Synthesis and biological evaluation of aryl phosphoramidate prodrugs of fosfoxacin and its derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.103012

Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized and investigated for their ability to target bacteria. No growth inhibition was observed neither for Mycobacterium smegmatis nor for Escherichia coli on solid medium,

Full text of DOI:10.1016/j.bioorg.2019.103012

Accepted Manuscript
Synthesis and biological evaluation of aryl phosphoramidate prodrugs of fos-
foxacin and its derivatives
Mathilde Munier, Denis Tritsch, Didier Lièvremont, Michel Rohmer, Catherine
Grosdemange-Billiard
PII:
S0045-2068(18)31362-2
https://doi.org/10.1016/j.bioorg.2019.103012
103012
DOI:
Article Number:
Reference:
YBIOO 103012
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
21 November 2018
22 May 2019
23 May 2019
Please cite this article as: M. Munier, D. Tritsch, D. Lièvremont, M. Rohmer, C. Grosdemange-Billiard, Synthesis
and biological evaluation of aryl phosphoramidate prodrugs of fosfoxacin and its derivatives, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103012
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Synthesis and biological evaluation of aryl phosphoramidate prodrugs of
fosfoxacin and its derivatives
Mathilde Munier, Denis Tritsch, Didier Lièvremont, Michel Rohmer, Catherine Grosdemange-Billiard*
Laboratoire Chimie et Biochimie de Molécules Bioactives – Université de Strasbourg/CNRS, UMR
7
*
177, Institut Le Bel, 4 rue Blaise Pascal, 67081 Strasbourg, France
Corresponding author: Tel.: +33-368-851-349; e-mail: grosdemange@unistra.fr
Abstract: Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized
and investigated for their ability to target bacteria. No growth inhibition was observed neither for
Mycobacterium smegmatis nor for Escherichia coli on solid medium, demonstrating the absence of
release of the active compounds in the bacterial cells. Investigation of the stability of the prodrugs
and their multienzymatic cleavage in abiotic and biotic conditions showed that the use of aryl
phosphoramidate prodrug approach to deliver non-nucleotides compounds is not obvious and might
not be appropriate for an antimicrobial drug.
OBn
N
O
O
O
O
O
H
H
R
OH
N
N P O
O
P O
O
N
R
O
O
O
1
5a R= H
O
O
8a R= H
8b R= CH3
15b R= CH3
Keywords: phosphate prodrug, antimicrobials, MEP pathway, DXR, Mycobacterium
tuberculosis
Abbreviations: DXP: 1-deoxy-D-xylulose 5-phosphate; DXR: 1-deoxy-D-xylulose 5-phosphate
reductoisomerase; MEP: 2-C-methyl-D-erythritol 4-phosphate; CPY: carboxypeptidase Y;
GlpT: glycerol 3-phosphate transporter.
1

1
. Introduction
Mycobacterium tuberculosis is a rod-shaped bacterium belonging to the family
Mycobacteriaceae. Bacterial cells are surrounded by an unusual, waxy mycolic acid lipid
complex coating on its cell surface. M. tuberculosis is the causative agent of tuberculosis
(TB), which is one of the major infectious diseases in the world, affecting 10.4 million
patients and responsible for 1.4 million deaths in 2016, among which 95% of the deaths
1
were reported in developing countries. Standard treatment for TB has been available for
more than 60 years, but such treatment, a combination of antibiotics taken daily for six
2
months, is constraining. Moreover, its efficiency is threatened due to the increasing
emergence of multidrug-resistant M. tuberculosis strains throughout the world. This
emphasizes the urgency and necessity to develop innovative drugs towards new therapeutic
targets. Isoprenoid biosynthesis in Mycobacterium represents such a target in this context.3,4
Indeed, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal
precursors of all isoprenoids, are synthesized in most bacteria including many pathogens,
plant plastids, unicellular green algae and apicomplexan parasites via the 2-C-methyl-D-
erythritol 4-phosphate (MEP) pathway, which is absent in human (Fig.1).
2+
Mg
OH
OPP
HO
O
DXR
IPP
OP(O)(OH)2
OP(O)(OH)2
NADPH
OH
OH OH
1
-deoxy-D-xylulose 5-phosphate
2-C-methyl-D-erythritol 4-phosphate
OPP
DXP
MEP
DMAPP
Fig. 1: Biosynthesis of IPP and DMAPP via the MEP pathway: second step catalyzed by the
deoxyxylulose 5-phosphate reductoisomerase (DXR).
The deoxyxylulose 5-phosphate reductoisomerase (DXR), the second enzyme of this
pathway, is effectively inhibited by fosmidomycin and its analogue FR900098, two natural
phosphono-retrohydroxamic acids 1a-b as well as by two synthetic phosphono-hydroxamic
5
-9
acids 2a-b (Fig.2). Unfortunately, due to a lack of uptake, fosmidomycin and its analogues
are inefficient and do not inhibit the growth of mycobacteria. This result is not surprising as
mycobacteria, in contrast to many other bacteria, do not possess a transporter for such
2

1
0
molecules and are surrounded by a hydrophobic waxy cell wall preventing passive diffusion
into the cells, making the bacteria naturally resistant to most antibiotics.
O
O
P
OH
N
O
P
OH
N
R
R
HO
HO
R'
O
O
O
O
O
O
O
1a R = H fosmidomycin
3a R = H, R' = n-Pr
3b R = CH , R' = n-Pr
R'
1b R = CH FR900098
3
3
O
P
O
O
O
O
HO
R
P
R
N
R'
O
O
N
HO
O
OH
OH
O
2
2
a R = H
b R = CH3
O
4
a R = H, R' = n-Pr
4b R = CH , R' = n-Pr
R'
3
Fig. 2: Phosphonate DXR inhibitors and examples of their prodrug analogues.
To overcome the absence of mycobacterial cell wall crossing, and to increase oral
bioavailability of these compounds, the prodrug approach was adopted. Hence, the
synthesis of prodrugs of fosmidomycin 3a-b and of its derivatives 4a-b has been developed
in which the phosphonate negative charge is masked by hydrophobic groups such as
acyloxymethyl or alkoxycarbonyloxymethyl moieties (Fig.2).11-13 Once the prodrug is inside
the bacterial cells, the biolabile protecting group is enzymatically cleaved, releasing the
parent drug in the cell and stopping the bacterial growth by inhibition of the DXR.
Numerous cell metabolites are phosphorylated compounds, but are rarely exploited as
potential antibiotics due to their limited cell penetration and their sensitivity towards the
hydrolytic activity of phosphatases. However, in the early 1990’s, it was reported that
fosfoxacin, the natural phosphate analogue of fosmidomycin, exhibited potent antibacterial
activity against Gram-negative and Gram-positive bacteria.14-15 Latter on, fosfoxacin 5a and
its N-acetylated analogue 5b were shown to be more potent inhibitors than fosmidomycin
on the DXR from Synechocystis sp. PCC6803 (Fig.3). Recently, we reported the synthesis and
biological evaluation of phosphate isosters of fosmidomycin and its analogues 6a-b as
inhibitors of Escherichia coli and Mycobacterium smegmatis DXRs. We pointed out that the
3

efficiency of phosphate compounds compared to their phosphonate homologs might be
notably different towards DXRs isolated from different microorganisms.16
O
O
P
OH
N
H
O
OH
N
N
R
O
P
HO
HO
R
O
O
O
O
O
5
a R = H, fosfoxacin
7
a R= H
5
b R = CH3
7b R= CH3
O
O
H
O
O
O
O
N
P
R
O
P
OH
HO
HO
O
N
O
N
R
O
OH
6a R = H
8a R= H
6b R = CH3
8b R= CH3
O
Fig. 3: Phosphate DXR inhibitors and aryl phosphoramidate prodrug analogues.
Even if the phosphate compounds are less efficient as compared to the phosphonates on E.
coli growth inhibition, they enter into the bacteria via the GlpT/UhpT transporters. They
cannot, however, cross the bacterial wall of M. smegmatis as this transporter is absent in
Mycobacteria and passive diffusion of the negatively charged inhibitor is prevented by the
hydrophobic coating of the cells. To increase the bioavailability in E. coli and circumvent the
lack of uptake of these compounds in the mycobacteria, we designed prodrugs in which the
polar phosphate group is masked. They are less hydrophilic and thus more susceptible to
cross the bacterial envelope.
We chose the phosphoramidate prodrug approach to allow the penetration of fosfoxacin
analogues into Mycobacterium spp. (Fig. 3). This method also called “ProTide approach” was
introduced and developed by McGuigan. This widely used approach is based on the delivery
to the target cell of monophosphorylated form nucleoside analogues and has been largely
applied to improve the pharmacological properties of antiviral and anticancer agents.17-19 To
our knowledge, this phosphoramidate prodrug approach was never applied to bacteria but
4

was once reported to improve the antiparasitic activity of 5-phospho erythronohydroxamic
acid against Trypanosoma brucei.19
In this work, we investigated the opportunity to use aryl phosphoramidate prodrug of
fosfoxacin derivatives 7 and 8 to inhibit the growth of M. smegmatis, which was used as a
mycobacterium model, and E. coli (Fig. 3). In a first attempt, we selected chemically stable
aryl phosphoramidate consisting of an L-alanine methyl ester and a 4-methoxyphenyl
moieties. After crossing the cell wall, prodrugs will undergo enzymatic conversions to release
the parent drug. The initial step requires the action of a bacterial carboxyl esterase (Scheme
1
), which hydrolyses the carboxyl ester bond of compounds 8a and 8b to form the free
carboxylate acid. This is followed by a putative nucleophilic attack of the phosphorus by the
oxygen of the carboxylate group, resulting in the elimination of the aryloxy group to form a
5
-membered cyclic intermediate B. A spontaneous hydrolysis of the cyclic phosphoramidate
gives the phosphoramidate compound C (Scheme 1). The last step involves a hydrolysis of
the P-N bond mediated by a phosphoramidase-type enzyme to release the inhibitor i.e. the
fosfoxacin analogues 6a or 6b able to inhibit the DXR and consequently the bacterial growth
(Scheme 1).
OH
N
OH
N
OH
N
O
O
O
O
O
O
R
R
R
Intramolecular
cyclization
O
O
O
O
Esterase
O-
O
O
P
HN
P
P
O
O
HN
O
spontaneous
O
HN
O
O
8
a R = H
A
B
8b R = CH3
OH
OH
O
N
O
N
R
R
H O
Phosphoramidase
2
O
O
O
O
O
P
OH
spontaneous
P
HO
HO
HN
OH
C
6a R = H
b R= CH3
6
Scheme 1: Potential bioactivation mechanism of phosphoramidate prodrugs 8a and 8b.
2
. Results
.1. Synthesis of prodrugs
2
5

The syntheses of 7 and 8 are outlined in Scheme 3. The key step to synthesize aryl
phosphoramidate prodrugs 7 and 8 is the coupling of the phosphochloridate 13 with the
protected hydroxamates 12 or 14. The synthesis of the O-benzyl hydroxamates 12a and 12b
were performed as outlined in Scheme 2.After a monoprotection of commercially available
ethyleneglycol into the silyl ether 9, the free alcohol was activated into the triflate with
trifluoromethanesulfonic anhydride, followed by substitution with commercially O-benzyl
hydroxylamine to give the protected hydroxylamine 10. Formylation with the mixed
acetyl/formyl anhydride generated in situ from a formic acid and acetic anhydride mixture
lead to the N-formylated compound 11a without purification as a mixture of two Z and E
diastereomers in a 40:60 ratio. Acetylation with a mixture of acetic anhydride and pyridine
gave the N-acetylated analog 11b as the sole E isomer without purification. After
deprotection of the silyl ether, the O-benzyl hydroxamates 12a and 12b were obtained after
purification as a mixture of two Z and E diastereoisomers in a 30:70 ratio for 12a and as the
sole E isomer for 12b. The O-benzyl hydroxamate 14 was synthesized according to previously
described methods.16
H
(a)
(b)
OH
OH
N
HO
TBDMSO
TBDMSO
OBn
9
10
OBn
OBn
N
(c)
(d)
N
R
R
TBDMSO
HO
O
O
1
1a R = H
12a R = H
12b R = CH3
11b R = CH3
Scheme 2: Synthesis of the O-benzyl hydroxamate 12a and 12b. Reagents and conditions. (a) NaH,
TBDMSCl, THF, 0 °C to rt, 85 %; (b) (i) Tf O, 2,6-lutidine, DCM, -78 °C; (ii) H NOBn, DCM, -78 °C to rt, 65 %; (c)
HCOOH/Ac O, THF, rt, 11a; Ac O, pyridine, rt, 11b; (d) TBAF.3(H O), THF, rt, 12a (82 %), 12b (93 %).
2
2
2
2
2
The phosphoroamidochloridate 13 was prepared from 4-methoxyphenol, phosphorus
oxychloride and the L-alanine methyl ester hydrochloride according to a previously
2
0
described method. The phosphoroamidochloridate was then coupled with the O-benzyl
hydroxamate 12 following conditions previously developed using N-methylimidazole in DCM
at -78 °C. 21,22 In these conditions the desired compounds 15a-b were obtained as a mixture
6

of two diastereomers ( = 3.05 and 3.30 ppm for 15a and  = 3.05 and 3.30 ppm for 15b) in
P
P
a 60:40 ratio due to the chirality of the phosphorus atom and contaminated with 50%
byproduct in the isolated compounds ( = 8.10 ppm). The byproduct might be a compound
P
originating from an addition/elimination reaction of two L-alanine methyl ester molecules on
the phosphorodichloridate, taking place throughout the synthesis of the
phosphoroamidochloridate 13. Even after several purification attempts, it was not possible
to obtain the pure compounds 15a-b and therefore the aryl phosphoramidate prodrugs 7.
No significant yield improvement of coupling reaction of compounds 16 with
phosphoroamidochloridate 13 in different basic conditions was obtained. In that case,
purification of the aryl phosphoramidate prodrugs 16 from the byproduct was achieved
followed by the removal of the protective benzyl groups of 16a and 16b by catalytic
hydrogenolysis with palladium over charcoal at atmospheric pressure and room temperature
in methanol.
7

O
O
H
N P Cl
O
O
O
13
OBn
N
O
R
R
1
2a R = H
HO
O
N
14a
14b
HO
R = H
R = CH3
(
a)
(b)
or (c)
12b R = CH3
OH
O
OBn
N
O
O
O
O
O
H
R
H
OBn
N P O
O
N P O
O
N
R
O
O
O
O
1
1
5a R = H
5b R = CH3
16a R = H
16b R = CH3
(d)
OH
O
O
O
O
O
O
O
H
N
R
H
OH
N P O
O
N P O
O
N
R
O
O
O
7a R = H
8a R = H
7b R = CH3
8b R = CH3
Scheme 3: Synthesis of the aryl phosphoramidate prodrugs 7 and 8. Reagents and conditions: (a) N-
methylimidazole, DCM, -78 °C, 15a (40 %), 15b (41 %); (b) pyridine, 14a, DCM, -40 °C, 16a (47 %); (c)
triethylamine, DMAP, 14b, DCM, -40 °C, 16b (48 %), (d) H , Pd/C, MeOH, rt, 8a (77 %), 8b (92 %).
2
2
.2. Biological properties
.2.1. Growth inhibition of E. coli and M. smegmatis with compound 15a-b, 16a-b and 8a-b
2
Growth inhibition of E. coli and M. smegmatis induced by the synthesized aryl
phosphoramidate prodrugs 8a-b as well as by the O-benzyl-protected intermediates 15a-b
and 16a-b was evaluated by the paper disc diffusion method. Isoniazid and fosmidomycin
were respectively used as reference compounds for positive M. smegmatis and E. coli
growth inhibition tests. None of the aryl phosphoramidate prodrugs 15a-b, 16a-b and 8a-b
8

blocked the growth of both M. smegmatis and E. coli at the highest tested dose (400
nmol/disk). The absence of growth inhibition lead to consider several hypotheses: i) no
membrane crossing of the aryl phosphoramidate prodrugs, ii) membrane crossing of aryl
phosphoramidate prodrugs followed by expulsion owing to efflux pumps, iii) no endogenous
enzymes able to release the phosphate drugs, iv) short life time of the prodrugs in the
culture medium.
2
.2.2. Stability assays of the prodrugs 16a-b and 8a-b in the buffer used for carboxyl esterase
enzymatic tests
Chemical stability of the prodrugs was evaluated by exposing them to aqueous 0.05 M
triethanolamine (TEOA) buffer (pH 7.5) at 25°C and monitored by 31P NMR, a sensitive
method to highlight possible modification(s) of the phosphorylated compounds (Supporting
information, Fig. S1). Except for the prodrug 8b, all the aryl phosphoramidate prodrugs are
3
1
stable in TEOA over the tested reaction time (48 h). Indeed, the P NMR spectra of these
compounds showed two signals (8a:   5.15 and 5.05 ppm; 16a:   4.85 and 4.70 ppm;
1
6b:   4.85 and 4.75 ppm) as the result of the presence of a mixture of diastereomers in
solution due to the chirality of the phosphorus atom. Surprisingly, the N-methylated prodrug
3
1
8
b is unstable in the TEOA buffer. The P NMR spectrum showed the absence of the two
characteristic signals (  4.85 and 4.75 ppm) of the diastereomers but the presence of two
new signals at   3.05 ppm and 4.25 ppm in a 85/15 ratio respectively, corresponding to
metabolites with an achiral phosphorus.
2
.2.3. Esterase activity on the aryl phosphoramidate prodrugs 16a-b and 8a-b with CPY
As shown in the scheme 1, the first step of the liberation of the parent drug in the cell
consists of an enzymatic hydrolysis of the aminoacyl moiety releasing the free carboxylate
acid C and involving a carboxypeptidase-type enzyme. To test the activation of the aryl
phosphoramidate prodrugs into the intermediate C, we performed an enzymatic hydrolysis
using carboxypeptidase-type enzyme and following the modifications by 31P NMR. To
determine whether the ester hydrolysis of the phosphoramidate prodrugs occurs or not, the
compounds 16a-b and 8a-b were incubated with carboxypeptidase Y (CPY), and their
3
1
possible modifications were monitored by P NMR (Supplementary information, Fig. S2-S5).
The assays were carried out by dissolving the compound in a H O/D O aqueous 0.05 M TEOA
2
2
9

buffer (pH 7.5), followed by incubation with the CPY enzyme. A blank is recorded for each
sample before addition of the CPY in D O. Upon the addition of CPY to the phosphoramidate
2
prodrug 8a, a fast hydrolysis occurred as two signals appeared at  = 5.70 and 5.50 ppm.
These signals correspond to the hydrolysed intermediate B present as two diastereomers,
which were partially converted after 2 h into the aminoacyl phosphoramidate intermediate
C according to McGuigan et al. (Scheme 1).23 A single signal at  = 7.45 ppm was finally
3
1
observed due to the achirality at the phosphorus atom. After 3 h, the P NMR spectra
displayed the increase of a signal at  = - 4.45 ppm concomitant with the decrease of the
signal at  = 7.45 ppm. This enzymatic assay showed a fast and complete conversion of the
prodrug 8a into the intermediate C. The half-life of prodrug 8a was 20 h (Supplementary
information, Fig. S2).
With the O-benzyl-protected precursor 16a, addition of CPY led to similar results as
previously described. The experiment showed a fast hydrolysis into the O-benzylated
aminoacyl C since the initial compound disappeared after 2 h and was replaced by a
3
1
compound with a P shift at 7.40 ppm (Supplementary information, Fig. S3). Nevertheless,
in contrast with previous results, longer incubation times showed a slow conversion of
3
1
intermediate C (half-life = 2 days) into two compounds with P shifts at -5.00 ppm and -7.50
ppm. The experiments with O-benzyl-N-methylated prodrugs 16b, showed a fast and total
hydrolysis after 2 h as the initial compound signals disappeared followed by the formation of
the stable intermediate C ( = 7.35 ppm). Longer incubation times did not lead to
3
1
modifications of the P NMR spectra. (Supplementary information, Fig. S4)
The N-methylated prodrug 8b was not stable in the buffer as two metabolites ( = 3.05 and
4
.25 ppm) are present. However, the incubation with the CPY rapidly showed an increase of
the signal at 4.25 ppm with the concomitant decrease of the signal at 3.05 ppm. After 24 h of
incubation the ratio was 80/20 in favour of the metabolite with a chemical shift at 4.25 ppm.
(Supplementary information, Fig. S5)
2
.2.4. Stability assays of the prodrugs 8a and 8b during M. smegmatis and E. coli cultures
To check the stability of the prodrugs in M. smegmatis and E. coli cultures, the bacterial cells
were removed from the culture medium by centrifugation at the end of the culture (24 h for
E. coli and 40 h for M. smegmatis). The compounds were incubated with a mixture of
1
0

resulting bacteria-free supernatants and D O in a 2/1 ratio and monitored over a 20 h time
2
3
1
period by P NMR (Supplementary information, Fig. S6-7).
The prodrug 8a is stable in both growth media as the two signals of the diastereoisomers at
3
1

= 5.15 and 5.10 ppm are still present in the P NMR spectrum after 20 h (Supplementary
information, Fig. S6), indicating that the bacteria do not excrete enzymes/metabolites
capable of degrading the prodrugs. The spectra of the N-methylated 8b in E. coli growth
medium displayed a single signal at 3.05 ppm over the experiment time period (20h), while
the spectra of 8b in M. smegmatis growth medium showed the presence of two signals at 
=
3.05 and 5.20 ppm in a 3/1 ratio respectively. The latter signal decreased after 20 h in
favour of the one at  = 3.05 ppm. The presence of this signal also observed in the TEOA
P
buffer medium suggests that 8b might not be stable in these conditions but is transformed
into a stable compound with a phosphorus signature at 3.05 ppm (Supplementary
information, Fig. S7).
2
.2.5. Esterase, phosphatase and phosphoramidase activities in bacterial crude cell-free
systems
Before investigating the bioactivation of the prodrugs in bacterial crude cell-free systems
containing the endogenous bacterial enzymes, it was essential to test the potential esterase
and phosphatase activities in both E. coli and M. smegmatis crude cell-free systems obtained
by sonication of the bacterial cells. Spectrophotometric enzymatic assays for esterase
activity were performed using p-nitrophenyl butyrate (NPB) as substrate.24 An intense
esterase activity was detected in the M. smegmatis crude cell-free system as shown by the
-1
-1
release of p-nitrophenol (404 nanomoles.min .mg protein) from NPB. With a crude E. coli
-1
-1
cell-free system, the esterase activity was lower, as only a 6.9 nanomoles.min .mg protein
release rate of p-nitrophenol was measured.
However, the phosphatase activity measured with p-nitrophenyl phosphate (NPP) as the
2
+
2+
+2
substrate, in the presence or absence of metal cations (Mn , Mg and Co ) was very low
-1
-1
-1
with a release rate of p-nitrophenol of 2.1 nanomoles .min .mg protein with the M.
-1
-1
smegmatis crude cell-free system. The maximum activity (4.3 nanomoles.min .mg protein)
was observed when CoCl was added to the cell-free system. Concerning the cell-free system
2
-1
-1
of E.coli, a phosphatase activity releasing p-nitrophenol at a 10 nanomoles.min .mg
1
1

protein rate was determined. These low phosphatase activities are not able to cleave the
phosphate of the fosfoxacin analogues 6a-b (Scheme 1)
When the prodrug 8a was incubated with the crude preparation of M. smegmatis, the same
modifications on the 31P NMR spectra were observed as those mediated with the CPY
enzyme (Supplementary information, Fig. S8). Additionally, the prodrug 8a was stable in a
crude cell-free system from E. coli as only the two signals corresponding to the two
diastereomers were still observed after 20 h (Supplementary information, Fig. S9). The
parent drug 6a was not formed, as no corresponding phosphorus signal was observed. Thus,
no phosphoramidase activity occurred within the crude cell-free systems of either M.
smegmatis or E. coli.
The N-methylated prodrug 8b with the crude cell-free system of M. smegmatis showed a
signal at 3.05 ppm complemented with four minor signals ( = 7.50, 5.20, 1.30 and -0.75
ppm). Thereafter, shifts at 7.50 and 5.20 ppm disappeared while the signal at -0.75 ppm
increased. Nevertheless, the ratio of both signals at 3.05 and -0.75 ppm remained constant,
even after a long incubation time (42 h) (Supplementary information, Fig. S10). In the crude
cell free system of E. coli, prodrug 8b showed three signals ( = 3.05, 5.20 and 1.30 ppm).
Initially, the 3.05 and 5.20 ppm signals showed a 55/45 ratio. Thereafter, the shift at 5.20
ppm disappeared after 20h (Supplementary information, Fig. S11).
3
. Discussion
Recently we reported the synthesis of fosmidomycin phosphate analogues, which showed to
be efficient inhibitors of the DXR. Unfortunately, the phosphate group being deprotonated
and negatively charged at physiological pH, these compounds do not readily enter into cells
in the absence of a transporter. To circumvent this penetration problem encountered with
such hydrophilic DXR inhibitors, they have to be converted into neutral lipophilic prodrugs.
Aryl phosphoramidate prodrugs represent such interesting candidates as this approach has
been widely used to deliver monophosphate nucleotide derivatives into cells and to improve
the pharmacological properties of antiviral and anticancer agents. However, this type of
prodrugs has never been developed for antibacterial purposes. The synthesized aryl
phosphoramidate prodrug 8 as well as the O-benzyl-protected intermediates 15a-b and 16a-
b do not inhibit the growth of M. smegmatis and E. coli on solid medium, even at the highest
tested dose (400 nmol/disk). Several hypotheses might be tested to explain this lack of
1
2

activity: i) either the prodrugs are not chemically stable in the incubation medium, ii) or they
do not cross the bacterial cell wall and/or the cytoplasmic membrane, iii) or the bacteria lack
the endogenous enzymes required to release the active free phosphate DXR inhibitors.
The correlation between the chemical stability of aryl phosphoramidate prodrugs in a buffer
and the biological activity has been notably emphasized for the in vitro trypanocidal activity
of the aryl phosphoramidate prodrugs of 2,3-O-isopropylidene-4-erythronohydroxamate. In
the buffer TEOA, pH 7.5, prodrug 8a as well as the O-benzyl-protected intermediates 15a-b
and 16a-b are stable for 24 h whereas surprisingly, the N-methylated prodrug 8b is rapidly
degraded into a compound with an apparently achiral phosphorus ( = 3.05 ppm). The
P
explanation for this discrepancy is not obvious as the stabilities are expected to be rather
similar. The difference of the biological activities between the N-methylated hydroxamic acid
and its non-methylated congener, is often attributed to the existence of different
conformations in solution. The ratio of the conformers depends upon experimental
conditions (solvent, concentration and temperature) but cannot explain the instability of 8b
2
5
in the buffer. . A hydroxamate can be considered as a potentially bidentate nucleophile,
using either the basic N atom or the O atom in a nucleophilic reaction with a variety of
electrophilic centers. They are known to be -effect nucleophiles and as such are effective
deacetylating and dephosphorylating agents. This hydrolyzing nucleophile activity is due to
-
the anionic N-O form, which cannot occur with the N-methyl O-benzyl intermediate 15b. As
in water the hydroxamic acids are present as the neutral OH rather than NH form, the pKa
-
for conversion of the N-OH into the negatively charged N-O form plays an important role for
cleavage of phosphate esters as well as the substitution on the N atom. In fact, due to lone-
electron pair repulsion, an N-methylated hydroxamic acid is more reactive in the anionic
form than the NH. Therefore, the degradation of the aryl phosphoramidate prodrug 8b
might be due to a fast dephosphorylation in the buffer conditions.26-28
The mechanism of bioactivation of the aryl phosphoramidate prodrugs involves a first
enzymatic step mediated by a carboxypeptidase Y. The esterase activity occurs with prodrug
8
a and the O-benzyl-protected intermediates 16a-b as they are hydrolyzed in the aminoacyl
phosphoramidate intermediate C, which appears as a single signal at ca.  =7.45 ppm in the
31P NMR spectra.
However, the aminoacyl intermediate of the N-H prodrug 8a and the NH-O-benzyl
intermediate 16a decomposes rapidly leading to new signals, and the disappearance of the
1
3

signal for the phosphate prodrugs is observed during the experiment time course. The
decomposition is probably due to the presence of the NH “acidic proton” as the aminoacyl
phosphoramidate intermediate C corresponding to the N-methyl O-benzyl intermediate 16b
is stable over the time course. In the case of the N-methyl prodrug 8b, the experiment
showed an activation of the starting material with the carboxypeptidase Y into a
phosphorylated compound. This result might suggest that the degraded compound ( =3.05
P
ppm) still contains the L-alanine methyl ester in its structural backbone. These experiments
indicated that the first activation step of prodrugs 8 may occur in the bacteria as long as the
compound enters into the cell and is hydrolyzed by an efficient intracellular
carboxypeptidase Y type enzyme.
In both growth media and as well as in E. coli cell-free system, prodrug 8a was stable.
However, in the M. smegmatis cell-free system, compound 8a was rapidly metabolized into
3
1
compounds having the same P NMR spectra profiles than those obtained with the CPY.
These results suggest that E. coli does not have the appropriate intracellular and
extracellular esterase in contrast with M. smegmatis. With the N-methylated prodrug 8b,
the results obtained are quite puzzling. In the growth media of E. coli and of M. smegmatis
and the cell-free system of E. coli, the degraded form ( = 3.05 ppm) was observed as the
P
sole or major compound indicating that no esterase activity is present in these conditions.
With a M. smegmatis cell-free system, the degraded form ( = 3.05 ppm) was still present
P
accompanied by minor metabolites, which are converted into a single metabolite ( = -0.75
P
ppm). It is thus quite difficult to conclude on the presence of an esterase activity in M.
smegmatis.
In the ProTides approach developed in eukaryotic cells, the enzymatic hydrolysis of the P-N
bound requires an enzyme with phosphoramidase activity and it has been proposed that it
2
9
belongs to the family of histidine triad nucleotide–binding proteins (Hint). In bacterial cells,
Hint homologs have been annotated from sequenced genomes, but the enzymes have not
been characterized. In E. coli a Hint homolog with a purine nucleoside phosphoramidase
activity has been found, and hint mutants were unable to grow.30 In our case, no
phosphoramidase activity has been detected with the cell-free system of both bacteria.
Either the bacteria do not have the endogenous phosphoramidase activity or the
intracellular phosphoramidase activity is substrate specific as it seems to be in E. coli.31
–
1
4

4
. Conclusion
The ProTides approach has largely been used to develop nucleoside monophosphate and
monophosphonates prodrugs. This technique has proved to be efficient to deliver nucleotide
therapeutics to treat viral infections and cancer. Even if a trypanocidal activity against the
protozoan Trypanosoma brucei using non nucleotide aryl phosphoramidate prodrugs has
been reported in the literature without decrypting action mechanisms of action has not be
proven, no study mentioned the use of such prodrugs to target bacteria.20 Our results
suggest that the use of aryl phosphoramidate prodrug approach to deliver non-nucleotide
compounds is not obvious and might not be the appropriate strategy for the design of an
antimicrobial drug. We showed that two main factors must be considered: i) the stability of
the aryl phosphoramidate prodrug in the buffer and the cell free system presumably linked
to the hydroxamate particular structural features, which can promote hydrolysis or/and side
reactions; ii) the presence of the release mechanism key enzymes activities i.e. CPY type
esterase and phosphoramidase in the target microorganisms.
5
. Experimental
5
5
.1. Chemistry
.1.1. General procedures
All non-aqueous reactions were run in dry solvents under an argon atmosphere. Commercial
grade reagents were purchased from Sigma-Aldrich or Acros Organics and used without
further purification. Petroleum ether 40-60 °C (Sigma-Aldrich) was used for purification.
Flash chromatography was performed on silica gel 60 230–400 mesh with the solvent system
as indicated. Automated flash chromatography was performed on a Combiflash® RfTM
(Serlabo Technologies) or on a Puriflash® 215 (Interchim).
TLC plates were revealed under UV light (254 nm) and/or by spraying with an ethanolic
solution of phosphomolybdic acid (20%) or an ethanolic solution of potassium permanganate
followed by heating.
1
13
The NMR spectra were recorded on a BRUKER Avance 300 ( H-NMR: 300 MHz; C-NMR,
3
1
19
1
7
5.5 MHz; P-NMR 121.5 MHz; F-NMR 282.4 MHz) or a BRUKER Avance 400 ( H-NMR: 400
1
3
31
1
MHz; C-NMR, 100.6 MHz; P-NMR 162 MHz) or a BRUKER Avance 500 ( H-NMR: 500 MHz;
1
3
1
C-NMR, 125.8 MHz). H-NMR experiments were performed in CDCl , D O, CD OD in CDCl
3
2
3
3
1
5

with CHCl ( = 7.26 ppm), DHO ( = 4.79 ppm), CD HOD ( = 3.31 ppm) as internal
3
2
1
3
references. C-NMR experiments were performed in CDCl with CDCl ( = 77.23 ppm),
3
3
3
1
CD HOD ( = 49.0 ppm) as internal references. For P-NMR reference, the spectrometer had
2
an external reference, corresponding to 80 % phosphoric acid in D O ( = 0 ppm). The
2
chemical shifts () are expressed in ppm. s, d, t, q, or bs are abbreviations for signal
multiplicity correspond to singlet, doublet, triplet and quadruplet or broad singlet. J-coupling
constants are expressed in Hz.
Most of the hydroxamate are present as two Z and E conformers in equilibrium. If only one
signal is described, it is common to both conformers. The evaluation of the relative amount
of the conformers was made by integration of the CH CO or CH N or OCH DMB proton
2
2
2
signals.
Negative or positive-mode electrospray MS were performed on a Bruker Daltonics microTOF
spectrometer (Bruker Daltonik GmgH, Bremen, Germany) equipped with an orthogonal
electrospray (ESI) interface. Calibration was performed using a 10 mM solution of sodium
formate. Sample solutions were introduced into the spectrometer source with a syringe
pump (Harvard type 55 1111: Harvard Apparatus Inc., South Natick, MA, USA) with a flow
−1
rate of 5 μL min .
5
.1.2. 2-((t-Butyldimethylsilyl)oxy)ethan-1-ol (9)
Sodium hydride (475 mg, 18.2 mmol) was suspended in THF (36 mL). The resulting mixture
was cooled to 0°C, the diol (1 mL, 17.7 mmol) was added dropwise and the mixture was
stirred for 1 h. A solution of t-butyldimethylsilyl chloride (2.74 g, 18.0 mmol) in THF (8 mL)
was added over a period of 10 min. The resulting mixture was stirred for 4 h at room
temperature. A saturated aqueous solution of NaHCO (40 mL) was added and the mixture
3
was extracted with EtOAc (2 x 40 mL). The aqueous layer was saturated with NaCl and
extracted with EtOAc (2 x 40 mL). The collected organic layers were dried over anhydrous
Na SO , filtered. The solvents were removed under reduced pressure. The crude was
2
4
purified by flash chromatography (EtOAc/petroleum ether, 2:8) to give the compound 9 as a
1
colorless oil (2.48 g, 80 %). Rf = 0.25 (EtOAc/petroleum ether, 1:9). H NMR (300 MHz, CDCl ):
3
δ 0.08 (6H, s, Si-CH ), 0.91 (9H, s, tBu), 1.99 (1H, bs, OH), 3.62-3.65 (2H, m, CH OTBDMS),
3
2
1
6

1
3
3
.70-3.73 (2H, m, CH OH); C NMR (75.5 MHZ, CDCl ): δ -5.1 (Si-CH ), 18.5 (C of t-Bu), 26.1
2
3
C
3
4°
(
CH of tBu), 63.9 (CH OTBDMS), 64.3 (CH OH).
3
2
2
5
.1.3. O-Benzyl-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)hydroxylamine (10)
To a solution of 9 (424 mg, 2.40 mmol) in DCM (76 mL) was added 2,6-lutidine (0.45 mL, 3.90
mmol). The solution was cooled down at -78 °C, and trifluoromethane sulfonic anhydride
(450 μL, 2.70 mmol) was added dropwise. The resulting mixture was stirred at -78 °C for 1 h,
and O-benzylhydroxylamine (625 mg, 5.07 mmol) in DCM (6 mL) was added dropwise. The
solution was stirred at -78 °C for 1 h, warmed up to room temperature and stirred for
another 2 h. The reaction mixture was diluted with DCM (30 mL) and washed with a
saturated aqueous solution of NH Cl (100 mL), a saturated solution of NaHCO (100 mL),
4
3
water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na SO ,
2
4
filtered and the solvents were removed under reduced pressure to give a pale yellow oil. The
crude was purified by flash chromatography (petroleum ether  EtOAc/petroleum ether,
5
:95) to give the product 10 as a colorless oil (438 mg, 65 %). Rf = 0.5 (EtOAc/petroleum
1
3
ether, 1:9). H NMR (500 MHz, CDCl ): δ 0.04 (6H, s, Si-CH ), 0.88 (9H, s, tBu), 3.02 (2H, t, J =
3
3
3
5
.2 Hz, CH N), 3.74 (2H, t, J = 5.1 Hz, CH OTBDMS), 4.72 (2H, s, OCH Ph), 7.29-7.37 (5H, m,
2
2
2
1
3
CH ); C NMR (125.8 MHZ, CDCl ): δ -5.2 (Si-CH ), 18.5 (C of tBu), 26.1 (CH of tBu), 54.1
Ar
3
C
3
4°
3
(
CH N), 59.5 (CH OTBDMS), 75.9 (OCH Ph), 127.9 (CH ), 128.5 (CH ), 128.6 (CH ), 138.2
2
2
2
Ar
Ar
Ar
+
+
(
C ); MS (EI) : m/z calculated for C H NO Si [M+H] 282.19, found 282.19.
Ar
15 28
2
5
.1.4. N-(Benzyloxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)formamide (11a)
A mixture of formic acid (2.75 mL, 73 mmol) and acetic anhydride (1.38 mL, 14.6 mmol) was
stirred at room temperature during 30 min. The reagent was cooled to 0 °C and a solution of
protected hydroxylamine 10 (410 mg, 1.46 mmol) in THF wad added dropwise. The reaction
mixture was stirred at 0 °C during 10 min, allowed to warm up at room temperature and
stirred overnight. The reaction was diluted with EtOAc and the organic layer was washed
twice with water and twice with a 0.1 M aqueous solution of KOH. The organic layer was
dried over Na SO , filtered and evaporated to dryness. The product 11a was obtained
2
4
without purification as a colorless oil (390 mg) and as a mixture of two Z and E conformers in
1
7

1
a 40:60 ratio. Rf = 0.61 (EtOAc/petroleum ether, 1:9). H NMR (500 MHz, CDCl ): δ 0.05 (6H,
3
s, Si-CH ), 0.88 (9H, s, tBu), 3.30 (4/10 of 2H, bs, CH N), 3.69 (6/10 of 2H, bs, CH N), 3.79
3
2
2
(
4/10 of 2H, bs, CH OTBDMS), 4.33 (6/10 of 2H, bs, CH OTBDMS), 4.89 (6/10 of 2H, bs,
2
2
OCH Ph), 4.98 (4/10 of 2H, bs, OCH Ph), 7.37 (5H, bs, CH ), 7.95 (4/10 of 1H, bs, CHO), 8.22
2
2
Ar
1
3
(
6/10 of 1H, bs, CHO); C NMR (125.8 MHZ, CDCl ): δ -5.2 (Si-CH ), 18.4 (C of tBu), 26.0
3
C
3
4°
(
CH of tBu), 48.1 (CH N), 52.1 (CH N), 58.6 (CH OTBDMS), 59.4 (CH OTBDMS), 76.7
3
2
2
2
2
(
OCH Ph), 78.1 (OCH Ph), 128.7 (CH ), 128.9 (CH ), 129.2 (CH ), 129.6 (CH ), 129.7 (CH ),
2
2
Ar
Ar
Ar
Ar
Ar
+
1
34.6 (C ), 138.5 (C ), 159.5 (CHO), 163.9 (CHO); MS (EI) : m/z calculated for C H NO SiNa
Ar
Ar
16 27
3
+
[
M+Na] 332.17, found 332.16.
5
.1.5. N-(Benzyloxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acetamide (11b)
To a solution of protected hydroxylamine 10 (298 mg, 1.06 mmol) in acetic anhydride (4.3
mL) was added dropwise pyridine (0.26 mL, 3.18 mmol). The reaction mixture was stirred
overnight at room temperature and the solvents were evaporated to dryness under reduced
pressure. The product 11b was obtained without purification as a colorless oil (311 mg) and
1
as the sole E conformer. Rf = 0.59 (EtOAc/petroleum ether 1:9). H NMR (400 MHz, CDCl ): δ
3
0
.06 (6H, s, Si-CH ), 0.89 (9H, s, tBu), 2.09 (3H, s, COCH ), 3.75-3.79 (4H, m, OCH CH N), 4.88
3
3
2
2
1
3
(
2H, s, OCH Ph), 7.38 (5H, s, CH ); C NMR (125.8 MHZ, CDCl ): δ -5.2 (Si-CH ), 18.5 (C of
2
Ar
3
C
3
4°
tBu), 20.7 (COCH ), 26.1 (CH of tBu), 49.7 (CH N), 59.8 (CH OTBDMS), 76.7 (OCH Ph), 78.1
3
3
2
2
2
+
(
OCH Ph), 128.9 (CH ), 129.1 (CH ), 129.4 (CH ), 134.8 (C ), 173.1 (COCH ); MS (EI) : m/z
2
Ar
Ar
Ar
Ar
3
+
calculated for C H NO Si [M+H] 324.20, found 324.20.
1
7
30
3
5
.1.6. N-(Benzyloxy)-N-(2-hydroxyethyl)formamide (12a)
The silyl ether 11a (376 mg, 1.21 mmol) in THF (18 mL) was treated with tetra-n-
butylammonium fluoride (633 mg, 2.42 mmol). The reaction was monitored by TLC (EtOAc)
and the solvent was removed under reduced pressure. The crude product was purified by
flash chromatography (EtOAc) to give 12a as a colorless oil (224 mg, 82 %) and as a mixture
1
of two Z and E conformers in a 30:70. Rf = 0.51 (EtOAc). H NMR (400 MHz, CDCl ): δ 3.70-
3
3
8
.80 (4H, m, OCH CH N), 4.90 (2H, bs, OCH Ph), 7.38 (5H, s, CH ), 7.99 (3/10 of 2H, bs, NH),
2
2
2
Ar
1
3
.25 (7/10 of 2H, bs, NH); C NMR (125.8 MHZ, CDCl ): δ 48.8 (CH N), 53.7 (CH N), 58.3
3
C
2
2
(
CH OH), 60.1 (CH OH), 76.6 (OCH Ph), 78.3 (OCH Ph), 128.7 (CH ), 129.0 (CH ), 129.5
2
2
2
2
Ar
Ar
1
8

+
(
CH ), 128.8 (CH ), 134.2 (C ), 159.7 (CHO), 164.5 (CHO); MS (EI) : m/z calculated for
Ar
Ar
Ar
+
C H NO K [M+K] 234.05, found 234.05.
1
0
13
3
5
.1.7. N-(Benzyloxy)-N-(2-hydroxyethyl)acetamide (12b)
The silyl ether 11b (311 mg, 0.81 mmol) in THF (12 mL) was treated with tetra-N-
butylammonium fluoride (424 mg, 1.62 mmol). The reaction was monitored by TLC (EtOAc)
and the solvent was removed under reduced pressure. The crude product was purified by
flash chromatography (EtOAc) to give 12b as a colorless oil (147 mg, 93 %) and as the sole E
1
conformer. Rf = 0.25 (EtOAc/petroleum ether, 7:3). H NMR (500 MHz, CDCl ): δ 2.13 (3H, s,
3
1
3
COCH ), 3.81 (4H, s, OCH CH N), 4.87 (2H, s, OCH Ph), 7.38 (5H, s, CH ); C NMR (125.8
3
2
2
2
Ar
MHZ, CDCl ): δ 20.6 (COCH ), 50.8 (CH N), 61.3 (CH OH), 77.1 (OCH Ph), 129.0 (CH ), 129.2
3
C
3
2
2
2
Ar
+
(
CH ), 129.5 (CH ), 134.3 (C ), 174.3 (COCH ); MS (EI) : m/z calculated for C H NO
Ar
Ar
Ar
3
11 16
3
+
[
M+H] 210.11, found 210.11.
5
.1.8. (4-Methoxy)phenyl (methoxy-L-alaninyl)phosphorochloridate (13)
To a solution of phosphorus oxychloride (0.75 mL, 8 mmol) and 4-methoxyphenol (1.00 g, 8
mmol) in dry diethyl ether (30 mL) was added at -78 °C triethylamine (1.12 mL, 8 mmol). The
resulting mixture was warmed up to room temperature and stirred overnight. The reaction
3
1
was monitored by P NMR. The tetraethylammonium chloride was filtered under argon and
the filtrate was concentrated under reduced pressure. The resulting yellow oil was used for
the next step without further purification. 31P NMR (162 MHz, CDCl ): δ 4.45. To (4-
3
P
methoxy)phenyl phosphorodichloridate was dissolved in dry DCM (31 mL) was added L-
alanine methyl ester hydrochloride (1.07 g, 7.65 mmol). The suspension was cooled to -78 °C
and triethylamine (2.4 mL, 16.9 mmol) was added dropwise. The mixture was stirred at -78
°
C for 15 min and stirred overnight at room temperature. The reaction mixture was
evaporated to dryness under reduced pressure, and the residue was poured into Et O (30
2
mL). The tetraethylammonium chloride was filtered off. The filtrate was evaporated to
dryness under reduced pressure to give a yellow oil 13, which was used for the next step
3
1
without further purification. P NMR (162 MHz, CDCl ): δ 8.25, 8.60.
3
P
1
9

5
.1.9. Methyl((2-(N-(benzyloxy)formamido)ethoxy)(4-methoxyphenoxy)phosphoryl)-L-
alaninate (15a)
To a solution of 12a (222 mg, 1.14 mmol), the phosphochloridate 13 (807 mg, 2.62 mmol) in
dry DCM (20 mL) was added at -78 °C N-methyl imidazole (0.45 mL, 5.7 mmol). The mixture
3
1
was warmed up at room temperature and monitored by TLC and P NMR and quenched
with MeOH. The organic layer was washed with 0.5 M aqueous solution of HCl (20 mL) and
the aqueous layer was extracted twice with DCM (2 x 20 mL). The combined organic layer
was dried over anhydrous Na SO , filtered and evaporated to dryness under reduced
2
4
pressure. A purification by flash chromatography gave a mixture (213 mg) of the desired
compound 15a and a byproduct (*) in a 50:50 ratio respectively, which was determined by
NMR spectroscopy. The product 15a was obtained as a mixture of two diastereoisomers in a
1
6
0:40 ratio. Rf = 0.35 (EtOAc/petroleum ether, 3:7). H NMR (500 MHz, CDCl ): δ 1.31-1.38
3
(
6/9 of 9H, m, NCHCH *, 3/9 of 9H, m, NCHCH ), 3.45 (2H, m, NCH ), 3.68-3.76 (9/15 of 15H,
3
3
2
m, OCH *, 6/15 of 15H, m, OCH ), 3.98-4.07 (2/3 of 3H, m, NCHCH *, 1/3 of 3H, m, NCHCH ),
3
3
3
3
4
4
.14 (4/10 of 2H, bs, POCH ), 4.23 (6/10 of 2H, bs, POCH ), 4.67 (4/10 of 2H, bs, OCH Ph),
2
2
2
.81 (6/10 of 2H, bs, OCH Ph), 6.77-6.82 (1/2 of 2H, m, CH *, 1/2 of 2H, m, CH ), 7.07-7.12
2
Ar
Ar
(
1/2 of 2H, m, CH *, 1/2 of 2H, m, CH ), 7.37 (5H, m, CHAr(Bn)), 7.81-7.85 (4/10 of 1H, bs,
Ar
Ar
1
3
CHO), 8.19-8.21 (6/10 of 1H, bs, CHO); C NMR (125.8 MHz, CDCl ): δ 21.1-21.4 (NCHCH *
3
C
3
and NCHCH ), 45.3 (NCH ), 46.5 (NCH ), 49.9-50.4 (NCHCH * and NCHCH ), 52.6 (CO CH *
3
2
2
3
3
2
3
and CO CH ), 55.8 (ArOCH * and ArOCH ), 61.9 (POCH ), 62.5 (POCH ), 76.9 (OCH Ph), 78.4
2
3
3
3
2
2
2
(
OCH Ph), 114.7 (CHAr(PMP)), 121.3 (CHAr(PMP)), 128.8 (CHAr(Bn)), 129.0 (CHAr(Bn)), 129.4 (CHAr(Bn)),
2
1
29.5 (CHAr(Bn)), 129.7 (CHAr(Bn)), 134.3 (C ), 144.5 (C ), 156.5 (C OCH ), 164.1 (CHO), 164.2
Ar
Ar
Ar
3
3
1
(
CHO),173.7 (CH CO), 174.1 (CO CH ), 174.3 (CO CH ); P-NMR (121.5 MHz, CDCl ): δ 3.05,
3
2
3
2
3
3
P
+
+
3
4
.30, 8.10*; HRMS (EI) : m/z calculated for C H N O PNa [M+Na] 489.1397, found
2
1 27 2 8
89.1376.
5
.1.10. Methyl((2-(N-(benzyloxy)acetamido)ethoxy)(4-methoxyphenoxy)phosphoryl)-L-
alaninate (15b)
To a solution of 12b (277 mg, 1.32 mmol), the phosphochloridate 13 (935 mg, 3.0 mmol) in
dry DCM (24 mL) was added at -78 °C N-methyl imidazole (0.53 mL, 6.6 mmol). The mixture
2
0

3
1
was warmed up at room temperature and monitored by TLC and P NMR and quenched
with MeOH. The organic layer was washed with 0.5 M aqueous solution of HCl (20 mL) and
the aqueous layer was extracted twice with DCM (2 x 20 mL). The combined organic layer
was dried over anhydrous Na SO , filtered and evaporated to dryness under reduced
2
4
pressure. Several purifications by flash chromatography and preparative TLC gave a mixture
31 mg) of the desired compound 15b and a byproduct (*) in a 50:50 ratio respectively,
(
which was determined by NMR spectroscopy. The product 15b was obtained as a mixture of
1
two diastereoisomers in a 60:40 ratio. Rf = 0.38 (EtOAc/petroleum ether, 3:7). H NMR (500
MHz, CDCl ): δ 1.37 (2/3 of 9H, m, NCHCH *, 2/3 of 9H, m, NCHCH ), 2.07 (6/10 of 2H, bs,
3
3
3
CH CO), 2.08 (4/10 of 2H, bs, CH CO), 3.41-3.46 (2H, m, NCH ), 3.68-3.76 (9/15 of 15H, m,
3
3
2
OCH *, 6/15 of 15 H, m, OCH *) 3.98-4.07 (2/3 of 1H, m, NCHCH *, 1/3 of 1H, m, NCHCH ),
3
3
3
3
4
6
.21-4.29 (2H, m, POCH ), 4.79 (4/10 of 2H, s, OCH Ph), 4.81 (6/10 of 2H, s, OCH Ph), 6.77-
2
2
2
.82 (1/2 of 2H, m, CH *, 1/2 of 2H, m, CH ), 7.07-7.12 (1/2 of 2H, m, CH *, 1/2 of 2H, m,
Ar
Ar
Ar
CH ), 7.37 (5H, m, CHAr(Bn)); 13C-NMR (125.8 MHz, CDCl ): δ 20.6 (CH CO), 21.1-21.4
Ar
3
C
3
(
NCHCH * and NCHCH ), 46.5 (NCH ), 46.7 (NCH ), 49.9-50.4 (NCHCH * and NCHCH ), 52.6
3
3
2
2
3
3
(
CO CH * and CO CH ), 55.8 (ArOCH * and ArOCH ), 62.9 (POCH ), 76.9 (OCH Ph), 114.6-
2
3
2
3
3
3
2
2
1
1
14.7 (CHAr(PMP)), 121.3-121.4 (CHAr(PMP)), 128.9 (CHAr(Bn)), 129.2 (CHAr(Bn)), 129.5 (CHAr(Bn)),
3
29.9 (CHAr(Bn)), 144.2-144.5 (C ), 156.5 (C OCH ), 173.7 (CH CO), 174.1 (d, J = 6.8 Hz,
Ar
Ar
3
3
C-P
3
31
CO CH ), 174.3 (d, J = 7.1 Hz, CO CH ); P-NMR (121.5 MHz, CDCl ): δ 3.05, 3.30, 8.10*;
2
3
C-P
2
3
3
P
+
+
HRMS (EI) : m/z calculated for C H N O P [M+H] 481.1734, found 481.1762.
2
2 30 2 8
5
.1.11. N-(Benzyloxy)-3-hydroxypropanamide-3-[(4-methoxy)phenyl(methoxyalaninyl)
phosphoramidate] (16a)
Alcohol 14a (200 mg, 1.02 mmol) was dissolved in anhydrous pyridine (2.8 mL). The mixture
was cooled to -40 °C and compound 13 (627 mg, 2.04 mmol) in toluene (2.5 mL) was added
dropwise. The reaction was stirred to -40 °C for 30 min and warmed up to room temperature
overnight. The pyridine was evaporated under reduced pressure and the crude product was
purified by flash chromatography (EtOAc/cyclohexane, 9:1) to give 16a as a colorless oil (222
mg, 47 %) and as a mixture of two diastereomers in a 50:50 ratio and two conformers Z and
1
E in a 80:20 ratio. Rf = 0.28 (EtOAc/petroleum ether, 8:2). H NMR (400 MHz, CDCl ): δ 1.31
3
3
3
(
5/10 of 3H, d, J = 6.8 Hz, NCHCH ), 1.35 (5/10 of 3H, d, J = 7.0 Hz, NCHCH ), 2.42 (8/10 of
3
H-H
3
2
1

2
H, bs, CH CO), 2.70 (2/10 of 2H, bs, CH CO), 3.52 (5/10 of 1H, m, NH), 3.67-3.73 (6H, m,
2
2
OCH ), 3.79 (5/10 of 1H, m, NH), 3.91-4.03 (1H, m, NCHCH ), 4.28-4.38 (2H, m, POCH ), 4.85-
3
3
2
4
.91 (2H, s, OCH Ph), 6.78-6.80 (2H, m, CH ), 7.06-7.09 (2H, m, CH ), 7.33-7.36 (5H, m,
2
Ar
Ar
1
3
CHAr(Bn)), 9.24 (5/10 of 1H, bs, N(H)OBn), 9.37 (5/10 of 1H, bs, N(H)OBn); C NMR (125.8
MHz, CDCl ): δ 21.1 (NCHCH ), 35.2 (CH CO), 35.8 (CH CO), 50.3 (NCHCH ), 50.5 (NCHCH ),
3
C
3
2
2
3
3
5
1
1
2.8 (CO CH ), 55.8 (ArOCH ), 63.4 (POCH ), 63.6 (POCH ), 78.4 (OCH Ph), 78.5 (OCH Ph),
2
3
3
2
2
2
2
14.8 (CHAr(PMP)), 121.2-121.4 (CHAr(PMP)), 128.8-129.0 (CHAr(Bn)), 129.3-129.4 (CHAr(Bn)), 135.4-
3
35.7 (CAr(Bn)), 144.2 (C OP), 156.9 (C O CH ), 167.8 (CONH), 174.2 (d, J = 6.8 Hz,
Ar
Ar
3
C-P
3
31
CO CH ), 174.5 (d, J = 6.9 Hz, CO CH ); P NMR (162.0 MHz, CDCl ): δ 2.70, 3.00; MS
2
3
C-P
2
3
3
P
+
+
(
EI) : m/z calculated for C H N O PNa [M+Na] 489.14, found 489.14.
2
1 27 2 8
5
.1.12. N-(Benzyloxy)-3-hydroxy-N-methylpropanamide-3-[(4-methoxy)phenyl
(methoxyalaninyl) phosphoramidate] (16b)
To a solution of alcohol 14b (194 mg, 0.93 mmol), triethylamine (0.15 mL, 1.02 mmol), DMAP
57 mg, 0.47 mmol) in dry DCM (2.8 mL) was treated with a solution of compound 13 (860
(
mg, 2.8 mmol) in DCM (2.2 mL) at -40 °C. The resulting mixture was warmed up to room
temperature and stirred overnight. A saturated aqueous solution of NH Cl (10 mL) was
4
added and the aqueous layer was extracted with DCM (2 x 15 mL). The organic layers were
collected, dried over anhydrous Na SO , filtered and solvents were removed under reduced
2
4
pressure. The crude product was purified by flash chromatography (EtOAc/cyclohexane, 9:1)
to give 16b as a colorless oil (213 mg, 48 %) and as a mixture of two diastereomers in a 50:50
1
ratio. Rf = 0.23 (EtOAc/petroleum ether, 6:4). H NMR (400 MHz, CDCl ): δ 1.35 (5/10 of 3H,
3
3
3
d, J = 7.0 Hz, NCHCH ), 1.39 (5/10 of 3H, d, J = 7.1 Hz, NCHCH ), 2.66-2.88 (2H, m, CH CO),
3
3
2
3
.19 (5/10 of 3H, s, NCH ), 3.20 (5/10 of 3H, s, NCH ), 3.70-3.72 (3H, m, OCH ), 3.75 (3H, m,
3
3
3
OCH ), 4.00-4.08 (1H, m, NCHCH ), 4.31-4.48 (2H, m, POCH ), 4.76-4.81 (2H, m, OCH Ph),
3
3
2
2
1
3
6
.79-6.81 (2H, m, CH ), 7.09-7.014 (2H, m, CH ), 7.34-7.39 (5H, m, CH
); C NMR (125.8
Ar
Ar
Ar(Bn)
MHz, CDCl ): δ 21.3 (NCHCH ), 33.2 (CH CO), 33.6 (NCH ), 50.3 (NCHCH ), 50.4 (NCHCH ),
3
C
3
2
3
3
3
5
2.6 (CO CH ), 55.8 (ArOCH ), 62.9 (POCH ), 76.6 (OCH Ph), 114.7 (CHAr(PMP)), 121.4-121.5
2
3
3
2
2
(CHAr(PMP)), 128.9 (CHAr(Bn)), 129.3 (CHAr(Bn)), 129.5 (CHAr(Bn)), 134.4 (CAr(Bn)), 144.5-144.6
4
3
(
C OP), 156.6 (C O CH ), 171.8 (d, J = 9.4 Hz, CONCH ), 174.2 (d, J = 7.4 Hz, CO CH ),
Ar
Ar
3
C-P
3
C-P
2
3
2
2

74.3 (d, 3JC-P = 7.7 Hz, CO CH ); P NMR (162.0 MHz, CDCl ): δ 2.75, 2.90; MS (EI) : m/z
31
+
1
2
3
3
P
+
calculated for C H N O PNa [M+Na] 503.16, found 503.16.
2
2 29 2 8
5
.1.13. Methyl((3-(hydroxyamino)-3-oxopropoxy)(4-methoxyphenoxy)phosphoryl)-L-
alaninate (8a)
The protected alcohol 16a (29 mg, 62 μmol), palladium on charcoal (10 % mol) in absolute
methanol (1.4 mL) was hydrogenolyzed at room temperature and at atmospheric pressure
under an atmosphere of H2. After total consumption of starting material (TLC) the catalyst
was removed by filtration over celite and the solvent was removed under reduced pressure.
The final product 8a was obtained without purification as a colorless oil (18 mg, 77 %) and as
1
a mixture of two diastereomers in a 55:45 ratio. Rf = 0.58 (EtOAc). H NMR (400 MHz, CDCl ):
3
δ 1.33 (3H, m, NCHCH ), 2.52 (2H, bs, CH CO), 3.68-3.70 (3H, m, OCH ), 3.74 (3H, m, OCH ),
3
2
3
3
3
.92-4.00 (1H, m, NCHCH ), 4.30-4.35 (2H, m, POCH ), 6.79-6.82 (2H, m, CH ), 7.06-7.09 (2H,
3
2
Ar
m, CH ), 10.0 (1H, bs, N(H)OH); 13C NMR (125.8 MHz, CDCl ): δ 20.7 (NCHCH ), 32.3
Ar
3
C
3
(
CH CO), 36.0 (NCH ), 50.3 (NCHCH ), 50.5 (NCHCH ), 52.7 (CO CH ), 52.8 (CO CH ), 55.8
2
3
3
3
2
3
2
3
(
ArOCH ), 63.5 (POCH ), 114.7 (CHAr(PMP)),114.8 (CHAr(PMP)), 121.2 (CHAr(PMP)), 121.3 (CHAr(PMP)),
3
2
1
21.4 (CHAr(PMP)), 121.5 (CHAr(PMP)), 144.1 (C OP), 156.8 (C OCH ), 167.8 (CONCH ), 167.9
Ar
Ar
3
3
3
3
31
(
CONCH ), 174.6 (d, J = 5.6 Hz, CO CH ), 174.8 (d, J = 6.6 Hz, CO CH ); P NMR (162.0 MHz,
3
2
3
2
3
+
+
CDCl ): δ 2.90, 3.40; HRMS (EI) : m/z calculated for C H N O PNa [M+Na] 399.0928, found
3
P
14 21 2 8
3
99.0918.
5
.1.14. Methyl ((3-(hydroxy(methyl)amino)-3-oxopropoxy)(4-methoxyphenoxy)phosphoryl)-
L-alaninate (8b)
The protected alcohol 16a (23 mg, 48 μmol), palladium on charcoal (10 % mol) in absolute
methanol (1 mL) was hydrogenolyzed at room temperature and at atmospheric pressure
under an atmosphere of H . After total consumption of starting material (TLC) the catalyst
2
was removed by filtration over celite and the solvent was removed under reduced pressure.
The final product 8b was obtained without purification as a colorless oil (18 mg, 92 %) and as
a mixture of two diastereomers in a 50:50 ratio and of two Z and E conformers in a 20:80
1
3
ratio respectively. Rf = 0.51 (EtOAc). H NMR (400 MHz, CDCl ): δ 1.35 (5/10 of 3H, d, J = 6.9
3
2
3

3
Hz, NCHCH ), 1.39 (5/10 of 3H, d, J = 6.4 Hz, NCHCH ), 2.70 (2/10 of 2H, bs, CH CO), 2.92
3
3
2
(
8/10 of 2H, bs, CH CO), 3.18 (8/10 of 2H, s, NCH ), 3.29 (2/10 of 2H, s, NCH ), 3.71 (5/10 of
2
3
3
3
H, s, OCH ), 3.72 (5/10 of 3H, s, OCH ), 3.78 (3H, m, OCH ), 3.98 (1H, m, NCHCH ), 4.37-4.45
3
3
3
3
4
3
(
2H, m, POCH ), 6.82 (2H, dd, J = 8.9 Hz, J = 1.4 Hz CH ), 7.07-7.10 (2H, m, CH ), 9.16 (1H,
2
Ar
Ar
1
3
bs, N(CH )OH); C NMR (125.8 MHz, CDCl ): δ 20.0 (NCHCH ), 32.2 (CH CO), 33.3 (CH CO),
3
3
C
3
2
2
2
5
3
6.0 (NCH ), 50.3 (d, J = 8.6 Hz, NCHCH ), 52.8 (pd, J = 3.4 Hz CO CH ), 55.8 (ArOCH ), 62.9
3
3
2
3
3
(
POCH ), 64.5 (POCH ), 114.8 (CHAr(PMP)), 121.2 (CHAr(PMP)), 121.3 (CHAr(PMP)), 121.4 (CHAr(PMP)),
2
2
1
21.5 (CHAr(PMP)), 144.1 (C OP), 156.9 (C OCH ), 170.5 (CONCH ), 170.6 (CONCH ), 174.1 (d,
Ar
Ar
3
3
3
3
3
31
J = 7.3 Hz, CO CH ), 174.2 (d, J = 7.8 Hz, CO CH ); P NMR (162.0 MHz, CDCl ): δ 2.85,
2
3
2
3
3
P
+
+
3
.10; HRMS (EI) : m/z calculated for C H N O PNa [M+Na] 413.1084, found 413.1048.
1
5 23 2 8
5
.2. Bacterial growth conditions
M. smegmatis DSM43756 (ATCC 19420) was obtained from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (Braunschweig, Germany). Bacteria were grown
aerobically at 30 °C in a liquid medium containing 0.4% yeast extract, 1% malt extract, 0.2%
CaCO and 0.4% D-glucose. They were plated on Petri dishes containing 0.05% yeast extract,
3
3
2
0
.05% malt extract, 0.1% CaCO , 0.1% D-glucose and 1.6% agar. E. coli XL1-blue was grown
3
in a liquid LB medium or on a solid LB-agar medium at 37 °C.
5
.3. Antibacterial activity of prodrugs
The antibacterial activity of the prodrugs was tested on M. smegmatis and E. coli XL1-blue by
the paper disc diffusion method. Plates were inoculated with bacteria (120 μL) of a tenfold-
diluted overnight culture. Prodrugs, not soluble in water, were dissolved at a 100 mM
concentration in DMSO. Isoniazid and fosmidomycin were used as positive controls for M.
smegmatis and E. coli respectively. Paper discs (Durieux no. 268, diameter 6 mm) were
impregnated with the prodrug solutions (8 µL, 800 nmoles) or with a 5 mM isoniazid or a
fosmidomycin (2 µL, 10 nanomoles) solution. Growth inhibition was examined after 24 to 48
h incubation.
5
.4. Stability assays of the prodrugs in buffered solution
2
4

The stability of the prodrugs in a 50 mM triethanolamine (TEOA) buffer (600 µL final volume,
3
1
pH 7.5) containing D O (200 µL) was studied over 48 h using P NMR. The prodrugs were
2
dissolved in DMSO at a concentration of 100 mM and diluted in the reaction medium to 3
mM.
5
.5. Stability assays of the prodrugs towards carboxypeptidase Y
3
1
The hydrolysis of the prodrugs by carboxypeptidase Y (Sigma-Aldrich) was studied using P
NMR. The experiments (600 µL final volume) were carried out in a TEOA 50 mM pH 7.5
containing D O (200 µL). The concentration of the prodrugs was 3 mM. Spectra were
2
recorded after addition of the enzyme (0.04 mg) and the hydrolysis was followed over 48 h.
5
.6. Stability assays of the prodrugs 8a and 8b during M. smegmatis and E. coli cultures
E. coli XL1-blue was grown in LB medium (50 mL) for 24 h. The culture was centrifuged (15
min at 7000 rpm), and the supernatant was filtered through a 0.22 µM sterile unit (Millex
Millipore). M. smegmatis was grown in medium (50 mL) during 40 h, and the culture was
then processed as described above.
3
1
The stability of the prodrugs in both resulting supernatants was followed over 48 h using P
NMR. The prodrugs (3 mM) were incubated with a mixture of used growth medium (400 µL)
and D O (200 µL).
2
5
.7. Esterase and phosphatase activity in bacterial crude cell-free extracts
Cells from a 500 mL culture were harvested by centrifugation (7000 rpm, 15 min) and the
pellet was frozen. Cells (about 1 g) were resuspended in a 50 mM TEOA buffer (2 mL, pH 7.5)
and sonicated (5×30 s with 2 min cooling in ice). After centrifugation (14ꢀ000 rpm, 25 min),
the supernatant was collected. Protein concentration was measured by the method of
Bradford using bovine serum albumin (BSA) as a standard.
3
1
The stability of the prodrugs towards enzymes of both crude extracts was studied using P
NMR. The experiments (600 µL final volume) were carried out in a TEOA 50 mM buffer (pH
7
.5) containing D O (200 µL) and crude cell-free system (150 µL). The concentration of the
2
prodrugs was 3 mM. Spectra were recorded after addition of the crude cell-free system and
the enzymatic reaction was followed over 20 h.
2
5

5
.8. Esterase activity in crude cell-free systems of M. smegmatis and E. coli
The esterase activity in the crude cell-free systems was measured with 4-nitrophenyl
butyrate (Sigma) as substrate. The assays (1 mL final volume) were performed at 30°C in a 50
mM TEOA buffer (pH 7.5). The concentration of the substrate was 1 mM. The reaction was
initiated by addition of crude cell-free system (5 µL) and followed at 405 nm with an Uvikon
9
33 spectrophotometer. The enzymatic activity was followed by the release of p-nitrophenol
-1
-1
−1
−1
(
 = 18000 M cm ) and expressed in μmoles min mg protein .
5
.9. Phosphatase activity in crude cell-free systems of M. smegmatis and E. coli
The phosphatase activity in the crude extracts was measured with 4-nitrophenyl phosphate
Sigma) as the substrate. The assays (1 mL final volume) were performed at 30°C in a 50 mM
TEOA buffer (pH 7.5). The influence of the presence of 5 mM dication (MgCl , MnCl and
(
2
2
CoCl ), on the hydrolysis, was also investigated. The enzymatic activity was determined as
2
for the esterase activity.
Acknowledgements
M. Munier acknowledges financial support from the ‘Ministère de la Recherche’. This work
was supported by a grant from the ‘Agence Nationale de la Recherche’ (Grant Nb ANR-13-
BS07-0013-01). The authors express their gratitude to B. Vincent and M. Coppe for NMR
measurements.
References
1
- World Health Organization 2017. Global Tuberculosis Report 2017.
http://www.who.int/tb/publications/global_report/en/
2
- World Health Organization 2008. Implementing the WHO Stop TB Strategy: A Handbook
for National Tuberculosis Control Programs
3
- M. Rohmer, C. Grosdemange-Billiard, M. Seemann and D. Tritsch, Curr. Opin. Investig.
Drugs, 2004, 5, 154–162.
4
5
- X. Wang, C.S. Dowd, ACS Infect Dis., 2018, 4, 278-290.
- M. Okuhara, Y. Kuroda, T. Goto, M. Okamoto, H. Terano, M. Kohsaka, H. Aoki and H.
Imanaka, J. Antibiot., 1980, 33, 13–17.
2
6

6
- M. Okuhara, Y. Kuroda, T. Goto, M. Okamoto, H. Terano, M. Kohsaka, H. Aoki and H.
Imanaka, J. Antibiot., 1980, 33, 24–28.
7
- Y. Kuroda, M. Okuhara, T. Goto, M. Okamoto, H. Terano, M. Kohsaka, H. Aoki and H.
Imanaka, J Antibiot, 1980, 33, 29–35.
8
7
- T. Kuzuyama, T. Shimizu, S. Takahashi and H. Seto, Tetrahedron Lett., 1998, 39, 7913–
916.
9
- L. Kuntz, D. Tritsch, C. Grosdemange-Billiard, A. Hemmerlin, A. Willem, T. J. Bach and M.
Rohmer, Biochem. J., 2005, 386, 127–135.
1
1
0- A.C. Brown, T. Parish, BMC Microbiol. 2008, 8, 78.
1- S. Ponaire, C. Zinglé, D. Tritsch, C. Grosdemange-Billiard and M. Rohmer, Eur. J. Med.
Chem., 2012, 51, 277–285.
1
2- R. Ortmanna, J. Wiesner, A. Reichenberg, D. Henschker, E. Beck, H. Jomaa and M.
Schlitzer, Bioorg Med. Chem. Lett. 2003, 13, 2163–2166.
1
3- K. Brücher, T. Gräwert, S. Konzuch, J. Held, C. Lienau, C. Behrendt, B. Illarionov, L. Maes,
A. Bacher, S. Wittlin, B. Mordmüller, M. Fischer and T. Kurz, J. Med. Chem., 2015, 58, 2025–
2
035.
1
2
4- N. Katayama, S. Tsubotani, Y. Nozaki, S. Harada and H. Ono, J. Antibiot., 1990, 43, 238–
46.
1
2
5- Y.-H. Woo, R. P. M. Fernandes and P. J. Proteau, Bioorg. Med. Chem., 2006, 14, 2375–
385.
1
6- M. Munier, D. Tritsch, F. Krebs, J. Esque, A. Hemmerlin, M. Rohmer, R.H. Stote, and C.
Grosdemange-Billiard, Bioorg. Med. Chem., 2017, 25, 684–689.
1
7- Y. Mehellou, J. Balzarini and C. McGuigan, ChemMedChem., 2009, 4, 1779-1791
1
5
8- M. Slusarczyk, S. Ferla, A. Brancale and C. McGuigan, Bioorg. Med. Chem., 2018, 26, 551–
65.
1
9- A. S. Alanazi, E. James, Y. Mehellou, ACS Med. Chem. Lett., 2019, 10, 2-5.
2
0- G. F. Ruda, P. E. Wong, V. P. Alibu, S. Norval, K. D. Read, M. P. Barrett and I. H. Gilbert, J.
Med. Chem., 2010, 53, 6071-6078.
2
1
1- C. McGuigan, R.N. Pathirana, J. Balzarini and E. De Clercq, J. Med. Chem., 1993, 36, 1048-
052.
2
7

2
2- J.H. van Boom, P.M.J. Burgers, R. Crea, W.C.M.M. Luyten, A.B.J. Vink and C. B. Reese,
Tetrahedron, 1975, 31, 2953–2959.
23- M. Derudas, D. Carta, A. Brancale, C. Vanpouille, A. Lisco, L. Margolis, Jan Balzarini,
C. McGuigan, J. Med. Chem., 2009, 52, 5520–5530.
2
2
4- P.G. Fallon, R.O. Cooper, A.J. Probert and M.J. Doenhoff, Parasitol., 1992, 105, 41-48.
5- A. Tsatsaroni, G. Zoidis, P. Zoumpoulakis, A. Tsotinis, M. C. Taylor, J. M. Kelly and G.
Fytas, Tetrahedron Lett., 2013, 54, 3238–3240.
2
2
6- J. D. Aubort and R. I. Hudson, J. Chem. Soc. D, 1970, 1378-1379.
7- K. Kl. Ghosh, M. L. Satnami and D. Sinha, Tetrahedron. Lett., 2004, 45 9103–9105.
2
8- K. Kl. Ghosh, D. Sinha, M. L. Satnami, D.K. Dubey, A. Shrivastava, R. M. Palepu and P.R.
Dafonte, J. Colloid Interface Sci., 2006, 301, 564–568.
2
9- C.McGuigan, A. Hassan-Abdallah, S. Srinivasan, Y. Wang, A. Siddiqui, S. M. Daluge, K. S.
Gudmundsson, H. Zhou, E.W. McLean, J.P. Peckham, T. C. Burnette, H. Marr, R. Hazen, L. D.
Condreay, L. Johnson and J. Balzarini, J. Med. Chem., 2006, 49, 7215-7226.
3
0- T.F. Chou, P. Bieganowski, K. Shilinski, J. Cheng, C. Brenner and C.R .Wagner, J. Biol.
Chem., 2005, 280, 15356-15361.
3
1- T.F. Chou and C.R .Wagner, J. Biol. Chem., 2007, 282 4719-4727.
3
1
2- S.R. Putra, A. Disch, J.M. Bravo and M. Rohmer, FEMS Microbiol. Lett., 1998, 164, 169-
75. https://doi.org/10.1111/j.1574-6968.1998.tb13082.x
2
8

Highlights



Application of the proTide technology to the synthesis of new antibacterial prodrug.
Synthesis of aryl phosphoramidate progrugs of fosfoxacin homologs.
ProTide strategy might not be appropriated for antibacterial drug design.
2
9