Pharmacomodulation of the antimalarial plasmodione: Synthesis of biaryl-and N-arylalkylamine analogues, antimalarial activities and physicochemical properties

molecules

Article

Pharmacomodulation of the Antimalarial

Plasmodione: Synthesis of Biaryl- and

N-Arylalkylamine Analogues, Antimalarial Activities

and Physicochemical Properties

Karène Urgin ¹, Mouhamad Jida ¹, Katharina Ehrhardt ^1,2,†, Tobias Müller ^1,3, Michael Lanzer ²,

Louis Maes ⁴, Mourad Elhabiri ¹and Elisabeth Davioud-Charvet ^1,

*

1

UMR 7509 CNRS-Université de Strasbourg, Bioorganic and Medicinal Chemistry Team,

European School of Chemistry, Polymers and Materials (ECPM), 25, rue Becquerel,

F-67087 Strasbourg, France; urginkarene@gmail.com (K.U.); mhmd_jida@yahoo.com (M.J.);

ehrhardt.ka@gmail.com or k.ehrhardt@ibmc-cnrs.unistra.fr (K.E.); tobias_mueller79@web.de (T.M.);

elhabiri@unistra.fr (M.E.)

2

Zentrum für Infektiologie, Parasitologie, Universität Heidelberg,

Im Neuenheimer Feld 324, 69120 Heidelberg, Germany; michael.lanzer@med.uni-heidelberg.de

Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical,

Biomedical and Veterinary Sciences, University of Antwerp,

3

4

Universiteitsplein 1, B-2610 Antwerp, Belgium; louis.maes@uantwerpen.be

Correspondence: elisabeth.davioud@unistra.fr; Tel.: +33-368-852-620; Fax: +33-368-852-742

Present Address: INSERM U963/CNRS UPR9022, Institut de Biologie Moléculaire et Cellulaire,

F-67084 Strasbourg, France.

*

†

Academic Editor: Thomas J. Schmidt

Received: 4 December 2016; Accepted: 12 January 2017; Published: 19 January 2017

Abstract: With the aim of increasing the structural diversity on the early antimalarial drug

plasmodione, an efﬁcient and versatile procedure to prepare a series of biaryl- and N-arylalkylamines

as plasmodione analogues is described. Using the naturally occurring and commercially available

menadione as starting material, a 2-step sequence using a Kochi-Anderson reaction and subsequent

Pd-catalyzed Suzuki-Miyaura coupling was developed to prepare three representative biphenyl

derivatives in good yields for antimalarial evaluation. In addition, synthetic methodologies to

afford 3-benzylmenadione derivatives bearing a terminal -N(Me)₂or -N(Et)₂in different positions

(ortho, meta and para) on the aryl ring of the benzylic chain of plasmodione were investigated through

reductive amination was used as the optimal route to prepare these protonable N-arylalkylamine

privileged scaffolds. The antimalarial activities were evaluated and discussed in light of their

physicochemical properties. Among the newly synthesized compounds, the para-position of the

substituent remains the most favourable position on the benzyl chain and the carbamate -NHBoc

was found active both in vitro (42 nM versus 29 nM for plasmodione) and in vivo in Plasmodium

berghei-infected mice. The measured acido-basic features of these new molecules support the

cytosol-food vacuole shuttling properties of non-protonable plasmodione derivatives essential for

redox-cycling. These ﬁndings may be useful in antimalarial drug optimization.

Keywords: antimalarial; N-arylalkylamines; atovaquone; biaryls; menadione; 1,4-naphthoquinone;

plasmodione; redox-cycling; reductive amination; Suzuki-Miyaura coupling

Molecules 2017, 22, 161; doi:10.3390/molecules22010161

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1. Introduction

Malaria is caused by four species of protozoan parasites of the genus Plasmodium in tropical

and subtropical areas and is curable if treated promptly and adequately. The most dangerous and

harmful species, P. falciparum is responsible for malignant malaria with complications such as cerebral

malaria or severe anaemia, and is responsible for signiﬁcant mortality and morbidity including the

death of approximately 0.6 million people worldwide by year, mostly African children younger than

5 years [1]. Artemisinin-based Combination Therapy (ACT) is currently the ﬁrst-line therapy for

malaria worldwide; however, a decrease in artemisinin sensitivity, mainly in Southeast Asia, has

recently been reported, thus underlining the need for alternative artemisinin-based combinations with

novel agents as drug partners [2]. New antimalarials must exhibit pleiotropic mechanisms-of-action or

target new metabolic pathways that counterbalance parasite resistance to current drugs. Additionally,

there is a continuous demand for new drugs that are not only affordable, safe, available and effective

but also easy to synthesize. Our approach is to target the thiol based redox-network of P. falciparum.

Recently, we synthesized a series of 3-benzylmenadione derivatives [3,4], which were identiﬁed

as potent antimalarial redox-active agents. Among these, the 3-[4-(triﬂuoromethyl)benzyl]-menadione

(called plasmodione, Figure 1 ) showed the most promising ‘early lead’ features for drug optimization [5 ,6 ]

Mechanistic studies revealed that plasmodione may enter a cascade of redox reactions for drug

bioactivation: in its oxidized state, the menadione derivative was proposed to be reduced by

the NADPH-dependent glutathione reductases of the Plasmodium-parasitized erythrocytes; in its

.

−

reduced state, the hydronaphthoquinone was shown to transfer 1e to methemoglobin (Fe^III) (metHb),

the major nutrient of the parasite [ ]. By redox-cycling NADPH and hemoglobin, it was postulated

3,4,7

that the increased oxidative stress leads to the death of the parasite, but only in parasitized erythrocytes.

In a previous study, it was found that the mitochondrial electron transport (mETC) chain

of P. falciparum blood stages is dispensable for the antimalarial activities of plasmodione and

methylene blue in contrast to the 2-hydroxy-naphthoquinone derivative atovaquone [5]. The latter

drug is given as a highly efﬁcacious ﬁxed-dose combination (atovaquone-proguanil marketed

as Malarone^®) both for prophylaxis and treatment of multidrug-resistant falciparum malaria.

Atovaquone inhibits complex III of the mETC system, inhibits the cell respiratory mechanism

and depolarizes the mitochondrial membrane potential [8]. However, the major function of the

mETC in P. falciparum blood stage cultures is to regenerate ubiquinone as the electron acceptor of

mitochondrial dihydroorotate dehydrogenase (DHODH) which catalyzes a key step in pyrimidine

biosynthesis [9]. Atovaquone has been shown to lose its antimalarial activity in the presence of a

yeast gene encoding an alternative cytosolic DHODH (yDHODH) that does not require the mETC as

electron acceptor [10]. Transgenic P. falciparum strains with or without yDHODH were established

providing an excellent tool for the analysis of potential inhibitors of the mETC. The previous

study [5] evaluated redox-active 1,4-naphthoquinones, including plasmodione and methylene blue,

and were shown to retain potency against the transgenic P. falciparum strain expressing yDHODH,

which had lost sensitivity to atovaquone. Furthermore, chemical modiﬁcation on the menadione

moiety of plasmodione derived analogues were functionalized with the atovaquone chain [11],

but these hybrid molecules were found inactive against the atovaquone-sensitive Dd2 strain of

P. falciparum because 1,4-naphthoquinones plasmodione and atovaquone do not share the same

mode-of-action and redox potential values. In addition, atovaquone and plasmodione were clearly

shown to result in different morphological changes during the intraerythrocytic parasitic cycle [5].

In agreement with previous reports [12], the morphologies of atovaquone-treated parasites were very

similar to the untreated controls. The growth of plasmodione-treated parasites was retarded with

a signiﬁcant increase in abnormal morphologies, predominantly pyknotic ring stages. In contrast

to atovaquone, the observations on morphology are in agreement with plasmodione’s pronounced

activity against ring stages compared to later stages. Other electrochemical parameters for lawsone

versus menadione (2-hydroxy versus 2-methyl-1,4-naphthoquinone, see Figure 1) reported in [11,13]

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support the distinct redox behaviour and mode-of-action of both 1,4-naphthoquinone antimalarials

atovaquone and plasmodione.

Figure 1. Structurally related antimalarial atovaquone (left) and the lead plasmodione (right

)

and envisaged synthetic 3-benzylmenadione/plasmodione derivatives (2nd line): biaryl- (left) and

N-arylalkylamine (right) analogues of plasmodione.

The bioavailability and activity/solubility of the plasmodione series is however poor, hindering

further drug development. Hence, investigations on its pharmaco-modulation are necessary to

determine whether the 4-triﬂuoromethyl group can be replaced or whether the substituent on the aryl

ring can be moved without sacriﬁcing the antimalarial activity. Based on the literature on the activity

of numerous.

4- or 8-aminoquinolines or phenothiazines, and as a part of our medicinal chemistry program

on 1,4-naphthoquinone scaffolds, the present study investigated the antimalarial activity of several

plasmodione derivatives in which the 4⁰-CF₃group was replaced either by a substituted aryl group

or by a N-dialkylamino group. Modiﬁcations on the menadione core previously provided various

plasmodione analogues with varying antiplasmodial activity [14]. In the present study the substitution

at the benzylic chain of plasmodione was explored, in particular by using Pd-catalyzed reactions to

prepare biphenyl analogues. Biaryls and their aryl-heteroaryl homologues represent an important

class of organic compounds found in natural products that were shown to be useful in various

applications, ranging from medicinal chemistry [15] to advanced materials and supramolecular

chemistry [16–18]. For example, the diphenylmethyl chain of the potent substituted quinolone CK-2-68

was replaced by a pyridylaryl chain in the initial lead compound SL-2-25 without sacriﬁcing its

antimalarial activity [19,20].

Herein, biaryl- and N-arylalkylamine analogues of plasmodione were synthesized to investigate

their antimalarial potential in comparison to plasmodione. For N-substitution, we incorporated

N-dimethyl or N-diethyl moieties as found in the approved drugs chloroquine (CQ) [21

related 4- or 8-aminoquinolines (e.g., ferroquine [23 24] or pamaquine [25 26]) and Methylene Blue

(MB) [27 29] (Figure 2) to learn the structural requirements for antimalarial activity of optimized

plasmodione derivatives. We further evaluated the impact of the free amino and N-dialkyl substituents

on the physicochemical properties of derivatives 10 11 and 17 19 in comparison to those of

,22] and

,

–

3

,

7,

,

–

two key reference drugs MB and CQ since pK_avalues are important for antimalarials that mainly

interact with Fe^IIIspecies.

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Figure 2. The most important clinically used antimalarial drugs presenting a terminal -N(Me)₂or

-N(Et)₂group in their side chains.

MB, the ﬁrst synthetic antimalarial dye discovered by Paul Ehrlich in 1891, has recently been

rediscovered for its promising activity in drug combinations for the control of transmission of

P. falciparum malaria in endemic regions [30] and against Plasmodium vivax [31]. MB is reduced by

various NADPH-dependent disulﬁde reductases, including glutathione reductases from the cytosols of

Plasmodium-infected erythrocytes [32]. Malarial parasites normally digest the host hemoglobin in the

metHb form in a specialized acidic compartment, called food vacuole, mainly at the intraerythrocytic

trophozoite stage. The blue dye is known to cycle between the oxidized form and its electron–reduced

leucomethylene blue (LMB) form that is involved in intricate redox processes, in particular conversion

of metHb to oxyhemoglobin (oxyHb) [29]. As oxyHb is not digested by the parasitic proteases,

MB has been proposed to exert its antimalarial action by depriving the parasite from its major

nutrient metHb. With its phenothiazine structure, MB displays redox cycling activity able to generate

bound (hemoglobin or heme) Fe^IIfrom Fe^IIIspecies, likely responsible for its potent activity in vivo

.

Upon reduction and different protonation states, MB has the ability to change its structure and display

different lipophilicities [29]. Due to its very low pK_a(pK_a= 0), MB does not accumulate in acidic

vesicles but may shuttle in and out the cell compartments depending on its redox state. The ﬁnal result

of MB-catalyzed redox-cycling is an arrest of parasite development [5].

In contrast, the broadly used CQ is a diprotic weak base (pK_a= 8.4 and 10.6), which accumulates

in the food vacuole and binds to Fe^III-heme leading to a stable 1:1

π

-

π

complex (K_D~2–15

pH 7.5) [33]. Intraerythrocytic parasites detoxify the exogenous deleterious Fe^III-heme by-product

(i.e., predominantly existing as a dimeric heme complex) of hemoglobin digestion through

µM at

π-π

biomineralization to an insoluble and much less active crystal, named hemozoin. In the presence of

CQ, the ﬁnal result is the elevation of toxic free heme and inhibition of hemozoin formation. Free heme

and heme CQ complexes are therefore thought to kill parasites by inducing severe oxidative stress [34],

which successively leads to peroxidation of the parasite membrane lipids, DNA damage, protein

oxidation and ultimately death of the parasite.

2. Results

2.1. Chemistry

Previously, we reported a new methodology for the preparation of 3-benzylmenadione

derivatives [14] and polysubstituted 6-methylquinoline-5,8-dione scaffolds [35] (i.e., 5- and

8-aza-menadione analogues) by the Kochi-Anderson reaction. The silver-catalyzed coupling

reaction represents an easy access and efﬁcient method to synthesize certain 3-benzyl-substituted

1,4-naphthoquinones derivatives starting from readily commercially available carboxylic acids [36].

This reaction has also been applied in the synthesis of naturally occurring quinones [37

,38] or

in the synthesis of 2- and 3-substituted menadiones as subversive substrates of trypanothione

reductase and lipoamide dehydrogenase from Trypanosoma cruzi [39,40] or of glutathione reductases of

Plasmodium-parasitized erythrocytes [41].

In continuation of our structure-activity relationships (SAR) studies on the plasmodione series

(Scheme 1), the plasmodione bromine derivatives 25a–c were prepared from menadione in moderate

to excellent yields (52%–88%) by the described silver-catalyzed decarboxylation reaction between

menadione 1 and the corresponding bromophenylacetic acid derivatives 24a–c.

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Scheme 1. Synthesis of 3-[4-(bromol)benzyl]-menadione intermediate 25 (a–c) via a Kochi-Anderson reaction.

Transition metal catalyzed cross-coupling reactions have revolutionized organic synthesis.

Many highly efﬁcient and mild methods have been developed over the last two decades allowing

rapid and versatile access to complex molecular structures [42–47]. In recent years, the Suzuki-Miyaura

reaction in particular has become one of the most important cross-coupling reactions, allowing not

only the formation of C(sp²)-C(sp²) and C(sp²)-C(sp³) but also C(sp³)-C(sp³) bonds [44–46].

Subsequently, the biaryl menadione derivatives 26–29 (Scheme 2 ) were successfully synthesized

in excellent yields (86%–97%) via a Suzuki-Miyaura coupling reaction. The reaction was performed

in dioxane/water (v/v = 4/1) at 80 ^◦C overnight under an atmosphere of argon. Aryl bromide 25c

was coupled with the corresponding para-substituted phenylboronic acid in the presence of K₂CO₃

as base and 4 mol % PdCl₂(dppf) as catalyst. K₂CO₃was chosen as a mild base because of the low

stability of benzylmenadione derivative 25c under basic conditions due to the acidic proton of the CH₃

group. Using these reaction conditions, no side product was observed compared to the treatment with

stronger bases leading to a rapid decomposition of the starting menadione derivatives. The expected

Suzuki-products 26–28 were isolated in excellent yield. This methodology is straightforward and

provides easy access to structurally diverse 1,4-naphthoquinones with a biphenyl motif, allowing to

study the inﬂuence of a second phenyl ring in plasmodione structure on its antimalarial activity.

Scheme 2. Synthesis of biphenyl derivatives of menadione by Suzuki-coupling reaction.

Regarding the 2nd series of N-arylalkylamines, we ﬁrst prepared the N-dialkylamine analogues

of 3-benzylmenadiones (Figure 2) using the Kochi-Anderson reaction. In earlier investigations

to synthesize compound

decarboxylation reaction of menadione

3

(Scheme 3), the reported conditions used in the silver-catalyzed

with 4-(dimethylamino)phenylacetic acid had proven

1

2

to be inappropriate and not compatible, leading mainly to degradation of the starting materials

because the N-substituent becomes oxidized in the reaction by Ag(II) leading to the formation of Ag(I)

and thereby destroying the competent catalyst Ag(II).

It was deemed of interest to explore a new pathway for this purpose. We attempted to achieve the

synthesis of para-N-substituted-3-benzylmenadione derivatives 3 via a N-alkylation procedure using

two synthetically N-protected routes through: (i) the N-tert-butyloxycarbonyl (N-Boc) and (ii) the

N-acetyl protection strategies (Scheme 4).

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Scheme 3. Silver-catalyzed decarboxylation reaction (Kochi-Anderson).

Following the N-Boc route (i), the commercial 4-aminophenylacetic acid (4) was treated with

Boc₂O in the presence of Na₂CO₃as base to give the corresponding N-Boc-protected phenylacetic

acid in 94% isolated yield (route I, Scheme 4 ). The 3-benzylmenadione intermediate was synthesized

in low yield (22%) by one-step Kochi-Anderson reaction between the phenylacetic acid derivative

and menadione ( ) in the presence of Ag(I) as catalyst and ammonium peroxodisulfate. After removal

of the N-Boc protecting group in the presence of triﬂuoroacetic acid (TFA), compound was isolated

in 84% yield as the precursor for N,N-dimethylation of the free amine group in the next step. Finally,

the desired product was obtained in 33% yield using Ph₃P/DDQ [48] in methanol as an efﬁcient

reagent system for the immediate and selective N-alkylation of aromatic amines. However, to improve

the synthesis and overall yields of the desired product , we studied the effect of the N-protecting

group by replacement of the Boc group by an acetyl group. Indeed, by following the N-acetyl

route (II), the N-acetyl-protected phenylacetic acid was obtained quantitatively by an acetylation

reaction between the free-amine 4 and acetic acid under microwave irradiation conditions [49]. Next,

the Kochi–Anderson reaction between the phenylacetic acid derivative and menadione ( ) gave

access to the desired compound in good yield (54%). The deprotection reaction of the acetyl group

was performed in the presence of hydrochloric acid (HCl) under microwave irradiation conditions,

affording the desired compound in 76% yield [50]. As shown in Scheme 4 , the expected compound

N,N-dimethyl-3-benzylmenadione was synthesized in good yield (59%) by one-step reductive

amination reaction between the intermediate and formaldehyde in the presence of acetic acid

5

6

5

1

7

3

8

1

9

7

3

7

and sodium cyanoborohydride as reducing reagent (59% with NaBH₃CN/AcOH versus 39% with

DDQ/PPh₃) [51]. Use of acetaldehyde instead of formaldehyde under the same reduction reaction

conditions (NaBH₃CN/AcOH) gave access to a separable mixture of para-N-ethyl-3-benzylmenadione

(10, 17%) and para-N,N-diethyl-3-benzylmenadione (11, 36%) (Scheme 4).

0

Scheme 4. Synthesis of new 3-[4 -N-alkylated-benzyl]-menadione derivatives via a reductive amination

procedure using the N-Boc or -acetyl protection strategies.

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We then applied the same N-alkylation procedures described in Scheme 4 (N-Boc and

-acetyl protection) for the synthesis of meta-N-substituted-3-benzylmenadione derivatives 18 and 19

(Scheme 5). Following route I and starting from the commercial 3-aminophenylacetic acid (12),

the meta-N-Boc-benzylmenadione intermediate 14 was obtained in two steps in low yield (7%) by

protection of the free benzylamine group with a Boc group followed by the Kochi–Anderson reaction.

As the desired compound 14 was obtained in low yield, we thought that the Boc protecting group

should be changed to an acetyl group in order to increase the efﬁcacy of this methodology when

following route II.

Indeed, the meta-N-acetyl-benzylmenadione intermediate 16 was ﬁrst isolated in moderate

yields (47%) in two steps by N-acetyl protection followed by a subsequent Kochi-Anderson

reaction. Then, an HCl-promoted acetyl removal followed by a reductive amination reaction of

free amine 17 with formaldehyde and acetaldehyde respectively, resulted in the formation of the

desired meta-N,N-dimethyl-3-benzylmenadione (18, 31%) and meta-N,N-diethyl-3-benzylmenadione

(19, 22%) respectively (route II, Scheme 5).

0

Scheme 5. Synthesis of new 3-[3 -N-alkylated-benzyl]-menadione derivatives via a reductive amination

procedure using the N-Boc or N-acetyl protection strategies.

Next, we synthesized the ortho-N-substituted-3-benzylmenadione derivatives by applying

the N-acetyl protection as depicted in Scheme 6 (route II), the ortho-N-acetyl-3-benzylmenadione

intermediate was isolated in moderate yield (39%) by a sequential opening of the oxoindole [52] ring 20

to aminoacid 21 followed by a N-acetyl protection [53] of the generated free amine 21 and a ﬁnal

Kochi-Anderson reaction. To synthesize compound 23, the optimal heating and microwave irradiation

conditions for the N-acetyl deprotection reaction of compound 22 proved to be inappropriate, leading

mainly to the degradation of the starting material 22 (Scheme 6).

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Scheme 6. Synthesis of new 3-[2-N-alkylated-benzyl]-menadione derivatives via an ortho-N-alkylation

procedure using the N-acetyl protection.

2.2. Biological Activities

2.2.1. In Vitro Antimalarial Activity

All ﬁnal isolated compounds were tested for their ability to inhibit the in vitro growth of

the multiresistant P. falciparum Dd2 strain by determining the concentration required to inhibit

parasite development by 50% (IC₅₀values). The protocols are described in the experimental section.

Two protocols to measure the growth inhibition were used: the tritiated hypoxanthine incorporation

method and the SYBR_greenmethod. This was due to the fact that the radioactive assay was abandoned

in the laboratory due to cost issues related to radioactive waste disposal. The growth inhibition

measured in the malarial parasite assays and the cytotoxicity against human ﬁbroblasts of all the new

compounds are listed in Tables 1 and 2. For comparison, data for plasmodione and its bromo analogue

described in our previous publications were also included in the tests. All data are mean values from

one to four independent experiments, each consisting of eight drug concentrations in duplicate.

Table 1. Mean IC₅₀values (nM) for plasmodione derivatives determined with the tritiated

hypoxanthine incorporation method against P. falciparum Dd2 ^a, and with the Alamar blue method

against the human MRC-5 ﬁbroblasts.

Compound Code

Structure

IC₅₀(nM)

Tox/MRC-5 (µM)

Plasmodione ^b

29 ± 2 (3)

>32.0

6

42.5 (2)

>64.0

>32.0

25c ^b

46 ± 4 (5)

26

223

>64.0

27

28

485

474

>64.0

51.5

Chloroquine ^b

110 ± 20 (4)

a

In vitro activity against P. falciparum Dd2 strain is presented as mean IC₅₀values of benzylmenadione derivatives

using the tritiated hypoxanthine incorporation method; ^bChloroquine and plasmodione were used as standard

drugs. The listed IC₅₀values of chloroquine, plasmodione and its bromo analogue 25c (named benzylMD 1c and 1a

in [3]) are previously reported values.

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Table 2. Mean IC₅₀values (nM) for plasmodione derivatives determined in the SYBR_greenmethod with

P. falciparum Dd2.

Compound Code

Structure

IC₅₀(nM) ± SD (n) ^a

567 ± 217(3)

Tox/MRC-5 (µM)

3

6

7

>64.0

109 ± 46 (3)

303 ± 138 (3)

>64.0

9

411 ± 220 (3)

>64.0

10

11

418 ± 166 (3)

2056 ± 1073 (3)

310 ± 134 (3)

1198 ± 1651 (3)

386 ± 151 (3)

236 ± 97 (3)

>64.0

29.3

16

4.5

17 *

18

>64.0

19

22

282 ± 86 (3)

4.4

25a

25b

86.3 (1)

127.6 (1)

>64.0

20.8

>32.0 ^b

25c

82 ± 2 (7)

58 ± 11 (9)

Plasmodione

51.5 ^b

Chloroquine

99 ± 19 (6)

a

In vitro activity against P. falciparum Dd2 is presented as mean IC₅₀values

±

standard deviation (SD) determined

from (n) independent growth inhibition assays in triplicate using the SYBR_greentechnique; ^bChloroquine was

used as reference drug. IC₅₀values of chloroquine, plasmodione and its bromo analogue 25c (named benzylMD 1c

and 1a in [3]) are previously reported values. * Precipitates in DMSO.

®

To explore the effect of modiﬁcations in plasmodione by replacing the substituted aryl by

biaryl moieties bearing three distinct functionalities on antiplasmodial activity and cytotoxicity, we

tested compounds 6, 25c, 26–28 in the hypoxanthine incorporation test (Table 1). Whatever the

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nature of the substituent (electron-donating t-Bu, electron-withdrawing NO₂, protonable substituent

NMe₂), the potency was lower than that of plasmodione itself. The most active compound was

the biaryl functionalized by a p-NMe₂exhibiting a 10-fold higher IC₅₀value (229 nM versus 29 nM

for plasmodione).

This la₀st result oriented the work towards the synthesis and testing of plasmodione analogues in

0

which the 4 -CF₃was replaced by 4 -NMe₂, 4 -NEt₂, 3 -NMe₂, or a 3 -NEt₂. All intermediates (bearing

NH₂, NHBoc, NHAc, Br groups) were tested in the SYBR_greendrug assay (Table 2). Interestingly, all

0

benzylmenadione analogues bearing a N-dialkyl or a N-monoalkyl group in 4 or 3 were >10-fold less

active than plasmodione itself, suggesting that the lysosomotropic compound concentration in acidic

compartments was not a favourable parameter to enhance the antiplasmodial activity of redox-cyclers.

In contrast, the non-protonable bromo- or the NH-protected amine analogues were much more potent

compared to the N-dialkyl derivatives. The para- and ortho-substitutions were also found the most

favourable in the bromo series (82 or 86 nM versus 127 nM for para- or ortho, versus -meta, compared to

58 nM for plasmodione). Unexpectedly, the 4⁰-NHBoc benzylmenadione was found very active, with

an IC₅₀value of 109 nM.

2.2.2. Cytotoxicity of Plasmodione Derivatives in Human Cell Assays

All compounds were tested for cytotoxicity on human lung MRC-5 ﬁbroblasts using the Alamar

Blue assay (Tables 1 and 2). Most of the benzylmenadione derivatives exhibited a low cytotoxicity as

evidenced by the high IC₅₀values above 32 µM.

2.2.3. In Vivo Antimalarial Activity

The in vitro most active compound

6 among the newly synthesized derivatives was subsequently

tested in the P. berghei mouse model using the chloroquine (CQ)-susceptible ANKA strain (Table 3).

For comparative purposes, CQ was also included and dosed at 10 mg/kg intraperitoneally (ip)

resulting in a decreased parasitemia by 86.3% and by 76.8% at days 4 and 7. Compound

6 showed

signiﬁcant activity following a 5-day treatment at 50 mg/kg ip resulting in decreased parasitaemia

by 75.5% and 81.8% at days 4 and 7. For comparison, the previously described 4-bromo derivative

25c showed 35.8% reduction of parasitemia in P. berghei strain ANKA-infected CD1 mice at day 4

(compound 1a in Table 4 of [3]).

2.3. Physicochemical Properties

Acid-Base Properties

The acid-base properties of the relevant plasmodione derivatives were evaluated by absorption

spectrophotometric titrations versus pH titrations. The derivatives are characterized by a set of three

main absorptions centered at ~340 nm, ~270 nm and ~250 nm, respectively. The electronic transitions

at low energies (>300 nm) were attributed to

π-π* transitions centered on the 1,4-napthoquinone

chromophore (benzoquinoidal structure), while those observed at higher energies (<300 nm) can be

ascribed to

compound

π

-

π

* benzene or benzyl subunits. Figure 3 depicts the spectral variations measured for

3

as a function of pH. The rise of pH does not affect the absorption of higher energies, while

those centered below 300 nm are signiﬁcantly affected by the acidity of the medium. These variations

are in agreement with (de)protonation of the ammonium group substituted on the benzylic subunit.

The absorption spectrophotometric titrations versus pH for the other plasmodione derivatives are

provided as Supplementary Materials (Figures S1–S4).

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Table 3. In vivo antimalarial activity in P. berghei ANKA-infected Swiss mice after a 5-day intraperitoneal dosing.

Treatment

(ip for 5 Days)

% Parasitaemia (dpi)

% Suppression (dpi)

Compound

Animals (n)

Health Status

4

7

11

14

4

7

11

14

From day 4 post-infection (p.i.) onwards:poor

appearance, tremor, body weight loss, rough hair,

4 animals died before day 7 p.i.

Untreated control

-

5

38.3

42.2

67.7

61.9

-

From day 4 p.i. onwards: poor appearance, tremor,

body weight loss, rough hair, one animal died before

day 14 p.i.

Chloroquine

Compound 6

10 mg/kg

50 mg/kg

5

4

5.2

9.4

9.8

7.7

32.1

41.9

56.2

69.3

86.3

75.5

76.8

81.8

52.7

38.1

9.2

From day 4 p.i. onwards: poor appearance

(intermittent), one animal died before day 14 p.i.

12.0

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2.0

1.5

1.0

0.5

0.0

0.4

0.3

0.2

0.1

O

N

3

0.35

0.30

0.25

2

1

3.H

3

4

5

6

7

pH

0.0

250

300

350

400

250

300

350

λ (nm)

400

λ (nm)

(a)

(b)

Figure 3.

) electronic spectra of

I = 0.1 M NaClO₄; T = 25.0

The charges have been omitted for clarity. The inset of (A) shows the spectral variation at 250 nm as a

function of pH. The UV-visible absorption spectra have not been corrected from dilution effects.

(

a

) Absorption spectrophotometric titration of compound

3

as a function of pH and

.H. Solvent: Water/DMSO (91/9 v/v);

10⁻⁵M; (1) pH = 3.06; (2) pH = 7.25.

(

b

3

and its _◦monoprotonated species

3

±

0.2 C; l = 1 cm; [

3]

tot

= 1.81

×

The statistical processing of the absorption and potentiometric datasets allowed the evaluation of a

single protonation constant related to the benzylic ionizable site (i.e., benzylamine and its N-methylated

analogues) whatever system considered (Table 4). As far as the plasmodione derivatives bearing diethyl

tertiary amines are concerned (i.e., 10 and 19), the pK_avalues could not be accurately evaluated due to

the precipitation at pH ~5 under our experimental conditions (H₂O/DMSO, 91/1 v/v). This is likely

due to the higher hydrophobic character brought by the ethyl substituent. The pK_avalues ranged from

4.3 (compound 17) to 5.07 (compound 11) and are in excellent agreement with the values determined

for adequate models such as aniline (pK_a= 4.72 [54] in 0.1 M KCl versus compound 7 pK_a= 4.74 ± 0.05

or compound 17 pK_a= 4.30

compound pK_a= 4.87

absence of any electronic effect of the menadione core on the acido-basic properties of the benzylamine

subunit due to the presence of the methylenic moiety which can be regarded as a poor electronic

relay. Taken as an example, Figure 4 depicts the distribution diagrams of the protonated species of

±

0.02) or N-phenyldiethanolamine (pK_a= 4.13 [55] in 0.4 M KCl versus

3

±

0.03 or compound 18 pK_a= 4.60 0.04). This feature emphasizes the

±

compound

3 clearly showing that below pH 5 (anticipated pH of the food vacuole of P. falciparum) the

plasmodione analogues bearing an amino substitution of a benzyl position exist predominantly in

their ammonium form. Table 4 provides the ratios of the protonated species at pH 5 and 7.4 clearly

evidencing accumulation in the food vacuole due to pH gradient.

100

O

N

O

H

N⁺

50

O

0

2

3

4

5

6

7

8

9

pH

Figure 4. Distribution diagrams of the protonated species of plasmodione analogue 3 as a function of

◦

−5

pH. Solvent: Water/DMSO (91/9 v/v); I = 0.1 M NaClO₄; T = 25.0 ± 0.2 C; [3]_tot= 2 × 10 M.

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Table 4. pK_avalues for selected plasmodione derivatives ^a.

Compound Code

Structure

pK_a± σ [LH⁺]_pH5/[LH⁺]_pH7.4

4.87 ± 0.03

3

144.7

4.74 ± 0.05

7 ^b

162.5

e

10

11

5.07 ± 0.06

112.0

4.30 ± 0.02

17 *^,b

209.6

4.60 ± 0.04

18

179.9

e

19

pK_a1= 1.7

pK_a2= 4.5

pK_a3= 5.9

Methylene blue ^c

Chloroquine ^d

pK_a1= 8.4 ± 0.2

pK_a2= 10.6 ± 0.2

1.1

a

Water/DMSO (0.91/0.09 v/v); I = 0.1 M NaClO₄; T = 25 ^◦C; ^bWater; I = 0.1 M NaClO₄; T = 25 ^◦C; ^cpK_avalues

were reported [

29]; ^dpK_avalues were measured in [56]; ^ePrecipitation prevented the accurate determination of

the pK_avalues which were estimated to be ~5–5.5. Error = 1 with = standard deviation. * Precipitates in DMSO.

4,

σ

3. Discussion

In an attempt to generate plasmodione analogues with improved activity/solubility proﬁles,

we developed synthetic methodologies to prepare a biaryl series through a 2-step sequence using

Kochi-Anderson reaction followed by a Pd-catalyzed Suzuki-Miyaura cross-coupling, and a second

3-benzylmenadione series bearing a terminal -N(Me)₂or -N(Et)₂in different positions (ortho, meta and

para) on the aryl ring of the benzylic chain of plasmodione through reductive amination, as the optimal

route to prepare these protonable N-arylalkylamines.

From the mechanistic point of view, the benzoylmenadiones were proposed to be the key

potential metabolites acting in oxidized form as the most efﬁcient subversive substrates of reduced GR

described so far and, in reduced form, to transfer electrons to methemoglobin. The reduced species of

benzoylmenadiones are assumed to be transported through Fe^IIIcomplexation into the food vacuole

where the electrons are transferred to oxidants (heme and methemoglobin). Regenerated in their

oxidized form, the benzoylmenadiones might be transported into the cytosol where they are reduced

by the cytosolic GR (either from human erythrocyte or from the parasite) in a continuous redox cycle

(Figure 5 ). Evidence for cytosol-food vacuole shuttling of plasmodione during redox-cycling is difﬁcult

to prove, however, replacing the 4⁰-CF₃group in plasmodione by protonable terminal -N(Me)₂or

-N(Et)₂, the aim was to vectorize the ﬁnal derivatives to the acidic compartments of the malarial

parasite, e.g., the food vacuole, where hemoglobin digestion takes place. By partly losing antimalarial

potency, these compounds provide indirect proof for the hypothesized cytosol-food vacuole shuttling

of plasmodione during redox-cycling. Worthy of mention is the fact that the redox-active MB, which

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possesses potent antimalarial activity, shares similar physico-biochemical properties to those found for

plasmodione and its active analogues, i.e., non-protonable N-dimethylamino groups at the acidic pH

of the food vacuole, redox potential values in the same range, NADPH-dependent methemoglobin

redox-cycling in the presence of the redox-active blue dye [3,29,33].

Figure 5. Putative model for cytosol-food vacuole shuttling of 3-benz(o)ylmenadione derivatives

during redox-cycling affecting the redox homeostasis in the cytosols of P. falciparum-parasitized

erythrocytes through a cascade of redox reactions accounting for the observed antimalarial activity.

Blue arrows indicate reduction; red arrows oxidation; dashed arrows uptake processes. The lead

3-benzylmenadione is proposed to be taken up by infected red blood cells (step 1), be reduced in the

cytosols of parasitized erythrocytes by human GR (step 2), and then be oxidized at the benzylic chain to

3-benz(o)ylmenadione (3-benz(o)ylMD) in the acidic vesicles or in the food vacuole in heme-catalyzed

reactions (not shown). The reduced species of 3-benz(o)ylmenadione are subsequently assumed to

be transported through Fe^IIIcomplexation into the acidic vesicles (step 3) where reduced species

of 3-benz(o)ylmenadione transfer the electrons to oxidants (hematin or metHb, step 4). The ﬁnal

result is an inhibition of hemozoin formation (step 5) and the arrest of parasite development as

shown previously [3]. Hence, the antimalarial benzylmenadione derivatives would act as prodrugs of

redox-active principles, being cycled in and out of the acidic vesicles in infected red blood cells, thereby

oxidizing major intracellular reductants (NADPH) and subsequently reducing oxidants like hematin or

metHb. HMS, hexose monophosphate shunt.

The most active derivatives presented in this work are the non-protonable plasmodione analogues,

suggesting that lysosomotropic properties are not favourable to optimize the redox-active compounds

that are cycled in and out of the food vacuole in parasitized erythrocytes.

4. Materials and Methods

4.1. General Information

4.1.1. Solvents and Reagents

Commercially available starting materials were purchased from Sigma-Aldrich (St. Louis, MO,

USA), ABCR GmbH & Co. KG (Karlsruhe, Germany), Alfa Aesar (Haverhill, MA, USA), and Apollo

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Scientiﬁc (Cheshire, UK) and were used without further puriﬁcation. Solvents were obtained from

Sigma-Aldrich and Carlos Erba (Val de Reuil, France). Unless noted, reagent grade chemicals were

used for reactions and analytical grade ones for column chromatography and recrystallizations.

When speciﬁed, anhydrous solvents were required; dichloromethane (DCM) was distilled over CaH₂

under argon. Tetrahydrofuran (THF) was distilled over sodium/benzophenone under argon or dried

by passage through an activated alumina column under argon. 1,4-Dioxane and dimethylformamide

(DMF) were purchased anhydrous over molecular sieves from Sigma-Aldrich. (St. Louis, MO,

USA) Triethylamine (Et₃N), diisopropylethyl amine (DIPEA), pyrrolidine, piperidine were distilled

over KOH under argon and stored over KOH. All reactions were performed in standard glassware.

Microwave reactions were carried out on two different apparatus (Biotage Initiator™, Uppsala, Sweden

and CEM, Orsay, France) with comparable results (cross compared); supplier standard microwave vials

were used. Crude mixtures were puriﬁed either by recrystallization or by ﬂash column chromatography.

The latter were performed using silica gel 60 (230–400 mesh, 0.040–0.063 mm) purchased from E. Merck

(Kenilworth, NJ, USA). Automatic ﬂash chromatographies were carried out in a Biotage Puriﬂash

apparatus (Uppsala, Sweden) with UV-Vis detection at 254 nm (unless otherwise speciﬁed). Monitoring

and primary characterization of products were achieved by TLC on aluminium 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 chemicals. Analytical TLC was carried out on pre-coated Sil G-25 UV₂₅₄plates

from Macherey Nagel (Hoerdt, France). Flash chromatography was performed using silica gel G60

(230–400 mesh) from Macherey Nagel.

4.1.2. Instruments

The Nuclear Magnetic Resonance (NMR, ¹H-NMR 300 MHz, ¹³C-NMR 75 MHz) spectra were

registered on a Bruker Avance 300 apparatus (Wissembourg, France). A Bruker Avance 400 apparatus

was used (¹H-NMR 400 MHz, ¹³C-NMR 100 MHz) for more complex spectra. All examples below are

labelled as 300/75 MHz except for a few ¹³C spectra (compounds

7, 18, 22). All chemical shifts (δ) are

quoted in parts per million (ppm). The chemical shifts are referred to the used partial deuterated-NMR

1

solvent (for CDCl₃: H-NMR, 7.26 ppm and ¹³C-NMR, 77.16 ppm). The coupling constants (J) and the

non-equivalence (∆ν) 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), AB (AB system), (ABX) (AB system of an ABX) and A₂B₂(A₂B₂aromatic system). In addition,

the following acronyms will be used: C=O carbonyl group; C_q: quaternary carbon; CH₂: secondary

carbon; CH₃: methyl group. Elemental analyses (EA) were obtained at “Service de Microanalyses”

at the Institut de Chimie de Strasbourg. Mass spectra (ESI-MS) were obtained on a microTOF LC

spectrometer (Bruker Daltonics, Bremen, Germany). High Resolution Mass (HRMS) spectra were

measured and ﬁtted with calculated data. Melting points (M.p.) were determined on a Büchi (Rungis,

France) melting point apparatus and were not corrected.

4.2. Protection of Aminophenylacetic acids with Boc Groups (Synthesis of 5 and 13)

2-(4-((tert-Butoxycarbonyl)amino)phenyl)acetic Acid (5). A solution of 4-aminophenylacetic acid (4, 4 g,

26.46 mmol, 1 equiv.) in a mixture of dioxane (52 mL) and water (52 mL), and sodium carbonate

(2.8 g, 26.42 mmol, 1 equiv., in 26 mL of water) was stirred and cooled in an ice bath. Di-tert-butyl

dicarbonate (BOC-anhydride, 6.24 g, 28.59 mmol, 1.1 equiv.) was added in one portion, and stirring

was continued at room temperature for 4 h. The dioxane was removed in vacuo and the aqueous

layer chilled, covered with a layer of ethyl acetate, and acidiﬁed to pH 4 with dilute KHSO₄. This was

followed by extraction (ethyl acetate) and puriﬁcation (1:1:1) of ethyl acetate–hexane–acetic acid; ethyl

1

acetate) to yield the compound

5

(Scheme 7 ; 7.07 g; 94%) as a white solid. H-NMR (300 MHz, CDCl₃):

7.29 (d, J = 8.40 Hz, 2H), 7.19 (d, J = 8.47 Hz, 2H), 3.70 (s, 1H), 3.58 (s, 2H), 1.52 (s, 9H).

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Scheme 7. Synthetic chemical route to compound 5.

2-(3-((tert-Butoxycarbonyl)amino)phenyl)acetic Acid (13). A solution of 3-aminophenylacetic acid (12, 4 g,

26.46 mmol, 1 equiv.) in a mixture of dioxane (52 mL) and water (52 mL), and sodium carbonate (2.8 g,

26.42 mmol, 1 equiv., in 26 mL of water) was stirred and cooled in an ice bath. Di-tert-butyl dicarbonate

(BOC-anhydride, 6.24 g, 28.59 mmol, 1.1 equiv.) was added in one portion, and stirring was continued

at room temperature for 4 h. The dioxane was removed in vacuo and the aqueous layer chilled, covered

with a layer of ethyl acetate, and acidiﬁed to pH 4 with dilute KHSO₄. This was followed by extraction

(ethyl acetate) and puriﬁcation by a ﬁltration on silica with dichloromethane followed by ethyl acetate

1

to yield the compound 13 (Scheme 8 ; 6.65 g; 85%) of a white solid. H-NMR (300 MHz, CDCl₃): 7.31

(d, 2H, J = 8.4 Hz), 7.20 (d, 2H, J = 8.4 Hz), 6.56 (br s, 1H), 3.59 (s, 2H), 1.5 (s, 9H).

Scheme 8. Synthetic chemical route to compound 13.

4.3. Protection of Aminophenylacetic Acids with Acetyl Groups (Synthesis of 8, 15 and 21a)

2-(4-Acetamidophenyl)acetic Acid (

8). According to a reported procedure [49], 4-aminophenylacetic acid

(4

, 2.0 g) was added to acetic acid (20 mL) under microwave irradiation (150 ^◦C) for 1 h. The reaction

mixture was poured into ethyl acetate then washed with water and then_◦allowed to dry to yield the

1

compound

8

δ

(Scheme 9; 2.252 g; 100%) as a white-grey solid. m.p. 166–168 C [57]. H-NMR (300 MHz,

DMSO-d₆):

= 9.92 (s, 1H), 7.51 (d, ³J = 8.6 Hz, 2H), 7.16 (d, ³J = 8.6 Hz, 2H), 3.49 (s, 1H), 2.03 (s, 3H).

¹³C-NMR (75 MHz, DMSO-d₆):

δ = 172.79 (C_q), 172.02 (C_q), 168.13 (C_q), 137.83 (C_q), 129.52 (CH),

118.87 (CH), 40.11 (CH₂), 23.87 (CH₃). IR: 3348 (w), 1709 (s), 1638 (m), 1601 (s), 1542 (s), 1538 (s), 1221

1

(m), 1195 (m), 968 (w), 806 (w), 723 (w). H-NMR (400 MHz, CD₃OD):

δ

7.48 (d, J = 8.8 Hz, 2H), 7.21

(d, J = 8.8 Hz, 2H), 3.55 (s, 2H), 2.10 (s, 3H); HRMS (FAB⁺): [M + H]⁺calcd. for C₁₀H₁₂O₃N, 194.0817;

found, 194.0770.

Scheme 9. Synthetic chemical route to compound 8.

2-(3-Acetamidophenyl)acetic Acid (15). 3-Aminophenylacetic acid (12, 200 mg) was added to acetic

acid (2 mL) under microwave irradiation (150 ^◦C) for 10 min. The reaction mixture was poured into a

mixture of ethyl acetate (1:50), then washed with water and then allowed to dry to yield the compound 15

(Scheme 10 ) (232 mg; 91%) as a white solid. m.p. 128–129 ^◦C. ¹H-NMR (300 MHz, DMSO-d₆):

δ

= 12.27

(s, 1H), 9.90 (s, 1H), 7.47 (dd, ³J = 2.32 Hz and 4.57 Hz, 1H), 7.24–7.19 (mc, 1H), 6.91 (d, ³J = 7.70 Hz, 1H),

3.51 (s, 2H), 2.03 (s, 3H). ¹³C-NMR (75 MHz, DMSO-d₆):

= 172.52 (C_q), 168.21 (C_q), 139.26 (C_q), 135.39

δ

(C_q), 128.48 (C_q), 124.00 (C_q), 119.76 (C_q), 117.32 (C_q), 40.86 (CH₂), 23.94 (CH₃). IR: 3298 (w), 1699 (s),

1662 (s), 1544 (m), 1492 (m), 1411 (m), 1308 (m), 1273 (m), 1232 (m), 886 (m), 782 (s), 714 (s).

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Scheme 10. Synthetic chemical route to compound 15.

2-(2-Acetamidophenyl)acetic Acid (21a). Oxindole 20 (2.5 g, 14.7 mmol, 1 equiv.) was diluted in 6 M HCl

(15 mL) and heated to reﬂux with stirring overnight. The solution was cooled and washed with

dichloromethane (3

×

100 mL). The aqueous phase was concentrated to give 21 as a white solid (2.73 g,

99%). The crude product was involved directly in the next step. A solution of 2-aminophenylacetic

acid hydrochloride (1.5 g, 8 mmol, 1 equiv.) in dichloromethane (110 mL) at room temperature was

treated with triethylamine (2.35 mL, 17.6 mmol, 2.2 equiv.) and acetyl chloride (682 µL, 9.6 mmol,

1.2 equiv.). The reaction mixture was stirred at r.t. for 3 h. The reaction mixture was diluted with

EtOAc and washed with water. The aqueous layer was extracted with ethyl acetate. The combined

extracts were rinsed with brine, dried over MgSO₄. Puriﬁcation on silica gel with cyclohexane/ethyl

◦

acetate (20:5 to 1:1) gives the desired product 21a (Scheme 11; 160 mg, 10%). m.p. 130–131 C. ¹H-NMR

(300 MHz, CDCl₃):

δ

= 8.21 (d, ³J = 8.29 Hz, 1H), 7.34–7.25 (m, 2H), 7.20–7.15 (m, 1H), 3.71 (s, 2H), 2.67

(s, 3H). ¹³C-NMR (75 MHz, CDCl₃):

δ

= 175.47 (C_q), 171.03 (C_q), 141.54 (C_q), 128.37 (CH), 125.09 (CH),

124.10 (CH), 123.57 (C_q), 116.82 (CH), 36.80 (CH₂), 26.83 (CH₃).

Scheme 11. Synthetic chemical route to compound 21a.

4.4. General Procedure for the Kochi-Anderson Reaction of Menadione with N-Protected Phenylacetic Acid

Derivatives (Synthesis of 6, 14, 9, 16, 22 and 25)

tert-Butyl(4-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl)phenyl) carbamate (

6

). A solution of

menadione (500 mg, 2.9 mmol, 1.0 equiv.) and phenylacetic acid

5 (1.455 g, 5.8 mmol, 2.0 equiv.) in

◦

acetonitrile (27 mL) and water (9 mL) was heated to 70 C. AgNO₃(50 mg, 0.29 mmol, 0.1 equiv.)

was added. (NH₄)₂S₂O₈(860 mg, 3.77 mmol, 1.3 equiv.) in acetonitrile (3 mL) and water (1 mL)

was added dropwise over a period of 45 min and then heated at reﬂux for 2 h. The acetonitrile was

removed in vacuo. The product was extracted with EtOAc (1 × 10 mL), dichloromethane (4 × 10 mL),

dried over MgSO₄and puriﬁed by chromatography on silica gel (cyclohexane/dichloromethane 30:5)

◦

to give the pure compound

¹H-NMR (300 MHz, CDCl₃):

6

δ

(Scheme 12 ; 243 mg; 22% yield) as a yellow solid. m.p. 148–149 C.

= 8.05–8.08 (m, 2H), 7.66–7.71 (m, 2H), 7.23 (d, ³J = 8.26 Hz, 2H), 7.12

(d, ³J = 8.57 Hz, 2H), 6.36 (s, 1H), 3.95 (s, 2H), 2.21 (s, 3H), 1.47 (s, 9H). ¹³C-NMR (75 MHz, CDCl₃):

δ = 185.42 (C_q), 184.68 (C_q), 152.73 (C_q), 145.38 (C_q), 144.21 (C_q), 136.70 (C_q), 133.47 (CH), 133.44 (CH),

132.61 (C_q), 132.10 (C_q), 132.03 (C_q), 129.14 (CH), 126.45 (CH), 126.25 (CH), 118.88 (CH), 80.49 (C_q),

31.74 (CH₂), 28.30 (CH₃), 13.22 (CH₃). EI MS (70 eV, m/z (%)): 377.2 ([M]⁺, 18), 321.1 (66), 305.9 (100),

261.1 (59), 201.3 (14), 160.1 (18), 121.1 (21). IR: 3439 (b, vs), 1704 (w), 1685 (w), 1660 (s), 1618 (m),

1596 (m), 1521 (m), 1370 (w), 1315 (m), 1296 (m), 1236 (w), 1162 (s), 709 (w). EA: obs. C, 72.94%; H,

6.16%; N, 3.74%, calcd. C, 73.19%; H, 6.14%, N, 3.71% for C₂₃H₂₃NO₄.

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Scheme 12. Synthetic chemical route to compound 6.

tert-Butyl (3-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl)phenyl) carbamate (14). A solution of

menadione (1.71 g, 9.93 mmol, 1.0 equiv.) and phenylacetic acid 13 (5.0 g, 19.9 mmol, 2.0 equiv.) in

acetonitrile (36 mL) and water (12 mL) was heated to 70 ^◦C. AgNO₃(173 mg, 0.29 mmol, 0.1 equiv.)

was added. (NH₄)₂S₂O₈(2.955 g, 3.77 mmol, 1.3 equiv.) in acetonitrile (11 mL) and water (3.5 mL)

was added dropwise over a period of 45 min and then heated at reﬂux for 3 h. The acetonitrile was

removed in vacuo. The product was extracted with EtOAc (2

dried over MgSO₄and puriﬁed by chromatography on silica gel (toluene) a second chromatography

×

10 mL), dichloromethane (4

×

10 mL),

on silica was necessary (cyclohexane/dichloromethane 10:1) to give the pure compound 14 (Scheme 13;

1

348 mg; 9% yield) as a yellow solid. H-NMR (300 MHz, CDCl₃):

δ = 8.10–8.05 (m, 2H), 7.72–7.66 (m,

2H), 7.28–7.26 (m, 1H), 7.20–7.14 (m, 2H), 6.86 (d, J = 7.52 Hz, 1H), 6.46 (s, 1H), 3.99 (s, 2H), 2.24 (s, 3H),

1.49 (s, 9H). ¹³C-NMR (75 MHz, CDCl₃):

= 185.45 (C_q), 184.73 (C_q), 152.73 (C_q), 145.24 (C_q), 144.70

δ

(C_q), 139.04 (C_q), 138.76 (C_q), 133.58 (CH), 133.57 (CH), 132.23 (C_q), 132.12 (C_q), 129.37 (CH), 126.60

(CH), 126.38 (CH), 123.31 (CH), 118.81 (CH), 116.79 (CH), 80.60 (CH), 32.48 (CH₂), 28.43 (CH₃), 13.44

(CH₃). HRMS-ESI (m/z): [M + Na]⁺calcd. for C₂₃H₂₃NO₄Na 400.1519; found 400.1539.

Scheme 13. Synthetic chemical route to compound 14.

N-(4-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl)phenyl)acetamide

(

9).

A

solution of

menadione (600 mg, 3.48 mmol, 1.0 equiv.) and phenylacetic acid

8 (1.3 g, 6.72 mmol, 2.0 equiv.) in

◦

acetonitrile (12 mL) and water (4 mL) was heated to 70 C. AgNO₃(59 mg, 0.35 mmol, 0.1 equiv.) was

added. (NH₄)₂S₂O₈(1.032 g, 4.52 mmol, 1.3 equiv.) in 4 mL acetonitrile and 2 mL water was added

dropwise over a period of 45 min and then heated at reﬂux for 3 h. The acetonitrile was removed in

vacuo. The product was extracted with dichloromethane (4

×

10 mL), dried over MgSO₄and puriﬁed

by chromatography on silica gel (cyclohexane/ethyl acetate 80:20 to cyclohexane/ethyl acetate 50:_◦50)

to give the pure compound

¹H-NMR (300 MHz, CDCl₃):

9

δ

(Scheme 14 ; 597 mg; 54% yield) as a yellow solid. m.p. 198–200 C.

= 8.12–8.05 (m, 2H), 7.72–7.67 (m, 2H), 7.38 (d, ³J = 8.40 Hz, 2H), 7.17

(d, ³J = 8.40 Hz, 2H), 3.99 (s, 2H), 2.24 (s, 3H), 2.14 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃): δ = 185.50 (C_q),

184.80 (C_q), 168.28 (C_q), 145.38 (C_q), 144.49 (C_q), 136.39 (C_q), 134.13 (C_q), 133.64 (CH), 132.27 (C_q),

132.17 (C_q), 129.29 (CH), 126.60 (CH), 126.44 (CH), 120.36 (CH), 32.01 (CH₂), 24.67 (CH₃), 13.40 (CH₃);

IR: 3359 (w), 1690 (s), 1661 (s), 1648 (s), 1593 (m), 1533 (s), 1411 (w), 1371 (m), 1334 (m), 1311 (s),

1295 (vs), 1259 (m), 972 (w), 818 (m), 735 (m), 709 (s), 679 (w).

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Scheme 14. Synthetic chemical route to compound 9.

N-(3-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl)phenyl)acetamide

(

16).

A

solution of

menadione (380 mg, 2.1 mmol, 1.0 equiv.) and phenylacetic acid 15 (812 mg, 4.2 mmol, 2.0 equiv.)

in acetonitrile (6 mL) and water (2 mL) was heated to 70 ^◦C. AgNO₃(36 mg, 0.21 mmol, 0.1 equiv.)

was added. (NH₄)₂S₂O₈(623 mg, 2.73 mmol, 1.3 equiv.) in acetonitrile (1.5 mL) and water (0.5 mL)

was added dropwise over a period of 45 min and then heated at reﬂux for 3 h. The acetonitrile

was removed in vacuo. The product was extracted with dichloromethane (4

MgSO₄and puriﬁed by chromatography on silica gel (cyclohexane/ethyl acetate 10:1) to give the

×

10 mL), dried over

◦

1

pure compound 16 (Scheme 15; 670 mg; 52% yield) as a yellow solid. m.p. 163–165 C. H-NMR

(300 MHz, CDCl₃):

= 8.10–8.03 (m, 2H), 7.71–7.65 (m, 2H), 7.41 (d, ³J = 8.30 Hz, 1H), 7.32 (s, 2H), 7.20

δ

(t, ³J = 7.82 Hz, 1H), 6.96 (d, ³J = 7.80 Hz, 1H), 3.99 (s, 2H), 2.24 (s, 3H), 2.12 (s, 3H). ¹³C-NMR (75 MHz,

CDCl₃): δ = 185.41 (C_q), 184.79 (C_q), 168.41 (C_q), 145.15 (C_q), 144.80 (C_q), 139.09 (C_q), 138.36 (C_q),

133.63 (CH), 132.25 (C_q), 132.12 (C_q), 129.41 (CH), 126.59 (CH), 126.43 (CH), 124.59 (CH), 119.99 (CH),

118.16 (CH), 32.50 (CH₂), 24.71 (CH₃), 13.44 (CH₃); IR: 1661 (m), 01609 (w), 1551 (w), 1490 (w), 1374 (w),

1319 (w), 1293 (m), 726 (w).

Scheme 15. Synthetic chemical route to compound 16.

N-(2-((3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl)phenyl)acetamide

(22). A solution of

2-aminophenylacetic acid hydrochloride (21a, 4.55 g, 24.25 mmol, 1 equiv.) in dichloromethane (110 mL)

at room temperature was treated with triethylamine (7.15 mL, 48.5 mmol, 2.2 equiv.) and acetyl chloride

(2.06 mL, 29.12 mmol, 1.2 equiv.). The reaction mixture was stirred at r.t. for 3 h. The reaction mixture

was diluted with EtOAc and washed with water. The aqueous layer was extracted with ethyl acetate.

The combined extracts were rinsed with brine, dried over MgSO₄. Thus, a solution of the crude and

menadione (890 mg, 5.17 mmol, 1.0 equiv.) in acetonitrile (32 mL) and water (11 mL) was heated to

70 ^◦C. AgNO₃(88 mg, 0.52 mmol, 0.1 equiv.) was added. (NH₄)₂S₂O₈(1.533 g, 6.72 mmol, 1.3 equiv.) in

acetonitrile (10 mL) and water (5 mL) was added dropwise over a period of 45 min and then heated at

reflux for 3 h. The acetonitrile was removed in vacuo. The product was extracted with dichloromethane

(4 × 10 mL), dried over MgSO₄and purified by chromatography on silica gel (cyclohexane/ethyl acetate

10:1 to cyclohexane/ethyl acetate 5:5) to give the pure compound 22 (Scheme 16 . 673 mg; 39% yield) as

a yellow solid. m.p. 218–220 ^◦C. ¹H-NMR (300 MHz, CDCl₃): δ = 8.99 (s, 1H), 8.11–8.05 (m, 2H), 7.86

(d, ³J = 8.01 Hz, 1H), 7.73–7.70 (m, 2H), 7.25–7.19 (m, 2H), 7.07–7.02 (m, 1H), 3.95 (s, 2H), 2.43 (s, 3H), 2.31

(s, 3H). ¹³C-NMR (100 MHz, CDCl₃): δ = 186.32 (C_q), 184.90 (C_q), 168.86 (C_q), 145.77 (C_q), 145.07 (C_q),

135.98 (C_q), 134.12 (CH), 133.70 (CH), 132.10 (C_q), 131.81 (C_q), 129.95 (CH), 129.24 (C_q), 127.72 (CH),

126.63 (CH), 125.08 (CH), 124.74 (CH), 28.43 (CH₂), 24.39 (CH₃), 14.14 (CH₃). IR: 3394 (w), 1674 (m),

1660 (s), 1505 (m), 1467 (m), 1295 (vs), 794 (m), 762 (s), 716 (s), 690 (m).

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Scheme 16. Synthetic chemical route to compound 22.

2-Methyl-3-(4-bromo-benzyl)-4a,8a-dihydro-[1,4]naphthoquinone (25c). A solution of menadione (5.81 mmol)

and a phenylacetic acid derivative 24c (11.58 mmol) in a mixture of (52.5 mL of acetonitrile and 17.5 mL

of water) was heated to 85 ^◦C. Then AgNO₃(90 mg, 0.58 mmol) was added and (NH₄)₂S₂O₈(1.72 g,

7.54 mmol) in 15 mL of acetonitrile and 5 mL of water was added dropwise over a period of 45 min and

then heated at reﬂux for two hours. The acetonitrile was removed in vacuo. The aqueous phase was

extracted with dichloromethane (4

on silica gel (petroleum ether/dichloromethane 1:1) to give the compound 25c (Scheme 17; 3.10 g;

9.12 mmol, 78% yield) as a yellow solid. m.p. 121–122 ^◦C. ¹H-NMR (300 MHz, CDCl₃):

= 8.03–8.10

(m, 2H), 7.66–7.71 (m, 2H), 7.36 (dt, J = 8.46 Hz, J = 1.95 Hz, 2H), 7.09 (d, J = 8.53 Hz, 2H), 3.96

(s, 2H), 2.22 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃):

= 185.20 (C_q), 184.54 (C_q), 144.75 (C_q), 144.57 (C_q),

×

10 mL), dried over MgSO₄and puriﬁed by ﬂash-chromatography

δ

3

4

3

δ

137.06 (C_q), 133.58 (CH), 132.08 (C_q), 131.94 (C_q), 131.71 (CH), 130.32 (CH), 126.50 (CH), 126.35 (CH),

120.31 (C_q), 31.93 (CH₂), 13.31 (CH₃). EI MS (70 eV, m/z (%)): 340.1 ([M]⁺, 13), 325.0 (100), 246.1 (63),

215.1 (41), 202.1 (49), 128.1 (72), 76.0 (74). IR (KBr): 3449 cm⁻¹(b, w), 3068 (w), 2962 (w), 1661 (vs),

1624 (m), 1618 (m), 1594 (s), 1486 (s), 1376 (m), 1332 (s), 1315 (s), 1294 (vs), 1071 (m), 1010 (s), 971 (w),

815 (m), 787 (s), 730 (m), 702 (m), 629 (w), 426 (w). EA: obs. C, 63.02%; H, 3.84%, calcd. C, 63.36%; H,

3.84% for C₁₈H₁₃BrO₂.

Scheme 17. Synthetic chemical route to compound 25c.

2-Methyl-3-(2-bromo-benzyl)-4a,8a-dihydro-[1,4]naphthoquinone (25a). After chromatography on silica gel

(petroleum ether/dichloromethane 1:1), 1.75 g (5.14 mmol, 88% yield) of 25a (Scheme 18) were isolated

◦

as a yellow solid. m.p. 94–95 C. ¹H-NMR (300 MHz, CDCl₃):

δ

= 8.06–8.11 (m, 2H), 7.66–7.72 (m, 2H),

7.56 (dd, ³J = 7.87 Hz, ⁴J = 1.32 Hz, 1H), 7.13 (dt, ³J = 7.47 Hz, ⁴J = 1.35 Hz, 1H), 7.04 (dt, ³J = 7.69 Hz

⁴J = 1.75 Hz, 1H), 6.89 (dd, ³J = 7.61 Hz, ⁴J = 1.56 Hz, 1H), 4.11 (s, 2H), 2.10 (s, 3H). ¹³C-NMR (75 MHz,

CDCl₃): = 184.98 (Cq), 184.30 (Cq), 145.85 (Cq), 144.53 (Cq), 137.31 (Cq), 133.55 (CH), 133.53 (CH),

132.87 (CH), 132.13 (Cq), 131.96 (Cq), 128.59 (CH), 127.93 (CH), 127.55 (CH), 126.53 (CH), 126.33

(CH), 124.67 (Cq), 32.65 (CH₂), 13.26 (CH₃). EI MS (70 eV, m/z (%)): 261.1 ([M

Br]⁺, 100), 231.1 (11),

,

δ

−

202.1 (11), 130.1 (8), 76.0 (10). IR: 3441 cm⁻¹(b, s), 3068 (w), 3017 (w), 2923 (w), 1660 (vs), 1621 (s),

1594 (s), 1467 (m), 1439 (m), 1376 (w), 1318 (m), 1296 (vs), 1263 (m), 1223 (w), 1184 (w), 1025 (m),

976 (m), 787 (w), 749 (s), 729 (m), 695 (w), 663 (w). EA: obs. C, 63.12%; H, 3.91%; Br, 23.31%, calcd. C,

63.36%; H, 3.84%; Br, 23.42% for C₁₈H₁₃BrO₂.

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Scheme 18. Synthetic chemical route to compound 25a.

2-Methyl-3-(3-bromo-benzyl)-4a,8a-dihydro-[1,4]naphthoquinone (25b). After chromatography on silica

gel (petroleum ether/dichloromethane 1:1)_◦, 328 mg (9.96 mmol, 52% yield) of 25b (Scheme 19 ) were

1

isolated as a yellow solid. M.p. 108–109 C. H-NMR (300 MHz, CDCl₃):

δ

= 7.98–8.02 (m, 2H),

7.59–7.65 (m, 2H), 7.28 (s, 1H), 7.24 (td, ³J = 6.78 Hz, ⁴J = 1.97 Hz, 1H), 7.02–7.09 (m, 2H), 3.92 (s, 2H),

2.16 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃):

= 185.16 (Cq), 184.45 (Cq), 144.78 (Cq), 144.50 (Cq), 140.35

δ

(Cq), 133.59 (CH), 132.10 (Cq), 131.94 (Cq), 131.52 (CH), 130.16 (CH), 129.65 (CH), 127.26 (CH), 126.54

(CH), 126.36 (CH), 122.72 (Cq), 32.09 (CH₂), 13.35 (CH₃). EI MS (70 eV, m/z (%)): 340.0 ([M]⁺, 28), 325.06

−1

(100), 246.13 (18), 215.14 (7), 202.12 (8), 184.99 (12), 76.0 (10). IR (KBr): 3430 cm (b, w), 1658 (vs), 1620

(vs), 1595 (vs), 1568 (s), 1474 (s), 1431 (m), 1381 (s), 1334 (vs), 1290 (vs), 1261 (s), 1180 (s), 1074 (m), 974

(s), 955 (s), 793 (s), 780 (vs), 728 (vs), 692 (s), 687 (s), 422 (m). EA: obs. C, 63.55%; H, 3.94%; Br, 23.69%,

calcd. C, 63.36%; H, 3.84%; Br, 23.42% for C₁₈H₁₃BrO₂.

Scheme 19. Synthetic chemical route to compound 25b.

4.5. Deprotection of the Benzylamine Acteyl Group (Synthesis of 7 and 17)

2-(4-Aminobenzyl)-3-methylnaphthalene-1,4-dione (7). 4-Acetamide derivative 9 (200 mg, 0.62 mmol) was

added to HCl (1 mL) in a mixture of water and methanol (6 mL, v/v, 1/1) under microwave irradiation

(150 ^◦C) for 2 h. The reaction mixture was poured into ethyl acetate then washed with a saturated

Na₂CO₃solution and then allowed to dry. 172 mg (76%) of compound

purple solid with no further puriﬁcation. m.p. 162–164 ^◦C. ¹H-NMR (300 MHz, CDCl₃):

(m, 2H), 7.69–7.67 (m, 2H), 7.03 (d, J = 8.30 Hz, 2H), 6.60 (d, J = 8.27 Hz, 2H), 3.91 (s, 2H), 2.24 (s, 3H).

¹³C-NMR (100 MHz, CDCl₃):

= 185.68 (C_q), 184.93 (C_q), 145.99 (C_q), 144.95 (C_q), 143.97 (C_q), 133.53

7

(Scheme 20) was isolated as a

δ

= 8.09–8.06

δ

(CH), 133.49 (CH), 132.27 (C_q), 132.25 (C_q), 129.67 (CH), 127.97 (C_q), 126.54 (CH), 126.33 (CH), 115.50

(CH), 31.69 (CH₂), 13.33 (CH₃). IR: 3474 (w), 3377 (w), 2932 (w), 1653 (s), 1617 (s), 1590 (m), 1515 (s),

1336 (m), 1293 (vs), 1182 (m), 973 (m) 816 (m), 768 (m), 708 (vs). HRMS-ESI (m/z): [M + H]⁺calcd. for

C₁₈H₁₆NO₂278.1176; found 278.1154.

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Scheme 20. Synthetic chemical route to compound 7.

2-(3-Aminobenzyl)-3-methylnaphthalene-1,4-dione (17). 3-Acetamide derivative 16 (278 mg, 0.87 mmol)

was added to HCl (1 mL) in a mixture of water and methanol (8 mL, v/v, 1/1) under microwave

◦

irradiation (150 C) for 20 min. The reaction mixture was poured into ethyl acetate then washed

with a saturated Na₂CO₃solution and then allowed to dry. After chromatography on silica gel

(cyclohexane/ethyl acetate 75:25 to cyclohexane/ethyl acetate 1:1), 150 mg (0.54 mmol, 62% yield)

of compound 17 (Scheme 21) was isolated as a purple solid. m.p. 159–160 ^◦C. ¹H-NMR (300 MHz,

CDCl₃):

6.56–6.48 (m, 2H), 3.95 (s, 3H), 2.24 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃):

δ

= 8.12–8.06 (m, 2H), 7.73–7.67 (m, 2H), 7.05 (t, ³J = 7.67 Hz, 1H), 6.62 (d, ³J = 7.56 Hz, 1H),

δ

= 185.67 (C_q), 184.95 (C_q),

146.88 (C_q), 145.63 (C_q), 144.66 (C_q), 139.46 (C_q), 133.69 (CH), 133.37 (CH), 132.40 (C_q), 132.32 (CH),

129.75 (CH), 126.72 (CH), 126.50 (CH), 119.19 (CH), 115.49 (CH), 113.54 (CH), 32.56 (CH₂), 13.52 (CH₃);

ESI MS: 278.12 (M⁺). IR: 3452 cm⁻¹(w), 3372 (w), 2921 (w), 1649 (m), 1615 (s), 1588 (s), 1492 (m),

1335 (m), 1292 (vs), 974 (m), 867 (m), 716 (s).

Scheme 21. Synthetic chemical route to compound 17.

4.6. Dialkylation of Benzylamine Menadione Derivatives (Synthesis of 3, 18, 11 and 19)

2-(4-(Dimethylamino)benzyl)-3-methylnaphthalene-1,4-dione (

0.82 mmol, 3 equiv.) was added by small portions to a stirred solution of

1 equiv.) in acetonitrile (5 mL) and 37% aqueous formaldehyde (220 L, 2.7 mmol, 10 equiv.) in

acetonitrile (1 mL) placed in a cool bath was then added. An additional glacial acetic acid portion

(45 L, 0.46 mmol, 1.7 equiv.) was added and stirring for 2 h. The reaction mixture was poured into

3). Sodium cyanoborohydride (52 mg,

7

(76 mg, 0.27 mmol,

µ

diethyl ether (10 mL) and then washed with 1N potassium hydroxide (10 mL) and brine. The organic

phase was dried and evaporated in vacuo. The crude product was puriﬁed by chromatography on silica

gel using a mixture of cyclohexane/ethyl acetate (10:1 to 5:5) affording the compound

3

(Scheme 22;

1

48 mg, 59%) as a purple solid. H-NMR (300 MHz, CDCl₃):

δ = 8.09–8.06 (m, 2H), 7.71–7.67 (m, 2H),

7.12 (d, ³J = 8.78 Hz, 2H), 6.67 (d, ³J = 7.73 Hz, 2H), 3.93 (s, 2H), 2.89 (s, 6H), 2.27 (s, 3H). ¹³C-NMR

(75 MHz, CDCl₃): = 185.72 (C_q), 185.00 (C_q), 145.96 (C_q), 143.65 (C_q), 142.94 (C_q), 133.51 (CH), 133.47

δ

(CH), 132.28 (C_q), 129.52 (CH), 126.55 (CH), 126.31 (CH), 113.26 (C_q), 40.96 (CH₃), 31.56 (CH₂), 13.35

(CH₃). IR: 2923 cm⁻¹(w), 2852 (w), 1660 (s), 1615 (m), 1594 (m), 1520 (s), 1291 (vs), 1130 (m), 945 (m),

814 (m), 689 (s).

Scheme 22. Synthetic chemical route to compound 3.

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2-(3-(Dimethylamino)benzyl)-3-methylnaphthalene-1,4-dione (18). Sodium cyanoborohydride (52 mg,

0.82 mmol, 4.5 equiv.) was added by small portions to a stirred solution of 17 (50 mg, 0.18 mmol,

1 equiv.) in acetonitrile (5 mL) and 37% aqueous formaldehyde (220

µL, 2.7 mmol, 15 equiv.) in

acetonitrile (1 mL) was then added. An additional glacial acetic acid portion (45

µL, 0.46 mmol,

5.1 equiv.) was added and stirring for 2 h. The reaction mixture was poured into diethyl ether (10 mL)

and then washed with potassium hydroxide 1 N (10 mL) and brine. The organic phase was dried and

evaporated in vacuo. The crude product was puriﬁed by chromatography on silica gel using a mixture

of cyclohexane/ethyl acetate (10:1 to 5:5) affording the compound 18 (Scheme 23; 17 mg, 31%) as a

1

purple solid. H-NMR (300 MHz, CDCl₃):

δ ,

= 8.12–8.06 (m, 2H), 7.72–7.66 (m, 2H), 7.12 (t, ³J = 7.89 Hz

1H), 6.65 (sl, 1H), 6.60–6.55 (m, 2H), 4.00 (s, 2H), 2.91 (s, 6H), 2.27 (s, 3H). ¹³C-NMR (100 MHz, CDCl₃):

= 185.58 (C_q), 184.82 (C_q), 150.93 (C_q), 145.74 (C_q), 144.35 (C_q), 138.95 (C_q), 133.51 (CH), 133.46 (CH),

δ

132.26 (C_q), 132.25 (C_q), 129.37 (CH), 126.56 (CH), 126.31 (CH), 116.97 (CH), 113.17 (CH), 110.94 (CH),

40.69 (CH₃), 32.90 (CH₂), 13.42 (CH₃). IR: 2920 cm⁻¹(w), 2807 (w), 1661 (vs), 1594 (vs), 1501 (m),

1361 (m), 1292 (m), 1292 (vs), 997 (m), 974 (m), 842 (m), 758 (s), 744 (s), 691 (vs).

Scheme 23. Synthetic chemical route to compound 18.

2-(4-(Diethylamino)benzyl)-3-methylnaphthalene-1,4-dione (11). Sodium cyanoborohydride (67 mg,

1.08 mmol, 3 equiv.) was added by small portions to a stirred solution of

1 equiv.) and acetaldehyde (200 L, 3.6 mmol, 10 equiv.) in acetonitrile (1 mL) placed in a cool

bath was added. Glacial acetic acid (60 L, 0.61 mmol, 1.7 equiv.) was then added and stirring for

7 (100 mg, 0.36 mmol,

µ

2 h. The reaction mixture was poured into diethyl ether (10 mL) and then washed with potassium

hydroxide 1N (10 mL) and brine. The organic phase was dried and evaporated in vacuo. The crude

product was puriﬁed by chromatography on silica gel using a mixture of cyclohexane/ethyl acetate

(10:1 to 5:5) affording the compounds 10 (17%) and 11 (Scheme 24; 36%) as a purple solid. m.p.

94–96 ^◦C. ¹H-NMR (300 MHz, CDCl₃):

2H), 6.58 (d, ³J = 8.70 Hz, 2H), 3.91 (s, 2H), 3.29 (q, ³J = 7.06 Hz, 4H), 2.28 (s, 3H), 1.11 (t, ³J = 7.06 Hz,

6H); ¹³C-NMR (75 MHz, CDCl₃):

= 185.81 (C_q), 185.02 (C_q), 146.60 (C_q), 146.21 (C_q), 143.69 (C_q),

δ ,

= 8.11–8.04 (m, 2H), 7.71–7.65 (m, 2H), 7.08 (d, ³J = 8.70 Hz

δ

133.48 (C_q), 133.43 (CH), 132.32 (CH), 129.73 (C_q), 129.62 (CH), 126.55 (CH), 126.30 (CH), 124.56 (C_q),

112.25 (CH), 44.48 (CH₂), 31.50 (CH₂), 13.37 (CH₃), 12.71 (CH₃). ESI MS: 333.17 (M⁺); EA: obs. C,

80.19%; H, 8.01%; N, 5.35%, calcd. C, 79.25%; H, 6.25%, N, 4.20% for C₂₂H₂₃NO₂; HRMS-ESI (m/z):

[M + H]⁺calcd. for C₂₂H₂₄NO₂334.1802; found 334.1765.

Scheme 24. Synthetic chemical route to compound 11.

2-(3-(Diethylamino)benzyl)-3-methylnaphthalene-1,4-dione (19). Sodium cyanoborohydride (52 mg,

0.82 mmol, 4.5 equiv.) was added by small portions to a stirred solution of 17 (50 mg, 0.18 mmol,

1 equiv.) and acetaldehyde (150

was added. Glacial acetic acid (45

The reaction mixture was poured into diethyl ether (10 mL) and then washed with 1N potassium

µL, 2.7 mmol, 15 equiv.) in acetonitrile (1 mL) placed in a cool bath

µL, 0.46 mmol, 5.1 equiv.) was then added and stirring for 2 h.

Molecules 2017, 22, 161

24 of 32

hydroxide (10 mL) and brine. The organic phase was dried and evaporated in vacuo. The crude

product was puriﬁed by chromatography on silica gel using a mixture of cyclohexane/ethyl acetate

1

(10:1 to 5:5) to afford the compound 19 (Scheme 25; 13 mg, 22%) as a purple solid. H-NMR (300 MHz,

CDCl₃):

2H), 3.99 (s, 2H), 3.31 (q, ³J = 7.06 Hz, 4H), 2.29 (s, 3H), 1.13 (t, ³J = 7.03 Hz, 6H). ¹³C-NMR (75 MHz,

CDCl₃): = 185.66 (C_q), 184.87 (C_q), 148.13 (C_q), 145.88 (C_q), 144.16 (C_q), 139.07 (C_q), 133.52 (CH),

δ

= 8.12–8.05 (m, 2H), 7.72–7.66 (m, 2H), 7.09 (t, ³J = 7.92 Hz, 1H), 6.60 (sl, 1H), 6.53–6.47 (m,

δ

133.45 (CH), 132.27 (C_q), 132.24 (C_q), 129.57 (CH), 126.54 (CH), 126.31 (CH), 115.67 (CH), 112.40 (CH),

110.10 (CH), 44.47 (CH₂), 32.98 (CH₂), 13.45 (CH₃), 12.70 (CH₃). IR: 2975 cm⁻¹(w), 2929 (w), 1658 (s),

1595 (vs), 1498 (m), 1290 (vs), 1023 (m), 975 (m), 755 (m), 731 (s), 693 (s). HRMS-ESI (m/z): [M + H]⁺

calcd. for C₂₂H₂₄NO₂334.1802; found 334.1819.

Scheme 25. Synthetic chemical route to compound 19.

4.7. General Procedure for the Suzuki-Miyaura Coupling Reaction (Synthesis of 26–28)

A Schlenk-tube was ﬂushed with argon and successively ﬁlled with 25 (100 mg, 0.29 mmol,

1 equiv.), boronic acid derivative (0.32 mmol, 1.1 equiv.), K₂CO₃(122 mg, 0.88 mmol, 3.0 equiv.) and

ﬁnally dissolved in dioxane/water (12 mL/3 mL). The solution was degassed by bubbling argon

through the mixture for 20 min. Then 4 mol % PdCl₂(dppf) (10 mg, 0.012 mmol) was added, the

Schlenk-tube was sealed and heated overnight at 80 ^◦C. The reaction was quenched by adding 10 mL

H₂O. All volatiles solvents were removed in vacuo, the product was extracted with CH₂Cl₂, dried

over MgSO₄and puriﬁed by ﬂash-chromatography.

0

2-(4 -Dimethylamino-biphenyl-4-ylmethyl)-3-methyl-[1,4]naphthoquinone (26). 4-Dimethylamino phenylboronic

acid was used as the boronic acid coupling partner for compound 25. After chromatography on silica

gel (dichloromethane:petroleum ether/3:1), 96 mg (0.25 mmol, 86% yield) of 26 (Scheme 26 ) was

◦

isolated as a black solid. m.p. 170–171 C. ¹H-NMR (300 MHz, CDCl₃):

δ

= 8.05–8.11 (m, 2H), 7.65–7.71

(m, 2H), 7.43 (d, ³J = 8.39 Hz, 4H), 7.24 (d, ³J = 8.17 Hz, 2H), 6.76 (d, ³J = 8.09 Hz, 2H), 4.03 (s, 2H),

2.96 (s, 6H), 2.27 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃):

= 185.47 (C_q), 184.74 (C_q), 145.43 (C_q), 144.34

δ

(C_q), 139.43 (C_q), 135.69 (C_q), 133.49 (CH), 133.46 (CH), 132.15 (C_q), 132.09 (C_q), 128.94 (CH), 127.58

(CH), 126.53 (CH), 126.52 (CH), 126.28 (CH), 112.75 (CH), 40.60 (CH₃), 32.08 (CH₂), 13.37 (CH₃). EI MS

(70 eV, m/z (%)): 381.2 ([M]⁺, 100), 366.1 (19). IR (KBr): 3432 cm⁻¹(b, m), 3028 (w), 2922 (w), 2855

(w), 2803 (w), 1660 (vs), 1612 (vs), 1595 (s), 1534 (w), 1504 (vs), 1444 (w), 1375 (w), 1357 (m), 1333 (m),

1294 (vs), 1226 (w), 1168 (w), 946 (w), 810 (s), 790 (s), 766 (w), 722 (w), 711 (w), 692 (w). EA: obs. C,

81.58%; H, 619%; N, 3.60%, calcd. C, 81.86%; H, 6.08%; N, 3.67% for C₂₆H₂₃NO₂.

Scheme 26. Synthetic chemical route to compound 26.

0

2-(4 -tert-Butyl-biphenyl-4-ylmethyl)-3-methyl-[1,4]naphthoquinone (27). tert-Butylphenylboronic acid was

used as the boronic acid coupling partner for compound 25. After chromatography on silica gel

Molecules 2017, 22, 161

25 of 32

(dichloromethane:petroleum ether/3:1), 113 mg (0.286 mmol, 97% yield) of 27 (Scheme 27 ) was isolated

◦

1

as a yellow solid. M.p. 112–114 C. H-NMR (300 MHz, CDCl₃):

(m, 2H), 7.41–7.50 (m, 6H), 7.24–7.30 (m, 2H), 4.06 (s, 2H), 2.28 (s, 3H), 1.35 (s, 9H). ¹³C-NMR (75 MHz,

CDCl₃): = 185.34 (C_q), 184.63 (C_q), 150.13 (C_q), 145.25 (C_q), 144.40 (C_q), 139.24 (C_q), 137.85 (C_q),

δ = 8.06–8.13 (m, 2H), 7.66–7.71

δ

136.75 (C_q), 133.45 (CH), 132.12 (C_q), 132.03 (C_q), 128.93 (CH), 127.21 (CH), 126.59 (CH), 126.46 (CH),

126.25 (CH), 125.64 (CH), 34.47 (C_q), 32.08 (CH₂), 31.32 (CH₃), 13.30 (CH₃). EI MS (70 eV, m/z (%)):

394.3 ([M]⁺, 42), 379.2 (100). IR: 3439 cm⁻¹(b, m), 2962 (w), 1659 (vs), 1620 (m), 1595 (m), 1498 (w),

1462 (w), 1377 (w), 1334 (w), 1295 (s), 1182 (w), 1114 (w), 976 (w), 815 (m), 790 (w), 713 (w), 568 (w).

EA: obs. C, 85.16%; H, 6.66%, calcd. C, 85.25%; H, 6.64% for C₂₈H₂₆O₂.

Scheme 27. Synthetic chemical route to compound 27.

2-(4⁰-nitro-Butyl-biphenyl-4-ylmethyl)-3-methyl-[1,4]naphthoquinone (28). 4-Nitrophenylboronic acid was

used as the boronic acid coupling partner for compound 25. After chromatography on silica gel

(dichloromethane:petroleum ether/3:1), 67 mg (0.175 mmol, 95% yield) of 28 (Scheme 28) was isolated

as a yellow solid. M.p. 197–199 ^◦C. ¹H-NMR (300 MHz, CDCl₃):

δ

= 8.23–8.26 (m, 2H), 8.05–8.11 (m,

2H), 7.65–7.73 (m, 4H), 7.51 (d, ³J = 8.27 Hz, 2H), 7.34 (d, ³J = 8.22 Hz, 2H), 4.08 (s, 2H), 2.27 (s, 3H).

¹³C-NMR (75 MHz, CDCl₃):

= 185.27 (C_q), 184.65 (C_q), 147.18 (C_q), 147.00 (C_q), 144.86 (C_q), 144.68

δ

(C_q), 139.16 (C_q), 136.95 (C_q), 133.63 (CH), 132.12 (C_q), 131.98 (C_q), 129.40 (CH), 127.65 (CH), 127.59

(CH), 126.52 (CH), 126.38 (CH), 124.12 (CH), 32.22 (CH₂), 13.40 (CH₃). EI MS (70 eV, m/z (%)): 383.3

([M]⁺, 41), 368.2 (100). IR (KBr): 3436 cm⁻¹(b, m), 3073 (w), 2934 (w), 1661 (vs), 1620 (m), 1596 (vs),

1513 (vs), 1485 (m), 1375 (w), 1344 (vs), 1295 (vs), 1261 (w), 1182 (w), 1111 (m), 974 (w), 852 (m), 821 (m),

787 (w), 745 (m), 711 (m), 693 (m), 555 (w). EA: obs. C, 72.95%; H, 4.47%; N, 3.56%, calcd. C, 72.79%;

H, 4.68%; N, 3.54% for C₂₄H₁₇NO₄0.7 H₂O.

Scheme 28. Synthetic chemical route to compound 28.

4.8. In Vitro Antimalarial Activity

4.8.1. Hypoxanthine Method Determination of IC₅₀Values of Dd2 Growth Inhibition

The IC₅₀values were determined using standard in vitro proliferation assays [58]. Erythrocytes

infected with ring-stage parasites (0.5% parasitemia, 2.5% hematocrit) were exposed to the compounds

for 48 h and then to radioactive hypoxanthine for 24 h in 96-well plates. The amount of radioactivity in

precipitable material served as an index of parasite proliferation.

4.8.2. SYBR_greenMethod for Determination of IC₅₀Values of P. falciparum Dd2

In vitro antiplasmodial activity is expressed as the 50% inhibitory concentration (IC₅₀) of

intraerythrocytic parasite development, using the SYBR_greenI assay as described before [

5,59].

Brieﬂy, synchronous ring-stage parasites were incubated for 72 h in the presence of decreasing drug

Molecules 2017, 22, 161

26 of 32

concentrations in microtiter plates (ﬁnal parasitemia 0.5%; ﬁnal hematocrit 1.5%). Each inhibitor

was analyzed in 3-fold serial dilutions in duplicate and in at least three independent experiments.

Parasite replication was assessed by ﬂuorescent SYBR_greenstaining of parasitic DNA [60] as previously

described [59]. IC₅₀values were calculated using SigmaPlot.

4.9. Cytotoxicity against Human MRC-5 Cells

MRC-5_SV2cells are cultured in Earls MEM +5% FCSi. Assays are performed in 96-well microtiter

plates, each well containing about 10⁴cells/well. After 3 days incubation, cell viability is assessed

ﬂuorimetrically after addition of resazurin and ﬂuorescence is measured (

λ

ex

550 nm,

λ

em

590 nm).

The results are expressed as % reduction in cell growth/viability compared to untreated control wells

and CC50 is determined. Compounds are tested at 5 concentrations (64–16–4–1–0.25 µM). When the

CC₅₀is lower than 16 µM, the compound is classiﬁed as toxic.

4.10. In Vivo Antimalarial Activity

4.10.1. Animals

Swiss mice (female—BW ~25 g; Janvier France, Le Genest Saint Isle, France) were allocated

randomly to ﬁve groups of four animals each. Drinking water and food were available ad libitum

throughout the experiment. The weight of the individual animals did not differ too much from the

group mean.

4.10.2. Infection and Drug Treatment of P. berghei-Infected Mice

P. berghei (ANKA-strain) is maintained in the laboratory by weekly mechanical subpassage in

Swiss mice. The infection inoculum was prepared by taking heparinized blood was collected from a

clinically ill donor mouse (approximately 20% parasitaemia) and diluted in PBS to obtain an infection

inoculum of 0.15 mL with about 4

×

10⁸infected erythrocytes. The infection-inoculum was given

intraperitoneally. The untreated infected controls developed severe malaria with most animals showing

severe clinical signs on day 4. Chloroquine was used as reference treatment (10 mg/kg ip for 5 days).

Treatment with chloroquine resulted in 100% survival until day 7 with low parasitaemia at day 4 post

infection. The tested compounds were evaluated after ip dosing at 50 mg/kg for 5 days.

4.11. Physicochemistry

4.11.1. Starting Materials and Solvents

Distilled water was further puriﬁed by passing it through a mixed bed of ion-exchanger

(R3-83002, M3-83006, Bioblock Scientiﬁc, Illkirch, France) and activated carbon (Bioblock Scientiﬁc

ORC-83005) and was de-oxygenated by CO₂- and O₂-free argon (Sigma Oxiclear cartridge) before use.

All the stock solutions were prepared in spectroscopic grade dimethylsulfoxide (DMSO, Bioreagent

for Molecular Biology, >>99.9%, Sigma) by weighing solid products using a XA105 Dual Range

(0.01/0.1 mg–41/120 g) balance Mettler Toledo (Viroﬂay, France) and the complete dissolution

was achieved using an ultrasonic bath. The ionic strength was maintained at 0.1 M with sodium

◦

perchlorate (NaClO₄.H₂O, Merck, p.a.), and all measurements were carried out at 25.0

±

0.2 C.

CAUTION! Perchlorate salts combined with organic ligands are potentially explosive and should be handled in

small quantities and with the adequate precautions [61].

4.11.2. Spectrophotometric Titrations Versus pH

The acido-basic properties of plasmodione derivatives

7 and 17 were performed in water

(0.1 M NaClO₄) while those of compounds 10 11 18 and 19 were performed in NaClO₄0.1 M/DMSO

3,

,

(91:9 v/v) for solubility reasons. Absorption spectrop₋ho₅tometric titrations versus pH titrations of

−5

plasmodione derivatives

3

(1.81

×

10 M),

7

(2.00

×

10 M), 10 (1.81

×

10 M), 11

(

2.18 × 10⁻⁵M),

Molecules 2017, 22, 161

27 of 32

17 (2.00

×

10⁻⁵M), 18 (1.84

×

10⁻⁵M) and 19 (1.79

×

10⁻⁵M) were carried out by diluting stock

solutions freshly prepared in spectroscopic grade DMSO in 40 mL of the corresponding solvent

contained in a jacketed cell (Metrohm, Villebon sur Yvette, France) maintained at 25.0

◦

±

0.2 C

(thermostat E200, Lauda, Roissy Charles de Gaulle, France). The free hydrogen ion concentration was

measured with a combined glass electrode (Metrohm 6.0234.500, Long Life) ﬁlled with 0.1 M NaCl in

water and an automatic titrator system 794 Basic Titrino (Metrohm) connected to a microcomputer

(Tiamo light 1.2 program for the acquisition of the potentiometric data). The combined glass electrode

was calibra₋te₁d as a hydrogen concentration probe by titrating known amounts of hydrochloric acid

(~1.06

(~9.35

×

10 M from HClO₄, normapur, 70% min Prolabo) with CO₂-free sodium hydroxide solution

10⁻²M from NaOH, BdH Prolabo Chemicals, AnalaR, via VWR distribution) [62]. The HClO₄

and NaOH solutions were freshly prepared just before use and titrated with sodium tetraborate

decahydrate (B₄Na₂O₇10H₂O, puriss, p.a, Fluka) and potassium hydrogen phthalate (C₈H₅KO₃,

·

Fluka, puriss, p.a.), respectively, using methyl orange (RAL) and phenolphthalein (Prolabo, purum)

as the indicators. The GLEE program [62] was applied for the glass electrode calibration (standard

electrode potential E₀/mV and slope of the electrode/mV pH⁻¹) and to check carbonate levels of

the NaOH solutions used (<5%). The cell was thermostated at 25.0

E200 thermostat. A stream of argon, pre-saturated with water vapour, was passed over the surface of

the solution. The initial pH was adjusted to ~2–3 with HClO₄(Prolabo, normapur, 70% min) and the

±

0.2 ^◦C by the ﬂow of a Lauda

absorption spectrophotometric titrations of the plasmodione derivatives versus pH (~3 < pH < ~9–10

were carried out by addition of known volumes of NaOH solutions using the automatic titrator

of the 794 Basic Titrino device (DET method). After each addition, an absorption spectra was

automatically and repeatedly recorded using a CARY 50 spectrophotometer (Varian, Les Ulis, France)

ﬁtted with Hellma optical ﬁbers (041.002-UV, Hellma, Plainview, NY, USA) and an immersion probe

made of quartz suprazil (Hellma, 661.500-QX) and interfaced (Cetrib) with the potentiometric unit.

The distribution curves of the protonated species of the plasmodione derivatives as a function of pH

were calculated using the Hyss program [63].

)

4.11.3. Analysis and Processing of the Spectroscopic Data

The spectrophotometric data were analyzed with the Specﬁt program [64–66] which adjusts the

absorptivities and the stability constants of the species formed at equilibrium. Specﬁt uses factor

analysis to reduce the absorbance matrix and to extract the eigenvalues prior to the multiwavelength

ﬁt of the reduced data set according to the Marquardt algorithm [67,68].

5. Conclusions

In summary, new synthetic procedures were successfully developed for the preparation of

biaryl analogues of the antimalarial plasmodione in a 2-step sequence based on Kochi-Anderson

and Suzuki-Miyaura coupling reactions. 3-Benzylmenadione derivatives bearing a terminal -N(Me)₂or

-N(Et)₂in the different position of the benzylic chain were prepared in a 4-step synthesis for antimalarial

drug evaluation using the multiresistant P. falciparum Dd2 strain. Interestingly, the non-protonable

4⁰-NHBoc-amino-3-benzylmenadione was selected for its potent in vitro activity, suggesting that

cytosol-food vacuole shuttling of redox-cyclers may not need to accumulate in the food vacuole

through lysosomotropic effects. This compound also revealed to be active in vivo in P. berghei-infected

mice. These observations encourage pursuing further optimization of 3-benzyl-menadiones to improve

the pharmacokinetic proﬁle of plasmodione that has been shown to fulﬁll the criteria required for early

antimalarial lead compounds.

Supplementary Materials: Supplementary materials can be accessed at: http://ww w .mdpi.com/1420-3049/22/

1/161/s1., on pages S2-S5: Physico-chemical data for compounds 7, 11, 17 , 18 (Figures S1–S4), and pages S6–S31:

¹H-NMR and ¹³C-NMR spectra of compounds 3, 5–9, 11, 14–19, 21–22, 25–28.

Molecules 2017, 22, 161

28 of 32

Acknowledgments: This work was made possible by grants of the ANR_émergence2010 program (grant SCHISMAL

to E.D.-C.), and of the Laboratoire d’Excellence ParaFrap (grant LabEx ParaFrap ANR-11-LABX-0024 to E.D.C.).

The Centre National de la Recherche Scientifique (CNRS France, via UMR 7509 to E.D.C.), the University of Strasbourg,

and the International Center for Frontier Research in Chemistry in Strasbourg (project entitled “Computer

−

aided

design of novel antimalarial naphthoquinones”, Acronym: “CAD NQ”, grant icFRC Innovation 2015 to E.D.-C.),

−

partly supported this work. L.M. and E.D.-C. (MC members) thank the COST Action CM1307, entitled “Targeted

Chemotherapy towards Diseases Caused by Endoparasites” for covering the exchanges and the costs to publish the

special issue of Molecules “COST Action CM1307—Proceedings in Medicinal and Natural Product Chemistry” in open

access. K.E. is grateful to CNRS, France, and to Alain van Dorsselaer for her co-funded CNRS doctoral fellowship

(BDI). E.D.-C. and M.E. acknowledge Matthieu Chessé for the measurement of pK_avalues.

Author Contributions: K.U. and E.D.-C. conceived and designed the experiments; K.U. and K.E. performed the

experiments; T.M., K.U., M.J., L.M., M.E. and E.D.-C. analyzed the data; M.L. contributed reagents/materials/

analysis tools; M.J., M.E., L.M. and E.D.-C. wrote the paper.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

The following abbreviations are used in this manuscript:

ACT

CQ

Artemisinin-based Combination Therapy

chloroquine

DHODH

DMSO

EA

dihydroorotate dehydrogenase

dimethylsulfoxide

Elemental analyses

equiv.

ESI-MS

HRMS

ip

equivalent

Electronspray-Mass spectrometry

High Resolution Mass Spectra

intraperitoneal

MB

methylene blue

mETC

M.p.

mitochondrial electron transport

Melting point

N-Boc

yDHODH

N-tert-butyloxycarbonyl

dihydroorotate dehydrogenase from yeast

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Article Doi

Pharmacomodulation of the antimalarial plasmodione: Synthesis of biaryl-and N-arylalkylamine analogues, antimalarial activities and physicochemical properties

DOI: 10.3390/molecules22010161

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