Synthesis of new chalcone-based homoserine lactones and their antiproliferative activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.12.014

Three series of new homoserine lactone analogs were efficiently synthesized starting from methionine and further evaluated for their antiproliferative activity against different cancer cell lines. Among these compounds, some of the chalcone containing compounds 6a-n showed acceptable antiproliferative activity against prostate cancer cells DU145 and PC-3 with the IC₅₀ values less than 10 μM. Compounds 6c, 6e and 6h inhibited growth of DU145 and PC-3 cells at low micromolar levels with the IC₅₀ values ranging from 3.0 to 5.0 μM, much more potent than natural OdDHL. Compound 6e concentration-dependently inhibited colony formation and cell migration of DU145 cells. A synergistic effect on the growth inhibition and the apoptosis of DU145 cells was observed when compound 6e was used in combination with TRAIL. OdDHL or 6e treatment concentration-dependently activated TRAIL death receptor DR5 which may account for the observed synergistic effect of 6e or OdDHL with TRAIL on the growth inhibition and cell apoptosis. Compound 6e also inhibited migration of DU145 cells in a time- and concentration-dependent manner. The data suggest that quorum sensing molecules OdDHL and 6e may improve the sensitivity of DU145 cells toward TRAIL via activating DR5, compound 6e may be used as a potential lead compound for developing new TRAIL receptor agonists.

Full text of DOI:10.1016/j.ejmech.2018.12.014

Accepted Manuscript
Synthesis of new chalcone-based homoserine lactones and their antiproliferative
activity evaluation
Bin Yu, Haoyue Liu, Xiaoyan Kong, Xinli Chen, Chunli Wu
PII:
S0223-5234(18)31051-1
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.014
EJMECH 10947
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2018
Revised Date: 4 December 2018
Accepted Date: 6 December 2018
Please cite this article as: B. Yu, H. Liu, X. Kong, X. Chen, C. Wu, Synthesis of new chalcone-based
homoserine lactones and their antiproliferative activity evaluation, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.014.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Three series of new homoserine lactones were synthesized from methionine, which
exhibited synergistic effects with TRAIL on the growth inhibition and cell apoptosis
of DU145 cells possibly via activating DR5.

ACCEPTED MANUSCRIPT
Synthesis of New Chalcone-Based Homoserine Lactones and Their
Antiproliferative Activity Evaluation
Bin Yu a, b, *, Haoyue Liu a, Xiaoyan Kong a, Xinli Chen a, Chunli Wu a, *
a
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR
China;
b State key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, Jiangsu, People’s Republic of China;
*Corresponding authors:
Dr. Bin Yu (zzuyubin@hotmail.com)
Dr. Chunli Wu (kedi2009@126.com);
Abstract: Three series of new homoserine lactone analogs were efficiently
synthesized starting from methionine and further evaluated for their antiproliferative
activity against different cancer cell lines. Among these compounds, some of the
chalcone containing compounds 6a-n showed acceptable antiproliferative activity
against prostate cancer cells DU145 and PC-3 with the IC50 values less than 10 µM.
Compounds 6c, 6e and 6h inhibited growth of DU145 and PC-3 cells at low
micromolar levels with the IC50 values ranging from 3.0 to 5.0 µM, much more potent
than natural OdDHL. Compound 6e concentration-dependently inhibited colony
formation and cell migration of DU145 cells. A synergistic effect on the growth
inhibition and the apoptosis of DU145 cells was observed when compound 6e was
used in combination with TRAIL. OdDHL or 6e treatment concentration-dependently
activated TRAIL death receptor DR5 which may account for the observed synergistic
effect of 6e or OdDHL with TRAIL on the growth inhibition and cell apoptosis.
Compound 6e also inhibited migration of DU145 cells in a time- and
concentration-dependent manner. The data suggest that quorum sensing molecules
OdDHL and 6e may improve the sensitivity of DU145 cells toward TRAIL via
activating DR5, compound 6e may be used as a potential lead compound for
developing new TRAIL receptor agonists.
Keywords: Homoserine lactones; AHLs; OdDHL; TRAIL; Death Receptor 5
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1. Introduction
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, also
known as Apo-2L and TNFSF10), a member of the TNF superfamily, was first
reported to be an apoptosis-inducing cytokine in 1995 and was subsequently proved to
be able to induce apoptosis of diverse cancer cell types in caspase-dependent manner
by cross-linking TRAIL death receptors DR4/TRAIL-R1 and DR5/TRAIL-R2 while
sparing vital normal cells [1-3]. TRAIL deficiency has been observed in a variety of
human cancers and is also associated with carcinogen-induced tumorigenesis and
metastasis in mice, and restoration of TRAIL can enhance sensitivity of tumor cells to
chemotherapeutic drugs [4-6]. Besides, treatment of recombinant TRAIL or TRAIL
receptor agonists effectively eliminates tumor cells in vivo. To date, several TRAIL
receptor agonistic antibodies (e.g. Dulanermin, MD5-1, etc.) are actively being
developed for clinical cancer therapy [7, 8]. However, TRAIL-induced cell death
involves complex extrinsic and intrinsic pathways, and some TRAIL receptor agonists
have basically failed to induce apoptosis because of the poor agonistic activity. All
these render tumor cells resistant to known TRAIL receptor agonists. Therefore, the
discovery of new agonists with increased bioactivity targeting TRAIL receptors or
novel chemotypes that can increase DR4/TRAIL-R1 and/or DR5/TRAIL-R2
expression has been highly pursued in last decades, aiming to overcome the resistance
observed. To date, some synthetic compounds including marketed drugs (e.g.
Bortezomib, etc.), natural products and nanoparticles being able to overcome
resistance have been identified to treat cancers [9-14]. Recently, Janda and co-workers
reported that N-acylhomoserine lactone (AHL) OdDHL (also known as C12), a
natural quorum sensing signaling molecule produced by Pseudomonas aeruginosa,
can result in a strong pro-apoptotic response to TNF or TRAIL and also lead to
MAPK p38 activation when combined with TRAIL, finally overcoming the TRAIL
resistance [15]. Natural products have long been recognized as rich sources to identify
new agents, over 100 natural products and analogs are currently being used in clinic
[16-18]. Inspired by the interesting biological profiles of OdDHL [19] and in
continuation with our previous work on the identification of new anticancer leads [20,
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21], we herein report the design and synthesis of a library of AHLs and their
underlying mechanisms of inducing cell death.
2. Results and discussion
2.1. Chemistry
Homoserine lactone hydrochloride (compound 3) was efficiently synthesized from
methionine following our previously reported methods (Scheme 1) [20, 21]. With this
key intermediate in hand, we next synthesized a focused library of AHL analogs by
introducing different terminal moieties. In our previous work, we found that the
incorporation of chalcone scaffold can improve the antiproliferative activity of the
AHL/chalcone hybrids [21-24]. Therefore, in this design, the chalcone scaffold was
also introduced to the AHL via suitable linker to further explore its effect on the
activity. In addition, the dithiocarbamate derivatives have been reported to possess
diverse and profound biological activities [25, 26]. The installation of such group may
endow the target compounds with improved or new biological profiles. Specifically,
treatment of compound 3 with chloroacetyl chloride in the presence of NaHCO3
yielded compound 4, which then reacted with various anilines to afford compounds
5a-j. Similarly, compound 4 reacted with hydroxylated chalcones in the presence of
K2CO3 and catalytic KI in MeCN to give 6a-n (Scheme 2). Compounds 8a-j were
efficiently synthesized from the corresponding phenols through two steps (Scheme 3).
Specifically, substituted phenols reacted with 1,2-dibromoethane in the presence of
NaOH in MeOH to give compounds 7a-j, which then reacted with CS2 and compound
3 in the presence of Na3PO4•12H2O in acetone to form compounds 8a-j (Scheme 3).
In order to compare the activity of the synthesized compounds with OdDHL and
explore further SARs, OdDHL were synthesized following the previously reported
procedures (Scheme 4) [20, 21]. S12 was also synthesized from compound 3 as
described for the synthesis of compounds 8a-j (Scheme 5).
Scheme 1. Synthesis of homoserine lactone hydrochloride 3. Reagents and conditions:
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(a) MeOH/H2O, CH3I, r.t., 20h; (b) NaHCO3 aq, rt~reflux 12h; (c) 6M HCl, 30%
H2O2, reflux, 12h.
Scheme 2. Synthesis of compounds 5a-j and 6a-n. Reagents and conditions: (a)
NaHCO3, DCM/H2O; (b) KI, DMF, 60 oC~under reflux; (c) K2CO3, KI, MeCN, r.t. to
under reflux, 6h.
Scheme 3. Synthesis of compounds 8a-j. Reagents and conditions: (a)
1,2-dibromoethane, aq. NaOH, MeOH, r.t.; (b) compound 3, Na3PO4•12H2O, CS2,
acetone.
Scheme 4. Synthesis of natural OdDHL. Reagents and conditions: (a) Ac2O, H2SO4, 0
oC-rt. (b) DCC, DMAP, r.t. (c) Compound 3, Et3N, CH3CN, reflux.
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Scheme 5. Synthesis of compound S12. Reagents and conditions: (a) Na3PO4•12H2O,
CS2, acetone.
2.2. The antiproliferative activity of synthesized AHLs, OdDHL and S12
The synthesized compounds were initially evaluated for their antiproliferative activity
against different cancer cell lines including MCF-7, MGC-803, DU145 and PC-3
using the MTT assay [27], OdDHL and S12 were used as the control. The primary
data are summarized in Table 1. Evidently, compounds 5a-j with the terminal aniline
group were inactive against the tested cancer cell lines (IC50 > 64 µM) regardless of
their substituents attached. The chalcone is a privileged scaffold that exists in a large
number of natural products and pharmaceutically relevant small molecules.
Chalcone-based hybridization/conjugation has yielded numerous structurally new and
biologically important compounds with interesting antiproliferative activity [26]. To
our delight, the incorporation of the chalcone scaffold improved the antiproliferative
activity. For MCF-7 and MGC-803 cells, compounds 6a-n exhibited weak to
moderate inhibitory activity, no clear SARs were observed. Compound 6h showed the
best potency against MCF-7 cells with an IC50 value of 15.25 µM, compounds 6h also
displayed moderate inhibitory activity against MGC-803 cells (IC50 =12.17 µM),
comparable to that of compound 6m (IC50 = 11.87 µM). Interestingly, some of
compounds 6a-n showed selective and potent growth inhibition toward prostate
cancer cells DU145 and PC-3 with the IC50 values less than 10 µM. Among these
compounds, halogenated compounds 6c, 6e and 6h displayed the best antiproliferative
activity with the IC50 values less than 5.0 µM, significantly more potent than OdDHL
and S12, unveiling the essential structural elements for the activity against prostate
cancer cells. In contrast, the hydroxylated chalcone fragment of compound 6e
inhibited growth of DU145 cells with an IC50 value of 23.67±2.52 µM, about 7.3-fold
less potent than 6e (IC50 = 3.24±0.16 µM), suggesting the importance of the
homoserine lactone/chalcone conjugate for the antiproliferative activity of 6e. The
toxicity of 6e toward normal cell line GES-1 was also examined, showing relatively
weak toxicity of 6e (IC50 = 13.37±1.13 µM). Conceivably, more analogs could be
obtained from readily accessible chalcone starting materials, the homoserine
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lactone/chalcone hybrid 6e could be used as a starting point for the development of
next-generation small-molecule compounds targeting prostate cancer cells.
The dithiocarbamate group, due to the prevalence in bioactive molecules and
synthetic simplicity, has been extensively used in the design of lead compounds or
probe molecules. As represented in Table 1, compounds 8a-j showed significantly
decreased activity against the tested cancer cell lines, some of them were much more
potent than S12, which was found to be devoid of the activity.
Table 1. The antiproliferative activity of synthesized N-acyl homoserine lactones
(AHLs) against four human cancer cell lines
IC50 (µM) a
Compound
MCF-7
>64
MGC-803
>64
DU145
>64
PC-3
>64
5a-5j
6a
6b
6c
6d
6e
6f
23.35±1.86
>64
16.84±1.43
>64
13.12±1.11
48.34±1.56
3.70±0.57
10.119±1.00
3.24±0.16
8.606±0.93
41.794±1.62
3.54±0.21
19.82±1.29
51.67±1.77
17.23±1.23
6.57±0.81
9.80±0.99
18.79±1.27
63.78±1.80
32.61±1.38
>64
4.66±0.66
56.77±1.75
4.99±0.49
7.24±0.86
4.61±0.33
8.04±0.90
56.25±1.71
3.26±0.34
21.89±1.34
>64
20.85±1.85
>64
29.09±1.69
39.40±1.84
27.07±1.75
26.47±1.56
>64
17.43±1.51
34.35±1.98
>64
6g
6h
6i
15.25±1.40
>64
12.17±1.51
>64
>64
50.96±1.71
>64
6j
>64
23.18±1.19
10.58±1.02
7.89±0.86
15.56±1.20
>64
6k
6l
16.44±1.56
28.34±1.45
>64
17.15±1.23
11.87±1.50
>64
6m
6n
8a
8b
8c
8d
8e
8f
>64
53.78±1.78
>64
>64
46.87±1.67
>64
39.49±1.05
>64
32.31±1.50
27.92±1.44
43.37±1.80
46.98±1.82
>100
>64
>64
>64
>64
>64
>64
33.42±1.52
31.01±1.49
>64
23.98±1.38
32.52±1.51
>64
>64
8g
8h
8i
50.53±1.56
>64
51.12±1.78
>64
>64
>64
>64
>64
28.25±1.45
24.12±1.24
8j
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26.24±1.68
>64
29.53±1.29
>64
19.33±0.98
>64
22.51±1.34
>64
OdDHL
S12
a Cells were treated with compounds for 72h, the data were presented as IC50 ± SD
and analyzed with the SPSS16.0 software.
2.3. Colony formation assay
The favorable antiproliferative activity of compound 6e against DU145 prompted us
to investigate the effect on colony formation and cell migration. The clone formation
of cancer cells represents an indirect estimation of neoplastic transformation [28]. As
shown in Fig. 1A and 1B, both OdDHL and compound 6e are able to inhibit colony
formation of DU145 cells concentration-dependently. Evidently, compound 6e
exhibited stronger inhibitory effect than OdDHL and completely inhibited colony
formation at 5.0 µM.
Figure 1. Effect of OdDHL and 6e on the colony formation of DU145 cells. (A)
Representative images of DU145 colonies after treatment at the indicated
concentrations for 21 d; (B) The percentage of colonies after treatment with 6e and
OdDHL at the indicated concentrations for 21 days.
2.4. The synergistic effect of 6e and TRAIL on the growth inhibition of DU145 cells
As shown in Fig. 2A, compound 6e and OdDHL inhibited growth of DU145 cells in a
time-dependent manner. After 24 and 48h treatment, these two compounds showed
comparable inhibitory effect toward DU145 cells. However, compound 6e showed
higher potency than OdDHL after 72h treatment (IC50 = 3.24 vs. 19.33 µM). Next, we
investigated the effect of the combination of compound 6e or OdDHL with TRAIL on
the growth inhibition of DU145 cells. As shown in Fig. 2B (c-e), OdDHL (5.0 µM)
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and TRAIL (100 ng/mL) showed a synergistic effect on the growth inhibition of
DU145 cells with around 20% of DU145 cells inhibited. In contrast, the combination
of 6e (2.5 µM) and TRAIL (100 ng/mL) displayed better growth inhibition of DU145
cells even at a lower concentration (2.5 µM) with the growth inhibition rate of about
50% (Fig. 2B-b), higher than that of 6e and TRAIL treatment alone (Fig. 2B, a & e).
Our data clearly suggest that homoserine lactone analogs and TRAIL have synergistic
effects on the growth inhibition of cancel cells, and homoserine lactone analogs, e.g.
6e, could be potentially used to overcome the resistance of tumor cells toward
TRAIL.
Figure 2. Growth inhibition of compound 6e and OdDHL alone and in combination
with TRAIL toward DU145 cells. (A) IC50 value (µM) of 6e and OdDHL of inducing
cell death of DU145 after treatment for 24, 48, and 72 h; (B) Effect of 6e, OdDHL or
their combination with TRAIL on the growth inhibition of DU145 cells (6e: 2.5 µM,
OdDHL: 5.0 µM, TRAIL: 100 ng/mL). *P < 0.05 was considered statistically
significant. Data are the mean ± SD. The experiment was repeated three times (n = 3).
2.5. The effect of homoserine lactone alone or its combination with TRAIL on
apoptosis of DU145 cells
In view of the ability of compound 6e, OdDHL or their combination with TRAIL
inhibiting growth of DU145 cells, we next investigated the potential effect on the
apoptosis of DU145 cells. As depicted in Fig. 3A, after treatment with OdDHL (10
µM), 6e (5.0 µM) and TRAIL (100 ng/mL) alone for 24 hours, characteristic
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apoptotic morphological changes of DU145 cells were observed, especially for the
group treated with the combination of OdDHL or 6e with TRAIL including cell
rounding, chromatin shrinkage and formation of apoptotic bodies. To further explore
the effect of the combination of OdDHL or 6e with TRAIL on cell apoptosis, the
apoptotic analysis was also performed with Annexin V-FITC/PI double staining.
Clearly, treatment of DU145 cells with the combination of OdDHL or compound 6e
with TRAIL induced apoptosis (Fig. 3B-C), the statistical analysis showed that the
corresponding apoptotic rate was higher than that of the group treated with 6e,
OdDHL and TRAIL alone (P < 0.01, Fig. 3C). Specifically, the percentage of
apoptotic cells treated with OdDHL+TRAIL and 6e+TRAIL, respectively at the
indicated concentrations was up to 21.9% and 15.1%, respectively (Fig. 3B).
Figure 3. Effect of 6e, OdDHL or their combination with TRAIL on the apoptosis of
DU145 cells. (A) Apoptosis analysis with Hoechst 33258 staining after 24 h treatment;
(B-C) Quantitative analysis of apoptotic cells using Annexin V-FITC/PI double
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staining and flow-cytometry calculation. **P < 0.01 was considered statistically
highly significant. Data are the mean ± SD. All experiments were carried out three
times (n = 3).
2.6. The effect of homoserine lactone alone or its combination with TRAIL on
expression changes of apoptosis-related proteins in DU145 cells
The observed ability of the combination of OdDHL or 6e with TRAIL in inducing
apoptosis of DU145 cells promoted us to further investigate the effect on the
apoptosis-related proteins (Fig. 4). Initially, we evaluated the effect of TRAIL,
OdDHL and 6e on the activity of caspase-3/7. Compared to the control group,
OdDHL and 6e marginally activated caspase-3/7 even at extremely high
concentrations (50 µM for OdDHL). In contrast, the combination of OdDHL (10 µM)
or 6e (5 µM) with TRAIL (100 ng/mL) led to about 2- and 3-fold activation of
caspase-3/7, respectively (P < 0.05, Fig. 4). The data indicate that OdDHL or 6e with
TRAIL are synergistic in activating caspase-3/7.
Figure 4. The effect of 6e, OdDHL or their combination with TRAIL on the activity
of caspase-3/7 in DU145 cells after 24 h treatment. All experiments were carried out
three times (n = 3). *P < 0.05 was considered statistically significant. Data are the
mean ± SD.
2.7. The effect of homoserine lactones OdDHL and 6e on the cell migration of DU145
cells
In light of the acceptable potency of 6e toward DU145 cells (IC50 = 3.24 µM), we
then examined the effect of 6e and OdDHL on the cell migration ability of DU145
cells by the wound healing assay. As shown in Fig. 5, both compound 6e and OdDHL
inhibited migration of DU145 cells in a time-and concentration-dependent manner,
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and 6e showed stronger migration inhibition than OdDHL.
Figure 5. Migration inhibition of DU145 cell upon treated with 6e and OdDHL. (A)
The effect of 6e and OdDHL on the cell migration of DU145 cells after 24 and 48 h
treatment at the indicated concentrations; (B) Statistic analysis of would gap closure
after 24 and 48 h treatment at different concentrations.
3. Conclusions
In summary, three series of new homoserine lactone analogs were efficiently
synthesized starting from methionine, the key intermediate homoserine lactone
hydrochloride (compound 3) and natural OdDHL were also synthesized following our
previously reported methods. The synthesized homoserine lactone analogs structurally
feature different terminal motifs, hoping to identify essential structural elements for
the activity. SARs studies showed that compounds 6a-n showed improved
antiproliferative activity against the tested cancer cell lines and were particularly
effective against prostate cancer cells DU145 and PC-3, underscoring the importance
of the chalcone scaffold for the observed activity. Among these compounds, some of
them were much more potent than natural OdDHL and S12 with the IC50 values less
than 10 µM. Especially, compounds 6c, 6e and 6h inhibited growth of DU145 and
PC-3 cells at low micromolar levels with the IC50 values ranging from 3.0 to 5.0 µM.
Compound 6e concentration-dependently inhibited colony formation and cell
migration of DU145 cells, being more potent than natural OdDHL. A synergistic
effect on the growth inhibition and the apoptosis of DU145 cells was observed when
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compound 6e was used in combination with TRAIL. OdDHL or 6e treatment
concentration-dependently activated TRAIL death receptor DR5 which may account
for the observed synergistic effect of 6e or OdDHL with TRAIL on the growth
inhibition and cell apoptosis. Compound 6e time and concentration-dependently
inhibited migration of DU145 cells. Compound 6e may be used as a potential lead
compound for developing new TRAIL receptor agonists. The development of new
small molecules that activate death receptors may be a viable strategy for cancer
therapy, especially for those resistant to known TRAIL receptor agonists.
4. Experimental section
4.1. General
Reagents and solvents were purchased from commercial sources and were used
without further purification. Melting points were determined on an X-5 micromelting
apparatus and are uncorrected. 1H NMR (400 MHz) and 13C NMR spectra (100 MHz)
were recorded on a Bruker (DPX-400) spectrometer. High resolution mass spectra
(HRMS) were recorded on a Waters Micromass Q-Tof of Micromass spectrometer by
electrospray ionizaton (ESI).
4.2. Synthesis of Homoserine lactone hydrochloride 3 and natural OdDHL
Homoserine lactone hydrochloride 3 and OdDHL were efficiently synthesized from
methionine following our previously reported methods [20, 21]. Therefore, the
synthetic details are not discussed here.
(S)-3-aminodihydrofuran-2(3H)-one hydrochloride (3), white solid, m.p. 218-220
1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 3H, NH2•HCl), 4.45 (t, J = 8.8 Hz, 1H,
-O-CH2), 4.37-4.21(m, 2H, -O-CH2, -CH-CO), 2.62-2.51 (m, 1H, -O-CH2-CH2-CH),
2.42-2.27 (m, 1H, -O-CH2-CH2-CH); 13C NMR (100 MHz, DMSO-d6) δ 173.81,
66.71, 48.12, 27.42. HRMS (ESI) Calcd for C4H8ClNO2 [M+H]+: 138.0322, found:
138.0325.
；
(S)-3-oxo-N-(2-oxotetrahydrofuran-3-yl)dodecanamide (OdDHL), white solid, m.p.
84-86 ℃；1H NMR (400 MHz, CDCl3) δ7.71 (d, J = 5.7 Hz, 1H, -CH-NH), 4.66-4.58
(m, 1H, -O-CH2), 4.49 (t, J = 9.0 Hz, 1H, -CH-NH), 4.34-4.15 (m, 1H, -O-CH2), 3.49
(s, 2H, -CO-CH2-CO), 2.82-2.72 (m, 1H, -O-CH2-CH2-CH), 2.57-2.52 (m, 2H,
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-CO-CH2-CH2), 2.34-2.20 (m, 1H, -O-CH2-CH2-CH), 1.65-1.55 (m, 2H,
-CO-CH2-CH2), 1.28 (m, 12H, -CO-CH2-(CH2)6), 0.89 (t, J = 6.6 Hz, 3H, -CH2-CH3);
13C NMR (100 MHz, CDCl3) δ 206.61, 174.84, 166.38, 65.89, 49.03, 48.11, 43.93,
31.83, 29.80, 29.37, 29.35, 29.24, 28.98, 23.35, 22.65, 14.10. HR-MS (ESI): Calcd.
C16H27NNaO4, [M+Na]+m/z: 320.1838, found: 320.1840.
4.3. Synthesis of S12
To a solution of homoserine lactone hydrochloride 3 (153 mg, 1.11 mmol) and
Na3PO4·12H2O (634 mg, 1.66 mmol) in acetone (15 mL) was added CS2 (141µL,
2.32 mmol) slowly. After 0.5h stirring, 1-bromo decane (219mg, 0.99mmol) was
added. Upon completion of the reaction indicated by TLC，acetone was removed
under vacuum, the resulting residue was dissolved with dichloromethane (DCM) and
washed with brine for 2-3 times, the aqueous layer was extracted with DCM for 2-3
times，the combined organic layers were evacuated to give the residue, which was
then subject to column chromatography, affording white solid, decyl
1
(S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate (S12)，m.p. 66-68 ℃；H NMR (400
MHz, CDCl3) δ 7.40 (d, J = 16.9 Hz, 1H, -CO-NH), 4.52 (t, J = 9.0 Hz, 1H, -CH-NH),
4.36 (ddd, J = 11.5, 9.4, 5.7 Hz, 1H, -CO-O-CH2), 3.36–3.13 (m, 3H, -CO-O-CH2,
-S-CH2), 2.18 (dd, J = 11.8, 9.2 Hz, 1H, -O-CH2-CH2-CH), 1.76-1.65 (m, 2H,
-O-CH2-CH2-CH, -S-CH2-(CH2)8), 1.50-1.21 (m, 15H, -S-CH2-(CH2)8), 0.90 (t, J =
6.8 Hz, 3H, -CH2-CH3). 13C NMR (100 MHz, CDCl3) δ 200.98, 174.61, 66.29, 54.97,
36.06, 31.89, 30.32, 29.53, 29.48, 29.30, 29.13, 28.89, 28.74, 22.68, 14.11. HRMS
(ESI): Calcd. C15H27NO2S2Na [M+Na]+ m/z:340.1381,found:340.1380.
4.4. Synthesis of compound 4
To a solution of homoserine lactone hydrochloride 3 (4.0 g, 22.52 mmol) in water (60
mL) was added NaHCO3 (5.67 g, 67.58 mmol). 5 min later, DCM (60 mL) was added
to the mixture followed by slow addition of chloroacetyl chloride (2.6 mL, 27.02
mmol). Upon completion of the reaction indicated by TLC, the DCM layer was
separated and evacuated under vacuum to give the white solid,
(S)-2-chloro-N-(2-oxotetrahydrofuran-3-yl)acetamide (4), yield: 72%, m.p.121-122 ℃；
1H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 8.0 Hz, 1H, -CO-NH), 4.63 (dd, J =
20.0 Hz, J = 8.0 Hz, 1H, -O-CH2), 4.35 (t, J = 8.0 Hz, 1H, -CH-NH), 4.21 (dd, J =
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ACCEPTED MANUSCRIPT
20.0 Hz, J= 8.0 Hz, 1H, -O-CH2), 4.14 (s, 2H, -CH2-Cl), 2.44-2.36 (m, 1H,
-O-CH2-CH2-CH), 2.24-2.15 (m, 1H, -O-CH2-CH2-CH); 13C NMR (100 MHz,
DMSO-d6) δ 175.30, 166.56, 65.80, 48.73, 42.71, 28.41. HRMS (ESI) Calcd for
C6H8ClNO3 [M+H]+: 178.0271, found: 178.0273.
4.5. General procedure for the synthesis of compounds 5a-j
To a solution of compound 4 (150 mg, 0.84 mmol) in DMF (5 mL) was added KI
o
(280 mg, 1.69 mmol). The resulting mixture was stirred at 60 C for 2 h, the
o
corresponding aniline (0.84 mmol) was added, and the mixture was stirred at 120 C
for 6 h. When the mixture was cooled to room temperature, the mixture was extracted
with EtOAc for several times, the combined EtOAc layers were washed with water
for several times and then dried under vacuum to give the residue, which was subject
to column chromatography, affording compounds 5a-j.
(S)-N-(2-oxotetrahydrofuran-3-yl)-2-(phenylamino)acetamide (compound 5a), yield:
65%, white solid, m.p.135-137 ；1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.1
Hz, 1H, -CO-NH), 7.16-7.00 (m, 2H, Ar-H), 6.66 -6.48 (m, 3H, Ar-H), 6.00 (t, J = 5.9
Hz, 1H, Ar-NH), 4.60 (dt, J = 10.8, 8.9 Hz, 1H, -O-CH2), 4.33 (td, J = 8.8, 1.7 Hz, 1H,
-CH-NH), 4.20 (ddd, J = 10.4, 8.7, 6.6 Hz, 1H, -O-CH2), 3.66 (d, J= 5.9 Hz, 2H,
-NH-CH2), 2.41-2.29 (m, 1H, -O-CH2-CH2-CH), 2.27-2.07 (m, 1H, -O-CH2-CH2-CH);
13C NMR (100 MHz, DMSO-d6) δ 175.64, 171.14, 148.87, 129.30, 117.04, 112.88,
65.71, 48.29, 47.53, 28.45. HR-MS (ESI): Calcd. C12H15N2O3, [M+H]+m/z: 235.1083,
found: 235.1081.
(S)-2-((4-fluorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound
5b), yield: 55%, white solid, m.p.162-163 ℃；1H NMR (400 MHz, DMSO-d6) δ 8.46
(d, J = 8.2 Hz, 1H, -CO-NH), 6.94 (t, J = 8.9 Hz, 2H, Ar-H), 6.57-6.51(m, 2H, Ar-H),
5.97 (s, 1H, Ar-NH), 4.60 (dt, J = 10.7, 8.9 Hz, 1H, -O-CH2), 4.33 (d, J = 1.6 Hz, 1H,
-CH-NH), 4.21 (ddd, J = 10.3, 8.8, 6.6 Hz, 1H, -O-CH2), 3.65 (d, J = 5.9 Hz, 2H,
-NH-CH2), 2.41-2.27 (m, 1H
,
-O-CH2-CH2-CH), 2.27-2.09 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, DMSO-d6) δ 175.65, 171.06, 156.37, 154.07,
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ACCEPTED MANUSCRIPT
145.57, 145.55, 115.78, 115.56, 113.68, 113.61, 65.71, 48.29, 47.96, 28.46. HR-MS
(ESI): Calcd. C12H13FN2NaO3, [M+Na]+m/z: 275.0808, found: 275.0802.
(S)-2-((4-chlorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound
5c), yield: 63%, light yellow solid, m.p.146-147 ; 1H NMR (400 MHz, DMSO-d6) δ
8.47 (d, J = 8.1 Hz, 1H, -CO-NH), 7.12 (d, J = 8.8 Hz, 2H, Ar-H), 6.55 (d, J = 8.8 Hz,
2H, Ar-H), 6.22 (s, 1H, Ar-NH), 4.60 (d, J = 10.4 Hz, 1H, -O-CH2), 4.33 (d, J = 1.6
Hz, 1H, -CH-NH), 4.21 (s, 1H, -O-CH2), 3.68 (d, J = 5.9 Hz, 2H, -NH-CH2),
2.41-2.30 (m, 1H, -O-CH2-CH2-CH), 2.19 (s, 1H, -O-CH2-CH2-CH). 13C NMR (100
MHz, DMSO-d6) δ 175.63, 170.77, 147.79, 128.97, 120.27, 114.22, 65.72, 48.31,
47.29, 28.48. HR-MS (ESI): Calcd. C12H13ClN2NaO3, [M+Na]+m/z: 291.0512, found:
291.0511.
(S)-2-((4-bromophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound
1
5d), yield: 71%, light yellow solid, m.p.156-158 ；H NMR (400 MHz, DMSO-d6) δ
8.47 (d, J = 8.1 Hz, 1H, -CO-NH), 7.23 (d, J = 8.8 Hz, 2H, Ar-H), 6.51 (d, J = 8.8 Hz,
2H, Ar-H), 6.25 (s, 1H, Ar-NH), 4.60 (dt, J = 10.7, 8.9 Hz, 1H, -O-CH2), 4.33 (dt, J =
8.8, 4.4 Hz, 1H, -CH-NH), 4.21 (ddd, J = 15.3, 8.8, 6.6 Hz, 1H,-O-CH2), 3.67 (d, J =
5.9 Hz, 2H, -NH-CH2), 2.40-2.29 (m, 1H, -O-CH2-CH2 -CH), 2.25-2.09 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, DMSO-d6) δ 175.63, 170.72, 148.14, 131.78,
114.79, 107.65, 65.72, 48.31, 47.17, 28.48. HR-MS (ESI): Calcd. C12H13BrN2NaO3,
[M+Na]+m/z: 335.0007, found: 335.0004 & 336.9978.
(S)-2-((3-chlorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound
5e), yield: 64%, white solid, m.p.142-144 ℃；1H NMR (400 MHz, DMSO-d6) δ 8.49
(d, J = 8.1 Hz, 1H, -CO-NH), 7.09 (t, J = 8.0 Hz, 1H, Ar-H), 6.58 (dt, J = 3.8, 1.6 Hz,
2H, Ar-H), 6.51 (s, 1H, Ar-H), 6.35 (s, 1H, Ar-NH), 4.59 (ddd, J = 34.8, 18.3, 11.2 Hz,
1H, -O-CH2), 4.34 (d, J = 1.7 Hz, 1H, -CH-NH), 4.27-4.14 (m, 1H,-O-CH2), 3.70 (d,
J = 5.9 Hz, 2H, -NH-CH2), 2.42-2.32 (m, 1H, -O-CH2-CH2-CH), 2.25-2.07 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, DMSO-d6) δ 175.63, 170.66, 150.33, 134.01,
130.80, 116.33, 112.14, 111.44, 65.74, 48.33, 46.90, 28.46. HR-MS (ESI): Calcd.
C12H13ClN2NaO3, [M+Na]+m/z: 291.0512, found: 291.0510.
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ACCEPTED MANUSCRIPT
(S)-2-((2-fluorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound
5f), yield: 55%, white solid, m.p.148-151 ；1H NMR (400 MHz, DMSO-d6) δ 8.50
(d, J = 8.1 Hz, 1H, -CO-NH), 6.97 (s, 2H, Ar-H), 6.55 (s, 2H, Ar-H), 5.82 (s, 1H,
Ar-NH), 4.60 (dd, J = 19.2, 8.9 Hz, 1H, -O-CH2), 4.33 (s, 1H, -CH-NH), 4.20 (dd, J =
12.8, 6.1 Hz, 1H, -O-CH2), 3.74 (d, J = 5.9 Hz, 2H, -NH-CH2), 2.36 (dt, J = 9.9, 7.6
Hz, 1H, -O-CH2-CH2-CH), 2.26-2.07 (m, 1H, -O-CH2-CH2-CH). 13C NMR (100 MHz,
DMSO-d6) δ 175.62, 170.71, 152.56, 150.19, 136.90, 136.79, 125.21, 125.18, 116.89,
116.83, 114.88, 114.70, 112.62, 112.59, 65.72, 48.32, 46.92, 28.44. HR-MS (ESI):
Calcd. C12H13FN2NaO3, [M+Na]+m/z: 275.0808, found: 275.0804.
(S)-2-((2-chlorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound
5g), yield: 80%, white solid, m.p.152-154 ℃；1H NMR (400 MHz, DMSO-d6) δ 8.54
(d, J = 8.0 Hz, 1H, -CO-NH), 7.28 (dd, J = 7.9, 1.3 Hz, 1H, Ar-H), 7.14 (s, 1H, Ar-H),
6.64 (d, J = 1.0 Hz, 1H, Ar-H), 6.54-6.48 (m, 1H, Ar-H), 5.75 (s, 1H, Ar-NH), 4.61 (dt,
J = 10.7, 8.9 Hz, 1H, -O-CH2), 4.34 (td, J = 8.8, 1.5 Hz, 1H, -CH-NH), 4.27-4.15 (m,
1H, -O-CH2), 3.80 (d, J = 5.7 Hz, 2H, -NH-CH2), 2.44-2.31 (m, 1H,
-O-CH2-CH2-CH), 2.26-2.11 (m, 1H, -O-CH2-CH2-CH). 13C NMR (100 MHz,
DMSO-d6) δ 175.60, 170.35, 144.22, 129.38, 128.49, 118.56, 117.72, 111.95, 65.75,
48.40, 46.83, 28.47. HRMS (ESI): Calcd. C12H13ClN2O3 [M+H]+ m/z:269.0693,
found: 269.0692.
(S)-2-((2,4-dichlorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide
(compound 5h), yield: 77%, white solid, m.p.135-137 ℃；1H NMR (400 MHz,
DMSO-d6) δ 8.53 (d, J = 8.1 Hz, 1H, -CO-NH), 7.40 (d, J = 2.4 Hz, 1H, Ar-H), 7.20
(dd, J = 8.8, 2.4 Hz, 1H, Ar-H), 6.50 (d, J = 8.8 Hz, 1H, Ar-H), 5.93 (s, 1H, Ar-NH),
4.61 (dt, J = 10.7, 8.9 Hz, 1H, -O-CH2), 4.34 (d, J = 1.5 Hz, 1H, -CH-NH), 4.21 (ddd,
J = 10.3, 8.8, 6.6 Hz, 1H, -O-CH2), 3.81 (d, J = 5.8 Hz, 2H, -NH-CH2), 2.42-2.33 (m,
13
1H, -O-CH2-CH2-CH), 2.24-2.12 (m, 1H, -O-CH2-CH2-CH). C NMR(100 MHz,
DMSO-d6) δ 175.60, 170.07, 143.41, 128.62, 128.26, 120.15, 119.02, 112.83, 65.75,
48.39, 46.71, 28.47. HR-MS (ESI): Calcd. C12H12Cl2N2NaO3, [M+Na]+m/z: 325.0123,
found: 325.0122.
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ACCEPTED MANUSCRIPT
(S)-2-((2-methyl-4-dichlorophenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide
(compound 5i), yield: 58%, white solid, m.p.131-133 ；1H NMR (400 MHz,
DMSO-d6) δ 8.49 (d, J = 8.1 Hz, 1H, -CO-NH), 6.99 (d, J = 7.9 Hz, 1H, Ar-H), 6.56
(dd, J = 7.8, 1.9 Hz, 1H, Ar-H), 6.30 (d, J = 1.8 Hz, 1H, Ar-H), 5.65 (s, 1H, Ar-NH),
4.60 (dd, J = 19.2, 8.9 Hz, 1H, -O-CH2), 4.35 (d, J = 1.3 Hz, 1H, -CH-NH), 4.25-4.17
(m, 1H, -O-CH2), 3.75 (d, J = 5.8 Hz, 2H, -NH-CH2), 2.37 (ddd, J = 12.4, 9.9, 7.2 Hz,
1H, -O-CH2-CH2-CH), 2.20 (dd, J = 20.8, 10.7 Hz, 1H, -O-CH2-CH2-CH), 2.11 (s, 3H,
Ar-CH3). 13C NMR (100 MHz, DMSO-d6) δ 175.59, 170.71, 147.82, 131.69, 131.31,
121.44, 116.10, 109.27, 65.73, 48.37, 47.08, 28.39, 17.50. HR-MS (ESI): Calcd.
C13H15ClN2NaO3, [M+Na]+m/z: 305.0669, found: 305.0670.
(S)-2-((4-isopropylphenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide
(compound 5j), yield: 74%, white solid, m.p.128-130 ℃；1H NMR (400 MHz,
DMSO-d6) δ 8.43 (d, J = 8.1 Hz, 1H, -CO-NH), 6.98 (d, J = 8.4 Hz, 2H, Ar-H), 6.49
(d, J = 8.4 Hz, 2H, Ar-H), 5.80 (s, 1H, Ar-NH), 4.59 (dd, J = 19.2, 8.9 Hz, 1H,
-O-CH2), 4.33 (d, J = 1.4 Hz, 1H, -CH-NH), 4.25-4.16 (m, 1H, -O-CH2), 3.63 (d, J =
5.8 Hz, 2H, -NH-CH2), 2.73 (dq, J = 13.7, 6.9 Hz, 1H, -O-CH2-CH2-CH), 2.35 (ddd, J
= 12.4, 9.8, 7.2 Hz, 1H, -O-CH2-CH2-CH), 2.21 (dt, J = 21.6, 10.8 Hz, 1H, Ar-CH),
1.14 (d, J = 6.9 Hz, 6H, Ar-CH-(CH3)2).13C NMR (100 MHz, DMSO-d6) δ 175.67,
171.39, 146.92, 137.12, 127.04, 112.93, 65.72, 48.29, 47.93, 33.00, 28.39, 24.72.
HR-MS (ESI): Calcd. C15H20N2NaO3, [M+Na]+m/z: 299.1372, found: 299.1376.
4.6. General procedure for the synthesis of compounds 6a-n
To a solution of compound 4 (150 mg, 0.85 mmol) and hydroxylated chalcone (0.85
mmol) in MeCN (5 mL) were added K2CO3 (350 mg, 3 mmol) and KI (42.3 mg, 0.3
mmol). The mixture was heated under reflux for 6 h and filtered upon completion of
the reaction indicated by TLC. The filtrate was evacuated under vacuum to give the
residue, which was then subject to column chromatography, affording the
corresponding 6a-n. The corresponding chalcone was prepared following the
literature reported methods [21].
(S,E)-2-((4-(3-(4-isopropylphenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3
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ACCEPTED MANUSCRIPT
-yl)acetamide (compound 6a), yield: 52%, light yellow solid, m.p.194-196 ；1H
NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 8.3 Hz, 1H, -CO-NH), 8.19 (d, J = 8.9 Hz,
2H, Ar-H), 7.91 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.81 (d, J = 8.2 Hz, 2H, Ar-H),
7.71 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.34 (d, J = 8.2 Hz, 2H, Ar-H), 7.14 (d, J =
8.9 Hz, 2H, Ar-H), 4.78-4.66 (m, 3H, -CH-NH, Ar-O-CH2), 4.39 (td, J = 8.7, 1.5 Hz,
1H, CH-CH2-CH2-O), 4.29-4.22 (m, 1H, CH-CH2-CH2-O), 2.94 (dt, J = 13.7, 6.9 Hz,
1H, -O-CH2-CH2-CH), 2.47-2.38 (m, 1H, -O-CH2-CH2-CH), 2.28 (m, 1H,
-CH(CH3)2), 1.23 (d, J = 6.9 Hz, 6H, -CH(CH3)2). 13C NMR (100 MHz, DMSO-d6) δ
187.88, 175.51, 167.91, 161.84, 151.76, 143.86, 132.98, 131.67, 131.26, 129.44,
127.35, 121.51, 115.29, 67.28, 65.80, 48.17, 33.89, 28.41, 24.09. HR-MS (ESI):
Calcd. C24H26NO5, [M+H]+m/z: 408.1811, found: 408.1807.
(S)-2-((4-cinnamoylphenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)acetamide
(compound 6b), yield: 60%, light yellow solid, m.p.179-183 ℃；1H NMR (400 MHz,
DMSO-d6) δ 8.74 (d, J = 8.2 Hz, 1H, -CO-NH), 8.21 (d, J = 8.8 Hz, 2H, Ar-H), 7.98
(d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.90 (dd, J = 6.4, 2.8 Hz, 2H, Ar-H), 7.73 (d, J =
15.6 Hz, 1H, -CO-HC=CH), 7.51-7.44 (m, 3H, Ar-H), 7.15 (d, J = 8.9 Hz, 2H, Ar-H),
4.77-4.66 (m, 3H, -CH-NH, Ar-O-CH2), 4.39 (dd, J = 8.8, 7.4 Hz, 1H,
CH-CH2-CH2-O), 4.25 (ddd, J = 15.3, 8.8, 6.6 Hz, 1H, CH-CH2-CH2-O), 2.42 (ddd, J
13
= 12.2, 9.8, 7.1 Hz, 1H, -O-CH2-CH2-CH), 2.35-2.22 (m, 1H, -O-CH2-CH2-CH). C
NMR (100 MHz, DMSO-d6) δ 187.89, 175.51, 167.90, 161.92, 143.79, 135.27,
131.56, 131.33, 130.94, 129.37, 129.29, 122.47, 115.32, 67.28, 65.80, 48.18, 28.41.
HR-MS (ESI): Calcd. C21H19NNaO5, [M+Na]+m/z: 388.1161, found: 388.1139.
(S,E)-2-((4-(3-(3-chlorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl
)acetamide (compound 6c), yield: 43%, light yellow solid, m.p.178-181 ℃；1H NMR
(400 MHz, DMSO-d6) δ 8.74 (d, J = 8.2 Hz, 1H, -CO-NH), 8.23 (dd, J = 12.3, 5.4 Hz,
3H, Ar-H), 8.03 (s, 2H, -CO-HC=CH), 7.60-7.56 (m, 1H, Ar-H), 7.52-7.44 (m, 2H,
Ar-H), 7.16 (d, J = 8.8 Hz, 2H, Ar-H), 4.77-4.68 (m, 3H, -CH-NH, Ar-O-CH2), 4.39
(t, J = 8.3 Hz, 1H, CH-CH2-CH2-O), 4.29-4.21 (m, 1H, CH-CH2-CH2-O), 2.42 (m, 1H,
-O-CH2-CH2-CH), 2.30 (m, 1H, -O-CH2-CH2-CH).13C NMR (100 MHz, DMSO-d6) δ
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ACCEPTED MANUSCRIPT
187.66, 175.51, 167.87, 162.11, 138.35, 134.76, 132.87, 132.33, 131.50, 131.26,
130.48, 129.06, 128.13, 125.21, 115.38, 67.29, 65.80, 48.18, 28.41. HR-MS (ESI):
Calcd. C21H18ClNNaO5, [M+Na]+m/z: 422.0771, found: 422.0771.
(S,E)-2-((4-(3-(2-methoxyphenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-
yl)acetamide (compound 6d), yield: 70%, light yellow solid, m.p.138-142 ；1H
NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 8.3 Hz, 1H, -CO-NH), 8.16 (d, J = 8.8 Hz,
2H, Ar-H), 8.04 (d, J = 15.7 Hz, 1H, -CO-HC=CH), 7.99 (dd, J = 8.0, 1.2 Hz, 1H,
Ar-H), 7.90 (d, J = 15.7 Hz, 1H, -CO-HC=CH), 7.49-7.43 (m, 1H, Ar-H), 7.13 (dd, J
= 8.4, 4.9 Hz, 3H, Ar-H), 7.04 (t, J = 7.5 Hz, 1H, Ar-H), 4.76-4.63 (m, 3H, -CH-NH,
Ar-O-CH2), 4.38 (td, J = 8.8, 1.4 Hz, 1H, CH-CH2-CH2-O), 4.25 (ddd, J = 10.4, 8.8,
6.6 Hz, 1H, CH-CH2-CH2-O), 3.91 (s, 2H, Ar-O-CH3), 2.46-2.37 (m, 1H,
-O-CH2-CH2-CH), 2.34-2.22 (m, 1H, -O-CH2-CH2-CH).13C NMR (100 MHz,
DMSO-d6) δ 188.05, 175.53, 167.92, 161.81, 158.66, 138.30, 132.65, 131.68, 131.22,
128.86, 123.50, 122.20, 121.16, 115.31, 112.26, 67.25, 65.81, 56.20, 48.17, 28.38.
HR-MS (ESI): Calcd. C22H21NNaO6, [M+Na]+m/z: 418.1267, found: 418.1265.
(S,E)-2-((4-(3-(2-chlorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl
1
)acetamide (compound 6e), yield: 59%, light yellow solid,，m.p.166-169 ℃；H NMR
(400 MHz, DMSO-d6) δ 8.75 (d, J = 8.2 Hz, 1H, -CO-NH), 8.27-8.19 (m, 3H, Ar-H),
8.02 (s, 2H, -CO-HC=CH), 7.60-7.55 (m, 1H, Ar-H), 7.47 (ddd, J = 6.3, 5.2, 3.5 Hz,
2H, Ar-H), 7.16 (d, J = 8.9 Hz, 2H, Ar-H), 4.77-4.67 (m, 3H, -CH-NH, Ar-O-CH2),
4.42-4.35 (td, 1H, CH-CH2-CH2-O), 4.25 (ddd, J = 10.4, 8.8, 6.5 Hz, 1H,
CH-CH2-CH2-O), 2.43 (m, 1H, -O-CH2-CH2-CH), 2.29 (m, 1H,
-O-CH2-CH2-CH).13C NMR (100 MHz, DMSO-d6) δ 187.67, 175.52, 167.88,
162.10, 138.36, 134.76, 132.87, 132.33, 131.50, 131.26, 130.47, 129.04, 128.13,
125.19, 115.38, 67.28, 65.81, 48.18, 28.40. HR-MS (ESI): Calcd. C21H18ClNNaO5,
[M+Na]+m/z: 422.0771, found: 422.0770.
(S,E)-2-((4-(3-(2,4-dichlorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-
3-yl)acetamide (compound 6f), yield: 66%, light yellow solid, m.p.193-195 ℃；1H
NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 8.2 Hz, 1H, -CO-NH), 8.29 (d, J = 8.6 Hz,
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ACCEPTED MANUSCRIPT
1H, Ar-H), 8.22 (d, J = 8.9 Hz, 2H, Ar-H), 8.07 (d, J = 15.5 Hz, 1H, -CO-HC=CH),
7.95 (d, J = 15.5 Hz, 1H, -CO-HC=CH), 7.77 (d, J = 2.1 Hz, 1H, Ar-H), 7.57 (dd, J =
8.5, 2.1 Hz, 1H, Ar-H), 7.15 (d, J = 8.9 Hz, 2H, Ar-H), 4.76-4.66 (m, 3H, -CH-NH,
Ar-O-CH2), 4.38 (dd, J = 8.8, 7.3 Hz, 1H, CH-CH2-CH2-O), 4.25 (ddd, J = 10.4, 8.7,
6.5 Hz, 1H, CH-CH2-CH2-O), 2.46-2.37 (m, 1H, -O-CH2-CH2-CH), 2.34-2.22 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, DMSO-d6) δ 187.51, 175.50, 167.86, 162.17,
137.09, 135.94, 135.54, 131.96, 131.54, 131.17, 130.30, 129.95, 128.39, 125.78,
115.39, 67.29, 65.80, 48.18, 28.40. HR-MS (ESI): Calcd. C21H17Cl2NNaO5,
[M+Na]+m/z: 456.0381, found: 456.0383.
(S,E)-2-((4-(3-(3-nitrophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)a
cetamide (compound 6g), yield: 40%, light yellow solid, m.p.185-188 ；1H NMR
(400 MHz, DMSO-d6) δ 8.80 (s, 1H, Ar-H), 8.74 (d, J = 8.2 Hz, 1H, -CO-NH), 8.35
(d, J = 7.9 Hz, 1H, Ar-H), 8.27 (m, 3H, Ar-H), 8.20 (d, J = 15.6 Hz, 1H,
-CO-HC=CH), 7.84 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.76 (t, J = 8.0 Hz, 1H,
Ar-H), 7.16 (d, J = 8.8 Hz, 2H, Ar-H), 4.77-4.66 (m, 3H, -CH-NH, Ar-O-CH2), 4.39
(td, J = 8.9, 1.3 Hz, 1H, CH-CH2-CH2-O), 4.29-4.21 (m, 1H, CH-CH2-CH2-O), 2.42
(m, 1H, -O-CH2-CH2-CH), 2.30 (m, 1H, -O-CH2-CH2-CH). 13C NMR (100 MHz,
DMSO-d6) δ 187.71, 175.51, 167.87, 162.13, 148.93, 141.26, 137.20, 135.55, 131.58,
131.27, 130.81, 125.24, 125.02, 123.42, 115.35, 67.29, 65.80, 48.18, 28.40. HR-MS
(ESI): Calcd. C21H18N2NaO7, [M+Na]+m/z: 433.1012, found: 433.0972.
(S,E)-2-((4-(3-(2-bromophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl
)acetamide (compound 6h), yield: 48%, light yellow solid, m.p.163-166 ℃；1H NMR
(400 MHz, DMSO-d6) δ 8.74 (d, J = 8.2 Hz, 1H, -CO-NH), 8.22 (d, J = 8.9 Hz, 3H,
Ar-H), 7.99 (s, 2H, -CO-HC=CH), 7.75 (d, J = 7.6 Hz, 1H, Ar-H), 7.53-7.47 (m, 1H,
Ar-H), 7.40 (td, J = 7.8, 1.5 Hz, 1H, Ar-H), 7.16 (d, J = 8.9 Hz, 2H, Ar-H), 4.78-4.68
(m, 3H, -CH-NH, Ar-O-CH2), 4.39 (td, J = 8.7, 1.3 Hz, 1H, CH-CH2-CH2-O), 4.25
(ddd, J = 10.4, 8.8, 6.6 Hz, 1H, CH-CH2-CH2-O), 2.42 (m, 1H, -O-CH2-CH2-CH),
2.28 (m, 1H, -O-CH2-CH2-CH).13C NMR (100 MHz, DMSO-d6) δ 187.64, 175.51,
167.87, 162.11, 141.10, 134.54, 133.75, 132.52, 131.51, 131.25, 129.24, 128.68,
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ACCEPTED MANUSCRIPT
125.78, 125.35, 115.38, 67.28, 65.80, 48.17, 28.40. HR-MS (ESI): Calcd.
C21H18BrNNaO5, [M+Na]+m/z: 466.0266, found: 466.0264 & 468.0244.
(S,E)-2-((4-(3-(3,4-difluorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3
-yl)acetamide (compound 6i), yield: 40%, light yellow solid, m.p.215-217 ；1H
NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 8.3 Hz, 1H, -CO-NH), 8.28-8.11 (m, 3H,
Ar-H), 8.00 (d, J = 15.5 Hz, 1H, -CO-HC=CH), 7.74 (d, J = 4.3 Hz, 2H, Ar-H), 7.70
(d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.59-7.47 (m, 1H, Ar-H), 7.15 (d, J = 8.8 Hz, 2H,
Ar-H), 4.78-4.66 (m, 3H, -CH-NH, Ar-O-CH2), 4.39 (td, J = 8.6, 1.1 Hz, 1H,
CH-CH2-CH2- O), 4.30-4.20 (m, 1H, CH-CH2-CH2-O), 2.47-2.37 (m, 1H,
-O-CH2-CH2-CH), 2.34-2.23 (m, 1H, -O-CH2-CH2-CH). 13C NMR (100 MHz,
DMSO-d6) δ 187.63, 175.52, 167.89, 162.03, 141.48, 133.31, 133.27, 133.25, 133.21,
131.43, 131.36, 127.27, 127.24, 127.21, 127.18, 123.71, 123.69, 118.50, 118.33,
117.48, 117.31, 115.33, 67.27, 65.81, 48.18, 28.39. HR-MS (ESI): Calcd.
C21H17F2NNaO5, [M+Na]+m/z: 424.0972, found: 424.1010.
(S,E)-2-((4-(3-(4-bromophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl
)acetamide (compound 6j), yield: 62%, light yellow solid, m.p.204-208 ℃；1H NMR
(400 MHz, DMSO-d6) δ 8.75 (d, J = 8.3 Hz, 1H, -CO-NH), 8.20 (d, J = 8.9 Hz, 2H,
Ar-H), 8.01 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.87 (d, J = 8.5 Hz, 2H, Ar-H), 7.68
(dd, J = 11.9, 10.1 Hz, 3H, -CO-HC=CH, Ar-H), 7.14 (d, J = 8.9 Hz, 2H, Ar-H),
4.76-4.66 (m, 3H, -CH-NH, Ar-O-CH2), 4.38 (td, J = 8.8, 1.5 Hz, 1H,
CH-CH2-CH2-O), 4.25 (ddd, J=10.3, 8.7, 6.5 Hz, 1H, CH-CH2-CH2-O), 2.47-2.37 (m,
13
1H, -O-CH2-CH2-CH), 2.34-2.22 (m, 1H, -O-CH2-CH2-CH). C NMR (100 MHz,
DMSO-d6) δ 187.77, 175.53, 167.91, 161.97, 142.41, 134.56, 132.33, 131.43, 131.39,
131.21, 124.28, 123.23, 115.32, 67.25, 65.82, 48.18, 28.38. HR-MS (ESI): Calcd.
C21H18BrNNaO5, [M+Na]+m/z: 466.0266, found: 466.0263 & 468.0234.
(S,E)-2-((4-(3-(3-fluorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)
acetamide (compound 6k), yield: 67%, light yellow solid, m.p.184-188 ℃；1H NMR
(400 MHz, DMSO-d6) δ 8.75 (d, J = 8.2 Hz, 1H, -CO-NH), 8.23 (d, J = 8.8 Hz, 2H,
Ar-H), 8.05 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.87 (d, J = 10.2 Hz, 1H, Ar-H),
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ACCEPTED MANUSCRIPT
7.76-7.68 (m, 2H, -CO-HC=CH, Ar-H), 7.51 (dd, J = 14.2, 7.9 Hz, 1H, Ar-H), 7.29
(td, J = 8.5, 2.2 Hz, 1H, Ar-H), 7.15 (d, J = 8.8 Hz, 2H, Ar-H), 4.80-4.66 (m, 3H,
-CH-NH, Ar-O-CH2), 4.39 ((td, J = 9.0, 1.5 Hz, 1H, CH-CH2-CH2-O), 4.32-4.21 (m,
1H, CH-CH2-CH2-O), 2.48-2.36 (m, 1H, -O-CH2-CH2-CH), 2.28 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, DMSO-d6) δ 187.75, 175.52, 167.89, 164.20,
162.03, 161.78, 142.32, 142.30, 137.90, 137.82, 131.45, 131.39, 131.33, 131.25,
126.03, 123.89, 117.69, 117.48, 115.33, 115.16, 114.94, 67.28, 65.80, 48.18, 28.40.
HR-MS (ESI): Calcd. C21H18FNNaO5, [M+Na]+m/z: 406.1067, found: 406.1065.
(S,E)-2-((4-(3-(4-t-butylphenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl
)acetamide (compound 6l), yield: 64%, light yellow solid, m.p.163-166 ；1H NMR
(400 MHz, DMSO-d6) δ 8.74 (d, J = 8.3 Hz, 1H, -CO-NH), 8.19 (d, J = 8.8 Hz, 2H,
Ar-H), 7.91 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.82 (d, J = 8.3 Hz, 2H, Ar-H), 7.71
(d, J = 15.5 Hz, 1H, -CO-HC=CH), 7.49 (d, J = 8.3 Hz, 2H, Ar-H), 7.15 (d, J = 8.8 Hz,
2H, Ar-H), 4.78-4.66 (m, 3H, -CH-NH, Ar-O-CH2), 4.39 (td, J = 8.8, 1.6 Hz, 1H,
CH-CH2-CH2-O), 4.25 (ddd, J = 15.4, 8.8, 6.6 Hz, 1H, CH-CH2-CH2-O), 2.47-2.38
(m, 1H, -O-CH2-CH2-CH), 2.34-2.22 (m, 1H, -O-CH2-CH2-CH), 1.32 (s, 9H, -C
13
(CH3)3). C NMR (100 MHz, DMSO-d6) δ 187.90, 175.51, 167.91, 161.85, 153.93,
143.75, 132.57, 131.66, 131.26, 129.17, 126.18, 121.63, 115.30, 67.28, 65.80, 48.17,
35.12, 31.39, 28.41. HR-MS (ESI): Calcd. C25H27NNaO5, [M+Na]+m/z: 444.1787,
found: 444.1784.
(S,E)-2-((4-(3-(3-bromophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl
)acetamide (compound 6m), yield: 75%, light yellow solid, m.p.207-211 ℃；1H NMR
(400 MHz, DMSO-d6) δ 8.74 (d, J = 8.3 Hz, 1H, -CO-NH), 8.27-8.20 (m, 3H, Ar-H),
8.06 (d, J = 15.6 Hz, 1H, -CO-HC=CH), 7.87 (d, J = 7.7 Hz, 1H, Ar-H), 7.69 (d, J =
15.6 Hz, 1H, -CO-HC=CH), 7.64 (d, J = 8.0 Hz, 1H, Ar-H), 7.42 (t, J = 7.9 Hz, 1H,
Ar-H), 7.15 (d, J = 8.8 Hz, 2H, Ar-H), 4.78-4.66 (m, 3H, -CH-NH ,Ar-O-CH2), 4.39
(td, J = 8.6, 1.3 Hz, 1H, CH-CH2-CH2-O), 4.30-4.21 (m, 1H, CH-CH2-CH2-O),
2.47-2.38 (m, 1H, -O-CH2-CH2-CH), 2.28 (dt, J = 22.3, 11.0 Hz, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, DMSO-d6) δ 187.21, 175.03, 167.40, 161.53,
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ACCEPTED MANUSCRIPT
141.54, 137.30, 132.88, 130.99, 130.91, 130.87, 130.70, 128.24, 123.43, 122.37,
114.82, 66.76, 65.31, 47.67, 27.89. HR-MS (ESI): Calcd. C21H18BrNNaO5,
[M+Na]+m/z: 466.0266, found: 466.0265 & 468.0257.
(S,E)-2-((4-(3-(4-fluorophenyl)acryloyl)phenyl)amino)-N-(2-oxotetrahydrofuran-3-yl)
acetamide (compound 6n), yield: 77%, light yellow solid, m.p.186-189 ；1H NMR
(400 MHz, DMSO-d6) δ 8.75 (d, J = 8.2 Hz, 1H, -CO-NH), 8.20 (d, J = 8.9 Hz, 2H,
Ar-H), 8.02-7.90 (m, 3H, Ar-H, -CO-HC=CH), 7.73 (d, J = 15.6 Hz, 1H,
-CO-HC=CH), 7.31 (t, J = 8.8 Hz, 2H, Ar-H), 7.14 (d, J = 8.9 Hz, 2H, Ar-H),
4.78-4.65 (m, 3H, -CH-NH, Ar-O-CH2), 4.38 (td, J = 8.7, 1.3 Hz, 1H,
CH-CH2-CH2-O), 4.25 (ddd, J = 10.4, 8.8, 6.6 Hz, 1H, CH-CH2-CH2-O), 2.46-2.38
(m, 1H, -O-CH2-CH2-CH), 2.28 (dt, J = 22.5, 11.1 Hz, 1H, -O-CH2-CH2-CH). 13C
NMR (100 MHz, DMSO-d6) δ 187.80, 175.52, 167.91, 165.04, 162.57, 161.92,
142.55, 131.96, 131.93, 131.67, 131.59, 131.52, 131.33, 122.35, 116.48, 116.26,
115.31, 67.27, 65.81, 48.18, 28.39. HRMS (ESI): Calcd. C21H18FNO5 [M+H]+ m/z:
384.1247, found: 384.1248.
4.7. General procedure for the synthesis of compounds 8a-j
Compounds 7a-7j were prepared following the literature reported methods in 54-83%
yields [29]. To a solution of compound 3 (102 mg, 0.74 mmol) and Na3PO4·12H2O
(423 mg, 1.11mmol) in acetone (10 mL) was added CS2 (94 µL, 1.55 mmol). After 0.5
h stirring, the corresponding compounds 7a-7j were added. About 1-2 h later, the
solvent was removed under vacuum, the resulting residue was dissolved with DCM
and then washed with brine. The organic layer was dried over MgSO4 and evacuated
under vacuum to give the residue, which was then subject to column chromatography,
affording the corresponding 8a-j.
2-Phenoxyethyl (S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate (compound 8a),
yield: 67%, white solid, m.p.89-90 ℃；1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 2.5
Hz, 1H, -CS-NH), 7.37-7.26 (m, 2H, Ar-H), 7.01-6.88 (m, 3H, Ar-H), 5.16 (m, 1H,
-CO-O-CH2), 4.50 (t, J = 9.0 Hz, 1H, -CH-NH), 4.34 (ddd, J = 11.5, 9.4, 5.7 Hz, 1H,
-CO-O-CH2), 4.23 (t, J = 6.2 Hz, 2H, Ar-O-CH2), 3.67 (t, J = 6.1 Hz, 2H, -CS-S-CH2),
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ACCEPTED MANUSCRIPT
3.24-3.08 (m, 1H, -O-CH2-CH2-CH), 2.27-2.07 (m, 1H, -O-CH2-CH2-CH);13C NMR
(100 MHz, CDCl3) δ 199.70, 174.45, 158.34, 129.65, 121.38, 114.80, 66.39, 55.29,
35.16, 30.22. HR-MS (ESI): Calcd. C13H15NNaO3S2, [M+Na]+m/z: 320.0391, found:
320.0389.
2-(4-Acetylphenoxy)ethyl
(S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate
1
(compound 8b), yield: 58%, white solid, m.p.107-109 ；H NMR (400 MHz, CDCl3)
δ 7.93 (d, J = 8.8 Hz, 2H, Ar-H), 7.76 (d, J = 2.2 Hz, 1H, -CS-NH), 6.96 (d, J = 8.8
Hz, 2H, Ar-H), 5.21 (t, J = 12.6 Hz, 1H, -CO-O-CH2), 4.51 (t, J = 9.1 Hz, 1H,
-CH-NH), 4.40-4.33 (m, 1H, -CO-O-CH2), 4.33-4.23 (m, 2H, -S-CH2), 3.70 (t, J = 6.2
Hz, 2H, -Ar-O-CH2), 3.23-3.07 (m, 1H, -O-CH2-CH2-CH), 2.56 (s, 3H, -CO-CH3),
2.29-2.11 (m, 1H, -O-CH2-CH2-CH);13C NMR (100 MHz, CDCl3) δ 199.37, 196.89,
174.33, 162.24, 130.72, 130.64, 114.33, 66.45, 66.28, 55.27, 34.65, 30.04, 26.37.
HR-MS (ESI): Calcd. C15H17NNaO4S2, [M+Na]+m/z: 362.0497, found: 362.0495.
2-(3-Benzylphenoxy)ethyl
(S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate
(compound 8c), yield: 56%, light yellow oil, 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J
= 14.8 Hz, 1H, -CS-NH), 7.29-7.08 (m, 7H, Ar-H), 6.97-6.82 (m, 2H, Ar-H), 5.11 (s,
1H, -CO-O-CH2), 4.48 (t, J = 9.0 Hz, 1H, -CH-NH), 4.32 (ddd, J = 11.5, 9.4, 5.7 Hz,
1H, -CO-O-CH2), 4.19 (t, J = 6.1 Hz, 2H ,Ar-O-CH2), 3.97 (s, 2H Ar-CH2-Ar), 3.64 (t,
J = 5.5 Hz, 2H, -S-CH2), 3.22-3.08 (m, 1H, -O-CH2-CH2-CH), 2.20-2.04 (m, 1H,
-O-CH2-CH2-CH).13C NMR (100 MHz, CDCl3) δ 199.88, 174.39, 156.09, 141.04,
130.59, 129.96, 128.98, 128.27, 127.47, 125.79, 121.08, 111.75, 66.38, 66.26, 55.20,
36.14, 35.33, 30.22. HR-MS (ESI): Calcd. C20H21NNaO3S2, [M+Na]+m/z: 410.0861,
found: 410.0860.
2-(4-Allyl-3-methoxyphenoxy)ethyl (S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate
(compound 8d), yield: 39%, light yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J
= 5.3 Hz, 1H, -CS-NH), 6.88 (d, J = 8.0 Hz, 1H, Ar-H), 6.75-6.68 (m, 2H, Ar-H), 5.95
(ddt, J = 16.9, 10.1, 6.7 Hz, 1H,-Ar-CH2-CH=CH2), 5.20 (dt, J = 19.1, 6.5 Hz, 1H,
-CO-O-CH2), 5.14-5.03 (m, 2H, -Ar-CH2-CH=CH2), 4.48 (t, J = 8.9 Hz, 1H,
-CH-NH), 4.40-4.22 (m, 3H, -CO-O-CH2, Ar-O-CH2), 3.85 (s, 3H, -O-CH3),
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ACCEPTED MANUSCRIPT
3.74-3.66 (m, 1H, -Ar-CH2-CH=CH2), 3.56 (dt, J = 8.3, 5.4 Hz, 1H-Ar-CH2-CH=
CH2), 3.33 (d, J = 6.6 Hz, 2H, -CS-S-CH2), 3.12-3.04 (m, 1H, -O-CH2-CH2-CH), 2.25
-2.08 (m, 1H, -O-CH2-CH2-CH).13C NMR (100 MHz, CDCl3) δ 199.50, 174.15,
149.47, 145.85, 137.50, 134.11, 120.68, 115.79, 114.44, 112.70, 68.41, 66.14, 56.00,
55.07, 39.82, 35.03, 29.79. HR-MS (ESI): Calcd. C17H21NNaO4S2, [M+Na]+m/z:
390.0810, found: 390.0812.
2-(2-Isopropyl-6-methylphenoxy)ethyl
(S)-(2-oxotetrahydrofuran-3-yl)
1
carbamodithioate (compound 8e), yield: 62%, light yellow oil; H NMR (400 MHz,
CDCl3) δ 7.48 (d, J = 16.7 Hz, 1H, -CS-NH), 7.09 (d, J = 7.7 Hz, 1H, Ar-H), 6.76 (d,
J = 7.6 Hz, 1H, Ar-H), 6.67 (s, 1H, Ar-H), 5.25-5.05 (m, 1H, -CO-O-CH2), 4.50 (t, J =
9.0 Hz, 1H, -CH-NH), 4.34 (ddd, J = 11.5, 9.4, 5.7 Hz, 1H, -CO-O-CH2), 4.22 (t, J =
6.0 Hz, 2H, Ar-O-CH2), 3.72 (t, J = 5.8 Hz, 2H, -CS-S-CH2), 3.28 (dt, J = 13.8, 6.8
Hz, 1H, Ar-CH-(CH3)2), 3.17 (dt, J = 13.1, 7.4 Hz, 1H, -O-CH2-CH2-CH), 2.31 (s, 3H
Ar-CH3), 2.22-2.08 (m, 1H, -O-CH2-CH2-CH), 1.20 (d, J = 6.9 Hz, 6H,
13
Ar-CH-(CH3)2). C NMR (100 MHz, CDCl3) δ 199.93, 174.26, 155.36, 136.39,
134.19, 126.04, 121.72, 112.61, 66.34, 66.25, 55.23, 35.59, 30.32, 26.64, 22.81, 21.28.
HR-MS (ESI): Calcd. C17H23NNaO3S2, [M+Na]+m/z: 376.1017, found: 376.1018.
2-(2-Nitrophenoxy)ethyl (S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate (compound
8f), yield: 47%, white solid, m.p.92-94 ；1H NMR (400 MHz, CDCl3) δ 7.93-7.70
(m, 2H, Ar-H,-CS-NH), 7.61-7.48 (m, 1H, Ar-H), 7.14 (d, J = 8.4 Hz, 1H, Ar-H), 7.06
(t, J = 7.8 Hz, 1H, Ar-H), 5.30-5.09 (m, 1H, -CO-O-CH2), 4.52 (t, J = 9.1 Hz, 1H,
-CH-NH), 4.37 (pd, J = 9.6, 6.4 Hz, 3H,-CO-O-CH2, Ar-O-CH2), 3.83-3.64 (m, 2H,
-CS-S-CH2), 3.18-3.01 (m, 1H, -O-CH2-CH2-CH), 2.34-2.16 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, CDCl3) δ 174.28, 151.72, 134.25, 125.70,
120.98, 114.99, 68.14, 66.31, 55.30, 34.49, 29.81. HRMS (ESI): Calcd. C13H14N2O5S2
[M+Na]+ m/z: 365.0242, found: 365.0240.
2-(4-Nitrophenoxy)ethyl (S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate (compound
8g), yield: 53%, white solid, m.p.98-100 ℃；1H NMR (400 MHz, DMSO-d6) δ 10.43
(d, J = 7.9 Hz, 1H, -CS-NH), 8.21 (d, J = 9.2 Hz, 2H, Ar-H), 7.20 (d, J = 9.3 Hz, 2H,
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ACCEPTED MANUSCRIPT
Ar-H), 5.55 (dt, J = 11.0, 8.7 Hz, 1H, -CO-O-CH2), 4.34 (dqd, J = 10.5, 8.8, 7.0 Hz,
4H, -CO-O-CH2, Ar-O-CH2), 3.77-3.61 (m, 2H, -CS-S-CH2), 2.64-2.53 (m, 1H,
-O-CH2-CH2-CH), 2.23 (dt, J = 22.8, 11.2 Hz, 1H, -O-CH2-CH2-CH). 13C NMR (100
MHz, DMSO-d6) δ 197.46, 173.53, 163.33, 141.03, 125.88, 115.10, 66.94, 65.49,
54.46, 33.14, 27.80. HR-MS (ESI): Calcd. C13H14N2NaO5S2, [M+Na]+m/z: 365.0242,
found: 365.0264.
2-(4-Chlorophenoxy)ethyl
(S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate
1
(compound 8h), yield: 64%, white solid, m.p.123-125 ；H NMR (400 MHz, CDCl3)
δ 7.62 (s, 1H, -CS-NH), 7.29-7.20 (m, 2H, Ar-H), 6.93-6.81 (m, 2H, Ar-H), 5.17 (t, J
= 12.5 Hz, 1H,-CO-O-CH2), 4.51 (t, J = 8.9 Hz, 1H, -CH-NH), 4.35 (ddd, J = 11.5,
9.4, 5.7 Hz, 1H, -CO-O-CH2), 4.20 (dt, J = 12.7, 6.4 Hz, 2H, Ar-O-CH2), 3.66 (t, J =
6.2 Hz, 2H, -CS-S-CH2), 3.23-3.09 (m, 1H, -O-CH2-CH2-CH), 2.25-2.09 (m, 1H,
-O-CH2-CH2-CH). 13C NMR (100 MHz, CDCl3) δ 199.50, 174.38, 156.91, 129.40,
126.14, 116.00, 66.67, 66.31, 55.25, 34.85, 30.12. HR-MS (ESI): Calcd.
C13H14ClNNaO3S2, [M+Na]+m/z: 354.0001, found: 353.9995.
2-(Naphthalen-1-yloxy)ethyl
(S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate
(compound 8i), yield: 66%, white solid, m.p.58-60 ℃；1H NMR (400 MHz, CDCl3) δ
8.25 (dd, J = 8.5, 4.2 Hz, 1H, Ar-H), 7.82-7.75 (m, 1H, Ar-H), 7.61-7.40 (m, 4H, Ar-H,
-CS-NH), 7.35 (t, J = 7.9 Hz, 1H, Ar-H), 6.83 (d, J = 7.5 Hz, 1H, Ar-H), 5.16 (d, J =
3.9 Hz, 1H, -CO-O-CH2), 4.43 (dt, J = 12.2, 7.6 Hz, 3H, -CH-NH, Ar-O-CH2), 4.31
(ddd, J = 11.3, 9.5, 5.7 Hz, 1H, -CO-O-CH2), 3.84 (t, J = 5.8 Hz, 2H, -CS-S-CH2),
3.19-3.07 (m, 1H, -O-CH2-CH2-CH), 2.20-2.07 (m, 1H, -O-CH2-CH2 -CH). 13C NMR
(100 MHz, CDCl3) δ 199.77, 174.63, 154.09, 134.52, 127.51, 126.52, 125.84, 125.55,
125.36, 122.03, 120.78, 105.19, 66.53, 66.34, 60.48, 55.24, 35.24, 29.85, 21.10, 14.23.
HRMS (ESI): Calcd. C17H17NO3S2, [M+Na]+ m/z: 370.0548, found: 370.0547.
2-(4-Ethoxyphenoxy)ethyl
(S)-(2-oxotetrahydrofuran-3-yl)carbamodithioate
1
(compound 8j), yield: 57%, white solid, m.p.94-96 ℃；H NMR (400 MHz, DMSO-d6)
δ 10.38 (d, J = 8.0 Hz, 1H, -CS-NH), 7.01 (dd, J = 7.5, 1.9 Hz, 1H, Ar-H), 6.96 (dd, J
= 7.7, 1.9 Hz, 1H, Ar-H), 6.93-6.84 (m, 2H, Ar-H), 5.55 (dt, J = 11.1, 8.7 Hz, 1H,
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-CO-O-CH2), 4.40 (dd, J = 8.9, 7.6 Hz, 1H，-CO-O-CH2), 4.30 (ddd, J = 10.6, 8.8, 6.3
Hz, 1H, -CH-NH), 4.16 (t, J = 6.6 Hz, 2H, Ar-O-CH2-CH2-S), 4.05-3.98 (m, J = 7.0
Hz, 2H, Ar-O-CH2-CH3 ), 3.61 (td, J = 6.6, 2.0 Hz, 2H, -CS-S-CH2), 2.62-2.52 (m, 1H,
-O-CH2-CH2-CH), 2.29-2.16 (m, J = 11.1, 9.2 Hz, 1H, -O-CH2-CH2-CH), 1.32 (t, 3H,
13
-O-CH2-CH3). C NMR (100 MHz, DMSO-d6) δ 197.86, 173.56, 148.39, 147.79,
121.59, 120.87, 114.49, 113.98, 66.98, 65.47, 63.86, 54.40, 33.60, 27.81, 14.78.
HRMS (ESI): Calcd. C15H19NO4S2, [M+Na]+ m/z: 364.0653, found: 364.0653.
4.8. Antiproliferative activity assay [23]
Human breast cancer cell MCF-7, gastric cancer cell MGC-803, prostate cancer cells
DU145 and PC-3 were purchased from the Cell Bank of Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, People’s
Republic of China) and cultured in Dulbecco’s modified Eagle medium (DMEM,
Gibco Invitrogen Corp., Carlsbad, CA, USA). Cell were seeded into 96-well plates at
a concentration of 3,000 cells per well. After 24 h of incubation, the culture medium
was removed and fresh medium containing different concentration (64, 32, 16, 8, 4, 2,
1, 0.5 µM) of the candidate compound was added to each well. The cells were then
incubated for 72 h, thereafter MTT assays were performed and cell viability was
assessed at 570 nm by a microplate reader (Biotech, Shanghai, China).
4.9. Colony formation inhibition assay [30]
DU145 cells (4000 cells/well) were seeded in a 6-well plate and incubated for 4 days,
then the media were replaced with fresh media containing different concentrations of
compound 6e and OdDHL. After 21 days of treatment, the cells were washed twice
with PBS, fixed with 4% paraformaldehyde, and colonies were visualized using 0.1%
crystal violet staining. The cells were imaged, and the number of colonies were
quantified by Image J software (Developed by National Institutes of Health). A group
of >10 cells was defined as one colony. Inhibition rate = (1-number
oftreatment/number of control) * 100%.
4.10. Hoechst 33258 staining [30]
DU145 cells were seeded into a 6-well plate (1×105/well) and incubated overnight for
adherent and treated with 6e, OdDHL or its combination with TRAIL at different
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concentrations for 24 h, and underwent Hoechst 33258 staining for 30 min in the dark.
The cells were observed under a Nikon Eclipse TE 2000-S fluorescence microscope
(Nikon, Japan).
4.11. Cell apoptosis assay [30]
DU145 cells were seeded into a 6-well plate (3×105/well) and incubated for 24 h.
Then the cells were treated with different concentrations of 6e, OdDHL or its
combination with TRAIL for 24 h. Thereafter, the cells were collected and the
Annexin-V-FITC/PI apoptosis kit (Biovision) was used according to the
manufacturer’s protocol. The cells were analyzed by high content screening system
(ArrayScan XTI, Thermo Fisher Scientific, MA).
4.12. Western blot analysis [30]
DU145 cells were treated with different concentrations of compound 6e, OdDHL or
its combination with TRAIL for 24 h, the cells were collected, lysed in RIPA buffer
contained a protease inhibitor cocktail for 30 min, followed by centrifugation at
o
14,000 rpm for 15 min at 4 C. After the collection of supernatant, the protein
concentration was detected using a bicinchonininc acid assay kit (Beyotmie
Biotechnology, Haimen, China). After added with loading buffer, cell lyses were
o
boiled for 10 min at 100 C for SDS- polyacrylamide gel electrophoresis (PAGE).
Proteins were transferred to nitrocellulose (NC) membranes. Then the membranes
were blocked with 5% skim milk at room temperature for 2 h, and then incubated
o
overnight at 4 C with primary antibodies. After washing the membrane with the
secondary antibody (1: 5000) at room temperature for 2 h. Finally, the blots were
washed in TBST/TBS. The antibody-reactive were revealed by enhanced
chemiluminescence (ECL) and exposed on Kodak radiographic film.
4.13. Caspase-3/7 activity detection
The caspase-3/7 activity of compound 6e, OdDHL or its combination with TRAIL
was detected according to the standard Caspase-Glo® 3/7 assay protocol.
4.14. Wound healing assay [30]
DU145 cells (3×105/well) were placed in a 6-well plate and incubated for 24 h, and
the cell surface was scratched using a 10 µL pipet tip. Then the cells were treated with
compound 6e or OdDHL at different concentrations, and then incubated for 24 or 48 h
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