Novel reverse thia-analogs of fosmidomycin: Synthesis and antiplasmodial activity

Article abstract of DOI:10.1016/j.ejmech.2019.07.058

Thia analogs of fosmidomycin are potent inhibitors of the non-mevalonate isoprenoid biosynthesis enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers displayed antiplasmodial in vitro activity

Full text of DOI:10.1016/j.ejmech.2019.07.058

Accepted Manuscript
Novel reverse thia-analogs of fosmidomycin: Synthesis and antiplasmodial activity
Claudia Lienau, Tobias Gräwert, Leandro A. Alves Avelar, Boris Illarionov, Jana Held,
Tanja C. Knaab, Beate Lungerich, Lasse van Geelen, Dieter Meier, Stefanie Geissler,
Holger Cynis, Ulrich Riederer, Mirko Buchholz, Rainer Kalscheuer, Adelbert Bacher,
Benjamin Mordmüller, Markus Fischer, Thomas Kurz
PII:
S0223-5234(19)30679-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.058
EJMECH 11555
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2019
Revised Date: 19 July 2019
Accepted Date: 20 July 2019
Please cite this article as: C. Lienau, T. Gräwert, L.A. Alves Avelar, B. Illarionov, J. Held, T.C. Knaab, B.
Lungerich, L. van Geelen, D. Meier, S. Geissler, H. Cynis, U. Riederer, M. Buchholz, R. Kalscheuer, A.
Bacher, B. Mordmüller, M. Fischer, T. Kurz, Novel reverse thia-analogs of fosmidomycin: Synthesis and
antiplasmodial activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.07.058.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Novel reverse thia-analogs of fosmidomycin: synthesis and
antiplasmodial activity
Claudia Lienau†, Tobias Gräwert,§,¥ Leandro A. Alves Avelar,† Boris Illarionov,§ Jana
Held,# Tanja C. Knaab, † Beate Lungerich,† Lasse van Geelen,¤ Dieter Meier,¤ Stefanie
‡
‡
Geissler,‡ Holger Cynis
Ulrich Riederer,∂ Mirko Buchholz
Rainer Kalscheuer,¤
Adelbert Bacher,§,& Benjamin Mordmüller,# Markus Fischer,§ Thomas Kurz*,†
†Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany, §Hamburg School of Food Science, University of
Hamburg, Grindelallee 117, 20146 Hamburg, Germany,# Institute of Tropical Medicine, University of
Tübingen, Wilhelmstr. 27, 72074 Tübingen Germany, Centre de Recherches Médicales de Lambaréné
¤
(CERMEL), Centre de Recherches Médicales de Lambaréné, B.P.: 242 Lambaréné, Gabon, Institute of
Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1,
40225 Düsseldorf, Germany, ∂Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146
‡
Hamburg, Germany, Fraunhofer Institute for Cell Therapy and Immunology, Department of Drug Design
and Target Validation, Weinbergweg 22, 06120 Halle (Saale), Germany, &Department of Chemistry,
¥
Technical University of Munich, Lichtenbergstr. 4, D-85748, Garching, Germany. Hamburg Outstation,
European Molecular Biology Laboratory, Notkestraße 85, 22607 Hamburg, Germany
KEYWORDS. fosmidomycin thia isosters, sulfones, IspC, antiplasmodial activity,
enantioselectivity
ABSTRACT. β-Thia analogs of fosmidomycin are potent inhibitors of the non-
mevalonate isoprenoid biosynthesis enzyme 1-deoxy-D-xylulose 5-phosphate
reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers
displayed antiplasmodial in vitro activity in the low nanomolar range, without apparent
cytotoxic effects in HeLa cells. The (S)-(+)-enantiomer of a typical representative
selectively inhibited IspC and the growth of P. falciparum in continuous culture. The
inhibitor was stable at pH 7.6 and room temperature, and no racemization was
observed under these conditions during a period of up to two days. Oxidation of
selected thioethers to sulfones reduced antiplasmodial activity and the inhibitory activity
against Escherichia coli, Mycobacterium tuberculosis and P. falciparum IspC orthologs.
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1.
Introduction
Malaria continues to be a major cause of morbidity and mortality, particularly in Sub-
Saharan Africa. Among human-pathogenic Plasmodium species, Plasmodium
falciparum is the clinically most relevant and responsible for almost all fatal cases.
Almost half of the world’s population is at risk and an estimated 435,000 deaths
occurred in 2017, most of them in early childhood. Infections and reinfections were
estimated at 219 million cases for the year 2017.[1] Malaria control and therapy is an
expensive and complex undertaking. Resistance of the pathogens and its vectors
against many antimalarials and insecticides is now widespread and progressing further.
Even the efficacy of artemisinins, nowadays the most used class of drugs, is
jeopardized by emerging of new resistance mechanisms.[2,3] In the last years, many
reports appeared about development of antimalarial vaccines, one undergoing
implementation trials in selected areas, though with moderate success.[4,5] There is
consensus in the expert community that new antimalarial drugs need to be introduced in
the next decade if malaria is to be contained.[6]
Plasmodia depend on the synthesis of isoprenoids via the non-mevalonate pathway that
was elucidated in the late 1990s.[7–9] The non-mevalonate isoprenoid biosynthesis
pathway is present in protozoa, eubacteria and plants but is absent in humans,
therefore inhibitors of the parasite’s isoprenoid biosynthesis enzymes should be exempt
from target-related toxicity. Fosmidomycin (1) is a hydroxamic acid natural product that
was discovered as an antibacterial agent in the late 1970s.[10] In 1999 it was shown
that 1 inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of P.
falciparum, the enzyme catalyzing the first committed step of the non-mevalonate
pathway.[8] The combination of fosmidomycin and piperaquine was very recently shown
to be effective and well tolerated in a phase II clinical trial in the treatment of
uncomplicated P. falciparum malaria.[11] However, despite these promising preliminary
results, fosmidomycin has hitherto not been implemented as a marketed antimalarial
drug. This is due to its pharmacokinetic and pharmacodynamic profile that only partially
matches the currently preferred target product profile.[12] Several research groups have
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therefore engaged in the development of fosmidomycin derivatives with improved
properties.[13-30]
Fosmidomycin’s mode of action is based on chelation of a divalent metal cation at the
IspC active site, which is crucially involved in the transformation of 1-deoxy-D-xylulose 5-
phosphate, (DOXP, DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). MEP is a key
intermediate in the biosynthesis of the essential isoprenoid precursors isopentenyl
pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (Figure 1).[31,32]
Attempts to replace the metal-chelating hydroxamate group and the phosphonate group
of 1 by (bio)isosteric functionalities have led to limited success against IspC orthologs of
Mycobacterium tuberculosis and Escherichia coli.[33] However, retro-inversion and
methylation of fosmidomycin’s hydroxamate motif afforded derivatives with improved
inhibitory properties (Figure 1).[15] Other important modifications were the introduction
of α-(hetero)aryl, α-fluoro and β-arylalkyl side chains and the isosteric replacement of
methylene groups. [33,34]. Furthermore, several types of phosphonate prodrugs with in
part improved antiplasmodial activity have been reported.[23,28,30]
Notably, an α-phenyl β-thia isoster showed strong enantioselectivity; specifically, the
inhibitory activity of the (S)-enantiomer exceeded that of the (R)-enantiomer by at least
three orders of magnitude.[18] To get additional insights into the SAR of this series of
reverse fosmidomycin analogs we synthesized and tested four novel 3,5-diphenyl-
substituted analogs (5e,f,h,i), two new para-substituted thioethers (5g,j) and for
comparison their cognate sulfones (7).
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Figure 1. IspC inhibition and reverse fosmidomycin analogs.
2.
Results
2.1. Synthesis of novel α-phenyl β-thia isosters
To further explore the influence of substituents at the phenyl ring of 5a, the synthesis of
six novel structural analogs (5e-j) using the previously published strategy was
performed (Scheme 1, Table 1).[18] To assess their inhibitory activity the novel
thioethers 5e-5j were first tested for inhibition of IspC from Escherichia coli,
Mycobacterium tuberculosis and Plasmodium falciparum using a photometric assay.
IspC inhibition data shown in Table 1 include the novel compounds 5e-5j and for
comparison the previously published compounds (5a-d).
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Table 1. IC50 values of thioethers versus IspC orthologs from P. falciparum, E. coli and
M. tuberculosis.
IspC
P. falciparum
IC50 [µM]a
IspC
E. coli
IC50 [µM]a
IspC
M. tuberculosis
IC50 [µM]a
R1
R2
5ab
5bb
5cb
5db
5e
5f
Ph
CH3
CH3
CH3
CH3
CH3
CH3
H
0.024±0.003
0.018±0.004
0.0082±0.0008
0.033±0.009
0.28±0.03
0.11±0.01
0.55±0.01
0.0092±0.0006
0.11±0.01
4.4±0.2
4-CH3-Ph
naphth-1-yl
3,4-Cl-Ph
3,5-F-Ph
0.0098±0.0009 0.14±0.01
0.0045±0.0004 0.0059±0.0006
0.013±0.004
0.020±0.004
0.032±0.006
0.024±0.004
0.011±0.002
0.029±0.008
0.16±0.02
0.018±0.003
0.16±0.03
3,5-OCH3-Ph
4-CH3-Ph
3,5-OCH3-Ph
3,5-F-Ph
5g
5h
5i
0.29±0.02
8.2±0.5
H
0.68±0.13
44±4
H
0.013±0.001
0.022±0.005
0.22±0.01
0.32±0.03
0.10±0.02
0.23±0.02
5j
4-SCH3-Ph
CH3
Fosmidomycin
aPhotometric assay.[18,35] Values are mean ± standard deviation. In general, two to
three independent determinations were performed. bIC50 values for this compound have
been published before.[18]
In line with earlier results, the novel thioethers 5e-5j inhibited IspC from P. falciparum
with IC50 values in the low nanomolar range. With IC50 values ranging from 0.0045 –
0.032 µM for the racemates, the structure activity relationship in the group of ten
structural analogs is relatively flat (Figure 2).
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Figure 2. Activity (IC50) of thioether compounds 5a-j in comparison to their cognate
sulfones 7a-h. Both types of compounds inhibit IspC enzyme from P. falciparum, E. coli
and M. tuberculosis. Red: Fosmidomycin. Light/dark green: Thioether compounds with
R2=CH3. Dark green: Compound 5a in its racemic form (a) and as the active enantiomer
((S)-5a). Grey: Thioether and Sulfone compounds with R2=H. Blue: Sulfone compounds
with R2=CH3. Pink arrow: IC50 greater than 1000 µM.
On the other hand, the IC50 values observed with IspC of E. coli (0.0059 – 0.68 µM)
resp. M. tuberculosis (0.0092 µM – 44 µM) span a considerably wider range, in
comparison with the Plasmodium enzyme (0.0045 – 0.032 µM). Both bacterial enzymes
are less sensitive to these inhibitors than the Plasmodium enzyme. Notably, however,
the strongest inhibitor, the 3,4-dichloro-N-methyl analogue 5d inhibited all three
enzymes with IC50 values in the single-digit nanomolar range (values of 4.5 to 9.2 nM).
2.2. Antiplasmodial in vitro activity of novel α-phenyl β-thia isosters (5e-j).
We next compared the antiplasmodial in vitro activity of earlier compounds and newly
synthesized compounds. Novel analogs 5e, 5f, 5h and 5j displayed nanomolar in vitro
antiplasmodial activity along with absence of cytotoxicity toward HeLa cells, even at
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millimolar concentrations (Table 2). However, the new compounds (5e-j) are somewhat
less active than the previously reported derivatives (5a-d). In line with the enzyme
assays, the N-methyl substituted derivatives demonstrated stronger antiplasmodial
activity toward strains 3D7 and Dd2 than the unsubstituted hydroxamic acids. The most
active novel thioethers (5e, j) showed IC50 values of 0.15 µM (5e) and 0.099 µM (5j)
against the Plasmodium 3D7 strain. However, compared to their inhibitory activity at the
enzyme level toward PfIspC the antiplasmodial in vitro activity of racemic thioethers 5a-j
is weaker (Table 1 vs Table 2). Since no stability problem has been observed (Figure
3), this is most likely due to the fact that the PfIspC inhibitors have to penetrate several
membranes in order to bind to the active site of PfIspC in the apicoplast. Table 2 also
shows that the chloroquine resistance has not much influence on the antiplasmodial
activity of compounds 5.
Table 2. IC50 values of thioethers 5a-j versus Plasmodium strain 3D7, Dd2 and versus
HeLa cells.
Pf3D7
PfDd2
HeLa
R1
R2
SIb
IC50 [µM]a
IC50 [µM]a
IC50 [µM]a
5ac
5bd
5cd
5dd
5e
5f
Ph
CH3
CH3
CH3
CH3
CH3
CH3
H
0.19±0.08
0.11±0.04
0.38±0.07
0.091±0.076
0.15±0.06
0.16±0.01
6.5±2.8
0.26±0.05
0.095±0.041
0.51±0.18
0.085±0.030
0.13±0.07
0.18±0.04
8.0±2.3
> 1000
n.d.
>37000
n.c.
4-CH3-Ph
naphth-1-yl
3,4-Cl-Ph
3,5-F-Ph
3,5-OCH3-Ph
4-CH3-Ph
3,5-OCH3-Ph
3,5-F-Ph
4-SCH3-Ph
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
n.d.
>2600
>11000
>6700
>6400
>150
>3000
>810
>10000
-
5g
5h
5i
H
0.33±0.17
1.2±0.5
0.27±0.13
0.87±0.18
0.18±0.003
0.81±0.16
H
5j
CH3
0.099±0.060
0.88±0.18
Fosmidomycin
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aIn vitro assay. Values are the mean of two independent double determinations.
bSelectivity Index (SI) shown as HeLa IC50/Plasmodium IC50 for strain 3D7. Values
c
larger than 1 indicate greater parasite selectivity. Previously published[18] values for
d
Pf3D7 (0.030 µM) and PfDd2 (0.075 µM) are herewith revised. IC50 values for this
compound have been published earlier.[18] n.d., not determined. n.c., not calculated.
2.3. Compound stability and absence of racemization.
Based on earlier results, it’s promising activity and synthetic accessibility compound 5a
was chosen to be further studied. First, the stability of 5a in phosphate buffer pH 7.6
was monitored by HPLC over a period of 72 h and almost no decomposition was
observed (>95% drug content). No net loss of inhibitory activity of racemic 5a occurred
over a period of two days (Fig. 3). In order to study potential racemization, (S)- resp.
(R)-enantiomer of 5a were incubated in 100 mM Tris hydrochloride, pH 7.6.
Figure 3. Stability of 5a in course incubated in phosphate buffer pH 7.6 at room
temperature (in % of the IC50 value ± SD for racemate or the respective enantiomer at
the beginning of incubation). Red, racemate; blue, (R)-5a, green, (S)-5a.
Since any racemization of the inactive (R)-enantiomer should generate the active (S)-
enantiomer, the inhibitory potency of the (R)-5a sample should increase with time. Any
significant formation of the inactive (R)-enantiomer from the active (S)-enantiomer
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should have resulted in a decrease of the apparent IC50 value of the (S)-5a sample (i.e.
a decrease of enzyme inhibition by generation of the inactive enantiomer). However,
apparent IC50 values for P. falciparum IspC determined at intervals with (S)- respectively
(R)-enantiomer of 5a in solution did not change significantly, neither over a period of two
°
days at room temperature (Fig. 3) nor over a period of seven days at +5 C (data not
shown).
2.4. Enantioselectivity in the P. falciparum replication assay.
Earlier, we had shown that the IspC orthologs from P. falciparum, E. coli, and M.
tuberculosis showed high enantiospecificity for the (S)-(+)-enantiomer of 5a. [18] Now
we studied their enantioselectivity in the P. falciparum replication assay. The
enantiomers of 5a showed a high degree of enantioselectivity as determined by
replication of P. falciparum in human erythrocytes (Figure 4, Table 3).
Table 3. Antiplasmodial in vitro activity of 5a racemate and enantiomers
Pf3D7
PfDd2
IC50 [µM]a
0.19 ± 0.08
IC50 [µM]a
5a racemateb
(S)-(+)-5a
0.26 ± 0.05
± 0.14 ± 0.05
0.087
0.033
(R)-(-)-5a
>130
>130
Fosmidomycin
0.88±0.18
0.81±0.16
b
aValues are the mean of two independent double determinations.
Previously
published [19] values for Pf3D7 (0.030 µM) and PfDd2 (0.075 µM) are herewith revised.
9

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Figure 4 compares the enantioselectivity observed in the enzyme assay and the
parasite replication assay. In the enzyme assay as well as the parasite assay, the (S)-
enantiomer has approximately double potency by comparison with the racemate. In
both assay systems, the activity of the (S)-enantiomer is lower than that of the racemate
by at least three orders of magnitude. Notably, the dose-response curves in the parasite
assay show a much steeper slope as compared to the enzyme assay.
Figure 4. Dose response curves. A, photometric assay of P. falciparum IspC; B, P.
falciparum asexual blood stages assay. green, (S)-(+)-5a; blue, (R)-(-)-5a; orange,
racemic 5a.
2.5. Conversion of thioethers to sulfones.
Based on the X-ray structure of PfIspC in complex with (S)-(+)-5a (PDB ID 4KP7) [18],
we performed a modeling study. [36] in order to analyze whether the active site of the
Plasmodium enzyme could accommodate derivatives of 5a with a higher oxidation level
of the sulfur (Figure 1A, SI). The sulfone analog 7a of thioether 5a could be
accommodated inside the active site without steric clashes.
Several sulfone derivatives (7a-h) were obtained in good yields (Scheme 1).
Specifically, diethyl phosphonates 4a-h were accessible via S-alkylation of the
respective α-mercaptophosphonates 3a-f, j as published before. [18] Novel diethyl
phosphonates 4e-j were deprotected by treatment with TMSBr and yielded thioethers
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5e, f, j as solids and 5g-i as oils.[37] Oxidation of diethyl phosphonates 4a-h with 3-
chlorobenzoperoxoic acid provided the partially protected sulfones 6a-h. [38] The
diethyl phosphonate moieties of intermediates 6a-h were deprotected with TMSBr and
afforded sulfones 7a-g as oils and 7h as white solid.
Scheme 1. Synthesis of thioethers 5e-j and sulfones 7a-h.
Reagents and conditions. I: n-BuLi, S, THF, -78 °C, 1 h; 2. -20 °C, THF, 1 h; 3. RT,
THF, 1 h; II: 4a-f, j: 2-chloro-N-hydroxy-N-methylacetamide, DMF, Na2CO3, 0 °C → RT;
4g-i: 2-chloro-N-hydroxyacetamide, DMF, Na2CO3,
0
°C
→
RT; III: 3-
chlorobenzoperoxoic acid, CH2Cl2, 0 °C, 1h; CH2Cl2, RT, 12 h; IV: 1. TMSBr, CH2Cl2, 0
°C, 1 h; 2. CH2Cl2, RT, 47 h; 3. THF/H2O, RT, 45 min. The synthesis of 2a-d and 5a-d
has been described previously.[18]
Inhibitory activities of the sulfone derivatives (7a-h) for IspC from P. falciparum, E. coli
and M. tuberculosis were determined using the photometric assay (Figure 4, Table 3).
[20] Data were collected at a substrate (DOXP) concentration of 500 µM.
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Table 3. IC50 values of sulfones 7a-h versus IspC orthologs from P. falciparum, E. coli
and M. tuberculosis.
IspC
IspC
IspC
R1
R2
P. falciparum
E. coli
M. tuberculosis
IC50 [µM]a
IC50 [µM]a
IC50 [µM]a
7a
7b
7c
7d
7e
7f
Ph
CH3
CH3
CH3
CH3
CH3
7.6±0.7
8.5±0.9
823±164
1.0±0.2
5.5±0.3
3.7±0.6
65±9
36±2
115±6
143±23
621±83
21±1
4-CH3-Ph
naphth-1-yl
3,4-Cl-Ph
3,5-F-Ph
36±3
15±0
11±1
4.3±0.3
80±5
44±4
3,5-OCH3-Ph CH3
>1000
>1000
194±12
0.23±0.02
7g
7h
4-CH3-Ph
H
H
64±7
3,5-OCH3-Ph
0.22±0.03
0.16±0.02
4.5±0.3
0.22±0.01
Fosmidomycin
aEnzyme assay. Values were calculated from at least eight data points. In general, two
to three independent determinations have been performed.
Without exception, the sulfone derivatives 7a-h are weaker IspC inhibitors as compared
to their cognate thioether analogs (Figures 2, 5). Typically, the oxidation of the thioether
motif to the sulfone level decreased the inhibitory activity by 2 to 3 orders of magnitude.
A notable exception occurred with the 3,5-dimethoxy compounds. The loss of inhibitory
activity of 7h in comparison with 5h is less than 1 order of magnitude for all three
enzyme orthologs.
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Figure 5. Inhibition of IspC orthologs (P. falciparum, blue; E. coli, green; M. tuberculosis,
orange) with sulfone 7h.
In contrast to their highly active cognate thioether analogs 5a-j, the sulfones 7a-d, f, g
showed little or no antiplasmodial in vitro activity toward Pf3D7 up to a concentration of
100 µM. Only sulfone 7h displayed at least some antiplasmodial activity with an IC50 2.7
µM.
Table 4. IC50 values of sulfones 7a-h versus Plasmodium strain 3D7 and Dd2.
Pf3D7
HeLa
R1
R2
IC50 [µM]a
IC50 [µM]a
7a
7b
7c
7d
7e
7f
Ph
CH3
CH3
CH3
CH3
CH3
CH3
H
>100
n.d.
4-CH3-Ph
naphth-1-yl
3,4-Cl-Ph
3,5-F-Ph
3,5-OCH3-Ph
4-CH3-Ph
3,5-OCH3-Ph
>100
> 50
>100
> 1000
> 1000
> 1000
> 1000
n.d.
>100
19± 3
>100
7g
7h
>100
H
2.7±0.4
0.88±0.18
> 500
n.d.
Fosmidomycin
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aIn vitro assay. Values are the mean of two independent double determinations. n.d.,
not determined.
2.6. Caco2 cell permeability assay.
To investigate the assumption of an insufficient transport through membranes in the P.
falciparum in vitro growth inhibition assays of 5a and 5j a Caco2-cell permeability assay
was performed. This assay is used in preclinical drug development for the assessment
of a compound transport through epithelial cell barriers, especially in the GI-tract. After
administration of the compounds to the polarized cell monolayer, the concentration of
the compound is measured time dependently on both sides, the donor and acceptor
medium, as well as in the cells themselves by using LC-MS/MS.[39] As a result for both
compounds very low Papp-values were calculated with even smaller values than for
atenolol, a compound used as a negative control in this assay.
Table 6. Caco2 cell assay of 5a and 5j
Papp [cm/s]a
Atenololb
0.45 · 10 -6± 0.02 · 10 -6
0.14 · 10 -6 ± 0.01 · 10 -6
0.15 · 10 -6 ± 0.01 · 10 -6
5a
5j
a Apparent permeability coefficient. bAtenolol is used as a
reference for substances with a low permeability
2.7. In vitro antibacterial activity.
Based on their inhibition of EcIspC and MtIspC (Table 1 and 2), thioethers and sulfones
were evaluated for antibacterial activity (Table 2, SI). With the exception of 5d, no
antibacterial activity was observed against M. tuberculosis and against a panel of Gram-
negative bacterial strains using concentrations up to 100 µM. Compound 5d showed
very weak activity against a multidrug-resistant strain of Acinetobacter baumannii (MIC
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= 100 µM; Table 2, SI). Thus, the high in vitro activity of thioethers 5 against purified
EcIspC and MtIspC did not translate into antibacterial whole cell activity, which suggests
that poor cell penetration rate could be the limiting factor.
3.
Discussion
The current antimalarial drug development pipeline urgently requires novel
antiplasmodial compounds.[40] The non-mevalonate isoprenoid biosynthesis is of
particular interest, because its enzymes are absent in humans. The interaction of the
first committed enzyme of the pathway, 1-deoxy- -xylulose 5-phosphate
D
reductoisomerases (IspC, Dxr), with the antibiotic fosmidomycin and its derivatives has
been addressed in numerous publications.[33] The introduction of aryl substituents into
the fosmidomycin skeleton has afforded compounds that showed significantly enhanced
inhibition of PfIspC, while also generating a chiral center.[34] X-ray structure analysis
had suggested preferential binding of the (S)-enantiomers of α-substituted reverse
fosmidomycin derivatives. Chromatographic enantiomer resolution of the α-phenyl β-
thia analog 5a has conclusively shown that the enantiomer selectivity of IspC from P.
falciparum exceeds three orders of magnitude.
In an earlier study, we have shown explicitly that the IspC orthologs from M.
tuberculosis and E. coli are also selectively inhibited by the (S)-(+)-enantiomer of 5a,
albeit at lower levels of enantioselectivity.[18] More importantly, we have now shown
that enantioselectivity also extends to the antiplasmodial activity against growth of P.
falciparum strains 3D7 and Dd2 (Table 3). The activity of the (S)-enantiomer exceeds
that of the (R)-enantiomer by at least 3 orders of magnitude, thus supporting the view
that IspC is the relevant target of fosmidomycin. The use of pure enantiomers would
then effectively double the efficacy of the compounds, while simultaneously avoiding the
risk of any potential toxicity effects of the inactive enantiomer.
Since no steric clashes were observed in the in silico studies with the sulfone
derivatives, the different electronic properties of the sulfone moiety compared to the
thioether group are one possible explanation for the decrease in the enzymatic and
cellular activities. Whereas the most potent type 5 compounds outperformed
15

ACCEPTED MANUSCRIPT
fosmidomycin by almost two orders of magnitude at the level of the molecular target,
only an advantage of about one order of magnitude remains at the level of the P.
falciparum growth inhibition assay. Inside the Plasmodium-infected erythrocyte, the
molecular target of fosmidomycin is shielded by not less than 7 membrane barriers (the
outer membrane of the erythrocyte, the membrane of the vacuole that contains the
parasite, the outer Plasmodium membrane and 4 membranes of the apicoplast). In light
of that extremely complex topology, it is not surprising that the IC50 value for each of the
study compounds is larger than the IC50 for the isolated enzyme.
4.
Conclusions
Reverse thioether analogs of fosmidomycin are highly active inhibitors of 1-deoxy-
D-
xylulose 5-phosphate reductoisomerases (IspC, Dxr) of Plasmodium falciparum,
Escherichia coli, and Mycobacterium tuberculosis. Several IspC inhibitors exert
antiplasmodial in vitro activity in the low nanomolar range without demonstrating
cytotoxic effects in HeLa cells even at millimolar concentrations. The (S)-(+)-enantiomer
of thioether 5a selectively inhibited IspC and the growth of P. falciparum in continuous
culture. None of the thioethers exhibited significant antibacterial in vitro activity against
M. tuberculosis and a spectrum of Gram-negative bacteria up to a concentration of 100
µM. Oxidation of selected thioethers to sulfones reduced the antiplasmodial activity and
the inhibitory activity toward E. coli, M. tuberculosis and P. falciparum IspC orthologs.
Currently our work focuses on phosphonamidate prodrugs as a promising new class of
phosphonate prodrugs and on reverse bissubstrate inhibitors, which target the substrate
and NADPH binding site of IspC.
5.
Experimental section
5.1. Chemistry
All solvents and chemicals were used as purchased without further purification. The
progress of all reactions was monitored on Merck precoated silica gel plates (with
fluorescence indicator UV254). Column chromatography was performed with Fluka silica
gel 60 (230-400 mesh ASTM) with the solvent mixtures specified in the corresponding
experiment. Spots were visualized by irradiation with ultraviolet light (254 nm). Melting
16

ACCEPTED MANUSCRIPT
points (m.p.) were taken in open capillaries on a Stuart melting point apparatus SMP11
and are uncorrected. Proton (1H) and carbon (13C) NMR spectra were recorded on a
1
Bruker Avance 500 (500.13 MHz for H; 125.76 MHz for 13C) using DMSO-d6 as
solvent. Chemical shifts are given in parts per million (ppm), (δ relative to residual
1
13
solvent peak for H and C). Elemental analysis was performed on a Perkin Elmer PE
2400 CHN elemental analyzer and a vario MICRO cube elemental analyzer, Elementar
Analysensysteme GmbH, Hanau Germany. Optical rotation was determined by a Krüss
P8000 polarimeter; specific rotations [α]ꢁꢀꢂ values are given in deg dm-1 cm3 g-1. High-
resolution mass spectrometry (HRMS) analysis was performed using a UHR-TOF
maXis 4G instrument (Bruker Daltonics, Bremen, Germany). If necessary, purity of
compounds was determined by high performance liquid chromatography (HPLC). The
purity of all novel final compounds (5e-j, 7a-h) was 95% or higher. Instrument: Elite
LaChrom system [Hitachi L-2130 (pump) and L-2400 (UV-detector)]; column:
Phenomenex Luna C-18(2) 5 µm particle size (250 mm × 4.6 mm), supported by
Phenomenex Security Guard Cartridge Kit C18 (4.0 mm × 3.0 mm).
5.1.1. Experimental data
5.1.1.1.
General procedure for the synthesis of phosphonic acids 5e-j and 7a-h:
The synthesis of 5a-d has been reported previously by Kunfermann et al.[18] The
appropriate phosphonic acid diethyl ester 4a-j, 6a-h (1eq., 1 mmol) was dissolved in dry
dichloromethane (10 mL) under an atmosphere of nitrogen. The solution was cooled to
0 °C and trimethylsilyl bromide (5 eq., 0.66 mL, 5 mmol) was added dropwise via a
syringe. After stirring for 1 h, the reaction mixture was allowed to warm up to RT and
was stirred for further 47 h. The solvent was removed under reduced pressure and
tetrahydrofurane (10 mL) was added to the residue. Next, the solution was treated with
water (3 eq., 0.05 mL, 3 mmol) and the reaction mixture was stirred for 45 min at RT.
Afterwards the solvent was evaporated and the residue was dried in vacuo overnight.
Addition of ethyl acetate afforded phosphonic acids 5e, f, j and 7h as solid compounds.
Target compounds 5g-i and 7a-g were obtained as oils after purification of the crude
products by column chromatography with a small amount of silica gel using ethyl
acetate/n-hexane (0:100 → 100:0).
17

ACCEPTED MANUSCRIPT
5.1.1.2.
((3,5-Difluorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)
1
phosphonic acid (5e): white solid; yield: 60%. M.p. 135 °C; H NMR (500.13 MHz, DMSO-
d6):
3
δ
=10.01 (br. s, OH), 7.17-7.12 (m, 1H, Harom.), 7.09 (d, J(H,H)=8.3 Hz, 2H, Harom.), 4.33 (d,
2
2
2J(H,P)=19.8 Hz, 0.87H, PCH), 4.25 (d, J(H,P)=19.8 Hz, 0.13H, PCH), 3.48 (d, J(H,H)=14.6
2
Hz, 1H, SCH2), 3.28 (d, J(H,H)=14.7 Hz, 1H, SCH2), 3.09 (s, 0.31H, NCH3), 3.05 (s, 2.53H,
NCH3), 3.01 ppm (s, 0.16H, NCH3); 13C NMR (125.76 MHz, DMSO-d6):
(dd, 1J(C,F)=246.8 Hz, 3J(C,F)=13.0 Hz, Carom.), 142.5-142.3 (m, Carom.), 112.3 (dd, 2J(C,F)=25.7
δ=168.5 (C=O), 161.8
4
1
Hz, J(C,F)=3J(C,P)=5.7 Hz, Carom.), 102.7-102.2 (m, Carom.), 45.4 (d, J(C,P)=137.5 Hz, PC),
3
35.7 (NCH3), 32.0 ppm (d, J(C,P)=7.8 Hz, SCH2); HPLC analysis: retention time=1.887 min,
peak area: 99.15%, eluent A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20
min with a flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.3.
((3,5-Dimethoxyphenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)
1
phosphonic acid (5f): yellow solid; yield: 18%. M.p. 108 °C; H NMR (500.13 MHz, DMSO-d6):
2
δ
=10.02 (br. s, OH), 6.61-6.55 (m, 2H, Harom.), 6.42-6.36 (m, 1H, Harom.), 4.16 (d, J(H,P)=19.1
2
2
Hz, 1H, PCH), 3.72 (s, 6H, OCH3), 3.50 (d, J(H,H)=14.6 Hz, 1H, SCH2), 3.27 (d, J(H,H)=14.6
Hz, 1H, SCH2), 3.07 ppm (s, 3H, NCH3); 13C NMR (125.76 MHz, DMSO-d6):
δ=168.6 (C=O),
3
159.9-159.8 (m, Carom.), 139.6 (d, 5J(C,P)=1.8 Hz, Carom.), 107.5 (d, J(C,P)=6.0 Hz, Carom.), 98.5
(Carom.), 55.1-54.9 (m, OCH3), 45.9 (d, 1J(C,P)=139.0 Hz, PC), 35.72 (NCH3), 32.1-31.9 ppm (m,
SCH2); HPLC analysis: retention time=1.887 min, peak area: 99.57%, eluent A: NH4OAc
solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and
detection at 254 nm.
5.1.1.4.
(((2-(Hydroxyamino)-2-oxoethyl)thio)(p-tolyl)methyl)phosphonic acid (5g): orange
oil; yield: 90%. 1H NMR (600.22 MHz, DMSO-d6):
δ
=10.54 (br. s, OH), 10.07 (br. s, OH), 10.03
3
4
(br. s, NH), 9.88 (br. s, NH), 7.27 (dd, J(H,H)=8.2 Hz, J(H,H)=1.5 Hz, 1H, Harom.), 7.25 (d,
3
3
3J(H,H)=7.9 Hz, 1H, Harom.), 7.11 (d, J(H,H)=7.7 Hz, 1H, Harom.), 7.11 (d, J(H,H)=7.8 Hz, 1H,
Harom.), 4.28 (d, 2J(H,P)=18.8 Hz, 0.29H, PCH), 4.23 (d, 2J(H,P)=18.3 Hz, 0.15H, PCH), 4.14 (d,
2
2
2J(H,P)=18.9 Hz, 0.27H, PCH), 4.11 (d, J(H,P)=19.0 Hz, 0.12H, PCH), 4.10 (d, J(H,P)=19.2
2
4
Hz, 0.17H, PCH), 3.39 (dd, J(H,H)=14.9 Hz, J(H,P)=1.4 Hz, 0.19H, SCH2), 3.37 (dd,
4
2J(H,H)=13.4 Hz, J(H,P)=1.4 Hz, 0.13H, SCH2), 3.33-3.32 (m, 0.04H, SCH2), 3.28 (dd,
4
2
4
2J(H,H)=14.9 Hz, J(H,P)=1.6 Hz, 0.31H, SCH2), 3.19 (dd, J(H,H)=15.3 Hz, J(H,P)=3.4 Hz,
0.09H, SCH2), 3.10 (d, 2J(H,H)=15.0 Hz, 0.20H, SCH2), 3.08 (d, 2J(H,H)=13.2 Hz, 0.10H, SCH2),
2
4
2
3.06 (dd, J(H,H)=13.7 Hz, J(H,P)=1.5 Hz, 0.27H, SCH2), 2.99 (d, J(H,H)=14.9 Hz, 0.32H,
18

ACCEPTED MANUSCRIPT
2
2
SCH2), 2.87 (d, J(H,H)=14.4 Hz, 0.02H, SCH2), 2.80 (d, J(H,H)=13.9 Hz, 0.33H, SCH2), 2.28
ppm (s, 3H, CH3arom.); 13C NMR (150.93 MHz, DMSO-d6):
δ=170.9 (C=O), 169.5 (C=O), 169.5
5
(C=O), 165.9 (C=O), 136.2-136.1 (m, Carom.), 136.0 (d, J(C,P)=1.5 Hz, Carom.), 134.0 (d,
2
2
2J(C,P)=4.3 Hz, Carom.), 133.7 (d, J(C,P)=4.5 Hz, Carom.), 133.5 (d, J(C,P)=4.8 Hz, Carom.),
1
129.2-129.0 (m, Carom.), 128.7-128.4 (m, Carom.), 45.9 (d, J(C,P)=139.5 Hz, PC), 45.9 (d,
1
1
1J(C,P)=139.8 Hz, PC), 45.9 (d, J(C,P)=139.7 Hz, PC), 45.7 (d, J(C,P)=139.7 Hz, PC), 34.6
(d, 3J(C,P)=5.6 Hz, SCH2), 33.0 (d, 3J(C,P)=6.7 Hz, SCH2), 32.8 (d, 3J(C,P)=7.2 Hz, SCH2), 31.4
3
(d, J(C,P)=6.4 Hz, SCH2), 20.7-20.5 ppm (m, CH3arom.); HPLC analysis: retention time=1.833
min, peak area: 99.83%, eluent A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1)
over 20 min with a flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.5.
((3,5-Dimethoxyphenyl)((2-(hydroxyamino)-2-oxoethyl)thio)methyl)phosphonic
1
acid (5h): light brown oil; yield: 16%. H NMR (500.13 MHz, DMSO-d6):
δ
=10.75-10.51 (br. s,
OH), 10.04 (br. s, OH), 8.94 (br. s, NH), 6.97-6.69 (m, 0.29H, Harom.), 6.67-6.48 (m, 1.71H,
Harom.), 6.47-6.33 (m, 0.85H, Harom.), 6.33-6.19 (m, 0.15H, Harom.), 4.13-3.95 (m, 1H, PCH), 3.82-
3.78 (m, 0.4H, OCH3), 3.77-3.74 (m, 0.92H, OCH3), 3.73-3-69 (m, 3.91H, OCH3), 3.69-3.66 (m,
2
0.36H, OCH3), 3.66-3.64 (m, 0.41H, OCH3), 3.37 (d, J(H,H)=15.0 Hz, 0.48H, SCH2), 3.29 (d,
2J(H,H)=14.7 Hz, 0.24H, SCH2), 3.24 (d, 2J(H,H)=15.6 Hz, 0.22H, SCH2), 3.15 (d, 2J(H,H)=15.0
2
2
Hz, 0.35H, SCH2), 3.07 (d, J(H,H)=13.1 Hz, 0.43H, SCH2), 2.86 ppm (d, J(H,H)=13.8 Hz,
0.28H, SCH2); 13C NMR (125.76 MHz, DMSO-d6):
=169.5 (C=O), 160.0-159.8 (m, Carom.),
139.4-138.8 (m, Carom.), 107.9-107.7 (m, Carom.), 107.7-107.4 (m, Carom.), 98.6-98.5 (m, Carom.),
δ
1
1
55.1-54.9 (m, OCH3), 46.4 (d, J(C,P)=138.6 Hz, PC), 46.4 (d, J(C,P)=138.6 Hz, PC), 33.0 (d,
3
3J(C,P)=7.5 Hz, SCH2), 31.5 ppm (d, J(C,P)=7.1 Hz, SCH2); HPLC analysis: retention
time=1.867 min, peak area: 98.94%, eluent A: NH4OAc solution (5 mM), eluent B: CH3CN,
isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.6.
((3,5-Difluorophenyl)((2-(hydroxyamino)-2-oxoethyl)thio)methyl)phosphonic acid
1
(5i): brown oil; yield: 82%. H NMR (600.22 MHz, DMSO-d6):
δ=11.47 (br. s, OH), 10.57 (br. s,
OH), 10.09 (br. s, OH), 8.94 (br. s, NH), 7.21-7.05 (m, 2.5H, Harom.), 7.05-6.92 (m, 0.5H, Harom.),
2
2
4.49 (d, J(H,P)=20.2 Hz, 0.16H, PCH), 4.37 (d, J(H,P)=19.8 Hz, 0.53H, PCH), 4.34-4.28 (m,
2
2
0.23H, PCH), 4.25 (d, J(H,P)=19.6 Hz, 0.06H, PCH), 4.25 (d, J(H,P)=19.6 Hz, 0.02H, PCH),
3.99-3.83 (m, 0.34H, SCH2), 3.73-3.65 (m, 0.08H, SCH2), 3.59-3.50 (m, 0.09H, SCH2), 3.47-
2
2
3.34 (m, 0.13H, SCH2), 3.29 (d, J(H,H)=15.3 Hz, 0.17H, SCH2), 3.25 (d, J(H,H)=13.4 Hz,
0.09H, SCH2), 3.22 (d, 2J(H,H)=15.7 Hz, 0.02H, SCH2), 3.19 (d, 2J(H,H)=14.7 Hz, 0.04H, SCH2),
19

ACCEPTED MANUSCRIPT
2
2
3.12 (d, J(H,H)=15.1 Hz, 0.13H, SCH2), 3.04 (d, J(H,H)=13.4 Hz, 0.16H, SCH2), 3.03 (d,
2
2J(H,H)=13.9 Hz, 0.37H, SCH2), 2.89 (d, J(H,H)=13.9 Hz, 0.11H, SCH2), 2.85 ppm (d,
13
2J(H,H)=13.9 Hz, 0.27H, SCH2); C NMR (150.93 MHz, DMSO-d6):
δ=165.3 (C=O), 161.8 (dd,
3
1J(C,F)=245.4 Hz, J(C,F)=12.3 Hz, Carom.), 142.3-141.7 (m, Carom.), 134.4-134.1 (m, Carom.),
2
1
112.3 (d, J(C,F)=20.3 Hz, Carom.), 102.9-102.1 (m Carom.), 45.9 (d, J(C,P)=137.3 Hz, PC), 31.5
3
ppm (d, J(C,P)=7.6 Hz, SCH2); HPLC analysis: retention time=1.883 min, peak area: 97.15%,
eluent A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate
of 1 mL min-1 and detection at 254 nm.
5.1.1.7.
(((2-(Hydroxy(methyl)amino)-2-oxoethyl)thio)(4-(methylthio)phenyl)methyl)phosp
=10.00
(br. s, OH), 7.33 (dd, J(H,H)=8.3 Hz, J(H,H)=1.6 Hz, 2H, Harom.), 7.20 (d, J(H,H)=8.3 Hz, 2H,
honic acid (5j): white solid; yield: 80%. M.p. 134 °C; 1H NMR (600.22 MHz, DMSO-d6):
δ
3
4
3
2
2
H
arom.), 4.23 (d, J(H,P)=19.2 Hz, 1H, PCH), 3.48 (d, J(H,H)=14.6 Hz, 1H, SCH2), 3.21 (d,
2J(H,H)=14.6 Hz, 1H, SCH2), 3.06 (s, 3H, NCH3), 2.46 ppm (s, 3H, SCH3); 13C NMR (150.93
2
MHz, DMSO-d6):
δ
=168.9 (C=O), 136.5 (Carom.), 133.9 (d, J(C,P)=4.1 Hz, Carom.), 129.8 (d,
1
3J(C,P)=5.8 Hz, Carom.), 125.5 (Carom.), 45.3 (d, J(C,P)=139.5 Hz, PC), 35.7 (NCH3), 31.8 (d,
3J(C,P)=6.5 Hz, SCH2), 14.6 ppm (SCH3); HPLC analysis: retention time=2.011 min, peak area:
98.90%, eluent A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 30 min with a
flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.8.
(((2-(Hydroxy(methyl)amino)-2-oxoethyl)sulfonyl)(phenyl)methyl)phosphonic acid
1
(7a): colorless oil; yield: 26%. H NMR (500.13 MHz, DMSO-d6):
δ
=10.37 (br. s, OH), 7.55-7.45
2
(m, 2H, Harom.), 7.43-7.30 (m, 3H, Harom.), 5.50 (d, J(H,P)=19.1 Hz, 0.9H, PCH), 5.42 (d,
2
2
2J(H,P)=19.1 Hz, 0.1H, PCH), 4.95 (d, J(H,H)=15.2 Hz, 0.92H, SCH2), 4.72 (d, J(H,H)=15.0
2
2
Hz, 0.08H, SCH2), 4.55 (d, J(H,H)=15.2 Hz, 0.08H, SCH2), 4.34 (d, J(H,H)=15.2 Hz, 0.92H,
SCH2), 3.19 (s, 0.15H, NCH3), 3.15 (s, 2.69H, NCH3), 3.11 ppm (s, 0.16H, NCH3); 13C NMR
(125.76 MHz, DMSO-d6): δ
=162.2 (C=O), 131.2 (d, 2J(C,P)=5.2 Hz, Carom.), 128.8 (d, 3J(C,P)=5.2
Hz, Carom.), 128.2 (Carom.), 128.2 (Carom.), 67.2 (d, 1J(C,P)=129.4 Hz, PC), 54.0 (SCH2), 35.6 ppm
(NCH3); HPLC analysis: retention time=1.790 min, peak area: 99.16%, eluent A: NH4OAc
solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and
detection at 254 nm.
5.1.1.9.
(((2-(Hydroxy(methyl)amino)-2-oxoethyl)sulfonyl)(p-tolyl)methyl)phosphonic acid
1
(7b): brown oil; yield: 61%. H NMR (600.22 MHz, DMSO-d6):
δ=10.36 (br. s, OH), 7.37 (d,
20

ACCEPTED MANUSCRIPT
3
2
3J(H,H)=7.3 Hz, 2H, Harom.), 7.18 (d, J(H,H)=7.9 Hz, 2H, Harom.), 5.4 (d, J(H,P)=19.1 Hz, 0.8H,
2
2
PCH), 5.35 (d, J(H,P)=18.9 Hz, 0.06H, PCH), 5.13 (d, J(H,P)=19.0 Hz, 0.06H, PCH), 5.12 (d,
2
2
2J(H,P)=19.0 Hz, 0.08H, PCH), 4.92 (d, J(H,H)=15.1 Hz, 0.78H, SCH2), 4.77 (d, J(H,H)=14.5
2
2
Hz, 0.04H, SCH2), 4.76 (d, J(H,H)=14.5 Hz, 0.04H, SCH2), 4.70 (d, J(H,H)=14.6 Hz, 0.14H,
2
2
SCH2), 4.52 (d, J(H,H)=15.1 Hz, 0.06H, SCH2), 4.48 (d, J(H,H)=14.4 Hz, 0.04H, SCH2), 4.44
(d, 2J(H,H)=14.5 Hz, 0.04H, SCH2), 4.31 (d, 2J(H,H)=15.1 Hz, 0.77H, SCH2), 4.24 (d,
2J(H,H)=14.6 Hz, 0.09H, SCH2), 3.14 (s, 3H, NCH3), 2.31 ppm (s, 3H, CH3arom.); 13C NMR
(150.93 MHz, DMSO-d6):
δ
=162.3 (C=O), 137.7 (Carom.), 131.1 (d, 2J(C,P)=5.4 Hz, Carom.), 128.8
3
1
(Carom.), 125.7 (d, J(C,P)=5.3 Hz, Carom.), 66.9 (d, J(C,P)=130.1 Hz, PC), 53.9 (SCH2), 35.5
(NCH3), 20.7 ppm (CH3arom.); HPLC analysis: retention time=1.870 min, peak area: 99.91%,
eluent A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate
of 1 mL min-1 and detection at 254 nm.
5.1.1.10.
(((2-(Hydroxy(methyl)amino)-2-oxoethyl)sulfonyl)(naphthalen-1-yl)methyl)
phosphonic acid (7c): beige-colored oil; yield: 34%. 1H NMR (600.22 MHz, DMSO-d6):
δ=10.46
3
(br. s, OH), 8.23 (d, J(H,H)=8.6 Hz, 0.8H, Harom.), 8.19-8.14 (m, 0.2H, Harom.), 8.08 (d,
3J(H,H)=7.2 Hz, 1H, Harom.), 8.01-7.94 (m, 2H, Harom.), 7.65-7.59 (m, 1H, Harom.), 7.58-7.52 (m,
2H, Harom.), 6.65 (d, 2J(H,P)=20.3 Hz, 0.79H, PCH), 6.55 (d, 2J(H,P)=21.0 Hz, 0.05H, PCH), 6.27
(d, 2J(H,P)=20.1 Hz, 0.07H, PCH), 6.18 (d, 2J(H,P)=20.1 Hz, 0.09H, PCH), 5.29 (d,
2
2
2J(H,H)=15.3 Hz, 0.78H, SCH2), 5.23 (d, J(H,H)=14.8 Hz, 0.1H, SCH2), 5.17 (d, J(H,H)=14.9
2
2
Hz, 0.12H, SCH2), 4.84 (d, J(H,H)=15.0 Hz, 0.04H, SCH2), 4.73 (d, J(H,H)=15.0 Hz, 0.07H,
2
2
SCH2), 4.60 (d, J(H,H)=14.8 Hz, 0.09H, SCH2), 4.51 (d, J(H,H)=15.4 Hz, 0.71H, SCH2), 4.38
2
(d, J(H,H)=14.8 Hz, 0.09H, SCH2), 3.34-3.29 (m, 0.16H, NCH3), 3.19 ppm (s, 2.84H, NCH3);
13C NMR (150.93 MHz, DMSO-d6):
δ=162.8 (C=O), 133.5 (Carom.), 132.7 (d, J(C,P)=6.2 Hz,
Carom.), 129.0 (d, J(C,P)=4.3 Hz, Carom.), 128.8 (Carom.), 128.6 (Carom.), 126.6 (Carom.), 125.6
(Carom.), 125.0-124.9 (m, Carom.), 124.3 (d, J(C,P)=5.0 Hz, Carom.), 123.4 (Carom.), 60.8 (d,
1J(C,P)=131.2 Hz, PC), 54.5 (SCH2), 35.7 (NCH3), 34.6 ppm (NCH3); HPLC analysis: retention
time=1.877 min, peak area: 99.95%, eluent A: NH4OAc solution (5 mM), eluent B: CH3CN,
isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.11.
((3,4-Dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)sulfonyl)methyl)
1
phosphonic acid (7d): brown oil; yield: 17%. H NMR (600.22 MHz, DMSO-d6):
δ
=10.47 (br. s,
OH), 7.79-7.76 (m, 0.03H, Harom.), 7.76-7.75 (m, 0.03H, Harom.), 7.75-7.71 (m, 0.94H, Harom.), 7.68
3
3
2
(d, J(H,H)=8.4 Hz, 1H, Harom.), 7.46 (d, J(H,H)=8.3 Hz, 1H, Harom.), 5.50 (d, J(H,P)=18.7 Hz,
21

ACCEPTED MANUSCRIPT
2
2
0.95H, PCH), 5.28 (d, J(H,P)=18.4 Hz, 0.02H, PCH), 5.21 (d, J(H,P)=18.3 Hz, 0.03H, PCH),
2
2
4.90 (d, J(H,H)=15.2 Hz, 1H, SCH2), 4.42 (d, J(H,H)=15.2 Hz, 0.9H, SCH2), 4.37 (d,
2
2J(H,H)=14.5 Hz, 0.07H, SCH2), 4.30 (d, J(H,H)=15.4 Hz, 0.03H, SCH2), 3.15 ppm (s, NCH3);
2
13C NMR (150.93 MHz, DMSO- d6):
δ
=161.9 (C=O), 132.5 (d, J(C,P)=4.7 Hz, Carom.), 131.6 (d,
3
3J(C,P)=4.8 Hz, Carom.), 131.3 (Carom.), 130.7 (Carom.), 130.4 (Carom.), 130.1 (d, J(C,P)=4.9 Hz,
1
C
arom.), 66.1 (d, J(C,P)=126.6 Hz, PC), 54.2 (SCH2), 35.5 ppm (NCH3); HPLC analysis:
retention time=1.817 min, peak area: 99.02%, eluent A: NH4OAc solution (5 mM), eluent B:
CH3CN, isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.12.
((3,5-Difluorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)sulfonyl)methyl)
1
phosphonic acid (7e): brown oil; yield: 80%. H NMR (600.22 MHz, DMSO- d6):
δ=10.48 (br. s,
NOH), 7.33-7.27 (m, 1H, Harom.), 7.23 (d, 3J(H,F)=7.6 Hz, 0.45H, Harom.), 7.19 (d, 3J(H,F)=7.1 Hz,
2
2
1.55H, Harom.), 5.51 (d, J(H,P)=18.6 Hz, 0.71H, PCH), 5.39 (d, J(H,P)=18.1 Hz, 0.07H, PCH),
2
2
5.29 (d, J(H,P)=18.5 Hz, 0.06H, PCH), 5.22 (d, J(H,P)=17.5 Hz, 0.16H, PCH), 4.97 (d,
2J(H,H)=15.0 Hz, 0.07H, SCH2), 4.88 (d, 2J(H,H)=15.1 Hz, 0.58H, SCH2), 4.71 (d, 2J(H,H)=14.1
2
2
Hz, 0.06H, SCH2), 4.63 (d, J(H,H)=14.4 Hiiz, 0.23H, SCH2), 4.57 (d, J(H,H)=14.5 Hz, 0.06H,
2
2
SCH2), 4.42 (d, J(H,H)=15.1 Hz, 0.78H, SCH2), 4.37 (d, J(H,H)=14.4 Hz, 0.22H, SCH2), 3.74
13
(br. s, POH), 3.15 ppm (NCH3); C NMR (150.93 MHz, DMSO- d6): δ=161.9 (C=O), 161.7 (dd,
3
1J(C,F)=245.5 Hz, J(C,F)=12.9 Hz, Carom.), 133.1-133.0 (m, Carom.), 114.3 (dd, 2J(C,F)=21.0 Hz,
1
4J(C,F)=3J(C,P)=5.2 Hz, Carom.), 104.2-103.8 (m, Carom.), 66.5 (d, J(C,P)=127.4 Hz, PC), 54.2
(SCH2), 35.6 ppm (NCH3); HPLC analysis: retention time=1.780 min, peak area: 99.51%, eluent
A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate of 1
mL min-1 and detection at 254 nm.
5.1.1.13.
((3,5-Dimethoxyphenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)sulfonyl)methyl)
1
phosphonic acid (7f): beige-colored oil; yield: 41%. H NMR (600.22 MHz, DMSO- d6):
δ
=10.54
(br. s, NOH), 6.76-6.72 (m, 0.2H, Harom.), 6.72-6.62 (m, 1.8H, Harom.), 6.55-6.46 (m, 1H, Harom.),
2
2
5.33 (d, J(H,P)=18.9 Hz, 0.61H, PCH), 5.21 (d, J(H,P)=18.3 Hz, 0.39H, PCH), 5.00 (d,
2
2
2J(H,H)=15.2 Hz, 0.28H, SCH2), 4.91 (d, J(H,H)=15.1 Hz, 0.5H, SCH2), 4.64 (d, J(H,H)=14.4
2
2
Hz, 0.22H, SCH2), 4.55 (d, J(H,H)=14.8 Hz, 0.17H, SCH2), 4.32 (d, J(H,H)=15.1 Hz, 0.47H,
2
2
SCH2), 4.31 (d, J(H,H)=14.6 Hz, 0.19H, SCH2), 4.23 (d, J(H,H)=13.9 Hz, 0.17H, SCH2), 3.76-
3.70 (m, 6H, OCH3), 3.65 (br. s, POH), 3.17-3.12 ppm (m, 3H, NCH3); 13C NMR (150.93 MHz,
DMSO- d6): δ
=162.2 (C=O), 159.8 (Carom.), 159.7 (Carom.), 109.6 (d, 3J(C,P)=3.1 Hz, Carom.), 99.60
(Carom.), 55.1-55.0 (m, OCH3), 54.1-54.0 (m, SCH2), 35.5 ppm (NCH3); HPLC analysis: retention
22

ACCEPTED MANUSCRIPT
time=1.853 min, peak area: 99.72%, eluent A: NH4OAc solution (5 mM), eluent B: CH3CN,
isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and detection at 254 nm.
5.1.1.14.
(((2-(Hydroxyamino)-2-oxoethyl)sulfonyl)(p-tolyl)methyl)phosphonic acid (7g):
=10.88 (br. s, OH), 10.54 (br. s, OH),
brown oil; yield: 92%. 1H NMR (600.22 MHz, DMSO- d6):
δ
10.10 (br. s, OH), 9.33 (br. s, NH), 7.42 (d, 3J(H,H)=7.7 Hz, 0.19H, Harom.), 7.40-7.38 (m, 0.25H,
Harom.), 7.36 (d, 3J(H,H)=7.7 Hz, 1.56H, Harom.), 7.22-7.20 (m, 0.12H, Harom.), 7.18 (d, 3J(H,H)=7.8
2
2
Hz, 1.88H, Harom.), 5.40 (d, J(H,P)=19.3 Hz, 0.7H, PCH), 5.27 (d, J(H,P)=19.0 Hz, 0.04H,
2
2
PCH), 5.25 (d, J(H,P)=18.6 Hz, 0.04H, PCH), 5.21 (d, J(H,P)=18.9 Hz, 0.02H, PCH), 5.16 (d,
2
2
2J(H,P)=19.0 Hz, 0.12H, PCH), 5.12 (d, J(H,P)=21.9 Hz, 0.04H, PCH), 5.09 (d, J(H,P)=22.1
2
2
Hz, 0.04H, PCH), 4.84 (d, J(H,H)=15.1 Hz, 0.05H, SCH2), 4.80 (d, J(H,H)=14.2 Hz, 0.11H,
SCH2), 4.70 (d, 2J(H,H)=14.6 Hz, 0.11H, SCH2), 4.59 (d, 2J(H,H)=14.2 Hz, 0.7H, SCH2), 4.48 (d,
2J(H,H)=14.3 Hz, 0.02H, SCH2), 4.44 (d, 2J(H,H)=14.5 Hz, 0.01H, SCH2), 4.25 (d, 2J(H,H)=14.7
2
2
Hz, 0.12H, SCH2), 4.21 (d, J(H,H)=15.2 Hz, 0.05H, SCH2), 4.09 (d, J(H,H)=14.4 Hz, 0.1H,
2
SCH2), 3.74 (d, J(H,H)=13.2 Hz, 0.73H, SCH2), 2.30 ppm (s, 3H, CH3arom.); 13C NMR (150.93
MHz, DMSO- d6):
δ=158.9 (C=O), 137.6 (Carom.), 131.14-130.9 (m, Carom.), 128.8 (Carom.), 125.8
3
1
(d, J(C,P)=5.3 Hz, Carom.), 66.8 (d, J(C,P)=130.2 Hz, PC), 55.2 (SCH2), 20.7 ppm (CH3arom.);
HPLC analysis: retention time=1.833 min, peak area: 99.75%, eluent A: NH4OAc solution (5
mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate of 1 mL min-1 and detection at
254 nm
5.1.1.15.
((3,5-Dimethoxyphenyl)((2-(hydroxyamino)-2-oxoethyl)sulfonyl)methyl)phosphoni
1
c acid (7h): white solid; yield: 80%. M.p. 167 °C; H NMR (600.22 MHz, DMSO- d6):
δ
=10.89
(br. s, NOH), 10.55 (br. s, NOH), 10.09 (br. s, NOH), 9.58 (br. s, NH), 9.35 (br. s, NH), 6.93-6.91
(m, 0.05H, Harom.), 6.80-6.75 (m, 0.07H, Harom.), 6.73-6.72 (m, 0.03H, Harom.), 6.72-6.70 (m,
0.05H, Harom.), 6.65 (s, 1.8H, Harom.), 6.59-6.56 (m, 0.11H, Harom.), 6.55-6.50 (m, 0.84H, Harom.),
2
2
6.41-6.38 (m, 0.05H, Harom.), 6.13 (d, J(H,P)=19.3 Hz, 0.02H, PCH), 5.82 (d, J(H,P)=19.2 Hz,
2
2
0.02H, PCH), 5.35 (d, J(H,P)=19.3 Hz, 0.77H, PCH), 5.26 (d, J(H,P)=19.6 Hz, 0.07H, PCH),
2
2
5.13 (d, J(H,P)=19.1 Hz, 0.12H, PCH), 5.02 (d, J(H,H)=14.2 Hz, 0.02H, SCH2), 4.80 (d,
2J(H,H)=15.0 Hz, 0.05H, SCH2), 4.77 (d, 2J(H,H)=13.9 Hz, 0.04H, SCH2), 4.64 (d, 2J(H,H)=14.6
2
2
Hz, 0.09H, SCH2), 4.53 (d, J(H,H)=14.2 Hz, 0.33H, SCH2), 4.44 (d, J(H,H)=14.2 Hz, 0.19H,
2
2
SCH2), 4.39 (br. s, POH), 4.28 (d, J(H,H)=14.6 Hz, 0.14H, SCH2), 4.23 (d, J(H,H)=16.2 Hz,
2
0.13H, SCH2), 3.78 (d, J(H,H)=14.2 Hz, 1H, SCH2), 3.74 (s, 5.6H, OCH3), 3.72 ppm (s, 0.4H,
OCH3); 13C NMR (150.93 MHz, DMSO- d6):
δ
=159.9 (C=O), 158.7 (Carom.), 130.7 (d, 2J(C,P)=5.2
23

ACCEPTED MANUSCRIPT
Hz, Carom.), 109.5 (d, 3J(C,P)=4.9 Hz, Carom.), 99.7 (Carom.), 67.0 (d, 1J(C,P)=128.7 Hz, PC), 55.3-
54.9 ppm (m, OCH3, SCH2); HPLC analysis: retention time=1.833 min, peak area: 99.79%,
eluent A: NH4OAc solution (5 mM), eluent B: CH3CN, isocratic (1:1) over 20 min with a flow rate
of 1 mL min-1 and detection at 254 nm.
5.1.2. Chiral separation of compound 5a
Chiral separation of compound 5a was performed with a Chiracel OZ-H column (5µM
particle size, 250 mm x 4.6 mm, Chiral Technologies, France) on an HP series 1100
HPLC (Agilent Technologies). After injection of 50 µL of 35 mM 5a in n-heptane /
propan-2-ol (70:30) at a flow rate of 1 mL min−1, the column was developed at the same
flow rate at 25 °C with n-heptane / propan-2-ol / (70:30) containing 0.075%
trifluoroacetic acid. 1 mL fractions were collected after 5 min (R)-5a = inactive
enantiomer) and after 7 min ((S)-5a = active enantiomer). Sodium phosphate (pH 7.4)
was added to a final concentration of 25 mM. The organic phase was discarded. The
aqueous phase was concentrated to 3 mM enantiomer with a nitrogen stream. A
Nucleosil 100-5 C18 HD column (250 x 10 mm, Machery-Nagel) on a 1260 Infinity
Preparative Scale HPLC (Agilent Technologies) was used to separate each enantiomer
from Na-phosphate and trifluoroacetic acid. After injection of 5 mL enantiomer
containing solution, the column was developed with water at a flow rate of 1 mL min−1 at
25 °C. Fractions were collected and lyophilized at -30 °C and subsequently stored at -80
°C.
5.1.3. Stability of 5a in the 100 mM Tris-HCl, pH 7.6.
Compound 5a (racemate, (R)-5a or (S)-5a, with concentrations between 0.6 mM and
o
o
0.7 mM) were incubated at room temperature (22 C – 23 C) for two days in the 100
mM Tris-HCl pH 7.6, 0.02% NaN3. At time points of 0 hours, 24 hours and 48 hours of
o
incubation aliquots were taken away from each sample and stored at -80 C. All
collected aliquots were tested for IC50 value in parallel in the photometric assay as
described in the previous paragraph.
5.1.4. Stability of compound 5a. Sample preparation and stability measurement.
24

ACCEPTED MANUSCRIPT
1.0 mg of 5a were dissolved with phosphate buffer pH 7.6 (prepared according to Ph.
Eur. 5.0) to obtain a final concentration of 1 mg/mL. The resulting solution was shaken
on an IKA KS 260 basic shaker at room temperature for 72 hours. 10 µl of this solution
were used for HPLC analysis at each time point (0, 1, 3, 6 24, 72h, n = 3).
Decomposition was determined by the relative reduction of the area under the curve
(AUC) at 254 nm.[41] For stability testing a Knauer HPLC with a Knauer UV detector K-
2600 (254nm) was used to identify decomposition. Column type Vertex Plus Column
(length 150 x 4 mm with precolumn, packing material of the column was Eurospher II
100-5 C18) was used with a linear gradient of acetonitrile containing 0.1% trifluoroacetic
acid (10 to 100 %) in water containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min
over 30 min, isocratic elution of acetonitrile containing 0.1% trifluoroacetic acid (100%
over 10 min. Retention time of 5a: 4.1 min.
5.2. Biological evaluation:
5.2.1.1.
IspC enzyme Assays
5.2.1.1.1.
Gene expression and protein purification
The ispC of P. falciparum was expressed in E. coli and the protein was purified as
described earlier.[20] IspC orthologs of E. coli or M. tuberculosis were isolated and
purified as follows. The recombinant E. coli strain M15 pREP4 containing pQEispC-Ec
(for the E. coli enzyme) or pQEispC-Mt (for the M. tuberculosis enzyme) was grown to
an optical density of 0.4-0.6 at 37 °C in LB medium supplemented with ampicillin (70 mg
L−1) and kanamycin (30 mg L−1). Isopropylthiogalactoside was added to a final
concentration of 1 mM, and the cell suspension was cultivated 36 hours at 20 °C under
shaking (140 rpm). Cells were harvested by centrifugation, washed with 0.9% NaCl
solution, and resuspended in loading buffer (50 mM Tris hydrochloride, pH 8.0,
containing 0.5 M NaCl, 20 mM imidazole and 0.02% NaN3). The suspension was
passed through a cell disruption device (FrenchPress, Constant Cell Disruption
o
Systems, Koenigswinter, Germany) and was then centrifuged (15 000 rpm, +4 C, 1h).
The supernatant was subjected to a Ni2+-chelating Sepharose fast flow column (volume
25

ACCEPTED MANUSCRIPT
of 30 mL) that had been pre-equilibrated with loading buffer. The column was washed
with 150 mL of the loading buffer and was then developed with imidazole gradient (20
mM – 500 mM) in laoding buffer. Fractions of eluent were combined and dialyzed
overnight against 50 mM Tris hydrochloride, pH 8.0, containing 2 mM DTT and 0.02%
NaN3. IspC batches were obtained with approximately 95% purity, as estimated by
SDS−PAGE analysis.
5.2.1.2.
Photometric Assay
Assays were conducted in 384-well plates with flat clear bottom (Nunc, Wiesbaden,
Germany). Assay mixtures with a total volume of 60 µL contained 100 mM Tris
hydrochloride, pH 7.6, 5 mM MnCl2, 5 mM DTT, 0.5 mM NADPH, 0.02 U of IspC
protein, and test compounds. Dilution series (1:3) of test compounds covered the
concentration range of 200 - 0.003 µM. The reaction was started by addition of 1-deoxy-
D-xylulose 5-phosphate in 100 mM Tris hydrochloride, pH 7.6, to a final concentration of
0.5 mM. The reaction was monitored photometrically (room temperature) at 340 nm
using a plate reader (SpectraMax M5, Molecular Devices, Biberach an der Riss,
Germany). Inhibition constant (Ki) and IC50 values were calculated with the program
Dynafit [35] as described earlier.[18]
5.2.2. In vitro antiplasmodial activity.
Acitivity against asexual blood stages was evaluated based on a histidine rich protein 2
(HRP2) enzyme linked immunosorbent assay (ELISA) as reported previously.[42,43]
The in vitro toxicity was tested via a spectrophotometric quantification of cytotoxicity
against HeLa cells.[44]
Author information
Corresponding Author
Thomas Kurz, Phone: +49 211 81 14984. Fax: +49 211 81 13847. E-mail:
thomas.kurz@hhu.de
Author Contributions
26

ACCEPTED MANUSCRIPT
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Acknowledgments
Funding Sources
RK and TK acknowledges support from the Deutsche Forschungsgemeinschaft (DFG)
research training group GRK2158. The Deutsche Forschungsgemeinschaft (DFG) is
acknowledged for funds used to purchase the UHR-TOF maXis 4G (Bruker Daltonics,
Bremen) HRMS instrument used in this research.
Appendix A. Supplementary data
Experimental procedures, analytical data for compounds 3e-f, 4a-j and 6a-h,
Computational Study of (S)-5a and (S)-7a and antibacterial activity are provided as
supplementary information.
Abbreviations
ABS, asexual blood stage; DMAPP, Dimethylallyl pyrophosphate, DOXP, 1-deoxy-D-
xylulose 5-phosphate, DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase,
EcIspC, Escherichia coli IspC enzyme; HeLa, Henrietta Lacks; HRP2, histidine rich
protein IPP, isopentenyl pyrophosphate mp, melting point; MSD, mean survival time in
days; MEP, 2-C-methyl-D-erythritol 4-phosphate; McIspC, Mycobacterium tuberculosis
IspC enzyme NMRI, Naval Medical Research Institute Pf3D7, Plasmodium strain 3D7;
PfDd2, Plasmodium strain Dd2; PfIspC, P. falciparum IspC enzyme; SI, selectivity
index; SD, Standard deviation TMSBr, Bromotrimethylsilane; UHR-TOF, ultrahigh
resolution – time of flight
Conflicts of interest
The authors declare that they have no conflict of interest.
27

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6.
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