Phosphonodiamidate prodrugs of N-alkoxy analogs of a fosmidomycin surrogate as antimalarial and antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2019.03.008

A series of N-alkoxy analogs of a L-leucine ethyl ester phosphonodiamidate prodrug of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. These compounds originate b

Full text of DOI:10.1016/j.bmcl.2019.03.008

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phosphonodiamidate prodrugs of N-alkoxy analogs of a fosmidomycin
surrogate as antimalarial and antitubercular agents
Charlotte Courtensa, Martijn Risseeuwa, Guy Caljonb, Paul Cosb, Anandi Martinc,
⁎
Serge Van Calenbergha,
a Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
b Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Universiteitsplein 1 (S7), B-2610 Wilrijk, Belgium
c Medical Microbiology, Institute of Experimental and Clinical Research, Université catholique de Louvain, Avenue Hippocrate 55, B-1200 Woluwe-Saint-Lambert, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of N-alkoxy analogs of a L-leucine ethyl ester phosphonodiamidate prodrug of a fosmidomycin surrogate
were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis.
These compounds originate by merging a previously reported successful phosphonate derivatisation with fa-
vorable modifications of the hydroxamate moiety. None of the synthesized compounds showed enhanced ac-
tivity against either P. falciparum or M. tuberculosis in comparison with the parent free hydroxamate analog.
Fosmidomycin
Prodrugs
Non-mevalonate pathway
Isoprenoid biosynthesis
Malaria
Tuberculosis
Despite international efforts, malaria and tuberculosis (TB) remain
among the most problematic infectious diseases worldwide. According
to the World Health Organization (WHO), malaria incidence has de-
creased significantly since 2010. Since 2014, however, the number of
malaria cases is steadily increasing, while the number of deaths remains
comparable.1 Resistance to antimalarial drugs is a persisting problem
and, alarmingly, elevated resistance to artemisinin combination
therapy (ACT) drugs has been observed in recent years.2,3 Despite a
significant drop in TB mortality rates since 2010, the proportion of
multidrug-resistant (MDR) TB cases is steadily increasing. Approxi-
mately 5% of active TB cases are multidrug-resistant (MDR), of which
6% are extensively drug-resistant (XDR). Treatment success rates of
drug-resistant TB cases are relatively low, being 50% for MDR-TB and
30% for XDR-TB.4,5 In 2009, the first totally drug-resistant (TDR)
strains have been detected in India.6 In order to halt the upsurge of
infections with drug-resistant pathogen strains, there is an urgent need
for antimalarial and antitubercular agents with a novel mechanism of
action (MOA). In this respect, the non-mevalonate pathway (NMP) for
isoprenoid biosynthesis represents an interesting potential drug target.
Isoprenoids form the largest class of natural compounds and are es-
sential to all living organisms. They are built up of the five-carbon
isoprene units isopentenyl pyrophosphate (IPP) and dimethylallyl pyr-
ophosphate (DMAPP). These building blocks can be synthesized via two
evolutionary distinct pathways: the mevalonate (MVA) pathway and
the NMP, also known as the methylerythritol phosphate (MEP)
pathway. Both malaria-causing Plasmodium parasites and Myco-
bacterium tuberculosis (Mtb), the causative agent of TB, rely entirely on
the NMP, while it is absent in humans. 1-Deoxy-D-xylulose-5-phosphate
reductoisomerase (DXR, also known as IspC), catalyzes the second step
of the MEP pathway and is the most extensively investigated enzyme of
this pathway.7,8
L-leucine ethyl ester based prodrug derivatives of N-alkoxy analogs
of a fosmidomycin surrogate are the focus of this work.
Fosmidomycin (1, Fig. 1) and FR900098 (2, Fig. 1), the N-acetyl
analog of fosmidomycin, are natural antibiotics originally isolated from
Streptomyces lavendulae and Streptomyces rubellomurinus, respectively.9
Fosmidomycin was originally evaluated for the treatment of urinary
tract infections. In 1998, however, fosmidomycin and FR900098 were
Abbreviations: AA, amino acid; ACT, artemisinin-based combination therapy; DCM, dichloromethane; DMAPP, dimethylallyl pyrophosphate; DXP, 1-deoxy-D-
xylulose 5-phosphate; DXR, 1-deoxyxylulose 5-phosphate reductoisomerase; EDC, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide; Et3N, triethylamine; GlpT,
glycerol-3-phosphate transporter; HOBt, hydroxybenzotriazole; HRMS, high-resolution mass spectrometry; MDR, multidrug-resistant; MEP, methylerythritolpho-
sphate; MIC, minimal inhibitory concentration; MOA, mechanism of action; Mtb, Mycobacterium tuberculosis; MVA, mevalonate; NADPH, nicotinamide adenine
dinucleotide phosphate; NMP, non-mevalonate pathway; PK, pharmacokinetic; POM, pivaloyloxymethyl; SD, standard deviation; SI, selectivity index; TB, tu-
berculosis; TDR, totally drug-resistant; TFA, trifluoroacetic acid; THF, tetrahydrofuran; WHO, world health organization; XDR, extensively drug-resistant
⁎ Corresponding author.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
https://doi.org/10.1016/j.bmcl.2019.03.008
Received 10 February 2019; Received in revised form 4 March 2019; Accepted 6 March 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:CharlotteCourtens,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.03.008

C. Courtens, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 1. Structural formulae of fosmidomycin, FR900098, previously reported prodrug derivatives 3–5, and N-alkoxy derivatives 6a-d.
Scheme 1. Synthesis of L-leucine ethyl ester based prodrug derivatives of N-alkoxy analogs of a fosmidomycin surrogate. aReagents and conditions. (a) (i) NaH, dry
THF; (ii) RX, 70 °C (82–95%); (b) TFA, DCM; (c) EDC.HCl, HOBt, Et3N, DCM (59–98% over 2 steps).
shown to be potent DXR inhibitors, resulting in regained interest in
these phosphonates, now as antimalarial agents.10 Fosmidomycin has
been shown to be safe and well-tolerated as an antimalarial agent in
combination therapy with clindamycin. However, fosmidomycin is a
highly polar compound, mainly due to its phosphonate functionality,
which exists mainly as its dianion at physiological pH. Primarily as a
result of its high polarity, fosmidomycin displays suboptimal pharma-
cokinetic (PK) properties including moderate oral bioavailability
(20–40%) and a short plasma half-life (1.87 h).11 Therefore, it needs to
be administered multiple times a day at relatively high doses.12–14
Furthermore, fosmidomycin displays poor permeation via passive dif-
fusion. This has important consequences not only for oral bioavail-
ability, but also for the inhibitor to reach its intracellular target. Fos-
midomycin uptake in E. coli has been shown to be dependent on the
presence of a glycerol 3-phosphate transporter (GlpT).15 Also P. falci-
parum infected erythrocytes have been shown to use parasite-induced
permeability pathways to facilitate uptake of fosmidomycin in infected
red blood cells.16 Mycobacteria lack an active fosmidomycin uptake
system and additionally have a highly lipophilic cell wall. As a result,
fosmidomycin is unable to penetrate Mycobacteria to reach its target.17
Lipophilic phosphonate prodrug derivatives of fosmidomycin (analogs)
have previously been reported to display significantly enhanced anti-
plasmodial and antitubercular activities.18,19 We previously reported
amino acid (AA) phosphonodiamidate derivatives of a fosmidomycin
surrogate (3 and 4, Fig. 1) with promising whole cell antitubercular
activities.20 The Dowd research group has recently reported N-acyl and
N-alkoxy analogs of FR900098 as bisubstrate DXR inhibitors. These
compounds were found to be competitive with both the natural sub-
strate (1-deoxy-D-xylulose 5-phosphate, DXP) and cofactor (NADPH).
Especially interesting are the antitubercular activities of the pivaloy-
loxymethyl (POM)-prodrugs of N-alkoxy analogs of FR900098 (6a-d,
Fig. 1) with MIC values up to 5.3–21 µM.21–23 These results demonstrate
that a free hydroxamate OH functionality is not essential for DXR in-
hibition. This is of interest as the heightened lipophilicity of the N-al-
koxy analogs is expected to be beneficial for penetration of the highly
lipophilic mycobacterial cell wall.
Compounds 3 and 4 (Fig. 1) display significantly more potent an-
titubercular activity than POM-prodrug 5 (Fig. 1).24 Mtb DXR in-
hibitory activity of the parent compound from prodrug 3 and 4 has
been reported to be 1.15 µM.25 Mtb DXR inhibitory activity of
FR900098, the parent compound from prodrug 5, has been reported to
be 2.39 µM.26 These inhibitory values are comparable, while the dif-
ference in antitubercular activities of compounds 3 and 4 in comparison
with compound 5 are up to 12-fold better. Therefore, we hypothesized
that the improvement in antitubercular activity could be mainly at-
tributed to the prodrug promoiety used. We envisaged that combining
the L-leucine ethyl ester phosphonodiamidate moiety with the hydro-
xamate substituents of 6a-d (Fig. 1) might further improve anti-
tubercular activity. To this end, we report the synthesis of a series of N-
alkoxy analogs of the L-leucine ethyl ester phosphonodiamidate deri-
vatives of a fosmidomycin surrogate with an inversed hydroxamate
group (see Scheme 1).
We gained access to all target compounds from the common inter-
mediate 10, which was synthesized as described previously (see
Supporting Information).20 Alkylation of Boc-protected N-methyl hy-
droxylamine 7 with the appropriate alkyl halides in the presence of
NaH provided alkoxy carbamates 8a-g, which were deprotected to give
the required hydroxylamine ethers 9a-g. Coupling of these diverse O-
substituted N-methylhydroxylamines with intermediate 10 using
EDC.HCl and HOBt yielded the desired compounds 11a-g in good
yields.
Final compounds 11a-g were screened for growth inhibition of
asexual blood stage parasites of P. falciparum (Pf-K1) and an avirulent
M. tuberculosis strain (H37Ra). Additionally, toxicity on MRC-5 fibro-
blasts was assessed (Table 1). While most compounds retained mod-
erate activity against P. falciparum, none surpassed the M. tuberculosis
activity of the free hydroxamate analog 4. The poor antitubercular
activities of the compounds were additionally confirmed against the
reference H37Rv M. tuberculosis strain, against which none of the N-
alkoxy analogs proved active up to the highest concentration tested
(50 µM). Furthermore, contrary to parent compound 4, all analogs
(except benzyl ether 11a) decreased cell viability of MRC-5 fibroblasts,
2

C. Courtens, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Table 1
Biological evaluation of L-leucine ethyl ester based prodrug derivatives of N-alkoxy analogs of a fosmidomycin surrogate.a
Compound
R
Pf-K1 IC50
(
SD) [µM]
H37Ra IC50
(
SD) [µM]
MRC-5 CC50
(
SD) [µM]
SIb (Pf-K1)
> 20
SIb (H37Ra)
> 2.2
4
H
4.83 ( 2.29)
29.4 ( 18.3)
> 64
> 64
11a
11b
11c
11d
11e
11f
Bn
4.22
> 64
> 15
(CH2)3Ph
8.93 ( 0.13)
6.72 ( 1.39)
5.28 ( 1.52)
7.12 ( 0.22)
6.22 ( 0.10)
5.25 ( 0.16)
> 64
23.1 ( 0.0)
7.23 ( 1.01)
7.22 ( 0.09)
18.4 ( 0.9)
5.47 ( 0.06)
5.94 ( 0.24)
2.6
(CH2)4Ph
50.3 ( 19.4)
49.1 ( 21.0)
49.0 ( 21.2)
48.2 ( 19.9)
35.2 ( 4.1)
1.1
0.14
0.15
0.37
0.11
0.17
4-iPr Bn
1.4
4-Cl Bn
2.6
CH2-(2-naphthyl)
CH2-4-(1,1′-biphenyl)
0.88
1.1
11 g
a
b
Values shown are the calculated mean values of at least two measurement results (except for 11a, which was tested only once).
SI = selectivity index.
indicating human cell toxicity. As a result, the selectivity index of all
isoprenoid biosynthesis. Annu Rev Biochem. 2013;82:497–530. https://doi.org/10.
1146/annurev-biochem-052010-100934.
target compounds was insufficient both with respect to antiplasmodial
and antitubercular activity, as shown in Table 1. Possibly, due to the
reverse orientation of the hydroxamate in comparison with FR900098,
the O-alkyl groups are no longer able to bind the NADPH binding
pocket, resulting in decreased DXR inhibitory activity and decreased in
vitro growth inhibition. In conclusion, in our hands, the O-alkyl sub-
stituents do not provide added value for antiplasmodial and/or anti-
tubercular activities in comparison with the corresponding free hy-
droxamate analog.
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Author contributions
All authors have given approval to the final version of the manu-
script.
Funding sources
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adenylate cyclase deficient (Cya) mutants of Escherichia Coli. Biosci Biotechnol
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The authors gratefully acknowledge funding by the Research Fund
Flanders (FWO).
Notes
The authors declare no competing financial interest.
17. Brown AC, Parish T. Dxr is essential in mycobacterium tuberculosis and
Fosmidomycin resistance is due to a lack of uptake. BMC Microbiol. 2008;8:78.
https://doi.org/10.1186/1471-2180-8-78.
Acknowledgment
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The authors would like to thank Izet Karalic for technical assistance.
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Experimental details and characterization data for the reported
compounds, NMR spectra, biological data (PDF). Supplementary data to
this article can be found online at https://doi.org/10.1016/j.bmcl.
2019.03.008.
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