Synthesis of urea analogues bearing N-alkyl-N’-(thiophen-2-yl) scaffold and evaluation of their innate immune response to toll-like receptors

Article abstract of DOI:10.1016/j.ejmech.2019.02.067

Previous high throughput virtual screening of 10 million-compound and following cell based validation led to the discovery of a novel, nonlipopeptide-like chemotype ZINC 6662436, as toll-like receptor 2 (TLR2) agonists. In this report, compounds belonging

Full text of DOI:10.1016/j.ejmech.2019.02.067

Accepted Manuscript
Synthesis of urea analogues bearing N-alkyl-N'-(thiophen-2-yl) scaffold and
evaluation of their innate immune response to toll-like receptors
Zhipeng Chen, Xiaohong Cen, Junjie Yang, Zhiman Lin, Meihuan Liu, Kui Cheng
PII:
S0223-5234(19)30191-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.067
EJMECH 11159
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 January 2019
Revised Date: 10 February 2019
Accepted Date: 25 February 2019
Please cite this article as: Z. Chen, X. Cen, J. Yang, Z. Lin, M. Liu, K. Cheng, Synthesis of urea
analogues bearing N-alkyl-N'-(thiophen-2-yl) scaffold and evaluation of their innate immune response
to toll-like receptors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.02.067.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of urea analogues bearing N-alkyl-N'-(thiophen-2-yl) scaffold and
evaluation of their innate immune response to toll-like receptors
Zhipeng Chen†, Xiaohong Cen†, Junjie Yang†, Zhiman Lin, Meihuan Liu, Kui
*
Cheng
Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key
Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School
of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515,
China.
Phone: +86 20 6164 7192, Fax: +86 20 6164 8533
Email address: Chengk@smu.edu.cn
†
These authors contributed equally to this work.
1

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Abstract:
Previous high throughput virtual screening of 10 million-compound and following
cell based validation led to the discovery of a novel, nonlipopeptide-like chemotype
ZINC 6662436, as toll-like receptor 2 (TLR2) agonists. In this report, compounds
belonging to four areas of structural modification of ZINC6662436 were evaluated for
biological activity using human HEK-Blue TLR2 reporter cells, and human THP-1
monocytic cells, yield SMU-C13 as an optimized, direct and high potent (EC =160
5
0
nM) agonist of human TLR2. Moreover, preliminary mechanism studies indicated that
SMU-C13 through activates TLR1 and TLR2 then stimulates the NF-κB activation to
trigger the downstream cytokines, such as TNF-α and secreted alkaline phosphatase
(SEAP).
Keywords: Toll-like receptor 2 (TLR2); Small molecule antagonist; Inflammatory;
Tumor immunity.
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1
. Introduction
Toll-like receptors (TLRs), which are expressed by various cells of the immune
system, such as macrophages, dendritic cells and epithelial cells, can recognize and
respond to molecules derived from bacterial, viral and fungal pathogens [1-5].
Generally, TLRs, particularly TLR2, are the major components of innate or adaptive
immunity by recognizing conserved patterns in diverse microbial molecules, and
TLR2 cooperation with TLR1 or TLR6 broads the range of recognition ligands [6-8].
In terms of function, recent evidence suggests that TLR2 is essential for sensing
Gram-positive bacteria including peptidoglycans, lipoteichoic acid and bacterial
lipoproteins. When activated, it can convey signals to transcription factors that can
influence the nature of the adaptive T- or B-cell response [9-11]. Because TLRs
specifically respond to the infection of microbial origin’s products and direct the
adaptive immune responses against antigens of microbial origin, just like other TLRs,
TLR2 also has become a desirable target for vaccine adjuvants and cancer treatment
in recent years. TLR2 agonists have been tested in vitro and in vivo for their ability to
activate an antitumor immune response against several cancers [12-14]. The
combination of TLR1/2 agonist (Diprovocim, EC50 110 pM in THP-1 cells) and
PD-L1 antibody showed great synergistic effect for tumor inhibition, and form
long-term antitumor memory effect [15, 16].
These potential tumor immune effects have spurred our enthusiasm for discovering
and developing new TLR2 selective agonists. While TLR2 recognizes a wide range of
ligands, many of which are from Gram-positive bacteria, and it signals as a
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heterodimer with either TLR1 or TLR6 [9], raising questions about the specificity of
small molecule agents. Besides, until today, none of the TLR2 agonists in clinical or
preclinical development are small molecules, and some contain mixtures with more
than one active ingredient. Our group has focused on small molecules that target the
TLR2 heterodimer protein-protein interaction [17, 18], specifically modulating the
TLR1/2 response [19, 20].
Recently, 10 million-compound (10,519,614) from ZINC drug database was screened
against the Pam CSK -binding domain of TLR1/2 (crystal structure PDB: 2Z7X) [9]
3
4
using the Glide 7.4 program and yield compound ZINC6662436 as potent TLR2
small molecule agonist probe [21]. In the current study, we report the results of
structure-activity
relationship
(SAR)
study
in
ethyl
2
-(4-methylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-car
boxylate scaffold with the goal of improving the potency of compound ZINC6662436.
Four categories of modification of ZINC6662436 (SMU127) ring system were
explored, while independently keeping all other structural features of the lead
compound constant as depicted in Figure 1: (I) Changing the piperazine ring with
bioisostere, such as piperidine, morpholine, or introducing different electron effect
substitutions on the piperazine ring; (II) Modifications of the urea moiety with
replacement of the oxygen by sulfur; (III) Modifications of the ethyl ester portion,
including changing other alkyl groups, or replacing the ethyl group with carboxyl
group; (IV) Ring enlargement derivatives in which the cyclopentyl ring portion was
either replaced by cyclohexyl, substituted cyclohexyl, or cycloheptyl.
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2
2
. RESULTS AND DISCUSSION
.1 Chemistry and SAR studies
The lead compound ZINC6662436 (SMU127) was synthesized from ethyl
2
-isocyanato-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate
A1
and
1
-methylpiperazine B1 as previously reported [21]. Moiety A1 was obtained by
mixture cyclopentanone, ethyl 2-cyanoacetate, sulfur and diethylamine at the sonicate
condition following the Gewald procedure. To explore more SAR studies, moiety
C2-12 were synthesized in the similar conditions by using assorted of ketone and
cyanoacetic ester (Scheme 1).
4
The synthesis of target analogues bearing substitution at the N -position of piperazine
(Figure 1, I) was undertaken as follows. Intermediate C1 reacts with
bis(trichloromethyl)carbonate in dimethyl carbonate at 90 °C to afford compound A1
without further purification, then N-substituted piperazine was added and stirred at
room temperature to yield target compounds 3-5 (Scheme 2). Synthesis of piperidine
or morpholine analogues was undertaken in similar reaction conditions only replacing
the N-substituted piperazine with piperidine or morpholine (compounds 1 and 2).
HEK-Blue hTLR2 cells were obtained by co-transfection of the human TLR2 and
secreted embryonic alkaline phosphatase (SEAP) genes into HEK293 cells and were
employed to test the compounds’ TLR2 activities. As shown in Table 1, when
piperizine was replaced by piperidine (1) or morpholine (2), the TLR2 activation
activity was lost. The results suggested that piperizine scaffold was necessary for the
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4
activity, next we performed a minor modification on the N of piperizine position and
investigated the substituent effects in the initial SAR study (Figure 1, part I). It was
found that electro-donating substituents on piperizine group had very positive effect
on TLR2 activity, while electro-withdraw substituents showed negative effect on the
4
TLR2 activation (Table 1, compound 3-5). The best substituent on N of piperizine
was methyl.
As the modification of position II (Figure 1), we replaced the oxygen with the
electronic isostere sulfur together with different substituents on piperizine. The sulfur
atom was generated from isothiocyanate (F) which obtained by reacting intermediates
C with thiophosgen and triethylamine in dichloromethane at 0 °C. Next,
isothiocyanates react with N-substituted piperazine to yield the target thiourea
compounds (Scheme 3). The SAR studies showed that the agonist activity was
decreased about 6 folds when oxygen atom replaced by sulfur atom (SMU127 vs 6).
4
As similar as the previous results, the TLR2 activity was disappeared when N of
piperizine was substituted by electro-withdraw group (Table 1, compounds 7, 8).
With these results in hand, we turn our attention to modify the third region (Figure 1,
III) and keep 1-methyl piperizine substituent and oxygen constant. We introduced
varying lengths of substituents from various aliphatic chains at the R position.
1
Compounds 9-11 were prepared by reacting 1-methyl piperazine with various A in
similar reaction conditions (Scheme 2). Hydrolyzing compound 9 with LiOH in
methanol/THF/H O solvent system provided the high yield hydrolyzed product 12
2
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(Table 1). The SAR for series 9-12 showed that methyl was the favorable substitution.
The activity sequence was methyl > ethyl > isopropyl > propyl. When the number of
carbon atoms was larger than 2 in the substitutions, the compounds showed less or no
activities. The activity was disappeared when the ester was hydrolyzed to carboxyl
group (Table 1, compound 12).
The varied size of the ring has been studied in site IV (Figure 1, Scheme 2). It was
found that six member ring fused[b]thiophene was the most favorable scaffold, and
5
-membered rings caused 5 times reduction of activity (13 vs SMU127). The activity
of compound 14 was decreased 10 folds than that of compound 13 when the six
member ring switched to seven. The size of substituent on 6-position of
4
,5,6,7-tetrahydrobenzo[b]thiophene was also crucial for the activity and the activity
decreased or disappeared with the size of substituents increasing (Table 1,
compounds 15, 16). To further investigate the SAR results, compounds 17-28 were
4
synthesized (Scheme 2, 3). When the substituent of N atom of piperizine was
replaced by phenyl or acetyl group, the activity disappeared (Table 1, compounds 20,
2
2, 24). Thiourea compounds, the oxygen replaced with sulfur, showed less potency
than that of urea (Table 1, compounds 17 vs 25, 18 vs 26). The methyl ester was
proved to be the favorable substituent on R position, and the activities of compounds
1
1
7 and 18 decreased 2 to 30 folds compared with compound 13 (SMU-C13). Over all,
with four positions of SAR studies, we obtained compound SMU-C13 possessed the
highest agonist TLR2 activity and was selected out for further evaluation.
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2
2
.2. Biological activity.
.2.1 Specificity validation of SMU-C13.
As we know, the TLR family contains both intracellular and extracellular members.
Here we selected the respective extracellular membrane protein (TLR4) and
intracellular member (TLR3) to verify the specificity of SMU-C13. As shown in
Figure 2A, the compound SMU-C13 can effectively active the TLR2 triggered SEAP
signaling in HEK-Blue hTLR2 cells with no activation to the TLR4 and TLR3
signaling in other HEK-Blue hTLRs (Fig. 2B). The Poly IC and LPS used as positive
controls which can significantly activate the signals of TLR4 and TLR3, respectively.
The experiment results showed that SMU-C13 has very good specificity to TLR2. We
also evaluated whether SMU-C13 binds to TLR2 on the cell surface and induces
TLR2 expression in the whole-cell environment. HEK-Blue hTLR2 cells, which
overexpress the human TLR2, were stimulated with SMU-C13. Cell lysates were
subjected to immunoprecipitation and immuno-detected using TLR2 antibody. As
shown in Figure 2C, with the dose of SMU-C13 increased, the expression of TLR2
was gradually increased. The TLR2 protein upregulated more than 5-fold when
treated with 0.1 µM SMU-C13 vs the blank control, and the highest TLR2 expression
level was achieved about 8-fold with the addition of 10 µM SMU-C13 (Fig. 2D).
These results indicate that SMU-C13 is a specific agonist of TLR2 and is capable of
binding to TLR2 on the cell surface to promote expression of TLR2 protein.
2
.2.2 Antibody specificity studies.
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TLR2 itself is unable to initiate an immune response, and it needs to form a
dimerization with another protein, TLR1 or TLR6, in order to function normally.
TLR1 and TLR6 are endogenously expressed in HEK-Blue hTLR2 cells, and whether
SMU-C13 is an activator of TLR1/2 or an activator of TLR2/6 is unclear. Here we
validate this result through antibodies experiments. When adding SMU-C13 to
HEK-Blue hTLR2, we added different antibodies, including anti-TLR1, anti-TLR2 or
anti-TLR6, to see which one or which TLRs were inhibited. The results showed that
anti-TLR1 and anti-TLR2 antibodies had a very significant inhibition on the SEAP
signal produced by SMU-C13, while anti-TLR6 antibody had no effect (Fig. 3). This
experiment confirmed that SMU-C13 was an activator of TLR1/2 but not TLR2/6.
2
.2.3 Downstream signaling evaluation.
Tumor necrosis factor α (TNF-α) is an important inflammatory cytokine produced
after TLR2 promoted NF-κB activation, also one of the most active bioactive factors
found to kill tumors so far. In this paper, the TNF-α was tested in phorbol
1
2-myristate 13-acetate (PMA)-differentiated human monocytic (THP-1) myeloid
cells [15]. As show in Figure 4A, SMU-C13 induced dose-dependent TNF-α
production after treat with the THP-1 cells for 4 hours. Meanwhile, SEAP assay data
in human HEK-Blue TLR2 cells showed that SMU-C13 can strongly trigger the
alkaline phosphatase secretion after NF-κB activation, and this activation can be
suppressed by the classical NF-κB inhibitor triptolide (Fig. 4B). In summary,
preliminary cytokine and protein levels studies have shown that SMU-C13 activates
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TLR1/2, which initiates NF-κB signaling pathways to activate downstream signaling
factors such as TNF and SEAP.
2
.2.4 In vitro cytotoxicity studies
The toxicity of the compound was evaluated by a CCK8 colorimetric method. From
the cell viability data (Fig. 5) we can confirm that SMU-C13 had no cytotoxicity as
concentration up to 100 µM. Considering compound SMU-C13 having the highest
TLR2 activation activity (EC =160±10 nM), low toxicity, as well as appropriate
5
0
molecular weight, it might own the higher drug-like potential ability.
2
.2.5 Molecular docking studies of SMU-C13.
The TLR1/2 agonist Pam3CSK4 binding to TLR1/TLR2 complex causes
hetrodimerization of the extracellular domains to form a TLR1/TLR2/Pam3CSK4
complex, a human complex crystal structure which has been solved by Lee et al [9].
Previous experiments have demonstrated that SMU-C13 can trigger downstream
cytokines, as well as directly active hTLR1 and hTLR2 protein directly in the
antibody assay. To examine possible explanations for the significantly TLR1/2
activation potency in human cells, we explored the predicted binding mode(s) of
compound SMU-C13 with the TLR1/TLR2 complex by conducting molecular
docking experiments in the human protein crystal complex (PDB 2Z7X). We selected
the most favorable energy configuration of SMU-C13 (shown in green) (Fig. 6A) and
compared the binding of Pam3CSK4 (shown in red) bound to the complex (Fig. 6B).
1
0

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Of particular interest is that SMU-C13 is well binding to the interface of TLR1
(purple) and TLR2 (yellow), tightly fit into the binding site of Pam3CSK4 and TLR1
and TLR2 (Fig. 6B), and shows significant interaction with the important residues,
such as Gly313, Gln316 on TLR1 and Phe349 on TLR2 (Fig. 6C). The six-membered
cyclohexane group and the methyl ester group are located on the moderately active
pocket of the protein, which explains why enlarging the size of the ring or extending
4
the ester chain results in a decrease in activity. Changes in N position on the
piperidine ring causing the interaction between the small molecule and the protein are
influenced, so the activity is also varying. From the computer simulation, we can see
that the small molecule well fit in the surface of dimer interface and greatly influence
the potency of SMU-C13 in the TLR1/2 activity.
3
. Conclusion
In this study, a series of ZINC 6662436 derivatives, were designed and synthesized,
and their potential TLR2 activity was evaluated in HEK-Blue hTLR2 cells, and
human THP-1 cells. The SAR results indicated that the piperidine ring, rather than the
piperazine ring or the morpholine ring, contributes to the activity of TLR2; further
4
studies have confirmed that the H at the N piperidine ring is substituted with a
methyl group as the optimal substituent. When the urea group in ZINC6662436 is
substituted by thiourea, its activity almost completely disappears. Meanwhile, when
the five-membered ring is expanded to a six-membered ring in the structure, the
activity is increased, while the activity is decreased when the six-member ring switch
1
1

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to seven. Considering the steric hindrance, electronegativity and electron effects of
various substituent groups, we obtained an optimal compound SMU-C13 with EC at
5
0
1
60±10 nM.
Following specificity assays in various HEK-Blue hTLRs indicated SMU-C13 only
active TLR2 signaling with no influence to TLR3 and TLR4. Notably, SMU-C13 can
also effectively up-regulate the TLR2 expression, up to 8 times. Antibodies
experiments showed that SMU-C13 active TLR2 through its associate with TLR1, not
TLR6. Elisa assays results indicated SMU-C13 could trigger the TNF-α expression
after NF-κB activation, and the NF-κB activation can be suppressed by the NF-κB
inhibitor triptolide. Computer simulation indicated SMU-C13 tightly fit into the
binding site of Pam3CSK4 and TLR1/2, and form important small molecule-protein
interactions. Taken together, our work enriched the situation of lacking of
small-molecule of TLR1/2 agonist family, and primary in vitro assays explores the
working mechanism of SMU-C13 and provides an novel molecule probe for potential
therapeutic applications, including vaccine adjuvants development and tumor
immunity therapies.
4
4
. Experimental section
.1. Chemistry
General procedure for synthesis of intermediate C
Compound C was synthesized following to the procedure of Gewald et al. [22], as
already reported with some minor modifications [21]. The mixture of ketone D (1
1
2

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mmol), alkyl cyanoacetate E (1.1 mmol), sulfur (1.1 mmol), and diethylamine (1.1
mmol) in ethanol/methanol (10 mL) was kept at room temperature in an ultrasonic
cleaner for 1-3 hours. After completion of the reaction, the solvent was evaporated to
dryness under reduced pressure. The crude product was purified by flash column
chromatography on silica gel (petroleum ether/ethyl acetate elute) to provide the
product C.
methyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (C2)
1
Yield: 96%. H NMR (400 MHz, CDCl
3
) δ 5.89 (s, 2H), 3.80 (s, 3H), 2.80-2.85 (m,
1
3
2
6
H), 2.71-2.76 (m, 2H), 2.91-2.36 (m, 2H). C NMR (101 MHz, DMSO-d ) δ 168.5,
+
1
65.5, 141.8, 119.5, 99.6, 50.7, 30.8, 28.8, 27.0. ESI-MS: m/z 198.1 ([M+H] ).
propyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (C3)
1
Yield: 90%. H NMR (400 MHz, CDCl ) δ 5.74 (s, 2H), 4.18 (t, J = 6.4 Hz, J = 12.8
3
1
2
Hz, 2H), 2.83-2.87 (m, 2H), 2.71-2.75 (m, 2H), 2.31-2.37 (m, 2H), 1.71-1.77 (m, 2H),
1
3
1
.02 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.8, 166.0,
1 2 3
+
1
42.5, 121.1, 102.5, 65.1, 30.9, 28.9, 27.2, 22.2, 10.8. ESI-MS: m/z 226.1 ([M+H] ).
isopropyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (C4)
1
Yield: 91%. H NMR (400 MHz, CDCl ) δ 5.77 (s, 2H), 5.12-5.18 (m, 1H), 5.82-5.86
3
13
(
m, 2H), 2.72-2.75 (m, 2H), 2.31-2.36 (m, 2H), 1.32 (d, J = 6.4 Hz, 6H). C NMR
(
101 MHz, CDCl ) δ 166.4, 165.3, 142.6, 121.0, 102.9, 66.5, 30.8, 28.8, 27.1, 22.1.
3
1
3

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+
ESI-MS: m/z 226.2 ([M+H] ).
methyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (C5)
1
Yield: 95%. H NMR (400 MHz, CDCl ) δ 5.97 (s, 2H), 3.80 (s, 3H), 2.69-2.72 (m,
3
1
3
2
H), 2.50-2.53 (m, 2H), 1.75-1.79 (m, 4H). C NMR (101 MHz, CDCl ) δ 166.4,
3
+
1
62.0, 132.3, 117.4, 105.3, 50.5, 26.8, 24.5, 23.2, 22.7. ESI-MS: m/z 212.1 ([M+H] ).
methyl 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylate (C6)
1
Yield: 90%. H NMR (400 MHz, CDCl
3
) δ 5.51 (s, 2H), 3.83 (s, 3H), 2.96-2.99 (m,
1
3
2
H), 2.58-2.61 (m, 2H), 1.82-1.85 (m, 2H), 1.61-1.68 (m, 4H). C NMR (101 MHz,
CDCl
3
) δ 166.4, 160.2, 137.8, 121.2, 107.2, 50.7, 32.1, 28.6, 28.6, 27.8, 27.0. ESI-MS:
+
m/z 226.1 ([M+H] ).
ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (C7)
1
Yield: 94%. H NMR (400 MHz, CDCl ) δ 5.96 (s, 2H), 4.28 (q, 2H), 2.71-2.74 (m,
3
1
3
2
H), 2.51-2.54 (m, 2H), 1.75-1.81 (m, 4H), 1.35 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C
1 2
NMR (101 MHz, CDCl ) δ 166.1, 162.0, 132.3, 117.4, 105.4, 59.3, 26.9, 24.5, 23.2,
3
+
2
2.8, 14.4. ESI-MS: m/z 226.1 ([M+H] ).
propyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (C8)
1
Yield: 91%. H NMR (400 MHz, CDCl ) δ 5.84 (s, 2H), 4.19 (t, J = 6.4 Hz, J = 12.8
3
1
2
Hz, 2H), 2.72-2.74 (m, 2H), 2.51-2.54 (m, 2H), 1.73-1.81 (m, 6H), 1.02 (t, J = 7.2 Hz,
1
13
J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.3, 162.1, 132.3, 117.4, 105.5,
2
3
1
4

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+
6
5.2, 27.0, 24.5, 23.3, 22.9, 22.2, 10.8. ESI-MS: m/z 240.1 ([M+H] ).
isopropyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (C9)
1
Yield: 90%. H NMR (400 MHz, CDCl ) δ 5.67 (s, 2H), 5.15-5.21 (m, 1H), 2.71-2.74
3
13
(
m, 2H), 2.50-2.54 (m, 2H), 1.74-1.81 (m, 4H), 1.34 (d, J = 7.2 Hz, 6H). C NMR
(
101 MHz, CDCl ) δ 165.6, 162.1, 132.2, 117.2, 105.4, 66.5, 27.0, 24.5, 23.2, 22.9,
3
+
2
2.1. ESI-MS: m/z 240.2 ([M+H] ).
isopropyl
C10)
Yield: 89%. H NMR (400 MHz, CDCl ) δ 5.76 (s, 2H), 5.16-5.23 (m, 1H), 2.98-3.00
2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylate
(
1
3
13
(
m, 2H), 2.50-2.53 (m, 2H), 1.63-1.83 (m, 6H), 1.34 (d, J = 6.4 Hz, 6H). C NMR
(
101 MHz, CDCl ) δ 165.3, 160.0, 137.7, 120.7, 107.3, 66.7, 32.0, 30.3, 27.8, 26.7,
3
+
2
4.2, 22.0. ESI-MS: m/z 254.1 ([M+H] ).
methyl 2-amino-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (C11)
1
Yield: 93%. H NMR (400 MHz, CDCl
3
) δ 5.96 (s, 2H), 3.80 (s, 3H), 2.84-2.90 (m,
1
1
1
H), 2.55-2.59 (m, 2H), 2.11-2.18 (m, 1H), 1.81-1.89 (m, 2H), 1.33-1.38 (m, 1H),
1
3
.06 (d, J = 6.4 Hz, 3H). C NMR (101 MHz, CDCl
3
) δ 166.5, 162.3, 131.9, 117.0,
+
05.1, 50.6, 32.6, 31.1, 29.5, 26.7, 21.5. ESI-MS: m/z 226.1 ([M+H] ).
methyl 2-amino-6-pentyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (C12)
1
Yield: 90%. H NMR (400 MHz, CDCl
3
) δ 5.93 (s, 2H), 3.80 (s, 3H), 2.84-2.90 (m,
1
5

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1
0
1
2
H), 2.57-2.62 (m, 2H), 2.13-2.19 (m, 1H), 1.73-1.91 (m, 2H), 1.31-1.38 (m, 9H),
1
3
1 2 3
.92 (t, J = 6.4 Hz, J = 12.8 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.5, 162.2,
32.2, 117.2, 105.2,50.5, 36.0, 34.5, 32.1, 30.9, 29.3, 26.7, 22.7, 14.1. ESI-MS: m/z
+
82.1 ([M+H] ).
General procedure for synthesis of urea compounds (compounds 1-5, 9-20)
The mixture of intermediate C (1 mmol) and triphosgene (0.35 mmol) in dimethyl
carbonate was kept at 90°C for 8h. After completion of the reaction, without further
purification, substituted piperizine B (1.1 mmol) was added and stirred at room
temperature [21]. After completion of the reaction, the solvent was evaporated to
dryness under reduced pressure. The crude product was purified by flash column
chromatography on silica gel (petroleum ether/ethyl acetate elute) to provide the urea
product.
General procedure for synthesis of thiourea compounds (compounds 6-8, 21-28)
The mixture of C (1 mmol) and triethylamine ( 1.1 mmol) in dichloromethane was
stirred at 0 °C. Thiophosgen (1.5 mmol) was dropped into the mixture to keep the
temperature lower than 5 °C. After completion of the reaction, the solvent was
evaporated to dryness under reduced pressure. Then, 50 mL dichloromethane was
added and washed with water (5 mL) 2 times [23]. The solvent was dried with
magnesium sulfate and evaporated to 10 mL, then substituted piperizine B (1.1 mmol)
was added and stirred at room temperature. After completion of the reaction, the
solvent was evaporated to dryness under reduced pressure. The crude product was
1
6

ACCEPTED MANUSCRIPT
purified by flash column chromatography on silica gel (petroleum ether/ethyl acetate
elute) to provide the thiourea product.
ethyl
2
-(piperidine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate
1)
Yield: 82%. H NMR (400 MHz, CDCl ) δ 10.78 (s, 1H), 4.31 (q, 2H), 3.52-3.53 (m,
(
1
3
4
H), 2.81-2.88 (m, 4H), 2.36-2.38 (m, 2H), 1.66 (s, 6H), 1.37 (t, J = 6.4 Hz, J = 12.8
1 2
13
Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.3, 155.6, 152.4, 140.5, 129.6, 105.4,
3
5
9.8, 44.7, 30.1, 28.6, 27.5, 25.4, 24.1, 14.0. HRMS (ESI): calcd for C H N O S
16 22 2 3
+
(
M+H) = 323.1429, found 323.1433.
ethyl
-(morpholine-4-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylat
e (2)
Yield: 85%. H NMR (400 MHz, CDCl ) δ 10.85 (s, 1H), 4.30 (q, 2H), 3.76-3.78 (m,
2
1
3
4
H), 3.54-3.57 (m, 4H), 2.82-2.91 (m, 4H), 2.36-2.39 (m, 2H), 1.38 (t, J = 7.2 Hz, J
1 2
1
3
=
14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.4, 155.0, 152.7, 140.6, 130.0,
3
1
05.8, 66.1, 60.0, 43.7, 30.1, 28.6, 275, 14.1. HRMS (ESI): calcd for C H N O S
15 20 2 4
+
(
M+H) = 325.1222, found 325.1226.
ethyl
1
7

ACCEPTED MANUSCRIPT
2
-(4-ethylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carb
oxylate (3)
1
Yield: 90%. H NMR (400 MHz, CDCl ) δ 10.82 (s, 1H), 4.30 (q, 2H), 3.59-3.61 (m,
3
4
1
1
H), 2.81-2.90 (m, 4H), 2.35-2.53 (m, 8H), 1.37 (t, J = 7.2 Hz, J = 14.4 Hz, 3H),
1 2
1
3
.12 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.5, 155.3,
1
2
3
52.5, 140.6, 129.9, 105.7, 59.9, 52.0, 52.0, 43.6, 30.2, 28.6, 27.5, 14.4, 11.7. HRMS
+
(
ESI): calcd for C H N O S (M+H) = 352.1695, found 352.1699.
1
7
25
3
3
ethyl
2
-(4-(2-hydroxyethyl)piperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiop
hene-3-carboxylate (4)
1
Yield: 80%. H NMR (400 MHz, CDCl ) δ 10.84 (s, 1H), 4.30 (q, 2H), 359-3.68 (m,
3
6
H), 2.81-2.90 (m, 4H), 2.59-2.63 (m, 7H), 2.35-2.38 (m, 2H), 1.37 (t, J = 7.2 Hz, J
1 2
1
3
=
14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 166.6, 155.2, 152.7, 140.7, 130.1,
3
1
06.0, 60.1, 59.5, 58.0, 525, 43.6, 30.2, 28.7, 27.6, 14.2. HRMS (ESI): calcd for
+
C H N O S (M+H) = 368.1644, found 368.1648.
17
25
3
4
ethyl
-(4-acetylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carb
oxylate (5)
Yield: 86%. H NMR (400 MHz, CDCl ) δ 10.75 (s, 1H), 4.18-4.19 (m, 2H),
2
1
3
3
.45-3.62 (m, 8H), 2.71-2.77 (m, 4H), 2.25-2.27 (m, 2H), 2.05 (s, 3H), 1.27 (t, J =
1
1
8

ACCEPTED MANUSCRIPT
1
3
7
.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 168.9, 166.2, 154.6, 152.3,
2 3
1
40.6, 129.9, 105.9, 60.0, 45.4, 43.1, 40.4, 30.0, 28.5, 27.4, 21.0, 14.0. HRMS (ESI):
+
calcd for C H N O S (M+H) = 366.1488, found 366.1492.
1
7
23
3
4
ethyl
-(4-methylpiperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
2
carboxylate (6)
1
Yield: 91%. H NMR (400 MHz, CDCl ) δ 12.11 (s, 1H), 4.33 (q, 2H), 4.12 (s, 4H),
3
2
.82-2.93 (m, 4H), 2.68 (s, 4H), 2.45 (s, 3H), 2.35-2.39 (m, 2H), 1.38 (t, J = 7.2 Hz,
1
1
3
J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 175.7, 167.1, 155.8, 140.7, 131.4,
2
3
1
08.3, 60.5, 54.3, 47.6, 45.6, 30.4, 28.8, 27.6, 14.2. HRMS (ESI): calcd for
+
C H N O S (M+H) = 354.1310, found 354.1315.
16
23
3
2 2
ethyl
-(4-acetylpiperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-c
2
arboxylate (7)
1
Yield: 88%. H NMR (400 MHz, CDCl
3
) δ 12.12 (s, 1H), 4.32 (q, 2H), 4.14-4.15 (m,
2
H), 3.99-4.01 (m, 2H), 3.81-3.84 (m, 2H), 3.66-3.69 (m, 2H), 2.82-2.93 (m, 4H),
1
3
2
1 2
.35-2.39 (m, 2H), 2.15 (s, 3H), 1.38 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C NMR
(101 MHz, CDCl
3
) δ 176.3, 139.2, 166.7, 155.1, 140.6, 131.1, 108.2, 60.4, 46.7, 44.8,
+
4
0.0, 30.2, 28.7, 27.4, 21.2, 14.1. HRMS (ESI): calcd for C17
82.1259, found 382.1263.
H
23
N
3
3
O S
2
(M+H) =
3
1
9

ACCEPTED MANUSCRIPT
ethyl
-(4-phenylpiperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
2
carboxylate (8)
1
Yield: 83%. H NMR (400 MHz, CDCl ) δ 12.14 (s, 1H), 7.31-7.34 (m, 2H),
3
6
.94-6.98 (m, 3H), 4.32-4.37 (m, 2H), 4.20-4.23 (m, 4H), 3.39-3.41 (m, 4H),
1
3
2
.84-2.95 (m, 4H), 2.36-2.40 (m, 2H), 1.40 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C
1
2
NMR (101 MHz, CDCl ) δ 175.8, 167.2, 155.9, 150.2, 140.8, 131.6, 129.3, 120.1,
3
115.9, 108.4, 60.6, 48.3, 47.5, 30.5, 28.9, 27.7, 14.3. HRMS (ESI): calcd for
+
C H N O S (M+H) = 416.1466, found 416.1471.
21
25
3
2 2
methyl
-(4-methylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-car
boxylate (9)
2
1
Yield: 92%. H NMR (400 MHz, CDCl ) δ 10.81 (s, 1H), 3.85 (s, 3H), 3.57-3.60 (m,
3
1
3
4
H), 2.82-2.84 (m, 4H), 2.46-2.49 (m, 4H), 2.34-2.36 (m, 5H). C NMR (101 MHz,
CDCl ) δ 166.8, 155.4, 152.4, 140.4, 129.9, 105.4, 54.3, 51.1, 45.8, 43.4, 30.0, 28.6,
3
+
2
7.5. HRMS (ESI): calcd for C H N O S (M+H) = 324.1382, found 324.1386.
1
5
21
3
3
propyl
2
-(4-methylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-car
boxylate (10)
1
Yield: 81%. H NMR (400 MHz, CDCl ) δ 10.86 (s, 1H), 4.22 (t, J = 6.4 Hz, J =
3
1
2
2
0

ACCEPTED MANUSCRIPT
1
5
2.8 Hz, 2H), 3.58-3.62 (m, 4H), 2.82-2.92 (m, 4H), 2.47-2.52 (m, 4H), 2.35-2.40 (m,
1
3
H), 1.75-1.80 (m, 2H), 1.04 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz,
1
2
CDCl ) δ 166.8, 155.5, 152.7, 140.7, 130.1, 105.9, 65.8, 54.4, 45.9, 43.6, 30.3, 28.7,
3
+
2
3
7.6, 22.0, 10.5. HRMS (ESI): calcd for C H N O S (M+H) = 352.1695, found
1
7
25
3
3
52.1698.
isopropyl
-(4-methylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-car
boxylate (11)
2
1
Yield: 82%. H NMR (400 MHz, CDCl ) δ 10.85 (s, 1H), 5.14-5.20 (m, 1H),
3
3
.59-3.61 (m, 4H), 2.81-2.88 (m, 4H), 2.48-2.51 (m, 4H), 2.35-2.38 (m, 5H), 1.35 (d,
1
3
J = 7.2 Hz, 6H). C NMR (101 MHz, CDCl ) δ 166.1, 155.1, 152.6, 140.8, 130.0,
3
1
06.2, 67.5, 54.4, 45.9, 43.5, 30.3, 28.7, 27.6, 21.9. HRMS (ESI): calcd for
+
C H N O S (M+H) = 352.1695, found 352.1699.
17
25
3
3
2
-(4-methylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-car
boxylic acid (12)
1
Yield: 78%. H NMR (400 MHz, DMSO-d ) δ 10.92 (s, 1H), 3.35-3.81 (m, 9H),
6
1
3
2
1
.74-2.82 (m, 7H), 2.78-2.33 (m, 2H). C NMR (101 MHz, DMSO-d ) δ 167.7, 154.3,
6
52.3, 141.5, 130.0, 107.2, 51.9, 42.4, 40.9, 30.3, 28.8, 27.7. HRMS (ESI): calcd for
-
C H N O S (M-H) = 308.1069, found 308.1065.
14
19
3
3
2
1

ACCEPTED MANUSCRIPT
methyl
-(4-methylpiperazine-1-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox
ylate (13)
Yield: 92%. H NMR (400 MHz, CDCl ) δ 11.12 (s, 1H), 3.84 (s, 3H), 3.57-3.60 (m,
2
1
3
1
3
4
H), 260-2.72 (m, 4H), 2.46-2.49 (m, 4H), 2.33 (s, 3H), 1.77-1.78 (m, 4H). C NMR
(
101 MHz, CDCl ) δ 167.8, 152.9, 151.7, 130.2, 125.0, 109.0, 54.4, 51.2, 45.9, 43.6,
3
+
2
6.2, 24.2, 22.9, 22.8. HRMS (ESI): calcd for C H N O S (M+H) = 338.1538,
1
6
23
3
3
found 338.1542.
methyl
2
-(4-methylpiperazine-1-carboxamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene
-3-carboxylate (14)
1
Yield: 88%. H NMR (400 MHz, CDCl ) δ 11.08 (s, 1H), 3.85 (s, 3H), 3.56-3.58 (m,
3
4
H), 2.98-2.99 (m, 2H), 2.67-2.68 (m, 2H), 2.45-2.48 (m, 4H), 2.33 (s, 3H), 1.81-1.83
1
3
(
m, 2H), 1.61-1.64 (m, 4H). C NMR (101 MHz, CDCl ) δ 167.9, 153.0, 149.9,
3
1
35.7, 129.1, 110.3, 54.4, 51.3, 45.9, 43.6, 32.1, 28.4, 28.2, 27.7, 27.0. HRMS (ESI):
+
calcd for C H N O S (M+H) = 324.1382, found 324.1386.
1
5
21
3
3
methyl
-methyl-2-(4-methylpiperazine-1-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophen
6
e-3-carboxylate (15)
1
Yield: 87%. H NMR (400 MHz, CDCl
3
) δ 11.30 (d, J = 6.4 Hz, 1H), 4.19-4.23 (m,
2
H), 3.83-3.88 (m, 5H), 3.57-3.60 (m, 2H), 2.85-2.94 (m, 6H), 2.64-2.69 (m, 2H),
2
2

ACCEPTED MANUSCRIPT
1
3
2
.21-2.23 (m, 1H), 1.82-1.88 (m, 2H), 133-1.36 (m, 1H), 1.05 (d, J = 6.4 Hz, 3H). C
NMR (101 MHz, DMSO-d
6
) δ 166.8, 152.4, 150.0, 130.1, 124.8, 109.5, 52.0, 51.9,
+
4
25 3 3
2.3, 40.9, 32.1, 30.9, 29.1, 26.0, 21.6. HRMS (ESI): calcd for C17H N O S (M+H)
=
352.1695, found 352.1698.
methyl
-(4-methylpiperazine-1-carboxamido)-6-pentyl-4,5,6,7-tetrahydrobenzo[b]thiophene
2
-3-carboxylate (16)
1
Yield: 80%. H NMR (400 MHz, CDCl ) δ 11.29 (s, 1H), 3.57-4.02 (m, 11H),
3
2
.85-2.89 (m, 5H), 2.59-2.73 (m, 2H), 2.22-2.25 (m, 1H), 1.88-1.92 (m, 1H), 1.71 (s,
1
3
1
H), 1.29-1.37 (m, 8H), 0.87-0.91 (m, 3H). C NMR (101 MHz, DMSO-d ) δ 166.8,
6
1
52.4, 150.3, 130.4, 124.9, 109.6, 52.1, 51.9, 43.5, 40.9, 35.8, 34.1, 31.9, 30.4, 29.0,
+
2
6.4, 26.0, 22.5, 14.3. HRMS (ESI): calcd for C H N O S (M+H) = 408.2321,
2
1
33
3
3
found 408.2326.
ethyl
2
-(4-methylpiperazine-1-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox
ylate (17)
Yield: 88%. H NMR (400 MHz, CDCl ) δ 11.16 (s, 1H), 4.32 (q, 2H), 3.59-3.61 (m,
1
3
4
H), 2.62-2.76 (m, 4H), 2.49-2.51 (m, 4H), 2.35 (s, 3H), 1.77-1.81 (m, 4H), 1.39 (t, J1
1
3
=
7.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 167.2, 152.9, 151.5,
2 3
1
30.2, 124.9, 109.1, 60.1, 54.4, 459, 43.5, 26.3, 24.1, 22.9, 22.8, 14.2. HRMS (ESI):
+
calcd for C H N O S (M+H) = 352.1695, found 352.1699.
1
7
25
3
3
2
3

ACCEPTED MANUSCRIPT
propyl
-(4-methylpiperazine-1-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox
2
ylate (18)
Yield: 83%. H NMR (400 MHz, CDCl
1
3
) δ 11.17 (s, 1H), 4.20-4.23 (m, 2H),
3
6
1
1
.56-3.58 (m, 4H), 2.62-2.75 (m, 4H), 2.45-2.48 (m, 4H), 2.33 (s, 3H), 1.74-1.80 (m,
1
3
1 2 3
H), 1.02 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 167.3,
52.8, 151.5, 130.1, 124.8, 109.0, 65.8, 54.4, 45.9, 43.6, 26.3, 24.1, 22.9, 22.8, 21.9,
+
0.6. HRMS (ESI): calcd for C18
H
27
N
3
O
3
S (M+H) = 366.1851, found 366.1856.
isopropyl
-(4-ethylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carb
oxylate (19)
2
1
Yield: 82%. H NMR (400 MHz, CDCl
3
) δ 10.84 (s, 1H), 5.14-5.18 (m, 1H),
3
.58-3.62 (m, 4H), 2.80-2.90 (m, 4H), 2.47-2.54 (m, 6H), 2.34-2.38 (m, 2H), 1.34 (d,
13
J = 6.4 Hz, 6H), 1.11-1.14 (m, 3H). C NMR (101 MHz, CDCl
3
) δ 166.1, 155.2,
1
52.6, 140.8, 130.0, 1062, 67.5, 52.1, 52.0, 43.6, 30.3, 28.7, 27.6, 21.9, 11.7. HRMS
+
(
27 3 3
ESI): calcd for C18H N O S (M+H) = 366.1851, found 366.1855.
isopropyl
2
-(4-phenylpiperazine-1-carboxamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-car
boxylate (20)
1
Yield: 81%. H NMR (400 MHz, CDCl ) δ 10.94 (s, 1H), 7.28-7.33 (m, 3H),
3
2
4

ACCEPTED MANUSCRIPT
6
2
.95-7.00 (m, 2H), 5.17-5.20 (m, 1H), 3.76-3.77 (m, 4H), 3.28-3.31 (m, 4H),
1
3
.83-2.90 (m, 4H), 2.37-2.40 (m, 2H), 1.37 (d, J = 6.0 Hz, 6H). C NMR (101 MHz,
CDCl ) δ 166.3, 155.2, 152.8, 150.8, 141.0, 130.2, 129.2, 120.3, 116.4, 106.5, 67.7,
3
+
4
9.0, 43.6, 30.5, 28.8, 27.7, 22.1. HRMS (ESI): calcd for C H N O S (M+H) =
2
2
27
3
3
4
14.1851, found 414.1856.
isopropyl
-(4-methylpiperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
2
carboxylate (21)
1
Yield: 88%. H NMR (400 MHz, CDCl
3
) δ 12.13 (s, 1H), 5.16-5.22 (m, 1H),
4
2
1
.09-4.12 (m, 4H), 2.91 (t, 2H), 2.84 (t, 2H), 2.65-2.67 (m, 4H), 2.42 (s, 3H), 2.37 (t,
1
3
3
H), 1.36 (d, J = 6.4 Hz, 6H). C NMR (101 MHz, CDCl ) δ 175.8, 166.7, 155.6,
40.8, 131.4, 108.8, 68.1, 54.2, 47.4, 45.5, 30.5, 28.8, 27.6, 22.0. HRMS (ESI): calcd
+
for C17
H
25
N
3
O
2
S
2
(M+H) = 368.1466, found 368.1468.
isopropyl
-(4-phenylpiperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-
2
carboxylate (22)
1
Yield: 80%. H NMR (400 MHz, CDCl
3
) δ 12.18 (s, 1H), 7.31-7.35 (m, 2H),
6
.95-6.99 (m, 3H), 5.18-5.24 (m, 1H), 4.21-4.24 (m, 4H), 3.39-3.41 (m, 4H),
1
3
2
.84-2.95 (m, 4H), 2.36-2.40 (m, 2H), 1.37 (d, J = 7.2 Hz, 6H). C NMR (101 MHz,
CDCl
3
) δ 175.9, 166.8, 155.6, 150.2, 140.9, 131.5, 129.3, 120.2, 115.9, 108.9, 68.1,
+
4
8.4, 47.5, 30.5, 28.8, 27.7, 22.0. HRMS (ESI): calcd for C22
30.1623, found 430.1627.
H
27
N
3
2
O S
2
(M+H) =
4
2
5

ACCEPTED MANUSCRIPT
isopropyl
-(piperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxyla
te (23)
Yield: 77%. H NMR (400 MHz, CDCl ) δ 12.07 (s, 1H), 5.17-5.21 (m, 1H), 4.29 (s,
2
1
3
1
1
1
H), 3.98-4.02 (m, 2H), 2.82-3.02 (m, 7H), 2.59-2.68 (m, 2H), 2.17-2.40 (m, 3H),
13
.36 (d, J = 5.6 Hz, 6H). C NMR (101 MHz, CDCl ) δ 175.6, 166.5, 155.7, 140.7,
3
31.1, 108.6, 68.0, 49.0, 45.6, 30.5, 28.8, 27.6, 22.0. HRMS (ESI): calcd for
+
C H N O S (M+H) = 354.1310, found 354.1315.
16
23
3
2 2
isopropyl
-(4-acetylpiperazine-1-carbothioamido)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-c
2
arboxylate (24)
1
Yield: 83%. H NMR (400 MHz, CDCl ) δ 12.14 (s, 1H), 5.14-5.20 (m, 1H),
3
3
3
1
.99-4.16 (m, 4H), 3.66-3.83 (m, 4H), 2.81-2.92 (m, 4H), 2.34-2.38 (m, 2H), 2.14 (s,
1
3
H), 1.35 (d, J = 6.0 Hz, 6H). C NMR (101 MHz, CDCl ) δ 175.6, 169.2, 166.5,
3
55.1, 140.8, 1313, 108.8, 68.1, 46.8, 46.8, 44.9, 40.1, 30.4, 28.7, 27.6, 21.9, 21.3.
+
HRMS (ESI): calcd for C H N O S (M+H) = 396.1416, found 396.1419.
1
8
25
3
3 2
ethyl
-(4-methylpiperazine-1-carbothioamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car
boxylate (25)
2
2
6

ACCEPTED MANUSCRIPT
1
Yield: 89%. H NMR (400 MHz, CDCl ) δ 12.44 (s, 1H), 4.34 (q, 2H), 4.04-4.05 (m,
3
4
H), 2.77-2.78 (m, 2H), 2.55-2.65 (m, 6H), 2.37 (s, 3H), 1.79 (s, 4H), 1.40 (t, J = 7.2
1
1
3
Hz, J = 14.4 Hz, 3H). C NMR (101 MHz, CDCl ) δ 176.1, 167.7, 152.2, 130.2,
2
3
1
25.7, 111.4, 60.5, 54.3, 47.7, 45.6, 26.3, 24.2, 22.9, 22.9, 14.2. HRMS (ESI): calcd
+
for C H N O S (M+H) = 368.1466, found 368.1469.
1
7
25
3
2 2
propyl
-(4-methylpiperazine-1-carbothioamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car
boxylate (26)
2
1
Yield: 84%. H NMR (400 MHz, CDCl ) δ 12.46 (s, 1H), 4.24 (t, J = 6.4 Hz, J =
3
1
2
1
1
2.8 Hz, 2H), 4.04-4.06 (m, 4H), 2.64-2.78 (m, 4H), 2.55-2.58 (m, 4H), 2.37 (s, 3H),
13
.78-1.80 (m, 6H), 1.04 (t, J = 7.2 Hz, J = 14.4 Hz, 3H). C NMR (101 MHz,
1
2
CDCl ) δ 175.9, 167.6, 152.1, 130.0, 125.3, 111.2, 66.2, 54.3, 47.6, 45.6, 26.2, 24.1,
3
+
2
2.9, 22.8, 21.9, 10.7. HRMS (ESI): calcd for C H N O S (M+H) = 382.1623,
18
27
3
2 2
found 382.1627.
isopropyl
2
-(4-methylpiperazine-1-carbothioamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car
boxylate (27)
1
Yield: 83%. H NMR (400 MHz, CDCl ) δ 12.47 (s, 1H), 5.20-5.23 (m, 1H),
3
4
.02-4.05 (m, 4H), 2.64-2.77 (m, 4H), 2.53-2.56 (m, 4H), 2.36 (s, 3H), 1.77-1.80 (m,
1
3
4
H), 1.36 (d, J = 6.0 Hz, 6H). C NMR (101 MHz, CDCl ) δ 176.2, 167.2, 152.0,
3
2
7

ACCEPTED MANUSCRIPT
1
30.3, 125.7, 111.8, 68.1, 54.4, 47.8, 45.7, 26.4, 24.2, 22.9, 22.0. HRMS (ESI): calcd
+
for C H N O S (M+H) = 382.1623, found 382.1628.
1
8
27
3
2 2
isopropyl
-(4-methylpiperazine-1-carbothioamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thioph
2
ene-3-carboxylate (28)
1
Yield: 80%. H NMR (400 MHz, CDCl ) δ 12.39 (s, 1H), 5.22-5.25 (m, 1H),
3
4
.00-4.03 (m, 4H), 3.02-3.05 (m, 2H), 2.71-2.74 (m, 2H), 2.52-2.55 (m, 4H), 2.35 (s,
13
3
H), 1.83-1.84 (m, 2H), 1.62-1.68 (m, 4H), 1.37 (d, J = 6.4 Hz, 6H). C NMR (101
MHz, CDCl ) δ 176.1, 167.3, 149.9, 135.8, 129.8, 113.1, 68.4, 54.3, 47.7, 45.6, 32.9,
3
+
2
3
8.4, 28.2, 27.7, 26.7, 21.9. HRMS (ESI): calcd for C H N O S (M+H) =
1
9
29
3
2 2
96.1779, found 396.1783.
4
4
.2. Biology assays
.2.1 QUANTI-Blue SEAPAssay.
4
HEK-Blue hTLR2 (or TLR3, TLR4) cells were cultured in 96-well plates (4 × 10
cells per well) of 200 µL DMEM (supplemented with 10% FBS, 10 ×
penicillin/streptomycin, and 10 × L-glutamine) at 37 °C for 24 hours before drug
treatment on the first day. In the next 24 hours of treatment, medium was removed
from the 96-well plate and substituted with 200 µL of DMEM containing indicated
concentrations of compounds, with/without different antibodies (0 to 10 µg/mL),
including anti-hTLR1–IgG, anti-hTLR2–IgA, or anti-hTLR6–IgA (InvivoGen) in the
2
8