1280 J ournal of Natural Products, 2001, Vol. 64, No. 10
Ito et al.
33.8 (t, C-7), 41.4 (s, C-8), 52.5 (d, C-9), 36.1 (s, C-10), 135.2
(d, C-11), 127.4 (d, C-12), 89.5 (s, C-13), 41.5 (s, C-14), 27.1 (t,
C-15), 21.3 (t, C-16), 44.0 (s, C-17), 50.5 (d, C-18), 37.3 (t, C-19),
31.4 (s, C-20), 30.4 (t, C-21), 25.4 (t, C-22), 26.0 (q, C-23), 20.8
(q, C-24), 17.3 (q, C-25), 18.6 (q, C-26), 18.1 (q, C-27), 179.9 (s,
C-28), 33.3 (q, C-29), 23.5 (q, C-30); EIMS m/z 452 (M+);
HRFABMS m/z 451.3195 [M - H]- (m/z 451.3212 calcd for
8.8 Hz, H-7), 2.52 (3H, s, H-9), 5.26, 5.33 (each 1H, brs, H-11),
4.63, 4.70 (each 1H, d, J ) 13.2 Hz, H-12), 2.01 (3H, s, CH3-
OCO-12); 13C NMR (CDCl3) δ 84.1 (d, C-2), 34.5 (t, C-3), 127.0
(s, C-3a), 130.5 (d, C-4), 130.8 (s, C-5), 125.5 (d, C-6), 109.0 (d,
C-7), 163.5 (s, C-7a), 196.6 (s, C-8), 26.4 (q, C-9), 142.4 (s, C-10),
114.9 (t, C-11), 63.6 (t, C-12), 170.5 (s, CH3OCO-12), 20.8 (q,
CH3OCO-12); EIMS m/z 260 (M+).21
C
30H43O3).
3â-Hyd r oxy-3-d eoxym or on ic Acid (9). To a stirred solu-
tion of moronic acid (2) (29.7 mg, 65.4 µmol) in anhydrous
tetrahydrofuran (THF) was added sodium borohydride (5.5 mg,
145.4 µmol) at room temperature. After 4 h, the mixture was
neutralized with 2% hydrochloric acid, diluted with brine, and
extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate and, after filtration, concentrated
under reduced pressure to give the target alcohol (9) (28.3 mg,
yield 94.9%) as a colorless amorphous solid: [R]D +39.8° (c
Mor on ic a cid (2): colorless powder; [R]D +59.3° (c 1.01,
CHCl3); 1H NMR (CDCl3) δ 5.10 (1H, s, H-19), 1.02 (3H, s,
H-23), 0.964 (3H, s, H-24), 0.89 (3H, s, H-25), 0.955 (3H, s,
H-26), 0.73 (3H, s, H-27), 0.94 (3H, s, H-29), 0.91 (3H, s, H-30);
13C NMR (CDCl3) δ 39.7 (t, C-1), 33.9 (t, C-2), 218.3 (s, C-3),
47.1 (s, C-4), 54.7 (d, C-5), 19.5 (t, C-6), 33.4 (t, C-7), 40.4 (s,
C-8), 50.3 (d, C-9), 36.8 (s, C-10), 21.4 (t, C-11), 25.9 (t, C-12),
41.3 (d, C-13), 42.4 (s, C-14), 29.2 (t, C-15), 33.6 (t, C-16), 47.8
(s, C-17), 136.4 (s, C-18), 133.0 (d, C-19), 31.9 (s, C-20), 33.3
(t, C-21), 33.2 (t, C-22), 26.7 (q, C-23), 20.8 (q, C-24), 16.4 (q,
C-25), 15.7 (q, C-26), 14.7 (q, C-27), 182.8 (s, C-28), 30.2 (q,
C-29), 29.0 (q, C-30); EIMS m/z 454 (M+).15
1
0.13, CHCl3); H NMR (CDCl3) δ 3.19 (1H, dd, J ) 5.5, 11.0
Hz, H-3), 5.13 (1H, s, H-19), 0.75 (3H, s, H-23)a, 0.95 (3H, s,
H-24)a, 0.84 (3H, s, H-25), 0.95 (3H, s, H-26), 0.74 (3H, s, H-27),
0.97 (3H, s, H-29), 0.96 (3H, s, H-30); 13C NMR (CDCl3) δ 38.94
(t, C-1), 27.4 (t, C-2), 79.0 (d, C-3), 38.87 (s, C-4), 55.5 (d, C-5),
18.2 (t, C-6), 34.6 (t, C-7), 40.7 (s, C-8), 51.2 (d, C-9), 37.2 (s,
C-10), 20.9 (t, C-11), 26.0 (t, C-12), 41.3 (d, C-13), 42.6 (s, C-14),
29.4 (t, C-15), 33.53 (t, C-16)b, 48.0 (s, C-17), 136.8 (s, C-18),
133.0 (d, C-19), 32.0 (s, C-20), 33.46 (t, C-21)b, 33.37 (t, C-22)b,
27.9 (q, C-23), 16.6 (q, C-24), 15.4 (q, C-25), 16.0 (q, C-26), 14.9
(q, C-27), 181.9 (s, C-28), 30.3 (q, C-29), 29.1 (q, C-30); EIMS
m/z 456 (M+).24 a,bAssignments may be reversed.
An w u w eizon ic a cid (3): yellow oil; [R]D -7.3° (c 2.95,
CHCl3); 1H NMR (CDCl3) δ 0.76 (3H, s, H-18), 1.05 (3H, s,
H-19), 0.93 (3H, d, J ) 6.0 Hz, H-21), 6.09 (1H, t, J ) 7.5 Hz,
H-24), 1.92 (3H, s, H-27), 0.89 (3H, s, H-28), 1.06 (3H, s, H-29),
1.10 (3H, s, H-30); 13C NMR (CDCl3) δ 35.5 (t, C-1), 34.5 (t,
C-2), 218.2 (s, C-3), 47.3 (s, C-4), 51.4 (d, C-5), 20.2 (t, C-6),
26.8 (t, C-7), 132.7 (s, C-8), 134.7 (s, C-9), 37.1 (s, C-10), 21.3
(t, C-11), 30.7 (t, C-12), 44.1 (s, C-13), 50.0 (s, C-14), 29.8 (t,
C-15), 28.0 (t, C-16), 50.0 (d, C-17), 15.5 (q, C-18), 19.7 (q, C-19),
36.4 (d, C-20), 18.5 (q, C-21), 35.8 (t, C-22), 27.4 (t, C-23), 147.3
(d, C-24), 125.8 (s, C-25), 173.3 (s, C-26), 20.5 (q, C-27), 24.2
(q, C-28), 26.7 (q, C-29), 21.2 (q, C-30); EIMS m/z 454 (M+).16,17
Betu lon ic a cid (4): colorless amorphous solid; [R]D +40.1°
(c 0.86, CHCl3); 1H NMR (CDCl3) δ 0.93, 0.98, 0.99, 1.02, 1.07
(each 3H, s, H-23, H-24, H-25, H-26, H-27), 4.62, 4.75 (each
1H, brs, H-29), 1.70 (3H, s, H-30); 13C NMR (CDCl3) δ 39.6 (t,
C-1), 34.1 (t, C-2), 218.2 (s, C-3), 47.3 (s, C-4), 54.9 (d, C-5),
19.6 (t, C-6), 33.6 (t, C-7), 40.6 (s, C-8), 49.8 (d, C-9), 36.9 (s,
C-10), 21.4 (t, C-11), 25.5 (t, C-12), 38.5 (d, C-13), 42.5 (s, C-14),
30.5 (t, C-15), 32.1 (t, C-16), 56.3 (s, C-17), 49.2 (d, C-18), 46.9
(d, C-19), 150.3 (s, C-20), 29.7 (t, C-21), 37.0 (t, C-22), 26.6 (q,
C-23), 21.0 (q, C-24), 15.9 (q, C-25), 15.8 (q, C-26), 14.6 (q,
C-27), 181.7 (s, C-28), 109.8 (t, C-29), 19.3 (q, C-30); EIMS m/z
454 (M+).18
4-Hyd r oxy-3-m eth oxyp r op iop h en on e (5): yellow amor-
phous solid; 1H NMR (CDCl3) δ 7.53 (1H, d, J ) 1.9 Hz, H-2),
6.92 (1H, d, J ) 8.8 Hz, H-5), 7.52 (1H, dd, J ) 8.8, 1.9 Hz,
H-6), 2.93 (2H, q, J ) 7.4 Hz, H-2′), 1.19 (3H, t, J ) 7.4 Hz,
H-3′), 3.93 (3H, s, CH3O-3), 6.08 (1H, s, HO-4); 13C NMR
(CDCl3) δ 129.9 (s, C-1), 113.7 (d, C-2), 146.6 (s, C-3), 150.1 (s,
C-4), 109.8 (d, C-5), 123.2 (d, C-6), 199.6 (s, C-1′), 32.3 (t, C-2′),
8.6 (q, C-3′), 56.0 (q, CH3O-3); EIMS m/z 180 (M+).19
4-Hyd r oxy-3-m eth oxyben za ld eh yd e (6): colorless amor-
phous solid; 1H NMR (CDCl3) δ 7.40 (1H, d, J ) 1.6 Hz, H-2),
7.02 (1H, d, J ) 8.5 Hz, H-5), 7.41 (1H, dd, J ) 8.5, 1.6 Hz,
H-6), 9.81 (1H, s, CHO-1), 3.95 (3H, s, CH3O-3), 6.19 (1H, s,
HO-4); 13C NMR (CDCl3) δ 129.9 (s, C-1), 114.4 (d, C-2), 147.1
(s, C-3), 151.6 (s, C-4), 108.7 (d, C-5), 127.5 (d, C-6), 190.9 (d,
CHO-1), 56.1 (q, CH3O-3); EIMS m/z 152 (M+). This compound
was identified by comparison to the 1H NMR spectrum of an
authentic sample.
3-O-(3′,3′-Dim eth ylsu ccin yl)m or on ic Acid (10). To a
mixture of 9 (21.7 mg, 47.6 µmol) and pyridine was added
4-(dimethylamino)pyridine (5.8 mg, 47.6 µmol) and 2,2-di-
methylsuccinic anhydride (18.2 mg, 147.8 µmol) at room
temperature and stirred under reflux. An additional 18.2 mg
of 2,2-dimethylsuccinic anhydride (totally 72.8 mg, 568.2 µmol)
was added to the reaction mixture each day for 5 days. Then
the mixture was diluted with water and extracted with
chloroform. The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and after filtration, concen-
trated under reduced pressure. The resulting residue was
subjected to silica gel CC using a gradient solvent system
(chloroform-acetone (20:1 to 4:1)) to give 2 fractions. The first
fraction was subjected to PTLC (chloroform-acetone (20:1))
to give 3-O-(3′,3′-dimethylsuccinyl)moronic acid (10) (16.8 mg,
yield 60.5%) as a colorless amorphous solid: [R]D +24.6° (c
1
1.28, CHCl3); H NMR (CDCl3) δ 4.50 (1H, dd, J ) 5.2, 10.4
Hz, H-3), 5.13 (1H, s, H-19), 0.78 (3H, s, H-23)a, 0.82 (3H, s,
H-24)a, 0.85 (3H, s, H-25), 0.95 (3H, s, H-26), 0.72 (3H, s, H-27),
0.98 (3H, s, H-29), 0.97 (3H, s, H-30), 2.74, 2.50 (each 1H, d,
J ) 15.7 Hz, H-2′), 1.25, 1.28 (each 3H, s, CH3-3′); 13C NMR
(CDCl3) δ 38.3 (t, C-1), 23.6 (t, C-2), 81.5 (d, C-3), 37.8 (s, C-4),
55.3 (d, C-5), 18.2 (t, C-6), 34.2 (t, C-7), 40.6 (s, C-8), 50.6 (d,
C-9), 37.1 (s, C-10), 21.0 (t, C-11), 25.7 (t, C-12), 41.2 (d, C-13),
42.5 (s, C-14), 29.4 (t, C-15), 33.5 (t, C-16)b, 48.1 (s, C-17), 136.6
(s, C-18), 133.1 (d, C-19), 32.0 (s, C-20), 33.4 (t, C-21)b, 33.3 (t,
C-22)b, 28.2 (q, C-23), 16.9 (q, C-24), 16.6 (q, C-25), 16.7 (q,
C-26), 14.7 (q, C-27), 182.9 (s, C-28), 30.3 (q, C-29), 29.0 (q,
C-30), 170.8 (s, C-1′), 45.1 (t, C-2′), 40.5 (s, C-3′), 183.1 (s, C-4′),
24.7, 26.1 (q, CH3-3′); EIMS m/z 540 (M - COOH)+; HR-
FABMS m/z 583.4003 [M - H]- (m/z 583.3999 calcd. for
C
36H55O6). a,bAssignments may be reversed.
An ti-HIV Assa y. The T cell line, H9, was maintained in
3-(3,4-Dim eth oxyp h en yl)-2-p r op en a l (7): yellow amor-
phous solid; 1H NMR (CDCl3) δ 7.06 (1H, d, J ) 1.9 Hz, H-2),
6.89 (1H, d, J ) 8.2 Hz, H-5), 7.14 (1H, dd, J ) 8.2, 1.9 Hz,
H-6), 7.40 (1H, d, J ) 15.7 Hz, H-1′), 6.59 (1H, dd, J ) 15.7,
7.7 Hz, H-2′), 9.64 (1H, d, J ) 7.7 Hz, H-3′), 3.91 (3H, s, CH3O-
3)a, 3.90 (3H, s, CH3O-4)a; 13C NMR (CDCl3) δ 127.0 (s, C-1),
111.1 (d, C-2), 149.4 (s, C-3), 152.0 (s, C-4), 109.8 (d, C-5), 126.7
(d, C-6), 152.8 (d, C-1′), 123.4 (d, C-2′), 193.5 (d, C-3′), 56.0 (q,
CH3O-3)b, 55.9 (q, CH3O-4)b; EIMS m/z 192 (M+).20 a,bAssign-
ments may be reversed.
12-Acetoxytr em eton e (8): yellow oil; [R]D -19.4° (c 0.41,
CHCl3); 1H NMR (CDCl3) δ 5.36 (1H, t, J ) 8.8 Hz, H-2), 3.16,
3.43 (each 1H, dd, J ) 8.8, 15.7 Hz, H-3), 7.81 (1H, d, J ) 1.9
Hz, H-4), 7.79 (1H, dd, J ) 8.8, 1.9 Hz, H-6), 6.81 (1H, d, J )
continuous culture with complete medium (RPMI 1640 with
10% fetal calf serum (FCS) supplemented with L-glutamine
at 5% CO2 and 37 °C). Aliquots of this cell line were used in
experiments only when in log-phase of growth. Test samples
were first dissolved in dimethyl sulfoxide (DMSO). The fol-
lowing were the final drug concentrations routinely used for
screening: 100, 20, 4, and 0.8 µ/mL, but for active agents,
additional dilutions were prepared for subsequent testing so
that an accurate EC50 value could be achieved. As the test
samples were being prepared, an aliquot of the T cell line, H9,
was infected with HIV-1 (IIIB isolate) while another aliquot
was mock-infected with complete medium. The mock-infected
was used for toxicity determinations (IC50). The stock virus
used for these studies typically had a TCID50 value of 104