Hyaluronidase inhibitory rosmarinic acid derivatives from Meehania urticifolia

Article abstract of DOI:10.1248/cpb.59.88

Nine new phenylpropanoids, rashomonic acids A-D (1-4) and meehaniosides A-E (5-9), along with four known compounds were isolated from Meehania urticifolia. The structure of each new compound was elucidated based on the results of spectroscopic analyses. Compounds 3-8 showed moderate hyaluronidase inhibitory activity with IC₅₀ values of 183-1049 μM.

Full text of DOI:10.1248/cpb.59.88

88
Regular Article
Chem. Pharm. Bull. 59(1) 88—95 (2011)
Vol. 59, No. 1
Hyaluronidase Inhibitory Rosmarinic Acid Derivatives from Meehania
urticifolia
a
Toshihiro MURATA, ,a Toshio MIYASE,b and Fumihiko YOSHIZAKI
*
a Department of Pharmacognosy, Tohoku Pharmaceutical University; 4–4–1 Komatsushima, Aoba-ku, Sendai 981–8558,
Japan: and b School of Pharmaceutical Sciences, University of Shizuoka; 52–1 Yada, Suruga-ku, Shizuoka 422–8526,
Japan. Received September 9, 2010; accepted October 3, 2010; published online October 8, 2010
Nine new phenylpropanoids, rashomonic acids A—D (1—4) and meehaniosides A—E (5—9), along with
four known compounds were isolated from Meehania urticifolia. The structure of each new compound was eluci-
dated based on the results of spectroscopic analyses. Compounds 3—8 showed moderate hyaluronidase in-
hibitory activity with IC50 values of 183—1049 mM.
Key words phenylpropanoid; rosmarinic acid; hyaluronidase inhibitor; Meehania urticifolia; Lamiaceae
Meehania urticifolia (MIQ.) MAKINO belongs to the family 134.8); and H-8ꢁ (d 4.98, dd, Jꢀ7.5, 7.5 Hz) was correlated
Lamiaceae. In the course of studying compounds from plants with C-9 (d 173.2). The absolute configurations of C-7 and
of the genus Meehania, we have identified spermidine alka- 8ꢂ were determined as 7R and 8ꢂR from the retention time of
loidal glycosides,1,2) spermidine alkaloids,3) and flavonoid the hydrolytic product of 1 and synthesized 1a (7R,8ꢁR) and
glycosides.3) Many phenylpropanoid oligomers and their de- 2a (7S,8ꢁR). Both 1 and 1a showed positive Cotton effects
rivatives, such as rosmarinic acid, have been found in Lami- around 240 nm in their circular dichroism (CD) spectra,
aceae plants.4) In this paper, four new phenylpropanoids (1— which supported this conclusion. The absolute configuration
4), and five new complexes of phenylpropanoid and of C-8ꢁ was determined to be R from the retention time of
phenylethanoid glycoside (5—9) were isolated along with the amide derivative of 3-(3,4-dihydroxyphenyl)-2-hydroxy-
rosmarinic acid (10), lithospermic acid B (11), conandroside propanoic acid, which was obtained by acidic hydrolysis of 1,
(12), and vervascoside (13) from M. urticifolia. Compound in (S)-2-phenylglycine methyl ester.6) These results suggested
105) and some of its derivatives3,6) have been recognized as that the structure of 1 was as shown in Fig. 1.
hyaluronidase inhibitors, and 3—8 also showed inhibitory
Rashomonic acid B (2) was concluded to have the molecu-
activity. M. fargesii, which have been used to traditional lar formula C27H26O13 based on HR-FAB-MS (m/z 557.1306,
1
Chinese medicine, have hyaluronidase inhibitory phenyl- Calcd for C27H25O13, 557.1295). Its H- and 13C-NMR spec-
propanoids as well.3)
tra were similar to those of 1. The NOE and HMBC spectra
The methanol extract of whole specimens of M. urticifolia suggested that 1 and 2 have the same planar configuration.
were dissolved in water and partitioned using ether. The After the acidic hydrolysis of 2, the absolute configurations
water layer was fractionated by multistep column chromatog- of C-7, 8ꢁ and 8ꢂ were determined as 7S, 8ꢁR and 8ꢂR from
raphy, and then 1—9 were isolated, as pale yellowish amor- the retention times of HPLC as in the case of 1 and a nega-
phous powders. Known compounds were also isolated and tive Cotton effect at 240 nm in the CD spectrum. The results
identified from spectroscopic data as references (10,6) 11,3) suggested the structure of 2 to be as shown in Fig. 1.
1
12,7) 138)).
The H- and 13C-NMR spectra of 3 and 4 were similar to
Rashomonic acid A (1) was concluded to have the molecu- those of 1 and 2, respectively. They also suggested that 3 and
lar formula C27H26O13 based on high resolution (HR)-FAB- 4 had another phenylpropanoid moiety. For 3, the molecular
MS (m/z 581.1255, Calcd for C27H26O13Na, 581.1270). The formula C36H32O16 was confirmed on the basis of HR-FAB-
1
1H-NMR and H–1H correlation spectroscopy (COSY) spec- MS (m/z 721.1743, Calcd for C36H33O16, 721.1768). In the
tra of 1 showed the presence of two sets of ABX spin system HMBC spectrum, a methine proton at d 5.26 (1H, dd, Jꢀ9.0,
protons and two singlet aromatic protons in the aromatic pro- 4.5 Hz, H-8ꢂ) was found to be correlated with a carboxyl car-
ton region, and three sets of methine–methylene protons in bon at d 168.8 (C-9ꢃ). The absolute configurations of C-7, 8ꢁ
the aliphatic proton region (Table 1). The 13C-NMR spectrum and 8ꢂ were determined to be 7R, 8ꢁR and 8ꢂR from the re-
of 1 showed the presence of three carboxyl carbons. In the tention times of HPLC as in the case of 1. The CD spectrum
nuclear Overhauser effect (NOE) spectra, a methine proton at was similar to that of 1, with positive Cotton effects of
d 4.64 (dd, Jꢀ8.0, 8.0 Hz, H-7) was found to be correlated around 240 nm. The structure of 3 was as shown in Fig. 1.
with aromatic protons at d 6.60 (d, Jꢀ2.0 Hz, H-2) and 6.45 For 4, the molecular formula C36H32O16 was confirmed on
(dd, Jꢀ8.0, 2.0 Hz, H-6), a methylene proton at d 2.91 (m, the basis of HR-FAB-MS (m/z 721.1786, Calcd for
H-7ꢁ) was correlated with protons at d 6.72 (d, Jꢀ2.0 Hz, H- C36H33O16, 721.1768). The absolute configurations of C-7, 8ꢁ
2ꢁ) and 6.55 (dd, Jꢀ8.0, 2.0 Hz, H-6ꢁ), and methylene pro- and 8ꢂ were determined as 7S, 8ꢁR and 8ꢂR from the retention
tons at d 2.65 (dd, Jꢀ14.0, 8.5 Hz, H-7ꢂ) and 3.15 (dd, times of HPLC as in the case of 2. The CD spectrum was
Jꢀ14.0, 4.5 Hz, H-7ꢂ) were correlated with the aromatic sin- similar to that of 2, with negative Cotton effects at around
glet proton (d 6.68, H-2). These results showed that 1 has 240 nm. The results suggested the structure of 4 to be as
three phenylpropanoid moieties. In the heteronuclear multi- shown in Fig. 1.
ple bond correlation (HMBC) spectrum, H-7 was found to be
Compound 5 showed a protonated ion peak at m/z
correlated with C-1ꢂ (d 128.1), C-5ꢂ (d 115.3), and C-6ꢂ (d 839.2387 in the HR-FAB-MS analysis, which indicated the
* To whom correspondence should be addressed. e-mail: murata-t@tohoku-pharm.ac.jp
© 2011 Pharmaceutical Society of Japan

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the HMBC spectrum, the H-Glc-3 proton signal at d 3.85
(1H, dd, Jꢀ9.5, 9.0 Hz) was found to be correlated with an
anomeric carbon of the xylose moiety at d 106.9. A sugar
analysis and the coupling constant of the anomeric proton (d
4.46, 1H, d, Jꢀ7.5 Hz) showed the presence of a b-D-xylose
unit. The absolute configurations of C-7 and C-8ꢁ were deter-
mined as 7R and 8ꢁR by HPLC and the CD spectrum as in
the case of 5. These results suggested that the structure of 7
was as shown in Fig. 1. Compound 8 was a diastereomer of
1
7. The H- and 13C-NMR spectra of 8 were similar to those
of 7. The absolute configuration of C-7 was concluded to be
S as in the case of 7. In the CD spectrum, a negative Cotton
effect around at 240 nm was detected. The structure of 8 is
shown in Fig. 1.
1
The H- and 13C-NMR spectra of 9 were similar to those
of 5. The molecular formula C32H36O16 was established by
HR-FAB-MS (m/z 699.1900, Calcd for C32H36O16Na,
699.1900), which was C9H6O3 less than that of 5, indicating
the absence of a caffeoyl moiety. The absolute configurations
of C-7 and C-8ꢁ were concluded to be 7R and 8ꢁR by the
Fig. 1. Structures of 1—9
molecular formula C41H42O19. The 1H-NMR and 1H–1H HPLC analyses and CD spectrum as in the case of 5. The
COSY spectra of 5 showed the presence of three sets of ABX structure of 9 is shown in Fig. 1.
spin system protons, two singlet aromatic protons, two sets
The absolute stereochemistry of C-7 in 1a, 2a, 5a, and 6a
of methine–methylene protons, and a set of ethylene protons was investigated by a modified version of the Mosher method
(Table 2). The ethylene protons at d 2.82 (2H, br t, Jꢀ8.0 Hz, for carboxylic acids having a chiral center at the b-position
H-7ꢂ), 3.37 (1H, m, H-8ꢂ), and 3.87 (1H, m, H-8ꢂ) suggested (Fig. 2).10) 1b and 2b were (R)-phenylglycine esters of 1a and
the presence of a phenylethanoid moiety. The H-7ꢂ proton 2a, and 1c and 2c were (S)-phenylglycine esters of 1a and 2a,
1
was correlated with a singlet proton at d 6.65, and another respectively. The H-NMR chemical shift difference [Dd
singlet proton at d 6.71 correlated with H-7 (d 4.47, 1H, (ppm)ꢀd 1bꢄd 1c] suggested that the absolute configuration
br dd, Jꢀ8.0, 7.5 Hz) in the NOE spectra. Two sets of ABX of C-7 in 1a was R (Table 3). However, chemical shift differ-
proton system protons and aliphatic protons including H-7 ences [Dd (ppm)ꢀd2bꢄd2c] for C-7 in 2a were not applica-
showed the presence of a C-7 substituted rosmarinic acid ble to this method (Table 3). The configuration of the C-7 in
moiety. An anomeric proton signal at d 4.24 (1H, d, 2a was established as S, in the CD spectrum of 2a by com-
Jꢀ7.5 Hz) and oxygenated carbons at d 104.3, 75.3, 75.8, parison to that of 1a which has an opposite curve. 5b and 6b
72.5, 75.9, and 62.3 suggested the presence of a glucose moi- were (R)-phenylglycine methylester (PGME) esters of 5a and
ety. A sugar analysis and the coupling constant of the 6a, and 5c and 6c were (S)-PGME esters of 5a and 6a, re-
anomeric proton showed the presence of a b-D-glucose unit.9) spectively. The 1H-NMR chemical shift differences [Dd
In the HMBC spectrum, a H-Glc-4 proton signal at d 4.86 (ppm)ꢀd5bꢄd5c] suggested that the absolute configuration
(1H, dd, Jꢀ9.5, 9.5 Hz) was found to be correlated with
carboxyl carbon of the caffeic acid moiety at d 168.7 (C-9ꢃ). that 6a had the S-configuration (Table 3).
The absolute configuration of C-7 was concluded to be R
a
of C-7 in 5a was R, and [Dd (ppm)ꢀd6bꢄd6c] suggested
Hyaluronidase inhibitory activity was measured for com-
from the retention time of the hydrolytic product of 5 and pounds 1—8, 10—13, 1a, and 2a as shown in Table 4. Dis-
synthesized 5a (7R) and 6a (7S). Both 5 and 5a showed posi- odium cromoglycate (DSCG, Wako Pure Chemical Indus-
tive Cotton effects at around 240 nm in their CD spectra, tries Ltd., Osaka, Japan; IC50 297 mM) was used as a positive
which supported this conclusion. The absolute configuration control. Rosmarinic acid dimers showed levels of activity
of C-8ꢁ was determined to be R as in the case of 1, in (S)-2- (IC50 3: 275 mM, 4: 183 mM) comparable to rosmarinic acid
phenylglycine methyl ester.6) These results suggested the (IC50 309 mM), and phenylpropanoid monomers, caffeic acid
structure of 5 to be as shown in Fig. 1.
and 3-(3,4)-dihydroxyphenyl)-2-hydroxy propanoic acid, had
Compound 6 was a diastereomer of 5. The molecular for- no activity. Although phenylethanoid glycosides (12, 13) did
mula C41H42O19 was confirmed on the basis of HR-FAB-MS not show inhibitory activity, complexes of rosmarinic acid
(m/z 839.2410, Calcd for C41H43O19, 839.2398). The 1H- and and phenylethanoid glycosides (5—8) showed moderate lev-
13C-NMR spectra of 6 were similar to those of 5. The ab- els of activity (IC50 1049, 873, 924, 781 mM, respectively).
solute configuration of C-7 was determined to be S as in the Compounds 1a and 2a had no activity. These results sug-
case of 5. In the CD spectrum, a negative Cotton effect at gested that the 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic
around 240 nm was detected. The structure of 6 is shown in acid moiety of oligomers was important to the hyaluronidase
Fig. 1.
inhibitory activity.
1
The H- and 13C-NMR spectra of 7 and 8 were similar to
those of 5 and 6, respectively. However, they showed that 7
and 8 had a xylose moiety. The molecular formula C46H50O23
was revealed based on HR-FAB-MS [m/z 971.2817 (7) and
Experimental
General Procedures Optical rotations were recorded on a Jasco P-2300
polarimeter. CD spectra were recorded on a Jasco J-700 spectropolarimeter;
and UV, on a Shimadzu MPS-2450. H- (400 MHz), 13C-NMR (100 MHz),
1
m/z 971.2813 (8), Calcd for C46H51O23, 971.2820]. For 7, in 1H–1H COSY, heteronuclear multiple quantum correlation (HMQC) (opti-

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50C-M, Yamazen, 37ꢆ100 mm; detector, UV at 210 nm). Preparative HPLC
was performed on a Jasco 2089 and detected with UV at 210 nm (columns,
ODS-100V, Tosoh, 20ꢆ250 mm; Capcell-Pak Ph, Shiseido, 20ꢆ250 mm;
Cosmosil AR-II, Nacalai Tesque, 20ꢆ250 mm; Cosmosil 5PE-MS, Nacalai
tesque, 20ꢆ250 mm).
Plant Material Meehania urticifolia was collected in July 2007 in
Sendai, Japan. The plant was identified by Dr. Koji Yonekura, Tohoku Uni-
versity, Sendai, Japan. A voucher specimen has been deposited in the
herbarium of Tohoku Pharmaceutical University, No. 20070727.
Extraction and Isolation Powdered whole specimens (760 g) of M. ur-
ticifolia were extracted with methanol (12 l) twice at room temperature for a
month. The methanol extract was concentrated at reduced pressure, sus-
pended in water (1.5 l), and subjected to extraction with ether (1.0 l) three
times. The aqueous layer (98.5 g) was dissolved in water and the solution
was passed through a porous polymer gel (Mitsubishi Diaion HP-20,
70ꢆ180 mm) eluted with water, 10%, 45%, and 90% MeOH and MeOH.
The 45% MeOH eluate (5.1 g) was chromatographed on a reversed-phase
column using ODS (Cosmosil 140C18-OPN, Nacalai Tesque, 150 g) eluting
with 10%, 20%, 30%, 40%, 50% and MeOH (fractions 1A—F). Fraction 1C
(1.3 g) was subjected to preparative LPLC [solvent, methanol–0.2% trifluo-
roacetic acid (TFA) (35 : 65)], to give 10 fractions (Frs. 2A—J). Fractions 2B
and 2C (220.6 mg) were subjected to preparative HPLC [column, Shiseido,
Tokyo, Japan, Capcell-Pak Ph; solvent, acetonitrile–0.2% TFA (12.5 : 87.5)]
to yield compounds 1 (7.7 mg), 2 (4.8 mg), and 9 (1.4 mg). Fractions 2D, 2E,
2F, and 2G (327.1 mg) were subjected to preparative HPLC [column, ODS-
100V; solvent, acetonitrile–water (25 : 75)], [column, Shiseido, Capcell-Pak
Ph; solvent, acetonitrile–0.2% TFA (20 : 80)] to yield compounds 3
(13.0 mg), 4 (5.7 mg), 5 (5.1 mg), 6 (6.5 mg), 7 (10.6 mg), 8 (8.7 mg), 12
(10.0 mg), and 13 (5.1 mg). Fraction 2J (446.1 mg) was subjected to prepara-
tive HPLC [column, ODS-100V; solvent, acetonitrile–0.2% TFA (25 : 75)] to
yield compounds 10 (52.4 mg) and 11 (73.5 mg).Rashomonic acid A (1):
Colorless amorphous powder; [a]D22 ꢄ17.6 (cꢀ0.74, MeOH); UV (MeOH)
lmax (log e) 285 (4.58); CD (cꢀ0.037, MeOH) l(q) 210 (ꢄ48600), 242
1
(17500), 294 (ꢄ1700) nm; H- and 13C-NMR, Table 1; HR-FAB-MS m/z
581.1255 [MꢅNa]ꢅ (Calcd for C27H26O13Na, 581.1270).
Rashomonic acid B (2): Colorless amorphous powder; [a]D23 ꢅ5.0
(cꢀ0.44, MeOH); UV (MeOH) lmax (log e) 285 (4.63); CD (cꢀ0.022,
MeOH) l(q) 226 (100), 240 (ꢄ19700), 261 (4300), 276 (2100), 294 (7800)
nm; 1H- and 13C-NMR, Table 1; HR-FAB-MS m/z 557.1306 [MꢄH]ꢄ
(Calcd for C27H25O13, 557.1295).
Fig. 2. Structure of Compounds for a Modified Version of the Mosher
Method
Table 4. Hyaluronidase Inhibitory Activity of Compounds 1—8, 10—13,
1a, 2a, and DSCG
Rashomonic acid C (3): Colorless amorphous powder; [a]D19 ꢅ27.7
(cꢀ1.14, MeOH); UV (MeOH) lmax (log e) 209 (5.67), 288 (5.19), 331
(5.05); CD (cꢀ0.057, MeOH) l(q) 242 (22900), 284 (100), 303 (6300) nm;
1H- and 13C-NMR, Table 1; HR-FAB-MS m/z 721.1743 [MꢅH]ꢅ (Calcd for
C36H33O16, 721.1768).
Compound
IC50 (mM)
1
2
3
N.D.a)
N.D.
275
Rashomonic acid D (4): Colorless amorphous powder; [a]D21 ꢅ43.1
(cꢀ0.58, MeOH); UV (MeOH) lmax (log e) 203 (5.92), 288 (5.22), 333
(5.08); CD (cꢀ0.029, MeOH) l(q) 223 (ꢄ800), 239 (ꢄ28500), 257 (3800),
4
183
1
278 (100), 296 (13000) nm; H- and 13C-NMR, Table 1; HR-FAB-MS m/z
5
6
1049
873
721.1786 [MꢅH]ꢅ (Calcd for C36H33O16, 721.1768).
Meehanioside A (5): Colorless amorphous powder; [a]D21 ꢄ13.2 (cꢀ0.44,
MeOH); UV (MeOH) lmax (log e) 205 (5.83), 288 (5.22), 331 (5.10); CD
(cꢀ0.029, MeOH) l(q) 211 (ꢄ48400), 242 (16000), 287 (ꢄ800), 304
(4200) nm; 1H- and 13C-NMR, Table 2; HR-FAB-MS m/z 839.2387
[MꢅH]ꢅ (Calcd for C41H43O19, 839.2398).
7
8
10
11
12
13
924
781
309b)
164b)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
297b)
Meehanioside B (6): Colorless amorphous powder; [a]D22 ꢄ4.9 (cꢀ0.53,
MeOH); UV (MeOH) lmax (log e) 208 (5.76), 289 (5.20), 331 (5.09); CD
(cꢀ0.053, MeOH) l(q) 213 (ꢄ9600), 226 (ꢄ2700), 238 (ꢄ12000), 255
(3400), 276 (600), 295 (4800) nm; 1H- and 13C-NMR, Table 2; HR-FAB-MS
m/z 839.2410 [MꢅH]ꢅ (Calcd for C41H43O19, 839.2398).
Caffeic acid
3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoic acid
1a
2a
DSCG
Meehanioside C (7): Colorless amorphous powder; [a]D20 ꢄ23.3 (cꢀ0.97,
MeOH); UV (MeOH) lmax (log e) 205 (5.77), 289 (5.14), 330 (5.03); CD
(cꢀ0.057, MeOH) l(q) 209 (ꢄ61300), 241 (14500), 288 (ꢄ1900), 303
(2400) nm; 1H- and 13C-NMR, Table 2; HR-FAB-MS m/z 971.2817
[MꢅH]ꢅ (Calcd for C46H51O23, 971.2820).
a) Not determined. b) Previously reported value.3)
mized for 1JC–Hꢀ145 Hz) and HMBC (optimized for nJC–Hꢀ8 Hz) spectra
were recorded on a Jeol JNM-AL400 FT-NMR spectrometer, and chemical
shifts were given as d values with tetramethylsilane (TMS) as an internal
standard. HR-FAB- and electron ionization-mass spectra (EI-MS) data were
obtained on a Jeol JMS700 mass spectrometer, using a m-nitrobenzyl alco-
hol or a glycerol matrix. A porous polymer gel (Mitsubishi Chemical, Di-
aion HP-20, 60ꢆ300 mm) and octadecyl silica (ODS) (Cosmosil 140 C18-
OPN, Nacalai Tesque, Kyoto, Japan, 150 g) were used for column chro-
matography. Preparative Yamazen Cartridge Column Chromatography
(YCCC) was performed on a Jasco 2089 (column, Ultra Pack ODS-SM-
Meehanioside D (8): Colorless amorphous powder; [a]D21 ꢄ17.7 (cꢀ0.79,
MeOH); UV (MeOH) lmax (log e) 203 (5.91), 289 (5.21), 330 (5.11); CD
(cꢀ0.040, MeOH) l(q) 215 (ꢄ9200), 225 (ꢄ1600), 239 (ꢄ14200), 262
(3500), 276 (700), 294 (5300) nm; 1H- and 13C-NMR, Table 2; HR-FAB-MS
m/z 971.2813 [MꢅH]ꢅ (Calcd for C46H51O23, 971.2820).
Meehanioside E (9): Colorless amorphous powder; [a]D21 ꢄ26.7 (cꢀ0.12,
MeOH); UV (MeOH) lmax (log e) 286 (5.00), 329 (4.70); CD (cꢀ0.012,
1
MeOH) l(q) 207 (ꢄ75600), 240 (23600) nm; H- and 13C-NMR, Table 2;
HR-FAB-MS m/z 699.1900 [MꢅNa]ꢅ (Calcd for C32H36O16Na, 699.1900).

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(R)-Phenylglycine Methyl Ester (PGME) Amide of Caffeic Acid: Color-
(60 mg) were dissolved in dioxan (10 ml) and added to p-toluenesulfonic
acid, then stirred for 3 h at 100 °C. After cooling, the reaction mixture was
concentrated and subjected to preparative HPLC [column, Capcell-Pak Ph;
less amorphous powder; [a]D19 ꢄ62.0 (cꢀ0.71, MeOH); 1H-NMR
(methanol-d4, 400 MHz) d: 7.02 (1H, d, Jꢀ2.0 Hz, H-2), 6.76 (1H, d,
Jꢀ8.5 Hz, H-5), 6.92 (1H, dd, Jꢀ8.5, 2.0 Hz, H-6), 7.43 (1H, d, Jꢀ16.0 Hz, solvent, acetonitrile–0.2% TFA (10 : 90)] to yield compounds 1a (6.8 mg)
H-7), 6.52 (1H, d, Jꢀ16.0 Hz), 5.59 (1H, s, CH), 7.30—7.45 (5H, m, and 2a (5.1 mg). The retention time of 1a was 8.40 min and that of 2a was
phenyl), 3.72 (3H, s, OMe); 13C-NMR (methanol-d4, 100 MHz) d: 138.2 (C-
1), 125.2 (C-2), 156.7 (C-3), 158.9 (C-4), 126.4 (C-5), 132.3 (C-6), 153.3
(C-7), 127.6 (C-8), 178.8 (C-9), 68.4 (CH), 182.7 (CꢀO), 138.8, 139.5,
139.9, 147.5 (phenyl), 62.9 (OMe); EI-MS m/z 327.
7.75 min [the analytical HPLC was performed on a Cosmosil AR-II column
(4.6ꢆ250 mm) using acetonitrile–0.2% TFA in water (10 : 90) as the solvent
(flow rate, 1 ml/min; detector, UV 210 nm)]. To caffeic acid (160 mg) in
DMF (6 ml) was added (R)-PGME (200 mg) or (S)-PGME (200 mg), and
then HOBt (200 mg), and N-methylmorpholine (300 ml) were added and the
mixture was stirred for 10 h at room temperature. The reactions gave the (R)-
PGME-amide of caffeic acid (53.2 mg) and (S)-PGME-amide of caffeic acid
(S)-PGME Amide of Caffeic Acid: Colorless amorphous powder; [a]D20
ꢅ50.9 (cꢀ1.93, MeOH); 1H- and 13C-NMR, identical with (R)-PGME
amide of caffeic acid; EI-MS m/z 327.
1a: Colorless amorphous powder; [a]D21 ꢄ30.5 (cꢀ1.24, MeOH); UV (49.6 mg). The (R)-PGME-amide of caffeic acid (15 mg) and 3-(3,4-dihy-
(MeOH) lmax (log e) 205 (5.66), 287 (4.78); CD (cꢀ0.012, MeOH) l(q) droxyphenyl)-2-hydroxypropanoic acid (15 mg) were dissolved in 7% HCl
1
206 (ꢄ95900), 241 (40800), 293 (ꢄ3400) nm; H- and 13C-NMR, Table 3;
(5 ml) and stirred for 1.5 h at 60 °C. After cooling, the reaction mixture was
concentrated and subjected to preparative HPLC [column, Capcell-Pak Ph;
EI-MS m/z 378.
(R)-Phenylglycine Amide of 1a (1b): Colorless amorphous powder; [a]D20 solvent, acetonitrile–0.2% TFA (10 : 90)] to yield compounds 1b (2.8 mg)
ꢄ41.9 (cꢀ0.43, MeOH); 1H-NMR, Table 3; FAB-MS m/z 513 [MꢅH]ꢅ.
(S)-Phenylglycine Amide of 1a (1c): Colorless amorphous powder; [a]D20 dihydroxyphenyl)-2-hydroxypropanoic acid (30 mg) were dissolved in 7%
and 2b (2.9 mg). The (S)-PGME-amide of caffeic acid (30 mg) and 3-(3,4-
ꢅ10.3 (cꢀ0.58, MeOH); 1H-NMR, Table 3; FAB-MS m/z 513 [MꢅH]ꢅ.
HCl (5 ml) and stirred for 1.5 h at 60 °C. After cooling, the reaction mixture
2a: Colorless amorphous powder; [a]D21 ꢅ31.8 (cꢀ1.39, MeOH); UV was concentrated and subjected to preparative HPLC [column, Capcell-Pak
(MeOH) lmax (log e) 206 (5.66), 287 (4.79); CD (cꢀ0.014, MeOH) l(q) Ph; solvent, acetonitrile–0.2% TFA (10 : 90)], yielding 1c (6.0 mg) and 2c
1
205 (13500), 240 (ꢄ34200), 294 (10100) nm; H- and 13C-NMR, Table 3;
(4.6 mg). Acid hydrolysis (7% HCl, 3 h, 100 °C) of 1b and 1c gave 1a. Acid
hydrolysis (7% HCl, 3 h, 100 °C) of 2b and 2c gave 2a.
EI-MS m/z 378.
(R)-Phenylglycine Amide of 2a (2b): Colorless amorphous powder; [a]D20
ꢄ12.6 (cꢀ0.57, MeOH); 1H-NMR, Table 3; FAB-MS m/z 513 [MꢅH]ꢅ.
(S)-Phenylglycine Amide of 2a (2c): Colorless amorphous powder; [a]D20
ꢅ37.4 (cꢀ0.46, MeOH); 1H-NMR, Table 3; FAB-MS m/z 513 [MꢅH]ꢅ.
Determination of the Stereochemistry of 5a and 6a 2-(3,4-Dihydroxy-
phenyl) ethyl alcohol (200 mg) (Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan) and caffeic acid (150 mg) were dissolved in 7% HCl (5 ml) and
stirred for 3 h at 80 °C. After cooling, the reaction mixture was concentrated
5a: Colorless amorphous powder; [a]D20 ꢄ44.3 (cꢀ0.14, MeOH); UV and subjected to preparative HPLC [column, Capcell-Pak Ph; solvent, ace-
(MeOH) lmax (log e) 208 (5.47), 287 (4.78); CD (cꢀ0.028, MeOH) l(q) tonitrile–0.2% TFA (10 : 90)] to yield a mixture of 5a and 6a (59.8 mg). To
1
207 (ꢄ54300), 240 (26400), 293 (ꢄ4700) nm; H- and 13C-NMR, Table 3; the mixture of 5a and 6a (27.2 mg) in DMF (3 ml) were added (R)-PGME
EI-MS m/z 334.
(R)-PGME Amide of 5a (5b): Colorless amorphous powder; [a]D20 ꢄ55.5 stirred for 10 h at room temperature. The solution was concentrated and sub-
(cꢀ0.71, MeOH); 1H-NMR, Table 3; EI-MS m/z 481.
jected to preparative HPLC [columns, Cosmosil AR-II and Cosmosil 5PE-
(S)-PGME Amide of 5a (5c): Colorless amorphous powder; [a]D20 ꢄ14.3 MS; solvent, acetonitrile–0.2% TFA (25 : 75)] to yield (R)-PGME-amide of
(cꢀ0.70, MeOH); 1H-NMR, Table 3; EI-MS m/z 481.
5a (5b, 11.4 mg) and (R)-PGME-amide of 6a (6b, 10.0 mg). To the mixture
6a: Colorless amorphous powder; [a]D20 ꢅ41.8 (cꢀ0.11, MeOH); UV of 5a and 6a (32.6 mg) in DMF (3 ml) were added (S)-PGME (60 mg),
(60 mg), HOBt (30 mg), and N-methylmorpholine (100 ml) and the solution
(MeOH) lmax (log e) 207 (5.52), 287 (4.75); CD (cꢀ0.022, MeOH) l(q)
205 (91000), 240 (ꢄ18700), 292 (7600) nm; 1H- and 13C-NMR, Table 3; EI-
MS m/z 334.
HOBt (30 mg), and N-methylmorpholine (100 ml) and the mixture stirred for
10 h at room temperature. The solution was concentrated and subjected to
preparative HPLC [columns, Cosmosil AR-II and Cosmosil 5PE-MS; sol-
(R)-PGME Amide of 6a (6b): Colorless amorphous powder; [a]D20 ꢅ56.1 vent, acetonitrile–0.2% TFA (25 : 75)] to yield (S)-PGME-amide of 5a (5c,
(cꢀ0.82, MeOH); 1H-NMR, Table 3; EI-MS m/z 481.
11.5 mg), and (S)-PGME-amide of 6a (6c, 12.7 mg). Acid hydrolysis (7%
HCl, 3 h, 100 °C) of 5b and 5c gave 5a (1.1 mg). Acid hydrolysis (7% HCl,
3 h, 100 °C) of 6b and 6c gave 6a (0.9 mg).
(S)-PGME Amide of 6a (6c): Colorless amorphous powder; [a]D20 ꢅ13.5
(cꢀ0.65, MeOH); 1H-NMR, Table 3; FAB-MS m/z 482 [MꢅH]ꢅ.
Acidic Hydrolysis of Compounds 1—9 Each compound [1 (2.6 mg), 2
(1.0 mg), 3 (2.6 mg), 4 (1.1 mg), 5 (2.5 mg), 6 (1.3 mg), 7 (5.4 mg), 8
(1.5 mg), and 9 (0.4 mg)] was dissolved in 7% HCl (1 ml) and stirred for 2 h
at 60 °C. After concentration, the residue of 1 and 3 was subjected to prepar-
ative HPLC [column, Capcell-Pak Ph; solvent, acetonitrile–0.2% TFA
(10 : 90)] to yield 1a and 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid.
The residue of compounds 2 and 4 was subjected to preparative HPLC [col-
umn, Capcell-Pak Ph; solvent, acetonitrile–0.2% TFA (10 : 90)], yielding 2a
and 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid. The residue of com-
pounds 5—9 was subjected to preparative HPLC [column, Capcell-Pak Ph;
solvent, acetonitrile–0.2% TFA (10 : 90)] to yield 5a or 6a, 3-(3,4-dihydroxy-
phenyl)-2-hydroxypropanoic acid, and a sugar fraction.
CD Spectra of the Products of Acidic Hydrolysis of 5—9, 5a, and 6a
Each complex of caffeic acid and 3-(3,4-dihydroxyphenyl)-2-hydroxy-
propanoic acid obtained by the acid hydrolysis of compounds 5, 7, and 9
showed a positive Cotton effect at 240 nm, identical to 5a. Each complex of
caffeic acid and 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid obtained
by acid hydrolysis of 6 and 8 showed a negative Cotton effect at 240 nm,
identical to 6a.
Sugar Identification Sugar fractions from 5—9 were dissolved in pyri-
dine (each 0.5 ml) and stirred with L-cysteine methyl ester (5 mg) before o-
tolyl isothiocyanate (20 ml) was added to the mixture using the same proce-
dures as in our previous report.1) The reaction mixtures were analyzed by
HPLC and detected at 250 nm. Analytical HPLC was performed on a Cos-
(S)-PGME and (R)-PGME Esters of 3-(3,4-Dihydroxyphenyl)-2-hy- mosil AR-II column (4.6ꢆ250 mm) at 25 °C using CH3CN–0.2% TFA in
droxypropanoic Acid To 2R-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic
acid (5 mg, each) obtained from rosmarinic acid6) in N,N-dimethylform-
amide (DMF) (1.0 ml) was added (S)-PGME or (R)-PGME (10 mg), and
then benzotriazol-1-yl-oxy-tris-pyrrolidinophonium hexafluorophosphate
(PyBOP) (15 mg), 1-hydroxybenzotriazole (HOBt) (5 mg), and N-methyl-
morpholine (20 ml) were added and the mixture was stirred for 10 h at room
H2O (25 : 75) as the solvent. Peaks were detected with a Tosoh UV8010 de-
tector. D-Glucose (tR 15.5 min) and D-xylose (tR 17.7 min) were identified as
the sugar moieties of 5—9 (5, 6, 9 were only D-glucose) by comparing their
retention times with those of authentic samples of D-glucose (tR 15.5 min), L-
glucose (tR 14.1 min), D-xylose (tR 17.7 min), and L-xylose (tR 16.5 min).10)
Assay of Hyaluronidase Inhibition The assay was carried out accord-
temperature. The reactions gave (S)-amide and (R)-amide.6) The retention ing to the Morgan–Elson method, which was modified by Davidson and
time of (S)-amide was 14.9 min and that of (R)-amide was 15.4 min. The an- Aronson.5,11,12) Each compound (final concentration: 1, 0.3, 0.1, 0.03 mM)
alytical HPLC was performed on a Shiseido Capcell Pak Ph column
(4.6ꢆ250 mm) using acetonitrile–0.2% TFA in water (15 : 85) as the solvent
was dissolved in 0.1 M acetate buffer as the sample solution. Hyaluronidase
activity was measured as described previously.3,6) DSCG was used as a posi-
(flow rate, 1 ml/min; detector, UV 210 nm). The retention time of (S)-PGME tive control. The final concentration of hyaluronidase was 400 unit/ml.
esters of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid obtained from
the acid hydrolysis of 1—9 was 14.9 min.
Determination of the Stereochemistry of 1a and 2a Acid hydrolysis
of rosmarinic acid (11) (300 mg) gave 3-(3,4-dihydroxyphenyl)-2-hydroxy-
Acknowledgments We thank Mr. S. Sato and Mr. T. Matsuki, Tohoku
Pharmaceutical University for assisting with the MS measurements and Mr.
H. Hayasaka and K. Ohba of the Department of Experimental Station for
propanoic acid (72.8 mg), and compounds 1a (11.9 mg) and 2a (13.4 mg). 3- Medicinal Plant Studies, Tohoku University, for supplying the plant mate-
(3,4-Dihydroxyphenyl)-2-hydroxypropanoic acid (20 mg) and caffeic acid rial.

January 2011
95
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