Collagenase inhibitors from Viola yedoensis collagenase assay

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Collagenase inhibitors from Viola yedoensis
Naohiro Oshima · Yuji Narukawa · Tadahiro Takeda ·
Fumiyuki Kiuchi
Received: 16 February 2012 / Accepted: 15 March 2012 / Published online: 13 April 2012
© The Japanese Society of Pharmacognosy and Springer 2012
Abstract Fractionation of acetone and methanol extracts
of Viola yedoensis, under the guidance of inhibition against
Clostridium histolyticum collagenase (ChC), resulted in the
isolation of esculetin (1) (IC50 12 μM) and scopoletin (2)
(IC50 1.8 μM) as the active constituents, together with
trans-p-coumaric acid (3), cis-p-coumaric acid (4), 3-O-β-
D-glucosyl-7-O-α-L-rhamnosylkaempferol (5), rutin (6), iso-
vitexin (7), isoorientin (8), vicenin-2 (9), isoscoparin (10),
vanillic acid (11) and adenosine (12). Modification of phe-
nolic hydroxy groups of 1 showed that small O-alkyl groups
largely increased the activity, whereas largerO-alkyl groups
decreased the activity, and 6,7-dimethoxycoumarin (scopa-
rone 13) potently inhibited ChC (IC50 24 nM).
Keywords Collagenase · Inhibitor · Viola yedoensis ·
Coumarin · Anti-inflammatory
Introduction
Viola herb (紫花地丁) is a crude drug traditionally con-
sidered effective for cooling toxic heat and removing
moisture and swelling. It has been used to treat inflam-
matory diseases such as sores, boils, jaundice, acute
nephritis, diarrhea, and snake bites [1]. Although several
members of the Violaceae family have been reported to be
used as origin plants of this crude drug [2], Viola yedoensis
Makino is listed as the solo origin plant of this crude drug
in the Chinese Pharmacopoeia [3]. V. yedoensis is a small
perennial herb with violet flowers distributed in China,
Japan and Korea. Phytosterols, coumarins, and flavonoid
glycosides have been reported as its chemical constituents
[4–7]. In addition, cylopeptides with potent anti-HIV
(human immunodeficiency virus) activity have been iso-
lated from this plant [8]. However, anti-inflammatory
constituents of this crude drug have not been reported.
Collagenase is a member of the matrix metalloprotein-
ases (MMPs), which are zinc-dependent endopeptidases
capable of degrading all kinds of extracellular matrix
proteins [9]. Collagenase causes bronchial inflammation,
especially status asthmaticus [10]. It is also known that
anti-inflammatory drugs (e.g. acetylsalicylic acid, flufen-
amic acid and indomethacin) inhibit collagenase [11, 12].
Thus, in this paper, we searched for collagenase inhibitors
of Viola herb.
Results and discussion
As a preliminary test, Viola herb (Viola yedoensis) was
successively extracted with hexane, chloroform, acetone,
methanol and 50 % ethanol, and the extracts were measured
for inhibitory activity against Clostridium histolyticum
collagenase (ChC) [13]. Of these extracts, acetone (IC50
27 μg/mL) and methanol (IC50 55 μg/mL) extracts showed
potent inhibitory activity. These extracts were thus frac-
tionated under the guidance of ChC inhibitory activity to
give esculetin (1) [14] and scopoletin (2) [15], together
with trans-p-coumaric acid (3) [16], cis-p-coumaric acid (4)
[17], 3-O-β-D-glucosyl-7-O-α-L-rhamnosylkaempferol (5)
[18], rutin (6) [19], isovitexin (7) [20], isoorientin (8) [21],
vicenin-2 (9) [22], isoscoparin (10) [23], vanillic acid (11)
[24] and adenosine (12) [25]. The structures of these com-
pounds were determined by comparison of their spectral data
with those reported (Fig. 1).
N. Oshima · Y. Narukawa · T. Takeda · F. Kiuchi (&)
Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen,
Minato-ku, Tokyo 105-8512, Japan
e-mail: kiuchi-fm@pha.keio.ac.jp
123
J Nat Med (2013) 67:240–245
DOI 10.1007/s11418-012-0665-8
Among these compounds, scopoletin (2, IC50 1.8 μM)
showed more potent activity than esculetin (1, IC50
12 μM). However, the isolated yield of 2 (5.5 mg) is far
less than that of 1 (876 mg), indicating 1 to be the major
collagenase inhibitor of this crude drug. Isoorientin (8, IC50
84 μM) and vicenin-2 (9, IC50 185 μM) also showed
moderate activity. However, similar C-glycosides 7 and 10
did not show the activity (IC50 [200 μM).
As scopoletin (2) showed stronger activity than esculetin
(1), the phenolic hydroxy groups of esculetin were modified
and the activities of the derivatives were compared (Fig. 2).
Coumarin did not show the activity (IC50 [200 μM). The
dimethyl derivative of esculetin (scoparone 13) showed
much stronger activity (IC50 24 nM) than 1 (IC50 12 μM).
However, replacement of the two O-methyl groups of 13
with longer alkyl groups greatly decreased the activity (15,
IC50 2.8 μM; 17, IC50 3.8 μM; 19, IC50 24 μM), whereas
replacement with acetyl groups or cyclic ether formation
retained appreciable activity (21, IC50 0.23 μM; 22, IC50
0.89 μM). The derivatives having O-methyl and O-ethyl
groups (23, IC50 0.56 μMand 24, IC50 0.70 μM) also showed
appreciable activity. Thus, it can be concluded that small
O-alkyl groups are necessary for the potent activity, whereas
larger O-alkyl groups decrease the activity. This was also
true among the 6-hydroxy-7-O-alkyl derivatives (14, IC50
3.8 μM; 16, IC50 10 μM; 18, IC50 43 μM; 20, IC50 56 μM).
In this study, we isolated esculetin (1) and scoporetin (2)
as collagenase inhibitors of V. yedoensis. These compounds
have been reported to have anti-inflammatory activity
through inhibiting the release of pro-inflammatory eicosa-
noid mediators such as prostaglandin E2 and leukotriene C4
from macrophages [26]. Thus, these compounds have plural
sites of action as anti-inflammatory agents and seem to be the
major anti-inflammatory constituents of V. yedoensis.
Experimental procedure
General
1H- and 13C-NMR spectra were recorded on Varian 400-MR,
Varian Unity Plus 500 and JEOL FT-NMR ECP-600 spec-
trometers, and the chemical shifts were expressed in δ (ppm)
O O
RO
HO
1
R
H (12 µM)
CH3 (1.8 µM)
CO2H
HO
CO2HHO
O
OOH
O
OH
Rha
O
Glc
O
OOH
HO
OH
O
Glc Rha
OH
6 (>200 µM)
3 (>200 µM)
4 (>200 µM)
5 (>200 µM)
O
OOH
Glc
HO
R1
OH
R2
7
R1
H
H
Glc
H
OH
HO2C OCH3
N
N
N
N
O
OH OH
HO
NH2
12 (>200 µM)
11 (>200 µM)
R2
H (>200 µM)
OH (84 µM)
H (185 µM)
OCH3 (>200µM)
8
9
10
2
Fig. 1 Structures and IC50
values of isolated compounds.
Positive control:
phosphoramidon
(IC50 = 16 μM)
O O
R1O
R2O
2
3
4
6
7
10
9
R1 R2 IC50 (µM)
1 H H 12
2 CH3 H 1.8
13 CH3 CH3 0.024
14 H CH3 3.8
15 C2H5 C2H5 2.8
16 H C2H5 10
17 C4H9 C4H9 3.8
18 H C4H9 43
19 C8H17 C8H17 24
20 H C8H17 56
21 Ac Ac 0.23
22 CH2-CH2 0.89
23 C2H5 CH3 0.56
24 CH3 C2H5 0.70
Fig. 2 Structures and IC50 values of esculetin derivatives. Positive
control: phosphoramidon (IC50 = 16 μM)
J Nat Med (2013) 67:240–245 241
123
with TMS as an internal standard. MALDI-TOF MS spectra
were recorded on a Voyager RP AK1 spectrometer. Column
chromatographies were carried out on silica gel (Silica gel 60,
Merck), Diaion HP-20 (Mitsubishi Chemical Co.) and Lobar
LiChroprep RP-18 (Merck). HPLC was performed on a Shi-
madzu LC-10VP system, equipped with SCL-10AVP system
controller, LC-10ADVP pumps, CTO-10AVP column oven
and SPD-10A detector, in a gradient mode using Capcell Pak
C18 column (4.6mm i.d.9 250mm, Shiseido FineChemicals).
Plant material
Viola herb (紫花地丁) was purchased from Tochimoto Co.
(Lot. 095907001, 095908001). The original plant was
identified as Viola yedoensis on the basis of morphological
features [2] by Dr. S. Nunome. Sample specimens were
deposited in the Laboratory of Natural Medicines, Faculty
of Pharmacy, Keio University (No. KYS-09001).
Collagenase inhibition assay
Collagenase assay was carried out by a modified Wunsch’s
method [13]. To a mixtureof a substrate (20 μM MOCAc-
Pro-Leu-Gly-Leu-A2pr-(DNP)Ala-Arg-NH2, Peptide Insti-
tute. Inc., 3163-v, 50 μL) and a sample dissolved in 50 μL
of 50 mM tris–HCl buffer (pH 7.6) in a well of a 96-well
plate (BD-Falcon Co., 353241), 100 μL of collagenase type
1-s (Sigma-Aldrich, C1639, 40 μg/mL) was added, and the
reaction mixture was incubated at 37 °C for 10 min.
The fluorescence intensity of liberated MOCAc-Pro-Leu in
the mixture was measured at Ex. 365 nm and Em. 465 nm
by a fluorescence spectrophotometer (Colona Electric Co.,
MTP-810Lab). Phosphoramidon (Peptide Institute Inc.)
was used as a positive control (IC50 = 16 μM).
Extraction and isolation
Preliminary test
Viola herb (10 g) was successively extracted at room
temperature for 24 h with 250 mL of hexane, chloroform,
acetone, methanol and 50 % ethanol to give hexane
(87 mg), chloroform (44 mg), acetone (86 mg), methanol
(65 mg), and 50 % EtOH (1.17 g) extracts. Collagenase
inhibitory activities of these extracts were IC50 [200, 88,
27, 55 and 61 μg/mL, respectively.
Fractionation of acetone extract
Viola herb (2 kg) was extractedwith hexane (6 L for 24 h9 8)
at room temperature and then the residue was extracted with
acetone (6 L for 24 h 9 15). The acetone extract was
concentrated under reduced pressure to give an acetone
extract (41 g) (IC50 27μg/mL).A part of the extract (30 g)was
dissolved in acetone, passed through an activated charcoal
(70 g) column, and eluted with acetone to give acetone elute
(26 g). The acetone elute (20 g)was fractionated by a silica gel
column with CHCl3–MeOH (1:0→ 50:1→ 25:1→ 15:1→
10:1→ 4:1→ 1:1→ 0:1) to afford eight fractions [fr. 1, 2.5 g
(IC50 40 μg/mL); fr. 2, 2.4 g (IC50 30 μg/mL); fr. 3, 1.9 g (IC50
110 μg/mL); fr. 4, 6.5 g (IC50 50 μg/mL); fr. 5, 2.2 g
(IC50 20 μg/mL); fr. 6, 1.7 g (IC50 71 μg/mL); fr. 7, 2.1 g (IC50
68 μg/mL); fr. 8, 2.2 g (IC50 170 μg/mL)]. Fr. 5 (2.2 g) was
further separated by silica gel column chromatography
(CHCl3–MeOH) to give 1 (687 mg) [14].
Fractionation of MeOH extract
The residue of the above acetone extraction was extracted
with MeOH (6 L for 24 h 9 5) at room temperature and the
extract was concentrated under reduced pressure to give
methanol extract (228 g) (IC50 55 μg/mL). A part of the
extract (138 g) was dissolved in water (600 mL), and frac-
tionated with a Diaion HP-20 column (15 9 45 cm) eluted
with H2O → MeOH → CHCl3 to afford three fractions
[Fr. DW, 100 g (IC50 165 μg/mL); Fr. DM, 19 g (IC50 18 μg/
mL); Fr. DC, 900 mg (IC50 72 μg/mL)]. Fr. DM (18 g) was
chromatographed on silica gel with CHCl3–MeOH
(1:0→ 100:1→ 50:1→ 20:1→ 10:1→ 1:1→ 1:2→ 1:4
→ 0:1) to afford five fractions [Fr. DM-1, 1.61 g (IC50 9.3 μg/
mL); Fr. DM-2, 2.70 g (IC50 2.2 μg/mL); Fr. DM-3, 14.0 g
(IC50 30 μg/mL); Fr. DM-4, 450 mg (IC50 60 μg/mL); Fr.
DM-5, 1.00 g (IC50 37 μg/mL)]. Repeated column chroma-
tography of Fr. DM-1 on silica gel (CHCl3–MeOH and
hexane–AcOEt) gave 1 (177 mg) and 2 (5.5 mg) [15].
Repeated column chromatography of Fr. DM-2
(700 mg) on Lobar RP-18 (H2O–MeOH) and silica gel
(CHCl3–MeOH), followed by Sephadex LH-20 (MeOH),
gave 1 (12.2 mg), 3 (1.5 mg) [16] and 4 (0.6 mg) [17].
Fractionation of CHCl3–MeOH extract
Viola herb (1 kg) was extracted with a mixture of
CHCl3/MeOH (1:1, 5 L) for 40 h at room temperature to
give CHCl3–MeOH extract (77 g). The extract (46 g) was
subjected to Diaion HP-20 column (6 9 45 cm) eluted with
H2O–MeOH–CHCl3 (1:1:0 → 0:1:0 → 0:0:1) to afford
three fractions [Fr. DMW, 22 g; Fr. DM, 10 g; Fr. DC, 4 g].
Fr. DMW (12 g) was subjected to Lobar RP-18 column
with H2O–MeOH (1:0 → 0:1) to afford twelve fractions
[Fr. 1, 5.8 g; Fr. 2, 130 mg; Fr. 3, 500 mg; Fr. 4, 330 mg;
Fr. 5; 710 mg; Fr. 6, 240 mg; Fr. 7, 150 mg; Fr. 8, 30 mg;
Fr. 9, 80 mg; Fr. 10, 75 mg; Fr. 11, 300 mg; Fr. 12, 41 mg].
Further purification of these fractions gave 11 (9.0 mg) [24]
from fr. 3, 12 (8.9 mg) [25] from fr. 4, 8 (34 mg) [21] and 9
242 J Nat Med (2013) 67:240–245
123
(46 mg) [22] from fr. 5, and 7 (1.5 mg) [20], 10 [23]
(7.0 mg), 5 (1.8 mg) [18] and 6 (20 mg) [19] from fr. 6.
Preparation of coumarin derivatives
Alkylation (general procedure)
A mixture of esculetin (1, purchased from Tokyo Chemical
Industry), Na2CO3 and alkyl iodide in DMF was stirred at
room temperature. The mixture was diluted with water and
extracted with ethyl acetate and the extract was concen-
trated under reduced pressure to dryness. The residue was
purified by silica gel column chromatography to give the
product(s). The compounds obtained, except for 19, were
crystallized from hexane–ethyl acetate.
6,7-Dimethoxycoumarin (13) [27] (y. 78 %): white
needles, mp 146 °C. MALDI-TOF MS m/z: 207 [M+H]+.
1H-NMR (400 MHz, CDCl3) δ: 7.63 (1H, d, J = 9.6 Hz),
6.86 (1H, s), 6.85 (1H, s). 6.29 (1H, d, J = 9.6 Hz), 3.95
(3H, s), 3.92 (3H, s).
6-Hydroxy-7-methoxycoumarin (14) [28] (y. 65 %):
white needles, mp 182 °C. MALDI-TOF MS m/z: 193
[M+H]+. 1H-NMR (400 MHz, acetone-d6) δ: 7.82 (1H, d,
J = 9.5 Hz), 7.06 (1H, s), 6.95 (1H, s), 6.21 (1H, d,
J = 9.5 Hz), 3.98 (3H, s).
6,7-Diethoxycoumarin (15) [y. 8 % (15) + 40 % (16)]:
white needles, mp 109 °C. MALDI-TOF MS m/z: 235 [M
+H]+. 1H-NMR (500 MHz, CDCl3) δ: 7.59 (1H, d,
J = 9.5 Hz, H-4), 6.87 (1H, s, H-5), 6.82 (1H, s, H-8). 6.26
(1H, d, J = 9.5 Hz, H-3), 4.15 and 4.11 (each 2H, q,
J = 7.0 Hz, OCH2CH3), 1.51 and 1.48 (each 3H, t,
J = 7.0 Hz, OCH2CH3).
6-Hydroxy-7-ethoxycoumarin (16): white needles, mp
149 °C. MALDI-TOF MS m/z: 207 [M+H]+. 1H-NMR
(500 MHz, CDCl3) δ: 7.59 (1H, d, J = 9.5 Hz, H-4), 6.97
(1H, s, H-5), 6.80 (1H, s, H-8), 6.27 (1H, d, J = 9.5 Hz,
H-3), 4.18 (2H, q, J = 7.0 Hz, –OCH2CH3), 1.51 (3H, t,
J = 7.0 Hz, –OCH2CH3),
13C-NMR (125 MHz, CDCl3) δ:
160.7 (C-2), 148.6 (C-7), 148.2 (C-9), 142.6 (C-6), 141.9
(C-4), 112.6 (C-3), 111.0 (C-10), 110.1 (C-5), 98.9 (C-8),
64.2 (–OCH2CH3), 13.5 (–OCH2CH3).
6,7-Dibutoxycoumarin (17) [y. 16 % (17) + 46 %
(18)]: white needles, mp 79 °C. MALDI-TOF MS m/z:
291 [M+H]+. 1H-NMR (500 MHz, CDCl3) δ: 7.59 (1H, d,
J = 9.5 Hz, H-4), 6.87 (1H, s, H-5), 6.81 (1H, s, H-8).
6.25 (1H, d, J = 9.5 Hz, H-3), 4.05 (2H, t, J = 6.5 Hz,
7-OCH2R), 4.01 (2H, t, J = 6.5 Hz, 6-OCH2R), 1.83 and
1.52 (each 4H, overlapped, 6,7-OCH2CH2CH2CH3), 1.00
and 0.99 (each 3H, t, J = 7.5 Hz, 6,7-OCH2CH2CH2CH3).
13C-NMR (125 MHz, CDCl3) δ: 161.6 (C-2), 153.2 (C-7),
150.1 (C-9), 146.1 (C-6), 143.4 (C-4), 113.2 (C-3),
111.3 (C-10), 110.6 (C-5), 101.0 (C-8), 69.7 and 69.0
(–OCH2R 9 2), 31.2 and 30.9 (–OCH2CH2CH2CH3 9 2),
19.2 and 19.1 (–OCH2CH2CH2CH3 9 2) 13.9 and 13.8
(–OCH2CH2CH2CH3 9 2).
6-Hydroxy-7-butoxycoumarin (18): white needles, mp
118–120 °C. MALDI-TOF MS m/z: 235 [M+H]+. 1H-
NMR (500 MHz, CDCl3) δ: 7.60 (1H, d, J = 9.5 Hz, H-4),
6.96 (1H, s, H-5), 6.81 (1H, s, H-8). 6.27 (1H, d,
J = 9.5 Hz, H-3), 4.11 (2H, t, J = 6.5 Hz, OCH2R), 1.85
and 1.52 (each 2H, m, OCH2CH2CH2CH3), 1.00 (3H, t,
J = 7.5 Hz, OCH2CH2CH2CH3).
13C-NMR (125 MHz,
CDCl3) δ: 161.6 (C-2), 149.6 (C-7), 149.2 (C-9), 143.5
(C-6), 142.9 (C-4), 113.7 (C-3), 112.1 (C-10), 111.0
(C-5), 99.9 (C-8), 69.3 (OCH2R), 30.9, 19.2, 13.8
(OCH2CH2CH2CH3).
6,7-Dioctyloxycoumarin (19) [y. 3.2 % (19) + 6.3 %
(20)]: white needles from MeOH, mp 66 °C. MALDI-TOF
MS m/z: 403 [M+H]+. 1H-NMR (500 MHz, CDCl3) δ:
7.58 (1H, d, J = 9.5 Hz, H-4), 6.86 (1H, s, H-5), 6.81 (1H,
s, H-8). 6.25 (1H, d, J = 9.5 Hz, H-3), 4.04 and 4.00 (each
2H, t, J = 6. 5 Hz, 7- and 6-OCH2R), 1.85 (4H, overlapped,
OCH2CH2R 9 2), 1.48 (4H, overlapped, O(CH2)2CH2R
9 2), 1.38–1.29 (16H, overlapped, -O(CH2)3(CH2)4CH3
9 2), 0.89 (6H, t, J = 7.0 Hz, –O(CH2)7CH3 9 2).
13C-
NMR (125 MHz, CDCl3) δ: 161.6 (C-2), 153.2 (C-7),
150.1 (C-9), 146.1 (C-6), 143.4 (C-4), 113.1 (C-3), 111.3
(C-10), 110.6 (C-5), 101.0 (C-8), 70.0 and 69.3 (7- and6-OCH2(CH2)6CH3), 31.8 and 31.7 (OCH2CH2R 9 2),
29.4, 29.3, 29.3, 29.2, 29.2, 28.9, 26.0, 25.9, 22.7, 22.6
(O(CH2)2(CH2)5CH3 9 2), 14.1 9 2 (O(CH2)7CH3 9 2).
6-Hydroxy-7-octyloxycoumarin (20): white needles,
mp 125 °C. MALDI-TOF MS m/z: 291 [M+H]+. 1H-NMR
(500 MHz, CDCl3) δ: 7.59 (1H, d, J = 9.5 Hz, H-4), 6.97
(1H, s, H-5), 6.81 (1H, s, H-8). 6.27 (1H, d, J = 9.5 Hz, H-
3), 5.65 (1H, br s, OH), 4.11 (2H, t, J = 6.5 Hz, 7-OCH2R),
1.85 (2H, m, OCH2CH2R), 1.47 (2H, m, O(CH2)2CH2R),
1.38–1.26 (8H, overlapped, O(CH2)3(CH2)4CH3), 0.90
(3H, t, J = 7.0 Hz, O(CH2)7CH3).
13C-NMR (125 MHz,
CDCl3) δ: 161.5 (C-2), 149.5 (C-7), 149.3 (C-9), 143.4
(C-6), 142.8 (C-4), 113.8 (C-3), 112.0 (C-10), 111.0 (C-5),
99.9 (C-8), 69.6 (OCH2R), 31.8 (OCH2CH2R), 29.3,
28.9, 26.0, 25.9 and 22.6 (O(CH2)2(CH2)5CH3), 14.0
(O(CH2)7CH3).
6,7-Diacethoxycoumarin (21) was prepared by an
acetylation of esculetin (1) with acetic anhydride/pyridine
(y. 99 %): white needles, mp 132 °C.MALDI-TOF MS m/z:
263 [M+H]+. 1H-NMR (500 MHz, CDCl3) δ: 7.62 (1H, d,
J = 9.6 Hz, H-4), 7.32 (1H, s, H-5), 7.19 (1H, s, H-8), 6.40
(1H, d, J = 9.6 Hz, H-3), 2.31 (3H, s, 7-OCOCH3), 2.29
(3H, s, 6-OCOCH3).
13C-NMR (125 MHz, CDCl3) δ: 168.1
J Nat Med (2013) 67:240–245 243
123
and 167.5 (OCOCH3), 160.0 (C-2), 151.7, 144.7, 142.3,
138.8, 121.6, 116.9, 116.8, 112.2, 20.6 and 20.5
(OCOCH3).
2,3-Dihydro-7H-pyrano[2,3-g]-1,4-benzodioxin-7-one (22)
[29] was prepared by a reaction of 1 with 1,2-dibromo-
ethane in DMF in the presence of Na2CO3 (y. 5.3 %): white
needles, mp 209 °C. MALDI-TOF MS m/z: 205 [M+H]+.
1H-NMR (500 MHz, CDCl3) δ: 7.56 (1H, d, J = 9.5 Hz,
H-4), 6.94 (1H, s, H-5), 6.85 (1H, s, H-8). 6.26 (1H, d,
J = 9.5 Hz, H-3), 4.34 (2H, m, 7-OCH2–), 4,28 (2H, m,
6-OCH2-).
13C-NMR (125 MHz, CDCl3) δ: 161.2 (C-2),
149.4 (C-9), 147.2 (C-7), 143.0 (C-4), 141.0 (C-6), 114.5
(C-5), 114.2 (C-3), 112.9 (C-10), 105.2 (C-8), 64.8 and
64.0 (7- and 6-OCH2–).
6-Ethoxy-7-methoxycoumarin (23) was prepared from
6-hydroxy-7-methoxycoumarin (14) as described in gen-
eral procedure (y. 29 %): white needles, mp 81 °C.
MALDI-TOF MS m/z: 221 [M+H]+. HR-FABMS m/z:
221.0830 [M+H]+, [calcd. for C12H13O4, 221.0814].
1H-
NMR (500 MHz, CDCl3) δ: 7.60 (1H, d, J = 9.5 Hz, H-4),
6.86 (1H, s, H-5), 6.84 (1H, s, H-8), 6.27 (1H, d,
J = 9.5 Hz, H-3), 4.11 (2H, q, J = 7.0 Hz, -OCH2CH3),
3.94 (3H, s, -OCH3), 1.49 (3H, t, J = 7.0 Hz, -OCH2CH3),
13C-NMR (125 MHz, CDCl3) δ: 161.5 (C-2), 153.2 (C-7),
150.0 (C-9), 145.6 (C-6), 143.4 (C-4), 113.4 (C-3), 111.4
(C-10), 109.4 (C-5), 100.1 (C-8), 65.0 (–OCH2CH3), 56.4
(–OCH3), 14.7 (–OCH2CH3).
7-Ethoxy-6-methoxycoumarin (24) was prepared from
7-ethoxy-6-hydroxycoumarin (16) as described in general
procedure (y. 62 %): white needles, mp 117–118 °C.
MALDI-TOF MS m/z: 221 [M+H]+. 1H-NMR (400 MHz,
CDCl3) δ: 7.62 (1H, d, J = 9.6 Hz, H-4), 6.86 (1H, s, H-5),
6.83 (1H, s, H-8), 6.26 (1H, d, J = 9.5 Hz, H-3), 4.16 (2H,
q, J = 7.2 Hz, -OCH2CH3), 3.91 (3H, s, -OCH3), 1.52 (3H,
t, J = 7.2 Hz, –OCH2CH3),
13C-NMR (100 MHz, CDCl3)
δ: 161.5 (C-2), 152.2 (C-7), 150.0 (C-9), 146.5 (C-6), 143.4
(C-4), 113.3 (C-3), 111.3 (C-10), 108.1 (C-5), 100.7 (C-8),
64.9 (–OCH2CH3), 56.4 (–OCH3), 14.4 (–OCH2CH3).
Acknowledgments The authors are grateful to Dr. Shinyu Nunome
for the identification of crude drug materials.
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123
	Collagenase inhibitors from Viola yedoensis
	Abstract
	Introduction
	Results and discussion
	Experimental procedure
	General
	Plant material
	Collagenase inhibition assay
	Extraction and isolation
	Preliminary test
	Fractionation of acetone extract
	Fractionation of MeOH extract
	Fractionation of CHCl3–MeOH extract
	Preparation of coumarin derivatives
	Alkylation (general procedure)
	Acknowledgments
	References