Non-thiol farnesyltransferase inhibitors: N-(4-aminoacylamino-3- benzoylphenyl)-3-[5-(4-nitrophenyl)-2 furyl]acrylic acid amides and their antimalarial activity

Article abstract of DOI:10.1016/j.ejmech.2004.09.019

Water solubility was previously found to be essential for in vivo-antimalarial activity of a novel type of benzophenone-based farnesyltransferase inhibitors. Introduction of a α-amino group into the phenylacetic acid substructure provided more soluble compounds with high farnesyltransferase inhibitory activity. The in vitro-antimalarial activity was detrimentally influenced by this structural modification.

Full text of DOI:10.1016/j.ejmech.2004.09.019

European Journal of Medicinal Chemistry 40 (2005) 93–101
www.elsevier.com/locate/ejmech
Short communication
Non-thiol farnesyltransferase inhibitors:
N-(4-aminoacylamino-3-benzoylphenyl)-3-[5-(4-nitrophenyl)-
2 furyl]acrylic acid amides and their antimalarial activity
Katja Kettler a, Jochen Wiesner b, Katrin Silber c, Peter Haebel c, Regina Ortmann a,
Isabel Sattler d, Hans-Martin Dahse d, Hassan Jomaa b, Gerhard Klebe c, Martin Schlitzer a,*
a Department für Pharmazie, Zentrum für Pharmaforschung, Ludwig-Maximilians Universität München,
Butenandtstraße 5-13, 81377 München, Germany
b Biochemisches Institut der Universität Gießen, Friedrichstraße 24, 35249 Gießen, Germany
c Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032 Marburg, Germany
d Hans-Knöll-Institut für Naturstoff-Forschung e. V., Beutenbergstraße 11, 07745 Jena, Germany
Received 22 March 2004; received in revised form 1 September 2004; accepted 6 September 2004
Available online 25 November 2004
Abstract
Water solubility was previously found to be essential for in vivo-antimalarial activity of a novel type of benzophenone-based farnesyltrans-
ferase inhibitors. Introduction of a a-amino group into the phenylacetic acid substructure provided more soluble compounds with high farne-
syltransferase inhibitory activity. The in vitro-antimalarial activity was detrimentally influenced by this structural modification.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Non-thiol farnesyltransferase inhibitors; Structure–activity relationships; Antimalarial activity
1. Introduction
Therefore, farnesyltransferase has been suggested as a novel
target in the development of drugs directed against parasite
infections [4]. P. falciparum is a target of particular impor-
tance since it is the causative agent of Malaria tropica, which
is responsible for 300–500 million clinical cases every year,
and 1–3 million deaths [12,13].
Farnesyltransferase catalyzes the transfer of a farnesyl resi-
due from farnesylpyrophosphate (FPP) to the thiol of a cys-
teine side chain of proteins, which carry at the C-terminus
the so-called CAAX-sequence. C represents a cysteine which
side chain is farnesylated; A, amino acids which normally,
but not necessarily, carry aliphatic side chains, and X mostly
methionine or serine [1–3].
Farnesyltransferase inhibitors have been developed as
novel anti-cancer agents. Several farnesyltransferase inhibi-
tors are in advanced stages of clinical trials for the therapy of
different types of cancer [4,5].
After farnesyltransferase inhibitors had already been tested
in advanced clinical trials for the treatment of tumor dis-
eases, the development of antiplasmodial farnesyltransferase
inhibitors has now been generally accepted as a new strategy
towards new drugs against malaria (Medicines for Malaria
Venture—annual report 2003). Development of a variety of
new antimalarial compounds directed against not-yet ex-
ploited molecular targets [14] is considered to be essential in
order to face the increasing threat by multi-resistant P. falci-
parum strains as well as the parasite’s ability to readily
develop resistance against new drugs. It has been argued [15]
that in clinical studies with cancer patients, the side effects of
farnesyltransferase inhibitors were acceptable and, thus,
should also be tolerable for antimalarial therapy. However,
while the antiproliferative activity of human farne-
However, farnesyltransferase has also been identified in
different parasites pathogenic to humans as for instance Plas-
modium falciparum [6,7], different Trypanosoma species
[8–10], Leishmania major [10] and Toxoplasma gondii [11].
* Corresponding author. Tel.:+49 89 2180 77804; fax: +49 89 2180 79992.
E-mail address: martin.schlitzer@cup.uni-muenchen.de (M. Schlitzer).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.019

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K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
Scheme 1
.
(I) TFAA, DCM/pyridine, 0 °C, 2 h; (II) SnCl2 × 2H2O, EtOAc, reflux 2 h; (III) 3-[5(4-nitrophenyl)-2-furyl]acrylic acid chloride, toluene/dioxane,
reflux, 2 h; (IV) K2CO3, dioxane/H2O, reflux, 3 h; (V) R–C6H4–CH(NHCOO-tert-Bu)–COOH, POCl3, pyridine, 0 °C, 0.5 h; (6) HCl (g), dioxane, RT, 1 h.
syltransferase inhibitors represents the desired therapeutic
principle in cancer therapy, such an activity precludes the use
in children and pregnant women, the main target populations
in malaria therapy. Therefore, we feel that in the long-term
perspective the development of parasite specific inhibitors is
mandatory.
In previous work we have developed benzophenone-type
non-thiol farnesyltransferase inhibitors with the nitrophenyl-
furylacryloyl residue at the 5-amino group, specifically de-
signed to utilize an aryl binding site of farnesyltransferase
[16]. Several of these benzophenone-based farnesyltrans-
ferase inhibitors (e.g. 8c) were highly active against a multi-
drug resistant P. falciparum strain in vitro [17–26]. However,
even though possessing in vitro IC50-values in the same
range as some standard antimalarials, these compounds were
completely inactive in a murine malaria model, presumably
due to poor water solubility. Introducing a methyl piperazine
residue resulted in soluble compounds (e.g. Schl-2171;
Fig. 1) with comparably low activity against cultured P.
falciparum parasites but representing the first farnesyltrans-
ferase inhibitors with in vivo-antimalarial activity described
in the literature, as demonstrated by curing mice infected
with the rodent malaria parasite P. vinckei [27]. In the present
study we addressed the question whether the piperazinyl
moiety of Schl-2171 can be replaced by a simple amino
group (Fig. 1). The activity of the novel inhibitors was as-
sayed against isolated farnesyltransferase and intraerythro-
cytic forms of P. falciparum.
2. Results and discussion
2.1. Chemistry
The key step in the preparation of the target compounds 7
was the acylation of the 2-amino-5-acylaminobenzophenone
5, which was prepared as described recently [28]. Briefly, the
2-amino group of 1 was protected as trifluoroacetamide 2.
After reduction of the 5-nitro group the resulting amine 3 was
acylated with 3-[5-(4-nitrophenyl)-2-furyl]arylic acid chlo-
ride. After removal of the protective group from 4 the result-
ing intermediate 5 could be acylated by appropriate Boc-
Fig. 1
. The piperazinyl moiety of Schl-2171 was formally reduced to an
amino group yielding aminoacyl-substituted benzophenones.

K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
95
protected phenylglycine derivatives, which were activated
using phosphorous oxychloride in pyridine yielding the
N-protected derivatives 6. Acidic removal of the Boc group
gave the target compounds 7 (Scheme 1).
incubation period using methylene blue staining and photo-
metric evaluation.
2.7. Structure–activity relationships
2.2. Farnesyltransferase inhibition assay
The underlying rationale for the design of piperazinyl-
substituted benzophenones (e.g. Schl2171; Fig. 1) was the
The inhibitory activity of the inhibitors was determined
using the fluorescence enhancement assay as described by
Pompliano et al. [29]. The assay employed yeast farnesyl-
transferase (FTase) fused to glutathione S-transferase at the
N-terminus of the 9-subunit [30].
FPP and the dansylated pentapeptide Ds-GlyCysVal-
LeuSer were used as substrates. Upon farnesylation of the
cysteine thiol the dansyl residue is placed in a lipophilic
environment resulting in an enhancement of fluorescence at
505 nm which is used to monitor the enzyme reaction.
introduction of a functional group, which is charged at physi-
ological pH in order to improve the water solubility of this
type of compounds. These compounds represent the first
farnesyltransferase inhibitors with in vivo-antimalarial activ-
ity described in the literature [27]. Here, we addressed the
question of the effect of the addition of an amino group
instead of a piperazinyl moiety to the a-position of the phe-
nylacetic acid substructure of our benzophenone-based farne-
syltransferase inhibitors as exemplified by compounds 8
.
Amino acid derivatives 7 proved to be more water soluble
than the parent compounds 8 (Table 1). Furthermore, these
inhibitors displayed farnesyltransferase inhibitory activity,
which is roughly in the same range as those of the parent
compounds 8 (Table 1). Only the S-phenylglycine derivative
7a is less active than the parent inhibitor 8a and the
R-enantiomer 7b.
2.3. Inhibition of the growth of P. falciparum
Compounds 6 and 7 were assayed for their inhibitory
activity against intraerythrocytic forms of the P. falciparum
strains Dd2 using a semi-automated microdilution assay as
described [31–33]. The growth of the parasites was moni-
tored through the incorporation of tritiumlabeled hypoxan-
thine. The Dd2 strain is resistant to several commonly used
antimalarial drugs (chloroquine, cycloguanile and py-
rimethamine) (Table 1). The comparability of measurements
of different series was granted by concurrent assay of stan-
dard compounds.
Flexible docking is ambiguous since for the same inhibi-
tor several solutions are obtained with marked differences in
the orientation of the aminoacyl substituent, but with very
similar energy values. However, some possible insight is
provided into the binding of the two enantiomers 7a and 7b
(Fig. 2). Main feature of the binding of the amino acid
derivatives is a hydrogen bond between the a-amino group
and the hydroxyl of Ser 99b mediated by a structural water.
In case of the more active R-enantiomer 7b, this hydrogen
bond is slightly shorter (3.3 Å) than with the less active
S-enantiomer 7a (3.6 Å). This difference is the only one
revealed by flexible docking. Whether flexible docking is
able to reveal all factors determining inhibitory activity, espe-
cially under the conditions described above, remains uncer-
tain. Docking has been performed using the structure of rat
farnesyltransferase. To address the question of structural
differences between rat and P. falciparum farnesyltrans-
ferases a homology model was prepared for P. falciparum
farnesyltransferase (Fig. 3). Although the sequences of rat
and plasmodial farnesyltransferases show considerable dif-
ferences, most active site residues are well conserved with a
local sequence identity of 72%. Sequence variation is found
at two opposite sides of the inhibitor binding site. The sub-
stitutions Tyr 166a to Phe, Met 193b to Val, Ala 129a to Ser,
Pro 152b to Thr and Cys 95b to Ser are located in the region
where the phenyl residue of the phenylglycine moiety is
bound. The conservative nature of these substitutions and the
fact that none of these residues is in direct contact with the
inhibitor suggests similar inhibitor binding modes for rat and
P. falciparum farnesyltransferase. The same holds true for
the opposite binding site, where the most conservative ex-
changesVal 43b to Ile, Thr 44b toAsn and Gln 48b to Pro are
too distant to affect inhibitor binding significantly. Only the
2.4. Flexible docking
The protein structure was taken from the PDB entry
1QBQ [34]. Ligands and solvent molecules were removed,
but the zinc ion and farnesyldiphosphate were included as
part of the protein. Docking was performed with AutoDock
3.0 [35,36]. Resulting ligand conformations with similar
structures (rms deviation <1) were clustered together and
represented by the conformer with the best docking energy.
2.5. Homology modeling
A homology model of P. falciparum farnesyltransferase
was prepared to analyze the differences between mammalian
and parasite enzymes. Sequences for alpha and beta subunits
of rat (Q04631 and Q02293) and P. falciparum (Q8I503 and
Q8IHP6) farnesyltransferase were retrieved from SWISS-
Prot [37] and aligned with T-COFFEE [38]. Homology mod-
els were computed based on the alignments and the rat
template structure (PDB entry: 1QBQ) using MODELLER
[39].
2.6. Cytotoxicity assay
Cytotoxicity of selected compounds was evaluated against
HeLa cells. Viability of the cells was determined after 72 h

96
K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
Table 1
Farnesyltransferase inhibitory activity and antimalarial activity a of compounds 6 and 7. Farnesyltransferase inhibitory activity [28] and antimalarial activity
[23] of parent compounds 8 lacking the a-substitution. Solubility of compounds 7 and 8a
R
FTase IC50
Pfal IC50
270 nM
CC50
>88 µM
Solubility in water
<0.06 mM
Solubility in buffer pH 7.4
<0.04 mM
8a
6a
6
3 nM
8
2 nM
150 nM
>73 µM
–
–
7a
6b
46 3 nM
10 2 nM
3200 nM
230 nM
>80 µM
>73 µM
>1.5 mM
0.2 mM
–
–
7b
8
2 nM
560 nM
>80 µM
>1.5 mM
0.2 mM
8c
6c
15 3 nM
230 nM
200 nM
–
–
–
–
–
100 9 nM
>73 µM
7c
25 3 nM
950 nM
>78 µM
–
–
8d
6d
26 6 nM
47 nM
>57 µM
–
–
–
–
–
8
2 nM
625 nM
(continued on next page)

K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
97
Table 1
(continued)
R
FTase IC50
17 4 nM
Pfal IC50
525 nM
CC50
–
Solubility in water
>1.5 mM
Solubility in buffer pH 7.4
0.08 mM
7d
a IC50 values (nM) for standard antimalarials were: chloroquine, 170; pyrimethamine, 2500; cycloguanile, 2200; quinine, 380; lumefantrine, 30; artemisinin,
18.
Fig. 2. Docking solutions for the S-phenylglycine derivative 7a (yellow carbons) and R-phenylglycine derivative 7b (white carbons). Both amino acid
derivatives show a hydrogen bridge to Ser 99b mediated by a structural water (dotted lines), which is slightly longer for the less active R-enatiomer. Lipophilic
(brown) and hydrophilic (green) properties of the active site are displayed on the Connolly surface. The structural zinc is represented by a violet sphere.
Fig. 3
. Homology model for P. falciparum farnesyltransferase with inhibitor 7a (green carbons). Amino acid residues which are different in rat and plasmodial
farnesyltransferase are indicated (rat: light blue; Pfal: magenta). The structural zinc is shown as brown sphere.

98
K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
replacement of the hydrogen bond acceptor Glu 47b by the
hydrogen bond donor Thr might affect the binding of inhibi-
tors carrying a terminal nitro group.
inhibitors provided the desired compounds with better solu-
bility preserving high farnesyltransferase inhibitory activity.
However, antimalarial activity was detrimentally influenced
by this structural modification.Although undesired, this find-
ing is important since it shows that it takes more for an
inhibitor to be an active antimalarial than to be a good
farnesyltransferase inhibitor, providing argument for the de-
velopment of specialized antimalarial farnesyltransferase in-
hibitors. Current work aims at the identification of alternative
modifications resulting in favorable pharmacokinetic proper-
ties required for in vivo activity while preserving or even
increasing the antimalarial activity of the parent
benzophenone-based farnesyltransferase inhibitors.
In summary, this homology model shows that the main
conclusions concerning the binding of such benzophenone-
based inhibitors derived from docking into the rat structure
are also valid for the plasmodial farnesyltransferase. How-
ever, the homology model reveals some differences, which
might be further exploited for inhibitor development. Inter-
estingly, three of the four Boc-protected precursors (6a, 6b,
6d) also displayed high farnesyltransferase inhibitory activ-
ity. A striking difference in the structure–activity relation-
ships is the lack of stereo-differentiation in case of the Boc-
protected derivatives 6a and 6b, probably due to a different
orientation of the acyl moiety. Unfortunately, no reliable
docking solutions could be obtained for the Boc-derivatives
due to the large number of rotatable bonds.
4. Experimental
In contrast to the activity against isolated farnesyltrans-
ferase the antimalarial activity of the amino derivatives 7a–d
was significantly decreased compared to their parent com-
pounds 8. This effect was most prominent with the most
active parent compound 8c, where introduction of the amino
group led to a 10-fold loss of activity, while only a 2.5-fold
decrease was observed in case of 8b, indicating that SARs
deduced from the parent compounds are not necessarily
relevant for the respective amino derivatives. Because of the
overall low antimalarial activity, no in vivo experiments
where performed.
Remarkably, with exception of the para trifluoromethyl
phenylglycine derivative 6d, the more lipophilic Boc-
protected precursors 6a–c were more active than the unpro-
tected phenylglycine derivatives. Thus, one may speculate
that the lower activity of the phenylglycine derivatives 7a–c
is in part attributable to a hindered membrane penetration of
the more polar amino acid derivatives. However, a variety of
established antimalarial compounds possesses amino
groups, and even a mechanism for the accumulation of basic
compounds in the parasite’s food vacuole has been sug-
gested. Nevertheless, membrane penetration seems to be a
factor to be considered, since structural differences between
mammalian and plasmodial farnesyltransferases as far as
revealed by homology modeling, cannot account for the
differences in farnesyltransferase inhibition and anitplasmo-
dial activity.
4.1. Synthesis
1H-NMR spectra were recorded on a Jeol JMN-GX-
400 and a Jeol JMN-LA-500 spectrometer. Mass spectra
were obtained with a Vacuum Generator VG 7070 H using a
Vector 1 data acquisition system from Teknivent or a Au-
toSpec mass spectrometer from Micromass. IR spectra were
recorded on a Nicolet 510P FT-IR spectrometer. Microanaly-
ses were obtained from a CH analyzer according to Dr. Salzer
from Labormatic and from a Hewlett–Packard CHN analyzer
type 185. Melting points were obtained with a Leitz micro-
scope and are uncorrected. Column chromatography was
carried out using silica gel 60 (0.062–0.200 mm) from
Merck.
4.1.1. General procedure 1: acylation of 5 by
Boc-protected amino acids
The various Boc-protected amino acids and equimolar
amounts of (E)-N-(4-amino-3-benzoylphenyl)-3-[5-(4-nitro-
phenyl)-2-furyl]acrylic acid amide (5) [28] were dissolved in
3 ml pyridine per mmol amino acid. Then, the mixture was
cooled to –15 °C and 0.1 ml POCl3 per mmol amino acid
were added dropwise. After 30 min at –15 °C, the mixture
was poured into ice/water and extracted three to four times
with EtOAc. The combined extracts were washed with satu-
rated NaHCO3 solution and brine, dried over Na2SO4, and
the solvent was evaporated to yield the crude product. The
following compounds were prepared accordingly.
When the antiplasmodial and the cytotoxic activity of the
compounds is compared (Table 1), there is a striking differ-
ence between the IC50-values against cultured parasites
which are ranging between 150 and 3200 nM and the CC50-
values, which are >70–80 µM. This can be interpreted in that
way that the farnesyltransferase inhibitors described here
indeed display a certain degree of selectivity towards ma-
larial parasites.
4.1.1.1.
(E-,S)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]-N-tert-butoxycarbonylphenyl-
glycine amide (6a). From Boc-L-phenylglycine (302 mg,
1.2 mmol) and (E)-N-(4-amino-3-benzoylphenyl)-3-[5-(4-
nitrophenyl)-2-furyl]acrylic acid amide (544 mg, 1.2 mmol).
Yield: 585 mg (71%), m.p. 163 °C; IR (KBr) m 3367, 1686,
1624, 1599, 1511, 1454, 1333, 1290, 1245, 1198, 1163,
1109, 1027, 967, 854, 796, 753, 696 cm–1; 1H-NMR
(DMSO-d6) d 1.34 (s, 9H), 5.19 (s, 1H), 6.74 (d, J = 16 Hz,
1H), 7.01–7.02 (m, 1H), 7.21–7.70 (m, 12H and d, J = 16 Hz,
3. Conclusion
Introduction of a a-amino group into the phenylacetic acid
substructure of our benzophenonebased farnesyltransferase

K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
99
1H), 7.82–7.84 (m, 2H), 7.88 (s, 1H), 7.97–8.00 (m, 2H),
8.30–8.32 (m, 2H), 10.37 (s, 1H), 10.48 (s, 1H); MS (FAB)
m/z 686 (100) [M+ + 1], 670 (2), 631 (23), 587 (90), 570 (18),
542 (8), 480 (63), 454 (63), 453 (38). Anal. (C39H34N4O8) C,
H, N.
removed in vacuo and the products were washed with ac-
etone. The following compounds were prepared accordingly.
4.1.2.1.
(E-,S)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]phenylglycine amide hydro-
chloride (7a). From (E-,S)-N-[2-benzoyl-4-[3-[5-(4-
nitrophenyl)-2furyl]acryloylamino]phenyl]-N-tert-butoxy-
carbonylphenylglycine amide (6a) (342 mg, 0.5 mmol).
Yield: 195 mg (66%), m.p. 211 °C; IR (KBr) m 3374, 1698,
1625, 1598, 1554, 1511, 1403, 1332, 1291, 1247, 1180,
1107, 966, 852, 751, 695 cm–1; 1H-NMR (DMSO-d6) d 5.17
(s, 1H), 6.80 (d, J = 16 Hz, 1H), 7.02–7.03 (m, 1H), 7.39–
7.77 (m, 14H and d, J = 16 Hz, 1H), 7.91–8.01 (m, 2H),
8.30–8.32 (m, 2H), 8.69 (s, 3H), 10.58 (s, 1H), 10.75 (s, 1H);
MS (ESI) m/z 587 (100) [M+], 569 (16), 454 (4), 106 (5);
Anal. (C34H26N4O6·HCl) C, H, N.
4.1.1.2.
(E-,R)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]-N-tert-butoxycarbonylphenyl-
glycine amide (6b). From Boc-D-phenylglycine (302 mg,
1.2 mmol) and (E)-N-(4-amino-3-benzoylphenyl)-3-[5-(4-
nitrophenyl)-2-furyl]acrylic acid amide (544 mg, 1.2 mmol).
Yield: 461 mg (56%), m.p. 163 °C; IR (KBr) m 3369, 1676,
1622, 1599, 1512, 1402, 1333, 1289, 1244, 1199, 1162,
1
1109, 854, 753, 695 cm–1; H-NMR (DMSO-d6) d 1.33 (s,
9H), 5.17 (s, 1H), 6.72 (d, J = 16 Hz, 1H), 7.00–7.01 (m, 1H),
7.24–7.69 (m, 12H and d, J = 16 Hz, 1H), 7.81–7.82 (m, 2H),
7.87 (s, 1H), 7.96–7.97 (m, 2H), 8.28–8.30 (m, 2H), 70.35 (s,
1H), 10.47 (s, 1H); MS (FAB) m/z 687 (46) [M+ + 1], 631
(18), 587 (76), 570 (28), 542 (8), 408 (68), 454 (100), 453
(23); Anal. (C39H34N4O8) C, H, N.
4.1.2.2.
(E-,R)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]phenylglycine amide hydro-
chloride (7b). From (E-,R)-N-[2-benzoyl-4-[3-[5-(4-nitro-
phenyl)-2furyl]acryloylamino]phenyl]-N-tert-butoxycarbo-
nylphenylglycine amide (6b) (342 mg, 0.5 mmol). Yield:
205 mg (82%), m.p. 211 °C; IR (KBr) m 3368, 1699, 1625,
1598, 1550, 1510, 1402, 1332, 1292, 1246, 1181, 751,
4.1.1.3. (E-,R,S)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]-N-tert-butoxycarbonyl-(4-fluo-
rophenyl)glycine amide (6c). From (R,S)-tert-butoxycarbo-
nylamino(4-fluorophenyl)acetic acid (323 mg, 1.2 mmol)
and (E)-N-(4-amino-3-benzoylphenyl)-3-[5(4-nitrophenyl)-
2-furyl]acrylic acid amide (544 mg, 1.2 mmol). Yield:
571 mg (68%), m.p. 196 °C; IR (KBr) m 3414, 1685, 1627,
1
695 cm–1; H-NMR (DMSO-d6) d 5.16 (s, 1H), 6.80 (d,
J = 16 Hz, 1H), 7.03–7.04 (m, 1H), 7.39–7.77 (m, 14H and d,
J = 16 Hz, 1H), 7.91–8.01 (m, 2H), 8.30–8.33 (m, 2H), 8.68
(s, 3H), 10.56 (s, 1H), 10.72 (m, 1H); MS (ESI) m/z 587
(100) [M+], 569 (17), 515 (4), 454 (4), 391 (4), 337 (4), 273
(6), 243 (3), 180 (3), 115 (27), 106 (9); Anal.
(C34H26N4O6·HCl) C, H, N.
1
1600, 1510, 1334, 1292, 1163, 1110, 854 cm–1; H-NMR
(DMSO-d6) d 1.34 (s, 9H), 5.20 (s, 1H), 6.74 (d, J = 16 Hz,
1H), 7.01–7.10 (m, 3H), 7.33–7.50 (m, 6H and d, J = 16 Hz,
1H), 7.60–7.82 (m, 5H), 7.89 (s, 1H), 7.97–8.00 (m, 2H),
8.29–8.31 (m, 2H), 10.37 (s, 1H), 10.44 (s, 1H); MS (FAB)
m/z 705 (100) [M+ + 1], 649 (18), 605 (45), 588 (25), 480
(30), 454 (55), 453 (20); Anal. (C39H33FN4O8) C, H, N.
4.1.2.3.
(E-,S)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]-(4-fluorophenyl)glycine amide
hydrochloride (7c). From (E-,S)-N-[2-benzoyl-4-[3-[5-(4-
nitrophenyl)-2-furyl]acryloylamino]phenyl]-N-tert-butoxy-
carbonyl-(4-fluorophenyl)glycine amide (6c) (342 mg,
0.5 mmol). Yield: 229 mg (75%), m.p. 211 °C; IR (KBr) m
3373, 1861, 1626, 1599, 1511, 1403, 1334, 1292, 1237,
4.1.1.4. (E-,R,S)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]-N-tert-butoxycarbonyl-(4-tri-
fluoromethylphenyl)glycine amide (6d). From (R,S)-tert-
butoxycarbonylamino-(4-trifluoromethylphenyl)acetic acid
(382 mg, 1.2 mmol) and (E)-N-(4-amino-3-benzoylphenyl)-
3-[5-(4-nitrophenyl)-2-furyl]acrylic acid amide (544 mg,
1.2 mmol). Yield: 562 mg (62%), m.p. 168 °C; IR (KBr) m
3314, 1691, 1627, 1599, 1513, 1403, 1333, 1314, 1247,
1
1197, 1165, 1109, 852, 752 cm–1; H-NMR (DMSO-d6): d
5.19 (s, 1H), 6.80 (d, J = 16 Hz, 1H), 7.01–7.21 (m, 3H),
7.39–7.78 (m, 10H and d, J = 16 Hz, 1H), 7.91–8.01 (m, 3H),
8.30–8.32 (m, 2H), 8.69 (s, 3H), 10.57 (s, 1H), 10.76 (s, 1H);
MS (ESI) m/z 605 (100) [M+], 588 (26), 525 (3), 515 (4), 481
(4), 454 (6), 437 (5), 393 (5), 349 (3), 271 (4), 186 (4), 123
(3); Anal. (C34H25FN4O6·HCl) C, H, N.
1
1157, 1109, 1037, 852, 752 cm–1; H-NMR (DMSO-d6) d
1.38 (s, 9H), 5.56 (d, J = 8 Hz, 1H), 6.75 (d, J = 16 Hz, 1H),
7.04–7.05 (m, 1H), 7.40–7.59 (m, 6H and d, J = 16 Hz, 1H),
7.64–7.79 (m, 4H), 7.89–8.05 (m, 4H), 8.17 (d, J = 8 Hz, 1H),
8.33–8.35 (m, 2H), 8.64 (m, 1H), 10.51 (s, 1H), 10.94 (s,
1H); MS (FAB) m/z 755 (21) [M+ + 1], 754 (20) [M+], 655
(47), 454 (100), 438 (18), 353 (11), 264 (27), 242 (100), 196
(59); Anal. (C40H33F3N4O8) C, H, N.
4.1.2.4.
(E-,S)-N-[2-benzoyl-4-[3-[5-(4-nitrophenyl)-2-
furyl]acryloylamino]phenyl]-(4-trifluoro-methylphenyl)gly-
cine amide hydrochloride (7d). From (E-,S)-N-[2-benzoyl-
4-[3-[5(4-nitrophenyl)-2-furyl]acryloylamino]phenyl]-N
-
tert-butoxycarbonyl-(4trifluoromethylphenyl)glycine amide
(6d) (342 mg, 0.5 mmol).Yield: 120 mg (41%); m.p. 224 °C;
IR (KBr) m 3371, 1698, 1678, 1624, 1599, 1547, 1510, 140,
1335, 1315, 1244, 1123, 1036, 852 cm–1; 1H-NMR (DMSO-
d6) d 5.14 (s, 1H), 6.84 (d, J = 16 Hz, 1H), 7.05 (m, 1H), 7.40
4.1.2. General procedure 2: Removal of the Boc-protective
group
The Boc-protected compounds 6 were dissolved in 10 ml
of 4 M HCl in dioxane. After stirring for 2 h, the solvent was

100
K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
(d, J = 16 Hz, 1H), 7.43–7.51 (m, 3H), 7.60–7.71 (m, 6H),
7.74–7.77 (m, 1H), 7.83–7.87 (m, 2H), 7.98–8.02 (m, 3H),
8.32–8.34 (m, 2H), 9.00 (s, 3H), 10.46 (s, 1H), 10.75 (s, 1H);
MS (FAB) m/z 654 (100) [M+], 480 (10), 454 (41), 391 (10),
242 (62), 212 (26); Anal. (C35H25F3N4O6·HCl) C, H, N.
plates were further incubated for 24 h. Cells were collected
on glass fiber filters with a cell harvester (Micromate 196,
Packard), and incorporated radioactivity was measured using
a b-counter (Matrix 9600, Packard).
4.1.6. Cytotoxicity assay
4.1.3. Enzyme preparation
HeLa (DSMACC 57) cells were grown in RPMI 1640 cul-
ture medium (GIBCO BRL 21875034) supplemented with
25 µg ml–1 gentamicin sulfate (BioWhittaker 17-528Z), and
10% heat inactivated fetal bovine serum (GIBCO BRL
10500-064) at 37 °C in high density polyethylene flasks
(NUNC 156340). The test substances are dissolved in DMSO
(10 mg ml–1) before being diluted in the cell culture medium
(1:200). The adherent HeLa cells were harvested at the loga-
rithmic growth phase after soft trypsinization, using 0.25%
trypsin in PBS containing 0.02% EDTA (Biochrom KG
L2163). For each experiment approximately 10,000 cells
were seeded with 0.1 ml RPMI 1640 (GIBCO BRL 21875-
034), containing 25 µg ml–1 gentamicin sulfate (BioWhit-
taker 17-528Z), but without HEPES per well of the 96-well
microplates (NUNC 167008). For the cytotoxic assay HeLa
cells were 48 h preincubated without the test substances. The
dilutions of the test substances were carried out carefully on
the monolayers of HeLa cells after the preincubation time.
Cells of HeLa were incubated for 72 h at 37 °C in a humidi-
fied atmosphere and 5% CO2. The adherent HeLa cells were
fixed by 25% glutaraldehyde and stained with a 0.05% solu-
tion of methylene blue for 15 min. After gently washing the
stain was eluted by 0.2 ml of 0.33 N HCl in the wells. The
optical densities were measured at 660 nm in SUNRISE
microplate reader (TECAN). Comparisons of the different
values were performed with software Magellan (TECAN).
Yeast farnesyltransferase was used as a fusion protein to
glutathione S-transferase at the Nterminus of the 9-subunit.
Farnesyltransferase was expressed in Escherichia coli DH59
grown in LB media containing ampicillin and chlorampheni-
col for co-expression of pGEX-DPR1 and pBC-RAM2 for
farnesyltransferase production [30]. The enzyme was puri-
fied by standard procedures with glutathione-agarose beads
for selective binding of the target protein.
4.1.4. Farnesyltransferase assay
The assay was conducted as described [29]. FPP was
obtained as a solution of the ammonium salt in methanol–
10 mM aqueous NH4Cl (7:3) from Sigma–Aldrich. Dansyl-
GlyCysValLeuSer (Ds-GCVLS) was custom-synthesized by
ZMBH, Heidelberg, Germany. The assay mixture (100 1 l
volume) contained 50 mM Tris–HCl pH 7.4, 5 mM MgCl2,
10 1 M ZnCl2, 5 mM dithiothreitol (DTT), 7 1 M Ds-
GCVLS, 20 1 M FPP and 5 nmol (approx.) yeast GST-
farnesyltransferase and 1% of various concentrations of the
test compounds dissolved in dimethylsulfoxide (DMSO).
The progress of the enzyme reaction was followed by moni-
toring the enhancement of the fluorescence emission at
505 nm (excitation 340 nm). The reaction was started by
addition of the enzyme and run in a Quartz cuvette thermo-
statted at 30 °C. Fluorescence emission was recorded with a
Perkin–Elmer LS50B spectrometer. IC50-values (concentra-
tions resulting in 50% inhibition) were calculated from initial
velocity of three independent measurements of four to five
different concentrations of the respective inhibitor.
4.2. Flexible docking
The protein structure was taken from the PDB entry
1QBQ [34]. Ligands and solvent molecules were removed,
but the zinc ion and farnesyldiphosphate were included as
part of the protein. For the use within AutoDock 3.0 [35,36],
polar hydrogens were added with the PROTONATE utility
from AMBER [40]. AMBER united atom force field charges
were assigned [41], and solvation parameters were added
using theADDSOL utility fromAutoDock 3.0. Ligand struc-
tures were built in mol2 format, Gasteiger partial atomic
charges were assigned [42], and all bonds except for amides
were kept rotatable. Docking runs were performed with the
Lamarckian genetic algorithm included in AutoDock 3.0
[43], performing 50 independent runs per ligand, using an
initial population of 50 randomly placed individuals, a maxi-
mum number of 1.5 × 106 energy evaluations, a mutation rate
of 0.02, a crossover rate of 0.80, and an elitism value of 1.
Resulting ligand conformations that differ by less than 1 Å
rmsd from each other were clustered together and were
represented by the solution with the best docking energy.
4.1.5. In vitro measurement of P. falciparum parasite
growth inhibition
Compounds were tested by a semi-automated microdilu-
tion assay against intraerythrocytic forms of P. falciparum
[31]. The P. falciparum strains Dd2 and 3D7 were cultivated
by a modification of the method described by Trager and
Jensen [32]. The culture medium consisted of RPMI
1640 supplemented with 10% human type 0+ serum and
25 mM HEPES. Human type 0+ erythrocytes served as host
cells. The cultures were kept at 37 °C in an atmosphere of 5%
O2, 3% CO2, and 92% N2.
Drug testing was carried out in 96-well microtiter plates.
The compounds were dissolved in DMSO (10 mM) and
prediluted in complete culture medium (final DMSO concen-
trations ≤1%). Infected erythrocytes (200 µl 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 [33]. After the addition of
0.8 µCi [3H]-hypoxanthine in 50 µl medium per well, the

K. Kettler et al. / European Journal of Medicinal Chemistry 40 (2005) 93–101
101
[21] J. Wiesner, A. Mitsch, P. Wißner, O. Krämer, H. Jomaa, M. Schlitzer,
Bioorg. Med. Chem. Lett. 12 (2002) 2681–2683.
Acknowledgements
[22] J. Wiesner, R. Ortmann, A. Mitsch, P. Wißner, I. Sattler, H. Jomaa,
M. Schlitzer, Pharmazie 58 (2003) 289–290.
[23] J. Wiesner, K. Kettler, J. Sakowski, R. Ortmann, H. Jomaa, M. Schl-
itzer, Bioorg. Med. Chem. Lett. 13 (2003) 361–363.
[24] J. Wiesner, K. Fucik, K. Kettler, J. Sakowski, R. Ortmann, H. Jomaa,
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The pGEX-DPR1 and pBC-RAM2 plasmids were kindly
provided by Professor F. Tamanoi (UCLA). I.S. wishes to
thank Professor Dr. S. Grabley for generous support and Ms.
S. Egner for technical assistance.
[26] J. Wiesner, A. Mitsch, M. Altenkämper, R. Ortmann, H. Jomaa,
M. Schlitzer, Pharmazie 58 (2003) 854–856.
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