Design, synthesis, in vitro antiproliferative activity evaluation of 2-alkanoylamidothiophene-3-carboxamide derivatives

Article abstract of DOI:10.3987/COM-16-13558

A series of 2-alkanoylamidothiophene-3-carboxamide derivatives were synthesized based on the hit compound 1. The anti-proliferative activity of all the compounds in vitro against MGC-803 (stomach) and HCT-116 (colon) cancer cell lines using SRB assays were tested. Several compounds showed improved anti-proliferative activity against MGC-803 and HCT-116. SAR study revealed that chlorine substituent in the 2-acetylamino part was important for anti-proliferative activity. 5a, 11b, 11c and 11d were the most potent compounds against MGC-803 (IC₅₀s = 2.32-2.95 μM), and 5a and 11c also showed good anti-proliferative activity against HCT-116 cells (IC₅₀s = 3.41-3.75 μM). In addition, the anti-proliferative activity of 11b and 11d could be attributed to the apoptosis in HCT116 cells via caspase 3 activation, confirmed by flow cytometry assay and western blot analysis. Meanwhile, 11b and 11d decreased the mitochondrial membrane potential (MMP) in HCT116 cells.

Full text of DOI:10.3987/COM-16-13558

HETEROCYCLES, Vol. 92, No. 12, 2016
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HETEROCYCLES, Vol. 92, No. 12, 2016, pp. 2145 - 2165. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 1st August, 2016, Accepted, 21st August, 2016, Published online, 9th November, 2016
DOI: 10.3987/COM-16-13558
DESIGN, SYNTHESIS, IN VITRO ANTIPROLIFERATIVE ACTIVITY
EVALUATION
OF
2-ALKANOYLAMIDOTHIOPHENE-3-
CARBOXAMIDE DERIVATIVES
Jiefeng Zhang, Fengjie Guan, Jiakun Qiu, Yanfen Fang, Lifang Yu,a
a
a
a
a,*
b
a
a
b
a,c,*
Jingya Li, Fan Yang, Xiongwen Zhang, Jia Li, and Jie Tang
a.Shanghai Engineering Research Center of Molecular Therapeutics and New
Drug Development, SCME, East China Normal University, Shanghai 200062,
China (phone: +86-21-62221232; e-mail: yffang@sat.ecnu.edu.cn; phone:
+86-21-62232764; fax: +86-21-62232100; e-mail: jtang@chem.ecnu.edu.cn)
b.National Center for Drug Screening, Shanghai Institute of Materia Medica,
Chinese Academy of Science, Shanghai 201203, China
c.Shanghai Key Laboratory of Green Chemistry and Chemical Processes, SCME,
East China Normal University, Shanghai 200062, China
Abstract – A series of 2-alkanoylamidothiophene-3-carboxamide derivatives were
synthesized based on the hit compound 1. The anti-proliferative activity of all the
compounds in vitro against MGC-803 (stomach) and HCT-116 (colon) cancer cell
lines using SRB assays were tested. Several compounds showed improved
anti-proliferative activity against MGC-803 and HCT-116. SAR study revealed that
chlorine substituent in the 2-acetylamino part was important for anti-proliferative
activity. 5a, 11b, 11c and 11d were the most potent compounds against MGC-803
(
IC50s = 2.32-2.95 μM), and 5a and 11c also showed good anti-proliferative activity
against HCT-116 cells (IC50s = 3.41-3.75 μM). In addition, the anti-proliferative
activity of 11b and 11d could be attributed to the apoptosis in HCT116 cells via
caspase 3 activation, confirmed by flow cytometry assay and western blot analysis.
Meanwhile, 11b and 11d decreased the mitochondrial membrane potential (MMP)
in HCT116 cells.
INTRODUCTION
The 2-acylaminothiophene-3-carboxamide derivatives have gained much interest because of their wide
1
2
3
range of biological activities, such as anti-diabetic, anti-virus (compound I, Figure 1), anti-bacterial,

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HETEROCYCLES, Vol. 92, No. 12, 2016
4
5
6
anti-inflammatory, anti-neurodegenerative (compound II, Figure 1), analgesic, cystic fibrosis
7
8
treatment and aspartic protease inhibiting activities which have been extensively reported in literature.
In addition, studies focus on their potentials as anticancer agents have also been performed. Recently, Cao
et al. developed
a
multi-target inhibitor (compound III, Figure 1) bearing
a
2-acylaminothiophene-3-carboxamide scaffold against ABL kinases and tubulin. Compound III showed
significant inhibition against both ABLs driven cell lines in the dose-response fashion while only
9
moderate effect on the parental cells. Compound IV (Figure 1) as the enzyme Fms-like tyrosine
kinase-3(FLT3) inhibitor (IC50 = 0.027 μM) blocked the proliferation of MV4-11 cells (IC50 = 0.41
μM).10 In addition, kinesin spindle protein (KSP) inhibitors which subsequently induced apoptosis
1
1
(
compound V, Figure 1) were also reported and compound V showed inhibition against A-549 cells
with the EC50 = 2.0 r 0.2 μM.
Figure 1. Biological active 2-acylaminothiophene-3-carboxamide derivatives
We have synthesized series of 2-(substituted acetamino)thiophene-3-carboxamides in our lab in recent
years. Through an anti-proliferative activity screening, compound 1 was identified as an anti-proliferative
agent with modest potency against MGC-803 and HCT-116 (IC50 = 5.96 r 0.08 and 19.80 r 0.65 μΜ,
Figure 2. Structure of compound 1 and antiproliferative activity of compound 1 in MGC-803 and
HCT-116 at different concentrations (Values are mean ± SD, n=2, *p<0.05, **p<0.01 and ***p<0.001
compared with DMSO (1‰) group. Differences between individual groups are analyzed by T-test).

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respectively) (Figure 2; entry 1 in Table 1). Song et al. suggested that halogen substituted acetylamino
chains at the 3-position of the benzoylurea derivatives played a significant role in regulating the
anti-proliferative activities. Their anti-proliferative activity in tumor cells was ranked in an order of the
nature of halogens: I > Br > Cl > F.12 Herein, we describe the synthesis of series novel
2-(chloroacetamido)thiophene-3-carboxamides or terminal chloro-subsituted alkanoylamidothiophene-3-
carboxamides based on hit compound 1, and in vitro evaluation of the anti-proliferative activity against
MGC-803 and HCT-116 cells is also discussed.
RESULTS AND DISCUSSION
CHEMISTRY
Although some aroyl substituted aminothiophenes at ortho-position have been developed (Figure 1), 2d, 6, 13
alkanoyl substituted corresponding compound has rarely been reported. We designed and synthesized
seventeen 2-alkanoylamidothiophene-3-carboxamide derivatives and the synthetic routes were outlined in
Schemes 1-4. With commercially available ketones as starting materials, 2a-2d were synthesized via
1
4
Gewald cyclization. Acylation of the 2-amino group of 2a-2d afforded 3a-3d which subsequently gave
1
5
4
a-4d after removing the tert-butyl group. HATU-facilitated condensations of 4a-4d with benzylamine
1
6
produced 5a-5c and compound 1 (Scheme 1).
Scheme 1
Acylation of the 2-amino group of 2a with corresponding acid chlorides in the presence of Et
3
N afforded
6
a-6f. In addition, 6g was prepared by condensation of 2a with dimethyl oxalate in the presence of
1
7
NaH. Subsequent deprotection of the tert-butyl group in 6a-6g afforded 7a-7g which underwent the

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HATU-facilitated condensations with benzylamine, giving 8a-8f and 9. Hydrolysis of 9 with LiOH/H
O18
2
1
9
afforded product 8g (Scheme 2). Nucleophilic substitution of 5a with potassium acetate followed by
deacetylation provided 8h (Scheme 3).
Scheme 2
Scheme 3
HATU-facilitated coupling reactions of 4a with corresponding amines produced 11a-11e (Scheme 4).
Scheme 4
Biological study
In vitro anti-proliferative activities and SAR
All the final products were tested for the anti-proliferative activity in vitro against MGC-803 and
HCT-116 for 72 h using SRB assays with cisplatin as positive control.20 As depicted in Table 1, 9
compounds (5a, 5c, 8d, 8f, 11a, 11b, 11c, 11d, 11e) showed anti-proliferative activity against MGC-803
(
IC50 < 10 μM) and 6 compounds (5a, 11a, 11b, 11c, 11d, 11e) showed anti-proliferative activity against
HCT-116 (IC50 < 10 μM).

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Compared to the hit compound 1 with two methyl groups at 4- and 5- positions, introduction of hindered
aliphatie cycles to the thiophene ring had different performances. In detail, the cyclopentyl compound 5a
exhibited increase of anti-proliferative activities on MGC-803 and HCT-116 with IC50 values of 2.53 r
0
.11 and 3.41 r 1.13 μM, respectively. However, the anti-proliferative activities decreased with the
expansion of ring size (5b showed a decreased activity on MGC-803 and 5c showed a decreased activity
on HCT-116). It is indicated that a five-member ring substituted at 4- and 5- positions of the thiophene
ring was beneficial to the activity potency.
When the cyclopentyl group was determinated to be the better fusing aliphatic ring of thiophene, we
focused our attention on the D-chloro at 2-acetylamino group which had the tendency of being
nucleophilically substituted. At first, terminal chlorinated alkyl chains with different lengths were
introduced, affording compounds 8a and 8b with lower nucleophilic substitution reaction activity.
Comparing with 5a (IC50 = 2.53 r 0.11 μM), IC50 of 8a in which the chain was extended with one carbon
was increasing to 14.63 r 5.18 μM against MGC-803. And 8b with further extended chain length showed
no anti-proliferative activity (IC50 > 30 μM). This tendency matched well with their anti-proliferative
activities against HCT-116. On the other hand, 8c with increased steric hindrance at E-carbon of terminal
chlorine, which decreased the tendency of being nucleophilically substituted, also showed no
anti-proliferative activity against both MGC-803 (IC50 > 30 μM) and HCT-116 (IC50 > 30 μM).
Compounds bearing one more terminal chlorine atom, such as 8d, although not as active as 5a, showed
considerable anti-proliferative activity against MGC-803 (IC50 = 5.75 r 0.32 μM). However, no
anti-proliferative activity against MGC-803 (IC50 > 30 μM) and HCT-116 (IC50 > 30 μM) was observed
when the terminal carbon was substituted with three chlorine atoms (8e). Similarly, 8g and 8h in which
the 2-acetylamino position was substituted with 3-oxopropanoic acid or 2-hydroxyacetamido group, also
showed no anti-proliferative activity against both MGC-803 (IC50 > 30 μM) and HCT-116 (IC50 > 30 μM).
While the anti-proliferative activity was maintained when the 2-acetylamino group was substituted with
electrophilic vinyl group (8f, IC50 = 7.88 r 0.54 μM against MGC-803). These results were consistent to
the suggestion that halogen substituted acetylamino chains at the 3-position in the benzoylurea derivatives
1
2
play a significant role in regulating the anti-proliferative activities. Thus, it is believed that the
electrophilicity of the 2-acetylamino moiety in the molecule would be in favor of its anti-proliferative
activity, and the chlorine substituent in the 2-acetylaminopart may play a key role as a leaving group.
At last, we tried to modify the chain length and the substituents on the aromatic ring as well. Compound
1
1a in which the chain length was prolonged to four carbons maintained anti-proliferative activity against
both MGC-803 (IC50 = 3.89 r 0.30 μM) and HCT-116 (IC50 = 8.03 r 0.63 μM). While 11b having a
-methyoxy group on the aromatic ring showed an increased anti-proliferative activity against both
4

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MGC-803 (IC50 = 2.32 r 0.39 μM) and HCT-116 (IC50 = 5.89 r 0.63 μM) compared with compound 1.
Meanwhile, 2-chloro (11c), 3-chloro (11d) and 4-chloro (11e) substituted on the aromatic ring also
increased the anti-proliferative activities against both MGC-803 and HCT-116. The results suggested that
both electron-donating groups and electron-withdrawing groups on the aromatic ring increase the
anti-proliferative activity. Thus, both the chain length of the 2-acylamino moiety and the substituents on
the aromatic ring play important roles for the anti-proliferative activity.
Table 1. Anti-proliferative activities of 2-alkanoylamidothiophene-3-carboxamide derivatives against
MGC-803 and HCT-116 cells
IC50 (μM)
Entry Compound
MGC-803
5.96 r 0.08
2.53 r 0.11
10.90 r 2.78
4.83 r 0.85
14.63 r 5.18
> 30
HCT-116
19.80 r 0.65
3.41 r 1.13
20.91 r 0.17
> 30
1
2
3
4
5
6
7
8
9
1
5a
5b
5c
8a
8b
8c
8d
8e
15.33 r 0.03
> 30
> 30
> 30
5.75 r 0.32
> 30
16.75 r 0.45
> 30
1
0
8f
8g
8h
7.88 r 0.54
> 30
16.12 r 0.30
> 30
1
1
2
3
4
5
6
7
8
1
1
1
1
1
1
1
> 30
> 30
11a
11b
11c
11d
11e
Cisplatin
3.89 r 0.30
2.32 r 0.39
2.95 r 0.05
2.76 r 0.45
3.01 r 0.09
6.04 r 0.20
8.03 r 0.63
5.89 r 0.63
3.75 r 0.68
6.23 r 3.04
7.71 r 2.69
3.57 r 0.23
Compounds 11b and 11d induce apoptotic cell death
Apoptosis is a programmed cell death process by which the body eliminates damaged or unnecessary
2
1
cells and is playing a vital role in cancer development and tumor cell survival. To characterize whether
the anti-proliferative activity was accomplished by inducing cell apoptosis, HCT116 cells were treated
with vehicle alone or with selected compound 11b or 11d for 48 h and then stained with FITC-annexin V
2
2
and propidium iodide (PI) (Figure 3). The results showed that compounds 11b and 11d induced 57.93%
and 42.58% apoptosis, respectively, comparing to 9.77% in the control group after treated for 48 h (10
μmol/L dose). Therefore, it is evident that the anti-proliferative activities of these compounds are related
to inducing apoptosis in HCT-116 cell lines.

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Figure 3. 11b and 11d induced apoptosis of HCT-116 cells. Cells were stained with Annexin V-FITC/PI
and quantitated by flow cytometry. The cells were treated with control (1‰ DMSO) or 10 μM compound
1
1b and 11d for 48 h.
Activation of caspase 3 was involved in the apoptosis induced by 11b and 11d
Caspase 3 is one of the key executioners of apoptosis, as it is either partially or totally responsible for the
proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase 1
23
(
PARP1). To investigate the molecular mechanisms involved in the observed apoptosis, we measured
the expression of cleaved-caspase 3 and PARP1 in HCT-116 cell line treated with 11b and 11d by
Western blotting. HCT-116 was treated with 15 μM of 11b and 11d for 24 h and 48 h, then all cells were
harvested and assayed for cleaved-caspase 3 and PARP1 using GAPDH as a loading control (Figure 4).
Comparing to the control, the relative activities of cleaved-caspase 3 were increased in HCT-116 cell line
after treated with 11b and 11d for 48 h, while we didn't observe the activation of cleaved-caspase 3 after
treating for 24 h. In addition, it was found that 11b and 11d significantly increased the cleavage of
PARP1 in HCT-116 cell line after 24 h and 48 h treatment. Together, these findings revealed that
compound 11b and 11d induced HCT-116 cells apoptosis through activation of caspase 3.
Figure 4. Effects of 11b and 11d on activation of caspase 3. HCT-116 was treated with 15 μM of 11b
and 11d for 24 h and 48 h and then the cells were harvested and assayed for caspase 3 activation and
PARP1 cleavage using GAPDH as a loading control.
Compounds 11b and 11d decrease the mitochondrial membrane potential (MMP)

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Decreasing of MMP during apoptosis has been reported in a number of studies, leading to the general
2
4
notion that depolarization of mitochondria is one of the first events to occur during apoptosis. The effect
of 11b and 11d on the MMP in HCT-116 cells was investigated with the fluorescent probe JC-1, a
2
5
mitochondrion-specific and voltage-dependent dye. Treatment of HCT-116 cells with 11b and 11d at 10
μM for 24 h resulted in the percentage of cells with depolarized MMP from 2.85% of control cells to
37.28% and 16.54%, respectively (Figure 5).
Figure 5. Effects of 11b and 11d on mitochondrial membrane potential. Incubation with 11b, 11d at 10
μM for 24 h, the mitochondrial membrane potential (MMP) was analyzed by JC-1 fluorescence with flow
cytometry analysis.
CONCLUSIONS
In summary, a series of 2-alkanoylamidothiophene-3-carboxamide derivatives were synthesized based on
the hit compound 1. The anti-proliferative activity of all the compounds against MGC-803 and HCT-116
cell lines was tested using SRB assays. Nine compounds (5a, 5c, 8d, 8f, 11a, 11b, 11c, 11d, 11e) showed
anti-proliferative activity against MGC-803 (IC50 < 10 μM) and 6 compounds (5a,11a, 11b, 11c, 11d, 11e)
showed anti-proliferative activity against HCT-116 (IC50 < 10 μM). The SAR study revealed that terminal
chloro-substituent at ortho position of alkanoylamidothiophene was important for anti-proliferative
activity. 5a, 11b, 11c and 11d were the most potent compounds against MGC-803 and HCT-116 cell lines
(
IC50 = 2.32-6.23 μM). Annexin V-FITC/Propidium iodide (PI) double staining assay in HCT-116 cells
suggested that the anti-proliferative activity of compound 11b and 11d occurred via apoptosis. Western
blot analysis showed that the anti-proliferative activity of 11b and 11d could be attributed to the apoptosis
in HCT116 cells via caspase 3 activation. In addition, 11b and 11d depolarized the mitochondrial
membrane in HCT116 cells. Further studies on improving the anti-proliferative activity and exploring the
action mechanism are ongoing.
EXPERIMENTAL
General comments

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2153
Starting materials, reagents and chemicals were purchased from commercial suppliers and used without
further purification unless otherwise stated. The progress of reactions was monitored by silica gel thin
layer chromatography (TLC) plates, visualized under UV. Flash column chromatography was performed
1
13
using Qingdao Haiyang silica gel (200 - 300) with distilled solvents. H (400 MHz) and C (100 MHz)
1
NMR spectra were recorded on a Bruker DRX-400 Fourier transform spectrometer. H chemical shifts are
reported in δ (ppm) using the δ 7.26 signal of CDCl
3
or the δ 2.50 signal of DMSO-d
standards. C chemical shifts are reported in δ (ppm) using the δ 77.23 signal of CDCl
standard or the δ 39.52 signal of DMSO-d
6
as internal
as internal
1
3
3
6
as internal standards. High-resolution mass data were obtained
on a MicrOTOF II spectrometer. All melting points were obtained on a Laboratory Devices MEL-TEMP
II melting apparatus and are uncorrected.
Chemical Synthesis
General procedure for preparation of compounds 2a–2d
To a mixture of ketone (1.0 mmol), tert-butyl cyanoacetate (1.0 mmol), and sulfur (1.1 mmol) in EtOH
(
20 mL) was added triethylamine (2 mmol) and the reaction mixture was refluxed for 16 h. Afterwards
the reaction mixture was concentrated and the residue was partitioned between water and EtOAc. The
organic layer was separated, dried over anhydrous Na SO , and concentrated. The crude product was
2
4
purified by silica gel column chromatography using a mixture of petroleum ether and EtOAc (20:1) as the
eluent.
tert-Butyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (2a). Prepared from
1
cyclopentanone, petroleum ether:EtOAc = 20:1, to give white solid (0.17 g, 72%). H NMR (400 MHz,
CDCl ): δ 5.79 (s, 2H), 2.78-2.81 (m, 2H), 2.69-2.72 (m, 2H), 2.26-2.33 (m, 2H), 1.53 (s, 9H).
3
tert-Butyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2b). Prepared from
1
cyclohexanone, petroleum ether:EtOAc = 20:1, to give white solid (0.13 g, 52%). H NMR (400 MHz,
CDCl ): δ 5.87 (s, 2H), 2.67 (t, J = 5.6 Hz, 2H), 2.49 (t, J = 5.6 Hz, 2H), 1.72-1.78 (m, 4H), 1.54 (s, 9H).
3
tert-Butyl 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylate (2c). Prepared from
cycloheptanone and the base is morpholine, petroleum ether:EtOAc = 20:1, to give white solid (0.17 g,
1
6
4%). H NMR (400 MHz, CDCl
3
): δ 5.67 (s, 2H), 2.93-2.96 (m, 2H), 2.56-2.58 (m, 2H), 1.77-1.83 (m,
2H), 1.59-1.66 (m, 4H), 1.55 (s, 9H).
tert-Butyl 2-amino-4,5-dimethylthiophene-3-carboxylate (2d). Prepared from butan-2-one, petroleum
1
ether:EtOAc = 20:1, to give white solid (0.13 g, 56%). H NMR (400 MHz, CDCl
3
): δ 5.83 (s, 2H), 2.14
(s, 6H), 1.55 (s, 9H).
General procedure for preparation of compounds 3a–3d
tert-Butyl 2-aminothiophene-3-carboxylate (1.0 mmol) was dissolved in CH
2
Cl (15.0 mL) and treated
2

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with triethylamine (3.0 mmol). To this mixture, the solution of acid chloride (1.2 mmol) in CH
was added dropwise under 0 °C. This mixture was stirred for 0.5 h at room temperature. The mixture was
diluted by CH Cl , and washed with water. The organic phase was dried over anhydrous Na SO , and
2
Cl
2
(5 mL)
2
2
2
4
concentrated. The crude product was purified by silica gel column chromatography using a mixture of
petroleum ether and EtOAc (20:1) as the eluent.
tert-Butyl
2-(2-chloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate
(3a).
Prepared from 2a and chloroacetyl chloride, petroleum ether:EtOAc = 20:1, to give pale yellow solid
1
(
0.29 g, 92%). H NMR (400 MHz, CDCl
m, 2H), 1.58 (s, 9H).
3
): δ 11.90 (s, 1H), 4.24 (s, 2H), 2.83-2.90 (m, 4H), 2.34-2.41
(
tert-Butyl 2-(2-chloroacetamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (3b). Prepared
1
from 2b, petroleum ether:EtOAc = 20:1, to give white solid (0.33 g, 99%). H NMR (400 MHz, CDCl
3
):
δ 12.17 (s, 1H), 4.24 (s, 2H), 2.75 (t, J = 5.2 Hz, 2H), 2.65 (t, J = 5.2 Hz, 2H), 1.75-1.79 (m, 4H), 1.59 (s,
H).
9
tert-Butyl 2-(2-chloroacetamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylate (3c).
1
Prepared from 2c, petroleum ether:EtOAc = 20:1, to give white solid (0.33 g, 95%). H NMR (400 MHz,
CDCl
3
): δ 11.97 (s, 1H), 4.22 (s, 2H), 3.00-3.03 (m 2H), 2.72-2.74 (m, 2H), 1.82-1.88 (m, 2H), 1.64-1.67
(m, 2H), 1.58-1.61(m, 11H).
tert-Butyl 2-(2-chloroacetamino)-4,5-dimethylthiophene-3-carboxylate (3d). Prepared from 2d,
1
petroleum ether:EtOAc = 20:1, to give white solid (0.25 g, 81%). H NMR (400 MHz, CDCl
3
): δ 12.19 (s,
1
H), 4.24 (s, 2H), 2.27 (s, 3H), 2.23 (s, 3H), 1.60 (s, 9H).
General procedure for preparation of compounds 4a–4d
tert-Butyl 2-acylaminothiophene-3-carboxylate was dissolved in 20% of trifluoroacetic acid in CH
This mixture was stirred for 2-3 h at room temperature and diluted by CH Cl , and washed with water.
The organic phase was dried over anhydrous Na SO , and concentrated. The crude product was purified
by silica gel column chromatography using a mixture of CH Cl and MeOH (20:1) as the eluent.
-(2-Chloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (4a). Prepared
2
Cl .
2
2
2
2
4
2
2
2
1
from 3a, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.24 g, 93%). H NMR (400 MHz, DMSO-d ): δ
6
1
2
3
1
2
1.69 (s, 1H), 4.58 (s, 2H), 2.79-2.85 (m, 4H), 2.28-2.35 (m, 2H).
-(2-Chloroacetamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (4b). Prepared from
1
b, CH Cl
2 2
:MeOH = 20:1, to give white solid (0.27 g, 99%). H NMR (400 MHz, DMSO-d
6
): δ 13.25 (br,
H), 11.92 (s, 1H), 4.57 (s, 2H), 2.73 (t, J = 5.6 Hz, 2H), 2.61 (t, J = 5.6 Hz, 2H), 1.71-1.73 (m, 4H).
-(2-Chloroacetamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic
acid
(4c).
1
Prepared from 3c, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.27 g, 93%). H NMR (400 MHz,

HETEROCYCLES, Vol. 92, No. 12, 2016
2155
DMSO-d
6
): δ 13.45 (br, 1H), 11.78 (s, 1H), 4.55 (s, 2H), 3.02-3.05 (m, 2H), 2.70-2.72 (m, 2H), 1.79-1.80
(m, 2H), 1.53-1.59 (m, 4H).
2
-(2-Chloroacetamino)-4,5-dimethylthiophene-3-carboxylic acid (4d). Prepared from 3d,
1
CH
H), 11.93 (s, 1H), 4.58 (s, 2H), 2.24 (s, 3H), 2.21 (s, 3H).
General procedure for preparation of compounds 5a–5c and 1.
-Acylaminothiophene-3-carboxylic acid (1.0 mmol) was dissolved in DMF (20 mL) and HATU (1.5
2
Cl
2
:MeOH = 20:1, to give white solid (0.23 g, 92%). H NMR (400 MHz, DMSO-d
6
): δ 13.38 (br,
1
2
mmol) was added. After stirring at room temperature for 10 min, amine (1.5 mmol) was added. The
mixture was stirred at room temperature for another 2-3 h. Then the reaction was quenched with water
and the aqueous solution was extracted with EtOAc (20 mLu3). The combined organic layer was washed
with brine, dried over anhydrous Na
2
SO and concentrated. The crude product was purified by silica gel
4
column chromatography using a mixture of petroleum ether and EtOAc (7:1-2:1) as the eluent.
N-Benzyl-2-(2-chloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
(5a).
1
Prepared from 4a, petroleum ether:EtOAc = 5:1-2:1, to white solid (0.1 g, yield 29%). H NMR (400
MHz, DMSO-d
6
): δ 12.41 (s, 1H), 7.74 (t, J = 5.9 Hz, 1H), 7.31-7.36 (m, 4H), 7.22-7.27 (m, 1H), 4.52 (s,
1
3
2
H), 4.51 (d, J = 5.9 Hz, 2H), 2.99 (t, J = 7.0 Hz, 2H), 2.82 (t, J = 7.0 Hz, 2H), 2.35-2.42 (m, 2H); C
NMR (100 MHzꢀ DMSO-d
6
): G 164.95, 163.48, 146.88, 139.37, 138.78, 132.69, 128.34, 126.98 (2C),
-
1
26.76 (2C), 112.00, 42.49, 42.35, 29.25, 28.30, 27.77. ESI-HRMS (m/z): [M-H] calcd. for
C
17
H16ClN
2
2
O S, 347.0626; found 347.0627. mp 195.0-195.7 °C.
N-Benzyl-2-(2-chloroacetamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (5b). Prepared
1
from 4b, petroleum ether:EtOAc = 3:1-2:1, to give white solid (0.23 g, yield 63%). H NMR (400 MHz,
CDCl ): δ 12.96 (s, 1H), 7.33-7.37 (m, 5H), 6.22 (br, 1H), 4.65 (d, J = 5.0 Hz, 2H), 4.23 (s, 2H),
3
1
3
2
.67-2.69 (m, 4H), 1.82 (s, 4H); C NMR (100 MHz, DMSO-d ): δ 164.95, 163.48, 139.99, 139.27,
6
128.95, 128.28 (2C), 127.14 (2C), 127.05, 126.73, 117.89, 42.54, 42.47, 25.05, 23.79, 22.45, 22.29,
+
ESI-HRMS (m/z): [M+Na] calcd. for C18
H19ClN
2
NaO S, 385.0748; found 385.0756. mp 196.4-197.2 °C.
2
N-Benzyl-2-(2-chloroacetamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (5c).
1
Prepared from 4c, petroleum ether:EtOAc = 7:1-3:1, to give white solid (0.09 g, yield 23%). H NMR
(
400 MHz, DMSO-d
6
): δ 10.87 (s, 1H), 8.53 (t, J = 5.8 Hz, 1H), 7.32-7.35 (m, 4H), 7.22-7.27 (m, 1H),
1
3
4
.45 (d, J = 5.8 Hz, 2H), 4.40 (s, 2H), 2.65-2.71 (m, 4H), 1.76-1.83 (m, 2H), 1.54-1.60 (m, 4H); C NMR
(
100 MHz, DMSO-d
6
): δ 164.80, 163.49, 139.22, 135.01, 133.66, 131.30, 128.24 (2C), 127.27 (2C),
+
1
26.76, 123.57, 42.66, 42.35, 31.61, 28.29, 28.02, 27.67, 27.09. ESI-HRMS (m/z): [M+Na] calcd. for
C
19
H21ClN
2
NaO
2
S, 399.0904; found 399.0904. m. p. 216.0-216.5 °C.
N-Benzyl-2-(2-chloroacetamino)-4,5-dimethylthiophene-3-carboxamide (1). Prepared from 4d,

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HETEROCYCLES, Vol. 92, No. 12, 2016
1
petroleum ether:EtOAc = 7:1-3:1, to give white solid (0.27 g, yield 81%). H NMR (400 MHz,
DMSO-d ): δ 11.47 (s, 1H), 8.32 (t, J = 5.6 Hz, 1H), 7.33-7.34 (m, 4H), 7.23-7.28 (m, 1H), 4.49 (d, J =
6
1
3
5
.6 Hz, 2H), 4.46 (s, 2H), 2.24 (s, 3H), 2.17 (s, 3H); C NMR (100 MHz, DMSO-d ): δ 164.98, 163.36,
6
1
39.16, 137.50, 128.19 (2C), 127.11 (2C), 127.05, 126.67, 124.08, 120.51, 42.50, 42.34, 13.08, 12.07.
+
ESI-HRMS (m/z): [M+Na] calcd. for C16
H17ClN
2
NaO S, 359.0591; found 359.0622. mp 160.2-161.5 °C.
2
General procedure for preparation of compounds 6a–6f
tert-Butyl 2-aminothiophene-3-carboxylate (2a) (1.0 mmol) was dissolved in CH
2
Cl
2
(15 mL) and treated
Cl (5 mL)
was added dropwise under 0 °C. This mixture was stirred for 0.5 h at room temperature. The mixture was
diluted by CH Cl (20 mL), and washed with water. The organic phase was dried over anhydrous Na SO ,
with triethylamine (3 mmol). To this mixture, the solution of acid chloride (1.2 mmol) in CH
2
2
2
2
2
4
and concentrated. The crude product was purified by silica gel column chromatography using a mixture
of petroleum ether and EtOAc (5:1-2:1) as the eluent.
tert-Butyl 2-(3-chloropropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (6a).
Prepared from 2a and 3-chloropropionyl chloride, triethylamine (3 mmol) was replaced by pyridine (1.5
1
mmol), petroleum ether:EtOAc = 2:1, to give pale yellow solid 0.267 g. Yield 81%. H NMR (400 MHz,
CDCl
3
): δ 11.16 (s, 1H), 3.88 (t, J = 6.64 Hz, 2H), 2.93 (t, J = 6.64 Hz, 2H), 2.82-2.88 (m, 4H), 2.33-2.40
(m, 2H), 1.57 (s, 9H).
tert-Butyl 2-(4-chlorobutanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (6b).
Prepared from 2a and 4-chlorobutyryl chloride, petroleum ether:EtOAc = 2:1, to give pale yellow solid
1
(
0.30 g, 88%). H NMR (400 MHz, CDCl
3
): δ 11.08 (s, 1H), 3.66 (t, J = 6.12 Hz, 2H), 2.82-2.88 (m, 4H),
2.67 (t, J = 6.12 Hz, 2H), 2.33-2.40 (m, 2H), 2.19-2.26 (m, 2H), 1.57 (s, 9H).
tert-Butyl
2-(3-chloro-2,2-dimethylpropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-ꢁ
carboxylate (6c). Prepared from 2a and 3-chloropivaloyl chloride, petroleum ether:EtOAc = 2:1, to give
1
pale yellow solid (0.31 g, 86%). H NMR (400 MHz, CDCl
3
): δ 11.55 (s, 1H), 3.71 (s, 2H), 2.82-2.88 (m,
4H), 2.34-2.40 (m, 2H), 1.57 (s, 9H), 1.45 (s, 6H).
tert-Butyl 2-(2,2-dichloroacetamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (6d).
Prepared from 2a and dichloroacetyl chloride, petroleum ether:EtOAc = 5:1, to give pale yellow solid
1
(
0.28 g, 79%). H NMR (400 MHz, CDCl
m, 2H), 1.58 (s, 9H).
3
): δ 12.08 (s, 1H), 6.13 (s, 1H), 2.84-2.91 (m, 4H), 2.34-2.42
(
tert-Butyl 2-(2,2,2-trichloroacetamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (6e).
Prepared from 2a and trichloroacetyl chloride, petroleum ether:EtOAc = 5:1, to give pale yellow solid
1
(
0.32 g, 82%). H NMR (400 MHz, CDCl
s, 9H).
3
): δ 12.45 (s, 1H), 2.88-2.91 (m, 4H), 2.36-2.43 (m, 2H), 1.58
(

HETEROCYCLES, Vol. 92, No. 12, 2016
2157
tert-Butyl 2-acrylamino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (6f). Prepared from
a and 3-chloropropanoyl chloride, petroleum ether:EtOAc = 2:1, to give pale yellow solid (0.27 g, 92%).
1H NMR (400 MHz, CDCl
): δ 11.22 (s, 1H), 6.48 (d, J = 16.96 Hz, 1H), 6.34 (dd, J = 16.96, 10.20 Hz,
H), 5.84 (d, J = 10.20 Hz, 1H), 2.83-2.89 (m, 4H), 2.33-2.41 (m, 2H), 1.57 (s, 9H).
2
3
1
tert-Butyl
2-(2-methoxy-2-oxoacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate
(6g). A mixture of 2a (1.0 g, 4.17 mmol) and dimethyl oxalate (1.97 g, 16.7mmol) in THF (30 mL) was
cooled to 0 °C and NaH (0.15 g, 6.25mmol) was added. The mixture was refluxed for 8 h. The reaction
was quenched with water. THF was removed in vacuo. The crude product was diluted with EtOAc (100
mL) and water (20 mL) and washed with a saturated aqueous NaCl solution (15 mL), dried (Na
2
SO ) and
4
filtered, and the solvent was removed in vacuo. The crude product was purified by silica gel column
1
chromatography using a mixture of petroleum ether:EtOAc = 5:1, to give white solid (1.07g, 79%). H
NMR (400 MHz, CDCl
H).
3
): δ 12.25 (s, 1H), 3.99 (s, 3H), 2.85-2.92 (m, 4H), 2.35-2.42 (m, 2H), 1.59 (s,
9
General procedure for preparation of compounds 7a–7g
tert-Butyl 2-acylaminothiophene-3-carboxylate (1.0 mmol) was dissolved in 20% of trifluoroacetic acid
(
5 mL) in CH
and washed with water. The organic phase was dried over anhydrous Na
crude product was purified by silica gel column chromatography using a mixture of CH
2
Cl
2
(20 mL). This mixture was stirred for 2-3 h at room temperature and diluted by CH
SO , and concentrated. The
Cl and MeOH
2
Cl ,
2
2
4
2
2
(20:1) as the eluent.
2
-(3-Chloropropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7a). Prepared
1
from 6a, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.26 g, 95%). H NMR (400 MHz, DMSO-d
6
): δ
1
2
2
2.99 (br, 1H), 11.06 (s, 1H), 3.88 (t, J = 6.20 Hz, 2H), 3.03 (t, J = 6.20 Hz, 2H), 2.77-2.83 (m, 4H),
.27-2.34 (m, 2H).
-(4-Chlorobutanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7b). Prepared
1
from 6b, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.24 g, 85%). H NMR (400 MHz, DMSO-d
6
): δ
1
2
2
2.97 (br, 1H), 11.01 (s, 1H), 3.69 (t, J = 6.60 Hz, 2H), 2.76-2.83 (m, 4H), 2.65 (t, J = 7.24 Hz, 2H),
.27-2.34 (m, 2H), 2.02-2.09 (m, 2H).
-(3-Chloro-2,2-dimethylpropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid
1
(
7c). Prepared from 6c, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.26 g, 86%). H NMR (400 MHz,
CDCl ): δ 11.26 (s, 1H), 3.71 (s, 2H), 2.96 (t, J = 6.88 Hz, 2H), 2.87 (t, J = 6.88 Hz, 2H), 2.38-2.45 (m,
3
2H), 1.45 (s, 6H).
2
-(2,2-Dichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7d). Prepared
1
from 6d, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.20 g, 79%). H NMR (400 MHz, DMSO-d
6
): δ
13.38 (br, 1H), 11.94 (s, 1H), 7.20 (s, 1H), 2.83-2.84 (m, 4H), 2.29-2.36 (m, 2H).

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HETEROCYCLES, Vol. 92, No. 12, 2016
2
-(2,2,2-Trichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic
acid
(7e).
1
Prepared from 6e, CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.30 g, 91%). H NMR (400 MHz,
DMSO-d
6
): δ 13.66 (br, 1H), 12.66 (s, 1H), 2.84-2.88 (m, 4H), 2.31-2.38 (m, 2H).
2
-Acrylamino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7f). Prepared from 6f,
1
CH
2
Cl
2
:MeOH = 20:1, to give white solid (0.21 g, 89%). H NMR (400 MHz, DMSO-d ): δ 13.02 (br,
6
1
1
2
H), 11.18 (s, 1H), 6.62 (dd, J = 16.96, 10.32 Hz, 1H), 6.30 (d, J = 16.96 Hz, 1H), 5.88 (d, J = 10.32 Hz,
H), 2.79-2.84 (m, 4H), 2.28-2.35 (m, 2H).
-(2-Methoxy-2-oxoacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7g).
1
Prepared from 6g, CH
DMSO-d
General procedure for preparation of compounds 8a-8f and 9
-Acylaminothiophene-3-carboxylic acid (1.0 mmol) was dissolved in DMF (20 mL) and HATU (1.5
2
Cl
2
:MeOH = 20:1, to give white solid (0.25 g, 93%). H NMR (400 MHz,
6
): δ 13.34 (br, 1H), 12.17 (s, 1H), 3.87 (s, 3H), 2.81-2.86 (m, 4H), 2.29-2.37 (m, 2H).
2
mmol) was added. After stirring at room temperature for 10 min, amine (1.5 mmol) was added. The
mixture was stirred at room temperature for another 2-3 h. Then the reaction was quenched with water
and the aqueous solution was extracted with EtOAc (3u20 mL). The combined organic layer was washed
with brine, dried over anhydrous Na
2
SO and concentrated. The crude product was purified by silica gel
4
column chromatography using a mixture of petroleum ether and ethyl acetate (7:1-2:1) as the eluent.
N-Benzyl-2-(3-chloropropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
(8a).
1
Prepared from 7a, petroleum ether:EtOAc = 5:1-2:1, to give white solid (0.21 g, yield 59%). H NMR
(
400 MHz, DMSO-d
6
): δ 11.74 (s, 1H), 7.73 (t, J = 5.2 Hz, 1H), 7.32-7.35 (m, 4H), 7.24-7.25 (m, 1H),
4.49 (d, J = 5.2 Hz, 2H), 3.86 (t, J = 6.08 Hz, 2H), 2.94-2.97 (m, 4H), 2.81 (t, J = 6.1 Hz, 2H), 2.33-2.41
1
3
(
m, 2H); C NMR (100 MHz, CDCl
3
) δ 166.46, 165.90, 149.70, 138.04, 137.14, 133.89, 129.12 (2C),
-
1
27.92, 127.60 (2C), 110.38, 43.55, 39.88, 39.45, 30.28, 28.73, 28.43. ESI-HRMS (m/z): [M-H] calcd.
for C18
H18ClN
2
2
O S, 361.0783; found 361.0791. mp 134.8-138.1 °C.
N-Benzyl-2-(4-chlorobutanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
(8b).
1
Prepared from 7b, petroleum ether:EtOAc = 2:1, to give white solid (0.30 g, yield 80%). H NMR (400
MHz, DMSO-d
6
): δ 11.72 (s, 1H), 7.69 (t, J = 5.7 Hz, 1H), 7.32-7.36 (m, 4H), 7.24-7.26 (m, 1H), 4.49 (d,
J = 5.7 Hz, 2H), 3.67 (t, J = 6.6 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.80 (t, J = 7.0 Hz, 2H), 2.58 (t, J = 7.3
13
Hz, 2H), 2.33-2.40 (m, 2H), 2.00-2.06 (m, 2H); C NMR (100 MHz, DMSO-d
6
): δ 168.41, 165.02,
1
2
0
47.62, 139.41, 138.53, 131.80, 128.25 (2C), 126.98 (2C), 126.68, 110.99, 44.56, 42.33, 32.86, 29.21,
+
8.22, 27.72, 27.70. ESI-HRMS (m/z): [M+Na] calcd. for C19
H21ClN
2
NaO S, 399.0904; found 399.
2
930. mp 83.1-86.4 °C.
N-Benzyl-2-(3-chloro-2,2-dimethylpropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbox-
amide (8c). Prepared from 7c, petroleum ether:EtOAc = 2:1, to give white solid (0.30 g, yield 76%).
1H

HETEROCYCLES, Vol. 92, No. 12, 2016
2159
NMR (400 MHz, DMSO-d
6
): δ 12.36 (s, 1H), 7.67 (t, J = 5.6 Hz, 1H), 7.32-7.34 (m, 4H), 7.24-7.26 (m,
1
2
1
H), 4.52 (d, J = 5.6 Hz, 2H), 3.75 (s, 2H), 2.98 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H), 2.36-2.40 (m,
13
H), 1.32 (s, 6H); C NMR (100 MHz, DMSO-d ): δ171.23, 165.41, 148.04, 139.27, 138.51, 132.16,
6
28.29 (2C), 126.91 (2C), 126.72, 111.07, 52.36, 44.22, 42.26, 29.19, 28.21, 27.74, 22.61 (2C).
+
ESI-HRMS (m/z): [M+Na] calcd. for C20
H23ClN
2
NaO S, 413.1061; found 413.1027. mp 149.6-150.0 °C.
2
N-Benzyl-2-(2,2-dichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8d).
1
Prepared from 7d, petroleum ether:EtOAc = 2:1, to give white solid (0.31 g, yield 82%). H NMR (400
MHz, DMSO-d
H), 4.52 (d, J = 6.0 Hz, 2H), 3.00 (t, J = 7.1 Hz, 2H), 2.85 (t, J = 7.1 Hz, 2H), 2.37-2.41 (m, 2H); C
NMR(100 MHz DMSO-d ): δ164.84, 160.50, 145.91, 139.15 (2C), 133.71, 128.29 (2C), 126.94 (2C),
26.74, 113.20, 66.40, 42.39, 29.15, 28.31, 27.71. ESI-HRMS (m/z): [M+Na]+ calcd. for
NaO S, 405.0202; found 405.0227. mp 142.4-143.2 °C.
6
): δ 12.72 (s, 1H), 7.86 (t, J = 6.0 Hz, 1H), 7.31-7.39 (m, 4H), 7.24-7.27 (m, 1H), 7.14 (s,
1
3
1
6
1
C
17
H
16
C
l2
N
2
2
N-Benzyl-2-(2,2,2-trichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8e).
1
Prepared from 7e, petroleum ether:EtOAc = 7:1-3:1, to give white solid (0.31 g, yield 73%). H NMR
(
400 MHz, DMSO-d
.54 (d, J = 5.3 Hz, 2H), 3.03 (t, J = 6.8Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 2.37-2.44 (m, 2H); C NMR
100 MHz DMSO-d ): δ 165.11, 157.90, 145.82, 139.47, 138.97, 134.57, 128.36 (2C), 126.94 (2C),
26.82, 113.71, 91.10, 42.42, 29.15, 28.40, 27.73. ESI-HRMS (m/z): [M+Na]+ calcd. for
NaO S, 438.9812; found 438.9824. mp 149.6-150.3 °C.
6
): δ 13.61 (s, 1H), 7.91 (t, J = 5.3 Hz, 1H), 7.31-7.36 (m, 4H), 7.23-7.27 (m, 1H),
1
3
4
(
6
1
C
17
H
15
C
l3
N
2
2
2
7
1
1
-Acrylamino-N-benzyl-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8f). Prepared from
1
f, petroleum ether:EtOAc = 2:1, to give white solid 0.23 g, yield 71%). H NMR (400 MHz, CDCl
3
): δ
2.22 (s, 1H), 7.33-7.37 (m, 5H), 6.47 (d, J = 16.9 Hz, 1H), 6.33 (dd, J = 16.9, 10.2 Hz, 1H), 6.22 (br,
1
3
H), 5.82 (d, J = 10.2 Hz, 1H), 4.62 (d, J = 5.2 Hz, 2H), 2.86-2.88 (m, 4H), 2.46-2.48 (m, 2H); C NMR
(
100 MHz, CDCl
3
): δ 164.98, 161.40, 147.27, 139.36, 138.86, 132.74, 130.45, 128.31, 128.27(2C),
+
1
26.97(2C), 126.69, 111.83, 42.36, 29.20, 28.28, 27.73. ESI-HRMS (m/z): [M+Na] calcd. for
C
18
H
18
N
2
NaO S, 349.0981; found 349.0961. mp 97.7-98.1 °C.
2
Methyl 2-((3-(benzylcarbamoyl)-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)amino)-2-oxoacetate
1
(
9). Prepared from 7g, petroleum ether:EtOAc = 5:1-2:1, to give white solid (0.26 g, 73%). H NMR (400
MHz, CDCl
5.64 Hz, 2H), 3.85(s, 3H), 3.01 (t, J = 6.68 Hz, 2H), 2.85 (t, J = 6.80 Hz, 2H), 2.38-2.43 (m, 2H).
-((3-(Benzylcarbamoyl)-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)amino)-2-oxoacetic acid (8g).
3
): δ 12.93 (s, 1H), 7.78 (t, J = 5.64 Hz, 1H), 7.32-7.34 (m, 4H), 7.24-7.26 (m, 1H), 4.52 (d, J
=
2
To a solution of 9 (0.28 g, 0.78 mmol) in 50 mL of MeOH was added a solution of LiOH (56 mg, 2.34
mmol) in water. After stirring the mixture at room temperature for 8 h, the solvent was evaporated and
adjusted the water pH to 3-4. Extracting with EtOAc, and concentrated the organic layer, the residue was

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HETEROCYCLES, Vol. 92, No. 12, 2016
purified by flash column chromatography using a mixture of CH
2
Cl : MeOH = 20:1, to give white solid
2
1
(
0.22 g), yield 82%. H NMR (400 MHz, DMSO-d
6
): δ 12.88 (s, 1H), 7.77 (t, J = 5.8 Hz, 1H), 7.32-7.33
(m, 4H), 7.24-7.25 (m, 1H), 4.51 (d, J = 5.8 Hz, 2H), 3.00 (t, J = 6.3 Hz, 2H), 2.85 (t, J = 6.7 Hz, 2H),
1
3
2
1
.38-2.41 (m, 2H); C NMR (100 MHz DMSO-d ): δ164.73, 160.33, 153.99, 146.19, 139.30, 139.19,
6
33.61, 128.29 (2C), 127.01 (2C), 126.72, 112.80, 42.37, 29.16, 28.31, 27.75. ESI-HRMS (m/z):
+
[
M+Na] calcd. for C17
H
16
N
2
NaO S, 367.0723; found 367.0730. mp 196.2-196.8 °C.
4
2
-((3-(Benzylcarbamoyl)-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)amino)-2-oxoethyl acetate (10).
To a solution of 5a (0.5 g, 1.43 mmol) in 20 mL of DMF was added potassium acetate (0.21 g, 2.14
mmol). Following reflux of the mixture for 3 h, the solution was evaporated, the mixture taken up in 70
ml of CH
2
Cl
2
and washed with saturated aqueous NaHCO
3
and brine. The organic layer was separated
and dried over anhydrous Na
2
SO , and then evaporated in vacuo. The crude product was purified by flash
4
column chromatography using a mixture of petroleum ether: EtOAc = 2:1 to give white solid (0.48 g,
1
9
7
2
0%). H NMR (400 MHz, DMSO-d
6
): δ 12.23 (s, 1H), 7.73 (t, J = 5.76 Hz, 1H), 7.32-7.34 (m, 4H),
.23-7.27 (m, 1H), 4.75 (s, 2H), 4.49 (d, J = 5.76 Hz, 2H), 2.97 (t, J = 6.96 Hz, 2H), 2.81 (t, J = 7.00 Hz,
H), 2.34-2.41 (m, 2H), 2.15 (s, 3H).
N-Benzyl-2-(2-hydroxyacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8h). To
a solution of 10 (0.3 g, 0.81 mmol) in 20 mL of MeOH was added a solution of Na CO (0.13 g, 1.21
2
3
mmol). After stirring the mixture at room temperature for 3 h, the solvent was evaporated and the residue
was purified by flash column chromatography using a mixture of petroleum ether: EtOAc = 1:1 to give
1
white solid (0.21 g, 78%). H NMR (400 MHz, DMSO-d
6
): δ 12.30 (s, 1H), 7.65 (t, J = 5.9 Hz, 1H),
7.32-7.33 (m, 4H), 7.22-7.26 (m, 1H), 6.08 (t, J = 5.8 Hz, 1H), 4.49 (d, J = 6.0 Hz, 2H ), 4.05 (d, J = 5.8
1
3
Hz, 2H), 2.98 (t, J = 7.1 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.36-2.41 (m, 2H); C NMR (100 MHz,
DMSO-d
6
): δ 169.64, 164.79, 147.16, 139.51, 138.54, 131.86, 128.26 (2C), 126.97 (2C), 126.66, 111.29,
+
6
1.04, 42.28, 29.26, 28.24, 27.74. ESI-HRMS (m/z): [M+Na] calcd. for C17
H
18
N
2
NaO S, 353.0930;
3
found 353.0944. mp 170.6-171.2 °C.
General procedure for preparation of compounds 11a-11e
2-Acylaminothiophene-3-carboxylic acid (1.0 mmol) was dissolved in DMF (20 mL) and HATU (1.5
mmol) was added. After stirring at room temperature for 10 min, amine (1.5 mmol) was added. The
mixture was stirred at room temperature for another 2-3 h. Then the reaction was quenched with water
and the aqueous solution was extracted with EtOAc (20 mLu3). The combined organic layer was washed
with brine, dried over anhydrous Na
column chromatography using a mixture of petroleum ether and EtOAc (7:1-3:1) as the eluent.
-(2-Chloroacetamino)-N-(4-phenylbutyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
2
SO and concentrated. The crude product was purified by silica gel
4
2
1
(
11a). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.29 g, yield 73%). H

HETEROCYCLES, Vol. 92, No. 12, 2016
2161
NMR (400 MHz, DMSO-d
6
): δ 12.46 (s, 1H), 7.25-7.29 (m, 2H), 7.15-7.21 (m, 4H), 4.53 (s, 2H),
3.27-3.32 (m, 2H), 2.91 (t, J = 6.8 Hz, 2H), 2.81 (t, J = 6.96 Hz, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.33-2.38
1
3
(
m, 2H), 1.52-1.63 (m, 4H); C NMR (100 MHz, DMSO-d
6
): δ 164.82, 163.42, 146.37, 142.16, 138.78,
132.60, 128.35 (2C), 128.28 (2C), 125.70, 112.36, 42.48, 38.67, 34.84, 29.10, 28.77, 28.46, 28.29, 27.75.
+
ESI-HRMS (m/z): [M+Na] calcd. for C20
H23ClN O
2 2
S, 413.1061; found 413.1030. mp 107.3-108.4 °C.
2
-(2-Chloroacetamino)-N-(4-methoxybenzyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxami-
1
de (11b). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.30 g, yield 79%). H
NMR (400 MHz, DMSO-d ): δ 12.41 (s, 1H), 7.65 (t, J = 5.9 Hz, 1H), 7.24 (d, J = 8.5 Hz, 2H), 6.89 (d, J
8.5 Hz, 2H), 4.52 (s, 2H), 4.43 (d, J = 6.0 Hz, 2H), 3.73 (s, 3H), 2.95 (t, J = 6.9 Hz, 2H), 2.82 (t, J = 7.0
6
=
1
3
Hz, 2H), 2.35-2.39 (m, 2H); C NMR (100 MHz, DMSO-d
6
): δ 164.82, 163.47, 158.21, 146.78, 138.76,
1
32.68, 131.27, 128.42 (2C), 113.73 (2C), 112.08, 55.06, 42.49, 41.81, 29.23, 28.29, 27.76. ESI-HRMS
+
(
m/z): [M+Na] calcd. for C18
H19ClN
2
3
NaO S, 401.0697; found 401.0670. mp 148.7-149.2 °C.
2
-(2-Chloroacetamino)-N-(2-chlorobenzyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
1
(
11c). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.25 g, yield 64%). H
NMR (400 MHz, DMSO-d ): δ 12.35 (s, 1H), 7.76 (t, J = 5.8 Hz, 1H), 7.47 (d, J = 7.32 Hz, 1H),
.29-7.34 (m, 3H), 4.56 (d, J = 5.8 Hz, 2H), 4.53 (s, 2H), 3.02 (t, J = 6.9 Hz, 2H), 2.84 (t, J = 7.0 Hz, 2H),
6
7
2
1
1
3
.38-2.42 (m, 2H); C NMR (100 MHz, DMSO-d ): δ 165.09, 163.55, 147.11, 138.79, 136.11, 132.80,
6
31.79, 129.16, 128.59, 128.31, 127.27, 111.79, 42.48, 40.49, 29.24, 28.32, 27.78. ESI-HRMS (m/z):
+
[
M+Na] calcd. forC17
H16Cl
2
N
2
NaO S, 405.0202; found 405.0211. mp 183.1-183.6 °C.
2
2
-(2-Chloroacetamino)-N-(3-chlorobenzyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
1
(
11d). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.30 g, yield 78%). H
NMR (400 MHz, DMSO-d
6
): δ 12.37 (s, 1H), 7.81 (t, J = 6.0 Hz, 1H), 7.35-7.39 (m, 2H), 7.28-7.32 (m,
2
2
1
H), 4.53 (s, 2H), 4.50 (d, J = 6.0 Hz, 1H), 2.99 (t, J = 7.0 Hz, 2H), 2.83 (t, J = 7.0 Hz, 2H), 2.35-2.42 (m,
1
3
H); C NMR: (100 MHz, DMSO-d
6
) δ165.02, 163.52, 147.02, 142.07, 138.77, 132.97, 132.74, 130.23,
+
26.91, 126.73, 125.73, 111.84, 42.49, 41.96, 29.25, 28.31, 27.75. ESI-HRMS (m/z): [M+Na] calcd. for
C
17
H
16
C
l2
N
2
NaO S, 405.0202; found 405.0227. mp 141.6-142.1 °C.
2
2
-(2-Chloroacetamino)-N-(4-chlorobenzyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
1
(
11e). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.35 g, yield 92%). H
NMR (400 MHz, DMSO-d
6
): δ 12.37 (s, 1H), 7.78 (t, J = 5.7 Hz, 1H), 7.39 (d, J = 8.2 Hz, 2H), 7.34 (d, J
=
8.2 Hz, 2H), 4.52 (s, 2H), 4.48 (d, J = 6.0 Hz, 2H), 2.98 (d, J = 6.3 Hz, 2H), 2.82 (t, J = 6.7 Hz, 2H),
1
3
2
.37-2.40 (m, 2H); C NMR (100 MHz DMSO-d
31.30, 128.93 (2C), 128.27 (2C), 111.87, 42.49, 41.80, 29.25, 28.31, 27.76. ESI-HRMS (m/z): [M+Na]+
NaO S, 405.0202; found 405.0198. mp 156.6-158.0 °C.
6
): δ 164.99, 163.51, 146.98, 138.76, 138.47, 132.73,
1
calcd. for C17
H16Cl
2
N
2
2

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HETEROCYCLES, Vol. 92, No. 12, 2016
Cell viability assay
Cell viability was measured by the SRB assay. MGC-803 cells (1500 cells/well) and HCT-116 (1800
cells/well) of 180 μL medium were seeded in 96-well plates and incubated overnight for cell adhering
before treatments. Cells were then treated with each compound at increasing concentrations ranging from
0
.03-30 μM giving the final volume of 200 μL each well and incubated for 72 h. Remove the cell culture
medium, gently, add 100 μL cold 10% TCA (20 g TCA in 200 mL ddH O) to each well, and incubate the
plates at 4 qC for 1 h. Washing the plates five times with ddH O, then allow them to air-dry at room
2
2
temperature. Adding 100 μL 0.4% SRB solution (0.8 g SRB in 200 mL 1% acetic acid) to each well,
drying at room temperature for 15 min and then quickly rinse the plates five times with 1% acetic acid
(
5ml acetic acid in 500ml ddH
2
O) to remove unbound dye then allow them to air-dry at room temperature.
O) to each well to solubilize the
Adding 150 μL 10 mM Tris base solution (0.6057 g Tris in 500 mL ddH
2
protein-bound dye. Absorbance was read at 570 nm with Spectra Max M 5 microplate spectrophotometer.
The IC50 value was defined as the concentration of drug that inhibits 50% cell growth compared with the
cisplatin (positive control).
Annexin V-FITC and propidium iodide (PI) double staining
HCT-116 (250000 cells/dish) of 5 mL medium were seeded in 60 mm dishes and incubated overnight for
cell adhering before treatments. Cells were then treated with 11b, 11d of 10 μM each dish and incubated
for 48 h, the control treated with DMSO. After 48 h, an Annexin V-FITC/PI binding assay was conducted
using a purchased kit and the manufacturer’s protocol (Dojindo, Japan). HCT-116 cells were collected,
6
washed twice with PBS and resuspended in Annexin V-FITC binding solution for 1u10 cells/ml, and
incubated with 5 μL Annexin V-FITC in the dark for 15 min at room temperature. Then added 400 μL
Annexin V-FITC binding solution and 5 μL propidium iodide (PI) and kept in the dark at room
temperature. Flow analysis was immediately performed using a flow cytometer (Guava easy Cyte
6HT-2L, Merck Millipore).
General method for western blot analysis
Cells were treated with compounds (10 μM) for 48 h, after treatment, the both floating and adherent cells
were collected, washed twice with PBS (pH 7.4) and suspended in RIPA lysis buffer containing 1X
protease inhibitor for 30 min on ice. Samples were centrifuged at 12000 rpm for 30 min at 4 °C and
supernatant was collected as total protein lysate. Protein concentrations were quantified with the DC
Protein Assay Kit and bovine serum albumin as standard, total protein was divided into the gel-loading
concentration of proteins (40 μg) and heated for 5 min at 100 °C. Equal amount of protein was loaded on
10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to a
PVDF membrane. After blocking with 5% skim milk at room temperature for 1 h, membranes were

HETEROCYCLES, Vol. 92, No. 12, 2016
2163
washed with TPBS and membrane was incubated with the desired antibody for 2 h either at room
temperature or at 4 °C overnight. The membrane was then incubated with corresponding
peroxidase-conjugated secondary antibody for 2 h (cleaved-caspase 3 1:1000, PARP1 1:500) at room
temperature, and protein expression was detected by ECL kit and visualized using a chemiluminescence
detection system (Amersham Imager 600, GE).
General method for evaluating MMP
HCT-116 cells were treated with compounds (10 μM) for 24 h, after treatment, the both floating and
adherent cells were harvested, and washed twice with PBS; 106 cells were incubated in 0.5 mL PBS
containing 10 μg JC-1 for 15 min at 37 °C in the dark. Stained cells were washed twice with PBS,
resuspended in 500 μL PBS, and used immediately with flow cytometer (Guava easy Cyte 6HT-2L,
Merck Millipore).
ACKNOWLEDGEMENTS
This research was financially supported by The National Key Technology R&D Program (No.
2015BAK45B00), National Natural Science Foundation of China (No. 81472788, 81272463), Major
State Basic Research Development Program of China (No. 2015CB910400), Shanghai Science and
Technology Council (No. 14DZ0511800, 14142201200). We also thank the Laboratory of Organic
Functional Molecules, Sino-French Institute of ECNU for support.
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