Development of a triclosan scaffold which allows for adaptations on both the A- and B-ring for transport peptides

Article abstract of DOI:10.1016/j.bmcl.2013.04.035

The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan parasites providing a significant challenge for drug delivery, which may be overcome through the addition of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of triclosan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which protrude into the solvent allowing for the future attachment of molecular transporters for delivery.

Full text of DOI:10.1016/j.bmcl.2013.04.035

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3551–3555
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a triclosan scaffold which allows for adaptations
on both the A- and B-ring for transport peptides q
b,
Stephen P. Muench a, , Jozef Stec , Ying Zhou c, Gustavo A. Afanador d, Martin J. McPhillie e,
⇑
Mark R. Hickman f, Patty J. Lee f, Susan E. Leed f, Jennifer M. Auschwitz f, Sean T. Prigge d,
David W. Rice e, Rima McLeod c
a School of Biomedical Sciences, University of Leeds, Leeds, UK
b Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States
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 Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
f 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:
Received 19 February 2013
Revised 9 April 2013
Accepted 13 April 2013
Available online 24 April 2013
The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan
parasites providing a significant challenge for drug delivery, which may be overcome through the addi-
tion of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of tri-
closan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers
challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which
protrude into the solvent allowing for the future attachment of molecular transporters for delivery.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords:
Enoyl reductase
Triclosan
Toxoplasma
Plasmodium
The Toxoplasma gondii (T. gondii) parasite and other apicomplex-
ans rely on the fatty acid synthesis type II pathway (FAS II), which is
prokaryotic-like and distinct from the eukaryotic fatty acid type I
pathway (FAS I). FAS II is carried out by discrete mono-functional
enzymes, whereas FAS I is typically carried out by one large poly-
peptide complex.1,2 This distinction has made this pathway a prom-
ising target for anti-microbial drug design.3,4 The FAS II pathway is
composed of four enzymes in an iterative process of fatty acid elon-
gation, in which the enoyl acyl-carrier protein reductase (ENR) has
gained the most attention with a range of drugs developed against
it. These include the anti-tuberculosis drug isoniazid, the diazabo-
rine family and triclosan which is a common antimicrobial found
in, amongst other things, toothpastes, mouthwashes and chopping
boards.5–8 Triclosan has been shown to be a very potent inhibitor
which binds at the core of the ENR enzyme, making p stacking inter-
actions with the reduced NAD+ cofactor.9 Its binding mode has been
characterized as a two state process, where it primarily interacts
with the NAD+ cofactor followed by an
a-helix packing over the tri-
closan, burying it away from the solvent forming a slow tight bind-
ing complex.10 Triclosan is a relatively simple scaffold which has
been extensively modified by a number of groups to improve its AD-
MET properties.
Significant progress has been made toward the development of
both T. gondii and Plasmodium falciparum medicines through the
discovery of a FAS II pathway residing within their apicoplast.11,12
This was particularly pertinent when it was discovered that the P.
falciparum, Eimeria tenella and T. gondii ENR enzyme could be
inhibited by the potent antibacterial triclosan.13–15 Since this dis-
covery, a number of groups have developed a range of triclosan
analogs which have shown potent inhibitory effects often with im-
proved ADMET properties.16–21 Although studies have reported
that FASII is not essential for blood stage survival of P. falciparum
it does play an important role in liver-stage development. More-
over, triclosan may have an off target effect within the blood stage
of its lifecycle.22,23
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel.: +44 (0) 113 3434279.
E-mail addresses: s.p.muench@leeds.ac.uk (S.P. Muench), jstec@csu.edu (J. Stec).
Present address: Chicago State University, College of Pharmacy, DH 203-3, 9501
South King Drive, Chicago, IL 60628, United States. Tel.: +1 773 821 2659; fax: +1 773
821 2595.
A significant problem with these inhibitors is the need to cross
several membranes imposed by the host cell, parasite and
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.035

3552
S. P. Muench et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3551–3555
Scheme 1. Synthesis of triclosan analogs bearing isoxazole group on ring B. Reagents and conditions: (a) 3-chloro-4-fluorobenzaldehyde, Cs2CO3, DMF, 95 °C, 16 h, 72%; (b)
H2O–EtOH–ice (1:1:2), H2NOHꢀHCl, 50% aq NaOH, rt, 1.5 h, 79%; (c) NCS, DMF, rt, 1.5 h, 100%; (d) sodium ascorbate, CuSO4ꢀ5H2O, KHCO3, 1-alkyne, t-BuOH–H2O (1:1), rt, 1 h.
For 4, R = CH2CH2OTBDPS, 51%; for 5, R = n-Pr, 50%; (e) for 6: CH2Cl2, BBr3 (4.0 equiv), ꢁ78 °C to rt, 3 h, 35%. For 7: CH2Cl2, BBr3 (8.0 equiv), ꢁ78 °C to rt, overnight, 61%.
Scheme 2. Synthesis of triclosan analogs bearing isoxazole groups on ring A and B. Reagents and conditions: (a) (1) LiAlH4, Et2O, ꢁ78 °C to 0 °C, 2.5 h, (2) H2O, 1.0 M NaOH,
50%; (b) 5-methylisoxazole-3-carboxylic acid, CH2Cl2, HOBt, EDCI, Et3N, rt, 17 h then H2O, 100% of crude material; (c) CH2Cl2, BBr3 (8.0 equiv), ꢁ78 °C to rt within 1 h, then rt
for 5 h, 32%.
apicoplast in order to reach the ENR enzyme target. This has been
aided with some success through the addition of a cleavable linker
and transductive peptide, although further work in this area is
needed.24 In order to establish if a more stable, non releasable
molecular transporter can be attached to the A- or B-ring of triclo-
san in a way that does not significantly alter binding to ENR, we
have taken two of our previously successful triclosan modifications
which resulted in extensions on the A- and B- ring and combined
them. In particular, isoxazole groups were chosen since they
retained good potency whilst improving the physiochemical prop-
erties (Stec et al., in press). This has resulted in a set of three com-
pounds with potent inhibitory effects and isoxazole extensions,
Table 1
Activity data for new diaryl ethers inhibitors of enoyl reductase
Notebook ID Structure
Parasite tissue challenge
TgENR Enzyme assay
P. falciparum blood stage
assay
dose response (ng/ml)
b
MIC50
(l
M) Toxicitya
(l
M) Conc.
98
(l
M) /Inhibition (%) IC50 (nM) D6
TM91C235
OH
Cl
O
Triclosan
5
>10
>10
15
29
N/A
N/A
A
B
Cl
Cl
4'
OH
Cl
O
6
ꢂ4
ꢂ8
ꢂ4
1/94
1/94
1/89
>2443
>N/A
NC
N
O
OH
Cl
O
7
>10
>10
34
>10,000
>10,000
>10,000
>10,000
NC
OH
N
O
OH
Cl
O
O
N
H
10
137
N
O
N
O
a
Toxicity to human foreskin fibroblasts.
At compound concentration (lM), enzyme inhibition percentage (%).
b

S. P. Muench et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3551–3555
3553
which allow through the incorporation of functional groups to be
further utilized for the addition of a linker and transductive pep-
tide or a non-releaseable linker.
The compounds were generated by reacting 4-hydroxy-3-
methoxybenzonitrile with 3-chloro-4-fluorobenzaldehyde to give
intermediate 1, which was readily converted to the imidoyl chlo-
ride 3 in a two-step protocol.25 Subsequent reaction of 3 with
TBDPS protected 3-butyn-1-ol and 1-pentyne afforded the corre-
sponding isoxazoles 4 and 5.26 The final compounds, 6 and 7, were
prepared by the modified demethylation procedure27 with BBr3,
employing 4 and 5 as the starting materials (Scheme 1).
Reduction of the nitrile 5 provided amine intermediate 8, which
was further elaborated through amide bond formation with 5-
methylisoxazole-3-carboxylic acid and demethylation to give the
final product 10 (Scheme 2).28 Full details on compound synthesis
are in the Supplementary data.
Inhibitory assays for parasite replication, toxicity against fibro-
blast host cell tests methods, and enzyme assays were performed
as previously described (Stec et al., in press).21,29–32
A major hurdle in targeting pathways which reside within the
apicoplast is the need for the inhibitor to cross several membrane
barriers. In order to avoid this difficulty, we adapted the triclosan
scaffold to contain bulky substituents on both the A- and B-rings
that were amenable to the addition of non-releasable transport
peptides ((Table 1) (Fig. 1A)). The relatively small binding pocket
means that these non-releasable linkers must sit outside the cav-
Figure 2. Structural formula of compound 33.
ity, exposed to the solvent to avoid any steric hindrance upon
inhibitor binding.
In the first instance, we used a previously identified modifica-
tion on the triclosan B-ring whereby a substituted-isoxazole group
was added at the 40-position.32 This group makes favorable interac-
tions around the entrance of the triclosan binding site and extends
out towards the solvent.
In order to test whether further modifications could be placed
at the exit of the binding site, the substituent on the isoxazole ring
was replaced with either a 5-propyl or 5-ethyl alcohol group.
Although only a minimal improvement in the MIC50; from 10
lM
to 4 M (6) and 7.5 M (7) is seen, no detrimental effect to the
l
l
enzymatic activity is observed. Importantly, docking studies have
shown that both of these extensions can clearly protrude from
the hydrophobic binding site towards a more solvent exposed area
of the enzyme (Fig. 1B). Further structural modification of the isox-
azole ring could allow for conjugation to a delivery peptide via
either a releasable or non-releasable linker.
Figure 1. (A) Overlay of triclosan and 10 within the ENR active site showing the similar mode of binding for the common A and B-ring motifs, colored red, blue, green and
yellow (10) or magenta (triclosan) for oxygen, nitrogen, chlorine and carbon, respectively. (B) Surface view of the modeled TgENR/NAD+/10 structure with the modified B-ring
protruding into the solvent. (C) Modeling of 10 within the TgENR/NAD+/triclosan crystal structure where Phe241 adopts a ‘closed’ position causing steric hindrance. (D)
Modeling of 10 within the modified TgENR crystal structure where Phe241 has adopted an ‘open’ position exposing the A-ring to the solvent.

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S. P. Muench et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3551–3555
A more challenging aspect of the project was to produce a mod-
that is, the A-ring would take the position of the B-ring and vice
versa, by the FLEXX docking program. This altered pose was observed
due to the large substituent on the A-ring causing severe steric
clashes within the binding site which could only be relieved
through the reverse binding mode. However, by allowing for flex-
ibility within the active site, in particular the movement of Phe243
about Cb within the TgENR/NAD+ complex using the Swiss PDB
Viewer the original binding mode was seen.34 Those orientations
that could accommodate the greater steric bulk of our hybrid com-
pounds resulted in a more open binding site such that the A-ring
modification is now exposed to the exterior solvent (Fig. 1C and
D). We have previously seen the movement of Phe243 about the
Cb, in a manner similar to that of the modeling in a TgENR co-crys-
tal structure for a different family of inhibitors (data not shown).
Subsequent docking of the compound series was carried out using
AUTODOCK 4.235 or MACROMODEL version 8.136 and PDB IDs 2O2S20 and
1LX637 available from the RCSB Protein DataBank.
ification on the A-ring of triclosan, which occupies an enclosed
hydrophobic region, resulting in its exposure to the outside solvent
thus allowing for its attachment to a delivery peptide. This is due
to the A-ring of triclosan being buried within the binding site,
whereas the B-ring is at the base of a channel which leads to the
solvent. The tight packing about the A-ring within the ENR enzyme
binding site often makes modifications about this ring difficult as
there are several residues predicted to make steric clashes with
these modified structures, as seen in docking simulations. It is
important to note however that most modeling programs do not
account for protein flexibility within the binding site.33
A solution to this problem was suggested through previous
studies of compound 33 (Fig. 2 (Stec et al., in press)). This com-
pound was predicted to bind in a reverse mode to that of triclosan,
A hybrid triclosan scaffold was then designed which contained
both A and B ring modifications allowing for the compound to be ex-
posed to the solvent on both ends of the molecular scaffold (10).
Modeling studies for this compound with increased bulk on both
the A- and B-rings does not permit the reverse mode binding seen
for the compound 33, but instead adopts the position shown in Fig-
ure 1. Importantly, this compound, despite its bulkier nature,
showed no decrease in MIC50 value but a slight increase in IC50 value
from 29 nM (6) and 19 nM (compound 33) to 137 nM (10). This in-
crease in IC50 to 137 nM is still therapeutically viable and more
importantly, the modifications to both the A and B-ring has resulted
in a compound which is amenable to further structural modifica-
tions to improve both binding and delivery via releasable/non-
releasable trans-peptide linkers. In vitro cytotoxicity tests also
showed no noticeable increase in toxicity based on the assay used.
Growth was measured using a type 1 T. gondii parasite tachyzoite
RH stably transfected with the yellow fluorescent protein (RH-YFP)
gene, with the relative fluorescence intensities of the parasites being
directly correlated with parasite viability and numbers (Fig. 3).
The activity of the 3 compounds (6, 7 and 10) were also tested
against two different strains of P. falciparum (D6 & TM91C235) in a
dose-response growth inhibition assay. Only 6 showed modest
activity against the drug sensitive strain, D6, but no activity against
the drug resistant strain, TM91C235 (Table 1). It is likely that the
non-essential nature of the FASII pathway within the blood stage
of the P. falciparum is responsible for the poor inhibitory effect of
these compounds within our assay.22 Further work will determine
the potency of these inhibitors against the liver stage parasite
which would be important in stopping recrudescence of the Plas-
modium parasite.
These results have shown how the triclosan scaffold can be
modified to result in both the A- and B-rings being exposed to
the exterior solvent without a significant loss in potency or detect-
able increase in toxicity. This is important since it allows for fur-
ther structural modifications to be made which are not
constrained by the size of the binding site. This also allows for
the addition of chemical functionalities which may aid in the deliv-
ery of triclosan into the apicoplast, a significant problem in current
drug design. Moreover, the bradyzoite form of T. gondii is currently
impossible to treat with current therapeutics due to the barriers
put in place by the cyst form of the parasite. Further work will
be carried out to use this scaffold as a basis for modifications by
various linker elements which may aid in drug delivery and target-
ing of a compound whose potency is in the nanomolar range.
Figure 3. Efficacy and absence of toxicity of compounds against T. gondii tachyzo-
ites. (A) Growth of RH-YFP in human Foreskin fibroblasts (HFF), measured as
fluorescence intensity. HFF infected with RH-YFP tachyzoites and fluorescence
intensities were measured after 72 h. Non infected fibroblasts that provided a
baseline control, HFF cells infected with 3200 RH-YFP tachyzoites treated with
pyrimethamine/sulfadiazine (p/s) or 0.1% DMSO serve as positive and negative
controls respectively. (B) Inhibitory effect of the compounds on RH-YFP. HFF cells
were infected with 3200 RH-YFP tachyzoites, compounds at various concentrations
were added 1 h after infection. The fluorescence intensities of the samples as
reflecting numbers of parasites were measured 72 h after addition of compounds.
(C) Effect of the compounds on HFF viability. The viability of host HFF cells was
assessed by Wst-1 staining, after 72 h of incubation of compounds at 10 mM
concentration. Effect of various concentrations of DMSO present in the HFF culture
medium shows varying amounts of toxicity.
Acknowledgments
We thank Dr. Alan Kozikowski for his contributions. The
authors are grateful for the financial support provided by NIAID

S. P. Muench et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3551–3555
3555
19. 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.
20. 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.
U01 AI082180-01 and the Rooney-Alden, Taub, Engel, Pritzker,
Harris, Kapnick, Mussilami and Samuel families and the Mann-
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Development fellowship (G1000567).
21. Perozzo, R.; Kuo, M.; Sidhu, A. b.; Valiyaveettil, J. T.; Bittman, R.; Jacobs, W. R.,
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Moura, P. A.; Lakshmanan, V.; Freundlich, J. S.; Valderramos, J. C.; Vilcheze, C.;
Siedner, M.; Tsai, J. H.; Falkard, B.; Sidhu, A. B.; Purcell, L. A.; Gratraud, P.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
035.
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