Design, synthesis, and application of novel triclosan prodrugs as potential antimalarial and antibacterial agents

Article abstract of DOI:10.1016/j.bmc.2008.04.006

A number of new triclosan-conjugated analogs bearing biodegradable ester linkage have been synthesized, characterized and evaluated for their antimalarial and antibacterial activities. Many of these compounds exhibit good inhibition against Plasmodium falciparum and Escherichia coli. Among them tertiary amine containing triclosan-conjugated prodrug (5) inhibited both P. falciparum (IC₅₀; 0.62 μM) and E. coli (IC₅₀; 0.26 μM) at lower concentrations as compared to triclosan. Owing to the presence of a cleavable ester moiety, these new prodrugs are hydrolyzed under physiological conditions and parent molecule, triclosan, is released. Further, introduction of tertiary/quaternary functionality increases their cellular uptake. These properties impart them with higher potency to their antimalarial as well as antibacterial activities. The best compound among them 5 shows close to four-fold enhanced activities against P. falciparum and E. coli cultures as compared to triclosan.

Full text of DOI:10.1016/j.bmc.2008.04.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5536–5546
Design, synthesis, and application of novel triclosan prodrugs
as potential antimalarial and antibacterial agents
Satyendra Mishra,a Krishanpal Karmodiya,b Prasanna Parasuraman,a
b,
Avadhesha Suroliaa,c, and Namita Surolia
*
*
aMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
bMolecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, India
cNational Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
Received 14 February 2008; accepted 4 April 2008
Available online 9 April 2008
Abstract—A number of new triclosan-conjugated analogs bearing biodegradable ester linkage have been synthesized, characterized
and evaluated for their antimalarial and antibacterial activities. Many of these compounds exhibit good inhibition against Plasmo-
dium falciparum and Escherichia coli. Among them tertiary amine containing triclosan-conjugated prodrug (5) inhibited both P. fal-
ciparum (IC50; 0.62 lM) and E. coli (IC50; 0.26 lM) at lower concentrations as compared to triclosan. Owing to the presence of a
cleavable ester moiety, these new prodrugs are hydrolyzed under physiological conditions and parent molecule, triclosan, is released.
Further, introduction of tertiary/quaternary functionality increases their cellular uptake. These properties impart them with higher
potency to their antimalarial as well as antibacterial activities. The best compound among them 5 shows close to four-fold enhanced
activities against P. falciparum and E. coli cultures as compared to triclosan.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
for drug development against malaria. Type II fatty acid
synthesis is brought about by FAS-II(fatty acid synthase
II), which is structurally different from FAS-I(fatty acid
synthase I) found in the human host as well as other
higher eukaryotes and yeast. While FAS-II has discrete
enzymes catalyzing individual reactions of the pathway,
FAS-I is a homodimer of a multifunctional polypeptide
with various domains catalyzing all the reactions of the
biosynthetic pathway.6 In P. falciparum, type II fatty
acid synthesis has been localized in the relict plastid
called apicoplast which is evolutionarily related to cya-
nobacteria.7 The striking difference in the organization
of the enzymes catalyzing fatty acid synthesis in P. falci-
parum and E. coli from that operating in the human host
makes it a potent drug target not only for treating ma-
laria but also for many bacterial infections. The iterative
cycle of fatty acid biosynthesis consists of four steps,
decarboxylative condensation, NADPH-dependent
reduction, dehydration, and NADH-dependent reduc-
tion.8 The NADH-dependent reduction step is carried
out by enoyl-acyl carrier protein (ACP) reductase
(ENR) which reduces the trans-2 enoyl bond of enoyl-
ACP substrates to saturated acyl-ACPs and plays a
deterministic role in completing the fatty acid elongation
cycles.9 ENR has been validated as a potential antima-
larial5,10 and antibacterial11 drug target by us and oth-
Malaria is a global health problem that threatens
300–500 million people and kills more than one million
people annually.1 Disease control is hampered by the
occurrence of multi-drug-resistant strains of the malaria
parasite Plasmodium falciparum.2,3 P. falciparum infec-
tion is the most widespread form of malaria and is the
predominant cause of severe disease and death. Tradi-
tional treatments with drugs, such as chloroquine and
sulfadoxine-pyrimethamine, are now much less effective
due to rampant resistance,4 hence harnessing drug tar-
gets unique to the malaria parasite such as the type II
fatty acid biosynthesis pathway5 opens new avenues
Abbreviations: DIPEA, N,N-diisopropylethylamine; DMAE, dimeth-
ylaminoethanol; CDI, 1,10-carbonyldiimidazole; MDMAEG, mono-
(dimethylaminoethanyl)glutaryl; P. falciparum, Plasmodium falcipa-
rum; E. coli, Escherichia Coli.
Keywords: Prodrug; Triclosan; Antimalarial; Antibacterial; Plasmo-
dium falciparum; Escherichia Coli.
*
Corresponding authors. Address: National Institute of Immunology,
Aruna Asaf Ali Marg, New Delhi 110067, India. Tel.: +91 11
26717102; fax: +91 11 26717104 (A.S.); tel.: +91 80 22082821; fax:
+91 80 22082766 (N.S.); e-mail addresses: surolia@nii.res.in; surolia
@jncasr.ac.in
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.006

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
5537
Figure 1. Inhibition of the growth of the parasites in red blood cell cultures. Synchronized parasites (5% parasitemia) were cultured in 96-well plates
at 2–3% hematocrit and at an initial parasitemia of 1–2%, with different concentrations of the inhibitors in DMSO (final concentration, 0.05%).
Parasite growth inhibition was assessed by microscopy of Giemsa-stained smears at 48 and 96 h. Inhibitions of the growth of parasites with
compound 5 (Fig. 1b) have been shown as representative example.
ers. We have extensively studied P. falciparum ENR
(PfENR) using biochemical as well as structural tools
and also worked out the mechanism of its inhibition
by triclosan.12–16 Triclosan was found to be effective in
killing P. falciparum in vitro and curing mice infected
with the rodent malaria species P. berghei as well as sys-
temic acute bacterial infection.5,17 Structure–activity
relationship (SAR)-based novel di-phenyl ethers have
been evaluated for inhibition of PfENR and EcENR
by us and others.18–20 Triclosan has been traditionally
used in consumer products such as toothpastes, mouth-
washes, deodorants, soaps, and lotions and it was be-
lieved earlier to act by non-specific disruption of the
bacterial cell wall. Now, with ENR having been identi-
fied as its target, triclosan provides us a promising scaf-
fold, around which we can design newer compounds
that can be tested for their possible therapeutic value
against malaria and bacterial infections.
across biological membranes. We focus on ester deriva-
tives, because the ubiquitous presence of enzymes with
esterase activity22–24 ensures the rapid regeneration of
the parent molecules ‘triclosan’. Bioavailability and/or
stability-improving ester prodrugs have been developed
for many drugs with alcoholic or phenolic hydrox-
yls.25,26 A releasable triclosan octaarginine derivatives
(prodrug) were recently shown to be effective in inhibit-
ing T. gondii tachyzoites with potency equivalent to that
of triclosan while its non-hydrolyzable derivatives were
devoid of activity.27
In the present paper, a series of prodrug esters were de-
signed and synthesized in order to improve the cell pene-
tration properties of the otherwise negatively charged
agents. These prodrugs contain triclosan conjugated with
appropriateacids, mono-(dimethylaminoethyl) glutarate,
succinamic acid, levulinicacid, and glutaric anhydride via
an enzyme-sensitive ester bond. Conjugated triclosan was
expected to target the parasite where the labile bond is
hydrolyzed so that the respective acids are released as
antimalarial and antibacterial principles at the site of ac-
tion. The literature survey suggests that triclosan ester-
based conjugates have so far not been evaluated for their
antimalarial and antimicrobial activities. Additionally,
incorporation of a weak base in the prodrug of triclosan
was expected to increase its uptake by the malaria parasite
and bacteria improving its potency (Fig. 1).
One approach towards improving the utility of such
drugs, is to develop prodrug analogs capable of selective
release of the parent triclosan. It is known that prodrug
design comprises approaches that help in enhancing the
efficacy and reducing the toxicity and unwanted effects
of drugs by controlling their absorption, metabolism,
and distribution. A prodrug is a derivative, which
undergoes two independent reactions in order to regen-
erate the parent drug. In case of the prodrugs, the inter-
mediate prodrug must be a chemically reactive entity,
which rapidly undergoes a chemical conversion to re-
lease the parent drug under physiological conditions.
However, this reactive molecule (viz. drug) is generated
only, subsequent to an enzyme-catalyzed reaction on an
otherwise chemically stable prodrug.21 However, pro-
drug approach applied to triclosan (an antimalarial
and antibacterial agent) indicates that a single chemical
modification (as required in prodrugs) is sufficient to
achieve the desired alteration in the biological properties
to enhance the activity. In this paper, we report the char-
acterization of a promising class of compounds designed
to facilitate the transport of hydrophobic molecules
2. Chemistry
2.1. Synthesis
Dimethylaminoethanol (DMAE) 2 reacts with an anhy-
dride (glutaric 1) to form a hemi-ester transferring its
free carboxylic acid group to an imidazolide for reaction
with the phenolic group of triclosan (Scheme 1). Mono-
(dimethylaminoethanyl)glutaryl ester (MDMAGE; 3)
thus obtained is a hybrid of the DMAE and DMG (di-
methyl glycine). Mono-(dimethylaminoethanyl)glutaryl

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S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
O
O
i
N
N
+
O
OH
HO
O
O
O
3
2
1
ii
O
O
O
O
N
Cl
O
O
N
Cl
O
O
O
iii
O
X
Cl
Cl
X = I
Cl
Cl
Sephadex
5
6
X = Cl
Scheme 1. Preparation of mono-(dimethylaminoethyl) glutarate (3), dimethylaminoethyl-glutaryl esters of triclosan (5) and trimethylaminoethyl-
glutaryl esters of triclosan (6). Reagent and conditions: (i) CH2Cl2, DIPEA;(ii) a—CDI, DMF;b—triclosan (4), DCM, 4h; (iii) methyl iodide, THF,
overnight.
imidazolide intermediate required for this purpose was
prepared by the reaction of mono-(dimethylaminoetha-
nol)glutaryl with CDI in dichloromethane. The corre-
sponding quaternary ammonium salt of triclosan (6)
was easily obtained by reaction of the tertiary amine
(5) with methyl iodide at room temperature in dry
THF. Subsequent ion exchange yielded the desired
quaternary ammonium (6) as the more stable chloride
salts.
derivative was made with levulinic acid which was ini-
tially converted to the corresponding acid chloride using
oxalyl chloride. This acid chloride was then coupled
with triclosan in the presence of base DIPEA to give
the levulinic acid ester of triclosan (9) (Scheme 2). In a
similar way ester of valeric acid (pentanoic acid) with tri-
closan (10) was synthesized. Acid chlorides of respective
acid, levulinic acid/valeric acid, were prepared by treat-
ment of oxalyl chloride in DCM as solvent.
Succinamic ester of triclosan (7) was synthesized by acti-
vating succinamic acid with CDI under N2 and the acti-
vated imidazolide derivative of succinamic acid was
coupled with triclosan. Triclosan glutaryl hemi ester
(8) was synthesized by derivatization of a glutaric anhy-
dride moiety to triclosan (4) in the presence of DIPEA.
Esterification of triclosan with succinic anhydride was
also attempted, but it was unsuccessful. Hence, a mini-
mum length of five methylene units is apparently re-
quired for the esterification of triclosan. Another
The nicotinoyl ester of triclosan (12) was synthesized by
treatment of nicotinoyl chloride hydrochloride (11) with
1 equivalent of triclosan (4) and 2.1 equivalent of trieth-
ylamine as base, to yield the pyridine esters in moderate
to good yields (Scheme 2). Quaternization of the pyrid-
inium nitrogen atom with afive-fold excess of methyl io-
dide in acetone afforded the methylated product in
quantitative yield. Resultant ion exchange yielded the
desired quaternary ammonium (13) as the more stable
chloride salts.
O
O
NH2
Cl
O
Cl
O
O
O
O
O
Cl
Cl
Cl
Cl
Cl
9
7
i
O
O
O
Cl
OH
Cl
O
OH
O
O
O
iv
O
ii
Cl
Cl
Cl
O
Cl
4
Cl
Cl
10
8
O
O
v
O
O
Cl
O
Cl
O
O
X = I
X = Cl
O
N
N
vi
Sephadex
X
Cl
Cl
Cl
Cl
13
12
Scheme 2. Reagents and conditions: (i) succinamic acid, CDI, DMF, N2; (ii) DCM, DIPEA; (iii) a—levulinic acid, (COCl)2, cat. DMF, CH2Cl2, rt;
b—triclosan; DIPEA, CH2Cl2; (iv) a—pentatonic acid, (COCl)2, CH2Cl2, rt; (b) triclosan; DIPEA, CH2Cl2; (v) nicotinoyl chloride hydrochloride,
Et3N, toluene, reflux; (vi) CH3I/acetone.

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
5539
Due to transesterification of the ester products with the
methanol eluent it was not possible to exchange the io-
dide for the chloride counter ion under the conditions
routinely applied (Dowex 1 · 8, 200–400 mesh). By the
use of the very mild ion exchange with Sephadex (DEAE
A25, chloride form), however, transesterification was
prevented despite the use of methanol as an eluent.
Moreover, it also led to a quantitative ion exchange.
IC50 reported represents the mean of at least three sets.
Hence, while triclosan inhibited E. coli cultures with an
IC50 of 0.75 lM, compounds 5–8 inhibited the cultures
with IC50 values of 0.26, 0.42, 0.22, and 0.33 lM, respec-
tively (Table 1). MIC (minimum inhibitory concentra-
tion) values of the compound (5– 8) were found to be
0.55, 1.0, 0.6, and 0.75 lM, respectively. Nicotinoyl
derivative of triclosan(compound 12; pyridine derivative
of triclosan), which is aromatic tertiary amine, inhibited
growth of E. coli with IC50 of 3.1 lM (MIC = 9.0 lM)
while pyridinium salt exhibits IC50 of 3.6 lM and MIC
of 10.2 lM. From the above results it is clear that ter-
tiary amine of aliphatic (5) is more active than an aro-
matic derivative (13).
3. Results
3.1. Effect of various triclosan esters on E. coli culture
All the synthesized prodrugs were checked on E. coli cul-
tures to determine the IC50 values of their inhibitory
abilities. Each test was performed in triplicate and the
The activity data (Table 1) suggest that compounds with
tertiary amine ester of triclosan 5 (–N(CH3)2) group and
Table 1. IC50 and minimum inhibitory concentration (MIC) values of triclosan and its derivatives on cultures of Escherichia coli and Plasmodium
falciparum
Compound ID
Structure
E. coli*
E. coli*
P. falciparum*
P. falciparum*
IC50 (lM)
MIC (lM)
IC50 (lM)
MIC (lM)
Cl
OH
O
4
0.75 0.1
1.0 0.1
2.29 0.3
0.62 0.1
17.6 2.1
7.9 0.7
Cl
Cl
Cl
Cl
O
O
O
N
Cl
O
O
5
0.26 0.05
0.55 0.07
O
O
N
Cl
Cl
Cl
Cl
O
O
6
7
8
9
0.42 0.07
0.22 0.03
0.33 0.02
77 12.3
1.0 0.1
0.6 0.12
0.75 0.2
nd
3.02 0.5
6.70 0.9
0.52 0.07
0.68 0.1
1 9.7 1.6
27.9 3.4
4.8 0.3
6.2 0.6
O
Cl
Cl
Cl
Cl
O
NH
2
O
O
O
Cl
O
O
O
O
OH
O
Cl
Cl
Cl
O
O
O
O
O
O
O
O
Cl
Cl
Cl
Cl
Cl
O
O
O
10
12
13
140 21
3.1 0.9
3.6 1.2
nd
8.90 1.1
1.15 0.3
2.07 0.3
33.8 4.7
9.7 1.1
13.7 1.3
Cl
Cl
Cl
9.0 2.3
N
Cl
O
10.2 3.5
N
Cl
Cl
*
Data are expressed as means SD from at least three different experiments in duplicate. nd, not determine.

5540
S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
succinamic acid ester of triclosan 7 (–CO–NH2) are
more active than the compound with quaternary ammo-
nium salt (6). This is because under assay conditions ter-
tiary amine derivatives of triclosan (5) and succinamic
ester of triclosan (7) do not undergo protonation and
this protecting group is hydrophilic.
potent as triclosan, perhaps due to its weak base
properties.
3.3. Stability
Stabilities of the prodrugs were measured by FPLC (UV
detection) in a Luria Broth at 37 ꢁC (pH 7.2). All the
prodrugs were found to be stable during 24 h of storage
between ꢀ10 to 40 ꢁC as no triclosan was liberated from
them under these conditions.
3.2. Effect of various ester of triclosan on P. falciparum
cultures
With P. falciparum cultures, compound 5 (tertiary amine
derivative of triclosan) showed IC50 of 0.62 lM and
MIC of 7.9 lM (Fig. 2B), which are more than 3.9 times
better than that of triclosan. Compound 6 (quaternary
ammonium salt of triclosan) showed IC50 of 3.02 lM
(Table 1). Similar pattern of outcome was obtained with
aromatic tertiary amine (nicotinoyl ester of triclosan 12)
and quaternary ammonium salt (compound 13). Com-
pound 7, succinamic acid derivative of triclosan, inhibits
P. falciparum growth at higher concentrations
(IC50 = 6.7 lM; MIC = 27.9 lM), whereas glutaryl ester
of triclosan (8) and levulinic ester of triclosan (9) inhib-
ited malaria parasite at lower concentrations as com-
pared to compound 7 as well as the parent molecule
(Table 1). Pentanoic acid-derived triclosan ester (pro-
drug) was active only at high micromolar concentrations
(Table 1). Compounds 12 and 13 showed MIC values of
9.7 and 13.7 lM, respectively. Compound 12 is twice as
3.4. FPLC assay of prodrug metabolism by E. coli and P.
falciparum cultures
The conversion of the pro-drug to parent molecule tri-
closan (drug) was checked by the protocol developed
in our laboratory,17 in which solid-phase extraction pro-
cedure was coupled with a fast performance liquid chro-
matography (FPLC) method to determine the level of
drug in the E. coli extract. The enzymatic hydrolyses
of prodrugs, lead to the appearance of only two peaks
characterized as triclosan and the given prodrug (Figs.
2A and 3A). The peak corresponding to the prodrug de-
creased until its complete disappearance while the par-
ent molecule (triclosan) reached
a plateau. The
retention time of the prodrug (compound 5 shown as a
representative example) was 31.9 min whereas for
triclosan it was 37.9 min. With P. falciparum cultures
A 150
125
Pro-drug
Drug
100
75
50
25
0
25
30
35
40
45
50
Retention Time (min)
B
C
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
10 20 30 40
Time (min)
50 60
0
5
10
15
20
Time (min)
Figure 2. (A) FPLC detection of conversion of pro-drug (5) to drug in E. coli extract with a C18 reverse-phase column by monitoring the absorbance
at 230 nm. Drug levels were detected at 0 (pink line), 2 min (black line), 5 min (red line), 10 min (green line), and 20 min (blue line) min after
treatment. (B) Enzymatic cleavage of prodrug. (C) Disappearance of the pro-drug 5 in E. coli culture.

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
5541
A
150
125
100
75
Drug
Pro-drug
50
25
0
25
30
35
40
45
50
Retention Time (min)
B
C
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
10
20
30
40
0
10
20
30
40
Time (h)
Time (h)
Figure 3. (A) FPLC detection of conversion of pro-drug (5) to drug in P. falciparum extract with a C18 reverse-phase column by monitoring the
absorbance at 230 nm. Drug levels were shown at 3 h (blue line), 12 h (red line), 18 h (green line), and 24 h (pink line) after treatment. (B) Enzymatic
cleavage of prodrug. (C) Disappearance of the pro-drug 5 in P. falciparum.
the half-life of prodrug disappearance for compound 5
was 9.45 h (Fig. 3C). For compounds 8 and 9 the half-
lives were ꢁ9.50 and 9.53 h, respectively (data not
shown). However, the half-life of prodrug disappearance
for compound 5 with E. coli cultures was approximately
ꢁ6 min (Fig. 2C). For compounds 6, 7, and 8 half-lives
were approximately 8, 12, and 9 min, respectively. Thus
a conversion of prodrug to parent molecule, triclosan,
by the esterases present in P. falciparum and E. coli cul-
tures is evident (Figs. 2A and 3A).
In order to achieve these objectives triclosan was cova-
lently linked to various ligands viz. succinamic acid, lev-
ulinic acid, nicotinic acid etc.
Compound 5 (dimethylaminoethyl-glutaryl esters of tri-
closan), which is a tertiary amine ester of triclosan,
exhibits the best growth inhibitory activity with an
IC50 of 0.26 lM, whereas the quaternary ammonium
salt derivative 6 (trimethylaminoethyl-glutaryl esters of
triclosan) shows activity at 0.42 lM. Thus the tertiary
amine (5) as well as the quaternary ammonium salt of
triclosan (6) showed more activity as compared to triclo-
san against E. coli.
4. Discussion
The goal of the work is development of permeability-
enhancing prodrug species for triclosan. Triclosan mol-
ecule has one active phenolic functional group, which is
ideal for modification. In the present work, the phenolic
group has been used to make prodrug candidates. The
main objective for preparing this prodrug was
For the inhibition of the growth of E. coli cultures suc-
cinamic ester of triclosan (compound 7) exhibited IC50
and MIC of 0.22 and 0.6 lM, respectively. It was noted
that compound 7 is 3.5 times more active than triclosan.
This may be due to the presence of a polar (–CO–NH2)
group at the end of succinamic ester of triclosan.
Replacing NH2, which is present at one of the termini
of succinamic acid of triclosan 7, with methyl group
(–CH3) or a non-polar polar group (levulinic acid, com-
pound 9) results in decrease in the activity. However,
when NH2 group of compound (7) is replaced by hydro-
xyl group (–OH; glutaryl ester of triclosan 8), the po-
tency exhibited is twice that of triclosan. This indicates
(i) to ease the transmembrane passage of triclosan, in or-
der to build its significant intracellular concentration,
(ii) to enhance the hydrophilicity of the molecule, and
(iii) to have a biodegradable linkage in the inhibitors,
which could be degraded by the cellular enzymes and tri-
closan released at the drug target site (pro-drug).

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S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
that the presence of polar groups at termini of the ali-
phatic esters of triclosan increases the activity of the
prodrug in E. coli culture. This is further confirmed by
the fact that when this terminus of ester is replaced by
non-polar group (s) the activity of compounds decreases
dramatically (Table 1). In case of E. coli compounds 5,
6, 7, and 8 have better inhibition as compared to triclo-
san. Improved inhibition in 5, 6, 7, and 8 was due to the
presence of tertiary amine (–N–(CH3)2), quaternary
amine (–N–(CH3)3), amido (–CONH2), and carboxy
(–COOH) groups, which are at the termini of respective
ester.
In P. falciparum pro-drug (5) entered the cell in 6 h
(Fig. 3B) and the observed half-life of the pro-drug disap-
pearance in Plasmodium is ꢁ9.45 h (Fig. 3C). Similarly
most of the other active compounds are permeable inside
the cell within 6 h after that conversion of prodrugs to
drug begins. The problem of delivery across the many
membranes enveloping the malaria parasite could be a
reason for the delay in their entry into the parasite.
We have tested these prodrugs for their inhibitory activ-
ity against ENRs and found that none of them possess
the activity. Incubation of these prodrugs with P. falci-
parum and E. coli culture results in hydrolysis of ester
linkage of these prodrugs releasing triclosan (Figs. 2A
and 3A). Triclosan thus released apparently targets FabI
inhibiting the growth of E. coli and the malaria parasite.
With P. falciparum cultures, while triclosan inhibited
growth of the cultures with an IC50 of 2.29 lM and
MIC = 17.6 lM, compounds 5, 8, 9, and 12 showed
inhibition in the low micromolar range (Table 1). Im-
proved inhibition in 5, 8, 9, and 12 was due to the pres-
ence of tertiary amine (–N–(CH3)2), carboxy (–COOH),
and keto (–COCH3) group, which are at the termini of
respective ester. Compounds 6, 7, 10, and 13 inhibited
cultures of the malaria parasite at high micromolar con-
centrations (Table 1).
5. Conclusion
Several derivatives of triclosan, to enhance their uptake
by malaria parasite and E. coli, have been synthesized.
All the covalent bonds in the prodrugs thus synthesized
are hydrolyzable by enzymes present in the target organ-
isms. This makes these derivatives promising prodrugs,
which can get accumulated inside the target organism
and hydrolyzed therein releasing the parent biocide.
Many of the compounds from the prodrug series (5–13 )
have shown promising in vitro biological activity against
malaria (P. falciparum) and bacteria (E. coli) establish-
ing that the triclosan could be converted to biologically
effective esterase-sensitive prodrug analogs. This in turn
opens up new avenues for exploring suitably designed
triclosan-based prodrugs as potential antimalarial and
antibacterial agents.
The tertiary amine conjugate of triclosan (5) displays en-
hanced inhibition against both E. coli and P. falciparum
cultures indicating that dimethylaminoethyl-glutaryl es-
ters of triclosan (tertiary amine conjugate of triclosan)
have the proclivity to diffuse rapidly across the biologi-
cal membranes and are transformed to mono-proton-
ated derivatives within E. coli and P. falciparum. Once
they are protonated they are less permeable, and are per-
haps trapped within the organisms increasing their accu-
mulation. These findings are consistent with the earlier
observations which show that accumulation of other
anti-malarials within the parasite leads to improvement
in their potencies.28
6. Experimental
Previously in the literature it is reported that 5 (and
6)-carboxy-2,7,-dichloro di-acetate carries only one
negative charge and is permeable to cells.29 Similarly
quinolone derivatives bearing free carboxylic acid have
also been reported (viz. quinoline-3-carboxylic acid)
that show more activity as compared to their un-
charged parent compounds against bacterial and
malarial strains.30,31 In our case also carboxylic deriv-
ative (8) has shown good activity against both E. coli
and P. falciparum cultures (Table 1). Although it is
premature at this stage to speculate about any specific
mechanism for the greater potency of compound (8),
its observed potency is not surprising in view of the
above reports.
All the starting materials were obtained from Aldrich or
Fluka and used as supplied. Solvents used for the chem-
ical synthesis acquired from commercial sources were of
analytical grade of the highest purity, and were used
without further purification unless otherwise stated.
Column chromatography was performed using 100–
200 mesh silica gel, whereas all TLC (silica gel) develop-
ment was performed on silica gel-coated sheets (Merck
1
Kiesel 60 F254, 0.2 mm thickness). Both H NMR and
13C NMR spectra were recorded on 300 and 400 MHz
Bruker NMR spectrometer using tetramethylsilane as
internal standard and the chemical shifts are reported
in (d) units. The sample concentration in each case
was approximately 10 mg in chloroform-d (0.6 mL).
Mass spectra were recorded on ESI-MS (Bruker Dalto-
nis, Esquire 3000plus) instrument.
Prodrugs were incubated with E. coli cultures for vari-
ous time intervals. It was found that most of the inhib-
itor is taken up by the bacteria within minutes and
hardly any amount of the ester used in these studies is
observed in the culture supernatant as evident from
the FPLC (Fig. 2B). On the contrary even after eight
hours very little of prodrugs (9 and 10) is able to pene-
trate the cell explaining their poor activity. It is evident
from the results that the better inhibition of these pro-
drugs is due to their increased permeability.
6.1. Pentanedioic acid mono-(2-dimethylamino-ethyl)
ester (3)
To an anhydrous dichloromethane solution (5 mL)
of dimethylaminoethanol (0.5 mL; 5 mmol) was added
dropwise at 0 ꢁC an anhydrous dichloromethane solu-
tion (5 mL) of glutaric anhydride (0.684 g; 6 mmol)

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
5543
and N,N-di-isopropylethylamine (DIPEA) (0.95 mL;
5.5 mmol) and stirred overnight at room temperature.
The reaction mixture was diluted with DCM (150) and
washed with water (3 · 50 mL). Organic layer was dried
over Na2SO4 and concentrated under reduced pressure,
giving compound (3 ), which was purified by column
chromatography on silica gel (eluent; chloroform/meth-
anol 9:1) to give desired product as a white syrupy mass.
Yield 0.792 g (78%), 1H NMR (400 MHz, CDCl3)d 1.94
(m, 2H), 2.3 (m, 4H), 2.26 (s, 6H), 3.15 (t, J = 7.6 Hz,
2H), 4.25 (t, J = 5.6 Hz, 2H), 10.46 (s, 1H, carboxy).
13CNMR (400 MHz, CDCl3) d 175.1, 171.0, 64.4,
63.66, 43.06, 32.3, 30.2, 17.4. Anal. Calcd for
C9H17NO4: C, 53.19; H, 8.43; N, 6.89. Found C,
52.93; H, 8.65; N, 6.86. ESI-MS m/z [M + H]+ 204.10.
Ar-H), 7.5 (d, J = 8.6 Hz, 1H, Ar-H). 13C NMR
(400 MHz, CDCl3) d 171.4, 169.6, 152.4, 147.9, 143.5,
131.4, 130.5, 129.6, 129.2, 128.3, 125.6, 121.5, 120.1,
118.5, 64.2, 63.8, 49.03, 33.9, 33.6, 20.8. Anal. Calcd
for C22H25Cl4NO5: C, 50.31; H, 4.80; N, 2.67. Found
+
C, 50.42; H, 4.74; N, 2.65. ESI-MS m/z [MꢀCl]
488.11.
6.4. Succinamic acid 5-chloro-2-(2,4-dichloro-phenoxy)-
phenyl ester (7)
To a solution of succinamic acid (5) (0.234 g, 2 mmol) in
dry DMF (5 mL) was added CDI (0.324 g, 2 mmol) with
stirring at room temperature under N2. After evolution
of CO2 was complete (2 h), triclosan (4) (0.579 g,
2 mmol) was added and the reaction proceeded for an
additional 24 h. The mixture was concentrated in vacuo
to a white solid and washed with H2O followed by CCl4.
The resulting solid was purified on a silica gel column;
elution withEtOAc/methanol (9:1) gave 7as a white crys-
6.2. Pentanedioic acid 5-chloro-2-(2,4-dichloro-phenoxy)-
phenyl ester 2-dimethylamino-ethyl ester (5)
Mono-(dimethyl
aminoethyl)glutatrate
(2.03
g,
1
10 mmol) and CDI (2.03 g, 10 mmol) were suspended
in dichloromethane (10 mL) with stirring at 25 ꢁC under
nitrogen for 1 h. Triclosan (2.895 g; 10 mmol) was added
to this reaction mixture and stirred for another 3 h.
Completion of the reaction was monitored on TLC
and after completion the reaction mixture was poured
in water and extracted with DCM. Organic layer was
dried over Na2SO4, and concentrated in vacuum. The
crude product was chromatographed on silica gel col-
umn, eluted with chloroform/methanol (9:1), Yield
talline solid (0.529 g, 68% yield). H NMR (400 MHz,
CDCl3) d 2.55 (t, J = 6.8 Hz, 2H), 2.82–2.92 (m, 2H),
5.6 (d, J = 8.4 Hz, 2H), 6.67–6.90 (m, 2H, Ar-H), 7.15–
7.26 (m, 4H, Ar-H). 13C NMR (400 MHz, CDCl3) d
172.4, 169.9, 152.0, 148.1, 143.2, 131.0, 129.5, 128.5,
126.9, 125.5, 121.6, 120.9, 119.9, 118.4, 32.6, 29.9. Anal.
Calcd for C16H12Cl3NO4: C, 49.45; H, 3.11; N,
3.60. Found C, 49.45; H, 3.10; N, 3.56. ESI-MS m/z
+
[M+Na] 410, [M + K]+ 427.9.
1
2.554 g (54%). H NMR (400 MHz, CDCl3) d 1.9(m,
6.5. Pentanedioic acid mono-[5-chloro-2-(2,4-dichloro-
phenoxy)-phenyl] ester (8)
2H), 2.27 (s, 6H), 2.3–2.56 (m, 4H), 3.18 (t, J = 7.2 Hz,
2H), 4.16 (t, J = 5.6 Hz, 2H), 6.82 (m, 2H, Ar-H), 7.15
(m, 3H, Ar-H), 7.4 (d, J = 8.0 Hz, 1H, Ar-H). 13CNMR
(400 MHz, CDCl3) d 170.1, 169.8, 148.4, 144.1, 139.2,
127.9, 127.0, 126.7, 125.6, 124.4, 123.4, 121.8, 117.8,
117.5, 64.6, 63.7, 43.0, 32.5, 30.0, 17.4. Anal. Calcd for
C21H22Cl3NO5: C, 53.13; H, 4.67; N, 2.95. Found C,
Triclosan (1.158 g; 4 mmol), glutaric anhydride (0.684 g;
6 mmol) and 4-(dimethyl amino)pyridine (16 mg;
0.13 mmol) were dissolved in 20 mL of dry dichloro-
methane and left overnight at room temperature. The
reaction mixture was diluted with dichloromethane
(100 mL) and washed with water. The organic layer
was dried over sodium sulfate and solvent removed by
vacuum evaporation. The white solid was purified by sil-
ica gel chromatography (eluent: chloroform/methanol
+
53.17; H, 4.69; N, 2.95. ESI-MS m/z [M+H] 474.10.
6.3. (2-{4-[5-Chloro-2-(2,4-dichloro-phenoxy)-phenoxy-
carbonyl]-butyryloxy}-ethyl)-trimethyl-ammonium chlo-
ride (6)
1
10:1 to 5:1). Yield 0.900 g (56%), H NMR (400 MHz,
CDCl3) d 1.98 (m, 2H), 2.40 (J = 4.8 Hz, 2H), 2.55
(t, J = 7.2 Hz), 6.8 (m, 2H, Ar-H), 7.1–7.4 (m, 4H, Ar-
H), 10.6 (s, 1H, carboxy). 13C NMR (400 MHz, CDCl3)
d 176.5, 170.2, 150.8, 146.5, 141.6, 130.4, 129.4, 128,
126.9, 125.8, 124.3, 121.3, 120.5, 119.5, 32.8, 32.5,
19.2. Anal. Calcd for C17H13Cl3O5: C, 50.59; H,
3.25. Found C, 50.45; H, 3.34. ESI-MS m/z
[M+K]+439.2.
The dimethylaminoethyl-glutaryl esters of triclosan
(compound 5, 0.946 g; 2 mmol) dissolved in the mini-
mum amount of dry THF was treated with methyl io-
dide ( 0.71 g; 0.32 mL; 10 mmol). Although a solid
product separated in the very first few minutes, stirring
at room temperature continued for another 3 h. The
reaction mixture was left overnight at 4 ꢁC, and then
the precipitate was filtered off, washed with several por-
tions of dry ether followed by drying in vacuum. Iodide
salts were used in ion exchange without additional puri-
fication. Ion exchange chromatography on a Sephadex
column (chloride form, DEAE, A25) with methanol as
eluent afforded white (waxy) solid. The residue was fur-
ther purified by flash chromatography over silica Merck-
60, eluting with a gradient (from 1:9 to 4:6) of methanol
6.6. 4-Oxo-pentanoic acid 5-chloro-2-(2,4-dichloro-phen-
oxy)-phenyl ester (9)
To a solution of levulinic (0.255 mL, 2.5 mmol), a few
drops of DMF, and 20 mL CH2Cl2 were added followed
by oxalyl chloride (0.435 mL, 5.0 mmol) dropwise at
room temperature. After stirring at room temperature
for 1 h, the mixture was concentrated to dryness. Then
solution of triclosan (868 mg, 3 mmol), DIPEA
(0.522 mL, 3.0 mmol), and 10 mL CH2Cl2 was added
dropwise, and the reaction mixture was stirred at room
1
in ethyl acetate to provide 6 (1.001 g, 82% yield). H
NMR (400 MHz, CDCl3) d 1.9(m, 2 H), 2.4–2.6
(m, 4H), 3.2 (s, 9H), 3.27 (t, J = 7.8 Hz, 2H), 4.15
(t, J = 5.6 Hz, 2H), 6.9 (m, 2H, Ar-H), 7.2–7.3 (m, 3H,

5544
S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
temperature overnight. After dilution with water and
extraction with ethyl acetate, the organic extracts were
dried over anhydrous Na2SO4 and evaporated to pro-
vide a light yellow syrupy mass. The residue was purified
by silica gel chromatography, eluting with a gradient
(from 1:1 to 6:4) of ethyl acetate in hexane to provide
6.9. 3-[5-Chloro-2-(2,4-dichloro-phenoxy)-phenoxy]-1-
methyl-pyridinium (13)
A solution of pyridine ester 11 (1 mmol) and methyl io-
dide (9 mmol) in dry acetone (20 mL) was refluxed
for 5–6 h under nitrogen, with the progress being mon-
itored by TLC. The mixture was taken to dryness and
the crude iodide salts were used in ion exchange without
additional purification. Ion exchange chromatography
on a Sephadex column (chloride form, DEAE, A25)
with methanol as eluent afforded light yellow (waxy) sol-
ids. That was purified on silica column chromatography
(Merck-60), eluting with a gradient (from 1:9 to 3:7) of
methanol in ethyl acetate to get desired compound.
1
9(0.730 g, 63% yield). H NMR (300 MHz, CDCl3) d
2.1 (s, 3H), 2.7–2.8 (m, 4H), 6.8 (m, 2H, Ar-H), 7.1–
7.2 (m, 3H, Ar-H), 7.4 (d, J = 2.4 Hz, 1H, Ar-H). 13C
NMR (300 MHz, CDCl3) d 205.9, 170.3, 150.9, 146.5,
141.7, 130.3, 129.4, 129.2, 128.1, 126.9, 125.7, 124.4,
120.2, 119.1, 36.7, 29.1, 27.6. Anal. Calcd for
C17H13Cl3O4: C, 52.67; H, 3.38. Found C, 52.65; H,
+
3.35. ESI-MS m/z [M+Na] 408.9.
1
Yields (70%). H NMR (400 MHz, CDCl3) d 4.06 (s,
6.7. Pentanoic acid 5-chloro-2-(2,4-dichloro-phenoxy)-
phenyl ester (10)
3H, –N CH3), 6.76–6.85 (m, 3H, Ar-H), 7.03–7.18 (m,
2H, Ar-H), 7.42 (d, J = 8.0, 1H, Ar-H), 7.58 (m, 1H,
py), 8.32 (d, J = 6.2 Hz, 1H, py), 9.15 (d, J = 8.08 Hz,
1H, py), 9.42 (m, 1H,py). 13C NMR (400 MHz, CDCl3)
d 164.4, 154.6, 151.2, 151.0, 146.2, 141.1, 136.8, 130.4,
129.5, 129.1, 128.4, 128.1, 126.2, 124.1, 129.9, 120.5,
120.2, 119.4, 49.2. Anal. Calcd for C19H13Cl4NO3: C,
51.27; H, 2.93; N, 3.15. Found C, 51.27; H, 2.93; N,
To a solution of pentanoic acid (0.432 mL, 4 mmol) and
20 mL CH2Cl2 was added oxalyl chloride (0.87 mL,
10 mmol) dropwise at room temperature. After stirring
at room temperature for 1 h, the mixture was concen-
trated to dryness. A solution of triclosan (1.158 g,
4 mmol), DIPEA (0.835 mL, 4.8 mmol), and 15 mL
CH2Cl2 was added dropwise, and the reaction mixture
was stirred at rt overnight. After dilution with water
and extraction with ethyl acetate, the organic extracts
were dried over anhydrous Na2SO4 . The solvent was
evaporated in vacuo, yielding viscous syrupy mass that
was purified on silica gel column eluted with ethyl ace-
+
3.17. ESI-MS m/z [M+H] 444.01.
6.10. Determination of antimicrobial activity
The whole-cell antimicrobial activity of the test com-
pounds was determined by micro-dilution method.
About 10 mM stock solutions of triclosan and its deriv-
atives were made in DMSO. Wild-type E. coli K12 was
grown in LB broth until the mid-log phase and then di-
luted hundred times in the same medium. One hundred
microliters of this cell suspension was used to inoculate
each tube containing 10 mL of the medium with the
inhibitors. The tubes were incubated at 37 ꢁC for 12 h.
Turbidity was measured by the OD taken at a time
gap of every 2 h at 600 nm. The absorbance values were
normalized to the solvent-treated control.
1
tate/hexane (15:85). Final yield was 1.116 g, (75%). H
NMR (300 MHz, CDCl3) d 0.9 (t, J = 7.5 Hz, 3H),
1.2–1.4 (m, 2H), 1.5–1.6 (m, 2H), 2.4 (t, J = 7.5 Hz,
2H), 6.8 (m, 2H, Ar-H), 7.1–7.2 (m, 3H, Ar-H), 7.4 (d,
J = 2.4 Hz, 1H, Ar-H). 13C NMR (300 MHz, CDCl3) d
170.1, 154.5, 148.6, 144.0, 128.9, 127.8, 126.8, 125.5,
124.3, 123.2, 121.9, 119.8, 117.4, 32.4, 26.2, 20.5, 12.8.
Anal. Calcd for C17H15Cl3O3: C, 54.64; H, 4.05.Found
C, 54.46; H, 4.67. ESI-MS m/z+ [M+H] 373.4.
6.8. 3-[5-Chloro-2-(2,4-dichloro-phenoxy)-phenoxy]-pyri-
dine (12)
6.11. Assessment of inhibition of growth of P. falciparum
and determination of IC50 of the compounds
Nicotinoyl hydrochloride 11 ( 3 mmol) was added in
small amounts to a refluxing solution of triclosan (4,
2.5 mmol)in toluene (20 mL). Triethylamine (0.7 mL;
7.0 mmol) was then added dropwise, and the mixture
was refluxed overnight. After filtration, the solvents
were removed and resulting yellow-white solid was
purified on Al2O3 (act. II–III) with a dichlorometh-
ane/hexane (1:1) to pure dichloromethane gradient,
followed by crystallization from acetonitrile to afford
esters 12 as white solid. Yield 0.903 g (92%). 1H
NMR (400 MHz, CDCl3) d 6.92 (m, 2H, Ar-H),
7.15–7.25 (m, 3H, Ar-H), 7.35 (d, J = 8.6 Hz, 1H,
Ar-H), 7.47 (m, 1H, py), 8.35 (d, J = 5.9 Hz, 1H, py),
8.9 (d, J = 7.9 Hz, 1H, py), 9.27(s, 1H, py). 13C
NMR (400 MHz, CDCl3) d 164.7, 154.2, 151.3,
150.8, 146.7, 141.3, 137.5, 130.4, 129.7, 129.2, 128.1,
127.3, 126.0, 124.4, 123.3, 120.5, 120.1, 119.1. Anal.
Calcd for C18H10Cl3NO3: C, 54.78; H, 2.55; N, 3.55.
Found C, 54.74; H, 2.77; N, 3.54. ESI-MS m/z
The experiments were performed using P. falciparum
FCK2 strain (chloroquine-sensitive, IC50, 18 nM), an
isolate from Karnataka, India. P. falciparum was cul-
tured using standard techniques32 and synchronized
using 5% sorbitol33 at 4% hematocrit in RPMI 1640
medium (Invitrogen, CA, USA) supplemented with
10% human serum, 0.225% sodium bicarbonate, and
0.01 mg/mL of gentamicin. Growth inhibition was mon-
itored using microscopic examination of the parasites by
standard Giemsa staining. Typically uninfected or in-
fected (1–2% parasitaemia, ring stage) red blood cells
(2% hematocrit) were added to the culture medium in
the wells of a 96-well plate (Nunc, Roskilde, Denmark),
and different concentrations of inhibitor in Me2SO did
not exceed 0.05%. The experiment started with the syn-
chronized parasite culture in the early trophozoite stage
and inhibitors were added up to the fourth day. Solvent
controls as well as triclosan as positive control were in-
cluded. P. falciparum growth was compared with solvent
control. The IC50 was calculated from a plot of relative
+
[M+H] 394.12.

S. Mishra et al. / Bioorg. Med. Chem. 16 (2008) 5536–5546
5545
percent parasitemia versus the log concentration of the
inhibitor by fitting it to non-linear regression analysis
using Sigma Plot 2000 software (Systat Software Inc.,
CA, USA).
young trophozoite stage were treated with pro-drug
for different time points (0, 0.5, 3, 6, 12, 18, 24, and
36 h). Parasites were isolated and resuspended in PBS.
Solid-phase extraction and FPLC assay have been done
using the above protocol for P. falciparum cultures also.
6.12. Assessment of inhibition of growth of P. falciparum
and determination of IC50 of the compounds by [3H]-
hypoxanthine uptake assay
Acknowledgments
IC50 was also determined by the [3H]-hypoxanthine up-
take assay. The parasites were cultured using standard
techniques as described above for the microscopic exam-
ination. The semi-automated microdilution technique of
Desjardins et al., which is based on [3H]-hypoxanthine
uptake by parasites, was used to assess the sensitivity
of the parasites to the various inhibitors.34 Briefly, syn-
chronized parasites were cultured in 96-well plates
(Nunc, Copenhagen, Denmark) at 2–3% hematocrit
and at an initial parasitemia of 1–2%, with varying con-
centrations of the inhibitors, and addition of the inhib-
itor in fresh medium every 24 h for 48 h. All additions
were done in duplicate. Inhibitor stocks were made in
sterile DMSO and dilutions made such that the final
concentration of DMSO in the parasite culture did not
exceed 0.1%. Parasites synchronized at the ring stage
were cultured in the presence of varying concentrations
of the inhibitors for the first 48 h and then incubated
with [3H]-hypoxanthine (1 lCi/well) for the next 36 h
and harvested. They were then harvested using a Nunc
cell harvester onto glass fiber filters, washed and sub-
jected to liquid scintillation counting (Hewlett–Pack-
ard). IC50s were calculated from plots of relative
percent parasitemia versus the log concentration of the
respective inhibitors, fitted to non-linear regression anal-
ysis using Sigma Plot 2000 software. The IC50 values
using this method were similar to those obtained using
microscopic examination.
This work was supported by a grant from the Depart-
ment of Science and Technology (DST, Government
of India) under PRDSF toN.S. and also by a grant from
the Centre of Excellence, DBT to A.S. A.S. is J.C. Bose
fellow of the Department of Science and Technology.
S.M. acknowledges DBT, Government of India, for
postdoctoral fellowship. K.K. acknowledges the CSIR,
Government of India, for a senior research fellowship.
We do not have any competing financial interests with
regard to this work.
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