E. E. Shults et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4228–4232
4231
5. Yang, H. O.; Kang, Y. H.; Suh, D.-Y.; Kim, Y. C.; Han,
B. H. Planta Med. 1995, 61, 519.
6. Han, B. H.; Go, H. J.; Song, W.-J.; Yang, H. O.; Kang,
Y.-H.; Kim, Y. C.; Suh, D.-Y.; Park, M. K. J. Med. Chem.
1998, 41, 2626.
7. Tolstikova, T. G.; Sorokina, I. V.; Dolgikh, M. P.;
Kharitonov, Y. V.; Chernov, S. V.; Shults, E. E.;
Tolstikov, G. A. Pharm. Chem. J. 2004, 38, 532.
8. Chernov, S. V.; Shults, E. E.; Shakirov, M. M.; Bag-
rjanskaja, I. Y.; Gatilov, Y. V.; Tolstikov, G. A. Arkivoc
2003, XIII, 172.
9. Tolstikov, G. A.; Tolstikova, T. G.; Shults, E. E.;
Sorokina, I. V.; Chernov, S. V.; Kharitonov, Y. V.
Bioactive Higher Furanoterpenoids and Their Derivatives.
In Oxygen and Sulfur-Containing Heterocycles; Kartsev,
V. G., Ed.; IBS Press: Moskow, 2003; Vol. 1, pp 112–127.
10. Kharitonov, Y. V.; Shults, E. E.; Shakirov, M. M.;
Tolstikov, G. A. Russ. J. Org. Chem. 2005, 41, 1145.
11. (a) Ross, S. D.; Finkelstein, M.; Uedel, J. J. J. Org. Chem.
1969, 34, 1018; (b) Hirsch, J. A.; Szur, A. J. J. Heterocycl.
Chem. 1972, 9, 523.
12. For a related method for the conversion of furanes into
(5H)-furan-2-one derivatives (using NBS and NaHCO3 in
CHCl3/EtOH and subsequent acidic hydrolysis), see:
Cenal, J. P.; Carreras, C. R.; Tonn, C. E.; Padron, J. I.;
Ramirez, M. A.; Diaz, D. D.; Garcia-Tellado, F.; Martin,
V. S. Synlett 2005, 1575.
dd, H-9, J = 11.1, 3.2 Hz), 1.62–1.78 (4H, m, H-1, H-2, H-
6, H-11), 1.80 (1H, m, H-7, Jgem = 12.7 Hz), 1.85 (1H, m,
H-6), 1.97 (1H, m, H-12), 2.05 (1H, m, H-3,
Jgem = 13.2 Hz), 2.29 (1H, ddd, H-7, J = 12.7, 4.0,
2.6 Hz), 2.33 (1H, m, H-12), 3.50 (3H, s, OCH3), 4.47
(1H, s, H-17), 4.77 (1H, s, H-17), 4.62 (1H, dd, H15,
J = 18.0, 1.6 Hz), 6.99 (1H, d, H-14, J = 1.6 Hz). 13C
NMR (CDCl3, 75 MHz): d [ppm] = 12.38 (C-20), 19.72
(C-2), 21.69 (C-11), 24.52 (C-12), 25.99 (C-6), 28.59 (C-
19), 38.04 (C-3), 38.49 (C-7) 39.00 (C-1), 40.08 (C-10),
43.98 (C-4), 50.71 (OCH3), 55.54 (C-9), 56.11 (C-5), 69.48
(C-15), 106.66 (C-17), 134.60 (C-14) 143.23 (C-13), 146.99
(C-8), 173.14 (C-16), 176.61 (C-18). MS (EI, 70 eV): m/z
(%) = 346 [M]+ (4), 314 (8), 286 (48), 271 (22), 217 (14),
189 (19), 161 (27), 121 (100), 109 (27), 81 (36). Calcd for
C21H30O4: 346.21439. Found: 346.21489.
14. X-ray crystallographic analysis of 1. A crystal of 1 was
chosen [0.80 mm · 0.20 mm · 0.20 mm]. The data were
collected on a Bruker P4 diffractometer with Mo Ka
radiation. The cell parameters and the orientation matrix
for data collection were obtained from least-squares
refinement in the range of 25–35ꢁ (2H). A total of 2442
unique reflections were collected. The structure was solved
by direct methods using the SHELXS-97 software. The
refining of structural parameters was carried out by the
least-squares procedure in the full-matrix anisotropic
approximation applying the program SHELXL-97 to
wR2 = 0.1202, S = 1.021 for all reflections (R = 0.0485
for 1768 F > 4r). Hydrogen atoms were included in the
refinement but restrained to ride on the atom to which
they are bonded. The X-ray data were submitted to the
Cambridge Crystallographic Data Centre (CCDC
13. Synthesis of pinusolide (1) from 3. Step 1: 4.7 g (21 mmol)
of the sodium salt of N-chloro-benzenesulfonamide
(‘chloramin-B’) was added portionwise (over 10 min) to
a stirred solution of 3.3 g (10 mmol) of methyl lamberti-
anate (3) in 25 ml of methanol and 1.5 ml of acetic acid at
0–5 ꢁC. After 30 min at 5 ꢁC, the reaction was quenched
by addition of 1–2 ml of 5% aqueous Na2SO3. The mixture
was concentrated by solvent evaporation in vacuo and the
residue was partitioned between 50 ml of water and 30 ml
of ethyl acetate. The aqueous layer was extracted once
again with dichloromethane and the combined organic
layers were washed with water and brine. After drying
over MgSO4 and filtration, the solvent was evaporated
and the residue purified by column chromatography
(petrol ether/t-BuOMe 1:1) to afford 3.5 g (90%) of a
15. Dauben, W. G.; German, V. F. Tetrahedron 1966, 22, 679.
16. Rajnikant, V. K.; Gupta, A.; Singh, M.; Lal, B. D.;
Gupta, B. V. Mol. Mater. (Mol. Cryst. Liq. Cryst., Sect.
C) 1996, 6, 227.
17. Medforth, C. J.; Chang, R. S.; Chen, G.-Q.; Olmstead, M.
M.; Smith, K. M. J. Chem. Soc., Perkin Trans. 2 1990, 1011.
18. Biological investigations: cell culture. The cells were
subcultured every 3–4 days by dilution of the cells to a
concentration of 1 · 105/ml. All experiments were per-
formed in RPMI 1640 supplemented with 10% heat-
inactivated fetal calf serum, 100 U/ml penicillin, 100 lg/ml
streptomycin, and 0.56 g/l L-glutamine. Twenty four hours
before the assay setup, cells were cultured at a concentra-
tion of 4 · 105/ml to ascertain standardized growth con-
ditions. For apoptosis induction, the cells were then
diluted to a concentration of 1 · 105/ml immediately
before addition of 1 (as a solution in DMSO).
1
mixture of compounds 4a–d as a colorless oil. H NMR
(CDCl3, 500 MHz): d = 0.47 (3H, s, CH3-20), 0.83–1.29
(3H, m, H-1,3,9), 1.14 (3H, s, CH3-19), 1.40–2.05 (10H, m,
H -1, H-2, H-3, H-5, H-6, H-7, H-11), 2.08–2.25 (2H, m,
H-7, H-12), 2.33–2.40 m (1H, H-12), 3.23 s, 3.24 s, 3.26 s,
3.27 s (ratio 1:1:1:1, all s 6H, 2CH3O), 3.52 (3H, s, OCH3–
C@O), 4.47 (1H, s, H-17), 4.82 (1H, s, H-17), 5.23 d, 5.27
s, 5.38 d (1H, H-16, for cis-isomers, J = 1.7 Hz), 5.48, 5.50,
5.59 (2H, all d, J = 1.7, 1.6, 1.6 Hz, H-14 and H-15).
Calcd. for C23H36O5: 392.22607. Found: 392.22225.
Determination of cell concentration and cell viability. Cell
viability was determined by CASYꢂ. Cell Counter + Ana-
lyzer System of Schaefer System GmbH (Reutlingen,
Germany). Settings were specifically defined for the
requirements of the used cells. With this system the cell
concentration is analyzed simultaneously in three different
size ranges: cell debris, dead cells, and viable cells were
determined in one measurement. BJAB cells were seeded
at a density of 1 · 105cells/ml and treated with different
concentrations of the pinustilbene compounds ranging
from 5 to 100 lM. After 24 and 48 h of incubation, cells
were resuspended properly and 100 ll of each well was
diluted in 10 ml CASYton (ready-to-use isotonic saline
solution) for an immediate automated count of the
cells.Measurement of the mitochondrial permeability tran-
sition. After incubation with different substance concen-
trations, BJAB cells were collected by centrifugation at
300g, 4 ꢁC for 5 min. Mitochondrial permeability
Step 2: to a solution of 1.95 g (5 mmol) of the mixture of
compounds 4a–d in 30 ml of dioxane was added 2.5 ml of
20% HCl. The mixture was stirred at rt for 30 min before
the reaction was quenched through addition of 15 ml of
3% aqueous K2CO3. The product was extracted with
dichloromethane and the combined extracts were washed
with water and brine. After drying over MgSO4 and
filtration, the solvent was evaporated and the residue
recrystallized from petrol ether to give 1.18 g (67%) of
+
20
pinusolide (1) as a yellow solid. Mp = 82–84 ꢁC; ½aꢁD
21.5 (c 7.1, CHCl3). {Lit.3: mp = 83–84 ꢁC; ½aꢁ20 +24 (c 2.0,
D
EtOH)}. IR (ATR): 758, 786, 874, 924, 1003, 1023, 1502,
1642, 1727, 1791, 3081 cmꢀ1
.
1H NMR (CDCl3,
500 MHz): d [ppm] = 0.39 (3H, s, CH3-20), 0.91 (1H, dt,
H-1, J = 13.3, 4.3 Hz), 0.96 (1H, ddd, H-3, J = 13.2, 12.8,
4.2 Hz), 1.07 (3H, s, CH3-19), 1.18 (1H, dd, H-5, J = 12.6,
2.9 Hz), 1.40 (1H, m, H-2), 1.47 (1H, m, H-11), 1.53 (1H,