Antiplasmodial and antitumor activity of dihydroartemisinin analogs derived via the aza-Michael addition reaction

Article abstract of DOI:10.1016/j.bmcl.2011.03.090

A series of dihydroartemisinin derivatives were synthesized via an aza-Michael addition reaction to a dihydroartemisinin-based acrylate and were evaluated for antiplasmodial and antitumor activity. The target compounds showed excellent antiplasmodial acti

Full text of DOI:10.1016/j.bmcl.2011.03.090

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2882–2886
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiplasmodial and antitumor activity of dihydroartemisinin analogs
derived via the aza-Michael addition reaction
Tzu-Shean Feng a,b, Eric M. Guantai c, Margo J. Nell d, Constance E. J. van Rensburg d,
Heinrich C. Hoppe b, Kelly Chibale a,e,
⇑
a Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
b Division of Pharmacology, University of Cape Town, Observatory 7925, South Africa
c Division of Pharmacology, School of Pharmacy, University of Nairobi, Box 19676-00202, Kenya
d Department of Pharmacology, University of Pretoria, Hatfield 0028, South Africa
e Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihydroartemisinin derivatives were synthesized via an aza-Michael addition reaction to a
dihydroartemisinin-based acrylate and were evaluated for antiplasmodial and antitumor activity. The
target compounds showed excellent antiplasmodial activity, with dihydroartemisinin derivatives 5, 7,
9 and 13 exhibiting IC50 values of 610 nM against both D10 and Dd2 strains of Plasmodium falciparum.
Received 30 January 2011
Revised 21 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Derivative 4d was the most active against the HeLa cancer cell line, with an IC50 of 0.37 lM and the high-
est tumor specificity.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Anticancer
Antiplasmodial
Aza-Michael addition reaction
Chloroquinoline
Dihydroartemisinin
Malaria remains the most dangerous and prevalent parasitic
disease that occurs in man. Over 200 million episodes of malaria
occur annually, occasioning approximately 1 million deaths every
year mainly among children and pregnant women in sub-Saharan
Africa.1–4 The main challenge to malaria control is that the malaria
parasite, and in particular the more virulent species Plasmodium
falciparum, has developed clinically significant resistance to many
classes of drugs,5,6 including possibly artemisinin-derived antimal-
arials.7,8 This situation has necessitated the current widespread ef-
forts aimed at developing new, highly efficacious drugs to treat
malaria.9
angiogenesis.16–18 On the other hand, chloroquine has been re-
ported as a notable apoptosis-inducing agent against MCF-7 hu-
man breast cancer cells, possibly by causing DNA damage and/or
suppression of the E2F1 protein.19 Related studies have revealed
the potential anticancer activity of chloroquinolinyl-chalcones
against human prostate LNCaP tumor cells.20
Our ongoing efforts in the direction of identifying new classes of
antimalarial and anticancer agents prompted us to undertake the
synthesis and biological evaluation of a variety of antimalarial
agents.21–23 The pharmacophore of chloroquine is generally ac-
cepted as the 4-amino-7-chloroquinoline unit24 (Fig. 1). As such
The antimalarial activities of artemisinins and chloroquine
(Fig. 1) have long been recognized and applied clinically.10–12 How-
ever, these compounds have also generated interest as potential
precursors for anticancer drug discovery. Artemisinins such as
artesunate have been found to be active against a variety of unre-
lated tumor cell lines, being quite active against leukemia and co-
lon cancer while moderately inhibiting cell growth of lung, breast,
ovarian, renal, prostate and CNS cancer cells.13–15 Other studies
have also shown that artemisinins can suppress the growth of hu-
man tumor xenografts in rats and mice, possibly by inhibition of
H
O
N
HN
O
O
2
1
3O
H
4O
Cl
N
Chloroquine
Artemisinin
⇑
Corresponding author. Tel.: +27 21 650 2553; fax: +27 21 689 7499.
E-mail address: Kelly.Chibale@uct.ac.za (K. Chibale).
Figure 1. Artemisinin and chloroquine (showing the 4-amino-7-chloroquinoline
pharmacophore).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.090

T.-S. Feng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2882–2886
2883
H
O
H
H
O
O
O
O
O
O
O
O
O
O
H
H
H
O
O
O
H
N
O
NH
N
n
O
Scheme 4. Reagents and conditions: appropriate amine (1.5 equiv), DBU
(0.5 equiv), CH3CN, N2, rt, 12 h, 54–80% yield.
Cl
Figure 2. General structure of some of the target compounds.
It has been reported that the length of the alkyl group between
the two nitrogen atoms on the 4-amino side chain may directly
influence the degree of biological activity of chloroquine-like com-
pounds. Chloroquine analogs where the alkyl chain between the
nitrogen atoms is shortened to 2–4 atoms, or lengthened to 10–
12 atoms, retained their activities against chloroquine resistant
strains, while those with intermediate chain lengths (5–8 carbon
atoms) had rather lower activities.25 Amines 2a–d (Scheme 2) were
therefore chosen such that the number of carbon atoms between
the two nitrogen atoms on the side chain spanned across the opti-
mal and intermediate biological activity range (2–6 carbon atoms).
The amine with the 5 carbon spacer between the nitrogen atoms
was not synthesized as the corresponding starting diamine was
not commercially available at the time of synthesis. Compound 3,
incorporating a piperazine moiety at the 4-position, was also
synthesized.
H
H
O
O
H
O
O
O
O
O
O
+
H
H
(i)
O
O
O
H
O
O
O
OH
1β (β−isomer)
1α (α−isomer)
Scheme 1. Reagents and conditions: (i) acryloyl chloride (1.2 equiv), Et3N
(1.2 equiv), CH2Cl2, N2, 0 °C to rt, 12 h.
derivatives 4a–d and 5 were synthesised by employing chloroquin-
oline amines in the aza-Michael addition reaction (Fig. 2).
The synthetic routes towards the target compounds were sim-
ple and straight forward, and commenced with the synthesis of
dihydroartemisinin acrylate (1). This was synthesized by reacting
dihydroartemisinin (DHA) with acryloyl chloride in CH2Cl2, in the
presence of Et3N (Scheme 1). The requisite acrylate was obtained
in 56% yield. 1H NMR of the crude material showed that the prod-
Using the protocol described by De et al.,26 2a–d were synthe-
sized in moderate to excellent yields from 4,7-dichloroquinoline
and an excess (approximately 20 equiv) of the corresponding alkyl
diamines (Scheme 2). The large excess of the diamine acted as a
base and as solvent, and was also necessary to avoid the formation
of terminally di-substituted amines. The synthesis of 3 was
achieved by reacting 4,7-dichloroquinoline with an excess of piper-
azine, using N-methylpyrrolidinone (NMP) as the solvent, with
Et3N as the base and a catalytic amount of K2CO3 (Scheme 2).27
The target compounds were synthesized via the conjugate addi-
uct was present as a 1:1 diastereomeric mixture of
mers, similar to the starting DHA which was also a 1:1 mixture
of - and b-anomers. Purification by column chromatography
a- and b-iso-
a
yielded the pure stereoisomers.
tion of 7-chloro-4-aminoquinoline amines 2a–d and 3 to the
a,b-
unsaturated system of DHA acrylate (1). Owing to its mildness
and operational simplicity, the aza-Michael addition reaction of
amines and Michael acceptors has recently attracted much atten-
tion as an alternative route for the synthesis of b-aminocarbonyl
compounds, of which the target compounds are examples
(Fig. 2). Yeom et al.28 reported a protocol for the aza-Michael addi-
tion employing 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a pro-
moter, and this mild method was chosen for the synthesis of our
target compounds from the DHA acrylate Michael acceptor 1 and
the amine intermediates 2a–d and 3 (Scheme 3).
H
N
NH2
Cl
N
HN
N
n
N
(i)
(ii)
Cl
Cl
Cl
N
n = 1: 2a
= 2: 2b
= 3: 2c
= 5: 2d
3
In order to prevent polymerization of the DHA acrylate 1,
1.5 equiv of the appropriate quinoline amine was used. Addition-
ally, the highly polar N,N0-dimethylformamide (DMF) was chosen
Scheme 2. Reagents and conditions: (i) piperazine (5.0 equiv), Et3N (1.2 equiv),
K2CO3 (0.5 equiv), NMP, N2, 150 °C, 4 h, 89% yield; (ii) H2N(CH2)nNH2 (20 equiv),
110–150 °C, 4 h, 64–95% yield.
H
H
O
O
N
O
O
O
H
O
H
O
O
O
O
Cl
(i)
H
N
O
(ii)
O
O
H
NH
N
N
n
O
O
Cl
n = 1: 4a
= 2: 4b
= 3: 4c
= 5: 4d
1β
5
Scheme 3. Reagents and conditions: DBU (0.5 equiv), DMF, N2, rt, 12 h; (i) 3 (1.5 equiv), (ii) 2a–d (1.5 equiv) (32–73% yields).

Table 1
Results from the in vitro biological evaluation of the intermediates and target compounds
H
O
O
O
O
H
O
6 - 13
Compd
C-10 stereochemistry
R
D10 IC50 (nM)
Dd2 IC50 (nM))
RIa
HeLa IC50
(l
M)
Primary lymphocytes IC50
(lM)
Tumor specificity
Resting
PHA-stimulated
1
a
a
b
—
—
—
—
—
—
—
—
—
—
—
—
5.85
7.54
9.27
12.4
1.6
1.6
4.9
5.2
4.7
0.9
1.1
1.0
1.3
1.1
0.9
1.0
6.37
2.88
7.22
9.83
11.24
15.69
27.58
9.47
5.58
10.13
0.37
39.58
30.53
ND
ND
ND
ND
ND
>50
21.52
32.40
19.03
44.61
11.47
9.22
ND
ND
ND
4.0
6.9
—
—
—
1b
2a
2b
2c
2d
3
4a
4b
4c
4d
5
—
—
—
—
—
b
b
b
b
b
231.1
240.3
482.9
2164.3
1463.2
20.6
18.7
24.0
55.6
10.1
1142.0
1251.7
2266.4
1861.4
1639.2
20.1
24.4
27.5
53.6
10.0
ND
ND
—
—
16.94
4.71
8.99
3.40
14.04
1.8
2.4
2.0
30.7
4.5
6.58
6
7
8
a
a
a
11.3
6.23
13.0
11.4
9.02
13.6
1.0
1.4
1.0
ND
ND
ND
-
N
N
N
O
4.80
6.02
>50
>50
27.27
30.45
5.7
5.0
9
a
5.3
6.16
1.2
4.48
>50
25.00
5.6
N
H
N
10
11
12
13
a
a
a
a
16.9
9.22
7.16
5.59
18.7
16.0
12.6
7.3
1.1
1.7
1.8
1.3
13.04
8.91
6.55
5.76
ND
ND
—
NH
>50
>50
>50
30.98
27.18
27.56
3.5
4.1
4.8
N
N
N
N
N
Artemisinin
DHA
Chloroquine
Cisplatin
25.5
4.1
30.6
ND
25.4
3.0
145.4
ND
1.0
0.75
4.7
-
ND
ND
9.67
0.142
a:b (1:1)
> 100
21.686
-
ND, not determined.
a
RI, resistance index, calculated as [IC50 (Dd2)]/[IC50 (D10)].

T.-S. Feng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2882–2886
2885
as solvent as it was capable of dissolving the extremely polar quin-
oline amines (a solvent that is unable to fully dissolve the quino-
line amine would create an environment where there was more
DHA acrylate than quinoline amine in the solution phase, which
would in turn encourage acrylate polymerization). The compounds
4a–d and 5 were synthesized using the DHA acrylate intermediate
1b, and hence all bore the b-stereochemistry at C-10. The effect of
the C10 orientation was envisaged to be studied by comparing the
mary resting and phytohaemagglutinin (PHA)-stimulated lympho-
cytes. Primary resting lymphocytes occur naturally in the body,
undergoing normal cell cycles. When these cells are stimulated
by PHA, the speed of their cell cycles is increased, mimicking that
of cancerous cells without being overtly cancerous themselves.
These therefore represent normal, primary cell lines commonly
used as controls for cancer cell lines. The activities of the selected
compounds are also tabulated in Table 1.
activities of acrylates 1
chromatography yielded the target compounds in low to moderate
yields.
a
and 1b. Purification by silica gel column
The selected compounds were found to be relatively less toxic
to the non-cancerous lymphocyte cell-lines, though they exhibited
low tumor specificity (calculated by taking the average of the IC50
in resting and PHA-stimulated primary lymphocytes and dividing
it by the IC50 in HeLa cells). Only one compound, 4d, displayed
good potential, with a tumor specificity of 31.
In summary, compounds, 7, 9 and 13 displayed the most potent
activities against D10 and Dd2 strains of P. falciparum. Their low
toxicity to primary human cell lines suggested a potentially large
therapeutic index for these compounds. In terms of development
as possible anticancer agents, 4d exhibited significant tumor spec-
ificity, and was identified as a good potential lead.
Apart from the target compounds where 7-chloro-4-amino-
quinoline amines were added to the DHA acrylate, commercially
available amines were also used in the conjugate addition
(Scheme 4). Acetonitrile was used as the solvent, as there were
no solubility problems with these low-molecular weight amines.
After the necessary reaction time, the solvent was simply removed
under reduced pressure, and the crude material purified by silica
gel column chromatography to furnish the final compounds 6–13
in moderate to good yields. These additional compounds were syn-
thesized using the DHA acrylate intermediate 1a, and hence all
bore the -stereochemistry at C-10.
a
Acknowledgments
Antiplasmodial results against D10 and Dd2 strains of P. falcipa-
rum (Table 1) showed that the target compounds 4-13 as well as
the DHA acrylate intermediates 1a and 1b displayed intermediate
activities relative to DHA and chloroquine, that is, more active than
chloroquine but less active to varying degrees than DHA in Dd2. In
D10, a number of derivatives (7, 9 and 13) showed comparable
activity to DHA.
This work was supported by the South African National Re-
search Foundation, NRF (T.-S.F., E.M.G.). Financial support received
from the South African Research Chair Initiative (SARChI) of the
Department of Science and Technology (DST) administered by
the NRF, as well as from the South African Medical Research Coun-
cil (MRC) and the University of Cape Town (UCT) is also gratefully
acknowledged (K.C.).
Also apparent was that the a- and b-acrylates (1a and 1b) syn-
thesized displayed comparable activities, suggesting that the ori-
entation at C-10 does not significantly influence the in vitro
activity of this type of compounds. This is unsurprising as both
Supplementary data
1
a and 1b show comparable activity; also, (b)-artesunate and
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.090.
(a)-artelinic acid have different orientations at C-10, but are both
active antimalarials.29
The activities of the quinoline amine precursors 2a–c against
the CQR Dd2 strain were much lower than against the CQS D10
strain (as indicated by the high resistance indices, RI, of >4.0), a
manifestation of cross-resistance with chloroquine. The activities
of intermediates 2a–d and 3 were also several times lower than
their corresponding DHA derivatives 4a–d and 5. The cross-resis-
tance noted above with some of the quinoline intermediates was
absent in these derivatives (RI values typically around 1), showing
that the incorporation of DHA aids 4-aminoquinolines in circum-
References and notes
1. Thwing, J.; Skarbinski, J.; Newman, R. D.; Barber, A. M.; Mali, S.; Roberts, J. M.;
Slutsker, L.; Arguin, P. M. Surveil. Summ. 2007, 56, 23.
2. WHO, World Malaria Report, 2008.
3. WHO Expert Committee on Malaria, WHO Technical Report Series 20th Report,
2000.
4. Kumar, A.; Katiyar, S. B.; Agarwal, A.; Chauhan, P. M. S. Curr. Med. Chem. 2003,
10, 1137.
5. Price, R. N.; Nosten, F. Drug Resist. Update 2001, 4, 187.
6. WHO, Guidelines for the Treatment of Malaria, 2006.
7. Noedl, H.; Se, Y.; Schaener, K.; Smith, B. L.; Socheat, D.; Fukuda, M. M. New Engl.
J. Med. 2008, 359, 2619.
8. Dondorp, A. M.; Norsten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong,
M.; Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.;
Day, N. P. J.; Lindegardh, N.; Socheat, D.; White, N. J. N. Engl. J. Med. 2009, 361,
455.
venting cross-resistance. Additionally,
a general decrease in
in vitro activities with increasing length of side-chains was ob-
served for the target derivatives 4a–d.
Interestingly, the conjugate adducts with commercially avail-
able amines were also quite active, and even exhibited better activ-
ities than those containing quinoline moieties. The most active
compounds out of this series were 7, 9 and 13, all exhibiting IC50
values of <10 nM against both strains of the malaria parasite. This
may indicate that the DHA component is generally responsible for
the majority of the antimalarial activity exhibited by all the conju-
gate adducts studied, and that the amine components of 6–13 (and
similarly the quinoline-amine components of 4a–d and 5) simply
serve to enhance this activity to varying extents.
9. Ridley, R. G. Nature 2002, 415, 686.
10. Coatney, G. R. Am. J. Trop. Med. Hyg. 1963, 12, 121.
11. Haynes, R. K. Curr. Top. Med. Chem. 2006, 6, 509.
12. Woodrow, C. J.; Haynes, R. K.; Krishna, S. Postgrad. Med. J. 2005, 81, 71.
13. Efferth, T.; Davey, M.; Olbrich, A.; Rücker, G.; Gebhart, E.; Davey, R. Blood Cells
Mol. Dis. 2002, 28, 160.
14. Efferth, T.; Dunstan, H.; Sauerbrey, A.; Miyachi, H.; Chitambar, C. R. Int. J.Oncol.
2001, 18, 767.
15. Krishna, S.; Bustamante, L.; Haynes, R. K.; Staines, H. M. Trends Pharmacol. Sci.
2008, 29, 520.
16. Oh, S.; Jeon, I. H.; Ahn, C. M.; Shin, W.-S.; Lee, S. Bioorg. Med. Chem. 2004, 12,
3783.
17. Dell’Eva, R.; Pfeffer, U.; Vené, R.; Anfosso, L.; Forlani, A.; Albini, A.; Efferth, T.
Biochem. Pharmacol. 2004, 68, 2359.
18. Moore, J. C.; Lai, H.; Li, J.-R.; Ren, R.-L.; McDougall, J. A.; Singh, N. P.; Chou, C.-K.
Cancer Lett. 1995, 98, 83.
19. Martirosyan, A. R.; Rahim-Bata, R.; Freeman, A. B.; Clarke, C. D.; Howard, R. L.;
Strobl, J. S. Biochem. Pharmacol. 2004, 68, 1729.
Cytotoxic activities of these compounds were assessed in HeLa
cells (human adenocarcinoma of the cervix cells), using cisplatin as
the control (Table 1). Compound 4d showed remarkable cytotoxic-
ity against the HeLa cell line, with an IC50 of 0.37 lM; the other
DHA conjugates displayed moderate cytotoxic activities, with
IC50 values in the low micromolar range. These results prompted
further tests against non-cancerous cells. Selected compounds
(based on cytotoxic activities in HeLa cells) were tested against pri-
20. Ferrer, R.; Lobo, G.; Gamboa, N.; Rodrigues, J.; Abramjuk, C.; Jung, K.; Lein, M.;
Charris, J. E. Sci. Pharm. 2009, 77, 725.

2886
T.-S. Feng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2882–2886
21. Walsh, J. J.; Bell, A. Curr. Pharm. Des. 2009, 15, 2970.
22. Guantai, E. M.; Ncokazi, K.; Egan, T. J.; Gut, J.; Rosenthal, P. J.; Smith, P. J.;
Chibale, K. Bioorg. Med. Chem. 2010, 18, 8243–8256.
26. De, D.; Byers, L. D.; Krogstad, D. J. J. Heterocycl. Chem. 1997, 34, 315.
27. Biot, C.; Glorian, G.; Maciejewski, L. A.; Brocard, J. S. J. Med. Chem. 1997, 40,
3715.
23. Olliaro, P. L.; Bloland, P. B.; Ed. Rosenthal, P. J. Humana Press 2001, 65.
24. Egan, T. J.; Hunter, R.; Kaschula, C. H.; Marques, H. M.; Mispolon, A.; Walden, J.
J. Med. Chem. 2000, 43, 283.
28. Yeom, C.-E.; Kim, M. J.; Kim, B. M. Tetrahedron 2007, 63, 904.
29. Li, Q. G.; Si, Y. Z.; Lee, P.; Wong, E.; Xie, L. H.; Kyle, D. E.; Dow, G. S. Parasitology
2003, 126, 283.
25. De, D.; Krogstad, F. M.; Byers, L. D.; Krogstad, D. J. J. Med. Chem. 1998, 41, 4918.