Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity

Article abstract of DOI:10.1016/S0960-894X(03)00354-8

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynt

Full text of DOI:10.1016/S0960-894X(03)00354-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2163–2166
Acyloxyalkyl Ester Prodrugs of FR900098 with Improved
In Vivo Anti-Malarial Activity
Regina Ortmann,a Jochen Wiesner,b Armin Reichenberg,b Dajana Henschker,b
Ewald Beck,b Hassan Jomaab and Martin Schlitzera,*
aDepartment fur Pharmazie, Ludwig-Maximilians-Universitat Munchen, Butenandtstraße 5-13, D-81377 Munchen, Germany
¨
¨
¨
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bBiochemisches Institut, Justus-Liebig-Universitat Gießen, Friedrichstrasse 24, D-35392 Gießen, Germany
¨
Received 6 November 2002; revised 14 March 2003; accepted 3 April 2003
Abstract—FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of
the 1-deoxy-d-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of
isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the
phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice
infected with the rodent malaria parasite Plasmodium vinckei.
# 2003 Elsevier Science Ltd. All rights reserved.
Malaria affects over 40% of the world’s population, and
1.5–3 million people die of the disease every year, most
of them children. The numbers are still rising since
available anti-malarials such as chloroquine and sulfa-
doxin-pyrimethamine increasingly lose efficacy due to
the spread of resistant parasite strains. As a result, there
is an urgent need for new efficient antimalarial agents.1
values.5 In previous work, we synthesized diaryl ester
prodrugs of FR900098 in order to increase gastro-
intestinal resorption.6 These prodrugs resulted in
improved oral efficacy in P. vinckei infected mice, but
concerns about the toxicity of the phenol derivative
generated through processing of the prodrugs prompted
us to investigate an alternative strategy. Therefore, a
series of acyloxyalkyl esters expected to be hydrolysed
by non-specific esterases has been prepared.
In malaria parasites, isoprenoids are synthesized by a
mevalonate independent pathway, the so-called DOXP
pathway, which is absent in humans. In previous stud-
ies, we demonstrated that fosmidomycin exerts potent
antimalarial activity by inhibition of DOXP reducto-
isomerase, the second enzyme in the reaction cascade of
the DOXP pathway.2,3 In recent clinical trials con-
ducted in Gabon and Thailand, fosmidomycin proved
to be efficient in the treatment of patients suffering from
acute, uncomplicated Plasmodium falciparum malaria.4
The acetyl derivative of fosmidomycin, FR900098, is
approx. twice as active against P. falciparum in vitro
and in the Plasmodium vinckei mouse model.2 However,
the oral bioavailability of both compounds is only
moderate, with a resorption rate of approx 30%, most
likely due to very low lipophilicity as a result of high
ionization of the phosphono moiety at physiological pH
Synthesis of the prodrugs was achieved starting from
the aldehyde 2, which is readily accessible from the
commercially available corresponding acetale 1 (Scheme
1). Treatment of the aldehyde 2 with O-benzylhydroxyl-
amine gave the oxime, which was then treated without
prior isolation with sodium cyanoborohydride and
hydrochloric acid to yield the O-(benzylhydroxyl-
amino)propylphosphonic acid diethyl ester 3.7 Acetyl-
ation to 4 was carried out with acetylchloride in
dichloromethane. Reaction of
4
with trimethyl-
bromosilane and hydrolysis of the resulting silylester
with water yielded the O-benzyl protected FR900098 5
(Fig. 1).8 Phosphonic acid 5 was then coupled with
chloroacetic acid methylester or chloromethyl pivalate,
respectively, in DMF in the presence of triethylamine
giving the phosphonic acid diesters 6a and 6b.9 This
method was unsuccessful in the case of 1-chloroethyl
pivalate and 1-chloroethyl benzoate. However, when the
phosphonic acid 5 and the chloroalkyl esters10 were
*Corresponding author. Tel.: +49-89-2180-77804; fax: +49-89-2180-
79992; e-mail: martin.schlitzer@cup.uni-muenchen.de
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00354-8

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R. Ortmann et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2163–2166
Scheme 1. Conditions and yields: (i) 2 N HCl, overnight, rt, 63%; (ii) O-benzylhydroxylamine in methanol, 3 h, 40 ꢀC and then NaCNBH3 in
methanol, HCl, 1 h, rt, 99%; (iii) acetylchloride in CH2Cl2, (H3C)3N, overnight, rt, 97%; (iv) trimethylbromosilane in CH2Cl2, 30 min, 0 ꢀC and then
H2O, overnight, rt, 82%; (v) (a and b) RCl in DMF, (H3C)3N, 6 h, 60 ꢀC; (c–g) RCl, DMPU, NaI, (CH3)3N, 6 h, 60 ꢀC; (vi) H2, Pd/C, MeOH.
5Â107 infected erythrocytes from a donor mouse. On
days 1 and 2, the mice were treated by oral administra-
tion of 40 mg/kg of the prodrugs.12 For comparison,
another group of mice was treated with an equivalent
dosage of FR900098. This dosage was estimated to
result in a partial reduction of parasitaemia. On the
following days, parasitaemia was monitored to assess
the efficacy of the compounds.
In the first experiment, the efficacy of 7a and 7b was
tested (Fig. 2). The treatment with the acetyl ester deri-
vative 7a resulted in a marked reduction of parasitaemia
compared with untreated control mice, but was less
Figure 1. Structures of fosmidomycin and FR900098.
effective than FR900098. In contrast, the pivaloyloxy-
methyl ester 7b was significantly more active in sup-
pressing the development of parasitaemia than the
parent compound. Based on this result, additional acyl-
oxyalkyl derivatives of FR900098 were investigated. In
order to avoid the release of formaldehyde, the dioxy-
methylen group was replaced by a 1,1-dioxyethylen
group generating the less toxic acetaldehyde. The piva-
loyloxyethyl ester 7c and the benzoyloxyethyl ester 7d
displayed activities comparable with but not superior to
FR900098. Therefore, two derivatives which carry less
bulky acyl residues were tested. Both the acetyloxyethyl
reacted in DMPU in the presence of triethylamine and
sodium iodide, the bis(acyloxy)alkyl esters 6c–g were
obtained as diastereomeric mixtures.9 Removal of the
protecting groups with hydrogen and 10% Pd/C in
methanol gave the prodrugs 7a–g.11
The anti-malarial activity of the prodrugs was tested in
the P. vinckei mouse model similar as described before.6
On day 0, Balb/c mice were inoculated by ip injection of

R. Ortmann et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2163–2166
2165
Figure 3. Antimalarial efficacy of 10 and 20 mg/kg 7e in comparison to
40 mg/kg FR900098. Mice were infected on day 0 and treated on days
1 and 2. Parasitaemia was monitored on days 3–8.
Figure 4. Plasma concentration of FR900098 in mice after oral
administration of 40 mg/kg FR900098 and 7e.
In a subsequent experiment, 7e as the most successful
compound of this series was characterized further. In
order to obtain a quantitative assessment of the increase
in efficacy, mice were treated with 10 mg/kg and 20 mg/
kg of 7e (Fig. 3), respectively, and compared with
another group of mice receiving 40 mg/kg FR900098.
The treatment with 10 mg/kg 7e resulted in a reduced
efficacy compared with 40 mg/kg FR900098. The treat-
ment with 20 mg/kg 7e, however, was slightly more
effective than 40 mg/kg FR900098.
In order to confirm whether the improved efficacy of 7e
correlates with an increased bioavailability, the plasma
levels of FR900098 were monitored at different time
points after oral administration of identical doses of
FR900098 and 7e. Since currently no chromatographic
methods for the determination of low amounts of
FR900098 are available, the inhibitory activity of the
plasma samples against recombinant DOXP reducto-
isomerase was measured in order to provide an estima-
tion of the FR900098 plasma concentration.13 As
expected, the FR900098 plasma concentration was sig-
nificantly higher after administration of the prodrug
(Fig. 4). At 0.5 h, after administration the plasma level
was 3.0 mM after administration of 7e in comparison to
Figure 2. Antimalarial efficacy of the prodrugs 7a–g in comparison to
FR900098. Mice were infected on day 0 and treated with 40 mg/kg of
the respective compound on day 1 and 2. Parasitaemia was monitored
on days 3–8.
ester 7e and propionyloxyethyl ester 7f proved to be
more effective than FR900098, with 7e being slightly
more active than 7f. In contrast, the propionyl-
oxyisobutyl derivative 7g did not lead to an improved
activity.

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R. Ortmann et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2163–2166
1.2 mM after administration of FR900098. After 2 h, the
plasma concentration fell to the limit of quantification
at approx. 0.4 mM in both groups.
6. Reichenberg, A.; Wiesner, J.; Weidemeyer, C.; Dreiseidler,
E.; Sanderbrand, S.; Altincicek, B.; Beck, E.; Schlitzer, M.;
Jomaa, H. Bioorg. Med. Chem. Lett. 2001, 11, 833.
7. Kurz, T.; Geffken, D.; Wackendorff, C. Z. Naturforsch., B:
Chem. Sci. 2003, 58b, 106.
8. McKenna, C. E.; Shen, P. J. Org. Chem. 1981, 46, 4573.
9. Serafinowska, H. T.; Ashton, R. J.; Bailey, S.; Harnden,
M. R.; Jackson, S. M.; Sutton, D. J. Med. Chem. 1995, 38,
1372.
In summary, prodrugs of FR900098 with increased oral
anti-malarial efficacy were obtained by masking the
polar phosphonate moiety as acyloxyalkyl esters. The
acetyloxyethyl ester 7e, which is expected to release only
acetic acid and acetaldehyde upon hydrolysis in addi-
tion to the active compound, was at least twice as active
than FR900098.14
10. (a) The chloroalkyl esters were prepared by reaction of
acetaldehyde (or isobutyraldehyde in case of 6g) with the
appropriate acid chloride. (a) Fleischmann, K.; Adam, F.;
Durckheimer, W.; Hertzsch, W.; Horlein, R.; Jendralla, H.;
Lefebvre, C.; Mackiewicz, P.; Roul, J.-M.; Wollmann, T. Lie-
bigs Ann. 1996, 1735. (b) Saari, W. S.; Freedman, M. B.;
Hartman, R. D.; King, S. W.; Raab, A. W.; Randall, W. C.;
Engelhardt, E. L.; Hirschmann, R. J. Med. Chem. 1978, 21,
746.
Acknowledgements
This study was supported by grants of the Bundesmi-
nisterium fur Bildung und Forschung (BioChance
0312588) and the European Commission (INCO-Dev,
5th Framework Programme, Contract No. ICA4-2000-
10290). We thank Matthias Eberl for critical reading of
the manuscript.
11. Target compounds 7a–g were characterized by IR, 1H
NMR, MS and microanalysis.
12. Prodrugs were dissolved in DMSO at 150 mg/mL and
further diluted in SSV (standard suspension vehicle; 0.9%
NaCl, 0.5% Na-carboxymethylcellulose, 0.5% benzyl alcohol,
0.4% Tween 80). FR900098 was dissolved in SSV. Para-
sitaemia was monitored by Giemsa-stained blood smears.
Mice were sacrificed when parasitaemia was exceeding approx
40%. Four mice were used for each treatment group, and
three mice for the control group. Results were expressed as
geometric mean values for each group.
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13. At each time point, one mouse of each group was sacri-
ficed, and the blood collected. The enzyme inhibition assay
was performed in a reaction mixture containing 100 mM Tris–
HCl (pH 7.5), 0.2% BSA, 1 mM MnCl2, 1 mM NADPH,
0.3 mM DOXP, and 1 mg/mL DOXP reductoisomerase from
Escherichia coli. The mixture was incubated with a dilution
series of the plasma samples on a 96-well microtiter plate, and
the reaction started by addition of DOXP. The decrease of
absorption was monitored at 340 nm using a SpectraMax
340PC microplate reader (Molecular Devices, Ismaning). The
plasma concentrations were calculated from the enzyme
inhibition values obtained with FR900098 standard solutions
analyzed in parallel.
14. When calculating the dosages in molar units instead of
mass units (40 mg/kg FR900098=0.18 mmol/kg; 20 mg/kg
7e=0.054 mmol/kg), 7e has 3-fold increased activity compared
with FR900098.