Design, synthesis, and characterization of novel apigenin analogues that suppress pancreatic stellate cell proliferation in vitro and associated pancreatic fibrosis in vivo

Article abstract of DOI:10.1016/j.bmc.2014.04.043

Accumulating evidence suggests that activated pancreatic stellate cells (PSC) play an important role in chronic pancreatitis (CP), and inhibition of the activated PSC is considered as a potential strategy for the treatment and prevention of CP. Herein, we disclose our findings that apigenin and its novel analogues suppress the proliferation and induce apoptosis in PSC, which reduce the PSC-mediated fibrosis in CP. Chemical modifications of apigenin have been directed to build a focused library of O-alkylamino-tethered apigenin derivatives at 4′-O position of the ring C with the attempt to enhance the potency and drug-like properties including aqueous solubility. A number of compounds such as 14, 16, and 24 exhibited potent antiproliferative effects as well as improved aqueous solubility. Intriguingly, apigenin, new analogues 23 and 24 displayed significant efficacy to reduce pancreatic fibrosis even at a low dose of 0.5 mg/kg in our proof-of-concept study using a preclinical in vivo mouse model of CP.

Full text of DOI:10.1016/j.bmc.2014.04.043

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and characterization of novel apigenin analogues
that suppress pancreatic stellate cell proliferation in vitro and
associated pancreatic fibrosis in vivo
Haijun Chen a, , Amy A. Mrazek b, , Xiaofu Wang b, Chunyong Ding a, Ye Ding a, Laura J. Porro b,
Huiling Liu a, Celia Chao b, Mark R. Hellmich b, , Jia Zhou a,
⇑
⇑
a Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, United States
b Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Accumulating evidence suggests that activated pancreatic stellate cells (PSC) play an important role in
chronic pancreatitis (CP), and inhibition of the activated PSC is considered as a potential strategy for
the treatment and prevention of CP. Herein, we disclose our findings that apigenin and its novel ana-
logues suppress the proliferation and induce apoptosis in PSC, which reduce the PSC-mediated fibrosis
in CP. Chemical modifications of apigenin have been directed to build a focused library of O-alkyl-
amino-tethered apigenin derivatives at 40-O position of the ring C with the attempt to enhance the
potency and drug-like properties including aqueous solubility. A number of compounds such as 14, 16,
and 24 exhibited potent antiproliferative effects as well as improved aqueous solubility. Intriguingly, api-
genin, new analogues 23 and 24 displayed significant efficacy to reduce pancreatic fibrosis even at a low
dose of 0.5 mg/kg in our proof-of-concept study using a preclinical in vivo mouse model of CP.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 18 February 2014
Revised 14 April 2014
Accepted 22 April 2014
Available online xxxx
Keywords:
Chronic pancreatitis
Pancreatic stellate cells
Fibrosis
Apigenin
Apigenin analogues
Therapy
1. Introduction
Within the last two decades, it has been well-established that
pancreatic stellate cells (PSC) are responsible for the fibrotic com-
Chronic pancreatitis (CP) is a progressive, non-curable disorder
of the pancreas.1 Pathologically, both the endocrine and exocrine
pancreas undergo progressive and often irreversible morphological
changes, including glandular fibrosis.2–5 In the United States, disor-
ders of the exocrine pancreas affect over 1 million patients and
result in a cost over $3.7 billion annually.6,7 Current treatment
options for CP are limited to supportive and palliative care;
patients with advanced disease can be managed with endoscopic
and/or surgical pancreatic decompression, denervation, resection,
bypass or transplantation.8,9 Overall, patients have a poor quality
of life and are burdened by chronic abdominal pain, increased hos-
pitalizations, impaired digestion, diarrhea, weight loss, diabetes,
complications like pseudocysts and an increased risk of pancreatic
cancer.9 Therefore, the development of effective, safe and afford-
able therapeutic agents remains a critical need.
ponent of CP, and suppressing PSC is therefore a potential thera-
peutic target for the disease.10–12 In the normal pancreas, PSC are
inactive/quiescent, whereas during tissue injury, PSC display an
activated, myofibroblastic phenotype, with increased proliferation,
motility, and secretion of extracellular matrix proteins.10–12 CP
favors the perpetual activation of PSC.13 Consequently, a promising
strategy for the prevention and treatment of CP involves limiting
the proliferation and inducing apoptosis of activated PSC.13–16
The sentinel acute pancreatitis event (SAPE) hypothesis pro-
vides a unified model for the pathogenesis of CP.17 After studying
cases of hereditary pancreatitis, Whitcomb et al. found that 50%
of patients with gain-of-function trypsinogen mutations experi-
enced repeated episodes of acute pancreatitis (AP) that later
developed into CP.1,17 Regardless of the inciting etiology(s) of the
sentinel event of AP, recurrent episodes of AP cause CP. AP is initi-
ated with acinar cell injury, characterized by premature acinar
zymogen activation, recruitment of inflammatory cells, auto-
digestion and necrosis of acinar and ductal cells, subsequent
anti-inflammatory response, and PSC-dependent scarring.1,5,17–19
Recurrent pancreatic injury overwhelms normal repair mecha-
nisms, favoring progressive irreversible fibrosis.1,5,17 We have
directed our efforts to develop novel compounds that would limit
⇑
Corresponding authors. Tel.: +1 (409) 772 1845; fax: +1 (409) 772 6368
(M.R.H.); tel.: +1 (409) 772 9748; fax: +1 (409) 772 9648 (J.Z.).
E-mail addresses: mhellmic@utmb.edu (M.R. Hellmich), jizhou@utmb.edu
(J. Zhou).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.04.043
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.043

2
H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
including limited aqueous solubility, and none of them has been
4'
OH
approved for clinical investigation.26,27 Herein, we report our
design and synthesis of novel apigenin analogues that suppress
the proliferation and promote apoptosis in activated PSC with
improved potency and favorable physicochemical properties. More
importantly, we also disclose that at a low dose, new analogues 23
and 24 are as efficacious as apigenin in reducing pancreatic fibrosis
in a preclinical animal model, providing the proof-of-concept as
potential therapeutics for CP.
C
7
HO
O
B
A
5
OH
O
Apigenin
Figure 1. Chemical structure of apigenin.
2. Results and discussion
2.1. Design
repeated pancreatic injury, and thus minimize the progression of
CP.
Natural products, especially common dietaries consumed on a
daily basis, continue to serve as a valuable source in developing
drug-like candidates for chemoprevention and chemotherapy.20–
Considerable efforts on the modifications of apigenin as the lead
compound in anticancer drug design have been made. Reported
structural-activity relationship (SAR) studies indicate that the A
ring of apigenin as well as its C ring are suitable for diverse modifi-
cations.28–31 In addition, increasing studies have demonstrated that
the polymethoxylated flavones or flavone analogues with nonpolar
and hydrophobic substituents on A ring generally exhibit more
potent antiproliferative activities against various human cancer cell
lines.28,32 Moreover, methylated flavones have dramatically higher
intestinal permeability and higher metabolic stability.33–35 Com-
bined with the structural features of the SAR trends, we directed
our initial optimization effort to discover novel apigenin derivatives
by introducing aqueous solubility-enhancing moieties at 40-O posi-
tion of apigenin with 5,7-dimethoxy groups on the A ring.
22
Flavonoids, which are ubiquitously distributed in many dietary
plant materials, have received a great deal of attention because
some of them have been shown to exert various beneficial effects
on human health.23 Apigenin (Fig. 1) is abundantly present in com-
mon fruits and vegetables, and has gained particular interest in
recent years as a beneficial and health-promoting agent because
of its low intrinsic toxicity.24 Apigenin has been demonstrated to
possess various clinically relevant properties such as anti-inflam-
matory, anti-oxidant, antiproliferative and pro-apoptotic activities
likely through multiple mechanisms.25 Our research efforts on this
natural product with low intrinsic toxicity have led to the discov-
ery that apigenin can ameliorate the stromal fibrosis characteristic
of CP. Despite its promising anti-fibrotic property, apigenin suffers
from poor aqueous solubility and low metabolic stability like most
flavonoids, limiting its clinical potential. While many research
groups focused on developing novel compounds based on the api-
genin structure as anticancer agents, most of these apigenin deriv-
atives suffered from unfavorable physicochemical properties,
2.2. Chemistry
The synthesis of new apigenin derivatives with chemical opti-
mizations on 40-hydroxyl group is outlined in Scheme 1. The key
intermediate 5 was prepared in a three-step synthesis starting
O
OH
O
OH
O
MeO
O
a
b
c
MeO
MeO
OMe
MeO
OMe
O
OMe O
2
4
5
N
O
OH
F
Br
R1
R1
=
MeO
O
O
d
7
N
8
9
O
N
N
N
OMe O
OMe O
O
6
7-13
13
12
10
11
O
O
Br
R2
N
R2
=
e
MeO
O
MeO
O
N
N
N
14
N
15
16
OMe O
OMe O
N
14-18
7
MeO2C
17
18
O
OH
R3
HN
MeO
O
MeO
O
f, g
R3
=
H2N
H2N
19
20
21
OMe O
19-21
OMe O
6
Scheme 1. Reagents and conditions: (a) 4-allyloxybenzaldehyde (3), 50% NaOH/H2O, EtOH, rt, 16 h, 76%; (b) cat. I2, DMSO, 140 °C, 4 h, 91%; (c) cat. Pd(PPh3)4, K2CO3, MeOH,
90 °C, 4 h, 95%; (d) R1OH, Ph3P, DIAD, THF, rt, 16 h, 81–94%; (e) R2H, KI, K2CO3, acetone, reflux, 18 h, 66–99%; (f) Boc-R3OH, Ph3P, DIAD, THF, rt, 2 h; (g) TFA, CH2Cl2, 0 °C to rt,
3 h, 59–90% (two steps).
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
with 1-(2-hydroxy-4,6-dimethoxyphenyl)ethanone (2) and 4-allyl-
2.3. Biology
oxybenzaldehyde (3) according to a literature procedure.36 As
shown in Scheme 1, base-catalyzed aldol condensation of 2 with
3 afforded the chalcone 4 in a yield of 76% with a simple purifica-
tion. The chalcone was cyclized in the presence of catalytic iodine
in dimethyl sulfoxide at 140 °C to provide the flavone 5 in high
yield. The allyl protecting group of flavones 5 was cleaved with a
catalytic amount of Pd(PPh3)4 in the presence of K2CO3 in MeOH
at reflux for 4 h to obtain the key intermediate 40-hydroxyflavone
6 for direct use without further purification.
New analogues 7–13 were conveniently synthesized by Mitsun-
obu reaction of the key intermediate 6 with appropriate substi-
tuted alcohols in high yields (81–94%). Alkylation of the bromide
intermediate 7 with the corresponding amine in the presence of
K2CO3 and KI in acetone introduced basic functionalities providing
final compounds 14–18 in 66–99% yields. Mitsunobu coupling of 6
with N-Boc-protected amino alcohols followed by the Boc-depro-
tection with the treatment of TFA in CH2Cl2 afforded analogues
19–21 with diversified O-alkylamino side chains in 59–90% yields
(two steps).
The calculated lipophilicity (cLogP) and topological polar sur-
face area (tPSA) values of all newly synthesized analogues are
listed in Table 1. The results indicate that all these new compounds
meet the criteria of Lipinski’s ‘Rule of Five’ and may have favorable
physicochemical properties. To explore a meaningful SAR and
examine how the substitutions on the key moieties affect biologi-
cal activities of new apigenin derivatives, we first evaluated the
in vitro antiproliferative effects of these analogues on transformed
PSC using AlamarBlue Cell Viability Assay (Life Technologies) as
described in the Section 4. AlamarBlue is a non-toxic reagent that
is converted to a highly fluorescent end product by viable cells. The
capabilities of these new analogues to inhibit the proliferation of
transformed PSC are summarized in Table 1.
The key intermediate 6 was found to display no significant anti-
proliferative effect even at 20 lM. After introduction of an O-bro-
moalkyl moiety or an O-fluoroalkyl moiety into the derivative 6
at 40-O position, compounds 7 and 8 showed a slightly increased
antiproliferative effects in comparison with 6, indicating that
appropriate modifications on 40-O position may regain the antipro-
liferative activity. O-Alkylamino-tethered derivatives 9, 10 and 12
The synthetic route to 3-amino-2-hydroxypropoxy-flavonesis
outlined in Scheme 2. The reaction of the key intermediate 6 with
excessive epichlorohydrin (22) and successive treatment of the
intermediate epoxide (23) with appropriate amines under reflux
afforded the desired derivatives 24–26 in yields of 71–94%. The
exhibited a moderate antiproliferative activity at 10 lM with
inhibitory effects of 49%, 51% and 59%, respectively. This finding
suggests that optimizations with a nitrogen-containing hydrophilic
moiety at 40-O position appears to be a viable strategy to yield
more potent compounds with a better aqueous solubility. To this
end, compounds 14 with a piperidinyl moiety and 16 with a pyrro-
Mitsunobu coupling of
6 with (2,2-dimethyl-1,3-dioxolan-4-
yl)methanol (solketal, 27) generated an intermediate which was
subjected to cleavage conditions using 0.5% HCl (aq) in EtOH to
produce the 1,2-diol derivative 28. For the synthesis of diversified
apigenin analogues on side chain of the ring C at para-position as
depicted in Scheme 3, the starting material 2 was condensed with
4-bromobenzaldehyde (29) to give chalcone 30, which was then
cyclized to give the key intermediate 31. Compound 33 was
obtained by Suzuki coupling reaction of 31 with 2-fluoropyri-
dine-5-boronic acid (32) in the presence of Pd(dppf)Cl2 catalyst
lidinyl group displayed potent antiproliferative activity at 10 lM
with inhibitory effects of 64% and 66%, respectively. Meanwhile,
we found that the tertiary amines with alkylated amino groups
appeared to be more favorable than secondary or primary amines
with free amino groups at the terminal of the side chains. For
instance, in comparison with compounds 14 and 16, analogues
19–21 only exhibited a moderate to low inhibitory effects. The
similar trend of SAR was also observed for derivatives 24–26 and
28. Compound 28 with a terminal OH group at the tail resulted
in a dramatic loss of activities compared with its according ana-
logues 24–26 with a terminal amino moiety. Interestingly, com-
pound 23 with an epoxide was identified as a highly potent
inhibitor suppressing PSC proliferation.
in
a yield of 85%. Palladium-catalyzed Buchwald–Hartwig
amination reaction of 31 with N,N-dimethylethylenediamine (34)
or 2-(pyrrolidin-1-yl)ethanamine (35) afforded the targeted com-
pounds 36 and 37 in yield of 64% and 57%, respectively.
Scheme 4 outlines the synthesis of demethylated derivatives 38
and 39. Generation of 38 was achieved in 79% yield by a mono-
demethylation of 16 using 2 equiv of boron tribromide at room
temperate for 2 h. Both methyl groups on A-ring of 16 were suc-
cessfully removed by treatment with 3 equiv of boron tribromide
for 24 h, leading to the demethylated analogue 39 in a yield of 73%.
Structural modifications of para-position on C-ring with
diversified substitutions were also investigated. Compound 33
with
a pyridinyl group on the para-position resulted in a
substantial loss of the activity. In contrast, introduction of
OH
O
O
OH
O
R4
MeO
O
MeO
O
a
b
MeO
O
OMe O
OMe O
OMe O
6
23
24-26
R4
=
N
N
N
24
25
26
OH
O
OH
OH
MeO
O
MeO
O
c,d
OMe O
OMe O
6
28
Scheme 2. Reagents and conditions: (a) epichlorohydrin (22), K2CO3, acetone, reflux, 24 h, 67%; (b) R4H, K2CO3, EtOH, reflux, 3 h, 71–94%; (c) Ph3P, DIAD, THF, (2,2-dimethyl-
1,3-dioxolan-4-yl)methanol (27), rt, 4 h; (d) 0.5 N HCl (aq), EtOH, reflux, 2 h, 71% (two steps).
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Br
OH
O
OH
O
MeO
O
a
b
MeO
OMe
MeO
OMe
Br
OMe O
31
30
2
N
F
c
MeO
O
OMe O
Br
33
H
N
R5
MeO
O
MeO
O
d
R5
=
N
N
OMe O
OMe O
36
37
31
36-37
Scheme 3. Reagents and conditions: (a) 4-bromobenzaldehyde (29), 50% NaOH/H2O, EtOH, rt, 16 h, 65%; (b) cat. I2, DMSO, 140 °C, 4 h, 92%; (c) 2-fluoropyridine-5-boronic
acid (32), Pd(dppf)Cl2, KOAc, THF/EtOH/H2O, 80 °C, 18 h, 85%; (d) NH2R5 [N,N-dimethylethylenediamine (34) for 36 or 2-(pyrrolidin-1-yl)ethanamine (35) for 37], Pd2(dba)3,
NaOtBu, ( )-BINAP, toluene, 80 °C, 48 h, 57–64%.
O
O
N
N
MeO
MeO
O
MeO
O
O
a
OMe O
OH
38
39
16
O
O
N
N
O
HO
O
O
b
OMe O
OH
16
Scheme 4. Reagents and conditions: (a) 1 N BBr3 (in CH2Cl2), CH2Cl2, rt, 2 h, 79%; (b) 1 N BBr3 (in CH2Cl2), CH2Cl2, rt, 24 h, 73%.
2-dimethylamino-ethylamino moiety or 2-pyrrolidin-1-yl-ethyl-
amino group displayed significantly better antiproliferative activi-
ties at 10 M with inhibitory effects of 60% and 51%, respectively.
Demethylation of compound 16 resulted in generation of two O-
demethylated compounds 38 and 39. We found that neither
mono-demethylation nor full demethylation of the methoxy
groups on the A-ring is favorable for the enhancement of activity,
and thus more extensive SAR study on the A-ring was not pursued.
The key intermediate 6 was found to display no significant anti-
instance, in comparison with compounds 14 and 16, analogues
19–21 only exhibited a moderate to low inhibitory effects. The
similar trend of SAR was also observed for derivatives 24–26 and
28. Compound 28 with a terminal OH group at the tail resulted
in a dramatic loss of activities compared with its according
analogues 24–26 with a terminal amino moiety. Interestingly,
compound 23 with an epoxide was identified as a highly potent
inhibitor suppressing PSC proliferation.
Structural modifications of para-position on C-ring with diversi-
fied substitutions were also investigated. Compound 33 with a
pyridinyl group on the para-position resulted in a substantial loss
of the activity. In contrast, introduction of 2-dimethylamino-ethyl-
amino moiety or 2-pyrrolidin-1-yl-ethylamino group displayed
l
proliferative effect even at 20 lM. After introduction of an O-bro-
moalkyl moiety or an O-fluoroalkyl moiety into the derivative 6
at 40-O position, compounds 7 and 8 showed a slightly increased
antiproliferative effects in comparison with 6, indicating that
appropriate modifications on 40-O position may regain the antipro-
liferative activity. O-Alkylamino-tethered derivatives 9, 10 and 12
significantly better antiproliferative activities at 10 lM with inhib-
itory effects of 60% and 51%, respectively. Demethylation of com-
pound 16 resulted in generation of two O-demethylated
compounds 38 and 39. We found that neither mono-demethyla-
tion nor full demethylation of the methoxy groups on the A-ring
is favorable for the enhancement of activity, and thus more exten-
sive SAR study on the A-ring was not pursued.
Since one goal of our drug discovery effort was to identify new
apigenin derivatives with improved water-solubility and oral bio-
availability, aqueous solubility of several selected analogues with
enhanced antiproliferative effects was determined by an HPLC anal-
ysis method. Since one goal of our drug discovery effort was to iden-
tify new apigenin derivatives with improved water-solubility and
exhibited a moderate antiproliferative activity at 10 lM with
inhibitory effects of 49%, 51% and 59%, respectively. This finding
suggests that optimizations with a nitrogen-containing hydrophilic
moiety at 40-O position appears to be a viable strategy to yield
more potent compounds with a better aqueous solubility. To this
end, compounds 14 with a piperidinyl moiety and 16 with a pyrro-
lidinyl group displayed potent antiproliferative activity at 10 lM
with inhibitory effects of 64% and 66%, respectively. Meanwhile,
we found that the tertiary amines with alkylated amino groups
appeared to be more favorable than secondary or primary amines
with free amino groups at the terminal of the side chains. For
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 1
tions using cell proliferation and cell death assays, as well as an
in vivo model of CP. As shown in Figure 3A, both analogues 23
and 24 inhibited PSC proliferation at lower doses than apigenin,
indicating their enhanced potency in vitro. With logarithmic trans-
formation and nonlinear regression, a best-fit curve was generated,
allowing determination of their IC50, representing the concentra-
tion at which the compound causes 50% inhibition of PSC prolifer-
ation (Fig. 3B). Compound 23 was identified as a highly potent
Effects of apigenin and newly synthesized apigenin analogues on PSC proliferation
Entry
cLogPa
tPSAb
Inhibitory effectc (%)
5
l
M
10
lM
20 lM
Apigenin (1)
6
7
8
2.33
2.73
3.86
3.35
3.29
2.73
2.45
3.06
2.69
3.84
2.66
3.48
3.54
3.22
3.08
1.93
2.43
2.50
3.03
2.66
2.13
1.77
3.80
4.05
2.72
3.13
3.28
2.98
90.9
68.9
57.9
57.9
61.2
61.2
70.4
61.2
78.2
61.2
64.4
61.2
61.2
87.5
69.9
83.9
83.9
70.4
81.4
81.4
81.4
98.4
48.7
61.6
63.9
63.9
72.1
83.1
11
34
NE
NE
NE
49
51
NE
59
14
64
42
66
60
37
21
35
39
89
65
37
60
11
10
6
64
NE
20
21
NEd
NE
NE
28
19
NE
36
7
9
NDe
65
NE
86
33
97
67
98
72
67
ND
70
ND
93
95
57
71
16
22
19
84
80
99
90
analogue with an IC50 value of 2.5 0.6
slightly less potent with an IC50 value of 8.0 1.8
apigenin was 18.6 1.6 M. The irreversible fibrosis that defines
lM. Compound 24 was
10
11
12
13
14
15
16
17
18
19
20
21
23
24
25
26
28
31
33
36
37
38
39
l
M, and that of
l
CP is mediated by activated PSC, which produce and remodel the
extracellular matrix (ECM).10 Therefore, the potent antiprolifera-
tive effect of these new analogues on PSC supports their further
development as anti-fibrotic agents for CP.
45
22
48
33
22
NE
6
22
69
34
21
42
NE
12
NE
30
21
NE
2
These two representative analogues were also investigated to
determine whether the growth inhibition induced by compounds
23 and 24 in PSC was attributed to apoptosis. Apoptosis was deter-
mined using the Cell Death Detection ELISAPLUS assay. This
sandwich-enzyme-immunoassay involved antibodies binding to
cytoplasmic nucleosomes, which were specific to the process of
apoptosis rather than necrosis. Analogue 23 was found to be quite
potent, inducing greater PSC cell death at lower concentrations
than apigenin (Fig. 4A). At low concentrations, compound 24 failed
to induce apoptosis; however, between the concentrations of 25–
60
51
39
71
35
lM, it produced a steep dose–response, and concentrations
beyond 35
l
M induced significant cell death. Next, the dose–
response curve was generated (Fig. 4B). The EC50 represented the
concentration at which the compound yielded half of the maximal
amount of cell death. The most potent analogue 23 exhibited the
a
b
c
cLogP: http://146.107.217.178/lab/alogps/start.html.
tPSA: http://www.molinspiration.com/cgi-bin/properties.
Values are mean of at least three independent experiments.
NE: no effect.
ND: not determined.
lowest EC50 value of 9.6 1.8
lM, while the EC50 value of apigenin
d
e
was 24.5 2.5 M, and that of 24 was 35.2 5.5
l
lM. The capacity
of the compounds to induce cell death in activated PSC would most
likely translate into reduced fibrosis in CP. Analogue 23 more
potently induced PSC apoptosis than apigenin, while 24 appeared
to act through a different cellular mechanism, inducing stellate cell
oral bioavailability, aqueous solubility of several selected analogues
with enhanced antiproliferative effects was determined by an HPLC
analysis method.37 As depicted in Figure 2, compounds 14, 16 and
24 (in the form of HCl salt) have demonstrated to possess more
favorable aqueous solubility, with a saturated concentration of
17.4, 13.7 and 84.1 mg/mL, respectively, while that of apigenin is
toxicity at concentrations greater than 25 lM.
These encouraging results prompted us to further test 23 and
24 as lead compounds for the development of a new series of
anti-fibrotic agents which may be useful for treating CP.38 In order
to evaluate the anti-fibrotic effect of these new compounds as a
proof-of-concept, we tested the compounds in a preclinical animal
model of CP. CP was induced in mice using the well-established
model of repeated cerulean (CR) injections.39,40 Treatment with
vehicle, apigenin or analogues (0.5 mg/kg daily gavage) was started
the second week of the experiment and continued with CP
only 2.16 l
g/mL.26
To investigate more detailed information about the antiprolifer-
ative effects of this series of apigenin derivatives against human
PSC, compounds 23 (HJC0561) and 24 (HJC05100) were selected
based on their potency or aqueous solubility for further evalua-
Figure 2. Aqueous solubility of apigenin and selected apigenin derivatives. Compounds 14, 16 and 24 (in the form of HCl salt) showed significantly improved solubility as
compared with apigenin.
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 3. Effect of apigenin and its analogues on PSC proliferation. Human PSC were treated with apigenin or analogues at various doses for 24 h. (A) Representative data
from one experiment: cell viability was measured using the AlamarBlue colorimetric assay. (B) The IC50 values of apigenin, 23, and 24 are 18.6 1.6 M, 2.5 0.6 M, and
8.0 1.8 M, respectively. The calculated IC50 values are derived from the mean SEM of at least three independent experiments.
l
l
l
Figure 4. Effect of apigenin and its analogues on PSC apoptosis. Human PSC were treated with apigenin or analogues at various doses for 14 h. (A) Representative data from
one experiment: Apoptosis was measured using the Cell Death Detection ELISAPLUS assay. (B) The EC50 values of apigenin, 23, and 24 are 24.5 2.5
M, 9.6 1.8 M, and
35.2 5.5 M, respectively. The calculated EC50 values are derived from the mean SEM of at least three independent experiments.
l
l
l
induction. After four weeks, the pancreata were processed and
stained for fibronectin, which is a major component of the ECM.
The 400ꢀ microscope image in Figure 5A showed a large amount
of periacinar and perilobular fibrosis (brown color), edema (the
space between lobules), and atrophic, irregularly shaped acini.41
Treatment with apigenin and analogues 23 and 24 significantly
decreased the stromal fibrosis of CP, reduced tissue edema, and
limited acinar cell damage (Fig. 5B–D). ImageJ analysis of the slides
quantified the significant decrease in fibrosis (p <0.001) (Fig. 5E).
Despite no statistical difference between apigenin and compounds
23 or 24 at the low dose of 0.5 mg/kg, these data demonstrated
that apigenin and new analogues significantly reduced fibrosis in
the pre-clinical animal model of CP. The present investigation
has provided a proof-of-concept study that apigenin and analogues
have the potential to function as anti-fibrotic agents in CP. The rel-
evant mechanistic studies and further dose–response investigation
of in vivo efficacy are currently ongoing.
solubility. A series of novel apigenin analogues have been synthe-
sized from the key intermediate 6, and a number of compounds
such as 14, 16, and 24 have been identified to exhibit potent anti-
proliferative effects as well as improved aqueous solubility. Even at
a low dose of 0.5 mg/kg, apigenin and new analogues 23 and 24
significantly reduced the fibrotic response in a preclinical animal
model of CP, providing a proof-of-concept study that supports their
development as promising therapeutics for CP.
4. Experimental section
4.1. Chemistry
4.1.1. General
All commercially available starting materials and solvents were
reagent grade, and used without further purification. Reactions
were performed under a nitrogen atmosphere in dry glassware
with magnetic stirring. Preparative column chromatography was
performed using silica gel 60, particle size 0.063–0.200 mm (70–
230 mesh, flash). Analytical TLC was carried out employing silica
gel 60 F254 plates (Merck, Darmstadt). Visualization of the devel-
oped chromatograms was performed with detection by UV
(254 nm). NMR spectra were recorded on a Bruker-600 (1H,
600 MHz; 13C, 150 MHz) spectrometer. 1H and 13C NMR spectra
were recorded with TMS as an internal reference. Chemical shifts
were expressed in ppm, and J values were given in Hz. High-
resolution mass spectra (HRMS) were obtained from Thermo Fisher
LTQ Orbitrap Elite mass spectrometer. Parameters include the fol-
lowing: Nano ESI spray voltage was 1.8 kV; Capillary temperature
was 275 °C and the resolution was 60,000; Ionization was achieved
by positive mode. Melting points were measured on a Thermo
3. Conclusion
Accumulating evidence suggests that activated PSC play an
important role in CP. We have proposed a potential strategy for
the prevention and treatment of CP by decreasing the proliferation
and inducing apoptosis of PSC. While a few flavone-based com-
pounds have previously been reported to possess antiproliferative
effects for cancer, we are the first to show that apigenin and its
novel analogues suppress the proliferation of PSC and reduce the
associated fibrosis. Chemical modifications of apigenin have been
directed to build a focused library of O-alkylamino-tethered apige-
nin derivatives at 40-O position of the ring C with the attempt to
enhance the potency and drug-like properties including aqueous
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 5. Effect of apigenin and its analogues in preclinical animal model of CP. CP was induced using repeated cerulein (CR) injections as outlined in the methods. Control
mice received PBS (n = 5), and mice injected with CR developed CP (n = 10–11). Treatment with vehicle (0.5% methylcellulose + 0.025% Tween 20 in ddH2O), apigenin or
analogues (0.5 mg/kg, daily gavage) started the second week and was continued 3 additional weeks while continuing CP induction. Pancreata were stained for fibronectin by
IHC, and counter-stained with hematoxylin. Representative 400ꢀ images of each group are shown: (A) CR + Vehicle; (B) CR + Apigenin; (C) CR + 24; and (D) CR + 23. ImageJ
⁄⁄⁄
analysis of the slides quantified the percent area of brown fibronectin stain in (E).
Represents p <0.001, comparing to the effect from apigenin at the same dosage.
Scientific Electrothermal Digital Melting Point Apparatus and
uncorrected. Purity of final compounds was determined by analyt-
ical HPLC, which was carried out on a Shimadzu HPLC system
(model: CBM-20A LC-20AD SPD-20A UV/VIS). HPLC analysis condi-
J = 2.4 Hz), 3.89 (s, 3H), 3.82 (s, 3H). HRMS (ESI) calcd for
C17H15O5 299.0914 (M + H)+, found 299.0917.
4.1.3. 2-(4-(2-Bromoethoxy)phenyl)-5,7-dimethoxy-chromen-
4-one (7)
tions: Waters
l
Bondapak C18 (300 ꢀ 3.9 mm); flow rate 0.5 mL/
min; UV detection at 270 and 254 nm; linear gradient from 30%
acetonitrile in water [0.1%, trifluoroacetic acid (TFA)] to 100% ace-
tonitrile (0.1% TFA) in 20 min followed by 30 min of the last-named
solvent. All biologically evaluated compounds are >95% pure.
To a solution of 6 (200 mg, 0.67 mmol) and Ph3P (351 mg,
1.34 mmol) in THF (10 mL) was added 2-bromoethanol (168 mg,
1.34 mmol) and diisopropylazodicarboxylate (DIAD, 271 mg,
1.34 mmol). The mixture was stirred at rt for 16 h. The reaction
mixture was diluted with ethyl acetate (EtOAc, 200 mL) and
extracted with H2O (40 mL). The organic layer was washed with
brine (10 mL), dried over anhydrous Na2SO4, and concentrated to
give the crude product. This residue was purified with silica gel
column (EtOAc) to provide 7 (240 mg, 88%) as a white solid (mp
202–203 °C). HPLC purity 99.6% (tR = 21.17 min). 1H NMR
4.1.2. 2-(4-Hydroxy-phenyl)-5,7-dimethoxy-chromen-4-one (6)
Starting from the commercially available 4-allyloxybenzalde-
hyde (3) and 1-(2-hydroxy-4,6-dimethoxy-phenyl)-ethanone (2),
the key intermediate 6 was prepared in three steps according to lit-
erature procedures.36 HPLC purity 97.0% (tR = 16.94 min). 1H NMR
(600 MHz, DMSO-d6)
d
7.88 (d, 2H, J = 9.0 Hz), 6.90 (d, 2H,
(600 MHz, DMSO-d6)
d 8.00 (d, 2H, J = 8.4 Hz), 7.12 (d, 2H,
J = 9.0 Hz), 6.83 (d, 1H, J = 2.4 Hz), 6.58 (s, 1H), 6.49 (d, 1H,
J = 9.0 Hz), 6.86 (d, 1H, J = 2.4 Hz), 6.68 (s, 1H), 6.50 (d, 1H,
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
J = 1.8 Hz), 4.43 (t, 2H, J = 5.4 Hz), 3.90 (s, 3H), 3.85(t, 2H,
J = 5.4 Hz), 3.83 (s, 3H). 13C NMR (150 MHz, DMSO-d6) d 175.6,
163.6, 160.4, 160.2, 159.5, 159.1, 127.8, 123.5, 115.1, 108.3,
106.9, 96.2, 93.4, 68.0, 56.1, 56.0, 31.2. HRMS (ESI) calcd for C19H18-
BrO5 405.0332 (M+H)+, found 405.0334.
J = 9.0 Hz), 6.59 (s, 1H), 6.56 (d, 1H, J = 2.4 Hz), 6.37 (d, 1H,
J = 2.4 Hz), 4.09 (t, 2H, J = 9.0 Hz), 3.95 (s, 3H), 3.91 (s, 3H), 2.48
(t, 2H, J = 7.2 Hz), 2.27 (s, 6H), 1.98–2.00 (m, 2H). 13C NMR
(150 MHz, CDCl3) d 177.8, 164.0, 161.7, 161.1, 160.9, 160.0,
127.7, 123.9, 115.0, 109.4, 108.0, 96.2, 93.0, 66.6, 56.6, 56.4, 55.9,
45.6, 27.6. HRMS (ESI) calcd for C22H26NO5 384.1806 (M+H)+, found
384.1808.
4.1.4. 2-(4-(2-Fluoroethoxy)phenyl)-5,7-dimethoxy-chromen-4-
one (8)
Compound 8 was prepared in 90% yield by a procedure similar
to that used to prepare 7. The title compound was obtained as a
white solid (mp 187–188 °C). HPLC purity 97.3% (tR = 19.56 min).
1H NMR (600 MHz, CDCl3) d 7.82 (d, 2H, J = 10.2 Hz), 7.03 (d, 2H,
J = 10.2 Hz), 6.59 (s, 1H), 6.55 (d, 1H, J = 2.4 Hz), 6.37 (d, 1H,
J = 1.8 Hz), 4.82–4.84 (m, 1H), 4.74–4.76 (m, 1H), 4.30–4.32 (m,
1H), 4.26–4.27 (m, 1H), 3.95 (s, 3H), 3.91 (s, 3H). 13C NMR
(150 MHz, CDCl3) d 177.8, 164.1, 161.0, 160.9, 160.6, 160.0,
127.8, 124.6, 115.1, 109.4, 108.0, 96.2, 93.0, 81.9 (d, J = 171.5 Hz),
67.4 (d, J = 20.3 Hz), 56.6, 55.9. HRMS (ESI) calcd for C19H18FO5
345.1133 (M+H)+, found 345.1135.
4.1.9. 1-(2-(4-(5,7-Dimethoxy-4-oxo-4H-chromen-2-
yl)phenoxy)ethyl)-pyrrolidin-2-one (13)
Compound 13 was prepared in 85% yield by a procedure similar
to that used to prepare 7. The title compound was obtained as a
white solid (mp 115–116 °C). HPLC purity 99.3% (tR = 18.44 min).
1H NMR (600 MHz, CDCl3) d 7.81 (d, 2H, J = 8.4 Hz), 6.97 (d, 2H,
J = 8.4 Hz), 6.58 (s, 1H), 6.55 (d, 1H, J = 2.4 Hz), 6.37 (d, 1H,
J = 1.8 Hz), 4.18 (t, 2H, J = 5.4 Hz), 3.95 (s, 3H), 3.91 (s, 3H), 3.71
(t, 2H, J = 5.4 Hz), 3.58 (t, 2H, J = 7.2 Hz), 2.39 (t, 2H, J = 8.4 Hz),
2.01–2.08 (m, 2H). 13C NMR (150 MHz, CDCl3) d 177.8, 175.6,
164.1, 161.0, 160.9, 160.7, 160.0, 127.8, 124.4, 114.9, 109.3,
107.9, 96.2, 92.9, 66.8, 56.6, 55.9, 49.1, 42.4, 30.9, 18.3. HRMS
(ESI) calcd for C23H24NO6 410.1598 (M+H)+, found 410.1601.
4.1.5. 5,7-Dimethoxy-2-(4-(1-methylpiperidin-4-yloxy)phenyl)-
chromen-4-one (9)
Compound 9 was prepared in 94% yield by a procedure similar
to that used to prepare 7. The title compound was obtained as a
white solid (mp 143–144 °C). HPLC purity 99.3% (tR = 15.86 min).
1H NMR (600 MHz, CDCl3) d 7.80 (d, 2H, J = 9.0 Hz), 6.99 (d, 2H,
J = 9.0 Hz), 6.58 (s, 1H), 6.55 (d, 1H, J = 2.4 Hz), 6.37 (d, 1H,
J = 3.0 Hz), 4.44 (s, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 2.72 (s, 2H),
2.38 (s, 2H), 2.34 (s, 3H), 2.04–2.08 (m, 2H), 1.88–1.92 (m, 2H).
13C NMR (150 MHz, CDCl3) d 177.8, 164.0, 161.0, 160.8, 160.1,
160.0, 127.8, 123.9, 116.2, 109.4, 107.8, 96.2, 93.0, 56.6, 55.9,
4.1.10. 5,7-Dimethoxy-2-(4-(2-piperidin-1-yl-ethoxy)phenyl)-
chromen-4-one (14)
To a solution of 7 (30 mg, 0.074 mmol), KI (25 mg, 0.15 mmol)
and K2CO3 (102 mg, 0.74 mmol) in acetone (5 mL) was added
piperidine (31 mg, 0.37 mmol) at 0 °C. The mixture was stirred at
75 °C for 18 h. The solution was diluted with EtOAc (100 mL),
washed with 0.1 N HCl (aq) (10 mL) and brine (10 mL). The organic
layer was dried over anhydrous Na2SO4, and then concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (EtOAc) to give the desired product 14
(30 mg, 99%) as a white solid (mp 79–80 °C). HPLC purity 95.1%
52.4, 46.1, 30.6. HRMS (ESI) calcd for
C23H26NO5 396.1806
(M+H)+, found 396.1808.
(tR = 16.24 min). 1H NMR (600 MHz, CDCl3)
d 7.83 (d, 2H,
4.1.6. 2-(4-(2-Dimethylaminoethoxy)phenyl)-5,7-dimethoxy-
chromen-4-one (10)
J = 6.6 Hz), 7.02 (d, 2H, J = 6.6 Hz), 6.62 (s, 1H), 6.58 (d, 1H,
J = 2.4 Hz), 6.39 (d, 1H, J = 2.4 Hz), 4.22 (t, 2H, J = 6.0 Hz), 3.98 (s,
3H), 3.94 (s, 3H), 2.85 (t, 2H, J = 6.0 Hz), 2.57–2.59 (m, 4H),
1.65–1.67 (m, 4H), 1.50–1.52 (m, 2H). 13C NMR (150 MHz, CDCl3)
Compound 10 was prepared in 81% yield by a procedure similar
to that used to prepare 7. The title compound was obtained as a
pale yellow solid (mp 153–154 °C). HPLC purity 98.8% (tR = 15.25 -
min). 1H NMR (600 MHz, CDCl3) d 7.81 (d, 2H, J = 9.0 Hz), 7.02 (d,
2H, J = 9.0 Hz), 6.59 (s, 1H), 6.55 (d, 1H, J = 1.8 Hz), 6.37 (d, 1H,
J = 1.8 Hz), 4.14 (t, 2H, J = 6.0 Hz), 3.95 (s, 3H), 3.91 (s, 3H), 2.78
(t, 2H, J = 6.0 Hz), 2.36 (s, 6H). 13C NMR (150 MHz, CDCl3) d
177.8, 164.0, 161.4, 161.0, 160.8, 160.0, 127.7, 124.1, 115.1,
109.4, 107.9, 96.2, 92.9, 66.3, 58.2, 56.6, 55.9, 46.0. HRMS (ESI)
calcd for C21H24NO5 370.1649 (M+H)+, found 370.1652.
d
177.8, 164.1, 161.4, 161.0, 160.8, 160.0, 127.7, 124.0,
115.1, 109.4, 107.8, 96.2, 93.0, 66.3, 57.8, 56.6, 55.9, 55.2, 25.9,
24.2. HRMS (ESI) calcd for C24H28NO5 410.1962 (M+H)+, found
410.1964.
4.1.11. 5,7-Dimethoxy-2-(4-(2-(4-methylpiperazin-1-
yl)ethoxy)phenyl)-chromen-4-one(15)
Compound 15 was prepared in 80% yield by a procedure similar
to that used to prepare 14. The title compound was obtained as a
white solid (mp 144–145 °C). HPLC purity 98.7% (tR = 16.43 min).
1H NMR (600 MHz, CDCl3) d 7.79–7.81 (m, 2H), 6.98–7.00 (m,
2H), 6.58 (s, 1H), 6.55 (d, 1H, J = 2.4 Hz), 6.36 (d, 1H, J = 2.4 Hz),
4.17 (t, 2H, J = 6.0 Hz), 3.95 (s, 3H), 3.90 (s, 3H), 2.85 (t, 2H,
J = 6.0 Hz), 2.50–2.65 (m, 8H), 2.30 (s, 3H). 13C NMR (150 MHz,
CDCl3) d 177.8, 164.0, 161.4, 161.0, 160.8, 160.0, 127.7, 124.1,
115.1, 109.4, 107.8, 96.2, 92.9, 66.3, 57.1, 56.6, 55.9, 55.1, 53.7,
46.1. HRMS (ESI) calcd for C24H29N2O5 425.2071 (M+H)+, found
425.2070.
4.1.7. 5,7-Dimethoxy-2-(4-(2-morpholin-4-yl-ethoxy)phenyl)-
chromen-4-one (11)
Compound 11 was prepared in 82% yield by a procedure similar
to that used to prepare 7. The title compound was obtained as a
white solid (mp 149–150 °C). HPLC purity 99.8% (tR = 15.26 min).
1H NMR (600 MHz, CDCl3) d 7.80 (d, 2H, J = 9.0 Hz), 6.99 (d, 2H,
J = 9.0 Hz), 6.58 (s, 1H), 6.55 (s, 1H), 6.37 (s, 1H), 4.18 (t, 2H,
J = 6.0 Hz), 3.95 (s, 3H), 3.90 (s, 3H), 3.74 (t, 4H, J = 3.0 Hz), 2.83
(t, 2H, J = 6.0 Hz), 2.59 (s, 4H). 13C NMR (150 MHz, CDCl3) d
177.8, 164.1, 161.3, 161.0, 160.7, 160.0, 127.7, 124.2, 115.1,
109.4, 107.9, 96.2, 93.0, 67.0, 66.2, 57.6, 56.6, 55.9, 54.3. HRMS
(ESI) calcd for C23H26NO6 412.1755 (M+H)+, found 412.1757.
4.1.12. 5,7-Dimethoxy-2-(4-(2-pyrrolidin-1-yl-ethoxy)phenyl)-
chromen-4-one (16)
Compound 16 was prepared in 85% yield by a procedure similar
to that used to prepare 14. The title compound was obtained as a
pale brown solid (mp 114–115 °C). HPLC purity 99.2% (tR = 15.91 -
min). 1H NMR (600 MHz, CDCl3) d 7.78(d, 2H, J = 6.6 Hz), 6.99 (d,
2H, J = 6.6 Hz), 6.55 (d, 1H, J = 3.6 Hz), 6.52 (d, 1H, J = 2.4 Hz),
6.34 (d, 1H, J = 2.4 Hz), 4.17 (t, 2H, J = 9.0 Hz), 3.92 (s, 3H), 3.88
(s, 3H), 2.93 (t, 2H, J = 9.0 Hz), 2.65–2.66 (m, 4H), 1.81–1.82 (m,
4.1.8. 2-(4-(3-Dimethylaminopropoxy)phenyl)-5,7-dimethoxy-
chromen-4-one (12)
Compound 12 was prepared in 91% yield by a procedure similar
to that used to prepare 7. The title compound was obtained as a
white solid (mp 105–106 °C). HPLC purity 98.2% (tR = 15.76 min).
1H NMR (600 MHz, CDCl3) d 7.81 (d, 2H, J = 9.0 Hz), 6.99 (d, 2H,
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
4H). 13C NMR (150 MHz, CDCl3) d 177.8, 164.0, 161.4, 161.0, 160.8,
160.0, 127.7, 123.9, 115.0, 109.3, 107.7, 96.2, 93.0, 67.3, 56.5, 55.8,
54.9, 54.8, 23.6. HRMS (ESI) calcd for C23H26NO5 396.1806 (M+H)+,
found 396.1809.
was obtained as a white solid (mp 165–166 °C). HPLC purity
98.4% (tR = 14.50 min). 1H NMR (600 MHz, CD3OD) d 7.87 (d, 2H,
J = 9.0 Hz), 7.05 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 9.0 Hz), 6.42 (s,
1H), 4.12 (t, 2H, J = 4.8 Hz), 3.94 (d, 6H, J = 2.4 Hz), 3.14 (s, 2H).
13C NMR (150 MHz, CD3OD) d 178.9, 164.7, 161.7, 161.6, 161.0,
160.1, 128.1, 124.0, 115.1, 108.8, 107.2, 96.5, 93.1, 69.1, 56.2,
56.0, 40.7. HRMS (ESI) calcd for C19H20NO5 342.1336 (M+H)+, found
342.1337.
4.1.13. 2-(4-(2-Diethylaminoethoxy)-phenyl)-5,7-dimethoxy-
chromen-4-one (17)
Compound 17 was prepared in 85% yield by a procedure similar
to that used to prepare 14. The title compound was obtained as a
pale yellow solid (mp 116–117 °C). HPLC purity 99.5% (tR = 16.09 -
min). 1H NMR (600 MHz, CDCl3) d 7.80 (d, 2H, J = 9.0 Hz), 7.00 (d,
2H, J = 9.0 Hz), 6.59 (d, 1H, J = 2.4 Hz), 6.56 (d, 1H, J = 1.8 Hz),
6.37 (d, 1H, J = 1.8 Hz), 4.12 (t, 2H, J = 6.0 Hz), 3.95 (s, 3H), 3.91
(s, 3H), 2.91 (t, 2H, J = 6.0 Hz), 2.65–2.68 (m, 4H), 1.09 (t, 6H,
J = 7.2 Hz). 13C NMR (150 MHz, CDCl3) d 177.8, 164.0, 161.5,
161.0, 160.8, 160.0, 127.7, 124.0, 115.1, 109.4, 107.8, 96.2, 93.0,
67.0, 56.6, 55.9, 51.8, 48.0, 12.0. HRMS (ESI) calcd for C23H28NO5
398.1962 (M+H)+, found 398.1964.
4.1.17. 2-(4-(3-Aminopropoxy)-phenyl)-5,7-dimethoxy-
chromen-4-one (21)
Compound 21 was prepared in 90% yield (two steps) by a pro-
cedure similar to that used to prepare 19. The title compound
was obtained as a white solid (mp 170–171 °C). HPLC purity
98.4% (tR = 15.07 min). 1H NMR (600 MHz, CD3OD) d 7.88 (d, 2H,
J = 9.0 Hz), 7.05 (d, 2H, J = 9.0 Hz), 6.66 (d, 1H, J = 1.8 Hz), 6.60 (s,
1H), 6.45 (d, 1H, J = 1.8 Hz), 4.18 (t, 2H, J = 6.0 Hz), 3.95 (d, 6H,
J = 3.0 Hz), 3.35 (s, 2H), 3.09 (t, 2H, J = 7.2 Hz), 2.14 (t, 2H,
J = 6.0 Hz). 13C NMR (150 MHz, CD3OD) d 179.3, 165.2, 162.2,
161.9, 161.1, 160.4, 128.3, 124.1, 115.3, 108.9, 107.1, 96.8, 93.4,
65.9, 56.3, 56.2, 38.2, 29.4. HRMS (ESI) calcd for C20H22NO5
356.1493 (M+H)+, found 356.1496.
4.1.14. 1-(2-(4-(5,7-Dimethoxy-4-oxo-4H-chromen-2-
yl)phenoxy)ethyl)-pyrrolidine-2-carboxylic acid methyl ester
(18)
Compound 18 was prepared in 66% yield by a procedure similar
to that used to prepare 14. The title compound was obtained as a
white solid (mp 121–122 °C). HPLC purity 98.4% (tR = 16.15 min).
1H NMR (600 MHz, CDCl3) d 7.83 (d, 2H, J = 9.0 Hz), 7.00 (d, 2H,
J = 9.0 Hz), 6.61 (s, 1H), 6.58 (d, 1H, J = 2.4 Hz), 6.40 (d, 1H,
J = 1.8 Hz), 4.18–4.24 (m, 2H), 3.98 (s, 3H), 3.94 (s, 3H), 3.66 (s,
3H), 3.39–3.41 (m, 1H), 3.32–3.35 (m, 1H), 3.11–3.14 (m, 1H),
3.04–3.07 (m, 1H), 2.59–2.62 (m, 1H), 2.20–2.22 (m, 1H), 1.96–
2.00 (m, 2H), 1.86–1.88 (m, 1H). 13C NMR (150 MHz, CDCl3) d
177.8, 174.9, 164.0, 161.2, 161.0, 160.8, 160.0, 127.7, 124.1,
115.0, 109.4, 107.9, 96.2, 93.0, 67.5, 66.2, 56.6, 55.9, 55.1, 53.6,
4.1.18. 5,7-Dimethoxy-2-(4-oxiranylmethoxyphenyl)-chromen-
4-one (23)
To a solution of 6 (200 mg, 0.67 mmol) and epichlorohydrin
(22) (617 mg, 6.7 mmol) in acetone (10 mL) was added K2CO3
(462 mg, 3.35 mmol). The mixture was stirred at 80 °C for 24 h,
and was then concentrated to give the crude product. The residue
was diluted with EtOAc (100 mL), and washed with 0.1 N HCl (aq)
(10 mL) followed by brine (10 mL). The organic layer was dried
over anhydrous Na2SO4, and then concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH = 20/1) to give the desired product 23
(160 mg, 67%) as a pale yellow solid (mp 191–192 °C). HPLC purity
98.9% (tR = 18.99 min). 1H NMR (600 MHz, CDCl3) d 7.80–7.82 (m,
2H), 7.00–7.03 (m, 2H), 6.58 (s, 1H), 6.55 (d, 1H, J = 2.4 Hz), 6.36
(d, 1H, J = 2.4 Hz), 4.31–4.33 (m, 1H), 3.99–4.02 (m, 1H), 3.95 (s,
3H), 3.90 (s, 3H), 3.37–3.39 (m, 1H), 2.93–2.94 (m, 1H), 2.78–
2.79 (m, 1H). 13C NMR (150 MHz, CDCl3) d 177.7, 164.1, 161.0,
161.0, 160.6, 160.0, 127.8, 124.6, 115.1, 109.4, 108.0, 96.2, 93.0,
52.0, 29.9, 23.5. HRMS (ESI) calcd for
C25H28NO7 454.1860
(M+H)+, found 454.1865.
4.1.15. 5,7-Dimethoxy-2-(4-(piperidin-4-yloxy)-phenyl)-
chromen-4-one (19)
To a solution of 6 (60 mg, 0.2 mmol) and Ph3P (210 mg,
0.8 mmol) in THF (5 mL) was added 4-hydroxy-piperidine-1-car-
boxylic acid tert-butyl ester (121 mg, 0.6 mmol) in THF (5 mL)
and DIAD (121 mg, 0.6 mmol). The mixture was stirred at rt for
2 h, and was then concentrated to give the crude product. This res-
idue was purified with silica gel column (EtOAc) to afford 80 mg of
the intermediate as a white solid. To the solution of the intermedi-
ate (80 mg) in CH2Cl2 (3 mL) was added TFA (1 mL) at 0 °C. The
mixture was stirred at rt for 3 h, and was then concentrated. The
residue was partitioned between EtOAc (250 mL) and 1 N NaHCO3
(aq, 10 mL). The organic layer was washed with H2O (10 mL) and
dried over Na2SO4. The organic layer was concentrated. The residue
was purified with silica gel column (EtOAc) to afford 19 (60 mg,
78%, two steps) as a pale yellow solid (mp 205–206 °C). HPLC pur-
ity 98.6% (tR = 15.43 min). 1H NMR (600 MHz, DMSO-d6) d 7.98–
8.00 (m, 2H), 7.15–7.17 (m, 2H), 6.85 (d, 1H, J = 1.8 Hz), 6.68 (s,
1H), 6.51 (d, 1H, J = 2.4 Hz), 4.77 (t, 1H, J = 4.2 Hz), 3.90 (s, 3H),
3.83 (s, 3H), 3.21–3.25 (m, 4H), 3.03–3.07 (m, 2H), 2.09–2.12 (m,
2H), 1.78–1.81 (m, 2H). 13C NMR (150 MHz, DMSO-d6) d 178.6,
164.5, 161.3, 160.9, 160.0, 159.4, 128.1, 124.4, 116.1, 108.9,
107.4, 96.4, 93.0, 69.2, 56.3, 55.9, 40.6, 28.0. HRMS (ESI) calcd for
69.1, 56.6, 55.9, 50.1, 44.7. HRMS (ESI) calcd for
C20H19O6
355.1176 (M+H)+, found 355.1180.
4.1.19. 2-(4-(2-Hydroxy-3-piperidin-1-yl-propoxy)phenyl)-5,7-
dimethoxy-chromen-4-one (24)
To a solution of 23 (30 mg, 0.085 mmol) and piperidine (72 mg,
0.85 mmol) in EtOH (5 mL) was added K2CO3 (117 mg, 0.85 mmol).
The mixture was stirred at 100 °C for 3 h, and was then concen-
trated to give the crude product. The residue was diluted with
EtOAc (100 mL), and washed with 0.1 N HCl (aq) (10 mL) followed
by brine (10 mL). The organic layer was dried over anhydrous Na2-
SO4, and then concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (CH2Cl2/
MeOH = 10/1) to give the desired product 24 (35 mg, 94%) as a
white solid (mp 109–110 °C). HPLC purity 98.5% (tR = 15.78 min).
1H NMR (600 MHz, CDCl3) d 7.79–7.81 (m, 2H), 7.00–7.03 (m,
2H), 6.58 (s, 1H), 6.54 (d, 1H, J = 3.0 Hz), 6.36 (d, 1H, J = 1.8 Hz),
4.11–4.15 (m, 1H), 4.02–4.06 (m, 2H), 3.95 (s, 3H), 3.90 (s, 3H),
2.66 (br s, 1H), 2.54–2.56 (m, 2H), 2.50–2.55 (m, 2H), 2.41–2.43
(m, 2H), 1.58–1.65 (m, 4H), 1.46–1.48 (m, 2H). 13C NMR
(150 MHz, CDCl3) d 177.8, 164.0, 161.4, 161.0, 160.8, 160.0,
127.7, 124.2, 115.1, 109.3, 107.8, 96.2, 92.9, 70.7, 65.3, 61.1, 56.6,
55.9, 54.9, 26.0, 24.2. HRMS (ESI) calcd for C25H30NO6 440.2068
(M+H)+, found 440.2071.
C
22H24NO5 382.1649 (M+H)+, found 382.1651.
4.1.16. 2-(4-(2-Aminoethoxy)-phenyl)-5,7-dimethoxy-chromen-
4-one (20)
Compound 20 was prepared in 59% yield (two steps) by a pro-
cedure similar to that used to prepare 19. The title compound
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10
H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.1.20. 2-(4-(2-Hydroxy-3-pyrrolidin-1-yl-propoxy)phenyl)-5,7-
dimethoxy-chromen-4-one (25)
4.1.24. 2-(4-(6-Fluoropyridin-3-yl)phenyl)-5,7-dimethoxy-
chromen-4-one (33)
Compound 25 was prepared in 94% yield by a procedure similar
to that used to prepare 24. The title compound was obtained as a
pale yellow solid (mp 137–138 °C). HPLC purity 99.9% (tR = 15.42 -
min). 1H NMR (600 MHz, CDCl3) d 7.82 (d, 2H, J = 9.0 Hz), 7.03 (d,
2H, J = 9.0 Hz), 6.59 (s, 1H), 6.55 (d, 1H, J = 2.4 Hz), 6.36 (d, 1H,
J = 2.4 Hz), 4.12–4.15 (m, 1H), 4.06 (d, 2H, J = 5.4 Hz), 3.95 (s, 3H),
3.91 (s, 3H), 3.10–3.30 (br s, 1H), 2.86–2.89 (m, 1H), 2.76–2.79
(m, 2H), 2.58–2.62 (m, 2H), 1.82–1.85 (m, 4H). 13C NMR
(150 MHz, CDCl3) d 177.8, 164.1, 161.3, 161.0, 160.7, 160.0,
127.7, 124.3, 115.1, 109.4, 107.9, 96.2, 93.0, 70.7, 67.2, 58.6, 56.6,
55.9, 54.4. HRMS (ESI) calcd for C24H28NO6 426.1911 (M+H)+, found
426.1915.
To a solution of 31 (90 mg, 0.25 mmol) and 2-fluoropyridine-5-
boronic acid (32) (42 mg, 0.3 mmol) in THF/EtOH/H2O (2 mL/2 mL/
2 mL) was added KOAc (94 mg, 0.75 mmol) and then Pd(dppf)Cl2
(20 mg, 0.025 mmol). The resulting mixture was deoxygenated
via five vacuum/N2-refill cycles. The mixture was stirred at 80 °C
for 18 h, and was then concentrated under vacuum. The residue
was partitioned between EtOAc (100 mL) and H2O (20 mL). The
organic layer was separated and washed with brine (10 mL), dried
over anhydrous Na2SO4, filtrated and concentrated to give an oily
residue. This residue was purified with silica gel column (CH2Cl2/
MeOH = 10/1) to obtain 33 (80 mg, 85%) as a pale red solid (mp
209–210 °C). HPLC purity 98.4% (tR = 20.91 min). 1H NMR
(600 MHz, CDCl3) d 8.48 (d, 1H, J = 1.8 Hz), 8.01–8.04 (m, 1H),
7.98 (s, 1H), 7.97 (s, 1H), 7.04–7.06 (m, 1H), 6.72 (s, 1H), 6.59 (d,
1H, J = 2.4 Hz), 6.40 (d, 1H, J = 2.4 Hz), 3.97 (s, 3H), 3.93 (s, 3H).
13C NMR (150 MHz, CDCl3) d 177.5, 164.5, 164.4, 162.9, 161.2,
160.1, 160.0, 146.2, 146.1, 139.8, 139.5, 133.8, 131.6, 127.6,
126.9, 110.0, 109.8, 109.6, 96.5, 93.1, 56.6, 55.9. HRMS (ESI) calcd
for C22H17FNO4 378.1136 (M+H)+, found 378.1138.
4.1.21. 2-(4-(3-Dimethylamino-2-hydroxypropoxy)-phenyl)-5,7-
dimethoxy-chromen-4-one (26)
Compound 26 was prepared in 71% yield by a procedure similar
to that used to prepare 24. The title compound was obtained as a
white solid (mp 92–93 °C). HPLC purity 99.6% (tR = 10.65 min). 1H
NMR (600 MHz, CDCl3) d 7.83 (d, 2H, J = 9.0 Hz), 7.04 (d, 2H,
J = 9.0 Hz), 6.61 (s, 1H), 6.58 (d, 1H, J = 2.4 Hz), 6.39 (d, 1H,
J = 2.4 Hz), 4.12–4.15 (m, 1H), 4.11–4.12 (m, 2H), 3.98 (s, 3H),
3.93 (s, 3H), 2.61–2.65 (m, 1H), 2.42–2.48 (m, 2H), 2.39 (s, 6H).
13C NMR (150 MHz, CDCl3) d 177.8, 164.1, 161.3, 161.0, 160.8,
160.0, 127.7, 124.2, 115.1, 109.3, 107.9, 96.2, 93.0, 70.7, 66.1,
61.8, 56.6, 55.9, 45.6. HRMS (ESI) calcd for C22H26NO6 400.1755
(M+H)+, found 400.1756.
4.1.25. 2-(4-(2-Dimethylaminoethylamino)phenyl)-5,7-
dimethoxy-chromen-4-one (36)
NaOtBu (43 mg, 0.45 mmol), Pd2(dba)3 (28 mg, 0.03 mmol) and
( )-BINAP (19 mg, 0.03 mmol) were placed into a flask and dis-
solved into distilled toluene (10 mL). To this solution was added
31 (108 mg, 0.3 mmol) and N,N-dimethylethylenediamine (34)
(40 mg, 0.45 mmol) dropwise with stirring at room temperature
and the mixture was refluxed at 80 °C for 48 h. After the mixture
was cooled, 20 mL of H2O was added and extracted with EtOAc
(100 mL). The organic layer was dried over anhydrous Na2SO4, fil-
tered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (CH2Cl2/MeOH = 10/1) to give the
desired product (65 mg, 64%) as a pale yellow solid (mp 159–
160 °C). HPLC purity 98.8% (tR = 15.10 min). 1H NMR (600 MHz,
CDCl3) d 7.70 (d, 2H, J = 9.0 Hz), 6.66 (d, 2H, J = 9.0 Hz), 6.54 (d,
1H, J = 1.8 Hz), 6.53 (s, 1H), 6.35 (d, 1H, J = 1.8 Hz), 4.84 (s, 1H),
3.95 (s, 3H), 3.90 (s, 3H), 3.22 (s, 2H), 2.60 (t, 1H, J = 6.0 Hz), 2.28
(s, 6H). 13C NMR (150 MHz, CDCl3) d 177.9, 163.8, 161.7, 161.0,
160.0, 151.1, 127.7, 119.4, 112.5, 109.4, 106.2, 96.0, 93.0, 57.7,
56.6, 55.8, 45.2, 40.6, 31.1. HRMS (ESI) calcd for C21H25N2O4
369.1809 (M+H)+, found 369.1811.
4.1.22. 2-(4-(2,3-Dihydroxypropoxy)phenyl)-5,7-dimethoxy-
chromen-4-one (28)
To a solution of 6 (50 mg, 0.17 mmol) and Ph3P (88 mg,
0.34 mmol) in THF (5 mL) was added (2,2-dimethyl-1,3-dioxolan-
4-yl)methanol (27) (44 mg, 0.34 mmol) in THF (5 mL) and DIAD
(68 mg, 0.34 mmol). The mixture was stirred at rt for 4 h, and
was then concentrated to give the crude product. This residue
was purified with silica gel column (CH2Cl2/MeOH = 20/1 to 10/1)
to afford 70 mg of the intermediate as a white solid. To the solution
of the intermediate (70 mg) in EtOH (5 mL) was added 0.5 N HCl
(aq, 0.5 mL) at 0 °C. The mixture was stirred at 100 °C for 2 h,
and was then concentrated. The residue was partitioned between
EtOAc (50 mL) and 1 N NaHCO3 (aq, 10 mL). The organic layer
was washed with H2O (10 mL) and dried over Na2SO4. The organic
layer was concentrated. The residue was purified with silica gel col-
umn (CH2Cl2/MeOH = 10/1) to afford 28 (45 mg, 71%, two steps) as
a white solid (mp 203–204 °C). HPLC purity 97.6% (tR = 15.51 min).
1H NMR (600 MHz, DMSO-d6) d 7.97–8.00 (m, 2H), 7.08–7.11 (m,
2H), 6.86 (d, 1H, J = 2.4 Hz), 6.66 (s, 1H), 6.50 (d, 1H, J = 2.4 Hz),
5.00 (d, 1H, J = 5.4 Hz), 4.70 (t, 1H, J = 6.0 Hz), 4.09–4.11 (m, 1H),
3.95–3.98 (m, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.80–3.83 (m, 1H),
3.46 (t, 2H, J = 6.0 Hz). 13C NMR (150 MHz, DMSO-d6) d 175.6,
163.6, 161.4, 160.2, 159.7, 159.1, 127.7, 122.9, 115.0, 108.3,
106.7, 96.2, 93.4, 69.9, 69.8, 62.6, 56.1, 56.0. HRMS (ESI) calcd for
4.1.26. 5,7-Dimethoxy-2-(4-(2-pyrrolidin-1-yl-
ethylamino)phenyl)-chromen-4-one (37)
Compound 37 was prepared in 57% yield by a procedure similar
to that used to prepare 36. The title compound was obtained as a
pale yellow solid (mp 148–149 °C). HPLC purity 98.2% (tR = 15.73 -
min). 1H NMR (600 MHz, CDCl3) d 7.69 (d, 2H, J = 9.0 Hz), 6.66 (d,
2H, J = 8.4 Hz), 6.53 (d, 1H, J = 2.4 Hz), 6.52 (s, 1H), 6.34 (d, 1H,
J = 2.4 Hz), 4.91 (s, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.27 (d, 2H,
J = 2.4 Hz), 2.78 (t, 1H, J = 6.0 Hz), 2.59 (s, 4H), 1.81 (s, 4H). 13C
NMR (150 MHz, CDCl3) d 177.9, 163.8, 161.7, 161.0, 160.0, 151.1,
127.6, 119.3, 112.5, 109.4, 106.1, 96.0, 93.0, 56.5, 55.8, 54.6, 54.0,
41.8, 23.6. HRMS (ESI) calcd for C23H27N2O4 395.1965 (M+H)+,
found 395.1968.
C
20H21O7 373.1282 (M+H)+, found 373.1284.
4.1.23. 2-(4-Bromophenyl)-5,7-dimethoxy-chromen-4-one (31)
Starting from the commercially available 4-bromobenzalde-
hyde (29) and 1-(2-hydroxy-4,6-dimethoxy-phenyl)ethanone (2),
31 was prepared in two steps according to literature procedures.36
HPLC purity 96.5% (tR = 21.53 min). 1H NMR (600 MHz, DMSO-d6) d
7.99 (d, 2H, J = 8.4 Hz), 7.75 (d, 2H, J = 9.0 Hz), 6.87 (d, 1H,
J = 2.4 Hz), 6.82 (s, 1H), 6.51 (d, 1H, J = 1.8 Hz), 3.90 (s, 3H), 3.83
(s, 3H). 13C NMR (150 MHz, DMSO-d6) d 175.6, 163.9, 160.3,
159.1, 158.5, 132.0, 130.2, 127.9, 125.1, 108.6, 108.4, 96.4, 93.4,
56.1, 56.0. HRMS (ESI) calcd for C17H14BrO4 361.0070 (M+H)+,
found 361.0070.
4.1.27. 5-Hydroxy-7-methoxy-2-(4-(2-pyrrolidin-1-yl-
ethoxyphenyl)-chromen-4-one (38)
To a solution of 16 (30 mg, 0.076 mmol) in 5 mL of CH2Cl2 was
added 1 N BBr3 in CH2Cl2 (0.15 mL, 0.15 mmol) at 0 °C. The result-
ing mixture was stirred at rt for 2 h. The solution was diluted with
CH2Cl2/MeOH (10/1, 50 mL), washed with H2O (15 mL) and brine
(10 mL). The organic layer was dried over anhydrous Na2SO4 and
then concentrated under reduced pressure. The residue was
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H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
purified by silica gel column chromatography (CH2Cl2/MeOH = 10/
1) to give the desired product (23 mg, 79%) as a pale yellow solid
(mp 127–129 °C). HPLC purity 99.7% (tR = 18.02 min). 1H NMR
(600 MHz, CDCl3) d 12.81 (s, 1H), 7.83 (d, 2H, J = 9.0 Hz), 7.03 (d,
2H, J = 9.0 Hz), 6.60 (s, 1H), 6.48 (d, 1H, J = 2.4 Hz), 6.36 (d, 1H,
J = 2.4 Hz), 4.21 (t, 2H, J = 9.0 Hz), 3.88 (s, 3H), 2.97 (t, 2H,
J = 9.0 Hz), 2.67–2.69 (m, 4H), 1.83–1.85 (m, 4H). 13C NMR
(150 MHz, CDCl3) d 182.6, 165.6, 164.2, 162.3, 162.0, 157.9,
128.2, 123.8, 115.2, 105.7, 104.5, 98.2, 92.8, 67.4, 55.9, 54.9, 54.8,
23.6. HRMS (ESI) calcd for C22H24NO5 382.1649 (M+H)+, found
386.1652.
apigenin or analogues for a 14 h incubation. The Cell Death Detec-
tion ELISAPLUS assay (Roche Applied Science, Indianapolis, IN) was
used and protocol followed (Version 11.0). Absorbance was mea-
sured at 405 nm, using the ELx800 Automated Microplate Reader
(Bio-TEK Instruments, Inc., Winooski, VT).
4.2.4. Chronic pancreatitis (CP) animal model
Animal experiments were conducted under an Institutional Ani-
mal Care and Use Committee-approved protocol. CP was induced
in C57BL/6 mice using supraoptimal pancreatic stimulation with
cerulein (CR), a cholecystokinin analogue (Bachem, Torrance, CA).
CP (50 lg CR/kg mouse weight) was administered via intraperito-
4.1.28. 5,7-Dihydroxy-2-(4-(2-pyrrolidin-1-yl-ethoxy)phenyl)-
chromen-4-one (39)
neal route hourly for 5 h for 3 d/week for a total of 4 weeks. Control
mice received phosphate buffered saline (PBS) injections following
the same schedule. Starting Week 2 of the experiment, apigenin,
analogue, or vehicle (0.5% methylcellulose + 0.025% Tween 20 in
ddH20) were administered (0.5 mg/kg/d, oral gavage, once daily,
6 d/week, for 3 week) while continuing CR injections. At the end
of Week 4, the mice were anesthestized with isoflurane and sacri-
ficed per protocol. The pancreata were quickly harvested and
processed.
To a solution of 16 (22 mg, 0.056 mmol) in 5 mL of CH2Cl2 was
added 1 N BBr3 in CH2Cl2 (0.17 mL, 0.17 mmol) at 0 °C. The result-
ing mixture was stirred at rt for 24 h. The solution was diluted with
CH2Cl2/MeOH (10/1, 50 mL), washed with H2O (5 mL) and brine
(5 mL). The organic layer was dried over anhydrous Na2SO4 and
then concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (CH2Cl2/MeOH = 10/1)
to give the desired product (15 mg, 73%) as a pale yellow solid
(mp 228–229 °C). HPLC purity 97.5% (tR = 16.04 min). 1H NMR
(600 MHz, CDCl3/CD3OD 2:1) d 7.80 (d, 2H, J = 9.0 Hz), 6.98 (d,
2H, J = 9.0 Hz), 6.50 (s, 1H), 6.39 (d, 1H, J = 2.4 Hz), 6.22 (d, 1H,
J = 2.4 Hz), 4.16 (t, 2H, J = 6.0 Hz), 2.94 (t, 2H, J = 6.0 Hz), 2.65–
2.67 (m, 4H), 1.80–1.83 (m, 4H). 13C NMR (150 MHz, CDCl3/CD3OD
2:1) d 182.6, 164.4, 164.3, 161.8, 161.7, 158.1, 128.2, 123.8, 115.1,
104.7, 103.9, 99.4, 94.5, 66.8, 54.8, 54.7, 23.4. HRMS (ESI) calcd for
4.2.5. Immunohistochemistry (IHC) and image analysis
Pancreata were formalin-fixed and paraffin-embedded. Prior to
staining, sections (5 lM) were deparaffinized with xylene, rehy-
drated with ethanol, and subjected to heat-mediated antigen
retrieval (DAKO, Carpinteria, CA) to optimize antigen immunore-
activity. Fibronectin antibody (1:600; Santa Cruz Biotechnology,
Dallas, TX), and biotinylated anti-goat IgG (1:400; Vector
Laboratories Inc., Burlingame, CA) were used. IHC staining was
completed with the VECTASTAIN Elite ABC kit (Vector Lab), color
development with DAB (DAKO), and counterstaining with
hematoxylin 7211 (Thermo Scientific, Kalamazoo, MI). Five non-
overlapping images of each pancreas (400ꢀ) were taken using
an Olympus BX51 microscope connected to a DP71 Olympus
digital camera. The percent area of brown fibronectin staining
was quantified using the Image Processing and Analysis in Java
(ImageJ) 1.46r software (NIH, Bethesda, MD) and a color deconvo-
lution plug-in.44,45
C
21H22NO5 368.1493 (M+H)+, found 368.1495.
4.2. Biology
4.2.1. Human pancreatic stellate cell culture and reagents
Under an IRB-approved tissue protocol, discarded human
resected pancreatic tissue (500 mm3) was attained fresh from the
operating room at the University of Texas Medical Branch. The tis-
sue was minced and plated on a collagen-coated flask (15
Invitrogen, Carlsbad, CA) with DMEM (VWR, Radnor, PA) supple-
mented with penicillin 200 U/mL, streptomycin 200 g/mL,
amphotericin B 0.25 g/mL and gentamicin 50 g/mL (Invitrogen,
lg/mL;
l
4.2.6. Statistical analysis
l
l
Dose–response curves were generated by plotting fluorescence
or absorbance versus log (compound concentration). A best-fit
curve was created using nonlinear regression, and the IC50 or
EC50 determined from the graph (GraphPad Prism 5; GraphPad
Software Inc., La Jolla, CA). SPSS (IBM, Armonk, NY) was used to
conduct statistical analysis, which included SEM, two-way ANOVA,
and one-way ANOVA with post-hoc Tukey-Kramer multiple com-
parisons test. Significance was set at p <0.05.
Carlsbad CA), 10% fetal bovine serum (Lonza, Walkersville, MD),
1% insulin-transferrin, selenium-ethanolamine (Gibco, Grand
Island, NY), and 1% non-essential amino acids (Sigma–Aldrich, St.
Louis, MO). The human PSC were isolated by the outgrowth
method.41–43 The purity of PSC culture was confirmed by immuno-
histochemical staining for vimentin,
a-smooth muscle actin, glial
fibrillar acidic protein, or Oil red O staining. Primary PSC were
transformed and immortalized for use in experiments using
lentiviral vectors containing SV40 Large T antigen and human
telomerase (plasmid # 12245 and 12246; Addgene, Cambridge,
MA). Cultures were maintained in DMEM with 10% FBS, at 37 °C
in a humidified 95% O2/5% CO2 atmosphere.
5. Notes
The authors declare no competing financial interest.
Acknowledgments
4.2.2. Cell proliferation assay
Transformed PSC (3 ꢀ 103) were plated in 96-well plates, in
sextuplicate. The next day, the media was changed to 1% FBS with
apigenin or analogues for 24–48 h. AlamarBlue reagent (10% sam-
ple volume, DAL1025) was added to each well per Invitrogen’s pro-
tocol. Fluorescence was recorded using excitation/emission
wavelengths of 544/590 nm and the SpectraMax M2 Microplate
Reader (Molecular Devices, Sunnyvale CA).
This work was supported by grants P50 CA097007, P30
DA028821, R21 MH093844 (J.Z.), T32 DK007639-21 (M.R.H.) and
K08 CA125209 (C.C.) from the National Institutes of Health, R.A.
Welch Foundation Chemistry and Biology Collaborative Grant from
the Gulf Coast Consortia (GCC), John Sealy Memorial Endowment
Fund, Institute for Translational Sciences (ITS), and the Center for
Addiction Research (CAR) at UTMB. We thank Drs. Lawrence C.
Sowers, Jacob A. Theruvathu, and Tianzhi Wang for the NMR spec-
troscopy assistance, and Dr. Carol Nilsson for mass spectrometry
assistance.
4.2.3. Cell death assay
Transformed PSC (8 ꢀ 103) were plated in 96-well plates in
triplicate. The next day, the media was changed to 1% FBS with
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12
H. Chen et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
22. Li, J. W.; Vederas, J. C. Science 2009, 325, 161.
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Please cite this article in press as: Chen, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.043