Synthesis of plasmodione metabolites and 13C-enriched plasmodione as chemical tools for drug metabolism investigation

Liwen Feng a, Don Antoine Lanfranchi a, Leandro Cotos a, Elena Cesar-Rodo a, Katharina Ehrhardt b, Alice-Anne Goetz b, Herbert Zimmermann c, François Fenaille d, Stephanie A. Blandin *b and Elisabeth Davioud-Charvet *a
aCNRS-Université de Strasbourg-Université de Haute-Alsace UMR 7042, Laboratoire d'Innovation Moléculaire et Applications (LIMA), Team Bioorganic and Medicinal Chemistry, ECPM 25 Rue Becquerel, 67087 Strasbourg, France. E-mail: elisabeth.davioud@unistra.fr
bUniversité de Strasbourg, CNRS, Inserm, UPR9022/U1257, Mosquito Immune Responses (MIR), F-67000 Strasbourg, France. E-mail: sblandin@unistra.fr
cMax Planck Institut für medizinische Forschung, Department of Biomolecular Mechanisms, Jahnstraße 29, D-69120 Heidelberg, Germany
dCEA Paris-Saclay, Laboratoire d'Etudes du Métabolisme des Médicaments (LEMM), Bât. 136, 91191 Gif-Sur-Yvette cedex, France

Received 26th January 2018 , Accepted 6th March 2018

First published on 6th March 2018


Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas. Resistance to antimalarial drugs has spread all over the world in the past 50 years, thus new drugs are urgently needed. Plasmodione (benzylmenadione series) has been identified as a potent antimalarial early lead drug, acting through a redox bioactivation on asexual and young sexual blood stages. To investigate its metabolism, a series of plasmodione-based tools, including a fully 13C-labelled lead drug and putative metabolites, have been designed and synthesized for drug metabolism investigation. Furthermore, with the help of UHPLC-MS/MS, two of the drug metabolites have been identified from urine of drug-treated mice.


Introduction

Malaria is a parasitic disease, threatening 3.2 billion people living in tropical and sub-tropical regions.1 According to the world health organization (WHO), 91 countries reported a total of 216 million cases of malaria, representing an increase of 5 million cases over the previous year and causing 445[thin space (1/6-em)]000 deaths in 2016, with 70% of malaria decedents being young children.1Plasmodium is a protozoan parasite that infects humans via the infectious bite of a female anopheles mosquito. Five plasmodium species are responsible for malaria in humans.2–4Plasmodium falciparum is the most dangerous parasite species causing severe disease such as cerebral malaria, hemolytic anemia and respiratory distress, and is responsible for 99% of fatal cases.1,5,6 The naturally occurring artemisinin with its peroxo bridge, identified in the 1970s (Youyou Tu, et al., Nobel Prize 2015 in Physiology or Medicine), acts with a specific mode of action via reactive oxygen species (ROS) and ferroptosis induction in malaria parasites. Combined with several drug partners artemisinin has been extremely useful in the treatment of multi-drug resistant infections, and artemisinin-based combination therapies (ACT) are currently recommended by the WHO as the first-line therapy to treat and cure malaria.1,7–9 Despite a spectacular drop in malaria-related morbidity observed in 2015 due to the massive use of insecticide-treated bednets and artemisinin-based combination therapies (ACT), there is a serious decrease in the efficacy of ACT treatment in South-East Asia due to the emergence of drug-resistant parasites since 2008, which threatens the world's malaria control and elimination efforts. Also, the intensity of climate change has an impact on the geographical distribution and proliferation of the vector, Anopheles mosquitoes. Given the lack of an effective vaccine, preventive measures to avoid mosquito bites and drug treatment of symptomatic cases remain crucial but are challenged by the emergence and spread of drug resistance in parasites and insecticide resistance in mosquitoes, urging for a global effort to develop novel antimalarial drugs and insecticides.

The early lead plasmodione, with its benzylmenadione core, has been identified as a potent and fast-acting antimalarial agent with pronounced activity against early sexual and asexual blood-stage parasites.10–13 This compound displays low and equally stable IC50 values (≈50 nM) whatever the degree of resistance of the parasite strains to chloroquine or quinine, synergy with dihydroartemisinin, and low toxicity to various human cell lines in vitro (IC50 > 50 μM) or in vivo in the murine model. Plasmodione was selected for further in vitro lead evaluation and characterisation. Recent investigations on the mode of action revealed that plasmodione is redox-active and enters a continuous redox-cycle reducing methemoglobin(FeIII) (metHb) and oxidizing the stock of NAD(P)H upon its reduction by a flavoenzyme, ultimately disturbing the redox homeostasis in parasitized red blood cells (pRBC). A direct proof of the disturbance of the parasite's redox balance in situ has been produced by imaging the glutathione redox potential in the cytosol of intact living pRBCs. This method used the recently developed genetically encoded real-time fluorescent biosensor (genetically encoded human glutaredoxin 1 fused to a redox-sensitive GFP [hGrx1-roGFP2]) and showed a relevant oxidation of the glutathione pool in pRBCs upon treatment with plasmodione.14 All biological studies suggested that the antimalarial selectivity of plasmodione comes largely from its specific bioactivation within pRBCs. A bioactivation pathway was proposed to start from a benzylic oxidation generating the benzoylmenadione metabolite (Scheme 1).10,13 Then, in the oxidized state, the benzoylmenadione can be reduced by both glutathione reductases (GRs) of the parasite and its host cell thus consuming NADPH into a redox cycle with metHb as an electron acceptor. Plasmodione redox cycling leads to the inhibition of glutathione regeneration, production of ROS and regeneration of hemoglobin(FeII) (Hb) that cannot be digested by the parasite.13,15 This NADPH-dependent redox cycle ends with the death of the parasites that appears pycnotic,12 and the enrichment of membrane-associated hemichrome,14 a biomarker of senescence of RBC responsible for their rapid removal by macrophagic phagocytosis in vivo.


image file: c8ob00227d-s1.tif
Scheme 1 Proposed plasmodione metabolism pathway in parasitized red blood cells.

Previous studies have proposed that additional metabolites could contribute to drug bioactivation and parasite killing. As an example, the benzoxanthone (metabolite III in Scheme 1) prevents heme detoxification as potently as chloroquine,14 and was proposed to be formed after the reduction of the benzoylmenadione metabolite via an oxidative C,O-coupling reaction in pRBC. However, the hypothesized bioactivation pathway is difficult to be proven in parasites because the lead plasmodione is quickly metabolized within cells and very limited structural information on drug metabolites was recorded. Furthermore, linking observations from models in solutions to observations in situ in parasites is one of the hardest steps in confirming the mode of action of any drug, especially of any antimalarial agent. The particular difficulties are associated with: (i) the complexity of the malaria parasite that oscillates between different stages of RBCs; (ii) the redox chemistry that involves very rapid reactions due to electron transfers,16,17 in a complex biological milieu (containing metals, sugars, proteins that can catalyze/modify the rate of these redox reactions at undefined rates). Consequently, an integrated perspective to unravel the drug mode of action in complex biochemical systems is clearly required. To make progress in understanding the mechanisms of antimalarial activity of plasmodione, it will be essential to decipher the drug interaction networks connecting the drug and/or drug metabolites to all the components of the biological systems (parasite, host cell) in the future. Toward this end, we designed and synthesized novel chemical tools, including 13C-enriched plasmodiones and additional unlabeled plasmodione metabolites, for future drug metabolism investigations. We further report the identification of drug metabolites in the urine of plasmodione-treated mice.

Results and discussion

The original plasmodione can be synthesized in one step from commercial starting materials, menadione and the corresponding p-trifluoromethylphenyl acetic acid (Scheme 2A), rendering the large-scale preparation easy, cheap and available for industrial production. However, because of the limited choice and high cost of commercially available 13C-enriched starting materials, the synthetic route of the fully 13C18-labeled plasmodione had to be built totally differently, with almost all C-enriched, except the –CF3 group. Based on the cheapest commercial available starting materials, 13C6-p-dibromobenzene, 13C6-benzene, 13C4-succinic anhydride, 13C1-dimethylformamide and 13C1-acetic acid, the synthesis of the almost fully 13C18-enriched plasmodione was designed through a convergent synthetic route via two key intermediates, tetralone and 4-trifluoromethyl-benzaldehyde, using a 10 step-long sequence (Scheme 2B). Each step of this total synthesis was set up and improved by using first, the unenriched compounds.
image file: c8ob00227d-s2.tif
Scheme 2 (A) The synthesis of unlabelled plasmodione. (B) The retrosynthesis of fully 13C-labelled plasmodione.

Synthesis of the 13C10-enriched tetralone 3b

The uniformly 13C10-labeled tetralone 3b was synthesized according to a reported procedure.18 The 13C10-tetralone 3b was produced by the Friedel–Crafts reaction (yield 96%), the Woff–Kishner reduction (yield 95%) and cycloaddition reaction (yield 95%) from 13C4-succinic acid and 13C6-benzene with excellent yields (total yield 87%, Scheme 3). All 1H and 13C NMR spectra of the tetralone precursors 1–2 are shown in Fig. S1-S6, ESI.
image file: c8ob00227d-s3.tif
Scheme 3 The synthesis of tetralone 3a/3b.

As such, 13C-enriched compounds represent an important instrumental tool in drug metabolism studies; they have very different profiles both in 1H NMR and 13C NMR spectra, especially in the case of multi-13C-enriched compounds. Because the 13C atom spin is ½, they can generate J-coupling with each other and with other atoms such as 1H and 19F. Normally, because of the low isotopic abundance of the 13C atom, isotopically-unlabeled products do not reveal any significant signal of J-coupling between 13C and 1H in the NMR spectra. However, when the fully 13C-enriched compounds are characterized by NMR spectra, this influence cannot be ignored.

As expected, the peaks of the 13C-enriched tetralone 3b in the 1H NMR spectrum were conspicuously different from those of the natural tetralone 3a (Fig. S5, ESI). Because of 1J13C–1H, the spin of both 13C and 1H atoms is ½ and the peaks of the protons that are bound to the 13C atom are split into two signals. Besides this, owing to additional J-coupling such as 3J or 4J between the proton and 13C, the shape of the peaks is wide and the signals have low sensitivity, making it difficult to identify the structure of 13C-enriched compounds by NMR spectra. Nevertheless, 13C-enriched and unenriched compounds with the same molecular structure have the same chemical properties, and thus display invariable chemical shifts in the NMR spectra. Thus, we can confirm the structure of 13C-enriched compounds by comparing the chemical shifts in the 1H NMR spectra profile of both labeled and unlabeled compounds.

Similarly to the 1H NMR spectrum, the 13C NMR of 13C10-tetralone 3b has also more complexity (Fig. S6, ESI). Because the fully 13C-enriched compounds have numerous adjacent and magnetically nonequivalent 13C atoms, the signal of each 13C is multiplied in several peaks. Still, as for the 1H NMR spectrum, the chemical shifts of both fully 13C-enriched and unenriched compounds are unchanged, and comparing both 13C NMR spectra can also profile the molecular structure of the fully 13C-enriched product. Because the synthesis of 13C10-tetralone 3b has been reported by Ball et al.,18 both 1H and 13C NMR spectra of the newly prepared 13C10-tetralone 3b were compared to the published data and analyzed carefully. Our detailed analyses confirmed the purity of 13C10-tetralone 3b.

Synthesis of the 13C-enriched 4-trifluoromethyl benzaldehyde 6b

The 13C6-p-trifluoromethylbenzaldehyde 6b was synthesized in 3 steps (total yield 23%, Scheme 4). First, the 13C6-p-bromoiodobenzene 4b was prepared according to an iodination reaction with I2 and n-BuLi under classical conditions (yield 75%).19 The limitation of the yield was due to the generation of o-bromoiodobenzene, a side product due to the resonance effect. Even upon varying the reaction temperature (from −78 °C to 25 °C), the o-bromoiodobenzene was produced with an ∼20% conversion of the starting material.
image file: c8ob00227d-s4.tif
Scheme 4 Synthesis of 4-trifluoromethyl benzaldehyde 6a/6b. aThe yield of the trifluoromethylation reaction was estimated by 1H NMR using trifluoromethoxytoluene as an internal standard. bYields of the 2 step-reactions using p-bromoiodobenzene 4a/4b as starting materials.

The trifluoromethyl-substitution is extensively explored in drug development due to the fluorine properties that increase the bio-solubility, bio-availability and metabolic resistance of the drug.20 In chemical processes, the [CuCF3] is usually used in trifluoromethylation as a catalyst.21 However, it is not stable in open air and thus not adapted to perform trifluoromethylation without a glove box.22 Generation of CuCF3in situ has been developed by using different generators such as NaCO2CF3 and MeCO2CF3 that need high temperature (∼150–160 °C) for their activation.23 However, as the desired trifluoromethylbenzene 5a/5b is volatile at these temperatures (b.p. 160 °C), we could not use these generators. Instead, we opted for trimethyl-(trifluoromethyl)silane (TMSCF3) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (MDFA). Indeed, TMSCF3 has been broadly used in trifluoromethylation with fluoride compounds under gentle conditions to form CF3.24,25N-Methyl-2-pyrrolidone (NMP) was chosen as a solvent for the reaction because of its b.p. 202 °C.

MDFA was originally studied first by Chen et al.26,27 First, the methyl group of MDFA is substituted by CuI. Subsequently, decarboxylation and desulfonylation take place to produce a carbene :CF2 by heating (∼80–100 °C), and then the :CF2 carbene reacts with F anions to form CF3 in equilibrium and coordinate with copper to form CuCF3in situ. With TMSCF3, the trifluoromethylation proceeded with similar yields when compared to MDFA (Table 1).

Table 1 Conditions for the trifluoromethylation reactions. NMP = N-methyl-2-pyrrolidone

image file: c8ob00227d-u1.tif

Conditions Yielda
a The yields were estimated by 1H NMR using trifluoromethoxytoluene as the internal standard.
CuI (1.5 equiv.), MDFA (5 equiv.), NMP, 16 h, 100 °C 95%
CuI (2 equiv.), TMSCF3 (2 equiv.), NMP, 16 h, 100 °C 90%


Due to the volatility of the desired product, the crude product was very hard to purify on a small quantity scale (less than 2 mL) by distillation. Thus, the resulting crude product had to be used in the next reaction without purification. In this case, it was necessary to ensure that all the starting material (p-bromoiodobenzene 4a/4b) was consumed. The trifluoromethylation was also found to be very sensitive to the quality of CuI (stored under argon).

In the next step, the hydroformylation with the Grignard reagent produced the p-trifluoromethylbenzaldehyde 6a (50%)/6b (30%) in two steps (overall yield). This methodology for benzaldehyde formation at a relatively high temperature (0 °C to 5 °C) was developed by Gallou et al.28 By using iPrMgBr, the bromoaryl group was transformed to the [phen]3-Mg Grignard reagent in situ, a reaction that was accelerated using n-BuLi. Subsequently, the resulting Grignard reagent underwent nucleophilic attack on DMF or 13C1-DMF to form desired benzaldehyde products. Because of the high water sensitivity of this reaction and the volatility of the starting material, the use of Na2SO4in situ was required to remove the maximum of water. The 1H and 13C NMR spectra of the synthetic 4-trifluoromethylbenzaldehyde precursors 4a/4b are shown in Fig. S7 and S8, ESI.

Based on the 1H and 13C NMR, we could verify the structure of 13C7-p-trifluoromethylbenzaldehyde 6b. In the 1H NMR spectrum of 6b, the peak of the proton at the aldehyde position at 10.1 ppm was split into a doublet–doublet signal with a 1J13C–1H constant of 177.0 Hz (Fig. S9, ESI), and the peaks of the protons in the phenyl ring were multiplied to doublet–triplet and doublet–quadruplet peaks at 8.01 ppm and 7.81 ppm, with 1J13C–1H constant 163.2 Hz and 163.8 Hz, respectively, in the 13C NMR spectrum of 6b, the peak of carbon at the aldehyde position at 191.2 ppm was divided into doublet–multiplet (Fig. S10, ESI). By comparing both 1H and 13C NMR spectra, we found that all the peaks of 13C7-p-trifluoromethylbenzaldehyde 6b retained the same chemical shifts as in the spectra of unlabeled trifluoromethylbenzaldehyde 6a. These 1H and 13C NMR data, along with the 19F NMR spectrum (Fig. S11, ESI), unambiguously assigned the molecular structure of the expected fully 13C-enriched key-intermediate 6b.

Synthesis of the 13C1-enriched plasmodione 10a and 13C18-enriched plasmodione 10b through the “tetralone express route”

The two resulting intermediates 13C10-tetralone 3b and 13C7-p-trifluoromethylbenzadehyde 6b were combined through an optimized “express tetralone route” to produce 13C18-plasmodione 10b in 4 steps as described in our previous study (Scheme 5).29 First, a condensation reaction from previously described p-trifluoromethylbenzaldehyde 6a/6b and tetralone 3a/3b under classical conditions (KOH as a base) led to the tetralone with an exo double bond 7a/7b with 90% and 75% yield, respectively. Then, the isomerization reaction with RhCl3 catalysis proceeded with similar yields (80%) to obtain the α-benzyl naphthols 8a/8b. The naphthols were subjected to an oxidation reaction with phenyliodonium diacetate (PIDA), generating the benzyl-1,4-napthoquinones 9a/9b with 90% and 75% yield, respectively. Finally, the Kochi–Anderson alkylation reaction was applied in the presence of 13C1-acetic acid with AgNO3/(NH4)2S2O8 to produce the 13C1-enriched plasmodione 10a and the fully 13C18-enriched lead plasmodione 10b with 50 and 55% yield, respectively.
image file: c8ob00227d-s5.tif
Scheme 5 The synthesis of 13C-enriched plasmodione 10a/10b from tetralone 3a/3b and p-trifluoromethylbenzaldehyde 6a/6b.

According to previous studies about radical methylation based on the Kochi–Anderson reaction, the methyl radical is less stable and more reactive, and might be the cause of the destruction of the desired product.29 In order to follow the reaction kinetics and to track the reaction products by 1H NMR spectroscopy (Fig. 1), we used unlabeled 2-(4-(trifluoromethyl)benzyl)-naphthalene-1,4-dione 9a and 13C1-acetic acid as starting materials in a model reaction. As observed, after 15 min stirring, the reaction conversion reached almost 50% and the desired product 10a was quickly generated. After 60 min, over 70% of the starting material 9a was consumed. The best conversion was obtained when the reaction was stopped at 75 min. Interestingly, for comparison, the reaction with the unlabeled acetic acid as the reaction partner took a shorter time (60 min) at the same stage of the reaction. After 90 min, the generation of side products was observed and the yield of the reaction decreased. The preparation of the fully 13C18-enriched-plasmodione 10b from 9b using the optimized process and 2 purification steps (chromatography followed by precipitation) led to a 55% yield. All 1H, 13C and 19F NMR spectra of the synthetic 13C-enriched plasmodione precursors 7–9 are shown in Fig. S12–S20, ESI.


image file: c8ob00227d-f1.tif
Fig. 1 Kinetics of the Kochi–Anderson reaction between 9a and 2-13C1-acetic acid studied by 1H NMR spectroscopy. The blue arrows indicate the peaks of the proton in the C2 position (blue box) and of the aromatic protons, respectively, which disappeared in 9a; the red arrows, the peaks of the aromatic protons, which appeared in 10a. The 1H NMR spectra were acquired in a mixture of CD3CN/D2O.

Based on the 1H and 13C NMR spectra, we confirmed the structure of 13C18-plasmodione 10b. In the 1H NMR spectra of 10b, the peaks of the proton at the naphthoquinone part were split into two signals, at 8.10 ppm and 7.71 ppm with 1J13C–1H constants 159.8 Hz and 136.0 Hz (Fig. S21, ESI). The peaks of the proton in the phenyl group were split at 7.52 ppm and 7.32 ppm with 1J13C–1H constants of 159.1 Hz and 147.5 Hz. The two protons at the benzyl chain were revealed as a doublet peak at 4.08 ppm with a 1J13C–1H constant of 130.3 Hz. The protons of the methyl group showed signals at 2.25 ppm with a 1J13C–1H constant of 128.9 Hz. In both 1H NMR and 13C NMR spectra, all the peaks of 13C18-plasmodione 10b can be recovered at the same chemical shifts as the unlabeled plasmodione and mono-labeled 10a (Fig. S22, ESI). It is noteworthy that the peak of the carbon of the CF3 group was covered by the intense signals of the other 13C-enriched carbons. The 19F NMR spectra of the synthetic 13C-enriched plasmodione 10 are shown in Fig. S23, ESI. All these NMR data corroborated the exact assignment to the molecular structure of the desired 13C-enriched plasmodiones 10b and 10a. Finally, the high resolution ESI spectrum with positive ionization recorded for the 13C18-enriched plasmodione 10b confirmed that the molecular mass peak of plasmodione increased by 18 m/z units and a >90% 13C18-enrichment was observed (Fig. S24, ESI). Overall, 50 mg of fully 13C-enriched-plasmodione 10b was obtained though the 10 step-long synthesis.

Synthesis of the 1-13C1-3-[(4-trifluoromethyl)benzyl]-menadione

In order to spare the expensive plasmodione 10b, we also prepared a one 13C1-enriched plasmodione 10c for preliminary metabolism analyses and for the optimization of the experimental conditions. The synthesis was performed in one step using the Kochi–Anderson reaction, starting from p-trifluoromethylphenylacetic acid and 50 mg of 13C1-menadione.30,31 (Scheme 6).
image file: c8ob00227d-s6.tif
Scheme 6 The synthesis of 1-13C1-plasmodione.

The identification and quantification of low-level impurities that are generated during the drug production processes gain more and more importance in medicinal chemistry research and development.32 Interestingly, during the synthesis of the mono-13C1-labeled compound, we discovered one of the trace amount nonchromophoric impurities that was undetectable in the NMR spectra.

The presence of an impurity containing a 1-13C1-enriched was revealed through an intense signal at 191.72 ppm in the 13C NMR spectrum. This signal did not belong to plasmodione 10c, and corresponded to an impurity generated in the Kochi–Anderson reaction. Using DOSY-NMR, we showed that the amount of this impurity was less than 4% (detection limit). By comparing both the 1H and 13C NMR spectra of potential candidate compounds, we confirmed that the structure of this impurity was the epoxide derivative 11 (Fig. S25 and Fig. S26, ESI). We verified that a trace amount of epoxide 11 did not change the antimalarial activity of plasmodiones, as shown in the next paragraph (Table 2).

Table 2 Antimalarial activities (presented as IC50 values) of plasmodione and 13C-enriched plasmodiones (10a–c) against murine P. berghei pRBCs determined in ex vivo cultures (two individual experiments each)

image file: c8ob00227d-u2.tif

Compound Synthetic route IC50 ± SD (nM)
(n steps) P. berghei
Plasmodione Kochi–Anderson (1) 181.4 ± 14.7
(13C1-methyl-plasmodione 10a) “Tetralone express” (4) 180.6 ± 5.2
(13C18-plasmodione 10b) “Tetralone express” (10) 186.1 ± 2.6
(13CC1-plasmodione 10c) Kochi–Anderson (1) 181.3 ± 3.2


Based on the mechanism of the Kochi–Anderson reaction, we presumed that the radical at the benzyl group generated in situ could transfer one electron to H2O and produce H2O2. This behaviour, which is similar to cytochrome P450 (CYP450) metabolism/oxidation, helped us to understand that the C2–C3 position of the menadione core is the most susceptible position to ROS, and that the epoxide derivative 11 should be considered as one of the putative plasmodione metabolites. Furthermore, the Ag content in several batches of plasmodione was determined as <1 mg kg−1 by inductively coupled plasma mass spectrometry (ICP-MS). Therefore, the plasmodione used in the biological study did not contain any residual amount of silver salts (coming from the Kochi–Anderson reaction) that could poison the drug even at a trace level.

Antimalarial activities of 13C-enriched plasmodiones

In order to evaluate the impact of 13C atom enrichment on the antimalarial activity of plasmodione, we compared the 50% inhibitory concentrations (IC50) of the different 13C-enriched (10a, 10b and 10c) and non-13C-enriched plasmodiones using ex vivo cultures of the rodent malaria parasite P. berghei (Table 2). All IC50 values were around 180 nM indicating that 13C-enrichment did not affect the antimalarial activity of plasmodiones.

Synthesis of several putative plasmodione metabolites

In order to study the metabolism and the mode of action of plasmodione, we further synthesized a series of putative plasmodione metabolites designed from the hypothesized plasmodione metabolism pathway (Scheme 1) as pure references for future investigations on drug metabolism and drug targets (Fig. 2). Although there are no CYP450 enzymes in the pRBC cultures in vitro, the Plasmodium parasites digest large amounts of metHb and release free heme iron III (FPIX FeIII), that can both catalyze oxidation reactions and metabolize the lead drug.33 These drug metabolites are supposed to perturb the redox homeostasis of the pRBC leading to the death of parasites. According to the mechanism of oxidation reactions catalyzed by CYP450 enzymes,34 the parent drug could be oxidized at different loci in the benzylmenadione core, i.e. the naphthoquinone ring, the methyl group or at the benzyl chain of plasmodione, generating the corresponding hydroxyl- 16a/16b, epoxyl- 11, benzhydrol 14, or benzoyl 12 derivatives. The synthesis of the putative drug metabolites 3-benzoylmenadione 12,10 benzhydrol 13,10 benzoxanthone 15,13 6-hydroxy 16a29 and 7-hydroxy 16b29 plasmodione derivatives had been reported in our previous publications. The synthesis of other putative drug metabolites 11, 14, 15 (via a new route) and 17 is described in the present work, along with the antimalarial activities of all synthetic metabolites.
image file: c8ob00227d-f2.tif
Fig. 2 Structures of plasmodione and its putative metabolites.

Synthesis of the epoxyl-plasmodione derivative 11

The epoxyl-plasmodione derivative 11 has been formed by an epoxidation reaction of plasmodione (Scheme 7). A similar reaction has been reported in the literature.35 In the mixture of H2O/MeOH (4/1), initial plasmodione was oxidized by hydrogen peroxide in the presence of NaOH at 0 °C leading to the formation of product 11 with 59% yield. 1H, 13C and 19F NMR spectra of the epoxyl-plasmodione derivative 11 are shown in Fig. S27–S29, ESI.
image file: c8ob00227d-s7.tif
Scheme 7 Synthesis of 1a-methyl-7a-(4-(trifluoromethyl)benzyl)-1a,7a-dihydronaphtho[2,3-b]oxirene-2,7-dione 11.

Synthesis of benzhydrol-plasmodione derivatives 14

Similar to the benzhydrol derivative 13 (R = –Br), the benzhydrol derivative 14 (R = –CF3) was obtained through the same synthetic pathway (Scheme 8).10 Its synthesis successively encompassed the lithiation of the 2-methyl-3-bromo-1,4-dimethoxy-naphthalene 18 and the addition of p-bromobenzaldehyde to give the benzhydrol intermediate 19. Oxidation with cerium ammonium nitrate (CAN) led to the benzhydrol 14. 1H, 13C and 19F NMR spectra of the benzhydrol derivative 14 are shown in Fig. S30–S32, ESI.
image file: c8ob00227d-s8.tif
Scheme 8 Synthesis of (±)-3-[4-trifluoromethyl-(phenyl)-hydroxy-methyl]menadione 14.

Synthesis of benzoxanthone 15

The first synthesis of benzoxanthone 15 was established via a benzophenone intermediate (dihydrobenzoylmenadione) through an SNAr reaction.13 However, the overall yield (≈10.8% from menadione, 6 steps) of the reported synthetic pathway was not satisfactory. We have therefore developed an optimized synthetic route (Scheme 9). Hence, bromomenadione 20 was first prepared by the bromination of menadione with bromine and pyridine with 93% yield. Subsequently, the quinone core of compound 20 was reduced by tin chloride/HCl as a reducing reagent, and then protected by a MOM-protection reaction with methoxymethyl chloride (MOM chloride) in the presence of a mild base, N,N-diisopropylethylamine (DIPEA) in dichloromethane at room temperature. During the work-up it was essential to isolate and dry the 2-bromo-3-methyl-dihydronapthoquinone under argon and with distillated toluene because this intermediate is highly sensitive to oxidation in open air. Then, the bromo-lithium exchange reaction was performed with naphthol 21 and n-BuLi, leading to the carbanion acting as a nucleophilic reagent in the next step. Then, the resulting lithium intermediate was allowed to react with 2-fluoro-4-(trifluoromethyl)benzoyl chloride affording the benzoylmenadione derivative 22. A selective mono-deprotection reaction removed one of the MOM groups of compound 22 producing compound 23. Noteworthy is that only the O-MOM group in the β-position to the C[double bond, length as m-dash]O of the benzoyl chain can be deprotected by MgBr2 because of the chelating effect.36 Of note, this deprotection of β-ketophenols had been successfully developed in flavone chemistry in the team. The aromatic nucleophilic substitution/cycloaddition of the benzoylmenadione derivative 23 with K2CO3 in acetone produced the O-MOM-benzoxanthone derivative 24 (35%) in combination with partially deprotected benzoxanthone 15 (35%) because the O-MOM group could be deprotected by HF generated during the reaction. Finally, O-MOM-benzoxanthone 24 was deprotected to give benzoxanthone 15 in a mixture of isopropanol/dichloromethane under acidic conditions with 95% yield. The overall yield of this synthetic route was 39.2% (from menadione, 6 steps) that is 3-fold higher than the former synthetic strategy. The 1H NMR spectra of compounds 22–24 and 15, the 13C NMR spectrum of compound 24, and the 19F NMR spectra of compounds 24 and 15 are shown in Fig. S33–S40, ESI.
image file: c8ob00227d-s9.tif
Scheme 9 Optimized synthesis of the benzoxanthone derivative 15.

Synthesis of the 2-hydroxymethyl-plasmodione derivative 17

As in the classical Kochi–Anderson reaction, the key intermediate hydroxymethyl radical (˙CH2[double bond, length as m-dash]O) was generated from MeOH in the presence of AgNO3 and (NH4)2S2O8.37 The reaction between the hydroxymethyl radical and the desmethyl-plasmodione 9a generated the hydroxymethylplasmodione 17 (Scheme 10). The product was purified by column chromatography (SiO2, toluene) and isolated with 49% yield. The 1H, 13C and 19F NMR spectra of the 2-hydroxymethyl-plasmodione derivative 17 are shown in Fig. S41–S43, ESI.
image file: c8ob00227d-s10.tif
Scheme 10 Synthesis of the 2-hydroxymethyl-plasmodione derivative 17.

Antimalarial activity of putative drug metabolites

We determined the antimalarial activity of each synthetic drug metabolite 11–17 against human P. falciparum pRBCs in in vitro cultures (Table 3). Some of the IC50 values were reported in our previous publications.10,14,29 Importantly, all compounds, except the benzoyl derivative 12, had IC50s below 1 μM. In particular, the 6-hydroxyl-plasmodione 16a and 2-hydroxymethyl-plasmodione 17 had low IC50 values around 100 nM, suggesting that the parent drug might have several effective drug metabolites. Still, the antimalarial activity of other drug metabolites was lower than that of the parent drug. For instance, the IC50 values of the benzhydrols 13 and 14 (∼735 nM) and benzoxanthone 15 (613 ± 79.0 nM) were more than 10 times higher than the IC50 value of the parent plasmodione. The low activity of these compounds does not necessarily rule out the fact that they can contribute to the antimalarial activity of the drug in infected cells. Indeed, they might have (1) a reduced extracellular stability compared to the parent drug (as shown for the benzoxanthone 1513), (2) a higher affinity to cell membranes or tissues, and/or (3) a lower ability to penetrate in pRBC. Thus, the drug metabolism investigations using 13C-enriched plasmodione and unenriched metabolites in pure form will be essential for the in-depth studies of plasmodione metabolism and mode of action through drug[thin space (1/6-em)]:[thin space (1/6-em)]target or drug metabolite[thin space (1/6-em)]:[thin space (1/6-em)]target interactions.
Table 3 Antimalarial activities (presented as IC50 values) of plasmodione and its putative drug metabolites against human P. falciparum pRBCs of the multi-drug resistant Dd2 strain determined in in vitro cultures using fluorescent SYBR® green stain to measure parasite survival except otherwise indicated
Compound Mean IC50 ± SD [nM] (n)a Compound Mean IC50 ± SD [nM] (n)a
a IC50 chloroquine = 99 ± 19 (n = 6), with n giving the number of repeats. b Data from ref. 10 were obtained using radioactive [3H]hypoxanthine to measure parasite survival. c The sensitive P. falciparum 3D7 strain was tested instead of the multidrug resistant P. falciparum Dd2 strain. d Data from ref. 13 were obtained using radioactive [3H]hypoxanthine to measure parasite survival.
image file: c8ob00227d-u3.tif 58 ± 11 (9) image file: c8ob00227d-u4.tif 613 ± 79.0 (2)d
image file: c8ob00227d-u5.tif 241 ± 131 (2) image file: c8ob00227d-u6.tif 107 ± 14 (3)
image file: c8ob00227d-u7.tif >1000 (3)b image file: c8ob00227d-u8.tif 273 ± 31 (3)
image file: c8ob00227d-u9.tif 13: 734b image file: c8ob00227d-u10.tif 102 ± 43 (3)
14: 737 (2)c


Identification of drug metabolites in the urine of plasmodione-treated mice

A preliminary investigation on drug metabolism was performed on urine collected from drug-treated mice using UHPLC-MS/MS. This analysis aimed at detecting plasmodione and its metabolites stemming from the reduction and/or CYP450-catalyzed oxidation reactions occurring in the liver. Such drug metabolites are possibly involved in the mode of action of the antimalarial plasmodione or can conversely accelerate the elimination of the drug from the circulation. Interestingly, the parent drug plasmodione was not detected in any of the urine samples collected at 24 h after 1, 2 or 3 daily injection(s) of the compound, but instead two peaks corresponding to a hydroxylated metabolite and its glucuronide were observed. Using negative electrospray ionization mass spectrometry (ESI-MS) under optimal conditions for glucuronide detection, a glucuronic acid-conjugated drug metabolite was detected at m/z = 522.11 (see Fig. S44 and S45, ESI). Based on mass spectrometry analysis of the glucuronide metabolite found in urine, a fragment ion with m/z 345.1 was detected, corroborating with the possible m/z fragment of a hydroxy-plasmodione. Two MRM transitions were used to study the fragmentation of each observed metabolite, the hydroxy-plasmodione at m/z = 344 → 317, 329, and the glucuronide at m/z 522 → 345, 317, 329. Considering the fragmentation pattern of all putative synthetic drug metabolites and using Metasite software,38 we predicted that the 6- and 7- hydroxy-plasmodiones (16a/16b) were the most likely metabolites of plasmodione and the precursors of the observed glucuronide.

The chemical properties of the two putative hydroxyl metabolites 16a/16b are very similar and they have the same retention time on the chromatography column (0.86 min). However, their fragmentation pattern is slightly different. The principal fragments of both hydroxy-plasmodiones were m/z 317.1 and m/z 329.0, but according to the position of the OH– group, the intensity ratio of these two peaks is different. For the 6-hydroxy-plasmodione 16a, the major ion fragment was 317 m/z, and the ratio of intensity was I317.1/I329 = 1.6; for the 7-hydroxy-plasmodione 16b, the major ion fragment was 329 m/z, and the ratio of intensity was I317.1/I329 = 0.4 (Scheme 11A, Fig. S46 and S47, ESI). In order to confirm the structure of the glucuronide metabolite in urine, we characterized by mass spectrometry both synthetic 6- and 7-hydroxylated metabolites of plasmodione and their corresponding glucuronides that were formed upon incubation of pure synthetic hydroxy-plasmodiones (16a/16b) with mouse liver microsomes (Scheme 11). After one hour, 39% and 46% unreacted 6-hydroxy-plasmodione 16a and 7-hydroxy-plasmodione 16b were recovered, respectively. Using UHPLC-MS/MS analysis, both hydroxylated metabolites were shown to be transformed to their corresponding glucuronide metabolites. Similarly to the results presented above, the MS/MS analysis revealed that the main peaks were found at m/z 345, 317 and 329, with the ratio I317.1/I329 = 2.8 for the 6-hydroxy-plasmodione glucuronide, and the ratio I317.1/I329 = 0.6 for the 7-hydroxy-plasmodione glucuronide. The mouse urine samples were re-analyzed using an optimized multiple-reaction monitoring (MRM) assay to quantify the various metabolites through the monitoring of their corresponding specific fragment ions (Scheme 11B and Fig. S48–S51, ESI). The intensity ratio of I317.1/I329 was 1.4 for the observed hydroxyl-metabolite (retention time: 0.86 min), suggesting that the major part of the hydroxy-metabolite found in urine was the 6-hydroxy-plasmodione 16a. The 6-hydroxy-plasmodione 16a concentration was evaluated to be 1 μM in the urine of plasmodione-treated mice. For the glucuronide metabolite, the intensity of the peaks at m/z 317 and 329 was too weak to predict its exact structure. However, it is conservative to assume that most of the glucuronide conjugate present in urine is obtained from the main 6-hydroxy-plasmodione 16a. In conclusion, based on UHPLC-MS/MS analysis, we showed that the main plasmodione metabolites present in the urine of plasmodione-treated mice are the major 6-hydroxy-plasmodione 16a and its glucuronide derivative produced by the conjugation of the hydroxyl metabolite with glucuronic acid.


image file: c8ob00227d-s11.tif
Scheme 11 Identification of drug metabolites in the urine of plasmodione-treated mice: 6- or 7-hydroxy-plasmodione and 6- or 7-hydroxy-plasmodione glucuronide derivatives. (a) The plasmodione-treated mouse urine was extracted using SPE and analyzed using UHPLC-MS/MS as described. (b) 50 μM of 6-hydroxy-plasmodione 16a and 7-hydroxy-plasmodione 16b were incubated with mouse liver microsomes (0.5 mg mL−1) and uridine diphosphate glucuronic acid (1 mM). The retention time of both 6- and 7- hydroxy-plasmodiones (16a and 16b) was 0.83 min. The retention time of both 6- or 7-hydroxy-plamodione glucuronides was 0.63 min. I317.1/I329 is the ratio of the intensity of the peak at m/z 317.1 and the intensity of the peak at m/z 329.

Conclusions

The work described here provides new insights into the synthesis of fully 13C-enriched plasmodiones and unlabelled putative metabolites. The 13C18-plasmodione 10b was synthesized by a 10 step-long sequence via an optimized “tetralone express route” with an overall yield of 5% for 10bversus 10.4% for the mono-13C1-methyl-plasmodione 10a. Besides this, another mono-13CC1-labeled plasmodione 10c was produced.

In the course of this work, we observed that some reactions produce 13C-enriched compounds and unlabeled compounds with different yields. For example, for some reactions like the rhodium-catalyzed isomerization reaction, both unlabeled and 13C-labeled compounds reacted similarly, while in other cases such as the oxidation reaction, the yields were different (90% for unlabeled 9aversus 75% for labeled 9b). Essentially, 13C atoms have the same number of electrons as 12C but different numbers of neutrons. The 13C-labeled compounds have the same chemical properties as unenriched compounds; however, the additional neutron in each 13C leads to an increased molecular weight and a decreased kinetic energy, around 10% in a reported study.39 It is therefore difficult to anticipate whether the reactivity of the 13C-enriched compounds would be different from that of the unlabeled compounds.

Besides this, we observed large differences in the signal patterns of the 1H and 13C NMR spectra of 13C-labeled versus unlabeled compounds. The most significant effect is the generation of 1J13C–1H coupling constants in the 1H NMR signals that ranged from 120 Hz to 170 Hz.40 Additional couplings further complexified the spectra of 13C-labeled compounds, however the chemical shifts were conserved between labeled and unlabeled compounds and we used this information to unambiguously characterize the 13C-labeled compounds. Also, in the mono-13C1-labeled plasmodione synthesis, we further discovered and identified an impurity generated in trace amounts in the Kochi–Anderson reaction, the epoxide 11.

The synthesis work presented here provides a solid basis for the investigation of plasmodione metabolism and mode of action. The IC50 values of the different 13C-enriched plasmodiones against P. berghei blood stage parasites confirmed that the 13C-enrichment did not affect the antimalarial activity of the lead drug. The uniformly 13C-enriched plasmodione will be used to elucidate the drug metabolism studies41 and the cellular localization of the drug and its metabolites using nanoSIMS imaging.42–44 In addition to the 13C-labeled compounds, we prepared a series of putative plasmodione metabolites. As expected, they presented variable parasite killing efficacies. Still, two of them had IC50s around 100 nM suggesting that plasmodione might generate several metabolites with potent antimalarial properties. Moreover, two of the synthetic plasmodione metabolites have been instrumental to identify the hydroxyl metabolite and its glucuronide detected in the urine of plasmodione-treated mice. Of note, the potent antimalarial 6-hydroxy-plasmodione 16a is the major metabolite found in mouse urine. It will be interesting to investigate whether this metabolite is also present in the serum of plasmodione-treated mice where it could mediate parasite killing, and how quickly it is excreted in urine. Future investigations will also aim at (1) optimizing the conditions to extract the different drug metabolites from pRBC samples; (2) confirming whether and where they are formed upon incubation of the lead drug with pRBC samples; and (3) identifying unknown plasmodione metabolites using the structure/fragmentation information of the synthetic metabolites.

Experimental

General

Nuclear magnetic resonance (NMR). The Nuclear Magnetic Resonance (NMR) spectra were recorded either with a Bruker Avance 400 apparatus (1H NMR 400 MHz, 13C NMR 100 MHz, 19F NMR 376 MHz, 31P NMR 81 MHz) or with a Bruker Avance 300 apparatus (1H NMR 300 MHz, 13C NMR 75 MHz, 19F NMR 281 MHz) at ECPM. All chemical shifts (δ) are quoted in parts per million (ppm). The chemical shifts are referred to the used partial deuterated NMR solvent (for CDCl3: 1H NMR, 7.26 ppm and 13C NMR, 77.36 ppm; for DMSO 1H NMR, 2.54 ppm and 13C NMR, 40.45 ppm). The coupling constants (J) are given in hertz (Hz). Resonance patterns are reported with the following notations: br (broad), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets). In addition, the following acronyms will be used: C[double bond, length as m-dash]O carbonyl group; Cq: quaternary carbon; CH2: secondary carbon; CH3: methyl group; ArH: aromatic proton of the menadione core; PhenylH: aromatic proton of the phenyl moiety. Elemental analysis: Elemental analyses (EA) were performed by “Service de Microanalyses at the Institut de Chimie de Strasbourg. Mass spectrometry: Mass spectrometry was performed at the Service de Spectrométrie de Mass e of the Université de Strasbourg. Mass spectra (ESI-MS) were obtained on a microTOF LC spectrometer (Bruker Daltonics, Bremen). High Resolution Mass (HRMS) spectra were recorded and fitted with the calculated data. The silver content determination was performed by inductively coupled plasma mass spectrometry (ICP-MS) in the department Reconnaissance et Procédés de Séparation Moléculaire (RePSeM) of Institute Pluridisciplinaire Hubert Curien (IPHC), ECPM, UMR 7178 CNRS, Strasbourg University. Melting point: Melting points were determined on a Büchi melting point apparatus and were not corrected.

Synthesis

Solvents and reagents. Commercially available starting materials were purchased from Sigma-Aldrich, ABCR GmbH & Co. KG, Alfa Aesar, and Apollo Scientific and were used without further purification. Solvents were obtained from Sigma-Aldrich and Carlos Erba. Unless noticed, reagent grade was used for reactions and column chromatography and analytical grade was used for recrystallizations. When specified, anhydrous solvents were required; tetrahydrofuran (THF) was distilled over sodium/benzophenone under argon or dried by passage through an activated alumina column under argon. All reactions were performed in standard glassware. Thin Layer Chromatography (TLC) was used to monitor the reactions (vide infra). Crude mixtures were purified either by recrystallization or by flash column chromatography. Monitoring and primary characterization of products were achieved by Thin Layer Chromatography on plastic sheets coated with silica gel 60 F254 purchased from E. Merck. Eluted TLCs were revealed under UV (325 nm and 254 nm) and with detection reagents. Alternatively, analytical TLC was carried out on pre-coated Sil G-25 UV254 plates from Macherey Nagel. Flash chromatography was performed using silica gel G60 (230–400 mesh) from Macherey Nagel. All 13C-enriched available chemical products were purchased by commercial sources without further purification. Chromatography: Generally, column chromatography was performed using silica gel 60 (230–400 mesh, 0.040–0.063 mm) or activated aluminum oxide, basic, and Brockmann Grade I (60 mesh, 58 Å) was purchased from E. Merck.

Synthesis of 4-oxo-4-phenylbutanoic acid ( 1a/1b), 4-phenylbutanoic acid (2a/2b), 3,4-dihydronaphthalen-1(2H)-one (3a/3b), followed the described procedures.18 Spectra related to 4-oxo-4-phenylbutanoic acid (1a), 1H NMR (400 MHz, CDCl3) (see Fig. S1, ESI), 13C NMR (100 MHz, CDCl3) (see Fig. S2, ESI); spectra related to 4-oxo-4-(phenyl-13C6)butanoic-1,2,3,4-13C4 acid (1b), 1H NMR (400 MHz, CDCl3) (see Fig. S1, ESI), 13C NMR (100 MHz, CDCl3) (see Fig. S2, ESI); spectra related to 4-phenylbutanoic acid (2a), 1H NMR (400 MHz, CDCl3) (see Fig. S3, ESI), 13C NMR (100 MHz, CDCl3) (see Fig. S4, ESI); spectra related to 4-(phenyl-13C6)butanoic-1,2,3,4-13C4 acid (2b), 1H NMR (400 MHz, CDCl3) (see Fig. S3, ESI), 13C NMR (100 MHz, CDCl3) (see Fig. S4, ESI); spectra related to 3,4-dihydronaphthalen-1(2H)-one (3a), 1H NMR (400 MHz, CDCl3) (see Fig. S5, ESI), 13C NMR (100 MHz, CDCl3) (see Fig. S6, ESI); spectra related to 3,4-dihydro-naphthalen-1(2H)-one-13C10 (3b), 1H NMR (400 MHz, CDCl3) (see Fig. S5, ESI), 13C NMR (100 MHz, CDCl3) (see Fig. S6, ESI), are shown in the ESI.

Synthesis of 1-bromo-4-iodobenzene (4a/4b). To a solution of 1,4-dibromobenzene-1,2,3,4,5,6-13C6 (1 equiv., 1000 mg, 4.13 mmol) in tetrahydrofuran (62.4 mL) at −78 °C was added dropwise over 15 min a solution of n-butyllithium (1.03 equiv., 1.6 M in hexane, 2.66 mL, 4.26 mmol) under an argon atmosphere. The reaction mixture was added dropwise over 15 min to a solution of iodine (1.2 equiv., 1259 mg, 1.23 mL, 4.96 mmol) in 10 mL of tetrahydrofuran and stirred for 15 min at −78 °C, and then for an additional 1 hour at room temperature. Saturated Na2S2O3 was added and stirred for 15 min and the resulting mixture became colorless. The reaction mixture was partitioned in 20 mL of water and 30 mL of diethylether. The resulting aqueous layer was extracted with diethylether (4 × 25 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (100%, cyclohexane Rf: 0.89), and a white solid product was obtained (1355 mg, 76%). 1-Bromo-4-iodobenzene (4a):1H NMR (400 MHz, CDCl3): δ 7.55 (d, 2H, J = 8.5 Hz, phenylH), 7.23 (d, 2H, J = 8.5 Hz, phenylH) ppm (see Fig. S7, ESI). 13C NMR (100 MHz, CDCl3): δ 139.2, 133.5, 122.3, 92.1 ppm (see Fig. S8, ESI). 1-Bromo-4-iodobenzene-1,2,3,4,5,6-13C6(4b):1H NMR (400 MHz, CDCl3): δ 7.54 (dq, 2H, J = 167.7 Hz, J = 8.5 Hz, phenylH), 7.22 (dq, 2H, J = 167.2 Hz, J = 8.5 Hz, phenylH) ppm (see Fig. S7, ESI). 13C NMR (100 MHz, CDCl3): δ 139.2 (ddd, J = 61.8 Hz, J = 54.1 Hz, J = 7.7 Hz), 133.5 (ddd, J = 62.4 Hz, J = 55.3 Hz, J = 7.7 Hz), 122.3 (td, J = 64.1 Hz, J = 11.1 Hz), 92.1 (td, J = 61.8 Hz, J = 11.1 Hz, J = 2.3 Hz) ppm (see Fig. S8, ESI).
Synthesis of 1-bromo-4-(trifluoromethyl)benzene (5a/5b). N-Methyl-2-pyrrolidone (NMP) (50 mL) was added to 1-bromo-4-iodobenzene-1,2,3,4,5,6-13C64b (1 equiv., 1278 mg, 4.42 mmol), copper(I) iodide (1.5 equiv., 1263 mg, 6.63 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (5 equiv., 4249 mg, 2.82 mL, 22.12 mmol). The brown reacting mixture was heated and stirred at 80 °C under an argon atmosphere for 16 hours. The reacting mixture was diluted with diethylether (25 mL) and filtered over Celite. To the filtrate was added water and the aqueous layer was extracted with diethylether (4 × 25 mL). The organic layer was washed with water (2 × 25 mL) and brine, dried over MgSO4 and concentrated under reduced pressure. The light-yellow oil crude was used in the next step. 1-Bromo-4-(trifluoromethyl)benzene (5a):1H NMR (400 MHz, CDCl3): δ 7.63 (d, 2H, J = 8.3 Hz, phenylH), 7.49 (d, 2H, J = 8.3 Hz, phenylH) ppm. 13C NMR (100 MHz, CDCl3): δ 132.2, 129.7 (q, J = 33.5 Hz), 127.0, 126.6, 124.0 (q, J = 275.9 Hz, CF3) ppm. 19F NMR (376 MHz, CDCl3): δ −62.79 ppm. 1-Bromo-4-(trifluoromethyl)benzene-1,2,3,4,5,6-13C6(5b):1H NMR (400 MHz, CDCl3): δ 7.63 (dq, 2H, J = 167.1 Hz, J = 7.3 Hz, phenylH), 7.49 (dq, 2H, J = 161.0 Hz, J = 9.1 Hz, phenylH) ppm. 13C NMR (100 MHz, CDCl3): δ 132.2 (tt, J = 59.9 Hz, J = 6.5 Hz), 129.7 (dm, J = 30.6 Hz, J = 8.3 Hz), 127.0 (tt, J = 56.3 Hz, J = 4.9 Hz), 126.4 (td, J = 56.2 Hz, J = 9.6 Hz), 124.0 (q, J = 275.9 Hz, CF3) ppm. 19F NMR (376 MHz, CDCl3): δ −62.78 (dtd, J = 32.0 Hz, J = 4.2 Hz, J = 1.5 Hz) ppm.
Synthesis of 4-(trifluoromethyl)benzaldehyde (6a/6b). To a solution of 1-bromo-4-(trifluoromethyl)benzene-1,2,3,4,5,6-13C65b (1 equiv., 1021 mg, 4.42 mmol) in tetrahydrofuran (4.42 mL) with anhydrous Na2SO4 (0.4 g), isopropylmagnesium bromide (0.53 equiv., 2.9 M in 2-methyltetrahydrofuran, 0.81 mL, 2.34 mmol) was added dropwise for 30 min under an argon atmosphere at 0 °C. After 10 min stirring, n-butyllithium (1.06 equiv., 1.6 M in hexane, 2.93 mL, 4.69 mmol) was added dropwise for 30 min, and the reaction mixture was stirred for 1 hour at −10 °C. A solution of N,N-dimethylformamide-13C (1.3 equiv., 426 mg, 0.45 mL, 5.75 mmol) in tetrahydrofuran (4.42 mL) was added dropwise for 30 min to the mixture at −10 °C and the reaction mixture was stirred for 1 hour at room temperature. 1 M citric acid solution (10 mL) was added to the mixture and the aqueous layer was extracted with diethylether (3 × 25 mL) and dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (pentane/Et2O, 9/1 Rf: 0.63), the colorless oil was obtained (249 mg, 31%). 4-(Trifluoromethyl)benzaldehyde (6a):1H NMR (400 MHz, CDCl3): δ 10.1 (s, 1H, CHO), 8.01 (d, 2H, J = 8.0 Hz, phenylH), 7.82 (d, 2H, J = 8.0 Hz, phenylH) ppm (see Fig. S9, ESI). 13C NMR (400 MHz, CDCl3): δ 191.2, 138.8, 135.8 (q, J = 34.3 Hz), 130.1, 126.3, 124.9 ppm (see Fig. S10, ESI). 19F NMR (376 MHz, CDCl3): δ −63.22 ppm (see Fig. S11, ESI). 4-(Trifluoromethyl)-benzaldehyde-1,2,3,4,5,6-13C6(6b):1H NMR (400 MHz, CDCl3): δ 10.1 (dd, 1H, J = 177.0 Hz, J = 24.1 Hz, CHO), 8.01 (dt, 2H, J = 163.2 Hz, J = 5.8 Hz, phenylH), 7.82 (dq, 2H, J = 164.0 Hz, J = 7.4 Hz, phenylH) ppm (see Fig. S9, ESI). 13C NMR (100 MHz, CDCl3): δ 191.2 (dt, J = 52.4 Hz, J = 4.0 Hz), 138.8 (dtd, J = 51.4 Hz, J = 58.7 Hz, J = 9.3 Hz), 135.8 (m, J = 31.1 Hz), 130.1 (tt, J = 58.0 Hz, J = 5.3 Hz), 126.2 (tq, J = 58.4 Hz, J = 3.7 Hz), 124.9 ppm (see Fig. S10, ESI). 19F NMR (376 MHz, CDCl3): δ −63.22 (dt, J = 32.7 Hz, J = 3.7 Hz) ppm (see Fig. S11, ESI).
Synthesis of 2-(4-(trifluoromethyl)benzylidene)-3,4-dihydro-naphthalen-1(2H)-one (7a/7b). A mixture of 3,4-dihydronaphthalen-1(2H)-one-13C103b (1 equiv., 159.5 mg, 1.02 mmol) and 4-(trifluoromethyl)benzaldehyde-1,2,3,4,5,6-13C66b (1 equiv., 185 mg, 1.02 mmol) was stirred at room temperature. Then, KOH (1.2 equiv., 68.8 mg, 1.23 mmol) in ethanol (4 mL) was added dropwise to the mixture. The mixture was poured in 30 mL ice cold water and the white pure product precipitated. The product was filtered, washed with ice cold water (2 × 5 mL) and dried under vacuum to yield a beige solid (248 mg, 76%) (TLC: cyclohexane/EtOAc, 8/2, Rf = 0.51). 2-(4-(Trifluoromethyl)benzylidene)-3,4-dihydronaphthalen-1(2H)-one (7a):1H NMR (400 MHz, CDCl3): δ 8.14 (1H, m, ArH), 7.85 (1H, s, olefinH), 7.68 (2H, d, J = 8.1 Hz, phenylH), 7.53 (2H, d, J = 8.1 Hz, phenylH), 7.52 (1H, m, ArH), 7.38 (1H, m, ArH), 7.27 (1H, m, ArH), 3.10 (2H, t, J = 6.5 Hz, CH2), 2.97 (2H, t, J = 6.3 Hz, CH2) ppm (see Fig. S12, ESI). 13C NMR (100 MHz, CDCl3): δ 187.9, 143.5, 139.8, 137.7, 135.0, 133.9, 133.6, 130.3, 128.7, 128.6,127.5, 125.7, 29.2, 27.5 ppm (see Fig. S13, ESI). 19F NMR (376 MHz, CDCl3): δ −62.70 ppm (see Fig. S14, ESI). HRMS (ESI) m/z: [M + H]+ calcd for C18H14F3O1: 303.0991; found 303.0971. 2-((4-(Trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methylene-13C)-3,4-dihydronaphthalen-1(2H)-one-13C10(7b):1H NMR (400 MHz, CDCl3): δ 8.14 (d, 1H, J = 157.7 Hz, ArH), 7.85 (d, 1H, J = 156.4 Hz, olefinH), 7.68 (d, 2H, J = 161.6 Hz, phenylH), 7.53 (d, 2H, J = 159.0 Hz, phenylH), 7.52 (m, 1H, ArH), 7.38 (d, 1H, J = 155.1 Hz, ArH), 7.27 (d, 1H, J = 162.8 Hz, ArH), 3.19 (d, 2H, J = 56.8 Hz, CH2), 2.88 (d, 2H, J = 56.8 Hz, CH2) ppm (see Fig. S12, ESI). 13C NMR (100 MHz, CDCl3): δ 187.9 (t, J = 52.0 Hz), 143.5 (q, J = 51.3 Hz), 139.8 (q, J = 57.1 Hz), 138.6–136.9 (m), 134.7 (q, J = 61.7 Hz), 134.0 (q, J = 55.4 Hz), 133.7 (t, J = 52.1 Hz), 130.3 (t, J = 57.9 Hz), 128.7 (m), 128.6 (m), 127.5 (m),125.7 (t, J = 60.8 Hz), 29.2 (t, J = 37.0 Hz), 27.5 (t, J = 37.0 Hz) ppm (see Fig. S13, ESI). 19F NMR (376 MHz, CDCl3): δ −62.70 (td, J = 31.3 Hz, J = 3.2 Hz) ppm (see Fig. S14, ESI). ESI-MS m/z: [M + H]+ calcd for 13C1712C1H14F3O1: 320.20; found 320.16.
Synthesis of 2-(4-(trifluoromethyl)benzyl)naphthalen-1-ol (8a/8b). 2-((4-(Trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methylene-13C)-3,4-dihydro-naphthalen-1(2H)-one-13C107b (1 equiv., 223 mg, 0.699 mmol) was solubilized in well degassed ethanol (15 mL). Trichlororhodium trihydrate (0.15 equiv., 27.6 mg, 0.105 mmol) was added. The mixture was reflux under an argon atmosphere and stirred for 5 hours. The mixture was evaporated and the resulting mixture was dissolved in ethyl acetate and extracted with water. The aqueous layer was washed with ethyl acetate (3 × 25 mL). The organic layer was combined and washed with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (cyclohexane/EtOAc, 9/1, Rf = 0.50) to give a light-yellow solid (175 mg, 78%). 2-(4-(Trifluoromethyl)benzyl)naphthalen-1-ol (8a):1H NMR (400 MHz, CDCl3): δ 8.05 (d, 1H, J = 8.0 Hz, ArH), 7.83 (d, 1H, J = 7.83 Hz, ArH), 7.54 (d, 2H, J = 7.7 Hz, phenylH), 7.51–7.45 (m, 3H, ArH), 7.36 (d, 2H, J = 7.9 Hz, phenylH), 7.28–7.23 (m, 1H, ArH), 5.26 (s, 1H, OH), 4.23 (s, 2H, CH2) ppm (see Fig. S15, ESI). 13C NMR (100 MHz, CDCl3): δ 149.0, 144.4, 134.2, 129.2, 129.1 (q, J = 33.0 Hz), 129.0, 128.3, 126.3, 126.0, 125.9 (q, J = 3.0 Hz), 125.0, 124.4 (q, J = 265.0 Hz), 121.3, 120.8, 120.0, 36.5 ppm (see Fig. S16, ESI). 19F NMR (376 MHz, CDCl3): δ −62.43 ppm (see Fig. S17, ESI). HRMS (ESI) m/z: [M + H]+ calcd for C18H14F3O1: 303.0991; found 303.0996. 2-((4-(Trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methyl-13C)naphthalen-1-ol-13C10(8b):1H NMR (400 MHz, CDCl3): δ 8.04 (dm, 1H, J = 154.5 Hz, ArH), 7.83 (dm, 1H, J = 154.0 Hz, ArH), 7.54 (dm, 2H, J = 156.1 Hz, phenylH), 7.46 (dm, 3H, J = 154.5 Hz, ArH), 7.35 (dm, 2H, J = 156.1 Hz, phenylH), 7.25 (dm, 1H, J = 154.5 Hz, ArH), 5.14 (q, 1H, J = 3.7 Hz, OH), 4.23 (d, 2H, J = 129.1 Hz, CH2) ppm (see Fig. S15, ESI). 13C NMR (100 MHz, CDCl3): δ 149.0 (t, J = 68.4 Hz), 144.3 (ddm, J = 99.6 Hz, J = 53.0 Hz), 134.1 (q, J = 54.3 Hz), 129.4 (tm, J = 58.3 Hz), 129.1 (q, J = 33.0 Hz), 129.0 (tm, J = 59.7 Hz), 128.3 (tm, J = 56.1 Hz), 126.4 (tm, J = 54.1 Hz), 125.7 (tm, J = 53.6 Hz), 125.9 (qm, J = 31.7 Hz), 124.9 (qm, J = 61.1 Hz), 124.4 (q, J = 265.0 Hz), 121.3 (tm, J = 59.8 Hz), 120.7 (tm, J = 56.5 Hz), 119.9 (qm, J = 61.3 Hz), 36.5 (t, J = 44.3 Hz) ppm (see Fig. S16, ESI). 19F NMR (376 MHz, CDCl3): δ −62.44 (dt, J = 32.1 Hz, J = 3.1 Hz) ppm (see Fig. S17, ESI). ESI-MS m/z: [M + H]+ calcd for 13C1712C1H14F3O1: 320.23; found 320.16.
Synthesis of 2-(4-(trifluoromethyl)benzyl)naphthalene-1,4-dione (9a/9b). 2-((4-(Trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methyl-13C) naphthalen-1-ol-13C108b (1 equiv., 160 mg, 0.501 mmol) was solubilized in 8.7 mL of a solution of acetonitrile–water (3[thin space (1/6-em)]:[thin space (1/6-em)]1). Phenyliodonium diacetate (2 equiv., 323 mg, 1.00 mmol) was added portionwise in 10 min at −5 °C. The reaction mixture was stirred at −5 °C for 30 min and warm to room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to evaporate acetonitrile and 10 mL of saturated NaHCO3 was added. The resulting mixture was extracted with diethylether (10 mL), the aqueous layer was washed with diethylether (3 × 10 mL) and the organic layers were combined and washed with brine (2 × 10 mL), dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (cyclohexane/EtOAc 8.5/1.5, Rf = 0.39) to yield a yellow solid (127 mg, 76%). 2-(4-(Trifluoromethyl)benzyl)naphthalene-1,4-dione (9a):1H NMR (400 MHz, CDCl3): δ 8.13–8.04 (m, 2H, ArH), 7.76–7.72 (m, 2H, ArH), 7.59 (d, 2H, J = 8.0 Hz, phenylH), 7.39 (d, 2H, J = 8.1 Hz, phenylH), 6.63 (s, 1H, ArH), 3.95 (s, 2H, CH2) ppm (see Fig. S18, ESI). 13C NMR (100 MHz, CDCl3): δ 185.2, 185.1, 150.1, 141.3, 136.2, 134.3, 134.2, 132.4, 130.1, 129.7 (q, J = 32.5 Hz), 127.1, 126.6, 126.1 (q, J = 3.83 Hz), 124.4 (q, J = 274.9 Hz) 36.0 ppm (see Fig. S19, ESI). 19F NMR (376 MHz, CDCl3): δ −62.55 ppm (see Fig. S20, ESI). HRMS (ESI) m/z: [M + H]+ calcd for C18H12F3O2: 317.0784; found 317.0791. 2-((4-(Trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methyl-13C)naphthalene-1,4-dione-13C10(9b):1H NMR (400 MHz, CDCl3): δ 8.08 (dm, 2H, J = 157.4 Hz, ArH), 7.74 (dm, 2H, J = 157.3 Hz, ArH), 7.59 (dm, 2H, J = 157.3 Hz, phenylH), 7.39 (d, 2H, J = 157.5 Hz, phenylH), 6.63 (dm, 1H, J = 159.0 Hz ArH), 3.96 (d, 2H, J = 130.4 Hz, CH2) ppm (see Fig. S18, ESI). 13C NMR (100 MHz, CDCl3): δ 185.2 (t, J = 49.0 Hz), 185.1 (t, J = 49.0 Hz), 150.1 (qm, J = 52.7 Hz), 141.3 (qm, J = 51.6 Hz), 136.9–135.4 (m), 135.0–133.4 (m), 132.4 (tm, J = 41.3 Hz), 130.1 (tm, J = 53.9 Hz), 129.2 (qd, J = 29.2 Hz, J = 8.6 Hz), 126.9 (tm, J = 59.1 Hz), 126.3 (tm, J = 53.8 Hz), 126.1 (tm, J = 55.7 Hz), 36.0 (t, J = 44.3 Hz) ppm (see Fig. S19, ESI). 19F NMR (376 MHz, CDCl3): δ −62.56 (dt, J = 31.8 Hz, J = 3.5 Hz) ppm (see Fig. S20, ESI). ESI-MS m/z: [M + H]+ calcd for 13C1712C1H12F3O2: 334.21; found 334.14.
Synthesis of 2-(methyl-13C)-3-(4-(trifluoromethyl)benzyl)naphthalene-1,4-dione (10a/10b). 2-((4-(Trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methyl-13C)naphthalene-1,4-dione-13C109b (1 equiv., 108.3 mg, 0.325 mmol) and acetic-2-13C acid (5 equiv., 99.2 mg, 0.095 mL, 1.63 mmol) were solubilized in 14 mL of a solution acetonitrile–water (3[thin space (1/6-em)]:[thin space (1/6-em)]1). The reaction mixture was heated at 85 °C and silver nitrate (0.35 equiv., 19.3 mg, 0.114 mmol) was added. Ammonium persulfate (1.3 equiv., 96.4 mg, 0.423 mmol) in a solution of acetonitrile–water (3[thin space (1/6-em)]:[thin space (1/6-em)]1) was added dropwise. The reaction mixture was stirred for 90 min. The resulting mixture was evaporated and 10 mL of dichloromethane was added. The aqueous layer was extracted with dichloromethane (3 × 10 mL) and the organic layers were combined and extracted with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (toluene, Rf = 0.51) and recrystallization (n-hexane, 50 mL) to give a pure yellow solid (52.1 mg, 46%). 2-(Methyl-13C)-3-(4-(trifluoromethyl)benzyl)naphthalene-1,4-dione (10a):1H NMR (400 MHz, CDCl3): δ 8.10–8.06 (m, 2H, ArH), 7.72–7.70 (m, 2H, ArH), 7.52 (d, 2H, J = 8.2 Hz, phenylH), 7.34 (d, 2H, J = 8.1 Hz, phenylH), 4.08 (s, 2H, CH2), 2.25 (s, 3H, CH3) ppm (see Fig. S21, ESI). 13C NMR (100 MHz, CDCl3): δ 185.2, 184.6 (d, J = 3.6 Hz), 145.0 (d. J = 45 Hz), 144.5, 142.3, 133.8, 132.2, 132.0, 129.0, 129.0 (q, J = 32.3 Hz, C–CF3), 126.6, 126.5, 125.7, 125.7, 124.3 (q, J = 273.0 Hz, CF3), 32.5 (d, J = 2.2 Hz), 13.5 (t, J = 21.5 Hz) ppm (see Fig. S22, ESI). 19F NMR (375 MHz, CDCl3): −62.49 ppm (see Fig. S23, ESI). ESI-MS m/z: [M + H]+ calcd for 13C12C18H14F3O2: 332.10; found 332.09. 2-(Methyl-13C)-3-((4-(trifluoromethyl)phenyl-1,2,3,4,5,6-13C6)methyl-13C)-naphthalene-1,4-dione-13C10(10b):1H NMR (400 MHz, CDCl3): δ 8.09 (dm, 2H, J = 167.0 Hz, ArH), 7.721 (dm, 2H, J = 148.5 Hz, ArH), 7.52 (dm, 2H, J = 160.8 Hz, phenylH), 7.35 (dm, 2H, J = 151.6 Hz, phenylH), 4.09 (d, 2H, J = 129.5 Hz, CH2), 2.25 (d, 3H, J = 131.6 Hz, CH3) ppm (see Fig. S21, ESI). 13C NMR (100 MHz, CDCl3): δ 185.2 (t, J = 48.8 Hz), 184.7 (t, J = 49.9 Hz), 146.1–143.4 (m), 143.3–141.3 (m), 134.6–132.8 (m), 132.8–130.9 (m), 129.9–128.1 (tm, J = 58.0 Hz), 127.4–125.8 (m), 125.6 (t, J = 56.1 Hz), 32.5 (t, J = 42.6 Hz), 13.9–13.1 (m) ppm (see Fig. S22, ESI). 19F NMR (376 MHz, CDCl3): −62.51 (dt, J = 31.6 Hz, J = 3.0 Hz) ppm (see Fig. S23, ESI). ESI-MS (Q-TOF) m/z: [M + H]+ calcd for 13C1812C1H14F3O2: 349.15; found 349.1546 (see Fig. S24, ESI).
Synthesis of 2-methyl-3-(4-(trifluoromethyl)benzyl)naphthalene-1,4-dione-1-13C (10c). 2-Methylnaphthalene-1,4-dione-1-13C (1 equiv., 108.3 mg, 0.325 mmol) and 2-(4-(trifluoromethyl) phenyl)acetic acid (5 equiv., 99.2 mg, 0.095 mL, 1.63 mmol) were solubilized in 14 mL of a solution acetonitrile–water (3[thin space (1/6-em)]:[thin space (1/6-em)]1). The reaction mixture was heated at 85 °C and silver nitrate (0.35 equiv., 19.3 mg, 0.114 mmol) was added. Ammonium persulfate (1.3 equiv., 96.4 mg, 0.423 mmol), in a solution of acetonitrile–water (3[thin space (1/6-em)]:[thin space (1/6-em)]1), was added dropwise. The reaction mixture was stirred for 3 hours. The resulting mixture was evaporated and 10 mL of dichloromethane was added. The aqueous layer was extracted with dichloromethane (3 × 10 mL) and the organic layers were combined and extracted with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (toluene, Rf = 0.51) and recrystallization (n-hexane, 50 mL) to give a pure yellow solid (52.1 mg, 46%). 1H NMR (400 MHz, CDCl3): δ 8.13–8.06 (m, 2H, ArH), 7.75–7.68 (m, 2H, ArH), 7.55 (d, 2H, J = 7.9 Hz, phenylH), 7.35 (d, 2H, J = 7.9 Hz, phenylH), 4.08 (s, 2H, CH2), 2.28 (d, 3H, J = 3.8 Hz, CH3) ppm (see Fig. S25, ESI). 13C NMR (100 MHz, CDCl3): δ 185.5 (t, J = 26.5 Hz), 184.8 (d, J = 5.8 Hz), 145.2 (d. J = 49.7 Hz), 144.7, 142.5, 134.0, 132.4 (d, J = 54.0 Hz), 132.2 (d, J = 2.0 Hz), 129.2, 129.2 (q, J = 32.5 Hz, C–CF3), 126.9 (d, J = 3.1 Hz), 126.7, 125.9, 125.9, 124.5 (q, J = 269.1 Hz, CF3), 32.7 (d, J = 3.4 Hz), 13.7 ppm (see Fig. S26, ESI). 19F NMR (376 MHz, CDCl3): −62.32 ppm. EI-MS m/z: [M]+ calcd for 13C12C18H13F3O2: 331.10; found 331.10.
Synthesis of 1a-methyl-7a-(4-(trifluoromethyl)benzyl)-1a,7a-dihydro-naphtho[2,3-b]oxirene-2,7-dione (11). 2-Methyl-3-{[4-(trifluoromethyl)phenyl]methyl}-1,4-dihydronaphthalene-1,4-dione or plasmodione (1 equiv., 300 mg, 0.908 mmol) was dissolved in a mixture of solvents: distilled water (1 mL) and methanol (4 mL) (MeOH/H2O 4[thin space (1/6-em)]:[thin space (1/6-em)]1). Sodium hydroxide (0.5 equiv., 3 M, 0.151 mL, 0.454 mmol) was added to the mixture at 0 °C. The resulting mixture was stirred for 10 min and subsequently H2O2 (1.5 eq., 132 mg, 0.13 mL, 1.36 mmol) was added. The reaction mixture was stirred at 0 °C for 3 hours. The reaction was quenched with 10 mL of distilled water. The resulting mixture was extracted with diethyl ether (3 × 10 mL), the organic phase was extracted with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by recrystallization (Et2O/n-hexane, 1/3, 20 mL) to give a pure white crystal (185 mg, 59%). M.p.: 117–119 °C. 1H NMR (400 MHz, CDCl3): δ 8.02–7.91 (m, 2H, ArH), 7.78–7.69 (m, 2H, ArH), 7.55 (d, J = 8.1 Hz, 2H, phenylH), 7.46 (d, J = 8.1 Hz, 2H, phenylH), 3.67 (d, J = 15.0 Hz, 1H, CH2), 3.40 (d, J = 15.0 Hz, 1H, CH2), 1.84 (s, 3H, CH3) ppm (see Fig. S27, ESI). 13C NMR (100 MHz, CDCl3): δ 192.5, 192.2, 140.0, 134.6, 134.5, 132.1, 132.0, 129.8, 129.3 (q, J = 32.3 Hz, C–CF3), 127.4, 127.3, 125.6, 125.5, 124.2 (q, J = 254.6 Hz, CF3), 67.3, 65.9, 31.9, 12.7 ppm (see Fig. S28, ESI). 19F NMR (376 MHz, CDCl3): −62.56 ppm (see Fig. S29, ESI). EA %: calcd for C19H13F3O3: C 65.90, H 3.78; found C 65.39 H 3.87. ESI-MS (Q-TOF) m/z: [M + H]+ calcd for C19H14F3O3: 347.08; found 347.0894.
Synthesis of 2-(hydroxy(4-(trifluoromethyl)phenyl)methyl)-3-methylnaphthalene-1,4-dione (14). A solution of 7.0 mL n-BuLi (1 equiv., 3.56 mmol, 1.6 M in hexane, 2.225 mL) in 10 mL distilled THF was added dropwise to a solution of 2-bromo-1,4-dimethoxy-3-methylnaphthalene 18 (1 equiv., 1000 mg, 3.56 mmol) in 30 mL distilled THF under an argon atmosphere at −78 °C. The yellow solution was stirred at −78 °C for 30 minutes. Then a solution of p-trifluoromethylbenzaldehyde (1.1 equiv., 0.53 mL, 681 mg, 3.916 mmol) in 7 mL distilled THF was added at −78 °C. The resulting mixture was stirred at −78 °C for 30 minutes and was then allowed to warm to room temperature. The color turned from yellow to orange to yellow. After stirring for 2 hours at room temperature, the reaction was quenched by the addition of 10 mL saturated NH4Cl solution (20 mL). Diethyl ether (10 mL) was added to the resulting mixture and the aqueous phase was extracted with diethyl ether (2 × 10 mL). The organic phases were collected, dried over MgSO4 and concentrated under reduced pressure to give a pale-yellow raw product The crude product was purified by silica chromatography (CH2Cl2/petroleum ether, 1/2) to afford a pale-yellow solid (1,4-dimethoxy-3-methylnaphthalen-2-yl)(4-(trifluoro-methyl)phenyl)methanol 19 (978 mg, 73%). (1,4-Dimethoxy-3-methylnaphthalen-2-yl)(4-(trifluoromethyl)phenyl)methanol (19):1H NMR (400 MHz, CDCl3): δ 8.10–8.08 (m, 1H, ArH), 7.98–7.96 (m, 1H, ArH), 7.54–7.47 (m, 6H, phenylH + ArH), 6.35 (d, J = 6 Hz, 1H, OH), 3.98 (d, J = 6 Hz, 1H, CH), 3.86 (s, 3H, OCH3), 3.53 (s, 3H, OCH3), 2.34 (s, 3H, CH3) ppm. 2-(Hydroxy(4-(trifluoromethyl)phenyl)methyl)-3-methyl-naphthalene-1,4-dione (14): To a solution of (1,4-dimethoxy-3-methylnaphthalen-2-yl)(4-(trifluoromethyl)phenyl)methanol 19 (1 equiv., 882.2 mg, 2.344 mmol) in 60 mL of a solution acetonitrile–water (3[thin space (1/6-em)]:[thin space (1/6-em)]1), CAN (3 equiv., 3874 mg, 7.07 mmol)) was added at room temperature and the resulting orange mixture was stirred overnight. The resulting mixture was evaporated under reduced pressure, and extracted with dichloromethane (4 × 15 mL). The organic layers were collected, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (CH2Cl2/PE, 10/1) to afford a yellow solid (702 mg, 86%). M.p.: 134–136 °C. 1H NMR (400 MHz, CDCl3): δ 8.15–8.10 (m, 1H, ArH), 8.03–7.99 (m, 1H, ArH), 7.79–7.69 (m, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, phenylH), 7.53 (d, J = 8.5 Hz, 2H, phenylH), 6.04 (d, J = 10.9 Hz, OH), 4.36 (d, J = 10.8 Hz, CH), 2.33 (s, 3H, CH3) ppm (see Fig. S30, ESI). 13C NMR (100 MHz, CDCl3): δ 186.8 (C[double bond, length as m-dash]O), 185.2 (C[double bond, length as m-dash]O), 146.0 (Cq), 145.4 (Cq), 144.0 (Cq), 134.6 (CH), 134.3 (CH), 132.1 (d, J = 3.8 Hz, Cq), 129.8 (q, J = 33.0 Hz, C–CF3), 127.0 (CH), 126.8 (CH), 126.0 (CH), 125.9 (CH), 124.3 (q, J = 273.8 Hz, CF3), 71.5 (CH–OH), 13.0 (CH3) ppm (see Fig. S31, ESI). 19F NMR (376 MHz, CDCl3): δ −62.56 ppm (see Fig. S32, ESI). HRMS (ESI) m/z: [M + H]+ calcd for C19H14F3O3: 345.0744; found 345.0750. EA %: calcd for C19H13F3O3: C 65.90, H 3.78; found C 65.82 H 3.90.
Synthesis of 2-bromo-3-methylnaphthalene-1,4-dione (20). A solution of Br2 (1.05 equiv., 24.4 g, 7.83 mL, 152 mmol) in 20 ml dichloromethane was added to a solution of menadione (1 equiv., 25 g, 145 mmol) in dichloromethane (100 mL) dropwise over a 30 min. The mixture was stirred for 1 hour and controlled by TLC. Subsequently, pyridine (1.05 equiv., 12.1 g, 12.3 mL, 152 mmol) was added to the reaction mixture and stirred overnight. The resulting mixture was poured in water, and the organic phase was washed with Na2S2O3, then brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by recrystallization in methanol to give a red solid (33.9 g, 93%). M.p.: 98–100 °C. 1H NMR (400 MHz, CDCl3): δ 7.51 (dd, J = 7.5 Hz, J = 2.7 Hz, 2H, ArH), 6.80 (dq, J = 7.5 Hz, J = 2.7 Hz, 2H, ArH), 2.39 (s, 3H, CH3) ppm. EA %: calcd for C11H7BrO2: C 52.62, H 2.81; found C 52.74 H 2.86.
Synthesis of 2-bromo-1,4-bis(methoxymethoxy)-3-methylnaphthalene (21). A solution of stannous chloride dihydrate (3.42 equiv., 7.7 g, 2.84 mL, 34.1 mmol) in HCl (7 mL) was added quickly to a solution of 2-bromo-3-methylnaphthalene-1,4-dione 20 (1 equiv., 2.5 g, 9.96 mmol) in ethanol (49.4 mL) at 0 °C. The reaction mixture was stirred for 10 min at 0 °C and then 1 hour at room temperature. Subsequently, the resulting mixture was poured into water. The white solid precipitate was filtered and water was removed by azeotropic distillation with toluene (3 × 80 ml) to afford a white-violet solid. The resulting violet solid was dissolved in distilled dichloromethane (60 mL) at 0 °C, under argon, chloroalkyl ether (4 equiv., 3.21 g, 3.03 mL, 39.8 mmol) and then DIPEA (2.4 equiv., 3.09 g, 3.95 mL, 23.9 mmol) was added dropwise. After stirring for 2 hours, 1 g of NaOH was added to destroy the excess MOMCl. The reaction mixture was washed with water. The aqueous layer was extracted with ether (2 × 20 mL). The organic layers were washed with brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The resulting crude product was purified by silica chromatography (cyclohexane/ethyl acetate: 6/1) to give a pure brown solid (3.13 g, 9.16 mmol, 92%). M.p.: 73–74 °C. 1H NMR (400 MHz, CDCl3): δ 8.14 (dd, J = 6.6 Hz, J = 2.1 Hz, 1H, ArH), 8.06 (dd, J = 6.6 Hz, J = 2.1 Hz, 1H, ArH), 7.52 (m, 2H, ArH), 5.23 (s, 2H, OCH2), 5.11 (s, 2H, OCH2), 3.74 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 2.58 (s, 3H, CH3) ppm. HRMS (ESI) m/z: [M + Na]+ calcd for C15H17BrNaO4 363.0202; found 363.0207. EA %: calcd for C15H17BrO4: C 52.80, H 5.02; found C 53.85 H 5.05.
Synthesis of (1,4-bis(methoxymethoxy)-3-methylnaphthalen-2-yl)(2-fluoro-4-(trifluoromethyl)phenyl)methanone (22). To a solution of 2-bromo-1,4-bis(methoxymethoxy)-3-methyl-naphthalene 21 (1.2 equiv., 1200 mg, 3.52 mmol) in THF (8 mL), n-BuLi (1.6 equiv., 1.6 M, 2.93 mL, 4.69 mmol) was added dropwise at −78 °C under argon and the mixture was stirred for 45 min. Subsequently, a solution of 2-fluoro-4-(trifluoromethyl)benzoyl chloride (1 equiv., 664 mg, 0.443 mL, 2.93 mmol) in THF (2 mL) was added dropwise to the resulting mixture. The reaction mixture was stirred for 1.5 hours at −78 °C. The mixture was poured onto a saturated NH4Cl solution (15 mL). The aqueous phase was extracted with ether (2 × 15 mL). Then the organic phases were combined and washed with saturated NaHCO3 solution, then saturated NaCl solution, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (cyclohexane/ethyl acetate: 95/5) to give a red foam (1259 mg, 95%). 1H NMR (400 MHz, CDCl3): δ 8.14 (d, J = 8.5 Hz, 1H, ArH), 8.07 (d, J = 8.5 Hz, 1H, ArH), 7.92 (t, J = 7.7 Hz, 1H, phenylH), 7.60 (t, J = 7.8 Hz, 1H, ArH), 7.53 (t, J = 7.5 Hz, 1H, ArH), 7.49 (d, 1H, J = 8.8 Hz, phenylH), 7.39 (d, 1H, J = 10.5 Hz, phenylH), 5.15 (s, 2H, OCH2), 5.00 (s, 2H, OCH2), 3.68 (s, 3H, OCH3), 3.35 (s, 3H, OCH3), 2.32 (s, 3H, CH3) ppm (see Fig. S33, ESI).
Synthesis of (2-fluoro-4-(trifluoromethyl)phenyl)(1-hydroxy-4-(methoxymethoxy)-3-methylnaphthalen-2-yl)methanone (23). To a solution of (1,4-bis(methoxymethoxy)-3-methylnaphthalen-2-yl) (2-fluoro-4-(trifluoromethyl)phenyl)methanone 22 (1 equiv., 1250 mg, 2.76 mmol) in benzene (15 mL), MgBr2.OEt2 (3 equiv., 2140 mg, 8.29 mmol) was added dropwise under argon at room temperature. The reaction mixture was stirred overnight. The resulting mixture was poured onto saturated NH4Cl solution (20 mL). The aqueous phase was extracted with ether (2 × 20 mL). The organic phase was combined and washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (cyclohexane/ethyl acetate: 4/1) to give a desired product (800 mg, 71%). 1H NMR (400 MHz, CDCl3 + D2O): δ 8.08 (t, J = 7.7 Hz, 1H, ArH), 8.01 (d, J = 7.8 Hz, 1H, ArH), 7.83 (t, J = 8.0 Hz, 1H, phenylH), 7.65 (t, J = 7.5 Hz, 1H, ArH), 7.52 (d, J = 8.2 Hz, 1H, phenylH), 7.47 (t, 1H, J = 7.6 Hz, ArH), 7.34 (d, 1H, J = 10.2 Hz, phenylH), 3.57 (dd, 2H, J = 14.3 Hz, J = 9.7 Hz, OCH2), 3.29 (s, 3H, OCH3), 2.20 (s, 3H, CH3) ppm (see Fig. S34, ESI). HRMS (ESI) m/z: [M + H]+ calcd for C21H17F4O4 409.1057; found 409.1056.
Synthesis of 5-(methoxymethoxy)-6-methyl-10-(trifluoromethyl)-7H-benzo[c]xanthen-7-one (24). To a solution of (2-fluoro-4-(trifluoromethyl)phenyl)(1-hydroxy-4-(methoxymethoxy)-3-methyl-naphthalen-2-yl)methanone 23 (1 equiv., 400 mg, 0.98 mmol) in acetone (5.52 mL), K2CO3 (2 equiv., 270 mg, 1.96 mmol) was added and the mixture was stirred at 50 °C for 2 hours. The resulting mixture was concentrated under reduced pressure. The crude product was purified by silica chromatography (hexane/EtOAc: 9/1) to give two pure desired products 24 (Rf = 0,37) and product 15 (Rf = 0,35) in a ratio 1[thin space (1/6-em)]:[thin space (1/6-em)]1 with 35% yield each. 5-(Methoxymethoxy)-6-methyl-10-(trifluoromethyl)-7H-benzo[c]xanthen-7-one (24): m.p.: 155–157 °C (from hexane/EtOAc). 1H NMR (400 MHz, CDCl3): δ 8.65 (d, J = 8.3 Hz, 1H, ArH), 8.46 (d, J = 8.5 Hz, 1H, ArH), 8.22 (d, J = 8.5 Hz, 1H, ArH), 7.95 (s, 1H, ArH), 7.79 (t, J = 7.5 Hz, 1H, ArH), 7.69 (t, J = 7.9 Hz, 1H, ArH), 7.66 (d, 1H, J = 8.1 Hz, ArH), 5.16 (s, 2H, OCH2), 3.71 (s, 3H, OCH3), 2.97 (s, 3H, CH3) ppm (see Fig. S35, ESI). 13C NMR (100 MHz, CDCl3): δ 178.0 (C[double bond, length as m-dash]O), 154.6 (Cq), 152.7 (Cq) 148.4 (Cq), 135.6 (C–CF3), 132.2 (Cq), 130.7 (CH), 128.3 (CH), 127.1 (CH), 126.4 (Cq), 125.6 (Cq), 123.8 (Cq), 123.6 (q, J = 274 Hz, CF3), 123.4 (CH), 123.0 (CH), 120.9 (CH), 117.8 (Cq), 115.8 (d, J = 4.6 Hz, CH), 100.6 (CH2), 58.5 (OCH3), 15.7 (CH3) ppm (see Fig. S36 and S37, ESI). 19F NMR (376 MHz, CDCl3): −62.99 ppm (see Fig. S38, ESI). EA %: calcd for C21H16F3O4 C 64.95, H 3.89; found C 64.68 H 3.96. HRMS (ESI) m/z: [M + H]+ calcd for C21H16F3O4 389.0995; found 389.0993.
Synthesis of 5-hydroxy-6-methyl-10-(trifluoromethyl)-7H-benzo[c]xanthen-7-one (15). To a solution of 10-(methoxymethoxy)-11-methyl-3-(trifluoromethyl)-12H-5-oxatetraphen-12-one 24 (1 equiv., 73 mg, 0.188 mmol) in isopropanol (11.4 mL), HCl (1.2 equiv., 1.25 M, 0.18 mL, 0.226 mmol) was added dropwise. The mixture was stirred for 24 hours, and then evaporated under vacuum. The resulting crude product was recrystallized in cyclohexane and ethyl acetate (5 mL) to give a yellow solid (60.7 mg, 94%). M.p.: 229–231 °C (from cyclohexane/EtOAc). 1H NMR (400 MHz, DMSO-D6): δ 9.37 (s, 1H, OH), 8.79 (d, 1H, J = 8.2 Hz, ArH), 8.42 (d, 2H, J = 8.1 Hz, ArH), 8.38 (d, 1H, J = 8.1 Hz, ArH), 7.91–7.77 (m, 3H, ArH), 2.88 (s, 3H, CH3) ppm (see Fig. S39, ESI). 19F NMR (376 MHz, DMSO-D6): δ 61.43 ppm (see Fig. S40, ESI). EA %: calcd for C19H11F3O3 C 66.28, H 3.22; found C 66.28 H 3.58. HRMS (ESI) m/z: [M + H]+ calcd for C19H12F3O3 345.0733; found 345.0770.
Synthesis of 2-(hydroxymethyl)-3-(4-(trifluoromethyl)benzyl)naphthalene-1,4-dione (17). 2-(4-(Trifluoromethyl)benzyl)naphthalene-1,4-dione 9a (1 equiv., 300 mg, 0.9485 mmol) was solubilized in methanol (35 mL). The reaction mixture was heated at 70 °C and silver nitrate (0.3 equiv., 48.3 mg, 0.2846 mmol) was added. Ammonium persulfate (2.7 equiv., 584.4 mg, 2.561 mmol), in a solution of methanol–water (3–1, 8 mL), was added dropwise. The reaction mixture was stirred for 3 hours. The resulting mixture was evaporated and 20 mL of dichloromethane was added. The aqueous layer was extracted with dichloromethane (2 × 20 mL) and the organic layers were combined and extracted with brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica chromatography (toluene, Rf = 0.1) to give a pure yellow solid (160 mg, 49%). M.p.: 93–95 °C (from toluene). 1H NMR (400 MHz, CDCl3): δ 8.13–8.05 (m, 2H, ArH), 7.77–7.71 (m, 2H, ArH), 7.53 (d, 2H, J = 8.2 Hz, phenylH), 7.4 (d, 2H, J = 8.1 Hz, phenylH), 4.78 (s, 2H, CH2), 4.15 (s, 2H), 2.74 (s, 1H, OH) ppm (see Fig. S41, ESI). 13C NMR (100 MHz, CDCl3): δ 186.7 (C[double bond, length as m-dash]O), 185.1 (C[double bond, length as m-dash]O), 145.5 (Cq), 144.1 (Cq), 142.2 (Cq), 134.5 (CH), 134.4 (CH), 132.2 (Cq), 132.1 (Cq), 129.4 (CH × 2), 129.4 (q, J = 32.1 Hz, C–CF3), 127.1 (CH), 126.7 (CH), 126.0 (CH), 126.0 (CH), 124.4 (q, J = 271.7 Hz, CF3), 58.6 (CH2), 32.1 (CH2) ppm (see Fig. S42, ESI). 19F NMR (376 MHz, CDCl3): −62.54 ppm (see Fig. S43, ESI). EA %: calcd for C19H13F3O3·0.3H2O: C 64.89, H 3.90; found C 65.27 H 4.22. HRMS (ESI) m/z: [M + H]+ calcd for C19H15F3O3: 347.0890; found 347.0897.

Protocol for antimalarial activity against P. berghei parasites determined in ex vivo cultures

The animal experiments described in this manuscript were performed at the Institut de Biologie Moléculaire et Cellulaire (Strasbourg, France), using facilities and protocols adhering to the national regulations of laboratory animal welfare in France. The murine malaria parasite P. berghei ANKA strain expressing constitutively GFP-luciferase45 was maintained within CD1 mice (in-house breeding) by regular passage of infected blood to a naïve mouse. For this, blood was taken by heart puncture from a donor CD1 mouse infected with the GFP-luciferase parasite with a parasitemia 3–8% and diluted in PBS to 2 × 108 parasitized erythrocytesper mL. 0.1 mL of this suspension was injected intravenously into mice. Parasitemia was monitored by using Giemsa-stained smears.

The sensitivity of the murine malaria parasite P. berghei to plasmodione and 13C-enriched plasmodiones was determined by measuring the activity of parasite-expressed luciferase after a 24 h of exposure to different concentrations of the compounds under ex vivo conditions as formerly described.45

Briefly, infected blood was drawn by heart-puncture from a mouse with 2–3% parasitemia. Blood was washed twice in RPMI 1640 culture medium supplemented with fetal calf serum (FCS) at 25% and resuspended in the same medium. Infected blood samples were exposed for 24 h to decreasing drug concentrations of plasmodione and 13C-enriched plasmodiones in microtiter plates (2.1% parasitemia, 2% hematocrit final). For this, the compounds were first dissolved in 100% DMSO at 6 mM and further diluted in culture medium to the desired concentrations. Each inhibitor was analyzed in a three-fold serial dilution (initial concentration: 5 μM) in triplicates. The plates were incubated for 24 h in 5% O2, 3% CO2, 92% N2 and 95% humidity at 37 °C and subsequently frozen at −80 °C. Parasite survival was assessed by measuring the activity of parasite-expressed luciferase with the Bright-Glo™ Luciferase Assay System (Promega) according to the manufacturer's protocol. Luciferase activity was measured on a luminescence plate reader (Promega). The mean of the luciferase activity was calculated for each technical triplicate and survival of drug-treated parasites (in percentage) was calculated based on the luciferase activity of non-treated controls (100% parasite survival). In vitro antiplasmodial activity is expressed as 50% inhibitory concentration (IC50) of parasite survival. IC50 values were calculated using Prism (GraphPad, log(inhibitor) vs. normalized response – variable slope) in two independent experiments.

Protocol for antimalarial activity against P. falciparum parasites determined in in vitro cultures

In vitro cultures of Plasmodium falciparum. Intra-erythrocytic stages of P. falciparum strains Dd2 and 3D7 were cultured according to standard protocols12,46 and maintained under controlled atmospheric conditions at 5% O2, 3% CO2, 92% N2 and 95% humidity at 37 °C. Synchronization of cultures was achieved by sorbitol treatment.47
In vitro anti-Plasmodium falciparum activity assays. The sensitivity of the human malaria parasite P. falciparum to new plasmodione derivatives 11, 14, 16a, 16b, and 17, and plasmodione and chloroquine as control agents was determined by exposing the parasite to different concentrations of the compounds in in vitro microtiter tests using the SYBR® green I assay as described before.12,48

Briefly, synchronous ring stage parasites were incubated for 72 h in the presence of decreasing drug concentrations in microtiter plates (0.5% parasitemia, 1.5% hematocrit final). For this, the compounds were first dissolved in 100% DMSO at 6 mM and further diluted in culture medium to the desired concentrations. Each inhibitor was analyzed in a three-fold serial dilution (initial concentrations: 3–10 μM) in duplicates. The plates were incubated for 72 h in 5% O2, 3% CO2, 92% N2 and 95% humidity at 37 °C and subsequently frozen at −80 °C. Parasite replication was assessed by fluorescent SYBR® green staining of parasitic DNA and measured in a fluorescence plate reader (BMG Labtech, Germany).

The mean of fluorescence intensity was calculated for each technical duplicate and normalized on the fluorescence of non-infected red blood cells (background). Parasite multiplication (in percentage) of drug-treated parasites was calculated based on the fluorescence intensity of non-treated control wells (100% parasite multiplication). In vitro antiplasmodial activity is expressed as 50% inhibitory concentration (IC50) of parasite multiplication. IC50 values were calculated using Prism (GraphPad, log(inhibitor) vs. normalized response – variable slope).

Protocol for drug metabolite analysis in mouse urine after the administration of plasmodione

Urine collection from plasmodione-treated mice. Plasmodione was resuspended in a solution of tween80/ethanol (7[thin space (1/6-em)]:[thin space (1/6-em)]3) at 300 mg mL−1, sonicated for 5–8 min and diluted (1[thin space (1/6-em)]:[thin space (1/6-em)]10) in water. Three doses of the final solution were administered daily intraperitoneally at 30 mg kg−1 to one naïve and one infected mouse starting 1 day post infection (infection procedure as described above). A control naïve mouse was injected daily with a 1[thin space (1/6-em)]:[thin space (1/6-em)]10 dilution of tween80/ethanol (7[thin space (1/6-em)]:[thin space (1/6-em)]3) in water without a compound (mock). Urine was collected (1) from non-infected and non-treated mice and pooled, and (2) individually (50–70 μL) from the mock- and plasmodione-treated mice 24 h after each injection and stored at −20 °C until analysis.
Sample analysis by LC-MS/MS. Plasmodione was added in the pooled urine sample from non-infected and non-treated mice to establish a standard curve (final concentrations: 10, 20, 100, 200 μM). The urine samples were diluted with 200 μL distilled water. The SPE (Strata C18-E, 55 μM, 70 A, 50 mg/1 mL) columns were conditioned with 1 mL of methanol and 1 mL of distilled water. The columns were subsequently loaded with the samples and washed with 1000 μL of distilled water and eluted with 800 μL of methanol. The eluent was dried under vacuum in a Speedvac® concentrator. The residue was re-suspended in 100 μL of water and acetonitrile (1[thin space (1/6-em)]:[thin space (1/6-em)]1, v[thin space (1/6-em)]:[thin space (1/6-em)]v) and 5 μL of this solution was subjected to LC-MS/MS analysis (UHPLC coupling with a triple quadrupole Shimadzu LC-MS 8030) to allow the detection of plasmodione and putative glucuronic acid-conjugated metabolites (Fig. S44 and S45; S48–S51, ESI).
Identification of drug metabolites detected in mouse urine samples. 5 mM solutions of 6- and 7-hydroxylplasmodione 16a/16b were prepared in DMSO and stored at 4 °C before experiment. Hydroxy-plasmodione (2 μL of the 5 mM stock solution) was added to a 100 mM phosphate buffer pH 7.4 containing 0.5 mg mL−1 of mouse liver microsomes and 1 mM of uridine diphosphate glucuronic acid. The reaction mixture was gently homogenized. The reaction was stopped by adding 200 μL of acetonitrile at 0 °C at 0 min and 60 min of incubation. All the samples were stored at −80 °C until analysis. After thawing, samples were vortexed for 5 min, placed in an ultrasonic bath for 1 min, and centrifuged (15[thin space (1/6-em)]000g, 5 min, 4 °C). Supernatants were transferred to small plastic vials and subjected to LC-MS/MS analysis (negative ESI-MS, UHPLC coupling with a triple quadrupole Shimadzu LC-MS 8030). Collision energies (CE) and quadrupole (Q1, Q3) voltages applied for the MRM signals 345 → 329, 317 of hydroxy-plasmodiones were: CE = 30 V, Q1 = 24 V, Q3 = 20 V; those for the MRM signals 521 → 345, 329, 317 of glucuronides were: CE = 35 V, Q1 = 40 V, Q3 = 20 V. The results are shown in Scheme 11 and in the ESI (Fig. S46 and S47, ESI).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the French Centre National de la Recherche Scientifique (E. D. C., S. A. B.), the Institut National de la Santé et de la Recherche Médicale (S. A. B.), the University of Strasbourg (E. D.-C., S. A. B.), the Laboratoire d'Excellence (LabEx) ParaFrap (grant LabEx ParaFrap ANR-11-LABX-0024 to E. D. C. and S. A. B., half PhD doctoral salary for L. F. and postdoctoral salary for K. E.), the Région Alsace (half PhD doctoral salary for L. F.), the ANR PRC2017 (grant PlasmoPrim), the Equipement d'Excellence (EquipEx) I2MC (grant ANR-11-EQPX-0022 to S. A. B.), the ANR MetaboHUB infrastructure (ANR-11-INBS-0010 grant to F. F.), and the ERC Starting Grant N°260918 (S. A. B.). The authors are indebted to Vrushali Khobragade for measuring the IC50 value of the benzhydrol 14 and Patrick Gizzi (UMS3286 CNRS-Université de Strasbourg, PCBIS Plate-forme de chimie biologie intégrative, Illkirch) for the drug metabolism studies with urine from plasmodione-treated mice.

Notes and references

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Footnote

Electronic supplementary information (ESI) available: 1H, 13C and 19F NMR spectrum, mass spectrum of compounds 1–17 and the UHPLC-MS/MS spectrum of drug metabolites from mouse urine. See DOI: 10.1039/c8ob00227d

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