Synthesis and evaluation of novel aza-caged Garcinia xanthones

Article abstract of DOI:10.1039/c2ob07088j

Inspired by the therapeutic potential of the simplified caged xanthones, we have developed a chemical strategy for synthesizing novel aza-caged Garcinia analogues through a regioselective Claisen/Diels-Alder cascade reaction. The origin of regioselectivity has been explained using the DFT method. We have further evaluated the cell proliferation and IKKβ inhibitory activities of these compounds and studied their binding mode with IKKβ by molecular docking. The results suggested that the aza-caged scaffold provides a suitable modification site and the introduction of a hydrophobic moiety leads to improvement in the cytotoxicity and IKKβ inhibitory activity. The aza-caged compound 6c exhibited an IC₅₀ value of 2.68, 2.10, 8.02 μM against the HepG2, A549 cells and IKKβ, respectively. Mechanism studies with 6c showed that the aza-caged compounds induce apoptosis and cell cycle S phase arrest in A549 cells.

Full text of DOI:10.1039/c2ob07088j

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PAPER
Synthesis and evaluation of novel aza-caged Garcinia xanthones†
Xiaojin Zhang,‡a Xiang Li,‡a Haopeng Sun,*b Zhengyu Jiang,b Lei Tao,c Yuan Gao,c Qinglong Guo*c and
Qidong You*a,c
Received 13th December 2011, Accepted 13th February 2012
DOI: 10.1039/c2ob07088j
Inspired by the therapeutic potential of the simplified caged xanthones, we have developed a chemical
strategy for synthesizing novel aza-caged Garcinia analogues through a regioselective Claisen/Diels–
Alder cascade reaction. The origin of regioselectivity has been explained using the DFT method. We have
further evaluated the cell proliferation and IKKβ inhibitory activities of these compounds and studied
their binding mode with IKKβ by molecular docking. The results suggested that the aza-caged scaffold
provides a suitable modification site and the introduction of a hydrophobic moiety leads to improvement
in the cytotoxicity and IKKβ inhibitory activity. The aza-caged compound 6c exhibited an IC50 value of
2.68, 2.10, 8.02 μM against the HepG2, A549 cells and IKKβ, respectively. Mechanism studies with 6c
showed that the aza-caged compounds induce apoptosis and cell cycle S phase arrest in A549 cells.
Introduction
Gamboge, the resin collected from the tropical trees of the genus
Garcinia, has been used as folk medicine for centuries in South-
east Asia.1 Efforts to identify the bioactive constituents of gam-
bogin have yielded a growing family of natural products defined
as “caged xanthones”, the structure features of which share an
intriguing 4-oxa-tricyclo[4.3.1.03,7]dec-8-en-2-one scaffold
merged into a common xanthone backbone.2 Among these
caged Garcinia xanthones, gambogic acid (GA, 1) (Fig. 1) is the
most prominent and representative example. Preclinical investi-
gation demonstrated that 1 possessed potent anti-tumor activity
both in vitro and in vivo and displayed an appropriate therapeutic
window for clinical applications as an anti-cancer agent.3 Practi-
cally, the gambogic acid injection has recently finished phase II
clinical trials in cancer patients in China.4
The significant anti-tumor activity of 1 has attracted increasing
worldwide research interest. Biological studies revealed that the
anti-tumor efficacy of 1 arose through multiple mechanisms,
Fig. 1 Chemical structures of selected caged Garcinia xanthones and
analogues.
including apoptosis induction,5 cell cycle regulation,6 anti-angio-
genesis,7 tumor cell adhesion inhibition etc.8 Wang et al.
reported a comprehensive proteome profiling which deduced
stathmin 1 (STMN1) as the target of 1.9 In addition, the surface
plasmon resonance studies showed that 1 bound to the N-term-
inal domain of heat shock protein 90 (Hsp90).10 Another study
showed that 1 inhibited the nuclear factor-κB (NF-κB) signaling
pathway11 and induced apoptosis through its interaction with the
transferrin receptor.12 Meanwhile, interaction studies using
biotin labeled GA suggested that 1 covalently bound to IκBα
kinase-β (IKKβ) protein through the oxa-caged moiety as a bio-
active Michael acceptor and thus suppressed NF-κB activation.13
aState Key Laboratory of Natural Medicines (China Pharmaceutical
University), Nanjing, 210009, China. E-mail: youqidong@gmail.com;
Tel: +86-25-83271351
bDepartment of Medicinal Chemistry, School of Pharmacy, China
Pharmaceutical University, Nanjing, 210009, China. E-mail:
sunhaopeng@163.com; Tel: +86-25-83271216
cJiangsu Key Laboratory of Carcinogenesis and Intervention, China
Pharmaceutical University, Nanjing, 210009, China. E-mail:
anticancerdrug@yahoo.cn; Tel: +86-25-83271440
†Electronic supplementary information (ESI) available: Experimental
procedures for 3–5. 1H and 13C NMR spectra for 11, 12, 13, 8, 7, 14,
17, 20a–e, 6a–e. 1H-1H COSY, HSQC, HMBC spectra for 6a, 17.
Energy data for 7, 15–17, 20a–23a, 6a, 25–27, 4–5. See DOI: 10.1039/
c2ob07088j
‡These two authors contributed equally to this work.
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Our recent studies further verified that IKKβ was the key regula-
tor of the NF-κB pathway affected by 1.
Results and discussion
Synthesis of aza-caged Garcinia xanthones and study on the
regioselective Claisen/Diels–Alder cascade reaction
Previous SAR studies indicated that the whole A ring moiety
and the carboxyl group of 1 could be removed thoroughly
without obvious loss of anti-tumor potency, while the intact
BCD ring containing the D-ring caged scaffold was the
minimum pharmacophoric motif that essential for its
activity.14,15 Recently, several simplified analogues were reported
to maintain the cytotoxicity exhibited by GA. For example, com-
pound 2 displayed comparable activity to GA (1) against
BGC-823 (gastric carcinoma) and HepG2 (hepatocellular carci-
noma) cells at the low micromolar levels.14 Cluvenone (3) was
also reported to possess similar activity to GA (1) against HL-60
(promyelocytic leukemia) and HL-60/ADR cells with an IC50
value of 1.5 and 1.4 μM respectively.15,16 Furthermore, com-
pound 4 with remarkable simple structure was still found to have
GI50 values of 1–2 μM in T47D (breast cancer), HCT116 (colon
cancer) and SNU398 (hepatocellular carcinoma) cells and to be
about 10–15 fold less active than GA (1).17
These caged analogues could be expediently obtained utilizing
a Claisen/Diels–Alder cascade reaction from their bis-allyloxy
precursors.18 However, in the pursuit of greatly simplified ana-
logues such as 3 and 4, the initial non-regioselective Claisen
rearrangement led to the formation of the regular oxa-caged scaf-
fold as well as a neo caged scaffold as in 5.14,16–17 The resulting
mixture of isomers shared similar properties and brought great
difficulties in separation. Moreover, current synthetic strategies
were limited in providing the oxa-caged scaffold,18,19 whereas
the oxa-caged moiety as in 4 lacked the suitable modification
sites to help to further understand the surrounding SAR and
improve the drug-like properties. Therefore, we sought to replace
the oxygen atom in the caged scaffold by a sp3 hybridized nitro-
gen atom to provide another modification site as shown in
Fig. 2. Herein, we present a regioselective chemical strategy that
allows to the synthesis of novel aza-caged Garcinia analogues
with the 4-aza-tricyclo[4.3.1.03,7]dec-8-en-2-one scaffold and
their biological evaluation.
Inspired by the synthesis of oxa-caged xanthones, our initial syn-
thetic approach toward aza-caged scaffold is shown in Fig. 2. We
expected aza-caged scaffold 6 to arise from regioselective
Claisen/Diels–Alder cascade reaction of precursor 7 followed by
a simple N-alkylation, the aza-caged ring system of which can
be traced to xanthone 8 that substituted with amino and hydroxyl
groups. With this in mind, our synthetic studies commenced
with a ZnCl2-mediated condensation of salicylic acid (9) with
resorcinol (10) in POCl3 to produce benzophenol adduct 11 in
72% yield. Initial efforts to convert 11 into xanthone 12 were
using a conventional thermal method by refluxing benzophenol
11 in water with NaOAc as a base catalyst. Nevertheless, only
8% conversion was observed upon refluxing for 24 h. On
account of the advantages of microwave irradiation (MWI) in
accelerating organic reactions,20 we tested this cyclization reac-
tion under MWI using the same reaction system and found that
12 could be successfully obtained in short time in quantitative
yield. The introduction of an amino group at C5 position was
accomplished by a two step procedure that involved nitration of
12 to form 13 followed by reduction of the nitro group to
provide 8 (57% combined yield). Subsequently, xanthone 8 was
allylated with allyl chloride in the presence of K2CO3 to afford
the desired di-allylated product 7 in 38% yield together with the
mono- and tri-allylated products 14 and 20b (93% yield in total)
(Scheme 1).
Scheme 1 Reagents and conditions: (a) ZnCl2, POCl3, 68 °C, 2 h,
71%; (b) 0.1 equiv. of NaOAc, H2O, MWI (120 °C, 150 W), 8 min,
99%; (c) HNO3, HOAc, 60 °C, 20 min, 58%; (d) H2, Pd/C, CH3OH–
THF 1 : 1, 25 °C, 3 h, 98%; (e) K2CO3, allyl chloride, acetone, 45 °C,
1 h, 7 (38%), 14 (45%) and 20b (10%).
Fig. 2 Design of aza-caged Garcinia analogues and the synthetic
approach.
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With the cascade precursor 7 in hand, the stage was set to
study the proposed Claisen/Diels–Alder reaction to construct the
designed aza-caged scaffold. Drawing on previous experience
with related substrates,21 we heated compound 7 in decalin at
180 °C with attempt to produce the regular aza-caged product 18
and its probable isomer 19 (or ketone 5 that likely formed upon
imine hydrolysis of 19) (Scheme 2). This reaction afforded an
unexpected product 17 in 58% yield with 33% recovery of 7.
None of the probable caged structures were isolated. By compar-
ing the structure of reactant 7 and product 17, it was supposed
that 17 was formed through a rearrangement involving the C16
allyl group migration from the C6 oxygen to the C5 nitrogen
of 7.
Why did the reaction proceed through C5 nitrogen allylation?
The answer may lie in the potential intramolecular hydrogen
bond between C6 oxygen and C5 amino group in precursor 7.
The hydrogen bond could accept electron density from the C6
oxygen atom. This contributed to a weakening of the ether bond
to the C16 allyl fragment. In addition, the C9 carbonyl group of
precursor 7 was para to the C6 allyloxy unit and this also facili-
tated the rupture of the C16 allyl group. We proposed that pre-
cursor 7 was prone to undergoing a hydrogen bond mediated
process from precursor 7 to product 17 by path C. However, in
the case of the non-observed Claisen rearrangement pathway
(path A and B), the states of the C5 or C6 atom changed from
sp2 hybridization in 7 into sp3 hybridization in 15 and 16. This
led to the destruction of the intramolecular hydrogen bond and
aromaticity of xanthone ring. As a result, both paths A and B
needed much more extra energy compared with path C. In
addition, molecular energies of the potential allyl rearrangement
products were further evaluated by the DFT (density functional
theory) method at the B3LYP/6-31G(d) level, computed using
Gaussian 03.22 The results were consistent with the above specu-
lation, and the calculated energies (relative to precursor 7) were
21.0, 33.8 and 0.5 kcal mol−1 for compounds 15, 16 and 17,
respectively.
Realizing the problem encountered, we decided to first alkyl-
ate the C5 amino group to eliminate the effect of hydrogen bond
and then perform the cascade reaction. As shown in Scheme 3,
compound 7 was first treated with methyl iodide in the presence
of K2CO3 to give precursor 20a in 99% yield. Encouragingly,
heating precursor 20a in decalin at 180 °C for 3 h successfully
furnished the desired aza-caged product 6a in 53% yield with
36% recovery of 20a. The structure of 6a was further confirmed
by two-dimensional correlation NMR spectroscopy. Since sub-
strate 20a without intramolecular hydrogen bonding could suc-
cessfully afford the caged scaffold, it was verified again that the
intramolecular hydrogen bond played an important role in hin-
dering the Claisen/Diels–Alder reaction in substrate 7.
six-membered ring transition state as shown in path
C
(Scheme 2). While the C16 allyl group migrated to the C5 nitro-
gen, the hydrogen on the C5 amino group shifted simultaneously
onto the C6 oxygen atom. Notably, the hydrogen bond and aro-
maticity of the xanthone ring were highly retained during the
Scheme 2 Reagents and conditions: (a) decalin, 180 °C, 3 h, 58%; (b)
RX, K2CO3, acetone. Energies given in square brackets are differences
of energies relative to precursor 7.
Scheme 3 Reagents and conditions: (a) K2CO3, acetone, 25 °C, over-
night, 98–99%; (b) decalin, 180 °C, 3 h, 46–65%. Energies given in
square brackets are the differences in energies relative to precursor 20a.
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In a similar manner, treating compound 7 with different alky-
lating agents afforded substrates 20b–e in 98–99% yields with
different substituents including allyl, benzyl and substituted
benzyl groups. These substrates were selected to further test the
cascade reaction and to tentatively detect the volume of the
binding pocket around the caged region. Substrates 20b–e were
then subjected to the same reaction conditions and gave rise to
the regular aza-caged compounds 6b–e as expected in 46–65%
yields with about 30% recovery of substrates 20b–e. Importantly,
we were not able to isolate any products containing the possible
neo caged scaffold as in compounds 23a–e or compound 5 that
could likely be formed upon imine hydrolysis of 23a–e. These
results suggested that the amino-xanthone precursors 20a–e with
a C–N bond at C5 position could engage in a regioselective
Claisen rearrangement pathway by the C5 allylation (A,
Scheme 3) to furnish the intermediates 21a–e. The intermediates
could then undergo intramolecular Diels–Alder reactions to
provide the designed regular caged analogues 6a–e selectively.
For comparison purpose, the precursor 25 with a C–O bond at
C5 position was synthesized by allylation of xanthone 24
(Scheme 4). Compound 24 was conveniently obtained according
to the method reported previously.15 When the dialloxy substi-
tuted precursor 25 was exposed to similar reaction conditions, it
produced the regular caged scaffold 4 together with the neo scaf-
fold isomer 5 in a ratio of about 1 : 1 with a combined yield of
61%. In this case, the precursor 25 ought to undergo an initial
non-regioselective Claisen rearrangement pathway through both
the C5 and C6 allylation (A, B, Scheme 4) and followed by the
Diels–Alder addition.
Why was the Claisen/Diels–Alder reaction regioselective in
precursors 20a–e but non-regioselective in the case of precursor
25? We supposed that this phenomenon was mainly due to the
energy differences between the Claisen products by path A and
path B. In the case of precursors 20a–e (Scheme 3), comparing
the two type of Claisen products 21a–e by path A and 22a–e by
path B, the former possessed a C–N single bond at C5 and a
CvO double bond at C6 while the latter had a CvN double
bond and a C–O single bond at corresponding positions. Accord-
ing to references,23 the general bond energies of C–N, CvO, C–
O and CvN were about 73, 191, 86 and 147 kcal mol−1
,
respectively. From this point of view, the energy of 21a–e was
greatly lower than 22a–e (about −31 kcal mol−1), which led to
the selectivity in the initial Claisen rearrangement. While in the
case of precursor 25 (Scheme 4), the similar bonds in the two
type of Claisen products 26 and 27 resulted in similar molecular
energies. Furthermore, to confirm the above assumptions, we
selected precursors 20a and 25 as representatives and calculated
the molecular energies of their rearrangement products by DFT
method. The results revealed that the energy differences between
21a/22a and 26/27 were −31.2 and −0.6 kcal mol−1. Moreover,
a large energy differences were also observed between the pair
of caged moieties (−9.1 kcal mol−1 for 6a/6a and −0.3 kcal
mol−1 for 4/5). These findings could explain the different selec-
tivity between 20a–e and 25 in the Claisen/Diels–Alder reaction.
Cell proliferation studies
The anti-proliferative activities of the synthesized caged Garci-
nia xanthone analogues were assessed by the tetrazolium-based
MTT assay using human hepatocellular carcinoma HepG2 cell
line, human lung carcinoma A549 cell line and human glioblas-
toma U251 cell line. Cancer cells were treated with increasing
concentrations of the respective compounds for 24 h, and then
the cell viability was evaluated through measurements of mito-
chondrial dehydrogenase activity. The anti-proliferative activi-
ties, expressed as IC50 values, were summarized in Table 1.
GA (1) was the most active compound among all the tested
compounds and exhibited an IC50 value of 0.89, 1.10 and
2.56 μM against HepG2, A549 and U251 cells, respectively. All
the analogues seemed to be less sensitive to U251 cells and
showed moderate or even inactivity against U251 cells. The
greatly simplified oxa-caged analogue 4 displayed relatively less
potency than cluvenone (3). Importantly, all the aza-caged
Table 1 Inhibition of cell proliferation by caged Garcinia xanthones
IC50/μM
Compound
HepG2
A549
U251
6a
6b
6c
6d
6e
4
5
10.9
6.51
2.68
4.29
3.89
16.7
24.2
6.77
0.89
6.84
4.34
2.10
3.61
5.02
18.9
12.7
1.93
1.10
16.7
24.9
16.4
29.1
26.2
>100
>100
18.7
2.56
Scheme 4 Reagents and conditions: (a) K2CO3, allyl chloride, DMF,
45 °C, 2 h, (b) decalin, 180 °C, 3 h, 4 (32%) and 5 (29%). Energies
given in square brackets are the differences in energies relative to precur-
sor 7.
Cluvenone (3)
GA (1)
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IKKβ inhibitory activity and binding pattern study by
molecular docking
compounds 6a–e were found to be more active than the parent
oxa-caged compound 4 against all the three cell lines. Further-
more, compounds 6c–e, containing a relatively large volume of
substituents, generally possessed a low micromolar activity
ranging between 2.10 to 5.02 μM against HepG2 and A549 cells
and were more potent than compounds 6a–b with smaller
groups. Especially, compound 6c, the most potent compound
among the aza-caged analogues, was almost 10 times more
active than the parent oxa-caged compound 4 and two fold more
potent than cluvenone (3) against HepG2 cells. These results
suggest that: (a) the nitrogen atom of the 4-aza-tricyclo
[4.3.1.03,7]dec-2-one caged scaffold is an accessible and suitable
modification site to derive novel potential anti-tumor agents; (b)
the introduction of hydrophobic moiety to the aza-caged region
leads to an obvious increase in cytotoxicity as compared to the
oxa-caged parent 4; (c) the substituent groups with relative large
volume are preferred during interacting with the putative
receptor.
Since GA was reported to inhibit the activity of IKKβ,13 a poten-
tial therapeutic target in cancer, we further tested the IKKβ
inhibitory activity of these analogues by using HTScan IKKβ
kinase assay kit. The IC50 values were summarized in Table 2.
Among all the analogues, compound 6c exhibited the most
potent IKKβ inhibitory effect with an IC50 value of 8.02 μM.
Although compound 6c was less active than GA (1), it was
about 3–5 times more effective than compound 4 and cluvenone
(3). The trend observed in the IKKβ inhibition revealed to be
consistent with the in vitro anti-tumor activity.
Recently, the crystal structure of IKKβ protein (PDB ID:
3DA8) has been reported by Xu’s groups.24 To further illustrate
the binding mode of IKKβ with the caged compounds, a mol-
ecular docking study was performed using the protein (3DA8)
and the program Gold 3.0. Goldscore Fitness was selected to
evaluate the binding affinity (Table 2). By analyzing the data, it
was evident that the values of the Goldscore Fitness were in cor-
relation with the experimental IKKβ inhibitory activities, indicat-
ing that the docking model was reliable for the binding study.
GA (1), the oxa-caged 4 and the aza-caged 6c were taken as
examples to depict the binding mode (Fig. 3). All the three com-
pounds were located in the ATP binding pocket of IKKβ and
possessed hydrogen bond with the hinge region of the enzyme,
which was considered to be a common binding pattern of IKKβ
inhibitors.25 Specifically, compound 4 and 6c formed a hydrogen
bond with Cys99 and GA (1) formed a hydrogen bond with
Gly102. There are mainly three important hydrophobic pockets
(Fig. 3) in the binding site of IKKβ. P1 is formed by Leu21,
Met96, Tyr98, Cys99, Val74, P2 is formed by Val152, Ile165
and Asn150, P3 is formed by Ile165, Asn150 and Gly22. As
Table 2 The inhibition and binding information of GA and its
analogues with IKKβ protein
Compound
IKKβ IC50/μM
Gold score
6a
6b
6c
6d
6e
4
5
26.30
19.80
8.02
10.12
9.55
42.12
44.78
24.16
3.07
47.55
49.78
52.24
52.77
52.36
47.23
46.02
47.83
61.04
Cluvenone (3)
GA (1)
Fig. 3 (A) Molecular docking simulations for GA (1) (yellow), 4 (blue) and 6c (red). Residues in the binding pocket were shown as gray sticks.
Hydrogen bonds were shown as green dotted lines; (B) solvent accessible surfaces of the binding pocket (solid, yellow), compound 4 (wire mesh,
blue) and 6c (wire mesh, red).
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Fig. 4 Cell morphological assessment of 6c on A549 cells. (A) Light microscopy detection (×200): (a) control group; (b) 2 μM of 6c; (c) 4 μM of
6c; (d) 8 μM of 6c. (B) Fluorescence microscopy detection (×400): (a) control group; (b) 2 μM of 6c; (c) 4 μM of 6c; (d) 8 μM of 6c. The white
arrows point the observed apoptotic bodies.
for 24 h resulted in phenotypic changes of A549 cells, such as
distortion, membrane blebbing and shrinkage, and a large pro-
portion of cells became round in shape and underwent necrosis
at high concentrations, while the untreated cells in the control
group displayed a normal, healthy shape as shown by their clear
cytoskeletons (Fig. 4). Significant morphological changes of
early apoptosis were observed under the fluorescence microscope
(×400) by DAPI staining (Fig. 5). Untreated A549 cells were
stained uniformly and emitted blue fluorescence which indicated
that the chromatin was equably distributed in the nucleolus.
After treatment with compound 6c, the bright nuclear conden-
sation and the apoptotic bodies appeared as the key markers of
cellular apoptosis. In detail, at 2 μM concentration, cell mem-
brane still displayed integrity, and the nuclei exhibited bright
condensed chromatin, at 4 μM and 8 μM concentration, typical
apoptotic bodies and shrinkage were observed.
Subsequently, the quantitative apoptosis-inducing activities of
compound 6c were assessed by flow cytometry using fluorescein
isothiocyanate annexin V/propidium iodide (PI) double staining
assay. The percentage of apoptotic cells (annexin V+/PI− stain-
ing) in the control group was 5.9% (Fig. 5A). After treatment
with 2 μM, 4 μM and 8 μM of compound 6c for 24 h, the per-
centages of apoptotic cells (lower right section of fluorocyto-
gram) increased to 9.1% (Fig. 5B), 18.3% (Fig. 5C) and 22.2%
(Fig. 5D), respectively. All the evidence revealed compound 6c
as a potent apoptosis inducer in the human lung cancer cell line
in a dose-dependent manner, the anti-proliferative activity of
which was associated with apoptosis rather than with a toxic
effect.
Furthermore, the contribution of compound 6c to cell cycle
progression was evaluated. Synchronized A549 cells were
treated with 2 μM, 4 μM and 8 μM of compound 6c for 24 h and
then subjected to flow cytometric analysis after DNA staining
with PI. The results of cell cycle distribution were illustrated in
Fig. 6. It was demonstrated that the exposure of A549 cells to
gradient concentrations of compound 6c led to a significant
decrease of cell population in S phase accompanied by a slight
increase in G1 phase in comparison to the control group. These
results suggested that the anti-proliferative activity and apopto-
sis-inducing effect of compound 6c were correlated with a sub-
stantial cell cycle arrest at S phase in A549 cells.
Fig. 5 Fluorescence-activated cell sorter analysis for 6c on A549 cells
by annexin-V/PI staining. Upper left: dead cells; upper right: late apop-
totic cells; lower left: fully viable cells; lower right: early apoptotic cells.
(A) Control group; (B) 2 μM of 6c; (C) 4 μM of 6c; (D) 8 μM of 6c.
depicted in Fig. 3A, GA occupied all three hydrophobic pockets
and especially fitted very well with the entail P1 pocket. Impor-
tantly, compound 6c also possessed the three pockets but only
inserted into the deep part of P1. However, compound 4
obviously missed the P3 pocket (Fig. 3B). These results
explained the weak binding affinity for IKKβ of compound 4
and the significant increase of IKKβ activity and cytotoxicity of
compound 6c compared to 4. The binding mode also revealed
that there is an appropriate chemical modification space around
the caged region as shown in P3 binding pocket.
Apoptosis and cell cycle distribution studies
To further study the mechanism of the aza-caged analogues, we
selected the most active compound 6c to study its effects on
apoptosis and cell cycle distribution. Firstly, compound 6c was
evaluated for its influence on cell skeleton by a morphological
observation study. The inverted light microscope (×200) demon-
strated that incubation of 2 μM, 4 μM and 8 μM of compound 6c
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Fig. 6 Effect of 11a on cell cycle progression on A549 cells. (A) Control group; (B) 2 μM of 6c; (C) 4 μM of 6c; (D) 8 μM of 6c; (E) quantitative
cell cycle distribution of 6c.
Rotavapor) below 45 °C under reduced pressure. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
0.25 mm silica gel plates (GF254) and visualized under UV
light. Silica gel (60 Å, 300–400 mesh) was used for flash
column chromatography. Melting points were determined with a
Melt-Temp II apparatus and were reported without any correc-
tion. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR
spectrometer using KBr film. The 1H NMR and 13C NMR
spectra were collected on Bruker AV-300 and/or Bruker AV-500
instruments using deuterated solvents with tetramethylsilane
(TMS) as internal standard. The multiplicity was defined by a s
(singlet), d (doublet), t (triplet) or m (multiplet). EI-MS was
recorded on Shimadzu GCMS-2010 apparatus. High resolution
mass spectra (HRMS) were recorded on a Water Q-Tof micro.
GA was isolated and purified according to the previously
reported methods.26 The cluvenone was synthesized according to
the previously reported methods. 15 The purity of GA used in all
experiments was 95% or higher. It was dissolved in PBS con-
taining 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-diphenyltetrazoliumbromide (MTT) was purchased from
Sigma (St Louis, MO). Annexin V-FITC Apoptosis Detection kit
was purchased from BioVision (Mountain View, CA). HTScan
IKKβ kinase assay kit was purchased from Cell Signaling Tech-
nology (Beverly, MA). Human IκBα and p-IκBα (Ser 32/36)
antibodies were bought from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). All drugs were diluted in the corresponding
culture medium to desired concentrations before use.
Conclusions
We present herein a regioselective chemical strategy that allows
the synthesis of novel aza-caged Garcinia analogues with the 4-
aza-tricyclo[4.3.1.03,7] dec-2-one scaffold and their biological
evaluation. Actually, this aza-caged scaffold provides a suitable
site for chemical modification that will help further understand-
ing of the SAR around the caged region and improving the
activity and drug-like properties. In the study of Clasien/Diels–
Alder reaction, we observed that precursor 7 containing an intra-
molecular hydrogen bond produces an unexpected allyl
migration product. We have also discovered that precursors 20a–
e with a C–N bond at the C5 position perform a regioselective
Claisen/Diels–Alder cascade reaction while corresponding pre-
cursor 25 with a C–O bond is not regioselective. These phenom-
ena are explained according to the DFT energy calculation. We
have also described the preliminary SAR by their anti-prolifera-
tive and IKKβ inhibitory activity and further illustrated the
binding mode of IKKβ with the analogues by molecular docking
simulation. The results suggest that introduction of hydrophobic
moiety to the caged region improves the cytotoxicity and IKKβ
inhibition. The aza-caged compound 6c has been shown to
induce apoptosis and cell cycle S phase arrest in A549 cells.
These findings enhance our understanding of the SAR and help
the discovery of the novel aza-caged Garinia xanthones as
potential anti-tumor agents.
Experimental
Human hepatocellular carcinoma HepG2 cell line, human
lung carcinoma A549 cell line and human glioblastoma U251
cell line were purchased from Cell Bank of Shanghai Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biologi-
cal Sciences, Chinese Academy of Sciences. HepG2, A549 and
U251 cells were cultured in DMEM which were supplemented
with 10% (v/v) fetal calf serum, 100 U mL−1 penicillin and
100 U mL−1 streptomycin in a 5% CO2 atmosphere.
General notes
All reagents were purchased from commercial sources and were
used without further purification unless otherwise noted. Micro-
wave irradiation was carried out with a single mode cavity Dis-
cover microwave synthesizer (CEM Corp., NC). Organic
solutions were concentrated by rotary evaporator (Büchi
3294 | Org. Biomol. Chem., 2012, 10, 3288–3299
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1H); 13C NMR (75 MHz, DMSO-d6): δ 173.76, 155.54, 154.82,
148.36, 135.42, 129.27, 128.25, 125.93, 125.13, 121.01, 117.91,
114.04, 113.61; IR (KBr, cm−1): 3085, 1614, 1534, 1449, 1322,
765; EI-MS (m/z): 257 (M+) (100); HRMS (ESI) calc. for
C13H7NO5 (M − H)− 256.0246, found 211.0250.
(2,4-Dihydroxyphenyl)(2-hydroxyphenyl)methanone (11)
POCl3 (60 mL) followed by anhydrous ZnCl2 (21.76 g,
160 mmol) was added to a round-bottomed flask containing sali-
cylic acid (9) (5.52 g, 40 mmol) and resorcinol (10) (5.28 g,
48 mmol). The reaction vessel was then equipped with a reflux
condenser and stirred under nitrogen at 68 °C for 2 h. The red
colored reaction mixture was then cooled to 25 °C and poured
into a beaker of about 1000 g of ice. The precipitate was filtered,
washed with ice brine (200 mL × 3) and dried. The crude
material was purified by flash column chromatography (30%
EtOAc–PE) to yield benzophenone 11 (6.62 g, 72%) as a yellow
solid. mp: 131–132 °C; Rf = 0.58 (20% EtOAc–hexane); 1H
NMR (300 MHz, DMSO-d6): δ 12.45 (s, 1H), 10.72 (s, 1H),
10.09 (s, 1H), 7.40–7.35 (m, 1H), 7.27–7.21 (m, 2H), 7.01–6.90
(m, 2H), 6.46–6.28 (m, 2H); 13C NMR (75 MHz, DMSO-d6): δ
199.95, 164.92, 164.23, 154.61, 135.64, 131.71, 128.86, 125.67,
118.85, 116.21, 113.33, 108.16, 102.27; IR (KBr, cm−1): 3384,
3196, 3090, 1629, 1580, 1486, 1281, 1234, 1159, 755; EI-MS
(m/z): 230 (M+) (24); HRMS (ESI) calc. for C13H10O4 (M −
H)− 229.0501, found 229.0504.
4-Amino-3-hydroxy-9H-xanthen-9-one (8)
Pd/C (250 mg) was added to a solution of compound 13 (2.57 g,
10 mmol) in THF (25 mL) and methanol (25 mL). The reaction
mixture was degassed using nitrogen and stirred under an atmos-
phere of hydrogen for 3 h at 25 °C, and then filtered through a
plug of silica gel. The filtration was concentrated and dried to
yield compound 8 (2.23 g, 98%) as a yellow solid. mp:
241–243 °C; Rf = 0.33 (50% EtOAc–hexane); 1H NMR
(300 MHz, DMSO-d6): δ 10.56 (br, 1H), 8.20 (d, J = 6.9 Hz,
1H), 7.98–7.81 (m, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.48–7.41 (m,
2H), 6.94 (d, J = 8.1 Hz, 1H), 5.06 (br, 2H); 13C NMR
(75 MHz, DMSO-d6): δ 175.53, 155.52, 148.98, 144.33, 134.50,
125.84, 124.12, 123.69, 120.78, 117.96, 114.53, 113.18, 112.15;
IR (KBr, cm−1): 3404, 3316, 3198, 1591, 1563, 1453, 1345,
1054; EI-MS (m/z): 227 (M+) (100); HRMS (ESI) calc. for
C13H9NO3 (M + H)+ 228.0661, found 228.0665.
3-Hydroxy-9H-xanthen-9-one (12)
A sealed glass tube containing a suspension of benzophenone 11
(690 mg, 3 mmol) and sodium acetate (24.6 mg, 0.3 mmol) in
water (5 mL) was introduced into the cavity of CEM microwave
reactor and irradiated at 150 W, 120 °C for 8 min under magnetic
stirring. After cooling to 25 °C by an air-flow, the tube was
removed from the rotor. The reaction mixture was diluted with
ice water (5 mL). The precipitate was filtered, washed with ice
water (5 mL × 3) and dried to yield xanthone 12 (630 mg, 99%)
as a white solid. mp: 254–255 °C; Rf = 0.32 (20% EtOAc–
hexane); 1H NMR (300 MHz, DMSO-d6): δ 11.02 (br, 1H), 8.15
(d, J = 7.8 Hz, 1H), 8.05 (d, J = 8.7 Hz, 1H), 7.82 (t, J = 7.8 Hz,
1H), 7.60 (d, J = 8.4 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H),
6.94–6.89 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 174.73,
164.00, 157.54, 155.54, 134.74, 129.97, 125.82, 124.07, 121.20,
117.83, 114.11, 113.99, 102.12; IR (KBr, cm−1): 3137, 1614,
1561, 1455, 1310, 844, 751; EI-MS (m/z): 212 (M+) (100);
HRMS (ESI) calc. for C13H8O3 (M − H)− 211.0395, found
211.0400.
4-(Allylamino)-3-(allyloxy)-9H-xanthen-9-one (7), 3-(allyloxy)-4-
amino-9H-xanthen-9-one (14), and 3-(allyloxy)-4-(diallylamino)-
9H-xanthen-9-one (20b)
Potassium carbonate (1.518 g, 11 mmol) was added to a solution
of compound 8 (1.135 g, 5 mmol) in dried acetone (20 mL). The
reaction vessel was then equipped with a reflux condenser, and
the mixture was stirred under nitrogen at 45 °C for 20 min. A
solution of allyl chloride (760 mg, 10 mmol) in dried acetone
(10 mL) was then added to the stirring reaction mixture dropwise
over 1 h and the mixture was further stirred at 45 °C for another
1 h. The reaction mixture was allowed to cool to 25 °C and then
partitioned between EtOAc (30 mL) and water (70 mL). The
water layer was back-extracted with EtOAc (30 mL × 2) and the
combined organic layers were dried over MgSO4, filtered and
concentrated. The residue was purified by flash column chrom-
atography (10–20% EtOAc–PE) to yield compound 7 (584 mg,
38%) as a yellow solid, compound 14 (601 mg, 45%) as a
yellow solid, and compound 20b (174 mg, 10%) as a yellow
solid, respectively. Compound 7: mp: 114–116 °C; Rf = 0.48
3-Hydroxy-4-nitro-9H-xanthen-9-one (13)
1
50% nitric acid (5.1 mL, 40 mmol) was added to a suspension
of xanthone 12 (4.24 g, 20 mmol) in acetic acid (100 mL) at
25 °C. The reaction mixture was stirred under nitrogen at 60 °C
for 30 min and the reaction suspension was turned into a red sol-
ution. The solution was stirred at 60 °C for next 20 min and a
yellow precipitate was formed. The reaction mixture was then
cooled to 25 °C and poured into ice water (500 mL). The pre-
cipitate was filtered, washed with ice water (100 mL × 3) and
dried. The crude material was purified by flash column chrom-
atography (60% CH2Cl2–PE) to yield compound 13 (2.98 g,
58%) as a pale-yellow solid. mp: 220–222 °C; Rf = 0.60 (50%
(20% EtOAc–hexane); H NMR (300 MHz, CDCl3): δ 8.32 (d,
J = 7.8 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.70 (t, J = 7.5 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 6.94 (d,
J = 9.0 Hz, 1H), 6.15–5.95 (m, 2H), 5.45 (d, J = 17.1 Hz, 1H),
5.35 (d, J = 10.5 Hz, 1H), 5.30 (s, 1H), 5.24 (d, J = 17.1 Hz,
1H), 5.10 (d, J = 10.2 Hz, 1H), 4.72 (d, J = 5.1 Hz, 2H), 4.13
(d, J = 5.7 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ 176.93,
155.99, 153.03, 147.88, 136.58, 134.33, 132.51, 126.73, 125.91,
123.76, 121.48, 118.39, 118.28, 117.80, 116.78, 116.09, 108.99,
69.89, 49.83; IR (KBr, cm−1): 3410, 3080, 2855, 1656, 1602,
1467, 1440, 1428, 1347, 1227, 1090, 758; EI-MS (m/z): 307
(M+) (100); HRMS (ESI) calc. for C19H17NO3 (M + H)+
308.1287, found 308.1292. Compound 14: mp: 121–122 °C; Rf
1
EtOAc–hexane); H NMR (300 MHz, DMSO-d6): δ 12.63 (br,
1H), 8.17 (d, J = 9.0 Hz, 1H), 8.14–8.11 (m, 1H), 7.83–7.79 (m,
1H), 7.59–7.55 (m, 1H), 7.49–7.44 (m, 1H), 7.12 (d, J = 9.0 Hz,
1
= 0.38 (20% EtOAc–hexane); H NMR (300 MHz, CDCl3): δ
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8.34 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.68 (d, J =
7.8 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H),
6.93 (d, J = 8.1 Hz, 1H), 6.15–6.07 (m, 1H), 5.46 (d, J = 17.4
Hz, 1H), 5.35 (d, J = 10.5 Hz, 1H), 4.73 (d, J = 1.8 Hz, 2H),
4.20 (br, 2H); 13C NMR (75 MHz, CDCl3): δ 176.42, 155.54,
149.26, 144.35, 133.75, 132.14, 126.34, 123.92, 123.21, 121.12,
117.86, 117.14, 115.86, 114.98, 108.05, 69.19; IR (KBr, cm−1):
3432, 3339, 3086, 3020, 2926, 1665, 1605, 1486, 1450, 1345,
1295, 906, 755; EI-MS (m/z): 267 (M+) (23); HRMS (ESI) calc.
for C16H13NO3 (M + H)+ 268.0974, found 268.00978. Com-
pound 20b: mp: 105–107 °C; Rf = 0.63 (20% EtOAc–hexane);
1H NMR (300 MHz, CDCl3): δ 8.31 (d, J = 7.8 Hz, 1H), 8.10
(dd, J = 9.0, 0.9 Hz, 1H), 7.72 (t, J = 8.4 Hz, 1H), 7.54 (d, J =
8.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H),
6.10–6.05 (m, 1H), 5.94–5.83 (m, 2H), 5.48 (d, J = 17.1 Hz,
1H), 5.33 (d, J = 10.5 Hz, 1H), 5.17 (d, J = 17.1 Hz, 2H), 4.98
(d, J = 9.9 Hz, 2H), 4.69 (d, J = 0.9 Hz, 2H), 3.87 (s, 2H), 3.85
(s, 2H); 13C NMR (75 MHz, CDCl3): δ 176.91, 161.24, 156.21,
154.92, 136.38 (2C), 134.29, 132.63, 126.56, 126.51, 123.70,
121.54, 117.98, 117.86, 116.64, 116.25, 109.48, 69.47, 55.92;
IR (KBr, cm−1): 3061, 2908, 2867, 1658, 1613, 1598, 1467,
1432, 1276, 1107, 1086, 917, 764, 751; EI-MS (m/z): 347 (M+)
(36); HRMS (ESI) calc. for C22H21NO3 (M + H)+ 348.1600,
found 348.1606.
J = 7.8, 1.5 Hz, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.71 (dt, J = 8.4,
1.5 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H),
6.95 (d, J = 9.0 Hz, 1H), 6.16–6.02 (m, 1H), 5.99–5.89 (m, 1H),
5.47 (dd, J = 17.4, 1.5 Hz, 1H), 5.34 (dd, J = 10.5, 1.2 Hz, 1H),
5.19 (d, J = 17.1 Hz, 1H), 5.06 (d, J = 9.9 Hz, 1H), 4.71 (d, J =
5.1 Hz, 2H), 3.83 (d, J = 6.3 Hz, 2H), 2.09 (s, 3H); 13C NMR
(75 MHz, CDCl3): δ 176.97, 160.71, 156.23, 154.51, 136.34,
134.28, 132.68, 128.29, 126.58, 123.72, 123.31, 121.52, 118.05,
117.99, 116.81, 116.42, 109.82, 69.65, 58.63, 40.76; IR (KBr,
cm−1): 3097, 2926, 2861, 1661, 1602, 1467, 1441, 1332, 1287,
1227, 1082, 768, 748; EI-MS (m/z): 321 (M+) (13); HRMS
(ESI) calc. for C20H19NO3 (M + H)+ 322.1443, found 322.1437.
4-(Allyl(benzyl)amino)-3-(allyloxy)-9H-xanthen-9-one (20c)
Compound 20c (395 mg, 99%) was prepared from compound 7
and benzyl bromide as a yellow solid. mp: 78–80 °C; Rf = 0.63
(20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ 8.31 (dd,
J = 7.8, 1.5 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.70 (dt, J = 7.8,
1.5 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 7.2 Hz, 2H),
7.34 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.2 Hz, 2H), 7.18–7.13 (m,
1H), 6.90 (d, J = 9.0 Hz, 1H), 6.13–6.02 (m, 1H), 6.96–5.85 (m,
1H), 5.48 (dd, J = 17.4, 1.5 Hz, 1H), 5.34 (dd, J = 10.8, 1.5 Hz,
1H), 5.13 (dd, J = 17.1, 1.5 Hz, 1H), 4.98 (d, J = 10.2 Hz, 1H),
4.66 (d, J = 5.1 Hz, 2H), 4.40 (s, 2H), 3.83 (d, J = 6.3 Hz, 2H);
13C NMR (75 MHz, CDCl3): δ 177.00, 161.46, 156.23, 155.00,
139.90, 136.23, 134.33, 132.63, 130.91, 128.85, 128.43, 127.97,
126.77, 126.60, 123.93, 123.72, 121.53, 117.94, 116.63, 116.43,
109.39, 69.49, 57.22, 55.71; IR (KBr, cm−1): 3068, 3032, 2926,
2849, 1658, 1614, 1598, 1466, 1432, 1277, 1219, 1104, 918,
752; EI-MS (m/z): 397 (M+) (7); HRMS (ESI) calc. for
C26H23NO3 (M + H)+ 398.1756, found 398.1764.
4-(Diallylamino)-3-hydroxy-9H-xanthen-9-one (17)
A solution of 7 (80 mg, 0.3 mmol) in decalin was stirred under
nitrogen at 180 °C for 3 h. The yellow reaction mixture was then
cooled to 25 °C and the residue was purified by flash column
chromatography (5–10% EtOAc–PE) to yield compound 17
(46.5 mg, 58%) as a yellow solid. mp: 109–111 °C; Rf = 0.48
1
(20% EtOAc–hexane); H NMR (300 MHz, CDCl3): δ 8.34 (d,
J = 7.8 Hz, 1H), 8.22 (d, J = 5.1 Hz, 1H), 7.32 (t, J = 7.5 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 6.99 (d,
J = 8.7 Hz, 1H), 5.88–5.76 (m, 2H), 5.15 (s, 1H), 5.10–5.04 (m,
4H), 3.90–3.69 (m, 4H); 13C NMR (75 MHz, CDCl3): δ 175.83,
160.08, 155.04, 155.01, 133.84, 133.77, 126.36, 126.04, 123.70,
121.33, 121.15, 118.30, 117.05, 115.09, 110.85, 56.72; IR (KBr,
cm−1): 3286, 3067, 2926, 1649, 1601, 1464, 1440, 1325, 1225,
754; EI-MS (m/z): 307 (M+) (59); HRMS (ESI) calc. for
C19H17NO3 (M + H)+ 308.1287, found 308.1292.
4-(Allyl(4-fluorobenzyl)amino)-3-(allyloxy)-9H-xanthen-9-one
(20d)
Compound 20d (408 mg, 98%) was prepared from compound 7
and 4-fluorobenzyl bromide as a yellow solid. mp: 84–86 °C; Rf
1
= 0.64 (20% EtOAc–hexane); H NMR (300 MHz, CDCl3): δ
8.31 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.74–7.69
(m, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.40–7.34 (m, 3H), 6.94–6.88
(m, 3H), 6.14–6.03 (m, 1H), 5.95–5.86 (m, 1H), 5.48 (d, J =
17.1 Hz, 1H), 5.36 (d, J = 10.5 Hz, 1H), 5.12 (d, J = 16.8 Hz,
1H), 4.98 (d, J = 10.2 Hz, 1H), 4.68 (d, J = 5.1 Hz, 2H), 4.36 (s,
2H), 3.80 (d, J = 6.3 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ
176.88, 161.87 (1JCF = 240.2 Hz), 161.43, 156.18, 154.97,
136.11, 135.52, 134.37, 132.55, 129.87 (3JCF = 7.5 Hz), 126.66,
126.49, 124.11, 123.79, 121.55, 118.01, 117.84, 116.64, 116.54,
114.72 (2JCF = 21.0 Hz), 109.32, 69.48, 56.40, 55.76; IR (KBr,
cm−1): 3085, 2932, 2844, 1657, 1599, 1508, 1466, 1433, 1223,
775, 760, 734; EI-MS (m/z): 415 (M+) (17); HRMS (ESI) calc.
for C26H22FNO3 (M + H)+ 416.1662, found 416.1656.
General procedure for the synthesis of compounds 20a–e
Potassium carbonate (276 g, 0.2 mmol) was added to a solution
of compound 7 (307 mg, 0.1) in dried acetone at 25 °C followed
by the corresponding halide (0.15 mmol). The reaction mixture
was stirred overnight at 25 °C and filtered. The filtration was
concentrated and the residue was purified by flash column
chromatography (10% EtOAc–PE) to yield compounds 20a–e,
respectively.
4-(Allyl(methyl)amino)-3-(allyloxy)-9H-xanthen-9-one (20a)
4-(Allyl(4-chlorobenzyl)amino)-3-(allyloxy)-9H-xanthen-9-one
(20e)
Compound 20a (318 mg, 99%) was prepared from compound 7
and methyl iodide as a yellow solid. mp: 110–112 °C; Rf = 0.55
(20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ 8.32 (dd,
Compound 20e (424 mg, 98%) was prepared from compound 7
and 4-chlorobenzyl bromide as a yellow solid. mp: 83–84 °C; Rf
3296 | Org. Biomol. Chem., 2012, 10, 3288–3299
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1
= 0.64 (20% EtOAc–hexane); H NMR (300 MHz, CDCl3): δ
HRMS (ESI) calc. for C22H21NO3 (M + H)+ 348.1600, found
348.1608.
8.32 (dd, J = 8.4, 1.5 Hz, 1H), 8.10 (d, J = 9.0 Hz, 1H), 7.72
(dt, J = 8.7, 1.5 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.40–7.36
(m, 3H), 7.20 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 9.0 Hz, 1H),
6.17–6.04 (m, 1H), 5.97–5.84 (m, 1H), 5.49 (dd, J = 17.1, 1.2
Hz, 1H), 5.37 (dd, J = 10.5, 0.9 Hz, 1H), 5.11 (d, J = 17.1 Hz,
1H), 4.98 (d, J = 9.9 Hz, 1H), 4.68 (d, J = 5.1 Hz, 2H), 4.37 (s,
2H), 3.80 (d, J = 6.0 Hz, 2H); 13C NMR (75 MHz, CDCl3): δ
176.87, 161.36, 156.17, 154.91, 138.51, 136.00, 134.41, 132.52,
132.40, 129.71, 128.13, 126.67, 126.46, 124.16, 123.83, 121.55,
118.07, 117.84, 116.66, 116.64, 109.33, 69.49, 56.48, 55.84; IR
(KBr, cm−1): 3068, 2926, 2855, 1658, 1598, 1466, 1432, 1277,
765, 752; EI-MS (m/z): 431 (M+) (9); HRMS (ESI) calc. for
C26H22ClNO3 (M + H)+ 432.1366, found 432.1371.
Aza-caged compound (6c)
Compound 6c (28.6 mg, 48%) was prepared from compound
20c (59.6 mg, 0.15 mmol) as a yellow solid. mp: 107–108 °C;
Rf = 0.55 (20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ
7.95 (dd, J = 7.8, 1.5 Hz, 1H), 7.55 (dt, J = 6.9, 1.5 Hz, 1H),
7.37–7.33 (m, 5H), 7.29 (m, 1H), 7.12–7.04 (m, 2H), 5.36–5.29
(m, 1H), 4.67 (d, J = 10.2 Hz, 1H), 4.52 (d, J = 17.1 Hz, 1H),
4.15 (d, J = 14.4 Hz, 1H), 3.68 (dd, J = 9.3, 4.5 Hz, 1H), 3.58
(d, J = 14.4 Hz, 1H), 3.48–3.45 (m, 1H), 2.74 (dd, J = 13.5, 5.1
Hz, 1H), 2.58–2.49 (m, 2H), 2.38–2.32 (m, 1H), 1.76–1.73 (m,
2H); 13C NMR (75 MHz, CDCl3): δ 201.26, 177.28, 160.19,
139.80, 136.35, 136.09, 133.03, 132.68, 128.38, 127.99, 127.24,
126.94, 121.78, 119.54, 118.65, 118.14, 89.81, 70.38, 58.20,
53.20, 46.44, 39.23, 33.49, 30.50; IR (KBr, cm−1): 3086, 2924,
2853, 1772, 1645, 1611, 1460, 1275, 1260, 762, 752; EI-MS
(m/z): 397 (M+) (8); HRMS (ESI) calc. for C26H23NO3
(M + H)+ 398.1756, found 398.1765.
General procedure for the synthesis of compounds 6a–e
A solution of 20a–e (0.15 mmol) in decalin was stirred under
nitrogen at 180 °C for 3 h. The yellow reaction mixture was then
cooled to 25 °C and the residue was purified by flash column
chromatography (5–10% EtOAc–PE) to yield aza-caged com-
pounds 6a–e, respectively.
Aza-caged compound (6d)
Aza-caged compound (6a)
Compound 6d (28.7 mg, 46%) was prepared from compound
20d (62.4 mg, 0.15 mmol) as a yellow solid. mp: 111–112 °C;
Rf = 0.56 (20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ
7.95 (d, J = 7.5, 1H), 7.55 (t, J = 7.5, 1H), 7.33–7.31 (m, 3H),
7.11–7.00 (m, 4H), 5.38–5.29 (m, 1H), 4.68 (d, J = 9.9 Hz, 1H),
4.53 (d, J = 17.1 Hz, 1H), 4.10 (d, J = 14.4 Hz, 1H), 3.70–3.61
(m, 1H), 3.56 (d, J = 14.4 Hz, 1H), 3.47–3.45 (m, 1H),
2.76–2.70 (m, 1H), 2.56–2.53 (m, 2H), 2.37–2.35 (m, 1H),
1.74–1.72 (m, 2H); 13C NMR (75 MHz, CDCl3): δ 201.16,
177.19, 163.42 (1JCF = 243.8 Hz), 160.07, 136.35, 136.02,
134.81, 132.97, 132.50, 129.47 (3JCF = 8.9 Hz), 127.25, 121.83,
119.56, 118.74, 118.10, 115.18 (2JCF = 21.1 Hz), 89.79, 70.39,
58.16, 52.58, 46.41, 39.18, 33.46, 30.52; IR (KBr, cm−1): 3088,
2962, 2852, 1776, 1635, 1465, 1266, 1213, 764; EI-MS (m/z):
415 (M+) (14); HRMS (ESI) calc. for C26H22FNO3 (M + H)+
416.1662, found 416.1655.
Compound 6a (25.6 mg, 53%) was prepared from compound
20a (48.2 mg, 0.15 mmol) as a yellow solid. mp: 126–128 °C;
Rf = 0.48 (20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ
7.93 (dd, J = 7.8, 1.5 Hz, 1H), 7.53 (dt, J = 6.9, 1.5 Hz, 1H),
7.31 (d, J = 6.9 Hz, 1H), 7.11–7.03 (m, 2H), 5.29–5.18 (m, 1H),
4.66 (d, J = 10.2 Hz, 1H), 4.51 (d, J = 16.5 Hz, 1H), 3.90–3.83
(m, 1H), 3.41 (dd, J = 6.6, 2.4 Hz, 1H), 2.72–2.67 (m, 2H), 2.47
(s, 3H), 2.41–2.33 (m, 2H), 1.75–1.71 (m, 2H); 13C NMR
(75 MHz, CDCl3): δ 200.55, 177.18, 160.05, 136.36, 136.12,
132.99, 132.69, 127.21, 121.82, 119.52, 118.73, 118.12, 89.85,
70.72, 60.52, 46.46, 39.26, 36.23, 33.01, 30.69; IR (KBr, cm−1):
3079, 2920, 2843, 1771, 1722, 1671, 1642, 1609, 1464, 1315,
1275, 1261,764; EI-MS (m/z): 321 (M+) (52); HRMS (ESI) calc.
for C20H19NO3 (M + H)+ 322.1443, found 322.1436.
Aza-caged compound (6b)
Aza-caged compound (6e)
Compound 6b (33.8 mg, 65%) was prepared from compound
20b (52.1 mg, 0.15 mmol) as a yellow solid. mp: 113–114 °C;
Rf = 0.54 (20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ
7.93 (dd, J = 7.8, 1.5 Hz, 1H), 7.53 (dt, J = 6.9, 1.5 Hz, 1H),
7.30 (d, J = 6.9 Hz, 1H), 7.08–7.02 (m, 2H), 5.88–5.79 (m, 1H),
5.29–5.21 (m, 2H), 5.11 (d, J = 10.2 Hz, 1H), 4.64 (d, J = 9.9
Hz, 1H), 4.49 (d, J = 16.8 Hz, 1H), 3.73 (dd, J = 9.6, 4.5 Hz,
1H), 3.53 (dd, J = 7.8, 1.5 Hz, 1H), 3.43–3.38 (m, 1H), 3.05
(dd, J = 14.7, 6.6 Hz, 1H), 2.71–2.64 (m, 2H), 2.44–2.40 (m,
1H), 2.37–2.31 (m, 1H), 1.73–1.70 (m, 2H); 13C NMR
(75 MHz, CDCl3): δ 201.04, 177.20, 160.12, 136.53, 136.26,
136.10, 132.88, 132.75, 127.17, 121.71, 119.53, 118.48, 118.10,
116.27, 89.84, 70.19, 57.77, 52.05, 46.36, 39.23, 33.31, 30.27;
IR (KBr, cm−1): 3062, 2927, 2850, 1731, 1676, 1642, 1608,
1464, 1269, 1260, 758, 744; EI-MS (m/z): 347 (M+) (15);
Compound 6e (29.7 mg, 46%) was prepared from compound
20e (64.6 mg, 0.15 mmol) as a yellow solid. mp: 110–112 °C;
Rf = 0.56 (20% EtOAc–hexane); 1H NMR (300 MHz, CDCl3): δ
7.95 (d, J = 7.8, 1H), 7.55 (t, J = 6.9, 1H), 7.35–7.33 (m, 1H),
7.33–7.28 (m, 4H), 7.10–7.04 (m, 2H), 5.36–5.24 (m, 1H), 4.67
(d, J = 10.2 Hz, 1H), 4.52 (d, J = 16.8 Hz, 1H), 4.14 (d, J =
14.7 Hz, 1H), 3.65 (dd, J = 9.3, 4.5 Hz, 1H), 3.54 (d, J = 14.4
Hz, 1H), 3.46–3.44 (m, 1H), 2.69–2.65 (m, 1H), 2.54–2.50 (m,
2H), 2.37–2.35 (m, 1H), 1.75–1.72 (m, 2H); 13C NMR
(75 MHz, CDCl3): δ 201.19, 177.17, 160.08, 138.22, 136.36,
136.03, 132.96, 132.65, 132.46, 129.32, 128.54, 127.26, 121.85,
119.55, 118.77, 118.09, 89.75, 70.35, 58.24, 52.66, 46.38,
39.20, 33.49, 30.52; IR (KBr, cm−1): 3080, 2924, 2861, 1764,
1644, 1608, 1464, 1257, 1225, 763; EI-MS (m/z): 431 (M+)
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3288–3299 | 3297

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(12); HRMS (ESI) calc. for C26H22ClNO3 (M + H)+ 432.1366,
found 432.1372.
IKKβ assay
12.5 μL of the 4 × reaction cocktail containing 50 ng IKKβ (sup-
plied from the HTScan IKKβ kinase assay kit, Cell Signaling
Technology, Beverly, MA) was incubated with 12.5 μL of test
sample for 5 min at room temperature. 25 μL 2× ATP/substrate
peptide cocktail was added to the preincubated reaction. After
incubation at room temperature for 30 min, a 50 μL stop solution
(50 mM EDTA, pH = 8) was added to stop the reaction. Then
25 μL of each reaction were transferred to a 96-well streptavidin-
coated plate (PerkinElmer Life Sciences, Boston). After adding
75 μL dH2O, the mixture was incubated at room temperature for
60 min. After thoroughly washing the wells 3 times with PBST,
100 μL primary antibody [phospho-IκBα (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 tempera-
ture, the wells were thoroughly washed 3 times with PBST.
100 μL diluted HRP-labeled anti-rabbit IgG (1 : 1000 in PBS/T
with 1% BSA) was added per well. After 30 min incubation at
room temperature, the wells were washed five times with PBST.
Then 100 μL TMB substrate was added into each well, and the
plate was incubated at room temperature for 15 min. After
adding the stop solution (100 μL per well), the plate was incu-
bated at room temperature for 15 min and then read at 450 nm
with Varioskan spectrofluorometer and spectrophotometer
(Thermo, Waltham, MA).
MTT assay
Cell viabilities were measured by a colorimetric assay using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT;
Roche, Ltd) as described previously.27 Experiments were
carried out in triplicate in a parallel manner for each concen-
tration of target compounds used and the results were presented
as mean
SE. Control cells were given only culture media.
After incubation for 24 h, absorbance (A) was measured at
570 nm. Survival ratio (%) was calculated using the following
equation: survival ratio (%) = (Atreatment/Acontrol) × 100%. IC50
was taken as the concentration that caused 50% inhibition of cell
viabilities and calculated by the SigmaPlot software (Systat Soft-
ware Inc., USA).
Cell morphological assessment
To detect morphological evidence of apoptosis, cell nuclei were
visualized following DNA staining with the fluorescent dye
DAPI. Briefly, cells were seeded at a concentration of 1 × 105
cells per well in 6-well tissue culture plates and treated with indi-
cated concentration of 6c. At the end of incubation, the mor-
phology of cells was monitored under an inverted light
microscope. Cells were then fixed with 4% paraform for 20 min
and washed with PBS, and then incubated with DAPI (1 μg
ml−1) for 10 min. After washing with PBS, cells were observed
using fluorescent microscopy (Olympus, Japan) with a peak
excitation wave length of 340 nm.
Acknowledgements
Financial support from National Natural Science Foundation of
China (No. 90713038, No. 21072231, No. 30873157) and
National Major Science and Technology Project of China (Inno-
vation and Development of New Drugs, No. 2008ZX09401–001
and No. 2009ZX09501–003) is acknowledged.
Annexin V/PI double staining assay
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