Incorporation of nitric oxide donor into 1,3-dioxyxanthones leads to synergistic anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2018.03.072

Fifty 1,3-dioxyxanthone nitrates (4a ～ i-n, n = 1–6) were designed and synthesized based on molecular similarity strategy. Incorporation of nitrate into 1,3-dioxyxanthones with electron-donating groups at 6–8 position brought about synergistic anticancer effect. Among them, compound 4g-4 was confirmed the most active agent against HepG-2 cells growth with an IC₅₀ of 0.33 ± 0.06 μM. It dose-dependently increased intramolecular NO levels. This activity was attenuated by either NO scavenger PTIO or mitochondrial aldehyde dehydrogenase (mtADH) inhibitor PCDA. Apoptosis analysis indicated different contributions of early/late apoptosis and necrosis to cell death for different dose of 4g-4. 4g-4 arrested more cells on S phase. Results from Western Blot implied that 4g-4 regulated p53/MDM2 to promote cancer cell apoptosis. All the evidences support that 4g-4 is a promising anti-cancer agent.

Full text of DOI:10.1016/j.ejmech.2018.03.072

Accepted Manuscript
Incorporation of nitric oxide donor into 1,3-dioxyxanthones leads to synergistic
anticancer activity
Jie Liu, Cao Zhang, Huailing Wang, Lei Zhang, Zhenlei Jiang, Jianrun Zhang, Zhijun
Liu, Heru Chen
PII:
S0223-5234(18)30315-5
10.1016/j.ejmech.2018.03.072
EJMECH 10337
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 January 2018
Revised Date: 24 March 2018
Accepted Date: 24 March 2018
Please cite this article as: J. Liu, C. Zhang, H. Wang, L. Zhang, Z. Jiang, J. Zhang, Z. Liu, H. Chen,
Incorporation of nitric oxide donor into 1,3-dioxyxanthones leads to synergistic anticancer activity,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.072.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Incorporation of nitric oxide donor into 1,3-dioxyxanthones leads to
synergistic anticancer activity
a,d
a
b
a
a
a
Jie Liu , Cao Zhang , Huailing Wang , Lei Zhang , Zhenlei Jiang , Jianrun Zhang ,
a
*a, c
Zhijun Liu , Heru Chen
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
5
c
10641, China
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New
Drugs Research,Jinan University, Guangzhou 510632, P. R. China
d
Department of Allergy, The Third Affiliated Hospital of Shenzhen University, Shenzhen 518020, 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
1

ACCEPTED MANUSCRIPT
Abstract Fifty 1,3-dioxyxanthone nitrates (4a~i-n, n = 1~6) were designed and
synthesized based on molecular similarity strategy. Incorporation of nitrate into
1
,3-dioxyxanthones with electron-donating groups at 6~8 position brought about
synergistic anticancer effect. Among them, compound 4g-4 was confirmed the most
active agent against HepG-2 cells growth with an IC50 of 0.33± 0.06 µM. It
dose-dependently increased intramolecular NO levels. This activity was attenuated by
either NO scavenger PTIO or mitochondrial aldehyde dehydrogenase (mtADH)
inhibitor PCDA. Apoptosis analysis indicated different contributions of early/late
apoptosis and necrosis to cell death for different dose of 4g-4. 4g-4 arrested more
cells on S phase. Results from Western Blot implied that 4g-4 regulated p53/MDM2
to promote cancer cell apoptosis. All the evidences support that 4g-4 is a promising
anti-cancer agent.
Keywords: xanthones; nitrates; anti-cancer; synergistic effect; p53/MDM2
2

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1
. Introduction
Nitric oxide (NO) is an omnipresent intercellular messenger in all vertebrates. It
is an important signaling and/or effector molecule involved in various physiological
and pathophysiological processes [1]. The biological production of NO is also very
important for nonspecific host defense. However, quite intriguing, in one hand,
overproduction of NO may create a microenvironment that enhances cell proliferation,
survival, and migration, as well as angiogenesis, thereby promotes tumor
development [2]. This observation is supported by the evidence that inducible nitric
oxide synthase (iNOS) knockout mice had an 80% reduction of urethane-induced lung
tumor formation [3]; While in the other hand, high levels of NO have strong
antitumor activity by inducing tumor cell apoptosis, inhibiting tumor metastasis, and
sensitizing drug-resistant tumor cells to chemotherapy, radiation, and immunotherapy
in vitro and in vivo [4-7].
It is well known that gaseous NO itself is generally ineffective when directly
delivered to tumor because of its short half-life and chemical instability [8]. Usually,
NO-donors are chosen as surrogates for NO in anticancer investigation [9]. We are
happy to see that numerous NO-donating hybrids reported so far were shown with
enhanced antitumor activity than sole NO donors, parent drugs and/or their
combinations [10-15]. Of particularly interest, NO-donating antitumor drugs generally
do not induce any drug resistance in tumor cells [12, 16].
As one of the common malignancies around the world, hepatocellular carcinoma
(HCC) account for over 80% of all liver cancers with extremely poor prognosis [17].
Recent investigations showed that the level of NO and the relative mRNA and protein
expression levels of iNOS in HCC were significantly lower than those in adjacent
non-tumor liver tissues. It was suggested that the decreased level of NO/iNOS may
partially contribute to overexpression of the mutant p53 protein and excessive
proliferation, which may be a potential mechanism in the development and
progression of HCC [18].
To our most concern, some xanthones were reported with anticancer activity and
3

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involved in the adjustment of NO/iNOS.
For example, DMXAA
(5,6-dimethylxanthone-4-acetic acid) is the first reported small-molecule vascular-
disrupting agent to enter phase III clinical trial stage [19]. Its action on vascular
endothelial cells seems to involve a cascade of events leading to induction of tumour
haemorrhagic necrosis. These events include both direct and indirect effects, the latter
involving the release of further vasoactive agents, such as serotonin, tumour necrosis
factor (TNF), other cytokines, and NO from host cells [20]. Another example is
mangiferin (MA), which is a naturally occurring 1,3-dihydroxyanthone chemically
named as 1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside. MA was shown with
antitumor activity [21]. It decreased mRNA levels of both iNOS and TNF-α in
activated macrophages [22].
Based on the principle of molecular similarity, which means similar molecule may
have similar biological activity [23], we postulated that some 1,3-dioxyxanthones may
possess similar function as MA, which might play a role as anticancer agent to
regulate NO/iNOS. Furthermore, when NO donor is incorporated into these xanthones,
it may probably bring about synergistic effects to enhance their anticancer activity.
Therefore, in the current paper, a series of 1,3-dioxyxanthone NO donor hybrids
were designed and synthesized. The screening of the inhibition activity of these
hybrids against three human cancer cell lines were carried on. Furthermore, the
mechanism of how this candidate hybrid induces the death of HepG-2 liver cancer
cells will also be disclosed thereafter.
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2
. Chemistry
Outlined in Scheme 1, the preparation of 1,3-dioxyxanthone nitrates 4a~i-n (n =
1
, 2, 3, 4, 5, 6) were started with the syntheses of 1,3-dihydroxyxanthones. That is, in
step i, by applying a modified method previously described [24], in which anhydrous
zinc chloride (ZnCl ) and phosphorus oxychloride (POCl ) were used as both catalyst
2
3
and solvent under the assistance of microwave radiation, condensation of salicyclic
acids 1a~i with 1,3,5-trihydroxybenzene (phloroglucinol), respectively, were
successfully carried on. It resulted in 1,3-dihydroxyxanthones. Yields were 70-90%.
Scheme 1. Syntheses of 1,3-dioxyxanthone nitrates. Conditions and Agents: (i) ZnCl /POCl ,
2
3
microwave (MW), 40 min; (ii) Br(CH2)n+1Br/K CO (n = 1, 2, 3, 4, 5, 6), DMF, r.t.; (iii)
2
3
AgNO /CH CN/dark.
3
3
Because 1-hydroxyl of compounds 2a~i may form intramolecular hydrogen bond
with 9-carbonyl in a manner of stable six-member ring, it is less reactive in alkylation
than 3-hydroxyl. Therefore, selective bromoalkylation at 3-O-position of
1
,3-dihydroxyxanthone was achieved under a mild conditions, in which potassium
carbonate (K CO ) was applied as base, leading to series of
-hydroxy-3-O-bromoalkylxanthones (3a~i-n, n = 1, 2, 3, 4, 5, 6). Yields were quite
a
a
2
3
1
high, which were almost 100%. This is the reason why we did not purify the crudes in
this step. The mixtures were used directly in step iii.
The title compounds 4a~i-n (n = 1, 2, 3, 4, 5, 6) were obtained in a yield of
2
6%-65% by the reactions of 3a~i-n with AgNO in dark, respectively. Formation of
3
AgBr was the driving force of this reaction. During this process, carbenium ion
5

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intermediate probably was produced through an intramolecular S 2 type mechansim.
N
The subsequent reaction of oxocarbenium with the nitrate ion leads to 4a-i~n. The
putative reaction pathway were shown below.
Scheme 2. Putative mechanism from 3a-i~n to 4a-i-n.
The structures of all 50 nitrates (4a~i-n, n = 1, 2, 3, 4, 5, 6) were characterized
by NMR and MS. Their purities were analyzed by RP-HPLC method.
3
. Results and discussion
3
.1. Growth inhibitory effects of 1,3-dioxyxanthone nitrates against human cancer cell
lines
Considering that breast cancer and liver cancer are very common cancer types,
we chose Michigan Cancer Foundation-7 (MCF-7), human breast MDA-MB-231, and
human liver Hep-G2 cells 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 1,3-dioxyxanthone nitrates on the growth inhibition
against cancer cells. The results were listed in Table 1. It can be seen that most of
1
,3-dioxyxanthone nitrates were inactive on MCF-7 cell line, except 4d-5, which
showed mild inhibition activity with IC value of 13.32 ± 1.51 µM. Interestingly, for
5
0
both HepG-2 and MDA-MB-231 cell lines, the incorporation of 3-O-alkyl nitrate
generally enhanced the anticancer activity of 1,3-dioxyxanthones with
electron-donating substituent groups at 6~8 positions (Entries 1~18 and 32~59);
However, when the substituent groups at 6~8 positions of 1,3-dioxyxanthones are
electron-withdrawal (4d~e), no enhancing inhibition effect was found (Entries 19~25
and 26~31). For each series of compounds, e.g. 4a-n (n = 2, 3, 4, 5, 6), the enhanced
inhibition effect increased with the elongation of alkyl chain. It was identified that
5
~6 carbon alkyl chain was the optimal length for increasing anticancer activity.
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We are pleased to find that 4g-4 and 4g-5 nitrates (Entries 43 and 44), respectively
exhibited significant inhibition enhancement against HepG-2 and MDA-MB-231.
Compared with 4g, the inhibition activity of 4g-4 against HepG-2 was increased by 98
times with an IC50 value of 0.33 ± 0.06 µM, which was the most active compound
against HepG-2 cancer cell amongst all the tested nitrates; While 4g-5 showed the
best inhibition activity against MDA-MB-231 cancer cells with an IC value of 0.45
5
0
±
0.04 µM, which was 147 times more active than 4g itself.
It was found that, NO donors themselves, which are HO(CH2)n+1ONO (Entries
2
6
1-65), were not active agents against the growth of HepG-2, MDA-MB-231, and
MCF-7 cancer cells. After the release of NO by enzyme, compound 4g-4 changed to
-hydroxy-3-(5-hydroxypentyl)oxy-8-methoxyxanthone (HHPX) (Entry 60, Table 1).
1
This compound was confirmed not an very active agent against the growth of all the
tested cancer cells, in which the IC value was close to that of 4g. This implied that
5
0
the enhanced inhibition activity of 4g-4 against HepG-2 cancer cell line was not from
the zymolytic product HHPX, but from the integrated molecule.
Table 1. Cell growth inhibition of 1,3-dioxyxanthone nitrates against cancer cell lines
a
IC50 (µM)
Entry Compds
HepG2
MDA-MB-231
MCF-7
#
#
#
#
#
##
##
1
2
3
4
5
6
7
8
9
2a
12.71 ± 1.82
12.77 ± 1.75
10.56 ± 1.26
2.953 ± 0.19
1.281 ± 0.33
8.62 ± 0.92
4.22±0.11
#
#
#
4a-2
4a-3
4a-4
4a-5
4a-6
2b
78.36 ± 9.27
62.78 ± 7.13
47.68 ± 7.26
#
#
62.87 ± 8.44
#
#
30.68 ± 4.70
39.45 ± 6.11
#
#
10.32 ± 1.26
35.13 ± 4.04
42.69 ± 7.25
> 200
#
#
#
8.76 ± 1.23
55.88 ± 7.41
#
93.21 ± 9.33
63.53 ± 6.59
52.08 ± 6.09
33.27 ± 3.66
13.59 ± 0.95
42.06 ± 5.11
> 200
> 200
#
4b-2
4b-3
4b-4
4b-5
4b-6
2c
35.56 ± 2.94
98.19 ± 10.05
83.25 ± 9.34
53.42 ± 7.12
#
#
22.28 ± 2.13
#
1
0
9.29 ± 1.01
#
#
##
#
1
1
2
3
4
5
6
7
3.05 ± 0.22
16.25 ± 1.82
> 200
41.13 ± 4.11
##
#
1
1
1
1
1
1
71.25 ± 6.33
> 200
#
#
#
#
4c-2
4c-3
4c-4
4c-5
33.04 ± 3.22
22.29 ± 2.68
9.910 ± 1.24
11.39 ± 1.18
133.7 ± 13.06
> 200
#
#
#
#
92.56 ± 9.63
143.4 ± 1.73
91.21 ± 9.17
53.73 ± 7.40
#
44.65 ± 5.33
#
18.49 ± 1.26
7

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#
#
#
#
#
#
#
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
4c-6
2d
15.25 ± 2.63
3.212 ± 0.20
43.06 ± 5.11
17.39 ± 1.29
10.09 ± 1.10
74.34 ± 7.56
112.3 ± 1.62
##
##
5.21 ± 0.43
33.27 ± 3.57
27.84 ± 2.59
11.22 ± 1.78
1.20±0.41
#
#
#
#
#
#
#
#
#
4d-1
4d-2
4d-3
4d-4
4d-5
4d-6
2e
74.32 ± 8.31
53.40 ± 6.43
42.92 ± 5.48
25.14 ± 3.17
13.32 ± 1.51
30.83 ± 4.23
#
#
#
#
#
##
5.32 ± 0.34
6.03 ± 0.52
8.13 ± 1.12
9.22 ± 1.01
7.79 ± 0.86
9.74 ± 0.87
#
#
#
10.25 ± 1.76
#
#
0.46 ± 0.04
3.41±0.12
#
#
#
#
#
#
#
4e-2
4e-3
4e-4
4e-5
4e-6
2f
13.96 ± 1.06
10.09 ± 1.10
43.56 ± 4.45
11.22 ± 1.78
11.05 ± 1.67
15.01 ± 1.68
> 200
#
#
#
#
42.92 ± 5.48
73.24 ± 6.32
43.46 ± 5.16
35.85 ± 3.46
#
#
#
#
2.65 ± 0.30
1.21 ± 0.17
7.28 ± 1.12
#
#
9.27 ± 1.65
#
#
#
#
#
#
11.71 ± 1.11
11.62 ± 1.22
90.90 ± 9.98
83.88 ± 7.84
56.60 ± 6.07
11.23±1.02
#
#
4f-1
4f-2
4f-3
4f-4
4f-5
4f-6
2g
20.52 ± 3.347
12.31 ± 1.906
> 200
#
> 200
#
6.55 ± 0.75
> 200
#
#
#
#
#
#
4.08 ± 0.32
3.17 ± 0.34
5.24 ± 0.63
40.67 ± 3.889
> 200
#
#
23.46 ± 2.12
93.14 ± 9.23
#
#
43.54 ± 5.71
153.32 ± 10.21
> 200
#
#
#
#
#
32.52 ± 3.46
21.54 ± 2.62
13.60 ± 2.01
10.23 ± 1.41
65.98 ± 6.37
#
4g-1
4g-2
4g-3
4g-4
4g-5
4g-6
2h
11.22 ± 1.10
103.7 ± 12.65
101.9 ± 10.86
#
#
9.68 ± 0.88
#
##
#
6.25 ± 0.58
62.95 ± 8.19
#
#
#
#
##
#
0.33 ± 0.06
0.61 ± 0.08
5.26 ± 1.33
0.57 ± 0.06
33.07 ± 4.80
#
##
#
0.45 ± 0.04
31.03 ± 4.33
#
##
#
4.29 ± 0.718
> 200
53.52 ± 7.21
#
122.8 ± 11.92
> 200
> 200
> 200
> 200
> 200
#
#
#
4h-1
4h-2
4h-3
4h-4
4h-5
4h-6
2i
91.67 ± 8.88
129.7 ± 11.66
#
#
53.59 ± 6.03
112.1 ± 9.30
#
#
#
21.16 ± 2.52
87.39 ± 10.69
#
6.353 ± 1.08
45.58 ± 5.56
#
#
#
1.83 ± 0.32
19.76 ± 1.95
98.16 ± 10.05
#
#
#
#
13.15 ± 1.46
> 200
67.58 ± 5.86
163.35 ± 9.23
> 200
> 200
> 200
> 200
> 200
#
#
#
4i-1
4i-2
4i-3
4i-4
4i-5
4i-6
159.6 ± 12.36
107.9 ± 11.29
#
116.2 ± 10.77
82.72 ± 8.86
#
#
96.03 ± 8.32
66.69 ± 7.08
#
#
#
62.31 ± 6.51
41.37 ± 4.91
111.6 ± 12.37
#
##
26.35 ± 3.66
10.55 ± 1.76
51.46 ± 6.12
> 200
> 200
#
#
76.15 ± 7.34
8

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#
#
#
6
0
23.65 ± 2.02
41.08 ± 5.11
>200
#
6
6
6
6
6
6
1
2
3
4
5
6
HO(CH ) ONO
127.81± 13.27
> 200
> 200
> 200
2
2
2
2
2
2
2
#
HO(CH ) ONO
110.98± 11.55
> 200
2
3
#
#
#
#
HO(CH ) ONO
90.79 ± 8.68
157.4 ± 17.83
140.3 ± 14.09
90.42 ± 10.50
48.42 ± 7.41
> 200
2
4
#
#
#
HO(CH ) ONO
72.80 ± 8.85
102.3 ± 10.54
89.73 ± 9.086
54.4±6.4
2
5
#
HO(CH ) ONO
52.55 ± 5.29
2
6
b
DMXAA
100.2 ± 11.21
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. Difference is considered significant at p < 0.05, p < 0.01 vs. control
group.
The current evidence support that the 4g-4 is probably a promising agent for the
treatment of cancer. Therefore the cytotoxicity against normal cell line from healthy
tissues must be evaluated. It was found that the cytotoxicities of 4g against human
liver cell line HL-7702 and mouse embryo fibroblast cell line NIH/3T3 are low, in
which the IC50 values are 486.9 ± 45.6 µM to HL-7702, and 476.5 ± 48.0 µM to
NIH/3T3. Although the incorporation of pentyl nitrate into 4g caused a little bit
increase in cytotoxicity, where the IC value was 376.9 ± 32.7 µM against HL-7702,
5
0
and 377.5 ± 32.9 µM against NIH/3T3. However, the cytotoxicity of 4g-4 against
normal cells was far less than that against cancer cell lines, in which the cytotoxicity
against HL-7702 cells is 1142 times less than that against HepG-2 cancer cells.
3
.2. Cell death induced by 4g-4
Because 4g-4 was confirmed as the promising agent against HepG-2 (Table 1),
together with the fact that HCC is the most common type of primary liver cancer in
adults, and is the most common cause of death in people with cirrhosis [25], we chose
HepG-2 cell line to explore the mechanisms involved in how this compound prevents
the cancer cell growth.
As we know, flow cytometry using propidium iodide (PI) and Annexin-V as dyes
to label viable and dead cells is one of the common techniques to study cell
apoptosis/necrosis, therefore it was applied here. As shown in Fig. 1, at a dose of 0.16,
9

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0
.33, and 0.66 µM, 4g-4 induced 4.6%, 76.6% and 86.8% HepG2 cells death,
respectively. Interestingly, treatment with middle dose of 4g-4 (0.33 µM) was found
causing more early apoptosis than that with high dose (0.66 µM), in which the rate
was 64.8%, and 45.1%, respectively. However, high dose of 4g-4 (0.66 µM) induced
far more late apoptosis/necrosis than middle dose (0.33 µM), where the rate was
4
1.7%, and 11.8%, respectively.
Fig. 1. 4g-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 0.16 (II),
0
.33 (III), and 0.66 µM (IV), 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 Apoptosis Detection Kit per manufacture’s protocol. And the quantitative results
were shown in (V). 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. Difference is
*
**
considered significant at p < 0.05, p < 0.01 vs. control group
In
-bromo-1,3-dihydroxyxanthone, which belongs to 1,3-dihydroxyxanthones, were
also confirmed the activity to induce cell death. Based on this fact, we suggested that
our
previous
work
[24],
the
reference
DMXAA
and
8
1
,3-dioxyxanthone scaffold in 4g-4 contributed to its ability to regulate cell death.
Nowadays, it is recognized that apoptosis is a highly regulated and controlled
process that confers advantages during an organism's lifecycle; while necrosis is a
form of traumatic cell death that results from acute cellular injury. 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. The exposition of
phosphatidylserine on the cell surface is an early marker of apoptosis, whereas it is
normally concentrated in the luminal layer of the cytoplasmic membrane [26]. It was
1
0

ACCEPTED MANUSCRIPT
displayed that different dose of 4g-4 promoted different rates of early, late
apoptosis/necrosis. We suggested that the reason might be the different NO content
released by different dose of 4g-4. Because different NO content may result in
different anticancer effect.
3
.3. NO release of 4g-4 and inhibition activity attenuated by PTIO and PCDA
To investigate the contribution of NO to the antiproliferative activity, NO-releasing
ability of 4g-4 in HepG-2 cells was detected by a Griess assay. As shown in Table 2,
treatment of 4g-4 caused higher levels of NO production in HepG-2 cells in a dose-
and time-dependent manner. It was found that the content of intramolecular NO came
to maximum after incubation of HepG-2 cells with 4g-4 for 24 h. After this time point,
NO levels reduced. At the same dose of 0.66 µM, NO donor HO(CH ) ONO also
2
5
2
increased intramolecular NO levels, but was shown with a little bit less active than
g-4. Interestingly, treatment of 0.66 µM 4g showed a slight activity to increase the
4
NO levels of HepG-2 cells, implying this 1,3-dioxyxanthone does possess a weak
ability to adjust NO levels.
It was of notice in Table 3 that either 4g-4 or HO(CH ) ONO also enhanced NO
2
5
2
levels in HL-7702 normal human liver cells. However, the releasing intramolecular
NO levels were far less than that in HepG-2 cells. We ascribed this phenomenon to
the different expression levels of mitochondrial aldehyde dehydrogenase (mtADH)
between HL-7702 and HepG-2 cells, in which mtADH was confirmed the key
enzyme to activate nitrates for releasing NO [27,28]. Obviously, mtADH expression
level is higher in HepG-2 cells than that in HL-7702 cells.
Table 2. Effects of NO produced by 4g-4 on HepG-2 cells
NO level (µM)
Groups
1
.0 h
3.0 h
6.0 h
12.0 h
24.0 h
48.0 h
60.0 h
Control (HepG-2) 12.9±1.5
12.8±1.5
13.3±1.4
12.6±1.1
14.8±1.6
12.7±1.8
15.4±1.5
12.8±1.7
14.0±1.4
12.8±1.7
12.9±1.4
12.8±1.5
12.7±1.3
#
#
#
##
#
#
#
0
.66 µM 4g
12.9±1.2
11

ACCEPTED MANUSCRIPT
0
.66 µM
HO(CH ) ONO
2
##
##
##
#
#
#
#
1
6.2±1.8
18.4±2.4
23.3±2.6
26.0±2.6
29.2±3.3
24.6±2.8
17.1±1.6
2
5
#
#
#
#
#
#
##
#
##
##
#
##
##
#
#
#
#
#
#
#
0
0
0
.16 µM 4g-4
.33 µM 4g-4
.66 µM 4g-4
15.6±1.7
19.3±1.9
21.6±2.1
18.8±2.1
23.2±2.1
25.7±2.7
22.9±2.3
29.6±2.8
31.4±3.2
25.7±2.6
31.5±3.0
36.0±3.6
26.2±2.8
35.1±3.4
39.1±4.1
21.3±2.5
26.6±2.9
28.1±2.9
15.7±1.5
18.2±1.7
20.2±2.2
#
##
#
##
Difference is considered significant at p < 0.05, p < 0.01 vs. control group; All values are
expressed as mean ± SD (n = 3).
Table 3. Effects of NO produced by 4g-4 on HL-7702 cells
NO level (µM)
Groups
1
.0
3.0
6.0
12.0
24.0
48.0
60.0
Control (HL-7702) 11.6±1.1
11..6±1.2
11.8±1.1
12.6±1.3
11.8±1.2
12.7±1.3
11.6±1.1
13.4±1.2
11.6±1.1
12.4±1.4
11.8±1.3
12.3±1.1
#
#
#
#
##
#
##
#
#
#
#
0
.66 µM 4g
.66 µM
HO(CH ) ONO
2
12.1±1.1
12.2±1.2
13.8±1.7
15.1±1.5
0
#
#
##
##
#
#
#
1
3.5±1.3
16.0±1.3
17.1±1.3
17.5±1.4
19.3±1.4
14.1±1.2
19.4±1.5
13.4±1.1
16.2±1.9
13.3±1.3
14.5±1.6
2
5
#
##
##
##
##
0
.66 µM 4g-4
14.2±1.1
#
##
Difference is considered significant at p < 0.05, p < 0.01 vs. control group; All values are
expressed as mean ± SD (n = 3).
Furthermore, compound 4g-4 was investigated for the effect of NO on its inhibitory
activity against HepG-2 cells. A NO scavenger carboxy-PTIO [29], which can
completely inhibit the effect of NO, was applied in the current study. We compared
the different inhibitory effects between treating HepG-2 cells with 0.66 µM 4g-4
alone and pretreating HepG-2 cells with set dose of PTIO following the treatment of
0
.66 µM 4g-4. It was displayed in Fig. 2A that PTIO dose-dependently attenuated the
inhibition activity of 4g-4, indicating the released NO did contribute to anticancer
effect.
1
2

ACCEPTED MANUSCRIPT
Fig. 2. PTIO (A) and PCDA (B) attenuated the inhibitory effect of 4g-4. (A) HepG-2 cells were
pretreated in triplicate with the set concentrations of carboxy-PTIO (0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0,
and 12.0 µM) for 1 h followed the treatment of 0.66 µM 4g-4 for 24 h; (B) HepG-2 cells were
pretreated in triplicate with the set concentrations of PCDA (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0,
1
6.0, 32.0, and 64.0 µM) for 2.5 h followed the treatment of 0.66 µM 4g-4 for 24 h. The results
are expressed as the percentage of cell survival relative to control cells. Difference is considered
#
significant at p < 0.05 vs. control group; Data are the mean value±SD obtained from three
independent experiments.
Further more, in order to confirm whether mtADH is involved in reducing nitrate to
release NO, a mtADH inhibitor, piperazine-1-carbothioic dithioperoxyanhydride
(
PCDA) [30,31] was used to probe the interaction. As disclosed in Fig. 2B, when the
dose was less than 8 µM, PCDA dose-dependently blocked the inhibition effect of
g-4; Interestingly, when the dose was greater than 8 µM, PCDA dose-dependently
4
inhibited the HepG-2 cells growth. This evidence implied that mtADH plays a key
role in reducing nitrate to NO. When the dose was less than 8 µM, PCDA mainly
acted as an inhibitor of mtADH, it blocked the release of NO from 4g-4, therefore
attenuated the inhibitory effect of 4g-4; However, when the dose becomes more than 8
µM, PCDA not only plays as an inhibitor of mtADH, but also an anticancer agent.
3
.4. Cell cycle analysis of 4g-4
To establish whether 4g-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. 3. Obviously,
compared with the control group, an increase in the S population was observed in
HepG-2 cells after the treatment with 4g-4 at a dose of 0.16, 0.33, and 0.66 µM,
1
3

ACCEPTED MANUSCRIPT
respectively. High dose of 4g-4 (0.66 µM) showed the best capability to arrest cells at
S state, in which the percentage of cells was 56.2%. Compared to the control group, It
increased S population by 93.1%.
This evidence implied that the cell cycle arrest is one of the inhibitory
mechanisms responsible for the anticancer activities of 4g-4. 4g-4 nitrate
dose-dependently altered the manner of cell cycle arrest to S state. We ascribed this to
the dose-dependently release of NO from 4g-4 nitrate.
Fig. 3. Cell cycle analysis of HepG-2 cells exposed to 4g-4. HepG-2 cells were pre-treated without
the addition of any samples, the control (C) (I); with a dose of 0.16 (II), 0.33 (III), and 0.66 µM
(IV) 4g-4, respectively for 24 h.. 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. Difference is considered significant at p < 0.05, p < 0.01 vs.
control group.
Quite interestingly, DMXAA and a 1,3-dihydroxyxanthones, 8-bromo-1,3-
dihydroxyxanthone were shown the ability to interrupt cell cycle. They increased the
G2/M population at a dose of 48.0 and 0.8 µM, respectively [24]. Therefore, the
ability of 4g-4 to interrupt cell cycle may ascribe to its 1,3-dioxyxanthone scaffold.
Generally, 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 observed evidence, it was concluded that 4g-4
may block the DNA replication and stop the cell cycle in S phase.
3
.5. Influence on the expression levels of proteins related to cell death by 4g-4
As we know, cell death includes apoptosis and necrosis. In order to explore how
1
4

ACCEPTED MANUSCRIPT
4
g-4 induced cell apoptosis, the expression levels of caspase-3, cleaved caspase-3,
caspase-9, cleaved caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP)
were determined by Western Blot. It was found that 4g-4 dose-dependently decreased
the levels of caspase-3, and caspase-9, respectively; while in the mean time
dose-dependently increased the expression of cleaved caspase-3, cleaved caspase-9
and cleaved PARP (Fig. 4 I-II). As a comparison, DMXAA and 8-bromo-1,3-
dihydroxyxanthone were reported the same functions in our previous work [24]
It is recognized today that caspase-3 is involved in the apoptotic process. It is
responsible for chromatin condensation and DNA fragmentation [32]; while caspase-9
is an initiator caspase [33]. 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 [34]
.
When PARP is cleaved by enzymes such as caspases or cathepsins, typically the
function of PARP is inactivated. Of no doubt, these data support that 4g-4 induce cell
apoptosis via the adjustment of caspase 3, caspase 9, and PARP, which closely
participate in programmed cell death.
Fig. 4. Effect of 4g-4 on proteins related to cell death. HepG-2 cells were treated with 4g-4 at a
1
5

ACCEPTED MANUSCRIPT
dose of 0, 0.16, 0.33, and 0.66 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. Difference is considered
*
**
significant at p < 0.05, p < 0.01 vs. control group.
Tumor protein p53 is recognized as a tumor suppressor which plays many roles
including the ability to induce cell cycle arrest, DNA repair, senescence, and
apoptosis [36,37]; 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 [38-40]. An appealing therapeutic
strategy for many wild-type p53 tumors with over expressed MDM2 is to restore p53
activity via inhibiting the p53/MDM2 interaction. We noticed that several xanthones,
including DMXAA and 8-bromo-1,3-dihydroxyxanthone, were reported as putative
small-molecule inhibitors of p53/MDM2 interaction [24,41]. Therefore, we examined
whether 4g-4 nitrate regulates p53/MDM2 interaction.
To our expect, it was found that 4g-4 up-regulated p53 expression in a
dose-dependent manner; while in the other hand, it down-regulated MDM2
expression, accordingly (Fig. 4 III-IV). Treatment of 0.66 µM 4g-4 changed the
p53/MDM2 ratio from 0.48 (control group) to 6.46. Based on this evidence, we
concluded that 4g-4 may regulate p53/MDM2 to promote cancer cell apoptosis.
4
. Conclusions
In a word, fifty 1,3-dioxyxanthone nitrates (4a~i-n, n = 1-6) were designed and
synthesized based on molecular similarity strategy. Their anticancer activities against
three human cells, Hep-G2, MDA-MB-231, and MCF-7 cell lines were determined.
4
g-4 was demonstrated as the most potent inhibitor against HepG-2 cell line with an
IC value of 0.33 ± 0.06; while 4g-5 was confirmed as the most potent agent against
5
0
MDA-MB-231 cell line with an IC value of 0.45 ± 0.04 µM.
5
0
The study of structure-activity relationship indicated that most of
,3-dioxyxanthone nitrates were inactive on MCF-7 cell line. For both the cases of
1
1
6

ACCEPTED MANUSCRIPT
HepG-2 and MDA-MB-231 cell lines, incorporation of 3-O-alkyl nitrate generally
enhanced the anticancer activity of 1,3-dihydroxyxanthones with electron-donating
substituent groups at 6~8 positions; However, when the substituent groups at 6~8
positions of 1,3-dihydroxyxanthones are electron-withdrawal, no enhanced inhibition
effect was found. Interestingly, the enhanced inhibition effect increased with the
elongation of alkyl chain. 5~6 Carbon alkyl chain was the optimal length for
increasing anticancer activity.
It was confirmed that 4g-4 can release high level of NO in HepG-2 cells. Its
inhibition activity was attenuated by either NO scavenger PTIO or mtADH inhibitor
PCDA.
4
g-4 induced HepG-2 cells death. The contribution of early apoptosis, necrosis,
and late apoptosis were different. It was found that middle dose of 4g-4 caused more
early apoptosis than that with high dose. However, high dose of 4g-4 induced far
more late apoptosis/necrosis than middle dose. Cell cycle analysis indicated that 4g-4
dose-dependently arrested more cells on S phase. Results in Western Blot indicated
that 4g-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. 4g-4 increases p53/MDM2 ratio to
promote cancer cell apoptosis. All the current evidences support that 4g-4 is a
promising agent for the treatment of liver cancer.
5
. Experimental section
5
.1. Materials
All chemicals, including salicylic acids (2a~i), 1,3,5-trihydroxybenzene, and
PTIO were purchased from Aldrich or Adamas. They were used directly 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 cancer cell line, MDA-MB-231, MCF-7 and Hep-G2 were obtained from the
Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and
1
7

ACCEPTED MANUSCRIPT
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
(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, caspase 3, caspase 9, cleaved-caspase 3, cleaved-caspase 9, cleaved PARP,
p53, MDM2, anti-mouse, and anti-rabbit horseradish peroxidase-conjugated secondary
antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).
5
5
.2. Synthetic process
.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.
1
8

ACCEPTED MANUSCRIPT
5
.2.2. Synthesis of 5,6-dimethylxanthone-4-acetic acid (DMXAA)
The synthesis was carried on referred to a procedure described in the literature
1
[
42]. Overall yield: 51% (four steps). Purity 98.6%. H NMR (DMSO-d , 300 MHz)
6
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 =
1
3
8
.0 Hz, 1H, H-2), 3.99 (s, 2H), 2.44 (s, 3H), 2.42 (s, 3H); C NMR (DMSO-d , 75
6
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),
20.5 (C-8b), 118.7 (C-8a), 35.4 (C-9), 20.1 (CH ), 11.0 (CH ); MS-ESI m/z: 283.2
(
1
3
3
+
[
M+H] .
5
.2.3. Synthesis of 1,3-dihydroxyxanthones (2a~i)
The synthesis refers to a procedure described in the literature [43]. To a 50-mL
flask 8 mL phosphorus oxychloride (POCl ) and anhydrous zinc chloride (6.8 g, 0.05
3
mol) were added. The suspension was stirred at 70°C until ZnCl was completely
2
dissolved into phosphorus oxychloride. The mixture was then cooled down to room
temperature (r.t.). Afterwards, salicylic acids (1a-i) (l.0 mmol) and
1
,3,5-trihydroxybenzene (1.1 mmol) were added, respectively, and the mixture was
heated with microwave reactor with a programmed procedure of 75°C for 30 min,
where the power was 200 W. Then 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.
5
.2.3.1. Synthesis of 1,3-dihydroxy-6-methyl-9H-xanthen-9-one (2a)
°
1
Light yellow powder, m.p. 193~195 C, yield 70.1 %. H NMR (DMSO-d , 300
6
MHz) δ: 10.85 (s, 1H, OH), 10.29 (s, 1H, OH), 7.96 (d, J = 8.1 Hz, 1H, H-8), 7.71 (d,
J = 2.1 Hz, 1H, H-5), 7.34 (t, J = 8.1 Hz, 1H, H-7), 6.43 (d, J = 1.8 Hz, 1H, H-4), 6.22
1
3
(
d, J = 1.8 Hz, 1H, H-2) , 2.42 (s, 3H, CH ). C NMR (DMSO-d , 75 MHz) δ: 180.4
3 6
1
9

ACCEPTED MANUSCRIPT
(C-9), 166.4 (C-3), 163.2 (C-1), 157.7 (C-4a), 154.1 (C-4b), 136.7 (C-6), 127.1 (C-8),
1
24.2 (C-7), 123.2 (C-8a), 120.1 (C-5), 102.4 (C-8b), 98.6 (C-2), 94.6 (C-4), 15.6
+
(
-CH ); MS-ESI m/z: 243.1 [M+H] .
3
5
.2.3.2. Synthesis of 1,3-dihydroxy-7-methoxy-9H-xanthen-9-one (2b)
°
1
Light yellow powder, m.p. 168~169 C, yield 76.2 %. H NMR (DMSO-d , 300
6
MHz) δ: 11.215 (s, 1H, OH), 10.23 (s, 1H, OH), 7.45 (d, J = 8.4 Hz, 1H, H-5), 7.41
dd, J = 8.4, 1.8 Hz, 1H, H-6), 7.24 (d, J = 1.8 Hz, 1H, H-8), 6.35 (d, J = 1.8 Hz, 1H,
(
13
H-1), 6.20 (d, J = 1.8 Hz, 1H, H-2), 3.65 (s, 3H, -OCH ); C NMR (DMSO-d , 75
3
6
MHz) δ: 179.4 (C-9), 165.3 (C-3), 162.5 (C-1), 157.2 (C-4a), 153.6 (C-7), 148.7
(C-4b), 124.1 (C-6), 120.1 (C-8a), 118.6 (C-5), 107.7 (C-8), 101.6 (C-8b), 97.5 (C-2),
+
9
3.4 (C-4), 55.8 (-OCH ); MS-ESI m/z: 259.1 [M+H] .
3
5
.2.3.3. Synthesis of 1,3,7-trihydroxy-9H-xanthen-9-one (2c)
°
1
Yellow powder, m.p. 179~181 C, yield 81.4 %. H NMR (DMSO-d , 300 MHz) δ:
6
1
7
1
2.86 (s, 1H, OH), 10.15 (s, 1H, OH), 9.23 (s, 1H, OH), 7.93 (d, J = 6.1 Hz, 1H, H-5),
.69 (d, J =6.1 Hz, 1H, H-6), 7.33 (td, J = 8.4, 2.1 Hz, 1H, H-8), 6.42 (d, J = 1.8 Hz,
1
3
H, H-4), 6.22 (d, J = 1.8 Hz, 1H, H-2); C NMR (DMSO-d , 75 MHz) δ: 180.5
6
(C-9), 166.2 (C-8), 163.5 (C-3), 160.6 (C-1), 157.6 (C-4a), 156.8 (C-4b), 136.3 (C-7),
110.4 (C-8a), 109.7 (C-5), 107.0 (C-6), 102.8 (C-8b), 98.6 (C-4), 93.8 (C-2); MS-ESI
+
m/z: 245.1 [M+H] . The data were the same with reference [24].
5
.2.3.4. Synthesis of 7-chloro-1,3-dihydroxy-9H-xanthen-9-one (2d)
°
1
Yellow powder, m.p. 187~189 C, yield 71.2 %. H NMR (DMSO-d , 300 MHz) δ:
6
1
2.85 (s, 1H,-OH), 10.31 (s, 1H, OH), 7.58 (d, J = 1.8 Hz, 1H, H-8), 7.48 (d, J = 8.4
Hz, 1H, H-5), 7.45 (dd, J = 8.4, 1.8 Hz, 1H, H-6), 6.40 (d, J = 0.9 Hz, 1H, H-4), 6.22
1
3
(
d, J = 0.9 Hz, 1H, H-2); C NMR (DMSO-d , 75 MHz) δ: 178.9 (C-9), 166.7 (C-3),
6
1
63.2 (C-1), 157.7 (C-4a), 154.3 (C-4b), 135.6 (C-6), 129.0 (C-7), 124.5 (C-8), 121.4
+
(
C-8a), 120.5 (C-5), 102.5 (C-8b), 98.8 (C-2), 94.6 (C-4); MS-ESI m/z: 263.1 [M+H] .
The data were the same with reference [24].
2
0

ACCEPTED MANUSCRIPT
5
.2.3.5. Synthesis of 7-bromo-1,3-dihydroxy-9H-xanthen-9-one (2e)
°
1
Light yellow powder, m.p. 179~180 C, yield 79.2 %. H NMR (DMSO-d , 300
6
MHz) δ: 12.93 (s, 1H,-OH), 10.34 (s, 1H, OH), 7.98 (d, J = 1.8 Hz, 1H, H-8), 7.69 (d,
J = 8.4 Hz, 1H, H-5), 7.46 (dd, J = 8.4, 1.8 Hz, 1H, H-6), 6.41 (d, J = 0.9 Hz, 1H,
1
3
H-4), 6.23 (d, J = 0.9 Hz, 1H, H-2); C NMR (DMSO-d , 75 MHz) δ: 178.5 (C-9),
6
1
66.2 (C-3), 162.8 (C-1), 157.2 (C-4a), 153.9 (C-4b), 135.2 (C-6), 128.6 (C-7), 124.0
(C-8), 121.0 (C-8a), 120.0 (C-5), 102.1 (C-8b), 98.4 (C-2), 94.2 (C-4); MS-ESI m/z:
+
3
06.1 [M+H] . The data were the same with reference [24].
5
.2.3.6. Synthesis of 1,3-dihydroxy-9H-xanthen-9-one (2f)
°
1
Light yellow powder, m.p. 196~198 C, yield 76.3 %. H NMR (DMSO-d , 300
6
MHz) δ: 12.92 (s, 1H, -OH), 10.35 (s, 1H, OH), 8.26 (dd, J = 8.4, 1.2 Hz, 1H, H-6),
.73 (dd, J = 5.4, 1.2 Hz, 1H, H-8), 7.43-7.38 (m, 2H, H-5, 7), 6.46 (d, J = 2.1 Hz, 1H,
7
1
3
H-4), 6.35 (d, J = 2.1 Hz, 1H, H-2); C NMR (DMSO-d , 75 MHz) δ:179.0 (C-9),
6
1
66.7 (C-1), 163.3 (C-3), 157.7 (C-4a), 154.4 (C-4b), 135.7 (C-6), 129.1 (C-8), 124.6
(C-7), 121.5 (C-8a), 120.5 (C-5), 102.6 (C-8b), 98.9 (C-2), 94.7 (C-4), MS-ESI m/z:
+
2
29.3 [M+H] . The data were the same with reference [24].
5
.2.3.7. Synthesis of 1,3-dihydroxy-8-methoxy-9H-xanthen-9-one (2g)
°
1
Light yellow powder, m.p. 189~191 C, yield 72.1 %. H NMR (DMSO-d , 300
6
MHz) δ: 13.03 (s, 1H, -OH), 10.94 (s, 1H, -OH), 7.92 (d, J = 5.4 Hz, 1H, H-6), 6.85
(d, J = 8.4 Hz, 1H, H-5), 6.78 (d, J = 8.4 Hz, 1H, H-7), 6.31 (d, J = 1.2 Hz, 1H, H-2),
1
3
6
1
.15 (d, J = 1.2 Hz, 1H, H-4), 3.78 (s, 3H, -OCH ); C NMR (DMSO-d , 75 MHz) δ:
3 6
79.1 (C-9), 165.2 (C-3), 164.3 (C-1), 162.9 (C-8), 157.5 (C-4a), 157.4 (C-4b) 127.2
(C-6), 114.0 (C-7), 112.4 (C-5), 102.1 (C-8a), 101.7 (C-8b), 98.0 (C-2), 94.0 (C-4),
+
5
9.9 (-OCH ); MS-ESI m/z: 259.1 [M+H] . The data were the same with reference
3
[
24].
5
.2.3.8. Synthesis of 1,3-dihydroxy-6,7-dimethoxy-9H-xanthen-9-one (2h)
2
1

ACCEPTED MANUSCRIPT
°
1
Light yellow powder, m.p. 185~187 C, yield 74.1 %. H NMR (DMSO-d , 300
6
MHz) δ: 11.90 (s, -OH), 10.04 (s, -OH), 7.54 (s, 1H, H-8), 6.87 (s, 1H, H-5), 6.23 (s,
1
3
1
H, H-4), 6.06 (s, 1H, H-1), 3.82 (s, 6H, -OCH ); C NMR (DMSO-d , 75 MHz) δ:
3 6
1
79.3 (C-9), 165.4 (C-8), 164.5 (C-6), 163.1 (C-4b), 157.7 (C-3), 157.6 (C-4a), 127.4
(C-2), 114.2 (C-8b), 112.6 (C-1), 102.3 (C-4), 101.9 (C-8a), 98.2 (C-7), 94.2 (C-5),
+
5
5
1
6.2 (-OCH ); MS-ESI m/z: 289.0 [M+H] .
3
.2.3.9. Synthesis of 1,3,6-trihydroxy-9H-xanthen-9-one (2i)
°
1
Yellow powder, m.p. 179~181 C, yield 84.0 %. H NMR (DMSO-d , 300 MHz) δ:
6
2.86 (s, 1H, OH), 10.25 (s, -OH), 9.85 (s, -OH), 7.93 (d, J = 2.1 Hz, 1H, H-5), 7.69
(dd, J =8.4, 2.1 Hz, 1H, H-7), 7.33 (td, J = 8.4, 2.1 Hz, 1H, H-8), 6.42 (d, J = 1.8 Hz,
1
3
1
H, H-4), 6.22 (d, J = 1.8 Hz, 1H, H-2); C NMR (DMSO-d , 75 MHz) δ: 180.5
6
(C-9), 166.2 (C-8), 163.5 (C-3), 160.6 (C-1), 157.6 (C-4a), 156.8 (C-4b), 136.3 (C-6),
110.4 (C-8a), 109.7 (C-5), 107.0 (C-7), 102.8 (C-8b), 98.6 (C-4), 93.8 (C-2); MS-ESI
+
m/z: 245.1 [M+H] .
5
.2.4. Synthesis of 1-hydroxy-(xanthen-3-yl)oxy alkyl bromide (3a~i-n)
To a 50-mL flask, 8 mL anhydrous DMF, 1.1 mmol K CO , and 1.0 mmol
2
3
1
,3-dihydroxyxanthones (2a~i) were added. The suspension was stirred at room
temperature for 30 minutes. Then 1.1 mmol alkyl dibromide (Br(CH2)n+1Br, n = 1, 2,
, 4, 5, 6) was added dropwise within 30 minutes. The mixture was stirred for
3
another 12 h. Afterwards, the mixture was poured into 50 ml ice-water with violently
stirring for 20 min. The mixed solution was filtered. The precipitates were collected
and washed with cold water. TLC analysis found the solids were pure. So we did not
purify the precipitates. The solid products (3a~i-n, n = 1, 2, 3, 4, 5, 6) were used
directly in next step.
5
.2.5. Synthesis of 1,3-dioxyxanthone nitrates (4a~i-n)
To a 50-mL round-bottom flask, 1.0 mmol compound 3a~i-n (n = 1, 2, 3, 4, 5, 6)
and 12 mL anhydrous acetonitrile were added and stirred for 30 minutes at room
2
2

ACCEPTED MANUSCRIPT
temperature. Afterwards, a mixture of 1.1 mmol silver nitrate (AgNO ) and 4 mL
3
anhydrous acetonitrile was added dropwise within 20 minutes. Keep the reaction in
dark and stirred for another 24 h. Then the mixture was pored into 20 mL ice-water
and stirred for 25 min. The mixed solution was filtered. The precipitates were
collected and washed with cold water to neutral. The residues were purified by flash
column chromatography (petroleum ether/ethyl acetate), leading to 4a~i-n (n = 1, 2, 3,
4
, 5, 6).
5
.2.5.1. 3-((1-hydroxy-6-methyl-9-oxo-9H-xanthen-3-yl)oxy)propyl nitrate (4a-2)
1
White powder, m.p. 165.4-165.8 °C; yield: 58.6 %; purity: 95.6%. H NMR
(
CDCl , 300 MHz) δ: 12.86 (s, 1H, OH), 8.14 (s, 1H, H-8), 7.15 (s, 1H, H-5), 7.13 (t,
3
J = 8.1 Hz, 1H, H-7), 6.34-6.48 (m, 2H, H-2, 4), 4.21 (m, 2H), 3.74 (m, 2H), 2.38 (s,
1
3
3
H), 2.11 (m, 2H); C NMR (CDCl , 75 MHz) δ: 180.7 (C-9), 165.8 (C-1), 163.6
3
(C-3), 157.8 (C-4b), 156.2 (C-4a), 146.8 (C-6), 125.7 (C-8), 125.6 (C-7), 118.4 (C-8a),
1
2
3
17.5 (C-2), 103.9 (C-5), 97.4 (C-4), 93.2 (C-8b), 76.7 (C-3'), 67.6 (C-1'), 33.4 (C-2'),
+
+
9.4 (-CH ); MS-ESI m/z: 346.1 [M+H] ; HRMS (m/z): calc. for [C H NO +H]
3
17 15
7
46.0927, found 346.0924.
5
.2.5.2. 4-((1-hydroxy-6-methyl-9-oxo-9H-xanthen-3-yl)oxy)butyl nitrate (4a-3):
1
White powder, m.p. 161.1-161.3 °C; yield: 55.1 %; purity: 96.1%. H NMR
(
CDCl , 300 MHz) δ: 12.90 (s, 1H, OH), 8.08 (d, J = 8.4 Hz, 1H, H-8), 7.17 (m, 2H,
3
H-5, 7), 6.36 (d, J = 1.2 Hz, 1H, H-4), 6.29 (d, J = 1.2 Hz, 1H, H-2), 4.06 (t, J = 5.7
1
3
Hz, 2H), 3.50 (t, J = 5.7 Hz, 2H), 2.48 (s, 3H, CH ), 2.01-2.10 (m, 4H); C NMR
3
(
CDCl , 75 MHz) δ: 180.8 (C-9), 165.9 (C-1), 163.6 (C-3), 157.8 (C-4b), 156.2
3
(C-4a), 146.8 (C-6), 125.7 (C-8), 125.6 (C-7), 118.4 (C-8a), 117.5 (C-2), 104.0 (C-5),
9
7.4 (C-4), 93.2 (C-8b), 76.8 (C-4'), 67.6 (C-1'), 33.4 (C-2'), 29.4 (C-3'), 27.8 (-CH3);
+
+
MS-ESI m/z: 360.1[M+H] ; HRMS (m/z): calc. for [C H NO +H] 360.1083, found
18
17
7
3
60.1086.
5
.2.5.3. 5-((1-hydroxy-6-methyl-9-oxo-9H-xanthen-3-yl)oxy)pentyl nitrate (4a-4):
2
3

ACCEPTED MANUSCRIPT
1
White powder, m.p. 168.3-169.2 °C; yield: 58.2 %; purity: 97.0%. H NMR (CDCl ,
3
3
6
3
1
00 MHz) δ: 12.93 (s, 1H, OH), 8.09-8.12 (m, 1H, H-8), 7.14-7.20 (m, 2H, H-5, 7),
.38 (d, J = 1.2 Hz, 1H, H-4), 6.31 (d, J = 1.2 Hz, 1H, H-2), 4.05 (t, J = 5.7 Hz, 2H),
.45 (t, J = 5.7 Hz, 2H), 2.50 (s, 3H, CH ), 1.94-1.99 (m, 2H), 1.80-1.85 (m, 2H),
3
13
.51-1.57 (m, 2H); C NMR (CDCl , 75 MHz) δ: 180.7 (C-9), 165.8 (C-1), 163.6
3
(
C-3), 157.7 (C-4b), 156.2 (C-4a), 146.7 (C-6), 125.6 (C-8), 125.6 (C-7), 118.3 (C-8a),
17.5 (C-2), 103.9 (C-5), 97.3 (C-4), 93.2 (C-8b), 76.7 (C-5'), 67.6 (C-1'), 33.3 (C-4'),
1
+
2
9.4 (C-3'), 27.7 (C-2'), 22.1 (-CH ); MS-ESI m/z: 374.1 [M+H] ; HRMS (m/z): calc.
3
+
for [C H NO +H] 374.1240, found 374.1237.
19
19
7
5
.2.5.4. 6-((1-hydroxy-6-methyl-9-oxo-9H-xanthen-3-yl)oxy)hexyl nitrate (4a-5)
1
White powder; m.p. 163.2-164.3 °C; yield: 64.4 %; purity: 98.3%. H NMR
(
CDCl , 300 MHz) δ: 12.90 (s, 1H, OH), 8.07-8.09 (m, 1H, H-8), 7.16-7.26 (m, 2H,
3
H-5, 7), 6.37 (d, J = 1.2 Hz, 1H, H-4), 6.30 (d, J = 1.2 Hz, 1H, H-2), 4.02 (t, J = 5.7
Hz, 2H), 3.41 (t, J = 5.7 Hz, 2H), 2.48 (s, 3H, CH ), 1.81-1.85 (m, 4H), 1.48-1.52 (m,
3
1
3
4
H); C NMR (CDCl , 75 MHz) δ: 180.7 (C-9), 166.1 (C-1), 163.6 (C-3), 157.8
3
(C-4b), 156.2 (C-4a), 146.7 (C-6), 125.7 (C-8), 125.6 (C-7), 118.4 (C-8a), 117.5 (C-2),
1
03.8 (C-5), 97.4 (C-4), 93.3 (C-8b), 76.9 (C-6'), 68.5 (C-1'), 33.9 (C-5'), 32.7 (C-2'),
+
2
8.9 (C-4'), 28.0 (C-3'), 25.3 (-CH ); MS-ESI m/z: 388.1 [M+H] ; HRMS (m/z): calc.
3
+
for [C H NO +H] 388.1386, found 388.1382.
20
21
7
5
.2.5.5. 7-((1-hydroxy-6-methyl-9-oxo-9H-xanthen-3-yl)oxy)heptyl nitrate (4a-6)
1
White powder; m.p. 160.6-162.2 °C; yield: 67.5%; purity: 96.3%. H NMR (CDCl ,
3
3
6
3
00 MHz) δ: 12.90 (s, 1H, OH), 8.07-8.09 (m, 1H, H-8), 7.16-7.26 (m, 2H, H-5, 7),
.37 (d, J = 1.2 Hz, 1H, H-4), 6.30 (d, J = 1.2 Hz, 1H, H-2), 4.02 (t, J = 5.7 Hz, 2H),
1
3
.41 (t, J = 5.7 Hz, 2H), 2.48 (s, 3H, CH ), 1.81-1.85 (m, 4H), 1.48-1.52 (m, 6H); C
3
NMR (CDCl , 75 MHz) δ: 180.7 (C-9), 166.1 (C-1), 163.6 (C-3), 157.8 (C-4b), 156.2
3
(C-4a), 146.7 (C-6), 125.7 (C-8), 125.6 (C-7), 118.4 (C-8a), 117.5 (C-2), 103.8 (C-5),
9
2
7.4 (C-4), 93.3 (C-8b), 76.9 (C-7'), 68.5 (C-1'), 33.9 (C-6'), 32.7 (C-2'), 30.3 (C-3'),
+
8.9 (C-5'), 28.0 (C-4'), 25.3 (-CH ); MS-ESI m/z: 402.2 [M+H] ; HRMS (m/z): calc.
3
2
4

ACCEPTED MANUSCRIPT
+
for [C H NO +H] 402.1385, found 402.1383.
21
23
7
5
.2.5.6. 3-((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)propyl nitrate (4b-2)
1
White powder; m.p. 161.5-162.2 °C; yield: 62.8 %; purity: 97.5%. H NMR
(
CDCl , 300 MHz) δ: 12.87 (s, 1H, OH), 7.61 (d, J = 1.5 Hz, 1H, H-8), 7.31-7.39 (m,
3
2
5
H, H-5, 6), 6.42 (d, J = 1.2 Hz, 1H, H-4), 6.34 (d, J = 1.2 Hz, 1H, H-2), 4.20 (t, J =
13
.7 Hz, 2H), 3.91 (s, 3H, CH ), 3.61 (t, J = 5.7 Hz, 2H), 2.36 (t, J = 5.7 Hz, 2H); C
3
NMR (CDCl , 75 MHz) δ: 180.0 (C-9), 165.2 (C-3), 162.7 (C-1), 157.2 (C-4a), 155.4
3
(C-7), 150.2 (C-4b), 124.3 (C-6), 120.2 (C-8a), 118.4 (C-5), 104.6 (C-2), 103.1 (C-4),
9
6.7 (C-8), 92.3 (C-8b), 66.9 (C-1'), 55.4 (C-3'), 52.6 (-OCH ), 29.1 (C-2'); MS-ESI
3
+
+
m/z: 362.1 [M+H] ; HRMS (m/z): calc. for [C H NO +H] 362.0876, found
1
7
15
8
3
62.0873.
5
.2.5.7. 4-((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)butyl nitrate (4b-3)
1
White powder; m.p. 163.1-164.2 °C; yield: 59.7 %; purity: 96.7%. H NMR
(
CDCl , 300 MHz) δ: 12.86 (s, 1H, OH), 7.60 (d, J = 1.5 Hz, 1H, H-8), 7.32-7.37 (m,
3
2
5
H, H-5, 6), 6.39 (d, J = 1.2 Hz, 1H, H-4), 6.32 (d, J = 1.2 Hz, 1H, H-2), 4.06 (t, J =
1
3
.7 Hz, 2H), 3.90 (s, 3H, CH ), 3.50 (t, J = 5.7 Hz, 2H), 1.96-2.00 (m, 4H); C NMR
3
(
CDCl , 75 MHz) δ: 180.7 (C-9), 165.9 (C-3), 163.5 (C-1), 157.9 (C-4a), 156.2 (C-7),
3
1
51.0 (C-4b), 125.0 (C-6), 121.0 (C-8a), 119.1 (C-5), 105.3 (C-2), 103.8 (C-4), 97.5
(
C-8), 93.0 (C-8b), 67.7 (C-1'), 56.1 (C-4'), 53.4 (-OCH ), 29.4 (C-2'), 27.8 (C-3');
3
+
+
MS-ESI m/z: 376.1[M+H] ; HRMS (m/z): calc. for [C H NO +H] 376.1032, found
18
17
8
3
76.1037.
5
.2.5.8. 5-((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)pentyl nitrate (4b-4)
1
White powder; m.p. 167.2-168.4 °C; yield: 51.7 %; purity: 97.3%. H NMR
(
CDCl , 300 MHz) δ: 12.88 (s, 1H, OH), 7.61 (d, J = 1.5 Hz, 1H, H-8), 7.28-7.37 (m,
3
2
5
H, H-5, 6), 6.40 (d, J = 1.2 Hz, 1H, H-4), 6.34 (d, J = 1.2 Hz, 1H, H-2), 4.07 (t, J =
.7 Hz, 2H), 3.92 (s, 3H, CH ), 3.49 (t, J = 5.7 Hz, 2H), 1.92-1.98 (m, 2H), 1.84-1.89
3
1
3
(
m, 2H), 1.62-1.67 (m, 2H); C NMR (CDCl , 75 MHz) δ: 180.3 (C-9), 165.5 (C-3),
3
2
5

ACCEPTED MANUSCRIPT
1
1
5
63.0 (C-1), 157.5 (C-4a), 155.7 (C-7), 150.5 (C-4b), 124.6 (C-6), 120.6 (C-8a),
18.7 (C-5), 104.9 (C-2), 103.4 (C-4), 97.0 (C-8), 92.6 (C-8b), 67.2 (C-1'), 55.7 (C-5'),
+
2.9 (-OCH ), 29.4 (C-2'), 29.0 (C-4'), 27.3 (C-3'); MS-ESI m/z: 390.1 [M+H] ;
3
+
HRMS (m/z): calc. for [C H NO +H] 390.1189, found 390.1187.
1
9
19
8
5
.2.5.9. 6-((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)hexyl nitrate (4b-5)
1
White powder; m.p. 162.1-163.3 °C; yield: 52.8 %; purity: 96.8%. H NMR
(
CDCl , 300 MHz) δ: 12.87 (s, 1H, OH), 7.61 (d, J = 1.5 Hz, 1H, H-8), 7.29-7.39 (m,
3
2
5
H, H-5, 6), 6.40 (d, J = 1.2 Hz, 1H, H-4), 6.34 (d, J = 1.2 Hz, 1H, H-2), 4.06 (t, J =
.7 Hz, 2H), 3.92 (s, 3H, CH ), 3.45 (t, J = 5.7 Hz, 2H), 1.86-1.91 (m, 4H), 1.50-1.55
3
1
3
(
m, 4H); C NMR (CDCl , 75 MHz) δ: 180.6 (C-9), 166.2 (C-3), 163.4 (C-1), 157.9
3
(C-4a), 156.1 (C-7), 150.9 (C-4b), 125.0 (C-6), 121.0 (C-8a), 119.1 (C-5), 105.3 (C-2),
1
03.7 (C-4), 97.5 (C-8), 93.0 (C-8b), 68.5 (C-1'), 56.1 (C-6'), 53.9 (-OCH ), 32.7
3
+
(
C-2'), 28.9 (C-5'), 28.0 (C-3'), 25.3 (C-4'). MS-ESI m/z: 404.1 [M+H] ; HRMS (m/z):
+
calc. for [C H NO +H] 404.1345, found 404.1347.
20
21
8
5
.2.5.10. 7-((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)heptyl nitrate (4b-6)
1
White powder; m.p. 161.3-162.5 °C; yield: 61.5%; purity: 97.2%. H NMR (CDCl ,
3
3
6
2
00 MHz) δ: 12.87 (s, 1H, OH), 7.61 (d, J = 1.5 Hz, 1H, H-8), 7.29-7.39 (m, 2H, H-5,
), 6.40 (d, J = 1.2 Hz, 1H, H-4), 6.34 (d, J = 1.2 Hz, 1H, H-2), 4.06 (t, J = 5.7 Hz,
H), 3.92 (s, 3H, CH ), 3.45 (t, J = 5.7 Hz, 2H), 1.86-1.91 (m, 4H), 1.50-1.55 (m, 6H);
3
1
3
C NMR (CDCl , 75 MHz) δ: 180.6 (C-9), 166.2 (C-3), 163.4 (C-1), 157.9 (C-4a),
3
1
56.1 (C-7), 150.9 (C-4b), 125.0 (C-6), 121.0 (C-8a), 119.1 (C-5), 105.3 (C-2), 103.7
(
C-4), 97.5 (C-8), 93.0 (C-8b), 68.5 (C-1'), 56.1 (C-7'), 53.9 (-OCH ), 32.7 (C-2'),
3
+
3
1.9 (C-3'), 28.9 (C-6'), 28.0 (C-5'), 25.3 (C-4'). MS-ESI m/z: 418.2 [M+H] ; HRMS
+
(
m/z): calc. for [C H NO +H] 418.1344, found 418.1346.
2
1
23
8
5
.2.5.11. 3-((1, 7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy) propyl nitrate (4c-2)
1
White powder; m.p. 166.5-167.4 °C; yield: 55.0 %; purity: 95.5%. H NMR
(
CDCl , 300 MHz) δ: 12.86 (s, 1H, OH), 9.23 (s, 1H, OH), 7.67 (d, J = 1.5 Hz, 1H,
3
2
6

ACCEPTED MANUSCRIPT
H-6), 7.24-7.34 (m, 2H, H-5, 8), 6.50 (d, J = 1.2 Hz, 1H, H-4), 6.36 (d, J = 1.2 Hz, 1H,
13
H-2), 4.24 (t, J = 5.7 Hz, 2H), 3.63 (t, J = 5.7 Hz, 2H), 2.31-2.36 (m, 2H); C NMR
CDCl , 75 MHz) δ: 175.2 (C-9), 164.0 (C-3), 161.3 (C-1), 159.8 (C-4a), 155.3 (C-7),
(
3
1
49.7 (C-4b), 123.8 (C-6), 123.4 (C-8a), 118.5 (C-5), 107.1 (C-2), 107.0 (C-8), 96.0
+
(
C-4), 93.2 (C-8b), 68.2 (C-1'), 67.6 (C-3'), 34.1 (C-2'); MS-ESI m/z: 348.1 [M+H] ;
+
HRMS (m/z): calc. for [C H NO +H] 348.0719, found 348.0721.
1
6
13
8
5
.2.5.12. 4-((1, 7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy) butyl nitrate (4c-3)
1
White powder; m.p. 175.1-175.7 °C; yield: 55.4 %; purity: 96.4%. H NMR
(
CDCl , 300 MHz) δ: 12.83 (s, 1H, OH), 9.21 (s, 1H, OH), 7.65 (d, J = 1.5 Hz, 1H,
3
H-8), 7.24-7.32 (m, 2H, H-5, 6), 6.46 (d, J = 1.2 Hz, 1H, H-4), 6.31 (d, J = 1.2 Hz, 1H,
1
3
H-2), 4.10 (t, J = 5.7 Hz, 2H), 3.52 (t, J = 5.7 Hz, 2H), 2.07-2.11 (m, 4H); C NMR
CDCl , 75 MHz) δ: 175.3 (C-9), 164.1 (C-3), 161.3 (C-1), 159.9 (C-4a), 155.3 (C-7),
(
3
1
49.7 (C-4b), 123.8 (C-6), 123.4 (C-8a), 118.5 (C-5), 107.1 (C-2), 107.0 (C-8), 96.1
(C-4), 93.2 (C-8b), 68.2 (C-1'), 67.6 (C-4'), 34.1 (C-2'), 33.5 (C-3'); MS-ESI m/z:
+
+
3
62.1 [M+H] . HRMS (m/z): calc. for [C H NO +H] 362.0876, found 362.0875.
17 15 8
5
.2.5.13. 5-((1,7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy)pentyl nitrate) (4c-4)
1
White powder; m.p. 172.3-173.9 °C; yield: 45.2 %; purity: 98.3%. H NMR
(
CDCl , 300 MHz) δ: 12.87 (s, 1H, OH), 9.25 (s, 1H, OH), 7.67 (d, J = 1.5 Hz, 1H,
3
H-8), 7.21-7.32 (m, 2H, H-5, 6), 6.46 (d, J = 1.2 Hz, 1H, H-4), 6.32 (d, J = 1.2 Hz, 1H,
H-2), 4.08 (t, J = 5.7 Hz, 2H), 3.47 (t, J = 5.7 Hz, 2H), 1.95-1.99 (m, 2H), 1.83-1.89
1
3
(
m, 2H), 1.62-1.67 (m, 2H); C NMR (CDCl , 75 MHz) δ: 175.3 (C-9), 164.1 (C-3),
3
1
61.4 (C-1), 159.9 (C-4a), 155.4 (C-7), 149.7 (C-4b), 123.8 (C-6), 123.5 (C-8a), 118.4
(
(
[
C-5), 107.0 (C-2), 106.0 (C-8), 96.1 (C-4), 93.1 (C-8b), 69.0 (C-1'), 68.3 (C-5'), 33.8
+
C-2'), 33.6 (C-4'), 32.5 (C-3'); MS-ESI m/z: 376.1 [M+H] ; HRMS (m/z): calc. for
+
C H NO +H] 376.1032, found 376.1036.
1
8
17
8
5
.2.5.14. 6-((1, 7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy) hexyl nitrate (4c-5)
1
White powder; m.p. 165.9-166.5 °C; yield: 40.8 %; purity: 97.2%. H NMR
2
7

ACCEPTED MANUSCRIPT
(
CDCl , 300 MHz) δ: 12.89 (s, 1H, OH), 9.26 (s, 1H, OH), 7.68 (d, J = 1.5 Hz, 1H,
3
H-8), 7.25-7.29 (m, 2H, H-5, 6), 6.46 (d, J = 1.2 Hz, 1H, H-4), 6.33 (d, J = 1.2 Hz, 1H,
H-2), 4.04-4.06 (m, 4H), 1.90-1.93 (m, 2H), 1.81-1.86 (m, 2H), 1.51-1.56 (m, 4H);
1
3
C NMR (CDCl , 75 MHz) δ:174.9 (C-9), 163.7 (C-3), 161.0 (C-1), 159.4 (C-4a),
3
1
55.0 (C-7), 149.3 (C-4b), 123.4 (C-6), 123.1 (C-8a), 118.0 (C-5), 106.6 (C-2), 106.6
(
(
[
C-8), 95.7 (C-4), 92.7 (C-8b), 68.6 (C-1'), 67.9 (C-6'), 33.4 (C-2'), 33.2 (C-5'), 32.2
+
C-3'), 28.0 (C-4'); MS-ESI m/z: 390.1 [M+H] ; HRMS (m/z): calc. for
+
C H NO +H] 390.1189, found 390.1193.
1
9
19
8
5
.2.5.15. 7-((1, 7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy) heptyl nitrate (4c-6)
1
White powder; m.p. 163.5-164.8 °C; yield: 46.7%; purity: 97.3%. H NMR (CDCl ,
3
3
7
4
00 MHz) δ: 12.89 (s, 1H, OH), 9.26 (s, 1H, OH), 7.68 (d, J = 1.5 Hz, 1H, H-8),
.25-7.29 (m, 2H, H-5, 6), 6.46 (d, J = 1.2 Hz, 1H, H-4), 6.33 (d, J = 1.2 Hz, 1H, H-2),
13
.04-4.06 (m, 4H), 1.90-1.93 (m, 2H), 1.81-1.86 (m, 2H), 1.51-1.56 (m, 6H);
C
NMR (CDCl , 75 MHz) δ:174.9 (C-9), 163.7 (C-3), 161.0 (C-1), 159.4 (C-4a), 155.0
3
(C-7), 149.3 (C-4b), 123.4 (C-6), 123.1 (C-8a), 118.0 (C-5), 106.6 (C-2), 106.6 (C-8),
9
3
5.7 (C-4), 92.7 (C-8b), 68.6 (C-1'), 67.9 (C-7'), 33.4 (C-2'), 33.2 (C-6'), 32.2 (C-4'),
+
1.3 (C-3'), 28.0 (C-5'); MS-ESI m/z: 404.2 [M+H] ; HRMS (m/z): calc. for
+
[
C H NO +H] 404.1188, found 404.1192.
2
0
21
8
5
.2.5.16. 2-((7-chloro-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy) ethyl nitrate (4d-1)
1
White powder; m.p.179.1-179.6 °C; yield: 38.8 %; purity: 97.2%. H NMR (CDCl ,
3
3
6
2
1
00 MHz) δ: 12.96 (s, 1H, -OH), 8.13 (d, J = 1.8 Hz, 1H, H-8), 7.20-7.32 (m, 2H, H-5,
), 6.43 (d, J = 1.2 Hz, 1H, H-4), 6.35 (d, J = 1.2 Hz, 1H, H-2), 4.38 (t, J = 5.7 Hz,
1
3
H), 3.69 (t, J = 5.7 Hz, 2H); C NMR (CDCl , 75 MHz) δ:174.7 (C-9), 163.6 (C-3),
3
60.8 (C-1), 159.3 (C-4a), 154.8 (C-4b), 149.1 (C-6), 123.2 (C-7), 122.9 (C-8), 117.8
(C-8a), 113.9 (C-5), 106.4 (C-2), 95.5 (C-4), 92.5 (C-8b), 76.8 (C-1'), 68.4 (C-2');
+
+
MS-ESI m/z: 352.0 [M+H] ; HRMS (m/z): calc. for [C H ClNO +H] 352.0224,
1
5
10
7
found 352.0221.
2
8