Synthesis and cytotoxicity studies of artemisinin derivatives containing lipophilic alkyl carbon chains

Article abstract of DOI:10.1021/ol050230o

(Chemical Equation Presented) Cytotoxic artemisinin derivatives have been synthesized by a modular approach of "artemisinin + linker + lipophilic alkyl carbon chain". A strong correlation between the length of the carbon chains and the cytotoxicities against human hepatocellular carcinoma (HepG2) was revealed. Notably, compared with artemisinin (IC₅₀ = 97 μM), up to 200-fold more potent cytotoxicity (IC₅₀ = 0.46 μM) could be achieved by attachment of a C₁₄H₂₉ carbon chain to artemisinin via an amide linker.

Full text of DOI:10.1021/ol050230o

ORGANIC
LETTERS
2005
Vol. 7, No. 8
1561-1564
Synthesis and Cytotoxicity Studies of
Artemisinin Derivatives Containing
Lipophilic Alkyl Carbon Chains
Yungen Liu, Vincent Kam-Wai Wong, Ben Chi-Bun Ko, Man-Kin Wong,* and
Chi-Ming Che*
Department of Chemistry, The UniVersity of Hong Kong, Pokfulam Road,
Hong Kong, China
cmche@hku.hk; mkwong@hkusua.hku.hk
Received February 3, 2005
ABSTRACT
Cytotoxic artemisinin derivatives have been synthesized by a modular approach of “artemisinin
+
linker
+
lipophilic alkyl carbon chain”. A
strong correlation between the length of the carbon chains and the cytotoxicities against human hepatocellular carcinoma (HepG2) was
revealed. Notably, compared with artemisinin (IC50 97 M), up to 200-fold more potent cytotoxicity (IC50 0.46 M) could be achieved by
)
µ
)
µ
attachment of a C14H29 carbon chain to artemisinin via an amide linker.
Artemisinin (qinghaosu, 1) is a sesquiterpene lactone en-
doperoxide isolated from an ancient Chinese herb Artemisia
annua (Sweet wormwood) (Figure 1).1,2 Artemisinin and its
derivatives are highly effective against multidrug-resistant
malaria caused by Plasmodium falciparum, and they have
been currently used for the clinical treatment of malaria.3-11
Although the exact mechanism of their antimalarial activities
is not clear, substantial experimental evidence indicates that
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10.1021/ol050230o CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/18/2005

“artemisinin + linker + lipophilic alkyl carbon chain”.
Through systematic investigations on these structurally
related artemisinin analogues, it was found that the lipophilic
carbon chain plays an important role in determining the
cytotoxicities of the modified artemisinin analogues.
To maximize the structural diversity of artemisinin ana-
logues and the efficiency of their synthesis, aldehyde 4 and
acid 5 derived from 1 were prepared as the key intermediates.
Starting from 4 and 5, amides 6, esters 7, alcohols 8, and
ketones 9, 10, 11, and 13 bearing alkyl carbon chains of
different length were prepared (Scheme 1, overall yields from
Figure 1. Artemisinin (qinghaosu, 1).
the peroxide moiety of artemisinin and its derivatives is
crucial to their antimalarial activities.3,4,8a,12,13 Given the
remarkable success of artemisinin in the treatment of malaria,
there is growing interest in exploring the other therapeutic
properties of artemisinin.
Scheme 1. Synthesis of Artemisinin Derivatives 6-11 and 13
A search of the literature revealed that cytotoxic artemisi-
nin analogues could be synthesized via chemical modifica-
tions of the relatively nontoxic artemisinin at its C10/C16
position by covalent attachment of alkyl/aryl groups.14-18 In
addition, by judicious selection of linkers, a variety of
dimeric, trimeric, and even tetrameric cytotoxic artemisinin
derivatives have been prepared.19-21 However, given the
diverse chemical structures of these cytotoxic artemisinin
analogues, identification of the key factors contributing to
their cytotoxicities and rational design of new classes of
cytotoxic artemisinin analogues is difficult. In this regard, it
is of importance to conduct structure-activity relationship
(SAR) studies to assist the design and synthesis of new
cytotoxic artemisinin derivatives.
Here we report the synthesis and cytotoxicity studies of
artemisinin derivatives modified by a modular approach of
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L.; Ward, S. A.; Roberts, S. M.; Scheinmann, F.; Stachulski, A. V.; Posner,
G. H.; Park, B. K. J. Med. Chem. 2001, 44, 58-68.
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ogy 2004, 71, 1-9. (c) Chen, H.-H.; Zhou, H.-J.; Wang, W.-Q.; Wu, G.-D.
Cancer Chemother. Pharmacol. 2004, 53, 423-432. (d) Chen, H.-H.; You,
L.-L.; Li, S.-B. Cancer Lett. 2004, 211, 163-173.
1 were shown in parentheses) (see the Supporting Informa-
tion for the detailed synthetic schemes).
Apart from the naturally occurring D-six-membered ring
artemisinin, a series of D-five-membered ring artemisinin
derivatives 19-21 were synthesized from key intermediates
17 and 18 (Figure 2).22
(17) 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, 469-479.
(18) Haynes, R. K.; Chan, H.-W.; Lam, W.-L.; Tsang, H.-W.; Hsiao,
W.-L. PCT Int. Appl. WO 2000004026 A1, 2000.
(19) (a) 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, 4275-4280. (b) Posner, G. H.; Northrop,
J.; Paik, I.-H.; Borstnik, K.; Dolan, P.; Kensler, T. W.; Xie, S.; Shapiro, T.
A. Bioorg. Med. Chem. 2002, 10, 227-232. (c) Posner, G. H.; Paik, I.-H.;
Sur, S.; McRiner, A. J.; Borstnik, K.; Xie, S.; Shapiro, T. A. J. Med. Chem.
2003, 46, 1060-1065. (d) Jeyadevan, J. P.; Bray, P. G.; Chadwick, J.;
Mercer, A. E.; Byrne, A.; Ward, S. A.; Park, B. K.; Williams, D. P.;
Cosstick, R.; Davies, J.; Higson, A. P.; Irving, E.; Posner, G. H.; O’Neill,
P. M. J. Med. Chem. 2004, 47, 1290-1298. (e) Posner, G. H.; McRiner,
A. J.; Paik, I.-H.; Sur, S.; Borstnik, K.; Xie, S.; Shapiro, T. A.; Alagbala,
A.; Foster, B. J. Med. Chem. 2004, 47, 1299-1301.
The in Vitro cytotoxicity of the artemisinin derivatives
against a human hepatocellular carcinoma cell line, HepG2,
were conducted by using the MTT assay.23 The cytotoxicities
(22) For selected references on the synthesis and biological studies of
D-five-membered ring artemisinins, see: (a) Venugopalan, B.; Bapat, C.
P.; Karnik, P. J. Bioorg. & Med. Chem. Lett. 1994, 4, 751-752. (b)
Venugopalan, B.; Bapat, C. P.; Karnik, P. J.; Chatterjee, D. K.; Iyer, N.;
Lepcha, D. J. Med. Chem. 1995, 38, 1922-1927. (c) Haynes, R. K.;
Vonwiller, S. C.; Wang, H.-J. Tetrahedron Lett. 1995, 36, 4641-4642. (d)
Grellepois, F.; Chorki, F.; Crousse, B.; Ourevitch, M.; Bonnet-Delpon, D.;
Begue, J.-P. J. Org. Chem. 2002, 67, 1253-1260. (e) Avery, M. A.; Alvim-
Gaston, M.; Rodrigues, C. R.; Barreiro, E. J.; Cohen, F. E.; Sabnis, Y. A.;
Woolfrey, J. R. J. Med. Chem. 2002, 45, 292-303.
(20) Jung, M.; Lee, S.; Ham, J.; Lee, K.; Kim, H.; Kim, S. K. J. Med.
Chem. 2003, 46, 987-994.
(21) Ekthawatchai, S.; Kamchonwongpaisan, S.; Kongsaeree, P.; Tarn-
chompoo, B.; Thebtaranonth, Y.; Yuthavong, Y. J. Med. Chem. 2001, 44,
4688-4695.
(23) Mosmann, T. J. Immunol. Methods 1983, 65, 55-63.
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Org. Lett., Vol. 7, No. 8, 2005

carbon chain length resulted in a slightly drop of cytotoxicity,
i.e., 6h (C16H33, IC50 ) 0.79 µM) and 6i (C18H37, IC50 ) 4.2
µM). As summarized in a plot of IC50 values versus the
length of carbon chains (Figure 3), 6f-h with carbon chains
of 12- to 16-carbon exhibited the most potent cytotoxicities
while lower cytotoxicities were obtained for analogues
bearing shorter or longer carbon chains. Notably, the cyto-
toxicity of 6g (IC50 ) 0.46 µM) bearing a C14H29 carbon
chain is almost 200-fold more potent than that of the
unmodified 1 (IC50 ) 97 µM).
Apart from the effect of carbon chain length, we have also
examined the effect of linkers on the cytotoxicities. The
cytotoxicities of esters 7, alcohols 8, and ketones 9 against
HepG2 cell line are summarized in Table 2. With a carbon
Figure 2. D-Five-membered ring artemisinin derivatives 17-21.
(IC50 values) of 1 and amides 6a-i bearing linear alkyl car-
bon chains of different length are summarized in Table 1.
As a control, unmodified artemisinin 1 was found to be
weakly cytotoxic (IC50 ) 97 µM) aganist a HepG2 cancer
Table 2. Cytotoxicities of 7-9 against HepG2 Cell Line
Table 1. Cytotoxicities of 1 and 6a-i against HepG2 Cell
Line
compd (IC50, µM)
7a (>100)
7b (0.72)
R-8b (2.8)
â-8b (4.4)
9b (1.4)
7c (>100)
R-8c (>100)
â-8c (>100)
9c (>100)
R-8a (>100)
â-8a (>100)
9a (>100)
compd
1
6a
6b
6c 6d 6e 6f 6g
6h 6i
chain of C12H25, comparable IC50 values were obtained for
ester 7b (0.72 µM), R-alcohol 8b (2.8 µM), â-alcohol 8b
(4.4 µM), and ketone 9b (1.4 µM). These IC50 values were
also similar to that of amide 6f (1.2 µM). These results
indicate that the nature of linkers has no significant effect
on their cytotoxicties. Similar cytotoxicities were observed
for alcohols R-8 and â-8 suggesting that their cytotoxicities
were independent of the stereochemistry of the alcohol linker.
No cytotoxicity (IC50 > 100 µM) was observed for 7a,c,
8a,c, and 9a,c bearing carbon chains of C2H5 and C18H37.
The above studies indicate that attachment of lipo-
philic carbon chains significantly enhances the cytotoxicity
of artemisinin derivatives. However, an increase in lipophi-
licity does lead to poor water solubility which is problemati-
cal for future development of these compounds as drug can-
didates. To circumvent this problem, we incorporated polar
groups to enhance the water solubility. In this regard,
artemisinin analogues 10, 11, and 13 incorporated with polar
hydroxyl and carboxylic acid groups were prepared and
examined. The results are summarized in Table 3.
IC50 (µM) 97 >100 >100 17.6 9.5 2.8 1.2 0.46 0.79 4.2
cell line. No cytotoxicities (IC50 > 100 µM) were obtained
for 6a and 6b bearing short carbon chains of C2H5 and C4H9.
Interestingly, 6c with a longer carbon chain of C6H13
exhibited good cytotoxicity with IC50 ) 17.6 µM. As the
length of the carbon chains increased, the cytotoxicities
increased gradually from 6d (C8H17, IC50 ) 9.5 µM), 6e
(C10H21, IC50 ) 2.8 µM) to 6f (C12H25, IC50 ) 1.2 µM).
Notably, the most potent cytotoxicity (IC50 ) 0.46 µM) was
exhibited by 6g with a C14H29 group. Further increase in
It is interesting to note that incorporation of polar hydroxyl
and carboxylic groups at the terminal ends of the carbon
chains remarkably reduces the cytotoxicities. As shown in
Table 3, the cytotoxicity of 10 (IC50 ) 42.3 µM) with a
terminal hydroxyl group and 11 (IC50 > 100 µM) with a
terminal carboxylic acid group was considerably lower than
that of their methyl analogue 9d (IC50 ) 1.8 µM). These
findings indicate that the lipophilic end of the carbon chain
is essential for their cytotoxicity. Yet, for 13 with a polar
Figure 3. Plot of IC50 values vs the carbon chain length for 6c-i.
Org. Lett., Vol. 7, No. 8, 2005
1563

To investigate the selectivity between the cancer cell line
and normal cells, the cytotoxicities of 6f, 6g, 7b, and 9b
against normal lung fibroblast (CCD-19Lu) were examined.
As shown in Table 5, the modified artemisinins displayed a
moderate to good selectivity between the cancer and the
normal cells. Note that 9b was around 10-fold less cytotoxic
toward normal lung fibroblast cells (IC50 ) 14.5 µM) than
the HepG2 cancer cell line (IC50 ) 1.4 µM).
Table 3. Cytotoxicities of 9d, 10, 11, and 13 against HepG2
Cell Line
To demonstrate the importance of the peroxide functional-
ity on the cytotoxicities, deoxygenated analogue 22 was
prepared from 6h (Figure 4). Compared with the peroxide-
compd
9d
10
11
13
IC50 (µM)
1.8
42.3
>100
3.5
dihydoxyl group located at the amide linker, a slightly more
potent cytotoxicity (IC50 ) 3.5 µM) than 6d (IC50 ) 9.5
µM) was obtained. These findings indicate that the position
of polar groups on the carbon chain has a significant effect
on the cytotoxicity.
As illustrated in Table 4, potent cytotoxicities (IC50
)
0.47-3.7 µM) against HepG2 cell line were observed for
Figure 4. Deoxygenated artemisinin analogue 22.
Table 4. Cytotoxicities of 19-21 against HepG2 Cell Line
containing 6h (IC50 ) 0.79 µM), significant loss of cyto-
toxicity (IC50 >100 µM) was observed for the deoxygenated
22.24 This result clearly indicated the importance of the
endoperoxide moiety to the cytotoxicty.25
In summary, we have synthesized and examined the
cytotoxicities of a series of artemisinin derivatives bearing
lipophilic alkyl carbon chains. There is a strong correlation
between the length of the carbon chains and the cytotoxicities
of the modified artemisinins. Notably, potent cytotoxicity
(IC50 ) 0.46 µM) against human hepatocellular carcinoma
cell line could be achieved by attachment of a C14H29 carbon
chain to the weakly cytotoxic artemisinin 1 (IC50 ) 97 µM)
via an amide linker. Our results also indicated that polar
groups in the carbon chain has a significant effect on the
cytotoxicity.26
compd
19a
19b
19c
20
21
IC50 (µM)
1.3
0.77
0.74
3.7
0.47
the D-five-membered ring artemisinin analogues 19-21.
These cytotoxicities were comparable to their D-six-
membered ring artemisinin analogues. These findings indi-
cate that structural differences in the D ring has no significant
effects on the cytotoxicities. Notably, an IC50 Value of 0.47
µM was obtained for 21 with a C12H25 group connected by
a ketone linker.
Acknowledgment. This work was supported by The
University of Hong Kong (University Development Fund).
Table 5. Cytotoxicities of Modified Artemisinins on
CCD-19Lu
Supporting Information Available: Synthesis, com-
pound characterization data, and cytotoxicity studies of
artemisinin derivativies. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL050230O
(24) Deoxygenated analogue 22 was synthesized according to literature
procedures; see refs 4 and 13.
compd
6f
6g
7b
9b
(25) The importance of endoperoxide moiety on the biological activities
of artemisinins has been reported in the literature; see refs 17 and 19b.
(26) For a literature report regarding the relationship between lipophilicity
and neurotoxicity of antimalarial artemisinin analogues, see: Bhattacharjee,
A. K.; Karle, J. M. Chem. Res. Toxicol. 1999, 12, 422-428.
IC50normal (µM)
IC50cancer (µM)
2.7
1.2
2.9
0.46
4.1
0.72
14.5
1.4
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Org. Lett., Vol. 7, No. 8, 2005