DMXAA-pyranoxanthone hybrids enhance inhibition activities against human cancer cells with multi-target functions

Article abstract of DOI:10.1016/j.ejmech.2017.10.074

Four 5,6-dimethylxanthone-4-acetic acid (D) and pyranoxanthone (P) hybrids (D-P-n) were design-synthesized based on multi-target-addressed strategy. D-P-4 was confirmed as the most active agent against HepG-2 cell line growth with an IC₅₀ of 0.216 ± 0.031 μM. Apoptosis analysis indicated different contributions of early/late apoptosis/necrosis to cell death for both monomers, the combination (D + P in 1:1 mol ratio) and D-P-4. They all arrested more cells on S phase. Western Blot implied that D-P-4 regulated p53/MDM2 to a better healthy state. Moreover, it improved Bax/Bcl-2 signaling pathway to increase cancer cell apoptosis. In all cases studied, D-P-4 showed the best activity and synergistic effect. All the evidences support that D-P-4 is a better anti-cancer therapy with multi-target functions.

Full text of DOI:10.1016/j.ejmech.2017.10.074

Accepted Manuscript
DMXAA-pyranoxanthone hybrids enhance inhibition activities against human cancer
cells with multi-target functions
Jie Liu, Fan Zhou, Lei Zhang, Huailing Wang, Jianrun Zhang, Cao Zhang, Zhenlei
Jiang, Yanbing Li, Zhijun Liu, Heru Chen
PII:
S0223-5234(17)30876-0
10.1016/j.ejmech.2017.10.074
EJMECH 9866
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 July 2017
Revised Date: 25 October 2017
Accepted Date: 27 October 2017
Please cite this article as: J. Liu, F. Zhou, L. Zhang, H. Wang, J. Zhang, C. Zhang, Z. Jiang, Y. Li, Z.
Liu, H. Chen, DMXAA-pyranoxanthone hybrids enhance inhibition activities against human cancer
cells with multi-target functions, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.074.
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ACCEPTED MANUSCRIPT

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DMXAA-Pyranoxanthone hybrids enhance inhibition activities
against human cancer cells with multi-target functions
Jie Liua, Fan Zhoua, Lei Zhanga, Huailing Wangb, Jianrun Zhanga, Cao Zhanga,
Zhenlei Jianga, Yanbing Lia, Zhijun Liua, Heru Chen*a, c
a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan
University, Guangzhou 510632, P. R. China
b School of Food Sciences and Engineering, South China University of Technology, Guangzhou
510641, China
c
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New
Drugs Research,Jinan University, Guangzhou 510632, P. R. China
* Corresponding authors:
Prof. Dr. Heru Chen
Huangpu Avenue West 601
Institute of Traditional Chinese Medicine and Natural Product
College of Pharmacy, Jinan University
Guangzhou 510632, P. R. China
Tel: +86-20-38375299; Fax: +86-20-85221559
E-mail: thrchen@jnu.edu.cn
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Abstract Four 5,6-dimethylxanthone-4-acetic acid (D) and pyranoxanthone (P)
hybrids (D-P-n) were design-synthesized based on multi-target-addressed strategy.
D-P-4 was confirmed as the most active agent against HepG-2 cell line growth with
an IC50 of 0.216±0.031 µM. Apoptosis analysis indicated different contributions of
early/late apoptosis/necrosis to cell death for both monomers, the combination (D+P
in 1:1 mol ratio) and D-P-4. They all arrested more cells on S phase. Western Blot
implied that D-P-4 regulated p53/MDM2 to a better healthy state. Moreover, it
improved Bax/Bcl-2 signaling pathway to increase cancer cell apoptosis. In all cases
studied, D-P-4 showed the best activity and synergistic effect. All the evidences
support that D-P-4 is a better anti-cancer therapy with multi-target functions.
Keywords: xanthones; anti-cancer; multi-targets-addressed ligand; synergistic effect;
p53/MDM2; Bax/Bcl-2
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1. Introduction
The multi-target approach has been suggested as particularly suitable to struggle
the heterogenity and the multifactorial nature of cancer [1]. This strategy involves the
concept of a single chemical entity with desirable activity at more than one biological
target. The designed multiple ligands (DMLs), which may also be described as
multiple-target directed ligands, heterodimers, promiscuous drugs and pan-agonists,
are drugs which act at multiple biomolecular targets. No double, the successful
treatment of these ligands often depends on pharmaceutical intervention at multiple
pathways, with a combination of different drugs in one single entity [2-5].
Many DMLs have been successfully applied to improve the anticancer efficacy.
For example, CS2164 (Fig. 1), named as (N-(2-aminophenyl)-6-6-[(7-methoxy-4-
quinolinyl)oxy]-1-naphthale-necarboxamide, was designed and evaluated as a novel
orally active multi-target inhibitor that simultaneously inhibits the angiogenesis-
related kinases (VEGFR2, VEGFR1, VEGFR3, PDGFRa and c-Kit), mitosis-related
kinase Aurora B and chronic inflammation-related kinase CSF-1R in a high potency
manner with the IC50 value at a single-digit nanomolar range [6]. Yan, ect. designed
and synthesised a series of benzoselenazole-stilbene hybrids by combining the
pharmacophores of resveratrol and ebselen, and showed that compound 6e (Fig. 1)
had the optimal activity against four human cell lines. It induces G2/M cell cycle
arrest and apoptosis of the human liver carcinoma Bel-7402 cell line and exhibits the
best thioredoxin reductase (TrxR) inhibitory activity amongst all the tested
compounds [7]. The oxime analog 7e (Fig. 1) was indicated as inhibitors of tubulin
polymerization, BRAFV600E, FAK and EGFR-TK; and it also exihibted multiple drugs
resistant (MDR) reversal activity [4].
Intriguing by the interesting scaffolds and pharmacological importance of
xanthones (Fig. 1), our group have been pursuing the search for novel xanthones by
either isolation from nature or synthesis design [8-10]. It is of notice that many
xanthone compounds are reported with anticancer activity [11]. Among them, two
xanthones, 5,6-dimethylxanthone-4-acetic acid (DMXAA) and 3,4-dihydro-12-
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hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one (pyranoxanthone) (Fig. 1)
[12,13] caught our eyes. As reported, DMXAA is the first small-molecule
vascular-disrupting agent to enter phase III clinical trial stage [14]; and
pyranoxanthone is a novel small-molecule inhibitor of p53-MDM2 interaction with a
xanthone scaffold, in which p53 is a tumor suppressor and MDM2 (murine double
minute 2) is the main endogenous negative regulator against p53.
Fig. 1. Chemical structures of some DMLs and xanthones.
However, DMXAA is not a particularly potent drug, with dose levels of up to 4.9
g/m2 reported in human trial [15]. Moreover, the side effects of paropsia, uracratia and
dysphoria limit its use in clinic [16]. How to improve the anti-cancer efficacy of
DMXAA is what we are concerning about. Recently, we disclosed that DMXAA can
regulate p53/MDM2 to a better healthy state, although the activity is weaker than that
of its combination [10]. Therefore, it is reasonable for us to suggest that
pyranoxanthone, which also has the same ability to adjust p53/MDM2 interaction,
may probably synergize with DMXAA to improve the anticancer efficacy by DML
design or in the form of combination.
Hence, here in the current study, a series of DMXAA (D) and pyranoxanthone (P)
hybrids were design-synthesized. The screening of the inhibition activity against
human cancer cell lines of these hybrids and the D/P combination were carried on.
Furthermore, the mechanism of how the candidate hybrid induces the death of
HepG-2 liver cancer cells will be also disclosed thereafter.
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2. Chemistry
As outlined in Scheme 1, the syntheses of DMXAA/pyranoxanthone hybrids
(D-P-n, n = 1, 2, 3, 4) firstly began with the syntheses of DMXAA and
pyranoxanthone. DMXAA was prepared based in a process early described [17].
While pyranoxanthone was made using salicyclic acid and pyrogallol as starting
materials. In step a, condensation of salicyclic acid with 1,2,3-trihydroxybenzene to
3,4-dihydroxyxanthone was succeffully carried on by applying a modified method
early described [10], in which anhydrous zinc chloride (ZnCl2) and phosphorus
oxychloride (POCl3) was used as both catalyst and solvent under the assistance of
microwave radiation. The yield was 79.5%. The construction of
2,2-dimethyl-2H,6H-pyrano[3,2-b] moiety was completed using montmorillonite K10
as catalyst assisted by microwave radiation, leading to pyranoxanthone with a yield of
38.5%. 4-Hydroxyalkylation of pyranoxanthone was realized under a mild conditions,
in which potassium carbonate (K2CO3) was applied as a base, resulted in a series of
4-hydroxyalkyl pyranoxanthone (P-n, n = 1, 2, 3, 4). Yields lied between 80-90%.
DMXAA condensated with P-n applied dicyclohexydiimide (DCC) as coupling
reagent to offer D-P-n hybrids in a yield of 48%-53%. The coupling reactions were
benefited from the addition of 4-N,N-dimethylaminopyridine (DMAP). All the four
hybrids were characterized by NMR and MS. And their purities were analyzed by
RP-HPLC method.
Scheme 1. Syntheses of DMXAA/pyranoxanthone hybrids. Conditions and Agents: (a)
ZnCl2/POCl3, microwave (MW), 40 min; (b) BrCH2CHC(CH3)2, MW/600 W, montmorillonite
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K10 (20 equiv.), CHCl3, 40 min; (c) Br(CH2)mOH (m = 3, 4, 5, 6) (dropwise, 30 min), K2CO3,
DMF, r. t., 24 h; (d) DCC/DMAP.
3. Results and discussion
3.1. Growth inhibitory effects of D-P hybrids and the combination against human
cancer cell lines
It’s the common sense that breast cancer, liver cancer, chronic myelogenous
leukemia, and colorectal adenocarcinoma are very popular cancer types, this is the
reason why Michigan Cancer Foundation-7 (MCF-7), human breast MDA-MB-231,
human liver Hep-G2, and chronic myelogenous leukemia K562 cancer cell lines were
chosen as materials in the current study. MTT assay, in which 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide was used as a dye, was applied to determine
the effects of D/P hybrids and D/P combination on the cellular growth inhibition. As
shown in Table 1, compared with DMXAA and Pyranoxanthone monomer, D/P
combination, which is the mixture of 1:1 molar ration of D and P, was showed greater
anti-proliferation activity against all the tested cancer cell lines. However, more
excitingly, the D-P-4 hybrid exhibited significant inhibitory activity enhancement
against all the tested cancer cell lines, particularly MCF-7 and HepG-2. Compared
with DMXAA, the inhibition activity of D-P-4 against MCF-7 was increased by 100
times with IC50 value of 0.534 ± 0.043 µM; and against HepG-2 by 460 times with
IC50 value of 0.216 ± 0.031 µM.
Generally speaking, all the hybrids were confirmed with enhancing effect
depending on the chain length of the spacer. The longer the carbon chain, the better
the inhibition activity. When the carbon is more than 5 (D-P-3 and D-P-4), the
inhibition activity of the hybrid against all the tested cancer cell lines is better that that
of D/P combination (in 1:1 molar ratio). This synergistic effect might be ascribed to
the conformation changes brought about by the spacer. When the spacer is a short
carbon chain (< 5), the hybrid is in a more compacted conformation and can only fit
into one pocket of the target protein; However, when the spacer is a long carbon chain
(> 5), the hybrid is in a flexible, strectched conformation, and may probably bind to
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more than one poket of the target protein, or bind to two targets. Whether the six
carbon chain spacer is the optimal needs further investigations.
Table 1. Cell growth inhibition of synthesized D/P hybrids and combination
IC50 a(µM)
Compound
MDA-MB-231
48.44 ± 7.41
59.32 ± 6.82
97.31 ± 9.97
12.12 ± 1.63
32.21 ± 3.31
19.63 ± 2.14
11.24 ± 1.37
1.129 ± 0.13
MCF-7
HepG2
K562
DMXAA (D)b
Pyranoxanthone (P)
P-4
54.41 ± 6.43
101.5 ± 11.72
138.7 ± 15.85
11.89 ± 1.23
51.74 ± 6.74
33.22 ± 3.72
8.723 ± 1.10
0.534 ± 0.043
100.2 ± 11.24
40.31 ± 5.12
99.07 ± 10.08
21.25 ± 2.73
32.72 ± 3.42
11.92 ± 1.52
1.529 ± 0.23
0.216 ± 0.031
57.44 ± 6.15
27.82 ± 3.20
109.51 ± 11.41
19.14 ± 2.53
32.31 ± 4.11
20.63 ± 2.27
15.03 ± 1.84
9.13 ± 1.34
D+P (1 : 1)
D-P-1
D-P-2
D-P-3
D-P-4
a IC50 values are shown as mean±standard error of the mean (SD), from at least three independent experiments.
b DMXAA was used as a positive control.
Since the existence of ester bond in D-P-n, they are expected to be hydrolyzed
easily by enzyme in vivo. Therefore, investigation on the stability of the hybrid was a
must. As the most active compound, the stability of D-P-4 in cell culture medium and
mouse serum was studied. It was found that the half life time (t1/2) of D-P-4 in cell
culture medium and mouse serum was 24.8 h, and 4.2 h, respectively (Fig. 2).
Obviously, D-P-4 has a certain degree of stability in either cell culture medium or
mouse serum. It is more stable in cell culture medium than that in mouse serum.
Fig. 2. Stability of D-P-4 in (A) in cell culture medium; and (B) mouse serum. 1 ml solution of 10
µM/L D-P-4 was suspended with 4 ml of mouse serum, and then incubated at 37°C. 10 µl of the
mixed solution was taken at the time point of (A) 0, 0.25, 0.5, 1, 2, 4, 8, 12, 20, 28, 36, 48 h,
repsectively; or (B) 0, 0.5, 1, 2, 4, 8, 12 h, repsectively. These samples were pretreated with
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methanol and 20% perchloric acid, centrifuged at 10000 r/min for 5 minutes. Then the supernatant
was submitted to HPLC for content analysis. Each experiments was repeated 4 times.
After hydolysis of D-P-4, the resulted products are DMXAA and P-4. The
inhibition activity of P-4 against four cancer cell lines was also determined. It was
shown that the introduction of 6-hydroxyhexyl group at 12-O position caused the
decreased of inhibition activity. This may exclude the possibility that the activity
enhancement of D-P-4 is depended on the release of P-4.
The current evidence support that the D-P-4 is probably a promising anticancer
therapy. Therefore, the cytotoxicity against normal cell line from healthy tissues must
be evaluated. Here, the inhibition against the growth of normal cell line was
determined. The testing method is the same as that applied to cancer cell line. It was
found that the cytotoxicities of DMXAA against human liver cell line HL-7702 and
mouse embryo fibroblast cell line NIH/3T3 are almost at the same level compared to
that against cancer cell lines, in which the IC50 values are 101.3±2.6 µM to HL-7702,
and 67.8±1.3 µM to NIH/3T3. However, the cytotoxicities of D-P-4 against normal
cells are far less than that against cancer cell lines. The IC50 value is 452.29 ± 41.20
µM (for 24 h), and 351.98 ± 31.26 µM (for 48 h) against HL-7702 cell line,
respectively; while the IC50 value is 378.03 ± 37.22 µM (for 24 h), and 293.22 ±
25.34 µM (for 48 h) against HL-7702 cell line, respectively. Compared the IC50 values
at the same incubation time (24 h), the cytotoxicity against HL-7702 cells is 2092
times less than that against HepG-2 cancer cells. Definitely, D-P-4 significantly
showed attenuated cytotoxicity effect against normal cells.
3.2. Cell death induced by pyranoxanthone, DMXAA, D/P combination and D-P-4
Since D-P-4 was indicated with the greatest enhancing effect against HepG-2
(Table 1), together with the fact that Hepatocellular carcinoma (HCC) is one of the
most common malignancies around the world, accounting for over 80% of all liver
cancers with extremely poor prognosis [18], the development for of novel and
effective therapies for this devastating disease is urgently needed. This is the reason
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why we chose HepG-2 cell line to explore the mechanisms involved in how these
agents prevent the cancer cell growth.
Flow cytometry using propidium iodide (PI) and Annexin-V as dyes to label
viable and dead cells is one of the common technique to study cell apoptosis/necrosis,
therefore it was applied here. As shown in Fig. 3, at the same dose of 0.2 µM,
pyranoxanthone caused less than 10% total apoptosis/necrosis rate; While DMXAA
(D) showed better inhibition activity than pyranoxanthone (P), in which about 20%
total apoptosis/necrosis rate (with 13% for early and 7% for late) was found.
Interestingly, the combination (D+P in 1:1 mol ratio) was found inducing 61% total
apoptosis/necrosis rate (with 15% for early and 46% for late). More excitingly, D-P-4
hybrid was indicated with the greatest inhibition activity, in which it led to 80% total
apoptosis/necrosis rate (with 38% for early and 42% for late). It was shown that
D-P-4 induced almost the same late apoptosis/necrosis rate but greater early
apoptosis/ necrosis rate than the combination (D+P).
Fig. 3. DMXAA (D), pyranoxanthone (P), the combination (D+P) and D-P-4 induced apoptosis in
HepG-2 cells. Representative scatter diagrams. HepG-2 cells were pre-treated without the addition
of any samples, the control (C) (I); with a dose of 0.2 µM D (II), P (III), D+P (1:1 mol ratio) (IV),
and D-P-4 (V), respectively for 24 h. Cells were stained with Annexin-V and PI. The apoptosis of
HepG-2 cells was detected by flow cytometry. The evaluation of apoptosis is via Annexin V: FITC
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Apoptosis Detection Kit per manufacture’s protocol. The quantitative results were shown in (VI). In
each scatter diagrams, the abscissa represents the fluorescence intensity of the cells dyed by Annexin
V; and the ordinate represents the fluorescence intensity of the cells dyed by PI. The lower left
quadrant shows the viable cells, the upper left shows necrotic cells, the lower right shows the early
apoptotic cells; while the upper right shows late apoptotic cells.
As we know, necrosis is a form of traumatic cell death that results from acute
cellular injury; while apoptosis is a highly regulated and controlled process that
confers advantages during an organism's lifecycle. Unlike necrosis, apoptosis
produces cell fragments called apoptotic bodies that phagocytic cells are able to
engulf and quickly remove before the contents of the cell can spill out onto
surrounding cells and cause damage to the neighboring cells. An early marker of
apoptosis is the exposition of phosphatidylserine on the cell surface, whereas it is
normally concentrated in the luminal layer of the cytoplasmic membrane [19]. D-P-4
hybrid promoted greater early apoptosis/necrosis than both monomers and the
combination, implying different anti-cancer mechanism compared with them.
3.3. Cell cycle analysis of DMXAA, pyranoxanthone, the combination, and D-P-4
To establish whether DMXAA (D), pyranoxanthone (P), the combination (D+P in
1:1 mol ratio), and D-P-4 inhibited cell growth by interrupting the cell cycle progress,
cellular DNA was analyzed and stained with propidium iodide (PI). The cells were
analyzed using flow cytometry. The profiles were shown in Fig. 4. Obviously,
compared with the control group, an increase in the S population was observed in
HepG-2 cells after the treatment with D, P, D+P, and D-P-4 at the dose of 0.2 µM,
respectively. D-P-4 showed the most capability to arrest cells at S state. It increased S
population by 90% compared to the control.
This fact suggests that the cell cycle arrest is one of the primary mechanisms
responsible for the anticancer activities of D, P, D+P, and D-P-4. The D-P-4 hybrid
alters the manner of cell cycle arrest to S state greater than both monomers and the
combination. This may possibly be one reason for the anti-cancer activity
enhancement of the hybrid.
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Fig. 4. Cell cycle analysis of HepG-2 cells exposed to DMXAA (D), pyranoxanthone (P), the
combination (D+P in 1:1 mol ratio), and D-P-4, respectively. HepG-2 cells were pre-treated with
D, P, D+P, and D-P-4 at a dose of 0.2 µM, respectively for 24 h. C was the control group, without
addition of any tested compounds. Cells were collected, fixed in 70% ethanol, and stained with
propidium iodide solution. G0/G1: quiescent state/growth phase; S: initiation of DNA replication;
G2/M: biosynthesis/mitosis phases.
Usually, the ensuing S phase starts when DNA synthesis commences; when this
phase is complete, all of the chromosomes have been replicated, i.e., each
chromosome has two (sister) chromatids. If DNA can not be replicated, cell cycle will
be stopped at this stage. Based on the current result, it was suggested that D-P-4 may
inhibit the DNA replication and make the cell cycle stop in S phase.
3.4. Influence on the expression levels of proteins related to cell death by
pyranoxanthone (P), the combination and D-P-4 hybrid
Cell death includes apoptosis and necrosis. In order to disclose how
pyranoxanthone (P), the combination (D+P in 1:1 mol ratio) and D-P-4 induced cell
apoptosis, we examined the expression levels of caspase-3, cleaved caspase-3,
caspase-9, cleaved caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP) by
Western Blotting. We found that P, D+P, and D-P-4 decreased the levels of caspase-3,
and caspase-9, respectively; while in the mean time increased the expression of
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cleaved caspase-3 and cleaved PARP (Fig. 5 I-II). Of notice, D+P and D-P-4
increased the ratios of cleaved caspase-3(9)/caspase-3(9) and the expression level of
cleaved PARP greater than P monomer. It was found that D-P-4 had the greatest effect.
As we know, caspase-3 is involved in the apoptotic process, where it is responsible for
chromatin condensation and DNA fragmentation [20]. Caspase-9 is an initiator
caspase [21]. The initiated caspase-9 will go on to cleave procaspase-3 and
procaspase-7. In other hand, PARP is a family of proteins involved in a number
of cellular processes involving mainly DNA repair and programmed cell death [22]
.
When PARP is cleaved by enzymes such as caspases or cathepsins, typically the
function of PARP is inactivated. Therefore, these data support that P, D+P, and D-P-4
induce cell apoptosis via the adjustment of caspase 3, caspase 9, and PARP, which
closely participate in programmed cell death. D-P-4 hybrid has better effect than
pyranoxanthone monomer or the combination.
Fig. 5. Effect of pyranoxanthone (P), the combination (D+P) and D-P-4 on proteins related to
cell death. HepG-2 cells were treated with P, D+P (in 1:1 mol ratio), and D-P-4 at a dose of 0.2
µM, respectively for 24 h. C was the control group, without addition of any tested compounds.
The proteins expression levels were measured using Western Blot. The density of each lane was
presented as mean ± standard deviation (SD) for at least three individual experiments. Blots were
quantified using Image J software.
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We noticed the fact that pyranoxanthone was reported as a putative
small-molecule inhibitor of p53/MDM2 interaction [23], in which p53 is a tumor
suppressor which plays many roles including the ability to induce cell cycle arrest,
DNA repair, senescence, and apoptosis [24,25]; while MDM2 (murine double
minute 2) is the main endogenous negative regulator. This oncoprotein MDM2 binds
p53 and negatively regulates p53 activity by direct inhibition of p53 transcriptional
activity and enhancement of p53 degradation via the ubiquitin-proteasome pathway
[26-28]. By inhibiting the p53/MDM2 interaction to restore p53 activity represents an
appealing therapeutic strategy for many wild-type p53 tumors with over expressed
MDM2. Therefore, we are interested in whether the combination D+P and D-P-4
hybrid can regulate the p53/MDM2 interaction.
To our expect, it was found that P, D+P, and D-P-4 up-regulated p53 expression,
respectively; while in the other hand, they down-regulated MDM2 expression,
respectively (Fig. 5 III-IV). Obviously, the combination and D-P-4 showed the
greater activity on the regulation of p53/MDM2 than the P monomer, which make
these two proteins in a better healthy state. And D-P-4 had the greatest effect, it
changed the p53/MDM2 ratio from 0.083 (control group) to 0.81; while the ratio was
0.67 and 0.19 for D+P and P group, respectively. This positive effect might be the key
mechanism of how D+P and D-P-4 induce cancer cell death. It might conclude based
on this evidence that the regulation of p53/MDM2 to a better healthy state is probably
the main reason for the synergetic effect in the form of combination or hybrid
between pyranoxanthone and DMXAA.
3.5. Pyranoxanthone, the combination, and D-P-4 influence Bcl-2 signaling pathway
The mitochondrial pathway plays a significant role in the apoptotic modulation as
a major signaling pathways [29]. The mitochondrial-mediated apoptotic pathway can
be triggered by several factors, including the expression of B-cell lymphoma 2 (Bcl-2)
family members such as Bcl-2, Bcl-2 associated protein X (Bax), B-cell lymphoma
-extra large (Bcl-xL) and BH3 interacting-domain death agonist (Bid), which BH
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means Bcl-2 homology. There are four BH domains named BH1, BH2, BH3, and
BH4. This proteins family can affect the permeability of the mitochondrial membrane
and trigger the opening of the mitochondrial permeability transition pore in the inner
mitochondrial membrane, resulting in the release of cytochrome c and mitochondrial
dysfunction [30].
In the current study, the down-regulation of anti-apoptotic protein Bcl-2 and
Bcl-xL, and up-regulation of the pro-apoptotic protein Bax and Bid were observed
after the treatment of 0.2 µM of pyranoxanthone (P), the combination D+P and D-P-4
hybrid in Fig. 6. Usually, the greater of Bax/Bcl-2 ratio, the more percentage of cell
apoptosis is involved. D-P-4 hybrid showed the greatest activity in regulating the
Bax/Bcl-2 ratio, in which it changed the ratio from 0.23 (the control group) to 0.87;
while the ratios were 0.58 (D+P group) and 0.36 (P group), respectively. This
evidence indicated that treatment of P, D+P, and D-P-4, respectively against HepG2
cells induce increased apoptosis via the mitochondrial-mediated apoptotic pathway.
D+P and D-P-4 showed better activity than P monomer, and the hybrid had the best
activity.
Fig. 6. Effect of pyranoxanthone (P), the combination (D+P) and D-P-4 on proteins related to
Bcl-2 signaling pathway. HepG-2 cells were treated with P, D+P (in 1:1 mol ratio), and D-P-4 at a
dose of 0.2 µM, respectively for 24 h. C was the control group, without addition of any tested
compounds. The proteins expression levels were measured using Western Blot. The density of
each lane was presented as mean ± standard deviation (SD) for at least three individual
experiments. Blots were quantified using Image J software.
Moreover, the increased expressions of Bid and decreased expressions of Bcl-xL
can also lead to the same conclusion as Bax/Bcl-2.
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Generally, cancer can be seen as a disturbance in the homeostatic balance between
cell growth and cell death. It is of notice that simultaneous over-expression of Bcl-2
and the proto-oncogene myc may produce aggressive B-cell malignancies including
lymphoma [31]. In follicular lymphoma, a chromosomal translocation commonly
occurs between the fourteenth and the eighteenth chromosomes-t (14 and 18), which
places the Bcl-2 gene from chromosome 18 next to the immunoglobulin heavy chain
locus on chromosome 14. This fusion gene is deregulated, leading to the transcription
of excessively high levels of Bcl-2 [32], and decreases the propensity of these cells
for apoptosis. Therefore, targeted or selective inhibited against Bcl-2 can be an
effective strategy to treat cancer. Several drugs including G3139, ABT-199, and
ABT-263 have been in clinical trial [34-35]. From this viewpoint, D-P-4 is certainly
an promising anti-cancer agent.
4. Conclusions
In a word, four DMXAA (D) and pyranoxanthone (P) hybrids (D-P-n, n = 1-4)
were design-synthesized based on multi-target-addressed ligand strategy. Their
anticancer activities against four human cells, MCF-7, MDA-MB-231, Hep-G2, and
K562 cell lines were evaluated. All the hybrids and the combination (D+P in 1:1 mol
ratio) exhibited greater inhibitory activities against the four tested cancer cell lines
growth with IC50 values between 0.2 and 52 µM. The D-P-4 hybrid demonstrated the
most potent inhibitory activity against MCF-7 and HepG-2 cell lines with IC50 values
of 0.534 ± 0.043, and 0.216± 0.031 µM, respectively.
The study of structure-activity relationship indicated that the length of carbon
chain between D and P is very important for the inhibitory activity, which in the
current study, the optimal carbon chain is 6. It was found that P enhanced the
inhibitory activity of D. The combination of D and P in 1:1 mol ratio mixture
increased 3-5 times the activity against the tested four cell lines growth than D itself.
At the same dose of 0.2 µM, D, P, the combination (D+P in 1:1 mol ratio) and
D-P-4 hybrid induced HepG-2 cells apoptosis. The contribution of early apoptosis,
necrosis, and late apoptosis were different among both monomers, the combination
15

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and the hybrid. D-P-4 showed the highest total apoptosis/necrosis rate, and caused far
more early apoptosis/necrosis rate than the others. Cell cycle analysis indicated that D,
P, D+P and D-P-4 all arrested more cells on S phase, in which the hybrid showed the
greatest effect. Results in Western Blot indicated that P, D+P, and D-P-4 decreased
the expression levels of caspase 3, caspase 9, and MDM2; while in the mean time
increased the expression levels of cleaved-caspase 3, cleaved-caspase-9, cleaved-
PARP, and p53. The hybrid showed the best regulation of p53/MDM2 to a better
healthy state, in which the p53/MDM2 ratio was 0.81 compared 0.083 for the control.
P, D+P, and D-P-4, respectively showed another activity to decrease Bcl-2 and
Bcl-xL expression levels, while in the mean time increase the expression levels of
Bax and Bid, indicating the ability to improve cancer cell apoptosis through Bcl-2
signaling pathway. D-P-4 again had the best activity to adjust the Bcl-2 protein family,
in which the Bax/Bcl-2 ratio was 0.87 compared 0.23 for the control. Better
improvement on p53/MDM2 and Bax/Bcl-2 might be the possible reasons for the
synergistic effect in the form of hybrid beween D and P. All the evidences support
that D-P-4 hybrid is a better anticancer therapy with multi-target function.
5. Experimental section
5.1. Materials
All chemicals salicylic acids and phenolics were purchased from Aldrich or
Adamas without further purification. Silica gel for column chromatography was
purchased from Qingdao Marine Chemicals Inc, China. Chromatographic grade
methanol was bought from Shandong YuWang Reagent Company (China).
The human CML cell line MDA-MB-231 and MCF-7, Hep-G2, and K562 were
obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).
MDA-MB-231 and MCF-7 cells were cultured in RPMI 1640 medium (Life
Technologies, Grand Island, NY, USA). HL-7702 and NIH/3T3 cells were grown in
DMEM medium (Life Technologies, Grand Island, NY, USA).
The reagents PI and JC-1 were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). PierceTM BCA Protein Assay Kit was obtained from Thermo Fisher Scientific
16

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(Rockford, IL, USA). MTT, TUNEL Apoptosis Detection Kit, dithiothreitol (DTT),
Nuclear and Cytoplasmic Extraction Kit, RIPA buffer and RNase were purchased from
Beyotime (Shanghai, China). Phosphatase inhibitor cocktail tablets and protease
inhibitor cocktail tablets were supplied by Roche (Mannheim, Germany). All other
chemicals and solvents were of reagent or HPLC grade.
β-Actin, GADPH, caspase 3, caspase 9, cleaved-caspase 3, cleaved-caspase 9,
cleaved PARP, Bax, Bid, Bcl-2, Bcl-xL, anti-mouse, and anti-rabbit horseradish
peroxidase-conjugated secondary antibodies were purchased from Cell Signaling
Technology (CST, Beverly, MA, USA).
5.2. Synthetic process
5.2.1. General chemical experimental procedures
Nuclear Magnetic Resonance (NMR) spectra were recorded on either a Bruker
AV-300 or a Bruker AV-400, or a Bruker AV-500 (Bruker Biospin, Swiss).
Tetramethylsilicane (TMS) was used as an internal standard. ESI-MS were recorded
on a Finnigan LCQ Advantage MAX mass spectrometer. HPLC was performed on
either a LC-100 liquid chromatograph equipped with a tunable LC-100 UV detector
(Shanghai Wufeng Inc., China) or an Agilent 1200 series liquid chromatograph
equipped with an Agilent 1200 Series UV detector (Agilent Technologies, USA).
Columns used were Cosmosil 5C18 (Nacalai Tesque Inc., Japan) for general
purification. Pre-coated thin-layer chromatography (TLC) plates (Institute of Yantai
Chemical Industry, China) were used for TLC. Spots on TLC plates were detected by
either a ZF-7A portable UV detector or spraying Bismuth potassium iodide solution
followed subsequent heating. Ethanol was refluxed over Fresh magnesium ribbon for
5 hours and redistilled.
5.2.2. Synthesis of 5,6-dimethylxanthone-4-acetic acid (DMXAA)
The synthesis was carried on referred to a procedure described in literature [17].
Firstly, 3,4-dimethylbenzoic acid was turned into 2,5-dibromo-3,4-dimethylbenzoic
17

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acid, which was named as compound 1. Compound 1 was then converted into its
potassium salt and dried thoroughly. This salt reacted with the anhydrous disodium
salt of 2-hydroxyphenylacetic acid under the interaction of tris[2-(2-methoxyethoxy)
ethyl]amine (TDA-1) and copper (I) to give 7-bromo-5,6-dimethyl-9-oxo-9H-
xanthen-4-yl acetic acid (2). Hydrogenation of compound 2 gave DMXAA in an
1
overall yield of 51% in four steps. Purity 98.5%. H NMR (DMSO-d6, 300 MHz)
δ:12.62 (s, 1H), 8.10 (dd, J = 8.0, 1.6 Hz, 1H, H-1), 7.93 (d, J = 8.4 Hz, 1H, H-8),
7.79 (dd, J = 7.2, 1.6 Hz, 1H, H-3), 7.42 (dd, J = 7.6, 1.6 Hz, 1H, H-7), 7.31 (d, J =
13
8.0 Hz, 1H, H-2), 3.99 (s, 2H), 2.44 (s, 3H), 2.42 (s, 3H); C NMR (DMSO-d6, 75
MHz) δ: 176.1 (-COOH), 171.7 (C-9), 153.7 (C-4a), 153.2 (C-4b), 144.6 (C-6), 136.5
(C-3), 125.9 (C-4), 125.2 (C-5), 125.1 (C-7), 124.6 (C-1), 123.6 (C-2), 122.5 (C-8),
120.5 (C-8b), 118.7 (C-8a), 35.4 (C-9), 20.1 (CH3), 11.0 (CH3); MS-ESI m/z: 283.2
[M+H]+.
5.2.3. Synthesis of 12-O-(n-hydroxyalkyl)pyranoxanthones
5.2.3.1. Synthesis of 3,4-dihydroxy-9H-xanthen-9-one
To a 50-ml flask, 8 ml phosphorus oxychloride (POCl3) and anhydrous zinc
chloride (6.8 g, 0.05 mol) were added. The suspension was stirred at 70°C until ZnCl2
was completely dissolved into phosphorus oxychloride. The mixture was then cooled
down to room temperature (r.t.). Afterwards, salicylic acid (0.138 g, l.0 mmol) and
pyrogallol (0.151 g, 1.2 mmol) were added. Then the mixture was heated with
microwave reactor with a programmed procedure of 75°C for 30 min. Afterwards, the
mixture was cooled down to r.t. and pulled into ice water stirring for 20 min. The
mixed solution was filtered, washed with cold water. The solid residues were
collected and purified by flash column liquid chromatography led to yellow powder
0.176 g, yield 77.1%. M.p. 220~221 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 7.38 (td, J
= 8.1, 0.9 Hz, 1H, H-8), 7.29 (dd, J = 8.1, 0.9 Hz, 1H, H-6), 6.91-6.95 (m, 2H, H-5, 7),
6.89 (d, J = 8.4 Hz, 1H, H-1), 6.36 (d, J = 8.4 Hz, 1H, H-2); 13C NMR (DMSO-d6, 75
MHz) δ: 202.62 (C-9), 157.41 (C-4b), 153.61 (C-3), 153.24 (C-4a), 133.64 (C-6),
133.54 (C-4), 131.06 (C-8), 127.04 (C-7), 126.18 (C-8a), 120.03 (C-1), 117.66 (C-5),
18

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115.24 (C-8b), 108.41 (C-2); MS-ESI m/z: 228.2 [M+H]+.
5.2.3.2. Synthesis of pyranoxanthone
To a 50-ml flask, 20 ml chloroform, 3,4-dihydroxy-9H-xanthen-9-one (0.228 g,
1.0 mmol), and montmorillonoid K10 (4.5 g, 20 eg) were added. The mixture was
radiated with a microwave reactor at 600 W under stirring for 40 min. After the end of
reaction, montmorillonoid K10 was filtered and washed. All the filtration was
combined. Removal of solvent was carried on by rotary evaporation under reduced
pressure. The solid residues were collected and purified by flash column liquid
chromatography led to slight yellow powder 0.13 g, yield 38.5%, Purity 97.6%. M.p.
°
1
176~178 C; H NMR (400 MHz, CDCl3) δ: 8.33 (1H, dd, J = 8.0 and 1.6 Hz, H-7),
7.70 (1H, ddd, J = 7.7, 8.0, 1.6 Hz, H-9), 7.70 (1H, s, H-5), 7.58 (1H, dd, J = 8.0, 1.0
Hz, H-8), 7.35 (1H, ddd, J = 7.3, 7.3, 1.0 Hz, H-10), 5.74 (1H, br s, OH), 2.93 (2H, t,
13
J = 6.7 Hz, H-4), 1.92 (2H, t, J = 6.7 Hz, H-3), 1.43 (6H, s, 2 -CH3); C-NMR (75
MHz, CDCl3) δ: 176.72 (C-6), 156.21 (C-10a), 146.33 (C-12a), 143.24 (C-11a),
134.30 (C-9), 132.42 (C-12), 126.60 (C-7), 123.62 (C-8), 121.53 (C-6a), 118.22
(C-10), 117.93 (C-5), 117.04 (C-4a), 115.41 (C-5a), 76.54 (C-2), 32.66 (C-3), 27.02
(C-4), 21.74 (C-1’); ESI-MS (m/z): 297.2[M+H]+.
5.2.3.3. Synthesis of 3,4-dihydro-12-O-(3′-hydroxypropyl)-2,2-dimethyl-2H,6H
-pyrano[3,2-b]xanthen-6-one (P-1)
To a 50-ml flask, pyranoxanthone (0.296 g, 1.0 mmol) and NaOH (44 mg, 1.1
mmol) in 15 ml of dried DMF. 3-Bromopropanol (0.208 g, 1.5 mmol) in 5 ml dried
DMF was then added dropwise with stirring. The reaction was lasted at r. t. for 24 h.
After the finish of reaction, the mixture was pulled into 50 ml ice-water with violently
stirring for 20 min. The precipitates were collected by filtrate, and were purified by
flash column liquid chromatography led to slight yellow powder 0.318 g, yield 89.6 %.
1
M.p.: 161-163 ; H-NMR (CDCl3, 400 MHz) δ: 8.35 (1H, dd, J = 8.0 and 1.6 Hz,
H-8), 7.73 (1H, ddd, J = 7.6, 8.0, 1.6 Hz, H-6), 7.71 (1H, s, H-1), 7.60 (1H, dd, J =
8.0 and 1.0 Hz, H-5), 7.37 (1H, ddd, J = 7.6, 7.6, and 1.0 Hz, H-4), 5.76 (1H, s, OH),
19

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4.21 (2H, t, J = 2.6 Hz, H-1"), 3.57 (2H, t, J = 2.6 Hz, H-3"), 2.95 (4H, m, H-2', 2"),
13
1.97 (2H, t, J = 2.6 Hz, H-1'), 1.43 (6H, s, H-4', 5'). C-NMR (CDCl3, 100 MHz) δ:
177.19 (C-9), 157.31 (C-4b), 147.32 (C-3), 144.27 (C-4a), 135.37 (C-4), 133.47 (C-6),
127.62 (C-8), 124.65 (C-7), 122.57 (C-1), 119.25 (C-8a), 118.95 (C-2), 118.05 (C-5),
116.44 (C-8b), 75.59 (C-3'), 62.45 (C-1"), 57.11 (C-3"), 32.75 (C-2'), 31.24 (C-2"),
26.44 (C-4'), 26.44 (C-5'), 22.62 (C-1'). MS-ESI m/z: 355.16 [M+H]+
.
5.2.3.4. Synthesis of 3,4-dihydro-12-O-(4′-hydroxybutyl)-2,2-dimethyl-2H,6H
-pyrano[3,2-b]xanthen-6-one (P-2)
Followed the same procedure described in 5.2.3.3. Led to light yellow powder
0.37 g, yield 83.4%. M.p.: 159-161 , 1H-NMR (CDCl3, 400 MHz) δ: 8.31 (1H, dd, J
= 8.0 and 1.6 Hz, H-8), 7.70 (1H, ddd, J = 7.6, 8.0, 1.6 Hz, H-6), 7.63 (1H, s, H-1),
7.52 (1H, dd, J = 8.0 and 1.0 Hz, H-5), 7.31 (1H, ddd, J = 7.6, 7.6, and 1.0 Hz, H-4),
5.70 (1H, s, OH), 4.15 (2H, t, J = 2.6 Hz, H-1"), 3.43 (2H, t, J = 2.6 Hz, H-4"), 2.91
13
(6H, m, H-2', 2", 3"), 2.02 (2H, t, J = 2.6 Hz, H-1'), 1.40 (6H, s, H-4', 5'). C-NMR
(CDCl3, 100 MHz) δ: 178.11 (C-9), 158.38 (C-4b), 148.35 (C-3), 145.48 (C-4a),
136.40 (C-4), 134.39 (C-6), 128.72 (C-8), 125.70 (C-7), 123.87 (C-1), 120.15 (C-8a),
119.44 (C-2), 119.15 (C-5), 117.43 (C-8b), 76.88 (C-3'), 63.49 (C-1"), 58.13 (C-4"),
33.87 (C-2'), 32.75 (C-2"), 31.64 (C-3"), 27.43 (C-4'), 27.43 (C-5'), 23.65 (C-1').
MS-ESI m/z: 369.18 [M+H]+.
5.2.3.5. Synthesis of 3,4-dihydro-12-O-(5′-hydroxypentyl)-2,2-dimethyl-2H,6H
-pyrano[3,2-b]xanthen-6-one (P-3)
Followed the same procedure described in 5.2.3.3. Led to light yellow powder
0.327 g, yield 85.6%. M.p.: 155-157℃; 1H-NMR (CDCl3, 400 MHz) δ: 8.37 (1H, dd,
J = 8.0 and 1.6 Hz, H-8), 7.76 (1H, ddd, J = 7.6, 8.0, 1.6 Hz, H-6), 7.69 (1H, s, H-1),
7.57 (1H, dd, J = 8.0 and 1.0 Hz, H-5), 7.38 (1H, ddd, J = 7.6, 7.6, and 1.0 Hz, H-4),
5.76 (1H, s, OH), 4.19 (2H, t, J = 2.6 Hz, H-1"), 3.48 (2H, t, J = 2.6 Hz, H-5"), 2.96
(8H, m, H-2', 2", 3", 4"), 2.07 (2H, t, J = 2.6 Hz, H-1'), 1.45 (6H, s, H-4', 5');
13C-NMR (CDCl3, 100 MHz) δ: 178.34 (C-9), 158.57 (C-4b), 148.65 (C-3), 145.59
20

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(C-4a), 136.67 (C-4), 134.58 (C-6), 128.89 (C-8), 125.85 (C-7), 123.92 (C-1), 120.34
(C-8a), 119.68 (C-2), 119.63 (C-5), 117.51 (C-8b), 76.96 (C-3'), 63.67 (C-1"), 58.45
(C-5"), 33.99 (C-2'), 32.94 (C-2"), 31.86 (C-3"), 29.86 (C-4"), 27.66 (C-4'), 27.66
(C-5'), 23.59 (C-1'); MS-ESI m/z: 383.21 [M+H]+.
5.2.3.6. Synthesis of 3,4-dihydro-12-O-(6′-hydroxyhexyl)-2,2-dimethyl-2H,6H
-pyrano[3,2-b]xanthen-6-one (P-4)
Followed the same procedure described in 5.2.3.3. Led to light yellow powder
1
0.329 g, yield 83.1%. M.p.: 153-155 ; H-NMR (CDCl3, 400 MHz) δ: 8.31 (1H, dd,
J = 8.0 and 1.6 Hz, H-8), 7.72 (1H, ddd, J = 7.6, 8.0, 1.6 Hz, H-6), 7.48 (1H, s, H-1),
7.46 (1H, dd, J = 8.0 and 1.0 Hz, H-5), 7.54 (1H, ddd, J = 7.6, 7.6, and 1.0 Hz, H-4),
5.56 (1H, s, OH), 4.26 (2H, t, J = 2.6 Hz, H-1"), 3.57 (2H, t, J = 2.6 Hz, H-6"), 2.92
(10H, m, H-2', 2", 3", 4", 5"), 2.11 (2H, t, J = 2.6 Hz, H-1'), 1.41 (6H, s, H-4', 5');
13C-NMR (CDCl3, 100 MHz) δ: 179.02 (C-9), 158.90 (C-4b), 149.25 (C-3), 145.93
(C-4a), 137.33 (C-4), 135.08 (C-6), 129.31 (C-8), 126.22 (C-7), 124.38 (C-1), 120.80
(C-8a), 119.95 (C-2), 119.97 (C-5), 117.96 (C-8b), 77.89 (C-3'), 64.32 (C-1"), 58.96
(C-6"), 34.62 (C-2'), 32.41 (C-2"), 31.49 (C-3"), 29.67 (C-4"), 28.48 (C-4"), 27.53
(C-4'), 27.53 (C-5'), 22.97 (C-1'); MS-ESI m/z: 397.23 [M+H]+.
5.2.4. Synthesis of DMXAA-pyranoxanthone hybrids
5.2.4.1. Synthesis of 3,4-dihydro-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one
-12-O-propyl 5,6-dimethylxanthone-4-acetate (D-P-1)
To a 100-ml flask, DMXAA (0.339 g, 1.2 mmol), DCC (0.248 g, 1.2 mmol) and
DMAP (15 mg, 0.12 mmol) were dissolved in 30 ml dried DMF. Compound P-1
(0.354 g, 1.0 mmol) in 10 ml dried DMF was then added dropwise with stirring. The
reaction was lasted at r. t. for 24 h. After the finish of reaction, the mixture was pulled
into 50 ml ice-water with violently stirring for 20 min. The precipitates were collected
by filtrate, and were purified by flash column liquid chromatography led to white
1
powder 0.324 g, yield 52.4%, purity 97.4%. Mp: 205-206℃; H NMR (DMSO-d6,
500 MHz) δ: 8.01-8.04 (m, 3H, H-1, 7′, 9′), 6.85-6.87 (m, 3H, H-3, 8, 10′), 6.73-6.75
21

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(m, 2H, H-5′, 8′), 6.28-6.31 (m, 1H, H-2), 6.21-6.24 (m, 1H, H-7), 4.00-4.03 (m, 2H,
H-5′′), 3.98 (m, 2H, H-3′′), 3.71 (s, 2H, H-1′′), 3.34-3.36 (m, 2H, H-4′′), 2.76-2.79 (m,
2H, H-4′), 2.62-2.64 (m, 2H, H-3′), 1.78-1.81 (s, 6H, 5,6-CH3), 1.41-1.44 (s, 6H,
2′-CH3); 13C NMR (DMSO-d6, 125 MHz) δ: 181.36 (C-9), 179.08 (C-6′), 176.54
(C-2′′), 166.76 (C-11a′), 164.13 (C-4b), 161.70 (C-10a′), 158.32 (C-3), 156.63 (C-4a),
151.97 (C-9′), 136.51 (C-4), 135.63 (C-6), 134.17 (C-12a′), 129.89 (C-12′), 128.48
(C-8), 127.48 (C-10′), 126.45 (C-8′), 125.63 (C-9′), 125.45 (C-7), 124.63 (C-4a′),
124.19 (C-5′), 123.39 (C-7′), 122.09 (C-5a′), 121.23 (C-8a), 118.20 (C-2), 104.48
(C-6a′), 98.03 (C-5), 93.83 (C-8b), 79.88 (C-2′), 68.93 (C-5′′), 64.24 (C-3′′), 34.07
(C-1′′), 33.98 (C-3′), 32.85 (C-4′), 28.32 (C-4′′), 25.17 (2×2′-CH3), 18.65 (6-CH3),
16.43 (5-CH3); MS-ESI m/z: 619.23 [M+H]+; HRMS (m/z): calc. for [C38H34O8+H]+
619.2332, found 619.2338.
5.2.4.2. Synthesis of 3,4-dihydro-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one
-12-O-butyl 5,6-dimethylxanthone-4-acetate (D-P-2)
Followed the same procedure described in 5.2.4.1. Led to 0.324 g, yield 51.3%,
purity 98.4%. M. p.: 209-211℃; 1H NMR (DMSO-d6, 500 MHz) δ: 8.13-8.16 (m, 3H,
H-1,7′, 9′), 6.96-6.99 (m, 3H, H-3, 8, 10′), 6.81-6.87 (m, 2H, H-5′, 8′), 6.40-6.44 (m,
1H, H-2), 6.31-6.36 (m, 1H, H-7), 4.13 (m, 2H, H-6′′), 4.10 (m, 2H, H-3′′), 3.74 (s,
2H, H-1′′), 3.49-3.429 (m, 2H, H-5′′), 3.03-3.08 (m, 2H, H-4′′), 2.81-2.86 (m, 4H,
H-3′, 4′), 1.90-1.95 (s, 6H, 5,6-CH3), 1.51-1.56 (s, 6H, 2′-CH3); 13C NMR (DMSO-d6,
125 MHz) δ: 182.23 (C-9), 179.95 (C-6′), 177.41 (C-2′′), 167.63 (C-11a′), 165.00
(C-4b), 162.57 (C-10a′), 159.19 (C-3), 157.50 (C-4a), 152.84 (C-9′), 137.38 (C-4),
136.50 (C-6), 135.04 (C-12a′), 130.76 (C-12′), 129.35 (C-8), 128.35 (C-10′), 127.33
(C-8′), 126.50 (C-9′), 126.32 (C-7), 125.50 (C-4a′), 124.26 (C-5′), 122.96 (C-7′),
122.68 (C-5a′), 122.10 (C-8a), 119.08 (C-2), 105.35 (C-6a′), 98.91 (C-5), 94.70
(C-8b), 80.75 (C-2′), 69.80 (C-6′′), 65.11 (C-3′′), 34.94 (C-1′′), 34.85 (C-3′), 33.72
(C-5′′), 33.37 (C-4′′), 26.23 (2′-CH3), 26.04 (2′-CH3), 16.88 (6-CH3), 14.69 (5-CH3);
MS-ESI m/z: 633.22 [M+H]+
;
HRMS (m/z): calc. for [C39H36O8+H]+ 633.2488, found
633.2492.
22

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5.2.4.3. Synthesis of 3,4-dihydro-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one
-12-O-pentyl 5,6-dimethylxanthone-4-acetate (D-P-3)
Followed the same procedure described in 5.2.4.1. Led to white powder 0.32 g,
1
yield 49.6%, purity 99.2%. M. p.: 211-213℃; H NMR (DMSO-d6, 500 MHz) δ:
8.15-8.11 (m, 3H, H-1, 7′, 9′), 6.97-6.94 (m, 3H, H-3, 8, 10′), 6.86-6.81 (m, 2H, H-5′,
8’), 6.45-6.41 (m, 1H, H-2), 6.36-6.32 (m, 1H, H-7), 4.10 (m, 2H, H-7′′), 4.07 (m, 2H,
H-3′′), 3.72 (s, 2H, H-1′′), 3.46-3.41 (m, 2H, H-6′′), 2.99-2.95 (m, 4H, H-3′, 4′′),
2.87-2.92 (m, 4H, H-4′, 5′′), 1.90-1.95 (s, 6H, 5,6-CH3), 1.50-1.54 (s, 6H, 2′-CH3);
13C NMR (DMSO-d6, 125 MHz) δ: 182.95 (C-9), 180.67 (C-6′), 178.14 (C-2′′),
168.35 (C-11a′), 165.72 (C-4b), 163.30 (C-10a′), 159.92 (C-3), 158.22 (C-4a), 153.56
(C-9′), 138.10 (C-4), 137.23 (C-6), 135.76 (C-12a′), 131.48 (C-12′), 130.08 (C-8),
129.07 (C-10′), 128.05 (C-8′), 127.22 (C-9′), 127.04 (C-7), 126.23 (C-4a′), 125.79
(C-5′), 124.99 (C-7′), 123.68 (C-5a′), 122.83 (C-8a), 119.80 (C-2), 106.08 (C-6a′),
99.63 (C-5), 9542 (C-8b), 81.47 (C-2′), 70.52 (C-7′′), 65.83 (C-3′′), 35.66 (C-1′′),
35.57 (C-3′), 34.44 (C-4′), 29.92 (C-6′′), 26.95 (C-4′′), 26.76 (C-5′′), 26.76
(2×2′-CH3), 17.61 (6-CH3), 15.42 (5-CH3); MS-ESI m/z: 647.28 [M+H]+; HRMS
(m/z): calc. for [C40H38O8+H]+ 647.2645, found 647.2642.
5.2.4.4. Synthesis of 3,4-dihydro-2,2-dimethyl-2H,6H-pyrano[3,2-b]xanthen-6-one
-12-O-hexyl 5,6-dimethylxanthone-4-acetate (D-P-4)
Followed the same procedure described in 5.2.4.1. Led to white powder 0.319 g,
1
yield 48.3%, purity 98.8%. M.p.: 215-217℃; H NMR (DMSO-d6, 500 MHz) δ:
8.14-8.10 (m, 3H, H-1, 7′, 9′), 6.96-6.92 (m, 3H, H-3, 8, 10′), 6.85-6.80 (m, 2H, H-5′,
8’), 6.38-6.33 (m, 1H, H-2), 6.35-6.30 (m, 1H, H-7), 4.10 (m, 2H, H-8′′), 4.07 (m, 2H,
H-3′′), 3.76 (s, 2H, H-1′′), 3.46-3.44 (m, 4H, H-3′, 7′′), 2.99-2.95 (m, 4H, H-4′′,6′′),
2.92-2.86 (m, 4H, H-4′, 5′′), 1.92-1.88 (s, 6H, 5,6-CH3), 1.48-1.53 (s, 6H, 2′-CH3);
13C NMR (DMSO-d6, 125 MHz) δ:183.25 (C-9), 180.98 (C-6′), 178.44 (C-2′′),
168.65 (C-11a′), 166.02 (C-4b), 163.60 (C-10a′), 160.22 (C-3), 158.52 (C-4a), 153.86
(C-9′), 138.40 (C-4), 137.53 (C-6), 136.07 (C-12a′), 131.79 (C-12′), 130.38 (C-8),
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129.38 (C-10′), 128.35 (C-8′), 127.52 (C-9′), 127.35 (C-7), 126.53 (C-4a′), 126.09
(C-5′), 125.29 (C-7′), 123.98 (C-5a′), 123.13 (C-8a), 120.10 (C-2), 106.38 (C-6a′),
99.93 (C-5), 95.73 (C-8b), 81.78 (C-2′), 70.82 (C-6′′), 66.14 (C-3′′), 35.96 (C-1′′),
35.88 (C-3′), 34.74 (C-8′′), 34.40 (C-4′′), 29.33 (C-7′′), 26.95 (C-6′′), 27.25 (C-5′′),
26.06 (2×2′-CH3), 17.91 (6-CH3), 15.72 (5-CH3); MS-ESI m/z: 661.31 [M+H]+;
HRMS (m/z): calc. for [C41H40O8+H]+ 662.2801, found 661.2805.
5. 3. Biological section
5.3.1. Cell culture
All the cell lines were grown in specific media supplemented with 10% fetal bovine
serum (FBS, Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen,
Carlsbad, CA, USA). The cells were grown in a 5% CO2 humidified atmosphere in
incubators maintained at 37◦C.
5.3.2. Cell proliferation assay
3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was
used to detect inhibition of cellular proliferation mediated by the drugs. This assay was
applied to all cell lines. The process was describe below: Cells in suspension were
plated in 96-well plates at a density of 5×103 cells/well and cultured for 24 h. Then the
medium was replaced with the respective medium containing drugs at different
concentrations and incubated for 24 h. The final DMSO concentration in all
experiments was less than 0.1% in medium. The concentration range of tested samples
was 0-200 µM and two-fold serial dilutions were applied. Afterwards, 10 µL MTT
solution (5 mg/ml) was added to each well, and the plate was incubated for an
additional 4 h. The absorbance of the converted dye in living cells was measured at a
wavelength of 570 nm using a microplate reader (Bio-Rad; Hercules, CA, USA) after
100 µL of DMSO added. IC50 values were determined by the nonlinear multipurpose
curve fitting program GraphPad Prism. All of the tests were repeated at least 3 times.
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5.3.3. Apoptosis and necrosis assay
HepG-2 cells (2×105 cells/ml) were plated in 6-well plates and then treated with
either vehicle or 0.2 µM of DMXAA (D), pyranoxanthone (P), D+P in 1:1 mol ratio,
and D-P-4. The cells were incubated at 37 ℃, 5% CO2 for 24 h. Then the cells were
collected by centrifugation at r.t. and washed twice with ice-cold PBS. Afterwards, the
cells were suspended in 100 µL annexin V binding buffer and 5 µL each of annexin V
and PI were added to these samples. Next, these samples were incubated for 30 min at
room temperature and then assayed by flow cytometric analysis (FACScan, Bection
Dickinson, San Jose, CA). All of the tests were repeated at least 3 times.
5.3.4. Western Blot
HepG-2 cells were collected and washed with PBS after the treatment with 0.2
µM of P, D+P (1:1 mol ratio), D-P-4, respectively. Then, the cells were lysed with
RIPA buffer for 45 min on ice and then centrifuged at 12000 g at 4°C for 15 min. Then
the total cellular protein were collected and the nuclear proteins were extracted using a
nuclear and cytoplasmic extraction kit. The protein concentration was measured using a
BCA protein assay kit. Equal amounts of protein (30 µg) were separated via 10-15%
gradient SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The
membranes were blocked with 5% BSA at room temperature for 1 h, incubated with
primary antibodies for at least 16 h at 4◦C, and then washed and incubated with
HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were
visualized using enhanced chemiluminescence detection reagents (Bio-Rad, USA).
The resulting images were scanned using a scanner (Epson V330 Photo, Japan).
5.3.5. Cell cycle assay
HepG-2 cells (2×105 cells/ml) were seeded into 6-well plates and treated with
vehicle or D, P, D+P (1:1 mol ratio), and D-P-4 at a dose of 0.2 µM, respectively for
28 h. Then the cells were collected and washed twice with PBS and fixed in cold 70%
ethanol (-20°C) for 12 h. The ethanol were carefully removed by centrifugation and the
cells were suspended in 1 ml staining reagent (100 mg RNase+50 mg PI/ml) and kept
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in darkness for 40 min at r.t. Cell cycle analysis was tested via a flow cytometry (BD
FACS Calibur, Franklin Lakes, CA, USA) with a excitation wavelength at 605 nm.
5.3.6. Stability of D-P-4
D-P-4 was dissolve in PBS to be a stock solution with a concentration of 10 µM/l.
Then l ml D-P-4 solution and 4 ml serum (Kunming mice from the animal research
center at medical laboratory animal center of Guangdong Province in China) were
suspended together for 1 min, and the suspension was put into the incubator at 37°C.
At the set time point, 10 µl sample was taken out, respectively, and pretreated with
methanol and perchloric acid 20% to eliminate the proteins. The mixture was
centrifuged at 10000 r/min for 5 minutes. Then the supernate was submitted for
RP-HPLC analysis. The instrument used was Agilent 1200 (Agilent Co., Germany).
Column: COSMOSIL 5C18-MS-II, 4.6×250 mm; Flow rate: 1.0 ml/min; Wavelength:
254 nm; Eluant: methanol : H2O = 55 : 45 (isocratic).
The content percentage of D-P-4 was set 100% at 0 h time point. The content
percentage at any time point was calculated as following: At/A0×100%, where At and
A0 are the peak area at t time point and 0 h time point, respectively. Each experiments
was repeated 4 times.
5.3.6. Statistical analysis
All data are expressed as the mean ± standard deviation (SD) of three independent
experiments. Statistical significance was assessed using Student’s t-test (for
comparisons of two treatment groups) or one-way ANOVA(for comparisons of three or
more groups). P-values < 0.05 were considered statistically significant.
Acknowledgements
This research was financially supported by Guangdong Provincial Project of
Science and Technology (No. 2015A030311012), the Guangzhou Project of Science
and Technology (No. 201607010215), and the Fundamental Research Funds for the
Central Universities (11615323).
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