Synthesis and antiplasmodial activity of highly active reverse analogues of the antimalarial drug candidate fosmidomycin

Article abstract of DOI:10.1002/cmdc.201000276

Inhibition of enzymes involved in the nonmevalonate pathway of isoprenoid biosynthesis represents a promising strategy for the development of novel antimalarial agents. A small series of reverse hydroxamate-based fosmidomycin analogues was synthesized and evaluated for their inhibitory activity against the recombinant 1-deoxy-D-xylulose 5-phosphate reductoisomerases (DXRs) of Escherichia coli and Plasmodium falciparum, as well as for their in vitro antiplasmodial activity and cytotoxicity.

Full text of DOI:10.1002/cmdc.201000276

DOI: 10.1002/cmdc.201000276
Synthesis and Antiplasmodial Activity of Highly Active Reverse Analogues
of the Antimalarial Drug Candidate Fosmidomycin
Christoph T. Behrendt,[a] Andrea Kunfermann,[b] Victoria Illarionova,[c] An Matheeussen,[d] Tobias Grꢀwert,[b]
Michael Groll,[b] Felix Rohdich,[b] Adelbert Bacher,[b] Wolfgang Eisenreich,[b] Markus Fischer,[c] Louis Maes,[d] and
Thomas Kurz*[a]
Although there are potent antimalarial drugs available, the
spread of drug resistance has led to an increase in morbidity
and mortality rates in malaria-endemic regions. To overcome
these problems, new antimalarial drugs with novel modes of
action have to be developed. The recently discovered nonme-
valonate pathway of isoprenoid biosynthesis (MEP pathway) is
an important metabolic pathway for the design of novel anti-
malarial drugs.[1] Various pathogenic bacteria (e.g., Mycobacteri-
um tuberculosis) and apicomplexan protozoa (e.g., Plasmodium
falciparum) use the MEP pathway for the production of isopen-
tenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP).[2–4] IPP and DMAPP are important precursors for the
biosynthesis of various essential isoprenoids. In contrast to the
latter microorganisms, humans produce IPP and DMAPP exclu-
sively via the mevalonate–acetate pathway. The second
enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate
reductoisomerase (DXR, IspC) represents a validated target
enzyme for the development of new antimalarial drugs.[4] In
1998, the natural product fosmidomycin was identified as a
potent inhibitor of P. falciparum DXR (PfDXR).[5] The DXR cata-
lyzes the rearrangement and reduction of 1-deoxy-d-xylulose
5-phosphate (DOXP) into 2-C-methyl-d-erythritol 4-phosphate
(MEP). While the first step of the reaction requires a divalent
cation (e.g., Mn2+ or Mg2+), the reduction step is NADPH-de-
pendent.[6] Clinical trials performed with fosmidomycin have al-
ready shown high efficiency in the treatment of acute, uncom-
plicated malaria tropica.[7] However, the oral bioavailability of
fosmidomycin is only moderate, with a resorption rate of
about 10–30%.[5,8] Various research groups are involved in the
design of novel DXR inhibitors.[3] Molecular field analyses, as
well as crystallographic studies, could already contribute in de-
fining structural requirements for the development of potent
DXR inhibitors.[9,10] Consequently, different types of DXR inhibi-
tors have been synthesized and biologically evaluated.[3,4,11] Im-
portant structural elements for potent antimalarial activity are
the hydroxamic acid functionality and the phosphonic acid
group.[4,11] The DXR–fosmidomycin complex crystal structure
clearly revealed that the hydroxamic acid functionality chelates
the divalent cation, while the phosphonic acid group occupies
the phosphate binding site.[10] Moreover, a defined distance be-
tween both functional groups is essential for potent antimalari-
al activity. Schlitzer and Klebe have shown that the two nega-
tive charges of the phosphonic acid group are necessary for
high inhibitory activity.[12] Furthermore, phosphonate prodrugs
have been designed to improve the bioavailability of fosmido-
mycin (1) and FR900098 (2).[13] A combinatorial approach for
the synthesis of nonhydrolyzable phosphate mimics was re-
ported by Link and co-workers,[14] while Song recently de-
scribed a coordination chemistry-based approach for the de-
velopment of lipophilic DXR inhibitors that exhibit good to
moderate activity against various pathogenic bacteria.[15] To
the best of our knowledge, a-aryl-substituted derivatives (4) of
fosmidomycin are among the most active analogues known so
far.[16] Recently, Van Calenbergh reported that b-oxaisosters
(3a,b) of fosmidomycin exhibit strong in vitro antimalarial ac-
tivity.[17] Interestingly, some of the new compounds contain an
N-methyl-substituted hydroxamic acid group with a reverse
orientation of the hydroxamate group (3b) compared to the
lead compounds (1 and 2).
Due to difficulties associated with the handling of PfDXR, no
data concerning the inhibitory activity of these analogues
toward PfDXR have been reported.[4,17] Furthermore, Rohmer
and co-workers presented several reverse fosmidomycin ana-
logues, which in part inhibit recombinant E. coli DXR
(EcDXR).[18] In order to improve the antiplasmodial activity and
[a] C. T. Behrendt, Prof. Dr. T. Kurz
Institut fꢀr Pharmazeutische und Medizinische Chemie
Heinrich-Heine-Universitꢁt Dꢀsseldorf
Universitꢁtsstr. 1, 40225 Dꢀsseldorf (Germany)
Fax: (+49)211-811-3847
E-mail: Thomas.Kurz@uni-duesseldorf.de
[b] A. Kunfermann, Dr. T. Grꢁwert, Prof. M. Groll, Dr. F. Rohdich, Prof. A. Bacher,
Dr. W. Eisenreich
Lehrstuhl fꢀr Biochemie, Center for Integrated Protein Science Mꢀnchen
Technische Universitꢁt Mꢀnchen
Lichtenbergstr. 4, 85747 Garching (Germany)
[c] Dr. V. Illarionova, Prof. Dr. M. Fischer
Institut fꢀr Lebensmittelchemie, Universitꢁt Hamburg
Grindelallee 117, 20146 Hamburg (Germany)
[d] A. Matheeussen, Prof. Dr. L. Maes
Laboratory for Microbiology, Parasitology and Hygiene (LMPH)
University of Antwerp, Groenenborgerlaan 171, 2020 Wilrijk (Belgium)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000276.
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lipophilicity of lead compounds 1 and 2, we investigated the
synthesis and antiplasmodial activity of reverse a-phenyl-sub-
stituted analogues of the natural products fosmidomycin (1)
and FR900098 (2). Our novel analogues contain the promising
a-phenyl substitution, as well as a reverse orientation of the
hydroxamic acid group.
Both lead compounds, fosmidomycin (1) and FR900098 (2),
were synthesized as mono-sodium salts according to estab-
lished literature procedures.[19] Diethyl benzylphosphonate 5
was chosen as the starting material for the preparation of
target compounds 12 and 14.[20] C-Alkylation of substrate 5
with [2-(1,3-dioxolan-2-yl)ethyl]bromide in the presence of
n-butyllithium provided 1,3-dioxolane 6 in 92% yield.
Acidic hydrolysis of the 1,3-dioxolane moiety of compound
6 furnished the expected aldehyde 7, which was smoothly oxi-
dized to the corresponding carboxylic acid 8 by treatment
with SeO2 and H2O2 (Scheme 1).[21] Conversion of 8 into phos-
phonic acid 20 was achieved by reacting 8 subsequently with
bromotrimethylsilane and water. The synthesis of O-benzyl-
protected hydroxamic acids 9 and 10 was accomplished by
1,1’-carbonyldiimidazole (CDI)-mediated coupling reactions of
compound 8 with O-benzyl-hydroxylamine and N-methyl-O-
benzyl-hydroxylamine, respectively.[22] Cleavage of the phos-
phonic acid ester group of intermediate 9 using bromotrime-
thylsilane led to the corresponding oily and hygroscopic phos-
phonic acid, which was directly converted into dibenzyl-
phosphonate 11.[23] In contrast to the behavior of 9, dealkyla-
tion of diethyl phosphonate 10 provided phosphonic acid 13
as white and crystalline solid. Finally, catalytic hydrogenation
of precursors 11 and 13 afforded target compounds 12 and 14
in good yields and high purity (Scheme 1). Starting from alde-
hyde 15,[24] the chain-shortened derivative 19 was accessible
using the same synthetic concept as described for compound
14 (Scheme 2). Structure elucidation was accomplished by IR,
1H and 13C NMR spectroscopy, as well as by mass spectrometry,
elemental analysis and HPLC analysis.
Scheme 1. Synthesis of reverse analogues 12 and 14. Reagents and condi-
tions: a) n-BuLi, [2-(1,3-dioxolan-2-yl)ethyl]bromide, toluene, À788C, 12h,
92%; b) 2m HCl, acetone, 508C, 3 h, 85%; c) SeO2, H2O2, THF, 658C, 4 h, 92%;
d) 1,1’-carbonyldiimidazole (CDI), BnONHR (R=H, CH3), CH2Cl2, RT, 12h, 80–
95%; e) 1. trimethylsilylbromide, CH2Cl2, RT, 24 h; 2. THF/H2O (100:1), RT, 1 h;
3. N,N’-dicyclohexylcarbodiimide (DCC), BnOH, benzene, 808C, 4 h, 12%
(three steps); f) H2, Pd-C, MeOH, RT, 3 h, 67%; g) 1. trimethylsilylbromide,
CH2Cl2, RT, 24 h; 2. THF/H2O (100:1), RT, 1 h, 74% (two steps); h) H2, Pd-C,
MeOH, RT, 1 h, 92%.
To prove the mode of action and to evaluate the biological
activity, the new analogues (12, 14, 19, 20) and both reference
compounds (1 and 2) were first tested for the inhibition of re-
combinant P. falciparum and E. coli DXRs. For that purpose, re-
combinant E. coli strain harboring a synthetic gene that was
optimized for expression under the control of a T5 promoter
and a lac operator direct the synthesis of the protozoan
enzyme in substantial amounts. The recombinant protein can
be isolated rapidly by affinity chromatography and fulfills the
stability requirements for the biological evaluation of new DXR
inhibitors. Both enzyme assays are based on the photometric
determination of NADPH turnover, which is associated with
the DXR-catalyzed reaction (for results see Table 1). Hydroxamic
acids 12 and 14 were found to be strong and selective inhibi-
tors of PfDXR. Compounds 12 and 14 exhibited stronger inhib-
itory activity against the plasmodial enzyme than the reference
compounds fosmidomycin (1) and FR900098 (2). With an IC50
value of 3.1 nm, the N-methyl-substituted hydroxamic acid 14
represents the most powerful PfDXR inhibitor described so far.
However, compound 14 is less active than FR900098 toward
EcDXR and approximately as active as fosmidomycin. Com-
pared to compounds 1, 2 and 14 the inhibitory potency of
compound 12 against the E. coli enzyme is significantly
weaker.
The Ki values of compounds 1, 2, 12 and 14, determined ex-
perimentally in case of PfDXR and calculated in case of EcDXR,
further support our findings (Table 2). In contrast, carboxylic
acid 20, as well as the chain-shortened derivative 19, did not
show any inhibitory activity against either recombinant DXRs.
Next, compounds 1, 2, 12 and 14 were evaluated for their in
vitro antiplasmodial activity against the multidrug-resistant K1
strain of P. falciparum and for their cytotoxicity on human
MRC-5 cells. Again, the N-methyl-substituted hydroxamic acid
14 demonstrated the highest inhibitory activity (IC50 =
0.59 mm), while the free hydroxamic acid 12 was approximately
as active as fosmidomycin. We assume that the high activity of
compound 14 is most likely due to the N-methyl group, which
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ChemMedChem 2010, 5, 1673 – 1676

Experimental Section
Enzyme assays
Materials: [3,4,5-13C3]1-Deoxy-d-xylulose 5-phosphate was pre-
pared as reported earlier.[26] Recombinant IspC protein from E. coli
was prepared as reported earlier.[27] The amino acid sequence of
the ispC gene from P. falciparum (GenBank: HQ018930), with omis-
sion of amino acid residues 1–73 was back-translated in line with
the codon usage of E. coli (http://www.kazusa.or.jp/codon/). The ar-
tificial open reading frame was cloned into the vector pQE30
(Qiagen; Hilden, Germany). The vector-derived amino acid se-
quence resulted in the extension MRGSHHHHHHGS preceding resi-
due 74 of the N terminus of the synthetic construct (pQEispCpla-
sart). The supernatant of E. coli XL1-pQEispCplasart cells (40 g) was
applied to a column of Ni-chelating Sepharose FF (Amersham Phar-
macia Biotech; Freiburg, Germany; column volume: 20 mL) and
the column was washed with 100 mm Tris hydrochloride (pH 7.5),
containing 0.5m sodium chloride, 20 mm imidazole hydrochloride
and 10% glycerol (v/v), and was then developed with a gradient of
20–500 mm imidazole in 100 mm Tris hydrochloride (pH 7.5), con-
taining 0.5m sodium chloride and 10% glycerol (v/v; total volume:
200 mL). Fractions were combined, concentrated by ultrafiltration
(10 kDa) and desalted with a HIPREP 26/10 column (Amersham) in
50 mm Tris hydrochloride (pH 7.5), containing 10% (v/v) glycerol.
After addition of dithiothreitol to give a final concentration of
5 mm, the protein was stored at À808C. NADPH was purchased
from BioMol (Hamburg, Germany). Fosmidomycin sodium salt was
purchased from Molekula Deutschland Ltd. (Taufkirchen, Germany).
Scheme 2. Synthesis of chain-shortened derivative 19. Reagents and condi-
tions: a) SeO2, H2O2, THF, 658C, 4h, 94%; b) CDI, BnONHCH3, CH2Cl2, RT, 12h,
99%; c) 1. Trimethylsilylbromide, CH2Cl2, RT, 24 h; 2. THF/H2O (100:1), RT, 1 h,
89% (two steps); d) H2, Pd-C, MeOH, RT, 1 h, 92%.
Table 1. In vitro activity[a] and cytotoxicity.[a]
Compd
IC50 [nm]
IC50 [mm]
Pf-K1[b]
PfDXR
EcDXR
MRC-5
1
2
12
14
19
20
143.7Æ15.5
15.3Æ1.2
11.6Æ3.1
3.1Æ0.3
inactive
221.3Æ14.4
131.3Æ2.9
592Æ25
3.71Æ2.47
1.48Æ0.84
3.87Æ1.81
0.59Æ0.2
inactive
>62
>59
>59
>60
>64
>64
243Æ29.6
inactive
inactive
inactive
inactive
IC50 determination using the photometric assay: Assays were
conducted in 96-well plates (Nunc) with a transparent flat bottom.
Assay mixtures (total volume: 200 mL) contained 100 mm Tris hy-
drochloride (pH 7.6), 4 mm MnCl2, 0.5 mm dithiothreitol (DTT),
0.5 mm NADPH, 10–30 nm of recombinant IspC protein (P. falci-
parum) or 100 mm Tris hydrochloride (pH 8.0), 10 mm MgCl2, 1 mm
NADPH, 27 nm of recombinant IspC protein (E. coli). Dilution series
(1:2) of inhibitors covered the concentration range of 200 mm to
1 nm. The reaction was started by addition of 1-deoxyxylulose 5-
phosphate to a final concentration of 1 mm. The reaction was
monitored photometrically (room temperature for P. falciparum or
378C for E. coli) at 340 nm in a microplate reader (P. falciparum:
Thermo Scientific, Multiskan Spectrum; E. coli: Molecular Devices,
SpectraMax M2). Initial rate values were evaluated with a nonlinear
regression method using the program Dynafit.[28]
[a] Values are the mean ÆSD of three or more independent experiments.
[b] Multidrug-resistant strain of P. falciparum
Table 2. Ki values of compounds 1, 2, 12 and 14, determined experimen-
tally for PfDXR and calculated for EcDXR.
Compd[a]
Ki [nm][a]
PfDXR
EcDXR[b]
1
2
12
14
8.4Æ0.6
2.6Æ0.2
2.1Æ0.1
0.42Æ0.03
21.2
12.6
56.7
23.3
[a] Values are the mean ÆSD of three or more independent experiments.
[b] Values calculated using the Cheng–Prusoff equation: Ki =IC50/(1+([S]/
Km)).[31]
Biological evaluation of antiplasmodial activity and
cytotoxicity
might mimic the methyl group present in FR900098. None of
the evaluated compounds showed cytotoxic effects on human
MRC-5 cells (Table 1).
Test plate production: The experiments were performed in 96-
well plates (Greiner) at four-fold dilutions in a dose-titration range
of 64 mgmLÀ1 to 0.25 mgmLÀ1. Dilutions were carried out by a pro-
grammable precision robotic station (Biomek 2000; Beckman,
USA). Each plate also contained medium controls (blanks: 0%
growth), infected untreated controls (negative control: 100%
growth) and reference controls (positive control: chloroquine).
Tests were run in triplicate.
In conclusion, we have developed a highly active and selec-
tive PfDXR inhibitor with a unique chemical structure. To the
best of our knowledge, compound 14 is the most active inhibi-
tor of PfDXR described so far. In contrast to both lead com-
pounds, compound 14 contains a lipophilic aromatic region in
the a-position relative to the phosphonic acid moiety, as well
as an N-methyl-substituted hydroxamic acid functionality. Up
to now, only very few N-substituted hydroxamic acids have
been identified as metalloenzyme inhibitors.[25] Therefore, com-
pound 14 represents a promising new lead compound for the
development of novel antimalarial drugs.
Biological screening tests: The integrated panel of microbial
screens used in this study and the standard screening methodolo-
gies were adopted from those described by Cos et al.[29]
In vitro P. falciparum culture and drug assay: The chloroquine-
resistant K1 strain was used. Parasites were cultured in human er-
ythrocytes A+ at 378C under a low oxygen atmosphere (3% O2,
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suspension of infected human red blood cells (1% parasitaemia,
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spectrophotometrically read at 655 nm (Biorad 3550-UV microplate
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Cytotoxicity test upon MRC-5 cells: MRC-5 SV2 cells, human fetal
lung fibroblast, were cultivated in minimum essential medium
(MEM) supplemented with l-glutamine (20 mm), 16.5 mm sodium
hydrogen carbonate and 5% fetal calf serum (FCS) at 378C and 5%
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We thank Proteros Biostructures GmbH (Martinsried, Germany)
for financial support to Andrea Kunfermann, the Hans-Fischer Ge-
sellschaft (Munich, Germany) for financial support to Wolfgang
Eisenreich and Michael Groll, as well as Krystina Kuna and Felix
Quitterer for experimental support.
Keywords: Antiprotozoal agents · DOXP reductoisomerase ·
Fosmidomycin · MEP pathway · Reverse analogues
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