Conformationally restrained aromatic analogues of fosmidomycin and FR900098

Article abstract of DOI:10.1002/ardp.200700013

The synthesis and in-vitro antimalarial activity of conformationally restrained bis(pivaloyloxymethyl) ester analogues of the natural product fosmidomycin is presented. In contrast to α-aryl-substituted analogues, conformationally restrained aromatic anal

Full text of DOI:10.1002/ardp.200700013

Arch. Pharm. Chem. Life Sci. 2007, 340, 339–344
T. Kurz et al.
339
Full Paper
Conformationally Restrained Aromatic Analogues of
Fosmidomycin and FR900098
Thomas Kurz1, Katrin Schlꢀter1, Miriam Pein1, Christoph Behrendt1, Bꢁrbel 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 conformationally restrained bis(pivaloyloxy-
methyl) ester analogues of the natural product fosmidomycin is presented. In contrast to a-aryl-
substituted analogues, conformationally restrained aromatic analogues exhibit only moderate
in-vitro antimalarial activity against the chloroquine-sensitive strain 3D7 of Plasmodium falcipa-
rum. The most active derivative displays an IC50 value of 47 lM.
Keywords: Conformationally Restrained Analogues / Fosmidomycin / Malaria tropica / Plasmodium falciparumphospho-
lipases / protein kinase C / SHIP / membrane fusion /
Received: January 26, 2007; accepted: May 4, 2007
DOI 10.1002/ardp.200700013
Introduction
Every year approximately 1.5 to 2.7 million people die of
malaria worldwide. Plasmodium falciparum, the causative
agent of malaria tropica, is responsible for most of the
fatal malaria cases. About 90% of the estimated 300–500
million malaria cases occur in Sub-Saharan Africa [1]. The
widespread resistance of Plasmodium falciparum against
currently used antimalarial drugs complicates an effi-
cient malaria therapy. Therefore, novel drugs based on
new modes of action are urgently needed. A promising
organelle for the development of new antimalarias repre-
sents the apicoplast of Plasmodium falciparum [2]. The plas-
tid-derived apicoplast harbours the DOXP/MEP pathway
of isoprenoid biosynthesis, which leads to isopentenyldi-
phosphate (IPP), an important isoprenoid precursor.
Since the DOXP/MEP pathway is absent in humans but
present in Plasmodium falciparum, several pathogenic bac-
Figure 1. Lead structures and target compounds 1–4.
teria and higher plants, it is an important target for drug
development [3].
the inhibition of the DOXP/MEP pathway of isoprenoid
synthesis by blocking the 1-desoxy-d-xylulose-5-phos-
phate-reductoisomerase (DXR), the second enzyme of the
non-mevalonate isoprenoid biosynthesis [4]. Later, fosmi-
domycin's antimalarial activity was confirmed in various
clinical trials [5]. Currently, 1 is in clinical phase-II trials
in combination with the lincosamide clindamycin [5].
In 1998, Jomaa discovered that the antibiotic fosmido-
mycin 1 (Fig. 1) displays its antimalarial activity through
Correspondence: Dr. 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
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340
T. Kurz et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 339–344
However, a drawback of 1 is its quite low oral bioavail-
ability of approximately 30% [5].
Various groups have contributed to the structural
modification of 1 and its more active analogue 2 (Fig. 1).
Important structural elements for potent antimalarial
activity are the hydroxamic acid functionality [7], the
phosphonic acid group [8] and the propyl spacer between
both pharmacophores. Improved in-vivo antimalarial
activity against Plasmodium vinckei was observed in case
of FR9000098 prodrugs [9]. Recently, modifications of the
propyl spacer have been reported. We have shown that a-
phenylfosmidomycin (3, IC50: 0.4 lM) demonstrates high
in-vitro activity against the chloroquine-resistant strain
Dd2 of Plasmodium falciparum [10]. Van Calenbergh et al.
confirmed our results and also reported on cyclopropane
analogues [11]. In addition, Schlitzer and our group have
shown that the hygroscopicity of phosphonohydroxamic
acids, which limitates their use and analysis, can be over-
come by masking the phosphonic acid moiety as bis(piva-
loyloxymethyl) (POM) esters [6]. In order to gain new
insights regarding the structure-activity relationships,
we investigated the synthesis and antimalarial activity of
conformationally restrained, aromatic bis(pivaloyloxy-
methyl) ester analogues of the natural products fosmido-
mycin 1 and FR900098 2.
Scheme 1. Synthesis of analogues 10a–c.
Results and discussion
Table 1. IC50 values of bis(pivaloyloxymethyl) ester analogues
10a–c.
Chemistry
Starting material 5 was synthesized according to a litera-
ture procedure [12]. Reaction of 5 with N-Boc-O-benzylhy-
droxylamine in presence of sodium hydride provided the
protected hydroxycarbamate 6. Removal of the Boc-pro-
tecting group with TFA in dichloromethane and formyla-
tion, acetylation or benzoylation of the instable hydrox-
ylamine 7 afforded hydroxamic acids 8a–c. The conver-
sion of ethyl phosphonates 8a–c into their correspond-
ing bis(pivaloyloxymethyl) esters 9a–c was accomplished
by treatment with bromotrimethylsilane/water and sub-
sequent alkylation with chloromethyl pivalate in the
presence of triethylamine. Finally, catalytic hydrogena-
tion furnished conformationally restrained analogues
10a–c (Scheme 1).
Compound
R
IC50 (lM)a)
n
SEMb)
10a
10b
10c
1-POM
2-POM
3-POM
Formyl
Acetyl
Benzoyl
H
Me
H
61
50
47
2.1
0.4
0.6
5
6
6
6
6
3
7.4
5.3
6.1
1.1
0.1
0.2
a)
Mean values of three (3) or six (6) independent determina-
tions.
b)
Standard errors of the means.
oxymethyl) esters of fosmidomycin 1, FR900098 2 and a-
phenylfosmidomycin 3 (see Table 1: 1-POM, 2-POM, 3-
POM). During the determination, the bis(pivaloyloxy-
methyl) esters were converted into free and active phos-
phonic acids by non-specific esterases.
The modification and rigidisation of fosmidomycin's
propyl spacer by a phenyl nucleus led to a significant
reduction of antimalarial activity. Analogues 10a–c dis-
play moderate antimalarial activity against the chloro-
quine-sensitive strain 3D7 in the range of 47–60 lM.
Biological activity
The in-vitro antimalarial activity of compounds 10a–c
was evaluated by an 8-[3H]hypoxanthine incorporation
assay [13]. For all experiments the chloroquine-sensitive
strain 3D7 of Plasmodium falciparum was used. IC50 values
for compounds 10a–c have been determined and their
antimalarial activity was compared to the bis(pivaloyl-
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Arch. Pharm. Chem. Life Sci. 2007, 340, 339–344
Analogues of Fosmidomycin and FR900098
341
quart., aromat.), 156.0 (C=O); Anal. calcd. for C23H32NO6P: C,
61.46; H, 7.18; N, 3.12. Found: C, 61.20; H, 7.23; N, 3.19.
Conclusions
The synthesis and in-vitro antimalarial activity of confor-
mationally restrained, aromatic bis(pivaloyloxymethyl)
ester analogues of fosmidomycin and FR900098 are
described. In contrast to the high antimalarial activity of
a-aryl-substituted fosmidomycin analogues as for
instance a-phenylfosmidomycin 3, the aromatic ana-
logues 10a–c display only moderate in-vitro antimalarial
activity. Since the bis(pivaloyloxymethyl) esters are trans-
formed into the corresponding phosphonic acids by non-
specific esterases during the determination, no animal
experiments were necessary to differentiate between
compounds 10a–c.
{2-(Benzyloxyamino-methyl]-phenyl}-phosphonic acid
diethyl ester 7
A solution of 6 (10 mmol) in dichloromethane (30 mL) was
treated with trifluoro acetic acid (10 mL) at 08C. The solution
was stirred at room temperature for 1 h. Afterwards, the solvent
was evaporated. The residue was dissolved in a solution of K2CO3
(10%, 30 mL) and successively extracted with diethyl ether
(3630 mL). The combined organic layers were dried over MgSO4
and the solvent was removed under reduced pressure. The
remaining oil was treated with an aqueous solution of HCl (1 M,
30 mL) and intensively stirred for 15 min before the mixture
was extracted with diethyl ether (2630 mL). The aqueous layer
was neutralised with a K2CO3-solution (10%) and again extracted
with diethyl ether (2630 mL). The organic layers were com-
bined, dried over MgSO4 and the solvent was evaporated. The res-
idue was purified by column chromatography on silica gel using
diethyl ether / ethyl acetate as an eluent to yield the instable
compound 7. Yield 92% (colourless oil); IR mmax (KBr) cm– 1: 3244
(N-H), 1240 (P=O); 1H-NMR (400 MHz, CDCl3) = d (ppm) 1.31 (t, J =
6.93 Hz, 6H, POCH2CH3), 3.99–4.21 (m, 4H, POCH2CH3), 4.32 (s,
2H, benzyl CH2), 4.76 (s, 2H, benzyl CH2), 7.25–7.41 (m, 6H, aro-
mat.), 7.47–7.56 (m, 2H, aromat.), 7.83–7.93 (m, 1H, aromat.);
13C-NMR (101 MHz, CDCl3) = d (ppm) 16.3 (d, JC,P = 6.68 Hz,
Experimental
Elemental analysis was carried out with a Heraeus CHN-O-Rapid
instrument (Heraeus, Hanau, Germany). IR spectra were
recorded on a Shimadzu FT-IR 8300 (Shimadzu, Tokyo, Japan).
1H-NMR (400 MHz) and13C-NMR (101 MHz) spectra were recorded
on a Bruker AMX 400 spectrometer (Bruker, Rheinstetten, Ger-
many) using tetramethylsilane as internal standard and DMSO-
d6 or CDCl3 as solvents. Mass spectra were recorded on a Micro-
mass VG 70-250S mass spectrometer (HRFAB) (Micromass, Man-
chester, UK).
POCH2CH3), 54.6 (d, JC,P = 3.74 Hz, benzyl CH2), 62.3 (d, JC,P
=
5.77 Hz, POCH2CH3), 75.9 (benzyl CH2), 127.3 (d, JC,P = 183.42 Hz,
PC quart., aromat.), 127.2 (d, JC,P = 14.59 Hz, tert., aromat.), 127.4,
128.3, 128.4 (tert., aromat.), 132.0 (d, JC,P = 14.72 Hz, tert., aro-
mat.), 132.4 (d, JC,P = 2.72 Hz, PCCH tert., aromat.), 133.6, 133.7
(tert., aromat.), 137.9 (quart., aromat.), 140.8 (d, JC,P = 11.32 Hz,
PCC quart., aromat.). Due to the instability of compound 7 no sat-
isfactory elementary analysis could be obtained. Compound 7
was immediately converted into stable hydroxamic acids 8a–c.
Chemistry
{2-[(Benzyloxy-tert-butyloxycarbonyl-amino)-methyl]-
phenyl}-phosphonic acid diethyl ester 6
N-Boc-O-benzylhydroxylamine (10mmol) was dissolved in anhy-
drous THF (30 mL) and the solution was cooled down to 08C.
Under nitrogen atmosphere, sodium hydride (11 mmol) was
added portionwise to the stirred solution while the temperature
was kept between 0–58C. Ice cooling was removed after gas for-
mation declined. The solution was treated with a catalytic
amount of NaI before (2-bromomethyl-phenyl)-phosphonic acid
diethyl ester 5 (10 mmol), dissolved in anhydrous THF (5 mL),
was added. The reaction mixture was stirred at room tempera-
ture over night. Afterwards, ice water was added and the mix-
ture was extracted with diethyl ether (3620 mL). The combined
organic layers were dried over MgSO4 and the solvent was
removed under reduced pressure. The residue was purified by
column chromatography on silica gel using diethyl ether/petro-
leum ether as an eluent to yield compound 6. Yield 72% (colour-
less oil); IR mmax (KBr) cm– 1: 1705 (C=O), 1252 (P=O); 1H-NMR
(400 MHz, CDCl3) = d (ppm) 1.29 (t, J = 6.95 Hz, 6H, POCH2CH3),
1.49 (s, 9H, (CH3)3C), 3.99–4.15 (m, 4H, POCH2CH3), 4.81 (s, 2H,
benzyl. CH2), 5.03 (s, 2H, benzyl CH2), 7.27–7.37 (m, 6H, aromat.),
7.45–7.55 (m, 2H, aromat.), 7.91–7.98 (m, 1H, aromat.); 13C-NMR
(101 MHz, CDCl3) = d (ppm) 16.7 (d, JC,P = 6.19 Hz, POCH2CH3), 28.7
((CH3)3C), 51.8 ((CH3)3C), 62.5 (d, JC,P = 5.54 Hz, POCH2CH3), 77.4
(benzyl. CH2), 82.0 (benzyl CH2), 126.2 (d, JC,P = 183.42 Hz, PC
quart., aromat.), 127.1 (d, JC,P = 13.73 Hz, tert., aromat.), 128.3 (d,
JC,P = 14.02 Hz, tert., aromat.), 128.7, 128.9, 129.8 (tert., aromat.),
133.0 (d, JC,P = 3.04 Hz, PCCH tert., aromat.), 134.4, 134.5 (tert.,
aromat.), 135.8 (quart., aromat.), 141.4 (d, JC,P = 10.36 Hz, PCC
{2-[(Benzyloxy-formyl-amino)-methyl]-phenyl}-
phosphonic acid diethyl ester 8a
Acetic acid anhydride (10 mmol) was treated with formic acid
(100 mmol) and stirred for 30 min at room temperature exclud-
ing humidity. The reaction mixture was cooled to 08C before a
solution of hydroxylamine 7 (1 mmol) in formic acid (5 mL) was
added dropwise. The mixture was allowed to warm up to room
temperature and was stirred for two more hours. The solution
was treated with ethyl acetate (200 mL) and washed with a satur-
ated solution of K2CO3 until the aqueous layer reacted alkaline.
The organic layer was then washed with water (100 mL) and
aqueous HCl (0.5 M, 36100 mL) and dried over MgSO4 The sol-
vent was removed under reduced pressure and the remaining
residue was purified by column chromatography on silica gel
using diethyl ether/ethyl acetate as an eluent to yield compound
8a. Yield 97% (pale yellow oil); IR mmax (KBr) cm– 1: 1662 (C=O),
1236 (P=O); 1H-NMR (400 MHz, CDCl3) = d (ppm) 1.33 (t, J =
7.05 Hz, 6H, POCH2CH3), 4.01–4.26 (m, 4H, POCH2CH3), 4.84 (s,
2H, benzyl. CH2), 4.91–5.37 (m, 2H, benzyl CH2), 7.24–7.49 (m,
7H, aromat.), 7.50–7.60 (m, 1H, aromat.), 7.86–7.98 (m, 1H, aro-
mat.), 8.09–8.38 (m, 1H, formyl.); 13C-NMR (101 MHz, CDCl3) = d
(ppm) 16.7 (d, JC,P = 6.85 Hz, POCH2CH3), 46.0 (benzyl. CH2), 62.8
(d, JC,P = 5.56 Hz, POCH2CH3), 78.0 (benzyl CH2), 125.4 (d, JC,P
134.85 Hz, PC quart., aromat.), 127.7 (d, JC,P = 15.77 Hz, tert., aro-
mat.), 129.0 (d, JC,P = 13.65 Hz, tert., aromat.), 129.5, 130.0 (tert.,
=
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T. Kurz et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 339–344
aromat.), 134.2 (d, JC,P = 2.99 Hz, PCCH tert., aromat.), 134.1, 134.2
(tert., aromat.), 134.6 (quart., aromat.), 139.6 (d, JC,P = 8.52 Hz,
PCC quart., aromat.), 163.6 (C=O); Anal. calcd. for C19H24NO5P: C,
60.47; H, 6.41; N, 3.71. Found: C, 60.42; H, 6.70; N, 3.46.
syringe. The mixture was stirred at room temperature for 24 h.
The solvent was removed under reduced pressure. Afterwards,
the remaining residue was dissolved in THF (3 mL), treated with
one drop of water and stirred at room temperature. After
10 min, the solvent was evaporated and remaining water was
removed in vacuo over night. The resulting phosphonic acid was
dissolved in anhydrous DMF (10 mL) and treated with TEA
(3 mmol) and chloromethyl pivalate (10 mmol). Under anhy-
drous conditions, the mixture was heated to 708C and stirred for
2 h. The solution was treated again with TEA (1 mmol) and chlor-
omethyl pivalate (2 mmol) and stirred for further 2 h. After-
wards, the procedure of adding TEA and chloromethyl pivalate
was repeated once again. The mixture was stirred for two more
hours at 708C, allowed to cool down to room temperature and
finally stirred over night. Diethyl ether (50 mL) was added and
the solution was successively washed with water (25 mL), an
aqueous saturated NaHCO3-solution (2625 mL) and again with
water (25 mL). The organic layer was dried over MgSO4 and the
solvent removed under reduced pressure. The residue was puri-
fied by column chromatography on silica gel using diethyl
ether/n-hexane as an eluent to yield compounds 9a–c.
General procedure for the synthesis of compounds 8b, c
Hydroxylamine 7 (3 mmol) was dissolved in anhydrous dichloro-
methane (20 mL), treated with triethylamine (4 mmol) and
cooled to 0–58C. Over a period of 10 min the corresponding acid
chloride (4 mmol in 5 mL dichloromethane) was added to the
stirred solution. The mixture was stirred for 2 h at room temper-
ature and the progress of the reaction was monitored by TLC.
The solvent was evaporated, the residue dissolved in ethyl ace-
tate (20 mL) and successively washed with aqueous HCl (1 M,
2620 mL) and with K2CO3 solution (10%, 2620 mL). The organic
layer was dried over MgSO4 and the solvent was removed under
reduced pressure. The residue was purified by column chroma-
tography on silica gel using n-hexane/ethyl acetate as an eluent
to yield compounds 8b, c.
{2-[(Acetyl-benzyloxy-amino)-methyl]-phenyl}-
phosphonic acid diethyl ester 8b
Yield 98% (pale yellow oil); IR mmax (KBr) cm– 1: 1668 (C=O), 1246
(P=O); 1H-NMR (400 MHz, CDCl3) = d (ppm) 1.32 (t, J = 6.65 Hz, 6H,
POCH2CH3), 2.19 (s, 3H, acetyl. CH3), 4.02–4.22 (m, 4H,
POCH2CH3), 4.84 (s, 2H, benzyl CH2), 5.32 (s, 2H, benzyl CH2),
7.25–7.44 (m, 7H, aromat.), 7.49–7.56 (m, 1H, aromat.), 7.88–
7.97 (m, 1H, aromat.); 13C-NMR (101 MHz, CDCl3) = d (ppm) 16.3
(d, JC,P = 6.23 Hz, POCH2CH3), 20.5 (acetyl CH3), 47.1 (benzyl CH2),
2,2-Dimethyl-propionic acid{2-[(benzyloxy-formyl-amino)-
methyl]-phenyl}-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 9a
Yield 33% (colourless oil); IR mmax (KBr) cm– 1: 1751 (C=O), 1680
(C=O, hydroxamic acid), 1259 (P=O); 1H-NMR (400 MHz, DMSO-d6)
= d (ppm) 1.02 (s, 18H, ((CH3)3C), 4.91 (s, 2H, benzyl. CH2), 5.02 (s,
2H, benzyl CH2), 5.62–5.82 (m, 4H, OCH2O), 7.23–7.57 (m, 7H,
aromat.), 7.60–7.89 (m, 2H, aromat.), 8.09–8.51 (m, 1H, formyl);
13C-NMR (101 MHz, DMSO-d6) = d (ppm) 27.1 ((CH3)3C), 39.1
((CH3)3C), 45.6 (benzyl CH2), 77.1 (benzyl CH2), 82.2 (OCH2O), 124.6
(d, JC,P = 135.32 Hz, PC quart., aromat.), 127.7 (d, JC,P = 15.12 Hz,
62.3 (d, JC,P = 5.41 Hz, POCH2CH3), 76.6 (benzyl CH2), 125.9 (d, JC,P
=
132.08 Hz, PC quart., aromat.), 126.9 (d, JC,P = 14.76 Hz, tert., aro-
mat.), 128.0 (d, JC,P = 14.76 Hz, tert., aromat.), 128.6, 128.9, 129.3
(tert., aromat.), 132.8 (d, JC,P = 2.84 Hz, PCCH tert., aromat.), 133.8,
133.9 (tert., aromat.), 134.3 (quart., aromat.), 140.3 (d, JC,P
=
tert., aromat.), 129.1, 129.5, 130.1 (tert., aromat.), 133.9 (d, JC,P
=
9.65 Hz, PCC quart., aromat.), 167.4 (C=O); Anal. calcd for
C20H26NO5P: C, 61.37; H, 6.70; N, 3.58. Found: C, 61.21; H, 6.69; N,
3.38.
2.97 Hz, PCCH tert., aromat.), 134.1 (d, JC,P = 10.84 Hz, tert., aro-
mat), 134.7 (quart., aromat.), 139.8 (d, JC,P = 10.92 Hz, PCC quart.,
aromat.), 163.5 (C=O, hydroxamic acid), 177.2 (C=O, acylal); Anal.
calcd. for C27H36NO9P: C, 59.01; H, 6.60; N, 2.55. Found: C, 58.79;
H, 6.77; N, 2.37.
{2-[(Benzoyl-benzyloxy-amino)-methyl]-phenyl}-
phosphonic acid diethyl ester 8c
Yield 91% (colourless oil); IR mmax (KBr) cm– 1: 1651 (C=O), 1246
(P=O); 1H-NMR (400 MHz, CDCl3) = d (ppm) 1.30 (t, 6H, J = 7.34 Hz,
POCH2CH3), 4.01–4.21 (m, 4H, POCH2CH3), 4.72 (s, 2H, benzyl
CH2), 5.43 (s, 2H, benzyl CH2), 6.88–7.03 (m, 2H, aromat.), 7.16–
7.25 (m, 3H, aromat.), 7.34–7.75 (m, 8H, aromat.), 7.91–7.99 (m,
2,2-Dimethyl-propionic acid{2-[(acetyl-benzyloxy-amino)-
methyl]-phenyl}-(2,2-dimethyl-propionyloxymethoxy)-
phophinoyloxymethyl ester 9b
Yield 37% (colourless oil); IR mmax (KBr) cm– 1: 1753 (C=O), 1668
(C=O, hydroxamic acid), 1259 (P=O); 1H-NMR (400 MHz, DMSO-d6)
= d (ppm) 1.02 (s, 18H, ((CH3)3C), 2.15 (s, 3H, acetyl. CH3), 4.90 (s,
2H, benzyl CH2), 5.12 (s, 2H, benzyl CH2), 5.66–5.80 (m, 4H,
OCH2O), 7.21–7.30 (m, 1H, aromat.), 7.32–7.50 (m, 6H, aromat.),
7.60–7.67 (m, 1H, aromat.), 7.75–7.86 (m, 1H, aromat.); 13C-NMR
(101 MHz, DMSO-d6) = d (ppm) 20.6 (acetyl CH3), 26.6 ((CH3)3C),
1H, aromat.); 13C-NMR (101 MHz, CDCl3) = d (ppm) 16.7 (d, JC,P
=
6.07 Hz, POCH2CH3), 48.8 (benzyl CH2), 62.7 (d, JC,P = 5.46 Hz,
POCH2CH3), 77.0 (benzyl CH2), 123.6 (d, JC,P = 183.41 Hz, PC quart.,
aromat.), 127.4 (d, JC,P = 13.96 Hz, tert., aromat.), 128.3 (d, JC,P
=
13.40 Hz, tert., aromat.), 128.5, 128.7, 128.8, 129.1, 129.9, 130.4,
131.0 (tert., aromat.), 133.3 (d, JC,P = 2.68 Hz, PCCH tert., aromat.),
134.3, 134.4 (tert., aromat.), 134.6 (quart., aromat.), 140.7 (d, JC,P
=
38.5 ((CH3)3C), 46.6 (benzyl CH2), 76.0 (benzyl CH2), 82.2 (d, JC,P
=
8.52 Hz, PCC quart., aromat.), 170.7 (C=O); Anal. Calcd for
C25H28NO5P: C, 66.22; H, 6.22; N, 3.09. Found: C, 66.22; H, 6.43; N,
3.03.
6.04 Hz, OCH2O), 125.4 (d, JC,P = 185.87 Hz, PC quart., aromat.),
127.4 (d, JC,P = 14.69 Hz, tert., aromat.), 127.6 (d, JC,P = 14.70 Hz,
tert., aromat.), 128.8, 129.2, 129.9, 133.5, 133.6 (tert., aromat.),
133.9 (d, JC,P = 2.88 Hz, PCCH tert., aromat.), 134.7 (quart., aro-
mat.), 140.7 (d, JC,P = 10.30 Hz, PCC quart., aromat.), 171.7 (C=O,
hydroxamic acid), 176.7 (C=O, acylal); Anal. calcd. for
C28H38NO9P: C, 59.67; H, 6.80; N, 2.49. Found: C, 59.53; H, 6.68; N,
2.51.
General procedure for the synthesis of compounds 9a–c
Trimethylsilyl bromide (6 mmol) was added dropwise to a
stirred solution of the respective phosphonic acid diethyl esters
8a–c (1 mmol) in anhydrous dichloromethane at 08C via a
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Arch. Pharm. Chem. Life Sci. 2007, 340, 339–344
Analogues of Fosmidomycin and FR900098
343
tert., aromat.), 133.5, 133.6 (tert., aromat.), 133.7 (d, JC,P = 2.35 Hz,
PCCH tert., aromat.), 140.7 (JC,P = 10.57 Hz, PCC quart., aromat.),
171.5 (C=O, hydroxamic acid), 176.3 (C=O, acylal); Anal. calcd. for
C21H32NO9P: C, 53.27; H, 6.81; N, 2.96. Found: C, 53.65; H, 7.20; N,
2.82.
2,2-Dimethyl-propionic acid{2-[(benzoyl-benzyloxy-
amino)-methyl]-phenyl}-(2,2-dimethyl-
propionyloxymethoxy)-phosphinoyloxymethyl ester 9c
Yield 41% (colourless oil); IR mmax (KBr) cm– 1: 1753 (C=O), 1649
(C=O, hydroxamic acid), 1257 (P=O); 1H-NMR (400 MHz, DMSO-d6)
= d (ppm) 1.02 (s, 18H, ((CH3)3C), 4.74 (s, 2H, benzyl. CH2), 5.28 (s,
2H, benzyl CH2), 5.68–5.79 (m, 4H, OCH2O), 6.90–6.98 (m, 2H,
aromat.), 7.18–7.31 (m, 3H, aromat.), 7.45–7.59 (m, 5H, aro-
mat.), 7.61–7.74 (m, 3H, aromat.), 7.79–7.88 (m, 1H, aromat.);
13C-NMR (101 MHz, DMSO-d6) = d (ppm) 26.2 ((CH3)3C), 38.0
((CH3)3C), 47.0 (benzyl. CH2), 75.5 (benzyl CH2), 81.7 (OCH2O), 125.1
(d, JC,P = 184.87 Hz, PC quart., aromat.), 127.1 (d, JC,P = 14.88 Hz,
tert., aromat.), 127.2 (d, JC,P = 14.41 Hz, tert., aromat.), 127.6,
128.0, 128.1, 128.6, 129.2, 130.4, 131.2, 133.3 (tert., aromat.),
133.6 (d, JC,P = 2.56 Hz, PCCH tert., aromat.), 134.1 (quart., aro-
mat.), 140.7 (d, JC,P = 11.00 Hz, PCC quart., aromat.), 169.4 (C=O,
hydroxamic acid), 176.7 (C=O, acylal); Anal. calcd. for
C33H40NO9P: C, 63.35; H, 6.44; N, 2.24. Found: C, 63.05; H, 6.56; N,
2.29.
2,2-Dimethyl-propionic acid {2-[(benzoyl-hydroxy-amino)-
methyl]-phenyl}-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 10c
Yield 86% (yellow oil); IR mmax (KBr) cm– 1: 3361 (O-H), 1751 (C=O),
1649 (C=O, hydroxamic acid), 1257 (P=O); 1H-NMR (400 MHz,
DMSO-d6) = d (ppm) 1.02-1.09 (m, 18H, ((CH3)3C), 4.72 (d, J =
6.37 Hz, 1.4H, benzyl CH2), 5.43 (s, 0.6H, benzyl CH2), 5.64–5.79
(m, 4H, OCH2O), 7.38–7.95 (m, 9H, aromat.), 8.98 (t, 0.7H, J =
5.90 Hz, OH), 10.2 (s, 0.3H, OH); 13C-NMR (101 MHz, DMSO-d6) = d
(ppm) 27.1 ((CH3)3C), 39.1 ((CH3)3C), 42.7, 42.8 (2d, JC,P = 4.50 Hz,
benzyl CH2), 82.3 (OCH2O), 126.2 (d, JC,P = 187.32 Hz, PC quart., aro-
mat.), 127.6 (tert. aromat.), 127.9 (d, JC,P = 14.97 Hz, tert., aromat.),
128.8, 131.8 (tert., aromat.), 132.6 (d, JC,P = 15.19 Hz, tert., aro-
mat.), 133.7, 133.8, 134.1 (tert., aromat.), 134.2 (d, JC,P = 3.32 Hz,
General procedure for the synthesis of compound 10a–c
The respective O-benzyl protected hydroxamic acids 9a–c
(1 mmol) were hydrogenated in MeOH (50 mL) using catalytic
amounts of 10% Pd/C (2 h, 2 bar). Afterwards, the suspension was
filtered through a SPE tube RP-18 purchased from Supelco
(Sigma-Aldrich, Munich, Germany) in order to remove the cata-
lyst. The filtrate was evaporated to yield the free hydroxamic
acids 10a–c.
PCCH tert., aromat.), 134.6 (quart., aromat.), 142.9 (d, JC,P
=
10.92 Hz, PCC quart., aromat.), 167.2 (C=O, hydroxamic acid),
177.4 (C=O, acylal); HRFAB-MS C26H34NO9P, MW 535.54, [M+H]+ cal-
culated 536.2051; found 536.2043.
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. [14].
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-
propionyloxymethoxy)-{2-[(formyl-hydroxy-amino)-
methyl]-phenyl}-phosphinoyloxymethyl ester 10a
Yield 81% (yellow oil); IR mmax (KBr) cm– 1: 3220 (O-H), 1755 (C=O),
1674 (C=O, hydroxamic acid), 1255 (P=O); 1H-NMR (400 MHz,
DMSO-d6) = d (ppm) 0.99–1.07 (m, 18H, ((CH3)3C), 4.55 (m, 0.25H,
benzyl CH2), 4.92 (m, 1.75H, benzyl CH2), 5.64–5.79 (m, 4H,
OCH2O), 7.30–7.52 (m, 2H, aromat.), 7.62–7.85 (m, 2H, aromat.),
8.10–8.22 (m, 0.45H, formyl), 8.39–8.52 (m, 0.55H, formyl), 9.83
(s, 0.45H, OH), 10.30 (s, 0.55H, OH); 13C-NMR (101 MHz, DMSO-d6)
= d (ppm) 26.7 ((CH3)3C), 38.5 ((CH3)3C), 48.4 (benzyl CH2), 82.1
(OCH2O), 125.4 (d, JC,P = 186.65 Hz, PC quart., aromat.), 127.4 (d,
JC,P = 13.34 Hz, tert., aromat.), 133.5 (d, JC,P = 13.44 Hz, tert., aro-
mat.), 133.6 (tert., aromat.), 133.8 (d, JC,P = 2.49 Hz, PCCH tert., aro-
mat.), 158.6, 161.7, 162.9 (C=O, hydroxamic acid), 176.4 (C=O, acy-
lal); Anal. Calcd. for C20H30NO9P: C, 52.29; H, 6.58; N, 3.05. Found:
C, 52.11; H, 6.87; N, 2.82.
Preparation of drug solutions
20 lmol of the respective compounds were 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. [14]. 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, 0.1lCi 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-Dimethyl-propionic acid {2-[(acetyl-hydroxy-amino)-
methyl]-phenyl}-(2,2-dimethyl-propionyloxymethoxy)-
phosphinoyloxymethyl ester 10b
Yield 79% (colourless oil); IR mmax (KBr) cm– 1: 3209 (O-H), 1757
(C=O), 1637 (C=O, hydroxamic acid), 1259 (P=O); 1H-NMR
(400 MHz, DMSO-d6) = d (ppm) 1.01–1.09 (m, 18H, ((CH3)3C), 1.95
(m, 0.4H, acetyl CH3), 2.14 (m, 2.6H, acetyl CH3), 4.49–4.64 (m,
0.2H, benzyl CH2), 4.86–5.08 (m, 1.8H, benzyl CH2), 5.62–5.81
(m, 4H, OCH2O), 7.25–7.51 (m, 2H, aromat.), 7.62–7.87 (m, 2H,
aromat.), 10.06 (s, 1H, OH); 13C-NMR (101 MHz, DMSO-d6) = d
(ppm) 20.6 (acetyl CH3), 26.7 ((CH3)3C), 38.5 ((CH3)3C), 49.8 (benzyl
CH2), 82.0 (OCH2O), 125.2 (d, JC,P = 184.58 Hz, PC quart., aromat.),
127.1 (d, JC,P = 15.86 Hz, tert., aromat.), 127.3 (d, JC,P = 14.09 Hz,
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

344
T. Kurz et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 339–344
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i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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