4388
X. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4385–4388
6. Aherne, S. A.; O’Brien, N. M. Nutrition 2002, 18, 75.
Table 2
7. Nowakowska, Z. Eur. J. Med. Chem. 2007, 42, 125.
The antiproliferative effects of DHA, 7c, 7k and adriamycin (Adr) in P388 and P388/
8. (a) Borges-Argáez, R.; Balnbury, L.; Flowers, A.; Giménez-Turba, A.; Ruiz, G.;
Waterman, P. G.; Peña-Rodríguez, L. M. Phytomedicine 2007, 14, 530; (b)
Saydam, G.; Aydin, H. H.; Sahin, F.; Kucukoglu, O.; Erciyas, E.; Terzioglu, E.;
Buyukkececi, F.; Omay, S. B. Leukocyte Res. 2003, 27, 57.
9. Song, L. L.; Kosmeder, J. W.; Lee, S. K.; Gerhäuser, C.; Lantvit, D.; Moon, R. C.;
Moriarty, R. B.; Pezzuto, J. M. Cancer Res. 1999, 59, 578.
10. (a) Lin, A. J.; Lee, M.; Klayman, D. L. J. Med. Chem. 1989, 32, 1249; (b) Lin, A. J.;
Zikry, A. B. J. Med. Chem. 1997, 40, 1396.
11. General procedure for the preparation of dihydroartemisinin aliphatic ethers (7a–
Adr cells
Compounds
IG50 (lM)
P388
P388/Adr
DHA
Adr
7c
2.30 0.48
1.51 0.32
3.73 0.19
0.155 0.001
0.178 0.013
0.019 0.002
0.098 0.019
0.096 0.008
7k
7k).
A
mixture of the p-hydroxyacetophenone (10 mmol) and the
corresponding aldehyde (11 mmol) in anhydrous MeOH (30 mL) was stirred
at room temperature for 5 min. Then, NaOH (40 mmol) was added. The
reaction mixture was stirred at room temperature for 2 h. Then water (30 mL)
was added and the mixture was acidified with HCl (5 mol/L) until pH 8 was
reached. When the chalcones precipitated, they were filtered and recrystallized
from EtOH (95%) to yield a yellow solid 3. K2CO3 (0.03 mol) was added to a
stirred solution of compound 3 (10 mmol) in MIBK (50 mL) at 50 °C for 30 min
followed by adding 2-bromoethanol (9 mmol) and KI as catalysts, and stirring
at 120 °C for another 6 h. The reaction mixture was washed with NaOH (5%)
and water. The organic layer was dried (Mg2SO4) and filtered to yield a crude
product 5 which was then purified by crystallization (cyclohexane–acetone).
To a CH2Cl2 solution of dihydroartemisinin (2 mmol) Et3N (2.4 mmol) and
TFAA (2.4 mmol) were added at 0 °C. The resulting mixture was stirred at room
temperature for 18 h, and then compound 5 was added and stired for another
24 h. The reaction was washed with NaOH (5%) and brine. The organic layer
was dried (MgSO4) and concentrated in vacuo to give the crude products. Pure
products were obtained by column chromatography (silica gel) using
petroleum ether–ethyl acetate (8–10:1 v/v) as eluent. Spectral data of the
potent compounds: Compound 7h: yield 16.4%; mp 110–112 °C; IR (KBr):
Data shown are average SD in three independent experiments. The cells were
treated for 72 h.
pounds 7c and 7k. The percentages of apoptotic cells were deter-
mined by morphological observation after staining with acridine
orange (AO) and ethidium bromide (EB) as we reported.15 Cells
with nuclear shrinkage, blebbing, and apoptotic bodies were
counted as apoptotic cells and percentage was calculated after
observing 300 cells. As shown in Figure 2, compounds 7c and 7k
at a concentration of 0.2
cells after 24 h treatment while DHA induced a similar apoptotic
effect only at a concentration of 2 M (Fig. 2). The apoptotic effects
lM induced apoptosis in about 50% of
l
of these compounds were further confirmed by subG1 induction
which was determined by flow cytometry and by DNA fragmenta-
tion determined by DNA gel electrophoresis as we previously re-
ported.15 As shown in Figure 3A, compounds 7c and 7k induced
more than 80% of cells in the subG1 phase (apoptotic cells) at a
3439, 2921, 1657, 1602, 1511, 1384, 1261, 1028, 984, 874, 828, 805, 766 cmÀ1
;
ESI-MS: 617.7 (M+Na)+; 1H NMR (600 MHz, CDCl3): d (ppm) 8.03 (2H, d,
J = 8.7 Hz, Ar-H), 7.77 (1H, d, J = 15.6 Hz, –CH@CH–), 7.41 (1H, d, J = 15.6 Hz, –
CH@CH–), 7.24 (1H, d, J = 8.3 Hz, Ar-H), 7.17 (1H, s, Ar-H), 6.99 (2H, d,
J = 8.7 Hz, Ar-H), 6.91 (1H, d, J = 8.3 Hz, Ar-H), 5.48 (1H, s, H-12), 4.91 (1H, d,
J = 3.3 Hz, H-10), 4.22 (3H, m, –OCH2CH2O–), 3.83 (1H, m, –OCH2CH2O–), 3.96
(3H, s, –OCH3), 3.94 (3H, s, –OCH3), 2.65 (1H, m, H-9), 2.37 (1H, m, H-4), 2.05
(1H, m, H-4), 1.86 (1H, m, H-5), 1.47 (3H, s, H-14), 0.93 (3H, d, J = 5.7 Hz, H-16),
0.91 (3H, d, J = 7.3 Hz, H-15); 13C NMR (150 MHz, CDCl3): d (ppm) 188.7, 162.6,
151.3, 149.2, 144.2, 131.4, 130.7, 128.0, 123.0, 119.8, 114.4, 111.1, 110.1, 104.1,
102.2, 87.9, 81.1, 67.5, 66.3, 56.0, 55.9, 52.5, 44.4, 37.5, 36.4, 34.6, 30.9, 26.2,
24.7, 24.4, 20.4, 13.0.
concentration of 0.8
lM after 24 h of treatment. Both compounds
7c and 7k induced DNA fragmentation (Fig. 3B). To obtain the sim-
ilar subG1 and DNA fragmentation induction ability, 4
was required.
lM of DHA
It has been shown that artemisinin and its derivatives are
equally active towards drug-sensitive and -resistant cell lines.
Using mouse lymphoma P388 and adriamycin-resistant P388/Adr
cells, we have compared the cell growth inhibitory and cytotoxic
activities of DHA, 7c and 7k. As shown in Table 2, compounds 7c
and 7k were more potent than DHA in inhibiting growth of both
P388 and P388/Adr cells.
In conclusion, our data indicate that (1) the DHA aliphatic
ethers are in the b-isomer configuration, while the DHA esters
are in the a-isomer configuration; (2) DHA derivatives containing
12. (a) Jung, M.; Lee, K.; Jung, H. Tetrahedron Lett. 2001, 42, 3997; (b) Hindley, S.;
Ward, S. A.; Storr, R. C.; Searle, N. L.; Bray, P. G.; Park, B. K.; Davies, J.; O’Neill, P.
M. J. Med. Chem. 2002, 45, 1052.
13. General procedure for the preparation of dihydroartemisinin esters (10a–10d): To
a stirred solution of compound 3 (10 mmol) in MIBK (50 mL) K2CO3 (0.03 mol)
was added at 50 °C for 30 min followed by adding ethyl chloroacetate (9 mmol)
and KI as catalysts and stirring at 120 °C for 6 h, and then water (50 mL) was
added. The mixture was washed with NaOH (5%) and water. The organic layer
was dried (Mg2SO4) and filtered to yield crude product 4 which was added to
25% NaOH (30 mL) for 1 h. The mixture was acidified with HCl (3 mol/L) until
acid and filtered to yield crude product 6 which was purified by crystallization
(ethanol–water). Et3N (2.4 mmol) was added to a CH2Cl2 of DHA (2 mmol),
freshly prepared compound 8 from compound 6 and sulfoxide chloride. The
resulting mixture was stirred at room temperature for 2 h. The reaction
mixture was washed with HCl (1 mol/L) and brine. The organic layer was dried
(MgSO4) and concentrated in vacuo to give the crude products. Pure products
were obtained by column chromatography (silica gel) using petroleum ether–
ethyl acetate (15:1 v/v) as eluent. Spectral data of the potent compounds:
Compound 10c: yield 11.7%; mp 137–139 °C; IR (KBr): 3448, 2928, 1772, 1658,
1600, 1510, 1028, 824 cmÀ1; ESI-MS: 579.2 (M+H)+, 601.2 (M+Na)+, 617.2
(M+K)+; 1H NMR (300 MHz, CDCl3): d (ppm) 8.03 (2H, d, J = 8.8 Hz, Ar-H), 7.78
(1H, d, J = 15.6 Hz, –CH@CH–), 7.61 (2H, d, J = 8.7 Hz, Ar-H), 7.42 (1H, d,
J = 15.6 Hz, –CH@CH–), 7.00 (2H, d, J = 8.8 Hz, Ar-H), 6.94 (2H, d, J = 8.7 Hz, Ar-
H), 5.90 (1H, d, J = 9.9 Hz, H-10), 5.46 (1H, s, H-12), 4.79 (2H, m, –COCH2O–),
3.86(3H, s, –OCH3), 2.62 (1H, m, H-9), 2.38 (1H, m, H-4), 2.05 (1H, m, H-4), 1.45
(3H, s, H-14), 0.97 (3H, d, J = 5.6 Hz, H-16), 0.84 (3H, d, J = 7.1 Hz, H-15); 13C
NMR (150 MHz, DMSO): d (ppm) 188.7, 167.9, 161.7, 161.6, 143.8, 131.8, 131.2,
127.9, 119.9, 115.0, 114.8, 104.1, 93.0, 91.2, 80.3, 65.0, 55.8, 51.5, 45.0, 36.4,
36.3, 34.1, 32.1, 26.0, 24.6, 21.4, 20.5, 12.1.
a chalcone are more potent in growth inhibition and cytotoxicity
than DHA; (3) DHA ethers are more effective than DHA esters in
growth inhibition and cytotoxicity.
Acknowledgment
This work was supported in part by the Joint Research Fund for
Overseas Chinese Young Scholars of the National Natural Science
Foundation of China (30328030).
References and notes
1. Klayman, D. L. Science 1985, 228, 1049.
2. Efferth, T. Drug Resist. Update 2005, 8, 85.
3. Li, Y.; Wu, J. M.; Shan, F.; Wu, G. S.; Ding, J.; Xiao, D.; Han, J. X.; Atassi, G.;
Leonce, S.; Caignard, D. H.; Renard, P. Bioorg. Med. Chem. 2003, 11, 977.
4. (a) Liu, Y.; Wong, V. K.; Ko, B. C.; Wong, M. K.; Che, C. M. Org. Lett. 2005, 7, 1561;
(b) Wu, J. M.; Shan, F.; Wu, G. S.; Li, Y.; Ding, J.; Xiao, D.; Han, J. X.; Atassi, G.;
Leonce, S.; Caignard, D. H.; Renard, P. Eur. J. Med. Chem. 2001, 36, 469.
5. Liu, Y. C.; Hsieh, C. W.; Wu, C. C.; Wung, B. S. Life Sci. 2007, 80, 1420.
14. Oh, S.; Jeong, I. H.; Ahn, C. M.; Shin, W. S.; Lee, S. Bioorg. Med. Chem. 2004, 12,
3783.
15. Liu, D.; Song, D.; Guo, G.; Wang, R.; Lv, J.; Jing, Y.; Zhao, L. Bioorg. Med. Chem.
2007, 15, 5432.