Synthesis, leishmanicidal and Cytotoxic activity of triclosan-chalcone, triclosan-chromone and triclosan-coumarin hybrids

Article abstract of DOI:10.3390/molecules190913251

Twelve hybrids derived from triclosan were obtained via Williamson etherification of O-triclosan alkyl bromide plus chalcone and O-coumarin or O-chromone alkyl bromide plus triclosan, respectively. Structures of the products were elucidated by spectroscop

Full text of DOI:10.3390/molecules190913251

Molecules 2014, 19, 13251-13266; doi:10.3390/molecules190913251
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Leishmanicidal and Cytotoxic Activity of
Triclosan-Chalcone, Triclosan-Chromone and
Triclosan-Coumarin Hybrids
Elver Otero 1, Sebastián Vergara 1, Sara M. Robledo 2,3, Wilson Cardona 1,*, Miguel Carda 4,
Ivan D. Vélez 2,3, Carlos Rojas 5 and Felipe Otálvaro 5
1
Química de Plantas Colombianas, Instituto de Química, Facultad de Ciencias Exactas y Naturales,
Universidad de Antioquia UdeA, Calle 70 No. 52-21, A.A 1226 Medellín, Colombia;
E-Mails: elverluisotero@yahoo.es (E.O.); sebitasnano@hotmail.com (S.V.)
2
PECET, Instituto de Investigaciones Médicas, Facultad de Medicina,
Universidad de Antioquia UdeA, Calle 62 No. 52-59, Lab 632, A.A 1226 Medellín, Colombia;
E-Mails: sara.robledo@udea.edu.co (S.M.R.); ivan.velez@udea.edu.co (I.D.V.)
3
CIDEPRO-Center for Development of Products against Tropical Diseases, Carrera 53 No 61-30,
Sede de Investigacion Universitaria, A.A 1226 Medellin, Colombia
4
Departamento de Química Inorgánica y Orgánica, Universidad Jaume I, E-12071 Castellón, Spain;
E-Mail: mcarda@qio.uji.es
5
SIN-BIO-ME-NA, Facultad de Ciencias Exactas y Naturales, Universidad de Antioquia UdeA,
Calle 70 No. 52-21, A.A 1226 Medellín, Colombia;
E-Mails: pipelion@quimica.udea.edu.co (F.O.); kimikrlos@hotmail.com (C.R.)
* Author to whom correspondence should be addressed; E-Mail: wilson.cardona1@udea.edu.co;
Tel.: +574-219-5653; Fax: +574-233-0120.
Received: 19 May 2014; in revised form: 17 July 2014 / Accepted: 7 August 2014 /
Published: 28 August 2014
Abstract: Twelve hybrids derived from triclosan were obtained via Williamson
etherification of O-triclosan alkyl bromide plus chalcone and O-coumarin or O-chromone
alkyl bromide plus triclosan, respectively. Structures of the products were elucidated by
spectroscopic analysis. The synthesized compounds were evaluated for antileishmanial
activity against L. (V) panamensis amastigotes. Cytotoxic activity was also evaluated
against mammalian U-937 cells. Compounds 7–9 and 17, were active against Leishmania
parasites (EC50 = 9.4; 10.2; 13.5 and 27.5 µg/mL, respectively) and showed no toxicity
toward mammalian cells (>200 µg/mL). They are potential candidates for antileishmanial
drug development. Compounds 25–27, were active and cytotoxic. Further studies using

Molecules 2014, 19
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other cell types are needed in order to discriminate whether the toxicity shown by these
compounds is against tumor or non-tumor cells. The results indicate that compounds
containing small alkyl chains show better selectivity indices. Moreover, Michael acceptor
moieties may modify both the leishmanicidal activity and cytotoxicity. Further studies are
required to evaluate if the in vitro activity against Leishmania panamensis demonstrated
here is also observed in vivo.
Keywords: leishmaniasis; antiprotozoal; triclosan; coumarin; chromone; chalcone; hybrids
1. Introduction
Leishmaniasis is a group of diseases caused by protozoan parasites of the genus Leishmania, which
infect and replicate inside macrophages of the vertebrate host. The diseases is a major health problem
because is present in 98 countries and three territories worldwide, affecting mostly low-income people
in rural areas of tropical and subtropical countries. Approximately 0.7 to 1.2 million cutaneous
leishmaniasis (CL) cases occur annually. Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia,
North Sudan, Costa Rica and Peru, together account for 70% to 75% of global estimated CL incidence [1].
Leishmania (Viannia) panamensis is one of the most important causal agent of CL in Central and
South America [2]. The different forms of leishmaniasis require expensive treatments, and the
currently used medicines, pentavalent antimonials, pentamidine isothianate and miltefosine, show high
toxicity and therefore severe side effects and therefore there is an urgent need for new drugs [3].
However, due to the lack of interest shown by the pharmaceutical industry to develop drugs against
neglected diseases, it is necessary to join forces to develop new and better drugs to manage the disease
and help patients to improve their quality of life [4].
Triclosan is an uncompetitive inhibitor of purified enoyl-acyl carrier protein reductase (ENR),
which has demonstrated inhibitory activity in vitro against Plasmodium falciparum [5–8]. A previous
study showed that triclosan and quinoline-triclosan hybrids with shorter spacers, that is three and five
methylene units, have in vitro activity against axenic and intracellular amastigotes with effective
concentration (EC50) below of 24 µg/mL) of Leishmania panamensis [9]. Similarly, antileishmanial
activity of several chalcones has been reported [10–12]. The most promising of this class of
compounds is licochalcone A, an oxygenated chalcone isolated from the roots of the Chinese plant
Glycyrrhiza spp., which inhibits the fumarate reductase, a selective target present in the mitochondria
of the parasite [13].
Coumarins and chromones are important classes of compounds having versatile biological
activities [14–22]. Both moieties are well known for their antiprotozoal activity. Some synthetic
chromones were effective against L. donovani [23] and L. major [24] in in vivo studies. Aurapten, a
7-geranyloxycoumarin, was extracted from the Rutaceae species Esenbeckia febrifuga. This compound
shows significant growth inhibition with a 50% inhibitory concentration (IC50) of 30 µM against
L. major [25]. Three coumarins isolated from the leaves of Galipea panamensis were tested against
axenic amastigote forms of L. panamensis and displayed 50% effective concentrations (EC50) of 9.9,
10.5, and 14.1 µg/mL, respectively [26]. In addition, several 4-arylcoumarins were found to strongly inhibit

Molecules 2014, 19
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the protozoan parasites of L. donovani, particularly 4-(3,4-dimethoxyphenyl)-6,7-dimethoxy-coumarin
that exhibits potent activity on intracellular amastigotes with a selectivity index (SI) of 265, twice that
shown by amphotericin B (SI = 140) [27].
The combination of two pharmacological agents into a single molecule, called hybrid molecule, is
an emerging strategy in medicinal chemistry and drug discovery research [28,29]. These hybrid
molecules may display dual activity but do not necessarily act on the same biological target [30–34].
In this work, four chalcone-triclosan, chromone-triclosan, and coumarin-triclosan hybrids
(Schemes 1 and 2) were synthesized and their cytotoxic and leishmanicidal activities determined in the
search for new therapeutic alternatives for the treatment of leishmaniasis.
2. Results and Discussion
2.1. Chemistry
Triclosan-chalcone hybrids 7–10 were obtained via microwave assisted Williamson
etherification [35] between bromoalkyltriclosan 2–5 and 3,4-dimethoxy-4'-hydroxychalcone (6).
Reaction yields ranged between 35% and 60%. Chalcone 6 was prepared using a previously described
method [36] (Scheme 1).
Scheme 1. Synthetic pathway to triclosan-chalcone hybrids.
Scheme 2. Synthetic pathway to triclosan-coumarin and triclosan-chromone hybrids.

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Triclosan-coumarin and triclosan-chromone hybrids were obtained following the same synthetic
strategy (Scheme 2). Initially, 7-hydroxy-4-methylcoumarin (11, obtained by microwave assisted
Pechmann reaction between resorcinol and ethyl acetoacetate [37]) or commercially available
7-hydroxychromone (20) were treated with potassium hydroxide and 1,ω-dibromoalkanes (ω = 3, 4, 5, 8)
to obtain the respective bromoalkyl derivatives in yields similar to previous reports [9,38,39] but in
significantly shorter times. These compounds were coupled with triclosan to produce compounds 16–19
and 25–28 in 50%–80% yields. Remarkably, low yields were obtained when bromoalkyltriclosan
analogs were used as tactical variants.
2.2. Antileishmanial and Cytotoxic Activities
The leishmanicidal activity and cytotoxicity of the synthesized compounds was evaluated following
previously reported method [40,41]. Leishmanicidal activity was reported as percentage of inhibition
at 20 µg/mL and 50% effective concentration (EC50) values. On the other hand, cytotoxicity was
reported as 50% lethal concentration (LC50) values. Results are summarized in Table 1.
Table 1. In vitro toxicity and anti-Leishmania activity of triclosan-chalcone,
triclosan-chromone and triclosan-coumarin hybrids.
Cytotoxicity
LC50 (μg/mL, µM) a
>200.0, >326.7
>200.0, >319.4
>200.0, >311,4
>200.0, >293.2
>200.0, >396.8
>200.0, >386.1
>200.0, >375.9
>200.0, >348.4
6.4 ± 0.8, 13.1
15.8 ± 4.3, 31.3
25.8 ± 4.2, 49.8
80.0 ± 18.5, 142.8
22.1 ± 3.1, 76.3
13.9 ± 1.4, 48.9
>200.0, >1234.6
98.2 ± 6.7, 557.4
495.9 + 55.6
Leishmanicidal activity
% Inhibition b EC50 (μg/mL, µM) c
Compound
SI d
7
67.4 ± 17.2
67.8 ± 19.0
69.5 ± 8.6
23.3 ± 5.2
34.0 ± 0.3
57.1 ± 11.5
0.0
9.4 ± 1.3, 15.4
10.2 ± 1.8, 16.3
13.5 ± 3.6, 21.1
NE e
>21.3
>19.6
>14.8
NC f
NC f
>7.3
NC f
NC f
2.4
8
9
10
16
NE e
17
27.5 ± 0.8, 53.1
NE e
18
19
0.0
NE e
25
94.4 ± 2.9
91.0 ± 9.6
75.5 ± 1.5
28.4 ± 2.1
61.8 ± 5.5
52.4 ± 6.5
14.8 ± 0.9
15.3 ± 0.1
79.4 ± 2.1 h
69.1 ± 1.3 i
2.7 ± 0.4, 5.5
7.5 ± 0.2, 14.9
16.0 ± 1.0, 30.9
NE e
26
2.1
27
1.6
NC f
28
Triclosan
18.3 ± 2.01, 63.2
20.03 ± 1.4, 70.5
NE e
1.3
3,4-Dimethoxy-4'-hydroxychalcone (6)
7-Hydroxychromone (20)
7-Hydroxy-4-methylcoumarin (11)
Sb(V) g
0.7
NC f
NC f
78.7
592
NE e
6.3 + 0.9
Amphotericin B
42.1 ± 2.0, 45.6
0.06 ± 0.01, 0.1
a
b
c
± Standard deviation; LC50: Lethal Concentration 50; % Inhibition at 20 µg/mL; EC50: Effective
d
e
Concentration 50; IS: Selectivity Index: = LC50/EC50; NE: Not evaluated because inhibition was below
f
g
50% at 20 µg/mL; NC: Not calculated because EC50 wasn’t determined; SbV: pentavalent antimonial
meglumine antimoniate; h Dose employed: 10 µg/mL; i Dose employed: 0.05 µg/mL.
According to the results shown in Table 1, triclosan, chalcone and compounds 7–9, 17 and 25–27
showed activity against intracellular amastigotes of L. (V) panamensis with more than 50% inhibition

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at 20 µg/mL. The most active compounds were 25–27, with inhibitions of 94.4%, 91.0% and 75.5%,
respectively. Compounds 7–9 and 17 showed a moderate activity with inhibitions of 67.4%, 67.8%,
69.5% and 57.1%, respectively. On the other hand, high toxicity was measured for compounds
25–27, with LC50 < 26 µg/mL. Lower toxic activity was obtained for compounds 7–9 and 17
(LC50 > 200.0 µg/mL). Weak to negligible leishmanicidal activity and no toxicity were detected for
compounds 10, 16, 18, and 19 (inhibition <50% at 20 µg/mL and LC50 higher than 200 µg/mL,
respectively). Compound 28 displayed very low activity and moderate cytotoxicity with values of
28.4% and 80.0 µg/mL, respectively.
The effective concentration against the intracellular forms of L. (V) panamensis was also measured
for those compounds showing percentages of inhibition higher than 50% at 20 µg/mL (Table 1). Thus,
compounds 10, 16, 18, 19 and 28 were not evaluated. The most active compounds for intracellular
parasites were 25, 26 and 7 with EC50 of 2.7, 7.5 and 9.4 µg/mL, respectively, followed by compounds
8, 9 and 27 with EC50 values of 10.2, 13.5 and 16.0 µg/mL, respectively (Table 1). All compounds
showed leishmanicidal activity higher than cytotoxicity and therefore, selectivity indexes higher than
1. The best SI´s were observed for compounds 7–9 and 17 with values higher than of 21.3, 19.6, 14.8
and 7.3, respectively. Although compound 25 showed better activity than meglumine antimoniate, its
SI is affected by the high cytotoxicity.
On a structure-activity relationship basis, it is interesting to note the synergistic effect of the parent
subunits in the hybrids in comparison with the unlinked cases. For example, chalcone 6 and triclosan
are less potent and more cytotoxic individually than their hybrids 7–9. This phenomenon can also be
observed in compounds 25–27 in which an increase in activity is evident in the hybrids at the expense
of cytotoxicity compared to the individual units. The length of the alkyl linker also plays a pivotal role,
with an inverse correlation between increased length and activity for the evaluated cases.
One possible mechanisms of action for these compounds may be formulated in terms of conjugated
addition of nucleophilic amino acid residues present in biomolecules of the natural receptors, in a
Michael type mechanism. This mechanism has been reported for other α,β-unsaturated compounds
such as lactones and cinnamic acid esters [42–44]. In this regard, the low reactivity shown by
triclosan-coumarin hybrids could be rationalized in terms of steric hindrance and electronic
deactivation caused by the methyl group at the β-position.
3. Experimental Section
3.1. Chemical Synthesis
3.1.1. General Remarks
The syntheses were carried out in a MW domestic oven adapted for the use of a reflux condenser
and magnetic stirrer, at constant power (400 W). NMR spectra were recorded as CDCl3 solutions on an
AMX 300 instrument (Bruker, Billerica, MA, USA) operating at 300 MHz for 1H and 75 MHz for 1C.
Chemical shifts (δ) are expressed in ppm with the solvent peak as reference and TMS as an internal
standard; coupling constants (J) are given in Hertz (Hz). High resolution mass spectra were recorded
using electrospray ionization mass spectrometry (ESI-MS). A QTOF Premier instrument with an
orthogonal Z-spray-electrospray interface (Waters, Manchester, UK) was used operating in the

Molecules 2014, 19
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W-mode. The drying and cone gas was nitrogen set to flow rates of 300 and 30 L/h, respectively.
Methanol sample solutions (ca. 1 × 10−5 M) were directly introduced to the ESI spectrometer at a flow
rate of 10 µL/min. A capillary voltage of 3.5 kV was used in the positive scan mode, and the cone
voltage set to Uc = 10 V. For the accurate mass measurements, a 2 mg/L standard solution of leucine
enkephalin was introduced via the lock spray needle at a cone voltage set to 85 V and a flow rate of
30 μL/min. IR spectra were recorded on a Spectrum RX I FT-IR system (Perkin-Elmer, Waltham, MA,
USA) in KBr disks. Elemental analysis were recorded on TruSpec Micro Series equipment
(LECO Corporation, St. Joseph, MI, USA). Commercially available reagents were used as received.
Silica gel 60 (0.063–0.200 mesh, Merck, Whitehouse Station, NJ, USA) was used for column
chromatography, and precoated silica gel plates (Merck 60 F254 0.2 mm) were used for thin layer
chromatography (TLC).
3.1.2. General Procedure for the Synthesis of Bromoalkyl Derivatives
Triclosan, chromone or coumarin (1 mmol, 1 eq.), potassium hydroxide (3 eq.) and acetonitrile
(10 mL), were placed in a 50 mL flat-bottomed flask equipped with a magnetic stirring bar. The
mixture was stirred and heated to reflux for a period of 5 min, under microwave irradiation. Then,
1,ω-dibromoalkane (1.1 eq.) was added to the reaction mixture which was refluxed for 30 min. The
crude reaction mixture was concentrated on a rotatory evaporator, and the residue was purified by
column chromatography over silica gel eluting with hexane and a mixture of hexane-ethyl acetate
(9:1 ratio) to obtain the bromoalkyl derivatives in yields ranging between 60%–85%. Monitoring of
the reaction progress and product purification was carried out by TLC.
7-[(8-Bromooctyl)oxy]-4-methyl-2H-chromen-2-one (15). Yield 60% (1.02 mmol, 375.3 mg); pale
yellow oil; IR (cm−1): νmax 2933 (C-H), 1737 (C=O), 1612 (C=C), 1463 (C=CAr), 1261 (C-O-C), 1199
1
((C=O)-O), 842 (C-HAr); H-NMR (CDCl3): δ 1.35–1.46 (4H, m), 1.46–1.58 (4H, m), 1.78–1.97 (4H,
m), 2.43 (S, CH3), 3.45 (CH2Br, t, J = 6.9 Hz), 4.05 (CH2O-, t, J = 6.6 Hz), 6.16 (H2, s), 6.83 (H7, d,
J = 2.4 Hz), 6.88 (H5, dd, J = 8.8, 2.4 Hz), 7.52 (H4, d, J = 8.8 Hz); 13C-NMR (CDCl3): δ 18.67 (CH3),
25.88 (CH2), 28.08 (CH2), 28.66 (CH2), 28.96 (CH2), 29.14 (CH2), 32.77 (CH2), 33.95 (CH2Br), 68.65
(CH2O), 101.37 (C7), 111.84 (C5), 112.68 (C2), 113.45 (C4a), 125.49 (C4), 152.61 (C7a), 155.33 (C3),
161.38 (C6), 162.23 (C=O).
7-(4-Bromobutoxy)-4H-chromen-4-one (22). Yield 82% (1.52 mmol, 450.78 mg); pale yellow oil; IR
(cm−1): νmax 2953 (C-H), 1639 (C=O), 1595 (C=C), 1438 (C=CAr), 1265 (C-O-C), 856 (C-HAr);
1H-NMR (CDCl3): δ 1.98–2.09 (2H, m), 2.09–2.18 (2H, m), 3.53 (CH2Br, t, J = 6.5 Hz), 4.12 (CH2O, t,
J = 6.0 Hz), 6.31 (H2, d, J = 6.1 Hz), 6.86 (H4, d, J = 2.3 Hz), 6.99 (H6, dd, J = 9.0, 2.2 Hz), 7.81 (H3, d,
J = 6.0 Hz), 8.14 (H7, d, J = 9.0 Hz); 13C-NMR (CDCl3): δ 27.63 (CH2), 29.29 (CH2), 33.17 (CH2Br),
67.58 (CH2O), 100.93 (C4), 112.97 (C6), 114.75 (C2), 118.83 (C7a), 127.26 (C7), 154.88 (C3), 158.24
(C4a), 163.35 (C5), 177.02 (C=O).
7-[(5-Bromopentyl)oxy]-4H-chromen-4-one (23). Yield 85% (1.57 mmol, 489.3 mg); pale yellow oil;
IR (cm−1): νmax 2929 (C-H), 1649 (C=O), 1595 (C=C), 1439 (C=CAr), 1267 (C-O-C), 856
(C-HAr); 1H-NMR (CDCl3): δ 1.61–1.74 (2H, m), 1.83–1.93 (2H, m), 1.93–2.04 (2H, m), 3.48 (CH2Br,

Molecules 2014, 19
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t, J = 6.7 Hz), 4.08 (CH2O, t, J = 6.3 Hz), 6.30 (H2, d, J = 6.1 Hz), 6.84 (H4, d, J = 2.3 Hz), 6.98 (H6,
13
dd, J = 8.9, 2.3 Hz), 7.80 (H3, d, J = 6.0 Hz), 8.12 (H7, d, J = 9.0 Hz); C-NMR (CDCl3): δ 24.74
(CH2), 28.17 (CH2), 32.37 (CH2), 33.51 (CH2Br), 68.29 (CH2O), 100.88 (C4), 112.94 (C6), 114.81
(C2), 118.72 (C7a), 127.18 (C7), 154.89 (C3), 158.25 (C4a), 163.49 (C5), 177.05 (C=O).
7-[(8-Bromooctyl)oxy]-4H-chromen-4-one (24). Yield 71% (1.31 mmol, 464 mg); yellow oil; IR
(cm−1): νmax 2939 (C-H), 1653 (C=O), 1596 (C=C), 1444 (C=CAr), 1227 (C-O-C), 856 (C-HAr);
1H-NMR (CDCl3): δ 1.34–1.46 (4H, m), 1.46–1.60 (4H, m), 1.77–1.97 (4H, m), 3.45 (CH2Br, t,
J = 6.8 Hz), 4.08 (CH2O, t, J = 6.5 Hz), 6.31 (H2, d, J = 6.1 Hz), 6.86 (H4, d, J = 2.3 Hz), 7.01 (H6, dd,
J = 8.9, 2.3 Hz), 7.81 (H3, d, J = 6.0 Hz), 8.14 (H7, d, J = 8.9 Hz); 13C-NMR (CDCl3): δ 25.88 (CH2),
28.07 (CH2), 28.66 (CH2), 28.93 (CH2), 29.13 (CH2), 32.76 (CH2), 33.95 (CH2Br), 68.65 (CH2O),
100.88 (C4), 112.92 (C6), 114.88 (C2), 118.64 (C7a), 127.17 (C7), 154.86 (C3), 158.31 (C4a), 163.70
(C5), 177.11 (C=O).
3.1.3. General Procedure for the Synthesis of Triclosan-Chalcone Hybrids
Chalcone (1.1 eq.), potassium hydroxide (2 eq.) and acetonitrile (10 mL), were placed in a 50 mL
flat-bottomed flask equipped with a magnetic stirring bar. The mixture was stirred and heated to reflux
for a period of 5 min, under microwave irradiation. Then, bromoalkyltriclosan (100 mg, 1 eq.) was
added to the reaction mixture which was then refluxed for 30 min. The crude reaction mixture was
concentrated on a rotatory evaporator, and the residue was purified by column chromatography over
silica gel eluting with hexane-ethyl acetate (9:1 ratio) to obtain the triclosan-chalcone hybrids in yields
between 35%–60%. Monitoring of the reaction progress and product purification was carried out by TLC.
(2E)-1-(4-{3-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]propoxy}phenyl)-3-(3,4-dimethoxyphenyl)
prop-2-en-1-one (7). Yield 35% (0.085 mmol, 52 mg); yellow solid, M.p. 129–130 °C; IR (cm−1): νmax
2935 (C-H), 1601 (C=O), 1512 (C=C), 1471 (C=CAr), 1265 (C-O-C), 1160 ((C=O)-O), 800 (C-HAr),
742 (C–Cl); 1H-NMR (CDCl3): δ 2.13–2.25 (2H, m), 3.96 (CH2O, t, J = 6.1 Hz), 3.98 (OCH3, s), 4.01
(OCH3, s), 4.18 (CH2O, t, J = 5.8 Hz), 6.62 (H3c, d, J = 8.8 Hz), 6.90 (H6d, d, J = 8.8 Hz), 6.95 (H3a, H5a,
d, J = 8.4 Hz), 6.99 (H4c, dd, J = 6.5, 2.5), 6.99–7.03 (H5b, H6b, m), 7.06 (H6c, d, J = 2.5 Hz), 7.21 (H2b,
d, J = 1.8 Hz), 7.29 (H5d, dd, J = 8.5, 1.8 Hz), 7.45 (H3d, d, J = 2.5 Hz), 7.46 (H2, d, J = 15.5 Hz), 7.81
(H3, d, J = 15.5 Hz), 8.06 (H2a, H6a, d, J = 8.9 Hz); 13C-NMR (CDCl3): δ 28.91 (CH2), 56.0 (2OCH3),
63.85 (CH2O), 65.10 (CH2O), 110.10 (C2b), 111.20 (C5b), 114.20 (C3a, C5a), 114.70 (C6c), 117.40 (C3c),
119.90 (C6b), 121.30 (C6d), 122.2 (C4c), 122.60 (C2), 123.0 (C2d), 124.10 (C1b), 127.70 (C5d), 128.10
(C5c), 130.20 (C4d), 130.80 (C2a, C6a), 130.90 (C1a), 131.4 (C3d), 142.70 (C3), 144.20 (C2c), 149.30 (C1c),
150.80 (C3b), 151.30 (C1d), 152.60 (C4b), 162.40 (C4a), 188.82 (C=O); EIMS: m/z 613.0946 [M + H]+,
for C32H28Cl3O6: 613.0951. Anal. Calc. for C32H27Cl3O6: C 62.61, H 4.43. Found C 63.15, H 4.72.
(2E)-1-(4-{4-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]butoxy}phenyl)-3-(3,4-dimethoxyphenyl)
prop-2-en-1-one (8). Yield 60% (0.142 mmol, 89.8 mg); yellow solid, M.p. 103–105 °C; IR (cm−1):
νmax 2954 (C-H), 1601 (C=O), 1512 (C=C), 1472 (C=CAr), 1262 (C-O-C), 1163 ((C=O)-O), 800
(C-HAr), 794 (C–Cl); 1H-NMR (CDCl3): δ 1.72–1.84 (2H, m), 1.84–1.97 (2H, m), 3.98 (OCH3, s), 4.01
(OCH3, s), 4.05 (2CH2O, t, J = 6.4 Hz), 6.68 (H3c, d, J = 8.8 Hz), 6.92–7.05 (H6c, H3a, H5a, H5b, H6b,

Molecules 2014, 19
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H6d), 7.12 (H4c, dd, J = 8.8, 2.5 Hz), 7.21 (H2b, d, J = 1.7), 7.28 (H5d, dd, J = 8.3, 1.6 Hz), 7.44 (H3d, d,
J = 2.4 Hz), 7.45 (H2, d, J = 15.5 Hz), 7.81 (H3, d, J = 15.5 Hz), 8.06 (H2a, H6a, d, J = 8.7 Hz);
13C-NMR (CDCl3): δ 25.55 (CH2), 25.64 (CH2), 56.03 (2OCH3), 67.46 (CH2O), 68.58 (CH2O), 110.11
(C2b), 111.15 (C5b), 114.24 (C3a, C5a), 114.80 (C6c), 117.80 (C3c), 119.70 (C6b), 121.16 (C6d), 122.25
(C4c), 122.60 (C2), 123.0 (C2d), 124.35 (C1b), 127.61 (C5c), 127.83 (C2c), 128.12 (C5d), 130.17 (C4d),
130.7 (C3d), 130.76 (C2a, C6a), 131.24 (C1a), 143.01 (C3), 144.35 (C2c), 149.25 (C1c), 150.85 (C3b),
151.30 (C1d), 152.60 (C4b), 162.68 (C4a), 188.80 (C=O); EIMS: m/z 627.1111 [M + H]+, Calcd for
C33H30Cl3O6: 627.1108. Anal. Calc. for C33H29Cl3O6: C 63.12, H 4.65. Found C 62.34, H 4.79.
(2E)-1-[4-({5-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]pentyl}oxy)phenyl]-3-(3,4-imethoxyphenyl)
prop-2-en-1-one (9). Yield 52% (0.119 mmol, 76 mg); yellow solid, M.p. 117–120 °C; IR (cm−1): νmax
2949 (C-H), 1603 (C=O), 1510 (C=C), 1472 (C=CAr), 1263 (C-O-C), 1159 ((C=O)-O), 862 (C-HAr),
742 (C–Cl); 1H-NMR (CDCl3): δ 1.35–1.51 (2H, m), 1.69–1.86 (4H, m), 3.98 (OCH3, s), 4.01 (OCH3, s),
3.81–4.17 (4H, m), 6.66 (H3c, d, J = 8.8 Hz), 6.94 (H4c , dd, J = 8.3, 1.4), 6.97–7.06 (H3a, H5a, H6d, H5b,
H6b, H6c, m), 7.08–7.15 (H5d, m), 7.21 (H2b, d, J = 1.7), 7.29 (H5d, dd, J = 8.5, 1.8 Hz), 7.45 (H3d , d,
J = 2.0 Hz), 7.46 (H2, d, J = 15.5 Hz), 7.81 (H3, d, J = 15.5 Hz), 8.06 (H2a, H6a, d, J = 8.8 Hz); 13C-NMR
(CDCl3): δ 22.45 (CH2), 28.67 (CH2), 28.73 (CH2), 56.02 (2OCH3), 67.94 (CH2O), 68.83 (CH2O),
110.12 (C2b), 111.15 (C5b), 114.27 (C3a, C5a), 114.71 (C6c), 117.61 (C3c), 119.89 (C6b), 121.05 (C6d),
122.41 (C4c), 122.20 (C2), 123.0 (C2d), 124.24 (C1b), 127.56 (C5c), 127.63 (C2c), 128.13 (C5d), 130.10
(C4d), 130.76 (C1a), 130.77 (C2a, C6a), 131.20 (C3d), 142.84 (C3), 144.12 (C2c), 149.25 (C1c), 151.0
(C3b), 151.27 (C1d), 152.72 (C4b), 162.82 (C4a), 188.82 (C=O); EIMS: m/z 641.1265 [M + H]+, Calcd
for C34H32Cl3O6: 641.1265. Anal. Calc. for C34H31Cl3O6: C 63.61, H 4.87. Found C 61.36, H 4.84.
(2E)-1-[4-({8-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]octyl}oxy)phenyl]-3-(3,4-dimethoxyphenyl)
prop-2-en-1-one (10). Yield 45% (0.094 mmol, 64 mg); yellow oil, IR (cm−1): νmax 2933 (C-H), 1601
(C=O), 1512 (C=C), 1471 (C=CAr), 1261 (C-O-C), 1166 ((C=O)-O), 804 (C-HAr), 740 (C–Cl);
1H-NMR (CDCl3): δ 1.19–1.35 (2H, m), 1.37–1.58 (6H, m), 1.73–1.99 (4H, m), 3.46 (CH2O, t,
J = 6.8 Hz), 3.98 (OCH3, s), 4.01 (OCH3, s), 4.08 (CH2O, t, J = 6.5 Hz), 6.66 (H3c, d, J = 8.8 Hz), 6.94
(H6d, d, J = 8.8 Hz), 6.97–7.05 (H3a, H5a, H5b, H6b, H6c, m), 7.11 (H4c, dd, J = 8.8, 2.5 Hz), 7.21 (H2b, d,
J = 1.8 Hz), 7.28 (H5d, dd, J = 8.5, 1.8 Hz), 7.45 (H3d, d, J = 2.5 Hz), 7.46 (H2, d, J = 15.5 Hz), 7.81
13
(H3, d, J = 15.5 Hz), 8.07 (H2a, H6a, d, J = 8.8 Hz); C-NMR (CDCl3): δ 25.70 (CH2), 25.93 (CH2),
28.10 (CH2),28.70 (CH2), 32.78 (CH2), 34.03 (CH2), 56.01 (2OCH3), 68.21 (CH2O), 69.0 (CH2O),
110.10 (C2b), 111.15 (C5b), 114.28 (C3a, C5a), 114.62 (C6c), 117.62 (C3c), 119.9 (C6b), 120.85 (C6d),
122.37 (C4c), 122.20 (C2), 123.0 (C2d), 124.22 (C1b), 127.51 (C5c), 127.61 (C2c), 128.12 (C5d), 130.10
(C4d), 130.76 (C1a), 130.77 (C2a, C6a), 131.12 (C3d), 142.83 (C3), 144.10 (C2c), 149.24 (C1c), 151.1
(C3b), 151.26 (C1d), 152.72 (C4b), 162.93 (C4a), 188.81 (C=O); EIMS: m/z 683.1736 [M + H]+, Calcd for
C37H38Cl3O6: 683.1734. Anal. Calc. for C37H37Cl3O5: C 64.97, H 5.45. Found C 64.07, H 6.04.
3.1.4. General Procedure for the Synthesis of Triclosan-Coumarin and Triclosan-Chromone Hybrids
Triclosan (1.1 eq.), potassium hydroxide (2 eq.) and acetonitrile (10 mL), were placed in a 50 mL
flat-bottomed flask equipped with a magnetic stirring bar. The mixture was stirred and heated to reflux
for a period of 5 min under microwave irradiation. Then, bromoalkylcoumarin or bromoalkylchromone

Molecules 2014, 19
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(1 eq.) was added to the reaction mixture which was refluxed for 30 min. The crude reaction mixture
was concentrated on a rotatory evaporator, and the residue was purified by column chromatography
over silica gel eluting with hexane-ethyl acetate (4:1 and then 3:2 ratio) to obtain the triclosan-chalcone
hybrids in yields between 50%–80%. Monitoring of the reaction progress and product purification was
carried out by TLC.
7-{3-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]propoxy}-4-methyl-2H-chromen-2-one (16). Yield 80%
(0.270 mmol, 136.8 mg); white solid, M.p. 139–141 °C; IR (cm−1): νmax 2968 (C-H), 1718 (C=O),
1
1610 (C=C), 1477 (C=CAr), 1263 (C-O-C), 1144 ((C=O)-O), 847 (C-HAr), 740 (C–Cl); H-NMR
(CDCl3): δ 1.14–1.25 (2H, m), 2.44 (CH3, s), 3.98 (CH2O, t, J = 6.6 Hz), 4.18 (CH2O, t, J = 5.8 Hz),
6.18 (H2, s), 6.61 (H3b, d, J = 8.8 Hz), 6.76 (H7, d, J = 2.5 Hz), 6.79 (H5, dd, J = 9.0, 2.5 Hz), 6.96 (H6b, d,
J = 2.5 Hz), 6.97–7.01 (H4b, H6c, m), 7.03–7.08 (H5c, m), 7.43 (H3c, d, J = 2.5 Hz), 7.51 (H4, d, J = 8.5 Hz);
13C-NMR (CDCl3): δ 18.67 (CH3), 28.82 (CH2), 64.21 (CH2O), 65.14 (CH2O), 101.76 (C7), 112.02
(C5), 112.10 (C2), 113.75 (C4a), 115.0 (C6b), 117.51 (C3b), 121.43 (C6c), 122.40 (C4b), 124.26 (C2c),
125.55 (C4), 127.58 (C5c), 127.80 (C5b), 130.15 (C4c), 130.82 (C3c), 142.91 (C2b), 150.74 (C1b), 152.50
(C1c), 155.22 (C7a), 158.0 (C3), 161.30 (C6), 161.70 (C=O); EIMS: m/z 505.0371 [M + H]+, Calcd for
C25H20Cl3O5: 505.0376. Anal. Calc. for C25H19Cl3O5: C 59.37, H 3.79. Found C 59.92, H 3.91.
7-{4-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]butoxy}-4-methyl-2H-chromen-2-one (17). Yield 53%
(0.170, 88 mg); white solid, M.p. 111–113 °C; IR (cm−1): νmax 2956 (C-H), 1735 (C=O), 1625 (C=C),
1474 (C=CAr), 1294 (C-O-C), 1153 ((C=O)-O), 851 (C-HAr), 742 (C–Cl); 1H-NMR (CDCl3): δ 1.72–1.84
(2H, m), 1.84–1.96 (2H, m), 2.44 (CH3, s), 4.01 (CH2O, t, J = 6.0 Hz), 4.05 (CH2O, t, J = 5.7 Hz), 6.17
(H2, s), 6.69 (H3b, d, J = 8.8 Hz), 6.80 (H7, d, J = 2.4 Hz), 6.86 (H5, dd, J = 8.8, 2.4 Hz), 6.97–7.0 (H4b,
H6b, m), 7.0–7.03 (H6c, m), 7.12 (H5c, dd, J = 8.9, 2.5), 7.43 (H3c, d, J = 2.5 Hz), 7.51 (H4, d, J = 8.6 Hz);
13C-NMR (CDCl3): δ 18.67 (CH3), 25.51 (CH2), 25.58 (CH2), 67.83 (CH2O), 68.62 (CH2O), 101.41 (C7),
112.0 (C5), 112.55 (C2), 113.55 (C4a), 114.82 (C6b), 118.0 (C3b), 121.2 (C6c), 122.08 (C4b), 124.46 (C2c),
125.50 (C4), 127.61 (C5c), 127.91 (C5b), 130.16 (C4c), 130.62 (C3c), 143.12 (C2b), 150.80 (C1b), 152.55
(C1c), 155.30 (C7a), 155.7 (C3), 161.35 (C6), 162.0 (C=O); EIMS: m/z 519.0536 [M + H]+, Calcd. for
C26H22Cl3O5: 519.0533. Anal. Calc. for C26H21Cl3O5: C 60.08, H 4.07. Found C 60.02, H 4.10.
7-({5-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]pentyl}oxy)-4-methyl-2H-chromen-2-one (18). Yield
67% (0.206 mmol, 110 mg); white solid, M.p. 84–86 °C; IR (cm−1): νmax 2936 (C-H), 1740 (C=O),
1
1611 (C=C), 1469 (C=CAr), 1285 (C-O-C), 1192 ((C=O)-O), 847 (C-HAr), 740 (C–Cl); H-NMR
(CDCl3): δ 1.36–1.51 (2H, m), 1.67–1.86 (4H, m), 2.43 (CH3, s), 3.98 (CH2O, t, J = 6.3 Hz), 3.99
(CH2O, t, J = 5.9 Hz), 6.16 (H2, s), 6.66 (H3b, d, J = 8.8 Hz), 6.82 (H7, d, J = 2.4 Hz), 6.87 (H5, dd,
J = 8.8, 2.4 Hz), 6.93–7.06 (H4b, H6b, H6c, m), 7.10 (H5c, dd, J = 8.8, 2.5), 7.43 (H3c, d, J = 2.5 Hz),
13
7.52 (H4, d, J = 8.6 Hz); C-NMR (CDCl3): δ 18.70 (CH3), 22.45 (CH2), 28.61 (CH2), 28.67 (CH2),
68.3 (CH2O), 68.84 (CH2O), 101.44 (C7), 111.90 (C5), 112.60 (C2), 113.52 (C4a), 114.75 (C6b), 117.66
(C3b), 121.05 (C6c), 122.36 (C4b), 124.27 (C2c), 125.51 (C4), 127.55 (C5c), 127.66 (C5b), 130.08 (C4c),
130.75 (C3c), 143.0 (C2b), 151.0 (C1b), 152.61 (C1c), 152.70 (C7a), 155.31 (C3), 161.40 (C6), 162.11
(C=O); EIMS: m/z 533.0692 [M + H]+, Calcd. for C27H24Cl3O5: 533.0689. Anal. Calc. for
C27H23Cl3O5: C 60.75, H 4.34. Found C 60.60, H 4.43.

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7-({8-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]octyl}oxy)-4-methyl-2H-chromen-2-one (19). Yield
75% (0.204 mmol, 117 mg); yellow pale oil; IR (cm−1): νmax 2936 (C-H), 1729 (C=O), 1613 (C=C),
1474 (C=CAr), 1268 (C-O-C), 1146 ((C=O)-O), 839 (C-HAr), 742 (C–Cl); 1H-NMR (CDCl3): δ 1.16–1.37
(6H, m), 1.39–1.55 (2H, m), 1.59–1.72 (2H, m), 1.77–1.94 (2H, m), 2.43 (CH3, s), 3.94 (CH2O, t,
J = 6.2 Hz), 4.05 (CH2O, t, J = 6.5 Hz), 6.16 (H2, s), 6.66 (H3b, d, J = 8.8 Hz), 6.84 (H7, d, J = 2.5 Hz),
6.89 (H5, dd, J = 8.8, 2.5 Hz), 6.95 (H4b, dd, J = 8.8, 2.5 Hz), 6.97–7.06 (H6b, H6c, m), 7.11 (H5c, dd,
13
J = 8.8, 2.5), 7.44 (H3c, d, J = 2.5 Hz), 7.52 (H4, d, J = 8.8 Hz); C-NMR (CDCl3): δ 18.71 (CH3),
25.70 (CH2), 25.90 (CH2), 28.91 (CH2), 29.0 (CH2), 29.14 (CH2), 29.24 (CH2), 68.57 (CH2O), 68.96
(CH2O), 101.36 (C7), 111.82 (C5), 112.71 (C2), 113.43 (C4a), 114.62 (C6b), 117.62 (C3b), 120.85 (C6c),
122.36 (C4b), 124.21 (C2c), 125.51 (C4), 127.51 (C5c), 127.60 (C5b), 130.06 (C4c), 130.73 (C3c), 142.82
(C2b), 151.1 (C1b), 152.66 (C1c), 152.71 (C7a), 155.32 (C3), 161.44 (C6), 162.25 (C=O); EIMS: m/z
575.1156 [M + H]+, Calcd. for C30H30Cl3O5: 575.1159.
7-{3-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]propoxy}-4H-chromen-4-one (25). Yield 79%
(0.279 mmol, 137 mg); yellow pale oil; IR (−1): νmax 2941 (C-H), 1651 (C=O), 1599 (C=C), 1496
1
(C=CAr), 1267 (C-O-C), 1190 ((C=O)-O), 812 (C-HAr), 740 (C–Cl); H-NMR (CDCl3): δ 2.15–2.25
(2H, m), 3.97 (CH2O, t, J = 6.0 Hz), 4.18 (CH2O, t, J = 5.8 Hz), 6.31 (H2, d, J = 6.0 Hz), 6.60 (H3b, d,
J = 8.8 Hz), 6.73 (H4, d, J = 2.3 Hz), 6.91 (H6, dd, J = 9.1, 2.3 Hz), 6.95 (H4b, dd, J = 8.7, 2.6 Hz),
6.98–7.02 (H6b, H6c, m), 7.02–7.06 (H5c, m), 7.42 (H3c, d, J = 2.5 Hz), 7.82 (H3, d, J = 6.0 Hz), 8.12
13
(H7, d, J = 9.0 Hz); C-NMR (CDCl3): δ 28.77 (CH2), 64.27 (CH2O), 67.58 (CH2O), 100.86 (C4),
112.97 (C6), 114.70 (C2), 114.83 (C6b), 117.38 (C3b), 118.87 (C7a), 121.45 (C6c), 122.52 (C4b), 124.0
(C2c), 127.21 (C7), 127.59 (C5c), 127.75 (C5b), 130.12 (C4c), 130.9 (C3c), 142.75 (C2b), 150.72 (C1b),
152.53 (C1c), 154.91 (C3), 158.20 (C4a), 163.13 (C5), 177.07 (C=O); EIMS: m/z 513.0032 [M + Na]+,
Calcd. for C24H17Cl3O5Na: 513.0039.
7-{4-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]butoxy}-4H-chromen-4-one (26). Yield 72% (0.243
mmol, 122 mg); yellow solid, M.p. 75–79 °C; IR (cm−1): νmax 2934 (C-H), 1662 (C=O), 1598 (C=C),
1471 (C=CAr), 1259 (C-O-C), 1190 ((C=O)-O), 812 (C-HAr), 740 (C–Cl); 1H-NMR (CDCl3): δ 1.73–1.85
(2H, m), 1.85–1.97 (2H, m), 4.03 (CH2O, t, J = 6.1 Hz), 4.05 (CH2O, t, J = 5.7 Hz), 6.32 (H2, d,
J = 6.1 Hz), 6.68 (H3b, d, J = 8.8 Hz), 6.82 (H4, d, J = 2.3 Hz), 6.92–7.0 (H6, H6b, m), 7.02–7.03 (H6c,
m), 7.12 (H5c, dd, J = 8.8, 2.5 Hz), 7.43 (H3c, d, J = 2.5 Hz), 7.81 (H3, d, J = 6.1 Hz), 8.13 (H7, d,
13
J = 8.9 Hz); C-NMR (CDCl3): δ 25.53 (2CH2), 67.93 (CH2O), 68.58 (CH2O), 100.84 (C4), 112.96
(C6), 114.76 (C2), 114.83 (C6b), 117.91 (C3b), 118.72 (C7a), 121.19 (C6c), 122.11 (C4b), 124.41 (C2c),
127.19 (C7), 127.62 (C5c), 127.89 (C5b), 130.16 (C4c), 130.65 (C3c), 143.04 (C2b), 150.76 (C1b), 152.51
(C1c), 154.86 (C3), 158.24 (C4a), 163.41 (C5), 177.07 (C=O); EIMS: m/z 505.0376 [M + H]+, Calcd. for
C25H20Cl3O5: 505.0376. Anal. Calc. for C25H19Cl3O5: C 59.37, H 3.79. Found C 59.58, H 3.79.
7-({5-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]pentyl}oxy)-4H-chromen-4-one (27). Yield 58%
(0.186 mmol, 96 mg); yellow solid, M.p. 74–77 °C; IR (cm−1): ν
2951 (C-H), 1649 (C=O), 1596
max
(C=C), 1475 (C=CAr), 1268 (C-O-C), 1195 ((C=O)-O), 809 (C-HAr), 740 (C–Cl); 1H-NMR (CDCl3): δ
1.35–1.51 (2H, m), 1.67–1.86 (4H, m), 3.98 (2CH2O, t, J = 6.1 Hz), 6.30 (H2, d, J = 6.3 Hz), 6.65 (H3b,
d, J = 8.9 Hz), 6.84 (H4, d, J = 2.0 Hz), 6.92–7.04 (H5c, H6, H6b, H6c, m), 7.09 (H4b, dd, J = 8.7,

Molecules 2014, 19
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2.6 Hz), 7.42 (H3c, d, J = 2.4 Hz), 7.80 (H3, d, J = 6.1 Hz), 8.13 (H7, d, J = 9.0 Hz); 13C-NMR (CDCl3):
δ 22.45 (CH2), 28.53 (CH2), 28.63 (CH2), 68.39 (CH2O), 68.82 (CH2O), 100.88 (C4), 112.93 (C6),
114.74 (C2), 114.83 (C6b), 117.60 (C3b), 118.70 (C7a), 121.10 (C6c), 122.41 (C4b), 124.22 (C2c), 127.15
(C7), 127.55 (C5c), 127.61 (C5b), 130.10 (C4c), 130.80 (C3c), 142.83 (C2b), 151.0 (C1b), 152.71 (C1c),
154.86 (C3), 158.27 (C4a), 163.54 (C5), 177.04 (C=O); EIMS: m/z 519.0538 [M + H]+, Calcd. for
C26H22Cl3O5: 519.0533. Anal. Calcd. for C26H21Cl3O5: C 60.08, H 4.07. Found C 59.39, H 4.08.
7-({8-[5-Chloro-2-(2,4-dichlorophenoxy)phenoxy]octyl}oxy)-4H-chromen-4-one (28). Yield 50%
(0.142 mmol, 79 mg); yellow pale oil; IR (cm−1): νmax 2932 (C-H), 1649 (C=O), 1599 (C=C), 1496
1
(C=CAr), 1268 (C-O-C), 1191 ((C=O)-O), 810 (C-HAr), 740 (C–Cl); H-NMR (CDCl3): δ 1.17–1.39
(4H, m), 1.40–1.58 (4H, m), 1.60–1.75 (2H, m), 1.79–1.94 (2H, m), 3.95 (CH2O, t, J = 6.1 Hz), 4.08
(CH2O, t, J = 6.4 Hz), 6.32 (H2, d, J = 6.1 Hz), 6.66 (H3b, d, J = 8.7 Hz), 6.87 (H4, d, J = 2.0 Hz),
6.93–7.06 (H5c, H6, H6b, H6c, m), 7.11 (H4b, dd, J = 8.9, 2.4 Hz), 7.45 (H3c, d, J = 2.4 Hz), 7.81 (H3, d,
J = 6.1 Hz), 8.15 (H7, d, J = 8.9 Hz).13C-NMR (CDCl3): δ 25.69 (CH2), 25.88 (CH2), 28.92 (CH2),
28.97 (CH2), 29.13 (CH2), 29.20 (CH2), 68.67 (CH2O), 69.0 (CH2O), 100.89 (C4), 112.95 (C6), 114.68
(C2), 114.87 (C6b), 117.64 (C3b), 118.66 (C7a), 120.88 (C6c), 122.35 (C4b), 124.25 (C2c), 127.18 (C7), 127.50
(C5c), 127.62 (C5b), 130.73 (C4c), 130.80 (C3c), 142.88 (C2b), 151.09 (C1b), 152.72 (C1c), 154.82 (C3),
158.29 (C4a), 163.71 (C5), 177.1 (C=O); EIMS: m/z 561.1003 [M + H]+, Calcd. for C29H28Cl3O5: 561.1002.
Solubility: Compounds 7–10, 16–19 and 25–28 are soluble in dichloromethane, ether and ethyl acetate,
which is in agreement with their low polarity.
3.2. Biological Activity Assays
The compounds were subjected to in vitro cytotoxic activity on mammalian cells and leishmanicidal
activity on intracellular amastigotes of L. panamensis.
3.2.1. In Vitro Cytotoxic Activity in Mammalian Cells
The cytotoxic activity of the compounds was assessed based on the viability of the human
promonocytic cell line U937 (ATCC CRL-1593.2TM) evaluated by the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay [41]. In brief, cells were grown in 96-well cell-culture
dishes at a concentration of 100,000 cells/mL in RPMI-1640 supplemented with 10% FBS and the
corresponding concentrations of the compounds, starting at 200 µg/mL in duplicate. The cells were
incubated at 37 °C with 5% CO2 for 72 h in the presence of the compounds, and then the effect was
determined using the MTT assay, incubating at 37 °C for 3 h. The effect of the compounds was
determined by measuring the activity of the mitochondrial dehydrogenase by adding 10 µL/well of
MTT solution (0.5 mg/mL) and incubating at 37 °C for 3 h. The reaction was stopped by adding a 50%
isopropanol solution with 10% sodium dodecyl sulfate for 30 min. Cell viability was determined based
on the quantity of formazan produced, according to the optical density (O.D) obtained at 570 nm in a
Bio-Rad (Hercules, CA, USA) ELISA instrument. Cultured cells in the absence of extracts were used
as viability controls, while meglumine antimoniate and amphotericin B were used as cytotoxicity
controls. Assays were performed twice with three replicates per each concentration tested.

Molecules 2014, 19
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3.2.2. Activity against Intracellular Amastigotes
The leishmanicidal activity of compounds was evaluated on intracellular amastigotes of
L. panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140pIR-GFP) [42].
Effect of each compound was determined according to the inhibition of the infection evidenced
by both decrease of the infected cells and decrease of intracellular parasite load. Briefly, U-937
human cells at a concentration of 3 × 105 cells/mL in RPMI 1640 and 0.1 μg/mL of PMA
(phorbol-12-myristate-13-acetate) were infected with promastigotes in stationary phase growth in a
15:1 parasites per cell ratio and incubated at 34 °C and 5% CO2 for 3 h. Cells were washed two times
with phosphate buffer solution (PBS) to eliminate not internalized parasites. Fresh RPMI 1640 1 mL
was added and cells were incubated during 24 h to guarantee multiplication of intracellular parasites.
After 24 h of infection, the culture medium was replaced by fresh culture medium containing each
compound at concentrations of 20 μg/mL. After 72 h, the inhibition of the infection progress was
determined. Cells were removed from the bottom plate with a trypsin/EDTA (250 mg) solution.
Recovered cells were centrifuged at 1100 rpm during 10 min at 4 °C, the supernatant was discarded
and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm during 10 min at 4 °C.
Supernatant was discarded and cells were suspended in 500 μL of PBS and analyzed by flow
cytometry (FC 500MPL, Cytomics, Brea, CA, USA) counting 20.000 events. All determinations for
each compound and standards were carried out in triplicate, in two isolated experiments [41,42].
Activity of tested compounds was carried out in parallel with infection progress in culture medium
alone and in culture medium with amphotericin B 0.05 μg/mL and meglumine antimoniate (10.0 μg/mL)
as control drugs. Compounds that showed growing inhibition percentages higher than 50% were then
evaluated at four additional concentrations to determine the 50% effective concentration (EC50). The
infected cells were exposed against each concentration of synthesized compounds during 72 h, then,
cells were removed and tested by flow cytometry as described before.
3.2.3. Statistical Analysis
Cytotoxicity was determined according to viability and mortality percentages obtained for each
isolated experiment (compounds, amphotericin B, meglumine antimoniate and culture medium alone).
The results were expressed as 50 lethal concentrations (LC50), that corresponds to the concentration
necessary to eliminate 50% of cells, calculated by Probit analysis [45]. Percentage of viability was
calculated by Equation (1), where the optical density (O.D) of control, corresponds to 100% of
viability. In turn, mortality percentage corresponds to 100% viability:
% Viability = (O.D Exposed cells)/(O.D Control cells) × 100
(1)
The degree of toxicity was established according to the LC50 value using the following scale: highly
toxic: LC50 < 50 μg/mL, toxic: LC50 > 50 to < 100 μg/mL; moderately toxic: LC50 > 100 to < 200 μg/mL
and potentially non-toxic: LC50 > 200 μg/mL.
Antileishmanial activity was determined according to percentage of infected cells and parasite load
obtained for each experimental condition by the cytometer. Percentage of infected cells was
determined as the number of positive events by double fluorescence (green for parasites and red for

Molecules 2014, 19
13263
cells) using dotplot analysis. On the other hand, the parasitic load was determined by analysis of mean
fluorescence intensity (MFI) [41].
The parasitemia inhibition was calculated by Equation (2), where the MFI of control, corresponds
to 100% of parasitemia. In turn, inhibition percentage corresponds to 100% Parasitemia. Results of
antileishmanial activity was expressed as 50% effective concentrations (EC50) determined by the
Probit method [45]:
% Parasitemia = (MFI Exposed parasites)/(MFI Control parasites) × 100
(2)
The degree of leishmanicidal activity was established according to the EC50 value, using the
following scale: Highly active: EC50 < 20 μg/mL, moderately active: EC50 > 20 to < 100 μg/mL;
potentially non active: EC50 > 100 μg/mL.
The selectivity index (SI), was calculated by dividing the cytotoxic activity and the leishmanicidal
activity using the following formula: SI = CL50/CE50. Cytotoxic compound: LC50 < 100 µg/mL.
Non-cytotoxic compound: LC50 > 200 µg/mL.
4. Conclusions
The design, synthesis, and antileishmanial screening of twelve triclosan derivatives are reported.
Several of the synthetic compounds have potential as templates for drugs development. Owing to the
high leishmanicidal activity and the low cytotoxicity we consider that compounds 7–9 and 17 are good
candidates. Studies on an animal model of leishmaniasis are needed to confirm the results observed
in vitro. On the other hand, compounds 25–27 that were active against Leishmania parasite but toxic
for mammalian cells still have potential to be considered as candidates for antileishmanial drug
development. However, more studies on toxicity using other cell lines are needed to discriminate
whether the toxicity shown by these compounds is against tumor or non-tumor cells. The results
indicate that compounds containing small alkyl chains show better selectivity indices. Moreover,
Michael acceptor moieties may modify both the leishmanicidal activity and cytotoxicity. The
mechanism of action of these promising compounds also needs to be addressed.
Acknowledgments
The authors thank COLCIENCIAS (contract No. 0333-2013, code: 111556933423) and the
Universidad de Antioquia (Estrategia de Sostenibilidad 2013–2014 and CIDEPRO) for financial support.
Author Contributions
Conceived, designed the synthesis and spectroscopy analysis: WC, MC and FO. Performed the
chemical experiments: EO, SV and CR. Designed, performed and analysis the biological experiments:
SMR and IDV. Wrote the paper: WC and SMR.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2014, 19
13264
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Sample Availability: Samples of the compounds are available from the authors.
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