MEPicides: α,β-unsaturated Fosmidomycin N-Acyl Analogs as Efficient Inhibitors of Plasmodium falciparum 1-Deoxy-d-xylulose-5-phosphate reductoisomerase

Abstract

Malaria, a mosquito-borne disease caused by several parasites of the Plasmodium genus, remains a huge threat to global public health. There are an estimated 0.5 million malaria deaths each year, mostly among African children. Unlike humans, Plasmodium parasites and a number of important pathogenic bacteria employ the methyl erythritol phosphate (MEP) pathway for isoprenoid synthesis. Thus, the MEP pathway represents a promising set of drug targets for antimalarial and antibacterial compounds. Here, we present new unsaturated MEPicide inhibitors of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. A number of these compounds have demonstrated robust inhibition of Plasmodium falciparum DXR, potent antiparasitic activity, and low cytotoxicity against HepG2 cells. Parasites treated with active compounds are rescued by isopentenyl pyrophosphate, the product of the MEP pathway. With higher levels of DXR substrate, parasites acquire resistance to active compounds. These results further confirm the on-target inhibition of DXR in parasites by the inhibitors. Stability in mouse liver microsomes is high for the phosphonate salts, but remains a challenge for the prodrugs. Taken together, the potent activity and on-target mechanism of action of this series further validate DXR as an antimalarial drug target and the α,β-unsaturation moiety as an important structural component.

Graphical Abstract

Malaria, caused by several parasites of the apicomplexan genus Plasmodium, is a huge threat to global public health, with an estimated 247 million new cases and 619,000 deaths in 2021, mostly among African children.¹ Plasmodium falciparum is responsible for the vast majority of malaria deaths and illness. Artemisinin combination therapies are currently the standard treatment for this type of malaria.¹ However, resistance to artemisinin has emerged in recent years, compromising this strategy for malaria treatment and control.² Thus, there is an urgent demand for new antimalarial drugs with novel modes of action to provide effective alternative chemotherapies.

Isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate, the C5 precursors of all essential isoprenoids, are synthesized via the mevalonate pathway in humans.³ In P. falciparum, as well as a number of other important pathogens, the methyl erythritol phosphate (MEP) pathway is employed instead (Figure 1).⁴ The second enzyme in the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR, IspC, EC 1.1.1.267), catalyzes its first committed step and is essential for survival of several pathogens including P. falciparum.^5,6 Because of its vital role in parasitic metabolites and absence in humans, DXR is considered a promising antimalarial drug target engaging a novel mode of action.

Figure 1.

The MEP pathway for the synthesis of isoprenoids.

DXR catalyzes the reduction and isomerization of DXP to produce MEP, with the assistance of the cofactor NADPH.⁷ Fosmidomycin (Figure 2, FOS, 1) and its N-acetylated analog FR-900098 (Figure 2, FR98, 2), natural products isolated from Streptomyces lavendulae and Streptomyces rubellomurinus, respectively, are well-characterized inhibitors that bind to the DXP site of the DXR enzyme.^8,9 These two DXR inhibitors also demonstrate potent antiparasitic activities against P. falciparum (IC₅₀ = 0.09–0.35 μM).¹⁰ Fosmidomycin failed in clinical trials for the treatment of uncomplicated malaria, due to poor pharmacokinetics and malaria recrudescence.¹¹ Because of the excellent safety profile and superior antimalarial activities of fosmidomycin and FR-900098, we have used this scaffold to design and synthesize new analogs to investigate their potential as antimalarial drugs. Previously, we found that α,β-unsaturation of the three-carbon backbone is crucial for fosmidomycin analogs to display potent antimalarial activities.^12,13 The most potent analogs from our previous work are the N-formyl α,β-unsaturated fosmidomycin analog (Figure 2, 3a) and its pivaloyloxymethyl (POM) prodrug (Figure 2, 3b), which showed inhibitory activities against P. falciparum in the low nanomolar range (IC₅₀ = 13–19 nM).¹² These fosmidomycin analogs were confirmed to target DXR, and the prodrug showed outstanding in vivo efficacy, comparable to chloroquine in a mouse model of malaria. These analogs have fairly high clearance rates in vivo, although potent antimalarial activity allows the drug concentration in plasma to be maintained above the minimum effective concentration at higher doses.¹² The goal of the present work was to develop potent analogs of compounds 3a/3b with improved pharmacokinetic profiles.

Figure 2.

Fosmidomycin and selected fosmidomycin analogs.

The DXR active site includes two adjacent pockets that are responsible for binding the substrate DXP and the cofactor NADPH.¹⁴ This allows the design of bisubstrate inhibitors, i.e., compounds that bind to both pockets, in an attempt to gain more potent binding and higher specificity for DXR.^15,16 We have previously reported a bisubstrate inhibitor 4 (Figure 2) to be competitive with both DXP and NADPH against recombinant Yersinia pestis DXR.¹⁶ Although this compound was only a moderate inhibitor of bacterial DXRs [IC₅₀ > 25 μM for the M. tuberculosis enzyme (MtDXR) and IC₅₀ = 4.45 μM for Y. pestis DXR], it confirmed the potential of the bisubstrate strategy.

In this work, we explored N-acyl fosmidomycin analogs now bearing α,β-unsaturation (Figure 2), aiming to develop potent inhibitors of DXR from P. falciparum and to learn if this extension can also result in compounds with better pharmacokinetics. Each structure maintains an unsaturated, three carbon backbone, connecting the phosphorus and nitrogen atoms. This pattern enables binding to the DXP site. The retrohydroxamic acid is extended using N-acyl substitution, designed to position an aromatic ring near the nicotinamide binding site of NADPH. The phenyl ring is linked to the retro-hydroxamate carbonyl by 0–3 methylene groups. Both the phosphonic acid compounds and the POM prodrugs were synthesized and evaluated as inhibitors against P. falciparum DXR (PfDXR) and asexual P. falciparum growth in red blood cells. The inclusion of an aromatic ring and addition of pivaloyl moieties increase the overall lipophilicity of the molecule, which by design should reduce renal excretion¹⁷ and possibly improve oral bioavailability and absorption.

RESULTS AND DISCUSSION

Synthesis.

Scheme 1 shows the synthesis of compounds 11a–d and compounds 13a, b, and d. Triethyl phosphite was reacted with allyl bromide to synthesize 5 via an Arbuzov reaction. Bromine addition was then implemented to prepare dibromide 6. Boc-protected O-benzyl hydroxylamine 7 was deprotonated with NaH and then substituted for the primary bromide of compound 6. A second equivalent of NaH eliminated the secondary bromide to yield α,β-unsaturated intermediate 8.¹² The Boc protecting group of 8 was cleaved by HCl generated in situ using acetyl chloride and methanol. The free amine was reacted with various acyl chlorides to prepare 9a–d. These compounds were deprotected with BCl₃ to remove the benzyl group, yielding 10a–d. The target phosphoric acid salts 11a–d were then synthesized by treating 10a–d with TMSBr and NaOH. For the three most potent salt analogs, the corresponding POM prodrugs were made. Amides 9a, b, and d were converted to the phosphoric acid salts, and then reacted with chloromethylpivalate to prepare POM intermediates 12a, b, and d. BCl₃ was used to remove the benzyl groups from 12a, b, and d to generate the desired prodrug target compounds 13a, b, and d.

Scheme 1. Synthesis of N-Acyl Analogs 10a–d, 11a–d, and 13a–b, d^a.

^aReagents and conditions: (a) allyl bromide, 60 °C, 2 days; (b) Br₂, CH₂Cl₂, 0 °C to rt, 2 h; (c) Boc₂O, TEA, H₂O, THF, rt, 2.5 h; (d) NaH, NaI, THF, 0 °C to rt, 20 h; (e) 1. AcCl, MeOH, rt, 30 min; 2. Na₂CO₃, RCOCl, 0 °C to rt, 30 min to 24 h; (f) BCl₃, CH₂Cl₂, −70 °C, 30 min to 3 h; (g) 1. TMSBr, CH₂Cl₂, 0 °C to rt, 24 h; 2. NaOH, H₂O, rt, 1 h; (h) chloromethylpivalate, TEA, NaI, DMF, 60 °C, 24 h.

Biological Evaluation.

The target salt compounds were evaluated as inhibitors of PfDXR, and all compounds were evaluated as inhibitors of asexual P. falciparum growth in red blood cells (Table 1). The data show the influence of backbone unsaturation, N-acyl substitution, and prodrug modification of the phosphonate on biological activity. The structures of compounds 4 and 11a are the same except for the level of saturation along the propyl backbone. Compound 4 is saturated while compound 11a bears a double bond adjacent to the phosphorus atom. Notably, this single change results in a 2-fold improvement in activity against the enzyme and a dramatic improvement in activity against P. falciparum (>300μM vs 0.369 μM). Extension of the acyl substituent by a single methylene (11b) is tolerated by DXR, but further elongation leads to decreased inhibition of both the enzyme and parasite growth (11c and 11d). Protection of 11a and 11b as the bisPOM prodrugs (compounds 13a and 13b, respectively) leads to improved activity against P. falciparum, a trend we have seen in prior compounds.^12,13 These most active compounds are 3–5-fold more potent against P. falciparum compared to the original parent compound, fosmidomycin (1). Selected compounds were also evaluated against HepG2 cells as a measure of mammalian cytotoxicity. For the compounds tested, cytotoxicity against HepG2 was negligible.

Table 1.

DXR Inhibition and Antimicrobial Activities of N-Acyl Analogs^a

cmpd	R1	R2	Pf DXR IC50 [μM]	Pf IC50 [μM]	HepG2 IC50 [μM]
1	Na/H (FOS, sat’d)	H	0.063	1.021
2	Na/H (FR98, sat’d)	CH3	0.022	0.511
3a	Na/H	H	0.092	0.01912
3b	POM	H	--	0.01312	>50 12
4	Na/H (satd)	Ph	0.232	>355.9
10a	Et	Ph	--	>319.4
10b	Et	CH2Ph	--	336.6
10c	Et	(CH2)2Ph	--	>293.1
10d	Et	(CH2)3Ph	--	31.25
11a	Na/H	Ph	0.1062	0.369	>50
11b	Na/H	CH2Ph	0.17	0.468
11c	Na/H	(CH2)2Ph	1.34	10.75
11d	Na/H	(CH2)3Ph	0.8976	4.983
13a	POM	Ph	--	0.200	>50
13b	POM	CH2Ph	--	0.304
13d	POM	(CH2)3Ph	--	4.552
14	Na/H	CH3	0.20612	0.20213

^aPf = P. falciparum; IC₅₀ = inhibitory concentration at 50%; -- = not determined.

The most active analogs, 11a and 13a, were further evaluated in two assays to determine their mechanism of action. The dose-responsive growth inhibition of parasites treated with compounds 11a and 13a, along with parent compound 1, is shown in Figure 3. When IPP, the product of the MEP pathway, is added to the culture media, the antiparasitic efficacy of the compounds is negated, and the parasites are rescued. These data strongly indicate that the antiparasitic effect of the compounds is mediated through inhibition of the MEP pathway intracellularly and is not the result of off-target cellular effects. In addition, compounds were evaluated against a had1 mutant strain of Plasmodium¹³ in which parasites maintain higher intracellular levels of DXP, the substrate of DXR. Parasites with elevated intracellular DXP are resistant to treatment with compounds 11a and 13a (Figure 4). Data from this experiment strongly suggests that all compounds function through direct inhibition of P. falciparum DXR. Taken together, data from these experiments indicate that compounds 11a and 13a, like fosmidomycin (1), act by inhibiting the MEP pathway via DXR.

Figure 3.

Isoprenoid precursors rescue P. falciparum treated with the DXR inhibitors. At a range of concentrations, 1, 11a, and 13a were used to treat P. falciparum strain 3D7. After 72 h, the parasitic growth was quantified by PicoGreen (Life Technologies), as previously described.¹⁸ IPP, the downstream precursor for isoprenoids, rescues the growth of parasites treated with both inhibitors 11a and 13a (open shapes), similar to the result with the control inhibitor 1. Thus, these compounds are proved to be specific MEP pathway inhibitors in P. falciparum. Shown are representative graphs from experiments performed in at least three independent biological replicates.

Figure 4.

High levels of the DXR substrate DXP grant P. falciparum strains resistance to DXR inhibitors. Dose-dependent parasitic growth inhibition by 1, 11a, and 13a was determined as previously described.¹⁹ HAD1 mutant P. falciparum strain expresses higher levels of DXR substrate DXP. This strain displays higher resistance to DXR inhibitors, as shown by a shift to the right in the IC₅₀ curve (had1; open shapes, black line) when compared to the wild-type P. falciparum strain (3D7; filled symbols, grey line). When a wild-type copy of HAD1 was supplied in the mutant strain (had1 + HAD1-GFP; closed symbols, black line), the sensitivity was restored, as shown by a shift to the left in the IC₅₀ curve. Data represent at least three independent biological experiments performed in duplicate.

A limitation of fosmidomycin is rapid clearance due to its polar structure.^11,20 We and others have adopted a prodrug strategy to increase lipophilicity and potentially improve antimicrobial activity and oral bioavailability.^{12,13,16,21–26} An important consideration when introducing such prodrug moieties is balancing the desired biological activity with compound stability. Prodrugs should be stable during absorption and distribution, but then activated once they reach the intended target. To understand the metabolic stability of compounds 11a and 13a, the compounds were subjected to mouse liver microsomes (MLM, Table 2). As was seen for prior analogs,¹² phosphonic acid salt 11a was found to be stable in MLM for over 1 h. However, prodrug 13a was unstable to MLM, displaying a half-life of less than 5 min. The improvement in activity of 13a over 11a (IC₅₀ values of 0.200 μM vs 0.369 μM) suggests that the bisPOM moiety improves uptake. The low half-life of the bisPOM, however, necessitates further studies to evaluate alternate prodrug moieties with improved stability.

Table 2.

Microsomal Stability and Computed Descriptors for N-Acyl Analogs^a

cmpd	R1	R2	cLog P	HA	HD	PSA	MLM t1/2 (min)
3a	Na/H	H	−0.28	6	2	87.07	>60
3b	POM	H	3.6	10	1	128.67	<512
4	Na/H (satd)	Ph	−1.27	6	2	87.07
11a	Na/H	Ph	1.73	6	2	87.07	>60
13a	POM	Ph	5.61	10	1	128.67	<5

^ac Log P and polar surface area (PSA) were calculated using ChemDraw 20.0.0.41. MLM = mouse liver microsomes.

The major pharmacokinetic liability of fosmidomycin is high kidney distribution and renal excretion (85% following iv dosing).²⁰ To start to address this issue, we previously developed the α,β-unsaturation strategy to increase compound lipophilicity. 11a has a much higher cLog P value than 4 (c Log P, Table 2). As mentioned above, this small change in structure was accompanied by a dramatic improvement in antiparasitic activity. By appending the aromatic group to the N-acyl position, 11a expectedly possesses superior lipophilicity over formyl analog 3a but suffers a decrease in potency. While the decrease in potency is noted, compound 11a remains a sub-micromolar inhibitor of P. falciparum. Thus, although the prodrug strategy requires further optimization, 11a stands out as a promising lead with its excellent activity and physicochemical properties.

CONCLUSIONS

As resistance to antimalarial agents continues, there remains a need for novel compounds working through unique mechanisms of action. Indeed, such compounds are crucial if we are to effectively treat malaria on a global scale. DXR is an essential enzyme in most bacteria and parasites and is used for the biosynthesis of isoprenoids. Because of its validation as a drug target, as well as the clinical history of natural product inhibitor fosmidomycin, we have focused on developing novel DXR inhibitors as a means of malarial treatment. Building on our prior compounds, this work explored extended N-acyl substitution on the retro-hydroxamate with an α,β-unsaturated backbone. These compounds, particularly 11a and 13a, displayed nM potency against Pf DXR and P. falciparum, while being non-toxic to mammalian HepG2 cells. The compounds act on-target intracellularly via the MEP pathway and through DXR specifically.

An important take home message from this work is the profound and somewhat surprising influence of backbone unsaturation on activity. Compounds bearing this unsaturation are consistently more active compared with their saturated counterparts. The effect against the parasite can be partly explained by the improvement in lipophilicity. We are attempting to gain further insight into the role of this pi bond through crystallography. Despite limited metabolic stability of the prodrugs, the overall activity of the series reiterates the importance of the MEP pathway, DXR, and this chemical family as promising leads in the antimalarial drug discovery pipeline.

METHODS

General.

¹H and ¹³C NMR spectra were recorded in CDCl₃, CD₃OD, or D₂O on Agilent spectrometer at 400 and 100 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), and m (multiplet). Mass spectra were measured in the ESI mode on an HPLC-MS (Agilent 1100) or in the EI mode on an GC–MS (Shimadzu GCMS-QP2010S). Thin layer chromatography was performed on Baker-flex Silica Gel IB2-F silica plates and flash column chromatography was carried out using SiliCycle SiliaFlash P60 silica gel (40–63 μm). All reagents were purchase from commercial suppliers and used without further purification. Anhydrous solvents were filtered by MBRAUN MB-SPS solvent purification system before use. All air sensitive reactions were carried out under a nitrogen atmosphere. Purity of synthesized compounds (>95%) was determined by ¹H/¹³C NMR in combination with HPLC-MS (Agilent 1100). Column: Thermo Fisher Scientific Hypersil GOLD aQ C-18 3 μm particle (250 × 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, followed by 100% acetonitrile was maintained for another 12.5 min. The UV detection wavelength was 210 and 254 nm. High-resolution mass spectroscopy spectra (HRMS) were recorded in positive or negative ESI mode on a Waters Q-TOF 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).

General Procedure for Synthesis of Amide 9a–d.

To a solution of MeOH (10.1 equiv) in dry CH₂Cl₂ (1 M) under N₂ was added acetyl chloride (10 equiv) dropwise at room temperature and the mixture was stirred for 10 min. To the reaction mixture was then added a solution of 8 (1 equiv) in dry CH₂Cl₂ (1 M) and was stirred at room temperature for 30 min. After the completion of deprotection, dry Na₂CO₃ (12 equiv) was added at 0 °C and the mixture was stirred at the same temperature for 10 min. The reaction mixture was then added RCOCl (2 equiv) slowly, 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. The crude was then purified by column chromatography on silica gel using EtOAc and CH₂Cl₂ to give the pure title compound.

Diethyl [(1E)-3-[N-(Benzyloxy)-1-phenylformamido]prop-1-en-1-yl]phosphonate (9a).

Light yellow oil (831 mg, 82%). ¹H NMR (400 MHz, CDCl₃): δ 7.71–7.00 (m, 10H), 6.89–6.70 (m, 1H), 6.24–5.98 (m, 1H), 4.64 (s, 2H), 4.48 (m, 2H), 4.20–3.98 (m, 4H), 1.37–1.23 (m, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 170.2, 145.8 (d, J = 6.1 Hz), 133.8, 133.6, 130.9, 129.4, 128.9, 128.5, 128.3, 128.1, 119.4 (d, J = 186.6 Hz), 62.1 (d, J = 5.7 Hz), 49.9 (d, J = 23.4 Hz), 16.4 (d, J = 6.5 Hz). LC–MS (ESI⁺): 404.2 m/z [M + H]⁺, 807.2 m/z [2M + H]⁺.

Diethyl [(1E)-3-[N-(Benzyloxy)-2-phenylacetamido]prop-1-en-1-yl]phosphonate (9b).

Light yellow oil (353 mg, 85%). ¹H NMR (400 MHz, CDCl₃): δ 7.43–7.11 (m, 10H), 6.63 (ddt, J = 22.3, 17.2, 5.2 Hz, 1H), 5.69 (ddt, J = 18.8, 17.2, 1.6 Hz, 1H), 4.73 (s, 2H), 4.38–4.28 (m, 2H), 4.03–3.90 (m, 4H), 3.71 (s, 2H), 1.23 (ddd, J = 5.6, 4.6, 2.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 173.2, 145.5 (d, J = 5.5 Hz), 134.4, 134.1, 129.3, 129.2, 129.1, 128.7, 128.5, 126.9, 119.9 (d, J = 187.1 Hz), 77.0, 61.8 (d, J = 5.6 Hz), 48.6 (d, J = 23.2 Hz), 39.5, 16.3 (d, J = 6.4 Hz). LC–MS (ESI⁺): 418.2 m/z [M + H]⁺, 835.2 m/z [2M + H]⁺.

Diethyl [(1E)-3-[N-(Benzyloxy)-3-phenylpropanamido]-prop-1-en-1-yl]phosphonate (9c).

Light yellow oil (224 mg, 69%). ¹H NMR (400 MHz, CDCl₃): δ 7.43–7.12 (m, 10H), 6.80–6.59 (m, 1H), 6.01–5.79 (m, 1H), 4.71 (s, 2H), 4.42–4.28 (m, 2H), 4.15–4.00 (m, 4H), 2.93 (t, J = 7.6 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 1.41–1.23 (m, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 174.5, 145.9 (d, J = 5.6 Hz), 141.0, 134.2, 129.1, 129.0, 128.7, 128.5, 128.4, 126.2, 118.1 (d, J = 117.8 Hz), 77.1, 62.0 (d, J = 5.7 Hz), 48.9 (d, J = 23.6 Hz), 34.1, 30.6, 16.4 (d, J = 6.4 Hz). LC–MS (ESI⁺): 432.2 m/z [M + H]⁺, 863.2 m/z [2M + H]⁺.

Diethyl [(1E)-3-[N-(Benzyloxy)-4-phenylbutanamido]-prop-1-en-1-yl]phosphonate (9d).

Light yellow oil (309 mg, 69%). ¹H NMR (CDCl₃, 400 MHz): δ (ppm): 7.37–7.16 (m, 10H), 6.76–6.64 (m, 1H), 5.85–5.75 (m, 1H), 4.73 (s, 2H), 4.34 (m, 2H), 4.12–4.00 (m, 4H), 2.68–2.62 (m, 2H), 2.45–2.41 (t, 1H, J = 7.2, 8.0 Hz), 2.36–2.32 (t, 1H, J = 7.2, 7.6 Hz), 1.99–1.91 (m, 2H), 1.30–1.27 (t, 6H, J = 6.8, 7.2 Hz). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm): 177.3, 146.1 (d, J = 5.8 Hz), 129.2, 129.1, 128.8, 128.7, 128.6, 128.6, 128.5, 128.5, 128.4, 126.3, 126.0, 119.7 (d, J = 187.4 Hz), 76.8, 62.0 (d, J = 5.9 Hz), 48.8 (d, J = 22.8 Hz), 35.1, 33.3, 31.5, 25.9, 16.3 (d, J = 6.0 Hz). LC–MS (ESI⁺): 446.2 m/z [M + H]⁺, 891.2 m/z [2M + H]⁺.

General Procedure for Synthesis of 10a–d and 13a,b, d.⁴

To a solution of 9a–d or 12a–b, d (1 equiv) in dry CH₂Cl₂ (0.1 M) under N₂ was added boron trichloride (1 M in CH₂Cl₂, 4 equiv) dropwise at −78 °C. The reaction mixture was stirred at −78 °C for 30 min to 3 h, quenched with saturated NaHCO₃ (aq), and extracted with EtOAc (5×). The combined organic layers were dried with anhydrous Na₂SO₄, filtered, and concentrated. The crude was then purified by column chromatography on silica gel using EtOAc and MeOH (EtOAc and CH₂Cl₂ for 13a–b, d) to give the pure title compound.

Diethyl [(1E)-3-(N-Hydroxy-1-phenylformamido)prop-1-en-1-yl]phosphonate (10a).

Light yellow oil (229 mg, 74%). ¹H NMR (400 MHz, CDCl₃): δ 9.85 (s, 1H), 7.67–7.26 (m, 5H), 6.89–6.65 (m, 1H), 6.04–5.78 (m, 1H), 4.52–4.32 (m, 2H), 3.97–3.86 (m, 4H), 1.36–1.06 (m, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 169.8, 146.9 (d, J = 5.9 Hz), 133.3, 130.7, 128.4, 127.9, 118.6 (d, J = 186.1 Hz), 62.1 (d, J = 5.7 Hz), 52.3 (d, J = 25.4 Hz), 16.2 (d, J = 6.5 Hz). LC–MS (ESI⁺): 314.2 m/z [M + H]⁺, 627.2 m/z [2M + H]⁺.

Diethyl [(1E)-3-(N-Hydroxy-2-phenylacetamido)prop-1-en-1-yl]phosphonate (10b).

Light yellow oil (224 mg, 81%). ¹H NMR (400 MHz, CDCl₃): δ 9.91 (s, 1H), 7.41–7.23 (m, 5H), 6.80–6.64 (m, 1H), 5.92–5.77 (m, 1H), 4.43–4.38 (m, 2H), 4.11–3.99 (m, 4H), 3.88 (s, 2H), 1.38–1.29 (m, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 172.9, 147.3 (d, J = 4.7 Hz), 135.2, 129.6, 128.4, 126.8, 118.1 (d, J = 188.9 Hz), 62.4 (d, J = 5.6 Hz), 50.7 (d, J = 24.9 Hz), 39.1, 16.3 (d, J = 6.3 Hz). LC–MS (ESI⁺): 328.2 m/z [M + H]⁺, 655.2 m/z [2M + H]⁺.

Diethyl [(1E)-3-(N-Hydroxy-3-phenylpropanamido)prop-1-en-1-yl]phosphonate (10c).

Light yellow oil (103 mg, 66%). ¹¹H NMR (400 MHz, CDCl₃): δ 9.70 (s, 1H), 7.31–7.12 (m, 5H), 6.67 (ddd, J = 22.1, 9.9, 4.9 Hz, 1H), 5.81 (ddd, J = 17.2, 10.9, 9.4 Hz, 1H), 4.43–4.32 (m, 2H), 4.04–3.91 (m, 4H), 2.97–2.89 (m, 2H), 2.87–2.79 (m, 1H), 1.29–1.21 (m, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 174.1, 147.7 (d, J = 4.6 Hz), 141.4, 128.4, 128.3, 126.0, 118.0 (d, J = 188.7 Hz), 62.3 (d, J = 5.7 Hz), 50.5 (d, J = 25.2 Hz), 34.1, 30.5, 16.2 (d, J = 6.4 Hz). LC–MS (ESI⁺): 342.2 m/z [M + H]⁺, 683.2 m/z [2M + H]⁺.

Diethyl [(1E)-3-(N-Hydroxy-4-phenylbutanamido)prop-1-en-1-yl]phosphonate (10d).

Light yellow oil (73 mg, 51%). ¹H NMR (400 MHz, CDCl₃): δ 9.52 (br s, 1H), 7.09–7.28 (m, 5H), 6.64–6.82 (m, 1H), 5.80–5.89 (m, 1H), 4.39 (br s, 2H), 3.93–4.12 (m, 4H), 2.66 (t, J = 7.2, 8.0 Hz, 2H), 2.51–2.58 (m, 2H), 1.93–2.00 (m, 2H), 1.28 (t, J = 7.2 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 174.8, 147.8 (d, J = 6.1 Hz), 128.3, 125.9, 118.0 (d, J = 189.5 Hz), 162.2, 62.2 (d, J = 5.3 Hz), 50.6 (d, J = 25.1 Hz), 35.2, 31.8, 26.0, 16.2 (d, J = 6.4 Hz). LC–MS (ESI⁺): 356.2 m/z [M + H]⁺, 711.2 m/z [2M + H]⁺.

[({[(2,2-Dimethylpropanoyl)oxy]methoxy}[(1E)-3-(N-hydroxy-1-phenylformamido)-prop-1-en-1-yl]phosphoryl)oxy]-methyl 2,2-dimethylpropanoate (13a).

Light yellow oil (27 mg, 30%). ¹H NMR (400 MHz, CDCl₃): δ 8.69 (s, 1H), 7.57–7.34 (m, 5H), 6.82 (ddt, J = 21.8, 17.2, 4.5 Hz, 1H), 6.04 (ddt, J = 20.6, 17.2, 1.7 Hz, 1H), 5.67–5.60 (m, 4H), 4.45–4.38 (m, 2H), 1.19 (s, 18H). ¹³C NMR (101 MHz, CDCl₃): δ 176.9, 167.7, 146.8 (d, J = 4.9 Hz), 131.3, 128.7, 128.5, 128.0, 118.7 (d, J = 192.4 Hz), 81.5 (d, J = 5.2 Hz), 52.7 (d, J = 24.3 Hz), 38.7, 26.8. LC–MS (ESI⁺): 486.2 m/z [M + H]⁺, 971.2 m/z [2M + H]⁺. HRMS (FAB⁺) calcd for C₂₂H₃₂NO₉P, 485.1815; found, 486.1877 [M + H]⁺

[({[(2,2-Dimethylpropanoyl)oxy]methoxy}[(1E)-3-(N-hydroxy-2-phenylacetamido)prop-1-en-1-yl]phosphoryl)oxy]-methyl 2,2-Dimethylpropanoate (13b).

Light yellow oil (54 mg, 49%).¹H NMR (400 MHz, CDCl₃): δ 8.94 (s, 1H), 7.34–7.17 (m, 5H), 6.79–6.61 (m, 1H), 5.90–5.76 (m, 1H), 5.64–5.54 (m, 4H), 4.38–4.31 (m, 2H), 3.81 (s, 2H), 1.18 (s, 18H). ¹³C NMR (101 MHz, CDCl₃): δ 177.0, 173.0, 148.4 (d, J = 4.6 Hz), 134.9, 129.5, 128.4, 126.7, 117.7 (d, J = 192.3 Hz), 81.5 (d, J = 5.3 Hz), 50.4 (d, J = 26.4 Hz), 39.3, 38.7, 26.8. LC–MS (ESI⁺): 500.2 m/z [M + H]⁺, 999.2 m/z [2M + H]⁺. HRMS (FAB⁺) calcd for C₂₃H₃₄NO₉P, 499.1971; found, 500.2037 [M + H]⁺.

[({[(2,2-Dimethylpropanoyl)oxy]methoxy}[(1E)-3-(N-hydroxy-4-phenylbutanamido)prop-1-en-1-yl]phosphoryl)-oxy]methyl 2,2-Dimethylpropanoate (13d).

Light yellow oil (9 mg, 26%). ¹H NMR (400 MHz, CDCl₃): δ 7.34–7.05 (m, 5H), 6.83–6.66 (m, 1H), 5.93–5.80 (m, 1H), 5.67–5.59 (m, 4H), 4.44–4.29 (m, 2H), 2.69–2.62 (m, 2H), 2.58–2.45 (m, 2H), 2.01–1.90 (m, 2H), 1.19 (s, 18H). ¹³C NMR (101 MHz, CDCl₃): δ 177.2, 148.5 (d, J = 4.5 Hz), 130.8, 128.4, 128.3, 125.9, 118.0 (d, J = 186.3 Hz), 81.5 (d, J = 5.0 Hz), 50.3 (d, J = 24.8 Hz), 38.7, 35.3, 31.7, 29.7, 26.8. LC–MS (ESI⁺): 528.2 m/z [M + H]⁺. HRMS (FAB⁺) calcd for C₂₅H₃₈NO₉P, 527.2284; found, 528.2352 [M + H]⁺.

General Procedure for Synthesis of 11a–d.

To a solution of 10a–d (1 equiv) in dry CH₂Cl₂ (0.1 M) under N₂ was added TMSBr (10 equiv) dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred overnight, concentrated. The mixture was dissolved in CH₂Cl₂, evaporated, and dried under vacuum. The crude was then stirred in 0.5 M NaOH (1 equiv) in H₂O at room temperature for 1 h, washed with Et₂O three times, and lyophilized to give the title compounds.

Diammonium [(1E)-3-(N-Hydroxy-1-phenylformamido)-prop-1-en-1-yl]phosphonate (11a).

Light yellow solids (23 mg, quantitative yield). ¹H NMR (400 MHz, CD₃OD): δ 7.52–7.38 (m, 5H), 6.45–6.31 (m, 1H), 6.13–6.00 (m, 1H), 4.43–4.26 (m, 2H). ¹³C NMR (101 MHz, CD₃OD): δ 170.1, 135.7 (d, J = 4.7 Hz), 134.2, 130.2, 129.0 (d, J = 173.7 Hz), 127.8, 127.7, 52.6 (d, J = 23.6 Hz). LC–MS (ESI⁻): 256 m/z [M-Na]⁻. HRMS (ESI⁻) calcd for C₁₀H₁₁NNaO₅P, 279.0273; found, 256.0377 [M – Na]⁻.

Sodium Hydrogen [(1E)-3-(N-Hydroxy-2-phenylacetamido)prop-1-en-1-yl]phosphonate (11b).

Light yellow solids (42 mg, 96%). ¹H NMR (400 MHz, D₂O): δ 7.46–7.25 (m, 5H), 6.52–6.37 (m, 1H), 5.98–5.80 (m, 1H), 4.44–4.31 (m, 2H), 3.92 (s, 2H). ¹³C NMR (101 MHz, D₂O): δ 174.5, 140.6 (d, J = 5.4 Hz), 134.9, 129.4, 128.9, 127.2, 123.2 (d, J = 179.0 Hz), 51.0 (d, J = 23.6 Hz), 38.8. LC–MS (ESI⁺): 272.0 m/z [M – Na + 2H]⁺, 543.2 m/z [2M – 2Na + 3H]⁺. HRMS (FAB⁺) calcd for C₁₁H₁₃NNaO₅P, 293.0429; found, 294.0508 [M + H]⁺.

Sodium Hydrogen [(1E)-3-(N-hydroxy-3-phenylpropanamido)prop-1-en-1-yl]phosphonate (11c).

Light yellow solids (51 mg, 92%). ¹H NMR (400 MHz, D₂O): δ 7.26–7.09 (m, 5H), 6.29–6.13 (m, 1H), 5.77–5.59 (m, 1H), 4.22–4.11 (m, 2H), 2.85–2.66 (m, 4H). ¹³C NMR (101 MHz, D₂O): δ 175.3, 140.7, 139.3 (d, J = 5.3 Hz), 128.7, 128.3, 126.4, 124.0 (d, J = 177.7 Hz), 50.8 (d, J = 23.6 Hz), 33.1, 30.2. LC–MS (ESI⁺): 571.2 m/z [2M – 2Na + 3H]⁺. HRMS (FAB⁺) calcd for C₁₂H₁₅NNaO₅P, 307.0586; found, 308.0655 [M + H]⁺.

Sodium Hydrogen [(1E)-3-(N-Hydroxy-4-phenylbutanamido)prop-1-en-1-yl]phosphonate (11d).

Light yellow solids (28 mg, 77%). ¹H NMR (400 MHz, CDCl₃): δ 7.12–7.25 (m, 5H), 6.30–6.41 (m, 1H), 5.75–5.85 (m, 1H), 4.22 (br s, 2H), 2.52–2.57 (m, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.76–1.83 (m, 2H). ¹³C NMR (101 MHz, D₂O): δ 176.5, 142.1 (d, J = 5.9 Hz), 133.0, 130.4, 128.8, 126.3, 121.9 (d, J = 180.7 Hz), 50.9 (d, J = 23.5 Hz), 34.6, 31.2, 26.1. LC–MS (ESI⁺): 300.0 m/z [M – Na + 2H]⁺, 599.2 m/z [2M – 2Na + 3H]⁺, 898.2 m/z [3M – 3Na + 4H]⁺. HRMS (FAB⁺) calcd for C₁₃H₁₇NNaO₅P, 321.0742; found, 322.0862 [M + H]⁺.

General Procedure for Synthesis of 12a–b, d.

To a solution of 9a–b, d (1 equiv) in dry CH₂Cl₂ (0.1 M) under N₂ was added TMSBr (10 equiv) dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred overnight, concentrated. The mixture was dissolved in CH₂Cl₂, evaporated, and dried under vacuum. The crude was then stirred in 0.5 M NaOH (2 equiv) in H₂O at room temperature for 1 h, washed with Et₂O (3×), and lyophilized to give disodium salts as white solids. The crude solids were then dissolved in dry DMF (0.1 M), added TEA (6 equiv), chloromethylpivalate (6 equiv), and NaI (0.1 equiv). The reaction mixture was stirred at 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. The crude was then purified by column chromatography on silica gel using Hexanes and EtOAc or CH₂Cl₂ and EtOAc to give the pure title compound.

({[(1E)-3-[N-(Benzyloxy)-1-phenylformamido]prop-1-en-1-yl]({[(2,2-dimethylpropanoyl)oxy]methoxy})phosphoryl}-oxy)methyl 2,2-Dimethylpropanoate (12a).

Light yellow oil (182 mg, 32%). ¹H NMR (400 MHz, CDCl₃): δ 7.74–7.01 (m, 10H), 6.95–6.77 (m, 1H), 6.08–5.93 (m, 1H), 5.75–5.58 (m, 4H), 4.65 (s, 2H), 4.51–4.46 (m, 2H), 1.20 (s, 18H). ¹³C NMR (101 MHz, CDCl₃): δ 176.7, 170.1, 147.2 (d, J = 6.0 Hz), 133.6, 133.5, 130.9, 129.4, 128.9, 128.4, 128.3, 128.0, 118.6 (d, J = 192.6 Hz), 81.5 (d, J = 5.4 Hz), 77.0, 49.5 (d, J = 25.4 Hz), 38.6, 26.7. LC–MS (ESI⁺): 576.2 m/z [M + H]⁺.

({[(1E)-3-[N-(Benzyloxy)-2-phenylacetamido]prop-1-en-1-yl]({[(2,2-dimethylpropanoyl)oxy]methoxy})phosphoryl}-oxy)methyl 2,2-Dimethylpropanoate (12b).

Light yellow oil (145 mg, 35%). ¹H NMR (400 MHz, CDCl₃): δ 7.43–7.15 (m, 10H), 6.80–6.65 (m, 1H), 5.77 (ddt, J = 17.2, 4.7, 1.7 Hz, 1H), 5.68–5.55 (m, 4H), 4.74 (s, 2H), 4.38–4.29 (m, 2H), 3.73 (s, 2H), 1.18 (s, 18H). ¹³C NMR (101 MHz, CDCl₃): δ 176.7, 147.2 (d, J = 5.7 Hz), 137.2, 129.3, 129.2, 129.1, 128.8, 128.5, 128.4, 127.0, 118.7 (d, J = 192.4 Hz), 81.4 (d, J = 5.4 Hz), 48.7 (d, J = 27.8 Hz), 39.5, 38.7, 26.8. LC–MS (ESI⁺): 590.2 m/z [M + H]⁺.

({[(1E)-3-[N-(Benzyloxy)-4-phenylbutanamido]prop-1-en-1-yl]({[(2,2-dimethylpropanoyl)oxy]methoxy})phosphoryl}-oxy)methyl 2,2-Dimethylpropanoate (12d).

Light yellow oil (50 mg, 18%). ¹H NMR (400 MHz, CDCl₃): δ 7.38–7.11 (m, 10H), 6.73 (ddt, J = 22.7, 17.4, 5.2 Hz, 1H), 5.88–5.75 (m, 1H), 5.66–5.59 (m, 4H), 4.70 (s, 2H), 4.34–4.28 (m, 2H), 2.62 (t, J = 7.5 Hz, 2H), 2.40 (t, J = 7.5 Hz, 2H), 1.92 (dt, J = 14.9, 7.6 Hz, 2H), 1.17 (s, 18H). ¹³C NMR (101 MHz, CDCl₃): δ 176.7, 147.5 (d, J = 5.6 Hz), 141.5, 134.1, 129.2, 129.0, 128.7, 128.5, 128.3, 125.9, 118.6 (d, J = 192.6 Hz), 81.5 (d, J = 5.4 Hz), 77.1, 48.7 (d, J = 26.9 Hz), 38.7, 35.2, 31.5, 29.6, 26.8. LC–MS (ESI⁺): 618.2 m/z [M + H]⁺.

P. falciparum DXR Enzyme Inhibition Assay.

As previously described, P. falciparum DXR activity was assayed at 37 °C by spectrophotometrically monitoring the enzyme-catalyzed oxidation of NADPH upon addition of 1-deoxy-d-xylulose 5-phosphate (DXP; Echelon Biosciences, Salt Lake City, UT) to the assay mixture.¹² Briefly, the assay system contained 100 mM Tris pH 7.8, 25 mM MgCl₂, 0.86 μM Pf DXR, and 150 μM NADPH. The reaction was initiated by adding 144 μM DXP to the complete assay mixture. One unit of P. falciparum DXR activity is defined as the amount of enzyme that catalyzes the oxidation of 1 μM NADPH per min. The oxidation of NADPH was monitored at 340 nm using an Agilent 8453 UV–visible spectrophotometer equipped with a temperature-regulated cuvette holder. All assays were performed in duplicate and the derived IC₅₀ value was validated by an independent analyst (by performing the assay at the IC₅₀ value and ensuring residual enzyme activity is within 5% of 1/2 V_max. If it was not, then the IC₅₀ was redetermined, then revalidated).

P. falciparum Culture.

P. falciparum strain 3D7 (wild-type, WT) was obtained through MR4 as part of the BEI Resources Repository, NIAID, NIH (www.mr4.org). A P. falciparum strain containing increased levels of MEP pathway metabolites, had1 (MRA-1257), and its isogenic compliment, had1 + PfHad1-GFP (MRA-1258), were generated in strain 3D7, as reported.¹⁹ Parasites were cultured in a 2% suspension of human erythrocytes and RPMI 1640 (Sigma) medium supplemented with 27 mM sodium bicarbonate, 11 mM glucose, 5 mM HEPES, 1 mM sodium pyruvate, 0.37 mM hypoxanthine, 0.01 mM thymidine, 10 μg/mL gentamicin, and 0.5% Albumax (Gibco) at 37 °C, 5% O₂/5% CO₂/90% N₂ atmosphere, as previously described.^27,28

P. falciparum Growth Inhibition Assays.

Asynchronous P. falciparum cultures were diluted to 1% parasitemia and treated with inhibitors at concentrations ranging from 0.5 ng/mL to 100 μg/mL. Growth inhibition assays were performed in opaque 96-well plates at 100 μL culture volume. After 3 days, parasite growth was quantified by measuring DNA content using PicoGreen (Life Technologies) as described.¹⁸ Fluorescence was measured on a FLUOstar Omega microplate reader (BMG Labtech) at 485 nm excitation and 528 nm emission. Half maximal inhibitory concentration (IC₅₀) values were calculated by nonlinear regression analysis using GraphPad Prism software. For IPP (Echelon) rescue experiments, 250 μM IPP was added to the appropriate wells for the duration of the experiment.

MLM Experimental methods.

In this protocol, the metabolic stability of compounds at 1 μM was determined in MLM. Each test compound was incubated in an aqueous reaction mixture consisting of 0.25 μM microsomal protein CYP450 activity, 1.2 mM NADPH, 3.3 mM MgCl₂, and 100 mM potassium phosphate buffer (pH 7.4). After incubation at 37 °C, a 50 μL aliquot of the reaction was transferred to 200 μL ice-cold acetonitrile containing internal standard (Enalapril, 100 ng/mL). The quenched reaction mixtures were centrifuged at 3200 rpm for 5 min, and 100 μL of the supernatant was transferred to a 96-well plate and analyzed by LC–MS/MS using an Applied Biosystems-Sciex API 4000. Analyte/internal standard peak area ratios were used to evaluate stability. The MRM transitions for enalapril, 3a, 3b, 11a, and 13a were m/z: 376.9 > 91.2, 283.3 > 102.1, 511.2 > 102.1, 257.9 > 104.8, and 486.0 > 626.2, respectively. An Armor C18 column (2.1 Å ~30 mm, 5 μm; Analytical Sales and Services, Pompton Plains, NJ) was used for chromatographic separation. Mobile phases were 0.1% formic acid, 1 mM triethylamine in water, and acetonitrile with a flow rate of 0.35 mL/min. The starting phase was 0% acetonitrile and increased to 100% acetonitrile over 3 min. Peak areas were integrated using Analyst Software (AB Sciex, Foster City, CA). Determination of in vitro half-life assumed first-order kinetics, where half-life is equal to −0.693/k, and −k is the slope of the linear regression of log percentage remaining versus incubation time.²⁹

HepG2 Method.

For cytotoxicity assays, HepG2 cells (ATCC HB-8065) were grown in DMEM/4 mM l-glutamine/4.5 g/L d-glucose (Gibco #11965–092) supplemented with 1 mM sodium pyruvate and 10% fetal bovine serum w or in DMEM/4 mM l-glutamine (Gibco #11966–025) supplemented with 1 mM sodium pyruvate, an additional 2 mM l-glutamine, 10% fetal bovine serum, and 10 mM d-galactose. Cells were trypsinized, resuspended in the respective medium (glucose-based DMEM or galactose-based DMEM) to 4 × 10⁵ cells/mL, and 50 μL/well transferred to flat-bottom white opaque tissue culture plates (Falcon #353296) containing 50 μL/well of the respective medium with test compound. Compound concentrations were twofold dilutions ranging from 100 to 0.10 μg/mL as well as the drug-free DMSO-only control. Positive control compounds were tamoxifen and antimycin that were tested from 250 to 0.24 μM. All concentrations were tested in duplicate for each carbon source. After 24 h incubation at 5% CO₂, 37 °C, 10 μL/well of Celltiter-Glo reagent (Promega #G9241) was added and luminescence recorded with a PerkinElmer Envision plate reader after 20 min incubation in the dark.