Antitumour and antimalarial activity of artemisinin-acridine hybrids

Article abstract of DOI:10.1016/j.bmcl.2009.02.028

Artemisinin-acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2-4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

Full text of DOI:10.1016/j.bmcl.2009.02.028

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2033–2037
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antitumour and antimalarial activity of artemisinin–acridine hybrids
Michael Jones a, Amy E. Mercer b, Paul A. Stocks e, Louise J. I. La Pensée a, Rick Cosstick a, B. Kevin Park b,
Miriam E. Kennedy c,d, Ivo Piantanida d, Stephen A. Ward e, Jill Davies e, Patrick G. Bray e, Sarah L. Rawe c,
,
*
Jonathan Baird f, Tafadzwa Charidza f, Omar Janneh b,f, Paul M. O’Neill a,
*
a Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, UK
b Department of Pharmacology, University of Liverpool, UK
c Focas Institute, Dublin Institute of Technology, Camden Row, Dublin, D8, Ireland
d Division of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, Zagreb, Croatia
e Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
f University of Ulster, Cromore Road, Coleraine, Co., Londonderry, BT52 1SA Northern Ireland, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Artemisinin–acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell
lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2–4-fold increase in
activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA)
and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells
was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 14 November 2008
Revised 5 February 2009
Accepted 6 February 2009
Available online 11 February 2009
Keywords:
Artemisinin
Acridine
Antimalarial
Anticancer
Apoptosis
DNA
Confocal microscopy
Artemisinin 1 and its semisynthetic and synthetic analogues are
effective antimalarial agents and are used to treat chloroquine
resistant strains of the disease. Since the early 1990s they have also
been shown to have antiproliferative and antitumour activity.1–5
Their activity is greatest against rapidly proliferating neoplastic
cells with high iron content, since their mode of action almost cer-
tainly involves iron(II) catalysed reductive cleavage of the peroxide
bond leading to the formation of C-centred radical or cationic
intermediates able to alkylate biomolecules and induce cell
death.6–10 We and others have postulated that a possible cellular
target of these alkylating intermediates is DNA10,11 and hoped to
enhance their antitumour properties by preparing DNA-targeted
1,2,4-trioxane-acridine hybrids. Acridines are known to intercalate
with DNA and have been employed as antibacterial, antiparasitic
and antitumour agents,12,13 moreover their fluorescent properties
allow the use of confocal microscopy to examine the accumulation
and cellular location of these drug hybrids.14
the aim of creating bitherapies that have improved biological activ-
ity and are less susceptible to the development of drug resis-
tance.15–17 We have synthesised a short series of artemisinin–
acridine hybrids in which a 1,2,4-trioxane derived from artemisi-
nin has been covalently linked to the 9-diaminoalkyl-6-chloro-2-
methoxyacridines 2-5, and evaluated these hybrids for their
in vitro antitumour and antimalarial activity (Fig. 1).
The hybrids were designed to incorporate a metabolically stable
C-10 carba linkage at the 1,2,4-trioxane moiety, therefore the car-
boxylic acid 6 was prepared from C-10 allyldeoxoartemisinin.18
Ozonolysis of the terminal double bond in methanol and reduction
of the intermediate ozonide with triphenylphosphine afforded the
aldehyde in 76 % yield, followed by oxidation with sodium chlorite
H
NH
NH2
HN
N
O
n
O
OMe
OMe
O
H
Hybrid drugs are formed by covalently linking two distinct
chemical moieties with differing biological modes of action with
O
Cl
N
Cl
N
5
2, n=1
3, n=2
4, n=3
O
1
* Corresponding authors.
E-mail address: p.m.oneill01@liv.ac.uk (P.M. O’Neill).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.028

2034
M. Jones et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2033–2037
Table 1
to give the carboxylic acid in quantitative yield. Treatment of the
acid with oxalyl chloride followed by addition of the appropriate
9-(diaminoalkyl)acridine afforded the hybrids 7–10 in moderate
to good yields (55–74 %), Scheme 1.19 The 9-(diaminoalkyl)acri-
dines 2–5 were prepared from 6,9-dichloro-3-methoxyacridine
and the corresponding diamines in the presence of phenol.
The hybrids were evaluated for their in vitro activity against
four tumour cell lines (HL60, Colon HT29-AK and Breast MDA-
MB-231 and MCF-7)20 and against a chloroquine sensitive strain
of P.falciparum (3D7)21 relative to DHA as the control (Tables 1
and 2).
The hybrids were cytotoxic in the cell lines evaluated, with the
exception of 10, towards HT29-AK (Table 1). The rank order of sen-
sitivity of the cells to the hybrids were as follows: HL60 > MCF-
7 > MDA-MB-231 > HT29-AK. The observed cytotoxicity of the hy-
brids and DHA against HL60 cells was anticipated since this cell
line is characterised by its rapid proliferation and high iron con-
tent. In HL60, MDA-MB-231 and MCF-7 cells, the hybrids displayed
activity 2–4-fold greater than DHA. In HT29-AK cells, only hybrid 8
was more active than DHA; the remaining hybrids displayed only
moderate activity with the acridine apparently inhibiting the activ-
ity of the 1,2,4-trioxane. In HL60 cell lines the side-chain acridine 2
Cytotoxicity results for compounds 7–10 and DHA against HL60, HT29-AK, MDA-MB-
231 and MCF-7 tumour cell lines
Compds
HL60
HT29-AK
MDA-MB-231
MCF-7
Cytotoxicity IC50
2
7
8
9
10
DHA
, l
Ma
12.5 ( 0.25)
1.17 ( 0.35)
3.08 ( 0.13)
1.58 ( 0.72)
0.56 ( 0.17)
2.41 ( 0.71)
6.57 ( 1.34)
193.55 ( 22.88)
9.91 ( 1.85)
247.27 ( 44.13)
>750
8.12 ( 1.13)
ND
48.98 ( 7.72)
11.64 ( 0.23)
43.30 ( 1.39)
21.14 ( 0.86)
99.76 ( 10.96)
13.69 ( 1.78)
11.85 ( 0.19)
11.70 ( 0.22)
3.51 ( 0.42)
45.23 ( 3.54)
16.12 ( 1.10)
a
Values are means of three experiments; standard deviation is given in paren-
theses. ND—not determined.
Table 2
Results for antimalarial activity of compounds 7–10 against chloroquine sensitive
3D7 P. falciparum
Compds
IC50, nMa
7
8
9
10
5.96 ( 1.50)
22.42 ( 1.97)
20.34 ( 3.11)
289.52 ( 10.52)
3.53 ( 1.91)
2.30 ( 1.50)
Artemether
DHA
was also examined and shown to have activity of 12.5
lM. This
indicates that the addition of the endoperoxide to this unit en-
hances cytotoxicity; in breast cancer cell lines HT29-AK and
MDA-MB-231 this is clearly not the case since the hybrids are less
potent than 2. The hybrids were also evaluated for their in vitro
antimalarial activity against chloroquine sensitive 3D7 strain of
P. falciparum but none of them were more active than the positive
controls (DHA and artemether). Indeed, only 7 had good activity,
while 8 and 9 displayed moderate activity and 10 was more than
a 100 times less active than DHA (Table 2).
a
Values are means of three experiments; standard deviation is given in
parentheses.
manner, reaching a significant level at 1
imum effect, 98 3% of cells depolarised, was observed at 10
after 24 h. Analysis of cellular DNA content with PI staining showed
concentration-dependent formation of a sub-G0/G1 population with
lM increasing until a max-
l
M
a maximum effect of 57 2% of cells at 10 lM after 24 h (Fig. 2A).
The ability of hybrid 8 to induce apoptosis was assessed in HL60
cells by flow cytometric measurement of mitochondrial membrane
depolarisation,22 DNA degradation23 and Western blot analysis of
caspase-3 activation.24 This compound was shown to induce mito-
chondrial membrane depolarisation in a concentration-dependent
Western blot analysis of caspase-3 activity also showed concentra-
tion-dependent appearance of the catalytically active subunit of
processed caspase-3 with a concomitant decrease of the inactive
32 kDa precursor (Fig. 2B). Together these results are strongly indic-
ative of the ability of hybrid 8 to induce cell death by apoptosis.
H
O
H
H
O
O
i, ii
iii
O
O
O
O
O
O
H
H
H
O
O
O
OH
O
O
6
iv
iv
H
H
O
O
O
O
O
O
H
H
O
O
O
O
NH
N
N
HN
N
7, n=1
8, n=2
9, n=3
n
OMe
OMe
Cl
Cl
N
10
Scheme 1. Reagents and conditions: (i) O3, MeOH, À78 °C, 1 h; (ii) PPh3, MeOH, À78 °C to rt; (iii) NaClO2, 2-methyl-2-butene, NaH2PO4, t-BuOH/H2O, rt, 2 h; (iv) (COCl)2,
CH2Cl2, 0 °C?rt, 90 min then diaminoacridine (2–5), NEt3, CH2Cl2, 0 °C?rt, 16 h.

M. Jones et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2033–2037
2035
Figure 2. (A) Concentration-dependent induction of mitochondrial depolarisation and DNA degradation by compound 8 in HL-60 cells (24 h). Key- Open circle: percentage of
cell population with depolarised mitochondria. Filled circles: percentage of cell population with degraded DNA (sub G0/G1 population). Results are the mean SD of three
independent sets of experiments. ***p < 0.001 and *p < 0.05 significance of data compared with drug blank as tested by the Mann-Whitney U test for nonparametric data. (B)
Concentration-dependent processing of caspase-3 induced by compound 8 in HL60 cells (24 h). Lane 1: control without 8, Lanes 2–5 increasing concentrations of 8: Lane 2,
0.5 lM, Lane 3: 1.0 lM, Lane 4: 5 lM and Lane 5: 10 lM.
Confocal microscopy was used to determine the cellular loca-
molecules (Fig. 3 D). Similar experiments were performed in the
presence of a ferric iron chelator (desferrioxamine (DFO), and un-
der these conditions the hybrid was removed from the cells on
washing with the buffer (data not shown).14 This result implies
that chelatable iron catalysed reductive cleavage of the peroxide
bond is required for covalent binding of the hybrid to the cellular
targets. (Since DFO is selective for ferric iron it is apparent that
intraparasitic reduction of iron to its ferrous form must be
achieved to facilitate endoperoxide bond cleavage.)
tion of both hybrid 7 and 9-(1,2-diaminoethyl)-6-chloro-2-meth-
oxyacridine 2 in treated parasite infected erythrocytes and MRC-
50 human lung fibroblast cells (7 was selected based on the fact
that this hybrid was the most potent antimalarial in the series).
Both hybrid 7 and 9-aminoacridine 2 were shown to accumulate
selectively in infected erthyrocytes and on the nuclear membrane
of MRC-5 cells (Fig. 3A–C). In malaria parasites acridine 2 was eas-
ily removed by washing with a buffer solution (Fig. 3B) whereas
the endoperoxide hybrid 7 remained, implying covalent binding
of the hybrid (and not the acridine) to intraparasitic cellular bio-
Finally, the in vitro affinity of hybrid 7 and acridine 2 for calf thy-
mus (ct)-DNA and poly A–poly U was investigated spectrophoto-
Figure 3. Confocal microscopy studies. (A) Accumulation of 2 in infected erythrocytes; (B) following perfusion of cells with buffer. (C) Accumulation of 7 in MRC-5 human
lung fibroblasts. (D) Accumulation of 7 in infected erythrocytes. Attempts to remove 7 by cell perfusion failed in line with previous studies.

2036
M. Jones et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2033–2037
lower concentrations, at which no precipitation was observed.
Compound 2
Compound 7
0.20
0.15
0.10
0 .05
0.00
Emission of both 2 and 7 was strongly quenched by addition of
ct-DNA and poly A–poly U and processing of the fluorimetric titra-
tion data by Scatchard equation26 gave similar binding constants (in
the range Ks ꢀ 106 MÀ1) and ratio binding n[compound]/[polynucleotide] of
about 0.2. In thermal melting studies addition of 2 generally yielded
a significantly stronger stabilisation effect on double stranded
polynucleotides than its hybrid analogue 7 (Table 3).
r = 0
r = 0
r = 0.22
r = 0.33
However, CD studies were the most elucidating. CD analysis of
ct-DNA (Fig. 5) and poly A–poly U in the presence of 2 showed an
increase in the CD of DNA/RNA (220–300 nm range) and a weak
negative induced CD at about 430 nm (absorption attributed to
the acridine), characteristic of intercalation.27 There was no change
in the CD spectra of either ct-DNA or poly A–poly U upon addition
of hybrid 7, indicating that the acridine moiety of the hybrid is not
intercalated into the double helix. That would imply that linking
the acridine moiety to the trioxane prevents intercalation in dou-
ble stranded polynucleotide or at least diminishes its role in bind-
ing, presumably due to the intramolecular aromatic stacking
interaction between acridine and trioxane moiety (visible from
comparison of the UV/vis spectrum of 2 and 7). Consequently,
changes in the electronic emission and absorption spectra of 7
upon addition of studied polynucleotides and the increase in melt-
ing temperature are caused either by counterion stabilisation or by
mixed binding modes including some electrostatic, hydrophobic
and van der Waals interactions as well as intercalation of the acri-
dine as a minor contribution.28
In summary, artemisinin–acridine hybrids display promising
antitumour activity in HL60, MDA-MB-231 and MCF-7 cells. They
have been shown to induce cell death by apoptosis and to cova-
lently bind to their intraparasitic cellular targets in the presence
of iron(II). Linking an acridine to an artemisinin derivative was
shown to enhance antitumour activity in the HL60 leukaemia cell
line (comparing 2 and 7), while it had a largely inhibitory effect
in HT29-AK and MDA-MB-231 cells. Compared to DHA the hybrids
had decreased in vitro antimalarial activity. Although hybrid 7 was
shown to have high affinity toward DNA/RNA, intercalation of hy-
brid 7 into DNA/RNA in vitro was not dominant binding mode, pre-
sumably due to the competitive intramolecular aromatic stacking
interaction between acridine and trioxane moiety. Future work
will involve the synthesis and evaluation of redesigned hybrids
in which longer linkers between the two moieties and a variety
of DNA targeting groups will be used in the hope of achieving
greater DNA affinity and enhanced antitumour activity.
350
400
450
500
λ
/ nm
Figure 4. UV/vis spectra of 2 and 7 (c = 5 Â 10-5 mol dmÀ3) and complexes 2/ctDNA
and 7/ctDNA at different ratios r = [2 or 7]/[ctDNA], pH 7, Na cacodylate buffer,
I = 0.05 M.
metrically using thermal melting analysis, electronic absorption,
fluorimetric binding titrations and circular dichroism (CD) analysis.
Addition of ct-DNA at concentrations close to equimolar in respect
of studied compounds yielded significant batochromic shifts in the
electronic absorption spectra of 7 and 2 (Fig. 4) at wavelengths
characteristic for acridine moiety.25 However, at higher excess of
ct-DNA over 2, 7 precipitation occurred, hampering collection of en-
ough data points for accurate processing by Scatchard equation.26
At variance to UV/vis titrations, strong fluorescence of 2 and 7 al-
lowed fluorimetric titrations with polynucleotides at significantly
Table 3
D
r
Tm valuesa (°C) of studied ds-polynucleotides upon addition of 2 and 7 at ratio
b = 0.3 at pH 7, Na cacodylate buffer, 0.02 M (I = 0.005 M)a
Polynucleotide
D
Tm w/2 (°C)
DTm w/7 (°C)
ctDNA
9.3
2.2
2.9
3.1
2.4
0.6
Poly A–poly U
Poly A–poly Tc
a
Error in
D
Tm
r = [compound]/[polynucleotide].
With the exception of this experiment (where I = 0.05 M) melting studies were
: 0.5 °C.
b
c
performed at I = 0.005 M due to the very weak effects at I = 0.05 M.
0.1
B
A
ΔAbs = +5 nm
3
2
265.4 nm
0.0
-0.1
-0.2
-0.3
-0.4
1
0
ct-DNA
r=0.05
r=0.1
r=0.2
r=0.3
3.0
2.5
2.0
1.5
-1
-2
-3
-4
0.0 0.1
0.2 0.3 0.4 0.5 0.6
r
230 240 250 260 270 280 290 300 310 320
390
400
410
420
λ / nm
430
440
450
λ / nm
Figure 5. (A) Changes in the CD spectrum of ct-DNA (c = 1Â10-5 mol dmÀ3) upon addition of 2 (inset: changes at k = 280 nm at various ratios r = [2]/[ctDNA]); (B) induced CD
band of acridine moiety at various ratios r (c(ct-DNA) = 1Â10-4 mol dmÀ3). Carried out at pH 7, Na cacodylate buffer, I = 0.05 M.

M. Jones et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2033–2037
2037
7.20 (1H, dd, J = 2.1 Hz, 9.3 Hz), 5.02 (1H, s), 4.0 (1H, s), 3.91 (1H, m), 3.70 (3H,
s), 3.43, (2H, m), 3.28 (2H, m), 2.48 (1H, m) 2.45–2.17 (3H, m) 2.07–1.20 (10H,
m) including 1.41 (3H, s) 0.96 (3H, d, J = 5.9 Hz) and 0.89 (3H, d, J = 7.67 Hz);
13C NMR: (CDCl3, 400 MHz) d 174.7, 156.8, 148.6, 148.0, 134.9, 131.0, 128.9,
128.0, 127.3, 121.9, 118.6, 104.6, 98.0, 96.2, 92.8, 84.5, 67.5, 56.2, 54.0, 44.6,
37.3, 36.3, 30.8, 30.4, 29.5, 25.3, 23.7, 22.8, 19.0, 18.4, 11.8, 11.4; HRMS (CI):
C33H40ClN3O6 [M+H]+ requires 611.1479 found: 611.1499; Anal. Calcd for
C33H40ClN3O6: C, 64.96; H, 6.61; N, 6.89. Found: C, 65.00; H, 6.78; N, 6.93.
20. Measurement of cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay: HL-60 Cells (2.5 Â 104/well) were
plated in triplicate, in flat bottom 96-well plates and were exposed to 0.01–
Acknowledgments
MJ would like to thank the BBSRC for a studentship (BBS/S/P/
2003/10353) and L.L. would like to thank the EPSRC for a DTA
award. T.C. was a recipient of undergraduate bursary from the Nuf-
field Foundation. S.R. would like to thank Science Foundation Ire-
land and the National Development Plan for funding (M.K.). I.P. is
grateful to Ministry of Science of Croatia (Project 098-0982914-
2918) for financial support.
100
viability measurements using the MTT assay were carried out by the addition
of 20 L of MTT solution (5 mg/mL in HBSS) to each well and incubating for 3 h
at 37 °C. Thereafter, 100 of lysing solution (20% w/v sodium
lM of each compound for 72 h (5% CO2 at 37 °C). Following incubation, cell
l
References and notes
lL
a
dodecylsulfate, 50% w/v N,N-dimethyl formamide) was added to each well to
dissolve the formazan crystals and incubated for an additional 4 hrs. The
absorbances of the samples were measured at a test wavelength of 570 nm and
1. Woerdenbag, H. J.; Moskal, T. A.; Pras, N.; Malingre, T. M.; Elferaly, F. S.;
Kampinga, H. H.; Konings, A. W. T. J. Nat. Prod. 1993, 56, 849.
2. Jung, M. Bioorg. Med. Chem. Lett. 1997, 7, 1091.
a
reference wavelength of 590 nm with a plate reader (MRX, Dynatech
3. Posner, G. H.; Ploypradith, P.; Hapangama, W.; Wang, D. S.; Cumming, J. N.;
Dolan, P.; Kensler, T. W.; Klinedinst, D.; Shapiro, T. A.; Zheng, Q. Y.; Murray, C.
K.; Pilkington, L. G.; Jayasinghe, L. R.; Bray, J. F.; Daughenbaugh, R. Bioorg. Med.
Chem. 1997, 5, 1257.
Laboratories). All results were expressed as a percentage of vehicle only cells.
The IC50 values were calculated from individual inhibition curves plotted by
Grafit software. The same method was used to determine cytotoxicity against
HT29-AK and MDA-MB-231 cells at drug concentrations up to 750 lM.
4. 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.
5. Efferth, T.; Dunstan, H.; Sauerbrey, A.; Miyachi, H.; Chitambar, C. R. Int. J. Oncol.
2001, 18, 767.
6. Efferth, T.; Benakis, A.; Romero, M. R.; Tomicic, M.; Rauh, R.; Steinbach, D.;
Hafer, R.; Stamminger, T.; Oesch, F.; Kaina, B.; Marschall, M. Free Radical Biol.
Med. 2004, 37, 998.
21. For in vitro antimalarial assessment versus the 3D7 strain of Plasmodium
falciparum the following protocol was employed. Parasites were maintained in
continuous culture using the method of Jensen and Trager: (a) Trager, W.;
Jensen, J. B. Science 1976, 193, 673–675; Antimalarial activity was assessed
with an adaption of the 48 hr sensitivity assay of Desjardins et al. using [3H]-
hypoxanthine incorporation as an assessment of parasite growth: (b)
Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710–718.
7. Efferth, T.; Oesch, F. Biochem. Pharmacol. 2004, 68, 3.
8. O’Neill, P. M.; Posner, G. H. J. Med. Chem. 2004, 47, 2945.
9. Efferth, T. Drug Res. Updates 2005, 8, 85.
10. Li, P. C. H.; Lam, E.; Roos, W. P.; Zdzienicka, M. Z.; Kaina, B.; Efferth, T. Cancer
Res. 2008, 68, 4347.
22. Mitochondrial membrane potential (MMP) was measured using
tetramethylrhodamine ethyl ester (TMRE) to quantify HL-60 cells with a high
MMP. Drug treated cells (5 Â 105 cells) were washed in HBSS and the resultant
cell pellet was resuspended in 500 lL of TMRE solution (50 nM in HBSS) and
11. 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.
12. Albert, A.; Goldacre, R. Nature 1948, 161, 95.
13. Denny, W. A. Curr. Med. Chem. 2002, 9, 1655.
14. (a) Eckstein-Ludwig, U.; Webb, R. J.; van Goethem, I. D. A.; East, J. M.; Lee, A. G.;
Kimura, M.; O’Neill, P. M.; Bray, P. G.; Ward, S. A.; Krishna, S. Nature 2003, 424,
957; (b) Stocks, P. A.; Bray, P. G.; Barton, V. E.; Al-Helal, M.; Jones, M.; Araujo, N.
C.; Gibbons, P.; Ward, S. A.; Hughes, R. H.; Biagini, G. A.; Davies, J.; Amewu, R.;
Mercer, A. E.; Ellis, G.; O’Neill, P. M. Angew. Chem., Int. Ed. 2007, 46, 6278.
15. Takahashi, T.; Tanaka, H.; Matsuda, A.; Doi, T.; Yamada, H.; Matsumoto, T.;
Sasaki, D.; Sugiura, Y. Bioorg. Med. Chem. Lett. 1998, 8, 3303.
16. Perez, J. M.; Lopez-Solera, I.; Montero, E. I.; Brana, M. F.; Alonso, C.; Robinson, S.
P.; Navarro-Ranninger, C. J. Med. Chem. 1999, 42, 5482.
incubated for 10 min at 37 °C. A minimum of 500 cells were measured by flow
cytometry on fluorescence channel FL-2 (Coulter Epics, XL Software). The data
was analysed using WinMDI, v2.8 software (Scripps Institute, California, USA).
23. The DNA major groovebinder PI, was used to quantify cellular DNA content to
measure the formation of a sub-G0/G1 population of HL-60 cells. Drug treated
cells (1 Â 106) were washed twice in HBSS, fixed in 1 mL of ice-cold 70%
ethanol, and frozen at À20 °C. After 2 h the 70% ethanol was removed and the
cell pellet was resuspended in 1 mL of PI stock solution (phosphate-buffered
saline containing 40 lg/mL PI, 0.1 mg/mL RNase, and 3.8 mM sodium citrate))
and incubated at 37 °C (30 min). A minimum of 5000 cells were analysed by
flow cytometry. PI fluorescence was measured on fluorescence channel FL-2.
The proportion of cells in each stage of the cell cycle was calculated from the
DNA content of the cell using WinMDI, v2.8 software.
24. Cell lysate samples were prepared, cells (3 Â 106) were washedbefore
17. Meunier, B. Acc. Chem. Res. 2008, 41, 69.
sonication with
a
sonic probe, (2 Â 5 s cycles). The samples were then
18. O’Neill, P. M.; Miller, A.; Bishop, L. P. D.; Hindley, S.; Maggs, J. L.; Ward, S. A.;
Roberts, S. M.; Scheinmann, F.; Stachulski, A. V.; Posner, G. H.; Park, B. K. J. Med.
Chem. 2001, 44, 58.
assayed for protein content using the Bradford assay. Lysates, with equal
amounts of protein (20 g per lane), were mixed with SDS–PAGE loading
l
buffer and denatured at 95 °C for 3 min prior to being resolved on 14% SDS–
PAGE. Proteins were transferred to nitrocellulose membrane for Western
blotting analysis as described previously.29
19. Synthesis of hybrid 7: To a stirred solution of artemisinin acid chloride (1 equiv)
and appropriate alkylaminoacridine (1 equiv) in anhydrous CH2Cl2 at 0 °C was
added anhydrous triethylamine (1.1 equiv). The reaction mixture was stirred at
low temperature (0 °C) for 18 h and then allowed to warm to ambient
temperature. The volatiles were removed in vacuo and the resultant yellow oil
was purified by flash column chromatography on silica gel to give a yellow
solid (20:80 = MeOH/CH2Cl2). Analysis for 7: mp 125 °C 1H NMR: (CDCl3,
400 MHz) d 8.40 ppm (1H, br s), 8.15 (1H, d, J = 9.2 Hz), 8.10 (1H, d, J = 2.5 Hz),
7.97 (1H, d, J = 9.1 Hz), 7.40 (1H, dd, J = 2.4 Hz, 9.3 Hz), 7.38 (1H, d, J = 2.1 Hz),
25. Zimmermann, H. W. Angew. Chem., Int. Ed. Engl. 1986, 25, 115.
26. McGhee, J. D.; von Hippel, P. H. J. Mol. Biol. 1974, 103, 679.
27. Eriksson, M.; Norden, B. In Drug-Nuc. Acid Int.; Chaires, J.B., Waring, M.J., Eds.;
Academic Press: London, 2001; Vol. 340, p 68.
28. Long, E. C.; Barton, J. K. Acc. Chem. Res. 1990, 23, 271.
29. Mercer, A. E.; Maggs, J. L.; Sun, X. M.; Cohen, G. M.; Chadwick, J.; O’Neill, P. M.;
Park, B. K. J. Biol. Chem. 2007, 13, 9372.