MEPicides: α,β-Unsaturated Fosmidomycin Analogues as DXR Inhibitors against Malaria

Article abstract of DOI:10.1021/acs.jmedchem.8b01026

Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,β-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC₅₀ = 13 nM) without significant inhibition of HepG2 cells (IC₅₀ > 50 μM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.

Full text of DOI:10.1021/acs.jmedchem.8b01026

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Article
MEPicides: #,#-Unsaturated Fosmidomycin
Analogs as DXR inhibitors against Malaria
Xu Wang, Rachel L Edwards, Haley Ball, Claire Johnson, Amanda Haymond, Misgina Girma, Michelle
Manikkam, R Carl Brothers, Kyle McKay, Stacy Arnett, Damon Osbourn, Sophie Alvarez, Helena
I. M. Boshoff, Marvin J Meyers, Robin D. Couch, Audrey Ragan Odom John, and Cynthia S. Dowd
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01026 • Publication Date (Web): 07 Sep 2018
Downloaded from http://pubs.acs.org on September 9, 2018
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MEPicides: α,βꢀUnsaturated Fosmidomycin Analogs as DXR inhibitors against Malaria
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Xu Wang†, Rachel L. Edwards§, Haley Ball#, Claire Johnson#, Amanda Haymond#, Misgina
Girma#, Michelle Manikkam‡, Robert C. Brothers†, Kyle T. McKay†, Stacy D. Arnett∆,
Damon M. Osbourn∏, Sophie Alvarez∞, Helena I. Boshoff‡, Marvin J. Meyers∆,∑, Robin D.
Couch#, Audrey R. Odom John§, Cynthia S. Dowd*,†
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†Department of Chemistry, George Washington University, Washington DC 20052
§Department of Pediatrics, Washington University School of Medicine, Washington
University, St. Louis, MO 63110
#Department of Chemistry and Biochemistry, George Mason University, Manassas, VA
20110
‡Tuberculosis Research Section, LCIM, NIAID/NIH, Bethesda, MD 20892
∆Department of Pharmacology and Physiology, Saint Louis University, St. Louis, MO 63104
∏Department of Molecular Microbiology and Immunology, Saint Louis University, St. Louis,
MO 63104
∞Proteomics & Metabolomics Facility, Center for Biotechnology, Department of Agronomy
and Horticulture, University of NebraskaꢀLincoln, Lincoln, NE 68588
∑Department of Chemistry, Saint Louis University, St. Louis, MO 63103
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Keywords: MEPicides, Plasmodium falciparum, MEP pathway, DXR, antibiotic,
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antimalarial, phosphonate prodrug
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Abstract
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Severe malaria due to Plasmodium falciparum remains a significant global health threat.
DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building
blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its
essentiality, as well as its absence in humans. In this study, we designed and synthesized a
series of α,βꢀunsaturated analogs of fosmidomycin, a natural product that inhibits DXR in P.
falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most
promising compound, 18a, displays onꢀtarget, potent inhibition against the growth of P.
falciparum (IC50 = 13 nM) without significant inhibition of HepG2 cells (IC50 > 50 ꢁM). 18a
was also tested in a luciferaseꢀbased P. berghei mouse model of malaria and showed
exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and
promising drug candidate for the treatment of malaria.
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INTRODUCTION
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Malaria is a severe, lifeꢀthreatening infectious disease with high mortality and morbidity
rates.1 In 2016, there was a substantially high incidence rate of malaria with 216 million new
malaria cases as well as 0.4 million deaths, 64% of which are children under 5 years of age.1
Malaria is caused by a group of Plasmodium parasites, with P. falciparum causing the
majority of deaths and severe infections.2 Parasites are transmitted to humans via the bites of
female Anopheles mosquitoes.3 After growing and replicating initially in human liver cells,
the parasites reach the blood and cause malarial symptoms such as fever, headache, chills, or
even death.4 Artemisininꢀbased combination therapy (ACT) is currently the best treatment
for malaria and is typically highly effective.3 Resistance to artemisinin, however, has already
spread in the Greater Mekong subregion.5 Thus, there is a pressing need for new therapeutics
for malaria with novel modes of action that could provide alternate chemotherapies to
combat sensitive and drugꢀresistant parasites.
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P. falciparum uses the methyl erythritol phosphate (MEP) pathway for the biosynthesis
of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP),
the C5 precursors of isoprenoids (Figure 1).6, 7 Humans, however, use an alternate
acetate/mevalonate pathway exclusively to synthesize these C5 isoprene building blocks.8
Blocking the MEP pathway terminates the biosynthesis of such important metabolites and
results in cell death of P. falciparum.3, 9 Thus, many enzymes in the MEP pathway could
become promising drug targets to design MEPicides that work against malaria through a new
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mechanism of action and one not found in humans.10
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Figure 1. The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis.
DXR, 1ꢀdeoxyꢀDꢀxylulose 5ꢀphosphate reductoisomerase, is the second enzyme in the
MEP pathway and catalyzes the first committed step.11 DXR converts DOXP to MEP with
the assistance of cofactor NADPH as well as a divalent metal cation (Figure 1).12 DXR is
essential for the viability of several pathogens and is validated as a promising drug target for
developing antitubercular agents13 and antimalarials14. Fosmidomycin (Figure 2, 1a),
isolated from Streptomyces lavendulae,15 is a potent inhibitor of P. falciparum DXR (IC50 =
0.034 ꢁM)16. FRꢀ900098(Figure 2, 1b), the Nꢀacetyl analog of fosmidomycin isolated from
Streptomyces rubellomurinus,15 is roughly equipotent to fosmidomycin (P. falciparum DXR
IC50 = 0.024 ꢁM)16. While these two natural products have submicromolar inhibition of P.
falciparum growth (IC50 = 0.09ꢀ0.35 ꢁM)7, their use as a single drug therapy is limited by
low bioavailability, short serum halfꢀlife, and malaria recrudescence.17 Despite these
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disadvantages, fosmidomycin is a remarkably safe drug as proven by several clinical trials
and a promising candidate for treating uncomplicated P. falciparum malaria in combination
therapies.18, 19 Thus, we selected fosmidomycin as the parent structure from which to design
analogs that would effectively inhibit Pf DXR, have improved pharmacokinetic properties,
and lead to promising drug candidates against malaria.
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We and others have previously evaluated the structureꢀactivity relationships (SAR) of
fosmidomycin and FRꢀ900098 as inhibitors of several DXR homologs, as well as various
microbial pathogens.20ꢀ29 Fosmidomycin binds to DXR competitively with substrate DOXP
and uncompetitively with cofactor NADPH.30 SAR studies on fosmidomycin analogs reveal
that the retroꢀhydroxamate or hydroxamate moiety should be retained to mimic the crucial
interaction of fosmidomycin with the divalent metal cation.21, 24, 25, 27ꢀ29, 31 Similarly, the
phosphonate moiety should be retained as it forms numerous hydrogen bonds with
neighboring amino acid residues.32ꢀ34 A three carbon linker between the two moieties is also
found to be crucial for DXR inhibition.24 As we reported earlier, the unsaturated FRꢀ900098
analog (Figure 2, 2) gained a twoꢀfold increase in potency against Mycobacterium
tuberculosis (Mtb) DXR (IC50 = 1.07 ꢁM) compared with parent compound FR900098.24 A
prodrug strategy was applied to this structure, and the corresponding pivaloyloxymethyl
(POM) phosphonate was synthesized (Figure 2, 3). Compound 3 displays an Mtb MIC99
value of 9.4 ꢁg/mL (22 ꢁM), thus gaining the needed lipophilicity to penetrate the Mtb cell
wall.24 This compound likely regenerates 2 inside the bacteria, and this acid inhibits DXR.24,
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27 Prodrug 3 also shows potent inhibition against P. falciparum growth with an IC50 value of
18.3 nM,35 nearly as potent as artemisinin (P. falciparum IC50 = 10.4 nM),35 a current
firstꢀline antimalarial drug. As expected, prodrug 3 displays potent in vivo antimalarial
activity.35
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O
O
P
O
P
OH
N
O
P
OH
N
R2O
R2O
NaO
HO
R2O
R2O
N
R1
O
O
O
R2 = Na/H, 2
R2 = Na/H, 4
5
R2 = POM,
R1 = H: fosmidomycin, 1a
1b
3
R2 = POM,
R2 = CH3; FR900098,
Figure 2. Fosmidomycin and selected previously reported analogs.
Since it was found that the NADPHꢀbinding pocket of DXR is druggable,36 and because
this pocket is adjacent to the cavity where the retroꢀhydroxamate moiety of fosmidomycin
binds,37 we previously synthesized analogs with extended aromatic groups on the Nꢀalkoxy
group of FRꢀ900098 to develop improved DXR inhibitors as antimicrobials (Figure 2, 4, 5).27,
28 These compounds are designed to act as bisubstrate inhibitors that could bind to both the
DOXP and NADPH binding sites.27, 28 The binding mode of compound 4 was then
determined using classical LineweaverꢀBurke double reciprocal plots. These experiments
showed that compound 4 is competitive with DOXP and NADPH, confirming bisubstrate
binding behavior.27 The POM prodrug of 4 was also synthesized (Figure 2, 5), showing
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effective Mtb growth inhibition (MIC99 = 18.75 ꢁg/mL or 33.2 ꢁM).27 The bisubstrate
strategy increases the overall lipophilicity of the analogs, which is likely beneficial for
penetration into several pathogens.
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The purpose of the current work is to synthesize a series of α,βꢀunsaturated Nꢀacyl
(Figure 3A) and Nꢀalkoxy (Figure 3B) fosmidomycin analogs and evaluate them as
antimalarial agents that work via DXR inhibition. For the Nꢀacyl analogs, we studied the
influence of electronic effects on DXR inhibition via introduction of CF3 and OCH3 groups
compared with the CH3 group of compound 2. The α,βꢀunsaturated Nꢀformyl analog was also
made to assess the effect of size and lipophilicity compared with the methyl group in 2. To
achieve the bisubstrate inhibitors that compete with both DOXP and NADPH, several
aromatic groups (with a linker of 1 carbon atom) were selected as Nꢀalkoxy analogs based on
previous results.27, 28 POM prodrugs of the initial hits that showed effective DXR inhibition,
and which were synthetically accessible, were synthesized. These prodrug compounds were
then evaluated as inhibitors of P. falciparum.
Figure 3. α,βꢀUnsaturated Nꢀacyl and Nꢀalkoxy fosmidomycin analogs.
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ꢀ RESULTS AND DISCUSSION
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Synthesis. Nꢀformyl analog 12a and Nꢀacyl analogs 12b and 12c were prepared in 7 steps
from commercially available starting materials shown in Scheme 1. First, allyl phosphonate
6 was synthesized from triethylphosphite and allyl bromide via a MichaelisꢀArbuzov
reaction.38 Subsequent addition of bromine to compound 6 resulted in production of
dibromide 7.39 O-Benzylhydroxylamine hydrochloride was neutralized in situ and protected
using diꢀtertꢀbutyldicarbonate to yield Bocꢀprotected 8,40 which was then reacted with
compound 7 and two equivalents of NaH to prepare compound 9.24 The first equivalent of
NaH deprotonated 8 and generated a nucleophile to attack the primary bromide of compound
7. The second equivalent of NaH was used to eliminate the βꢀbromide to furnish
α,βꢀunsaturated phosphonate 9. Under acidic conditions, compound 9 was hydrolyzed and
generated the deprotected amine in situ that acted as a building block to be acylated with an
acyl chloride or anhydride to synthesize Nꢀacyl intermediates 10b-c. For compound 10a, the
electrophilic reagent Nꢀformylimidazole was prepared using formic acid and
1,1’ꢀcarbonyldiimidazole.41 Removal of the benzyl group using BCl3 yielded
retroꢀhydroxamic acids 11a-c.27 Treatment of these acids with TMSBr, followed by either
NaOH or NH3 gave monosodium salts 12a-b or diammonium salt 12c, respectively.21, 27 In
the case of 11c, synthesis of the sodium salt resulted in an unstable compound, leading us to
make the ammonium salt.
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O
P
O
P
Br
EtO
EtO
EtO
EtO
b
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Br
P(OEt)3
O
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OBn
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EtO
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NH2HCl
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O
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OH
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NH4O
NaO
EtO
EtO
g
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b:
P
N
R
P
R
R
HO
R = CF3
c: R = OCH3
O
O
O
12c
12a-b
11a-c
h
Scheme 1. Synthesis of N-acyl analogs 11a-c, 12a-c.a aReagents and conditions: a) Allyl bromide, 60 C, 2
o
o
days (85%); b) Br2, CH2Cl2, 0 C to rt, 2 h (88%); c) Boc2O, TEA, H2O, THF, rt, 2.5 h (90%); d) NaH, NaI,
THF, 0 oC to rt, 20 h (77%); e) i. AcCl, MeOH, rt, 30 min; ii. Na2CO3, RCOCl or (RCO)2O, 0 oC to rt, 30 min
to 24 h (10a: Na2CO3, HCOOH, 1,1’ꢀCarbonyldiimidazole, 0 C, 30 min) (50ꢀ73%); f) BCl3, CH2Cl2, ꢀ70 oC,
o
o
30 min to 3 h (19ꢀ85%); g) i. TMSBr, CH2Cl2, 0 C to rt, 24 h; ii. NaOH, H2O, rt, 1 h (quant.); h) i. TMSBr,
CH2Cl2, 0 oC to rt, 24 h; ii. NH4OH, H2O, rt, 1 h (quant.).
Scheme 2 shows the 4ꢀstep synthesis used to prepare the Nꢀalkoxy analogs, starting
from intermediate 9. Acetyl chloride was used to synthesize compound 13 following the
method described above. Subsequent debenzylation of 13 by BCl3 generated 14, the diethyl
ester of FRꢀ900098.24 This compound acted as a core intermediate from which to synthesize
the series of the Nꢀalkoxy analogs. The synthesis of 15d-g was initially attempted using
Williamson ether synthesis with either NaH or sodium tertꢀbutoxide in polar solvents such as
THF and DMF.27 Unfortunately, these reactions failed because of the instability of 14 in the
presence of such harsh bases. Weaker bases, such as Na2CO3 or Et3N, were then applied to
the reaction, and yet an overwhelming amount of side products were generated
concomitantly, possibly due to the high polarity of the solvents. The reaction conditions were
optimized using Na2CO3 with CH2Cl2 as a relatively nonꢀpolar solvent. The reaction was
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carried out in a sealed tube and heated at 60 °C for 48 h to yield 15d-g, which were then
converted to monosodium salts 16d-g in a manner similar to that used to prepare 12a-c.
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Scheme 2. Synthesis of N-hydroxyl analogs 15d-g, 16d-g. a aReagents and conditions: a) i. AcCl, MeOH, rt,
o
o
30 min; ii. Na2CO3, CH3COCl, 0 C to rt, 24 h (85%); b) BCl3, CH2Cl2, ꢀ70 C, 30 min to 3 h (77%); c)
o
o
R’CH2Br or R’CH(CH3)Br, Na2CO3, NaI, CH2Cl2, 60 C, 2 days (25ꢀ45%); d) i. TMSBr, CH2Cl2, 0 C to rt,
24 h; ii. NaOH, H2O, rt, 1 h (51ꢀ95%).
The prodrugs of selected analogs were made (Scheme 3). To obtain the diPOM esters
for Nꢀacyl analogs (17a-c), compounds 10a-c were treated with TMSBr and then
transformed to the esters using POM chloride and Et3N.27 Due to the low yield of 17b, only
17a and 17c were carried to the next reaction. Removal of the benzyl group using BCl3 gave
the diPOM prodrugs 18a and 18c. Similarly, the diPOM esters of Nꢀalkoxy analogs 19e-g
were prepared from 16e-g via treatment with POM chloride and Et3N.
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Scheme 3. Synthesis of POM prodrugs 18a, c and 19e-g. a aReagents and conditions: a) i. TMSBr, CH2Cl2,
o
o
0 C to rt, 24 h; ii. NaOH, H2O, rt, 1 h (1ꢀ30%). b) Chloromethylpivalate, TEA, NaI, DMF, 60 C, 24 h
(8ꢀ15%); c) BCl3, CH2Cl2, ꢀ70 oC, 30 min to 3 h (34ꢀ55%).
Because of the low yields of Nꢀacyl analog 18a (overall yield 2.1%) and 17b (overall
yield 0.42%), an optimized synthesis for Nꢀacyl prodrugs was developed (Scheme 4). This
route to synthesize diPOM esters was brought forward since the stability of compound 6
enabled a longer reaction time. By this method, compound 20 was obtained in higher yield
(52%) compared with previous esterification reactions, and the synthetic attrition was greatly
reduced. Bromine addition of the resulting product 20 yielded dibromide 21. Because of the
instability of the POM group to acidic conditions, use of Bocꢀprotected
O-benzylhydroxylamine was inadvisable due to the acidic conditions that would be required
to cleave the Boc group in the future steps. Thus, free amine 23 was prepared to react with
monoꢀbromide 22, the eliminated product from 21 and NaH, to yield 24. This conversion
enabled a variety of diPOM Nꢀacyl analogs. Compound 24 was converted to acylated
compounds 17a and 17b, with the latter compound obtained in a 17ꢀfold increase in yield
(overall yield 7.0%) versus the previous synthetic route (overall yield 0.42%). The final
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Nꢀacyl prodrugs 18a (overall yield 3.9%) and 18b were subsequently obtained via a
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debenzylation reaction as in Scheme 3.
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a
Scheme 4. Optimized synthesis of N-acyl analog prodrugs 18a-b.
aReagents and conditions: a) i.
o
o
TMSBr, CH2Cl2, 0 C to rt, 24 h; ii. CH3OH, rt, 1 h; iii. Chloromethylpivalate, TEA, NaI, DMF, 60 C, 24 h
o
(52%); b) Br2, CH2Cl2, 0 C to rt, 2 h (78%); c) NaH, THF, rt, 24h (70%); d) NaOH, Et2O, H2O, rt, 30 min
(97%); e) TEA, THF, reflux, 3 h (34%); f) TEA, (CF3CO)2O, 0 oC, 30 min or TEA, HCOOH,
1,1’ꢀCarbonyldiimidazole, 0 oC, 30 min (73ꢀ82%); g) BCl3, CH2Cl2, ꢀ70 oC, 30 min to 3 h (34ꢀ55%).
Evaluation of 12a-c, 16d-g as DXR inhibitors. The phosphonic acid salts were
evaluated as inhibitors of DXR from P. falciparum, and the results are shown in Table 1.
Initially, the percent remaining enzyme activity was measured by treating the enzyme with
each compound at a single concentration of 100 ꢁM. This data shows the intrinsic activity of
the compounds. Halfꢀmaximal inhibitory concentrations (IC50 values) were determined for
compounds showing greater than 75% inhibition of DXR.
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Table 1. Inhibition of P. falciparum DXR by phosphonic acid salts
Pf DXR
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Compound
R1
R2
R3
IC50 [µM]a
0.064
0.023
0.206
0.092
(37)
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1a
1b
2
Na/H
Na/H
NH4
H
H
12a
12b
12c
16d
16e
16f
16g
H
CF3
H
OCH3
CH3
CH3
CH3
CH3
14.45
2.54
Na/H
Na/H
Na/H
Na/H
CH(CH3)Ph
CH2(4ꢀiprPh)
CH2(2ꢀnaphthyl)
CH2(4ꢀbiphenyl)
2.11
(25)
4.53
aValues in parentheses are percent remaining enzyme activity at 100 ꢁM. Pf = P. falciparum; IC50 =
halfꢀmaximal inhibitory concentration
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Of the new compounds, the most potent P. falciparum DXR inhibitor is 12a with an IC50
value of 92 nM, slightly more potent than parent unsaturated compound 2. Within the
Nꢀalkoxy series of bisubstrate inhibitors 16d-g, compound 16e (4ꢀiprPh) displays the most
potent inhibition of the enzyme with an IC50 value of 2.11 ꢁM. Interestingly, the Nꢀalkoxy
substituent of 16e was also found among the more active substituents in the saturated
series.27 Compounds 16d and 16g with phenethyl and biphenyl substituents, respectively,
were also active against the enzyme, displaying low ꢁM inhibition.
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Compounds 12b-c explore the influence of electronic effects in DXR inhibition,
comparing an electronꢀwithdrawing group (CF3, 12b) and electronꢀdonating group (OCH3,
12c) with parent formyl analog 12a and acetyl analog 2. Compounds 12b and 12c display
only weak and moderate inhibition of P. falciparum DXR, respectively. This result shows
that electronic effects on the Nꢀacyl group do not sway DXR inhibition as neither the CF3
group (12b) or OCH3 group (12c) improved the activity, compared with the CH3 group of 2
which is a potent inhibitor of DXR.
In vitro effects on pathogen growth by 12a-c, 16d-g, 18a-c, 19e-g. POM prodrugs of
selected analogs were synthesized in an effort to improve their cellular activity (and possibly
bioavailability). All target compounds were tested for growth inhibition against P. falciparum
following reported procedures (Table 2).35 This data indicates the inhibitory concentration of
compound required to decrease growth of P. falciparum by 50% (Pf IC50).
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Table 2. Growth inhibition of the analogs against P. falciparum
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Compound
R1
R2
R3
Pf IC50 [µM]
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12
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1.087 ± 0.064
0.511 ± 0.0535
0.202 ± 0.01535
0.018 ± 0.00235
0.019 ± 0.002
113.3 ± 22.4
28.6 ± 5.0
1a
1b
Na/H
POM
Na/H
Na/H
NH4
H
CH3
CH3
H
2
H
3
H
12a
12b
12c
16d
16e
16f
16g
18a
18b
18c
19e
19f
19g
H
H
CF3
OCH3
CH3
CH3
CH3
CH3
H
Na/H
Na/H
Na/H
Na/H
POM
POM
POM
POM
POM
POM
CH(CH3)Ph
CH2(4ꢀiprPh)
CH2(2ꢀnaphthyl)
CH2(4ꢀbiphenyl)
H
1.2 ± 0.1
1.1± 0.0
26.1 ± 1.1
2.3 ± 0.4
0.013 ± 0.002
14.3 ± 2.5
H
CF3
OCH3
CH3
CH3
CH3
H
23.7± 6.6
CH2(4ꢀiprPh)
CH2(2ꢀnaphthyl)
CH2(4ꢀbiphenyl)
55.2 ± 1.9
50.5 ± 1.1
44.1 ± 1.1
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Pf = P. falciparum; IC50 = concentration giving 50% inhibition of Pf growth
Due to the penetrable cell membrane of eukaryotic parasite P. falciparum, as well as the
significant remodeling of host cell membranes by malaria parasites, we expect substantial
cellular uptake into P. falciparum.42 In Table 2, the polar phosphonic acid salts show
significant activity against P. falciparum parasites. Compound 12a is the most active
compound of the phosphonate salts, with an activity surpassing that of parent compound (and
clinically evaluated candidate) fosmidomycin (1a). The data also shows that the inhibition of
P. falciparum growth corresponds well to the activities of these compounds against the
enzyme target P. falciparum DXR. Of the salts, compounds 12a and 16e were the most active
DXR inhibitors. These compounds are also the most active inhibitors of P. falciparum growth
among the salts.
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The cellular activity of the POM prodrugs is also shown in Table 2. As was the case with
the phosphonic acid salts, several of the POM prodrugs are highly active against P.
falciparum. Of the Nꢀacyl series, compound 12a was the most potent P. falciparum DXR
inhibitor. Its prodrug, compound 18a, is the most potent prodrug inhibitor of P. falciparum
(IC50 = 13 nM) from the POM series. In the Nꢀalkoxy series of compounds, compound 16e
was the most potent DXR inhibitor and also shows the highest potency (IC50 = 1.1 µM)
against P. falciparum parasites. Interestingly, addition of the prodrug did not improve the
activity of this compound.
As is evident from the data in Table 2, several compounds show potent antimalarial
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activity. Much of our work focuses on analogs of fosmidomycin, which is itself a reasonably
potent inhibitor of P. falciparum growth (1a, IC50 = 634 nM). Modification of fosmidomycin
with the sole change of added α,βꢀunsaturation yields compound 12a as a highly potent P.
falciparum inhibitor with an IC50 value of 19 nM. Its prodrug 18a also potently inhibits P.
falciparum with an IC50 value of 13 nM. This value is comparable to the inhibitory activity of
current firstꢀline antimalarial drug artemisinin (P. falciparum IC50 = 10.4 nM)35.
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The cLogP, cytotoxicity, and selective indices of 12a and 18a. Due to the remarkable
activities of compounds 12a and 18a, additional studies were pursued. The computed
cLogP43, inhibition of HepG2, and selectivity indices (ratio of the antimicrobial activity to
the human cell toxicity) for compounds 12a and 18a are shown in Table 3. As expected,
compound 12a has a low cLogP value of ꢀ5.7. The prodrug strategy significantly increased
the lipophilicity of the compound, yielding 18a with a cLogP of 0.89 (an increase of over six
orders of magnitude). Neither the phosphonic acid salt 12a nor the prodrug 18a show toxicity
against HepG2 cell lines, with IC50 values > 50 ꢁM. Thus, these compounds have excellent
selectivity indices of 2632 and 3846 for 12a and 18a, respectively, against P. falciparum.
These compounds show promise as safe drug candidates for malaria.
Compound 18a is rapidly converted to 12a in vitro and in vivo. Compound 18a was
designed to be a prodrug for 12a. To determine the rate of hydrolysis by plasma and hepatic
esterases, compounds 12a and 18a were incubated in mouse liver microsomes (MLM) and in
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mouse plasma (Table 3). Compound 12a is stable in plasma (t1/2 > 120 min) and in mouse
liver microsomes (t1/2 > 60 min). In contrast, POM prodrug 18a is very rapidly converted to
compound 12a in plasma and microsomes (t1/2 < 5 min for both).
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Table 3. Computed cLogP, Cytotoxicity, and Selectivity Index
HepG2
IC50
Metabolic
stability
Plasma
Compound
R
cLogP
SIPf
stability
[ꢁM]
>50
MLM (t1/2)
>60 min
<5 min
Na/H
POM
ꢀ5.7
2632
3846
>120 min
<5 min
12a
18a
0.89
>50
cLogP calculated by DataWarrior44; SIPf = Selectivity Index for P. falciparum (HepG2
IC50/P.falciparum IC50); MLM = mouse liver microsomes
Compounds 12a and 18a inhibit isoprenoid synthesis in P. falciparum. Due to their
significant activities against P. falciparum growth, we examined compounds 12a and 18a in
further assays to determine their intracellular mechanism of action. First, we asked if
parasites treated with these inhibitors could be rescued by MEP pathway product IPP
supplementation. If the inhibitors target the MEP pathway, parasite growth should be
restored if exogenous IPP were added to the growth media.35 As is shown in Figure 4,
addition of exogenous IPP effectively rescues growth of P. falciparum treated with 12a or
18a. This pattern is similar to the restoration effect observed in fosmidomycin (1a)ꢀtreated
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parasites supplied with IPP.35 In addition, P. falciparum growth inhibited by the Nꢀalkoxy
analog 16e is also restored by IPP supplementation (Figure S3). This data hints that these
α,βꢀunsaturated fosmidomycin analogs might act on target to inhibit parasitic growth by
blocking the MEP pathway, the targeted intracellular pathway.
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Figure 4. Isoprenoid precursors rescue MEPicideꢀtreated P. falciparum. P. falciparum strain 3D7 was treated
with inhibitors at a range of concentrations and growth was quantified by PicoGreen (Life Technologies) after
72 h, as previously described.35 IPP, the product of the MEP pathway, rescues growth of drugꢀtreated parasites
(open shapes) hinting the compounds might be inhibitors of the MEP pathway in P. falciparum. Shown are
representative graphs from 3 independent experiments. Data from remaining experiments is shown in Figure
S2.
Similarly, the proposed DXR inhibitor 18a should deplete MEP pathway
intermediates beyond DXR from the treated P. falciparum. Thus, we employed a mass
spectrometryꢀbased method to quantify the MEP metabolites from untreated P.
falciparum versus parasites treated with this compound (Figure 5).45 These metabolites
include the DXR substrate (1ꢀdeoxyꢀDꢀxylulose 5ꢀphosphate, DOXP), the DXR product
(2ꢀCꢀmethylerythritol 4ꢀphosphate, MEP), and the downstream metabolites
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(4ꢀdiphosphocytidylꢀ2ꢀCꢀmethylerythritol (CDPꢀME) and 2ꢀCꢀmethylꢀDꢀerythritol
2,4ꢀcyclopyrophosphate (MEcPP)). Such metabolite levels were measured by LCꢀMS/MS
after the 3D7 parasites were treated +/ꢀ 18a after 10 h. The DOXP levels did not show
significant difference between the untreated and treated parasites, while the metabolites
downstream of DXR displayed reduced levels in treated parasites. This metabolic profiling
data suggests that 18a inhibits DXR, the first committed enzyme in the MEP pathway from P.
falciparum.
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Figure 5. Treating P. falciparum with 18a results in diminished concentrations of MEP pathway metabolites.
Intracellular concentrations of MEP pathway metabolites were measured, comparing untreated (UNT)
parasites and those treated with 18a at 5X the IC50 value of 13 nM. The cultures were saponinꢀlysed after
treatment for 10 h, and subsequently analyzed by LCꢀMS/MS. Shown are the metabolite levels from 3
independent experiments.
To further elucidate the mode of action of these analogs, compounds 12a and 18a were
evaluated for efficacy against a unique mutated P. falciparum strain, using reported
procedures.46 Due to a mutation in the metabolic regulator HAD1 (PF3D7_1033400), this P.
falciparum strain produces high levels of DOXP, the substrate for DXR. Increased DOXP
levels make it more difficult to inhibit DXR. For example, using this mutant, the (competitive)
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inhibition of DXR by fosmidomycin (1a) is impeded, resulting in fosmidomycin (1)ꢀresistant
parasites (had1 parasites).35 As shown in Figure 6, had1 parasites (had1; open shapes, black
line) were 3.4ꢀfold and 2.5ꢀfold more resistant to 12a and 18a, respectively, when compared
with wildꢀtype P. falciparum (3D7; closed shapes, grey line). Notably, the sensitivity of 12a
and 18a were restored if the mutant strain was supplied with a wildꢀtype copy of HAD1
(had1 + HAD1ꢀGFP; closed shapes, black line). Similar results were observed with Nꢀalkoxy
analog 16e, where had1 parasites were more resistant to these compounds than wildꢀtype
parasites (Figure S2). These data corroborate earlier findings that these α,βꢀunsaturated
fosmidomycin analogs inhibit P. falciparum growth via inhibition of the DXR enzyme in the
MEP pathway.35
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Figure 6. P. falciparum had1 mutant strains resistant to fosmidomycin (1a) are also resistant to MEPicides.
Doseꢀdependent growth inhibition was determined for P. falciparum strains treated with inhibitors as
previously described.35 The had1 mutation results in higher levels of the DXR substrate DOXP, and mutants
are resistant to DXR inhibition as indicated by a shift in the IC50 curve (had1; open shapes, black line) when
compared to WT P. falciparum (3D7; closed shapes, grey line). Sensitivity was restored if a WT copy of had1
was supplied in the mutant strain (had1 + HAD1ꢀGFP; closed shapes, black line). Data are representative of
at least 3 independent biological replicates performed in duplicate.
In vivo evaluation of 18a in a mouse model of malaria infection. Compound 18a
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stands out among these analogs and appears to be a promising antimalarial agent due to its
potent inhibition of P. falciparum growth. Thus, compound 18a was further evaluated for in
vivo efficacy in a P. berghei–infected mouse model of malaria using reported procedures.35
Groups of mice were infected with luciferaseꢀbased bloodꢀstage P. berghei ANKA by
intraperitoneal (ip) injection. After being infected for two days, the mice were treated daily
for five days with vehicle, 20 mg/kg chloroquine, 20 mg/kg 18a, or 50 mg/kg 18a via ip
injection. Seven days after infection, intensity of the luciferin signal was measured,
correlating to the parasitemia burden. As shown in Figure 7, parasitemia was greatly reduced
in mice treated with the control drug chloroquine (20 mg/kg), lowering the luciferin signal
intensity to 2.13×103. Interestingly, mice treated with 18a at the same dose showed a similar
result as chloroquine, with a 3ꢀlog drop in luciferin signal intensity (2.87×103) when
compared to the vehicle (2.62×106). When a higher dose of 18a was administered, the
average luciferin signal intensity of the P. bergheiꢀinfected mice is 2.34×103, not
significantly different from the result with the lower dose. Additionally, 18a was well
tolerated in mice at these dosages, as no adverse effects were observed, corroborating our
results with the HepG2 cells.
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Figure 7. Compound 18a is effective in an in vivo mouse model of efficacy. Swiss Webster mice in groups of
2ꢀ5 were infected with 103 P. berghei parasites expressing luciferase. From day 2 to day 7, mice were dosed
daily with vehicle, 20 mg/kg chloroquine, 20 mg/kg 18a, or 50 mg/kg 18a. Mice were imaged using an IVIS
imager at 7 days postꢀinfection (A) and parasitemia was quantified (B). All doses were administered ip.
Based on our in vitro stability studies, we anticipated that 18a would be rapidly
converted to 12a in the in vivo study. We designed a compound exposure study to determine
plasma concentration of 12a under similar conditions to the efficacy study. Compound 18a
was dosed at 20 mg/kg i.p. in Swiss Webster mice (n=3) and plasma samples were removed
at select time points over the course of 8 h (Figure 8). Over the course of 8 h, 12a was
observed at high concentrations, with a concentration of 485 ng/mL (2.8 µM) at 8 h which is
approximately 200ꢀfold above the Pf IC50 of 18a in vitro.
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10000
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Time (h)
Figure 8. Compound 18a is rapidly converted to 12a in vivo. Swiss Webster mice were dosed with 18a at 20
mg/kg ip. Plasma samples were collected over 8 h and analyzed for concentration of 12a by LCꢀMS/MS.
ꢀ CONCLUSIONS
With a growing number of drugꢀresistant cases of malaria, there is an urgent demand for
new antimalarial agents with novel modes of action. DXR, an essential enzyme in the
causative pathogens of malaria, while completely absent in humans, appears to be a
promising drug target for the treatment of this disease. We aim to develop compounds that
selectively inhibit this crucial enzyme to eliminate P. falciparum, while avoiding toxicity in
humans. Thus, several α,βꢀunsaturated analogs of fosmidomycin were designed and
synthesized, including the Nꢀalkoxy and Nꢀacyl series. Our lead compound 18a, stands out as
a superior antimalarial agent. The excellent safety profile, transparent mode of action, and
encouraging in vivo efficacy of 18a in eliminating Plasmodium spp. parasites establish 18a
as a promising drug candidate for the treatment of malaria. Encouraging activities of these
Nꢀalkoxy and Nꢀacyl fosmidomycin analogs suggests a new direction for the development of
more prospective drug candidates in the fight against this important human pathogen.
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ꢀ EXPERIMENTAL SECTION
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General. H and C NMR spectra were recorded in CDCl3, CD3OD or D2O on Agilent
spectrometer at 400 and 101 MHz, respectively, with TMS, H2O or solvent signal as
internal standard. Chemical shifts are given in parts per million (ppm). Spin multiplicities
are given with the following abbreviations: s (singlet), br s (broad singlet), d (doublet), dd
(doublet of doublets), ddd (doublet of doublets of doublets), t (triplet), dt (doublet of triplets),
ddt (doublet of doublet of triplets), q (quadruplet), qt (quintuplet), m (multiplet). Mass
spectra were measured in the ESI mode on an HPLCꢀMS (Agilent 1100) or in the EI mode
on an GCꢀMS (Shimadzu GCMSꢀQP2010S). Thin layer chromatography (TLC) was
performed on Bakerꢀflex Silica Gel IB2ꢀF silica plates and flash column chromatography
was carried out using SiliCycle SiliaFlash P60 silica gel (40ꢀ63 µm). All reagents were
purchase from commercial suppliers and used without further purification. Anhydrous
solvents were purified by MBRAUN MBꢀSPS solvent purification system before use. All air
sensitive reactions were carried out under nitrogen atmosphere. The purity of synthesized
compounds (>95%) was determined by H/13C NMR in combination with HPLCꢀMS
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(Agilent 1100). Column: Thermo Fisher Scientific Hypersil GOLD aQ Cꢀ18 3 ꢁm particle
(250 mm × 4.6 mm). Mobile phase (containing 0.1% formic acid as the additive): linear
gradient of acetonitrile (50%ꢀ100%) in water at a flow rate of 0.8 mL/min over 12.5 min,
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followed by 100% acetonitrile that was maintained for another 12.5 min. The UV detection
wavelength was 210 nm and 254 nm. Highꢀresolution mass spectroscopy spectra (HRMS)
were recorded in positive or negative ESI mode on a Waters QꢀTOF Ultima mass
spectrometer (UIUC Mass Spectrometry Laboratory) or in positive FAB mode on a VG
Analytical VG70SE magnetic sector mass spectrometer (JHU Mass Spectrometry Facility).
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General procedure for synthesis of amides 10a-c and 13. To a solution of MeOH (10.1 eq)
in dry CH2Cl2 (1 M) under N2 was added acetyl chloride (10 eq) dropwise at room
temperature and the mixture was stirred for 10 min. The reaction mixture was then added a
solution of 9 (1 eq) in dry CH2Cl2 (1 M) and stirred at room temperature for 30 min. After the
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completion of deprotection, dry Na2CO3 (12 eq) was added at 0 C and the mixture was
stirred at the same temperature for 10 min. The reaction mixture at 0 oC was added dropwise
RCOCl, (CF3CO)2O or Nꢀformylimidazole* (2 eq). The mixture was then warmed up to
room temperature and stirred for 30 min to 24 h, quenched with saturated NaHCO3 (aq) and
extracted with CH2Cl2 (3×). The combined organic layers were dried with anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The crude concentrate was then purified by
column chromatography on silica gel using EtOAc and CH2Cl2 (with a ratio from 1/10 to 2/1)
to give the pure title compound.
General procedure for synthesis of 11a-c, 14 and 18a-c.24 To a solution of 10, 13 or 17 (1
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eq) in dry CH2Cl2 (0.1 M) under N2 was added boron trichloride (1 M in CH2Cl2, 4 eq) at ꢀ78
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oC dropwise. The reaction mixture was stirred at ꢀ78 C for 30 min to 3 h, quenched with
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saturated NaHCO3 (aq) and extracted with EtOAc (5×). The combined organic layers were
dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude
residue was then purified by column chromatography on silica gel using EtOAc and MeOH
(EtOAc and CH2Cl2 for 18a-c) to give the pure title compound.
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General procedure for synthesis of 12a-b and 16d-g. To a solution of 11a-b or 15d-g (1 eq)
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in dry CH2Cl2 (0.1 M) under N2 was added TMSBr (10 eq) dropwise at 0 C. The reaction
mixture was warmed to room temperature, stirred overnight, and then concentrated under
reduced pressure. The mixture was dissolved in CH2Cl2, evaporated under reduced pressure
and dried under vacuum. The crude residue was then stirred in 0.5 M NaOH (1 eq) in H2O at
room temperature for 1 h, washed with Et2O three times and lyophilized to give the title
compounds.
General procedure for synthesis of 17a-c. To a solution of 10a-c (1 eq) in dry CH2Cl2 (0.1
M) under N2 was added TMSBr (10 eq) dropwise at 0 oC. The reaction mixture was warmed
to room temperature, stirred overnight, and then concentrated under reduced pressure. The
mixture was dissolved in CH2Cl2, evaporated under reduced pressure and dried under
vacuum. The crude residue was then stirred in 0.5 M NaOH (2 eq) in H2O at room
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temperature for 1 h, washed with Et2O (3×) and lyophilized to give disodium salts as white
solids. The crude solid was then dissolved in dry DMF (0.1 M), and TEA (6 eq),
chloromethylpivalate (6 eq) and NaI (0.1 eq) were added. The reaction mixture was stirred at
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60 C for 24 h, quenched with H2O, and extracted with Et2O (3×). The combined organic
layers were dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure.
The crude product was then purified by column chromatography on silica gel using hexanes
and EtOAc or CH2Cl2 and EtOAc to give the pure title compound.
Diethyl [(1E)-3-[N-(benzyloxy)formamido]prop-1-en-1-yl]phosphonate (10a) Light
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yellow oil (594 mg, 73%). H NMR (400 MHz, cdcl3) δ 8.24 (s, 1H), 7.41 – 7.29 (m, 5H),
6.72 – 6.57 (m, 1H), 5.82 (ddd, J = 12.4, 10.0, 6.3 Hz, 1H), 4.84 (s, 2H), 4.30 – 4.20 (m, 2H),
4.13 – 3.98 (m, 4H), 1.32 – 1.27 (m, 6H). 13C NMR (101 MHz, cdcl3) δ 163.3, 144.40 (d, J =
5.7 Hz), 134.1, 129.4, 129.2, 128.8, 120.8 (d, J = 188.7 Hz), 78.3, 61.9 (d, J = 5.6 Hz), 47.2 (d,
J = 26.7 Hz), 16.3 (d, J = 6.3 Hz). LCꢀMS (ESI+): 328.2 m/z [M+H]+, 655.2 m/z [2M+H]+.
Diethyl [(1E)-3-(N-hydroxyformamido)prop-1-en-1-yl]phosphonate (11a) Light yellow
oil (106 mg, 55%). 1H NMR (400 MHz, cdcl3) δ 10.06 (s, 1H), 8.32 (s, 1H), 6.75 – 6.55 (m,
1H), 5.99 – 5.74 (m, 1H), 4.32 – 4.25 (m, 2H), 4.09 – 3.92 (m, 4H), 1.25 (t, J = 7.1 Hz, 6H).
13C NMR (101 MHz, cdcl3) δ 172.5, 162.78, 146.3 (d, J = 5.5 Hz), 118.7 (d, J = 189.1 Hz),
62.3 (d, J = 5.7 Hz), 48.
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