Discovery and Structure-Activity Relationships of Quinazolinone-2-carboxamide Derivatives as Novel Orally Efficacious Antimalarials

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Discovery and Structure−Activity Relationships of Quinazolinone-2-

carboxamide Derivatives as Novel Orally Eﬃcacious Antimalarials

Beno î t Laleu,* Yuichiro Akao, Atsuko Ochida, Sandra Du ﬀ y, Leonardo Lucantoni, David M. Shackleford,

Gong Chen, Kasiram Katneni, Francis C. K. Chiu, Karen L. White, Xue Chen, Angelika Sturm,

Koen J. Dechering, Benigno Crespo, Laura M. Sanz, Binglin Wang, Sergio Wittlin, Susan A. Charman,

Vicky M. Avery, Nobuo Cho, and Masahiro Kamaura

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ABSTRACT: A phenotypic high-throughput screen allowed discovery of quinazolinone-2-carboxamide derivatives as a novel

antimalarial scaﬀold. Structure−activity relationship studies led to identiﬁcation of a potent inhibitor 19f, 95-fold more potent than

the original hit compound, active against laboratory-resistant strains of malaria. Proﬁling of 19f suggested a fast in vitro killing proﬁle.

In vivo activity in a murine model of human malaria in a dose-dependent manner constitutes a concomitant beneﬁt.

maximize structural diversity (molecular weight: range 107−

1449 g/mol, mean 390 g/mol; cLog P: range −8.91 to 10.44,

mean 3.61; cLog D at pH 7.4: range −9.71 to 10.17, mean 2.99;

sp3 character: range 0.00−1.00, mean 0.287), was screened

against P. falciparum using a phenotypic growth inhibition

assay.⁹As the most potent hit series were previously abandoned,

we retrospectively re-evaluated the results of the HTS to rescue

hits with potency in the micromolar range that appeared readily

chemically tractable and possessed favorable physicochemical

properties. Among those, compound 1 (ligand eﬃciency and

lipophilic eﬃciency of 0.21 and 3.45, respectively) caught our

attention since it was previously reported by Takeda as a

selective matrix metalloproteinase (MMP)-13 inhibitor¹⁰and

exhibited acceptable physicochemical properties (e.g., calcu-

lated Log D at pH 7.4 = 2.01, molecular weight 491.5) and

lipophilic eﬃciency (LipE). Resynthesis allowed activity

conﬁrmation from a fresh solid sample, with no signiﬁcant

shift against the resistant malaria strain Dd2 (IC₅₀values of 2.38

INTRODUCTION

■

Malaria is a mosquito-borne disease that remains a critical public

health challenge in the world with 229 million cases of malaria

and 409 000 deaths reported in 2019.¹Human malaria is caused

by ﬁve diﬀerent species of parasites belonging to the Plasmodium

genus, with Plasmodium falciparum being the most lethal species.

The rise of resistance to current antimalarial drugs, including

artemisinin combination therapies (ACTs), threatens eﬀorts

toward malaria elimination and eradication.¹⁻³This under-

scores the need to discover chemotypes with novel modes of

action active against resistant parasites to replace or combine

with existing malaria medicines.⁴⁻⁶In that regard, antimalarial

hit and lead criteria as well as target candidate proﬁles have been

established to deﬁne the attributes that need to be demonstrated

by new antimalarial molecules.^7,8

Herein, we describe the identiﬁcation of a novel antimalarial

scaﬀold. Structure−activity relationship (SAR) studies allowed

one to achieve 95-fold improvement of potency from the hit

compound with identiﬁcation of a potent inhibitor demonstrat-

ing in vivo eﬃcacy in a humanized SCID mouse model of P.

falciparum malaria.

Received: March 10, 2021

RESULTS AND DISCUSSION

■

Hit Identiﬁcation. In search for novel antimalarials, a

Takeda proprietary library of 20 064 compounds, designed to

American Chemical Society

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A

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and 4.46 μM against 3D7 and Dd2, respectively) and no

cytotoxicity observed against HEK293 mammalian cells (Figure

1).

thoroughly investigated, as well as the inﬂuence of substituents

on the phenyl moiety of the quinazolone ring (Scheme 3). The

appropriate nitrobenzoic acid of type 11 (aa−ah) was used as

the starting material and readily converted into the correspond-

ing methyl ester 12aa−ah following standard methods.

Bromination was then performed using 1,3-dibromo-5,5-

dimethyl-imidazolidine-2,4-dione (DBMH) or N-bromosucci-

nimde (NBS)¹³with benzoyl peroxide (BPO) or 2,2-azobis-

(isobutyronitrile) (AIBN) as the radical initiator to aﬀord

intermediates 13aa−ah. At this stage, bromine displacement by

a variety of nucleophiles of type 14 (alcohols, phenols, amines,

or thiols) was performed under the appropriate reaction

conditions. This allowed the coverage of a broad range of

diﬀerent spacers with alteration of both chain length and

functionality with intermediates 15a−n. Reduction of the nitro

group followed by saponiﬁcation generated anthranilic acids

17a−n. The following step consisted of a condensation reaction

with ethyl cyanoformate to give quinazolinones 18a−n. Finally,

the ester at the 2-position was reactive enough for introduction

of (3-ethylphenyl)methanamine in the presence of triethyl-

amine to aﬀord quinazolinone-2-carboxamides 19a−n in 9−

30% overall yields.

Next, we decided to alter the position of the carboxylic acidic

functionality on the optimal linker (compound 19o) as well as

the site of linkage onto the quinazolinone ring (19p−r).

Synthetic routes followed the one used in Scheme 3, with the

main modiﬁcation being the use of silver oxide to force the

displacement of the bromine by the appropriate hydroxyme-

thylbenzoic acid in the third step¹⁴(Scheme 4).

Having determined the potential optimal linker, we

synthesized a few compounds by varying the LHS of the

molecule. Starting from intermediate 15f, benzylamines were

readily introduced to aﬀord compounds 20a−g (Scheme 5, vide

infra Table 3 for more details) in excellent yields (91−96%). An

amide derivative of 19f was also synthesized following standard

conditions (compound 21, Scheme 6).

Figure 1. Antimalarial hit compound 1.

The most frequent side eﬀect associated with the clinical trials

of MMP inhibitors was reported to be a musculoskeletal

syndrome (MSS) that manifested as pain and immobility in the

shoulder joints, arthralgias, and contractures in the hands.

MMP-1, 2, and 14 were prime candidates as the possible causes

of MSS but with no deﬁnitive answer.¹¹It has been recently

demonstrated that selective MMP inhibitors targeting MMP-2,

MMP-9, or MMP-13 were not involved in MSS.¹²Until the

possible relationship between MSS and inhibition of MMP-1

and -14 is fully explained, avoiding the use of this class of MMP

inhibitors in chronic scenarios seems wise. Given this

uncertainty and the short antimalarial treatment dosing

timeframe, greater than 100-fold selectivity against MMP-1

and -14 was arbitrarily set as the ultimate goal for a potential

clinical candidate. As the hit compound 1 was initially designed

as a selective MMP-13 inhibitor (IC₅₀0.0039 nM, >41 000-fold

selectivity over other MMPs) and beneﬁts from low inhibitory

activity against MMP-1 and -14 (IC₅₀s > 10 μM),¹⁰we decided

to proﬁle this chemical series against MMP-1 and MMP-14 at a

later stage while focusing on antimalarial treatment (and not

prophylaxis) as a possible indication.

Antiplasmodial Activity and Cytotoxicity Assessment.

All compounds were tested in a phenotypic 72 h growth

inhibition assay for P. falciparum⁹(3D7 strain) and a 72 h

HEK293 assay¹⁵to assess the intraerythrocytic antiplasmodial

activity and mammalian cytotoxicity, respectively. Activity

against the Dd2 strain was determined only for key compounds

to ensure no obvious concern against resistance (mutated loci:

Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps; choloroquine-, meﬂoquine-,

pyrimethamine-, and sulfadoxine-resistant, respectively). Pyr-

imethamine, chloroquine, artesunate, DHA, and puromycin

were used as reference antimalarial drugs in each P. falciparum

growth inhibition assay run. None of the compounds showed

any sign of cytotoxicity (CC₅₀> 20 μM) against HEK293

mammalian cells. For clarity, these cytotoxicity data were not

included in the SAR Tables.

Despite modest potency, the absence of a signiﬁcant shift

against Dd2 and the therapeutic index against HEK293 cells

suggesting the absence of obvious safety ﬂags around MMP-13

inhibition encouraged us to evaluate this chemical class further.

Chemistry. As close structural analogues previously

reported¹⁰as MMP-13 inhibitors covered modiﬁcations of the

acidic part (vide infra, Table 1, compounds 2 and 3), initial

synthetic eﬀorts were directed toward the left-hand side (LHS)

of the molecule. This part appeared indeed readily amenable to a

variety of modiﬁcations through introductions of various

amines. To establish rudimentary structure−activity relation-

ships (SARs), quinazolinone-2-carboxamide derivatives of type

9 were prepared following a slightly modiﬁed procedure of the

one previously reported¹⁰(Scheme 1). The synthetic route was

not optimized, with emphasis on purity of the product rather

than quantity. Chemical diversity was introduced in the fourth

step, by varying the amine moieties to be introduced, before

conducting nucleophilic aromatic substitution with 4-(2-

hydroxyethyl)benzoic acid to yield the ﬁnal compounds 9a−m

(see Table 1 for more details).

STRUCTURE−ACTIVITY RELATIONSHIPS

■

SAR on the Left-Hand Side. Our primary goal was to

establish rudimentary SAR around the hit scaﬀold. Data on a

representative set of compounds are summarized in Table 1.

Initially, the need for the acidic moiety was investigated.

Removal of this functionality (compound 2) or its replacement

by an aniline (compound 3) resulted in complete loss of

antiplasmodial activity, whereas subnanomolar potencies were

reported with respect to human MMP-13 inhibition.¹⁰These

results highlight the importance of the carboxylic acid for

antiplasmodial potency and discouraged us from immediate

As 9h was identiﬁed as the potent derivative among the ones

tested, condensation with ammonium chloride or ethylamine

was performed to give amide derivatives 10a and 10b to probe

further the essentiality of the acidic functional group (Scheme

2).

Once the potential optimal LHS was identiﬁed, the linker

between the quinazolone core and the acidic moiety was

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Table 1. Antiplasmodial Rudimentary SAR around the Left-Hand Side (R¹and R²Modiﬁcations)

a

N = 1 (unless otherwise speciﬁed), duplicate points against Pf 3D7 (DAPI 72 h inhibition growth assay); compound 9h was used as the internal

control when testing compounds 9i−m and 10a,b; antimalarial drugs pyrimethamine, chloroquine, artesunate, DHA, and puromycin were used as

b

references (mean IC₅₀values of 0.009, 0.025, 0.001, 0.0004, and 0.077 μM, respectively) in all experiments. Conﬁdence interval or standard

deviation; NA, not applicable. Mean value, N = 9, duplicate points.

c

additional investigations targeting this part of the molecule. The

data suggested possible SAR divergence between activity against

malaria parasites and human MMP-13 inhibition.

To generate rudimentary SAR in a short timeframe, close

structural analogues of the hit compound (9a−m) were

synthesized through variations of the LHS while ﬁxing R²=

COOH. The inﬂuence of R¹substitution started with evaluation

of the unsubstituted benzyl derivative (9a) and its cyclohexyl

structural analogue (9b). 9a demonstrated activity in the same

range as the antiplasmodial hit, indicating a most likely marginal

eﬀect of the methoxy substituent on potency. This corroborated

further the hypothesis of divergent SAR between human MMP-

13 inhibition and antiplasmodial activity. In contrast, the

inactivity of 9b underscored the importance of the LHS benzyl

group, which encouraged us to investigate its substitution

pattern. Methyl (9d) or a larger alkoxy group (9e) appeared to

be beneﬁcial over the methoxy substituent harbored by the

original hit. These ﬁndings directed us toward further

C

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a

Scheme 1. Synthesis of Quinazolinone-2-Carboxamides 9a−m

a

Reagents and conditions: (a) Boc₂O, THF, 60 °C; (b) s-BuLi, CO2, THF, −78 °C; (c) CNCOOEt, HCl/dioxane, 80 °C; (d) RR′NH, AlMe₃,

toluene, 0−25 °C; (e) 4-(2-hydroxyethyl)benzoic acid, NaH, DMF, 25 °C; then, 60−80 °C.

a

Scheme 2. Synthesis of Amides 10a and 10b

a

Reagents and conditions: (a) DIEA, NH₄Cl, HATU, CH₃CN, 25 °C; (b) DIEA, ethylamine, HATU, CH₃CN, 20 °C.

a

Scheme 3. Synthesis of Quinazolinone-2-Carboxamides 19a−n

a

Reagents and conditions: (a) Method A: SOCl₂, CH₃OH; Method B: K₂CO₃, CH₃I; (b) Method A: DBDMH, BPO; Method B: DBDMH,

AIBN; (c) Method A: K₂CO₃; Method B: NaH; (d) Fe, NH₄Cl; (e) Method A: NaOH, CH₃OH/H₂O; Method B: LiOH, CH₃OH/H₂O; (f)

CNCOOEt, HCl/dioxane; (g) Et₃N, (3-ethylphenyl)methanamine.

D

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a

Scheme 4. Synthesis of Quinazolinones of Types 19o−r

a

Reagents and conditions: (a) Method B: K₂CO₃, CH₃I; (b) Method B: DBDMH, AIBN; (c) Method C: Ag₂O, 3-(hydroxymethyl)benzoic acid or

4-(hydroxymethyl)benzoic acid; (d) Fe, NH₄Cl; (e) Method B: LiOH, CH₃OH/H₂O; (f) CNCOOEt, HCl/dioxane; (g) Et₃N, (3-

ethylphenyl)methanamine.

a

Scheme 5. Synthesis of Compounds of Types 20a−e

a

Reagents and conditions: (a) RNH₂(3 equiv.), Et₃N, EtOH, 50 °C.

a

Scheme 6. Synthesis of Compound 21

a

Reagents and conditions: (a) DIEA, NH₄Cl, EDCI, CH₃CN, 25 °C.

exploration of alkyl substituents by varying their size and length

(9f−h). Among those, meta-ethyl revealed to be the best

substituent with 5.7-fold improvement in potency when

compared to the hit compound (9h vs 1). It appeared therefore

logical to alter the position of this ethyl substituent on the benzyl

moiety (9i,j). para-Substitution was relatively well-tolerated,

whereas ortho-substitution resulted in complete loss of

inhibitory eﬀect. Compound 9k, the aniline analogue of 9h,

showed ∼7-fold loss of potency. On the other hand,

incorporation of a methyl group at the benzylic position (9l)

or on the amine nitrogen (9m) gave disappointing results.

Finally, amide derivatives 10a and 10b of the most potent

compound 9h were found to be totally inactive, which

reinforced the initial ﬁndings with respect to the need for the

carboxylic acid moiety.

steric constraints. The submicromolar potency demonstrated by

9h was further conﬁrmed by testing against the Dd2-resistant

strain (IC₅₀values of 0.42 and 0.21 μM against 3D7 and Dd2,

respectively), which convinced us to pursue investigation on this

chemotype as a novel antimalarial scaﬀold.

SAR on the Right-Hand Side. After probing the SAR on

the LHS, we next turned our attention toward the linker between

the quinazolinone ring and the acidic function. A variety of

spacers was evaluated while ﬁxing the LHS as meta-ethylbenzyl-

amine to allow direct comparison with 9h. Results are presented

in Table 2.

Initially, R⁴was set as ﬂuorine. Modiﬁcations focused on

walking the ether oxygen atom and altering the length of the

linker not only between the quinazolinone and the phenyl but

also between the phenyl and the carboxylic acid moiety. All

tested changes (19a−c) were found detrimental. Despite these

disappointing results, the same variations were considered with

R⁴= H. While compounds 19d−e were found to be totally

inactive, 19f showed double-digit nanomolar potency (3D7 IC₅₀

In summary, this initial SAR investigation shed light on the

meta-ethylbenzylamine moiety as being the potential optimal

LHS. SAR data suggest involvement of this alkyl substituent in

van der Waals and/or hydrophobic interactions with associated

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Table 2. Antiplasmodial SAR: Linker and Right-Hand-Side Investigation (R³, R⁴, R⁵, R⁶Modiﬁcations)

F

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Table 2. continued

a

N = 1 (unless otherwise speciﬁed), duplicate points against Pf 3D7 (DAPI 72 h inhibition growth assay); compound 9h was used as the internal

control when testing compounds 19a−f; compound 19f was used as the internal control when testing compounds 19g−r; antimalarial drugs

pyrimethamine, chloroquine, artesunate, DHA, and puromycin used as references (mean IC₅₀values of 0.008, 0.035, 0.0004, 0.0002, and 0.108 μM,

b

c

d

respectively) in all experiments. Conﬁdence interval or standard deviation; NA, not applicable. Mean value, N = 2, duplicate points. Curve too

steep to get conﬁdence limits. Mean value, N = 13, duplicate points.

e

Table 3. Antiplasmodial SAR with Potential Optimal Linker (R¹and R²Modiﬁcations)

a

N = 1 (unless otherwise speciﬁed), duplicate points against Pf 3D7 (DAPI 72 h inhibition growth assay); compound 19f (mean IC₅₀0.025 μM)

was used as the internal control; antimalarial drugs pyrimethamine, chloroquine, artesunate, DHA, and puromycin were used as reference controls

b

(mean IC₅₀values of 0.006, 0.028, 0.0006, 0.0003, and 0.087 μM, respectively) in all experiments. Conﬁdence interval or standard deviation.

c

Mean value, N = 6, duplicate points.

= 25 nM). Remarkably, this constituted a 378- and 95-fold

improvement in potency versus its ﬂuorinated analogue 19c and

the original hit 1, respectively. Such a dramatic impact was not

observed with respect to human MMP-13 SAR.¹⁰Surprised by

the huge diﬀerence in activity between 19c and 19f, we decided

to test close structural analogues by varying substituents at

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Table 4. In Vitro ADME Characterization for Selected Compounds

a

b

c

d

e

CL h-LM

CL r-LM

CL m-LM

Caco-2 (Papp)

(10⁻⁶cm/s)

solubility

f

Cpd.

(μL/(min mg) protein)

(μg/mL)

cLog D

9a

9b

9h

19f

20e

20g

21

7

123

740

485

36

42

78

12

62

69

16

32

44

35

6.3−12.5

<1.6

12.5−25

<1.6

1.0

1.4

2.0

1.5

1.4

0.47

35

7

13

83

183

1.6−3.1

<3.1

636

3.8

a

b

c

d

Intrinsic clearance in human liver microsomes. Intrinsic clearance in rat liver microsomes. Intrinsic clearance in mouse liver microsomes. Caco-

e

2 cell permeability assay. Papp, permeability coeﬃcient in the apical to basolateral direction. Aqueous kinetic solubility measured at pH = 6.5.

f

Calculated Log D at pH 7.4 (Chem Axon JChem, version 21.2).

position R⁴. The resulting compounds (19g−i) were found

inactive. This advocates for a deleterious eﬀect associated with a

substituent in the ortho position (R⁴), which might induce a

detrimental repulsive conformational change.

than isopropyl group.¹⁶⁻¹⁸In that regard, the observed 16-fold

potency loss was somewhat disappointing and suggested that

alkyl substituents were favored and/or that tight steric

constraints were present around meta substitution of the benzyl

group. In addition, picolyl analogues of 19f (compounds 20f−g)

were considered to enhance the aqueous solubility but showed

unsatisfactory potencies. Lastly, the primary amide analogue of

19f was found to be moderately potent (21, IC₅₀= 310 nM).

This result suggests potential for replacement of the acidic

moiety provided the associated potency loss could be

compensated. Indeed, the acidic nature of the frontrunner

compound 19f can possibly constitute a concern for its

development as an antimalarial due to the expected low volume

of distribution. If conﬁrmed, this would pose challenges in terms

of achieving the long half-life required for single-dose treatment

of malaria^8,19and would require clearance to be as low as

possible. Moreover, acyl glucuronide metabolite formation²⁰⁻²²

would need be to be investigated to assess whether this could be

a concern for further development of this acidic chemotype.

Overall, 19f appeared as the most potent compound with

acceptable ligand eﬃciency and lipophilic eﬃciency (0.30 and

5.22, respectively) among those tested.

Encouraged by the promising result obtained with 19f,

variations of the R⁶substituent were undertaken (19j−l) to ﬁne-

tune the best possible combinations. Among the substituents

evaluated, only a ﬂuorine atom was well accommodated (19j,

IC₅₀= 54 nM), which indicates possible steric constraints at this

position. In addition, alternatives to the ether oxygen atom of the

spacer were envisaged. Its replacement by an N-methyl or thiol

ether resulted in considerable loss of potency (19m,n), whereas

the sulfone analogue was totally inactive (data not shown).

Shifting the acidic function to the meta position in the optimal

linker gave disappointing activity (19o). Lastly, as 4-

(methoxymethyl)benzoic acid seemed to be the optimal RHS,

it appeared sensible to walk this group through positions R⁴, R⁵,

and R⁶. At best, the R⁴position provided antimalarial potency

only in the micromolar range.

In total, 4-(methoxymethyl)benzoic acid at position R³with

R⁴= R⁵= R⁶= H appeared as the optimal RHS. In-depth

investigation of the RHS of the molecule shed light on a strongly

beneﬁcial modiﬁcation by positioning the ether oxygen atom at

the central position of the three-atom linker between the

quinazolinone and the benzoic acid moieties while removing the

ﬂuorine at position R⁴. SAR data suggest establishment of key

interaction(s) through this ether oxygen atom, most likely of the

hydrogen bond and/or dipole bond type(s) with a well-deﬁned

geometry.

Rudimentary SAR with Potential Optimal Linker. The

favorable contribution of 4-(methoxymethyl)benzoic acid as

RHS was then combined with LHS substituents previously

investigated to check whether the LHS SAR remained similar.

The antiplasmodial activities of representative compounds are

combined in Table 3. Comparable shifts in potency to that

observed with compounds of type 9 were noted when

comparing 19f with unsubstituted benzyl, meta-methoxybenzyl,

and para-ethylbenzyl analogues (compounds 20a−d). Methyl-

ation at the benzylic position remained not tolerated. These

ﬁndings appeared convincing enough to posit that the LHS SAR

was following a similar trend with this new linker. Finally, a few

simple changes were envisaged to check whether potential

liabilities could be readily solved without recourse to thorough

optimization of 19f. To address potential metabolic stability

issues related to the ethyl substituent, its replacement by a

triﬂuoromethyl group was undertaken in 20e. The bioisosterism

of the triﬂuoromethyl group has been questioned recently with

studies arguing for its steric eﬀect being rather similar to an ethyl

In Vitro ADMET. 19f and representative compounds from

this chemical class (9a, 9b, 9h, 20d, 20e) were subjected to in

vitro ADME proﬁling, with respect to oxidative metabolism in

liver microsomes, Caco-2 cell monolayer permeation, and

aqueous solubility. Results are combined in Table 4.

9a and 9b were used as representative compounds of the

original chemotype and demonstrated greater stability in human

liver microsomes than compounds of types 19−20. Interest-

ingly, direct comparison of the close structural analogues 9h and

19f did not show any obvious detrimental impact associated

with the shift of the ether oxygen atom at the central position of

the linker, thereby creating two potential metabolic hot spots.

This was further conﬁrmed by the good metabolic stability

demonstrated by 20e in human liver microsomes. Despite

disappointing antiplasmodial activity, the beneﬁcial impact of

the replacement of the ethyl substituent by a triﬂuoromethyl

designates this position as a metabolic soft spot with respect to

oxidative metabolism and underlines the possibility of achieving

acceptable stability through structural modiﬁcations. Moreover,

this modiﬁcation did not change markedly the calculated Log D

(pH 7.4), which remains in a favorable range (∼2.0). Overall,

lower Log D was associated with improved microsomal stability

as exempliﬁed by compounds 20e and 20g. It is noteworthy to

mention that the acidic moiety appears to have a strongly

beneﬁcial impact on Log D as illustrated by the comparison

between 19f and its amide analogue 21, which is likely linked to

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the poor microsomal stability of the latter. On the other hand,

microsomal stability was moderate to poor in rat species for all

tested compounds, with the best stability obtained with 19f.

We were encouraged to observe good permeability for this

chemical series in spite of the unfavorable polar surface area

(PSA), relatively high molecular weight (MW), and the

presence of an acidic moiety (e.g., Papp 35 × 10⁻⁶cm/s with

PSA = 117 Å²and MW = 471 g/mol for 19f). In terms of

aqueous solubility, we hoped that the linker harbored by 19f

would be beneﬁcial, when compared to compounds of type 9,

thanks to the two ether oxygen lone pairs readily accessible to

form hydrogen bonds with water. On the contrary, results

showed a detrimental impact with solubility below 1.6 μg/mL.

These data are in line with the higher calculated Log D for

compounds of type 19 (1.5 and 2.0 for 19f and 9h, respectively).

Moreover, introduction of an unﬂanked pyridine nitrogen atom

with 20g resulted only in a marginal improvement of the kinetic

solubility within the range of 1.6−3.1 μg/mL at pH 6.5 despite

the lower calculated Log D. This ﬁnding was particularly

surprising, and we hypothesized that the poor aqueous solubility

might be due to the tight crystal packing related to the high

molecular planarity²³induced by the quinazolinone ring. To

conﬁrm this, melting points were measured for compounds 19f

and 20e (238.5 and 249.5 °C, respectively). These high melting

points advocate for more drastic chemical modiﬁcations to be

performed during optimization of 19f by targeting disruption of

crystal packing.

cytotoxicity against mammalian cells based on the lack of

inhibition of hERG and HEK293 cellular assays, respectively.

In summary, despite the poor aqueous solubility and poor

microsomal stability in mouse, 19f demonstrated some

encouraging in vitro ADMET properties with good permeability,

moderate metabolic stability in humans and rats, no major CYP

inhibition risk, and no risk of hERG.

Inhibition of MMP-1 and MMP-14. Although this

chemical series is still at an early stage, the frontrunner was

tested against MMP-1 and MMP-14 to assess any potential risks.

At 10 μM, 19f showed 11.3 and 10.8% inhibition of MMP-1 and

MMP-14, respectively (N = 2). The low level of inhibition

observed against these two MMPs is reassuring at this stage but

would need to be monitored during the optimization course of

this chemical series.

Antimalarial Proﬁle. The eﬃcacy of 19f was proﬁled

against various life-cycle stages. Beyond the intraerythrocytic

stage, the malaria parasite life cycle in the human host involves

two additional major stages with the liver and sexual blood

stages. In vitro testing in a Pf liver schizont 96 h assay using

primary human hepatocytes²⁴led to an IC₅₀of ∼75 nM but with

only 50% as E_max. Precipitation issues were reported and could

be a possible explanation for the observed limited maximum

eﬃcacy, as well as metabolism due to moderate stability of 19f in

human hepatocytes. Nevertheless, this liver stage activity

appeared to be real, as no toxicity was observed against

hepatocytes, suggesting that further investigation by testing

more stable and soluble compounds was warranted. In addition,

no activity was observed against late-stage gametocytes.^25,26

These results underscore promises for the prophylactic activity

but a lack of the transmission-blocking potential.

Table 5 summarizes additional ADMET data gathered on 19f.

First, rat and human hepatocyte stability data were collected.

Table 5. Additional In Vitro ADMET Proﬁling of Compound

The in vitro killing proﬁle was determined in a parasite

reduction ratio (PRR) assay that uses a limiting dilution

technique to quantify the number of asexual blood-stage

parasites that remain viable after drug treatment.²⁷Results are

depicted in Figure 2 with comparison to reference antimalarials

19f

a

CL h-heps

CL r-heps

15 μL/min/10⁶cells

6 μL/min/10⁶cells

100%

93.4%

0.2%

<10% @ 10 μM

55% @ 10 μM

<10% @ 10 μM

>30 μM

b

c

h-plasma stability

r-plasma stability

d

e

h-Fu

f

CYP1A2

f

CYP2C9

f

CYP2D6

f

CYP3A4

g

hERG IC₅₀

h

HEK293 CC₅₀

>40 μM

a

b

Intrinsic clearance in human hepatocytes. Intrinsic clearance in rat

c

hepatocytes. Human plasma stability at 37 °C (% remaining after 2

d

e

h). Rat plasma stability at 37 °C (% remaining after 2 h). Plasma

protein binding assay, rapid equilibrium dialysis. Fu, fraction

f

g

unbound. Inhibition of cytochrome P450 isoforms. QPatch

automated patch clamp. 72 h assay, N = 4.

h

Microsome and hepatocyte-predicted hepatic extraction ratios

were found to be in the same range (0.5−0.6 and 0.3−0.4 in

humans and rats, respectively, without correction for binding).

This was reassuring since phase II metabolism could have been a

concern for such a molecule harboring an acid function and four

benzylic positions. Moreover, 19f demonstrated good stability in

rat and human plasma, removing concerns regarding the

possible degradation of the amide moiety by hydrolytic enzymes

present in plasma. Plasma protein binding was found to be

relatively high (99.8%). No inhibition was noted against

CYP450s at 10 μM except for 2C9, which is not surprising

due to the acidic nature of the molecule. Finally, 19f exhibited a

low probability to generate QT prolongation and no potential

Figure 2. Killing proﬁle of 19f compared with reference antimalarial

drugs (all tested at 10× their respective IC₅₀s).

covering a broad range of killing rates. 19f beneﬁts from a fast-

killing proﬁle similar to chloroquine with a PRR of 4.6 log units

over a period of 48 h, a parasite clearance time (PCT_99.9%) of 32

h, and no lag phase.

Assessing the risk of resistance at an early stage is critical when

developing novel malaria therapeutic agents. In that regard, 19f

was tested against various laboratory-resistant P. falciparum

strains and was found to potently inhibit growth in all strains

tested, irrespective of their geographical origin and resistance

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proﬁle, with respect to the sensitive strain NF54 (Table 6).²⁸On

the other hand, 19f showed no cross-resistance when tested

Table 6. In Vitro Intraerythrocytic Antiplasmodial Activity of

19f against Laboratory Strains (Chloroquine and Artesunate

as Controls)

compound

artesunate

laboratory

strains

19f IC₅₀

chloroquine

a

IC

⁵⁰a

a

mutated loci

(μM)

IC₅₀(μM)

(μM)

b

NF54

Dd2

0.060

0.033

0.010

0.144

0.006

0.004

Pfcrt, Pfmdr1,

Pfdhfr, Pfdhps

Figure 3. Plasma concentration versus time proﬁles for 19f (Na·4H₂O

salt) in male Sprague Dawley rats (n = 3). IV vehicle: 50% DMA, 50%

PEG400; PO vehicle: 0.5% methyl cellulose in water. The black

symbols represent the IV proﬁles and the red, the PO.

K1

Pfcrt, Pfmdr1,

Pfdhfr, Pfdhps

Pfcrt, Pfmdr1,

Pfdhfr, Pfdhps

0.031

0.016

0.031

0.243

0.067

0.160

0.003

0.002

0.004

7G8

TM90C2b Pfcrt, Pfmdr1,

Pfdhfr, Pfdhps,

Pfcytb_Q0

the in vitro metabolic stability in rat liver microsomes (15 vs 32

mL/(min kg), respectively). This could possibly be explained by

not considering the unbound fraction in blood and microsomes

in the in vitro prediction (rat plasma and microsomal protein

binding was not available). Recovery of the unchanged

compound in urine was negligible, suggesting that direct urinary

excretion was not a major contributor to in vivo elimination. The

volume of distribution was moderate (blood V_ss= 1.4 L/kg).

Although this volume of distribution is in a high range for acidic

compounds, a very low clearance will be needed to achieve a

half-life commensurate with the single-dose cure targeted for

treatment of uncomplicated malaria. Therefore, dramatic

improvement of the metabolic stability is a major goal for the

development of this chemical series for the delivery of an

antimalarial preclinical candidate.

Following oral administration (PO), maximum plasma

concentrations of 19f were observed 0.25−1 h post dose and

the apparent half-life was comparable to that after IV dosing.

The apparent oral bioavailability (F) ranged from 30 to 40%.

Given that the in vivo blood clearance was equivalent to 20% of

the nominal hepatic blood ﬂow in the rat, hepatic ﬁrst-pass

elimination is unlikely to pose a substantial limitation to the oral

bioavailability. Rather, oral exposure is likely to be limited by

poor absorption due to incomplete dissolution and poor

solubility. Indeed, 19f exhibited very low aqueous solubility at

pH 6.5 but good membrane permeability across Caco-2 cells.

A search for potential metabolites of 19f in plasma and urine

samples detected a putative O-dealkylation metabolite (M-120)

in pooled urine samples after both intravenous and oral dosing.

Based on high-resolution mass measurement and high-collision-

energy MS data, this metabolite is likely derived from cleavage of

the ether linkage of the methylbenzoic acid moiety. Potential

metabolites due to glucuronidation, oxygenation, N-deal-

kylation, and amide hydrolysis were not observed.

Cam3.I

Pfcrt, Pfmdr1,

Pfdhfr, Pfdhps,

Pfkelch13

0.024

0.126

0.006

a

72 h [³H]hypoxanthine incorporation assay, mean values from two

independent biological replicates; the majority of the individual values

varied not more than 10% (maximum 17% for Dd2). sensitive strain.

b

against a panel of laboratory strains with mutations in recently

identiﬁed antimalarial targets from compounds currently in

development^6,29(Table 7).

Table 7. In Vitro Antiplasmodial Activity of 19f against

Laboratory-Generated Strains

a

b

strain generated

mutated locus

mutation references

IC₅₀(μM)

Dd2

wt

0.033

0.023

0.020

0.019

0.018

0.027

Dd2 DSM265

Dd2 ELQ-300

Dd2 DDD498

Dd2 KAF156

Pfdhodh

PfcytB

Pfeef2

Pfcarl

Pfpi4k

30

31

32

33

34

Dd2 MMV048

a

Genetically engineered P. falciparum strains. Mutations were

b

introduced in the Dd2 wild-type strain. 72 h [³H]hypoxanthine

incorporation assay, mean values from two independent biological

replicates; the majority of the individual values varied not more than

10% (maximum 17% for Dd2 wt); chloroquine and artesunate as

controls (mean IC₅₀values of 0.161 and 0.003 μM, respectively).

Overall, the proﬁle of 19f appears attractive with a fast in vitro

killing proﬁle, potent activity against resistant strains of malaria,

and no cross-resistance against known antimalarials.

In Vivo Pharmacokinetics. On the basis of the in vitro

DMPK data gathered, we assessed the in vivo pharmacokinetic

proﬁle of 19f in rats. Given data previously reported on related

chemotypes,¹⁰19f was administered as its sodium salt to

enhance the dissolution rate and thereby optimize oral

bioavailability. The plasma concentration versus time proﬁles

are shown in Figure 3, and pharmacokinetic parameters are

presented in Table 8. The apparent in vitro rat whole-blood-to-

plasma partitioning ratio of 19f was determined to be 0.62,

indicating limited distribution into erythrocytes, possibly due to

the high plasma protein binding.

Encouraged by this in vivo PK proﬁle in rats, we considered an

IV PK study in mice. Despite the rather disappointing in vitro

metabolic stability in this species, further understanding was

considered important before testing 19f in an in vivo murine

model of malaria. The plasma concentration versus time proﬁles

are reported in Figure 4 and pharmacokinetic parameters in

Table 9. The apparent in vitro mouse whole-blood-to-plasma

partitioning ratio of 19f was determined to be 0.53. Following IV

administration, plasma concentrations remained above the

analytical limit of quantitation for the duration of the 24 h

sampling period and the apparent terminal half-life was ∼14 h.

Assuming that in vivo clearance occurs predominantly via

After intravenous (IV) administration of 19f, plasma

concentration−time proﬁles exhibited an apparent terminal

half-life (t_1/2) of ∼5 h. In vivo blood clearance (plasma CL/

blood-to-plasma ratio) was lower than that predicted based on

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Table 8. In Vivo Pharmacokinetic Proﬁle for Compound 19f (Na·4H₂O Salt) in Male Sprague Dawley Rats (n = 3)

route

dose (mg/kg)

AUC_0−∞(h·μM)

F (%)

34

C

_max(μM)

t

_max(h)

t

_1/2(h)

plasma CL (mL/(min kg))

9.5 0.6

plasma V_ss(L/kg)

IV

PO

1

5.6

3.7 0.2

6.9 1.1

5.3 1.7

3.2 0.9

0.88 0.08

5

1.7 0.7

0.5 0.4

Figure 4. Plasma concentration versus time proﬁles for 19f (Na·4H₂O

salt) in male Swiss outbred mice (n = 3) following IV administration.

Vehicle: 5% DMSO in a 0.9% saline base containing 5% solutol HS15.

Figure 5. Therapeutic eﬃcacy of 19f (Na·4H₂O salt) in the in vivo P.

falciparum NODscidIL2Rγnull mouse model of malaria. The arrows

indicate the days of oral treatment at 50 and 100 mg/kg in the 4-day test

by Peters. Values correspond to the average parasitemia in peripheral

blood of n = 2 mice per group.

Table 9. In Vivo Pharmacokinetic Proﬁle for Compound 19f

(Na·4H₂O Salt) in Male Swiss Outbred Mice (n = 3)

dose

route (mg/kg)

AUC_0−∞

(h·μM)

plasma CL

(mL/(min kg))

blood CL

(mL/(min kg))

IV

0.95

1.1

31

57

metabolic stability and aqueous solubility. In that regard,

modiﬁcations targeting possible metabolic soft spots would

beneﬁt from metabolite identiﬁcation studies, and crystal

packing disruption could also be envisaged. Overall, the

quinazolinone-2-carboxamide series deserves further investiga-

tion to fully assess its potential with respect to the attributes to

be demonstrated by novel preclinical candidates for treatment of

uncomplicated malaria.

hepatic elimination, the apparent blood clearance corresponds

to approximately 50% of the nominal hepatic blood ﬂow in a

mouse (120 mL/(min kg)). This is lower than the predicted

blood clearance based on metabolic stability data in mouse liver

microsomes (predicted hepatic extraction ratio 0.80). The

higher in vivo clearance in the mouse compared to the rat

conﬁrms interspecies diﬀerences in the elimination of this

compound in line with microsomal stability data collected in

vitro.

In Vivo Eﬃcacy in a Humanized Murine Model of

Malaria. Due to unfavorable DMPK properties in mice, we

tested 19f at relatively high doses in a P. falciparum humanized

SCID mouse model³⁵with the objective of obtaining proof-of-

principle of antimalarial eﬃcacy for this chemical series. The

compound was administered as its monosodium tetrahydrate

salt orally at 50 and 100 mg/kg over 4 consecutive days. The

therapeutic eﬃcacy illustrated in Figure 5 demonstrated that at

day 7 post infection, 65 and 83% reductions in activity were

measured compared to the untreated control mice at 50 and 100

mg/kg, respectively (n = 2 mice per group). No adverse events

were reported. Although the level of eﬃcacy remained modest

for such a dose regimen, it was encouraging to see that

parasitemia was reduced in a dose-dependent manner despite

the suboptimal short exposure exhibited by 19f in mice (see

blood concentration−time proﬁles on page S12 of the

Supporting Information).

EXPERIMENTAL SECTION

■

Chemistry. Solvents and chemicals were of reagent grade or better

and were obtained from commercial sources. All experiments dealing

with moisture-sensitive compounds were conducted under nitrogen.

NMR spectra were recorded on Bruker 400 MHz spectrometers.

Chemical shifts were referenced with respect to the residual solvent

signals (DMSO: 2.50 ppm). Data were reported as follows: chemical

shift (δ), integration, multiplicity (br, broad; s, singlet; d, doublet; t,

triplet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt,

doublet of triplets; td, triplet of doublets; q, quadruplet; m, multiplet),

and coupling constants (J) in hertz. The signal of the amide proton of

quinazolinone-2-carboxamide derivatives was reported only when

visible (exchange with deuterated solvent prevented its observation in

a number of ﬁnal compounds). Liquid chromatography-mass

spectrometry (LC-MS) data provided were obtained using a Shimadzu

LC-MS-2020 detector operated in positive or negative electrospray.

Analytical HPLC data were obtained using a Shimadzu LC-20AB

separation module (Alliance) equipped with a Kinetex EVO C-18

column (3 mm × 2.1 mm, 5 μm). Detection was conducted at 220 nm,

254 nm, and full scan at 210−260 nm with a photodiode array detector.

The mobile phase was a linear gradient with a ﬂow rate of 1.5 mL/min

using a 95:5 A/B to 5:95 A/B mixture over 1.5 min. Solvent A was

0.0375% TFA in water, and solvent B was 0.01875% TFA in

acetonitrile. Silica ﬂash was performed with LCGY-FP0100. For thin-

layer chromatography (TLC), alumina sheets from WuXi AppTec

coated with silica gel 60 F254 were used. All tested compounds

possessed a purity of at least 95% established by HPLC. When the

purity of a compound was less than 95%, the percent of purity was

speciﬁed. Reported yields were not optimized, with emphasis on purity

of the product rather than quantity. Melting points were measured with

CONCLUSIONS

■

We have identiﬁed quinazolinone-2-carboxamide derivatives as

novel antimalarial scaﬀolds. SAR studies led to identiﬁcation of a

double-digit nanomolar inhibitor, 19f, 95-fold more potent than

the original hit compound. Despite suboptimal DMPK and

physicochemical properties, 19f demonstrated oral eﬃcacy in a

murine model of human malaria in a dose-dependent manner.

The next major goals for this series should focus on improving

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CAS-WH-MPA-A instrument (type M70, Mettler Toledo). Amines

were purchased from commercial suppliers or synthesized according to

literature procedures. Synthesis and characterization of compounds 1,¹⁰

2,¹⁰3,¹⁰9c,³⁶9d,³⁶and 20a¹⁰have already been reported in the

literature as well as the corresponding synthetic intermediates.^10,36

General Procedure A for the Synthesis of Compounds of

Type 9. Step 1: Synthesis of tert-Butyl(3,4-diﬂuorophenyl)-

carbamate (5). To a solution of compound 4 (17.00 g, 131.67

mmol) in THF (200.00 mL) was added Boc₂O (30.17 g, 138.25 mmol,

31.76 mL). The mixture was stirred at 60 °C for 16 h. TLC (PE/EA =

10:1) indicated disappearance of the starting material and formation of

a new spot. The reaction mixture was concentrated under reduced

pressure. The residue was diluted with ethyl acetate (100 mL) and

washed with saturated citric acid solution (2 × 30 mL) and then

saturated with a NaHCO₃solution (30 mL), dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The resulting

residue was recrystallized from ethyl acetate/petroleum ether (50 mL/

150mL) to give compound 5 as a white solid (28.00 g, 122.15 mmol,

92.8% yield).

Green ODS 150 × 30 mm, 5 μm; mobile phase: water with 0.225%

formic acid/CH₃CN, 55/82%; 10 min) to aﬀord the desired compound

9 in 8−17% yields.

Following the above-described general procedure A, using the

appropriate amine, compounds 9a−l were obtained with 4−9% overall

yields.

4-(2-((2-(Benzylcarbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydroquinazo-

lin-5-yl)oxy)ethyl)benzoic Acid (9a). ¹H NMR (400 MHz, DMSO-d₆)

δ 9.53 (t, J = 6.0 Hz, 1H), 7.88−7.85 (d, J = 8.4 Hz, 2H), 7.79−7.60 (m,

1H), 7.55−7.51 (m, 1H), 7.55−7.42 (m, 2H),7.35 (m, 4H), 7.34 (m,

1H), 4.49−4.47 (d, J = 6.4 Hz, 2H), 4.32 (t, J = 6.8 Hz, 2H), 4.32 (t, J =

6.8 Hz, 2H), 3.17 (t, J = 6.8 Hz, 2H). LC-MS m/z 462.0 (M + H)⁺.

4-(2-((2-((Cyclohexylmethyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihy-

droquinazolin-5-yl)oxy)ethyl)benzoic Acid (9b). ¹H NMR (400 MHz,

DMSO-d₆) δ 12.02 (br s, 1H), 8.92 (t, J = 6.0 Hz, 1H), 7.87 (d, J = 8.0

Hz, 2H), 7.78 (t, J = 10.0 Hz, 1H), 7.54 (dd, J = 4.8, 8.8 Hz, 1H), 7.45

(d, J = 8.8 Hz, 2H), 4.32 (t, J = 6.8 Hz, 2H), 3.17 (t, J = 6.8 Hz, 2H),

3.13 (br t, J = 6.8 Hz, 2H), 1.70−1.60 (m, 6H), 1.23−1.012 (m, 3H),

1.01−0.85 (m, 2H). LC-MS m/z 468.1 (M + H)⁺.

Step 2: Synthesis of 6-((tert-Butoxycarbonyl)amino)-2,3-diﬂuor-

obenzoic Acid (6). Compound 5 (10.60 g, 46.24 mmol) was degassed

and purged with N₂three times, and then, THF (10.00 mL) was added.

The mixture was stirred at −78 °C under a N₂atmosphere, added

dropwise in s-BuLi (1.3 M, 78.26 mL), and then stirred at −78 °C for 3

h under a N₂atmosphere. After that, CO₂(6.11 g, 138.73 mmol) was

added and the resulting reaction mixture was stirred at −78 °C for 1 h

under a N₂atmosphere. TLC (PE/EA = 50:1) indicated disappearance

of the starting material and formation of a new spot only. The reaction

mixture was quenched by addition of H₂O (20 mL) at −78 °C and

stirred at 25 °C for 10 min. The resulting mixture was then washed with

t-BuOMe (2 × 10 mL). The combined water layers were treated with a

1 M aqueous HCl solution (20 mL) to give a mixture that was extracted

with t-BuOMe (2 × 40 mL). The combined organic layers were dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure to give

compound 6 as a white solid without further puriﬁcation (9.00 g, 32.94

mmol, 71.2% yield).

4-(2-((2-((3-(Cyclopropylmethoxy)benzyl)carbamoyl)-6-ﬂuoro-4-

oxo-3,4-dihydroquinazolin-5-yl)oxy)ethyl)benzoic Acid (9e). ¹H

NMR (400 MHz, DMSO-d₆) δ 9.48 (br t, J = 6.2 Hz, 1H), 7.86 (d, J

= 8.0 Hz, 2H), 7.78 (t, J = 9.7 Hz, 1H), 7.54 (dd, J = 4.7, 9.0 Hz, 1H),

7.43 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 8.0 Hz, 1H), 6.91−6.86 (m, 2H),

6.83−6.78 (m, 1H), 4.44 (br d, J = 6.3 Hz, 2H), 4.32 (t, J = 6.9 Hz, 1H),

4.36−4.28 (m, 1H), 3.79 (d, J = 7.0 Hz, 2H), 3.29−2.87 (m, 2H),

1.27−1.14 (m, 1H), 1.25−1.14 (m, 1H), 0.60−0.52 (m, 2H), 0.35−

0.24 (m, 2H). LC-MS m/z 532.1 (M + H)⁺.

4-(2-((6-Fluoro-2-((3-isopropylbenzyl)carbamoyl)-4-oxo-3,4-di-

hydroquinazolin-5-yl)oxy)ethyl)benzoic Acid (9f). ¹H NMR (400

MHz, DMSO-d₆) δ 9.47 (br s, 1H), 8.37 (s, 1H), 7.86 (d, J = 8.0 Hz,

2H), 7.77 (br t, J = 9.7 Hz, 1H), 7.52 (br dd, J = 4.5, 8.8 Hz, 1H), 7.39

(br d, J = 7.8 Hz, 2H), 7.27−7.20 (m, 2H), 7.18−7.11 (m, 2H), 4.46 (br

d, J = 6.3 Hz, 2H), 4.30 (br t, J = 6.9 Hz, 2H), 3.16 (br t, J = 7.0 Hz, 1H),

2.87 (td, J = 6.9, 13.7 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H). LC-MS m/z

504.3 (M + H)⁺.

Step 3: Synthesis of Ethyl 5,6-Diﬂuoro-4-oxo-3,4-dihydroquinazo-

line-2-carboxylate (7). To a solution of compound 6 (5.00 g, 18.30

mmol) in HCl/dioxane (150.00 mL) was added ethyl cyanoformate

(2.15 mL, 2.18 g, 21.96 mmol). The resulting mixture was stirred at 80

°C for 3 h. As LC-MS indicated that compound 6 was consumed

completely, the reaction mixture was quenched by addition of a

saturated Na₂CO₃solution (50 mL) at 25 °C, diluted with

dichloromethane (50 mL), and extracted with DCM (3 × 100 mL).

The combined organic layers were washed with H₂O (2 × 20 mL),

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure

to give compound 7 as a white solid (4.50 g, 17.70 mmol, 96.7% yield).

¹H NMR (400 MHz, DMSO-d₆) δ = 12.80 (s, 1H), 8.00−7.95 (m, 1H),

4-(2-((2-((3-Cyclopropylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-

1

dihydroquinazolin-5-yl)oxy)ethyl)benzoic Acid (9g). H NMR (400

MHz, DMSO-d₆) δ 9.48 (t, J = 6.4 Hz, 1H), 7.94−7.72 (m, 3H), 7.53

(dd, J = 4.7, 9.0 Hz, 1H), 7.42 (br d, J = 8.3 Hz, 2H), 7.23−7.16 (m,

1H), 7.13−7.06 (m, 2H), 6.94 (d, J = 7.6 Hz, 1H), 4.43 (br d, J = 6.2 Hz,

2H), 4.31 (t, J = 7.0 Hz, 2H), 3.17 (br t, J = 6.9 Hz, 2H), 1.94−1.85 (m,

1H), 0.97−0.91 (m, 2H), 0.67−0.61 (m, 2H). LC-MS m/z 502.1 (M +

H)⁺.

4-(2-((2-((3-Ethylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydro-

quinazolin-5-yl)oxy)ethyl)benzoic Acid (9h). (92% HPLC purity) ¹H

NMR (400 MHz, DMSO-d₆) δ 9.50 (t, J = 6.0 Hz, 1H), 7.81 (m, 2H),

7.79 (m, 1H), 7.55 (m, 1H), 7.55−7.53 (m, 2H), 7.24−7.09 (m, 4H),

4.33 (d, J = 6.4 Hz, 2H), 4.30 (t, J = 6.8 Hz, 2H), 3.30 (t, J = 6.0 Hz,

2H), 2.56 (m, 2H), 1.24 (m, 3H). LC-MS m/z 490.1 (M + H)⁺.

4-(2-((2-((2-Ethylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydro-

7.71−7.68 (m, 1H), 4.4−4.25 (m, 2H), 1.36−1.27 (m, 3H). LC-MS m/

z 255.2(M + H)⁺.

Step 4: Synthesis of Compounds of Type 8. At 0 °C under a N₂

atmosphere, AlMe₃(283.61 mg, 3.93 mmol) was added dropwise to a

solution of the appropriate amine (1.57 mmol) in toluene (20.0 mL).

The resulting mixture was stirred at 0 °C for 30 min; then, compound 7

(200.00 mg, 0.786 mmol) was added at 25 °C for 12 h. LC-MS showed

consumption of the starting material and formation of 60% of the

desired product. The solvent was removed and the crude mixture was

puriﬁed by chromatography over silica gel (PE/EA = 1:1) to aﬀord the

desired compound 8 in 76−82% yields.

Step 5. At 0 °C under a N₂atmosphere, the appropriate compound 8

(120.00 mg, 0.350 mmol) and 4-(2-hydroxyethyl)benzoic acid (87.12

mg, 0.524 mmol) were added to a suspension of NaH (60% in mineral

oil, 83.88 mg, 2.10 mmol) in DMF (20.0 mL). The resulting mixture

was stirred at 25 °C for 1 h and then at 80 °C for 11 h. LC-MS revealed

that the starting material was almost consumed and 15% of the desired

product was formed. The reaction was quenched with citric acid (50

mL) and extracted with ethyl acetate (3 × 40 mL). The combined

organic layers were washed with water (20 mL) and brine (20 mL),

dried over Na₂SO₄, and concentrated under reduced pressure. The

resulting residue was puriﬁed by preparative HPLC (column: Boston

1

quinazolin-5-yl)oxy)ethyl)benzoic Acid (9i). H NMR (400 MHz,

DMSO-d₆) δ 8.36 (s, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.78 (t, J = 9.7 Hz,

1H), 7.54 (dd, J = 4.6, 9.0 Hz, 1H), 7.41 (br d, J = 8.1 Hz, 2H), 7.29 (d, J

= 7.2 Hz, 1H), 7.23−7.12 (m, 3H), 4.51 (br d, J = 6.2 Hz, 2H), 4.31 (br

t, J = 7.0 Hz, 2H), 3.23−3.08 (m, 2H), 2.71 (q, J = 7.5 Hz, 2H), 1.19 (t, J

= 7.5 Hz, 3H). LC-MS m/z 490.1 (M + H)⁺.

4-(2-((2-((4-Ethylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydro-

quinazolin-5-yl)oxy)ethyl)benzoic Acid (9j). (90% HPLC purity). ¹H

NMR (400 MHz, DMSO-d₆) δ 9.48 (br t, J = 6.1 Hz, 1H), 7.86 (br d, J

= 7.9 Hz, 2H), 7.78 (br t, J = 9.7 Hz, 1H), 7.53 (br dd, J = 4.5, 9.0 Hz,

1H), 7.44 (br d, J = 7.9 Hz, 2H), 4.43 (br d, J = 6.1 Hz, 2H), 4.31 (br t, J

= 6.7 Hz, 2H), 3.17 (br t, J = 6.7 Hz, 2H), 2.61−2.55 (m, 2H), 1.15 (br

t, J = 7.5 Hz, 3H). LC-MS m/z 490.0 (M + H)⁺.

4-(2-((6-Fluoro-4-oxo-2-(phenylcarbamoyl)-3,4-dihydroquinazo-

lin-5-yl)oxy)ethyl)benzoic Acid (9k). ¹H NMR (400 MHz, DMSO-d₆)

δ 10.63 (s, 1H), 7.90−7.79 (m, 3H), 7.75−7.61 (m, 3H), 7.47 (d, J =

8.0 Hz, 2H), 7.31 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H), 4.35 (t, J

= 6.8 Hz, 2H), 3.20 (br t, J = 6.9 Hz, 2H), 2.65−2.60 (m, 2H), 1.21 (t, J

= 7.6 Hz, 3H). LC-MS m/z 476.1 (M + H)⁺.

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4-(2-((2-((1-(3-Ethylphenyl)ethyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-

5.877 g, 41.40 mmol) and K₂CO₃(57.22 g, 414.03 mmol) in one

portion at 20 °C. The resulting mixture was stirred at 50 °C for 12 h.

TLC (PE/EA = 10:1) showed disappearance of the starting material

and formation of a new main spot with a lower polarity. The reaction

mixture was ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The residue was dissolved in DCM (500 mL), washed with

water (100 mL) and brine (100 mL), dried under Na₂SO₄, and

concentrated under reduced pressure to aﬀord compounds of type 12 in

91−96% yields.

Step 2, Method A: Bromination with DBDMH/BPO to Aﬀord

Compounds of Type 13. Under a N₂atmosphere at 20 °C, to a mixture

of compound of type 12 (11.74 mmol) in chlorobenzene (100.00 mL)

were added 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (4.03 g,

14.09 mmol) and benzyl peroxide (BPO) (568.76 mg, 2.35 mmol) in

one portion. The mixture was stirred at 70 °C for 12 h. TLC (PE/EA =

5:1) showed disappearance of the starting material and a new main spot

with a higher polarity. The solvent was removed under reduced

pressure. The residue was puriﬁed by chromatography over silica gel

(PE/EA = 50:1 to 20:1) to aﬀord the desired compound of type 13 in

38−66% yields.

Step 2, Method B: Bromination with DBDMH/AIBN to Aﬀord

Compounds of Type 13. Under a N₂atmosphere, to a mixture of

compound 12 (7.173 mmol) in CCl₄(15 mL) were added AIBN (0.236

g, 1.435 mmol) and DBDMH (2.256 g, 7.890 mmol) in one portion at

25 °C. The resulting mixture was stirred at reﬂux for 3−12 h. TLC (PE/

EA = 10:1) showed disappearance of the starting material and

formation of a new main spot with a higher polarity. The reaction

mixture was ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The residue was puriﬁed by chromatography over silica gel

(PE/EA = 20:1 to 10:1) to aﬀord the desired compound of type 13 in

42−68% yields.

1

dihydroquinazolin-5-yl)oxy)ethyl)benzoic Acid (9l). H NMR (400

MHz, DMSO-d₆) δ 9.23 (br d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.2 Hz, 2H),

7.78 (t, J = 9.7 Hz, 1H), 7.58 (dd, J = 4.7, 9.0 Hz, 1H), 7.42 (br d, J = 7.9

Hz, 2H), 7.30−7.22 (m, 3H), 7.13−7.07 (m, 1H), 5.10 (m, 1H), 4.31

(t, J = 6.9 Hz, 2H), 3.20−3.12 (m, 1H), 2.64−2.60 (m, 2H), 1.53 (d, J =

7.0 Hz, 3H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS m/z 504.1 (M + H)⁺.

4-(2-((2-((3-Ethylbenzyl)(methyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-

dihydroquinazolin-5-yl)oxy)ethyl)benzoic Acid (9m). ¹H NMR (400

MHz, DMSO-d₆) δ 7.87 (br d, J = 6.9 Hz, 2H), 7.79−7.70 (m, 1H),

7.49−7.37 (m, 3H), 7.34−7.25 (m, 1H), 7.24−7.10 (m, 3H), 4.62 (br

d, J = 12.5 Hz, 2H), 4.40−4.25 (m, 2H), 3.21−3.12 (m, 2H), 2.96−2.84

(m, 3H), 2.68−2.54 (m, 2H), 1.18 (td, J = 7.6, 18.6 Hz, 3H). LC-MS

m/z 504.1 (M + H)⁺.

5-(4-Carbamoylphenethoxy)-N-(3-ethylbenzyl)-6-ﬂuoro-4-oxo-

3,4-dihydroquinazoline-2-carboxamide (10a). To a solution of

compound 9h (50.00 mg, 0.102 mmol) in CH₃CN (2.00 mL) were

added DIEA (53.5 μL, 39.60 mg, 0.306 mmol), NH₄Cl (109.28 mg,

2.04 mmol, 71.42 μL), and HATU (77.68 mg, 0.204 mmol) at 25 °C.

The resulting mixture was stirred at 25 °C for 12 h. LC-MS showed

disappearance of the starting material 9h and formation of the desired

product. The reaction mixture was ﬁltered and concentrated under

reduced pressure. The resulting residue was puriﬁed by preparative

HPLC (column: Boston Green ODS 150 mm × 30 mm, 5 μm; mobile

phase: water (0.225% formic acid)/ CH₃CN, 48/72%, 10 min) to

aﬀord compound 10a as a white solid (33.10 mg, 0.064 mmol, 63.0%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 9.47−9.45 (t, J = 6.8 Hz,

1H), 8.39 (s, 1H), 7.92−7.81 (m, 2H), 7.79−7.75 (m, 1H), 7.51−7.39

(m, 1H), 7.27−7.22 (m, 2H), 7.17−7.10 (m, 4H), 4.45−4.44 (d, J = 6.4

Hz, 2H), 4.31−4.28 (t, J = 13.6 Hz, 2H), 3.16−3.13 (t, J = 14 Hz, 2H),

2.61−2.55 (m, 2H), 1.18−1.10 (t, J = 7.6 Hz, 3H). LC-MS m/z 489.1

(M + H)⁺.

Step 3, Method A: Bromine Displacement to Aﬀord Compounds

of Type 15. To a solution of compound of type 13 (1.12 mmol) in DMF

(10.00 mL) were added K₂CO₃(309.67 mg, 2.24 mmol) and the

appropriate compound of type 14 (1.34 mmol). The mixture was

stirred at 25 °C for 3−16 h. LC-MS showed disappearance of the

starting material and formation of the desired product. The reaction

mixture was quenched by addition of water (20 mL), diluted with EA

(20 mL), and extracted with EA (3 × 40 mL). The combined organic

layers were washed with H₂O (4 × 15 mL), dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure. The resulting residue was

either puriﬁed by silica gel (PE/EA = 20:1 to 3:1) or by preparative

HPLC (column: Phenomenex Synergi C18 Max-RP 250x50mm, 10

μm; mobile phase: water (0.225% formic acid)/CH₃CN, 55/80%, 8

min) and washed with saturated NaHCO₃solution to aﬀord the desired

compound of type 15 in 66−88% yields.

Step 3, Method B: Bromine Displacement to Aﬀord Compounds

of Type 15. To a suspension of NaH (1.04 g, 25.96 mmol, 60%

dispersion in mineral oil) in THF (50.00 mL) was added dropwise at 0

°C a mixture of compound 13 (4.33 mmol) and the appropriate

compound of type 14 (10.82 mmol) in solution in THF (2 mL). The

resulting mixture was stirred at 25 °C for 1−3 h. TLC (PE/EA = 5:1)

showed disappearance of the starting material and formation of a new

main spot. The reaction mixture was quenched with citric acid (100

mL) and extracted with EA (100 mL). The organic layer was washed

with water (20 mL) and brine (20 mL), dried over Na₂SO₄, and

concentrated under reduced pressure. The resulting residue was

puriﬁed by chromatography over silica gel (PE/EA = 30:1 to 1:1) to

aﬀord the desired compound of type 15 in 44−67% yields.

N-(3-Ethylbenzyl)-5-(4-(ethylcarbamoyl)phenethoxy)-6-ﬂuoro-4-

oxo-3,4-dihydroquinazoline-2-carboxamide (10b). To a mixture of

compound 9h (30.00 mg, 0.061 mmol) and ethylamine (25.8 mg, 0.613

mmol, 10.0 equiv) in acetonitrile (3.00 mL) were added HATU

(116.52 mg, 0.306 mmol) and DIEA (107 μL, 79.21 mg, 0.613 mmol)

in one portion at 20 °C, and then, the mixture was stirred at 20 °C for 12

h. LC-MS showed disappearance of the starting material and 15% of the

desired product. The solvent was removed to give a residue that was

puriﬁed by preparative HPLC (column: Phenomenex Synergi C18

Max-RP 250 mm × 50 mm, 10 μm; mobile phase: water (0.1%

triﬂuoracetic acid)/CH₃CN, 44/74%, 12 min) to aﬀord compound 10b

as a white solid. (10.00 mg, 0.019 mmol, 30.6% yield). ¹H NMR (400

MHz, DMSO-d₆) δ 12.16 (s, 1H), 9.51 (br t, J = 6.3 Hz, 1H), 8.42 (br t,

J = 5.4 Hz, 1H), 7.88−7.72 (m, 3H), 7.54 (dd, J = 4.6, 9.0 Hz, 1H), 7.41

(d, J = 8.0 Hz, 2H), 7.30−7.07 (m, 4H), 4.45 (br d, J = 6.4 Hz, 2H), 4.30

(t, J = 7.0 Hz, 2H), 3.30−3.24 (m, 2H), 3.16 (br t, J = 6.8 Hz, 2H),

2.63−2.57 (m, 2H), 1.26−1.02 (m, 6H). LC-MS m/z 517.2 (M + H)⁺.

General Procedure B for the Synthesis of Compounds of

Type 19. Step 1, Method A: Esteriﬁcation to Aﬀord Compounds of

Type 12. Under a N₂atmosphere at 25 °C, SOCl₂(6.56 mL, 10.75 g,

90.39 mmol) was added to the appropriate acid of type 11 (30.13

mmol) dissolved in a mixture of DMF/dichloromethane (1.41/100

mL). The reaction mixture was stirred at 50 °C for 2 h. Then, the

solvent was removed to give a residue that was added dropwise at 25 °C

to a mixture of MeOH (12.22 mL, 9.65 g, 301.30 mmol) and Et₃N

(20.88 mL, 15.24 g, 150.65 mmol) in dichloromethane (20 mL). The

resulting mixture was stirred at 25 °C for 8 h. TLC (PE/EA = 10:1)

showed disappearance of the starting material was consumed and a new

main spot with a lower polarity. The reaction was diluted with

dichloromethane and quenched with water (200 mL). The organic

layer was washed with brine (100 mL), dried over Na₂SO₄, and

concentrated under reduced pressure. The resulting residue was

puriﬁed by chromatography over silica gel (petroleum ether/ethyl

acetate = 30:1 to 10: 1) to aﬀord the desired compound of type 12 in

76−86% yields.

Step 3, Method C: Bromine Displacement to Aﬀord Compounds

of Type 15. Under N₂at 20 °C, to a mixture of compound 13 (0.55 g,

2.01 mmol) and the appropriate hydroxymethylbenzoic acid (542.44

mg, 3.01 mmol) in solution in DCM/n-hexane (2/8 mL) were added

Ag₂O (1.40 g, 6.02 mmol) and 4Å molecular sieves (0.5 g) in one

portion. The resulting mixture was stirred at 60 °C for 12 h in complete

darkness under a N₂atmosphere. TLC (PE/EA = 3:1) showed

disappearance of the starting material and formation of a new main spot

with higher polarity. The reaction mixture was ﬁltered, and the ﬁltrate

was concentrated under reduced pressure. The residue was puriﬁed by

Step 1, Method B: Methylation to Aﬀord Compounds of Type 12.

Under a N₂atmosphere, to a mixture of compound of type 11 (13.80

mmol) in acetone (50.00 mL) were added methyl iodide (2.578 mL,

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chromatography over silica gel (PE/EA = 20:1 to 10:1) to aﬀord

compounds of type 15 in 80−89% yields.

(m, 6H), 6.94 (d, J = 8.4 Hz, 2H), 5.71 (s, 2H), 4.46 (d, J = 6.4 Hz, 2H),

3.48 (s, 2H), 2.58 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS

m/z 490.1 (M + H)⁺.

Step 4: Reduction of the Nitro Group to Aﬀord Anilines of Type 16.

Under a N₂atmosphere at 20 °C, to a mixture of compound 15 (8.04

mmol) in EtOH (100.00 mL) were added Fe (4.49 g, 80.40 mmol) and

NH₄Cl (2.81 mL, 4.30 g, 80.40 mmol) in one portion. The resulting

mixture was stirred at 70 °C for 1 h. LC-MS showed disappearance of

the starting material was consumed and formation of the desired

product (note: 20−30% was the corresponding acid resulting from

hydrolysis of the ester moiety). The reaction mixture was ﬁltered, and

the ﬁltrate was concentrated under reduced pressure. The residue was

dissolved in EA/THF (50/50 mL), washed with water (100 mL) and

brine (100 mL), dried over Na₂SO₄, and concentrated under reduced

pressure to give a mixture of the desired compound of type 16 and the

corresponding acid that was used as such for the next step.

4-((2-((3-Ethylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydroqui-

nazolin-5-yl)methoxy)benzoic Acid (19b). 19b (94% HPLC purity)

was obtained starting from 3-ﬂuoro-2-methyl-6-nitrobenzoic acid using

method A in step 1, method A in step 2, method A in step 3 using

1

methyl 4-hydroxybenzoate, and method A in step 5. H NMR (400

MHz, DMSO-d₆) δ 9.49 (br s, 1H), 8.33 (s, 1H), 7.94−7.73 (m, 4H),

7.27−7.21 (m, 1H), 7.20−7.13 (m, 2H), 7.12−7.04 (m, 3H), 5.81 (br s,

2H), 4.46 (br d, J = 6.4 Hz, 2H), 2.58 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6

Hz, 3H). LC-MS m/z 476.1 (M + H)⁺.

4-(((2-((3-Ethylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)methyl)benzoic Acid (19c). 19c was

obtained starting from 3-ﬂuoro-2-methyl-6-nitrobenzoic acid using

method A in step 1, method A in step 2, method B in step 3 using ethyl

Step 5, Method A: Saponiﬁcation with NaOH to Aﬀord

Compounds of Type 17. To a solution of compound of type 16

(0.562 mmol) in THF (4.00 mL) were added H₂O (4.00 mL), MeOH

(4.00 mL), and NaOH (1.97 g, 49.26 mmol). The resulting mixture was

stirred at 25 °C for 16 h. LC-MS showed disappearance of the starting

material. The reaction mixture was quenched with 3N aqueous HCl (30

mL) and extracted with EA (4 × 40 mL). The combined organic layers

were dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure to aﬀord the desired compound of type 17 in 92−96% yields

for steps 4 and 5.

Step 5, Method B: Saponiﬁcation with LiOH to Aﬀord Compounds

of Type 17. To a mixture of compound of type 16 (2.17 mmol) in H₂O

(10.00 mL), MeOH (10.00 mL), and THF (10.00 mL) was added

LiOH.H₂O (910.53 mg, 21.70 mmol) in one portion at 25 °C. The

resulting mixture was stirred at 25 °C for 16−20 h. LC-MS showed

disappearance of the starting material and formation of the desired

product. The reaction was quenched with citric acid (200 mL) and

extracted with EA (3 × 50 mL). The organic layer was washed with

water (50 mL) and brine (50 mL), dried over Na₂SO₄, and

concentrated under reduced pressure to aﬀord the desired compound

of type 17 in 91−96% yields for steps 4 and 5.

Step 6: Cyclization to Aﬀord Quinazolinones of Type 18. To a

solution of compound of type 17 (0.396 mmol) in dioxane (20.00 mL)

were added HCl/dioxane (4M, 20 mL) and ethyl cyanoformate

(116.46 μL, 117.62 mg, 1.19 mmol). The reaction mixture was stirred at

60 °C and followed by LC-MS. After 6−16 h, LC-MS showed

disappearance of the starting material and formation of the desired

product. The reaction mixture was quenched by addition of H₂O (20

mL) and extracted with EA or DCM (3 × 40 mL). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure to aﬀord the desired compound of type 18 in 72−86%

yields.

Step 7: Reaction with (3-Ethylphenyl)methanamine to Aﬀord

Final Compounds of Type 19. To a solution of compound of type 18

(0.250 mmol) in EtOH (10.00 mL) were added Et₃N (1.04 mL, 7.50

mmol) and (3-ethylphenyl)methanamine (101.32 mg, 0.750 mmol).

The mixture was stirred at 25 °C for 16 h. LC-MS showed

disappearance of the starting material and formation of the desired

compound. The reaction mixture was quenched by addition of H₂O (15

mL) and extracted with EA (3 × 40 mL). The combined organic layers

were washed with H₂O (3 × 10 mL), dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was

puriﬁed by preparative HPLC (column: Boston Green ODS 150 mm ×

30 mm, 5 μm; mobile phase: water (0.225% formic acid)/CH₃CN, 30/

60%, 10 min) to aﬀord the desired ﬁnal compound of type 19 in 91−

96% yields.

1

4-(hydroxymethyl)benzoate, and method B in step 5. H NMR (400

MHz, DMSO-d₆) δ 9.51 (br s, 1H), 7.88 (d, J = 8.2 Hz, 2H), 7.85−7.74

(m, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.28−7.22 (m, 1H), 7.21−7.14 (m,

2H), 7.11 (d, J = 7.3 Hz, 1H), 5.27 (s, 2H), 4.66 (s, 2H), 4.47 (d, J = 6.4

Hz, 2H), 2.63−2.58 (m, 2H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS m/z

490.1 (M + H)⁺.

2-(4-((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazo-

lin-5-yl)methoxy)phenyl)acetic Acid (19d). 19d was obtained starting

from 2-methyl-6-nitrobenzoic acid using method B in step 1, method B

in step 2, method A in step 3 using methyl 2-(4-hydroxyphenyl)acetate,

and method A in step 5. ¹H NMR (400 MHz, DMSO-d₆) δ 12.32 (br s,

2H), 9.61 (br t, J = 6.2 Hz, 1H), 7.96−7.89 (m, 1H), 7.79 (d, J = 7.8 Hz,

2H), 7.34−7.29 (m, 1H), 7.28−7.21 (m, 4H), 7.17 (d, J = 7.6 Hz, 1H),

7.01 (d, J = 8.6 Hz, 2H), 5.81 (s, 2H), 4.53 (d, J = 6.4 Hz, 2H), 3.56 (s,

2H), 2.69−2.64 (m, 2H), 1.24 (t, J = 7.6 Hz, 3H). LC-MS m/z 472.1

(M + H)⁺.

4-((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

5-yl)methoxy)benzoic Acid (19e). 19e was obtained starting from 2-

methyl-6-nitrobenzoic acid using method B in step 1, method B in step

2, method A in step 3 using methyl 4-hydroxybenzoate, and method A

in step 5. ¹H NMR (400 MHz, DMSO-d₆) δ 12.35 (br s, 2H), 9.56 (br t,

J = 6.1 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.87 (br t, J = 7.8 Hz, 1H), 7.73

(br dd, J = 7.6, 15.3 Hz, 2H), 7.28−7.23 (m, 1H), 7.21 (s, 1H), 7.17 (br

d, J = 7.7 Hz, 1H), 7.10 (br d, J = 8.8 Hz, 2H), 7.15−7.05 (m, 1H), 5.84

(s, 2H), 4.48 (br d, J = 6.4 Hz, 2H), 2.63−2.60 (m, 2H), 1.18 (t, J = 7.6

Hz, 3H). LC-MS m/z 458.1 (M + H)⁺.

4-(((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

5-yl)methoxy)methyl)benzoic Acid (19f). 19f was obtained starting

from 2-methyl-6-nitrobenzoic acid using method B in step 1, method B

in step 2, method B in step 3 using ethyl 4-(hydroxymethyl)benzoate,

and method B in step 5. Oﬀ-white solid. Mp 238.5 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 12.92 (br s, 1H), 12.17 (s, 1H), 9.52 (t, J = 6.0 Hz,

1H), 7.96 (d, J = 8.0 Hz, 2H), 7.91−7.80 (m, 2H), 7.70 (d, J = 7.6 Hz,

1H), 7.55 (d, J = 8.0 Hz, 2H), 7.28−7.22 (m, 1H), 7.21−7.14 (m, 2H),

7.11 (br d, J = 7.6 Hz, 1H), 5.24 (s, 2H), 4.80 (s, 2H), 4.46 (d, J = 6.4

Hz, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS m/z

472.1 (M + H)⁺.

4-(((6-Chloro-2-((3-ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)methyl)benzoic Acid (19g). 19g was

obtained starting from 3-chloro-2-methyl-6-nitrobenzoic acid using

method A in step 1, method A in step 2, method B in step 3 using ethyl

1

4-(hydroxymethyl)benzoate, and method B in step 5. H NMR (400

MHz, DMSO-d₆) δ 12.88 (br s, 1H), 12.38 (br s, 1H), 9.56 (br t, J = 6.4

Hz, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.77 (d, J =

8.7 Hz, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.28−7.22 (m, 1H), 7.20 (s, 1H),

7.18−7.14 (m, 1H), 7.11 (d, J = 7.5 Hz, 1H), 5.38 (s, 2H), 4.71 (s, 2H),

4.46 (d, J = 6.4 Hz, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz,

3H). LC-MS m/z 530.1 (M + H)⁺.

Following the above-described general procedure B, using the

appropriate compounds and methods, compounds 19a−r were

obtained with 9−30% overall yields.

4-(((6-Bromo-2-((3-ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)methyl)benzoic Acid (19h). 19h was

obtained starting from 3-bromo-2-methyl-6-nitrobenzoic acid using

method A in step 1, method A in step 2, method B in step 3 using ethyl

4-(hydroxymethyl)benzoate, and method B in step 5. H NMR (400

MHz, DMSO-d₆) δ 9.51 (br s, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.89 (d, J =

2-(4-((2-((3-Ethylbenzyl)carbamoyl)-6-ﬂuoro-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)phenyl)acetic Acid (19a). 19a was obtained

starting from 3-ﬂuoro-2-methyl-6-nitrobenzoic acid using method A in

step 1, method A in step 2, method A in step 3 using methyl 2-(4-

hydroxyphenyl)acetate, and method A in step 5. ¹H NMR (400 MHz,

DMSO-d₆) δ 9.51 (br t, J = 6.4 Hz, 1H), 7.93−7.75 (m, 2H), 7.27−7.10

1

N

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8.2 Hz, 2H), 7.67 (d, J = 8.7 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.29−

7.07 (m, 4H), 5.40 (s, 2H), 4.70 (s, 2H), 4.46 (br d, J = 6.4 Hz, 2H),

2.60 (br s, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS m/z 550.2 (M + H)⁺.

4-(((2-((3-Ethylbenzyl)carbamoyl)-6-methoxy-4-oxo-3,4-dihy-

droquinazolin-5-yl)methoxy)methyl)benzoic Acid (19i). 19i was

obtained starting from 3-methoxy-2-methyl-6-nitrobenzoic acid using

method A in step 1, method A in step 2, method B in step 3 using ethyl

method B in step 5. ¹H NMR (400 MHz, DMSO-d₆) δ 9.53 (br t, J = 6.3

Hz, 1H), 8.00 (s, 1H), 7.94−7.85 (m, 2H), 7.84−7.77 (m, 1H), 7.69

(br t, J = 7.2 Hz, 2H), 7.57−7.48 (m, 1H), 7.28−7.21 (m, 1H), 7.21−

7.14 (m, 2H), 7.10 (d, J = 7.5 Hz, 1H), 5.23 (s, 2H), 4.79 (s, 2H), 4.46

(d, J = 6.3 Hz, 2H), 2.59 (q, J = 7.6 Hz, 3H), 1.17 (t, J = 7.6 Hz, 3H).

LC-MS m/z 472.2 (M + H)⁺.

4-(((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

6-yl)methoxy)methyl)benzoic Acid (19p). 19p was obtained starting

from 5-methyl-2-nitrobenzoic acid using method B in step 1, method B

in step 2, method C in step 3 using 4-(hydroxymethyl)benzoic acid, and

method B in step 5. ¹H NMR (400 MHz, DMSO-d₆) δ 12.90 (br s, 1H),

12.28 (br s, 1H), 9.53 (br t, J = 6.2 Hz, 1H), 8.17 (s, 1H), 7.95 (d, J = 8.3

Hz, 2H), 7.91−7.85 (m, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.81−7.75 (m,

1H), 7.51 (d, J = 8.3 Hz, 2H), 7.28−7.22 (m, 1H), 7.20 (s, 1H), 7.19−

7.15 (m, 1H), 7.11 (d, J = 7.3 Hz, 1H), 4.75 (s, 2H), 4.69 (s, 2H), 4.46

(d, J = 6.4 Hz, 2H), 3.30 (br s, 2H), 2.63−2.58 (m, 3H), 1.18 (t, J = 7.6

Hz, 3H). LC-MS m/z 472.1 (M + H)⁺.

4-(((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

7-yl)methoxy)methyl)benzoic Acid (19q). 19q was obtained starting

from 4-methyl-2-nitrobenzoic acid using method B in step 1, method B

in step 2, method C in step 3 using 4-(hydroxymethyl)benzoic acid, and

method B in step 5. ¹H NMR (400 MHz, DMSO-d₆) δ 12.95 (br s, 1H),

12.28 (s, 1H), 9.55 (t, J = 6.2 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.96 (d,

J = 8.2 Hz, 2H), 7.77 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.4

Hz, 2H), 7.27−7.22 (m, 1H), 7.20 (s, 1H), 7.18−7.14 (m, 1H), 7.11 (d,

J = 7.7 Hz, 1H), 4.79 (s, 2H), 4.71 (s, 2H), 4.46 (d, J = 6.3 Hz, 2H),

2.62−2.58 (m, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS m/z 472.1 (M +

H)⁺.

1

4-(hydroxymethyl)benzoate, and method B in step 5. H NMR (400

MHz, DMSO-d₆) δ 12.89 (br s, 1H), 11.96 (s, 1H), 9.46 (t, J = 6.5 Hz,

1H), 7.89 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 9.2

Hz, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.28−7.22 (m, 1H), 7.20 (s, 1H),

7.18−7.14 (m, 1H), 7.11 (d, J = 7.4 Hz, 1H), 5.27 (s, 2H), 4.65 (s, 2H),

4.46 (d, J = 6.3 Hz, 2H), 3.93 (s, 3H), 2.59 (q, J = 7.6 Hz, 2H), 1.17 (t, J

= 7.6 Hz, 3H). LC-MS m/z 502.3 (M + H)⁺

4-(((2-((3-Ethylbenzyl)carbamoyl)-8-ﬂuoro-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)methyl)benzoic Acid (19j). 19j was ob-

tained starting from 3-ﬂuoro-6-methyl-2-nitrobenzoic acid using

method B in step 1, method B in step 2, method B in step 3 using

1

ethyl 4-(hydroxymethyl)benzoate, and method B in step 5. H NMR

(400 MHz, DMSO-d₆) δ 9.41 (br t, J = 6.3 Hz, 1H), 7.95 (d, J = 8.1 Hz,

2H), 7.79−7.73 (m, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.28−7.06 (m, 4H),

5.16 (s, 2H), 4.78 (s, 2H), 4.46 (br d, J = 6.4 Hz, 2H), 2.58 (q, J = 7.6

Hz, 2H), 1.16 (t, J = 7.5 Hz, 3H). LC-MS m/z 509.2 (M + H)⁺.

4-(((8-Chloro-2-((3-ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)methyl)benzoic Acid (19k). 19k was

obtained starting from 3-chloro-6-methyl-2-nitrobenzoic acid using

method B in step 1, method B in step 2, method B in step 3 using ethyl

1

4-(hydroxymethyl)benzoate, and method B in step 5. H NMR (400

MHz, DMSO-d₆) δ 12.55 (br s, 1H), 9.24 (t, J = 6.6 Hz, 1H), 8.03 (d, J

= 8.3 Hz, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.79 (d, J = 8.3 Hz, 1H), 7.56 (s,

1H), 7.29−7.24 (m, 1H), 7.22 (s, 1H), 7.18 (d, J = 7.5 Hz, 1H), 7.12 (d,

J = 7.8 Hz, 1H), 5.18 (s, 2H), 4.80 (s, 2H), 4.51 (d, J = 6.4 Hz, 2H), 2.60

(q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS m/z 506.2 (M +

H)⁺.

4-(((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

8-yl)methoxy)methyl)benzoic Acid (19r). 19r was obtained starting

from 3-methyl-2-nitrobenzoic acid using method B in step 1, method B

in step 2, method C in step 3 using 4-(hydroxymethyl)benzoic acid, and

method B in step 5. ¹H NMR (400 MHz, DMSO-d₆) δ 9.46 (t, J = 6.4

Hz, 1H), 8.17−8.09 (m, 1H), 7.99 (d, J = 6.5 Hz, 1H), 7.93 (d, J = 8.2

Hz, 2H), 7.63 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.27−7.21

(m, 1H), 7.20 (s, 1H), 7.18−7.14 (m, 1H), 7.11 (d, J = 7.3 Hz, 1H),

5.19 (s, 2H), 4.75 (s, 2H), 4.52 (d, J = 6.4 Hz, 2H), 2.58 (q, J = 7.5 Hz,

2H), 1.16 (t, J = 7.6 Hz, 3H). LC-MS m/z 472.2 (M + H)⁺.

General Procedure C for the Synthesis of Compounds of

Type 20. A mixture of compound 15f (70 mg, 0.183 mmol), the

appropriate amine (0.549 mmol), and Et₃N (127.41 μL, 92.62 mg,

0.915 mmol) in EtOH (5.00 mL) was stirred at 50 °C for 24 h. LC-MS

showed disappearance of the starting material and formation of the

desired product. The reaction mixture was quenched with HCl (50 mL)

and then extracted with EA (3 × 40 mL). The combined organic layers

were washed with water (20 mL) and brine (20 mL), dried over

Na₂SO₄, and concentrated under reduced pressure. The crude mixture

was triturated with CH₃CN (20 mL) to aﬀord the desired compound of

type 20.

4-(((2-((3-Ethylbenzyl)carbamoyl)-8-methoxy-4-oxo-3,4-dihy-

droquinazolin-5-yl)methoxy)methyl)benzoic Acid (19l). 19l was

obtained starting from 3-methoxy-6-methyl-2-nitrobenzoic acid using

method B in step 1, method B in step 2, method B in step 3 using ethyl

1

4-(hydroxymethyl)benzoate, and method B in step 5. H NMR (400

MHz, DMSO-d₆) δ 12.22 (br s, 1H), 9.19 (br t, J = 6.3 Hz, 1H), 7.95 (d,

J = 8.2 Hz, 2H), 7.73 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.46

(d, J = 8.5 Hz, 1H), 7.28−7.23 (m, 1H), 7.22−7.14 (m, 2H), 7.12 (d, J

= 7.5 Hz, 1H), 5.14 (s, 2H), 4.76 (s, 2H), 4.48 (d, J = 6.4 Hz, 2H), 3.93

(s, 3H), 2.63−2.58 (m, 2H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS m/z 502.3

(M + H)⁺.

4-((((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

5-yl)methyl)(methyl)amino)methyl)benzoic Acid (19m). 19m was

obtained starting from 2-methyl-6-nitrobenzoic acid using method B in

step 1, method B in step 2, method A in step 3 using methyl 4-

((methylamino)methyl)benzoate, and method A in step 5. ¹H NMR:

(400 MHz, DMSO-d₆) δ 9.63−9.40 (m, 1H), 8.35 (s, 2H), 7.96−7.79

(m, 4H), 7.67 (dd, J = 1.9, 7.0 Hz, 1H), 7.43 (br d, J = 8.1 Hz, 2H),

7.29−7.08 (m, 4H), 4.46 (br d, J = 6.2 Hz, 2H), 4.27 (s, 2H), 3.69 (br s,

1H), 2.63−2.57 (m, 2H), 2.18 (s, 3H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS

m/z 483.3 (M + H)⁺.

Following the above-described general procedure C, using the

appropriate compounds and methods, compounds 20b−g were

obtained with 65−80% yields.

4-(((2-(Benzylcarbamoyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-

1

methoxy)methyl)benzoic Acid (20b). H NMR (400 MHz, DMSO-

d₆) δ 9.56 (br t, J = 5.6 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.91−7.79 (m,

2H), 7.70 (d, J = 7.2 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.39−7.30 (m,

3H), 7.39−7.30 (m, 1H), 7.29−7.21 (m, 1H), 5.22 (s, 2H), 4.79 (s,

2H), 4.53−4.44 (m, 2H). LC-MS m/z 444.1 (M + H)⁺.

4-(((2-((4-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

5-yl)methoxy)methyl)benzoic Acid (20c). ¹H NMR (400 MHz,

DMSO-d₆) δ 9.52 (br s, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.91−7.86

(m, 1H), 7.85−7.80 (m, 1H), 7.70 (br d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.2

Hz, 2H), 7.29−7.24 (m, 2H), 7.19−7.15 (m, 2H), 5.23 (s, 2H), 4.80 (s,

2H), 4.44 (d, J = 6.3 Hz, 2H), 2.61−2.55 (m, 2H), 1.16 (t, J = 7.6 Hz,

3H). LC-MS m/z 472.2 (M + H)⁺.

4-(((2-((3-Ethylbenzyl)(methyl)carbamoyl)-4-oxo-3,4-dihydro-

quinazolin-5-yl)methoxy)methyl)benzoic Acid (20d). ¹H NMR (400

MHz, DMSO-d₆) δ 9.24 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.1 Hz, 2H),

7.85−7.80 (m, 1H), 7.79−7.74 (m, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.47

(d, J = 8.2 Hz, 2H), 7.24−7.18 (m, 3H), 7.07−7.02 (m, 1H), 5.16 (s,

4-(((2-((3-Ethylbenzyl)carbamoyl)-8-methoxy-4-oxo-3,4-dihy-

droquinazolin-5-yl)methoxy)methyl)benzoic Acid (19n). 19n was

obtained starting from 2-methyl-6-nitrobenzoic acid using method B in

step 1, method B in step 2, method A in step 3 using methyl 4-

1

(mercaptomethyl)benzoate, and method A in step 5. H NMR (400

MHz, DMSO-d₆) δ 12.88 (br s, 1H), 12.17 (s, 1H), 9.52 (br t, J = 6.4

Hz, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.80−7.72 (m, 1H), 7.70−7.64 (m,

1H), 7.45−7.33 (m, 3H), 7.29−7.22 (m, 1H), 7.21−7.14 (m, 2H), 7.11

(br d, J = 7.6 Hz, 1H), 4.47 (d, J = 6.2 Hz, 2H), 4.34 (s, 2H), 3.76 (s,

2H), 2.63−2.58 (m, 2H), 1.18 (t, J = 7.6 Hz, 3H). LC-MS m/z 530.1

(M + H)⁺.

3-(((2-((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-

5-yl)methoxy)methyl)benzoic Acid (19o). 19o was obtained starting

from 2-methyl-6-nitrobenzoic acid using method B in step 1, method B

in step 2, method C in step 3 using 3-(hydroxymethyl)benzoic acid, and

O

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2H), 5.12−4.99 (m, 1H), 4.73 (s, 2H), 2.54 (q, J = 7.7 Hz, 2H), 1.49 (d,

J = 7.0 Hz, 3H), 1.12 (t, J = 7.6 Hz, 3H). LC-MS m/z 486.2 (M + H)⁺.

4-(((4-Oxo-2-((3-(triﬂuoromethyl)benzyl)carbamoyl)-3,4-dihy-

supplemented with hypoxanthine, HEPES, 2.5 mg/mL

AlbuMAX II, and 5% human serum. Compounds were

incubated in the presence of 2% sorbitol synchronized ring-

stage parasites in a total assay volume of 50 μL, for 72 h at 37 °C

and 5% CO₂, 5% O₂in poly-D-lysine-coated imaging plates.

After incubation, the plates were stained with DAPI in the

presence of saponin and Triton X-100 and incubated for a

further 8 h at room temperature in the dark before imaging using

the Opera (PerkinElmer). The digital images obtained from

each well were analyzed using spot detection software.

Pyrimethamine, chloroquine, artesunate, DHA, and puromycin

were used as internal controls in all experiments.

HEK293 Alamar Blue Viability Estimation Assay. It was

performed as previously reported.¹⁵Incubation times, com-

pound additions, and plate read were as per the Pf 3D7 growth

assay, with the exception that Alamar Blue was diluted in

HEK293 growth media before addition, and incubation of

Alamar Blue at 37 °C, in 5% CO₂, was for 4 h, followed by

incubation at room temperature for 20 h. Puromycin

(Calbiochem) was used as the control.

1

droquinazolin-5-yl)methoxy)methyl)benzoic Acid (20e). H NMR

(400 MHz, DMSO-d₆) δ 12.18 (s, 1H), 9.69 (br t, J = 5.6 Hz, 1H), 7.95

(d, J = 8.0 Hz, 2H), 7.92−7.80 (m, 2H), 7.75−7.57 (m, 5H), 7.55 (br d,

J = 8.2 Hz, 2H), 5.23 (s, 2H), 4.80 (s, 2H), 4.56 (br d, J = 6.4 Hz, 2H).

LC-MS m/z 512.0 (M + H)⁺.

4-(((2-(((4-Ethylpyridin-2-yl)methyl)carbamoyl)-4-oxo-3,4-dihy-

1

droquinazolin-5-yl)methoxy)methyl)benzoic Acid (20f). H NMR

(400 MHz, DMSO-d₆) δ 12.23 (br s, 1H), 9.52 (br s, 1H), 8.43 (br d, J

= 5.1 Hz, 1H), 7.96 (br d, J = 7.9 Hz, 2H), 7.92−7.80 (m, 2H), 7.72 (br

d, J = 7.9 Hz, 1H), 7.55 (br d, J = 8.1 Hz, 2H), 7.31−7.14 (m, 2H),

5.27−5.20 (m, 2H), 4.80 (s, 2H), 4.58 (br d, J = 6.0 Hz, 2H), 2.63 (br s,

2H), 1.17 (t, J = 7.5 Hz, 3H). LC-MS m/z 473.2 (M + H)⁺.

4-(((2-(((5-Ethylpyridin-3-yl)methyl)carbamoyl)-4-oxo-3,4-dihy-

droquinazolin-5-yl)methoxy)methyl)benzoic Acid (20g). Oﬀ-white

1

solid. Mp 249.5 °C. H NMR (400 MHz, DMSO-d₆) δ 12.69−11.61

(m, 1H), 9.71 (t, J = 5.9 Hz, 1H), 8.68 (d, J = 7.0 Hz, 2H), 8.29 (s, 1H),

7.95 (d, J = 7.9 Hz, 2H), 7.91−7.78 (m, 2H), 7.70 (d, J = 7.8 Hz, 1H),

7.54 (d, J = 7.8 Hz, 2H), 5.22 (s, 2H), 4.78 (s, 2H), 4.63 (d, J = 6.0 Hz,

2H), 2.77 (q, J = 7.4 Hz, 2H), 1.22 (t, J = 7.5 Hz, 3H). LC-MS m/z

473.3 (M + H)⁺.

In Vitro Antimalarial Activity against Pf Liver Schiz-

onts. It was performed as previously reported.²⁴In brief,

primary human hepatocytes were cultured for 2 days and then

overlaid with P. falciparum NF54 sporozoites and compounds.

The supernatant was refreshed daily with fresh compounds.

Four days post infection, hepatocytes are immunostained for the

presence of Hsp70-positive liver stage parasites.

5-(((4-Carbamoylbenzyl)oxy)methyl)-N-(3-ethylbenzyl)-4-oxo-

3,4-dihydroquinazoline-2-carboxamide (21). To a mixture of 19f

(0.06 g, 0.127 mmol) and aqueous solution of NH₄Cl (89 μL, 136.14

mg, 2.55 mmol) in DMF (5 mL) were added EDCI (73.18 mg, 0.382

mmol), HOBt (51.58 mg, 0.382 mmol), and DIEA (221.65 μL, 164.46

mg, 1.27 mmol) in one portion. The mixture was stirred at 25 °C for 12

h. LC-MS showed disappearance of the starting material and formation

of the desired product. The reaction mixture was poured into water (20

mL), and the resulting solid was collected by ﬁltration. The solid was

triturated with CH₃CN (10 mL × 2) and MTBE (20 mL) to aﬀord

compound 21 (16 mg, 0.032 mmol, 25% yield, 94% HPLC purity) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.0 (br s, 1H), 9.53 (t, J

= 1.6 Hz, 2H), 7.97 (br s, 1H), 7.91−7.85 (m, 3H), 7.85−7.80 (m, 1H),

7.70 (br d, J = 7.6 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.35 (br s, 1H),

7.27−7.22 (m, 1H), 7.19 (s, 1H), 7.16 (br d, J = 7.6 Hz, 1H), 7.11 (br d,

J = 7.6 Hz, 1H), 5.22 (s, 2H), 4.77 (s, 2H), 4.46 (d, J = 6.4 Hz, 2H), 2.61

(q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS m/z 471.3 (M +

H)⁺.

Preparation of the Sodium Salt of Compound 19f: Sodium 4-(((2-

((3-Ethylbenzyl)carbamoyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-

methoxy)methyl)benzoate Tetrahydrate (19f Na·4H₂O). A total of

0.01 M aqueous NaOH (24.93 mL, 0.249 mmol) was added dropwise

at 20 °C to compound 19f (0.115 g, 0.244 mmol) in solution in THF/

CH₃OH (20/20 mL). The resulting mixture was stirred at 20 °C for 1 h.

The organic solvents were then removed under reduced pressure, and

methanol (10 mL) was added to the residual suspension. The resulting

suspension was stirred at room temperature for 30 min and cooled to 10

°C. The precipitated solid was collected and washed with cold

methanol to aﬀord the desired sodium salt as a white solid. Elemental

analysis was consistent with a monosodium tetrahydrate salt (0.118 g,

0.209 mmol, 86% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 9.47 (br t, J

= 6.1 Hz, 1H), 7.93 (d, J = 7.9 Hz, 2H), 7.86−7.76 (m, 2H), 7.69 (d, J =

8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.33−7.27 (m, 1H), 7.26−7.19

(m, 2H), 7.16 (d, J = 7.5 Hz, 1H), 5.29 (s, 2H), 4.77 (s, 2H), 4.51 (d, J =

6.1 Hz, 2H), 2.65 (q, J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). LC-MS

m/z 472.3 (M + H)⁺. Elemental analysis calculated for C₂₇H₂₄N₃O₅Na·

4H₂O: C, 57.34; H, 5.70; N, 7.43; O: 25.46; Na, 4.07. Found: C, 57.37;

H, 5.55; N, 7.29; Na, 4.22.

Pf Late-Stage Gametocyte Assay. It was run as previously

described.^25,26In brief, NF54-pfs16-LUC-GFP parasites were

cultured in RPMI1640 media supplemented with hypoxanthine,

HEPES, 2.5 mg/ml AlbuMAX II, 5% human serum, and

blasticidin for selection. The parasites were processed through a

synchronous single cycle of “stress” to induce gametocyto-

genesis. Under N-acetyl-glucosamine (NAG) treatment, asexual

parasites were removed, and resultant stage IV gametocytes were

isolated using VarioMACS separator columns, adjusted to 10%

gametocytsaemia and added to poly-^D-lysine-coated imaging

plates containing the compounds of interest. After 72 h

incubation at 37 °C and 5% CO₂, 5% O₂, MitoTracker Red

CM-H2Xros was added to all wells and the plates were

incubated for a further 16 h and imaged using an Opera High

Content Imaging System to identify elongated gametocytes with

demonstrated mitochondrial viability. The percent inhibition of

gametocyte inhibition was calculated using 0.4% DMSO (0%

inhibition) and 5 μM Puromycin (100% inhibition) to

normalize all of the activity data.

Parasite Reduction Ratio Assay. The assay used the

limiting dilution technique to quantify the number of parasites

remaining viable after drug treatment following a method

published previously.²⁷P. falciparum strain 3D7A (MR4) was

treated with compound 19f at a concentration corresponding to

10x IC₅₀. Conditions of parasites exposed to treatment are

identical to the ones used in the IC₅₀determination (2%

hematocrit, 0.5% parasitemia). Parasites were exposed to the

drug for 120 h, with the drug being renewed daily over the entire

treatment period. Samples of parasites were taken from the

treated culture every 24 h (24, 48, 72, 96, and 120 h time points),

the drug was washed out, and drug-free parasites were cultured

in 96-well plates by adding fresh erythrocytes and new culture

media. To quantify the number of viable parasites after

treatment, 3-fold serial dilutions were used for samples after

removing the drug. Parasites were cultured in 96-well microtiter

plates to allow all wells with viable parasites to render detectable

BIOLOGICAL ASSAYS

■

Intraerythrocytic Pf 3D7 and Dd2 Inhibition Growth

Assay. It utilizes the DNA-intercalating dye 4′,6′,-diamidino-2-

phenylindole (DAPI) to monitor changes in the parasite

number observed within infected erythrocytes. This assay was

performed as previously reported.⁹In brief, Pf parasites (3D7

and Dd2 strains) were cultured in RPMI1640 media

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parasitemia. Four independent serial dilutions were performed

with each sample to correct for experimental variation. After 21

days of culturing, samples were taken to examine growth.

Additional sampling was done after 28 days to conﬁrm growth/

no growth. The number of viable parasites was determined by

counting the number of wells with growth. Chloroquine,

pyrimethamine, atovaquone, and artesunate were used as

controls. The following reagent was obtained through BEI

Resources, NIAID, NIH: P. falciparum, Strain 3D7A, MRA-151,

contributed by David Walliker. The human biological samples

were sourced ethically, and their research use was in accord with

the terms of the informed consents under an IRB/EC-approved

protocol.

utilizing a substrate-speciﬁc interaction approach in human liver

microsomes as recently described³⁷with the exception that the

test compound and reference inhibitors were included at only

two assay concentrations (1 and 10 μM for 19f and furafylline

(CYP1A2 reference inhibitor), 0.2 and 2 μM for sulfaphenazole

(CYP2C9 reference inhibitor), 0.0075 and 0.075 μM for

quinidine (CYP2D6 reference inhibitor), and 0.01 and 0.1 μM

for ketoconazole (CYP3A4 reference inhibitor)). Concentra-

tions of the substrate-speciﬁc metabolites in acetonitrile-

quenched samples were determined by UPLC-MS relative to

calibration standards prepared in a quenched microsomal

matrix. The percent reduction (% inhibition) in the extent of

formation of the speciﬁc metabolite was assessed at each assay

concentration relative to the incubation in the absence of the

inhibitor.

Caco-2 Permeability. Apical-to-basolateral (A−B) perme-

ability across Caco-2 cell monolayers was assessed according to

methods recently described³⁷using an aqueous transport buﬀer

(pH 7.4 Hanks balanced salt solution containing 20 mM

HEPES) in both the apical and basolateral chambers. Donor

solutions were prepared by spiking a DMSO stock solution of

19f into transport buﬀer at a nominal concentration of 20 μM

(ﬁnal DMSO concentration in the donor solution was 0.1% v/

v), and the compound ﬂux was assessed over a period of 120

minutes. Concentrations of 19f were quantiﬁed in acceptor and

donor chamber samples by LC-MS and mass balance, and

apparent permeability (Papp) was calculated as previously

described.

Kinetic Solubility Estimation Using Nephelometry.

Test compounds in DMSO were spiked into either pH 6.5

phosphate buﬀer or 0.01 M HCl (approx pH 2.0) with the ﬁnal

DMSO concentration being 1%. After 30 minutes had elapsed,

samples were then analyzed via Nephelometry to determine a

solubility range.³⁸A pH of 6.5 was used to represent the pH of

the fasted-state upper small intestine as previously reported.³⁹

hERG Assay. The test compound was tested using the

QPatch automated patch clamp system (Sophion, Denmark) at

four concentrations (using 0.5-log unit dilutions) from a top

concentration of 30 μM, against a minimum of two separate

cells. Each four-point concentration−response curve was

constructed using cumulative double sample additions of each

concentration to the same cell. Verapamil was used as the

reference positive control compound.

Pf-Resistant Laboratory Strains and Cross-Resistance.

The testing was performed with the modiﬁed [³H]-hypoxan-

thine incorporation assay, as previously reported.²⁸

In Vitro Metabolism. Metabolic stability was assessed by

incubating test compounds (at a concentration of 0.25 or 0.5

μM) with human, rat, and mouse liver microsomes at 37 °C and

0.4 mg/mL microsomal protein supplemented with an

NADPH-regenerating buﬀer system as described recently.³⁷

The hepatocyte stability assay was performed by incubating 19f

(0.25 μM) with human or rat cryopreserved hepatocytes

suspended in Krebs−Henseleit buﬀer at 37 °C (1 × 10⁶cells/

mL). Microsome and hepatocyte samples were quenched with

the addition of acetonitrile containing diazepam as the internal

standard.

Following protein precipitation, samples were centrifuged and

the supernatant was removed for quantitative analysis by LC-

MS. The in vitro CL_intvalues were calculated from the ﬁrst-order

degradation rate constant.

Sample analysis was conducted using a Waters Xevo G2

QTOF mass spectrometer coupled to a Waters Acquity UPLC

system. The column was a Supelco Ascentis Express C8 column

(50 mm × 2.1 mm, 2.7 μm), and the mobile phase was a water/

acetonitrile (each containing 0.05% formic acid) gradient

delivered with a 4 min cycle time. The ﬂow rate was 0.4 mL/

min, and the injection volume was 5 μL. Detection was

conducted via positive electrospray ionization in the multiple

reaction monitoring mode with a cone voltage of 30 V. Samples

were quantiﬁed by comparison to the response for spiked

calibration standards prepared in a prequenched blank matrix

and analyzed along with the samples.

Stability in Rat and Human Plasma. Human blood was

procured from the Australian Red Cross Blood Service. Rat

blood was obtained from male Sprague Dawley rats procured

from the Animal Resources Centre (Perth). Plasma from each

species was separated and pooled (n = 3 donors for human;

multiple animals for rat) and stored frozen at −80 °C. On the

day of the experiment, plasma was thawed, spiked with DMSO/

acetonitrile/water solutions of 19f to a nominal compound

concentration of 1000 ng/mL (maximum ﬁnal DMSO and

acetonitrile concentrations of 0.2% (v/v) and 0.4% (v/v),

respectively), and vortexed to mix. Spiked plasma was

maintained at 37 °C for 4 h with aliquots taken at various

time points and snap-frozen in dry ice. Concentrations of 19f

were quantiﬁed by LC-MS relative to calibration standards

prepared in blank human or rat plasma, using diazepam as the

internal standard and acetonitrile to precipitate plasma proteins.

The percentage of test compound remaining in the plasma at

each time point was calculated relative to samples taken at 5 min.

Cytochrome P450 Inhibition. Inhibition of cytochrome

P450 (CYP) enzymes 1A2, 2C9, 2D6, and 3A4 was conducted

Whole-Blood-to-Plasma Ratio. The compound was

spiked to a nominal concentration of 1000 ng/mL, into fresh

heparinized whole blood collected from male Sprague Dawley

rats approximately 30 min prior to compound spiking and

maintained at 37 °C. Aliquots of the spiked whole blood were

transferred into fresh microcentrifuge tubes and maintained at

37 °C under a 5% CO₂atmosphere to maintain a stable pH. At

30 min, aliquots of whole blood were collected, the remaining

whole blood was centrifuged for 3 min, and aliquots of the

plasma fraction were collected for assessment of the whole-

blood-to-plasma partitioning ratio. The whole blood and plasma

fraction samples were diluted with an equal volume of the

opposite blank matrix (such that both contained a 1:1 ratio of

blood/plasma) and immediately snap-frozen in dry ice. All

samples were stored frozen at −80 °C until analysis by LC-MS

against standards prepared in the same 1:1 blood/plasma matrix.

The apparent whole-blood-to-plasma partitioning ratio was

calculated as the ratio of the concentration of compound in

blood to that in the plasma fraction of the whole blood sample.

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In Vivo Pharmacokinetic (PK) Studies. Studies in rats and

mice were conducted using established procedures in

accordance with the Australian Code of Practice for the Care

and Use of Animals for Scientiﬁc Purposes, and study protocols

were reviewed and approved by the Monash Institute of

Pharmaceutical Sciences Animal Ethics Committee.

Rat PK studies were carried out using six overnight-fasted

male Sprague Dawley rats weighing 273−290 g. Rats had access

to water ad libitum throughout the pre- and postdose sampling

period, and access to food was reinstated 4 h post dose.

The IV dose was administered as a 6 min constant-rate

infusion via an indwelling jugular vein cannula in vehicle

containing 50% (v/v) DMA/50% (v/v) PEG400 (0.3 mL dose

volume), which had been ﬁltered through a 0.22 μm syringe-tip

ﬁlter prior to dosing. The average measured concentration of 19f

in aliquots of the ﬁltered solution was 1.0 mg/mL. The oral dose

was administered by gavage (5 mL/kg dose volume) as a

suspension in 0.5% (w/v) methyl cellulose in water. The average

measured concentration of 19f in aliquots of suspension

formulation was 1.1 mg/mL. All animals were dosed within

0.5 h of formulation preparation.

Samples of arterial blood and total urine were collected over

24 h post dose. Blood was collected directly into borosilicate

vials (at 4 °C) containing heparin, Complete (a protease

inhibitor cocktail), and potassium ﬂuoride to minimize the

potential for ex vivo degradation. Once collected, blood samples

were centrifuged, and supernatant plasma was transferred to

fresh tubes and stored frozen (−80 °C) until analysis by LC-MS.

The mouse IV PK study was conducted in three male Swiss

outbred mice (25−34 g). Mice had access to food and water

throughout the pre- and postdose sampling period. The IV dose

was administered as a bolus injection into the lateral tail vein (2

mL/kg dose volume) in a solution of 5% DMSO in a base vehicle

of 5% (w/v) Solutol HS-15 in 0.9% (w/v) saline solution, which

was ﬁltered through a 0.22 μm syringe-tip ﬁlter prior to dosing.

The average measured concentration of 19f in aliquots of the

ﬁltered solution was 0.48 mg/mL.

Blood was collected via either submandibular bleed or cardiac

puncture while mice were under gaseous isoﬂurane anesthesia,

into tubes containing heparin, Complete, and potassium

ﬂuoride. A maximum of two samples was taken from each

mouse, and three samples were collected at each time point.

Blood was separated by centrifugation, and the plasma fraction

was analyzed by LC-MS.

metabolite discovery module of the Waters UNIFI operating

system (UNIFI v1.8.0.0).

Plasma concentration vs time proﬁles from individual animals

(for rats) or for the mean at each sample time (mice) were

analyzed using noncompartmental methods (PKSolver Version

2.0).

In Vivo SCID Mouse Model. The animal experiments

adhered to local and national regulations of laboratory animal

welfare in Switzerland (awarded permission no. 2303).

Protocols are regularly reviewed and revised following approval

̈

by the local authority (Veterinaramt Basel Stadt). Studies were

performed following a protocol published previously³⁵using the

Pf 3D70087/N9 strain. Mice were infected intravenously with 2

× 107 Pf-infected erythrocytes on day 0. The test compound 19f

(Na·4H₂O salt) was solubilized (suspended) in a solution

consisting of 70% Tween-80 (d = 1.08 g/mL) and 30% ethanol

(d = 0.81 g/mL), followed by a 10-fold dilution in H₂O.

Experimental mice (NODscidIL2Rγnull female) were treated

orally at days 3, 4, 5, and 6 post infection with the test compound

19f (Na·4H₂O salt) at two diﬀerent doses (50 and 100 mg/kg)

and compared to an infected control group for parasitemia

reduction on day 7. In total, six animals were used (n = 2 mice

per group). Peripheral blood samples (20 μL) were taken at

diﬀerent time points, mixed with 20 μL of H₂O MilliQ,

immediately frozen on dry ice, and stored at −80 °C until

analysis. Blood samples were analyzed by LC-MS/MS for

quantiﬁcation to determine pharmacokinetic parameters.

MMP-1 Human Matrix Metallopeptidase Enzymatic

Assay. The test compound 19f (tested at 10 μM), reference

compound, or water (control) was added to a buﬀer containing

50 mM Hepes (pH 7.0), 10 mM CaCl₂, 0.05% Brij35, and 20 U

MMP-1. The ﬂuorescence intensity was then measured at λ_ex=

340 nm and λ_em= 460 nm using a microplate reader (Envision,

Perkin Elmer). This measurement at t = 0 allows the detection of

any compound interference with the ﬂuorimetric detection

method at these wavelengths. Thereafter, the reaction was

initiated by adding 10 μM of the substrate DNP-Pro-Cha-Gly-

Cys(Me)-His-Ala-Lys(n-Me-Abz)-NH2 and the mixture was

incubated for 20 min at 37 °C. After incubation, the ﬂuorescence

intensity emitted by the reaction product Cys(Me)-His-Ala-

Lys(n-Me-Abz)-NH2 was measured at the same wavelengths (t

= 40). The enzyme activity was determined by subtracting the

signal measured at t = 0 from that measured at t = 40. The results

are expressed as a percent inhibition of the control activity. The

standard inhibitory compound is GM6001, which is tested in

each experiment at several concentrations to obtain an

inhibition curve from which its IC₅₀value is calculated.

LC-MS analysis of 19f in biological samples (plasma, urine, or

mixed matrix samples) was conducted using a Waters Xevo TQ

mass spectrometer coupled to a Waters Acquity UPLC system.

The column was a Supelco Ascentis Express amide column (50

× 2.1 mm, 2.7 μm), and the mobile phase was a water/

acetonitrile (each containing 0.05% formic acid) gradient

delivered with a 4 min cycle time. The ﬂow rate was 0.4 mL/

min, and the injection volume was 3 μL. Detection was

conducted via positive electrospray ionization in multiple

reaction monitoring mode monitoring the transition 472.15 >

320.19 m/z. The cone and CID voltages were 35V and 15 V,

respectively. Samples were quantiﬁed by comparison to the

response for spiked calibration standards prepared in the same

matrix and analyzed along with the study samples.

MMP-14 Human Matrix Metallopeptidase Enzymatic

Assay. The test compound 19f (tested at 10 μM), reference

compound, or water (control) are mixed with 4 ng of MMP-14

in a buﬀer containing 50 mM Tris−HCl (pH 7.5) and 10 mM

CaCl₂. The ﬂuorescence intensity was then measured at λ_ex

=

340 nm and λ_em= 405 nm using a microplate reader (Envision,

Perkin Elmer). This measurement at t = 0 allows the detection of

any compound interference with the ﬂuorimetric detection

method at these wavelengths. Thereafter, the reaction was

initiated by adding 2 μM of the substrate Mca-Pro-Leu-Ala-

Cys(p-OmeBz)-Trp-Ala-Arg(Dpa)-NH2 and the mixture was

incubated for 60 min at 22 °C. After incubation, the ﬂuorescence

intensity emitted by the reaction product Mca-Pro-Leu-Ala was

measured at the same wavelengths (t = 15). The enzyme activity

was determined by subtracting the signal measured at t = 0 from

that measured at t = 15. The results are expressed as a percent

A preliminary search for metabolites in selected plasma and

urine samples from the in vivo rat PK study was performed using

high-resolution mass spectrometry operating in the MS^Emode.

Metabolite identiﬁcation was aided using a software-assisted

R

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inhibition of the control activity. The standard inhibitory

compound is GM6001, which is tested in each experiment at

several concentrations to obtain an inhibition curve from which

its IC₅₀value is calculated.

Susan A. Charman − Centre for Drug Candidate Optimisation,

Monash Institute of Pharmaceutical Sciences, Monash

University, Parkville, Victoria 3052, Australia; orcid.org/

0000-0003-1753-8213

Vicky M. Avery − Discovery Biology, Griﬃth University,

Nathan 4111 Queensland, Australia

Nobuo Cho − Takeda Pharmaceutical Company Limited,

Fujisawa, Kanagawa 251-8555, Japan

Masahiro Kamaura − Takeda Pharmaceutical Company

Limited, Fujisawa, Kanagawa 251-8555, Japan

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00441.

■

sı

¹H NMR and LC-MS data for compounds 9a, 9h, 19f, and

20e; Pf liver dose−response curve for compound 19f; and

blood concentration−time proﬁle of compound 19f in

SCID mice (PDF)

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00441

Author Contributions

Molecular formula strings (CSV)

B.L.: project leader, compound design, writing, original draft;

M.K., N.C.: compound design; B.L., M.K., N.C.: conceptualiza-

tion; S.A.C., V.M.A.: supervisors at CDCO and Griﬃth,

respectively; M.K., N.C., Y.A., A.O., K.L.W., S.D., L.L.: project

team; V.M.A., S.D., L.L.: 3D7, Dd2, LSG, and HEK assays;

S.A.C., D.M.S., G.C., K.K., F.C.K.C., K.L.W.: DMPK.; X.C.,

B.W.: chemistry; A.S., K.J.D.: Pf liver; B.C., L.M.S.: P.R.R.; S.W.:

cross-resistance and SCID. Edits and contributions are from all

authors who read and approved the ﬁnal manuscript.

AUTHOR INFORMATION

Corresponding Author

■

Benoît Laleu − Medicines for Malaria Venture, ICC, 1215

Geneva, Switzerland; orcid.org/0000-0002-7530-2113;

Email: laleub@mmv.org

Authors

Yuichiro Akao − Takeda Pharmaceutical Company Limited,

Fujisawa, Kanagawa 251-8555, Japan

Atsuko Ochida − Takeda Pharmaceutical Company Limited,

Fujisawa, Kanagawa 251-8555, Japan

Sandra Duﬀy − Discovery Biology, Griﬃth University, Nathan

4111 Queensland, Australia

Leonardo Lucantoni − Discovery Biology, Griﬃth University,

Nathan 4111 Queensland, Australia; orcid.org/0000-

0002-7246-0677

David M. Shackleford − Centre for Drug Candidate

Optimisation, Monash Institute of Pharmaceutical Sciences,

Monash University, Parkville, Victoria 3052, Australia

Gong Chen − Centre for Drug Candidate Optimisation,

Monash Institute of Pharmaceutical Sciences, Monash

University, Parkville, Victoria 3052, Australia

Kasiram Katneni − Centre for Drug Candidate Optimisation,

Monash Institute of Pharmaceutical Sciences, Monash

University, Parkville, Victoria 3052, Australia

Francis C. K. Chiu − Centre for Drug Candidate Optimisation,

Monash Institute of Pharmaceutical Sciences, Monash

University, Parkville, Victoria 3052, Australia

Karen L. White − Centre for Drug Candidate Optimisation,

Monash Institute of Pharmaceutical Sciences, Monash

University, Parkville, Victoria 3052, Australia

Xue Chen − WuXi AppTec (Wuhan) Company Ltd., Wuhan

430075, China

Angelika Sturm − TropIQ Health Sciences, 6534 AT Nijmegen,

The Netherlands

Koen J. Dechering − TropIQ Health Sciences, 6534 AT

Nijmegen, The Netherlands

Benigno Crespo − Global Health, GlaxoSmithKline R&D,

Tres Cantos 28760 Madrid, Spain

Laura M. Sanz − Global Health, GlaxoSmithKline R&D, Tres

Cantos 28760 Madrid, Spain; orcid.org/0000-0003-

0796-5211

Binglin Wang − WuXi AppTec (Wuhan) Company Ltd.,

Wuhan 430075, China

Sergio Wittlin − Swiss Tropical and Public Health Institute,

4002 Basel, Switzerland; University of Basel, 4002 Basel,

Switzerland

Funding

Research reported in this publication was supported by the

Global Health Innovative Technology (GHIT) Fund under the

project title “New Hit-to-Lead Activity for New Anti-malarials

between MMV and Takeda” (Project ID: H2016−205)

Notes

The authors declare the following competing ﬁnancial

interest(s): The authors declare the following ﬁnancial

interest(s): BL is a MMV employee; YA, AO are Takeda

employees; BC, LMS are GSK employees.

ACKNOWLEDGMENTS

■

The authors wish to thank the Australian Red Cross Blood

Service for the provision of human red blood cells in accordance

with agreement 19-05QLD-21. Dr. Mitsuyuki Shimada and Dr.

Brice Campo are thanked for facilitating project transfer from

Takeda to MMV.

ABBREVIATIONS USED

■

ADME, absorption-distribution-metabolism-excretion;

ADMET, absorption-distribution-metabolism-excretion-toxic-

ity; CL, clearance; DHA, dihydroartemisinin; DIPEA, N,N-

diisopropylethylamine; DMPK, drug metabolism pharmacoki-

netics; EA, ethyl acetate; Fu, fraction unbound; F, bioavail-

̀

ability; hERG, human ether-a-go-go related gene; HPLC, high-

performance liquid chromatography; HTS, high-throughput

screening; MS, mass spectroscopy; PE, petroleum ether; rt,

room temperature; SAR, structure−activity relationship; THF,

tetrahydrofuran

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Discovery and Structure-Activity Relationships of Quinazolinone-2-carboxamide Derivatives as Novel Orally Efficacious Antimalarials

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