Reverse fosmidomycin derivatives against the antimalarial drug target IspC (Dxr)

Article abstract of DOI:10.1021/jm200694q

Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial target, were synthesized and biologically evaluated. The binding mode of one derivative in complex with EcIspC

Full text of DOI:10.1021/jm200694q

ARTICLE
pubs.acs.org/jmc
Reverse Fosmidomycin Derivatives against the Antimalarial Drug
Target IspC (Dxr)†,‡
Christoph T. Behrendt,§,∞ Andrea Kunfermann,||,∞ Victoria Illarionova,^ An Matheeussen,#
Miriam K. Pein,§ Tobias Gr€awert,|| Johannes Kaiser,|| Adelbert Bacher,|| Wolfgang Eisenreich,||
Boris Illarionov,^ Markus Fischer,^ Louis Maes,# Michael Groll,|| and Thomas Kurz*,§
§Institut f€ur Pharmazeutische und Medizinische Chemie, Heinrich Heine Universit€at, Universit€atsstrasse 1, 40225 D€usseldorf, Germany
Center for Integrated Protein Science M€unchen, Lehrstuhl f€ur Biochemie, Technische Universit€at M€unchen, Lichtenbergstrasse 4,
85747 Garching, Germany
^Institut f€ur Lebensmittelchemie, Universit€at Hamburg, Grindelallee 117, 20146 Hamburg, Germany
#Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Groenenborgerlaan 171, 2020 Wilrijk,
Belgium
S Supporting Information
b
ABSTRACT: Reverse hydroxamate-based inhibitors of IspC, a key enzyme of the
non-mevalonate pathway of isoprenoid biosynthesis and a validated antimalarial
target, were synthesized and biologically evaluated. The binding mode of one
derivative in complex with EcIspC and a divalent metal ion was clarified by X-ray
analysis. Pilot experiments have demonstrated in vivo potential.
’ INTRODUCTION
1, initially isolated from Streptomyces lavendulae, resembles the
structure of the IspC substrate but is endowed with a metabo-
lically stable phosphonate group and a hydroxamic acid motif
that is perfectly suited for formation of a chelate complex with the
essential divalent metal ion of the enzyme.10 Derivatives of 1 have
been synthesized with the intention to provide drug candidates with
improved pharmacokinetic and pharmacodynamic properties.11
We now report on the synthesis and antiplasmodial activity of
novel reverse fosmidomycin analogues. In addition, we provide
kinetic and crystallographic evidence for their mode of action.
Each year, malaria causes several hundred million infections
resulting in approximately one million fatalities.1 All currently
used antimalarial drugs are subject to rapidly progressing attri-
tion by resistance development.2 The urgent need for novel
therapeutic agents is universally acknowledged, but the resources
for their development are still sadly limited.
IspC (Dxr) protein of Plasmodium falciparum is a clinically
validated antimalarial target.3 The antibiotic fosmidomycin (1),
which acts as a slow-binding IspC inhibitor,4 has been used
successfully in clinical malaria trials.5 However, its therapeutic use
is hampered by the requirement for large and frequent doses, due to
its unsatisfactory pharmacokinetic properties.6
IspC catalyzes the first committed step of the non-mevalonate
pathway,7 supplying essential isoprenoids in apicomplexan pro-
tozoa including Plasmodium but not in mammals that use the
mevalonate pathway for that purpose.8 Specifically, IspC con-
verts a carbohydrate (1-deoxy-D-xylulose 5-phosphate, 2) into a
branched polyol (2C-methyl-D-erythritol 4-phosphate, 4) by a
skeletal rearrangement via 3 followed by hydride transfer.9 The
enzyme requires NADPH and a divalent cation as cofactors
(Scheme 1).
’ RESULTS AND DISCUSSION
Chemistry. C-Alkylation of starting materials 5aꢀd with 2-
(2-bromoethyl)-1,3-dioxolane in the presence of n-butyllithium
provided 1,3-dioxolanes 6aꢀd in good yields of 60ꢀ92%. Acidic
hydrolysis of 6aꢀd afforded the corresponding aldehydes 7aꢀd,
which were converted into carboxylic acids 8aꢀd by oxidation
with SeO2 and H2O2.12 The synthesis of O-benzyl-protected
hydroxamic acids 9a,b, 10aꢀc, and 11 was accomplished by
Received: June 1, 2011
Published: August 25, 2011
r
2011 American Chemical Society
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Scheme 1. Target Compounds and IspC-Catalyzed Reaction
Scheme 3. Synthesis of Target Compounds 16ꢀ20a
Scheme 2. Synthesis of Intermediates 9ꢀ12a
a Reagents and conditions: (f) (1) TMSBr, CH2Cl2, RT, 24 h; (2) THF/
H2O, RT, 1 h; (3) DCC, BnOH, benzene, 80 ꢀC, 4 h; (g) H2, PdꢀC,
MeOH, 3 h; (h) (1) TMSBr, CH2Cl2, RT, 24 h; (2) THF/H2O, RT, 1 h;
(i) H2, PdꢀC, MeOH, 1 h.
Table 1. In Vitro Inhibition Potencya and Cytotoxicityc of
Target Compounds
IC50 (μM)
compd
PfIspCa
EcIspCa
PfK1a,b
MRC-5c
1d
0.14 ( 0.02
0.22 ( 0.01
7.4 ( 0.2
3.7 ( 2.5
2.4 ( 1.1
0.38 ( 0.17
1.3( 1.5
>62
>64
>64
>64
>64
>62
>64
>64
16a
16b
17
0.037 ( 0.003
0.003 ( 0.001
0.015 ( 0.002
inactive
0.21 ( 0.02
15 ( 0.4
18
inactive
inactive
20a
20b
20c
0.009 ( 0.001
0.003 ( 0.001
0.004 ( 0.001
3.8 ( 0.2
0.97 ( 0.79
0.29 ( 0.20
0.41 ( 0.25
0.12 ( 0.07
0.18 ( 0.02
a Reagents and conditions: (a) n-BuLi, 2-(2-bromoethyl)-1,3-dioxolane,
toluene, ꢀ78 ꢀC; (b) HCl, acetone, 50 ꢀC, 3 h; (c) SeO2, H2O2, THF,
65 ꢀC, 4 h; (d) 1,10-carbonyldiimidazole, BnONHR2 (R2 = H, Me, Et),
CH2Cl2, RT; (e) C2O2Cl2/DMF, BnONH-i-Pr, RT.
a Enzyme assay. Values are the mean ( SD of three or more independent
experiments. b Replication of P. falciparum in human erythrocytes.
c Cytotoxicity test with MRC-5 cells. d IC50 value according to ref 11a.
1,10-carbonyldiimidazole-mediated coupling reactions of carboxylic
acids 8aꢀd with O-benzylhydroxylamine, N-methyl-O-benzylhy-
droxylamine, and N-ethyl-O-benzylhydroxylamine, respectively.13
However, the acylation of N-isopropyl-O-benzylhydroxylamine
under similar reaction conditions failed. Finally, the N-isopropyl-
substituted hydroxamic acid 12 was accessible in 81% yield by
subsequent treatment of 8d with oxalyl chloride, DMF, and N-
isopropyl-O-benzylhydroxylamine (Scheme 2).
and 12 provided O-benzyl protected compounds 13a,b, 14, and
15, which were deprotected by catalytic hydrogenation to afford
target compounds 16a,b, 17, and 18 (Scheme 3).15
Biological Evaluation. As shown in Table 1, several com-
pounds under study inhibit IspC protein of P. falciparum (PfIspC)
with IC50 values in the single-digit nanomolar range, whereas the
IC50 values for the Escherichia coli enzyme (EcIspC) exceed those
for the P. falciparum enzyme by 1ꢀ3 orders of magnitude.
Some of the compounds studied are strong inhibitors of P.
falciparum replication in erythrocytes in vitro, with IC50 values in
the low micromolar range (Table 1, column 4); notably, com-
pounds 16b, 20b, and 20c have about 10-fold higher potency
than 1 against the chloroquine-resistant K1 strain of P. falcipar-
um. Moreover, their toxicity in cell culture experiments is low,
with IC50 values above 60 μM (Table 1, column 5).
Next, we studied the two-step deprotection of N-methyl-
substituted benzyloxyamides 10aꢀc. Their reactions with bro-
motrimethylsilane (TMSBr) afforded crystalline phosphonic
acids 19aꢀc in good yields and high purity.14
Catalytic hydrogenation of 19aꢀc finally yielded the required
N-methyl-substituted hydroxamic acids 20aꢀc in 87ꢀ98% yield.
In contrast to the smooth dealkylation of substrates 10aꢀc, the
cleavage of ethyl phosphonates 9a,b, 11, and 12 in the presence
of TMSBr was incomplete, and only crude product mixtures were
obtained. However, transesterification of diethyl esters 9a,b, 11,
In summary, the in vitro evaluation has demonstrated that not
only free but also N-methyl-substituted hydroxamic acids are
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Figure 1. (a) Stereoview of the EcIspC with bound 20b (PDB code 3R0I). (b) Close-up view of the active site with bound 20b. The hydrogen-bonding
networks at the EcIspC active site including protein, ligand, and water molecules are indicated by dashed black lines. Electron densities represented in
purple are contoured at 0.9 Å with 2FO ꢀ FC coefficients. (c) Schematic overview of the interactions (distances are in Å) of 20b (green) bound to the
active site of EcIspC as well as intramolecular van der Waals interactions (light blue) of the N-methyl group with the difluorophenyl ring and the main
chain atoms of 20b.
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Figure 2. (a) Structural superposition of 20b and 1 at the active site of EcIspC in the open conformation (PDB codes 3R0I (20b) and 1ONP16).
(b) Superposition of 20b and 1/EcIspC/NADPH (closed conformation) complex (PDB codes 3R0I and 1Q0L17).
promising candidates for further investigation. In contrast, the
bulky N-isopropyl substituent of compound 17 caused a com-
plete loss of in vitro activity.
Referring to the selection of aryl substituents in the α-position
of the connecting spacer, it is an interesting outcome that the
α-naphthyl derivative 20a inhibits PfIspC in the low nanomolar
range with an IC50 value of 9.4 nM.
Structure elucidation of EcIspC in complex with 20b at 2.1 Å
showed that the space group of the protein can vary according to
the crystallization conditions (Figures 1 and 2).16ꢀ18 The overall
structure is virtually identical with other published EcIspC struc-
tures except for a flexible loop region (amino acids Arg208ꢀ
Ser213), which is distorted most likely because of the lack of
NADPH. In its closed conformation, this loop stabilizes the
Figure 3. KaplanꢀMeier analysis of experiments with mice that had
substrate or fosmidomycin by hydrophobic interactions.
been artificially infected with P. berghei: red, control; black, chloroquine
A structural overlay of the 1/EcIspC complex with 20b/
(10 mg kgꢀ1); green, 1 (80 mg kgꢀ1); light blue, 16b (80 mg kgꢀ1); dark
EcIspC complex (Figure 2a) documents the consistency of the
blue, 20b (80 mg kgꢀ1).
ligand binding mode.16 It reveals that, irrespective of the hydro-
xamic acid in 1 or the reverse hydroxamic acid in 20b, the
coordination of the divalent metal ion is octahedral in both cases.
This is reminiscent of the catalytic mechanism of the isomeriza-
tion of 2, in which an alcohol and a hydroxyketone are converted
into a hydroxyaldehyde (a “reverse hydroxycarbonyl analogue”).
Structural superpositioning of the 20b/EcIspC complex with
the 1/EcIspC/NADPH (closed conformation) complex17 clearly
shows that some amino acid side chains (Figure 2b; only Asn211,
Trp212, and Met214 are shown) of the loop region would clash
with the difluorophenyl ring of 20b. This explains why the loop
cannot adopt the closed conformation in the 20b complex where
binding of the inhibitor necessitates a major reorientation of the
amino acid side chains of the protein. In contrast, binding of
1 occurs in the closed conformation because it perfectly fits into
the closed active site. Interestingly, the binding of 20b in the
open conformation might not have suggested that 20b is, in fact,
a stronger inhibitor of the enzyme than 1, which can bind to the
closed conformation.
The 20b/EcIspC complex structure reveals intramolecular van
der Waals interactions of the N-methyl group of 20b with the
difluorophenyl ring (3.9 Å) as well as with the main chain atoms
of the ligand. The N-methyl group though has no contact to the
enzyme.
Thus, 20b is enthalpically and entropically stabilized despite
the missing stabilization through the loop, and ligand binding
may thereby be favored. This observation is in good agreement
with the obtained in vitro data for compounds 16b and 20b.
Although the residues in the active site of IspC are conserved
between E. coli and P. falciparum, the overall sequence homology
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Table 2. In Vivo Activity in the P. berghei Acute Mouse
tetramethylsilane). Elemental analysis was performed on a Perkin-Elmer
PE 2400 CHN elemental analyzer. IR spectra were recorded on a Varian
800 FT-IR Scimitar series. Analytical high pressure liquid chromatogra-
phy (HPLC) was performed in analogy to a previously reported
procedure.11k The instrument was an Elite LaChrom system [Hitachi
L-2130 (pump) and L-2400 (UV-detector)]. The column was a Phe-
nomenexLuna C-18(2), 1.8 μm particle (250 mm ꢁ 4.6mm), supported
by Phenomenex Security Guard Cartridge Kit C18 (4.0 mm ꢁ 3.0 mm).
The purity of all final compounds was 95% or higher.
Modela
treatmentb
mean % suppression of infected RBC (day 4)
vehicle
0
98
92
89
78
chloroquine (10 mg/kg)c
1 (80 mg/kg)c
16b (80 mg/kg)c
20b (80 mg/kg)c
Experimental Data for Compounds. Experimental data are
listed below for selected compounds 10b, 19b, and 20b.
a GFP ANKA strain. b ip for 5 consecutive days. c Dose was divided into
two administrations of 40 mg kgꢀ1 dayꢀ1. Ritonavir was coadministered
orally at 10 mg kgꢀ1 (CYP3A4 inhibitor to minimize possible metabolic
degradation.)
General Procedure for the Synthesis of O-Bn-Protected
Hydroxamic Acids (9a,b, 10aꢀc, 11, 12). To a solution of the
respective carboxylic acid 8aꢀd (1 equiv, 5 mmol) in dry CH2Cl2
(50 mL) was added 1,10-carbonyldiimidazole (1.1 equiv, 0.9 g, 5.5 mmol)
in small portions. After the mixture was stirred at room temperature for
45 min, the appropriate hydroxylamine was added in one portion. The
solution was stirred overnight, and the solvent was removed under
reduced pressure. The remaining residue was dissolved in ethyl acetate
(30 mL), and the organic layer was washed three times with an aqueous
solution of citric acid (10%, 10 mL) and once with a saturated aqueous
solution of NaHCO3 (10 mL). The organic layer was dried over MgSO4,
filtered, and concentrated under reduced pressure. Hydroxamic acids
(9a, 10b, and 10c) were obtained by crystallization from diethyl ether
at 7 ꢀC. Hydroxamic acids 9b, 10a, and 11 were purified by column
chromatography on silica gel using ethyl acetate/n-hexane (1:1) as the eluent.
Diethyl {4-[(Benzyloxy)(methyl)amino]-1-(3,4-difluoro-
phenyl)-4-oxobutyl}phosphonate (10b). White solid (2.10 g,
92%). Mp: 60.2 ꢀC. 1H NMR (500.13 MHz, DMSO-d6): δ = 7.61ꢀ7.28
(m, 5H), 7.28ꢀ7.01 (m, 3H), 4.71 (PhCH2, s, 2H), 4.25ꢀ3.92
(CH3CH2, m, 2H), 3.92ꢀ3.70 (CH3CH2, m, 2H), 3.34ꢀ3.22 (PCH,
dd, J1 = 11.0 Hz, J2 = 24.0 Hz, 1H), 3.10 (NCH3, s, 3H), 2.42ꢀ2.07
(CH2, m, 3H), 2.08ꢀ1.76 (CHCH2, m, 1H), 1.20 (CH3, t, J=6.9 Hz,
3H), 1.06 (CH3, t, J = 7.0 Hz, 3H) ppm. 13C NMR (125.76 MHz,
CDCl3): δ = 172.65 (CdO), 149.07 (dd, 2JCꢀF = 14.1 Hz, 1JCꢀF = 246.9
Hz), 148.43 (dd, 2JCꢀF = 12.0 Hz, 1JCꢀF = 242.7 Hz), 134.38, 133.97,
129.27, 128.58, 128.24, 125.92, 117.87 (dd, 3JCꢀF = 6.5 Hz, 2JCꢀF = 17.4
is very low. The considerable degree of species specificity that is
revealed by our kinetic data emphasizes the necessity to actually
use IspC from P. falciparum in screening programs for antimalar-
ials directed at IspC.
Compounds 16b and 20b were tested in the Plasmodium
berghei mouse model. Drug treatment of animals was initiated 2 h
after artificial infection by intraperitoneal injection of infected
erythrocytes and was continued for 5 days. The in vivo efficacy of
16b was similar to that of 1 and chloroquine (Figure 3). On the
other hand, 20b had less effect on survival, although the percen-
tage of infected erythrocytes was significantly reduced (Figure 3,
Table 2).
Thus, whereas the novel compounds show high potency in the
nanomolar range when assayed against P. falciparum in human
erythrocytes and against IspC protein from that pathogen, the
small-scale animal study using a different Plasmodium species
gave less impressive results. It should be noted, however, that in
vitro studies on IspC protein of P. berghei are not available, and
species differences may well be relevant for the different out-
comes of the in vitro assays and the animal experiments.
3
2
’ CONCLUSION
Hz), 117.26 (dd, JCꢀF = 1.4 Hz, JCꢀF = 16.7 Hz), 75.08 (PhCH2),
61.73 (POCH2, d, 2JCꢀP = 6.8 Hz), 61.45 (POCH2, d, 2JCꢀP = 7.1 Hz),
In summary, we provide kinetic and crystallographic evidence
for the mode of action of a series of novel hydroxamate-based
IspC inhibitors. Several new analogues inhibit IspC protein of
P. falciparum with IC50 values in the low nanomolar range.
In contrast, the new compounds are significantly weaker inhibi-
tors of the E. coli enzyme. Finally, pilot in vivo experiments have
shown that compound 16b has some potential and is believed to
be a promising candidate for further pharmacological studies,
structural modifications, and the design of prodrugs.
41.28 (CHP, d, 1JCꢀP = 136.9 Hz), 32.63 (NCH3), 29.17 (CH2, d, 2JCꢀP
=
15.5 Hz), 24.31 (CH2), 16.14 (CH3, d, 3JCꢀP = 5.5 Hz), 15.99 (CH3, d,
3JCꢀP = 5.4 Hz) ppm. IR (KBr): ~ν = 3037 (CꢀHarom.), 2985 (CꢀHaliph.),
1673 (CdO), 1233 (PdO), 1053 (PꢀO) cmꢀ1. Anal. Calcd for
C22H28F2NO5P: C 58.02, H 6.20, N 3.08. Found: C 58.29, H 6.29, N 2.78.
General Procedure for the Synthesis of Phosphonic Acids
(19aꢀc). To a solution of the respective phosphonic acid diethyl ester
10aꢀc (1 equiv, 3 mmol) in dry dichloromethane (10 mL) was added
trimethylsilyl bromide (5 equiv, 1.99 mL, 15 mmol) at 0 ꢀC. After 1 h,
the solution was allowed to warm to room temperature and stirred for an
additional 23 h. The solvent was removed under reduced pressure. The
remaining residue was dissolved in THF (10 mL), and water (0.1 mL)
was added. After 30 min the solvent was evaporated and the resulting
residue dried in vacuo overnight. Pure phosphonic acids (19aꢀc) were
obtained as white solids at ꢀ20 ꢀC after digestion with ethyl acetate.
{4-[(Benzyloxy)(methyl)amino]-1-(3,4-difluorophenyl)-
4-oxobutyl}phosphonic Acid (19b). White solid (0.480 g,
40%). Mp: 143.5 ꢀC. 1H NMR (500.13 MHz, DMSO-d6): δ =
7.62ꢀ7.15 (m, 7H), 7.08 (s, 1H), 4.70 (PhCH2, s, 2H), 3.09
(NCH3, s, 3H), 2.94 (PCH, dd, J1 = 11.0 Hz, J2 = 20.2 Hz, 1H),
2.22 (CH2, s, 3H), 1.94 (CH2, m, 1H) ppm. 13C NMR (125.76 MHz,
’ EXPERIMENTAL SECTION
General Procedures. All solvents and chemicals were used as
purchased without further purification. The progress of all reactions was
monitored on Merck precoated silica gel plates (with fluorescence
indicator UV254) usingethyl acetate/n-hexaneas solventsystem. Column
chromatography was performed with Fluka silica gel 60 (230ꢀ400 mesh
ASTM) with the solvent mixtures specified in the corresponding
experiment. Spots were visualized by irradiation with ultraviolet light
(254 nm). Melting points (mp) were taken in open capillaries on a
Mettler FP 5 melting-point apparatus and are uncorrected. Proton (1H)
and carbon (13C) NMR spectra were recorded on a Bruker Avance 500
(500.13 MHz for 1H; 125.76 MHz for 13C) using DMSO-d6 and CDCl3
as solvents. Chemical shifts are given in parts per million (ppm)
DMSO-d6): δ = 172.66 (CdO), 149.02 (dd, 2JCꢀF = 12.7 Hz, 1JCꢀF
=
233.9 Hz), 148.12 (2JCꢀF = 13.3 Hz, 1JCꢀF = 228.1 Hz), 136.11 (d,
2JCꢀP = 8.8 Hz), 134.39, 129.29, 128.58, 128.23, 125.80, 117.57 (dd,
3JCꢀF = 5.7 Hz, 2JCꢀF = 16.5 Hz), 116.84 (dd, 2JCꢀF = 16.4 Hz), 75.06
1
(δ relative to residual solvent peak for H and 13C and to external
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ARTICLE
(PhCH2), 43.53 (CHP, d, 1JCꢀP = 133.4 Hz), 32.68 (NCH3), 29.57
(CH2, d, 2JCꢀP = 16.8 Hz), 24.90 (CH2, d, 3JCꢀP = 2.1 Hz) ppm. IR
(KBr): ν~ = 3437 (NꢀH), 3037 (CꢀHarom.), 2944 (CꢀHaliph.), 1607
(CdO), 1277 (PdO), 999 (PꢀO) cmꢀ1. Anal. Calcd for C18H20F2NO5P:
C 54.14, H 5.05, N 3.51. Found: C 54.02, H 5.06, N 3.23.
General Procedure for the Synthesis of Target Compounds
(16a,b, 17, 18, and 20aꢀc). To a solution of the appropriate O-Bn-
protected acid (1 mmol) in freshly distilled methanol (20 mL) was added
PdꢀC catalyst (10%, 40 mg). The mixture was hydrogenated at a pressure
of 2 bar for 1.5 h (in the case of compounds 13a,b, 14, and 15) or 3 h (in
the case of compounds 19aꢀc). The suspension was filtered through an
SPE tube RP-18, and the solvent was removed under reduced pressure.
Whereas compounds 17, 18, and 20c were obtained as hygroscopic oils,
compounds 16a,b and 20a,b were crystallized from appropriate solvents as
described below.
D-xylulose 5-phosphate; Dxr (IspC), 1-deoxy-D-xylulose 5-phos-
phate reductoisomerase; ip, intraperitoneal; MEP, 2C-methyl-D-
erythritol 4-phosphate; MRC-5, human fetal lung fibroblast;
NADPH, nicotinamide adenine dinucleotide phosphate; PdꢀC,
palladium on activated carbon; RBC, red blood cell; RT, room
temperature; SD, standard deviation; THF, tetrahydrofuran;
TMSBr, bromotrimethylsilane
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A.; Gr€awert, T.; Groll, M.; Rohdich, F.; Bacher, A.; Eisenreich, W.; Fischer,
M.; Maes, L.; Kurz, T. Synthesis and antiplasmodial activity of highly active
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{1-(3,4-Difluorophenyl)-4-[hydroxy(methyl)amino]-4-
oxobutyl}phosphonic Acid (20b). White solid (0.258 g, 84% after
1
recrystallization in ethyl acetate). Mp: 116.8 ꢀC. H NMR (500.13 MHz,
DMSO-d6): δ= 9.67 (NOH, s, 1H), 7.35 (dd, J1 =8.9 Hz, J2 =19.0Hz, 1H),
7.30ꢀ7.22 (m, 1H), 7.18ꢀ6.86 (m, 1H), 3.02 (NCH3, s, 3H), 2.97 (PCH,
dd, J1 = 9.6 Hz, J2 = 21.7 Hz, 1H), 2.20 (CH2, s, 3H), 1.98ꢀ1.82 (CHCH2,
m, 1H) ppm. 13C NMR (125.76 MHz, DMSO-d6): δ=172.20 (CdO),
149.76 (dd, 2JCꢀF = 10.8 Hz, 1JCꢀF = 242.9 Hz), 148.96 (dd, 2JCꢀF = 13.9
1
3
Hz, JCꢀF = 250.6 Hz), 136.13, 125.82, 117.54 (dd, JCꢀF = 5.8 Hz,
2JCꢀF=16.9 Hz), 116.77 (dd, JCꢀF = 1.5 Hz, JCꢀF = 16.6 Hz), 43.61
(CHP, d, 1JCꢀP = 133.4 Hz), 35.54 (NCH3), 29.69 (CH2, d, 2JCꢀP = 15.1
Hz), 25.00 (CH2) ppm. IR (KBr): ~ν = 3405 (NꢀH), 1615 (CdO), 1282
(PdO), 1019 (PꢀO) cmꢀ1. Anal. Calcd for C11H14F2NO5P: C 42.73, H
4.56, N 4.53. Found: C 43.00, H 4.77, N 4.50.
3
2
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures, ana-
b
lytical data, enzyme assays, biological evaluation of in vitro
antiplasmodial activity and cytotoxicity, in vivo mouse model
with P. berghei, and experimental data regarding crystallization
and structure determination. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
‡PDB code for EcIspC with bound 20b is 3R0I.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (+49)21181-14984. Fax: (+49)21181-13847. E-mail:
thomas.kurz@uni-duesseldorf.de.
Author Contributions
∞These authors contributed equally.
’ ACKNOWLEDGMENT
We thank Proteros Biostructures GmbH, Martinsried, Germany,
for financial support to A.K., and we thank Krystina Kuna and Felix
Quitterer for experimental support.
’ DEDICATION
†Dedicated to Dr. Viktoriya Illarionova, in memoriam.
’ ABBREVIATIONS USED
n-BuLi, n-butyllithium; BW, body weight; DCC, dicyclohexylcar-
bodiimide; DMF, N,N-dimethylformamide; DOXP, 1-deoxy-
6801
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Journal of Medicinal Chemistry
ARTICLE
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(18) All animal experiments were approved by the ethical committee
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pathogens: ref LA-1100158.
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