Discovery of a novel lead structure for anti-malarials

Article abstract of DOI:10.1016/S0968-0896(00)00296-0

From a library of 61 compounds available from former studies, 2,5-bis-acylaminobenzophenone 1 was identified as a lead structure for a novel class of anti-malaria agents active against multi-resistant Plasmodium falciparum strain Dd2. Some structural modifications of this initial lead demonstrated the potential for further improvement of the anti-plasmodial activity of this novel class of anti-malarials. Copyright

Full text of DOI:10.1016/S0968-0896(00)00296-0

Bioorganic & Medicinal Chemistry 9 (2001) 785±792
Discovery of a Novel Lead Structure for Anti-Malarials
Jochen Wiesner,b Pia Wiûner,a Hans-Martin Dahse,c Hassan Jomaab
and Martin Schlitzera,Ã
aInstitut fur Pharmazeutische Chemie, Philipps-Universitat Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
È
È
bBiochemisches Institut der Universitatsklinik Gieûen, Friedrichstraûe 24, D-35249 Gieûen, Germany
È
cHans-Knoll-Institut fur Natursto€-Forschung e. V., Beutenbergstraûe 11, D-07745 Jena, Germany
È
È
Received 30 August 2000; accepted 2 November 2000
AbstractÐFrom a library of 61 compounds available from former studies 2,5-bis-acylaminobenzophenone 7p was identi®ed as a
lead structure for a novel class of anti-malaria agents active against multi-resistant Plasmodium falciparum strain Dd2. Some
structural modi®cations of this initial lead demonstrated the potential for further improvement of the anti-plasmodial activity of
this novel class of anti-malarials. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
The cinnamic acid derivatives 11a±e have been prepared
from 172e and the correspondingcinnamic acids which
have been prepared as described.2b
Malaria is one of the most threateningtropical diseases
causingbetween 1.5 and 2.7 million fatal cases per year,
particularly amongchildren and women, primarily in
Africa.1 Nearly all fatal cases are caused by Plasmodium
falciparum, the causative agent of Malaria tropica. This is
largely due to the widespread emergence of P. falciparum
strains which are resistant to presently available drugs.
Therefore, there is an urgent need for new agents active
against multi-resistant Plasmodium strains.1
Biological testing
Compounds were evaluated for their inhibitory activity
against intraerythrocytic forms of P. falciparum strains
Dd2 and 3D7 usinga semi-automated microdilution assay
as described.3 The growth of the parasites was monitored
through the incorporation of tritium-labeled hypo-
xanthine. For initial assessment of the anti-plasmodial
activity of the test compounds, growth inhibition (expres-
sed as percent inhibition in comparison to an untreated
control) was determined at test compound concentrations
of 100, 10 and 1.0 mM. For selected compounds IC50
values were determined. All values are estimated to be
correct within Æ30%.
We have assayed a small library of altogether 61 com-
pounds which were available from di€erent studies2 for
potential activity against P. falciparum.
Chemistry
The synthesis of the library compounds has been descri-
bed before.2 The synthesis of the derivatives of the initial
lead compound 7p is outlined in Scheme 1. Brie¯y,
appropriate 4-nitroaniline derivatives 12 and 14 have
been acylated usingphenylacetyl and acetyl chloride,
respectively, in boilingtoluene or toluene/dioxane. The
nitro groups of the resulting amides have been reduced
to amino functions usingtin-II-chloride as reductive
agent. Finally, the newly formed amino groups of 13
and 16 were acylated with 3-phenylpropionyl chloride.
Compounds 7p and 11a were assayed against cell lines
K-562 (human chronic myeloid leukaemic cell line), THP-
1 (human acute monocytic leukaemic cell line), L-929
(mouse ®broblast cell line) and HeLa (human cervix car-
cinoma cell line) for their antiproliferative and cytotoxic
e€ects. The cells were incubated with 10 concentrations of
the test compounds ranging from 50 to 0.1mg/mL.
Results and Discussion
ÃCorrespondingauthor. Tel.: +49-6421-28-25825; fax: +49-6421-28-
27052; e-mail: schlitze@mailer.uni-marburg.de
A library of 61 compounds of di€erent structural classes
(Figs 1±3) which were available from other studies2 were
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00296-0

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J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
Scheme 1. (I) Ph±CH2±COCl, toluene, 2 h, re¯ux; (II) SnCl2Â2H2O, ethyl acetate, 2 h, re¯ux; (III) Ph-CH2-CH2-COCl, toluene, 2 h, re¯ux; (IV)
H3C-COCl, toluene, 2 h, re¯ux; R1R2C6H3±HCˆCH±COCl, toluene/dioxane, 2 h, re¯ux.
assayed for their inhibitory activity against P. falciparum
(Table 1). Forty seven of these compounds showed an
inhibition of P. falciparum growth at a concentration of
100 mM. However, only seven compounds which are dis-
tributed over all structural classes retained signi®cant
inhibitory activity when assayed at a concentration of
10 mM. No rationale can be provided for the distribution
of the activity within the library. Compound 7p, which
inhibited the growth of P. falciparum by 93% at a con-
centration of 10 mM, failed to inhibit the growth of the
parasites at 1.0 mM. Nevertheless, compound 7p appears
to be the most active compound of the whole library and
therefore, and because of its chemical structure which
allows a variety of structural modi®cations, it was fur-
ther evaluated as a potential lead structure for novel anti-
malaria agents. First, an exact IC50 value was deter-
mined. The value of 2.7 mM against the wild strain 3D7
seems not to be particularily impressive if compared to
the values of some standard drugs (Table 3). However,
compound 7p retained its activity if assayed against the
multi-resistant strain Dd2, while most of the standard
therapeutics showed a marked reduction in activity.
although this target has yet to be determined. As all
three substituents of the central benzene have been
demonstrated to be important for activity, all three
positions are open for structural variations to possibly
improve anti-plasmodial activity. In the ®rst line, we
decided to concentrate our e€orts on the acylamino
substituent in the 5-position. We replaced the 3-phenyl-
propionyl residue in 7p by some substituted cinnamic
acid derivatives (Fig. 4, Table 2). In this relatively small
series of cinnamic acid derivatives, clear structure±
activity relationships can be delineated. The 4-phenyl
and the 4-benzyloxy derivative showed a signi®cant
improvement in activity compared to the 3-phenylpro-
pionyl derivative 7p, while the other two showed no
improvement or were even less active. For 11a and 11c,
exact IC50 values were determined (Table 3). Both
compounds are approximately 5-fold more active than
compound 7p initially identi®ed by random screen.
Although their IC50 values of 650 and 500 nM, respec-
tively, are still 10-fold higher than the values of some
active anti-malarials as for example me¯oquine (see
Table 3), the improvement in activity upon structural
modi®cation demonstrates that this class of compounds
possesses a potential for further development.
Next, some drastical structural modi®cations were per-
formed with compound 7p (Fig. 4). Each of the three
derivatives (8±10) is lackingone of the substituents of
the central benzene core. As shown in Table 2, all three
modi®cations resulted in a marked decrease in anti-
plasmodial activity demonstratingthat the complete
structure is necessary for activity. This argues for a
rather speci®c interaction of 7p with a molecular target
Altogether, the activity against a multi-resistant strain
of P. falciparum and the potential for further improve-
ment of activity by structural modi®cations demon-
strated by compounds 11a and 11c quali®es compound
7p as a lead structure for a novel class of anti-malarial
agents.

J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
787
Figure 1. Structures of the library compounds.
One major concern in this stage of development was
that the activity of the compounds investigated against
P. falciparum may be due to an unspeci®c cytotoxicity.
Therefore, compounds 7p and 11a were assayed for
potential cytotoxic e€ects against some cultured cell
lines (Table 4). Indeed, compound 7p turned out to be
cytotoxic but the 5-fold more active compound 11a
showed no cytotoxic activity at concentrations up to
180 mM which is more than 300 times the concentration
at which the compound inhibits the growth of malaria
parasites. Therefore, it has been demonstrated that the
inhibitory activity against P. falciparum can be separated
from cytotoxicity by structural modi®cations which at
the same time improve anti-plasmodial ecacy.
In summary, we report a novel lead structure for anti-
malaria agents with interesting activity against a multi-
resistant strain of P. falciparum. This class of com-

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J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
Figure 2. Structures of the library compounds, continued.
pounds possesses a potential for further development
which will be necessary to obtain useful anti-malarials.
Progress will be reported in due course.
VG 7070 H usinga Vector 1 data acquisition system
from Teknivent or a AutoSpec mass spectrometer from
Micromass. IR spectra were recorded on a Nicolet 510P
FT-IR-spectrometer. Microanalyses were obtained from
a CH analyzer accordingto Dr. Salzer from Labormatic
and from a Hewlett Packard CHN-analyzer type 185
and are within Æ0.4% of the theoretical values. Melting
points were obtained with a Leitz-microscope and are
uncorrected. Column chromatography was carried out
usingsilica gel 60 (0.062±0.200mm) from Merck. Medium
Experimental
1H and 13C NMR spectra were recorded on a Jeol
JMN-GX-400 and a Jeol JMN-LA-500 spectrometer.
Mass spectra were obtained with a Vacuum Generators

J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
789
Figure 3. Structures of the library compounds, continued.
pressure liquid chromatography (MPLC) was per-
formed usinga pump type 688 from Bu chi and a col-
umn of 3.5 cm diameter and 45 cm length ®lled with
silica gel 60 (0.015±0.040 mm) from Merck.
acetate phase was dried over MgSO4 and evaporated to
dryness. The product 13 (384 mg, 1.7 mmol, 50%) was
used without further puri®cation for the next acylation
step accordingto general procedure 1 employing3-phe-
nylpropionic acid chloride (286 mg, 1.7 mmol). Puri-
®cation: recrystallization from toluene. Yield: 444 mg
(73%); mp 223 ꢀC. IR (KBr): n=3250, 3060, 1700, 1670,
General procedure for the acylation of aromatic amines
(procedure 1)
1
1600, 1555, 1505 cmÀ1. H NMR (CDCl3): d=2.59 (t,
The various carboxylic acids were dissolved in toluene
and 0.1 mL SOCl2 per mmol acid was added. The mix-
ture was heated under re¯ux for 2 h and the volatiles
were evaporated in vacuo. The resultingacyl chlorides
were dissolved in toluene or dioxane (approx. 10 mL)
and added to a solution of the appropriate aromatic
amine in hot toluene (approx. 50 mL). The mixtures
were heated under re¯ux for 2 h. Then, the solvent was
removed in vacuo to give the crude products.
J=8 Hz, 2H), 2.90 (t, J=8 Hz, 2H), 3.60 (s, 2H), 7.10±
7.32 (m, 10H), 7.48 (m, 4H), 9.72 (s, 1H), 9.96 (s, 1H).
MS: m/z (%) 358 (100) [M+], 226 (41), 108 (41). Anal.
calcd for C23H22N2O2 (358.44): C, 77.07; H, 6.19; N,
7.82; found: C, 77.38; H, 6.56; N, 8.11.
N-(4-Acetylamino-3-benzoylphenyl)propionic acid amide
10. 2-Amino-5-nitrobenzophenone 14 (1.21 g, 5 mmol)
was acylated by acetylchloride (393 mg, 5 mmol) accord-
ingto general procedure 1. Yield: 978 mg(68%). The
product 15 was dissolved in ethyl acetate (50 mL) and
SnCl2Â2H2O (3.83 g) was added. After 2 h under re¯ux,
the solution was diluted with water and the pH was
adjusted to 8 by the addition of satd NaHCO3 solution.
The aqueous mixture was extracted with ethyl acetate
(3Â100 mL) and the combined organic extracts were
washed with brine. The ethyl acetate phase was dried
over MgSO4 and evaporated to dryness. The product 16
(381 mg, 1.5 mmol, 44%) was used without further puri-
®cation for the next acylation step accordingto general
procedure 1 employing3-phenylpropionic acid chloride
(252 mg, 1.5 mmol). Puri®cation: recrystallization from
toluene. Yield: 301 mg(52%); mp 178 ꢀC. IR (KBr):
n=3255, 3060, 1665, 1595, 1555cmÀ1. 1H NMR (CDCl3):
N-[4-(Phenylacetylamino)phenyl]-3-phenylpropionic acid
amide 9. 4-Nitroaniline 12 (690 mg, 5 mmol) was acy-
lated by phenylacetyl chloride (773 mg, 5 mmol)
accordingto general procedure 1. Yield: 870 mg(68%).
1H NMR (CDCl3): d=3.79 (s, 2H), 7.34 (m, 2H), 7.41
(m, 1H), 7.43 (m, 2H), 7.60 (m, 2H), 8.16 (m, 2H).
The product from the step described above was dissolved
in ethyl acetate (50 mL) and SnCl2Â2H2O (3.83 g) was
added. After 2 h under re¯ux, the solution was diluted
with water and the pH was adjusted to 8 by the addition
of satd NaHCO3 solution. The aqueous mixture was
extracted with ethyl acetate (3Â100 mL) and the com-
bined organic extracts were washed with brine. The ethyl

790
J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
Figure 4. Structural variations of lead compound 7p and cinnamic acid amides 11a±e.
d=2.19 (s, 3H), 2.60 (t, J=8 Hz, 2H), 2.99 (t, J=8 Hz,
2H), 7.19 (m, 4H), 7.25 (m, 2H), 7.40 (m, 1H), 7.49 (m,
2H), 7.61 (m, 1H), 7.72 (m, 2H), 7.81 (m, 1H), 8.49 (m,
1H), 10.56 (s, 1H). MS: m/z (%) 386 (100) [M+], 344
(29), 254 (23), 212 (52). Anal. calcd for C24H22N2O3
(386.45): C, 74.59; H, 5.74; N, 7.29; found: C, 74.50; H,
5.57; N, 7.76.
(m, 3H), 7.96 (m, 1H), 8.43 (m, 1H), 10.46 (s, 1H). MS:
m/z=550 (98) [M+], 207 (100). Anal. calcd for
C37H30N2O3 (550.66) C, 80.70; H, 5.49; N, 5.09; found
C, 80.53; H, 5.36; N, 5.12.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-
styrylcinnamic acid amide 11b. From 4-styrylcinnamic
acid (0.360 g, 1.5 mmol) and N-(4-amino-2-benzoylphe-
nyl)-(4-methylphenyl)acetamid 17 (0.413 g, 1.2 mmol)
accordingto general procedure 1. Puri®cation: recrys-
tallization from acetone/n-hexane. Yield: 437 mg(63%);
mp 163 ꢀC. IR (KBr): n=3310, 3025, 2930, 1650, 1600,
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-
phenylcinnamic acid amide 11a. From 4-phenylcinnamic
acid (0.270 g, 1.2 mmol) and N-(4-amino-2-benzoylphe-
nyl)-(4-methylphenyl)acetamid 17 (0.344 g, 1.0 mmol)
accordingto general procedure 1. Puri®cation: recrys-
tallization from toluene. Yield: 456 mg(83%); mp
200 ꢀC. IR (KBr): n=3445, 1700, 1685, 1655, 1640,
1
1560, 1510 cmÀ1. H NMR (CDCl3): d=2.31, (s, 3H),
3.67, (s, 2H), 6.47 (d, J=16 Hz, 1H), 7.07 (s, 1H), 7.11
(s, 1H), 7.13±7.15 (m, 2H), 7.22±7.23 (m, 2H), 7.25±7.28
(m, 1H), 7.33±7.39 (m, 4H), 7.42±7.45 (m, 4H), 7.49±
7.51 (m, 2H), 7.52±7.56 (m, 2H), 7.64 (d, J=16 Hz, 1H),
7.69±7.70 (m, 2H), 7.96 (s, br, 1H), 8.06 (s, br, 1H), 8.49
1
1550, 1505 cmÀ1. H NMR (CDCl3): d=2.24 (s, 3H),
3.60 (s, 2H), 6.42 (d, J=16 Hz, 1H), 7.08 (m, 2H), 7.16
(m, 3H), 7.28 (m,1H), 7.38 (m, 5H), 7.48 (m, 6H), 7.61

J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
791
Table 1. Activity of library compounds 1±7 against P. falciparum
Dd2 strain
Table 2. Activity of derivatives 8±11 of the lead structure 7p against
P. falciparum Dd2 strain
Compound
% Inhibitiona at
Compound
% Inhibition at
100 mM
10 mM
100 mM
10 mM
1.0 mM
1a
1b
1c
1d
1e
1f
1g
1h
1i
96
0
89
0
30
58
0
78
50
38
0
0
0
0
0
0
0
0
0
0
0
0
0
0
84
0
0
0
0
0
0
0
63
0
0
0
0
0
0
0
0
0
36
0
0
0
0
0
0
0
0
0
0
0
33
0
0
0
19
0
0
0
0
0
0
7p
8
9
10
11a
11b
11c
11d
11e
99
92
0
95
99
99
97
98
99
93
0
0
0
0
0
0
96
0
85
0
0
0
99
99
96
36
99
1j
1k
2a
2b
2c
3a
3b
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
0
Table 3. IC50 values (mM) of selected compounds and some standard
anti-malarials
57
99
76
83
91
17
0
Compound
P. falciparum strain
3D7
Dd2
7p
11a
11c
2.7
0.49
0.40
0.022
0.10
0.024
0.003
0.008
2.7
0
0.65
0.50
0.17
0.38
0.057
2.5
73
93
0
Chloroquine
Quinine
Me¯oquine
Pyrimethamine
Cycloguanil
98
76
98
98
59
96
91
98
97
98
89
ndb
99
99
99
32
92
98
98
67
98
53
38
59
95
29
98
nd
96
83
nd
93
99
nd
0
2.2
5g
5h
5i
5j
5k
5l
Table 4. Cytotoxic and anti-proliferative e€ect of compounds 7p and
11a
Compound
IC50 (mM)
GI50 (mM)
L929
HeLa
K-562
THP-1
5m
5n
5o
6a
6b
6c
6d
6e
6f
6g
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7p
11a
17.8
>180
36.9
>180
13.6
>180
14.6
>180
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-4-
benzyloxycinnamic acid amide 11c. From 4-benzyloxy-
cinnamic acid (0.381g, 1.5 mmol) and N-(4-amino-2-
benzoylphenyl)-(4-methylphenyl)acetamid 17 (0.413g, 1.2
mmol) accordingto egneral procedure 1. Puri®cation:
recrystallization from toluene. Yield: 482 mg(69%); mp
183 C. IR (KBr): n=3285, 1675, 1600, 1540 cmÀ1. H
NMR (CDCl3): d=2.31 (s, 3H), 3.67 (s, 2H), 5.06 (s,
2H), 6.33 (d, J=16 Hz, 1H), 6.90±6.93 (m, 2H), 7.14±
7.16 (m, 2H), 7.22±7.26 (m, 2H), 7.31±7.46 (m, 9H),
7.53±7.54 (m, 2H), 7.60 (d, J=16 Hz, 1H), 7.67±7.72 (m,
2H), 7.78 (s, br, 1H), 8.03 (s, br, 1H), 8.49 (m, 1H),
10.52 (s, br, 1H). MS: m/z (%) 580 (0.3) [M+], 581 (81),
345 (55), 237 (100). Anal. calcd for C38H32N2O4
(580.72): C, 78.60; H, 5.55; N, 4.82; found: C, 78.85; H,
5.59; N, 4.94.
ꢀ
1
0
0
0
0
0
67
93
nd
89
99
aNone of the compounds was active at 1.0 mM.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-
3,4-bis-(benzyloxy)cinnamic acid amide 11d. From 3,4-
bis-(benzyloxy)cinnamic acid (0.541 g, 1.5 mmol) and N-
(4-amino-2-benzoylphenyl)-(4-methylphenyl)acetamid 17
(0.413 g, 1.2 mmol) according to general procedure 1.
Puri®cation: MPLC ethyl acetate n-hexane, 1:1. Yield:
502 mg(61%); mp 102 ꢀC. IR (KBr): n=3265, 3030,
bnd: not determined.
(d, J=9 Hz, 1H), 10.53 (s, br, 1H). MS: m/z (%) 576
(80) [M+], 577 (35), 574 (22), 344 (37), 233 (100). Anal.
calcd for C39H32N2O3 (576.69): C, 81.21; H, 5.60; N,
4.86; found: C, 81.21; H, 5.63; N, 4.92.

792
J. Wiesner et al. / Bioorg. Med. Chem. 9 (2001) 785±792
1
1665, 1630, 1595, 1550, 1505 cmÀ1. H NMR (CDCl3):
d=2.31, (s, 3H), 3.67, (s, 2H), 5.09 (s, 2H), 5.15 (s, 2H),
6.25 (d, J=16 Hz, 1H), 6.85±6.86 (m, 1H), 6.96±7.00 (m,
1H), 7.02±7.03 (m, 1H), 7.14±7.15 (m, 2H), 7.22±7.45 (m,
14H), 7.50±7.53 (m, 3H), 7.67±7.69 (m, 3H), 8.00 (s, br,
1H), 8.49 (m, 1H), 10.53 (s, br, 1H). MS: m/z (%) 686
(10) [M+], 344 (12), 129 (25), 105 (41), 91 (100), 57 (48),
44 (78). Anal. calcd for C45H38N2O5 (686.85): C, 78.69;
H, 5.59; N, 4.08; found: C, 78.53; H, 5.61; N, 4.28.
RPMI 1640 culture medium (Gibco BRL 21875-034)
supplemented with 100 U/mL penicillin G sodium salt
and 100 mg/mL streptomycin sulfate (Gibco BRL 15140-
114), 10% heat inactivated fetal bovine serum (Gibco
BRL 10500-064), and 10 mL/L non-essential amino acid
(Gibco BRL 11140-035) at 37 ꢀC in high density poly-
ethylene ¯asks (Nunc 156340).
For each experiment with K-562 and THP-1 cells,
approximately 10,000 cells were seeded with 0.1 mL RPMI
1640 culture medium (Gibco BRL 21875-34) but without
HEPES, into 96-well microplates (Nunc 163320). The
adherent cells of L-929 and HeLa were harvested at the
logarithmic growth phase after soft trypsinization, using
0.25% trypsin in PBS containing0.02% EDTA. L929
and HeLa cells were seeded with approximately 10,000
cells per 0.1 mL RPMI 1640 per well of the 96-well
microplates. The plates were previously prepared with
ten dilutions of the test compounds in 0.1 mL RPMI
1640. The cells were incubated for 72 h at 37 ꢀC in a
humidi®ed atmosphere and 5% CO2.
N-[3-Benzoyl-4-[(4-methylphenyl)acetylamino]phenyl]-3-
phenoxycinammic acid amide 11d. From 3-phenoxy-
cinnamic acid (0.360 g, 1.5 mmol) and N-(4-amino-2-
benzoylphenyl)-(4-methylphenyl)acetamid 17 (0.413 g, 1.2
mmol) accordingto general procedure 1. Puri®cation:
MPLC ethyl acetate n-hexane, 1:1. Yield: 428 mg(63%);
mp 133 ꢀC. IR (KBr): n=3240, 3085, 1670, 1640, 1595,
1580, 1550 cmÀ1. H NMR (CDCl3): d=2.31, (s, 3H),
1
3.67, (s, 2H), 6.38 (d, J=16 Hz, 1H), 6.93±7.01 (m, 3H),
7.08±7.16 (m, 5H), 7.21±7.35 (m, 5H), 7.42±7.46 (m, 2H),
7.51±7.61 (m, 3H), 7.64±7.69 (m, 3H), 7.99 (s, br, 1H),
8.49 (d, J=9Hz, 1H), 10.51 (s, br, 1H). MS: m/z (%) 566
(100) [M+], 567 (42), 434 (31), 344 (31), 223 (35), 212 (30).
Anal. calcd for C37H30N2O4 (566.69): C, 78.42; H, 5.35;
N, 4.94; found: C, 78.20; H, 5.31; N, 5.22.
Suspension cultures of K-562 and THP-1 cells in micro-
plates were analysed by an electronic cell analyser system
CASY1 (Scharfe, Reutlingen, Germany). The principle of
measurement and evaluation of data were described.4 The
0.2 mL content of each well in the microplate was diluted
1:50 with CASYTON (Scharfe). Every count/mL was
automatically calculated from the arithmetic mean of
three successive counts of 0.4 mL each. From the dose
response curves the GI50 values (concentration which
inhibited cell growth by 50%) were calculated with the
software for data evaluation CASYSTAT (Scharfe). The
monolayer of the adherent cell lines L-929 and HeLa
were ®xed by glutaraldehyde and stained with a 0.05%
solution of methylene blue for 15 min. After gentle
washingthe stain was eluted by 0.2 mL of 0.33 N HCl in
the wells. The optical densities were measured at 630 nm
in a Dynatech MR 7000 microplate reader. Comparison
of di€erent values was performed with Microsoft
EXCEL.
In vitro measurement of P. falciparum parasite growth
inhibition
Compounds were tested by a semiautomated microdilu-
tion assay against intraerythrocytic forms of P. falcipar-
um.3a The P. falciparum strains Dd2 and 3D7 were
cultivated by a modi®cation of the method described by
Trager and Jensen.3b The culture medium consisted of
RPMI 1640 supplemented with 10% human type 0+
serum and 25mM HEPES. Human type 0+ erythrocytes
served as host cells. The cultures were kept at 37ꢀC in an
atmosphere of 5% O2, 3% CO2, and 92% N2.
Drugtestingwas carried out in 96-well microtiter plates.
The compounds were dissolved in DMSO (10 mM) and
prediluted in complete culture medium (®nal DMSO con-
centrations ꢁ1%). Infected erythrocytes (200mL per well,
with 2% hematocrit and 0.4% parasitemia, predominantly
ring-stage parasites) were incubated in duplicate with a
serial dilution of the drugs for 48 h.3c After the addition of
0.8 mCi [3H]-hypoxanthine in 50mL medium per well, the
plates were further incubated for 24 h. Cells were col-
lected on glass ®ber ®lters with a cell harvester (Micro-
mate 196, Packard) and incorporated radioactivity
measured usinga b-counter (Matrix 9600, Packard).
Acknowledgements
M. Schlitzer thanks Professor Dr. W. Hanefeld for
generous support and Ms. K. Burk and Mr. G. Hohage
for technical assistance.
References
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Cytotoxicity assay
Cells of established suspended cell lines K-562, THP-1,
and L-929 were cultured in RPMI 1640 medium (Gibco
BRL 42402-016), supplemented with 100 U/mL peni-
cillin G sodium salt and 100 mg/mL streptomycin sulfate
(Gibco BRL 15140-114) and 10% heat inactivated fetal
bovine serum (Gibco BRL 10500-064), and l-glutamine
(Gibco BRL 25030-024) at 37 ꢀC in high density poly-
ethylene ¯asks (Nunc 156340). HeLa cells were grown in