Prodrugs of reverse fosmidomycin analogues

Article abstract of DOI:10.1021/jm5019719

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical

Full text of DOI:10.1021/jm5019719

Article
pubs.acs.org/jmc
Prodrugs of Reverse Fosmidomycin Analogues
Karin Brucher,† Tobias Grawert,‡ Sarah Konzuch,† Jana Held,§ Claudia Lienau,† Christoph Behrendt,†
̈
̈
Boris Illarionov,‡ Louis Maes,∥ Adelbert Bacher,‡,⊥ Sergio Wittlin,# Benjamin Mordmuller,§,○
̈
Markus Fischer,‡ and Thomas Kurz*,†
†Institut fur Pharmazeutische und Medizinische Chemie, Heinrich Heine Universitat, Universitatsstr. 1, 40225 Dusseldorf, Germany
̈
̈
̈
̈
‡Hamburg School of Food Science, Universitat Hamburg, Grindelallee 117, 20146 Hamburg, Germany
̈
§Institut fur Tropenmedizin, Eberhard Karls Universitat Tubingen, Wilhelmstr. 27, 72074 Tubingen, Germany
̈
̈
̈
̈
∥Laboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Groenenborgerlaan 171, 2020 Wilrijk,
Belgium
⊥Department Chemie, Technische Universitat Munchen, Lichtenbergstr. 4, 85747 Garching, Germany
̈
̈
#Swiss Tropical and Public Health Institute, Socinstr. 57, 4051 Basel, Switzerland
○Centre de Recherches Med
́ ́ ́
icales de Lambarene (CERMEL), 13901 Libreville, Gabon
S
* Supporting Information
ABSTRACT: Fosmidomycin inhibits IspC (Dxr, 1-deoxy-D-xylulose 5-
phosphate reductoisomerase), a key enzyme in nonmevalonate
isoprenoid biosynthesis that is essential in Plasmodium falciparum. The
drug has been used successfully to treat malaria patients in clinical
studies, thus validating IspC as an antimalarial target. However,
improvement of the drug’s pharmacodynamics and pharmacokinetics
is desirable. Here, we show that the conversion of the phosphonate
moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can
increase the in vitro activity against asexual blood stages of P. falciparum
by more than 1 order of magnitude. We also synthesized double
prodrugs by additional esterification of the hydroxamate moiety.
Prodrugs with modified hydroxamate moieties are subject to
bioactivation in vitro. All prodrugs demonstrated improved antiplasmo-
dial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo
in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.
reasonably high level of protection for extended periods cannot
be predicted.3
INTRODUCTION
■
Despite substantial international efforts, malaria remains a
major source of global morbidity and mortality. Published
estimates for the number of fatal cases in the year 2013 range
from 367 000 to 755 000, and the number of nonfatal malaria
episodes are orders of magnitude higher.1 Optimistic reports
about a progressive decrease in malaria mortality may need to
be reviewed in light of the apparent uncertainty of the numbers
quoted above. Even more importantly, progressing attrition of
the antimalarial drug arsenal by the emergence and spread of
drug-resistant Plasmodium parasites and of insecticide-resistant
vectors is an acute danger. Development of resistance against
antimalarials and insecticides arises as a natural consequence of
selection pressure on parasites and mosquitoes.2 Resistance
affects all antimalarial drug classes; recently, resistance to drugs
of the artemisinin family, our main line of defense, has been
confirmed in several Southeast Asian countries.
There is universal consensus about the urgent need for a
continuous influx of novel antimalarials, and, indeed, the
malaria drug pipeline of new preclinical and clinical drug
candidates is somewhat stronger than it was 5 years ago.4
However, there is still much scope for improvement. In fact, no
major novel antimalarial has been implemented since the
introduction of atovaquone in 1992.5 The total number of
druggable targets among the approximately 5300 gene products
of Plasmodium falciparum,6 the clinically most important
Plasmodium species, is not known; in fact, it may be smaller
than initially expected. In any case, it would appear sensible to
work with any known clinically validated targets that are likely
to be exempt from cross-resistance with currently used
antimalarial drugs. The proteins of the nonmevalonate
isoprenoid biosynthesis pathway, and, most notably, IspC
protein catalyzing the first committed step of that pathway,
In light of the drug resistance problems, a protective vaccine
would be a preferable approach. However, the time frame for
the development and deployment of a vaccine with a
Received: December 21, 2014
© XXXX American Chemical Society
A
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Scheme 1. IspC Inhibitors and Prodrugs with Antibacterial and Antiplasmodial Activity
Scheme 2. Prodrugs of Reverse Fosmidomycin Derivatives 3−5
fulfill these requirements.7 Specifically, fosmidomycin, an IspC
inhibitor originally found as an antibacterial product of
Streptomyces lavendulae, has been validated as a well-tolerated,
safe, and efficacious combination partner in antimalarial
treatment regimes in several clinical trials conducted in Africa
and South Asia.8 Moreover, since humans generate isoprenoids
via the mevalonate pathway, antimalarials directed at IspC
should be exempt from target-related toxicity.
Fosmidomycin inhibits IspC from P. falciparum with an IC50
in the range of 21−160 nM9,10 and is active against the blood
stage of the parasite with an IC50 in the single-digit micromolar
range (literature reports varying from 0.4 to 3.7 μM).9a,11,12
Unfortunately, its pharmacokinetic properties are less than
ideal, with only moderate oral bioavailability and a short plasma
half-life.9
Fosmidomycin acts as a slow, tight-binding substrate-
analogue inhibitor whose hydroxamate motif complexes a
catalytically essential divalent metal ion at the active site of
IspC.10 Enzyme inhibition has been significantly enhanced by
the introduction of aromatic substituents at fosmidomycin’s α-
position.11 Other reported structural modifications included the
modification of the N-acyl group,12 the modification and
inversion of the hydroxamate motif,13 and the modification of
the aliphatic linker that connects the phosphonate motif
(mimicking the phosphate moiety of the IspC substrate DXP,
1-deoxy-D-xylulose 5-phosphate) to the hydroxamate motif that
interacts with the essential divalent cation of the enzy-
me.11c,13b,14 Fosmidomycin prodrugs have been obtained by
conversion of the phosphonate moiety into phosphonate esters.
(Scheme 1;15 for review, see Jackson and Dowd 201216).
This article reports on a systematic comparison of
phosphonate and phosphonate-hydroxamate double prodrugs
of reverse fosmidomycin derivatives using two chloroquine-
sensitive and one multidrug-resistant P. falciparum strains of
different geographic origin (3D7, D10, and Dd2). Representa-
tive prodrugs and parent compounds were also screened for
their inhibition of IspC and their cytotoxicity toward HeLa cells
as well as in vivo in a Plasmodium berghei malaria model and in
the SCID mouse P. falciparum model.
RESULTS
■
The reverse fosmidomycin derivative 3, its N-methyl derivate 4,
and the N-methyl-substituted oxa-analogue 5 have been shown
earlier to inhibit P. falciparum IspC (Pf IspC) with IC50 values
in the low nanomolar range (cf. Table 1 and Scheme 2).17
However, in vitro growth inhibition of asexual blood stages of P.
falciparum by these compounds was weaker. With the aim to
improve penetration of the numerous biomembranes that the
inhibitors must pass before reaching their target protein inside
the apicoplast (Figure 2B), we decided to combine the
B
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a
Scheme 3. Synthesis of Target Compounds
a
Indices a−c and A−E indicate phosphonate- and hydroxamate-prodrug functionalities of target compounds, respectively. Reagents and conditions:
(a) (1) TMSBr, CH2Cl2, rt 24 h; (2) THF/H2O, RT, 1 h; (3) n-butyl chloromethyl carbonate (3aF−5aF), isopropyl chloromethyl carbonate (3bF−
5bF), or chloromethyl pivalate (4cF, 5cF), TEA, DMF, 80 °C, 6 h; (b) H2, Pd−C, MeOH, 1 atm, rt, 2 h; (c) acetyl chloride (4bA, 4cA), ethyl
chloroformate (4bB, 4cB, 5cB), ethyl isocyanate (4bC, 4cC, 5cC), or pivaloylchloride (4bD, 4cD), TEA, CH2Cl2, rt, 1 h or CDI, CH2Cl2, 4 °C, 1 h;
morpholine, CDI, DCM, rt, 12 h (4cE).
modification of the phosphonate group and the hydroxamate
motif (Scheme 2).
NMR spectra. Their purity was determined by HPLC and
elemental analysis, respectively.
Biological Studies. The study compounds were assayed for
inhibition of Pf IspC as described earlier.17c None of the
prodrugs (e.g., 3a,b, 4a,b) caused detectable enzyme inhibition.
This is in line with expectations based on X-ray structure data,
since all prodrugs appear too bulky to be able to be
accommodated at the IspC active site. Moreover, the double
prodrugs (e.g., 4bB−D) are unable to chelate the essential
divalent metal ion of IspC. The calculated logP values of the
prodrugs are within the limits of Lipinski’s rule of five. Hence,
we expected the prodrugs to be cell-permeable (Table 1).
The study compounds were also assayed for growth
inhibition of asexual blood stage parasites of three P. falciparum
strains kept in continuous culture (Table 1). Specifically, we
used the chloroquine-sensitive 3D7 and D10 strains as well as
the multiresistant Dd2 strain. Parasite proliferation was
monitored by an enzyme-linked immunoassay (ELISA) for
histidine-rich protein 2 (HRP2).
Several prodrugs under study, phosphonate prodrugs (3a, 4a,
5a) and double prodrugs (4bA, 4cC), inhibit the multiplication
of P. falciparum blood stages with IC50 values in the single-digit
nanomolar range (IC50 values vs PfDd2: 4−9 nM, Table 1).
Among them, the most active prodrugs are phosphonates 4a
and 4b and double prodrug 4cC (Table 1). Their inhibitory
efficacy exceeds that of the parent free phosphonic acid 4 by
about 1 order of magnitude. Notably, the antiparasite IC50
values of prodrugs 3a, 4a, 4b, and 4cC are similar to the IC50
values for IspC inhibition by their respective mother
compounds, 3 and 4. To the best of our knowledge, 4a, 4b,
For the modification of the phosphonate motif, we used
acyloxymethyl- and alkoxycarbonyloxymethyl ester groups.
Numerical estimates predicted that the designed modifications
should increase the lipophilicity (clogP) by approximately 4 to
6 orders of magnitude to values that would be in line with
Lipinski’s rule of five (Table 1).
As starting materials, we selected phosphonic esters 3dF−
5dF (Scheme 3). Their dealkylation with bromotrimethylsilane
provided the corresponding crude phosphonic acids (structures
not shown). Subsequent conversion into acyloxymethyl and
alkoxycarbonyloxymethyl esters 3aF−3bF, 4aF−4cF, and
5aF−5cF was accomplished without prior purification by
treatment with chloromethyl pivalate and alkyl chloromethyl
carbonates in the presence of triethylamine. After column
chromatography, all intermediates except 4bF were obtained in
good purity. Catalytic hydrogenation of O-benzyl-protected
precursors afforded the corresponding hydroxamic acids 3a,b,
4a−c, and 5a−c. With the exception of compound 4c, all
alkoxycarbonyloxyalkyl ester prodrugs were obtained in good
purity by column chromatography. Reactions of compounds 4b
and 4c with acetyl chloride, ethyl chloroformiate, ethyl
isocyanate, and pivaloyl chloride yielded bis-prodrugs 4bA−
4bD and 4cA−4cD; treatment of crude 4c with CDI (1,1′-
carbonyldiimidazole) and morpholine afforded prodrug 4cE.
The conversion of β-oxa-isosteric prodrug 5c into bis-prodrugs
5cB and 5cC has been described elsewhere.17c The structures
1
of all novel compounds were confirmed by IR and H and 13C
C
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Table 1. Inhibition of PfIspC, in Vitro Antiplasmodial Activity, and Calculated logP
a
b
Values (mean SD calculated from nine or more data points) were determined using nonlinear regression analysis as described earlier.18 Values
c
d
are the mean of at least two independent experiments conducted in duplicate, each using 12 serial dilutions. n.d., not determined. logP values were
calculated using ACD/ChemSketch freeware, version 12.01. Data from Behrendt et al.17b Data from Behrendt et al.17a Data from Brucher et al.17c
e
f
g
̈
and 4cC are the most potent fosmidomycin derivatives with
regard to inhibition of P. falciparum blood stage proliferation. A
more detailed comparison of the biological activities of the
study compounds is described below.
prodrugs. Notably, parent compound 5 showed the highest
respective enhancement by conversion into prodrugs. More-
over, the impact of the respective prodrug functionality is larger
with the 3D7 strain than with the Dd2 strain. (v) The
modification of the hydroxamic acid motif introduces an
additional level of complexity; simple esters, carbonates, and
carbamates have been synthesized and tested. Specifically,
results obtained with acetyl, pivaloyl, ethoxycarbonyl, and
ethylaminocarbonyl residues are summarized in Table 1 and
Figures 1 and 2A (in Figure 1, the double prodrugs are marked
by colored rings around the data points). In parallel to Figure 1,
the abscissa of Figure 2A represents the IC50 value of double-
derivatized prodrugs when assayed with one of the three P.
falciparum strains under study. The ordinate shows the
difference of the log10 (IC50) values of the phosphonate-
modified (single-modified) and the double-modified com-
pounds.
With few exceptions (e.g., 4cC, the most active compound
under study), the additional modification of the hydroxamic
acid group led to reduced antiplasmodial activity. Only the
morpholine derivative had no detectable antiparasite activity.
We also determined the cytotoxicity and selectivity of parent
compounds (4, 5) and prodrugs (4a, 5a, 4bA−4bC, 4cA−4cD,
5cC) for the parasite versus mammalian cells (Table 2). Parent
compounds 4 and 5 and all prodrugs tested displayed no
cytotoxicity toward mammalian cells and are characterized by
excellent selectivity indices (SIs, Table 2).
Figure 1 compares the impact of different phosphonate and
hydroxamate prodrug functionalities applied to three α-
substituted mother compounds with regard to their activity
against three different P. falciparum strains. As phosphonate
prodrug functionalities, we used n-butyloxycarbonyloxymethyl
group a, isopropyloxycarbonyloxymethyl group b, and
pivaloyloxymethyl group c, which have been used earlier for
antiviral prodrug strategies and, to some extent, in earlier work
on IspC inhibitors. The ordinate of each graph represents the
specific activity gain resulting from the modification of the
respective parent compound (defined as the difference of the
log10 of the IC50 values of parent compound and prodrug
derivative). For each data point, the abscissa value represents
the IC50 value of the respective prodrug when applied to the
respective strain.
The three-way comparison affords the following conclusions:
(i) Whereas the overall sensitivity of strains 3D7 and Dd2
against the study compounds are similar, the D10 strain appears
to be somewhat less sensitive. (ii) With few exceptions, n-
butyloxycarbonyloxymethyl group a showed the largest activity
enhancement of the prodrugs studied. (iii) The impact of the
prodrug functionality differs substantially among the tested P.
falciparum strains. Enhancements up to 50-fold are observed
with the 3D7 laboratory strain. On the other hand, the
maximum observed enhancement with the D10 strain was by
an approximate factor of 17. (iv) Parent compounds differ with
regard to the activity enhancement caused by conversion into
DISCUSSION
■
Phosphonates play an important role in pharmacology as stable
mimics of naturally occurring phosphates, most notably as
D
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Figure 1. Inhibitory effect of prodrug minilibraries versus three P. falciparum strains. Minilibraries derived from a given parent compound are
connected by dashed lines. Double prodrugs feature colored rings.
was followed by work with more complex groups such as the
isopropyloxycarbonyloxymethyl group.
Table 2. Cytotoxicity and Selectivity Indices
a
HeLa IC50
[μM]
selectivity
b
The most potent prodrug candidates reported in this article
exceed the efficacy of authentic fosmidomycin by almost 2
orders of magnitude, as judged by their in vitro IC50 values
against the asexual blood stage of P. falciparum. Moreover,
efficacy enhancement of reverse analogues 3−5 by prodrug
derivatization is about 1 order of magnitude higher than in the
case of fosmidomycin and FR900098. The additional
derivatization of the hydroxamic acid motif yielded IC50 values
between 4 and 70 nM vs strain Dd2. In some cases, the
antiplasmodial activity of the double prodrugs is reduced;
however, the most active prodrug under study is double-
prodrug 4cC (IC50 value vs PfDd2: 4 nM). The introduction of
a morpholine-containing carbamate resulted in complete loss of
antiplasmodial activity (4cE).
The prodrugs under study are too bulky to access the active
site of IspC, and the compounds with modifications of the
hydroxamic acid motif would be unable to complex the divalent
metal of the enzyme. Not surprisingly, single and double
prodrugs fail to inhibit isolated IspC protein. However, the
inhibitory activity in the parasite assay shows that several
hydroxamate prodrug functionalities can be hydrolyzed in
viable P. falciparum asexual blood stage cultures. Possibly, their
hydrolytic cleavage may be a limiting factor that is responsible
for the observed reduction of antiplasmodial activity caused by
several modifiers of the hydroxamate motif.
entry
R
R1
R2
index
4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
>1000
>1000
190
190
188
205
168
175
157
219
69
>2500
>7692
38 000
21 111
23 500
11 389
14 000
3977
5
4a
n-BuO
n-BuO
i-PrO
i-PrO
i-PrO
t-Bu
5a
4bA
4bB
4bC
4cA
4cB
4cC
4cD
5cC
COCH3
CO2C2H5
CONHC2H5
COCH3
t-Bu
CO2C2H5
8722
t-Bu
CONHC2H5
COC(CH3)3
CONHC2H5
54 750
986
t-Bu
t-Bu
158
7182
a
Cytotoxicity test with HeLa cells. Values are the mean of two
b
duplicate determinations. Selectivity indices were calculated as the
HeLa cell IC50/Pf Dd2 IC50; larger values indicate greater parasite
activity.
antiviral drugs. However, the highly polar phosphonate motif
frequently causes difficulties in uptake. Extensive literature has
accumulated about the masking of the phosphonate group by
prodrug groups that are designed to be enzymatically removed
at the desired site of drug action (for review, see refs 19a and
19b).
Modification of the phosphonate motif of fosmidomycin and
the analogous natural product FR900098 has been addressed
repeatedly over the past decade15,20 (for review, see also
Jackson and Dowd 201216). Initial work on substituted
phenylphosphonate esters of fosmidomycin and FR900098
How Are the Phosphonate Prodrugs Bioactivated in
the Cell Culture Assay? The first-generation prodrugs of
FR900098 were structurally simple aryl phosphonates.21
Obviously, their hydrolysis would have to involve hydrolytic
E
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Scheme 4. Hypothetical Sequence of Reactions for Bioactivation of Phosphonate Prodrugs
Figure 2. (A) Impact of hydroxamate modification on inhibitory activity. Double prodrugs derived from a given phosphonate prodrug (4b, 5b) are
connected by dashed lines. (B) Passage of an IspC inhibitor across membrane barriers in Plasmodium-infected red blood cells.
F
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cleavage of oxygen phosphorus bonds by phosphatases or
similar hydrolases. Interestingly, ordinary alkyl phosphonates
are not active against P. falciparum, most likely due to a lack of
bioactivation. However, alkyl phosphonates have been reported
to be active against Mycobacterium tuberculosis in vitro.20
The more complex acyloxyalkyl and alkoxycarbonyloxyalkyl
esters offer additional opportunities for cleavage by esterase
type enzymes (Scheme 4).19 The concept of phosphonate drug
modification, especially the alkoxycarbonyloxymethyl and
pivaloyloxyalkyl derivatives, have resulted in the success of
antiviral drugs Tenofovir disoproxil fumarate22 and Adefovir
dipivoxil,23 whose pharmacological properties have been
investigated in considerable detail.
In the present study, the best results were obtained with the
n-butyloxycarbonyloxymethyl group. This may indicate a
preferred cleavage of the alkoxycarbonyloxymethyl motif,
where the n-butyl residue would be preferable to the isopropyl
residue for steric reasons. The enzymatic step could be followed
by spontaneous release of carbon dioxide, followed by
formaldehyde.
apicoplast. Partial or terminal prodrug hydrolysis could be
proceeding in any or all of the following compartments: (i) the
erythrocyte cytoplasm, (ii) the parasitophorous vacuole, (iii)
the parasite cytoplasm, and/or (iv) inside the apicoplast
(Figure 2B).
Since the activity ratio of prodrugs and their mother
compounds is different for the three P. falciparum strains
used, it appears unlikely that the compounds undergo complete
hydrolysis in the erythrocyte cytoplasm. Hence, at least a
significant fraction of the prodrugs must be able to access the
parasite without prior hydrolysis and must then be hydrolyzed
at some point inside the parasite to yield the actual inhibitor.
Notably, however, it remains unknown whether hydrolysis
proceeds in the parasite cytoplasm and/or inside the apicoplast.
In Vivo Antimalarial Studies with Prodrugs 4bC and
4cC and Its Parent Compound 4 in Two Mouse Models
of Malaria. On the basis of their promising antiplasmodial in
vitro properties and excellent selectivity indices, compounds 4
and 4cC were selected and tested for their in vivo activity in P.
berghei-infected NMRI mice (Table 3).27 A standard Peters test
How Are the Hydroxamic Acid Prodrugs Bioacti-
vated? It appears obvious that the hyxdroxamate prodrugs
would have to be activated by esterase-like enzymes of the host
or parasite cells, although details are not known. Because of the
presence of different phosphonate ester groups and the
relatively small number of double prodrugs tested, it is difficult
to derive comprehensive structure− activity relationships.
However, the data show that O-acetyl hydroxamates (4bA,
4cA) are more active than the quite bulky O-pivaloyl
hydroxamates (4dD, 4cD) and that N-ethyl substituted
carbamates (4bC, 4cC, 5cC) are stronger P. falciparum growth
inhibitors than ethyl carbonates (4bB, 4cB, 5cB). One
exception is the bulky morpholine based carbamate 4cE,
which is inactive in vitro toward all P. falciparum strains used in
this study. While the structure−metabolism relationships of
various carbamate prodrugs has recently been reviewed by
Testa and co-workers,24 no comparable studies have yet been
published on carbamate prodrugs of hydroxamic acids.
However, it appears that steric restrictions are responsible for
the lack of in vitro activity of 4cE. It remains unknown whether
the hydrolysis of the dual-modified prodrugs begins at the
hydroxamate or phosphonate motif. Compounds with exclusive
modification of the hydroxamate motif would be useful for
further studies but have not yet been obtained.
Table 3. In Vivo Studies Using Plasmodium berghei-Infected
Mice
a
,
28
b
parasitized erythrocytes [%]
activity [%] survival [days]
c
control
35
15
13
15
11
0
4.0
6.0
6.7
6.3
6.3
4 (p.o.)
4 (i.p.)
57
62
58
68
4cC (p.o.)
4cC (i.p.)
a
A standard Peters test using daily oral or intraperitoneal doses of 50
mg/kg body weight was conducted for 4 days. Briefly, compounds
were dissolved or suspended in 70:30 Tween 80/ethanol and diluted
10× with water. Experimental groups (n = 3 mice) were administered
4× by the oral (p. o.) or intraperitoneal (i.p.) route (once per day; 4,
b
24, 48, and 72 h postinfection). Blood was collected on day 4 (96 h
after infection). Parasitemia reduction (activity) and mean survival
time in days (MSD) for multidose regimens are reported. Activity was
calculated as the difference between the mean percent parasitaemia for
the control and treated groups expressed as a percent relative to the
c
control group. Mice were euthanized on day 4 postinfection in order
to prevent death otherwise occurring on day 6. For comparison,
clinically used antimalarial drugs such as artesunate and mefloquine,
administered in this P. berghei model at four daily oral doses of 30 mg/
kg, showed a reduction in parasitemia of 99.0 and 99.9% with a mean
survival time of 10 and 29 days.29
Where Are the Phosphonate Prodrugs Bioactivated?
By comparison with antiviral drugs and prodrugs, the
antimalarial activity involves a much more complex situation
at the level of cellular topology. Specifically, oral antiviral drugs
must be capable of gastrointestinal absorption and of
permeation into the nuclear and/or cytoplasmic compartments
of target cells, the site of viral replication and assembly. By
contrast, in order to attack IspC, which is located in the
apicoplast compartment of the erythrocyte stage of Plasmodium,
an oral drug must be capable of gastrointestinal absorption,
passage through the erythrocyte membrane (whose properties
are known to be modified by the parasite),25 and passage
through up to six additional membranes.26 More specifically, in
the in vitro parasite assay, inhibitors (i.e., mother compounds,
prodrugs, and/or the products resulting from partial or
complete hydrolysis or unmodified fosmidomycin type
compounds) must pass through the erythrocyte membrane,
the parasitophorous vacuole membrane (PVM), the Plasmo-
dium cell membrane, and the four membrane layers of the
using daily oral or intraperitoneal doses of 50 mg/kg body
weight was conducted for 4 days. To preliminarily assess their
efficacy, pharmacokinetic properties, and compound stability in
vivo, both compounds were administered via the oral and
intraperitoneal routes. However, regardless of the method of
application, neither mother compound 4 nor prodrug 4cC
displayed pronounced efficacy in this in vivo model, evidenced
by activities of 56.9−68.4% and MSD of 4.0−6.7 days, which is
comparable to the MSD of control mice (∼6.0 days) (Table 3).
While parent compound 4 and prodrug 4cC showed high
potency in the cell culture assays against sensitive and
multidrug-resistant strains of P. falciparum, the outcome of
our initial in vivo studies was less promising (Table 3). Both
compounds displayed <70% in vivo efficacy with a mean
survival time of 6.3 to 6.7 days, which is comparable with the
mean survival time of untreated, infected control mice (6 days,
Table 3). There are several explanations for the weak in vivo
G
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activity of 4 and 4cC in the P. berghei mouse model such as
chemical instability, metabolic instability, insufficient bioactiva-
tion of 4cC, and structural differences between the targets
PfIspC and PbIspC. It should be noted, however, that species
differences cannot be addressed in vitro since studies on IspC
protein of P. berghei are not available.
To exclude differences between both Plasmodium species
(and both target enzymes), the in vivo antimalarial activity of
parent compound 4 and prodrug 4bC was also studied in the
SCID mouse P. falciparum model. This recently established
model uses SCID mice engrafted with human erythrocytes,
offering the possibility to investigate the actual target parasite P.
falciparum in vivo. Parasitemia reduction (activity) is reported in
Table 4 and is in accordance with the P. berghei model; neither
mother compound 4 nor prodrug 4bC displayed significant in
vivo activity in the SCID mouse P. falciparum model.30,31
development of double prodrugs failed to provide a significant
activity gain in all cases. Only a few double prodrugs were
highly active with IC50 values in the single-digit nanomolar
range; among them, the most active prodrug under study was
4cC (IC50 value vs Pf Dd2: 4 nM). Additional experiments
demonstrated that parent compounds 4 and 5 and the most
active prodrugs showed no cytotoxicity against mammalian cells
and are characterized by excellent selectivity indices. To
preliminarily assess their activity in vivo, prodrug 4cC and its
parent compound 4 were administered via the oral and
intraperitoneal routes in the P. berghei malaria model. However,
neither mother compound 4 nor prodrug 4cC demonstrated
pronounced in vivo activity in the P. berghei-infected mouse
model. To exclude species differences and thereby structural
differences of the drug targets Pf IspC and PbIspC, the in vivo
activity of parent compound 4 and a structurally related
prodrug 4bC was also tested in the SCID mouse P. falciparum
model. Again, neither mother compound 4 nor prodrug 4bC
displayed in vivo efficacy.
Table 4. In Vivo Studies Using the P. falciparum Mouse
a
Model
b
parasitized erythrocytes [%]
activity [%]
EXPERIMENTAL SECTION
■
control (n = 5 mice)
4bC (p.o.) (n = 2 mice)
4 (i.p.) (n = 2 mice)
12
14
14
0
0
0
Chemistry. 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/n-hexane as solvent
system. Column chromatography was performed with Fluka silica gel
60 (230−400 mesh ASTM) with the solvent mixtures specified in the
corresponding experiment. Spots were visualized by irradiation with
ultraviolet light (254 nm). IR spectra were recorded on a Varian 800
FT-IR Scimitar series. Proton (1H) and carbon (13C) NMR spectra
a
A standard Peters test using daily oral or intraperitoneal doses of 50
mg/kg body weight was conducted for 4 days. Compounds were
dissolved or suspended in 70:30 Tween 80/ethanol and diluted 10×
b
with water. Blood was collected on day 4 after the first treatment. All
mice were euthanized after blood collection in order to prevent death
otherwise occurring 3−5 days later. Experimental and control groups
(n = 2 and 5 mice, respectively) were treated 4× by the oral (p.o.) or
intraperitoneal (i.p.) route (once per day 3, 4, 5, and 6 days
postinfection). For comparison, clinically used antimalarial drugs such
as artesunate and chloroquine, administered in this P. falciparum
model, showed a reduction in parasitemia of 90% at four daily oral
doses of 13 and 5 mg/kg.30
1
were recorded on a Bruker Avance 500 (500.13 MHz for H; 125.76
MHz for 13C) and a Bruker Avance III-600 (600.22 MHz for H;
1
150.93 MHz for 13C) using DMSO-d6 and CDCl3 as solvents.
Chemical shifts are given in parts per million (ppm) (δ relative to
residual solvent peak for 1H and 13C and to external tetramethylsilane).
Elemental analysis was performed on a PerkinElmer 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.1 Instrument:
Varian ProStar HPLC System [Varian ProStar 210 (pump), Varian
ProStar 320 (UV-detector), and Varian ProStar 410 (autosampler)];
column: Phenomenex Luna C-18(2) 5 μm particle size (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.
Since both prodrugs 4cC and 4bC are not cytotoxic to
mammalian cells and highly active against different strains of P.
falciparum in vitro, we assume that the lack of significant in vivo
antiplasmodial activity in both mouse models of malaria
(regardless the way of administration) may be attributed to
the metabolic and/or chemical instability of the parent
compound 4, which was inactive in both animal models. One
possible explanation is the hydrolysis of the essential
hydroxamic acid pharmacophore of 4 into the corresponding
carboxylic acid that is known to be inactive toward Pf IspC and
PfK1 strain.17b
Experimental Data for Compounds. Experimental data are
listed below for selected compounds: 5bF, 4b, and 4bB.
General Procedure for the Synthesis of O-Bn-Protected
Prodrugs 3aF−3bF, 4aF−4cF, and 5aF−5bF. To a solution of
protected phosphonohydroxamic acids 3dF−5dF (1 eq., 3 mmol) in
dry dichloromethane (20 mL) was added trimethylsilyl bromide (5
equiv, 15 mmol, 2.0 mL) at 0 °C. After 1 h, the solution was allowed to
warm to room temperature and stirred for a 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) and treated
with triethylamine (3 equiv, 9 mmol, 1.25 mL); after stirring for 10
min at room temperature, n-butyl chloromethyl carbonate (3aF−5aF,
10 equiv, 30 mmol, 5.0 g), isopropyl chloromethyl carbonate (3bF−
5bF, 10 equiv, 30 mmol, 4.6 g), or chloromethyl pivalate (4cF, 10
equiv, 30 mmol, 4.5 g) was added, and the solution was heated at 70
°C for 2 h. The mixture was treated again with triethylamine (1 equiv,
3 mmol, 0.4 mL) and n-butyl chloromethyl carbonate (3aF−5aF, 1.5
equiv, 4.5 mmol, 0.75 g), isopropyl chloromethyl carbonate (3bF−
5bF, 1.5 equiv, 4.5 mmol, 0.69 g), or chloromethyl pivalate (4cF, 1.5
equiv, 4.5 mmol, 0.68 g) and stirred for a further 2 h at 70 °C. The
CONCLUSIONS
■
The P. falciparum in vitro proliferation assay is a valid tool for
the in vitro characterization of preclinical antimalarial drug
candidates. In this study, we used chloroquine-sensitive and
multidrug-resistant P. falciparum strains of different geographic
origin (3D7, D10, and Dd2). We showed that the in vitro
efficacy of reverse fosmidomycin derivatives can be enhanced
by more than an order of magnitude by esterification of the
hydrophilic phosphonate motif. The most active phosphonate
prodrugs are n-butyloxycarbonyloxymethyl prodrugs with IC50
values in the single-digit nanomolar range. We also modified
the hydroxamate pharmacophore by esterification. With the
exception of the morpholine-based carbamate group, all
hydroxamate prodrugs were activated in the experimental
system under study. In light of the present, initial study, the
H
DOI: 10.1021/jm5019719
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
procedure of adding triethylamine and alkylating reagent was repeated
once again. After two more hours at 70 °C, the reaction mixture was
allowed to cool 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 × 60 mL),
and once again with water (60 mL). The organic layer was dried over
MgSO4 and filtered, and the solvent was evaporated in vacuo.
Purification of crude products 3aF−3bF, 4aF−4cF, and 5aF−5bF was
accomplished by column chromatography on silica gel with diethyl
ether as the eluent.
min, the reaction mixture was allowed to warm to room temperature
and stirred for 30 min. The solution was concentrated under reduced
pressure, and the residue was dissolved in 20 mL of ethyl acetate. The
organic layer was washed with water (2 × 5 mL), dried over MgSO4,
and filtered, and the solvent was removed under reduced pressure.
Compounds 4bA−C, 4cA−C, and 5cB−C were purified by column
chromatography using ethyl acetate as the eluent.
(((1-(3,4-Difluorophenyl)-4-(((ethoxycarbonyl)oxy)(methyl)-
amino)-4-oxobutyl)-phosphoryl)bis(oxy))bis(methylene)diisopropyl
Dicarbonate (4bB). Light yellow oil (0.07 g, 38%); 1H NMR (500.13
MHz, DMSO-d6): δ 1.19−1.31 (m, 15H, CH3), 1.92−2.05 (m, 1H,
CH2), 2.05−2.15 (m, 1H, CH2), 2.15−2.29 (m, 2H, CH2), 3.17 (s, 3H,
NCH3), 3.49 (dd, J = 24.6, 10.1 Hz, 1H, PCH), 4.24 (q, J = 7.1 Hz,
2H, OCH2CH3), 4.77 (td, J = 12.5, 6.3 Hz, 1H, OCH), 4.84 (td, J =
12.5, 6.3 Hz, 1H, OCH), 5.42−5.51 (m, 2H, OCH2O), 5.53−5.65 (m,
2H, OCH2O), 7.07−7.14 (m, 1H), 7.29 (dd, J1 = 10.6 Hz, J2 = 8.8 Hz,
1H,), 7.42 (dd, J1 = 19.1 Hz, J2 = 8.7 Hz, 1H) ppm; 13C NMR (125.76
MHz, DMSO-d6): δ 21.54 (CH3), 21.58 (CH3), 21.62 (CH3), 24.08
((((3,4-Difluorophenyl)(2-(hydroxy(methyl)amino)-2-oxoethoxy)-
methyl)phosphoryl)bis(oxy))bis(methylene)diisopropyl Dicarbonate
1
(5bF). Yellow oil (0.39 g, 20%); H NMR (500.13 MHz, CDCl3): δ
1.30 (d, J = 6.3 Hz, 6H, CH(CH3)2), 1.31 (d, J = 6.2 Hz, 6H,
CH(CH3)2), 3.18 (s, 3H, NCH3), 4.02 (d, J = 16.2 Hz, 1H, OCH2C
O), 4.35 (dd, J1 = 16.1 Hz, J2 = 0.9 Hz, 1H, OCH2CO), 4.74 (dd, J1
= 15.7 Hz, J2 = 10.9 Hz, 2H, OCH2Ph), 4.90 (dq, J1 = 12.1 Hz, J2 = 6.1
2
Hz, CH(CH3)2), 4.97 (d, JH,P = 14.0 Hz, 1H, PCH), 5.62 (dd, J1 =
2
(m, CH2CH2), 29.20 (d, JC,P =17.5 Hz, CHCH2), 35.30 (NCH3),
12.4 Hz, J2 = 5.3 Hz, 1H, POCH2), 5.68 (dd, J1 = 11.6 Hz, J2 = 5.5 Hz,
1H, POCH2), 5.68 (d, J = 11.7 Hz, 2H, POCH2), 7.10−7.18 (m, 2H,
C6H3F2), 7.22−7.36 (m, 6H, C6H5, C6H3F2) ppm; 13C NMR (125.76
1
35.95 (OCH2CH3), 41.67 (d, JC,P = 137.8 Hz, PCH), 73.09 (OCH),
73.23 (OCH), 84.38 (d, 2JC,P = 6.6 Hz, OCH2O), 84.47 (d, 2JC,P = 7.0
Hz, OCH2O), 117.95 (d, 2JC,F = 15.4 Hz), 118.41 (dd, 2JC,F = 17.6 Hz,
3JC,F = 6.7 Hz), 126.41 (m), 132.49 (m), 149.17 (dd, 1JC,F1 = 245.2 Hz,
MHz, CDCl3): δ 21.61 (CH(CH3)2), 33.44 (NCH3), 66.85 (d, 3JC,P
=
12.3 Hz, OCH2CO), 73.25 (CH(CH3)2), 76.25 (OCH2Ph), 76.62
1
2
2JC,F2 = 12.4 Hz), 149.57 (dd, JC,F1 = 244.2 Hz, JC,F2 = 12.3 Hz),
1
2
(d, JC,P = 156.6 Hz, PCH), 84.67 (d, JC,P = 6.5 Hz, POCH2), 84.80
2
152.87, 152.95, 153.52 (CO, carbonate), 175.99 (CO, hydrox-
(d, JC,P = 6.5 Hz, POCH2), 117.40 (m), 124.57 (m), 128.75, 129.23,
1
2
amate) ppm; IR (NaCl): v = 2987 (C−Haliph.), 1765 (CO,
̃
129.44, 129.94 (m), 133.91, 150.32 (ddd, JC,F = 248.2 Hz, JC,F
=
5
1
2
hydroxamate), 1684 (CO, carbonate), 1267 (PO), 1030 (P−O)
cm−1; Anal. Calcd for C24H34F2NO13P: C, 46.99; H, 5.59; N, 2.28.
Found: C, 47.18; H, 5.59; N, 2.32.
12.08 Hz, JC,P = 2.4 Hz), 150.66 (ddd, JC,F = 251.1 Hz, JC,F = 12.2
4
Hz, JC,P = 3.4 Hz, C6H3F2: C3), 153.03 (CO, carbonate), 153.06
(CO, carbonate), 170.51 (CO, hydroxamate) ppm; IR (NaCl): v
= 2986 (C−Haliph.), 1760 (CO), 1266 (PO), 1030 (P−O) cm−1;
Anal. Calcd For C27H34F2NO12P: C, 51.19; H, 5.41; N, 2.21. Found:
C, 51.17; H, 5.27; N, 2.27.
̃
ASSOCIATED CONTENT
* Supporting Information
■
S
General Procedure for the Synthesis of Prodrugs 3a,b, 4a−c,
and 5a,b. Bn-protected prodrugs 3aF−3bF, 4aF−4cF, and 5aF−5bF
(0.4 mmol) were dissolved in methanol (30 mL). Pd−C calalyst (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 target compounds
3a,b, 4a−c, and 5a,b.
Additional experimental procedures for syntheses, enzyme
assays, and evaluation of biological activity; analytical data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+49)21181-14985. Fax: (+49)21181-13847. E-mail:
thomas.kurz@uni-duesseldorf.de.
■
(((1-(3,4-Difluorophenyl)-4-(hydroxy(methyl)amino)-4-oxobutyl)-
phosphoryl)bis(oxy))bis(methylene)diisopropyl Dicarbonate (4b).
1
Colorless oil (0.21 g, 97%); H NMR (500.13 MHz, DMSO-d6): δ
1.38−1.15 (m, 12H, CH(CH3)2), 1.87−1.72 (m, 1H, CHCH2), 1.97
(q, J1 = 14.2 Hz, J2 = 7.9 Hz, 1H, CHCH2), 2.20 (tdd, J1 = 19.9 Hz, J2
= 17.2 Hz, J3 = 14.2 Hz, J4 = 8.4 Hz, 2H, CH2CO), 3.50−3.40 (m, 1H,
PCH), 4.79 (ddq, J1 = 30.9 Hz, J2 = 12.1 Hz, J3 = 6.1 Hz, 2H, OCH),
5.45 (d, J = 12.7 Hz, 2H, OCH2O), 5.81−5.50 (m, 2H, OCH2O),
7.21−6.98 (m, 1H), 7.31 (dd, J1 = 11.9 Hz, J2 = 7.7 Hz, 1H), 7.41 (q, J
= 9.1 Hz, 1H), 9.70 (s, 1H, NH) ppm; 13C NMR (125.76 MHz,
DMSO-d6): δ 21.10 (CH(CH3)2), 21.14 (CH(CH3)2), 21.18 (2C,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was in part supported by the Hans-Fischer-
■
Gesellschaft e.V., Munchen.
̈
2
CH(CH3)2), 23.76 (CH2CH2), 29.10 (d, JC,P = 16.8 Hz, CHCH2),
ABBREVIATIONS USED
35.53 (NCH3), 41.54 (d, 1JC,P = 137.0 Hz, PCH), 72.62 (CH(CH3)2),
■
72.76 (CH(CH3)2), 83.90 (d, 2JC,P = 6.5 Hz, POCH2), 83.99 (d, 2JC,P
=
CDI, 1,1′-carbonyldiimidazole; DOXP/DXP, 1-deoxy-D-xylu-
lose 5-phosphate; Dxr/IspC, 1-deoxy-D-xylulose 5-phosphate
reductoisomerase; ELISA, enzyme linked immunoassay; HRP2,
histidine-rich protein 2; Pd−C, palladium on activated carbon;
Pf IspC, P. falciparum IspC; PVM, parasitophorous vacuole
membrane; SD, standard deviation; TEA, triethylamine;
TMSBr, trimethylsilyl bromide
6.8 Hz, POCH2), 117.44 (d, 2JC,F = 17.0 Hz), 117.97 (dd, 2JC,F = 17.6
3
1
2
Hz, JC,F = 6.7 Hz), 126.01, 132.32, 148.62 (dd, JC,F = 244.9, JC,F
=
12.0 Hz), 149.09 (dd, 1JC,F = 248.9 Hz, 2JC,F = 12.6 Hz), 152.42 (C
O, carbonate), 152.51 (CO, carbonate), 171.57 (CO, hydrox-
amate) ppm; IR (NaCl): v = 3217 (O−H), 3061 (C−Haromat.), 2988
̃
(C−Haliph.), 1762 (CO, carbonate) 1647 (CO, hydroxamate),
1272 (PO), 1032 (P−O) cm−1; Anal. Calcd for C21H30F2NO11P: C,
46.59; H, 5.58; N, 2.59. Found: C, 46.67; H, 5.61; N, 2.43.
REFERENCES
General Procedure for the Synthesis of Double Ester
Prodrugs 4bA−D, 4cA−D, and 5cB−C. Prodrugs 4b, 4c, and 5c
(1 equiv, 0.3 mmol) were dissolved in dry dichloromethane (5 mL)
and treated with triethylamine (1 equiv, 0.3 mmol, 0.05 mL). At 0 °C,
solutions of acetyl chloride (4bA, 4cA, 1.1 equiv, 0.33 mmol, 0.026 g),
ethyl chloroformiate (4bB, 4cB, 5cB, 1.1 equiv, 0.33 mmol, 0.013 g),
ethyl isocyanate (4bC, 4cC, 5cC, 1 equiv, 0.3 mmol, 0.021 g), or
pivaloyl chloride (4bD, 4cD, 1 equiv, 0.3 mmol, 0.036 g) in dry
dichloromethane (2 mL) were added dropwise, respectively. After 10
■
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