Synthesis and Antiproliferative Activity of Natural and Non-Natural Polymethoxychalcones and Polymethoxyflavones

Article abstract of DOI:10.1007/s10600-019-02605-x

Two series of polymethoxychalcones and polymethoxyflavones, including the natural products 2′-hydroxy-3,4,5,4′,6′-pentamethoxychalcone (8c), 5,6,7,8,3′,4′,5′-heptamethoxyflavone (6), 5,7,3′,4′,5′-pentamethoxyflavone (9c), 3-hydroxy-5,6,7,8,3′,4′,5′-heptam

Full text of DOI:10.1007/s10600-019-02605-x

DOI 10.1007/s10600-019-02605-x
Chemistry of Natural Compounds, Vol. 55, No. 1, January, 2019
SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY OF NATURAL
AND NON-NATURAL POLYMETHOXYCHALCONES
AND POLYMETHOXYFLAVONES
1,2
1
Kingsadingthongkham Vongdeth, Peipei Han,
Wei Li, and Qiu-An Wang
1
1*
Two series of polymethoxychalcones and polymethoxyflavones, including the natural products 2′-hydroxy-
3,4,5,4′,6′-pentamethoxychalcone (8c), 5,6,7,8,3′,4′,5′-heptamethoxyflavone (6), 5,7,3′,4′,5′-pentamethoxyflavone
(9c), 3-hydroxy-5,6,7,8,3′,4′,5′-heptamethoxyflavone (7), and 3-hydroxy-5,7,3′,4′,5′-pentamethoxyflavone
(10), were synthesized. The antiproliferative activity in vitro was evaluated against a panel of three human
cancer cell lines (HeLa, HCC1954, and SK-OV-3) by the cell counting kit-8 (CCK-8) assay. The results
showed that most of the synthetic compounds exhibited moderate to potent antiproliferative activities.
Some compounds displayed equal or higher potential than the positive control drug cisplatin. In particular,
compounds 4c, 4e, 8a, and 9a possess IC values equal to or below 10 μM and are worthy of further
50
investigation.
Keywords: polymethoxychalcone, polymethoxyflavone, synthesis, antiproliferative activity.
Flavones are a large and well-known family of compounds of natural and synthetic origin with great structural variety
responsible for a wide range of biological activities and pharmacological effects [1–3]. Chalcones are prominent secondary
metabolite precursors of flavonoids and isoflavonoids in plants. Chemically, they consist of two aromatic rings linked by a
three-carbon α,β-unsaturated system and are known to exhibit an impressive array of biological properties [4, 5].
Over the past two decades, increasing attention has been given to polymethoxyflavones (PMFs) [6, 7]. It was reported
that flavones and chalcones containing a high number of methoxy groups are effective as colorectal cancer chemopreventive
agents, and polymethoxylated flavonoids and chalcones in general appear to have great potential as cancer chemopreventive/
chemotherapeutic agents in contrast with their nonmethylated analogues, even though they occur in much lower concentrations
[8–10].
However, systematic studies on the synthesis and structure–activity relationships of PMFs have not been widely
reported. Due to our continuous interest in discovering and developing flavones and chalcones as potential anticancer compounds
[11–13], in the present study, we developed a versatile method for the synthesis of natural and non-natural polymethoxychalcones
and polymethoxyflavones, and evaluated in vitro their antiproliferative activities on a panel of three human cancer cell lines,
including HeLa (cervical carcinoma), HCC1954 (breast cancer), and SK-OV-3 (ovarian cancer) by the CCK-8 assay.
H C
3
Br
Br
OCH
3
OCH
3
H CO
3
OCH
Br
OCH
O
HO
H CO
OCH
OCH
3
3
3
3
3
CuBr, NaOCH
3
Br Cu
Br
O
OCH
3
CH
3
I
1
1) College of Chemistry and Chemical Engineering, Hunan University, 410082, Changsha, P. R. China, e-mail:
wangqa@hnu.edu.cn; 2) Department of Chemistry, Faculty of Natural Sciences, National University of Laos, Dong Dok
Campus, North 13 rd, Vientiane Capital, Lao PDR, P. O. Box 7322. Published in Khimiya Prirodnykh Soedinenii, No. 1,
January–February, 2019, pp. 12–17. Original article submitted July 13, 2017.
0009-3130/19/5501-0011 ©2019 Springer Science+Business Media, LLC
11

Br
OCH
OCH
3
3
H CO
3
OCH
H CO
3
OCH
Br
HO
H CO
OCH
H CO
OCH
OCH
3
3
3
3
3
3
3
a
c
d
b
Br
OCH
H CO
3
3
5
1
2
R
1
CH
R
R
-
3
2
H CO
Cl
3
OCH
OCH
3
3
OH
Cl
Cl
O
+
OH
O
H CO
3
H CO
H CO
3
Al
3
-CH Cl
3
e
3
H O
2
O
3
H CO
3
H CO
3
H CO
3
OCH
O
3
CH
H CO
3
H CO
3
II
3
4a-e
OCH
OCH
3
3
OCH
OCH
OCH
OCH
3
3
3
OCH
OCH
3
O
3
H CO
H CO
O
3
3
f
g
3
4e
H CO
3
OH
H CO
3
O
O
H CO
3
H CO
3
6
7
OCH
OCH
3
3
R
R
R
4
4
R
4
OH
O
O
H CO
3
OCH
O
H CO
3
R
5
H CO
3
H CO
3
3
f
R
5
5
h
+
OH
CHO
O
H CO
3
H CO
3
8a-c
9a-c
OCH
3
OCH
3
3
O
O
H CO
3
OCH
4a: R = R = H, R = OH; 4b: R = OCH , R = OH, R = H
1
3
2
1
3
2
3
g
4c: R = OH, R = OCH , R = H; 4d: R = R = OH, R = OCH
9c
OH
10
a. Br , CH Cl , r.t; b. CuBr, NaOCH , DMF, microwave, 120°C; c. (CH ) SO , K CO , acetone, reflux; d. CH COCl, AlCl , ether, r.t;
1
2
3
3
1
2
3
3
4e: R = R = R = OCH ; 8a, 9a: R = R = OH
1
2
3
3
4
5
H CO
3
8b, 9b: R = OH, R = OCH ; 8c, 9c: R = R = OCH
4
5
3
4
5
3
2
2
2
3
3 2
4
2
3
3
3
e. substituted benzaldehydes, 50% NaOH (aq), C H OH, r.t; f. I , DMSO, H SO , reflux; g. oxone, acetone–CH Cl , Na CO –NaHCO ,
2
5
2
2
4
2
2
2
3
3
5°C; h. 50% NaOH (aq), C H OH, r.t.
2
5
Scheme 1.
Base on the results, we proposed a reaction mechanism that includes the intermediate I, in which CuBr coordinates
with the two ortho-position methoxy groups. The complex is the subsequent thermal elimination of volatile CH Br to form the
3
salt of 1, which is converted under acidic conditions into 1. Although we did not find in the literature the detailed mechanism
of this reaction, it seems that nucleophilic attack of the bromide anion on the methyl group is involved in the demethylation
reaction. Such a mechanism has been proposed for the catalytic demethoxylation of aryl ethers at the position ortho to the
carbonyl group by magnesium iodide [14].
Methoxylation of 1 was achieved using dimethylsulfate as methylating agent in the presence of anhydrous potassium
carbonate and acetone to yield pentamethoxybenzene (2) (Scheme 1). 2-Hydroxy-3,4,5,6-tetramethoxyacetophenone (3),
prepared from 2 with Friedel-Crafts acetylation and demethylation with aluminium trichloride, proceeded in a highly
regioselective manner including the intermediate II. Claisen-Schmidt aldol condensation of the acetophenone 3 or 2-hydroxy-
4,6-dimethoxyacetophenone with various hydroxyl or methoxyl-substituted benzaldehyde yielded a series of
polymethoxychalcones 4a–e and 8a–c. All synthesized chalcones were purified by recrystallization or chromatography, and
the analytical and spectroscopic data confirmed their structures, as detailed in the experimental section. The coupling constant
of J = 15–16 Hz observed in olefinic protons Hα and Hβ indicates the formation of only the expected E isomers. Compound 8c
is a natural product isolated from Merrillia caloxylon (Rutaceae) [15].
12

TABLE 1. Half-inhibitory Concentrations of Compounds 4a–e, 8a–c, 6, 9a–c, 7, and 10 on the Human Cancer Cell Lines
(IC , μM)
50
Compound
Hela
HCC1954
SK-OV-3
34.09
29.23
1.44
32.95
4.99
33.85
25.25
9.28
19.47
15.98
8.26
> 100
15.05
> 100
8.58
> 100
53.84
62.33
> 100
29.32
0.009
0.037
56.41
32.53
1.60
4a
4b
4c
48.40
10.53
12.97
> 100
23.76
63.04
10.64
> 100
30.17
31.19
> 100
18.66
0.0028
0.0031
4d
4e
8a
8b
8c
6
9a
9b
9c
7
5.14
> 100
> 100
35.80
4.83
> 100
40.82
28.56
> 100
13.30
0.0055
0.0112
10
Cisplatina
Paclitaxela
Staurosporina
______
a
Cisplatin, paclitaxel, and staurosporin were employed as positive controls.
The synthesis of polymethoxychalcones and polymethoxyflavones was accomplished according to the general pathway
in Scheme 1. The approaches rely on polymethoxyacetophenone 3 as a key compound. However, efficient synthesis of such a
highly oxygenated acetophenone derivative remains to be resolved. With this mind, we commenced with the development of
an alternative access to key compound 4. We chose commercially available 1,3-dimethoxybenzene as a starting material.
Our efforts concentrated on methoxylation of the 2,4,6-positions of 1,3-dimethoxybenzene using an Ulmann-type reaction.
1,3-Dimethoxybenzene was brominated with bromide in methylene chloride to give 1,3,5-tribromo-2,4-dimethoxybenzene (5).
Compound 5 was subjected to a CuBr-mediated Ulmann-type reaction with a large excess amount of sodium methoxide,
which gave rise to 2-hydroxy-1,3,4,5-tetramethoxybenzene instead of the anticipated pentamethoxybenzene (2).
The treatment of chalcone 4e and 8a–c with concentrated H SO and a catalytic amount of iodine in DMSO under
2
4
reflux gave the desired flavones 6 and 9a–c in good yield. Compound 6 is a natural product isolated from Eupatorium coelestinum
[16]. Introduction of a 3-OH group to flavones 6 and 9c was achieved using dimethyldioxirane (DMDO), followed by opening
of the formed epoxide with catalytic p-toluenesulfonic acid to afford the desired flavonols 7 and 10 in 63–65% yield. Compound 7
is another natural product isolated from Athrixia phylicoides (Asteraceae) [17]. Compounds 9c and 10 are also natural products
isolated from Merrillia caloxylon (Rutaceae) [18].
The synthesized target chalcones and flavones were investigated for their antiproliferative activity in vitro employing
the CCK-8 assay with cisplatin, paclitaxel, and staurosporin as positive control against three human cancer cell lines (HeLa,
HCC1954, and SK-OV-3). The antiproliferative activities of compounds, as indicated by IC values, were calculated by
50
linear regression analysis of the concentration-response curves obtained for each compound. The results from the antiproliferation
assay are summarized in Table 1.
The result indicated that most of these polymethoxychalcones and flavonoids exhibited moderate to potent
antiproliferative activities, except compounds 8b, 9b, and 10, toward all three cells. The activity of compound 8c to HeLa cells
and compound 6 to HCC1954 was negative at the highest tested concentration (> 100 μM). Some compounds displayed
equal or higher (lower IC values) potential than the positive control drug cisplatin. Compounds 4c, 4e, 8a, and 9a were
50
more potent against HeLa cells, with IC values of 1.44–5.13 μM, than the positive control cisplatin (IC 13.30 μM).
50
50
Compounds 4b–e, 8a, 8c, and 9a were more potent against HCC1954 cells, with IC values of 8.26–25.25 μÌ, than the
50
positive control cisplatin (IC 29.32 μM), and compounds 4c, 4e, 8a, and 9a were more potent against SK-OV-3 cells, with
50
IC values of 1.60–12.97 μM, than the positive control cisplatin (IC 18.66 μM).
50
50
It is interesting to note that several compounds showed promising cytotoxicity, such as 4c, 4e, 8a, and 9a, which give
IC values equal to or below 10 μM. Compound 4c is the most potent among all the tested compounds, with the highest
50
13

potency toward HeLa cells (IC 1.44 μM) and SK-OV-3 cells (IC 1.60 μM), which are 9-fold and 12-fold greater compared
50
50
to the positive control cisplatin, respectively. The antiproliferative activity of compound 8c to HCC1954 and SK-OV-3 and
compound 6 to HeLa and SK-OV-3 was selective.
Preliminary structure–activity relationship analysis revealed that the position of the hydroxyl or methoxyl group and
the number of methoxyl groups may be closely linked with their antiproliferative activity against these cancer cells. These
compounds contain a high number of methoxyl groups and thus exhibit higher antiproliferative activity. Compound 8a and 9a,
which contain two hydroxyl groups (at the ortho position) in the B-ring, significantly increase the antiproliferative activity,
with IC values in the range of 4.83–12.97 μM. Chalcone 8b and flavone 9b, having 3,5-dimethoxy-4-hydroxy group in the
50
B-ring, did not demonstrate any antiproliferative activity below 100 μM concentration. Compound 10, which has a hydroxyl
group at the 3-position, also showed inactivity, while the presence of 3,4-dihydroxyl-5-methoxyl phenyl or 3,4,5-trimethoxyl
aryl ring moieties was very important with respect to the inhibitory activity against these three cancer cell lines. Clarification
of the structural determinants of potency will guide the design of novel potent molecules for future development.
To the best of our knowledge, such observations on the antiproliferative activity against these three cells have not
been reported previously. Although we have limited information on the structure–activity relationship (SAR), the findings of
this study provide important information for the exploitation and utilization of PMFs as anticancer agents.
In summary, eight polymethoxychalcones 4a–e and 8a–c and six polymethoxyflavones 6, 9a–c, 7, and 10 were
synthesized. Among the synthesized target compounds, 8c, 6, 9c, 7, and 10 are natural products. The majority of the target
compounds exhibited moderate to potent antiproliferative activities against human cancer cell lines (HeLa, HCC1954, and
SK-OV-3). Among them, compounds 4c, 4e, 8a, and 9a were the most active with IC values ranging from 1.44 to 15.98 μM
50
against all three cancer cell lines and are worthy of further development.
EXPERIMENTAL
General Experimental Procedures. Melting points were measured on an XRC-I apparatus and were uncorrected.
Microwave reaction was performed with an XH-MC-1 microwave reactor (Beijing Xianghua Science and Technology
1
13
Development Co., China). H and C NMR spectra were recorded on a Bruker AM-400 instrument using tetramethylsilane as
an internal standard. Chemical shifts (δ) are in ppm, and coupling constants (J) are in Hz. Mass spectra (MS) or high-resolution
mass spectrometry (HR-MS) was determined with a VG Autospec-3000 or Mat 95 XP spectrometer by the ESI or FAB
method. Column chromatography was carried out on 200–300 mesh silica gel (Qingdao Ocean Chemical Products of China).
Commercially available AR or chemically pure reagents and anhydrous solvents with the water removed and redistilled were
employed.
3,4-Dihydroxy-5-methoxybenzaldehyde and 3,4,5-trimethoxybenzaldehyde were prepared according to our previous
methods [19]. 2-Hydroxy-4,6-dimethoxyacetophenone was prepared from resorcinol via Friedel-Crafts acetylation and selective
O-methylation [20].
Synthesis of 1,3,5-Tribromo-2,4-dimethoxybenzene (5). To a solution of 1,3-dimethoxybenzene (5 g, 36 mmol) in
CH Cl (30 mL) at room temperature was added bromine (5 mL), and the reaction mixture was stirred for 1 h at room
2
2
temperature. The reaction mixture was taken up in water and extracted with CH Cl . Standard workup of the organic layer
2
2
1
provided a white crystallized residue 13.13 g, yield 98%; mp 70–71°C [lit. [21] 70–71°C]. H NMR (400 MHz, CDCl ,
3
δ, ppm): 7.63 (1H, s, Ar-H), 3.88 (6H, s, OCH ).
3
Synthesis of 2,3,4,6-Tetramethoxyphenol (1). To a solution of 5 (1 g, 2.7 mol) in DMF (20 mL) at room temperature
were added CuBr (1.79 g, 12.62 mmol) and NaOCH (0.44 g, 8.1 mmol) in CH OH (15 mL), and the reaction mixture was
3
3
exposed to microwave irradiation (700 W) at 120°C for 1 h and then cooled to room temperature. The cooled reaction mixture
was taken up in water (50 mL) and extracted at pH 6 with ethyl acetate (3 × 50 mL), and the organic layer was dried over
anhydrous Na SO . The solvent was evaporated to yield a residue that was chromatographed on silica gel using petroleum
2
4
1
ether–ethyl acetate (7:3) as eluent to afford a white solid 430 mg, yield 75%; mp 85–86°C [lit. [22] 84–85°C] H NMR
(400 MHz, CDCl , δ, ppm): 6.84 (1H, s, Ar-H), 5.25 (1H, s, OH), 3.89, 3.86, 3.83, 3.65 (each 3H, s, OCH ). MS (FAB )
m/z 215 [M + 1] .
Synthesis of 1,2,3,4,5-Pentamethoxybenzene (2). A solution of 1 (1 g, 4.6 mmol) and anhydride K CO (2 g,
+
3
3
+
2
3
13.53 mmol) in dry acetone (20 mL) was refluxed for 30 min. Then dimethyl sulfate (0.5 mL, 5.19 mmol) was added dropwise.
The reaction mixture was stirred at 30°C for 4 h, filtered, and the filtrate evaporated to afford a crude solid, which was
14

crystallized from petroleum ether–ethyl acetate (3:1) to afford a white solid (950 mg, yield 91%, mp 57–58°C [lit. [21]
1
57–58°C]). H NMR (400 MHz, CDCl , δ, ppm): 3.83 (6H, s, OCH ), 3.85 (6H, s, OCH ), 3.96 (3H, s, OCH ), 6.30 (1H, s,
3
3
3
3
+
+
Ar-H). MS (FAB ) m/z 229 [M + 1] .
Synthesis of 2-Hydroxy-3,4,5,6-tetramethoxyacetobenzene (3). To a solution of 2 (1 g, 4.4 mmol) and AlCl
3
anhydride (1.15 g, 9 mmol) in dry ether (20 mL) was added dropwise acetyl chloride (1.05 g, 15 mmol). The reaction mixture
was stirred at room temperature for 5 h and acidified with 2 N aqueous HCl. The mixture was stirred for a further 2 h and
extracted with EtOAc (3 × 50 mL). The organic phase was washed with 5% NaOH (aq), then neutralized with 2N HCl (aq) and
dried over anhydrous Na SO . The solvent was evaporated, and the crude residue was chromatographed on silica gel using
2
4
1
petroleum ether–ethyl acetate (8:2) to afford a yellow oil 0.35 g, yield 31%. H NMR (400 MHz, CDCl , δ, ppm): 13.08 (1H,
s, OH), 4.00, 3.88, 3.78, 3.73 (each 3H, s, OCH ), 2.60 (3H, s, C(O)CH ). ESI-MS m/z 255 [M – H] .
3
–
3
3
General Procedure for the Synthesis of Chalcones 4a–e and 8a–c. To a stirred solution of substituted benzaldehyde
(2.0 g, 15.62 mmol) and 2-hydroxy-3,4,5,6-tetramethoxyacetophenone or 2-hydroxy-4,6-dimethoxyacetophenone (2.5 g,
15.62 mmol) in ethanol (30 mL) was added 20 mL of 20% aqueous KOH, and the reaction mixture was stirred at room
temperature for 48 h. The reaction mixture was cooled to 0°C (ice-water bath) and acidified with 10% aqueous HCl. A yellow
precipitate was formed, which was filtered and washed with 10% aqueous HCl. The yellow solid was recrystallized from
petroleum ether–EtOAc to give chalcone 4a–e and 8a–c.
1
4,2′-Dihydroxy-3′,4′,5′,6′-tetramethoxychalcone (4a). Yellow solid, yield 84%, mp 156–158°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 13.22 (1H, s, 3-OH), 7.77 (1H, d, J = 15.5, H-β), 7.71 (1H, d, J = 15.6, H-α), 7.46 (2H, d, J = 7.6, H-2, 6),
3
6.82 (2H, d, J = 7.7, H-3, 5), 6.22 (1H, s, 2′-OH), 4.02 (3H, s, 3′-OCH ), 3.82 (3H, s, 4′-OCH ), 3.81 (3H, s, 5′-OCH ), 3.79
3
3
3
–
(3H, s, 6′-OCH ). ESI-MS m/z 359 [M – H] .
3
1
4,2′-Dihydroxy-3,3′,4′,5′,6′-pentamethoxychalcone (4b). Yellow solid, yield 87%, mp 94–96°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 13.18 (1H, s, 4-OH), 7.74 (1H, d, J = 15.5, H-β), 7.69 (1H, d, J = 15.5, H-α), 7.14 (1H, dd, J = 8.2, 1.5,
3
H-6), 7.03 (1H, d, J = 1.4, H-2), 6.86 (1H, d, J = 8.2, H-5), 6.11 (1H, s, 2′-OH), 4.01 (3H, s, 3′-OCH ), 3.85 (3H, s, 4′-OCH ),
3
3
13
3.80 (6H, s, 5′, 6′-OCH ), 3.78 (3H, s, 3-OCH ). C NMR (100 MHz, CDCl , δ, ppm): 192.50, 153.86, 152.27, 149.84,
3
3
3
147.42, 145.89, 143.49, 137.42, 136.26, 126.74, 122.85, 122.34, 114.01, 110.13, 109.33, 61.21, 60.60, 60.33, 60.03, 54.95.
+
ESI-MS m/z 391 [M + H] .
1
3,2′-Dihydroxy-4,3′,4′,5′,6′-pentamethoxychalcone (4c). Yellow solid, yield 92%, mp 94–96°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 13.23 (1H, s, 3-OH), 7.68 (1H, d, J = 15.5, H-β), 7.65 (1H, d, J = 15.5, H-α), 7.14 (1H, d, J = 1.5, H-2),
3
6.98 (1H, dd, J = 8.3, 1.5, H-6), 6.72 (1H, d, J = 8.3, H-5), 6.26 (1H, s, 2′-OH), 3.96 (3H, s, 3′-OCH ), 3.76 (6H, s, 5′, 6′-OCH ),
3
3
13
3.75 (3H, s, 4′-OCH ), 3.74 (3H, s, 4-OCH ). C NMR (100 MHz, CDCl , δ, ppm): 193.55, 154.82, 153.28, 0.97, 149.25,
3
3
3
146.06, 144.30, 138.40, 137.11, 128.59, 124.26, 122.87, 113.30, 111.07, 110.74, 62.03, 61.50, 61.29, 60.96, 55.90. ESI-MS
+
m/z 391 [M + H] .
1
3,4,2′-Trihydroxy-5,3′,4′,5′,6′-pentamethoxychalcone (4d). Yellow solid, yield 90%, mp 76–78°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 13.20 (1H, s, 4-OH), 7.73 (1H, d, J = 15.5, H-β), 7.68 (1H, d, J = 15.5, H-α), 7.12 (1H, d, J = 8.2, 1.4,
3
H-2), 7.02 (1H, d, J = 1.1, H-5), 6.85 (1H, d, J = 8.2, 3-OH), 6.23 (1H, s, 2′-OH), 4.00 (3H, s, 3′-OCH ), 3.83 (3H, s, 4′-OCH ),
3
3
13
3.80 (6H, s, 5, 5′-OCH ), 3.78 (3H, s, 6′-OCH ). C NMR (100 MHz, CDCl , δ, ppm): 193.51, 154.87, 153.29, 150.86,
3
3
3
148.50, 146.96, 144.56, 138.43, 137.26, 127.72, 123.81, 123.36, 115.07, 111.13, 110.36, 62.22, 61.61, 61.34, 61.04, 55.93.
+
+
MS (FAB ) m/z 407 [M + 1] .
2′-Hydroxy-3,4,5,3′,4′,5′,6′-heptamethoxychalcone (4e). Yellow solid, yield 75%, mp 89–91°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 7.82 (1H, d, J = 15.5, H-β), 7.77 (1H, d, J = 15.6, H-α), 6.87 (2H, s, H-2, 6), 4.10 (3H, s, OCH ), 3.92
1
3
3
13
(6H, s, OCH ), 3.89 (9H, s, OCH ), 3.87 (3H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm): 190.1, 157.7, 151.2, 150.1,
147.5, 140.7, 137.1, 135.1, 133.9, 127.4, 122.4, 107.7, 102.4, 59.0, 58.4, 58.1, 57.8, 57.7, 52.9. ESI-MS m/z 433 [M – H] .
3
3
3
3
–
1
3,4,2′-Trihydroxy-5,4′,6′-trimethoxychalcone (8a). Yellow solid, yield 74%, mp 162–164°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 14.23 (1H, s, 2′-OH), 7.67 (1H, d, J = 15.5, H-β), 7.56 (1H, d, J = 15.5, H-α), 7.32 (1H, s, H-2), 6.93
3
13
(1H, s, H-6), 6.15 (1H, s, H-3′), 6.03 (1H, s, H-5′), 5.89 (1H, s, 2′-OH), 3.88, 3.84, 3.76 (each 3H, s, OCH ). C NMR (100 MHz,
3
CDCl , δ, ppm): 191.14, 167.37, 165.21, 161.38, 146.20, 143.84, 140.10, 127.85, 125.48, 124.13, 108.80, 107.69, 105.23,
3
+
92.80, 90.29, 55.34, 54.89, 54.59. ESI-MS m/z 347 [M + H] .
1
4,2′-Dihydroxy-3,5,4′,6′-tetramethoxychalcone (8b). Yellow solid, yield 76%, mp, 89–91°C. H NMR (400 MHz,
DMSO-d , δ, ppm, J/Hz): 13.51 (1H, s, 2′-OH), 9.09 (1H, s, 4-OH), 7.66 (1H, d, J = 15.6, H-β), 7.63 (1H, d, J = 15.5, H-α),
6
13
7.02 (2H, s, H-2, 6), 6.13 (2H, d, J = 7.9, H-3′, 5′), 3.90, 3.85, 3.82 (12H, each s, 4 × OCH ). C NMR (100 MHz, DMSO-d ,
3
6
δ, ppm): 192.71, 165.71, 165.60, 162.15, 148.59, 144.30, 139.12, 125.64, 125.10, 106.94, 106.76, 94.34, 91.41, 56.53, 56.48,
–
56.04. ESI-MS m/z 359 [M – H] .
15

2
′
-Hydroxy-3,4,5,4
′
,6′-pentamethoxychalcone (8c). Yellow solid, yield 85%, mp 188–190°C [lit. [18] 186–187°C].
1
H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 14.33 (1H, s, OH), 7.82 (1H, d, J = 15.5, H-β), 7.72 (1H, d, J = 15.5, H-α), 6.86
3
(2H, s, H-2, 6), 6.13 (1H, d, J = 2.3, H-5′), 5.98 (1H, d, J = 2.3, H-3′), 3.93 (9H, s, 3, 4, 5-OCH ), 3.91 (6H, s, 4′, 6′-OCH ).
3
3
13
C NMR (100 MHz, CDCl , δ, ppm): 192.2, 168.3, 166.1, 162.3, 153.3, 142.3, 139.0, 131.0, 126.8 106.2, 105.5, 93.8, 91.2,
3
–
61.0, 56.0, 55.7. ESI-MS m/z 373 [M – H] .
General Procedure for Synthesis of Polymethoxyflavones 6, 9a–c. Polymethoxychalcones 4e, 8a–c (0.46 mmol)
and iodine (18.5 mg, 0.074 mmol) were added to a solution of conc. H SO (0.07 mL) in DMSO (5 mL). The mixture was
2
4
warmed to 80–85°C and stirred for 24 h. When the reaction was over (TLC analysis), saturated aqueous Na S O was added.
2 2
3
Then a mixture of 1N HCl (aq)/ethyl acetate (30 mL:30 mL) was added at room temperature. The organic layer was discarded,
and concentrated ammonium hydroxide was added the aqueous solution until it was clearly basic (pH > 9). The product was
extracted with ethyl acetate (3 × 30 mL). The extract was washed with brine and dried over anhydrous Na SO , and the solvent
2
4
was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluent, petroleum ether–
EtOAc, 8:2–6:4) to afford the desired products.
5,6,7,8,3′,4′,5′-Heptamethoxyflavone (6). Yellow solid, yield 62%, mp 105–106°C [lit. [16]: 101.5–102.5°C].
1
H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.12 (2H, s, H-2′, 6′), 6.67 (1H, s, H-3), 4.12, 4.03, 3.99 (each 3H, s, OCH ), 3.87
3
3
13
(6H, s, OCH ), 3.85 (3H, s, OCH ), 3.79 (3H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm): 189.8, 176.0, 160.3, 158.9,
152.5, 147.7, 144.1, 125.4, 112.7, 111.0, 108.7, 103.3, 91.6, 59.9, 59.8, 58.1, 58.1, 57.9, 57.7. ESI-MS m/z 433 [M + H] .
3
3
3
3
+
1
4′,5′-Dihydroxy-5,7,3′,-trimethoxyflavone (9a). Yellow solid, yield 75%, mp 241–243°C. H NMR (400 MHz,
DMSO-d , δ, ppm, J/Hz): 9.20 (1H, s, OH), 9.04 (1H, s, OH), 7.15 (1H, s, H-2′), 7.07 (1H, s, H-6′), 6.65, 6.57 (1H, d, J = 2.3,
6
13
H-8), 6.43 (1H, d, J = 2.3, H-6), 6.34 (1H, s, H-3), 3.93, 3.89, 3.82 (each 3H, s, OCH ). C NMR (100 MHz, DMSO-d ,
3
6
δ, ppm): 179.26, 169.01, 168.34, 159.25, 148.76, 146.35, 146.20, 137.38, 122.76, 112.68, 111.72, 108.05, 104.78, 94.76,
+
90.14, 56.92, 56.56, 56.47. ESI-MS m/z 345 [M + H] .
1
4′-Hydroxy-5,7,3′,5′-tetramethoxyflavone (9b). Yellow solid, yield 75%, mp 240–242°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 7.07 (2H, s, H-2′, 6′), 6.62 (1H, d, J = 2.4, H-8), 6.26 (1H, d, J = 2.4, H-6), 6.06 (1H, s, H-3), 5.77 (1H,
3
13
s, OH), 3.89, 3.88, 3.85 (12H, each s, 4 × OCH ). C NMR (100 MHz, CDCl , δ, ppm): 179.47, 167.75, 167.69, 158.39,
146.11, 145.79, 135.63, 122.98, 110.63, 107.35, 104.40, 92.98, 88.18, 55.38, 55.19, 55.12. ESI-MS m/z 359 [M + H] .
3
3
+
1
5,7,3′,4′,5′-Pentamethoxyflavone (9c). Yellow solid, yield 65%, mp 200–202°C [lit. [18]: 195–196°C]. H NMR
(400 MHz, CDCl , δ, ppm): 7.01 (2H, s, H-2′, 6′), 6.57 (1H, s, H-3), 6.51 (1H, d, J = 2.3, H-8), 6.33 (1H, d, J = 2.3, H-6), 3.90
3
13
(3H, s, OCH ), 3.89 (6H, s, OCH ), 3.86 (3H, s, OCH ), 3.85 (3H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm): 177.5,
164.2, 160.9, 160.6, 159.8, 153.5, 140.9, 130.9, 128.8, 126.6, 103.4, 96.2, 92.9, 61.0, 56.3, 55.8. MS (FAB ) m/z 373 [M + 1] .
Synthesis of Polymethoxyflavones 7 and 10. A solution of flavones 6 or 8c (1.34 mmol) in acetone–CH Cl (6:5,
3
3
3
3
3
+
+
2
2
70 mL) was mixed with a buffer of Na CO (8 g) and NaHCO (4 g) in water (100 mL), and the mixture was vigorously stirred
2
3
3
at 5°C. A solution of oxone (6.0 g, 9.8 mmol) in 70 mL water was added dropwise over 2 h after the addition was complete,
and the pH of the reaction mixture was adjusted to pH 8. Then the mixture was kept again at 5°C for another 12 h. The water
layer was extracted with dichloromethane (3 × 50 mL). The combined organic extracts were washed with saturated aqueous
Na S O and saturated aqueous NaCl and dried over anhydrous Na SO . The solution was filtered, and p-tuluenesulfonic acid
2 2
3
2
4
(5 mg, 0.03 mmol) was added. After stirring for 1 h, the solvent was removed under reduced pressure, and the solid residue
was recrystallized from methanol to afford flavonol 7 or 10 as yellow crystals.
1
3-Hydroxy-5,6,7,8,3′,4′,5′-heptamethoxyflavone (7). Yellow solid, yield 63%, mp 110–112°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 13.96 (1H, s, OH), 5.98 (1H, d, J = 2.3, H-2′), 5.85 (1H, d, J = 2.3, H-6′), 3.78 (9H, s, OCH ), 3.75 (6H,
3
3
13
s, OCH ), 2.54 (6H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm): 194.2, 162.3, 158.7, 158.0, 153.3, 144.3, 140.0, 130.4,
128.5, 111.9, 105.4, 91.4, 91.0, 61.0, 56.7, 56.1, 55.9, 55.4, 52.6. MS (FAB ) m/z 449 [M + 1] .
3
3
3
+
+
3-Hydroxy-5,7,3′,4′,5′-pentamethoxyflavone (10). Yellow solid, yield 65%, mp 218–220°C [lit. [17]: 232–234°C].
1
H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 12.66 (1H, s, OH), 7.02 (1H, s, H-2′), 6.54 (1H, s, H-6′), 6.44 (1H, d, J = 2.2, H-8),
3
13
6.32 (1H, d, J = 2.2, H-6), 3.89 (9H, s, OCH ), 3.87 (3H, s, OCH ), 3.83 (3H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm):
3
3
3
3
180.5, 168.9, 168.8, 159.4, 153.2, 147.3, 139.5, 128.0, 111.0, 108.5, 105.2, 94.0, 89.2, 61.0, 56.2, 55.5. ESI-MS m/z 389
+
[M + H] .
For in vitro antiproliferative activity screening, HeLa, HCC1954, and SK-OV-3 cell lines were maintained in
DMEM + 5% fetal bovine serum (FBS), RPMI 1640 + 5% FBS, and Mccoy′s 5 A + 10% FBS medium in a humidified
atmosphere of 5% CO at 37°C, respectively. Stock solutions of the tested compounds (20 mL) including controls, were
2
16

prepared with DMSO. The highest DMSO concentration of medium (0.5%) did not have a significant effect on the determined
cellular functions.
Proliferation of HeLa, HCC1954, and SK-OV-3 cells was evaluated by the CCK-8 (Cell Counting Kit-8, Dojindo
Laboratories, Kumamoto, Japan) assay [23]. This assay was based on the cleavage of the tetrazolium salt WST-8 by mitochondrial
3
dehydrogenase in viable cells. Briefly, 5 × 10 cells/well were incubated with 40 μL culture medium in 96-well plates. After
adherence to the well, the cells were treated with 5 μL of the tested compounds at different concentrations at 37°C; absorbance
at 450 nm of each well was measured with a Bio-Rad 680 microplate read. Each experiment was repeated three times.
Mean absorbance for each drug dose was expressed as the percentage of the control and plotted versus drug
concentration. Dose-response curves were fitted, and IC values were calculated by SPSS statistical software. IC values
50
50
represent drug concentrations that reduce the mean absorbance at 450 nm to 50%.
ACKNOWLEDGMENT
We thank the National Natural Science Foundation of China (No. J1210040, 21173074) for financial support.
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