Synthesis, biological evaluation and molecular docking analysis of 2-phenyl-benzofuran-3-carboxamide derivatives as potential inhibitors of Staphylococcus aureus Sortase A

Article abstract of DOI:10.1016/j.bmc.2016.12.030

In Gram-positive bacteria, Sortase A (Srt A) is a critical cysteine transpeptidase that is responsible for recognizing and assembling surface virulence proteins through the recognition of a LPXTG (leucine, proline, X, threonine, and glycine, where X is an

Full text of DOI:10.1016/j.bmc.2016.12.030

Accepted Manuscript
Synthesis, Biological Evaluation and Molecular Docking Analysis of 2-phenyl-
benzofuran-3-carboxamide derivatives as Potential Inhibitors of Staphylococ-
cus aureus Sortase A
Wan He, Yong Zhang, Jian Bao, Xinxian Deng, Jennifer Batara, Shawn Casey,
Qiuyuan Guo, Faqin Jiang, Lei Fu
PII:
S0968-0896(16)31454-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.030
BMC 13457
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 October 2016
15 December 2016
18 December 2016
Please cite this article as: He, W., Zhang, Y., Bao, J., Deng, X., Batara, J., Casey, S., Guo, Q., Jiang, F., Fu, L.,
Synthesis, Biological Evaluation and Molecular Docking Analysis of 2-phenyl-benzofuran-3-carboxamide
derivatives as Potential Inhibitors of Staphylococcus aureus Sortase A, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.12.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, Biological Evaluation and Molecular Docking Analysis of
2
-phenyl-benzofuran-3-carboxamide derivatives as Potential
Inhibitors of Staphylococcus aureus Sortase A
1
,a
1,b
a
a
a
a
Wan He , Yong Zhang , Jian Bao , Xinxian Deng , Jennifer Batara , Shawn Casey , Qiuyuan
a
*,a
*,a
Guo , Faqin Jiang , Lei Fu
School of Pharmacy, Shanghai Jiao Tong University, No. 800 Dongchuan Rd., Shanghai,
00240, PR China;
a
2
b
School of Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240,
PR China；
1
Co-first authors
*
Corresponding author. Dr. Faqin Jiang, Tel.:+86 21 34204787, fax:+86 21 34204787, E-mail
address :jfq2008@sjtu.edu.cn; Dr. Lei Fu, Tel.:+86 21 34204791, fax:+86 21 34204791,
E-mail address: leifu@sjtu.edu.cn.
Abstract:
In Gram-positive bacteria, Sortase A (Srt A) is a critical cysteine transpeptidase that is
responsible for recognizing and assembling surface virulence proteins through the
recognition of a LPXTG (leucine, proline, X, threonine, and glycine, where X is any
amino acid) signal. Mutants lacking genes for Srt A attenuate infections without
affecting microbial viability. Here a series of 2-phenyl- benzofuran-3-carboxamide
derivatives were synthesized and identified as potent Srt A inhibitors. Activity assays
revealed that multiple compounds exhibited excellent inhibitory activity against Srt A
compared with known Sortase A inhibitor pHMB (IC50=130 µM). Structural activity
relationships (SARs) demonstrated that the amide group at 3-position was essential
for inhibitory activity. Replacement of the hydroxyl group at the 2-phenyl position of
benzofuran with other substitutions such as a methoxyl, halogen or nitro group
reduced the enzyme inhibitory activity in most cases. The compound Ia-22 was found
to be the most potent inhibitor against the enzyme with an IC50 value of 30.8 µM.
Molecular docking studies showed Ia-22 shared similar binding pattern with substrate
LPXTG in the binding pocket of Srt A (PDB: 2KID) including i-butyl stretching,

L-shape pattern kinking, and H-bond interaction with Srt A functional site residues
Cys184, Trp194 and Arg197.
Keywords: Staphylococcus aureus, Sortase A, Inhibitor, Benzofuran
1. Introduction
Staphylococcus aureus (S. aureus) is one
of the most common pathogenic bacteria
which clinically leads to purulent
infections, such as pneumonia, pseudo
transpeptidase that is responsible for
anchoring surface virulence proteins to
peptidoglycan through recognizing the
LPXTG signal labeled on their
5
-7
membranous
septicemia
enteritis,
and sepsis
pericarditis,
systemic
C-terminal.
These surface proteins
display a critical role in the infection
process, including bacterial adhesion
and invasion of host tissues, and evasion
of the host immune system. Mutants
lacking genes for Srt A display
attenuated virulence, but did not affect
1,2
infection. With the wide application of
antibiotics clinically, S. aureus resistant
strains appear constantly. An example of
a clinically problematic infection is the
main pathogenic bacteria of nosocomial
infections and multiple drug resistance,
8, 9
the growth of the bacteria. Therefore,
Srt A has been considered as an ideal
target for the design of novel
anti-virulence as they may be less likely
to induce a selective pressure that leads
methicillin-resistant
S.
aureus
3
,4
(MRSA).
This strain has developed
resistance to antibiotics currently in
clinical use, including vancomycin,
which was once considered the best
therapy for MRSA. Therefore, in order to
cope with the increasingly serious drug
resistance problems, it is urgent to
develop new antibacterial targets and
corresponding inhibitors.
to drug resistance.
Previously, progress towards this
goal has been made and several distinct
Srt A inhibitors have been identified,
10,11
including
diarylacrylonitriles,
1
2
aryl(β-amino)ethyl
bis(indole)alkaloids,
ketones,
13
1
4,15
substrate-modified peptide,
vinyl
Srt A is
a
critical cysteine
16
sulfones,
and
other
natural

1
7-19
compounds.
However,
these
carboxamide at C-7 position exhibited
compounds have not been developed
into drugs or clinical applications
because of their low activity or poor
specificity. It is suggested that potent
inhibitors may result from utilizing the
specific recognition of Srt A to its
highly inhibitory activity against S.
22
aureus Srt A. Based on our published
results of benzo[d]oxazole derivatives,
i-butyl amide group and the benzoxazole
core shaped the whole molecule into a
L-shape mode to be specific recognition
by Srt A. We speculate similar structures
should be specific recognized by Srt A
2
0,21
substrate (LPXTG).
Usually, Srt A can specifically
recognize a substrate with an L-shape
mode, which is afforded by Leucine and
Proline side chains. If compounds with
an L-shape like the substrate by
Leucine/Proline residues can be
tolerated by Srt A. Based on the
principle, early, we discovered that the
benzo[d]oxazole derivatives bearing aryl
substituents at the C-2 position and
and exhibited highly inhibitory activity
.
Therefore, we designed a series of novel
2-phenyl-benzofuran-3-carboxamide
derivatives (Fig.1) as potential Srt A
inhibitors. The whole molecule is
constructed into a kinked pattern like
LPATG, with a benzofuran core and a
3-carboxamide mimicking the Proline
and Leucine side chains, respectively.
Fig. 1. The design strategy of target compounds.
2
2
. Results and discussion
.1 Chemistry
The
2,3-substituted-benzofuran
derivatives were prepared according to

the synthetic route in Scheme 1. Firstly,
p-methylbenzyl chloride was used as the
starting material, which was then treated
Subsequently, coupling compound XII
with 2-hydroxybenzaldehyde via
Wittig reaction yielded intermediate XI.
a
3
with PPh to yield intermediate XII.
a
PPh3
HCl
b
c
Cl
O
OH
XII
XI
X
CHO
COOH
COOCH3
e
f
d
g
O
O
O
VII
IX
VIII
COOCH3
COOCH3
COOH
Br
h
OOCCH3
i
OH
O
O
O
VI
V
IV
R1
NH
R1
NH
O
O
j
k
OH
Cl
O
O
III
II
l
m
R1
R1
NH
O
O
NH
O
O
m
O
R2
R2
m
O
O
Ia
Scheme 1 Reagents and conditions: a) PPh , CH CN, reflux, 70%; b) DBU, CH CN, reflux, 61%; c) I ,
Ib
3
3
3
2
K CO , THF, r.t. 94%; d) DMF, POCl , 1,2-dichloroethane, reflux, 78.2%; e) NH SO H, NaClO ,
2
3
3
2
3
2
CH OH/H O, r.t., 86%; f) H SO , CH OH, reflux, 90%; g) NBS, AIBN, CCl , reflux, 75%; h) NaOAc,
3
2
2
4
3
4
0
DMF, 90 C; i) NaOH, CH OH/H O, reflux, 86%; j) HATU, Et N, r.t., 90%; k) SOCl , CH Cl , reflux, 78%;
3
2
3
2
2
2
0
l) NaOH, DMF, 45 C, 30% - 71%; m) K CO , CH CN, reflux, 45% - 90%.
2
3
3
reaction of an aromatic acid or phenol.
The chemical structures were confirmed
Compound
intra-molecular cyclization in the
presence of K CO and I to produce
benzofuran X. Compound X was then
treated with POCl and DMF by a
XI
underwent
1
13
by spectral data. The H NMR,
C
2
3
2
NMR and mass spectra results are
consistent with the proposed structures.
3
2
.2 Inhibition of Sortase A
Vilsmeier reaction to produce compound
IX. Following oxidation, esterification,
bromination, hydrolysis, amidation and
chloridization reactions, 2,3-substituted
intermediate II was obtained. Eventually,
the target molecules Ia and Ib were
easily synthesized by substitution
Thirty-seven new compounds were
evaluated for their in vitro inhibitory
activities against Srt A according to a
previously reported HPLC assay
procedure with known Sortase
A

2
3,
inhibitor pHMB as positive compound.
compounds exhibited stronger activity
than pHMB (130 ± 4.3 µM) against the
Srt A of S. aureus. Especially, compound
Ia-22 was found to be the most potent
inhibitor against the enzyme with an
IC50 value of 30.8 µM, closely followed
by compounds Ia-4, Ia-5 and Ia-7.
2
4
The inhibitory potency of the tested
compounds, presented as IC50 values,
are shown in the Table 1 and are
compared with that of a known Srt A
10
inhibitor,
pHMB.
The
results
demonstrated that most of the
Table 1. Inhibitory activity of the target compounds against Sortase A
a
m
b
Structure index R1
R2
IC (µM)
50
c
pHMB (positive control)
130.0 ± 4.3
*
Ia-1
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
H
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
158.4 ± 1.0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Ia-2
2-OH
76.4 ± 2.7
50.6 ± 1.0
43.0 ± 0.7
34.5 ± 2.4
62.0 ± 0.8
39.6 ± 1.7
55.2 ± 4.9
Ia-3
3-OH
Ia-4
4-OH
Ia-5
3,5-di-OH
3,5-di-OCH
3,4-di-OH
Ia-6
3
Ia-7
Ia-8
3,4,5-tri-OCH
4-N(CH
2,4-di-OH
3
Ia-9
3
)
2
>200
**
Ia-10
Ia-11
Ia-12
Ia-13
Ia-14
Ia-15
Ia-16
Ia-17
Ia-18
Ia-19
Ia-20
57.0 ± 0.7
2-NO
2
>200
>200
4-F
**
**
**
**
**
**
4-SH
89.1 ± 1.2
60.6 ± 0.3
52.3 ± 1.4
89.0 ± 0.6
76.3 ± 0.8
84.0 ± 2.4
3-OH,4-NO
2
4-NH
2-OH
3-OH
4-OH
4-NO
2
2
>200
>200
3-CF
3

Ia-21
Ia-22
i-Bu
i-Bu
4-CF
3
1
1
>200
**
3,4-di-OH
4-OH
30.8 ± 2.3
(
C=
**
Ia-23
i-Bu
158.5 ± 3.8
C)
0
**
**
**
Ia-24
Ia-25
Ib-26
Ib-27
Ib-28
Ib-29
Ib-30
Ib-31
Ib-32
Ib-33
n-Bu
n-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
4-OH
186.4 ± 5.8
66.0 ± 1.4
86.4 ± 1.7
3,4-di-OH
3-OH
0
0
0
0
0
0
0
0
0
4-CH
4-Cl
3
>200
>200
*
4-CH
OH
120.8 ± 2.6
2
*
*
*
*
3-CHO
3-COCH
4-CN
89.0 ± 1.3
3
147.9 ± 0.8
*
97.0 ± 0.5
4-SO
3
CH
3
131.6 ± 1.3
**
Ib-34
Ib-35
Ib-36
Ib-37
3-NHCOCH
4-NHCOCH
3
3
0
0
0
0
148.0 ± 1.8
170.7 ± 4.6
*
i-Bu
i-Bu
n-Bu
4-COOCH
4-SO CH
3
>200
>200
3
3
a
"
m" represents double bond for Ia-23 and number of carbon for the rest.
b
c
IC50 values are means ± SD (n = 3). pHMB : p-Hydroxymercuribenzoic acid.
*Significantly different from control, p <0.05; ** Significantly different from control, p <0.001.
Preliminary
relationships were deduced as follows.
Firstly, the R substituent at the
-position of benzofuran has an obvious
influence on inhibition. Compounds Ia-4
IC50=43.0 µM) and Ia-7 (IC50=39.6 µM) as well. For example, introduction of a
structural
activity
3-position is indispensable for activity
which is consistent with our design.
Secondly, R
2
substituents at the C ring of
derivatives have
1
3
benzofuran
a
significant impact on inhibitory activity
(
showed significantly higher inhibitory
activity, as compared to those of
compounds Ia-24 (IC50 = 186.4 µM) and
3 2 3
-F, -Cl, -CF , -NO or -COOCH group
instead of a -OH, -SH, -NH or -OCH is
2
3
significantly
detrimental
to
the
Ia-25 (IC50
=
60.0 µM). Ib-33
inhibitory activity (Ia-2 vs Ia-11 and
Ia-12; Ia-13 to Ia-15 vs Ia-12; Ia-16 to
Ia-18 vs Ia-19 to Ia-21; Ia-7 vs Ia-14;
Ib-26 vs Ib-28 to Ib-37). Meanwhile,
(IC50=131.6 µM) showed higher activity
than Ib-37(IC50>200 µM). This indicates
that the i-butylamide substituent at the

we noted that the inhibitory activity is
proportional to the number of hydroxyl
groups as compounds Ia-5 and Ia-22
boost of inhibitory activity against Srt A
compared to an ether linker (Ia-3 vs
Ib-26).
(IC50=34.5 µM, 30.8 µM), which contain
2
.3 Molecular docking simulation
two hydroxyl groups, showed higher
To further elucidate the possible
activity than compounds Ia-3 and Ia-18
binding mechanism of our compounds,
molecular docking of some of the most
potent compounds was done using Gold
Suite v5.0.1 (CCDC, Cambridge, U.K.,
(
IC50=50.6 µM, 84.0 µM) with a single
hydroxyl group. Thirdly, the length of
the linker between the B and C rings has
little influence on inhibitory activity.
The activity slightly decreased with an
increase in linker length (Ia-16 to Ia-18
vs Ia-2 to Ia-4) except in the case of
compound Ia-22 (IC50=30.8 µM).
Finally, introduction of an ester linker
between the B and C rings resulted in a
2
010 ).The initial structure of Srt A was
obtained from the 3D-NMR structure of
the Srt A - LPAT complex (PDB code:
2
4
2
KID). The docking conformations of
our ligands (Ia-22 and Ib-26) and the
overlay of the ligands and LPAT within
the binding pocket are shown in Fig. 2.

Fig. 2. A, B: Docking of compo
situated less than 4 Å from the inhibitor have been shown and colored yellow; C: Ov ee rlap of the
LPAT and compounds (Ia-22 an Ib-26) in the binding pocket of Srt A. The protein ii s colorized
by electrostatic surface potential. LPAT is colored by yellow, Ia-22 colored by blue, Ib -- 26 colored
by magenta. The figures were cr ated using the program PYMOL
uu nds Ia-22 and Ib-26 to Srt A (PBD code: 2KID). Only residues
dd
ee
group stretches to the channel aa nd forms
hydrophobic interactions wit hh Thr164,
Val166, Val168, Leu169 and Il ee 182.
The model showed a special L-shape
binding mode which was si ilar to that
of the peptide substrate LPAT binding to
mm
The binding arrangement e xx plains the
21
Srt A. As depicted in Fig. 2C, the two
ligands bind to Srt A with a special
L-shape pattern similar to that of the
activity change. H-bonds formed
between the carbonyl oxyge nn atom of
the ester and the two active sit ee residues,
LPAT binding. To our knowl ee dge, the Ile
Cys184 and Arg197, may al ss o help to
stabilize the key conformation. In
addition, two -OH groups sub ss tituted at
the C ring of compound Ia- 22 2 formed
two additional H-bonds to th ee residues
Trp194 and Arg197. It ex pp lains the
of LPAT binding to the β6/β7 loop-β8
channel exerts decisive effect for
compound activity. We design our
ligands mimicking such c
o
o
mponent to
simulate the similar
binding
conformation. Indeed, the i-butyl amide
significant reduction in activit yy when the

3
3
. Experimental
-
OH group was replaced with a methyl
and halogen. As shown in Fig. 2B, the
OH group at the C ring of compound
.1 General methods for synthesis
-
The moisture sensitive reactions were
carried out under a nitrogen atmosphere
with the solvents distilled from
appropriate drying agents prior to use.
Ib-26 formed a H-bond to the basic
residue Arg197. However, compared to
the ester linker of compound Ia-22, the
ether linker of compound Ib-26
significantly reduced binding affinity,
which led to a slight reduction in activity
compared to that of compound Ia-22.
These results may explain why the ester
linker contributes to more positive
impacts in activity than that of the ether
linker.
Other
solvents
and
commercial
chemicals were purchased and used
directly without purification. Reactions
were monitored by TLC (silica gel
aluminum sheets 60 F254) with a UV
indicator. The NMR spectra were
obtained using an Agilent NMR Systems
1
4
00 MHz Spectrometer ( H NMR at 400
The 2,3-substituted benzofuran
plays a key role in shaping the whole
molecule in a L-shape conformation
which fits the corresponding active site
pocket of Srt A. An overlay of
compounds Ia-22, Ib-26 and LPAT in
1
3
MHz; C NMR at 100 MHz). Chemical
shifts were designated as follows in ppm
from the internal standard (chemical
shift in δ, ppm). Splitting patterns are
designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. Coupling
constants (J values) were measured in
Hertz (Hz). High resolution mass spectra
the
binding pocket (Fig.
2C)
demonstrates reoccurring binding motifs
shared by these ligands and Srt A:
i-butyl side chain, and the L-shape
pattern. Since the disulfide bond
between LPAT and Cys184 greatly
were
obtained
on
an
Agilent
Technologies Q-TOF 6230. Melting
points were determined on a Mel-Temp
II melting point apparatus and are
uncorrected.
strengthens
its
binding,
further
modification shall focus on the
interactions between Cys184 and
functional groups of the ligand.
3.1.1 Synthesis procedures and
analytical data

achieve a neutral pH (7) before
concentration by pressure reduction. The
residue obtained was dissolved in
EtOAc (150 mL) and washed with water
(2×75 mL), brine (1×75 mL) and dried
over anhydrous Na SO . The drying
2
4
agent was filtered off and the solvent
was removed in vacuo to afford the
crude product which was purified by
column chromatography to give
Triphenyl-(4-methylbenzyl) phosphonic
chloride (XII)
To a solution of p-methylbenzyl chloride
3
(50 g, 356 mmol) in CH CN (200 mL),
intermediate XI as a white solid (31.8 g,
triphenylphosphine (93.3 g, 356 mmol)
was added and refluxed for 6 h. Then the
reaction was cooled to room temperature
before filtered. The residue was washed
with ether (3×30 mL) and dried in vacuo
to afford intermediate XII as a white
solid (102.3 g, 70 % yield) which was
used in the next step without further
purification.
1
6
9
7
1
7
6
1 % yield); H NMR (DMSO-d
6
) δ
.67 (s, 1H), 7.52 (d, J = 7.40 Hz, 1H),
.40 (d, J = 7.80 Hz, 2H), 7.32 (d, J =
6.82 Hz, 1H), 7.09-7.17 (m, 3H), 7.01 -
.08 (m, 1H), 6.83 (d, J = 7.80 Hz, 1H),
.77 (t, J = 7.40 Hz, 1H), 2.27 (s, 3H).
2
-(4-methylphenyl) benzofuran (X)
To a solution of compound XI (5.4 g,
5.7 mmol) and anhydrous K CO (21.2
g, 154.2 mmol) in THF (150 mL), I2
39.4 g, 154.2 mmol) was added and
2
2
3
(E)-2-(4-methylstyryl)phenol (XI)
To a solution of compound XII (100 g,
(
2
2
48.2
-hydroxybenzaldehyde (30.3 g, 248.2
CN (350 mL), DBU (91.3
mmol)
and
stirred at room temperature for 1 h. After
the reaction was completed, aqueous
mmol) in CH
3
Na
2
S
2
O
3
was added to eliminate the
The mixture was
mL, 595.7 mmol) was added dropwise
slowly and refluxed overnight. The
reaction was cooled to room temperature.
Subsequently, HCl (1 M) was added to
remaining
2
I .
concentrated. The residue obtained was
dissolved in EtOAc (100 mL) and
washed with water (2×50 mL), brine

(1×50 mL) and dried over anhydrous
by column chromatography to give
Na SO . The drying agent was filtered
intermediate IX as a white solid (3.5 g,
2
4
1
off and the solvent was removed in
vacuo to afford the crude product which
was purified by column chromatography
78 % yield). H NMR (DMSO-d ) δ
6
10.23 (s, 1H), 8.10 - 8.14 (m, 1H), 7.87
(d, J = 8.20 Hz, 2H), 7.71 (d, J = 7.40
Hz, 1H), 7.36 - 7.46 (m, 4H), 2.39 (s,
3H).
to give intermediate X as a white solid
1
(
5.1 g, 94 % yield). H NMR (DMSO-d
6
)
δ 7.78 (d, J = 8.20 Hz, 2H), 7.57 (d, J =
8
7
7
.60 Hz, 1H), 7.60 (d, J = 7.40 Hz, 1H),
.32 (s, 1H), 7.28 (d, J = 8.20 Hz, 2H),
.24 (m, 1H), 7.19 - 7.22 (m, 1H), 2.32
2-(4-methylphenyl)benzofuran-3-carbox
ylic acid (VIII)
To a solution of intermediate IX (3.5 g,
14.8 mmol) in acetone (40 mL), NaClO2
(s, 3H).
(
3.3 g, 36.5 mmol) and NH
2
SO
3
H (2.86
2-(4-methylphenyl)benzofuran-3-carbal
0
g, 29.5 mmol) were added at 0 C and
dehyde (IX)
stirred at room temperature for 4 h. Then
To a solution of DMF (11.8 mL, 153.6
mmol) in 1,2-dichloroethane (50 mL),
aqueous Na
2
S
2
O
3
was added to eliminate
before being poured
remaining NaClO
2
POCl
3
(23.5 g, 153.6 mmol) was added
onto water (20 mL) which yielded a
large amount of solid precipitation. The
mixture was filtered to give intermediate
VIII as yellow solid (3.0 g, 86 % yield)
which was used in the next step without
further purification.
0
dropwise at 0 C. The reactions were
allowed to reach room temperature
before it was stirred for 1h. Compound
X (4.0 g, 19.2 mmol) was then added
and refluxed overnight. The reaction was
cooled to room temperature before
poured onto 350 g of crushed ice and
stirred for an additional 1 h. The
aqueous solution was basified to pH 9
with aqueous NaOH (1M) and extracted
with CH Cl . The combined extracts
2
-(4-methylphenyl)benzofuran-3-carbox
ylate (VII)
To a solution of intermediate VIII (3.0 g,
11.9 mmol) in MeOH (50 mL),
2
2
2 4
concentrated H SO (5 mL) was added
were dried, concentrated, and purified
0
dropwise at 0 C. The reaction was then

1
refluxed overnight before being cooled
to room temperature and concentrated
by reduced pressure. Then the residue
was dissolved in EtOAc (30 mL) and
6
(6.8 g, 75 % yield); H NMR (DMSO-d )
δ 8.00 (d, J = 5.50 Hz, 1H), 7.95 (d, J =
8.60 Hz, 2H), 7.69 (d, J = 7.40 Hz, 1H),
7.59 (d, J = 8.20 Hz, 2H), 7.36 - 7.44 (m,
washed with aqueous NaHCO , brine
2H), 4.77 (s, 2H), 3.85 (s, 3H).
3
2 4
and dried over anhydrous Na SO . The
solvent was removed and the residue
was concentrated and purified by
column chromatography to give
2-(4-(acetoxymethyl)phenyl)benzofuran
-3-carboxylate (V)
To a solution of intermediate VI (1.5 g,
4.3 mmol) in DMF (10 mL), anhydrous
NaOAc (1.78 g, 21.5 mmol) was added
intermediate VII as a white solid (2.85 g,
1
9
7
2
2
3
0 % yield); H NMR (DMSO-d
6
) δ
0
.94-7.98 (m, 1H), 7.85 (d, J = 8.20 Hz,
H), 7.63 - 7.68 (m, 1H), 7.34 - 7.42 (m,
H), 7.32 (d, J = 7.80 Hz, 2H), 3.83 (s,
H), 2.36 (s, 3H).
and heated at 120 C for 3 h. After
cooling to room temperature the mixture
was extracted with EtOAc. The
combined extracts were washed with
water, brine, dried over anhydrous
2
-(4-bromomethylphenyl)benzofuran-3-
Na
intermediate V as white solid (1.2 g,
6.3 % yield) which was used in the
next step without further purification.
2
SO
4
and concentrated to give
carboxylate (VI)
To a solution of intermediate VII (7.0 g,
8
26.3 mmol) in CCl
4
(100 mL), NBS
(5.14 g, 28.9 mmol) and AIBN (0.2 g,
1
1
.2 mmol) were added and refluxed for
2 h. The reaction was subsequently
2
-(4-(hydroxymethyl)phenyl)benzofuran
3-carboxylic acid (IV)
To a solution of intermediate V (2.0 g,
.17 mmol) in THF (10 mL), aqueous
-
cooled to room temperature before
filtration. The filtrate was extracted with
EtOAc and the organic phase was
washed with water and brine, dried over
6
NaOH (3M, 10 mL) was added and
refluxed for 3 h. The reaction was
cooled to room temperature and
acidified by HCl (1M) to be pH 4 before
being poured onto water (10 ml). The
2 4
anhydrous Na SO , concentrated and
purified by column chromatography to
give intermediate VI as a white solid

precipitate was filtered to give
To a solution of intermediate III (1.0 g,
3.1 mmol) in CH Cl (20 mL), SOCl
2
intermediate IV as yellow solid (1.65 g,
2
2
95 % yield) which was used in the next
(0.78 mL, 9.3 mmol) was added
0
step without further purification.
dropwise at 0 C and stirred at room
temperature for 1 h. Then the mixture
was poured onto ice water and extracted
with EtOAc. The combined extracts
2
-(4-(hydroxymethyl)phenyl)-N-isobutyl
benzofuran-3-carboxamide (III)
To a solution of intermediate IV (1.0 g,
2 3
were washed with aqueous Na CO and
3
.7 mmol) in CH
i-butylamine (0.54 g, 7.4 mmol), HATU
2.1 g, 5.6 mmol), triethylamine (0.75 g,
4.8 mmol) was added and stirred at
room temperature for 2 h. Then HCl
1M) was added to achieve acidic pH 5
and extracted with CH Cl . The
2
Cl
2
(20 mL),
brine, dried, concentrated and purified
by column chromatograph to give
intermediate IIa as yellow solid (525 mg,
(
1
1
40.6 % yield); H NMR (CDCl ) δ 8.01
3
(d, J = 8.60 Hz, 2H), 7.78 (d, J = 7.40
Hz, 1H), 7.49 - 7.55 (m, 3H), 7.29 - 7.39
(m, 2H), 5.01 (br. s., 1H), 4.27 (s, 2H),
3.31 (t, J = 6.4 Hz, 2H), 1.87 (m, 1H),
0.95 (d, J = 6.60 Hz, 6H).
(
2
2
combined extracts were washed with
aqueous NaHCO and brine, dried,
3
concentrated and purified by column
chromatograph to give intermediate III
2-(4-(chloromethyl)phenyl)-N-n-butylbe
nzofuran-3-carboxamide (IIb)
1
as yellow solid (1.2 g, 90 % yield); H
NMR (DMSO-d
6
) δ 7.79 - 7.88 (d, J =
.20 Hz, 2H), 7.64 (d, J = 8.20 Hz, 1H),
.57 (d, J = 7.40 Hz, 1H), 7.39 - 7.45 (d,
Compound IIb was synthesized,
following a similar procedure as the one
used for preparation of IIa as yellow
solid (11.3 % totally yield).
8
7
J = 8.20 Hz, 2H), 7.27 - 7.39 (m, 2H),
.30 (br. s., 1H), 4.53 (br. s., 2H), 3.11 (t,
J = 6.4 Hz, 2H), 1.83 (m, 1H), 0.89 (d, J
6.60 Hz, 6H).
5
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl benzoate (Ia-1)
=
To a solution of intermediate IIa (200
mg, 0.496 mmol) in DMF (10 mL),
NaOH (99.2 mg, 2.48 mmol) and
2
-(4-(chloromethyl)phenyl)-N-i-butylbe
nzofuran-3-carboxamide (IIa)

benzoic acid (302.6 mg, 2.48 mmol) was
added and stirred at room temperature
Hz, 1H), 7.77 - 7.82 (m, 1H), 7.49 - 7.56
(m, 3H), 7.43 - 7.49 (m, 1H), 7.27 - 7.37
(m, 2H), 6.98 (d, J = 8.60 Hz, 1H), 6.88
(t, J = 7.60 Hz, 1H), 5.96 (br. s., 1H),
5.41 (s, 2H), 3.28 (t, J = 6.46 Hz, 2H),
1.84 (m, 1H), 0.92 (d, J = 6.60 Hz, 6H);
2
for 3 h before H O (10 mL) was added.
The resulting solid was filtered, washed
with ethyl acetate and 95 % ethanol, and
2
recrystallized in DMF/H O to give
1
3
compound Ia-1 as white solid (150 mg,
3
C NMR (CDCl ) δ 169.8, 163.9, 161.7,
0
7
1.5 % yield); m.p. 168-170 C,
153.8, 153.7, 136.9, 136.0, 129.9, 129.6,
128.4, 128.4, 127.5, 125.4, 123.8, 120.8,
119.3, 117.6, 113.4, 112.2, 111.3, 66.4,
1
H-NMR (CDCl ) δ 8.07 (m, 2H), 7.94
3
(
d, J=8.4 Hz, 2H), 7.81 (m, 1H), 7.54 (m,
4
H), 7.44 (m, 2H), 7.32 (m, 2H), 5.94
br. s., 1H), 5.41 (s, 2H), 3.28 (t, J =
.4Hz, 2H), 1.82 (m, 1H), 0.91 (d, J =
47.2, 28.5, 20.2; HRMS-ESI C27H25NO
5
(
calcd [M+H] 444.1805, found 444.1801.
6
6
1
1
1
2
13
.60 Hz, 6H); C NMR (CDCl
3
) δ 166.3,
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-3’-hydroxybenzoate (Ia-3)
64.0, 154.0, 153.7, 137.8, 133.2, 129.8,
29.7, 129.3, 128.4, 128.3, 127.6, 125.4,
23.8, 120.8, 113.3, 111.3, 66.1, 47.1,
Compound Ia-3 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIa and 3-hydroxybenzoic
acid to obtain the product as white solid
8.5, 20.2; HRMS-ESI C27
4
H25NO calcd
[
M+H] 428.1856, found 428.1859.
0
1
4-(3-(isobutylcarbamoyl)benzofuran-2-y
(64 % yield); m.p. 185-187 C. H NMR
l)benzyl-2’-hydroxybenzoate (Ia-2)
(DMSO-d ) δ 9.81 (s, 1H), 8.60 (t, J =
6
Compound Ia-2 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIa and 2-hydroxybenzoic
acid to obtain the product as yellow
5.60 Hz, 1H), 7.89 (d, J = 8.20 Hz, 2H),
7.65 (d, J = 7.80 Hz, 1H), 7.54 - 7.59 (m,
3H), 7.24 - 7.45 (m, 5H), 7.01 (d, J =
8.60 Hz, 1H), 5.32 (s, 2H), 3.11 (t, J =
6.4 Hz, 2H), 1.83 (m, 1H), 0.88 (d, J =
0
13
solid (55.3 % yield); m.p. 180-182 C.
6.60 Hz, 6H); C NMR (DMSO-d ) δ
6
1
H NMR (CDCl ) δ 10.70 (s, 1H), 7.97
165.9, 163.6, 158.0, 153.3, 152.0, 137.9,
131.1, 130.4, 129.4, 128.8, 128.0, 127.2,
3
(d, J = 8.20 Hz, 2H), 7.88 (d, J = 8.20

1
1
25.9, 124.1, 121.0, 120.9, 120.4, 116.1,
15.0, 111.7, 66.1, 47.1, 28.5, 20.7;
Compound Ia-5 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
HRMS-ESI C27
H
25NO
5
calcd [M+H]
4
44.1805, found 444.1811.
compound
,5-dihydroxybenzoic acid to obtain the
product as white solid (45.9 % yield);
IIa
and
3
4-(3-(isobutylcarbamoyl)benzofuran-2-y
0
1
l)benzyl-4’-hydroxybenzoate (Ia-4)
m.p. 184-186 C. H NMR (DMSO-d ) δ
6
Compound Ia-4 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIa and 4-hydroxybenzoic
acid to obtain the product as white solid
9.61 (s, 2H), 8.57 (t, J = 5.80 Hz, 1H),
7.90 (d, J = 8.20 Hz, 2H), 7.65 (d, J =
8.20 Hz, 1H), 7.53 - 7.60 (m, 3H), 7.35 -
7.40 (m, 1H), 7.29 - 7.35 (m, 1H), 6.85
(s, 2H), 6.43 (s, 1H), 5.32 (s, 2H), 3.12
(t, J = 6.4 Hz, 2H), 1.83 (m, 1H), 0.89 (d,
0
1
(
52.6 % yield); m.p. 185-188 C. H
NMR (DMSO-d ) δ 10.34 (s, 1H), 8.57
t, J = 5.67 Hz, 1H), 7.89 (d, J = 8.20 Hz,
13
6
J = 6.65 Hz, 6H); C NMR (DMSO-d )
6
(
δ 166.0, 163.6, 159.0, 153.3, 152.0,
137.9, 131.6, 129.4, 128.8, 128.0, 127.3,
125.9, 124.1, 120.9, 115.0, 111.7, 107.8,
107.6, 66.1, 47.1, 28.5, 20.7;
2H), 7.84 (d, J = 9.00 Hz, 2H), 7.65 (d, J
=
7.80 Hz, 1H), 7.54 - 7.59 (m, 3H),
7
6
3
0
.35 - 7.38 (m, 1H), 7.31 - 7.34 (m, 1H),
.84 (d, J = 8.60 Hz, 2H), 5.32 (s, 2H),
.11 (t, J = 6.4 Hz, 2H), 1.82 (m, 1H),
HRMS-ESI C27
6
H25NO calcd [M+H]
460.1755, found 460.1759.
1
3
.88 (d, J = 6.60 Hz, 6H); C NMR
) δ 163.8, 163.6, 162.6, 153.3,
(DMSO-d
6
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-3’,5’-dimethoxybenzoate (Ia-6)
Compound Ia-6 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
1
1
1
4
52.0, 138.2, 132.0, 129.3, 128.6, 128.0,
27.2, 127.1, 126.9, 125.9, 124.1, 120.9,
20.8, 120.5, 115.9, 114.9, 111.7, 65.6,
7.1, 28.5, 20.7; HRMS-ESI C27H25NO
4
calcd [M+H] 444.1805, found 444.1807.
compound
,5-dimethoxybenzoic acid to obtain the
product as white solid (54.5 % yield);
IIa
and
3
4-(3-(isobutylcarbamoyl)benzofuran-2-y
0
1
l)benzyl-3’,5’-dihydroxybenzoate (Ia-5)
m.p. 181-182 C. H NMR (DMSO-d ) δ
6

1
3
8
8
7
7
1
.56 (t, J = 5.80 Hz, 1H), 7.90 (d, J =
.60 Hz, 2H), 7.65 (d, J = 8.20 Hz, 1H),
.55 - 7.60 (m, 3H), 7.35 - 7.40 (m, 1H),
.29 - 7.34 (m, 1H), 7.09 (s, 2H), 6.77 (s,
H), 5.38 (s, 2H), 3.77 (s, 6H), 3.11 (t, J
6
C NMR (DMSO-d ) δ 165.9, 163.6,
153.3, 152.0, 151.1, 145.6, 138.3, 129.3,
128.7, 128.0, 127.2, 125.9, 124.1, 122.4,
120.9, 120.7, 116.7, 115.8, 114.9, 111.7,
65.6, 47.1, 28.5, 20.7; HRMS-ESI
=
6.4 Hz, 2H), 1.83 (m, 1H), 0.88 (d, J =
C
27
H
25NO
6
calcd [M+H] 460.1755,
1
3
6
1
1
1
6
.65 Hz, 6H); C NMR (DMSO-d
6
) δ
found 460.1749.
65.7, 163.6, 161.0, 153.3, 152.0, 137.8,
31.9, 129.4, 128.7, 128.0, 127.3, 125.9,
24.0, 120.9, 115.0, 111.7, 107.3, 105.8,
6.3, 56.0, 47.1, 28.4, 20.7; HRMS-ESI
4-(3-(isobutylcarbamoyl)
benzofuran-2-yl)
benzyl-3’,4’,5’-trimethoxybenzoate
(Ia-8)
C H NO calcd [M+H] 488.2068,
29
29
6
found 488.2060.
Compound Ia-8 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
4
-(3-(isobutylcarbamoyl)
benzofuran-2-yl)
’-dihydroxybenzoate (Ia-7)
benzyl-3’,
compound
IIa
and
4
3,4,5-trimethoxybenzoic acid to obtain
the product as white solid (28.9 % yield);
Compound Ia-7 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
0
1
m.p. 190-192 C. H NMR (DMSO-d ) δ
6
8.57 (t, J = 5.8 Hz, 1H), 7.90 (d, J =
8.20 Hz, 2H), 7.65 (d, J = 7.80 Hz, 1H),
7.54 - 7.61 (m, 3H), 7.35 - 7.40 (m, 1H),
7.29 - 7.34 (m, 1H), 7.27 (s, 2H), 5.39 (s,
2H), 3.81 (s, 6H), 3.71 (s, 3H), 3.11 (t, J
= 6.26 Hz, 2H), 1.82 (m, 1H), 0.88 (d, J
compound
,4-dihydroxybenzoic acid to obtain the
product as white solid (34 % yield); m.p.
IIa
and
3
0
1
1
80-182 C. H NMR (DMSO-d
6
) δ 9.81
(s, 1H), 9.38 (s, 1H), 8.59 (t, J = 5.80 Hz,
1
3
1
7
7
5
1
H), 7.87 - 7.93 (m, 2H), 7.65 (d, J =
.80 Hz, 1H), 7.52 - 7.60 (m, 3H), 7.29 -
.40 (m, 4H), 6.80 (d, J = 8.20 Hz, 1H),
.30 (s, 2H), 3.12 (t, J = 6.4 Hz, 2H),
.83 (m, 1H), 0.89 (d, J = 6.60 Hz, 6H);
= 6.6 Hz, 6H); C NMR (DMSO-d ) δ
6
165.6, 163.6, 153.5, 153.3, 152.0, 142.4,
138.0, 129.3, 128.6, 128.0, 127.3, 125.9,
125.0, 124.1, 120.9, 115.0, 111.7, 107.1,
66.2, 60.6, 56.5, 47.1, 28.4, 20.7;

HRMS-ESI C30
H
31NO
7
calcd [M+H]
4’-dihydroxybenzoate (Ia-10)
5
18.2173, found 518.2167.
Compound Ia-10 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
4-(3-(isobutylcarbamoyl)
benzofuran-2-yl)
compound
IIa
and
benzyl-4’-dimethylaminobenzoate (Ia-9)
Compound Ia-9 was synthesized,
following a similar procedure as the one
used for preparation of Ia-1 starting
2,4-dihydroxylbenzoic acid to obtain the
product as white solid (27 % yield); m.p.
0
1
173-175 C. H NMR (DMSO-d ) δ
6
10.64 (s, 1H), 10.48 (s, 1H), 8.58 (t, J =
from
-dimethylaminobenzoic acid to obtain
the pure product as white solid (65 %
compound
IIa
and
5.8 Hz, 1H), 7.90 (d, J = 8.20 Hz, 2H),
7.65 (d, J = 8.20 Hz, 1H), 7.68 (d, J =
9.00 Hz, 1H), 7.54-7.60 (m, 3H), 7.37 (t,
J = 7.40 Hz, 1H), 7.32 (t, J = 7.20 Hz,
1H), 6.36 (d, 8.80 Hz, 1H), 6.29 (s, 1H),
5.37 (s, 2H), 3.11 (t, J = 6.4 Hz, 2H),
1.83 (m, 1H), 0.88 (d, J = 6.6 Hz, 6H);
4
0
1
yield); m.p. 180-183 C. H NMR
DMSO-d ) δ 8.57 (br. s., 1H), 7.89 (d, J
7.80 Hz, 2H), 7.77 - 7.83 (m, 2H),
(
6
=
7
3
.65 (d, J = 7.80 Hz, 1H), 7.52 - 7.61 (m,
H), 7.37 (t, J = 7.40 Hz, 1H), 7.32 (t, J
1
3
C NMR (DMSO-d ) δ 169.2, 164.8,
6
=
7.20 Hz, 1H), 6.67 - 6.74 (m, 2H),
163.6, 163.3, 153.3, 152.0, 137.6, 132.2,
129.4, 128.8, 128.0, 127.3, 125.9, 124.1,
120.9, 115.0, 111.7, 108.9, 104.4, 103.0,
66.0, 47.1, 28.5, 20.7; HRMS-ESI
C H NO calcd [M+H] 460.1755,
5
2
6
1
1
1
1
.30 (s, 2H), 3.12 (t, J = 6.0 Hz, 2H),
.97 (s, 6H), 1.83 (m, 1H), 0.89 (d, J =
1
3
6
.6 Hz, 6H); C NMR (DMSO-d ) δ
66.1, 163.6, 153.8, 153.3, 152.1, 138.6,
31.4, 129.2, 128.5, 128.1, 127.2, 125.8,
24.0, 120.9, 115.9, 114.9, 111.7, 111.3,
10.0, 65.2, 47.1, 28.5, 20.7;
27
25
6
found 460.1755.
4-(3-(isobutylcarbamoyl)
HRMS-ESI C27
H
25NO
5
calcd [M+H]
benzofuran-2-yl)
4
71.2206, found 471.2249.
benzyl-2’-nitrobenzoate (Ia-11)
Compound Ia-11 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
4-(3-(isobutylcarbamoyl)
benzofuran-2-yl)
benzyl-2’,

compound IIa and 2-nitrobenzoic acid
4H), 5.38 (s, 2H), 3.11 (t, J = 6.4 Hz,
to obtain the product as yellow solid (55 % 2H), 1.82 (m, 1H), 0.88 (d, J = 6.6 Hz,
0
1
13
yield); m.p. 173-175 C. H NMR
DMSO-d ) δ 8.59 (t, J = 5.4 Hz, 1H),
.06 (d, 8.20 Hz, 2H), 7.90 (d, J = 7.80
Hz, 2H), 7.65 (d, J = 7.80 Hz, 1H), 7.58
d, J = 8.20 Hz, 3H), 7.29 - 7.41 (m, 4H),
6H); C NMR (DMSO-d ) δ 166.9,
6
(
6
165.1, 164.4, 163.6, 153.3, 152.0, 137.8,
132.6 (d, JC-F = 9), 129.4, 128.8, 128.0,
127.2, 126.5, 125.9, 124.1, 120.9, 116.5,
116.3, 115.0, 111.7, 66.3, 47.1, 28.5,
8
(
5
1
.38 (s, 2H), 3.11 (t, J = 6.4 Hz, 2H),
.82 (m, 1H), 0.88 (d, J = 6.6 Hz, 6H);
20.7; HRMS-ESI C27
H
24FNO
4
calcd
[M+H] 446.1762, found 446.1768.
1
3
6
C NMR (DMSO-d ) δ 166.9, 165.1,
1
1
1
1
64.4, 163.6, 153.3, 152.0, 137.8, 132.7,
32.6, 129.4, 128.8, 128.0, 127.2, 126.5,
25.9, 124.1, 120.9, 116.5, 116.3, 115.0,
11.7, 66.3, 47.1, 28.5, 20.7;
4-(3-(isobutylcarbamoyl)
benzofuran-2-yl)
benzyl-4’-thiolbenzoate (Ia-13)
Compound Ia-13 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIa and 4-thiolbenzoic acid to
obtain the product as white solid (18 %
HRMS-ESI C H FNO calcd [M+H]
27
24
4
4
73.1634, found 473.1677
4
-(3-(isobutylcarbamoyl)
0
1
benzofuran-2-yl)
yield); m.p. 179-181 C. H NMR
benzyl-4’-fluorobenzoate (Ia-12)
(DMSO-d ) δ 8.58 (t, J = 5.8 Hz, 1H),
6
Compound Ia-12was
synthesized,
7.71 - 7.86 (m, 4H), 7.62 (d, J = 7.8 Hz,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIa and 4-fluorobenzoic acid
1H), 7.55 (d, J = 7.4 Hz, 1H), 7.47 (d, J
= 8.2 Hz, 2H), 7.27 - 7.38 (m, 2H), 7.23
(d, J = 7.8 Hz, 2H), 4.28 (s, 2H), 3.09
to obtain the product as white solid (55 % (br. s., 2H), 1.81 (m, 1H), 0.87 (d, J =
0
1
13
yield); m.p. 173-175 C. H NMR
DMSO-d ) δ 8.59 (t, J = 5.4 Hz, 1H),
.06 (d, J = 8.20 Hz, 2H), 7.90 (d, J =
6.26 Hz, 6H); C NMR (DMSO-d ) δ
6
(
6
170.0, 163.7, 153.2, 152.1, 139.6, 137.9,
137.1, 130.1, 129.7, 128.4, 128.1, 127.3,
127.1, 126.5, 125.8, 124.0, 120.8, 114.6,
111.6, 110.0, 66.8, 47.1, 28.5, 20.7;
8
7
7
.80 Hz, 2H), 7.65 (d, J = 7.80 Hz, 1H),
.58 (d, J = 8.2 Hz, 3H), 7.29 - 7.41 (m,

HRMS-ESI C27
H
25NO
4
S calcd [M+H]
Compound Ia-15 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIa and 4-aminobenzoic acid
to obtain the product as white solid (45 %
460.1577, found 460.1568.
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-3’-hydroxy-4’-nitrobenzoate
Ia-14)
0
1
(
yield); m.p. 221-223 C. H NMR
Compound Ia-14 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
(DMSO-d ) δ 10.34 (s, 2H), 8.57 (t, J =
6
5.6 Hz, 1H), 7.89 (d, J = 8.2 Hz, 2H),
7.84 (d, J = 9.00 Hz, 2H), 7.65 (d, J =
7.8 Hz, 1H), 7.54-7.59 (m, 3H),
7.35-7.38 (m, 1H), 7.31-7.34 (m, 1H),
6.84 (d, J = 8.6 Hz, 2H), 5.32 (s, 2H),
3.11 (t, J = 6.4 Hz, 2H), 1.82 (m, 1H),
compound
-hydroxy-4-nitrobenzoic acid to obtain
the product as white solid (38 % yield);
IIa
and
3
0
1
m.p. 199-201 C. H NMR (DMSO-d
6
) δ
.60 (t, J = 5.6 Hz, 1H), 7.87-7.97 (m,
H), 7.68-7.72 (m, 1H), 7.66 (d, J = 8.2
1
3
8
3
0.88 (d, J = 6.6 Hz, 6H); C NMR
(DMSO-d ) δ 165.5, 163.6, 163.1, 153.3,
6
Hz, 1H), 7.55-7.62 (m, 3H), 7.48-7.53
m, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.32 (t,
J = 7.2 Hz, 1H), 5.39 (s, 2H), 3.12 (t, J =
152.0, 151.7, 138.3, 138.1, 129.2, 128.6,
(
128.1, 127.2, 125.8, 124.1, 120.9, 114.9,
113.4, 111.7, 107.6, 65.3, 47.1, 28.5,
6
6
1
1
1
1
.4 Hz, 2H), 1.83 (m, 1H), 0.89 (d, J =
20.7; HRMS-ESI C27
H
26
N
2
O
4
calcd
13
6
.6 Hz, 6H); C NMR (DMSO-d ) δ
[M+H] 443.1965, found 443.1953.
65.5, 163.6, 163.1, 153.3, 152.0, 151.7,
38.3, 138.1, 129.2, 128.6, 128.1, 127.2,
25.8, 124.1, 120.9, 114.9, 113.4, 111.7,
07.6, 65.3, 47.1, 28.5, 20.7;
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-2’-(2-hydroxyphenyl)acetate
(Ia-16)
HRMS-ESI C H N O calcd [M+H]
Compound Ia-16 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
2
7
24
2
7
489.1656, found 489.1661.
4-(3-(isobutylcarbamoyl)
compound
IIa
and
benzofuran-2-yl)
2-(2-hydroxyphenyl)acetic acid to obtain
the product as white solid (42.4 % yield);
benzyl-4’-aminobenzoate (Ia-15)

0
1
m.p. 179-181 C. H NMR (CDCl
.87 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 7.4
Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.40
d, J = 8.2 Hz, 2H), 7.26 - 7.36 (m, 2H),
.15 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 7.4
Hz, 1H), 6.81 - 6.94 (m, 2H), 6.01 (br. s.,
H), 5.17 (s, 2H), 3.71 (s, 2H), 3.27 (t, J
6.4 Hz, 2H), 1.84 (m, 1H), 0.92 (d, J =
3
) δ
J = 7.2 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H),
6.57 - 6.74 (m, 3H), 5.14 (s, 2H), 3.63 (s,
2H), 3.12 (t, J = 6.4 Hz, 2H), 1.83 (m,
7
13
(
1H), 0.89 (d, J = 6.6 Hz, 6H); C NMR
7
(DMSO-d ) δ 171.4, 163.6, 157.8, 153.3,
6
151.9, 137.9, 135.8, 129.7, 129.2, 128.5,
1
128.0, 127.1, 125.9, 124.1, 120.9, 120.3,
116.7, 114.9, 114.3, 111.7, 65.7, 47.1,
=
1
3
6
1
1
1
6
.6 Hz, 6H); C NMR (CDCl ) δ 170.7,
40.8, 28.5, 20.7; HRMS-ESI C28H27NO
5
3
61.5, 152.5, 151.5, 151.1, 134.4, 128.5,
26.8, 126.5, 125.8, 125.7, 124.8, 122.8,
21.2, 118.1, 118.0, 114.5, 110.6, 108.8,
4.1, 44.6, 34.9, 25.9, 17.7; HRMS-ESI
calcd [M+H] 458.1962, found 458.1959.
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-2’-(4-hydroxyphenyl)acetate
(Ia-18)
C
28
H
27NO
5
calcd [M+H] 458.1962,
found 458.1954.
Compound Ia-18 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
4
-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-2’-(3-hydroxyphenyl)acetate
Ia-17)
compound
IIa
and
(
2-(4-hydroxyphenyl)acetic acid to obtain
the product as white solid (33.0 %
Compound Ia-17 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
0
1
yield); m.p. 212-214 C. H NMR
(DMSO-d ) δ 9.28 (s, 1H), 8.55 (t, J =
6
compound
IIa
and
5.8 Hz, 1H), 7.81-7.89 (m, 2H), 7.65 (d,
2-(3-hydroxyphenyl)acetic acid to obtain
J = 8.2 Hz, 1H), 7.55-7.61 (m, 1H),
the product as white solid (39.4 % yield); 7.40-7.46 (m, 2H), 7.35-7.40 (m, 1H),
0
1
m.p. 197-199 C. H NMR (DMSO-d ) δ
7.28-7.34 (m, 1H), 7.02-7.08 (m, 2H),
6.65-6.72 (m, 2H), 5.13 (s, 2H), 3.59 (s,
2H), 3.12 (t, J = 6.4 Hz, 2H), 1.83 (m,
6
9
7
1
.40 (br. s., 1H), 8.59 (t, J = 5.6 Hz, 1H),
.80-7.90 (m, 2H), 7.65 (d, J = 8.2 Hz,
H), 7.58 (d, J = 7.8 Hz, 1H), 7.41-7.48
13
1H), 0.90 (d, J = 6.6 Hz, 6H); C NMR
(m, 2H), 7.37 (t, J = 7.6 Hz, 1H), 7.32 (t,
(DMSO-d ) δ 171.4, 163.6, 157.8, 155.3,
6

1
1
1
4
51.9, 137.9, 135.8, 129.7, 129.2, 128.5,
28.0, 127.1, 125.9, 124.1, 120.9, 120.3,
16.7, 112.9, 114.3, 111.7, 65.5, 47.1,
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-2’-(3-trifloromethyl-phenyl)ace
tate (Ia-20)
0.8, 28.5, 20.7; HRMS-ESI C28
H
27NO
5
Compound Ia-20 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
calcd [M+H] 458.1962, found 458.1960.
4
-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-2’-(4-nitrophenyl)acetate
Ia-19)
compound
IIa
and
2-(3-trifloromethyl-phenyl)acetic acid to
obtain the product as white solid (61.5 %
(
0
1
Compound Ia-19 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
yield); m.p. 211-213 C. H NMR
(CDCl ) δ 7.86-7.96 (m, 2H), 7.78 (d, J
3
= 7.4 Hz, 1H), 7.43-7.57 (m, 5H),
compound
IIa
and
7.36-7.41 (m, 2H), 7.26-7.36 (m, 2H),
5.99 (br. s., 1H), 5.17 (s, 2H), 3.74 (s,
2-(4-nitrophenyl)acetic acid to obtain
the product as white solid (42.0 % yield); 2H), 3.27 (t, J = 6.4 Hz, 2H), 1.83 (m,
0
1
13
m.p. 233-235 C. H NMR (DMSO-d ) δ
1H), 0.92 (d, J = 6.6 Hz, 6H); C NMR
6
8
2
.56 (t, J = 5.6 Hz, 1H), 8.13-8.22 (m,
H), 7.83-7.89 (m, 2H), 7.65 (d, J = 8.2
(CDCl ) δ 170.5, 164.0, 153.8, 153.7,
3
137.1, 134.5, 132.8, 129.4, 129.1, 128.3,
Hz, 1H), 7.57 (d, J = 9.00 Hz, 3H),
128.2, 127.5, 126.2, 126.1, 125.4, 124.1
(q, JC-F=365), 123.8, 120.8, 113.4, 111.5,
111.3, 110.0, 66.3, 47.1, 40.9, 28.5, 20.2;
HRMS-ESI C H F NO calcd [M+H]
7
7
2
1
.42-7.48 (m, 2H), 7.38 (m, 1H),
.29-7.35 (m, 1H), 5.17 (s, 2H), 3.97 (s,
H), 3.11 (t, J = 6.4 Hz, 2H), 1.83 (m,
29
26
3
4
13
H), 0.89 (d, J = 6.6 Hz, 6H); C NMR
) δ 170.6, 163.6, 160.6, 153.3,
510.1887, found 510.1876.
(
DMSO-d
6
1
1
1
2
51.9, 147.0, 142.7, 137.6, 131.4, 129.3,
28.6, 128.0, 127.1, 125.9, 124.1, 123.8,
20.9, 115.0, 111.7, 66.0, 47.1, 40.6,
4-(3-(isobutylcarbamoyl)benzofuran-2-y
l)benzyl-2’-(4-trifloromethyl-phenyl)ace
tate (Ia-21)
8.5, 20.7; HRMS-ESI C28
H
26
N
2
O
6
Compound Ia-21 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
calcd [M+H] 487.1864, found 487.1862.

compound
IIa
and
2H), 7.66 (d, J = 8.2 Hz, 1H), 7.58 (d, J
2-(4-trifloromethyl-phenyl)acetic acid to
= 7.4 Hz, 1H), 7.40-7.47 (m, 2H), 7.37 (t,
obtain the product as white solid (56.2 % J = 7.4 Hz, 1H), 7.32 (t, J = 7.4 Hz, 1H),
0
1
yield); m.p. 213-215 C. H NMR
6.66 (br. s., 1H), 6.64 (d, J = 8.2 Hz,
1H), 6.50 (d, J = 7.8 Hz, 1H), 5.12 (s,
2H), 3.52 (s, 2H), 3.12 (t, J = 6.0 Hz,
2H), 1.83 (m, 1H), 0.89 (d, J = 6.6 Hz,
(CDCl
3
) δ 7.92 (d, J = 8.2 Hz, 2H), 7.79
(
d, J = 7.4 Hz, 1H), 7.58 (d, J = 7.8 Hz,
2
7
2
2
6
1
1
H), 7.51 (d, J = 7.8 Hz, 1H), 7.26 -
.45 (m, 6H), 5.96 (br. s., 1H), 5.17 (s,
H), 3.74 (s, 2H), 3.28 (t, J = 6.4 Hz,
H), 1.84 (m, 1H), 0.92 (d, J = 6.6 Hz,
1
3
6H); C NMR (DMSO-d ) δ 171.8,
6
163.6, 153.3, 151.9, 145.5, 144.7, 138.0,
129.2, 128.5, 128.1, 127.1, 125.9, 125.2,
124.1, 120.9, 120.5, 117.1, 115.9, 114.9,
111.7, 65.5, 47.1, 40.2, 28.5, 20.7;
1
3
3
H); C NMR (CDCl ) δ 170.5, 164.0,
53.8, 153.7, 137.1, 133.6, 129.9, 129.7,
29.5, 128.4, 128.3, 127.5, 125.5(q,
HRMS-ESI C28
6
H27NO calcd [M+H]
J
C-F=370), 123.8, 120.7, 113.4, 111.3,
6.3, 47.2, 41.0, 28.5, 20.2; HRMS-ESI
NO calcd [M+H] 510.1887,
found 510.1877.
474.1911, found 474.1912.
6
C
29
H
26
F
3
4
(E)-4-(3-(isobutylcarbamoyl)benzofuran
-2-yl)benzyl-3’-(4-hydroxyphenyl)
acrylate (Ia-23)
4
-(3-(isobutylcarbamoyl)benzofuran-2-y
Compound Ia-23 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
l)benzyl-2’-(3,4-dihydroxy-phenyl)aceta
te (Ia-22)
Compound Ia-22 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound
IIa
and
2-(4-hydroxyphenyl)acetic acid to obtain
the product as white solid (37.0 % yield);
0
1
compound
-(3,4-dihydroxy-phenyl)acetic acid to
obtain the product as white solid (23.2 % 7.85-7.91 (m, 2H), 7.60-7.69 (m, 2H),
IIa
and
m. p. 173-175 C. H NMR (DMSO-d
6
)
2
δ 10.02 (s, 1H), 8.60 (t, J = 5.8 Hz, 1H),
0
1
yield); m.p. 232-234 C. H NMR
DMSO-d ) δ 8.87 (s, 1H), 8.78 (s, 1H),
7.57 (d, J = 8.6 Hz, 2H), 7.54 (d, J =
2.35 Hz, 2H), 7.51 (s, 1H), 7.37 (t, J =
7.24 Hz, 1H), 7.32 (t, J = 7.4 Hz, 1H),
(
6
8
.60 (t, J = 5.6 Hz, 1H), 7.81-7.89 (m,

6
1
2
6
1
1
1
1
.74-6.79 (m, 2H), 6.46 (d, J = 15.65 Hz,
H), 5.23 (s, 2H), 3.11 (t, J = 6.4 Hz,
H), 1.83 (m, 1H), 0.89 (d, J = 6.6 Hz,
120.5, 116.7, 115.8, 114.8, 111.7, 65.9,
39.1, 31.4, 20.1, 14.1; HRMS-ESI
C
27
H
25NO
5
calcd [M+H] 444.1805,
13
H); C NMR (DMSO-d
6
) δ 166.9,
found 444.1807.
63.6, 160.4, 153.3, 152.0, 145.7, 138.2,
30.9, 129.2, 128.8, 128.0, 127.2, 125.9,
25.5, 124.1, 120.9, 116.2, 114.9, 114.2,
11.7, 65.3, 47.1, 28.5, 20.7;
4-(3-(butylcarbamoyl)benzofuran-2-yl)b
enzyl-3’,4’-dihydroxybenzoate (Ia-25)
Compound Ia-25 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
HRMS-ESI C29
H
27NO
5
calcd [M+H]
4
70.1962, found 470.1955.
compound
IIb
and
4
-(3-(butylcarbamoyl)benzofuran-2-yl)b
3,4-dihydroxybenzoic acid to obtain the
product as white solid (32.0 % yield);
enzyl-4’-hydroxybenzoate (Ia-24)
0
1
Compound Ia-24 was synthesized,
following a similar procedure as the
preparation of Ia-1 starting from
compound IIb and 4-hydroxybenzoic
acid to obtain the product as white solid
m.p. 187-189 C. H NMR (DMSO-d ) δ
6
9.83 (br. s., 1H), 9.39 (br. s., 1H), 8.55 (t,
J = 5.2 Hz, 1H), 7.89 (d, J = 7.8 Hz, 2H),
7.65 (d, J = 8.2 Hz, 1H), 7.52-7.61 (m,
3H), 7.28-7.41 (m, 4H), 6.80 (d, J = 8.2
Hz, 1H), 5.30 (s, 2H), 3.28 (m, 2H), 1.50
(m, 2H), 1.31 (m, 2H), 0.87 (t, J = 7.2
0
1
(
49.6 % yield); m.p. 158-160 C. H
NMR (DMSO-d ) δ 10.37 (br. s., 1H),
.55 (t, J = 5.6 Hz, 1H), 7.85 (d, J =
6
13
8
9
7
3
1
2
1
Hz, 3H); C NMR (DMSO-d ) δ 165.9,
6
.00 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H),
.65 (d, J = 7.8 Hz, 1H), 7.53 - 7.61 (m,
H), 7.34-7.40 (m, 1H), 7.28-7.34 (m,
H), 6.84 (d, J = 9.00 Hz, 2H), 5.33 (s,
H), 3.24-3.31 (m, 2H), 1.49 (m, 2H),
.31 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H);
163.4, 153.3, 152.1, 151.1, 145.6, 138.3,
129.2, 128.6, 128.0, 127.2, 125.9, 124.1,
122.4, 120.9, 120.7, 116.7, 115.8, 114.8,
111.7, 65.6, 39.1, 31.4, 20.1, 14.1;
HRMS-ESI C27
6
H25NO calcd [M+H]
460.1755, found 460.1754.
1
3
6
C NMR (DMSO-d ) δ 165.8, 163.4,
1
1
62.9, 153.3, 152.1, 138.2, 132.0, 129.2,
28.9, 128.0, 127.2, 125.9, 124.0, 120.9,
2-(4-((3-hydroxyphenoxy)methyl)
phenyl)-N-isobutylbenzofuran-3-carbox

amide (Ib-26)
following a similar procedure as the
preparation of Ib-26 starting from
compound IIa and 4-methylphenol to
obtain the product as white solid (82.3 %
To a solution of compound IIa (250 mg,
0
2
.62 mmol) in CH
3
CN (10 mL), 1,
-dihydroxybenzene (68.2 mg, 0.62
0
1
mmol) and anhydrous K CO (171.12
yield); m.p. 176-179 C. H NMR
2
3
mg, 1.24 mmol) were added and
refluxed overnight before cooled to
(DMSO-d ) δ 8.60 (t, J = 5.8 Hz, 1H),
6
7.88 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.4
Hz, 1H), 7.50- 7.60 (m, 3H), 7.35-7.40
(m, 1H), 7.29-7.34 (m, 1H), 7.02-7.10
(m, 2H), 6.85-6.92 (m, 2H), 5.10 (s, 2H),
3.12 (t, J = 6.4 Hz, 2H), 2.19 (s, 3H),
1.83 (m, 1H), 0.89 (d, J = 6.8 Hz, 6H);
2
room temperature. H O (10 mL) was
then added. The resulting solid was
filtered, washed with ethyl acetate, and
2
recrystallized in MeOH/H O to yield
compound Ib-26 as yellow solid (210
0
1
13
mg, 42.5 % yield). m.p. 201-203 C. H
NMR (DMSO-d ) δ 9.39 (s, 1H), 8.59
br. s., 1H), 7.89 (d, J = 7.8 Hz, 2H),
C NMR (DMSO-d ) δ 163.7, 156.5,
6
153.3, 152.1, 139.1, 130.3, 129.9, 129.0,
128.3, 128.1, 127.1, 125.8, 124.0, 120.9,
115.1, 114.8, 111.7, 69.1, 47.1, 28.5,
6
(
7
3
.65 (d, J = 7.8 Hz, 1H), 7.49 - 7.61 (m,
H), 7.28 - 7.42 (m, 2H), 7.03 (t, J = 7.8
20.7, 20.5; HRMS-ESI C27
3
H27NO calcd
Hz, 1H), 6.30 - 6.48 (m, 3H), 5.08 (br. s.,
H), 3.12 (br. s., 2H), 1.83 (m, 1H), 0.89
[M+H] 414.2064, found 414.2034.
2
1
3
(
d, J = 6.4 Hz, 6H);
DMSO-d ) δ 170.0, 163.7, 153.2, 152.1,
39.6, 137.9, 137.1, 130.1, 129.7, 128.4,
C NMR
2-(4-((4-chlorophenoxy)methyl)phenyl)-
N-isobutylbenzofuran-3-carboxamide(Ib
-28)
(
6
1
1
1
28.1, 127.3, 127.1, 125.8, 124.0, 120.8,
14.6, 111.6, 47.1, 36.5, 28.5, 20.7;
Compound Ib-28 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
compound IIa and 4-chlorophenol to
obtain the product as white solid (76.6 %
HRMS-ESI C H NO calcd [M+H]
2
6
25
4
4
16.1856, found 416.1832.
0
1
2-(4-((4-tolyloxy)methyl)phenyl)-N-isob
yield); m.p. 183-185 C. H NMR
utyl benzofuran-3-carboxamide (Ib-27)
Compound Ib-27 was synthesized,
(DMSO-d ) δ 8.59 (t, J = 5.8 Hz, 1H),
6
7.86-7.92 (m, 2H), 7.65 (d, J = 8.4 Hz,

1
3
1
7
7
7
H), 7.58 (d, J = 8.0 Hz, 1H),
.588-7.533 (d, J = 8.2 Hz, 2H),
.35-7.40 (m, 1H), 7.28-7.35 (m, 3H),
.00-7.05 (m, 2H), 5.14 (s, 2H), 3.11 (t,
6.4 Hz, 6H); C NMR (DMSO-d ) δ
6
163.7, 157.5, 153.3, 153.2, 152.1, 139.1,
135.3, 129.0, 128.4, 128.3, 128.2, 128.1,
127.1, 126.9, 125.8, 124.0, 120.9, 114.9,
114.8, 111.7, 69.1, 62.9, 47.1, 28.5, 20.7;
J = 6.4 Hz, 2H), 1.82 (m, 1H), 0.88 (d, J
13
=
6.4 Hz, 6H); C NMR (DMSO-d ) δ
HRMS-ESI C27
4
H27NO calcd [M+H]
6
1
1
1
1
63.6, 157.5, 153.3, 152.0, 138.5, 129.7,
29.6, 129.2, 128.4, 128.1, 127.2, 125.8,
25.0, 124.1, 120.9, 117.3, 117.0, 114.9,
11.7, 110.0, 69.4, 47.1, 28.5, 20.7;
430.2013, found 430.2045.
2-(4-((3-formylphenoxy)methyl)
phenyl)-N-isobutylbenzofuran-3-carbox
amide(Ib-30)
3
HRMS-ESI C26H24ClNO calcd [M+H]
4
34.1517, found 434.1536.
Compound Ib-30 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
2-(4-((4-(hydroxymethyl)phenoxy)
methyl)phenyl)-N-isobutylbenzofuran-3-
carboxamide (Ib-29)
compound
IIa
and
3-hydroxybenzaldehyde to obtain the
product as white solid (42.1 % yield);
Compound Ib-29 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
0
1
m.p. 166-168 C, H NMR (DMSO-d ) δ
6
9.95 (s, 1H), 8.60 (t, J = 5.6 Hz, 1H),
compound
-hydroxymethylphenol to obtain the
product as white solid (33.4 % yield);
IIa
and
7.90 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 8.2
Hz, 1H), 7.58 (d, J = 8.6 Hz, 3H), 7.49 -
7.54 (m, 3H), 7.31 - 7.39 (m, 3H), 5.24
(s, 2H), 3.11 (t, J = 6.4 Hz, 2H), 1.82 (m,
4
0
1
m.p. 203-205 C. H NMR (DMSO-d
.60 (t, J = 5.8 Hz, 1H), 7.88 (d, J = 8.2
Hz, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.52 -
6
) δ
13
8
1H), 0.88 (d, J = 6.4 Hz, 6H); C NMR
(DMSO-d ) δ 193.3, 163.6, 159.1, 153.3,
6
7
1
2
1
.60 (m, 3H), 7.37 (m, 1H), 7.32 (m,
H), 7.18 - 7.23 (m, 2H), 6.93 - 6.97 (m,
H), 5.13 (s, 2H), 5.03 (t, J = 5.6 Hz,
H), 4.38 (d, J = 5.4 Hz, 2H), 3.11 (t, J
152.0, 138.4, 138.1, 130.9, 129.2, 128.4,
128.1, 127.2, 125.9, 124.1, 123.3, 122.1,
120.9, 114.9, 114.4, 111.7, 69.4, 47.1,
28.5, 20.7; HRMS-ESI C27
4
H25NO calcd
=
6.4 Hz, 2H), 1.83 (m, 1H), 0.89 (d, J =
[M+H] 428.1856, found 428.1831.

following a similar procedure as the
preparation of Ib-26 starting from
compound IIa and 4-cyanophenol to
obtain the product as white solid (51.0 %
2
-(4-((3-acetylphenoxy)methyl)phenyl)-
N-isobutylbenzofuran-3-carboxamide
Ib-31)
(
0
1
Compound Ib-31 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
compound IIa and 3-acetylphenol to
yield); m.p. 176-178 C. H NMR
(DMSO-d ) δ 8.60 (t, J = 6.0 Hz, 1H),
6
7.905 - 7.884 (d, J = 8.4 Hz, 2H), 7.774
- 7.752 (d, J = 8.8 Hz, 2H), 7.662 -
obtain the product as white solid (47.0 % 7.642 (d, J = 8.0 Hz, 1H), 7.589 - 7.554
0
1
yield); m.p. 168-170 C. H NMR
DMSO-d ) δ 8.60 (t, J = 5.8 Hz, 1H),
.90 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 8.2
Hz, 1H), 7.58 (d, J = 8.6 Hz, 3H), 7.52 -
(m, 3H), 7.302 - 7.396 (m, 2H), 7.190 -
7.167 (d, J = 9.2 Hz, 1H), 5.251 (s, 2H),
3.11 (t, J = 6.4 Hz, 2H), 1.82 (m, 1H),
(
6
7
1
3
0.88 (d, J = 6.4 Hz, 6H); C NMR
7
7
7
6
0
.56 (m, 2H), 7.43 (t, J = 7.8 Hz, 1H),
.35 - 7.40 (m, 1H), 7.33 (m, 1H), 7.26 -
.31 (m, 1H), 5.23 (s, 2H), 3.11 (t, J =
.4 Hz, 2H), 2.55 (s, 3H), 1.82 (m, 1H),
(DMSO-d ) δ 163.6, 162.1, 153.3, 152.0,
6
137.9, 134.7, 129.4, 128.6, 128.0, 127.2,
125.9, 124.1, 120.9, 119.5, 116.4, 115.0,
111.7, 103.6, 69.6, 47.1, 28.5, 20.7;
13
.88 (d, J = 6.8 Hz, 6H); C NMR
DMSO-d ) δ 198.1, 163.6, 158.8, 153.3,
HRMS-ESI C27
24 2 3
H N O calcd [M+H]
(
425.1860, found 425.1879.
6
1
1
1
2
52.0, 138.7, 138.6, 130.4, 129.2, 128.4,
28.1, 127.2, 125.9, 124.1, 121.5, 120.9,
20.4, 114.9, 114.2, 111.7, 69.3, 47.1,
8.5, 27.3, 20.7, 47.1, 28.5, 20.7;
N-isobutyl-2-(4-((4-(methylsulfonyl)
phenoxy)methyl)phenyl)benzofuran-3-c
arboxamide (Ib-33)
HRMS-ESI C28
H
27NO
4
calcd [M+H]
Compound Ib-33 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
442.2013, found 442.2045.
2-(4-((4-cyanophenoxy)methyl)phenyl)-
compound
IIa
and
N-isobutylbenzofuran-3-carboxamide
Ib-32)
Compound Ib-32 was synthesized,
4-(methylsulfonyl)phenol to obtain the
product as white solid (60.2 % yield);
(
0
1
m.p. 203-205 C. H NMR (DMSO-d ) δ
6

8.60 (t, J = 5.6 Hz, 1H), 7.911-7.891 (d,
2H), 2.00 (s, 3H), 1.83 (m, 1H), 0.89 (d,
1
3
J = 8.0 Hz, 2H), 7.8430 - 7.8226 (d, J =
J = 6.6 Hz, 6H); C NMR (DMSO-d ) δ
6
8
1
7
.4 Hz 2H), 7.664-7.644 (d, J = 8.0 Hz,
H), 7.588 - 7,569 (d, J = 7.6 Hz, 3H),
.395 - 7.303 (m, 2H), 7.243 - 7.221 (d,
168.8, 163.7, 158.9, 153.3, 152.1, 141.0,
138.9, 129.9, 129.1, 128.3, 128.1, 127.1,
125.8, 124.0, 120.9, 114.8, 112.0, 111.7,
110.0, 109.5, 106.2, 69.0, 47.1, 28.5,
J = 8.8 Hz, 2H), 5.27 (s, 2H), 3.131 -
3.110 (m, 5H), 1.83 (m, 1H), 0.89 (d, J
28 2 4
24.5, 20.7; HRMS-ESI C28H N O
13
=
6.8 Hz, 6H); C NMR (DMSO-d
6
) δ
calcd [M+H] 457.2122, found 457.2100.
1
1
1
4
63.6, 162.4, 153.3, 152.0, 138.0, 133.3,
29.7, 129.3, 128.5, 128.0, 127.2, 125.9,
24.1, 120.9, 115.8, 114.9, 111.7, 69.6,
7.1, 44.4, 28.5, 20.7; HRMS-ESI
2-(4-((4-acetamidophenoxy)methyl)
phenyl)-N-isobutylbenzofuran-3-carbox
amide(Ib-35)
C
27
H
27NO
5
S calcd [M+H] 478.1683,
Compound Ib-35 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
compound IIa and 4-acetamidophenol to
obtain the product as white solid (49.0 %
found 478.1661.
2-(4-((3-acetamidophenoxy)methyl)
phenyl)-N-isobutylbenzofuran-3-carbox
amide(Ib-34)
0
1
yield); m.p. 158-160 C. H NMR
Compound Ib-34 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
compound IIa and 3-acetamidophenol to
(DMSO-d ) δ 9.90 (br. s., 1H), 8.60 (br.
6
s., 1H), 7.89 (d, J = 8.2 Hz, 2H), 7.65 (d,
J = 7.8 Hz, 1H), 7.49 - 7.61 (m, 3H),
7.28 - 7.41 (m, 3H), 7.16 (t, J = 8.0 Hz,
obtain the product as white solid (45.0 % 1H), 7.07 (d, J = 7.8 Hz, 1H), 6.68 (d, J
0
1
yield); m.p. 166-168 C. H NMR
DMSO-d ) δ 9.91 (br. s., 1H), 8.60 (t, J
5.6 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H),
= 7.4 Hz, 1H), 5.10 (s, 2H), 3.12 (t, J =
(
6
6.0 Hz, 2H), 2.00 (s, 3H), 1.83 (m, 1H),
1
3
=
0.89 (d, J = 6.4 Hz, 6H); C NMR
7
3
.65 (d, J = 7.8 Hz, 1H), 7.52 - 7.60 (m,
H), 7.29 - 7.40 (m, 3H), 7.17 (t, J = 8.2
(DMSO-d ) δ 168.8, 163.7, 158.9, 153.3,
6
152.1, 141.0, 138.9, 129.9, 129.1, 128.3,
Hz, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.68
m, 1H), 5.10 (s, 2H), 3.12 (t, J = 6.4 Hz,
128.1, 127.2, 125.8, 124.0, 120.9, 114.8,
112.0, 111.7, 109.5, 106.2, 69.0, 47.1,
(

2
8.5,
24.5,
20.7;
HRMS-ESI
Compound Ib-37 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
C H N O calcd [M+H] 457.2122,
28
28
2
4
found 457.2178.
compound
IIb
and
N-butyl-2-(4-((4-(methoxycarbonyl)
phenoxy)methyl)phenyl)benzofuran-3-c
arboxamide (Ib-36)
4-(methylsulfonyl)phenol to obtain the
product as white solid (61.0 % yield);
0
1
m.p. 234-236 C. H NMR (DMSO-d ) δ
6
Compound Ib-36 was synthesized,
following a similar procedure as the
preparation of Ib-26 starting from
8.56 (t, J = 5.6 Hz, 1H), 7.90 (d, J = 8.2
Hz, 2H), 7.81 - 7.86 (m, 2H), 7.65 (d, J
= 7.8 Hz, 1H), 7.58 (d, J = 8.2 Hz, 3H),
7.34 - 7.40 (m, 1H), 7.29 - 7.34 (m, 1H),
7.20 - 7.26 (m, 2H), 5.28 (s, 2H), 3.28
compound
IIb
and
4-(methoxycarbonyl)phenol to obtain
the product as white solid (37.0 % yield); (m, 2H), 3.13 (s, 3H), 1.50 (m, 2H), 1.26
0
1
m.p. 168-170 C. H NMR (DMSO-d
.60 (t, J = 5.4 Hz, 1H), 7.90 (d, J = 8.2
Hz, 4H), 7.65 (d, J = 7.8 Hz, 1H), 7.53 -
.61 (m, 3H), 7.28 - 7.42 (m, 2H), 7.12
d, J = 8.6 Hz, 2H), 5.23 (s, 2H), 3.78 (s,
6
) δ
- 1.38 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H);
1
3
8
6
C NMR (DMSO-d ) δ 163.4, 162.4,
153.3, 152.0, 138.0, 133.3, 129.7, 129.3,
128.5, 128.0, 127.2, 125.9, 124.1, 120.9,
115.8, 114.9, 111.7, 69.7, 44.4, 39.1,
7
(
3
1
H), 3.12 (t, J = 6.4 Hz, 2H), 1.83 (m,
31.4,
20.1,
14.1;
HRMS-ESI
13
H), 0.88 (d, J = 6.6 Hz, 6H); C NMR
) δ 166.3, 163.6, 162.5, 153.3,
27 5
C H27NO S calcd [M+H] 478.1683,
(DMSO-d
6
found 478.1651.
1
1
1
2
52.0, 138.2, 131.7, 129.3, 128.5, 128.1,
27.2, 125.9, 124.1, 122.6, 120.9, 115.3,
14.9, 111.7, 110.0, 69.5, 52.3, 47.1,
8.5, 20.7; HRMS-ESI C H NO calcd
3.2 Biological assays
3.2.1 Purification of Srt A Protein
The Srt A protein was purified
2
8
27
5
2
5
[
M+H] 458.1962, found 458.1921.
according to the known method. The
pET28-SrtA△59 was transformed into
Escherichia coli strain BL21 (Novagen).
The pET28-SrtA59 transformed cells
were grown in 1 L of Luria broth media
N-butyl-2-(4-((4-(methylsulfonyl)pheno
xy)methyl)phenyl)benzofuran-3-carboxa
mide (Ib-37)

at 37 °C until the OD600 reached 0.6.
The culture was then induced with 1
µM) was breifly pre-incubated with
varying concentrations of inhibitor
(dissolved in DMSO, final concentration
of 12.5~200 µM) in 96-well plates
containing assay buffer (150 mM NaCl,
mM
isopropyl
(IPTG;
β-D-1-thiogalactopyranoside
Invitrogen, Carlsbad, CA) and grown for
another 8 h at 37 °C. The cells were
harvested and incubated on ice for 30
2
5 mM CaCl , 50 mM Tris-HCl, 0.05%
Tween 20, pH 7.5) at 37 0C for 1 h.
Reactions were then initiated by adding
min with lysis buffer (50 mM NaH
2
PO
00 mM NaCl, 20 mM imidazole, pH
.0) containing 1 mg/mL lysozyme.
4
,
3
8
substrate
Abz-LPATG-Dnp
(final
concentration 10 µM). Reactions were
performed in a total volume of 100 µL
with all reagents. 30 min later, the
reaction was quenched by adding 50 µL
HCl (1 M). 20 µL quenched reaction
mixture was then injected onto a reverse
phase Agilent XDB-C 18(4.6×150 mm,
3.5 µm) HPLC column. The reaction
components were separated using a
linear gradient from 20% to 60%
acetonitrile/0.1% trifluoroacetic acid
applied over a period of 25 mins. For
each concentration, the area percentage
of G-Dnp compared with those in the
absence of inhibitor was calculated at
After a brief sonication, the lysate was
centrifuged and the supernatant was
applied to 0.8 mL of Ni-NTA agarose
beads pre-equilibrated with lysis buffer
(Qiagen, Madison, WI). After washing
off the unbound contaminating proteins,
the His6-tagged protein was eluted with
2 4
elution buffer (50 mM NaH PO , 300
mM NaCl, 250 mM imidazole, pH 8.0).
All samples were analyzed using
Coomassie Plus Protein Assay (Pierce
Biotechnology, Rockford, IL) measuring
the absorbance at wavelength 595 nm to
determine the protein concentration.
3
55 nm. IC50 values were calculated as
3
.2.2 Inhibition assays
the concentration associated 50%
decrease of the area percentage of
G-Dnp. Each experiment was repeated
three times.
The activities of compounds were tested
using previously reported high
performance liquid chromatography
HPLC) assay.23, 24 Purified Srt A (5
(