Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle

Article abstract of DOI:10.1016/S0960-894X(00)00578-3

Modification of artemisinin structure led us to the discovery of a novel class of antitumor compounds. These artemisinin derivatives containing cyano and aryl groups showed potent antiproliferative effect in vitro against P388 and A549 cells. This activit

Full text of DOI:10.1016/S0960-894X(00)00578-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 5±8
Novel Antitumor Artemisinin Derivatives Targeting G1 Phase
of the Cell Cycle
Ying Li,a,* Feng Shan,a Jin-Ming Wu,a Guang-Shao Wu,a
Jian Ding,b Dong Xiao,b Wei-Yi Yang,b Ghanem Atassi,c
Stephane Leonce,c Daniel-Henri Caignardd and Pierre Renardd
aDepartment of Synthetic Chemistry, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
bDepartment of Pharmacology, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
cInstitut de Recherche Servier, Suresnes, France
dADIR ET COMPAGNIE, Courbevoie, France
Received 9 August 2000; accepted 2 October 2000
AbstractÐModi®cation of artemisinin structure led us to the discovery of a novel class of antitumor compounds. These artemisinin
derivatives containing cyano and aryl groups showed potent antiproliferative e€ect in vitro against P388 and A549 cells. This
activity was re¯ected in P388 murine leukemia by an accumulation of cells in G1 phase, and induction of apoptosis. # 2000
Elsevier Science Ltd. All rights reserved.
Artemisinin (qinghaosu, 1) and its derivatives artem-
ether (3), artesunate (5), dihydroartemisinin (2) and
arteether (4) have been developed as new kinds of
antimalarial drugs active against multidrug-resistant
Plasmodium falciparum strains.1 Their rapid action,
powerful e€ect and good tolerance may be attributed to
the 1,2,4-trioxane moiety in their molecules. Such a
novel chemical structure from rich natural resources
encourages chemists and pharmacologists to attempt
further exploration. It was reported that artemisinin and
many of its analogues possessed not only antiparasitic
e€ect against P. falciparum, Schistosoma japonicum
and Clonorchis sinensi but also immuno-modulation
e€ects.2,3 Moreover, the cytotoxic e€ect of some
artemisinin derivatives attracted many investigators.4 16
and L1210 murine leukemia cell lines. Hence, a research
program on this series of compounds (6: R=H, CH3,
R0=substituted or unsubstituted aryl) was launched. In
this paper, the synthesis and the antiproliferative e€ect
of these compounds against P388 and A549 tumor cell
lines are brie¯y reported.
Chemistry
These compounds were synthesized in moderate yield by
acid catalyzed condensation of dihydroartemisinin and
corresponding cyanohydrin. Generally, a pair of iso-
mers were produced, but individual pure isomer could
be separated by careful column chromatography. Here-
with, the preparation of 6d and 6e (Fig. 1) as a typical
example and their antitumor activity is described. In
order to test whether the peroxy group is essential for
antitumor activity, 6f was prepared too.
In the early nineties, it was found in our laboratory that
cyano artemether (6a, which was ®rst synthesized by
researchers in Hoechest Company17) possessed inhibi-
tory e€ect against P. falciparum and no cytotoxic e€ect
against P388 cells in vitro. However, a pair of isomers (6b
and 6c: R=H, R0=phenyl) in test of antiproliferative
potential displayed in vitro cytotoxicity against P388
Under stirring, boron tri¯uoride diethyl etherate
(0.1 mL, 0.8 mmol) was added at 20 ꢀC to a dichloro-
methane solution (20 mL) of dihydroartemisinin (1.5 g,
5.3 mmol) and 2-(4-bromophenyl)-2-hydroxyacetonitrile
newly prepared from 4-bromobenzaldehyde (1.9 g,
10 mmol) with potassium cyanide. The reaction tem-
perature was then allowed to rise to room temperature.
*Corresponding author. Fax: +86-021-64370269; e-mail: yli@
mail.shcnc.ac.cn
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00578-3

6
Y. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 5±8
Figure 1. Chemical structures of artemisinin derivatives.
After 24 h, the solution was washed with aqueous
sodium bicarbonate, water and brine. The organic layer
was dried (MgSO4), ®ltered and the solvent evaporated.
The residue was chromatographed on silica gel to yield
0.57 g of 6d (22%) and 0.63 g of 6e (25%).18 From their
1H NMR spectra, both isomers were shown to be 12-b
derivatives of artemisinin, their sole di€erence was the
con®guration of C-16. The R con®guration of C-16 in
6d was determined by X-ray di€raction of 6d (Fig. 2).19
6f was prepared from 6e by reduction with zinc in acetic
acid according to the general procedure in 60% yield.20
Pharmacological Studies
Cell culture and cytotoxicity
The murine P388 leukemia and human lung A549 car-
cinoma cell lines were cultivated in RPMI 1640 medium
supplemented with 10% fetal calf serum, 2 mM l-glu-
tamine, 100 units/mL penicillin, 100 mg/mL streptomy-
cin, and 10 mM HEPES bu€er (pH: 7.4). Cytotoxicity
was measured by the microculture tetrazolium assay as
described.21 Brie¯y, P388 and A549 cells were exposed
to graded concentrations of drug for 48 and 96 h,
respectively (four doubling times). Results are expressed
as IC50, the concentration which reduced by 50% the
optical density of treated cells with respect to the density
of untreated cells.
Figure 2. X-ray crystal structure of 6d.
Cell cycle and apoptosis
P388 cells (2.5Â105/mL) were incubated for 21 h with
various concentrations of drugs then cells were ®xed by
70% ethanol and incubated for 30 min in PBS containing
100 mg/mL RNAse and 50 mg/mL propidium iodide (PI,
Sigma). For each sample, 104 cells were analyzed on an
Epics XL/MCL ¯ow cytometer (Beckman counter,

Y. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 5±8
Table 1. Inhibition of cell proliferation, cell cycle e€ect and induction of apoptosis
7
Compound
IC50 (nM)
A549
Cell cycle and apoptosis (P388)
% apoptose
P388
NIH-3T3
% G1
nM
6a
6b
6c
6d
6e
6f
VCR
Control
1855
238
48
12
11
79,432
1227
662
47
39
41,990
18
aNT
NT
65
67
61
62
63
NT
7
53
45
49
51
10,000
500
100
25
NT
3,050,000
5,120,000
NT
25
24,200
2
42
4
aNT, not tested.
France). Results are expressed as the percentage of cells
in G1 phase of the cell cycle and in the sub-G1 phase
(apoptotic cells). Apoptotic cells were also quanti®ed by
¯ow cytometry using the annexin-V-FITC labeling.
7. Liang, J.; Li, Y. Chin. J. Med. Chem. 1996, 6, 22.
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9. Posner, H. Bioorg. Med. Chem. Lett. 1997, 7, 1257.
10. Beekman, A. C. J. Pharm. Pharmacol. 1997, 49, 1254.
11. Beekman, A. C.; Barentsen, A. R. W.; Woerdenbag,
H. J.; Uden, W. V.; Pras, N.; Konings, A. W. T.; El-Feraly,
F. S.; Galal, A. M.; Wikstrom, H. V. J. Nat. Prod. 1997, 60,
325.
The results are shown in Table 1.
12. Yang, X.-P.; Pan, Q.-C.; Liang, Y.-G.; Zhang, Y.-L.
Cancer 1997, 16, 186 (in Chinese).
13. Tan, R.-X.; Zheng, W.-F.; Tang, H.-Q. Planta Medica
1998, 64, 295.
14. Beekman, A. C.; Wierenga, P. K.; Woerdenbag, H. J.;
Uden, W. V.; Pras, N.; Konings, A. W. T.; El-Feraly, F. S.;
Gala, A. M.; Wikstrom, H. V. Planta Medica 1998, 64, 615.
15. Zhang, X.; Yang, X. P.; Pan, Q. C. Zhong Cao Yao 1998,
29, 467.
Results and Discussion
As shown in Table 1 and other data to be published
later, 6d and 6e were the most potent to inhibit the
proliferation of P388 and A549 cells as compared to
other compounds. Furthermore, 6d and 6e induced an
accumulation of P388 cells in the G1 phase of the cell
cycle and approximately 50% of apoptotic cells.
16. Posner, G. H.; Ploypradith, P.; Parker, M. H.; O'Dowd,
H.; Woo, S.-H.; Northrop, J.; Krasavin, M.; Dolan, P.;
Kensler, T. W.; Xie, S.; Shapiro, T. A. J. Med. Chem. 1999,
42, 4275.
17. Venugopalan, B.; Bapat, C. P.; Karnik, P. J.; Lal, B.;
Chatterjee, D. K.; Iyer, S. N.; Rupp, R. H. Eur. Patent EP
362730, 1989; Chem. Abstr. 1991, 115, 86.
The structure±activity relationships can be drawn from
these results: (i) The aryl group seems to be necessary
for the potent antiproliferation. 6a without a phenyl
group was much less potent when compared to 6b and
6c. (ii) The peroxy group appears to be essential for
cytotoxicity as in the case of antimalarial activity. 6f
without a peroxy group showed poor antiproliferation.
(iii) The con®guration of C-16 has insigni®cant in¯u-
ence on the activity, 6d and 6e were equipotent for the
inhibition of the proliferation and the cell cycle pro-
gression, and for the apoptosis.
18. Data for 6b: mp 135±137 ꢀC. 1H NMR (400 MHz), d: 0.87
(3H, d, J=6.02 Hz, 10-CH3), 1.01 (3H, d, J=7.27 Hz, 11-
CH3), 1.43 (3H, s, 4-CH3), 5.24 (1H, d, J=3.62 Hz, 12-H),
5.28 (1H, s, 16-H), 5.68 (1H, s, 5-H), 7.44 (5H, m, aromatic
H). IR (KBr): 1457.9, 1103.1, 875.5 cm 1. Anal calcd for (C23
H29NO5): C 69.15, H 7.32, N 3.51. Found: C 68.86, H 7.35, N
3.33. 6c: mp 98±100 ꢀC. H NMR (400 MHz), d: 0.84 (3H, d,
1
J=7.48 Hz, 10-CH3), 0.95 (3H, d, J=6.59 Hz, 11-CH3), 1.44
(3H, s, 4-CH3), 4.81 (1H, d, J=3.43 Hz, 12-H), 5.50 (1H, s, 16-
H), 5.60 (1H, s, 5-H), 7.42 (5H, m, aromatic H). IR (KBr):
1452.2, 1101.2, 877.5 cm 1. Anal. calcd for (C23H29NO5): C
69.15, H 7.32, N 3.51. Found: C 68.85, H 7.42, N 3.18. 6d: mp
References and Notes
1. Meshnick, S. R.; Taylor, T. E.; Kamchonwongpaisan, S.
Microbiol. Rev. 1996, 60, 301.
128±129 ꢀC. H NMR (400 MHz), d: 0.90 (3H, d, J=6.10 Hz,
1
2. Wu, Y. L.; Li, Y. Med. Chem. Res. 1995, 5, 569.
3. (a) Merali, S.; Meshnick, S. R. Antimicrob. Agents Che-
mother. 1991, 35, 1225. (b) Ou-Yang, K.; Krug, E. C.; Marr,
J. J.; Berens, R. L. Antimicrob. Agents Chemother. 1990, 34,
1961. (c) Chen, Y. T.; Ma, L.; Mei, Q.; Tang, Y.; Liao, X.-G.
Chin. Med. J. 1994, 107, 673. (d) Liu, C.-M.; Qu-Yang, K.
Hunan Med. J. 1998, 15, 264 (in Chinese).
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1990, 56.
5. Woerdenbag, H. J.; Moskal, T. A.; Pras, N.; Malingre, T.
M.; El-Feraly, F. S.; Kampinga, H. H.; Konings, A. W. T. J.
Nat. Prod. 1993, 56, 849.
10-CH3), 1.00 (3H, d, J=7.36 Hz, 11-CH3), 1.43 (3H, s, 4-
CH3), 5.22 (1H, d, J=3.66 Hz, 12-H), 5.25 (1H, s, 16-H), 5.63
(1H, s, 5-H), 7.33 (2H, d, J=8.48 Hz, aromatic H), 7.57 (2H,
d, J=8.48 Hz, aromatic H). IR (KBr): 1592.9, 1488.8, 1375.0,
1101.2, 1031.7, 1010.5, 954.6, 875.5 cm 1. Anal. calcd for
(C23H28BrNO5): C 57.75, H 5.90, N 2.93. Found: C 57.80, H
6.07, N 2.85. 6e: mp 144±145 ꢀC. 1H NMR (400 MHz), d: 0.85
(3H, d, J=7.32 Hz, 10-CH3), 0.96 (3H, d, J=6.32 Hz, 11-
CH3), 1.44 (3H, s, 4-CH3), 4.79 (1H, d, J=3.40 Hz, 12-H),
5.46 (1H, s, 16-H), 5.58 (1H, s, 5-H), 7.32 (2H, d, J=8.40 Hz,
aromatic H), 7.55 (2H, d, J=8.30 Hz, aromatic H). IR (KBr):
1592.9, 1486.9, 1103.1, 1035.6, 954.6, 879.4 cm 1. Anal. calcd
for (C23H28BrNO5): C 57.75, H 5.90, N 2.93. Found: C 57.80,
H 5.89, N 2.96.
6. Yang, Y. H.; Li, Y.; Shi, Y. L.; Yang, J. D.; Wu, B. A.
Bioorg. Med. Chem. Lett. 1995, 5, 1791.

8
Y. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 5±8
aromatic H), 7.54 (2H, d, J=8.49 Hz, aromatic H). IR (KBr):
19. The X-ray crystallographic data have been deposited at the
Cambridge Crystallographic Data Centre, University Chemical
Laboratory, Lens®eld Road, Cambridge CB2 1EW, UK.
20. Data for 6f: mp 122±122.5 ꢀC. 1H NMR (400 MHz), d: 0.88
(3H, d, J=6.37 Hz, 10-CH3), 0.91 (3H, d, J=7.25 Hz, 11-
CH3), 1.47 (3H, s, 4-CH3), 4.78 (1H, d, J=4.06 Hz, 12-H),
5.34 (1H, s, 16-H), 5.47 (1H, s, 5-H), 7.31 (2H, d, J=8.46 Hz,
1
1592.9, 1486.9, 1394.3, 1211.1, 1103.0, 929.8, 867.8 cm
.
Anal. calcd for (C23H28BrNO4): C 59.74, H 6.10, N 3.03.
Found: C 59.85, H 5.78, N 3.31.
21. Leonce, S.; Perez, V.; Casablanca-Pignede, M. R.; Anstett,
M.; Bisagni, E.; Pierre, A.; Atassi, G. Investigational New
Drugs 1996, 14, 169.