Practical Synthesis and Biological Evaluation of Bergenin Analogs

Article abstract of DOI:10.1111/j.1747-0285.2011.01194.x

Here, we describe the practical synthesis and biological properties of bergenin and its structural analogs. Synthetic bergenin compounds were prepared by acylation of bergenin. These compounds were then evaluated for suppression of lipopolysaccharide-indu

Full text of DOI:10.1111/j.1747-0285.2011.01194.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01194.x
Chem Biol Drug Des 2011; 78: 725–729
Research Letter
Practical Synthesis and Biological Evaluation of
Bergenin Analogs
Jae-Chul Jung1,†, Eunyoung Lim2,†, Seung
properties and characteristic fused ring molecular architecture
(Figure 1) (7,8).
Hwan Kim3, Nam Soo Kim4, Mankil Jung2,*
and Seikwan Oh1,*
Bergenin moieties possess important pharmacological properties
including analgesic (9), anti-arthritis (10), anti-arrhythmic, antioxi-
1Department of Neuroscience, TIDRC and Medical Research
dative antimicrobial, hepatoprotective, anti-inflammatory, and anti-
cancer effects (11–15). They are also used to enhance wound
healing and improve symptoms of Alzheimer's disease and senes-
cence.
Institute, School of Medicine, Ewha Womans University, Seoul 158–
710, South Korea
2Department of Chemistry, Yonsei University, Seoul 120–749, Korea
3College of Physical Education, Kyunghee University, Yongin, Korea
4Department of Sports and Leisure Studies, Kyungwon University,
Songnam, Korea
Bergenin-containing herbs were traditionally used for cardiac ar-
rhythmias, and a number of current comparative pharmacological
investigations of bergenin and its derivatives have found good anti-
oxidant activity with low side effects and little toxicity.
*Corresponding authors: Mankil Jung, mjung @yonsei.ac.kr and
Seikwan Oh, skoh@ewha.ac.kr
These authors contributed equally to this work.
Here, we describe the practical synthesis and bio-
logical properties of bergenin and its structural
analogs. Synthetic bergenin compounds were pre-
pared by acylation of bergenin. These compounds
were then evaluated for suppression of lipopoly-
saccharide-induced nitric oxide (NO) generation in
cultured cells and anti-narcotic effects on mor-
phine-dependent mice. We found that bergenin
derivatives showed potent anti-inflammatory
activity (suppression of NO generation) at concen-
trations ranging from 20 to 30 lM in vitro, and
bergenin derivatives (10–20 mg ⁄ kg) exhibited sig-
nificant anti-narcotic effects on morphine depen-
dence in mice. These results suggest the potential
utility of bergenin and its analogs as anti-narcotic
agents and the design of more potent anti-inflam-
matory compounds.
A recent report (16–18) described the simple synthesis of bergenin
derivatives and their biological properties. The Fukuyama and other
groups have researched the synthesis of synthetic bergenin moieties
and their antioxidant and neuroprotective activities (19). Our medici-
nal research program is focused on the synthesis of biologically
active bergenin derivatives, and we report here the simple derivati-
zation of bergenin by an acylation reaction. The biological activities
of bergenin and its structural analogs in suppression of lipopolysac-
charide (LPS)-induced nitric oxide (NO) generation in vitro suggest
their possible usefulness as novel antioxidant and anti-inflammatory
lead compounds.
Experimental Section
Key words: bergenin, methylation, acylation reaction, NO genera-
tion, neurotoxicity
General procedure
Reactions requiring anhydrous conditions were performed with stan-
dard precautions for rigorous exclusion of air and moisture. Thin
layer chromatography was performed on precoated silica gel G and
GP uniplates from Analtech and visualized with 254-nm UV light.
Flash chromatography was carried out on Merck silica gel 60 [parti-
Received 10 July 2010, revised 14 July 2011 and accepted for publica-
tion 16 July 2011
1
cle size 230–400 lm, pore size 60 ꢀ]. H NMR and 13C NMR spec-
Natural polyphenolic products are often used as intermediates for
industrial products and phytochemical applications including food
sciences, as well as for traditional drugs (1). These compounds act
as scavengers for free radicals that are correlated with the progres-
sion of various pathological conditions. Isocoumarin derivatives nor-
bergenin 1 and bergenin 2 have been isolated from the roots of
Bergenia crassifolia (2,3), bark of Corylopsis spicata (4), heartwood
of Shorea leprosula (5), and roots of Caesalpinia digyna (6) and
have evoked a great deal of interest because of their biological
tra were recorded on a Bruker DPX 250 (Bruker, Germany) at 250
and 63 MHz, respectively. The chemical shifts are reported in parts
per million (ppm) downfield from tetramethylsilane, and J-values
are in Hz. Infrared (IR) spectra were obtained on an JASCO FT ⁄ IR-
430 spectrometer (USA). Mass spectra were recorded with an
Applied Biosystems (USA) 4700 proteomics analyzer spectrometer.
When necessary, chemicals were purified as previously described
(20).
725

Jung et al.
General procedure for the preparation of
compounds 3–6 by condensation of bergenin 2
and various acid chlorides
992, 851 ⁄ cm; 1H NMR (250 MHz, CDCl3) d 8.20 (d, J = 7.58 Hz,
3H), 7.95 (d, J = 7.90 Hz, 5H), 7.81 (d, J = 7.27 Hz, 2H), 7.68–7.23
(m, 16 H), 6.05 (t, J = 9.64 Hz, 1H), 5.72 (brs, 1H), 5.16 (d,
J = 9.16 Hz, 1H), 4.73 (t, J = 10.11 Hz, 1H), 3.97–4.05 (m, 2 H) 3.93
(s, 3 H), 3.48 (d, J = 10.16 Hz, 1 H); 13C NMR (63 MHz, CDCl3) d
165.6, 165.0, 161.7, 144.6, 134.2, 133.5, 133.4, 133.1, 130.4, 130.0,
129.8, 129.7, 129.5, 129.0, 128.9, 128.6, 128.4, 77.7, 77.2, 76.7,
76.6, 72.6, 68.8, 61.9; MALDI-TOF-MS calcd. for C49H36O14 871.1997
[M+Na]+, found: 871.5660.
A stirred solution of bergenin 2 (1 g, 3.05 mmol) in pyridine (10 mL)
was added dropwise to appropriate acid chlorides or ethyl chloro-
formate (16.0 mmol) at 0 ꢀC, and the reaction mixture was warmed
to room temperature and then stirred for 16 h. The reaction was
quenched with slow addition of cold water (20 mL) and extracted
with ethyl acetate (20 mL). The organic phase was separated and
washed with saturated aqueous NaHCO3 solution (20 mL) and brine
(20 mL). The organic layer was dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure to provide crude
product, which was purified by flash silica chromatography to
obtain pure bergenin derivatives 3–6.
BV-2 microglia culture
The murine BV-2 microglia cell line was maintained in DMEM sup-
plemented with 10% fetal bovine serum and penicillin ⁄ streptomycin
at 37 ꢀC in a humidified incubator under 5% CO2. For all experi-
ments, cells were plated at a density of 1 · 105 cells ⁄ mL in 24-
well plates and then treated with 100 ng ⁄ mL LPS alone or with
various concentrations of compounds for 24 h at 37 ꢀC.
Penta-O-acetyl bergenin (3)
White solid, mp 206–207 ꢀC; IR mmax (KBr) 3024, 2950, 1755, 1748,
1732, 1609, 1486, 1455, 1428, 1372, 1328, 1196, 1091, 1032, 962,
892 ⁄ cm; 1H NMR (250 MHz, CDCl3) d 7.77 (s, 1H), 5.48 (t,
J = 9.48 Hz, 1H), 5.13 (t, J = 9.64 Hz, 1 H), 4.82 (d, J = 10.74 Hz,
1H), 4.40–4.29 (m, 2H), 4.14 (dd, J = 12.64, J = 3.79 Hz, 1H), 3.91
(s, 3H), 3.85–3.79 (m, 1H), 2.34 (d, J = 2.53 Hz, 6H), 2.11–2.07 (m,
9H); 13C NMR (63 MHz, CDCl3) d 170.5, 170.0, 169.7, 168.3, 167.7,
161.7, 150.2, 144.4, 141.5, 129.7, 124.1, 118.8, 77.7, 77.2, 76.8,
76.7, 73.0, 72.3, 68.5, 62.0, 61.6, 20.8, 20.7; MALDI-TOF-MS calcd.
for C24H26O14 561.1215 [M+Na]+, found: 561.3566.
Nitric oxide assay
The Griess reaction was used to perform nitrite assays. Cells were
incubated with LPS (100 ng ⁄ mL) and various concentrations of
bergenin derivatives for 24 h at 37 ꢀC. The culture media were
then mixed with an equal volume of reagent (one part 0.1% N-1-
naphthylethylenediamine dihydrochloride, one part 1% sulfanilamide
in 5% phosphoric acid) in 96-well plates. The absorbance was
determined at 540 nm using a microplate reader. Data are reported
as the mean € the SD of three observations.
Penta-O-caproyl bergenin (4)
Light yellow liquid, IR mmax (KBr) 2956, 2891, 1747, 1609, 1484,
1
1459, 1425, 1378, 1327, 1217, 1165, 1095, 1016, 964, 885 ⁄ cm; H
Animals and drug administration
NMR (250 MHz, CDCl3) d 7.75 (s, 1H) 5.50 (t, J = 9.47, 1H) 5.17(t,
J = 9.55 Hz, 1H), 4.84 (d, J = 10.58 Hz, 1H), 4.23–4.41 (m, 2H),
4.07–4.18 (m, 1 H), 3.88 (s, 3H), 3.82–3.78 (m, 1H), 2.60 (t,
J = 7.42 Hz, 4H), 2.42–2.23 (m, 6H), 1.82–1.55 (m, 10H), 1.43–1.28
(m, 20H), 0.95–0.85 (m, 15H); 13C NMR (63 MHz, CDCl3) d 173.3,
172.7, 172.2, 171.2, 170.6, 161.7, 150.1, 144.4, 141.6, 129.6, 124.0,
118.8, 77.4, 76.9, 73.0, 71.9, 67.9, 61.5, 34.0, 34.0, 33.9, 33.8, 31.2,
31.2, 24.5, 24.4, 24.4, 22.4, 22.3; MALDI-TOF-MS calcd. for
C44H66O14 841.4345 [M+Na]+, found: 841. 5308.
C57BL ⁄ 6 mice were obtained from Daehan Biolink (Eumsung,
Korea). Mice were housed with a 12-h light ⁄ dark cycle and main-
tained at 24 € 3 ꢀC. All animal procedures were performed in
accordance with the Institutional Animal Care and Use Committee
of Ewha Womans University. The mice (male, 20 € 2 g) were ran-
domly divided into each group (n = 10) and were given saline, mor-
phine, or both morphine and bergenin derivatives. The morphine
chloride (10 mg ⁄ kg ⁄ day, Myungmun Pharm., Seoul) was dissolved
in saline, and bergenin derivatives (10 or 20 mg ⁄ kg ⁄ day) were dis-
solved in 10% cremophor solution containing 2% dimethyl sulfoxide.
Morphine was administered daily for 7 days intraperitoneally (i.p.),
and bergenin derivatives were administered orally 30 min prior to
injection of morphine. Naloxone hydrochloride (5 mg ⁄ kg, i.p.) was
injected 6 h after the final morphine injection for induction of mor-
phine withdrawal syndrome in mice.
Penta-O-ethoxycarbonyl bergenin (5)
White solid, mp 69–72 ꢀC; IR mmax (KBr) 2985, 2912, 1762, 1612,
1488, 1467, 1430, 1394, 1371, 1335, 1281, 1126, 1094, 1008, 876,
758 ⁄ cm; 1H NMR (250 MHz, CDCl3) d 7.85 (s, 1H), 5.38 (t,
J = 9.48 Hz, 1H), 5.34–4.97 (m, 2H), 4.47–4.16 (m, 13H), 3.98 (s,
3H), 3.93 (t, J = 3.16 Hz, 1H), 1.42–1.28 (m, 15H); 13C NMR
(63 MHz, CDCl3) d 161.2, 154.7, 154.2, 153.9, 152.4, 151.9, 150.1,
144.4, 141.5, 129.4, 123.5, 118.4, 77.7, 77.2, 76.7, 76.4, 76.0, 75.8,
72.3, 72.1, 65.7, 65.1, 64.9, 64.8, 64.4, 61.8, 14.1, 14.0; MALDI-TOF-
MS calcd. for C29H36O19 711.1743 [M+Na]+, found: 711.4653.
Measurement of morphine withdrawal
syndrome
Morphine withdrawal syndrome was induced by injection of nalox-
one (5 mg ⁄ kg), a competitive antagonist with high opioid receptor
affinity. Immediately after the naloxone injection, mice were placed
into individual observation cylinders (24 cm in diameter and 50 cm
high), and the frequency of jumps of each mouse was observed for
30 min. The data were expressed as mean € SE. Statistical differ-
ences were analyzed by Student's t-test.
Penta-O-benzoyl bergenin (6)
White solid, mp 145–147 ꢀC; IR mmax (KBr) 3064, 3021, 2949, 1731,
1601, 1584, 1485, 1451, 1427, 1370, 1330, 1314, 1279, 1065, 1025,
726
Chem Biol Drug Des 2011; 78: 725–729

Biological Evaluation of Bergenin Analogs
Table 1: Synthesis of bergenin derivatives 3–6
Product
Compound
Formula
Yielda (%)
71
Figure 1: Structures of norbergenin 1 and bergenin 2.
3
C24H26O14
compounds contain three stereogenic centers and secondary or pri-
mary alcohol groups on the tetrahydropyran ring (21).
This simple one-step condensation reaction produced novel deriva-
tives 3–6 as listed in Table 1. All physical and spectral data for
synthetically prepared compound 3 were identical to those reported
(19), and all new compounds 4–6 have given satisfactory analytical
and spectral data.
4
5
6
C44H66O14
C29H36O19
C49H36O14
59
69
95
Biology
Nitrite was used as a measure of NO production. The in vitro
suppression of LPS-induced NO generation by the prepared berge-
nin derivatives was evaluated as previously described (22). Berge-
nin and most of the bergenin 3–6 derivatives inhibited nitrite
accumulation in LPS-stimulated microglia BV-2 cells at a concen-
tration of 20–30 lM (Figure 2). In addition, bergenin derivatives
acted well as anti-narcotics for morphine dependence. Interest-
ingly, compounds 3–6 showed more favorable anti-narcotic activity
compared with bergenin. These compounds seemed to be enhanc-
ing bioavailability by increasing lipophilicity in the in vitro experi-
ment in cells.
aIsolated pure yield.
Results and Discussion
Chemistry
A series of bergenin derivatives was prepared from naturally
extracted bergenin 2-(4-methoxy-2-[(1S,2R,3S,4S,5R)-3,4,5,6-tetrahy-
dro-3,4,5-trihydroxy-6-(hydroxymethyl)-2H-pyran-2-yl]-a-resorcylic acid
d-lactone), which was treated with several acyl chlorides or ethyl
chloroformate (acetyl chloride, caproyl chloride, ethyl chloroformate,
and benzoyl chloride) in the presence of two equivalents of base in
dichloromethane to provide bergenin analogs 3–6 at high yields
(Table 1). Even though bergenin analogs 3–6 were modified by acyl
groups on hydroxyl groups of the bergenin skeleton, their structural
composition remained as a six-membered ring, aromatic ring, gluco-
pyranose ring, and annellated d-lactone ring. In addition, these
Figure 2: Effect of bergenin and bergenin derivatives 3–6 on
nitrite production in lipopolysaccharide (LPS)-stimulated BV-2 micro-
glia cells. Cells were treated with 100 ng ⁄ mL LPS. Various concen-
trations of bergenin compounds (5–30 lM) were added for 24 h at
37 ꢀC. Values indicate nitrite production from culture supernatants
of LPS-treated cells with or without bergenin compounds. Data rep-
resent the mean € SD of three observations.
Chem Biol Drug Des 2011; 78: 725–729
727

Jung et al.
(MRC, 2010-0029355) and a research grant from the Ministry of
Health & Welfare (Project No B040002), the Republic of Korea. E.
L. received fellowship from the BK 21 program of the Ministry of
Education, the Republic of Korea. This study was supported by the
Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education, Sci-
ence, and Technology (2010-0021716).
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