Discovery of benzofuran-3(2H)-one derivatives as novel DRAK2 inhibitors that protect islet β-cells from apoptosis

Article abstract of DOI:10.1016/j.ejmech.2017.02.048

Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is a serine/threonine kinase that plays a key role in a wide variety of cell death signaling pathways. Inhibition of DRAK2 was found to efficiently protect islet β-cells from apoptosis and hence DRAK2 inhibitors represent a promising therapeutic strategy for the treatment of diabetes. Only very few chemical entities targeting DRAK2 are currently known. We carried out a high throughput screening and identified compound 4 as a moderate DRAK2 inhibitor with an IC₅₀value of 3.15?μM. Subsequent SAR studies of hit compound 4 led to the development of novel benzofuran-3(2H)-one series of DRAK2 inhibitors with improved potency and favorable selectivity profiles against 26 selected kinases. Importantly, most potent compounds 40 (IC₅₀?=?0.33?μM) and 41 (IC₅₀?=?0.25?μM) were found to protect islet β-cells from apoptosis in dose-dependent manners. These data support the notion that small molecule inhibitors of DRAK2 represents a promising strategy for the treatment of diabetes.

Full text of DOI:10.1016/j.ejmech.2017.02.048

European Journal of Medicinal Chemistry 130 (2017) 195e208
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of benzofuran-3(2H)-one derivatives as novel DRAK2
inhibitors that protect islet -cells from apoptosis
b
,
,
, c
Sheng Wang a 1, Lei Xu b 1, Yu-Ting Lu b, Yu-Fei Liu a, Bing Han d, Ting Liu a, Jie Tang a
,
Jia Li b, Jiangping Wu d ****, Jing-Ya Li b ***, Li-Fang Yu a **, Fan Yang a
,
,
,
, *
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China
Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
b Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
c Shanghai Key Laboratory of Green Chemistry and Chemical Process, School of Chemistry and Molecular Engineering, East China Normal University, 3663
North Zhongshan Road, Shanghai 200062, China
d
ꢀ
ꢀ
ꢀ
ꢀ
Laboratory of Immunology and Cardiovascular Research, Centre Hospitalier de l'Universite de Montreal, 900 rue St-Denis, Montreal, Quebec, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is a serine/threonine ki-
nase that plays a key role in a wide variety of cell death signaling pathways. Inhibition of DRAK2 was
found to efficiently protect islet b-cells from apoptosis and hence DRAK2 inhibitors represent a prom-
ising therapeutic strategy for the treatment of diabetes. Only very few chemical entities targeting DRAK2
are currently known. We carried out a high throughput screening and identified compound 4 as a
Received 11 January 2017
Received in revised form
17 February 2017
Accepted 18 February 2017
Available online 21 February 2017
moderate DRAK2 inhibitor with an IC50 value of 3.15 mM. Subsequent SAR studies of hit compound 4 led
to the development of novel benzofuran-3(2H)-one series of DRAK2 inhibitors with improved potency
and favorable selectivity profiles against 26 selected kinases. Importantly, most potent compounds 40
Keywords:
DRAK2 inhibitor
Diabetes
Islet b-cell protection
Apoptosis
(IC50 ¼ 0.33
m
M) and 41 (IC50 ¼ 0.25
mM) were found to protect islet b-cells from apoptosis in dose-
dependent manners. These data support the notion that small molecule inhibitors of DRAK2 repre-
sents a promising strategy for the treatment of diabetes.
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
regulation of signal transduction pathways [2].
DRAK2 is constitutively expressed in multiple tissues and or-
Death-associated protein kinase-related apoptosis-inducing
kinase-2 (DRAK2, also known as STK17B) is a serine/threonine ki-
nase, belonging to the family of death-associated protein kinase
(DAPK). DAPK plays a key role in a wide variety of cell death
signaling pathways. In addition to DRAK2, there are four other DAP
kinase family members identified to date, including DAPK1, DAPK2,
DAPK3, and DRAK1 [1]. The sequence homology of the DAPK family
members is restricted to the N-terminal kinase domain, while the
C-terminal kinase domains are diverse and relate to their different
gans, such as the thymus, suprachiasmatic nuclei, pituitary, olfac-
tory lobes, adrenal gland medulla, stomach, skin and testes [3]. It is
also rapidly induced in islet b-cells by inflammatory cytokines [4]
and free fatty acids [5]. It is conceivable that such induced
expression could occur in other types of cells or tissues. Therefore,
the initial notion that DRAK2 has T cell-specific expression is
incorrect. Inflammatory cytokines are pathogenic in type I diabetes.
We have demonstrated that transgenic DRAK2 overexpression
along with the cytokine assaults leads to aggravated apoptosis of
cells [4]. Free fatty acids (FFA) cause insulin resistance and islet
b
b
-
-
cell apoptosis in type II diabetes. DRAK2 overexpression increases
-cell apoptosis after FFA stimulation [5]. Conversely, the cytokine
and FFA-induced -cell apoptosis can be inhibited by DRAK2 small
interfering RNA (siRNA) [4,5]. Our in vivo studies confirm that the
DRAK2 transgenic mice are prone to low-dose streptozotosin-
induced diabetes (a type I diabetes model) [4] and high-fat diet-
induced glucose intolerance (a type II diabetes model) [5]. In
b
* Corresponding author.
** Corresponding author.
*** Corresponding author.
**** Corresponding author.
b
E-mail addresses: jianping.wu@umontreal.ca (J. Wu), jyli@simm.ac.cn (J.-Y. Li),
lfyu@sat.ecnu.edu.cn (L.-F. Yu), fyang@chem.ecnu.edu.cn (F. Yang).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2017.02.048
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

196
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
consistent with our findings in transgenic mice, DRAK2 deficient
mice are less prone to type I diabetes [3]. Collectively, these results
indicate that DRAK2 inhibition can improve islet survival and
DRAK2 is a therapeutic target for early stage type I and type II
diabetes.
reaction with 3,4-dihydroxybenzaldehyde in the presence of po-
tassium carbonate to afford compound 26. Condensation of the
benzyl protected 3,4-dihydroxybenzaldehyde (28) [16] with com-
pound 6 and subsequent hydrogenation reaction afforded com-
pound 29.
Only very few inhibitors targeting DRAK2 have been reported to
date. SC82510 was reported to be a DRAK2 inhibitor that induced
axon branching of adult sensory neurons at a low concentration of
1 nM [6]. The thieno [2,3-b]pyridine (1) were identified as dual
The synthetic routes for 4- or 5-substituted 2-
benzylidenebenzofuran-3(2H)-one derivatives were listed in
Scheme 2. 4-Methoxybenzofuran-3(2H)-one (32) was obtained
from commercially available 1-(2,6-dihydroxyphenyl) ethan-1-one
(30) via reported cyclization and methylation reactions [17,18].
Compound 33 was obtained via condensation reaction of 32 with 3-
ethoxy-4-hydroxybenzaldehyde in the presence of aluminum oxide
in dichloromethane at room temperature. Similarly, 5-
methoxybenzofuran-3(2H)-ones (40e43) were obtained from 5-
methoxy, 5-hydroxy, or 5-OAc substituted benzofuran-3(2H)-ones
(37e39) in the presence of aluminum oxide in dichloromethane or
potassium hydroxide in methanol and water under reflux condi-
tion. Compound 39 was obtained from 38 under a standard acyla-
tion conditions. Compounds 37 and 38 were synthesized from
commercially available 4-methoxyphenol 34 in 2e3 steps utilizing
reported procedures. Likewise, compounds 45e47 were synthe-
sized from the corresponding 4-ethoxyphenol, 4-butoxyphenol,
and 4-methyphenol. 4-Substituted anisole (48e52) underwent a
Friedel-Crafts acylation with chloroacetyl chloride in the presence
of aluminum chloride followed by cyclization with alkali to give 5-
substituted benzofuran-3(2H)-ones (53e57) [19]. Nitro compound
57 was reduced with iron powder in glacial acetic acid to afford
aniline 58 [10]. Condensation reaction of compounds 53e56, 58
with 3-ethoxy-4-hydroxybenzaldehyde afforded final compounds
59e63 respectively.
The procedures for the synthesis of 6- and 7-substituted 2-
benzylidenebenzofuran-3(2H)-ones were listed in Scheme 3. 6-
Hydroxybenzofuran-3(2H)-one (65) was synthesized from
commercially available resorcinol (64) using a reported procedure
[20]. Subsequent methylation and condensation reaction with 3-
ethoxy-4-hydroxybenzaldehyde or 3,4-dihydroxybenzaldehyde in
the presence of aluminum oxide in dichloromethane or potassium
hydroxide in methanol and water under reflux condition afforded
compounds 67 and 68. Compounds 73 and 74 were synthesized
from commercially available 1-(4-fluoro-2-hydroxyphenyl)ethan-
1-one (69) and 1-(4-chloro-2-hydroxyphenyl)ethan-1-one (70)
utilizing similar procedures as those for compound 7. Friedel-Crafts
acylation of carboxylic acid 76 [21], followed by a condensation
reaction with 3-ethoxy-4-hydroxybenzaldehyde afforded (Z)-2-(3-
ethoxy-4-hydroxybenzylidene)-7-methoxybenzofuran-3(2H)-one
(78).
DRAK1 and DRAK2 inhibitors with IC50 values of 2.25 and 0.82 mM
respectively (Fig. 1) [7]. 5-Arylthieno [2,3-b]pyridine (2) was re-
ported to be a more potent inhibitor of DRAK2 with an IC50 value of
29 nM. Meanwhile, compound 2 was also a strong binder of DRAK1
(Kd ¼ 99 nM) and DAPK1 (Kd ¼ 54 nM) [8]. The indirubin 3 was
recently reported as a high-efficient DRAK2 inhibitor with an IC50
value of 3 nM, while showing a high selectivity over the other 4
kinase members in DAPK family and selected protein kinases, such
as, tyrosine-protein kinase Met (c-MET), anaplastic lymphoma ki-
nase (ALK), activated CDC42 kinase 1 (Ack1), maternal embryonic
leucine zipper kinase (MELK), inwardly rectifying potassium
channels IRK, and epidermal growth factor receptor (EGFR) [9].
In our efforts to discover novel DRAK2 inhibitors, we identified
2-(3,4-dihydroxybenzylidenebenzofuran-3(2H)-one (4) which dis-
plays a moderate DRAK2 inhibitory activity with an IC50 value of
3.15 mM. Compound 4 belongs to the family of aurones which have
been shown to have a wide range of biological and pharmacological
activities. With the aim to develop potent and selective novel
DRAK2 inhibitors, we carried out structure-activity relationship
(SAR) studies. Selected compounds were further assessed for their
selectivity against 26 kinases and effects on the glucose-stimulated
insulin secretion of primary islets.
1.1. Chemistry
The synthetic routes for compounds 4, 7e20, 23, 26, and 29
were described in Scheme 1. Benzofuran-3(2H)-one (6) was syn-
thesized from commercially available hydroxyacetophenone (5)
using a reported procedure [10]. Aldol condensation reaction of 6
with appropriate substituted benzaldehyde in the presence of
aluminum oxide [11] or sodium hydroxide [12] afforded com-
pounds 7e20. Indan-1-one 23 was synthesized in two steps ac-
cording to the reported procedure using 3-phenylpropanoic acid
[13,14]. 1-(3,4-Dihydroxyphenyl)ethan-1-one (24) was bromi-
nated to afford compound 25 [15] which underwent cyclization
All final 2-benzylidenebenzofuran-3(2H)-one compounds were
prepared via condensation reactions of benzofuran-3(2H)-ones
with benzaldehydes in the presence of aluminum oxide in
dichloromethane, or potassium hydroxide in methanol and water.
These conditions are prone to yield mainly the Z isomers according
to the literature [22e27]. The
d values of olefinic carbon for Z
configurations determined by 13C NMR spectroscopy were reported
around 111 ppm, while the olefinic carbon for E configurations were
generally between 120 and 130 ppm. In the case of 2-
benzylidenebenzofuran-3(2H)-ones presented in this work,
d
values for olefinic carbons were 101e114 ppm, suggesting Z
geometries.
2. Results and discussion
All final compounds were initially screened using a DRAK2-GST
autophosphorylation assays for their DRAK2 inhibitory activities
and their IC50 values were listed in Tables 1 and 2. Keeping the 2-
benzylidenebenzofuran-3(2H)-one skeleton intact, we first
Fig. 1. Structures of selected DRAK2 inhibitors.

S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
197
Scheme 1. Reagents and conditions: (a) 1) CuBr2, CHCl3/EtOAc (1:1), reflux, 10 h; 2) Et3N, CH3CN, rt, 5 h; (b) Al2O3, CH2Cl2, rt, 5 h; (c) KOH, MeOH, H2O, reflux, 3 h; (d) TfOH, CH2Cl2,
0
ꢀC to rt, 6 h; (e) 3,4-dihydroxybenzaldehyde, NaOH, MeOH, rt, 5 h (f) CuBr2, EtOAc, reflux, 5 h; (g) 2-hydroxybenzaldehyde, K2CO3, acetone, reflux, 20 h; (h) BnBr, K2CO3, DMF,
70 ꢀC, 6 h; (i) 1) benzofuran-3(2H)-one, Al2O3, CH2Cl2, rt, 5 h; 2) cat. Pd/C, H2, MeOH, rt, 5 h.
focused on assessing the effects of substitutions on the benzene
ring. Deletion of 30-hydroxyl (8) or both 30- and 40-hydroxyl groups
(7) resulted in an 18- or 8-fold decreased in inhibitory activities
compared to hit compound 4. Methylation of 40-hydroxyl and 30,40-
dihydroxyl groups resulted in a 4e7-fold decrease in activity,
substituents at the benzofuran-3(2H)-one ring on DRAK2 inhibition
(Table 2). A 4-methoxy substitution yielded a 2-fold reduction of
activity compared to the unsubstituted compound 14, while
introduction of a methoxy group at 5-, 6-, and 7-position (40, 41, 68,
and 78) of benzofuran-3(2H)-one resulted in an increase in activ-
indicating the importance of the hydrogen bond donor at position
40. Alkylation of the 30-hydroxyl group with methyl (10) or ethyl
(14) maintained the activity, while bulkier groups such as n-propyl,
i-propyl, n-butyl, as seen in compounds 15e17, were found to be
detrimental to the activity compared to smaller groups. Replace-
ment of 40-hydroxyl with fluoro or hydroxymethyl decreased the
inhibitory activity against DRAK2. A decrease of activity was also
found when the 3,4-dihydroxybenzyl moiety was replaced by other
heterocycles such as 30-pyridyl (19) and 20-furyl (20).
We next looked at the effects of linker part on the inhibitory
activity. Replacing the oxygen of benzofuran-3(2H)-one compound
4 with CH2 afforded a weaker inhibitor 23 with a 4-fold drop in IC50
value. Benzofuran-2-methanone (26) and 2-(3,4-dihydroxybenzyl)
benzofuran-3(2H)-one (29) showed weak inhibitory activity with
ities (IC50 ¼ 0.25e2.84
m
M) with compounds 40 and 41 displaying
the best activities with IC50 values of 0.33 and 0.25
mM. More EDGs
and EWGs at 5-position were next accessed including OH (42),
OCOCH3 (43), OCH2CH3 (45), OnBu (46), CH3 (47), F (59), Cl (60), Br
(61), and I (62). EDGs were generally found to be beneficial to the
activities with the exception of amino compound 63 which showed
a 2-fold decrease in activity compared to the corresponding
unsubstituted compound 14, indicating the lack of importance of
hydrogen bond donors at this position. Increasing steric bulk from
methoxy to ethoxy and acetoxy resulted in submicromolar in-
hibitors (41, 43, 45) but the n-butoxy analog was found to be 10-
fold less potent, with an IC50 value of 3.66 mM. The same trend
was observed in the 5-halogen substituted analogs 59e62, where
the iodo-substituted compound 62 was the least potent among
them and the 5-fluoro substituted compound 59 showing a 3-fold
IC50 values over 20
mM. We then examined the effects of electron
withdrawing group (EWG) and electron donating group (EDG)
increase in activity (IC50 ¼ 1.05
mM). It was found that as the

198
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
Scheme 2. Reagents and conditions: (a) 1) (CH3)3SiCl, Lithium diisopropylamide (LDA), THF, ꢁ78 ꢀC, 4 h; 2) NBS, ꢁ78 ꢀC to rt, 1.5 h; 3) NaOH, H2O, rt, overnight; (b) CH3I, K2CO3,
DMF, 80 ꢀC, 3 h; (c) 3-ethoxy-4-hydroxybenzaldehyde, Al2O3, CH2Cl2, rt, 5 h; (d) BCl3/CH2Cl2, ClCH2CN, AlCl3, 0 ꢀCe25 ꢀC, 6 h; (e) BBr3/CH2Cl2, CH2Cl2, ꢁ78 ꢀCe0 ꢀC, 2 h; (f) AcONa,
EtOH, reflux, 2 h; (g) Ac2O, Et3N, CH2Cl2, rt, 1 h; (h) 3,4-dihydroxybenzaldehyde, KOH, MeOH, H2O, reflux, 3 h; (i) 1) ClCH2COCl, AlCl3, CH2Cl2, 25 ꢀCe40 ꢀC to 25 ꢀC, 72 h; 2) AcONa,
EtOH, reflux, 2 h; (j) Fe, AcOH, 70 ꢀC, 3 h.
electron-donating ability goes up (OCH3 > OCOCH3 > CH3), the
inhibitory activities against DRAK2 also increased in sequence
(40 > 43 > 47). At 6-position, the EWGs F and were found to be
selectivity for DRAK2 over the other four kinases (>4 fold for
compound 40; >23 fold for compound 41), while compound 67
showed extensive inhibitory activities against the other kinases of
DAPK family.
detrimental to the activities with IC50 values of 60 and 40
mM.
Respectively, while the 6-methoxy compounds 67 and 68 main-
tained activities (IC50 values of 1.02 and 2.44 mM).
We further investigated the selectivity of compounds 40, 41, and
67 against protein kinases available in house (Table 3, including B
lymphoid tyrosine kinase (BLK), IL2-inducible T-cell kinase (ITK),
tunica interna endothelial cell kinase (TEK), Janus kinase (JAK),
spleen tyrosine kinase (SYK), checkpoint kinases 1 (CHK1), v-akt
murine thymoma viral oncogene homolog 1 (AKT1), Aurora kinase
A (Aurora-A), EGFR, Bruton agammaglobulinemia tyrosine kinase
(BTK), cyclin-dependent kinase (CDK), maternal embryonic leucine
zipper kinase (MELK), inhibitor of nuclear factor kappa-B kinase
2.1. Selectivity studies at 26 kinases
DRAK2 belongs to DAPK family, which consists of DAPK1 DAPK2,
DAPK3, DRAK1 and DRAK2. Most potent compounds 40, 41, and a
natural product sulphuretin (67), were selected to evaluate their
ability to inhibit DRAK1, DAPK1, DAPK2, and DAPK3 in enzyme
assays. As shown in Table 3, compound 1 showed equal potency for
subunit beta (IKK
b
), glycogen synthase kinase 3 beta (GSK3
b),
mitogen-activated protein kinase kinase
1
(MEK1), mitogen-
both DRAK1 (IC50 ¼ 1.72
m
M) and DRAK2 (IC50 ¼ 1.31
mM) with very
activated protein kinase 3 (ERK1). Sulphuretin 67 was found to be
a highly promiscuous compound with extensive activities against
these protein kinases. Compound 41 showed moderate inhibitory
weak inhibitory activities if any, for DAPK1, DAPK2, and DAPK3,
which were consistent to the reported data [7]. Compounds 40 and
41 showed similar selectivity profiles with moderate to good

S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
199
Scheme 3. Reagents and conditions: (a) 1) ClCH2CN, ZnCl2, HCl (g), Et2O, 4 h; 2) H2O, reflux, 5 h; 3) AcONa, EtOH, reflux, 2 h; (b) CH3I, K2CO3, DMF, 80 ꢀC, 3 h; (c) 3-ethoxy-4-
hydroxybenzaldehyde, Al2O3, CH2Cl2, rt, 5 h; (d) 3,4-dihydroxybenzaldehyde, KOH, MeOH, H2O, reflux, 3 h; (e) 1) CuBr2, CHCl3/EtOAc (1:1), reflux, 10 h; 2) Et3N, CH3CN, rt, 5 h;
(f) ClCH2COOH, NaH, DMF, 0 ꢀC to rt, 12 h; (g) 1) SOCl2, reflux; 2) AlCl3, CH2Cl2, 0 ꢀC to rt, 1.5 h.
Table 1
The inhibitory activities of compounds 1, 4, 7e20, 23, 26, and 29 against DRAK2.a
Compound
R1
R2
R3
DRAK2 IC50, mM
1
4
7
e
e
e
H
H
1.31 0.08
3.08 0.40
24.64 8.65
OH
H
OH
H
8
9
H
H
OMe
OH
OMe
OMe
OEt
OnPr
OiPr
OnBu
H
OH
CH2OH
OH
OMe
OMe
OH
OH
OH
OH
OH
F
H
H
H
H
H
OMe
H
H
H
H
H
e
e
e
e
e
53.03 10.46
11.09 1.45
3.11 1.90
12.10 2.19
21.43 0.28
6.65 1.77
10
11
12
13
14
15
16
17
18
19
20
23
26
29
3.24 0.05
5.37 0.17
17.39 1.95
29.10 9.02
40.55 14.37
39.39 8.85
63.96 14.21
13.29 1.59
22.91 1.05
27.81 1.99
3-pyridyl
2-furyl
e
e
e
e
e
e
e
e
a
See Experimental Section.
activities against BLK, ITK, TEK, and Aurora-A, with IC50 values of
0.5e5 M. Compound 40 showed greatest selectivity among three
tested compounds with relatively weak inhibitory activities against
BLK, Aurora-A, BTK, IKK
5 mM.
b, and GSK3b, with IC50 values no less than
m

200
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
Table 2
as a kind of free fatty acid, caused mitochondria functional
impairment then induced -cell dysfunction and apoptosis via ER
stress [28,29]. And previously studies shown that palmitate-
induced
-cell death was caused by Ca2þ influx; the Ca2þ would
trigger apoptotic signals subsequently [30]. Palmitate lipotoxicity
mediated cell death involving caspase activation and sub-
sequentially cell failure [31]. When human islets were cultured
The inhibitory activities of compounds 33, 40e43, 45e47, 59e63, 67, 68, 73, 74, and
b
78 against DRAK2 kinase.a
b
b
b
with palmitate together, the intracellular insulin content would
decreased as the extension of time and cleaved-caspase 3 increased
Compound
R1
R2
DRAK2 IC50, mM
in the process [32]. Thus palmitate would induce
and damaged insulin secretion capacity of cell. When treated is-
lets with FFA, the mRNA level and protein level of DRAK2 signifi-
cantly increased and promotes the apoptosis of cell; further more
cell apoptosis were aggravated in DRAK2 transgenic mice islets. If
b cell apoptosis
1
e
e
1.31 0.80
7.26 3.93
0.33 0.10
0.25 0.01
0.99 0.07
0.69 0.23
0.41 0.02
3.66 0.20
0.96 0.12
1.05 0.06
13.71 0.92
28.15 4.61
42.52 4.09
7.16 2.23
1.02 0.06
2.44 0.56
b
33
40
41
42
43
45
46
47
59
60
61
62
63
67
68
73
74
78
Et
Et
H
4-OMe
5-OMe
5-OMe
5-OH
5-OAc
5-OEt
5-OnBu
5-Me
5-F
5-Cl
5-Br
5-I
5-NH2
6-OH
6-OMe
6-F
6-Cl
7-OMe
b
H
b
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
knockdown DRAK2 in NIT-1 cell, it can effectively reduce the FFA
induced apoptosis [5].
In our study, two most potent compounds 40 and 41 were
selected to assess their effect on the glucose-stimulated insulin
secretion of primary islets. Isolated islets (10 islets/well) were
cultured in RPMI 1640 with 10% FBS, palmitate (PA, 0.4 mM) was
added to the wells at the beginning of the culture. The glucose-
stimulated insulin secretion (GSIS) ability of islets were signifi-
cantly impaired by palmitate incubation as shown in the reduction
of 16.7 mM glucose stimulated insulin secretion compared to the
dramastic response after 16.7 mM glucose stimulated in control
group (Fig. 2A); while adding compound 40 or 41 to the palmitate
co-culture islets, the reduced GSIS were partially recovered in a
dose-dependent manner compared to the DMSO-treated control,
suggesting that compounds 40 and 41 may protect islets function
from palmitate-impairment. The result suggests that the palmitate-
induced GSIS capacity of primary islets could be reversed by the
DRAK2 inhibitors.
Et
Et
Et
Et
60.40 13.23
40.54 10.69
1.29 0.10
a
See Experimental Section.
2.2. Effect of DRAK2 inhibitors on the glucose-stimulated insulin
secretion of primary islets and apoptosis protection of INS-1 cell
Type I and type II diabetes share a common pathological feature
of pancreatic cell apoptosis and hyperglycemia-induced cell
apoptosis has been implicated mainly in type II diabetes. Palmitate,
To explore the possible mechanism of protection from palmitate
impairment, INS-1 cells were applied to co-culture with palmitate
b
b
Table 3
The inhibitory activities of 4 selected compounds against the in house protein kinases.a
Compound
1
40
41
67
IC50
,
m
M
DRAK2
DRAK1
DAPK1
DAPK2
DAPK3
BLK
ITK
TEK
JAK1
1.31 0.08
0.33 0.10
1.41 0.16
NA
22.72 2.40
18.88 2.85
5.26 0.32
NA
0.25 0.01
5.81 0.77
NA
6.34 1.62
12.15 1.94
3.05 0.78
2.19 0.15
0.51 0.01
NA
1.02 0.06
5.85 1.37
13.15 0.71
3.07 0.30
2.33 0.15
2.88 0.92
2.02 0.16
0.95 0.17
NA
1.72 0.03
NAb
55.00 8.54
39.82 0.96
NTc
NT
NT
NT
NA
NA
JAK2
JAK3
SYK
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NA
NA
NA
NA
NA
9.92 1.75
NA
5.00 0.23
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.05 0.20
NA
5.03 0.43
NA
NA
9.32 2.87
4.26 0.32
4.63 1.38
NA
NA
0.85 0.02
NA
CHK1
AKT1
Aurora-A
EGFR
BTK
CDK2
CDK4
CDK5
CDK6
CDK9
MELK
6.02 0.02
NA
2.82 0.27
1.50 0.10
2.44 0.03
2.16 0.16
NA
8.84 0.52
NA
NA
NA
IKK
GSK3
MEK1
ERK1
b
NT
NT
NT
NT
6.23 1.00
5.01 0.01
NA
NA
5.30 0.98
NA
NA
1.95 0.21
NA
b
NA
NA
NA
a
See Experimental Section.
NA: not active, defined as < 50% inhibitory activity at 10 mM in the primary assay.
NT: not tested.
b
c

S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
201
Fig. 2. Effect of DRAK2 inhibitors on the glucose-stimulated insulin secretion of primary islets and apoptosis protection of INS-1 cell. Asterisks indicate p values (*P < 0.05,
**P < 0.01, ***P < 0.001) of control versus treated groups, Student's t-test. Croisillons indicate p-values (#P < 0.05, ##P < 0.01, ###P < 0.001) of PA and compounds co-treated groups
versus PA treated DMSO group, Student's t-test. All datas were shown as means SD of three replications.
for 48 h to trigger the INS-1 apoptosis. As shown in Fig. 2B, the
cleaved poly (ADP-ribose) polymerase (PARP), cleaved-caspase 9
and cleaved-caspase 3 were increased with palmitate co-culture
compared to the control group without palmitate incubation.
Adding the tested compound 40 or 41 together with palmitate, the
above cleaved-PARP, -caspase 3, and -caspase 9 content were
decreased compared to the palmitate co-cultured control. These
data suggest that compounds 40 and 41 may protect INS-1 cells
from palmitate-induced impairment by reducing cell apoptosis.
The partially function recovery of primary islets may be rendered
from the inhibition of DRAK2.
were recorded on a Nicolet Nexus 670 Fourier transform infrared
spectrometer using KBr discs. 1H and 13C NMR spectra were tested
with a Bruker Avance 400 spectrometer with tetramethylsilane
(TMS) as an internal standard (400 MHz for 1H, 100 MHz for 13C). 1H
and 13C chemical shifts were reported in parts per million (ppm,
d).
The high-resolution mass spectra were recorded on a Bruker ESI-
TOF high-resolution mass spectrometer. Melting points of the
products were recorded on a WRR-Y drug melting point measure-
ment apparatus and were uncorrected. The purities of the final
compounds were recorded with Waters e2695 HPLC system with a
ZORBAX SB-C18 column, with detection at 260 nm and 360 nm on a
variable wavelength detector 2998 PDA; Gradient I of 50e95%
acetonitrile in water in 21min, flow rate ¼ 1.2 mL/min. Gradient II of
10e90% acetonitrile in water (5% formic acid in water) in 23min,
flow rate ¼ 1.0 mL/min.
3. Conclusions
A total of 36 compounds were synthesized and their inhibitory
activities against DRAK2 were evaluated. Key SAR findings included
that 1) 30,40-dihydroxyl phenyl was crucial for the DRAK2 inhibitory
activity with 30-methoxy or 30-ethoxy analog tolerated; 2) intro-
duction of a methoxy group at 5-, 6-, and 7-position of benzofuran-
3(2H)-one resulted in a significant increase in activities. The most
potent compounds 40 and 41 exhibited favorable selectivity pro-
files against the other protein kinases of DAPK family as well as 22
other in house protein kinases. In a GSIS assay, compounds 40 and
4.1.1. General procedure for the synthesis of 2-
benzylidenebenzofuran-3(2H)-one (method A)
To a solution of benzofuran-3(2H)-one (1.0 mmol) and benzal-
dehyde (1.0 mmol) in dichloromethane (6 mL) was added
aluminum oxide (30.0 mmol) at room temperature. After stirring
for 6 h, the reaction mixture was filtered off. The filtrate was
concentrated under vacuum and the residue was purified by flash
chromatography on silica gel to give the desired compound.
41 were found to protect islet b-cell from palmitate impairment in a
dose-dependent manner. Western blotting studies in INS-1 cells
showed elevated cleavage of PARP, caspase 9 and 3, which can be
inhibited by these two novel DRAK2 inhibitors dose-dependently.
Overall, this study supports the notion that DRAK2 inhibitors
could be a promising strategy for the treatment of diabetes.
Continued medicinal chemistry efforts should be made to obtain
potent and selective drug-like DRAK2 inhibitors for potential use in
clinic.
4.1.2. General procedure for synthesis of 2-benzylidenebenzofuran-
3(2H)-one (method B)
To a solution of 3,4-dihydroxybenzaldehyde (1.0 mmol) and the
benzofuran-3(2H)-one derivatives (1.0 mmol) in methanol (20 mL)
was added an aqueous solution of 50% potassium hydroxide
(15.0 mmol). After refluxing for 3 h, the reaction mixture was
cooled to rt and concentrated under vacuum. The residue was
diluted with water (40 mL) and extracted with ethyl acetate
(3 ꢂ 50 mL). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4 and concentrated under vacuum. The
residue was purified by flash chromatography on silica gel to pro-
vide the desired compound.
4. Experimental
4.1. General methods
All reagents and solvents (analytical grade) were purchased
from commercial suppliers and were used directly without further
purification in addition to the special instructions. All reactions
were carried out under nitrogen atmosphere in addition to the
special instructions. The progress of reactions was monitored by
silica gel thin layer chromatography (TLC), visualized under ZF-20D
black box ultraviolet analyzer. Flash column chromatography was
performed using Yantai Kangbinuo silica gel (200e300). IR spectra
4.1.3. (Z)-2-(3,4-Dihydroxybenzylidene)benzofuran-3(2H)-one (4)
This compound was obtained from
6
[10] and 3,4-
dihydroxybenzaldehyde employing method B. Yellow solid, yield
45%; purity (gradient II) 99.1%; m.p. 223e225 ꢀC; IR (KBr) nmax
3440, 3276, 3038, 1697, 1648, 1584, 1529, 1449, 1298, 1259, 1188,
1162, 1134, 1101, 886, 809, 744 cmꢁ1; 1H NMR (400 MHz, DMSO-d6)
d
9.79 (s, 1H), 9.32 (s, 1H), 7.83e7.75 (m, 2H), 7.54e7.51 (m, 2H),

202
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
7.37e7.27 (m, 2H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.82 (s, 1H); 13C NMR
150.0, 146.6, 145.0, 137.2, 124.8, 124.6, 124.1, 123.7, 121.2, 117.6, 113.3,
113.0, 112.1, 55.6; HRMS (ESI): calcd for C16H12NaO4 [MþNa]þ,
291.0633; found, 291.0628.
(100 MHz, DMSO-d6)
d 183.0, 164.9, 148.6, 145.6, 144.6, 137.1, 125.1,
124.1, 123.7, 123.2, 121.3, 118.2, 116.0, 113.9, 113.0; HRMS (ESI): calcd
for C15H10NaO4 [MþNa]þ, 277.0477; found, 277.0463.
4.1.9. (Z)-2-(3,4-Dimethoxybenzylidene)benzofuran-3(2H)-one
4.1.4. (Z)-2-Benzylidenebenzofuran-3(2H)-one (7)
(12)
This compound was obtained from
6
and benzaldehyde
This compound was obtained from
6
and 3,4-
employing method A. Yellow solid, yield 88%; purity (gradient I)
95.1%; m.p. 106e107 ꢀC; IR (KBr) nmax 3057, 3022, 1709, 1658, 1600,
1478, 1458, 1301, 1191, 1127, 1098, 883, 744, 686, 558 cmꢁ1; 1H NMR
dimethoxybenzaldehyde employing method A. Yellow solid, yield
77%; purity (gradient I) 95.1%; m.p. 147e149 ꢀC; IR (KBr) nmax 3061,
2999, 2938, 2835, 1703, 1645, 1594, 1523, 1462, 1327, 1298, 1272,
1204, 1153, 1124, 1014, 883, 805, 757, 558 cmꢁ1; 1H NMR (400 MHz,
(400 MHz, CDCl3)
d
7.93 (s, 1H), 7.92 (s, 1H), 7.81 (d, J ¼ 7.6 Hz, 1H),
7.70e7.61 (m, 1H), 7.52e7.44 (m, 2H), 7.43e7.37 (m, 1H), 7.33 (d,
DMSO-d6)
1H), 7.36e7.29 (m, 1H), 7.12 (d, J ¼ 8.4 Hz, 1H), 6.95 (s, 1H), 3.86 (s,
6H); 13C NMR (100 MHz, DMSO-d6)
183.2, 165.1, 150.8, 148.8,
d
7.85e7.76 (m, 2H), 7.69e7.61 (m, 2H), 7.58 (d, J ¼ 8.4 Hz,
J ¼ 8.3 Hz, 1H), 7.25e7.18 (m, 1H), 6.90 (s, 1H); 13C NMR (100 MHz,
CDCl3)
d
184.8, 166.2, 146.9, 136.9, 132.3, 131.6 (2C), 129.9, 128.9
d
(2C), 124.7, 123.5, 121.6, 113.1, 113.0; HRMS (ESI): calcd for
C
145.2, 137.2, 125.7, 124.5, 124.1, 123.8, 121.1, 114.4, 113.2, 113.1,
112.00, 55.6, 55.5; HRMS (ESI): calcd for C17H14NaO4 [MþNa]þ,
305.0790; found, 305.0794.
15H10NaO2 [MþNa]þ, 245.0578; found, 245.0582.
4.1.5. (Z)-2-(4-Hydroxybenzylidene)benzofuran-3(2H)-one (8)
This compound was obtained from and
6
4-
4.1.10. (Z)-2-(4-Hydroxy-3,5-dimethoxybenzylidene)benzofuran-
3(2H)-one (13)
hydroxybenzaldehyde employing method A. Yellow solid, yield
85%; purity (gradient II) 98.7%; m.p. 252e254 ꢀC; IR (KBr) vmax
3135, 3019, 2954, 1687, 1639, 1613, 1571, 1510, 1449, 1288, 1172,
This compound was obtained from 6 and 4-hydroxy-3,5-
dimethoxybenzaldehyde employing method A. Yellow solid, yield
69%; purity (gradient II) 98.6%; m.p. 151e153 ꢀC; IR (KBr) nmax 3511,
3070, 3009, 2938, 2842, 1687, 1642, 1606, 1513, 1455, 1294, 1220,
1130, 1101, 886, 828, 754 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
;
d
10.25 (s,1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.79 (d, J ¼ 7.1 Hz, 2H), 7.56 (d,
J ¼ 8.4 Hz, 1H), 7.38e7.26 (m, 1H), 7.01e6.80 (m, 3H); 13C NMR
1134, 1111, 879, 838, 747, 564 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
;
(100 MHz, DMSO-d6)
d
183.1, 165.0, 159.7, 144.7, 137.1, 133.7 (2C),
d
9.29 (s, 1H), 7.85e7.75 (m, 2H), 7.59 (d, J ¼ 8.4 Hz, 1H), 7.38 (s, 2H),
7.35e7.28 (m, 1H), 6.93 (s, 1H), 3.86 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) 183.00, 164.9, 148.00 (2C), 144.9, 138.7, 137.1, 124.0,
124.1, 123.7, 122.8, 121.2, 116.1 (2C), 113.4, 113.1; HRMS (ESI): calcd
for C15H10NaO3 [MþNa]þ, 261.0528; found, 261.0526.
d
123.7, 122.0, 121.2, 114.0, 113.3, 109.6 (2C), 56.0 (2C); HRMS (ESI):
4.1.6. (Z)-2-(4-(Hydroxymethyl)benzylidene)benzofuran-3(2H)-one
(9)
calcd for C17H14NaO4 [MþNa]þ, 321.0739; found, 321.0733.
This compound was obtained from 6 and 4-(hydroxymethyl)
benzaldehyde employing method A. Yellow solid, yield 70%; purity
(gradient II) 98.8%; m.p. 118e121 ꢀC; IR (KBr) nmax 3392, 3321, 2929,
2868, 1703, 1651, 1597, 1478, 1458, 1301, 1188, 1130, 1040, 886, 818,
4.1.11. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)benzofuran-3(2H)-
one (14)
This compound was obtained from
6 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
77%; purity (gradient I) 99.9%; m.p. 121e122 ꢀC; IR (KBr) nmax 3305,
3061, 2980, 2868, 1690, 1642, 1584, 1513, 1478, 1442, 1285, 1253,
1220, 1182, 1130, 1101, 876, 805, 754, 574 cmꢁ1; 1H NMR (400 MHz,
757 cmꢁ1
;
1H NMR (400 MHz, CD3OD)
d
7.92 (d, J ¼ 7.8 Hz, 2H),
7.84e7.66 (m, 2H), 7.58e7.36 (m, 3H), 7.35e7.20 (m, 1H), 6.87 (s,
1H), 4.65 (s, 2H); 13C NMR (100 MHz, CD3OD)
186.2, 167.5, 148.1,
d
145.4, 138.7, 132.8 (2C), 132.4, 128.3 (2C), 125.4, 124.9, 122.6, 114.2,
114.1, 64.8; HRMS (ESI): calcd for C16H12NaO3 [MþNa]þ, 275.0684;
found, 275.0693.
CDCl3)
d
7.81 (dd, J ¼ 7.6, 1.4 Hz, 1H), 7.68e7.61 (m, 1H), 7.52e7.44
(m, 2H), 7.35e7.28 (m, 1H), 7.24e7.18 (m, 1H), 7.03e6.98 (m, 1H),
6.86 (s, 1H), 6.08 (s, 1H), 4.22 (q, J ¼ 7.0 Hz, 2H), 1.51 (t, J ¼ 7.0 Hz,
3H); 13C NMR (100 MHz, CDCl3)
d 184.5, 165.7, 147.9, 146.0, 145.7,
4.1.7. (Z)-2-(4-Hydroxy-3-methoxybenzylidene)benzofuran-3(2H)-
one (10)
136.5, 126.5, 124.8, 124.6, 123.3, 121.9, 114.9, 114.2, 114.0, 112.8, 64.6,
14.8; HRMS (ESI): calcd for C17H14NaO4 [MþNa]þ, 305.0790; found,
305.0792.
This compound was obtained from
6 and 4-hydroxy-3-
methoxybenzaldehyde employing method A. Yellow solid, yield
73%; purity (gradient I) 98.9%; m.p. 200e201 ꢀC; IR (KBr) nmax 3312,
3061, 2935, 1690, 1639, 1581, 1523, 1365, 1301, 1204, 1117, 1034, 886,
4.1.12. (Z)-2-(4-Hydroxy-3-propoxybenzylidene)benzofuran-
3(2H)-one (15)
809, 754, 574 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6)
d
9.91 (s, 1H),
This compound was obtained from 6 and 4-hydroxy-3-
7.88e7.75 (m, 2H), 7.63 (s,1H), 7.58 (d, J ¼ 8.5 Hz, 2H), 7.39e7.29 (m,
propoxybenzaldehyde employing method A. Yellow solid, yield
74%; purity (gradient I) 96.9%; m.p. 105e107 ꢀC; IR (KBr) nmax 3389,
3064, 2961, 2922, 2851, 1703, 1642, 1584, 1513, 1462, 1282, 1191,
1H), 7.10e6.84 (m, 2H), 3.90 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
d
183.0, 164.9, 149.4, 147.8, 144.8, 137.0, 126.0, 124.0, 123.7, 123.2,
121.2, 116.1, 115.3, 113.7, 113.2, 55.6; HRMS (ESI): calcd for
1124, 883, 805, 747 cmꢁ1 1H NMR (400 MHz, CDCl3)
; d 7.84e7.78
C
16H12NaO4 [MþNa]þ, 291.0633; found, 291.0633.
(m, 1H), 7.67e7.60 (m, 1H), 7.52e7.45 (m, 2H), 7.34e7.29 (m, 1H),
7.25e7.18 (m, 1H), 7.04e6.98 (m, 1H), 6.86 (s, 1H), 6.06 (s, 1H), 4.11
(t, J ¼ 6.6 Hz, 2H),1.96e1.85 (m, 2H),1.09 (t, J ¼ 7.5 Hz, 3H); 13C NMR
4.1.8. (Z)-2-(3-Hydroxy-4-methoxybenzylidene)benzofuran-3(2H)-
one (11)
(100 MHz, CDCl3) d 183.4, 164.7, 147.0, 145.1, 144.7, 135.5, 125.4,
This compound was obtained from
6
and 3-hydroxy-4-
123.8, 123.6, 122.3, 120.9, 113.9, 113.3, 113.0, 111.8, 69.5, 21.5, 9.5;
HRMS (ESI): calcd for C18H16NaO4 [MþNa]þ, 319.0946; found,
319.0934.
methoxybenzaldehyde employing method A. Yellow solid, yield
72%; purity (gradient II) 96.1%; m.p. 185e187 ꢀC; IR (KBr) nmax 3244,
3019, 2958, 2839, 1693, 1639, 1590, 1503, 1455, 1278, 1246, 1188,
1127, 1098, 1024, 879, 802, 754 cmꢁ1; 1H NMR (400 MHz, DMSO-d6)
4.1.13. (Z)-2-(4-Hydroxy-3-isopropoxybenzylidene)benzofuran-
d
9.35 (s, 1H), 7.89e7.74 (m, 2H), 7.60e7.51 (m, 2H), 7.44 (d,
J ¼ 8.4 Hz, 1H), 7.36e7.29 (m, 1H), 7.07 (d, J ¼ 8.4 Hz, 1H), 6.86 (s,
1H), 3.86 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
183.2, 165.0,
3(2H)-one (16)
This compound was obtained from
isopropoxybenzaldehyde employing method A. Yellow solid, yield
6 and 4-hydroxy-3-
d

S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
203
70%; purity (gradient I) 97.4%; m.p. 110e111 ꢀC; IR (KBr) nmax 3386,
indanone [13] (22, 2.0 mmol) in ethanol (30 mL) was added 5%
aqueous solution of sodium hydroxide (10.0 mmol) dropwise at
room temperature. After stirring for 5 h, the reaction mixture was
diluted with water (40 mL) and extracted with ethyl acetate
(3 ꢂ 30 mL). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4 and evaporated under vacuum. The
residue was purified by flash chromatography on silica gel (petro-
leum ether/ethyl acetate ¼ 1/1) to provide the title compound.
Yellow solid, yield 42%; purity (gradient II) 95.2%; m.p. > 250 ꢀC; IR
(KBr) nmax 3463, 3215, 3048, 1677, 1594, 1571, 1532, 1446, 1333,
1291, 1265, 1179, 1124, 1108, 986, 928, 786, 735, 677, 564 cmꢁ1; 1H
3067, 2964, 2913, 2871, 1703, 1651, 1603, 1581, 1513, 1462, 1298,
1275, 1191, 1124, 883, 805, 744 cmꢁ1 1H NMR (400 MHz, CDCl3)
;
d
7.81 (d, J ¼ 7.4 Hz, 1H), 7.69 (d, J ¼ 2.1 Hz, 1H), 7.67e7.62 (m, 1H),
7.38e7.31 (m, 2H), 7.24e7.18 (m, 1H), 6.91 (d, J ¼ 8.4 Hz, 1H), 6.84 (s,
1H), 5.78 (s,1H), 4.75e4.64 (m,1H),1.42 (d, J ¼ 6.0 Hz, 6H); 13C NMR
(100 MHz, CDCl3)
d 184.6, 165.9, 146.5, 146.4, 146.1, 136.6, 125.6,
125.3, 124.6, 123.3, 121.9, 117.0, 113.6, 113.0, 112.6, 71.7, 22.1 (2C);
HRMS (ESI): calcd for C18H16NaO4 [MþNa]þ, 319.0946; found,
319.0940.
4.1.14. (Z)-2-(3-Butoxy-4-hydroxybenzylidene)benzofuran-3(2H)-
NMR (400 MHz, DMSO-d6) d 9.64 (s, 1H), 9.35 (s, 1H), 7.80 (d,
one (17)
J ¼ 7.5 Hz, 1H), 7.71 (s, 2H), 7.57e7.47 (m, 1H), 7.43 (s, 1H), 7.27 (s,
This compound was obtained from
6
and 3-butoxy-4-
1H), 7.16 (d, J ¼ 8.6 Hz, 1H), 6.91 (d, J ¼ 8.4 Hz, 1H), 4.06 (s, 2H); 13
C
hydroxybenzaldehyde employing method A. Yellow solid, yield
65%; purity (gradient I) 98.9%; m.p. 126e128 ꢀC; IR (KBr) nmax 3212,
2954, 2922, 2864, 1693, 1642, 1577, 1510, 1436, 1288, 1179, 1130,
NMR (100 MHz, DMSO-d6) d 193.1, 149.7, 148.0, 145.6, 137.6, 134.4,
133.8, 131.3, 127.5, 126.6,126.4,124.3, 123.3,117.5, 116.0, 32.0; HRMS
(ESI): calcd for C16H11O3 [MꢁH]ꢁ, 251.0708; found, 251.0711.
883, 815, 760 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
; d 9.76 (s, 1H),
7.84e7.74 (m, 2H), 7.59 (d, J ¼ 2.0 Hz, 1H), 7.58e7.50 (m, 2H),
7.34e7.29 (m, 1H), 6.99e6.88 (m, 2H), 4.06 (t, J ¼ 6.5 Hz, 2H),
1.83e1.70 (m, 2H), 1.56e1.43 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H); 13C
4.1.19. Benzofuran-2-yl(3,4-dihydroxyphenyl)methanone (26)
To a solution of 2-bromo-1-(3,4-dihydroxyphenyl) ethan-1-one
(25, 1.0 mmol) and 2-hydroxybenzaldehyde (1.0 mmol) in acetone
(40 mL) was added anhydrous potassium carbonate (3.0 mmol).
After refluxing for 20 h the reaction mixture was cooled to rt
concentrated under vacuum. The residue was added water (30 mL)
and extracted with ethyl acetate (3 ꢂ 20 mL). The combined organic
phases were washed with brine, dried over anhydrous Na2SO4 and
evaporated under vacuum. The residue was purified by flash
chromatography on silica gel to provide the title compound
(dichloromethane/methanol ¼ 80/1). Brown solid, yield 35%; purity
(gradient II) 98.3%; m.p. 165e168 ꢀC; IR (KBr) nmax 3463, 3305, 3119,
NMR (100 MHz, CDCl3)
d 183.4, 164.7, 146.9, 145.1, 144.7, 135.5,
125.4, 123.8, 123.6, 122.3, 120.9, 113.9, 113.2, 113.0, 111.8, 67.8, 30.1,
18.2, 12.8; HRMS (ESI): calcd for C19H18NaO4 [MþNa]þ, 333.1103;
found, 333.1081.
4.1.15. (Z)-2-(4-Fluorobenzylidene)benzofuran-3(2H)-one (18)
This compound was obtained from 6 and 4-fluorobenzaldehyde
employing method A. Yellow solid, yield 76%; purity (gradient I)
97.3%; m.p. 156e157 ꢀC; IR (KBr) nmax 3064, 1713, 1658, 1597, 1510,
1458,1301,1236,1188,1124,1098, 879, 831, 751, 696, 571, 503 cmꢁ1
;
1594, 1545, 1446, 1294, 1191, 937, 847, 773, 738 cmꢁ1
(400 MHz, DMSO-d6)
;
1H NMR
1H NMR (400 MHz, DMSO-d6)
d
8.13e8.02 (m, 2H), 7.88e7.76 (m,
d
10.05 (s, 1H), 9.54 (s, 1H), 7.86 (d, J ¼ 7.8 Hz,
2H), 7.57 (d, J ¼ 8.3 Hz,1H), 7.44e7.29 (m, 3H), 6.99 (s, 1H); 13C NMR
1H), 7.76 (d, J ¼ 8.5 Hz, 1H), 7.72 (s, 1H), 7.63e7.46 (m, 3H),
(100 MHz, DMSO-d6)
d
183.5, 165.4, 162.7 (J
¼ 250.8 Hz, 1C),
7.44e7.33 (m, 1H), 6.94 (d, J ¼ 8.2 Hz, 1H); 13C NMR (100 MHz,
C-F
146.0 (J C-F ¼ 2.6 Hz, 1C), 137.7, 133.7 (J C-F ¼ 8.7 Hz, 2C), 128.6 (J C-
DMSO-d6) d 181.6,154.9,152.0,151.1,145.4,128.0,127.9,126.8,123.9,
¼ 3.2 Hz, 1C), 124.3, 124.0, 120.8, 116.1 (J
¼ 11.8 Hz, 2C), 113.2,
123.5, 122.8, 116.3, 115.3, 115.2, 112.1; HRMS (ESI): calcd for
F
C-F
111.1; HRMS (ESI): calcd for C15H9FNaO2 [MþNa]þ, 263.0484;
C
15H10NaO4 [MþNa]þ, 277.0477; found, 277.0464.
found, 263.0480.
4.1.20. 2-(3,4-Dihydroxybenzyl)benzofuran-3(2H)-one (29)
4.1.16. (Z)-2-(Pyridin-3-ylmethylene)benzofuran-3(2H)-one (19)
This compound was obtained from 6 and nicotinaldehyde
employing method B. Yellow solid, yield 83%; purity (gradient I)
99.1%; m.p. 123e124 ꢀC; IR (KBr) nmax 3041, 1713, 1664, 1600, 1478,
1455, 1423, 1298, 1179, 1117, 1024, 883, 760, 696, 561 cmꢁ1; 1H NMR
To a solution of 6 (1.0 mmol) and 3,4-bis(benzyloxy)benzalde-
hyde (28, 1.0 mmol) in dichloromethane (6 mL) was added
aluminum oxide (30.0 mmol) at room temperature. After stirring
for 5 h, the reaction mixture was filtered off. The filtrate was
concentrated under vacuum to give crude product. In a round-
bottom flash, the residue was dissolved in methanol (20 mL), and
10% Pd/C (0.1 mmol) was added. A H2 balloon was connected after
the atmosphere was exchanged three times with nitrogen. After
stirring for 5 h, the catalyst was filtered off. The filtrate was
concentrated in vacuo and the residue was purified by flash chro-
matography on silica gel (petroleum ether/ethyl acetate ¼ 3/1) to
afford the title compound. Light yellow solid, yield 72%; purity
(gradient II) 96.4%; m.p. 130e132 ꢀC; IR (KBr) nmax 3299, 2919, 1693,
(400 MHz, DMSO-d6)
J ¼ 8.0 Hz, 1H), 7.98e7.72 (m, 2H), 7.69e7.48 (m, 2H), 7.43e7.29 (m,
1H), 7.01 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
183.4, 165.5, 151.9,
d
9.11 (s, 1H), 8.62 (d, J ¼ 4.7 Hz, 1H), 8.40 (d,
d
150.1,147.4,137.9, 137.6,128.2,124.4, 124.1, 124.0,120.6,113.3,108.6;
HRMS (ESI): calcd for
224.0700.
C
14H10NO2 [MþH]þ, 224.0712; found,
4.1.17. (Z)-2-(Furan-2-ylmethylene)benzofuran-3(2H)-one (20)
This compound was obtained from 6 and furan-2-carbaldehyde
employing method B. Yellow solid, yield 80%; purity (gradient I)
98.6%; m.p. 118e119 ꢀC; IR (KBr) nmax 3131, 3106, 3057, 1706, 1651,
1600, 1468, 1298, 1201, 1114, 1101, 1018, 963, 879, 747, 696 cmꢁ1; 1H
1610, 1516, 1481, 1462, 1330, 1288, 1195, 1114, 764 cmꢁ1
;
1H NMR
(400 MHz, DMSO-d6) 8.74 (s, 1H), 8.68 (s, 1H), 7.72e7.64 (m, 1H),
d
7.62e7.53 (m, 1H), 7.25e7.18 (m, 1H), 7.13e7.04 (m, 1H), 6.64 (d,
J ¼ 2.1 Hz, 1H), 6.60e6.55 (m, 1H), 6.52e6.47 (m, 1H), 4.96 (dd,
J ¼ 7.8, 4.0 Hz, 1H), 3.08 (dd, J ¼ 14.7, 4.0 Hz, 1H), 2.80 (dd, J ¼ 14.7,
NMR (400 MHz, DMSO-d6)
J ¼ 8.3 Hz, 1H), 7.38e7.29 (m, 1H), 7.25 (d, J ¼ 3.4 Hz, 1H), 6.92 (s,
1H), 6.79 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
182.9,165.1, 148.0,
d 8.03 (s, 1H), 7.88e7.75 (m, 2H), 7.56 (d,
7.8 Hz, 1H); 13C NMR (100 MHz, CD3OD)
d 203.6, 174.4, 146.0, 145.2,
d
139.5, 128.4, 124.8, 122.9, 122.3, 122.0, 117.8, 116.1, 114.5, 87.4, 37.6;
HRMS (ESI): calcd for C15H12NaO4 [MþNa]þ, 279.0633; found,
279.0627.
146.8,144.1, 137.4, 124.1, 123.9,121.2,118.2,113.5, 113.1, 100.8; HRMS
(ESI): calcd for C13H8NaO3 [MþNa]þ, 235.0371; found, 235.0376.
4.1.18. 2-(3,4-Dihydroxybenzylidene)-2,3-dihydro-1H-inden-1-one
(23)
4.1.21. 4-Methoxybenzofuran-3(2H)-one (32)
To a solution of 4-hydroxybenzofuran-3(2H)-one (31, 2.0 mmol)
in anhydrous DMF (10 mL) was added successively anhydrous
To a solution of 3,4-dihydroxybenzaldehyde (2.0 mmol) and 1-

204
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
potassium carbonate (3.0 mmol) and iodomethane (2.2 mmol).
After stirring for 3 h at 80 ꢀC, the reaction mixture was cooled to rt,
quenched with water (30 mL), and extracted with dichloromethane
(3 ꢂ 20 mL). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4 and evaporated under vacuum. The
residue was purified by flash chromatography on silica gel (petro-
leum ether/ethyl acetate ¼ 30/1) to afford the title compound.
Bright yellow solid, yield 93%; 1H NMR (400 MHz, CDCl3)
1117, 905, 809, 735 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
; d 9.77 (s,
1H), 7.57 (s, 1H), 7.54e7.45 (m, 2H), 7.40e7.33 (m, 1H), 7.26e7.20
(m, 1H), 6.92 (dd, J ¼ 8.3, 2.6 Hz, 1H), 6.88 (d, J ¼ 2.6 Hz, 1H), 4.11 (q,
J ¼ 6.7 Hz, 2H), 3.82 (s, 3H), 1.38 (t, J ¼ 6.7 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) d 183.0, 159.8, 155.7, 150.4, 147.0, 145.4, 126.2,
125.4, 122.8, 121.5, 116.6, 116.3, 114.1, 114.0, 105.2, 63.9, 55.8, 14.7;
HRMS (ESI): calcd for C18H16NaO5 [MþNa]þ, 335.0895; found,
335.0881.
d
7.49e7.40 (m, 1H), 6.61 (d, J ¼ 8.4 Hz, 1H), 6.40 (d, J ¼ 8.4 Hz, 1H),
4.52 (s, 2H), 3.89 (s, 3H).
4.1.26. (Z)-2-(3,4-Dihydroxybenzylidene)-5-methoxybenzofuran-
3(2H)-one (41)
4.1.22. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-4-
methoxybenzofuran-3(2H)-one (33)
This compound was obtained from 37 and 3,4-
dihydroxybenzaldehyde employing method B. Yellow solid, yield
33%; purity (gradient II) 99.8%; m.p. 234e236 ꢀC; IR (KBr) nmax
3466, 3312, 2977, 2938, 2845, 1684, 1639, 1584, 1526, 1487, 1429,
This compound was obtained from 32 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
73%; purity (gradient I) 97.3%; m.p. 177e178 ꢀC; IR (KBr) nmax 3302,
3009, 2977, 2925, 1693, 1642, 1581, 1510, 1491, 1433, 1294, 1256,
1320, 1278, 1201, 1166, 1127, 1021, 899, 809, 741 cmꢁ1
;
1H NMR
9.76 (s, 1H), 9.25 (s, 1H), 7.68 (d, J ¼ 8.5 Hz,
1H), 7.47 (s, 1H), 7.31 (d, J ¼ 8.3 Hz, 1H), 7.08 (s, 1H), 6.95e6.78 (m,
2H), 6.71 (s, 1H), 3.93 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
183.1,
(400 MHz, DMSO-d6)
d
1169, 1079, 886, 796, 760 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
;
d
9.69 (s, 1H), 7.77e7.65 (m, 1H), 7.54 (s, 1H), 7.48 (d, J ¼ 8.2 Hz, 1H),
d
7.04 (d, J ¼ 8.2 Hz, 1H), 6.92 (d, J ¼ 8.2 Hz, 1H), 6.82 (d, J ¼ 8.2 Hz,
1H), 6.76 (s, 1H), 4.11 (q, J ¼ 6.9 Hz, 2H), 3.93 (s, 3H), 1.39 (t,
159.9, 155.7, 148.5, 145.6, 145.4, 125.5, 125.1, 123.3, 121.5, 118.2,
116.0, 113.9 (2C), 105.3, 55.8; HRMS (ESI): calcd for C16H12NaO5
[MþNa]þ, 307.0582; found, 307.0582.
J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 180.5, 165.8, 157.8,
149.2, 146.8, 144.7, 138.6, 125.5, 123.3, 116.4, 116.1, 112.1, 110.1, 105.8,
104.6, 63.9, 56.0, 14.7; HRMS (ESI): calcd for C18H16NaO5 [MþNa]þ,
335.0895; found, 335.0890.
4.1.27. (Z)-2-(3,4-Dihydroxybenzylidene)-5-hydroxybenzofuran-
3(2H)-one (42)
This compound was obtained from 38 and 3,4-
dihydroxybenzaldehyde employing method B. Yellow solid, yield
28%; purity (gradient II) 96.2%; m.p. >270 ꢀC; IR (KBr) nmax 3447,
3250, 1690, 1639, 1590, 1513, 1494, 1391, 1291, 1230, 1191, 1159, 1121,
4.1.23. 2-Chloro-1-(2-hydroxy-5-methoxyphenyl)ethan-1-one (35)
To a dichloromethane solution of boron trichloride (12.0 mmol)
was added dropwise
a solution of 4-methoxyphenol (34,
10.0 mmol) in dichloromethane (20 mL) at 0 ꢀC under nitrogen. And
then, chloroacetonitrile (12.0 mmol) was added dropwise, followed
by aluminum chloride (5.0 mmol) in one portion. The reaction
mixture was allowed to warm to room temperature and was stirred
for 6 h until the starting material disappeared completely. The re-
action mixture was quenched with 1 N hydrochloric acid at 0 ꢀC.
After stirring for 10 min, the aqueous layer was extracted with
dichloromethane (3 ꢂ 30 mL). The combined organic layers were
washed with brine, dried with anhydrous Na2SO4 and evaporated
under reduced pressure. The crude mixture was purified by silica
gel column chromatography (petroleum ether/ethyl acetate ¼ 30/1)
to afford the desired compound 35. Yellow solid, yield 66%; 1H NMR
895, 818 cmꢁ1; 1H NMR (400 MHz, DMSO-d6)
d 9.76 (s, 2H), 9.31 (s,
1H), 7.48 (d, J ¼ 2.1 Hz, 1H), 7.35 (d, J ¼ 8.8 Hz, 1H), 7.29 (dd, J ¼ 8.3,
2.1 Hz, 1H), 7.21 (dd, J ¼ 8.8, 2.7 Hz, 1H), 7.01 (d, J ¼ 2.7 Hz, 1H), 6.85
(d, J ¼ 8.3 Hz, 1H), 6.74 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
d
183.3, 158.8, 153.6, 148.4, 145.5, 145.4, 125.2, 125.0, 123.3, 121.6,
118.1, 116.0, 113.6, 113.4, 107.5; HRMS (ESI): calcd for C15H9O5
[MꢁH]ꢁ, 269.0450; found, 269.0453.
4.1.28. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-3-oxo-2,3-
dihydrobenzofuran-5-yl acetate (43)
This compound was obtained from 39 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
63%; purity (gradient I) 99.1%; m.p. 185e187 ꢀC; IR (KBr) nmax 3466,
3070, 2974, 2922, 2877, 1751, 1697, 1642, 1581, 1516, 1478, 1368,
(400 MHz, CDCl3)
J ¼ 2.4 Hz, 1H), 6.98 (dd, J ¼ 9.1, 1.9 Hz, 1H), 4.70 (s, 2H), 3.81 (s, 3H).
d
11.32 (s, 1H), 7.18 (dd, J ¼ 9.1, 2.8 Hz, 1H), 7.11 (d,
1301, 1265, 1227, 1175, 1111, 1040, 908, 828 cmꢁ1
(400 MHz, DMSO-d6) d
;
1H NMR
9.83 (s, 1H), 7.85e7.35 (m, 5H), 7.07e6.80
(m, 2H), 4.10 (q, J ¼ 7.0 Hz, 2H), 2.28 (s, 3H), 1.37 (t, J ¼ 7.0 Hz, 3H);
13C NMR (100 MHz, DMSO-d6)
182.5, 169.3, 162.2, 149.8, 146.9,
4.1.24. 3-Oxo-2,3-dihydrobenzofuran-5-yl acetate (39)
To a solution of 5-hydroxybenzofuran- 3(2H)-one (38, 1.0 mmol)
in dichloromethane (20 mL) was added acetic anhydride
(1.5 mmol) and triethylamine (2.0 mmol) at the rt. After stirring for
1 h, the reaction mixture was poured into water (30 mL) and
extracted with ethyl acetate (3 ꢂ 15 mL). The combined organic
phases were washed with saturated sodium bicarbonate solution,
brine, dried over anhydrous Na2SO4, and evaporated under vacuum.
The residue was purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate ¼ 20/1) to afford the title com-
d
146.2, 145.3, 130.8, 126.2, 123.1, 121.7, 116.7 (2C), 116.2, 114.4, 114.0,
63.9, 20.8, 14.7; HRMS (ESI): calcd for C19H16NaO6 [MþNa]þ,
363.0845; found, 363.0841.
4.1.29. (Z)-5-Ethoxy-2-(3-ethoxy-4-hydroxybenzylidene)
benzofuran-3(2H)-one (45)
This compound was synthesized from 4-ethoxyphenol and 3-
ethoxy-4-hydroxybenzaldehyde according to the methodology
described for 40. Yellow solid, yield 27%; purity (gradient I) 99.0%;
m.p. 164e166 ꢀC; IR (KBr) nmax 3537, 3070, 2980, 2929, 2874, 1684,
1639, 1577, 1516, 1491, 1468, 1310, 1278, 1208, 1182, 1114, 1047, 908,
pound. Yellow solid, yield 88%; 1H NMR (400 MHz, CDCl3)
d 7.39 (d,
J ¼ 2.5 Hz, 1H), 7.33 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.14 (d, J ¼ 8.9 Hz, 1H),
4.67 (s, 2H), 2.31 (s, 3H); 13C NMR (100 MHz, CDCl3)
169.5, 145.2, 131.7, 121.5, 116.3, 114.3, 75.6, 21.0.
d 199.1, 171.4,
818, 738, 503 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
; d 9.79 (s, 1H),
4.1.25. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-5-
methoxybenzofuran-3(2H)-one (40)
This compound was obtained from 37 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
78%; purity (gradient I) 98.0%; m.p. 149e151 ꢀC; IR (KBr) nmax 3527,
3064, 2980, 2935, 1690, 1642, 1581, 1516, 1484, 1310, 1275, 1195,
7.56 (d, J ¼ 2.0 Hz, 1H), 7.52 (dd, J ¼ 8.2, 2.0 Hz, 1H), 7.49e7.42 (m,
1H), 7.37e7.29 (m, 1H), 7.19 (d, J ¼ 2.8 Hz, 1H), 6.94 (d, J ¼ 8.3 Hz,
1H), 6.87 (s, 1H), 4.17e4.00 (m, 4H), 1.46e1.29 (m, 6H); 13C NMR
(100 MHz, DMSO-d6) d 183.1, 159.8, 154.9, 149.6, 146.9, 145.6, 126.0,
125.9, 123.3, 121.5, 116.6, 116.1, 114.1, 113.7, 105.9, 63.9, 63.9, 14.7,
14.5; HRMS (ESI): calcd for C19H18NaO5 [MþNa]þ, 349.1052; found,

S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
205
349.1036.
1H), 7.06 (d, J ¼ 8.8 Hz, 1H), 4.67 (s, 2H).
4.1.35. 5-Iodobenzofuran-3(2H)-one (56)
4.1.30. (Z)-5-Butoxy-2-(3-ethoxy-4-hydroxybenzylidene)
benzofuran-3(2H)-one (46)
This compound was obtained from 51 according to the meth-
This compound was obtained from 4-butoxyphenol and 3-
ethoxy-4-hydroxybenzaldehyde according to the methodology
described for 40. Yellow solid, yield 25%; purity (gradient I) 98.8%;
m.p. 182e185 ꢀC; IR (KBr) nmax 3389, 3340, 3057, 2961, 2929, 2871,
1703, 1645, 1584, 1520, 1487, 1426, 1394, 1282, 1182, 1108, 1043, 912,
odology described for 53. Bright yellow solid, yield 62%; 1H NMR
(400 MHz, CDCl3)
1H), 6.96 (d, J ¼ 8.7 Hz, 1H), 4.65 (s, 2H).
d
7.98 (d, J ¼ 2.0 Hz, 1H), 7.85 (dd, J ¼ 8.7, 2.0 Hz,
4.1.36. 5-Nitrobenzofuran-3(2H)-one (57)
828 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6)
d
9.73 (s, 1H), 7.69e7.41
This compound was obtained from 52 according to the meth-
(m, 3H), 7.38e7.30 (m,1H), 7.19 (d, J ¼ 2.7 Hz,1H), 6.94 (d, J ¼ 8.4 Hz,
1H), 6.86 (s, 1H), 4.10 (q, J ¼ 7.2 Hz, 2H), 4.00 (t, J ¼ 6.5 Hz, 2H),
1.79e1.61 (m, 2H), 1.51e1.31 (m, 5H), 0.93 (t, J ¼ 7.2 Hz, 3H); 13C
odology described for 53. Bright yellow solid, yield 22%; 1H NMR
(400 MHz, DMSO-d6)
J ¼ 2.5 Hz, 1H), 7.52 (d, J ¼ 9.1 Hz, 1H), 5.04 (s, 2H).
d
8.56 (dd, J ¼ 9.2, 2.5 Hz, 1H), 8.40 (d,
NMR (100 MHz, DMSO-d6)
d 183.1, 159.8, 155.1, 149.9, 146.9, 145.5,
126.0, 125.9, 123.1, 121.5, 116.6, 116.2, 114.1, 113.7, 105.8, 68.0, 63.9,
30.6, 18.7, 14.7, 13.6; HRMS (ESI): calcd for C21H22NaO5 [MþNa]þ,
377.1365; found, 377.1362.
4.1.37. 5-Aminobenzofuran-3(2H)-one (58)
To a solution of 5-nitrobenzofuran-3(2H)-one (57, 2.0 mmol) in
acetic acid (20 mL) was added iron (10.0 mmol). After stirring for
3 h at 70 ꢀC, the reaction mixture was cooled to rt and filtered off.
The filtrate was neutralized by adding the saturated sodium bi-
carbonate solution until pH ¼ 10 and extracted with ethyl acetate
(3 ꢂ 40 mL). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, and evaporated under vacuum. The
crude mixture was purified by flash chromatography on silica gel
4.1.31. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-5-
methylbenzofuran-3(2H)-one (47)
This compound was obtained from p-cresol and 3-ethoxy-4-
hydroxybenzaldehyde according to the methodology described
for 40. Yellow solid, yield 28%; purity (gradient I) 99.1%; m.p.
172e174 ꢀC; IR (KBr) nmax 3530, 3073, 2977, 2938, 1700, 1639, 1613,
1577, 1513, 1487, 1471, 1301, 1278, 1191, 1153, 1124, 1043, 908, 802,
(petroleum ether/ethyl acetate ¼ 4/1) to afford the title compound.
1
Yellow solid, yield 77%; H NMR (400 MHz, DMSO-d6)
d 7.08e6.94
735, 500 cmꢁ1
;
1H NMR (400 MHz, DMSO-d6)
d
9.78 (s, 1H),
(m, 2H), 6.69 (d, J ¼ 2.3 Hz, 1H), 5.06 (s, 2H), 4.66 (s, 2H).
7.65e7.48 (m, 4H), 7.47e7.38 (m,1H), 6.94 (d, J ¼ 8.2 Hz,1H), 6.86 (s,
1H), 4.12 (q, J ¼ 6.9 Hz, 2H), 2.37 (s, 3H), 1.40 (t, J ¼ 6.9 Hz, 3H); 13
C
4.1.38. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-5-
fluorobenzofuran-3(2H)-one (59)
NMR (100 MHz, DMSO-d6)
d
183.0, 163.3, 149.9, 146.9, 145.1, 137.9,
132.9, 126.0, 123.4, 123.1, 121.2, 116.5, 116.2, 113.5, 112.7, 63.9, 20.2,
14.7; HRMS (ESI): calcd for C18H16NaO4 [MþNa]þ, 319.0946; found,
319.0951.
This compound was obtained from 53 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
78%; purity (gradient I) 99.3%; m.p. 157e158 ꢀC; IR (KBr) nmax 3517,
2987, 2942, 2893, 1693, 1645, 1577, 1516, 1481, 1436, 1307, 1285,
4.1.32. 5-Fluorobenzofuran-3(2H)-one (53)
1259, 1175, 1105, 1037, 902, 812, 783, 738, 503 cmꢁ1 1H NMR
;
To a solution of 1-fluoro-4-methoxybenzene (48, 10.0 mmol) in
dichloromethane (30 mL) was added chloroacetyl chloride
(11.0 mmol) at 0 ꢀC. After stirring for 15 min, the reaction mixture
was added anhydrous aluminium chloride (11.0 mmol) over
30 min. After stirring for 6 h at 40 ꢀC, the reaction mixture was cool
to rt. After stirring for 72 h at rt, the reaction mixture was poured
into ice water (50 mL) and extracted with dichloromethane
(3 ꢂ 30 mL). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, and evaporated under vacuum
without further purification. To a solution of the residue in ethanol
(30 mL) was added anhydrous sodium acetate (20.0 mmol). After
refluxing for 2 h, the reaction mixture was cooled to rt and
concentrated under vacuum. The residue was dissolved in water
(50 mL) and extracted with dichloromethane (3 ꢂ 40 mL). The
combined organic phases were washed with brine, dried over
anhydrous Na2SO4, and evaporated under vacuum. The crude
mixture was purified by flash chromatography on silica gel (pe-
troleum ether/ethyl acetate ¼ 40/1) to afford the title compound.
Bright yellow solid, yield 60%; 1H NMR (400 MHz, CDCl3)
(400 MHz, DMSO-d6) d 9.87 (s, 1H), 7.74e7.48 (m, 5H), 7.01e6.87
(m, 2H), 4.12 (q, J ¼ 6.9 Hz, 2H), 1.39 (t, J ¼ 6.9 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6)
d
182.5 (J
¼ 3.2 Hz, 1C), 161.1, 158.2 (J
C-F
C-
¼ 242.1 Hz,1C),149.8,146.9,145.4,126.2,124.3 (J C-F ¼ 26.1 Hz,1C),
F
123.1,122.0 (J C-F ¼ 8.3 Hz,1C),116.6,116.1,114.9,114.8 (J C-F ¼ 8.3 Hz,
1C), 109.6 (J
¼ 24.4 Hz, 1C), 63.9, 14.7; HRMS (ESI): calcd for
C-F
C
17H13FNaO4 [MþNa]þ, 323.0696; found, 323.0702.
4.1.39. (Z)-5-Chloro-2-(3-ethoxy-4-hydroxybenzylidene)
benzofuran-3(2H)-one (60)
This compound was obtained from 54 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
24%; purity (gradient I) 99.9%; m.p. 202e203 ꢀC; IR (KBr) nmax 3389,
3070, 2971, 2925, 2880, 1706, 1648, 1577, 1513, 1458, 1317, 1262,
1175, 1127, 1047, 918, 805, 709, 625, 502 cmꢁ1; 1H NMR (400 MHz,
DMSO-d6) d 9.86 (s, 1H), 7.78 (s, 2H), 7.69e7.42 (m, 3H), 6.92 (s, 2H),
4.10 (q, J ¼ 7.4 Hz, 2H), 1.37 (t, J ¼ 7.4 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6)
d 181.8, 163.3, 149.9, 146.9, 145.0, 136.4, 127.9, 126.3,
123.3, 123.0, 122.7, 116.7, 116.2, 115.1, 114.9, 63.9, 14.7; HRMS (ESI):
d
7.36e7.16 (m, 2H), 7.04 (d, J ¼ 8.8 Hz, 1H), 4.61 (s, 2H).
calcd for C17H13ClNaO4 [MþNa]þ, 339.0400; found, 339.0407.
4.1.33. 5-Chlorobenzofuran-3(2H)-one (54)
4.1.40. (Z)-5-Bromo-2-(3-ethoxy-4-hydroxybenzylidene)
benzofuran-3(2H)-one (61)
This compound was obtained from 49 according to the meth-
odology described for 53. Bright yellow solid, yield 55%; 1H NMR
This compound was obtained from 55 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
30%; purity (gradient I) 99.0%; m.p. 207e209 ꢀC; IR (KBr) nmax 3376,
3067, 2971, 2925, 2877, 1709, 1648, 1600, 1581, 1513, 1462, 1282,
1201, 1182, 1130, 1047, 921, 805, 696, 622 cmꢁ1; 1H NMR (400 MHz,
(400 MHz, CDCl3)
1H), 7.11 (d, J ¼ 8.8 Hz, 1H), 4.67 (s, 2H).
d
7.64 (d, J ¼ 2.4 Hz, 1H), 7.56 (dd, J ¼ 8.8, 2.3 Hz,
4.1.34. 5-Bromobenzofuran-3(2H)-one (55)
This compound was obtained from 50 according to the meth-
CDCl3)
d
7.92 (d, J ¼ 2.2 Hz, 1H), 7.72 (dd, J ¼ 8.7, 2.2 Hz, 1H),
odology described for 53. Bright yellow solid, yield 53%; 1H NMR
7.51e7.39 (m, 2H), 7.23 (d, J ¼ 8.7 Hz, 1H), 7.00 (d, J ¼ 8.3 Hz, 1H),
(400 MHz, CDCl3)
d
7.79 (d, J ¼ 2.2 Hz, 1H), 7.69 (dd, J ¼ 8.8, 2.2 Hz,
6.87 (s, 1H), 6.06 (s, 1H), 4.21 (q, J ¼ 7.0 Hz, 2H), 1.51 (t, J ¼ 7.0 Hz,

206
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
3H); 13C NMR (100 MHz, DMSO-d6)
d
181.6, 163.6, 149.9, 146.9,
7.10e6.91 (m, 1H), 4.86 (s, 2H).
144.8, 139.1, 126.3 (2C), 123.2, 123.0, 116.6, 116.1, 115.6, 115.5, 114.9,
63.8, 14.7; HRMS (ESI): calcd for C17H13BrNaO4 [MþNa]þ, 382.9895;
found, 382.9897.
4.1.46. 6-Chlorobenzofuran-3(2H)-one (72)
This compound was obtained from 70 according to the meth-
odology described for the synthesis of 6. Yellow solid, yield 45%; 1H
4.1.41. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-5-iodobenzofuran-
3(2H)-one (62)
NMR (400 MHz, CDCl3)
1H), 7.08 (dd, J ¼ 8.3, 1.6 Hz, 1H), 4.66 (s, 2H).
d
7.60 (d, J ¼ 8.3 Hz, 1H), 7.17 (d, J ¼ 1.6 Hz,
This compound was obtained from 56 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
24%; purity (gradient I) 95.0%; m.p. 215e216 ꢀC; IR (KBr) nmax 3534,
3070, 2974, 2929, 1700, 1642, 1597, 1581, 1516, 1458, 1265, 1195,
1175, 1127, 1040, 918, 812, 693, 587, 500 cmꢁ1; 1H NMR (400 MHz,
4.1.47. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-6-
fluorobenzofuran-3(2H)-one (73)
This compound was obtained from 71 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
80%; purity (gradient I) 96.6%; m.p. 141e142 ꢀC; IR (KBr) nmax 3553,
3488, 3090, 2977, 2797, 1693, 1645, 1600, 1590, 1516, 1436, 1410,
1288, 1265, 1172, 1124, 1095, 1043, 963, 895, 809, 760, 657,
DMSO-d6)
d 9.88 (s, 1H), 8.14e7.98 (m, 2H), 7.58 (s, 1H), 7.52 (d,
J ¼ 8.3 Hz,1H), 7.43 (d, J ¼ 8.5 Hz,1H), 6.93 (s, 2H), 4.11 (q, J ¼ 7.0 Hz,
2H), 1.39 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 181.4,
164.2,149.9,146.9,144.7,144.4,132.1,126.3,123.7,123.0,116.5,116.1,
115.8, 114.6, 87.3, 63.9, 14.7; HRMS (ESI): calcd for C17H13INaO4
[MþNa]þ, 430.9756; found, 430.9734.
509 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 7.88e7.71 (m,1H), 7.54e7.36
(m, 2H), 7.10e6.88 (m, 3H), 6.85 (s, 1H), 6.06 (s, 1H), 4.22 (q,
J ¼ 7.0 Hz, 2H), 1.52 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
181.7, 167.1 (J
¼ 243.5 Hz, 1C), 165.8, 147.1, 145.1, 145.0, 125.6,
C-F
4.1.42. (Z)-5-Amino-2-(3-ethoxy-4-hydroxybenzylidene)
benzofuran-3(2H)-one (63)
125.4 (J C-F ¼ 11.8 Hz, 1C), 123.4, 117.6, 114.0, 113.3, 113.2, 110.8 (J C-
¼ 24.0 Hz, 1C), 99.7 (J
¼ 26.7 Hz, 1C), 63.7, 13.8; HRMS (ESI):
F
C-F
This compound was obtained from 58 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
60%; purity (gradient II) 97.4%; m.p. 213e216 ꢀC; IR (KBr) nmax 3386,
3308, 2980, 2909, 2874, 1693, 1642, 1584, 1513, 1494, 1436, 1391,
1288, 1259, 1198, 1153, 1105, 1047, 895, 860, 812, 786, 738 cmꢁ1; 1H
calcd for C17H13FNaO4 [MþNa]þ, 323.0696; found, 323.0676.
4.1.48. (Z)-6-Chloro-2-(3-ethoxy-4-hydroxybenzylidene)
benzofuran-3(2H)-one (74)
This compound was obtained from 72 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
80%; purity (gradient I) 99.1%; m.p. 175e176 ꢀC; IR (KBr) nmax 3534,
3067, 2977, 2929, 1690, 1645, 1603, 1581, 1513, 1423, 1304, 1269,
NMR (400 MHz, DMSO-d6) d 9.70 (s, 1H), 7.55 (s, 1H), 7.52e7.45 (m,
1H), 7.25 (d, J ¼ 8.7 Hz, 1H), 7.08e7.00 (m, 1H), 6.92 (d, J ¼ 8.2 Hz,
1H), 6.83 (d, J ¼ 2.3 Hz, 1H), 6.77 (s, 1H), 5.23 (s, 2H), 4.10 (q,
J ¼ 7.0 Hz, 2H),1.38 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
1191, 1137, 1121, 1063, 921, 857, 805, 770, 622, 500 cmꢁ1 1H NMR
;
d
183.7, 157.7, 149.2, 146.8, 145.6, 145.3, 125.6, 124.2, 123.6, 121.4,
(400 MHz, CDCl3)
d
7.72 (d, J ¼ 8.1 Hz, 1H), 7.53e7.39 (m, 2H), 7.34
116.4, 116.1, 113.1, 112.4, 105.3, 63.9, 14.7; HRMS (ESI): calcd for
(s, 1H), 7.20 (d, J ¼ 8.2 Hz, 1H), 7.00 (d, J ¼ 8.1 Hz, 1H), 6.86 (s, 1H),
C
17H15NNaO4 [MþNa]þ, 320.0899; found, 320.0895.
6.08 (s, 1H), 4.21 (q, J ¼ 6.9 Hz, 2H), 1.51 (t, J ¼ 6.9 Hz, 3H); 13C NMR
(100 MHz, CDCl3)
d 182.9, 165.8, 148.2, 146.0, 145.8, 142.4, 126.7,
4.1.43. (Z)-2-(3,4-Dihydroxybenzylidene)-6-hydroxybenzofuran-
3(2H)-one (67)
125.3, 124.4, 124.2, 120.6, 115.0, 114.8, 114.2, 113.5, 64.7, 14.8; HRMS
(ESI): calcd for C17H13ClNaO4 [MþNa]þ, 339.0400; found, 339.0401.
This compound was obtained from 65 and 3,4-
dihydroxybenzaldehyde employing method B. Yellow solid, yield
44%; purity (gradient II) 98.0%; m.p. > 270 ꢀC; IR (KBr) nmax 3305,
3154, 1680, 1597, 1584, 1510, 1307, 1282, 1191, 1130, 1108, 989, 892,
4.1.49. 7-Methoxybenzofuran-3(2H)-one (77)
2-(2-Methoxyphenoxy) acetic acid (76, 5.0 mmol) was dissolved
in thionyl chloride (15 mL) at rt. After refluxing for 4 h, the reaction
mixture was cooled to rt and concentrated under vacuum. The
residue was dissolved in dichloromethane (50 mL) and aluminum
chloride (10.0 mmol) was added slowly at 0 ꢀC. After stirring for
0.5 h at 0 ꢀC and 1 h at rt, the reaction mixture was then poured into
ice water (60 mL) and extracted with ethyl acetate (3 ꢂ 30 mL). The
combined organic phases were washed with brine, dried over
anhydrous Na2SO4, and evaporated under vacuum. The residue was
purified by flash chromatography on silica gel (petroleum ether/
ethyl acetate ¼ 30/1) to afford the title compound. Bright yellow
831, 767, 661, 513 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
; d 11.11 (s,
1H), 9.68 (s,1H), 9.28 (s,1H), 7.61 (d, J ¼ 8.4 Hz,1H), 7.46 (s,1H), 7.25
(d, J ¼ 8.2 Hz,1H), 6.85 (d, J ¼ 8.2 Hz,1H), 6.80e6.67 (m, 2H), 6.64 (s,
1H); 13C NMR (100 MHz, DMSO-d6)
d 181.1, 167.4, 166.0, 147.9, 145.6,
145.5, 125.7, 124.5, 123.4, 117.9, 116.0, 113.2, 112.8, 111.9, 98.3; HRMS
(ESI): calcd for C15H9O5 [MꢁH]ꢁ, 269.0450; found, 269.0454.
4.1.44. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-6-
methoxybenzofuran-3(2H)-one (68)
This compound was obtained from 66 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
72%; purity (gradient I) 99.7%; m.p. 182e183 ꢀC; IR (KBr) nmax 3215,
2971, 2922,1687, 1642, 1594, 1510,1429, 1301, 1272, 1191, 1137,1098,
solid, yield 27%; 1H NMR (400 MHz, CDCl3)
d 7.22e7.17 (m, 1H), 7.05
(d, J ¼ 7.9 Hz, 1H), 7.00e6.93 (m, 1H), 4.61 (s, 2H), 3.89 (s, 3H).
4.1.50. (Z)-2-(3-Ethoxy-4-hydroxybenzylidene)-7-
methoxybenzofuran-3(2H)-one (78)
1011, 912, 821 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d
7.75e7.67 (m, 1H),
7.47 (dd, J ¼ 8.3, 1.9 Hz, 1H), 7.44e7.39 (m, 1H), 6.99 (d, J ¼ 8.3 Hz,
This compound was obtained from 77 and 3-ethoxy-4-
hydroxybenzaldehyde employing method A. Yellow solid, yield
69%; purity (gradient I) 99.8%; m.p. 155e157 ꢀC; IR (KBr) nmax 3427,
3073, 2971, 2925, 2832, 1703, 1651, 1603, 1581, 1516, 1310, 1272,
1H), 6.83e6.70 (m, 3H), 6.01 (s, 1H), 4.22 (q, J ¼ 7.0 Hz, 2H), 3.93 (s,
3H), 1.51 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 181.8, 167.1,
166.1, 146.7, 145.6, 144.9, 125.1, 124.7, 123.8, 114.1, 113.9, 113.2, 111.7,
111.0, 95.6, 63.6, 55.0, 13.8; HRMS (ESI): calcd for C18H16NaO5
[MþNa]þ, 335.0895; found, 335.0877.
1208, 1124, 1069, 912, 879, 805, 744 cmꢁ1
;
1H NMR (400 MHz,
CDCl3)
d
7.57 (d, J ¼ 1.9 Hz,1H), 7.47 (dd, J ¼ 8.3, 1.9 Hz,1H), 7.39 (dd,
J ¼ 6.8, 2.0 Hz,1H), 7.18e7.09 (m, 2H), 7.00 (d, J ¼ 8.3 Hz,1H), 6.88 (s,
1H), 6.04 (s, 1H), 4.24 (q, J ¼ 7.0 Hz, 2H), 4.02 (s, 3H), 1.51 (t,
4.1.45. 6-Fluorobenzofuran-3(2H)-one (71)
This compound was obtained from 69 according to the meth-
J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d 183.1, 154.4, 149.7,
odology described for the synthesis of 6. Yellow solid, yield 42%; 1H
146.9, 145.5, 144.8, 126.0, 124.2, 123.2, 122.6, 119.1, 116.6, 116.2,
114.9, 114.2, 63.9, 56.4, 14.7; HRMS (ESI): calcd for C18H16NaO5
NMR (400 MHz, DMSO-d6)
d 7.77e7.66 (m, 1H), 7.28e7.19 (m, 1H),

S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
207
[MþNa]þ, 335.0895; found, 335.0906.
impairment as indicated in each experiment.
4.1.56. Insulin secretion/content measurement
4.1.51. DRAK2 in vitro kinase assay
Recombinant GST fused DRAK2 protein were prepared as pre-
vious reported [6].DRAK2 kinase assays was carried out based on
ADP produced during the DRAK2 autophosphorylation process,
with Promega ADP-Glo kits (Promega, Madison, WI). The kinase
Glucose-stimulated insulin secretion was determined as previ-
ously described in detail [35]. Islets were cultured for 48 h in
complete RPMI 1640 medium with 10% FBS in the absence or
presence of various stimulants; then pick out 10 islets/well trans-
ferred to 48-well plates. The islets were incubated in Kreb's buffer
(135 mM NaCl, 3.6 mM KCl, 5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM
CaCl2, 2 mM NaHCO3, 10 mM HEPES (pH 7.4), and 0.1% BSA) for 1 h,
and then incubated in Kreb's buffer containing 2.8 mM or 16.7 mM
glucose for another hour at 37 ꢀC. One hundred microliters of su-
pernatant was removed for determination of insulin levels. Insulin
was determined by HTRF kit (Cisbio).
reaction were conducted in a volume of 5
50 mM (pH7.0), NaN3 0.02%, Orthovanadate 0.1 mM, 5 mM MgCl2,
0.01% (w/v) bovine serum albumin), contained 0.5 g DRAK2-GST,
mL 1X Buffer (HEPES
m
20
mM ATP, and was reacted for 2 h at room temperature. The
florescence signal was read on an EnVision® Multilabel Reader to
calculate the DRAK2 kinase activity. Inhibition data was shown as
means SD of three replicates.
4.1.52. Protein kinase profiling
4.1.57. Antibodies and immunoblotting
Protein kinase profiling of compounds 1, 40, 41, and 67 was
performed within a panel of 27 human protein kinases. Percentage
of kinase activities were determined for compounds 1, 40, 41 and
Cleaved PARP, cleaved-caspase3 and cleaved-caspase9 anti-
bodies were purchased from Cell Signaling Technologies. b-actin
antibody was purchased from ABGENT (San Diego, CA, USA).
Western immunoblotting was performed as described previously
[36].
67 at 10
enzyme control without inhibitor. IC50 values were calculated via
four-parameter nonlinear regression using Prism software.
mM. The percentage inhibition was calculated relative to an
6
Profiling data was shown as means SD of three replicates.
Acknowledgments
4.1.53. Primary pancreatic islet isolation
This work was supported by grants from the National Key
Technology R&D Program (2015BAK45B00), Science and Technol-
ogy Commission of Shanghai Municipality (16430711700 and
Mouse pancreatic islets were isolated using the Liberase diges-
tion method [33]. Euthanize C57BL/6J mice (20e24 g) by cervical
dislocation, saturate the mouse torso with 70% EtOH, open
abdominal cavity completely. Find the hepatic artery, portal vein
and bile duct bundle, tie tight close to the liver as close as possible,
insert the cannula into the bile duct up to half the length of the bile
duct, and then gently pull the suture tight around the cannula.
Digestion solution (2 mL HBSS containing 20 mM HEPES and 1 mg/
mL collagenase P (Roche) enzyme solution) was injected into the
common bile duct. After inflation of the pancreas is completed,
remove the pancreas from spleen. Harvest the pancreas and
transfer it to a 15 mL tube containing 2 mL cold collagenase P
enzyme solution. Incubate 10 min in a 37 ꢀC water bath. After in-
cubation, shake the tube by hand in 37 ꢀC water bath for about
8 min. Wash the conical tube with 5 mL Quenching buffer (HBSS
containing 10% FBS), spin at RT 300 g for 3 min. Take out super-
natant and wash pellet with 10 mL cold Quenching buffer transfer
the mixture into a 15 mL plastic tube. Spin at RT 300 g for 3 min.
Add 2 mL of 25% Ficoll solution to the pellet and mix by pipette up
and down. Slowly add 1 mL each of the next three layers one by
one: 23%, 21%, 13% to form the gradient. Put tube on ice until all of
the tubes are ready to spin. Spin at 2500 g for 20 min at 4 ꢀC. The
islet layer should be visible between the 13% and 21% gradient
which near the 4.5 mL mark on the tube. Collect islets and place it
into a 5e10 cm cell culture dish with 3e5 mL regular cell culture
media. Wash the islet layer in 5 mL HBSS buffer, 1000 g 5 min. Re-
suspended the islets in 1 mL RPMI 1640 complete medium, and
then used for experiment.
16JC1405000),
and
Chinese
Academy
of
Sciences
(CASIMM0120162021), and Shanghai Science and Technology
Council (No 16DZ2280100 and 15DZ2291600). This work was also
supported by Juvenile Diabetes Foundation (17-2013-440) and J.-
Louis Levesque Foundation to J.W. We also thank the Laboratory
of Organic Functional Molecules, Sino-French Institute of ECNU for
support.
Abbreviations used
c-MET tyrosine-protein kinase Met
ALK
anaplastic lymphoma kinase
Ack1
MELK
IRK
activated CDC42 kinase 1
maternal embryonic leucine zipper kinase
inwardly rectifying potassium channels
mTORC1 mammalian target of rapamycin complex 1
Bcl
B-cell lymphoma
Bcl-xl
Flip
LDA
DMF
NBS
THF
rt
B-cell lymphoma-extra large
FLICE inhibitory protein
Lithium diisopropylamide
N,N-dimethylformamide
N-bromobutanimide
tetrahydrofuran
room temperature
DRAK1 DAP kinase-related apoptosis-inducing protein kinase
DAPK1 death-associated protein kinase 1
DAPK2 death-associated protein kinase 2
4.1.54. Cell culture
DAPK3 death-associated protein kinase 3
The rat insulinoma INS-1 cell line was kindly provided by Dr. Liu
Yong (Institute for Nutritional Sciences, Chinese Academy of Sci-
ences) and maintained in RPMI 1640 as described [34]. INS-1 cells
between passages 20e30 were used.
BLK
ITK
TEK
JAK
B lymphoid tyrosine kinase
IL2-inducible T-cell kinase
tunica interna endothelial cell kinase
Janus kinase
SYK
CHK1
AKT1
spleen tyrosine kinase
checkpoint kinases 1
v-akt murine thymoma viral oncogene homolog 1
4.1.55. Treatment of islets and INS-1 with palmitate
Isolated islets were cultured in 48-well plates for about 10 islets/
well in RPMI 1640 with 10% FBS. INS-1 cells were cultured in 48-
well plates in RPMI 1640 with 10% FBS. Palmitate (0.4 mM;
Sigma-Aldrich) was added to the wells to induce the islets function
Aurora-A aurora kinase A
EGFR
BTK
epidermal growth factor receptor
bruton agammaglobulinemia tyrosine kinase

208
S. Wang et al. / European Journal of Medicinal Chemistry 130 (2017) 195e208
1436e1445.
CDK
MELK
IKK
GSK3
MEK1
ERK1
HBSS
cyclin-dependent kinase
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maternal embryonic leucine zipper kinase
inhibitor of nuclear factor kappa-B kinase subunit beta
glycogen synthase kinase 3 beta
mitogen-activated protein kinase kinase 1
mitogen-activated protein kinase 3
b
b
Hank's balanced salt solution
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
FBS
RPMI
INS-1
BSA
fetal bovine serum
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Racemic ([2s[2r*[r[r*]]]] and ([2r[2s*[s[s*]]]]-( )- a,
a0 -[imino-bis(methylene)]
bovine serum albumin
bis[6-fluoro-chroman-2-methanol]
and
its
Pure
[2s[2r*[r[r*]&,
HTRF
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homogeneous time resolved fluorescence
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.02.048.
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