7
24 Letters
Compound
Cytotoxic and syncytium assays
Reverse transcriptase assay
% inhibition at 200 µg/mL
Table 2 Anti-HIV‑1 activities of
the isolated compounds, as de-
termined by using cell-based
MC99 virus and 1A2 cell line
and reverse transcriptase assays.
IC50 (µg/mL)
> 125
> 125
42.7
EC50 (µg/mL)
SI
IC50 (µg/mL)
1
2
3
4
5
6
7
8
9
0
1
2
I
NA
> 1.1
3.1
3.1
6.1
NA
47.1
3.1
ND
ΔTat/Rev
108.7
ND
13.7
97.9
20.5
11.8
66.1
25.2
8.2
34.1
ND
104.8
189.1
< 3.9
33.9
30.8
ND
T
ND
> 125
> 125
107.5
5.2
> 125
> 125
I
NA
ND
66.5
> 1.9
3.6
NA
ND
29.6
71.1
46.8
22.0
7.7
108.4
ND
1
1
1
T
I
NA
ND
I
NA
ND
Note: Cytotoxic assay: IC50 = dose of extract, fraction, or compound that inhibited 50% metabolic activity of uninfected cells; AZT, aver-
2
aged from three experiments; IC50 < 2.67 × 10 µg/mL. Syncytium assay: EC50 = dose of extract, fraction, or compound that reduced 50%
syncytium formation by ΔTat/RevMC99 virus in 1A2 cells; AZT, averaged from three experiments; EC50 9.9 × 10−4 µg/mL; I, less than 50%
reduction of syncytium formation at the highest nontoxic concentration; T, toxic. Selectivity index (SI): IC50/EC50; NA, nonapplicable or the
value could not be estimated. RT assay: Compounds were prescreened at 200 µg/mL, and only those that were very active at this con-
centration were further determined for IC50, the dose that inhibited 50% HIV-1 RT activity; ND = not determined. Positive controls were
averaged from two experiments; IC50 fagaronine chloride, 10.1 µg/mL; IC50 nevirapine, 2.1 µg/mL. The 50% endpoint assay was carried
2
out by using six concentrations in duplicate. The coefficients of determination, R , were 0.88–0.97, 0.91–0.99, and 0.86–0.98, respec-
tively, for cytotoxic, syncytium, and RT assays
MeOH–EtOAc (1:1, 1.5 L) followed by solvent removal yielded
fraction 1 (333 g), fraction 2 (61 g), and the residue (161 g), re-
spectively.
(SiO , 250 g, 5.1 × 30.5 cm, acetone–hexane gradient) to give frs
H1–H8. Fr. H6 (2.83 g, eluted with 11–18% acetone–hexane) gave
9 (101.7 mg) after recrystallizaton (MeOH). Fr. H7 (2.78 g) was
2
The bioactive fraction 1 (332 g) was separated by CC (SiO , 1.8 kg,
further purified by CC (SiO , 100 g, 3.5 × 26.5 cm, EtOAc–hexane
2
2
1
6 × 18 cm), eluting with 80%, 90%, and 100% CH Cl –hexane (2 L
gradient) to afford frs. I1–I6. Fr. I3 (335 mg, eluted with 28%
EtOAc–hexane) provided 10 (75.1 mg) after recrystallization
(EtOH–CH Cl ). Fr. A10 (49.7 g, eluted with 10–20% MeOH–
2
2
each), then with 2%, 3%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
6
0%, 80%, and 100% EtOAc–CH Cl (2 L each), followed by 1%,
2 2
2
2
10%, 20%, 30%, 50%, 70%, and 100% MeOH–EtOAc (4 L each). Frac-
EtOAc) was separated by CC (SiO , 700 g, 10 × 19 cm, MeOH–
2
tions (500 mL each) were combined (based on TLC behavior) to
CH Cl gradient) to give frs. J1–J7. Fr. J7 (17.7 g, eluted with 25–
2
2
give frs. A1–A12. Fr. A3 (46.5 g, eluted with 90% CH Cl –hexane)
100% MeOH–CH Cl ) was rechromatographed by CC (SiO ,
2 2 2
2
2
and fr. A4 (8.51 g, eluted with CH Cl ) were recrystallized from
300 g, 7 × 17.0 cm MeOH–EtOAc gradient) to give frs. M1–M7. Fr.
M4 (555.7 mg, eluted with 20% MeOH–EtOAc) and fr. M5 (1.28 g,
eluted with 25–40% MeOH–EtOAc) gave 11 (75.6 mg) and 12
(35.6 mg), respectively, after recrystallization (MeOH).
2
2
EtOH–CH Cl to give 3 (38.8 g and 3.79 g, respectively). The resi-
2
2
due of A4 (4.54 g) yielded frs. B1–B4 after CC (SiO , 200 g,
2
5.1 × 24.5 cm, acetone–hexane gradient). Fr. B4 (1.71 g, eluted
with 16–100% acetone–hexane) was rechromatographed (Se-
phadex LH 25 g, 2.5 × 28 cm), eluting with 90% CH Cl –hexane,
The bioactive fraction 2 (61 g) was separated by CC (SiO , 900 g,
2
10 × 24 cm), eluting with 50%, 60%, 80%, and 100% CH Cl –hex-
2
2
2
2
followed by recrystallization (EtOH–CH Cl ) to give 7 (31.8 mg).
ane (2 L each) and then with 10%, 20%, 30%, 40%, 50%, 60%, 80%,
2
2
Fr. A5 (47.9 g, eluted with 2–3% CH Cl –EtOAc) afforded 5
and 100% EtOAc–CH Cl (1 L each). Fractions (500 mL each) were
2
2
2
2
(
10.3 g) after recrystallization (EtOH–CH Cl ). The residue of A5
combined (based on TLC behavior) to give frs. N1–N9. Fr. N4
(4.4 g, eluted with 50% CH Cl –hexane) provided 3 (2.67 g) after
2
2
(35.7 g) was further separated by CC (SiO , 700 g, 8.5 × 30.5 cm,
2
2
2
CH Cl –hexane and MeOH–CH Cl gradients) to yield frs. C1–C8.
recrystallization (EtOH–CH Cl ). Fr N5 (8.77 g, eluted with 60–
2 2
2
2
2
2
Fr. C2 (8.51 g, eluted with 86–90% CH Cl –hexane) was rechro-
100% CH Cl –hexane) gave 4 (918.9 mg) after recrystallization
2 2
2
2
matographed by CC (SiO , 250 g, 6 × 20.5 cm, acetone–hexane
(EtOH–CH Cl ). The residue (7.6 g) was further separated by CC
2 2
2
gradient) to provide frs. D1–D12. Fr. D2 (3.5 g, eluted with 4% ace-
tone–hexane) was further separated by CC (SiO2, 100 g,
(SiO , 200 g, 5.1 × 24.5 cm, EtOAc–hexane gradient) to afford frs.
2
O1–O6. Fr. O3 (1.91 g, eluted with 10–13% EtOAc–hexane) pro-
vided 5 (504.7 mg) after recrystallization (EtOH–CH Cl ). Fr. O5
3
.5 × 26.5 cm, CH Cl –hexane gradient) to give frs. E1–E5. Fr. E3
2 2
2
2
(
1.16 g, eluted with 90–100% CH Cl –hexane) afforded
2
(779.7 mg, eluted with 25–40% EtOAc–hexane) was separated by
CC (SiO , 30 g, 2.5 × 16 cm), eluting with MeOH–CH Cl –hexane
2
2
(178.7 mg, R = 0.51, 20% EtOAc–hexane) after TLC (20% EtOAc–
f
2
2
2
hexane) and recrystallization (EtOH–CH Cl ). Fr. C4 (6.81 g,
2:16:82, followed by recrystallization (MeOH–CH Cl ) to give 8
2 2
2
2
eluted with 98–100% CH Cl –hexane) was separated by CC
(109.8 mg). Fr. N6 (2.47 g, eluted with 20–40% EtOAc–CH Cl ) was
2 2
2
2
(
SiO , 200 g, 5.1 × 24.5 cm, acetone–hexane gradient) to yield frs.
further separated CC (SiO , 80 g, 3.5 × 28 cm, EtOAc–hexane gra-
2
2
G1–G10. Fr. G6 (639.4 mg, eluted with 3.5–4% acetone–hexane)
gave 6 (20.5 mg) after recrystallization (EtOH–CH Cl ). Fr. A6
dient) to give frs. P1–P8. Fr. P5 (89.9 mg, eluted with 16% EtOAc–
hexane) gave 9 (5.3 mg) after recrystallization (MeOH).
2
2
(
24.3 g, eluted with 6–10% CH Cl –EtOAc) provided 4 (10.8 g)
Ent-kaur-sclerodimer (1): Colorless plates; m.p. 370–372°C
2
2
26
after recrystallization (EtOH–CH Cl ). Fr A7 (13.5 g, eluted with
1
(
(DMSO), dec.; [α]589: – 79.6 (c 0.5, THF). FTIR (KBr): ν
= 3690–
2
2
max
−1
1
13
5–35% EtOAc–CH Cl ) afforded 1 (4.41 g) after recrystallization
2350, 1732, 1687, 1635, 1471, 1447 cm ; H- and C‑NMR (pyr-
2
2
"
+
DMSO). The residue (9.28 g) was rechromatographed by CC
idine-d ): see l Table 1; EI‑MS: m/z (rel. int.) = 618 [M] (100),
5
Saepou S et al. Anti-HIV‑1 Diterpenoids from… Planta Med 2010; 76: 721–725