Salicylanilide inhibitors of Toxoplasma gondii

Article

pubs.acs.org/jmc

Salicylanilide Inhibitors of Toxoplasma gondii

Alina Fomovska,^†,▽Richard D. Wood,*^,‡,▽Ernest Mui,^†Jitenter P. Dubey,^§Leandra R. Ferreira,^§

Mark R. Hickman,^∥Patricia J. Lee,^∥Susan E. Leed,^∥Jennifer M. Auschwitz,^∥William J. Welsh,*^,‡,⊥

Caroline Sommerville,^#Stuart Woods,^#Craig Roberts,^#and Rima McLeod*^,†

^†Department of Ophthalmology and Visual Sciences, Pediatrics (Infectious Diseases), Committees on Genetics, Immunology, and

Molecular Medicine, Institute of Genomics and Systems Biology, and The College, The University of Chicago, Chicago,

Illinois 60637, United States

^‡Snowdon, Inc., Monmouth Junction, New Jersey, United States

^§United States Department of Agriculture, Agricultural Research Service, Animal Natural Resources Institute, Animal Parasitic Disease

Laboratory, BARC-East, Building 1001, Beltsville, Maryland 20705-2350, United States

^∥Department of Discovery, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Rm 2N61,

503 Robert Grant Avenue, Silver Spring, Maryland 20910, United States

^⊥Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey,

Piscataway, New Jersey 08854, United States

^#Strathclyde Institute Of Pharmacy And Biomedical Sciences, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom

ABSTRACT: Toxoplasma gondii (T. gondii) is an apicomplexan parasite that can cause eye disease,

brain disease, and death, especially in congenitally infected and immune-compromised people.

Novel medicines eﬀective against both active and latent forms of the parasite are greatly needed.

The current study focused on the discovery of such medicines by exploring a family of potential

inhibitors whose antiapicomplexan activity has not been previously reported. Initial screening eﬀorts

revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in

vitro against T. gondii. This observation inspired the evaluation of the activity of a series of

salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no

appreciable in vitro toxicity to human cells were identiﬁed. An initial structure−activity relationship

was explored. Four compounds were selected for evaluation in an in vivo model of infection, and

two derivatives with potentially enhanced pharmacological parameters demonstrated the best

activity proﬁles.

Niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxy-

benzamide, 4) is a well-established FDA-approved anthel-

minthic drug⁴whose activity in the tapeworm is thought to

involve the uncoupling of oxidative phosphorylation.⁵It is not

toxic at high concentrations when administered orally.⁶Earlier

unpublished studies by our group had shown that niclosamide

was a potential inhibitor of T. gondii (MIC₅₀250−200 nM).

Although niclosamide has the disadvantage of low solubility and

low bioavailability,^7,8its promising activity against T. gondii

inspired the preparation and testing of a series of salicylanilides

and derivatives in the anticipation of potentially improving

potency and physicochemical and pharmacological properties.

These were evaluated for activity against T. gondii tachyzoites

and for toxicity toward host cells in vitro. Experiments were

conducted to determine whether the observed activity was

due to static or cidal eﬀects. The most promising inhibitors

that emerged from this study were the carbamate derivatives

14a and 14b, which possess an ionizable moiety appended to

the salicylanilide core.

INTRODUCTION

■

The apicomplexan family of parasites, which includes members

such as Plasmodium, Babesia, and Toxoplasma, are protozoa of

great medical and economic signiﬁcance.¹T. gondii is one of the

most successful parasites on earth, infecting all warm-blooded

animals and one-third to one-half of the human population.

This parasite can cause disease, toxoplasmosis, with eye and

neurological damage, systemic illness, and death. Toxoplasmo-

sis can be especially devastating in those infected congenitally,

immune-compromised persons, or those with postnatally acquired

infection.

Although the only deﬁnitive hosts of this obligate, intra-

cellular parasite are members of the Felidae (cat) family, in

humans and other intermediate hosts, T. gondii exists in two life

stages: the rapidly proliferating tachyzoite form and the latent

encysted bradyzoite form, which remains in the body for the

duration of the lifetime of the host, maintaining the risk of

recurrence.²There are currently no eﬀective treatments against the

bradyzoite form, and those medicines which target the tachyzoite

form (pyrimethamine and sulfadiazine are the most eﬀective) can

be associated with toxicity and hypersensitivity.³Novel, nontoxic

anti-Toxoplasma agents are greatly needed.

Received: May 31, 2012

Published: September 12, 2012

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Scheme 1. Preparation of 3a−3 ae

As an apicomplexan parasite, T. gondii is often used as a

model organism to study other members of this Order, such as

Plasmodium, Babesia, and Eimeria. Because the inhibitory

activity of the subject compounds against other apicomplexans

is of great interest, selected compounds were also tested for

eﬃcacy against both drug-sensitive and drug-resistant strains of

Plasmodium falciparum, the causative agent of the most virulent

form of malaria in humans, and were found to be eﬀective as

described herein.

were simultaneously evaluated for eﬃcacy and toxicity against

human cells.

Selected compounds exhibiting the ability to inhibit

tachyzoite growth were evaluated to determine whether their

activity was due to a static or cidal eﬀect.

Compound activity against P. falciparum, a causative agent of

malaria, was assessed using the Malaria SYBR Green I-Based

Fluorescence (MSF) Assay. The assay is a microtiter plate drug

sensitivity assay that uses the presence of malarial DNA as a

measure of parasitic proliferation in the presence of antimalarial

drugs or experimental compounds based on modiﬁcations of

previously described methods by Plouﬀe et al.¹⁰and Johnson

et al.¹¹As the intercalation of SYBR Green I dye and its resulting

ﬂuorescence is relative to parasite growth, a test compound

that inhibits the growth of the parasite will result in a lower

ﬂuorescence.

CHEMISTRY

■

Commercially available salicylic acids 1 were coupled with

commercially available anilines 2 in hot xylenes in the presence

of PCl₃to furnish salicylanilides 3⁹(Scheme 1). R₁−R₆are

deﬁned in Table 1.

Reduction of niclosamide 4 with Zn dust in methanol and

acetic acid followed by salt formation gave amino salicylanilide

hydrochloride 5 (Scheme 2).

Simple ester or carbamate derivatives of 4 were obtained

through treatment of 4 with various carbonyl chlorides 6 to

provide acylated derivatives 7 (Scheme 3).

Selected compounds were examined for activity against two

strains of P. falciparum: D6 (CDC/Sierra Leone), a drug-

sensitive strain readily killed by chloroquine, and TM91-C235,

a multidrug resistant strain resistant to chloroquine.

IN VIVO BIOASSAYS

■

The ﬂuorine-containing salicylanilide methyl ethers 10 were

synthesized by HATU-mediated condensation of 5-ﬂuoro-2-

methoxybenzoic acid 8 with nitroanilines 9 (Scheme 4).

Sarcosine tert-butyl ester hydrochloride 11 was transformed

into the free base and treated with phosgene in toluene to provide

tert-butyl 2-((chlorocarbonyl)(methyl) amino)acetate 12. We

found that triphosgene and a solution of phosgene in toluene are

essentially equivalent for this transformation. 12 reacted with 4

smoothly in warm pyridine under DMAP catalysis to furnish

the expected carbamate ester. Sequential removal of the ester

function by treatment with triﬂuoroacetic acid, condensation of

the resulting carboxylic acid 13a with tert-butyl carbazate using

EDCI, and treatment of the resulting protected acid hydrazide

with HCl in dioxane furnished the sarcosine hydrazide hydro-

chloride 14a. In a similar fashion, salicylanilide 3j was converted

to the corresponding sarcosine hydrazide hydrochloride 14b

(Scheme 5).

Selected compounds were evaluated for in vivo eﬃcacy against

T. gondii. To assess toxic eﬀects when administered orally,

selected compounds were administered orally to mice daily for

nine days at a dose of 100 mg/kg. At the end of the 10 days, the

animals were evaluated for toxic eﬀects.

Compounds 14a and 14b were tested for eﬃcacy against

T. gondii in the oocyst stage following per oral challenge in

mice. Mice were infected by oral gavage with ME49 or

TgGoatUS4 oocysts. Mice were treated with either 100 or

25 mg/kg of test substance. Diluent was DMSO (1.0%)/PEG

400 (5.0%)/0.5% CMC (94.0%), where PEG 400 is polyethylene

glycol, average MW 400, 0.5% CMC = carboxymethylcellulose

0.5% in water.

RESULTS AND DISCUSSION

■

A limited medicinal chemistry eﬀort was undertaken to probe

the eﬀects of the variation of ring substituents and the derivatiza-

tion of the phenolic oxygen of the core salicylanilide structure.

Salicylanilides 3a−3ae, 4, and 5 and derivatives 7a−7d, 10a,

10b, 14 a, and 14b were tested for in vitro eﬃcacy against

T. gondii tachyzoites. It was decided that only those compounds

possessing MIC₅₀≤ 1 μM would be considered active against

T. gondii. The eﬃcacy and corresponding cellular toxicity data

appear in Table 1. Of the 39 compounds assayed, 16 (41%) had

MIC₅₀≤ 1 μM, 12 (31%) had MIC₅₀≤ 500 nM, 6 (15%) had

MIC₅₀≤ 250 nM, and 4 (10%) had MIC₅₀≤ 125 nM.

IN VITRO BIOASSAYS

■

Parasite proliferation was monitored using stably transfected

type I RH-YFP parasites, which constitutively express yellow

ﬂuorescent protein. Proliferation also was tested using a [³H]-

uracil incorporation assay, as uracil is incorporated into nucleic

acids of T. gondii tachyzoites, but not mammalian cells, as

they divide. Complementary challenge assays ensured that the

observed ﬂuorescence data was due to parasite inhibition and

not to quenched ﬂuorescence. Pyrimethamine and sulfadiazine

were used as positive controls, and DMSO at a concentration

of 0.1% was used as the negative control. Because T. gondii is an

obligate parasite, compounds that are toxic to host cells will

appear to inhibit parasite growth. Therefore, all test compounds

Initially, the phenolic functionality was maintained, and two

alterations to the A (salicyl) ring were made while retaining the

2′-chloro-4′-nitro B (anilide) ring. Replacement of the 5-chloro

of 4 with methyl (3m) decreased potency, while replacement

with H (3k) eliminated activity altogether. Compounds with a

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Table 1. Activity of Salicylanilides against T. gondii

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The activity of a few of the 3′-monosubstituted salicylanilides

prompted the evaluation of four compounds with substituents

at both the 3′ and 5′ positions. Compounds 3o (3′,5′-diﬂuoro)

and 3s (3′,5′-dimethyl) were inactive, and 3n (3′,5′-dichloro)

showed slight activity. Interestingly, the 3′,5′-bis(triﬂuoro-

methyl) derivative 3j displayed promising activity.

Scheme 2

A cursory study of the eﬀect of capping the phenol of 4 via

acylation was undertaken. The acylated derivatives 7a−d were

prepared and tested. The carbamates 7b, 7c, and 7d impart

altered polarity and hydrogen bonding capabilities compared to 4.

7d showed modest activity, but 7c and 7d were devoid of activity.

We were surprised to learn that benzoate ester 7a showed an

apparent increase in potency over 4. In other studies, we found

that 7a and other carboxylic esters of 4 were hydrolytically labile

when incubated in a mixture of THF and buﬀer (pH 7.4 or

pH 8.5), returning 4 at rates dependent on the nature of the ester

moiety (data not shown). Any diﬀerential activity of 7a over 4

therefore may be due to altered solubility and permeability

parameters which 7a may possess and that eventual liberation of

active 4 may be responsible for enhancing the observed activity.

The intrinsic activity of intact 7a cannot yet be ruled out, and this

interesting phenomenon is currently under study. The carbamates

7b, 7c, and 7d are much more stable against hydrolysis when

incubated in a mixture of THF and buﬀer (pH 7.4 or pH 8.5)

(data not shown) and are not expected to yield free 4 during

bioassay. The fact that these derivatives have no activity suggests

that either the increased steric demand of the carbamate groups,

or the capping of the phenolic oxygen, renders these compounds

inactive.

Scheme 3

4-ﬂuoro substituent (3ac, 10a, and 10b) or 3,5-diiodo substitu-

tion (3aa) demonstrated no activity. The decision was made to

proceed with the study of 4-chloro A-ring analogues with a

variety of B ring substituents.

Altering the electron-withdrawing character of the B ring

substituents of 4 had a profound eﬀect on activity. When a nitro

group was replaced with an amino group at position 4′, (5) all

activity was lost. Likewise, compounds possessing O-alkyl

electron-donating substituents at 2′ or 4′ (3q, 3v, 3ab) were

devoid of activity. It was surprising to note that replacement of

the 2′ chloro with the more electronegative ﬂuoro substituent,

while simultaneously replacing the 4′ nitro with the less

powerful electron withdrawing chloro group (3p), removed all

activity.

A series of 3′monosubstituted compounds were examined.

Various activities were observed, and it is clear that the nature

of the substituent at this position has a profound eﬀect. In this

series, the activity range shows ^tBu (3i) ≫ Et (3c) ≈ CH₂CH₂

(3e) ≈ CF₃(3f) ≈ F (3h) > Br (3b) ≈ CH₂Ph (3l). All other

3′ substituents resulted in compounds with no activity. Clearly

the introduction of 3′-alkoxy or aryloxy substitution resulted in

no increase in activity and actually may even be detrimental.

When compared to 3i, the best in the 3′-monosubstituted

series, both electronegative (halo, CF₃) and modestly electron-

donating (alkyl) substitutions provided moderate activity.

Two 5-ﬂuorosalicylanilide methyl ethers (10a and 10b) were

synthesized, tested, and proved to be inactive. This series of

salicylanilide ethers was not further pursued.

In an attempt to improve the activity of compound 4 and one

of the most promising salicylanilide analogues 3j, we prepared

ionizable derivatives of each compound. The acid hydrazide

salts 14a and 14b were designed to possess enhanced solubility

and bioavailability relative to the parent structures. We were

delighted to learn that 14a and 14b possess compelling in vitro

and at least minimal in vivo activity against a highly virulent

challenge. It is not yet clear whether the ionized derivatives have

innate activity or whether these compounds provide, by virtue of

increased solubility and permeability, a more eﬀective delivery of

the parent salicylanilides. It is possible that the sarcosine hydrazide

moiety of these derivatives provides an initial enhancement of

concentration by virtue of increased solubility and ultimately

cleaves to liberate the active salicylanilide. Research is currently

underway to elucidate this mode of enhancement.

The limited number of compounds studied allows a number

of observations that suggest a primitive structure−activity

relationship. It is appreciated that salicylanilides have been

studied for decades, and that it is known that the electronic

Scheme 4

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Scheme 5

environment of one aromatic ring strongly inﬂuences that of

the other. The evaluation of eﬀects on activity of rationally

modifying a given substituent while holding the others constant

was limited by the scope of the research project and the relative

availability of starting materials. The main observation that

emerged is that there is a strong interplay between all of the

substituents and that a complex relationship of interaction

across the amide bond exists. For example, whereas the activity

of 4 (MIC₅₀= 250−200 nM, R6 = nitro) is destroyed in 5

(R6 = amino), 3i (MIC₅₀= 16−8 nM, R6 = H, R5 = t-butyl),

which possesses neither the strong electron-withdrawing group

of 4 or the strong electron-donating group of 5 is active.

Further research to elaborate on the observations and trends pres-

ented below is planned. The compounds studied are repre-

sented by generic structure 15.

of these compounds to give additional test concentrations were

made and studied to identify inhibitory IC₅₀and IC₉₀values.

Graphical presentation of parasite inhibition is in Figure 1,

while graphical display of toxicity to HFF cells is in Figure 2.

The measured IC₅₀and IC₉₀ranges and the corresponding

toxicity data are compared in Table 3.

Because the ideal antiparasitic agent would have cidal activity,

it is of interest whether potential antiparasitic drugs exhibit a

static (inhibition of growth and/or replication) or cidal (lethal)

eﬀect. To determine whether leading compounds in this study

inhibited parasite proliferation by either a cidal or static

mechanism, four were selected (3i, 3j, 7a, and 14a) and applied

at four to eight times MIC₅₀to parasites. In this assay, RH-YFP

tachyzoites were treated with each compound at 1 μM under

various dosing conditions:

• Condition A: Parasites were treated for four days, then

compound was removed

• Condition B: Parasites were treated for 10 days, then

compound was removed

• Condition C: Compound was refreshed at four days then

removed at 10 days

• Condition D: Compound was maintained for the

duration of the experiment

The four and 10 day time points were taken to reveal the

impact of extended exposure of the parasites to the test

substance. Compounds were refreshed at four days to examine

whether compound degradation could contribute to an

observed static eﬀect. Parasite growth was assessed at days

Observations of eﬀects of substituents on activity and

possible trends in Table 1 are summarized in Table 2.

These screening eﬀorts revealed that six of the compounds

were the most eﬀective inhibitors. Of these, 3i, 3j, 7a, 14a, and

14b were selected for further in vitro evaluation. Serial dilutions

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Figure 1. Inhibition of T. gondii RH-YFP ﬂuorescence in the presence of compounds 3i, 3j, 7a, 14a, and 14b. FIBS, host ﬁbroblasts alone, not

infected; P/S, infected control treated with pyrimethamine and sulfadiazine in combination; RH-YFP, untreated infected ﬁbroblast control; 0.1%

DMSO (vehicle) infected ﬁbroblast control; [nM], concentration of inhibitor dissolved in 0.1% DMSO. Ordinate axes: relative ﬂuorescence units.

11, 17, and 25. The growth data, as a function of RH-YFP

ﬂuoresence, is expressed in Figure 3.

cidal eﬀect after 10 days of treatment, comparable to treatment

with the combination of pyrimethamine and sulfadiazine.

The eﬀect of selected compounds on other apicomplexan

parasites was also explored. Compounds 3i, 3j, 7a, and 14a

were examined for activity against two strains of P. falciparum,

a causative agent of malaria. One of these strains, D6 (CDC/

Sierra Leone), is drug-sensitive and readily killed by

chloroquine, while the second strain, TM91-C235, is multidrug

resistant and shows resistance to chloroquine. The activity of

these compounds was assessed using the Malaria SYBR Green

I-Based Fluorescence (MSF) Assay. This microtiter plate drug

sensitivity assay uses the presence of malarial DNA as

a measure of parasitic proliferation. As shown in Table 4,

Treatment with 3i under condition A reveals that the parasite

burden is roughly equivalent to untreated controls at day 11. At

day 17, condition C dosing of 3i also shows renewed growth.

Application of 3i under condition D demonstrates inhibition.

These data suggest that 3i is parasitostatic with 4 and 10 days of

exposure to the compounds in vitro. In contrast, compounds 3j

and 7a inhibited growth under all conditions employed in this

experiment. No parasite growth observed after the removal of

these compounds, even at day 25, suggesting that their activity

is parasitocidal. Compounds 3j and 7a demonstrated a cidal

eﬀect after four days of treatment, while 14a demonstrated a

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Figure 2. Eﬀect of selected compounds on survival of HFF cells. Ordinate: optical density.

eﬀect upon oral administration at the dosage studied. Thus,

compounds 14a and 14b were tested for eﬃcacy in a mouse

model of T. gondii oocyst infection. The oocyst form of the

parasite is excreted by cats and is often the form by which

people and other animals become infected. This oocyst infection

in mice is highly virulent and often fatal, making even limited

survival following such a virulent challenge of importance. Mice

were infected by oral gavage with ME49 or TgGoatUS4 oocysts.

Mice were treated with either a high dose (100 mg/kg) or low

dose (25 mg/kg) of 14a or 14b 1 mL suspension via oral gavage

or were not treated. All uninfected mice dosed with compound

alone remained asymptomatic, whereas all mice inoculated

orally with oocysts of either strain died of acute toxoplasmosis

8−9 days post infection, and tachyzoites were found in smears

of their mesenteric lymph nodes. Treatment with 14a and 14b

increased survival by 1 day (Table 5, Figure 4).

Table 3. Comparison of IC₅₀and IC₉₀Values of Lead

Compounds Characterized Further in Vitro

a

compd

IC₅₀(nM)

IC₉₀(nM)

toxicity (nM)

3i

3j

16−8

31−16

125−61

31−16

250−125

31−16

>1000

250−125

7a

14a

14b

a

The HFF used as the host cells in the parasite assay also were used

for the toxicity assay.

all compounds demonstrated activity against both P. falciparum

strains, with 7a the most eﬀective (D6 chloroquine sensitive

IC₅₀= 770 nM) and TM91-C235 chloroquine resistant IC₅₀

=

690 nM). Compounds 7a, 3j, and 14a were equally eﬀective

against the chloroquine-sensitive D6 and the multidrug

resistant Thai strain, TM91-C235, while compound 3i had a

2-fold higher IC₅₀against TM91-C235 (D6 IC₅₀= 3100 nM

and TM91-C235 IC₅₀> 6500 nM). The lack of cross-resistance

in compounds 7a, 3j, and 14a is an encouraging ﬁnding for a

novel scaﬀold and a valuable lead quality compound attribute

given the rapid development of drug resistance against many

antimalarials in the ﬁeld. This initial ﬁnding is the basis for

future research directed to the development of agents eﬀective

against P. falciparum.

The in vitro data for T. gondii prompted the selection of 3i,

3j, 7a, 14a, and 14b for evaluation in mouse models of T. gondii

infection. Initial diﬃculties were encountered with the

formulation and preliminary safety studies of 3i, 3j, and 7a,

presumably due to limited aqueous solubility. Compound 3i,

although slightly more active as a static agent in vitro, was not

further derivatized herein because it was not cidal and because

of its poorer solubility. It was anticipated that 14a and 14b, by

virtue of their polar, ionizable appended functionality, may

possess improved physicochemical proﬁles. Consequently, 14a

and 14b were chosen for evaluation in a mouse model of

T. gondii oocyst infection.

Kaplan−Meier curves in Figure 4 permit direct comparison

of the detailed data set in Table 5.

This is a highly virulent oocyst challenge lethal to all mouse

strains tested so far. Although the protection is of modest

biological signiﬁcance, adding only a day of increased survival, it is

protection against an otherwise lethal infection and was consistent

across doses and strains of parasites and replicate experiments.

These dosages of compounds also were not harmful.

In an attempt to discover the molecular target of selected

compounds, insertional mutagenesis experiments were per-

formed. The goal of this study was the identiﬁcation of one or

more genes which, when disrupted, confer resistance to the

parasite, thus potentially identifying the gene product which,

upon interaction with the active compound, inhibits the growth

of the parasite. THdhxgTRP tachyzoites were successfully

transfected with pLK47 vector plasmid to create parasites with

random gene mutations. No parasite growth was observed

after prolonged incubation in the presence of 3i, 3j, 7a, or 14b

(data not shown). The value of this approach to elucidate

molecular targets or target pathways of T. gondii inhibitors was

recently demonstrated. In an unrelated study conducted in our

laboratory, this methodology has successfully identiﬁed the

T. gondii traﬃcking pathway inhibited by a series of N-benzoyl-

2-hydroxybenzamides.¹²This insertional mutagenesis approach

was used successfully in parallel studies (ref 12) which served as

controls. One interpretation of the data reported herein is that

the molecular target of the active inhibitors of the study may be

essential. Speciﬁcally, the fact that it was not possible to identify

To explore whether 14a or 14b exerted toxic eﬀects when

dosed orally, each compound was administered by gavage to

mice daily for nine days at a dose of 100 mg/kg. At the

beginning, throughout and at the end of the 10 days, all mice

were alive and appeared healthy using well-being criteria

including sleek fur, normal activity pattern, and movement,

suggesting that neither compound had any observable toxic

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Figure 3. Eﬀect of various dosing conditions on prolonged survival of RH-YFP tachyzoites, measured by inhibition of RH-YFP ﬂuorescence.

Abscissa: 4 days, condition A; 10 days, renewed at 4, condition B; 10 days, condition C; all time, condition D. Ordinate: relative ﬂourescence units.

Table 4. Inhibition of P. falciparum D6 and C235 by Selected

Compounds

Table 5. Eﬃcacy of Compounds 14a and 14b in B7 Mice

Infected with T. gondii Me-49 or Tg-Goat-US4 Oocysts

compd

D6 IC₅₀(nM)

D6 R²

C235 IC₅₀(nM)

C235 R²

compd

dose (mg/kg)

challenge

no. of mice

day of death

chloroquine

7

3100

1400

770

89

>6000

1300

690

14a

none

14b

none

100

25

none

Me-49

5

none

3i

0.93

0.96

0.97

0.95

0.67

0.97

0.87

8, 9, 9, 9, 9

none

3j

none

7a

14a

25

Me-49

8, 9, 9, 9, 9

8, 8, 8, 8, 8

none

2700

none

100

25

Me-49

none

the molecular target with an insertional mutagenesis strategy,

which has been successful in other work with other targets,

indicates that it is likely that when this target is mutated

it is lethal for the parasites (i.e., that the target is essential).

TgGoatUS4

none

8, 9, 9, 9, 9

none

25

TgGoatUS4

8, 9, 9, 9, 9

8, 8, 8, 8, 8

none

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Figure 4. Eﬀect of compound 14a (left panel) on survival in a mouse model following challenge with T. gondii oocysts of the Me-49 strain. On day 8,

p < 0.014. N = 5 per group. Doses 25 and 100 mg/kg (dose 25/100) are shown with a single line as both have the same results. Eﬀect of compound

14b (right panel) on survival in a mouse model following challenge with T. gondii oocysts of the TgGoatUS4 strain. On day 8, p < 0.014. N = 5 per

group. Doses 25 and 100 mg/kg (dose 25/100) are shown with a single line as both have the same results.

The cidal activity of the lead compounds also indicates this is the

case and that it is not possible to identify the target by prolonged

culture of the parasite in the presence of the concentration of the

inhibitor we utilized. Future approaches to target and interacting

molecule identiﬁcation might also include prolonged growth at

subinhibitory concentrations to try to identify a resistant mutant

followed by sequencing of that mutant. Expression proﬁling by

determining transcriptome of treated parasites early after their

treatment to identify the signature of pathways perturbed by the

compounds using the connectivity map might also be useful.

Also, proteomics with a similar approach, and trying pull down

experiments with candidate proteins from T. gondii that are

homologues of known targets of niclosamide, determining

compound eﬀects on these targets, and whether overexpression

of the targets could rescue inhibition of parasite growth are other

approaches to target identiﬁcation which could be used in the

future.

As T. gondii and P. falciparum are both members of the same

phylum Apicomplexa, it is gratifying that the activity proﬁle of

the inhibitors is similar for both parasites. It will be of interest in

the future to characterize utility of these compounds as agents to

treat toxoplasmosis and malaria and perhaps diseases caused by

other apicomplexan parasites as well. When molecular targets

are identiﬁed in the future, it will be of interest to determine

whether selectivity or broader utility for multiple apicomplexan

parasites with similar targets will be the strengths of this new

family of inhibitors. Discoveries of safe and highly cidal leads as

potent as the compounds we have created for diseases such as

toxoplasmosis and malaria that are worldwide scourges and

harm many children are rare.

showed no signs of toxicity. Diluent alone had no eﬀect. It will

next be important to explore the activity of these compounds

against the latent encysted bradyzoite life stage.

3i, 3j, 7a, and 14a were examined for activity against two

drug-resistant strains of P. falciparum, the causative agent of the

most virulent form of malaria in humans. All compounds

demonstrated activity against P. falciparum, with 7a as the most

eﬀective.

The next steps in the exploration of this series will include

detailed investigation of structure−activity relationships, optimiz-

ing potency and pharmacodynamic properties, and the

identiﬁcation of the molecular target of this family of compounds.

The failure to create drug-resistant insertional mutants in this

study suggests the possibility that the target of the compounds

studied may be essential. This is a promising characteristic for

novel medicines given the risk of emergence of drug-resistant

strains. Identiﬁcation of the molecular target of these compounds

would enhance the optimization of activity through structural

modiﬁcation.

EXPERIMENTAL SECTION

■

Chemistry. Synthesis of Potential Inhibitors. Unless otherwise

stated, all solvents and reagents were used as received from vendors.

¹H NMR spectra were measured at either 400 MHz (Varian) or

500 MHz (Varian Inova AS500) in DMSO-d₆or CDCl₃. HPLC-MS

analyses were carried out with a Shimadzu LCMS 2020 using a

Phenomenex CHO-8463 C18 column (50 mm × 3.0 mm) with a

gradient of 10% acetonitrile:90% water (0.1% formic acid) to 100%

acetonitrile (0.1% formic acid) over 5 min. Retention times (T_R) are

reported in minutes (min). Mass spectra (ESI) are reported in positive

(m/z⁺) and/or negative (m/z⁻) mode. The calculated exact mass is

denoted as EM. Unless otherwise stated, all compounds were obtained at

≥95% purity (HPLC-MS). Niclosamide (compound 4) was purchased

from Sigma-Aldrich (St. Louis, MO). Reactants and reagents were used

as received from the vendor except as noted.

CONCLUSIONS

■

Herein, the inhibitory properties of the antihelminthic drug

niclosamide inspired the synthesis and evaluation of a series of

salicylanilides. The initial in vitro screen yielded ﬁve promising

agents, compounds 3i, 3j, 7a, 14a, and 14b, which were active

at low nanomolar concentrations and were not toxic to human

host cells. Compound 3i had a static eﬀect on parasite prolifera-

tion, which resumed after drug pressure was removed, while the

activity of compounds 3j, 7a, and 14a was cidal. Compounds

14a and 14b compounds were eﬀective at prolonging very

slightly survival of mice infected with T. gondii oocysts and

General Method of Salicylanilide Synthesis.⁹A suspension of

the salicylic acid derivative and aniline in xylenes (0.1−0.5 M) was

warmed to reﬂux, and then a solution of phosphorus trichloride in

CH₂Cl₂or xylenes was introduced dropwise. When the reaction was

complete, as determined by TLC or HPLC-MS, the reaction mixture

was rapidly transferred while hot by pipet, cannulation, or decanting to

a beaker and allowed to cool under rapid stirring. This action removed

tarry residue which may accumulate on the reaction vessel walls during

the reaction. Typically, the product crystallized from the reaction

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solvent as it cooled or was induced to crystallize upon the slow

addition of hexanes when the temperature of the reaction solvent

reached 75−80 °C.

[M + CH₃CN + H]⁺, 272.90 [M + H]⁺; m/z⁻= 542.95 [2M − H]⁻,

270.85 [M − H]⁻(EM = 272.04).

N-(3-Fluorophenyl)-5-chloro-2-hydroxybenzamide (3h). Using

the method described for compound 3a, 5-chlorosalicylic acid (2.42

g, 14.02 mmol) reacted with 3-amino benzonitrile (1.35 mL, 14.02

mmol) and 2 M PCl₃in CH₂Cl₂(2.80 mL, 5.61 mmol) in xylenes (30

mL). The crude product was recrystallized from 2-methyl-1-propanol.

¹H NMR (500 MHz, DMSO-d₆) δ 6.995 (m, 2H), 7.433 (m, 3H),

N-(3-Methylphenyl)-5-chloro-2-hydroxybenzamide (3a). A boil-

ing solution of 5-chlorosalicylic acid (0.72 g, 4.17 mmol) in xylenes

(10 mL) was treated dropwise with a 2.0 M solution of PCl₃in

CH₂Cl₂(0.83 mL, 1.67 mmol). After 2 h, the reaction solution was

transferred via pipet to a beaker and was allowed to cool to room

temperature under rapid stirring. The product separated as oﬀ-white

7.706 (m, 1H), 7.897 (d, J = 2.8 Hz, 1H), 10.519 (s, 1H), 11.641

(s, 1H). HPLC T_R2.639 min; m/z⁺265.90 [M + H]⁺; m/z⁻263.85

(EM = 265.03).

1

crystals. The crude product was recrystallized from EtOAc. H NMR

(400 MHz, DMSO-d₆) δ 2.291 (s, 3H), 6.942 (d, J = 7.2 Hz, 1H),

6.988 (d, J = 8.8 Hz, 1H), 7.227 (M, 1H), 7.425−7.508 (M, 3H),

7.943 (d, J = 2.24 Hz, 1H), 10.322 (s, 1H). HPLC T_R2.70 min; m/z⁺

N-(3-tert-Butylphenyl)-5-chloro-2-hydroxybenzamide (3i). Using

the method described for compound 3a, 5-chlorosalicylic acid (2.04 g,

11.82 mmol) reacted with 3-tert-butylaniline (1.76 g, 11.82 mmol) and

2 M PCl₃in CH₂Cl₂(2.336 mL, 4.73 mmol) in xylenes (30 mL). At

completion of reaction, the hot xylenes solvent was decanted, cooled

to room temperature, and then diluted with hexanes (30 mL). This

was stored at 4 °C for 30 h, during which time an oﬀ-white crystalline

solid separated. The product was recrystallized from EtOAc/hexanes

to give a mixture of the title compound (89.9% and an unidentiﬁed

impurity (10.1%). ¹H NMR of the major component (400 MHz,

DMSO-d₆) δ 1.280 (S, 9H), 7.172 (dq, J = 8.0, 0.8 Hz, 1H), 7.283 (dd,

J = 8.0, 0.8 Hz, 1H), 7.455 (dd, J = 8.8, 2.6 Hz, 1H), 7.560 (dd, J = 8.0,

0.8 Hz, 1H), 7.679 (M, 1H), 7.982 (d, J = 2.6 Hz, 1H), 10.345

(S, 1H), 11.903 (S, 1H). HPLC T_R3.095 min; m/z⁺303.95 [M + H]⁺;

m/z⁻= 301.85 [M − H]⁻(EM = 303.10).

+

261.95 [M + H] ; m/z⁻259.85, [M − H]⁻(EM = 261.06).

N-(3-Bromophenyl)-5-chloro-2-hydroxybenzamide (3b). Using

the method described for compound 3a, 5-chlorosalicylic acid (0.57 g,

3.30 mmol) reacted with 3-bromoaniline (0.36 mL, 3.30 mmol) and

2 M PCl₃in CH₂Cl₂(0.66 mL, 1.32 mmol) in xylenes (8 mL). The

crude product was recrystallized from EtOAc/hexanes. ¹H NMR

(500 MHz, DMSO-d₆) δ 7.024 (m, 2H), 7.354 (m, 3H), 7.474-

(m, 1H), 7.660 (m, 1H), 7.895 (m, 1H), 8.056 (s, 1H), 10.482 (s,

1H). HPLC T_R2.84 min; m/z⁺327.85 [M + H]⁺; m/z⁻325.75 [M − H]⁻

(EM = 324.95).

N-(3-Ethylphenyl)-5-chloro-2-hydroxybenzamide (3c). Using the

method described for compound 3a, 5-chlorosalicylic acid (0.63 g,

3.65 mmol) reacted with 3-ethylaniline (0.45 mL, 3.65 mmol) and

2 M PCl₃in CH₂Cl₂(0.73 mL, 1.45 mmol) in xylenes (9 mL). The

crude product was recrystallized from EtOAc/hexanes. ¹H NMR (400

MHz, DMSO-d₆) δ 1.198 (t, J = 7.6 Hz, 3H), 2.618 (q, J = 7.6 Hz,

2H), 7.012 (m, 2H) 7.254 (m, 1H), 7.457 (m, 3H), 7.983 (m, 1H),

N-(3,5-Bis(triﬂouromethyl)phenyl-5-chloro-2-hydroxybenzamide

(3j). Using the method described for compound 3a, 5-chlorosalicylic

acid (0.94 g, 5.48 mmol) reacted with 3,5-bis(triﬂuoromethyl)aniline

(0.85 mL, 5.48 mmol) and 2 M PCl₃in CH₂Cl₂(1.10 mL, 2.19 mmol)

in xylenes (15 mL). At completion of reaction, the hot xylenes solvent

was decanted, cooled to room temperature, and then diluted with

hexanes (50 mL). This was stirred at room temperature for 14 h,

+

10.357 (s, 1H). HPLC T_R2.86 min; m/z⁺275.95 [M + H] ; m/z⁻

273.80 [M − H]⁻(EM = 275.07).

N-(3-Ethynylphenyl)-5-chloro-2-hydroxybenzamide (3d). Using

the method described for compound 3a, 5-chlorosalicylic acid (0.75 g,

4.35 mmol) reacted with 3-ethynyllaniline (0.45 mL, 4.35 mmol)

and 2 M PCl₃in CH₂Cl₂(0.87 mL, 1.74 mmol) in xylenes (19 mL).

The crude product was recrystallized from 2-methyl-1-propanol.

¹H NMR (400 MHz, DMSO-d₆) δ 4.191 (s, 1H), 6.997 (d, J = 8.8

Hz, 1H), 7.231 (d, J = 7.6 Hz, 1H), 7.366 (dd, J = 8.0, 8.0 Hz, 1H),

7.442 (dd, J = 8.8, 2.8 Hz, 1H), 7.692 (m, 1H), 7.889 (m, 2H),

10.423 (s, 1H). HPLC T_R2.67 min; m/z⁺271.90 [M + H]⁺; m/z⁻

269.80 [M − H]⁻(EM = 271.04).

1

during which time pure product separated as white crystals. H NMR

(400 MHz, DMSO-d₆) δ 7.048 (d, J = 8.7 Hz, 1H), 7.493 (dd, J = 9.0,

2.7 Hz, 1H), 7.845 (M, 2H), 8.449 (S, 2H), 10.851 (S, 1H), 11.427

(S, 1H). HPLC T_R3.118 min; m/z⁻381.80 (EM = 383.01).

N-(2-Chloro-4-nitrophenyl)-2-hydroxybenzamide (3k). Using the

method described for compound 3a, salicylic acid (1.03 g, 7.46 mmol)

reacted with 2-chloro-5-nitroaniline (1.29 g, 7.46 mmol) and 2 M PCl₃

in CH₂Cl₂(1.50 mL, 2.98 mmol) in xylenes (20 mL). The crude

1

product was recrystallized from EtOH. H NMR (400 MHz, DMSO-

N-(3-Vinylphenyl)-5-chloro-2-hydroxybenzamide (3e). Using the

method described for compound 3a, 5-chlorosalicylic acid (0.56 g,

3.24 mmol) reacted with 3-vinyllaniline (0.37 mL, 3.24 mmol) and

2 M PCl₃in CH₂Cl₂(0.65 mL, 1.30 mmol) in xylenes (10 mL). The

d₆) δ 7.055 (M, 1H), 7.495 (M, 1H) 8.053 (dd, J = 8.0, 1.5 Hz, 1H)

8.283 (dd, J = 9.0, 2.5 Hz, 1H), 8.415 (d, J = 2.5 Hz, 1H), 8.854 (d, J =

9.0 Hz, 1H). HPLC T_R2.482 min; m/z⁺292.95 [M + H]⁺; m/z⁻

290.80 [M − H]⁺(EM = 292.03).

1

crude product was recrystallized from EtOH. H NMR (400 MHz,

N-(3-Benzyl phenyl)-5-chloro-2-hydroxybenzamide (3l). Using

the method described for compound 3a, 5-chlorosalicylic acid (0.81

g, 4.69 mmol) reacted with 3-benzylaniline (0.86 g, 4.69 mmol) and

2 M PCl₃in CH₂Cl₂(0.94 mL, 1.88 mmol) in xylenes (15 mL). The

DMSO-d₆) δ 5.305 (dd, J = 0.8, 11.6 Hz, 1H), 5.825 (dd J = 0.8, 17.6

Hz, 1H), 6.750 (dd, J = 11.6, 17.6 Hz, 1H) 7.024 (d, J = 8.8 Hz, 1H),

7.272 (d, J = 7.6 Hz, 1H), 7.360 (dd, J = 7.6, 8.0 Hz, 1H), 7.478 (dd,

J = 2.8, 8.8 Hz, 1H), 7.618 (d, J = 8.0 Hz, 1H), 7.804 (s, 1H), 7.972

(d, J = 2.8, 1H), 10.416 (s, 1H). HPLC T_R2.77 min; m/z⁺273.90

[M + H]⁺; m/z⁻271.80 [M − H]⁻(EM = 273.06).

N-(3-(Triﬂouromethyl)phenyl)-5-chloro-2-hydroxybenzamide

(3f). Using the method described for compound 3a, 5-chlorosalicylic

acid (2.19 g, 12.69 mmol) reacted with 3-triﬂuoromethylaniline (1.58

mL, 12.69 mmol) and 2 M PCl₃in CH₂Cl₂(2.54 mL, 5.08 mmol)

in xylenes (32 mL). The crude product was recrystallized from EtOH.

¹H NMR (500 MHz, DMSO-d₆) δ 7.035 (d, J = 9.0 Hz, 1H), 7.488

1

crude product was recrystallized from toluene. H NMR (500 MHz,

DMSO-d₆) δ 3.951 (S, 2H), 7.017 (m, 2H), 7.248 (m, 6H), 7.460 (dd,

J = 8.5, 2.5 Hz, 1H), 7.550 (m, 2H), 7.948 (d, J = 2.0 Hz, 1H) 10.363

(s, 1H), 11.846 (s, 1H), HPLC T_R3.017 min; m/z⁺337.95 [M+H]⁺;

m/z⁻335.85 (EM = 337.09).

N-(2-Chloro-5-nitrophenyl)-5-methyl-2-hydroxybenzamide (3m).

Using the method described for compound 3a, 5-methylsalicylic acid

(0.77 g, 5.06 mmol) reacted with 2-chloro-4-nitroaniline (0.87 g, 5.06

mmol) and 2 M PCl₃in CH₂Cl₂(1.01 mL0, 2.02 mmol)in xylenes

(18 mL). The desired product (64.5% pure) was found to contain

5.5% of an unidentiﬁed contaminant after collection upon cooling of

the reaction solvent. ¹H NMR (500 MHz, DMSO-d₆) δ 2.261 (s, 3H),

6.949 (d, J = 8.5 Hz, 1H), 7.272 (dd, J = 8.5, 2.0 Hz, 1H), 7.812 (d, J =

2.0 Hz, 1H), 8.257 (dd, J = 9.0, 2.5, 1H), 8.384 (d, J = 2.5, 1H), 8.824

(d, J = 9.0 Hz, 1H). HPLC T_R2.628 min; m/z⁺306.95 [M + 1]⁺;

m/z⁻304.85 [M − H]⁻(EM = 306.04).

(m, 2H), 7.619 (dd, J = 8.0, 8.0 Hz, 1H), 7.934 (m, 2H), 8.209

(s, 1H), 10.624 (s, 1H). HPLC T_R2.843 min; m/z⁺315.90 [M + H]⁺;

m/z⁻628.80 [2M − H]⁻, 314.80 [M − H]⁻(EM = 315.03).

N-(3-Cyanophenyl)-5-chloro-2-hydroxybenzamide (3g). Using

the method described for compound 3a, 5-chlorosalicylic acid (2.06

g, 11.94 mmol) reacted with 3-amino benzonitrile (1.41 g, 11.94

mmol) and 2 M PCl₃in CH₂Cl₂(2.39 mL, 4.78 mmol) in xylenes

1

(30 mL). The crude product was recrystallized from EtOH. H NMR

N-(2,4-Dichlorophenyl)-5-chloro-2-hydroxybenzamide (3n). A

suspension of 5-chlorosalicylic acid (1.73 g, 10.0 mmol) and 2,4-

dichloraniline (1.62 g, 10.0 mmol) in xylenes (50 mL) was heated to

reﬂux and a solution of PCl₃(0.35 mL, 4.0 mmol) in xylenes (5.0 mL)

(500 MHz, DMSO-d₆) δ 7.012 (dd, J = 1.4, 8.5 Hz, 1H), 7.459 (dd,

J = 1.8, 8.5 Hz, 1H), 7.580 (m, 2H), 7.856 (m, 1H), 7.916 (m, 1H),

8.182 (s, 1H), 10.588 (s, 1H). HPLC T_R2.479 min.; m/z⁺= 313.95

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was introduced in a dropwise manner. After 90 min, the reaction

mixture was transferred to a beaker via pipet and was allowed to cool

to room temperature under rapid stirring. The crude product was

(1.73 g, 10.0 mmol) and 3-ethoxyaniline (1.37 g, 10.0 mmol) reacted

in reﬂuxing xylenes (25 mL) in the presence of PCl₃(0.35 mL, 4.0

mmol). The crude product was puriﬁed by column chromatography

1

recrystallized from EtOAc. H NMR (500 MHz, DMSO-d₆) δ 7.075

(silica gel, 35 EtOAc:65 hexanes). H NMR (500 MHz, DMSO-d₆) δ

(dd, J = 8.8, 1.2 Hz, 1H), 7.473 (m, 2H), 7.693 (dd, J = 2.4, 1.2 Hz,

1H), 7.982 (dd, J = 2.8, 1.2 Hz, 1H), 8.457 (dd, J = 8.8, 1.2 Hz, 1H),

10.925 (s, 1H), 12.241 (s, 1H). HPLC T_R2.889 min; m/z⁺315.85

[M + H]⁺; m/z⁻313.70 [M − H]⁻(EM = 314.96).

N-(2,4-Diﬂuorophenyl)-5-chloro-2-hydroxybenzamide (3o).

Using the method described for compound 3n, 5-chlorosalicylic acid

(1.73 g, 10.0 mmol) and 2,4-diﬂuoroaniline (1.29 g, 10.0 mmol)

reacted in reﬂuxing xylenes (25 mL) in the presence of PCl₃(0.35 mL,

4.0 mmol). The crude product was recrystallized from EtOH. ¹H

NMR (500 MHz, DMSO-d₆) δ 7.045 (d, J = 9.0 Hz, 1H), 7.143

(M, 1H), 7.410 (M, 1H), 7.498 (dd, J = 9.0, 2.5 Hz, 1H), 7.973 (d, J =

2.5 Hz, 1H), 8.116 (M, 1H), 10.624 (s, 1H), 12.139 (s, 1H). HPLC

T_R2.523 min; m/z⁺283.90 [M + H]⁺; m/z⁻281.75 [M − H]⁻(EM =

283.02).

5-Chloro-N-(4-chloro-2-ﬂuorophenyl)-2-hydroxybenzamide (3p).

Using the method described for compound 3n, 5-chlorosalicylic acid

(1.73 g, 10.0 mmol) and 2,4-diﬂuoroaniline (1.46 g, 10.0 mmol)

reacted in reﬂuxing xylenes (25 mL) in the presence of PCl₃(0.35 mL,

4.0 mmol). The crude product was recrystallized from EtOAc. ¹H

NMR (500 MHz, DMSO-d₆) δ 7.057 (d, J = 8.8 Hz, 1H), 7.335 (dd,

J = 8.8, 1.2 Hz), 7.508 (dd, J = 8.8, 2.8 Hz, 1H), 7.577 (dd, J = 10.4,

2.0 Hz, 1H), 7.959 (d, J = 2.8 Hz, 1H), 8.245 (dd, J = 8.8, 8.8 Hz),

10.704 (s, 1H), 12.166 (s, 1H). HPLC T_R2.756 min; m/z⁺299.90

[M + H]⁺; m/z⁻297.75 [M − H]⁻(EM = 298.99).

5-Chloro-2-hydroxy-N-(3,4,5-trimethoxyphenyl)benzamide (3q).

Using the method described for compound 3n, 5-chlorosalicylic acid

(1.73 g, 10.0 mmol) and 3,4,5-trimethoxaniline (1.83 g, 10.0 mmol)

reacted in reﬂuxing xylenes (20 mL) in the presence of PCl₃(0.35 mL,

4.0 mmol). The crude product was dissolved in 100 mL of boiling

toluene:heptane (1:1 v/v), and the resulting solution was decanted

from a dark insoluble residue. White crystalline product separated

upon cooling. ¹H NMR (500 MHz, DMSO-d₆) δ 11.88−11.84

(s, 1H), 10.33−10.29 (s, 1H), 7.99−7.95 (d, J = 2.7 Hz, 1H), 7.51−

7.45 (dd, J = 8.8, 2.6 Hz, 1H), 7.15−7.11 (s, 2H), 7.05−6.99 (d, J = 8.8

Hz, 1H), 3.81−3.77 (s, 6H), 3.68−3.64 (s, 3H), 3.35−3.31 (s, 3H).

HPLC T_R2.400 min; m/z⁺337.95 [M + H]⁺; m/z⁻335.85 [M − H]⁻

(EM = 337.07).

12.03−11.61 (s, 1H), 10.62−10.19 (s, 1H), 8.09−7.86 (d, J = 2.7 Hz,

1H), 7.61−6.51 (m, 6H), 4.47−3.72 (q, J = 7.0 Hz, 2H), 1.69−0.97

(t, J = 7.0 Hz, 3H). HPLC T_R2.729 min; m/z⁺307.90 [M + H]⁺;

m/z⁻305.85 [M − H]⁻(EM = 307.06).

5-Chloro-N-(3,4-dimethoxyphenyl)-2-hydroxybenzamide (3v).

Using the method described for compound 3n, 5-chlorosalicylic acid

(1.73 g, 10.0 mmol) and 3,4-dimethoxyaniline (1.53 g, 10.0 mmol)

reacted in reﬂuxing xylenes (25 mL) in the presence of PCl₃(0.35 mL,

4.0 mmol). The crude product was puriﬁed by column chromatog-

raphy (silica gel, 35 EtOAc:65 hexanes) then recrystallized from

EtOAc. ¹H NMR (500 MHz, DMSO-d₆) δ 12.04−12.00 (s, 1H),

10.33−10.29 (s, 1H), 8.03−7.99 (d, J = 2.6 Hz, 1H), 7.51−7.45 (dd,

J = 8.8, 2.7 Hz, 1H), 7.41−7.37 (d, J = 2.4 Hz, 1H), 7.28−7.22 (dd, J =

8.7, 2.4 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H).

NMR HPLC T_R2.359 min; m/z⁺307.90 [M + H]⁺; m/z⁻305.85

[M − H]⁻(EM = 307.06).

5-Chloro-2-hydroxy-N-(3-isopropoxyphenyl)benzamide (3w).

Using the method described for compound 3n, 5-chlorosalicylic acid

(1.73 g, 10.0 mmol) and 3-isopropoxyaniline (1.51 g, 10.0 mmol)

reacted in reﬂuxing xylenes (25 mL) in the presence of PCl₃(0.35 mL,

4.0 mmol). The crude product was puriﬁed by column chromatog-

1

raphy (silica gel, 1 EtOAc:1 hexanes). H NMR (500 MHz, DMSO-

d₆) δ 12.23−11.64 (s, 1H), 10.87−10.01 (s, 1H), 7.96−7.92 (d, J = 2.7

Hz, 1H), 7.50−7.43 (dd, J = 8.8, 2.7 Hz, 1H), 7.39−7.34 (t, J = 2.2 Hz,

1H), 7.27−7.18 (m, 2H), 7.03−6.98 (d, J = 8.8 Hz, 1H), 6.72−6.67

(ddd, J = 7.8, 2.5, 1.4 Hz, 1H), 4.71−4.43 (hept, J = 6.0 Hz, 1H),

1.34−1.13 (d, J = 6.0 Hz, 6H). HPLC T_R2.843 min; m/z⁺305.95

[M + H]⁺; m/z⁻303.85 [M − H]⁻(EM = 305.08).

5-Chloro-2-hydroxy-N-(5-methoxy-2-methylphenyl)benzamide

(3x). Using the method described for compound 3n, 5-chlorosalicylic

acid (0.645 g, 3.74 mmol) and 5-methoxy-2-methylaniline (0.513 g,

3.74 mmol) reacted in reﬂuxing xylenes (15 mL) in the presence of

PCl₃(0.13 mL, 1.50 mmol). The crude product was recrystallized

1

from EtOH/H₂O. H NMR (400 MHz, DMSO-d₆) δ 3.331 (s, 1H),

3.742 (s, 1H), 6.708 (dd, J = 8.4, 1.2, Hz), 7.053 (d, J = 8.4 Hz, 1H),

7.175 (d, J = 8.4 Hz, 1H), 7.188 (m, 1H), J = 7.617 (s, 1H), 8.018

(s, 1H). HPLC T_R2.510 min; m/z⁺291.90 [M + H]⁺; m/z⁻289.85

[M − H]⁻(EM = 291.07).

5-Chloro-2-hydroxy-N-(3-phenoxyphenyl)benzamide (3y). Using

the method described for compound 3n, 5-chlorosalicylic acid (0.919

g, 5.33 mmol) and 3-phenoxyaniline (0.987 g, 5.33 mmol) reacted in

reﬂuxing xylenes (20 mL) in the presence of PCl₃(0.19 mL, 2.13

mmol). The crude product was recrystallized from EtOAc/hexanes. ¹H

NMR (500 MHz, CDCL₃) δ 6.858 (m, 1H), 6.982 (d, J = 8.8 Hz, 1H),

7.057 (d, J = 7.6 Hz, 2H), 7.151 (dd, J = 7.6, 7.2 Hz, 1H), 7.355 (m,

6H), 7.462 (d, J = 2.4 Hz, 1H), 7.794 (s, 1H), 11.776 (s, 1H). HPLC

T_R2.999 min; m/z⁺339.95 [M + H]⁺; m/z⁻337.85 [M − H]⁻(EM =

339.07).

5-Chloro-N-(3-(diﬂuoromethoxy)phenyl)-2-hydroxybenzamide

(3z). Using the method described for compound 3n, 5-chlorosalicylic

acid (0.86 g, 4.98 mmol) and 3-diﬂuoromethoxyaniline (0.0.79 g, 4.98

mmol) reacted in reﬂuxing xylenes (15 mL) in the presence of PCl₃

(0.17 mL, 1.99 mmol). The crude product was recrystallized from

EtOAc. ¹H NMR (500 MHz, DMSO-d₆) δ 11.82−11.49 (s, 1H),

10.63−10.27 (s, 1H), 7.92−7.87 (d, J = 2.7 Hz, 1H), 7.69−7.65 (t, J =

2.2 Hz, 1H), 7.56−7.51 (ddd, J = 8.2, 2.0, 1.0 Hz, 1H), 7.49−7.45 (dd,

J = 8.8, 2.7 Hz, 1H), 7.44−7.39 (t, J = 8.2 Hz, 1H), 7.39−7.06 (t, J =

74.0 Hz, 1H), 7.04−6.99 (d, J = 8.8 Hz, 1H), 6.98−6.93 (dd, J = 8.1,

2.3 Hz, 1H). NMR HPLC T_R2.665 min; m/z⁺313.85 [M + H]⁺;

m/z⁻311.80 [M − H]⁻(EM = 313.03).

N-(3-Chlorophenyl)-2-hydroxy-3,5-diiodobenzamide (3aa). A

mixture of 5-chlorosalicylic acid (0.573 g, 1.47 mmol) and 2-hydroxy-

3,5-diiodobenzoic acid (0.154 mL, 1.47 mmol) in xylenes (15 mL) was

brought to reﬂux whereupon all solids dissolved. A solution of PCl₃

(0.051 mL, 0.588 mmol) in xylenes (5 mL) was introduced in a

dropwise fashion. After 1 h, the reaction mixture was allowed to cool to

5-Chloro-N-(2-chloro-5-cyanophenyl)-2-hydroxybenzamide (3r).

Using the method described for compound 3n, 5-chlorosalicylic acid

(0.86 g, 5.0 mmol) and 3-amino-4-chlorobenzonitrile (0.76 g, 5.0

mmol) reacted in reﬂuxing xylenes (10 mL) in the presence of PCl₃

(0.18 mL, 2.0 mmol). The crude product was recrystallized from

EtOH/water to provide a tan-colored solid. ¹H NMR (500 MHz,

DMSO-d₆) δ 7.053 (d, J = 8.5 Hz), 7.345 (dd, J = 8.5, 3.0 Hz), 7.407

(dd, J = 8.5, 2.5 Hz), 7.598 (d, J = 8.5 Hz), 8.079 (d, J = 3.0 Hz), 8.979

(s, J = 2.5 Hz), 11.105 (s, 1H), 11.700 (s, 1H.) HPLC T_R2.551 min;

m/z⁺306.90 [M + H]⁺; m/z⁻= 304.75 [M − H]⁻(EM = 306.00).

5-Chloro-N-(3,5-dimethylphenyl)-2-hydroxybenzamide (3s).

Using the method described for compound 3a, 5-methylsalicylic acid

(1.04 g, 6.03 mmol) reacted with 3,5-dimethylaniline and 2 M PCl₃in

CH₂Cl₂(1.21 mL, 2.42 mmol) and 2 M in reﬂuxing xylenes (15 mL).

1

Pure white crystalline product resulted. H NMR (500 MHz, DMSO-

d₆) δ 2.260 (s, 6H), 6.780 (s, 1H), 7.015 (M, 2H), 7.315 (s, 2H),

7.451 (dd, J = 9.0, 2.5 Hz, 1H), 7.965 (d, J = 2.5 Hz, 1H), 10.265

(s, 1H). HPLC T_R2.848 min; m/z⁺275.95, m/z⁻273.80 (EM = 275.07).

5-Chloro-N-(3-cyano-4-ﬂuorophenyl)-2-hydroxybenzamide (3t).

Using the method described for compound 3n, 5-chlorosalicylic acid

(1.73 g, 10.0 mmol) and 3-ethynyl-4-ﬂuoroaniline (1.36 g, 10.0 mmol)

reacted in reﬂuxing xylenes (25 mL) in the presence of PCl₃(0.35 mL,

4.0 mmol). The crude product was recrystallized from EtOAc/

hexanes. ¹H NMR (500 MHz, DMSO-d₆) δ 11.57 (s, 1H), 10.64

(s, 1H), 8.24 (m, 1H), 8.03 (m, 1H), 7.87 (m, 1H), 7.64 (m, 1H), 7.48

(m, 1H), 7.04 (m, 1H). HPLC T_R2.546 min; m/z⁺290.85 [M + H]⁺;

m/z⁻288.80 [M − H]⁻(EM = 289.03).

5-Chloro-N-(3-ethoxyphenyl)-2-hydroxybenzamide (3u). Using

the method described for compound 3n, 5-chlorosalicylic acid

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40 °C and was decanted into rapidly stirring hexanes (40 mL) at rt.

White crystals separated, and after 1 h the product was collected by

ﬁltration, washed well with hexanes, and air-dried. ¹H NMR (500 MHz,

DMSO-d₆) δ 12.87−12.83 (s, 1H), 10.72−10.68 (s, 1H), 8.38−8.34 (d,

J = 2.0 Hz, 1H), 8.27−8.22 (d, J = 1.9 Hz, 1H), 7.86−7.81 (t, J = 2.1

Hz, 1H), 7.67−7.61 (ddd, J = 8.2, 2.1, 1.0 Hz, 1H), 7.47−7.40 (t, J =

8.1 Hz, 1H), 7.29−7.23 (ddd, J = 8.1, 2.1, 0.9 Hz, 1H). HPLC T_R3.351

min; m/z⁺499.70 [M + H]⁺; m/z⁻497.65 [M − H]⁻(EM = 498.83).

N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-hydroxybenzamide

(3ab). A mixture of 5-chlorosalicylic acid (1.73 g, 10.0 mmol) and 3,4-

methylenedioxyaniline (1.51 g, 10.0 mmol) in xylenes (20 mL) was

brought to reﬂux, whereupon all solids dissolved. A solution of PCl₃

(0.35 mL, 4.0 mmol) in xylenes (5 mL) was introduced in a dropwise

fashion. After 1 h, the reaction solution was decanted from a dark

residue which had accumulated in the reaction vessel. After cooling,

decolorizing carbon (0.25 g) was introduced, and the mixture was

heated at 80 °C for 30 min. Decolorizing carbon was removed by

ﬁltration through a ﬁlter aid. The solution was washed with water,

dried over MgSO₄, and concentrated. Pure product was obtained by

Reaction continued at this temperature for 2 h. The cooled reaction

mixture was diluted with EtOAc (100 mL) and was washed

successively with 1N HCl until the aqueous wash was acidic (about

pH 1) to litmus. The EtOAc phase was washed with brine, dried

(MgSO₄), and concentrated to an oﬀ-white solid. The crude product

was recrystallized from EtOAc/hexanes. ¹H NMR (500 MHz, DMSO-

d₆) δ 10.56 (s, 1H), 8.31 (m, 1H), 8.25 (m, 1H), 8.08 (m, 2H), 7.90

(m, 2H), 7.25 (m, 1H), 7.54 (m, 3 H).). HPLC T_R2.990 min; m/z⁺

430.95 [M + H]⁺; m/z⁻428.80 [M − H]⁻(EM = 430.01).

(S)-1-(4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl) 2-

Methyl Pyrrolidine-1,2-dicarboxylate (7b). (Step A) (S)-Methyl

1-(chlorocarbonyl)pyrrolidine-2-carboxylate. ^L-Proline methyl ester

hydrochloride (2.62 g, 15.82 mmol) was partitioned between saturated

NaHCO₃(50 mL) and CH₂Cl₂(50 mL). The CH₂Cl₂phase was dried

(MgSO4) and concentrated to an oil. This was dissolved in THF

(20 mL), and DIEA (3.03 mL, 17.40 mmol) was added. The resulting

solution was added dropwise to a solution of phosgene in toluene

(12.5%, 24.0 mL, 20.88 mmol) previously cooled to 0 °C. The reaction

mixture was allowed to warm to room temperature over 1 h, then was

ﬁltered and concentrated to provide an oil. This was used in the next

step without puriﬁcation. (Step B) (S)-1-(4-Chloro-2-((2-chloro-4-

nitrophenyl)carbamoyl)phenyl) 2-methyl pyrrolidine-1,2-dicarboxylate.

A solution of (S)-methyl 1-(chlorocarbonyl)pyrrolidine-2-carboxylate

(from step A, 0.397 g, 2.07 mmol) and DMAP (10 mg) in CHCl₃

(15 mL) was added to a slurry of niclosamide (645 mg, 1.973 mmol) in

pyridine (15 mL). This was warmed to 80 °C (all solids dissolved) and

maintained 1 h. The cooled solution was diluted with EtOAc (100 mL).

The resulting mixture was washed successively with 1N HCl until the

aqueous wash was acidic (about pH 1) to litmus. The EtOAc solution

was then washed with brine, dried (MgSO₄), and concentrated to an

oﬀ-white solid. The crude product was recrystallized from EtOAc/

1

column chromatography (silica gel, 35 EtOAc:65 hexanes. H NMR

(500 MHz, DMSO-d₆) δ 11.99−11.90 (s, 1H), 10.30−10.22 (s, 1H),

8.01−7.94 (d, J = 2.7 Hz, 1H), 7.50−7.43 (dd, J = 8.8, 2.7 Hz, 1H),

7.34−7.29 (d, J = 2.5 Hz, 1H), 7.14−7.08 (dd, J = 8.7, 2.5 Hz, 1H),

7.04−6.98 (d, J = 8.8 Hz, 1H), 6.89−6.82 (d, J = 8.7 Hz, 1H), 4.29−

4.21 (qd, J = 3.7, 2.0 Hz, 4H). HPLC T_R2.482 min; m/z⁺305.90 [M +

H]⁺; m/z⁻303.80 [M − H]⁻(EM = 305.05).

Preparation of N-(3-ethoxyphenyl)-5-ﬂuoro-2-hydroxybenzamide

(3ac). 5-Fluorosalicylic acid (0.312 g, 2.0 mmol), 3-ethoxyaniline

(0.274 g, 2.0 mmol), PCl₃(0.174 mL, 2.0 mmol), and xylenes (15 mL)

were placed in a sealed tube and heated to 150 °C for 2 h. Upon cooling

to room temperature, the reaction solution was diluted with EtOAc and

washed successively with saturated NaHCO₃, water, and brine and then

dried (MgSO₄). Solvents were evaporated, and the residue was puriﬁed

by preparative TLC (silica gel, 35 EtOAc:65 hexanes). Signiﬁcant

decomposition was observed during the chromatography. Pure product

(0.027 g, 4.9% yield) was obtained. ¹H NMR (500 MHz, DMSO-d₆) δ

11.59 (s, 1H), 10.36 (s, 1H), 7.74 (dd, J = 9.7, 3.2 Hz, 1H), 7.39 (t, J =

2.1 Hz, 1H), 7.28 (m, 3H), 7.00 (dd, J = 9.0, 4.6 Hz, 1H), 6.71 (ddd, J =

7.8, 2.5, 1.4 Hz, 1H), 4.03 (q, J = 6.9 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H).

HPLC T_R2.532 min; m/z⁺275.95 [M + H]⁺; m/z⁻549.00 [2M − H]⁻,

273.80 [M − H]⁻(EM = 275.10).

N-(4-Amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide Hy-

drochloride (5). A slurry of niclosamide (4, 1.435 g, 4.38 mmol) in

1MeOH: 1 HOAc (20 mL) was treated with portions of Zn dust

(3.0 g total) over 3 h at rt. A mild exotherm was observed. As the

reaction progressed, precipitates caused a thickening of the reaction

mixture. MeOH was added from time to time to facilitate magnetic

stirring. The reaction mixture was ﬁltered through a plug of Celite and

concentrated to an oﬀ-white residue. This was partitioned between

EtOAc and saturated NaHCO₃(copious precipitate appeared in the

aqueous phase). Water was added to the mixture to facilitate phase

separation. The EtOAc phase was washed with brine, dried (MgSO₄),

and concentrated to a tan solid. This was recrystallized from EtOAc/

hexanes to provide tan crystals (≥99% pure by LC-MS). The aniline

product was dissolved in EtOAc by warming a suspension (0.2 M) to

50 °C, and HCl in dioxane (4M, 2 equiv) was added in a dropwise

manner. The resulting slurry was diluted with 1 volume of EtOAc,

cooled to room temperature, and stirred for an additional 10 min.

The product was collected by ﬁltration and washed well with EtOAc.

¹H NMR (500 MHz, DMSO-d₆) δ 12.37 (s, 1H), 10.81 (t, J = 2.0 Hz,

1

hexanes. The H NMR spectrum is complex and reveals the existence

of rotational isomers, presumably due to restricted rotation about the

pyrolidine-1-carbamoyl bond. Analysis of HPLC and MS data reveals

1

a single compound in ≥95% purity. HNMR (300 MHz, DMSO-d₆)

δ 1.823−2.003 (m, 3H, pyrrolidine C4 methylene and pyrrolidine

C3 syn-H), 2.213 (m, 1H, pyrrolidine C3 anti-H), 3.450 (m, 1H,

pyrrolidine C5), 3.599 (m, 1H, pyrrolidine C5), 3.600 (s, 1.5 H, methyl

ester), 3.621 (s, 1.5 H, methyl ester), 4.252 (m, 0.5 H, pyrrolidine

methine), 4.541 (m, 0.5H, pyrrolidine methine), 7.108 (d, J = 8.5 Hz,

0.33 H, H3), 7.216 (d, J = 8.5 Hz, 0.33 H, H3), 7.361 (d, J = 9 Hz,

0.33H, H3), 7.545 (m, 0.33H, H4), 7.665 (m, 0.66H, H4), 7.763 (m,

0.66H, H6), 7.974 (m, 0.33H, H6), 8.133 (m, 0.66H, H6′), 8.287 (m,

1H, H5′), 8.302 (m, 0.33H, H3′), 8.539 (m, 0.33H, H3′), 8.446 (m,

0.33H, H3′), 8.822 (d, J = 9.5 Hz, 0.33H, H6′), 10.313 (s, 0.33H, NH),

10.385 (s, 0.33H, NH), 11.367 (br s, 0.17H, NH), 12.519 (br s, 0.17H,

NH). HPLC T_R2.743 min; m/z⁺482.00 [M + H]⁺; m/z⁻479.90

[M − H]⁻; (EM = 481.04). Note: The optical purity of this material

was not determined.

4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl Morpho-

line-4-carboxylate (7c). 4-Morpholine carbonyl chloride (0.353 mL,

3.08 mmol) was added dropwise to a suspension of niclosamide 4

(0.503 g, 1.54 mmol) in pyridine (8.0 mL) containing DMAP (10

mg). This was raised to reﬂux (all solids dissolved) and maintained

3 h. The hot solution was admitted via pipet dropwise into rapidly

stirring distilled water (100 mL), whereupon a ﬁne white solid pre-

cipitated. This mixture was stirred at room temperature for 2 h.

The crude product was collected by ﬁltration, washed well with water

(100 mL) and 1N HCl (200 mL), and then dried under a stream of

room air for 2 h. The product was then dried in a vacuum oven (28″

Hg, 50 °C) for 60 h. The oﬀ-white product was recrystallized from

EtOAc to give white crystals. ¹H NMR (500 MHz, CDCl₃) δ 8.60 (m,

2H), 8.45 (m, 1H), 8.33 (m, 1H), 7.96 (m, 1H), 7.62 (m, 1H), 7.24

(m, 1H), 3.78−3.87 (m, 8H). HPLC T_R2.569 min; m/z⁺440.00

[M + H]⁺; m/z⁻437.85 [M − H]⁻(EM = 439.03).

1H), 8.26 (m, 1H), 7.99 (d, J = 2.8 Hz, 1H), 7.51 (dd, J = 8.8, 2.8 Hz,

1H), 7.38 (m, 1H), 7.16 (m, 2H). HPLC T_R1.125 min; m/z⁺339.85

[M + CH₃CN + 2]⁺, 337.90 [M + CH₃CN + H]⁺, 298.85 [M + 2]⁺

296.85 [M + H]⁺; m/z⁻296.80 [M + 2 − H]⁻294.80 [M − H]⁻

(EM = 296.01).

4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl Benzoate

(7a). Benzoyl chloride (0.49 mL, 4.27 mmol) was added dropwise

to a suspension of niclosamide (1.27 g, 3.88 mmol) in a solution of

4-dimethylaminopyridine (DMAP, 30 mg) in pyridine (15 mL) at rt.

The suspension was warmed to 80 °C, whereupon all solids dissolved.

4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl 4-Methyl-

piperazine-1-carboxylate (7d). 4-Methyl-1-piperazinecarbonyl chlor-

ide (0.603 g, 3.02 mmol) was added to a suspension of niclosamide

4 (0.495 g, 1.51 mmol) in pyridine (8.0 mL) containing DMAP

(10 mg). The mixture was raised to reﬂux for 1 h. The hot solution

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was introduced by pipet to rapidly stirring water at rt. After 1 h, the

solids were collected by ﬁltration and added to 50 mL of rapidly stirred

1N HCl. This was stirred for 30 min, and then the crude product was

collected by ﬁltration and dried in a vacuum oven (28″ Hg, 50 °C) for

18 h, then recrystallized from EtOH. A small portion of the product

was converted to the free base by partitioning between EtOAc and

saturated NaHCO₃. TLC (SiO₂₁, 5 MeOH:95 CHCl₃) demonstrated

a single component, R_f= 0.37. H NMR (400 MHz, CDCl₃) δ 8.91

(s, 1H), 8.81 (d, J = 9.2 Hz), 8.34 (d, J = 2.4 Hz), 8.22 (dd, J = 9.2, 2.4

Hz), 7.87 (d, J = 2.8 Hz), 7.51 (dd, J = 2.8, 8.4 Hz), 7.13 (d, J = 8.4

Hz), 3.68 (m, 2H), 3.56 (m, 2H), 2.39 (m, 4H), 2.28 (s, 3H). HPLC

T_R1.664; m/z⁺452.95 [M + H]⁺; m/z⁻450.90 [M − H]⁻(EM =

452.07).

N-(2-Chloro-4-nitrophenyl)-5-ﬂuoro-2-methoxybenzamide (10a).

A solution of 5-ﬂuoro-2-methoxybenzoic acid (0.340 g, 2.0 mmol),

2-chloro-4-nitroaniline (0.173 g, 1.0 mmol), and O-(7-azabenzotriazol-

1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuorophosphate (HATU,

0.76 g, 2.0 mmol) in MeCN (4.0 mL) was treated with DIEA (0.10

mL), and the resulting solution was stirred at rt for 16 h. EtOAc

(10 mL) was added, and the organic phase was washed with 50% 1N

HCl in saturated NaCl and then brine, dried over MgSO₄, and

concentrated to aﬀord a yellow solid. This was recrystallized from

EtOAc:hexanes. ¹H NMR (500 MHz, DMSO-d₆) δ 13.78 (s, 1H), 12.88

(s, 1H), 8.76 (dd, J = 4.4, 1.4 Hz, 1H), 8.53 (dd, J = 8.4, 1.4 Hz, 1H),

7.51 (dd, J = 8.4, 4.4 Hz, 1H), 7.37 (m, 2H), 7.13 (dd, J = 9.1, 4.3 Hz,

1H), 3.79 (s, 3H). HPLC T_R2.176 min; m/z⁺288.90 [M − Cl]⁺(EM =

324.03).

(15 mL) was added. After 16 h, the reaction solution was concentrated

to an oily residue. This was dissolved in CHCl₃(25 mL) and con-

centrated. The CHCl₃chase was repeated to leave a sinterable foam.

This was layered with 35 EtOAc:65 hexanes (50 mL) and warmed to

45 °C under rapid stirring for 30 min. Hexane (30 mL) was added, and

the stirring mixture was allowed to cool to room temperature over

20 min. The product was collected by ﬁltration. The ¹H NMR

spectrum was complex and revealed the existence of rotational isomers,

presumably due to restricted rotation about the sarcosine-1-carbamoyl

bond. Analysis of HPLC and MS data reveals a single compound

in ≥95% purity. T_R2.382 min; m/z⁺441.85 [M + H]⁺; m/z⁻324.75

[M − sarcosine − H]⁻(EM = 441.01).

4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl (2-Hydra-

zinyl-2-oxoethyl)(methyl) Carbamate Hydrochloride (13b). (Step A)

tert-Butyl 2-(2-(((4-chloro-2-((2-chloro-4-nitrophenyl) carbamoyl)-

phenoxy)carbonyl)(methyl)amino)acetyl)hydrazinecarboxylate. tert-

Butyl carbazate (417 mg, 3.16 mmol) was added to a solution of 13a

(1.27 g, 2.87 mmol) in THF (8.7 mL) with stirring at rt. A solution

of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

(660 mg, 3.44 mmol) in CH₂Cl₂(17.4 mL) was introduced dropwise

at rt. The homogeneous solution was stirred at room temperature for

14 h, then concentrated. The residue was partitioned between EtOAc

and 1 N HCl. The EtOAc phase was washed with water and then with

brine, dried (MgSO₄), and concentrated to an oﬀ-white solid 1.20 g

(75.1%). This was used without further manipulation. (Step B)

4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenyl (2-hydrazinyl-

2-oxoethyl)(methyl)carbamate hydrochloride. The product from step A

(2.52 g, 5.70 mmol) was dissolved in a solution of HCl in dioxane

(4.0 M, 10 mL), and the solution was stirred at room temperature for

30 min, then concentrated to an oﬀ-white solid which was washed well

with EtOAc and then hexanes and dried under a stream of air to

5-Fluoro-N-(2-ﬂuoro-4-nitrophenyl)-2-methoxybenzamide (10b).

A solution of 5-ﬂuoro-2-methoxybenzoic acid (0.510 g, 3.0 mmol),

2-ﬂuoro-4-nitroaniline (0.156 g, 1.0 mmol), and O-(7-azabenzotriazol-

1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuorophosphate (HATU,

1.140 g, 3.0 mmol) in MeCN (6.0 mL) was treated with DIEA

(0.150 mL), and the resulting solution was stirred at room temperature

for 2 h. The reaction mixture was added to rapidly stirring water, and

the resulting precipitate was washed well with water and then dissolved

in EtOAc, dried over MgSO₄, and concentrated to a yellow solid. The

1

provide a white solid (2.50 g, 89%). H NMR spectrum was complex

and revealed the existence of two rotational isomers, presumably due to

1

restricted rotation about the sarcosine-1-carbamoyl bond. H NMR

(500 MHz, DMSO-d₆) δ 11.09 (s, 1H), 10.45 (s, 1H), 10.40 (s, 1H),

8.41 (dd, J = 2.3, 1.5 Hz, 1H), 8.31−8.25 (m, 1H), 8.10 (m, 1H), 7.80

(dd, J = 6.2, 2.6 Hz, 1H), 7.71−7.65 (m, 1H), 7.36 (d, J = 8.7 Hz, 1H),

7.26 (d, J = 8.7 Hz, 1H), 4.21 (s, 1H), 4.05 (s, 1H), 3.07 (s, 1.5H), 2.92

(s, 1.5H). HPLC T_R2.03 min; m/z⁺455.95 [M + H]⁺; m/z⁻453.95

[M − H]⁻(EM = 308.06).

1

crude product was recrystallized from EtOAc/hexanes. H NMR (500

MHz, DMSO-d₆) δ13.78 (s, 1H), 12.89 (s, 1H), 8.76 (dd, J = 4.4, 1.4

Hz, 1H), 8.53 (dd, J = 8.4, 1.4 Hz, 1H), 7.51 (dd, J = 8.4, 4.4 Hz, 1H),

7.40 (dd, J = 8.8, 3.3 Hz, 1H), 7.35 (ddd, J = 9.1, 8.1, 3.3 Hz, 1H), 3.79

(s, 3H). HPLC T_R2.190 min; m/z⁺288.90 [M − F]⁺; m/z⁻292.85

[M − CH₃]⁻(EM = 308.06).

2-(((2-((2,4-Bis(triﬂuoromethyl)phenyl)carbamoyl)-4

Chlorophenoxy)carbonyl) (Methyl) amino) Acetic Acid (14a). (Step A)

tert-Butyl 2-(((2-((2,4-bis(triﬂuoromethyl)phenyl)carbamoyl)-4-

chlorophenoxy)carbonyl)(methyl)amino)acetate. N-(3,5-Bis(triﬂouro-

methyl)phenyl-5-chloro-2-hydroxybenzamide (3j, 6.47 g, 16.86 mmol)

was added to a solution of 12 (4.20 g, 20.22 mmol) in pyridine

(35 mL), and the mixture was warmed to 80 °C for 3 h. The reaction

solution was cooled to room temperature, diluted with EtOAc

(200 mL), and washed four times with 1N HCl (ﬁnal wash ph about 1

to litmus), once with brine, and dried (MgSO₄). Concentration

aﬀorded an oﬀ-white solid, which was triturated with hexanes to

provide the product (7.01 g, 75%), which was used without further

puriﬁcation. (Step B) 2-(((2-((2,4-Bis(triﬂuoromethyl)phenyl)-

carbamoyl)-4-chlorophenoxy)carbonyl)(methyl)amino) acetic acid.

The product from step A (5.43 g, 9.70 mmol) was dissolved in

CH₂Cl₂(15 mL), and CF₃COOH (7.5 mL) was added. The reaction

mixture was stirred at room temperature for 14 h, and then the reaction

mixture was concentrated and the residue was chased twice with CHCl₃

(25 mL). The ¹H NMR spectrum was complex and revealed the

existence of rotational isomers, presumably due to restricted rotation

about the sarcosine-1-carbamoyl bond. Analysis of HPLC (T_R= 2.65

min) and MS data (m/z⁺499.05, [M + H]⁺, m/z⁻381.80, [M −

C₄H₆NO]⁻) revealed a single compound in ≥95% purity.

tert-Butyl 2-((Chlorocarbonyl)(methyl)amino)acetate (12). A

solution of sarcosine tert-butyl ester hydrochloride 11 (4.214 g,

23.20 mmol) in CH₂Cl₂(40 mL) was shaken with saturated NaHCO₃

in a separatory funnel. The organic phase was dried over MgSO₄and

concentrated to a clear oil (2.298 g, 68% yield). A solution of phosgene

in toluene (20%, 10.9 mL, 23.75 mmol) was cooled to −25 °C, and a

solution of sarcosine tert-butyl ester (2.298 g, 15.78 mmol) and DIEA

(5.5 mL, 31.66 mmol) in CH₂Cl₂(10 mL) was introduced in a

dropwise fashion. The solution was allowed to warm to room

temperature over 1 h and was then washed with 1N HCl (50 mL)

and EtOAc suﬃcient to form two layers was added. The organic phase

was washed with water and then brine and dried over MgSO₄. This

solution was used without further manipulation.

2-(((4-Chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenoxy)-

carbonyl) (Methyl)amino)acetic Acid (13a). (Step A) tert-Butyl 2-

(((4-chloro-2-((2-chloro-4-nitrophenyl)carbamoyl)phenoxy)carbonyl)-

(methyl)amino)acetate. A solution of 12 (15.78 mmol) in EtOAc (ca.

0.5M) was introduced dropwise to a reﬂuxing solution of niclosamide

(2.56 g, 7.92 mmol) in pyridine (80 mL containing DMAP (50 mg).

After 30 min, 100 mL of solvent was distilled from the reaction mixture.

The remaining reaction mixture was cooled to room temperature,

diluted with EtOAc (50 mL), and washed successively with 1N HCl

until the aqueous wash was acidic (about pH 1) to litmus. The organic

phase was washed with brine, dried (MgSO₄), and concentrated to

an oﬀ-white solid. This was used without further puriﬁcation. (Step B)

2-(((4-Chloro-2-((2-chloro-4-nitrophenyl) carbamoyl) phenoxy) car-

bonyl) (methyl)amino) acetic acid. The solid from step A (542 mg,

1.09 mmol) was dissolved in CH₂Cl₂(15 mL), and CF₃COOH

2-((2,4-Bis(triﬂuoromethyl)phenyl)carbamoyl)-4-chlorophenyl(2-

hydrazinyl-2-oxoethyl) (Methyl)carbamate Hydrochloride (14b).

(Step A) tert-Butyl 2-(2-(((2-((2,4-bis (triﬂuoromethyl) phenyl)-

carbamoyl)-4-chlorophenoxy) carbonyl) (methyl)amino) acetyl) hydra-

zine carboxylate. A solution of 2-(((2-((2,4-bis(triﬂuoromethyl)phenyl)-

carbamoyl)-4-chlorophenoxy)carbonyl) (methyl)amino)acetic acid (14a,

540 mg,1.08 mmol) in CH₂Cl₂(5.0 mL) was treated with tert-butyl

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carbazate (172 mg, 1.30 mmol) and N-(3-dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (249 mg, 1.30 mmol). The solution was

stirred at room temperature for 4 h, then concentrated, and the residue

was dissolved in EtOAc (15 mL), washed with 1N HCl and then with

brine, dried over MgSO₄, and concentrated to a scinterable foam. The ¹H

NMR spectrum was complex and revealed the existence of rotational

isomers, presumably due to restricted rotation about the sarcosine-1-

carbamoyl bond. Analysis of HPLC (T_R= 2.81 min) and MS data (m/z⁺

634.90 [M + Na]⁺, m/z⁻610.85, [M − H]⁻) reveals a single compound

in ≥95% purity. (Step B) 2-((2,4-Bis(triﬂuoromethyl)phenyl)-

carbamoyl)-4-chlorophenyl (2-hydrazinyl-2-oxoethyl)(methyl) carbamate

hydrochloride (14b, 2.263 g, 3.69 mmol) was dissolved in a solution of

HCl in dioxane (15 mL). After stirring 2 h at room temperature, the

reaction mixture was concentrated and the residue was chased twice with

15 mL portions of CHCl₃, providing a scinterable foam (1.90 g, 94%). ¹H

NMR (500 MHz, DMSO-d₆) δ 11.03 (s, 1H), 10.95 (s, 1H), 9.15 (s,

1H), 9.08 (s, 1H), 8.36 (s, 4H), 7.84 (s, 2H), 7.80 (dd, J = 5.2, 2.7 Hz,

2H), 7.70−7.64 (m, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 8.7 Hz,

1H), 4.17 (q, J = 18.2, 13.8 Hz, 3H), 3.97 (s, 2H), 3.79 (s, 2H), 3.00

(s, 3H), 2.83 (s, 3H). HPLC T_R2.38 min; m/z⁺512.90 [M + H]⁺; m/z⁻

510.80 [M − H]⁻(EM = 512.07).

Biology. In Vitro Evaluation of Inhibition of T. gondii

Tachyzoites. Test compounds were dissolved in DMSO to make a

10 mM solution, and subsequently diluted with IMDM-C to the

concentrations used in bioassay. In the in vitro experiments, DMSO

concentration was not greater than 0.1% unless otherwise speciﬁed.

RH-YFP parasites, which stably express yellow ﬂuorescent protein,

were used. Tachyzoites were extracted from HFF cells by double

passage through a 25 gauge¹²needle, centrifuged for 15 min at 1500

rpm, and resuspended in IMDM-C. Conﬂuent monolayers of HFF

cells were infected with parasites in 96-well plates (Falcon 96 Optilux

Flat-bottom) with 3500 parasites in 100 μL per well. One hour after

inoculation, test compounds and control media were added for a ﬁnal

volume of 200 μL per well. Parasite proliferation was assessed using

[³H]-uracil incorporation¹³or YFP ﬂuorescence assay.

P. falciparum strains in late-ring or early trophozoite stages were cultured

in predosed 384-well microtiter drug assay plates in 38 μL culture volume

per well at a starting parasitemia of 0.3% and a hematocrit of 2%.

Predosed, sterile, 384-well black optical bottom microtiter drug plates for

use in the MSF assay were produced using a Tecan EVO Freedom liquid

handling system (Tecan US, Durham, NC). Dose response plates were

produced at a ﬁnal concentration ranging from 0.5−10000 ng/mL in

quadruplicate (12 2-fold serial dilutions of each test compound or

chloroquine control in DMSO). Each run was validated by a batch

control plate with chloroquine (Sigma-Aldrich Co., catalogue no. C6628)

at a ﬁnal concentration of 2000 ng/mL. The cultures were incubated for

72 h at 37 °C, 5% CO₂, 5% O₂, and 90% N₂. Lysis buﬀer (38 μL per

well), consisting of 20 mM Tris HCl, 5 mM EDTA, 1.6% Triton X,

0.016% saponin, and SYBR green I dye at a 20× concentration

(Invitrogen, catalogue no. S-7567), was then added to the assay plates

utilizing the Tecan EVO Freedom system for a ﬁnal SYBR Green

concentration of 10×. Plates were incubated in the dark at room

temperature in the dark for 24 h. Compound activity was assessed by

examining for the relative ﬂuorescence units (RFU) per well using the

Tecan Genios Plus (Tecan US, Inc., Durham, NC). GraphPad Prism

(GraphPad Software Inc., San Diego, CA) using the nonlinear regression

(sigmoidal dose−response/variable slope) equation was used to

determine IC₅₀values.

Determination of Static or Cidal Eﬀects. RH-YFP tachyzoites were

treated with each compound at 1 μM under one of four conditions:

(a) parasites were treated for four days, then compound was removed;

(b) parasites were treated for 10 days, then compound was removed;

(c) compound was refreshed at four days and removed at 10 days; or

(d) compound was maintained for the duration of the experiment. The

4 and 10 day time points were taken to reveal the impact of extended

exposure of the parasites to the test substance. Compounds were

refreshed at four days to examine whether compound degradation

could contribute to an observed static eﬀect. Parasite growth was

assessed at 11, 17, and 25 days.

In Vivo Toxicity and Oocyst Assays. HLA B07 transgenic mice

were produced at Pharmexa-Epimmune (San Diego, CA, USA) and

bred at the University of Chicago. All studies were conducted with

Institutional Animal Care and Use Committee at the USDA, the

University of Chicago, and the University of Strathclyde.

T. gondii Parasite and Cell Culture. Human foreskin ﬁbroblast

(HFF) cells were maintained in conﬂuent monolayers in Iscoves’s

Modiﬁed Dulbecco’s Medium supplemented with 10% fetal bovine

serum, 1% GlutaMAX, and 1% penicillin−streptomycin−fungizone

(IMDM-C). T. gondii tachyzoites were cultivated in HFF mono-

layers.^12,13Parasites and cells were maintained at 33 or 37 °C and 5%

CO₂. The strains of parasite used in this study include RH, RH-YFP,

Me49 strain,¹⁴andTgGoatUS4 isolate.¹⁵

Infection of Mice with T. gondii Oocysts. Oocysts were obtained

by feeding infected tissues of Swiss Webster mice to cats, sporulated in

2% sulfuric acid on a shaker for one week, and stored at 4 °C until

used (Dubey 2010). Oocysts were counted in a disposable hemo-

cytometer and diluted 10-fold from 10⁻¹to 10⁻⁷to reach an end point of

≅1 oocyst. All 10-fold dilutions were made in 50 mL tubes with 2%

sulfuric acid (5 mL aliquot + 45 mL sulfuric acid), and dilutions were

stored at 4 °C to avoid variability in inocula preparations. For inoculation

of mice, oocysts from the designated dilution were neutralized with 3.3%

sodium hydroxide with neutral red as indicator (approximately the same

volume as the inoculum). The resultant mixture was inoculated orally

into ﬁve mice for each dilution (unless indicated otherwise) via a gastric

needle with a blunt bulb (22 gauge, 50 mm long, Cadence Science

catalogue no. 7920), without washing to avoid variability of the inocula

during washing procedure. All orally inoculated mice were housed in

autoclavable rodent cages with biohazard signs to incinerate bedding and

food for 10 days to avoid spread of T. gondii because some oocysts pass

unencysted in mouse feces.¹⁶

Bioassay of T. gondii in Mice. Mice were observed daily for the

duration of the experiment. All mice were examined for T. gondii

infection. Impression smears of tissues (usually mesenteric lymph

nodes or lungs) were examined microscopically for tachyzoites.

Survivors were bled 6−8 weeks later, and 1:25 dilution of their sera

were tested for T. gondii antibodies using the (MAT) as described.¹⁷

The last infective dilution was considered to have one viable organism.

The inoculated mice were considered infected with T. gondii when

tachyzoites or tissue cysts were found in tissues. Seroconversion at

6 weeks was considered as indication of the presence of live parasites in the

inocula. However, brains of all mice that survived 6 weeks were examined

for tissue cysts, irrespective of serological results. With the strains of T.

gondii used here, tissue cysts are found in all seropositive mice.

[³H]-Uracil and [³H]-Thymidine Incorporation Assays. First, 25 μL

of 0.1mCi/mL [³H]-uracil or [³H]-thymidine was added to each well

24 h before reading plates. Then at harvesting, contents of the wells

were transferred onto a 96-well UniFilter GF/C ﬁlter plates using a

Filtermate 196 cell harvester (Packard). [³H]-Uracil and [³H]-

thymidine incorporation was measured using a Microplate scintillation

luminescence counter (Packard).^12,13

YFP Fluorescence Assay. After 72 h of initiation of the in vitro

challenge assay, parasite proliferation was assessed by reading ﬂuore-

scence of YFP parasites with a Synergy H4 Hybrid Reader (BioTek) and

Gen5 1.10 software, using a bottom optics positions, excitation

wavelength of 514 nm, and emission wavelength of 540 nm.¹²

In Vitro Toxicity Assay. HFF cells were grown to ∼30% conﬂuence

in 96-well plates. Inhibitory compounds and control media were added

to wells in concentrations equal to those being tested in challenge assays.

After 72 h, [³H]-thymidine incorporation assay was conducted to assess

cell growth.^12,13Alternatively, toxicity was assessed using WST-1 cell

proliferation reagent (Roche). Conﬂuent HFF cells were treated with

inhibitory and control compounds. On the ﬁnal day of experiment,

10 μL of WST-1 reagent was added to each well. Plates were incubated

for 1 h in the dark at 37 °C, and absorbance was measured using

Synergy H4 hybrid reader (BioTek) ﬂuorometer at 420 nM.¹²

Antiplasmodial SYBR Green I-Based Fluorescence (MSF) Assay.

D6 (CDC/Sierra Leone) and TM91-C235 (WRAIR, Thailand)

laboratory strains of P. falciparum were used for each drug sensitivity

assessment. The parasite strains were maintained continuously in

long-term cultures as previously described in Johnson et al,¹¹and

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Journal of Medicinal Chemistry

Article

Evaluation of Eﬃcacy of 14a in a Mouse Model of T.gondii

Oocyst Infection. Five groups of B7 female mice weighing approxi-

mately 25 g were used. Group 1 received only 14a (100 mg/kg,

2.5 mg/mL, 1.0 mL) by oral gavage. Group 2 received only 14a

(25 mg/kg, 0.5 mg/mL, 1.0 mL) by oral gavage. Group 3 received 14a

(100 mg/kg, 2.5 mg/mL, 1.0 mL) and 100 ME49 oocysts by oral

gavage. Group 4 received 14a (25 mg/kg, 0.5 mg/mL, 1.0 mL) and

100 ME49 oocysts by oral gavage. Group 5 received only 100 ME49

oocysts by oral gavage.

Evaluation of Eﬃcacy of 14b in a Mouse Model of T.gondii

Oocyst Infection. Five groups of B7 female mice weighing approxi-

mately 25 g were used. Group 1 received only 14b (100 mg/kg,

2.5 mg/mL, 1.0 mL) by oral gavage. Group 2 received only 14b

(25 mg/kg, 0.5 mg/mL, 1.0 mL) by oral gavage. Group 3 received 14b

(100 mg/kg, 2.5 mg/mL, 1.0 mL) and 100 TgGoatUS4 oocysts by

oral gavage. Group 4 received 14a (25 mg/kg, 0.5 mg/mL, 1.0 mL)

and 100 TgGoatUS4 oocysts by oral gavage. Group 5 received only

100 TgGoatUS4 oocysts by oral gavage.

Insertional Mutagenesis Experiments. THdhxgTRP tachyzoites

were transfected with pLK47 vector plasmid.¹²Parasites were extracted

from HFF cells and resuspended in 1 mL of Cytomix electroporation

buﬀer solution (120 mM KCl, 150 μM CaCl₂, 5 mM K₂HPO₄, 5 mM

KH₂PO₄, 25 mM HEPES, 2 mM EDTA, and 5 mM MgCl₂in sterile

H₂O). Equivalent amounts of plasmid DNA and Cytomix solution

containing 10 × 10⁷parasites were combined for a total volume of

400 μL and electroporated using BioRad electroporator at 1.5k, 25fF,

and 100 Ω. This resulted in a random insertion of the plasmid in the

parasite genome and random disruption of a gene. Successfully

transfected parasites were selected by month-long treatment with

mycophenolic acid (25 μg/mL) and xanthine (50 μg/mL). After one

month, the mixed mutant population was exposed to 3i, 3j, 7a, or 14b

to select for those mutants whose gene disruption conferred resistance

to the drug. No parasite growth was noted after six weeks of exposure

to mycophenolic acid, xanthine, and any of the four test compounds.

providing RH-YFP parasites. This work was supported by

NIAID-AT-SBIR 1R43AI078763-01A1 and by gifts from the

Mann and Cornwell, Taub, Rooney-Alden, Engel, Pritzker,

Harris, Zucker, Morel, Samuel, and Mussilami families. We

thank K. Wroblewski for assistance with statistical analyses.

ABBREVIATIONS USED

■

CDC, Centers for Disease Control and Prevention; DIEA,

diisopropylethylamine; DMAP, dimethylaminopyridine;

DMSO, dimethylsulfoxide; ESI, electrospray interface; EtOAc,

ethyl acetate; FDA, Food and Drug Administration; FIBS,

ﬁbroblasts; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium hexaﬂuorophosphate; HFF, human fore-

skin ﬁbroblasts; IC, inhibitory concentration; IC₅₀, half-maximal

inhibitory concentration; IC₉₀, concentration necessary to

achieve 90% inhibition; IMDM-C, Iscove’s Modiﬁed Dulbec-

co’s Media; LC-MS, high performance liquid chromatography−

mass spectral analysis; m/z, mass to charge ratio; MAT,

modiﬁed agglutination test; MeCN, acetonitrile; MIC,

minimum inhibitory concentration; MSF, Malaria SYBR

Green I-Based Fluorescence Assay; OD, optical density; P.

falciparum, Plasmodium falciparum; P/S, pyrimethamine and

sulfadiazine in combination; RH-YFP, tachyzoites expressing

YFP; SYBR, SYBR Green Dye I; T. gondii, Toxoplasmosis gondii;

T_r. chromatographic retention time; WST-1, water-soluble

tetrazolium salt 1; YFP, yellow ﬂuorescent protein

REFERENCES

■

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AUTHOR INFORMATION

Corresponding Author

■

*For R.M.: phone, +1-773-834-4130; fax, +1-773-834-3577;

E-mail, rmcleod@midway.uchicago.edu. For R.D.W.: phone,

+1-732-230-3796; fax, +1-732-230-3956; E-mail, rwood@

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^▽A.F. and R.D.W. contributed equally to this work.

Funding

The project described was supported by award no.

R43AI078763 from the National Institute of Allergy and

Infectious Diseases. The content is solely the responsibility of

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of the National Institute of Allergy and Infectious Diseases or

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Dr. Laura Knoll (University of Wisconsin) for

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analyses of in vivo studies, Daniel Lee for assistance with

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