Efficient and divergent enantioselective syntheses of DHPVs and anti-inflammatory effect on IEC-6 cells

Article abstract of DOI:10.3390/molecules25092215

Despite numerous reports on the beneficial effects of catechin or epicatechin contained in tea and cacao extract on human health, a conclusive and precise molecular mechanism has not been elucidated. Metabolism of chemical compounds in gut microbiota recently gained significant attention, and extensive studies have been devoted in this field. In conjunction with these results, our group focused on the anti-inflammatory effects of both enantiomers of DHPV (5-(3',4'-dihydroxyphenyl)-γ-valerolactone), produced in the intestine by microbiota metabolism, on IEC-6 cells. Divergent and efficient enantioselective synthesis of (S)- and (R)-DHPV was efficiently achieved by cross-metathesis and Sharpless asymmetric dihydroxylation as a key reaction for four steps in 16% and 14% overall yields, respectively. The anti-inflammatory effects of two enantiomers were tested on IEC-6 cells, and we found that (S)-DHPV was more active than (R)-DHPV. This result implicates that the metabolite produced in the gut has beneficial effects on IEC-6 cells of rat intestines, and the chirality of the metabolite is important for its anti-inflammatory activity. This also provided information for the future discovery of novel small molecular therapeutics for the treatment of inflammatory bowel disease.

Full text of DOI:10.3390/molecules25092215

molecules
Article
Efficient and Divergent Enantioselective Syntheses of
DHPVs and Anti-Inflammatory Effect on IEC-6 Cells
Hyun Su Kim 1,†, Sungkyun Chung 1,†, Moon-Young Song 1,†, Changjin Lim 2, Hyeyoung Shin 1,
Joonseong Hur 3, Hyuk Kwon 1, Young-Ger Suh 1, Eun-Hee Kim 1,
*
, Dongyun Shin 4,* and
Seok-Ho Kim 1,
*
1
Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University,
120 Haeryong-ro, Pocheon 11160, Korea; khs8812@snu.ac.kr (H.S.K.); wjdtjdrbs123@naver.com (S.C.);
wso219@naver.com (M.-Y.S.); onam54@gmail.com (H.S.); sed0guitar@naver.com (H.K.);
ygsuh@cha.ac.kr (Y.-G.S.)
School of Pharmacy, Jeonbuk National University, Jeonju 54896, Korea; limcj@jbnu.ac.kr
Natural Products Research Institute, Korea Institute of Science and Technology (KIST), 679 Saimdang-ro,
Gangneung 25451, Korea; hjs1120@kist.re.kr
College of Pharmacy, Gachon University, 191 Hambangmoe-ro, Yeonsu-gu, Incheon 21936, Korea
Correspondence: ehkim@cha.ac.kr (E.-H.K.); dyshin@gachon.ac.kr (D.S.); ksh3410@cha.ac.kr (S.-H.K.);
Tel.: +82-31-881-7179 (E.-H.K.); +82-032-820-4945 (D.S.); +82-31-881-7169 (S.-H.K.)
These authors contributed equally to this work.
2
3
4
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 April 2020; Accepted: 6 May 2020; Published: 8 May 2020
Abstract: Despite numerous reports on the beneficial effects of catechin or epicatechin contained
in tea and cacao extract on human health, a conclusive and precise molecular mechanism has
not been elucidated. Metabolism of chemical compounds in gut microbiota recently gained
significant attention, and extensive studies have been devoted in this field. In conjunction with
these results, our group focused on the anti-inflammatory effects of both enantiomers of DHPV
(5-(30,40-dihydroxyphenyl)-
γ-valerolactone), produced in the intestine by microbiota metabolism, on
IEC-6 cells. Divergent and efficient enantioselective synthesis of (S)- and (R)-DHPV was efficiently
achieved by cross-metathesis and Sharpless asymmetric dihydroxylation as a key reaction for four
steps in 16% and 14% overall yields, respectively. The anti-inflammatory effects of two enantiomers
were tested on IEC-6 cells, and we found that (S)-DHPV was more active than (R)-DHPV. This
result implicates that the metabolite produced in the gut has beneficial effects on IEC-6 cells of rat
intestines, and the chirality of the metabolite is important for its anti-inflammatory activity. This also
provided information for the future discovery of novel small molecular therapeutics for the treatment
of inflammatory bowel disease.
Keywords: divergent synthesis; asymmetric dihydroxylation; DHPV; anti-inflammatory effect;
NF-κB; IκBα
1. Introduction
Inflammatory bowel disease (IBD) is a chronic and multifactorial inflammatory condition of
the gastrointestinal tract, related to an infectious and immunological imbalance of the intestinal
mucosa [
1
,
2
]. The incidence and prevalence of IBD has relentlessly climbed for past several decades [
3
4
],
].
and it is known to be one of the most important risk factors for developing colorectal cancer [
IBD is most commonly classified into two principal types: Crohn’s disease (CD) and ulcerative
colitis (UC). CD can involve any segment of the gastrointestinal tract and causes non-continuous
whole layer inflammation, while UC occurs in the inner lining of the large intestine or rectum and
causes continuous inflammation of the colonic mucosa [
5
,6
]. This involves immunity, damage, and
Molecules 2020, 25, 2215; doi:10.3390/molecules25092215
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2215
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patient’s symptoms such as diarrhea and hematochezia [
7
]. These responses are controlled by nuclear
factor kappa-light-chain-enhancer of activated B cells (NF-
inflammatory reaction. Dysregulation of NF- B signaling is well known to patients with IBD and are
associated with the rapid and acute production of diverse pro-inflammatory mediators [ ]. Therefore,
B signaling are needed to develop new
κB), which are master regulators of the
κ
8
pharmacological attempts to block the activation of NF-
κ
therapeutic strategies in IBD.
Metabolism in the body plays an important role in the elimination of xenobiotics absorbed by
diverse route. Among them, the liver is the major organ contributing to these transformations of phase
I and phase II metabolisms [9]. Sometimes, metabolites produced in these biological processes can be
newly discovered therapeutics, for example cetirizine or oxazepam. Recently, growing interests in
metabolism on the intestine and its effects on brain have also been reported.
Flavanols, such as (
−
)-catechin, ( )-epicatechin, and each enantiomer, are widely found in red
−
wine, green tea, and cocoa extract (Figure 1). Catechins exist in four isomers, and the enantiomers
can be separated by chiral HPLC chromatography [10]. Many research groups have investigated the
molecular mechanism, signaling pathway, and metabolisms of polyphenols due to their well-known
antioxidant and free radical scavenging activities. However, beneficial effects of these compounds
on human health were not conclusive, because these compounds are not readily absorbed into the
body as their original structures. Rather, these flavanols undergo metabolism into smaller structures in
the gut by microbiota. These metabolites, produced in the intestine, were smoothly absorbed into the
bloodstream, even though their potency was decreased compared to the parent molecule.
OH
OH
OH
OH
O
O
HO
O
HO
O
OH
OH
OH
OH
O
O
OH
OH
OH
OH
(S)-DHPV (1a)
(R)-DHPV (
1b)
(+)-Catechin
(-)-Catechin
OR1
OR2
via metabolism in the gut
by microbiota
O
HO
O
OH
caffeic acid, R1=OH, R2=OH
ferulic acid, R1=OH, R2=OCH3
isoferulic acid, R1=OCH3, R2=OH
Flavanol
OH
OH
OH
OH
OH
HO
O
HO
O
HO
HO
( )n
OH
( )n
OH
O
O
OH
OH
n=1, 3,4-dihydroxyphenylpropionic acid
n=2, 3,4-dihydroxyphenylpropionic acid
n=3, 3,4-dihydroxyvaleric acid
n=1, 3-hydroxyphenylpropionic acid
n=2, 3-hydroxyphenylpropionic acid
n=3, 3-hydroxyvaleric acid
OH
OH
(+)-Epicatechin
(-)-Epicatechin
Figure 1.
Structures of catechin,
epicatechin,
and metabolites,
including
5-(30,40-dihydroxyphenyl)-γ-valerolactones (DHPVs).
Catechin and epicatechin undergo intestinal metabolism, producing diverse metabolites including
caffeic acid, ferulic acid, isoferulic acid, and so on (Figure 1) [11]. Among the various microbial
metabolites from polyphenols in the human body, 5-(30,40-dihydroxyphenyl)-
-valerolactone (DHPV)
γ
is the representative metabolite of flavanols containing the intact catechin and epicatechin moiety.
DHPV, well-known for its antioxidant activities, such as its protective effect against UVB induced
wrinkle formation and neuroprotective effect in the brain, can exist as two enantiomers by the
stereocenter in γ-position of butyrolactone moiety. However, their structures are represented in
ambiguous or racemic forms in numerous literatures, and we became interested in confirming which
DHPV enantiomer exhibited more potent biological activities, because there are numerous reports on
the completely different biological activities of enantiomers [12,13].

Molecules 2020, 25, 2215
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Herein, we reported the enantioselective synthesis of (S)- and (R)-DHPV by the divergent and
efficient approach. We also evaluated the effects of both enantiomers on lipopolysaccharide (LPS)
induced phosphorylation and degradation of inhibitor of κB alpha (IκBα), the major key regulators of
NF-κB signaling pathway, to test the mechanism of anti-inflammatory effects on IEC-6 cells.
2. Results and Discussion
2.1. Retrosynthetic Analysis of 5-(30,40-Dihydroxyphenyl)-γ-Valerolactone (DHPV)
Until now, four groups completed the racemic or enantioselective total synthesis of DHPV. The
Watanabe [14] group reported the first racemic synthesis of DHPV from benzyl epoxide as a key
intermediate in a four step synthesis, and the Lambert [15] group also reported the racemic synthesis
of DHPV via Heck reaction and dehydrative lactonization for a total of eight synthetic steps. The first
enantioselective synthesis of DHPV was completed by the Nakajima [16,17] group from optically pure
epoxide in seven steps, and the Curti [18] group achieved the relatively recent asymmetric synthesis of
DHPV via the concise and catalytic enantioselective Mukaiyama aldol reaction in six steps. All of the
previous reports of DHPV were accomplished in four to eight steps, but the only racemic DHPV was
obtained through the minimal synthetic pathway.
Our group planned the divergent synthesis and biological evaluation of both enantiomers.
Representative retrosynthetic analysis of DHPV was summarized in Scheme 1. (S) or (R)-DHPV
could be obtained by deoxygenation of benzylic alcohol and demethylation from 2a or 2b
-Hydroxy- -valerolactone (2a 2b) could be formed by Sharpless Asymmetric Dihydroxylation
(SAD) [21] and spontaneous lactonization from common intermediate alkene . Alkene can be
obtained from by cross metathesis, and styrene was used to lower homo-dimerization of terminal
olefin [22]. Although we planned the synthesis of -unsaturated ester by Johnson–Claisen
[19,20].
δ
γ
,
3
3
4
4
γ
,
δ
3
rearrangement [15] instead of cross metathesis at the beginning of this project, we couldn’t obtain
meaningful result.
Scheme 1. Retrosynthetic analysis of DHPV.
2.2. Divergent Synthesis of DHPV
Synthesis of (S)- and (R)-DHPV began with construction of the phenylpentanoate scaffold
(Scheme 2). Cross metathesis of commercially available (E)-3,4-dimethoxyphenylpropene (
methyl 4-pentenoate ( ) by Grubbs 2nd generation catalyst afforded the desired product in 59% yield.
Interestingly, only (E)-olefinic isomer was obtained, which was identified through 1H-NMR analysis
(Supplementary Materials). One pot Sharpless asymmetric dihydroxylation (AD-mix- and AD-mix-
respectively) and concomitant lactonization of alkene provided 2a and 2b in 62% and 55% for each
4) and
5
6
α
β,
6
isomer, respectively. Then, alcohols 2a and 2b were subjected to hydrogenolysis, providing 7a and
7b by Pearlman’s catalyst in good yields of 72% and 68%, which were transformed to 1a and 1b by
demethylation with BBr3 in 60% and 62%, respectively. The 1H- and 13C-NMR spectral data of both

Molecules 2020, 25, 2215
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enantiomers were compared and matched well. A detailed summary of the spectrum was represented
in Supplementary Materials.
Scheme 2. Divergent and enantioselective synthesis of DHPV. (
CH2Cl2, reflux, 59% (70% BORSM); ( ) AD-mix- , MeSO2NH2, t-BuOH/H2O (1/1), 62%; (
MeSO2NH2, t-BuOH/H2O (1/1), 55%; ( ) Pd(OH)2, H2, MeOH, r.t. 72% (7a) and 68% (7b); and (
CH2Cl2, 0 ◦C to r.t., 60% (1a) and 62% (1b).
a
)
4
,
5
, Grubbs 2nd generation catalyst,
) AD-mix-
) BBr3,
b
α
c
β,
d
e
The enantiomeric purities of asymmetric dihydroxylation products, 2a and 2b, and final (S)- and
(R)-DHPV, 1a and 1b, were confirmed by the analytical chiral HPLC experiment (Supplementary
Materials), according to previous report of Suh group [23]. The synthetic final enantiomers were pure
enough for further biological study, with the enantioselectivity of 97.3:2.7 for 1a and 95.1:4.9 for 1b.
2.3. Anti-Inflammatory Activity of (R)- and (S)-DHPV on IEC-6 Cells
NF-
and carcinogenesis [
of IBD, and the transcription factor, NF-
in this complex scenario [25]. It is well known that the expression and activation of NF-
strongly induced in the inflamed gut of IBD patients. In particular, macrophages and epithelial
cells isolated from inflamed gut specimens from IBD patients showed increased levels of the NF-
subunit [26]. Interestingly, the amount of activated NF- B significantly correlated with the severity
of intestinal inflammation [27]. Classic activation of NF- B can be initiated by various stimuli,
κ
B is a major regulator of the immune system, and the activation of NF-
24]. Dysregulated immune responses were implicated in the pathogenesis
B, was found to be one of the key regulatory components
B was
κB induces inflammation
6,8,
κ
κ
κ
B
κ
κ
including bacterial cell wall components like lipopolysaccharide (LPS), pro-inflammatory cytokines,
viruses, and DNA damaging agents. These triggering substances stimulate a phosphorylation of
I
κβs, and the translocation of NF-
inflammation-related proteins [28 29]. Therefore, the measurement of phosphorylation and degradation
of I could be the simple but valid assay for the anti-inflammatory ability of synthesized compounds
κB into the nucleus promotes the transcription of genes that encode
,
κBα
through the suppression of NF-κB in IBD.
To evaluate the preventive effects of DHPVs against inflammatory responses, we pretreated rat
intestinal epithelial IEC-6 cells with each DHPV (25
The inhibitory effect of (R)-DHPV (1b) and (S)-DHPV (1a) on phosphorylation and degradation of I
expression were tested under the same conditions for comparison. We observed that the inhibitory
effect of (S)-DHPV on the phosphorylation and degradation of I was more significant than that of
(R)-DHPV in LPS-treated IEC-6 cells (Figure 2A). Comparing the anti-inflammatory effects of (S)-DHPV
with (R)-DHPV, in terms of suppression of phosphorylation and degradation of I , (S)-DHPV
was found to be more effective in suppressing NF- B signaling than (R)-DHPV. We also observed
µM) for 1 h prior to the LPS stimulation (Figure 2A).
κBα
κBα
κBα
κ

Molecules 2020, 25, 2215
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that (S)-DHPV (1a) downregulated LPS-induced phosphorylation and degradation of I
κ
Bα
in a dose
dependent manner (Figure 2B). These results indicated that (S)-DHPV can protect against LPS-induced
inflammation by inhibiting NF-κB activation through repression of IκBα degradation.
Figure 2. Biological activity of synthesized (R)- and (S)-DHPV. (
and 1a (S)-DHPV (25 M) on the phosphorylation and degradation of I
Western blot analysis in IEC-6 cells exposed to lipopolysaccharides (LPS; 10 ng/mL) for 1 h. (
A
) The effects of 1b (R)-DHPV (25
were determined by
) The
was analyzed
µM)
µ
κBα
B
effect of 1a (S)-DHPV on the expression of phosphorylation and degradation of I
κBα
dose dependently by Western blot analysis in LPS (10 ng/mL)-treated IEC-6 cells.
3. Materials and Methods
3.1. General Information
3.1.1. General Methods and Materials
Unless otherwise noted, all the reactions were run under nitrogen (N2) atmosphere in anhydrous
condition. DHPVs were dissolved in dimethyl sulfoxide (DMSO) as stock solution and stored at
20 ◦C.
DMSO was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lipopolysaccharide (LPS) was
−
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies for Phosphorylated-IkBα and
total IkBα were purchased from Cell Signaling Technology (Danvers, MA, USA).
3.1.2. Materials
All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) or TCI (Tokyo, Japan),
and solvents were purchased from Daejung (Siheung, Korea) and used without further purification.
Dichloromethane (CH2Cl2) was distilled from calcium hydride (CaH2). Methanol (MeOH) was distilled
from magnesium sulfate (MgSO4).
3.1.3. Instrumentation
1H and 13C-spectra were recorded on a 500 MHz JNM-ECZ 500R (JEOL, Tokyo, Japan). Chemical
shifts are recorded as a
δ value (δ δ
7.26 for 1H and 77.0 for 13C). Data were represented as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br =
broad), integration, coupling constant in Hz, and assignment. Infrared (IR) spectra were measured on
a 1600 FT–IR spectrometer (Perkin-Elmer, Waltham, MA, USA). High resolution mass spectrometry
data were recorded by JMS-700 (JEOL, Tokyo, Japan) (FAB or ESI), and values were calculated to four
decimal places from the molecular formula. Melting point (m.p.) was obtained using MEL-TEMP®
(Cole-Parmer, Staffordshire, UK).
3.2. Experimental Part
(E)-Ethyl-5-(3,4-dimethoxyphenyl)pent-4-enoate (
6). To a stirred solution of ethyl pent-4-enoate
(967mg, 7.54 mmol) and 4-(1-propenyl)-1,2-dimethoxybenzene (2.67 g, 15.1 mmol, 2.0 equiv.) in CH2Cl2

Molecules 2020, 25, 2215
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(50 mL) was added Grubbs 2nd generation catalyst (310 mg, 0.38 mmol, 0.05 equiv.) and then heated to
reflux for 6 h. Then, the reaction mixture was cooled to room temperature, and solvent was removed
under reduced pressure. EtOAc/n-hexane (1:10, 30 mL) was added, and the solid was filtered to
provide the 1.76 g dimer of styrene. The filtrate was purified by silica gel column chromatography
to afford
(70% BORSM); 1H-NMR (500 MHz, CDCl3)
6.78 (d, J = 8.5 Hz, 1H), 6.35 (d, J = 15.5 Hz, 1H), 6.06 (dt, J = 16.0, 6.6 Hz, 1H), 4.13 (q, J = 7.3 Hz, 2H),
3.88 (s, 3H), 3.86 (s, 3H), 2.52–2.44 (m, 4H), and 1.24 (t, J = 7.2 Hz, 3H); 13C-NMR (125 MHz, CDCl3)
173.1, 149.0, 148.5, 130.6, 130.6, 126.6, 119.1, 111.1, 108.6, 60.4, 55.9, 55.8, 34.2, 28.3, and 14.3; FT-IR (thin
6
(1.17 g, 4.44 mmol), a colorless oil with recovered ethyl pent-4-enoate (160 mg); Yield 59%
δ
6.88 (d, J = 1.7 Hz, 1H), 6.85 (dd, J = 8.0, 1.7 Hz, 1H),
δ
film, neat)
ν
2980, 2358, 2341, 2040, 1732, 1514, 1263, 1024, and 966 cm−1; and HRMS (ESI+) found
max
265.1444 (calculated for C15H21O4 ([M + H]+): 265.1434).
(S)-5-((S)-(3,4-dimethoxyphenyl)(hydroxy)methyl)dihydrofuran-2(3H)-one (2a). To a stirred solution
of AD-mix-
t-BuOH/H2O (1:1, 30 mL) was added
α
(1.47 g, 1.40g/mmol) and methane sulfonamide (120.9 mg, 1.27 mmol, 1.21 equiv.) in
6
(280 mg, 1.05 mmol) in t-BuOH (10 mL) and stirred at 0 ◦C for
72 h. Then, EtOAc (100 mL) was added to the reaction mixture and the organic layer was separated.
The aqueous layer was extracted again with EtOAc (two times, 50 mL) and the combined organic
layer was washed with H2O (two times, 10 mL). Then, the organic layer was dried over MgSO4 and
the solvent was removed under reduced pressure. The lactone was purified by silica gel column
chromatography (EtOAc/n-hexane, 2:1) to afford 2a (164 mg, 0.65 mmol) as a white solid; Yield 62%;
m.p. 138–140 ◦C; 1H-NMR (500 MHz, CDCl3)
6.83 (d, J = 8.0 Hz, 1H), 4.63–4.59 (m, 2H), 3.87 (s, 3H), 3.86 (s, 3H), 2.50–2.43 (m, 3H), and 2.03–1.95 (m,
2H); 13C-NMR (125 MHz, CDCl3)
177.0, 149.3, 149.2, 130.8, 119.4, 111.0, 109.7, 83.5, 76.3, 56.0, 55.9,
δ 6.91 (d, J = 1.7 Hz, 1H), 6.89 (dd, J = 8.0, 1.7 Hz, 1H),
δ
28.5, and 24.0; FT–IR (thin film, neat)
ν
3477, 2939, 1770, 1514, 1263, and 1095 cm−1; and HRMS
max
(ESI+) found 252.0979 (calculated for C13H16O5 ([M]+): 252.0992).
(R)-5-((R)-(3,4-dimethoxyphenyl)(hydroxy)methyl)dihydrofuran-2(3H)-one (2b). This reaction was
conducted with AD-mix-
(500 MHz, CDCl3) 6.93 (d, J = 2.3 Hz, 1H), 6.91 (dd, J = 8.6, 1.7 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H),
4.70–4.62 (m, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 2.75 (bs, 1H), 2.49–2.43 (m, 2H), and 2.03–1.99 (m, 2H);
13C-NMR (125 MHz, CDCl3)
177.2, 149.2, 149.2, 130.9, 119.4, 111.0, 109.8, 83.6, 76.2, 56.0, 55.9, 28.5,
β, instead of AD-mix-α
. Yield 55%, white solid; m.p. 129–131 ◦C; 1H-NMR
δ
δ
and 24.0; FT–IR (thin film, neat)
ν
3477, 2939, 1770, 1516, 1263, 1184, and 1141 cm−1; HRMS (ESI+)
max
found 252.1003 (calculated for C13H16O5 ([M]+): 252.0992).
(S)-5-(3,4-dimethoxybenzyl)dihydrofuran-2(3H)-one (7a). To a stirred solution of 2a (150 mg,
0.603 mmol) in MeOH (20 mL) and N2 atmosphere was added Pd(OH)2 on activated charcoal
(20 mg). Then, nitrogen gas was removed under reduced pressure, and the reaction mixture was
charged with a hydrogen gas balloon. After 6 h, the reaction mixture was filtered through a Celite
pad, and solvent was removed under reduced pressure. The crude product was purified by silica gel
column chromatography (EtOAc/n-hexane, 1:1) to afford 2a (102 mg, 0.43 mmol), a colorless oil; Yield
72%; 1H-NMR (500 MHz, CDCl3)
1H), 3.85 (s, 3H), 3.84 (s, 3H), 2.96 (dd, J = 14.4, 5.8 Hz, 1H), 2.89 (dd, J = 14.3, 6.3 Hz, 1H), 2.50–2.22
(m, 3H), and 1.96–1.89 (m, 1H); 13C-NMR (125 MHz, CDCl3)
177.2, 149.0, 148.1, 128.3, 121.6, 112.6,
111.4, 111.3, 80.9, 55.9, 55.9, 28.7, and 27.0; FT-IR (thin film, neat) max 3522, 2937, 1770, 1734, 1514, 1261,
δ 6.79 (d, J = 8.1 Hz, 1H), 6.75–6.71 (m, 2H), 4.71 (quintet, J = 6.4 Hz,
δ
ν
1178, 1157, 1141, and 1026 cm−1; HRMS (ESI+) found 237.1129 (calculated for C13H17O4 ([M + H]+):
237.1121).
(R)-5-(3,4-dimethoxybenzyl)dihydrofuran-2(3H)-one (7b). Yield 68%, colorless oil; 1H-NMR (500 MHz,
CDCl3)
3H), 2.97 (dd, J = 14.3, 5.8 Hz, 1H), 2.89 (dd, J = 14.4, 5.8 Hz, 1H), 2.47–2.23 (m, 3H), and 1.97–1.89
(m, 1H); 13C-NMR (125 MHz, CDCl3)
177.2, 149.0, 148.2, 128.4, 121.6, 112.7, 113.4, 80.9, 56.0, 55.9,
40.9, 28.7, and 27.0; FT–IR (thin film, neat)
3477, 2939, 1772, 1516, 1263, 1184, 1143, and 1026 cm−1
δ 6.80 (d, J = 8.0 Hz, 1H), 6.76–6.73 (m, 2H), 4.71 (quintet, J = 6.9 Hz, 1H), 3.86 (s, 3H), 3.85 (s,
δ
ν
;
max
HRMS (ESI+) found 237.1125 (calculated for C13H17O4 ([M + H]+): 237.1121).

Molecules 2020, 25, 2215
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(S)-5-(3,4-dihydroxybenzyl)dihydrofuran-2(3H)-one (1a). To a stirred solution of 7a (50 mg, 0.212 mmol)
in CH2Cl2 (10 mL) was added to BBr3 (100
L, 1.06 mmol, 5.0 equiv.) at 0 ◦C, and the reaction mixture
µ
was warmed to room temperature. Then, the reaction mixture was quenched with H2O (10 mL) at 0 ◦C,
and the organic layer was separated. The aqueous layer was extracted again with CH2Cl2 (two times,
50 mL), and the combined organic layer was washed with H2O (two times, 10 mL). Then, the organic
layer was dried over MgSO4, and the solvent was removed under reduced pressure. The lactone was
purified by silica gel column chromatography (CH2Cl2/MeOH = 10:1) to afford 1a (26 mg, 0.127 mmol),
a white solid. Yield 60%; m.p. 163–165 ◦C; 1H-NMR (500 MHz, DMSO-d6)
δ 8.74 (bs, 2H), 6.60 (d, J =
8.0 Hz, 1H), 6.58 (d, J = 2.3 Hz, 1H), 6.44 (dd, J = 8.0, 2.3 Hz, 1H), 4.58 (quintet, J = 6.7 Hz, 1H), 2.73 (dd,
J = 13.8, 6.9 Hz, 1H), 2.66 (dd, J = 13.7, 5.8 Hz, 1H), 2.41 (dd, J = 17.8, 8.6 Hz, 1H), 2.31 (ddd, J = 17.8, 9.8,
5.2 Hz, 1H), 2.14–2.07 (m, 1H), and 1.83–1.76 (m, 1H); 13C-NMR (125 MHz, DMSO-d6)
δ
177.6, 145.5,
max 3404, 1575, 1517,
1273, and 1186 cm−1; HRMS (ESI+) found 209.0811 [calculated for C11H13O4 ([M + H]+): 209.0808].
(R)-5-(3,4-dihydroxybenzyl)dihydrofuran-2(3H)-one (1b). Yield 62%, white solid; m.p. 154
156 ◦C;
1H-NMR (500 MHz, DMSO-d6)
8.78 (bs, 2H), 6.60 (d, J = 7.5 Hz, 1H), 6.57 (d, J = 1.7 Hz, 1H), 6.44 (dd,
144.4, 128.0, 120.5, 117.2, 115.9, 81.3, 40.2, 28.7, and 27.1; FT–IR (thin film, neat)
ν
−
δ
J = 8.0, 2.3 Hz, 1H), 4.58 (quintet, J = 6.7 Hz, 1H), 2.73 (dd, J = 14.3, 6.8 Hz, 1H), 2.65 (dd, J = 14.3, 6.5
Hz, 1H), 2.41 (dd, J = 17.8, 9.2 Hz, 1H), 2.31 (ddd, J = 17.2, 9.2, 4.6 Hz, 1H), 2.13–2.08 (m, 1H), and
1.83–1.75 (m, 1H); 13C-NMR (125 MHz, DMSO-d6)
δ 177.6, 145.6, 144.4, 127.9, 120.5, 117.2, 115.9, 81.3,
40.2, 28.7, and 27.1; FT-IR (thin film, neat) ν ;
max 3331, 3055, 1762, 1517, 1444, 1265, 1180, and 736 cm−1
HRMS (ESI+) found 209.0808 (calculated for C11H13O4 ([M + H]+): 209.0808).
3.3. Cell Culture and Treatments
Rat intestinal epithelial IEC-6 cells were obtained from the American Type Culture Collection
(ATCC, Rockville, MD, USA) and maintained according to the ATCC’s instructions. IEC-6 cells were
grown as a monolayer in Dulbecco’s modified Eagle’s medium (HyClone, GE Healthcare, Logan,
UT, USA), containing 10% (v/v) fetal bovine serum (ATCC), 100 U/mL penicillin, and 100 µg/mL
streptomycin and maintained at 37 ◦C in a humidified atmosphere containing 5% CO2. The cells were
treated with or without LPS for 1 h, in the presence or absence of DHPV.
3.4. Cell Lysates and Western Blot Analysis
Cell monolayers were washed twice with PBS, scraped on ice into cold cell lysis buffer containing
protease inhibitor (Roche Applied Science, Mannheim, Germany), and centrifuged to remove the pellet
and debris. Western blot analysis was performed as previously described [1]. Proteins were separated
by SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were incubated with the
primary antibodies, washed, incubated with peroxidase conjugated secondary antibodies, rewashed,
and then visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham,
MA, USA).
4. Conclusions
Our results, focused on the metabolite produced by metabolism of the catechins in the intestine
and subsequent effects on the intestine, has been disclosed here. In this context, we synthesized both
enantiomers of DHPV via the efficient and divergent approach and confirmed their biological activity.
The synthesis was satisfactorily achieved with overall yield of 14% to 16% and the enantioselectivity of
97.3:2.7 (1a) and 95.1:4.9 (1b), respectively. The main cause of the IBD was related to inflammation on
the intestine. Therefore, the anti-inflammatory effects of these compounds on rat intestinal epithelial
IEC-6 cells were examined. (S)-DHPV proved to more efficiently inhibit inflammation induced by LPS
by inhibiting the phosphorylation of IκBα, dose-dependently.
Our findings indicate that there was a new rationale to use (S)-DHPV as a new therapeutic agent
for the prevention and treatment of IBD. Due to the complex mucosal immune system and very broad

Molecules 2020, 25, 2215
8 of 9
panel of genes controlled by NF-
κ
B, which contribute to the pathogenesis of IBD, it is necessary to
carefully explore the further effects of (S)-DHPV on NF-κB signaling.
Supplementary Materials: The following are available online: 1H and 13C-NMR spectra and the chiral
HPLC analysis.
Author Contributions: Conceptualization and methodology, S.-H.K., D.S., and E.-H.K.; investigation, H.S.K.,
S.C., M.-Y.S., H.S., and H.K.; formal analysis, H.S.K., C.L., J.H., H.S., H.K., S.-H.K., and E.-H.K.; writing—original
draft preparation, S.-H.K., D.S., and E.-H.K.; writing—review and editing, Y.-G.S., S.-H.K., D.S., and E.-H.K.;
supervision, Y.-G.S., S.-H.K., and E.-H.K.; funding acquisition, S.-H.K.; All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by S.-H. Kim, National Research Foundation of Korea grant
NRF-2017R1D1A1B03034612, and the GRRC program of Gyeonggi province GRRC-CHA2018-B02, Development
of Effective Substance from Region-specific Resources.
Conflicts of Interest: The authors declare no conflict of interest.
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