Antimalarial dual drugs based on potent inhibitors of glutathione reductase from Plasmodium falciparum

1260

J. Med. Chem. 2008, 51, 1260–1277

Antimalarial Dual Drugs Based on Potent Inhibitors of Glutathione Reductase from

Plasmodium falciparum

Wolfgang Friebolin,^§Beate Jannack,^§Nicole Wenzel,^§Julien Furrer,^†Thomas Oeser,^†Cecilia P. Sanchez,^#Michael Lanzer,^#

Vanessa Yardley,^¶Katja Becker, and Elisabeth Davioud-Charvet*^,§,‡

Biochemie-Zentrum der UniVersität Heidelberg, Im Neuenheimer Feld 504, 69120 Heidelberg, Germany, Organisch-Chemisches Institut der

UniVersität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany, Hygiene Institut, Abt. Parasitologie, Im Neuenheimer Feld 324,

69120 Heidelberg, Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E

7HT, U.K., Interdisciplinary Research Center, Justus-Liebig-UniVersity, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany, and Centre National de

la Recherche Scientiﬁque, 3 Rue Michel-Ange, 75794 Paris Cedex 16, France

ReceiVed July 30, 2007

Plasmodium parasites are exposed to higher ﬂuxes of reactive oxygen species and need high activities of

intracellular antioxidant systems providing a steady glutathione ﬂux. As a future generation of dual drugs,

18 naphthoquinones and phenols (or their reduced forms) containing three different linkers between the

4-aminoquinoline core and the redox active component were synthesized. Their antimalarial effects have

been characterized in parasite assays using chloroquine-sensitive and -resistant strains of Plasmodium, alone

or in drug combination, and in the Plasmodium berghei rodent model. In particular, two tertiary amides 34

and 36 showed potent antimalarial activity in the low nanomolar range against CQ-resistant parasites. The

ability to compete both for (Fe^III)protoporphyrin and for chloroquine transporter was determined. The data

are consistent with the presence of a carrier for uptake of the short chloroquine analogue 2 but not for the

potent antimalarial amide 34, suggesting a mode of action distinct from chloroquine mechanism.

Introduction

belongs to the drug/metabolite superfamily^13,14and is thought

to act as a channel^15–17or a carrier of chloroquine.^7,9,17–22

During the past decades chloroquine (CQ^a) was one of the

most successful antimalarial drugs since its introduction 60 years

ago. This success was based on both its high antimalarial

effectiveness and its cheapness and safety. It was shown that

the antimalarial 4-aminoquinolines, like CQ, accumulate in the

acidic food vacuole of the parasites and inhibit heme

biomineralization.^1–6Consequently toxic heme builds up in

the vacuole, subsequently killing the parasite. However, the

use of CQ as a standard therapeutic drug became more and

more restricted in the world because of the dissemination of

resistant malaria strains of which Plasmodium falciparum is

the most deadly species. CQ resistance in Plasmodium

falciparum is associated with mutations in the digestive

vacuole transmembrane protein PfCRT^7–11and with eleva-

tions of intracellular glutathione concentrations.¹²Plasmo-

dium falciparum chloroquine resistance transporter (PfCRT)

Evidence from structure–activity relationships in the

4-aminoquinoline series has demonstrated that CQ resistance

can be overcome by subtly altering the length^23–26and the

basic nature of the CQ side chain.²⁷The level of accumulation

depends upon the pK_aof the quinoline side chain nitrogen.^28,29

However, CQ analogues with a shortened N-alkyl side chain,

although very active against sensitive as well as resistant

malarial strains in vitro, were less active in vivo tests because

of rapid N-dealkylation in the parasite/cell.²⁶Modiﬁcations

of the substituents on the aromatic nucleus,^24,25,30,31e.g.,

exchange of the 7-chloro atom, or the deaza-bioisosteres of

CQ^32,33have only little inﬂuence toward resistant strains or

are inactive. In addition, introduction of structural diversity

in the side chain of 4-aminoquinolines was recently shown

to enhance activity against drug-resistant P. falciparum^34,35

or to affect the partition coefﬁcient leading to distinct

localization of the 4-aminoquinoline in the parasites.

Plasmodium parasites are exposed to higher ﬂuxes of

reactive oxygen species and need high activities of intracel-

lular antioxidant systems. They do not develop in glucose-

6-phosphate dehydrogenase deﬁcient red blood cells or in

erythrocytes depleted in glutathione reductase activity.^36,37

High ﬂux of reactive oxygen species results both from the

host immune response to infection and from hemoglobin

digestion. In sensitive parasites, CQ prevents heme detoxi-

ﬁcation, resulting in free heme accumulation and conse-

quently in parasite death and erythrocyte lysis. Free heme in

concert with oxygen species is thought to catalyze oxidation

reactions and protein damage. The small amount of free heme

(Fe³⁺) released from the food vacuole in the cytosol is rapidly

reduced into heme (Fe²⁺) by the reducing milieu including

glutathione. Heme (Fe²⁺) can enter the Fenton reaction (eq

1), which participates in the protophorphyrin protein destruc-

tion due to the formation of hydroxyl radical or some higher

* To whom correspondence should be addressed. Phone: +49-6221-54-

4188. Fax: +49-6221-54-5586. E-mail: elisabeth.davioud@gmx.de.

§

Biochemie-Zentrum der Universität Heidelberg.

†

Organisch-Chemisches Institut der Universität Heidelberg.

Hygiene Institut.

London School of Hygiene and Tropical Medicine.

#

¶

Justus-Liebig-University.

Centre National de la Recherche Scientiﬁque. E.D.-C. is delegate in

‡

the frame of a French-German cooperation with Heidelberg University,

Germany.

a

Abbreviations: CQ, chloroquine; DIEA, diisopropylethylamine; DMA,

N,N-dimethylacetamide; DMF, N,N-dimethylformamide; DMSO, dimethyl

sulfoxide; EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; ESI-MS,

electrospray ionization mass spectrometry; GR, glutathione reductase;

HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuoro-

phosphate; HOBt, 1-hydroxybenzotriazole; M5, 6-[2′-(3′-methyl)-1′,4′-

naphthoquinolyl]hexanoic acid; PfCRT, Plasmodium falciparum chloroquine

resistance transporter; TLC, thin layer chromatography; TFA, triﬂuoroacetic

acid.

10.1021/jm7009292 CCC: $40.75

2008 American Chemical Society

Published on Web 02/09/2008

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1261

Chart 1. Structures of 1,4-Naphthoquinones and 1,4-Dimethoxynaphthalenes and Their Related 4-Aminoquinoline Derivatives

Chart 2. Structures of 4-Aminoquinoline Derivatives Based on Glutathione Reductase Inhibitors and Their Precursors

oxidation state transition metal species (eq 2), which remain

bound to the heme iron and can initiate attack on the

porphyrin ring.

included the 4-aminoquinoline-based isatin derivatives,⁵⁰the

peroxide-based trioxaquine derivatives,^51,52ferrocene-chloroquine

analogues,^27,53the 4-aminoquinolines based on inhibitors of a

neutral zinc aminopeptidase,⁵⁴or primaquine based on plasme-

psin inhibitors.⁵⁵These compounds were identiﬁed as dual drugs

or Trojan horse drugs in literature.

Fe²⁺+ H₂O₂f Fe³⁺+ HO^•+ OH^-

Fe²⁺+ H₂O₂f FeO²⁺+ H₂O

(1)

(2)

Since 4-aminoquinolines accumulate in infected erythrocytes,

we hypothesized that exploiting the heme-catalyzed oxidation

reactions might provide a general and speciﬁc approach to

design new 4-aminoquinolines as antimalarial agents. Oxidative

N-dealkylation of amides and amines is reported in numerous

drug detoxiﬁcation pathways or in drug bioactivation.⁵⁶There-

fore, we designed and synthesized three series of 4-amino-

quinolines (Chart 2) containing the GR inhibitors, the naph-

thoquinones, or their related deprotected and reduced precursors,

the dimethoxynaphthalene. These motifs were linked to the basic

side chain via an amide, an amine, or an ether bond (Chart 1),

which might be labile under pathological conditions found in

the parasite, i.e., the heme-catalyzed oxidation reactions. The

1,4-dimethoxynaphthalenes are inactive as GR inhibitors (Davi-

oud-Charvet, unpublished data) but chemically more stable and

easier to handle than the naphthoquinones. Under speciﬁc

conditions found in the food vacuole of the parasites, the 1,4-

dimethoxynaphthalenes might be oxidized to the active naph-

thoquinone species. So the function of the 4-aminoquinoline

part within the dual drug is, next to its own antiparasitic activity,

the accumulation and the transport of the second molecule part,

the naphthoquinones, or its naphthalene precursor within the

infected/parasitic cells.

For this purpose we ﬁrst prepared new short CQ derivatives

with alterations in the side chain to lower the molecular weight

of the ﬁnal dual drug while maintaining the antimalarial activity.

We also prepared new 1,4-dimethoxynaphthalenes in order to

generate their corresponding 1,4-naphthoquinones upon biooxi-

dation. These new inhibitors include introduction of the triﬂu-

oromethyl group to reinforce the oxidant character of the parent

menadione. Introduction of ﬂuorine at the methyl group of

menadione was recently shown to affect the redox potential

values of the ﬂuoro analogue versus menadione and to increase

the GR inhibitory capacity.⁵⁷From both components the dual

This reaction is well-known in cytochrome c chemistry,³⁸and

autoxidation of hemoglobin in the presence of glutathione has

been investigated at the molecular level.^39–41Glutathione

indirectly contributes to heme degradation (via the Fenton

reaction) in the cytosol by redox-cycling the heme Fe³⁺in heme

Fe^{2+ 42}

. The glutathione-dependent heme degradation was shown

to be inhibited by 4-aminoquinolines,⁴³likely by slowing the

overall rate of the heme redox-cycling. This observation indeed

reveals the cofacial π-π sandwich-type complex formed

between CQ (and the related 4-aminoquinolines) and heme Fe³⁺

in the form of two hematin µ-oxo dimers, an interaction that

was exploited in numerous screening assays to measure the heme

binding afﬁnity in the presence of potential drugs.^44,45

One of the most important antioxidative systems consists of

(di)thiols (e.g., glutathione), which are recycled by disulﬁde

reductases, namely, both glutathione reductases (GR) of the

malarial parasite P. falciparum and of man and the thioredoxin

reductase of P. falciparum.⁴⁶An elevation of glutathione content

in parasites leads to increased resistance to CQ,⁴⁷while GSH

depletion in resistant P. falciparum strains is expected to restore

the sensitivity to CQ. An important class of inhibitors for the

glutathione and thioredoxin reductases are the 1,4-naphtho-

quinones. Two representatives are the menadione and its

hexanoic acid derivative 6-[2′-(3′-methyl)-1′,4′-naphthoquinolyl]

hexanoic acid, or M₅(Chart 1).^48,49As previously demonstrated,

they are uncompetitive inhibitors of GR with respect to both

NADPH and glutathione disulﬁde. The ﬁrst generation of

double-headed prodrugs based on the GR inhibitor M₅and

4-aminoquinolines was designed, and the compounds proved

to be potent antimalarial agents both in vitro and in vivo.⁴⁸

Alternatively the link developed between the 4-aminoquino-

line and the drug was also designed in other laboratories as not

being labile under physiological conditions. Such approach

1262 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Scheme 1. Synthesis of Short Chloroquine Analogues 1-9^a

Friebolin et al.

tryptophan.⁶²Reaction of glycin-tert-butylamide with 4,7-

dichloroquinoline giving 8 as reported was followed by

BH₃·THF complex reduction to the amine 9.

For all 1,4-dimethoxynaphthalene derivatives bearing a tri-

ﬂuoromethyl group the 2-bromomethyl-1,4-dimethoxy-3-trif-

luoromethylnaphthalene 13 was the central intermediate (Scheme

2). The bromide 13 was accessible from 1,4-dimethoxy-2-

methylnaphthalene 10 through bromination at the aromatic ring

with Br₂in CH₂Cl₂at 0 °C to the bromide 11 (86%) and then

replacement of the bromine by the CF₃group using (triﬂuo-

romethyl)copper.⁶³This reagent can be produced from

CF₃CO₂Na and CuI by decarboxylation in toluene/DMA at high

temperatures.⁶⁴After 8 h at 150-170 °C the 1,4-dimethoxy-

2-methyl-3-triﬂuoromethylnaphthalene 12 was isolated in 46%

yield next to 33% of the unreacted bromide 11. It was not

possible for us to increase this yield through longer reaction

times, higher temperatures, or the use of DMF instead of DMA.

In all cases we recovered the unreacted starting material, which

was then recycled. Finally, the bromination of the methyl group

in 12 with N-bromosuccinimide in CCl₄and benzoyl peroxide

as initiator^63,64gave 55% of the desired 2-bromomethyl-1,4-

dimethoxy-3-triﬂuoromethylnaphthalene 13. For the synthesis

of the dual drugs 14 and 15 (Scheme 2), amodiaquine and

paracetamol were respectively deprotonated with cesium hy-

droxide⁶⁵in DMF together with molecular sieves and then

coupled with the bromide 13. This procedure was superior to

the use of NaH as a base. Both 2-alkoxymethyl-3-triﬂuorom-

ethylhydronaphthoquinol methyl ethers 14 and 15 were produced

in 82% and 93% yields, respectively.

^aConditions: (i) (1) 1 equiv of ketone or aldehyde, 2 equiv of Ti(OⁱPr)₄,

8 h, room temp; (2) 3 equiv of NaBH₄, EtOH, 15 h; (ii) Bu-NH₂, CsOH,

4 Å molecular sieves, DMF, 20 h, room temp; (iii) 1.05 equiv of 1 M

BH₃·THF, dry THF, 30 min, 0 °C, and then 2 h, room temp.

t

The synthesis of the structural related compounds 19 and 20

started from the 1,4-dimethoxynaphthalenes 16 and 10 (Scheme

3A). The carbaldehyde group was ﬁrst introduced by the reaction

with dichloromethyl methyl ether and SnCl₄⁶⁶to afford the 1,4-

dimethoxynaphthalen-2-carbaldehyde 17 (92%) or the 1,4-

dimethoxynaphthalen-3-methyl-2-carbaldehyde 18 (89%). Sub-

sequent reductive amination with the 4-aminoquinoline 1 and

NaBH(OAc)₃in CH₂Cl₂gave 19 (44%) or 20 (90%). The

triﬂuoromethyldimethoxynaphthalene analogue 21 could only

be prepared by substituting the bromide 13 by the terminal

primary amine in the 4-aminoquinoline 1 in DMF/EtOH with

an excess of NEt₃(85%) (Scheme 3B). Its dihydrochloride salt

21 was prepared after reaction with TMSCl in methanol. The

synthesis of the ﬁnal tertiary amines was performed by a high-

yielding reductive amination of acetone, isobutyraldehyde, or

pivalaldehyde in the presence of NaBH(OAc)₃as the reducing

agent. Use of the secondary starting amines 19, 20, or 21

respectively gave the N-isopropyl-substituted derivatives 22

(98%), 23 (80%), or 24 (87%), the N-isobutyl-substituted

derivatives 25 (99%), 26 (97%), or 27 (90%), and the N-

neopentyl-substituted derivatives 28 (97%), 29 (86%), or 30

(68%). This approach was considerably more effective than the

direct formation of the N-isopropyl- and the N-isobutyl-

substituted derivatives by reductive amination of the carbalde-

hydes 17 and 18 starting from the corresponding secondary

amines 2 and 3 under various conditions, e.g., NaBH(OAc)₃

drugs were created by linking them together in three different

ways. This linkage is, in the case of the carboxylic acid M₅

and its analogues, an amide bond and, in the other cases, an

amine bridge. A few nonquinoline dual drugs were also designed

with the view to establishing preliminary structure–activity

relationships with respect to the role of the 1,4-dimethoxynaph-

thalene moiety as naphthoquinone precursor. In this case, we

coupled the 1,4-dimethoxynaphthalene with two phenols known

to deplete the glutathione content in the cell, paracetamol or

amodiaquine, by an ether linkage. All new derivatives were

tested for their antimalarial potency against two P. falciparum

strains expressing different degrees of resistance with respect

to CQ. The most active dual drugs were tested in the Plasmo-

dium berghei rodent model. Additional results about the

mechanism of potent antimalarial tertiary amides are presented.

Results

Chemistry. The short CQ analogues 2-5 were synthesized

by reductive amination⁵⁸of the respective carbonyl compound

by N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine 1 in the presence

of NaBH₄and Ti(OⁱPr)₄(Scheme 1A). In our hands the synthesis

of the tert-butyl substituted 4-aminoquinoline 9 as previously

described²⁵did not work because the preparation of the amino

side chain, the N-tert-butylethylene diamine,⁵⁹mainly generates

aziridine from 2-bromoethylamine by thermal elimination.

Alternatively, another route was developed. We ﬁrst attempted

to substitute the bromo atom in the N-bromoethyl-4-amino-

quinoline 6 with tert-butylamine in the presence of CsOH in

DMF over molecular sieves⁶⁰(Scheme 1B). But instead of the

desired product, we exclusively obtained the aziridine 7 (93%).

Its X-ray structure is given in the Supporting Information. The

tert-butyl substituted 4-aminoquinoline 9 was then accessible

in two steps (Scheme 1C). First, glycin-tert-butylamide⁶¹was

produced according to the reported protocol developed for

67,58

(also under acidic conditions), Ti(OⁱPr)₄/NaBH(OAc)_3,

TiCl₄/NaBH₄, or NaBH(OAc)₃. The Mannich base 32 was

prepared from the CQ analogue 31, 1-(7-chloroquinolin-4-

yl)propane-3-isopropyl-1,3-diamine, and paracetamol through

the Mannich reaction in the presence of paraformaldehyde under

reﬂux for 3 days (Scheme 3C).

For the synthesis of the amides 34-36, derived from

naphthoquinones and the short CQ analogues 1 and 2

(Scheme 4), the starting acids 6-(3-methyl-1,4-dioxo-1,4-

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1263

Scheme 2. Synthesis of 2-Alkoxymethyl-3-triﬂuoromethylhydronaphthoquinol Methyl Ethers 14 and 15^a

^aConditions: (i) 2.5 equiv of SnCl₂, concentrated HCl, EtOH, 40 °C, 10 min; 2.5 equiv of KOH, 3 equiv (CH₃O)₂SO₂, acetone, 2.5 h, room temp; (ii)

1.05 equiv of Br₂, CH₂Cl₂, 2 h; (iii) 3 equiv of CF₃COONa, 2 equiv of CuI, DMA/toluene, 170 °C, 12 h; (iv) 1.0 equiv of NBS, benzoyl peroxide, CCl₄,

reﬂux, 5 h; (v) (1) phenol, CsOH, 4 Å molecular sieves, DMF, 40 min, room temp at 40 °C; (2) bromide 13, DMF, 40 °C, 6.5 h.

Scheme 3. Synthesis of Phenolic Mannich Bases 19–30 and 32^a

^aConditions: (i) 2.5 equiv of SnCl₂, concentrated HCl, EtOH, 40 °C, 10 min; 2.5 equiv of KOH, 3 equiv of (CH₃O)₂SO₂, acetone, room temp, 2.5 h; (ii)

SnCl₄, CHCl₂OCH₃; (iii) (1) amine 1 in CH₂Cl₂, dry methanol, 2 h, room temp; (2) 3 equiv of NaBH(OAc)₃, 24 h, room temp; (iv) (1) excess acetone or

aldehyde in CH₂Cl₂, dry methanol, 2.5 h, room temp; (2) 3 equiv of NaBH(OAc)₃, 48-72 h, room temp; (v) 3 equiv of amine 1 in DMF, anhydrous EtOH,

3 equiv of NEt₃, 18 h, room temp; (v) 0.5 equiv of acetamidophenol, 1.25 equiv of aqueous formaldehyde, EtOH, reﬂux, 79 h.

68

dihydronaphthalen-2-yl)hexanoic acid M₅and [4-(1,4-

dimethoxy-3-methylnaphthalen-2-ylmethyl)phenyl]acetic acid

33⁵⁷were activated through standard coupling conditions

using HOBt/EDC (34, 62.1%; 35, 19%; 36, 23.2%). When

the coupling reaction was applied in the presence of the

secondary amine 2 in the presence of the starting menadione-

derived carboxylic acids M₅and 33, low to moderate yields

of the desired compound were obtained because of the

formation of a mixture consisting of two main products. The

mixture was separated by several fractionations through silica

gel chromatography columns. The main product (highest R_f,

yellow powder after desiccation) was the desired tertiary

amide. The second product (lowest R_f, brown gum after

desiccation) was assigned as a polymer formed upon proton

abstraction at the methyl group of the menadione moiety by

the basic secondary amine 2 (addition to the quinone

1264 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Friebolin et al.

Scheme 4. Synthesis of Tertiary Amides 34–36 and 39–41

Based on M₅, Its Benzyl Analogue 33, and Their Deprotected

Reduced Precursors 37 and 38^a

^aConditions: (i) 1 equiv of HOBt, 1.3 equiv of EDC, 1.5 equiv of amine,

DMF, overnight, room temp; (ii) (1) 3.7 equiv of SnCl₂, concentrated HCl,

EtOH, 40 °C, 2.5 h; (2) 4.4 equiv of (CH₃O)₂SO₂, acetone, 5.4 equiv of

KOH dropwise, 60 °C, 3.5 h; (iii) (1) 5.0 equiv of SOCl₂; (2) 1.2 equiv of

amine, NEt₃, CH₂Cl₂, 1 h, 0 °C, then overnight, room temp.

Figure 1. Inhibition of ꢀ-hematin formation by the 4-aminoquinolines,

chloroquine, the short chloroquine analogue 2, the tertiary amide 34,

and the glutathione reductase inhibitor M₅. IC₅₀values for ꢀ-hematin

inhibition were determined from the absorbance at 405 nm versus drug

(equiv)/hematin (equiv). The solid line is a dose-response curve resulting

from interpolated experimental data points measured in duplicate for

CQ (red circle), the short CQ analogue 2 (green circle), the tertiary

amide 34 (blue square), the GR inhibitor M₅(black square). As controls,

(i) the formation of ꢀ-hematin in the absence of the drug (red triangle,

100% polymerization) and (ii) the hematin content without preincu-

bation for 1 h at 60 °C (blue triangle), 0% polymerization) are shown

on the Y-axis.

methide). The corresponding 1,4-dimethoxynaphthalene de-

rivatives 39-41 were also prepared as potential dual drugs.

6-(1,4-Dimethoxy-3-methylnaphthalen-2-yl)hexanoic acid 37

and [4-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)phe-

nyl]acetic acid 38 were ﬁrst transformed into their respective

acid chloride with thionyl chloride in CH₂Cl₂and then

coupled in a one-pot reaction to the appropriate 4-amino-

quinoline 1 or 2. The yields of the coupling reaction were

low to moderate 39 (7%), 40 (61.4%), and 41 (14.6%). The

tertiary amide 36 was alternatively prepared by oxidation of

40 with cerium ammonium nitrate (CAN) in acetonitrile/water

in 62% yield.

Inhibition Assay of ꢀ-Hematin Formation. Four represen-

tative compounds, CQ, the short CQ analogue 2, and the

tertiary amide 34, were tested for their ability to inhibit the

polymerization of hematin in vitro using the colorimetric

ꢀ-hematin inhibition screening assay⁴⁵with slight modiﬁca-

tions described in the experimental procedure. We selected

DMF as the solvent because the tertiary amide 34 precipitated

in other solvents (aqueous HCl and MeOH or aqueous HCl

and n-butanol). CQ was used as reference inhibitor of

ꢀ-hematin formation. The GR inhibitor M₅was also tested

to evaluate separately the interference of the non-quinoline

portion of the dual drug 34 with the formation of ꢀ-hematin

As controls, we also measured (i) the formation of ꢀ-hematin

in the absence of the drug with or without DMF (0%

polymerization) and (ii) the hematin content without prein-

cubation for 1 h at 60 °C with or without DMF (100%

polymerization). The results of the experiment were expressed

as IC₅₀values representing the molar equivalents of tested

compounds, relative to 1 equiv of hematin, required to inhibit

the ꢀ-hematin formation by 50% (reproducible data from

three experiments). Considering the dose-response curves of

CQ and the short CQ analogue 2, the absorbance ﬁrst

decreased from 0 to 1 for CQ (red curve, Figure 1), and from

0 to 2 for 2 (green curve, Figure 1), revealing the shift of

the equilibrium between the dimer H₂O/HO-Fe(III)PPIX in

aqueous solution⁶⁹to the µ-oxo dimer. This shift is ac-

companied by the release of free sites of hematin because

water bound to HO-Fe(III)PPIX likely prevents the binding

of pyridine in accordance with the A405 decrease as the drug

equivalent number increased from 0. Then, when the drug

has saturated all the sites of the µ-oxo dimer, the inhibition

of the hematin polymerization reached the maximum level

in agreement with the A405 value to the highest point. Under

the conditions described, CQ showed an IC₅₀value of 1.6

and the maximal inhibition was reached at a drug/hematin

ratio of ∼2:1; these values are similar to the reported values.⁴⁵

The short CQ analogue 2 and the tertiary amide 34 displayed

IC₅₀values of 2.6 and 0.5 with a drug/hematin ratio of ∼3:1

and 0.9:1, respectively, while the GR inhibitor M₅displayed

no inhibition of the ꢀ-hematin formation. Thus, the tertiary

amide 34 displayed a potent ꢀ-hematin inhibitory activity

when compared with CQ and the short CQ analogue 2 and

with the non-quinoline portion M₅(Figure 1), suggesting that

both components of the tertiary amide are involved in the

hematin interaction.

Antimalarial Activity and Cytotoxicity of the Compounds

in Vitro. All compounds synthesized were tested for their

antiparasitic activity on the CQ-sensitive P. falciparum strain

3D7 and the CQ-resistant strain K1. In parallel, cytotoxicity

of the compounds was determined on human KB cells. The

results are expressed as IC₅₀and TD₅₀values representing

the drug concentration required to inhibit the growth of

parasites and human cells, respectively, by 50%. The ratio

(IC₅₀of K1)/(IC₅₀of 3D7) was also determined and is known

to increase with the cross-resistance toward chloroquine. Data

for the short CQ analogues are summarized in Table 1, for

dual drugs based on amodiaquine and paracetamol in Table

2, for the phenolic Mannich bases in Table 3, and for the

tertiary amides in Table 4. Among the short CQ analogues

(Table 1) the N-isobutyl- and the N-neopentyl-substituted

derivatives 3 and 4 exhibited the most potent antimalarial

effects with the highest activity for 4 against the CQ-resistant

strain K1. The excellent activity of both compounds is

consistent with the activity of short CQ analogues containing

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1265

Table 1. Antimalarial Activity of Short Chloroquine Analogues 1–9 and 31

compd

R

IC₅₀3D7^a(nM)

IC₅₀K1^a(nM)

(IC₅₀K1)/(IC₅₀3D7)^b

TD₅₀KB^c(µM)

1

2

3

4

5

7

8

9

-H

4.5

10.2

22.8

20.6

2.7

1800

96.7

72.0

400

9.5

3.1

0.3

53.7

2.4

1.4

6.0

40

49.6

89.8

22.5

62.1

71.9

101.6

194.0

142.9

47.9

-ⁱPr

-CH₂-ⁱPr

-CH₂-^tBu

-CH₂-^cPr

6.8

145.0

39235

1,439

127.8

144.0

519.3

16000

993.9

21.2

3.6

-^tBu

31

CQ

15.0

18.7

127.33

^aIn these assays, 3D7 and K1 are CQ-sensitive and -resistant Plasmodium falciparum strains, respectively. The standard drugs chloroquine (CQ) and

amodiaquine (AQ) served as positive controls for CQ-sensitive P. falciparum 3D7 strain and CQ-resistant P. falciparum K1 strain, respectively. ^bThe ratio

increases with the cross-resistance toward CQ. ^cThe cytotoxicity is evaluated against KB cells. Podophyllotoxin exhibited an IC₅₀value of 0.07 µM against

the human KB cell line.

Table 2. Antimalarial Activity of Potential Dual Drugs 14 and 15 Based on Amodiaquine and Paracetamol

compd

type

IC₅₀3D7^a

IC₅₀K1^a

(IC₅₀K1)/(IC₅₀3D7)^b

TD₅₀KB^c(µM)

14

15

AQ

dual drug

reference

32.0

6438

9.7

>198400

32.0

15737

6.4

1.0

2.4

0.6

15.0

>715

nd

paracetamol

86700

<0.4

nd

^aIn these assays, 3D7 and K1 are CQ-sensitive and -resistant Plasmodium falciparum strains, respectively. The standard drug amodiaquine (AQ) served

as positive control for chloroquine-sensitive P. falciparum 3D7 strain and chloroquine-resistant P. falciparum K1 strain. ^bThe ratio increases with the

cross-resistance toward CQ. ^cThe cytotoxicity is evaluated against KB cells. Podophyllotoxin exhibited an IC₅₀value of 0.07 µM against the human KB cell

line.

an ethylenediamine-based side chain and an increased anti-

malarial activity against the CQ-resistant strain K1.^25,26The

design of the dual drugs built in this study from both phenols,

amodiaquine and paracetamol, and the 2-alkoxymethyl-3-

triﬂuoromethyl-1,4-dimethoxynaphthalene did not afford

improved antimalarial effects with respect to the parent

phenols (Table 2). The data of the second series of dual drugs

based on phenolic Mannich bases (Table 3) revealed signiﬁ-

cant antimalarial effects, depending on the substitution both

of the N-alkyl (R₂) side chain of the 4-aminoquinoline and

the substitution of the 1,4-dimethoxynaphthalene (R₁). The

most potent antimalarial dual drugs are the 4-aminoquinolines

with an unbranched side chain (R₂) H), but they are also

the most cytotoxic ones against the human cells. Noteworthy

is the fact that they are more active than the parent short CQ

analogue 1. Among these three derivatives the most active

and the less toxic is the derivative 21 built from the

3-triﬂuoromethyl-1,4-dimethoxynaphthalene. In each sub-

series of 1,4-dimethoxynaphthalenes (R₁) H, Me, CF₃) the

cytotoxicity decreased with the branching of the N-alkyl

substituent (R₂) Pr) of the 4-aminoquinoline but also

i

coincided with a decrease of the antiplasmodial activity.

When the branching of the alkyl chain was moved from the

N atom by adding a methylene group, both the antimalarial

effects and the cytotoxicity increased suggesting a common

origin to both effects, likely through the generation of a

quinone methide upon oxidation followed by Michael addi-

tion (data not shown). Noteworthy is that oxidation of the

ﬁnal phenolic Mannich bases by CAN resulted in a rapid

degradation of the ﬁnal naphthoquinone upon elimination of

the amine moiety. For this reason the phenolic Mannich bases

under their 1,4-dimethoxynaphthalene forms were tested as

prodrugs (precursors) of naphthoquinones against the para-

sites in cultures. Finally tertiary amides were prepared starting

from the short CQ analogues 1 and 2 and the carboxylic acids

M₅and 33, identiﬁed earlier as potent GR inhibitors, or their

1266 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Friebolin et al.

Table 3. Antimalarial Activity of Phenolic Mannich Bases 19–30 and 32

compd

R₁

R₂

IC₅₀3D7^a(nM)

IC₅₀K1^a(nM)

TD₅₀KB^b(µM)

CQ

19

22

25

28

20

23

26

29

21

24

27

30

32

12.3

23.7

596.7

28.4

344.8

188.3

154.1

22.9

104.6

40.6

134.2

16.0

127.3

3.8

67.0

43.8

8.8

-H

H

-H

ⁱPr

517.2

146.4

237.2

229.4

83.7

182.9

155.7

127.5

183.8

162.1

146.0

22.7

-H

CH₂-ⁱPr

CH₂-^tBu

H

-Me

-CF₃

5.1

ⁱPr

85.8

30.6

10.6

28.5

35.4

11.9

12.0

180.5

CH₂-ⁱPr

CH₂-^tBu

H

ⁱPr

298.9

198.3

115.7

317.5

CH₂-ⁱPr

CH₂-^tBu

^aIn these assays, 3D7 and K1 are CQ-sensitive and -resistant Plasmodium falciparum strains, respectively. The standard drug chloroquine (CQ) served

as positive control for chloroquine-sensitive P. falciparum 3D7 strain and chloroquine-resistant P. falciparum K1 strain, respectively. ^bThe cytotoxicity is

evaluated against KB cells. Podophyllotoxin exhibited an IC₅₀value of 0.07 µM against the human KB cell line.

Table 4. Antimalarial Activity of Tertiary Amides 34–36 and 39–41 Based on M₅, Its Benzyl Analogue 33, and Their Deprotected Reduced Precursors

37 and 38

compd

type

IC₅₀3D7^a(nM)

IC₅₀K1^a(nM)

(IC₅₀K1)/(IC₅₀3D7)^b

TD₅₀KB^c(µM)

1

2

CQ analogue

dual drug

reference

4.5

10.2

7.5

183.9

12.4

1,800

96.7

9.4

367.3

5.3

302.4

268.3

505.3

571.0

400

9.5

1.1

2.0

0.4

5.7

0.8

1.7

18.7

49.6

89.8

37.0

22.2

318.32

6.9

1.7

11.51

127.33

34

35

36

39

40

41

CQ

52.8

318.7

288.8

5.8 -15.0

^aIn these assays, 3D7 and K1 are CQ-sensitive and -resistant Plasmodium falciparum strains, respectively. The standard drug chloroquine (CQ) served

as positive control for CQ-sensitive P. falciparum 3D7 strain and CQ-resistant P. falciparum K1 strain. ^bThe ratio increases with the cross-resistance toward

CQ. ^cThe cytotoxicity is evaluated against KB cells. Podophyllotoxin exhibited an IC₅₀value of 0.07 µM against the human KB cell line.

reduced and deprotected forms. Both tertiary amides 34 and

36 showed a higher activity than the parent 4-aminoquinoline

2 and the parent 1,4-naphthoquinones to equally kill sensitive

and resistant parasites. Because these tertiary amides do not

possess weak base properties as CQ and its short analogues,

another mechanism of action is strongly suggested to be

involved in their antimalarial activity.

In Vivo Antimalarial Activity Against P. berghei in Mice.

Three of the most active compounds (4, 21, 34), representa-

tive of each class, were tested in P. berghei infected mice

by intraperitoneal administration. Results of in vivo screens

for the three compounds conducted against chloroquine-

sensitive P. berghei ANKA BALB/c mice according to the

Peters’s 4-day test⁷⁰are given in Table 5. For comparative

purposes, data acquired in the same screens for CQ and the

three derivatives are included. The most active drug 4, the

short CQ analogue with a side chain containing a N-

neopentylamine group, caused a 94% reduction in parasitemia

at 30 mg/kg with no overt toxicity. Both dual drugs 21 and

34, based on 1,4-dimethoxy-2-triﬂuoromethylnaphthalene and

M₅, respectively, showed moderate activity (18% reduction

in parasitemia) on a 4-day treatment with a daily dose of 21

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1267

Table 5. In Vivo Antimalarial Response of P. berghei Infected Mice Treated with 4-Aminoquinolines 4, 21, and 34

control untreated

CQ, 10 mg/kg po, ×4

4,^a30 mg/kg ip, ×4

21,^a30 mg/kg ip, ×4

34, 21 mg/kg ip, ×4

mean % parasitemia

95% clearance

standard deviation

% inhibition

15.28

1.64

1.18

0

0.13

0.10

0.08

99.2

0.86

0.31

0.35

94.4

12.48

2.82

3.21

18.3

12.50

2.65

3.03

18.2

^aThe chlorohydrate salt was used.

Table 6. Drug Combination Assays Using Newly Synthesized

4-Aminoquinolines and Clinically Used Antimalarials^a

drug

4

14

20

34

36

40

CQ

1:3

1:1

A

nd

A

nd

A

1.1

1.2

A

1.1

1.2

1.1

A

1.1

A

3:1

methylene blue

A

1:3

1:1

3:1

meﬂoquine

1.3

A

1.3

1.7

2.2

A

1.3

1.4

1.6

A

1.0

1.3

nd

1.1

1.2

A

1.3

1.2

nd

1:3

1:1

3:1

1.2

1.1

D

1.2

1.0

nd

1.4

2.1

A

1.2

1.3

nd

1.4

1.8

1.7

A

1.1

1.2

1.3

A

1.2

1.4

A

1.4

1.3

1.5

A

1.3

1.5

A

1.1

1.5

1.7

1.1

1.0

nd

amodiaquine

1:3

1:1

3:1

piperaquine

1:3

1:1

3:1

artemisinin

1:3

1:1

3:1

artemether

1:3

1:1

3:1

artesunate

1:3

1:1

nd

Figure 2. Effect of preloaded 4-aminoquinolines 2 (A) and 34 (B) on

labeled CQ accumulation by erythrocytes infected with the CQ-resistant

P. falciparum parasite Dd2. The level of labeled CQ accumulation,

given as the ratio of the intracellular vs extracellular CQ concentration

(CQ_in/CQ_out), is analyzed as a function of the extracellular concentration

of 2 and 34 used for preloading. The data points were ﬁtted using

previously described equations,¹⁸which assume as carrier-model for 2

or a simple binding model for 34. The mean (SEM) of four or more

independent determinations is shown.

nd

A

S

A

1.3

1.2

A

1.3

1.2

A

nd

0.9

0.8

0.9

nd

0.8

0.7

0.8

nd

1.2

1.3

1.4

nd

antagonistically in the drug combinations tested. The excep-

tions were 34 and 36, which showed a slight synergy with

artemisinin. Interestingly, both compounds 34 and 36 are

tertiary amides with related structures. These structure–ac-

tivity relationships are promising, and the mechanisms of

action of these tertiary amides are being studied in further

detail and will be reported elsewhere. It should be mentioned,

however, that besides a promising synergistic action in cell

culture, the success of a drug combination therapy is largely

deﬁned by absorption, distribution, metabolization, and

excretion, deﬁning, for example, the half-life of a compound

in vivo.

A

1.5

1.6

2.0

A

1.1

1.5

2.2

nd

1.3

1.7

1.5

3:1

^aIn columns 2–7 FIC₅₀values are given that were obtained at ﬁxed ratio

drug combinations listed in column 1. FIC₅₀values were determined

according to the reported method.⁹⁰FIC₅₀< 1 indicates synergistic drug

action. FIC₅₀) 1 indicates additive action. FIC₅₀> 1 indicates antagonistic

action. FIC₅₀(A) ) [IC₅₀(A+B)]/IC₅₀(A). FIC₅₀(B) ) [IC₅₀(B+A)]/IC₅₀(B).

FIC₅₀) FIC₅₀(A) + FIC₅₀(B). IC₅₀values determined for the clinically

used antimalarials (monotherapy) in the experiments were as follows: CQ,

155 ( 11.4 nM; methylene blue, 5.4 ( 0.6 nM; meﬂoquine, 4.85 ( 1.0

nM; amodiaquine, 9.5 ( 1.2 nM; piperaquine, 14.6 ( 2.2 nM; artemisinin,

4.5 ( 0.7 nM; artemether, 2.8 ( 0.5 nM; artesunate, 2.3 ( 0.4 nM.

Competition Studies on Chloroquine Transport. Previous

studies have demonstrated trans-stimulated chloroquine move-

ment in CQ-resistant parasites;^18,19,22i.e., CQ present on the

trans face of the membrane was able to stimulate CQ movement

from the cis to the trans site. This phenomenon was linked to

the PfCRT polymorphisms associated with CQ resistance.^21,22,71

These data have led to the hypothesis that CQ resistance is based

on the acquisition of a PfCRT-mediated CQ efﬂux system.^21,22

In order to determine whether the short CQ analogue 2 and the

tertiary amide 34, which has no basic function at the side chain,

interfere with the putative CQ transport, the compounds were

investigated in a trans-stimulation assay¹⁹in which preloaded

2 and 34 were used to stimulate labeled CQ uptake into the

CQ-resistant P. falciparum parasite Dd2 (Figure 2). To this end,

erythrocytes infected with trophozoites of Dd2 were preloaded

with different concentrations of 2 or 34, ranging from 0 to 10

µM, for 15 min before the cells were washed and placed in

medium containing 43 nM [³H]CQ. The cellular CQ accumula-

tion ratio (intracellular versus extracellular CQ concentration)

was determined and analyzed as a function of the preloaded 2

or 34 concentration. In the case of 2, the CQ analogue with a

short aminoalkyl side chain, the curve ﬁrst rises and then falls

with increasing preloaded 2, consistent with trans stimulation

mg/kg (34) or 30 mg/kg (21). This might partially result from

the poor bioavailability of the compound and the low

solubility in organic and aqueous solvents.

Drug Combination Assays with Clinically Used Antima-

larials. Because of the increasing resistance of Plasmodium

to presently available drugs, the development of efﬁcient drug

combination therapies is currently favored. To test the

potential of the newly developed GR inhibitors as components

of combination therapies, we carried out drug combination

assays with chloroquine, methylene blue, meﬂoquine, amo-

diaquine, piperaquine, artemisinin, artemether, and artesunate.

For these assays we used at least one compound with good

antimalarial activity from each class, namely, 4, 14, 20, 34,

36, and 40 (Tables 1–4). The results are summarized in Table

6. The short CQ analogue 4 showed slight antagonistic action

in combination with basically all drugs tested. Only for

amodiaquine was a tendency toward additive action observed.

Most of the dual drugs tested were found to act mainly

1268 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Friebolin et al.

of labeled CQ uptake by 2. In the case of the tertiary amide 34

no such trans stimulation was observed. The curve falls with

increasing preloaded concentrations of 34. This behavior is

distinct from that seen with 2 and suggests a simple binding

process in which both 34 and CQ compete for binding to

common sites, possibly heme.

2 showed an activity similar to CQ to inhibit the formation of

ꢀ-hematin in vitro. Thus, the antimalarial activity of 2 (3D7,

∼10 nM; K1, ∼100 nM), in the same range as CQ, can correlate

with the ꢀ-hematin inhibitory activity. However, under the same

conditions, the tertiary amide 34 was found to inhibit the

ꢀ-hematin formation at very low equivalent number versus

hematin. In addition, the behavior of this compound in this test,

in accordance with the curve shapes, by comparison to CQ and

its analogues, strongly suggests a mode of action distinct from

the CQ mode of action. Noteworthy is the fact that the

antimalarial activity of 2 and 34 was in the nanomolar range

despite the loss of the basic character at the terminal side chain

of 34. These results proved that the terminal tertiary nitrogen

is not essential to kill resistant parasites and might be associated

with drug resistance.

Discussion

Our strategy is aimed at the design of antimalarial dual drugs

based on a short CQ analogue attached to a GR inhibitor or a

glutathione depletor. Our previous studies with GR enzymes

revealed two types of mechanism-based inhibitors: the reversible

uncompetitive inhibitors like the carboxylic acids M₅and 33;

the subversive substrates like menadione and the 1,4-naphtho-

quinone. A more recent work with a ﬂuoromethyl analogue of

M₅showed the inﬂuence of the ﬂuorine atom at the methyl

group of menadione on the redox potential value of the ﬁnal

molecule, resulting in an increased oxidant character. Thus, in

the present work, we produced dual drugs based on the two

types of inhibitors: (i) the carboxylic acids and (ii) the 1,4-

naphthoquinone, menadione, and the triﬂuoromenadione or their

reduced dimethoxynaphthalene forms. For glutathione depletors,

we used paracetamol and amodiaquine as starting phenols.

First, we focused on the preparation of short CQ analogues

(Scheme 1) that retain the activity of CQ and are not too bulky

to allow the chemical attachment of the redox active moiety.

Among the short CQ analogues, the N-neopentyl-substituted

4-aminoquinoline 4 was found to be the most active both in

vitro and in vivo. However, we selected the less bulky

N-isopropyl analogue 2 instead of the N-^tBu- or the N-CH₂-^tBu

because of (i) its effective preparation via reductive amination

of acetone, (ii) the lower hindrance observed in the reactions

involving the secondary amine function of the side chain, (iii)

its antimalarial activity similar to that of CQ, and (iv) the low

molecular weight (MW) allowing the preparation of dual drugs

with a ﬁnal MW approaching 500. From the selected short CQ

analogue 2, we synthesized two effective series of dual drugs,

the phenolic Mannich bases (Scheme 3) and the tertiary amides

(Scheme 4). All phenolic Mannich bases (Table 3) showed

signiﬁcant antimalarial activities in the nanomolar range. The

N-unsubstituted 4-aminoquinolines (R₂) H, 19–21) gave potent

dual drugs with the lowest IC₅₀values in the nanomolar range

but also the lowest TD₅₀values against human cells. In order

to limit the cytotoxicity, we synthesized the N-alkyl analogues

(R₂) ⁱPr, 22–24) resulting in lower cytotoxicity but also lower

antimalarial activities. When the branched alkyl chain was

extended with one methylene group at the N atom (R₂) CH₂-

ⁱPr, CH₂-^tBu), both the antimalarial and the cytotoxic effects

increased again. In this series of dual drugs we were unable to

dissociate these two effects suggesting a common mechanism

likely involving the formation of a quinone methide.

The different behavior of the short CQ analogue 2 and the

tertiary amide 34 in a CQ trans-stimulation assay is consistent

with this hypothesis. Since polymorphisms within PfCRT have

been linked with altered responses to a range of structurally

and functionally distinct antimalarial drugs,^9–11,74we wondered

whether PfCRT can act on 2 and 34. By use of the established

trans-stimulation protocol,¹⁸the short CQ analogue 2 revealed

rather complex kinetics. The stimulated CQ uptake at low

concentration of preloaded 2 to levels above the zero-trans

control (cells not preloaded with 2) suggests the presence of a

carrier-mediated transport process and can be interpreted as

follows: the preloaded 2 competes with incoming labeled CQ

for efﬂux by the same carrier, presumably PfCRT.¹⁸Only at

high concentrations of preloaded 2, when the carrier is fully

saturated, does the uptake curve fall, possibly because of both

2 and CQ competing for binding to heme. It is worth mentioning

that 2 is a derivative of CQ with a shortened aminoalkyl side

chain. Thus, a mode of action for short CQ analogues similar

to that of CQ leading to cross-resistance with CQ is possible.

Cross-resistance with CQ has been described for a number of

side chain variants of CQ.^23,24In the case of 34, no evidence

for stimulated CQ uptake was found. The decline of the uptake

curve with increasing amounts of intracellularly preloaded 34

is consistent with a model in which incoming labeled CQ

competes with preloaded 34 for common binding sites, possibly

heme. Alternatively, the tertiary amide 34 may affect the pH in

the parasite’s digestive vacuole, which in turn would reduce

acidotropic partitioning of the amphiphilic diprotic weak base

CQ in this organelle. Consequently no cross-resistance with CQ

is expected for the tertiary amide 34 in accordance with high

antimalarial effects against the CQ-resistant K1 strain.

In the present study, we designed various series of dual drugs

built from a short CQ analogue and a GR inhibitor (or a

precursor) linked together via different linkages. In the absence

of evidence for amide/amine cleavage in vivo, mechanisms by

which tertiary amines or tertiary amides exert their antimalarial

action remain elusive. Possibly, after cleavage, the simultaneous

inhibition of GR and one (several) target(s) may result in

potentiation of the antimalarial activity by interfering with the

redox equilibrium and parasite development. In this context,

cleavage of a tertiary amide bond under speciﬁc biomimetic

conditions found in the food vacuole of the parasites mimicking

the cytP₄₅₀-catalyzed oxidation reactions might be possible.⁷⁵

Hydrolysis of tertiary amides is also known upon complexation

with copper(II) ions through a Lewis acid effect.^76,77Different

members of the protease families involved in hemoglobin

degradation could be candidates for the proteolysis of the tertiary

amide bond of 34 in P. falciparum. Thus, the antimalarial action

of the newly synthesized tertiary amides deserves to be discussed

The second series of dual drugs is illustrated with the amides

(Table 4) built from the short CQ analogues 1 and 2 and the

carboxylic acids or their reduced and deprotected forms. The

most effective were the tertiary amides 34 and 36 with excellent

antimalarial activities against CQ-sensitive and -resistant para-

sites in the very low nanomolar range (3D7, 7-12 nM; K1,

5-10 nM). Some tertiary amides in the side chain of 4-ami-

noquinolines were also reported to display signiﬁcant antima-

larial activities.^72,73In the case of the tertiary amide 34, in

comparison to the parent short CQ analogue 2, we examined

its ability to compete for the same intraparasitic receptor of CQ,

the (Fe^III)protoporphyrin, and for the CQ transporter. In the

hematin polymerization test, as expected, the short CQ analogue

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1269

1 equiv) in 15 mL of anhydrous THF was stirred for 8 h at room

temperature under N₂. NaBH₄(15 mmol, 3 equiv) and absolute

ethanol were added, and the resulting mixture was stirred for 15 h.

The mixture was then poured into 20 mL of 2 M NH₄OH, and the

resulting inorganic precipitate was ﬁltered and washed with Et₂O

(50 mL).

with cautious considerations until a complete study is done in

the future. Further work is aimed at the evaluation of the

reactivity of the tertiary amide 34, its rate of hydrolysis under

both chemical and biological conditions, and/or its complexation

properties with the biologically relevant iron(II) or iron(III)

species.

When tested for in vivo efﬁcacy in a P. berghei infected

mouse model, the 4-aminoquinolines failed to cure the infection

at 30 mg/kg given intraperitoneally. No toxicity in infected mice

was observed. While compound 4 is similar to other short CQ

analogues, it might be considered as a dual drug because after

oxidative N-dealkylation it is expected to release trimethylac-

etaldehyde. Trimethylacetaldehyde was reported to be deformy-

lated at high rate by cytP₄₅₀isoforms and to release isobuty-

lene,⁷⁸which could elicit metabolic activation to become

potentially harmful⁷⁹for the parasite.

Taken together, our strategy can serve for the rational design

of more potent and less toxic antimalarial dual agents after the

link between both entities (a 4-aminoquinoline and a GR

inhibitor) is optimized. Furthermore, the drug combination

studies showed an interesting trend with the synergistic action

of the tertiary amides with artemisinin that deserves to be

exploited in the future drug development against malaria.

N′-(7-Chloroquinolin-4-yl)-N-isopropylethane-1,2-diamine 2.

GP1 from N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine 1 (1.3 g, 5.8

mmol), acetone (0.51 mL, 7.0 mmol), Ti(OⁱPr)₄(3.50 mL, 11.6

mmol), and NaBH₄(0.66 g, 17.4 mmol) yielded N′-(7-chloroquino-

lin-4-yl)-N-isopropylethane-1,2-diamine 2 (0.67 g, 44%) as a

colorless solid following puriﬁcation by ﬂash chromatography

(SiO₂, CH₂Cl₂/methanol 4:1). ¹H NMR (300 MHz, CD₃OD): δ 8.33

(d, 1H, H-2, ³J ) 5 Hz), 8.05 (d, 1H, H-5, J ) 9 Hz), 7.75 (d, 1H,

H-8, J ) 2 Hz), 7.37 (dd, 1H, H-6, J ) 2 Hz, J ) 9 Hz), 6.52 (d,

3

1H, H-3, J ) 5 Hz), 3.46 (t, 2H, NCH₂, J ) 6 Hz) 2.91 (t, 2H,

CH₂N^tBu, J ) 6 Hz), 2.89 (sept, 1H, CH, J ) 6 Hz), 1.09 (d,

6H, CH₃, ³J ) 6 Hz). ¹³C NMR (75 MHz, CD₃OD): δ 152.7, 152.5,

149.7, 136.4, 127.6, 126.1, 124.3, 118.8, 99.7, 49.8, 46.0, 43.6,

22.5. MS (ES+): 263 (M⁺). Anal. (C₁₄H₁₈N₃Cl) C, H, N.

3

N′-(7-Chloroquinolin-4-yl)-N-isobutylethane-1,2-diamine 3.

GP1 from N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine 1 (1.0 g, 4.5

mmol), isobutyraldehyde (0.39 g, 0.49 mL, 5.4 mmol), Ti(OⁱPr)₄

(2.68 mL, 9.0 mmol), and NaBH₄(0.51 g, 13.5 mmol) yielded N′-

(7-chloroquinolin-4-yl)-N-isobutylethane-1,2-diamine 3 (0.585 g,

46.5%) as a pale-yellow solid after ﬂash chromatography (SiO₂,

1

CH₂Cl₂/methanol, 4:1), mp 116 – 118 °C. H NMR (250 MHz,

Experimental Section

3

CDCl₃): δ 8.43 (d, 1H, H-2, J ) 5 Hz), 7.86 (d, 1H, H-8, J ) 2

Hz), 7.64 (d, 1H, H-5, J ) 9 Hz), 7.27 (dd, 1H, H-6, J ) 2 Hz, J

Chemistry. Chemicals. The starting compounds N′-[7-chloro-

quinolin-4-yl)ethane-1,2-diamine 1,⁸⁰4-(2-bromoethylamino)-7-

chloroquinoline 6,⁸¹N-tert-butyl-2-(quinolin-4-ylamino)acetamide

8,⁸²1,4-dimethoxy-2-methylnaphthalene 10,⁶³2-bromo-3-methyl-

1,4-dimethoxynaphthalene 11,⁸³1,4-dimethoxynaphthalene 16,⁸⁴

1,4-dimethoxynaphthalene-2-carboxaldehyde 17,⁶⁶1,4-dimethoxy-

naphthalene-3-methyl-2-naphthalenecarboxaldehyde 18,⁸⁵N-(7-

chloroquinolin-4-yl)propane-3-isopropyl-1,3-diamine 31,¹and 4-(3-

methyl-1,4-dioxo-1,4-dihydronaphthalen-2-ylmethyl)phenyl]acetic acid

33⁵⁷were synthesized according to reported procedures. N′-(7-

Chloroquinolin-4-yl)-N-isopropylethane-1,2-diamine 2,¹4-(1-aziri-

dinyl)-7-chloroquinoline 7,⁶⁰and N′-(7-chloroquinolin-4-yl)-N-tert-

butylethane-1,2-diamine 9²⁵are known compounds but were

produced according to new procedures. All commercially available

chemicals were purchased from Acros and Aldrich and used without

further puriﬁcation. All solvents for the reaction were dried before

use (solvents in p.a. quality). CH₂Cl₂was distilled under nitrogen

atmosphere from CaCl₂. The used petroleum ether had a bp of

40–60 °C.

3

) 9 Hz), 6.29 (d, 1H, H-3, J ) 5 Hz), 6.01 (s, 1H, NH), 3.25 (t,

2H, N_QnCH₂, ³J ) 6 Hz), 2.96 (t, 2H, CH₂N, ³J ) 6 Hz), 2.41 (d,

3

2H, CH₂, J ) 7 Hz), 2.10 (s, 1H, NH), 1.70 (m, 1H, CH), 0.50

i

(m, 2H, CH₂), 0.87 (d, 6H, Pr). MS (FAB+): 278.1 (M⁺+ 1).

Anal. (C₁₅H₂₀ClN₃) C, H, N.

N′-(7-Chloroquinolin-4-yl)-N-(2,2-dimethylpropyl)ethane-1,2-

diamine 4. GP1 from N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine

1 (0.20 g, 0.9 mmol), trimethylacetaldehyde (0.12 mL, 1.1 mmol),

Ti(OⁱPr)₄(0.54 mL, 1.8 mmol), and NaBH₄(0.10 g, 2.7 mmol)

yielded N′-(7-chloroquinolin-4-yl)-N-(2,2-dimethylpropyl)ethane-

1,2-diamine 4 (130 mg, 0.44 mmol, 40%) as a pale-yellow solid

after ﬂash chromatography (SiO₂, CH₂Cl₂/methanol, 4:1), mp

139–141 °C. ¹H NMR (250 MHz, CD₃OD): δ 8.38 (d, 1H, H-2, ³J

) 5 Hz), 8.08 (d, 1H, H-5, J ) 9 Hz), 7.80 (d, 1H, H-8, J ) 2

Hz), 7.39 (dd, 1H, H-6, J ) 2 Hz, J ) 9 Hz), 6.60 (d, 1H, H-3, ³J

3

) 5 Hz), 3.51 (t, 2H, N_QnCH₂, J ) 6 Hz), 2.97 (t, 2H, CH₂N, J

) 6 Hz), 2.45 (s, 2H, CH₂), 0.97 (s, 9H, ^tBu). MS (FAB+): 292.1

(M⁺+ 1). Anal. (C₁₆H₂₂ClN₃·0.5H₂O) C, H, N.

General Methods. New derivatives were isolated by silica gel

chromatography (silica gel 60, Merck 230–400 mesh) of the crude

reaction mixture. Thin-layer chromatography (TLC) was carried

out on ﬂexible Machery & Nagel Polygram SIL G/UV-254 and

Alugram SIL G/UV-254 silica gel sheets (0.2 mm). The components

were detected by their absorption at 254 nm, followed by staining

with 10% ninhydrine in acetone and heating. The purity of the

isolated compounds was checked by elemental analysis. Elemental

analyses were performed on an Elementar vario EL at Organisch-

Chemisches Institut, University of Heidelberg. Electrospray ioniza-

tion mass spectra (ESI-MS) of the compounds were recorded by

using a Finnigan-MAT (San Jose, CA) model TSQ 7000 at the

Biochemie-Zentrum of Heidelberg University. Melting points were

determined on a Büchi melting point apparatus and were not

N′-(7-Chloroquinolin-4-yl)-N-cyclopropylmethylethane-1,2-di-

amine 5. GP1 from N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine 1

(0.20 g, 0.9 mmol), cyclopropanecarboxaldehyde (0.10 mL, 1.1

mmol), Ti(OⁱPr)₄(0.54 mL, 1.8 mmol), and NaBH₄(0.10 g, 2.7

mmol) yielded N′-(7-chloroquinolin-4-yl)-N-cyclopropylmethyl-

ethane-1,2-diamine 5 (60 mg, 24%) as a pale-yellow solid after

ﬂash chromatography (SiO₂, CH₂Cl₂/methanol, 4:1), mp 117–118

°C. ¹H NMR (300 MHz, CD₃OD): δ 8.35 (d, 1H, H-2, ³J ) 5 Hz),

8.09 (d, 1H, H-5, J ) 9 Hz), 7.76 (d, 1H, H-8, J ) 2 Hz), 7.38

(dd, 1H, H-6, J ) 2 Hz, J ) 9 Hz), 6.55 (d, 1H, H-3, ³J ) 5 Hz),

3

3.48 (t, 2H, N_QnCH₂, J ) 6 Hz), 2.95 (t, 2H, CH₂N, J ) 6 Hz),

3

2.50 (d, 2H, CH₂, J ) 7 Hz), 0.96 (m, 1H, CH), 0.50 (m, 2H,

CH₂), 0.16 (m, 2H, CH₂). MS (FAB+): 276.1 (M⁺+ 1). Anal.

(C₁₅H₁₈ClN₃) C, H, N.

1

corrected. H (300 MHz) and ¹³C NMR (75 MHz) spectra were

4-(1-Aziridinyl)-7-chloroquinoline 7. CsOH·H₂O (1.61 g, 9.6

mmol) in 25 mL of anhydrous DMF was stirred over molecular

sieves (4Å) for 10 min. tert-Butylamine (0.35 g, 4.8 mmol) was

added and the reaction mixture stirred for 30 min. Then 4-(2-

bromoethylamino)-7-chloroquinoline 6 (1.5 g, 5.8 mmol) was

added, and after 20 h the solvent was concentrated in vacuo. The

insoluble salt was removed by ﬁltration and washed with ethyl

acetate. The ﬁltrate was concentrated in vacuo, treated with 50 mL

of 1 N NaOH, and extracted with ethyl acetate (3×). The combined

organic layers were washed with brine and dried over Na₂SO₄. Then

recorded on Bruker AC 300 and DRX-300 MHz spectrometers,

and the chemical shifts were expressed in ppm relative to TMS.

Multiplicity was indicated as s (singlet), d (doublet), t (triplet), q

(quartet), sept (septet), and m (multiplet). The following abbrevia-

tions were used to assign the proton in NMR spectra: Qn, quinine;

Np, naphthalene; Nq, naphthoquinone; Ph, phenyl.

General Procedure 1 (GP1) for the Synthesis of 2-5. A

mixture of the ketone (6 mmol, 1.2 equiv), Ti(OⁱPr)₄(10 mmol, 2

equiv), and N-(7-chloroquinolin-4-yl)ethyl-1,2-diamine 1 (5 mmol,

1270 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Friebolin et al.

the solvent was evaporated. Flash chromatography (SiO₂, ethyl

water, mp 46–47 °C. ¹H NMR (250 MHz, CDCl₃): δ 8.15 (d, 1H,

Ar-H, J ) 8.0 Hz), 8.08 (d, 1H, Ar-H, J ) 8.0 Hz), 7.57 (m,

2H, Ar-H), 3.96 (s, 3H, -OCH₃), 3.86 (s, 3H, -OCH₃), 2.52 (q,

acetate) afforded 4-(1-aziridinyl)-7-chloroquinoline 7 (1.11 g, 5.4

1

mmol, 93%) as a colorless solid, mp 99–100 °C. H NMR (300

3

5

MHz, CDCl₃): δ 8.73 (d, 1H, H-2, J ) 5 Hz), 8.23 (d, 1H, H-5,

3H, J ) 2.7 Hz, -CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 152.7,

1

J ) 9 Hz), 8.08 (d, 1H, H-8, J ) 2 Hz), 7.52 (dd, 1H, H-6, J ) 2

150.9, 130.4, 128.4, 127.7, 126.4, 124.7 (q, J ) 275.8 Hz, CF₃),

3

Hz, J ) 9 Hz), 6.85 (d, 1H, H-3, J ) 5 Hz), 2.40 (s, 4H, 2 ×

124.5, 123.7, 123.6, 122.9, 122.6, 122.3, 119.0, 79.0, 63.9, 61.4,

CH₂). ¹³C NMR (75 MHz, CDCl₃): δ 158.1, 151.6, 149.1, 135.1,

128.4, 126.4, 123.8, 122.1, 109.9, 27.9. MS (FAB+): 205.0 (M⁺

+ 1). Anal. (C₁₁H₉ClN₂) C, H, N.

12.8. MS (FAB+): 270.1 (M⁺). Anal. (C₁₄H₁₃F₃O₂) C, H.

2-Bromomethyl-1,4-dimethoxy-3-triﬂuoromethyl-naphtha-

lene 13. To a solution of 1,4-dimethoxy-2-methyl-3-triﬂuorometh-

ylnaphthalene (1.34 g, 5.0 mmol) in 18 mL of CCl₄was added

N-bromosuccinimide (0.89 g, 5.0 mmol) and then benzoyl peroxide

(0.12 g, 0.5 mmol). After the mixture was heated to reﬂux for 5 h

and cooled to room temperature, the succinimide was ﬁltered

off and the ﬁltrate was washed with sodium dithionite and 2 N

NaOH and then dried over MgSO₄. After evaporation of the solvent

the residue was puriﬁed by ﬂash chromatography (SiO₂, CH₂Cl₂/

petroleum ether, 1:3) to give 2-bromomethyl-1,4-dimethoxy-3-

triﬂuoromethylnaphthalene 13 (0.97 g, 2.78 mmol, 55%) as colorless

crystals after recrystallization from methanol/water, mp 72–73 °C.

¹H NMR (250 MHz, CDCl₃): δ 8.22 (dd, 1H, Ar-H, J ) 7.3 and

1.8 Hz), 8.15 (dd, 1H, Ar-H, J ) 7.3 and 1.8 Hz), 7.68 (m, 2H,

Ar-H), 4.94 (s, 2H, CH₂Br), 4.14 (s, 3H, -OCH₃), 4.04 (s, 3H,

-OCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 153.5, 152.7, 130.2, 129.4,

N-tert-Butyl-2-(quinolin-4-ylamino)acetamide 8. N-R-(tert-

Butoxycarbonyl)glycin-tert-butylamide (1.20 g, 5.2 mmol) and

triﬂuoracetic acid (4.7 mL, 35 mmol) in 4.7 mL of CH₂Cl₂were

stirred at room temperature for 3 h. The reaction mixture was

concentrated in vacuo, 10 N NaOH (1 mL) was added to the residual

oil, and again the mixture was evaporated. The resulting crude

2-amino-N-tert-butylacetamide was used without further puriﬁcation

for the following reaction. ¹H NMR (250 MHz, CDCl₃): δ 2.83 (t,

2H, CH₂), 2.69 (t, 2H, CH₂), 1.95 (s_br, 3H, NH₂, NH), 1.16 (s, 9H,

^tBu). 4,7-Dichloroquinoline (1.02 g, 5.2 mmol) and the crude

2-amino-N-tert-butylacetamide were heated to reﬂux overnight.

After the mixture was cooled to room temperature, the crude product

was ﬁltered and washed with warm water. After ﬂash chro-

matograpy (SiO₂, CH₂Cl₂/methanol, 12:1) N-tert-butyl-2-(quinolin-

4-ylamino)acetamide 8 (950 mg, 3.3 mmol, 63%) was obtained as

a colorless solid, mp 185 °C. ¹H NMR (250 MHz, CD₃OD): δ

8.39 (d, 1H, H-2, ³J ) 5 Hz), 8.20 (d, 1H, H-5, J ) 9 Hz), 7.80 (d,

1H, H-8, J ) 2 Hz), 7.50 (dd, 1H, H-6, J ) 2 Hz, J ) 9 Hz), 6.52

(d, 1H, H-3, ³J ) 5 Hz), 4.09 (s, 2H, CH₂), 1.37 (s, 9H, tBu). MS

(ES+): 292 (M⁺+ 1), 157.

1

128.9, 127.9, 124.5, 124.4 (q, J ) 275.9 Hz, CF₃), 123.9, 123.2,

117.9 (q, ²J ) 29.09 Hz, C-CF₃), 79.0, 64.2, 62.9, 24.5. MS

(FAB+): calcd 349.0 (M⁺); found 348.0, 350.0 (M⁺). Anal.

(C₁₄H₁₂BrF₃O₂) C, H, Br.

7-Chloro-N-(3-((diethylamino)methyl)-4-((1,4-dimethoxy-3-

(triﬂuoromethyl)naphthalen-2-yl)methoxy)phenyl)quinolin-4-

amine 14. To a solution of amodiaquine dihydrochloride (256 mg,

0.55 mmol) in dry DMF (17 mL) was added, under argon, cesium

hydroxide hydrate (360 mg, 2.14 mmol) and then powdered

molecular sieves (4 Å). After the mixture was stirred at room

temperature for 40 min, the bromide 13 (160 mg, 0.46 mmol) in 5

mL of DMF was added dropwise. After being further stirred for

6 h, the brown reaction mixture was poured into 2 N NaOH solution

(100 mL). A yellow solid precipitated. Then the mixture was

extracted with CH₂Cl₂(2×) and the combined organic layers were

washed with brine and dried over MgSO₄. The solvent and DMF

were removed in vacuo and the crude product was puriﬁed by ﬂash

chromatography on SiO₂(CH₂Cl₂/methanol, 10:1) and then recrys-

tallized from methanol/water to give 7-chloro-N-(3-((diethyl-

amino)methyl)-4-((1,4-dimethoxy-3-(tri-ﬂuoromethyl)naphthalen-

2-yl)methoxy)phenyl)quinolin-4-amine 14 (234 mg, 0.37 mmol,

82%) as pale-yellow crystals, mp 161–162 °C. ¹H NMR (300 MHz,

N′-(7-Chloroquinolin-4-yl)-N-tert-butylethane-1,2-diamine 9.

To N-tert-butyl-2-(quinolin-4-ylamino)acetamide 8 (0.15 g, 0.51

mmol) in 1 mL of anhydrous THF was added dropwise a 1 M BH₃/

THF solution (5.1 mL) under N₂at 0 °C. After the mixture was

stirred at 0 °C for 30 min and then at room temperature for 2 h,

the excess of BH₃was hydrolyzed by 1 mL of 1 M NaHCO₃and

the solvent was evaporated. The residue was treated with brine and

extracted with CH₂Cl₂. The combined organic layers were evapo-

rated. The residue was treated with 20 mL of THF, and the insoluble

salt was removed by ﬁltration and washed with THF. The solution

was concentrated to about 20 mL and then 4 N HCl (0.5 mL) was

added. After the mixture was stirred for 2 h, the solvent was

evaporated and the residue was treated with 1 N NaOH and

extracted with CH₂Cl₂. After evaporation of the solvent the N′-(7-

chloroquinolin-4-yl)-N-tert-butylethane-1,2-diamine 9 was puriﬁed

by preparative TLC (SiO₂, CH₂Cl₂/methanol, 6:1) and obtained as

3

1

CDCl₃): δ 8.49 (d, 1H, QnH-2, J ) 5 Hz), 8.21 (m, 1H, NpH-5/

a colorless solid (32 mg, 0.11 mmol, 22%). H NMR (300 MHz,

CD₃OD): δ 8.37 (d, 1H, H-2, ³J ) 5 Hz), 8.10 (d, 1H, H-5, J ) 9

Hz), 7.78 (d, 1H, H-8, J ) 2 Hz), 7.41 (dd, 1H, H-6, J ) 2 Hz, J

8), 8.20 (m, 1H, NpH-5/8), 8.00 (d, 1H, QnH-8, J ) 2 Hz), 7.85

3

(d, 1H, QnH-5, J ) 9 Hz), 7.69 (m, 2H, NpH-6/7), 7.46 (d, 1H,

2

3

J ) 3 Hz, PhH-2′), 7.43 (dd, 1H, J ) 9 Hz, J ) 2 Hz, QnH-6),

7.19 (dd, 1H, J ) 3 Hz, J ) 9 Hz, PhH-6′), 7.15 (d, 1H, J ) 9 Hz,

) 9 Hz), 6.58 (d, 1H, H-3, J ) 5 Hz), 3.50 (t, 2H, NCH₂, J )

6 Hz), 2.94 (t, 2H, CH₂N^tBu, J ) 6 Hz), 1.17 (s, 9H, Bu). ¹³C

NMR (75 MHz, CD₃OD): δ 152.7, 152.5, 149.7, 136.5, 127.7,

126.2, 124.3, 118.8, 99.8, 52.4, 43.8, 41.6, 28.4. MS (ES+): 278

(M⁺).

3

t

3

PhH-5′), 6.82 (d, 1H, QnH-3, J ) 5 Hz), 6.62 (s, 1H, NH), 5.38

(d, 1H, OCH₂, J ) 0.9 Hz), 4.05 (s, 3H, OCH₃), 4.00 (s, 3H, OCH₃),

3

3.53 (s, 2H, NCH₂), 2,46 (q, 4H, 2 × CH₂, J ) 7 Hz), 0.93 (t,

3

6H, 2 × CH₃, J ) 7 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 154.6,

1,4-Dimethoxy-2-methyl-3-triﬂuoromethylnaphthalene 12. 2-

Bromo-3-methyl-1,4-dimethoxynaphthalene 11 (9.13 g, 32.54 mmol),

sodium triﬂuoracetate (13.95 g, 102.56 mmol), and copper iodide

(13.02 g, 68.37 mmol) were suspended under argon in 40 mL of

dry toluene in a three-necked ﬂask equipped with a Dean–Stark

apparatus and a thermometer. About one-third of toluene was

distilled off to remove traces of water. After addition of 50 mL of

dry dimethylacetamide, the solvent was distilled until the temper-

ature of the mixture increased to 170 °C. After the mixture was

stirred for 8 h at this temperature, the solvent was evaporated at

75 °C. CH₂Cl₂and then petroleum ether were added, and the

subsequent precipitated copper iodide was ﬁltered out through silica

gel. The precipitate was washed with CH₂Cl₂/petroleum ether

(1:2), and then the solvent was removed in vacuo. Flash chroma-

tography (SiO₂, CH₂Cl₂/petroleum ether, 1:5) yielded pure 1,4-

dimethoxy-2-methyl-3-triﬂuoromethylnaphthalene 12 (4.10 g, 15.17

mmol, 46%). A fraction of the starting bromide 11 (3.02 g, 33%)

was also isolated. The product 12 was recrystallized from methanol/

153.5, 153.2, 152.0, 149.7, 148.8, 135.1, 131.9, 130.8, 130.4, 129.5,

1

129.0, 128.8, 127.9, 126.0, 124.4 (q, J ) 275.3 Hz, CF₃), 125.7,

2

123.8, 123.2, 123.1, 122.5, 121.0, 119.2 (q, J ) 30 Hz, C-CF₃),

111.8, 101.6, 64.1, 62.1, 62.0, 50.6, 47.4, 12.0. MS (FAB+): 623

(M⁺), 552, 354, 269, 200. Anal. (C₃₄H₃₃ClF₃N₃O₃· 1H₂O) C,

H, N.

N-[4-(1,4-Dimethoxy-3-triﬂuoromethylnaphthalen-2-yl-

methoxy) phenyl]acetamide 15. To 4-acetamidophenol (178 mg,

1.14 mmol) in dry DMF (7.5 mL) was added, under argon, cesium

hydroxide hydrate (400 mg, 2.38 mmol) and then powdered

molecular sieves (4 Å). After the mixture was stirred at 40 °C for

30 min and cooled to room temperature, the bromide 13 (200 mg,

0.57 mmol) in 2.5 mL of DMF was added dropwise. The reaction

mixture was stirred at 40 °C for a further 6 h and 30 min. Then

CH₂Cl₂was added, and the mixture was poured into brine. Phase

separation, extraction of the aqueous layer with CH₂Cl₂(3×), drying

of the combined organic layers over MgSO₄, and evaporation of

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1271

the solvent and DMF in vacuo led to the product, which was puriﬁed

by ﬂash chromatography (SiO₂, CH₂Cl₂/methanol, 100:1) and then

recrystallized from methanol/water to afford N-[4-(1,4-dimethoxy-

3-triﬂuoromethylnaphthalen-2-ylmethoxy)phenyl]acetamide 15 (223

chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuoromethylnaphtha-

len-2-ylmethyl)ethane-1,2-diamine 21 (1.81 g, 3.70 mmol, 85%)

as a pale-yellow oil/foam. ¹H NMR (250 MHz, CDCl₃): δ 8.51 (d,

1H, QnH-2, ³J ) 5 Hz), 8.21 (m, 1H, NpH-5/8), 8.08 (m, 1H, NpH-

5/8), 7.95 (d, 1H, QnH-8, J ) 2 Hz), 7.73 (d, 1H, QnH-5, J ) 9

Hz), 7.68 (m, 2H, NpH-6/7), 7.34 (dd, 1H, QnH-6, J ) 2 Hz, J )

1

mg, 0.53 mmol, 93%) as colorless crystals, mp 156–158 °C. H

NMR (300 MHz, CDCl₃): δ 8.15 (m, 2H, NpH-5/8), 7.63 (m, 2H,

NpH-6/7), 7.40 (m, 2H, PhH-2/6), 6.97 (m, 2H, PhH-3/5), 5.29 (s,

2H, OCH₂), 4.00 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 2.13 (s, 3H,

CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 156.1, 153.7, 131.8,

130.8, 129.9, 129.1, 128.2, 126.6, 124.2, 123.5, 123.4, 123.0, 122.4,

119.6, 115.5, 105.7, 64.4, 62.63, 62.6, 24.7. MS (EI): 419 (M⁺),

269, 200, 185, 151. Anal. (C₂₂H₂₀F₃NO₄) C, H, N.

General Procedure 2 (GP2) for the Reductive Amination of

1,4-Dimethoxynaphthalene-2-carbaldehyde and 3-Methyl-1,4-

dimethoxynaphthalene-2-carbaldehyde with (7-Chloroquinolin-

4-yl)ethane-1,2-diamine. (7-Chloroquinolin-4-yl)ethane-1,2-di-

amine 1 (1 equiv) and aldehyde (1.00 g, 1 equiv) were dissolved

in 50 mL of dry CH₂Cl₂and 5 mL of dry methanol and stirred

under argon atmosphere at room temperature. After 2 h, sodium

triacetoxyborohydride (3 equiv) was added, and the reaction mixture

was stirred for another 24 h. Then the mixture was poured into

100 mL of 2 N NaOH solution, the organic layer was separated,

and the aqueous layer was extracted with CH₂Cl₂(3×). The

combined organic layers were dried over MgSO₄, and the solvent

was evaporated in vacuo. Flash chromatography (SiO₂, CH₂Cl₂/

methanol, 5:1) afforded the products as pale-yellow oils.

N-(7-Chloroquinolin-4-yl)-N′-(1,4-dimethoxynaphthalen-2-

ylmethyl)ethane-1,2-diamine 19. GP2 from 1,4-dimethoxynaph-

thalene-2-carboxaldehyde 17 and N-(7-chloro-4-quinolinyl)-1,2-

ethanediamine 1 afforded N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-

naphthalen-2-ylmethyl)ethane-1,2-diamine 19 (0.86 g, 2.04 mmol,

44%) as a pale-yellow foam, mp 48–50 °C. ¹H NMR (300 MHz, CDCl₃):

δ 8.52 (d, 1H, QnH-2, ³J ) 5 Hz), 8.27 (m, 1H, NpH-5/8, J ) 8 Hz),

8.06 (m, 1H, NpH-5/8, J ) 8 Hz), 7.96 (d, 1H, QnH-8, J ) 2 Hz), 7.69

(d, 1H, QnH-5, J ) 9 Hz), 7.55 (m, 2H, NpH-6/7), 7.34 (dd, 1H, QnH-6,

J ) 2 Hz, J ) 9 Hz), 6.78 (s, 1H, NpH-3), 6.40 (d, 1H, QnH-3, ³J ) 5

Hz), 5.91 (s_br, 1H, NH), 4.06 (s, 2H, NCH₂Np), 3.98 (s, 3H, OCH₃), 3.92

(s, 3H, OCH₃), 3.38 (m, 2H, N_QnCH₂), 3.10 (m, 2H, CH₂N), 2.00 (s_br,

1H, NH). ¹³C NMR (75 MHz, CDCl₃): δ 152.0, 151.6, 149.7, 148.6,

147.4, 134.7, 128.3, 127.0, 126.6, 126.0, 125.3, 125.0, 122.2, 121.5, 121.1,

117.1, 104.5, 99.9, 62.2, 55.5, 47.7, 46.4, 41.7. MS (FAB+): 422 (M⁺).

Anal. (C₂₄H₂₄ClN₃O₂·0.5H₂O) C, H, N.

N-(7-Chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-methylnaph-

thalen-2-ylmethyl)ethane-1,2-diamine 20. GP2 from 1,4-dimethoxy

naphthalene-3-methyl-2-naphthalenecarboxaldehyde 18 and N-(7-

chloro-4-quinolinyl)-1,2-ethanediamine1affordedN-(7-chloroquinolin-

4-yl)-N′-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)ethane-1,2-

diamine 20 (1.71 g, 3.92 mmol, 90%) as a pale-yellow foam, mp

51–53 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.52 (d, 1H, QnH-2, ³J

) 5 Hz), 8.08 (m, 2H, NpH-5/8), 7.96 (d, 1H, QnH-8, J ) 2 Hz),

7.67 d, 1H, QnH-5, J ) 9 Hz), 7.53 (m, 2H, NpH-6/7), 7.34 (dd,

1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.39 (d, 1H, QnH-3, ³J ) 5 Hz),

5.92 (s_br, 1H, NH), 4.06 (s, 2H, NCH₂Np), 3.94 (s, 3H, OCH₃),

3.89 (s, 3H, OCH₃), 3.38 (m, 2H, N_QnCH₂), 3.13 (m, 2H, CH₂N),

2.53 (CH₃), 1.77 (s_br, 1H, NH). ¹³C NMR (75 MHz, CDCl₃): δ

152.0, 150.9, 150.5, 149.9, 149.1, 134.8, 128.6, 128.4, 128.3, 127.1,

126.3, 126.2, 125.6, 125.2, 122.4, 122.3, 121.2, 117.3, 99.2, 62.8,

61.3, 47.1, 44.7, 41.8), 12.3 (CH₃). MS (FAB+): 436 (M⁺), 215.

Anal. (C₂₅H₂₆ClN₃O₂·0.5H₂O) C, H, N.

3

9 Hz), 6.38 (d, 1H, QnH-3, J ) 5 Hz), 6.05 (s_br, 1H, NH), 4.15

(s, 2H, NCH₂Np), 4.01 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.35

(m, 2H, HNCH₂), 3.11 (m, 2H, CH₂N), 2.10 (s_br, 1H, NH). ¹³C

NMR (75 MHz, CDCl₃): δ 153.2, 152.0, 151.7, 150.0, 148.7, 134.7,

130.1, 128.7, 128.6, 128.2, 127.2, 126.5, 126.1, 125.0, 124.7 (q,

-CF₃), 123.7, 122.6, 121.5, 118.2 (q, C-CF₃), 117.3, 99.0, 64.0,

62.8, 46.9, 44.2, 41.7. MS (EI): 488.9 (M⁺), 269, 192, 156.

Preparation of the Dihydrochloride Salt 21. To a solution of

N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuoromethylnaph-

thalen-2-ylmethyl)ethane-1,2-diamine 21 (1.40 g, 2.86 mmol) in

methanol (10 mL) was added trimethylchlorosilane (5.72 mmol,

0.73 mL, 2 equiv). The reaction mixture was stirred for 15 min at

room temperature. Then the solid was ﬁltered and washed with

methanol and diethyl ether to give the dihydrochloride salt of N-(7-

chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuoromethylnaphtha-

len-2-ylmethyl)ethane-1,2-diamine 21 (1.05 g, 1.86 mmol, 65%),

mp 213–215 °C. A second batch was obtained by further addition

of diethyl ether to the ﬁltrate. Anal. (C₂₅H₂₃ClF₃NO₂·2HCl ·1H₂O)

C, H, N.

Geneal Procedure 3 (GP3) for the Reductive Amination of

the Chloroquinoyl-1,4-dimethoxynaphthalene Derivatives 22–

30 with Acetone, Isobutyraldehyde, or Pivalaldehyde. The amine

derivative (1 equiv) and the ketone or aldehyde (14–30 equiv of

acetone, 3–6 equiv of isobutyraldehyde, or 6 equiv of pivalaldehyde)

in dry CH₂Cl₂(10–30 mL) were stirred under argon atmosphere at

room temperature for 2 h and 30 min. Then sodium triacetoxy-

borohydride (3 equiv) was added and the reaction mixture was

stirred for further 2–3 days. If isobutyraldehyde is used, the reaction

time is 2–5 h. The reaction mixture was poured into 2 N NaOH

solution, the organic layer separated, and the aqueous layer extracted

with CH₂Cl₂(3×). After drying of the combined organic layers

over MgSO₄, the solvent was removed in vacuo and the crude

product puriﬁed by ﬂash chromatography (SiO₂, CH₂Cl₂/methanol,

1:1).

N-(7-Chloroquinolin-4-yl)-N′-(1,4-dimethoxynaphthalen-2-yl-

methyl)-N-isopropylethane-1,2-diamine 22. GP3 from N-(7-

chloroquinolin-4-yl)-N′-(1,4-dimethoxynaphthalen-2-ylmethyl)ethane-

1,2-diamine 19 (0.25 g, 0.59 mmol), acetone (0.48 g, 0.61 mL, 8.3

mmol), and NaBH(OAc)₃(0.38 g, 1.78 mmol) after 48 h yielded

N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxynaphthalen-2-ylmethyl)-

N-isopropylethane-1,2-diamine 22 (0.27 g, 0.58 mmol, 98%) as a

colorless oil, mp 139–141 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.44

3

(d, 1H, QnH-2, J ) 5 Hz), 8.22 (m, 1H, NpH-5/8, J ) 8 Hz),

8.02 (m, 1H, NpH-5/8, J ) 8 Hz), 7.93 (d, 1H, QnH-8, J ) 2 Hz),

7.59–7.47 (m, 2H, NpH-6/7), 7.44 (d, 1H, QnH-5, J ) 9 Hz), 7.27

(dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.78 (s, 1H, NpH-3), 6.27

(d, 1H, QnH-3, ³J ) 5 Hz), 5.88 (s_br, 1H, NH), 3.88 (s, 3H, OCH₃),

3.83 (s, 2H, NCH₂Np), 3.80 (s, 3H, OCH₃), 3.22 (m, 2H, N_QnCH₂),

3.11 (sept, 1H, CH, ³J ) 7 Hz), 2.96 (m, 2H, CH₂N), 1.20 (d, 6H,

3

CH₃, J ) 7 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 152.2, 151.6,

150.0, 148.6, 148.0, 134.9, 128.6, 128.2, 127.3, 126.8, 126.1, 125.5,

125.1, 122.4, 121.7, 121.1, 117.2, 105.0, 99.0, 62.4, 55.4, 49.8,

47.7, 47.0, 40.2, 18.1. MS (FAB+): 464 (M⁺). Anal.

(C₂₇H₃₀ClN₃O₂·0.5H₂O) C, H, N.

N-(7-Chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuorometh-

ylnaphthalen-2-ylmethyl)ethane-1,2-diamine 21. To (7-chloro-

quinolin-4-yl)ethane-1,2-diamine 1 (2.90 g, 13.06 mmol) in dry

DMF (70 mL) and anhydrous ethanol (20 mL) was added

triethylamine (1.32 g, 1.82 mL, 13.06 mmol) and then 2-bromo-

methyl-1,4-dimethoxy-3-triﬂuoromethylnaphthalene 13 (1.52 g, 4.35

mmol) in dry DMF (5 mL) under argon atmosphere. After being

stirred for 18 h at room temperature, the reaction mixture was

poured into brine, the organic layer was separated, and the aqueous

layer was extracted with CH₂Cl₂(3×). The combined organic layers

were dried over MgSO₄, and the solvent was removed in vacuo.

Flash chromatography (SiO₂, CH₂Cl₂/methanol, 5:1) yielded N-(7-

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-methylnaph-

thalen-2-ylmethyl)-N-isopropylethane-1,2-diamine 23. GP3 from

N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-methylnaphthalen-

2-ylmethyl)ethane-1,2-diamine 20 (0.40 g, 0.92 mmol), acetone

(1.60 g, 2.0 mL, 27.5 mmol), and NaBH(OAc)₃(0.59 g, 2.80 mmol)

after 66 h yielded N′-(7-chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-

methylnaphthalen-2-ylmethyl)-N-isopropylethane-1,2-diamine 23

(0.35 g, 0.73 mmol, 80%) as a colorless solid, mp 147–148 °C. ¹H

NMR (300 MHz, CDCl₃): δ 8.35 (d, 1H, QnH-2, ³J ) 5 Hz),

8.10–8.00 (m, 2H, NpH-5/8), 7.92 (d, 1H, QnH-8, J ) 2 Hz),

7.55–7.47 (m, 2H, NpH-6/7), 7.46 (d, 1H, QnH-5, J ) 9 Hz), 7.36

1272 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Friebolin et al.

3

(dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.11 (d, 1H, QnH-3, J )

117.0, 98.7, 63.9, 62.3, 61.1, 51.7, 51.0, 40.2, 26.2, 20.8, 12.4. MS

(FAB+): 492 (M⁺). Anal. (C₂₉H₃₄ClN₃O₂·0.5Et₂O) C, H, N.

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-triﬂuorometh-

ylnaphthalen-2-ylmethyl)-N-isobutylethane-1,2-diamine 27. GP3

from N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuorometh-

ylnaphthalen-2-ylmethyl)ethane-1,2-diamine 21 (0.40 g, 0.81 mmol),

isobutyraldehyde (0.22 mL, 0.18 g, 2.5 mmol), and NaBH(OAc)₃

(0.52 g, 2.50 mmol) after a 6-day-long reaction yielded N′-(7-

chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-triﬂuoromethylnaphthalen-

2-ylmethyl)-N-isobutylethane-1,2-diamine 27 (0.40 g, 7.32 mmol,

90%) as colorless crystals following puriﬁcation by ﬂash chroma-

tography (SiO₂, CH₂Cl₂/methanol, 20:1) and recrystallization from

5 Hz), 5.64 (s_br, 1H, NH), 3.90 (s, 3H, OCH₃), 3.88 (s, 2H,

NCH₂Np), 3.78 (s, 3H, OCH₃), 3.10 (sept, 1H, CH, J ) 7 Hz),

3

3.07 (m, 2H, N_QnCH₂), 2.88 (m, 2H, CH₂N), 2.54 (s, 3H, CH₃),

1.20 (d, 6H, CH₃, ³J ) 7 Hz). ¹³C NMR (75 MHz, CDCl₃): δ

151.6, 151.5, 150.5, 150.0, 148.6, 134.8, 128.4, 128.1, 127.5, 127.1,

127.0, 126.2, 125.6, 125.3, 122.4, 122.3, 121.3, 117.2, 98.9, 62.6,

61.2, 50.7, 46.3, 45.7, 40.6, 18.0, 12.4. MS (FAB+): 478 (M⁺).

Anal. (C₂₈H₃₂ClN₃O₂·0.5H₂O) C, H, N.

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-triﬂuorometh-

ylnaphthalen-2-ylmethyl)-N-isopropylethane-1,2-diamine 24.

GP3 from N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuo-

romethylnaphthalen-2-ylmethyl)ethane-1,2-diamine 21 (0.40 g, 0.81

mmol)), acetone (1.80 mL, 1.42 g, 24.5 mmol), and NaBH(OAc)₃

(0.52 g, 2.50 mmol) after a 6-day-long reaction yielded N′-(7-

chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-triﬂuoromethylnaphthalen-

2-ylmethyl)-N-isopropylethane-1,2-diamine 24 (0.38 g, 7.14 mmol,

87%) following puriﬁcation by ﬂash chromatography (SiO₂, CH₂Cl₂/

methanol, 20:1) and recrystallization from diethyl ether/petroleum

ether (colorless crystals), mp 87–88 °C. ¹H NMR (250 MHz,

1

diethylether/petroleum ether, mp 91–92 °C. H NMR (300 MHz,

CDCl₃): δ 8.37 (d, 1H, QnH-2, ³J ) 5 Hz), 8.14 (d, 1H, J ) 8 Hz,

NpH-5/8), 8.06 (d, 1H, J ) 8 Hz, NpH-5/8), 7.91 (d, 1H, QnH-8,

J ) 2 Hz), 7.80 (d, 1H, QnH-5, J ) 9 Hz), 7.66–7.56 (m, 2H,

NpH-6/7), 7.38 (dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.18 (d, 1H,

3

QnH-3, J ) 5 Hz), 6.00 (s_br, 1H, NH), 3.98 (s, 2H, NCH₂Np),

3.95 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.15 (m, 2H, HN_QnCH₂),

2.73 (m, 2H, CH₂N), 2.24 (d, 2H, CH₂, J ) 7 Hz), 1.73 (m, 1H,

CH), 0.76 (d, 6H, ³J ) 6 Hz, 2 × CH₃). ¹³C NMR (75 MHz,

CDCl₃): δ 153.8, 152.6, 151.6, 150.1, 148.8, 134.8, 130.2, 128.8,

3

CDCl₃): δ 8.34 (d, 1H, QnH-2, J ) 5 Hz), 8.05 (m, 2H, NpH-5/

8), 7.92 (d, 1H, QnH-8, J ) 2 Hz), 7.71 (d, 1H, QnH-5, J ) 9

Hz), 7.60 (m, 2H, NpH-6/7), 7.38 (dd, 1H, QnH-6, J ) 2 Hz, J )

1

128.7, 128.2, 127.4, 127.3 (q, J ) 270 Hz, CF₃), 126.5, 125.2,

125.0, 123.8, 122.7, 121.7, 119.3 (q, ²J ) 28.3 Hz, C-CF₃), 117.4,

98.9, 64.1, 63.6, 62.5, 52.5, 51.1, 40.2, 26.3, 20.9. MS (FAB+):

546 (M⁺), 532, 354. Anal. (C₂₉H₃₁ClF₃N₃O₂) C, H, N.

3

9 Hz), 6.09 (d, 1H, QnH-3, J ) 5 Hz), 5.93 (s_br, 1H, NH), 4.09

(s, 2H, CH₂Np), 3.93 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 3.00–2.94

(m, 3H, HNCH₂+ CH), 2.81 (m, 2H, CH₂N), 1.10 (d, 6H, ³J ) 6

Hz, 2 × CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 153.9, 152.5, 151.7,

150.0, 148.9, 134.7, 130.1, 128.7, 128.6, 128.2, 127.3, 126.6, 125.8,

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxynaphthalen-2-yl-

methyl)-N-isopentylethane-1,2-diamine 28. GP3 from N-(7-chlo-

roquinolin-4-yl)-N′-(1,4-dimethoxynaphthalen-2-ylmethyl)ethane-

1,2-diamine 19 (0.16 g 0.37 mmol), pivalaldehyde (0.25 mL, 2.28

mmol), and NaBH(OAc)₃(0.25 g, 1.14 mmol) after an 80-min-

long reaction yielded N′-(7-chloroquinolin-4-yl)-N-(1,4-dimeth-

oxynaphthalen-2-ylmethyl)-N-isopentylethane-1,2-diamine 28 (0.18

g, 0.37 mmol, 97%) as a pale-yellow oil following puriﬁcation by

ﬂash chromatography (SiO₂, CH₂Cl₂/methanol, 40:1), mp 53–56

1

125.2 (q, J ) 270 Hz, CF₃), 125.0, 123.7, 122.9, 122.7, 121.6,

119.0 (q, ²J ) 30 Hz, C-CF₃), 117.5, 98.9, 63.9, 62.8, 50.8, 46.7,

46.4, 40.2, 17.9. MS (FAB+): 532 (M⁺), 340. Anal. (C₂₈H₂₉F₃ClN₃O₂)

C, H, N.

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxynaphthalen-2-yl-

methyl)-N-isobutylethane-1,2-diamine 25. GP3 from N-(7-chlo-

roquinolin-4-yl)-N′-(1,4-dimethoxynaphthalen-2-ylmethyl)ethane-

1,2-diamine 19 (0.25 g, 0.59 mmol), isobutyraldehyde (0.24 g 3.30

mmol), and NaBH(OAc)₃(0.38 g, 1.78 mmol) after 2 h yielded

N′-(7-chloroquinolin-4-yl)-N-(1,4-dimethoxynaphthalen-2-ylmethyl)-

N-isobutylethane-1,2-diamine 25 (0.28 g, 0.59 mmol, 99%) as a

pale-yellow oil, mp 48–49 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.46

1

3

°C. H NMR (250 MHz, CDCl₃): δ 8.37 (d, 1H, QnH-2, J ) 5

Hz), 8.23 (m, 1H, NpH-5/8), 7.99 (m, 1H, NpH-5/8), 7.92 (d, 1H,

QnH-8, J ) 2 Hz), 7.50 (m, 3H, NpH-6/7, QnH-5), 7.33 (dd, 1H,

QnH-6, J ) 2 Hz, J ) 9 Hz), 6.91 (s, 1H, NpH-3), 6.22 (d, 1H,

QnH-3, J ) 5 Hz), 5.75 (s, 1H, NH), 3.88 (s, 3H, OCH₃), 3.84 (s,

2H, NpCH₂), 3.81 (s, 3H, OCH3), 3.18 (m, 2H, NCH₂), 2.85 (m,

2H, NCH₂), 2.45 (s, 2H,CH₂tBu), 0.90 (s, 9H, ^tBu). ¹³C NMR (75

MHz, CDCl₃): δ 152.1, 151.3, 150.1, 148.2, 148.1, 135.0, 128.6,

128.1, 127.1, 126.9, 126.2, 125.6 (NpC-6/7), 125.2, 122.4, 121.8,

121.3, 117.2, 105.4, 98.9, 77.2, 67.8, 62.4, 55.7, 55.2, 54.4, 40.9,

32.9, 28.5. MS (FAB+): 492 (M⁺). Salt 28, mp 178–180 °C. Anal.

(C₂₉H₃₄ClN₃O₂·2HCl·1H₂O) C, H, N.

3

(d, 1H, QnH-2, J ) 5 Hz), 8.26 (m, 1H, NpH-5/8, J ) 8 Hz),

8.05 (m, 1H, NpH-5/8, J ) 8 Hz), 7.94 (d, 1H, QnH-8, J ) 2 Hz),

7.61–7.46 (m, 2H, NpH-6/7), 7.53 (d, 1H, QnH-5, J ) 9 Hz), 7.32

(dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.83 (s, 1H, NpH-3), 6.29

(d, 1H, QnH-3, ³J ) 5 Hz), 5.78 (s_br, 1H, NH), 3.92 (s, 3H, OCH₃),

3.89 (s, 3H, OCH₃), 3.82 (s, 2H, NCH₂Np), 3.27 (m, 2H, N_QnCH₂),

3

2.88 (m, 2H, CH₂N), 2.38 (d, 2H, CH₂, J ) 7 Hz), 2.00 (m, 1H,

CH, ³J ) 7 Hz), 0.95 (d, 6H, 2 × CH₃, ³J ) 7 Hz). ¹³C NMR (75

MHz, CDCl₃): δ 152.1, 151.8, 149.9, 148.8, 148.1, 134.8, 128.6,

128.5, 126.9, 126.8, 126.2, 125.6, 125.1, 122.4, 121.8, 121.2, 117.3,

105.3, 99.0, 63.3, 62.4, 55.6, 53.3, 52.4, 40.2, 26.5, 18.8. MS

(FAB+): 478 (M⁺). Anal. (C₂₈H₃₂Cl₃N₃O₂) C, H, N.

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-methylnaph-

thalen-2-ylmethyl)-N-isopentylethane-1,2-diamine 29. GP3 from

N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-methylnaphthalen-

2-ylmethyl)ethane-1,2-diamine 20 (0.21 g, 0.48 mmol), pivalalde-

hyde (0.25 g, 0.31 mL, 2.89 mmol), and NaBH(OAc)₃(0.31 g,

1.44 mmol) after a 2.5-h-long reaction yielded N′-(7-chloroquinolin-

4-yl)-N-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)-N-isopen-

tylethane-1,2-diamine 29 (0.21 g, 0.41 mmol, 86%) as a colorless

solid following puriﬁcation by ﬂash chromatography (SiO₂, CH₂Cl₂/

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-methylnaph-

thalen-2-ylmethyl)-N-isobutylethane-1,2-diamine 26. GP3 from

N-(7-chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-methylnaphthalen-

2-ylmethyl)ethane-1,2-diamine 20 (0.40 g, 0.92 mmol), isobutyral-

dehyde (0.20 g, 0.25 mL, 2.75 mmol), and NaBH(OAc)₃(0.59 g,

2.80 mmol) after a 5-h-long reaction yielded N′-(7-chloroquinolin-

4-yl)-N-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)-N-isobu-

tylethane-1,2-diamine 26 (0.44 g, 0.89 mmol, 97%) as colorless

crystals following recrystallization (diethylether/petroleum ether),

1

methanol, 20:1), mp 63–65 °C. H NMR (300 MHz, CDCl₃): δ

3

8.31 (d, 1H, QnH-2, J ) 5 Hz), 7.98 (m, 2H, NpH-5/8), 7.89 (d,

1H, QnH-8, J ) 2 Hz), 7.71 (d, 1H, QnH-5, J ) 9.0 Hz), 7.42 (m,

2H, NpH-6/7), 7.34 (dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.16 (d,

1H, QnH-2, J ) 5 Hz), 6.01 (s, 1H, NH), 3.80 (s, 2H, NCH₂Np),

3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.18 (m, 2H, NCH₂), 2.80

(m, 2H, NCH₂), 2.47 (s, 3H, CH₃), 2.32 (s, 2H, CH₂tBu), 0.72 (s,

9H, ^tBu). ¹³C NMR (75 MHz, CDCl₃): δ 151.9, 151.3, 150.5, 150.2,

148.4, 135.1, 128.5, 128.0, 127.4, 127.0 126.3, 125.7, 125.4, 122.5,

122.4, 121.7, 117.3, 98.9, 73.5, 67.6, 62.3, 61.3, 54.7, 52.0, 40.7,

32.8, 28.6, 13.2. MS (FAB+): 506 (M⁺). Anal. (C₃₀H₃₆ClN₃O₂·0.4H₂O)

C, H, N.

1

mp 88–90 °C. H NMR (300 MHz, CDCl₃): δ 8.40 (d, 1H, QnH-

3

2, J ) 5 Hz), 8.14–8.04 (m, 2H, NpH-5/8), 7.92 (d, 1H, QnH-8,

J ) 2 Hz), 7.55 (d, 1H, QnH-5, J ) 9 Hz), 7.56–7.48 (m, 2H,

NpH-6/7), 7.40 (dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 6.20 (d, 1H,

3

QnH-3, J ) 5 Hz), 5.62 (s_br, 1H, NH), 3.92 (s, 3H, OCH₃), 3.87

(s, 3H, OCH₃), 3.86 (s, 2H, NCH₂Np), 3.16 (m, 2H, N_QnCH₂), 2.81

(m, 2H, CH₂N), 2.59 (s, 3H, CH₃), 2.37 (d, 2H, CH₂, ³J ) 6 Hz),

1.92 (m, 1H, CH), 0.88 (d, 6H, CH₃, J ) 6 Hz). ¹³C NMR (75

N′-(7-Chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-triﬂuoromethyl-

naphthalen-2-ylmethyl)-N-isopentylethane-1,2-diamine 30. N-(7-

Chloroquinolin-4-yl)-N′-(1,4-dimethoxy-3-triﬂuoromethylnaphtha-

3

MHz, CDCl₃): δ 151.5, 151.4, 150.4, 149.7, 148.5, 134.6, 128.3,

128.1, 126.9, 126.8, 126.7, 126.1, 125.5, 125.0, 122.3, 122.1, 121.2,

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1273

len-2-ylmethyl)ethane-1,2-diamine dihydrochloride salt 21 (0.14 g,

0.26 mmol) was treated with 2 N NaOH solution to liberate the

free base, and the mixture was subsequently extracted with CH₂Cl₂

(3×). The combined organic phases were dried over MgSO₄, and

then the solvent was removed in vacuo. The product 21 was used

without further puriﬁcation for the reaction following GP3. The

reaction involving the free base N-(7-chloroquinolin-4-yl)-N′-(1,4-

dimethoxy-3-triﬂuoromethylnaphthalen-2-ylmethyl)ethane-1,2-di-

amine 21, pivalaldehyde (0.17 mL, 0.14 g, 1.57 mmol), and

NaBH(OAc)₃(0.17 g, 0.78 mmol) after 3 days yielded N′-(7-

chloroquinolin-4-yl)-N-(1,4-dimethoxy-3-triﬂuoromethylnaphthalen-

2-ylmethyl)-N-isopentylethane-1,2-diamine 30 (0.10 g, 0.18 mmol,

68%) as a colorless solid following puriﬁcation by ﬂash chroma-

thalen-2-yl)hexanoic acid [2-(7-chloroquinolin-4-ylamino)ethyl]

amide 34 (3.47 g, 62.1%) as an amorphous yellow powder, mp

1

65–67 °C. H NMR (300 MHz, CD₃OD): δ 8.27 (d, 1H, QnH-2,

³J ) 6 Hz), 7.94–7.88 (m, 3H, QnH-5, NqH-5/8), 7.69 (d, 1H,

QnH-8, J ) 2 Hz), 7.66–7.63 (m, 2H, NqH-6/7), 7.38 (dd, 1H,

3

QnH-6, J ) 2 Hz, J ) 9 Hz), 6.65 (d, 1H, QnH-3, J ) 6 Hz),

4.24 (sept, 1H, CH), 3.58–3.52 (m, 2H, N_QnCH₂), 3.48–3.43 (m,

2H, CH₂N), 2.52–2.45 (m, 4H, CH₂), 2.05 (s, 3H, CH₃), 1.72–1.67

(m, 2H, CH₂), 1.45–1.42 (m, 4H, CH₂), 1.24 (d, 6H, iPr). ¹³C NMR

(75 MHz, CD₃OD): δ 186.1, 185.6, 176.8, 153.5, 150.8, 148.1,

147.5, 144.4, 137.2, 134.5, 133.3, 127.0, 126.6, 126.2, 124.4, 118.1,

99.6, 50.3, 44.4, 40.3, 34.2, 30.5, 29.4, 27.6, 26.4, 21.4, 12.7. MS

(FAB+): 532.2 (M⁺). HRMS (FAB+, NBA): calcd (M⁺+ 1)

532.2367; found: 532.2396. Anal. (C₃₁H₃₄ClN₃O₃·1.2 H₂O) C, H,

N.

1

tography (SiO₂, CH₂Cl₂/methanol, 40:1), mp 71–72 °C. H NMR

3

(300 MHz, CDCl₃): δ 8.36 (d, 1H, QnH-2, J ) 5 Hz), 8.10 (m,

2H, NpH-5/8), 8.00 (d, 1H, QnH-8, J ) 2 Hz), 7.90 (d, 1H, QnH-

5, J ) 9 Hz), 7.55 (m, 2H, NpH-6/7), 7.40 (dd, 1H, QnH-6, J ) 2

6-(3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)hexanoic

Acid [2-(7-Chloroquinolin-4-ylamino)ethyl]amide 35. GP4 from

6-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)hexanoic acid

M₅(200 mg, 0.7 mmol) and N-(7-chloroquinolin-4-yl)ethyl-1,2-

diamine 1 (233 mg, 1.05 mmol) afforded 6-(3-methyl-1,4-dioxo-

1,4-dihydronaphthalen-2-yl)hexanoic acid [2-(7-chloroquinolin-4-

ylamino)ethyl]amide 35 (70 mg, 0.05 mmol, 19%) as a yellow solid,

mp 58–60 °C. ¹H NMR (250 MHz, CD₃OD): δ 8.31 (d, 1H, QnH-

2, ³J ) 6 Hz), 8.03 (d, 1H, QnH-5, J ) 9 Hz), 7.99–7.94 (m, 2H,

NqH-5/8), 7.72–7.68 (m, 3H, QnH-8, NqH-6/7), 7.44 (dd, 1H, QnH-

6, J ) 2 Hz, J ) 9 Hz), 6.60 (d, 1H, QnH-3, ³J ) 6 Hz), 3.52 (m,

4H, NCH₂CH₂N), 2.48 (m, 2H, CH₂), 2.23 (m, 2H, CH₂), 2.05 (s,

3H, CH₃), 1.65 (m, 2H, CH₂), 1.39 (m, 4H, CH₂). ¹³C NMR (75

MHz, CDCl₃): δ 185.1, 184.6, 176.2, 154.0, 147.0, 145.0, 143.2,

141.6, 138.1, 133.3, 132.0, 128.0, 126.1, 124.8, 122.1, 118.2, 115.8,

111.2, 97.6, 45.7, 38.1, 36.1, 29.4, 28.2, 26.8, 25.3, 12.6. MS

(FAB+): 490 (M⁺+ 1). HRMS (FAB+, NBA): calcd (M⁺+ 1)

490.1897; found 490.1876.

3

Hz, J ) 9 Hz), 6.58 (s_br, 1H, NH), 6.22 (d, 1H, QnH-3, J ) 5

Hz), 3.94 (s, 2H, CH₂Np), 3.90 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃),

t

3.18 (m, 2H, NCH₂), 2.79 (m, 2H, CH₂N), 2.26 (s, 2H, CH₂Bu),

t

0.84 (s, 9H, Bu). ¹³C NMR (75 MHz, CDCl3): δ 153.6, 153.5,

152.9, 152.5, 151.1, 135.9, 130.1, 129.0, 128.8, 127.5, 127.0, 125.6,

125.2, 123.9, 122.5, 122.0, 119.2 (q, C-CF₃), 117.2, 98.8, 64.1,

62.3, 54.8, 51.4, 40.5, 32.6, 28.5. MS (FAB+): 560 (M⁺). Anal.

(C₃₀H₃₃ClF₃N₃O₂·0.4H₂O) C, H, N.

N-[4-Hydroxy-3-({isopropyl-[3-(7-chloroquinolin-4-ylamino)

propyl]amino}methyl)phenyl]acetamide 32. Acetamidophenol

(0.48 g, 3.17 mmol) was subjected to the Mannich reaction with

N-(7-chloroquinolin-4-yl)propane-3-isopropyl-1,3-diamine 31 (1.77

g, 6.37 mmol) and 37% aqueous formaldehyde (0.65 mL, 8 mmol)

in 5 mL of ethanol. After the mixture was heated to reﬂux for 79 h,

the solvent was removed under reduced pressure and the residue

was dissolved in dichloromethane (10 mL). The organic solution

was extracted with dilute hydrochloric acid (0.1 M, 2 × 20 mL).

This solution was basiﬁed (pH 9–10) with 0.65 mL of 0.1 N NaOH

and extracted with dichloromethane (5 × 20 mL). The combined

extracts were washed with water (1 × 20 mL) and dried over

MgSO₄. The solvent was evaporated under reduced pressure to give

the product as a crude oil, mp 108–109 °C. Flash chromatography

(SiO₂, CH₂Cl₂/methanol, 6:1) afforded the product 32 (0.25 g, 0.59

mmol, 18.5%) as colorless crystals. ¹H NMR (250 MHz, CDCl₃):

δ 8.44 (d, 1H, QnH-2, ³J ) 5 Hz), 7.91 (d, 1H, QnH-5, J ) 2 Hz),

7.63 (d, 1H, QnH-3, J ) 9 Hz), 7.54 (s_br, 1H, NHCO), 7.37 (d,

1H, PhH-2′, J ) 2 Hz), 7.30 (dd, 1H, QnH-6, J ) 2 Hz, J ) 9

Hz), 7.03 (dd, 1H, PhH-6′, J ) 2 Hz, J ) 9 Hz), 6.74 (d, 1H,

PhH-5′, J ) 9 Hz), 6.33 (d, 1H, QnH-3, ³J ) 5 Hz), 5.70 (s_br, 1H,

N-[2-(7-Chloroquinolin-4-ylamino)ethyl]-N′-isopropyl-2-[4-(3-

methyl-1,4-dioxo-1,4-dihydronaphthalen-2-ylmethyl)phenyl]ac-

etamide 36. GP4 from [4-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-

2-ylmethyl)phenyl]acetic acid 33 (3 g, 9.36 mmol, 1 equiv) and

N′-(7-chloroquinolin-4-yl)-N-isopropylethane-1,2-diamine 2 (3.70

g, 14.04 mmol, 1.5 equiv) yielded N-[2-(7-chloroquinolin-4-

ylamino)ethyl]-N′-isopropyl-2-[4-(3-methyl-1,4-dioxo-1,4-dihy-

dronaphthalen-2-ylmethyl)phenyl]acetamide 36 (1.23 g, 23.2%) as

1

a yellow solid, mp 157–158 °C. H NMR (300 MHz, CD₃OD): δ

3

8.28 (d, 1H, QnH-2, J ) 6 Hz), 7.96 (m, 2H, NqH-5/8), 7.86 (d,

1H, QnH-5, J ) 9 Hz), 7.71–7.65 (m, 3H, QnH-8, NqH-6/7), 7.29

(dd, 1H, QnH-6, J ) 2 Hz, J ) 9 Hz), 7.14 (m, 4H, PhH), 6.62 (d,

1H, QnH-3, ³J ) 6 Hz), 4.21 (sept, 1H, CH), 4.20 (s, 2H, COCH₂),

3.94 (s, 2H, CH₂), 3.78 (s, 2H, CH₂), 3.52 (m, 4H, NCH₂CH₂N),

2.80 (s, 3H, CH₃), 1.06 (d, 6H, ⁱPr). ¹³C NMR (75 MHz, CDCl₃):

δ 185.2, 184.5, 173.8, 151.0, 149.9, 147.1, 145.0, 144.4, 136.8,

135.5, 133.5, 132.7, 132.0, 131.9, 129.0, 128.8, 126.7, 126.4, 126.2,

125.7, 122.7, 116.9, 97.8, 49.5, 45.8, 40.9, 39.2, 31.9, 21.1, 13.2

(CH₃). ¹³C NMR (75 MHz, CD₃OD): δ 186.3, 185.8, 174.8, 152.5,

149.6, 146.5, 145.6, 138.4, 136.3, 134.7, 134.6, 134.5, 133.4, 133.3,

130.1, 130.1, 130.0, 127.6, 127.2, 127.1, 126.1, 124.1, 118.5, 99.6,

50.8, 43.8, 41.5, 40.5 (CH₂), 38.9, 32.6, 21.1, 13.2. MS (FAB+):

566.4 (M⁺+ 1). HRMS (FAB+, NBA) calcd (M⁺+ 1) 566.2210;

found 566.2207. Anal. (C₃₄H₃₂ClN₃O₃·0.5H₂O) C, H, N.

3

NH), 3.73 (s, 2H, PhCH₂N), 3.35 (t, 2H, NCH₂, J ) 6 Hz), 3.15

(sept, 1H, CH, ³J ) 7 Hz), 2.61 (t, 2H, NCH₂, ³J ) 6 Hz), 2.15 (s,

3H, COCH₃), 1.93 (t, 2H, CH₂, ³J ) 6 Hz), 1.11 (d, 6H, 2 × CH₃,

³J ) 7 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 168.4, 154.6, 151.1,

149.9, 148.2, 135.1, 129.8, 127.8, 125.3, 122.1, 121.6, 120.9, 117.0,

116.0, 98.7, 52.9, 49.4, 46.6, 40.7, 26.2, 24.3, 17.1. MS (FAB+):

441.1 (M⁺+ 1). HRMS (FAB+, NBA): calcd (M⁺+ 1) 441.2057;

found 441.2043. Anal. (C₂₄H₂₉ClN₄O₂·2H₂O) C, H, N.

General Procedure 4 (GP4) for the Synthesis of Tertiary

Amides 34–36, Illustrated with 6-(3-Methyl-1,4-dioxo-1,4-dihy-

dronaphthalen-2-yl)hexanoic Acid [2-(7-Chloroquinolin-4-ylam-

ino)ethyl]-N-isopropylamide 34. To a solution of 6-(3-methyl-

1,4-dioxo-1,4-dihydronaphthalen-2-yl)hexanoic acid M₅(3 g, 10.5

mmol, 1 equiv) in DMF (35 mL) was added HOBT (1.4 g, 10.5

mmol, 1 equiv) and EDC (2.1 g, 2.4 mL, 13.65 mmol, 1.3 equiv)

dropwise at 0 °C. The reaction mixture was allowed to stir at 0 °C

for 1 h. Then the amine N′-(7-chloroquinolin-4-yl)-N-isopropyle-

thane-1,2-diamine 2 (4.1 g, 15.75 mmol, 1.5 equiv) in 30 mL of

DMF was added at 0 °C and the reaction mixture was stirred

overnight at room temperature. The reaction mixture was extracted

with CH₂Cl₂and successively washed with brine, 0.1 N HCl, brine,

3% NaHCO₃solution, and brine. The combined organic layers were

dried over MgSO₄and evaporated. Puriﬁcation of the crude product

was done by two ﬂash chromatography steps (SiO₂, CH₂Cl₂/

CH₃OH, 12:1) yielding 6-(3-methyl-1,4-dioxo-1,4-dihydronaph-

General Procedure 5 (GP5) for the Synthesis of Tertiary

Amides 39–41 Illustrated with N-[2-(7-Chloroquinolin-4-ylam-

ino)ethyl]-2-[4-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)phe-

nyl]-N-isopropylacetamide 40. [4-(3-Methyl-1,4-dioxo-1,4-dihy-

dronaphthalen-2-ylmethyl)phenyl]acetic acid 33 (3.50 g, 9.99 mmol)

in 350 mL of ethanol was heated at 65 °C until most of the

compound was dissolved. At 50 °C SnCl₂(7.00 g, 36.92 mmol) in

7 mL of concentrated HCl was added slowly and further stirred

for 2.5 h. During this period the reaction mixture became almost

colorless. The ethanol was evaporated, and the residue was slowly

poured into ice–water. The pale-yellow precipitate was ﬁltered,

washed with cold water, dissolved in acetone, dried over MgSO₄,

and concentrated in vacuo. The residue was dissolved in 100 mL

of acetone, and dimethyl sulfate (5.50 g, 4.20 mL, 43.6 mmol) was

1274 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Friebolin et al.

added. Then the solution was heated at 60 °C and a KOH solution

(3.06 g, 54.5 mmol) in 15 mL of CH₃OH was added dropwise and

very slowly (CAUTION: exothermic reaction). Then the mixture

was heated under reﬂux for 3.5 h and afterward stirred overnight

at room temperature. The reaction mixture was poured into 300

mL of 5% NaOH solution and stirred for 30 min. Then concentrated

HCl was added dropwise until the solution became acidic. The

acetone was evaporated and the aqueous residue extracted with

diethyl ether. The combined organic layers were washed once with

water, dried over MgSO₄, and then evaporated. Flash chromatog-

raphy (SiO₂, petroleum ether/ethyl acetate, 5:2) afforded [4-(1,4-

dimethoxy-3-methylnaphthalen-2-ylmethyl)phenyl]acetic acid 38

(2.98 g, 8.5 mmol, 78%) as a colorless solid. ¹H NMR (250 MHz,

CD₃OD): δ 8.06 (m, 2H, NqH-5/8), 7.49 (m, 2H, NqH-6/7), 7.15

(d, 2H, PhH), 7.04 (d, 2H, PhH), 4.25 (s, 2H, CH₂), 3.81 (s, 3H,

OCH₃), 3.80 (s, 3H, OCH₃), 3.53 (s, 2H, CH₂), 3.78 (s, 2H, CH₂),

2.20 (s, 3H, CH₃). To [4-(1,4-dimethoxy-3-methylnaphthalen-2-

ylmethyl)phenyl]acetic acid 38 (200 mg, 0.56 mmol, 1 equiv) in

0.2 mL of CH₂Cl₂was added thionyl chloride (0.2 mL, 2.80 mmol,

5 equiv), and then the reaction mixture was heated to reﬂux for

1.5 h at 50 °C. After the mixture was cooled at 0 °C, a mixture of

N-(7-chloroquinolin-4-yl)-N-isopropylethyl-1,2-diamine 2 (177.2

mg, 0.70 mmol, 1.2 equiv) and triethylamine (0.16 mL, 1.20 mmol,

2 equiv) in 2 mL of CH₂Cl₂was added. After the mixture was

stirred for 1 h at 0 °C and for a further 12 h at room temperature,

the solvent was evaporated and the residue was puriﬁed by ﬂash

chromatography (SiO₂, CH₂Cl₂/CH₃OH, 12:1) to afford N-[2-(7-

chloroquinolin-4-ylamino)ethyl]-2-[4-(1,4-dimethoxy-3-methyl-

naphthalen-2-ylmethyl)phenyl]-N-isopropylacetamide 40 (205 mg,

N-[2-(7-Chloroquinolin-4-ylamino)ethyl]-2-[4-(1,4-dimethoxy-

3-methylnaphthalen-2-ylmethyl)phenyl]acetamide 41. GP5 from

[4-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)phenyl]acetic acid

38 (0.50 g, 1.43 mmol, 1 equiv) and N-(7-chloroquinolin-4-yl)ethyl-

1,2-diamine 1 (0.38 g, 1.72 mmol, 1.2 equiv) yielded N-[2-(7-

chloroquinolin-4-ylamino)ethyl]-2-[4-(1,4-dimethoxy-3-methyl-

naphthalen-2-ylmethyl)phenyl]acetamide 41 (116 mg, 0.21 mmol,

14.6%) as a colorless solid, mp 108–110 °C. ¹H NMR (250 MHz,

3

CD₃OD): δ 8.30 (d, 1H, QnH-2, J ) 5 Hz), 8.04 (m, 2H, NqH-

5/8), 7.93 (d, 1H, QnH-5, J ) 9 Hz), 7.75 (d, 1H, QnH-8, J ) 2

Hz), 7.48 (m, 2H, NqH-6/7), 7.35 (dd, 1H, QnH-6, J ) 2 Hz, J )

9 Hz), 7.07 (d, 2H, PhH), 6.95 (d, 2H, PhH), 6.56 (d, 1H, QnH-3,

³J ) 5 Hz), 4.20 (s, 2H, COCH₂), 3.78 (s, 3H, OCH₃), 3.77 (s, 3H,

OCH₃), 3.50–3.45 (m, 4H, NCH₂), 3.44 (s, 2H, PhCH₂), 2.14 (s,

3H, CH₃). ¹³C NMR (75 MHz, CD₃OD): δ 175.5, 154.5, 151.7,

151.6, 149.1, 145.8, 140.5, 138.0, 134.2, 130.4, 130.1, 129.4, 129.2,

128.5, 128.0, 127.0, 126.8, 126.6, 124.7, 124.6, 123.4, 123.2, 117.8,

99.5, 62.3, 61.7, 44.5, 43.4, 39.1, 33.2, 12.8. MS (FAB+): 554.1

(M⁺+ 1). HRMS (FAB+, NBA): calcd (M⁺+ 1) 554.2210; found

554.2219.

Assay of ꢀ-Hematin Inhibition in Eppendorf Tubes. We

determined the IC₅₀values for inhibition of ꢀ-hematin formation

using Egan’s test⁴⁵with very slight modiﬁcations as described

below. Increasing numbers of equivalents of the 4-aminoquino-

line derivatives were prepared using aqueous HCl for CQ and

DMF instead of methanol for all other compounds. Hematin

stock solution (1.68 mM) was prepared by dissolving bovine

hemin in 0.1 M NaOH. The solution was incubated at room

temperature for 60 min. In a series of 2 mL Eppendorf tubes

2.0 µL of drug solution (or solvent for the blank) and 2.0 µL of

1 M HCl (when DMF was used, or 2.0 µL of water for CQ)

were dispensed. The Eppendorf tubes were placed in an incubator

at 60 °C, and then 12.9 M sodium acetate solution, pH 5.0 (11.7

µL) and preincubated at 60 °C, was added. The ꢀ-hematin

formation process was initiated by addition of hematin stock

solution (20.2 µL) prepared above. The ﬁnal hematin concentra-

tion was 1 mM, and the ﬁnal drug concentrations were 5, 4, 3,

2.5, 2, 1.5, 1, 0.5, and 0 mM for CQ, 2, and M₅and were 5,3,

1.5, 1, 0.75, 0.6, 0.5, 0.25, and 0 mM for amide 34. The pH of

the resulting acetate solution was checked to be 4.5 after

addition of the hematin solution. The reaction mixtures were

incubated at 60 °C for 60 min. Then the reaction mixtures were

quenched at room temperature by adding 900 µL of 200 mM

HEPES 5% (v/v) pyridine solution, pH 8.2, to adjust the ﬁnal

pH of the mixtures to a value between 7.2 and 7.5. Then 1100

µL of 20 mM HEPES 5% (v/v) pyridine solution, pH 7.5, was

added. The Eppendorf tubes were shaken, and the precipitate of

ꢀ-hematin was scrapped from the walls of the Eppendorf tubes

to ensure complete dissolution of hematin. The ꢀ-hematin was

allowed to settle at room temperature for at least 15 min. The

supernatant was carefully transferred to a cuvette without

disturbing the precipitate, and absorption was measured at 405

nm.

Parasite Cultures. The CQ-sensitive 3D7 clone of the NF54

isolate⁸⁶of Plasmodium falciparum and the chloroquine-,

pyrimethamine-, and cycloguanil-resistant K1 strain (Thailand)

were acquired from MR4 (Malaria Research and Reference

Reagent Resource Center, Manassas, VA). P. falciparum in vitro

culture was carried out using standard protocols⁸⁷with modiﬁca-

tions. Brieﬂy, parasites were maintained in tissue culture ﬂasks

in human A Rh+ erythrocytes at 5% hematocrit in RPMI 1640

supplemented with 25 mM HEPES, 24 mM NaHCO₃, 0.2% (w/

v) glucose, 0.03% L-glutamine, 150 µM hypoxanthine, and 0.5%

Albumax II (Invitrogen) in a 5% CO₂/air mixture at 37 °C, and

the medium was changed daily.

In Vitro Antiparasitic Bioassays. Drug susceptibility of P.

falciparum was studied using a modiﬁed method⁸⁸of the protocol

described previously.⁸⁹All assays included CQ diphosphate (Sigma,

U.K.) as a standard and control wells with untreated infected and

uninfected erythrocytes. IC₅₀values were derived by sigmoidal

regression analysis (Microsoft xlﬁt). All data from in vitro tests

1

0.34 mmol, 61.4%) as a colorless solid, mp 95–97 °C. H NMR

3

(300 MHz, CD₃OD): δ 8.31 (d, 1H, QnH-2, J ) 6 Hz), 8.03 (m,

2H, NqH-5/8), 7.91 (d, 1H, QnH-5, J ) 9 Hz), 7.77 (d, 1H, QnH-

8, J ) 2 Hz), 7.46 (m, 2H, NqH-6/7), 7.34 (dd, 1H, QnH-6, J ) 2

Hz, J ) 9 Hz), 7.11 (d, 2H, PhH), 7.00 (d, 2H, PhH), 6.69 (d, 1H,

3

QnH-3, J ) 6 Hz), 4.21 (sept, 1H, CH), 4.20 (s, 2H, COCH₂),

3.77 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.52 (m, 4H, NCH₂CH₂N),

2.15 (s, 3H, CH₃), 1.04 (d, 6H, ⁱPr). ¹³C NMR (75 MHz, CD₃OD):

δ 174.9, 152.9, 152.0, 151.8, 151.7, 148.9, 140.6, 136.6, 133.9,

130.4, 129.8, 129.6, 129.3, 128.6, 128.0, 127.2, 126.9, 126.6, 126.3,

124.2, 123.4, 123.2, 118.4, 99.7, 62.7, 61.7, 50.8, 43.8, 41.7, 40.5,

33.1, 21.1, 12.8. MS (FAB+): 596.8 (M⁺

(C₃₆H₃₈ClN₃O₃·1H₂O) C, H, N.

+ 1). Anal.

6-(1,4-Dimethoxy-3-methylnaphthalen-2-yl)hexanoic Acid [2-(7-

Chloroquinolin-4-ylamino)ethyl]-N-isopropylamide 39. 6-(1,4-

Dimethoxy-3-methylnaphthalen-2-yl)hexanoic acid 37 was pro-

duced using the same protocol as for [4-(1,4-dimethoxy-3-

methylnaphthalen-2-ylmethyl)phenyl]acetic acid 38. GP5 from

[4-(1,4-dimethoxy-3-methylnaphthalen-2-ylmethyl)phenyl]acetic acid

38 (0.50 g, 1.6 mmol) and N′-(7-chloroquinolin-4-yl)-N-isopropyl

ethane-1,2-diamine 2 (0.51 g, 1.90 mmol) yielded 6-(1,4-dimethoxy-

3-methylnaphthalen-2-yl)hexanoic acid [2-(7-chloroquinolin-4-

ylamino)ethyl]isopropylamide 39 (63 mg, 0.01 mmol, 7%) as a

colorless solid after ﬂash chromatography (SiO₂, CH₂Cl₂/CH₃OH,

20:1 and 1:1) and preparative TLC (SiO₂, CH₂Cl₂/CH₃OH, 12:1),

mp 57–59 °C. ¹H NMR (300 MHz, CD₃OD): δ 8.28 (d, 1H, QnH-

2, ³J ) 5 Hz), 8.02 (d, 1H, QnH-5, J ) 9 Hz), 7.95–7.90 (m, 2H,

NqH-5/8), 7.75 (d, 1H, QnH-8, J ) 2 Hz), 7.49 (dd, 1H, QnH-6,

J ) 2 Hz, J ) 9 Hz), 7.40–7.35 (m, 2H, NqH-6/7), 6.75 (d, 1H,

QnH-3, ³J ) 6 Hz), 4.27 (sept, 1H, CH), 3.80 (s, 3H, OCH₃), 3.78

(s, 3H, OCH₃), 3.57–3.46 (m, 4H, N_QnCH₂), 2.75 (m, 2H, CH₂),

2.49 (m, 2H, CH₂), 2.32 (s, 3H, CH₃), 1.73–1.62 (m, 2H, CH₂),

i

1.60–1.44 (m, 4H, CH₂), 1.20 (d, 6H, Pr). ¹³C NMR (75 MHz,

CDCl₃): δ 175.9, 153.8, 150.1, 149.8, 145.4, 142.0, 138.0, 131.4,

127.4, 127.1, 126.9, 126.3, 125.4, 125.3, 123.4, 122.7, 122.2, 116.0,

97.8, 62.1, 61.3, 49.1, 45.6, 39.0, 33.7, 30.0, 29.7, 27.3, 27.1, 25.4,

21.2, 12.3. MS (FAB+): 562.3 (M⁺). HRMS (FAB+, NBA): calcd

for (M⁺+ 1) 562.2836; found 562.2859.

Potent Antimalarial 1,4-Naphthoquinones

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1275

were carried out in triplicate and were given with the 95%

conﬁdence limits.

mixture was held at 37 °C, and the amount of [³H]CQ accumulation

was monitored at 4 min. The intracellular CQ concentration was

calculated from the amount of [³H]CQ taken up by the cells and

by assuming that the volume of a trophozoite-infected erythrocyte

is 75 fL.⁹¹CQ accumulation was then expressed as the ratio of the

intracellular CQ concentration to the extracellular CQ concentration

(CQ_in/CQ_out). The data points were ﬁtted using previously described

equations,¹⁸which, for 2, assume the presence of a carrier and, for

34, a simple binding model.

Effect of Drug Combinations with Glutathione Reductase

Inhibitors on P. falciparum. Methylene blue was obtained from

Roth (Karlsruhe, Germany), CQ and amodiaquine were from Sigma-

Aldrich (Steinheim, Germany), meﬂoquine was from Roche (Man-

nheim, Germany), and artemisinin was from Aldrich Chem. Co.

(Milwaukee, WI). Artemisinin derivatives (artemether and artesu-

nate) and piperaquine tetraphosphate were kindly provided by the

Swiss Tropical Institute (Basel, Switzerland) and by Dr. J. Carl

Craft, Medicines for Malaria Venture (Geneva, Switzerland),

respectively. Isotopic drug sensitivity assays based on the semiau-

tomated microdilution technique⁸⁹were employed to study the

effects of selected GR inhibitors in combination with other clinically

used antimalarials. The method that depends on the incorporation

of radioactive [³H]hypoxanthine, which is taken up by the parasite

as a precursor of purine deoxynucleotides for DNA synthesis, was

performed according to reported modiﬁcations.⁹⁰In 96-well mi-

crotiter plates (Nunc), a 2-fold serial dilution of the starting

concentration of each drug to be tested was carried out. Two drugs

to be tested in combination were applied alone and in ﬁxed

concentration ratios of 1:1, 1:3, and 3:1 as described.⁹⁰Parasites

were incubated at a parasitemia of 0.125% (>70% ring forms) and

1.25% hematocrit in hypoxanthine-free medium. After 48 h, 0.5

µCi [³H]hypoxanthine was added into each well and the plates were

incubated for another 24 h. The cells of each well were harvested

on a glass ﬁber ﬁlter (Perkin-Elmer, Rodgau-Jügesheim, Germany),

washed, and dried. Their radioactivity in counts per minute was

considered to be proportional to the respective growth of P.

falciparum in the well. IC₅₀values (drug concentrations that produce

50% reduction in the uptake of [³H]hypoxanthine) and IC₉₀values

were calculated; the fractional inhibitory concentrations (FIC) of

the respective drugs were determined on the basis of the following

deﬁnitions: FIC₅₀(A) ) [IC₅₀(A + B)]/IC₅₀(A); FIC₅₀(B) ) [IC₅₀(B

+ A)]/IC₅₀(B); FIC₅₀) FIC₅₀(A) + FIC₅₀(B).⁹⁰

Acknowledgment. Drs. Jens Pfannstiel and Johannes Lech-

ner, Biochemistry Centre, Heidelberg University, are acknowl-

edged for the electrospray-ionization mass spectra of the

4-aminoquinolines derivatives. The excellent technical assistance

of Margit Brückner in performing the hematin polymerization

assays is gratefully acknowledged. Our work is supported by

CNRS-DFG program from the Centre National de la Recherche

Scientiﬁque (E.D.-C., W.F.) and by the Deutsche Forschungs-

gemeinschaft (Project SFB 544 “Control of Tropical Infectious

Diseases”, Project B14 (E.D.-C., W.F.), Project B12 (M.L.,

C.P.S.), and Grant BE 1540/4-4 (K.B.)). W.F. is CNRS Junior

Fellow from the CNRS-DFG program. This investigation

received ﬁnancial support from the UNICEF/UNDP/World

Bank/WHO Special Programme for Research and Training in

Tropical Diseases (TDR).

Supporting Information Available: X-ray diffraction analysis

of the 4-aminoquinoline 7, including summary of data collection,

tables of crystallographical and geometrical data, and elemental

analysis results of new compounds 2–5, 7, 12–15, 19–30, 32, 34,

36, and 40. This material is available free of charge via the Internet

at http://pubs.acs.org.

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JM7009292

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Antimalarial dual drugs based on potent inhibitors of glutathione reductase from Plasmodium falciparum

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