Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase

Article abstract of DOI:10.1016/j.bmcl.2013.02.019

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC₅₀ value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

Full text of DOI:10.1016/j.bmcl.2013.02.019

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2035–2043
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological activity of diaryl ether inhibitors
of Toxoplasma gondii enoyl reductase
Gang Cheng a, , Stephen P. Muench b, , Ying Zhou c, , Gustavo A. Afanador d, , Ernest J. Mui c,
Alina Fomovska c, Bo Shiun Lai c, Sean T. Prigge d, Stuart Woods e, Craig W. Roberts e, Mark R. Hickman g,
Patty J. Lee g, Susan E. Leed g, Jennifer M. Auschwitz g, David W. Rice f, Rima McLeod c,
⇑
a Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States
b School of Biomedical Sciences, University of Leeds, LS2 9JT Leeds, UK
c 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, IL 60637, United States
d Johns Hopkins School of Public Health Rm. E5132, 615 N. Wolfe St. Baltimore, MD 21205, United States
e Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
f Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
g Department of Discovery, Division of Experimental Therapeutics Walter Reed Army Institute of Research, Rm. 2N61 503 Robert Grant Avenue, Silver Spring, MD 20910, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme
for parasite survival. In view of triclosan’s poor druggability, which limits its therapeutic use, a new set of
B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives
were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display
improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these
inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifi-
cations have additional interactions with the strongly conserved Asn130.
Received 15 December 2012
Revised 24 January 2013
Accepted 1 February 2013
Available online 13 February 2013
Keywords:
TgENR
Triclosan
ADMET
Ó 2013 Elsevier Ltd. All rights reserved.
Toxoplasma
Toxoplasma gondii is an obligate intracellular, protozoan para-
site belonging to the phylum Apicomplexa. It infects over one third
of the world’s population. It can cause substantial morbidity and
mortality.1–6 People are mainly infected by unrecognized and inad-
vertent ingestion of materials contaminated with oocysts from the
feces of cats4,5 or eating undercooked meat containing bradyzoite-
stage parasites.6,7 Generally most infections in healthy individuals
are asymptomatic and self-limiting. However, in immunocompro-
mised persons, T. gondii infection can cause eye and brain disease
such as toxoplasmic encephalitis, chorioretinitis, and death.8,9
Infection acquired during pregnancy can be especially severe; it
can transmit from mother to the fetus, leading to congenital toxo-
plasmosis which may result in abortion, neonatal death, or fetal
abnormalities.2,10–16 Currently, there is no vaccine available to pre-
vent human infection from this pathogen. Antifolate agents, sulfa-
diazine and pyrimethamine, are two primary medicines for
treatment of T. gondii infection in humans.2,15 Although these med-
icines are effective against tachyzoites in the acute stage of the
disease, they do not eradicate encysted, latent bradyzoites. Fur-
thermore, these therapies can be associated with side effects such
as bone marrow depression, hypersensitivity and skin rashes.15,16
There is an urgent need to develop new anti-T. gondii medicines
that are both efficacious and nontoxic to humans.
One attractive target for chemotherapeutic intervention against
apicomplexan parasites is the prokaryotic-like type II fatty acid
biosynthesis (FAS-II) pathway.17–21 In T. gondii, this apicoplast-
localized FAS-II pathway has been proved to be essential for para-
site survival by both in vitro and in vivo studies.21 Type II FAS is
fundamentally divergent from the analogous FAS I pathway in
mammals. In eukaryotes, fatty acid biosynthetic enzymes integrate
on a single multifunctional polypeptide (FAS-I), whereas fatty acid
synthesis in prokaryotes utilizes a set of distinct enzymes compos-
ing the FAS-II pathway.22 Fatty acid biosynthesis is an iterative
process beginning with condensation of acetyl-coenzyme
A
(acetyl-CoA) with a growing fatty acid chain. In T. gondii, enoyl–
acyl carrier protein (ACP) reductase (ENR) is responsible for the fi-
nal reductive step in each round of the fatty acid chain elongation,
the NADH-dependent reduction of trans-2-enoyl-ACP to acyl-
ACP.23 There are a number of known bacterial and parasitic ENR
inhibitors such as diazaborines, isoniazid and as well as triclosan
⇑
Corresponding author. Tel.: +1 773 834 4152; fax: +1 773 834 3577.
E-mail address: rmcleod@midway.uchicago.edu (R. McLeod).
These authors contributed equally.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.019

2036
G. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2035–2043
which is a slow, tight-binding inhibitor of ENR.24–26 Moreover, tri-
closan inhibits TgENR enzymatic activity with an IC50 value of less
than 20 nM28 and inhibits the growth of T. gondii parasites with an
IC50 value of ꢀ200 nM.18
pound 3 with a trifluoromethyl substituent at the 40 position was
obtained from phenol 1 and 2-chloro-1-fluoro-4-(trifluoromethyl)
benzene via nucleophilic substitution and demethylation. Cu-pro-
moted Chan–Lam coupling29 of the phenol 1 with the 4-bromo-
phenylboronic acid and 4-formylphenylboronic acid gave the
corresponding intermediate bromide 4a and aldehyde 4b, respec-
tively. Palladium-catalyzed amination30 of the bromoarene 4a
and reductive amination of the benzaldehyde 4b afforded, after
deprotection, the piperazine or morpholine inhibitors 5a–d. The
40-sulfonamide derivatives 7a, b were prepared by Cu-catalyzed
arylation of phenol 1 with 4-iodo-benzenesulfonamides31 and sub-
sequent demethylation. Compound 10 was prepared by coupling of
2-amino-5-chlorophenol 8 with 1-methyl-3-(trifluoromethyl)-1H-
pyrazole-4-carboxylic acid followed by demethylation. Similar to
the procedure for 7a, Compound 19 was obtained by treatment
of phenol 1 with 6-bromoquinoline and subsequent demethyla-
tion. The furyl phenyl ether analogs (13, 14a–c) were obtained in
2–4 steps utilizing the synthetic routes shown in Scheme 2. Reac-
tion of phenol 1 with 5-nitrofuran-2-carbaldehyde provided the
furyl phenyl ether aldehyde 11.32 This intermediate was subjected
to NaClO2 oxidation and BBr3 mediated demethylation to afford the
furan-2-carboxylic acid 13. Next, using 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide (EDCI), 1-hydroxy-benzotriazole (HOBt)
and N,N-diisopropylethylamine (DIEA) carboxylic acid 13 was cou-
pled with three different amines to generate the desired ligands
14a–c.
Although triclosan is a potent inhibitor of TgENR, the diphenyl
ether has low water solubility and a high ClogP value. Another major
challenge for the development of medicines against targets which
reside within the apicoplast of apicomplexan parasites is the need
for the inhibitors to cross the four membranes of the parasite-spe-
cific organelle, in addition to the barriers set by both host cell and
the parasite.27 In order to overcome these structural drawbacks
and address the uptake problem, structure-based modification of
triclosan was directed by improving the ADMET (absorption, distri-
bution, metabolism, excretion and toxicity) profiles with special fo-
cus on the increase of aqueous solubility and permeability. We have
previously shown that the A-ring of triclosan can be modified to ex-
ploitanadditionalspaceatthebaseoftheinhibitorbindingpocket.28
Furthermore, we have shown that substitution on the B-ring may
also be tolerated to produce effective triclosan analogues {Stec
et al., in preparation}. This study has now been extended to show
that modifications of the B-ring can exploit the channel which leads
from the substrate binding pocket to the outside solvent. We report
herein on our efforts in developing novel 40-substituted and B-ring
substituted triclosan analogs with better physico-chemical
properties.
Triclosan analogs substituted at the 40 position were generated
from the commercially available phenol 1 using the methods out-
lined in Scheme 1.
The naphthyl phenyl ether derivative 2 was synthesized by
Cu-promoted Chan–Lam coupling29 of the phenol 1 with the
2-naphthyl boronic acid and Subsequent O-demethylation. Com-
Compounds 17a–c were prepared in an analogous manner as
described for 14a, starting from 1 via a sequence of steps involving
nucleophilic aromatic substitution, oxidation, demethylation and
amide bond formation (Scheme 3). Compound 18 was obtained from
15 through reductive amination and subsequent demethylation.
OMe
OH
N
O
O
N
N
O
N
c, b
R2
=
N
N
Me
Cl
R1
R2
N
Me
Cl
4a, b
O
Me
5a-d
R
1 = Br or CHO
a
c
b
d
a
OMe
OH
OMe
O
O
OH
d
b
O
O
Cl
S
Cl
S
R3
R3
Cl
O
O
1
7a-b
6
a, b
e, b
or(d, b)
O
N
N
R3 =
OH
OH
Cl
a
b
O
X
Cl
CF3
Cl
2
X = CH
X = N
3
19
F3C
F3C
HO
N
OH
OH
N
H
N
CH3
NH2
f, b
N
N
CH3
O
Cl
O
Cl
10
8
9
Scheme 1. Synthesis of 40-substituted derivatives and compound 10. Reagents and conditions: (a) arylboronic acid, Et3N, Cu(OAc)2, O2, CH2Cl2, room temp, 24 h. For 4a,
R = Br, 70%; for 4b, R = CHO, 58%; (b) BBr3, CH2Cl2, ꢁ78 °C to room temp, 6 h 30–80%; (c) for R = Br:amine, 3 mol % Pd2(dba)3, 9 mol % xantphos, t-BuOK, toluene, 110 °C, 5 h;
for R = CHO: amine, NaBH(OAc)3, AcOH, CH2Cl2, 0 °C to room temp, 1 h; (d) 4-iodobenzenesulfonamide, 30 mol % picolinic acid, 10 mol % CuI, DMSO, K3PO4, 85 °C, 24 h, 60–
70%; (e) 2-chloro-1-fluoro-4-(trifluoromethyl)benzene, K2CO3, DMF, 120 °C, 10 h; (f) HOBt, EDCI, DIEA, CH2Cl2, 0 °C to rt, overnight.

G. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2035–2043
2037
OCH3
OCH3
OH
OCH3
O
O
O
O
g
h
CO2H
CHO
Cl
Cl
Cl
12
11
1
O
N
OH
OH
CH3
O
N
O
O
O
O
b
O
N
f
CO2H
R
CH3
Cl
Cl
N
R =
H
14a-c
13
a
b
c
Scheme 2. Synthesis of furanyl phenyl ethers. Reagents and conditions: (b) BBr3, CH2Cl2, ꢁ78 °C to room temp, 6 h, 30–80%; (f) amine, HOBt, EDCI, DIEA, CH2Cl2, 0 °C to rt,
overnight, 40–60%; (g) 5-nitrofuran-2-carbaldehyde, NaH, DMSO, 35%; (h) NaClO2, DMSO, NaH2PO4, THF/tert-BuOH/H2O.
OH
OH
O
O
O
S
S
f
CO2H
R
Cl
Cl
N
N
16
17a-c
R =
CH3
N
H
N
BOC
h, b
a
c
b
OH
OCH3
OH
OCH3
O
S
O
i
b, j
S
CHO
N
Cl
N
CH3
Cl
Cl
O
18
1
15
Scheme 3. Synthesis of thienyl phenyl ethers. Reagents and conditions: (b) BBr3, CH2Cl2, ꢁ78 °C to room temp, 6 h; (f) amine, HOBt, EDCI, DIEA, CH2Cl2, 0 °C to rt, overnight,
40–60%; (h) NaClO2, DMSO, NaH2PO4, THF/t-BuOH/H2O; (i) 5-bromothiophene-2-carbaldehyde, K2CO3, DMSO, 80 °C, overnight, 60%; (j) amine, NaBH(OAc)3, AcOH, CH2Cl2,
0 °C to room temperature, 1 h.
Compounds were tested against TgENR, T. gondi in vitro and in
vivo, and Plasmodium falciparum in vitro using methods that have
been described in the recent literature28,33 For comparison of
parasite burden between treatment groups, analysis of variance
(ANOVA) was performed with group and run as factors. Due to
evidence of non-normality, natural log-transformed parasite
burden was used in the analysis (also, see Supplementary data).
The crystal structure of TgENR in complex with NAD+ and triclo-
san,34 showed that the 4-chloro phenoxy ring (A-ring) of triclosan
Modeling studies indicated that these inhibitors should have
similar binding positions as triclosan with respect to the A-ring.
While the changes on the B-ring in these derivatives might pro-
trude out from the triclosan binding pocket using the natural chan-
nel which the fatty acid substrate uses when attached to the acyl
carrier protein (Fig. 1A). An attractive additional interaction which
these substituted inhibitors may acquire is with Asn130 (TgENR
numbering) which is fully conserved within the apicomplexan
family (Fig. 1A and B). The inhibitory properties and cytotoxicity
data of the tested inhibitors are shown in Table 1.
participates in a p–p
stacking interaction with NAD+, and a hydro-
gen bond forms between the hydroxyl group and Tyr189. However,
the 2,4-dichlorophenoxy ring (B-ring) engages only in van der
Waals interactions within a pocket encompassed by the peptide
backbone of residues Leu128 to Ala131, the pyrophosphate and
nicotinamide moieties of NAD+, and the side chains of Val134,
Met193, Ala231 and Ile235. Moreover, there remains some addi-
tional space around the B-ring that could be exploited. In particular
the B-ring is exposed to the outside solvent via a channel which
would allow the fatty acyl substrate attached to the acyl carrier
protein to enter the active site (Fig. 1A).
In view of the space around the B-ring which could be exploited,
we devised a modification strategy to change the B-ring, incorpo-
rating additional polar groups to optimize the physico-chemical
properties (such as permeability and solubility) while keeping
the A-ring intact. These modifications were guided by computer-
aided ADMET prediction (ADMET suite from ACD/labs). In total,
19 triclosan analogs have been designed with either substitutions
at the 40-position of the B-ring or with the complete replacement
of its benzene ring by heteroaromatic groups. The biological test
data are shown in Table 1 along with some calculated ADMET
properties.
Initially, the B-ring of triclosan was substituted with a bulkier
naphthalene ring system (compound 2) to explore the size of the
pocket around the B-ring. This had the adverse effect of making
the inhibitor even less soluble than triclosan itself, which is
unpractical for a therapeutic drug. Despite the good activity of this
compound, it was therefore not consider worthwhile to develop
this family of compounds further. This result did however, confirm
that the B-ring of triclosan can accommodate significant change
and still be an effective ENR inhibitor. To increase the solubility,
we prepared compound 19 in which the naphthalene ring was
replaced by a quinoline ring. However, it lost some enzyme activ-
ity. In order to investigate the role of the oxygen atom in the ether
linker and to further understand the role of the B-ring, we prepared
compound 10. Despite the A-ring system being intact, compound
10 was relatively inactive compared to triclosan and thus further
modifications of this compound were not attempted. The low
activity of compound 10 is primarily due to the loss of the bridging
oxygen which has been shown to play an important role in
triclosan’s efficacy.35 Excellent activity was observed for com-
pound 3 in which the chlorine atom attached to the C4 of triclosan
was replaced by a trifluoromethyl group. The comparable activity

2038
G. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2035–2043
Figure 1. (A) The TgENR/NAD+/triclosan crystal structure shown in a cartoon representation covered by a transparent surface showing the channel which leads from the
triclosan inhibitor to the outside solvent. TgENR Asn130 which has been targeted for the design of new inhibitors is shown in stick format close to the B-ring of triclosan. (B) A
multiple sequence alignment in the vicinity of TgENR Asn130 (shown by an asterisk) of a number of parasitic and plant ENR enzymes showing full sequence conservation
across parasite and plant families.
of this derivative to triclosan is not surprising due to the close sim-
ilarity in size between the trifluoromethyl and chloro substituent.
Since compound 3 has similar physico-chemical properties to tri-
closan, and therefore is thought to share many of the known prob-
lems, we did not further optimize this compound. Instead, we
exploited the fact that we were able to substitute the 4-chlorine
atom of the B-ring without significant loss of efficacy, and we made
a series of substitutions at this position of the triclosan B-ring.
One of the most promising compound families developed in-
volved the replacement of chlorine at the C4-position of the B-ring
with a piperazine moiety (5a and 5c). Incorporation of this polar
group would greatly improve the water solubility according the AD-
MET prediction (360 mg/L for 5a and 160 mg/L for 5c vs 4.6 mg/L for
triclosan). Furthermore, placing an acetyl group onto the N4 position
of the piperazine derivative 5c improved its inhibitory properties in
modeling of these inhibitors showed no steric clashes within the
binding pocket which would cause a decrease in binding affinity.
However, the sulfonyl groups would be able to form a hydrogen
bond with the main chain nitrogen of Asn130 and Gly131. This
may inadvertently cause a shift of the B-ring moiety which has a
secondary effect on the A-ring such that it no longer has optimum
stacking interactions with the NAD+ co-factor. Further crystallo-
graphic studies would be required to test this hypothesis.
The next set of compounds was designed with a 2-furyl group to
replace the B-ring of triclosan. The furan ring was then further
substituted at C5 with several chemical motifs. Attachment of an
N-acetylpiperazine ring through a carboxyl group (14c) would
greatly increase the compound’s solubility to 1600 mg/L according
to the calculated ADMET predication. However, a significant loss of
potency was observed when 14c was tested in the TgENR assay. A
second approach was to attach a carboxyl group (13) or related
amide group (14a) to the furan ring at its C5-position. These inhib-
itors showed similar results in the parasite tissue culture assay and
shared similar calculated solubility values. However, they varied in
their ability to inhibit TgENR (compound 13 displayed 79% inhibi-
the enzyme assay (81% inhibition at 1 lM for 5c vs 25% for 5a) (Table
1). However, this increased potency is offset by a slight difference in
solubility between 5a and 5c. This highlights the fine balance be-
tween the inhibitory properties and other important features such
as solubility and cellular permeability. Modeling of these two com-
pounds using the TgENR/NAD+/triclosan complex crystal structure34
as a guide indicated that these compounds should be able to form a
hydrogen bond between the N4 of the piperazine ring and the oxy-
gen atom of the Asn130 side chain (Fig. 2).
The replacement of N4 with oxygen (5b) results in a significant
decrease in efficacy (Table 1). This is likely due to the loss of a hydro-
gen bond to the O4 atom through the subtle differences in a mor-
pholine ring structure when compared to a piperazine structure.
Insertion of a flexible carbon linker between the piperazine group
and the B-ring (5d) resulted in a significant loss of potency when
compared to 5a. Since the only difference between these inhibitors
is the methylene linker, the reduction in potency can be explained
by the shift of the piperazine group further out of the binding pocket
so that it can no longer maintain a hydrogen bond with Asn130.
These results show that the N4 position of the rigidly attached
piperazine group is important for good inhibitor activity and that
replacement of the chlorine atom at the C40-position on the B-ring
of triclosan by a large piperazine group yields a scaffold suited for
further development of TgENR inhibitors.
tion at 1 lM whereas compound 14a displayed only 46% inhibi-
tion). The improved inhibitory activity of compound 13 is likely
to be due to the carboxyl group. However, the best compound of
this family, 14b, contains the amide motif at this position with
an additional isoxazole group. We conclude that either a carboxyl
or amide group is tolerated at this position, with the substituent
on the amide nitrogen playing an important role in inhibitor effi-
cacy, as an amide group not bearing additional functionality is
not sufficient to improve inhibitor binding. In order to further
understand the possible role that the additional isoxazolyl group
at the amine nitrogen might play, this inhibitor was modeled into
the TgENR/NAD+/triclosan complex crystal structure. The modeling
result showed that as with the piperazine based inhibitors (5a, 5c),
Asn130 may play an important role in inhibitor binding by forming
a hydrogen bond to the inhibitor. Inhibitor 14b also showed a
greatly improved solubility (210 mg/L) when compared to triclo-
san (4.6 mg/L) without a significant effect on its predicted cellular
permeability properties. This compound family has shown that not
just substituents added to the B-ring of triclosan are tolerated but
that the B-ring itself can be replaced without significant loss of po-
tency. This is an important insight when trying to avoid some of
the major downsides of triclosan such as low solubility, stability.
Since triclosan can accommodate the replacement of the B-ring
with a 2-furyl group, our next step in designing novel ENR inhibi-
tors was to replace the B-ring with a 2-thienyl group, to which a
variety of chemically distinct groups were attached at its C5-posi-
tion. The smallest group that we attached to the thiophene ring
was carboxyl (16), which did not produce an effective inhibitor like
Two compounds were designed that feature a replacement of
the chlorine atom at the C4-position on the B-ring of triclosan by
a pyrrolidinylsulfonyl (7a) and piperidinylsulfonyl group (7b).
These inhibitors displayed similar activities in both the enzyme-
based and cell-based assays (Table 1) but were significantly less ac-
tive than compounds 5b and 5c. We concluded that the observed
reduction in potency is unlikely to be an effect of their piperidine
or pyrrole ring motifs but instead an effect of their shared sulfonyl
motifs suggesting that this is an unsuitable substitution. The

Table 1
Activity data and ADMET parameter predictions for new diaryl ethers inhibitors of TgENR
Compound
Structure
Parasite tissue challenge assay
TgENR enzyme assay
Inhibitionb (%)at 1
Calculated ADMET properties (ACD/Labs)d
MIC50
(
lM)
Toxicitya
(lM)
lM
IC50 (nM)
ClogPc
TPSA (Å2)
Sw (mg/L)
Caco-2 (ꢂ10ꢁ6 cm/s)
F >30%
PPB (%)
99
OH
Cl
O
O
O
Triclosan
5
>10
98
15
5.53
53.25
4.6e
174
0.81
A
B
Cl
Cl
Cl
4' Cl
OH
2
10
4
>10
>10
91
95
41
30
5.51
5.70
29.46
29.46
2.8
3.1
188
192
0.81
0.81
99
Cl
OH
OH
3
99.6
CF3
O
O
O
5a
10-1
>10
>10
>10
25
74
—
—
3.72
4.15
35.94
41.93
360
78
211
236
0.35
0.35
95
97
Cl
Cl
N
N
OH
OH
5b
5c
N
O
1.5
>10
81
225
2.32
53.01
160
232
0.70
97
Cl
N
N
O
OH
OH
O
O
N
5d
7a
>10
10
>10
>10
32
78
—
—
4.64
4.16
35.94
75.22
1300
15
188
227
0.35
0.94
89
97
N
Cl
Cl
N
O
S
O
OH
O
7b
10
>10
81
—
4.72
75.22
8.3
234
0.94
98
N
Cl
S
O O
(continued on next page)

Table 1 (continued)
Compound
Structure
Parasite tissue challenge assay
TgENR enzyme assay
Inhibitionb (%)at 1
IC50 (nM)
Calculated ADMET properties (ACD/Labs)d
MIC50
(
lM)
Toxicitya
(lM)
lM
ClogPc
TPSA (Å2)
Sw (mg/L)
Caco-2 (ꢂ10ꢁ6 cm/s)
F >30%
PPB (%)
93
F3C
N
OH
OH
OH
H
N
CH3
N
10
>10
>10
27
—
1.54
67.15
270
173
0.95
O
Cl
Cl
O
O
O
O
13
>10
>10
>10
>10
79
46
—
—
3.25
2.23
79.90
71.70
710
680
18
0.91
0.95
98
95
OH
O
O
14a
162
HN
Cl
O
N
O
O
HO
N
O
O
Me
H
14b
14c
10
>10
>10
90
45
58
3.07
1.72
97.73
83.22
210
202
140
0.95
98
95
Cl
O
O
N
HO
N
>10
—
1600
0.7
O
Cl
Cl
OH
OH
O
O
O
S
S
16
10
10
>10
10
48
75
—
—
3.84
3.42
95.00
96.80
610
290
17
0.91
0.85
98
97
OH
O
17a
195
HN
Cl
CH3
O
S
HO
N
O
N
O
O
17b
17c
>10
>10
>10
>10
76
60
—
—
4.71
2.73
107.55
90.04
130
233
31
0.35
0.51
96
93
Cl
Cl
HO
O
S
O
1900
N
NH

G. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2035–2043
2041
the similar compound 13. This is likely the case because no addi-
tional inhibitor–enzyme interactions can be formed to the small
carboxyl group. The most promising of the thiophene-based inhib-
itors is compound 18 which showed a good inhibitory effect in the
parasite tissue cell culture assay and moderate activity in the en-
zyme assay, coupled with a significant improvement in solubility
compared to triclosan. Modeling of compound 18, which contains
a 4-acetylpiperazin-1-yl group attached to the thiophene ring
through a methylene linker, showed again the importance of
Asn130 on the efficacy of these inhibitors as it may hydrogen bond
to the piperazine moiety (Table 1, Fig. 2). Interestingly, 14c is sim-
ilar to 18 in that it also contains a N-acetylated piperazine ring at
its terminal. However, this group is attached through a furan ring
and carbonyl group in compound 14c rather than a thiophene ring
and methylene group in compound 18. Since the furan and thio-
phene ring systems are similar, it is conceivable that the flexible
methylene linker permits the piperazine ring in 18 to adopt a
favorable position with respect to hydrogen bonding with
Asn130 while the rigid carbonyl linker in 14c does not. The car-
bonyl linker in the effective furan-based compound 14b does not
have this negative effect due to the different orientation of the
hydrogen bond acceptors in its isoxazolyl group compared to the
N4-atom of the piperazinyl group in compound 18. Moreover,
compounds 17a, 17b, and 17c, which contain a carbonyl rather
than methylene linker between their B rings and terminal amine
moieties, also exhibit significant decreases in activity.
Compound 5c was selected for further biological evaluation in a
T. gondii murine infection model (Fig. 3). In 3 separate, replicate
experiments mice treated with compound 5c had fewer parasites
on average when compared with the DMSO control on the 6th
day after infection. Although parasite number varied between indi-
vidual mice, these modest differences reached significance (p
<0.05), in two of the three replicate experiments. These data are
shown pooled in Figure 3 with a separate symbol for each experi-
ment. When the data were pooled the difference between the
amount of parasite burden in DMSO control and compound 5c
treated mice was significant. The p-value for the treatment group
comparison was 0.003. A lower dose of 50 mg/kg was not effective
and a higher dose of 100 mg/kg was not completely soluble. The
compound was toxic at 75 mg/kg by the seventh day after infec-
tion. Thus, although the predicted ADMET properties were superior
to those of triclosan, this compound did not prove to be superior in
these in vivo tests where triclosan is active at 25 mg/kg. The rela-
tively lower efficacy may be due to metabolic instability of com-
pound 5c.
Compound 5c was also tested against P. falciparum strain D6
(CDC/Sierra Leone) and TM91C235 (WRAIR, Thailand). Results are
in Table 2.
Compound 5c was inactive against the chloroquine sensitive D6
and the chloroquine resistant C235 strains. Modeling of 5c within
the PfENR active site shows that there is one subtle difference
which may affect 5c binding whereby ASN which H-bonds to the
inhibitor is followed by Gly-Pro in TgENR and Ala-Lys in PfENR.
This change alters the main chain position about the exit of 5c such
that it makes less favorable packing interactions. However, it is
unlikely that this small change would abolish activity and it is
more likely that this can be explained by the non-essential nature
of ENR within P. falciparum during its blood stage development37
A
crystal structure would be needed to determine if that prediction is
correct and explains the lack of activity of 5c for P. falciparum. Since
the fatty acid biosynthesis enzyme ENR plays a key role in the
development of liver stage malarial parasites, it also will be of
interest in the future to learn whether the new inhibitors that uti-
lize the space in the enzyme that accepts B ring modifications
would improve the activity of such inhibitors against the liver
stage organisms.

2042
G. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2035–2043
Figure 2. Modeling of the designed inhibitors into the TgENR/NAD+/triclosan crystal structure. TgENR is shown in cartoon format with inhibitors, NAD+ cofactor, Asn130,
Ala231 and Ile235 shown in stick format and colored red, blue, orange, pale blue, magenta, gold, yellow and green for oxygen, nitrogen, phosphorus, fluorine, chlorine, sulfur,
carbon (TgENR) and carbon (modeled inhibitors), respectively.
inhibitors bearing modifications to the B-ring of the triclosan scaf-
p value = 0.003
fold. Compounds 2 and 3 showed that, as previously reported,36
the B-ring of triclosan can be modified without a significant reduc-
tion in inhibitor efficacy. The structural changes that were ex-
plored were based on chemical units comprised of piperazine,
furan and thiophene ring systems. These added chemical groups
were designed to occupy the channel which leads from the triclo-
san binding site to the area exposed to outside solvent; this area is
thought to be the portal through which insertion of fatty acid sub-
strates by the ACP takes place.
109
108
107
106
105
Run 1
Run 2
Run 3
Because of the important role that this portal appears to play, as
it is lined by relatively well conserved amino acid residues, the use
of this region in the design of new therapeutics appears justified. In
particular, the strongly conserved amino acid residue Asn130
(Fig. 1A) may play an important role in binding a number of these
ligands. For those compounds which are incapable of forming an
H-bond interaction with Asn130, only poor inhibitory activity is
observed. Of the compounds disclosed herein, three compounds,
namely 14b, 5c, and 18 may serve as leads candidates for further
rounds of SAR optimization, as these compounds appear to show
drug-like properties along with the lack of toxicity to human
fibroblasts in comparison to their toxicity to the parasite.
DMSO
Compound 5c
Figure 3. Compound 5c protected mice from T. gondii in vivo. Mice were infected
intraperitoneally with 2 ꢂ 104 tachyzoites (Pru strain expressing firefly luciferase)
and imaged daily following intraperitonal injection of luciferin (3 mg) in an IVIS
spectrum (Caliper Biosciences).
Table 2
Effects on Plasmodium falciparum strain D6 (Sierra Leone) and C235 (Thailand;
chloroquine resistant)
Acknowledgments
Compound
SYBR Green D6 IC50 (ng/ml)
>10,000
SYBR Green C235 IC50 (ng/ml)
>10,000
The authors are grateful for the financial support provided by
NIAID grant U01 AI082180-0101 and the Wellcome Trust and the
support of the Mann and Cornwell, Rooney-Alden, Taub, Engel
and Mussilami families. SPM is funded by an MRC Career Develop-
ment Fellowship (G1000567). The authors also thank Alan
Kozikowski for his contributions. They also thank K. Wroblewski
for her assistance with statistical analyses of the in vivo data.
The opinions of the authors from Walter Reed Army Institute of Re-
search are their own and do not reflect the official views of the
Department of Defense.
5c
The use of triclosan-based compounds as possible inhibitors of
apicomplexan parasites has become an intense area of study since
the parasitic ENR was validated as a novel target.17,18 However,
despite its potency, triclosan displays a range of poor ADMET
properties. In particular, it is very poorly soluble in water. In order
to address some of these problems we synthesized a range of

G. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2035–2043
2043
18. McLeod, R.; Muench, S. P.; Rafferty, J. B.; Kyle, D. E.; Mui, E. J.; Kirisits, M. J.;
Mack, D. G.; Roberts, C. W.; Samuel, B. U.; Lyons, R. E.; Dorris, M.; Milhous, W.
K.; Rice, D. W. Int. J. Parasitol. 2001, 31, 109.
19. Ben Mamoun, C.; Prigge, S. T.; Vial, H. Drug Dev. Res. 2010, 71, 44.
20. Lu, J. Z.; Muench, S. P.; Allary, M.; Campbell, S.; Roberts, C. W.; Mui, E.; McLeod,
R. L.; Rice, D. W.; Prigge, S. T. Parasitology 1949, 2007, 134.
Supplementary data
Supplementary data (experimental procedures and spectral
data) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmcl.2013.02.019.
21. Mazumdar, J.; Wilson, E.; Masek, K.; Hunter, C.; Striepen, B. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 13192.
22. Magnuson, K.; Jackowski, S.; Rock, C. O.; Cronan, J. E. Microbiol. Rev. 1993, 57,
522.
References and notes
23. Massengo-Tiasse, R. P.; Cronan, J. E. Cell Mol. Life Sci. 2009, 66, 1507.
24. Ward, W. H.; Holdgate, G. A.; Rowsell, S.; McLean, E. G.; Pauptit, R. A.; Clayton,
E.; Nichols, W. W.; Colls, J. G.; Minshull, C. A.; Jude, D. A.; Mistry, A.; Timms, D.;
Camble, R.; Hales, N. J.; Britton, C. J.; Taylor, I. W. Biochemistry 1999, 38, 12514.
25. Rozwarski, D. A.; Grant, G. A.; Barton, D. H.; Jacobs, W. R.; Sacchettini, J. C.
Science 1998, 279, 98.
26. Grassberger, M. A.; Turnowsky, F.; Hildebrandt, J. J. Med. Chem. 1984, 27, 947.
27. Samuel, B. U.; Hearn, B.; Mack, D.; Wender, P.; Rothbard, J.; Kirisits, M. J.; Mui,
E.; Wernimont, S.; Roberts, C. W.; Muench, S. P.; Rice, D. W.; Prigge, S. T.; Law,
A. B.; McLeod, R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14281.
28. Tipparaju, S. K.; Muench, S. P.; Mui, E. J.; Ruzheinikov, S. N.; Lu, J. Z.; Hutson, S.
L.; Kirisits, M. J.; Prigge, S. T.; Roberts, C. W.; Henriquez, F. L.; Kozikowski, A. P.;
Rice, D. W.; McLeod, R. L. J. Med. Chem. 2010, 53, 6287.
29. Evans, D. A.; Katz, J. L.; West, T. R. Tetrahedron Lett. 1998, 39, 2937.
30. Guari, Y.; van Es, D. S.; Reek, J. N. H.; Kamer, P. C. J.; van Leeuwen, P. W. N. M.
Tetrahedron Lett. 1999, 40, 3789.
31. Maiti, D.; Buchwald, S. L. J. Org. Chem. 2010, 75, 1791.
32. Kuo, S.-C.; Wu, J.-S.; Huang, L.-J.; Wu, C.-H.; Huang, S.-C.; Chou, T.-C. J.
Heterocycl. Chem. 1989, 26, 605.
33. Fomovska, A.; Huang, Q.; El Bissati, K.; Mui, E. J.; Witola, W. H.; Cheng, G.; Zhou,
Y.; Sommerville, C.; Roberts, C. W.; Bettis, S.; Prigge, S. T.; Afanador, G. A.;
Hickman, M. R.; Lee, P. J.; Leed, S. E.; Auschwitz, J. M.; Pieroni, M.; Stec, J.;
Muench, S. P.; Rice, D. W.; Kozikowski, A. P.; McLeod, R. Antimicrob. Agents
Chemother. 2012, 56, 2666.
34. Muench, S. P.; Prigge, S. T.; McLeod, R.; Rafferty, J. B.; Kirisits, M. J.; Roberts, C.
W.; Mui, E. J.; Rice, D. W. Acta. Crystallogr. Sect. D 2007, 63, 328.
35. Heath, R. J.; Li, J.; Roland, G. E.; Rock, C. O. J. Biol. Chem. 2000, 275, 4645.
36. Freundlich, J. S.; Anderson, J. W.; Sarantakis, D.; Shieh, H. M.; Yu, M.;
Valderramos, J. C.; Lucumi, E.; Kuo, M.; Jacobs, W. R., Jr.; Fidock, D. A.;
Schiehser, G. A.; Jacobus, D. P.; Sacchettini, J. C. Bioorg. Med. Chem. Lett. 2005,
15, 5247.
[37]. Yu, M.; Santha Kumar, T. R.; Nkrumah, L. J.; Coppi, A.; Retzlaff, S.; Li, C. D.;
Kelly, B. J.; Moura, P. A.; Lakshmanan, V.; Freundlich, J. S.; Vilcheze, J.-C.;
Siedner, S.; Tsai, J. H.-C.; Falkard, B.; Sidhu, B. S.; Purcell, L. A.; Gratraud, P.;
Kremer, L.; Waters, A. P.; Schiehser, G.; Jacobus, D. P.; Janse, C. J.; Ager, A.;
Jacobs, W. R., Jr.; Sacchettini, J. C.; Heussler, V.; Sinnis, P.; Fidock, D. A. Cell
Host Microbe 2008, 4, 567.
1. Hill, D.; Dubey, J. P. Clin. Microbiol. Infect. 2002, 8, 634.
2. McLeod, R.; Boyer, K.; Karrison, T.; Kasza, K.; Swisher, C.; Roizen, N.;
Jalbrzikowski, J.; Remington, J.; Heydemann, P.; Noble, A. G.; Mets, M.;
Holfels, E.; Withers, S.; Latkany, P.; Meier, P. Clin. Infect. Dis. 2006, 42, 1383.
3. Boyer, K.; Marcinak, J.; McLeod, R. In Principles and Practice of Pediatric Infectious
Diseases; Long, S., Pickering, L. K., Prober, C. G., Eds., 4th ed.; Churchill
Livingstone: New York, 2009. section 274.
4. Hill, D.; Coss, C.; Dubey, J. P.; Wroblewski, K.; Sautter, M.; Hosten, T.; Munoz-
Zanzi, C.; Mui, E.; Withers, S.; Boyer, K.; Hermes, G.; Coyne, J.; Jagdis, F.;
Burnett, A.; McLeod, P.; Morton, H.; Robinson, D.; McLeod, R. J. Parasitol. 2011,
97, 328.
5. Boyer, K.; Hill, D.; Mui, E.; Wroblewski, K.; Karrison, T.; Dubey, J. P.; Sautter, M.;
Noble, A. G.; Withers, S.; Swisher, C.; Heydemann, P.; Hosten, T.; Babiarz, J.; Lee,
D.; Meier, P.; McLeod, R. Clin. Infect. Dis. 2011, 53, 1081.
6. Desmonts, G.; Couvreur, J. N. Engl. J. Med. 1974, 290, 1110.
7. Mead, P. S.; Slutsker, L.; Dietz, V.; McCaig, L. F.; Bresee, J. S.; Shapiro, C.; Griffin,
P. M.; Tauxe, R. V. Emerg. Infect. Dis. 1999, 5, 607.
8. Luft, B. J.; Remington, J. S. Clin. Infect. Dis. 1992, 15, 211.
9. Porter, S. B.; Sande, M. A. N. Engl. J. Med. 1992, 327, 1643.
10. Swisher, C. N.; Boyer, K.; McLeod, R. Semin. Pediatr. Neurol. 1994, 1, 4.
11. Olariu, T. R.; Remington, J. S.; McLeod, R.; Alam, A.; Montoya, J. G. Pediatr. Infect.
Dis. J. 2011, 30, 1056.
12. McLeod, R.; Mack, D. G.; Boyer, K.; Mets, M.; Roizen, N.; Swisher, C.; Patel, D.;
Beckmann, E.; Vitullo, D.; Johnson, D., et al J. Lab. Clin. Med. 1990, 116, 623.
13. McGee, T.; Wolters, C.; Stein, L.; Kraus, N.; Johnson, D.; Boyer, K.; Mets, M.;
Roizen, N.; Beckman, J.; Meier, P., et al Otolaryngol. Head Neck Surg. 1992, 106,
75.
14. McAuley, J.; Boyer, K. M.; Patel, D.; Mets, M.; Swisher, C.; Roizen, N.; Wolters,
C.; Stein, L.; Stein, M.; Schey, W., et al Clin. Infect. Dis. 1994, 18, 38.
15. Remington, J. S.; McLeod, R.; Thulliez, P.; Desmonts, G. Toxoplasmosis. In
Infectious Diseases of the Fetus and Newborn Infant; Remington, J., Klein, J., Eds.,
7th ed.; W.B. Saunders: Philadelphia, 2011.
16. McLeod, R.; Khan, A. R.; Noble, G. A.; Latkany, P.; Jalbrzikowski, J.; Boyer, K.
Pediatr. Infect. Dis. J. 2006, 25, 270.
17. Zuther, E.; Johnson, J. J.; Haselkorn, R.; McLeod, R.; Gornicki, P. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 13387.