Discovery, synthesis, and optimization of antimalarial 4(1 H)-quinolone-3-diarylethers

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Discovery, Synthesis, and Optimization of Antimalarial

4(1H)‑Quinolone-3-Diarylethers

Aaron Nilsen,*^,‡Galen P. Miley,^‡Isaac P. Forquer,^‡Michael W. Mather,^§Kasiram Katneni,^∥Yuexin Li,^‡

Sovitj Pou,^‡April M. Pershing,^§Allison M. Stickles,^⊥Eileen Ryan,^∥Jane Xu Kelly,^‡J. Stone Doggett,^‡

Karen L. White,^∥David J. Hinrichs,^‡Rolf W. Winter,^‡Susan A. Charman,^∥Lev N. Zakharov,^#

Ian Bathurst,^∇,†Jeremy N. Burrows,^∇Akhil B. Vaidya,^§and Michael K. Riscoe*^,‡,○

^‡VA Medical Center, 3710 SW US Veterans Hospital Road, Portland, Oregon 97239, United States

^§Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia,

Pennsylvania 19129, United States

^∥Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052,

Australia

^⊥Department of Physiology and Pharmacology, Oregon Health & Science University, 3181 SW Sam Jackson Boulevard, Portland,

Oregon 97239, United States

^#Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1253, United States

^∇Medicines for Malaria Venture, 20 route de Pre-

́

Bois, PO Box 1826, 1215 Geneva 15, Switzerland

^○Department of Molecular Microbiology and Immunology, Oregon Health & Science University, 3181 Sam Jackson Boulevard,

Portland, Oregon 97239, United States

S

* Supporting Information

ABSTRACT: The historical antimalarial compound endochin served as a structural lead for optimization. Endochin-like

quinolones (ELQ) were prepared by a novel chemical route and assessed for in vitro activity against multidrug resistant strains of

Plasmodium falciparum and against malaria infections in mice. Here we describe the pathway to discovery of a potent class of

orally active antimalarial 4(1H)-quinolone-3-diarylethers. The initial prototype, ELQ-233, exhibited low nanomolar IC₅₀values

against all tested strains including clinical isolates harboring resistance to atovaquone. ELQ-271 represented the next critical step

in the iterative optimization process, as it was stable to metabolism and highly eﬀective in vivo. Continued analoging revealed that

the substitution pattern on the benzenoid ring of the quinolone core signiﬁcantly inﬂuenced reactivity with the host enzyme. This

ﬁnding led to the rational design of highly selective ELQs with outstanding oral eﬃcacy against murine malaria that is superior to

established antimalarials chloroquine and atovaquone.

contain and eliminate malaria from the many countries where it

remains endemic.²

INTRODUCTION

■

Malaria is a deadly disease that has plagued human civilization

down through recorded history and has been responsible for the

deaths of millions, particularly infants, young children, and

expectant mothers.¹While progress has been made over the past

decade to lessen the impact of the disease on global health,

widespread multidrug resistance threatens to reverse recent gains

brought about by the use of insecticide-treated bed nets and

artemisinin-combination therapies. New drugs that are active

against multiple stages of the Plasmodium parasite’s life cycle,

including transmissible forms in the host and mosquito vector,

will be important for pressing forward with a worldwide eﬀort to

Interest in the antimalarial properties of 3-alkyl-4(1H)-

quinolones dates to the 1940s and the pioneering research led

by Hans Andersag at Bayer IG Farbenindustrie in Elberfeld,

Germany.³Their work on this particular class of molecules

focused principally on endochin (see Figure 1) because it was

highly active against blood and tissue stages of avian malaria

(Plasmodium cathemerium in canaries), a preclinical model

Received: January 28, 2014

Published: April 10, 2014

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Figure 1. Lead optimization.

system in use at the time.⁴However, their attempts to

demonstrate eﬃcacy with endochin in humans failed. Recently

we showed that endochin is extensively metabolized (including

O-demethylation as well as hydroxylation of the ring nitrogen

and heptyl side chain) to poorly active metabolites by human

hepatic microsomal enzymes, thus providing one possible

explanation for the patient treatment failures over six decades ago.⁵

With intent to develop an endochin-like quinolone (ELQ) that

targets multiple stages of the malaria life cycle, we set out to

design derivatives with enhanced metabolic stability and potent

activity against drug resistant malaria. Our early work⁵showed

that endochin analogues with a halogen at the 6-position, such as

ELQ-130 (1) (Figure 1), were equipotent against Plasmodium

falciparum strains resistant to chloroquine, quinine, meﬂoquine,

and the folate antagonists, pyrimethamine and sulfadoxine, as

well as the antirespiratory compound atovaquone (Figure 2).

containing pyridones that, like atovaquone, mimic the structure

of coenzyme Q and target the Plasmodium cytochrome bc₁

complex.^7,8A late lead from this series, GW844520, appears in

Figure 2. Another clue to this modiﬁcation came from earlier

work by Fieser and colleagues, who described an antimalarial

hydroxynaphthoquinone containing a 3-alkyl-diphenylether side

chain (designated M-2309, Figure 2) with enhanced metabolic

stability.⁹We report here the discovery of 4(1H)-quinolone-3-

diarylethers and the in vitro and in vivo structure−activity

proﬁles that signiﬁcantly impacted the optimization path toward

the design and selection of a preclinical candidate with the

potential for prevention and treatment of malaria and for

interfering with disease transmission.

CHEMISTRY

■

Iodination of commercially available 4(1H)-quinolone 9a with

iodine in saturated aqueous potassium iodide solution and

n-butylamine provided the 3-iodo-4(1H)-quinolone 10a (Scheme 1).

Direct Suzuki−Miyaura reaction of 10a with 4-phenoxyphenyl

boronic acid resulted in an inseparable mixture of products. 4(1H)-

Quinolones suﬀer from low solubility in almost all solvents and are

thus diﬃcult to isolate chromatographically. Mphahlele and

Mtshemla showed that 3-aryl-4(1H)-quinolones could be prepared

via Suzuki−Miyaura reaction of 3-iodo-4-methoxyquinolones.¹⁰

Conversion of 10a to the corresponding 4-chloro-3-iodoquinoline

in neat phosphorus oxychloride followed by nucleophilic displace-

ment of the chloro substituent with sodium methoxide provided 3-

iodo-4-methoxyquinolone 11a. Suzuki−Miyaura reaction of 11a

with phenoxyphenylboronic acid in the presence of palladium

tetrakis triphenyphosphine (0) and aqueous potassium carbonate

provided 4-methoxyquinolone-3-diarylether 12a. Removal of the

methyl ether-protecting group with 3 equiv of BBr₃provided the

desired 4(1H)-quinolone-3-diarylether ELQ-233 (3) (Figure 1) in

excellent overall yield.

Figure 2. Chemical structures of compounds important to the design

and optimization of endochin analogues (ELQs).

Our studies revealed that ELQs eﬀectively shut down the parasite

respiratory processes within minutes following drug addition.⁶A

carbonate prodrug of a potent endochin analogue, 5,7-diﬂuoro-3-

heptyl-2-methylquinolin-4(1H)-one, was curative against patent

malaria infections in mice, albeit at relatively high doses of

50−100 mg/kg/day.⁵

At this point, we joined a research consortium supported by

the Medicines for Malaria Venture (Geneva, Switzerland) for

chemical optimization of endochin. We proposed to replace the

metabolically labile heptyl side chain at position 3 of endochin

with a substituted diarylether, taking a cue for this from Yeates

and co-workers,⁷who described a series of diphenylether

Because the lead compound, endochin, has a methoxy group at

the 7-position, it was important to demonstrate that the

quinolone 4-methoxy ether could be selectively cleaved in the

presence of an aryl methoxy moiety. Thus, the synthesis of

4(1H)-quinolone-3-diarylether ELQ-262 (13) was undertaken.

Condensation of commercially available 4-ﬂuoro-3-methoxyani-

line 8b and ethyl acetoacetate followed by thermal cyclization

provided 2-methyl-4(1H)-quinolone 9b via Conrad−Limpach

synthesis.¹¹Iodination using the method described above

followed by selective oxygen alkylation using methyl iodide

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a

Scheme 1

a

(a) (1) Ethyl acetoacetate, p-TsOH, benzene, reﬂux, (2) DOWTHERM A, 250 °C; (b) iodine, aq KI, n-butylamine, DMF; (c) MeI or EtI, K₂CO₃,

DMF; (d) 4-phenoxyphenyl boronic acid, Pd(PPh₃)₄, aq K₂CO₃, DMF, 85 °C; (e) BBr₃, CH₂Cl₂or 48% aq HBr, AcOH, 90 °C.

a

Scheme 2

a

(a) 4-Bromophenol, Cu(OAc)₂, DIPEA, pyridine, DCM; (b) Pd(dppf)₂Cl₂, bis(pinacolato)diboron, KOAc, DMF, 80 °C; (c) 16a, Pd(PPh₃)₄, aq

K₂CO₃, DMF, 85 °C; (d); 48% aq HBr, AcOH, 90 °C; (e) 11g, Pd(PPh₃)₄, aq K₂CO₃, DMF, 85 °C; (f) 48% aq HBr, AcOH, 90 °C.

and potassium carbonate provided the 3-iodo-4-methoxyquino-

lone 11b. 4-Methoxyquinolone-3-diarylether 12b was obtained

from 11b using the Suzuki reaction described above. However,

deprotection of 12b with 3 equiv of BBr₃resulted in the cleavage

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a

Table 1. Optimization of Benzenoid Ring Substituents

a

clogP values were calculated using ChemDraw Ultra software (version 12). EC₅₀values are the average of at least three determinations, each carried

out in triplicate. Cytotoxicity assays (IC₅₀values) were carried out with human hepatoma derived HepG2 cells and performed in triplicate across an

initial concentration range of 2.5 nM to 10 μM. Full details of each of these biological and biochemical assays can be found in the Experimental

Section. ND = not determined.

of both methyl ethers. Various selective deprotection methods

were evaluated by HPLC including less BBr₃(1.5 equiv),

reﬂuxing 48% aqueous HBr, and reﬂuxing 1:1 48% aqueous HBr

and acetic acid. However, it was not possible to selectively

deprotect the 4-methoxy position in the presence of the 7-

methoxy moiety. Considering that an ethyl group would form a

more stable carbocation intermediate under acidic cleavage

conditions compared to the corresponding methyl ether, we

prepared 4-ethoxyquinolone-3-diarylether 12c. Deprotection of

12c in 1:2 48% aqueous HBr and acetic acid at 90 °C gave

complete and selective conversion to 4(1H)-quinolone-3-

diarylether 13.

The boronic ester of the triﬂuoromethoxy diarylether side

chain 16a was then prepared (Scheme 2). Copper-mediated

coupling of commercially available 4-bromophenol and 4-

triﬂuoromethoxyphenyl boronic acid 14a with Hunig’s base

and pyridine aﬀorded the bromo diarylether 15a.¹²Palladium-

mediated coupling of 15a and bis(pinacolato) diboron with

potassium acetate gave the desired boronic ester 16a. A series of

benzenoid ring substitituted 4(1H)-quinolone-3-diarylethers

(Table 1) was prepared from the appropriate 3-iodo-4-ethoxy-

quinolones 11c−h and boronic ester 16a using the above

method. A series of 4(1H)-quinolone-3-diarylethers varying in

the side chain portion of the molecule (Table 3) was prepared

from 3-iodo-4-ethoxy-quinolone 11g and the appropriate

boronic esters 16b−g also using the above method.

Preparation of heterocyclic diarylethers using the copper (II)

acetate coupling described above proved problematic. However,

a series of heterocyclic diarylether compounds (22a−c and 28)

was prepared by coupling of commercially available aryl iodides

21a−c with 4-triﬂuoromethoxy phenol (Scheme 3) or by

coupling of 4-bromophenol with 1-iodo-4-triﬂuoromethylpyr-

idine (Scheme 4) in the presence of copper (I) iodide, picolinic

acid, and tribasic potassium phosphate.¹³A series of 4(1H)-

quinolone-3-diarylethers with heterocyclic side chains (Table 4)

was prepared using the above method.

RESULTS AND DISCUSSION

■

Here we describe some of the key compounds and assays that

helped to guide us through the discovery phase and to accelerate

the optimization process leading to highly active molecules of the

4(1H)-quinolone-3-diarylether class of antimalarials. At the

outset of research activities with the Medicines for Malaria

Venture, the primary challenges to endochin optimization were

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Table 2. Optimization of Benzenoid Ring Substituents: Metabolic Stability and in Vivo Eﬃcacy

a

Detailed procedures for assay of microsomal stability and for in vivo assessment of antimalarial activity against P. yoelii infected mice can be found in

the Experimental Section. ND = not determined.

clear: (1) enhance metabolic stability, (2) enhance aqueous

solubility, (3) eliminate cross-resistance in clinical strains of

P. falciparum, and (4) diminish the propensity for resistance, i.e.,

relative to atovaquone for which resistance arises quickly.

Early in the analoging work we obtained X-ray diﬀraction

analysis of the crystal structure for the endochin derivative

5,7-diﬂuoro-3-heptyl-2-methylquinolin-4(1H)-one.⁵The crystal

structure revealed extensive π−π stacking in the Z plane and

an extensive network of intermolecular H-bonds in the X−Y

plane (see Supporting Information Figure S1). A decision was

made to place an aryl group at position 3 because the adjacent

2-position CH₃would force an out-of-plane movement of the

bulky aromatic ring, thereby potentially disrupting π−π interac-

tions and reducing the crystal lattice energy. We chose the

lipophilic diphenylether moiety because it was a key structural

element for the highly active antimalarial GSK pyridones^7,14

and earlier 2-hydroxy-naphthoquinones from Fieser’s research

program.^9,15

All of the compounds were evaluated for parasite growth

inhibitory activity in vitro by a microplate-based assay in which

SyBr Green 1 ﬂuorescent dye was utilized to quantitate parasite

double stranded DNA following a 72 h incubation period.¹⁶In

vivo eﬃcacy was determined in a murine Plasmodium yoelii model

in which animals were randomly placed in groups of four and

administered test drugs by oral gavage on four sequential days

following the day of inoculation. The in vivo data are expressed

as ED₅₀values and reﬂect the dose (estimated from dose−

response curves) for suppression of parasitemia by 50% relative

to vehicle-only controls as assessed on day 5 of each study. Drug

treated animals that were parasite free on day 30 of the

experiment are deﬁned as “cures”, and the amount of drug that

was needed to achieve a cure is referred to as the “non-

recrudescence dose” (NRD).

Compounds 3 and 13 were prepared as described above from

commercially available phenoxyphenylboronic acid. Unsubsti-

tuted in the benzenoid ring of the quinolone core, compound 3,

the ﬁrst compound in this subseries, showed equipotency (EC₅₀

values 10 nM) against all three tested strains including multidrug

resistant strains and an atovaquone-resistant clinical isolate

Tm90-C2B (Table 1). Compound 13, with a ﬂuorine atom at

position 6 and a methoxy group at position 7, also exhibited

impressive low nanomolar EC₅₀values against all three strains.

Both of these compounds were metabolically unstable in the

presence of rat and human liver microsomes (Table 2) with

in vitro degradation half-lives of 10.6−92.7 min and microsome-

predicted hepatic extraction ratios (E_H) in the intermediate

to high range of 0.5−0.8. In contrast, there was no detectable

degradation in the microsomal matrix with ELQ-271 (4)

(Figure 1), and the intrinsic antiplasmodial activity was enhanced

about 2-fold over its progenitors. Clearly, the introduction of an

OCF₃group at the para position of the outermost ring of the

diarylether side chain stabilized the drug against liver derived

microsomal enzymes.

The eﬀect of substituents on the benzenoid ring of the

quinolone core was further explored to optimize the scaﬀold for

potency and lack of cross-resistance, focusing especially on the

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a

Table 3. Optimization of Diarylether Side Chain: Substituents

a

clogP values were calculated using ChemDraw Ultra software (version 12). EC₅₀values are the average of at least three determinations, each carried

out in triplicate. Cytotoxicity assays (IC₅₀values) were carried out with human hepatoma derived HepG2 cells and performed in triplicate across an

initial concentration range of 2.5 nM to 10 μM. Full details of each of these biological and biochemical assays can be found in the Experimental

Section. ND = not determined.

a

Scheme 3

a

(a) 4-Triﬂuoromethoxy phenol, CuI, picolinic acid, K₃PO₄, DMSO, 80 °C; (b) Pd(dppf)₂Cl₂, bis(pinacolato)diboron, KOAc, DMF, 80 °C;

(c) 11g, Pd(PPh₃)₄, aq K₂CO₃, DMF, 85 °C; (d) 48% aq HBr, AcOH, 90 °C; (e) 19a, Pd(PPh₃)₄, aq K₂CO₃, DMF, 85 °C; (f) 48% aq HBr, AcOH,

90 °C.

atovaquone resistant isolate. On the basis of earlier work by

Andersag relating to endochin (which exhibits modest cross

resistance vs Tm90-C2B), our earlier discovery of 6-position

halogenated variants 1 and 6-ﬂuoro-3-heptyl-2-methylquinolin-

4(1H)-one (i.e., lack of atovaquone cross resistance),⁵as well as

the 6-Cl/7-OCH₃combination provided by WR-109,878 (2) as

published previously by Cross et al.¹⁷(and also lacking

atovaquone cross resistance), we completed the synthesis of

ELQ-296 (5), ELQ-298 (6), ELQ-300 (7), ELQ-314 (18a), and

ELQ-316 (18b) (Scheme 2). In vitro microsomal assays showed

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Scheme 4

a

(a) CuI, picolinic acid, K₃PO₄, DMSO, 80 °C; (b) Pd(dppf)₂Cl₂, bis(pinacolato)diboron, KOAc, DMF, 80 °C; (c) 11g, Pd(PPh₃)₄, aq K₂CO₃,

DMF, 85 °C; (d) 48% aq HBr, AcOH, 90 °C.

a

Table 4. Optimization of Diarylether Side Chain: N-Heterocycles

a

clogP values were calculated using ChemDraw Ultra software (version 12). EC₅₀values are the average of at least three determinations, each carried

out in triplicate. Cytotoxicity assays (IC₅₀values) were carried out with human hepatoma derived HepG2 cells and performed in triplicate across an

initial concentration range of 2.5 nM to 10 μM. Full details of each of these biological and biochemical assays can be found in the Experimental

Section. ND = not determined.

that 6 was metabolized at a moderate to intermediate rate in both

rat and human hepatic microsomal mixtures, while the other

compounds in this set were highly stable to metabolism. All of

these molecules exhibited low nanomolar EC₅₀values against

P. falciparum strains D6, Dd2, and Tm90-C2B. It is noteworthy

that compound 7 was also highly active against P. falciparum

strain Tm93-C1088 with an EC₅₀value of 1.0 nM (see

Supporting Information Table S2). Like Tm90-C2B, this

parasite¹⁸was isolated from a Thai patient who experienced a

recrudescence of parasitemia after treatment with atovaquone.

The inhibitory eﬀect of this subseries of molecules was highly

speciﬁc, as evidenced by the lack of cytotoxicity exhibited by any

member of the set against proliferating HepG2 cells (a human

hepatoma cell line) at concentrations as high as 10 μM.

observation that it is metabolically unstable, endochin was

inactive in vivo while the estimated ED₅₀value for chloroquine

was 2.2 mg/kg/day (CQ was not curative in this model even at

doses as high as 64 mg/kg/day). With the minimum structural

elements needed for potency and metabolic stability, 4 showed a

dramatic enhancement of in vivo eﬃcacy over endochin with an

ED₅₀value of 0.1 mg/kg/day and parasite-free cures, established

on day 30, at 3 mg/kg/day. The 6-chloro analogue (5) was much

less eﬀective than 4, which may relate to low aqueous solubility

and poor oral bioavailability. Compound 7 (delivery vehicle:

PEG400), with the 6-Cl/7-OCH₃substitution pattern, provided

excellent in vivo values against P. yoelii in mice with ED₅₀and

ED₉₀values of 0.02 and 0.06 mg/kg/day, respectively, and a

nonrecrudescence cure dose of 0.3 mg/kg/day. It is noteworthy

that 7 was shown previously to be 30-fold more eﬀective than

atovaquone against murine malaria in side-by-side tests.¹⁹The

6-F and 6-F/7-OCH₃analogues (i.e., 18a and 18b) within this

series, with reduced cLogP values relative to 7, were also highly

The in vivo antimalarial eﬃcacy of this initial series of ELQ

derivatives is summarized in Table 2 and compared to endochin,

the original lead, as well as chloroquine, a 4-aminoquinoline

antimalarial drug. Consistent with earlier reports and our

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Table 5. In Vivo Activity of Selected ELQs

a

ND = not determined.

active in vitro and stable to metabolism. However, they were

somewhat less eﬃcacious against malaria in mice, which may

relate to diﬀerences in pharmacokinetics.

It is important to note that a prodrug of the GSK pyridone

progressed until toxicology results revealed unexpected acute

toxicity in rats that was putatively linked to inhibition of

mammalian cytochrome bc₁complex.¹⁴Because of the close

structural similarity between the pyridone and 4(1H)-quinolone-

diarylethers and out of concern for potential host toxicity, each of

the 4(1H)-quinolone-3-diarylethers in the original set (4−7 and

18a−b) was screened for inhibition of human cytochrome bc₁

complex (derived from HEK-293 cells) activity (Figure 3). An

interesting SAR trend emerged from the in vitro inhibition

proﬁle. For 4, in which each position of the core benzenoid ring is

occupied by an H atom, the IC₅₀level against host cytochrome

bc₁was 1.85 μM. ELQs with a 6-position halogen atom (e.g.,

compound 5 and 18a) or a −OCH₃group in position 7 (e.g.,

compound 6), or both (e.g., compounds 7 and 18b), were not

inhibitory toward the human enzyme at concentrations as high as

10 μM. Evaluation of 7 for inhibition of P. falciparum cytochrome

bc₁complex provided an EC₅₀value of 0.56 nM, thereby

highlighting the remarkable parasite selectivity (≥20000-fold) of

this analogue as well as its superiority over 4 (IC₅₀= 8.9 nM) and

atovaquone (IC₅₀= 2 nM) as inhibitory molecules in this assay.¹⁹

Overall, we observed that the degree of host vs parasite selectivity

increased as the size of the core increased from the pyridone to

the quinolone ring system. We assume that this is due to steric

hindrance in binding of the larger quinolone core to the more

restrictive Q_isite of the human cytochrome bc₁. The evidence

further shows that addition of bulky substituents to the 6- or 7-

position widens the selective advantage and minimizes the

potential developmental risk associated with the inhibition of

host cytochrome bc₁complex.

Figure 3. Inhibition of P. falciparum cytochrome bc₁complex by 4

(squares) and 7 (circles).

Other structural variations were explored including the meta-

OCF₃congener of 7. This compound, ELQ-307 (20a), exhibited

spectacular activity in vitro against P. falciparum; however, it was

metabolically unstable in the presence of hepatic microsomes

and ineﬀective in vivo. In an attempt to capture the high intrinsic

activity of 20a in a metabolically stable form, we prepared ELQ-

352 (20e), which added a ﬂuoro substituent ortho to the

triﬂuoromethoxy group in 20a. While 20e had an excellent

antiplasmodial proﬁle with subnanomolar EC₅₀values against all

three reference strains, it was both less potent than 20a in vitro

and far less eﬀective than 7 in vivo. This result indicated that

there is sensitivity to the SAR in this region, and the ﬂuoro

substituent diminished exposure and eﬃcacy as a consequence.

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To demonstrate this, ELQ-353 (20f) was prepared, which added

a meta-ﬂuoro substituent to 7. With decreased in vitro potency

and in vivo eﬃcacy relative to 7, compound 20f conﬁrmed the

ﬁndings that even a small modiﬁcation to 7 results in a decrease

in activity.

reaction conditions that would allow for the selective

deprotection of 4-O-methylether quinoline intermediates in

the presence of an essential core methoxy group, we devised a

successful route through the corresponding 4-O-ethylethers. The

parallel convergent approach that is described is straightforward,

utilizes inexpensive and readily available reagents, yields the

desired quinolone in high purity (>99%), and is amenable to

large-scale production. All of these factors are important given

that antimalarial drugs need to be inexpensive so that they can be

made available to populations in need in the developing world

where the disease is endemic and resources are limited.

One of the greatest challenges to the clinical development of 7

for human use against malaria is the identiﬁcation of a clinical

formulation that improves exposures at higher doses needed to

establish safety and tolerability in preclinical species and humans.

Higher doses are also needed to establish whether and if it would

be feasible to use 7 to achieve one-dose cures of falciparum

malaria. Conventional formulation technologies such as particle

size reduction,²²use of solubilizing excipients or complexation

agents,²³as well as the development of amorphous spray-dried

dispersions²⁴and prodrugs²⁵are all part of an ambitious program

of research that is currently underway to resolve this issue so that

the clinical potential for this new drug can be fully explored.

Finally, the 4(1H)-quinolone-3-diarylether series described

here includes molecules with a high selectivity index against the

parasite mitochondrial electron transport chain. Potent against

all life cycle stages of P. falciparum including infections in the liver

and bloodstream and with particular sensitivity exhibited by

developing forms within the mosquito vector, 7 has been selected

as a preclinical candidate.²⁰In preclinical animal studies, 7

demonstrated nonlinear pharmacokinetics, i.e., at low doses

required for a therapeutic eﬀect oral bioavailability was good to

excellent from the PEG400 solution formulation, however, it fell

oﬀ rapidly at higher doses, presumably due to solubility limited

absorption.¹⁹A focused medicinal chemistry plan was developed

to address the poor aqueous solubility and oral bioavailability

issues that limit exposures of 7 at higher doses that are needed to

establish a therapeutic safety window. Side chain analogues with

reduced cLogP values in which the terminal OCF₃moiety was

replaced by CF₃(ELQ-309, 20b), Cl (ELQ-323, 20c), and F

(ELQ-333, 20d) exhibited impressive antiplasmodial activity

with EC₅₀values in the low to subnanomolar range. In vitro tests

revealed that they were metabolically stable in the presence of

human and rat microsomes. The introduction of nitrogen atoms

in the form of pyridyl or pyrimidyl rings at either the inner or

outermost rings of the diarylether yielded ELQs with excellent,

e.g., ELQs 310 (25a) and 313 (25b), to poor ELQ-329 (25c), in

vitro activity against P. falciparum. Unfortunately, none of the

compounds that were selected for further study from this series

(25a and 20c) provided a signiﬁcant enhancement over the

blood levels achieved with 7 at oral doses of 1 and 10 mg/kg, and

they were at least 3−10 times less eﬀective than 7 against murine

malaria.

EXPERIMENTAL SECTION

■

General Chemistry. Anhydrous solvents and reagents were

purchased from various ﬁne chemical suppliers and were used without

further puriﬁcation. Inert atmosphere operations were conducted under

argon in ﬂame-dried glassware. ¹H NMR spectra were taken on a Bruker

400 MHz instrument. Data reported were calibrated to internal TMS

(0.0 ppm) for all solvents and are reported as follows: chemical shift,

multiplicity (bs, broad singlet; s, singlet; d, doublet; t, triplet; q, quartet;

and m, multiplet), coupling constant, and integration. High-resolution

mass spectrometry (HRMS) using electrospray ionization was

performed by the Portland State University BioAnalytical Mass

Spectrometry Facility. Final compounds were judged to be >95% pure

by HPLC analysis using an HP1100 HPLC at 254 nm with Phenomenex

Luna C8(2) reverse phase column (5 mm, 50 mm × 2 mm i.d) at 40 °C

and eluted with methanol/water with 0.5% TFA and acetonitrile/water

with 0.5% TFA at 0.4 mL/min. Further information is provided in the

Supporting Information accompanying this report in which we have

employed this separation system to characterize the relative hydro-

phobicity for each of the synthesized 4(1H)-quinolone-3-diarylethers

(i.e., retention time) and correlated these results to cLogP values

calculated with ChemDraw Ultra software (Cambridgesoft, version 12).

General Procedure A. A solution of aniline (63.5 mmol), ethyl

acetoacetate (63.5 mmol), and catalytic para-toluene sulfonic acid

(1.59 mmol) in 65 mL of benzene was stirred 6 h at reﬂux with a Dean−

Stark trap. The reaction mixture was then concentrated in vacuo and the

resulting Schiﬀ base added to 65 mL of boiling (250 °C) DOWTHERM

A and stirred 20 min at 250 °C. The reaction mixture was cooled to

room temperature. After trituration with ethyl acetate, the product

4(1H)-quinolone was collected by ﬁltration.

SUMMARY AND CONCLUDING REMARKS

■

Our interest in endochin derived from earlier work on the larger

acridone scaﬀold.²¹The hydrolysis of quinacrine produced an

acridone (3-chloro-7-methoxyacridone) with impressive anti-

plasmodial and antirespiratory properties, and this same

molecule also served as the catalyst for Hans Andersag’s

optimization program that spotlighted endochin for its

pronounced eﬀectiveness against multiple stages of malaria

infection.³Our eﬀorts to chemically alter the structure of

endochin to enhance its clinical utility have shown that additions

to the benzenoid ring of the quinolone core have the greatest

inﬂuence on intrinsic antiplasmodial activity, cross-resistance,

and selective inhibition of host and parasite cytochrome bc₁

complexes. The 6-Cl/7-OCH₃substitution pattern yielded

analogues that were not cross resistant to atovaquone, a highly

desirable attribute given that atovaquone is a key component of a

clinical formulation with proguanil that is used in treatment of

malaria. Replacement of endochin’s extended alkyl group with a

diphenylether at position 3 resulted in >1000-fold greater

eﬃcacy in vivo compared to the original lead molecule.

Presumably, the overall boost in antimalarial eﬃcacy in going

from endochin to 7 is due to the presence of the 3-position

diphenylether side chain that imparts enhanced metabolic

stability while maintaining acceptable, albeit low, aqueous

solubility.¹⁹

General Procedure B. To a stirred solution of 4(1H)-quinolone

(62.2 mmol) and n-butylamine (622 mmol) in dimethylformamide

(125 mL) cooled by a room temperature water bath was added iodine

(62.2 mmol) in a saturated solution of aqueous potassium iodide

(62 mL). The reaction mixture was stirred 12 h at room temperature.

Residual iodine was quenched with excess 0.1 M aqueous sodium

thiosulfate, and the resulting solution was concentrated in vacuo. The

residue was resuspended in water and ﬁltered to give the product 3-iodo-

4(1H)-quinolone.

General Procedure C. To a stirred solution of 3-iodo-4(1H)-

quinolone (5.72 mmol) in dimethylformamide (57 mL) was added

potassium carbonate (11.4 mmol) at room temperature. The resulting

suspension was stirred 0.5 h at 50 °C. Ethyl iodide (8.58 mmol) was

added dropwise at room temperature, and the reaction mixture was

In summary, we discovered a novel route to synthesis of 3-

diarylether substituted 4(1H)-quinolones. Unable to ﬁnd

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stirred 8 h at 50 °C. The solvent was removed in vacuo, and the resulting

residue was resuspended in ethyl acetate and water and ﬁltered. The

organic layer was extracted with brine, dried over magnesium sulfate,

and concentrated in vacuo to give the product 3-iodo-4(1H)-quinolone

O-ethyl ether.

(400 MHz, DMSO-d₆) δ 11.65 (s, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.64

(td, J = 7.6, 1.4 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.5 Hz,

2H), 7.27−7.31 (m, 3H), 7.15−7.18 (m, 2H), 7.07−7.09 (m, 2H), 2.27

+

(s, 3H). HRMS (ESI) m/z for [C₂₃H₁₇F₃NO₃]: calculated 412.1155,

found 412.1158.

General Procedure D. Using a method adapted from Hart et al.,¹²

to a solution of boronic acid (36.5 mmol) and 4-bromophenol

(24.3 mmol) in dichloromethane (250 mL) over heat-activated 3 Å

molecular sieves was added copper (II) acetate (24.3 mmol),

diisopropylethylamine (121 mmol), and pyridine (121 mmol). The

reaction mixture was stirred 12 h at room temperature under positive

pressure of dry air and concentrated in vacuo. The resulting residue was

resuspended in ethyl acetate and water. The organic layer was extracted

with 0.5 M HCl and brine, dried over magnesium sulfate, and

concentrated in vacuo. Puriﬁcation by silica gel chromatography (ethyl

acetate/hexanes) provided product bromo diaryl ether.

General Procedure E. Using a method adapted from Maiti and

Buchwald,¹³to a ﬂask containing aryl iodide (5.00 mmol), phenol

(6.00 mmol), copper (I) iodide (0.500 mmol), picolinic acid

(1.00 mmol), and potassium phosphate (10.0 mmol) was added dry

DMSO (10 mL). The reaction mixture was heated to 80 °C, stirred for

18 h, and allowed to cool. The reaction was then diluted with ethyl

acetate (50 mL) and water (5 mL) and separated. The aqueous phase

was extracted with ethyl acetate (2 × 50 mL). The combined organic

phases were treated with brine, dried over magnesium sulfate, ﬁltered,

and concentrated in vacuo. The resulting residue was puriﬁed by ﬂash

chromatography (ethyl acetate/hexanes).

General Procedure F. To a solution of bromo diaryl ether

(33.0 mmol) in DMF (130 mL) over heat-activated 3 Å molecular sieves

(13 g) was added 1,1′-bis(diphenylphosphino)ferrocene dichloropalla-

dium (II) (1.65 mmol), bis(pinacolato) diboron (36.3 mmol), and

potassium acetate (99.0 mmol). The reaction mixture was heated to

80 °C, stirred for 18 h, cooled, and ﬁltered. The ﬁltrate was concentrated

in vacuo, resuspended in ethyl acetate and water, and separated. The

organic layer was extracted with brine and dried over magnesium sulfate.

Puriﬁcation by silica gel chromatography (ethyl acetate/hexanes)

provided the product boronic ester.

General Procedure G. To a solution of 3-iodo-4(1H)-quinolone

O-ethyl ether (3.35 mmol), boronic acid/ester (5.02 mmol), and

palladium (0) tetrakis triphenylphosphine (0.168 mmol) in degassed

dimethylformamide (17 mL) was added 6.7 mL of a 2 N aqueous

potassium carbonate solution. The reaction mixture was stirred 18 h at

85 °C, ﬁltered through celite, and concentrated in vacuo. The resulting

residue was resuspended in ethyl acetate and water and separated. The

organic layer was extracted with brine, dried over magnesium sulfate,

and concentrated in vacuo. Puriﬁcation by silica gel chromatography

(ethyl acetate/dichloromethane) provided the product 4(1H)-quino-

lone O-ethyl ether.

6-Chloro-2-methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)quinolin-4(1H)-one (5). The title compound was prepared

from 6-chloro-4-ethoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl)quinoline 17b according to general procedure H.

Yield: 73%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 8.00−8.03

(m, 1H), 7.66−7.70 (m, 1H), 7.57−7.61 (m, 1H), 7.39−7.45 (m, 2H),

7.28−7.32 (m, 2H), 7.15−7.20 (m, 2H), 7.06−7.11 (m, 2H), 2.27 (s,

+

3H). HRMS (ESI) m/z for [C₂₃H₁₆ClF₃NO₃]: calculated 446.0765,

found 446.0766.

7-Methoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)quinolin-4(1H)-one (6). The title compound was prepared

from 4-ethoxy-7-methoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl)quinoline 17c according to general procedure H.

Yield: 40%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.65 (s, 1H), 8.09 (d, J =

7.2 Hz, 1H), 7.64 (td, J = 7.6, 1.4 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.42

(d, J = 8.5 Hz, 2H), 7.27−7.31 (m, 3H), 7.15−7.18 (m, 2H), 7.07−7.09

+

(m, 2H), 2.27 (s, 3H). HRMS (ESI) m/z for [C₂₄H₁₉F₃NO₄]:

calculated 442.1261, found 442.1263.

6-Chloro-7-methoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl) quinolin-4(1H)-one (7). The title compound was

prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-(4-

(triﬂuoromethoxy)phenoxy)phenyl) quinoline 17d according to

1

general procedure H. Yield: 84%. H NMR (400 MHz, CDCl₃) δ

11.97 (s, 1H), 8.05 (s, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.3 Hz,

2H), 7.17 (d, J = 8.3 Hz, 2H), 7.12 (s, 1H), 7.08 (d, J = 8.7 Hz, 2H), 3.97

(s, 3H), 2.26 (s, 3H). HRMS (ESI) m/z for [C₂₄H₁₇ClF₃NO₄]:

calculated 475.0798, found 475.0801.

6-Fluoro-7-methoxy-2-methylquinolin-4(1H)-one (9b). The

title compound was prepared from commercially available 4-ﬂuoro-3-

1

methoxyaniline 8b according to general procedure A. Yield: 51%. H

NMR (400 MHz, DMSO-d₆) δ 12.21 (bs, 1H), 7.62 (d, J = 11.8 Hz,

1H), 7.02 (d, J = 7.4 Hz, 2H), 5.81 (s, 1H), 3.89 (s, 3H), 2.28 (s, 3H).

6-Chloro-2-methylquinolin-4(1H)-one (9c). The title compound

was prepared from commercially available 4-chloroaniline 8c according

to general procedure A. Yield: 34%. ¹H NMR (400 MHz, DMSO-d₆) δ

11.72 (bs, 1H), 7.67 (dd, J = 8.9, 2.5 Hz, 2H), 7.48 (d, J = 8.7 Hz, 1H),

7.35 (d, J = 8.9 Hz, 1H), 6.37 (s, 1H), 2.06 (s, 3H), 1.86 (s, 1H).

7-Methoxy-2-methylquinolin-4(1H)-one (9d). The title com-

pound was prepared from commercially available 3-methoxyaniline 8d

1

according to general procedure A. Yield: 36%. H NMR (400 MHz,

DMSO-d₆) δ 11.78 (s, 1H), 7.89 (d, J = 9.5 Hz, 1H), 7.35−7.49 (m, 1H),

6.85 (dq, J = 4.6, 2.4 Hz, 1H), 5.79 (s, 1H), 3.81 (s, 3H), 2.34 (s, 1H),

2.27 (s, 3H).

6-Chloro-7-methoxy-2-methylquinolin-4(1H)-one (9e). The

General Procedure H. To a solution of 4(1H)-quinolone O-ethyl

ether (2.30 mmol) in acetic acid (10 mL) was added a 50% aqueous

hydrobromic acid solution (5 mL). The reaction mixture was stirred

24 h at 90 °C, cooled, and neutralized with a saturated potassium

hydroxide solution. The product 4(1H)-quinolone-3-diaryl ether was

triturated in ethyl acetate and recrystallized from DMF/methanol.

2-Methyl-3-(4-phenoxyphenyl)quinolin-4(1H)-one (3). To a

solution of 4-methoxy-2-methyl-3-(4-phenoxyphenyl)quinoline 12a

(1.60 mmol) in anhydrous dichloromethane (12 mL) was added

boron tribromide (0.23 mL) at room temperature. The reaction mixture

was stirred 18 h at room temperature, quenched with 10 mL of water,

and ﬁltered. The ﬁlter cake was triturated in ethyl acetate and

recrystallized from methanol to give the title compound as a ﬂaky oﬀ-

title compound was prepared from commercially available 4-chloro-3-

1

methoxyaniline 8e according to general procedure A. Yield: 60%. H

NMR (400 MHz, DMSO-d₆) δ 11.54 (bs, 1H), 7.94 (s, 1H), 7.02 (s,

1H), 5.86 (s, 1H), 3.94 (s, 3H), 2.31 (s, 3H).

6-Fluoro-2-methylquinolin-4(1H)-one (9f). The title compound

was prepared from 4-ﬂuoroaniline 8f according to general procedure A.

Yield: 20%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.73 (s, 1H), 7.68 (dd,

J = 9.3, 2.8 Hz, 1H), 7.46−7.62 (m, 2H), 5.93 (s, 1H), 2.35 (s, 3H).

4-Hydroxy-3-iodo-2-methylquinolone (10a). The title com-

pound was prepared from commercially available 4-hydroxy-2-

methylquinolone 9a according to general procedure B. Yield: 99%. ¹H

NMR (400 MHz, DMSO-d₆) δ 12.13 (1H, bs), 8.08 (1H, d, J = 8.3 Hz),

7.68 (1H, m), 7.55 (1H, d, J = 8.3 Hz), 7.35 (1H, m), 2.65 (3H, s).

6-Fluoro-3-iodo-7-methoxy-2-methylquinolin-4(1H)-one

(10b). The title compound was prepared from 6-ﬂuoro-7-methoxy-2-

methylquinolin-4(1H)-one 9b according to general procedure B.

1

white solid (314 mg, 60% yield). H NMR (400 MHz, DMSO-d₆) δ

12.87 (s, 1H), 8.20−8.25 (m, 1H), 7.74−7.83 (m, 2H), 7.39−7.52 (m,

3H), 7.33 (d, J = 8.3 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 7.04−7.12 (m,

+

4H), 2.37 (s, 3H). HRMS (ESI) m/z for [C₂₂H₁₈NO₂]: calculated

1

Yield: 42%. H NMR (400 MHz, DMSO-d₆) δ 12.11 (bs, 1H), 7.70

328.1332, found 328.1329.

(d, J = 11.7 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H), 3.95 (s, 3H), 2.61 (s, 3H).

6-Chloro-3-iodo-2-methylquinolin-4(1H)-one (10c). The title

compound was prepared from 6-chloro-2-methylquinolin-4(1H)-one

9c according to general procedure B. Yield: 48%. ¹H NMR (400 MHz,

2-Methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)phenyl)-

quinolin-4(1H)-one (4). The title compound was prepared from

4-ethoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)phenyl)-

quinoline 17a according to general procedure H. Yield: 84%. ¹H NMR

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1

DMSO) δ 8.02 (dd, J = 7.9, 2.3 Hz, 1H), 7.71 (dd, J = 8.8, 2.5 Hz, 1H),

7.60 (d, J = 8.8 Hz, 1H), 2.64 (s, 3H).

procedure C. Yield: 99%. H NMR (400 MHz, CDCl₃) δ 7.99 (s,

1H), 7.40 (s, 1H), 4.19 (q, J = 7.1 Hz, 2H), 4.02 (s, 3H), 2.92 (s, 3H),

1.61 (t, J = 7.1 Hz, 3H).

3-Iodo-7-methoxy-2-methylquinolin-4(1H)-one (10d). The

title compound was prepared from 7-methoxy-2-methylquinolin-

4(1H)-one 9d according to general procedure B. Yield: 88%. ¹H

NMR (400 MHz, DMSO-d₆) δ 11.96 (bs, 1H), 7.97 (d, J = 8.91 Hz,

1H), 6.91−6.96 (m, 2H), 3.86 (s, 3H), 2.60 (s, 3H).

6-Chloro-3-iodo-7-methoxy-2-methylquinolin-4(1H)-one

(10e). The title compound was prepared from 6-chloro-7-methoxy-2-

methylquinolin-4(1H)-one 9e according to general procedure B. Yield:

89%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.65 (bs, 1H), 7.59 (s, 1H),

6.41 (s, 1H), 3.91 (s, 3H), 2.18 (s, 3H).

4-Ethoxy-6-ﬂuoro-3-iodo-2-methylquinoline (11h). The title

compound was prepared from 6-ﬂuoro-3-iodo-2-methylquinolin-

4(1H)-one 10f according to general procedure C. Yield: 69%. ¹H

NMR (400 MHz, CDCl₃) δ 8.00 (dd, J = 9.2, 5.2 Hz, 1H), 7.60 (dd, J =

9.2, 2.8, 1H), 7.45 (ddd, J = 9.1, 8.3, 2.8 Hz, 1H), 4.20 (q, J = 7.0 Hz,

2H), 2.94 (s, 3H), 1.62 (t, J = 7.0 Hz, 3H).

4-Methoxy-2-methyl-3-(4-phenoxyphenyl)quinoline (12a).

The title compound was prepared from 3-iodo-4-methoxy-2-methyl-

quinoline 11a according to general procedure G. Yield: 80%. ¹H NMR

(400 MHz, DMSO) δ 8.09 (dd, J = 8.3, 1.0 Hz, 1H), 7.96 (d, J = 8.3 Hz,

1H), 7.74 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.58 (ddd, J = 8.1, 6.9, 1.1 Hz,

1H), 7.37−7.49 (m, 4H), 7.16−7.23 (m, 1H), 7.09−7.15 (m, 4H), 3.58

(s, 3H), 2.42 (s, 3H).

6-Fluoro-4,7-dimethoxy-2-methyl-3-(4-phenoxyphenyl)-

quinoline (12b). The title compound was prepared from 6-ﬂuoro-

3-iodo-4,7-dimethoxy-2-methylquinoline 11b according to general

procedure G. Yield: 84%. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J =

11.8 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.35−7.44 (m, 2H), 7.29−7.35

(m, 2H), 7.20−7.08 (m, 5H), 4.03 (s, 3H), 3.57 (s, 3H), 2.50 (s, 3H).

4-Ethoxy-6-ﬂuoro-7-methoxy-2-methyl-3-(4-phenoxyphenyl)-

quinoline (12c). The title compound was prepared from 4-ethoxy-6-

ﬂuoro-3-iodo-7-methoxy-2-methylquinoline 11c according to general

procedure G. Yield: 91%. ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J =

11.8 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.4, 7.5 Hz, 2H),

7.27−7.33 (m, 3H), 7.07−7.13 (m, 4H), 4.03 (s, 3H), 3.70 (q, J = 7.0

Hz, 2H), 2.50 (s, 3H), 1.18 (t, J = 7.0 Hz, 3H).

6-Fluoro-7-methoxy-2-methyl-3-(4-phenoxyphenyl)-

quinolin-4(1H)-one (13). The title compound was prepared from 4-

ethoxy-6-ﬂuoro-7-methoxy-2-methyl-3-(4-phenoxyphenyl)quinoline

12c according to general procedure H. Yield: 89%. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.69 (s, 1H), 7.70 (d, J = 11.8 Hz, 1H), 7.42 (t, J = 8.0 Hz,

2H), 7.25 (d, J = 8.6 Hz, 2H), 6.96−7.20 (m, 6H), 3.95 (s, 3H), 2.24 (s,

3H). HRMS (ESI) m/z for [C₂₃H₁₈FNO₃]: calculated 375.1271, found

375.1270.

1-Bromo-4-(4-(triﬂuoromethoxy)phenoxy)benzene (15a).

The title compound was prepared from commercially available 4-

(triﬂuoromethoxy)phenylboronic acid 14a according to general

procedure D. Yield: 63%. ¹H NMR (400 MHz, CDCl₃) δ 7.45 (d, J =

9.3 Hz, 1H), 7.19 (d, J = 8.9 Hz, 1H), 6.99 (d, J = 9.3 Hz, 1H), 6.89

(d, J = 8.9 Hz, 1H).

1-(4-Bromophenoxy)-3-(triﬂuoromethoxy)benzene (15b).

The title compound was prepared from commercially available 3-

(triﬂuoromethoxy)phenylboronic acid 14b according to general

procedure D. Yield: 69%. H NMR (400 MHz, CDCl₃) δ 7.16−7.69

(m, 2H), 6.54−7.14 (m, 2H), 1.54 (s, 2H), 1.26 (s, 2H).

1-Bromo-4-(4-(triﬂuoromethyl)phenoxy)benzene (15c). The title

compound was prepared from commercially available 4-(triﬂuoromethyl)-

phenylboronic acid 14c according to general procedure D. Yield: 62%.

¹H NMR (400 MHz, CDCl₃) δ 7.37−8.00 (m, 2H), 6.68−7.15 (m, 2H),

6-Fluoro-3-iodo-2-methylquinolin-4(1H)-one (10f). The title

compound was prepared from 6-ﬂuoro-2-methylquinolin-4(1H)-one 9f

1

according to general procedure B. Yield: 97%. H NMR (400 MHz,

DMSO-d₆) δ 12.25 (bs, 1H), 7.73 (dd, J = 9.3, 2.4 Hz, 1H), 7.56−7.67

(m, 2H), 2.64 (s, 3H).

3-Iodo-4-methoxy-2-methylquinoline (11a). A solution of 4-

hydroxy-3-iodo-2-methylquinolone 10a in phosphorus oxychloride

(80 mL) was reﬂuxed 3 h. The reaction mixture was cooled to 0 °C,

quenched with a cold, saturated sodium hydroxide solution, and

extracted with ethyl acetate. The organic layer was extracted with brine,

dried over magnesium sulfate, ﬁltered, and concentrated in vacuo to give

4-chloro-3-iodo-2-methylquinoline (6.68 g, 88% yield) as an oﬀ-white

solid. 4-Chloro-3-iodo-2-methylquinoline (1.55 g, 5.11 mmol) was

added to a solution of sodium metal (1.00 g, 43.5 mmol) in dry

methanol (40 mL).¹⁰The reaction mixture stirred 18 h at reﬂux and

concentrated in vacuo. The resulting residue was resuspended in ethyl

acetate and water. The organic layer was extracted with water and brine,

dried over magnesium sulfate, ﬁltered, and concentrated in vacuo. The

resulting oﬀ-white solid was recrystallized from hexanes and ethyl acetate to

give white crystals (0.866 g, 56% yield). ¹H NMR (400 MHz, DMSO) δ

7.94−8.12 (m, 2H), 7.58−7.83 (m, 2H), 3.99 (s, 3H), 2.86 (s, 3H).

6-Fluoro-3-iodo-4,7-dimethoxy-2-methylquinoline (11b). To

a stirred solution of 10b (5.72 mmol) in dimethylformamide (57 mL)

was added potassium carbonate (11.4 mmol) at room temperature. The

resulting suspension was stirred 0.5 h at 50 °C. Methyl iodide

(8.58 mmol) was added dropwise at room temperature, and the reaction

mixture was stirred 8 h at 50 °C. The solvent was removed in vacuo, and the

resulting residue was resuspended in ethyl acetate and water and ﬁltered.

The organic layer was extracted with brine, dried over magnesium sulfate,

and concentrated in vacuo to give the title compound (30% yield) as an oﬀ-

white solid. ¹H NMR (400 MHz, DMSO) δ 7.78 (d, J = 11.8 Hz, 1H), 7.56

(d, J = 8.2 Hz, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 2.82 (s, 3H).

4-Ethoxy-6-ﬂuoro-3-iodo-7-methoxy-2-methylquinoline

(11c). The title compound was prepared from 6-ﬂuoro-3-iodo-7-

methoxy-2-methylquinolin-4(1H)-one 10b according to general

procedure C. Yield: 77%. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J =

11.5 Hz, 1H), 7.42 (d, J = 8.0, 1H), 4.18 (q, J = 7.0 Hz, 2H), 2.92 (s, 3H),

1.60 (t, J = 7.0 Hz, 3H).

1

4-Ethoxy-3-iodo-2-methylquinoline (11d). The title compound

was prepared from 4-hydroxy-3-iodo-2-methylquinolone 10a according

1

to general procedure C. Yield: 99%. H NMR (400 MHz, CDCl₃) δ

1.55 (s, 2H), 1.26 (s, 2H).

8.00−8.03 (m, 2H), 7.68−7.72 (m, 1H), 7.48−7.52 (m, 1H), 4.21 (q, J =

7.0 Hz, 2H), 2.96 (s, 3H), 1.62 (t, J = 7.0 Hz, 3H).

1-Bromo-4-(4-chlorophenoxy)benzene (15d). The title com-

pound was prepared from commercially available 4-chlorophenylbor-

onic acid 14d according to general procedure D. Yield: 59%. ¹H NMR

(400 MHz, CDCl₃) δ 7.41−7.46 (m, 2H), 7.27−7.32 (m, 2H), 6.91−

6.95 (m, 2H), 6.85−6.89 (m, 2H).

1-Bromo-4-(4-ﬂuorophenoxy)benzene (15e). The title com-

pound was prepared from commercially available 4-ﬂuorophenylbor-

onic acid 14e according to general procedure D. Yield: 46%. ¹H NMR

(400 MHz, CDCl₃) δ 7.39−7.43 (m, 2H), 7.01−7.07 (m, 2H), 6.94−

6.99 (m, 2H), 6.82−6.86 (m, 2H).

4-(4-Bromophenoxy)-1-ﬂuoro-2-(triﬂuoromethoxy)benzene

(15f). The title compound was prepared from commercially available 4-

ﬂuoro-3-(triﬂuoromethoxy)phenylboronic acid 14f according to general

procedure D. Yield: 66%. ¹H NMR (400 MHz, CDCl₃) δ 7.42−7.49 (m,

2H), 7.17 (t, 1H, J = 9.26), 6.96−6.98 (m, 1H), 6.86−6.92 (m, 3H).

4-(4-Bromophenoxy)-2-ﬂuoro-1-(triﬂuoromethoxy)benzene

(15g). The title compound was prepared from commercially available

6-Chloro-4-ethoxy-3-iodo-2-methylquinoline (11e). The title

compound was prepared from 6-chloro-3-iodo-2-methylquinolin-

4(1H)-one 10c according to general procedure C. Yield: 96%. ¹H

NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 9.0 Hz,

1H), 7.62 (dd, J = 9.0, 2.4 Hz, 1H), 4.20 (q, J = 7.0 Hz, 2H), 2.95 (s, 3H),

1.63 (t, J = 7.0 Hz, 3H).

4-Ethoxy-3-iodo-7-methoxy-2-methylquinoline (11f). The

title compound was prepared from 3-iodo-7-methoxy-2-methylquino-

1

lin-4(1H)-one 10d according to general procedure C. Yield: 99%. H

NMR (400 MHz, CDCl₃) δ 7.89 (d, J = 9.1 Hz, 1H), 7.33 (d, J = 2.5 Hz,

1H), 7.13 (dd, J = 9.1, 2.5 Hz, 1H), 4.19 (q, J = 7.0 Hz, 2H), 3.93 (s, 3H),

2.93 (s, 3H), 1.60 (t, J = 7.0 Hz, 3H).

6-Chloro-4-ethoxy-3-iodo-7-methoxy-2-methylquinoline

(11g). The title compound was prepared from 6-chloro-3-iodo-

7-methoxy-2-methylquinolin-4(1H)-one 10e according to general

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Article

3-ﬂuoro-4-(triﬂuoromethoxy)phenylboronic acid 14g according to

general procedure D. Yield: 45%. H NMR (400 MHz, CDCl₃) δ

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-(4-(triﬂuoro-

methoxy)phenoxy)phenyl) quinoline (17d). The title compound

was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-methylqui-

noline 11g and 4,4,5,5-tetramethyl-2-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)-1,3,2-dioxaborolane 16a according to general procedure G. Yield:

85%. ¹H NMR (400 MHz, CDCl₃) δ 8.11 (s, 1H), 7.43 (s, 1H), 7.31−7.36

(m, 2H), 7.22−7.26 (m, 2H), 7.07−7.14 (m, 4H), 4.04 (s, 3H), 3.71 (q, J =

7.1 Hz, 2H), 2.49 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H).

4-Ethoxy-6-ﬂuoro-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl)quinoline (17e). The title compound was prepared

from 4-ethoxy-6-ﬂuoro-3-iodo-2-methylquinoline 11h and 4,4,5,5-

tetramethyl-2-(4-(4-(triﬂuoromethoxy)phenoxy)phenyl)-1,3,2-dioxa-

borolane 16a according to general procedure G. Yield: 84%. ¹H NMR

(400 MHz, CDCl₃) δ 8.01 (dd, J = 9.2, 5.2 Hz, 1H), 7.72 (dd, J = 9.5, 2.9

Hz, 1H), 7.45 (td, J = 8.7, 2.9 Hz, 1H), 7.33−7.36 (m, 2H), 7.23−7.26

(m, 2H), 7.08−7.15 (m, 4H), 3.72 (q, J = 7.0 Hz, 2H), 2.51 (s, 3H), 1.19

(t, J = 7.0 Hz, 3H).

1

7.47−7.51 (m, 2H), 7.36−7.40 (m, 1H), 6.91−6.95 (m, 2H), 6.81

(dd, 1H, J = 10.92, 2.75), 6.75 (ddd, 1H, J = 9.00, 2.87, 1.63).

4,4,5,5-Tetramethyl-2-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)-1,3,2-dioxaborolane (16a). The title compound was

prepared from 1-bromo-4-(4-(triﬂuoromethoxy)phenoxy)-benzene

15a according to general procedure F. Yield: 92%. ¹H NMR

(400 MHz, CDCl₃) δ 7.80 (d, J = 8.9 Hz, 2H), 7.19 (d, J = 8.9 Hz,

2H), 6.97−7.04 (m, 2H), 1.35 (s, 12H).

4,4,5,5-Tetramethyl-2-(4-(3-(triﬂuoromethoxy)phenoxy)-

phenyl)-1,3,2-dioxaborolane (16b). The title compound was

prepared from 1-(4-bromophenoxy)-3-(triﬂuoromethoxy)benzene

15b according to general procedure F. Yield: 90%. ¹H NMR

(400 MHz, CDCl₃) δ 7.49 (d, J = 9.0 Hz, 2H), 7.36 (t, J = 8.3 Hz,

1H), 6.99 (dm, J = 8.2, 1.1 Hz, 1H), 6.94 (d, J =9.0 Hz, 2H), 6.88 (s, 1H),

6.69 (d, J = 8.9 Hz, 1H).

4-Ethoxy-6-ﬂuoro-7-methoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)-phenyl)quinoline (17f). The title compound was prepared

from 4-ethoxy-6-ﬂuoro-3-iodo-7-methoxy-2-methylquinoline 11c and

4,4,5,5-tetramethyl-2-(4-(4-(triﬂuoromethoxy)-phenoxy)phenyl)-

1,3,2-dioxaborolane 16a according to general procedure G. Yield: 87%.

¹H NMR (400 MHz, DMSO) δ 8.06 (s, 1H), 7.87 (d, J = 8.6 Hz, 1H),

7.75 (d, J = 11.9 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.42 (ddd, J = 16.3,

7.1, 1.6 Hz, 2H), 7.17−7.27 (m, 2H), 7.14 (d, J = 9.1 Hz, 1H), 7.01

(d, J = 8.6 Hz, 1H), 4.01 (s, 3H), 3.70 (q, J = 7.0 Hz, 2H), 2.41 (s, 3H),

1.09 (t, J = 7.0 Hz, 3H).

4,4,5,5-Tetramethyl-2-(4-(4-(triﬂuoromethyl)phenoxy)-

phenyl)-1,3,2-dioxaborolane (16c). The title compound was

prepared from 1-bromo-4-(4-(triﬂuoromethyl)phenoxy)benzene 15c

1

according to general procedure F. Yield: 85%. H NMR (400 MHz,

CDCl₃) δ 7.83 (dt, J = 8.57 Hz, 2H), 7.58 (dt, J = 8.47 Hz, 2H), 7.06

(d, J = 8.38 Hz, 2H), 7.03 (dt, J = 8.59 Hz, 2H), 1.35 (s, 12H).

2-(4-(4-Chlorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-di-

oxaborolane (16d). The title compound was prepared from 1-bromo-

4-(4-chlorophenoxy)benzene 15d according to general procedure

1

F. Yield: 97%. H NMR (400 MHz, CDCl₃) δ 7.77−7.80 (m, 2H),

7.27−7.32 (m, 2H), 6.93−6.98 (m, 4H), 1.34 (s, 12H).

6-Fluoro-2-methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)quinolin-4(1H)-one (18a). The title compound was prepared

from 4-ethoxy-6-ﬂuoro-2-methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)quinoline 17e according to general procedure H. Yield: 67%. ¹H

NMR (400 MHz, DMSO-d₆) δ 11.83 (s, 1H), 7.71−7.74 (m, 1H), 7.62

(dd, J = 9.1, 4.7 Hz, 1H), 7.56 (td, J = 8.6, 3.0 Hz, 1H), 7.42 (d, J = 8.5

Hz, 2H), 7.28−7.31 (m, 2H), 7.15−7.19 (m, 2H), 7.06−7.10 (m, 2H),

2-(4-(4-Fluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-di-

oxaborolane (16e). The title compound was prepared from 1-bromo-

4-(4-ﬂuorophenoxy)benzene 15e according to general procedure

1

F. Yield: 72%. H NMR (400 MHz, CDCl₃) δ 7.75−7.79 (m, 2H),

6.97−7.06 (m, 4H), 6.92−6.95 (m, 2H), 1.34 (s, 12H).

2-(4-(4-Fluoro-3-(triﬂuoromethoxy)phenoxy)phenyl)-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane (16f). The title compound was

prepared from 4-(4-bromophenoxy)-1-ﬂuoro-2-(triﬂuoromethoxy)-

+

2.27 (s, 3H). HRMS (ESI) m/z for [C₂₃H₁₆F₄NO₃]: calculated

430.1061, found 430.1059.

1

benzene 15f according to general procedure F. Yield: 75%. H NMR

6-Fluoro-7-methoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl)quinolin-4(1H)-one (18b). The title compound

was prepared from 4-ethoxy-6-ﬂuoro-7-methoxy-2-methyl-3-(4-(4-

(triﬂuoromethoxy)phenoxy)phenyl)quinoline 17f according to general

procedure H. Yield: 68%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.78 (s,

1H), 7.69−7.74 (m, 1H), 7.40−7.43 (m, 2H), 7.29 (d, J = 8.5 Hz, 2H),

7.15−7.19 (m, 2H), 7.10−7.15 (m, 1H), 7.06−7.09 (m, 2H), 3.96 (s,

3H), 2.25 (s, 3H). HRMS (ESI) m/z for [C₂₄H₁₇F₄NO₄]: calculated

459.1094, found 459.1093.

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-(3-(triﬂuoro-

methoxy)phenoxy)-phenyl)quinoline (19a). The title compound

was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-methylqui-

noline 11g and 4,4,5,5-tetramethyl-2-(4-(3-(triﬂuoromethoxy)-

phenoxy)phenyl)-1,3,2-dioxaborolane 16b according to general proce-

dure G. Yield: 84%. ¹H NMR (400 MHz, CDCl₃) δ 8.11 (s, 1H), 7.34−

7.43 (m, 4H), 7.14−7.17 (m, 2H), 6.97−7.02 (m, 2H), 6.90−6.93 (m,

2H), 4.04 (s, 3H), 3.71 (q, J = 7.02 Hz, 2H), 2.50 (s, 3H), 1.18 (t, J =

7.02 Hz, 3H).

(400 MHz, CDCl₃) δ 7.77−7.83 (m, 2H), 7.14−7.18 (m, 1H), 6.88−

7.02 (m, 4H), 1.35 (s, 12H).

2-(4-(3-Fluoro-4-(triﬂuoromethoxy)phenoxy)phenyl)-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane (16g). The title compound was

prepared from 4-(4-bromophenoxy)-2-ﬂuoro-1-(triﬂuoromethoxy)-

1

benzene 15g according to general procedure F. Yield: 81%. H NMR

(400 MHz, CDCl₃) δ 7.80−7.85 (m, 2H), 7.21−7.27 (m, 1H), 7.00−

7.04 (m, 2H), 6.80−6.86 (m, 1H), 6.75−6.79 (m, 1H), 1.35 (s, 12H).

4-Ethoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)phenoxy)-

phenyl)quinoline (17a). The title compound was prepared from

4-ethoxy-3-iodo-2-methylquinoline 11d and 4,4,5,5-tetramethyl-2-

(4-(4-(triﬂuoromethoxy)phenoxy)-phenyl)-1,3,2-dioxaborolane 16a

1

according to general procedure G. Yield: 84%. H NMR (400 MHz,

CDCl₃) δ 8.12−8.14 (m, 1H), 8.01−8.04 (m, 1H), 7.70 (ddd, J = 8.5,

6.9, 1.5 Hz, 1H), 7.51 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.34−7.37 (m, 2H),

7.22−7.25 (m, 2H), 7.07−7.15 (m, 4H), 3.76 (q, J = 7.0 Hz, 2H), 2.54

(s, 3H), 1.19 (t, J = 7.0 Hz, 3H).

6-Chloro-4-ethoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl)quinoline (17b). The title compound was prepared

from 6-chloro-4-ethoxy-3-iodo-2-methylquinoline 11e and 4,4,5,5-

tetramethyl-2-(4-(4-(triﬂuoromethoxy)phenoxy)phenyl)-1,3,2-dioxa-

borolane 16a according to general procedure G. Yield: 76%. ¹H NMR

(400 MHz, CDCl₃) δ 8.09 (dd, J = 2.4, 0.4 Hz, 1H), 7.95 (dd, J = 9.0, 0.3

Hz, 1H), 7.61 (dd, J = 9.0, 2.4 Hz, 1H), 7.32−7.36 (m, 2H), 7.22−7.25

(m, 2H), 7.12−7.14 (m, 2H), 7.09−7.11 (m, 2H), 3.73 (q, J = 7.0 Hz,

2H), 2.52 (s, 3H), 1.19 (t, J = 7.0 Hz, 3H).

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-(4(triﬂuoromethyl)-

phenoxy)phenyl)-quinoline (19b). The title compound was prepared

from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-methylquinoline 11g and

4,4,5,5-tetramethyl-2-(4-(4-(triﬂuoromethyl)-phenoxy)phenyl)-1,3,2-

1

dioxaborolane 16c according to general procedure G. Yield: 70%. H

NMR (400 MHz, CDCl₃) δ 8.11 (s, 1H), 7.63 (d, J = 8.5, 2H), 7.43

(s, 1H), 7.36−7.39 (m, 2H), 7.15−7.18 (m, 2H), 7.13 (d, J = 8.6 Hz,

2H), 4.05 (s, 3H), 3.72 (q, J = 7.0 Hz, 2H), 2.50 (s, 3H), 1.19

(t, J = 7.0 Hz, 3H).

4-Ethoxy-7-methoxy-2-methyl-3-(4-(4-(triﬂuoromethoxy)-

phenoxy)phenyl)quinoline (17c). The title compound was prepared

from 4-ethoxy-3-iodo-7-methoxy-2-methylquinoline 11f and 4,4,5,5-

tetramethyl-2-(4-(4-(triﬂuoromethoxy)phenoxy)phenyl)-1,3,2-dioxa-

borolane 16a according to general procedure G. Yield: 72%. ¹H NMR

(400 MHz, DMSO) δ 8.00 (d, J = 9.1 Hz, 1H), 7.35−7.48 (m, J = 14.3,

11.1, 1.6 Hz, 5H), 7.12−7.24 (m, 5H), 3.92 (s, 3H), 3.71 (q, J = 7.0 Hz,

2H), 2.41 (s, 3H), 1.09 (t, J = 7.0 Hz, 3H).

6-Chloro-3-(4-(4-chlorophenoxy)phenyl)-4-ethoxy-7-me-

thoxy-2-methylquinoline (19c). The title compound was prepared

from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-methylquinoline 11g and

2-(4-(4-chlorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaboro-

1

lane 16d according to general procedure G. Yield: 78%. H NMR

(400 MHz, CDCl₃) δ 8.06−8.19 (m, 1H), 7.21−7.63 (m, 5H), 6.92−

7.18 (m, 4H), 4.04 (s, 3H), 3.70 (q, 2H), 2.49 (s, 3H), 1.17 (t, J = 6.9,

2.1 Hz, 3H).

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6-Chloro-4-ethoxy-3-(4-(4-ﬂuorophenoxy)phenyl)-7-me-

thoxy-2-methylquinoline (19d). The title compound was prepared

from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-methylquinoline 11g and

2-(4-(4-ﬂuorophenoxy)-phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaboro-

19f according to general procedure H. Yield: 84%. ¹H NMR (400 MHz,

DMSO) δ 11.67 (s, 1H), 8.00 (s, 1H), 7.60 (s, 1H), 7.19−7.39 (m, J =

22.3, 9.4 Hz, 3H), 7.00−7.19 (m, 3H), 6.94 (d, J = 5.4 Hz, 1H), 3.97 (s,

3H), 2.25 (s, 3H). HRMS (ESI) m/z for [C₂₄H₁₆ClF₄NO₄]: calculated

493.0704, found 493.0707.

5-Bromo-2-(4-(triﬂuoromethoxy)phenoxy)pyridine (22a).

The title compound was prepared from commercially available 5-

bromo-2-iodopyridine 21a according to general procedure E. Yield:

91%. ¹H NMR (400 MHz, CDCl₃) δ 8.21 (dd, J = 2.55, 0.62 Hz, 1H),

7.78−7.81 (m, 1H), 7.22−7.27 (m, 2H), 7.13−7.18 (m, 2H), 6.87

(dd, J = 8.70, 0.64 Hz, 1H).

5-Bromo-2-(4-(triﬂuoromethoxy)phenoxy)pyrimidine (22c).

The title compound was prepared from commercially available 5-

bromo-2-iodopyrimidine 21c according to general procedure E. Yield:

79%. ¹H NMR (400 MHz, CDCl₃) δ 8.58 (s, 2H), 7.25−7.30 (m, 2H),

7.19−7.23 (m, 2H).

5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4-(triﬂuoro-

methoxy)phenoxy)pyridine (23a). The title compound was prepared

from 5-bromo-2-(4-(triﬂuoromethoxy)phenoxy)pyridine 22a accord-

ing to general procedure F. Yield: 63%. ¹H NMR (400 MHz, CDCl₃) δ

8.54−8.56 (m, 1H), 8.05−8.08 (m, 1H), 7.20−7.26 (m, 2H), 7.12−7.18

(m, 2H), 6.88−6.91 (m, 1H), 1.34 (s, 12H).

1

lane 16e according to general procedure G. Yield: 95%. H NMR

(400 MHz, CDCl₃) δ 8.10 (s, 1H), 7.42 (s, 1H), 7.25−7.34 (m, 3H),

7.07 (m, 5H), 4.04 (s, 3H), 3.70 (q, J = 7.0 Hz, 2H), 2.49 (s, 3H), 1.18

(t, J = 7.0 Hz, 3H).

6-Chloro-4-ethoxy-3-(4-(4-ﬂuoro-3-(triﬂuoromethoxy)-

phenoxy)phenyl)-7-methoxy-2-methylquinoline (19e). The title

compound was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-

methylquinoline 11g and 2-(4-(4-ﬂuoro-3-triﬂuoromethoxy)phenoxy)-

phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 16f according to gen-

eral procedure G. Yield: 82%. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (s,

1H), 7.59 (ddd, J = 9.5, 6.9, 5.7 Hz, 1H), 7.29−7.49 (m, 4H), 6.93−7.28

(m, 3H), 4.04 (s, 3H), 3.71 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H), 1.17

(t, J = 7.0 Hz, 3H).

6-Chloro-4-ethoxy-3-(4-(3-ﬂuoro-4-(triﬂuoromethoxy)-

phenoxy)phenyl)-7-methoxy-2-methylquinoline (19f). The title

compound was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-

methylquinoline 11g and 2-(4-(3-ﬂuoro-4-(triﬂuoromethoxy)phenoxy)-

phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 16g according to general

procedure G. Yield: 70%. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (s, 1H),

7.34−7.46 (m, 4H), 7.30 (td, J = 8.7, 1.0 Hz, 1H), 7.12−7.19 (m, 2H),

6.89 (dd, J = 14.4, 3.6 Hz, 1H), 4.05 (s, 3H), 3.71 (q, J = 7.0 Hz, 2H), 2.50

(s, 3H), 1.18 (t, J = 7.0 Hz, 3H).

2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(4-(triﬂuoro-

methoxy)phenoxy)pyridine (23b). 2-Bromo-5-(4-(triﬂuoromethoxy)-

phenoxy)pyridine 22b was prepared from commercially available

2-bromo-5-iodopyridine 21b according to general procedure E and

used without further puriﬁcation. The title compound was prepared

from compound 22b according to general procedure F. Yield (over two

steps): 38%. ¹H NMR (400 MHz, CDCl₃) δ 8.17−8.22 (m, 1H), 7.69−

7.74 (m, 1H), 7.20−7.28 (m, 2H), 7.12−7.19 (m, 2H), 7.00−7.04 (m,

1H), 1.34 (s, 12H).

6-Chloro-7-methoxy-2-methyl-3-(4-(3-(triﬂuoromethoxy)-

phenoxy)phenyl)quinolin-4(1H)-one (20a). The title compound

was prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-

(3-(triﬂuoromethoxy)phenoxy)-phenyl)quinoline 19a according to

1

general procedure H. Yield: 88%. H NMR (400 MHz, DMSO-d₆) δ

12.41 (s, 1H), 8.03−8.17 (m, 1H), 7.51−7.60 (m, 1H), 7.28−7.38 (m,

2H), 7.04−7.25 (m, 6H), 3.99 (s, 3H), 2.28 (s, 3H). HRMS (ESI) m/z

for [C₂₄H₁₇ClF₃NO₄]: calculated 475.0798, found 475.0796.

6-Chloro-7-methoxy-2-methyl-3-(4-(4-(triﬂuoromethyl)-

phenoxy)phenyl)quinolin-4(1H)-one (20b). The title compound

was prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-(4-

(triﬂuoromethyl)phenoxy)-phenyl)quinoline 19b according to general

procedure H. Yield: 88%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.68 (s,

1H), 8.01 (s, 1H), 7.77 (d, J = 8.7 Hz, 2H), 7.31−7.35 (m, 2H), 7.20

(d, J = 8.6 Hz, 2H), 7.13−7.17 (m, 2H), 7.08 (s, 1H), 3.95 (s, 3H), 2.24

(s, 3H). HRMS (ESI) m/z for [C₂₄H₁₇ClF₃NO₃]: calculated 459.0849,

found 459.0850.

5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4-(triﬂuoro-

methoxy)phenoxy)pyrimidine (23c). The title compound was

prepared from 5-bromo-2-(4-(triﬂuoromethoxy)phenoxy)pyrimidine

22c according to general procedure F. Yield: 38%. ¹H NMR (400 MHz,

CDCl₃) δ 8.84 (s, 2H), 7.21−7.30 (m, 4H), 1.35 (s, 12H).

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(6-(4-(triﬂuoro-

methoxy)phenoxy)pyridin-3-yl)quinoline (24a). The title com-

pound was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-

2-methylquinoline 11g and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)-2-(4-(triﬂuoromethoxy)phenoxy)pyridine 23a according to gen-

1

eral procedure G. Yield: 47%. H NMR (400 MHz, CDCl₃) δ 8.18

(dd, J = 2.4, 0.6 Hz, 1H), 8.08 (s, 1H), 7.74 (dd, J = 8.4, 2.4 Hz, 1H), 7.43

(s, 1H), 7.25−7.30 (m, 4H), 7.09 (dd, J = 8.4, 0.6 Hz, 1H), 4.04 (s, 3H),

3.74 (q, J = 7.0 Hz, 2H), 2.50 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H).

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(5-(4-(triﬂuoro-

methoxy)phenoxy)pyridin-2-yl)quinoline (24b). The title com-

pound was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-

methylquinoline 11g and 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

5-(4-(triﬂuoromethoxy)phenoxy)pyridine 23b according to general

procedure G. Yield: 22%. ¹H NMR (400 MHz, CDCl₃) δ 8.16−8.21 (m,

1H), 8.08 (s, 1H), 7.74 (dd, J = 8.4, 2.4 Hz, 1H), 7.40−7.45 (m, 1H),

7.08 (dd, J = 8.4, 0.4 Hz, 1H), 4.04 (d, J = 2.8 Hz, 3H), 3.74 (q, J = 7.0

Hz, 2H), 2.50 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H).

6-Chloro-3-(4-(4-chlorophenoxy)phenyl)-7-methoxy-2-

methylquinolin-4(1H)-one (20c). The title compound was prepared

from 6-chloro-3-(4-(4-chlorophenoxy)phenyl)-4-ethoxy-7-methoxy-2-meth-

1

ylquinoline 19c according to general procedure H. Yield: 89%. H NMR

(400 MHz, DMSO-d₆) δ 11.65 (s, 1H), 8.00 (s, 1H), 7.44−7.48 (m, 2H),

7.25−7.28 (m, 2H), 7.03−7.11 (m, 5H), 3.96 (s, 3H), 2.24 (s, 3H). HRMS

(ESI) m/z for [C₂₃H₁₇Cl₂NO₃]: calculated 425.0585, found 425.0582.

6-Chloro-3-(4-(4-ﬂuorophenoxy)phenyl)-7-methoxy-2-

methylquinolin-4(1H)-one (20d). The title compound was prepared

from 6-chloro-4-ethoxy-3-(4-(4-ﬂuorophenoxy)phenyl)-7-methoxy-2-

methylquinoline 19d according to general procedure H. Yield: 92%.

¹H NMR (400 MHz, DMSO-d₆) δ 12.14 (bs, 1H), 8.08 (s, 1H), 7.24−

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(2-(4-(triﬂuoro-

methoxy)phenoxy)pyrimidin-5-yl)quinoline (24c). The title

compound was prepared from 6-chloro-4-ethoxy-3-iodo-7-methoxy-2-

methylquinoline 11g and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)-2-(4-(triﬂuoromethoxy)phenoxy)pyrimidine 23c according to

general procedure G. Yield: 51%. ¹H NMR (400 MHz, CDCl₃) δ 8.61

(s, 2H), 8.06 (s, 2H), 7.44 (s, 1H), 7.23−7.39 (m, 4H), 4.05 (s, 3H),

3.79 (q, J = 7.0 Hz, 2H), 2.54 (s, 3H), 1.62 (s, 3H), 1.24

(t, J = 7.0 Hz, 3H).

7.29 (m, 4H), 7.12−7.17 (m, 3H), 6.99−7.03 (m, 2H), 3.98 (s, 3H),

2.27 (s, 3H). HRMS (ESI) m/z for [C₂₃H₁₇ClFNO₃]: calculated

409.0881, found 409.0879.

6-Chloro-3-(4-(4-ﬂuoro-3-(triﬂuoromethoxy)phenoxy)-

phenyl)-7-methoxy-2-methylquinolin-4(1H)-one (20e). The title

compound was prepared from 6-chloro-4-ethoxy-3-(4-(4-ﬂuoro-3-

(triﬂuoromethoxy)phenoxy)-phenyl)-7-methoxy-2-methylquinoline

19e according to general procedure H. Yield: 84%. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.68 (s, 1H), 8.00 (s, 1H), 7.56 (t, J = 9.6 Hz, 1H), 7.31−

7.35 (m, 2H), 7.26−7.30 (m, 2H), 7.15 (dt, J = 9.1, 3.4 Hz, 1H), 7.06−

7.08 (m, 3H), 3.97 (s, 3H), 2.23 (s, 3H). HRMS (ESI) m/z for

[C₂₄H₁₆ClF₄NO₄]: calculated 493.0704, found 493.0706.

6-Chloro-3-(4-(3-ﬂuoro-4-(triﬂuoromethoxy)phenoxy)-

phenyl)-7-methoxy-2-methylquinolin-4(1H)-one (20f). The title

compound was prepared from 6-chloro-4-ethoxy-3-(4-(3-ﬂuoro-4-

(triﬂuoromethoxy)phenoxy)-phenyl)-7-methoxy-2-methylquinoline

6-Chloro-7-methoxy-2-methyl-3-(6-(4-(triﬂuoromethoxy)-

phenoxy)pyridin-3-yl)quinolin-4(1H)-one (25a). The title com-

pound was prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(6-

(4-(triﬂuoromethoxy)-phenoxy)pyridin-3-yl)quinoline 24a according

1

to general procedure H. Yield: 76%. H NMR (400 MHz, DMSO-d₆)

δ 11.78 (s, 1H), 8.03 (d, J = 2.1 Hz, 1H), 8.01 (s, 1H), 7.78 (dd, J = 8.4,

2.4 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.30−7.34 (m, 2H), 7.14

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(d, J = 7.0 Hz, 1H), 7.08 (s, 1H), 3.97 (s, 3H), 2.26 (s, 3H). HRMS

(ESI) m/z for [C₂₃H₁₆ClF₃N₂O₄]: calculated 476.0751, found

476.0749.

2H), 7.30−7.36 (m, 2H), 7.15 (d, J = 8.4 Hz, 1H), 2.31 (s, 3H). HRMS

+

(ESI) m/z for [C₂₂H₁₅F₄N₂O₃]: calculated 431.1013, found 431.1015.

2-(4-Bromophenoxy)-5-(triﬂuoromethyl)pyridine (28). The

title compound was prepared from commercially available 2-iodo-5-

triﬂuoromethylpyridine according to general procedure E. Yield: 99%.

¹H NMR (400 MHz, CDCl₃) δ 8.43 (td, J = 1.69, 0.84 Hz, 1H), 7.92

(ddd, J = 8.67, 2.55, 0.55, 1H), 7.52−7.55 (m, 2H), 7.01−7.06 (m, 3H).

2-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-

5-(triﬂuoromethyl)pyridine (29). The title compound was prepared

from 2-(4-bromophenoxy)-5-(triﬂuoromethyl)pyridine 28 according to

general procedure F. Yield: 99%. ¹H NMR (400 MHz, CDCl₃) δ 8.41−

8.45 (m, 1H), 7.87−7.91 (m, 2H), 7.12−7.16 (m, 2H), 6.99−7.06 (m,

2H), 1.35 (s, 12H).

6-Chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-((5-

(triﬂuoromethyl)pyridin-2-yl)oxy)phenyl)quinoline (30). The

title compound was prepared from 2-(4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)phenoxy)-5-(triﬂuoromethyl)pyridine 29 according

to general procedure G. Yield: 82%. ¹H NMR (400 MHz, CDCl₃) δ 8.49

(s, 1H), 8.12 (s, 1H), 7.95 (dd, J = 8.7, 2.4 Hz, 2H), 7.38−7.47 (m, 1H),

7.24−7.32 (m, 2H), 7.08 (d, J = 8.6 Hz, 2H), 4.05 (s, 3H), 3.72 (q, J = 7.0

Hz, 2H), 2.53 (s, 3H), 1.19 (t, J = 7.0 Hz, 3H).

6-Chloro-7-methoxy-2-methyl-3-(5-(4-(triﬂuoromethoxy)-

phenoxy)pyridin-2-yl)quinolin-4(1H)-one (25b). The title com-

pound was prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(5-

(4-(triﬂuoromethoxy)phenoxy)pyridin-2-yl)quinoline 24b according to

1

general procedure H. Yield: 98%. H NMR (400 MHz, DMSO-d₆) δ

11.87 (s, 1H), 8.02−8.03 (m, 2H), 7.77−7.80 (m, 1H), 7.42−7.47 (m,

2H), 7.30−7.34 (m, 2H), 7.10−7.14 (m, 2H), 3.97 (s, 3H), 2.27 (s, 3H).

HRMS (ESI) m/z for [C₂₃H₁₆ClF₃N₂O₄]: calculated 476.0751, found

476.0755.

6-Chloro-7-methoxy-2-methyl-3-(2-(4-(triﬂuoromethoxy)-

phenoxy)pyrimidin-5-yl)quinolin-4(1H)-one (25c). The title com-

pound was prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(2-

(4-(triﬂuoromethoxy)phenoxy)pyrimidin-5-yl)quinoline 24c according

1

to general procedure H. Yield: 51%. H NMR (400 MHz, DMSO) δ

8.07 (s, 4H), 7.96 (s, 2H), 7.13 (s, 2H), 3.92 (s, 3H), 2.29 (s, 3H).

HRMS (ESI) m/z for [C₂₂H₁₅ClF₃N₃O₄]: calculated 477.0703, found

477.0705.

6-Chloro-4-ethoxy-2-methyl-3-(6-(4-(triﬂuoromethoxy)-

phenoxy)pyridin-3-yl)quinoline (26a). The title compound was

prepared from 6-chloro-4-ethoxy-3-iodo-2-methylquinoline 11e and

5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4-(triﬂuoromethoxy)-

phenoxy)pyridine 23a according to general procedure G. Yield: 31%. ¹H

NMR (400 MHz, CDCl₃) δ 8.19 (dd, J = 2.4, 0.6 Hz, 1H), 8.06 (d, J = 2.3

Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 7.74 (dd, J = 8.4, 2.4 Hz, 1H), 7.64 (dd,

J = 9.0, 2.4 Hz, 1H), 7.24−7.29 (m, 4H), 7.10 (dd, J = 8.4, 0.6 Hz, 1H), 3.75

(q, J = 7.0 Hz, 2H), 2.53 (d, J = 3.3 Hz, 3H), 1.22 (t, J = 7.0 Hz, 3H).

4-Ethoxy-2-methyl-3-(6-(4-(triﬂuoromethoxy)phenoxy)-

pyridin-3-yl)quinoline (26b). The title compound was prepared from

4-ethoxy-3-iodo-2-methylquinoline 11d and 5-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)-2-(4-(triﬂuoromethoxy)phenoxy)pyridine

23a according to general procedure G. Yield: 32%. ¹H NMR (400 MHz,

CDCl₃) δ 8.20−8.21 (m, 1H), 8.11 (dd, J = 8.3, 0.8, 1H), 8.04 (d, J = 8.4,

1H), 7.69−7.77 (m, 2H), 7.50−7.55 (m, 1H), 7.25−7.33 (m, 4H), 7.07−

7.10 (m, 1H), 3.78 (q, J = 7.0 Hz, 2H), 2.54 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H).

4-Ethoxy-6-ﬂuoro-2-methyl-3-(6-(4-(triﬂuoromethoxy)-

phenoxy)pyridin-3-yl)quinoline (26c). The title compound was

prepared from 4-ethoxy-6-ﬂuoro-3-iodo-2-methylquinoline 11h and 5-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4-(triﬂuoromethoxy)-

phenoxy)pyridine 23a according to general procedure G. Yield: 38%. ¹H

NMR (400 MHz, CDCl₃) δ 8.19 (dd, J = 2.4, 0.6 Hz, 1H), 8.02 (dd, J =

9.2, 5.2 Hz, 1H), 7.72 (ddd, J = 12.2, 8.9, 2.6 Hz, 2H), 7.47 (ddd, J = 9.2,

8.3, 2.9 Hz, 1H), 7.25−7.30 (m, 4H), 7.10 (dd, J = 8.4, 0.6 Hz, 1H), 3.75

(q, J = 7.0 Hz, 2H), 2.52 (s, 3H), 1.21 (t, J = 7.0 Hz, 3H).

6-Chloro-7-methoxy-2-methyl-3-(4-((5-(triﬂuoromethyl)-

pyridin-2-yl)oxy)phenyl)quinolin-4(1H)-one (31). The title com-

pound was prepared from 6-chloro-4-ethoxy-7-methoxy-2-methyl-3-(4-

((5-(triﬂuoromethyl)-pyridin-2-yl)oxy)phenyl)quinoline 30 according

to general procedure H. Yield: 66%. ¹H NMR (400 MHz, DMSO-d₆) δ

11.69 (s, 1H), 8.61−8.64 (m, 1H), 8.25 (dd, J = 8.7, 2.5, 1H), 8.01 (s,

1H), 7.30−7.33 (m, 2H), 7.28 (d, J = 8.8 Hz, 1H), 7.20−7.25 (m, 2H),

7.08 (s, 1H), 3.97 (s, 3H), 2.26 (s, 3H). HRMS (ESI) m/z for

[C₂₃H₁₆ClF₃N₂O₃]: calculated 460.0802, found 460.0802.

Biology. Parasite Culture and Drug Sensitivity. P. falciparum

parasite lines D6 (chloroquine sensitive, MRA-285) and Dd2

(multidrug resistant, MRA-156) were obtained from MR4, ATCC

Collection, Manassass, Virginia, and deposited by D. E. Kyle and T. E.

Wellems, respectively. Atovaquone resistant clinical isolates, Tm90-C2B

and Tm93-C1088, each containing a Y_(TAT)268 ⇒ S_(TCT)268 trans-

formation in the cytochrome b gene, were originally collected from Thai

patients with recrudescent parasites following atovaquone therapy.^18,26

These two isolates were obtained from the frozen parasite repository of

WRAIR, Division of Experimental Therapeutics (Silver Spring,

Maryland) and were kindly provided by Victor Melendez.

Laboratory strains of P. falciparum were cultured in human

erythrocytes by standard methods under a low oxygen atmosphere

(5% O₂, 5% CO₂, 90% N₂) in an environmental chamber.²⁷The culture

medium was RPMI-1640, supplemented with 25 mM HEPES buﬀer,

25 mg/L gentamicin sulfate, 45 mg/L hypoxanthine, 10 mM glucose,

2 mM glutamine, and 0.5% Albumax II (complete medium). The

parasites were maintained in fresh human erythrocytes suspended at a

2% hematocrit in complete medium at 37 °C. Stock cultures were

subpassaged every 3−4 days by transfer of infected red cells to a ﬂask

containing complete medium and uninfected erythrocytes.

EC₅₀Determination by the Fluorescence-Based SyBr Green Assay.

In vitro antimalarial activity of the 4(1H)-quinolone-3-diarylether

derivatives was assessed by the SYBR Green I ﬂuorescence-based

method described previously by us¹⁶with minor modiﬁcations.²¹

Brieﬂy, experiments were set up in triplicate in 96-well plates (Costar,

Corning) with 2-fold dilutions of each drug across the plate in a total

volume of 100 μL and at a ﬁnal red blood cell concentration of 2% (v/v).

The dilution series was initiated at a concentration of 1 μM, and the

experiment was repeated beginning with a lower initial concentration for

those compounds in which the EC₅₀value was below 10 nM. Automated

pipetting and dilution was carried out with the aid of a programmable

Precision 2000 robotic station (BioTek, Winooski, VT). An initial

parasitemia of 0.2% was attained by addition of normal uninfected red

cells to a stock culture of asynchronous parasite infected red cells

(PRBC). The plates were incubated for 72 h at 37 °C in an atmosphere

of 5% CO₂, 5% O₂, and 90% N₂. After this period, the SYBR Green I

dye−detergent mixture (100 μL) was added and the plates were

incubated at room temperature for an hour in the dark and then placed

in a 96-well ﬂuorescence plate reader (Spectramax Gemini-EM,

6-Chloro-2-methyl-3-(6-(4-(triﬂuoromethoxy)phenoxy)-

pyridin-3-yl)quinolin-4(1H)-one (27a). The title compound was

prepared from 6-chloro-4-ethoxy-2-methyl-3-(6-(4-(triﬂuoromethoxy)-

phenoxy)pyridin-3-yl)quinoline 26a according to general procedure H.

Yield: 71%. ¹H NMR (400 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.03 (dd,

J = 8.9, 2.4 Hz, 2H), 7.80 (dd, J = 8.3, 4.1 Hz, 1H), 7.69−7.72 (m, 1H),

7.61 (d, J = 8.9 Hz, 1H), 7.42−7.47 (m, 2H), 7.30−7.35 (m, 2H), 7.12−

+

7.16 (m, 1H), 2.29 (s, 3H). HRMS (ESI) m/z for [C₂₂H₁₅ClF₃N₂O₃]:

calculated 447.0718, found 447.0718.

2-Methyl-3-(6-(4-(triﬂuoromethoxy)phenoxy)pyridin-3-yl)-

quinolin-4(1H)-one (27b). The title compound was prepared from 4-

ethoxy-2-methyl-3-(6-(4-(triﬂuoromethoxy)phenoxy)pyridin-3-yl)-

quinoline 26b according to general procedure H. Yield: 80%. ¹H NMR

(400 MHz, DMSO) δ 8.06 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 1.8 Hz, 1H),

7.78 (dd, J = 8.4, 2.4 Hz, 1H), 7.52 (t, J = 8.5 Hz, 2H), 7.43 (d, J = 8.3 Hz,

2H), 7.26−7.33 (m, 2H), 7.21 (t, J = 7.3 Hz, 1H), 7.10 (d, J = 8.9 Hz,

+

1H), 2.26 (s, 3H). HRMS (ESI) m/z for [C₂₂H₁₆F₃N₂O₃]: calculated

413.1108, found 413.1110.

6-Fluoro-2-methyl-3-(6-(4-(triﬂuoromethoxy)phenoxy)-

pyridin-3-yl)quinolin-4(1H)-one (27c). The title compound was

prepared from 4-ethoxy-6-ﬂuoro-2-methyl-3-(6-(4-(triﬂuoromethoxy)-

phenoxy)pyridin-3-yl)quinoline 26c according to general procedure H.

Yield: 82%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.15 (s, 1H), 8.05 (d, J =

2.2 Hz, 1H), 7.79−7.84 (m, 1H), 7.75 (dd, J = 9.3, 2.9 Hz, 1H), 7.69 (dd,

J = 9.1, 4.7 Hz, 1H), 7.61 (dd, J = 8.6, 2.9 Hz, 1H), 7.44 (d, J = 8.6 Hz,

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Molecular Diagnostics) for analysis, with excitation and emission

wavelength bands centered at 497 and 520 nm, respectively. The

ﬂuorescence readings were plotted against the logarithm of the drug

concentration, and curve ﬁtting by nonlinear regression analysis (GraphPad

Prism software) yielded the drug concentration that produced 50% of the

observed decline relative to the maximum readings in drug-free control wells

(EC₅₀). Chloroquine and atovaquone were used as internal controls to

establish zero percent viability and cross-resistance.

50 mM Tricine, 100 mM KCl, 4 mM KCN, 50 μM ferric cytochrome c

(horse heart, Sigma), 0.1 mg/mL n-dodecyl β-^D-maltoside, and 50 μM

decylubiquinol (prepared freshly before each experiment by reduction

of decylubiquinone with sodium borohydride followed by HCl quenching),

pH = 8.0. Cytochrome c reductase measurements were made at 550−542

nm at 30 °C. Measurements were initiated by the addition of decylubiquinol,

and a baseline was collected for approximately 20 s to account for the

nonenzymatic reduction of cytochrome c by decylubiquinol. Once the

baseline collection was complete, enzyme was added to the mixture and the

reaction was allowed to proceed. Once the kinetic trace had been collected,

the baseline was subtracted from the initial rate of enzymatic activity. The

activity of each kinetic trace is reported as the fraction of activity with respect

to control uninhibited enzyme activity under identical conditions.

HepG2 Cytotoxicity Assay. Drugs were dissolved in DMSO to make

10 mM stock solutions. Human hepatocarcinoma cells (HepG2) were

maintained on RPMI-1640 medium supplemented with 10% fetal

bovine serum at 37 °C in a humidiﬁed 5% CO₂atmosphere. Cells were

seeded at a density of 2 × 10⁴per well in 96-well ﬂat-bottom tissue

culture plates containing complete medium in a total volume of 160 μL/well.

The cells were allowed to attach at 37 °C overnight. On the following day,

drug solutions (40 μL/well) were serially diluted with complete culture

medium across the plate to achieve a concentration range of 2.5 nM to

10 μM. The plates were then incubated at 37 °C and 5% CO₂for another

24−36 h. Afterward, the medium was aspirated and replaced with complete

RPMI medium (200 μL/well), and the plates were incubated for an

additional 24 h at 37 °C and 5% CO₂. An aliquot of a stock solution of

resazurin (Alamar Blue, prepared in 1× PBS) was then added at 20 μL per

well (ﬁnal concentration 10 μM), and the plates were returned to the

incubator for 3 h. After this period, ﬂuorescence in each well, indicative of

cellular redox activity, was measured in a Gemini EM plate reader with

excitation wavelength at 560 nm and emission wavelength at 590 nm.²⁸IC₅₀

values were determined by nonlinear regression analysis of logistic

concentration−ﬂuorescence intensity curves (GraphPad Prism software).

In Vivo Eﬃcacy against Murine Malaria. The in vivo activity of

selected ELQ derivatives was assessed against the blood stages using a

modiﬁed 4-day test.²⁹Mice (female, CF1, Charles River Laboratories)

were infected intravenously with 2.5−5.0 × 10⁵P. yoelii (Kenya strain,

MR4 MRA-428) parasitized erythrocytes from a donor animal. Drug

administration commenced the day after the animals were inoculated

(day 1). The test compounds were dissolved in PEG-400 and

administered by oral gavage once daily for four successive days;

chloroquine phosphate was used as a positive control. On the ﬁfth day,

blood ﬁlms were prepared and the extent of parasitemia was determined

by microscopic examination of Giemsa stained smears. Initially, ELQ

analogues were administered at 0.1, 0.3, 1.0, 3.0, and 10 mg/kg/day.

ED₅₀values (mg/kg/day) were derived graphically from the dose

required to reduce parasite burden by 50% relative to drug-free controls.

If necessary, the initial dose range was adjusted to include higher or

lower dosages for accurate assessment of the ED₅₀. Animals remaining

parasite free 30 days after the last drug dose were considered cured of

their infection. The malaria infection in this model system was rapidly

fulminated, producing average parasitemias of ≈30% in untreated

control animals by day 5. The procedures involved, together with all

matters relating to the care, handling, and housing of the animals used in

this study, were approved by the Portland VA Medical Center

Institutional Animal Care and Use Committee.

Isolation of P. falciparum Mitochondria. Mitochondria from the

trophozoite stage of P. falciparum were prepared according to the

protocol published by Mather et al.³⁰A particular care was taken to

minimize contamination with hemozoin in mitochondrial preparations

from parasites.

Ubiquinol−Cytochrome c Oxidoreductase (Cytochrome bc₁)

Activity: P. falciparum. Cytochrome c reductase activity was assayed

by a modiﬁcation of the method of Trumpower and Edwards.³¹The

assay was performed at 35 °C in a stirred cuvette with a ﬁnal volume of 1

mL containing various amounts of mitochondrial preparation (generally

∼6−12 μL), 100 μM 2,3-dimethoxy-5-methyl-6-decyl-1,4 benzohydro-

quinone (Q_DH₂) (5 μL 20 mM), 100 μM horse heart cytochrome c

(Sigma Aldrich), 0.1 mg/mL n-dodecyl-β-^D-maltoside, 60 mM HEPES

(pH 7.4), 10 mM sodium malonate, 1.0 mM EDTA, and 2.0 mM KCN.

The reduction of cytochrome c was recorded with a modiﬁed SLM-

AMINCO DW2C dual wavelength spectrophotometer (Online Instrument

Systems, Inc., Bogart, GA, USA) in dual mode (550−541 nm). The short

chain ubiquinol analogue Q_DH₂was prepared by reducing Q_Din dimethyl

sulfoxide with sodium borohydride and acidifying the mixture with

concentrated HCl and stored under argon in aliquots at −80 °C.

Methods to Assess in Vitro Microsomal Stability. Compounds were

incubated at 37 °C and 1 μM concentration in human liver microsomes

(BD Gentest, Discovery Labware Inc., Woburn, MA) suspended in

0.1 M phosphate buﬀer (pH 7.4) at a ﬁnal protein concentration of

0.4 mg per mL. Metabolic reactions were initiated by the addition of an

NADPH-regenerating system (1 mg/mL NADP, 1 mg/mL glucose-

6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl₂

(0.67 mg/mL) and were quenched at various time points up to 60 min

by the addition of ice-cold acetonitrile. Quenched samples were

centrifuged, and the relative loss of parent compound over the course of

the incubation was monitored by LC-MS using either a Micromass

single quadrupole, triple quadrupole, or TOF mass spectrometer

(Waters Corporation, Milford, MA). Concentration versus time data for

each compound were ﬁtted to an exponential decay function to

determine the ﬁrst-order rate constant for substrate depletion, which

was then used to calculate the degradation half-life, an in vitro intrinsic

clearance value, and a predicted in vivo intrinsic clearance (CL_int) value

according to the methods of Obach.³²In vivo CL_intvalues were

converted to a predicted in vivo hepatic extraction ratio (E_H) using the

following equation: E_H= CL_int/Q + CL_int, where Q is liver blood ﬂow

which was assumed to be 20.7, 55.2, and 90 mL/min/kg for humans,

rats, and mice, respectively.³³

Enzymology (Assay for Inhibition of Human Cytochrome

bc₁). Isolation of HEK-293 Derived Mitochondria. HEK-293 cells were

grown in DMEM containing 10% FCS using standard methods. When

cell monolayers were conﬂuent, ﬂasks were treated with trypsin and the

detached cells were pelleted and washed twice in ice-cold PBS. The

washed cells were resuspended in PBS containing 1 mM PMSF and

processed three times with an ice-cold Dounce homogenizer. Large or

insoluble matter was removed from the broken cells by centrifugation at

800g for 10 min. The pellet was discarded, and the supernatant was

centrifuged at 20000g for 40 min. The pellet containing mitochondria

was resuspended in a minimum of PBS and made 30% (v/v) glycerol for

storage at −80 °C until needed for enzyme assays.

ASSOCIATED CONTENT

■

S

* Supporting Information

Additional details describing the crystal structure of 5,7-diﬂuoro-

3-heptyl-2-methylquinolin-4(1H)-one and relative log P values

of selected ELQs. This material is available free of charge via the

Internet at http://pubs.acs.org.

Measurement of Cytochrome bc₁Complex Inhibition (Derived

from Human HEK-293 Cells). Mitochondrial fraction was diluted to a

concentration that yielded suitable activity levels (usually 10⁻⁴−10⁻³

absorbance units per second; see below) and dispersed in 2 mg/mL

n-dodecyl β-^D-maltoside. The mixture was allowed to incubate for

45 min on ice and then clariﬁed by microcentrifugation at 10000g.

Enzymatic activity was measured in the following reaction buﬀer:

AUTHOR INFORMATION

■

Corresponding Authors

*For A.N.: phone, 503-220-8262 ext 53398; E-mail, nilsena@

ohsu.edu.

*For M.K.R.: phone, 503-721-7885; E-mail, riscoem@ohsu.edu.

3832

dx.doi.org/10.1021/jm500147k | J. Med. Chem. 2014, 57, 3818−3834

Journal of Medicinal Chemistry

Article

Tropical Medicine and Malaria, Jomtien, Pattaya, Thailand, November

29−December 4, 1992, Abstract 149.

Notes

The authors declare no competing ﬁnancial interest.

^†Deceased 26 June 2011.

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Co-authors M.K.R., A.N., J.X.K., Y.L., D.J.H., J.N.B., and R.W.W.

are listed as coinventors on a U.S. patent that is relevant to this

work (US Patent 2014/0045888 A1 published February 13,

2014). The authors have no relevant aﬃliations or ﬁnancial

involvement with any organization or entity with a ﬁnancial

interest in or ﬁnancial conﬂict with the subject matter or

materials discussed in the manuscript.

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as substrates for the synthesis of 2,3-diaryl-4-methoxyquinolines. J.

Chem. Res. 2008, 437−440.

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2311−2323.

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Ettlinger, M.; Fawaz, G.; Fields, M.; Fieser, M.; Heidelberger, C.;

Heymann, H.; Seligman, A.; Vaughan, W.; Wilson, A.; Wilson, E.;

Leffler, M.; Hamlin, K.; Hathaway, R.; Matson, E.; Moore, E.; Moore,

M.; Rapala, T.; Zaugg, H. Naphthoquinone antimalarials. I. General

survey. J. Am. Chem. Soc. 1948, 70, 3156−3165.

ACKNOWLEDGMENTS

■

This project was supported by the Medicines for Malaria Venture

(0095/08), and by the National Institutes of Health AI079182

and AI100569 (M.K.R.) and AI028398 (M.W.M., A.M.P., and

A.B.V.). M.K.R. also receives funding from the United States

Department of Veterans Aﬀairs and the Stanley Medical

Research Institute. All of the authors would like to recognize

the outstanding leadership provided by Ian Bathurst (deceased,

26 June 2011) who served as a codirector for this project. This

project was established as a collaboration by the MMV with the

University of South Florida, and all of the authors would like to

recognize the signiﬁcant contributions to the overall 4(1H)-

Quinolone Optimization Project provided by Roman Manetsch

and Dennis Kyle and members of their respective laboratories.

(16) Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe,

M. Simple and inexpensive fluorescence-based technique for high-

throughput antimalarial drug screening. Antimicrob. Agents Chemother.

2004, 48, 1803−1806.

(17) Cross, R. M.; Monastyrskyi, A.; Mutka, T. S.; Burrows, J. N.; Kyle,

D. E.; Manetsch, R. Endochin optimization: structure−activity and

structure−property relationship studies of 3-substituted 2-methyl-

4(1H)-quinolones with antimalarial activity. J. Med. Chem. 2010, 53,

7076−7094.

(18) Korsinczky, M.; Chen, N.; Kotecka, B.; Saul, A.; Rieckmann, K.;

Cheng, Q. Mutations in Plasmodium falciparum cytochrome b that are

associated with atovaquone resistance are located at a putative drug-

binding site. Antimicrob. Agents Chemother. 2000, 44, 2100−2108.

(19) Nilsen, A.; Lacrue, A. N.; White, K. L.; Forquer, I. P.; Cross, R. M.;

Marfurt, J.; Mather, M. W.; Delves, M. J.; Shackleford, D. M.; Saenz, F.

E.; Morrisey, J. M.; Steuten, J.; Mutka, T.; Li, Y.; Wirjanata, G.; Ryan, E.;

Duffy, S.; Kelly, J. X.; Sebayang, B. F.; Zeeman, A. M.; Noviyanti, R.;

Sinden, R. E.; Kocken, C. H.; Price, R. N.; Avery, V. M.; Angulo-

Barturen, I.; Jimenez-Diaz, M. B.; Ferrer, S.; Herreros, E.; Sanz, L. M.;

Gamo, F. J.; Bathurst, I.; Burrows, J. N.; Siegl, P.; Guy, R. K.; Winter, R.

W.; Vaidya, A. B.; Charman, S. A.; Kyle, D. E.; Manetsch, R.; Riscoe, M.

K. Quinolone-3-diarylethers: a new class of antimalarial drug. Sci. Transl.

Med. 2013, 5, 177ra37.

(20) Riscoe, M. K.; Kelly, J. X.; Winter, R. W.; Hinrichs, D. J.;

Smilkstein, M. J.; Nilsen, A.; Burrows, J. N.; Kyle, D. E.; Manetsch, M.;

Cross, R. M.; Monastyrskyi, A.; Flanigan, D. L. Compounds having

antiparasitic or anti-infectious activity. (Oregon Health & Science

University, USA; Medicines for Malaria Venture, Switzerland;

University of South Florida, USA). US Patent US8598354 B2, Dec 3,

2013.

ABBREVIATIONS USED

■

ACT, artemisinin combined therapies; CQ, chloroquine; EC50,

50% eﬀective concentrations (relative to drug-free controls); E_H,

predicted human hepatic extraction ratio based on in vitro

clearance values; ELQ, endochin-like quinolone; HFF, human

foreskin ﬁbroblasts; MMV, Medicines for Malaria Venture;

NRD, nonrecrudescence dose; OHSU, Oregon Health &

Science University; SAR, structure−activity relationship;

WRAIR, Walter Reed Army Institute of Research

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Discovery, synthesis, and optimization of antimalarial 4(1 H)-quinolone-3-diarylethers

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