Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2019.03.009

A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was c

Full text of DOI:10.1016/j.bmcl.2019.03.009

Accepted Manuscript
Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular
agents
Charlotte Courtens, Martijn Risseeuw, Guy Caljon, Louis Maes, Paul Cos,
Anandi Martin, Serge Van Calenbergh
PII:
S0960-894X(19)30135-0
https://doi.org/10.1016/j.bmcl.2019.03.009
BMCL 26323
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 December 2018
10 February 2019
5 March 2019
Please cite this article as: Courtens, C., Risseeuw, M., Caljon, G., Maes, L., Cos, P., Martin, A., Van Calenbergh,
S., Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.03.009
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Double prodrugs of a fosmidomycin surrogate as antimalarial and
antitubercular agents.
Charlotte Courtensa, Martijn Risseeuwa, Guy Caljonb, Louis Maesb, Paul Cosb, Anandi Martinc and Serge
Van Calenbergha,*
aLaboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium
bLaboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Universiteitsplein 1 (S7), B-2610 Wilrijk,
Belgium
cMedical Microbiology, Institute of Experimental and Clinical Research, Université catholique de Louvain, Avenue Hippocrate
55, B-1200 Woluwe-Saint-Lambert, Belgium
Keywords: Fosmidomycin, prodrugs, non-mevalonate pathway, isoprenoid biosynthesis, malaria, tuberculosis
ABSTRACT: A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their
ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was
combined with various prodrug derivatisations of the hydroxamate moiety. The majority of compounds showed activity comparable
with or inferior to fosmidomycin against P. falciparum. N-benzyl substituted carbamate prodrug 6f was the most active antimalarial analog
with an IC50 value of 0.64 µM. Contrary to fosmidomycin and parent POM-prodrug 5, 2-nitrofuran and 2-nitrothiophene prodrugs 6i
and 6j displayed promising antitubercular activities.
Malaria and tuberculosis (TB) are two of the most lethal infectious diseases worldwide, being together
responsible for over 2 million deaths in 2016.1,2 The malaria targets set by the WHO aim at reducing
malaria incidence and mortality by 90% by 2030, eliminating malaria in at least 35 additional countries
and preventing resurgence of malaria in malaria-free countries.1 Challenges for achieving these goals are
numerous. The major challenge today is rapid emergence of insecticide and drug resistance. Especially
the rise of resistance to artemisinin derivatives in the Greater Mekong region is problematic as no
alternative first-line antimalarial treatments are currently available.3–5 TB mortality rates have dropped
significantly in recent years. However, the proportion of multidrug-resistant (MDR) TB-cases is steadily
increasing.2 The WHO End TB Strategy aims at eradication of TB by 2030 with TB mortality reduced by
95% and TB incidence by 90%.6,7 The current annual decline in TB incidence worldwide is far off from
what is necessary to reach the targets set by the WHO.7 The reasons for the failing TB pushback in recent
years are numerous and include failing health care systems in TB endemic countries, the emergence and
rise of HIV/AIDS and the expansion of resistant TB strains, at least partially due to the lengthy treatment
regimens and their consequent low compliance rates.8–10 In order to halt the rise of resistant pathogen

strains, drugs with a novel mechanism of action (MOA) against both malaria-causing Plasmodium
parasites and Mycobacterium tuberculosis are urgently required.
Figure 1. Structural formulae of fosmidomycin, FR900098, their reverse analogs 3 and 4, and previously
11
reported double prodrug derivatives. Double prodrugs of 4, with a POM phosphonate prodrug
derivatisation and various modifications of the hydroxamate moiety, are the focus of this work.
In this respect, fosmidomycin (1, Figure 1) is an interesting lead compound. Fosmidomycin (1, Figure
12
1) and FR900098 (2, Figure 1) are natural phosphonate antibiotics, produced by Streptomyces bacteria.
They are slow, tight-binding inhibitors of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the
13,14
second enzyme of the non-mevalonate isoprenoid biosynthesis pathway. Isoprenoids or terpenoids are
essential molecules for all living organisms. They are built up of five-carbon building blocks, which can
be metabolically derived from two evolutionary distinct pathways, the mevalonate (MVA) and the non-
mevalonate or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Humans exclusively utilize the
MVA pathway, whereas both Plasmodium parasites and Mycobacterium tuberculosis rely solely on the
MEP pathway. The absence of homologous enzymes in humans and the essentiality of the pathway in
15,16
pathogenic organisms renders the enzymes of the MEP pathway highly interesting drug targets. DXR
is by far the most extensively investigated enzyme of the non-mevalonate pathway.14,17 Ki values of
18,19
fosmidomycin against P. falciparum DXR and Mtb DXR are 20nM and 140nM, respectively. Several
phase II clinical trials of fosmidomycin in combination therapy with clindamycin as a potential treatment
of acute uncomplicated P. falciparum malaria have been completed and have repeatedly demonstrated
their good synergistic profile. However, the most recently completed study (Mozambique, 2012) found a
low cure rate on day 28, resulting in a slowdown of further development of this combination. Possible
explanations for these discrepant results are differences in the age of the patients involved and the

formulation used.20,21 Despite its overall promising activity and safety profile, fosmidomycin has several
shortcomings. Fosmidomycin is highly polar. This is mainly due to the phosphonate functionality, which
is negatively charged at physiological pH. This results in poor passive permeability characteristics and
suboptimal pharmacokinetic (PK) properties, including moderate oral bioavailability (20-40%) and a
22
short plasma half-life (1.87h). Furthermore, P. falciparum induced permeability pathways are required
23
for active uptake of fosmidomycin in infected red blood cells. Mycobacteria do not have this transporter,
and additionally have a highly lipophilic cell wall, resulting in intrinsic resistance of Mycobacteria against
24
this phosphonate antibiotic.
In recent years, fosmidomycin research has mainly focused on the development of prodrug derivatives,
especially in the interest of improving PK properties and obtaining antibacterial activity. Furthermore,
enabling fosmidomycin uptake independent of transporters would be interesting as sole dependence of
drug uptake on one transporter increases the chances of resistance development significantly.25 Prodrugs
of fosmidomycin (analogs) have been obtained, mainly by conversion of the phosphonate moiety into
acyloxyalkyl and alkoxycarbonyloxyalkyl phosphonate esters and have been shown to possess increased
antimalarial activity.11,26,27 However, sufficiently potent antitubercular analogs have not been obtained. Not
only the phosphonate, but also the (retro)hydroxamate functionality contributes to the high polarity of
fosmidomycin. Therefore, additionally masking the (retro)hydroxamate might further enhance uptake via
passive diffusion. Furthermore, masking the hydroxamate functionality might also result in enhanced
plasma stability as hydroxamates are known to be susceptible to plasma hydrolysis.28,29 Few hydroxamate
prodrug modifications have been described in the literature. There have been reports of increased cellular
uptake with carbamate30,31 and p-acetoxybenzyl ester32 prodrug derivatives of hydroxamates. For the p-
acetoxybenzyl ester prodrugs, improved in vivo oral PK in mice has been demonstrated, with five-fold
higher plasma levels of the parent drug after administration as its prodrug.32 A number of double prodrugs
of fosmidomycin analogs has been reported, of which a selection is shown in Figure 1.11 Inspired by the
promising antiplasmodial activities of these compounds, we decided to further expand the double prodrug
series. The previously reported double prodrugs are derived from the reverse hydroxamate α-3,4-
difluorophenyl analog, which compared to fosmidomycin is more active against PfDXR (IC50 = 4 nM),

but less active against MtbDXR (IC50 = 3.4 µM).33 As we are interested in not only antiplasmodial, but
also antitubercular activities, fosmidomycin surrogate 4 (Figure 1) has been used as a starting point for
the development of prodrugs. The hydroxamate counterparts of fosmidomycin and FR900098 (3-4, Figure
1) have equal inhibitory activity as fosmidomycin against both PfDXR and MtbDXR. Also IC50 values
against the P. falciparum K1 strain have been shown to be comparable.34 We combined a POM
phosphonate prodrug modification with various prodrug derivatisations of the hydroxamate moiety.
Besides simple ester, carbonate and carbamate prodrug derivatisation, 3 prodrug promoieties were
selected based on bioreductive properties. The bioreductive prodrug approach in general has mainly been
investigated in the interest of the development of anticancer agents which are activated selectively in
hypoxic tumor tissue. We hypothesized that this prodrug strategy might also allow targeting to hypoxic
TB lesions. The most commonly used prodrug promoieties in this approach are 4-nitrobenzyl, 1-methyl-
2-nitroimidazole, 2-nitrothiophene and 2-nitrofuran.35,36 We have synthesized the synthetically easily
accessible 4-nitrobenzyl, 2-nitrothiophene and 2-nitrofuran hydroxamate prodrug derivatives of
fosmidomycin surrogate 4. In view of the results obtained with the p-acetoxybenzyl ester prodrug
approach by Rais et al., and our own research on acyloxybenzyl prodrug derivatives of a fosmidomycin
surrogate as antitubercular agents, we also synthesized the p-nonanoyloxybenzyl ester prodrug derivative
of 4.32,37
Scheme 1. Synthesis of double prodrugsa
aReagents and conditions. esters: R(C=O)Cl, Et3N, DCM (63-90%); carbonates: benzylchloroformate, Et3N, DCM (76%); carbamates: R-
isocyanate, Et3N, THF (85-96%); ethers: RCl or RBr, Na2CO3, MeCN, 80°C (18-24%).
The synthesis of double prodrugs 6a-j started from POM prodrug 5 (Scheme 1), which was reported by us previously.37 Ester prodrugs
6a-c were synthesized by reaction with the appropriate acid chloride and Et3N as base. Carbonate prodrug 6d was synthesized by reaction
with benzylchloroformate and Et3N. Carbamate prodrugs 6e-g were synthesized by reaction with the appropriate isocyanate and Et3N.
Ether prodrugs 6h-j were synthesized by heating 5 with the appropriate alkyl halide and Na2CO3 as base. For the synthesis of double

prodrug 16, alkylation of 5 with nonanoyloxybenzyl halide was attempted using a range of different reaction conditions, but failed to
1
produce the desired product in acceptable yield. HRMS and H-nmr analysis of the obtained products revealed that both the POM-
moiety and the nonanoyloxybenzyl moiety degraded during the reaction, resulting in a complex mixture of undesired reaction products.
Therefore, an alternative synthesis route was designed.
Scheme 2. Synthesis of nonanoyloxybenzyl hydroxamate prodruga
aReagents and conditions: (a) (i) NaH, DMF (ii) nonanoyloxybenzyl iodide 8 (17%); (b) TFA, DCM; (c) BnBr, Na2CO3, MeCN, 60°C (q); (d)
(i) TMSBr, DCM; (ii) H2O, THF; (e) POM-Cl, Et3N, DMF, 80°C (33%); (f) H2, Pd/C, MeOH (74%); (g) EDC.HCl, HOBt, Et3N, DCM
(84%).
The synthesis of hydroxylamine ether 10 started from Boc-protected hydroxylamine 7 (Scheme 2). Alkylation with nonanoyloxybenzyl
iodide 8 in the presence of NaH as a base, yielded Boc-protected hydroxylamine ether 9 in low, but acceptable yield. Boc deprotection
with TFA produced 10. The synthesis of phosphonate intermediate 15 started from carboxylic acid 11. Benzyl protection of the
carboxylic acid group afforded 12. TMSBr-mediated phosphonate deprotection and subsequent hydrolysis of the phosphonate esters,
provided free phosphonate 13. Alkylation with POM-Cl gave 14, which was hydrogenated to the free carboxylic acid 15. EDC-coupling
of 10 and 15 afforded final compound 16.
Table 1. Biological evaluation of double prodrugs of a fosmidomycin surrogate.
H37Rv
Pf-K1
MRC-5
Compound
MIC [µM]
IC50 (±SD) [µM]
CC50 (±SD) [µM]
1
1.7338
>64
>50
[>64]a
50
>64
5
0.73
>64
6a
6b
6c
6d
6e
6f
6g
6h
6i
1.34 (±0.52)
1.41
>64
>64
1.08 (±0.80)
>36
50
>64
>50
>50
>50
[>64]
>50
12.5
12.5
50
>42
2.52 (±2.76)
0.64 (±0.02)
3.34 (±3.86)
8.79 (±4.88)
4.42
25.9 (±1.7)
27.9 (±1.6)
33.4 (±6.9)
26.5 (±1.7)
26.3
6j
4.69
20.5
16
19.0
>64
avalues in square brackets are IC50 (µM) values, determined against avirulent M. tuberculosis strain H37Ra
Final compounds 6a-j and 16 were screened for growth inhibition of asexual blood stage parasites of
P. falciparum (Pf-K1) and an avirulent M. tuberculosis strain (H37Ra) and/or a virulent M. tuberculosis
strain (H37Rv). Additionally, toxicity on MRC-5 fibroblasts was assessed (Table 1). Ester prodrugs 6a-c

displayed antiplasmodial activities comparable to both fosmidomycin and parent prodrug 5, while
carbonate prodrug 6d was inactive. Only N-benzyl substituted carbamate prodrug 6f displayed good
antiplasmodial activity with an IC50 value of 0.64 µM. Remarkably, all ester prodrugs 6a-c, carbonate 6d
and carbamates 6e-g were inactive or only very weakly active against M. tuberculosis. Also
nonanoyloxybenzyl ester prodrug 16 displayed poor antiplasmodial activity and a poor antitubercular
MIC value of 50 µM. It is unclear whether this is due to inadequate stability of the prodrug, inefficient
release of the parent drug or poor permeation by passive diffusion. Compared to parent prodrug 5,
prodrugs 6i and 6j displayed significantly improved antitubercular activity. Unfortunately, these analogs
also decreased cell viability of MRC-5 fibroblasts. Despite the observed toxicity, it would be interesting
to expand this prodrug series in order to see if superior activity and enhanced selectivity can be obtained.
Additionally, it would be valuable to evaluate the activity of the hypothetical bioreductive prodrugs under
non-replicating conditions and to validate their mechanism of bioactivation. At this point, it is unclear
whether derivatives 6h-j are being activated by reduction and/or simple hydrolysis, or whether they are
binding as such in the active site. It is also possible that reactive metabolites formed during the reduction
of the nitro heterocycles at least contribute to the observed activity. We did not further investigate the lack
of activity of 4-nitrobenzyl prodrug 6h. However, it is known that 2-nitrofuran and 2-nitrothiophene
prodrug moieties are more easily reducible than 4-nitrobenzyl moieties.35 We hypothesize that as a result,
the 4-nitrobenzyl prodrug moiety is not activated as efficiently, resulting in insufficient parent drug
release.
In conclusion, we report on the synthesis, antiplasmodial and antimycobacterial activity of a series of
11 double prodrug derivatives of fosmidomycin surrogate 4 (Figure 1). N-benzyl substituted carbamate
derivative 6f displayed good antiplasmodial activity. Hypothetical bioreductive 2-nitrofuran and 2-
nitrothiophene derivatives 6i and 6j displayed promising antitubercular activities. These findings suggest
that a bioreductive prodrug approach may enable uptake and release of fosmidomcyin surrogate 4 in
mycobacterial cells. Further research is necessary to confirm these findings.
ASSOCIATED CONTENT
Supporting Information

The supporting information is available free of charge at DOI: xxx.
Experimental details and characterization data for the reported compounds, 1H NMR, 13C NMR and LC-MS spectra of the final compounds,
biological data (PDF).
AUTHOR INFORMATION
Corresponding Author
*Serge Van Calenbergh. Telephone: +32 (0)9 264 81 24. Fax: +32 (0)9 264 81 46. E-mail: serge.vancalenbergh@ugent.be
Author Contributions
All authors have given approval to the final version of the manuscript.
Funding Sources
The authors gratefully acknowledge funding by the Research Fund Flanders (FWO).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors would like to thank Izet Karalic for technical assistance.
ABBREVIATIONS
AIDS, acquired immunodeficiency syndrome; DCM, dichloromethane; DMF, dimethylformamide; DXR, 1-deoxyxylulose 5-phosphate
reductoisomerase; EDC, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide; Et3N, triethylamine; HIV, human immunodeficiency virus; HOBt,
hydroxybenzotriazole; HRMS, high-resolution mass spectrometry; MDR, multidrug-resistant; MEP, methylerythritolphosphate; MIC, minimal
inhibitory concentration; MOA, mechanism of action; Mtb, Mycobacterium tuberculosis; MVA, mevalonate; PK, pharmacokinetic; POM,
pivaloyloxymethyl; TB, tuberculosis; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSBr, trimethylsilyl bromide; WHO, world health
organization
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TABLE OF CONTENTS GRAPHIC
Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.
Charlotte Courtens, Martijn Risseeuw, Guy Caljon, Louis Maes, Paul Cos, Anandi Martin and Serge Van Calenbergh