Med Chem Res (2017) 26:1585–1592
DOI 10.1007/s00044-017-1871-4
REVIEW ARTICLE
Synthesis of Citrus polymethoxyflavonoids and their
antiproliferative activities on Hela cells
Van-Son Nguyen1,2 Wei Li1 Yue Li1 Qiuan Wang1
● ● ●
Received: 11 May 2015 / Accepted: 8 March 2017 / Published online: 17 March 2017
© Springer Science+Business Media New York 2017
Abstract A series of polymethoxyflavonoids (3–16) were
synthesized through dehydrogenation, O-methylation, gly-
coside hydrolysis, bromination, microwave-assisted aro-
matic nucleophilic substitution, dimethyldioxirane
oxidation and regioselective demethylation, starting from
abundant and inexpensive natural sources naringin and
hesperidin. All the synthetic compounds were test for
antiproliferative activities on human cervical carcinoma
Hela cell line by the standard CCK-8 assay, the result
showed that most of the target compounds exhibited mod-
erate to potent antiproliferative activities on Hela cells
comparable with the positive control cis-Platin. Among
them, 5-hydroxypolymethoxy flavonoid 13 showed the
strongest activity (IC50 0.791 μM).
Graphical Abstract
Keywords Polymethoxyflavonoids Synthesis ● ●
Antiproliferative activity Hela cells
●
* Qiuan Wang
wangqa@hnu.edu.cn
1
College of Chemistry and Chemical Engineering, Hunan
University, Changsha 410082, China
2
Technology Faculty of Thanh Hoa Campus, Industrial University
of Ho Chi Minh City, Ho Chi Minh City, Vietnam
MEDICINAL
CHEMISTRY
RESEARCH
Introduction
Polymethoxyflavonoids (PMFs) are a class of natural pro-
ducts, which almost exclusively exist in Citrus species,
particularly in the peel of sweet orange [Citrus sinensis (L.)
Osbeck] and mandarin (Citrus reticulata Blanco) (Lewin
et al. 2010). Recently, quite a few studies focused on the
PMFs in Citrus plants because they were found to possess
distinguished anticarcinogenic, anti-inflammatory, and
antiviral activities (Miyata et al. 2013; Li et al. 2012; Chen
et al. 2007).
Over the past decade, many biological studies have
focused on two of the most abundant PMFs in Citrus peels:
tangeretin and nobiletin. For example, tangeretin (5,6,7,8,4′-
pentamethoxyflavone, 5) was demonstrated to be an anti-
proliferative agent against a variety of tumor types (Buisson
et al. 2007), to induce G1 cell-cycle arrest in human col-
orectal carcinoma and induce colon cancer cells apoptosis
(Pan et al. 2002), it was identified as an effective Multiple
Drug Resistance modulator (Olga et al. 2012). Nobiletin
(5,6,7,8,3′,4′-hexamethoxyflavone, 6), another abundant
PMFs in Citrus peel, has been reported to exhibit anti-
proliferative activity on HL-60 cell line (Li et al. 2007), to
inhibit tyrosinase activity and to exhibit antimutagenic
activity (Okuno and Miyazawa 2004). In particular,
1586
Scheme 1 Reagents and conditions: a I2/pyridine, reflux; b
(CH3)2SO4, K2CO3, acetone, reflux; c concentrated H2SO4, ethanol,
reflux; d NBS, TFA, r.t; e CH3ONa, CuBr, DMF, microwave heating
increasing attention has been paid to its antitumor metastatic
activity due to the inhibition of gene expression and pro-
duction of some matrix metalloproteinases (MMP-1, -3, and
-9) (Oshitari et al. 2011).
PMFs generally present as minor components in the
Citrus plants. The synthesis of these compounds have been
much less studied, the full potential of this group of com-
pounds to be used as drugs or bioactive molecules has not
been realized. Recently we has reported the total synthesis
of some PMFs such as nobiletin, tangeretin and so on, as a
continuation of our investigation of bioactive flavonoids
and development of new antitumor activity compounds (Cai
et al. 2012; Wang et al. 2010). We reported the new
synthesis of a series of Citrus PMFs. Furthermore, all
synthesized compounds were evaluated for their anti-
proliferative activities on human cervical carcinoma Hela
cells by the standard CCK-8 assay. The synthetic pathways
adopted for the synthesis of a series of Citrus PMFs are
illustrated in Scheme 1.
Results and discussion
First, 7-hydroxy-5,4′-dimethoxyflavone (1) and 7-hydroxy-
5,3′,4′-trimethoxy flavone (2) were prepared from abundant
Med Chem Res (2017) 26:1585–1592
(700 W); f AlCl3, CH3CN, reflux; g Oxone, acetone, CH2Cl2,
NaHCO3, Na2CO3, 5 °C; h prenyl bromide, K2CO3, acetone, r.t
and commercially low-cost naringin or hesperidin by
dehydrogenation with I2/Py followed by base-induced
elimination to rhoifolin or diosmin, respectively, then
O-methylation of phenol groups and acid hydrolysis of the
neohesperidose residue.
Next, our efforts were concentrated on the methoxylation
of the 6,8-positions of 1 or 2 using the Ulmann-type reac-
tion. Upon treatment with 2 equiv N-bromosuccinimide
(NBS) in trifluoroacetic acid, 1 or 2 was brominated to give
6,8-dibromoflavones 17 or 18 in good yield. Bromination at
both C-6 and C-8 were confirmed by 1H nuclear magnetic
resonance (NMR) spectroscopy (loss of H-6 and H-8 sig-
nals of A-ring, unchanged signals for the AA’BB’ or ABX
spin system of the B-ring). The next step was substitution of
the bromines at C-6 and C-8 by CuBr-mediated Ulmann-
type reaction with a large excess amount of sodium meth-
oxide, according to Guillaumet reported (Besson et al.
1995), methoxylation of 6,8-dibromoflavones 17 or 18 by
heating it at 120 °C in Dimethyl Formamide (DMF)-MeOH
with MeONa (10 equiv) and CuBr (0.03 equiv) for 22 h led
to nucleophilic aromatic substitution at both C-6 and C-8
giving the expected flavone 3 or 4 only 25% yield in this
procedure, we suspected that decomposition of DMF caused
by prolonged heating at 120 °C had affected the yield,
therefore, the microwave-assisted reaction was tried in an
Med Chem Res (2017) 26:1585–1592 1587

attempt to shorten the reaction time. Gratifyingly, a very
good yield (85–90%) of 6,8-dimethoxy group-substituted
flavones 3 or 4 were obtained under microwave heating
(700 W) within 1 h. For the catalyst CuCl, CuCl2, and CuI
were found to work as well as CuBr to give the same 6,8-
dimethoxylated products. On the other hand, no reaction
occurred in the absence of the Cu catalyst. O-methylation of
7-hydroxyl group of 3 or 4 with (CH3)2SO4/ K2CO3 in
acetone provided the PMFs tangeretin 5 and nobiletin 6,
respectively. The spectroscopic data for compounds 5 and 6
the obtained were identical to those reported previously by
us (Cai et al. 2012). Oxidation of flavones to the 3-
hydroxyflavones (flavonols) proved to be challenging. The
method was oxidation of polymethoxyflavone 5 or 6 with
dimethyldioxirane generated in situ from Oxone and acet-
one (Wu et al. 2012; Adam et al. 1987), followed by acid-
induced rearrangement that gave the 3-hydroxy poly-
methoxyflavone 7 or 8 in comparable yield. O-methylation
of 7 or 8 gave PMFs 9 or 10. Then promoted by a lewis acid
aluminum chloride and with anhydrous acetonitrile as the
solvent, the reaction of compounds 5, 6, 9, and 10 under-
went regioselective demethylation of the C-5 methoxy
group to give 5-hydroxypolymethoxyflavone 11–14 in good
yields due to the neighboring group participation of C-4
carbonyl oxygen. Prenyl moiety can potentially substitute
for conventional lipids as lipophilic carriers of bioactive
molecules. The introduction of a prenyl moiety in bioactive
molecules is an important post-translational modification
centre to many cellular processes (Michelle et al. 2007).
Next, 3-hydroxylated polymethoxyflavone 7 and 8 were
reacted, respectively, with commercially available prenyl
bromide in the presence of anhydrous potassium carbonate
and anhydrous acetone at ambient temperature to give O-
prenyl-substituted PMF derivatives 15 and 16. The structure
of the synthesized compounds were confirmed by 1H NMR,
13
C NMR, mass spectra (MS), and infrared (IR) spectra.
Antiproliferative activities on human cervical carcinoma
Hela cells of these PMFs 3–16, as well as 7-hydroxy-5,4′-
dimethoxyflavone (1), 7-hydroxy-5,3′,4′-dimethoxyflavone
(2) and intermediates 17, 18, were evaluated by the CCK-8
[2H-tetrazolium-5-(2,4-disulfophenyl)-3-(2-methoxy-4-
nitrophenyl) -2-(4-nitrophenyl)-, inner salt, sodium salt
(1:1)] assay. The results are listed in Table 1. The
dose–response curve for CCK-8 assay of compounds 9, 10,
11, 13, 17and 18 on Hela cells proliferation are shown in
Fig. 1. cis-Platin and paclitaxel, two anticancer drug, were
used as the positive control. The results showed that most of
the target compounds exhibited moderate to potent anti-
proliferative activities on Hela cells.
Compounds 11, 12, and 13, with a 5-OH in A-ring dis-
played higher Hela cells inhibition activity than the com-
pounds 5, 6, and 9, where the C-5 position on the A-ring
were occupied by methoxy group, among them,
Table 1 Half-inhibitory concentration [IC50 (μM)] of compounds
1–18 on the Hela cells
Compounds IC50 (μM) Compounds IC50 (μM)
1 81.242 11 6.788
2 59.623 12 27.014
3 >100 13 0.791
4 >100 14 18.243
5 >100 15 97.391
6 46.185 16 37.946
7 >100 17 20.533
8 69.763 18 21.084
9 11.763 cis-Platina 41.253
10 16.847 Paclitaxela 0.003
a
cis-Platin and paclitaxel were employed as positive control
5-hydroxypolymethoxy flavonoid 13 showed the strongest
activity (IC500.791 μM), which suggested that 5-OH was an
important functional group. Recently 5-OH-PMFs have
gained more attention, as considerable evidence suggests
that 5-OH-PMFs have much stronger health-promoting
biological activities than permethoxylated PMFs because of
the presence of 5-OH (Pan et al. 2007; Lai et al. 2007). The
other four monohydroxylated PMFs, 3 and 4, having a
hydroxyl group at position 7 did not demonstrate any
antiproliferative activity below 100 μM concentration.
Compounds 7 and 8, having a hydroxyl group at position 3,
also showed the lowest activities. This suggested that the
position of the hydroxyl group affects the antiproliferative
activity on Hela cells of PMFs. Compounds 9, 10 exhibited
stronger antiprotiferative activity than 5,6,7,8,4′-penta-
methoxyflavone (5) and nobiletin (6), with the only struc-
tural difference between these four compounds being that 9,
10 have a methoxyl group at 3-position in the C-ring, while
5, 6 have none. Compounds 7, 8, with a hydroxyl group at
position 3, the antiproliferative were poor in comparison to
compounds 9, 10, with a methoxyl group at position 3.
These indicates that the methoxyl group at 3-position in the
C-ring contributes to the antiproliferative of PMFs. Our data
also showed that compound 9, 10, characterized by more
methoxy group were more potent. Compounds 17 and 18,
which possess two bromo substituents on the 6 and 8-
position of flavones 1 and 2, were found to confer an
increase in antiproliferative activity. Compounds 15 and 16,
having an O-prenyl group at position 3, showed slightly
higher antiproliferative activity than 7 and 8. This showed
the presence of the lipophilic substituents appears to
improve the antiproliferative activity of PMFs by interac-
tion between PMFs and cell membrane.
The structure–activity relationship (SAR) analysis
revealed that the position of the hydroxyl group of PMFs
1588 Med Chem Res (2017) 26:1585–1592

Fig. 1 The dose–response curve for CCK-8 assay of compound 9, 10, 11, 13, 17, and 18 on Hela cells proliferation

and number of methoxyl group may be closely linked with
their antiproliferative activity against Hela cells, for good
biological effect, the 5-OH or 3-OCH3 functionalities were
essential, while the presence of 3-OH or 7-OH greatly
reduced the activity. To the best of our knowledge, such
observations on the antiproliferative activity of Hela cells
have not been reported previously. Although we have lim-
ited information on the SAR, the findings of this study
provide important information for the exploitation and uti-
lization of Citrus PMFs as natural anticancer agents.
Med Chem Res (2017) 26:1585–1592
Experimental
General methods
Melting points were measured on a XRC-I apparatus and
were uncorrected. Microwave reaction was performed with
XH-MC-1 microwave reactor (Beijing Xianghu Science and
Technology Development Co., China). 1H NMR and 13C
NMR spectra were recorded on a Bruker AM-400 instru-
ment, using tetramethylsilane as an internal standard che-
mical shifts (δ) in ppm, and coupling constants (J) in Hz.
MS or high-resolution mass spectrometry (HRMS) was
determined with VG Autospec-3000 or Mat 95 XP spec-
trometer by the EI method. Column chromatography was
carried out on silica gel using 200–300 mesh (Qingdao
Ocean Chemical Products of china). Commercially avail-
able AR or chemical pure reagents, and anhydrous solvent
removed water and redistilled were employed.
Rhoifolin and Diosmin were synthesized and purified
according to previous method by us (Liu et al. 2012; Shan
et al. 2008). 3-Hydroxytangeretin (3-hydroxy-5,6,7,8,4′-
pentamethoxyflavone) (7), 3-hydroxynobiletein (3-
hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone) (8),
3,5,6,7,8,4′-hexamethoxyflavone (9), 3,5,6,7,8,3′,4′-hepta-
methoxyflavone (10), 5-hydroxy-6,7,8,4′-tetramethoxy-
flavone (11), 5-hydroxy-6,7,8,3′,4′-pentamethoxy-flavone
(12), 5-hydroxy-3,6,7,8,4′-pentamethoxyflavone (13) and 5-
hydroxy- 3,6,7,8,3′,4′-hexamethoxyflavone (14) were syn-
thesized and purified according to previous method by us
(Cai et al. 2012; Li et al. 2014).
Synthesis of 7-hydroxy-5,4′-dimethoxyflavone (1)
The solution of rhoifolin (10 g, 17.3 mmol) in 7% NaOH (150
mL) and (CH3)2SO4 (17 mL, 63.05 mmol) was stirred at room
temperature for 6 h. Then 10 mL of concentrated sulfuric acid
was added dropwise, The reaction mixture was stirred under
reflux for 4 h, then reaction mixture cooled and poured into
cold water, the solids was filtered, washed with water, dried
and then crystallized in ethyl acetate to give 1 (4.6 g, yield:
89%) as yellow solid, m.p. 296–297 °C (lit (Shizuo et al.
1952), 298 °C); 1H NMR (400 MHz, DMSO-d6): δ 7.93 (d, J
= 8.9 Hz, 2H, H-2′ and H-6′), 7.07 (d, J = 8.9 Hz, 2H, H-3′,
and H-5′), 6.58 (s, 1H, H-3), 6.54 (d, J = 2.0 Hz, 1H, H-8),
6.37 (d, J = 2.0 Hz, 1H, H-6), 3.82, 3.78 (each, s, each 3H,
–OCH3); 13C NMR (100 MHz, DMSO-d6): δ 176.1, 162.9,
162.0, 160.9, 159.9, 159.4, 127.9, 123.3, 114.7, 107.3, 106.7,
96.8, 95.5, 56.1, 55.7; EIMS: m/z 298 [M]+.
Synthesis of 7-hydroxy-5,3′, 4′-trimethoxyflavone (2)
According to a similar procedure as described for the pre-
paration of 1. 2 was obtained (87% yield from diosmin) as
1589
yellow solid, m.p. 283–284 °C (lit (Shizuo and Hiroaki
1954), 284 °C); 1H NMR (400 MHz, DMSO-d6): δ 10.72 (s,
1H, 7-OH), 7.58 (d, J = 8.4 Hz, 1H, H-6′), 7.48 (s, 1H, H-
5′), 7.08 (d, J = 8.7 Hz, 1H, H-2′), 6.69 (s, 1H, H-3), 6.56
(s, 1H, H-8), 6.37 (d, J = 2.5 Hz, 1H, H-6), 3.86, 3.83, 3.79
(each, s, each 3H, –OCH3);13C NMR (100 MHz, DMSO-
d6): δ 178.8, 161.6, 160.6, 160.2, 151.7, 149.2, 124.1,
119.2, 111.2, 108.5, 107.6, 104.2, 101.8, 96.8, 58.8, 56.3,
56.1; EIMS: m/z 328 [M]+.
Synthesis of 6,8-dibromo-7-hydroxy-5,4′-
dimethoxyflavone (17)
To a solution of compound 1 (3 g, 10 mmol) in 35 mL of
trifluoroacetic acid (TFA), N-Bromosuccinimide (3.57 g,
20.1 mmol) was added dropwise, and the reaction mixture
was stirred at room temperature for 5 h, then the mixture
was poured into cold water, the solids was filtered, washed
with water, dried. The crude product was purified by silica
gel column chromatography (petroleum ether/EtOAc, v/v,
3:1) to afford 17 (4.3 g, yield: 95%) as white solid, m.p.
197–198 °C; 1H NMR (400 MHz, CDCl3): δ 10.25 (s, 1H,
7-OH), 7.93 (d, J = 8.9 Hz, 2H, H-2′ and H-6′), 7.09 (d, J
= 8.8 Hz, 1H, H-3′, and H-5′), 6.65 (s, 1H, H-3), 3.87 (d, J
= 3.5 Hz, 3H, 5-OCH3), 3.81 (s, 3H, 4′-OCH3); 13C NMR
(100 MHz, CDCl3): δ 176.8, 161.2, 158.4, 156.8, 156.2,
131.7, 124.4, 114.4, 112.9, 110.8, 109.2, 106.7, 61.7, 57.3;
EIMS: m/z 454 [M]+.
Synthesis of 6,8-dibromo-7-hydroxy-5, 3′, 4′-
trimethoxyflavone (18)
According to a similar procedure as described for the pre-
paration of 17. 18 was obtained (yield: 95%) as yellow
soild, m.p. 229–231 °C;1H NMR (400 MHz, CDCl3): δ 7.67
(d, J = 8.6 Hz, 1H, H-6′), 7.54 (d, J = 2.3 Hz, 1H, H-2′),
6.96 (d, J = 8.6 Hz, 1H, H-5′), 6.62 (s, 1H, H-3), 4.01, 3.98,
3.96 (each, s, each 3H, –OCH3); 13C NMR (100 MHz,
CDCl3): δ 178.2, 162.3, 160.2, 158.6, 154.7, 149.8, 147.6,
123.8, 118.8, 116.6, 112.3, 109.3, 105.6, 101.1, 60.2, 56.2;
EIMS: m/z 484 [M]+.
Synthesis of 7-hydroxy-5,6,8,4′-tetramethoxyflavone (3)
To a solution of compound 17 (500 mg) in 10 mL DMF,
CuBr (28 mg, 0.28 mmol) and NaOCH3 (0.22 g) in 5 mL
CH3OH were added under nitrogen protection. The reaction
mixture was stirred under microwave heating (700 W) for
1 h. Then the reaction mixture was poured into ice-water
and adjusted to pH 6 using 2% HCl (aq), and extracted
by ethyl acetate (20 mL × 3), dried over anhydrous
Na2SO4. The solvent was removed, and the residue was
purified by silica gel column chromatography (petroleum
1590
ether / EtOAc, v/v, 5:1) to afford 3 (yield: 62%) as yellow
solid, m.p. 182–184 °C; 1H NMR (400 MHz, CDCl3): δ
7.60 (d, J = 8.9 Hz, 2H, H-2′, and H-6′), 6.93 (d, J = 8.9
Hz, 2H, H-3′, and H-5′), 6.53 (s, 1H, H-3), 4.01, 3.92, 3.85,
3.82 (each, s, each 3H, –OCH3); 13C NMR (100 MHz,
CDCl3): δ 178.8, 162.5, 159.6, 147.3, 146.7, 143.0, 142.3,
137.1, 136.5, 128.7, 122.8, 114.9, 105.7, 103.6, 61.3, 61.0,
60.2, 55.2; EIMS: m/z 358 [M]+.
Synthesis of 7-hydroxy-5,6,8,3′, 4′-pentamethoxyflavone
(4)
According to a similar procedure as described for the pre-
paration of 3. 4 was obtained (yield: 76%) as yellow soild,
m.p. 115–117 °C; 1H NMR (400 MHz, CDCl3): δ 7.58 (d, J
= 8.3 Hz, 1H, H-6′), 7.46 (d, J = 7.6 Hz, 1H, H-2′), 7.06 (d.
J = 7.3 Hz, 1H, H-5′), 6.68 (s, 1H, H-3), 4.12, 4.03, 3.83
(each, s, each 3H, –OCH3), 3.96 (s, 6H, –OCH3); 13C NMR
(100 MHz, CDCl3): δ 177.8, 162.6, 158.6, 149.3, 147.6,
146.5, 143.0, 137.3, 136.7, 123.6, 119.6, 116.5, 112.6,
105.9, 103.3, 61.0, 60.3, 60.0, 56.2; EIMS: m/z 388 [M]+.
Synthesis of 5,6,7,8,4′-pentamethoxyflavone (tangeretin)
(5)
Me2SO4 (0.1 mL, 3 equiv.) was added dropwise to stirred
solution of 3 (135 mg) in acetone and K2CO3 (0.91 g, 6.48
mmol) at 65 °C. The reaction was terminated after 5 h, and
the mixture was filtered. The solution was evaporated to
afford a solid residue. The crude solid was chromato-
graphed on silica gel using petroleum ether/ethyl acetate (v/
v, 3:1) as eluent to afford light yellow crystals of 5 (115 mg,
yield: 86%), m.p. 151–153 °C (lit[12]. 152–153 °C); 1H
NMR (CDCl3): δ 7.80 (d, J = 8.8 Hz, 2H, H-2′ and 6′), 6.94
(d, J = 8.8 Hz, 2H, H-3′ and 5′), 6.52 (s, 1H, H-3), 4.03,
3.94, 3.87, 3.81, (each, s, each 3H, –OCH3); 13C NMR
(CDCl3): δ 176.3, 161.3, 160.1, 150.3, 147.3, 146.7, 143.0,
137.1, 126.7, 122.8, 113.8, 113.5, 105.6, 61.2, 61.0, 60.8,
60.6, 54.5; IR (KBr): ν 2946, 2843, 1650, 1607, 1587,
1512, 1462, 1363, 1265, 1181, 1074, 968, 830 cm–1; MS
(m/z, EI): 372 (M+), 327, 315, 287, 259, 194, 135, 77, 57;
HRMS (EI): m/z [M]+ calcd for C20H20O7: 372.1209,
found: 372.1204.
Synthesis of nobiletin (5,6,7,8,3′, 4′-hexamethoxyflavone,
6)
According to a similar procedure as described for the pre-
paration of 5. 6 was obtained (yield: 80%) as yellow soild;
m.p. 112–113 °C (lit (Wang et al. 2010), 112–113 °C); 1H
NMR (CDCl3) δ 7.50 (dd, J = 8.5, 1.9 Hz, 1H, H-6′), 7.34
(s, 1H, H-2′), 6.92 (d, J = 8.5 Hz, 1H, H-5′), 6.55 (s, 1H, H-
3), 4.03, 3.96, 3.91, 3.89 (each, s, each 3H, –OCH3), 3.88
Med Chem Res (2017) 26:1585–1592
(s, 6H, –OCH3); 13C NMR (CDCl3): δ 177.3, 161.1, 151.9,
151.5, 149.2, 148.4, 147.7, 144.1, 123.9, 119.9, 114.7,
111.2, 108.5, 106.7, 62.3, 62.0, 61.8, 61.7, 56.1, 55.9; MS
(m/z, EI): 402 (M+), 387 (100), 371, 344, 326, 298, 283,
239, 225, 198, 197, 182, 162, 153, 139, 91, 83, 77; HRMS
(EI): m/z [M]+ calcd for C21H22O8: 402.1315, found:
402.1314.
Synthesis of 3-O-prenyl-5,6,7,8,4′-pentamethoxyflavonol
(15)
To a mixture of compound 7 (100 mg, 0.26 mmol) and
anhydrous K2CO3 (110 mg, 0.8 mmol) in dry acetone
(20 mL), a solution of prenyl bromide (0.06 mL,
0.26 mmol) in acetone (3 mL) was added dropwise upon
stirring for 3 h, then the reaction mixture was filtered and
evaporated. The residue was subjected to chromatography
on silica gel with petroleum ether-ethyl acetate (v/v,5:1) as
eluent to give 114 mg of compound 15 as a yellow soild
(yield: 95%), m.p. 123–125 °C; 1H NMR (400 MHz,
CDCl3): δ 8.19 (d, J = 8.8 Hz, 2H, H-2′ and 6′), 7.03 (d,
J = 8.8 Hz, 2H, H-3′ and 5′), 5.47 (s, 1H, CH), 4.57 (s, 2H,
CH2), 4.11, 4.01, 3.98, 3.96, 3.91 (each, s, each 3H,
-OCH3), 1.71 (s, 3H, CH3), 1.66 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ 173.0, 160.2, 152.8, 150.1, 147.1,
145.8, 142.7, 138.3, 137.92, 136.8, 129.2. 122.6, 119.1,
114.0, 112.8, 98.9, 67.6, 61.3, 61.0, 60.8, 60.6, 54.4, 24.8,
17.1; IR (KBr) v 3417.03, 3234.66, 2029.75, 1635.74,
1509.74, 1453.52, 1360.70, 1257.00, 1171.23, 1050.70,
977.21, 801.96, 753.41, 621.55, 481.87 cm−1.
Synthesis of 3-O-prenyl-5,6,7,8,3′,4′-hexamethoxyflavonol
(16)
Compound 16 was prepared from compound 8 and prenyl
bromide as described for the preparation of compound 15
from compound 7. Yellow solid, (yield: 94%), m.p.
140–143 °C; 1H NMR (400 MHz, CDCl3): δ 7.83 (d, J =
2.0 Hz, 1H, H-6′), 7.77 (dd, J = 8.6, 2.1 Hz, 1H, H-5′), 6.92
(d, J = 8.6 Hz, 1H, H-2′), 5.42 (t, J = 7.3 Hz, 1H, CH), 4.52
(d, J = 7.3 Hz, 2H, CH2), 4.02, 3.93, 3.90, 3.88, 3.87 (s,
each, s, each 3H, –OCH3), 1.63 (s, 3H, CH3), 1.56 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3): δ 173.0, 152.4, 150.2,
149.8, 147.5, 147.2, 145.7, 142.7, 138.6, 137.8, 136.8,
122.7, 120.8, 119.3, 114,0, 110.4, 109.7, 67.9, 61.3, 60.9,
60.8, 60.6, 54.9, 54.8, 24.7, 17.1; EIMS: m/z 486 [M]+
Assay for antiproliferative activity
The antiproliferative activity was tested using a CCK-8
assay on Human cervix carcinoma cell line (Hela) (Xuan
et al. 2015). Briefly, cells (5 × 103 per well in a 96-well
plate) were treated with different concentrations of
Med Chem Res (2017) 26:1585–1592
compounds 1–18 (100, 25, 6.25, 1.56, 0.39, 0.0976, 0.0244,
0.0061 µM) for 48 h. Then 5% CCK-8-solution was added
into each well and incubated with 90% humidity and 5%
CO2 for another 1–3 h. Color development was quantified
photometrically at 450 nm, and used an EL × 808 (Bio-Rad
680) absorbance microplate reader to determine the con-
centration that killed 50% of cells (IC50). To stop the color
reaction by add 10 µL of 1% sodium dodecyl sulfate (SDS)
[(dissolve 0.1 g SDS with phosphate buffer saline (PBS)
buffer to prepare 10 mL solution)] or add 10 µL of 0.1 mol/
L acid such as hydrochloric acid.
Conclusion
In summary, we succeeded in developing a new synthetic
route for PMFs from abundant and commercially low-cost
naringin or hesperidin with CuBr-catalysted and
microwave-assisted aromatic nucleophilic substitution of
bromide by methoxy group as the key step. Their anti-
proliferative activities on human cervical carcinoma Hela
cell line were evaluated by the standard CCK-8 assay, the
result showed that most of the target compounds exhibited
moderate to potent antiproliferative activities on Hela cells
comparable with the positive control cis-Platin. Among
them, 5-hydroxy PMF 13 showed the strongest activity
(IC50 0.791 μM). The result indicated that this compound is
a potential antitumor agent and is promising for further
development.
Acknowledgements We thank the National “Twelfth Five-year” plan
for Science & Technology Support (NO. 2012BAD31B02) and the
Hunan Provincial Natural Science Foundation of China (NO.
14JJ2048) for financial support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interest.
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