Synthesis of Novel Quaternary Ammonium Salts and Their in Vitro Antileishmanial Activity and U-937 Cell Cytotoxicity

Article abstract of DOI:10.3390/molecules21040381

This work describes the synthesis of a series of quaternary ammonium salts and the assessment of their in vitro antileishmanial activity and cytotoxicity. A preliminary discussion on a structure-activity relationship of the compounds is also included. Three series of quaternary ammonium salts were prepared: (i) halomethylated quaternary ammonium salts (series I); (ii) non-halogenated quaternary ammonium salts (series II) and (iii) halomethylated choline analogs (series III). Assessments of their in vitro cytotoxicity in human promonocytic cells U-937 and antileishmanial activity in axenic amastigotes of L. (Viannia) panamensis (M/HOM/87/UA140-pIR-eGFP) were carried out using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) micromethod. Antileishmanial activity was also tested in intracellular amastigotes of L. (V) panamensis using flow cytometry. High toxicity for human U937 cells was found with most of the compounds, which exhibited Lethal Concentration 50 (LC₅₀ ) values in the range of 9 to 46 μg/mL. Most of the compounds evidenced antileishmanial activity. In axenic amastigotes, the antileishmanial activity varied from 14 to 57 μg/mL, while in intracellular amastigotes their activity varied from 17 to 50 μg/mL. N-Chloromethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (1a), N-iodomethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (2a), N,N,N-trimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (3a) and N,N,N-trimethyl-N-(5,5-diphenylpent-4-en-1-yl)ammonium iodide (3b) turned out to be the most active compounds against intracellular amastigotes of L. (V) panamensis, with EC₅₀ values varying between 24.7 for compound 3b and 38.4 μg/mL for compound 1a. Thus, these compounds represents new "hits" in the development of leishmanicidal drugs.

Full text of DOI:10.3390/molecules21040381

molecules
Article
Synthesis of Novel Quaternary Ammonium Salts and
Their in Vitro Antileishmanial Activity and U-937
Cell Cytotoxicity
Sandra M. Duque-Benítez 1, Luz Amalia Ríos-Vásquez 1,*, Rogelio Ocampo-Cardona 1,
David L. Cedeño 2,†, Marjorie A. Jones 2, Iván D. Vélez 3 and Sara M. Robledo 3,
*
1
Department of Chemistry, Universidad de Caldas, AA275 Manizales, Colombia;
samidube@gmail.com (S.M.D.-B.); rogelio.ocampo@ucaldas.edu.co (R.O.-C.)
Department of Chemistry, Illinois State University, Normal, 61790 IL, USA; daleceme@gmail.com (D.L.C.);
majone3@ilstu.edu (M.A.J.)
PECET, Medical Research Institute, School of Medicine, University of Antioquia, 0500100 Medellín,
Colombia; ivan.velez@udea.edu.co
2
3
*
Correspondence: amalia.rios@ucaldas.edu.co (L.A.R.-V.); sara.robledo@udea.edu.co (S.M.R.);
Tel.: +57-311-733-9680 (L.A.R.-V.); +57-304-576-2059 (S.M.R.)
†
Present/Permanent Address: Millennium Pain Center, Basic Science, Bloomington, 61701 IL, USA.
Academic Editor: Alessandro Palmieri
Received: 25 February 2016 ; Accepted: 14 March 2016 ; Published: 29 March 2016
Abstract: This work describes the synthesis of a series of quaternary ammonium salts and the assessment
of their in vitro antileishmanial activity and cytotoxicity. A preliminary discussion on a structure-activity
relationship of the compounds is also included. Three series of quaternary ammonium salts were
prepared: (i) halomethylated quaternary ammonium salts (series I); (ii) non-halogenated quaternary
ammonium salts (series II) and (iii) halomethylated choline analogs (series III). Assessments
of their in vitro cytotoxicity in human promonocytic cells U-937 and antileishmanial activity in
axenic amastigotes of L. (Viannia) panamensis (M/HOM/87/UA140-pIR-eGFP) were carried out
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) micromethod.
Antileishmanial activity was also tested in intracellular amastigotes of L. (V) panamensis using
flow cytometry. High toxicity for human U937 cells was found with most of the compounds,
which exhibited Lethal Concentration 50 (LC50) values in the range of 9 to 46
the compounds evidenced antileishmanial activity. In axenic amastigotes, the antileishmanial
activity varied from 14 to 57 g/mL, while in intracellular amastigotes their activity varied from
µg/mL. Most of
µ
17 to 50 µg/mL. N-Chloromethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (1a),
N-iodomethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (2a), N,N,N-trimethyl-
N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (3a) and N,N,N-trimethyl-N-(5,5-diphenylpent-4-en-
1-yl)ammonium iodide (3b) turned out to be the most active compounds against intracellular
amastigotes of L. (V) panamensis, with EC50 values varying between 24.7 for compound 3b and
38.4 µg/mL for compound 1a. Thus, these compounds represents new “hits” in the development of
leishmanicidal drugs.
Keywords: leishmaniasis; halomethylated quaternary ammoniun salts; halomethylated choline
analogs; Leishmania (V) panamensis; antileishmanial activity; cytotoxicity
1. Introduction
Leishmaniasis is an infectious disease caused by trypanosomatid parasites of the Leishmania
genus. At least 20 Leishmania species are known to cause different clinical forms of leishmaniasis
in humans and they are present in different locations worldwide [
1
]. There are three main clinical
Molecules 2016, 21, 381; doi:10.3390/molecules21040381
www.mdpi.com/journal/molecules

Molecules 2016, 21, 381
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manifestations: visceral (VL), mucocutaneous (ML), and cutaneous leishmaniasis (CL), respectively.
These vary according to the Leishmania species, the geographical region, and the host s immune
´
response. Leishmaniases are endemic in 98 countries over five continents, with CL being the
most prevalent around the world. It is estimated that 12 million people are infected, with 0.7 to
1.2 million new cases occurring every year and 350 million people at risk of infection [
disproportionately affects economically and socially disadvantaged people and causes psychological
suffering due to the social stigma associated to it [ ]. Considering that an effective vaccine is not
yet available to protect against leishmaniasis [ ], most current research aims at finding alternative
2]. The disease
3
4
treatments of the illness. This has been focused on the development of improved chemotherapies
because current medications are not fully satisfactory. Pentavalent antimonials, such as meglumine
antimoniate (MA) or sodium stibogluconate are the most widely used antileishmanial drugs, while
other medications including pentamidine isothionate, miltefosine, and amphotericin B (AmB) are also
available. Although these are reasonably effective, they have serious side effects and require prolonged
treatments. Additionally, Leishmania parasites are increasingly developing resistance. Altogether, these
drawbacks are affecting the adherence of patients to treatments [5]. On the other hand, pharmaceutical
companies have demonstrated little motivation to invest in the generation and production of new
treatments, thus, since 2002 leishmaniasis has been considered a neglected disease by the World Health
Organization [6]. Therefore, academic centers have taken the challenge to investigate and develop
therapies that are more accessible, safer, easier to administer, while more effective at low dose with
short-length treatments, and at a reasonable cost.
It is well known that certain quaternary ammonium salts are widely used by the pharmaceutical
industry for their recognized activity against fungi, bacteria, viruses and parasites. For example,
there are reports on their antimicrobial activity against Cryptococcus neoformans, Candida albicans,
Saccharomyces cerevisiae, Aspergillus fumigatus, A. niger, Mucor mucedo [
virus-1 [ ], Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Proteus vulgaris [
falciparum [10], L. donovani and L. infantum [11,12].
7
,
8
]. Human immunodeficiency
9
8], Plasmodium
Miltefosine (hexadecylphosphocholine), a particular example of a quaternary ammonium salt
bearing an apolar C16 chain and a phosphoester group, was initially used as antitumor drug and
subsequently was approved for the oral treatment of both cutaneous and visceral leishmaniasis [13,14].
Its biochemical mechanism of action, however, is only partially known. It is generally accepted that the
biosynthesis of phosphatidylcholine in Leishmania spp. parasites is strongly dependent on choline [15];
thus, quaternary ammonium salts, acting as choline structural analogs, might reduce the content of
phosphatidylcholine and phosphatidyl ethanolamine of the parasites, metabolites that are important
to synthesize and preserve the structure of the lipid membranes of eukaryotic cells [16].
Based on the fact that the biosynthesis of phosphatidylcholine in Leishmania spp. parasites
is strongly dependent on choline [15], and on the in vitro and in vivo antileishmanial activity
demonstrated by quaternary ammonium salts such as edelfosine, ilmofosine and miltefosine [17], a
series of N-halomethylated quaternary ammonium salts with C4 or C5 side chains bearing monophenyl-
and biphenyl-substituted double bonds (Figure 1) were screened in vitro as potential antileishmanial
agents. The screening revealed that these compounds are active against promastigotes of L. tarentolae
(unpublished data). Although L. tarentolae is a Leishmania species that affects certain reptiles but does
not infect humans, it has been demonstrated that L. tarentolae offer a suitable model during the in vitro
screening of compounds for activity against Leishmania parasite and the identification of compounds
with potential as antileishmanial drugs [18].
In this work, various series of quaternary ammonium salts were prepared with the purpose
to advance in the identification of “hit” and “lead” compounds as potential candidates for
new antileishmanial agents, and trying to establish a possible structure-activity relationship on
leishmanicidal activity, especially the effect of the chain length and the presence or absence of a halogen
in place of one of the hydrogens of the N-methyl. Thus, three series of quaternary ammonium salts
were prepared: (i) halomethylated quaternary ammonium salts
1 and 2 (series I), (ii) non-halogenated

Molecules 2016, 21, 381
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quaternary ammonium salts
3
(series II) and (iii) halomethylated choline analogs 4a and 4b (series
III). The assessment of their in vitro cytotoxicity in the human promonocytic U-937 cell line and their
antileishmanial activity in axenic and intracellular amastigotes of L. (V) panamensis was performed,
taking into account that this parasite is one of the most pathogenic species for humans in the Americas.
A
A
X
X
A
X
( )
n
( )
n
N
N
N
Z
-
-
-
7 : n = 3 , X = I , A = BF4
6 a : n = 3 , X = I , A = BF4 , Z = H
b : n = 2 , X = Br , A = BF4- , Z = H
c : n = 3 , X = I , A = BF4- , Z = OCH3
d : n = 3 , X = I , A = BF4- , Z = CH3
e : n = 3 , X = I , A = BF4- , Z = Cl
f : n = 3 , X = I , A = BF4- , Z = CF3
5 a : X = I , A = BF4
b : X = Br , A = Br-
Figure 1. Compounds screened for in vitro efficacy against Leishmania tarentolae parasites.
2. Results
2.1. Synthesis
Eleven compounds (
1
,
2,
3,
4a and 4b) and choline 4c were prepared (Schemes 1 and 2). Eight
of them are new: N-chloromethyl-N,N-dimethyl-N-(4,4-diphenyl-3-buten-1-yl)ammonium iodide
1a), N-chloromethyl-N,N-dimethyl-N-(5,5-diphenyl-4-penten-1-yl)ammonium iodide (1b), N-chloro-
methyl-N,N-dimethyl-N-(6,6-diphenyl-5-hexen-1-yl)ammonium iodide 1c N-iodomethyl-N,
(
(
)
N-dimethyl-N-(4,4-diphenyl-3-buten-1-yl)ammonium iodide (2a), N-iodomethyl-N,N-dimethyl-N-
(6,6-diphenyl-5-hexen-1-yl)ammonium iodide (2c), N,N,N-trimethyl-N-(4,4-diphenyl-3-buten-1-yl)-
ammonium iodide (3a), N,N,N-trimethyl-N-(5,5-diphenyl-4-penten-1-yl)ammonium iodide (3b) and
N,N,N-trimethyl-N-(6,6-diphenyl-5-hexen-1-yl)ammonium iodide (3c). The other three: N-iodo-
methyl-N,N-dimethyl-N-(5,5-diphenyl-4-penten-1-yl)ammonium iodide (2b), N-chloromethyl-
N-(2-hydroxyethyl)-N,N-(dimethyl)ammonium iodide (4a) and N-(2-hydroxyethyl)-N-iodomethyl-N,
N-dimethyl)ammonium iodide (4b), are known compounds.
I
Ph
Ph
O
Ph
X
b)
a)
d)
Br
Ph
n
Ph
Br
Cl
n
n
Ph
Z
n
N
HO
Me
Me
10 a : n = 2 , Z = Br
b : n = 3 , Z = Br
c : n = 4 , Z = Br
8 a : n = 2
b : n = 3
c : n = 4
9 a : n = 2
b : n = 3
c : n = 4
1 a : X = Cl ; n = 2
b : X = Cl ; n = 3
c : X = Cl ; n = 4
2 a : X = I ; n = 2
b : X = I ; n = 3
c : X = I ; n = 4
3 a : X = H ; n = 2
b : X = H ; n = 3
c : X = H ; n = 4
c)
11 a : n = 2 , Z = N(CH3)2
b : n = 3 , Z = N(CH3)2
c : n = 4 , Z = N(CH3)2
Reagents and conditions: a) PhMgBr (2.5 eq), dry ether, 0ºC to r.t; b) TsOH.H2O 1.5% molar, benzene, reflux, 6 h.
c) 40% aqueous (CH3)2NH (25 eq of amine), r.t 24 - 48 h. d) XCH2I where X=H, Cl or I (4.0 eq), acetonitrile, r.t, 24 h.
Scheme 1. Synthesis of quaternary ammonium salts 1, 2 and 3 (series I and II).

Molecules 2016, 21, 381
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I
X
OH
OH
a)
N
N
12
4 a : X = Cl
b : X = I
c : X = H
Reagents and conditions: a) XCH2I (with X= Cl, I or H), THF, stirring in the dark for 24 h.
Scheme 2. Synthesis of choline 4c and choline analogs 4a and 4b.
Compounds 1a
,
1b
,
1c
,
2a,
2b
,
2c
,
3a
,
3b and 3c were prepared according to the sequence
19] from -bromoacyl chlorides
by a four-step sequence involving: (i) a Grignard reaction to obtain -bromo- -diphenyl
with yields ranging from 68% to 78%; (ii) an acid-catalized dehydration of alcohols
-bromo- -diphenyl olefins
10 with yields beteween 82% and 90%; (iii) a nucleophilic displacement of bromide from the
-bromo- -diphenyl olefins 10 by dimethylamine to produce -(N,N-dimethylamino)- -diphenyl
olefins 11 with yields ranging from 50% to 68%; (iv) a nucleophilic displacement of iodide from
iodomethane, chloroiodomethane or diiodomethane by the -(N,N-dimethylamino)- -diphenyl
shown in Scheme 1, following or adapting literature methods [18
,
ω
8
ω
α,α
alcohols
9
9
using p-toluenesulfonic acid in benzene to form the corresponding
ω
α,α
ω
α
,
α
ω
α,α
ω
α,α
olefins 11 to obtain the target quaternary ammonium salts (series I and II) with yields between
61% and 85%. Choline 4c and choline analogues 4a and 4b were prepared as shown in Scheme 2,
following the method previously reported [20]. The halomethylated cholines iodides 4a and 4b were
synthesized from N,N-dimethyl-2-hydroxyethanamine (12), which was treated with 1.5 equivalents of
the corresponding dihalomethane.
2.2. In Vitro Cytotoxicity of Quaternary N-(Halomethylated) Ammonium Salts 1 and 2,
Non N-(Halomethylated) Ammonium Salts 3 and Halogenated Cholines 4
Compounds were tested first for toxicity on U937 human macrophages at four serial dilution
concentrations (200, 50, 12.5 and 3.25
4c was also tested as a reference compound. Based on our own classification all of the compounds
showed LC50 < 100 g/mL and were therefore classified as highly cytotoxic, with 2a being the
most cytotoxic (LC50 = 46 g/mL). As expected, choline 4c showed a very low level of cytotoxicity,
µg/mL) and their LC50 values were determined (Table 1). Choline
µ
µ
with LC50 = 372 µg/mL. AmB showed high cytotoxicity, while MA was potentially non-cytotoxic to
U-937 cells.
Antileishmanial activity of quaternary N-(halomethylated) ammonium salts
1 and 2,
non-N-(halomethylated) ammonium salts and halogenated cholines 4a and 4b was assessed in both
3
intracellular (inside mammalian U-937 cells) and axenic amastigotes of L. (V) panamensis and compared
to the activity of choline 4c. All compounds showed antileishmanial activity against both amastigotes
(intracellular and axenic) of L. (V) panamensis with EC50 values <50
an EC50 = 56.3 g/mL in axenic amastigotes (Table 1). Compound 2c showed the highest activity
with EC50 values of 17.6 g/mL and 14.0 g/mL in intracellular and axenic amastigotes, respectively.
Compounds 2b 3b and 4b were also active against both types of amastigotes with EC50 values of
g/mL and 23.9 g/mL, respectively (in intracellular amastigotes) and 19.7 g/mL,
g/mL and 19.6 g/mL, respectively (in axenic amastigotes) (Table 1). Activity of compounds
µg/mL, except 1b that exhibited
µ
µ
µ
,
24.0
25.0
µ
g/mL, 24.7
µ
µ
µ
µ
µ
3a and 4a was high in axenic amastigotes (EC50 25.0 and 24.3 µg/mL, respectively) but moderate in
intracellular amastigotes (EC50 30.0 and 29.6 g/mL, respectively). The remaining compounds 1a
2a and 3c showed moderate activity with EC50 values varying from 27.8 µg/mL to 49.4 g/mL in
intracellular amastigotes and from 34.1 g/mL to 40.5 g/mL in axenic amastigotes. As expected,
µ
–c,
µ
µ
µ

Molecules 2016, 21, 381
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compound 4c (choline) showed almost no activity against intracellular parasites (EC50 = 149.5 µg/mL)
and moderate activity against axenic amastigotes (EC50 < 50 µg/mL) AmB, one of the used
antileishmanial drug, was active against both amastigote types with EC50 values <1.0 µg/mL while
.
MA was active only against intracellular amastigotes (EC50 = 6.33
axenic parasites (Table 1).
µg/mL) and was not active against
Table 1. Cytotoxicity against U-937 cells and antileishmanial activity of N-(halomethylated) ammonium
salts, non N-(halomethylated) ammonium salts and choline and halogenated cholines.
Cytotoxicity
LC50
Intracellular Amastigotes
Axenic Amastigotes
Compound
EC50
SI
EC50
SI
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
44.4 ˘ 4.9
21.0 ˘ 2.0
21.0 ˘ 0.5
46.0 ˘ 5.4
10.1 ˘ 0.2
9.5 ˘ 0.3
38.4 ˘ 0.2
46.8 ˘ 1.2
49.4 ˘ 3.1
27.8 ˘ 3.9
24.0 ˘ 0.7
17.6 ˘ 1.0
30.0 ˘ 1.4
24.7 ˘ 1.9
41.6 ˘ 2.8
29.6 ˘ 2.8
23.9 ˘ 1.0
149.5 ˘ 17.9
0.05 ˘ 0.001
6.33 ˘ 0.86
1.2
0.5
0.4
1.7
0.4
0.5
1.3
1.2
0.3
0.6
0.7
2.5
560
56.6
35.0 ˘ 5.7
56.3 ˘ 3.7
40.5 ˘ 3.3
36.2 ˘ 0.1
19.7 ˘ 1.5
14.0 ˘ 0.9
25.0 ˘ 0.7
25.0 ˘ 0.5
34.1 ˘ 5.1
24.3 ˘ 1.3
19.6 ˘ 1.8
47.2 ˘ 7.1
0.041 ˘ 0.001
312.1 ˘ 18.6
1.3
0.4
0.5
1.3
0.5
0.7
1.5
1.1
0.4
0.7
0.9
7.9
690
1.4
39.2 ˘ 4.8
27.2 ˘ 0.7
12.5 ˘ 0.8
17.9 ˘ 0.02
17.8 ˘ 0.2
372.0 ˘ 43.1
28.2 ˘ 0.5
358.2 ˘ 1.7
AmB
MA
Results reported as the mean value
Amphotericin B; MA: meglumine antimoniate; the Selectivity Index (SI) = LC50/EC50. Bold data represent
˘
standard deviation of the half-maximum concentration in
µ
g/mL. AmB:
compounds with high activity against amastigotes of L. (V) panamensis.
3. Discussion
Based on the facts that: (i) choline is important to the biosynthesis of phosphatidylcholine in
Leishmania parasites [15]; (ii) some quaternary ammonium salts such as edelfosine, ilmofosine and
miltefosine have antileishmanial activity [17] and; (iii) some quaternary ammonium compounds
may efficiently inhibit choline transport in other intracellular parasites such as P. falciparum [19],
this work reports the synthesis of a series of new quaternary ammonium salts and their in vitro
effectiveness against the human pathogen Leishmania (V.) panamensis, which causes cutaneous and
mucocutaneous leishmaniasis.
As shown in Table 1, chlorinated compounds of the series I characterized by the general structure
[ClCH2N(CH3)3(CH2)nCH=C(Ph)2]+I´ with n = 2, 3 and 4 (1a
,
1b, and 1c, respectively) showed
moderate to low activity against axenic amastigotes of L. (V) panamensis, with EC50 values ranging
between 35 and 57 g/mL, and moderate activity against intracellular amastigotes with EC50 values
between 38 and 50 g/mL. These compounds also exhibited high cytotoxicity in U-937 cells with
LC50 values ranging from 21 to 45 g/mL. Compound 1a (n = 2) was the most active against axenic
µ
µ
µ
amastigotes while 1b (n = 3) was less active than 1c (n = 4). Similarly, 1a (n = 2) exhibits the best activity
against intracellular amastigotes while 1c (n = 4) is the worst. In contrast, 1a (n = 2) exhibited the
lowest cytotoxicity, while 1b (n = 3) and 1c (n = 4) exhibit similar toxicity.
The selectivity index (SI) is the ratio of the toxicity and activity of a compound, thus it provides
a measurement of the balancing effect of these opposing effects. Large values of SI indicate that a
compound is relatively active and presents a low toxicity. SI values ranging from 0.4 to 1.3 were
found for these three compounds related to their activity against axenic amastigotes. Similar SI
values were found in relation to their activity against intracellular amastigotes. In both cases, 1a
(n = 2) has the best SI. A comparison of compounds 1a
, 1b and 1c with respect to chlorocholine 4a
(from series III), reveals that 4a is more effective against both axenic and intracellular amastigotes

Molecules 2016, 21, 381
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(EC50 decreases), but the toxicity of 4a (LC50 = 17.9
µg/mL) is more than two times larger than that
for 1a (LC50 = 44.4 µg/mL). The results imply that the terminal diphenylethylene moiety in the
tail of the N-chlorometyl-N,N-dimethyl quaternary salts moderates both toxicity and activity with
respect to chlorocholine 4a. Thus, when taking both activity and toxicity into account, compound
1a (n = 2) is the N-chloromethyl quaternary salt that exhibits the best anti-leishmanial profile in this
study. An interesting trend observed for these chlorinated salts is that, upon lengthening the chain,
the activity against both axenic amastigotes and intracellular amastigotes decreases but the toxicity
steadily increases.
The iodinated compounds
2
of the series
I
are salts with the structure
2b and 2c respectively) exhibit
[ICH2N(CH3)3(CH2)nCH=C(Ph)2]+I´ with n = 2, 3 and 4 (2a
,
high to moderate activity against axenic amastigotes, with EC50 values varying between 14.0 and
36.0 µg/mL and high activity against intracellular amastigotes with EC50 values ranging between 17.0
and 28.0
salt of series I turned out to be the one with the longest aliphatic chain between the quaternary
ammonium head and the diphenyl tail, 2c (n = 4), with an EC50 value of 14.0 g/mL against axenic
amastigotes and 17.6 g/mL against intracellular amastigotes. Also, these iodinated salts were found
to have high toxicity with LC50 values ranging between 9 and 46 g/mL. Interestingly, a comparative
µg/mL. Contrary to the chlorinated analog salts 1, the most active N-iodomethyl quaternary
µ
µ
µ
analysis of N-chloromethyl and N-iodomethyl quaternary salts implies that the chlorine atom exerts
an opposite effect with respect to the iodine atom on the activity against both axenic and intracellular
amastigotes but a parallel effect on the toxicity in terms of the effect of the length of the tether chain.
However, the effect of lengthening the chain on toxicity is more sensitive when an iodine atom rather
than a chlorine atom is present in the cationic ammonium head. For instance, upon lengthering the
chain from n = 2 to n = 4, the LC50 value goes from 46
(n = 3) and 9.5 g/mL for 2c (n = 4). In general, iodomethylated salts were both more active against
Leishmania parasite and toxic to cells. In terms of the balance between toxicity and antileishmanial
activity, the SI of the N-iodomethyl quaternary salts of series I (2a 2b, and 2c) varies from 0.5 to
µg/mL for 2a (n = 2) to 10.1 µg/mL for 2b
µ
,
1.3 for axenic amastigotes and from 0.4 to 1.7 for intracellular amastigotes, with the higher values
corresponding to 2a (n = 2). So, 2a (n = 2) would be the most appropriate if we were to choose a lead
compound on the basis of its SI value only.
N-(2-Hydroxyethyl)-N-iodomethyl-N,N-dimethylammonium iodide (4b) of series III (iodocholine
for short) was also tested. The EC50 for iodocholine 4b is similar to that determined for 2b (n = 3)
against both axenic and intracellular amastigotes. However, 4b is a slightly less toxic than 2b and
2c (LC50 = 17.8
µg/mL for 4b, 10.1 µg/mL for 2b and 9.5 µg/mL for 2c), and more toxic than 2a
(LC50 = 46 µg/mL).
Although in general the iodomethylated salts of the series I behave similar to iodocholine 4b, we
can observe higher bioactivity of the N-iodomethyl quaternary salt bearing a diphenylethylene unit in
the tail and a carbon tether of n = 4 (i.e., 2c).
Compounds of the series II, characterized by the general structure [CH3N(CH3)2(CH2)nCH=C(Ph)2]+I´
with n = 2, 3 and 4 (3a
, 3b and 3c respectively) exhibit high toxicity with the lowest being for 3a (n = 2,
LC50 = 39.2 g/mL) while increasing when going to n = 3 and n = 4, with 3c (n = 4) being the most
µ
toxic. These compounds exhibited moderate activity against both axenic and intracellular amastigotes,
and it seems that the activity is sensitive to the length of the tether chain in going from n = 3 to n = 4.
The results clearly indicate that halogen atoms and the length of the tether chain modify the
biological profile of the quaternary salts. In general, the toxicity increases upon lengthening the chain
for a particular X = H (series II), Cl or I (Series I). Interestingly, for both X = Cl and I, the toxicity largely
decreases with the addition of a methylene unit in the tethering chain from n = 2 to n = 3 but remains
approximately the same with the increase of an additional methylene unit to n = 4. As previously
mentioned, there is a larger decrease when X = I as LC50 decreases four-fold, while when X = Cl the
LC50 is halved. In slight contrast, when X = H the toxicity seems to step-up as an additional methylene
unit is added to the tethering chain (LC50 decreases about 12–14 µg/mL per additional methylene

Molecules 2016, 21, 381
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unit). The changes in the antileishmanial activity are less clear although trends can be discerned as
previously noted.
We hypothesize that the molecular structure of the salt (i.e., diphenylethylene moiety and its
proximity to the ammonium head) as well as the presence and identity of the halogen in the ammonium
head influence the activity and toxicity of N-halomethylated quaternary salts. Further knowledge
of the interactions between the compounds and proteins or enzymes of the parasite will be key to
understanding the relationship between structure and the differential cytotoxicity and anti-leishmanial
activity. Indeed, the conformational structure of each compound constitutes a piece of information in
the overall picture of the biological behavior of these salts. For that purpose, a recent X-ray diffraction
study of the solid state structures of the iodinated compounds of the series I (2a, 2b and 2c) was carried
out [20]. Interestingly, the solid state conformation of 2a (n = 2) differs from that of the 2b (n = 3) and
2c (n = 4). The study revealed that conformations may be influenced by supramolecular interactions
as well as intramolecular interactions. The understanding of these interactions will be important in
both establishing mechanisms of bioavailability of the compounds as they go from the solid state to
a liquid/solution phase and on how the most stable conformation of the compounds interact with
enzymes or proteins of the parasite. A patent has been granted for compounds 1a, 1c, 2a and 2c [21].
This patent is one of the at least 10 patents that have been granted during the last 10 years to various
cores and their derivatives that have been reported to possess antileishmanial activity [22].
4. Materials and Methods
4.1. Structural Characterization
1
Structural characterization of compounds was carried out by H-NMR, 13C-NMR, elemental
1
analysis and mass spectrometry. H-NMR and 13C-NMR spectra were obtained at 300 MHz, 400, 500
and 126 MHz and 75 MHz respectively on a VXR-300, a Gemini-300 both Varian Medical System
(Palo Alto, CA, USA) or a Bruker (Billerica, MA, USA) 500 MHz spectrometer. Elemental analyses of
carbon, hydrogen and nitrogen were performed at the microanalysis laboratory at the University of
Illinois (Urbana-Champaign, IL, USA). Mass spectra were obtained on a HP Series 1100 MSD ESI-MS
(Hewlett Packard, Palo Alto, CA, USA) electrospray ionization/mass spectrometer,) using a solution of
0.1% formic acid in acetonitrile as solvent injection. Samples were dissolved in acetonitrile (4 mg/mL)
prior to injection into the instrument. Melting points were determined on a capillary Thomas-Hoover
melting point apparatus (Swedesboro, NJ, USA).
4.2. Synthesis
4.2.1. General Procedure for the Preparation of ω-Bromo-α,α-diphenyl Alcohols 9
The methodology used was according to Horner et al. [23]. All glassware was flame-dried under an
inert atmosphere, and reactions were carried out under argon or dry nitrogen. A commercially available
ethereal 3M solution of phenylmagnesium bromide (an appropriate volume for 2.6 equivalents) was
poured into a three-necked, round-bottomed flask that was equipped with an addition funnel and a
vertical condenser. The solution was cooled with an ice-salt bath and then an ethereal solution of the
respective
ω-bromoacyl chloride 8 (1 equivalent) was slowly added from the addition funnel with
continuous magnetic stirring while the reaction mixture was kept at about 0 ˝C. Once the addition
was finished, the reactant mixture was kept at 0 ˝C during 15 min and then allowed to slowly reach
room temperature. After that, a saturated aqueous NH4Cl solution was carefully added and the
whole mixture was transferred to a separatory funnel. The organic layer was separated, washed with
saturated aqueous NaCl solution and dried with anhydrous MgSO4. After filtration, solvent was
removed by rotary evaporation under reduced pressure. The products were purified by flash column
chromatography (hexane:ethyl acetate 5:1 as eluent). White solids or yellow oils were usually obtained.

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4-Bromo-1,1-diphenylbutanol (9a). Starting from 4-bromobutanoyl chloride 8a (5.4 mmol), 9a was
obtained as a yellow oil (78% yield). 1H-NMR (CDCl3, 300 MHz)
δ 1.80–1.90 (m, 2H), 2.54–2.94
(m, 2H), 3.33 (t, J = 6.9 Hz, 2H), 7.01–7.41 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz)
δ 22.92, 30.91,
36.18, 75.34, 123.10–142.24 ppm.
5-Bromo-1,1-diphenylpentanol (9b). Starting from 5-bromopentanoyl chloride 8b (5.0 mmol), 9b was
obtained as a white solid (68% yield). 1H-NMR (CDCl3, 300 MHz)
δ 1.53–1.63 (m, 2H), 2.02–2.12
(m, 2H), 2.31–2.41 (m, 2H), 3.47 (t, J = 6.9 Hz, 2H), 7.23–7.63 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz)
δ 23.12, 28.97, 33.62, 33.97, 41.51, 126.53–143.53 ppm.
6-Bromo-1,1-diphenylhexanol (9c). Starting from 6-bromohexanoyl chloride 8c (11.7 mmol), 9c was
1
obtained as a yellow oil (72% yield). H-NMR (CDCl3, 300 MHz)
δ
1.47–1.57 (m, 2H), 1.91–2.01 (m, 2H),
2.03–2.13 (m, 2H), 2.27–2.37 (m, 2H), 3.39–3.49 (m, 2H), 7.17–7.57 (m, 10H) ppm. 13C-NMR (CDCl3,
75 MHz) δ 23.07, 28.90, 33.53, 33.56, 33.87, 41.46, 126.45–143.48 ppm.
4.2.2. General Procedure for the Preparation of ω-Bromo-α,α-diphenyl Olefins 10
The methodology was according to Horner et al. [18]. A round-bottomed flask was equipped
with a Dean-Stark trap and a vertical condenser. Alcohols
9 (a, b or c) and p-toluenesulfonic acid
monohydrate (p-TsOH H2O) in a molar ratio of about 60:1 were dissolved in benzene and refluxed
¨
during 6 h. The approximate concentration of the solution was 5 M. After cooling down, the crude
mixture was washed with a saturated NaHCO3 aqueous solution and then with a saturated NaCl
solution. The organic layer was dried with anhydrous MgSO4 and filtered, and the solvent was
removed by rotary evaporation under reduced pressure. The products were purified by flash column
chromatography using hexane as eluent.
4-Bromo-1,1-diphenyl-1-butene (10a). Starting from 9a (3.9 mmol), 10a was obtained as a yellow oil
1
(90% yield). H-NMR (CDCl3, 300 MHz)
δ 2.54–2.64 (m, 2H), 3.32 (t, J = 6.9 Hz, 2H), 5.99 (t, J = 7.2 Hz,
1H), 7.04–7.34 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz) δ 30.57, 30.82, 123.60–142.20 ppm.
5-Bromo-1,1-diphenyl-l-pentene (10b). Starting from 9b (3.1 mmol), 10b was obtained as a yellow oil
1
(82% yield). H-NMR (CDCl3, 300 MHz)
δ 1.81–1.97 (m, 2H), 2.08–2.22 (m, 2H), 3.27 (t, J = 6.9 Hz,
2H), 5.94 (t, J = 7.5 Hz, 1H), 7.01–7.31 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz)
δ 26.35, 30.97, 31.23,
124.97–141.23 ppm.
6-Bromo-1,1-diphenyl-l-hexene (10c). Starting from 9c (8.2 mmol), 10c was obtained as a yellow oil
1
(84% yield). H-NMR (CDCl3, 300 MHz)
δ 1.66–1.74 (m, 2H), 1.91–2.01 (m, 2H), 2.26 (c, J = 7.4 Hz, 2H),
3.47 (t, J = 6.9 Hz, 2H), 6.18 (t, J = 7.4 Hz, 1H), 7.24–7.54 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz)
δ
28.85, 29.26, 32.75, 34.12, 127.42–143.05 ppm.
4.2.3. General Procedure for the Preparation of ω-(N,N-Dimethylamino)-α,α-diphenyl Olefins 11
The synthesis of
methodology reported by Ríos et al. [24]. The reaction of
ω
-(N,N-dimethylamino)-
α
,
α
-diphenyl olefins 11 was carried out according to the
-bromo- -diphenyl olefins 10 with a 40%
ω
α,α
aqueous dimethylamine solution was carried out using the appropriate volume for a molar ratio 26:1
of amine to olefin 10. The aqueous dimethylamine solution was poured into a round-bottomed flask,
and then a tetrahydrofuran (THF) solution of 10(a, b or c) was slowly added to the amine solution.
The mixture was stirred at room temperature overnight and then it was transferred to a separatory
funnel and extracted with diethyl ether. The organic layer was separated and extracted with aqueous
10% HCl. The aqueous extract was neutralized with aqueous 10% NaOH and it was extracted again
with diethyl ether. The ether extract was dried with anhydrous MgSO4 and filtered. The solvent was
removed by rotary evaporation under reduced pressure, yielding the expected amines as yellow oils.
N,N-Dimethyl-4,4-diphenyl-3-butenylamine (11a). Starting from 10a (3.1 mmol), 11a was obtained as
yellow oil (50% yield). 1H-NMR (CDCl3, 300 MHz)
δ 2.07 (s, 6H), 2.12–2.22 (m, 2H), 2.23–2.33

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(m, 2H), 5.97 (t, J = 7.3 Hz, 1H), 7.00–7.30 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz)
δ
26.14, 43.25,
57.52, 124.76–140.43 ppm.
N,N-Dimethyl-5,5-diphenyl-4-pentenylamine (11b). Starting from 10b (2.2 mmol), 11b was obtained as
1
yellow oil (68% yield). H-NMR (CDCl3, 300 MHz)
δ 1.44–1.54 (m, 2H), 2.00–2.10 (m, 2H), 2.14 (s, 6H),
2.21–2.31 (m, 2H), 5.98 (t, J = 7.5 Hz, 1H), 7.00–7.30 (m, 10H) ppm. 13C-NMR (CDCl3, 75 MHz)
δ 25.34,
26.26, 43.23, 57.71, 124.28–140.60 ppm.
N,N-Dimethyl-6,6-diphenyl-5-hexenylamine (11c). Starting from 10c (6.5 mmol), 11c was obtained as
1
yellow oil (61% yield). H-NMR (CDCl3, 300 MHz)
δ
1.25–1.34 (m, 2H), 1.48–1.57, (m, 2H), 2.18–2.29
(m, 2H), 2.20–2.29 (m, 2H), 2.26 (s, 6H), 6.16 (t, J = 7.5 Hz, 1H), 7.20–7.51 (m, 10H) ppm. 13C-NMR
(CDCl3, 75 MHz) δ 27.78, 28.24, 30.06, 45.94, 60.10, 127.21–143.26 ppm.
4.2.4. General Procedure for the Preparation of Quaternary Ammonium Salts 1–3 (Series I and II)
Synthesis of series I and II of quaternary ammonium salts was according to the methodology
reported by Ríos et al. [24]. The reaction of
ω-(N,N-dimethylamino)-α,α-diphenyl olefins 11 a, b or c)
with a 50% acetonitrile solution of XCH2I (with X= Cl or I for the series I, and X = H for the series II)
was carried out using a 4:1 molar ratio of XCH2I to amine 11. The starting tertiary amine 11 and the
acetonitrile solution of the respective XCH2I substrate were mixed in a round-bottomed flask, and the
reactions were allowed to run overnight in the dark. The precipitated salts were filtered off and then
washed several times with diethyl ether or ethyl acetate, and then recrystallized from water. The pure
quaternary ammonium salts were obtained as white solids.
N-Chloromethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (1a). Starting from 11a and
˝
ClCH2I, salt 1a (85% yield) was obtained as a white solid, m.p. 140–142 C. 1H-NMR (D2O, 300 MHz)
δ
2.31–2.53 (m, 2H), 3.02 (s, 6H), 3.37–3.55 (m, 2H), 5.11 (s, 2H), 5.99 (t, J = 7.3 Hz, 1H), 7.04–7.47
(m, 10H) ppm. 13C-NMR (D2O, 75 MHz)
δ
23.31, 49.42, 61.43, 68.13, 121.97, 127.18–129.46, 138.76,
141.46, 145.22 ppm. Elemental analysis: calc. for C19H23NICl: C, 53.35%; H, 5.42%; N, 3.27%; found C,
53.35%; H, 5.34%; N, 3.28%. MS-ESI m/z calc. for [C19H23NCl]+: 300.15 amu, found: 300.20 amu.
N-Chloromethyl-N,N-dimethyl-N-(5,5-diphenylpent-4-en-1-yl)ammonium iodide (1b). Starting from 11b
and ClCH2I, salt 1b (84% yield) was obtained as a white solid, m.p. 121–124 ˝C. 1H-NMR (DMSO,
300 MHz)
δ
1.83–1.93 (m, 2H), 2.06–2.16 (m, 2H), 3.12 (s, 6H), 3.38–3.45 (m, 2H), 5.33 (s, 2H), 6.12
24.96, 29.09, 52.01, 64.92,
(t, J = 7.2 Hz, 1H), 7.07–7.53 (m, 10H) ppm. 13C-NMR (DMSO, 75 MHz)
δ
71.35, 129.93–132.46, 142.13, 144.69, 145.58 ppm. Elemental analysis: calc. for C20H25NICl: C, 54.37%;
H, 5.70%; N, 3.17%; found C, 53.39%; H, 5.57%; N, 3.17%. MS-ESI m/z calc. for [C20H25NCl]+: 314.17
amu, found: 314.20 amu.
N-Chloromethyl-N,N-dimethyl-N-(6,6-diphenylhex-5-en-1-yl)ammonium iodide (1c). Starting from 11c and
˝
ClCH2I, salt 1c (61% yield) was obtained as a white solid, m.p. 119–121 C. 1H-NMR (CDCl3, 400 MHz)
δ
1.53–1.63 (m, 2H), 1.70–1.80 (m, 2H), 2.18–2.26 (m, 2H), 3.46 (s, 6H), 3.60–3.68 (m, 2H), 5.64 (s, 2H),
6.05 (t, J = 7.4 Hz, 1H), 7.12–7.42 (m, 10H) ppm. 13C-NMR (CDCl3, 101 MHz)
δ
22.23, 26.25, 28.91, 49.96,
63.15, 68.92, 127.16–129.82, 139.76, 142.25, 142.97 ppm. Elemental analysis: calc. for C21H27NICl: C,
55.34%; H, 5.97%; N, 3.07%; found C, 55.10%; H, 5.95%; N, 3.10%. MS-ESI m/z calc. for [C21H27NCl]+:
328.18 amu, found: 328.20 amu.
N-Iodomethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (2a). Starting from 11a and
1
CH2I2, salt 2a (74% yield) was obtained as a white solid, m.p. 152–154 ˝C. H-NMR (DMSO,
300 MHz
δ
, ppm.): 2.43–2.53 (m, 2H), 3.12 (s, 6H), 3.45–3.55 (m, 2H), 5.05 (s, 2H), 6.07 (t, J = 7.4 Hz,
1H), 7.15–7.58 (m, 10H) ppm. 13C-NMR (DMSO, 75 MHz,
δ
, ppm) 23.70, 31.49, 51.66, 63.58, 121.92,
δ
127.19–129.51, 138.79, 141.62, 145.03 ppm. Elemental analysis: calculated for C19H23NI2: C, 43.95%;
H, 4.46%; N, 2.70%; found, C, 43.48%; H, 4.35%; N, 2.68%. MS-ESI m/z calculated for cation
[C19H23NI]+: 392.09 amu, found: 391.95 amu.

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N-Iodomethyl-N,N-dimethyl-N-(5,5-diphenyl-4-penten-1-yl)ammonium iodide (2b).˝Starting from 11b and
1
CH2I2, salt 2b (77% yield) was obtained as a white solid, m.p. 157–158 C. H-NMR (DMSO,
300 MHz δ, ppm): 1.80–1.90 (m, 2H), 2.07–2.17 (m, 2H), 3.15 (s, 6H), 3.31–3.40 (m, 2H), 5.18 (s, 2H), 6.14
(t, J = 7.2 Hz, 1H), 7.11–7.51 (10H). 13C-NMR (DMSO, 75 MHz,
δ, ppm): 22.30, 25.91, 39.01, 51.19, 63.84,
126.84–141.68. ESI-MS m/z calc. for [C20H25NI]+: 406.10 amu, found: 406.20 amu.
N-Iodomethyl-N,N-dimethyl-N-(6,6-diphenylhex-5-en-1-yl)ammonium iodide (2c). Starting from 11c and
˝
CH2I2, salt 2c (72% yield) was obtained as a white solid, m.p. 156–158 C. 1H-NMR (DMSO, 300 MHz
δ
, ppm): 1.42–1.46 (m, 2H), 1.64–1.72 (m, 2H), 2.08–2.15 (m, 2H), 3.10 (s, 6H), 3.33–3.44 (m, 2H), 5.14
(s, 2H), 6.14 (t, J = 7.3 Hz, 1H), 7.06–7.51 (m, 10H) ppm. 13C-NMR (DMSO, 75 MHz,
δ
, ppm): 25.07,
28.91, 31.91, 35.35, 54.29, 67.34, 129.89–132.50, 130.19, 142.45, 144.34, 145.02. Elemental analysis: calc.
for C21H27NI2: C, 46.09%; H, 4.97%; N, 2.56%; found C, 45.91%; H, 4.93%; N, 2.58%. MS-ESI m/z calc.
for [C21H27NI]+: 420.12 amu, found: 420.20 amu.
N,N,N-Trimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (3a). Starting from 11a and CH3I, salt 3a
(82% yield) was obtained as a white solid, m.p. 190–192 ˝C. 1H-NMR (DMSO, 300 MHz)
δ
2.54–2.63
(m, 2H), 3.11 (s, 9H), 3.53–3.63 (m, 2H), 6.13 (t, J = 7.1 Hz, 1H), 7.18–7.66 (m, 10H) ppm. 13C-NMR
(DMSO 75 MHz,) δ 24.03, 52.52, 64.92, 123.28–144.69 ppm. Elemental analysis: calc. for C19H24NI: C,
58.02%; H, 6.15%; N, 3.56%; found C, 56.71%; H, 6.23%; N, 3.52%. MS-ESI m/z calc. for [C19H24N]+:
266.19 amu, found: 266.20 amu.
N,N,N-Trimethyl-N-(5,5-diphenylpent-4-en-1-yl)ammonium iodide (3b). Starting from 11b and CH3I, salt
˝
3b (92% yield) was obtained as a white solid, m.p. 196–197 C. 1H-NMR (DMSO, 400 MHz)
δ 1.87–1.93
(m, 2H), 2.24 (c, J = 7.1 Hz, 2H), 3.37 (s, 9H), 3.43–3.55 (m, 2H), 6.08 (t, J = 7.1 Hz, 1H), 7.10–7.45
(m, 10H) ppm. 13C-NMR (DMSO, 101 MHz,) δ 23.41, 26.19, 53.32, 66.67, 125.90, 127.14–128.92, 139.39,
141.82, 143.96 ppm. Elemental analysis: calc. for C20H26NI: C, 58.97%; H, 6.43%; N, 3.44%; found C,
55.93%; H, 6.10%; N, 3.23%. MS-ESI m/z calc. for [C20H26N]+: 280.21 amu, found: 280.20 amu.
N,N,N-Trimethyl-N-(6,6-diphenylhex-5-en-1-yl)ammonium iodide (3c). Starting from 11c and CH3I, salt 3c
(72% yield) was obtained as a white solid, m.p. 202–204 ˝C. 1H-NMR (DMSO, 500 MHz)
δ
1.40–1.46
(m, 2H), 1.64–1.72 (m, 2H), 2.11 (c, J = 7.4 Hz, 2H), 3.03 (s, 9H), 3.20–3.30 (m, 2H), 6.16 (t, J = 7.4 Hz, 1H),
7.10–7.49 (m, 10H) ppm. 13C-NMR (DMSO, 126 MHz)
22.22, 26.22, 29.12, 52.69, 65.82, 127.10–129.12,
δ
129.71, 139.78, 141.76, 142.26 ppm. Elemental analysis: calc. for C21H28NI: C, 59.86%; H, 6.70%;
N, 3.32%; found C, 59.49%; H, 6.74%; N, 3.35%. MS-ESI m/z calc. for [C21H28N]+: 294.22 amu, found:
294.20 amu.
4.2.5. General Procedure for the Preparation of Choline 4c and Choline Analogs 4a and 4b (Series III)
The compounds 4a, 4b and 4c were prepared following the procedure reported by Mistry et al. [25]
starting from N,N-dimethylethanolamine 12 and XCH2I in THF (with X = H, Cl or I), using a 1.5:1
molar ratio of XCH2I to ethanolamine. The reaction mixture was stirred in the dark for 24 h at room
temperature. The gummy crude product was washed with dry THF, and the resulting salts were
filtered off to obtain the products as white solids.
N-Choromethyl-N,N-dimethyl-N-(2-hydroxyet-1-yl)ammonium iodide (4a). Starting from N,N-dimethyl-
˝
ethanolamine (12) and ClCH2I, 4a (51.2% yield) was obtained as a white solid, m.p. 101–103 C
1H-NMR (D2O, 300 MHz)
δ 3.32 (s, 6H), 3.62–3.74 (m, 2H), 4.01–4.10 (m, 2H), 5.28 (s, 2H) ppm.
13C-NMR (D2O, 75 MHz) δ 33.11, 53.14, 55.95, 66.46 ppm.
N-Iodomethyl-N,N-dimethyl-N-(2-hydroxyet-1-yl)ammonium iodide (4b). Starting from N,N-dimethyl-
˝
ethanolamine (12) and CH2I2, 4b (61.5% yield) was obtained as a white solid, m.p. 96–97 C. 1H-NMR
(D2O, 300 MHz)
(D2O, 75 MHz) δ 33.32, 53.22, 56.14, 66.51 ppm.
δ
3.33 (s, 6H), 3.65–3.74 (m, 2H), 4.01–4.13 (m, 2H), 5.29 (s, 2H) ppm. 13C-NMR

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(2-Hydroxyethyl)trimethylammonium iodide (choline) (4c). Starting from N,N-dimethylethanolamine (12
and CH3I, 4c (88% yield) was obtained as a white solid, m.p. 268–270 ˝C. 1H-NMR (D2O, 300 MHz),
3.20 (s, 9H), 3.49–3.55 (m, 2H), 4.00–4.10 (m, 2H) ppm. 13C-NMR (D2O; 75 MHz)
54.39, 55.63,
67.98 ppm.
)
δ
δ
The spectral data of the synthesized -bromo-
ω
α,α-diphenyl alcohols 9, ω-bromo-α,α-diphenyl
olefins 10 and -(N,N-dimethylamino)-α,α-diphenyl olefins 11 were in agreement with those reported
ω
previously by Horner et al. [18]. All compounds were stored at room temperature until use. Prior to the
biological testing, each compound was solubilized in 0.5% dimethyl sulfoxide (DMSO; Sigma-Aldrich,
St. Louis, MO, USA), and then diluted to the appropriate concentration in culture media.
4.3. Biological Activity
4.3.1. Cells and Culture Conditions
U-937 promonocytes (CRL1593.2™) were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA), cultured in complete medium containing RPMI 1640 medium (Sigma)
supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY, USA)
and 1% antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin) (Sigma). Cells were maintained
˝
in standard conditions at 37 C, 5% CO2 with change of medium every three days until use.
4.3.2. Leishmania Strain and Cultivation of Parasites
Virulent L. (V) panamensis MHOM/CO/87/UA140 strain transfected with green fluorescent
protein gene called L. (V) panamensis-pIR-eGFP was maintained by successive passaging in golden
hamsters (Mesocricetus auratus). Briefly, amastigotes were aspirated from cutaneous lesions of hamsters
experimentally infected with promastigotes of L. (V) panamensis-pIR-eGFP. Samples were cultured in
biphasic culture with Novy-MacNeal-Nicholle (NNN) medium as solid phase and PBS plus glucose,
pH 6.9 as liquid phase and incubated at 26 ˝C to obtain promastigotes. Medium was changed every
week until 2 months.
The axenic amastigotes were obtained from promastigotes cutured in Schneider’s medium, as
described elsewhere [26]. Briefly, promastigotes of L. (V) panamensis-pIR-eGFP at 5
ˆ
106 parasites/mL
were cultured in Schneider medium (Sigma), pH 5.4 supplemented with 20% FBS and 1% antibiotics
˝
and incubation at 26 C. Every three days, medium was changed and temperature was increased one
degree each time until it reached 32 ˝C at which the transformation of the promastigotes into axenic
amastigotes is achieved. After three days of growing as axenic amastigotes, parasites were ready to be
used in the antileishmanial testing assay as described below.
Intracellular amastigotes were obtained after infection of U-937 cells with promastigotes. Briefly,
U-937 cells were dispensed in 24-well plates at a concentration of 300,000 cells/well and were treated
with 1 µ
M of phorbol myristate acetate (PMA) (Sigma) for 48 h at 37 ˝C. Then, cells were infected
with promastigotes in stationary growth phase (day 5) at a ratio of 1:15 cell/parasite and incubated for
˝
3 h at 34 C in 5% CO2. Cells were washed twice with PBS to eliminate not internalized parasites and
fresh RPMI-1640 was added into each well (1 mL); plates were incubated again at 34 ˝C and 5% CO2
to allow intracellular differentiation of promastigotes to amastigotes form. After 24 h of infection, cells
were ready to be used in antileishmanial testing assay as described below.
4.3.3. In Vitro Cytotoxicity Using U-937 Cells
The cytotoxicity of compounds was assessed based on the viability of U-937 cells and evaluated
by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method as described by
others [26]. Briefly, into each well of a 96-well cell-culture dishes 100,000 cells/100
in RPMI-1640 supplemented with 10% FBS and 100 L of the corresponding concentrations of the
corresponding product. Six two fold serial dilutions were were tested starting at 200 g/mL (200,
g/mL). Cells were incubated at 37 C with 5% CO2 for 72 h in the presence
µL were dispensed
µ
µ
˝
100, 50, 25, 12.5 and 6.25
µ

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of compounds, and then the effect was determined by measuring the activity of the mitocho˝ndrial
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 100 µL /well of 50% isopropanol solution with 10% sodium
dodecyl sulfate for 30 min. Cell growth was determined based on the quantity of formazan produced,
which was measured at 570 nm in a plate reader spectrophotometer (Bio-Rad, Hercules, CA, USA). Cells
cultured in the absence of products were used as cell growth controls (negative control), while cells
cultured in presence of amphotericin B (AmB, Sigma) and meglumine antimoniate (MA, Sanofi-Aventis,
Bogotá, Colombia) were used as cytotoxicity controls (positive control). Each concentration was tested
in triplicate in at least two independent experiments.
4.3.4. In Vitro Leishmanicidal Activity on Intracellular Amastigotes
The effect of compounds against intracellular amastigotes of L. (V) panamensis-pIR-eGFP was
evaluated by flow cytometry using the methodology described by others [27]. After 24 h of infection
of U-937 cells, culture medium was replaced by fresh complete RPMI 1640 medium containing
each compound at the corresponding concentration (four serial dilution were prepared starting at
a concentration n˝ot exceeding the LC50 previously determined). Infected and treated cells were
maintained at 34 C and 5% CO2 for 72 h. After 72 h, cells were washed twice with 1 mL of cold
PBS and cells were removed from the bottom plate with a trypsin/EDTA (250 mg/mL) solution.
˝
Recovered cells were centrifuged at 1100 rpm for 10 min at 4 C, the supernatant was discarded
˝
and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm for 10 min at 4 C.
Supernatant was discarded and cells were suspended in 500 L of PBS and analyzed by flow cytometry
µ
(FC 500MPL, Cytomics, Brea, CA, USA) using histogram analyses (Figure 2). Infected cells treated with
AmB or MA were used as control for antileishmanial activity (positive control) while infected cells
incubated in culture RPMI 1640 medium alone were used as control for infection (negative control).
Each concentration of compounds was tested in triplicate in two independent experiments.
Figure 2. Flow cytometry analysis of GFP expressing parasites exposed to active and non-active
N-(halomethylated) ammonium salts, non N-(halomethylated) ammonium salts and choline and
halogenated cholines. The figure shows a representative fluorescence intensity signal from U-937
cells infected with L. (V) panamensis-pIR-eGFP exposed to any of the N-(halomethylated) ammonium
salts, non N-(halomethylated) ammonium salts and choline and halogenated cholines compound.
(a) shift of the relative increase in the average fluorescence for L. (V) panamensis-pIR-eGFP compared
to plain parasites; (b) black histogram represents the signal produced by Leishmania exposed to non
active compounds and; (c) green histogram represents the signal produced by parasites exposed to
active compounds.

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4.3.5. In Vitro Leishmanicidal Activity with Axenic Amastigotes
The ability of compounds to kill axenic amastigotes of L. (V) panamensis was determined based
on the viability of the parasites after exposure to each compound evaluated by the MTT method
following the methodology described by others [26]. Axenic amastigotes obtained as described above
were harvested, washed, and adjusted at 2
20% FBS. Into each well of a 96-well plate 100
were dispensed and then 100 L of each concentration of the corresponding compound were added.
Four serial dilutions were prepared starting at 100 g/mL (100, 25, 6.25 and 1.56 g/mL). Plates were
ˆ
106 parasites/mL in fresh Schneider’s medium with
L of parasite suspension (2
105 axenic amastigotes)
µ
ˆ
µ
µ
µ
incubated at 32 ˝C. After 72 h of incubation, the effect of each compound was determined by adding
10 µL/well of MTT and incubating at 32 ˝C for 3 h. The reaction was stopped as described above
and the quantity of formazan produced was measured in a plate reader spectrophotometer (Bio-Rad)
set at 570 nm. Axenic amastigotes cultivated in the absence of any compound but maintained under
the same conditions were used as controls for growth and viability (negative control) while parasites
cultivated in the presence of MA were used as controls for leishmanicidal activity (positive control).
Assays were performed at least twice with three replicates per each concentration tested.
4.3.6. Data Analysis
Cytotoxicity was determined according to the percentages of cell growth (viability) obtained
for each tested compound or medium alone. Percentages of viability were calculated first using
Equation (1):
% Viability “ pO.D. of treated cellsq{pO.D. of untreated cellsq ˆ 100
(1)
where the O.D. of the untreated cells corresponds to 100% viability. Then, the percentage of cell growth
inhibition was calculated using Equation (2):
% Cell growth inhibition “ 100 ´ % Viability
(2)
Both % viability and % cell growth inhibition were used to calculate the Lethal Concentration
50 (LC50) that corresponds to the concentration of drug that gives the half-maximal reduction of the
cell growth using the Probit model [28]. The degree of cytotoxicity was established as convenient
according to the LC50 values, using our own scale: high cytotoxicity: LC50 < 100 µg/mL, moderate
cytotoxicity: 100 µg/mL < LC50 < 200 µg/mL; and potential non-cytotoxicity: LC50 > 200 µg/mL.
The antileishmanial activity in intracellular amastigotes was determined according to the
reduction of infected cells and parasite load (viable parasites inside infected cells) obtained for
each experimental condition. The percentage of infected cell inhibition and parasite load for each
concentration of each compound were calculated first using Equation (3):
% infection “ p% infected and treated cells ˜ % infected and untreated cellsq ˆ 100
And then, the percentage of infection inhibition was calculated using Equation (4):
% Inhibition “ 100 ´ % infection
(3)
(4)
In turn, activity of compounds with axenic amastigotes was determined according to the reduction
of parasite growth percentages obtained for each experimental condition. The percentage of parasite
growth was calculated first using Equation (5):
% parasite growth “ pO.D. treated ax. amastigotesq{pO.D. of untreated ax. amastigotesq ˆ 100 (5)

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where the O.D. of the untreated axenic amastigotes corresponds to 100% parasite growth. Then, the
percentage of parasite growth inhibition was calculated using Equation (6):
% parasite growth inhibition “ 100 ´ % parasite growth
(6)
The % of infection in U-937 cells and the % of inhibition of axenic amastigotes growth were used
to calculate the Effective Concentration 50 (EC50) that corresponds to the concentration of drug that
gives the half-maximal reduction of the infected cells and parasites using the Probit model [28].
The degree of leishmanicidal activity was also established as convenient according to the EC50
values, using our own scale: high leishmanicidal activity: EC50 < 25 µg/mL, moderate activity:
25 µg/mL < EC50 < 50 µg/mL; and potential non-activity: EC50 > 50 µg/mL.
The Selectivity Index (SI) was calculated by comparing the cytotoxicity and the leishmanicidal
activity using Equation (7):
SI “ LC50{EC50
(7)
5. Conclusions
Based on in vitro activity (EC50,
µg/mL) against L. (V) panamensis, it was determined that the
most active compound against L. (V) panamensis is compound 2c, which has the longest carbon chain
and a iodomethyl group on the nitrogen. When comparing the halomethylated salts 1 and 2 (series I)
it is observed that their activity decreases with decreasing the carbon number of the salts that bear
an iodomethyl group as a substituent, such that 2c is more effective than 2b and this, in turn, is more
effective than 2a. On the other hand, as a general trend, the salts bearing an iodomethyl group are more
effective against L. (V) panamensis parasites than those bearing a chloromethyl group as a substituent.
Currently, the structure-activity relationships via structural variations is being carried out.
Acknowledgments: This work was funded by Universidad de Caldas, Universidad de Antioquia (Codi-CIIEs)
and Illinois State University.
Author Contributions: L.A.R.-V., S.M.R., D.L.C. and R.O.-C. conceived and designed the experiments; S.M.D.-B.
and S.M.R. performed the experiments; L.A.R.-V., R.O.-C., S.M.D.-B., S.M.R., I.D.V., D.L.C., and M.A.J. analyzed
the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
VL
ML
CL
MA
AmB
EC50
LC50
FBS
Visceral leishmaniasis
Mucocutaneous leishmaniasis
Cutaneous leishmaniasis
Meglumine antimoniate
Amphotericin B
Effective Concentration 50
Lethal Concentration 50
Fetal Bovine Serum
NNN
PMA
RPMI
O.D.
THF
DMSO
Novy-MacNeal-Nicholle
Phorbol 12-myristate 13-acetate
Roswell Park Memorial Institute
Optical Density
Tetrahydrofuran
Dimethylsulfoxide

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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).