3
002
M. Tischer et al. / Bioorg. Med. Chem. 18 (2010) 2998–3003
antimalarial activity, further variation of this moiety is in
progress.
4.4. General synthesis procedure for compounds 3e and 3f
One equivalent of the corresponding
a,x-diaminoalkane,
6
.5 equiv of methyliodide and 4 equiv of N-ethyldiisopropylamine
4
. Experimental
were dissolved in 30 mL of chloroform and stirred at room temper-
ature for two days. The precipitate was filtered off and extracted
with acetone in a Soxleth extractor for 10 h to give 3e and 3f. For
spectroscopic and analytical data see Supplementary data.
1H and 13C NMR spectra were recorded on an Avance 400 nucle-
ar magnetic resonance spectrometer, Bruker Biospin GmbH, Rhein-
1
13
stetten, Germany ( H 400.132 MHz, C 100.613 MHz). As internal
standard the signals of the deuterated solvents were used (DMSO-
1
13
1
13
4
.5. General synthesis procedure for compounds 6 and 7
d
6
:
H 2.5 ppm, C 39.52 ppm, methanol-d
4
:
H 3.31 ppm,
C
4
9.0 ppm). The following abbreviations describing the multiplicity
0
.2 Mmol of 4 and 5, respectively, were dissolved in a mixture of
are used: (s) singlet, (d) doublet, (t) triplet, (dd) doublet of dou-
blets, (br) broad signal, (m) multiplet. Coupling constants are given
in hertz. IR-spectra were obtained with a Biorad PharmalyzIR FT-IR
spectrometer (Biorad, Cambridge, MA, USA). Melting points were
measured using an apparature Sanyo Gallenkamp (Sanyo Gallenk-
amp, Loughborough, UK), and are not corrected. TLC was carried
out on TLC aluminum sheets, silica gel F254 (Merck KGaA, Darms-
tadt, Germany). All chemicals were purchased from Sigma–Aldrich
Chemicals (Deisenhofen, Germany), Acros Organics (Geel, Belgium)
and VWR International (Darmstadt, Germany) and were used with-
out further purification.
4
0 mL ethanol, 20 mL water and 5 mL of acetic acid. To this solu-
tion 0.08 g of palladium 10% on activated carbon were added and
heated in a synthetic microwave hydrogenation reactor (12.5 bar
H , 800 W, 30 °C/min, 90 °C) for 1.5 h. The catalyst was filtered
2
and the solvent removed in vacuo. For spectroscopic and analytical
data see Supplementary data.
4
.6. Malstat assay
The compounds were screened on the human malaria pathogen
P. falciparum at concentrations between 10 pM and 100
antimalarial activity was determined for the human malaria path-
lM. The
The syntheses of compounds 1 and 12, and 2d, 8 and 9 were
performed according to Refs. 16,14, respectively, of compounds
1
9
ogen P. falciparum in vitro, using chloroquine-sensitive strain
D7 and in selected cases chloroquine-resistant strain Dd2. Syn-
chronized ring stages of P. falciparum strain 3D7 were plated in
6-well-plates at a parasitemia of 1% in the presence of the com-
pounds (dissolved either in H O or in dimethyl sulfoxide, DMSO).
Incubation of parasites with DMSO alone at a concentration of
.5% was used for negative control. The parasites were cultivated
in triplicates in vitro as described for 72 h. Nineteen parasite viabil-
ity were screened subsequently using the Malstat assay, which
measures the activity of the Plasmodium-specific enzyme lactate
3
d, 3f, 3h and 3k according to Ref. 18 and of compound 3e accord-
3
ing to Ref. 26.
9
4
.1. General synthesis procedure for compounds 1 and 12–14
2
One equivalent of a substituted 1,8-naphthalenedicarboxylic
0
acid anhydride was suspended in 100 mL of toluene abs. One equiv
of the correspondingly substituted N,N-dimethylpropane-1,3-dia-
mine and a catalytic amount of p-toluenesulfonic acid was added
and the mixture was refluxed for 6 h. The reaction mixture was al-
lowed to cool to room temperature and extracted with 300 mL
2
0,21
dehydrogenase as described.
to four times and the IC50 inhibitory effect and the standard devi-
ation were calculated.
Each compound was tested two
(
9
6 ꢁ 50 mL) of aqueous sodium hydrogencarbonate solution pH
. Precipitated p-toluenesulfonic acid was filtered off, the solution
dried over anhydrous Na SO and the solvent removed in vacuo to
2
4
4
.7. Cytotoxicity assay
give compounds 1 and 12–14. Recrystallization from ethanol may
be applied. Spectroscopic and analytical data see Supplementary
data.
The cytotoxicities of all compounds were measured in the mac-
rophage cell line J774.1 using the AlamarBlue-based cytotoxicity
2
2
4
.2. General synthesis procedure for compounds 2a–k, 4, 5, 8,
test. Macrophages were cultured in NaHCO
ium containing 10% FCS, 2 mM glutamine, 10 mM Hepes pH 7.2,
00 U/mL penicillin, 50 g/mL gentamicin, and 50 M 2-mercap-
3
-buffered RPMI med-
and 9
1
l
l
One equivalent of 1, 12, 13, and 14, respectively, 0.5 equiv of
the corresponding -dibromoalkane and a catalytic amount of
KI/K CO were dissolved in 20–40 mL of acetonitrile. The reac-
tion mixture was refluxed for several days or in an overpressure
tube in a synthetic microwave oven (30 °C/min, 500 W, 90 °C)
and than cooled to 4 °C for 2 h. The precipitate was filtered off
and washed with acetonitrile and pentane, and dried over
toethanol without phenol red in the absence or presence of
a,x
increasing concentrations of the compounds at a cell density of
5
1 ꢁ 10 cells/mL for 24 h at 37 °C, 5% CO
2
, and 95% humidity. Fol-
2
3
lowing the addition of 10% of AlamarBlue solution, the plates were
incubated for 24 and 48 h and the optical densities measured with
a Multiscan Ascent enzyme-linked immunosorbent assay (ELISA)
reader (Thermo Electron Corporation, Dreieich, Germany) using a
4
P O
10. For spectroscopic and analytical data see Supplementary
test wavelength of 540 nm and a reference wavelength of
data.
630 nm. The final concentration of DMSO in the medium never ex-
ceeded 1% (v/v) and had no effect on the proliferation of extracel-
lular or intracellular parasites. Each compound was tested in
duplicate. None of the compounds showed any cytotoxic activity
4
.3. General synthesis procedure for compounds 3d and 3g–k
One equivalent of the corresponding -dibromoalkane was
a
,x
(IC50 value higher than 100 lM after 48 h).
suspended in 50 mL of methanol. A fivefold excess of 2 M solution
of triethylamine in methanol was added and the mixture stirred
at room temperature for two days. The volume was reduced in
vacuo and the obtained oil cooled to 0 °C. The precipitation was
initiated by adding some drops of acetone. The precipitate was fil-
tered and washed with acetone and pentane, and dried over
4.8. Electron microscopy
Blood stages of in vitro cultivated P. falciparum parasites were
incubated with compounds 5 and 8 at IC50 concentrations or with
0.5 vol % DMSO for control for 72 h and fixed in 1% glutaraldehyde
and 4% paraformaldehyde in PBS for five days. The samples were
post fixed in 1% osmium tetroxide and 1.5% K [Fe(CN) ] in PBS
4
P O
10. For spectroscopic and analytical data see Supplementary
data.
3
6