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and E. Davioud-Charvet, Org. Biomol. Chem., 2018, DOI: 10.1039/C8OB00227D.

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COMMUNICATION

Takeharu Haino et al.

Solvent-induced emission of organogels based on tris(phenylisoxazolyl)

benzene

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_DOI:1₁₀3_{.1039/C8OB00227D}

Synthesis of plasmodione metabolites and

C-enriched

plasmodione as chemical tools for drug metabolism investigation

Liwen Feng,^aDon Antoine Lanfranchi,^aLeandro Cotos-Munoz,^aElena Cesar-Rodo,^aKatharina

Ehrhardt,^bAlice-Anne Goetz,^bHerbert Zimmermann,^cFrançois Fenaille,^dStephanie Blandin^b* and

Elisabeth Davioud-Charvet^a*

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

Abstract: Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas. Resistance to

antimalarial drugs 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

fully ¹³C-labelled lead drug and putative metabolites, have been designed and synthetized for drug metabolism

investigation. Furthermore, with the help of UHPLC-MS/MS, two of the drug metabolites have been identified from urines

of drug-treated mice.

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effective vaccine, preventive measures to avoid mosquito bites and

drug treatment of symptomatic cases remain crucial but are

challenged by 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.^10,11,12,13The compound displays low and equally stable

IC₅₀values (≈ 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 (IC₅₀

> 50 µM) or 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

Introduction

Malaria is a parasitic disease, threatening 3.2 billion people living

in the tropical and sub-tropical regions.¹According 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 445,000 deaths in 2016, with

70% malaria decedents being young children.1 Plasmodium is a

protozoan parasite that infects humans via the infectious bite of a

female anopheles mosquito. Five plasmodia species are responsible

for malaria in Humans.^2,3,4Plasmodium falciparum is the most

dangerous parasite species causing severe disease such as cerebral

malaria, hemolytic anemia and respiratory distress, and is

,5,6

responsible for 99% of fatal cases.1 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 malarial parasites. Combined with several

drug partners artemisinin has been extremely useful in treatment of

multi-drug resistant infections, and artemisinin-based combinations

therapies (ACT) are currently recommended by WHO as first-line

therapy to treat and cure malaria.1^,7,8,9Despite 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 changes has an impact on the geographical distribution and

proliferation of vector Anopheles mosquitoes. Given the lack of an

redox-active and enters

a continuous redox-cycle reducing

methemoglobin (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. The 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.¹⁴All 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).^10,13Then, in 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

methemoglobin(Fe^III) as electron acceptor. Plasmodione redox

cycling leads to the inhibition of glutathione regeneration,

production of ROS and regeneration of hemoglobin(Fe^II) that cannot

be digested by the parasite.^13,15This NADPH-dependent redox cycle

ends with the death of the parasites that appears pycnotic,¹²and

^a.Université de Strasbourg, Université de Haute-Alsace, CNRS, LIMA, UMR 7042,

Team Bioorganic and Medicinal Chemistry, ECPM 25 Rue Becquerel, 67087

Strasbourg, France. E-mail: elisabeth.davioud@unistra.fr

^b.Université de Strasbourg, CNRS, Inserm, MIR UPR9022/U1257, F-67000

Strasbourg, France. E-mail: sblandin@unistra.fr

^c.Max Planck Institut für medizinische Forschung, Department of Biomolecular

Mechanisms, Jahnstraße 29, D-69120 Heidelberg.

^d.CEA Paris-Saclay, Laboratoire d’Etudes du Métabolisme des Médicaments

(LEMM), Bât. 136, 91191 Gif-Sur-Yvette cedex France.

the enrichment of membrane-associated hemichrome,¹⁴

biomarker of senescence of RBC responsible for their rapid removal

by macrophagic phagocytosis in vivo.

a

† Electronic Supplementary Information (ESI) available: ¹H, ¹³C and ¹⁹F NMR

spectrum, mass spectrum of compounds 1-17 and UHPLC-MS/MS spectrum of

drug metabolites from mice urine. See DOI: 10.1039/x0xx00000x

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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

Results and discussion

example, the benzoxanthone (metabolite III on Scheme 1)

prevents heme detoxification as potently as chloroquine,¹⁴and

was proposed to be formed after 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 to: i)

the complexity of the malarial parasite that oscillates between

different stages RBCs; ii) the redox chemistry that involves very

rapid reactions due to electron transfers,^16,17in 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 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.

The original plasmodione can be synthetized in one step from

commercial starting materials, menadione and the

corresponding p-trifluoromethylphenyl acetic acid (Scheme 2.A),

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

13

starting materials, the synthetic route of the fully C₁₈-labeled

plasmodione had to be built totally differently, with almost all C-

enriched, except the -CF₃group. Based on the cheapest

commercial available starting materials, ¹³C₆-p-dibromobenzene,

¹³C₆-benzene, ¹³C₄-succinic anhydride, ¹³C₁-dimethylformamide

13

and ¹³C₁-acetic acid, the synthesis of the almost fully C₁₈-

enriched plasmodione was designed through a convergent

synthetic route via two key intermediates, the tetralone and the

4-trifluoromethyl-benzaldehyde, using a 10 step-long sequence

(Scheme 2.B). Each step of this total synthesis was set up and

improved by using first, the unenriched compounds.

Synthesis of the ¹³C₁₀-enriched tetralone 3b

13

The uniformly C₁₀-labeled tetralone 3b was synthesized

13

according to the reported procedure.¹⁸The C₁₀-tetralone 3b

was produced by Friedel-Crafts reaction (yield 96%), Woff-

Kishner reduction (yield 95%) and cycloaddition reaction (yield

95%) from ¹³C₄-succinic acid and ¹³C₆-benzene with excellent

1

yields (total yield 87%, Scheme 3). All H and ¹³C NMR spectra of

the tetralone precursors 1-2 are shown in Fig. S1-S, ESI†.

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Scheme 2. A) The synthesis of unlabelled plasmodione. B) The retrosynthesis of fully ¹³C-labelled plasmodione.

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 atom, 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

1

13

both H and ¹³C NMR spectra of the newly prepared C₁₀-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

Scheme 3. The synthesis of the tetralone 3a/3b.

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 I₂and n-BuLi under classical conditions (yield 75%).¹⁹

The limitation of the yield was due to generation of o-

bromoiodobenzene, a side product due to the resonance effect. No

matter how varying the reaction temperature (from -78 °C to 25 °C),

the o-bromoiodobenzene was produced at a ~ 20% conversion of

the starting material.

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.²⁰In

chemical processes, the [CuCF₃] is usually used in

trifluoromethylation as catalyst.²¹However, it is not stable in open

air and thus not adapted to prepare the trifluoromethylation

without a glove box.²²Generation of CuCF₃in situ has been

developed by using different generators such as NaCO₂CF₃and

MeCO₂CF₃that need high temperature (~ 150-160 °C) for their

activation.²³However, 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, TMSCF₃has been broadly

used in trifluoromethylation with fluoride compounds under gentle

conditions to form CF₃^-.^24,25The N-Methyl-2-pyrrolidone (NMP) was

chosen as solvent for the reaction because of its b.p. 202 °C.

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. Owing to 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 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

1

natural tetralone 3a (Fig. S5, ESI†). Because of J_13C-1H, the spin of

1

both ¹³C and H atoms are ½ and the peaks of the protons that are

bound to the ¹³C atom are split into two signals. Besides, owing to

additional J-coupling such as ³J ⁴J between proton and ¹³C, the

shape of the peaks is wide and the signals have low sensitivity,

making difficult to identify the structure of ¹³C-enriched compounds

by NMR spectra. Nevertheless, ¹³C-enriched and unenriched

compounds with the same molecular structure keep the same

chemical properties, and thus display invariable chemical shifts in

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.

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In the next step, the hydroformylation with Grignard reagent

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DOI: 10.1039/C8OB00227D

produced the p-trifluoromethylbenzaldehyde 6a (50%) /6b (30%) in

two steps (overall yield). This methodology for benzaldehyde

formation in relatively high temperature (0 °C to 5 °C) was

developed by Gallou et al²⁸. By using iPrMgBr, the bromoaryl group

was transformed to [phen]₃-Mg Grignard reagent in situ, a reaction

that was accelerated using n-BuLi. Subsequently, the resulting

Grignard reagent underwent nucleophilic attack to DMF or ¹³C₁-

DMF to form desired benzaldehyde products. Because of the highly

water sensitivity of this reaction and the volatility of starting

material, the use of Na₂SO₄in situ was required to remove the

maximum of water. ¹H and ¹³C NMR spectra of the synthetic 4-

trifluoromethylbenzaldehyde precursors 4a/4b are shown in Fig.

S7-S8, ESI†.

1

Based on the H and ¹³C NMR, we could verify the structure of

1

¹³C₇-p-trifluoromethylbenzaldehyde 6b. In the H NMR spectrum of

6b, the peak of the proton at the aldehyde position at 10.1 ppm

was split to a doublet-doublet signal with a ¹J_13C-1Hconstant 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

1

Scheme 4. Synthesis of the 4-trifluoromethyl benzaldehyde 6a/6b.

[a] The yield of trifluoromethylation reaction was estimated by H

NMR using trifluoromethoxytoluene as an internal standard. [b]

Yields of the 2 step-reactions using p-bromoiodobenzene 4a/4b as

starting materials.

8.01 ppm and 7.81 ppm, with J_13C-1Hconstant 163.2 Hz and 163.8

1

Hz, respectively, in the ¹³C NMR spectrum of 6b, the peak of carbon

at the aldehyde position at 191.2 ppm was divided to 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.

The MDFA was originally studied first by Chen et al.^26,27First, the

methyl group of MDFA is substituted by CuI. Subsequently,

decarboxylation and desulfonylation take place to produce a

carbene :CF₂by heating (~ 80-100 °C), and then the :CF₂carbene

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).

1

These H 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”

13

The two resulting intermediates C₁₀-tetralone 3b and ¹³C₇-p-

trifluoromethylbenzadehyde 6b were combined through an

13

optimized “express tetralone route” to produce C₁₈-plasmodione

10b in 4 steps as described in our previous study (Scheme 5).²⁹First,

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 RhCl₃catalysis proceeded with similar

yields (80%) to obtain the α-benzyl naphthols 8a/8b. The naphthols

were submitted 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)₂S₂O₈to produce the ¹³C₁-enriched plasmodione

Conditions

Yield^*

CuI (1.5 equiv.), MDFA (5 equiv.), NMP, 16 h, 100 °C

CuI (2 equiv), TMSCF₃(2 equiv.), NMP, 16 h, 100 °C

95%

90%

Table 1: Conditions for the trifluoromethylation reactions. NMP =

N-Methyl-2-pyrrolidone. *The yields were estimated by ¹H NMR

using trifluoromethoxytoluene as internal standard.

13

10a and the fully C₁₈-enriched lead plasmodione 10b with 50 and

Due to the volatility of the desired product, the crude product

was very hard to purify in 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 starting material (p-bromoiodobenzene 4a/4b) was

consumed. The trifluoromethylation was also found very sensitive

to the quality of CuI (stored under argon).

55% yield, respectively.

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.²⁹In order to follow the reaction kinetics and to

track the reaction products by H NMR spectroscopy (Figure 1), we

used unlabeled 2-(4-(trifluoromethyl)benzyl)-naphthalene-1,4-

1

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dione 9a and ¹³C₁-acetic acid as starting materials in a model the CF₃group was covered by the intense signals of _Vt_ih_ewe _Ao_rtt_ih_cle_er_OnlinC_e-

19

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DOI: 10.1039/C8OB00227D

reaction. As observed, after 15 min stirring, the reaction conversion enriched carbons. F NMR spectra of the synthetic C-enriched

reached almost 50% and the desired product 10a was quickly plasmodione 10 are shown in Fig. S23, ESI†. All these NMR data

generated. After 60 min, over 70% of the starting material 9a was corroborated the exact assignment to the molecular structure of

consumed. The best conversion was obtained when the reaction desired ¹³C-enriched plasmodione 10b and 10a. Finally, the high

was stopped at 75 min. Interestingly, for comparison, the reaction resolution ESI spectrum with positive ionization recorded for the

13

with the unlabeled acetic acid as reaction partner took a shorter

C -enriched plasmodione 10b confirmed both the molecular

18

time (60 min) at the same stage of the reaction. After 90 min, the mass peak of plasmodione increased by 18 m/z units and a >90%

13

generation of side products was observed and the yield of the

C -enrichment (Fig. S24, ESI†). Overall, 50 mg of fully ¹³C-

18

13

reaction decreased. The preparation of the fully C₁₈-enriched- enriched-plasmodione 10b was obtained though the 10 step-long

plasmodione 10b from 9b using the optimized process and 2 synthesis.

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†.

Figure 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 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.

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

Scheme 5. The synthesis of ¹³C-enriched plasmodione 10a/10b from

tetralone 3a/3b and p-trifluoromethylbenzadehyde 6a/6b.

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 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).

Based on the ¹H and ¹³C NMR spectra, we confirmed the structure

13

1

of C₁₈-plasmodione 10b. In the H NMR spectra of 10b, the peaks

of the proton at the naphthoquinone part were split into two

1

signals, at 8.10 ppm and 7.71 ppm with J_13C-1Hconstants 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 ¹J_13C-1Hconstants of

159.1 Hz and 147.5 Hz. The two protons at the benzyl chain were

1

revealed as a doublet peak at 4.08 ppm with J_13C-1Hconstant 130.3

Hz. The protons of methyl group showed signals at 2.25 ppm with

1

¹J_13C-1Hconstant 128.9 Hz. In both H NMR and ¹³C NMR spectra, all

13

the peaks of C₁₈-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

Scheme 6. The synthesis of 1-¹³C₁-plasmodione.

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The identification and quantification of low-level impurities that

are generated during the drug production processes gain more and revealed through an intense signal at 191.^D72^OIp^{: 1}p⁰m^.10i³n^9/t^Ch⁸e^OBC⁰⁰N²²M^7DR

more importance in medicinal chemistry research and spectrum. This signal did not belong to plasmodione 10c, and

development.³²Interestingly, during the synthesis of the mono ¹³C₁- corresponded to an impurity generated in the Kochi-Anderson

labeled compound, we discovered one of the trace amount reaction. Using DOSY-NMR, we showed that the amount of this

nonchromophoric impurities that was undetectable in the NMR impurity was less than 4% (detection limit). By comparing both the

ARTICLE

The presence of an impurity containing a 1-¹³C₁-enriched was

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13

spectra.

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).

Based on the mechanism of 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 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 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 trace level.

IC₅₀± SD

(nM)

Compound

Synthetic route

(n steps)

P. berghei

plasmodione

Kochi-Anderson (1)

181.4 ±14.7

(¹³C_1-methyl-plasmodione 10a) “tetralone express” (4) 180.6 ± 5.2

Antimalarial activities of ¹³C-enriched plasmodiones

In order to evaluate the impact of ¹³C atoms 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

were around 180 nM indicating that ¹³C-enrichment did not affect

the antimalarial activity of plasmodiones.

(¹³C₁₈-plasmodione 10b)

(¹³C _C1—plasmodione 10c)

“tetralone express” (10) 186.1 ± 2.6

Kochi-Anderson (1) 181.3 ± 3.2

Table 2. Antimalarial activities (presented as IC₅₀values) of

plasmodione and ¹³C-enriched plasmodiones (10a-c) against murine

P. berghei pRBCs determined in ex vivo cultures (two individual

experiments each).

Figure 2. Structures of plasmodione and its putative metabolites.

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Synthesis of several putative plasmodione metabolites

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†.

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

(Figure 2). Although there are no CYP450 enzymes in the pRBC

cultures in vitro, the Plasmodium parasites digest large amounts of

methemoglobin and release free heme iron III (FPIX Fe^III), 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,³⁴the 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 12¹⁰, benzhydrol 13¹⁰,

benzoxanthone 15¹³, 6-hydroxy 16a²⁹and 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.

Scheme 8. Synthesis of the (±)-3-[4-trifluoromethyl-(phenyl)-

hydroxy-methyl)menadione 14.

Synthesis of benzoxanthone 15

The first synthesis of the benzoxanthone 15 was established via

a benzophenone intermediate (dihydrobenzoylmenadione) through

S_NAr reaction.¹³However, 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, the bromomenadione 20 was first

prepared by bromination of menadione with bromine and pyridine

with a 93% yield. Subsequently, the quinone core of compound 20

was reduced by tin chloride/HCl as reduction reagent, and then

protected by 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-

Synthesis of epoxyl-plasmodione derivative 11

Epoxyl-plasmodione derivative 11 has been formed by

epoxidation reaction of plasmodione (Scheme 7). A similar reaction

has been reported in the literature.³⁵In 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 a 59 % yield. ¹H, ¹³C and ¹⁹F NMR spectra of epoxyl-

plasmodione derivative 11 are shown in Fig. S27-S29, ESI†.

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

fluoro-4-(trifluoromethyl)benzoyl

chloride

affording

the

Similar to benzhydrol derivative 13 (R = -Br), the benzhydrol

derivative 14 (R = -CF₃) was obtained through the same synthetic

pathway (Scheme 8).¹⁰Its synthesis successively encompassed the

benzoylmenadione derivative 22. A selective mono-deprotection

reaction removed one of the MOM groups of compound 22

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Scheme 9. Optimized synthesis of the benzoxanthone derivative 15.

producing compound 23. Noteworthy is that only the O-MOM

group in β-position to the C=O of the benzoyl chain can be deprotec

ted by MgBr₂because of the chelating effect.³⁶Of note, this

deprotection of β-ketophenols had been successfully set-up in

flavones chemistry in the team. The aromatic nucleophilic

substitution/cycloaddition of benzoylmenadione derivative 23 with

K₂CO₃in acetone produced the O-MOM-benzoxanthone derivative

24 (35%) in mixture with partially deprotected benzoxanthone 15

(35%) because the O-MOM group could be deprotected by HF

Scheme 10. Synthesis of the 2-hydroxymethyl-plasmodione

derivative 17.

generated during the reaction. Finally, O-MOM-benzoxanthone 24 Antimalarial activity of putative drug metabolites

was deprotected to give benzoxanthone 15 in the mixture of

We determined the antimalarial activity of each synthetic drug

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

metabolite 11-17 against human P. falciparum pRBCs in in vitro

cultures (Table 3). Some of the IC₅₀values were reported in our

previous publications.^10,14,29Importantly, 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 IC₅₀values 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 IC₅₀values of the benzhydrols 13 and 14

(~735 nM) and benzoxanthone 15 (613 ± 79.0 nM) were more than

10 times higher than the IC₅₀value 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 parental 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:target or drug metabolite:target interactions.

1

strategy. The H 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†.

Synthesis of 2-hydroxymethyl-plasmodione derivative 17

As in the classical Kochi-Anderson reaction, the key

intermediate hydroxymethyl radical (^•CH₂=O) was generated from

MeOH in the presence of AgNO₃and (NH₄)₂S₂O₈. ³⁷Reaction

between the hydroxymethyl radical and the desmethyl-

plasmodione 9a generated the hydroxymethylplasmodione 17

(Scheme 10). The product was purified by chromatography of

1

column (SiO₂, Toluene) and isolated with 49% yield. H, ¹³C and ¹⁹F

NMR spectra of 2-hydroxymethyl-plasmodione derivative 17 are

shown in Fig. S41-S43, ESI†.

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Compound

Mean IC₅₀± SD [nM] (n)^a

Compound

Mean IC₅₀± SD [nM] (n)^a

58 ± 11 (9)

613 ± 79.0 (2)^e

241 ± 131 (2)

107 ± 14 (3)

273 ± 31 (3)

>1000 (3)^b

13 : 734^b

102 ± 43 (3)

14 : 737 (2)^c

Table 3. Antimalarial activities (presented as IC₅₀values) 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. [a] IC₅₀chloroquine = 99 ± 19 (n=6), with (n) giving the number of repeats. [b] data from ref. 10, were

obtained using radioactive [³H]hypoxantine 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]hypoxantine to measure parasite

survival.

Identification of drug metabolites in urines of plasmodione- detected at m/z = 522.11 (see Fig. S44-S45, ESI†). Based on mass

treated mice

spectrometry analysis of the glucuronide metabolite found in urines,

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,³⁸we predicted that the 6- and 7-

hydroxy-plasmodiones (16a/16b) were the most likely metabolites

of plasmodione and 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

A preliminary investigation on drug metabolism was performed

on urines 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

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Inten.(x10,000)

Inten.(x1,000)

1.00

7.5

5.0

2.5

0.0

329.0

317.1

345.1

0.75

0.50

0.25

317.1

329.0

330

0.00

320.0

325.0

m/z

320

340 m/z

Inten.(x10,000)

3.0

317.1

345.1

3.0

2.0

1.0

0.0

329.0

2.0

1.0

0.0

317.1

329.0

330

320.0

325.0

m/z

320

340 m/z

Inten.(x10,000)

329.0

345.1

2.5

317.1

2.5

0.0

317.1

329.0

0.0

320.0

325.0

m/z

320

330

340 m/z

Scheme 11. Identification of drug metabolites in urines 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 by SPE and analyzed by UHPLC-MS/MS as

described. b) 50 μM of 6-hydroxy-plasmodione 16a and 7-hydroxy-plasmodione 16b were incubated with mouse liver microsome (0.5

mg/mL) and uridine diphosphate glucuronic acid (1 mM). The retention time of both 6- and 7- hydroxy-plasmodione (16a and 16b) was

0.83 min. The retention time of both 6- or 7-hydroxy-plamodione glucuronides was 0.63 min. I_317.1/I₃₂₉is the ratio of the intensity of the

peak at m/z 317.1 and the intensity of the peak at m/z 329.

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¹³C-labeled compounds keep the same chemical properties as

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 I_317.1/I₃₂₉= 1.6; for the 7-hydroxy-plasmodione

16b, the major ion fragment was 329 m/z, and the ratio of intensity

was I_317.1/I₃₂₉= 0.4 (Scheme 11.A, Fig. S46-S47, ESI†). In order to

confirm the structure of the glucuronide metabolite in urines, 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 results presented above, the

MS/MS analysis revealed that the main peaks were found at m/z

345, 317 and 329, with the ratio I_317.1/I₃₂₉= 2.8 for the 6-hydroxy-

plasmodione glucuronide, and the ratio I_317.1/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 11.B and Fig.

S48-S51, ESI†). The intensity ratio of I_317.1/I₃₂₉was 1.3 for the

observed hydroxyl-metabolite (retention time: 0.86 min),

suggesting that the major part of the hydroxy-metabolite found in

urines was the 6-hydroxy-plasmodione 16a. The 6-hydroxy-

plasmodione 16a concentration was evaluated to be 1 µM in the

urines 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 urines is derived

from the main 6-hydroxy-plasmodione 16a. In conclusion, based on

UHPLC-MS/MS analysis, we showed that the main plasmodione

metabolites present in urines 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.

unenriched compounds; however, the additional ne^Vu^iet^wro^An^rticin^{le O}eⁿa^licⁿh^e

DOI: 10.1039/C8OB00227D

¹³C leads to an increased molecular weight and a decreased kinetic

energy, around 10% in a reported study.³⁹It is therefore difficult to

not anticipate whether the reactivity of the ¹³C-enriched

compounds would be different from that of unlabeled compounds.

Besides, we observed large differences in the signal patterns of

¹H and ¹³C NMR spectra of ¹³C-labeled versus unlabeled compounds.

1

The most significant effect is the generation of J_13C-1Hcoupling

constants in the ¹H NMR signals that ranged from 120 Hz to 170

Hz.⁴⁰Additional 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

IC₅₀values 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 studies⁴¹and the cellular localization of the drug

and its metabolites using nanoSIMS imaging^42,43,44. 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 100nM

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 urines of

plasmodione-treated mice. Of note, the potent antimalarial 6-

hydroxy-plasmodione 16a is the major metabolite found in urines. 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 urines.

Future investigations will also aim at (1) optimizing the conditions

to extract the different drug metabolites from pRBCs 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.

Conclusions

The work described here provides new insights into the synthesis of

fully ¹³C-enriched plasmodiones and unlabelled putative

metabolites. The C₁₈-plasmodione 10b was synthesized by a 10

steps-long sequence via an optimized “tetralone express route”

with an overall yield of 5 % for 10b versus 10.4 % for the mono-¹³C₁-

_methyl-plasmodione 10a. Besides, another mono-¹³C_C1-labeled

plasmodione 10c was produced.

Experimental

General

13

Nuclear Magnetic Resonance (NMR): The Nuclear Magnetic

Resonance (NMR) spectra were registered 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,

³¹P NMR 60 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: H NMR, 7.26 ppm and

¹³C NMR, 77.36 ppm; for DMSO H 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

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 9a

versus 75% for labeled 9b). Essentially, ¹³C atoms have the same

number of electrons as ¹²C but different number of neutrons. The

1

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(broad), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), NMR (400 MHz, CDCl₃) (see Fig. S3, ESI†), ¹³C NMR (100 MHz, CDCl₃)

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13

DOI: 10.1039/C8OB00227D

dd (doublet of doublets). In addition, the following acronyms will be (see Fig. S4, ESI†); spectra related to 4-(phenyl- C₆)butanoic-

used: C=O carbonyl group; C_q: quaternary carbon; CH₂: secondary 1,2,3,4-¹³C₄acid (2b), ¹H NMR (400 MHz, CDCl₃) (see Fig. S3, ESI†),

carbon; CH₃: methyl group; ArH: aromatic proton of the menadione ¹³C NMR (100 MHz, CDCl₃) (see Fig. S4, ESI†); spectra related to 3,4-

1

core; PhenylH: aromatic proton of the phenyl moiety; PyH: aromatic dihydronaphthalen-1(2H)-one (3a), H NMR (400 MHz, CDCl₃) (see

proton of the pyridine moiety. Elemental analysis: Elemental Fig. S5, ESI†), ¹³C NMR (100 MHz, CDCl₃) (see Fig. S6, ESI†); spectra

analyses (EA) were performed by “Service de Microanalyses” at the related to 3,4-dihydro-naphthalen-1(2H)-one-¹³C₁₀(3b), ¹H NMR

Institut de Chimie de Strasbourg. Mass spectrometry: Mass (400 MHz, CDCl₃) (see Fig. S5, ESI†), ¹³C NMR (100 MHz, CDCl₃) (see

spectrometry was performed at Service de Spectrométrie de Mass- Fig. S6, ESI†), are shown in the supplementary information.

Université de Strasbourg. Mass spectra (ESI-MS) was obtained on a

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 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 Na₂S₂O₃was 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 MgSO₄and concentrated under reduced pressure. The crude

product was purified by silica chromatography (100%, cyclohexane

Rf: 0.89), 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

microTOF LC spectrometer (Bruker Daltonics, Bremen). High

Resolution Mass (HRMS) spectra were measured and fitted with

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

1

(4b): H NMR (400 MHz, CDCl₃): δ 7.54 (dq, 2H, J = 167.7 Hz, J = 8.5

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 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 TLC's were revealed under UV (325 nm and 254 nm) and

with detection reagents. Analytical TLC was carried out on pre-

coated Sil G-25 UV₂₅₄plates 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 Aluminum oxide activated, basic, Brockmann Grade I (60 mesh,

58 Å) purchased from E. Merck. Analytical TLC was carried out on

pre-coated Silica gel 60 F₂₅₄aluminum plates from Merck.

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 argon atmosphere for 16 hours. The reacting mixture

was diluted by 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 MgSO₄and concentrated under

reduced pressure. The light-yellow oil crude was used in the next

1

step. 1-bromo-4-(trifluoromethyl)benzene (5a): H 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

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.¹⁸Spectra related to 4-

(376

MHz,

CDCl₃):

δ

-62.79

ppm.

1-bromo-4-

(trifluoromethyl)benzene-1,2,3,4,5,6-¹³C₆(5b): H 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.

1

oxo-4-phenylbutanoic acid (1a), H NMR (400 MHz, CDCl₃) (see Fig.

S1, ESI†), ¹³C NMR (100 MHz, CDCl₃) (see Fig. S2, ESI†); spectra

1

related to 4-oxo-4-(phenyl-¹³C₆)butanoic-1,2,3,4-¹³C₄acid (1b), H

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

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 11

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Organic & Biomolecular Chemistry

Page 13 of 18

ARTICLE

Synthesis of 4-(trifluoromethyl)benzaldehyde (6a/6b): To

Journal Name

a

(m),125.7 (t, J = 60.8 Hz), 29.2 (t, J = 37.0 Hz), 27.5 (_Vt_i,_ewJ _A=_rt3_ic7_le.0_OnH_linz_e)

solution of 1-bromo-4-(trifluoromethyl)benzene-1,2,3,4,5,6-¹³C₆5b ppm (see Fig. S13, ESI†). ¹⁹F NMR (376 MHz_D, _OCD_I:C₁₀l₃_.)₁:₀δ₃₉-_/6_C2₈._O70_B0(₀td₂,₂₇J_D=

(1 equiv., 1021 mg, 4.42 mmol) in tetrahydrofuran (4.42 mL) with 31.3 Hz, J = 3.2 Hz) ppm (see Fig. S14, ESI†). ESI-MS m/z: [M+H]⁺

Na₂SO₄anhydrous (0.4 g), isopropylmagnesium bromide (0.53 calcd for ¹³C₁₇¹²C₁H₁₄F₃O₁: 320.20; found 320.16.

equiv., 2.9 M in 2-methyltetrahydrofuran, 0.81 mL, 2.34 mmol) was

added dropwise for 30 min under 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. 1M citric acid solution (10 mL) was added to the

mixture and the aqueous layer was extracted with diethylehter

(3×25 mL) and dried over MgSO₄and 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-

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 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

MgSO₄and concentrated under reduced pressure. The crude

product was purified by silica chromatography (cyclohexane/EtOAc,

9/1, Rf = 0.50) to give the light-yellow solids (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-

1

1,2,3,4,5,6-¹³C₆(6b): H 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†).

1

ol-¹³C₁₀(8b): H 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 =

Synthesis

naphthalen-1(2H)-one

of

2-(4-(trifluoromethyl)benzylidene)-3,4-dihydro- 156.1 Hz, phenylH), 7.25 (dm, 1H, J = 154.5 Hz, ArH), 5.14 (q, 1H, J =

(7a/7b): mixture of 3,4- 3.7 Hz, OH), 4.23 (d, 2H, J = 129.1 Hz, CH₂) ppm (see Fig. S15, ESI†).

A

dihydronaphthalen-1(2H)-one-¹³C₁₀3b (1 equiv., 159.5 mg, 1.02 ¹³C NMR (100 MHz, CDCl₃): δ 149.0 (t, J = 68.4 Hz), 144.3 (ddm, J =

mmol) and 4-(trifluoromethyl)benzaldehyde-1,2,3,4,5,6-¹³C₆6b (1 99.6 Hz, J = 53.0 Hz), 134.1 (q, J = 54.3 Hz), 129.4 (tm, J = 58.3 Hz),

equiv., 185 mg, 1.02 mmol) was stirred at room temperature. Then, 129.1 (q, J = 33.0 Hz), 129.0 (tm, J = 59.7 Hz), 128.3 (tm, J = 56.1 Hz),

KOH (1.2 equiv., 68.8 mg, 1.23 mmol) in ethanol (4 mL) was added 126.4 (tm, J = 54.1 Hz), 125.7 (tm, J = 53.6 Hz), 125.9 (qm, J = 31.7

dropwise to the mixture. The mixture was poured in 30 mL ice cold Hz), 124.9 (qm, J = 61.1 Hz), 124.4 (q, J = 265.0 Hz), 121.3 (tm, J =

water and the white pure product precipitated. The product was 59.8 Hz), 120.7 (tm, J = 56.5 Hz), 119.9 (qm, J = 61.3 Hz), 36.5 (t, J =

filtrated, washed with ice cold water (2×5 mL) and dried under 44.3 Hz) ppm (see Fig. S16, ESI†). ¹⁹F NMR (376 MHz, CDCl₃): δ -

vacuum to yield

cyclohexane/EtOAc, 8/2, Rf

a

beige solid (248 mg, 76%). (TLC: 62.44 (dt, J = 32.1 Hz, J = 3.1 Hz) ppm (see Fig. S17, ESI†). ESI-MS

=

0.51). 2-(4-(trifluoromethyl) m/z: [M+H]⁺calcd for ¹³C₁₇¹²C₁H₁₄F₃O₁: 320.23; found 320.16.

1

benzylidene)-3,4-dihydronaphthalen-1(2H)-one (7a): H 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):

δ187.9 (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

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 (3:1).

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 NaHCO₃was 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 MgSO₄and 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

1

(9a): H 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

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Organic & Biomolecular Chemistry

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Page 14 of 18

Journal Name

ARTICLE

Hz, phenylH), 6.63 (s, 1H, ArH), 3.95 (s, 2H, CH₂) ppm (see Fig. S18, Synthesis of 2-methyl-3-(4-(trifluoromethyl)benzyl)naphthalene-

View Article Online

ESI†). ¹³C NMR (100 MHz, CDCl₃): δ 185.2, 185.1, 150.1, 141.3, 1,4-dione-1-¹³C (10c): 2-methylnaphthalene-1,4-dione-1-¹³C (1

DOI: 10.1039/C8OB00227D

136.2, 134.3, 134.2, 132.4, 130.1, 129.7 (q, J = 32.5 Hz), 127.1, equiv., 108.3 mg, 0.325 mmol) and 2-(4-(trifluoromethyl)

126.6, 126.1 (q, J = 3.83 Hz), 124.4 (q, J = 274.9 Hz) 36.0 ppm (see phenyl)acetic acid (5 equiv., 99.2 mg, 0.095 mL, 1.63 mmol) were

Fig. S19, ESI†). ¹⁹F NMR (376 MHz, CDCl₃): δ -62.55 ppm (see Fig. solubilized in 14 mL of a solution acetonitrile-water (3:1). The

S20, ESI†). HRMS (ESI) m/z: [M+H]⁺calcd for C₁₈H₁₂F₃O₂: 317.0784; reaction mixture was heated at 85°C and silver nitrate (0.35 equiv.,

found 317.0791. 2-((4-(trifluoromethyl)phenyl-1,2,3,4,5,6-¹³C₆) 19.3 mg, 0.114 mmol) was added. Ammonium persulfate (1.3

methyl-¹³C)naphthalene-1,4-dione-¹³C₁₀(9b): ¹H NMR (400 MHz, equiv., 96.4 mg, 0.423 mmol), in a solution of acetonitrile-water

CDCl₃): δ 8.08 (dm, 2H, J = 157.4 Hz, ArH), 7.74 (dm, 2H, J = 157.3 (3:1), was added dropwise. The reaction mixture was stirred for 3

Hz, ArH), 7.59 (dm, 2H, J = 157.3 Hz, phenylH), 7.39 (d, 2H, J = 157.5 hours. The resulting mixture was evaporated and 10 mL of

Hz, phenylH), 6.63 (dm, 1H, J = 159.0 Hz ArH), 3.96 (d, 2H, J = 130.4 dichloromethane was added. The aqueous layer was extracted with

Hz, CH₂) ppm (see Fig. S18, ESI†). ¹³C NMR (100 MHz, CDCl₃): δ dichloromethane (3×10 mL) and the organic layer were combined

185.2 (t, J = 49.0 Hz), 185.1 (t, J = 49.0 Hz), 150.1 (qm, J = 52.7 Hz), and extracted with brine, dried over MgSO₄and concentrated

141.3 (qm, J = 51.6 Hz), 136.9-135.4 (m), 135.0-133.4 (m), 132.4 under reduced pressure. The crude product was purified by silica

(tm, J = 41.3 Hz), 130.1 (tm, J = 53.9 Hz), 129.2 (qd, J = 29.2 Hz, J = chromatography (toluene, Rf=0.51) and recrystallization (n-hexane,

8.6 Hz), 126.9 (tm, J = 59.1 Hz), 126.3 (tm, J = 53.8 Hz), 126.1 (tm, J 50 mL) to give a pure yellow solid (52.1 mg, 46%). ¹H NMR (400

= 55.7 Hz), 36.0 (t, J = 44.3 Hz) ppm (see Fig. S19, ESI†). ¹⁹F NMR MHz, CDCl₃): δ 8.13-8.06 (m, 2H, ArH), 7.75-7.68 (m, 2H, ArH), 7.55

(376 MHz, CDCl₃): δ -62.56 (dt, J = 31.8 Hz, J = 3.5 Hz) ppm (see Fig. (d, 2H, J = 7.9 Hz, phenylH), 7.35 (d, 2H, J = 7.9 Hz, phenylH), 4.08 (s,

13

S20, ESI†). ESI-MS m/z: [M+H]⁺calcd for

found 334.14.

C

¹²C₁H₁₂F₃O₂: 334.21; 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),

17

145.2 (d. J = 49.7 Hz), 144.7, 142.5, 134.0, 132.4 (d, J = 54.0 Hz),

Synthesis of 2-(methyl-¹³C)-3-(4-(trifluoromethyl)benzyl) naphtha-

lene-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

(3:1). The reaction mixture was heated at 85 °C and silver nitrate

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.

(0.35 equiv., 19.3 mg, 0.114 mmol) was added. Ammonium Synthesis

of

1a-methyl-7a-(4-(trifluoromethyl)benzyl)-1a,7a-

persulfate (1.3 equiv., 96.4 mg, 0.423 mmol) in a solution of dihydro-naphtho[2,3-b]oxirene-2,7-dione (11): 2-methyl-3-{[4-

acetonitrile-water (3:1) was added dropwise. The reaction mixture (trifluoromethyl)phenyl]methyl}-1,4-dihydronaphthalene-1,4-dione

was stirred for 90 min. The resulting mixture was evaporated and or plasmodione (1 equiv., 300 mg, 0.908 mmol) was dissolved in a

10 mL of dichloromethane was added. The aqueous layer was mixture of solvent: distillated water (1 mL) and methanol (4 mL)

extracted with dichloromethane (3×10 mL) and the organic layers (MeOH/H₂O 4:1). Then sodium hydroxide (0.5 equiv., 3 M, 0.151

were combined and extracted with brine, dried over MgSO₄and mL, 0.454 mmol) was added to the mixture at 0 °C. The resulting

concentrated under reduced pressure. The crude product was mixture was stirred for 10 min and subsequently H₂O₂(1.5 eq., 132

purified by silica chromatography (toluene, Rf

= 0.51) and mg, 0.13 mL, 1.36 mmol) was added. The reaction mixture was

recrystallization (n-hexane, 50 mL) to give a pure yellow solid (52.1 stirred at 0°C for 3 hours. The reaction was quenched by 10 mL of

mg, 46%). 2-(methyl-¹³C)-3-(4-(trifluoromethyl)benzyl) naphtha- distillated water. The resulting mixture was extracted by diethyl

lene-1,4-dione (10a): ¹H NMR (400 MHz, CDCl₃): δ 8.10-8.06 (m, 2H, ether (3×10 mL), the organic phase was extracted with brine, dried

ArH), 7.72-7.70 (m, 2H, ArH), 7.52 (d, 2H, J = 8.2 Hz, phenylH), 7.34 over MgSO₄and concentrated under reduced pressure. The crude

(d, 2H, J = 8.1 Hz, phenylH), 4.08 (s, 2H, CH₂), 2.25 (s, 3H, CH₃) ppm product was purified by recrystallization (Et₂O/n-hexane, 1/3, 20

(see Fig. S21, ESI†). ¹³C NMR (100 MHz, CDCl₃): δ 185.2, 184.6 (d, J = mL) to give a pure white crystal. (185 mg, 59%). m.p.: 117-119 °C.

3.6 Hz), 145.0 (d. J = 45 Hz), 144.5, 142.3, 133.8, 132.2, 132.0, 129.0, ¹H NMR (400 MHz, CDCl₃): δ 8.02-7.91 (m, 2H, ArH), 7.78-7.69 (m,

129.0 (q, J = 32.3 Hz, C-CF₃), 126.6, 126.5, 125.7, 125.7, 124.3 (q, J = 2H, ArH), 7.55 (d, J = 8.1 Hz, 2H, phenylH), 7.46 (d, J = 8.1 Hz, 2H,

273.0 Hz, CF₃), 32.5 (d, J = 2.2 Hz), 13.5 (t, J = 21.5 Hz) ppm (see Fig. phenylH), 3.67 (d, J = 15.0 Hz, 1H, CH₂), 3.40 (d, J = 15.0 Hz, 1H,

S22, ESI†). ¹⁹F NMR (375 MHz, CDCl₃): -62.49 ppm (see Fig. S23, CH₂), 1.84 (s, 3H, CH₃) ppm (see Fig. S27, ESI†). ¹³C NMR (100 MHz,

ESI†). ESI-MS m/z: [M+H]⁺calcd for ¹³C¹²C₁₈H₁₄F₃O₂: 332.10; found CDCl₃): δ 192.5, 192.2, 140.0, 134.6, 134.5, 132.1, 132.0, 129.8,

332.09. 2-(methyl-¹³C)-3-((4-(trifluoromethyl) phenyl-1,2,3,4,5,6- 129.3 (q, J = 32.3 Hz, C-CF₃), 127.4, 127.3, 125.6, 125.5, 124.2 (q, J =

¹³C₆)methyl-¹³C)-naphthalene-1,4-dione-¹³C₁₀(10b): ¹H NMR (400 254.6 Hz, CF₃), 67.3, 65.9, 31.9, 12.7 ppm (see Fig. S28, ESI†). ¹⁹F

MHz, CDCl₃): δ 8.09 (dm, 2H, J = 167.0 Hz, ArH), 7.721 (dm, 2H, J = NMR (376 MHz, CDCl₃): -62.56 ppm (see Fig. S29, ESI†). EA %: calcd

148.5 Hz, ArH), 7.52 (dm, 2H, J = 160.8 Hz, phenylH), 7.35 (dm, 2H, J for C₁₉H₁₃F₃O₃: C 65.90, H 3.78; found C 65.39 H 3.87. ESI-MS (Q-

= 151.6 Hz, phenylH), 4.09 (d, 2H, J = 129.5 Hz, CH₂), 2.25 (d, 3H, J = TOF) m/z: [M+H]⁺calcd for C₁₉H₁₄F₃O₃: 347.08; found 347.0894.

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-

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 distillated

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

distillated THF under 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

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-

13

TOF) m/z: [M+H]⁺calcd for

349.1546 (see Fig. S24, ESI†).

C

¹²C₁H₁₄F₃O₂: 349.15; found

18

J. Name., 2013, 00, 1-3 | 13

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mmol) in 7 mL distillated THF was added at –78 °C. The resulting mixture was stirred 10 min at 0°C and then 1 hour at room

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mixture was stirred at –78 °C for 30 minutes and was then allowed temperature. Subsequently, the resulting mixture was poured into

DOI: 10.1039/C8OB00227D

to warm to room temperature. The color turned from yellow to water. The white solid precipitate was filtrated and water was

orange to yellow. After stirring for 2 hours at room temperature, removed by azeotropic distillation with toluene (3x80ml) to afford a

the reaction was quenched by addition of 10 mL saturated NH₄Cl white-violet solid. The resulting violet solid was dissolved in distilled

solution (20 mL). Diethyl ether (10 mL) was added to the resulting dichloromethane (60 mL) at 0°C, under argon, chloroalkyl ether (4

mixture and the aqueous phase was extracted with diethyl ether equiv., 3.21 g, 3.03 mL, 39.8 mmol) and then DIPEA (2.4 equiv., 3.09

(2×10 mL). The organic phases were collected, dried over MgSO₄g, 3.95 mL, 23.9 mmol) was added dropwise. After 2 hour stirring,

and concentrated under reduced pressure to give a pale-yellow raw 1g of NaOH was added to destroy the excess MOMCl. The reaction

product The crude product was purified by silica chromatography mixture was washed with water. The aqueous layer was extracted

(CH₂Cl₂/ petroleum ether, 1/2) to afford a pale-yellow solid (1,4- with ether (2×20 mL). The organic layers were washed with brine

dimethoxy-3-methylnaphthalen-2-yl)(4-(trifluoro-methyl)phenyl)

(20 mL), dried over Na₂SO₄and concentrated under reduced

methanol 19 (978 mg, 73%). 1,4-dimethoxy-3-methylnaphthalen-2- pressure. The resulting crude product was purified by silica

yl)(4-(trifluoromethyl)phenyl)methanol (19): ¹H NMR (400 MHz, chromatography (cyclohexane/ethyl acetate: 6/1) to give pure

1

CDCl₃): δ 8.10-8.08 (m, 1H, ArH), 7.98-7.96 (m, 1H, ArH), 7.54-7.47 brown solid (3.13 g, 9.16 mmol, 92%). m.p.: 73-74 °C. H NMR (400

(m, 6H, phenylH+ArH), 6.35 (d, J=6 Hz, 1H, OH), 3.98 (d, J= 6Hz, 1H, MHz, CDCl₃): δ 8.14 (dd, J = 6.6 Hz, J = 2.1 Hz, 1H, ArH), 8.06 (dd, J =

CH), 3.86 (s, 3H, OCH₃), 3.53 (s, 3H, OCH₃), 2.34 (s, 3H, CH₃) ppm. 2- 6.6 Hz, J = 2.1 Hz, 1H, ArH), 7.52 (m, 2H, ArH), 5.23 (s, 2H, OCH₂),

(hydroxy(4-(trifluoromethyl)phenyl)methyl)-3-methyl-naphthale-

5.11 (s, 2H, OCH₂), 3.74 (s, 3H, OCH₃), 3.68 (s, 3H, OCH₃), 2.58 (s, 3H,

ne-1,4-dione (14): To

a

solution of (1,4-dimethoxy-3- CH₃) ppm. HRMS (ESI) m/z: [M+Na]⁺calcd for C₁₅H₁₇BrNaO₄

methylnaphthalen-2-yl)(4-(trifluoromethyl)phenyl)me-thanol 19 (1 363.0202; found 363.0207. EA %: calcd for C₁₅H₁₇BrO₄: C 52.80, H

equiv., 882.2 mg, 2.344 mmol) in 60 mL of a solution acetonitrile- 5.02; found C 53.85 H 5.05.

water (3:1), 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, extracted with dichloromethane (4×15 mL). The organic

layers were collected, dried over MgSO₄and concentrated under

reduced pressure. The crude product was purified by silica

chromatography (CH₂Cl₂/PE, 10/1) to afford a yellow solid (702 mg,

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-naphtha-

lene 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) THF (2 mL) was added dropwise to

the resulting mixture. The reaction mixture was stirred 1.5 hour 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

NaHCO₃solution, then saturated NaCl solution, dried over MgSO₄,

concentrated under reduced pressure. The crude produce was

purified by silica 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),

1

86%). m.p.: 134-136 °C. H 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=O), 185.2

(C=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 20ml 3.35 (s, 3H, OCH₃), 2.32 (s, 3H, CH₃) ppm (see Fig. S33, ESI†).

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 the overnight. The

resulting mixture was poured in water, and the organic phase was

washed with Na₂S₂O₃, then brine, dried over MgSO₄and

concentrated under reduced pressure. The crude product was

purified by recrystallization in methanol to give red solid (33.9 g,

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 the 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 Na₂SO₄and

concentrated under reduced pressure. The crude product was

purified by silica chromatography (cyclohexane/ethyl acetate: 4/1)

1

93%). m.p.: 98-100 °C. H 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.

1

to give desired product (800 mg, 71%). H NMR (400 MHz, CDCl₃+

Synthesis of 2-bromo-1,4-bis(methoxymethoxy)-3-methyl-naph- D₂O): δ 8.08 (t, J = 7.7 Hz, 1H, ArH), 8.01 (d, J = 7.8 Hz, 1H, ArH),

thalene (21): A solution of stannous chloride dihydrate (3.42 7.83 (t, J = 8.0 Hz, 1H, phenylH), 7.65 (t, J = 7.5 Hz, 1H, ArH), 7.52 (d,

equiv., 7.7 g, 2.84 mL, 34.1 mmol) in HCl (7 mL) was added quickly J = 8.2 Hz, 1H, phenylH), 7.47 (t, 1H, J = 7.6 Hz, ArH), 7.34 (d, 1H, J =

to a solution of 2-bromo-3-methylnaphthalene-1,4-dione 20 (1 10.2 Hz, phenylH), 3.57 (dd, 2H, J = 14.3 Hz, J = 9.7 Hz, OCH₂), 3.29

equiv., 2.5 g, 9.96 mmol) in ethanol (49.4 mL) at 0 °C. The reaction

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(s, 3H, OCH₃), 2.20 (s, 3H, CH₃) ppm (see Fig. S34, ESI†). HRMS (ESI) (C=O), 145.5 (Cq), 144.1 (Cq), 142.2 (Cq), 134.5 (CH), 134.4 (CH),

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m/z: [M+H]⁺calcd for C₂₁H₁₇F₄O₄409.1057; found 409.1056.

132.2 (Cq), 132.1 (Cq), 129.4 (CH×2), 129.4 (q, J = 32.1 Hz, C-CF ),

DOI: 10.1039/C8OB00227D

3

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

C₁₉H₁₃F₃O_{3 •}0.3 H₂O: 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.

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 Protocol for antimalarial activity against P. berghei parasites

mixture was concentrated under reduced pressure. The crude determined in ex vivo cultures

product was purified by silica chromatography (hexane/EtOAc: 9/1)

to give two pure desired product 24 (Rf= 0,37) and product 15 (Rf=

0,35) in a ratio 1:1 with 35% yield each. 5-(methoxymethoxy)-6-

methyl-10-(trifluoromethyl)-7H-benzo[c]xanthen-7-one (24): m.p.:

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. Murine

1

155-157 °C (from hexane/EtOAc). H 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=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),

malaria parasite P. berghei ANKA strain expressing constitutively

45

GFP-luciferase

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 2x10⁸parasitized erythrocytes/mL. 0.1

mL of this suspension was injected intravenously into mice.

Parasitemia was monitored by Giemsa-stained smears.

100.6 (CH₂), 58.5 (OCH₃), 15.7 (CH₃) ppm (see Fig. S36-S37, ESI†). ¹⁹

F

NMR (376 MHz, CDCl₃): -62.99 ppm (see Fig. S38, ESI†). EA %: calcd

for C₂₁H₁₆F₃O₄C 64.95, H 3.89; found C 64.68 H 3.96. HRMS (ESI)

m/z: [M+H]⁺calcd for C₂₁H₁₆F₃O₄389.0995; found 389.0993.

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 an

exposure of 24 h to different concentrations of the compounds in

ex vivo conditions as formerly described.⁴⁵

Synthesis of 5-hydroxy-6-methyl-10-(trifluoromethyl)-7H-benzo[c]

xanthen-7-one (15): To a solution of 10-(methoxy methoxy)-11-

methyl-3-(trifluoromethyl)-12H-5-oxatetraphen-12-one

24

(1

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M)

in triplicates. The plates were incubated for 24 h in 5% O₂, 3% CO₂,

92% N₂and 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 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. IC₅₀values were calculated using Prism (GraphPad,

log(inhibitor) vs. normalized response – variable slope) in two

independent experiments.

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, then evaporated in 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

1

°C (from cyclohexane/EtOAc). H 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 C₁₉H₁₁F₃O₃C 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 layer were combined and extracted with brine, dried

over MgSO₄and 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

Protocol for antimalarial activity against P. falciparum parasites

determined in in vitro cultures

1

toluene). H NMR (400 MHz, CDCl₃): δ 8.13-8.05 (m, 2H, ArH), 7.77-

In vitro cultures of Plasmodium falciparum

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=O), 185.1

Intra-erythrocytic stages of P. falciparum strains Dd2 and 3D7 were

cultured according to standard protocols^12,46and maintained under

J. Name., 2013, 00, 1-3 | 15

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controlled atmospheric conditions at 5% O₂, 3% CO₂, 92% N₂and analysis (UHPLC coupling with triple quadripole Shi_Vm_iea_wd_Az_ru_ticleLC_O-_nM_linS_e

95% humidity at 37 °C. Synchronization of cultures was achieved by 8030) to allow the detection of plasmodione and

_{DOI: 10.1039/C8O}p_B0u₀ta₂t₂i₇v_De

glucuronic acid-conjugated metabolites (Fig. S44-S45; S48-S51,

sorbitol treatment.⁴⁷

ESI†).

In vitro anti-Plasmodium falciparum activity assays

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 of mice

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

ultrasonic bath for 1 min, and centrifugated (15000g, 5 min, 4°C).

Supernatants were transferred to small plastic vials and subjected

to LC-MS/MS analysis (negative ESI-MS, UHPLC coupling with triple

quadripole Shimadzu LC-MS 8030). Collision energies (CE) and

quadripole (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 supplementary information (Fig.

S46-S47, ESI†).

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M) in duplicates. The plates were incubated for 72 h in 5% O₂,

3% CO₂, 92% N₂and 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. IC₅₀values

were calculated using Prism (GraphPad, log(inhibitor) vs.

normalized response – Variable slope).

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. and S.A.B), the Laboratoire d’Excellence (LabEx)

ParaFrap (grant LabEx ParaFrap ANR-11-LABX-0024 to E.D.C. and

S.A.B., PhD doctoral salary for L.F. and postdoctoral salary for K.E.),

the ANR PRC2017 (grant PlasmoPrim), the Equipement d’Excellence

(EquipEx) I2MC (grant ANR-11-EQPX-0022 to S.A.B.), and the ERC

Starting Grant Nº260918 (S.A.B.). The authors are indebted to

Vrushali Khobragade for measuring the IC₅₀value 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 urines from plasmodione-treated

mice.

Protocol for drug metabolite analysis in mouse urine after

administration of plasmodione

Urine collection from plasmodione-treated mice: Plasmodione was

resuspended in a solution of tween80/ethanol (7:3) at 300 mg/mL,

sonicated for 5-8 min and diluted 1:10 in water. Three doses of the

final solution were administrated daily intraperitoneally at 30mg/kg

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 1:10 dilution of tween80/ethanol (7:3) in

water without compound (mock). Urines were 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

(1:1, v:v) and 5 μL of this solution was subjected to LC-MS/MS

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Article Doi

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

DOI: 10.1039/c8ob00227d

Source and publish data:

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Article abstract of DOI:10.1039/c8ob00227d

Full text of DOI:10.1039/c8ob00227d

Products guided by the article

R&D Labs maybe for 877203-61-7

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Iron(III) bound by hydrosulfide anion ligands: NO-promoted stabilization of the [Fe^III-SH] motif