Studies on gambogic acid (IV): Exploring structure-activity relationship with IκB kinase-beta (IKKβ)

Article abstract of DOI:10.1016/j.ejmech.2012.02.029

Previously we have reported a series of gambogic acid's analogs and have identified a compound that possessed comparable in vitro growth inhibitory effect as gambogic acid. However, their target protein as well as the key pharmacophoric motifs on the target have not been identified yet. Herein we report that gambogic acid and its analogs inhibit the activity of IκB Kinase-beta (IKKβ) through suppressing the activation of TNFα/NF-κB pathway, which in turn induces A549 and U251 cell apoptosis. IKKβ can serve as one of gambogic acid's targets. The preparation of the compounds was carefully discussed in the article. Caged 4-oxa-tricyclo[4.3.1.0^3,7]dec-2-one xanthone, which was identified as the pharmacophoric scaffold, represents a promising therapeutic agent for cancer and useful probe against NF-κB pathway.

Full text of DOI:10.1016/j.ejmech.2012.02.029

European Journal of Medicinal Chemistry 51 (2012) 110e123
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Studies on gambogic acid (IV): Exploring structureeactivity relationship with I
kB
kinase-beta (IKK
b)
,
b
,
,
c
,
,
,
b
Haopeng Sun a 1, Feihong Chen a 1, Xiaojian Wang b, Zongliang Liu a b, Qian Yang b, Xiaojin Zhang a
,
Jia Zhu a b, Lei Qiang a c, Qinglong Guo a
, Qidong You
,
,
,
c
,
a,b,c,
*
**
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, China
b School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously we have reported a series of gambogic acid’s analogs and have identified a compound that
possessed comparable in vitro growth inhibitory effect as gambogic acid. However, their target protein as
well as the key pharmacophoric motifs on the target have not been identified yet. Herein we report that
Received 14 March 2011
Received in revised form
14 February 2012
Accepted 14 February 2012
Available online 3 March 2012
gambogic acid and its analogs inhibit the activity of I
k
B Kinase-beta (IKK
b
) through suppressing the
can serve
activation of TNF /NF- B pathway, which in turn induces A549 and U251 cell apoptosis. IKKb
a
k
as one of gambogic acid’s targets. The preparation of the compounds was carefully discussed in the
article. Caged 4-oxa-tricyclo[4.3.1.03,7]dec-2-one xanthone, which was identified as the pharmacophoric
Keywords:
scaffold, represents a promising therapeutic agent for cancer and useful probe against NF-kB pathway.
IKKb
Ó 2012 Elsevier Masson SAS. All rights reserved.
NF-kB signaling pathway
Caged xanthone
Pharmacophoric motif
Inhibitor
1. Introduction
mechanism of GA’s anti-tumor activities is still debatable. Multiple
mechanisms have been proposed by research groups worldwide,
such as apoptosis induction [8], cell cycle regulation [9], telomerase
depression [10,11], angiogenesis inhibition [12], reactive oxygen
species (ROS) generation [13] etc. Wang et al. reported a compre-
hensive proteome profiling which deduced STMN1 as the target of
GA [14]. Very recent studies further reported that GA inhibited NF-
Caged Garcinia xanthones, a special category of polyprenylated
xanthones which are naturally discovered in Garcinia plants, have
received increasing attention among phytochemical, pharmaco-
logical, synthetic, and biological communities in recent years
because of their potent bioactivity and unique 4-oxa-tricyclo
[4.3.1.03,7]dec-2-one scaffold, in which a highly substituted tetra-
hydrofuran core [1,2]. Among these noticeable natural products,
gambogic acid (GA, Fig. 1) is one of the best representatives.
Previous studies showed that GA possessed strong growth inhibi-
tory effect against a broad panel of cancer cell lines with IC50
k
B signaling pathway [15,16] and induced apoptosis through its
interaction with the transferrin receptor [17].
As a transcription factor, NF- B participates in the regulation of
diverse biological processes, including immune, inflammatory and
apoptotic response [18e20]. Under normal conditions, NF- B binds
to its natural inhibitory protein, I and locates at cytoplasm. In
response to an activation signal, the I subunit is phosphorylated
k
k
ranging from 1 to 5
mM [3e6]. Due to its prominent growth
kBa
inhibitory effect and good tolerance in different animal tests, GA
has been approved by the Chinese State Food and Drug Adminis-
tration (SFDA) to enter phase II clinical trial [7]. However, the
kBa
at serine residues 32 and 36, ubiquitinated at lysine residues 21 and
22, and then degraded through the ubiquitin-proteasome pathway,
thus exposing the nuclear localization signals on the p50-p65 het-
erodimer. The p65 is then phosphorylated, leading to nuclear trans-
location. It binds to a specific sequence in DNA, which in turn results
* Corresponding author. State Key Laboratory of Natural Medicines, China Phar-
maceutical University, China. Tel./fax: þ86 25 83271351.
** Corresponding author. Jiangsu Key Laboratory of Carcinogenesis and Interven-
tion, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China. Tel./
fax: þ86 25 83271440.
in gene transcription. The phosphorylation of I
kinase (IKKs), which is essential for NF- B activation. IKKs consists of
3 subunits named IKK , IKK , and IKK
deletion studies have indicated that IKK
canonical (classical) pathway of NF-
kBa is catalyzed by IkB
k
a
b
g
b
(also called NEMO). Gene
is the key regulator in the
E-mail addresses: anticancerdrug@yahoo.cn (Q. Guo), youqidong@gmail.com
(Q. You).
1
kB activation [21,22].
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.029

H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
111
Fig. 1. The structure of gambogic acid (GA) and ten designed derivates.
Several strategies focusing on the modification of GA have
already been reported [23,24]. Previously, we have reported a series
of GA’s analogs and discovered the importance of “caged”
xanthones for the growth inhibitory effect through a strict “step by
step” modification strategy [24]. Among these analogs, compound
19 (Fig. 1) possessed comparable in vitro growth inhibitory effect as
GA, while its chemical structure was remarkably simplified.
However, the mechanism by which GA and its analogs exert their
anti-tumor effects, particularly their target protein, remains to be
clarified. Considering that previously suggested targets such as
route for xanthone-based caged compounds
4
and
5
were
summarized in Scheme 1. Briefly, xanthone 1 was O-alkylated with
2-chloro-2-methyl butyne in the presence of potassium carbonate
and a catalytic amount of copper iodide in acetone to afford alkyne
2. Reduction of 2 with Lindlar catalyst produced 3,5,6-tris-(1,1-
dimethyl-allyloxy)-9H-xanthen-9-one (3). The desired target
compounds 4 and 5 were obtained from Claisen/DielseAlder
cascade reaction of 3 in DMF at 120 ꢀC for 1 h.
The preparation of flavanone-based caged compounds 14 and 15
was outlined in Scheme 2. 2,3,4-trihydroxyacetophenone was
selected as starting material. Benzyl bromide was employed to
protect the hydroxyl groups of 6 to afford 7 in 85% yield. Selective
debenzylation of the phenolic group adjacent to the ethanone
moiety was achieved by the action of trifluoroacetic acid in toluene,
leading to O-hydroxyacetophenone derivative 8 in 70% yield.
Chalcone compound derivative 9a or 9b was prepared by conden-
sation of 8 with benzaldehyde or 4-benzyloxy benzaldehyde,
respectively. 10a or 10b was obtained by cyclization of 9a or 9b in
a solution of DMSO at 110 ꢀC in w70% yield. Deprotection of benzyl
groups using 10% Pd/C in THF/CH3OH led to 11a or 11b. The resulted
flavones were then O-alkylated with 2-chloro-2-methyl butyne and
reduced with Lindlar catalyst to produce 13a and 13b. The desired
targeted compound 14 or 15 was obtained from Claisen/
DielseAlder cascade reaction of 13a or 13b in DMF at 120 ꢀC for 1 h.
The preparation of 17 was described in Scheme 3. Compound 16
was obtained through the condensation of phloroglucinol and
salicylic acid mediated by ZnCl2 in POCl3 in 43% yield. It was then
alkylated with isopentenyl bromide in the condition of NaOCH3 and
CH3OH in 11% yield.
telomerase, CDK7 and STMN1 are all client proteins of NF-
signaling pathway, in the present study, we tried to identify the key
regulator of NF- B signaling pathway affected by GA and its
analogs. Our results suggested that GA inhibited the activation of
NF- B signaling pathway through suppression the activity of IKK
kB
k
k
b
and further induced the apoptosis of A549 and U251 cancer cell
lines. Furthermore, because of the complexed structure and huge
difficulties in total synthesis of GA, we tried to identify the phar-
macophoric motif of GA to inhibit IKK
described in our previous paper [24]. The 4-oxa-tricyclo[4.3.1.03,7
dec-2-one was certified to be the key scaffold of GA to exert its
inhibition effect on IKK . Additionally, we also studied the effects of
b using the same strategy
]
b
the substituents such as hydroxyl and isopentenyl groups. Mecha-
nistic studies for the most potent compound 20 showed that it
possessed a very similar mechanism with GA. Its structure provides
a new skeleton for developing novel IKK
b
inhibitors. It is also
signaling
a
promising chemical probe to understand NF-kB
pathway as well as the biological functions of IKK
b.
The preparation of flavanone-based caged compounds 20 was
outlined in Scheme 4. Xanthone 24 wasobtainedfrom xanthone1 via
selective protection of 5,6-bihydroxyl with Ph2CCl2, 3-hydroxyl with
MOMCl (Methyl chloromethyl ether) and 1-hydroxyl with Ac2O in
76% yieldover threesteps. Deprotectionof the5,6-biphenyl group via
hydrogenolysis at the presence of Pd/C (95%) lead to compound 25.
2. Results and discussion
2.1. Chemistry
In order to identify the pharmacophoric motif of GA, we
designed and synthesized ten GA’s analogs (Fig. 1). The synthetic

112
H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
Scheme 1. The synthetic route for compound 4 and 5. Reagents and conditions: (a) ClC(CH3)2C^CH2, K2CO3, KI, CuI, CH3COCH3, 50 ꢀC, 5 h; (b) 10% Pd/BaSO4, CH3COOC2H5/C2H5OH
(1:3, v/v), rt, 2 h; (c) DMF, 120 ꢀC, 1 h.
5,6-dihydroxy groups were alkylated simultaneously with 2-chloro-
2-methyl butyne in the presence of KI and K2CO3 under CuI catalysis
in 70% yield and lead to compound 26. Compound 27 was easily
formed from compound 26 via hydrogenolysis at the presence of Pd/
BaSO4 and Claisen/DielseAlder cascade reaction in 65% yield over
two steps. Caged compound 20 was obtained via deprotection of Ac
and MOM groups simultaneously under the condition of 3 M HCl in
acetone with the yield of 60%.
signaling should inhibit the translocation of p65 to the nucleus.
We first investigated the effect of GA on TNF induced NF-
nuclear translocation by using high content screening. The results
a
kB
confirmed that GA suppressed the translocation of NF-
nucleus (Fig. 2A). GA dose-dependently inhibited TNF
NF- B nuclear translocation, with 50.2% inhibition at 5
We also determined the effect of GA on TNF induced nuclear
translocation of p65 by Western blot analysis. GA obviously sup-
pressed TNF induced translocation of p65 in a dose-dependent
k
B to the
a
induced
k
mM (Fig. 2B).
a
Compound 18, 19 and 21 was synthesized using the method
previously reported by our laboratory [24].
a
manner (Fig. 3).
2.2. GA inhibits TNF-induced phosphorylation and nuclear
translocation of p65
2.3. GA inhibits TNF-dependent I
phosphorylation
kBa degradation and
In response to an activation signal, the I
phorylated at serine residues 32 and 36, then ubiquitinated at
lysine residues 21 and 22, and degraded through the proteasomal
pathway, thus exposing nuclear localization signals on the NF-
leading to its interaction with a specific sequence in DNA, which in
turn results in gene transcription [15]. Suppression of NF-
k
B
a
subunit is phos-
The translocation of NF-
phosphorylation, ubiquitination, and proteolytic degradation of
. To determine whether inhibition of TNF -induced NF-
translocation was due to inhibition of I degradation, cells were
pretreated with 4 or 8 M GA and then exposed to TNF for various
time periods. We then examined the I degradation in the
kB to the nucleus is preceded by the
IkB
a
a
kB
k
B,
kBa
m
a
k
B
kBa
O
O
O
O
d
g
e
f
BnO
OH
X
HO
OH
BnO
OH
BnO
OBn
OBn
OH
OBn
8
OBn
7
9a x=H
9b x=OBn
6
O
O
O
O
O
O
h
i
j
HO
BnO
O
OH
OBn
O
X
X
X
10a
10b
11a
11b
12a
12b
O
O
O
O
O
O
k
O
14
O
X
O
O
13a
13b
O
O
HO
15
Scheme 2. The synthetic route for compound 11a, 14 and 15. Reagents and conditions: (d) BnBr, K2CO3, KI, DMF, 50 ꢀC, 8 h; (e) trifluoroacetic acid/toluene (1:5, v/v), rt, 1 h; (f) 20%
aqueous KOH/EtOH, rt, 36 h; (g) I2, DMSO, 110 ꢀC, 12 h; (h) 10% Pd/C, THF/CH3OH (1:1, v/v), rt, 3 h; (i) ClC(CH3)2C^CH2, K2CO3, KI, CuI, CH3COCH3, 50 ꢀC, 4 h; (j) 10% Pd/BaSO4,
CH3COOC2H5/C2H5OH (1:3, v/v), rt, 2 h; (k) DMF, 120 ꢀC, 1 h.

H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
113
caged 4-oxa-tricyclo[4.3.1.03,7]dec-2-one xanthone scaffold (4, 5,
18, 19 and 20) showed similar activity to GA (Table 1). Among all the
derivatives, compound 19 and 20 showed remarkable IKK
b inhib-
itory effect, with IC50 8.17 M and 3.52 M, respectively. Consid-
m
m
ering its potent cell viability inhibitory effects, we thus concluded
that this structure contained the key pharmacophoric motif of GA.
To further study the mechanism of GA and its derivatives to
inhibit the viability of cancer cells, we chose GA and compound 20
to study their effects on apoptosis and cell cycle distribution. The
substantial morphological changes observed in GA-treated and 20-
treated U251 cells were examined and photographed by an inver-
ted light microscope. Damaged cells became round and shrunken,
while the untreated cells retained the normal size and shape
(Fig. 5A). Identified by DAPI staining, the bright nuclear conden-
sation and the apoptotic bodies appeared after treatment with 20
(Fig. 5B). To further confirm the apoptosis induced by GA and 20,
Annexin V/PI staining assay was used. After treated with 4, 8 and
Scheme 3. The synthetic route for compound 17. Reagents and conditions: (l) ZnCl2,
POCl3, 70 ꢀC, 3 h; (m) BrCH2CHC(CH3)2, NaOMe, CH3OH, rt, 12 h.
cytoplasm by Western blot analysis. TNF
tion in the control cells in 15 min, but it had no effect on I
degradation in GA pretreated cells (Fig. 4). Moreover, the phos-
phorylation of I was stimulated by TNF in 15 min, and it was
suppressed by GA. These results indicated that GA inhibited TNF-
induced NF- B activation through inhibition of I phosphoryla-
tion and suppression of I degradation.
a induced IkBa degrada-
kBa
k
Ba
a
k
kBa
kBa
2.4. Identification of caged 4-oxa-tricyclo[4.3.1.03,7]dec-2-one
xanthones as a new scaffold of IKK inhibitor
16 mM 20 and 8 mM GA for 24 h, the early and median apoptotic cells
(right low section of fluorocytogram) were increased (from 2.75% to
58.21%, Fig. 5C and D) and the late apoptotic and necrotic cells
(right upper section of fluorocytogram) were also increased strik-
ingly (from 1.39% to 34.24%, Fig. 5B and C). These results suggested
that apoptosis induction by 20 was involved in its anti-tumor effect.
20 induced apoptosis in a very similar manner to GA, which further
confirmed that 20 possessed the key pharmacophoric motifs of GA.
Based on immunofluorescence experiment, we next studied
whether 20 exhibited similar mechanism to GA in inhibiting the
b
GA is a natural product isolated from the resin of Garcinia han-
buryi. The complicated structure of GA brings huge hindrance for its
further usage. To simplify the structure skeleton and to find out the
key pharmacophoric motifs that account for GA’s biological func-
tion, 10 derivatives of GA were designed and synthesized (Fig. 1).
MTT assays were first performed to ascertain inhibitory effect on
cell viability by GA and its derivatives in A549 and U251 cells.
Following 24 h treatment, GA inhibited the viability of A549 and
activation of NF-kB signaling pathway. We found that 20 inhibited
TNF -induced NF-
a
k
B nuclear translocation in a dose-dependent
U251 cells and the IC50 value was 1.10 mM and 2.56 mM, respectively
(Table 1). Compounds 19 and 20 showed equivalent in vitro growth
inhibitory effects to GA.
manner (Fig. 6). This further demonstrated that 20 exhibited its
anti-cancer effect in a very similar mechanism to GA.
Since GA inhibited the phosphorylation and degradation of I
kBa,
we further tested the effect of GA and its derivatives on the activity
2.5. Structureeactivity relationships
of IKK
degradation and NF-
b
, an enzyme that was reported to play crucial role in I
k
B
a
b
k
B activation [25]. By using HTScan IKK
To further identify the pharmacophoric motif of GA, we explored
kinase assay kit, we found that GA remarkably suppressed the
activity of IKK . The phosphorylation of I by IKK was inhibited
M. Additionally, compounds contained
structureeactivity relationships (SAR) using IKKb kinase assay
b
k
Ba
b
and cellular assay. Generally, compounds possessed caged 4-oxa-
by GA with IC50 of 3.07
m
tricyclo[4.3.1.03,7]dec-2-one xanthone scaffold (4, 5, 18, 19 and 20)
OH
O
OH
O
OH
O
o
p
n
MOMO
O
O
Ph
HO
O
O
Ph
HO
O
1
OH
O
Ph
O
Ph
OH
22
23
OAc O
O
OAc O
O
OAc O
O
q
s
t
r
MOMO
O
MOMO
OH
MOMO
O
Ph
O
OH
O
Ph
26
25
24
O
OH
O
O
O
OAc O
u
O
O
HO
MOMO
O
Scheme 4. The synthetic route for compound 20. Reagents and conditions: (n) Ph2CCl2, DIPEA, Ph2O, 170 ꢀC, 0.5 h; (o) MOMCl, K2CO3, acetone, 25 ꢀC, 5 h; (p) Ac2O, DMAP, CH2Cl2,
25 ꢀC, 4 h; (q) H2, Pd/C, THF/MeOH, 50 ꢀC, 12 h; (r) (CH3)2CClC^CH, KI, K2CO3, CuI, acetone, reflux, 1.5 h; (s) H2, Pd/BaSO4, EtOH/EtOAc, 35 ꢀC, 4 h; (t) DMF, 120 ꢀC, 1 h; (u) 3M HCl,
acetone, 40 ꢀC, 6 h.

114
H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
Fig. 2. High content analysis of NF-
k
B localization. (A) A549 cells were first treated with indicated dose of GA for 2 h at 37 ꢀC and then exposed to mixed stimulators for 30 min.
Then the cells were underwent high content analysis. (B) The ratio of NF-kB fluorescence in nuclei and cytoplasm, indicating the inhibition rate of NF-kB translocation by GA.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
exhibited similar activity to GA on not only A549 and U251 cancer
cell lines but also IKK , indicating this scaffold was the key phar-
activity and 8e10 fold less active on cancer cells than compound 20.
Although flavone compound 11a and xanthone compound 17
b
macophoric motif of GA. The activities of compounds that were lack
of either xanthone structure (14, 15 and 21) or caged 4-oxa-tricyclo
[4.3.1.03,7]dec-2-one ring (11a and 17) remarkably decreased in
both cellular and kinase test. Compound 14 and 15 with caged
flavone scaffold was approximately 10e15 fold less active on kinase
possessed moderate activity on IKKb (3e4 fold less active than 20),
their activity on cancer cells dramatically decreased.
Additionally, the substituted groups on 4-oxa-tricyclo[4.3.1.03,7
]
dec-2-one xanthone scaffold were also important to their activities.
We have previously demonstrated that C1 hydroxy group was
indispensable for the growth inhibitory effect of 19 [24]. This group
was crucial for the inhibitory activity on IKK
b. Compounds
possessed this group such as 19 and 20 were most effective on IKK
b
,
while compound lacking this group such as 18 was approximately
8-fold less active than 20. It has been reported that the formation of
H-bond interactions between inhibitors and the hinge region of
IKKb were very important [26]. The missing of hydroxy groups in 18
might cause the decrease in its activity. The results also verified the
importance of hydroxy groups in this series of compounds. The
activities of compound 4 and 5 on both cancer cell lines and IKK
b
enzyme were a little bit lower than 19 and 20. These results sug-
gested that the isopentenyl motif on the phenyl ring of these
compounds was not necessary for the bioactivity, removal of this
motif would not affect the inhibitory effect, and thus would
simplify the structure.
We also compared the most potent compounds 19 and 20. 20
possessed a C3 hydroxy group. Although it did not affect the activity
remarkably, more modifications such as O-alkylation could be
Fig. 3. GA inhibits TNF
untreated or pretreated with indicated dose of GA for 4 h at 37 ꢀC and then treated
with 50 ng/mL TNF for 30 min. Cytoplasmic and nuclear extracts were prepared and
analyzed by western blotting with antibodies against p65. For loading control of
a-induced nuclear translocation of p65. A549 cells were either
a
cytoplasmic and nuclear protein, the membrane was blotted with
antibody, respectively.
b-actin and histone

H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
115
Fig. 4. Effect of GA on TNF
a
-induced degradation of I
k
B
a. A549 cells were incubated with 4
m
M (A) and 8
mM (B) GA for 4 h and treated with 0.1 ng/mL TNF for the indicated times.
Cytoplasmic extracts were prepared and analyzed by western blotting with antibodies against I
k
B
a
and phorspho-IkBa.
carried out on this position, and thus might lead to more potent
molecules. Additionally, this group might also enhance the solu-
for binding mode study. GA, compound 14, 20 and 21 were taken as
examples to depict the binding mode (Fig. 7). All 4 compounds
bility of the caged xanthone compounds and improve the physi-
cochemical properties.
could located in the ATP binding pocket of IKK
H-bonds with the hinge region of the enzyme, which is considered
to be a common binding pattern of IKK and its inhibitors [28e33].
To be more specific, GA formed H-bonds with Asp103 and Lys106,
compound 20 formed H-bonds with Cys99 and Lys106. However,
compound 14 and 21 only formed one H-bond with Lys106, which
b and make several
In summary, (a) caged 4-oxa-tricyclo[4.3.1.03,7]dec-2-one
xanthone scaffold was the key pharmacophoric motif of GA, (b)
this scaffold possessed potent inhibitory effect on cancer cell
proliferation and similar molecular mechanism to GA, (c) this
b
scaffold could serve as a new skeleton of IKK
groups on this scaffold especially at C1 position enhanced the IKK
inhibitory effect, while the isopentenyl group at the aromatic ring
was not important.
b
inhibitor, (d) hydroxy
may explain the decrease of their IKK
the anti-tumor effect. Our results agreed with Nagarajan et al. in
that as the key residues within the ATP binding pocket of IKKb,
Cys99 and Asp103 could form H-bond with ATP and frequently
interact with the inhibitors [26]. Besides, there are 3 hydrophobic
b inhibitory activity as well as
b
pockets (Fig. 7) in the binding site of IKKb. P1 is formed by Leu21,
2.6. Molecular binding pattern study of GA and its derivatives with
Gly22, Thr23 and Lys46, P2 is formed by Met96, Cys99, Ile165 and
Glu172, P3 is formed by Asp103, Glu149 and Asn150 [26]. As
depicted in Fig. 7, GA occupied P1 and P3 (Fig. 7D), 14 and 20
occupied P2 and P3 (Fig. 7A and B, respectively), while 21 obviously
missed P1 and P2. The result indicated a lower binding affinity of
this compound, and explained the decrease of the inhibitory effect.
Very recently, the co-crystal structure was reported by Xu, G.
et al [41]. We compared the binding pattern of GA with homology
model (Fig. 8A) to the pattern with the crystal structure (PDB id:
3QAD, Fig. 8B). We can see that the two binding model were very
similar. In both patterns, GA formed H-Bond with Asp103 and
Lys106. Asn109 and Glu149 were also important for GA’s binding.
Compared to the co-crystal structure, GA located well in the ATP
IKKb by molecular docking
To further illustrate the binding mode of IKK
we built up the protein homology model of IKK
work has demonstrated that GA covalently modified IKK
[16]. However, the MS analysis of this modification was based on
a short peptide around the binding site of IKK , it was not clear
whether the spatial structure of this peptide was in accord with the
“real” structure under physiological environment. No crystal
structure has been reported until now. As a result, the computa-
tional method is meaningful and can help us to form a deeply
insight into the binding patterns of this series of compounds with
b
with its inhibitors,
[27]. Previously
at Cys179
b
b
b
IKKb. The binding modes were studied using the docking program
binding pocket of IKK
bind to Cys179 (Fig. 8B). However, this residue obviously locates at
the edge loop region of IKK , which not correlate to the ATP binding
pocket. It needs to be emphasize that Cys99, a residue located at the
ATP binding pocket of IKK , has been mentioned to be crucial for
the activity of IKK . The double bond at isopentenyl group of GA is
b. Previous study indicated that GA covalently
Gold 3.0. Gold score Fitness was selected to evaluate the binding
affinity (Table 2). By analyzing the data, it was evident that the
Glodscore Fitnesses of most compounds were in well correlation
with the experimental data, indicating that the model was reliable
b
b
b
Table 1
Inhibition of IKK
b and cell proliferation by GA and its analogs.
close to this residue, indicating the possibility of covalent binding.
Thus, it may be Cys99, not Cys179, that contributes to the covalent
Comp.
IKK
b
IC50
(m
M)
A549 IC50
(m
M)
U251 IC50 (mM)
binding of GA with IKKb.
4
5
6.60
10.78
9.82
41.65
25.30
13.14
24.16
8.17
3.58
14.41
37.25
27.46
15.51
34.89
1.93
0.70
2.67
60.26
1.10
8.57
22.44
50.11
40.24
18.74
46.76
18.70
9.05
11a
14
15
17
18
19
20
21
GA
3. Conclusion
Inspired by the previous study by our group [24] and the
molecular mechanism research of GA on NF- B pathway described
by Pandey, Ujjawal and colleagues [15,16], we verified that the
k
3.52
40.27
3.07
7.32
82.43
2.56
anti-tumor effects of GA were mediated through modulation of the
NF-kB pathway and IKKb was confirmed as the the key regulator of
the pathway affected by GA. However, the complicated structure of

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H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123

H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
117
Table 2
The binding information of GA and its analogs with IKK
b
protein homology model.
Compound
Gold Score
IKKb IC50 (mM)
4
5
59.46
54.25
48.87
41.21
49.20
50.22
46.67
54.57
57.67
38.75
60.46
6.60
10.78
9.82
41.65
25.30
13.14
24.16
8.17
11a
14
15
17
18
19
20
21
GA
3.52
40.27
3.07
GA brings huge hindrance for its further usage. To simplify the
structure skeleton and to find out the key pharmacophoric motifs
that account for GA’s biological function, ten derivatives of GA were
synthesized and evaluated for their inhibitory effect on IKKb as well
as cancer cell proliferation. The caged 4-oxa-tricyclo[4.3.1.03,7]dec-
2-one xanthone scaffold was confirmed as the key pharmacophoric
motif of GA. This scaffold possessed potent inhibitory effect on
cancer cell proliferation similar to GA and could serve as a new
skeleton of IKK
of this scaffold with IKK
b
inhibitor. We also investigated the binding pattern
through molecular docking. The resulted
b
information would help us design more potent derivatives based on
this scaffold.
This is the first report to identify 4-oxa-tricyclo[4.3.1.03,7]dec-2-
one xanthone scaffold as a new skeleton of IKK
b inhibitor. Our
results provided the molecular basis for the antiproliferative effects
of GA and its derivatives. Further preclinical and clinical trials are
required to determine the anti-tumor efficacy of these compounds.
4. Experimental protocols
4.1. General materials and methods
All reagents were purchased from commercial sources and were
used without further purification unless otherwise noted. Melting
points were determined by XT-4 melting point apparatus and were
reported without any correction. IR spectra were recorded on
a Nicolet Impact 410 spectrometer using KBr film. The 1H NMR and
13C NMR spectra were collected on Bruker AV-300 instruments
using deuterated solvents with tetramethylsilane (TMS) as internal
standard. EI-MS was recorded on Shimadzu GCMS-2010 apparatus.
Each of the target compounds were purified by silica gel (60 Å,
70e230 mesh) column chromatography. Concentration and evap-
oration of the solvent after reaction or extraction was carried out on
a rotary evaporator (Büchi Rotavapor) operated at reduced pressure.
GA was isolated and purified according to the previously
reported methods [34]. The purity of GA used in all experiments
was 95% or higher. It was dissolved in PBS containing arginine to
a concentration of 10 mM as the primary stock solution and stored
at ꢁ20 ꢀC. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-ium-
bromide (MTT) was purchased from Sigma (St. Louis, MO). Annexin
V-FITC Apoptosis Detection kit was purchased from BioVision
(Mountain View, CA). Cellomics NF-
was obtained from Thermo Scientific (Pittsburgh, PA). HTScan IKK
kinase assay kit was purchased from Cell Signaling Technology
(Beverly, MA). Human I and p-I (ser 32/36) antibodies were
kB Activation HCS Reagent Kit
Fig. 6. Immunofluorescence staining for Rel A (p65) and nucleus in U251 cells: green
represents p65, blue represents nuclei, and merge represents both. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version
of this article.)
b
kB
a
kBa
Fig. 5. 20 induced apoptosis in U251 cells. (A) The morphology of U251 cells after exposure to different doses of 20 and GA for 24 h was examined and photographed by an inverted
light microscope (200 ꢂ ). The cell damage appeared after treatment with 20, comparing with the control plate (GA). (B) Fluorescence microscope detection of 20 (400 ꢂ ).
(C) Annexin V/PI double-staining assay of U251 cells. Y axis showed PI labeled population and X axis shows FITC-labeled Annexin V positive cells. (D) The apoptotic rates of U251
cells induced by 20 and GA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

118
H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Histone antibody was bought from Bioworld Technology, Inc.
(Bioworld, MN). All drugs were diluted in the corresponding culture
medium to desired concentrations before use.
(m, 1H), 6.08 (s, 1H), 6.60 (br s, 1H), 7.40 (d, J ¼ 6.8 Hz, 1H), 12.80 (s,
1H); m/z (ESIeMS): 463 [M ꢁ H]ꢁ [35].
4.2.4. 2,3,4-Trisbenzyloxyacetophenone (7)
Human U251 glioma cell lines and A549 lung epithelial cell lines
were purchased from Cell Bank of Shanghai Institute of Biochem-
istry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences. U251 and A549 cells were cultured in
DMEM which were supplemented with 10% (v/v) fetal calf serum,
100 U/mL penicillin and 100 U/mL streptomycin in a 5% CO2
atmosphere.
Compound 6 (1.7 g, 10 mmol) was added to the suspension of
benzyl bromide (6.0 g, 35.0 mmol), K2CO3 (8.0 g, 58.0 mmol) and KI
(0.3 g, 1.5 mmol) in DMF (20.0 mL). The reaction mixture was
heated to 50 ꢀC for 8 h. Then the reaction mixture was cooled to
room temperature and poured into ethyl acetate (100 mL) and
washed with brine. The organic layer was dried with Na2SO4 fol-
lowed concentration under reduced pressure. The residue was
purified through column chromatography (eluent, PE/EtOAc ¼ 4:1)
to afford 3.7 g 7 as light yellow solid. Yield: 85%. m.p.: 71e73 ꢀC 1H
4.2. Chemistry
NMR (300 MHz, CDCl3): d 2.30 (s, 3H), 5.02 (s, 2H), 5.08 (s, 2H), 5.14
4.2.1. 1-Hydroxy-3,5,6-tris(2-methylbut-3-yn-2-yloxy)-9H-
xanthen-9-one (2)
(s, 2H), 6.62 (d, J ¼ 9.0 Hz, 1H), 7.28e7.33 (m, 6H), 7.34e7.38 (m,
10H); m/z (EIeMS): 438 [M]þ.
To a solution of 1 (2.9 g, 10.8 mmol) in acetone (100.0 mL),
potassium iodide (5.9 g, 35.7 mmol), potassium carbonate
(4.9 g, 35.7 mmol) and CuI (0.2 g, 1.1 mmol) were added. The
reaction mixture was stirred at room temperature for 10 min, then
2-chloro-2-methylbut-3-yne (9.7 mL, 86.4 mmol) was added and
the resulted mixture was stirred at 50 ꢀC for 5 h. Water (100.0 mL)
and EtOAc (50.0 mL) were added to the mixture and stirred for
30 min. The organic layer was separated, dried over sodium sulfate,
and evaporated. The residue was purified through column chro-
matography (eluent, PE/EtOAc ¼ 8:1) to afford 1.2 g 2 as yellow oil.
4.2.5. 2-Hydroxy-3,4-bisbenzyloxyacetophenone (8)
To a solution of 7 (2.0 g, 4.6 mmol) in toluene (20.0 mL) was
added trifluoroacetic acid (4.0 mL). The dark reaction mixture was
stirred at room temperature for 2 h. Then the mixture was
neutralized with 10% NaOH aqueous and extracted with ethyl
acetate. The organic layer was dried with Na2SO4 followed
concentration under reduced pressure. The residue was purified
through column chromatography (eluent, PE/EtOAc ¼ 4:1) to afford
1.1 g 8 as light yellow solid. Yield: 70%. m.p.: 122e123 ꢀC 1H NMR
Yield: 25%. 1H NMR(300 MHz, CDCl3):
d
1.76 (s, 6H),1.78 (s, 6H),1.82
(300 MHz, CDCl3): d 2.22 (s, 3H), 5.10 (s, 2H), 5.15 (s, 2H), 6.58 (d,
(s, 6H), 2.34 (s, 1H), 2.68 (s, 1H), 2.70 (s, 1H), 6.72 (d, J ¼ 2.2 Hz, 1H),
6.82 (d, J ¼ 2.2 Hz, 1H), 7.64 (d, J ¼ 9.0 Hz, 1H), 7.97 (d, J ¼ 9.0 Hz),
12.85 (s, 1H); m/z (EIeMS): 458 [M]þ.
J ¼ 10.2 Hz, 1H), 7.28e7.34 (m, 6H), 7.35e7.40 (m, 5H), 11.12 (s, 1H);
m/z (EIeMS): 348 [M]þ.
4.2.6. Chalcone compounds (9a) and (9b)
4.2.2. 1-Hydroxy-3,5,6-tris(2-methylbut-3-en-2-yloxy)-9H-
xanthen-9-one (3)
To a cold solution of 8 (500.0 mg, 1.4 mmol) and appropriate
benzaldehyde in 3 mL H2O/EtOH (1:4, v/v), 3.0 mL 20% KOH solu-
tion was added. The resulting mixture was stirred at room
temperature for 36 h. The mixture was poured into ice, acidified to
pHw5 with 1 N HCl, and extracted with ethyl acetate. The organic
phase was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified through column
chromatography (eluent, PE/EtOAc ¼ 6:1) to afford 9a or 9b.
9a, reagent: benzaldehyde (168.5 mg, 1.6 mmol). 388.4 mg;
Yield: 62%; yellow solid; m.p.: 164e165 ꢀC 1H NMR (300 MHz,
To a solution of 2 (100 mg, 0.2 mmol) in ethanol (10.0 mL) and
ethyl acetate (3.3 mL) was added 10% Pd/BaSO4 (10.0 mg). The
mixture was stirred under an atmosphere of hydrogen for 2 h at
room temperature and filtered through a plug of silica gel. The
filtration was concentrated and purified through column chroma-
tography (eluent, PE/EtOAc ¼ 8:1) to afford 81.0 mg 3 as brown oil.
Yield: 80%. 1H NMR (300 MHz, CDCl3): 1.55 (s, 6H), 1.60 (s, 6H), 1.66
(s, 6H), 5.17e5.20 (m, 3H), 5.41e5.46 (m, 3H), 5.63e5.70 (m, 3H),
6.42 (d, J ¼ 2.1 Hz, 1H), 6.51 (d, J ¼ 2.1 Hz, 1H), 6.92 (d, J ¼ 10.8 Hz,
1H), 7.35 (d, J ¼ 10.8 Hz, 1H), 12.78 (s, 1H); m/z (EIeMS): 464 [M]þ.
CDCl3):
d
5.10 (s, 2H), 5.17 (s, 2H), 6.47 (d, J ¼ 10.8 Hz, 1H), 7.08 (d,
J ¼ 9.6 Hz, 2H), 7.34e7.58 (m, 12H), 7.73 (d, J ¼ 9.6 Hz, 2H), 7.80 (d,
J ¼ 10.8 Hz, 1H); 13.03 (s, 1H); m/z (EIeMS): 436 [M]þ.
4.2.3. Caged xanthone (4) and (5)
9b, reagent: 4-benzyloxy benzaldehyde (335.0 mg, 1.6 mmol).
529.5 mg; Yield: 68%; yellow solid; m.p.: 161e162 ꢀC 1H NMR
A solution of 3 (100.0 mg, 0.2 mmol) in DMF (3.0 mL) was heated
at 120 ꢀC for 1 h. The yellow reaction mixture was cooled to 25 ꢀ
C
(300 MHz, CDCl3): d 5.12 (s, 2H), 5.14 (s, 2H), 5.18 (s, 2H), 6.52 (d,
and purified by column chromatography (eluent, PE/EtOAc ¼ 5:1)
to yield the caged xanthone 4 (50.0 mg, Yield: 50%) and 5 (15.0 mg,
Yield: 15%).
J ¼ 9.0 Hz, 1H), 7.02 (d, J ¼ 9.0 Hz, 2H), 7.28e7.67 (m, 13H), 7.73 (d,
J ¼ 9.0 Hz, 2H), 7.74 (d, J ¼ 9.0 Hz,1H), 7.86 (d, J ¼ 15.0 Hz,1H); 13.33
(s, 1H); m/z (EIeMS): 542 [M]þ.
4: yellow solid; m.p. > 200 ꢀC 1H NMR (300 MHz, CDCl3):
d 1.04
(s, 3H), 1.24e1.26 (m, 4H), 1.38 (s, 3H), 1.69 (s, 3H), 1.76 (s, 3H), 1.81
(s, 3H), 2.35 (dd, J ¼ 13.2 Hz, J ¼ 4.4 Hz, 1H), 2.46 (d, J ¼ 9.2 Hz, 1H),
2.56e2.59 (m, 2H), 3.41e3.49 (m, 2H), 3.50e3.53 (m, 1H),
4.30e4.45 (m, 1H), 5.22e5.27 (m, 1H), 6.04 (s, 1H), 6.09 (br s, 1H),
7.46 (d, J ¼ 6.8 Hz, 1H), 12.60 (s, 1H); 13C NMR (CDCl3, 75 MHz)
4.2.7. Flavone compounds (10a) and (10b)
A stirred solution of corresponding Flavones 9a (500.0 mg,
1.1 mmol) or 9b (600.0 mg, 1.1 mmol) and iodine (28.0 mg,
0.1 mmol) in DMSO (10.0 mL) was heated to 110 ꢀC for 12 h. The
mixture was cooled and poured into cold water. The mixture was
extracted with ethyl acetate. The combined organic phase was
washed with saturated sodium thiosulfate and water, successively.
Then the organic layer was dried with anhydrous Na2SO4 and
concentrated. The residue was purified by silica gel column chro-
matography (eluent, PE/EtOAc ¼ 6:1) to afford 10a or 10b.
10a: 355.0 mg; Yield: 71%; yellow solid; m.p. > 200 ꢀC 1H NMR
d
16.9, 18.2, 22.3, 25.6, 25.7, 25.9, 29.0, 29.2, 30.3, 47.0, 49.2, 83.2,
84.5, 90.5, 97.0, 101.0, 105.6, 117.7, 121.1, 133.3, 133.9, 134.9, 135.4,
157.9,163.0, 163.9,179.4, 203.1; m/z (ESIeMS): 463 [M ꢁ H]ꢁ; HRMS
(ESIeTOF) calcd. for C28H32O6 [M þ Na]þ 487.2097, found 487.2104.
5: yellow solid; m.p. > 200 ꢀC 1H NMR (300 MHz, CDCl3):
d 1.12
(s, 3H),1.30 (s, 3H),1.36 (dd, J ¼ 13.6 Hz, J ¼ 10.5 Hz,1H),1.40 (s, 3H),
1.67 (s, 3H), 1.77 (d, J ¼ 1.1 Hz, 3H), 1.82 (s, 3H), 2.33 (dd, J ¼ 13.6 Hz,
J ¼ 4.5 Hz, 1H), 2.39 (d, J ¼ 9.6 Hz, 1H), 2.61e2.59 (m, 2H), 3.37 (m,
2H), 3.48 (dd, J ¼ 6.8, J ¼ 4.3 Hz, 1H), 4.48e4.44 (m, 1H), 5.26e5.23
(300 MHz, CDCl3):
d 5.22 (s, 2H), 5.30 (s, 2H), 6.91 (s, 1H), 7.15 (d,
J ¼ 9.0 Hz, 1H), 7.31e7.40 (m, 3H), 7.42e7.53 (m, 11H), 7.75 (d,
J ¼ 8.4 Hz, 2H), 7.90 (d, J ¼ 9.0 Hz, 2H) m/z (EIeMS): 434 [M]þ.

H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
119
Fig. 7. Molecular docking simulations for GA and its derivatives with IKKb. (A) Compound 20; (B) Compound 14; (C) Compound 21; (D) GA. Residues in the binding pocket were
shown as gray stick. H-bonds were shown as red dot line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
10b: 410.0 mg; Yield: 69%; yellow solid; m.p. > 200 ꢀC 1H NMR
(300 MHz, CDCl3): 5.20 (s, 2H), 5.24 (s, 2H), 5.35 (s, 2H), 6.84 (s,
4.2.8. Flavone compounds (11a) and (11b)
d
To a solution of compound 10a (4.3 g, 10.0 mmol) or 10b (5.4 g,
10.0 mmol) in THF/CH3OH (40.0 mL/40.0 mL) was added 10% Pd/C
(0.5 g). The reaction mixture was stirred under an atmosphere of
hydrogen overnight. The reaction mixture was filtered and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (eluent, PE/EtOAc ¼ 6:1) to
afford 11a or 11b.
1H), 7.15 (d, J ¼ 8.4 Hz, 2H), 7.31e7.52 (m, 16H), 7.75 (d, J ¼ 8.1 Hz,
1H), 7.90 (d, J ¼ 8.4 Hz, 2H) m/z (EIeMS): 540 [M]þ.
11a: 2.1 g; Yield: 83%; yellow solid; m.p. > 200 ꢀC 1H NMR
(300 MHz, CDCl3):
d
6.90 (s, 1H), 6.96 (d, J ¼ 8.4 Hz, 1H), 7.41 (d,
J ¼ 8.4 Hz, 1H), 7.58e7.60 (m, 3H), 8.14e8.17 (m, 2H), 9.50 (s, 1H),
10.35 (s, 1H). m/z (EIeMS): 254 [M]þ.
11b: 2.2 g; Yield: 81%; yellow solid; m.p. > 200 ꢀC 1H NMR
(300 MHz, CDCl3):
d
6.70 (s, 1H), 6.92 (d, J ¼ 7.8 Hz, 2H), 7.37 (d,
J ¼ 8.4 Hz, 2H), 8.00 (d, J ¼ 8.4 Hz, 2H), 9.40 (s, 1H),10.21 (s, 2H). m/z
(EIeMS): 270 [M]þ.
4.2.9. Flavone compounds (12a) and (12b)
To a solution of 11a (500.0 mg, 2.1 mmol) or 11b (555.0 mg,
2.1 mmol) in acetone (20.0 mL), potassium iodide (1.7 g,
10.5 mmol), potassium carbonate (1.5 g, 10.5 mmol) and CuI
(114.6 mg, 0.6 mmol) were added. The reaction mixture was stirred
at room temperature for 10 min, then 2-chloro-2-methylbut-3-yne
(1.72 g, 16.8 mmol) was added and the resulted mixture was stirred
at 50 ꢀC for 4 h. Water (30.0 mL) and EtOAc (30.0 mL) were added to
the mixture and stirred for 30 min. The organic layer was separated,
dried over sodium sulfate, and evaporated. The residue was purified
through column chromatography (eluent, PE/EtOAc ¼ 8:1) to afford
12a or 12b.
12a: 470.1 mg; Yield: 62%; white solid; m.p.: 163e165 ꢀC 1H
NMR (300 MHz, CDCl3): d 1.74 (s, 6H), 1.83 (s, 6H), 2.29 (s, 1H), 2.65
(s, 1H), 6.74 (s, 1H), 7.49 (m, 3H), 7.66 (d, J ¼ 8.7 Hz, 1H), 7.92 (d,
J ¼ 8.7 Hz, 1H), 8.02 (m, 2H). m/z (EIeMS): 386 [M]þ.
12b: 577.2 mg; Yield: 60%; white solid; m.p.: 152e153 ꢀC 1H
NMR (300 MHz, CDCl3): d 1.68 (s, 6H), 1.73 (s, 6H), 1.86 (s, 6H), 2.29
(s, 1H), 2.51 (s, 1H), 2.65 (s, 1H), 6.72 (s, 1H), 6.80 (d, J ¼ 10.5 Hz, 1H),
7.05 (d, J ¼ 11.7 Hz, 2H), 7.49 (d, J ¼ 10.5 Hz, 1H), 7.82 (d, J ¼ 11.7 Hz,
2H). m/z (EIeMS): 468 [M]þ.
Fig. 8. The binding pattern comparison of GA with IKK
b. (A) GA with the homology
model of IKK . (B) GA with the crystal structure of IKK . Residues in the binding pocket
b
b
were shown as gray stick. H-bonds were shown as red dot line. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)

120
H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
4.2.10. Flavone compounds (13a) and (13b)
was added dropwise at 0 ꢀC and the resulted mixture was stirred
at room temperature for 12 h. After adding 50 mL dichloro-
methane, the mixture was neutralized with 2N HCl. The organic
layer was separated, dried over sodium sulfate, and evaporated.
The residue was purified through column chromatography (eluent,
PE/EtOAc ¼ 16:1) to afford 277.0 mg 17 as light yellow solid. Yield:
To a solution of 12a (200.0 mg, 0.5 mmol) or 12b (242.4 mg,
0.5 mmol) in ethanol (10.0 mL) and ethyl acetate (3.3 mL) was
added 10% Pd/BaSO4 (20.0 mg). The mixture was stirred under an
atmosphere of hydrogen for 2 h at room temperature and filtered
through a plug of silica gel, the filtration was concentrated and
purified through column chromatography (eluent, PE/EtOAc ¼ 8:1)
to afford 13a or 13b.
11%; m.p.: 172e174 ꢀC; 1H NMR (300 MHz, CDCl3):
d 1.72 (s, 3H),
1.15e1.30 (m, 7H), 1.86 (s, 3H), 3.59 (d, J ¼ 7.2 Hz, 2H), 5.30 (t,
J ¼ 7.2 Hz, 1H), 6.15 (s, 1H), 6.31 (s, 1H), 7.38 (dd, J ¼ 7.8 Hz,
J ¼ 1.5 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 1H), 7.70 (dd, J ¼ 7.8 Hz,
J ¼ 1.5 Hz, 1H), 8.26 (d, J ¼ 1.5 Hz, 1H), 12.91 (s, 1H); m/z (EIeMS):
296 [M]þ.
13a: 155.6 mg; Yield: 77%; light yellow oil. 1H NMR (300 MHz,
CDCl3):
d
1.54 (s, 6H), 1.58 (s, 6H), 4.88 (dd, J ¼ 10.8 Hz, J ¼ 1.8 Hz,
1H), 5.10e5.22 (m, 3H), 6.34 (m, 2H), 6.66 (s, 1H), 7.10 (d, J ¼ 9.0 Hz,
1H), 7.42 (m, 3H), 7.73 (dd, J ¼ 9.0 Hz, J ¼ 0.9 Hz, 1H), 7.86 (m, 2H);
m/z (EIeMS): 390 [M]þ.
13b: 198.5 mg; Yield: 81%; light yellow oil. 1H NMR (300 MHz,
4.2.14. Caged xanthone (18)
CDCl3):
d
1.53e1.56 (m, 18H), 4.99 (d, J ¼ 10.8 Hz, 1H), 5.12e5.29 (m,
The synthesis method of 18 was reported previously by our
5H), 6.11e6.31 (m, 3H), 6.67 (s, 1H), 7.10 (d, J ¼ 9.0 Hz, 2H), 7.15 (d,
J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 9.0 Hz, 1H), 7.85 (d, J ¼ 9.0 Hz, 2H); m/z
(EIeMS): 474 [M]þ.
group [24]. 18: yellow solid; m.p.: 124e126 ꢀC 1H NMR (300 MHz,
CDCl3)
d 0.84 (s, 3H), 1.15e1.30 (m, 7H), 1.66 (s, 3H), 2.27 (dd,
J ¼ 13.5 Hz, J ¼ 4.5 Hz,1H), 2.39 (d, J ¼ 9.6 Hz,1H), 2.55 (d, J ¼ 9.3 Hz,
2H), 3.41e3.47 (m, 1H), 4.35 (t, 1H), 6.97e7.02 (m, 2H), 7.36 (d,
J ¼ 6.9 Hz,1H), 7.45 (dd, J ¼ 8.4 Hz, J ¼ 7.2 Hz,1H), 7.87 (d, J ¼ 8.4 Hz,
1H); 13C NMR (CDCl3): 16.69, 25.14, 25.33, 29.12, 30.33, 30.93,
46.84, 48.83, 83.52, 84.62, 90.37, 118.10, 118.97, 119.14, 121.87,
126.99, 133.76, 134.78, 134.94, 136.21, 159.67, 176.52, 203.02. m/z
(ESIeMS): 363 [M ꢁ H]ꢁ; HRMS (ESIeTOF) calc. for C23H24O4
[M þ H]þ 365.1753, found 365.1766.
4.2.11. Caged flavones (14) and (15)
To a solution of 13a (100.0 mg, 0.3 mmol) or 13b (100.0 mg,
0.2 mmol) in DMF (3.0 mL) was heated at 120 ꢀC for 1 h. The yellow
reaction mixture was cooled to 25 ꢀC and the mixture was purified
by column chromatography (eluent, PE/EtOAc ¼ 4:1) to yield the
caged flavone 14 or 15.
14: 51.0 mg; Yield: 51%; white solid; m.p.: 146e148 ꢀC 1H NMR
(300 MHz, CDCl3):
d
1.32 (s, 6H), 1.38 (m, 1H), 1.42 (s, 3H), 1.71 (s,
4.2.15. Caged xanthone (19)
3H), 2.35 (dd, J ¼ 13.3, J ¼ 4.5,1H), 2.53 (d, J ¼ 9.5 Hz,1H), 2.57e2.66
(m, 2H), 3.48 (dd, J ¼ 7.0, J ¼ 4.5, 1H), 4.69 (m, 1H), 6.13 (s, 1H), 7.30
(d, J ¼ 7.0, 1H), 7.57e7.46 (m, 3H), 7.85e7.82 (m, 2H); 13C NMR
Compound 19 was synthesized in our laboratory [24]. 19: yellow
solid; m.p.: 130e132 ꢀC 1H NMR (300 MHz, CDCl3):
d 0.95 (s, 3H),
1.18e1.25 (m, 4H), 1.30 (s, 3H), 1.61 (s, 3H), 2.26 (dd, J ¼ 13.5 Hz,
J ¼ 4.5 Hz, 1H), 2.37 (d, J ¼ 9.6 Hz, 1H), 2.54 (d, J ¼ 7.8 Hz, 2H), 3.44
(dd, J ¼ 6.9 Hz, J ¼ 4.5 Hz, 1H), 4.34 (m, 1H), 6.43 (dd, J ¼ 8.1 Hz,
J ¼ 0.9 Hz, 1H), 6.45 (dd, J ¼ 8.1 Hz, J ¼ 0.9 Hz, 1H), 7.32 (t, J ¼ 8.1 Hz,
(75 MHz, CDCl3):
d 17.9, 25.2, 25.8, 29.3, 29.4, 30.5, 46.6, 49.7, 83.6,
84.3, 92.6, 100.9, 117.8, 126.6, 128.9, 131.0, 131.8, 132.8, 134.2, 134.6,
168.5, 177.2, 203.1; m/z (ESIeMS): 389 [M ꢁ H]ꢁ; HRMS (ESIeTOF)
calcd. for C25H27O4 [M þ H]þ 391.1909, found 391.1914.
1H), 7.41 (d, J ¼ 9.6 Hz, 1H), 12.00 (s, 1H). 13C NMR (CDCl3):
d 16.68,
15: 44.5 mg; Yield: 45%; light yellow solid; 152e154 ꢀC 1H NMR
24.95, 25.49, 29.05, 29.14, 30.27, 46.98, 48.82, 83.53, 84.46, 90.02,
106.12, 107.38, 109.37, 118.64, 133.88, 134.90, 135.30, 138.75, 159.59,
162.90, 181.33, 202.69. m/z (ESIeMS): 379 [M ꢁ H]ꢁ; HRMS
(ESIeTOF) calc. for C23H24O6 [M þ Na]þ 403.4337, found 403.4342.
(300 MHz, CDCl3): d 1.31 (s, 6H), 1.37 (s, 3H), 1.68e1.77 (m, 9H), 1.71
(s, 3H), 2.34 (dd, J ¼ 13.2, J ¼ 4.2, 1H), 2.54 (d, J ¼ 9.6, 1H), 2.58e2.67
(m, 2H), 3.44 (d, J ¼ 7.8, 2H), 3.47 (dd, J ¼ 7.0, J ¼ 4.2 Hz,1H), 4.70 (m,
1H), 5.33 (m, 1H), 6.06 (s, 1H), 6.37 (br-s, 1H),6.92 (d, J ¼ 7.8, 1H),
7.30 (s, 1H), 7.59e7.62 (m, 2H); 13C NMR (75 MHz, CDCl3):
d
8.85,
4.2.16. Caged chromanone (21)
21.24, 25.86, 29.07, 29.10, 30.12, 48.80, 48.95, 63.16, 83.47, 84.36,
91.05, 101.90, 106.87, 134.81, 135.76, 139.75, 150.23, 153.68, 162.07,
164.57, 170.08, 175.43, 197.08, 203.93; m/z (ESIeMS): 473 [M ꢁ H]ꢁ;
HRMS (ESIeTOF) calcd. for C25H27O4 [M þ H]þ 475.2484, found
475.2496.
Compound 21 was synthesized using method reported by our
laboratory [24]. 21: yellow solid; m.p.: 155e156 ꢀC 1H NMR
(CDCl3): 1.18 (s, 3H), 1.24e1.36 (m, 4H), 1.42 (s, 3H), 1.51 (s, 3H), 1.54
(s, 3H), 2.09 (dd, J ¼ 18 Hz, J ¼ 16 Hz, 1H), 2.23e2.41 (m, 3H),
2.52e2.66 (m, 2H), 3.23e3.30 (m,1H), 4.0e4.07 (m, 1H), 4.33 (m,
1H), 7.17 (d, J ¼ 7.2 Hz, 1H); 13C NMR (CDCl3): 17.55, 20.66, 25.05,
27.39, 28.38, 28.42, 29.76, 44.4, 44.77, 45.74, 66.08, 82.37, 83.88,
86.58, 118.99, 133.06, 134.48, 136.18, 191.96, 203.33. m/z (ESI): 353
[M þ Na]þ. Anal. Calcd for C20H26O4 (%): C,72.70; H, 7.93; Found: C,
72.87; H, 7.93.
4.2.12. 1,3-Bishydroxy-9H-xanthen-9-one (16)
To a round-bottomed flask containing phloroglucinol (10.0 g,
79.3 mmol), salicylic acid (10.9 g, 79.3 mmol) and ZnCl2 (70.0 g,
515.0 mmol) was added POCl3 (120.0 mL). The reaction mixture was
stirred for 3.5 h at 70 ꢀC under N2. It was then cooled to 25 ꢀC and
poured into a beaker of ice. The reaction mixture was filtered and
the filter cake was washed with saturated NaCl to get the crude
material which was purified by silica gel column chromatography
(eluent, PE/EtOAc ¼ 4:1) to afford 7.8 g 16 as yellow solid. Yield:
4.2.17. 7,9-Dihydroxy-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]xanthen-
6-one (22)
Compound 1 (0.13 g, 0.5 mmol) was added to diphenyl ether
(10 mL) and then dichlorodiphenylmethane (0.18 g, 0.75 mmol)
was added. The reaction mixture was heated to 175 ꢀC for 0.5 h
under N2. The reaction mixture cooled to room temperature was
poured into petroleum ether (100 mL). The precipitate was
collected by filtration and washed with petroleum ether, which was
purified by silica gel column chromatography (petroleum ether/EA
8:1). Compound 22 (0.18 g, 85%) was obtained : light yellow solid;
43%; m.p.>200 ꢀC; 1H NMR (300 MHz, CDCl3):
6.41 (d, J ¼ 1.8, 1H), 7.47 (t, J ¼ 7.5 Hz, 1H), 7.60 (d, J ¼ 8.4 Hz, 1H),
7.86 (t, J ¼ 7.5 Hz, 1H), 8.13 (d, J ¼ 8.4 Hz, 1H), 11.14 (br-s, 1H), 12.83
(s, 1H); m/z (EIeMS): 228 [M]þ.
d
6.22 (d, J ¼ 1.8, 1H),
4.2.13. 1,3-Dihydroxy-4-(3-methylbut-2-enyl)-9H-xanthen-9-one
(17)
m.p.: 212e214 ꢀC; 1H NMR (300 MHz, CD3COCD3):
d 6.26 (d,
To a solution of 16 (2.0 g, 8.8 mmol) in methanol (70.0 mL),
sodium methoxide (2.5 g, 43.9 mmol) was added slowly and then
stirred for 15 min. 1-bromo-3-methyl-2-butene (2.0 g, 13.2 mmol)
J ¼ 2.2 Hz, 1H), 6.46 (d, J ¼ 2.2 Hz, 1H), 7.13 (d, J ¼ 8.5 Hz, 1H), 7.48
(m, 6H), 7.66 (m, 4H), 7.82 (d, J ¼ 8.5 Hz, 1H), 9.83 (s, 1H), 12.99 (s,
1H); EIeMS (m/z) 424 (M)þ.

H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
121
4.2.18. 7-Hydroxy-9-(methoxymethoxy)-2,2-diphenyl-6H-[1,3]
dioxolo[4,5-c]xanthen-6-one (23)
pressure and the residue was purified by silica gel column chro-
matography (petroleum ether/EtOAc 8:1) to give the compound 27
(0.14 g, 65%) as a yellow solid: m.p: 169e171 ꢀC; 1H NMR (300 MHz,
At room temperature K2CO3 (1.1 g, 8 mmol) was added to the
solution of compound 22 (1.70 g, 4 mmol) in acetone (50 mL). After
stirred for 15 min, to this mixture was added MOMCl (0.46 mL,
6 mmol). The reaction mixture was stirred for 6 h and poured into
H2O (150 mL). The precipitate was collected by filtration and
washed with water, which was purified by silica gel column chro-
matography (petroleum ether/EtOAc 8:1) to give the compound 23
(1.68 g, 90%) as a white solid: m.p: 125e127 ꢀC; 1H NMR (300 MHz,
CDCl3):
d
1.41 (s, 3H), 1.31 (s, 3H), 1.35 (d, 1H, J ¼ 9.9 Hz), 1.53 (s, 3H),
1.69 (s, 3H), 2.30 (dd,1H, J ¼ 13.5 Hz, J ¼ 4.5 Hz), 2.38 (s, 3H), 2.42 (d,
1H, J ¼ 9.6 Hz), 2.60 (d, 2H, J ¼ 8.1 Hz), 3.49 (m, 4H), 4.47e4.48 (m,
1H), 5.22 (s, 2H), 6.41 (d, 1H,J ¼ 2.4 Hz), 6.58 (d, 1H, J ¼ 2.4 Hz), 7.30
(d, 1H, J ¼ 6.9 Hz); 13C NMR (CDCl3, 75 MHz)
d 17.0, 21.1, 25.4, 29.0,
30.4, 46.8, 48.7, 56.6, 83.3, 84.4, 90.5, 94.2, 102.1, 106.3, 107.3, 118.3,
133.1, 135.1, 135.1, 152.3, 162.1, 163.2, 169.4, 173.9, 203.1; m/z (EI):
505M þ Naþ, 483M þ Hþ; HRMS(ESIeTOF) found 505.1834 (calcd
for C27H30O8þNaþ 505.1838).
CDCl3):
d
3.50 (s, 3H), 5.24 (s, 2H), 6.45 (d, J ¼ 2.2 Hz, 1H), 6.65 (d,
J ¼ 2.2 Hz, 1H), 6.97 (d, J ¼ 8.5 Hz, 1H), 7.40 (m, 6H), 7.64 (m, 4H),
7.87 (d, J ¼ 8.5 Hz, 1H), 12.89 (s, 1H); EIeMS(m/z): 468 (M)þ.
4.2.23. Caged xanthone (20)
4.2.19. 9-(methoxymethoxy)-6-oxo-2,2-diphenyl-6H-[1,3]dioxolo
[4,5-c]xanthen-7-yl acetate (24)
To a solution of xanthone 27 (0.48 g, 1.0 mmol) in acetone
(3.0 mL) was added 3M HCl (1.0 mL). The reaction mixture was
stirred under 40 ꢀC for 6 h at room temperature. The reaction
mixture was concentrated under reduce pressure. The residue was
purified by silica gel column chromatography (petroleum ether/
EtOAc 6:1) to give the compound 20 (0.24 g, 60%): yellow solid;
Ac2O (1.7 g, 16.7 mmol) was added to a solution of compound 23
(6.0 g, 12.8 mmol) and DMAP (2.35 g, 0.193 mmol) in dichlor-
methane (100 mL). The reaction mixture was stirred for 0.5 h at
room temperature. Another dichlormethane (100 mL) was added to
dilute and the reaction mixture was washed with H2O (100 mL*3).
The organic layer was dried with Na2SO4 and concentrated under
reduce pressure. The residue was purified by silica gel column
chromatography (petroleum ether/EtOAc 4:1) to give the
compound 24 (5.9 g, 91%) as a white solid: m.p: 192e193 ꢀC; 1H
m.p: 168e170 ꢀC 1H NMR (300 MHz, CDCl3):
d 1.11 (s, 3H), 1.23 (s,
3H), 1.33 (s, 3H), 1.61 (s, 3H), 2.26 (dd, J ¼ 13.5 Hz, J ¼ 4.5 Hz, 1H),
2.37 (d, J ¼ 9.6 Hz, 1H), 2.54 (d, J ¼ 7.8 Hz, 2H), 3.44 (m, 2H), 4.37 (m,
1H), 5.96 (d, J ¼ 2.1 Hz, 1H), 5.98 (d, J ¼ 2.1 Hz, 1H), 7.35 (d,
J ¼ 6.9 Hz, 1H), 12.40 (s, 1H); 13C NMR (CDCl3):
d 16.80, 26.20, 26.60,
NMR (300 MHz, CDCl3):
d
2.46 (s,3H), 3.51 (s, 3H), 5.27 (s, 2H), 6.67
30.06, 30.14, 32.22, 48.68, 49.94, 84.63, 85.46, 91.13, 107.14, 108.34,
109.40, 120.64, 135.84, 136.95, 137.30, 140.75, 159.52, 165.61, 182.36,
206.60. m/z (ESIeMS): 395 [M ꢁ H]ꢁ; HRMS (ESIeTOF) calc. for
C23H24O6 [M þ Na]þ 419.1471, found 419.1478.
(d, J ¼ 2.1 Hz, 1H), 6.94 (d, J ¼ 8.4 Hz, 1H), 7.09(d, J ¼ 2.1 Hz, 1H), 7.26
(m, 6H), 7.41 (m, 4H), 7.87 (d, J ¼ 8.4 Hz, 1H); EIeMS(m/z): 510 (M)þ.
4.2.20. 5,6-Dihydroxy-3-(methoxymethoxy)-9-oxo-9H-xanthen-
1-yl acetate (25)
4.3. Assays for cell viability, apoptosis, and cell cycle distribution
To a solution of compound 24 (5.9 g, 11.6 mmol) in THF/MeOH
(40 mL/40 mL) was added 10% Pd/C (0.59 g). The reaction mixture
was stirred at 50 ꢀC under an atmosphere of hydrogen overnight.
The reaction mixture was filtered and concentrated under reduce
pressure. The residue was pulped with petroleum ether/EtOAc
(50 mL/10 mL) and filtered to give the compound 25 (3.6 g, 90%) as
Cells viabilities were measured by a colorimetric assay using
MTT as described previously [37]. Vitality Index (%) was calculated
using the following equation: Vitality Indexð%Þ ¼ ðAtreatment
=
AcontrolÞ ꢂ 100%. Apoptotic cells were assayed according to the
method described previously [36,38]. Apoptotic or necrotic cells
were identified by dual staining with recombinant fluorescein
isothiocyanate (FITC)-conjugated with Annexin V and propidium
iodide (PI) (Sigma, St. Louis, MO). The experiment was performed
according to manufacturer’s instructions (Becton Dickinson,
Franklin Lakes, NJ). For cell cycle distribution assay, cells were
trypsinized, washed with PBS, and fixed in 1.5 mL 95% ethanol at
white solid: m.p: 149e151 ꢀC; 1H NMR (300 MHz, CDCl3):
d 2.50 (s,
3H), 3.51 (s, 3H), 5.22 (s, 2H), 6.65 (d, J ¼ 2.4 Hz, 1H), 6.86
(d, J ¼ 9 Hz, 1H), 6.94 (d, J ¼ 2.4 Hz, 1H) 7.64 (d, J ¼ 9 Hz, 1H);
EIeMS(m/z): 346 (M)þ.
4.2.21. 3-(methoxymethoxy)-5,6-bis(2-methylbut-3-yn-2-yloxy)-
9-oxo-9H-xanthen-1-yl acetate (26)
4
ꢀC overnight followed by incubation with RNase and staining by
To a solution of xanthone 25 (0.55 g, 1.6 mmol) in dried acetone
(5 mL) was added KI (0.79 g, 4.8 mmol), K2CO3 (0.66 g, 4.8 mmol),
CuI (0.03 g, 0.16 mmol) and 2-chloro-2-methylbut-3-yne (1.6 g,
16 mmol). The reaction mixture was heated at reflux for 1.5 h under
nitrogen. It was then cooled to 25 ꢀC and filtered. The filtration was
concentrated and the residue was purified by silica gel column
chromatography (petroleum ether/EtOAc 8:1) to give the
compound 26 (0.56 g, 70%) as a light yellow solid: m.p: 133e136 ꢀC;
propidium iodide (PI). Data acquisition was performed with a Bec-
ton Dickinson FACS Calibur flow cytometer (Franklin Lakes, NJ).
4.4. NF-
kB translocation detection using high-content screening
The NF-
k
B translocation experiment was performed according
to Thermo’s instructions. Briefly, A549 cells were plated into 96-
well plates at 9000 cells per well and then serum-starved for
18 h. Different concentrations of GA was added and the plate was
1H NMR (300 MHz, CDCl3):
d 1.75 (s,6H), 1.80 (s,6H), 2.34(s,1H),
2.48(s, 3H), 2.64 (s, 1H), 3.51 (s, 3H), 5.26 (s, 2H), 6.69 (d, J ¼ 2.1 Hz,
1H), 7.03 (d, J ¼ 2.1 Hz, 1H), 7.57 (d, J ¼ 9 Hz, 1H), 7.93 (d, J ¼ 9 Hz,
1H); ESIeMS(m/z): 479M þ Hþ.
incubated at 37 ꢀC for 2 h. Mixed stimulators containing TNF
a
(50 ng/mL), IFN-
the plate was incubated at 37 ꢀC for 30 min. Culture medium was
aspirated and 100 l of pre-warmed fixation solution was added to
each well. After incubation at room temperature for 15 min, fixa-
tion solution was aspirated and the plate was washed twice. 100
g (100 U/mL) and IL-1b (100 U/mL) were added and
m
4.2.22. Caged xanthone (27)
To a solution of xanthone 26 (0.22 g, 0.46 mmol) in alcohol
(10 mL) was added 10% Pd/BaSO4 (22 mg). The reaction mixture
was degassed using hydrogen and stirred under an atmosphere of
hydrogen for 0.5 h at room temperature. The reaction mixture was
filtered and concentrated under reduce pressure. The residue need
not be purified and dissolved in DMF (5 mL). The solution was
heated at 120 ꢀC for 1 h under N2. DMF was removed under reduce
ml
of 1 ꢂ permeabilization buffer was then added and incubated for
15 min. Permeabilization buffer was aspirated and washed twice.
50
for 1 h, followed by two washes. 100
added, and after 15 min incubation, 50
solution was added and incubated for 45 min protected from light.
m
l of primary antibody solution was then added and incubated
l of blocking buffer was then
l of secondary antibody
m
m

122
H. Sun et al. / European Journal of Medicinal Chemistry 51 (2012) 110e123
After another two washes, the plate was sealed and evaluated on
spectrofluorometer and spectrophotometer (Thermo, Waltham,
KineticScan HCS Reader (Thermo Scientific, Pittsburgh, PA).
MA) at 562 nm. The cytoplasmic and nuclear fractions were
prepared as described above. Protein (20e100 mg) prepared from
4.5. Preparation of cytoplasmic and nuclear extracts
the indicated cells was loaded per lane and electrophoresed in 8%
or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), and then transferred onto polyvinylidene difluoride
(PVDF) Immobilon-P membrane (Bio-Rad, Hercules, CA) using
a transblot apparatus (Bio-Rad, Hercules, CA). The membranes were
blocked with 5% (w/v) non-fat milk at 30 min at 37 ꢀC, followed by
overnight incubation at 4 ꢀC with primary antibodies diluted
To determine the effect of GA on TNF-dependent p65 trans-
location, cytoplasmic or nuclear extracts were prepared as
described previously [40]. To reduce the constitutive signal for NF-
k
B activation, exponentially growing U251 cells were serum-
starved overnight and then preincubated with GA for 4 h before
being stimulated with TNF for 30 min. Cells were washed once
with ice-cold PBS and scraped into 1 ml of PBS. Cells were centri-
fuged at 14,000 g/min and resuspended in 200 l buffer A, which
was composed of (in mM) 10 HEPES, pH 7.9, 10 KCl, 0.1 EDTA, 0.2
EGTA, 1 DTT, and 0.5 PMSF for 15 min, before 13 l of 10% Nonidet P-
a
in PBST (1:2000 for b-actin; 1:500 for IkBa; 1:500 for p-IkBa
(ser 32/36), Santa Cruz, CA). After washing with PBST, the
membranes were incubated for 1 h with an IRDyeTM 800 conju-
gated secondary antibody diluted 1:20000 in PBST, and the labeled
proteins were detected with an Odyssey Scanning System (LI-COR.,
Lincoln, Nebraska, USA).
m
m
40 was added. Cells were then vortexed for 10 s before being
centrifuged again at 14,000 g/min for 30 s. The cytoplasmic
supernatant was removed, and the nuclear pellet was resuspended
4.9. IKK
b
assay
in 30 ml of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA,
1 mM EGTA, 25% glycerol, 1 mM DTT, and 1 mM PMSF) and shaken
for 15 min before being centrifuged for 5 min at 14,000 g/min. The
supernatant was retained, and the protein concentration was
determined by the BCA assay method with Varioskan spectrofluo-
rometer and spectrophotometer (Thermo, Waltham, MA) at
562 nm. Samples were stored at ꢁ80 ꢀC until use.
12.5
m
L of the 4 ꢂ reaction cocktail containing 50 ng IKK
b
(supplied from the HTScan IKK
b kinase assay kit, Cell Signaling
Technology, Beverly, MA) was incubated with 12.5
5 min at room temperature. 25
L 2 ꢂ ATP/substrate peptide cocktail
was added to the preincubated reaction. After incubation at room
temperature for 30 min, a 50
L stop solution (50 mM EDTA, pH ¼ 8)
was added to stop the reaction. Then 25 L of each reaction were
transferred to a 96-well streptavidin-coated plate (PerkinElmer Life
Sciences, Boston). After adding 75 L dH2O, the mixture was incu-
bated at room temperature for 60 min. After thoroughly washing the
wells 3 times with PBST, 100 L primary antibody [Phospho-I
(Ser32) (14D4) Rabbit mAb, 1:1000 in PBS/T with 1% bovine serum
albumin (BSA)] was added per well. After 120 min incubation at
room temperature, the wells were thoroughly washed 3 times with
PBST. 100 mL diluted HRP-labeled anti-rabbit IgG (1:1000 in PBS/T
with 1% BSA) was added per well. After 30 min incubation at room
mL test sample for
m
m
m
4.6. Flow cytometry
m
Annexin V-FITC apoptosis detection kit (BioVision, Mountain
View, CA) assay was performed according to the manufacturer’s
protocol. Briefly, cells were treated with compound 20 (4, 8 and
m
kBa
16 mM) and GA (8 mM) for 24 h and washed with PBS. Then the cells
were collected, resuspended in binding buffer (pH 7.5, 10 mM
HEPES, 2.5 mM CaCl2 and 140 mM NaCl), and incubated with
Annexin V-FITC and then PI for 10 min in the dark at room
temperature. Cells were then analyzed by flow cytometry (FACS
Calibur, Becton Dickinson, Franklin Lakes, NJ) and a computer
station running Cell-Quest software (Becton Dickinson, Franklin
Lakes, NJ).
temperature, the wells were washed five times with PBST. Then
100
incubated at room temperature for 15 min. After adding the stop
solution (100 L/well), the plate was incubated at room temperature
mL TMB substrate was added into each well, and the plate was
m
for 15 min and then read at 450 nm with Varioskan spectrofluo-
rometer and spectrophotometer (Thermo, Waltham, MA).
4.7. Immunofluorescence
Immunostaining was performed as described previously [39].
Briefly, U251 cells were pretreated with compound 20 (1, 2, 4, 8, 16
Acknowledgments
and 20 mM) for 4 h, stimulated with 50 ng/ml TNFa for 30 min and
This work was supported by the Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical University
(No. JKGQ201103), 2008ZX09401-001 and 2009ZX09501-003 of
National Major Science and Technology Project of China (Innova-
tion and Development of New Drugs), 90713038 of National Natural
Science Foundation of China (Key Program).
then incubated at 4 ꢀC overnight with p65 primary antibodies
(1:200; Santa Cruz Biotechnology, Santa Cruz, CA). After washing
with PBS, cells were incubated at 37 ꢀC for 1 h with FITCelabeled
secondary goat anti-mouse IgG antibodie. Cells were then stained
with fluorochrome dye DAPI (Santa Cruz Biotechnology, Santa Cruz,
CA) to visualize the cell nuclei, and observed under a fluorescence
microscopy (OlympusIX51, Japan) with a peak excitation wave
length of 340 nm.
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