Synthesis and biological evaluation of cyclopropyl analogues of fosmidomycin as potent Plasmodium falciparum growth inhibitors

Article abstract of DOI:10.1021/jm051177c

A series of fosmidomycin analogues featuring restricted conformational mobility has been synthesized and evaluated as inhibitors of 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase and as growth inhibitors of P. falciparum. The enantiomerically pure

Full text of DOI:10.1021/jm051177c

2656
J. Med. Chem. 2006, 49, 2656-2660
Synthesis and Biological Evaluation of Cyclopropyl Analogues of Fosmidomycin as Potent
Plasmodium falciparum Growth Inhibitors
Vincent Devreux,‡,∇ Jochen Wiesner,§ Jan L. Goeman,∇ Johan Van der Eycken,∇ Hassan Jomaa,§ and Serge Van Calenbergh‡,*
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent UniVersity, Harelbekestraat 72, B-9000 Gent, Belgium,
Laboratory of Organic and Bioorganic Chemistry, Department of Organic Chemistry, Faculty of Sciences, Ghent UniVersity,
Krijgslaan 281 (S.4), B-9000 Gent, Belgium, and Institut fu¨r Klinische Chemie und Pathobiochemie, UniVersita¨tsklinkum Giessen und Marburg,
Gaffkystrasse 11, 35392 Giessen, Germany
ReceiVed NoVember 22, 2005
A series of fosmidomycin analogues featuring restricted conformational mobility has been synthesized and
evaluated as inhibitors of 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase and as growth inhibitors
of P. falciparum. The enantiomerically pure trans-cyclopropyl N-acetyl analogue 3b showed comparable
inhibitory activity as fosmidomycin toward E. coli DOXP reductoisomerase and proved equally active when
tested in vitro for P. falciparum growth inhibition. Conversely, the R-phenyl cis-cyclopropyl analogue 4
showed virtually no inhibition of the enzyme.
Malaria is fatal for approximately 1.5 to 2.7 million people
each year. Among other reasons, the increasing resistance to
prevalent drugs such as chloroquine allows for a widespread
emergence of Plasmodium falciparum, the causative agent of
malaria tropica.
The discovery of the 2C-methyl-D-erithrytol 4-phosphate
phosphate (MEP) pathway as a mevalonate-independent path-
way for the biosynthesis of isoprenoids fueled research activities
dealing with the characterization of the enzymes involved in
this pathway and the search for inhibitors of these novel
targets.1,2 This MEP pathway is of particular interest since it is
present in most bacteria, plants, and the malaria parasite P.
falciparum, but is absent in humans.
The natural antibiotics fosmidomycin (1) and FR-900098 (2)
(Figure 1) possess high activities toward P. falciparum in vitro.3
In addition, the antimalarial activity of fosmidomycin (1) was
demonstrated in several recent clinical Phase II trials.4-8
The activity of these compounds is based on the inhibition
of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR),
the second enzyme in the non-mevalonate pathway. This enzyme
mediates the conversion of 1-deoxy-D-xylulose 5-phosphate into
2C-methyl-D-erithrytol 4-phosphate.
Until now, several X-ray crystallographic structures of DXR
have been reported.9,10 Additionally, Silber et al. recently
published a study of the AFMoC force field as an enhanced in
silico predictor of the binding affinity of ligands to DXR.11
Structural changes of fosmidomycin to enhance the activity
of this lead toward P. falciparum would provide critical
information concerning structure-activity relationships of fos-
midomycin. Much synthetic work has been dealing with
phosphonate moiety alterations, such as ester prodrugs,12-14
biphosphonates,15 or carboxylic acids.16 Alternatively, several
reports address hydroxamate moiety modifications, including
benzoxazolone, benzothione, oxazopyridone, or hydroxamic acid
functionalities.17,18 Interestingly, variations affecting the three-
Figure 1. Targeted fosmidomycin analogues and synthetic strategy.
carbon spacer are scarce. An important strategy to gather more
knowledge of the structure-activity relationships of fosmido-
mycin involves the restriction of the rotational freedom of this
carbon chain. This approach could be realized by incorporating
the C-1 and C-2 atoms in a three-membered ring. Here we
present the synthesis and biological activity of such novel
cyclopropyl analogues (3a-d, 4) of fosmidomycin. The syn-
thetic strategy to generate the three-membered ring involves the
intramolecular opening of an epoxide. This route was chosen
above more obvious cyclopropanation schemes because it
permits readily available chiral epoxides for the synthesis of
enantiomerically pure analogues. Moreover, even though the
number of reports mentioning the catalytic enantioselective
cyclopropanation of unsaturated phosphonates is growing, there
is still no generally applicable highly enantioselective method
to date. As it was envisioned that the use of a benzyl
phosphonate would allow a clean final deprotection, dibenzyl
methylphosphonate (7)19 was chosen as starting material
(Scheme 1). Regioselective opening of epoxide 8 with 7 under
Lewis acid conditions afforded alcohol 9. Special caution had
to be taken to prevent side reactions; hence, the temperature
had to be monitored closely during the reaction. Deprotonation
of alcohol 9 and subsequent elimination of the tosylate
functionality yielded epoxide 5 as precursor for the formation
of the three-membered ring. Ring closing was performed using
a Lewis acid-assisted intramolecular epoxide ring opening,
yielding alcohol 10. Unfortunately, during this step an unavoid-
able partial benzyl deprotection occurred, which was also
observed in step a. In accordance with literature data,20 the
cyclopropane ring possessed the trans-configuration, which was
* To whom correspondence should be addressed. Phone: +32 9 264 81
24. Fax + 32 9 264 81 46. E-mail: Serge.VanCalenbergh@UGent.be.
‡ Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sci-
ences, Ghent University.
1
proven via H NMR coupling constants. Next, alcohol 10 was
3 Laboratory of Organic and Bioorganic Chemistry, Department of
Organic Chemistry, Ghent University.
converted into tosylate 11 using a mild but very effective
§ Universita¨tsklinkum Giessen und Marburg.
procedure published by Yoshida et al.,21 which prevents further
10.1021/jm051177c CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/24/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2657
Table 1. Inhibition of Recombinant E. coli DXR
Scheme 1. Synthesis of the Unsubstituted trans-Cyclopropyl
Fosmidomycin Analoguesa
compound
IC50 (µM)
fosmidomycin
FR900098
0.048
0.058
0.160
0.050
0.313
>3.0
3a
3b
3c
3d
4
>30
Table 2. In Vitro Growth Inhibition of the P. falciparum Strains Dd2
and 3D7
IC50 (µM)
compound
Dd2
3D7
fosmidomycin
FR900098
3b
3c
0.48
0.28
0.48
2.0
0.40
0.24
0.32
2.1
with the handling of the highly analogous P. falciparum enzyme.
The conversion of DOXP to MEP by the enzyme was
determined in an assay based on the NADPH dependency of
the reaction.1 The results are summarized in Table 1. The
initially synthesized racemic trans-cyclopropane 3a displayed
activity in the submicromolar range, which prompted us to
prepare the enantiomerically pure (1R,2S)-analogue 3b. Com-
pared to the racemic mixture this enantiomer displayed a
remarkable activity enhancement, which indicates the preferred
trans-cyclopropane stereochemistry to be (1R,2S). In fact, this
eutomer represents the first synthesized analogue of fosmido-
mycin that exhibits similar activity on the DXR enzyme.
Replacing the acetamide moiety of 3b by a formamide moiety
(3c) caused an 8-fold increase of the IC50 value, while
homologation to the propionamide (3d) reduced the binding
affinity by a factor of more than 150. The racemic R-phenyl-
substituted cis-cyclopropane analogue 4 had no significant
binding activity toward the DXR-enzyme. On the basis of the
observed activity in the enzyme assay, compounds 3b and 3c
were evaluated for their inhibitory effect against intra-
erythrocytic forms of P. falciparum (strains Dd2 and 3D7) using
a semiautomated microdilution assay as described.3 The growth
of the parasites was monitored through the incorporation of
tritium-labeled hypoxanthine. The results obtained are sum-
marized in Table 2.
The (1R,2S)-trans-cyclopropane 3b showed very promising
in vitro antimalarial activity with both P. falciparum strains,
performing equally as well as fosmidomycin. In accordance with
the results obtained in the enzyme inhibition assay, the forma-
mide analogue 3c showed a 5-fold decreased in vitro antimalarial
activity.
In summary a number of conformationally restricted fosmi-
domycin analogues have been synthesized. From this series, the
(1R,2S)-trans-analogue with an N-hydroxy-N-acetamide group
emerged as the best inhibitor of the E. coli DXR enzyme and
proved equally as potent as fosmidomycin in inhibiting P.
falciparum growth.
a Reagents and conditions: (a) (i) n-BuLi, THF, <-70 °C, (ii) 8, <-70
°C, (iii) BF3‚OEt2; (b) KOtBu, THF, 0 °C; (c) (i) n-BuLi, THF, -78 °C,
(ii) BF3‚OEt2, -78 °C; (d) TsCl, Me3N‚HCl, Et3N, 0 °C; (e) BocNHOBn,
NaH, DMF, 50 °C; (f) TFA, CH2Cl2, 0 °C; (g) Ac2O, pyridine, rt; (h)
HCOOH, DCC, pyridine, CHCl3, 0 °C to room temperature; (i) (C2H5CO)2O;
pyridine; rt; (j) H2, Pd/C, rt.
Scheme 2. Synthesis of Substituted cis-Cyclopropyl
Fosmidomycin Analoguea
a Reagents and conditions: (a) (i) n-BuLi, THF, <-70 °C, (ii) 8, <-70
°C, (iii) BF3‚OEt2 <-70 °C; (b) KOtBu, THF, 0 °C; (c) (i) n-BuLi, THF,
-78 °C, (ii) BF3‚OEt2, -78 °C; (d) TsCl, Me3N‚HCl, Et3N, 0 °C; (e)
BocNHOBn, NaH, DMF, 50 °C; (f) TFA, CH2Cl2, 0 °C; (g) Ac2O, pyridine,
rt; (h) H2, Pd/C, rt.
chlorine-assisted deprotection of the phosphonate. Subsequently,
tosylate 11 was converted into Boc-benzyl-protected hydroxyl-
amine 12, which, upon treatment with trifluoroacetic acid in
dichloromethane, gave 13. Acylation of the benzyloxyamine
moiety using different reagents yielded protected hydroxyla-
mides 14a-d. Finally, hydrogenolysis of 14a-d afforded the
free phosphonates 3a-d, which were purified via reversed phase
HPLC.
Application of the above-mentioned strategy starting from
the dibenzylbenzylphosphonate22 led to the synthesis of the
R-phenyl-substituted cyclopropane analogue 4 (Scheme 2). As
opposed to the unsubstituted cyclopropane ring only the cis-
adduct was formed, based on both 1H NMR coupling constants
and a NOESY experiment. This result is in disagreement with
a former study, which claims epoxide opening to yield exclusive
formation of the trans-isomer irrespective of the R-substituent.20
The synthesized compounds were tested for inhibition of
recombinant E. coli DXR because of the difficulties associated
Experimental Section
General Methods and Materials. Melting points were deter-
mined on a Reichert Heizbank type 184321 melting point apparatus
calibrated with acetanilide (mp 114.5 °C) or an Electrothermal
AI9100 digital melting point apparatus and are uncorrected. The
1H, 13C, and 31P NMR spectra were recorded in CDCl3, C6D6,
MeOD, or D2O on a Bruker Avance 300 MHz spectrometer.
Chemical shifts are in parts per million with respect to TMS.
Reversed phase chromatograms were recorded on a Agilent 1100

2658 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Brief Articles
series HPLC system spectrometer with quaternary pump and DAD
detector. Mass spectroscopy spectra were recorded on an Agilent
1100 series single quadrupole spectrometer type VL with API-ES
source. Optical rotations were measured on a Perkin-Elmer 241
polarimeter. Silica gel (60 Å, 0.063-0.200 mm) was purchased
from BioSolve. Preparative reversed phase chromatography was
performed on a Phenomenex Luna C-18 (2) 5 µm particle (21.20
× 250 mm) column using 5 mM NH4OAc solution and MeCN as
solvents. Enantiomeric excess calculation was performed on a
Diacel Chiralcel OD-H (4.6 × 250 mm) column using hexane and
absolute EtOH as isocratic solvents (flow rate ) 1 mL/min).
Tetrahydrofuran was distilled from sodium and benzophenone;
dichloromethane, pyridine, and Et3N were distilled from CaH2
unless otherwise stated. All other solvents and chemicals were used
as purchased unless otherwise stated. n-BuLi was titrated from
diphenylacetic acid prior to use. Reactions were performed under
Ar atmosphere in oven-dried flasks unless otherwise stated.
Dibenzyl 3-Hydroxy-4-(p-toluenesulfonyloxy)butanephos-
phonate (9a). A 2.3 M solution of n-BuLi in hexanes (20.6 mL,
47.4 mmol) was added dropwise to a stirred solution of dibenzyl
methylphosphonate (13.1 g, 47.4 mmol) in dry THF (94 mL) while
the temperature was kept below -70 °C. After 15 min of stirring,
a solution of glycidyl tosylate (7.2 g, 34.5 mmol) in dry THF (16
mL) was added dropwise, followed by BF3‚OEt2 (8.0 mL, 63.2
mmol) with continuous monitoring of the temperature (below -70
°C). The mixture was stirred for 7 h at -78 °C, after which the
reaction was quenched with a NH4Cl solution (200 mL) and allowed
to warm to room temperature. The aqueous phase was extracted
three times with CH2Cl2 (200 mL). The combined organic fractions
were dried on anhydrous MgSO4, the solids were filtered, and
solvents were removed under reduced pressure. The residual oil
was purified via column chromatography (pentane/CH2Cl2/ac-
etone: 6/3/1) giving 8.7 g of a thick gray-white oil in a yield of
twice with CH2Cl2 (250 mL) and twice with CHCl3 (250 mL). The
combined organic fractions were dried on anhydrous MgSO4,
filtered, and evaporated under reduced pressure. The residual oil
was purified via column chromatography (hexane/CH2Cl2/ac-
etone: 2/9/9), giving 1.56 g of a thick colorless oil (46%). 1H NMR
(C6D6, 300.13 MHz) δ 0.46 (1H, dddd, J ) 11.6, 9.2, 5.7 and 4.4
Hz), 0.74 (1H, dddd, J ) 9.2, 5.5, 5.5 and 5.2 Hz), 1.10 (1H, dddd,
J ) 17.7, 8.4, 5.9 and 4.4 Hz), 1.69 (dddddd, J ) 16.0, 8.4, 6.4,
5.4, 5.4 and 5.4 Hz), 1.79 (1H, br s), 3.03 (1H, dd, J ) 11.5 and
6.5 Hz), 3.35 (1H, ddd, J ) 11.5, 5.4 and 2.0 Hz), 4.89 (1H, dd,
J ) 11.8 and 8.5 Hz), 4.95 (1H, dd, J ) 11.8 and 8.5 Hz), 4.96
(1H, dd, J ) 11.9 and 8.5 Hz), 5.04 (1H, dd, J ) 12.2 and 8.5
Hz), 7.00-7.27 (10H, m); 13C NMR (75.47 MHz, C6D6) δ 8.3 (CH2,
1
2
2JPC ) 5.5 Hz), 9.4 (P-CH, JPC ) 195.9 Hz), 19.7 (CH, JPC
)
4.4 Hz), 64.8 (OCH2, 3JPC ) 3.8 Hz), 67.6 (OCH2, 2JPC ) 6.1 Hz),
2
67.7 (OCH2, JPC ) 6.5 Hz), 128.1 (dCH), 128.6 (dCH), 128.8
(dCH), 136.6 (dC); 31P NMR (121.50 MHz, C6D6) δ 45.1; ESMS
m/z 333 ([M] + H+), 665 ([2M] + H+), 687 ([2M] + Na+).
Dibenzyl 2-(p-Toluenesulfonyloxymethyl)cyclopropylphos-
phonate (11a). To a solution of alcohol 10a (380 mg, 1.143 mmol)
in dry CH2Cl2 (5 mL) at 0 °C were added dry Et3N (480 µL, 3.43
mmol) and Me3N.HCl (33 mg, 0.343 mmol). A suspension of TsCl
(327 mg, 1.715 mmol) in dry CH2Cl2 was added at 0 °C, and the
mixture was stirred for 1 h at the same temperature. Saturated
aqueous NH4Cl (70 mL) was added, and the aqueous layer was
extracted three times with CH2Cl2 (70 mL). The combined organic
fractions were dried on anhydrous MgSO4 and filtered, and the
solvents were removed under reduced pressure. The residual oil
was purified via column chromatography (hexane/CH2Cl2/ac-
etone: 3/1/1) yielding 450 mg of a viscous colorless oil (81%). 1H
NMR (300.13 MHz, CDCl3) 0.60-0.82 (2H, m), δ 0.96-1.08 (1H,
m), 1.68 (1H, dddddd, J ) 15.4, 8.4, 6.9, 6.8, 5.4 and 5.4 Hz),
2.34 (3H, s), 3.67 (1H, dd, J ) 10.5 and 7.0 Hz), 3.81 (1H, ddd,
J ) 10.6, 6.7 and 1.6 Hz), 4.89 (2H, dd, J ) 11.9 and 8.4 Hz),
4.95 (1H, dd, J ) 11.4 and 8.4 Hz), 4.96 (1H, dd, J ) 11.4 and
8.3 Hz), 7.22 (12H, m), 7.65 (2H, d, J ) 8.3 Hz); 13C NMR (75.47
1
50%. H NMR (300.13 MHz, CDCl3) δ 1.57-1.97 (4H, m), 2.42
(3H, s), 3.77-3.92 (3H, m), 4.91 (1H, dd, J ) 11.9 and 8.4 Hz),
4.92 (1H, dd, J ) 11.9 and 8.3 Hz), 5.01 (2H, dd, 11.9 and 9.2),
7.29-7.33 (12H, m), 7.76 (2H, d, J ) 8.3 Hz); 13C NMR (75.47
2
1
MHz, CDCl3) 8.7 (CH2, JPC ) 4.9 Hz), δ 10.1 (P-CH, JPC
)
1
2
MHz, CDCl3) δ 21.6 (CH3), 21.9 (PCH2, JPC ) 142.2 Hz), 25.8
195.9 Hz), 15.9 (CH, JPC ) 3.9 Hz), 21.6 (CH3), 67.5 (OCH2,
2
2
2JPC ) 6.1 Hz), 67.7 (OCH2, JPC ) 6.1 Hz), 72.1 (OCH2, JPC
)
2
3
(CH2, JPC ) 4.9 Hz), 67.4 (OCH2, JPC ) 6.0 Hz), 68.8 (OCH,
3JPC ) 13.2 Hz), 72.9 (OCH2), 128.0 (dCH), 128.5 (dCH), 128.6
4.0 Hz), 127.9 (dCH), 128.5 (dCH), 128.6 (dCH), 129.9 (dCH),
3
3
3
(dCH), 129.9 (dCH), 132.7 (dCH), 136.2 (dC, JPC ) 5.5 Hz),
133.0 (dC), 136.2 (dC, JPC ) 6.1 Hz), 136.3 (dC, JPC ) 6.0
Hz), 145.0 (S-Cd); 31P NMR (121.50 MHz, CDCl3) δ 29.0; ESMS
m/z 487 ([M] + H+).
145.0 (dC); 31P NMR (121.50 MHz, CDCl3) δ 33.6; ESMS m/z
505 ([M] + H+).
Dibenzyl 3,4-Epoxybutanephosphonate (5a). To a stirred
solution of 9a (2.4 g, 4.76 mmol) in dry THF (35 mL) was added
KOt-Bu (694 mg, 6.18 mmol) in one portion at 0 °C, and the
mixture was stirred for 3 h at 0 °C. Saturated aqueous NH4Cl (80
mL) was added, and the mixture was allowed to warm to room
temperature. The aqueous layer was extracted three times with
CH2Cl2 (80 mL), the combined organic fractions were dried on
anhydrous MgSO4 and filtered, and the solvents were removed
under reduced pressure. The resulting oil was purified via column
chromatography (hexane/acetone: 3/2) yielding 1.17 g of a pale
Dibenzyl 2-[N-(Benzyloxy),N-(t-butoxycarbonyl)aminomethyl]-
cyclopropylphosphonate (12a). NaH (47.3 mg, 0.987 mmol, 50%
in mineral oil) was added to a solution of tert-butyl N-(benzyloxy)-
carbamate (220 mg, 0.987 mmol) in anhydrous DMF (5 mL), and
the mixture was stirred for 30 min at room temperature. A solution
of 11a (400 mg, 0.822 mmol) in dry DMF (5 mL) was added
dropwise to the solution containing the deprotonated carbamate,
and the reation mixture was stirred for 3 h at 50 °C. The reaction
was quenched with saturated aqueous NH4Cl (40 mL), and the water
phase was extracted three times with CH2Cl2 (40 mL). The
combined organic fractions were dried on anhydrous MgSO4 and
filtered, and the solvents were removed under reduced pressure.
The residual oil was purified via column chromatography (hexane/
CH2Cl2/acetone: 4/1/1) yielding 352 mg of compound 12a as a
1
yellow oil (74%). H NMR (300.13 MHz, CDCl3) δ 1.75-1.93
(4H, m), 2.41 (1H, dd, J ) 4.8 and 2.6 Hz), 2.68-2.70 (1H, m),
2.90 (1H, ddt, J ) 6.1, 4.1 and 2.3 Hz), 4.96 (1H, dd, J ) 11.8
and 8.6 Hz), 4.96 (1H, dd, 11.8 and 8.1 Hz), 5.05 (2H, dd, J )
11.8 and 9.0 Hz), 7.34 (10H, m); 13C NMR (75.47 MHz, CDCl3)
1
colorless oil (80%). H NMR (300.13 MHz, CDCl3) δ 0.72-0.89
1
2
δ 22.3 (P-CH2, JPC ) 142.7 Hz), 25.5 (CH2, JPC ) 4.4 Hz),
(2H, m), 1.10 (1H, dddd, J ) 18.2, 8.5, 5.6 and 4.2 Hz), 1.46 (9H,
s), 1.73 (1H, dddddd, J ) 15.7, 8.3, 6.9, 6.9, 5.5 and 5.5 Hz), 3.29
(2H, d, J ) 6.7 Hz), 4.83 (2H, s), 4.97 (2H, dd, J ) 11.8 and 7.9
Hz), 5.03 (1H, dd, J ) 11.9 and 8.3 Hz), 5.03 (1H, dd, J ) 12.0
and 7.9 Hz), 7.30-7.38 (15H, m); 13C NMR (75.47 MHz, CDCl3)
3
2
47.0 (OCH2), 51.7 (OCH, JPC ) 19.8 Hz), 67.2 (OCH2, JPC
)
6.6 Hz), 67.3 (OCH2, 2JPC ) 6.5 Hz), 128.0 (dCH), 128.5 (dCH),
128.7 (dCH), 136.3 (dC, 3JPC ) 6.0 Hz); 31P NMR (121.50 MHz,
CDCl3) δ 32.2; ESMS m/z 333 ([M] + H+), 355([M] + Na+), 687
([2M] + Na+).
2
1
δ 9.3 (CH2, JPC ) 4.9 Hz), 10.0 (P-CH, JPC ) 195.4 Hz), 15.6
2
3
Dibenzyl 2-(Hydroxymethyl)cyclopropylphosphonate (10a).
A 2.4 M solution of n-BuLi in hexanes (5.11 mL, 12.3 mmol) was
added dropwise to a stirred solution of 5a (3.4 g, 10.2 mmol) in
dry THF at -78 °C. After 5 min of stirring BF3‚OEt2 (2.58 mL,
20.5 mmol) was added dropwise, after which the mixture was stirred
for 15 min at -78 °C. The reaction was quenched with saturated
aqueous NH4Cl (250 mL), and the aqueous phase was extracted
(CH, JPC ) 3.8 Hz), 28.3 (CH3), 52.8 (NCH2, JPC ) 4.8 Hz),
2 2
67.3 (OCH2, JPC ) 6.0 Hz), 67.4 (OCH2, JPC ) 5.5 Hz), 77.2
(OCH2), 81.6 (O-C), 127.8 (dCH), 127.9 (dCH), 128.3 (dCH),
128.5 (dCH), 128.6 (dCH), 129.4 (dCH), 135.6 (dC), 136.5 (d
3
3
C, JPC ) 6.6 Hz), 136.6 (dC, JPC ) 6.6 Hz), 156.6 (N-CdO);
31P NMR (121.50 MHz, CDCl3) δ 30.7; ESMS m/z 438 ([M] -
Boc + 2H+), 538 ([M] + H+).

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2659
Dibenzyl 2-[N-(Benzyloxy)aminomethyl]cyclopropylphosphon-
ate (13a). TFA (2.58 mL, 33.6 mmol) was added dropwise to a
solution of 12a (350 mg, 0.651 mmol) in dry CH2Cl2 (6 mL) at 0
°C, and the mixture was stirred for 45 min at 0 °C. The mixture
was transferred into a separation funnel, and saturated aqueous
NaHCO3 (40 mL) was added. The aqueous layer was extracted
twice with CH2Cl2 (40 mL) and twice with CHCl3 (40 mL). The
combined organic layers were dried on MgSO4 and filtered, and
the solvents were removed under reduced pressure yielding 256
mg of pale yellow solid in a quantitative yield. mp 40-41 °C; 1H
NMR (300.13 MHz, CDCl3) δ 0.61-0.74 (2H, m), 1.04-1.15 (1H,
m), 1.60 (1H, dddddd, J ) 16.1, 8.4, 6.7, 6.7, 5.4 and 5.4 Hz);
2.66 (1H, dd, J ) 13.2 and 6.8 Hz), 2.75 (1H, ddd, J ) 13.2, 6.7
and 1.7 Hz), 4.62 (2H, s), 4.94 (2H, dd, J ) 11.8 and 8.1 Hz),
5.00 (1H, dd, J ) 11.9 and 8.3 Hz), 5.02 (1H, dd, J ) 11.9 and
8.3 Hz), 7.21-7.31 (15H, m); 13C NMR (75.47 MHz, CDCl3) δ
Hz), 173.6 (N-CdO); 31P NMR (121.50 MHz, D2O) δ 22.7; ESMS
m/z 210 ([M] + H+).
Acknowledgment. This study was supported by grants from
the European Commission (QLK2-CT-2002-00887) and INTAS
(03-51-4077). Technical assistance was provided by Dajana
Henschker.
Supporting Information Available: Experimental details (1H,
13C, 31P NMR, MS, HPLC) for intermediates (9b, 5b, 10b-13b,
14b-d, 6, 16-19, and 21) and final products (3b-d, 4); deter-
mination of the relative configuration of the cyclopropane ring. This
material is available free of charge via Internet at http://pubs.acs.org.
References
2
1
9.3 (CH2, JPC ) 5.5 Hz), 10.0 (P-CH, JPC ) 196.0 Hz), 15.9
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(2) Zeidler, J.; Schwender, J.; Muller, C.; Wiesner, J.; Weidemeyer, C.;
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2
3
(CH, JPC ) 3.8 Hz), 55.3 (NCH2, JPC ) 3.8 Hz), 67.3 (OCH2,
2JPC ) 6.0 Hz), 67.4 (OCH2, JPC ) 6.1 Hz), 76.3 (OCH2), 127.8
2
(dCH), 128.3 (dCH), 128.4 (dCH), 128.5 (dCH), 136.6 (dC,
3JPC ) 6.1 Hz), 136.6 (dC, 3JPC ) 6.1 Hz), 137.8 (dC); 31P NMR
(121.50 MHz, CDCl3) δ 31.1; ESMS m/z 438 ([M] + H+), 460
([M] + Na+), 875 ([2M] + H+).
Dibenzyl 2-[N-Acetyl,N-(benzyloxy)aminomethyl]cyclopro-
pylphosphonate (14a). Amine 13a (256 mg, 0.586 mmol) was
dissolved in dry pyridine (6 mL). Ac2O (615 µL, 6.51 mmol) was
added dropwise, and the mixture was stirred overnight at room
temperature. The mixture was transferred to a separation funnel,
and a 1 M HCl solution (70 mL) was added. The aqueous layer
was extracted twice with CH2Cl2 (70 mL) and twice with CHCl3
(70 mL). The combined organic fractions were dried on anhydrous
MgSO4 and filtered, and the solvents were removed on rotary
evaporation. The residual oil was dissolved in CH2Cl2 (40 mL),
saturated aqueous NaHCO3 (40 mL) was added, the layers were
separated, and the aqueous phase was extracted twice with CH2Cl2
(40 mL) and once with CHCl3 (40 mL). The combined organic
layers were dried on anhydrous MgSO4 and filtered, and the solvents
were removed under reduced pressure. The residual oil was purified
via column chromatography (hexane/CH2Cl2/MeOH: 50/48/2)
giving 253 mg of a pale yellow oil in an overall yield of 90%. 1H
NMR (300.13 MHz, CDCl3) δ 0.81 (1H, dddd, J ) 12.0, 9.4, 5.3,
4.3 Hz), 0.89-0.97 (1H, m), 1.11 (1H, dddd, J ) 18.2, 8.4, 5.7
and 4.4 Hz), 1.73 (1H, dddddd, J ) 15.7, 8.4, 7.0, 6.8, 5.4 and 5.4
Hz), 2.00 (3H, s), 3.47 (1H, dd, J ) 14.9 and 7.4 Hz), 3.53 (1H,
ddd, J ) 14.9, 6.6 and 1.8 Hz), 4.79 (2H, s), 4.95 (1H, dd, J )
12.1 and 7.7 Hz); 4.97 (1H, dd, J ) 11.8 and 8.4 Hz), 5.02 (1H,
dd, J ) 11.8 and 8.4 Hz), 5.03 (1H, dd, J ) 12.3 and 8.4 Hz),
7.19-7.30 (15H, m); 13C NMR (75.47 MHz, CDCl3) δ 9.4 (CH2,
(3) Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.; Weidemeyer,
C.; Hintz, M.; Turbachova, I.; Eberl, M.; Zeidler, J.; Lichtenthaler,
H. K.; Soldati, D.; Beck, E. Inhibitors of the nonmevalonate pathway
of isoprenoid biosynthesis as antimalarial drugs. Science 1999, 285,
1573-1576.
(4) Lell, B.; Ruangweerayut, R.; Wiesner, J.; Missinou, M. A.; Schindler,
A.; Baranek, T.; Hintz, M.; Hutchinson, D.; Jomaa, H.; Kremsner,
P. G. Fosmidomycin, a novel chemotherapeutic agent for malaria.
Antimicrob. Agents Chemother. 2003, 47, 735-738.
(5) Missinou, M.; Borrmann, S.; Schindler, A.; Issifou, S.; Adegnika,
A. A.; Matsiegui, P. B.; Binder, R.; Lell, B.; Wiesner, J.; Baranek,
T.; Jomaa, H.; Kremsner, P. G. Fosmidomycin for malaria. Lancet
2002, 360, 1941-1942.
(6) Borrmann, S.; Issifou, S.; Esser, G.; Adegnika, A. A.; Ramharter,
M.; Matsiegui, P. B.; Oyakhirome, S.; Mawili-Mboumba, D. P.;
Missinou, M. A.; Kun, J. F. J.; Jomaa, H.; Kremsner, P. G.
Fosmidomycin-clindamycin for the treatment of Plasmodium falci-
parum malaria. J. Infect. Dis. 2004, 190, 1534-1540.
(7) Borrmann, S.; Adegnika, A. A.; Matsiegui, P. B.; Issifou, S.;
Schindler, A.; Mawili-Mboumba, D. P.; Baranek, T.; Wiesner, J.;
Jomaa, H.; Kremsner, P. G. Fosmidomycin-clindamycin for Plas-
modium falciparum infections in African children. J. Infect. Dis. 2004,
189, 901-908.
(8) Borrmann, S.; Adegnika, A. A.; Moussavou, F.; Oyakhirome, S.;
Esser, G.; Matsiegui, P. B.; Ramharter, M.; Lundgren, I.; Kombila,
M.; Issifou, S.; Hutchinson, D.; Wiesner, J.; Jomaa, H.; Kremsner,
P. G. Short-course regimens of artesunate-fosmidomycin in treatment
of uncomplicated Plasmodium falciparum malaria. Antimicrob Agents
Chemother. 2005, 49, 3749-3754
(9) Steinbacher, S.; Kaiser, J.; Eisenreich, W.; Huber, R.; Bacher, A.;
Rohdich, F. Structural basis of fosmidomycin action revealed by the
complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC)
- Implications for the catalytic mechanism and anti-malaria drug
development. J. Biol. Chem. 2003, 278, 18401-18407.
(10) MacSweeney, A.; Lange, R.; Fernandes, R. P. M.; Schulz, H.; Dale,
G. E.; Douangamath, A.; Proteau, P. J.; Oefner, C. The crystal
structure of E. coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase
in a ternary complex with the antimalarial compound fosmidomycin
and NADPH reveals a tight-binding closed enzyme conformation.
J. Mol. Biol. 2005, 345, 115-127.
(11) Silber, K.; Heidler, P.; Kurz, T.; Klebe, G. AFMoC enhances
predictivity of 3D QSAR: A case study with DOXP-reducto-
isomerase. J. Med. Chem. 2005, 48, 3547-3563.
(12) Ortmann, R.; Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck,
E.; Jomaa, H.; Schlitzer, M. Alkoxycarbonyloxyethyl ester prodrugs
of FR900098 with improved in vivo antimalarial activity. Arch.
Pharm. 2005, 338, 305-314.
(13) Ortmann, R.; Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck,
E.; Jomaa, H.; Schlitzer, M. Acyloxyalkyl ester Prodrugs of
FR900098 with improved in vivo anti-malarial activity. Bioorg. Med.
Chem. Lett. 2003, 13, 2163-2166.
2JPC ) 5.0 Hz), 10.4 (P-CH, 1JPC ) 185.4 Hz), 15.8 (CH, 2JPC
)
2
3.8 Hz), 20.4 (CH3), 50.0 (NCH2), 67.3 (OCH2, JPC ) 6.0 Hz)
2
67.4 (OCH2, JPC ) 6.0 Hz), 76.9 (OCH2), 127.7 (dCH), 127.8
(dCH), 128.3 (dCH), 128.5 (dCH), 128.7 (dCH), 129.0 (dCH),
3
129.1 (dCH), 134.4 (dC), 136.5 (dC, JPC ) 6.1 Hz), 136.6 (d
3
C, JPC ) 6.6 Hz), 167.8 (N-CdO); 31P NMR (121.50 MHz,
CDCl3) δ 30.5; ESMS m/z 480 ([M] + H+).
2-[N-(Acetyl),N-(hydroxy)aminomethyl]-cyclopropylphospho-
nic Acid (3a). To a solution of 14a (253 mg, 0.528 mmol) in MeOH
(5 mL) was added Pd/C (100 mg, 10%). The reaction was placed
under H2 (1 atm) and was stirred overnight at room temperature.
The mixture was filtered over Celite, and the Celite was washed
with portions of MeOH. The filtrate was evaporated under reduced
pressure; the residual oil was dissolved in a minimal amount of
water and lyophilized. The resulting solid was purified via reversed
phase HPLC using a gradient elution of 5 mM NH4OAc solution
to MeCN in 20 min. The appropriate fractions were lyophilized,
giving 80 mg of an amber amorphous solid in a yield of 72%. mp
100-102 °C;1H NMR (300.13 MHz, MeOD) δ 0.45 (1H, m), 0.57
(1H, m), 0.77 (1H, m), 1.27 (1H, m), 1.95 (3H, s), 3.14-3.21 (1H,
m), 3.53 (1H, dd, J ) 14.3 and 5.3 Hz); 13C NMR (75.47 MHz,
(14) Reichenberg, A.; Wiesner, J.; Weidemeyer, C.; Dreiseidler, E.;
Sanderbrand, S.; Altincicek, B.; Beck, E.; Schlitzer, M.; Jomaa, H.
Diaryl ester prodrugs of FR900098 with improved in vivo antimalarial
activity. Bioorg. Med. Chem. Lett. 2001, 11, 833-835.
2
1
MeOD) δ 9.6 (CH2, JPC ) 5.1 Hz), 14.6 (CH, JPC ) 177.8 Hz),
2
3
15.8 (CH, JPC ) 6.1 Hz), 20.2 (CH3), 52.7 (NCH2, JPC ) 3.3

2660 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Brief Articles
(15) Ling, Y.; Sahota, G.; Odeh, S.; Chan, J. M. W.; Araujo, F. G.;
Moreno, S. N. J.; Oldfield, E. Bisphosphonate inhibitors of Toxo-
plasma gondi growth: In vitro, QSAR, and in vivo investigations.
J. Med. Chem. 2005, 48, 3130-3140.
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Convenient catalysts for the synthesis of methylphosphonates. J. Org.
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(20) Hah, J. H.; Gil, J. M.; Oh, D. Y. The stereoselective synthesis of
cyclopropylphosphonate analogs of nucleotides. Tetrahedron Lett.
1999, 40, 8235-8238.
(21) Yoshida, Y.; Sakakura, Y.; Aso, N.; Okada, S.; Tanabe, Y. Practical
and efficient methods for sulfonylation of alcohols using Ts(Ms)Cl/
Et3N and catalytic Me3N‚HCl as combined base: Promising
alternative to traditional pyridine. Tetrahedron 1999, 55, 2183-2192.
(22) Saady, M.; Lebeau, L.; Mioskowski, C. First Use of Benzyl
Phosphites in the Michaelis-Arbuzov Reaction - Synthesis of
Monophosphate, Diphosphate, and Triphosphate Analogs. HelV.
Chim. Acta 1995, 78, 670-678.
(16) Kurz, T.; Geffken, D.; Wackendorff, C. Carboxylic acid analogues
of fosmidomycin. Z. Naturforsch., B: Chem. Sci. 2003, 58, 457-
461.
(17) Courtois, M.; Mincheva, Z.; Andreu, F.; Rideau, M.; Viaud-Massuard,
M. C. Synthesis and biological evaluation with plant cells of new
fosmidomycin analogues containing a benzoxazolone or oxazolopy-
ridinone ring. J. Enzyme Inhib. Med. Chem. 2004, 19, 559-565.
(18) Kuntz, L.; Tritsch, D.; Grosdemange-Billiard, C.; Hemmerlin, A.;
Willem, A.; Bacht, T. J.; Rohmer, M. Isoprenoid biosynthesis as a
target for antibacterial and antiparasitic drugs: phosphonohydroxamic
acids as inhibitors of deoxyxylulose phosphate reducto-isomerase.
Biochem. J. 2005, 386, 127-135.
JM051177C