3372
R. Tanaka et al. / European Journal of Medicinal Chemistry 46 (2011) 3368e3375
acid and other reagents for the bioassay were purchased from
Nacalai Tesque, Inc. (Kyoto, Japan). TPA and DMBA were obtained
from Sigma Chemical Co. (St. Louis, MO, USA).
J ¼ 2.3 Hz, hesperetin H-20), 7.25 (1H, dd, J ¼ 8.4, 2.3 Hz, hesperetin
H-60), 12.01 (1H, d, J ¼ 1.4 Hz, OH); 13C NMR
d 29.1 and 29.5
-OeCOeCH2eCH2eCOeOe), 43.1 (hesperetin C-3), 77.2 (hesper-
etin C-2), 78.3 (C-21), 85.9 (C-3), 95.5 (hesperitin C-8), 96.8 (hes-
peretin C-6), 103.0 (hesperetin C-10), 112.5 (hesperetin C-50), 121.0
(hesperetin C-20), 121.7 (C-15), 124.8 (hesperetin C-60), 130.9 (hes-
peretin C-30), 138.7 (C-14), 151.4 (hesperetin C-10), 163.0 (hesperetin
C-9), 164.3 (hesperetin C-5), 164.9 (hesperetin C-7), 170.6, 171.6
(eOeCOeCH2eCH2eCOeOe) 195.6 (hesperetin C-4); MS (FAB)
m/z: 841 [M þ H]þ; HRFABMS (C51H69O10, 841.4893; calculated for
841.4891) [M þ H]þ.
3.2. Test compounds
The natural serratane-type triterpenoids, 3
a
b
-methoxyserrat-14-
-ol (PJ-2) were iso-
en-21 -ol (PJ-1), 3 -methoxyserrat-14-en-21
b
b
lated from the stem bark of P. jezoensis Carr. var. jezoensis (Pinaceae)
collected in Sapporo City, Japan. The isolation and characterization
of PJ-1 and PJ-2 were reported previously [10]. A voucher specimen
was deposited in the Laboratory of Medicinal Chemistry, Osaka
University of Pharmaceutical Sciences.
3.2.4. PJ-2ehesperetin conjugate (4)
PJ-2ehesperetin conjugate (4) (137 mg, 68%) was synthesized
from hesperidine (108 mg, 0.43 mmol) and PJ-2 succinate (200 mg,
0.35 mmol), similarly to compound 1. Mp 136e138 ꢂC
3.2.1. PJ-1enaringenin conjugate (1)
Naringenin (174 mg, 0.62 mmol), WSC,HCl (79 mg, 0.42 mmol),
and a small amount of N,N-dimethylaminopyridine (DMAP) were
added to a solution of PJ-1 succinate ester (180 mg, 0.32 mmol) in
tetrahydrofuran(2.0mL), andstirredat60ꢂCfor2.5hunderanitrogen
atmosphere. A small amount of distilled water was added to the
reaction mixture, which was extracted with ethyl acetate. The organic
layer was washed with brine, dried over MgSO4, and condensed in
vacuo to give an amorphous powder, which was purified by silica gel
column chromatography (n-hexane : AcOEt ¼ 5/1e4/1), followed by
HPLC (n-hexane : AcOEt ¼ 2/1) to afford compound 1 (133.6 mg, 51%)
(MeOHeCHCl3); ½a D23
ꢃ
ꢄ3.2 (c 0.11, CHCl3). 1H NMR
d 2.64 (1H, dd,
J ¼ 11.9, 4.3 Hz, H-3 ), 2.76 (1H, dd, J ¼ 3.2, 17.0 Hz, hesperetin
a
H-3
J ¼ 12.8,17.0 Hz, hesperetin H-3
(1H, t, J ¼ 2.5 Hz, H-21
a
), 2.80e2.97 (4H, m, -OeCOeCH2eCH2eCOeOe), 3.02 (1H, dd,
), 3.84 (3H, s hesperetin OMe), 4.73
), 5.30 (1H, dd, J ¼ 3.2, 12.8 Hz, hesperetin
b
a
H-2), 5.31 (1H, m, H-15), 5.94 (1H, br s, hesperetin H-8), 5.97 (1H, br
s, hesperetin H-6), 6.88 (1H, br s, hesperetin OH), 6.98 (1H, d,
J ¼ 8.5 Hz, hesperetin H-50), 7.16 (1H, d, J ¼ 2.0 Hz, hesperetin H-20),
7.25 (1H, dd, J ¼ 2.0, 8.5 Hz, hesperetin H-60), 12.0 (1H, s, hesperetin
as a colorless powder. Mp 145e147 ꢂC (MeOHeCHCl3); ½a 2D3
ꢃ
ꢄ57.4 (c
H-5-OH); 13C NMR
d 29.1 and 29.5 eOeCOeCH2eCH2eCOeOe),
0.05, CHCl3). 1H NMR
d
2.75e2.85 (6H, m, H-3
b
, naringenin H-3
a
, and
43.1 (hesperetin C-3), 57.5 (OMe), 78.3 (hesperetin C-2), 79.1 (C-21),
88.7 (C-3), 95.5 (hesperetin C-8), 96.8 (hesperetin C-6), 102.9
(hesperetin C-10), 112.5 (hesperetin C-50), 121.0 (hesperetin C-20),
121.9 (C-15), 124.8 (hesperetin C-60), 130.9 (hesperetin C-30), 138.5
(C-14), 151.3 (hesperetin C-10), 162.9 (hesperetin C-9), 164.2 (hes-
peretin C-5), 165.1 (hesperetin C-7), 195.5 (hesperetin C-4); MS
(FAB) m/z: 841 [M þ H]þ; HRFABMS (C51H69O10, 841.4899; calcu-
lated for 841.4891) [M þ H]þ.
eOeCOeCH2eCH2eCOeOe), 3.03 (1H, dd, J ¼ 13.0, 16.5 Hz, nar-
ingenin H-3 ),5.31(1H,
b), 3.31(3H, s,OMe), 4.73(1H, brt,J¼ 2.6,H-21
a
m, H-15), 5.39 (1H, dt, J ¼ 13.0, 3.2 Hz, naringenin H-2), 5.98 (1H, br s,
naringenin H-8), 6.00 (1H, br s, naringenin H-6), 6.85 (1H, br s, nar-
ingenin OH), 7.15(2H, d, J¼ 8.7 Hz, naringenin H-30 and H-50), 7.44 (2H,
d, J ¼ 8.7 Hz, naringenin H-20 and H-60), 12.02 (1H, s, naringenin OH);
13C NMR
d 29.4 and 29.5 (eOeCOeCH2eCH2eCOeOe), 43.3 (nar-
ingenin C-3), 57.1 (OMe), 78.6 (naringenin C-2), 79.3(C-21), 86.0 (C-3),
95.5 (naringenin C-8), 96.9 (naringenin C-6), 121.7 (C-15), 121.9 (nar-
ingenin C-30 and C-50), 127.3 (naringenin C-20 and C-60), 138.7 (C-14),
171.0and171.8(eOeCOeCH2eCH2eCOeOe),195.4(naringeninC-4);
MS (FAB) m/z: 779 [M e OMe]þ; HRFABMS (C49H63O8, 779.4515;
calculated for 779.4523) [M e OMe]þ.
3.2.5. PJ-1egenistein conjugate (5)
PJ-1egenistein conjugate (5) (221.3 mg, 76%) was synthesized
from genistein (126 mg, 0.47 mmol) and PJ-1 succinate (205 mg,
0.36 mmol), similarly to compound 1. Mp 143e145 ꢂC
(MeOHeCHCl3); ½a D23
ꢃ
ꢄ32.7 (c 0.11, CHCl3). 1H NMR
d 2.77 (1H, t,
a), 5.31 (1H, m, H-15),
J ¼ 2.5 Hz, H-3
b
), 4.74 (1H, t, J ¼ 2.6 Hz, H-21
3.2.2. PJ-2enaringenin conjugate (2)
6.33 (1H, d, J ¼ 2.3 Hz, genistein H-6), 6.38 (1H, d, J ¼ 2.3 Hz,
genistein H-8), 7.16 (2H, dt, J ¼ 8.9, 2.4 Hz, genistein H-30 and H-50),
7.53 (2H, dt, J ¼ 8.9, 2.4 Hz, genistein H-20 and H-60), 7.84 (1H, s,
PJ-2enaringenin conjugate (2) (151 mg, 51%) was synthesized
from naringenin and PJ-2 succinic acid (187 mg, 0.34 mmol),
similarly to compound 1. Mp 155e157 ꢂC (MeOHeCHCl3);
genistein H-2), 12.79 (1H, s, genistein OH); 13C NMR
d 79.2 (C-21),
½
a 2D3
H-3
ꢃ
ꢄ12.4 (c 0.11, CHCl3). 1H NMR
d
2.64 (1H, dd, J ¼ 11.7, 4.1 Hz,
85.8 (C-3), 94.2 (genistein C-8), 99.8 (genistein C-6), 105.9 (genis-
tein C-10), 121.7 (C-15, genistein C-30 and C-50), 123.0 (genistein
C-3), 128.5 (genistein C-10), 130.0 (genistein C-20 and C-60), 138.7
(C-14), 150.7 (genistein C-40), 153.1 (genistein C-2), 157.0 (genistein
C-5), 158.0 (genistein C-9), 162.9 (genistein C-7), 171.0, 171.7
(eOeCOeCH2CH2eCOeOe), 180.3 (C-4); MS (FAB) m/z: 809 [M]þ;
HRFABMS (C49H61O8, 777.4363; calculated for 777.4366)
[M e OMe]þ.
a
), 4.74 (1H, dif t, H-21a), 5.33 (1H, m, H-15), 5.40 (1H, dd,
J ¼ 13.0, 3.0 Hz, naringenin H-2), 5.97e6.10 (2H, m, naringenin H-6
and H-8), 7.16 (2H, dd, J ¼ 8.8 Hz, naringenin H-30, 50), 7.45 (2H, d,
J ¼ 8.8 Hz, naringenin H-20, 60), 12.0 (1H, d, J ¼ 1.4 Hz, naringenin
OH); 13C NMR
d
79.2 (C-21), 88.7 (C-3), 121.9 (C-30, C-50, C-15, and
C-40), 127.2, 127.3 (C-20 and C-60), 136.0 (C-10), 138.5 (C-14), 195.4
(naringenin C-4); MS (FAB) m/z: 779 [M e OMe]þ; HRFABMS
(C49H63O8, 779.4515; calculated for 779.4523) [M e OMe]þ.
3.2.6. PJ-2egenistein conjugate (6)
3.2.3. PJ-1ehesperetin conjugate (3)
PJ-2egenistein conjugate (6) (122 mg, 41%) was synthesized
from PJ-2 succinate (200 mg, 0.35 mmol) and genistein (126 mg,
0.47 mmol), similarly to compound 1. Mp 261e263 ꢂC
PJ-1ehesperidine conjugate (2) (151 mg, 51%) was synthesized
from hesperetin (108 mg, 0.43 mmol) and PJ-1 succinate (200 mg,
0.35 mmol), similarly to compound 1. Mp 138e140 ꢂC
(MeOHeCHCl3); ½a D22
ꢃ
ꢄ136.9 (c 0.10, CHCl3). 1H NMR
d
2.63 (1H, dd,
(MeOHeCHCl3); ½a D23
ꢃ
ꢄ36.1 (c 0.11, CHCl3). 1H NMR
d
2.77 (1H, m,
), 2.78e2.96
J ¼ 11.8, 4.0 Hz, H-3
b
), 4.74 (1H, t, J ¼ 2.6 Hz, H-21
a), 5.33 (1H, m,
H-3
b
), 2.77 (1H, dd, J ¼ 17.0, 3.2 Hz, hesperetin H-3
a
H-15), 6.35 (1H, br s, genistein H-6), 6.40 (1H, br s, H-8), 7.16 (2H, d,
J ¼ 8.7 Hz, genistein H-30 and H-50), 7.53 (2H, d, J ¼ 8.7 Hz, genistein
H-20 and H-60), 7.82 (1H, br s, genistein H-2), 12.77 (1H, s, genistein
(4H, m, eOeCOeCH2CH2eCO2), 3.02 (1H, m, hesperetin H-3
b), 4.73
(1H, t, J ¼ 2.7 Hz, H-21
a
), 5.32 (2H, m, H-15 and hesperetin H-2),
5.94 (1H, d, J ¼ 2.0 Hz, hesperetin H-8), 5.97 (1H, d, J ¼ 2.0 Hz,
OH); 13C NMR
d
79.1 (C-21), 88.4 (C-3), 94.2 (genistein C-8), 99.9
hesperetin H-6), 6.98 (1H, d, J ¼ 8.4 Hz, hesperetin H-50), 7.16 (1H, d,
(genistein C-6), 105.7 (genistein C-10), 121.9 (C-15), 122.9 (C-30 and