Efficient total synthesis and biological activities of 6-deoxyisojacareubin

Article abstract of DOI:10.1002/ardp.201200440

6-Deoxyisojacareubin was directly synthesized in a six-step route with an overall yield of about 20%. In this route, the excellent site selectivity of this Claisen rearrangement-cyclization reaction cascade was achieved by inserting a bulky p-tosyl group

Full text of DOI:10.1002/ardp.201200440

314
Arch. Pharm. Chem. Life Sci. 2013, 346, 314–320
Full Paper
Efficient Total Synthesis and Biological Activities of
6-Deoxyisojacareubin
Ming Dai1*, Xing Yuan1*, Zhi-Jun Zhu1, Lei Shan1, Run-Hui Liu1, Qing-Yan Sun2,
and Wei-Dong Zhang1
1 Department of Phytochemistry, School of Pharmacy, Second Military Medical University, Shanghai,
P. R. China
2 Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, Shanghai,
P. R. China
6-Deoxyisojacareubin was directly synthesized in a six-step route with an overall yield of about 20%.
In this route, the excellent site selectivity of this Claisen rearrangement-cyclization reaction cascade
was achieved by inserting a bulky p-tosyl group into the free 1-OH, and in the last step, some efficient
demethylation methods were explored. Furthermore, all synthesized intermediates including
6-deoxyisojacareubin were evaluated for their inhibitory activity against the QGY-7703 cell line.
Of these, compound 1 and 6-deoxyisojacareubin showed moderate activities with IC50 values of
39.61 and 9.65 mM, respectively, when compared to the positive control 5-fluorouracil with an
IC50 value of 11.24 mM. Further investigation using non-radioactive detection of protein kinase C
(PKC) suggested that these two compounds possessed potency in the inhibition of PKC.
Keywords: Antitumor / 6-Deoxyisojacareubin / Protein kinase C / Xanthone
Received: November 21, 2012; Revised: December 27, 2012; Accepted: January 18, 2013
DOI 10.1002/ardp.201200440
Additional supporting information may be found in the online version of this article at the publisher’s
:
web-site.
Introduction
promotion [7]. Therefore, PKC has become an attractive target
for anticancer treatment.
Recently, some studies have reported that natural and
synthetic xanthones mediate several important biological activi-
ties such as antitumor [1], anti-inflammatory [2], antithrombotic
[3], and neuropharmacological effects [4]. Furthermore, it has
been suggested that these compounds may interact with
protein kinase C (PKC) and cause effects compatible not only
with PKC activation [5] but also with PKC inhibition [6]. PKC is
a family of serine-threonine kinases with significant roles in
carcinogenesis and the maintenance of a malignant pheno-
type, and it is one of the key elements of signal transduction
and intimately involved in cell growth regulation and tumor
6-Deoxyisojacareubin (6) is a natural product with a xan-
thone scaffold. Fu et al. [8] isolated this compound from
Hypericum japonicum Thunb. ex Murray. Niu et al. [9] measured
this compound against the human leukemic cell line HL-60
with GI50 values of 27.3 ꢀ 1.4 mM in vitro. Up to now, the
synthesis of 6-deoxyisojacareubin has been reported only a
few times. The first total synthesis of 6-deoxyisojacareubin
was accomplished by Gujral and Gupta [10]. 1,3,5-
Trihydroxyxanthone was treated with 2-methylbut-3-en-2-ol
in the presence of a catalytic amount of BF3-etherate to yield a
mixture of prenylated xanthone derivatives in poor yield,
and one of these prenylated derivatives along with 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ) in dry toluene
afforded 6-deoxyisojacareubin. Helesbeux et al. [11] prepared
Correspondence: Prof. Wei-Dong Zhang, Department of
Phytochemistry, School of Pharmacy, Second Military Medical University,
325 Guohe Road, Shanghai 200433, P. R. China.
E-mail: wdzhangy@hotmail.com
*These two authors contributed equally to this work.
Additional Correspondence: Prof. Qingyan Sun, E-mail: sqy_2000@163.com;
Prof. Runhui Liu, E-mail: lyliurh@126.com.
Fax: þ86 21 81871244
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Arch. Pharm. Chem. Life Sci. 2013, 346, 314–320
Synthesis and Biological Activities of 6-Deoxyisojacareubin
315
6-deoxyisojacareubin in a different way in 2004. They applied
the photooxygenation-reduction sequence to synthesize sev-
eral natural secondary allylic alcohol derivatives (three in the
coumarin series and two novel compounds in the xanthone
series); our target compound 6-deoxyisojacareubin was iso-
lated in the reduction step, and the total yield to synthesize 6-
deoxyisojacareubin was just about 1.68%. In the above two
synthesis schemes, no regioselectivity was achieved when
introducing a prenyl side chain on the xanthone skeleton,
and many other byproducts were produced, leading to a low
yield. In the present study, we sought to develop a chemical
strategy that would allow an efficient synthetic access to 6-
deoxyisojacareubin and thus facilitate its biological evalu-
ation regarding its cytotoxicity and inhibitory activity on
PKC.
P2O5/CH3SO3H) [13] as the coupling agent afforded the oxy-
genated xanthone nucleus 1 in high yield (90%) without
further purification. As the 1-OH can form a hydrogen bond
with the adjacent carbonyl group, by controlling the reaction
time and temperature, we can selectively get xanthone 2
without the 1-OH substitute product. So propargylic ether
2 was easily prepared in the presence of KI and K2CO3 with a
catalytic amount of CuI, with a yield of 63%.
According to the reports, the following Claisen rearrange-
ment-cyclization may have two cyclization directions [14, 15],
which could lead to the angular or the linear isomer. Almost
equal amounts of the two isomers were prepared if the 1-OH
was exposed. Then, we observed that methylation of the 1-OH
group prior to ring closure improves the percentage of the
angular isomer, presumably due to steric hindrance, but a
small amount of linear isomer was still monitored in our
reaction. Then, we inserted the bulky p-tosyl group into the
free 1-OH of propargylic ether 2 to provide the ester 3, which
was subjected to thermal cyclization to furnish selectively
the angular isomer 4; this was easily hydrolyzed to the
required methylated derivative 5 (Scheme 2). Treatment of
2 with 1.5 equiv. of TsCl in acetone at room temperature for
1.5 h and purification via a simple chromatography on silica
gel afforded a white powdery product 3 in 83% yield. Then,
the xanthone sulfonate 3 was heated in DMF at 1208C via
Claisen-cyclization reaction to form the angular isomer 4 in
85% yield. Just one spot in TLC was monitored. 1H NMR,
13C NMR, and HMBC analysis were used to determine its
structure and confirm that it was the angular isomer
(Scheme 3). The key signals were outlined to confirm the
right cyclization direction. The carbon–carbon double
bond proton [H-1] correlated with the oxygenated aromatic
Result and discussion
Chemistry
The retrosynthetic analysis is outlined in Scheme 1. In this
route, the important intermediate methyl ether of 6-deoxy-
isojacareubin (5) was firstly synthesized by just five steps,
from the commercially available starting materials 3-
methoxysalicylic acid and phloroglucinol, in around 30%
yield. Then an efficient demethylation method was explored
to complete the total synthesis of 6-deoxyisojacareubin with
about 20% overall yield.
The synthesis approach for the construction of the xan-
thone nucleus was based on the method of Grover et al. [12]
and involves the condensation between phloroglucinol
and 2-hydroxy-3-methoxybenzoic acid. The use of Eaton’s
reagent (phosphorus pentoxide and methanesulfonic acid:
Scheme 1. Retrosynthetic analysis of 6-deoxyisojacareubin.
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316
M. Dai et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 314–320
Scheme 2. Synthetic route of 6-deoxyisojacareubin.
carbons [C-12a] and [C-4a]; the aromatic proton [H-5] corre-
lated with [C-6] and [C-6a] (Scheme 3 and Table 1). Then,
the subsequent hydrogenolysis of the Ts group led to the
methylated derivative 5 in 72% yield.
With our methylated derivative 5 in hand, we directed our
efforts toward the demethylation. According to the litera-
ture, a Lewis acid could be selected as our demethylation
condition [16, 17]. Treating methylated derivative 5 with BBr3
in CH2Cl2 at ꢁ78 to 08C for 4 h, 6-deoxyisojacareubin 6 was
obtained in a low yield (about 25%). Furthermore, more than
one kind of demethylation conditions were tested. AlCl3 and
pyridine hydrochloride were employed by us and the results
were the same as those with BBr3. At last, the generation of
6-deoxyisojacareubin from its methylated derivative was
effected by exposure to AlBr3–EtSH in methylene chloride
at 08C (65% yield).
Biological activity
Aimed at discovering potential antitumor agents, 6-deoxy-
isojacareubin and another five intermediates were employed
Scheme 3. Long-range correlation in the HMBC spectrum of
compound 4.
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Synthesis and Biological Activities of 6-Deoxyisojacareubin
317
Table 1. HMBC spectroscopic data of compound 4.
Position
dH (ppm)
dC (ppm)
HMBC
1
2
3
6.80 d
5.99 d
113.85
128.52
79.01
C-12a, C-3, C-4a
C-13, C-14, C-3
4
4a
5
6
6a
7
7a
8
156.62
108.22
148.03
109.46
172.62
128.89
122.33
124.26
116.23
147.05
145.76
6.42
C-6a, C-6, C-4a
7.53 dd
7.37–7.31 m
7.45 dd
C-9, C-10, C-7
C-10, C-8, C-11
C-11a, C-9, C-11
9
10
11
11a
12
12a
12b
13, 14
151.78
108.55
27.61, 27.3
3.96, 2.35
C-13(14), C-3, C-2
in the preliminary screen to identify cytotoxicity against the
QGY-7703 cell line. Compared with the positive control agent
5-fluorouracil (5-FU; Sigma), compound 1 and 6-deoxyisoja-
careubin showed the highest activities in the preliminary
cytotoxicity screen (Fig. 1A); therefore, these two compounds
were evaluated further. The inhibition rates for the QGY-7703
cells pretreated with compound 1, 6-deoxyisojacareubin
and 5-FU at different concentrations are shown in Fig. 1B.
Both compound 1 and 6-deoxyisojacareubin inhibited the
proliferation of QGY-7703 cells in a concentration-dependent
manner, as compared with the positive control 5-FU. The IC50
values of compound 1, 6-deoxyisojacareubin, and 5-FU
were 39.61, 9.65, and 11.24 mM, respectively. Furthermore,
6-deoxyisojacareubin presented a remarkable inhibition
proliferation ability in comparison with 5-FU in the concen-
tration range of 5–10 mg/mL.
Figure 1. Cytotoxicity against the QGY-7703 cell line. The plotted
data were averaged from three independent experiments. (A)
Preliminary cytotoxicity screen of 6-deoxyisojacareubin and five
intermediates. (B) Inhibition rates of compound 1 and 6-deoxyisoja-
careubin against the QGY-7703 cell line.
Interestingly, our study showed that 6-deoxyisojacareubin
had better proliferation inhibition ability, while compound
1 possessed better inhibitory potency regarding the PKC
activity. Based on this result, other target proteins besides
PKC may be the acting substrates of 6-deoxyisojacareubin and
its synthetic intermediates, which requires further research.
Conclusion
The activity of PKC in QGY-7703 cells was assessed 3 h after
the addition of compound 1, 6-deoxyisojacareubin, and the
PKC inhibitor GF109203X (GFX; Tocris Bioscience). Compared
to the basal control (in the absence of any compound), the
PKC activity, as measured by the phosphorylation state of
a PKC-specific target peptide, was inhibited by compound 1
and 6-deoxyisojacareubin. Both compound 1 and 6-deoxyiso-
jacareubin presented better inhibition potency at the high
concentration of 20 mg/mL, as compared with 10 mg/mL,
especially compound 1 at 20 mg/mL (Fig. 2A, lane 5).
Compared with the basal control, the inhibition rate of
GFX was 63.4%, and those of compound 1 and 6-deoxyisoja-
careubin (20 mg/mL) were 59.3% and 32.4%, respectively
(Fig. 2B).
We showed a concise methodology for the preparation of
6-deoxyisojacareubin in about 20% overall yield and explored
an efficient way to control the Claisen-cyclization direction.
In the last step, several demethylation methods were
screened and EtSH–AlBr3 provided a satisfactory yield to
complete this total synthesis. Our exploration of these effects
could be helpful in the synthesis of other natural products or
their designed analogs containing an analogous structural
constitution in a more efficient way. Furthermore, the cyto-
toxicities of 6-deoxyisojacareubin and another five inter-
mediates were evaluated and the results revealed that the
derivatives of polyoxygenated xanthone gave a positive
response against QGY-7703 cells. The PKC activity assay
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Arch. Pharm. Chem. Life Sci. 2013, 346, 314–320
benzoic acid (10.08 g, 60 mmol) was added slowly 100 mL of
Eaton’s reagent (P2O5/CH3SO3H, Aldrich). The mixture was
warmed up to 808C for 20 min, under stirring. After cooling
to room temperature, the reaction mixture was poured into ice
(300 g) and stirred for 2 h. The resulting solid was collected by
filtration, washed with water until pH 6, and dried at 608C to give
a reddish brown solid 1 (14.09 g, 91%) without further purifi-
cation. Rf ¼ 0.71 (hexane/ethyl acetate 2:1); mp 304–3058C;
1H NMR (600 MHz, DMSO-d6) d 12.81 (s, 1H), 11.11 (s, 1H), 7.66
(d, J ¼ 8.0 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.38 (t, J ¼ 8.0 Hz, 1H),
6.42 (d, J ¼ 2.0 Hz, 1H), 6.22 (d, J ¼ 2.0 Hz, 1H), 4.03–3.93 (m, 3H)
ppm; 13C NMR (150 MHz, DMSO-d6) d 182.30, 168.11, 165.02,
159.43, 150.19, 147.64, 126.32, 122.83, 118.93, 117.71, 104.44,
100.49, 96.37, 58.27 ppm; HRMS: m/z (MþHþ) calcd. for C14H10O5
259.0601, found 259.0606.
1-Hydroxy-5-methoxy-3-(2-methylbut-3-yn-2-yloxy)-9H-
xanthen-9-one (2, C19H16O5)
To a round-bottom flask containing xanthone 1 (377 mg,
1.46 mmol), KI (400 mg, 2.41 mmol), K2CO3 (220 mg, 1.59 mmol)
and CuI (14.0 mg, 73.8 mmol) were added dry acetone (8.00 mL)
and 2-chloro-2-methylbut-3-yne (164 mg, 1.61 mmol). The reac-
tion vessel was then equipped with a reflux condenser and the
reaction was stirred under argon while heating at 458C. The
reaction was monitored by TLC until complete. After 2 h,
the reaction mixture was allowed to cool to room temperature
and acidified with AcOH. The reaction mixture was then parti-
tioned between EtOAc and water. The water layer was back-
extracted once and the combined EtOAc layers were dried over
MgSO4, filtered, and concentrated. The crude material was col-
umn chromatographed (hexane/ethyl acetate 4:1) to yield a yel-
low solid 2 (300 mg, 63.3%); Rf ¼ 0.68 (hexane/ethyl acetate 4:1);
mp 167.9–1698C; IR (KBr, film): n (cmꢁ1) 3246, 3008, 2983, 2961,
2934, 2843, 2665, 2105, 1837, 1674, 1607, 1565, 1505, 1494,
1458, 1437, 1351, 1315, 1269, 1233, 1102, 1017, 1004, 967,
881, 834, 805, 762, 722, 688, 656, 583, 544, 523, 464. 1H NMR
(600 MHz, DMSO-d6) d 12.69 (s, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.53
(d, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 6.88 (d, J ¼ 2.1 Hz, 1H),
6.55 (d, J ¼ 2.1 Hz, 1H), 3.98 (s, 3H), 3.94 (s, 1H), 1.72 (s, 6H) ppm;
13C NMR (150 MHz, DMSO-d6) d 182.16, 164.52, 163.77, 158.25,
149.80, 147.37, 126.09, 122.41, 118.73, 117.52, 105.48, 102.86,
98.61, 86.40, 80.27, 74.77, 58.07 ppm; HRMS m/z (MþHþ) calcd.
for C19H16O5 325.1071, found 325.1074.
Figure 2. Effects of compound 1 and 6-deoxyisojacareubin (Com
6) on PKC activity in QGY-7703 cells. Protein cell lysates were pre-
pared from QGY-7703 cells and measured for the ability to phos-
phorylate a PKC-specific peptide substrate (PLSRTLSVAAK) in a
non-radioactive assay. (A) Lanes 1 and 2 show the negative (water
only) and positive controls (with 20 ng purified PKC). Supernatants
from QGY-7703 cells were subjected to the PKC activity assay in the
absence (basal control, lane 3) or presence (lanes 4–8) of com-
pounds. The PKC inhibitor GFX (10 mg/mL, lane 4) or compound 1
(lanes 5 and 6) and 6-deoxyisojacareubin (lanes 7 and 8) at 20 and
10 mg/mL were added separately. (B) Quantitation of (A); ꢂp < 0.05
versus the basal control. Data are shown as mean ꢀ SD of three
independent experiments.
suggested that compound
1 and 6-deoxyisojacareubin
possessed potency in the inhibition of PKC. The present
study shows that 1 and 6-deoxyisojacareubin are of interest
as prospective antitumor agents, which requires further
research.
5-Methoxy-3-(2-methylbut-3-yn-2-yloxy)-9-oxo-9H-
Experimental
xanthen-1-yl-4-methylbenzenesulfonate (3, C26H22O7S)
To a solution of the xanthone 2 (410 mg, 1.26 mmol) in dry
acetone (10 mL) were added p-toluenesulfonyl chloride
(777 mg, 4.08 mmol) and potassium carbonate (938 mg,
6.8 mmol), and the resulting mixture was heated at reflux under
argon for 90 min. The inorganic precipitate was then filtered off
and the filtrate was evaporated to dryness. Flash chromatog-
raphy on silica gel using a mixture of hexane/ethyl acetate
80:20 as the eluent provided the white solid 3 (500 mg, 83%).
Rf ¼ 0.21 (hexane/ethyl acetate 4:1); mp 140.9–141.28C; 1H NMR
(600 MHz, DMSO-d6) d 7.81 (d, J ¼ 8.3 Hz, 2H), 7.58–7.54 (m, 1H),
7.47 (d, J ¼ 8.0 Hz, 1H), 7.43 (d, J ¼ 8.3 Hz, 2H), 7.36
(t, J ¼ 8.0 Hz, 1H), 7.32 (d, J ¼ 2.3 Hz, 1H), 6.85 (d, J ¼ 2.3 Hz,
1H), 3.97 (s, 3H), 3.94 (s, 1H), 2.35 (s, 3H), 1.67 (s, 6H) ppm; 13C NMR
(75 MHz, DMSO-d6) d 174.84, 161.79, 159.42, 150.12, 149.91,
148.04, 146.80, 133.99, 132.21, 130.70, 126.48, 124.76, 118.50,
Chemistry
Column chromatography: silica gel (200–300 mesh). 1H, 13C, and
2D-NMR spectra (see also Supporting Information): Bruker DRX-
500 spectrometer; d in ppm rel. to Me4Si, J in Hz. MS: Agilent-
1100-LC/MSD-Trap (ESI-MS) and Agilent Micro-Q-Tof (HR-ESI-MS)
spectrometer; in m/z. Melting points (mp) were recorded on a
WRS-1B digital melting point apparatus (Shenguan, Shanghai,
China).
1,3-Dihydroxy-5-methoxy-9H-xanthen-9-one
(1, C14H10O5)
To a mixture containing phloroglucinol dehydrate (7.56 g,
60 mmol, dried at 1208C overnight) and 2-hydroxy-3-methoxy-
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Arch. Pharm. Chem. Life Sci. 2013, 346, 314–320
Synthesis and Biological Activities of 6-Deoxyisojacareubin
319
113.06, 112.26, 107.83, 86.21, 81.12, 75.87, 58.50, 31.20,
21.33 ppm; HRMS m/z (MþHþ) calcd. for C26H22O7S 479.1159,
found 479.1180.
solids were combined and stirred in water (100 mL) for 1 h.
Recovery by filtration and drying under vacuum yielded the
product as a yellow solid (77 mg, 0.25 mmol, 25%). Rf ¼ 0.51
(hexane/ethyl acetate 4:1); mp 222.5–223.28C; 1H NMR (600 MHz,
DMSO-d6) d 13.00 (s, 1H), 10.40 (s, 1H), 7.56 (dd, J ¼ 7.9, 1.5 Hz,
1H), 7.34 (dd, J ¼ 7.8, 1.5 Hz, 1H), 7.28 (t, J ¼ 7.9 Hz, 1H), 6.99 (d,
J ¼ 10.0 Hz, 1H), 6.24 (s, 1H), 5.80 (d, J ¼ 10.0 Hz, 1H), 3.30 (s, 3H),
1.45 (s, 6H) ppm; 13C NMR (151 MHz, DMSO-d6) d 182.42, 164.07,
162.05, 152.95, 148.02, 146.64, 129.43, 126.27, 123.03, 122.65,
116.53, 116.43, 104.80, 102.78, 100.35, 80.26), 29.70 ppm; HRMS
m/z (MþHþ) calcd. for C18H14O5 311.0914, found 311.0915.
Method II (EtSH–AlBr3): In a dry 10-mL round-bottom flask with
a stir bar under Ar was added AlBr3 solution (1 M in CH2Br2,
3 mL, 3 mmol). The flask was cooled to 08C and EtSH (5.0 mL) was
added dropwise with stirring. After 10 min, compound 5 (81 mg,
0.25 mmol) was added. After 1 h of stirring, the reaction mixture
was cooled to ꢁ308C, quenched with sat. aq. NaHCO3 (15 mL) and
allowed to warm to ambient temperature. Concentration of the
reaction mixture was followed by filtration through a short plug
of alumina (EtOAc and CH2Cl2 as eluent). Purification of the
crude product obtained after evaporation by column chroma-
tography afforded a yellow solid at 50 mg (65% yield).
11-Methoxy-3,3-dimethyl-7-oxo-3,7-dihydropyrano[2,3-c]-
xanthen-6-yl-4-methylbenzenesulfonate (4, C26H22O7S)
A solution of compound 3 (220 mg, 0.46 mmol) in dry DMF
(20 mL) was heated at reflux for 2 h. The solvent was then
vacuum-evaporated and the residue was purified by column
chromatography (silica gel, hexane/ethyl acetate 8:2) to provide
compound 4 (187 mg, 85%), a yellow solid. Rf ¼ 0.31 (hexane/
ethyl acetate 4:1); 1H NMR (400 MHz, DMSO-d6) d 7.79 (d, J ¼ 8.2,
2H), 7.53 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.45 (dd, J ¼ 8.1, 1.4 Hz, 1H),
7.42 (d, J ¼ 8.2 Hz, 2H), 7.37–7.31 (m, 1H), 6.80 (d, J ¼ 10.1 Hz,
1H), 6.42 (s, 1H), 5.99 (d, J ¼ 10.1 Hz, 1H), 3.99–3.94 (m, 3H), 2.35
(s, 3H), 1.46 (s, 6H) ppm; mp 166.8–168.28C; 13C NMR (101 MHz,
DMSO-d6) d 172.62 156.62, 151.78, 148.03, 147.05, 145.76, 144.57–
143.35, 131.73, 131.41, 129.89, 128.52, 124.26, 122.33, 116.23,
113.85, 109.46, 108.55, 108.22, 79.01, 56.48, 27.67, 21.12 ppm;
HRMS: m/z (MþHþ) calcd. for C26H22O7S 479.1159, found
479.1163.
6-Hydroxy-11-methoxy-3,3-dimethylpyrano[2,3-c]-
xanthen-7(3H)-one (5, C19H16O5)
Biology
Cell culture
A solution of potassium hydroxide (600 mg) in water (10 mL) and
ethanol (10 mL) was prepared. The alkaline solution (20 mL) was
added to compound 4 (80 mg, 0.17 mmol) in three 5-mL portions
at 15-min intervals. After refluxing for 1 h, the solution was
cooled, neutralized with acetic acid, and concentrated under
diminished pressure. The mixture was extracted with ether;
the combined organic layer was washed successively with aque-
ous sodium hydrogen carbonate and 3% aqueous sodium hydrox-
ide solution and finally dried over magnesium sulfate. After
removal of the solvent, the residue was purified by column
chromatography (silica gel, hexane/ethyl acetate 4:1). At last,
we obtained a white solid 5 (41 mg, 72.69%). Rf ¼ 0.75 (hex-
The QGY-7703 cell line was obtained from the Cell Resource
Center, Institute of Life Sciences, Chinese Academy of
Medical Science. QGY-7703 cells were maintained in 1640
medium (Gibco) supplemented with 10% fetal bovine serum
and 100 U/mL penicillin/streptomycin at 378C, 5% CO2.
Cell proliferation/viability assay
QGY-7703 cells were seeded in 96-well microplates (2 ꢃ 105 cells/
well in 100 mL medium) for 24 h. For the viability assay, varying
concentrations of compounds or 1% DMSO vehicle control were
added to the wells; the cells were then maintained at 378C
for 48 h. After incubation, the cell viability was measured by
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-
2H-tetrazolium, monosodium salt (WST-8) using the Cell
Counting Kit-8 (Dojindo, Tokyo, Japan), which reflects mitochon-
drial succinate dehydrogenase function [18, 19]. In brief, 10 mL of
CCK-8 solution was added to each well of the plate. Then, the
plate was incubated at 378C for an additional 3 h. The absorb-
ance of the WST-8 formazan dye was measured at 450 nm by an
Epoch microplate spectrophotometer. The relative cell viability
was presented as % inhibition calculated using the formula:
ane/ethyl acetate 4:1); mp 180–1828C; IR (KBr, film): n (cmꢁ1
)
3443, 3420, 2024, 1650, 1613, 1578, 1498, 1482, 1437, 1410,
1383, 1355, 1321, 1293, 1273, 1226, 1192, 1172, 1151, 1118,
1096, 1076, 973, 891, 825, 794, 752, 722, 701, 626, 422.
1H NMR (600 MHz, DMSO-d6) d 12.90 (s, 1H), 7.67 (dd, J ¼ 8.0,
1.4 Hz, 1H), 7.55–7.53 (m, 1H), 7.41 (dd, J ¼ 8.0 Hz, 1.4 Hz, 1H),
6.75 (d, J ¼ 10.0 Hz, 1H), 6.27 (d, J ¼ 0.6 Hz, 1H), 5.84
(d, J ¼ 10.0 Hz, 1H), 4.00 (s, 3H), 1.46 (s, 6H) ppm; 13C NMR
(151 MHz, DMSO-d6) d 182.10, 163.98, 162.04, 152.79, 149.97,
147.16, 129.79, 126.15, 122.30, 118.82, 117.52, 115.87, 104.86,
102.69, 101.34, 100.43, 80.36, 58.31, 29.66 ppm; HRMS: m/z
(MþHþ) calcd. for C19H16O5 325.1071, found 325.1067.
ꢀ
ꢁ
ODtreated
ODcontrol
% Inhibition ¼ 1 ꢁ
ꢃ 100
6,11-Dihydroxy-3,3-dimethylpyrano[2,3-c]xanthen-7(3H)-
one (6, C18H14O5)
Method I: Compound 5 (324 mg, 1 mmol) was dissolved in DCM
(16 mL) and stirred under argon. BBr3 (4 mL, 1 M in 100 mmol
DCM) was added dropwise to the solution and the mixture was
allowed to stir at room temperature for 4 h. The reaction mix-
ture was poured slowly into a stirred solution of saturated
aqueous sodium bicarbonate (400 mL) and allowed to stir for
1 h. The resulting colorless precipitate was recovered by filtra-
tion. The filtrate was neutralized with HCl (1 M) to pH 7, to yield
further product, which was also recovered by filtration. Both
PKC activity assay
The PKC activity assay was carried out according to the instruc-
tions of the PepTag non-radioactive PKC assay kit (Promega,
Madison, WI, USA) [20]. Briefly, test compounds were added to
the serum-free medium, and the cells with or without the com-
pounds were incubated at 378C for 3 h. The medium was
removed; then, the cells were homogenized in lysis buffer
(25 mM Tris–HCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.1% Triton
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

320
M. Dai et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 314–320
X-100, 10 mM b-mercaptoethanol, 1 mg/L leupeptin, 1 mM phenyl-
methanesulfonyl fluoride). Reactions were then performed at 308C
in a final volume of 25 mL and terminated after 30 min by
incubation of the reaction mixture at 958C for 10 min. After
adding 1 mL of 80% glycerol, each sample was loaded onto an
agarose gel (0.8% agarose in 50 mM Tris–HCl, pH 8.0) and sep-
arated at 120 V for 15 min. For quantification, bands were photo-
graphed by the Syngene GBOX-iCHEMI-XR system and the
phosphorylated bands were cut out for measurement at
570 nm. The results are expressed in OD units and differences
between two groups were analyzed by Student’s t-test.
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The work was supported by program NCET Foundation, NSFC (81230090),
partially supported by the Global Research Network for Medicinal Plants
(GRNMP) and King Saud University, Shanghai Leading Academic
Discipline Project (B906), FP7-PEOPLE-IRSES-2008 (TCMCANCER Project
230232), the Key Laboratory of Drug Research for Special Environments,
PLA, the Shanghai Engineering Research Center for the Preparation of
Bioactive Natural Products (10DZ2251300) and the Scientific Foundation of
Shanghai China (09DZ1975700, 09DZ1971500, 10DZ1971700). National
Major Project of China (2011ZX09307-002-03). National Key Technology
R&D Program of China (2012BAI29B06).
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www.archpharm.com