Anti-glioma effects of 2-aminothiophene-3-carboxamide derivatives, ANO1 channel blockers

Article abstract of DOI:10.1016/j.ejmech.2020.112688

Anoctamin1 (ANO1), a calcium-activated chloride ion channel (CaCC), is associated with various physiological functions including cancer progression and metastasis/invasion. ANO1 has been considered as a promising target for cancer therapeutics as ANO1 is over-expressed in a variety of cancers including glioblastoma (GBM) and inhibition of ANO1 has been reported to suppress cell proliferation, migration and invasion in GBM. GBM is one of the most common and aggressive cancers with poor prognosis with median survival for 15 months. Lack of effective treatment options against GBM emphasizes urgent necessity of effective GBM therapeutics. In an effort to discover potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and synthesized a series of new 2-aminothiophene-3-carboxamide derivatives and performed SAR studies using both fluorescent cellular membrane potential assay and whole-cell patch-clamp recording. We observed that among these substances, 9c and 10q strongly suppress ANO1 channel activities and possess remarkable selectivity over ANO2. Unique structural feature of 10q, a cyclopentane-fused thiophene-3-carboxamide derivative, is the presence of benzoylthiourea functionality which dramatically contributes to activity. Both 9c and 10q suppress more strongly proliferation of GBM cells than four reference compounds including 3, Ani-9 and are also capable of inhibiting much more strongly colony formation than reference compounds in both 2D colony formation assay and 3D soft agar assay using U251 glioma cells. In addition, 9c and 10q suppress far more strongly migration/invasion of GBM cells than reference compounds. We, for the first time, found that the combination of ANO1 inhibitor (9c or 3) and temozolomide (TMZ) brings about remarkable synergistic effects in suppressing proliferation of GBM cells. Our study may provide an insight into designing selective and potent ANO1 inhibitors aiming at GBM treatment.

Full text of DOI:10.1016/j.ejmech.2020.112688

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Anti-glioma effects of 2-aminothiophene-3-carboxamide derivatives, ANO1 channel
blockers
Seunghye Choi, SeongShick Ryu, Kyoungmi Sim, Chiman Song, Injae Shin, Seong-
Seop Kim, Young-Sun Lee, Jae-Yong Park, Taebo Sim
PII:
S0223-5234(20)30660-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112688
EJMECH 112688
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 June 2020
Revised Date: 15 July 2020
Accepted Date: 21 July 2020
Please cite this article as: S. Choi, S. Ryu, K. Sim, C. Song, I. Shin, S.-S. Kim, Y.-S. Lee, J.-Y. Park,
T. Sim, Anti-glioma effects of 2-aminothiophene-3-carboxamide derivatives, ANO1 channel blockers,
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Anti-glioma effects of 2-aminothiophene-3-carboxamide derivatives, ANO1 channel blockers
Seunghye Choia,1 SeongShick Ryua,1, Kyoungmi Simb, Chiman Songc,d, Injae Shina,e Seong-Seop Kimb,
Young-Sun Leeb, Jae-Yong Parkb and Taebo Sima,c,e,*
a KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro,
Seongbuk-gu, Seoul 02841, Republic of Korea.
b
School of Biosystems and Biomedical Sciences, College of Health Sciences, Korea University,
Seoul 02841, Republic of Korea.
c
Chemical Kinomics Research Center, Korea Institute of Science and Technology, 5 Hwarangro 14-
gil, Seongbuk-gu, Seoul 02792, Republic of Korea.
d Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National
University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
e
Severance Biomedical Science Institute, Yonsei University College of Medicine, 50-1 Yonsei-ro,
Seodaemun-gu, Seoul 03722, Republic of Korea.
1 These authors are equally contributed to this work.
ABSTRACT
Anoctamin1 (ANO1), a calcium-activated chloride ion channel (CaCC), is associated with
various physiological functions including cancer progression and metastasis/invasion. ANO1 has been
considered as a promising target for cancer therapeutics as ANO1 is over-expressed in a variety of
cancers including glioblastoma (GBM) and inhibition of ANO1 has been reported to suppress cell
proliferation, migration and invasion in GBM. GBM is one of the most common and aggressive
cancers with poor prognosis with median survival for 15 months. Lack of effective treatment options
against GBM emphasizes urgent necessity of effective GBM therapeutics. In an effort to discover
potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and
synthesized a series of new 2-aminothiophene-3-carboxamide derivatives and performed SAR studies
using both fluorescent cellular membrane potential assay and whole-cell patch-clamp recording. We
observed that among these substances, 9c and 10q strongly suppress ANO1 channel activities and
possess remarkable selectivity over ANO2. Unique structural feature of 10q, a cyclopentane-fused
thiophene-3-carboxamide derivative, is the presence of benzoylthiourea functionality which
dramatically contributes to activity. Both 9c and 10q suppress more strongly proliferation of GBM

cells than four reference compounds including 3, Ani-9 and are also capable of inhibiting much more
strongly colony formation than reference compounds in both 2D colony formation assay and 3D soft
agar assay using U251 glioma cells. In addition, 9c and 10q suppress far more strongly
migration/invasion of GBM cells than reference compounds. We, for the first time, found that the
combination of ANO1 inhibitor (9c or 3) and temozolomide (TMZ) brings about remarkable
synergistic effects in suppressing proliferation of GBM cells. Our study may provide an insight into
designing selective and potent ANO1 inhibitors aiming at GBM treatment.
Keywords: ANO1 chloride ion channel, ANO1 inhibitors, glioblastoma (GBM), 2-aminothiophene-
3-carboxamides, combination of TMZ and ANO1 inhibitor
1. INTRODUCTION
Anotamin-1 (ANO1), a Ca2+-activated chloride ion channel (CaCCs), plays important roles
in cellular physiological processes including smooth muscle excitability, cardiac excitability and
nociception, and epithelial secretion in various cell types[1, 2]. ANO1 has been considered as a
promising target for targeted cancer therapeutics as ANO1 is amplified and over-expressed in various
cancers such as breast[3], pancreas[4], prostate[5], urinary bladder[6], oesophagus[7], lung[8], head-
and-neck squamous cell carcinoma (HNSCC)[9], gastrointestinal stromal tumors (GIST)[10], and
glioblastoma (GBM)[11]. It has been reported that ANO1 is overexpressed in various GBM cells
including U87MG[12], U138[13], and U251[14] and decrease of ANO1 expression by the treatment
of siRNA suppresses proliferation[12], invasion and migration[14] of GBM cells. ANO2 (TMEM16B)
has about ~60% sequence homology with ANO1 and is expressed in the cilia of olfactory sensory
neurons[15] and mediates olfactory amplification[16]. Loss of ANO2 expression leads to markedly
diminished action potential firing of inferior olivary neurons and ANO2 knockout mice exhibits
severe cerebellar motor learning deficits[17]. It is therefore necessary to selectively inhibit ANO1
rather than ANO2 for anti-ANO1 drug discovery.
GBM is one of the most common and aggressive human cancers[18] and accouts for over 60%
of malignant glioma. Only around 5% of GBM patients survive for 5 years. GBM cell invasion occurs
in the parenchyma of the brain and surgical resection inevitably leads to recurrent disease[11].
Surgical resection followed by radiotherapy and concomitant and adjuvant chemotherapy with
temozolomide (TMZ) has become the standard first-line therapy against GBM with a small increase
(around 2.5 months) of the median survival[19]. Despite tremendous efforts to overcome GBM,
effective therapeutics against GBM are still very limited. No standard treatment has been established
for GBM recurrence which is almost inevitable and median survival is measured in months[20].

Figure 1. Representative ANO1 channel blockers
To date, a few small molecule inhibitors (Fig. 1) of ANO1 have been described including
CaCCinh-A01 (1), T16Ainh-A01 (2), Ani-9 (3), and 10bm (4). Non-selective ANO1 ion channel
blocker 1 (IC50 = 2.1 µM), a cyclohexa[b]thiophene derivative, is capable of decreasing ANO1 protein
expression level by ER-associated proteasomal degradation, which causes appreciable suppressive
effects on ANO1-dependent cellular proliferation in head and neck squamous cell carcinoma (HNSCC)
and esophageal squamous cell carcinoma (ESCC) cells[21]. T16Ainh-A01 2 (IC50 = 1.39 µM)
discovered by high-throughput screening campaign inhibits the functional channel activity of ANO1
in vascular smooth muscle cells and relaxes human and mouse blood vessels[22].
Ani-9 (3), discovered from screening of 54,400 synthetic small molecules, is a selective and
potent ANO1 inhibitor with an IC50 value of 77 nM[23]. Ani-9 (3), a benzylideneacetohydrazide
derivative, has low metabolic stability with a half-life value of 32 minutes[24]. Another potent ANO1
inhibitor 4 belonging to one of the 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides
(AACTs) has an IC50 value of 30 nM and displays over 10-fold improved metabolic stability
compared with 3[25]. As part of a program to perform GBM drug discovery, we have decided to
explore effective ANO1 channel blockers and been interested in the AACT analogue 4, the most
potent ANO1 inhibitor although activity of 4 against GBM has not been reported. In an effort to
discover potent and selective ANO1 inhibitors capable of inhibiting GBM cells, we have designed and
synthesized a series of new 2-aminocycloalkylthiophene-3-carboxamide derivatives based on AACT
analogue 4 and carried out an SAR study exploring inhibitory activities on GBM cells as well as
against ANO1 ion channel. We observed that among the 33 synthetic 2-aminocycloalkylthiophene-3-
carboxamide derivatives prepared for this SAR study, 9c and 10q completely block ANO1 channel
activity with appreciable selectivity and significantly suppress proliferation and migration/invasion of
GBM cells. Herein, we report that 9c and 10q are potent and selective ANO1 inhibitors as valuable hit
compounds for GBM drug discovery.
2. RESULT AND DISCUSSION
2.1. Synthesis

All the 2-aminothiophene-3-carboxamide derivatives were synthesized in a very
straightforward manner as described in Scheme 1. In order to obtain C-4 and C-5 unsubstituted 2-
aminothophene-3-carboxamide 6 and 2-aminocycloalkylthophene-3-carboxamide 8a-c, we carried out
Gewald reactions[26] using commercially available 2-cyanoacetamide 5. The stable dimer of α-
mercapto acetaldehyde, 1,4-dithiane-2,5-diol was condensed with 5 in the presence of triethylamine to
afford 6 in 75% yield. Compound 8a-c were also readily obtained by the condensation of the
corresponding ketones with 5 in the presence of elemental sulfur and morpholine base in 65-73%
yield. The Gewald condensation products 6 and 8a-c were directly utilized for the next step without
further purification. The amine 6 and 8a-c were coupled with a variety of acyl chlorides to produce
the corresponding amides 7a-c and 9a-i in 80-95% yield. The coupling of 8a-c with various acyl
chlorides and ammonium thiocyanate afforded the desired N-acylated thiourea derivatives[27] 10a-q
(65-88%).
Scheme 1. Synthesis of 2-aminothiophene-3-carboxamide derivativesa
o
aReagent and condition: (a) 1,4-dithiane-2,5-diol, Et3N, EtOH, 70 C, 6 h, 75%; (b) various acyl
o
chlorides, pyridine, rt, 1 h, 80-95%; (c) various ketones, sulfur, morpholine, EtOH, 70 C, 12 h, 65-
73%; (d) various acyl chlorides, ammonium thiocyanate, acetone, reflux, 2 h, 65-88%.
2.2. Structure-Activity Relationships

Among several methods[28] for measuring ion channel modulation including
electrophysiology patch-clamp assay, competitive binding assay with radiolabeled ligand, ion flux
assay using radioactive tracers or fluorescence sensors, measurement of ionic current using patch-
clamp has been considered to be most reliable indicator but it suffers from low-throughput.
Fluorescent cellular membrane potential assay (FLIPR format) using fluorescence sensors offers high
throughput efficiency and reproducible data. We have therefore adopted fluorescent cellular
membrane potential assay[29] as a primary assay, followed by ion current assessment using patch
clamp for secondary validation assay. We observed fluorescent cellular membrane potential assay data
for the three references (1, 2, and 3) are in good agreement with patch clamp assay data, which is also
accordance with reported IC50 values of 1 (2.1 µM)[30], 2 (1.1-1.8 µM)[23, 30], and 3 (77 nM)[23].
In the performance of the fluorescent cellular membrane potential assay, percent inhibitions at a single
concentration (10 μM) were measured for all the derivatives, followed by estimation of IC50 values for
the selected active (over 60% inhibiton) derivatives.
Table 1. Inhibitory-activities of 6, 7a-c, 8a-c and 9a-i against ANO1
% inhibition (%)a
IC50 (µM)
R1
n
R2
Entry
FLIPR
format
FLIPR
format
Patch
clamp
-
-
-
-
-
-
-
-
-
92.88 ± 0.24
63.85 ± 0.28
73.77 ± 5.17
NAc
1.31 ± 0.77
2.60 ± 0.76
0.06 ± 0.02
NAc
1.53 ± 0.52
2.74 ± 0.78
0.03 ± 0.01
0.05 ± 0.01
1
2
3
4
-
-
-
-
inactiveb
6
tert-butyl
-
inactiveb
7a
7b
7c
8a
8b
8c
2-furyl
-
-
39.54 ± 5.24
inactiveb
phenyl
-
-
-
-
-
0
2
1
H
H
15.44 ± 0.41
inactiveb
tert-butyl
80.09 ± 0.75
2.95 ± 0.05
2.50 ± 0.19

tert-butyl
tert-butyl
tert-butyl
2-furyl
0
2
1
0
2
1
0
2
1
H
12.51 ± 3.17
67.94 ± 0.78
86.87 ± 2.01
51.49 ± 1.55
inactiveb
9a
9b
9c
9d
9e
9f
H
4.79 ± 1.10
3.10 ± 1.23
4.07 ± 0.37
2.56 ± 0.05
tert-butyl
H
2-furyl
H
tert-butyl
H
2-furyl
inactiveb
phenyl
inactiveb
9g
9h
9i
phenyl
H
12.13 ± 9.65
21.99 ± 0.12
phenyl
tert-butyl
a Average % inhibition value at 10 μM with S.D. are shown (mean ± S.D., n = 2)
b Maximum inhibition is less than 10% at 10 μM
c Not applicable
As shown in Table 1, among four C-4 and C-5 unsubstituted 2-aminothophene-3-
carboxamides tested, three derivatives (6, 7a, and 7c) are inactive and 7b having 2-furyl amide
functional group exhibits a moderate ANO1 inhibitory activity (39.54% inhibition at 10 µM) in
fluorescent cellular membrane potential assay. Fusion of 2-aminothophene-3-carboxamide (6) with
unsubstituted cyclopentane (8a) or cycloheptane (8b) has little or no influence on activity as 8a
exhibits little activity and 8b is inactive like 6. In contrast, tert-butyl group substituted cyclohexane-
fused 2-aminothophene-3-carboxamide (8c) displays excellent activity with IC50 value of 2.95 µM in
fluorescent cellular membrane potential assay, which is confirmed in electrophysiology patch clamp
assay (IC50 = 2.50 µM). It is worth noting that this fusion with tert-butylcyclohexane was inspired by
1 and ANO1 inhibitory activities of 8c are comparable to those (IC50 = 1.31 µM from fluorescent
cellular membrane potential assay, 1.53 µM from patch clamp assay) of 1. We next introduced a
variety of acyl groups on 2-aminocycloalkylthiophene-3-carboxamides to generate 9a-i and
investigated the effects of the acylations. In contrast to the observation that installation of tert-butyl
amide on 8a having cyclopentane ring has no influence on activity (9a), the incorporation of tert-butyl
amide on inactive 8b possessing cycloheptane ring was found to lead dramatically to a highly active
derivative (9b) with IC50 values of around 4 µM from both fluorescent cellular membrane potential
and patch clamp assays. Meanwhile, no differences in potency were observed between very potent 8c
bearing tert-butylcyclohexane group and 9c obtained by introduction of tert-butyl amide on 8c. It is
worthwhile to note that 9c exhibits the highest percentage (86.87%) of inhibition at single
concentration (10 µM) in fluorescent cellular membrane potential assay among all our derivatives
tested and displays great IC50 values of 3.10 µM and 2.56 µM in fluorescent cellular membrane
potential and patch clamp assays, respectively. We next adopted 2-furyl amide group incorporated in 1
and explored the effects of 2-furyl amide functionality. In contrast to the contribution of tert-butyl

amide group to ANO1 inhibitory activity exemplified by 9b, introduction of 2-furyl amide group on
8b has no influence on activity (9e) even though the activity of 9d installed with 2-furyl amide is
increased by around two-folds compared with 8a. To our surprise, incorporation of 2-furyl amide on
potent 8c causes complete abolition of activity (9f).
Our attention next turned to an evaluation of the effects of acylthiourea functionality on 2-
aminocycloalkylthiophene-3-carboxamide in comparison with amide functionality. Acylthiourea
group has been adopted for a variety of medicinal chemistry researches including PBD inhibitors[31],
15-lipoxygenase inhibitors[27], and histone deacetylase-8 activators[32]. Benzoylthiourea is
absolutely superior to benzamide functionality in terms of the degree of contribution to ANO1
inhibitory activities of 2-aminocycloalkylthiophene-3-carboxamides. To our surprise, inactive 9g and
almost inactive 9i were converted into a potent derivative 10a (IC50 = 2.83 µM from fluorescent
cellular membrane potential assay, 2.63 µM from patch clamp assay) and an active derivative 10c by
substitution of benzamide group with benzoylthiourea group. The contribution of benzoylthiourea
group to ANO1 inhibitory activity was not observed on 2-aminothiophene-3-carboxamide fused with
cycloheptane ring (10b) in comparison between activities of 9h (12.13% inhibition at 10 µM) and 10b
(21.13% inhibition at 10 µM). Encouraged by the contribution of benzoylthiourea group to activity
exemplified with 10a and 10c, we next investigated the effects of 2-furoylthiourea and thiazole-5-
carbonylthiourea. In contrast to benzoylthiourea group, substitutions of furan-2-amide group on 9d
and 9e with 2-furoylthiourea group result in decreased activity (10d) and almost unaltered activity
(10e). Likewise, thiazole-5-carbonylthiourea functionality on 10g is not capable of enhancing activity.
Our attention next focused on investigating effects of substituents on the benzoylthiourea group in
order to improve potency of 10a. Unfortunately, neither electron-donating substituents (10g-l) nor
electro-withdrawing substituents (10m-q) increase ANO1 inhibitory in comparison to the potency of
10a having unsubstituted benzoylthiourea functionality. The electron-donating substituents (10g-l)
and trifluoromethyl substituent (10m-n) cause little to no activities but 2-chloro (10o) and 4-chloro
(10q) substituents result in maintained activities compared with the activities of 10a. Interestingly, 3-
chloro substituent (10p) causes abolished activity. It is worth recalling that replacement of benzamide
group on inactive 9g with benzoylthiourea group dramatically causes high potency indicating that
benzoylthiourea functionality substantially contributes to ANO1 inhibitory activity. The activity (IC50
= 1.75 µM from patch clamp assay) of 10q bearing 4-chloro substituted benzoylthiourea group in
patch clamp assay is only marginally higher than that (IC50 = 2.63 µM from patch clamp assay) of 10a.
Table 2. Inhibitory-activities of 10a-q against ANO1

% inhibition (%)a
IC50 (µM)
R1
n
R2
Entry
FLIPR
format
FLIPR
format
Patch
clamp
phenyl
phenyl
0
2
H
H
70.50 ± 0.00
21.13 ± 16.94
70.28 ± 8.76
inactiveb
2.83 ± 0.96 2.63 ± 0.47
10a
10b
10c
10d
10e
10f
phenyl
1 tert-butyl
3.09 ± 1.07 5.66 ± 0.57
2-furyl
0
2
H
H
2-furyl
21.75 ± 4.32
13.79 ± 3.75
inactiveb
2-furyl
1 tert-butyl
4-methylthiazol-5-yl
3,4-dimethoxyphenyl
3,5-dimethoxyphenyl
3,4,5-trimethoxyphenyl
1,3-benzodioxol-5-yl
0
0
0
0
0
0
0
0
0
0
0
H
H
H
H
H
H
H
H
H
H
H
10g
10h
10i
inactiveb
46.07 ± 0.92
inactiveb
10j
16.33 ± 14.22
12.28 ± 3.85
26.69 ± 7.61
inactiveb
10k
10l
2,3-dihydro-1,4-
benzodioxin-6-yl
3-(trifluoromethyl)phenyl
4-(trifluoromethyl)phenyl
2-chlorophenyl
10m
10n
10o
10p
10q
76.70 ± 10.75
inactiveb
2.29 ± 0.30 3.12 ± 0.27
2.36 ± 0.79 1.75 ± 0.30
3-chlorophenyl
4-chlorophenyl
72.25 ± 11.78
a Average % inhibition value at 10 μM with S.D. are shown (mean ± S.D., n = 2)
b Maximum inhibition is less than 10% at 10 μM
2.3. Endogenous ANO1 expression level in both normal cells and GBM cells
It has been reported that ANO1 is highly expressed in GBM cell lines such as U87MG[12],
U138[13] and U251[14]. Glioma is divided into I-IV according to the pathological grade. The degree
of mRNA and protein expression of ANO1 is increased with increasing disease grade[12]. It has been

reported that inhibition of ANO1 expression by siRNA inhibits cell proliferation[12], invasion and
migration[14] in GBM cell line U87MG. In order to select GBM cell lines with high expression level
of ANO1, we measured the mRNA level and protein expression level of ANO1 using qRT-PCR and
western blot analysis.
Figure 2. Expression level of endogenous ANO1 in various GBM cell lines and normal cells. (A)
Expression level of endogenous ANO1 mRNA. (B) Quantification for expression level of endogenous
ANO1 mRNA using ImageJ. (C) Expression level of endogenous ANO1 protein. (D) Quantification
for expression level of endogenous ANO1 protein using ImageJ. (B, D) statistics analysis was
evaluated by using Prism version 6.0. Band densities were measured using ImageJ software and
shown in the bar graph (mean ± SD, two-tailed Student’s t-test, **** p < 0.0001, *** p < 0.001, ** p
< 0.01, ns: not significant).
We also compared the levels of mRNA and protein of ANO1 between normal IM-PHFA
(human fetal astrocyte) cells and three GBM cell lines (U87MG, U251 and U138) (Fig. 2). The
expression level of ANO1 mRNA turned out to be very low in normal brain cells, IM-PHFA (0.13 ±
0.06). The mRNA expression level of ANO1 in U251 (1.20 ± 0.17) and U87MG (1.30 ± 0.21) divided
into Grade IV is slightly higher than that (1.06 ± 0.15) in U138 (Fig. 2A-B). As expected, the
expression level of ANO1 protein in IM-PHFA cells is very low (0.12 ± 0.01) and the level of ANO1
protein expression in both U251 (1.21 ± 0.06) and U87MG (1.27 ± 0.12) is higher than that (1.03 ±
0.10) in U138 (Fig. 2C-D).
2.4. 2D and 3D Anti-proliferative effects of 9c and 10q in GBM cell lines
Inhibition of ANO1 by siRNA suppresses cancer cell viability in GBM[12], breast cancer[3],
prostate carcinoma[5] and ovarian cancer[33]. In addition, ANO1 channel blocker 1 significantly
suppresses the growth of breast cancer cells[3]. Based on the ANO1 inhibitory activities estimated in
both fluorescent cellular membrane potential and patch clamp assays, we selected seven potent
derivatives and assessed their anti-proliferative activities on three GBM cell lines having high
expression level of ANO1 mRNA and protein.

Table 3. Anti-proliferative effects of selected derivatives
GI50 (µM)a
Entry
Mouse primary
astrocyte
U251
U87MG
U138
inactiveb
inactiveb
inactiveb
inactiveb
41.89 ± 3.38
79.27 ± 8.09
36.48 ± 0.54
45.33 ± 0.59
inactiveb
inactiveb
89.38 ± 7.06
inactiveb
1
2
3
4
32.98 ± 2.16
32.44 ± 2.23
48.00 ± 5.18
29.40 ± 1.22
inactiveb
83.24 ± 5.07
inactiveb
12.58 ± 0.75
inactiveb
inactiveb
inactiveb
inactiveb
8c
9b
9c
inactiveb
94.83 ± 4.13
31.49 ± 0.60
22.24 ± 0.65
inactiveb
inactiveb
inactiveb
24.37 ± 1.61
70.18 ± 0.74
94.60 ± 3.11
12.04 ± 0.20
25.96 ± 2.23
82.35 ± 3.29
inactiveb
17.05 ± 0.59
inactiveb
inactiveb
10a
10c
10o
10q
45.15 ± 0.85
23.77 ± 10.39
13.22 ± 0.72
a Cell Titer-glo assay results. Average GI50 values with S.D. (n = 3, duplicate) are shown.
b Maximum inhibition <50% at 100 μM.
Among the selected seven derivatives, anti-proliferative activities of 9c, 10a and 10q in three
GBM cell lines (U251, U87MG and U138) turned out to be higher than those of the three reference
compounds (1, 2 and 3). As shown in Table 3, the GI50 values of 9c, 10a and 10q in U251 glioma cells
are 12.58 µM, 24.37 µM and 12.04 µM, respectively, which are superior to those (41.89 µM, 79.27
µM, 36.48 µM and 45.33 µM, respectively) of four references 1, 2, 3 and 4. We also examined
whether our ANO1 inhibitors possess differential cytotoxic effects between glioma cancer cells and
normal brain cells, primary astrocytes derived from adult mouse brain. It was found that 9c, 10a and
10q have little to no effects on primary astrocytes and are capable of discriminating between glioma
cells and normal brain cells (Table 3). It is worthwhile recalling that ANO1 inhibitory activities of 9c,
10a and 10q are inferior to those of 3 and 4 and comparable to those of both 1 and 2. It is not likely
that ANO1 inhibitory activities of all compounds tested in this study are well translated into anti-
proliferative effects in GBM cells. Reference compound 3 and 4 possess potent ANO1 inhibitory
activities but display moderate GI50 values of 36.48 µM and 45.33 µM, respectively on U251 cells.
Also, anti-proliferative activities of both 1 and 2 on U251 glioma cells appear to be low in comparison
to their ANO1 inhibitory potencies with IC50 values of around 2 µM. Meanwhile, it is worth noting
that 3 has low plasma stability[24], which might contribute to its moderate anti-proliferative activity
in GBM cells.
Based on the growth inhibitory activities (GI50s), endogenous ANO1 expression level and
glioma cell morphology, we selected U251 among three GBM cell lines to perform further

experiments. In addition, both 9c and 10q were elected as representative compounds as they
significantly suppress the proliferation of U251 glioma cells with GI50 values of less than 20 μM
(Table 3).
Anchorage independent growth (AIG) is one of the hallmarks of tumorigenesis and AIG
assay has been considered as the most reliable in vitro method to detect cell transformation. The
theoretical basis for this technique relies on extracellular matrix (ECM) contact for normal cell growth
and division, but cancer cells have capability to grow and differentiate without affecting the
surrounding environment. Inhibition of ANO1 by siRNA results in a reduction of soft agar colony
formation on ovarian cancer[33] and colorectal cancer[34] cells. To investigate if both 9c and 10q are
capable of blocking tumorigenesis of ANO1-expressed GBM cells, we carried out colony formation
assay (2D) and soft agar assay (3D) in U251 glioma cells. Incubation with each compound at three
different concentrations (1, 3 and 10 μM) for 14 days suppressed anchorage-independent growth in
terms of colony number of U251 cells.
Figure 3. Inhibition of ANO1 suppresses tumor cell growth in U251 GBM cells. (A) Colony
formation assay (2D) in U251 GBM cells. (C) Anchorage independent growth (3D) of U251 GBM
cells. Cells embedded in 0.3% top agar were incubated with indicated concentrations of compounds
for 14 days and colonies were observed (n = 3) (B, D) The results were compared to DMSO control.
Average number of colonies per well was automatically counted using ImageJ software and shown in

the bar graph (mean ± SD, two-tailed Student’s t-test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns:
not significant).
In contrast to both 9c and 10q, none of three reference compounds (2, 3 and 4) exhibited
effects at even 10 μM in two-dimensional colony formation analysis. However, 10 μM of 1 showed an
inhibitory effect. The degree (72.71%) of inhibition by 1 (10 μM) is similar to that promoted by 3 μM
of 9c or 10q. Compound 9c and 10q at 10 μM concentration showed an inhibitory effect of 96.58%
and 96.40%, respectively, which indicates that both 9c and 10q possess a concentration-dependent
inhibitory effect (Fig. 3A-B, Fig. S1). In the soft agar analysis (3D), colony formation was not
suppressed at 10 μM concentration of all compounds tested and three-dimensional soft agar assays
were carried out with increased concentration (30 and 100 μM) of test compounds. None of three
reference compounds (2, 3 and 4) displayed an inhibitory effect even at 100 μM but 1 showed an
inhibitory effect (72.65%) at 100 μM even though 30 μM of 1 exhibited no effect. Derivative 9c and
10q at 100 μM concentration strongly suppressed colony formation by 95.70% and 88.05%,
respectively and showed dose-dependency in three-dimensional soft agar assay (Fig. 3C-D, Fig. S1).
Taken together, both 9c and 10q turned to be superior to the three reference compounds (1, 2 and 3) in
terms of capability of inhibiting colony formation in both 2D colony formation assay and 3D soft agar
assay using U251 glioma cells.
2.5. Inhibitory effects of 9c and 10q on migration and invasion of U251 GBM cells
Inhibition of ANO1 by shRNA or siRNA blocks cellular migration and invasion in GBM,
anaplastic thyroid cancer[35] and ovarian cancer[33]. In addition, ANO1 channel inhibitor 2
suppresses migration and invasion of U251 GBM cells[13]. In order to identify derivatives possessing
remarkable migration-inhibitory activity, a typical scratch wound healing assay was conducted with 5
μM (Fig. S2) of selected seven derivatives of which IC50 values are less than 3 μM in the Cell Titer-
Glo assay. Among these seven derivatives, both 9c and 10q significantly suppressed migration of
U251 cells. We next assessed dose-dependent effects of 9c and 10q and observed that both 9c and 10q
significantly blocked migration of U251 cells in a dose-dependent manner (Fig. 4A-B). In addition,
both 9c and 10q also strongly suppressed invasion of U251 cells at 10 μM in Boyden chamber
invasion assay (Fig. 4C-D). Indeed, migration and invasiveness of U251 cells were decreased up to
90% by 9c (10 μM). It was observed that 1, 2, 3 and 4 at 10 μM concentration reduced migration of
U251 cells by 34.83%, 26.53%, 10.45% and 16.74%, respectively (Fig. 4A-B) and decreased invasion
of U251 cells by 28.33%, 19.17%, 12.10% and 11.73%, respectively (Fig. 4C-D), which indicates that
both 9c and 10q are superior to 1, 2, 3 and 4 in terms of capability of suppressing both migration and
invasion of U251 cells. Based on the report that knockdown of ANO1 using siRNA is related to

increase the expression E-cadherin in breast cancer cells[36], we investigated if the level of E-
cadherin in U251 glioma cells is increased by the treatment of 9c and 10q and observed that both
substances cause increase of E-cadherin in concentration-dependent fashion (Fig. 4E-F). We also
investigated the effects of both 9c and 10q on ANO1 protein level in U251 glioma cells. The western
blot analysis reveals that both 9c and 10q cause ANO1 protein degradation in U251 cells and this
ANO1 degradation is blocked by the treatment of MG132, a proteasome inhibitor (Fig. 4G).
Figure 4. Derivative 9c and 10q suppress migration and invasion of U251 GBM cells. (A) Each
compound treated at 10 µM concentration, were incubated for 18 h after scratching cell monolayer.
Migration ratio was calculated using migration area using ImageJ. (C) Invasion assays were
performed using CHEMICON QCM 24 well invasion assay kit. Invasion assay ratio were calculated
using colorimetric reading of OD at 560 nm. (E) Representative western marker related ANO1.
Analysis of EMT (Epithelial–mesenchymal transition) marker, E-cadherin. Quantification of western

band was analysis using band density using ImageJ. (G) U251 cells were incubated with 10
μM
concentration of the inhibitors (1, 9c or 10q) for 24 h. Also, U251 cells were incubated with 10
M concentration of the inhibitors (1, 9c or 10q) for 6 h, followed by the treatment of MG132
μ
(20
μM) for an additional 18 h. (B, D, F) Significant differences were calculated using a one-way
ANOVA (* p <0.05; ** p <0.01; *** p< 0.001; **** p< 0.0001).
2.6. Electrophysiologic effects of 9c and 10q

Figure 5. Both 9c and 10q suppress ANO1 channel activities in HEK293 cells transfected with ANO1.
(A, E) inhibitory effects of 9c and 10q on ANO1 current amplitude at +80 mV. Error bars represent
SD. (B, F) normalized current density at +80 mV. The n values for each statistical analysis are
indicated at histograms. (C, G) inhibitory effects of 9c (10 μM) and 10q (10 μM) on ANO1 current
amplitude at +80 mV. Error bars represent SD. (D, H) normalized current density at +80 mV. The n
value for each statistical analysis is 3. Significant differences were calculated using a one-way
ANOVA. (* p <0.05; ** p <0.01; *** p< 0.001; **** p< 0.0001).

In order to evaluate the effects of 9c and 10q on ANO1 channel currents, whole-cell
recordings were carried using HEK293A cells transfected with GFP-mANO1 channel plasmids (Fig.
5). As depicted in Figure 5B and 5F, both 9c and 10q effectively inhibit ANO1-mediated currents in
dose-dependent manner. ANO1 currents were inhibited by 50.64% and 57.63% at 10 μM
concentration of 9c and 10q. IC50 values of 9c and 10q were estimated to be 2.56 μM and 1.75 μM
respectively for inhibition of ANO1-mediated currents. These results clearly indicate that both 9c and
10q are capable of inhibiting channel activity of ANO1. In addition, to examine the reversibility of
ANO1 inhibition caused by 9c and 10q, we measured the ANO1-mediated currents after washout of
compound 9c and 10q. Reduced ANO1-mediated currents by 9c were partially recovered after
washout. The amplitude of ANO1-mediated currents was recovered up to 85.80% compared to the
currents prior to the treatment of 9c (Fig. 5C-D), which reveals that 9c possesses reversible inhibitory
effect on ANO1-mediated currents. However, reduced ANO1-mediated currents by 10q were not
recovered after washout, indicating that 10q irreversibly inhibits ANO1 channel activity. In addition
to 9c and 10q, other derivatives 8c, 9b, 10a, 10c and 10o were also submitted to whole-cell patch
clamp assay using GFP-mANO1 transfected HEK293A cells and their effects on ANO1 channel
currents are described in Figure S3.
On the basis of a previous report that ANO1 is highly expressed in U251 glioma cells[13].
We next examined the effects of 9c and 10q on ANO1 currents in U251 cells and observed that
ANO1-mediated currents are inhibited about 40 % and 60% respectively by 10 μM of 9c and 10q,
ANO1 channel blockers (Fig. S4), which demonstrates that 9c and 10q have capability of inhibiting
ANO1 currents in a glioma cells as well as in HEK293A cells transfected with ANO1.
Our attention next turned into the evaluation of selectivity of both 9c and 10q in inhibiting
ion channel activities. In order to make comparison between inhibitory effects of 9c and 10q on
ANO1, ANO2 and TTYH-1 channels which belongs to a family of human high-conductance chloride
ion channels, we obtained current-voltage (I-V) curves using HEK293A cells transfected with GFP-
mANO1, GFP-mANO2 or GFP-mTTYH-1. As shown in Figure 6A and B, apical membrane current
measurement in HEK293A cells expressing GFP-mANO1, GFP-mANO2 or GFP-mTTYH-1 revealed
that ANO1 is more strongly inhibited rather than ANO2 and TTYH-1 is not inhibited by both 9c and
10q. Both 9c and 10q strongly inhibit ATP-induced ANO1 chloride currents in a dose dependent
manner with IC50 values of 2.56 µM and 1.75 µM but slightly suppress ANO2 with IC50 values of
15.43 µM and 7.43 µM. It is worth recalling that ANO2 knockout mice exhibits severe cerebellar
motor learning deficits[17] and selective ANO1 blockers over ANO2 are obviously favarable to avoid
potential toxicities. It is challenging to obtain selectivity for ANO1 inhibition over ANO2 inhibition as

ANO1 and ANO2 are highly homologous. It should be noted that both 9c and 10q are selective ANO1
blockers over ANO2 and TTYH-1 chloride ion channel.
Figure 6. Selective inhibition of ANO1 over ANO2 and TTYH-1 by 9c and 10q. (A, D) inhibition of
ANO1 channel activity by 9c and 10q. (B, E) inhibition of ANO2 channel activity by 9c and 10q (C,
F) inhibition of TTYH-1 channel activity by 9c and 10q. The n value for each statistical analysis is 3.
(G) IC50 values of 9c and 10q.
2.7. Combination treatment of 9c with temozolomide (TMZ) in U251 cells
As aforementioned, effective therapeutics against recurrent GBM are still very limited even though
surgical resection followed by radiotherapy and chemotherapy with temozolomide (TMZ)[37] has
become the first-line therapy against GBM. It was reported that the level of mRNA and protein
expression of ANO1 increases with increasing disease grade in GBM patients[12]. We hypothesized
that the combination of ANO1 inhibitors and TMZ would result in synergistic effects in suppressing
proliferation of glioma cells and for the first time, to the best of our knowledge, examined synergistic
effect of combination of an ANO1 inhibitor (9c or 3) and TMZ in U251 glioma cells.

a Cell Titer-glo assay results. Average GI50 values with S.D. (n = 3, duplicate) are shown.
Figure 7. Synergistic effects of combination of ANO1 inhibitor (9c or 3) and TMZ in suppressing
proliferation of U251 cells. (A, D) full titration curve for 3 or 9c combined with TMZ (B, E)
histogram for GI50 values (C, F) effects of 3 or 9c combined with TMZ on the proliferation of U251
cells. (G) GI50 and CI values of 3 and 9c combined with TMZ. Significant differences were calculated
using a one-way ANOVA (* p <0.05; ** p <0.01; *** p< 0.001; **** p< 0.0001).
As depicted in figure 7, appreciable synergistic effects of combination of ANO1 inhibitor
and TMZ were observed and effect of 9c combined with TMZ is superior to that of 3 combined with
TMZ. GI50 value (5.32 μM) of 9c combined with TMZ is decreased by more than 50% compared with
that (GI50 = 12.58 μM) of 9c alone. CI value of 9c combined TMZ (TMZ : 9c = 1:1) is 0.63 ± 0.03
(0.4< CI < 0.7, synergism). GI50 value of 3 combined with TMZ is decreased by more than 40% with

CI value of 0.70 ± 0.03 (0.7< CI < 0.85, moderate synergism). CI values were estimated using
compusyn software.
3. Conclusions
Glioblastoma (GBM) is one of the most aggressive cancers with poor prognosis with median
survival for 15 months. Lack of effective treatment options against GBM emphasizes urgent necessity
of effective GBM therapeutics. ANO1, a calcium-activated chloride ion channel (CaCC), is over-
expressed in GBM and inhibition of ANO1 has been reported to suppress cell proliferation, migration
and invasion in GBM. In order to identify potent and selective ANO1 inhibitors capable of inhibiting
GBM cells, we have designed and synthesized a series of new 2-aminothiophene-3-carboxamide
derivatives. We performed SAR study using fluorescent cellular membrane potential membrane
potential assay as a primary assay, followed by ion current assessment using patch clamp for
secondary validation assay and selected seven derivatives that effectively inhibit ANO1 channel
activity with IC50 values ranging from 1.75 μM to 5.66 μM. It should be noted that manual whole-cell
patch clamp, the gold standard of electrophysiological method, requires a high expertise and a lot of
efforts with a very low throughput. These seven substances were submitted to anti-proliferation assay
using GBM cells and two lead compounds (9c and 10q) were identified. Unique structural feature of
10q, a cyclopentane-fused thiophene-3-carboxamide derivative, is the presence of benzoylthiourea
functionality which dramatically contributes to activity. Both 9c and 10q suppress more strongly
proliferation of GBM cells than four reference compounds including 3, Ani-9. ANO1 blocker 9c and
10q are also capable of inhibiting much more strongly colony formation than reference compounds in
both 2D colony formation assay and 3D soft agar assay using U251 glioma cells. In addition, 9c and
10q suppress far more strongly migration/invasion of GBM cells than reference compounds. Whole-
cell patch clamp recordings were carried out to confirm activities of seven inhibitors selected from
fluorescent cellular membrane potential assay. ANO2 knockout mice was reported to exhibit severe
cerebellar motor learning deficits and selective ANO1 blockers over ANO2 are obviously favarable to
avoid potential toxicities. Satisfyingly, both 9c and 10q turned to be selective ANO1 blockers over
ANO2 and TTYH-1 chloride ion channel. It is challenging to obtain selectivity for ANO1 inhibition
over ANO2 inhibition as ANO1 and ANO2 are highly homologous. We, for the first time, found that
the combination of ANO1 inhibitor (9c or 3) and TMZ brings about remarkable synergistic effects in
suppressing proliferation of GBM cells. Our study may provide an insight into designing selective and
potent ANO1 inhibitors as pharmacological tools as well as promising leads for novel GBM drug
discovery.
4. Experimental Section

4.1 Chemistry
Unless otherwise described, all commercial reagents and solvents were purchased from
commercial suppliers and used without further purification. All reactions were performed under
N2 atmosphere in flame-dried glassware. Reactions were monitored by TLC with 0.25 mm E.
Merck precoated silica gel plates (60 F254). Reaction progress was monitored by TLC analysis
using a UV lamp, ninhydrin, or p-anisaldehyde stain for detection purpose. All solvents were
purified by standard techniques. Purification of reaction products was carried out by silica gel
column chromatography using Kieselgel 60 Art. 9385 (230–400 mesh). The purity and of all
compounds was over 95% and mass spectra and purity of all compounds was analyzed using
Waters LCMS system (Waters 2998 Photodiode Array Detector, Waters 3100 Mass Detector,
Waters SFO System Fluidics Organizer, Water 2545 Binary Gradient Module, Waters Reagent
Manager, Waters 2767 Sample Manager) using SunFireTM C18 column (4.6 × 50 mm, 5 μm
particle size): solvent gradient = 90% A at 0 min, 0% A at 5 min. Solvent A = 0.1% TFA in H2O;
Solvent B = 0.1% TFA in MeCN; flow rate : 2.0 mL/min. 1H and 13C NMR spectra were obtained
1
using a Bruker 400 MHz FT-NMR (400 MHz for H, and 100 MHz for 13C) spectrometer.
Standard abbreviations are used for denoting the signal multiplicities.
2-Aminothiophene-3-carboxamide (6) To a suspension of 1,4-dithiane-2,5-diol (9.1 g, 59.8 mmol)
and 2-cyanoacetamide (5.0 g, 59.8 mmol) in EtOH (60.0 mL) was added triethylamine (16.3 mL,
o
119.6 mmol) at room temperature. The reaction mixture was stirred at 70 C for 6 h. The reaction
mixture was cooled to room temperature, diluted with EtOAc and quenched with H2O. The organic
layer was washed with brine, dried over MgSO4, filtered and concentrated. The residue was dissolved
in CH2Cl2 and solidified by adding Et2O. The resulting solid was collected to afford 6 (6.4 g, 75%) as
a brown solid. 1H NMR (400 MHz, DMSO-d6) δ 7.21 (s, 2H), 7.21 (brs, 1H), 7.04 (d, J = 5.7 Hz, 1H),
13
6.71 (brs, 1H), 6.22 (d, J = 5.7 Hz, 1H); C NMR (100 MHz, DMSO-d6) δ 167.68, 161.80, 124.94,
107.20, 105.56; LRMS (ESI) m/z 143 [M + H]+.
2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (8a) To a suspension of 2-
cyanoacetamide (3.0 g, 35.7 mmol), cyclopentanone (3.2 mL, 35.7 mmol), and morpholine (7.1 mL,
71.4 mmol) in EtOH (60.0 mL) was added sulfur (1.1 g, 35.7 mmol) at room temperature. The
o
reaction mixture was stirred at 70 C for 12 h. The reaction mixture was cooled to room temperature,
diluted with EtOAc and quenched with H2O. The organic layer was washed with brine, dried over
MgSO4, filtered and concentrated. The residue was dissolved in CH2Cl2 and solidified by adding Et2O.
1
The resulting solid was collected to afford 8a (3.7 g, 73%) as a brown solid. H NMR (400 MHz,

DMSO-d6) δ 7.15 (s, 2H), 6.43 (brs, 2H), 2.77 (t, J = 7.0 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H), 2.25 (m,
2H); 13C NMR (100 MHz, DMSO-d6) δ 167.69, 165.63, 139.94, 119.59, 103.02, 29.81, 28.27, 26.95;
LRMS (ESI) m/z 227 [M + H]+.
2-Amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (8b) To a suspension of
2-cyanoacetamide (3.0 g, 35.7 mmol), cycloheptanone (4.2 mL, 35.7 mmol), and morpholine (7.1 mL,
71.4 mmol) in EtOH (60.0 mL) was added sulfur (1.1 g, 35.7 mmol) at room temperature. The
o
reaction mixture was stirred at 70 C for 12 h. The reaction mixture was cooled to room temperature,
diluted with EtOAc and quenched with H2O. The organic layer was washed with brine, dried over
MgSO4, filtered and concentrated. The residue was dissolved in CH2Cl2 and solidified by adding Et2O.
1
The resulting solid was collected to afford 8b (4.9 g, 65%) as a brown solid. H NMR (400 MHz,
DMSO-d6) δ 6.81 (brs, 1H), 6.03 (s, 1H), 2.67 (m, 2H), 2.52 (m, 2H), 1.73 (m, 2H), 1.55 (m, 4H); 13C
NMR (100 MHz, DMSO-d6) δ 167.81, 153.47, 136.20, 119.88, 113.29, 31.57, 28.43, 28.21, 27.71,
27.09; LRMS (ESI) m/z 237 [M + H]+.
2-Amino-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (8c) To a suspension
of 2-cyanoacetamide (3.0 g, 35.7 mmol), 4-tert-butylcyclohexanone (5.8 mL, 35.7 mmol), and
morpholine (7.1 mL, 71.4 mmol) in EtOH (60.0 mL) was added sulfur (1.1 g, 35.7 mmol) at room
o
temperature. The reaction mixture was stirred at 70 C for 12 h. The reaction mixture was cooled to
room temperature, diluted with EtOAc and quenched with H2O. The organic layer was washed with
brine, dried over MgSO4, filtered and concentrated. The residue was dissolved in CH2Cl2 and
collected by adding Et2O. The resulting solid was collected to afford 8c (5.2 g, 69%) as a brown solid.
1H NMR (400 MHz, DMSO-d6) δ 6.91 (s, 1H), 6.48 (brs, 1H), 2.66 (m, 1H), 2.59 (m, 1H), 2.44 (m,
1H), 2.23 (m, 1H), 1.91 (m, 1H), 1.39 (m, 1H), 1.17 (m, 2H), 0.89 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 167.87, 159.34, 129.94, 116.08, 107.08, 44.55, 32.14, 27.10, 26.99, 25.62, 24.18; LRMS
(ESI) m/z 247 [M + H]+. HRMS (ESI) m/z calculated for C13H21N2OS+ [M + H] +: 253.13. Found:
253.1505.
General procedure A for the synthesis of compounds 7a-c and 9a-i. To a solution of 6 or 8a-c (1
equiv) in pyridine (0.2 M) was added various acyl chloride (1.1 equiv) at 0 oC. The reaction
mixture was stirred for 1 h at room temperature, quenched with H2O and diluted with EtOAc. The
organic layer was washed with water and brine, dried over MgSO4, filtered and concentrated. The
resulting residue was purified by flash column chromatography on silica gel.

2-Pivalamidothiophene-3-carboxamide (7a) Compound 6 (100 mg, 0.70 mmol) was transformed to
the target compound using general procedure A. The resulting crude product was purified by flash
1
column chromatography on silica gel (25% EtOAc/hexane) to afford 7a (108 mg, 84%). H NMR
(400 MHz, DMSO-d6) δ 12.65 (s, 1H), 7.94 (brs, 1H), 7.53 (brs, 1H), 7.42 (d, J = 5.8 Hz, 1H), 6.94 (d,
J = 5.8 Hz, 1H), 1.23 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 174.76, 167.24, 146.27, 122.94,
115.90, 114.98, 38.56, 30.67, 26.94; LRMS (ESI) m/z 183 [M + H]+.
N-(3-Carbamoylthiophen-2-yl)furan-2-carboxamide (7b) Compound 6 (100 mg, 0.70 mmol) was
transformed to the target compound using general procedure A. The resulting crude product was
purified by flash column chromatography on silica gel (25% EtOAc/hexane) to afford 7b (122 mg,
82%). 1H NMR (400 MHz, DMSO-d6) δ 13.12 (s, 1H), 8.03 (dd, J = 1.7, 0.7 Hz, 1H), 8.01 (brs, 1H),
7.60 (brs, 1H), 7.47 (d, J = 5.7 Hz, 1H), 7.32 (dd, J = 3.5, 0.7 Hz, 1H), 7.04 (d, J = 5.7 Hz, 1H), 6.77
(dd, J = 3.5, 1.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 166.98, 153.84, 146.68, 146.09, 145.13,
123.20, 116.64, 116.32, 115.85, 112.97; LRMS (ESI) m/z 211 [M + H]+.
2-Benzamidothiophene-3-carboxamide (7c) Compound 6 (100 mg, 0.70 mmol) was transformed o
the target compound using general procedure A. The resulting crude product was purified by flash
1
column chromatography on silica gel (25% EtOAc/hexane) to afford 7c (162 mg, 91%). H NMR
(400 MHz, DMSO-d6) δ 13.44 (s, 1H), 8.06 (brs, 1H), 7.93 (m, 2H), 7.64 (m, 4H), 7.50 (d, J = 5.7 Hz,
1H), 7.05 (d, J = 5.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 167.37, 162.46, 146.20, 132.64,
132.13, 129.17, 126.98, 123.17, 116.44, 115.64; LRMS (ESI) m/z 253 [M + H]+.
2-Pivalamido-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (9a) Compound 8a (100
mg, 0.55 mmol) was transformed to the target compound using general procedure A. The resulting
crude product was purified by flash column chromatography on silica gel (25% EtOAc/hexane) to
1
afford 9a (117 mg, 80%). H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H), 7.61 (brs, 1H), 6.65 (brs,
1H), 2.90 (t, J = 7.0 Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.34 (m, 2H), 1.22 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 174.50, 167.50, 149.16, 138.86, 131.33, 110.42, 38.65, 29.03, 28.17, 27.67, 26.94;
LRMS (ESI) m/z 267 [M + H]+.

2-Pivalamido-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (9b) Compound 8b
(100 mg, 0.48 mmol) was transformed to the target compound using general procedure A. The
resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9b (121 mg, 86%). 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 2.67 (m,
4H), 1.81 (m, 2H), 1.58 (m, 4H), 1.23 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 176.31, 144.52,
135.85, 132.10, 114.75, 98.22, 31.38, 28.35, 28.33, 27.62, 26.92, 26.73; LRMS (ESI) m/z 295 [M +
H]+. HRMS (ESI) m/z calculated for C15H22N2NaO2S+ [M + Na] +: 317.13. Found: 317.1227.
6-(tert-Butyl)-2-pivalamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (9c) Compound
8c (100 mg, 0.40 mmol) was transformed to the target compound using general procedure A. The
resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9c (119 mg, 89%). 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 7.52 (brs,
1H), 6.86 (brs, 1H), 2.76 (m, 2H), 2.64 (m, 1H), 2.36 (m, 1H), 1.97 (m, 1H), 1.40 (m, 1H), 1.21 (m,
1H), 1.21 (s, 9H), 0.91 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 174.46, 167.87, 144.07, 128.64,
126.28, 114.48, 44.35, 38.56, 32.14, 27.07, 26.94, 26.27, 2540, 24.11; LRMS (ESI) m/z 337 [M + H]+.
HRMS (ESI) m/z calculated for C18H29N2O2S+ [M + H] +: 337.19. Found: 337.1890.
N-(3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)furan-2-carboxamide
(9d)
Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound using general procedure
A. The resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9d (144 mg, 95%). 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 8.02 (d,
J = 1.7 Hz, 1H), 7.68 (brs, 1H), 7.29 (d, J = 3.2 Hz, 1H), 6.76 (dd, J = 3.2, 1.7 Hz, 1H), 6.76 (brs, 1H),
13
2.94 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.37 (m, 2H); C NMR (100 MHz, DMSO-d6) δ
167.22, 153.58, 147.72, 146.51, 146.18, 139.23, 132.22, 116.05, 112.91, 111.40, 29.01, 28.22, 27.68;
LRMS (ESI) m/z 276 [M + H]+.
N-(3-Carbamoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-2-yl)furan-2-carboxamide
(9e)
Compound 8b (100 mg, 0.48 mmol) was transformed to the target compound using general procedure
A. The resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9e (129 mg, 89%). 1H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 7.98 (d,
J = 1.0 Hz, 1H), 7.50 (brs, 2H), 7.27 (d, J = 3.3 Hz, 1H), 6.74 (dd, J = 3.3, 1.0 Hz, 1H), 2.81 (m, 2H),
2.71 (m, 2H), 1.79 (m, 2H), 1.60 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 167.55, 153.74, 146.32,

146.18, 136.91, 134.99, 130.87, 121.06, 115.80, 112.77, 31.46, 28.21, 28.13, 27.48, 27.04; LRMS
(ESI) m/z 305 [M + H]+.
N-(6-(tert-Butyl)-3-carbamoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)furan-2-carboxamide (9f)
Compound 8c (100 mg, 0.40 mmol) was transformed to the target compound using general procedure
A. The resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9f (126 mg, 92%). 1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.99 (d,
J = 1.5 Hz, 1H), 7.59 (brs, 1H), 7.28 (d, J = 3.3 Hz, 1H), 6.96 (brs, 1H), 6.74 (dd, J = 3.3, 1.5 Hz, 1H),
2.79 (m, 2H), 2.68 (m, 1H), 2.39 (m, 1H), 1.97 (m, 1H), 1.42 (m, 1H), 1.23 (m, 1H), 0.90 (s, 9H); 13C
NMR (100 MHz, DMSO-d6) δ 167.57, 153.63, 146.44, 146.26, 142.53, 129.03, 127.26, 115.97,
115.68, 112.86, 44.31, 32.14, 27.04, 26.25, 25.44, 24.02; LRMS (ESI) m/z 347 [M + H]+.
2-Benzamido-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (9g) Compound 8a (100 mg,
0.55 mmol) was transformed to the target compound using general procedure A. The resulting crude
product was purified by flash column chromatography on silica gel (25% EtOAc/hexane) to afford 9g
1
(143 mg, 91%). H NMR (400 MHz, DMSO-d6) δ 13.28 (s, 1H), 7.90 (d, J = 7.2 Hz, 2H), 7.65 (m,
13
4H), 6.78 (brs, 1H), 2.96 (t, J = 6.9 Hz, 2H), 2.83 (t, J = 7.0 Hz, 2H), 2.37 (m, 2H); C NMR (100
MHz, DMSO-d6) δ 167.59, 162.15, 148.87, 139.23, 132.57, 132.22, 132.04, 129.18, 126.91, 111.16,
29.02, 28.23, 27.70; LRMS (ESI) m/z 287 [M + H]+.
2-Benzamido-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (9h) Compound 8b
(100 mg, 0.48 mmol) was transformed to the target compound using general procedure A. The
resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9h (124 mg, 83%). 1H NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 7.86 (m,
13
2H), 7.65 (m, 2H), 7.58 (m, 3H), 2.81 (m, 2H), 2.73 (m, 2H), 1.80 (m, 2H), 1.60 (m, 4H); C NMR
(100 MHz, DMSO-d6) δ 167.74, 162.63, 137.82, 135.13, 132.57, 132.33, 130.82, 128.97, 127.03,
121.17, 31.54, 28.28, 28.12, 27.57, 27.13; LRMS (ESI) m/z 315 [M + H]+.
2-Benzamido-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (9i) Compound
8c (100 mg, 0.40 mmol) was transformed to the target compound using general procedure A. The
resulting crude product was purified by flash column chromatography on silica gel (25%
EtOAc/hexane) to afford 9i (123 mg, 87%). 1H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H), 7.89 (m,
2H), 7.63 (m, 3H), 2.76 (m, 3H), 2.42 (m, 1H), 2.00 (m, 1H), 1.42 (m, 1H), 1.25 (m, 1H), 0.93 (s, 9H);

13C NMR (100 MHz, DMSO-d6) δ 167.92, 162.24, 143.70, 132.53, 132.35, 129.14, 129.08, 127.07,
126.93, 115.39, 44.35, 32.17, 27.08, 26.29, 25.48, 24.06; LRMS (ESI) m/z 357 [M + H]+.
General procedure B for the synthesis of compounds 10a-q. To a solution of ammonium
thiocyanate (1.5 equiv) in acetone (0.2 M) was added various acyl chloride (1.0 equiv) at room
temperature. The reaction mixture was refluxed for 1 h and then treated with 8a-c (1.0 equiv). The
reaction mixture was allowed to reflux for an additional 1 h and cooled to room temperature and
solidified by adding H2O. The resulting solid was collected and purified by flash column
chromatography on silica gel.
2-(3-Benzoylthioureido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
(10a)
Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound using general procedure
B. The resulting crude product was purified by flash column chromatography on silica gel (5%
MeOH/CH2Cl2) to afford 10a (159 mg, 84%). 1H NMR (400 MHz, DMSO-d6) δ 14.84 (s, 1H), 11.56
(s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.65 (m, 1H), 7.53 (m, 1H), 7.53 (brs, 1H), 6.89 (brs, 1H), 2.96 (t, J
= 6.9 Hz, 2H), 2.85 (t, J = 7.3 Hz, 2H), 2.36 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.41,
166.28, 147.00, 139.64, 133.91, 132.95, 132.15, 128.65, 128.38, 116.40, 29.17, 28.47, 27.62; LRMS
+
(ESI) m/z 346 [M + H]+. HRMS (ESI) m/z calculated for C16H16N3O2S2 [M + H] +: 346.06. Found:
346.0614.
2-(3-Benzoylthioureido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide
(10b)
Compound 8b (100 mg, 0.48 mmol) was transformed to the target compound using general procedure
B. The resulting crude product was purified by flash column chromatography on silica gel (5%
MeOH/CH2Cl2) to afford 10b (139 mg, 78%). 1H NMR (400 MHz, DMSO-d6) δ 13.96 (s, 1H), 11.70
(s, 1H), 7.96 (d, J = 7.3 Hz, 1H), 7.70 (s, 2H), 7.66 (m, 1H), 7.53 (m, 2H), 2.72 (m, 4H), 1.81 (m, 2H),
1.59 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 173.76, 167.70, 166.50, 134.96, 134.23, 133.15,
132.25, 131.86, 128.69, 128.43, 128.18, 31.78, 28.39, 28.05, 27.78, 27.19; LRMS (ESI) m/z 374 [M +
H]+.
2-(3-Benzoylthioureido)-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (10c)
Compound 8c (100 mg, 0.40 mmol) was transformed to the target compound using general procedure
B. The resulting crude product was purified by flash column chromatography on silica gel (5%
MeOH/CH2Cl2) to afford 10c (134 mg, 81%). 1H NMR (400 MHz, DMSO-d6) δ 14.40 (s, 1H), 11.62

(s, 1H), 7.96 (m, 2H), 7.65 (m, 1H), 7.60 (brs, 1H), 7.53 (m, 2H), 7.25 (brs, 1H), 2.73 (m, 3H), 2.41
13
(m, 1H), 1.99 (m, 1H), 1.44 (m, 1H), 1.24 (m, 1H), 0.93 (s, 9H); C NMR (100 MHz, DMSO-d6) δ
173.92, 166.87, 166.34, 140.05, 133.04, 132.04, 129.43, 128.80, 128.67, 128.42, 122.35, 44.52, 32.22,
27.10, 25.87, 25.30, 24.08; LRMS (ESI) m/z 416 [M + H]+. HRMS (ESI) m/z calculated for
C21H26N3O2S2 [M + H] +: 416.14. Found: 416.1385.
+
N-((3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)carbamothioyl)furan-2-
carboxamide (10d) Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound
using general procedure B. The resulting crude product was purified by flash column chromatography
1
on silica gel (5% MeOH/CH2Cl2) to afford 10d (138 mg, 75%). H NMR (400 MHz, DMSO-d6) δ
14.70 (s, 1H), 11.31 (s, 1H), 8.06 (d, J = 1.1 Hz, 1H), 7.80 (d, J = 3.5 Hz, 1H), 7.57 (brs, 1H), 6.88
(brs, 1H), 6.75 (dd, J = 3.5, 1.1 Hz, 1H), 2.95 (t, J = 7.0 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.35 (m,
2H); 13C NMR (100 MHz, DMSO-d6) δ 172.90, 166.27, 155.80, 148.18, 147.06, 144.77, 139.62,
133.93, 118.37, 116.33, 112.59, 29.20, 28.47, 27.61; LRMS (ESI) m/z 336 [M + H]+.
N-((3-Carbamoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-2-yl)carbamothioyl)furan-2-
carboxamide (10e) Compound 8b (100 mg, 0.48 mmol) was transformed to the target compound
using general procedure B. The resulting crude product was purified by flash column chromatography
1
on silica gel (5% MeOH/CH2Cl2) to afford 10e (128 mg, 74%). H NMR (400 MHz, DMSO-d6) δ
13.75 (s, 1H), 11.47 (s, 1H), 8.08 (d, J = 1.7 Hz, 1H), 7.83 (d, J = 3.7 Hz, 1H), 7.69 (brs, 2H), 6.76
(dd, J = 3.7, 1.7 Hz, 1H), 2.70 (m, 4H), 1.81 (m, 2H), 1.59 (m, 4H); 13C NMR (100 MHz, DMSO-d6)
δ 173.41, 166.39, 157.17, 148.49, 144.49, 135.04, 134.18, 132.29, 127.33, 118.73, 112.70, 31.79,
28.39, 28.02, 27.78, 27.19; LRMS (ESI) m/z 364 [M + H]+.
N-((6-(tert-Butyl)-3-carbamoyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamothioyl)furan-2-
carboxamide (10f) Compound 8c (100 mg, 0.40 mmol) was transformed to the target compound
using general procedure B. The resulting crude product was purified by flash column chromatography
1
on silica gel (5% MeOH/CH2Cl2) to afford 10f (142 mg, 88%). H NMR (400 MHz, DMSO-d6) δ
14.23 (s, 1H), 11.35 (s, 1H), 8.06 (d, J = 1.7 Hz, 1H), 7.80 (d, J = 3.5 Hz, 1H), 7.57 (brs, 1H), 7.24
(brs, 1H), 6.75 (dd, J = 3.5, 1.7 Hz, 1H), 2.70 (m, 3H), 2.39 (m, 1H), 1.99 (m, 1H), 1.43 (m, 1H), 1.23
(m, 1H), 0.92 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 173.46, 166.26, 156.35, 148.30, 144.65,
140.04, 129.45, 128.78, 122.31, 118.52, 112.63, 44.51, 32.20, 27.08, 25.86, 25.28, 24.06; LRMS (ESI)
m/z 406 [M + H]+.

N-((3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)carbamothioyl)-4-methylthiazole-
5-carboxamide (10g) Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound
using general procedure B. The resulting crude product was purified by flash column chromatography
1
on silica gel (5% MeOH/CH2Cl2) to afford 10g (153 mg, 76%). H NMR (400 MHz, DMSO-d6) δ
14.69 (s, 1H), 11.57 (s, 1H), 9.20 (s, 1H), 7.60 (brs, 1H), 6.88 (brs, 1H), 2.96 (t, J = 6.6 Hz, 2H), 2.84
(t, J = 6.7 Hz, 2H), 2.62 (s, 3H), 2.35 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 172.69, 166.36,
160.72, 157.79, 156.43, 147.10, 139.65, 134.00, 124.38, 116.24, 29.17, 28.47, 27.62, 17.24; LRMS
(ESI) m/z 367 [M + H]+.
2-(3-(3,4-Dimethoxybenzoyl)thioureido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
(10h) Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound using general
procedure B. The resulting crude product was purified by flash column chromatography on silica gel
1
(5% MeOH/CH2Cl2) to afford 10h (180 mg, 81%). H NMR (400 MHz, DMSO-d6) δ 14.87 (s, 1H),
11.43 (s, 1H), 7.69 (dd, J = 8.4, 1.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.51 (brs, 1H), 7.09 (d, J = 8.4
Hz, 1H), 6.90 (brs, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 2.95 (t, J = 7.0 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H),
2.35 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.63, 166.25, 165.44, 152.91, 148.20, 146.98,
139.64, 133.85, 123.67, 122.86, 116.44, 111.54, 110.94, 55.74, 55.67, 29.18, 28.48, 27.62; LRMS
(ESI) m/z 406 [M + H]+.
2-(3-(3,5-Dimethoxybenzoyl)thioureido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide
(10i) Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound using general
procedure B. The resulting crude product was purified by flash column chromatography on silica gel
1
(5% MeOH/CH2Cl2) to afford 10i (185 mg, 83%). H NMR (400 MHz, DMSO-d6) δ 14.86 (s, 1H),
11.56 (s, 1H), 7.55 (brs, 1H), 7.15 (s, 1H), 6.89 (brs, 1H), 6.75 (s, 1H), 3.83 (s, 6H), 2.96 (t, J = 7.0
13
Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.35 (m, 2H); C NMR (100 MHz, DMSO-d6) δ 173.30, 166.30,
165.70, 160.24, 147.03, 139.65, 133.96, 116.40, 106.27, 105.41, 55.60, 29.20, 28.49, 27.64; LRMS
(ESI) m/z 406 [M + H]+.
2-(3-(3,4,5-Trimethoxybenzoyl)thioureido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
carboxamide (10j) Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound
using general procedure B. The resulting crude product was purified by flash column chromatography
1
on silica gel (5% MeOH/CH2Cl2) to afford 10j (187 mg, 78%). H NMR (400 MHz, DMSO-d6) δ

14.90 (s, 1H), 11.57 (s, 1H), 7.53 (brs, 1H), 7.36 (s, 2H), 6.91 (brs, 1H), 3.88 (s, 6H), 3.75 (s, 3H),
13
2.96 (t, J = 7.5 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H), 2.35 (m, 2H); C NMR (100 MHz, DMSO-d6) δ
173.46, 166.28, 165.38, 152.56, 146.96, 141.53, 139.67, 133.95, 126.66, 116.48, 106.32, 60.15, 56.16,
29.19, 28.50, 27.64; LRMS (ESI) m/z 436 [M + H]+.
N-((3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-
yl)carbamothioyl)benzo[d][1,3]dioxole-5-carboxamide (10k) Compound 8a (100 mg, 0.55 mmol)
was transformed to the target compound using general procedure B. The resulting crude product was
purified by flash column chromatography on silica gel (5% MeOH/CH2Cl2) to afford 10k (184 mg,
86%). 1H NMR (400 MHz, DMSO-d6) δ 14.82 (s, 1H), 11.36 (s, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.53 (s,
1H), 7.53 (brs, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.88 (brs, 1H), 6.15 (s, 2H), 2.95 (t, J = 6.8 Hz, 2H),
2.84 (t, J = 6.7 Hz, 2H), 2.34 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.46, 166.28, 165.13,
151.39, 147.42, 147.02, 139.63, 133.85, 125.60, 124.72, 116.37, 108.42, 107.99, 102.14, 29.18, 28.48,
27.62; LRMS (ESI) m/z 390 [M + H]+.
N-((3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)carbamothioyl)-2,3-
dihydrobenzo[b][1,4]dioxine-6-carboxamide (10l) Compound 8a (100 mg, 0.55 mmol) was
transformed to the target compound using general procedure B. The resulting crude product was
purified by flash column chromatography on silica gel (5% MeOH/CH2Cl2) to afford 10l (164 mg,
74%). 1H NMR (400 MHz, DMSO-d6) δ 14.81 (s, 1H), 11.35 (s, 1H), 7.55 (s, 1H), 7.54 (d, J = 9.0 Hz,
1H), 7.54 (brs, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.89 (brs, 1H), 4.32 (m, 4H), 2.95 (t, J = 6.9 Hz, 2H),
2.84 (t, J = 7.1 Hz, 2H), 2.35 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.49, 166.25, 165.15,
147.75, 146.98, 142.98, 139.62, 133.82, 124.61, 122.52, 117.81, 116.95, 116.39, 64.52, 63.93, 29.16,
28.47, 27.61; LRMS (ESI) m/z 404 [M + H]+.
2-(3-(3-(Trifluoromethyl)benzoyl)thioureido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
carboxamide (10m) Compound 8a (100 mg, 0.55 mmol) was transformed to the target compound
using general procedure B. The resulting crude product was purified by flash column chromatography
1
on silica gel (5% MeOH/CH2Cl2) to afford 10m (157 mg, 69%). H NMR (400 MHz, DMSO-d6) δ
14.88 (s, 1H), 11.93 (s, 1H), 8.32 (s, 1H), 8.23 (d, J = 7.9 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.77 (dd,
J = 7.9 Hz, 1H), 7.57 (brs, 1H), 6.90 (brs, 1H), 2.97 (t, J = 7.2 Hz, 2H), 2.85 (t, J = 7.2 Hz, 2H), 2.36
13
(m, 2H); C NMR (100 MHz, DMSO-d6) δ 173.19, 166.32, 164.99, 147.04, 139.66, 134.02, 133.27,