Isolation and synthesis of melodamide A, a new anti-inflammatory phenolic amide from the leaves of melodorum fruticosum

Article abstract of DOI:10.1055/s-0032-1328131

Together with twelve known compounds (2-13), melodamide A (1), a new phenolic amide possessing p-quinol moiety, was purified and characterized from the methanolic extracts of the leaves of Melodorum fruticosum. The structure of melodamide A (1) was established with a combination of 2D NMR experiments, HR-ESI-MS and X-ray analyses. The other known compounds were identified by comparison of their spectroscopic and physical data with those reported in the literature. Moreover, some isolated compounds were examined for their inhibitory activity towards superoxide anion generation and elastase release in human neutrophils. Among the tested compounds, 1, 3, and 5 exhibited strong inhibition of superoxide anion generation with IC₅₀ values ranging from 5.25 to 8.65 M. Furthermore, synthesis and biological evaluation of melodamide A (1) and its analogs (14a-p) were described. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0032-1328131

288 Original Papers
Isolation and Synthesis of Melodamide A, a New
Anti-inflammatory Phenolic Amide from the Leaves
of Melodorum fruticosum
Authors
Hsiu-Hui Chan1*, Tsong-Long Hwang2,3*, Tran Dinh Thang4*, Yann-Lii Leu2,3, Ping-Chung Kuo5, Bui Thi Minh Nguyet4,
Do Ngoc Dai6, Tian-Shung Wu7
Affiliations
The affiliations are listed at the end of the article
Key words
Abstract
examined for their inhibitory activity towards
superoxide anion generation and elastase release
in human neutrophils. Among the tested com-
pounds, 1, 3, and 5 exhibited strong inhibition of
superoxide anion generation with IC50 values
ranging from 5.25 to 8.65 µM. Furthermore, syn-
thesis and biological evaluation of melodamide A
(1) and its analogs (14a–p) were described.
"
l Melodorum fruticosum
!
"
l Annonaceae
Together with twelve known compounds (2–13),
melodamide A (1), a new phenolic amide possess-
ing p-quinol moiety, was purified and character-
ized from the methanolic extracts of the leaves of
Melodorum fruticosum. The structure of meloda-
mide A (1) was established with a combination
of 2D NMR experiments, HR‑ESI‑MS and X‑ray
analyses. The other known compounds were
identified by comparison of their spectroscopic
and physical data with those reported in the liter-
ature. Moreover, some isolated compounds were
"
l melodamide A
"
l superoxide anion generation
"
l elastase release
"
l anti‑inflammatory
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
the leaves of M. fruticosum. Herein we wish to re-
port the isolation, structural elucidation, and bio-
logical activity of melodamide A (1) as well as
synthesis of 16 counterparts with different sub-
stitutions (halide, -NO2, -OCH3, -CH3) on ring A
of melodamide A (1).
!
Inflammation is a complex biological response of
vascular tissues to harmful stimuli such as patho-
gens, damaged cells, or irritants. Inflammation
can be classified as either acute or chronic.
Although inflammation is a protective attempt
by the organism to remove the injurious stimuli,
chronic inflammation can also lead to a host of
diseases such as hay fever, atherosclerosis, rheu-
matoid arthritis, and cancer [1–3]. From the point
of view of exploring new lead compounds for the
treatment of these diseases, it is valuable to iden-
tify the anti-inflammatory principles from the
natural sources, and several phytochemical and
biological studies on this subject were reported
by our group recently [4–7]. Melodorum frutico-
sum (Annonaceae) is widely distributed in South-
East Asia and commonly known as devil tree,
white cheesewood, and lamduan [8]. Its flowers
and barks have been reported to have antifungal,
antioxidant, and cytotoxic activities [8–10]. In
this study, a new phenolic amide along with
twelve known compounds were identified from
received
revised
accepted
June 26, 2012
Nov. 22, 2012
Dec. 14, 2012
Materials and Methods
!
General experimental procedures
Bibliography
Melting point was determined using a Fisher Sci-
entific melting point measuring apparatus with-
out corrections. The UV spectrum was recorded
on an Agilent UV‑VIS recording spectrophotome-
ter. The IR spectrum was obtained, as KBr discs, on
a Bruker type spectrometer. Optical rotation was
measured with a Jasco DIP-1000 KUY polarimeter.
The electrospray ionization (ESI) mass spectrum
was determined using an Agilent 1200 LC‑MSD
Trap spectrometer, and the HR‑ESI‑MS was com-
pleted with the aid of the Bruker APEX II mass
spectrometer. 1H- and 13C‑NMR, COSY, NOESY,
HMQC, and HMBC spectra were recorded on the
Bruker AV-500, Avance-III 400, and Avance 300
NMR spectrometers, using tetramethylsilane
(TMS) as the internal standard. X‑ray structure
DOI http://dx.doi.org/
10.1055/s-0032-1328131
Published online January 23,
2013
Planta Med 2013; 79: 288–294
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Tian-Shung Wu
Department of Chemistry
National Cheng Kung University
1 Ta-Hsueh Road
Tainan 70101
Taiwan
Phone: + 88662757575,
ext. 65333
Fax: + 88662740552
tswu@mail.ncku.edu.tw
* These authors provided equal contributions to this work.
Chan H-H et al. Isolation and Synthesis… Planta Med 2013; 79: 288–294

Original Papers 289
analyses were performed on a Bruker APEX DUO diffractometer.
Standard pulse sequences and parameters were used for the NMR
experiments, and all chemical shifts were reported in parts per
million (ppm, δ). All chemicals (reagent grade) used were pur-
chased from Sigma-Aldrich. Column chromatography (CC) was
performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400
mesh; E. Merck).
tography with silica gel (300 g, 80 × 2 cm) using elution mixtures
of chloroform-methanol-water (9:1:0.05) and further purified
by repeated silica gel column chromatography to result in β-si-
tosteroyl-3-O-β-D-glucoside (13) (97 mg). Silica gel column
chromatography (80 g, 60 × 3 cm) of fraction 7 (10.4 g) with the
eluent mixtures of chloroform-methanol-water (4:1:0.05) and
step gradient with methanol afforded 3-O-β-D-apiofuranosyl-
(1 → 2)-O-[α-L-rhamnopyranosyl-(1 → 6)]-β-D-glucopyranoside
(12) (12 mg).
Plant materials
The leaves of Melodorum fruticosum (Annonaceae) were collected
from Hue, Vietnam, during May 2009, and the plant materials
were identified and authenticated by Dr. Tran Huy Thai, Institute
of Ecology and Biological Resources, Vietnamese Academy of Sci-
ence and Technology. A voucher specimen (20090515) was de-
posited in the Herbarium of the Institute of Ecology and Biologi-
cal Resources, Vietnamese Academy of Science and Technology,
Hanoi, Vietnam.
Melodamide A (1): Colorless powder; mp 183–185°C; UV (MeOH)
λ
max (log ε) 272 (4.9), 217 (4.8), 204 (4.7) nm; IR (KBr) max: 3290,
2920, 2851, 1713, 1663, 1620, 1551, 1450, 1227 cm−1 1H‑NMR
;
(DMSO-d6, 500 MHz) δ 8.09 (1H, br t, J = 4.5 Hz, NH), 7.55 (2H, d,
J = 8.5 Hz, H-5, H-9), 7.39 (3H, m, H-3, H-6, H-8), 7.39 (1H, d,
J = 15.5 Hz, H-3), 6.94 (2H, d, J = 8.5 Hz, H-4′, H-8′), 6.56 (1H, d,
J = 15.5 Hz, H-2), 6.09 (2H, d, J = 8.5 Hz, H-5′, H-7′), 5.83 (1H, br s,
OH), 3.18 (2H, dd, J = 14.0, 7.0 Hz, H-1′), 1.83 (2H, t, J = 7.0 Hz, H-
2′); 13C‑NMR (DMSO-d6,125 MHz) δ 185.2 (C-6′), 164.8 (C-1),
152.8 (C-4′, C-8′), 138.6 (C-3), 134.9 (C-4), 129.4 (C-7), 128.9 (C-
6, C-8), 127.5 (C-5, C-9), 126.8 (C-5′, C-7′), 122.1 (C-2), 67.8 (C-3′),
39.0 (C-2′), 34.2 (C-1′). ESI‑MS m/z 284 ([M + Na]+); HRESI‑MS m/z
284.1289 [M + Na]+ (calcd. for C17H17NO3Na, 284.1287).
Extraction and isolation
The leaves of Melodorum fruticosum (9.8 kg) were powdered and
soaked with methanol (40 L × 3) at room temperature, and the
combined extracts were concentrated under reduced pressure
to give a deep brown syrup (716 g). The crude extract was sus-
pended into water and partitioned with n-hexane, ethyl acetate,
and n-butanol, successively to afford n-hexane (38 g), ethyl ace-
tate (267 g), n-butanol (192 g), and water soluble (49 g) fractions,
respectively.
Crystallographic data of 1
Crystal system monoclinic, space group P121/n 1 a = 8.6116 (3) Å,
b = 10.3693 (3) Å, c = 17.8352 (6) Å, V = 1559.44 (9) Å3, T = 100 (2)
K, Z = 4, Dc = 1.283 mg/m3, absorption coefficient 0.751 mm−1
,
The n-hexane soluble (38 g) fraction was chromatographed over
a silica gel column (300 g, 120 × 5 cm) by gradient elution with n-
hexane and increasing concentrations of acetone (9:1, 5:1, 3:1,
1:1, each 2.0 L) to afford eight fractions (fraction 1–6). Fraction 2
(3 g) was further subjected into silica gel column chromatogra-
phy (100 g, 60 × 3.5 cm) eluted with n-hexane/acetone (19:1) to
yield β-sitosterol (2) (321 mg). The ethyl acetate soluble fraction
(267 g) was chromatographed over a silica gel column (2 kg,
150 × 10 cm) by gradient elution with n-hexane and increasing
concentrations of acetone (19:1, 12:1, 7:1, 3:1, 1:1, each 2.0 L)
to afford eight fractions (fraction 1–12). Fraction 6 (14.2 g) was
further subjected into silica gel column chromatography (150 g,
80 × 3.5 cm) eluted with n-hexane/acetone (19:1) to yield flavo-
kawain-A (3) (52 mg). Silica gel column chromatography (500 g,
110 × 5 cm) of fraction 9 (35.7 g) with step gradient elution of n-
hexane/acetone (15:1, 10:1, 7:1, 5:1, each 1 L) led to the isola-
tion of melodamide A (1) (99 mg), (−)-7,4′-dihydroxy-5-me-
thoxyflavanone (4) (35 mg), 2′,6′-dihydroxy-4′-methoxychalcone
(5) (70 mg), and 2′,4′-dihydroxy-4,6′-dimethoxydihydrochalcone
(6) (71 mg), successively. Purification of fraction 10 (15.9 g), by
column chromatography with silica gel (150 g, 90 × 4 cm) eluted
by n-hexane/acetone (7:1), afforded 4′,5-dimethoxy-7-hydroxy-
flavanone (7) (58 mg). The n-butanol soluble residues (143 g)
were subjected to silica gel column chromatography (1.5 kg,
120 × 8 cm) and purified by step gradient elution with chloro-
form and increasing concentrations of methanol (0%, 5%, 10%,
20%, 30%, 50%, 70%, 90%, and 100%, each 2 L) to afford ten frac-
tions (fraction 1–10). Silica gel column chromatography (80 g,
80 × 2 cm) of fraction 3 (5.0 g) with the eluent mixtures of chloro-
form-methanol (19:1) and step gradient with methanol afforded
ponciretin (8) (39 mg) and oxoanolobine (11) (14 mg). Separation
of fraction 4 (18.6 g) by silica gel column chromatography (200 g,
100 × 4 cm) with chloroform-methanol (15:1) yielded rutinic ac-
id (9) (87 mg) and 3,3′,4-tri-O-methylellagic acid (10) (45 mg),
successively. Fraction 6 (30.6 g) was subjected to column chroma-
crystal size 0.18 × 0.15 × 0.12 mm3. 8662 reflections collected,
2619 independent reflections (Rint = 0.0199). Data were 95.8%
complete to 66.38° θ. The final R1 values were 0.0320 with I > 2σ
(I), and the final wR (F2) values were 0.0857. Goodness-of-fit on
F2 was 1.036. Crystallographic data for 1 (CCDC 872187) have
been deposited with the Cambridge Data Centre.
General procedure for the synthesis of compounds 1
and 14a–p
The general procedure is illustrated immediately below with
compound 1 as a specific example. N-(4-hydroxyphenethyl)cin-
namamide was prepared by mixing cinnamoyl chloride (1.8 g,
8.0 mmol), tyramine (1.0 g, 7.3 mmol), and triethylamine
(2.0 mL) in CH2Cl2 at 0°C under nitrogen. The reaction mixture
was stirred for 2 h, extracted by EtOAc and washed with saturat-
ed aqueous NaHCO3. The organic phases were combined and
dried over Na2SO4. After the solvent was removed under vacuum,
the residue was purified by column chromatography eluting with
hexanes/EtOAc (1:1) to yield N-(4-hydroxyphenethyl)cinnama-
mide (15) as a colorless crystalline solid (0.675 g, 84%), identified
by mp, UV, IR, 1H-, 13C‑NMR, and mass spectral analysis [11]. To a
solution of 200 mg (0.748 mmol) of 15 in 2 mL of THF/CH3CN/H2O
(1:1:1) was added phenyliodine (III) diacetate 289 mg
(0.898 mmol) at 0°C. After stirring for 1 h, phenyliodine (III) diac-
etate 121 mg (0.374 mmol) was added at 0°C and kept for one
more hour. The reaction mixture was extracted by EtOAc and
washed with saturated aqueous NaHCO3. The organic phases
were combined and dried over Na2SO4. After the solvent was re-
moved under vacuum, the crude product was purified by column
chromatography eluting with CH2Cl2/EtOAc (1:1) to yield melo-
damide A (1) as a colorless solid (47.0 mg, 23%). The structure
was identified by comparison of its spectral and physical data
with those of the isolated compound.
Chan H-H et al. Isolation and Synthesis… Planta Med 2013; 79: 288–294

290 Original Papers
Preparation of human neutrophils
Blood was taken from healthy human donors (20 ~ 32 years old)
by venipuncture, using a protocol approved by the institutional
review board at Chang Gung Memorial Hospital. Neutrophils
were isolated using a standard method of dextran sedimentation
prior to centrifugation in a Ficoll Hypaque gradient and the hypo-
tonic lysis of erythrocytes. Purified neutrophils that contained
> 98% viable cells, as determined by the trypan blue exclusion
method, were resuspended in a calcium (Ca2+)-free HBSS buffer
at pH 7.4 and maintained at 4°C before use.
Fig. 1 Chemical structure of melodamide A (1).
Measurement of O2•− generation
The assay of O2•− generation was based on the SOD-inhibitable re-
duction of ferricytochrome c [12]. In brief, after supplementation
with 0.5 mg/mL ferricytochrome c and 1 mM Ca2+, neutrophils
were incubated with drugs for 5 min at 37°C. Cells were then ac-
tivated with 100 nM FMLP for 10 min. Cytochalasin B (CB, 1 µg/
mL) was incubated for 3 min before activation by the peptide
(FMLP/CB). Changes in absorbance with the reduction of ferricy-
tochrome c at 550 nm were continuously monitored in a double-
beam, six-cell positioner spectrophotometer with constant stir-
ring (Hitachi U-3010). Sorafenib tosylate (Selleck Chemicals, pu-
rity 99%) and SB202190 (Sigma, purity > 98%) were used as the
positive control.
Statistical analysis
Results were expressed as mean SD. Calculation of 50% inhib-
itory concentration (IC50) was computer-assisted (PHARM/PCS
v.4.2). Statistical comparisons were made between groups using
Studentʼs t test. Values of p less than 0.05 were considered to be
statistically significant.
Supporting information
The full characterization of compounds 14a–p, 15, and 16a–p are
available as Supporting Information.
Measurement of elastase release
Results and Discussion
!
Experiments were performed using MeO‑Suc-Ala-Ala-Pro-Val-p-
nitroanilide as the elastase substrate [13]. Briefly, after
supplementation with MeO‑Suc-Ala-Ala-Pro-Val-p-nitroanilide
(100 µM), neutrophils (5 × 105/mL) were equilibrated at 37°C for
2 min and incubated with drugs for 5 min. Cells were activated by
100 nM FMLP and 0.5 µg/mL CB. Changes in absorbance at
405 nm were continuously monitored to assay elastase release.
The methanol extract of the leaves of Melodorum fruticosum
yielded a new compound, melodamide A (1) (l Fig. 1), together
"
with 12 known compounds identified as β-sitosterol (2) [15], fla-
vokawain-A (3) [16], (-)-7,4′-dihydroxy-5-methoxyflavanone (4)
[17], 2′,6′-dihydroxy-4′-methoxychalcone (5) [18], 2′,4′-dihy-
droxy-4,6′-dimethoxydihydrochalcone (6) [19], 4′,5-dimethoxy-
7-hydroxyflavanone (7) [20], ponciretin (8) [21], rutinic acid (9)
[22], 3,3′,4-tri-O-methylellagic acid (10) [23], oxoanolobine (11)
[24], kaempferol 3-O-β-D-apiofuranosyl-(1 → 2)-O-[α-L-rham-
nopyranosyl-(1 → 6)]-β-D-glucopyranoside (12) [25], and β-si-
tosteroyl-3-O-β-D-glucoside (13) [26] by comparing their spec-
troscopic data with those reported in the literature.
Lactate dehydrogenase (LDH) release
Cytotoxicity was represented by LDH release in culture medium.
The total LDH released was determined by lysing cells with 0.1%
Triton X-100 for 30 min at 37°C. LDH concentrations were deter-
mined according to the manufacturerʼs instructions (Promega).
Melodamide A (1) was isolated as colorless powder and showed a
[M + Na]+ ion peak at m/z 284.1289 in its HRESIMS, corresponding
to the molecular formula C17H17NO3Na. The UV spectrum of 1
displayed absorption maxima at 272 and 217 nm, and the IR
spectrum exhibited strong absorption peaks for OH (3290 cm−1),
a conjugated carbonyl group (1713 cm−1), and amide (1663,
1450 cm−1). In the 1H‑NMR spectrum, there were signals ob-
served for a D2O exchangeable proton at δ 8.11 (1H, br s, NH), a
set of five mutually aromatic protons at δ 7.55 (2H, d, J = 7.5 Hz,
H-5 and H-9) and 7.39 (3H, m, H-3, H-4, H-5), trans-olefinic pro-
tons at δ 7.39 (1H, d, J = 15.5 Hz, H-3) and δ 6.56 (1H, d, J = 15.5 Hz,
H-2), four conjugated olefinic protons at δ 6.94 (2H, d, J = 8.5 Hz,
H-4′, H-8′) and 6.09 (2H, d, J = 8.5 Hz, H-5′, H-7′), and upfield sig-
nals for four mutually coupled protons at δ 3.18 (2H, dd, J = 14.0,
7.0 Hz, H-8′) and 1.83 (2H, t, J = 7.0 Hz, H-2′). The 13C‑NMR and
DEPT spectra combined with HMQC experiment indicated 17 sig-
nals including a conjugated ketone (δ 185.2), a conjugated amide
(δ 164.8), two olefinic carbon resonances (δ 138.6, 122.1), two
conjugated alkenes (δ 152.8, 152.8, 126.8, 126.8), six aromatic
signals (δ 134.9, 129.4, 129.4, 128.9, 127.5, 127.5), an oxygen-
bearing quaternary carbon (δ 67.8), and two methylenes (δ 39.0
and 34.2). The HMBC correlations of H-2 (δ 6.56) to C-1 (δ 164.8)/
C‑4 (δ 134.9) showed the cinnamic amide sequence in the mole-
Western analysis
Neutrophils were incubated with drugs for 5 min at 37°C before
being stimulated with FMLP for 0.5 min. The reaction was
stopped by adding 5× Laemmliʼs sample buffer [14]. Samples
were centrifuged at 14000 × g for 20 min at 4°C to yield whole-
cell lysates. Proteins derived from whole-cell lysates were sepa-
rated by SDS-polyacrylamide gel electrophoresis (PAGE) using
12% polyacrylamide gels and blotted onto nitrocellulose mem-
branes. Immunoblotting was performed using the indicated anti-
bodies and horseradish peroxidase (HRP)-conjugated secondary
anti-rabbit antibodies (Cell Signaling Technology). The immuno-
reactive bands were visualized by an enhanced chemilumines-
cence system (ECL; Amersham Biosciences) and detected by UVP
imaging.
Measurement of intracellular calcium concentration
([Ca2+]i)
Neutrophils were loaded with 2 µM fluo-3 AM at 37°C for 45 min.
The change in fluorescence was monitored using a Hitachi F-
4500 spectrofluorometer in a quartz cuvette with a thermostat
(37°C) and continuous stirring. The excitation wavelength was
488 nm, and the emission wavelength was 520 nm [13].
Chan H-H et al. Isolation and Synthesis… Planta Med 2013; 79: 288–294

Original Papers 291
activation of phospholipase C [27]. No significant inhibition of
FMLP-induced increase in [Ca2+]i mobilization by compound 1
"
was observed in human neutrophils (l Fig. 4), suggesting that
compound 1 might not inhibit phospholipase C. In addition, stim-
ulation of human neutrophils by FMLP resulted in the rapid phos-
phorylation of several proteins, including mitogen-activated pro-
tein kinases (MAPKs) and protein kinase B (Akt). Activation of
these signal transduction pathways is known to be responsible
for various neutrophil activations [13,27]. Our data showed that
the FMLP-induced phosphorylation of p38 MAPK, but not ERK
"
and Akt, was diminished by compound 1 (l Fig. 5). To investigate
whether the activation of p38 MAPK has a significant role on
superoxide anion generation and elastase release in FMLP/CB-in-
duced human neutrophils, the effects of SB202190, a selective
p38 MAPK inhibitor, in superoxide anion generation and elastase
release were assayed. SB202190 diminished FMLP/CB-induced
superoxide anion generation with an IC50 value of 6.73
0.65 µM. However, it did not show significant inhibition of elas-
Fig. 2 HMBC correlations for melodamide A (1).
"
cule of 1 (l Fig. 2). In addition, the B-ring possessing a p-quinol
"
moiety was established by the signals of H-5′/H-7′ (δ 6.09) and
H-4′/H-8′ (δ 6.94) related with C-3′ (δ 67.8) and C-6′ (δ 185.2), re-
spectively. Furthermore, the cross peaks of H-1′ (δ 3.18) to C-1 (δ
164.8)/C‑2′ (δ 39.0)/C-3′ (δ 67.8) and H-2′ (δ 1.83) to C-1′ (δ 34.2)/
C-3′ (δ 67.8)/C‑4′ (δ 152.8)/C‑8′ (δ 152.8) in the HMBC spectrum
indicated the conjugated amide and p-quinol B-ring were con-
nected with C-1′ and C-2′, respectively. Besides, the structure of
1 was confirmed by an X‑ray diffraction crystallographic study
tase release (IC50 value > 10 µM) (l Table 1). These data sug-
gested that p38 MAPK signaling pathway is a significant compo-
nent in FMLP/CB-induced superoxide anion generation in human
neutrophils. Taken together, our results demonstrate that the
suppressive effects of compound 1 are associated with inhibition
of p38 MAPK signaling pathway. In addition, p38 MAPK is a com-
ponent of the oxidant stress-sensitive signaling kinases [28].
Therefore, whether the reduction of oxidant stress involved inhi-
bition of p38 MAPK phosphorylation will need to be further in-
vestigated in future studies.
"
(l Fig. 3). On the basis of these data, 1 was identified as shown.
The isolated pure compounds 1, 3–5, and 7–10 (purity > 92%)
were evaluated for their inhibitory effects on superoxide anion
generation and elastase release by human neutrophils in re-
sponse to formyl-L-methionyl-L-leucyl-L-phenylalanine/cyto-
In order to investigate the synthesis of natural products as anti-
inflammatory agents, melodamide A (1) was designed and syn-
"
thesized (l Fig. 6). To begin with, N-(4-hydroxyphenethyl)cinna-
"
chalasin B (FMLP/CB) (l Table 1). Among them, compound 1 ex-
mamide (15) was obtained in 84.4% yield through the nucleo-
philic reaction of tyramine and cinnamoyl chloride. Oxidation of
15 with phenyliodine (III) diacetate afforded the new compound
1 with p-quinol B-ring (22.5%). The spectral (1H-, 13C‑NMR, and
MS) and physical data of the synthetic compound 1 coincided
well with those of the natural melodamide A. In addition, the
synthesis of different substituents (halide, NO2, OCH3, CH3) on
hibited more potent inhibition in response to FMLP/CB induced
superoxide anion generation with IC50 values of 5.25 µM. Cultur-
ing with compound 1 (up to 30 µM) did not affect cell viability, as
assayed by LDH release (data not shown). Compound 1 at concen-
trations of up to 30 µM failed to induce DPPH reduction. α-Toco-
pherol (3, 10, and 30 µM) was used as a positive control in the
DPPH assay (data not shown). These results rule out the possibil-
ity that the inhibitory effect of compound 1 on superoxide anion
generation occur through the direct radical scavenging activity.
The action mechanisms of compound 1 were further investigated
in this study. The intracellular signaling mechanisms responsible
for neutrophil activations are very complex. FMLP activates neu-
trophils by binding to the GPCR and induces the Ca2+ signal via
1
"
ring A of melodamide A (1) is also presented in l Fig. 6. IR, H-,
13C‑NMR, and MS spectra were used for identifying the analogs
structures (14a–p). The synthesized compounds (14a–p, purity
> 96%) were evaluated for their inhibitory effects against the re-
lease of both O−2 and elastase by human neutrophils. As shown in
"
l Table 1, compounds 14a, 14 d, and 14 f displayed inhibitory ac-
tivity on O− as well as 1, with IC50 values of 7.49, 5.59, and
2
Fig. 3 Single-crystal X‑ray structure of meloda-
mide A (1).
Chan H-H et al. Isolation and Synthesis… Planta Med 2013; 79: 288–294

292 Original Papers
Compounds
Superoxide anion
Elastase release
(Inh %)
Table 1 Effects of compounds 1,
3-5, 7–10, and 14a–p on superox-
ide anion generation and elastase
release by human neutrophils in
response to FMLP/CB.
IC50 (µM)a or (Inh %)
ꢀ1
5.25 0.48
(39.17 6.87)**
(40.22 6.72)**
(2.11 7.91)
ꢀ3
8.65 1.93
ꢀ4
(2.20 1.68)
8.40 2.19
ꢀ5
(25.14 5.22)**
(− 4.54 1.44)*
(10.14 6.49)
(10.06 4.75)
(14.91 5.97)
(14.20 4.72)*
(18.69 2.98)**
(10.53 4.99)
(33.26 6.60)**
(5.82 1.84)*
(18.36 2.26)**
(24.16 3.05)**
(15.74 4.50)*
(23.55 5.04)**
(16.69 3.61)**
(10.72 4.94)
(− 0.32 4.42)
(1.54 4.72)
ꢀ8
(7.69 5.72)
(2.50 1.43)
(39.46 2.29)***
(3.08 0.88)*
7.49 1.78
ꢀ7
ꢀ9
10
14a
14b
14c
(27.06 4.53)**
(26.33 6.05)*
5.59 0.26
14 d
14e
14 f
14 g
14 h
14i
(8.26 6.05)
5.19 0.39
(23.82 6.42)*
(21.47 5.12)*
(34.13 6.40)**
(30.10 8.33)*
(19.62 3.43)**
(1.39 0.98)
(8.06 6.73)
(4.55 3.30)
(31.80 8.59)*
(12.30 5.53)
3.44 0.29
14j
14 k
14 l
14 m
14 n
14o
14 p
Sorafenibb
SB202190b
(10.93 6.32)
(26.99 4.99)**
(12.88 0.21)***
1.00 0.60
6.73 0.65
(5.53 3.05)
Percentage of inhibition (Inh %) at 10 µM concentration. Results are presented as mean SEM (n = 3).* p < 0.05, ** p < 0.01, *** p < 0.001
compared with the control value. a Concentration necessary for 50% inhibition (IC50). b Sorafenib tosylate and SB202190 were used as the
positive control
(1) and the analogs of 1 possessing p-quinol were synthesized
and evaluated for their anti-inflammatory activity.
Acknowledgements
!
The authors would like to thank Dr. Tran Huy Thai, Institute of
Ecology and Biological Resources, Vietnam Academy of Science
and Technology, for the plant identification. This study was sup-
ported by the National Science Council to T.S. Wu (Taiwan) and
by NAFOSTED (104.01–2010.27) (Vietnam).
Fig. 4 Effect of compound 1 on Ca2+ mobilization in FMLP-activated hu-
man neutrophils. Fluo 3-loaded neutrophils were incubated with DMSO (as
the control) or compound 1 (30 µM) for 5 min and then activated by 0.1 µM
FMLP in 1 mM Ca2+-containing HBSS. The traces shown are from four dif-
ferent experiments.
Conflict of Interest
!
The authors have no conflict of interest to report.
Affiliations
1
Department of Pharmacy, China Medical University, Taichung, Taiwan
Graduate Institute of Natural Products, Chang Gung University, Taoyuan,
Taiwan
Chinese Herbal Medicine Research Team, Healthy Aging Research Center,
Chang Gung University, Taoyuan, Taiwan
Department of Chemistry, Vinh University, Vinh City, Nghean Province,
Vietnam
Department of Biotechnology, National Formosa University, Yunlin, Taiwan
Institute of Ecology and Biological Resources, Vietnam Academy of Science
and Technology, Hanoi, Vietnam
Department of Chemistry, National Cheng Kung University, Tainan, Taiwan
5.19 µM, while showing the presence of 2-chloro, 3-fluoro, and 2-
bromo substitutions on ring A, respectively. However, all test
compounds exhibited no inhibitory activity of elastase release.
The results indicated that the substitutions on ring A were not of
much benefit to the anti-inflammatory activity.
In summary, a new amide possessing novel p-quinol, meloda-
mide A (1), was isolated from the leaves of M. fruticosum. The
structure of 1 was fully elucidated by X‑ray and 2D‑NMR analy-
ses. Moreover, 1 showed potent inhibitory effect on superoxide
anion generation in fMLP/CB-activated human neutrophils via in-
hibiting p38 MAPK signaling pathway. In addition, melodamide A
2
3
4
5
6
7
Chan H-H et al. Isolation and Synthesis… Planta Med 2013; 79: 288–294

Original Papers 293
Fig. 5 Effects of compound 1 on phosphorylation
of p38 MAPK, ERK, and Akt in FMLP-activated hu-
man neutrophils. Human neutrophils were incu-
bated with DMSO or compound 1 (3, 10, and
30 µM) for 5 min before stimulation with FMLP
(0.1 µM) for 0.5 min at 37°C. Phosphorylation of
p38 MAPK, p42/44 ERK, and Akt were analyzed by
an immunoblot analysis using antibodies against
the phosphorylated form and the total of each pro-
tein. Quantitation of the p-MAPK/MAPK and p-Akt/
Akt ratios is shown. Representative images from
one of three or four experiments are shown.
Fig. 6 Synthesis of compounds 1 and 14a–p. Reagents and conditions: (a) tyramine, triethylamine, 0°C, 2 h, 84%; (b) phenyliodine (III) diacetate, THF/CH3CN/
H2O, 0°C, 1 h, 23%.
7 Wu SJ, Leu YL, Chen CH, Chao CH, Shen DY, Chan HH, Lee EJ, Wu TS, Wang
YH, Shen YC, Qian K, Bastow KF, Lee KH. Camphoratins A−J, potent cyto-
toxic and Anti-inflammatory triterpenoids from the fruiting body of
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