Novel TypeII Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents

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DOI: 10.1002/cmdc.201200407

Novel Type II Fatty Acid Biosynthesis (FAS II) Inhibitors as

Multistage Antimalarial Agents

Florian C. Schrader,^[a]Serghei Glinca,^[a]Julia M. Sattler,^[b]Hans-Martin Dahse,^[c]

Gustavo A. Afanador,^[d]Sean T. Prigge,^[d]Michael Lanzer,^[b]Ann-Kristin Mueller,^[b]

Gerhard Klebe,^[a]and Martin Schlitzer*^[a]

Malaria is a potentially fatal disease caused by Plasmodium par-

asites and poses a major medical risk in large parts of the

world. The development of new, affordable antimalarial drugs

is of vital importance as there are increasing reports of resist-

ance to the currently available therapeutics. In addition, most

of the current drugs used for chemoprophylaxis merely act on

parasites already replicating in the blood. At this point, a pa-

tient might already be suffering from the symptoms associated

with the disease and could additionally be infectious to an

Anopheles mosquito. These insects act as a vector, subsequent-

ly spreading the disease to other humans. In order to cure not

only malaria but prevent transmission as well, a drug must

target both the blood- and pre-erythrocytic liver stages of the

parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) re-

ductase (ENR) is a key enzyme of plasmodial type II fatty acid

biosynthesis (FAS II). It has been shown to be essential for

liver-stage development of Plasmodium berghei and is there-

fore qualified as a target for true causal chemoprophylaxis.

Using virtual screening based on two crystal structures of

PfENR, we identified a structurally novel class of FAS inhibitors.

Subsequent chemical optimization yielded two compounds

that are effective against multiple stages of the malaria para-

site. These two most promising derivatives were found to in-

hibit blood-stage parasite growth with IC₅₀values of 1.7 and

3.0 mm and lead to a more prominent developmental attenua-

tion of liver-stage parasites than the gold-standard drug, pri-

maquine.

Introduction

Over two billion people in approximately 100 countries are at

risk of contracting malaria, a disease that often turns out to be

fatal for young children.^[1]Their chances to survive depend not

least on the development of new, affordable drugs, as increas-

ing resistance against the currently used therapeutic agents is

observed. Malaria is caused by the protozoan Plasmodium and

is transmitted by the Anopheles mosquito. When this insect

feeds on an infected individual, it can ingest Plasmodium ga-

metocytes along with the blood meal. These represent the

only stages in the life cycle of Plasmodium that are infectious

to the mosquito. Inside the mosquito host, Plasmodium sporo-

zoites are subsequently formed. These motile stages travel to

the salivary gland of the mosquito and can be injected along

with its saliva while feeding on another human host. There,

Plasmodium sporozoites migrate actively into the circulation

and finally end up in the liver, where they infect hepatocytes.

It takes around seven to ten days for P. falciparum sporozoites

to subsequently develop into several thousand first-generation

merozoites.^[2]As a consequence, the liver stage is characterized

by huge metabolic demands. Merozoites are then released

into the blood to begin their pathological blood-stage devel-

opment by infecting erythrocytes. Subsequently, the patient

begins to suffer from symptoms like fever, pain and nausea.^[3]

Given the life cycle, antimalarial drug development should be

directed towards compounds that are able to kill the silent

liver stages. In addition, these desired compounds must also

be able to kill blood stage parasites.

[a] Dr. F. C. Schrader, Dr. S. Glinca, Prof. Dr. G. Klebe, Prof. Dr. M. Schlitzer

Institut fꢀr Pharmazeutische Chemie

Philipps Universitꢁt Marburg

Marbacher Weg 6, 35032 Marburg (Germany)

E-mail: schlitzer@staff.uni-marburg.de

Replicating in the liver as well as in erythrocytes, Plasmodi-

um parasites require vast amounts of fatty acids (FA). Metabo-

lism in these two stages of the life cycle, however, is funda-

mentally different.^[4,5]In the blood stage, the vast majority of

the FAs are acquired from the host.^[6]In earlier studies, it was

assumed that Plasmodium acquires FAs merely by scavenging,

however, Plasmodium was recently found to be capable of

type II fatty acid biosynthesis (FAS II). FAS enzymes are target-

ed to the apicoplast, a relict, non-photosynthetic plastid of

algal origin. In most plants as well as in bacteria, discrete en-

zymes catalyze the distinct steps in plasmodial FAS II

[b] Dr. J. M. Sattler, Prof. Dr. M. Lanzer, Dr. A.-K. Mueller

Dept. fꢀr Infektiologie: Parasitologie

Universitꢁtsklinikum Heidelberg

Im Neuenheimer Feld 324, 69120 Heidelberg (Germany)

[c] Dr. H.-M. Dahse

Leibniz-Institut fꢀr Naturstoff-Forschung und Infektionsbiologie

Hans-Knçll-Institut

Beutenbergstraße 11, 07745 Jena (Germany)

[d] G. A. Afanador, Prof. Dr. S. T. Prigge

Department of Molecular Microbiology and Immunology

Johns Hopkins Bloomberg School of Public Health

615 N. Wolfe Street, E4628 Baltimore, MD 21205 (USA)

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Table 1. Inhibitory effects (IC₅₀values) of existing PfENR inhibitors on iso-

lated enzyme and blood-stage cultured parasites.^[a]

(ꢀ)-Catechin gallate

0.3 mm

Oroidin

0.8 mm

Triclosan

48 nm

PfENR (FabI)

P. falciparum

3.2 mm (NF54)

0.4 mm (K1)

10 mm

3.3 mm

[a] Data taken from Ref. [17] for (ꢀ)-catechin gallate and Ref. [18] for oroi-

din; triclosan (own data). (ꢀ)-Catechin gallate was evaluated against

a chloroquine-sensitive (NF54) and -resistant (K1) strain, ornidin against

the K1 strain, and triclosan against DD2.

Scheme 1. Type II fatty acid biosynthesis. The most vital precursor to fatty

acids in Plasmodium is acetyl-CoA, which is provided by acetyl-CoA synthase

or pyruvate dehydrogenase. Fatty acid biosynthesis begins with the carbox-

ylation of acetyl-CoA by acetyl-CoA carboxylase (ACC). The resulting prod-

uct, malonyl-CoA, is then converted to malonyl-acyl carrier protein (ACP) by

malonyl-CoA–ACP transacylase (FabD). ACP is a small, acidic protein that

binds acyl intermediates as thioesters during fatty acid synthesis and carries

them between enzymes. The first reaction is a condensation catalyzed by b-

ketoacyl-ACP synthase III (FabH), which uses acetyl-CoA and malonyl-ACP as

substrates. Next is a NADPH-dependent reduction of b-ketoacyl-ACP to b-hy-

droxyacyl-ACP catalyzed by b-ketoacyl-ACP reductase (FabG). b-Hydroxyacyl-

ACP dehydratase (FabZ) then forms trans-2-enoyl-ACP by removing a water

molecule from the acyl chain. trans-Enoyl-ACP is finally NADH-dependently

reduced by enoyl-ACP reductase (FabI), which presents the rate-limiting step

in fatty acid chain elongation. Another molecule of malonyl-ACP can subse-

quently be used to add two more carbon atoms to the nascent acyl chain

by b-ketoacyl-ACP synthase II. This cycle continues with the length of the

acyl chain increasing by two carbons until the desired fatty acid is pro-

duced.

tured parasites as well (see Table 1). Hence, it is tempting to

speculate on a common off-target.^[12,13]The genome of Plasmo-

dium appears to encode for three different fatty acid elongases

(ELOs). In contrast to FAS type I and II, ELO pathways use CoA

rather than ACP as an acyl carrier. Importantly, ELO pathways

contain an enoyl-CoA reductase (EnCR) that catalyzes a similar

reaction to that of PfENR. Although ELO pathways typically

elongate long-chain FAs such as palmitate,^[14]trypanosomes

were recently shown to synthesize most of their FAs from bu-

tyryl-CoA precursors.^[15]So, we speculated that enzymes able

to catalyze similar reactions to ENR and EnCR might also have

active sites of similar topology, and therefore, ENR could serve

as a model for the as yet uncharacterized EnCR—in other

words, compounds that inhibit ENR should also, due to the

presumed similarity of these two enzymes, inhibit EnCR and

should therefore display the mandatory activity against blood-

stage parasites, which was verified by an assay against these

stages.

(Scheme 1). In contrast to this, in mammals, FAS is performed

by a multi-enzyme complex, handling all four of the enzymatic

steps of the elongation of fatty acids (FAS I). Although there is

no difference in mechanism in the elongation of fatty acid

chains, this fundamentally distinct setup of enzymes makes

FAS I insensitive to a number of FAS II inhibitors and qualifies

fatty acid biosynthesis in Plasmodium as a potential drug

target.

FAS II, and ENR in particular, has been shown to play a key

role in the development of liver-stage malaria parasites.^[4,12]

ENR-deficient Plasmodium berghei sporozoites are markedly

less infective to mice and typically fail to complete liver-stage

development in vitro. This defect is characterized by an inabili-

ty to form intrahepatic merosomes, which normally initiate

blood-stage infections.^[12]Even though it is not clear how tri-

closan and other FAS II inhibitors act upon blood-stage para-

sites, present data suggest that FAS II inhibitors might provide

true causal chemoprophylaxis and could simultaneously cure

blood-stage Malaria.^[4,12]

Triclosan is an antibacterial and antifungal agent commonly

used in a large variety of consumer products such as tooth-

pastes and pillowcases. In 1998, it was shown to be an Esche-

richia coli FAS inhibitor and to specifically inhibit E. coli enoyl

acyl carrier protein (ACP) reductase (ENR).^[7]The contemporary

discovery of the plastidial origin of the apicoplast and its sug-

gestion as a drug target^[8]prompted efforts to assay triclosan

as an antiplasmodial agent. Triclosan proved to inhibit P. falcip-

arum (Pf) ENR with a K_ivalue of 0.4 nm^[9]and, in addition, to

prevent the growth of blood-stage P. falciparum at a low mi-

cromolar concentration.^[10,11]Subsequent work by Yu et al.^[4]

showed that FAS II is essential in liver-stage parasites but not

in blood-stage parasites. Nevertheless, triclosan and other in-

hibitors of plasmodial FAS, and PfENR inhibitors in particular,

frequently do show an inhibitory effect on blood-stage cul-

Results and Discussion

In an effort to find structurally novel, potent FAS inhibitors, we

performed a virtual screen based on the two crystal structures

of PfENR currently available. The inhibitor binding pocket of

both PfENR and Toxoplasma gondii ENR (TgENR) are highly con-

served with only one amino acid difference (Figure 1).^[16]There-

fore, we used a Toxoplasma homologue that was available to

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Figure 1. The inhibitor binding sites of a) PfENR and b) TgENR shown here in

complex with triclosan (PDB: 1NHG^[22]and 2O2S^[16]).

us for the enzyme inhibition assay. Of compounds derived

from virtual screening, eight molecules were selected with re-

spect to sufficient drug-likeness, chemical diversity, synthetic

accessibility and easy scope of variation. In a first step, minor

modifications were performed to the chemical structure of

these eight promising screening hits in order to overcome ob-

vious metabolic instability or to facilitate convenient synthesis

(shown in bold in Table 2). The intended modifications were

validated by subsequent docking to ensure predicted binding

to the target protein and consistency with the derived phar-

macophore were still fulfilled.

Figure 2. Co-crystal structures of PfENR and NAD⁺with bound inhibitor:

a) triclosan (PDB: 2O2Y^[16]) and b) 5-substituted triclosan derivative (PDB:

2OOS^[19]). Introduction of a large hydrophobic substituent at the 5-position

of triclosan induces a conformational transition of Phe368. Hydrogen-bond

interactions of the inhibitor phenolic hydroxy group with Tyr277 and the

2’-hydroxy group of the nicotinamide ribose are depicted as red dashed

lines. For clarity, Ile323 and Val222 are not shown.

house fragment-like library (Table 3) were docked into the re-

spective pocket using GOLD.^[20]Results were reranked applying

the DSX scoring function.^[21]Eight chemically diverse hits were

selected (Table 2), which satisfied the pattern of interactions

supposed to be essential for inhibitor binding as indicated by

the reference crystal structures.^[22]Docked compounds were re-

quested to exhibit stacking interactions with the nicotinamide

moiety of NAD⁺and to form hydrogen bonds with Tyr277 and

the 2’-hydroxy group of the nicotinamide ribose. The docking

poses obtained are depicted in Figure 3a–h.

Virtual screening

Docking experiments were performed using two different

PfENR crystal structures (PDB codes: 2O2Y^[16]and 2OOS^[19]), as

the structures indicate that the active site residue Phe368 can

adopt two alternative conformations (Figure 2). The PfENR

binding site hosts an NAD⁺molecule, which was retained

during docking as an integral part of the pocket, since it is

tightly bound. Deleting NAD⁺would have led to undesirably

large hits. A total of 13200 compounds retrieved from an in-

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accessed via compound 5c, pre-

pared as depicted in Scheme 2.

Acidic hydrolysis of the acetyl

ester group of 5c yielded salicyl-

ic acid amide derivative 1a.

Compounds 1b–e were ob-

tained by the formation of their

Table 2. Suggested and actual structures of amide-containing salicylic acid derivatives 1a–h, their antimalarial

activity against P. falciparum (IC₅₀) and their cytotoxicity (CC₅₀) in HeLa cells.

Compd

IC₅₀^[a][mm]

CC₅₀^[b][mm]

suggested

synthesized

0a

1a

7.9ꢁ0.3

59.3

corresponding

imines

2b–

e using aromatic aldehydes and

the respective amino or hydra-

zino derivatives. All imines were

isolated as aromatic Schiff bases

prior to their reduction to the

corresponding amines using

sodium cyanoborohydride. Com-

pounds 1 f and 1g were conven-

iently prepared in a single step

1b

1c

1b as suggested

1c as suggested

>50

<189.2

<185.7

via

a

Mannich condensation

using benzotriazole and phthal-

imide, respectively, formalde-

hyde and the appropriate aniline

derivative. Benzoxazole 1h was

synthesized by first condensing

the appropriate aldehyde and

amino derivative to form imine

2g. From there, we attempted

a number of literature methods

to access the desired benzoxa-

zole by oxidative cyclization of

the 2-(benzylideneamino)phenol

using one of the following

agents or procedures: 2,3-di-

chloro-5,6-dicyano-1,4-benzoqui-

none (DDQ), UV irradiation,

active charcoal/O₂, lead(IV) ace-

tate. The only procedure that

was successful used lead(IV) ace-

tate as the oxidizing agent.

1d

0e

1d as suggested

>50

<185.6

1e

136.4

0 f

1 f

>50

<185.7

159.2

1g

0h

1g as suggested

1h

26.11

[a] Data represent the mean of n=3 independent experiments performed in triplicate. [b] Data are the result

of four experiments.

Chemical synthesis

For compound 1a, the virtual

screening hit (0a; Table 2 ) suggested an ester function to link

The eight most promising hits were synthesized in one to four

steps. Full details are given in the Experimental Section and

compound structures are given in Table 2. Compound 1a was

the benzoic acid and the aryloxyalkyl moiety. However, since

Table 3. Physicochemical properties of the fragment-like library used for

screening.

Property

Min

Max

no. of heavy atoms

8

122

0

1

ꢀ1.2

0

20

360

4

8

7.6

7

MW^[a][gmol^ꢀ1

]

Lipinski donor

Lipinski acceptor

ClogP^[b]

free rotatable bonds

TPSA^[c][ꢁ²]

Scheme 2. Synthesis of aryloxyalkylbenzamide derivatives. Full structures of

all compounds tested can be found in Tables 4–6. Reagents and conditions:

a) 2-Chloroacetonitrile, K₂CO₃, KI, 2-butanone, reflux, 6 h; b) LiAlH₄, Et₂O, RT,

2 h; then HCl/Et₂O (2m), 08C, to precipitation; c) Substituted acyl chloride,

Et₃N, THF, 08C!RT, 2 h.

12

126

[a] Molecular weight (MW); [b] calculated (C) logP (octanol/water);

[c] total polar surface area (TPSA).

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Figure 3. Top-ranked docking poses of the eight most promising hits (0a, e, f, h and 1b–d, g) from virtual screening (a–h). All compounds comprise an aro-

matic moiety, which is able to establish stacking interactions with the nicotinamide portion of NAD⁺. Hydrogen bonding with Tyr277 and/or the 2’-hydroxy

group of the ribose can be formed by a carbonyl oxygen (a, b, g, h) or by nitrogen atoms (c–f).

ester groups are prone to intracellular cleavage by nonspecific

esterases, we decided to replace this group with a more stable

amide function. In a subsequent docking experiment, no con-

siderable disadvantage in binding compared with the ester an-

alogue was suggested. Scheme 2 illustrates the synthetic route

for the preparation of the aryloxyalkylbenzamide derivatives

(1a, 5c–g, 5j–k, 6a–g, 6i–x). The reaction of bromoethylbenz-

amide derivatives with appropriate phenolates under various

conditions did not result in the formation of the expected

ether; instead, we could only isolate the elimination product.

The same was observed using bromoalkylphthalimide deriva-

tives. The reaction of 2-substituted acetonitrile derivatives

(2-chloro and 2-bromoacetonitril), however, led to the forma-

tion of the desired ether derivatives in good yields. No differ-

ence in yield was observed when either 2-bromoacetonitrile or

2-chloroacetonitrile was used. Although the reaction proceed-

ed markedly faster when using the bromo derivative, we de-

cided to use the more readily available chloro derivative. Com-

pared with acetone, 2-butanone as a solvent generally gave

rise to a 10% higher yield. The obtained nitrile derivatives

were reduced by lithium aluminum hydride to form the corre-

sponding amino derivatives. These were isolated as their hy-

drochloride salts by the addition of a solution of HCl in ether

and were subsequently acylated. For acylation, freshly pre-

pared or commercially available acyl chlorides were used in

tetrahydrofuran. In case of salicylic acid derivatives, acetylsali-

cylic acid chloride was used for acylation. Cleavage of the

acetyl ester under acidic conditions furnished the appropriate

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derivative of salicylic acid (1a,

5d, 5j). This synthetic route al-

lowed us to use virtually any of

the easily available phenol and

benzoic acid derivatives to pro-

duce a series of inhibitors ready

for testing.

Table 4. Structures of amide-containing salicylic acid derivatives 1a, 5c–g, and 5j–k, their antimalarial activity

against P. falciparum (IC₅₀) and their cytotoxicity (CC₅₀) in HeLa cells.

Compd

R₁

R₂

IC₅₀^[a][mm]

7.9ꢁ0.3

CC₅₀^[b][mm]

SI^[c]

1a

59.3

12.1

Biological evaluation and struc-

ture–activity relationships

5c

7.7ꢁ0.4

66.7

14.2

Activity against the asexual

blood-stage parasite is consid-

ered to be a mandatory property

of the intended compounds.

Therefore, all synthesized com-

pounds were first tested for their

inhibitory activity against cul-

tured blood-stage P. falciparum

(multidrug-resistant Dd2 isolate).

This way, the compounds could

also prove themselves to be suf-

ficiently cell-permeable, which

poses a critical hurdle to over-

come by appropriate drug

design of compounds targeting

living parasites intracellularly.

5d

5e

5 f

13.4ꢁ0.6

24.3ꢁ2.2

8.4ꢁ0.4

137.2

>195.8

133.5

10.2

8.0

15.9

5g

>50

73.1

ND

5j

3.0ꢁ0.2

>162.7

>54

5k

25.0ꢁ1.8

19.5

0.8

Out of our eight promising

hits from virtual screening, the

aryloxyalkylbenzamide derivative

(1a) inhibited the growth of

blood stage Dd2 malaria para-

sites in the cell-based assay with

[a] Data represent the mean of n=3 independent experiments performed in triplicate. [b] Data are the result

of four experiments. [c] Selectivity index (SI): CC₅₀(HeLa)/IC₅₀(P. falciparum). ND=not determined.

an IC₅₀value of 7.9 mm (Table 4). When evaluated against isolat-

ed enzyme at a concentration of 1 mm, inhibition of TgENR by

1a was rather poor (10%). Nevertheless, in the following ex-

periments, we tried to evaluate whether this scaffold could be

chemically optimized to obtain a dual inhibitor of both blood-

stage Plasmodium and TgENR.

The docking pose of 1a suggests that its polar 2-hydroxy

substituent does not specifically interact with the inhibitor

binding site of ENR (Figure 4). At this position, nonpolar sub-

stituents might lead to an enhanced affinity toward the

enzyme as they could potentially interact with the hydropho-

bic amino acids Ile323 and Ala320. Therefore, we synthesized

derivatives 6a and 5e, which either bear such a nonpolar

2-substituent (6a) or lack this group entirely (5e). Interestingly,

both derivatives inhibit the TgENR enzyme (Figure 5).

In the cell-based assay, removal of the 2-substituent results

in a compound (5e) that exhibits a threefold decrease in activi-

ty (IC₅₀=24.3 mm) compared with 1a. However, 2-chloro deriv-

ative 6a shows no decrease in activity against blood-stage par-

asites (IC₅₀=7.5 mm) and displays an even lower cytotoxicity

(CC₅₀>172.6 mm, Table 5; c.f. CC₅₀=59.3 mm for 1a, Table 4). As

the 2-chlorobenzoic acid derivative (6a) inhibits both blood-

stage parasites and TgENR, it qualifies as a lead structure for

subsequent optimization.

Figure 4. Docking pose for 1a.

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Table 6. Structures of various aryl amide derivatives, their antimalarial ac-

tivity against P. falciparum (IC₅₀) and their cytotoxicity (CC₅₀) in HeLa cells.

Compd

R¹

IC₅₀^[a][mm]

10.5ꢁ0.9

CC₅₀^[b][mm]

SI^[c]

6i

170.0

16.2

Figure 5. Inhibition of TgENR by 1a and its derivatives at a concentration of

1 mm.

6j

43.1ꢁ1.3

26.6ꢁ0.8

>50

>154.7

132.9

ND

5.0

ND

6l

Table 5. Structures of amide derivatives of 2-chloro-benzoic acid 6a–g,

their antimalarial activity against P. falciparum (IC₅₀) and their cytotoxicity

(CC₅₀) in HeLa cells.

6m

>156.5

6n

6o

6p

>50

>142.9

124.6

ND

Compd

R¹

IC₅₀^[a][mm]

7.5ꢁ0.5

CC₅₀^[b][mm]

>172.6

SI^[c]

ND

>50

6a

50.0ꢁ0.8

>149.4

6b

6c

6d

>50

113.7

127.9

60.8

ND

3.9

1.9

6q

6r

10.5ꢁ0.8

>50

>164.1

ND

32.6ꢁ1.7

32.0ꢁ1.9

117.5

6s

6t

>50

154.1

119.4

ND

6.7

ND

6e

22.0ꢁ0.5

105.4

4.8

17.8ꢁ1.0

>50

6 f

2.5ꢁ0.1

1.7ꢁ0.1

60.1

89.6

24.4

52.7

6u

6v

>172.0

6g

[a] Data represent the mean of n=3 independent experiments performed

in triplicate. [b] Data are the result of four experiments. [c] Selectivity

index (SI): CC₅₀(HeLa)/IC₅₀(P. falciparum). ND=not determined.

>50

6w

6x

>50

>133.3

>140.9

ND

Initially, we evaluated whether selected heteroatoms are

mandatory in this compound. We subsequently varied the ap-

parently important 2-substituent introducing a fluoro- and tri-

fluoromethyl group. For the 2-fluoro (IC₅₀=6i, 10.5 mm) and

2-trifluoromethyl (6j, IC₅₀=43.1 mm) derivatives, there was no

considerable improvement in the cell-based assay (Table 6). We

also prepared a derivative with a 3-chloro-substituted benzoic

acid moiety (6l) instead of attachment at the 2-position. Com-

pared with the 2-chloro-substituted derivative (6a), this

change led more than a threefold decrease in activity (IC₅₀=

26.6 mm) in the cell-based assay. These data led us to conclude

that the 2-position of the benzoic acid moiety is optimal for

substitution. Apparently, the substituent does not necessarily

[a] Data represent the mean of n=3 independent experiments performed

in triplicate. [b] Data are the result of four experiments. [c] Selectivity

index (SI): CC₅₀(HeLa)/IC₅₀(P. falciparum). ND=not determined.

act as a hydrogen-bond donor or acceptor, as either the hy-

droxy- or chloro-substituted derivatives show equipotent in-

hibition. We prepared the sulfur homologue (6d) by using

thiocresolate as a nucleophile in the ether synthesis. As there

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was about a fourfold decrease in activity (IC₅₀=32.0 mm;

Table 5) in the cell-based assay, we kept the original composi-

tion.

Based on the 2-chloro benzoic acid moiety (6a), a series of

derivatives were prepared to optimize the lead structure. Dock-

ing of 6a suggested a hydrogen-bonding interaction with the

hydroxy group of Tyr267 within the binding site. Therefore,

a series incorporating hydrogen-bond acceptor and/or donor

functionalities in the 3- and 4-positions of the benzoic moiety

were synthesized and tested (6m–n, 6p–r, 6t). None of these

compounds showed improved activity in the cell-based assay

compared with lead compound 6a. Strikingly, introduction of

a polar group in almost all cases led to a complete loss of ac-

tivity in the cell-based assay. The only derivatives that at least

partially maintained activity were the 4-amino (6q, IC₅₀=

10.5 mm) and the 4-hydroxy (6t, IC₅₀=17.8 mm). We therefore

abandoned the idea of introducing polar groups at the 4-posi-

tion.

In an attempt to increase the activity through an entropic

gain in binding energy, we conformationally rigidified the mol-

ecule by incorporating a fixed linker between the aromatic

rings by using as a 3-substituted azetidine (6c, IC₅₀=32.6 mm)

and a 3-subsitituted piperidine (6b, IC₅₀>50 mm) heterocycles.

As these modifications were clearly detrimental to the activity

in the cell-based assay, it seems that the most favorable con-

formations required for binding of the inhibitors are not easily

accessible by incorporation of the two tested rigid linkers. This

prompted us to retain the original oxyethylamide moiety as

a linker.

Figure 6. Docking pose for 5j.

and naphthyloxy derivative 6g (IC₅₀=1.7 mm), we observed

a three- to fourfold improvement in activity as determined in

a cell-based assay. At a concentration of 1 mm, 6g showed

slightly higher inhibition (58%) in the TgENR enzyme assay

when compared with lead compound m-tolyloxy 6a (41%)

(Figure 7). In addition, 6g displayed a selectivity index (SI) of

more than 50 (Table 5).

Combinatorial library

Encouraged by this observation, we also prepared derivative

5j, which includes both a naphthoxy- and a salicylic acid

moiety connected by an ethylamine linker. Compound 5j is

the most active derivative in our series of salicylic acid amides

in the cell-based assay (IC₅₀=3.0 mm; Table 4). Compound 5j

also inhibits TgENR activity by 58% at 1 mm and displays an SI

value of more than 50.

To elaborate the chemical space and the potential of aryloxyal-

kylbenzamide derivatives 1a, 5c–g, 5j–k, 6a–g, 6i–x, a combi-

natorial virtual library was generated considering commercially

available agents. This library contained about 430000 mole-

cules with a molecular weight (MW) of less than 600 Da. A sub-

group of 10000 compounds was preselected from the com-

puted library using a coarse-grained docking procedure, fol-

lowed by a second, more extensive docking run. The results

were reranked by DSX and visually inspected. The most prom-

ising derivatives were selected for synthesis and subsequent

biological testing. The top-ranked compound comprised

a naphthaleneoxy (6g) instead of the m-tolyloxy moiety found

in 6a. In our model, this derivative is predicted to exhibit the

same interaction patterns as the initial compound (1a) but the

increased molecular surface of the naphthyl moiety likely pro-

vides more van der Waals interactions with hydrophobic resi-

dues Val222, Ala319, Ala322, Ile323, and additionally Asn218

(Figure 6).

In order to confirm that this improvement in activity can be

attributed to a directional interaction rather than to increased

Docking into the crystal structure suggested that the oxyaryl

moiety of 4-isopropylbenzoxy 6e, 5,6,7,8-tetrahydronaphtha-

leneoxy 6 f, and naphthaleneoxy 6g interacts with lipophilic

amino acid residues. We synthesized these compounds and

observed a decline in activity in the cell-based assay for the

isopropylbenzeneoxy derivative (6e; IC₅₀=22.0 mm). In con-

trast, for tetrahydronaphthyloxy derivative 6 f (IC₅₀=2.5 mm)

Figure 7. Inhibition of TgENR by structurally optimized derivatives of 1a at

a concentration of 1 mm.

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lipophilicity, we also prepared 1-naphthoxy derivative 5k. This

compound showed an eightfold decrease in activity (IC₅₀=

25.0 mm) compared with 2-naphthoxy derivative 5j. Therefore,

compounds 6g and 5j represent the most promising deriva-

tives in this series so far and were tested for plasmodial sporo-

zoite- and liver-stage inhibition.

cal microscopy with subsequent image processing, we mea-

sured the diameter of the maturing intrahepatic liver-stage

parasites as an indication of the developmental status. Striking-

ly, we found that at reasonably early time points (24 h) after

sporozoite inoculation, treatment with 6g at 40 mm caused

a significant developmental delay compared with the infection

control (Figure 9d; p<0.0001); representative confocal images

are shown in Figure 10. More importantly, at later time points

(40 h and 60 h) during liver-stage development, we observed

a significant attenuation of liver-stage growth even at lower

concentrations (2 mm) for both compounds 6g and 5j (Fig-

ure 9e,f; p<0.0001).

Evaluation of the effect on pre-erythrocytic parasites

In order to address the question of whether compounds 6g

and 5j have an effect on the motor machinery of the parasite,

crucial for the invasion of liver cells (that is to say the infective

sporozoites), we first performed two-color host-cell invasion

assays. Salivary gland sporozoites of P. berghei were applied to

immortalized human hepatoma cell lines (HuH7) and allowed

to invade for 90 min in the presence of different concentra-

tions of either compound 6g or 5j. Quantification of the num-

bers of extracellular versus intracellular parasites revealed that

we could not observe any effect on the invasion of liver cells

compared with the DMSO-treated control (Figure 8).^[23,24]

Interestingly, when compared with primaquine, a member of

the 8-aminoquinoline group of antimalarial agents exclusively

active against the intrahepatic stages, at a standard concentra-

tion of 10 mm, compounds 6g and 5j exert more potent inhibi-

tion of malarial liver-stage growth with determined IC₉₀values

at 60 h of 2.79 mm and 3.14 mm, respectively. The activity of pri-

maquine in liver cells relies on metabolism of the parent com-

pound to an active metabolite, while our compounds presum-

ably act in the nonmetabolized state. One could argue that

the low in vitro activity of primaquine could be due to poor

metabolism to the active agent in this cell system. However, it

has been shown that, in HuH7 cells, DMSO significantly in-

creases the mRNA levels of most drug-metabolizing enzymes

and liver-specific proteins.^[26]As described above, compounds

6g and 5j inhibit blood-stage parasites with IC₅₀values of

1.7 mm and 3.0 mm, respectively, making them more active

against these parasites than primaquine, for which the IC₅₀is

reported to be in the range of 10–20 mm.^[27]

Next we tested the inhibitory effect of compounds 6g and

5j on the development of the exoerythrocytic form of the par-

asite (the clinically silent liver stage) by applying 6g or 5j to

the culture medium after invasion of infectious P. berghei spor-

ozoites into HuH7 cells. After early- (24 h), mid- (40 h) and late-

(60 h) intrahepatic development, cells were fixed and liver

stage parasites were visualized by immunostaining of intracel-

lular malarial HSP70.^[24,25]Interestingly, the number of liver

stage parasites determined by immunofluorescence microsco-

py was not significantly different under the various conditions

tested (Figure 9a–c), except for a decrease in liver-stage para-

site numbers when compound 6g was applied at a concentra-

tion of 40 mm (Figure 9a). However, growth inhibition is caused

by compound incubation, resulting in fairly small-sized liver-

stage parasites, and as such, we most likely failed to incorpo-

rate every single fluorescent liver-stage parasite. Using confo-

Conclusions

We have presented novel type II fatty acid biosynthesis (FAS II)

inhibitors that inhibit the growth of both blood-stage and pre-

erythrocytic (liver-stage) malarial parasites. The lead com-

pounds identified are structurally different from other inhibi-

tors of FAS in malaria parasites (for a recent Review, see

Ref. [28]). Compared with primaquine, the gold-standard, com-

pounds 6g and 5j show at least a fivefold enhanced inhibitory

effect on both the development of clinically silent liver-stage

as well as disease-inducing erythrocytic blood-stage P. falcipa-

rum parasites. Due to their low cytotoxicity in human (HeLa)

cells and their overall drug-like chemical properties (Table 7),

these compounds can be considered as promising candidates

for further development and evaluation in animal models of

malaria infection. Altogether, this work provides evidence for

novel concepts in chemical treatment of pre-erythrocytic ma-

larial parasites and the pharmacological management of Plas-

modium infections.

Experimental Section

Figure 8. Compounds 6g and 5j have no effect on the invasion of HuH7

cells in vitro. Infectious P. berghei sporozoites were pre-incubated with either

0.5% DMSO, 10 mm cytochalasin D (CytD), or test compound at different

concentrations (0.2 mm, 2 mm or 40 mm) and allowed to invade for 90 min

under drug cover. After sporozoite double-staining, 50 sporozoites were

counted and classified as invaded (inside: &) or non-invaded (outside: &).

¹H NMR and ¹³C NMR spectra were recorded on a Jeol ECA-500 and

a Jeol EX-400 spectrometer. Mass spectra (MS) were recorded on

a Varian MAT CH7a and a S.I.S VG 7070. Infrared (IR) spectra were

recorded on an Bruker Alpha-P attenuated total reflectance fourier

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Figure 9. Establishment of exoerythrocytic stages is not impaired but the development is significantly compromised in vitro. 24 h, 40 h or 60 h after infection

of human hepatoma HuH7 cells with infectious P. berghei sporozoites, cells were fixed, stained with anti-PbHSP70 and analyzed by immunofluorescence mi-

croscopy. We could not observe any difference in numbers of exoerythrocytic forms (a–c); only compound 6g applied at a concentration of 40 mm caused

a prominent decrease in the number of liver stage parasites at 40 h and 60 h after infection (b and c). Measurements of liver-stage growth over time revealed

that already at 24 h, compound 6g at 40 mm caused significant developmental delay compared with the DMSO control (d). At later time points, even 2 mm of

either compound 6g or 5j could arrest the growth of exoerythrocytic stages significantly (e and f). Primaquine (PQ) at 10 mm was used as a positive control

(p<0.0001).

Figure 10. Representative confocal pictures of malarial exoerythrocytic stages at 24 h, 40 h and 60 h after infection with infectious PbANKA sporozoites (scale

bar=10 mm). Primaquine (PQ) at 10 mm was used as a positive control.

transform (ATR FT) spectrometer. Melting points (mp) were mea-

sured on a Barnstead Thermolyne MEL-TEMP-II microscope and are

uncorrected. Reagents and solvents were purchased from ABCR,

Alfa Aesar, Fluorochem, Matrix Scientific, Merck, Sigma–Aldrich and

Thermo Fisher Scientific, and were purified by distillation or recrys-

tallization, if necessary. Chromatography refers to column flash

chromatography using the indicated eluent and Macherey–Nagel

silica gel (0.040–0.063 mm).

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for 30 min and then poured into H₂O (250 mL). A solid was sepa-

rated and recrystallized.

Table 7. Physicochemical properties of compound 6g.

Property

General procedure 7: Preparation of acyl chlorides: The respec-

tive carboxylic acid was dissolved in CH₂Cl₂(sufficient for complete

dissolution) and oxalyl chloride (1.5 equiv) was added. The reaction

mixture was stirred for 2 h at RT, and the solvent and excess oxalyl

chloride were evaporated in vacuo.

no. of heavy atoms

MW^[a][gmol^ꢀ1

23

325.8

1

]

Lipinski donor

Lipinski acceptor

ClogP

free rotatable bonds

TPSA^[b][ꢁ²]

3

4.6260

6

38.33

2-Hydroxy-N-(2-(m-tolyloxy)ethyl)benzamide (1a): Prepared ac-

cording to general procedure 6 from 5c (410 mg, 1.31 mmol), gla-

cial AcOH (25.0 mL, 0.44 mol) and 32% aq HCl (1.0 mL, 10.3 mmol).

[a] Molecular weight (MW); [b] calculated (C) logP (octanol/water);

[c] total polar surface area (TPSA).

Recrystallization from toluene gave

a white crystalline solid

1

(327 mg, 92%); mp: 928C; H NMR (400 MHz, [D₆]DMSO): d_H=2.26

(s, 3H), 3.67 (ps q, J=5.7 Hz, 2H), 4.11 (t, J=5.8 Hz, 2H), 6.74–6.78

(m, 3H), 6.87–6.92 (m, 2H), 7.13–7.17 (m, 1H), 7.38–7.42 (m, 1H),

7.86–7.89 (m, 1H), 9.02 (t, J=5.8 Hz, 1H), 12.45 ppm (s, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 38.6, 65.6, 111.4, 115.1,

115.3, 117.3, 118.6, 121.4, 127.9, 129.2, 133.7, 139.0, 158.3, 159.7,

168.9 ppm; IR (ATR): n˜ =3356, 3054, 2943, 1645, 1583, 1539, 1490,

1251, 1162, 752 cm^ꢀ1; MS (ESI): m/z (%): 272 [M+H]⁺(100), 294

[M+Na]⁺(84), 565 [2M+Na]⁺(20); HRMS (EI): m/z calcd for [M]⁺:

271.1208, found: 271.1193.

General procedure 1: Preparation of imine derivatives: The re-

spective amino or hydrazino derivative and the appropriate alde-

hyde were dissolved in MeOH (20 mL). The mixture was heated at

reflux for 3 h and then poured into H₂O (250 mL). A solid was sepa-

rated and recrystallized.

General procedure 2: Reduction of imine derivatives: The re-

spective imino derivative, NaBH₃CN (2 equiv) and glacial AcOH

(3 equiv) where suspended in 1,2-dichloroethane (15 mL). The reac-

tion mixture was stirred for 2 h at RT. The reaction was quenched

with saturated aq Na₂CO₃and extracted with CH₂Cl₂(3ꢂ30 mL).

The organic layer was separated and dried over MgSO₄, the solvent

was removed in vacuo, and the crude product was purified by

chromatography.

3-((m-Tolylamino)methyl)chinolin-2-ol (1b): Prepared according

to general procedure 2 from 2 f (302 mg, 1.15 mmol), NaBH₃CN

(145 mg, 2.3 mmol) and glacial AcOH (0.21 mL, 3.45 mmol). Purifi-

cation by chromatography (Et₂O) gave a white crystalline solid

(238 mg, 78%); mp: 1858C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=

2.15 (s, 3H), 4.13 (d, J=5.4 Hz, 2H), 5.99 (t, J=6.0 Hz, 1H), 6.35–

6.41 (m, 3H), 6.92–6.95 (m, 1H), 7.11–7.15 (m, 1H), 7.30–7.59 (m,

3H), 7.72 (s, 1H), 11.84 ppm (s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO):

d_C=21.3, 41.9, 109.3, 112.8, 114.8, 116.9, 119.1, 121.8, 127.4, 128.8,

129.5, 131.1, 134.6, 137.7, 137.8, 148.4, 161.7 ppm; IR (ATR): n˜ =

3340, 3015, 2971, 2822, 2445, 1645, 1603, 1568, 1425, 770, 747,

696 cm^ꢀ1; MS (ESI): m/z (%): 265 [M+H]⁺(96), 287 [M+Na]⁺(100);

HRMS (EI): m/z calcd for [M]⁺: 264.1263, found: 264.1265.

General procedure 3: Preparation of aryl ether derivatives: The

respective phenol derivative and K₂CO₃(2 equiv) were suspended

in 2-butanone (20 mL). 2-Chloroacetonitrile (1 equiv) and KI

(20 mg) were added, and the reaction mixture was heated at reflux

for 6 h. The same volume of a 1m aq NaOH solution was added,

and the mixture was extracted with twice the volume of Et₂O (3ꢂ

40 mL). The organic layers were separated and dried over MgSO₄.

The solvent was evaporated in vacuo, and the product was used

without further purification.

5-(4-Methoxybenzylamino)-1H-benzo[d]imidazol-2-(3H)-one (1c):

Prepared according to general procedure 2 from 2e (403 mg,

1.51 mmol), NaBH₃CN (190 mg, 3.02 mmol) and glacial AcOH

(0.26 mL, 4.53 mmol). Purification by chromatography (Et₂O) gave

a white crystalline solid (159 mg, 39%); mp: 2678C; ¹H NMR

(400 MHz, [D₆]DMSO): d_H=3.72 (s, 3H), 4.13 (d, J=5.7 Hz, 2H), 5.71

(t, J=5.7 Hz, 1H), 6.20–6.22 (m, 2H), 6.60–6.62 (m, 1H), 6.86–6.88

(m, 2H), 7.25–7.27 (m, 2H), 10.01 (s, 1H), 10.15 ppm (s, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=46.7, 54.9, 93.8, 105.3, 108.7,

113.6, 120.5, 128.2, 130.5, 132.2, 143.8, 155.4, 157.9 ppm; IR (ATR):

n˜ =3397, 3010, 2955, 2836, 1747, 1607, 1509, 1460, 1179, 1029,

734, 715 cm^ꢀ1; MS (ESI): m/z (%): 121 (68), 270 [M+H]⁺(100), 292

[M+Na]⁺(31); HRMS (EI): m/z calcd for [M]⁺: 269.1164, found:

269.1175.

General procedure 4: Reduction of nitrile groups: The respective

nitrile derivative was dissolved in Et₂O and cooled to 08C. A 4m so-

lution of LiAlH₄(0.5 equiv) in Et₂O was added, and the reaction

mixture was stirred for 2 h. The reaction mixture was cooled to

08C, and water was added dropwise. The organic layer was sepa-

rated, dried over MgSO₄and evaporated to a volume of 20 mL.

The mixture was cooled to 08C, and a 2m HCl solution in Et₂O was

added dropwise until further formation of a solid ceased. The solid

was collected and washed with Et₂O.

General procedure 5: Acylation of amino derivatives: a) The re-

spective amino derivative and Et₃N (1 equiv; or 2 equiv when a hy-

drochloride is used in the reaction) was dissolved in THF (20 mL)

and the respective acyl chloride (1 equiv) was added. The reaction

mixture was stirred for 2 h at RT and then poured into H₂O

(250 mL). A solid was separated and recrystallized.

N’-Benzyl-2-(m-tolylamino)acetohydrazide (1d): Prepared accord-

ing to general procedure

2 from 2c (444 mg, 1.66 mmol),

NaBH₃CN (209 mg, 3.32 mmol) and glacial AcOH (0.30 mL,

4.98 mmol). Purification by chromatography (Et₂O) gave a white

crystalline solid (272 mg, 61%); mp: 768C; ¹H NMR (400 MHz,

[D₆]DMSO): d_H=2.17 (s, 3H), 3.59 (d, J=6.2 Hz, 2H), 3.85 (d, J=

5.0 Hz, 2H), 5.21–5.26 (m, 1H), 5.71 (t, J=6.2 Hz, 1H), 6.32–6.41 (m,

3H), 6.93–6.97 (m, 1H), 7.23–7.38 (m, 5H), 9.29 ppm (d, J=6.1 Hz,

1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.3, 45.3, 54.5, 109.5,

112.9, 117.2, 126.9, 128.1, 128.5, 128.6, 137.7, 138.6, 148.2,

169.5 ppm; IR (ATR): n˜ =3316, 3230, 3028, 2920, 1645, 1605, 1509,

1481, 746, 688 cm^ꢀ1; MS (ESI): m/z (%): 270 [M+H]⁺(49), 292 [M+

b) The respective amino derivative and Et₃N (1 equiv; or 2 equiv

when a hydrochloride is used in the reaction) were dissolved in

CH₂Cl₂(20–50 mL). The respective acyl chloride (1 equiv) was

added, and the reaction mixture was stirred for 2 h at RT. After the

solvent was removed in vacuo, the crude product was purified by

chromatography.

General procedure 6: Hydrolysis of ester groups: The respective

ester derivative was dissolved in glacial AcOH (25 mL) and 32% aq

HCl (1 mL) was added. The reaction mixture was heated at reflux

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Na]⁺(100), 561 [2M+Na]⁺(22); HRMS (EI): m/z calcd for [M]⁺:

269.1528, found: 269.1548.

Purification by chromatography (Et₂O) gave a light brown crystal-

line solid (1.48 g, 89%); H NMR (400 MHz, [D₆]DMSO): d_H=2.17 (s,

1

3H), 3.64 (s, 3H), 3.88 (d, J=6.5 Hz, 2H), 5.90 (t, J=6.4 Hz, 1H),

6.31–6.40 (m, 3H), 6.93–6.97 ppm (m, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=21.2, 44.5, 51.4, 109.2, 112.7, 117.2, 128.6, 137.7,

147.9, 171.7 ppm. Analytical data are consistent with previous find-

ings.^[31]

4-Cyclohexyl-1-(4-hydroxybenzyl)thiosemicarbazide (1e): Pre-

pared according to general procedure

2 from 2d (351 mg,

1.27 mmol), NaBH₃CN (160 mg, 2.54 mmol) and glacial AcOH

(0.22 mL, 3.81 mmol). Purification by chromatography (Et₂O) gave

a white crystalline solid (103 mg, 29%); mp: 1658C; ¹H NMR

(400 MHz, [D₆]DMSO): d_H=1.07–1.68 (m, 10H), 3.67 (d, J=4.4 Hz,

2H), 3.86–3.95 (m, 1H), 5.18 (t, J=4.3 Hz, 1H), 6.67–6.69 (m, 2H),

7.09–7.11 (m, 2H), 7.34 (d, J=8.6 Hz, 1H), 8.54 (br s, 1H), 9.26 ppm

(s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=24.5, 25.1, 32.0, 51.1,

53.9, 115.0, 128.2, 130.1, 156.6, 179.3 ppm; IR (ATR): n˜ =3321, 3181,

3021, 2917, 2851, 1568, 1514, 1445, 1211, 831 cm^ꢀ1; MS (ESI): m/z

(%): 280 [M+H]⁺(100), 302 [M+Na]⁺(77), 581 [2M+Na]⁺(21);

HRMS (EI): m/z calcd for [M]⁺: 279.1405, found: 279.1436.

2-(m-Tolylamino)acetohydrazide (2b): Prepared as previously de-

scribed^[32]from 2a (1.04 g, 5.80 mmol) and a 64% aq N₂H₄·H₂O so-

lution (0.66 mL, 8.70 mmol). Recrystallization from toluene gave

a

white crystalline solid (936 mg, 90%); ¹H NMR (400 MHz,

[D₆]DMSO): d_H=2.17 (s, 3H), 3.59 (d, J=6.2 Hz, 2H), 4.23 (br s, 2H),

5.73 (t, J=6.1 Hz, 1H), 6.32–6.39 (m, 3H), 6.936.97 (m, 1H),

9.04 ppm (br s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.2, 45.3,

109.5, 112.9, 117.2, 128.6, 137.7, 148.2, 169.4 ppm.

N-((1H-Benzo[d][1,2,3]triazol-1-yl)methyl)-3-chloroaniline

(1 f):

(E/Z)-N’-Benzylidene-2-(m-tolylamino)acetohydrazide (2c): Pre-

3-Chloroaniline (0.30 mL, 2.84 mmol), paraformaldehyde (94 mg,

3.12 mmol) and 1H-benzotriazole were dissolved in EtOH (20 mL).

The reaction mixture was heated at reflux for 2 h. Upon cooling,

a solid was separated and recrystallized from EtOH to give a white

crystalline solid (485 mg, 66%); mp: 1818C (1818C); ¹H NMR

(400 MHz, [D₆]DMSO): d_H=6.14 (d, 1H), 6.61–6.64 (m, 1H), 6.77–

6.79 (m, 1H), 6.88–6.89 (m, 1H), 7.07–7.11 (m, 1H), 7.37–7.41 (m,

1H), 7.52–7.58 (m, 2H), 8.01–8.04 ppm (m, 2H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=56.3, 111.0, 111.5, 112.2, 117.2, 119.0, 124.0, 127.2,

130.5, 132.0, 133.6, 145.3, 147.3 ppm; IR (ATR): n˜ =3299, 3051,

2955, 1699, 1589, 1270, 1151, 735, 681 cm^ꢀ1; MS (ESI): m/z (%): 120

(100), 281 [M+Na]⁺(32); HRMS (EI): m/z calcd for [M]⁺: 258.0672,

found: 258.0638. Analytical data are consistent with previous find-

ings.^[29]

pared according to general procedure

1 from 2b (504 mg,

2.38 mmol) and 4-hydroxybenzaldehyde (291 mg, 2.38 mmol). Re-

crystallization from Et₂O gave a yellow crystalline solid (528 mg,

80%); ¹H NMR (400 MHz, [D₆]DMSO): d_H=2.18 (s, 3H), 3.78/4.21

(2 d, J=6.1, 5.9 Hz, 0.8/1.2H), 5.61/5.90 (2 t, J=5.9, 6.1 Hz, 0.4/

0.6H), 6.37–6.45 (m, 3H), 6.94–6.99 (m, 1H), 7.42–7.48 (m, 3H),

7.66–7.74 (m, 2H), 8.02/8.25 (2 s, 0.4/0.6H), 11.42/11.51 ppm (2 s,

0.4/0.6H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.2, 43.7/46.0,

109.5, 112.9/113.0, 117.0/117.4, 126.8/126.9, 128.6/128.7, 129.8/

129.9, 134.0/134.1, 137.7/137.8, 143.4/146.8, 148.2/148.3, 167.0/

171.4 ppm.

(E/Z)-4-Cyclohexyl-1-(4-hydroxybenzaldehyde)thiosemicarba-

zone (2d): Prepared according to general procedure 1 from 4-cy-

clohexylthiosemicarbazide (504 mg, 2.38 mmol) and 4-hydroxy-

benzaldehyde (291 mg, 2.38 mmol). Recrystallization from Et₂O

gave a yellow crystalline solid (528 mg, 80%); ¹H NMR (400 MHz,

[D₆]DMSO): d_H=1.09–1.88 (m, 10H), 4.13–4.22 (m, 1H), 6.79–6.81

(m, 2H), 7.57–7.61 (m, 2H), 7.84 (d, 1H), 7.97 (s, 1H), 9.88 (br s,

1H), 11.19 ppm (s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=24.8,

25.1, 31.8, 52.4, 115.5, 124.9, 129.0, 142.5, 159.2, 175.3 ppm. Analyti-

cal data are consistent with previous findings.^[33]

2-((p-Tolylamino)methyl)isoindoline-1,3-dione (1g): p-Toluidine

(0.50 mL, 4.54 mmol), paraformaldehyde (150 mg, 5.00 mmol) and

phthalimide were dissolved in EtOH (20 mL). The reaction mixture

was heated at reflux for 2 h. Upon cooling, a solid was separated

and recrystallized from EtOH to give a white crystalline solid

(932 mg, 77%); mp: 1718C (174.5–175.58C); ¹H NMR (400 MHz,

[D₆]DMSO): d_H=2.10 (s, 3H), 4.97 (s, 2H,), 6.70–6.73 (m, 2H), 6.87–

6.89 (m, 2H), 7.82–7.89 ppm (m, 4H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=19.9, 47.3, 112.7, 123.1, 129.3, 134.2, 134.6, 143.5,

167.9 ppm; IR (ATR): n˜ =3370, 3040, 2962, 1707, 1519, 1395, 1243,

723, 509 cm^ꢀ1; MS (ESI): m/z (%): 120 (100), 267 [M+H]⁺(56), 289

[M+Na]⁺(68), 555 [2M+Na]⁺(18); HRMS (EI): m/z calcd for [M]⁺:

266.1055, found: 266.1059. Analytical data are consistent with pre-

vious findings.^[30]

(E/Z)-5-(4-Methoxybenzylideneamino)-1H-benzo[d]imidazol-2-

(3H)-one (2e): Prepared according to general procedure 1 from

5-amino-1H-benzo[d]imidazol-2(3H)-one (507 mg, 3.40 mmol) and

4-methoxybenzaldehyde (0.41 mL, 3.40 mmol). Recrystallization

from toluene gave a yellow crystalline solid (654 mg, 72%);

¹H NMR (400 MHz, [D₆]DMSO): d_H=3.83 (s, 3H), 6.88–7.06 (m, 5H),

7.85–7.87 (m, 2H), 8.54 (s, 1H), 10.62 ppm (br s, 2H); ¹³C NMR

(100 MHz, [D₆]DMSO): d_C=55.3, 100.9, 108.5, 114.1, 114.3, 128.0,

129.2, 130.0, 130.3, 145.0, 155.5, 157.3, 161.5 ppm.

4-(5-Methylbenzo[d]oxazol-2-yl)benzene-1,3-diol (1h): Diol 2g

(303 mg, 1.25 mmol) was dissolved in glacial AcOH (10 mL) and

treated with Pb(OAc)₄(612 mg, 1.38 mmol). The reaction mixture

was stirred for 5 min at RT and then poured into H₂O (100 mL). A

solid was separated and purified by chromatography (Et₂O) to give

(E/Z)-3-((m-Tolylimino)methyl)quinolin-2-ol (2 f): Prepared accord-

ing to general procedure 1 from m-toluidine (0.26 mL, 2.40 mmol)

and 2-hydroxyquinolin-3-carbaldehyde (415 mg, 2.40 mmol). Re-

crystallization from toluene gave a yellow crystalline solid (345 mg,

55%); ¹H NMR (400 MHz, [D₆]DMSO): d_H=2.36 (s, 3H), 7.05–7.10

(m, 3H), 7.22–7.37 (m, 3H), 7.56–7.61 (m, 1H), 7.88–7.91 (m, 1H),

8.67 (s, 1H), 8.78 (s, 1H), 12.12 ppm (s, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=20.8, 115.1, 118.1, 118.8, 121.2, 122.3, 126.3, 126.9,

129.0, 129.6, 131.8, 137.5, 138.6, 139.7, 151.4, 154.9, 161.4 ppm.

a

red crystalline solid (147 mg, 49%); mp: 2208C; ¹H NMR

(400 MHz, [D₆]DMSO): d_H=2.42 (s, 3H), 6.46–6.54 (m, 2H), 7.18–

7.23 (m, 1H), 7.54–7.82 (m, 3H), 10.40 (s, 1H), 11.30 ppm (s, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=20.9, 101.9, 102.7, 108.6, 110.0,

118.3, 125.8, 128.6, 134.4, 139.6, 146.6, 159.7, 162.6, 162.9 ppm; IR

(ATR): n˜ =3357, 2923, 1634, 1600, 1480, 1415, 1244, 1145, 796 cm^ꢀ1

;

MS (ESI): m/z (%): 242 [M+H]⁺(100); HRMS (EI): m/z calcd for

[M]⁺: 241.0739, found: 241.0742.

(E/Z)-4-((2-Hydroxy-5-methylphenylimino)methyl)benzene-1,3-

Methyl-2-(m-tolylamino)acetate (2a): m-Toluidine (2.00 mL,

18.5 mmol) was dissolved in acetone and treated with methyl

2-bromoacetate (0.86 mL, 9.25 mmol). The reaction mixture was

stirred for 2 h at RT, and the solvent was then removed in vacuo.

diol (2g): Prepared according to general procedure 1 from

2-amino-4-methylphenol (630 mg, 5.12 mmol) and 2,4-dihydroxy-

benzaldehyde (707 mg, 5.12 mmol). Recrystallization from toluene

gave a yellow crystalline solid (872 mg, 70%); ¹H NMR (400 MHz,

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[D₆]DMSO): d_H=2.23 (s, 3H), 6.21–6.22 (m, 1H), 6.23–6.34 (m, 1H),

6.80–6.88 (m, 2H), 7.13–7.14 (m, 1H), 7.34–7.36 (m, 1H), 8.77 (s,

1H), 9.46 (s, 1H), 10.18 (s, 1H), 14.33 ppm (s, 1H); ¹³C NMR

(100 MHz, [D₆]DMSO): d_C=20.1, 102.6, 107.4, 112.2, 116.1, 119.3,

127.4, 128.2, 133.9, 134.0, 148.0, 159.8, 162.3, 165.1 ppm.

2-(m-Tolyloxy)ethanaminium chloride (4a): Prepared according to

general procedure 4 from 3a (4.82 g, 32.8 mmol) and LiAlH₄

(4.10 mL, 16.4 mmol). Washing the precipitate with Et₂O (1ꢂ5 mL)

gave a white crystalline solid (5.53 g, 90%); ¹H NMR (400 MHz,

D₂O): d_H=2.30 (s, 3H), 3.39 (t, J=4.8 Hz, 2H), 4.23 (t, J=4.8 Hz,

2H,), 6.81–6.91 (m, 3H), 7.23–7.27 ppm (m, 1H); ¹³C NMR (100 MHz,

D₂O): d_C=20.5, 39.0, 63.8, 111.6, 115.3, 122.6, 129.8, 140.6,

155.7 ppm.

2-(m-Tolyloxy)acetonitrile (3a): Prepared according to general

procedure 3 from m-cresol (5.01 mL, 47.7 mmol), K₂CO₃(13.2 g,

95.4 mmol), chloroacetonitrile (3.00 mL, 47.7 mmol) and KI

(238 mg, 1.44 mmol). Evaporation of the solvent in vacuo gave

2-(m-Tolylsulfanyl)ethanaminium chloride (4b): Prepared accord-

ing to general procedure 4 from 3b (758 mg, 4.64 mmol) and

LiAlH₄(0.58 mL, 2.32 mmol). Washing the precipitate with Et₂O (1ꢂ

5 mL) gave a white crystalline solid (823 mg, 87%); ¹H NMR

(400 MHz, D₂O): d_H=2.39 (s, 3H), 3.24 (t, J=6.4 Hz, 2H), 3.31 (t, J=

6.2 Hz, 2H), 7.24–7.40 ppm (m, 4H); ¹³C NMR (100 MHz, D₂O): d_C=

20.3, 30.6, 38.1, 127.4, 128.3, 129.4, 130.8, 132.6, 134.0 ppm.

1

a reddish oil (4.84 g, 69%); H NMR (400 MHz, CDCl₃): d_H=2.36 (s,

3H), 4.73 (s, 2H), 6.78–6.80 (m, 2H), 6.91 (d, J=7.5 Hz, 1H),

7.23 ppm (t, J=7.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): d_C=21.4,

53.5, 111.7, 115.2, 115.7, 123.9, 129.5, 140.1, 156.5 ppm. Analytical

data are consistent with previous findings.^[34]

2-(m-Tolylsulfanyl)acetonitrile (3b): NaH (60% dispersion in min-

eral oil; 280 mg, 6.99 mmol) was dissolved in DMF (15 mL) and

cooled to 08C. m-Thiocresol (0.76 mL, 6.35 mmol) was added, and

the reaction mixture was stirred for 30 min while it warmed to RT.

Chloroacetonitrile (0.4 mL, 6.35 mL) and KI (32 mg, 0.19 mmol)

were added, and the reaction mixture was stirred at 608C for 2 h.

The solution was concentrated to a volume of about 5 mL and

then diluted with Et₂O (100 mL). Aq NaOH (1m) was added, and

the mixture was extracted with Et₂O (2ꢂ50 mL). Evaporation of the

solvent in vacuo gave a reddish oil (798 mg, 77%); ¹H NMR

(400 MHz, CDCl₃): d_H=2.14 (s, 3H), 4.42 (s, 2H), 6.79–6.92 (m, 3H),

7.41–7.43 ppm (m, 1H); ¹³C NMR (100 MHz, CDCl₃): d_C=22.0, 22.5,

117.1, 124.9, 126.1, 126.3, 129.1, 136.6, 137.6 ppm.

2-(4-Isopropylphenoxy)ethanaminium chloride (4c): Prepared ac-

cording to general procedure 4 from 3c (960 mg, 5.48 mmol) and

LiAlH₄(0.69 mL, 2.74 mmol). Washing the precipitate with Et₂O (1ꢂ

5 mL) gave a white crystalline solid (959 mg, 81%); ¹H NMR

(400 MHz, D₂O): d_H=1.16 (d, J=6.2 Hz, 6H), 3.12–3.14 (m, 1H), 3.47

(t, J=5.7 Hz, 2H), 4.76 (t, J=5.7 Hz, 2H,), 6.94–6.99 (m, 2H), 7.53–

7.59 ppm (m, 2H); ¹³C NMR (100 MHz, D₂O): d_C=23.1, 32.5, 41.3,

64.2, 114.6, 125.9, 140.6, 155.2 ppm.

2-(5,6,7,8-Tetrahydronaphthalen-2-yloxy)ethanaminium chloride

(4d): Prepared according to general procedure 4 from 3d (944 mg,

5.04 mmol) and LiAlH₄(0.63 mL, 2.52 mmol). Washing the precipi-

tate with Et₂O (1ꢂ5 mL) gave a white crystalline solid (1.01 g,

88%); ¹H NMR (400 MHz, D₂O): d_H=1.77–1.80 (m, 4H), 2.72–2.77

(m, 4H), 3.46 (t, J=5.7 Hz, 2H), 4.28 (t, J=5.7 Hz, 2H,), 6.84–6.87

(m, 2H), 7.13–7.15 ppm (m, 1H); ¹³C NMR (100 MHz, D₂O): d_C=22.6,

22.9, 28.0, 29.1, 39.0, 64.1, 112.6, 114.6, 130.3, 130.9, 139.1,

155.4 ppm.

2-(4-Isopropylphenoxy)acetonitrile (3c): Prepared according to

general procedure 3 from 4-isopropylphenol (1.08 g, 7.95 mmol),

K₂CO₃(2.20 g, 15.9 mmol), chloroacetonitrile (0.50 mL, 7.95 mmol)

and KI (40 mg, 0.24 mmol). Evaporation of the solvent in vacuo

1

gave a reddish oil (975 mg, 70%); H NMR (400 MHz, CDCl₃): d_H=

1.25 (d, 6H), 3.07 (m, 1H), 4.81 (s, 2H), 6.87–6.92 (m, 2H), 7.41–

7.45 ppm (m, 2H); ¹³C NMR (100 MHz, CDCl₃): d_C=22.1, 30.0, 57.7,

115.0, 127.7, 111.6, 139.1, 154.2 ppm.

2-(Naphthalen-1-yloxy)ethanaminium chloride (4e): Prepared ac-

cording to general procedure 4 from 3e (918 mg, 5.01 mmol) and

LiAlH₄(0.63 mL, 2.50 mmol). Washing the precipitate with Et₂O (1ꢂ

5 mL) gave awhite crystalline solid (930 mg, 83%); ¹H NMR

(400 MHz, D₂O): d_H=3.34 (t, J=5.2 Hz, 2H), 4.14 (t, J=5.1 Hz, 2H,),

6.81–6.89 (m, 2H), 7.31–7.50 (m, 3H), 7.80–7.86 ppm (m, 2H);

¹³C NMR (100 MHz, D₂O): d_C=41.3, 69.9, 106.7, 118.6, 118.8, 120.3,

123.7, 125.6, 126.0, 127.2, 134.4, 153.8 ppm.

2-(5,6,7,8-Tetrahydronaphthalen-2-yloxy)acetonitrile (3d): Pre-

pared according to general procedure 3 from 2-(5,6,7,8-tetrahydro-

naphthalen-2-ol (1.18 g, 7.95 mmol), K₂CO₃(2.20 g, 15.9 mmol),

chloroacetonitrile (0.50 mL, 7.95 mmol) and KI (40 mg, 0.24 mmol).

Evaporation of the solvent in vacuo gave a reddish oil (967 mg,

1

65%); H NMR (400 MHz, CDCl₃): d_H=1.69–1.73 (m, 4H), 2.65–2.70

2-(Naphthalen-2-yloxy)ethanamine (4 f): 3 f (8.73 g, 47.7 mmol)

was dissolved in Et₂O, and the solution was cooled to 08C. A 4m

solution of LiAlH₄in Et₂O (6.00 mL, 23.9 mmol) was added, and the

reaction mixture was stirred for 2 h at RT. The reaction mixture was

again cooled to 08C, and water was added dropwise. The organic

layer was separated and dried over MgSO₄. The solvent was evapo-

rated to give a reddish solid (4.84 mg, 69%); ¹H NMR (400 MHz,

D₂O): d_H=1.48 (br s, 2H), 3.14 (t, J=5.2 Hz, 2H), 4.10 (t, J=5.2 Hz,

2H,), 7.14–7.19 (m, 2H), 7.32–7.46 (m, 2H), 7.72–7.78 ppm (m, 3H);

¹³C NMR (100 MHz, D₂O): d_C=41.6, 70.2, 106.7, 118.8, 123.6, 126.3,

126.7, 127.6, 129.0, 129.4, 134.5, 156.8 ppm.

(m, 4H), 5.08 (s, 2H), 6.75–6.80 (m, 2H), 7.01–7.03 ppm (m, 1H);

¹³C NMR (100 MHz, CDCl₃): d_C=23.1, 23.3, 28.5, 29.6, 54.0, 113.1,

115.2, 117.4, 130.5, 131.2, 138.6, 154.6 ppm.

2-(Naphthalen-1-yloxy)acetonitrile (3e): Prepared according to

general procedure 3 from 1-naphthol (1.15 g, 7.95 mmol), K₂CO₃

(2.20 g, 15.9 mmol), chloroacetonitrile (0.50 mL, 7.95 mmol) and KI

(40 mg, 0.24 mmol). Evaporation of the solvent in vacuo gave a red-

1

dish oil (946 mg, 65%); H NMR (400 MHz, CDCl₃): d_H=4.87 (s, 2H),

6.81–6.90 (m, 2H), 7.33–7.51 (m, 3H), 7.81–7.88 ppm (m, 2H);

¹³C NMR (100 MHz, CDCl₃): d_C=54.5, 104.5, 115.3, 120.6, 122.6,

126.7, 126.9, 127.2, 129.8, 132.0, 135.1, 155.5 ppm.

2-(2-Phenoxyethylcarbamoyl)phenyl acetate (5a): Prepared ac-

cording to general procedure 5a from 2-phenoxyethanamine

(0.19 mL, 1.46 mmol), acetyl salicylic acid chloride (290 mg,

1.46 mmol) and Et₃N (0.20 mL, 1.46 mmol). Recrystallization from

toluene gave a white crystalline solid (359 mg, 82%); ¹H NMR

(400 MHz, [D₆]DMSO): d_H=2.18 (s, 3H), 3.57 (ps q, J=5.7 Hz, 2H),

4.07 (t, J=5.8 Hz, 2H), 6.92–7.00 (m, 3H), 7.17–7.19 (m, 1H), 7.28–

7.25 (m, 3H), 7.45–7.59 (m, 2H), 8.48 ppm (t, J=5.4 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=20.7, 38.6, 65.8, 114.4, 120.6,

2-(Naphthalen-2-yloxy)acetonitrile (3 f): Prepared according to

general procedure 3 from 2-naphthol (1.15 g, 7.95 mmol), K₂CO₃

(2.20 g, 15.9 mmol), chloroacetonitrile (0.50 mL, 7.95 mmol) and KI

(40 mg, 0.24 mmol). Evaporation of the solvent in vacuo gave a red-

1

dish oil (976 mg, 67%); H NMR (400 MHz, CDCl₃): d_H=4.87 (s, 2H),

7.18–7.25 (m, 2H), 7.41–7.53 (m, 2H), 7.78–7.83 ppm (m, 3H);

¹³C NMR (100 MHz, CDCl₃): d_C=53.4, 107.7, 115.1, 118.1, 124.8,

126.9, 127.1, 127.7, 129.8, 130.2, 133.9, 154.4 ppm.

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123.2, 125.7, 129.0, 129.0, 129.5, 131.2, 147.8, 158.3, 165.4,

168.7 ppm.

(183 mg, 49%); mp: 1438C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=

2.26 (s, 3H), 3.74 (ps q, J=5.6 Hz, 2H), 4.16 (t, J=5.7 Hz, 2H), 6.74–

6.81 (m, 3H), 7.14–7.18 (m, 1H), 7.26 (s, 1H), 7.32–7.36 (m, 1H),

7.48–7.51 (m, 1H), 7.73–7.75 (m, 1H), 7.85–7.87 (m, 1H), 8.54 (s,

1H), 9.22 (t, J=5.4 Hz, 1H), 11.93 ppm (s, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=20.9, 38.7, 65.7, 110.6, 111.4, 115.1, 118.6, 121.4,

123.6, 125.6, 126.5, 128.1, 128.6, 129.1, 129.8, 135.9, 138.9, 154.9,

158.3, 167.9 ppm; IR (ATR): n˜ =3379, 2943, 1651, 1580, 1542, 1260,

1231, 1169, 777, 754 cm^ꢀ1; MS (EI, 70 eV): m/z (%): 171 (49), 214

(100), 321 [M]⁺(38); HRMS (EI): m/z calcd for [M]⁺: 321.1365,

found: 321.1393.

2-(2-(Naphthalen-2-yloxy)ethylcarbamoyl)phenyl acetate (5b):

Prepared according to general procedure 5a from 2-phenoxyethan-

amine (0.19 mL, 1.46 mmol), acetyl salicylic acid chloride (290 mg,

1.46 mmol) and Et₃N (0.20 mL, 1.46 mmol). Recrystallization from

toluene gave a white crystalline solid (359 mg, 82%); ¹H NMR

(400 MHz, [D₆]DMSO): d_H=2.19 (s, 3H), 3.67 (ps q, J=5.63 Hz, 2H),

4.22 (t, J=5.7 Hz, 2H), 8.50 (t, J=5.4 Hz, 1H), 7.17–7.19 (m, 2H),

7.31–7.53 (m, 5H), 7.60–7.63 (m, 1H), 7.80–7.84 ppm (m, 3H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=20.6, 38.6, 66.1, 106.9, 118.6,

123.2, 123.5, 125.7, 126.3, 126.6, 127.4, 127.9, 128.5, 129.0, 129.2,

131.2, 133.6, 147.9, 156.2, 165.4, 168.7 ppm.

1-Hydroxy-N-(2-(m-tolyloxy)ethyl)-2-naphthamide (5g): Prepared

according to general procedures 7 and 5b from 1-hydroxy-2-naph-

thoic acid (426 mg, 2.26 mmol), oxalyl chloride (0.29 mL,

2-(2-(m-Tolyloxy)ethylcarbamoyl)phenyl acetate (5c): Prepared

according to general procedure 5b from 2-(m-tolyloxy)ethanamini-

um chloride (150 mg, 0.79 mmol), acetylsalicylic acid chloride

(143 mg, 0.79 mmol) and Et₃N (0.22 mL, 1.58 mmol). Purification by

chromatography (CH₂Cl₂/MeOH, 19:1) gave a white crystalline solid

3.39 mmol),

2.26 mmol) and Et₃N (0.63 mL, 4.52 mmol). Purification by chroma-

tography (CH₂Cl₂/MeOH, 19:1) gave white crystalline solid

2-(m-tolyloxy)ethanaminium

chloride

(424 mg,

a

1

(4.52 mg, 41%); mp: 928C; H NMR (400 MHz, [D₆]DMSO): d_H=2.26

(s, 3H), 3.73 (ps q, J=5.7 Hz, 2H), 4.16 (t, J=5.8 Hz, 2H), 6.73–6.80

(m, 3H), 7.13–7.17 (m, 1H), 7.37–7.40 (m, 1H), 7.53–7.67 (m, 2H),

7.86–7.92 (m, 2H), 8.26–8.28 (m, 1H), 9.20 (t, J=5.4 Hz, 1H),

14.53 ppm (s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 38.8,

65.4, 106.8, 111.4, 115.1, 117.5, 121.4, 122.5, 122.9, 124.6, 125.7,

127.4, 128.8, 129.2, 135.7, 138.9, 158.3, 159.6, 170.8 ppm; IR (ATR):

n˜ =3452, 3054, 2942, 1593, 1536, 1258, 783, 760 cm^ꢀ1; MS (FAB):

m/z (%): 170 (64), 214 (100), 321 [M]⁺(62); HRMS (EI): m/z calcd for

[M]⁺: 321.1365, found: 321.1394.

1

(221 mg, 89%); mp: 688C; H NMR (400 MHz, [D₆]DMSO): d_H=2.18

(s, 3H), 2.27 (s, 3H), 3.56 (ps q, J=5.7 Hz, 2H), 4.05 (t, J=5.8 Hz,

2H), 6.73–6.78 (m, 3H), 7.14–7.19 (m, 2H), 7.31–7.35 (m, 1H), 7.49–

7.53 (m, 1H), 7.57–7.59 (m, 1H), 8.46 ppm (t, J=5.7 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=20.6, 21.0, 38.7, 65.8, 111.4,

115.1, 121.3, 123.1, 125.7, 128.9, 129.0, 129.2, 131.2, 138.9, 147.9,

158.4, 165.4, 168.7 ppm; IR (ATR): n˜ =3331, 3079, 2990, 2925, 1750,

1641, 1538, 1264, 1198, 1174, 658 cm^ꢀ1; MS (ESI): m/z (%): 314 [M+

H]⁺(36), 336 [M+Na]⁺(100), 649 [2M+Na]⁺(23); HRMS (EI): m/z

calcd for [M]⁺: 313.1314, found: 313.1298.

2-Hydroxy-N-(2-(naphthalen-2-yloxy)ethyl)benzamide (5h): Pre-

pared according to general procedures 7 and 5b from salicylic acid

(310 mg, 2.24 mmol), oxalyl chloride (0.29 mL, 3.36 mmol), 2-(naph-

thalen-2-yloxy)ethanamine (420 mg, 2.24 mmol) and Et₃N (0.31 mL,

2.24 mmol). Purification by chromatography (CH₂Cl₂/MeOH, 19:1)

2-Hydroxy-N-(2-phenoxyethyl)benzamide (5d): Prepared accord-

ing to general procedure 6 from 5a (410 mg, 1.31 mmol), glacial

AcOH (25.0 mL, 0.44 mol) and 32% aq HCl solution (1.0 mL,

10.3 mmol). Recrystallization from toluene gave a white crystalline

solid (283 mg, 94%); mp: 1208C; ¹H NMR (400 MHz, [D₆]DMSO):

d_H=3.69 (ps q, J=5.7 Hz, 2H), 4.13 (t, J=5.8 Hz, 2H), 6.87–6.98 (m,

5H), 7.27–7.31 (m, 2H), 7.38–7.42 (m, 1H), 7.88–7.90 (m, 1H), 9.03

(t, J=5.3 Hz, 1H), 12.46 ppm (s, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=38.5, 65.6, 114.4, 115.2, 117.2, 118.5, 120.6, 127.9,

129.4, 133.6, 158.2, 159.6, 168.8 ppm; IR (ATR): n˜ =3389, 2949,

1638, 1593, 1543, 1488, 1242, 1231, 752 cm^ꢀ1; MS (EI, 70 eV): m/z

(%): 121 (85), 164 (100), 257 [M]⁺(15); HRMS (EI): m/z calcd for

[M]⁺: 257.1052, found: 257.1091.

1

gave a white crystalline solid (393 mg, 57%); mp: 1908C; H NMR

(400 MHz, [D₆]DMSO): d_H=3.77 (ps q, J=5.7 Hz, 2H), 4.28 (t, J=

5.8 Hz, 2H), 6.88–6.93 (m, 2H), 7.18–7.20 (m, 1H), 7.32–7.47 (m,

4H), 7.79–7.91 (m, 4H), 9.06 (t, J=5.3 Hz, 1H), 12.43 ppm (s, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=38.6, 65.9, 106.8, 115.3, 117.3,

118.5, 118.6, 123.6, 126.3, 126.6, 127.4, 127.9, 128.5, 129.3, 133.7,

134.2, 156.1, 159.7, 168.9 ppm; IR (ATR): n˜ =3386, 1638, 1592, 1550,

1337, 1256, 1215, 1177, 836, 750 cm^ꢀ1; MS (ESI): m/z (%): 208 (33),

308 [M+H]⁺(100), 330 [M+Na]⁺(24); HRMS (EI): m/z calcd for

[M]⁺: 307.1208, found: 307.1196.

N-(2-(m-Tolyloxy)ethyl)benzamide (5e): Prepared according to

general procedure 5b from benzoic acid chloride (0.16 mL,

2-Hydroxy-N-(2-(naphthalen-1-yloxy)ethyl)benzamide (5i): Pre-

pared according to general procedures 7 and 5b from salicylic acid

(297 mg, 2.15 mmol), oxalyl chloride (0.28 mL, 3.23 mmol), 2-(m-tol-

yloxy)ethanaminium chloride (481 mg, 2.15 mmol) and Et₃N

(0.60 mL, 4.30 mmol). Purification by chromatography (CH₂Cl₂/

MeOH, 19:1) gave a white crystalline solid (350 mg, 53%); mp:

1318C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=3.86 (ps q, J=5.6 Hz,

2H), 4.32 (t, J=5.5 Hz, 2H), 6.87–7.01 (m, 3H), 7.37–7.53 (m, 5H),

7.84–7.91 (m, 2H), 8.24–8.26 (m, 1H), 9.10 (t, J=5.5 Hz, 1H),

12.44 ppm (s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=38.0, 66.3,

105.1, 115.4, 117.3, 118.6, 120.0, 121.6, 124.8, 125.1, 126.1, 126.4,

127.3, 127.9, 133.6, 133.9, 153.8, 159.7, 168.9 ppm; IR (ATR): n˜ =

3426, 1638, 1593, 1536, 1484, 1270, 1240, 1101, 752 cm^ꢀ1; MS (ESI):

m/z (%): 308 [M+H]⁺(100), 325 [M+NH₄]⁺(32), 330 [M+Na]⁺

(40); HRMS (EI): m/z calcd for [M]⁺: 307.1208, found: 307.1180.

1.34 mmol),

1.34 mmol) and Et₃N (0.37 mL, 2.68 mmol). Purification by chroma-

tography (CH₂Cl₂/MeOH, 19:1) gave white crystalline solid

2-(m-tolyloxy)ethanaminium

chloride

(252 mg,

a

1

(285 mg, 83%); mp: 598C; H NMR (400 MHz, [D₆]DMSO): d_H=2.26

(s, 3H), 3.62 (ps q, J=5.9 Hz, 2H), 4.10 (t, J=6.0 Hz, 2H), 6.73–6.78

(m, 3H), 7.31–7.17 (m, 1H), 7.44–7.54 (m, 3H), 7.857.87 (m, 2H),

8.67 ppm (t, J=5.3 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=

21.0, 38.9, 65.7, 111.4, 115.0, 121.3, 127.1, 128.2, 129.2, 131.2, 134.2,

138.9, 158.4, 166.4 ppm; IR (ATR): n˜ =3309, 3038, 2934, 2879, 1633,

1534, 1487, 1309, 1261, 1154, 1064, 768, 688 cm^ꢀ1; MS (EI, 70 eV):

m/z (%): 105 (59), 148 (100), 255 [M]⁺(6); HRMS (EI): m/z calcd for

[M]⁺: 255.1259, found: 255.1293.

3-Hydroxy-N-(2-(m-tolyloxy)ethyl)-2-naphthamide (5 f): Prepared

according to general procedures 7 and 5b from 3-hydroxy-2-naph-

thoic acid (219 mg, 1.16 mmol), oxalyl chloride (0.15 mL,

2-Chloro-N-(2-(m-tolyloxy)ethyl)benzamide (6a): Prepared ac-

cording to general procedure 5a from 2-(m-tolyloxy)ethanaminium

chloride (202 mg, 1.08 mmol), 2-chlorobenzoic acid chloride

(0.14 mL, 1.08 mmol) and Et₃N (0.30 mL, 2.16 mmol). Recrystalliza-

1.74 mmol),

1.16 mmol) and Et₃N (0.32 mL, 2.32 mmol). Purification by chroma-

tography (CH₂Cl₂/MeOH, 19:1) gave white crystalline solid

2-(m-tolyloxy)ethanaminium

chloride

(203 mg,

a

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tion from toluene gave a white crystalline solid (278 mg, 89%);

mp: 838C; H NMR (400 MHz, [D₆]DMSO): d_H=2.27 (s, 3H), 3.59 (ps

7.16 (m, 2H), 7.35–7.50 (m, 4H), 8.66 ppm (t, J=5.4 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=24.0, 32.4, 38.6, 65.9, 114.3,

126.9, 127.0, 128.7, 129.4, 129.8, 130.6, 136.7, 140.5, 156.4,

166.4 ppm; IR (ATR): n˜ =3261, 2959, 2869, 1637, 1551, 1510, 1305,

1244, 1042, 828, 700, 543 cm^ꢀ1; MS (ESI): m/z (%): 318 [M+H]⁺

(51), 335 [M+NH₄]⁺(100), 342 [M+Na]⁺(37); HRMS (ESI): m/z

calcd for [M+H]⁺: 318.1261, found: 318.1243.

1

q, J=5.7 Hz, 2H), 4.07 (t, J=5.8 Hz, 2H), 6.74–6.78 (m, 3H), 7.14–

7.18 (m, 1H), 3.36–7.50 (m, 4H), 8.68 ppm (t, J=5.4 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 38.6, 65.7, 111.5, 115.1,

121.3, 126.9, 128.7, 129.1, 129.5, 129.8, 130.6, 136.7, 138.9, 158.4,

166.5 ppm; IR (ATR): n˜ =3248, 3061, 2931, 1640, 1561, 1292, 1257,

1159, 1058, 692 cm^ꢀ1; MS (EI, 70 eV): m/z (%): 139 (57), 182 (100),

289 [M]⁺(7); HRMS (EI): m/z calcd for [M]⁺: 289.0870, found:

289.0911.

2-Chloro-N-(2-(5,6,7,8-tetrahydronaphthalen-2-yloxy)ethyl)benz-

amide (6 f): Prepared according to general procedure 5b from

2-(5,6,7,8-tetrahydronaphthalen-2-yloxy)ethanamime

(196 mg,

(R,S)-N,N-(2-(m-Tolyloxy))-pentamethylene-2-chlorobenzamide

0.86 mmol), 2-chlorobenzoic acid chloride (0.11 mL, 0.86 mmol)

and Et₃N (0.24 mL, 1.72 mmol). Purification by chromatography

(CH₂Cl₂/MeOH, 19:1) gave a white crystalline solid (252 mg, 88%);

mp: 658C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=1.68–1.72 (m, 4H),

2.63–2.68 (m, 4H), 3.56 (ps q, J=5.8 Hz, 2H), 4.04 (t, J=5.8 Hz, 2H),

6.64–6.69 (m, 2H), 6.93–6.96 (m, 1H), 7.37–7.49 (m, 4H), 8.60 ppm

(t, J=5.4 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=22.6, 22.9,

27.9, 29.0, 38.7, 65.8, 112.4, 114.2, 127.0, 128.6, 128.8, 129.5, 129.7,

129.8, 130.7, 136.8, 137.5, 156.1, 166.5 ppm; IR (ATR): n˜ =3336,

2943, 2926, 1651, 1531, 1502, 1264, 1059, 735, 647 cm^ꢀ1; MS (ESI):

m/z (%): 330 [M+H]⁺(52), 347 [M+NH₄]⁺(100), 352 [M+Na]⁺

(59); HRMS (ESI): m/z calcd for [M+H]⁺: 330.1261, found:

330.1234.

(6b):

A

solution of (R,S)-3-(p-tolyloxy)piperidine (202 mg,

1.11 mmol) and 2-chlorobenzoic acid chloride (0.14 mL, 1.11 mmol)

in pyridine was heated at reflux for 3 h. The solvent was removed,

and the residue was purified by chromatography (CH₂Cl₂/MeOH,

19:1) to give a white crystalline solid (320 mg, 87%); mp: 918C;

¹H NMR (400 MHz, [D₆]DMSO): d_H=1.51–1.71 (m, 2H), 1.86–2.04 (m,

2H), 2.26 (s, 3H), 3.09–3.53 (m, 3H), 3.97–4.05 (m, 1H), 4.60–4.65

(m, 1H), 6.73–6.80 (m, 3H), 7.13–7.17 (m, 1H), 7.37–7.54 ppm (m,

4H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 30.0, 30.7, 38.0, 43.3,

71.2, 112.7, 116.5, 121.5, 127.5, 127.6, 127.7, 129.0, 129.2, 129.3,

129.4, 130.3, 135.9, 139.0, 156.7, 165.3 ppm; IR (ATR): n˜ =2954,

2861, 1627, 1584, 1440, 1255, 1150, 1061, 765, 739 cm^ꢀ1; MS (EI,

70 eV): m/z (%): 330 [M+H]⁺(100), 347 [M+NH₄]⁺(33); HRMS (EI):

m/z calcd for [M]⁺: 330.1261, found: 330.1241.

2-Chloro-N-(2-(naphthalen-2-yloxy)ethyl)benzamide (6g): Pre-

pared according to general procedure 5a from 2-(naphthalen-2-

yloxy)ethanaminium chloride (212 mg, 1.13 mmol), 2-chlorobenzoic

acid chloride (0.14 mL, 1.13 mmol) and Et₃N (0.16 mL, 1.13 mmol).

Recrystallization from toluene/pentane (1:20) gave a white crystal-

line solid (244 mg, 66%); mp: 1168C; ¹H NMR (400 MHz,

[D₆]DMSO): d_H=3.69 (ps q, J=5.7 Hz, 2H), 4.24 (t, J=5.7 Hz, 2H),

7.17–7.20 (m, 1H), 7.33–7.39 (m, 3H), 7.42–7.50 (m, 4H), 7.80–7.85

(m, 3H), 8.70 ppm (t, J=5.3 Hz, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=38.6, 66.0, 106.7, 118.7, 123.5, 126.3, 126.6, 127.0,

127.4, 128.4, 128.8, 129.2, 129.5, 129.8, 130.7, 134.2, 136.8, 156.3,

166.6 ppm; IR (ATR): n˜ =3274, 1645, 1547, 1258, 1118, 815, 747,

733, 474 cm^ꢀ1; MS (ESI): m/z (%): 326 [M+H]⁺(22), 343 [M+NH₄]⁺

(100); HRMS (ESI): m/z calcd for [M+H]⁺: 326.0948, found:

326.0928.

N,N-(2-(m-Tolyloxy))trimethylene-2-chlorobenzamide (6c): A solu-

tion of 3 m-tolyloxy-azetidine (205 mg, 1.26 mmol) and 2-chloro-

benzoic acid chloride (0.16 mL, 1.26 mmol) in pyridine was heated

at reflux for 3 h. The solvent was removed, and the residue was pu-

rified by chromatography (CH₂Cl₂/MeOH, 19:1) to give a colorless

resin (316 mg, 83%); ¹H NMR (400 MHz, CDCl₃): d_H=2.31 (s, 3H),

4.00–4.04 (m, 1H), 4.24–4.31 (m, 2H), 4.56–4.61 (m, 1H), 4.97 (tt,

J=6.5, 4.2 Hz, 1H), 6.51–6.56 (m, 2H), 6.80–6.81 (m, 1H), 7.13–

7.41 ppm (m, 5H); ¹³C NMR (100 MHz, CDCl₃): d_C=21.4, 55.5, 57.9,

65.3, 111.2, 115.3, 122.6, 127.0, 128.4, 129.4, 129.8, 130.5, 130.8,

133.9, 139.9, 156.2, 167.9 ppm; IR (ATR): n˜ =3057, 2942, 2873, 1638,

1584, 1422, 1256, 1180, 1156, 771, 748, 691 cm^ꢀ1; MS (ESI): m/z (%):

302 [M(35Cl)+H]⁺(100), 319 [M+NH₄]⁺(95); HRMS (ESI): m/z

calcd for [M+H]⁺: 302.0948, found: 302.0925

2-Chloro-N-(2-(7-chloroquinolin-4-ylamino)ethyl)benzamide (6h):

Prepared according to general procedure 5a from N’-[7-chloroqui-

nolin-4-yl)ethane-1,2-diamine (194 mg, 0.83 mmol), 2-chlorobenzoic

acid chloride (0.11 mL, 0.83 mmol) and Et₃N (0.12 mL, 0.83 mmol).

2-Chloro-N-(2-(phenylsulfanyl)ethyl)benzamide (6d): Prepared

according to general procedure 5b from 2-(m-tolylsulfanyl)ethan-

aminium chloride (198 mg, 0.97 mmol), 2-chlorobenzoic acid chlo-

ride (0.12 mL, 0.97 mmol) and Et₃N (0.27 mL, 1.93 mmol). Purifica-

tion by chromatography (CH₂Cl₂/MeOH, 19:1) gave a white crystal-

Recrystallization from toluene gave

a white crystalline solid

(268 mg, 89%); mp: 2138C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=

3.37 (s, 1H), 3.48–3.55 (m, 4H), 6.60–6.62 (m, 1H), 7.37–7.50 (m,

5H), 7.79–80 (m, 1H), 8.19–8.21 (m, 1H), 8.42–8.44 (m, 1H),

8.64 ppm (t, J=5.0 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=

37.7, 41.8, 98.6, 117.3, 123.8, 124.1, 127.0, 127.4, 128.8, 129.5, 129.8,

130.7, 133.3, 136.6, 148.9, 149.9, 151.8, 166.7 ppm; IR (ATR): n˜ =

3251, 1639, 1579, 1536, 1418, 1314, 1229, 810, 749, 688 cm^ꢀ1; MS

(ESI): m/z (%): 360 [M+H]⁺(100); HRMS (ESI): m/z calcd for [M+

H]⁺: 360.0670, found: 360.0705.

1

line solid (251 mg, 85%); mp: 518C; H NMR (400 MHz, [D₆]DMSO):

d_H=2.29 (s, 3H), 3.11 (t, J=8.0, 6.3 Hz, 2H), 3.42 (td, J=7.5, 6.0 Hz,

2H), 7.01–7.03 (m, 1H), 7.19–7.25 (m, 3H), 7.38–7.51 (m, 4H),

8.66 ppm (t, J=6.0 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=

20.8, 31.2, 38.6, 125.2, 126.5, 127.0, 128.6, 128.8, 128.9, 129.5, 129.8,

130.7, 135.3, 136.7, 138.4, 166.3 ppm; IR (ATR): n˜ =3253, 3084,

2913, 1639, 1555, 1427, 1314, 1291, 755, 683 cm^ꢀ1; MS (ESI): m/z

(%): 306 [M+H]⁺(35), 323 [M+NH₄]⁺(100), 328 [M+Na]⁺(34);

HRMS (ESI): m/z calcd for [M+H]⁺: 306.0719, found: 306.0686.

2-Fluoro-N-(2-(m-tolyloxy)ethyl)benzamide (6i): Prepared accord-

ing to general procedure 5b from 2-(m-tolyloxy)ethanaminium

chloride (191 mg, 1.02 mmol), 2-fluorobenzoic acid chloride

(0.12 mL, 1.02 mmol) and Et₃N (0.28 mL, 2.04 mmol). Purification by

chromatography (CH₂Cl₂/MeOH, 19:1) gave a white crystalline solid

2-Chloro-N-(2-(4-isopropylphenoxy)ethyl)benzamide (6e): Pre-

pared according to general procedure 5b from 2-(4-isopropylphe-

noxy)ethanaminium chloride (194 mg, 0.90 mmol), 2-chlorobenzoic

acid chloride (0.11 mL, 0.90 mmol) and Et₃N (0.25 mL, 1.80 mmol).

Purification by chromatography (CH₂Cl₂/MeOH, 19:1) gave a white

crystalline solid (240 mg, 84%); mp: 468C; ¹H NMR (400 MHz,

[D₆]DMSO): d_H=1.17 (d, J=6.9 Hz, 6H), 2.78–2.88 (m, 1H), 3.59 (ps

q, J=5.7 Hz, 2H), 4.07 (t, J=5.8 Hz, 2H), 6.87–6.89 (m, 2H), 7.14–

1

(222 mg, 79); mp: 378C; H NMR (400 MHz, [D₆]DMSO): d_H=2.27 (s,

3H), 3.63 (ps q, J=5.8 Hz, 2H), 4.09 (t, J=5.9 Hz, 2H), 6.74–6.79 (m,

3H), 7.14–7.18 (m, 1H), 7.25–7.30 (m, 2H), 7.50–7.65 (m, 2H),

8.46 ppm (t, J=6.1 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=

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21.0, 38.8, 65.6, 111.5, 115.1, 116.0 (d, J_C–F=22 Hz), 121.4, 123.8 (d,

6.75 (m, 3H), 6.90–7.02 (m, 2H), 7.11–7.15 (m, 1H), 7.33–7.35 (m,

1H), 8.44 ppm (t, J=5.5 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO):

d_C=21.0, 38.7, 55.6, 65.7, 111.4, 112.7, 114.8, 115.1, 121.3, 128.8,

129.1, 130.0, 131.1, 138.8, 158.4, 160.3, 166.2 ppm; IR (ATR): n˜ =

3343, 2958, 1643, 1601, 1490, 1265, 1237, 1169, 1024, 789 cm^ꢀ1; MS

(ESI): m/z (%): 320 [M+H]⁺(100), 342 [M+Na]⁺(31); HRMS (ESI):

m/z calcd for [M+H]⁺: 320.1054, found: 320.1077.

J

_C–F=14 Hz), 124.4 (d, J_C–F= =3 Hz), 129.6, 130.0 (d, J_C–F=3 Hz),

132.3 (d, _C–F=9 Hz), 138.9, 158.4, 159.1 (d, _C–F=249 Hz),

J

164.5 ppm; IR (ATR): n˜ =3374, 2924, 1644, 1523, 1480, 1261, 1166,

1158, 788, 757, 516 cm^ꢀ1; MS (ESI): m/z (%): 274 [M+H]⁺(68), 291

[M+NH₄]⁺(100), 296.14 [M+Na]⁺(49); HRMS (ESI): m/z calcd for

[M+Na]⁺: 296.1063, found: 296.1102.

N-(2-(m-Tolyloxy)ethyl)-2-(trifluoromethyl)benzamide (6j): Pre-

pared according to general procedures 7 and 5b from 2-trifluoro-

methylbenzoic acid (199 mg, 1.05 mmol), oxalyl chloride (0.13 mL,

2-Chloro-4,5-dimethoxy-N-(2-(m-tolyloxy)ethyl)benzamide (6n):

Prepared according to general procedures 7 and 5a from 2-chloro-

4,5-dimethoxybenzoic acid (208 mg, 0.96 mmol), oxalyl chloride

1.57 mmol),

1.05 mmol) and Et₃N (0.29 mL, 2.10 mmol). Purification by chroma-

tography (CH₂Cl₂/MeOH, 19:1) gave white crystalline solid

2-(m-tolyloxy)ethanaminium

chloride

(196 mg,

(0.12 mL,

1.44 mmol),

2-(m-tolyloxy)ethanaminium

chloride

(180 mg, 0.96 mmol) and Et₃N (0.27 mL, 1.92 mmol). Recrystalliza-

tion from toluene gave a white solid (252 mg, 75%); mp: 1128C;

¹H NMR (400 MHz, [D₆]DMSO): d_H=2.27 (s, 3H), 3.57 (ps q, J=

5.8 Hz, 2H), 3.76 (s, 3H), 3.80 (s, 3H), 4.08 (t, J=5.9 Hz, 2H), 6.74–

6.78 (m, 3H), 7.00–7.03 (m, 2H), 7.14–7.18 (m, 1H), 8.45 ppm (t, J=

5.5 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 38.7, 55.7,

55.9, 65.7, 111.4, 112.0, 112.8, 115.1, 121.3, 121.5, 128.0, 129.1,

139.0, 147.2, 145.0, 158.4, 166.1 ppm; IR (ATR): n˜ =3358, 1646,

1598, 1503, 1260, 1213, 1158, 1033, 860, 790 cm^ꢀ1; MS (ESI): m/z

(%): 350 [M+H]⁺(100), 372 [M+Na]⁺(29); HRMS (ESI): m/z calcd

for [M+H]⁺: 350.1159, found: 350.1149.

a

1

(301 mg, 89%); mp: 758C; H NMR (400 MHz, [D₆]DMSO): d_H=2.28

(s, 3H), 3.59 (ps q, J=5.7 Hz, 2H), 4.06 (t, J=5.7 Hz, 2H), 6.73–6.77

(m, 3H), 7.15–7.19 (m, 1H), 7.48–7.50 (m, 1H), 7.62–7.78 (m, 3H),

8.72 ppm (t, J=5.4 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=

21.0, 38.7, 65.6, 111.4, 115.0, 121.3, 123.6 (q, J_C–F=ꢀ270 Hz), 125.8

(q, J_C–F=ꢀ31 Hz), 128.4, 129.2, 129.6, 132.3, 136.3, 138.9, 158.4,

167.3 ppm; IR (ATR): n˜ =3277, 2942, 1638, 1553, 1313, 1267, 1170,

1107, 1031, 779 cm^ꢀ1; MS (ESI): m/z (%): 324 [M+H]⁺(15), 341

[M+NH₄]⁺(100), 346 [M+Na]⁺(18); HRMS (ESI): m/z calcd for

[M+Na]⁺: 346.1031, found: 346.1030.

2,4-Dichloro-N-(2-(m-tolyloxy)ethyl)benzamide (6o): Prepared ac-

cording to general procedures 7 and 5b from 2,4-dichlorobenzoic

acid (148 mg, 0.78 mmol), oxalyl chloride (0.10 mL, 1.17 mmol), 2-

(m-tolyloxy)ethanaminium chloride (146 mg, 0.78 mmol) and Et₃N

(0.22 mL, 1.56 mmol). Purification by chromatography (CH₂Cl₂/

2-Methoxy-N-(2-(m-tolyloxy)ethyl)benzamide (6k): Prepared ac-

cording to general procedures 7 and 5b from 2-methoxybenzoic

acid (207 mg, 1.36 mmol), oxalyl chloride (0.18 mL, 2.04 mmol),

2-(m-tolyloxy)ethanaminium chloride (256 mg, 1.36 mmol) and Et₃N

(0.38 mL, 2.72 mmol). Purification by chromatography (CH₂Cl₂/

1

MeOH, 19:1) gave a white solid (189 mg, 74%); mp: 758C; H NMR

MeOH, 19:1) gave

a

colorless resin (315 mg, 81%); ¹H NMR

(400 MHz, [D₆]DMSO): d_H=2.27 (s, 3H), 3.59 (ps q, J=5.7 Hz, 2H),

4.08 (t, J=5.7 Hz, 2H), 6.73–6.78 (m, 3H), 7.14–7.18 (m, 1H), 7.42–

7.49 (m, 2H), 7.66–7.67 (m, 1H), 8.70 ppm (t, J=5.5 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.1, 38.7, 65.7, 111.5, 115.1,

121.4, 127.2, 129.1, 129.2, 130.2, 131.1, 134.4, 135.6, 138.9, 158.4,

165.7 ppm; IR (ATR): n˜ =3253, 2931, 1651, 1590, 1540, 1262, 1176,

1104, 786, 691 cm^ꢀ1; MS (ESI): m/z (%): 324 [M+H]⁺(30), 341 [M+

NH₄]⁺(100); HRMS (ESI): m/z calcd for [M+H]⁺: 324.0558, found:

324.0538.

(400 MHz, [D₆]DMSO): d_H=2.33 (s, 3H), 3.93 (s, 3H), 3.88 (ps q, J=

5.5 Hz, 2H), 4.15 (t, J=5.2 Hz, 2H), 6.73–6.80 (m, 3H), 6.95–6.97 (m,

1H), 7.05–7.20 (m, 2H), 7.42–7.48 (m, 1H), 8.21–8.23 (m, 1H),

8.36 ppm (br s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.5, 39.2,

55.9, 66.7, 111.3, 111.4, 115.2, 121.2, 121.3, 121.8, 129.3, 132.2,

132.8, 139.6, 157.5, 158.6, 165.4 ppm; IR (ATR): n˜ =3400, 2944,

1650, 1599, 1531, 1484, 1292, 1239, 1159, 758 cm^ꢀ1; MS (ESI): m/z

(%): 286 [M+H]⁺(100), 308 [M+Na]⁺(22); HRMS (ESI): m/z calcd

for [M+Na]⁺: 308.1263, found: 308.1291.

2-Chloro-4-nitro-N-(2-(m-tolyloxy)ethyl)benzamide (6p): Prepared

according to general procedures 7 and 5b from 2-chloro-4-nitro-

benzoic acid (220 mg, 1.09 mmol), oxalyl chloride (0.14 mL,

3-Chloro-N-(2-(m-tolyloxy)ethyl)benzamide (6l): Prepared accord-

ing to general procedures 7 and 5b from 3-chlorobenzoic acid

(202 mg, 1.29 mmol), oxalyl chloride (0.17 mL, 1.93 mmol), 2-(m-tol-

yloxy)ethanaminium chloride (242 mg, 1.29 mmol) and Et₃N

(0.36 mL, 2.58 mmol). Purification by chromatography (CH₂Cl₂/

1.64 mmol),

2-(m-tolyloxy)ethanaminium

chloride

(205 mg,

1.09 mmol) and Et₃N (0.30 mL, 2.18 mmol). Purification by chroma-

tography (CH₂Cl₂/MeOH, 19:1) gave a white solid (260 mg, 72%);

1

MeOH, 19:1) gave a white solid (306 mg, 82%); mp: 458C; H NMR

mp: 948C; H NMR (400 MHz, [D₆]DMSO): d_H=2.28 (s, 3H), 3.63 (ps

(400 MHz, [D₆]DMSO): d_H=2.26 (s, 3H), 3.63 (ps q, J=5.8 Hz, 2H),

4.10 (t, J=5.9 Hz, 2H), 6.73–6.78 (m, 3H), 7.13–7.17 (m, 1H), 7.48–

7.60 (m, 2H), 7.82–7.91 (m, 2H), 8.80 ppm (t, J=5.3 Hz, 1H);

¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 39.0, 65.6, 111.4, 115.0,

121.3, 125.9, 127.0, 129.2, 130.3, 131.0, 133.1, 136.2, 138.9, 158.3,

165.0 ppm; IR (ATR): n˜ =3280, 3054, 2939, 1633, 1543, 1261, 1172,

1063, 779, 689 cm^ꢀ1; MS (ESI): m/z (%): 290 [M+H]⁺(50), 307 [M+

NH₄]⁺(100); HRMS (ESI): m/z calcd for [M+Na]⁺: 312.0767, found:

312.0795.

q, J=5.6 Hz, 2H), 4.10 (t, J=5.6 Hz, 2H), 6.75–6.79 (m, 3H), 7.15–

7.19 (m, 1H), 7.67–7.69 (m, 1H), 8.21–8.33 (m, 2H), 8.93 ppm (t, J=

5.5 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 38.7, 65.7,

111.5, 115.1, 121.4, 122.3, 124.4, 129.2, 129.8, 131.0, 138.9, 142.5,

148.1, 158.3, 165.1 ppm; IR (ATR): n˜ =3299, 1645, 1531, 1349, 1269,

1176, 1065, 880, 778, 690 cm^ꢀ1; MS (ESI): m/z (%): 352 [M+NH₄]⁺

(100), 357 [M+Na]⁺(44); HRMS (ESI): m/z calcd for [M+Na]⁺:

357.0618, found: 357.0596.

4-Amino-2-chloro-N-(2-(m-tolyloxy)ethyl)benzamide (6q): A solu-

tion of 6p (305 mg, 0.91 mmol) and SnCl₂·2H₂O (1.03 g, 4.55 mmol)

in EtOAc (30 mL) was heated at reflux for 3 h. The reaction mixture

was adjusted to pH 8 by addition of saturated aq NaHCO₃, and the

organic layer was separated and saved. The aqueous layer was ex-

tracted with EtOAc (3ꢂ50 mL), and the organic layers were com-

bined and dried over MgSO₄. The solvent was removed, and the

residue was purified by recrystallization from toluene/pentane (9:1)

2-Chloro-4-methoxy-N-(2-(m-tolyloxy)ethyl)benzamide (6m): Pre-

pared according to general procedures 7 and 5b from 2-chloro-4-

methoxybenzoic acid (204 mg, 1.09 mmol), oxalyl chloride

(0.14 mL,

1.64 mmol),

2-(m-tolyloxy)ethanaminium

chloride

(205 mg, 1.09 mmol) and Et₃N (0.30 mL, 2.18 mmol). Purification by

chromatography (CH₂Cl₂/MeOH, 19:1) gave a white solid (203 mg,

58%); mp: 658C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=2.25 (s, 3H),

3.54 (ps q, J=5.8 Hz, 2H), 3.77 (s, 3H), 4.04 (t, J=5.9 Hz, 2H), 6.71–

1

to give a white solid (199 mg, 71%); mp: 888C; H NMR (400 MHz,

ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2013, 8, 442 – 461 457

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[D₆]DMSO): d_H=2.27 (s, 3H), 3.53 (ps q, J=5.9 Hz, 2H), 4.04 (t, J=

5.9 Hz, 2H), 5.68 (br s, 2H), 6.47–6.59 (m, 2H), 6.74–6.77 (m, 3H),

7.14–7.18 (m, 2H), 8.15 ppm (t, J=5.5 Hz, 1H); ¹³C NMR (100 MHz,

[D₆]DMSO): d_C=21.0, 38.6, 65.8, 111.5, 111.5, 113.6, 115.1, 121.3,

122.3, 129.2, 130.4, 131.1, 138.9, 151.1, 158.4, 166.6 ppm; IR (ATR):

n˜ =3351, 3215, 2941, 1603, 1551, 1490, 1290, 1263, 851, 688,

597 cm^ꢀ1; MS (ESI): m/z (%): 305 [M+H]⁺(100), 327 [M+Na]⁺(27);

HRMS (ESI): m/z calcd for [M+Na]⁺: 327.0877, found: 327.0862.

s, 1H), 7.15–7.19 (m, 1H), 7.30–7.33 (m, 1H), 8.05–8.08 (m, 1H),

8.43–8.55 ppm (m, 1H); ¹³C NMR (100 MHz, CDCl₃): d_C=21.5, 39.7,

66.2, 111.3, 115.3, 122.1, 122.7, 129.3, 131.0, 139.7, 147.2, 150.9,

158.3, 164.7 ppm; IR (ATR): n˜ =3271, 3067, 2918, 2871, 1646, 1581,

1398, 1260, 1158, 1062, 772 cm^ꢀ1; MS (ESI): m/z (%): 291 [M+H]⁺

(31), 308 [M+NH₄]⁺(100); HRMS (ESI): m/z calcd for [M+Na]⁺:

313.0720, found: 313.0697.

3-Chloro-N-(2-(m-tolyloxy)ethyl)isonicotinamide (6v): Prepared

according to general procedures 7 and 5b from 3-chloroisonicotin-

ic acid (210 mg, 1.33 mmol), oxalyl chloride (0.17 mL, 2.00 mmol),

2-(m-tolyloxy)ethanaminium chloride (250 mg, 1.33 mmol) and Et₃N

(0.37 mL, 2.67 mmol). Purification by chromatography (CH₂Cl₂/

2-Chloro-4-(dimethylamino)-N-(2-(m-tolyloxy)ethyl)benzamide

(6r): Reductive alkylation of 6q (150 mg, 0.49 mmol) was carried

out as described previously^[11]using 37% aq formaldehyde solution

(0.25 mL, 4.90 mmol), NaBH₃CN (92 mg, 1.47 mmol) and glacial

AcOH (0.08 mL, 1.47 mmol). The residue was purified by recrystalli-

zation from toluene/pentane (9:1) to give a white solid (141 mg,

86%); mp: 618C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=2.27 (s, 3H),

2.93 (s, 6H), 3.55 (ps q, J=5.9 Hz, 2H), 4.06 (t, J=5.9 Hz, 2H), 6.64–

6.78 (m, 5H), 7.14–7.18 (m, 1H), 7.30–7.32 (m, 1H), 8.23 ppm (t, J=

5.5 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0, 38.7, 39.6,

65.8, 109.8, 111.5, 112.0, 115.1, 121.3, 122.5, 129.2, 130.2, 131.4,

138.9, 151.6, 158.4, 166.5 ppm; IR (ATR): n˜ =3289, 2934, 1609, 1514,

1260, 1156, 1061, 808, 764, 686 cm^ꢀ1; MS (ESI): m/z (%): 333 [M+

H]⁺(100), 355 [M+Na]⁺(50); HRMS (ESI): m/z calcd for [M+H]⁺:

333.1370, found: 333.1392.

MeOH, 19:1) gave

a

colorless resin (322 mg, 83%); ¹H NMR

(400 MHz, CDCl₃): d_H=2.33 (s, 3H), 3.88 (ps q, 5.57 Hz, 2H), 4.15 (t,

J=5.9 Hz, 2H), 6.69–6.81 (m, 3H), 6.91 (br s, 1H), 7.15–7.19 (m,

1H), 7.54–7.55 (m, 1H), 8.54–8.55 (m, 1H), 8.63 ppm (s, 1H);

¹³C NMR (100 MHz, CDCl₃): d_C=21.5, 39.6, 66.2, 111.3, 115.3, 122.2,

123.6, 127.8, 129.3, 139.7, 141.3, 148.3, 150.4, 158.2, 164.2 ppm; IR

(ATR): n˜ =3247, 3042, 2941, 2872, 1650, 1534, 1260, 1157, 775,

690 cm^ꢀ1; MS (ESI): m/z (%): 291 [M+H]⁺(86), 308 [M+NH₄]⁺

(100); HRMS (ESI): m/z calcd for [M+Na]⁺: 313.0720, found:

313.0750.

3,7-Dichloro-N-(2-(m-tolyloxy)ethyl)quinoline-8-carboxamide

(6w): Prepared according to general procedures 7 and 5a from 3,7-

dichloroquinoline-8-carboxylic acid (208 mg, 0.86 mmol), oxalyl

chloride (0.11 mL, 1.29 mmol), 2-(m-tolyloxy)ethanaminium chloride

(161 mg, 0.86 mmol) and Et₃N (161 mg, 0.86 mmol). Recrystalliza-

tion from toluene gave a white solid (251 mg, 78%); mp: 1708C;

¹H NMR (400 MHz, CDCl₃): d_H=2.28 (s, 3H), 3.69 (ps q, J=5.7 Hz,

1H), 4.13 (t, J=5.7 Hz, 2H), 6.75–6.79 (m, 3H), 7.15–7.19 (m, 1H),

7.74–7.76 (m, 1H), 8.02–8.04 (m, 1H), 8.66–8.67 (m, 1H), 8.75 (t, J=

4.7 Hz, 1H), 8.90–8.91 ppm (m, 1H); ¹³C NMR (100 MHz, CDCl₃):

d_C=21.1, 38.7, 65.9, 111.5, 115.2, 121.3, 126.6, 128.1, 128.8, 129.0,

129.2, 131.0, 134.3, 135.9, 138.9, 143.3, 150.3, 158.4, 164.9 ppm; IR

(ATR): n˜ =3328, 1672, 1548, 1255, 1157, 1059, 896, 772, 658,

458 cm^ꢀ1; MS (ESI): m/z (%): 375 [M+H]⁺(100); HRMS (ESI): m/z

calcd for [M+H]⁺: 375.0667, found: 375.0708.

2-Chloro-4-methyl-N-(2-(m-tolyloxy)ethyl)benzamide (6s): Pre-

pared according to general procedures 7 and 5a from 2-chloro-4-

methylbenzoic acid (203 mg, 1.19 mmol), oxalyl chloride (0.15 mL,

1.79 mmol),

2-(m-tolyloxy)ethanaminium

chloride

(223 mg,

1.19 mmol) and Et₃N (0.33 mL, 2.38 mmol). Recrystallization from

toluene gave a white solid (195 mg, 54%); mp: 808C; ¹H NMR

(400 MHz, [D₆]DMSO): d_H=2.27, (s, 3H), 2.32 (s, 3H), 3.58 (ps q, J=

5.8 Hz, 2H), 4.07 (t, J=5.8 Hz, 2H), 6.74–6.78 (m, 3H), 7.14–7.31 (m,

4H), 8.58 ppm (t, J=5.5 Hz, 1H); ¹³C NMR (100 MHz, [D₆]DMSO):

d_C=20.4, 21.1, 38.7, 65.7, 111.5, 115.1, 121.4, 127.5, 128.7, 129.2,

129.7, 129.8, 133.8, 138.9, 140.8, 158.4, 166.6 ppm; IR (ATR): n˜ =

3257, 3085, 3052, 2913, 2860, 1643, 1558, 1257, 1052, 689 cm^ꢀ1

;

MS (ESI): m/z (%): 304 [M+H]⁺(100), 321 [M+NH₄]⁺(97), 326

[M+Na]⁺(41); HRMS (ESI): m/z calcd for [M+Na]⁺: 326.0924,

found: 326.0954.

7-Chloro-3-methyl-N-(2-(m-tolyloxy)ethyl)quinoline-8-carboxam-

ide (6x): Prepared according to general procedures 7 and 5a from

7-chloro-3-methyl-quinoline-8-carboxylic acid (204 mg, 0.92 mmol),

oxalyl chloride (0.12 mL, 1.38 mmol), 2-(m-tolyloxy)ethanaminium

chloride (173 mg, 0.92 mmol) and Et₃N (0.26 mL, 1.84 mmol). Re-

crystallization from toluene gave a white solid (219 mg, 67%); mp:

116 8C; ¹H NMR (400 MHz, CDCl₃): d_H=2.29 (s, 3H), 2.48 (s, 3H),

3.69 (ps q, J=5.8 Hz, 2H), 4.13 (t, J=5.8 Hz, 2H), 6.76–6.80 (m, 3H),

7.15–7.19 (m, 1H), 7.60–7.63 (m, 1H), 7.93–7.95 (m, 1H), 8.18–8.19

(m, 1H), 8.67 (t, J=5.6 Hz, 1H), 8.78–8.79 ppm (m, 1H); ¹³C NMR

(100 MHz, CDCl₃): d_C=18.1, 21.1, 38.6, 66.0, 111.5, 115.2, 121.3,

126.1, 127.3, 128.9, 129.2, 129.4, 131.2, 134.4, 135.7, 138.9, 143.6,

153.3, 158.4, 165.4 ppm; IR (ATR): n˜ =3316, 2917, 1628, 1575, 1487,

1258, 1159, 1062, 895, 785, 690 cm^ꢀ1; MS (ESI): m/z (%): 335 [M+

H]⁺(100); HRMS (ESI): m/z calcd for [M+H]⁺: 355.1213, found:

355.1239.

2-Chloro-4-hydroxy-N-(2-(m-tolyloxy)ethyl)benzamide (6t): Pre-

pared according to general procedures 7 and 5b from 2-chloro-4-

hydroxybenzoic acid (209 mg, 1.21 mmol), oxalyl chloride (0.16 mL,

1.82 mmol),

2-(m-tolyloxy)ethanaminium

chloride

(228 mg,

1.21 mmol) and Et₃N (0.34 mL, 2.42 mmol). Purification by chroma-

tography (CH₂Cl₂/MeOH, 19:1) gave a white solid (152 mg, 41%);

mp: 1548C; ¹H NMR (400 MHz, [D₆]DMSO): d_H=2.27 (s, 3H), 3.55

(ps q, J=5.8 Hz, 2H), 4.05 (t, J=5.9 Hz, 2H), 6.73–6.83 (m, 5H),

7.14–7.18 (m, 1H), 7.26–7.29 (m, 1H), 8.48 (t, J=5.6 Hz, 1H),

10.17 ppm (br s, 1H); ¹³C NMR (100 MHz, [D₆]DMSO): d_C=21.0,

38.7, 65.7, 111.5, 113.9, 115.1, 116.1, 121.3, 127.2, 129.2, 130.3,

130.9, 138.9, 158.4, 158.9, 166.4 ppm; IR (ATR): n˜ =3277, 3115,

1598, 1569, 1492, 1264, 1170, 1119, 855, 783, 689 cm^ꢀ1; MS (ESI): m/

z (%): 306 [M+H]⁺(100), 328 [M+Na]⁺(36); HRMS (ESI): m/z calcd

for [M+Na]⁺: 328.0716, found: 328.0723.

2-Chloro-N-(2-(m-tolyloxy)ethyl)nicotinamide (6u): Prepared ac-

Virtual screening and docking

cording

2-chloronicotinic acid (207 mg, 1.32 mmol), oxalyl chloride

(0.17 mL, 1.98 mmol), 2-(m-tolyloxy)ethanaminium chloride

(247 mg, 1.32 mmol) and Et₃N (0.37 mL, 2.64 mmol). Purification by

chromatography (CH₂Cl₂/MeOH, 19:1) gave colorless resin

to

general

procedures

7

and

5b

from

The fragment-like library was designed according to the methodol-

ogy reported by Koester et al.^[35]The number of Lipinski acceptors,

calculated (C) logP, and total polar surface area (TPSA) parameters

were slightly modified in order to expand the chemical space of

the library; in particular, the number of lipophilic compounds was

increased. All properties were calculated within the Molecular Op-

a

1

(287 mg, 75%); H NMR (400 MHz, CDCl₃): d_H=2.32 (s, 3H), 3.88 (ps

q, J=5.5 Hz, 2H), 4.15 (t, J=5.7 Hz, 2H), 6.70–6.80 (m, 3H), 7.05 (br

ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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erating Environment (MOE) software.^[36]A total of 13200 ligands

were energy minimized and protonated using the standard force

field (MMFF94x) implemented in MOE. Ligands were docked into

the binding sites of two PfENR X-ray crystal structures (PDB:

2O2Y^[16]and 2OOS^[19]) using the GOLD program.^[20]For the docking

procedure, solvent molecules were removed, and the binding site

was defined by a radius of 7 ꢁ around the inhibitor, considering

NAD⁺as a part of the protein. Docking runs were performed by

applying 50 genetic algorithm (GA) runs, a search efficiency of

50%, and the astex statistical potential (ASP) scoring function.^[37]

For each compound, five best-scored solutions were further evalu-

ated. After clustering the poses according to root-mean-square de-

viation (RMSD)<2 ꢁ, the results were locally minimized, rescored

and reranked by DSX using the per-atom-score as indicator. The

200 top-ranked solutions were visually inspected. Graphical repre-

sentations of protein–ligand interactions were prepared using

PyMOL (version 1.2r2).^[38]

independent determinations. Data were analyzed using the Sigma-

Plot (version 9.0; Hill function, three parameters) and Sigma Stat

programs.

TgENR purification and inhibition assay

Recombinant TgENR was purified as described previously.^[45]An

assay of TgENR enzymatic activity used previously^[46]was adapted

for use in 96-well plates. The activity of TgENR was monitored by

consumption of NADH (e340=6220m^ꢀ1cm^ꢀ1) with a SpectraMax

M2 plate reader. Reactions were carried out with a final volume of

100 mL in 96-well Corning UV plates. Initially, 10 mL of 1 mm croton-

yl-CoA (Sigma) was placed in each well with 1 mL of DMSO (con-

trol) or compound dissolved in DMSO. Then a reaction mixture

containing a final concentration of 5 nm TgENR, 100 mm Na/K

phosphate (pH 7.5), 150 mm NaCl and 100 mm NADH was added

row by row with a 12-channel pipetter to initiate the assay. Follow-

ing a brief mixing period, the absorbance in each well was moni-

tored at 35 s intervals for 15 min. The first column of each plate

contained blank reactions with 1% DMSO, which served as base-

line activities for each row of the assay plate. The last column of

each plate contained the potent inhibitor triclosan in concentra-

tions ranging from 10 mm to 610 pm (serial dilutions by factors of

4), which served as a positive control for TgENR inhibition. Enzy-

matic activity was determined by comparing the slopes of the ab-

sorbance curves for each well to those of the blanks in the first

column of the plate.

For the creation of a combinatorial library of aryloxyalkylbenzam-

ides, the ZINC database (version 11)^[39]was screened for commer-

cially available derivatives of salicylic acid and bromoethyl ethers

applying the fconv^[40]substructure search. We were able to retrieve

3068 salicylic acid and 190 bromoethylether non-redundant deriva-

tives. The hydroxy group of the carboxy moiety was defined as

linker L1 and the bromine atom as linker L2. All fragments were

connected via a nitrogen atom using the combinatorial routine of

CoLibri.^[41]The computed library of about 580000 molecules was

filtered using a molecular weight (MW) threshold (MW<450 Da)

resulting in nearly 110000 compounds ready for screening. The pa-

rameters for the initial docking were set to 30 GA runs, search effi-

ciency 30%, ASP scoring function, and only the best-scored pose

per compound was considered for further evaluation. The DSX per-

atom-score was considered in the re-ranking procedure. The top

10000 compounds were used for the subsequent docking run. All

settings for the subsequent docking were set to and the analysis

of results was performed as those described for the fragment-like

library screening.

Anopheles mosquito infection cycle and isolation of infectious

salivary gland sporozoites

All animal experiments were performed according to FELASA cate-

gory B and GV-SOLAS standard guidelines and approved by

German authorities (Regierungsprꢃsidium Karlsruhe, Germany), § 8

Abs. 1 Tierschutzgesetz (TierSchG).

Female NMRI outbred mice were obtained from Charles River Lab-

oratories. For analysis of sporozoite invasion and liver-stage devel-

opment, we used Plasmodium berghei ANKA parasites (MRA-871,

ANKA cl15cy1). For P. berghei sporozoite production, Anopheles ste-

phensi mosquitoes were fed on gametocyte-infected outbred NMRI

mice. Then, sporozoites were dissected from the salivary glands of

mosquitoes 17–21 days after the infectious blood meal and were

immediately used for in vitro infection studies. HuH7 cells were

kindly provided by Ralf Bartenschlager (Molecular Virology, Univer-

sity Hospital Heidelberg, Germany).

Cell culture and blood-stage drug assays

The multidrug-resistant P. falciparum clone Dd2 was cultured as de-

scribed previously^[42]and synchronized using the sorbitol

method.^[43]Cultures were maintained in type A-positive human er-

ythrocytes suspended at a hematocrit of 5% in RPMI 1640 supple-

mented with 2 mm l-glutamine, 25 mm HEPES, 20 gmL^ꢀ1gentami-

cin, and 10% human type A-positive serum. Cultures were incubat-

ed at 378C under controlled atmospheric conditions of 5% O₂, 3%

CO₂, and 92% N₂. Cell proliferation assays were performed in the

presence of different drug concentrations as described previous-

ly,^[44]with the following modifications: 100 mL aliquots of a cell sus-

pension containing highly synchronized rings (2–6 h post-invasion)

at a parasitemia of 0.5% and a hematocrit of 1.25% were placed in

the wells of 96-well microtiter plates, and the plates were incubat-

ed for 72 h in the presence of different drug concentrations. Sub-

sequently, 100 mL of lysis buffer (40 mm Tris, pH 7.5, 10 mm EDTA,

0.016% saponin, 0.08% Triton X-100) containing 8.3 mm SYBR

green I was added to each well. The content was mixed, and the

microtiter plates were incubated for 1 h in the dark at RT before

fluorescence (l_exc=485 nm; l_em=520 nm) was determined using

a microtiter plate fluorescence reader (FLUOstar; BMG Labtech).

Drugs were titrated 1:3, and each drug concentration was exam-

ined in triplicate. As controls, uninfected erythrocytes (hematocrit,

1.25%) and infected erythrocytes without drug were investigated

in parallel. The IC₅₀values reported are the means of at least three

Liver-stage cell culture and assay

HuH7 cells were cultured in Dulbecco’s modified Eagle medium

(Invitrogen) supplemented with 10% fetal calf serum (Invitrogen)

and 1% penicillin/streptomycin (Invitrogen) at 378C and 5% CO₂.

For both the sporozoite invasion assay and liver-stage growth

assay, 25000 HuH7 hepatoma cells were seeded in 8-well chamber

slides (Lab-Tek slides, NUNC) one day before the infection experi-

ment was started. Prior to invasion of HuH7 cells, sporozoites were

incubated for 15 min with either 0.5% DMSO (Carl Roth), 10 mm cy-

tochalasin D (Sigma–Aldrich) or different concentrations of com-

pound 6g or 5j. 10000 salivary gland sporozoites were applied

per well and allowed to invade for 90 min. In order to distinguish

extracellular from intracellular parasites, cells were then fixed with

4% paraformaldehyde in phosphate-buffered saline (AppliChem)

and stained with anti-PbCSP and goat anti-mouse Alexa Fluor 488

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secondary antibody (Becton, Dickinson and Co.) followed by per-

meabilization with ice-cold MeOH (AppliChem) and further staining

with anti-PbCSP and goat anti-mouse Alexa Fluor 546 secondary

antibody (Becton, Dickinson and Co.), according to the two-color-

host-cell invasion method described previously.^[25]

Keywords: antimalarial agents

molecular modeling multistage inhibitors

falciparum · virtual screening

·

fatty acid biosynthesis

·

Plasmodium

Liver-stage development assays were performed as previously de-

scribed.^[23]Briefly, on day 0, each well of a confluent Lab-Tek slide

layered with HuH7 cells was inoculated with 10000 freshly pre-

pared P. berghei ANKA infectious sporozoites. 24 h, 40 h and 60 h

after infection, the assay was fixed by applying ice-cold MeOH (Ap-

pliChem), followed by staining with monoclonal PbHSP70 anti-

body^[24]and anti-mouse Alexa Fluor 488 (Becton, Dickinson and

Co.) as a secondary antibody. Counting of liver-stage parasites was

carried out using a Zeiss fluorescence microscope. Confocal pic-

tures were taken with a Zeiss Axiovert 100M microscope. Zeiss

Image Examiner software was utilized for image processing and

measurement of liver-stage sizes. One-way ANOVA statistical analy-

sis and calculation of IC₉₀values from variable-slope sigmoidal

dose–response curves was performed with Prism 5 for Mac OS X

and Microsoft Excel for Mac 2011.

[1] C. J. L. Murray, L. C. Rosenfeld, S. S. Lim, K. G. Andrews, K. J. Foreman, D.

Haring, N. Fullman, M. Naghavi, R. Lozano, A. D. Lopez, Lancet 2012 ,

379 , 41 3 –4 31.

[2] A. Sturm, Science 2006 , 313 , 128 7 –1 290.

[3] World Malaria Report 2010, World Health Organization (WHO) Global

Malaria Programme, 2010; available here: http://www.who.int/malaria/

world_malaria_report_2010/worldmalariareport2010.pdf (Last accessed:

December 8, 2012) .

[4] M. Yu, T. R. S. Kumar, L. J. Nkrumah, A. Coppi, S. Retzlaff, C. D. Li, B. J.

Kelly, P. A. Moura, V. Lakshmanan, J. S. Freundlich, J.-C. Valderramos, C.

Vilcheze, M. Siedner, J. H.-C. Tsai, B. Falkard, A. bir Singh Sidhu, L. A. Pur-

cell, P. Gratraud, L. Kremer, A. P. Waters, G. Schiehser, et al., Cell Host Mi-

crobe 2008 , 4 , 56 7 –5 78.

[5] A. S. Tarun, A. M. Vaughan, S. H. I. Kappe, Trends Parasitol. 2009 , 25 ,

54 5 –5 50.

[6] H. J. Vial, M. J. Thuet, J. R. Philippot, J. Protozool. 1982, 29, 258–263.

[7] L. M. McMurry, M. Oethinger, S. B. Levy, Nature 1998 , 394 , 53 1 –5 32.

[8] a) G. I. McFadden, M. E. Reith, J. Munholland, N. Lang-Unnasch, Nature

1996 , 381 , 482; b) S. Kohler, Science 1997 , 275 , 148 5 –1 489.

[9] M. Kapoor, M. J. Dar, A. Surolia, N. Surolia, Biochem. Biophys. Res.

Commun. 2001 , 289 , 83 2 –8 37.

Cytotoxicity assay

[10] N. Surolia, A. Surolia, Nat. Med. 2001 , 7 , 16 7 –1 73.

[11] R. McLeod, S. P. Muench, J. B. Rafferty, D. E. Kyle, E. J. Mui, M. J. Kirisits,

D. G. Mack, C. W. Roberts, B. U. Samuel, R. E. Lyons, M. Dorris, W. K Mil-

hous, D. W. Rice, Int. J. Parasitol. 2001 , 31 , 10 9 –1 13.

[12] A. M. Vaughan, M. T. O’Neill, A. S. Tarun, N. Camargo, T. M. Phuong,

A. S. I. Aly, A. F. Cowman, S. H. I. Kappe, Cell. Microbiol. 2009 , 11 , 50 6 –

520.

[13] M. D. Spalding, S. T. Prigge, Cell Host Microbe 2008 , 4 , 50 9 –5 11.

[14] S. D. Kohlwein, S. Eder, C.-S. Oh, C. E. Martin, K. Gable, D. Bacikova, T.

Dunn, Mol. Cell. Biol. 2001 , 21 , 10 9 –1 25.

[15] S. H. Lee, J. L. Stephens, K. S. Paul, P. T. Englund, Cell 2006 , 126 , 69 1 –

699.

[16] S. P. Muench, S. T. Prigge, R. McLeod, J. B. Rafferty, M. J. Kirisits, C. W.

Roberts, E. J. Mui, D. W. Rice, Acta Crystallogr. Sect. D 2007 , 63 , 32 8 –

338.

[17] D. Tasdemir, G. Lack, R. Brun, P. Rꢄedi, L. Scapozza, R. Perozzo, J. Med.

Chem. 2006 , 49 , 334 5 –3 353.

[18] D. Tasdemir, B. Topaloglu, R. Perozzo, R. Brun, R. O’Neill, N. M. Carbal-

leira, X. Zhang, P. J. Tonge, A. Linden, P. Rꢄedi, Bioorg. Med. Chem. 2007 ,

15 , 683 4 –6 845.

[19] J. S. Freundlich, F. Wang, H.-C. Tsai, M. Kuo, H.-M. Shieh, J. W. Anderson,

L. J. Nkrumah, J.-C. Valderramos, M. Yu, T. R. S. Kumar, J. Biol. Chem.

2007 , 282 , 2543 6 –2 5444.

[20] G. Jones, P. Willett, R. C. Glen, J. Mol. Biol. 1995 , 245 , 4 3 –5 3.

[21] G. Neudert, G. Klebe, J. Chem. Inf. Model. 2011 , 51 , 273 1 –2 745.

[22] R. Perozzo, M. Kuo, A. b. S. Sidhu, J. T. Valiyaveettil, R. Bittman, W. R.

Jacobs, D. A. Fidock, J. C. Sacchettini, J. Biol. Chem. 2002 , 277 , 1310 6 –

13114.

HeLa (DSM ACC 57) cells were grown in RPMI 1640 culture

medium (LONZA, BE 12–167F) supplemented with 10 mLL^ꢀ1ultra-

glutamine 1 (CAMBREX 17-605E/U1), 550 mLL^ꢀ1gentamicin sulfate

(CAMBREX 17-518Z), and 10% heat-inactivated fetal bovine serum

(PAA A15-104) at 378C in high-density polyethylene flasks (NUNC

156340). The cells were preincubated for 48 h without the test sub-

stances. Dilutions of the compounds were carried out carefully on

the subconfluent monolayers of HeLa cells after the preincubation

time. The compound-treated HeLa cells were further incubated for

72 h at 378C in a humidified atmosphere and 5% CO₂. After incu-

bation, adherent HeLa cells were fixed by glutaraldehyde (MERCK

1.04239.0250) and stained with a 0.05% solution of methylene

blue (SERVA 29198) for 15 min. After gently washing, the stain was

eluted by 0.2 mL of 0.33n HCl in the wells. The optical densities

(OD) were measured at 660 nm in a microplate reader (Sunrise,

Tecan, Mꢃnnedorf, Switzerland). Under the experimental conditions

described, the methylene blue signal is proportional to the

number of viable cells. For data analysis, the Magellan software

(Tecan) was used. The 50% cytotoxicity concentration (CC₅₀) was

defined as the test compound concentration required for destruc-

tion of 50% of the cell monolayer compared with untreated con-

trol. The CC₅₀values reported are the means of four replicates.

[23] M. R. Hollingdale, P. Leland, A. L. Schwartz, Am. J. Trop. Med. Hyg. 1983,

32, 682–684.

[24] M. Tsuji, D. Mattei, R. S. Nussenzweig, D. Eichinger, F. Zavala, Parasitol.

Res. 1994 , 80 , 1 6 –2 1.

Acknowledgements

Financial support from the US National Institute of Allergy and

Infectious Diseases (NIAID) (grant U01 AI082180-0101 to J.T.P.)

and the LOEWE program “Synmikro” of the State of Hesse (Ger-

many) (grants to S.G. and G.K.) is gratefully acknowledged. J.M.S.

is a fellow of the Rahel-Goitein-Strauss-Program of the University

Hospital Heidelberg (Germany). The authors would like to thank

Jennifer Schahn and Miriam Ester for practical assistance and

mosquito breeding, and Felix Terwesten for calculating the mo-

lecular properties of compound 6g.

[25] C. Pinzon-Ortiz, J. Friedman, J. Esko, P. Sinnis, J. Biol. Chem. 2001 , 276 ,

2678 4 –2 6791.

[26] S. Choi, B. Sainz Jr., P. Corcoran, S. Uprichard, H. Jeong, Xenobiotica

2009 , 39 , 20 5 –2 17.

[27] J. L. Vennerstrom, E. O. Nuzum, R. E. Miller, A. Dorn, L. Gerena, P. A.

Dande, W. Y. Ellis, R. G. Ridley, W. K. Milhous, Antimicrob. Agents Chemo-

ther. 1999, 43, 598–602.

[28] T. Qidwai, F. Khan, Chem. Biol. Drug Des. 2012 , 80 , 15 5 –1 72.

[29] A. R. Katritzky, S. Rachwal, J. Wu, Can. J. Chem. 1990 , 68 , 44 6 –4 55.

[30] M. B. Winstead, H. W. Heine, J. Am. Chem. Soc. 1955 , 77 , 191 3 –1 914.

[31] M. C. Cruz, F. Jimꢅnez, F. Delgado, J. Tamariz, Synlett 2006, 749–755.

ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2013, 8, 442 – 461 460

CHEMMEDCHEM

FULL PAPERS

www.chemmedchem.org

[32] G. C. Finger, D. R. Dickerson, L. D. Starr, D. E. Orlopp, J. Med. Chem.

1965 , 8 , 40 5 –4 07.

[33] P. Jꢄtten, W. Schumann, A. Hꢃrtl, H.-M. Dahse, U. Grꢃfe, J. Med. Chem.

2007 , 50 , 366 1 –3 666.

[34] P. E. Morris Jr., A. J. Elliott, S. P. Walton, C. H. Williams, J. A. Montgomery,

Nucleosides Nucleotides Nucleic Acids 2000 , 19 , 37 9 –4 04.

[35] H. Kçster, T. Craan, S. Brass, C. Herhaus, M. Zentgraf, L. Neumann, A.

Heine, G. Klebe, J. Med. Chem. 2011 , 54 , 778 4 –7 796.

[36] Molecular Operating Environment (MOE), version 2010.10, Chemical

Computing Group Inc. (Montreal, Canada), 2010.

[37] W. T. M. Mooij, M. L. Verdonk, Proteins 2005 , 61 , 27 2 –2 87.

[38] The PyMOL Molecular Graphics System, version 1.2r2, Schrçdinger, LLC;

http://www.pymol.org/.

[42] W. Trager, J. B. Jensen, J. Parasitol. 2005 , 91 , 48 4 –4 86.

[43] C. Lambros, J. P. Vanderberg, J. Parasitol. 1979 , 65 , 41 8 –4 20.

[44] a) R. E. Desjardins, C. J. Canfield, J. D. Haynes, J. D. Chulay, Antimicrob.

Agents Chemother. 1979 , 16 , 71 0 –7 18; b) M. Smilkstein, N. Sriwilaijar-

oen, J. X. Kelly, P. Wilairat, M. Riscoe, Antimicrob. Agents Chemother.

2004 , 48 , 180 3 –1 806.

[45] S. P. Muench, S. T. Prigge, L. Zhu, M. J. Kirisits, C. W. Roberts, S. Werni-

mont, R. McLeod, D. W. Rice, Acta Crystallogr. Sect. F 2006 , 62 , 60 4 –6 06.

[46] S. K. Tipparaju, S. P. Muench, E. J. Mui, S. N. Ruzheinikov, J. Z. Lu, S. L.

Hutson, M. J. Kirisits, S. T. Prigge, C. W. Roberts, F. L. Henriquez, A. P. Ko-

zikowski, D. W. Rice, R. L. McLeod, J. Med. Chem. 2010 , 53 , 628 7 –6 300.

[39] J. J. Irwin, B. K. Shoichet, J. Chem. Inf. Model. 2005 , 45 , 17 7 –1 82.

[40] G. Neudert, G. Klebe, Bioinformatics 2011 , 27 , 102 1 –1 022.

[41] BioSolveIT, BioSolveIT GmbH (Sankt Augustin, Germany); http://

www.biosolveit.de/.

Received: September 5, 2012

Published online on January 22, 2013

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ChemMedChem 2013, 8, 442 – 461 461

Article Doi

Novel TypeII Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents

DOI: 10.1002/cmdc.201200407

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Article abstract of DOI:10.1002/cmdc.201200407

Full text of DOI:10.1002/cmdc.201200407

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