Synthesis and biological activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 2: 2′-Substituted triclosan derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.01.051

2′-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC ₅₀ < 500 nM) against

Full text of DOI:10.1016/j.bmcl.2006.01.051

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2163–2169
Synthesis and biological activity of diaryl ether inhibitors
of malarial enoyl acyl carrier protein reductase.
Part 2: 20-Substituted triclosan derivatives
Joel S. Freundlich,a,* Min Yu,b Edinson Lucumi,d Mack Kuo,d Han-Chun Tsai,d
Juan-Carlos Valderramos,b Luchezar Karagyozov,b William R. Jacobs, Jr.,b,c
Guy A. Schiehser,a David A. Fidock,b 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 26 October 2005; revised 13 January 2006; accepted 13 January 2006
Available online 8 February 2006
Abstract—20-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium fal-
ciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC50 < 500 nM) against
in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and modest (IC50 = 1–20 lM) potency
against purified PfENR enzyme. Compared to triclosan, this survey of 20-substituted derivatives has afforded gains in excess of
20- and 30-fold versus the 3D7 and Dd2 strains of parasite, respectively.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Treatment of Plasmodium falciparum malaria has
depended for decades on the use of the aminoquinoline
chloroquine or the antifolate combination pyrimeth-
amine-sulfadoxine. P. falciparum infection is the most
widespread and the deadliest form of malaria—a disease
that afflicts 300–600 million people worldwide resulting
in 1–3 millions deaths per annum.1 The occurrence
and spread of drug-resistance necessitates new, highly
potent antimalarials that are not encumbered by existing
resistance mechanisms and are orally biovailable, afford-
able, and safe.
ont.3 Given the prokaryotic nature of the apicoplast,
its metabolic machinery differs significantly from that
of mammalian cells.4 For example, higher eukaryotes
and yeast utilize fatty acid biosynthetic machinery resid-
ing on a single multifunctional polypeptide (FAS-I),
whereas fatty acid synthesis in Plasmodium is reliant
on a dissociative process that utilizes a set of distinct en-
zymes composing a FAS-II pathway. This observation
hints at selectivity with regard to the human host in a
therapeutic strategy.
The enoyl reductase enzyme (PfENR) is responsible for
the final step in fatty acid synthesis: NADH-dependent
reduction of trans-2-enoyl-ACP to acyl-ACP (ACP =
acyl carrier protein). Triclosan (Fig. 1) is a known
The inhibition of fatty acid synthesis in P. falciparum
has been a recent focus of drug discovery efforts to find
novel antimalarials.2 This synthetic pathway is believed
to be crucial to parasite survival as a result of its key role
in membrane construction and energy production. Syn-
thesis of fatty acids is regulated within the apicoplast—
an organelle derived from a cyanobacterial endosymbi-
Keywords: Antimalarial; Diaryl ether; Phenol.
*
Figure 1. Chemical structure of triclosan with demarcation of key
B-ring sites.
Corresponding author. Tel.: +1 6099217447; fax: +1 6097991176;
e-mail: J_Freundlich@jacobuspharm.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.051

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J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2163–2169
inhibitor of PfENR, as determined with native enzyme
purified from P. falciparum or recombinant enzyme ex-
pressed in Escherichia coli.5,6 Furthermore, triclosan is
potent in the low micromolar range against P. falcipa-
rum5,7–10 and is reported to effectively treat P. berghei
in vivo infections in mice.5 A direct relation between tri-
closan binding to and inhibition of PfENR in vitro and
its whole cell properties is supported by the demonstra-
tion that triclosan also inhibits fatty acid synthesis with
cultured parasites or parasite extracts.5 Given triclosan’s
abundance (U.S. annual production > 1 · 106 lbs),11 its
safety as demonstrated by its widespread use in personal
care and household products, and the absence of a lipid
synthesis inhibitor in the antimalarial arsenal, we and
others have chosen to pursue the discovery of a triclo-
san-derived therapeutic for malaria.6,8,10,12–16
able interactions with proximal enzyme residues such as
Ala-319 and Arg-318. Compared to triclosan, 20-analogs
with these features could have enhanced interactions
with both the bound co-factor and enzyme, thus confer-
ring greater binding affinity for the co-factor:enzyme
complex and potentially enhanced anti-parasitic activi-
ty. Amino derivatives off the 20-position could also have
improved physiochemical properties.
The synthesis of triclosan analogs began with diaryl
ether anisoles 1a–d, prepared from commercially avail-
able materials via nucleophilic aromatic substitution
(Scheme 1). Aniline 2 was synthesized via hydrogenation
of nitroarene 1a followed by boron(III) bromide-medi-
ated cleavage of the methyl ether. Benzonitriles 1c and
1d were converted to the 20-aminomethyl compounds 3
and 4 via reduction with lithium aluminum hydride
and then methyl ether cleavage. In all cases, final com-
This work recently culminated in the X-ray crystal struc-
ture of triclosan and NAD+ co-factor bound to
PfENR.8 The structure demonstrated the critical bind-
ing element of triclosan to be the phenol moiety, while
suggesting vectors off the triclosan A and B rings where
additional functionality could be placed to enhance
binding affinity. We have pursued a strategy to build
off these aromatic ring positions to enhance enzymatic
and anti-parasitic activity. A significant gain in potency
could facilitate replacement or removal of the phenol,
due to its metabolic liability.17 Previously, we reported
efforts to explore SAR at the 40-position which resulted
in minor improvements in enzyme and anti-parasite
potency.16 This report details our further exploration
of the triclosan B-ring, focusing specifically on the 20-
position.
1
pounds were characterized by H NMR and LC–MS.
As shown in Scheme 2, reductive amination, utilizing
solid-phase reagents/scavengers18 and various alde-
hydes, allowed the functionalization of aminomethyl
derivatives 3 and 4 to provide 5–10, 11, and 14–18.
Interestingly, the reductive aminations proceeded more
cleanly (i.e., less dialkylation) with the corresponding
phenol than with the anisole (free base or hydrochloride
salt). Compound 4 was also coupled with benzoic acid
to afford amide 12 and sulfonylated with benzenesulfo-
nyl chloride to yield sulfonamide 13.
Anisole 1c could be converted to benzylic amine 19 via
nitrile reduction with lithium aluminum hydride fol-
lowed by reductive amination using the protocol of
Bhattacharyya.19
Examination of the triclosan:co-factor:PfENR structure
showed the 20-chloro is pointed toward the pyrophos-
+
phate portion of NAD and is approximately 4 A from
˚
Analogs of 14 were prepared as shown in Scheme 3.
Benzonitrile 1d was transformed to a,a-dimethylamino
derivative 20 via a three-step sequence, featuring reduc-
two of the negatively charged oxygens (Fig. 2). We
hypothesized that a properly placed positively charged
amine off the 20-position could favorably interact with
one or both of these oxygens. In addition, a hydropho-
bic substituent on the amine could participate in favor-
Figure 2. X-ray crystal structure of triclosan (stick drawing) bound to
PfENR (ribbon and tube with key residues in stick format) in the
presence of NAD+ (space-fill). The 20-chloro is shown pointing
towards Ala-319 and Arg-318.
Scheme 1. Reagents and conditions: (a) K2CO3, DMSO, D; (b) Ra-Ni,
H2, EtOH/EtOAc; (c) BBr3, DCM, 0 ꢁC to rt; (d) LiAlH4, THF.
Ar = 4-chloro-2-methoxyphenyl.

J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2163–2169
2165
Scheme 2. Reagents and conditions: (a) i—MP-Triacetoxyborohydride, RCHO, THF; ii—PS-PhCHO; iii—MP-TsOH; (b) PhCO2H, ACTU,
PS-HOBt, DIEA, DMF; (c) PhSO2Cl, pyridine, DCM/THF; (d) LiAlH4, THF; (e) i—Ti(O-i-Pr)4, p-ClC6H4CHO, THF/EtOH; ii—NaBH4, EtOH.
Scheme 3. Reagents and conditions: (a) CeCl3, MeLi, THF, ꢀ78 ꢁC to rt; (b) BBr3, DCM, 0 ꢁC to rt; (c) i—Ti(O-i-Pr)4, p-ClC6H4CHO, THF/EtOH;
ii—NaBH4, EtOH; (d) Ti(O-i-Pr)4, NaBH4, (HCHO)n, EtOH; (e) Ac2O, DCM. Ar = 4-chloro-2-methoxyphenyl.
tive alkylation of the nitrile using an excess of in situ
prepared methylorganocerium reagent.20 N-methylation
of 14 and 20 was achieved via a Ti(O-i-Pr)4/NaBH4 med-
iated reaction with paraformaldehyde to provide 21 and
22, respectively.21 N-acetylation, however, was only
attainable with 16 to afford acetamide 23.
in duplicate. Two parasite strains were utilized: 3D7,
which is drug-sensitive, and Dd2, which is resistant
to chloroquine and pyrimethamine-sulfadoxine. Both
systems were previously described in detail.8,16 To date,
fourteen independent determinations of the IC50 of tri-
closan have been made in the biochemical screen with
purified PfENR, resulting in a mean of 73 nM with a
standard error of 21 nM. For the parasite whole-cell
assay, 46 independent runs of triclosan have been con-
ducted. Against the 3D7 strain, the EC50 was deter-
mined 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 for triclosan was 3.8 lM with a stan-
dard error of 0.2 lM.
Reductive aminations to afford tertiary amines were
initiated from benzaldehyde 1b. In contrast to reduc-
tive aminations with aminomethyl analogs 3 and 4,
1b and various amines were observed to react most
smoothly with regard to a reductive amination path-
way in the presence of two equivalents of sodium tri-
acetoxyborohydride in 1,2-dichloroethane (Eq. 1).22
This synthetic protocol, followed by boron(III) bro-
mide-promoted methyl ether cleavage, afforded tertiary
amines 24–30.
Final compounds 2–30 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
account three independent determinations performed
ð1Þ

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J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2163–2169
Table 1. Inhibitory properties of selected 20-substituted derivatives
derivatives are less active than 11 versus the enzyme and
parasite. Whereas utilization of polar moieties was not
tolerated, proper placement of hydrophobic functional-
ity off the phenyl ring of 11 generally afforded more po-
tent anti-parasitic compounds while leading to reduced
enzyme inhibition. For example, the 2-naphthyl deriva-
tive 17 exhibits EC50 values of 210 and 140 nM against
the two parasite strains, while the 5-(2,3-dihydrobenzof-
uranyl) analog 18 displays EC50 values of 180 and
110 nM against 3D7 and Dd2. Compound 18 exhibits
the most potent activity against Dd2 in the 20-substitut-
ed class.
Compound R1
R2 EC50a3D7/Dd2
PfENRa IC50
(lM)
(lM)
Triclosan
Cl
NH2
Cl 2.9 0.2/3.8 0.2 0.073 0.021
2
3
H
H
83 15/140 20
9.7 1.5/7.2 0.8 >50
7
2
CH2NH2
a Values reported as means standard error.
Analogs of 6 and 14, containing a p-chlorobenzyl-20-
aminomethyl substituent, were investigated further and
the biological data are shown in Table 4. The impor-
tance of the phenol is underscored by the observation
that anisole 19 is much less potent than phenol 6 against
both the enzyme and parasite. Comparison of the data
for 6 and 14 shows that alteration of the 40-substituent
from hydrogen to chloride confers slightly more potency
against PfENR, while not significantly affecting anti-
parasitic activity. Interestingly, a,a-dimethylation of
the 20-benzylic carbon (cf. 20) slightly reduces the para-
site potency, while having no effect in the enzyme assay.
N-methylation (cf. 21) leads to a slight gain in the para-
site assay and a loss in enzyme inhibition. The combina-
tion of these two structural alterations in 22 leads to
increased anti-parasitic potency against the 3D7 strain
but not the Dd2 strain, with a slight loss in enzyme
activity. 22 exhibits the most potent activity in the 20-
substituted chemotype against the 3D7 strain of parasite
(EC50 = 140 nM). N-acetylation to afford 23 produces
losses in efficacy in both assay systems.
The first set of 20-derivatives prepared is shown in Table
1 where the 40-position was chosen to be hydrogen.
Although aminomethyl 3 is much less potent than ani-
line 2 against the purified enzyme, it is significantly more
efficacious against the cultured parasites.
In keeping with our design hypothesis, we examined the
effect of placing hydrophobic groups off the amine ter-
minus of 3 in order to improve its enzyme and anti-par-
asitic potencies over those of triclosan. As shown in
Table 2, compounds 5–10 achieve approximately an
order of magnitude increase in potency over 3 in the par-
asite assay. The 1-naphthyl 9, phenylpropyl 8, and p-Cl-
benzyl 6 derivatives are the most potent members of this
series against the parasite. In general, the compounds
also display an improvement in enzyme activity over 3,
although their micromolar level IC50 values against
PfENR appear incongruent with their nanomolar level
anti-parasitic potencies. It is interesting to note that of
the 20-substituted derivatives prepared to date, pheneth-
yl analog 7 is the most potent against purified enzyme
(IC50 = 2.5 lM).
In light of the enhanced anti-parasitic activity of 21 due
to its N-methylation, a series of 20-N-methylamines was
synthesized and evaluated in both screening assays (Ta-
ble 5). Mono- and dimethylamino analogs 24 and 25
show enhanced activity compared to that of parent ami-
no 4 in both assays. The two N-methyl substituents were
next constrained in a ring system. Cyclic analogs (26–27)
display slight losses in parasite activity and are inactive
against PfENR. The N4-substituent of piperazines 28–
30 modulates potency against both cultured parasites
Subsequently, triclosan derivatives with a 40-chloro were
prepared in an effort to improve enzyme and anti-para-
sitic potency while adhering more strictly to the triclosan
scaffold (Table 3). While 4 is observed to be more potent
than 3 against cultured parasites, it fails to offer an ob-
servable improvement in the enzyme assay. Surprisingly,
introduction of the 40-chloro group does not consistent-
ly improve the performance of these analogs in both as-
say systems. Phenylamide 12 and phenylsulfonamide 13
Table 2. Effect of secondary amine substituent with 40-hydrogen
a
a
Compound
R
EC50 3D7/Dd2 (lM)
PfENR IC50 (lM)
3
H
CH2Ph
9.7 1.5/7.2 0.8
>50
5
0.49 0.02/0.42 0.03
0.33 0.04/0.30 0.04
0.78 0.05/0.75 0.07
0.32 0.08/0.39 0.08
0.30 0.02/0.37 0.07
0.45 0.05/0.33 0.09
10
35
3
4
6
CH2(p-ClC6H4)
(CH2)2Ph
(CH2)3Ph
1-Naphthyl
2-Naphthyl
7
2.5 0.9
8
19
11
5
4
9
10
46 14
a Values reported as means standard error.

J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2163–2169
Table 3. Effect of secondary amine substituent with 40-chloride
2167
PfENRa IC50 (lM)
a
Compound
R
EC50 3D7/Dd2 (lM)
4
H
CH2Ph
2.7 0.1/2.0 0.5
0.77 0.05/0.83 0.01
>50
3.3 1.5
11
12
13
14
15
16
17
C(O)Ph
SO2Ph
8.6 1.8/12
11 1/17
1
18
15
8
7
1
CH2(p-ClC6H4)
CH2(p-PhC6H4)
1-Naphthyl
2-Naphthyl
O
0.32 0.08/0.27 0.10
0.23 0.06/0.15 0.04
2.6 0.1/2.2 0.1
7.2 1.4
35 15
>50
0.21 0.06/0.14 0.03
26
7
18
0.18 0.03/0.11 0.01
9
3
a Values reported as means standard error.
Table 4. Inhibitory properties of p-Cl-benzylamine derivatives
PfENRa IC50 (lM)
a
Compound
Chemical structure
EC50 3D7/Dd2 (lM)
Cl
Cl
Cl
H
N
OH
6
0.33 0.04/0.30 0.04
35
4
O
Cl
H
N
O
19
14
19 2/24
1
>50
O
Cl
H
N
OH
0.32 0.08/0.27 0.10
7.2 1.4
O
Cl
Cl
Cl
Cl
Cl
Cl
H
N
OH
20
21
22
0.41 0.03/0.35 0.02
7.2 1.6
O
Cl
N
OH
0.25 0.06/0.19 0.03
22
27
2
4
O
Cl
Cl
N
OH
0.14 0.01/ 0.38 0.02
O
Cl
Cl
O
Cl
N
OH
23
3.5 0.2/5.9 0.1
28
3
O
Cl
Cl
a Values reported as means standard error.

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J. S. Freundlich et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2163–2169
Table 5. Inhibitory properties of selected amines
NR1R2
EC50 3D7/Dd2 (lM)
PfENRa IC50 (lM)
a
Compound
24
25
N(H)Me
NMe2
0.58 0.09/0.47 0.13
0.18 0.01/0.22 0.01
27
19
9
5
26
27
28
0.50 0.03/0.45 0.03
>50
N
N
0.73 0.21/0.48 0.26
>50
Ph
Ph
N
14 2/15
3
6.1 1.0
N
N
N
N
Ph
Ph
29
30
0.63 0.05/0.62 0.03
2.1 0.1/1.4 0.1
11
2
>50
N
a Values reported as mean standard error.
and purified enzyme. Compared to piperidine 27, signif-
icant gains in enzymatic potency against the enzyme are
realized with 28 (IC50 = 6.1 lM). The corresponding
anti-parasitic activities of these piperazines do not im-
prove significantly upon that of 27.
These include studies on whether PfENR is the primary
target (indeed, it may very well not be the intracellular
target for these compounds) and parallel genetic investi-
gations to assess whether this target is essential. This will
be done in conjunction with probing other positions
along the diaryl ether scaffold of triclosan.
In conclusion, a series of 20-substituted triclosan
derivatives has been prepared and assayed for inhibi-
tion of PfENR and intracellular parasite growth. The
effort provided a number of compounds that are sig-
nificantly more potent than triclosan against the cul-
tured parasites. 5-(2,3-dihydrobenzofuranyl) analog
18 was 16 and 34 times more active than triclosan
against the 3D7 and Dd2 strains, respectively. Meth-
ylated analog 22 displayed anti-parasitic efficacy 21
and 10 times greater than triclosan against 3D7 and
Dd2, respectively. Studies are now required to assess
whether these compounds are cytotoxic or cytostatic.
Preliminary indications that this compound class is
cytotoxic come from the study of Waller et al.,9
who exposed synchronized parasites at different stages
to triclosan for 16 h periods. With ring stage para-
sites, susceptibility to triclosan was similar to that ob-
served when parasites were exposed for the full 48 h
of intracellular development, suggesting that the drug
action was toxic and not static. Nevertheless, further
experiments are required to rigorously address this,
including ‘washout’ experiments and these will be per-
formed with the more potent compounds from the tri-
closan series.
Acknowledgment
This work has been supported by funding from the
Medicines for Malaria Venture.
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