Discovery of flavonoids from scutellaria baicalensis with inhibitory activity against PCSK 9 expression: Isolation, synthesis and their biological evaluation

Article abstract of DOI:10.3390/molecules23020504

Nine flavonoids were isolated and identified from a chloroform-soluble fraction of the roots of Scutellaria baicalensis through a bioactivity-guided fractionation using a proprotein convertase subtilisin/kexin type 9 (PCSK9) monitoring assay in HepG2 cell

Full text of DOI:10.3390/molecules23020504

molecules
Article
Discovery of Flavonoids from Scutellaria baicalensis
with Inhibitory Activity Against PCSK 9 Expression:
Isolation, Synthesis and Their Biological Evaluation
Piseth Nhoek †, Hee-Sung Chae †, Jagadeesh Nagarajappa Masagalli, Karabasappa Mailar,
Pisey Pel, Young-Mi Kim, Won Jun Choi * and Young-Won Chin *
College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk University-Seoul,
32 Dongguk-lo, Ilsandong-gu, Goyang-si, Gyeonggi-do 10326, Korea; piseth2306@gmail.com (P.N.);
chaeheesung83@gmail.com (H.-S.C.); mnjagadeesh123@gmail.com (J.N.M.); Kitty_1506@yahoo.com (K.M.);
jully.christ07@gmail.com (P.P.); 0210121@hanmail.net (Y.-M.K.)
*
Correspondence: mp89@dongguk.edu (W.J.C.); f2744@dongguk.edu (Y.-W.C.); Tel.: +82-31-961-5218 (Y.-W.C.)
† These authors contributed equally to this work.
Received: 23 January 2018; Accepted: 22 February 2018; Published: 24 February 2018
Abstract: Nine flavonoids were isolated and identified from a chloroform-soluble fraction of the
roots of Scutellaria baicalensis through a bioactivity-guided fractionation using a proprotein convertase
subtilisin/kexin type 9 (PCSK9) monitoring assay in HepG2 cells. All structures were established
by interpreting the corresponding spectroscopic data and comparing measured values from those
in the literature. All compounds were assessed for their ability to inhibit PCSK9 mRNA expression;
compounds
suppress PCSK9 mRNA via SREBP-1. Furthermore, compound
lipoprotein receptor protein expression. Also, synthesis of compound
form (1a) was completed for the first time. Natural compound and synthetic racemic 1a were
1
(3,7,20-trihydroxy-5-methoxy-flavanone) and
4
(skullcapflavone II) were found to
was found to increase low-density
as a racemic mixture
1
1
1
evaluated for their inhibitory activities against PCSK9 mRNA expression and the results confirmed
the stereochemistry of 1 was important.
Keywords: Scutellaria baicalensis; flavonoid; PCSK9; SREBP-1; low density lipoprotein receptor
1. Introduction
Cardiovascular disease (CVD) is a class of well-recorded diseases that lead to prominent adult
mortality worldwide. High cholesterol level in the plasma has been identified as a major cause
of CVD [
in biosynthesis, uptake, or storage, is tremendously associated to heart-related diseases. Over the
past 20 years, statin therapy has been the standard treatment for successful cholesterol reduction [ ].
However, some patients with familial hypercholesterolemia (FH) fail to reach the desired cholesterol
concentration using common statin therapy [ ] even at maximal statin dose. Low density lipoprotein
receptors (LDL-R) are expressed on the surface of hepatocytes and bind to LDL particles [ ].
1]. Cholesterol is an essential substance to cell membranes, but excess levels of it, either
2
3,4
5,6
This LDL-R/LDL complex is primarily internalized into the cell through clathrin-coated vesicles,
after which the complex dissociates and LDL-C gets degraded into lipids and amino acids. After this
process, LDL-R moves back to the cell surface, suggesting that the recycling of LDL-R seems to be
significant in lowering LDL-C levels in the plasma [7,8]. In patients with FH, this recycling system is
impaired by high concentrations of proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is
known to bind LDL-R and prevent its recycling, which decreases LDL-R expression on cell surfaces
and consequently results in high levels of LDL-C in the plasma, leading to hypercholesterolemia [
9,10].
Therefore, PCSK9 inhibition would increase LDL-R expression, which in turn decreases cholesterol
Molecules 2018, 23, 504; doi:10.3390/molecules23020504
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levels in the plasma. Thus far, two antibody drugs have been approved as PCSK9 inhibitors, while no
small molecules have ever reached at the clinical trials [11 12]. Moreover, only a few natural products
,
in either extracts or individual chemicals have been explored for PCSK9 regulation [13–18].
Scutellaria baicalensis belongs to the Lamiaceae family [19] and is native to the soil of Asian
countries. The plant has been used as a food additive and for traditional medicine [20]; its roots have
been used for the treatment of allergies, inflammation [21
,22], fever, dysentery [23], pneumonia,
influenza [24], diarrhea [25], and have potential anticancer activities [26
,
27]. Phytochemical
investigations of S. baicalensis roots have disclosed flavonoids, phenylethanoids and sterols as the main
chemical constituents [28].
2. Results and Discussion
2.1. Inhibitory Activity against PCSK9 mRNA Expression of Root Extract from S. baicalensis
In the preliminary screening assay to assess medicinal plant extracts for their inhibitory activity
against PCSK9 mRNA expression using HepG2 cells, a methanolic extract of S. baicalensis was found
to inhibit PCSK9 mRNA expression. Thus, subsequent partitioning with organic solvents (hexane,
chloroform, butyl alcohol and water-soluble extracts) was conducted and the resultant solvent-soluble
extracts were tested again using the same bioassay (Figure 1). A chloroform-soluble extract
demonstrated inhibitory activities on PCSK9 mRNA expression. Therefore, the chloroform-soluble
fraction was further investigated to discover potential molecules in the extract that are responsible for
the inhibitory effects on PCSK9 expression.
Figure 1. The effects of (total) methanol extracts, hexane (Hex), chloroform (CHCl3), butyl
alcohol (BuOH) and aqueous fractions of S. baicalensis on PCSK9 mRNA expression in HepG2 cells.
(Atorva = Atorvastatin).
2.2. Identification of Isolates 1–9 from S. baicalensis
In the present study, nine known structures were isolated (Figure 2), with structures
confirmed by spectroscopic data analyses. The isolated compounds
follows: 3,7,20-trihydroxy-5-methoxy-flavanone ( ) [29], 3,5,7,20,60-pentahydroxyflavanone (
2-methyl-6-phenylpyran-4-one ( ) [31 32], skullcapflavone II (
) [33], 5,7,20,60-tetrahydroxyflavone
30], 2,3-dihydro-7-hydroxy-2-(2-hydroxyphenyl)-5-methoxy-benzopyran-4-one
5,7,20-trihydroxy-60-methoxyflavone ( ) [35], 5,7,20-trihydroxyflavone (
) [36] and wogonin (
1–
9
were identified as
1
2
) [30],
3
,
4
(5
)
[
(6)
[34],
7
8
9
) [37,
38],
by extensive NMR experiments (1H, 13C, HSQC, and HMBC) and comparisons with values previously
reported in the literature. Since there are no reports regarding full assignments for compound
all assignments were provided in the isolation method.
3,

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HO
4'
1
O
7
1'
5
HO
O
3
R2
OH
O
3
R1
R1
1 OCH3
2 OH
O
R2
H
OH
R5
R4
R3
R2
O
R3
R6
R2
O
R1
O
OH
R3
R1
R1
O
R1 R2
R3
R4
R5
R6
4
7
8
9
OH OCH3 OCH3 OCH3 OCH3 OH
R2
OH
OH
OH
H
H
H
OH
OH
OH
H
H
OCH3
OCH3 OH
H
H
5 OH OH OH
6 OCH3 OH
OH
H
H
Figure 2. Chemical structures isolated from S. baicalensis.
2.3. Stereochemistry Determination of Compound 1
The stereochemistry of C-2 and C-3 in was determined to be in a trans orientation based on
the observation that the vicinal coupling constant between H-2 and H-3 was 11.2 Hz, consistent with
what is known in the literature regarding compound 39 40]. Furthermore, circular dichroism (CD)
spectroscopy of revealed a positive Cotton effect at 328 nm and a negative Cotton effect at 299 nm,
1
2
[ ,
1
suggesting that the configurations of C-2 and C-3 were 2R and 3R, respectively [41]. The structure of
1
was thus characterized as (2R,3R), 3,7,20-trihydroxy-5-methoxyflavanone.
2.4. Inhibitory Activity against PCSK9 mRNA Expression of Isolates from S. baicalensis
The isolated
HepG2 cells. Compound
II) exhibited inhibitory activity at 20
some compounds from the same plant that have been previously isolated in our laboratory [26] were
tested in the same assay at 20 M in order to assess structure-activity relationship (Supplementary
Materials Figure S15). None of them were active in this assay system. From the structures (active)
and (inactive), it was inferred that a hydroxyl group at C-3 position might provide the different
activities. When the structure of skullcapflavone II ( ) was compared with the similar structures of
1–
6,
8
and
was excluded for cytotoxicity (Figure 3A). Only
M (84.4% and 42.4%, respectively) (Figure 3B). In addition,
9
from this study were evaluated for their PCSK9 mRNA expression in
7
1
and (skullcapflavone
4
µ
µ
1
6
4
molsoflavone and alnetin in Supplementary Materials Figure S15, no conclusive result was reached
due to the limited number of structures.
Out of the active compounds, compound
expression and it was found that compound
expression, respectively (Figure 3C). As mentioned earlier, PCSK9 facilitates LDL-R degradation
and prevents LDL-R recycling. Taking into consideration this function of PCSK9, compound may
1
was further tested for its PCSK9 and LDL-R protein
1
was able to inhibit PCSK9 and increase LDL-R protein
1
potentially lower cholesterol levels by decreasing PCSK9 expression and concomitantly increasing
LDL-R expression.

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Figure 3. Effects of compounds from S. baicalensis on Cell viability, PCSK9 and LDL-R expression in
HepG2 cells by MTT, qRT-PCR and western blot analysis. ( ) Cells grown were treated with 20
of compounds for 24 h, and cell viability was assessed by the MTT assay; ( ) Expression of PCSK9
was assayed by qRT-PCR in cells treated with compounds except for , and berberine (50 M) for
24 h; ( ) Expression of PCSK9 and LDL-R were assayed by western blots in cells treated with ( ) and
atorvastatin for 24 h. (Ber50, berberine 50 µM).
A
µM
B
1
–9
7
µ
C
1
2.5. Comparison of Inhibitory Activity against PCSK9 mRNA Expression between Compound 1 and 1a
Due to its activity, we selected compound for synthesis and designed the synthetic
1
scheme shown in Scheme 1. Our approach to synthesize racemic 1a began with di-MOM
protected 1-(2-hydroxy-4,6-bis(methoxymethyl)phenyl)ethanone (11), which was readily prepared
from the commercially available 20,40,60-trihydroxyacetophenone (10) by treatment with MOMCl
and ethyldiisopropylamine [42]. Compound 11 was converted to chalcone 14 through methylation
using dimethyl sulfate in acetone, followed by Claisen-Schmidt aldol condensation with
2-(methoxymethyl)benzaldehyde (13) in the presence of aqueous base [43,44]. Epoxidation of
compound 14 with alkaline hydrogen peroxide at room temperature produced 15 in 89% yield [45].
Treatment of the epoxide 15 employing various acidic conditions for the purpose of instantaneous
MOM deprotection and intramolecular 6-endo opening of epoxide afforded flavanol 1a, however,

Molecules 2018, 23, 504
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the yield of the product was limited to 3–5%, with a variety of undesired products [44
,46]. Finally,
treatment of 15 with 12% conc HCl in methanol produced compound 1a as a racemic mixture in 11%
yield as shown in Scheme 1 [47].
OH
O
OH
O
OMe O
a
c
b
CH3
OH
CH3
CH3
OMOM
O
OMOM
HO
MOMO
OMOM
OMe O
MOMO
H
10
11
(13)
12
OMe O
O
OMe O
OMe O
OH
OH
OMOM
OMOM
O
+
e
d
HO
HO
O
MOMO
MOMO
OMOM
OMOM
14
HO
HO
15
1a
Reactions and conditions: a) MOMCl, DMF, DIPEA, DCM, 4 h. b) K2CO3, (CH3)2SO4, (CH3)2CO, 65 oC, 2 d. c) aldehyde 13,
30% KOH (aq.), EtOH, 55 oC, 6 h. d) K2CO3, H2O2, MeOH, 1 h. e) 12% HCl in MeOH, MeOH, 55 oC, 30 min. f) Isolated
yields: compound 11 (71.4%), 12 (99.0%), 14 (85.8%), and 15 (89.0%).
Scheme 1. Synthesis of racemic compound 1a.
To compare bioactivity of natural
1 and racemic 1a, these two compounds were tested in HepG2
cells for their inhibitory activities against PCSK9 mRNA expression. As expected (Figure 4), natural
1
seemed to be more potent than racemic 1a because racemic 1a are composed of two isomers. From this
result, PCSK9 mRNA expression might be modulated by 1 with specific configurations.
Figure 4. Expressions of PCSK9 (A) LDLR (B) and SREBF1 (C) were assayed by qRT-PCR in cells
treated with compounds 1a, 1, 4 and Ber (berberine) at a indicated concentrations.

Molecules 2018, 23, 504
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Further analysis demonstrated that downregulation of SREBP-1 mRNA was detected in
compounds and , suggesting inhibition of PCSK9 mRNA expression was mediated by SREBP-1 as
1
4
reported in the literature [48]. Thus, it merits enantioselective synthesis of (2R,3R) compound
1 and its
derivatives for further chemical modifications and in vivo studies.
3. Materials and Methods
3.1. General Information
Nuclear magnetic resonance (NMR) spectra were obtained using a Varian 400 spectrometer
1
(Varian, Palo Alto, CA, USA) 400 MHz spectrometer operated at 400 MHz for H-NMR and at
100 MHz for 13C-NMR. High-resolution mass spectra data were measured on a Xevo G2 Q-TOF
mass spectrometer (Waters, Milford, MA, USA). Fourier Transform Infrared (FT-IR) recorded
on a Nicolet
™ iS™ 5 FT-IR spectrometer (ThermoFisher Scientific, Madison, WI, USA) were
used. Ultraviolet visible spectroscopy was performed using a DU 730 UV/Vis spectrophotometer
(Beckman Coulter GmbH, North Rhine-Westphalia, Germany). Optical rotation and CD data are also
given in the Supplementary file. Semi-preparative high performance liquid chromatography (HPLC)
was performed on a system equipped with a Gilson 321 pump and Gilson 172 Diode Array Detector
(
Gilson, Madison, WI, USA) using YMC-pack Ph (250
×
20 mm) and YMC-pack Ph (250
×
10 mm)
HPLC columns (YMC, Kyoto, Japan). Water was purified using a Milli-Q system (Waters Corporation,
Milford, MA, USA). Column chromatography on C-18 RP silica gel (Cosmosil, Kyoto, Japan) and
Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) was conducted, and TLC analysis on silica gel 60
F254 plates (Merck, Darmstadt, Germany) was done. The spots were visualized by spraying with 10%
aqueous H2SO4.
3.2. Cell Culture and Chemical Reagents
The HepG2 human hepatocellular liver cell line was obtained from the Korea Research Institute
of Bioscience and Biotechnology (Daejeon, Korea) and grown in Eagle’s Minimum Essential Medium
(EMEM) containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin sulfate. Cells were
incubated in a humidified 5% CO2 atmosphere at 37 ◦C. EMEM, penicillin, and streptomycin were
purchased from Hyclone (Logan, UT, USA). Bovine serum albumin was purchased from Sigma-Aldrich
(St. Louis, MO, USA). Antibodies against PCSK9, LDL-R, and β-actin were purchased from Abcam, Inc.
(Cambridge, MA, USA). PCSK9, LDL-R, SREBP1, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) oligonucleotide primers were purchased from Bioneer Corp. (Daejeon, Korea). The solvents
for extraction and isolation (methanol, ethyl acetate, n-butyl alcohol, chloroform, n-hexane, etc.) were
purchased from SK Chemical (Seoul, Korea). The solvents for HPLC-grade acetonitrile (MeCN) and
methanol were also purchased from SK Chemical. The solvent for NMR (CD3OD) was obtained from
Cambridge Isotope Laboratories, Inc. (Andover, MA, USA).
3.3. Extraction, Isolation and Synthetic Method
3.3.1. Isolation Method
The roots of S. baicalensis (3 kg) was extracted with MeOH three times (18 L
×
3) at room
temperature and evaporated in vacuo. The concentrated MeOH extract (558.1 g) was suspended in
H2O and successively partitioned between hexane, chloroform, and butyl alcohol, to give the inactive
residues of hexane-soluble fraction, butyl alcohol-soluble fraction, and water-soluble fraction, and
the active residue of chloroform-soluble fraction (34.7 g). The chloroform-soluble fraction (33.8 g,
SBC) was chromatographed over a silica gel column chromatography (CC) (5
×
90 cm, 685 g) using
a gradient of increasing polarity with chloroform:MeOH (200:0–1:1) as eluted solvents system and
was fractioned into 50 sub-fractions (SBC1~50). SBC29 (8.8 g) was subjected to medium pressure
liquid chromatography (MPLC), carried out with the binary system of MeOH–H2O (0:100, 100:0)

Molecules 2018, 23, 504
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to give 8 sub-fractions (SBC 29-1~8). SBC 29-6 was purified by size exclusion chromatography on
Sephadex LH-20 CC by using chloroform:MeOH (1:1) as eluent, and washing by acetone to give
11 sub-fractions (SBC 29-6A~K), including compound
to subject to semi-preparative high performance liquid chromatography (HPLC) performed on a
250 × 21.2 i.d.mm, Acclaim Polar Advantage 2, Thermo Scientific column (5 m, Life Technologies
Korea LCC, Seoul, Korea), using MeCN:H2O (45:65) as solvent system, flow rate 3 mL/min by isocratic
elution method for 30 min and MeCN 100% for 5 min to afford compound (tR 17.3 min, 1.3 mg).
Sub-fraction SBC 44 and 45 were mixed together and separated on Sephadex LH-20 column with
chloroform:MeOH (1:1), to give 14 sub-fractions (SBC 44A~N), includes compound (10.3 mg).
Sub-fraction SBC 44-J was subjected to HPLC separation (250 20 i.d.mm, YMC-Pack Pro C18 RS,
m), using MeCN: H2O as eluted solvent, flow rate 5 mL/min, by isocratic elution, using MeCN 30%
for 25 min, then increase directly to MeCN 35% and then maintained in the same isocratic mode for
15 min and MeCN 100% for 5 min to give compound (tR 23.2 min, 2.4 mg). SBC 44-I was subjected
to HPLC separation (250 21.2 i.d.mm, Acclaim Polar Advantage 2, 5 m) using MeCN:H2O, flow
rate 3 mL/min, eluted with MeCN 30% for 25 min, then MeCN 35% for 15 min and MeCN 100% for
5 min to give compound (tR 29.7 min, 1 mg), compound (tR 41.7 min, 2.4 mg) and compound
4 (227.2 mg). SBC 29-7 was taken out 100 mg
µ
9
5
×
5
µ
1
×
µ
6
7
8
(tR 43.8 min, 1 mg). Sub-fraction SBC 42 was combined with sub-fraction SBC 41 and purified on
Sephadex LH-20 CC to give 10 sub-fractions (SBC 42A~J). Sub-fraction SBC 42-I was further purified
by HPLC (250
×
21.2 i.d.mm, Acclaim Polar Advantage 2, 5
µ
m), with MeCN:H2O (30:70), flow rate
3 mL/min by isocratic mode for 30 min and MeCN 100% for 5 min to afford compound
3
(tR 22.5 min,
3.2 mg). Sub-fraction SBC 48 was mixed with sub-fraction SBC 46 and 47 and separated on Sephadex
LH-20 CC by using chloroform: MeOH (1:1) solvent system and washing by acetone 100% to afford
8 sub-fractions (SBC 48A~H). Compound
2
(tR 12.5 min, 1.6 mg) was isolated from sub-fraction
m) with MeCN:H2O
SBC48-G by HPLC separation (250 21.2 i.d.mm, Acclaim Polar Advantage 2, 5
×
µ
(25:75), flow rate 3 mL/min by isocratic elution mode for 20 min and MeCN 100% for 5 min.
1
2-Methyl-6-phenyl-4H-pyran-4-one (
3
): H-NMR (400 MHz, CD3OD)
δ
: 6.30 (1H, d, J = 2.0 Hz, H-3), 6.82
0
0
0
0
0
(1H, d, J = 2.0 Hz, H-5), 7.55 (3H, m, H-3 , H-4 , H-5 ), 7.90 (2H, dd, J = 4.5, 1.6 Hz, H-2 , H6 ), 2.43 (3H,
s, H-7). 13C-NMR (100 MHz, CD3OD)
δ 169.2 (C-2), 114.4 (C-3), 183.0 (C-4), 110.7 (C-5), 166.3 (C-6),
19.7 (C-7), 132.2 (C-10), 127.1 (C-20, C-60), 130.2 (C-30, C-40, C-50).
3.3.2. Synthesis Method
Phloroacetophenoe monohydrate (>98% purity) and hydrogen peroxide (35% in water) were
purchased from TCI Co., Ltd., (Tapei, Taiwan). Salicylaldehyde (97%) was purchased from Junsei
Chemicals Co., Ltd. (Chuo-ku, Tokyo, Japan). Methoxymethyl chloride (>95%) was purchased from
Kanto Chemicals (Chuo-ku, Tokyo, Japan) with yield. Potassium carbonate (99.5%) was purchased
from Samchun Chemicals (Gangnam-gu, Seoul, South Korea). Terahydrofuran (>99.9%) was purchased
from Sigma-Aldrich (St. Louis, MO, USA). N-Ethyldiisopropylamine (99%) was purchased from Alfa
Aesar (Gangnam-gu, Seoul, South Korea).
1-(2-Hydroxy-4,6-bis(methoxymethyl)phenyl)ethanone
(
11).
To
a
stirred
solution
of
1-(2,4,6-trihydroxyphenyl)ethanone (10, 1 g, 5.9 mmol) in CH2Cl2 (36 mL), was added DMF
(1.2 mL). The reaction mixture was cooled to 0 ◦C, DIPEA (3.1 mL, 17.7 mmol) was added and stirred
for 5 min. Chloro(methoxy)methane (1 g, 12.4 mmol) in CH2Cl2 (5.8 mL) was next added dropwise to
the reaction mixture under a N2 atmosphere. The mixture was warmed to room temperature and
stirred for 4 h. The reaction mass was quenched with sat. aq. NH4Cl, and the organic layer was
separated. The aqueous layer was further extracted with CH2Cl2 (2
layer was washed with brine solution and dried over MgSO4, filtered, and evaporated under vacuum.
×
50 mL), the combined organic
The residue was purified by column chromatography (petroleum ether/ethyl acetate, 8:2, v/v) to
1
afford compound 11 as a colorless oil (0.95 g, 71.4%). H-NMR (CDCl3)
δ 13.74 (s, 1H), 6.26 (s, 1H),

Molecules 2018, 23, 504
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6.24 (s, 1H), 5.26 (s, 2H), 5.17 (s, 2H), 3.52 (s, 3H), 3.47 (s, 3H), 2.66 (s, 3H). 13C-NMR (CDCl3)
166.8, 163.5, 160.4, 106.9, 97.1, 94.5, 94.0, 56.7, 56.5, 33.0.
δ
203.2,
1-(2-Methoxy-4,6-bis(methoxymethyl)phenyl)ethanone (12). To a stirred solution of compound 11 (0.95 g,
3.7 mmol) in acetone (20 mL), K2CO3 (1.5 g, 11.1 mmol) and (CH3)2SO4 (2 0.26 mL, 5.5 mmol)
×
were added at 15 min intervals at room temperature. The reaction mixture was heated to reflux
and maintained for 2 d. The reaction mass was cooled to room temperature, filtered and the filtrate
was evaporated to dryness. The crude product was purified by column chromatography (petroleum
1
ether/ethyl acetate, 8:2, v/v) to afford compound 12 as a pale brown oil (1 g, 99.0%). H-NMR (CDCl3)
δ
6.45 (d, J = 2.0 Hz, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.16 (s, 2H), 5.14 (s, 2H), 3.78 (s, 3H), 3.48 (s, 3H), 3.46
(s, 3H), 2.48 (s, 3H). 13C-NMR (CDCl3)
δ 201.8, 159.7, 157.9, 155.4, 115.8, 95.8, 94.8, 94.4, 93.8, 56.3, 56.2,
55.8, 32.5.
(E)-1-(2-Methoxy-4,6-bis(methoxymethyl)phenyl)-3-(2-(methoxymethyl)phenyl)prop-2-en-1-one (14). To a
stirred solution of compound 12 (0.45 g, 1.66 mmol) in ethanol (10 mL) at 0 ◦C, was added aq. KOH
(4.5 mL, 30% solution) dropwise. After stirring for 10 min then 2-(methoxymethyl) benzaldehyde
◦
(
13, 0.415 g, 2.49 mmol) was added in one portion. The reaction mass was heated to 55 C a◦nd
maintained for 6 h. The reaction progress was monitored by TLC. The reaction mass was cooled to 0 C,
acidified to pH 5–6 using 2 N HCl, and extracted with CH2Cl2 (3
layer was washed with brine solution, dried over MgSO4, filtered and evaporated under vacuum.
×
25 mL). The combined organic
The crude product was purified by column chromatography (petroleum ether/ethyl acetate, 7:3, v/v)
1
to afford compound 14 as a low melting solid (0.6 g, 85.8%
)
. H-NMR (CDCl3)
δ
7.75 (d, J = 16 Hz, 1H),
7.56 (d, J = 7.6, 1.2 Hz, 1H), 7.31 (t, J = 8.8, 1.6, Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 16.4 Hz, 1H),
7.00 (t, J = 7.6 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H), 5.20 (s, 2H), 5.19 (s, 2H), 5.11
(s, 2H), 3.76 (s, 3H), 3.50 (s, 3H), 3.44 (s, 3H), 3.40 (s, 3H). 13C-NMR (CDCl3)
δ 194.6, 159.8, 158.5, 156,2,
156.0, 139.8, 131.5, 129.5, 128.6, 124.7, 122.0, 115.0, 114.0, 95.9, 94.7, 94.6, 94.5, 94.0, 56.2, 55.9.
(2-Methoxy-4,6-bis(methoxymethyl)phenyl)(3-(2-(methoxymethyl)phenyl)oxiran-2-yl)methanone (15). To a
stirred solution of compound 14 (0.07 g, 0.18 mmol) in methanol (6 mL), was added K2CO3 (0.069 g,
0.5 mmol) followed by dropwise addition of H2O2 (0.13 mL, 1.3 mmol, 35% aq. solution) at room
temperature. The reaction mass was stirred for 1 h. After completion of starting material, mixture
was diluted with ether and washed twice with saturated aq. NH4Cl solution. The combined organic
layer was dried over MgSO4, filtered and evaporated under vacuum. The crude product was purified
by column chromatography (petroleum ether/ethyl acetate, 7:3, v/v) to afford compound 15 as a
1
light green oil (0.065 g, 89.0%). H-NMR (CDCl3)
δ 7.25 (m, 2H), 7.19 (d, J = 8.0, 2.0, Hz, 1H), 7.09
(
d, J = 8.0 Hz, 1H), 6.99 (t, J = 8.0 Hz, 1H), 6.45 (d, J = 2.0 Hz, 1H), 6.31 (d, J = 2.0 Hz, 1H), 7.39
(d, J = 2.0 Hz, 1H), 3.87 (d, J = 2.0 Hz, 1H), 3.77 (s, 3H), 3.47 (s, 3H), 3.45 (s, 3H), 3.38 (s, 3H), 13C-NMR
(CDCl3) 196.8, 161.0, 159.4, 157.1, 155.9, 129.4, 125.4, 125.3, 122.0, 114.1, 111.7, 95.7, 94.8, 94.6, 94.3,
δ
93.7, 63.7, 56.3, 56.3, 56.2, 55.9, 55.0.
2,3-Dihydro-3,7-dihydroxy-2-(2-hydroxyphenyl)-5-methoxychromen-4-one (1a). To a stirred solution of
compound 15 (0.05 g, 0.11 mmol) in MeOH (2 mL), was added HCl in methanol (0.2 mL, 12% Wt)
at room temperature. The reaction mass was heated to 55 ◦C and stirred for 30 min. After cooling,
the reaction mixture was concentrated in vacuo, the crude product was then purified by prep. TLC
(silica gel, CH2Cl2/MeOH, 9.5:0.5, v/v) to yield 1a (4 mg, 11.7%) as a white solid of m.p. 124–126 ◦C.
1H-NMR (CD3OD)
6.88 (1H, d, J =7.6 Hz), 6.09 (d, J = 2.0 Hz, 1H), 5.97 (d, J = 2.0 Hz, 1H), 5.48 (1H, d, J = 11.2 Hz), 4.61 (1H,
δ 7.41 (1H, dd, J =7.6, 1.5 Hz), 7.17 (1H, td, J =7.6, 1.5 Hz), 6.82 (1H, d, J =8.5 Hz),
d, J = 11.2 Hz), 3.83 (3H, s). 13C-NMR (CD3OD)
δ 192.2, 166.6, 165.7, 164.0, 156.4, 130.3, 128.8, 123.6,
119.8, 115.9, 102.9, 96.2, 93.5, 78.3, 72.7, 55.5. HRESIMS m/z [M + H]+ 303.0869 (calcd for C16H14O6
303.0869).
Supplementary Materials: Supplementary materials are available online.

Molecules 2018, 23, 504
9 of 11
Acknowledgments: This study was supported by the National Research Foundation of Korea (NRF) grant funded
by the Korea government (MSIT) (NRF-2015R1A2A2A01006736, Y.-W. Chin).
Author Contributions: Y.-W. Chin and W. J. Choi conceived and designed the experiments; P. Nhoek,
J. N. Masagalli, K. Mailar, H.-S. Chae, P. Pel, and Y.-M. Kim performed the experiments; Y.-W. Chin and W. J. Choi
analyzed the data; Y.-W. Chin and W. J. Choi contributed reagents/materials/analysis tools; Y.-W. Chin and
W. J. Choi wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).