Article

pubs.acs.org/jnp

Anisucoumaramide, a Bioactive Coumarin from Clausena anisum-

olens
Yun-Song Wang,† Bi-Tao Li,‡ Shi-Xi Liu,† Zheng-Qi Wen,‡ Jing-Hua Yang,*,† Hong-Bin Zhang,†
and Xiao-Jiang Hao*,§
†
Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology,
Yunnan University, Kunming 650091, People’s Republic of China
‡
First Affiliated Hospital of Kunming Medical University, Kunming 650031, People’s Republic of China
§
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, 132 Lanhei Road, Kunming 650201, People’s Republic of China
*
S Supporting Information

ABSTRACT: A new coumarin, anisucoumaramide (1), and a new δ-truxinate derivative, anisumic acid (2), were isolated from
Clausena anisum-olens. Their structures were elucidated from extensive NMR and MS data. The absolute configurations of the
coumarins were assigned using the experimental and calculated electronic circular dichroism data. Anisucoumaramide (1)
represents the first example of a naturally occurring coumarin of which the terpenoidal side chain does not comply with the
biosynthesis isoprene rule due to the presence of an unprecedented acetamido motif directly connected with the terpenoidal side
chain. The δ-truxinate derivative was isolated from Clausena species for the first time. Compound 1 showed high selectivity for
the MAO-B isoenzyme and inhibitory activity in the nanomolar range. Putative biosynthesis pathways toward 1 and 2 are
proposed.

T he genus Clausena (Rutaceae) is known for being a rich

source of coumarins. Some species of this genus have
been used in folk medicine to treat various diseases in Asia for a
structural elucidation, and putative biosynthesis pathways
toward the new compounds, as well as the inhibitory activity
of 1 on human monoamine oxidase (MAO), are described.

long time.1−13 Naturally occurring coumarins derived from
Clausena have exhibited a variety of biological activities
including antioxidant,8 hepatoprotective,9 anti-inflammatory,10
antibacterial and antifungal,4,8 neuroprotective,5 anti-HIV,11,12
and antimycobacterial,13 as well as potent inhibitory effects on
Epstein−Barr virus early antigen activation induced by 12-O-
tetradecanoylphorbol-13-acetate in Raji cells.2 Clausena anisum-
olens Merr. is a perennial evergreen shrub or small tree that is
mainly distributed in the Philippines, South China, and
throughout Southeast Asia. The fresh fruits are edible, and
the dried fruits are used for preventing phlegm formation and
stopping coughing. The leaves and twigs have traditionally been
used for the treatment of dysentery and arthritis in folk
medicine.7
Earlier phytochemical investigations of this species have led
to the discovery of new monoterpenoid coumarins.14−17 A
reinvestigation of the minor constituents of the ethanol extract
of C. anisum-olens afforded a new coumarin, anisucoumaramide
(1), and a new 1,2,3,4-tetrasubstituted cyclobutane derivative,
anisumic acid (2), as well as the known 8-methoxycapnolactone
(3) and trans-4-hydroxycinnamic acid (4). Herein, the isolation,
© 2017 American Chemical Society and
American Society of Pharmacognosy
■
RESULTS AND DISCUSSION
Compound 1 was isolated as a colorless oil, [α]21.5D −13 (c 1.5,
MeOH). The molecular formula, C22H23NO7, was established
by 13C NMR and HRFABMS data (414.1620 [M + Na]+, calcd
for 414.1613), suggesting 12 indices of hydrogen deficiency.
Analysis of the 13C NMR data (Table 1) and the HMQC
spectrum shows that the 22 carbons comprise four aliphatic
methylenes (including one oxygenated methylene), seven
methines (six olefinic/aromatic carbons and one oxygenated
aliphatic carbon), nine sp2 nonprotonated carbons (one amide
carbonyl, two ester carbonyls, six olefinic/aromatic carbons), a
methyl group, and a methoxy group. Strong UV bands at λmax
261 and 319 nm, an IR band at 1728 cm−1, and two AB
coupling systems at δH 6.33 and 7.77 and δH 7.01 and 7.25 in
the 1H NMR data (Table 1) indicated the presence of a 7,8-
dioxygenated coumarin nucleus.2 Comparison of the 1H and
Received: May 2, 2016
Published: April 3, 2017
798 DOI: 10.1021/acs.jnatprod.6b00391
J. Nat. Prod. 2017, 80, 798−804
Journal of Natural Products Article

Table 1. NMR Spectroscopic Data (500 MHz, Pyridine-d5) of Compounds 1 and 3

1 3
position δH (J in Hz) δC, type HMBC NOESY δH (J in Hz) δC, type
2 160.6, C 162.9, C
3 6.33, d (9.5) 113.6, CH C-2, C-4, C-9 H-4 6.25, d (9.4) 114.9, CH
4 7.77, d (9.5) 144.3, CH C-2, C-5, C-8, C-9, C-10 H-3, H-5 7.62, d (9.4) 144.9, CH
5 7.25, d (8.6) 123.6, CH C-4, C-7, C-8, C-9, C-10 H-6, H-4 7.16, d (8.6) 124.7, CH
6 7.01, d (8.6) 110.6, CH C-5, C-7, C-8, C-9, C-10 6.84, d (8.6) 111.5, CH
7 155.3, C 154.7, C
8 137.9, C 136.8, C
9 114.3, C 115.2, C
10 149.4, C 148.2, C
1′ 4.70, d (6.2) 66.2, CH2 C-7, C-2′, C-3′, C-4′, C-10′ H-10′, H-6 4.69, d (6.2) 67.2, CH2
2′ 5.68, t (6.2) 123.8, CH C-1′, C-4′, C-10′ H-4′ 5.62, t (6.2) 124.0, CH
3′ 136.5, C 138.0, C
4′a 2.39, dd (14.3, 5.0) 43.1, CH2 C-2′, C-3′, C-5′, C-6′, C-10′ H-2′, H-5′ 2.42, d (9.1) 44.5, CH2
4′b 2.27, dd (14.3, 7.8)
5′ 5.08, m 80.0, CH C-3′, C-4′, C-6′, C-7′ H-10′, H-4′, H-6′ 5.00, m 80.8, CH
6′ 7.28, d (1.5) 150.1, CH C-5′, C-7′, C-8′, C-9′ H-5′ 7.02, d (1.6) 149.5, CH
7′ 133.6, C 130.9, C
8′ 173.1, C 175.2, C
9′ 2.88, t (7.0) 21.2, CH2 C-6′, C-7′, C-8′, C-11′, C-12′ H-11′ 1.83, s 12.0, CH3
10′ 1.73, s 17.3, CH3 C-2′, C-3′, C-4′ H-1′ 1.90, s 18.8, CH3
11′ 2.77, t (7.0) 33.5, CH2 C-7′, C-9′, C-12′
12′ 173.7, C
OMe 3.95, s 61.1, CH3 C-8 3.97, s 62.8, CH3
NHa 8.32, br s
NHb 7.83, br s

13
C NMR data of compound 1 and 8-methoxycapnolactone
(3), a known coumarin isolated from the same plant, indicated
that these two compounds differed only in the side chain. The
location of the side chain at C-7 was confirmed by the long-
range C−H correlation between the methylene protons at δH
4.70 (H-1′) and the oxygenated carbon (δC 155.3, C-7). In the
HMBC experiment (Figure 2), the three-bond cross-peak

Figure 2. 1H−1H COSY, key HMBC, and ROESY correlations for 1.

was identified from the NMR signals and IR band at 1730

Figure 1. Structures of compounds 1−4.
between OMe (δH 3.95) and C-8 at δC 137.9 indicated the
location of the methoxy group at C-8. The E configuration of
the C-2′/C-3′ double bond was deduced from the 13C NMR
chemical shift value of C-10′ (δC 17.3) and HMBC analysis
(Figure 2, Table 1). The NOE correlations between H-l′ and
H-10′ and between H-2′ and H-4′ further supported this
assignment. Additionally, the α,β-unsaturated γ-lactone moiety
799
cm−1.2,10
The EIMS spectra of 1 did not exhibit the expected
molecular ion, although it showed characteristic 8-methox-
ycoumarin fragment ions at m/z 193 and 176, corresponding to
the loss of the side chain (Scheme S1, Supporting
Information).18 The successive loss of CO afforded fragments
at m/z 164 and 148. Coumarins have been the subject of
numerous mass spectrometric investigations due to their
pharmacological relevance.19 The mass spectrometer employed
in the present investigation was optimized using positive-ion EI,
FAB, and ESI conditions. The positive-ion mode was found to
be the most sensitive. The FAB mass spectrum of 1 exhibited a
protonated molecular ion, [M + H]+, at m/z 414, and ions were
observed at m/z 221, 207, and 115. The ESIMS exhibited the
sodium adduct [M + Na]+ and protonated molecular ions [M +
H]+ at m/z 436 and 414, respectively. Thus, the FAB and
ESIMS data of 1 suggested the presence of a nitrogen atom. In
the ESIMS data of compound 1, cleavage of the bond between
the oxygen atom at C-7 and the side chain resulted in the
formation of a stable 7-hydroxy-8-methoxycoumarin ion at m/z
DOI: 10.1021/acs.jnatprod.6b00391
J. Nat. Prod. 2017, 80, 798−804
Journal of Natural Products Article

193, further confirming the presence of a 7,8-dioxygenated

coumarin moiety. The fragment ion at m/z 222, corresponding
to the side chain, originated from the loss of the coumarin
nucleus. The characteristic fragment ion at m/z 205 indicated
the loss of NH3. The subsequent loss of 28 mass units (CO)
was evidenced by the ion at m/z 177.
Observation of the 1H NMR high-field region at δH 2.2−2.9
in methanol-d4 is often complicated by overlapping signals.
However, the signals can be shifted by pyridine-d5 as the
solvent. Change of the NMR solvent produced a dramatic effect
on the 1H NMR signals of 1. Two well-resolved sharp N−H
proton signals at δH 8.32 and 7.83 (each 1H, br s) were
observed in the 1H NMR spectra of 1. The difference between
the 13C NMR spectra of 1 and 3 involved the presence of a
characteristic acyl carbonyl signal at δC 173.3, two extra
methylene carbon signals at δC 21.2 and 33.5 (Table 1), and the
absence of the C-9′ methyl resonance at δC 12.0. Characteristic
IR signals at 3450 cm−1 observed in 1 are indicative of the N−
H stretching of the primary amide, and the strong bands at
1690 cm−1 are typical for the N−CO stretching region of
amides. The 1H−1H COSY cross-peaks of H-9′/H-11′
suggested the linkage of C-9′ and C-11′. The HMBC cross-
peaks from H-9′ to C-6′, C-7′, C-8′, C-11′, and C-12′ and from
H-11′ to C-7′, C-9′, and C-12′ indicated the linkage of an
acetamido unit to the terpenoidal side chain at C-9′, signals that
are absent in the case of compound 3.
Compound 1 was isolated as an oil, making it impossible to
obtain a single crystal for X-ray diffraction analysis. The limited
amount of 1 also made it challenging to obtain a derivative for
further crystallographic analysis. Thus, the relative configu-
ration was established by examining the cross-peaks in the
ROESY spectrum. Correlations between H2-1′ and H3-10′ and
between H-2′ and H-4′a/b indicated that the C-2′/C-3′ double
bond was E-configured. Monoterpenoid coumarins isolated
from Clausena species tend to have an E double bond in the C- Figure 3. Calculated ECD spectra for (5′R)- and (5′S)-enantiomers
7 or C-8 side-chain moiety.2,5,6,10 The C-5′ absolute and experimental ECD spectra of 1 and 3 in MeOH.
configurations of 1 and 3 were assigned by comparing the
calculated electronic circular dichroism (ECD) spectra of the
to oxidation, deprotonation, and amidation to give compound
5′R- and 5′S-enantiomers with the experimental spectra of both
1.
coumarins. The experimental ECD spectra of 1 and 3 exhibited
Anisumic acid (2) was obtained as a white powder, [α]21.5D
negative Cotton effects at 204, 208, and 216 and at 205, 210, +13 (c 2, MeOH). Its molecular formula, C19H18O6, was
and 217 nm in methanol, respectively, indicating a 5′R determined on the basis of the molecular ion observed in
configuration.10 The good agreement between the experimental negative-ion HRESI at m/z 341.1012 [M − H]− (calcd
and calculated ECD spectra of the (5′R)-enantiomers led to the 341.1025), which corresponds to 11 indices of hydrogen
unequivocal absolute configuration assignment (Figure 3). deficiency. The UV spectrum of 2 in MeOH showed absorption
Therefore, the absolute configurations of 1 and 3 were maxima at 205, 230, 279, and 360 nm. The IR spectrum
identified as 5′R. Collectively, the data showed that the showed absorption bands for phenolic (3442, 1244 cm−1),
structure of anisucoumaramide (1) was as shown in Figure 1. carbonyl (1716 cm−1), and aromatic functionalities (1614,
The occurrence of coumarins featuring a C10 terpenoid 1516, 720 cm−1).
moiety has been demonstrated in the genus Clausena and other The resolution of overlapped proton signals was improved
genera;2,5,6,10,20−23 however, it is noteworthy that anisucoumar- when the 1H NMR spectrum of 2 was recorded in pyridine-d5.
amide (1) represents the first example of a naturally occurring Using HMQC data analysis, the 13C NMR and DEPT spectra
coumarin of which the terpenoidal side chain does not comply resolved 19 carbon signals that were ascribed to one methoxy
with the biosynthesis isoprene rule because an unprecedented (δC 51.9), 12 methines [four sp3 methines, δC 48.6, 48.4, 46.3,
acetamido motif is directly connected with a terpenoidal side 45.9, eight sp2 methines, 128.8 (×4), 116.5 (×4)], and six
chain. Anisucoumaramide (1) is possibly biosynthesized from nonprotonated carbons (including four sp2 carbons, δC 132.8,
8-methoxycapnolactone (3), a coumarin occurring in the same 133.1, 158.1, 158.2, and two carbonyls, δC 173.9 and 175.6)
plant. As shown in Scheme 1, the putative biosynthesis of 1 (Table 2). In addition, 2 revealed 1H NMR signals in three
starts from a double-bond migration (C6′/C7′ to C7′/C9′) of distinct regions: aromatic protons ascribed to two 1,4-
3. The enoyl moiety of 5 then undergoes decarboxylative disubstituted benzene rings [δH 7.51 (2H, d, J = 8.0 Hz),
Michael addition with malonyl CoA under radical conditions to 7.46 (2H, d, J = 8.1 Hz), 7.16 (4H, d, J = 8.1 Hz)], four sp3
yield the intermediate radical 6,24,25 which in turn is susceptible methines [δH 4.08, 3.97, 3.96, 3.94 (each 1H, m)], and a
800 DOI: 10.1021/acs.jnatprod.6b00391
J. Nat. Prod. 2017, 80, 798−804
Journal of Natural Products Article

Scheme 1. Putative Biosynthesis Pathway toward Anisucoumaramide (1)

Table 2. NMR Spectroscopic Data (500 MHz, Pyridine-d5)

of Compound 2
position δH (J in Hz) δC, type HMBC
1 3.96, m 45.9, CH C-2, C-3, C-4, C-7′
2 4.08, m 48.6, CH C-1, C-3, C-4, C-1′, C-2′/6′, C-7′
3 3.94, m 48.4, CH C-2, C-4, C-1, C-1″, C-2″/6″,
C-8′
4 3.97, m 46.3, CH C-1, C-2, C-3, C-8′
1′ 133.1, C
2′ 7.51, d (8.0) 128.8, CH C-2, C-3′, C-4′
3′ 7.16, d (8.1) 116.5, CH C-1′, C-2′, C-4′
4′ 158.2, C
5′ 7.16, d (8.1) 116.5, CH C-1′, C-4′, C-6′ Figure 4. 1H−1H COSY, key HMBC, and ROESY correlations of 2.
6′ 7.51, d (8.0) 128.8, CH C-2, C-4′, C-5′
1″ 132.8, C an HMBC experiment based on the following key correlations:
2″ 7.46, d (8.1) 128.8, CH C-3, C-3″, C-4″ from H-1 to C-2, C-3, C-4, and C-7′; H-2 to C-1, C-3, C-4, C-
3″ 7.16, d (8.1) 116.5, CH C-1″, C-2″, C-4″ 1′, C-2′, and C-7′; H-3 to C-2, C-4, C-1, C-1″, C-2″, and C-8′;
4″ 158.1, C and H-4 to C-1, C-2, C-3, and C-8′. This analysis produced
5″ 7.16, d (8.1) 116.5, CH C-1″, C-3″, C-4″, C-6″ four possible structures, A−D, based on the position of the
6″ 7.46, d (8.1) 128.8, CH C-3, C-4″, C-5″ hydroxycarbonyl and the methoxycarbonyl groups linked to the
7′ 175.6, C cyclobutane core (Scheme 2). The HMBC cross-peaks between
8′ 173.9, C the methoxy protons (δH 3.64) and C-8′ (δC 173.9) excluded
OMe 3.64, s 51.9, CH3 C-8′ the possibility of structures C and D. The analysis of the EIMS
fragmentation pattern of 2 confirmed that only structure A was
in accordance with the NMR and MS data (Figure 4). The
methoxy group (δH 3.64). The 1H−1H COSY spectrum also successive fragmentation of the key ion of structure A at m/z
showed five separate spin systems (shown in bold in Figure 4) 212 (55), 178 (100), 164 (80), 147 (87), and 131 (20)
that comprised two 1,4-disubstituted benzene moieties and four confirmed the 7′-COOH and 8′-COOMe groups. The HMBC
contiguous sp3 methines. Thus, the direct connections between cross-peaks between H-1 (δH 3.96) and C-7′ (δC 175.6) and
C-1, C-2, C-3, and C-4 implied the presence of a cyclobutane from H-2 (δH 4.08) to C-1′, C-2′, and C-7′ (δC 175.6)
moiety. The two carbonyl carbons and eight aromatic carbons supported the location of the hydroxycarbonyl group at C-1.
represented 10 indices of hydrogen deficiency. The remaining On the basis of these data, the 2D structure of 2 was defined as
index of hydrogen deficiency further supports that 2 possesses a 2,3-bis(4-hydroxyphenyl)-4-(methoxycarbonyl)-
four-membered ring system. Analyses of 1D and 2D NMR data cyclobutanecarboxylic acid as shown in Figure 1.
indicated that 2 was a cyclobutane derivative, possibly Attempts at obtaining a single crystal for X-ray diffraction
assembled by two phenylpropanoid subunits: part A and part analysis failed, and the limited quantity of 2 made it difficult to
B (Figure 4). The connection of parts A and B was inferred by produce a derivative for further crystallographic analysis. Thus,
801 DOI: 10.1021/acs.jnatprod.6b00391
J. Nat. Prod. 2017, 80, 798−804
Journal of Natural Products
Scheme 2. Possible Structures and EIMS Fragmentation Analysis of 2
the relative configurations of the stereogenic centers in 2 were
assigned by analysis of the ROESY correlations of the
cyclobutyl and aromatic proton signals (Figure 4). The
ROESY spectrum of 2 showed NOE correlations between H-
2 (δH 4.08) and H-6′ (δH 7.51)/H-2″ (δH 7.46) and
correlations between H-1/H-2′ (δH 7.51) and H-3/H-2′ (δH
7.51). No diagnostic NOE correlation was observed among the
four mutually coupled proton signals of the cyclobutane
moiety. Therefore, the key NOE correlations suggested that
H-1 and H-3 occupied the face of the cyclobutane moiety
opposite that of H-2 and H-4, thus confirming the 1,2-trans-3,4-
trans relative configuration of the cyclobutane moiety.26
Considering the remaining possible δ- or μ-truxinic-type
structures, only the former conformed to these conditions.
The deshielded methoxy resonance (δH 3.64) also accounted
for the adjacent trans-oriented aromatic groups on the
cyclobutane ring.27,28 Thus, the relative configuration of
compound 2 was established, and the compound was named
anisumic acid (2). Compound 2 is a new δ-truxinate derivative
possessing a cyclobutane core. This δ-truxinate derivative was
isolated from Clausena species for the first time. The relative
configuration of the cyclobutyl unit is in accord with those of
recently reported δ-truxinic acid derivatives.27,28 Compound 2
is similar to monomethyl 3,3′,4,4′-tetrahydroxy-δ-truxinate, a δ-
truxinate derivative isolated from Lysimachia clethroides.28 The
difference is that the latter possesses 1,3,4-trisubstituted
aromatic rings instead of the para-substituted rings in
compound 2.
Cyclobutane-containing organic compounds, including nat-
ural products and/or drugs, present an intriguing group of
metabolites with a variety of biological activities and may serve
as potential drug leads or provide new ideas for the study of
enzymatic mechanisms and/or organic synthesis.29,30 It is
generally presumed that cyclobutane derivatives originate from
the coupling of two phenylpropenoids. Thus, compound 2 may
originate from (E)-4-hydroxycinnamic acid (4), which was
isolated from the same plant (Scheme 3). A key intermolecular
[2+2] cycloaddition of 4 formed the anti-head-to-head dimer
(8), followed by monoesterification to afford compound 2. (E)-
4-Hydroxycinnamic acid (4) is also a biogenetically significant
802
Article
Scheme 3. Putative Biosynthesis Pathway toward Anisumic
Acid (2)
constituent for 7-O-isoprenylcoumarins. The (E)- and (Z)-p-
hydroxycinnamic acids are the precursors for all known
rutaceous coumarins that are oxygenated at C-7.31
Coumarins have drawn considerable attention in recent years
as an important group of organic compounds that exhibits
significant biological activities associated with neurological
disorders. Both natural and synthesized coumarin analogues
showed potent MAO-B inhibitory activity, particularly for the
3-, 4-, and 7-substituted coumarin analogues.32−34 Thus, the
potential inhibitory effects of the new coumarin 1 were
evaluated on human recombinant monoamine oxidase
(hMAO) isoforms. The inhibitory effects were assessed by
measuring the production of H2O2 from p-tyramine using the
Amplex Red MAO assay kit with selegiline and iproniazide as
reference drugs. The IC50 values and MAO-B selectivity indices
for the inhibitory effects of both the new compound and
reference inhibitors were calculated (Table 3). Compound 1
inhibited MAO-B with an IC50 value of 143.65 ± 0.90 nM
(MAO-B selectivity index >696), but it was inactive at 100 μM
to the MAO-A, demonstrating that compound 1 shows high
selectivity for the MAO-B isoenzyme and inhibitory activity in
the nanomolar range.
DOI: 10.1021/acs.jnatprod.6b00391
J. Nat. Prod. 2017, 80, 798−804
Journal of Natural Products
Table 3. MAO-A and MAO-B Inhibitory Activity Results for
Compound 1 and Reference Compounds
compound MAO-A IC50 MAO-B IC50 SIb
1 − a
143.65 ± 0.90 nM >696
selegiline 67.25 ± 1.02 μM 19.60 ± 0.86 nM 3431
iproniazide 6.56 ± 0.76 μM 7.54 ± 0.36 μM 0.87
a
Inactive at 100 μM (highest concentration tested). Each IC50 value is
the mean ± SEM from three experiments. bSI: MAO-B selectivity
index = IC50(MAO-A)/IC50 (MAO-B).
■
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
determined with a JASCO P-1020 polarimeter. UV spectra were
measured on a Shimadzu UV-2401PC spectrophotometer. ECD
spectra were acquired on a Chirascan instrument. IR spectra were
obtained on a Bio-Rad FTS-135 infrared spectrophotometer. 1D and
2D NMR spectra were obtained at 500 and 125 MHz for 1H and 13C,
respectively, on a Bruker DRX-500 spectrometer with tetramethylsi-
lane as an internal standard. MS data were recorded on a VG
Autospec-3000 mass spectrometer. Commercially available silica gel
(100−200 mesh or 200−300 mesh, Qingdao Haiyang Chemical Co.),
Lobar LiChroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20
(Pharmacia) were used for open-column chromatography. All the
solvents were distilled prior to use.
Plant Material. The leaves and twigs of Clausena anisum-olens
Merr. were collected in Hekou County of Yunnan Province, People’s
Republic of China, in May 2003 and identified by Prof. De-Ding Tao
of the Kunming Institute of Botany. A voucher specimen (No.
02041705) was deposited in the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences.
Extraction and Isolation. The powdered leaves and twigs of
C. anisum-olens Merr. (22.5 kg) were repeatedly extracted with 90%
aqueous EtOH (3 × 80 L) at room temperature. The extract was
concentrated under reduced pressure to give a brown syrup, which was
partitioned into H2O (15 L) and extracted successively with petroleum
ether (5 × 10 L), EtOAc (5 × 10 L), and n-BuOH (5 × 10 L); the
extracts were kept separately. The EtOAc extract (110.5 g) was
subjected to silica gel column chromatography, eluting with petroleum
ether−EtOAc (4:1, 2:1, 1:1, 2:3), EtOAc, EtOAc−MeOH (8:2, 7:3,
6:4, 1:1), and finally MeOH to afford nine fractions (I−IX). Fraction
II (18 g) was resubjected to silica gel column chromatography and
Pharmadex LH-20 (MeOH) to give compound 4 (9 mg). Fraction III
(25.8 g) was resubjected to silica gel column chromatography,
Pharmadex LH-20 (MeOH), and RP C18 to yield compounds 2 (3
mg) and 3 (52 mg). Fraction IV (4.8 g) was resubjected to silica gel
column chromatography, Pharmadex LH-20 (MeOH), and RP C18 to
afford compound 1 (2 mg).
Anisucoumaramide (1): colorless oil; [α]21.5D −13 (c 1.5, MeOH);
UV (MeOH) λmax 208, 215, 261, and 319 nm; ECD (c 0.44 mM,
MeOH) λmax (Δε) 204 (−5.3), 208 (−5.3), 216 (−2.2) nm; IR (KBr)
νmax 3450, 2925, 2856, 1730, 1728, 1690, 1621 cm−1; 1H and 13C
NMR data see Table 1; HRFABMS [M + Na]+ m/z 414.1620 (calcd
for C22H23NO7, 414.1613); FABMS m/z 414 [M + H]+ (95), 207
(100), 115 (74); ESIMS m/z 436 [M + Na]+, 414 [M + H]+, 222, 205,
193, 187, 177, 159, 131.
Anisumic Acid (2): white powder; [α]21.5D +13 (c 3, MeOH); UV
(MeOH) λmax 205, 230, 279, and 360 nm; IR (KBr) νmax 3442, 2955,
1716, 1614, 1516, 1244 cm−1; 1H and 13C NMR data see Table 2;
HRESIMS [M − H]− m/z 341.1012 (calcd for C19H18O6, 341.1025);
EIMS m/z 342 [M]+ (10), 316 (1), 306 (27), 263 (7), 237 (27), 212
(55), 178 (100), 164 (80), 147 (87), 131 (20).
8-Methoxycapnolactone (3): colorless semisolid; UV (MeOH)
λmax 204 and 319 nm; ECD (c 0.22 mM, MeOH) λmax (Δε) 205
(−8.1), 210 (−5.0), 217 (−9.2) nm; 1H and 13C NMR data see Table
1; EIMS m/z 356 [M]+, 192, 163, 147, 97.
803
Article
Computational Methods. The geometries of compounds 1 and 3
were calculated using the DFT (B3LYP) method at the 6-31G(d) level
by Gaussian 09.35 The ECD computation was performed using time-
dependent density-functional theory at the same level with the PCM
solvent model for MeOH. The ECD spectra were generated by the
program GaussView using a Gaussian band shape with 0.333 eV
exponential half-width from dipole-length dipolar and rotational
strengths.
Bioassay Methods. The effects of 1 on the hMAO isoform
enzymatic activity were evaluated by measuring the effects on the
production of H2O2 from p-tyramine using a fluorimetric method.
Selegiline and iproniazide served as reference inhibitors. Briefly, the
study medium comprised 0.1 mL of Na3PO4 buffer (0.05 M, pH 7.4),
various concentrations of compound 1 or reference compounds, and
adequate amounts of recombinant hMAO-A or hMAO-B (Sigma-
Aldrich) required to oxidize (in the control group) 165 pmol of p-
tyramine/min (hMAO-A, 1.1 μg of protein; specific activity: 150 nmol
of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg
protein; hMAO-B, 7.5 μg of protein; specific activity: 22 nmol of p-
tyramine transformed/min/mg protein). This mixture was incubated
for 15 min at 37 °C in a flat-black-bottom 96-well microtest plate
placed in the dark multimode microplate reader chamber. After this
incubation period, the reaction was initiated by adding 200 μM
Amplex Red reagent, 1 U/mL horseradish peroxidase (HRP), and 1
mM p-tyramine as a common substrate for both hMAO-A and hMAO-
B. The production of H2O2 catalyzed by MAO isoforms was detected
using Amplex Red reagent, a nonfluorescent probe reacting with H2O2
in the presence of HRP. The fluorescent product resorufin was
quantified at 37 °C in a multidetection microplate fluorescence reader
with excitation at 545 nm and emission at 590 nm for 15 min. The
specific fluorescence emission was calculated after subtraction of the
background activity. Control experiments were carried out simulta-
neously by replacing the test drugs (compound 1 and reference
inhibitors) with appropriate dilutions of the vehicles. In addition, the
possible capacity of the above-mentioned test drugs to directly react
with Amplex Red reagent was determined by adding these drugs to
solutions containing only the Amplex Red reagent in a sodium
phosphate buffer.36,37
■ ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00391.
HRESIMS and 1D and 2D NMR spectra of compounds
1 and 2; proposed MS fragmentation pathway of 1
(PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*Tel: +86 871 65033715. E-mail: yangjh@ynu.edu.cn.
*Tel: +86 871 65219684. E-mail: haoxj@mail.kib.ac.cn.
ORCID
Jing-Hua Yang: 0000-0001-5835-2445
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by the National Natural
Science Foundation of China (Grant Nos. 21462048,
21162038, 21262040, and 21662040) and the China Scholar-
ship Council (CSC) Fund No. 201508535020, and Grant No.
2007PY01-23. We are grateful to the High Performance
Computing Center of Yunnan University for providing the
calculation resources.
DOI: 10.1021/acs.jnatprod.6b00391
J. Nat. Prod. 2017, 80, 798−804
Journal of Natural Products

■
Article

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804 DOI: 10.1021/acs.jnatprod.6b00391

J. Nat. Prod. 2017, 80, 798−804