Double ester prodrugs of FR900098 display enhanced in-vitro antimalarial activity

Article abstract of DOI:10.1002/ardp.200700069

Fosmidomycin and FR900098 are inhibitors of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; IspC), a key enzyme of the mevalonate-independent isoprenoid biosynthesis pathway. We have determined the in-vitro antimalarial activity of two double ester prodrugs 2, 3 in direct comparison with the unmodified FR900098 1 against intraerythrocytic forms of Plasmodium falciparum. Temporarily masking the polar properties of the phosphonate moiety of the DXR inhibitor FR900098 1 enhanced not only its oral bioavailability but also the intrinsic activity of this series against the parasites.

Full text of DOI:10.1002/ardp.200700069

Arch. Pharm. Chem. Life Sci. 2007, 340, 667–669
J. Wiesner et al.
667
Short Communication
Double Ester Prodrugs of FR900098 Display Enhanced
In-Vitro Antimalarial Activity
Jochen Wiesner1, Regina Ortmann2, Hassan Jomaa1, and Martin Schlitzer2
1 Institut fꢀr Klinische Chemie und Pathobiochemie, Universitꢁtsklinikum Giessen und Marburg GmbH,
Gießen, Germany
2 Institut fꢀr Pharmazeutische Chemie, Philipps-Universitꢁt Marburg, Marburg, Germany
Fosmidomycin and FR900098 are inhibitors of the 1-deoxy-D-xylulose 5-phosphate reductoiso-
merase (DXR; IspC), a key enzyme of the mevalonate-independent isoprenoid biosynthesis path-
way. We have determined the in-vitro antimalarial activity of two double ester prodrugs 2, 3 in
direct comparison with the unmodified FR900098 1 against intraerythrocytic forms of Plasmo-
dium falciparum. Temporarily masking the polar properties of the phosphonate moiety of the
DXR inhibitor FR900098 1 enhanced not only its oral bioavailability but also the intrinsic activ-
ity of this series against the parasites.
Keywords: 1-Deoxy-D-xylulose 5-phosphate reductoisomerase inhibitors / Mevalonate-independent isoprenoid bio-
synthesis / Prodrug /
Received: June 20, 2007; accepted: August 24, 2007
DOI 10.1002/ardp.200700069
FR900098 1 is a structurally closely related derivative
of fosmidomycin, and is about twice as active as fosmido-
mycin against cultured parasites and in animal models
[8]. To enhance oral bioavailability we have developed
double ester prodrugs [9, 10].
Introduction
Malaria is the most important tropical disease and one of
the most important infectious diseases altogether. Due
to the widespread occurrence of Plasmodium falciparum
strains resistant against most common antimalarial
agents development of new drugs directed against tar-
gets hitherto unexploited is mandatory [1].
Results and discussion
Fosmidomycin is an inhibitor of the 1-deoxy-D-xylulose
5-phosphate reductoisomerase (DXR; IspC), a key enzyme
of the mevalonate-independent isoprenoid synthesis.
This biosynthetic pathway is used in most eubacteria as
well as by the plastids of algae and higher plants. It is also
used exclusively in P. falciparum, while being absent in
the human host [2–4]. In three different clinical studies,
fosmidomycin in combination with the antibiotic clinda-
mycin has shown efficacy and good tolerability [5–7].
However, due to its high polarity, oral bioavailability is
only about 30% [8].
Synthesis of the prodrugs [10] was achieved starting from
the aldehyde 2, which is readily accessible from the com-
mercially available diethyl acetal (Scheme 1). Treatment
of the aldehyde 2 with O-benzylhydroxylamine and subse-
quent reduction with sodium cyanoborohydride gave
the O-(benzylhydroxylamino)propylphosphonic acid
diethyl ester 3. Acetylation to 4 was carried out with ace-
tylchloride in dichloromethane. Reaction of 4 with trime-
thylbromosilane and hydrolysis of the resulting silylester
with water yielded the O-benzyl protected FR900098 5.
Phosphonic acid 5 was then coupled with commercially
available chloromethyl pivalate and 2-chloroethyl ace-
tate respectively yielding 6a and b. Removal of the pro-
tecting groups with hydrogen and 10% Pd/C in methanol
gave the prodrugs 7a and b.
Correspondence: Martin Schlitzer, Institut fꢀr Pharmazeutische Chemie,
Philipps-Universitꢁt, Marbacher Weg 6, D-35037 Marburg, Germany
E-mail: Schlitzer@staff.uni-marburg.de
Fax: +49 6421 28-25978
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

668
J. Wiesner et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 667–669
Conditions and yields: (i) O-benzylhydroxylamine in methanol, 3 h, 408C, NaCNBH3 in methanol, HCl, 1 h, rt, 99%; (ii) acetylchloride in CH2Cl2, (H5C2)3N, overnight, rt, 97%; (iii)
trimethylbromosilane in CH2Cl2, 30 min, 08C, overnight, H2O, rt, 82%; (iv) 6a: RCl in DMF, (H5C2)3N, 6 h, 608C, 6b: RCl, DMPU, NaI, (H5C2)3N, 6 h, 608C; (v) H2, Pd/C, MeOH.
Scheme 1. Synthesis of compounds 2–7.
Double ester prodrugs displayed 2-3fold higher activ- the incorporation of tritium-labeled hypoxanthine. The
ities in comparison to the unmodified FR900098 1 when enzyme inhibition assay was performed in a reaction
dosed orally to malaria-infected mice [10].
mixture containing 100 mM Tris HCl (pH 7.5), 0.2% BSA,
It has been anticipated that these prodrugs are 1 mM MnCl2, 1 mM NADPH, 0.3 mM DOXP, and 1 lg/mL
resorbed from the gastrointestinal tract and cleaved by recombinant DOXP reductoisomerase from E. coli. The
plasma esterases into the active FR900098 and the corre- mixture was incubated with a dilution series of the test
sponding aldehyde and acid. Using the activity of plasma compounds on a 96-well plate, and the reaction started
samples against DXR, the concentration of free FR900098 by addition of DOXP. The decrease of absorption was
was estimated to be 3 lM 0.5 h after oral administration monitored at 340 nm using a SpectraMax 340PC micro-
of 40 mg/kg of prodrug 7b into mice in comparison to plate reader (Molecular Devices, Ismaning, Germany).
1.2 lM after administration of FR900098 (40 mg/kg). It
was further anticipated that free phosphonate FR900098 2-5fold more active against the cultured parasites of the
1 then should enter and subsequently kill the parasites. chloroquine sensitive 3D7 and the multi-resistant Dd2 P.
Unexpectedly, the two double esters 7a, b were about
Independently, it was also shown that double ester pro- falciparum strain (Table 1). Both double ester prodrugs 7a
drugs of several fosmidomycin derivatives display in-vitro and b did not show any activity against E. coli DXR at
antimalarial activity [11, 12]. Hitherto, it has been antici- 30 lM, the highest concentration tested (Table 1). Since
pated that the ester prodrugs are readily cleaved under the activities of a particular compound against DXR form
assay conditions to yield the free phosphonates which E. coli and P. falciparum are usually well correlated [16],
then enter and subsequently kill the parasites, so that we conclude that the antiparasitic activity of our esters
the in-vitro activity of the double ester prodrugs is practi- 7a and b is caused by the action of the free FR900098 mol-
cally the same as the activity of the unmodified parent ecule on DXR. Would the esters have been cleaved already
phosphonic acid. If this is true, there should be no differ- in the assay medium as previously has been anticipated,
ence in the in-vitro activity between the double ester pro- their activity should not be higher than that of the
drugs of FR900098 and the unmodified FR900098.
unmodified FR900098. But since the esters 7a and b are 2-
To test this hypothesis, we have tested the activity of 5fold more active than the unmodified FR900098, we
two double ester prodrugs 7a, b in direct comparison conclude that the double ester prodrugs possess consider-
with the unmodified FR900098 1 against intraerythro- able stability under the assay conditions. We propose the
cytic forms of the P. falciparum strains 3D7 and Dd2 using following explanation for the higher antiparasitic activ-
a semi-automated microdilution assay as described [13–
ity of the ester prodrugs. These uncleaved ester prodrugs
15]. The growth of the parasites was monitored through are much more lipophilic than the free phosphonate
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Arch. Pharm. Chem. Life Sci. 2007, 340, 667–669
Double Ester Prodrugs of FR900098
669
Table 1. In-vitro activity of FR900098 and two of its double ester prodrugs.
IC50 (nM)
IC50 (nM)
E.coli DXR
3D7
Dd2
FR900098 (1)
Schl-7150 (7a)
50
868
893
A30000
328
272
174
Schl-7168 (7b)
A30000
–
[2] J. Wiesner, F. Seeber, Expert Opin. Ther. Targets 2005, 9,
drugs, and therefore could more readily enter the para-
site via diffusion through the parasites' membranes. Pos-
sibly, the prodrugs circumvent the glycerol-3-phosphate
transport system which, at least in bacteria, is thought to
facilitate the uptake of the unmodified phosphonate
drug fosmidomycin [17]. Inside the parasites, the double
ester prodrugs are then cleaved (since the esters are inac-
tive, see above) by the action of esterases to liberate the
free FR900098.
23–44.
[3] J. Wiesner, H. Jomaa, Curr. Drug Targets 2007, 8, 3–13.
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Conclusion
[8] J. Wiesner, S. Borrmann, H. Jomaa, Parasitol. Res. 2003, 90,
71–76.
In conclusion, temporarily masking the polar properties
of the phosphonate moiety of the DXR inhibitor
FR900098 1 enhances not only the oral bioavailability as
shown previously [9, 10], but also the intrinsic activity of
these less polar derivatives against the parasites, most
probably by achieving a higher intraparasitic drug con-
centration through enhanced drug uptake. To what
degree this enhanced intrinsic antiparasitic activity con-
tributes to the observed superior in vivo antimalarial
activity remains to be investigated.
[9] R. Ortmann, J. Wiesner, A. Reichenberg, D. Henschker, et
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The authors have declared no conflict of interest.
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i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com