Synthesis, biological activity, and X-ray crystal structural analysis of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 1: 4′-Substituted triclosan derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.08.044

A structure-based approach has been taken to develop 4′-substituted analogs of triclosan that target the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit nanomolar potency against purified PfENR enzyme and modest (2-10 μM) potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite. X-ray crystal structures of nitro 29, aniline 30, methylamide 37, and urea 46 demonstrate the presence of hydrogen-bonding interactions with residues in the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular parasites.

Full text of DOI:10.1016/j.bmcl.2005.08.044

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5247–5252
Synthesis, biological activity, and X-ray crystal structural
analysis of diaryl ether inhibitors of malarial enoyl acyl
carrier protein reductase. Part 1: 40-Substituted triclosan derivatives
Joel S. Freundlich,a,* John W. Anderson,a Dimitri Sarantakis,a Hong-Ming Shieh,a
Min Yu,b Juan-Carlos Valderramos,b Edinson Lucumi,d Mack Kuo,d
William R. Jacobs, Jr.b,c David A. Fidock,b Guy A. Schiehser,a
David P. Jacobusa and James C. Sacchettinid
aDepartment of Medicinal Chemistry, Jacobus Pharmaceutical Company, 37 Cleveland Lane, Princeton, NJ 08540, USA
bDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
cHoward Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY 10461, USA
dDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA
Received 1 June 2005; revised 9 August 2005; accepted 17 August 2005
Available online 29 September 2005
Abstract—A structure-based approach has been taken to develop 40-substituted analogs of triclosan that target the key malarial
enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit nanomolar potency
against purified PfENR enzyme and modest (2–10 lM) potency against in vitro cultures of drug-resistant and drug-sensitive strains
of the P. falciparum parasite. X-ray crystal structures of nitro 29, aniline 30, methylamide 37, and urea 46 demonstrate the presence
of hydrogen-bonding interactions with residues in the active site and point to future rounds of optimization to improve compound
potency against purified enzyme and intracellular parasites.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The development of novel anti-malarials is critical as
malaria worldwide afflicts 300–600 million people result-
ing in 1–3 million deaths per year.1,2 Plasmodium falci-
parum infection is the most widespread form of
malaria and is the predominant cause of severe disease
and death. Traditional treatments with drugs, such as
chloroquine and sulfadoxine-pyrimethamine, are now
much less effective due to rampant resistance.3
is reliant on a dissociative process that utilizes a set of
distinct enzymes composing a FAS-II pathway.4 This
pathway has been localized to the parasite apicoplast,
an essential organelle ancestrally related to cyanobacte-
ria.5 Fatty acid biosynthesis is an iterative process begin-
ning with the condensation of acetyl CoA with the
growing fatty acid chain. P. falciparum enoyl acyl carrier
protein (ACP) reductase (PfENR) is responsible for the
final step in each fatty acid synthesis cycle: the NADH-
dependent reduction of trans-2-enoyl-ACP to acyl-ACP.
The inhibition of fatty acid synthesis in P. falciparum
holds significant promise in potentially enabling the dis-
covery and development of an orally dosed therapeutic
that is affordable, safe, and efficacious against drug-re-
sistant strains. Fatty acid synthesis plays a key role in
membrane construction and energy production. Unlike
in higher eukaryotes and yeast where fatty acid biosyn-
thetic machinery resides on a single multifunctional
polypeptide (FAS-I), fatty acid synthesis in Plasmodium
We and others have shown that triclosan inhibits
PfENR,6,7 in addition to the related ENRs for Esche-
richia coli,8,9 Staphylococcus aureus,10 and mycobacte-
ria.11 This compound was also found to be effective in
killing P. falciparum in vitro6,7,12 and curing mice of
infection with the rodent malaria species Plasmodium
berghei.6 Given triclosanÕs abundance13 (U.S. annual
production > 1 · 106 lbs), its safety as demonstrated by
its widespread use in personal care and household prod-
ucts, and the absence of a lipid synthesis inhibitor in the
anti-malarial arsenal, we and others have chosen to
Keywords: Anti-malarial; Phenol; Diaryl ether.
*
Corresponding author. Tel.: +1 609 9217447; fax: +1 609 7991176;
e-mail: j_freundlich@jacobuspharm.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.044

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J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5247–5252
pursue the discovery of a triclosan-derived therapeutic
for malaria.7,14–19
Prior elucidation of the X-ray crystal structure of triclo-
san and its NAD+ co-factor bound to PfENR revealed
the phenol moiety to be the critical binding element of
triclosan.7 Structural analysis suggested vectors off the
triclosan A and B rings where additional functionality
could be placed to enhance binding affinity. A strategy
was developed to build off these aromatic ring positions
to enhance enzymatic and anti-parasitic activity. A sig-
nificant gain in potency could facilitate replacement or
removal of the phenol, which is a metabolic liability as
in vivo conjugation of the phenol leads to rapid clear-
ance of triclosan.20 With this goal in mind, this report
details our work on the 40-position of the triclosan
pharmacophore.
Examination of the PfENR:NAD+:triclosan structure
revealed the 40-chloro group to be in van der Waals con-
tact with the hydrophobic sidechains of Val-222 and
Met-281. The halogen is directed toward the sidechain
of Asn-218 and backbone carbonyl of Ala-219. Our
hypothesis was that replacement of the chloride with a
variety of groups with both hydrophobic and hydro-
gen-bonding characteristics could enhance the enzyme
binding affinity of triclosan derivatives. A recently
reported triclosan analog utilizes the hydroxyl group
of a naphthol to make these proposed hydrogen bonds.7
Scheme 2. Synthesis of various 40-derivatives from the corresponding
aldehyde and nitrile derivatives, where Ar = 4-chloro-2-methoxyphe-
nyl. Reagents: (a) i—LiAlH4, THF, ii—TsCl, NEt3, DCM, iii—NaCN,
DMSO; (b) NaN3, ZnBr2, H2O, D; (c) NaOH, H2O2, THF, 70–90 ꢁC;
(d) i—HBTU, HNR1R2, DIEA, DMF/NMP or ii—cat. DMF, SOCl2
then HNR1R2.
yielded 11–13. Activation of the aniline with triphosgene
in the presence of base followed by aminolysis afforded
ureas 14–15. Diazotization of the aniline followed by
acid hydrolysis of the corresponding diazonium salt
afforded 40-phenol 16.21
0
˚
The 4 -chloro is approximately 4 A from the solvent-ac-
cessible surface and, thus, the 40-position could also
serve to append functionality to alter inhibitor physio-
chemical properties.
As shown in Scheme 2, aldehyde 2 was reacted in three
steps to produce 17 via a sequence of conventional
methods consisting of lithium aluminum hydride reduc-
tion, tosylation, and displacement with sodium cyanide.
The method of Sharpless was implemented to prepare
tetrazole 18 from nitrile 3.22 Sodium hydroperoxide
was generated in situ and utilized to hydrolyze 3 to car-
boxylic acid 19.23 The carboxylic acid was taken on to
afford amides 20–27 using either acid chloride methodol-
ogy or HBTU-mediated coupling.
Compound syntheses began with nitroaromatic 1, alde-
hyde 2, and nitrile 3, prepared from commercially avail-
able starting materials via nucleophilic aromatic
substitution. Elaboration of the nitro derivative 1 is
depicted in Scheme 1. Compound 1 was hydrogenated
to afford aniline 4 which could be acylated or sulfonylat-
ed to afford 5–10. Reductive amination with aldehydes
The intermediate anisoles 1 and 3–27 were treated with
an excess of boron tribromide or aluminum trichloride
to derive the final product phenols 28–53. All final com-
pounds were characterized by 1H NMR, LC–MS, and in
some cases elemental analysis.
Final compounds 28–53 were tested in two assay sys-
tems to determine their inhibition of PfENR enzymatic
activity and inhibition of parasite whole cell growth. For
both assays, the reported inhibitory concentrations are
tabulated as means standard error, taking into ac-
count three independent determinations performed in
duplicate. The enzyme assay has been previously de-
scribed7 and was modified slightly to allow for a higher
throughput.25 Briefly, the assay measured the NADH-
dependent enzymatic reduction of crotonoyl-CoA sub-
strate by following a decrease in absorbance at 340 nm
resulting from the conversion of NADH to NAD+. To
date, 14 independent determinations of the IC50 of tri-
closan have been made, resulting in a mean of 73 nM
with a standard error of 21 nM. The parasite whole cell
Scheme 1. Synthesis of various 40-derivatives from the corresponding
parent aniline where Ar = 4-chloro-2-methoxyphenyl. Reagents and
conditions: (a) H2, Ra-Ni, AcOH/EtOH, (b) Ac2O or PhC(O)Cl or
RSO2Cl in NEt3, DCM; (c) ArCHO, NaBH3CN, HOAc, MeOH; (d)
i—(Cl3CO)2CO, NEt3, DCM, ꢀ78 ꢁC rt, ii—R1R2NH; (e) i—H2SO4,
NaNO2, 0 ꢁC, ii—tol, 110 ꢁC.

J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5247–5252
5249
assay, previously described,7 uses [3H]hypoxanthine
incorporation into nucleic acids in cultured P. falcipa-
rum parasites (exposed to drug for 72 h) as a marker
of growth inhibition in the presence of drug. In the cur-
rent study, two parasite strains were utilized: 3D7, which
is drug-sensitive, and Dd2, which is resistant to the anti-
malarials chloroquine and pyrimethamine-sulfadox-
ine.24 To date, 43 independent runs of triclosan have
been conducted with the following results. Against the
3D7 strain, the EC50 was determined to have a mean
value of 2.9 lM and a standard error of 0.2 lM. Versus
the Dd2 strain, the EC50 mean value was 3.9 lM with a
standard error of 0.3 lM.
35, however, suffers a significant loss against the para-
site, while nitro derivative 29 exhibits potent anti-para-
site activity. The significant anti-parasitic efficacy of 29
is not commensurate with its enzyme inhibition and
off-target activity must be considered as an explanation.
Utilizing the criteria of enzymatic potency and ease of
synthetic elaboration, the amide 35 core was chosen
for analog synthesis with the dual goals of improving en-
zyme and parasite assay potency. Aniline 30, although
less potent than 35 in both assay systems, was also
selected for derivatization due to the facile nature of
the synthetic chemistry. Not only could an increase in
compound hydrophobicity potentially aid parasite
membrane permeability, but the potential of interacting
with other hydrophobic moieties (i.e., Val-222, Tyr-277,
and Met-281) within the PfENR active site held
promise.
Table 1 shows the results from biological testing of the
initial round of compounds (28–36), which targeted
replacement of the 40-chloro group with simple organic
functionality capable of hydrogen bonding with Asn-
218 and Ala-219. All compounds in this subset display
IC50 values <400 nM, except for molecules featuring a
relatively acidic proton: carboxylic acid 33, tetrazole
34, and hydroxamic acid 36. Sharing the common fea-
ture of a hydrogen bond acceptor, amide 35 and nitrile
31 are among the most potent of these derivatives
against the enzyme. Consistent with its enzymatic activ-
ity, 31 exhibits parasite activity equipotent to triclosan.
Inspection of the biological data in Table 2 for amide
derivatives 35, 37–42 does not afford a clear SAR trend.
With regard to enzyme inhibition, the parent amide 35 is
most potent, with the morpholino amide 42 being most
preferable amongst the tertiary amides tested. This trend
is not reflected in the parasite assay, where the benzyl-
amide 38 and piperidino amide 41 are the most
Table 1. Inhibitory properties of selected 40-substituted derivatives.
OH
Cl
O
Cl
R
PfENRa IC50 (nM)
a
Compound
R
EC50 3D7/Dd2 (lM)
Triclosan
Cl
OH
2.9 0.2/3.9 0.3
65.3 9.2/87.8 20.6
2.1 0.1/3.2 0.6
120 21/140 24
3.9 0.7/5.4 0.7
23.1 5.1/36.4 3.7
73 21
220 25
180 22
360 92
120 43
190 52
550 20
413 49
120 35
1200 240
28
29
30
31
32
33
34
35
36
NO2
NH2
CN
CH2CN
COOH
1H-tetrazol-5-yl
C(O)NH2
C(O)NHOH
110 27/130
120 10/>150
4
96.3 11.7/100 16
31.2 10.8/30.6 2.2
a Values reported as means standard error.
Table 2. Inhibitory properties of selected 40-amide derivatives.
OH
Cl
O
Cl
R
PfENRa IC50 (nM)
a
Compound
R
EC50 3D7/Dd2 (lM)
35
37
38
39
40
41
42
NH2
N(H)Me
96.3 11.7/100 16
77.8 25.3/100 22
7.5 2.8/20.6 3.6
68.4 13.2/95.7 11.6
20.7 6.0/42.6 12.8
11.7 2.2/18.0 3.0
63.5 14.1/94.5 10.0
120 35
310 66
N(H)Bn
NMe2
3000 800
460 110
810 300
680 120
200 10
N-Pyrrolidine
N-Piperidine
N-Morpholine
a Values reported as means standard error.

5250
J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5247–5252
efficacious. Clearly, gains in anti-parasitic activity can be
made by substituting for one or more of the parent
amideÕs N–H groups. Further rounds of compound
synthesis and assay evaluation are necessary to clarify
the structural requirements for attaining both enzyme
and parasite assay activity. It is interesting to note that
the substituted amides 37–42 are significantly more
potent against the 3D7 strain than the Dd2 strain.
lone pair may be engaged in a favorable interaction
with the main chain N–H group of Ala-219 (dN–N
=
˚
2.7 A). Compound 29 binds in a conformation similar
to 30. The only significant change in the enzyme ac-
tive site is the rotation of the Asn-218 sidechain to al-
low its carboxamide N–H to interact with one of the
˚
nitro oxygens (dN–O = 3.4 A). The same nitro oxygen
forms a hydrogen bond with the main chain N–H
˚
group of Ala-219 (dN–O = 3.2 A), while the other oxy-
gen makes no direct interactions with the enzyme.
The assay data for aniline derivatives 43–53 are shown
in Table 3. Acyl-based derivatives such as the acetyl 43
and benzoyl 44 analogs display modest improvements
in both assay systems. The sulfonamide analog of 44 is
equipotent against cultured parasites, while being less
active against purified enzyme. Alteration of the sulfon-
amide aryl group from phenyl (47) to 1- or 2-naphthyl
(48 and 49) offers slight improvements in parasite assay
performance while resulting in decreased enzyme
inhibition.
Methylamide derivative 37, shown in Figure 2, utilizes
its amide carbonyl to form a hydrogen bond with the
˚
sidechain N–H of Asn-218 (dN–O@C = 3.2 A) and the
carbonyl may also make a weak hydrogen bond with
Benzylamine derivative 51 exhibits greater potency than
aniline 30 against purified enzyme and cultured para-
sites. Introduction of an ortho-substituent in 52 and 53
affords more potent compounds than 51 in the parasite
assay, while not improving enzyme inhibition. Ortho-
phenol 53 is much more potent against the parasite
(EC50 = 4.5 (3D7) and 5.6 (Dd2) lM) than 30, while
not exhibiting enhanced enzyme inhibition.
To understand the molecular basis of these results, we
have solved the X-ray crystal structures of nitro 29, ani-
line 30, methylamide 37, and urea 46 bound to PfENR
in the presence of co-factor. As expected, the only signif-
icant difference in all of these structures is the interaction
of the 40-substituent with the surrounding enzyme
residues. Other interactions of the triclosan core with
co-factor and enzyme are preserved. Displayed in
Figure 1, 30 utilizes its aniline hydrogens to form
hydrogen bonds to the side-chain carbonyl of Asn-
Figure 1. Overlay of the X-ray structures of 29 (stick drawing with
carbons in yellow) and 30 (stick drawing with carbons in purple) in the
PfENR active site (ribbon and tube with key residues in stick format)
with bound co-factor (space-fill). One of the nitro oxygens of 29 is
hydrogen-bonded to the side-chain amide N–H of Asn-218 and the
backbone N–H of Ala-219. The 40-anilino hydrogens of 30 are
hydrogen-bonded to the side-chain carbonyl of Asn-218 and the main
chain carbonyl of Ala-219. The aniline lone pair may be participating
in a hydrogen bond with the main chain N–H of Ala-219.
˚
218 (dC@O–N = 3.4 A) and the main chain carbonyl
˚
of Ala-219 (dC@O–N = 3.7 A). The aniline nitrogenÕs
Table 3. Inhibitory properties of selected 40-aniline derivatives.
PfENRa IC50 (nM)
a
Compound
R
EC50 3D7/Dd2 (lM)
30
43
44
45
46
47
48
49
50
51
52
53
H
Ac
120 21/140 24
52.2 17.3/50.9 19.2
57.2 14.7/83.9 15.0
110 24/120 22
360 92
57 24
Bz
170 16
160 45
250 49
370 100
590 88
2500 900
140 34
140 69
560 120
320 59
C(O)NH2
C(O)N-Morpholine
SO2Ph
66.8 14.1/86.4 14.9
55.1 10.2/77.5 8.0
34.3 5.6/43.7 12.2
23.5 3.2/33.9 9.6
>120/>120
SO2(1-Naphthyl)
SO2(2-Naphthyl)
SO2CF3
CH2Ph
CH2(2-CNPh)
CH2(2-HOPh)
35.8 5.3/47.5 12.2
26.7 2.9/45.4 6.5
4.5 0.5/5.6 1.3
a Values reported as means standard error.

J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5247–5252
5251
or ureido may result in the loss of hydrophobic interac-
tions with Val-222 and Met-281. Subtle rearrangements
in the enzyme complex with co-factor and ligand may
also be responsible for the observed decreases in ligand
affinity.
In conclusion, a series of 40-substituted triclosan deriv-
atives have been prepared and assayed for inhibition
of purified PfENR and cultured P. falciparum. While
the effort did not result in compounds significantly
more potent than triclosan against both the enzyme
and the parasite, it did provide an understanding of
which groups could be substituted for the 40-chloro
to provide nanomolar inhibitors of PfENR with dem-
onstrated anti-parasitic activity. X-ray crystallography
studies demonstrate the ability of some of the pre-
pared compounds to expand upon triclosanÕs interac-
tions with PfENR via hydrogen-bonding networks
with Asn-218 and Ala-219. The examination of the
40-position will aid future efforts guided toward
improving the potency and pharmacokinetic profiles
of this diaryl ether class of anti-malarials. This will
be done in conjunction with probing other positions
along the diaryl ether scaffold.
Figure 2. X-ray structure of 37 (stick drawing) in the PfENR active
site (ribbon and tube with key residues in stick format) with bound co-
factor (space-fill). The 40-methylamide carbonyl group makes
a
hydrogen bond with the side-chain N–H of Asn-218 and it may also
be engaged in a weak hydrogen bond with the backbone N–H of Ala-
219. The amide methyl group is interacting with the Val-222 sidechain.
Acknowledgment
This work has been supported by funding from the
Medicines for Malaria Venture.
Supplementary data
Supplementary data associated with this article can be
found in the online version, at doi:10.1016/
j.bmcl.2005.08.044
Figure 3. X-ray structure of 46 (stick drawing) in the PfENR active
site (ribbon and tube with key residues in stick format) with bound co-
factor (space-fill). The 40-urea carbonyl group is hydrogen bonded with
the sidechain N–H of Asn-218 and the backbone N–H of Ala-219.
References and notes
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˚
4.0 A). Its N-methyl group appears to interact favorably
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˚
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FLUOstar OPTIMA multifunctional microplate reader
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using 50 nM PfENR 400 lM NADH and 40 lM NAD+
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