation of compounds 5a ~ 5n with acryloyl chloride, which were synthesized by the reaction of
compounds 4a-4n and vinylmagnesium chloride. Compounds 4a-4n was prepared by the reaction of substituted
phenols (2a-2n) and S-epoxy chloropropane (3)[20]
.
Scheme 2 Synthesis of (R)-6-((1H-indol-1-yl)methyl)-5, 6-dihydro-2H-pyran-2-ones (7a ~ 7d)
Compounds 7a ~ 7d were synthesized in a similar way with compounds 1a ~ 1n，and the only difference was
in the first step where indol analogues instead of the phenyl derivatives were used as the start materials to react
with S-epoxy chloropropane (3) (Scheme 2). Compound 12 with phenyl sulfane was synthesized by the same
strategy, which was oxidized using mCPBA to yield compound 13 (Scheme 3).
2

Scheme 3 Synthesis of (R)-6-(((4-chlorophenyl)sulfinyl) methyl)-5, 6-dihydro-2H- pyran-2- ones (12, 13)
Compounds 18a-18d and 19a ~ 19d with a linker of 3 atoms length were synthesized from compound 20
which can be obtained commercially (Scheme 4). Compound 20 was transfer to compounds 21a-d by the acylation.
Compounds 18a-18d and 19a ~ 19d were prepared by an intramolecular esterification in toluene under refluxing in
the presence of p-TSA.
18, 19
Comp.
a
b
c
d
R
Scheme 4 Synthesis of compounds 18a-d and 19a-d
2.2. Biology
The anti-proliferation of all synthesized compounds were evaluated on MGC803 by MTT assay (Table 1).
Compounds (1a ~ 1n) containing benzyl groups and a two atoms length linker showed moderate anti-proliferation
activity on MGC-803 with IC50 of 2.3 - 47.7 μM. The most potent compound 1l had an IC50 of 2.3 μM. Compounds
7a ~ 7e with aniline or indole groups and compounds 18a ~ 18d with a linker of 3 atoms length linker led to
significantly decreasing of efficacy with IC50>50μM. Compounds 19a ~ 19d did not inhibit the proliferation of
MGC803. The SAR data clearly suggested that the benzyl groups and the two atoms length linker were very
important for their anti-proliferation on MGC803. The steric hindrance at the terminal of the linker and the more
atoms length linkers (compounds 7a-7d and 18a-18d) were not beneficial for efficacy. Thus compound 1l was
chosen for further study based on all the above data.
Table 1 the anti-proliferation of synthesized compounds on MGC803
Comp. MGC803/IC50(µM) Comp. MGC803/IC50(µM)
1a
1b
1c
1d
1e
1f
10.353±1.015
24.159±1.383
22.978±1.36
>50
7a
>50µM
7b
7c
25.620±1.409
>50µM
7d
7e
>50µM
12.425±1.094
16.950±1.229
10.593±1.025
>50µM
>50µM
13
>50µM
1g
1h
18a
18b
>50µM
>50µM
3

1i
8.489±0.929
35.439±1.549
>50µM
18c
18d
19a
19b
19c
19d
>50µM
>50µM
>50µM
>50µM
>50µM
>50µM
1j
1k
1l
2.314±0.364
47.687±1.678
11.976±1.078
24.803±1.395
1m
1n
5-Fu
The anti-proliferation activity of compound 1l (Fig 1A) was then evaluated on human gastric cancer cell lines.
Two gastric cancer cell lines (MGC803 and HGC27) and human gastric epithelial cells lines (GES1) were
incubated with it (24 h) at different concentrations, and then the effects of compound 1l on reducing cell viabilities
were assayed by MTT. As shown in Fig.1B, the viability of the gastric cancer cell lines decreased in a dose
dependent manner after treatment with compound 1l. MGC803 and HGC27 cells lines showed the sensitivity to
compound 1l in related to control treatment, causing 40 ~ 60% viability reduction at 10 μM for 24 h. However, it is
almost no toxicity to human gastric epithelial cells (GES1) (Fig. 1B). Taken together, these results suggested that
compound 1l has selective anti-proliferation on gastric cancer cell lines versus normal human gastric epithelial cell
lines.
B
A
Figure 1. the structure of compound 1l and its anti-proliferation activity on MGC803 and HGC27
2.2.1 Compound 1l induced apoptosis by the activation of Bcl-2 family proteins in gastric cancer cell lines
Further experiments were conducted to determine whether the anti-proliferation of compound 1l on the gastric
cancer cell lines were the result of apoptotic cell death. The inducing apoptosis of compound 1l on MGC803 and
HGC27 were evaluated by Annexin V and PI (Propidium Iodide) staining. As shown in Fig.2A, the numbers of
Annexin V positive cells gradually increased in a dose dependent manner in these two cell lines. The results
exhibited that exposure to compound 1l for 24 h significantly upregulated the levels of p53 and apoptosis-related
proteins including cleaved PARP and cleaved caspase-3, whereas downregulated CRM1 in MGC803 and HGC27
cells were observed.
The MMP (ΔΨ) was measured by using JC-1 to confirm the function of mitochondrial in the induction of
apoptosis caused by compound 1l on MGC 803 and HGC27 cell lines. After treatment with compound 1l in a dose
dependent manner, MMP (ΔΨ) was decreased in MGC803 and HGC27 cell lines comparing with controls, as
shown in Fig.2B. The pro-apoptotic and anti-apoptotic members of the Bcl-2 super family play primary roles in
regulating mitochondrial associated apoptosis. After treatment with compound 1l, the levels of the pro-apoptotic
protein Bax was increased and the levels of the anti-apoptosis proteins such as Bcl-2 and Bcl-xL were decreased in
MGC803 and HGC27 cell ines comparing with controls (Fig. 2B). In addition, it was observed that compound 1l
increased the expression of cleaved caspase-3 and cleaved caspase-9 in MGC803 and HGC27 cells in comparison
with controls. These results indicated that the mitochondrial pathway is an important factor in compound 1l
mediated apoptosis in gastric cancer cells.
4

B
A
Figure 2. Apoptosis induced by compound 1l involves Mitochondrial pathway in gastric cancer cell lines.
(A) Cell apoptosis of MGC803 and HGC27 was detected by flow cytometry after treatment by compound 1l for 24
h. The protein levels of CRM1, PARP, Cleaved PARP, Cleaved caspase-3 and p53 were detected at different
concentrations by Western blot in MGC803 cells. (B) MMP (ΔΨ) was decreased by compound 1l treatment.
Membrane potential was measured by JC-1 dye retention using Flow Cytometry. The protein levels of Bcl-2,
Bcl-xL, Bax and Cleaved caspase-9 were determined by Western blot after compound 1l treatment.
2.2.2 Compound 1l-mediated CRM1 depletion depends on ubiquitin-proteasome pathway
C
A
B
Figure 3. Mitochondrial p53 pathway is involved in the inducing apoptosis of compound 1l in MGC803 cells
and compound 1l induces CRM1 protein degradation via the ubiquitin-proteasome pathway.
(A)CRM1 bound with p53 detected by immunoprecipitation assay in MGC803 cell lines after treatment with
compound 1l at 10 µM at various time points. Accumulation of Ranbp1 or p53 in nucleus was observed in
MGC803 cells with treatment of compound 1l at 10 μM for 6 h. The treated and untreated MGC803 cells with
compound 1l at 10 µM for 0 and 6 h are stained with Ranbp1 antibody (Green) and DAPI (Blue) and analyzed by
confocal microscopy. Westernblot analysis for expression of Ranbp1, p53 and CRM1 in the nucleus and cytoplasm.
(B) MTT assay detected the effect of siRNA targeting to p53 on the survival rate of MGC803 cells, *P<0.05 vs
control group. (C) MGC803 cells were treated with compound 1l (10 μM) in the presence or absence of MG132
(0.05 μM ), MG132 (0.1 μM ) and bortezomib (2.5nM) for 24 h. Westernblot was used to detect the expression
levels of CRM1.
5

The levels of CRM1 protein in MGC803 and HGC27 cell lines with treatment of compound 1l were analyzed.
The results showed that compound 11 decreased CRM1 protein level in a dose dependent manner and the levels of
the p53 were increased in MGC803 and HGC27 cell lines comparing with controls (Fig. 2A), which suggested that
compound 1l was possible a CRM1 degrader. Therefore, we detected whether compound 1l could regulate the
expression of p53 by acting on the CRM1 protein. Immuno precipitation indicated that CRM1 bound with p53
dramatically decreased at 6 h (Fig. 3A). As revealed by immunofluorescence staining, notable nuclear
accumulation of Ranbp1 and p53 occurred at 6 h after compound 1l treatment. Whereas the cytoplasmic level of
Ranbp1 and CRM1 decreased, as shown in Fig. 3A. In addition, the utilization of p53 siRNA with MGC803 cell
lines, the cell death led by compound 1l were decreased evidently, as shown in Fig. 3B. The results showed that
p53 is required for compound 1l mediated apoptosis.
The degradation of CRM1 proceeds by the ubiquitin-proteasome pathway. To investigate this, MG132, a
proteasome inhibitor, was used to block the CRM1 degradation led by compound 1l. MGC803 and HGC27 cells
were treated with compound 1l (5 μM) for 24h in the presence or absence of MG132 (0.05 μM). Reduced CRM1
protein levels were observed in compound 1l-treated cell lines. In contrast, in the presence of MG132, depletion of
CRM1 protein by compound 1l was almost completely abolished (Fig. 3C). Similar results were obtained with the
using of bortezomib, another proteasome inhibitor. These data demonstrated that compound 1l mediated CRM1
depletion depends on ubiquitin-proteasome pathway.
2.2.3. Compound 1l synergize with MG132 to increase the apoptosis of MGC803 and HGC27 cell lines
Some of CRM1 inhibitors have been investigated in clinical trials, while they have not demonstrated adequate
potency in the clinical settings as a single agent and they are more potency in combination with proteasome
inhibitor such as bortezomib[16]. In Fig. 4A, pretreatment with MG132 significantly increased apoptosis in
MGC803 and HGC27 cells, whereas treatment with compound 1l or MG132 alone decreased the activity of gastric
cancer cells only slightly or not at all. In addition, the data showed that pretreatment with MG132 increased
expression of cleaved PARP, cleaved caspase-3, p53 and Bax in two gastric cancer cells (Fig.4A). To investigate the
role of p53 in MG132 induced enhancement of compound 1l mediated apoptosis. Fig. 4B revealed that p53
translocated in cytoplasm and bound with anti-apoptotic Bcl family proteins including Bcl-2, Bcl-xl. These data
indicated that, combination of compound 1l with MG132 increased apoptosis via accumulate p53 in the
cytoplasmic, p53 translocated in cytoplasm and bound with Bcl-2 and Bcl-xl, which increased Bax expression and
induced apoptosis, despite degradation of CRM1 protein by compound 1l was completely blocked.
A
B
Figure 4. Compound 1l work synergistically with MG132increased apoptosis
(A) MGC803 and HGC27 cells were pretreated with/without 0.05 μM of MG132 for 1 h before exposure to 10 μM
of compound 1l for 24 h. Cell viability was determined by MTT assays. ***P < 0.01 vs. untreated group.MGC803
6

and HGC27 cells were pretreated with/without 0.05 μM of MG132 for 1 h before exposure to 10 μM of compound
1lfor 24 h. Cells apoptosis of MGC803 and HGC27 was detected by flow cytometry. Western blot assay was used
to detect the expression levels of CRM1. Cleaved PARP, Cleaved caspase-3, p53 and Bax after pretreatment with
the MG132 (0.05 μM) in MGC803 and HGC27 cells for 1 hour, followed by 24 h of compound 1l(10 μM)
incubation. (B) MGC803 cells were pretreated with 0.05 μM of MG132 for 1 h before exposure to 10 μM of
compound 1lfor 24 h. The indicated proteins were detected by immunoprecipitation with an antibody for
p53.MGC803 cells were pretreated with/without 0.05 μM of MG132 for 1 h before exposure to 10 μM of
compound 1l for 6 h. p53 in the nucleus or cytoplasm was detected by Immunofluorescence analysis. The treated
and untreated samples are stained with p53antibody (Green) and DAPI (Blue) and analyzed by confocal
microscopy.
3. Discussion
Gastric cancer is one of highly aggressive malignant tumor, while impactful chemotherapies for that are
limited[21]. Therefore, development of the novel therapeutic drugs is an attractive topic. The over expression of
CRM1 has been detected in gastric cancer and caused the dysfunction of cell fate controls[22], which became a
promising therapeutic target for cancer[23]. In this research, compound 1l showed anti-proliferative activity via the
degradation of CRM1 in gastric cancer cell lines in vitro. For the first time, a new degradation of CRM1, compound
1l suppresses proliferation and induces apoptosis on MGC803 and HGC27 cell lines in a dose dependent manner
through mitochondrial pathway.
Up to present, CRM1 mediates the transport of more than 230 proteins, many of which are tumor suppressors
[24]. Therefore, the inhibition or degradation of CRM1 induced the accumulation of tumor suppressor proteins in the
nucleus and reduced cytoplasmic degradation of these proteins. This has been thought to be as significant
mechanism of the anti-gastric cancer activity of CRM1 inhibitor or degrader. In our study, the expression levels of
CRM1 was decreased and p53 accumulated in the nuclear of MGC803 and HGC27 cell lines treated by compound
1l. In addition, the mitochondrial apoptotic pathway induced by compound 1l was decreased evidently in MGC803
cell lines with knockdown of p53 by specific siRNA. The result suggest that the anti-proliferation activity of
compound 1l is associate with the accumulation of the tumor suppressor proteins p53 in the nuclear. The
ubiquitin-proteasome system (UPS) and autophagy are major intracellular protein degradation systems[25]. The UPS
pathway is essential to the cell cycle, transcription and cell survival. Numerous studies have revealed that blockage
of proteasome mediated protein degradative pathway stabilized the tumor suppressor p53 and the proapoptotic
proteins Bax[16, 26]. MG132, a 26S proteasome inhibitor, whereas inhibition of the 26S proteasome lead to
accumulation of non-degraded ubiquitinated proteins in the cytoplasmic. In our study, it was observed that the
degradation of CRM1 protein induced by compound 1l was almost completely blocked by co-incubation with the
proteasome inhibitor MG132 or bortezomib. The combination treatment with MG132 and compound 1l enhanced
expression of p53, cleaved PARP, cleaved caspase-3 and Bax, and induced more apoptosis in two gastric cancer
cells compared to cell lines treated with compound 1l or proteasome inhibitors alone. p53 transcription-independent
apoptosis is initiated by interacts with anti-apoptotic Bcl family proteins including Bcl-2, Bcl-xl[27]. In this study,
p53 translocated in cytoplasm and bound with Bcl-2 and Bcl-xl by pretreatment with MG132, which indicated that
the treatment by combination with compound 1l and proteasome inhibitors significantly increased apoptosis via
accumulate p53 in the cytoplasmic, despite degradation of CRM1 protein by compound 1l was completely blocked.
A critical component of any degrader/drug is permeability. Passive diffusion permeability is the most
important permeability for small molecules. Cell permeability of a drug depends on its molecular weight, polar
surface area and clogP. The permeability of the lipophilic molecules is better than that of polar molecules. Neutral
molecules are much more permeable than their charged forms. Compound 1l is a neutral and lipophilic molecule
with clogP of 3.32 and polar surface area of 35.5. Its molecular weight is 260.4. The small molecule structure and
the reasonable clogP suggest that compound 1l has a good permeability possibly.
In summary, the results in this work show that compound 1l is a novel CRM1 degrader that has major
cytotoxicity in gastric cancer cells and have little toxicity in human gastric epithelial cells. It is a potential
pro-apoptotic agent for prevention and treatment of gastric cancer.
4. Experimental section
4.1. General information
Reagents and solvents were purchased from Bide Pharmatech Ltd, Aladdin, Sinopharm Chemical Reagent Co,
1
Ltd. with purities of at least 97%. H NMR (400 MHz) and 13C NMR (100 MHz) spectrawere recorded with a
Bruker spectrometer. All reactions were monitored by thin-layer chromatography (TLC) on 25.4 mm× 76.2 mm
silica gel plates (GF-254) and UPLC-Mass on Waters ACQUITY UPLC H-Class or Q-Tof Micro HRMS on waters.
Melting points were determined on a Beijing Keyi XT4A apparatus. The silica gel used for column
chromatography was 200～300 mesh or recrystallization with solvents specified in the corresponding experiments.
7

4.2. General synthetic procedure for the compounds
4.2.1. General synthesis of (R)-6-((2-ethoxyphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1a)
Grubbs-2 (188 mg, 0.221 mmol) was dissolved in 20 mL anhydrous DCM in an oven dried flask. Subsequently,
compound 6a (612 mg, 2.21 mmol) was dissolved in 2 mL DCM and added drop wise under an nitrogen
atmosphere. The reaction was stirred for 15 hours at 50℃ ~ 60℃. The crude product was purified by
chromatography on silica gel with petroleumether/ethylacetate (10:1) to afford 1a as a white solid (173 mg,
31.46%). 1H NMR (400 MHz, CDCl3) δ 7.06 – 6.84 (m, 5H), 6.14 – 6.04 (m, 1H), 4.95 – 4.75 (m, 1H), 4.30 (dd, J
= 10.2, 4.5 Hz, 1H ), 4.21 (dd, J = 10.2, 6.2 Hz, 1H), 4.19 – 4.00 (m, 2H), 2.79 – 2.58 (m, 2H), 1.45(t, J = 7.0 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 163.48, 148.11, 144.86, 122.71, 121.34, 121.09, 116.01, 113.97, 75.69, 70.53,
64.49, 26.41, 14.96.
4.2.2. Synthesis of (R)-6-((2-methoxyphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1b)
Compound 6b (520 mg, 1.98 mmol) was converted to 1b (145 mg, 31.22%) as a white solid by the same
procedure as described for compound 1a. m.p. 90.2 ~ 91.6℃; 1H NMR (400 MHz, CDCl3) δ 7.06 – 6.84 (m, 5H),
6.10 – 6.04 (m, 1H), 4.88 – 4.80 (m, 1H), 4.28 (dd, J = 10.2, 4.6 Hz, 1H), 4.19 (dd, J = 10.2, 6.2 Hz, 1H), 3.85 (s,
3H), 2.75 – 2.53 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 163.42, 149.95, 147.78, 144.81, 122.48, 121.36, 121.00,
115.03, 112.31, 75.62, 70.19, 55.93, 26.45. HRMS: calcd for C13H14NaO4 [M+Na]+m/z: 257.0790, found:
257.0792.
4.2.3. Synthesis of (R)-6-((2-benzylphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1c)
Compound 6c (550 mg, 1.71 mmol) was converted to 1c (220mg, 43.81%) as a white solid by the same procedure
1
as described for compound 1a. m.p. 86.5 ~ 87.2℃; H NMR (400 MHz, DMSO-d6) δ 7.27 – 7.21m, 4H), 7.21 –
7.13 (m, 3H), 7.12 – 7.04 (m, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.91 (t, J = 7.4 Hz, 1H), 6.02-5.97 (m, 1H), 4.85 – 4.77
(m, 1H), 4.25 – 4.14 (m, 2H), 3.91 (s, 2H), 2.50 – 2.44 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.26 , 155.68,
146.80, 140.85, 130.28, 129.45, 128.65, 128.16 , 127.58, 125.71, 120.82, 119.95, 111.85, 75.63, 68.57, 35.45, 25.10.
HRMS: calcd for C19H18NaO3 [M+Na]+ m/z: 317.1154, found: 317.1153.
4.2.4. Synthesis of (R)-6-((4-(methylthio)phenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1d)
Compound 6d (500 mg, 1.80 mmol) was converted to 1d (200mg, 44.32%) as a gray solid by the same procedure
as described for compound 1a. m.p. 100.5 ~ 101.7℃; 1H NMR (400 MHz, CDCl3) δ 7.26 (s, 2H), 7.02 – 6.91 (m,
1H), 6.86 (d, J = 7.6 Hz, 2H), 6.08 (d, J = 9.6 Hz, 1H), 4.84 – 4.74 (m, 1H), 4.22 – 4.08 (m, 2H), 2.77 – 2.50 (m,
2H), 2.45 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.27, 156.63, 144.61, 129.99, 129.89, 121.38, 115.30, 75.47,
68.72, 26.24, 17.77. HRMS: calcd for C13H14NaO3S [M+Na]+m/z: 273.0561, found: 273.0561.
4.2.5. Synthesis of (R)-6-((4-ethylphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1e)
Compound 6e (560 mg, 2.15 mmol) was converted to 1e (213 mg, 42.63%) as a white solid by the same procedure
as described for compound 1a. m.p. 65.1 ~ 66.7℃; 1H NMR (400 MHz, CDCl3) δ 7.12 (d, J = 8.5 Hz, 2H), 6.98 –
6.90 (m, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.07 (dd, J = 9.8, 1.7 Hz, 1H), 4.83 – 4.73 (m, 1H), 4.19 (dd, J = 10.0, 4.5
Hz, 1H), 4.13 (dd, J = 10.0, 5.6 Hz, 1H), 2.71 – 2.59 (m, 2H), 2.59 – 2.49 (m, 2H), 1.21 (t, J = 7.6 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ 163.43 , 156.21, 144.81, 137.30, 128.85, 121.30, 114.47, 75.60, 68.62, 27.99, 26.29,
15.87. HRMS: calcd for C14H16NaO3 [M+Na]+m/z: 255.0997, found: 255.0998.
4.2.6. Synthesis of (R)-6-((4-pentylphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1f)
Compound 6f (550 mg, 1.82 mmol) was converted to 1f (246 mg, 49.30%) as a white solid by the same procedure
as described for compound 1a. m.p. 68.0 ~ 69.1℃; 1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 8.5 Hz, 2H), 6.98 –
6.92 (m, 1H), 6.83 (d, J = 8.6 Hz, 2H), 6.14 – 6.02 (m, 1H), 4.85 – 4.75 (m, 1H), 4.19 (dd, J = 10.0, 4.5 Hz, 1H),
4.13 (dd, J = 10.0, 5.7 Hz, 1H), 2.72 – 2.50 (m, 4H), 1.62 – 1.56 (m, 2H), 1.39 – 1.24 (m, 4H), 0.89 (t, J = 6.9 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 156.15, 144.75, 135.97, 129.37, 121.32, 114.33, 77.33, 75.56, 68.55, 35.00,
31.43, 31.38, 26.31, 22.54, 14.05. HRMS: calcd for C17H22NaO3 [M+Na]+m/z: 297.1467, found: 297.1467.
8

4.2.7. Synthesis of (R)-6-((4-cyclohexylphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1g)
Compound 6g (470 mg, 1.49 mmol) was converted to 1g (196 mg, 45.79%) as a white solid by the same
1
procedure as described for compound 1a. m.p. 85.5 ~ 86.3℃; H NMR (400 MHz, CDCl3) δ 7.13(d, 7.5Hz, 2H),
6.98 –6.89(m, 1H), 6.84 (d, J = 8.0 Hz, 2H), 6.07 (d, 9.7Hz, 1H), 4.85 – 4.75 (m, 1H), 4.25 – 4.07 (m, 2H), 4.13
(dd, J = 10.0, 5.7 Hz, 1H), 2.72 – 2.50 (m, 1H), 2.50 – 2.36 (m, 1H), 1.93 – 1.79 (m, 4H), 1.73 (d, J = 13.0 Hz, 1H),
1.46 – 1.30 (m, 4H), 1.30 – 1.15 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 163.37, 156.22, 144.71, 141.28, 127.76,
121.34, 114.36, 75.55, 68.56, 43.70, 34.68, 26.91, 26.33, 26.14. HRMS: calcd for C18H22NaO3 [M+Na]+ m/z:
309.1467, found: 309.1466.
4.2.8. Synthesis of (R)-6-((4-(2-methoxyethyl)phenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1h)
Compound 6h (556mg, 1.91 mmol) was converted to 1h (223 mg, 44.40%) as a white solid by the same procedure
as described for compound 1a. m.p. 47.3 ~ 48.1℃; 1H NMR (400 MHz, CDCl3) δ 7.14 (d, J = 8.5 Hz, 2H), 7.03 –
6.90 (m, 1H), 6.84 (d, J = 8.6 Hz, 2H), 6.06 (dd, J = 9.8, 1.7 Hz, 1H), 4.84 – 4.74(m, 1H), 4.18 (dd, J = 10.0, 4.6
Hz, 1H), 4.13 (dd, J = 10.0, 5.6 Hz, 1H), 3.56 (t, J = 7.0 Hz, 2H), 3.35 (s, 3H), 2.82 (t, J = 7.0 Hz, 2H), 2.70 – 2.59
(m, 1H), 2.58 – 2.48 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 163.40, 156.62, 144.83, 131.91, 129.86, 121.22,
114.48, 75.56, 73.72, 68.53, 58.64, 35.26, 26.21. HRMS: calcd for C15H18NaO4 [M+Na]+m/z: 285.1103, found:
285.1104.
4.2.9. Synthesis of (R)-6-((4-(trifluoromethoxy)phenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1i)
Compound 6i (570 mg, 1.80 mmol) was converted to 1i (250 mg, 48.13%) as a white solid by the same procedure
as described for compound 1a. m.p. 71.1 ~ 72.7℃; 1H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 8.7 Hz, 2H), 7.01 –
6.95 (m, 1H), 6.95 – 6.90 (m, 2H), 6.11 (dd, J = 9.4, 2.1 Hz, 1H), 4.88 – 4.78 (m, 1H), 4.26 – 4.13 (m, 2H), 2.75 –
2.64 (m, 1H), 2.63 – 2.50 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 163.22, 156.63, 144.59, 143.26, 122.57, 121.37,
119.24, 115.41, 75.39, 68.96, 26.15. HRMS: calcd for C13H11F3NaO4 [M+Na]+ m/z: 311.0507, found: 311.0506.
4.2.10. Synthesis of (R)-6-((4-(benzyloxy)phenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1j)
Compound 6j (530 mg, 1.57 mmol) was converted to 1j (210 mg, 43.20%) as a white solid by the same procedure
1
as described for compound 1a. m.p. 146.0 ~ 147.3℃; H NMR (400 MHz, CDCl3) δ 7.47 – 7.28 (m, 5H), 6.98 –
6.93 (m, 1H), 6.92 – 6.88 (m, 2H), 6.88 – 6.82 (m, 2H), 6.11 –6.04 (m, 1H), 5.02 (s, 2H), 4.83 – 4.73 (m, 1H), 4.16
(dd, J = 10.0, 4.5 Hz, 1H), 4.11 (dd, J = 10.0, 5.6 Hz, 1H), 2.71 – 2.59 (m, 1H), 2.58 – 2.48(m, 1H). 13C NMR (100
MHz, CDCl3) δ 163.42, 153.53, 152.53, 144.74, 137.14, 128.57, 127.94, 127.47, 121.34, 115.91, 115.64, 75.62,
70.66, 69.27, 29.70. HRMS: calcd for C19H18NaO4 [M+Na]+ m/z: 333.1103, found: 333.1102.
4.2.11. Synthesis of (R)-6-((3-(diethylamino)phenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1k)
Compound 6k (360 mg, 1.19 mmol) was converted to 1k (150 mg, 45.91%) as a white solid by the same procedure
as described for compound 1a. m.p. 50.2 ~ 51.6℃; 1H NMR (400 MHz, CDCl3) δ 7.11 (t, J = 8.2 Hz, 1H), 6.98 –
6.91 (m, 1H), 6.34 (dd, J = 8.4, 1.8 Hz, 1H), 6.26 – 6.15 (m, 2H), 6.11 – 6.04 (m, 1H), 4.85 – 4.75 (m, 1H), 4.21
(dd, J = 10.0, 4.5 Hz, 1H), 4.13 (dd, J = 10.0, 6.0 Hz, 1H), 3.33 (q, J = 7.1 Hz, 4H), 2.71 – 2.50 (m, 2H), 1.15 (t, J
= 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 163.46, 159.56, 144.79, 130.01, 121.35, 105.72, 100.50, 98.87,
75.63, 68.30, 44.36, 29.70, 26.41, 12.58.
4.2.12. Synthesis of (R)-6-((2-isopropyl-5-methylphenoxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1l)
Compound 6l (460 mg, 1.60 mmol) was converted to 1l (186 mg, 44.79%) as a colorless oily liquid by the same
procedure as described for compound 1a. 1H NMR (400 MHz, CDCl3) δ 7.11 (d, J = 7.7 Hz, 1H), 7.05 – 6.91 (m,
1H), 6.79 (d, J = 7.7 Hz, 1H), 6.65 (s, 1H), 6.09 (d, J = 11.4 Hz, 1H), 4.90 – 4.80 (m, 1H), 4.21 (dd, J = 9.9, 4.3 Hz,
1H), 4.16 (dd, J = 9.9, 5.7 Hz, 1H), 3.30 – 3.20 (m, 1H), 2.76 – 2.50 (m, 2H), 2.32 (s, 3H), 1.19 (d, J = 6.9 Hz, 6H).
13C NMR (100 MHz, CDCl3) δ 163.47, 155.02, 144.76, 136.49, 134.06, 126.06, 121.93, 121.33, 112.14, 75.63,
68.51, 26.54, 26.43, 22.76, 22.73, 21.31. HRMS: calcd for C16H20NaO3 [M+Na]+ m/z: 283.1310, found: 283.1311.
4.2.13. Synthesis of (R)-6-((naphthalen-1-yloxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1m)
9

Compound 6m (480 mg, 1.70 mmol) was converted to 1m (153 mg, 35.39%) as a white solid by the same
1
procedure as described for compound 1a. m.p. 87.0 ~ 88.1℃; H NMR (400 MHz, DMSO-d6) δ 8.33 – 8.10 (m,
1H), 7.93 –7.87 (m, 1H), 7.61 – 7.48 (m, 3H), 7.44 (t, J = 7.9 Hz, 1H), 7.20 – 7.13 (m, 1H), 7.03 (d, J = 7.5 Hz,
1H), 6.08 – 6.02 (m, 1H), 5.05 – 4.95 (m, 1H), 4.46 – 4.34 (m, 2H), 2.78 – 2.60 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 163.36, 153.53, 146.95, 133.99, 127.47, 126.53, 126.14, 125.43, 124.80, 121.48, 120.39, 120.03,
105.45, 75.68, 68.90, 25.30. HRMS: calcd for C16H14NaO3 [M+Na]+ m/z: 277.0841, found: 277.0842.
4.2.14. Synthesis of (R)-6-((benzo[d][1, 3]dioxol-5-yloxy)methyl)-5, 6-dihydro-2H-pyran-2-one (1n)
Compound 6n (530 mg, 1.92 mmol) was converted to 1n (198 mg, 41.58%) as a white solid by the same procedure
as described for compound 1a. m.p. 96.5 ~ 97.4℃; 1H NMR (400 MHz, CDCl3) δ 7.00 – 6.90 (m, 1H), 6.71 (d, J =
8.4 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 6.33 (dd, J = 8.5, 2.5 Hz, 1H), 6.10 – 6.06 (m, 1H), 5.93 (s, 2H), 4.83 – 4.72
(m, 1H), 4.13 (dd, J = 10.0, 4.6 Hz, 1H), 4.08 (dd, J = 10.0, 5.4 Hz, 1H), 2.70 – 2.46 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 163.36, 153.66, 148.35, 144.68, 142.25, 121.33, 107.95, 105.79, 101.28, 98.34, 75.53, 69.59, 26.22.
HRMS: calcd for C13H12NaO5 [M+Na]+ m/z: 271.0582, found: 271.0582.
4.3. General synthesis procedure for analogues of (R)-2-(phenoxymethyl)oxirane (4a ~ 4n)
Phenol derivatives 2a ~ 2n (1 equvi) and NaOH (3 equvi) was dissolved in 5 mL H2O in a flask. Subsequently, the
reaction mixture was stirred at room temperature for 30 min. The reaction system was added drop wise to a 50 mL
round bottom flask containing (S)-epoxy chloropropane (3 equvi) and tetrabutyl ammonium bromide (0.05 equvi).
The reaction mixture was stirred at room temperature. After the reaction completed, the resulting mixture was
diluted with water (10 mL) and extracted with ethylacetate (20 mL × 3). The combined organic phases were dried
with Na2SO4, concentrated to afford a crude product. The crude product was purified by chromatography on silica
gel with petroleum ether and ethylacetate to afford 4a ~ 4n.
4.4. General Synthesis Procedure for analogues of (R)-1-phenoxypent-4-en-2-ol (5a ~ 5n)
(R)-2-(phenoxymethyl) oxirane derivatives 4a ~ 4n (1 equvi) and CuI (0.2 equvi) was dissolved in 20 mL
anhydrous THF in a flask. The reaction mixture was stirred at -10 ~ 0℃ for 10 min and ethylene magnesium
chloride (1.5 equvi) was added drop wise under an argon atmosphere. After the reaction completed, the resulting
mixture was diluted with saturated ammonium chloride (50 mL) and the system was filtered with celite under
reduced pressure, and then evaporated and extracted with ethylacetate (15 mL × 3). The combined organic phases
were dried with Na2SO4, concentrated to afford a crude product. The crude product was purified by chromatography
on silica gel with petroleum ether and ethylacetate to afford 5a ~ 5n.
4.5. General Synthesis Procedure for analogues of (R)-1-phenoxypent-4-en-2-yl acrylate (6a ~ 6n)
A solution of 5a ~ 5n (1 equvi), anhydrous MgSO4 (2 equvi), and TEA (1.5 equvi) in Anhydrous CH2Cl2 (20 mL)
was stirred at room temperature. Acryloylchloride (1.5 equvi) was added drop wise. The reaction mixture was
stirred at room temperature for 2h. After the reaction completed, the resulting mixture was diluted with water (20
mL) and extracted with CH2Cl2 (20 mL × 3), dried (MgSO4), and concentrated to afford a crude product. The crude
product was purified by chromatography on silica gel with petroleum ether/ethylacetate to afford 6a ~ 6n.
4.6. General Synthesis of (R)-6-((1H-indol-1-yl)methyl)-5, 6-dihydro-2H-pyran-2-one (7a)
Grubbs-2 (126 mg, 0.219 mmol) was dissolved in 20 mL anhydrous DCM in adried flask. Subsequently, compound
11a (380 mg, 1.49 mmol) was dissolved in 2 mL DCM and added drop wise under a nitrogen atmosphere. The
reaction was stirred for 15 hours at 50℃ ~ 60℃. The crude product was purified by chromatography on silica gel
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with petroleum ether/ethylacetate (10:1) to afford 7a as a colorless oily liquid (143 mg, 42.28%). H NMR (400
MHz, CDCl3) δ 7.63 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.26 – 7.19 (m, 1H), 7.19 – 7.04 (m, 2H), 6.89 –
6.66 (m, 1H), 6.54 (d, J = 2.5 Hz, 1H), 5.99 (d, J = 9.7 Hz, 1H), 4.90 – 4.68 (m, 1H), 4.46 (dd, J = 15.1, 4.8 Hz,
1H), 4.39 (dd, J = 15.0, 5.5 Hz, 1H), 2.36 – 2.17 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 163.17 (s), 144.32,
136.28 128.66, 128.57, 122.02, 121.30, 121.22, 119.83, 109.06, 102.46, 76.35, 48.99, 26.88.
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