Benzothiophene-2-carboxamide derivatives as SENPs inhibitors with selectivity within SENPs family

Article abstract of DOI:10.1016/j.ejmech.2020.112553

The SUMO (small ubiquitin-related modifier)-specific proteases (SENPs) are responsible for the cleavage of SUMO from its target proteins, thus play important roles in the dynamic SUMOylation and deSUMOylation processes. SENPs are related to a variety of human diseases including cancer and represent a new class of potential therapeutic targets with mechanism of action that is likely to be different from that of current clinically used drugs. However, potent inhibitors that are selective within the SENPs family members still remain a challenge due to their high homology. In order to demonstrate the feasibility of developing selective inhibitors within the SENPs family, we chose SENP1/2/5 as representatives, aiming to identify inhibitors with selectivity among the members. Starting from a hit compound ZCL951 from virtual screening, a series of benzothiophene-2-carboxamide inhibitors were designed based on the protein structures of SENP1, 2, and 5. First, an unoccupied hydrophobic pocket was first identified which led to IC₅₀ as low as 0.56 μM. Furthermore, the ethylacetate 77 gave both submicromolar inhibitory activity and 33-fold selectivity for SENP2 versus SENP5. They are the most potent and selective nonpeptidic inhibitor reported so far for the SENPs family, as far as we are aware. Their structure-activity relationship was also discussed.

Full text of DOI:10.1016/j.ejmech.2020.112553

Journal Pre-proof
Benzothiophene-2-Carboxamide Derivatives as SENPs Inhibitors with Selectivity
within SENPs Family
Zhongli Wang, Yunqi Liu, Jianchen Zhang, Shafi Ullah, Ning Kang, Yaxue Zhao,
Huchen Zhou
PII:
S0223-5234(20)30525-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112553
EJMECH 112553
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 March 2020
Revised Date: 2 June 2020
Accepted Date: 7 June 2020
Please cite this article as: Z. Wang, Y. Liu, J. Zhang, S. Ullah, N. Kang, Y. Zhao, H. Zhou,
Benzothiophene-2-Carboxamide Derivatives as SENPs Inhibitors with Selectivity within SENPs Family,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112553.
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Graphical Abstract
O
S
H
N
S
N
H
OBn
O
O
O
HN
Compound 77
SENP1 IC50 = 1.3 µM
SENP2 IC50 = 0.69 µM
SENP5 IC50 = 22.7 µM

Benzothiophene-2-Carboxamide Derivatives as SENPs
Inhibitors with Selectivity within SENPs Family
#
#
Zhongli Wang, Yunqi Liu, Jianchen Zhang, Shafi Ullah, Ning Kang,
∗
Yaxue Zhao, Huchen Zhou
State Key Laboratory of Microbial Metabolism, School of Pharmacy, Shanghai Jiao
Tong University, Shanghai 200240, People’s Republic of China
*
Corresponding author. E-mail address: hczhou@sjtu.edu.cn; Tel.: +86 21 34206721.
These authors contributed equally to this work.
#
Abstract
The SUMO (small ubiquitin-related modifier)-specific proteases (SENPs)
are responsible for the cleavage of SUMO from its target proteins, thus
play important roles in the dynamic SUMOylation and deSUMOylation
processes. SENPs are related to a variety of human diseases including
cancer and represent a new class of potential therapeutic targets with
mechanism of action that is likely to be different from that of current
clinically used drugs. However, potent inhibitors that are selective within
the SENPs family members still remain a challenge due to their high
homology. In order to demonstrate the feasibility of developing selective
inhibitors within the SENPs family, we chose SENP1/2/5 as
representatives, aiming to identify inhibitors with selectivity among the
members. Starting from a hit compound ZCL951 from virtual screening,
a series of benzothiophene-2-carboxamide inhibitors were designed based
on the protein structures of SENP1, 2, and 5. First, an unoccupied

hydrophobic pocket was first identified which led to IC as low as 0.56
5
0
µM. Furthermore, the ethylacetate 77 gave both submicromolar inhibitory
activity and 33-fold selectivity for SENP2 versus SENP5. They are the
most potent and selective nonpeptidic inhibitor reported so far for the
SENPs family, as far as we are aware. Their structure-activity relationship
was also discussed.
Keywords:
SUMOylation;
SENPs;
cancer;
selectivity;
benzothiophene-2-carboxamide; structure-activity relationship.
Abbreviations: SUMO, small ubiquitin-related modifier; SENPs,
SUMO-specific proteases.
Highlights:
ò Starting from ZCL951, a series of benzothiophene-2-carboxamide
SENPs inhibitors were designed
ò An unoccupied hydrophobic pocket was first identified which led to
IC as low as 0.56 µM
5
0
ò Compound 77 gave a 33-fold selectivity of SENP2 vs. SENP5 and
these compunds represent the most potent and selective nonpeptidic
inhibitors reported so far for the SENPs family
1
. Introduction
SUMOylation, a post-translational modification, involves the covalent
attachment of SUMO, an Ubl (ubiquitin-like protein), to the ε-amino

group of lysine residues in target proteins through an enzymatic cascade
that requires the sequential action of an E1 activating enzyme, an E2
conjugating enzyme, and E3 ligases [1,2]. SUMOylation has been
discovered to play critical roles in a vast array of cellular processes such
as maintenance of protein stability, transcriptional regulation, and cell
proliferation [3]. SUMO modification is a dynamic and reversible process
with SUMOylation/deSUMOylation cycles. The deconjugation of SUMO
from its target proteins is catalyzed by a family of cysteine proteases
which were first characterized in yeast as Ulp 1/2 [4]. Their human
homologs were later characterized to be SUMO-specific proteases
(
SENPs) including SENP1, SENP2, SENP3, SENP5, SENP6, and SENP7
5,6]. SENPs play critical roles in maintaining normal cellular physiology
[
by preserving the balance between SUMOylated and deSUMOylated
proteins, thus have been found to be involved in various diseases
including prostate cancer [7,8], thyroid cancer [9], colon cancer [10], lung
cancer [11], oral squamous cell carcinomas [12] and osteosarcoma [13],
and cardiovascular disease [14,15]. As an important class of therapeutic
targets, cysteine proteases have been studied extensively in the past and
the discovery of their inhibitors as useful therapeutic agents has been
reviewed elsewhere [23-25].
The ubiquitin/Ubl pathways have recently evoked great interest in drug
discovery, and are believed to be leading us toward the emergence of a

new wave of novel targets. With their pivotal role in the development of
various diseases, SENPs represent a promising class of potential
therapeutic targets with mechanism of action that is likely to be different
from that of current clinically used drugs. The first SENPs inhibitors, i.e.
our group’s benzodiazepine-based SENP1 inhibitors (A) (SENP1 IC =
5
0
9
1
.2 µM) [16] and Bogyo’s nonnatural peptidyl inhibitors (B) (SENP1
3.8; SENP2 7.0 µM) [17], were reported in 2011 (Figure 1). Since then,
benzoates (C) (SENP1 1.08 µM) [18], sulfonyl benzenes (D) (SENP1
.88; SENP2 1.1 µM) [19], and oxadiazole (E) (SENP1 9.7; SENP2 5.9
1
µM) [20] inhibitors have been reported during the past five years.
However, an in-depth study of the structure-activity relationship of
SENPs inhibitors had remained lacking. At the same time, the six
members of human SENPs family share high sequence homology, but
differ in their substrate specificity and subcellular localization. Although
inhibitors that are selective within the family are urgently needed for the
development of new therapeutic agents and as probes to help elucidate
the exact function of individual SENP, medicinal chemistry work
dedicated to this goal had remained lacking.
In a previous study, we have identified a number of new scaffolds as
SENP1 inhibitors [21] including compound ZCL951. In this report, using
ZCL951 as a starting point we identified an unoccupied hydrophobic
pocket adjacent to SENP1’s catalytic site, which led to the discovery of a

series of SENP1 inhibitors with IC as low as 0.56 µM. Furthermore,
50
three members of SENPs, i.e. SENP1, SENP2, and SENP5, were studied
to demonstrate the feasibility of identifying SENPs inhibitors that are
selective within the family. A series of benzothiophene-2-carboxamides
were designed and synthesized by exploiting the subtle amino acid
differences among the SENPs members, and selective inhibitors were
achieved. The structure-activity relationship will also be discussed.
2
. Chemistry
As shown in Scheme 1, the common intermediate 3 was obtained
from 3-aminomethylaniline (1) via Boc protection, coupling with benzoic
acid, and deprotection, sequentially. At the same time, the thiosalicylic
acid 4 was converted to methyl ester 5 which underwent subsequent
S-alkylation and Dieckmann condensation to afford benzothiophene 6.
Alkylation of the 3-hydroxyl with R I or R Br in the presence of K CO
1
1
2
3
gave compounds 7-13 which were hydrolyzed to give carboxylic acids
4-20. Finally, compounds 21-27 were obtained by coupling with
1
intermediate 3 in the presence of EDCI and DMAP.
The synthetic route of compounds 29-60 was shown in Scheme 2.
First, aniline 28 was prepared by coupling carboxylic acid 16 with
3
-nitro-benzylamine followed by reduction of the nitro group using
iron/acetic acid. Coupling of aniline 28 with various acyl chlorides in the

presence of Et N or with carboxylic acids in the presence of
3
EDCI/DMAP gave compounds 29-60.
Synthesis of compounds 65-77 from Table 4 were outlined in Scheme
3
. The commercially available carboxylic acids 61-64 were coupled with
N-(3-(aminomethyl)phenyl)thiophene-2-carboxamide in the presence of
EDCI/DMAP to give derivatives 65-68. Nitrobenzothiophene 68 was
hydrogenated by iron in acetic acid to give aminobenzothiophene 69
which was subsequently coupled with acyl chlorides in the presence of
Et N or with iodides/bromides in the presence of K CO to give
3
2
3
derivatives 70-77.
3
. Results and discussion
The six members of the human SENPs family, SENP1-3 and
SENP5-7, all share a conserved C-terminal catalytic domain containing a
His-Asp-Cys catalytic triad (Figure 2a) [3,4]. Their N-terminal regions
varies in sequence and are essential for the different cellular localization.
They are divided into three subfamilies, SENP1/2, SENP3/5, and
SENP6/7. SENP1 is located in the nucleus and was found to shuttle
between nucleus and cytoplasm. SENP2 was found in nuclear pore
complex. Although SENP1 and SENP2 are closely related and both
deconjugate SUMO1-3, they possess different functions and are unable to
compensate for each other. SENP3 and SENP5 are located in nucleolus

and deconjugate SUMO2-3. SENP6 and SENP7 have an extra loop
inserted in the catalytic domain, thus are distinct from other SENPs. They
are located in nucleoplasm and catalyze the deconjugation of SUMO2-3.
Although SENPs have been proven to play important roles in various
diseases including a number of cancers, the exact understanding of the
cellular functions of each member and their corroborative mechanism
remains lacking. Thus it is urgent to develop inhibitors that are selective
within the family in order to facilitate this understanding and eventually
provide novel therapeutic targets and drug candidates.
In order to demonstrate the feasibility of developing selective
inhibitors within the SENPs family, we chose SENP1/2/5 as
representatives, aiming to identify inhibitors that are selective among
these three members. SENP1 and SENP2 are the two most closely related
sharing 60% identical sequence in their catalytic domain. Among their
eighteen substrate-contacting residues, only two residues are different
between SENP1 and SENP2 (SENP1/SENP2: K455/G400 and
G531/K476) (Figure 2b). SENP5 is chosen as a relatively distant member
which shares 39% sequence identity with SENP1, and five out of the
eighteen substrate-contacting residues are different between SENP1 and
SENP5 (SENP1/SENP5: K455/D572, T495/S608, G531/E644,
M552/Q665, and G600/D710). Thus, the identification of SENPs

inhibitors that are selective within the family represents a challenging
task due to the high resemblance of their catalytic sites.
The catalytic core of SENP1, 2, 5 and RanGAP-SUMO were
expressed in E.coli strain BL21 (DE3) and purified. The inhibitory
activity of compounds were measured using the quantitative assay based
on SDS-PAGE-Coomassie system and RanGAP-SUMO2 substrate as
described in our previous work [16,21,22].
We first studied the predicted binding pose of the hit compound
ZCL951 at SENP1’s catalytic site using the crystal structure of SENP1
from PDB database (PDB code: 2IY1). As shown in Figure 3a, ZCL951
interacted with the same binding site of the endogenous peptide
EQTGGH by forming extensive hydrophobic interactions and three
hydrogen bonds with Gln597, Val531, and His529. However, there was
an adjacent site consisting of Phe496 and His529 which was occupied by
the E88 and Q89 of the endogenous peptide but remained unoccupied by
ZCL951 (pink area in Figure 3a). Molecular modeling showed that the
C(3) position of the benzothiophene ring is pointed toward the
unoccupied pocket. This observation led to the design of 3-substituted
benzothiophene derivatives in order to exploit this unoccupied site. As
shown in Table 1, the SENP1 inhibitory activity of the methoxy
derivative 21 showed a 2-fold increase as measured by IC value. An
5
0
amide group was introduced in compound 22 to mimic the endogenous

Q89, the inhibitory IC of SENP1 was further improved to 6.9 µM. The
5
0
benzyl compound 23 gave a significantly reduced IC of 2.4 µM which
5
0
is an 11-fold improvement as compared to the unsubstituted ZCL951. It
also showed good inhibitory activity for SENP2 and SENP5. Nitration or
methylation of the benzyl group did not further improve the inhibitory
activity. Docking of compound 23 at SENP1's catalytic site predicted that
new π–π interactions were formed with Phe496 and His529 by the benzyl
group that inserted into the hydrophobic pocket consisting of Thr495,
Phe496, and His529 (Figure 3b). These compounds also showed
comparable inhibitory activity against SENP2 and SENP5 with poor
selectivity.
In order to develop selective inhibitors among the three SENPs
members, we studied the structural differences of SENP1/2/5 around the
binding site of compound 23 as shown in Figure 4a. The crystal structure
of SENP2 was withdrawn from PDB database (PDB code: 2IO0). The
homology model of SENP5 was built using Glide based on the known
SENP1 structure. The terminal phenyl group of compound 23 formed
hydrophobic interaction with the M552/M497 residues from SENP1/2,
and dipole-dipole interaction with Q665 from SENP5. Above the phenyl
group, the G600/G545/D710 residues corresponding to SENP1/2/5
provide an opportunity to develop inhibitors that are selective towards
SENP1/2 versus SENP5. Thus we designed and synthesized a series of

phenyl derivatives as shown in Table 2. First, various substituents at the
para-position were explored. The halogenated derivatives 29-31 and the
methoxy and methyl derivatives 34-35 did not give any significant
improvement of selectivity for the three SENPs members. However, the
electron-withdrawing nitro and trifluoromethyl compounds 32 and 36
gave 6-fold SENP1 vs. SENP5 selectivity. Furthermore, the amino
compound 33 gave a significant >10 fold selectivity for SENP1/2 vs.
SENP5 although its inhibitory potency was moderately diminished.
Among the meta-substituted phenyl compounds 37-42, the bromo, amino,
and methoxy compounds 39, 41, and 42 showed around 6-fold selectivity
for SENP1 vs. SENP5. The ortho-substituted phenyl derivatives 43-49
did not give any significant selectivity. The difluoro compound 47
showed a 5-fold selectivity for SENP1 vs. SENP5. Due to the high
similarity between SENP1 and SENP2, the selectivity between these two
members was not observed in these modified phenyl derivatives in Table
2
.
We next explored the effect of replacing the terminal phenyl group
with various aromatic rings or aliphatic groups. As shown in Table 3, the
five-membered heterocyclic pyrrolyl and furanyl derivatives 50 and 51
showed decreased activity. However, the thiophenyl compound 52 not
only gave improved SENP1/2 inhibitory activity (IC50 SENP1 = 1.3 µM;
IC50 SENP2 = 2.5 µM), but also gave good selectivity for SENP1/2 versus

SENP5 (IC50 SENP5 = 16.9 µM) with as much as 13-fold selectivity.
Methylthiophenyl (53) and pyridinyl (54) compounds showed decreased
activity and selectivity. The bulky naphthyl (55) and benzothiophenyl (56)
compounds also gave poor selectivity. The aliphatic derivatives 57-60
showed significantly diminished activity and poor selectivity, which
corroborated with the observation in the predicted binding model that the
phenyl group in compound 23 was needed to form π–π interaction with
His533 (Figure 3b).
Thus, the thiophenyl compound 52 with the best inhibitory activity
and selectivity for SENP1/2 versus SENP5 so far was used as the starting
structure for further structure-activity relationship exploration. In order to
exploit the structural difference of SENP1/2/5 near the benzothiophenyl
group, modification on the benzothiophenyl group was carried out as
shown in Table 4. Replacement of the C(6) of the benzothiophenyl
group with a nitrogen atom in compound 65 led to significant decrease of
inhibitory activity. Introduction of chloro or methoxy group in
compounds 66 and 67 also gave comparable or decreased activity.
However, modification at the C(5) position gave promising improvement
as shown by compunds 68-77. Nitro and amino compounds 68 and 69
gave comparable activity with moderate selectivity. The amino compound
6
9 was further alkylated to give compounds 70-77. Although the
N-methyl compound 70 gave SENP1/2 inhibitory activity comparable to

compound 52, the dimethyl compound 71 showed submicromolar
inhibitory activity for the first time ((IC50 SENP1 = 0.79 µM; IC50 SENP2
.56 µM) and represented the most potent inhibitor in this study. The
=
0
N-ethyl compound 72 also gave good inhibitory activity. However the
diethyl compound 73 showed significantly reduced activity, which is
likely due to steric hindrance. Compound 70 also showed 5-fold
selectivity of SENP1 versus SENP5. Further exploration of amide
derivatives 74-77 gave retained activity and much improved selectivity.
Although the acetamide 74 showed significantly reduced activity, its
α-chloro derivative 75 gave activity comparable to compound 52. Thus,
bulkier acyl groups were introduced to give compounds 76 and 77. The
ethylacetate 77 gave both submicromolar inhibitory activity and an
excellent 33-fold selectivity for SENP1/2 versus SENP5 (IC50 SENP1 = 1.3
µM; IC50 SENP2 = 0.69 µM; IC50 SENP5 = 22.7 µM). The docking model
showed the formation of a hydrogen bond between K455-SENP1 and the
carbonyl of compound 77, while SENP5 has a D572 at this position
instead. They are the most potent and selective nonpeptidic inhibitor
reported so far for the SENPs family, as far as we are aware.
Examination of the predicted binding pose of compound 77 in
SENP1/2/5 revealed a few interesting observations. As shown in Figure
4
b, the lysine (K455) residue from SENP1 formed a new hydrogen bond
with the carbonyl group from the α-aminoethylacetate chain on the

benzothiophene, while the SENP2 protein has a much smaller glycine
G400) residue at the same position which allows for more space and
(
flexibility for the α-aminoethylacetate chain to take a favorable
conformation in order to form two new hydrogen bonds between the
carbonyl/amino groups and Asn412/Asp413 residues respectively. Thus,
compound 77 has a moderately higher inhibitory activity for SENP2 over
SENP1. However, in the case of SENP5, the aspartate (D572) residue at
this position did not have the potential to form hydrogen bond with the
α-aminoethylacetate chain, at the same time, the conformation of
α-aminoethylacetate chain was significantly more restricted than that in
SENP2 due to the steric hindrance of D572 as compared to G400/SENP2.
Thus, in SENP5 the α-aminoethylacetate chain was not able to form any
hydrogen bond, which resulted in its poor inhibition as compared to
SENP1/2. In summary, the steric and polarity variation among
SENP1/2/5 at this corner provided a useful strategy for the future
development of inhibitors with further improved activity and selectivity.
Unfortunately, our extensive efforts to obtain the cocrystal structure
of compound 77 and SENP1 have been unsuccessful so far. While
continuing the cocrystal efforts, we undertook a site-directed mutagenesis
study to survey the amino acids residues that were predicted to have
interactions with compound 77. The five amino acids, K455, Q597, H529,
W465, and F496, have either hydrogen bond or hydrophobic interactions

with compound 77 in the docked structure (Figure 5). Thus, each of them
was mutated to alanine to give five mutant SENP1 proteins. The
inhibitory potency of compound 77 on all five mutants and the wild type
SENP1 were compared to see if the mutation of the interacting residue
diminished the potency of the ligand. As shown in Table 5, mutation of
each of the five residues diminished the potency of compound 77, while
W465 showed the most significant difference. This observation indirectly
demonstrated that compound 77 interacts with this region of SENP1.
In summary, the family of SENPs represents a promising class of
potential therapeutic targets with mechanism of action that is likely to be
different from that of current clinically used drugs. However, an in-depth
study of the structure-activity relationship of SENPs inhibitors had
remained lacking. At the same time, inhibitors that are selective within
the SENPs family are urgently needed for the development of new
therapeutic agents and as probes to help elucidate the functions of
individual SENPs members. Based on our previously reported SENP1
inhibitor ZCL951, we hereby identified an unoccupied hydrophobic
pocket which led to IC as low as 0.56 µM. Furthermore, we for the first
5
0
time demonstrated the feasibility of exploiting the subtle amino acid
difference to design inhibitors that are selective within the SENPs family
by achieving 33-fold selectivity. This selectivity may better target
8,11
prostate cancer and lung cancer. This work will facilitate the eventual

discovery of novel therapeutics targeting SENPs family.
4
4
. Material and methods
.1. Chemistry
All solvents and reagents were purchased from commercial sources
and used without further purification unless otherwise noted. NMR
spectra were recorded on Bruker Avance III 400 MHz or 600 MHz.
Chemical shifts are expressed in parts per million (ppm) relative to
residual solvent as an internal reference (DMSO-d : 2.50). High
6
resolution mass spectra were obtained on Agilent 6530 Accurate Mass
Q-TOF LC-MS. Column chromatography was performed using Huanghai
silica gel (45-75 µm). HPLC analysis was performed on an Agilent1200
with a flow rate of 1 mL/min and a gradient of 10% v/v MeOH in H O (t
2
=
0.0 min) to 100% MeOH (t = 15.0 min) stopping at 20 or 25 min using
a DAD detector. An Agilent Eclipse XDB-C18 column (4.6 mm × 150
mm, 5 µm) was used. Purity was based on the integrated UV
chromatogram (254 nm). The purity of all biological assay compounds
was > 95.0 %.
N-(3-(Aminomethyl)phenyl)benzamide (3). To a mixture of benzoic
acid (1.0 g, 8.19 mmol) and tert-butyl-3-aminobenzyl-carbamate (2) (2.0
g, 9.01 mmol) in 20 mL DCM at 0 °C were added EDCI (3.14 g, 16.38
mmol) and DMAP (100 mg, 0.82 mmol), and the mixture was warmed to

r.t. and stirred overnight. The mixture was washed with 1 M NaOH (20
mL), 1 M HCl (20 mL), and saturated NaHCO (10 mL), dried over
3
Na SO , and concentrated under reduced pressure. The residue was
2
4
dissolved in DCM (5 mL), and TFA (5 mL) was added. After stirring for
h at r.t., solvent was removed in vacuo and the residue was purified by
2
column chromatography over silica gel followed by recrystallization
1
(
Et O) to obtain the title compound as a white solid (1.2 g, 64.7%). H
2
NMR (600 MHz, DMSO-d ): δ 10.42 (s, 1H), 8.34 (s, 3H), 8.01-7.96 (m,
6
3
H), 7.70 (d, J = 7.8 Hz, 1H), 7.61 (m, 1H), 7.54 (t, J = 7.8 Hz, 2H), 7.42
1
3
(
t, J = 7.8 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 4.05 (s, 2H). C NMR (150
MHz, DMSO-d ): δ 166.1, 139.9, 135.2, 134.9, 132.1, 129.4, 128.8,
6
1
28.1, 124.4, 121.3, 121.0, 42.9.
-Methoxy-1-benzothiophene-2-carboxylic acid (14). To a solution of
methyl iodide (71 µL, 1.15 mmol) and methyl
-hydroxybenzothiophene-2-carboxylate (6) (200 mg, 0.96 mmol) in
3
3
anhydrous DMF (5 mL) potassium carbonate (586 mg, 4.2 mmol) was
added and the mixture was stirred at r.t. overnight. The mixture was
filtered and DMF was evaporated under reduced pressure, the residue was
purified by column chromatography on silica gel to give compound 7
(
180 mg, 84.3%) which was dissolved in THF (3 mL) and H O (1 mL).
2
LiOH·H O (102 mg, 2.43 mmol) was added and stirred at r.t. overnight.
2
THF was evaporated under reduced pressure to afford a white residue

which was taken up in water and acidified with conc. HCl to give a white
precipitation which was collected by filtration to afford compound 14 as a
1
white solid (110 mg, 65.2%). H NMR (600 MHz, DMSO-d ): δ 13.35 (s,
6
1
H), 7.96 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 8.4
1
3
Hz, 1H), 7.47 (t, J = 8.4 Hz, 1H), 4.08 (s, 3H). C NMR (150 MHz,
DMSO-d ): δ 163.1, 155.9, 137.4, 133.9, 128.5, 125.3, 123.8, 122.9,
6
+
1
2
3
16.8, 62.8. HRMS-ESI: [M+H] C H O S calcd 209.0272, found
1
0
9
3
09.0269.
-(2-Amino-2-oxoethoxy)-1-benzothiophene-2-carboxylic acid (15).
To a solution of compound 6 (200 mg, 0.96 mmol) in anhydrous DMF
5 mL), potassium carbonate (398 mg, 2.9 mmol) was added and the
mixture was stirred at 60 °C for 30 min. After 2-bromoacetamide (159 mg,
.15 mmol) was added and stirred at 60 °C overnight. After filtered and
(
1
evaporated under reduced pressure, the residue was purified by column
chromatography on silica gel to give compound 8 (170 mg, 66.7%). To a
solution of compound 8 (170 mg, 0.64 mmol) in THF (3 mL) and H O (1
2
mL) LiOH·H O (166 mg, 3.96 mmol) was added and stirred at r.t.
2
overnight. The mixture was evaporated under reduced pressure to afford a
white residue which was taken up in water and acidified with conc. HCl
to give a white precipitation which was collected by filtration to afford
1
compound 15 as a white solid (151 mg, 93.8%). H NMR (600 MHz,
DMSO-d ): δ 13.45 (s, 1H), 8.28 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.97 (d,
6

J = 8.4 Hz, 1H), 7.87 (s, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 8.4 Hz,
1
3
1
1
H), 5.13 (s, 2H). C NMR (150 MHz, DMSO-d ): δ 171.4, 162.8, 149.3,
6
37.2, 132.8, 127.4, 125.3, 124.1, 123.3, 123.2, 70.5. HRMS-ESI:
+
[
M+H] C H NO S calcd 252.0331, found 252.0329.
11
10
4
3
-Benzyloxy-1-benzothiophene-2-carboxylic acid (16). Compound 16
was prepared following a similar procedure to compound 15 as a white
1
solid (160 mg, 54.5%) in two steps. H NMR (600 MHz, DMSO-d ): δ
6
1
7
3.39 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H),
1
3
.53-7.52 (m, 3H), 7.44-7.35 (m, 4H), 5.35 (s, 2H). C NMR (150 MHz,
DMSO-d ): δ 163.2, 154.7, 137.4, 134.4, 128.9, 128.9, 128.7, 128.5,
6
+
1
25.3, 123.7, 123.1, 117.5, 76.8. HRMS-ESI: [M+Na] C H O SNa
1
6
12
3
calcd 307.0405, found 307.0403.
3
-(4-Nitrobenzyloxy)-1-benzothiophene-2-carboxylic
acid
(17).
Compound 17 was prepared following a similar procedure to compound
1
1
5 as a white solid (175 mg, 56.4%) in two steps. H NMR (400 MHz,
DMSO-d ): δ 13.49 (s, 1H), 8.29 (d, J = 8.8 Hz, 2H), 7.98 (d, J = 8.0 Hz,
6
1
1
H), 7.86 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.56 (t, J = 8.0 Hz,
1
3
H), 7.46 (t, J = 8.0 Hz, 1H), 5.51 (2H, s). C NMR (100 MHz,
DMSO-d ): δ 164.8, 147.3, 146.9, 146.7, 136.1, 135.9, 129.9, 129.1,
6
+
1
25.9, 124.1, 123.8, 123.0, 121.7, 73.9. HRMS-ESI: [M+H] C H NO S
1
6
12
5
calcd 330.0436, found 330.0441.
3
-(3-Nitrobenzyloxy)-1-benzothiophene-2-carboxylic
acid
(18).

Compound 18 was prepared following a similar procedure to compound
1
1
5 as a white solid (220 mg, 66.3%) in two steps. H NMR (600 MHz,
DMSO-d ): δ 13.49 (s, 1H), 8.46 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 8.02 (d,
6
J = 7.2 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.72 (t,
J = 7.8 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 5.48 (s,
1
3
2
1
H). C NMR (150 MHz, DMSO-d ): δ 163.1, 154.3, 148.2, 139.7, 137.4,
6
35.2, 134.0, 130.5, 128.6, 125.5, 123.8, 123.5, 123.3, 123.1, 118.0, 75.4.
+
HRMS-ESI: [M+H] C H NO S calcd 330.0436, found 330.0440.
1
6
12
5
3
-(2-Nitrobenzyloxy)-1-benzothiophene-2-carboxylic
acid
(19).
Compound 19 was prepared following a similar procedure to compound
1
1
5 as a white solid (182 mg, 57.1%) in two steps. H NMR (600 MHz,
DMSO-d ): δ 13.44 (s, 1H), 8.14 (dd, J = 8.4, 1.2 Hz, 1H), 8.05 (d, J =
6
7
.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.86 (td, J = 7.8, 1.2 Hz, 1H), 7.81
(d, J = 7.8 Hz, 1H), 7.66 (td, J = 8.4, 1.2 Hz, 1H), 7.56 (td, J = 7.8, 1.2
1
3
Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 5.72 (s, 2H). C NMR (150 MHz,
DMSO-d ): δ 163.1, 154.0, 147.8, 137.4, 134.6, 133.9, 133.0, 130.2,
6
1
29.8, 128.6, 125.4, 125.2, 123.8, 122.9, 117.3, 73.1. HRMS-ESI:
+
[
M+H] C H NO S calcd 330.0436, found 330.0439.
1
6
12
5
3
-(4-Methylbenzyloxy)-1-benzothiophene-2-carboxylic acid (20).
Compound 20 was prepared following a similar procedure to compound
1
1
5 as a white solid (173 mg, 53.9%) in two steps. H NMR (600 MHz,
DMSO-d ): δ 13.27 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.4 Hz,
6

1
2
H), 7.53 (t, J = 7.8 Hz, 1H), 7.42 (t, J = 8.4 Hz, 1H), 7.41 (d, J = 7.8 Hz,
13
H), 7.19 (d, J = 7.8 Hz, 2H), 5.31 (s, 2H), 2.31 (s, 3H). C NMR (150
MHz, DMSO-d ): δ 163.2, 154.7, 138.0, 137.4, 134.4, 134.4, 129.4,
6
+
1
29.0, 128.5, 125.3, 123.7, 123.1, 76.7, 21.3. HRMS-ESI: [M+Na]
C H O SNa calcd 321.0561, found 321.0565.
1
7
14
3
N-(3-Benzamidobenzyl)-3-methoxybenzothiophene-2-carboxamide
21). To a mixture of compound 3 (120 mg, 0.53 mmol) and compound
(
1
4 (100 mg, 0.48 mmol) in 5 mL DCM at 0 °C were added EDCI (230
mg, 1.2 mmol) and DMAP (29 mg, 0.24 mmol), and the mixture was
warmed to r.t. and stirred overnight. The mixture was quenched with
water and extracted with DCM. The organic layer was washed with 1 M
NaOH (10 mL), 1 M HCl (10 mL), and saturated NaHCO (10 mL), dried
3
over Na SO , and concentrated under reduced pressure. The residue was
2
4
purified by column chromatography over silica gel followed by
recrystallization (Et O) to obtain compound 21 (67 mg, 33.0%) as a white
2
1
solid, m.p. 203-205 °C. H NMR (400 MHz, DMSO-d ): δ 10.27 (s, 1H),
6
8
.58 (t, J = 6.0 Hz, 1H), 8.00-7.93 (m, 4H), 7.80 (s, 1H), 7.69 (d, J = 8.0
Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.54-7.46 (m,
H), 7.33 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 4.56 (d, J = 6.0
4
1
3
Hz, 2H), 4.13 (s, 3H). C NMR (100 MHz, DMSO-d ): δ 166.0, 161.4,
6
1
1
51.0, 140.5, 139.8, 137.2, 135.5, 133.0, 132.0, 129.0, 128.8, 128.1,
27.4, 125.3, 124.2, 123.0, 122.6, 119.5, 119.4, 62.6, 43.1. HRMS-ESI:

+
[
M+H] C H N O S calcd 417.1273, found 417.1275. HPLC: purity
2
4
21
2
3
9
9.3% (14.6 min).
N-(3-Benzamidobenzyl)-3-(2-amino-2-oxoethoxy)benzothiophene-2-c
arboxamide (22). Compound 22 was prepared following a similar
procedure to compound 21 as a white solid (22 mg, 37.8%), m.p. 246-248
1
°
C. H NMR (400 MHz, DMSO-d ): δ 10.26 (s, 1H), 8.91 (t, J = 6.0 Hz,
6
1
H), 8.52 (s, 1H), 8.00-7.92 (m, 4H), 7.81 (s, 1H), 7.74 (s, 1H), 7.69 (d, J
8.0 Hz, 1H), 7.60-7.44 (m, 5H), 7.32 (t, J = 8.0 Hz, 1H), 7.05 (d, J =
=
13
8
.0 Hz, 1H), 4.99 (s, 2H), 4.38 (d, J = 6.0 Hz, 2H). C NMR (100 MHz,
DMSO-d ): δ 168.4, 166.0, 162.7, 149.4, 139.7, 137.2, 135.4, 132.9,
6
1
32.4, 129.1, 128.8, 128.1, 127.4, 125.2, 124.2, 123.2, 123.0, 119.9,
+
1
19.6, 72.5, 42.7. HRMS-ESI: [M+H] C H N O S calcd 460.1331,
2
5
22
3
4
found 460.1331. HPLC: purity 99.6% (13.1 min).
N-(3-Benzamidobenzyl)-3-benzyloxybenzothiophene-2-carboxamide
(23). Compound 23 was prepared following a similar procedure to
1
compound 21 as a white solid (28 mg, 30.6%), m.p. 178-180 °C. H
NMR (400 MHz, DMSO-d ): δ 10.27 (s, 1H), 8.34 (t, J = 6.0 Hz, 1H),
6
7
.99 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.6 Hz, 3H), 7.78 (s, 1H), 7.72 (d, J
8.0 Hz, 1H), 7.59 (m, 1H), 7.54-7.44 (m, 4H), 7.39-7.30 (m, 6H), 7.03
=
1
3
(
d, J = 7.6 Hz, 1H), 5.35 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H). C NMR (100
MHz, DMSO-d ): δ 165.5, 160.8, 148.9, 139.4, 139.3, 136.5, 135.9,
6
1
34.9, 132.8, 131.5, 128.7, 128.6, 128.6, 128.4, 128.3, 127.6, 127.0,

+
1
24.9, 123.6, 122.8, 122.6, 119.3, 119.1, 76.2, 42.7. HRMS-ESI: [M+H]
C H N O S calcd 493.1586, found 493.1586. HPLC: purity 98.6% (16.0
3
0
25
2
3
min).
N-(3-Benzamidobenzyl)-3-(4-nitrobenzyloxy)benzothiophene-2-carbo
xamide (24). Compound 24 was prepared following a similar procedure
1
to compound 21 as a white solid (54 mg, 38.5%), m.p. 218-220 °C. H
NMR (400 MHz, DMSO-d ): δ 10.26 (s, 1H), 8.49 (t, J = 6.0 Hz, 1H),
6
8
.14 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.95-7.93 (m, 3H), 7.82
(s, 1H), 7.69-7.66 (m, 3H), 7.61-7.48 (m, 5H), 7.29 (t, J = 8.0 Hz, 1H),
13
7
.03 (d, J = 7.6 Hz, 1H), 5.51 (s, 2H), 4.50 (d, J = 6.0 Hz, 2H). C NMR
(
100 MHz, DMSO-d ): δ 165.9, 161.3, 149.1, 147.7, 144.2, 139.9, 139.8,
6
1
1
36.9, 135.4, 133.0, 132.0, 129.5, 129.0, 128.8, 128.1, 127.5, 125.5,
24.2, 123.9, 123.3, 123.1, 122.8, 119.7, 119.4, 75.0, 43.3. HRMS-ESI:
+
[
M+H] C H N O S calcd 538.1437, found 538.1435. HPLC: purity
3
0
24
3
5
9
8.1% (15.3 min).
N-(3-Benzamidobenzyl)-3-(3-nitrobenzyloxy)benzothiophene-2-carbo
xamide (25). Compound 25 was prepared following a similar procedure
1
to compound 21 as a white solid (137 mg, 25.4%), m.p. 160-162 °C. H
NMR (400 MHz, DMSO-d ): δ 10.22 (s, 1H), 8.48 (t, J = 6.0 Hz, 1H),
6
8
.34 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.92 (t, J
7.6 Hz, 3H), 7.84 (d, J = 7.6 Hz, 1H), 7.76 (s, 1H), 7.65 (d, J = 8.0 Hz,
H), 7.61-7.45 (m, 6H), 7.26 (t, J = 7.6 Hz, 1H), 6.69 (d, J = 7.6 Hz, 1H),
=
1

1
3
5
1
1
1
.51 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ
6
65.9, 161.3, 149.1, 148.1, 139.9, 139.8, 138.8, 136.9, 135.4, 135.3,
33.1, 131.9, 130.4, 128.9, 128.8, 128.1, 127.6, 125.5, 124.1, 123.7,
+
23.4, 123.1, 122.9, 119.5, 119.4, 75.0, 43.2. HRMS-ESI: [M+H]
C H N O S calcd 538.1437, found 538.1435. HPLC: purity 97.4% (15.4
3
0
24
3
5
min).
N-(3-Benzamidobenzyl)-3-(2-nitrobenzyloxy)benzothiophene-2-carbo
xamide (26). Compound 26 was prepared following a similar procedure
1
to compound 21 as a white solid (94 mg, 30.9%), m.p. 163-165 °C. H
NMR (400 MHz, DMSO-d ): δ 10.19 (s, 1H), 8.47 (t, J = 6.0 Hz, 1H),
6
8
2
1
.08 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.6 Hz,
H), 7.88 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.73 (t, J = 7.6 Hz,
H), 7.71 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.62-7.56 (m, 2H), 7.55-7.43
(
m, 4H), 7.24 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 5.66 (s, 2H),
1
3
4
1
1
1
5
.49 (d, J = 6.0 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ 165.9, 161.3,
6
49.1, 147.6, 139.8, 139.8, 136.9, 135.4, 134.6, 133.0, 132.2, 130.3,
29.9, 128.9, 128.8, 128.1, 127.6, 125.5, 125.3, 124.2, 123.1, 123.0,
+
22.6, 119.6, 119.4, 72.9, 43.2. HRMS-ESI: [M+H] C H N O S calcd
3
0
24
3
5
38.1437, found 538.1437. HPLC: purity 97.1% (15.6 min).
N-(3-Benzamidobenzyl)-3-(4-methylbenzyloxy)benzothiophene-2-car
boxamide (27). Compound 27 was prepared following a similar
procedure to compound 21 as a white solid (45 mg, 32.4%), m.p. 178-180

1
°
C. H NMR (400 MHz, DMSO-d ): δ 10.28 (s, 1H), 8.31 (t, J = 6.0 Hz,
6
1
H), 7.98 (t, J = 8.0 Hz, 2H), 7.96 (d, J = 7.8 Hz, 2H), 7.79 (s, 1H), 7.73
d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.56-7.48 (m, 4H), 7.32 (t, J
8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 7.03 (d,
(
=
13
J = 7.8 Hz, 1H), 5.31 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H), 2.25 (s, 3H). C
NMR (100 MHz, DMSO-d ): δ 166.0, 161.3, 149.5, 139.9, 138.5, 137.1,
6
1
1
35.4, 133.3, 133.2, 132.0, 129.5, 129.4, 129.1, 128.8, 128.1, 127.4,
25.3, 124.1, 123.4, 123.3, 123.1, 119.9, 119.5, 76.5, 43.2, 21.3.
+
HRMS-ESI: [M+H] C H N O S calcd 507.1742, found 507.1745.
3
1
27
2
3
HPLC: purity 99.0% (16.3 min).
N-(3-Aminobenzyl)-3-benzyloxybenzothiophene-2-carboxamide (28).
To a mixture of compound 16 (870 mg, 3.1 mmol) and
3
-nitrobenzylamine (466 mg, 3.1 mmol) in 20 mL DCM at 0 °C were
added EDCI (1.17 g, 6.12 mmol) and DMAP (37 mg, 0.31 mmol) and the
mixture was warmed to r.t. and stirred overnight. It was quenched with
water and extracted with DCM. The organic layer was washed with 1 M
NaOH (20 mL), 1 M HCl (20 mL), and saturated NaHCO (10 mL), dried
3
over Na SO , and concentrated under reduced pressure. The residue was
2
4
dissolved in AcOH (5 mL) and Fe (581 mg, 10.4 mmol) was added. After
stirring overnight, the mixture was filtered and AcOH was evaporated
under reduced pressure, and the residue was purified by column
chromatography on silica gel to give compound 28 (750 mg, 63.1%) as a

1
white solid. H NMR (400 MHz, DMSO-d ): δ 8.17 (t, J = 6.0 Hz, 1H),
6
7
7
.98 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.54-7.46 (m, 2H),
.39-7.36 (m, 5H), 6.97 (t, J = 8.0 Hz, 1H), 6.52 (s, 1H), 6.47 (d, J = 8.0
Hz, 1H), 6.42 (d, J = 7.6 Hz, 1H), 5.31 (s, 2H), 5.07 (s, 2H), 4.29 (d, J =
1
3
6
1
1
.0 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ 149.3, 139.8, 136.9,
6
36.3, 133.2, 129.4, 129.3, 129.1, 129.0, 127.4, 125.4, 124.1, 123.5,
22.9, 115.4, 113.4, 113.2, 76.7, 43.3.
N-(3-(4-Fluorobenzamido)benzyl)-3-benzyloxybenzothiophene-2-carb
oxamide (29). To the solution of compound 28 (60 mg, 0.15 mmol) and
4
-fluorobenzoyl chloride (27 mg, 0.19 mmol) in 5 mL DCM at 0 °C TEA
(64 µL, 0.46 mmol) was added, and the reaction was stirred at r.t. for 2 h.
After quenched with water and extracted with DCM, the organic layer
was washed with 1 M NaOH (10 mL), 1 M HCl (10 mL), and saturated
NaHCO (10 mL), dried over Na SO , and concentrated under reduced
3
2
4
pressure. The residue was purified by column chromatography on silica
gel to give compound 29 (53 mg, 67.2%) as a white solid; m.p. 170-172
1
°
C. H NMR (400 MHz, DMSO-d ): δ 10.29 (s, 1H), 8.36 (t, J = 6.0 Hz,
6
1
H), 8.05-7.97 (m, 3H), 7.94 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.70 (d, J
8.0 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.40-7.26
=
(
m, 8H), 7.04 (d, J = 7.6 Hz, 1H), 5.35 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H).
1
3
C NMR (100 MHz, DMSO-d ): δ 164.9, 161.3, 149.5, 139.9, 139.7,
6
1
37.0, 136.4, 133.2, 130.9, 130.8, 129.2, 129.1, 129.1, 128.9, 127.5,

1
25.4, 124.1, 123.4, 123.3, 122.9, 119.8, 119.6, 115.9, 115.7, 76.6, 43.7.
+
HRMS-ESI: [M+H] C H FN O S calcd 511.1492, found 511.1489.
3
0
24
2
3
HPLC: purity 99.5% (16.0 min).
N-(3-(4-Chlorobenzamido)benzyl)-3-benzyloxybenzothiophene-2-car
boxamide (30). Compound 30 was prepared following a similar
procedure to compound 29 as a white solid (26 mg, 41.3%), m.p. 165-167
1
°
C. H NMR (400 MHz, DMSO-d ): δ 10.32 (s, 1H), 8.34 (t, J = 6.0 Hz,
6
1
H), 7.98 (d, J = 7.6 Hz, 2H), 7.96 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.75
s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.50-7.46 (m,
H), 7.38-7.34 (m, 2H), 7.32-7.26 (m, 4H), 7.03 (d, J = 7.6 Hz, 1H), 5.33
(
2
1
3
(
s, 2H), 4.44 (d, J = 6.0 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ
6
1
1
1
64.9, 161.3, 149.4, 139.9, 139.6, 137.0, 136.8, 136.3, 134.0, 133.2,
30.1, 129.2, 129.2, 129.1, 128.9, 127.5, 125.4, 124.1, 123.5, 123.3,
+
23.0, 119.9, 119.6, 76.6, 43.2. HRMS-ESI: [M+H] C H ClN O S
3
0
24
2
3
calcd 527.1196, found 527.1198. HPLC: purity 97.1% (16.3 min).
N-(3-(4-Bromobenzamido)benzyl)-3-benzyloxybenzothiophene-2-car
boxamide (31). To a solution of compound 28 (40 mg, 0.13 mmol) and
4
-bromobenzoic acid (42 mg, 0.18 mmol) in DCM (5 mL) at 0 °C were
added EDCI (64 mg, 0.33 mmol) and DMAP (3 mg, 0.026 mmol), and
the mixture was warmed to r.t. and stirred overnight. After washed by 1
M HCl (5 mL) and saturated NaHCO solution (5 mL), dried with
3
Na SO , and concentrated in vacuo, the residue was purified by column
2
4

chromatography on silica gel to give compound 31 (48 mg, 81.6%) as a
1
white solid, m.p. 159-161 °C. H NMR (400 MHz, DMSO-d ): δ 10.34 (s,
6
1
1
7
2
1
1
1
H), 8.36 (t, J = 6.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.6 Hz,
H), 7.90 (d, J = 8.4 Hz, 2H), 7.76-7.73 (m, 3H), 7.70 (d, J = 8.0 Hz, 1H),
.54-7.46 (m, 2H), 7.39-7.30 (m, 6H), 7.04 (d, J = 7.6 Hz, 1H), 5.35 (s,
1
3
H), 4.46 (d, J = 6.0 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ 165.0,
6
61.3, 149.5, 139.9, 139.6, 137.0, 136.3, 134.4, 133.2, 131.8, 130.3,
29.2, 129.1, 129.0, 128.9, 127.5, 125.8, 125.4, 124.1, 123.5, 123.2,
+
22.9, 119.9, 119.6, 76.6, 43.2. HRMS-ESI: [M+H] C H BrN O S
3
0
24
2
3
calcd 571.0691, found 571.0692. HPLC: purity 98.1% (16.6 min).
N-(3-(4-Nitrobenzamido)benzyl)-3-benzyloxybenzothiophene-2-carbo
xamide (32). Compound 32 was prepared following a similar procedure
1
to compound 29 as a white solid (86 mg, 86.7%), m.p. 163-165 °C. H
NMR (400 MHz, DMSO-d ): δ 10.59 (s, 1H), 8.38-8.36 (m, 3H), 8.17 (d,
6
J = 8.4 Hz, 2H), 8.00 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.77 (s,
1
H), 7.72 (d, J = 7.6 Hz, 1H), 7.52-7.48 (m, 2H), 7.41-7.29 (m, 6H), 7.08
1
3
(
d, J = 7.6 Hz, 1H), 5.35 (s, 2H), 4.47 (d, J = 6.0 Hz, 2H). C NMR (100
MHz, DMSO-d ): δ 164.4, 161.3, 149.6, 149.5, 141.0, 140.1, 139.3,
6
1
1
37.0, 136.4, 133.2, 129.7, 129.2, 129.2, 129.1, 128.9, 127.5, 125.4,
24.1, 123.9, 123.8, 123.2, 123.0, 119.9, 119.6, 76.6, 43.1. HRMS-ESI:
+
[
M+H] C H N O S calcd 538.1437, found 538.1437. HPLC: purity
3
0
24
3
5
9
8.4% (16.0 min).

N-(3-(4-Aminobenzamido)benzyl)-3-benzyloxybenzothiophene-2-carb
oxamide (33). To the solution of compound 32 (60 mg, 0.11 mmol) in
AcOH (2 mL) was added Fe (31 mg, 0.56 mmol) and stirred overnight.
After filtered and evaporated under reduced pressure the residue was
purified by column chromatography on silica gel to give compound 33
1
(
21 mg, 37.1%) as a white solid, m.p. 184-186 °C. H NMR (400 MHz,
DMSO-d ): δ 9.79 (s, 1H), 8.32 (t, J = 6.0 Hz, 1H), 8.00 (d, J = 8.0 Hz,
6
1
H), 7.95 (d, J = 7.6 Hz, 1H), 7.76 (s, 1H), 7.71-7.69 (m, 3H), 7.52-7.48
m, 2H), 7.40-7.35 (m, 2H), 7.34-7.30 (m, 4H), 7.27 (t, J = 8.0 Hz, 1H),
.96 (d, J = 7.6 Hz, 1H), 6.58 (d, J = 8.4 Hz, 2H), 5.75 (s, 2H), 5.35 (s,
(
6
2
1
1
1
3
H), 4.44 (d, J = 6.0 Hz, 2H). C NMR (100 MHz, DMSO-d ): δ 165.3,
6
60.8, 152.1, 148.9, 139.9, 139.1, 136.5, 135.8, 132.7, 129.3, 128.7,
28.6, 128.5, 126.9, 124.9, 123.6, 122.8, 122.5, 122.0, 121.1, 119.1,
+
1
18.8, 112.5, 76.2, 42.8. HRMS-ESI: [M+H] C H N O S calcd
3
0
26
3
3
5
08.1695, found 508.1696. HPLC: purity 97.0% (14.9 min).
N-(3-(4-Methoxybenzamido)benzyl)-3-benzyloxybenzothiophene-2-ca
rboxamide (34). Compound 34 was prepared following a similar
procedure to compound 29 as a white solid (27 mg, 50.6%), m.p. 161-163
1
°
C. H NMR (400 MHz, DMSO-d ): δ 10.12 (s, 1H), 8.35 (t, J = 6.0 Hz,
6
1
H), 8.00 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.8 Hz, 3H), 7.77 (s, 1H), 7.71
d, J = 8.0 Hz, 1H), 7.52-7.48 (m, 2H), 7.41-7.35 (m, 2H), 7.34-7.27 (m,
H), 7.05 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 7.6 Hz, 1H), 5.35 (s, 2H), 4.46
(
4