Novel arylalkylamine compounds exhibits potent selective antiparasitic activity against Leishmania major

Article abstract of DOI:10.1016/j.bmcl.2015.09.041

Leishmania major (L. major) is a protozoan parasite causal agent of Leishmaniasis. It is estimated that 12 million people are currently infected and around 2 million infections occur each year. Current treatments suffer of high toxicity for the patient, low efficacy toward the parasite, high cost, and are losing effectiveness due to parasite resistance. Discovering novel small molecule with high specificity/selectivity and drug-like properties for anti-leishmanial activity remains a significant challenge. The purpose of this study is to communicate the design and synthesis strategies of novel chemical compounds based of the arylalkylamine scaffold with selective toxicity towards L. major and less toxicity to human cells in vitro. Here, we have developed a structure activity relationship (SAR) study of arylalkylamine AA1 in order to study their anti-parasitic effect in L. major. Overall, 27 arylalkylamine compounds derived from AA1 were synthesized and purified by silica gel column chromatography. The purity of each analog was confirmed by spectroscopic methods (¹H, ¹³C NMR and LC/MS). Among these analogs, the compound AA9 showed the best toxic activity on L. major (LD₅₀ = 3.34 μM), which represents a 9 fold higher lethality as compared with its parental AA1 (Fer-1) compound (LD₅₀ = 28.75 μM). In addition, AA9 showed no significant toxicity at 80 μM on U20S Human Osteoblasts, Raw 264.7 Macrophages or intraperitoneal macrophages. In summary, our combined SAR study and biological evaluation data of AA1-AA27 compounds allow the identification of novel arylalkylamine compound AA9 that exhibits potent cytotoxicity against L. major promastigote with minimum toxic effect on human cells.

Full text of DOI:10.1016/j.bmcl.2015.09.041

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel arylalkylamine compounds exhibits potent selective
antiparasitic activity against Leishmania major
a,b,c,
Eva A. Iniguez a, Andrea Perez b, Rosa A. Maldonado a,c, , Rachid Skouta
⇑
⇑
a Department of Biological Sciences, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79902, USA
b Department of Chemistry, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79902, USA
c Border Biomedical Research Center, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79902, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Leishmania major (L. major) is a protozoan parasite causal agent of Leishmaniasis. It is estimated that 12
million people are currently infected and around 2 million infections occur each year. Current treatments
suffer of high toxicity for the patient, low efficacy toward the parasite, high cost, and are losing effective-
ness due to parasite resistance. Discovering novel small molecule with high specificity/selectivity and
drug-like properties for anti-leishmanial activity remains a significant challenge. The purpose of this
study is to communicate the design and synthesis strategies of novel chemical compounds based of
the arylalkylamine scaffold with selective toxicity towards L. major and less toxicity to human cells
in vitro. Here, we have developed a structure activity relationship (SAR) study of arylalkylamine AA1
in order to study their anti-parasitic effect in L. major. Overall, 27 arylalkylamine compounds derived
from AA1 were synthesized and purified by silica gel column chromatography. The purity of each analog
was confirmed by spectroscopic methods (1H, 13C NMR and LC/MS). Among these analogs, the compound
Received 30 July 2015
Revised 14 September 2015
Accepted 16 September 2015
Available online xxxx
Keywords:
Leishmania major
Amphotericin B
Arylalkylamine compounds
Mammalian cells
AA9 showed the best toxic activity on L. major (LD50 = 3.34
as compared with its parental AA1 (Fer-1) compound (LD50 = 28.75
nificant toxicity at 80 M on U20S Human Osteoblasts, Raw 264.7 Macrophages or intraperitoneal
lM), which represents a 9 fold higher lethality
l
M). In addition, AA9 showed no sig-
l
macrophages. In summary, our combined SAR study and biological evaluation data of AA1–AA27 com-
pounds allow the identification of novel arylalkylamine compound AA9 that exhibits potent cytotoxicity
against L. major promastigote with minimum toxic effect on human cells.
Ó 2015 Elsevier Ltd. All rights reserved.
Leishmaniasis caused by protozoan parasites Leishmania major,
affect millions of people worldwide.1 This disease is among the
World Health Organization’s Neglected Tropical Diseases (NTD)
list.2 It has a wide range of clinical symptoms and over 20 species
and subspecies of Leishmania that can infect humans, causing three
different diseases: visceral (VL), cutaneous (CL), or mucocutaneous
leishmaniasis (MCL).3 This infection is encountered in tropical and
subtropical regions of the world. Approximately 350 million people
are at risk in 88 countries around the world.4 An estimated 12 mil-
lion people are currently infected and around 2 million infections
occur each year.5 Currently, there is no human vaccine available
to prevent or treat these parasitic infections despite numerous
efforts.6 Several drugs were utilized to cure Leishmaniasis; for
example, the chemotherapeutic pentavalent antimonial drugs
(meglumine antimoniate and stibogluconate, Scheme 1) are still
used for treatment.7 Nevertheless, this treatment requires daily
injections for 28 days and recently an increase of therapeutic fail-
ure cases were reported.8,9 The 2-(hexadecoxy-oxido-phosphoryl)
oxylethyl-trimethyl-azanium (miltefosine, Scheme 1) was
reported to be the first oral drug and showed low toxicity in the
treatment of leishmaniasis, though there is no evidence that
miltefosine is better than meglumine antimony.10–12 On the other
hand, the polyene antibiotic amphotericin B (Scheme 1) is the most
extensively used drugs for this disease.13,14 Recently, Wyllie and
co-workers reported that the 1-methyl-2-((4-(methylthio)
phenoxy)methyl)-5-nitro-1H-imidazole (fexinidazole, Scheme 1),
a drug currently in phase 1 clinical trials for treating African try-
panosomiasis, displays potential for treating visceral leishmania-
sis.15 The absence of highly efficient and potent new drugs or
vaccines to control L. major makes the search for active chemother-
apeutic agents an ‘urgent priority’ in parasitic research.16,17
Maldonado and co-workers reported that the cationic steroid
antibiotics (ceragenins) containing the sterol backbone bear-
ing amino acids were effective compounds with submicromolar
potency against L. major promastigotes.18 The same research group
⇑
Corresponding authors.
E-mail addresses: ramaldonado@utep.edu (R.A. Maldonado), rskouta@utep.edu
(R. Skouta).
http://dx.doi.org/10.1016/j.bmcl.2015.09.041
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Iniguez, E. A.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.041

2
E. A. Iniguez et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
HO
OH
O
H2N
HO
HO
OH
HO
O
OH
OH
OH
O
O
Sb
OH
O
Sb
O
O
OH
OH
O
O
HO
OH
OH
OH
O
O
HN
Meglumine antimoniate
O
Stibogluconate
OH
OH
O
O
O
HO
OH
P
N
O
HO
O
OH
OH
Amphotericin B
Miltefosine
OH
OH
OH
OH
HO
O
O-
N+
O
N
HO
O
O
O
N
O
OH
OH
OH
OH
S
OH
O
Quercetin
Epigallocatechin gallate
Fexinidazole
OH
Scheme 1. Chemical structures of known anti-leishmania compounds.
NO2
NO2
described the synthesis and the high biological activity of new
ruthenium(II) complexes of ketoconazole against L. major nontoxic
to human or murine normal cells.19,20 A recent study suggested
that biological compounds that involve the production of reactive
oxygen species (ROS) may induce cell death in various leishmani-
asis.21,22 Almeida-Amaral and co-workers reported that the
flavonol quercetin and the epigallocatechin gallate (Scheme 1),
known potent antioxidants, induced death in Leishamania amazo-
nensis and braziliensis, respectively.21,22 Recently, Stockwell and
co-workers reported the discovery of an arylalkylamine AA1-type
compound (Fer-1) that was shown to be a potent and specific inhi-
bitor of a unique form of non-apoptotic, iron-dependent, oxidative
cell death, known as ferroptosis.23,24 The Fer-1 compound inhibits
the lipid ROS production through an anti-oxidant mechanism.
Here we demonstrate, for the first time, that the arylalkylamines
AA1–AA27 compounds with drug-like properties, particularity
the compound AA9, are lethal to the L. major promastigotes para-
sites. Using the high-content imaging assay, we indicated that
compound AA9 inhibits the proliferation of L. major amastigotes
propagated in macrophages. Our mechanistic investigation
showed that AA9 mediate the cell death through the production
of ROS.
Cl
R2OH
Cl
DCC, DMAP
X
O
X
OH
R2
DCM, r.t., 17h
66-85%
R1
O
R1
O
1
2
R2 = C2H5, C10H22, t-Bu,
1a: X= C; R1 = H
N
1b: X= C; R1 = F
O
1C: X= N; R1 = F
R3NH2, K2CO3
DMSO, 60 oC, 17 h
68-90%
R4
X
R5
N
NO2
H
N
H
N
1. H2, Pd (OH)2, MeoH, r.t. 17h
68-90%
R3
R3
O
X
O
,
R2
R2
R1
AA1-AA27
O
2. condition a or b
56-83%
R1
O
3
R3
t-Bu
=
R
4 = benzyl, aryl, pyrimidine, methylester
,
O
R5= H, benzyl.
H2N
AA1: X = C; R1 = H; R2 = Et
,
R
3 = cyclohexyl; R4 = benzyl; R5 = H
Scheme 2. General synthetic route of arylalkylamine compounds (AA1–AA27).
Reagents and conditions: (a) aldehydes, NaBH(OAc)3, DCE, 60 °C, 17 h; (b) ClCO2
CH3, DIPEA, DC, rt, 17 h.
Discovering small molecule with drug-like properties (highly
lethal to parasite, highly soluble and less toxic to human cells)
remains a significant challenge. We have previously reported the
design and synthesis of Pictet–Spengler condensation derivatives
(tetrahydro-b-varboline) that exhibit a specific cell death in cancer
cells.25 The purpose of this study is to communicate the design and
synthesis strategies of novel chemical compounds with selective
toxicity towards L. major and less toxicity to human cells in vitro.
We therefore developed potent small molecules based on the ary-
lalkylamine AA1 (Fer-1) scaffold. This serves towards creating
novel molecules with better antiparasitic activity and cytotoxicity.
We modified our previously reported synthesis to create 27
arylalkylamine (AA1–AA27) compounds (Scheme 2).
First, various esters (2) were prepared with a good yield (66–85%
yield) starting from commercially available acids [4-chloro-3-
nitrobenzoic acid (1a), 4-chloro-2-fluoro-5-nitrobenzoic acid (1b)
and 6-chloro-5-nitronicotinic acid (1c)] and alcohols [ethanol,
dodecanol and morpholinoethanol] using dicyclohexylcarbodi-
imide (DCC) and dimethylamine pyridine (DMAP) coupling
reagents. Second, a nucleophilic aromatic substitution reaction
(SNAr) between the prepared esters (2) and cyclohexylamine in
the presence of potassium carbonate in DMSO at 60 °C provided
68–90% yield of the nitro derivatives (3). Third, hydrogenolysis of
the nitro derivatives (3) provided the desired aniline derivatives
at 70–94% yield. Finally, the aniline of the latter compounds was
reacted through either reductive amination condition in the pres-
ence of sodium triacetoxyborohydride in the presence of arylalde-
hydes or acylation reaction to generate the arylalkylamine
derivatives (4) at 58–82% yield. All the compounds were purified
using column chromatography on silica gel and characterized using
nuclear magnetic resonance (NMR) and liquid chromatography
coupled mass spectrometry (LC/MS) (see Supporting information).
In order to evaluate the efficacy of the arylalkylamine AA1 (Fer-1)
as potential anti-parasite scaffold, we first tested its lethality in
L. major as a lead compound. Our evaluation of AA1 compound for
72 h showed modest activity in L. major strain Friedlin clone V1 Luci-
ferase promastigotes (LD50 = 28.75
lM) compared to the known
positive controls treated with Amphotericin B (at 25
lM) (Fig. 1).
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E. A. Iniguez et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
synthesized 26 additional (AA2–AA27) analogs of AA1 (Scheme 2)
to evaluate their lethality specifically in L. major promastigotes.
The lethality data (LD50) of the AA1 and analogs are shown in
Tables 1 and 2.
The SAR study of AA1 compounds and their lethality data in
L. major allow us to drive the following conclusions: First, the nitro
analog AA2 was not potent (Table 1). Second, the dodecyl and mor-
pholinoethyl esters (AA5 and AA7, Table 1) did not improve the
potency. Third, modification on the N-cyclohexyl (AA3 and AA6,
Table 1) as well on the benzene ring (AA4 and AA8 Table 1) did
not improve the potency of AA1. Fourth, reductive amination reac-
tion between AA1 and various aryl aldehydes generated additional
analogs (AA9–AA24, Tables 1 and 2). Among these analogs, the
compound AA9 (Table 1) showed the best activity in L. major
Figure 1. Evaluation of the antiparasitic activity and cytotoxicity of AA1
compound.26
(LD50 = 3.34 lM) which represent a higher lethality (up to 9 fold)
than the lead molecule (AA1) (Fig. 2).
In addition, no significant cytotoxicity of compound AA9 was
shown at a concentration up to 80 lM in U20S Human Osteoblasts,
Raw 264.7 Macrophages or intraperitoneal macrophages (Fig. 2).
Fifth, the carbamate AA25 (Table 2) was not potent which confirm
While AA1 compound has a modest activity in L. major (Fig. 1),
we were surprised to observe that AA1 is not cytotoxic in mam-
malian cells (LLC-MK2) at the highest concentration of 240 lM
(Fig. 1). Encouraged by the cytotoxicity data of AA1, we decided
to develop a SAR study of AA1 compound. We designed and
Table 1
LD50 of arylalkylamine (AA1) and analogs (AA2–AA16)
Name/structure
LD50
(lM)
Name/structure
LD50
3.34
(lM)
28.75
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
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E. A. Iniguez et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
LD50 of arylalkylamine (AA9) and analogs (AA17–AA27)
Name/structure
LD50
3.34
(lM)
Name/structure
LD50
>10
(lM)
>10
>10
>10
>10
>10
4.98
>10
>10
4.92
9.49
>10
macrophages. First, starch activated intraperitoneal macrophages
were placed in a 96 well microplate. After 24 h, macrophages were
infected with L. major metacyclic promastigote, at a density of 10
parasites per 1 cell, followed by the treatment with AA9 for 48 h.
The cells were fixed and stained with annexin-V 488 and DAPI.
The number of infected cells and amastigotes were determined
by HCIA, using BD pathway bio-imager microplate confocal micro-
scope. As observed in Figure 3A, compound AA9 successfully inhi-
bit 80% of the intracellular proliferation of the parasite at a
concentration of 5
lM as compared with negative control treated
with 1% DMSO. To confirm the efficacy of the AA9, we compared
its efficacy with the known Amphotericin B (Amp B, 25
as a positive control (Fig. 3B).
lM) used
Figure 2. Evaluation of antiparasitic activity and cytotoxicity of the lead compound
AA9.27
We hypothesized, based on the recent study21,22 that AA9 com-
pound may induce cell death in L-major through the production of
ROS. We then investigated the intracellular ROS in L-major after
treatment with AA9 compound. To do so, we assessed the reactive
oxygen species produced within the cells including superoxide,
hydrogen peroxide, and other reactive oxygen intermediates in
L. major promastigotes cells. Cells were incubated with AA9 for
the importance of the NH-benzyl moiety. Finally, the di-benzyl
AA26 and the acid AA27 analogs (Table 2) showed no improvement
of the potency. Based on our SAR study, the NH-benzyl moiety
(a hydrophobic moiety) of the AA9 compound seems very crucial
for the potency.
We then, evaluated the AA9 compound in the intracellular
proliferation of L. major using the well-established high-content
imaging assay.28,29 The effect of AA9 was determined against the
intracellular proliferation of L. major amastigotes propagated in
24 h at 3 lM. After incubation period, H2DCFDA was added and
the cells were incubated for an additional 20 min at 37 °C.
Fluorescence intensity was evaluated using a fluorescent micro-
plate reader. The levels of ROS in AA9 (3 lM) treated cells after
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E. A. Iniguez et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Figure 3. Proliferation assay of the lead compound AA9.30
by spectroscopic methods (1H, 13C NMR and LC/MS). The lead
optimized compound AA9 showed the best toxic activity on
L. major (LD50 = 3.34
lethality as compared with its parental AA1 (Fer-1) compound
(LD50 = 28.75 M) (Table 3). In contrast, AA9 was not significantly
toxic at 80 M on U20S Human Osteoblasts, Raw 264.7 Macro-
lM), which represents a 9 fold higher
l
l
phages or intraperitoneal macrophages (Table 3). We are currently
developing the synthesis of additional analogs with drug-like prop-
erties (e.g., better solubility and selective potency) for mechanism
evaluation and in vivo efficacy study in BALB/c mice infected with
transgenic L. major.
Acknowledgments
We thank Dr. Armando Varela-Ramirez from the Cell Culture
and High Throughput Screening Core Facility of the University of
Texas at El Paso for his thoughtful feedback during the preparation
of this manuscript. We would like to thank the staff of the Cytom-
etry, Imaging and Screening Core Facility (CSI) and the Biomolecule
Analysis Core Facility (BACF) for services and facilities provided.
We thank the Border Biomedical Research Center-UTEP for a Pilot
Grant (NIMHD/G12MD007592) to R.S. and the University of Texas
URI for a Grant to R.S.
Figure 4. Evaluation of reactive oxygen species (ROS) in L. major promastigotes
treated with the lead compound AA9.31
Table 3
Antiparasitic activity and cytotoxicity of compounds AA1 and AA932
Supplementary data
Compound
Mammalian cells IC50
(
l
M)
L. major
LD50 M)
Promastigotes
LD50 M)[TI]
(
l
Supplementary data (bioassays, protocols, synthesis of analogs
and their 1H NMR and LC–MS) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2015.09.041.
U2OS
Human
LLC-MK2 Raw 264.7
Macrophages
I PU
(l
Osteoblasts
AA1
AA9
N/A
>80
>240
N/A
N/A
>80
N/A
>80
28.75 [8.35]
3.34 [23.95]
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for 24 h with AA9 at 3 M. Negative controls treated with 1% DMSO and
Medium only. Positive control treated with hydrogen peroxide at 100 M. As
lM. (B) Representative pictures of macrophages
l
l
l
l
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Please cite this article in press as: Iniguez, E. A.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.041