Antimicrobial activity of stereoisomers of morinols A and B, tetrahydropyran sesquineolignans

Article abstract of DOI:10.1271/bbb.80536

The antimicrobial activity of all stereoisomers of morinols A and B was tested. All stereoisomers of morinols A and B showed antifungal activity against Alternaria alternata, especially (-)-morinol B which showed the strongest activity. The natural compon

Full text of DOI:10.1271/bbb.80536

Biosci. Biotechnol. Biochem., 73 (1), 129–133, 2009
Antimicrobial Activity of Stereoisomers of Morinols A and B,
Tetrahydropyran Sesquineolignans
y
Koichi AKIYAMA,3 Satoshi YAMAUCHI,1;2; Masafumi MARUYAMA,1 Takuya SUGAHARA,1;2
1
Taro KISHIDA,1;2 and Yojiro KOBA
1Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan
2South Ehime Fisheries Research Center, 1289-1 Funakoshi, Ainan, Ehime 798-4292, Japan
3Integrated Center for Sciences, Tarumi Station, Ehime University,
3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan
Received August 4, 2008; Accepted October 1, 2008; Online Publication, January 7, 2009
[doi:10.1271/bbb.80536]
The antimicrobial activity of all stereoisomers of
morinols A and B was tested. All stereoisomers of
morinols A and B showed antifungal activity against
Alternaria alternata, especially (ꢀ)-morinol B which
showed the strongest activity. The natural com-
ponent, (+)-morinol A, and unnatural stereoisomer,
(7S,70S,8R,80R)-morinol B, showed antibacterial activity
against the gram-positive bacteria, Bacillus subtilis and
Listeria denitrificans.
the unique tetrahydropyran type of neolignan and shows
the important stereochemistry for this activity.
Materials and Methods
Melting point (mp) data are uncorrected. NMR data were measured
by a JNM-EX400 spectrometer, using TMS as a standard (0 ppm), MS
data were measured with a JMS-MS700V spectrometer, and optical
rotation values were evaluated with a Horiba SEPA-200 instrument.
Elemental analysis was carried out with Yanako CHN MT-5 coder.
The silica gel used was Wakogel C-300 (Wako, 200–300 mesh).
Key words: neolignan; tetrahydropyran neolignan; anti-
microbial activity
The NMR, MS data, and specific rotation values of A-1, A-2, A-7,
B-1, B-2, and B-5 have been presented in the previous work.3)
Morinols A and B have been isolated from the
Chinese medicinal herb, Morina chinensis,1,2) as enan-
tiomeric mixtures. The stereochemistry of morinols A
and B was determined by an enantioselective synthetic
study.3) The biosynthesis of lignans and neolignans as
enantiomeric mixtures has recently been reported.1,2,4)
This possibility means that an experiment on biological
activity using isolated lignans and neolignans is not
sufficient to clarify the biological activity of one
stereoisomer, and also the plants do not biosynthesis
the all stereoisomers. To examine the relationship
between the stereochemistry of lignans and neolignans
and their biological activity, all optically pure stereo-
isomers should be synthesized. Lignans and neolignans
are large groups of natural products, because many types
of bonding of the C6-C3 units have been found and each
type has many kinds of oxidation. Although the many
biological activities were have been reported for the
lignans and neolignans,5–8) the relationship between
their stereochemistry and biological activity is not clear.
Our efforts are continuing to collate a library for the
structure and stereochemistry of lignans and neolignans.
The stereospecific antifungal and antibacterial activ-
ities of tetra-substituted tetrahydrofuran lignan9,10) and
7,70-oxo-matairesinol have already been reported.11)
Morinols A and B have a unique tetrahydropyran
structure. The inhibition of cytokines has been reported
by using enantiomeric mixtures,2) although there is no
report on the biological activity of optically pure
morinols A and B. In our presented studies, the anti-
microbial activity was tested by employing all the synthe-
sized stereoisomers of morinols A and B (Table 1).
This article compares the activity of all stereoisomers of
(2S,3R,5S)-3-Allyl-2-(3,4-dimethoxyphenyl)-5-[(S)-(3,4-dimethoxy-
phenyl)(hydroxy)methyl]tetrahydropyran (2). After a reaction solution
of silyl ether 13) (0.15 g, 0.26 mmol) and n-Bu4NF (0.28 ml, 1 M in
THF, 0.28 mmol) in THF (10 ml) was stirred at room temperature for
30 min, sat. aq. NH4Cl solution and EtOAc were added. The organic
solution was separated, washed with brine, and dried (Na2SO4).
Concentration followed by silica gel column chromatography (EtOAc/
hexane = 2/1) gave alcohol 2 (86 mg, 0.20 mmol, 77%) as a colorless
20
oil, ½ꢀꢀ
+55 (c 1.3, CHCl3). 1H NMR (CDCl3) ꢁ 1.54 (1H, ddd,
D
J ¼ 14:2, 8.3, 8.3 Hz, 4-HH), 1.64 (1H, ddd, J ¼ 14:2, 5.4, 5.4 Hz,
4-HH), 1.86 (1H, m, CH2=CHCHH), 1.96 (1H, m, CH2=CHCHH),
2.02 (1H, m, CH), 2.10 (1H, m, CH), 2.22 (1H, br. s, OH), 3.86–3.88
(1H, overlapped, 6-HH), 3.86 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.88
(6H, s, OCH3), 4.05 (1H, dd, J ¼ 12:0, 6.6 Hz, 6-HH), 4.61 (1H, d,
J ¼ 8:8 Hz, ArCHOH), 4.66 (1H, d, J ¼ 4:9 Hz, 2-H), 4.74 (1H, d,
J ¼ 17:1 Hz, HHC=C), 4.79 (1H, d, J ¼ 10:3 Hz, HHC=C), 5.45 (1H,
m, CH=CH2), 6.81–6.85 (4H, m, ArH), 6.87–6.92 (2H, m, ArH);
13C NMR (CDCl3) ꢁ 27.9, 35.6, 38.7, 41.5, 55.75, 55.80, 65.4, 75.8,
79.2, 109.6, 110.5, 110.8, 111.6, 115.6, 119.2, 120.0, 132.4, 135.5,
137.0, 148.0, 148.56, 148.59, 149.0. Anal. Found: C, 69.83; H, 7.54.
20
Calcd. for C25H32O6: C, 70.12; H 7.53%. An enantiomer of 2: ½ꢀꢀ
D
ꢁ55 (c 0.8, CHCl3).
(2S,3R,5S)-3-Allyl-2-(3,4-dimethoxyphenyl)-5-[(R)-(3,4-dimethoxy-
phenyl)(triisopropylsilyloxy)methyl]tetrahydropyran (3). Compound 3
was obtained from (R)-4-benzyl-3-{(R)-2-[(R)-(3,4-dimethoxyphe-
nyl)(hydroxy)methyl]-5-hexenoyl}-2-oxazolidinone as a colorless oil
by the same synthetic method as that described in the literature,3)
20
½ꢀꢀ
+32 (c 1.2, CHCl3). 1H NMR (CDCl3) ꢁ 0.94–1.02 (21H, m,
D
iso-Pr), 1.55 (1H, m, 4-HH), 1.84 (1H, ddd, J ¼ 12:8, 7.9, 7.9 Hz,
4-HH), 1.90–1.99 (2H, m), 2.13–2.25 (2H, m), 3.49 (1H, dd, J ¼ 11:1,
11.1 Hz, 6-HH), 3.55 (1H, dd, J ¼ 11:1, 4.6 Hz, 6-HH), 3.73 (3H, s,
OCH3), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.88 (3H, s, OCH3),
4.62 (1H, d, J ¼ 5:8 Hz, 2-H), 4.67 (1H, d, J ¼ 6:1 Hz, ArCHOSi),
4.85 (1H, dd, J ¼ 17:1, 1.9 Hz, CHH=CH), 4.92 (1H, dd, J ¼ 17:1,
y
To whom correspondence should be addressed. Tel: +81-89-946-9846; Fax: +81-89-977-4364; E-mail: syamauch@agr.ehime-u.ac.jp

130
K. AKIYAMA et al.
1.9 Hz, CHH=CH), 5.67 (1H, m, CH2=CH), 6.73–6.80 (4H, m, ArH),
6.83–6.87 (2H, m, ArH); 13C NMR (CDCl3) ꢁ 12.5, 16.8, 18.0, 18.1,
27.9, 33.6, 37.5, 39.0, 44.7, 55.7, 55.8, 62.5, 77.1, 78.1, 110.0, 110.2,
110.5, 113.2, 116.0, 119.2, 122.0, 131.6, 135.5, 136.6, 148.2, 148.3,
108.7, 109.1, 110.8, 115.7, 117.3, 118.5, 134.0, 135.1, 137.5, 147.6,
148.6, 149.1. Anal. Found: C, 69.79; H, 7.57. Calcd. for C25H32O6: C,
20
70.12; H 7.53%. An enantiomer of 8: ½ꢀꢀ
+66 (c 0.4, CHCl3).
D
148.5, 148.6. Anal. Found: C, 69.59; H, 8.95. Calcd. for C34H52O6Si:
20
(2S,3R,5S)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(S)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (A-3).
C, 69.82; H, 8.96%. An enantiomer of 3: ½ꢀꢀ
ꢁ32 (c 2.3, CHCl3).
D
Method A: A reaction mixture of alkene 2 (56 mg, 0.13 mmol),
1-bromo-3,4-dimethoxybenzene (56 mg, 0.26 mmol), Et3N (56 ml,
0.40 mmol), and PdCl2(PPh3)2 (9 mg, 0.013 mmol) in DMF (0.5 ml)
was heated for 5 h at 90 ꢂC under N2 gas. After additions of H2O and
EtOAc, the organic solution was separated, washed with brine, and
dried (Na2SO4). Concentration followed by silica gel column chro-
matography (EtOAc/hexane = 1/1) gave A-3 (11 mg, 0.019 mmol,
15%) as a colorless oil. Method B: A reaction mixture of alkene 2
(40 mg, 0.093 mmol), 3,4-dimethoxyphenylboronic acid (21 mg,
0.12 mmol), Cu(OAc)2 (35 mg, 0.19 mmol), LiOAc (19 mg,
0.29 mmol), and Pd(OAc)2 (21 mg, 0.094 mmol) in DMF (0.5 ml)
was heated at 100 ꢂC for 2 h under N2 gas. After additions of EtOAc
and H2O, the mixture was filtered. The organic solution was separated,
washed with brine, and dried (Na2SO4). Concentration followed by
(2S,3R,5S)-3-Allyl-2-(3,4-dimethoxyphenyl)-5-[(R)-(3,4-dimethoxy-
phenyl)(hydroxy)methyl]tetrahydropyran (4). Benzyl alcohol 4 was
obtained from 3 by the same method as that described for 2 in 70%
20
yield as a colorless oil, ½ꢀꢀ
ꢁ40 (c 0.7, CHCl3). 1H NMR (CDCl3) ꢁ
D
1.90–2.06 (3H, m), 2.06–2.20 (3H, m), 3.49 (1H, dd, J ¼ 11:9, 4.1 Hz,
6-HH), 3.63 (1H, dd, J ¼ 11:9, 5.0 Hz, 6-HH), 3.88 (9H, s, OCH3),
3.90 (3H, s, OCH3), 4.66 (1H, d, J ¼ 3:2 Hz, 2-H), 4.71 (1H, d,
J ¼ 8:7 Hz, ArCHOH), 4.93–4.97 (2H, m, CH2=CH), 5.71 (1H, m,
CH2=CH), 6.82–6.85 (2H, m, ArH), 6.89–6.93 (4H, m, ArH);
13C NMR (CDCl3) ꢁ 26.5, 35.1, 38.6, 41.9, 55.8, 55.85, 55.88, 66.8,
75.5, 80.2, 109.3, 110.6, 110.8, 110.9, 115.9, 119.2, 119.4, 132.8,
135.9, 138.0, 148.60, 148.63, 149.1. Anal. Found: C, 69.80; H, 7.60.
20
Calcd. for C25H32O6: C, 70.12; H 7.53%. An enantiomer of 4: ½ꢀꢀ
D
+40 (c 0.4, CHCl3).
silica gel column chromatography (EtOAc/hexane = 3/1) gave A-3
20
(22 mg, 0.039 mmol, 42%) as a colorless oil, ½ꢀꢀ
+84 (c 0.55,
D
CHCl3). 1H NMR (CDCl3) ꢁ 1.55–1.70 (2H, m), 1.78 (1H, m), 1.90–
2.17 (3H, m), 3.83–3.86 (1H, overlapped, HH-6 (HH-90)), 3.83 (6H, s,
OCH3), 3.858 (6H, s, OCH3), 3.864 (6H, s, OCH3), 4.22 (1H, dd,
J ¼ 11:7, 5.4 Hz, HH-6 (HH-90)), 4.68 (1H, d, J ¼ 2:0 Hz, H-2 (H-7)),
4.74 (1H, d, J ¼ 8:8 Hz, ArCHOH, H-70), 5.59 (1H, m, ArCH=CH
(H-800)), 5.94 (1H, d, J ¼ 16:1, ArCH=CH (H-700)), 6.64–6.65 (2H, m,
ArH), 6.73 (1H, d, J ¼ 8:8 Hz, ArH), 6.78–6.93 (6H, m, ArH);
13C NMR (CDCl3) ꢁ 28.3, 34.3, 39.5, 41.1, 55.7, 55.8, 55.9, 66.3, 75.6,
79.9, 108.4, 109.8, 110.7, 111.0, 111.1, 111.2, 118.7, 119.6, 127.2,
130.4, 130.7, 132.9, 135.5, 148.1, 148.3, 148.8, 148.9, 149.2; EIMS
m=z 564 (Mþ, 84), 151 (100); HREIMS m=z 564.2723 (calcd for
C33H40O8, 564.2723).
(2R,3S,5S)-3-Allyl-2-(3,4-dimethoxyphenyl)-5-[(S)-(3,4-dimethoxy-
phenyl)(hydroxy)methyl]tetrahydropyran (6). Benzyl alcohol 6 was
obtained from silyl ether 53) by the same method as that described for 2
20
in 73% yield as a colorless oil, ½ꢀꢀ
+37 (c 1.6, CHCl3). 1H NMR
D
(CDCl3) ꢁ 1.33 (1H, ddd, J ¼ 12:8, 12.8, 4.2 Hz, 4-HH), 1.47 (1H, m,
4-HH), 1.75 (1H, m, CH2=CHCHH), 1.80–1.89 (1H, m, CH2=
CHCHH), 2.05 (1H, m, 3-H), 2.22 (1H, m, 5-H), 3.48 (1H, dd,
J ¼ 11:2, 11.2, 6-HH), 3.85 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.876
(3H, s, OCH3), 3.883 (3H, s, OCH3), 4.25 (1H, d, J ¼ 8:5 Hz,
ArCHOH), 4.50 (1H, d, J ¼ 2:2 Hz, 2-H), 4.56 (1H, m, 6-HH), 4.77
(1H, d, J ¼ 9:9 Hz, CHH=CH), 4.83 (1H, dd, J ¼ 17:0 Hz, CHH=
CH), 5.35 (1H, m, CH2=CH), 6.76–6.86 (6H, m, ArH); 13C NMR
(CDCl3) ꢁ 29.85, 29.92, 36.6, 38.5, 55.80, 55.82, 55.84, 55.9, 72.3,
81.0, 108.8, 109.2, 110.7, 110.8, 115.6, 117.3, 119.1, 134.1, 135.3,
(2R,3S,5R)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(R)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (A-4). A-4
137.3, 147.6, 148.6, 148.7, 149.1. Anal. Found: C, 69.81; H, 7.55.
20
was obtained from an enantiomer of 2. The NMR data agreed with
20
Calcd. for C25H32O6: C, 70.12; H 7.53%. An enantiomer of 6: ½ꢀꢀ
D
those for A-3. ½ꢀꢀ
ꢁ84 (c 0.34, CHCl3).
ꢁ37 (c 0.7, CHCl3).
D
(2R,3R,5S)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(R)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (A-5). A-5
(2S,3R,5R)-3-Allyl-2-(3,4-dimethoxyphenyl)-5-[(S)-(3,4-dimethoxy-
phenyl)(triisopropylsilyloxy)methyl]tetrahydropyran (7). Compound 7
was obtained as a colorless oil from (S)-4-benzyl-3-{(S)-2-[(S)-(3,4-
dimethoxyphenyl)(hydroxy)methyl]-5-hexenoyl}-2-oxazolidinone by
was synthesized by the same method as that described for A-7 in the
20
literature,3) ½ꢀꢀ
+28 (c 0.22, CHCl3). The NMR data agreed with
20
D
those for A-7.3) An enantiomer of A-5 (A-7), ½ꢀꢀ
ꢁ28 (c 0.3,
the same synthetic method as that described in the literatures,3)
D
CHCl3) in the literature.3)
20
½ꢀꢀ
ꢁ62 (c 0.7, CHCl3). 1H NMR (CDCl3) ꢁ 0.97–1.03 (21H, m,
D
iso-Pr), 1.45 (1H, ddd, J ¼ 12:6, 12.6, 4.2 Hz, 4-HH), 1.78 (1H, m,
4-HH), 1.92 (1H, m, 3-H), 2.05 (1H, m, CH2=CHCHH), 2.15 (1H, m,
CH2=CHCHH), 2.20 (1H, m, 5-H), 3.28 (1H, dd, J ¼ 11:2, 11.2 Hz,
6-HH), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.88 (6H, s, OCH3),
4.01 (1H, br. d, J ¼ 11:2 Hz, 6-HH), 4.39 (1H, d, J ¼ 0:6 Hz, 2-H),
4.51 (1H, d, J ¼ 5:7 Hz, ArCHOSi), 4.88–4.92 (2H, m, CH2=CH),
5.53 (1H, m, CH2=CH), 6.72–6.74 (2H, m, ArH), 6.78–6.82 (3H, m,
ArH), 6.86 (1H, d, J ¼ 1:8 Hz, ArH); 13C NMR (CDCl3) ꢁ 12.4, 18.0,
18.1, 29.8, 30.0, 38.4, 38.8, 55.75, 55.80, 55.83, 70.9, 81.2, 108.7,
109.7, 110.1, 110.8, 115.7, 117.2, 118.9, 134.2, 135.3, 137.5, 147.5,
(2S,3R,5S)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(R)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (A-6). A-6
was obtained from alkene 4 by employing method B described for the
20
synthesis of A-3 in 37% yield as a colorless oil, ½ꢀꢀ
ꢁ41 (c 0.24,
D
CHCl3). 1H NMR (CDCl3) ꢁ 1.95–2.09 (3H, m), 2.11–2.30 (4H, m),
3.51 (1H, dd, J ¼ 11:8, 4.0 Hz, HH-6 (HH-90)), 3.66 (1H, dd, J ¼ 11:8,
4.7 Hz, HH-6 (HH-90)), 3.85 (3H, s, OCH3), 3.867 (3H, s, OCH3),
3.873 (3H, s, OCH3), 3.878 (3H, s, OCH3), 3.884 (3H, s, OCH3), 3.90
(3H, s, OCH3), 4.70 (1H, d, J ¼ 2:2 Hz, H-2 (H-7)), 4.77 (1H, d,
J ¼ 8:7 Hz, ArCHOH (H-70)), 5.92 (1H, m, ArCH=CH (H-800)), 6.18
(1H, d, J ¼ 15:7 Hz, ArCH=CH (H-700)), 6.75–6.96 (9H, m, ArH);
13C NMR (CDCl3) ꢁ 27.1, 34.3, 39.4, 41.8, 55.86, 55.90, 67.1, 75.6,
80.4, 108.5, 109.3, 110.7, 110.8, 110.9, 111.1, 118.8, 119.3, 119.4,
127.8, 130.7, 133.0, 136.0, 148.1, 148.7, 149.0, 149.2; EIMS m=z 564
(Mþ, 30), 546 (84), 151 (100); HREIMS m=z 564.2723 (calcd. for
148.1, 148.5, 148.6. Anal. Found: C, 69.63; H, 8.92. Calcd. for
20
C34H52O6Si: C, 69.82; H, 8.96%. An enantiomer of 7: ½ꢀꢀ
(c 0.5, CHCl3).
+61
D
(2S,3R,5R)-3-Allyl-2-(3,4-dimethoxyphenyl)-5-[(S)-(3,4-dimethoxy-
phenyl)(hydroxy)methyl]tetrahydropyran (8). The compound 8 was
C33H40O8, 564.2723).
obtained from 7 by the same method as that described for 2 in 78%
20
yield as a colorless oil, ½ꢀꢀ
ꢁ67 (c 0.7, CHCl3). 1H NMR (CDCl3) ꢁ
D
(2R,3S,5R)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(S)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (A-8).
1.58 (1H, ddd, J ¼ 12:6, 12.6, 4.4 Hz, 4-HH), 1.64 (1H, s, OH), 1.81
(1H, m, 4-HH), 1.96 (1H, m, 3-H), 2.07 (1H, m, CH2=CHCHH), 2.18
(1H, m, CH2=CHCHH), 2.25 (1H, m, 5-H), 3.33 (1H, dd, J ¼ 11:3,
11.3 Hz, 6-HH), 3.82–3.90 (1H, overlapped, 6-HH), 3.85 (3H, s,
OCH3), 3.86 (3H, s, OCH3), 3.88 (3H, s, OCH3), 3.90 (3H, s, OCH3),
4.36 (1H, d, J ¼ 7:0 Hz, ArCHOH), 4.50 (1H, d, J ¼ 2:2 Hz, 2-H),
4.88–4.92 (2H, m, CH2=CH), 5.54 (1H, m, CH2=CH), 6.76 (1H, d,
J ¼ 8:4 Hz, ArH), 6.81–6.87 (5H, m, ArH); 13C NMR (CDCl3) ꢁ 29.5,
30.0, 37.0, 38.6, 55.79, 55.84, 55.90, 55.91, 71.2, 76.1, 76.7, 100.5,
A-8 was obtained from an enantiomer of 4. The NMR data agreed
20
with those for A-6. ½ꢀꢀ
+41 (c 0.15, CHCl3).
D
(2R,3S,5S)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(S)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (B-3). B-3
was obtained from alkene 6 in 35% yield as a colorless oil by
20
employing method B described for the synthesis of A-3, ½ꢀꢀ
ꢁ46
D

Antimicrobial Activity of Neolignans
131
(c 0.13, CHCl3). 1H NMR (CDCl3) ꢁ 1.38 (1H, ddd, J ¼ 12:8, 12.8,
4.3 Hz, HH-4 (HH-9)), 1.55 (1H, m, HH-4 (HH-9)), 1.76 (1H, br. s,
OH), 1.88–1.98 (2H, m, ArCH=CHCH2, H2-900), 2.17 (1H, m, H-3
(H-8)), 2.29 (1H, m, H-5 (H-80)), 3.52 (1H, dd, J ¼ 11:1, 11.1 Hz, HH-
6 (HH-90)), 3.76 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.85 (6H, s,
OCH3), 3.86 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.27 (1H, d,
J ¼ 8:6 Hz, ArCHOH (H-70)), 4.55 (1H, s, H-2 (H-7)), 4.61 (1H, dd,
J ¼ 11:1, 2.9 Hz, H-6 (HH-90)), 5.59 (1H, m, ArCH=CH (H-800)), 6.10
(1H, d, J ¼ 15:8 Hz, ArCH=CH (H-700)), 6.61–6.67 (3H, m, ArH),
6.75–6.89 (6H, m, ArH); 13C NMR (CDCl3) ꢁ 29.2, 30.5, 37.0, 39.3,
55.7, 55.8, 55.9, 72.3, 77.2, 81.0, 108.4, 108.9, 109.6, 110.9, 111.1,
117.4, 118.7, 118.8, 127.4, 130.7, 130.8, 134.2, 135.2, 147.7, 148.2,
148.7, 148.8, 148.9, 149.0; FABMS m=z 565 [ðM þ HÞþ, 1], 154 (100);
HRFABMS m=z 565.2798 (calcd. for C33H41O8, 565.2802).
15 ml of 50 mM of the tested compound was put on to the agar plate.
The plate was incubated for 24 h at 30 ꢂC (L. denitrificans and
P. fluorescens) or at 37 ꢂC (B. subtilis, S. aureus, E. coli, S. choler-
aesuis and Y. intermedia), and the diameter of any halo of inhibition
around the paper disc was measured. The MIC value was determined
from a two-fold dilution series for any strain that showed a halo of
inhibition.
Antifungal assay. The paper disc method was adopted for the first
screening. Briefly, fungal mycelia were spotted on the center of the
PDA plate (’100-mm dish) and incubated at 28 ꢂC until the colony
diameter had became 4–5 cm. A paper disc soaked in dimethyl
sulfoxide containing a test chemical was then placed at the edge of
the colony. Growth inhibition was observed after culturing at 28 ꢂC
for 7–10 d. A further inhibition test was performed with the active
compounds. Each compound was added to three aliquots each
containing 3 ml of PDA at 50 ꢂC, mixed rapidly and poured on
to the PDA plates (’50-mm dish). Dimethyl sulfoxide only served
as the control. After incubating at 28 ꢂC for 7–10 d, the area of
the mycelial colony was measured by a caliper, the assays being
triplicated.
(2S,3R,5R)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(R)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (B-4).
B-4 was obtained from an enantiomer of 6. The NMR data agreed with
20
those for B-3. ½ꢀꢀ
+46 (c 0.22, CHCl3).
D
(2S,3R,5R)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(S)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (B-6). B-6
was obtained from alkene 8 by employing method A (8% yield) and
Results and Discussion
method B (52% yield) described for the synthesis of A-3 as colorless
20
crystals, mp 84–85 ꢂC, ½ꢀꢀ
ꢁ60 (c 1.7, CHCl3). 1H NMR (CDCl3) ꢁ
Preparation of the stereoisomers of morinols A and B
The syntheses of A-1, A-2, A-7, B-1, B-2, and B-5
have already been reported.3) A-5 and B-7 were
synthesized by employing the synthetic method reported
for A-7 and B-5. In this project, two Heck-Mizorogi
reaction conditions were tested to give the other stereo-
isomers. After desilylation of compounds 1, 3, 5, and 7,
the resulting olefin was subjected to the Heck-Mizorogi
reaction. Although the treatment of 2 and 8 with 1-
D
1.64 (1H, ddd, J ¼ 12:8, 12.8, 4.2 Hz, HH-4 (HH-9)), 1.80 (1H, s, OH),
1.90–2.08 (2H, m), 2.10–2.31 (3H, m), 3.38 (1H, dd, J ¼ 11:2,
11.2 Hz, HH-6 (HH-90)), 3.83–3.88 (1H, overlapped, HH-6 (HH-90)),
3.83 (3H, s, OCH3), 3.84 (6H, s, OCH3), 3.85 (3H, s, OCH3), 3.86 (3H,
s, OCH3), 3.88 (3H, s, OCH3), 4.39 (1H, d, J ¼ 7:5 Hz, ArCHOH (H-
70)), 4.52 (1H, s, H-2 (H-7)), 5.73 (1H, m, ArCH=CH (H-800)), 6.11
(1H, d, J ¼ 15:7 Hz, ArCH=CH (H-700)), 6.59–6.86 (9H, m, ArH);
13C NMR (CDCl3) ꢁ 29.2, 29.5, 37.1, 39.4, 55.6, 55.7, 55.8, 71.2, 75.6,
81.1, 108.3, 108.7, 109.0, 110.7, 110.8, 111.0, 117.2, 118.3, 118.6,
127.5, 130.3, 130.8, 134.0, 135.1, 147.5, 148.0, 148.4, 148.6, 148.8,
148.9; EIMS m=z 564 (Mþ, 100), 510 (88), 151 (75); HREIMS m=z
564.2723 (calcd. for C33H40O8, 564.2723).
bromo-3,4-dimethoxybenzene,
triethylamine,
and
PdCl2(PPh3)2 in DMF at 90 ꢂC gave A-3 and B-6 in
low yields (15% and 8%), respectively, the yield was
improved by reaction with 3,4-dimethoxyphenylboronic
acid, Cu(OAc)2, LiOAc, and Pd(OAc)2 in DMF12) at
100 ꢂC (A-3, 42% yield; B-6, 52% yield). A-6 and B-3
were obtained from 4 and 6 by employing 3,4-
dimethoxyphenylboronic acid in 37% and 35% yields,
respectively (Scheme 1). A-4, A-8, B-4, and B-8 were
respectively obtained from the enantiomers of 1, 3, 5,
and 7 by the same method. The syntheses of all 16
stereoisomers were achieved.
(2S,3S,5S)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(R)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (B-7).
B-7 was obtained by the same method as that described for B-5,3)
20
½ꢀꢀ
ꢁ34 (c 0.88, CHCl3). The NMR data agreed with those for
D
20
B-5.3) An enantiomer of B-7 (B-5), ½ꢀꢀ
+34 (c 0.4, CHCl3) in
D
reference.3)
(2R,3S,5S)-3-(3,4-Dimethoxycinnamyl)-2-(3,4-dimethoxyphenyl)-5-
[(R)-(3,4-dimethoxyphenyl)(hydroxy)methyl]tetrahydropyran (B-8).
20
B-8 was obtained from an enantiomer of 8, mp 84–85 ꢂC, ½ꢀꢀ
D
+60 (c 1.1, CHCl3). The NMR data agreed with those for B-6.
Antifungal activity of the stereoisomers of morinols A
and B
Organisms. Bacillus subtilis subsp. subtiis NBRC 13719T, Pseudo-
monas fluorescens NBRC 14160T, and Staphylococcus aureus subsp.
aureus NBRC 14462 were purchased from the National Institute of
Technology and Evaluation (NITE), Biological Resource Center,
Japan. Escherichia coli JCM 1649, Listeria denitrificans JCM 11481,
Salmonella choleraesuis subsp. choleraesuis JCM 6977 and Yersinia
intermedia JCM 7579 were obtained from RIKEN, Japan. The
phytopathogenic fungi, Colletotrichum lagenarium, Bipolaris oryzae,
Fusarium solani, and Alternaria alternata, had been isolated from a
farm at Ehime University and were kindly presented by Dr. Ohguchi.
Each fungus was cultured on potato dextrose agar (PDA, Sigma-
Aldrich, Canada).
Since (ꢁ)-virgatusin, which is a tetra-substituted
tetrahydrofuran lignan, and 3,4-dibenzoyltetrahydrofur-
an have respectively shown antifungal activity against
Colletotrichum lagenarium and Bipolaris oryzae,9,11)
these fungal strains were included for this test. All
stereoisomers showed antifungal activity against only
Alternaria alternata at 0.5 mM. In particular, (ꢁ)-
morinol B (B-2), which is one of the natural stereo-
isomers bearing a (7R, 8R, 70R, 80R) structure, showed
the strongest activity (Table 1). The dependence on
stereochemistry was weak, however, and specificity
against fungal species was apparent in this test. This is
the first report on the antifungal activity of morinols A
and B. In our previous study,9) the antifungal activity
of tetra-substituted tetrahydrofuran lignans depended
on the stereochemistry; in contrast, all stereoisomers of
the tetrahydropyran sesquineolignan morinols showed
activity. It is noteworthy that one of the natural
stereoisomers had the strongest activity and the
optimum stereochemistry for antifungal activity against
A. alternata.
Antibiotic spectra of morinols A and B and stereoisomers. The
ability of each compound to inhibit the growth of a variety of Gram-
positive and Gram-negative bacterial strains was assessed by the paper
disc method (Advantec Toyo, Japan; 6-mm thin paper disc), and the
minimum inhibitory concentration (MIC) was determined for those
strains that showed sensitivity to the tested compounds. The paper disc
test used agar plates containing Nissui nutrient broth (Nissui
Pharmaceutical Co.). A hundred microliter of an exponential culture
of each respective strain was made into molten agar containing the
nutrient broth. After the agar had solidified, a paper disc containing

132
K. AKIYAMA et al.
OTIPS
Ar
OH
OTIPS
Ar
OH
H
H
H
H
a
a
R
Ar
R
Ar
Ar
O
Ar
O
Ar
O
Ar
O
1
3
2: R = H
4: R = H
b or c
c
Mor A-3: R = Ar
Mor A-6: R = Ar
OTIPS
Ar
OH
H
OH
OTIPS
Ar
H
H
H
a
a
R
Ar
R
Ar
Ar
O
Ar
O
Ar
O
Ar
O
7
5
8: R = H
6: R = H
b or c
c
Mor B-6: R = Ar
Mor B-3: R = Ar
Scheme 1. Preparation of Morinols.
a) n-Bu4NF, THF, r.t., 30 min (2, 77% yield; 4, 70% yield; 6, 73% yield; 8, 78% yield); b) 1-Br-3,4-(CH3O)2C6H3, Et3N, PdCl2(PPh3)2, DMF,
90 ꢂC, 5 h (A-3, 15% yield; B-6, 8% yield); c) 3,4-(CH3O)2PhB(OH)2, Cu(OAc)2, LiOAc, Pd(OAc)2, DMF, 100 ꢂC, 2 h (A-3, 42% yield; A-6,
37% yield; B-3, 35% yield; B-6, 52% yield). Ar, 3,4-dimethoxyphenyl
Table 1. Growth Rate (% ꢃ ꢂ, n ¼ 3) of Alternaria alternata at 0.5 mM of the Morinol A and B Stereoisomers
Compound
Structure
Compound
Structure
Growth %
Growth %
No.
No.
OH
OH
H
H
8
8
7'
7'
Ar
Ar
Ar
Ar
Ar
Ar
A-1
B-1
8'
56.6 + 0.94
8'
65.5 + 3.29
O
O
7
Ar
7
Ar
(-)-morinol B
(-)-morinol A
OH
OH
OH
H
H
Ar
Ar
Ar
Ar
A-2
A-3
A-4
B-2
B-3
43.0 + 0.41
60.0 + 0.90
O
Ar
O
Ar
(+)-morinol A
(+)-morinol B
OH
OH
H
H
Ar
Ar
Ar
Ar
71.5 + 0.69
66.5 + 4.05
55.5 + 2.77
62.5 + 4.43
O
O
O
O
Ar
Ar
Ar
OH
OH
OH
OH
H
H
H
H
H
B-4
B-5
B-6
B-7
B-8
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
Ar
OH
OH
OH
H
H
H
A-5 Ar
Ar
Ar
Ar
72.5 + 2.10
54.3 + 2.15
66.1+ 6.55
84.1 + 6.67
61.0 + 2.96
O
O
O
O
O
O
Ar
Ar
Ar
A-6
A-7
Ar
Ar
75.2 + 1.29
59.4 + 1.97
OH
OH
H
H
A-8 Ar
Ar
Ar
Ar
78.3 + 1.08
O
O
Ar
Ar
Ar, 3,4-dimethoxyphenyl; growth (%), (colony diameter of sample/colony diameter of control) ꢄ 100

Antimicrobial Activity of Neolignans
133
Table 2. Antibacterial Activity of the Morinols (MIC, mM)
activity, although the antibacterial activity was influ-
enced by the stereochemistry.
Species
A-2
B-6
Bacillus subtilis NBRC 13719
Staphylococcus aureus NBRC 14462
Listeria denitrificans JCM 11481
50
>50
25
25
>50
50
Acknowledgments
We measured the 400 MHz NMR, MS and elemental
analysis data at INCS of Ehime University. Our thanks
to the president of Ehime University for supporting this
project. We are also grateful to Marutomo Co., for
financial support.
Antibacterial activity of the stereoisomers of morinols
A and B
In the test of the antibacterial activity of natural
component (ꢁ)-morinol A (A-1), (+)-morinol A (A-2),
(+)-morinol B (B-1) and (ꢁ)-morinol B (B-2), no
activity was shown against gram-negative bacteria, and
only (+)-morinol A (A-2), which is a (7S,70R,8S,80R)-
isomer, showed activity against the gram-positive
bacteria, B. subtilis (MIC, 50 mM) and L. denitrificans
(MIC, 25 mM). The growth of S. aureus was not
inhibited by (+)-morinol A (A-2). To determine the
important stereochemistry of morinols A and B for
antibacterial activity, the activity of all other stereo-
isomers was tested by using synthesized compounds
A-3–A-8 and B-3–B-8 (Table 2). The stereoisomers of
morinol A, which is 8-80 cis form, did not show activity,
however, one of the stereoisomers of morinol B type
bearing the 8-80 trans form showed activity. Thus, the
(7S,70S,8R,80R)-isomer (B-6) showed activity against
B. subtilis (MIC, 25 mM) and L. denitrificans (MIC,
50 mM). The growth of S. aureus and gram-negative
bacteria was not inhibited by B-6. Both A-2 and B-6
have the (7S,80R) structure. Although A-7 and B-4 also
have this (7S,80R) structure, no antibacterial activity was
observed. This fact means that the antibacterial activity
of morinols A and B depends on the whole stereo-
chemistry of these compounds. The antibacterial activity
of the tetrahydropyran sesquineolignans, morinols A and
B and their stereoisomers, was clarified for the first time.
In our previous study, the tetra-substituted tetrahydro-
furan lignan,10) di-substituted tetrahydrofuran lignan,
and di-substituted ꢃ-butyrolactone lignan11) showed
antibacterial activity against gram-positive bacteria,
but not against gram-negative bacteria. In this study of
the tetrahydropyran sesquineolignan, the same result
was apparent, showing activity against gram-positive
bacteria. The bonding of the C6-C3 unit would be
effective against gram-positive bacteria, but not against
gram-negative bacteria.
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