γ-substituted bis(pivaloyloxymethyl)ester analogues of fosmidomycin and FR900098

Article abstract of DOI:10.1002/ardp.200700107

The synthesis and in-vitro antimalarial activity of γ-substituted bis(pivaloyloxymethyl)ester analogues of the drug candidate fosmidomycin have been investigated. In contrast to the high antimalarial activity of α-aryl substituted fosmidomycin analogues l

Full text of DOI:10.1002/ardp.200700107

Arch. Pharm. Chem. Life Sci. 2007, 340, 661–666
T. Kurz et al.
661
Full Paper
c-Substituted Bis(pivaloyloxymethyl)ester Analogues of
Fosmidomycin and FR900098
Thomas Kurz1, Christoph Behrendt1, Miriam Pein1, Uwe Kaula1, Bꢀrbel Bergmann2 and
Rolf D. Walter2
1 Institute of Pharmacy, University of Hamburg, Hamburg, Germany
2 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
The synthesis and in-vitro antimalarial activity of c-substituted bis(pivaloyloxymethyl)ester ana-
logues of the drug candidate fosmidomycin have been investigated. In contrast to the high anti-
malarial activity of a-aryl substituted fosmidomycin analogues like a-phenylfosmidomycin, c-
substituted derivatives display only weak to moderate activity against the chloroquine-sensitive
strain 3D7 of Plasmodium falciparum.
Keywords: Fosmidomycin / Malaria tropica / Plasmodium falciparum / c-Substituted analogues /
Received: May 18, 2007; accepted: August 24, 2007
DOI 10.1002/ardp.200700107
(DXR), the second enzyme of the DOXP/MEP pathway [3].
Clinical trials conducted with fosmidomycin in combina-
tion with clindamycin [4] have already demonstrated
high efficiency in the treatment of acute, uncomplicated
Malaria tropica. However, a disadvantage of fosmidomy-
cin is its quite poor oral bioavailability of approximately
30% [2]. Recently, Schlitzer et al. and our group have
shown that the hygroscopicity of phosphonohydroxamic
acids can be overcome by masking the phosphonic acid
moiety as bis(pivaloyloxymethyl) esters [5, 6].
In order to improve fosmidomycin's 1 (Fig. 1) antima-
larial activity and physicochemical properties, various
chemical modifications have been realised. The hydroxa-
mic acid group [7], the phosphonic acid moiety [8] and
the propyl chain are key structural elements for distinct
antimalarial activity [9]. In addition, Schlitzer and Ort-
mann have demonstrated, that prodrugs of FR900098 2
(Fig. 1) exhibit improved in-vivo antimalarial activity
against Plasmodium vinckei in the mouse model [6].
Recently, modifications of the carbon spacer have been
reported. We have discovered that a-phenylfosmidomy-
cin (3, IC50: 0.4 lM; Fig. 1) displays potent antimalarial
activity against the chloroquine-resistant strain Dd2 of
Plasmodium falciparum [10]. Van Calenbergh et al. con-
firmed the high activity of aryl substituted fosmidomy-
cin analogues and also described cyclopropyl analogues
with potent activity [11]. In a previous publication [5], we
reported that the non-hygroscopic bis(pivaloyloxy-
Introduction
The discovery of the (1-deoxy-D-xylulose 5-phosphate/2-C-
methyl-D-erythritol 4-phosphate) DOXP/MEP pathway of
isoprenoid biosynthesis, which is for instance present in
the malaria parasite Plasmodium falciparum and in several
pathogenic bacteria or higher plants but not in humans,
has stimulated intensive research activities in medicinal
and agricultural chemistry [1]. In Plasmodium falciparum
the DOXP/MEP pathway is located in a plastid-like organ-
elle termed apicoplast that is surrounded by four mem-
branes [2]. The DOXP/MEP pathway leads to isopentenyl
diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP), the common precursors of all isoprenoids [2].
Since the resistance of Plasmodium falciparum to estab-
lished antimalarial drugs steadily increases, new classes
of compounds displaying novel modes of action have to
be developed. The phosphonohydroxamic acid antibiot-
ics fosmidomycin and FR900098 are potent inhibitors of
the
1-deoxy-D-xylulose-5-phosphate-reductoisomerase
Correspondence: Thomas Kurz, Institute of Pharmacy, University of
Hamburg, Bundesstraße 45, 20146 Hamburg, Germany.
E-mail: kurz@chemie.uni-hamburg.de
Fax: +49 40 4283 86573
Abbreviations: (1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol
4-phosphate) DOXP/MEP; 1-deoxy-D-xylulose-5-phosphate-reductoiso-
merase (DXR); bis(pivaloyloxymethyl) ester analogue (3-POM)
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T. Kurz et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 661–666
Figure 1. Fosmidomycin and analogues.
methyl) ester analogue (3-POM, Table 1) of a-phenylfosmi-
domycin 3 displays an IC50 value of 0.6 lM towards the
chloroquine-sensitive strain 3D7 of Plasmodium falcipa-
rum, which is in good accordance with the activity of the
free acid [5, 10]. However, the most active aryl-analogues
reported so far bear a 3,4-dihalophenyl-substituent in the
a-position of the propyl-spacer [5, 10, 11]. Furthermore,
we have shown that a 3,4-dichlorobenzyl-substituent in
the a-position of fosmidomycin's propyl chain leads to
high in-vitro antimalarial activity against the 3D7 strain
(IC50 value of 0.9 lM), while the introduction of electron-
donating substituents on the phenyl nucleus diminishes
the activity [5]. Conformationally restrained bis(pivaloy-
loxymethyl) ester analogues of 1 and 2 recently investi-
gated exhibit IC50 values between 47–61 lM against
strain 3D7 [12]. In order to obtain further insights of the
structure-activity relationships, we now describe the syn-
thesis and in-vitro antimalarial activity of c-substituted
bis(pivaloyloxymethyl) ester analogues of the natural
products fosmidomycin 1 and FR900098 2.
Scheme 1. Synthesis of c-substituted analogues.
Table 1. Inhibition of P. falciparum growth.
Compound
R
R1
100 lM [%]a)
3-POM
11a
11b
12a
12b
H
H
Me
H
Me
H
100
38
50
Me
Me
Ph
Ph
60
26
a)
Mean values of two independent determinations.
phosphite in ethanol followed by acidic hydrolysis
according to a procedure of Harvey [13]. Reactions of 5
with O-benzylhydroxylamine and subsequent reduction
of the resulting oximes with sodium cyanoborohydride
in presence of hydrochloric acid provided O-benzyl pro-
tected hydroxylamines 6. Acetylation and formylation of
6 furnished aceto- and formohydroxamic acids (7 and 8).
Transformation of compounds 7 and 8 into the corre-
sponding bis(pivaloyloxymethyl) esters (9 and 10) was
accomplished by ester cleavage with trimethylsilyl bro-
mide and subsequent alkylation with chloromethyl piva-
late in DMF using triethylamine as a base. Finally, cata-
lytic hydrogenation afforded the target compounds 11,
12 in almost quantitative yields.
Results and discussion
Chemistry
Since a-substituted analogues of 1 and 2 are accessible
from a,b-unsaturated aldehydes [5], we have chosen a,b-
unsaturated ketones as starting materials for the prepa-
ration of c-substituted phosphonohydroxamic acids 11
and 12 (Scheme 1). Starting materials 5 were synthesized
by treatment of a,b-unsaturated ketones with triethyl
Biological activity
The in-vitro antimalarial activity of compounds 11a, b
and 12a, b was determined by an 8-[3H]hypoxanthine
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Arch. Pharm. Chem. Life Sci. 2007, 340, 661–666
c-Substituted Fosmidomycin and FR900098 Analogues
663
resulting solution treated with NaCNBH3 (90 mmol). HCl (37%,
30 mL) was added dropwise under ice cooling over a period of
30 min. The mixture was allowed to warm up to room tempera-
ture, followed by treatment with additional NaCNBH3
(20 mmol). After an overall time of 2 h, the solution was concen-
trated and a solution of KOH (10%) was added under ice cooling
until an alkaline pH was detected. The aqueous solution was
extracted with CH2Cl2 (3650 mL). The organic layers were com-
bined, dried with MgSO4 and evaporated. The residue was puri-
fied by column chromatography on silica gel using EtOAc/MeOH
(90 : 10) as an eluent to yield compounds 6a and 6b as colourless
oils.
incorporation assay [14] using the chloroquine-sensitive
strain 3D7 of Plasmodium falciparum. Inhibition of para-
site growth at 100 lM has been evaluated. The bis(piva-
loyloxymethyl) ester of a-phenylfosmidomycin 3 was
used as reference compound (Table 1: 3-POM). All bis(pi-
valoyloxymethyl) esters were converted into phosphonic
acids by non-specific esterases during the experiments.
The in-vitro studies have demonstrated that the intro-
duction of a methyl group as well as a phenyl substituent
in the c-position of the propyl chain of 1 and 2 led to a
significant reduction of antimalarial activity. Only
incomplete inhibition of Plasmodium falciparum growth
was detected at a concentration of 100 lM (Table 1). The
low activity may be due to a sterically hindered coordina-
tion of the divalent cation in the active site of the DXR by
the hydroxamic acid functionality of compounds 11 and
12.
(3-Benzyloxyamino-butyl)-phosphonic acid diethyl ester
6a
Yield 93%, IR mmax (KBr) cm-1: 3236 (N-H) and 1246 (P=O); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 7.36-7.25 (m, 5H), 6.44 (d, 1H, J =
5.9 Hz), 4.60 (s, 2H), 4.01-3.92 (m, 4H), 2.99-2.90 (m, 1H), 1.79-1.60
(m, 3H), 1.54-1.43 (m, 1H), 1.21 (t, 6H, J = 7.1 Hz), 0.96 (d, 3H, J =
6.4 Hz); 13C-NMR (100 MHz, DMSO-d6), d (ppm): 138.74, 128.46,
2
3
128.36, 127.75, 75.97, 61.15 (d, JC-P 6.1 Hz), 55.53 (d, JC-P
=
=
17.3 Hz), 26.39 (d, JC-P = 4.6 Hz), 21.35 (d, 1JC-P = 139 9 Hz), 17.50,
2
16.64 (d, JC-P = 5.6 Hz); 31P-NMR (202 MHz, DMSO-d6), d (ppm):
3
Conclusions
33.7; Found: C 56.91, H 8.45, N 4.33%. C15H26NO4P requires C
57.13, H 8.31, N 4.44%.
This paper describes the synthesis and in-vitro antimalar-
ial activity of stable and non-hygroscopic, c-substituted
bis(pivaloyloxymethyl) ester analogues of fosmidomycin
1 and FR900098 2 and contributes to ongoing structure-
activity studies. In contrast to a-aryl substituted ana-
logues, which display high activity, c-substituted deriv-
atives possess only weak antimalarial activity. The bis(pi-
valoyloxymethyl) esters are converted into the corre-
sponding acids by non-specific plasma esterases during
the determination as shown in previous publications.
(3-Benzyloxyamino-3-phenyl-propyl)-phosphonic acid
diethyl ester 6b
Yield 81%, IR mmax (KBr) cm– 1: 3234 (N-H) and 1243 (P=O); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 7.35–7.20 (m, 10H), 6.94 (d, 1H, J =
6.62 Hz), 4.53 (dd, 2H, JAB = 11.5 Hz), 4.0–3.86 (m, 5H), 2.01–1.90
(m, 1H), 1.74–1.61 (m, 2H), 1.50–1.38 (m, 1H), 1.18 (t, 6H, J =
7.1 Hz); 13C-NMR (100 MHz, DMSO-d6), d (ppm): 142.11, 138.49,
128.48, 128.42, 128.40, 127.92, 127.77, 127.49, 75.77, 64.86 (d,
3JC-P = 17.3 Hz), 61.23 (d, 2JC-P = 6.6 Hz), 26.56 (d, 2JC-P = 4.1 Hz), 21.93
1
3
(d, JC-P = 140.4 Hz), 16.60 (d, JC-P = 5.6 Hz); 31P-NMR (202 MHz,
DMSO-d6), d (ppm): 32.7; Found: C 63.38, H 7.40, N 3.38%.
C20H28NO4P requires C 63.65, H 7.48, N 3.71%.
The authors have declared no conflict of interest.
General procedure for the synthesis of compounds 7a, b
Formic acid (500 mmol) was treated with acetic acid anhydride
(50 mmol) and stirred under exclusion of humidity. After
20 min, the solution was cooled to 08C, and the respective
hydroxylamine 6a, b (10 mmol), dissolved in dry THF (20 mL),
was added dropwise. After 10 min, the mixture was allowed to
warm up to room temperature and stirred for another hour. The
solution was treated with EtOAc (200 mL) and successively
washed with water (3650 mL), with aqueous KOH (0.1 M, 3625
mL) and once again with water. The organic layer was dried over
MgSO4 and the solvent was removed under reduced pressure.
The residue was purified by column chromatography on silica
gel with EtOAc as an eluent to yield compound 7a as a pale yel-
low and compound 7b as colourless oil.
Experimental
Elemental analysis was carried out with a Heraeus CHN-O-Rapid
instrument (W.C. Heraeus, Hanau, Germany). IR spectra were
recorded on a Shimadzu FT-IR 8300 (Shimadzu, Tokyo, Japan).
1H-NMR (400 MHz) and13C-NMR (100 MHz) spectra were recorded
on a Bruker AMX 400 spectrometer (Bruker Bioscience, Billerica,
MA, USA) using tetramethylsilane as internal standard and
DMSO-d6 or CDCl3 as solvents. Mass spectra were recorded on a
Micromass VG 70-250S mass spectrometer (HRFAB; Micromass,
Manchester, UK), a Finnegan MAT 311 A mass spectrometer (EI;
Thermo Electron Corporation, Bremen, Germany) or a Varian
MS 1200L mass spectrometer (ESI; Varian Inc., Palo Alto, CA,
USA).
[3-(Benzyloxy-formyl-amino)-butyl]-phosphonic acid
Chemistry
diethyl ester 7a
Yield 96%, IR mmax (KBr) cm– 1: 1679 (C=O) and 1243 (P=O); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 8.51–8.01 (m, 1H), 7.46–7.36 (m,
5H), 4.96 (s, 2H), 4.33–3.75 (m, 5H), 1.93–1.53 (m, 4H), 1.24 (d,
3H, J = 6.6 Hz), 1.20 (t, 6H, J = 7.1 Hz);13C-NMR (100 MHz, CDCl3), d
General procedure for the synthesis of compounds 6a, b
A solution of the respective ketone 5 (30 mmol) in MeOH (20 mL)
was reacted with O-benzylhydroxylamine (30 mmol) and stirred
at 608C for 1 h. Afterwards, MeOH (430 mL) was added and the
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Arch. Pharm. Chem. Life Sci. 2007, 340, 661–666
(ppm): 164.77, 134.55, 129.27, 129.02, 128.70, 80.82, 61.69 (d, 2JC-P
= 6.1 Hz), 53.62, 26.83 (d, 2JC-P = 3.1 Hz), 22.95 (d, 1JC-P = 145.98 Hz),
18.38, 16.44 (d, 3JC-P = 6.1 Hz); HRFAB-MS C16H26NO5P MW 343.36,
[M+H]+: calculated 344,1627; found 344, 1653.
08C for one hour and was allowed to warm up to room temper-
ature. After 23 h reaction time, the solvent was evaporated and
the residue was dissolved in THF (10 mL). The solution was
treated with water (0.1 mL) and stirred for further 5 min before
the solvent was removed under reduced pressure to yield the
free phosphonic acids.
The resulting crude phosphonic acids were dissolved in anhy-
drous DMF (20 L), treated with TEA (30 mmol) and chloromethyl
pivalate (9 mmol). The mixture was stirred at 708C for 2 h
excluding atmospheric moisture. Another 3 mmol of TEA were
added and the solution was stirred over night. The reaction mix-
ture was treated with Et2O (100 mL) and washed with water
(50 mL), with a saturated aqueous NaHCO3-solution (50 mL) and
once again with water (50 mL). The organic layer was dried over
Na2SO4, the solvent evaporated and the residue purified by col-
umn chromatography on silica gel with Et2O as an eluent to
yield compounds 9a, b and 10a, b as colourless oils.
[3-(Benzyloxy-formyl-amino)-3-phenyl-propy]-
phosphonic acid diethyl ester 7b
Yield 91%, IR mmax (KBr) cm– 1: 1679 (C=O) and 1238 (P=O); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 8.40 (s, 1H), 7.44–7.29 (m, 10H),
5.24–4.97 (m, 1H), 4.93 (d, 1H, J = 9.2 Hz), 4.61–4.46 (m, 1H),
4.03–3.93 (m, 4H), 2.41–2.30 (m, 1H), 2.19–2.06 (m, 1H), 1.79–
1.67 (m, 2H), 1.21 (t, 6H, J = 7.1 Hz); 13C-NMR (100 MHz, CDCl3), d
(ppm): 164.15, 137.53, 134.39, 129.37, 128.97, 128.86, 128.58,
128.27, 80.14, 61.72 (d, 2JC-P = 6.1 Hz), 59.58, 23.62, 22.78 (d, 1JC-P
=
142.4 Hz), 16.43 (d, 3JC-P = 6.1 Hz); Found: C 61.86, H 7.00, N 3.56%.
C21H28NO5P requires C 62.21, H 6.96, N 3.45%.
General procedure for the synthesis of compounds 8a, b
Acetic acid anhydride (20 mmol) was added to a solution of the
respective hydroxylamine 6a, b (10 mmol) in dry THF (10 mL)
and stirred at room temperature for 2 h. After addition of EtOAc
(100 mL), the organic layer was washed with aqueous KOH
(0.1 M, 2650 mL), water (50 mL) and with aqueous HCl (1 M,
3620 mL). The organic layer was dried over MgSO4, evaporated,
and the residue was purified by column chromatography on
silica gel with EtOAc as an eluent to yield compound 8a as a pale
yellow and compound 8b as colourless oil.
2,2-Dimethyl-propionic acid [3-(benzyloxy-formyl-amino)-
butyl]-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 9a
Yield 34%, IR mmax (KBr) cm– 1: 1753 (C=O, ester), 1680 (C=O,
1
hydroxamic acid) and 1252 (P=O); H-NMR (400 MHz, CDCl3), d
(ppm): 8.32–8.04 (m, 1H), 7.47–7.33 (m, 5H), 5.69–5.63 (m, 4H),
4.97–4.86 (m, 2H), 4.40–3.72 (m, 1H), 2.05–1.73 (m, 4H), 1.32
(m, 3H), 1.22 (s, 18H); 13C-NMR (100 MHz, CDCl3), d (ppm): 176.86,
2
164.80, 134.46, 129.35, 129.09, 128.73, 81.44 (d, JC-P = 5.1 Hz) ,
81.38 (d, 2JC-P = 6.1 Hz), 80.90, 53.25, 38.73, 26.85, 26.33, 23.84 (d,
1JC-P = 141.4 Hz), 18.28; ESI-MS C24H38NO9P, MW 515.55, [M+Na]+:
calculated 538; found 538.
[3-(Acetyl-benzyloxy-amino)-butyl]-phosphonic acid
diethyl ester 8a
Yield 85%, IR mmax (KBr) cm– 1: 1665 (C=O) and 1246 (P=O); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 7.46–7.36 (m, 5H), 4.93 (dd, 2H, JA-B
= 22.1 Hz), 4.39–4.21 (m, 1H), 4.01–3.91 (m, 4H), 2.11 (s, 3H),
1.95–1.83 (m, 1H), 1.79–1.61 (m, 3H), 1.21 + 1.20 (2t, 6H, J =
7.1 Hz); 13C-NMR (100 MHz, CDCl3), d (ppm): 174.24, 134.59,
128.88, 128.75, 128.70, 79.08, 61.60 (d, 2JC-P = 6.1 Hz), 61.57 (d, 2JC-P
= 6.1 Hz), 54.60 (d, 3JC-P = 16.3 Hz), 27.00 (d, 2JC-P = 4.6 Hz), 23.02 (d,
2,2-Dimethyl-propionic acid [3-(benzyloxy-formyl-amino)-
3-phenyl-propyl]-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 9b
Yield 27%, IR mmax (KBr) cm– 1: 1753 (C=O, ester), 1680 (C=O,
1
hydroxamic acid) and 1256 (P=O); H-NMR (400 MHz, CDCl3), d
(ppm): 8.23 (s, 1H), 7.47–7.30 (m, 8H), 7.23–7.13 (m, 2H), 5.67 (d,
3
1JC-P = 142.4 Hz), 21.20, 18.47, 16.43 (d, JC-P = 6.1 Hz); HRFAB-MS
3
4H, JH-P = 13 Hz), 5.50–5.20 (m, 1H), 4.60–4.31 (m, 2H), 2.55–
C17H28NO5P MW 357.39, [M+H]+: calculated 358.1783; found
358,1773.
2.28 (m, 2H), 1.94–1.72 (m, 2H), 1.19 + 1.18 (2s, 18H); 13C-NMR
(100 MHz, CDCl3), d (ppm): 176.87, 176.84, 163.97, 137.11,
134.32, 129.45, 129.00, 128.92, 128.69, 128.62, 128.32, 81.45 (d,
2JC-P = 6.1 Hz), 79.96, 59.34, 38.70, 26.81, 23.82 (d, 1JC-P = 142.4 Hz),
23.23; ESI-MS C29H40NO9P, MW 577.62, [M+H]+: calculated 578;
found 578.
[3-(Acetyl-benzyloxy-amino)-3-phenyl-propyl]-
phosphonic acid diethyl ester 8b
Yield 88%, IR mmax (KBr) cm– 1: 1665 (C=O) and 1242 (P=O); 1H-NMR
(400 MHz, DMSO-d6), d (ppm): 7.43–7.29 (m, 10H), 5.44 (t, 1H, J =
7.7 Hz), 4.81 (d, 1H, J = 9.4 Hz), 4.54 (d, 1H, J = 9.4 Hz), 4.06–3.91
(m, 4H), 2.43–2.31 (m, 1H), 2.26–2.09 (m, 4H), 1.84–1.64 (m, 2H),
1.21 (t, 6H, J = 7.1 Hz);13C-NMR (100 MHz, CDCl3), d (ppm): 173.60,
138.36, 134.34, 128.86, 128.81, 128.69, 128.58, 128.51, 128.21,
78.75, 61.66 (d, 2JC-P = 7.1 Hz), 60.21 (d, 3JC-P = 19.3 Hz), 23.40 (d, 2JC-P
= 3.1 Hz), 22.95 (d, 1JC-P = 142.4 Hz), 21.05, 16.42 (d, 3JC-P = 6.1 Hz);
Found: C 62.75, H 7.17, N 3.27%. C22H30NO5P calc. C 63.00, H 7.21,
N 3.34%.
2,2-Dimethyl-propionic acid [3-(acetyl-benzyloxy-amino)-
butyl]-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 10a
Yield 41%, IR mmax (KBr) cm– 1: 1752 (C=O, ester), 1668 (C=O,
hydroxamic acid) and 1261 (P=O);1H-NMR (400 MHz, DMSO-d6), d
(ppm): 7.45–7.37 (m, 5H), 5.63–5.55 (m, 4H), 4.91 (dd, 2H, JAB
=
19.7 Hz), 4.36–4.23 (m, 1H), 2.10 (s, 3H), 1.89–1.78 (m, 3H),
1.73–1.62 (m, 1H), 1.21 (d, 3H, J = 6.9 Hz), 1.14 (s, 18H); 13C-NMR
(100 MHz, CDCl3), d (ppm): 176.86, 174.28, 134.46, 128.93,
2
2
General procedure for the synthesis of
bis(pivaloyloxymethyl)esters 9a-b, 10a-b
To a stirred solution of the respective phosphonic acid diethyl
esters 7 or 8 (3 mmol) in anhydrous CH2Cl2 (20 mL) was added tri-
methylsilyl bromide (1.5 mL) at 08C. The solution was stirred at
128.81, 128.74, 82.42 (d, JC-P = 7.1 Hz), 81.35 (d, JC-P = 6.1 Hz),
3
2
79.21, 54.28 (d, JC-P = 21.4 Hz), 38.71, 26.84, 26.49 (d, JC-P
=
4.1 Hz), 23.94 (d, 1JC-P = 141.4 Hz), 21.19, 18.38; MW 529.57, Anal.
Calc. for C25H40NO9P: C 56.70, H 7.61, N 2.64%. Found: C 56.48, H
7.72, N 2.77%.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 661–666
c-Substituted Fosmidomycin and FR900098 Analogues
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1H), 1.97 (s, 3H), 1.84–1.48 (m, 4H), 1.17 (s, 18H), 1.03 (d, 3H, J =
6.6 Hz); 13C-NMR (100 MHz, CDCl3), d (ppm): 176.95, 176.88,
2,2-Dimethyl-propionic acid [3-(acetyl-benzyloxy-amino)-
3-phenyl-propyl]-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 10b
2
3
173.05, 81.56 (d, JC-P = 6.1 Hz), 51.39 (d, JC-P = 8.1 Hz), 38.78,
2
1
38.75, 26.86, 26.83, 24.36 (d, JC-P = 5.1 Hz), 22.64 (d, JC-P
=
Yield 60%, IR mmax (KBr) cm– 1: 1753 (C=O, ester), 1667 (C=O,
hydroxamic acid) and 1257 (P=O); 1H-NMR (400 MHz, DMSO-d6), d
(ppm): 7.41–7.30 (m, 10H), 5.64–5.56 (m, 4H), 5.41 (t, 1H, J =
7.6 Hz), 4.78 (d, 1H, J = 9.7 Hz), 4.48 (d, 1H, J = 9.7 Hz), 2.37–2.16
(m, 2H), 2.13 (s, 3H), 1.99–1.73 (m, 2H), 1.12 (s, 9H), 1.10 (s, 9H);
13C-NMR (100 MHz, CDCl3), d (ppm): 176.83, 173.56, 137.84,
134.25, 128.94, 128.84, 128.76, 128.60, 128.59, 128.33, 81.42 (d,
2JC-P = 6.1 Hz), 78.83, 60.03 (d, 3JC-P = 20.3 Hz), 38.70, 38.67, 26.84,
140.4 Hz), 20.87, 17.25; MW 439.45, Anal. Calc. for C18H34NO9P: C
49.20, H 7.80, N 3.19%. Found: C 48.60, H 8.00, N 3.08%.
2,2-Dimethyl-propionic acid [3-(acetyl-hydroxy-amino)-3-
phenyl-propyl]-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 12b
Yield 99% (colourless oil), IR mmax (KBr) cm– -1: 1754 (C=O, ester),
1618 (C=O, hydroxamic acid) and 1254 (P=O); 1H-NMR (400 MHz,
DMSO-d6), d (ppm): 9.66 (s, 1H), 7.35–7.25 (m, 5H), 5.63–5.56 (m,
4H), 5.49–5.42 (m, 1H), 2.19–2.11 (m, 1H), 2.03-1.89 (m, 5H),
1.78–1.61 (m, 1H), 1.14+1.13 (2s, 18H); 13C-NMR (100 MHz,
CDCl3), d (ppm): 176.95, 176.89, 173.26, 138.72, 128.45, 127.84,
127.61, 81.60 (d, 2JC-P = 5.1 Hz), 58.52 (d,3JC-P = 9.2 Hz), 38.75, 26.83,
23.05 (d, 1JC-P = 140.4 Hz), 22.43, 20.87; MW 501.52, Anal. Calc. for
C23H36NO9P: C 55.08, H 7.24, N 2.79%. Found: C 54.59, H 7.32, N
2.93%.
1
2
26.81, 24.01 (d, JC-P = 142.4 Hz), 22.95 (d, JC-P = 3.6 Hz), 21.05;
HRFAB-MS C30H42NO9P, MW 591.64, [M+H]+: calculated 592.2675;
found 592.2653.
General procedure for the synthesis of compounds 11a, b
and 12a, b
The respective O-benzyl protected hydroxamic acids 9a, b and
10a, b (3 mmol) were dissolved in freshly distilled MeOH (50 mL).
A catalytic amount of Pd/C was added before the mixture was
hydrogenated under 3 bar for one hour. To remove the catalyst,
the suspension was filtered through a SPE tube RP-18 purchased
from Supelco (Sigma-Aldrich Chemie GmbH, Munich, Germany).
The filtrate was evaporated to give the free hydroxamic acids
11a, b and 12a, b.
Determination of in-vitro antimalarial activity
Culture of P. falciparum
The P. falciparum 3D7 strain was maintained in continuous cul-
ture, according to Trager and Jensen and DasGupta et al. [15].
The parasites were grown in human red blood cells (RBCs blood
group A positive), RPMI 1640 medium supplemented with
25 mM HEPES, 20 mM sodium bicarbonate and 0.5% AlbuMAX
(Invitrogen, Karlsruhe, Germany) at 5% hematocrit. The flasks
were gassed with 90% N2, 5% O2 and 5% CO2 and incubated at
378C. The development of the cultures and the percentage of
infected RBC`s were determined by light microscopy of Giemsa-
stained thin smears.
2,2-Dimethyl-propionic acid (2,2-dimethyl-propionyloxy-
methoxy)-[3-(formyl-hydroxy-amino)-butyl]-phosphinoy-
loxymethyl ester 11a
Yield 99% (pale yellow oil), IR mmax (KBr) cm– 1: 1753 (C=O, ester),
1669 (C=O, hydroxamic acid) and 1239 (P=O); 1H-NMR (400 MHz,
CDCl3), d (ppm): 8.54 (s, 1H), 7.95 (s, 1H), 5.70–5.59 (m, 4H), 4.47–
4.39 (m, 0.5H), 3.88–3.79 (s, 0.5H), 2.16–1.72 (m, 4H), 1.26–1.22
(s, 21H); 13C-NMR (100 MHz, CDCl3), d (ppm): 176.98, 176.95,
163.77, 81.62 (d, 2JC-P = 7.1 Hz), 81.50 (d, 2JC-P = 6.1 Hz), 51.27 (d, 3JC-P
= 7.1 Hz), 38.76, 26.86, 26.82, 24.15 (d, 2JC-P = 5.1 Hz), 22.60 (d, 1JC-P
= 140.4 Hz), 17.36; MW 425.42, Anal. Calc. for C17H32NO9P: C
48.00, H 7.58, N 3.29%. Found: C 47.53, H 7.71, N 3.39%.
Preparation of drug solutions
There were 20 lmol of the respective compounds dissolved in
400 lL DMSO and further diluted with water/ethanol (50 / 50) to
obtain the particular concentration.
Determination of parasite growth inhibition
The tests were carried out in 96-well microtiter plates under
strict aseptic conditions, according to DasGupta et al. [15]. Dilu-
tions of each compound were added to 250 lL of a suspension of
P. falciparum infected erythrocytes (1.5% hematocrit, 1.5–2% par-
asitemia). The plates were flushed with a gas mixture consisting
of 90% N2, 5% O2 and 5% CO2, closed tightly and incubated at
378C for 24 h. Afterwards, 0.1 lCi of 8-[3H]-hypoxanthine was
added to each well. The plates were flushed with the above men-
tioned gas mixture, incubated for additional 24 h at 378C and
subsequently harvested with a cell harvester system (Inotech,
Dottikon, Switzerland). Infected erythrocytes were washed four
times with distilled water before they were analysed for incorpo-
rated radioactivity in a multidetector liquid scintillation coun-
ter (Wallac, Turku, Finland).
2,2-Diemthyl-propionic acid (2,2-dimethyl-
propionyloxymethoxy)-[3-(formyl-hydroxy-amino)-3-
phenyl-propyl]-phosphinoyloxymethyl ester 11b
Yield 99% (pale yellow solid), IR mmax (KBr) cm– 1: 1753 (C=O, ester),
1672 (C=O, hydroxamic acid) and 1257 (P=O); 1H-NMR (400 MHz,
CDCl3), d (ppm): 8.48 (s, 1H), 8.09 (s, 1H), 7.41–7.28 (m, 5H), 5.72–
5.60 (m, 4H), 5.47–5.40 (m, 0.5H), 4.83–4.75 (m, 0.5H), 2.64–2.43
(m, 1H), 2.28–1.80 (m, 3H), 1.24–1.20 (m, 18H); 13C-NMR
(100 MHz, CDCl3), d (ppm): 176.98, 176.94, 163.83, 138.10,
2
128.84, 128.55, 128.10, 127.58, 127.07, 81.46 (d, JC-P = 6.1 Hz),
58.51 (d, 3JC-P = 10.2 Hz), 38.76, 26.83, 23.19 (d, JC-P = 143.4 Hz),
1
22.38 (d, 2JC-P = 4.1 Hz); ESI-MS C22H34NO9P, MW 487.49, [M+Na]+:
calculated 510; found 510.
2,2-Dimethyl-propionic acid [3-(acetyl-hydroxy-amino)-
butyl]-(2,2dimethyl-propionyloxymethoxy)-
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