HETEROCYCLES

Paper | Regular issue | Vol. 92, No. 12, 2016, pp. 2145-2165
Received, 1st August, 2016, Accepted, 21st October, 2016, Published online, 9th November, 2016.

■ Design, Synthesis, in vitro Antiproliferative Activity Evaluation of 2-Acylaminothiopene-3-carboxamide Derivatives

Jiefeng Zhang, Fengjie Guan, Jiakun Qiu, Yanfen Fang, Lifang Yu, Jingya Li, Fan Yang, Xiongwen Zhang, Jia Li, and Jie Tang*

Department of Chemistry, East China Normal University, 3663 North Zhongshan Road, 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 range of biological activities, such as anti-diabetic,1 anti-virus (compound I, Figure 1),2 anti-bacterial,3 anti-inflammatory,4 anti-neurodegenerative (compound II, Figure 1),5 analgesic,6 cystic fibrosis treatment7 and aspartic protease inhibiting activities8 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 moderate effect on the parental cells.9 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 (compound V, Figure 1)11 were also reported and compound V showed inhibition against A-549 cells with the EC50 = 2.0 0.2 μM.

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 ± 0.08 and 19.80 ± 0.65 μΜ,

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 Gewald cyclization.14 Acylation of the 2-amino group of 2a-2d afforded 3a-3d which subsequently gave 4a-4d after removing the tert-butyl group.15 HATU-facilitated condensations of 4a-4d with benzylamine produced 5a-5c and compound 116 (Scheme 1).

Acylation of the 2-amino group of 2a with corresponding acid chlorides in the presence of Et3N afforded 6a-6f. In addition, 6g was prepared by condensation of 2a with dimethyl oxalate in the presence of NaH.17 Subsequent deprotection of the tert-butyl group in 6a-6g afforded 7a-7g which underwent the HATU-facilitated condensations with benzylamine, giving 8a-8f and 9. Hydrolysis of 9 with LiOH/H2O18 afforded product 8g (Scheme 2). Nucleophilic substitution of 5a with potassium acetate19 followed by deacetylation provided 8h (Scheme 3).

HATU-facilitated coupling reactions of 4a with corresponding amines produced 11a-11e (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).
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 ± 0.11 and 3.41 ± 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 α-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 ± 0.11 μM), IC50 of 8a in which the chain was extended with one carbon was increasing to 14.63 ± 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 β-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 ± 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 ± 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 play a significant role in regulating the anti-proliferative activities.12 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 11a in which the chain length was prolonged to four carbons maintained anti-proliferative activity against both MGC-803 (IC50 = 3.89 ± 0.30 μM) and HCT-116 (IC50 = 8.03 ± 0.63 μM). While 11b having a 4-methyoxy group on the aromatic ring showed an increased anti-proliferative activity against both MGC-803 (IC50 = 2.32 ± 0.39 μM) and HCT-116 (IC50 = 5.89 ± 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.

Compounds 11b and 11d induce apoptotic cell death
Apoptosis is a programmed cell death process by which the body eliminates damaged or unnecessary cells and is playing a vital role in cancer development and tumor cell survival.21 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 and propidium iodide (PI)22 (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.

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 (PARP1).23 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.

Compounds 11b and 11d decrease the mitochondrial membrane potential (MMP)
Decreasing of MMP during apoptosis has been reported in a number of studies, leading to the general notion that depolarization of mitochondria is one of the first events to occur during apoptosis.24 The effect of 11b and 11d on the MMP in HCT-116 cells was investigated with the fluorescent probe JC-1, a mitochondrion-specific and voltage-dependent dye.25 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).

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
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 using Qingdao Haiyang silica gel (200 - 300) with distilled solvents. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker DRX-400 Fourier transform spectrometer. 1H chemical shifts are reported in δ (ppm) using the δ 7.26 signal of CDCl3 or the δ 2.50 signal of DMSO-d6 as internal standards. 13C chemical shifts are reported in δ (ppm) using the δ 77.23 signal of CDCl3 as internal standard or the δ 39.52 signal of DMSO-d6 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 Na2SO4, and 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-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (2a). Prepared from cyclopentanone, petroleum ether:EtOAc = 20:1, to give white solid (0.17 g, 72%). 1H NMR (400 MHz, CDCl3): 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).
tert-Butyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2b). Prepared from cyclohexanone, petroleum ether:EtOAc = 20:1, to give white solid (0.13 g, 52%). 1H NMR (400 MHz, CDCl3): 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).
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, 64%). 1H NMR (400 MHz, CDCl3): 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 ether:EtOAc = 20:1, to give white solid (0.13 g, 56%). 1H NMR (400 MHz, CDCl3): 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 CH2Cl2 (15.0 mL) and treated with triethylamine (3.0 mmol). To this mixture, the solution of acid chloride (1.2 mmol) in CH2Cl2 (5 mL) was added dropwise under 0 °C. This mixture was stirred for 0.5 h at room temperature. The mixture was diluted by CH2Cl2, and washed with water. The organic phase was dried over anhydrous Na2SO4, and 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 (0.29 g, 92%). 1H NMR (400 MHz, CDCl3): δ 11.90 (s, 1H), 4.24 (s, 2H), 2.83-2.90 (m, 4H), 2.34-2.41 (m, 2H), 1.58 (s, 9H).
tert-Butyl 2-(2-chloroacetamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (3b). Prepared from 2b, petroleum ether:EtOAc = 20:1, to give white solid (0.33 g, 99%). 1H NMR (400 MHz, CDCl3): 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, 9H).
tert-Butyl 2-(2-chloroacetamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylate (3c). Prepared from 2c, petroleum ether:EtOAc = 20:1, to give white solid (0.33 g, 95%). 1H NMR (400 MHz, CDCl3): 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, petroleum ether:EtOAc = 20:1, to give white solid (0.25 g, 81%). 1H NMR (400 MHz, CDCl3): δ 12.19 (s, 1H), 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 CH2Cl2. This mixture was stirred for 2-3 h at room temperature and diluted by CH2Cl2, and washed with water. The organic phase was dried over anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography using a mixture of CH2Cl2 and MeOH (20:1) as the eluent.
2-(2-Chloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (4a). Prepared from 3a, CH2Cl2:MeOH = 20:1, to give white solid (0.24 g, 93%). 1H NMR (400 MHz, DMSO-d6): δ 11.69 (s, 1H), 4.58 (s, 2H), 2.79-2.85 (m, 4H), 2.28-2.35 (m, 2H).
2-(2-Chloroacetamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (4b). Prepared from 3b, CH2Cl2:MeOH = 20:1, to give white solid (0.27 g, 99%). 1H NMR (400 MHz, DMSO-d6): δ 13.25 (br, 1H), 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-(2-Chloroacetamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid (4c). Prepared from 3c, CH2Cl2:MeOH = 20:1, to give white solid (0.27 g, 93%). 1H NMR (400 MHz, DMSO-d6): 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, CH2Cl2:MeOH = 20:1, to give white solid (0.23 g, 92%). 1H NMR (400 MHz, DMSO-d6): δ 13.38 (br, 1H), 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.
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 mL×3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The crude product was purified by silica gel 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). Prepared from 4a, petroleum ether:EtOAc = 5:1-2:1, to white solid (0.1 g, yield 29%). 1H NMR (400 MHz, DMSO-d6): 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, 2H), 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); 13C NMR (100 MHz, DMSO-d6): δ 164.95, 163.48, 146.88, 139.37, 138.78, 132.69, 128.34, 126.98 (2C), 126.76 (2C), 112.00, 42.49, 42.35, 29.25, 28.30, 27.77. ESI-HRMS (m/z): [M-H]- calcd. for C17H16ClN2O2S, 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 from 4b, petroleum ether:EtOAc = 3:1-2:1, to give white solid (0.23 g, yield 63%). 1H NMR (400 MHz, CDCl3): 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), 2.67-2.69 (m, 4H), 1.82 (s, 4H); 13C NMR (100 MHz, DMSO-d6): δ 164.95, 163.48, 139.99, 139.27, 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 C18H19ClN2NaO2S, 385.0748; found 385.0756. mp 196.4-197.2 °C.
N-Benzyl-2-(2-chloroacetamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (5c). Prepared from 4c, petroleum ether:EtOAc = 7:1-3:1, to give white solid (0.09 g, yield 23%). 1H NMR (400 MHz, DMSO-d6): 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), 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); 13C NMR (100 MHz, DMSO-d6): δ 164.80, 163.49, 139.22, 135.01, 133.66, 131.30, 128.24 (2C), 127.27 (2C), 126.76, 123.57, 42.66, 42.35, 31.61, 28.29, 28.02, 27.67, 27.09. ESI-HRMS (m/z): [M+Na]+ calcd. for C19H21ClN2NaO2S, 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, petroleum ether:EtOAc = 7:1-3:1, to give white solid (0.27 g, yield 81%). 1H NMR (400 MHz, DMSO-d6): 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 = 5.6 Hz, 2H), 4.46 (s, 2H), 2.24 (s, 3H), 2.17 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 164.98, 163.36, 139.16, 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 C16H17ClN2NaO2S, 359.0591; found 359.0622. mp 160.2-161.5 °C.
General procedure for preparation of compounds 6a–6f
tert-Butyl 2-aminothiophene-3-carboxylate (2a) (1.0 mmol) was dissolved in CH2Cl2 (15 mL) and treated with triethylamine (3 mmol). To this mixture, the solution of acid chloride (1.2 mmol) in CH2Cl2 (5 mL) was added dropwise under 0 °C. This mixture was stirred for 0.5 h at room temperature. The mixture was diluted by CH2Cl2 (20 mL), and washed with water. The organic phase was dried over anhydrous Na2SO4, 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 mmol), petroleum ether:EtOAc = 2:1, to give pale yellow solid 0.267 g. Yield 81%. 1H NMR (400 MHz, CDCl3): 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 (0.30 g, 88%). 1H NMR (400 MHz, CDCl3): 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 pale yellow solid (0.31 g, 86%). 1H NMR (400 MHz, CDCl3): 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 (0.28 g, 79%). 1H NMR (400 MHz, CDCl3): δ 12.08 (s, 1H), 6.13 (s, 1H), 2.84-2.91 (m, 4H), 2.34-2.42 (m, 2H), 1.58 (s, 9H).
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 (0.32 g, 82%). 1H NMR (400 MHz, CDCl3): δ 12.45 (s, 1H), 2.88-2.91 (m, 4H), 2.36-2.43 (m, 2H), 1.58 (s, 9H).
tert-Butyl 2-acrylamino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (6f). Prepared from 2a and 3-chloropropanoyl chloride, petroleum ether:EtOAc = 2:1, to give pale yellow solid (0.27 g, 92%). 1H NMR (400 MHz, CDCl3): δ 11.22 (s, 1H), 6.48 (d, J = 16.96 Hz, 1H), 6.34 (dd, J = 16.96, 10.20 Hz, 1H), 5.84 (d, J = 10.20 Hz, 1H), 2.83-2.89 (m, 4H), 2.33-2.41 (m, 2H), 1.57 (s, 9H).
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 (Na2SO4) and filtered, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether:EtOAc = 5:1, to give white solid (1.07g, 79%). 1H NMR (400 MHz, CDCl3): δ 12.25 (s, 1H), 3.99 (s, 3H), 2.85-2.92 (m, 4H), 2.35-2.42 (m, 2H), 1.59 (s, 9H).
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 CH2Cl2 (20 mL). This mixture was stirred for 2-3 h at room temperature and diluted by CH2Cl2, and washed with water. The organic phase was dried over anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography using a mixture of CH2Cl2 and MeOH (20:1) as the eluent.
2-(3-Chloropropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7a). Prepared from 6a, CH2Cl2:MeOH = 20:1, to give white solid (0.26 g, 95%). 1H NMR (400 MHz, DMSO-d6): δ 12.99 (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), 2.27-2.34 (m, 2H).
2-(4-Chlorobutanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7b). Prepared from 6b, CH2Cl2:MeOH = 20:1, to give white solid (0.24 g, 85%). 1H NMR (400 MHz, DMSO-d6): δ 12.97 (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), 2.27-2.34 (m, 2H), 2.02-2.09 (m, 2H).
2-(3-Chloro-2,2-dimethylpropanamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7c). Prepared from 6c, CH2Cl2:MeOH = 20:1, to give white solid (0.26 g, 86%). 1H NMR (400 MHz, CDCl3): 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, 2H), 1.45 (s, 6H).
2-(2,2-Dichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7d). Prepared from 6d, CH2Cl2:MeOH = 20:1, to give white solid (0.20 g, 79%). 1H NMR (400 MHz, DMSO-d6): 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).
2-(2,2,2-Trichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7e). Prepared from 6e, CH2Cl2:MeOH = 20:1, to give white solid (0.30 g, 91%). 1H NMR (400 MHz, DMSO-d6): 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, CH2Cl2:MeOH = 20:1, to give white solid (0.21 g, 89%). 1H NMR (400 MHz, DMSO-d6): δ 13.02 (br, 1H), 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, 1H), 2.79-2.84 (m, 4H), 2.28-2.35 (m, 2H).
2-(2-Methoxy-2-oxoacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (7g). Prepared from 6g, CH2Cl2:MeOH = 20:1, to give white solid (0.25 g, 93%). 1H NMR (400 MHz, DMSO-d6): 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).
General procedure for preparation of compounds 8a-8f and 9
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 (3×20 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The crude product was purified by silica gel 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). Prepared from 7a, petroleum ether:EtOAc = 5:1-2:1, to give white solid (0.21 g, yield 59%). 1H NMR (400 MHz, DMSO-d6): 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 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 166.46, 165.90, 149.70, 138.04, 137.14, 133.89, 129.12 (2C), 127.92, 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 C18H18ClN2O2S, 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). Prepared from 7b, petroleum ether:EtOAc = 2:1, to give white solid (0.30 g, yield 80%). 1H NMR (400 MHz, DMSO-d6): 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 Hz, 2H), 2.33-2.40 (m, 2H), 2.00-2.06 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 168.41, 165.02, 147.62, 139.41, 138.53, 131.80, 128.25 (2C), 126.98 (2C), 126.68, 110.99, 44.56, 42.33, 32.86, 29.21, 28.22, 27.72, 27.70. ESI-HRMS (m/z): [M+Na]+ calcd. for C19H21ClN2NaO2S, 399.0904; found 399. 0930. 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 NMR (400 MHz, DMSO-d6): δ 12.36 (s, 1H), 7.67 (t, J = 5.6 Hz, 1H), 7.32-7.34 (m, 4H), 7.24-7.26 (m, 1H), 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, 2H), 1.32 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ171.23, 165.41, 148.04, 139.27, 138.51, 132.16, 128.29 (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 C20H23ClN2NaO2S, 413.1061; found 413.1027. mp 149.6-150.0 °C.
N-Benzyl-2-(2,2-dichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8d). Prepared from 7d, petroleum ether:EtOAc = 2:1, to give white solid (0.31 g, yield 82%). 1H NMR (400 MHz, DMSO-d6): 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, 1H), 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); 13C NMR(100 MHz DMSO-d6): δ164.84, 160.50, 145.91, 139.15 (2C), 133.71, 128.29 (2C), 126.94 (2C), 126.74, 113.20, 66.40, 42.39, 29.15, 28.31, 27.71. ESI-HRMS (m/z): [M+Na]+ calcd. for C17H16Cl2N2NaO2S, 405.0202; found 405.0227. mp 142.4-143.2 °C.
N-Benzyl-2-(2,2,2-trichloroacetamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8e). Prepared from 7e, petroleum ether:EtOAc = 7:1-3:1, to give white solid (0.31 g, yield 73%). 1H NMR (400 MHz, DMSO-d6): 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), 4.54 (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); 13C NMR (100 MHz DMSO-d6): δ 165.11, 157.90, 145.82, 139.47, 138.97, 134.57, 128.36 (2C), 126.94 (2C), 126.82, 113.71, 91.10, 42.42, 29.15, 28.40, 27.73. ESI-HRMS (m/z): [M+Na]+ calcd. for C17H15Cl3N2NaO2S, 438.9812; found 438.9824. mp 149.6-150.3 °C.
2-Acrylamino-N-benzyl-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8f). Prepared from 7f, petroleum ether:EtOAc = 2:1, to give white solid 0.23 g, yield 71%). 1H NMR (400 MHz, CDCl3): δ 12.22 (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, 1H), 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); 13C NMR (100 MHz, CDCl3): δ 164.98, 161.40, 147.27, 139.36, 138.86, 132.74, 130.45, 128.31, 128.27(2C), 126.97(2C), 126.69, 111.83, 42.36, 29.20, 28.28, 27.73. ESI-HRMS (m/z): [M+Na]+ calcd. for C18H18N2NaO2S, 349.0981; found 349.0961. mp 97.7-98.1 °C.
Methyl 2-((3-(benzylcarbamoyl)-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)amino)-2-oxoacetate (9). Prepared from 7g, petroleum ether:EtOAc = 5:1-2:1, to give white solid (0.26 g, 73%). 1H NMR (400 MHz, CDCl3): 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 = 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).
2-((3-(Benzylcarbamoyl)-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)amino)-2-oxoacetic acid (8g). 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 purified by flash column chromatography using a mixture of CH2Cl2: MeOH = 20:1, to give white solid (0.22 g), yield 82%. 1H NMR (400 MHz, DMSO-d6): 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), 2.38-2.41 (m, 2H); 13C NMR (100 MHz DMSO-d6): δ164.73, 160.33, 153.99, 146.19, 139.30, 139.19, 133.61, 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 C17H16N2NaO4S, 367.0723; found 367.0730. mp 196.2-196.8 °C.
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 CH2Cl2 and washed with saturated aqueous NaHCO3 and brine. The organic layer was separated and dried over anhydrous Na2SO4, and then evaporated in vacuo. The crude product was purified by flash column chromatography using a mixture of petroleum ether: EtOAc = 2:1 to give white solid (0.48 g, 90%).1H NMR (400 MHz, DMSO-d6): δ 12.23 (s, 1H), 7.73 (t, J = 5.76 Hz, 1H), 7.32-7.34 (m, 4H), 7.23-7.27 (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, 2H), 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 Na2CO3 (0.13 g, 1.21 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 white solid (0.21 g, 78%). 1H NMR (400 MHz, DMSO-d6): 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 Hz, 2H), 2.98 (t, J = 7.1 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.36-2.41 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 169.64, 164.79, 147.16, 139.51, 138.54, 131.86, 128.26 (2C), 126.97 (2C), 126.66, 111.29, 61.04, 42.28, 29.26, 28.24, 27.74. ESI-HRMS (m/z): [M+Na]+ calcd. for C17H18N2NaO3S, 353.0930; 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 mL×3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and EtOAc (7:1-3:1) as the eluent.
2-(2-Chloroacetamino)-N-(4-phenylbutyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (11a). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.29 g, yield 73%). 1H NMR (400 MHz, DMSO-d6): 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 (m, 2H), 1.52-1.63 (m, 4H); 13C NMR (100 MHz, DMSO-d6): 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 C20H23ClN2O2S, 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-de (11b). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.30 g, yield 79%). 1H NMR (400 MHz, DMSO-d6): 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 Hz, 2H), 2.35-2.39 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 164.82, 163.47, 158.21, 146.78, 138.76, 132.68, 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 C18H19ClN2NaO3S, 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 (11c). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.25 g, yield 64%). 1H NMR (400 MHz, DMSO-d6): δ 12.35 (s, 1H), 7.76 (t, J = 5.8 Hz, 1H), 7.47 (d, J = 7.32 Hz, 1H), 7.29-7.34 (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), 2.38-2.42 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 165.09, 163.55, 147.11, 138.79, 136.11, 132.80, 131.79, 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. for C17H16Cl2N2NaO2S, 405.0202; found 405.0211. mp 183.1-183.6 °C.
2-(2-Chloroacetamino)-N-(3-chlorobenzyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (11d). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.30 g, yield 78%). 1H NMR (400 MHz, DMSO-d6): δ 12.37 (s, 1H), 7.81 (t, J = 6.0 Hz, 1H), 7.35-7.39 (m, 2H), 7.28-7.32 (m, 2H), 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, 2H); 13C NMR: (100 MHz, DMSO-d6) δ 165.02, 163.52, 147.02, 142.07, 138.77, 132.97, 132.74, 130.23, 126.91, 126.73, 125.73, 111.84, 42.49, 41.96, 29.25, 28.31, 27.75. ESI-HRMS (m/z): [M+Na]+ calcd. for C17H16Cl2N2NaO2S, 405.0202; found 405.0227. mp 141.6-142.1 °C.
2-(2-Chloroacetamino)-N-(4-chlorobenzyl)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (11e). Prepared from 4a, petroleum ether: EtOAc = 7:1-3:1, to give white solid (0.35 g, yield 92%). 1H NMR (400 MHz, DMSO-d6): 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), 2.37-2.40 (m, 2H); 13C NMR (100 MHz DMSO-d6): δ 164.99, 163.51, 146.98, 138.76, 138.47, 132.73, 131.30, 128.93 (2C), 128.27 (2C), 111.87, 42.49, 41.80, 29.25, 28.31, 27.76. ESI-HRMS (m/z): [M+Na]+ calcd. for C17H16Cl2N2NaO2S, 405.0202; found 405.0198. mp 156.6-158.0 °C.
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 ddH2O) to each well, and incubate the plates at 4 °C for 1 h. Washing the plates five times with ddH2O, then allow them to air-dry at room 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 ddH2O) to remove unbound dye then allow them to air-dry at room temperature. Adding 150 μL 10 mM Tris base solution (0.6057 g Tris in 500 mL ddH2O) to each well to solubilize the 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, washed twice with PBS and resuspended in Annexin V-FITC binding solution for 1×106 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 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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