α-Substituted β-oxa isosteres of fosmidomycin: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm300652f

Specific inhibition of enzymes of the non-mevalonate pathway is a promising strategy for the development of novel antiplasmodial drugs. α-Aryl-substituted β-oxa isosteres of fosmidomycin with a reverse orientation of the hydroxamic acid group were synthes

Full text of DOI:10.1021/jm300652f

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Article
#-Substituted #-oxa isosteres of Fosmidomycin:
Synthesis and biological evaluation
Karin Brücher, Boris Illarionov, Jana Held, Serena Tschan, Andrea Kunfermann, Miriam Pein, Adelbert
Bacher, Tobias Gräwert, Louis Maes, Benjamin Mordmueller, Markus Fischer, and Thomas Kurz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300652f • Publication Date (Web): 25 Jun 2012
Downloaded from http://pubs.acs.org on June 26, 2012
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αꢀSubstituted βꢀoxa isosteres of Fosmidomycin:
Synthesis and biological evaluation
Karin Brücher,† Boris Illarionov§ Jana Held,# Serena Tschan,# Andrea Kunfermann,‡ Miriam K. Pein,†
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∇
Adelbert Bacher,‡ Tobias Gräwert§, Louis Maes,* Benjamin Mordmüller,# Markus Fischer§ and
Thomas Kurz*,†
†Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität, Universitätsstr. 1,
40225 Düsseldorf, Germany, §Hamburg School of Food Science, Universität Hamburg, Grindelallee
117, 20146 Hamburg, Germany, #Institut für Tropenmedizin, Eberhard Karls Universität Tübingen,
Wilhelmstr. 27, 72074 Tübingen, Germany, ‡Center for Integrated Protein Science Munich, Lehrstuhl
für Biochemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany,
*Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,
Groenenborgerlaan 171, 2020 Wilrijk, Belgium, and ∇Medical Research Laboratory, Albert Schweitzer
Hospital, Lambaréné, Gabon.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
†
CORRESPONDING AUTHOR FOOTNOTE Institut für Pharmazeutische und Medizinische Chemie,
HeinrichꢀHeineꢀUniversität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany. Phone:
(+49)21181ꢀ14985, Fax ꢀ13847, Eꢀmail: thomas.kurz@uniꢀduesseldorf.de.
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ABSTRACT Specific inhibition of enzymes of the nonꢀmevalonate pathway is a promising strategy for
the development of novel antiplasmodial drugs. αꢀAryl substituted βꢀoxa isosteres of fosmidomycin
with a reverse orientation of the hydroxamic acid group were synthesized and evaluated for their
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inhibitory activity against recombinant 1ꢀdeoxyꢀ ꢀxylulose 5ꢀphosphate synthase (IspC) of Plasmodium
D
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falciparum and for their in vitro antiplasmodial activity against chloroquineꢀsensitive and resistant
strains of P. falciparum. The most active derivative inhibits IspC protein of P. falciparum (PfIspC) with
an IC50 value of 12 nM and shows potent in vitro antiplasmodial activity. In addition, lipophilic ester
prodrugs demonstrated improved P. falciparum growth inhibition in vitro.
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Introduction
The spread of Plasmodium falciparum resistance to most malarial drugs is a major challenge in the
control of malaria.1 Today, resistance affects nearly all main classes of antimalarial drugs and until now
artemisinin derivatives and artemisinin combination therapy remain the workhorse for the treatment of
severe and uncomplicated malaria, respectively. Currently, the situation is alarming because decelerated
parasite elimination after artemisinin therapy is reported from Southeast Asia, a possible indicator for
the development of resistance.2 Despite the urgent need of new antimalarials, only very few nonꢀ
artemisinin drug candidates are currently in clinical development. Most of these drug candidates are
variations of existing antimalarial drugs.3 Hence, the development of new classes that exhibit novel
modes of action is of utmost importance. Currently, only very few drug candidates meet these criteria.
Among them and of particular interest is the phosphonohydroxamic acid antibiotic fosmidomycin (1),
which was isolated from Streptomyces lavendulae in the late 1970’s.4 Due to its unique mechanism of
action, crossꢀresistance between 1 and other commonly used antimalarials is unlikely. Clinical studies
conducted with 1 in combination with clindamycin, a slow acting partner drug, have already
demonstrated good efficacy in the treatment of acute uncomplicated P. falciparum malaria.5 However,
due to its high polarity and its deprotonation at physiological pH values, 1 has low oral bioavailability of
approximately 10ꢀ30 % and a short plasma halfꢀlife of approximately 1.9 hours.6
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Scheme 1. Methylerythritol phosphate (MEP) pathway and chainꢀmodified target compounds.
1 exerts its antiplasmodial activity by inhibition of 2CꢀmethylꢀDꢀerythritol synthase (IspC/Dxr), which
catalyzes the first committed step of the nonꢀmevalonate isoprenoid biosynthesis pathway.7 Since that
pathway is absent in mammalians, which produce isoprenoid precursors via the mevalonate pathway,
targetꢀbased adverse effects are not to be expected.8 To improve the properties of 1, bioisosteric
replacements of both functional groups, modification of the aliphatic spacer as well as the design of
phosphonate prodrugs have been reported.9 Recently, some analogues with a reverse orientation of the
hydroxamic acid functionality have been described (3aꢀd), which inhibit IspC protein of P. falciparum
(PfIspC) with IC50 values in the low nanomolar range (Scheme 1).10 In a previous publication, we
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provided kinetic and crystallographic evidence for their mode of action. Pilot in vivo experiments have
also demonstrated in vivo potential for compound 3a.10b
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In an attempt to find derivatives of 1 with further improved activity, we synthesized a small library of
reverse βꢀoxa isosteres (4aꢀk) and determined their inhibitory activity in the PfIspC enzyme assay and
in continuous P. falciparum in vitro culture.
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Results
Earlier studies had shown that the introduction of differently substituted phenyl residues at the αꢀ
position of 1 can significantly increase the inhibitory potency towards PfIspC.10a, b It has also been
shown that the replacement of the βꢀmethylene group of 1 by oxygen resulted in improved efficacy
against P. falciparum laboratory isolates.9j It was therefore appropriate to investigate whether the
combination of these respective structural modifications (i.e. aromatic substituents at the αꢀposition of
βꢀoxa isosteric compounds) results in improved activity at the level of the isolated enzyme target and at
the level of the erythrocyte in vitro assay.
In order to analyze whether the replacement of the βꢀmethylene group of the previously reported
compound 3b10b by oxygen might enable the formation of an additional hydrogen bond with PfIspC and
a consecutive increase in affinity, we performed a modeling study using the Xꢀray structure of PfIspC in
complex with 1, NADPH and a metal ion as template (RCSB accession code, 3AU9; the use of the
quaternary complex including inhibitor, coenzyme and metal ion appeared crucial for a comparison
between kinetic and structural data since kinetic studies are by necessity performed in presence of
coenzyme and metal ions). 1 in 3AU9 was replaced by compound 3b in the conformation that it
assumes in the complex with IspC of Escherichia coli (EcIspC; PDB accession code, 3R0I),10b and the
βꢀmethylene group of the ligand was replaced by oxygen (affording the hypothetical ligand structure
4e). Minimization of the hybrid model supported the potential formation of a hydrogen bond between
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the oxygen in the βꢀposition of the hypothetical 4e ligand and the highly conserved Ser306 (P.
falciparum numbering) side chain (Fig. 1). On this basis, we decided to synthesize a small library of
βꢀoxa isosteres (4) of previously published derivatives of 1 with known inhibitory activity (3).10
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Figure 1. Stereo representation of a hypothetical model of compound 4e into the active site of PfIspC.
4e (white), NADPH (white), and S306 (green) are shown in stick representation. Additional amino acid
residues at the active site are shown in line representation. A hypothetical Hꢀbond (2.9 Å) between Oγ
of S306 and the βꢀoxa group of 4e is shown as dashed red line. Atomic coordinates for the carba
analogue of 4e were taken from PDB file 3R0I, and the β−methylene group was replaced with oxygen.
The coordinates of 4e were then merged with 3AU9, replacing the coordinates of 1. The ensemble was
minimized with SYBYLX 2.0.
Synthesis of βꢀoxa isosteres. A retrosynthetic analysis led to αꢀhydroxyphosphonates (6) and
commercially available benzyl bromoacetate as suitable building blocks for the synthesis of target
compounds 4aꢀk. Previously reported and unreported αꢀhydroxyphosphonates 6aꢀg were accessible by
Pudovik reactions of aromatic aldehydes 5aꢀg with diethyl phosphite in presence of triethylamine
according to established literature procedures.11 OꢀAlkylation of 6aꢀg with benzyl bromoacetate and
neutral silver oxide led to completely protected synthones 7aꢀj in good yields.12 Next, benzyl esters 7aꢀg
were deprotected by catalytic hydrogenation to afford carboxylic acids 8aꢀg. 1,1´ꢀCarbonyldiimidazole
(CDI)ꢀmediated activation of 8aꢀg followed by treatment with various Oꢀbenzylꢀprotected
hydroxylamines provided the protected phosphonohydroxamic acids 9aꢀh and 10aꢀc.13 The
corresponding free hydroxamic acids 11aꢀh were obtained in good yields and purity by catalytic
hydrogenation of 9aꢀh.
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Scheme 2. Synthesis of target compounds 4.a
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aReagents and conditions: a) DEP, TEA, RT, 12 h, 74ꢀ99 %; b) BrCH2CO2Bn, Ag2O, DMF, RT, 24 h,
40ꢀ83 %; c) H2, PdꢀC, MeOH or ethyl acetate, 1 atm, RT, 2 h, 75ꢀ99 %; d) CDI, BnONHR2 (R2 = H,
CH3, C2H5), CH2Cl2, RT, 12 h, 85ꢀ98 %; e) H2, PdꢀC, MeOH or ethyl acetate, 1 atm, RT, 2 h, 73ꢀ99 %;
f) 1. TMSBr, CH2Cl2, RT, 24 h; 2. THF/H2O, RT, 1 h; 3. DCC, BnOH, benzene, 80 °C, 4 h, 25ꢀ26 % (3
steps); g) 1. TMSBr, CH2Cl2, RT, 24 h; 2. THF/H2O, RT, 1 h, 21ꢀ85 % (2 steps); h) H2, PdꢀC, MeOH, 1
atm, RT, 2 h, 99 %.
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Cleavage of the phosphonic ester group of compounds 11 was accomplished by dealkylation with
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bromotrimethylsilane and subsequent aqueous hydrolysis.14 In case of the Nꢀalkylꢀsubstituted
derivatives 11aꢀh, the desired target compounds 4aꢀh were obtained as pure solids, whereas compounds
4iꢀk were isolated as oily and hygroscopic mixtures of 4 and 11. Therefore, the phosphonic acid diethyl
ester moieties of substrates 10aꢀc were converted into the corresponding dibenzyl phosphonate moieties
using a transesterification method.15 Finally, catalytic hydrogenation of Bnꢀprotected precursors 12aꢀc
afforded free hydroxamic acids 4iꢀk.
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Synthesis of prodrugs of 4e. Deprotection of the phosphonic acid moiety of intermediate 9e was
accomplished by dealkylation and subsequent hydrolysis as described before. Without prior purification,
the crude phosphonic acid was alkylated by reaction with chloromethyl pivalate in the presence of
triethylamine according to published procedures.9i, j
Scheme 3. Synthesis of prodrugs 14, 15.a
aReagents and conditions: i) 1. TMSBr, CH2Cl2, RT, 24 h; 2. THF/H2O, RT, 1 h; 3. chloromethyl
pivalate, TEA, DMF, 80 °C, 6 h, 37 % (3 steps); j) H2, PdꢀC, MeOH, 1 atm, RT, 2 h, 99 %; k) ethyl
chloroformate or ethyl isocyanate, TEA, CH2Cl2, RT, 1h, 93ꢀ98 %.
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Next, catalytic hydrogenation of benzyl ester 13 led to prodrug 14. Finally, the hydroxamic acid
moiety of 14 was reacted with ethyl chloroformate or ethyl isocyanate in the presence of triethylamine
to yield a new type of lipophilic ester prodrugs 15a, b.16
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Biological studies. The βꢀoxa isoster minilibrary was assayed with recombinant PfIspC using
published procedures.10a, b The experimental data can be described by a competitive kinetic model
without cooperative interactions (Fig. 2).17 IC50 values are summarized in Table 1 and show a range of
0.012 ꢁM to > 50 ꢂM (Fig. 2). The table also shows IC50 values obtained by growth assay with P.
falciparum using the chloroquineꢀsensitive strain 3D7 and the multiꢀresistant strain Dd2.
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Figure 2. Inhibition of PfIspC; 1 ( ), 4b ( ), 4c ( ), 4f ( ), 4h ( ) and 4k ( ).
IC50 values against the chloroquineꢀsensitive strain 3D7 and the chloroquineꢀresistant strain Dd2 are
visualized in Fig. 3A. The differences between the results obtained with the respective strains are quite
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small; in the double logarithmic plot, all values lie close to the line of best fit (dashed red line in Fig.
3A).
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Table 1. Inhibition of PfIspC and in vitro antiplasmodial activity.
PfIspCa,c
PfDd2b,c
IC50 [ꢁM] IC50 [ꢁM]
Pf3D7b,c
Compound R1
R2
X
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IC50 [ꢁM]
0.16 +/ꢀ 0.02
1
0.81
0.70
0.35
0.35
1.2
0.88
1.2
Ph
CH3
0.037 +/ꢀ 0.002
0.039 +/ꢀ 0.004
0.027 +/ꢀ 0.001
0.065 +/ꢀ 0.005
0.012 +/ꢀ 0.001
0.014 +/ꢀ 0.001
0.025 +/ꢀ 0.003
0.21 +/ꢀ 0.01
1.5 +/ꢀ 0.1
4a
4b
4c
naphthꢀ1ꢀyl CH3
0.52
0.48
1.3
4ꢀFꢀPh
CH3
CH3
CH3
CH3
CH3
C2H5
H
2,4ꢀFꢀPh
3,4ꢀFꢀPh
3,4ꢀClꢀPh
4ꢀCH3ꢀPh
Ph
4d
4e
0.13
0.14
0.19
3.8
0.54
0.24
0.18
8.1
4f
4g
4h
4i
Ph
n. d.
n. d.
n. d.
0.022
0.019
0.022
n. d.
n. d.
n. d.
> 50
> 50
> 50
0.013
0.023
0.031
n. d.
n. d.
n. d.
2,4ꢀFꢀPh
4ꢀCH3ꢀPh
3,4ꢀFꢀPh
3,4ꢀFꢀPh
3,4ꢀFꢀPh
3,4ꢀFꢀPh
Ph
H
1.7 +/ꢀ 0.1
4j
H
1.8 +/ꢀ 0.1
4k
14
15a
15b
3b
3c
CH3
CH3
CH3
CH3
CH3
CH3
n.d.
O
N
n.d.
n.d.
0.0034 +/ꢀ 0.0004
0.0031 +/ꢀ 0.0003
0.014 +/ꢀ 0.002
4ꢀCH3ꢀPh
3d
aValues (mean +/ꢀ SD calculated from nine or more data points) were determined using nonlinear
regression analysis as described earlier.18 bValues are the mean of at least two independent experiments
c
conducted in duplicate, each using 12 serial dilutions. n.d., not determined; antiplasmodial in vitro
activity of 3bꢀd towards PfK1 strain was published previously.10
The IC50 values for the enzyme assay and the in vitro growth assay with P. falciparum are correlated
graphically in Fig. 3B. Notably, for the βꢀoxa analogues of 1, the IC50 values of the enzyme assay are
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lower than those from the growth inhibition assay; the values show an excellent correlation coefficient
of 0.98 and can be roughly correlated by a parallel to the image diagonal.
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Figure 3. (a), activities against the chloroquine resistant strain Dd2 versus activities against strain 3D7.
The line of best fit is close to the image diagonal; black, fomidomycin. (b), Correlation of enzyme assay
and cellꢀbased growth inhibition assay. Most substances lie close to a red line that is shifted from the
image diagonal (blue) by one order of magnitude in y direction (no line of best fit is shown in this
graph); black, 1. (c & d), All values originate from IspC enzyme assays. C, Difference between the log10
of the IC50 value of a given oxa substance and the log10 of the IC50 of the respective carba derivative
versus log10 of the IC50 of the oxa substance; red, methyl or ethyl group in position R2; green, hydrogen
in position R2. (d), Difference between the log10 of the IC50 value of a given Nꢀmethylꢀsubstituted
compound (R2) and the log10 of the IC50 of the cognate nonꢀalkylated substance (R2) versus log10 of the
IC50 of the respective Nꢀmethylated substance. Light blue: carba analogues; magenta: oxa analogues.
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In order to unequivocally assess the impact of the βꢀoxa replacement, it is of course crucial to
compare the data in table 1 with those for the cognate derivatives of 1 with methylene groups in the β
position (subsequently designated as “carba” analogues, 3aꢀd). The data for the “carba series” (from our
previous work: scheme 1, table 1) were published previously.10 3bꢀd are also shown graphically in
Figure 3C. The abscissa in this graph is the log10 of the IC50 value of the βꢀoxa series. The ordinate is
the difference between the logarithm of the IC50 values of the cognate oxa:carba pairs. The data leave no
doubt that, contrary to our initial hypothesis, the oxa compounds are weaker inhibitors as compared to
the cognate carba compounds. The losses in binding free enthalpy caused by the oxygen substitution are
in the range of about 1 kcal molꢀ1 for the compounds shown in red, and above of 1 kcal molꢀ1 for the
compounds shown in green in Fig. 3C (even though we are using IC50 data rather than Ki values, the
ordinate values are approximately equivalent to ꢀꢀGbinding, i.e. the free enthalpy difference between
each respective oxa and carba compound in the bound state). The derivatives differ by the presence
(red) or absence (green) of an alkyl substituent (typically methyl). The marked difference between these
groups prompted further analysis, which was possible since the minilibrary contained several pairs of
compounds which differ only by the presence or absence of the Nꢀalkyl substituent. In Figure 3D, the
abscissa represents IC50 values of compounds from either the carba or oxa series. The ordinate
represents the difference of the logarithms of IC50 of the Nꢀmethylated and nonꢀmethylated compound,
respectively (which is related to ꢀꢀGbinding of methylated and nonꢀmethylated partner). The values for
cognate pairs in the carba series are shown as triangles, and the values for the oxa pairs are shown as
squares. The data show that Nꢀmethyl substitution causes a gain in binding enthalpy ranging from about
0 – 2 kcal molꢀ1.
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Earlier, we could show that the efficacy of αꢀaryl substituted analogs of 1 could be improved by
conversion into phosphonate ester prodrugs.9f, 9h Based on that experience, we prepared several prodrugs
of compound 4e which documented impressively improved efficacy in P. falciparum growth assays.
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Discussion
1
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9
1 is exceptional since it is the only new nonꢀartemsinin drug candidate in clinical development that
may complement artemisinin combinations in the treatment of uncomplicated malaria. In an attempt to
improve the affinity of existing derivatives of 1 for PfIspC, we prepared a series of βꢀoxa isosteres with
the expectation that the formation of an additional hydrogen bond might afford an increase in affinity.
However, the comparison of two minilibraries whose cognate members differed only by the presence of
oxygen or methylene groups in the β position showed that the β oxygen reduced the binding free
enthalpy by about 1 kcal molꢀ1. Attempts to cocrystallize one of the oxa derivatives with PfIspC were as
yet unsuccessful. Thus, it remains unknown whether the expected additional hydrogen bond fails to be
formed, or whether it is indeed formed but does not improve the affinity due to some structural penalty,
possibly due to interference with the network of other hydrogen bonds at the active site. As a side issue,
the data also show clearly that the Nꢀmethyl derivatives are significantly better inhibitors than the
unmethylated analogs.
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After conversion to lipophilic ester prodrugs (14, 15a, b), the best oxa isosteres had significantly
better IC50 values (0.013ꢀ0.031 ꢁM) in the P. falciparum growth assay than 1 (Table 1). This is in line
with earlier favorable reports on pivaloyloxymethyl ester prodrugs of derivatives of 1. However, many
details of the successful prodrug concept are still incompletely understood. As a surrogate marker for
the efficacy of derivatives of 1 as antimalarials, enzyme assays with the molecular target PfIspC have
the advantage of high reproducibility and accuracy. When this biochemical precision tool can be applied
to families of structurally similar compounds, the impact of minimal structural changes on the binding
enthalpy can be assessed in minute detail. Using this approach, we found that, contrary to expectation,
the replacement of the methylene group in the βꢀposition of the linker between the phosphonate and
hydroxamate motifs by an oxygen atom is accompanied by an affinity penalty in the range of about
1 kcal molꢀ1. We also found that methylation of the hydroxamic acid pharmacophore is accompanied by
a gain in affinity of similar size. Notably, the effect of introducing oxygen in the βꢀposition and of
methylation are not correlated in a simple way. The impact of methylation is much larger in case of the
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oxa series as compared to the carba series. The combination of methylene in the βꢀposition and methyl
at the hydroxamic acid motif gives the highest affinity. Clearly, only the detailed kinetic analysis of
multiple analogs has the potential to unravel these details which are “below the observational horizon”
of a purely structureꢀbased approach, even in case of optimal crystallographic resolution.
1
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9
The antimalarial activity of derivatives of 1 cannot be predicted solely by PfIspC enzyme assays
because the situation in the intact organism may have a strong impact on activity and onꢀtarget
concentration. Since the enzyme is located inside the apicoplast, a plastid that arose through secondary
endosymbiosis in apicomplexan protozoa, the inhibitor has to pass the erythrocyte membrane and 5 to 7
lipid membranes of the parasite in order to reach its molecular target.19 Notably, it is as yet unknown
how and where the prodrugs are hydrolytically converted to the free phosphonic acids.
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Moreover, recent work suggests that the membrane of the erythrocyte host cell allows the passage of
(genuine, unmodified) 1 only after it has been modulated by the presence of the intracellular pathogen.20
Enteral resorption and bioavailability are further factors that must be taken into account. Even though
enzyme inhibition at singleꢀdigit nM IC50 values has been achieved with certain derivatives of 1, the
challenges for their clinical success remain high. On the other hand, the drugability of the nonꢀ
mevalonate pathway enzymes in apicoplasts in general and specifically in Plasmodium spp. has received
additional support by recent findings. Since it has been shown that the antiplasmodial effect of 1 can be
reversed by IPP, the final product of the nonꢀmevalonate pathway, it now appears safe to assume that
PfIspC is really the one and only target of 1 in the parasite.21 It has also been shown that the elimination
of the apicoplast is lethal in case of Toxoplasma gondii.19, 22 However, whereas inhibition studies with
recombinant PfIspC can be used for the fineꢀtuning of the inhibitorꢀtarget interaction, the progression of
chemically modified derivatives of 1 to the clinical level depends crucially on the combination of the
enzymeꢀbased analysis with other preclinical tools.
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Experimental Section
1
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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) using ethyl acetate/nꢀhexane as solvent system. 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 meltingꢀpoint apparatus and are
uncorrected. IR spectra were recorded on a Varian 800 FTꢀIR Scimitar series. Proton (1H) and carbon
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1
13
(13C) NMR spectra were recorded on a Bruker Avance 500 (500.13 MHz for H; 125.76 MHz for C)
using DMSOꢀd6 and CDCl3 as solvents. Chemical shifts are given in parts per million (ppm), (δ relative
1
to residual solvent peak for H and 13C and to external tetramethylsilane). Elemental analysis was
performed on a Perkin Elmer PE 2400 CHN elemental analyzer. If necessary the purity was determined
by HPLC. Analytical high pressure liquid chromatography (HPLC) was performed in analogy to a
previously reported procedure.23 Instrument: Elite LaChrom system [Hitachi Lꢀ2130 (pump) and Lꢀ2400
(UVꢀdetector)]; column: Phenomenex Luna Cꢀ18(2) 1.8 ꢁm particle (250 mm × 4.6 mm), supported by
Phenomenex Security Guard Cartridge Kit C18 (4.0 mm × 3.0 mm). The purity of all final compounds
determined by HPLC was 95% or higher.
Experimental Data for compounds. Experimental Data are listed below for selected compounds 4e,
9e, 11e, 13, and 14.
General procedure for the synthesis of OꢀBnꢀprotected hydroxamic acids 9aꢀh, 10aꢀc. To a
solution of the respective carboxylic acid 8aꢀg (1 eq, 5 mmol) in dry dichloromethane (20 mL)
1,1´ꢀcarbonyldiimidazole (1.1 eq, 5.5 mmol, 0.89 g) was added in small portions. After stirring 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
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solution of citric acid (10%, 10 mL), once with a saturated aqueous solution of NaHCO3 (10 mL) and
once with water (10 mL). The organic layer was dried over MgSO4, filtered and the solvent was
removed under reduced pressure. Hydroxamic acids 9aꢀk and 10aꢀc were purified by column
chromatography on silica gel using ethyl acetate as the eluent.
1
2
3
4
5
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7
8
9
Diethyl ((2ꢀ((benzyloxy)(methyl)amino)ꢀ2ꢀoxoethoxy)(3,4ꢀdifluorophenyl)methyl)ꢀphosphonate
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(9e). Yellow oil (6.11 g, 89%); H NMR (500.13 MHz, CDCl3): δ=1.25 (t, J=7.1 Hz, 3H, POCH2CH3),
1.28 (t, J=7.1POCH2CH3), 3.19 (s, 3H, NCH3), 3.96 (d, J=16.2 Hz, 1H, OCH2C=O), 4.05ꢀ4.18 (m, 4H,
POCH2CH3), 4.33 (dd, J1=16.11 Hz, J2=1.0 Hz, 1H, OCH2C=O), 4.72 (dd, J1=10.8 Hz, J2=9.4 Hz, 2H,
OCH2Ph), 4.91 (d, J=14.7 Hz, 1H, PCH), 7.11ꢀ7.19 (m, 2H), 7.22ꢀ7.28 (m, 3H), 7.22ꢀ7.35 (m, 3H)
13
3
3
ppm; C NMR (125.76 MHz, CDCl3): δ=16.36 (d, JC,P=6.6 Hz, POCH2CH3), 16.41 (d, JC,P=6.5 Hz,
2
2
POCH2CH3), 33.45 (NCH3), 63.20 (d, JC,P=6.9 Hz, POCH2CH3), 63.46 (d, JC,P=6.4 Hz, POCH2CH3),
3
1
66.37 (d, JC,P=14.1 Hz, OCH2C=O), 76.25 (OCH2Ph), 76.82 (d, JC,P=167.8 Hz, PCH), 117.21 (dd,
2JC,F=17.4 Hz, JC,F=1.7 Hz), 117.29 (dd, JC,F=18.0 Hz, JC,F=5.3 Hz), 124.50 (ddd, JC,P=3JC,F=6.1 Hz,
3
2
3
3
4JC,F=3.6 Hz), 128.73 (2C), 129.21, 129.37 (2C), 131.34 (m), 133.91, 150.32 (ddd, JC,F=249.0 Hz,
1
2JC,F=15.1 Hz, JC,P=3.0 Hz), 150.43 (ddd, JC,F=248.9 Hz, JC,F=12.3 Hz, JC,P=2.7 Hz), 170.90 (C=O)
ppm; IR (NaCl): ṽ=2984 (CꢀHaliph.), 1682 (C=O), 1285, 1253 (P=O), 1051, 1028 (PꢀO) cmꢀ1; Anal.
calcd. for C21H26F2NO6P: C 55.14, H 5.73, N 3.06, found: C 54.90, H 5.66, N 3.08.
4
1
2
5
General procedure for the synthesis of carboxylic acids 8aꢀg, hydroxamic aids 11aꢀh, target
compounds 4iꢀk and prodrug 14. Benzyl esters 7aꢀg, Bnꢀprotected compounds 9aꢀh, 12aꢀc and Bnꢀ
protected prodrug 13 (1 mmol) were dissolved in methanol (30 mL), respectively. In case of 7f and 11f,
ethyl acetate was used as a solvent. PdꢀC catalyst (10%, 15% w/w) was added and the respective
mixture was hydrogenated for 2 h at atmospheric pressure. The catalyst was removed by filtration and
the solvent was evaporated in vacuo to yield crystalline compounds 8a,b,d,e,g 11aꢀe,g and 4i.
Carboxylic acids 8c,f and hydroxamic acids 11f,h and 14 were obtained as colourless or yellow oils. In
case of target compounds 4j,k, catalytic hydrogenation yielded hygroscopic resins.
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Diethyl
((3,4ꢀdifluorophenyl)(2ꢀ(hydroxy(methyl)amino)ꢀ2ꢀoxoethoxy)methyl)ꢀphosphonate
1
2
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8
9
1
(11e). Yellow oil (1.84 g, 99%); H NMR (500.13 MHz, [D6]DMSO): δ=1.26 (t, J=7.2 Hz, 3H,
POCH2CH3), 1.29 (t, J=7.1POCH2CH3), 3.27 (s, 3H, NCH3), 4.05ꢀ4.20 (m, 4H, POCH2CH3), 4.12 (d,
J=14.6 Hz, 1H, OCH2C=O), 4.61 (d, J=14.6 Hz, 1H, OCH2C=O), 4.89 (d, J=15.9 Hz, 1H, PCH), 7.08ꢀ
7.29 (m, 3H), 9.36 (br.s, 1H, OH) ppm; 13C NMR (125.76 MHz, [D6]DMSO): δ=16.31 (d, 3JC,P=5.6 Hz,
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POCH2CH3), 16.33 (d, JC,P=5.4 Hz, POCH2CH3), 35.99 (NCH3), 63.66 (d, JC,P=7.2 Hz, POCH2CH3),
2
3
1
64.17 (d, JC,P=6.7 Hz, POCH2CH3), 68.42 (d, JC,P=13.5 Hz, OCH2C=O), 76.84 (d, JC,P=170.9 Hz,
2
3
2
3
PCH), 116.83 (dd, JC,F=18.4 Hz, JC,F=4.7 Hz), 117.37 (dd, JC,F=17.3 Hz, JC,F=1.14 Hz), 124.09 (m),
1
2
5
1
131.04 (m), 150.38 (ddd, JC,F=248.5 Hz, JC,F=12.7 Hz, JC,P=2.1 Hz), 150.48 (ddd, JC,F=248.5 Hz,
2JC,F=12.5 Hz, JC,P=2.5 Hz), 169.36 (C=O) ppm; IR (NaCl): ṽ=3154 (OꢀH), 2985, 2913 (CꢀHaliph.),
4
1665 (C=O), 1286 (P=O), 1050, 1028 (PꢀO) cmꢀ1; Anal. calcd. for C14H20F2NO6P: C 45.78, H 5.49, N
3.81, found: C 46.00, H 5.38, N 3.68.
((((3,4ꢀDifluorophenyl)(2ꢀ(hydroxy(methyl)amino)ꢀ2ꢀoxoethoxy)methyl)phosphoryl)bis(oxy))ꢀ
1
bis(methylene) bis(2,2ꢀdimethylpropanoate) (14). Yellow oil (0.27 g, 99%); H NMR (500.13 MHz,
CDCl3): δ=1.20, 1.22 (2s, 18H, C(CH3)3), 3.26 (s, 3H, NCH3), 4.16 (d, J=14.5 Hz, 1H, OCH2C=O),
4.57 (d, J=14.5 Hz, 1H, OCH2C=O), 4.95 (d, J=15.2 Hz, 1H, PCH), 5.58ꢀ5.73 (m, 4H, POCH2), 7.09ꢀ
13
7.20 (m, 2H), 7.22ꢀ7.32 (m, 1H), 8.74 (br.s, 1H, OH) ppm; C NMR (125.76 MHz, CDCl3): δ=26.76,
3
27.07 (2s, C(CH3)3), 36.04 (NCH3), 38.72 (C(CH3)3), 68.68 (d, JC,P=12.9 Hz, OCH2C=O), 76.59 (d,
1JC,P=163.1 Hz, PCH), 82.28 (d, JC,P=6.6 Hz, POCH2), 82.36 (d, JC,P=7.2 Hz, POCH2), 117.10 (dd,
2
2
2JC,F=18.5 Hz, JC,F=5.6 Hz), 117.50 (d, JC,F=16.7 Hz), 124.35 (m), 129.86 (m), 150.39 (dd,
3
2
1JC,F=245.8 Hz, JC,F=11.4 Hz), 150.70 (dd, JC,F=249.4 Hz, JC,F=12.1 Hz), 169.06 (C=O,
hydroxamate), 176.81 (C=O, ester), 176.90 (C=O, ester) ppm; IR (KBr): ṽ=3196 (OꢀH), 2978
(CꢀHaliph.), 1756 (C=O, ester), 1666 (C=O), 1283 (P=O), 1024, 1007 (PꢀO) cmꢀ1; Anal. calcd. for
C22H32F2NO10P: C 48.98, H 5.98, N 2.60, found: C 49.19, H 6.17, N 2.51.
2
1
2
General procedure for the synthesis of target compounds 4aꢀh. To a solution of the respective
phosphonic acid diethyl ester 11aꢀh (1 eq, 3 mmol) in dry dichloromethane (10 mL), trimethylsilyl
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bromide (5 eq, 15 mmol, 1.99 mL) was added at 0 °C. After 1 h, the solution was allowed to warm up
to room temperature and stirred for 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 removed under reduced pressure and the resulting residue was dissolved in ethyl acetate
(20 mL). The organic layer was washed twice with 1M hydrochloric acid (2 mL), dried over MgSO4 and
concentrated. Phosphonic acids 4aꢀh were treated with ethyl acetate and stored at 7 °C to yield pure
white solids.
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((3,4ꢀDifluorophenyl)(2ꢀ(hydroxy(methyl)amino)ꢀ2ꢀoxoethoxy)methyl)phosphonic acid (4e).
1
White solid (0.86 g, 55%); mp: 160 °C; H NMR (500.13 MHz, [D6]DMSO): δ=3.07 (s, 3H, NCH3),
4.26 (d, J=16.1 Hz, 1H, OCH2C=O), 4.36 (d, J=16.1 Hz, 1H, OCH2C=O), 4.72 (d, J=14.1 Hz, 1H,
PCH), 7.19ꢀ7.25 (m, 1H), 7.34ꢀ7.46 (m, 2H), 9.88 (br.s, 1H, OH) ppm; 13C NMR (125.76 MHz,
3
1
[D6]DMSO): δ=35.65 (NCH3), 67.14 (d, JC,P=8.5 Hz, OCH2C=O), 77.51 (d, JC,P=158.7 Hz, PCH),
1
2
5
116.63 (m), 124.68 (m), 134.42 (m), 148.73 (ddd, JC,F=244.2 Hz, JC,F=12.6 Hz, JC,P=2.6 Hz), 148.89
1
2
4
(ddd, JC,F=244.9 Hz, JC,F=12.6 Hz, JC,P=2.0 Hz), 169.27 (C=O) ppm; IR (KBr): ṽ=3138 (OꢀH), 2877
(CꢀHaliph.), 1621 (C=O), 1206 (P=O), 1031 (PꢀO) cmꢀ1; Anal. calcd. for C10H12F2NO6P: C 38.60, H 3.89,
N 4.50, found: C 38.85, H 4.08, N 4.58.
((((2ꢀ((benzyloxy)(methyl)amino)ꢀ2ꢀoxoethoxy)(3,4ꢀdifluorophenyl)methyl)phosphoryl)bis(oxy))ꢀ
bis(methylene) bis(2,2ꢀdimethylpropanoate) (13). To a solution of protected hydroxamic acid 9e
(1 eq., 4 mmol, 1.83 g) in dry dichloromethane (20 mL), trimethylsilyl bromide (5 eq, 20 mmol,
2.64 mL) was added at 0 °C. After 1 h, the solution was allowed to warm up to room temperature and
stirred for further 23 h. The solvent was removed under reduced pressure; the residue was dissolved in
THF (20 mL) and treated with water (0.2 mL). After 30 min, the solvent was evaporated and the residue
was dried in vacuo overnight. The residue was dissolved in anhydrous DMF (15 mL), treated with
triethylamine (3 eq, 12 mmol, 1.66 mL) and after stirring for 10 min at room temperature chloromethyl
pivalate (10 eq., 40 mmol, 5.80 mL) was added. The solution was heated to 70 °C for 2 h. The mixture
was treated again with triethylamine (1 eq, 4 mmol, 0.55 mL) and chloromethyl pivalate (1.5 eq,
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6 mmol, 0.87 mL) and stirred for further 2 h at 70 °C. The procedure of adding triethylamine and
chloromethyl pivalate was repeated once again. After two more hours at 70 °C, the reaction mixture was
allowed to cool down to room temperature and stirred overnight. The solution was diluted with diethyl
ether (120 mL) and washed with water (60 mL), saturated aqueous solution of NaHCO3 (2 x 60 mL) and
once again with water (60 mL). The organic layer was dried over MgSO4, filtered and the solvent was
evaporated in vacuo. Purification of crude 13 was accomplished by column chromatography on silica
gel with diethyl ether as the eluent to yield a yellow oil (0.93 g, 37%). 1H NMR (500.13 MHz, CDCl3):
δ=1.20, 1.23 (2s, 18H, C(CH3)3), 3.18 (s, 3H, NCH3), 3.99 (d, J=16.2 Hz, 1H, OCH2C=O), 4.33 (d,
J=16.1 Hz, 1H, OCH2C=O), 4.74 (dd, J1=16.3 Hz, J2=10.9 Hz, 2H, CH2Ph), 4.95 (d, J=14.2 Hz, 1H,
PCH), 5.60ꢀ5.73 (m, 4H, POCH2), 7.11ꢀ7.18 (m, 2H), 7.22ꢀ7.28 (m, 3H), 7.30ꢀ7.35 (m, 3H) ppm;
13C NMR (125.76 MHz, CDCl3): δ=26.79, 27.05 (2s, C(CH3)3), 33.41 (NCH3), 38.67 (C(CH3)3), 66.77
1
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1
(d, JC,P=11.8 Hz, OCH2C=O), 76.23 (OCH2Ph), 76.61 (d, JC,P=158.7 Hz, PCH), 82.13 (d,
2
2JC,P=6.3 Hz, POCH2), 82.41 (d, JC,P=6.5 Hz, POCH2), 117.37 (m, 2C), 124.56 (m), 128.75 (2C),
129.36, 129.44 (2C), 130.17 (m), 133.88, 150.31 (d, 1JC,F=251.2 Hz), 150.65 (d, 1JC,F=249.7 Hz), 170.54
(C=O, hydroxamate), 176.71 (C=O, ester), 176.75 (C=O, ester) ppm; IR (KBr): ṽ=2977 (CꢀHaliph.), 1754
(C=O, ester), 1681 (C=O, hydroxamate), 1281 (P=O), 1025 (PꢀO) cmꢀ1; Anal. calcd. for
C29H38F2NO10P: C 55.32, H 6.08, N 2.22, found: C 55.49, H 6.06, N 2.17.
ABBREVIATIONS
CDI, 1,1‘ꢀcarbonyldiimidazole, DCC, dicyclohexylcarbodiimide; DEP, diethylphosphite; DMF, N,Nꢀ
dimethylformamide; DOXP, 1ꢀdeoxyꢀ
D
ꢀxylulose 5ꢀphosphate; Dxr, 1ꢀdeoxyꢀ ꢀxylulose 5ꢀphosphate
D
reductoisomerase; NADPH, nicotinamide adenine dinucleotide phosphate; PdꢀC, palladium on activated
carbon; PfIspC, P. falciparum IspC; RT, room temperature; SD, standard deviation; TEA, triethylamine;
THF, tetrahydrofuran; TMSBr, trimethylsilyl bromide.
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Journal of Medicinal Chemistry
ASSOCIATED CONTENT
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Supporting Information. Experimental procedures, analytical data, enzyme assays, and biological
evaluation of in vitro antiplasmodial activity. This material is available free of charge via the Internet at
http://pubs.acs.org.
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