Synthesis of 3-substituted isocoumarins and their inhibitory effects on degranulation of RBL-2H3 cells induced by antigen

Article abstract of DOI:10.1248/cpb.56.1264

Eleven 3-substituted isocoumarins and a benzylidenephthalide were synthesized through thermal cyclization reaction of δ- and γ-ketoamides, respectively. Subsequent deprotection of the hydroxyl groups of the resulting isocoumarin and benzylidenephthalide compounds afforded thunberginols A, B, and F., respectively, which originated from the processed leaves of Hydrangea macrophylla Seringe var. thunbergii MAKINO. The synthesized isocoumarins and thunberginols were evaluated for their anti-allergic activity, in which thunberginol B exhibited the highest inhibitory potency on the degranulation of RBL-2H3 cells induced by antigen. Structure - activity relationship studies were carried out to determine the necessary substituents on the 3-phenylisocoumarin skeleton for inhibitory activity.

Full text of DOI:10.1248/cpb.56.1264

1264
Chem. Pharm. Bull. 56(9) 1264—1269 (2008)
Vol. 56, No. 9
Synthesis of 3-Substituted Isocoumarins and Their Inhibitory Effects on
Degranulation of RBL-2H3 Cells Induced by Antigen
a
a
Ai KURUME, Yasuhiro KAMATA, Masayuki YAMASHITA, ,a Qilong WANG,b Hisashi MATSUDA,b
*
Masayuki YOSHIKAWA,b Ikuo KAWASAKI,a,c and Shunsaku OHTA
a
a Department of Functional Molecular Chemistry, Kyoto Pharmaceutical University; b Department of Pharmacognosy, 21st
Century COE Program, Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8414, Japan: and c School
of Pharmaceutical Sciences, Mukogawa Women’s University; 11–68 Koshien Kyuban-cho, Nishinomiya 663–8179, Japan.
Received March 3, 2008; accepted July 4, 2008; published online July 9, 2008
Eleven 3-substituted isocoumarins and a benzylidenephthalide were synthesized through thermal cycliza-
tion reaction of d- and g-ketoamides, respectively. Subsequent deprotection of the hydroxyl groups of the result-
ing isocoumarin and benzylidenephthalide compounds afforded thunberginols A, B, and F, respectively, which
originated from the processed leaves of Hydrangea macrophylla SERINGE var. thunbergii MAKINO. The synthesized
isocoumarins and thunberginols were evaluated for their anti-allergic activity, in which thunberginol B exhibited
the highest inhibitory potency on the degranulation of RBL-2H3 cells induced by antigen. Structure–activity re-
lationship studies were carried out to determine the necessary substituents on the 3-phenylisocoumarin skeleton
for inhibitory activity.
Key words thunberginol; antiallergy; isocoumarin
Although excellent anti-allergic agents have been devel- as the electrophile. The maximum yield of d-ketoamide 3a
oped, recent increases in the number of patients in Japan with was obtained when N,N-diethyl-2-toluamide was lithiated
allergies such as pollinosis and asthma necessitate more ef- with tert-BuLi at ꢀ65 °C. The generality of these reactions
fective and safe anti-allergic agents. In a previous study, anti- under optimized conditions was proved as shown in Table 1.
allergic thunberginols A (1a), B (1b), and F (1c), which were
Among the various cyclization conditions for the conver-
isolated from Hydrangeae Dulcis Folium, the processed sion of ketoamide 3a to 3-phenylisocoumarin 4a, as summa-
leaves of Hydrangea macrophylla SERINGE var. thunbergii rized in Table 2, the maximum yield (91%) was obtained
MAKINO,1—6) were shown to substantially inhibit the degranu- under only refluxing AcOH for 3 h (entry 3).7) Under the
lation of rat peritoneal mast cells and rat basophilic leukemia same reaction conditions, N,N-diethyl-d-ketoamides 3b—j
(RBL-2H3) cells stimulated by calcium ionophore A23187 were readily cyclized to the corresponding 3-substituted iso-
or antigen. Of the three, 1b was reported to be a potential coumarins 4b—j in good to excellent yields (99—76%,
anti-allergic compound.5,6) Because 1a, 1b, and 1c can be Table 1), which can be attributed to the thermodynamic sta-
isolated from the herbs in only extremely low yields (1a: bility of the resulting aromatic isocoumarin nucleus.
0.0086%, 1b: 0.0016%, 1c: 0.0028%), investigations of the
As shown in Table 3, demethylation of 4c, 4h and 4i was
compounds’ anti-allergic effects, metabolism, and safety performed via treatment with BBr3. Deprotection of 4c and
using animal models require an efficient synthetic methodol- 4h proceeded smoothly under relatively mild conditions (6 eq
ogy. In a previous study, we reported the synthesis of 3-sub- of BBr3, ꢀ78 °C to rt) to give 1d (entry 1, 94% yield) and
stituted isocoumarin derivatives and its application towards thunberginol A (1a, entry 2, 92% yield), respectively. In con-
the total synthesis of thunberginol A.7) Herein, we report the trast, treatment of 4i with BBr3 under similar conditions gave
preparation of 3-substituted isocoumarins and an improved the partially deprotected 6-methylated thunberginol B (1e,
synthetic chart of thunberginols. Moreover, the resulting entry 3, 97% yield). Despite the report of complete demethyl-
compounds are evaluated for their inhibitory effects on de- ation of 4i using BBr3 (10 eq) at rt by Rossi et al.,9) in our
granulations induced by an antigen in RBL-2H3 cells.
hands, 1b was obtained in low yields, along with some de-
Chemistry Designed as precursors for isocoumarins 4, composition compounds, only after treatment of 4i with ex-
d-ketoamides 3 were prepared via benzylic metalation in- cess BBr3 (10 eq) in refluxing 1,1,2,2-tetrachloroethane.
volving the directing effects of the amide group.8) Lithiation
To improve the yield of 1b, our synthetic methodology
conditions for 2a were investigated using various lithiating was modified using 4-MOM-protected d-ketoamide 3k
agents such as lithium diisopropylamide (LDA), sec- and (Table 1, entry 11) as the precursor (Chart 1). The two-step
tert-BuLi, followed by treatment of N,N-dimethylbenzamide isocoumarin synthesis, as described above, was applied to
amide 2d (Table 1, entry 11) to afford 6-deprotected iso-
coumarin 4k (79% overall yield from 2d). Deprotection of
4k using BBr3 (6 eq) at ꢀ78 °C to rt proceeded as expected
to give 1b in 84% yield (Table 3, entry 4).
Subsequently, the synthesis of 1c, a (Z)-3-benzylidene-
phthalide that was co-isolated with thunberginols A and B
from the same plant,1) was carried out using the above reac-
tion chart. As shown in Chart 2, N,N-diethyl-2-methoxybenz
amide 9 was acylated via the ortho-lithiation process8) using
Fig. 1. Chemical Structure of Thunberginols A, B and F
∗ To whom correspondence should be addressed. e-mail: yamasita@mb.kyoto-phu.ac.jp
© 2008 Pharmaceutical Society of Japan

September 2008
1265
Table 1. Synthesis of d-Ketoamide 3 and Isocoumarin 4
2
N,N-Dimethylamide
d-Ketoamide 3;
Isocoumarin 4;
yield (%)
Entry
yield (%)
R1
R2
R3
1
2
3
4
5
6
2a:
2a:
2a:
2a:
2a:
2a:
H
H
H
H
H
H
H
H
H
H
H
H
Ph-
4-MeO–C6H4–
3,4-diMeO–C6H3–
Me
3a; 60
3b; 82
3c; 82
3d; 81
3e; 73
3f; 66
4a; 91
4b; 91
4c; 98
4d; 87
4e; 99
4f; 76
n-Hexyl
Cyclohexyl
7
2a:
H
H
3g; 82
4g; 96
8
9
2b:
2c:
H
MeO
MeO
MeO
3,4-diMeO–C6H3–
3,4-diMeO–C6H3–
3h; 73
3i; 68
4h; 81
4i; 99
10
2c:
MeO
MeO
3j; 93
4j; 91
11
2d:
MOMO
MeO
3,4-diMeO–C6H3–
3k; 85
4ka); 93
a) 4k: R1ꢁOH; R2ꢁMeO; R3ꢁ3,4-diMeO–C6H3–.
Table 2. Cyclization of d-Ketoamide 3a to Isocoumarin 4a
Temp.
(°C)
Time
(h)
Yield of
4a (%)
Entry
Additive
Solvent
1
2
3
4
5
6
7
—
—
—
AcOH
AcOH
AcOH
THFa)
THF
50
100
Reflux
50
Rt
100
6
3
3
6
12
3
22
88
91
13
0c)
25
86
HCla)
t-BuOLib)
—
Toluene
Xylene
Reagents and conditions: (a) tert-BuLi, 2-(3,4-dimethoxyphenyl)-N,N-dimethylac-
etamide, TMEDA, THF, ꢀ78 °C to rt, 11%; (b) AcOH, reflux, 81%; (c) BBr3, DCM,
ꢀ78 °C to rt, 98%.
—
Reflux
2
a) 10% HCl (aq.)/THFꢁ1 : 1. b) 1 eq of t-BuOLi was used. c) Recovery of 3a.
Chart 2
native method of evaluating anti-allergic compounds using
passive cutaneous anaphylaxis (PCA) reactions in laboratory
animals.5,11,12) In the present study, synthetic isocoumarins
1d, 1e, 4a—c, 4f—j, and 3-benzylidenephthalide 11 evalu-
ated for their effects on the release of b-hexosaminidase
from RBL-2H3 cells in order to investigate the relationship
between substituent groups on the 3-phenylisocoumarin
skeleton and biological activities. The results were compared
to those of naturally isolated 1a (IC50ꢁ17 mM), 1b (5.7 mM),
Reagents and conditions: (a) K2CO3, MOMCl, 87%; (b) NaH, MeI, 99%; (c) KOH,
MeOH, 94%; (d) EDAC, DMAP, Et2NH, 63%.
Chart 1
2-(3,4-dimethoxyphenyl)-N,N-dimethylacetamide to give g- and 1c (19 mM),5) of which, 1b possessed the highest potency.
ketoamide 10 in 11% yield. This low yield can be attributed Among the synthetic compounds, 1e exhibited substantial in-
to anion migration of lithiated 9 to the benzylic position of hibition of degranulation, which was nearly equivalent to
the N,N-dimethylacetamide. Next, g-ketoamide 10 was read- those of 1a and 1c (Table 4). Furthermore, to confirm that the
ily converted to (Z)-3-benzylidenephthalide 11 (81% yield), inhibitory effects of the active compounds 4a—c, 4h, and
then smoothly demethylated to give 1c in 98% yield.
1a—e are due to the inhibition of degranulation, but not the
Biological Study Along with the release of histamine, false positive due to the inhibition of enzyme activity of b-
degranulation of granules in mast cells or basophils also hexosaminidase, effects of the compounds on the enzyme ac-
causes the release of an enzyme, b-hexosaminidase. Accord- tivity were examined. As a result, each compound showed
ingly, determination of b-hexosaminidase activity, as a only a weak inhibition against enzyme activity of b-hexos-
marker of mast cell or basophil degranulation, offers an alter- aminidase less than 10% at 100 mM (data not shown).

1266
Vol. 56, No. 9
Table 3. Demethylation of 4a)
4
1
Entery
X
Y
Xꢂ
Yꢂ
Yield of 1 (%)
1
2
3
4
4c:
4h:
4i:
H
H
MeO
OH
H
1d:
H
H
MeO
OH
H
94
92
97
84
MeO
MeO
MeO
1a (thunberginol A):
1e:
1b (thunberginol B):
OH
OH
OH
4k:
a) All reaction was carried out with an excess BBr3 in CH2Cl2 at ꢀ78 °C to rt.
Table 4. Effects of Synthetic Compounds on DNP-BSA-Induced Degranulations in RBL-2H3 Cells Sensitized with Anti-DNP IgE
Conc.
(mM)
Inhibition
(%)
IC50
(mM)
Conc.
(mM)
Inhibition
(%)
IC50
(mM)
Compounds
Compounds
4a
0
30
60
100
0
0.0ꢅ2.9
ꢀ1.4ꢅ3.4
25.3ꢅ2.9**
53.3ꢅ3.1**
0.0ꢅ4.5
4h
0
10
30
100
0
10
0.0ꢅ2.9
100
100
26.6ꢅ3.6**
36.4ꢅ3.0**
49.4ꢅ1.4**
0.0ꢅ4.3
26.3ꢅ2.7**
36.6ꢅ5.4**
44.1ꢅ2.3**
100
4b
4c
4i
3
12.6ꢅ6.3
ꢄ100
10
30
100
22.4ꢅ4.2**
40.9ꢅ4.7**
50.3ꢅ2.9**
30
100
4j
0
100
0.0ꢅ4.2
8.1ꢅ3.9
0
3
0.0ꢅ4.5
3.8ꢅ5.0
ꢄ100
10
30
100
33.5ꢅ3.5**
69.5ꢅ2.4**
79.6ꢅ1.3**
20
1d
0
10
30
0.0ꢅ1.6
5.44ꢅ2.1
15.2ꢅ2.2**
63.3ꢅ2.1**
84
100
4f
0
0.0ꢅ4.2
100
ꢀ10.3ꢅ5.6
ꢄ100
ꢄ100
1e
11
0
10
30
100
0
0.0ꢅ4.4
28.6ꢅ5.7**
87.6ꢅ2.6**
104.7ꢅ0.8**
0.0ꢅ2.3
18
4g
0
100
0.0ꢅ4.2
26.6ꢅ4.3**
ꢄ100
100
30.8ꢅ1.5**
Each value represents the meanꢅS.E.M. (nꢁ4). Significantly different from control, ∗∗ pꢃ0.01.
A previous study based on structure–activity relationships reduce activity, synthetic 1a and 1b can be utilized as the
(SAR)3,5) has demonstrated the necessity of the hydroxyl substrates for the development of photolabile ligands to in-
groups at the 3ꢂ-, 4ꢂ-, 6-, and 8-positions for activity. Ac- vestigate target molecules.
tivities of our synthetic isocoumarins showed a similar trend
[4a (100 mM)ꢃ1d (84 mM)ꢃ1a (17 mM)ꢃ1b (5.7 mM)]. In
addition, permethylation of the hydroxyl groups in 1a and
Experimental
General Melting point was measured using a Yanaco MP micro-melt-
ing-point apparatus and are uncorrected. IR spectra were taken using a
1b as well as 1c significantly reduced their activities
Shimadzu IR-435 spectrophotometer. NMR spectra were measured using
JEOL-EX 270 (1H: 270 MHz), Varian XL-300 (1H: 300 MHz; 13C: 75 MHz),
[4h (100 mM)ꢃ1a (17 mM), 4i (ꢄ100 mM)ꢃ1b (5.7 mM), 11
or Varian UNITY INOVA 400NB (1H: 400 MHz; 13C: 100 MHz); the chemi-
(ꢄ100 mM)ꢃ1c (19 mM)], with the exception of the stronger
cal shifts are expressed in parts per million (ppm) downfield from tetra-
methylsilane as the internal standard. MS and HR-MS (EI) were measured
using a Hitachi M-80 or JEOL JMS BU-20 spectrometer. Purifications using
column chromatography was carried out on silica gel (Merck Art. 7737).
General Procedure for the Synthesis of d-Ketoamides (3) and the g-
Ketoamide 10, Synthesis of N,N-Diethyl-2-(2-oxo-2-phenylethyl)benz-
amide (3a) as an Example tert-BuLi (1.9 M in pentane; 1.1 ml, 2.1 mmol)
was added to a stirred solution of 2a (383 mg, 2 mmol) in THF (4 ml) under
N2 at ꢀ65 °C. After stirring for 1 h at the same temperature, a solution of
N,N-dimethylbenzamide (298 mg, 2 mmol) in THF (2 ml) was added to the
activity of 4c than that of 1d [4c (20 mM)ꢄ1d (84 mM)]. Al-
though methylation of the 6-hydroxyl group in 1b reduced its
activity, the activity of 1e remained equipotent to that of 1a
[1e (18 mM)ꢁ1a (17 mM)ꢃ1b (5.7 mM)].
To date, detailed mechanisms of 1a and 1b, including
those of the target molecules, have yet to be clarified. Re-
cently, photolabile ligands have been employed in drug dis-
covery.13) Because an isocoumarin structure is fluorescent,
and because substitution at the 6-position does not markedly reaction mixture and the whole was stirred for 4 h at ambient temperature.

September 2008
1267
H2O (5 ml) was added to the mixture, and after evaporation of the solvent MS m/z: 415.2008 (Calcd for C23H29NO6: 415.2000).
the products were extracted with AcOEt (20 mlꢆ2). The organic layer was
dried over anhydrous Na2SO4 and evaporated to give an oily residue, which amide (3j) Starting with 2c15) and N,N-dimethyl-3-(4-methoxyphenyl)-
was purified by column chromatography (AcOEt/n-hexaneꢁ1/2) to give 3a
propanamide, 3j was obtained as a viscous oil (771 mg, 93%). 1H-NMR
N,N-Diethyl-2,4-dimethoxy-6-[4-(4-methoxyphenyl)-2-oxobutyl]benz-
1
as a viscous oil (356 mg, 60%). H-NMR (270 MHz, CDCl3) d: 1.02 and (270 MHz, CDCl3) d: 1.00 and 1.18 (3H each, each t, Jꢁ7.1 Hz,
1.07 (3H each, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 3.08—3.62 (4H, br, 2ꢆNCH2CH3), 2.75—2.83 (4H, m, CO(CH2)2), 2.92—3.74 (4H, br m,
2ꢆNCH2CH3), 4.41 (2H, br s, COCH2), 7.23—7.59 (7H, m, Ar), 8.00—8.03 2ꢆNCH2CH3), 3.76 (6H, s, 2ꢆOMe), 3.77 (3H, s, OMe), 3.79 (2H, s,
(2H, m, Ar). IR (CHCl3) cmꢀ1: 1683, 1615. HR-MS m/z: 295.1547 (Calcd
COCH2Ar), 6.23 (1H, d, Jꢁ2.0 Hz, Ar), 6.35 (1H, d, Jꢁ2.0 Hz, Ar), 6.80
for C19H21NO2: 295.1570).
(2H, d, Jꢁ8.6 Hz, Ar), 7.07 (2H, d, Jꢁ8.6 Hz, Ar). IR (CHCl3) cmꢀ1: 1708,
N,N-Diethyl-2-[2-(4-methoxyphenyl)-2-oxoethyl]benzamide (3b) 1605. MS m/z: 413 (Mꢇ), 236, 179, 121.
Starting with 2a and N,N-dimethyl-4-methoxybenzamide, 3b was obtained
as a viscous oil (534 mg, 82%). H-NMR (270 MHz, CDCl3) d: 1.04 and phenyl)-2-oxoethyl]benzamide (3k) Starting with 2d and N,N-dimethyl-
1.07 (3H each, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 3.02—3.67 (4H, br, 3,4-dimethoxybenzamide in DME, 3k was obtained as colorless crystals
N,N-Diethyl-2-methoxy-4-methoxymethoxy-6-[2-(3,4-dimethoxy-
1
1
2ꢆNCH2CH3), 3.86 (3H, s, OMe), 4.34 (2H, br s, COCH2), 6.93 (2H, d, (754 mg, 85%). mp: 107—110 °C (AcOEt–n-hexane). H-NMR (400 MHz,
Jꢁ8.9 Hz, Ar), 7.20—7.36 (4H, m, Ar), 8.00 (2H, d, Jꢁ8.9 Hz, Ar). IR
(CHCl3) cmꢀ1: 1676, 1610. HR-MS m/z: 325.1679 (Calcd for C20H23NO3: 3.72 (4H, m, 2ꢆNCH2CH3), 3.46 (3H, s, OMe), 3.79 (3H, s, OMe), 3.92
325.1680). (3H, s, OMe), 3.93 (3H, s, OMe), 4.15 and 4.26 (1H each, each d,
CDCl3) d: 1.02 and 1.05 (3H each, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 2.99—
N,N-Diethyl-2-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]benzamide (3c) Jꢁ16.5 Hz, COCH2), 5.12 and 5.15 (1H each, each d, Jꢁ6.8 Hz, OCH2O),
Starting with 2a and N,N-dimethyl-3,4-dimethoxybenzamide, 3c was ob- 6.51 (1H, d, Jꢁ2.1 Hz, Ar), 6.52 (1H, d, Jꢁ2.1 Hz, Ar), 6.89 (1H, d,
1
tained as colorless powders (584 mg, 82%). H-NMR (270 MHz, CDCl3) d: Jꢁ8.6 Hz, Ar), 7.54 (1H, d, Jꢁ2.0 Hz, Ar), 7.71 (1H, dd, Jꢁ8.4, 2.0 Hz, Ar).
1.07 (6H, t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 3.03—3.68 (4H, br, 2ꢆNCH2CH3), 13C-NMR (100 MHz, CDCl3) d: 12.6, 13.4, 38.2, 42.1, 42.6, 55.4, 56.0ꢆ2,
3.92 (3H, s, OMe), 3.94 (3H, s, OMe), 4.34 (2H, br s, COCH2), 6.89 (1H, d, 56.1, 94.5, 98.4, 109.8, 110.1, 110.4, 120.7, 123.6, 129.7, 134.3, 148.9,
Jꢁ8.6 Hz, Ar), 7.22—7.36 (4H, m, Ar), 7.54 (1H, d, Jꢁ2.0 Hz, Ar), 7.71
153.3, 156.5, 158.3, 167.9, 195.7. IR (CHCl3) cmꢀ1: 1670, 1600. HR-MS
(1H, dd, Jꢁ8.6, 2.0 Hz, Ar). IR (CHCl3) cmꢀ1: 1673, 1614. HR-MS m/z: m/z: 445.2097 (Calcd for C24H31NO7: 445.2100). Anal. Calcd for
355.1768 (Calcd for C21H25NO4: 355.1780).
C24H31NO7: C, 64.70; H, 7.01; N, 3.14. Found: C, 64.61; H, 7.10; N, 3.18.
N,N-Diethyl-2-methoxy-6-[2-(3,4-dimethoxyphenyl)-1-oxoethyl]benz-
amide (10) Starting with 9 (5 mmol) and N,N-dimethyl-3,4-(dimethoxy-
benzene)acetamide (5 mmol), 10 was obtained as a pale yellow viscous oil
N,N-Diethyl-2-(2-oxopropyl)benzamide (3d) Starting with 2a and
N,N-dimethylacetamide, 3d was obtained as a viscous oil (376 mg, 81%).
1H-NMR (270 MHz, CDCl3) d: 1.08 and 1.22 (3H each, each t, Jꢁ7.1 Hz,
2ꢆNCH2CH3), 2.19 (3H, s, Me), 3.17 (2H, q, Jꢁ7.1 Hz, NCH2CH3), 3.40— (208 mg, 11%). 1H-NMR (270 MHz, CDCl3) d: 1.02 and 1.27 (3H each,
3.62 (2H, br, NCH2CH3), 3.81 (2H, br s, COCH2), 7.18—7.37 (4H, m, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 2.98—3.17 (2H, m, NCH2CH3), 3.42—
Ar). IR (CHCl3) cmꢀ1: 1717, 1616. HR-MS m/z: 233.1426 (Calcd for
3.73 (2H, m, NCH2CH3), 3.83 (3H, s, OMe), 3.85 (6H, s, 2ꢆOMe), 4.07
and 4.21 (1H each, each d, Jꢁ16.2 Hz, COCH2), 6.78—6.84 (3H, m, Ar),
C14H19NO2: 233.1420).
N,N-Diethyl-2-(2-oxooctyl)benzamide (3e) Starting with 2a and N,N- 7.05 (1H, t, Jꢁ4.6 Hz, Ar), 7.35 (2H, d, Jꢁ4.3 Hz, Ar). IR (CHCl3) cmꢀ1
:
1
dimethylheptanamide, 3e was obtained as a viscous oil (444 mg, 73%). H- 1683, 1617. HR-MS m/z: 385.1869 (Calcd for C22H27NO5: 385.1890).
NMR (270 MHz, CDCl3) d: 0.87 (3H, t, Jꢁ6.6 Hz, (CH2)5CH3), 1.08 and
1.21 (3H each, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 1.19—1.35 (6H, m, 1(3H)-Isobenzofuranone (11), Synthesis of 3-Phenylisocoumarin (4a) as
(CH2)3CH3), 1.48—1.62 (2H, m, COCH2CH2), 2.47 (2H, t, Jꢁ7.4 Hz, an Example A solution of 3a (295 mg, 1 mmol) in AcOH (2 ml) was re-
General Procedure for the Synthesis of Isocoumarins (4) and the
COCH2CH2), 3.17 (2H, q, Jꢁ7.1 Hz, NCH2CH3), 3.38—3.62 (2H, br, fluxed under N2 for 3 h. After evaporation of the solvent, the crude material
NCH2CH3), 3.77 (2H, br s, COCH2Ar), 7.16—7.37 (4H, m, Ar). IR (CHCl3) was purified by column chromatography (AcOEt/n-hexaneꢁ1/2) and recrys-
cmꢀ1: 1710, 1616. HR-MS m/z: 303.2218 (Calcd for C19H29NO2: 303.2200).
N,N-Diethyl-2-(2-cyclohexyl-2-oxoethyl)benzamide (3f) Starting with
tallization from AcOEt–n-hexane to give 4a as colorless needles (202 mg,
1
91%). mp: 88—89 °C (lit.16) mp: 87—88 °C). H-NMR (270 MHz, CDCl3)
2a and, N,N-dimethylcyclohexanecarboxamide, 3f was obtained as a viscous
d: 6.96 (1H, s, 4-H), 7.40—7.53 (5H, m, Ar), 7.72 (1H, td, Jꢁ7.3, 1.3 Hz,
1
oil (397 mg, 66%). H-NMR (270 MHz, CDCl3) d: 1.08 and 1.21 (3H each, Ar), 7.86—7.91 (2H, m, Ar), 8.31 (1H, d, Jꢁ7.3 Hz, Ar). IR (CHCl3) cmꢀ1
:
each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 1.13—1.92 (10H, m, CH2 in cyclohexane), 1723. MS m/z: 222 (Mꢇ), 194, 165.
2.35—2.48 (1H, m, COCH), 3.04—3.26 (2H, br, NCH2CH3), 3.37—3.64
(2H, br, NCH2CH3), 3.87 (2H, br s, COCH2), 7.16—7.37 (4H, m, Ar). IR
3-(4-Methoxyphenyl)isocoumarin (4b) Starting with 3b, 4b was ob-
tained as colorless needles (229 mg, 91%). mp: 114—115 °C (AcOEt–n-
(CHCl3) cmꢀ1: 1706, 1615. HR-MS m/z: 301.2023 (Calcd for C19H27NO2: hexane) (lit.17) mp: 119—121 °C). 1H-NMR (270 MHz, CDCl3) d: 3.87 (3H,
301.2040).
s, OMe), 6.83 (1H, s, 4-H), 6.97 (2H, d, Jꢁ8.9 Hz, Ar), 7.43—7.48 (2H, m,
N,N-Diethyl-2-[4-(4-methoxyphenyl)-2-oxobutyl]benzamide (3g) Ar), 7.69 (1H, td, Jꢁ7.3, 1.0 Hz, Ar), 7.82 (2H, d, Jꢁ8.6 Hz, Ar), 8.29 (1H,
Starting with 2a and, N,N-dimethyl-3-(4-methoxyphenyl)propanamide, 3g
was obtained as a viscous oil (579 mg, 82%). H-NMR (270 MHz, CDCl3)
d, Jꢁ7.9 Hz, Ar). IR (CHCl3) cmꢀ1: 1724. MS m/z: 252 (Mꢇ), 224.
3-(3,4-Dimethoxyphenyl)isocoumarin (4c) Starting with 3c, 4c was
1
d: 1.06 and 1.19 (3H each, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 2.69—2.86 obtained as colorless needles (276 mg, 98%). mp: 120—121 °C (AcOEt–n-
(4H, m, CO(CH2)2), 3.12 (2H, q, Jꢁ7.1 Hz, NCH2CH3), 3.37—3.58 (2H, br, hexane) (lit.17) mp: 116 °C). 1H-NMR (270 MHz, CDCl3) d: 3.94 (3H, s,
NCH2CH3), 3.74 (2H, br s, COCH2Ar), 3.77 (3H, s, OMe), 6.80 (2H, d,
OMe), 3.99 (3H, s, OMe), 6.85 (1H, s, 4-H), 6.94 (1H, d, Jꢁ8.2 Hz, Ar),
7.38 (1H, d, Jꢁ2.3 Hz, Ar), 7.45—7.50 (3H, m, Ar), 7.71 (1H, td, Jꢁ7.3,
Jꢁ8.6 Hz, Ar), 7.07 (2H, d, Jꢁ8.6 Hz, Ar), 7.12—7.36 (4H, m, Ar). IR
(CHCl3) cmꢀ1: 1711, 1613. HR-MS m/z: 353.1993 (Calcd for C22H27NO3: 1.3 Hz, Ar), 8.30 (1H, d, Jꢁ8.6 Hz, Ar). IR (CHCl3) cmꢀ1: 1724. MS m/z:
353.1990).
282 (Mꢇ), 254.
N,N-Diethyl-2-methoxy-6-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]benz-
3-Methylisocoumarin (4d) Starting with 3d, 4d was obtained as color-
amide (3h) Starting with 2b14) and, N,N-dimethyl-3,4-dimethoxybenzam- less needles (139 mg, 87%). mp: 71 °C (n-hexane) (lit.18) mp: 71—72 °C).
1
ide, 3h was obtained as a viscous oil (559 mg, 73%). H-NMR (270 MHz,
1H-NMR (270 MHz, CDCl3) d: 2.29 (3H, s, Me), 6.26 (1H, s, 4-H), 7.32—
CDCl3) d: 1.02 and 1.08 (3H each, each t, Jꢁ7.1 Hz, 2ꢆNCH2CH3), 2.90— 7.48 (2H, m, Ar), 7.67 (1H, td, Jꢁ7.9, 1.3 Hz, Ar), 8.24 (1H, d, Jꢁ7.9 Hz,
3.78 (4H, m, 2ꢆNCH2CH3), 3.81 (3H, s, OMe), 3.92 (3H, s, OMe), 3.93 Ar). IR (CHCl3) cmꢀ1: 1720. MS m/z: 160 (Mꢇ), 145, 118, 89.
(3H, s, OMe), 4.23 (2H, s, COCH2), 6.78—6.91 (3H, m, Ar), 7.25 (1H,
3-Hexylisocoumarin (4e)19) Starting with 3e, 4e was obtained as a col-
1
d, Jꢁ7.9 Hz, Ar), 7.54 (1H, d, Jꢁ2.0 Hz, Ar), 7.73 (1H, dd, Jꢁ8.3, 2.0 Hz, orless oil (229 mg, 99%). bp: 146 °C/3 mmHg. H-NMR (270 MHz, CDCl3)
Ar). IR (CHCl3) cmꢀ1: 1671, 1610. HR-MS m/z: 385.1914 (Calcd for
d: 0.89 (3H, t, Jꢁ6.6 Hz, (CH2)5CH3), 1.20—1.48 (6H, m, (CH2)3CH3),
1.63—1.77 (2H, m, ArCH2CH2), 2.53 (2H, t, Jꢁ7.6 Hz, ArCH2), 6.25 (1H,
C22H27NO5: 385.1890).
N,N-Diethyl-2,4-dimethoxy-6-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]- s, 4-H), 7.32—7.47 (2H, m, Ar), 7.67 (1H, td, Jꢁ7.9, 1.3 Hz, Ar), 8.25 (1H,
benzamide (3i) Starting with 2c15) and N,N-dimethyl-3,4-dimethoxy- d, Jꢁ7.9 Hz, Ar). IR (CHCl3) cmꢀ1: 1718. HR-MS m/z: 230.1301 (Calcd for
benzamide, 3i was obtained as a viscous oil (561 mg, 68%). 1H-NMR C15H18O2: 230.1310).
(270 MHz, CDCl3) d: 1.01 and 1.07 (3H each, each t, Jꢁ7.1 Hz, 2ꢆ
3-Cyclohexylisocoumarin (4f) Starting with 3f, 4f was obtained as col-
NCH2CH3), 2.93—3.75 (4H, m, 2ꢆNCH2CH3), 3.76 (3H, s, OMe), 3.78 orless crystals (174 mg, 76%). mp: 94 °C (n-hexane) (lit.19) mp: 91—93 °C).
(3H, s, OMe), 3.93 (6H, s, 2ꢆOMe), 4.23 (2H, d, Jꢁ1.7 Hz, COCH2), 1H-NMR (270 MHz, CDCl3) d: 1.16—2.19 (10H, m, CH2 in cyclohexane),
6.33—6.39 (2H, m, Ar), 6.89 (1H, d, Jꢁ8.6 Hz, Ar), 7.55 (1H, d, Jꢁ1.7 Hz,
2.23—2.53 (1H, m, ArCH), 6.23 (1H, s, 4-H), 7.32—7.47 (2H, m, Ar), 7.67
Ar), 7.75 (1H, dd, Jꢁ8.3, 2.0 Hz, Ar). IR (CHCl3) cmꢀ1: 1669, 1602. HR- (1H, td, Jꢁ7.6, 1.3 Hz, Ar), 8.24 (1H, d, Jꢁ7.6 Hz, Ar). IR (CHCl3) cmꢀ1
:

1268
Vol. 56, No. 9
1718. HR-MS m/z: 228.1165 (Calcd for C15H16O2: 228.1155). Anal. Calcd (60% in mineral oil; 4.138 g, 103 mmol) was added to a stirred solution of 6
for C15H16O2: C, 78.92; H, 7.06. Found: C, 78.64; H, 7.13.
3-[2-(4-Methoxyphenyl)ethyl]isocoumarin (4g) Starting with 3g, 4g
(16.569 g, 69 mmol) in THF (300 ml) under N2 at 0 °C. After stirring for
30 min at the same temperature, MeI (12.9 ml, 207 mmol) was added to the
was obtained as colorless needles (269 mg, 96%). mp: 87—88 °C (n-hexane) reaction mixture and the whole was stirred for 22 h at ambient temperature.
1
(lit.20) mp: 84—86 °C). H-NMR (270 MHz, CDCl3) d: 2.74—3.03 (4H, m, H2O (100 ml) was added to the mixture, and after evaporation of the solvent
CO(CH2)2), 3.78 (3H, s, OMe), 6.20 (1H, s, 4-H), 6.83 (2H, d, Jꢁ8.6 Hz,
the products were extracted with AcOEt (100 mlꢆ3). The organic layer was
Ar), 7.12 (2H, d, Jꢁ8.6 Hz, Ar), 7.31—7.69 (3H, m, Ar), 8.26 (1H, d, dried over anhydrous Na2SO4 and evaporated to give a residue, which was
Jꢁ7.9 Hz, Ar). IR (CHCl3) cmꢀ1: 1718. HR-MS m/z: 280.1088 (Calcd for
purified by column chromatography (AcOEt/n-hexaneꢁ1/10 to 1/5) to give
1
C18H16O3: 280.1100). Anal. Calcd for C18H16O3: C, 77.12; H, 5.75. Found: 7 as a colorless oil (17.378 g, 99%). H-NMR (400 MHz, CDCl3) d: 1.36
C, 77.30; H, 5.78.
(3H, t, Jꢁ7.1 Hz, CH2CH3), 2.28 (3H, s, 6-Me), 3.46 (3H, s, OMe), 3.79
(3H, s, OMe), 4.36 (2H, q, Jꢁ7.1 Hz, CH2CH3), 5.16 (2H, s, OCH2O), 6.45
(1H, d, Jꢁ2.0 Hz, Ar), 6.48 (1H, dd, Jꢁ2.1, 0.5 Hz, Ar). 13C-NMR
8-Methoxy-3-(3,4-dimethoxyphenyl)isocoumarin (4h)1) Starting with
3h, 4h was obtained as colorless crystals (254 mg, 81%). mp: 153—154 °C
(AcOEt–n-hexane) (lit.9) mp: 153—154 °C). 1H-NMR (270 MHz, CDCl3) d: (100 MHz, CDCl3) d: 14.2, 19.6, 55.8, 56.0, 60.9, 94.2, 97.8, 109.2, 117.7,
3.93 (3H, s, OMe), 3.98 (3H, s, OMe), 4.02 (3H, s, OMe), 6.74 (1H, s, 4-H),
6.89—6.94 (2H, m, Ar), 7.01 (1H, d, Jꢁ7.9 Hz, Ar), 7.36 (1H, d, Jꢁ1.7 Hz,
Ar), 7.45—7.63 (2H, m, Ar). IR (CHCl3) cmꢀ1: 1724. MS m/z: 312 (Mꢇ),
284.
6,8-Dimethoxy-3-(3,4-dimethoxyphenyl)isocoumarin (4i) Starting
with 3i, 4i was obtained as colorless crystals (339 mg, 99%). mp: 156—
157 °C (AcOEt–n-hexane) (lit.9) mp: 145—146 °C). 1H-NMR (270 MHz,
137.8, 157.9, 158.8, 168.1. IR (CHCl3) cmꢀ1: 2945, 1715. HR-MS m/z:
254.1151 (Calcd for C13H18O5: 254.1154).
2-Methoxy-4-methoxymethoxy-6-methylbenzoic Acid (8) A mixture
of 7 (5.159 g, 20 mmol) and KOH (12.112 g, 216 mmol) in MeOH (75 ml)
was refluxed for 12 h. The reaction mixture was cooled to 0 °C and neutral-
ized by adding of 10% HCl aq., and after evaporation of the solvent the prod-
ucts were extracted with Et2O (100 mlꢆ3). The organic layer was dried over
CDCl3) d: 3.91 (3H, s, OMe), 3.93 (3H, s, OMe), 3.98 (6H, s, 2ꢆOMe), anhydrous Na2SO4 and evaporated to give a crystalline residue, which was
6.42—6.46 (2H, m, Ar), 6.68 (1H, s, 4-H), 6.91 (1H, d, Jꢁ8.6 Hz, Ar), 7.35
purified by recrystallization from AcOEt–n-hexane to give 8 as colorless
prisms (4.305 g, 94%). mp: 117—119 °C. H-NMR (400 MHz, DMSO-d6)
1
(1H, d, Jꢁ2.0 Hz, Ar), 7.45 (1H, dd, Jꢁ8.4, 2.0 Hz, Ar). IR (CHCl3) cmꢀ1
:
1713. HR-MS m/z: 342.1091 (Calcd for C19H18O6: 342.1100). Anal. Calcd d: 2.20 (3H, s, 6-Me), 3.38 (3H, s, OMe), 3.74 (3H, s, OMe), 5.20 (2H, s,
for C19H18O6: C, 66.66; H, 5.30. Found: C, 66.33; H, 5.30. OCH2O), 6.49 (1H, d, Jꢁ2.0 Hz, Ar), 6.53 (1H, d, Jꢁ2.2 Hz, Ar), 12.70
6,8-Dimethoxy-3-[2-(4-methoxyphenyl)ethyl]isocoumarin (4j) Start- (1H, br s, OH). 13C-NMR (100 MHz, DMSO-d6) d: 19.4, 55.9ꢆ2, 93.9,
ing with 3j, 4j was obtained as colorless needles (309 mg, 91%). mp: 147 °C
98.1, 109.1, 119.1, 136.4, 157.1, 158.1, 168.8. IR (KBr) cmꢀ1: 3387, 2951,
(AcOEt–n-hexane) (lit.21): reported as an oil). 1H-NMR (270 MHz, CDCl3) 1717. HR-MS m/z: 226.0844 (Calcd for C11H14O5: 226.0841). Anal. Calcd
d: 2.27 (2H, t, Jꢁ7.7 Hz, CH2CH2), 2.95 (2H, t, Jꢁ7.7 Hz, CH2CH2), 3.77 for C11H14O5: C, 58.40; H, 6.24. Found: C, 58.61; H, 6.12.
(3H, s, OMe), 3.81 (3H, s, OMe), 3.96 (3H, s, OMe), 6.02 (1H, s, 4-H), 6.27
N,N-Diethyl-2-methoxy-4-methoxymethoxy-6-methylbenzamide (2d)
(1H, d, Jꢁ2.0 Hz, Ar), 6.42 (1H, d, Jꢁ2.0 Hz, Ar), 6.81 (2H, d, Jꢁ8.6 Hz, EDAC (1.457 g, 7.6 mmol) and DMAP (928 g, 7.6 mmol) was added to a
Ar), 7.11 (2H, d, Jꢁ8.6 Hz, Ar). IR (CHCl3) cmꢀ1: 1710. HR-MS m/z: stirred solution of 8 (1.420 g, 6.3 mmol) in CHCl3 (11 ml) under N2. After
340.1324 (Calcd for C20H20O5: 340.1310). Anal. Calcd for C20H20O5: C,
70.58; H, 5.92. Found: C, 70.59; H, 5.95.
stirring for 30 min at rt, diethylamine (3.26 ml, 31.5 mmol) was added to the
reaction mixture and the whole was stirred for 23 h at rt. H2O (20 ml) was
added to the mixture, and the products were extracted with AcOEt
(30 mlꢆ3). The organic layer was dried over anhydrous Na2SO4 and evapo-
6-Hydroxy-8-methoxy-3-(3,4-dimethoxyphenyl)isocoumarin (4k)
A
solution of 3k (1.337 g, 3 mmol) in AcOH (3 ml) and xylene (3 ml) was re-
fluxed under N2 for 10 h. After evaporation of the solvent, the crude material rated to give a residue, which was purified by column chromatography
1
was dissolved in AcOH (4.5 ml) and refluxed for 5 h. After evaporation of (AcOEt/n-hexaneꢁ2/1) to give 2d as a pale yellow oil (1.107 g, 63%). H-
the solvent, the crude drystalls were washed with toluene and recrystalliza-
NMR (400 MHz, CDCl3) d: 1.02 and 1.24 (3H each, each t, Jꢁ7.1 Hz,
2ꢆNCH2CH3), 2.20 (3H, s, 6-Me), 3.13 (2H, q, Jꢁ7.1 Hz, NCH2CH3),
tion from DMF–H2O to give pure 4k as colorless needles (912 mg, 93%).
mp: 272—276 °C (dec.). 1H-NMR (400 MHz, DMSO-d6) d: 3.82 (3H, s, 3.36—3.81 (2H, m, NCH2CH3), 3.49 (3H, s, OMe), 3.76 (3H, s, OMe), 5.15
OMe), 3.86, 3.87 (3H each, s each, OCH3ꢆ2), 6.48 (1H, d, Jꢁ2.0 Hz, Ar),
6.52 (1H, d, Jꢁ2.2 Hz, Ar), 7.07 (1H, d, Jꢁ8.6 Hz, 5ꢂ-H), 7.16 (1H, s, 4-H),
7.40 (1H, d, Jꢁ2.2 Hz, 2ꢂ-H), 7.44 (1H, dd, Jꢁ2.0, 8.4 Hz, 6ꢂ-H), 10.77 (1H,
br s, OH). 13C-NMR (100 MHz, DMSO-d6) d: 55.8, 55.9, 56.0, 99.2, 100.6,
and 5.17 (1H each, each d, Jꢁ6.9 Hz, OCH2O), 6.43 (1H, d, Jꢁ2.2 Hz, Ar),
6.49 (1H, dd, Jꢁ2.2, 0.5 Hz, Ar). 13C-NMR (100 MHz, CDCl3) d: 12.8,
13,8, 19.0, 38.5, 42.4, 55.4, 56.0, 94.4, 97.5, 109.2, 120.4, 136.5, 156.4,
158.0, 168.3. IR (CHCl3) cmꢀ1: 2953, 1604. HR-MS m/z: 281.1625 (Calcd
100.7, 103.5, 108.3, 111.8, 118.2, 124.4, 142.2, 149.1, 150.6, 152.9, 157.5, for C15H23NO4: 281.1627).
163.4, 164.4. IR (KBr) cmꢀ1: 3256, 1684, 1586, 1260. HR-MS (EI) m/z:
328.0945 (Calcd for C18H16O6: 328.0947). Anal. Calcd for C18H16O6: C,
65.85; H, 4.91, Found: C, 65.57; H, 4.90.
General Procedure of Demethylation of 4c, 4h, 4i, 4k and 11, Synthe-
sis of Thunberginol A (1a) as an Example BBr3 (1.0 M in CH2Cl2; 3.0 ml,
3.0 mmol) was added to a stirred solution of the isocoumarin 4h (156 mg,
(3Z)-7-Methoxy-3-[(3,4-dimethoxyphenyl)methylene]-1(3H)-isobenzo- 0.5 mmol) in CH2Cl2 (25 ml) under N2 at ꢀ78 °C. The reaction mixture was
furanone (11)1) Starting with 10, 11 was obtained as yellow crystals stirred for 12 h at ambient temperature. H2O (5 ml) was added to the mix-
(253 mg, 81%). mp: 185 °C (AcOEt–n-hexane). 1H-NMR (270 MHz,
ture, and after evaporation of the solvent the products were extracted with
CDCl3) d: 3.93 (3H, s, OMe), 3.97 (3H, s, OMe), 4.03 (3H, s, OMe), 6.34 AcOEt (40 mlꢆ2). The organic layer was dried over anhydrous Na2SO4 and
(1H, s, 8-H), 6.88—6.93 (2H, m, Ar), 7.29 (1H, d, Jꢁ7.9 Hz, Ar), 7.39 (1H,
dd, Jꢁ8.6, 2.0 Hz, Ar), 7.46 (1H, d, Jꢁ2.0 Hz, Ar), 7.63 (1H, t, Jꢁ7.9 Hz,
Ar). IR (CHCl3) cmꢀ1: 1767. MS m/z: 312 (Mꢇ), 297, 269.
evaporated to give a residue, which was purified by column chromatography
(AcOEt) and recrystallization from DMF–H2O to give 1a as yellow crystals
1
(124 mg, 92%). mp: 248—249 °C (lit.1) mp: 240 °C). H-NMR (300 MHz,
Ethyl 2-Hydroxy-4-methoxymethoxy-6-methylbenzoate (6) MOMCl
(1.3 ml, 18 mmol) was added to a stirred solution of 5 (2.667 g, 14 mmol)
and K2CO3 (2.442 g, 18 mmol) in acetone (30 ml) under N2 at 0 °C. The re-
DMSO-d6) d: 6.88 (1H, d, Jꢁ8.3 Hz, 5ꢂ-H), 6.93 (1H, d, Jꢁ8.3 Hz, 7-H),
7.10 (1H, d, Jꢁ7.8 Hz, 5-H), 7.23 (1H, s, 4-H), 7.24 (1H, dd, Jꢁ8.3, 2.1 Hz,
6ꢂ-H), 7.30 (1H, d, Jꢁ2.1 Hz, 2ꢂ-H), 7.69 (1H, t, Jꢁ8.0 Hz, 6-H), 9.32 (1H,
action mixture was stirred for 15 h at ambient temperature. H2O (20 ml) was s, OH), 9.59 (1H, s, OH), 10.86 (1H, s, OH). 13C-NMR (75 MHz, DMSO-d6)
added to the mixture, and after evaporation of the solvent the products were d: 100.6, 105.2, 112.2, 114.0, 116.0, 116.6, 117.0, 122.3, 137.6, 138.5,
extracted with AcOEt (20 mlꢆ3). The organic layer was dried over anhy- 145.6, 147.8, 152.8, 160.4, 165.2. IR (KBr) cmꢀ1: 3412, 1663. MS m/z: 270
drous Na2SO4 and evaporated to give a residue, which was purified by col- (Mꢇ), 242, 213.
umn chromatography (AcOEt/n-hexaneꢁ1/30) and recrystallization from n-
3-(3,4-Dihydroxyphenyl)isocoumarin (1d)22) Starting with 4c (54 mg,
0.19 mmol), 1d was obtained as pale yellow plates (46 mg, 94%). mp: 236—
237 °C (MeOH–H2O). 1H-NMR (400 MHz, DMSO-d6) d: 6.87 (1H, d,
1
hexane to give 6 as colorless crystals (2.842 g, 87%). mp: 47—48 °C. H-
NMR (400 MHz, CDCl3) d: 1.41 (3H, t, Jꢁ7.1 Hz, CH2CH3), 2.52 (3H, s, 6-
Me), 3.46 (3H, s, OMe), 4.40 (2H, q, Jꢁ7.1 Hz, CH2CH3), 5.17 (2H, s, Jꢁ8.2 Hz, 5ꢂ-H), 7.22 (1H, s, 4-H), 7.25 (1H, dd, Jꢁ2.3, 8.3 Hz, 6ꢂ-H), 7.31
OCH2O), 6.38 (1H, dq, Jꢁ2.6, 0.7 Hz, Ar), 6.49 (1H, d, Jꢁ2.4 Hz, Ar),
(1H, d, Jꢁ2.2 Hz, 2ꢂ-H), 7.54 (1H, dt, Jꢁ1.1, 7.6 Hz, 5-H), 7.66 (1H, d,
Jꢁ7.7 Hz, 6-H), 7.82 (1H, dt, Jꢁ1.3, 7.6 Hz, 7-H), 8.13 (1H, td, Jꢁ0.6,
8.1 Hz, 8-H), 9.43 (2H, br s, 2ꢆOH). 13C-NMR (100 MHz, DMSO-d6) d:
11.75 (1H, s, OH). 13C-NMR (100 MHz, CDCl3) d: 14.2, 24.4, 56.2, 61.3,
93.8, 101.5, 106.4, 111.7, 143.3, 161.3, 165.2, 171.6. IR (CHCl3) cmꢀ1
:
2952 (br), 1645, 1613, 1575. HR-MS m/z: 240.0995 (Calcd for C12H16O5: 99.8, 112.4, 116.2, 117.1, 119.4, 123.0, 126.4, 128.0, 129.0, 135.5, 138.1,
240.0998). Anal. Calcd for C12H16O5: C, 59.99; H, 6.71. Found: C, 60.00; H, 145.8, 147.9, 153.4, 161.7. IR (KBr) cmꢀ1: 3222, 1687, 1599. MS m/z: 254
6.69.
Ethyl 2-Methoxy-4-methoxymethoxy-6-methylbenzoate (7) NaH
(Mꢇ), 226, 89, 76, 63. Anal. Calcd for C15H10O4·1/2H2O: C, 68.44; H, 4.21.
Found: C, 68.37; H, 4.34.

September 2008
1269
and incubated with 50 ml of the substrate solution at 37 °C for 1 h. The reac-
tion was stopped by adding 200 ml of the stop solution and the absorbance
was measured using a microplate reader at 405 nm.
Each inhibition (%) was represents the meanꢅS.E.M. (nꢁ4). A one-way
analysis of variance followed by Dunnett’s test for multiple comparisons was
used for the statistical analysis. Probability (p) values of less than 0.05 were
considered significant.
8-Hydroxy-3-(3,4-dihydroxyphenyl)-6-methoxyisocoumarin (1e)
Starting with 4i, 1e was obtained as pale yellow crystals (145 mg, 97%).
mp: 231—232 °C (DMF–H2O). 1H-NMR (270 MHz, DMSO-d6) d: 3.87
(3H, s, OMe), 6.51 (1H, d, Jꢁ2.3 Hz), 6.67 (1H, d, Jꢁ2.3 Hz), 6.87
(1H, d, Jꢁ8.3 Hz), 7.15 (1H, s), 7.21 (1H, dd, Jꢁ8.3, 2.0 Hz), 7.26
(1H, d, Jꢁ2.0 Hz), 9.22—9.78 (2H, br, 2ꢆOH), 10.96 (1H, br s, OH). IR
(KBr) cmꢀ1: 3335, 1676. MS m/z: 300 (Mꢇ), 272. Anal. Calcd for
C16H12O6·3/5H2O: C, 61.78; H, 4.27. Found: C, 61.78; H, 4.24.
Acknowledgements We are grateful for financial support in part by the
Frontier Research Program and the 21st Century COE Program ‘Develop-
ment of Drug Discovery Frontier Integrated from Traditional to Proteome’
from the Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan, and a Grant-in-Aid for the Promotion of the Advance-
ment of Education and Research in Graduate Schools in Subsidies for ordi-
nary expenses of private schools from the Promotion and Mutual Aid Cor-
poration for Private Schools.
Thunberginol B (1b) Starting with 4k, 1b was obtained as pale yellow
crystals (240 mg, 84%). mp: 288—293 °C (MeOH–H2O). (lit.9) mp: 281—
1
285 °C, lit.1) mp: 244 °C). H-NMR (400 MHz, DMSO-d6) d: 6.30 (1H, d,
Jꢁ2.0 Hz, 5-H), 6.47 (1H, d, Jꢁ2.0 Hz, 7-H), 6.83 (1H, d, Jꢁ8.4 Hz, 5ꢂ-H),
7.09 (1H, s, 4-H), 7.20 (1H, dd, Jꢁ8.2, 2.2 Hz, 6ꢂ-H), 7.25 (1H, d,
Jꢁ2.2 Hz, 2ꢂ-H), 9.28 (1H, br s, OH), 9.55 (1H, br s, OH), 10.84 (1H, br s,
OH), 10.92 (1H, br s, OH). 13C-NMR (100 MHz, DMSO-d6) d: 98.0, 100.6,
101.4, 103.2, 112.2, 116.0, 117.0, 122.4, 140.1, 145.6, 147.7, 152.8, 162.6
165.0, 165.6. IR (KBr) cmꢀ1: 3332, 3149, 2834, 1667, 1614, 1521, 1241.
HR-MS m/z: 286.0478 (Calcd for C15H10O6: 286.0477).
References
1) Yoshikawa M., Harada E., Naitoh Y., Inoue K., Matsuda H., Shimoda
H., Yamahara J., Murakami N., Chem. Pharm. Bull., 42, 2225—2230
(1994).
2) Yamahara J., Matsuda H., Shimoda H., Wariishi N., Yagi N., Mu-
rakami N., Yoshikawa M., Folia Pharmacol. Jpn., 105, 365—379
(1995).
3) Yoshikawa M., Matsuda H., Shimoda H., Shimada H., Harada E.,
Naitoh Y., Miki A., Yamahara J., Murakami N., Chem. Pharm. Bull.,
44, 1440—1447 (1996).
4) Matsuda H., Shimoda H., Yoshikawa M., Bioorg. Med. Chem., 7,
1445—1450 (1999).
5) Wang Q., Matsuda H., Matsuhira K., Nakamura S., Yuan D.,
Yoshikawa M., Biol. Pharm. Bull., 30, 388—392 (2007).
6) Matsuda H., Wang Q., Matsuhira K., Nakamura S., Yuan D.,
Yoshikawa M., Phytomedicine, 15, 177—184 (2008).
7) Ohta S., Kamata Y., Inagaki T., Masuda Y., Yamamoto S., Yamashita,
M., Kawasaki I., Chem. Pharm. Bull., 41, 1188—1190 (1993).
8) Snieckus V., Chem. Rev., 90, 879—933 (1990).
9) Rossi R., Carpita A., Bellina F., Stabile P., Mannina L., Tetrahedron,
59, 2067—2081 (2003).
10) Gramatica P., Gianotti M. P., Speranza G., Manitto P., Heterocycles,
24, 743—750 (1995).
11) Cheong H., Choi E.-J., Yoo G.-S., Kim K.-M., Ryu S.-Y., Planta Med.,
64, 577—578 (1998).
Thunberginol F (1c) Starting with 11, 1c was obtained as yellow crys-
tals (132 mg, 98%). mp: 220—240 °C (AcOEt–n-hexane) (lit.1) mp: 242—
1
243 °C). H-NMR (270 MHz, DMSO-d6) d: 6.62 (1H, s, 8-H), 6.79 (1H, d,
Jꢁ8.3 Hz, 5ꢂ-H), 6.91 (1H, d, Jꢁ8.2 Hz, 6-H), 7.02 (1H, br d, Jꢁ8.3 Hz, 6ꢂ-
H), 7.38 (1H, br s, 2ꢂ-H), 7.41 (1H, d, Jꢁ7.9 Hz, 4-H), 7.58 (1H, t,
Jꢁ7.9 Hz, 5-H), 9.25 (2H, br, 2ꢆOH), 11.02 (1H, br, OH). IR (KBr) cmꢀ1
3204, 1745. MS m/z: 270 (Mꢇ), 213, 168.
:
Bioassay Method As a marker of the degranulation of RBL-2H3 cells,
release of b-hexosamindase into the medium was determined as described
previously.5,23) Briefly, RBL-2H3 cells [Cell No. JCRB0023, obtained from
Health Science Research Resources Bank (Osaka, Japan)] in Eagle’s mini-
mum essential medium (MEM) containing 10% fetal calf serum (FCS) and
penicillin (100 units/ml) and streptomycin (100 mg/ml) were seeded into 24-
well multiplates at the density of 2ꢆ105 cells per well and were incubated
with anti-dinitrophenyl (DNP) IgE antibody (0.45mg/ml, monoclonal anti-
DNP, Sigma) for sensitization of the cells. Then, the cells were washed twice
with Siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 25 mM
PIPES, and 40 mM NaOH, pH 7.2) supplemented with 5.6 mM glucose, 1 mM
CaCl2, and 0.1% bovine serum albumin (BSA) and incubated in 160 ml of
buffer for 10 min at 37 °C. Then, cells were added with 20 ml of test sample
solution, and were stimulated with 20 ml of dinitrophenylated bovine serum
albumin (DNP-BSA)4) (final conc. 10 mg/ml) as an antigen for 10 min. The
reaction was stopped by cooling in an ice bath for 10 min. The supernatant
(50 ml) was transferred into a 96-well microplate and incubated with 50 ml of
substrate (1 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide) in 0.1 M citrate
buffer (pH 4.5) at 37 °C for 1 h. The reaction was stopped by adding 200 ml
of stop solution (0.1 M Na2CO3/NaHCO3, pH 10.0). The absorbance was
measured with a microplate reader at 405 nm. The test sample was dissolved
in dimethylsulfoxide (DMSO), and the solution was added to incubation
buffer (final DMSO conc. was 0.1%). The inhibition (%) of the release of b-
hexosaminidase by the test sample was calculated by the following equation,
and IC50 values were determined graphically:
12) Kaul S., Hoffmann A., ALTEX., 18, 55—58 (2001).
13) Dorman G., Prestwich G. D., Trends Biotechnol., 18, 64—77 (2000).
14) Watanabe M., Sahara M., Kubo M., Furukawa S., Billedeau R. J.,
Snieckus V., J. Org. Chem., 49, 742—747 (1984).
15) Superchi S., Pini D., Salvadori P., Marinelli F., Rainaldi G., Zanelli U.,
Nuti-Ronchi V., Chem. Res. Toxicol., 6, 46—49 (1993).
16) Larock R. C., Varaprath S., Lau H. H., Fellows C. A., J. Am. Chem.
Soc., 106, 5274—5284 (1984).
17) Bradsher C. K., Wallis T. G., J. Org. Chem., 43, 3817—3820 (1978).
18) Tirodkar R. B., Usgaonkar R. N., J. Ind. Chem. Soc., 46, 935—944
(1969).
19) Ogawa Y., Maruno M., Wakamatsu T., Heterocycles, 41, 2587—2599
(1995).
20) Rama N. H., Iqbal R., Zamani K., Saeed A., Choudhary M. I., Indian
J. Chem., 37B, 480—483 (1998).
21) Saeed A., Rama N. H., Arfan M., J. Heterocycl. Chem., 40, 519—522
(2003).
22) Ahmad H. B., Rama N. H., Hussain M., Hussain M. T., Qasim M. M.,
Hameed S., Malana M. A., Malik A., Indian J. Chem., 42B, 611—615
(2003).
23) Matsuda H., Tewtrakul S., Morikawa T., Nakamura A., Yoshikawa M.,
Bioorg. Med. Chem., 12, 5891—5898 (2004).
inhibition (%)ꢁ[1ꢀ(TꢀBꢀN)/(CꢀN)]ꢆ100
Control (C): DNP-BSA (ꢇ), test sample (ꢀ);
Test (T): DNP-BSA (ꢇ), test sample (ꢇ);
Blank (B): DNP-BSA (ꢀ), test sample (ꢇ);
Normal (N): DNP-BSA (ꢀ), test sample (ꢀ).
Under the same conditions, IC50 values of reference compounds, tranilast
and ketotifen fumarate were 282 and 158 mM as reported previously.23) In
order to clarify that the anti-allergic effects of samples were due to the inhi-
bition of degranulation, but not the false positive by the inhibition of b-hex-
osaminidase activity, the following assay was carried out.23) Briefly, the cell
suspension of PBS was sonicated, and the solution was then centrifuged. The
supernatant was diluted with Siraganian buffer and adjusted to equal the en-
zyme activity of the degranulation tested above. The enzyme solution (45 ml)
and test sample solution (5 ml) were transferred into a 96-well microplate