Synthesis and evaluation of α-halogenated analogues of 3-(acetylhydroxyamino)propylphosphonic acid (FR900098) as antimalarials

Article abstract of DOI:10.1021/jm100211e

Three α-halogenated analogues of 3-(acetylhydroxyamino) propylphosphonic acid (FR900098) have been synthesized from diethyl but-3-enylphosphonate using a previously described method for the α-halogenation of alkylphosphonates. These analogues were evaluated for antimalarial potential in vitro against Plasmodium falciparum and in vivo in the P. berghei mouse model. All three analogues showed higher in vitro and/or in vivo potency than the reference compounds.

Full text of DOI:10.1021/jm100211e

5342 J. Med. Chem. 2010, 53, 5342–5346
DOI: 10.1021/jm100211e
Synthesis and Evaluation of r-Halogenated Analogues of 3-(Acetylhydroxyamino)propylphosphonic
Acid (FR900098) as Antimalarials
Thomas Verbrugghen,† Paul Cos,‡ Louis Maes,‡ and Serge Van Calenbergh*,†
†Laboratory for Medicinal Chemistry (FFW), Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium, and ‡Laboratory for Microbiology,
Parasitology, and Hygiene, Faculty of Pharmaceutical, Biomedical, and Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171,
B-2020 Antwerp, Belgium
Received February 17, 2010
Three R-halogenated analogues of 3-(acetylhydroxyamino)propylphosphonic acid (FR900098) have
been synthesized from diethyl but-3-enylphosphonate using a previously described method for the
R-halogenation of alkylphosphonates. These analogues were evaluated for antimalarial potential in
vitro against Plasmodium falciparum and in vivo in the P. berghei mouse model. All three analogues
showed higher in vitro and/or in vivo potency than the reference compounds.
Chart 1
Introduction
Despite huge efforts already taken, malaria still poses a
major threat to public health and the economy of affected
countries.1 With resistance emerging to virtually all available
therapeutics, new antimalarials directed against new targets
are highly awaited.2 In this respect, the non-mevalonate path-
way for isoprenoid biosynthesis (also known as the MEPa
pathway) and considered essential in all malaria-causing Plas-
modium species constitutes a promising target.3 This pathway
is unrelated to the classical mevalonate pathway (HMG-CoA
reductase pathway) present in all higher eukaryotes and starts
with the condensation of pyruvate and glyceraldehyde 3-phos-
phate to 1-deoxy-D-xylulose 5-phosphate (DOXP). Thus far,
the second enzyme DOXP reductoisomerase (DXR), which
catalyzes the conversion of DOXP into 2-C-methyl-D-erythri-
tol 3-phosphate (MEP), is the best investigated target in the
search for new antibiotics, antimalarials, and herbicides tack-
ling the non-mevalonate pathway (Chart 1).
Fosmidomycin (1) and its acetyl congener 2 (FR900098),4
structurally simple antibiotics isolated from Streptomyces
cultures, are selective inhibitors of DXR,5 and the former
has been advanced to clinical trials in Gabon and Cameroon.6
Since the discovery of their antimalarial activity, different
analogues of fosmidomycin/2 have been synthesized to explore
the SAR and to develop more potent inhibitors. From these
studies, it has become clear that neither the phosphonate4,7,8
nor the (retro)hydroxamate moiety9 can be replaced without
drastic loss of activity. On the other hand, modification of the
three-carbon spacer has resulted in DXR inhibitors that surpass
fosmidomycin’s potency. For example, some R-aryl substituted
fosmidomycin analogues proved to be more potent than
fosmidomycin in inhibiting the growth of P. falciparum.10,11
This advantage was especially apparent with electron with-
drawing substituents, such as the 3,4-dichlorophenyl group in 3.
One possible explanation for the observed SAR in this series is
that electron withdrawing substituents in R-position decrease
the second pKa of the phosphonate group, which for that
reason appears in its double-ionized form. Indeed, earlier
SAR studies at the enzyme level indicate that the presence
of two ionizable groups on the phosphonate or phosphate
probably plays a key role in the highly potent inhibition of
DXR.7,12 Consonant with the hypothesis that increasing
the acidity of the phosphonate moiety might confer im-
proved activity, fosfoxacin (4), the phosphate analogue of
fosmidomycin, and its acetyl congener were found to be
more potent inhibitors of Synechocystis sp. PCC6803
DXR than fosmidomycin.7
*To whom correspondence should be addressed. Phone: þ32 9 264 81
24. Fax: þ 32 9 264 81 46. E-mail: Serge.vancalenbergh@ugent.be.
a Abbreviations: BAIB, iodobenzene I,I-diacetate; CDI, 1,10-carbonyl-
diimidazole; DOXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-
xylulose 5-phosphate reductoisomerase; HMG-CoA, 3-hydroxy-3-methyl-
glutarylcoenzyme A; MEP, 2-C-methyl-D-erythritol 3-phosphate; MST,
mean survival time; NFBS, N-fluorobenzenesulfonimide; NMO, 4-methyl-
morpholine N-oxide; SAR, structure-activity relation; TBAF, tetra-
n-butylammonium fluoride; TBDMS, tert-butyldimethylsilyl; TEMPO,
2,2,6,6-tetramethylpiperidine-1-oxyl; TMS, trimethylsilyl; TMSBr, bromo-
trimethylsilane.
Since the metabolic liability of the phosphate precludes its
in vivo use as a DXR inhibitor, we decided to investigate if the
phosphonate groupof fosmidomycin couldbemanipulated to
more accurately mimic the electronic nature of the phosphate
moiety present in fosfoxacin and the DOXP substrate.
Nieschalk et al.13 showed that a monofluoromethylenephos-
phonate moiety can be a better phosphate mimic than the
r
pubs.acs.org/jmc
Published on Web 06/22/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5343
Scheme 1. Substrates Unsuccessfully Tested in the Halogenation Reaction
Scheme 2. Halogenation Strategy with Standard Desilylation Conditions (EtOLi/EtOH) Resulting in Elimination Product 9 and Alter-
native Acidic Procedure (TBAF/AcOH) towards 10a
more popular difluoromethylenephosphonates often used for
this goal, as the former one has a second pKa essentially equal
to that of an organophosphate, whereas a difluoromethylene-
phosphonate is more acidic. Hence, we undertook the synth-
esis of R-halogenated fosmidomycin analogues 5a and 5b in
which the required electron withdrawing effect comes from
the halogen insteadof the stericallymoredemanding aromatic
group as in 3, possibly resulting in a better fit into the active
pocket of DXR.
As stated above, modification of the N-formyl or N-acetyl
retrohydroxamate in these structures usually results in a total
loss of inhibitory activity.9 A notable exception to this “rule”
is the inversion of the retrohydroxamate into a hydroxamate
as proven by Rohmer and co-workers.12,14 A β-oxa analogue
bearing an N-methylhydroxamate functionality showed even
better DXR inhibiting properties;15 hence, we also envisaged
the synthesis of R-fluorohydroxamate 6.
azide group. Here again, the halogenation conditions caused
decomposition of the starting materials and were also un-
successful when applied on TBDMS-protected 3-hydroxy-
propylphosphonate. These failures led us to explore a simpler
phosphonate precursor, i.e., commercially available diethyl
but-3-enyl phosphonate (7),18 which could be halogenated in
satisfying yields without noticeable breakdown to afford 10b.
One side reaction observed in the synthesis of the R-chloro
derivative 10a was the formation of conjugated diene 9 caused
by elimination of chlorine when using the standard lithium
ethoxide-ethanol deprotection for the TMS group of inter-
mediate 8. Hence, we decided to remove the installed TMS
group by means of an acidic procedure (TBAF in acetic acid),
resulting in the desired product 10 (Scheme 2).
With the R-halogenated precursors 10a,b in hand, we then
assembled the hydroxamate headgroup (Scheme 3). First the
double bond was oxidized with NMO and osmium tetroxide
as a catalyst. The resulting vicinal diol was then cleaved
oxidatively with sodium periodate and the resulting aldehyde
reduced with sodium borohydride to give alcohols 12a,b.
Subsequently, the alcohols were converted into tosylates 13a,
b, which were substituted with N-Boc-O-benzylhydroxylamine.
Treatment of 14a,b with trifluoroacetic acid in dichloro-
methane gave hydroxylamines 15a,b, which were acetylated
with acetic anhydride. Finally, debenzylation of the retro-
hydroxamate by hydrogenation on palladium on carbon
followed by TMSBr deprotection of the phosphonate esters
and basic workup gave 5a,b as bisammonium salts. The
synthesis of R-fluorohydroxamate 6 could easily be elabo-
rated from the R-fluoro precursor 10b (Scheme 2). Hydro-
boration of 10b gave rise to alcohol 18 which was oxidized
with TEMPO-BAIB to carboxylic acid 19. This acid was
activated with CDI followed by coupling with N-methyl-O-
benzylhydroxylamine to give 20, which was then depro-
tected in the same way as 16a,b to give 6 as the bisammo-
nium salt (Scheme 4).
Results
For the introduction of the respective halogens into 2, we
adopted the strategy of Iorga et al.,16 based on the attack of a
deprotonated R-monosilylated alkylphosphonate on an elec-
trophilic halogenation reagent, hexachloroethane or N-fluoro-
benzenesulfonimide (NFBS).17 This straightforward one-pot
strategy allowed for the synthesis of both envisaged precursors
10a and 10b with just a minor modification of the desilylation
conditions (vide infra) and has several advantages such as high
yield, high speed, ready availability of electrophilic halogenat-
ing reagents, and easy elimination of byproducts. Unfortu-
nately, this strategy proved to be very sensitive toward func-
tionalities in the starting alkyl phosphonate, implying that
all attempts to introduce a halogen onto a suitably protected
fosmidomycin precursor using Iorga’s conditions remained
unsuccessful (Scheme 1).
First, three differently protected hydroxyamines, synthe-
sized from diethyl 3-bromopropylphosphonate were tested.
Unfortunatelyallreactions startedtoturnblackafteraddition
of the halogenation reagent, resulting in complex reaction
mixtures from which the desired compounds could not or
only in very low yield be isolated. Assuming that it was the
protected hydroxylamine functionality that did not withstand
the reaction conditions, we turned to chemically more resis-
tant (hydroxyl)amine precursors such as a phthalimide or
To assess the influence of the introduced halogens on the
ionization of these phosphonates, the pKa values of 5a and 5b
and reference 2 were estimated from the pH dependence of
their 31P chemical shifts (Figure 1). Therefore, the 31P chemi-
cal shift of each compound was measured at different pH and
plotted as a function of these pH values. The pKa of each
compound is estimated to be at the pH of the inflection point

5344 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Verbrugghen et al.
Scheme 3. Synthesis of R-Chloro and R-Fluoro analogues of FR900098a
a Reagents and conditions: (a) (i) LDA, TMSCl, C2Cl6 or (PhSO2)2NF, THF, -78 °C; (ii) TBAF, AcOH, THF; (b) (i) OsO4, dioxane, (ii) NaIO4;
(c) NaBH4, MeOH; (d) TsCl, Et3N, CH2Cl2; (e) BocNH(OBn), NaH, DMF; (f) TFA, CH2Cl2; (g) Ac2O, Et3N, DMAP, CH2Cl2; (h) H2, Pd/C, EtOAc;
(i) TMSBr, CH2Cl2, (ii) NH4OHaq, THF
Scheme 4. Synthesis of R-Fluorohydroxamate 6a
a Reagents and conditions: (a) (i) BH3 THF, (ii) NaOH, H2O2; (b) TEMPO, BAIB, CH3CN, H2O; (c) 1,10-carbonyldiimidazole, Me-NH-OBn;
(d) H2, Pd/C, EtOAc; (e) (i) TMSBr, CH2Cl2, (ii) NH4OH, THF.
3
Figure 1. Titration curves of compounds 2, 5a, and 5b around their second equivalence point.
of its titration curve. Of special interest here is the second pKa
of each molecule, as the value of this pKa determines whether
the phosphonate willbeinits single- or double-ionizedform at
physiological pH. Therefore, a cutout of the titration curves is
displayed from which the pKa2 can be estimated (for the
complete curves see Supporting Information).
From these figures, a pKa2 of 7.35 can be estimated for reference
2, whereas both the R-chlorinated analogue 5a and the R-fluo-
rinated analogue 5b show a pKa2 of around 6. It can thus be
concluded that introduction of a halogen in R-position of the
phosphonate moiety indeed lowered the pKa2 of those compounds
to a pKa2 comparable to that of a phosphate13 and that at a phy-
siological pH they will be present in their double-ionized form.
The title compounds were tested in duplicate for their
inhibitory effect against intraerythrocytic forms of P. falci-
parum (strains GHA and K1) using a microdilution assay.19
The results are summarized in Table 1. All three analogues
show submicromolar activity on both strains and appear to be
Table 1. In Vitro Growth Inhibition of the P. falciparum Strains GHA
and K1
IC50 (μM)
compd
Pf-GHA
Pf-K1
fosmidomycin (1)
2
1.73 ( 0.89
0.42 ( 0.17
0.16 ( 0.01
0.30 ( 0.06
0.29 ( 0.06
0.31 ( 0.07
3
0.60 ( 0.01
0.82 ( 0.10
0.70 ( 0.08
0.73 ( 0.11
5a
5b
6
5- to 6-fold more active than the parent compound fosmido-
mycin and slightly superior to 2 on the K1 strain.
The initial R-aryl derivative 3 and the fluorinated analogues
5b and 6 were subsequently evaluated in vivo in the P. berghei
(GFP ANKA strain) acute mouse model after intraperitoneal
dosing at 50 mg/kg for 5 consecutive days. Chloroquine
(10 mg/kg for 5 days) was included as reference treatment.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5345
Table 2. Survivors and Mean Survival Time (MST in Days) in the P. berghei (GFP ANKA Strain) Acute Mouse Model
survivors
treatment (ip for 5 consecutive days)
parasitemia suppression (day 4)
day 7
day 11
day 14
day 25
MST
vehicle
0
1/6
6/6
6/6
6/6
4/5
4/4
2/3
1/6
6/6
4/6
3/6
0/5
2/4
2/3
1/6
6/6
3/6
2/6
0/5
2/4
1/3
0/6
3/6
nd
8.5
chloroquine (10 mg/kg)
fosmidomycin (1) (50 mg/kg)
2 (50 mg/kg)
100
82
93
46
88
85
20.7
11.5
10.8
7.0
nd
3 (50 mg/kg)
5b (50 mg/kg)
6 (50 mg/kg)
0/5
0/4
0/3
15.8
11.7
The animals were observed for the occurrence/presence of
clinical or adverse effects during the course of the experi-
ment. In case of very severe clinical signs, either due to
toxicity or malaria, animals were euthanized for welfare
reasons. Parasitemia was determined on days 4, 7, and 14 on
surviving animals using flow cytometry (10 μL of blood in
2000 μL of PBS). Percentage reduction of parasitemia com-
pared to vehicle-treated infected controls is used as a measure
for drug activity, and the mean survival time (MST) was
calculated (Table 2). Compound 3 did not show any relevant
activity. On the other hand, 6 resulted in 85% suppression of
parasitemia at 4 dpi, which dropped to 42% at 7 dpi and 41%
at 14 dpi. The mean survival time was 11.7 days. Compound
5b resulted in 88% suppression of parasitaemia at 4 dpi, which
after ending the treatment also dropped to 62% at 7 dpi and
32% at 14 dpi. The overall MST was 15.8 days. These data
clearly demonstrate that all three synthesized compounds
have promising in vitro activity and that 5b and 6 surpass
the antimalarial activity of 2 in vivo.
In summary, three R-halogenated analogues of 2 were
synthesized and surpass or equal fosmidomycin or 2 in in
vitro and in vivo antimalarial activity. These findings con-
solidate the assumption that electron withdrawing substitu-
ents, causing a decrease in phosphonate pKa, favor the anti-
malarial activity of fosmidomycin analogues. Furthermore,
we provided a new example of a fosmidomycin analogue in
which the (N-formyl-N-hydroxy)amino moiety, involved in a
chelating interaction with a Mn2þ cation, can be replaced by a
N-hydroxy-N-methylamide group as found in 6. This opens
new perspectives to combine other favorable R-modifications
with a hydroxamate moiety.
time-of-flight spectrometer with API-ES source. High resolu-
tion mass spectroscopy spectra for all compounds were also
recorded on a Waters LCT Premier XE orthogonal time-of-
flight spectrometer with API-ES source. Purity of all final
compounds was 95% or higher.
(()-3-(N-Hydroxyacetamido)-1-chloropropylphosphonic Acid,
Bisammonium Salt (5a). To a solution of 17a (150 mg, 0.52 mmol)
in dry dichloromethane (5 mL) was added TMSBr (0.7 mL, 5.20
mmol) while stirring at 0 °C. After 45 min the ice bath was removed
and stirring was continued at room temperature. After 3 days, 31
P
NMR revealed the presence of incompletely deprotected material,
so another 0.2 mL of TMSBr was added. After another 3 days of
stirring at room temperature, the volatiles were removed in vacuo
and the residue was redissolved in 5% aqueous ammonia and
washed with diethyl ether. The aqueous phase was then lyophilized
to give 138 mg of a very hygroscopic, off-white powder. 1H NMR
(300.01 MHz, D2O) δ 1.92-2.57 (2H, m), 2.16 (3H, s), 3.71-3.92
(2H, m), 3.92-4.06 (1H, m); 13C NMR (75.44 MHz, D2O) δ 19.5
(CH3), 30.7 (CH2), 45.9 (CH2, d, 3JPC = 13.0 Hz), 54.8 (CClH, d,
1JPC = 139.0 Hz), 174.0 (CdO); 31P NMR (121.45 MHz, DMSO-
d6) δ 11.85; HRMS (ESI) m/z 232.0135 [(M þ H)þ, calcd for
C5H12ClNO5Pþ 232.0136].
(()-3-(N-Hydroxyacetamido)-1-fluoropropylphosphonic Acid,
Bisammonium Salt (5b). To a solution of 17b (223 mg, 0.82 mmol)
in dry dichloromethane (8 mL) was added TMSBr (1.1 mL,
8.2 mmol) while stirring at 0 °C. After 45 min the ice bath was
removed and stirring was continued at room temperature. After
3 days, 31P NMR revealed the presence of incompletely deprotected
material, so another 0.2 mL of TMSBr was added. After another
4 days of stirring at room temperature, the volatiles were removed
in vacuo and the residue was redissolved in 5% aqueous ammonia
and washed with diethyl ether. The aqueous phase was then lyophi-
lized to give 207 mg of 5b as a very hygroscopic, off-white powder.
1H NMR (300.01 MHz, D2O) δ 1.96-2.22 (2H, m), 2.11 (3H, s),
3.58-4.00 (2H, m), 4.33-4.64 (1H, m); 13C NMR (75.44 MHz,
D2O) δ 19.5 (CH3), 28.4 (CH2, d, 2JCF = 20.2 Hz), 45.0 (CH2, d,
3JPC = 3.6 Hz), 90.7 (CHF, dd, 1JCF = 171.0 Hz, 1JPC = 154.0
Hz), 174.0 (CdO); 31P NMR (121.45 MHz, D2O) δ 11.80 (d,
2JPF = 62.3 Hz); HRMS (ESI) m/z 216.0455 [(MþH)þ, calcd for
C5H12FNO5Pþ 216.0432].
An important outcome of the current study is that the
promising in vitro activity of the R-fluorinated analogues 5b
and 6 is reflected in the P. berghei acute mouse model, while
the R-aryl fosmidomycin analogue 3 failed to show significant
in vivo activity despite its promising in vitro activity.
(()-3-(N-Hydroxy-N-methylcarbamoyl)-1-fluoropropylphos-
phonic Acid, Bisammonium Salt (6). 21 (119 mg, 0.44 mmol) was
dissolved in dry dichloromethane under inert atmosphere and
cooled to 0 °C. TMSBr (0.6 mL, 4.4 mmol) was added dropwise
while stirring. The ice bath was removed, and the mixture was
stirred at room temperature. After 24 h another 0.3 mL of
TMSBr was added and the mixture was stirred for another 4
days. The volatiles were removed in vacuo. The crude material
was dissolved in 5% aqueous ammonia and washed with diethyl
ether. Lyophilization of the ammonia solution yielded the pro-
Experimental Section
Synthesis. General. 1H, 13C, 19F, and 31P NMR spectra were
recorded in CDCl3, acetone-d6, DMSO-d6, or D2O on a Varian
Mercury 300 spectrometer. Chemical shifts are given in parts
per million (ppm) (δ relative to residual solvent peak for 1H and
13C and to external D3PO4 for P). Silica gel (60 A, 0.063-
31
˚
0.200 mm) was purchased from Biosolve. All solvents and
chemicals were used as purchased unless otherwise stated. Purity
of the final compounds was deduced from clean 1H, 13C, and 31
P
1
duct as a brown solid in quantitative yield. H NMR (300.01
NMR spectra and high resolution mass spectra and assessed by
LC-DAD-MS. Reversed phase chromatograms were recorded
ona Phenomenex LunaC-18 2.5μm particle(100 mmꢀ 2.00 mm)
column or a Phenomenex Luna HILIC 200A 3 μm particle
(100 mm ꢀ2.00 mm) column in a Waters Alliance 2695 XE
HPLC system spectrometer with quaternary pump and DAD
detector, coupled to a Waters LCT Premier XE orthogonal
MHz, D2O) δ 1.82-2.00 (2H, m), 2.30-2.62 (2H, m), 3.07 (3H,
s), 4.18-4.45 (1H, m); 13C NMR (75.44 MHz, D2O) δ 27.0
(CH2, d, 2JCF = 19.6 Hz), 28.5 (CH2, d, 3JPC = 1.0 Hz), 36.1
1
1
(CH3), 92.9 (CHF, dd, JCF = 171.1 Hz, JPC = 153.7 Hz),
=
175.5 (CdO); 31P NMR (121.45 MHz, D2O) δ 11.74 (d, 2JPF
63.27 Hz); HRMS (ESI) m/z 216.0437 [(M þ H)þ, calcd for
C5H12FNO5Pþ 216.0432].

5346 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Verbrugghen et al.
Acknowledgment. T.V. is a Fellow of the Agency for
Innovation by Science and Technology of Flanders (IWT
Vlaanderen). P.C. is a postdoctoral fellow of the Fund for
ScientificResearch-Flanders(F.W.O.-Vlaanderen). Financial
support by F.W.O.-Vlaanderen is gratefully acknowledged.
Wealsothank AnMatheeussenforrunning all the in vitroand
in vivo biological evaluation work.
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Supporting Information Available: Experimental details and
spectral information for intermediates 11a,b-17a,b, 18, 19, 20,
21; pKa determination for 2, 5a, and 5b; 1H, 31P, and 13C spectra
for 5a, 5b, and 6. This material is available free of charge via the
Internet at http://pubs.acs.org.
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