In vitro evaluation and in vivo efficacy of nitroimidazole-sulfanyl ethyl derivatives against Leishmania (V.) braziliensis and Leishmania (L.) mexicana

Article abstract of DOI:10.1007/s00436-021-07266-w

The aim of this study was to synthesize several small molecules of the type 5-nitroimidazole-sulfanyl and evaluate biological properties against the main Leishmania species that cause cutaneous leishmaniasis in Venezuela. Final compounds 4–7 were generated through simple nucleophilic substitution of 1-(2-chloroethyl)-2-methyl-5-nitroimidazole 3 with 2-mercaptoethanol, 1-methyl-2-mercaptoethanol, and 2-thyolacetic acid derivative. Compound 8 was synthesized via a coupling reaction between 7 and (S)-Methyl 2-amino-4-methylpentanoate hydrochloride. The inhibitory concentrations of (3, 4, 7, 8) against Leishmania (L.) mexicana and (V.) braziliensis in promastigotes and experimentally infected macrophages were determined by in vitro activity assays. Compounds 7 and 8 shown high activity against both species of Leishmania and were selected for the in vivo evaluation. Animals were infected with promastigotes of the two species and divided into four groups of ten (10) animals and a control group. Intralesional injection way was used for the treatment. The parasitological diagnostic after treatment was obtained by PCR using species specific oligonucleotides. The two Leishmania species were susceptible to compounds 7 and 8 in vivo assays. The results indicated that both compounds reduce significantly (96%) the size of the lesion and cure 63% of the mice infected with L (L) mexicana or L (V) braziliensis as was determined by PCR. The results are indicating that both compounds may represent an alternative treatment for these two Leishmania species.

Full text of DOI:10.1007/s00436-021-07266-w

Parasitology Research (2021) 120:3307–3317
https://doi.org/10.1007/s00436-021-07266-w
TREATMENT AND PROPHYLAXIS - ORIGINAL PAPER
In vitro evaluation and in vivo efcacy of nitroimidazole‑sulfanyl ethyl
derivatives against Leishmania (V.) braziliensis and Leishmania (L.)
mexicana
Zuleima Blanco1 · Michael R. Mijares2 · Hegira Ramírez1,3 · Esteban Fernandez‑Moreira4 · Henry J. Oviedo5 ·
Noris M. Rodríguez5 · Jaime E. Charris1
Received: 12 April 2021 / Accepted: 21 July 2021
/ Published online: 9 August 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
The aim of this study was to synthesize several small molecules of the type 5-nitroimidazole-sulfanyl and evaluate biological
properties against the main Leishmania species that cause cutaneous leishmaniasis in Venezuela. Final compounds 4–7 were
generated through simple nucleophilic substitution of 1-(2-chloroethyl)-2-methyl-5-nitroimidazole 3 with 2-mercaptoetha-
nol, 1-methyl-2-mercaptoethanol, and 2-thyolacetic acid derivative. Compound 8 was synthesized via a coupling reaction
between 7 and (S)-Methyl 2-amino-4-methylpentanoate hydrochloride. The inhibitory concentrations of (3, 4, 7, 8) against
Leishmania (L.) mexicana and (V.) braziliensis in promastigotes and experimentally infected macrophages were determined
by in vitro activity assays. Compounds 7 and 8 shown high activity against both species of Leishmania and were selected
for the in vivo evaluation. Animals were infected with promastigotes of the two species and divided into four groups of
ten (10) animals and a control group. Intralesional injection way was used for the treatment. The parasitological diagnostic
after treatment was obtained by PCR using species specifc oligonucleotides. The two Leishmania species were susceptible
to compounds 7 and 8 in vivo assays. The results indicated that both compounds reduce signifcantly (96%) the size of the
lesion and cure 63% of the mice infected with L (L) mexicana or L (V) braziliensis as was determined by PCR. The results
are indicating that both compounds may represent an alternative treatment for these two Leishmania species.
Keyword Leishmania · Metronidazole · Nitroimidazole · Sulfanyl · PCR
Introduction
Section Editor: Sarah Hendrickx.
Leishmaniasis is a neglected tropical disease (NTD) caused
by protozoa of the genus Leishmania; the disease is endemic
in 98 countries with an overall prevalence of 12 million
cases and an annual mortality rate of more 59.000 deaths
(WHO 2019). Over 20 Leishmania species are known to
be infective to humans and are transmitted by the bite of
infected female phlebotomine sandfies, thus giving three
main clinical manifestations of leishmaniasis: visceral (VL),
cutaneous (CL), and mucocutaneous (MCL). It is estimated
that approximately 0.2 to 0.4 million of new VL cases and
0.7 to 1.2 million of new CL cases occur each year (WHO
2019). Since there are currently no efective vaccines that
protect against the disease, leishmaniasis control relies on
chemotherapy. The recommended frst-line therapies include
pentavalent antimony compounds, such as sodium stiboglu-
conate and meglumine antimoniate, although these drugs
present several disadvantages, such as toxicity, high costs,
* Jaime E. Charris
jaime.charris@ucv.ve
Noris M. Rodríguez
nmrodric@gmail.com
1
Organic Synthesis Laboratory, Faculty of Pharmacy,
Central University of Venezuela, Los Chaguaramos 1041-A,
Caracas 47206, Venezuela
2
Biotechnology Unit, Faculty of Pharmacy, Central University
of Venezuela, Los Chaguaramos 1041-A, Caracas 47206,
Venezuela
3
4
5
Facultad de Medicina, Universidad de Las Américas, Quito,
Ecuador
Escuela de Medicina, Universidad Espíritu Santo,
Samborondón, Ecuador
Institute of Biomedicine, Faculty of Medicine, Central
University of Venezuela, 1010-A, 4043 Caracas, Venezuela
Vol.:(0123456789)
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prolonged treatment, and parenteral or intralesional routes of
administration. The second-line treatments include pentami-
dine and amphotericin B, but their use is limited because of
toxicity and cost, even though lipid and liposomal formula-
tions of amphotericin B have been developed to reduce this
toxicity. Recently, the oral administration of miltefosine has
been used for the treatment of VL in some countries, but
despite its great efcacy, miltefosine is not free either from
toxicity as it shows teratogenic potential (Singh et al. 2016;
Sangshetti et al. 2015; Dietze et al. 2001; Agrawal and Singh
2006; Soto et al. 2001; Monge and López 2015). Several
compounds that show leishmanicidal activity are currently in
diferent stages of development. Among them, a few classes
of compounds, such as the 8-aminoquinolinic sitamaquine
(Loiseau et al. 2011), the 7-aminoimidazoquinolinic imiqui-
mod (Seeberger et al. 2003), the triazolic posaconazole
(Al-Abdely et al. 1999), as well as some natural product
derivatives, such as licochalcone A (Zhai et al. 1999), have
been revealed as potential new drugs for antileishmania
therapy. The synthesis of several molecules showing leish-
manicidal activity and that of new lead compounds, such
as β-carboline alkaloids (Stefanello et al. 2014), piper-
oylaminoacid conjugates (Singh et al. 2010), heteroretinoid-
bis(benzylidene)ketones (Tiwari et al. 2015), and bispyri-
dinium cyclophanes, has also been described (Gómez-Pérez
et al. 2015). The Drugs for Neglected Diseases initiative
(DNDi) and Takeda will work on the “lead optimization”
of the aminopyrazole series (DNDi. Org. 2021). Consider-
ing toxicity issues, increasing parasite resistant, increasing
failure rates of current treatments, and the lack of efective
clinic agents against leishmaniasis, there is an urgent need in
the development of efective and safe new drugs for treating
leishmaniasis in their diferent clinical forms. In this sense,
also, attention has been directed to those drugs that con-
tain a nitro group, for their use for the treatment of a wide
variety of diseases, including antineoplastic, antibacterial,
antiparasitic, tranquilizer, fungicide, and insecticide as well
as herbicide agents (Ang et al. 2017; Nepali et al. 2019). In
particular, nitro containing imidazol like 5-nitroimidazole,
metronidazole clinically, or fexinidazole and its sulfonic
metabolite now in clinical trial were shown to have efective
antileishmanial efcacy (Somaratne et al. 2019; Wyllie et al.
2012; de Morais-Texaira et al. 2019; Burrows et al. 2014).
Another example is related to delamanid, a nitroimidazooxa-
zole demonstrated efective antileishmanial activity resulting
in a total cure in a mouse model of VL (Patterson et al. 2016;
Wyllie et al. 2016; Andrews et al. 2014).
(FMN) or favin adenine dinucleotide (FAD) as prosthetic
groups and nicotinamide adenine dinucleotide (NADH) or
nicotinamide adenine dinucleotide phosphate (NADPH) as
reducing agents (Macherey and Dansette 2015).
In the search for more efective alternatives to the cur-
rently used antileishmanial drugs, some research groups
have been introducing the hybridization strategy to obtain
new structures with potential activity. The results have led
to some leading compounds (Otero et al. 2017; Baquedano
et al. 2014; Upadhyay et al. 2019). To throw light on this
scenario, our research group has been synthesized a set of
novel metronidazole analogues featuring an heterocyclic
and basic 5-nitroimidazole head linked to a substituted
benzoic acid through a dialkyl sulfur chain (Rodríguez
et al. 2020). The choice of this sulfur-containing spacer
was related to our previous successful experience with
7-chloroquinolin-4-ylthio derivatives (Rodrigues et al.
2012). In view of the above considerations, here, we are
reporting the synthesis and the biological results related
to a series of metronidazole analogues against Leishmania
(V.) braziliensis and Leishmania (L.) mexicana in vitro and
their efcacy in vivo.
Materials and methods
Chemicals
All solvents and reagents used for the synthesis were of
analytical grade and were purchased from diferent chemi-
cal suppliers. Thin-layer chromatography (TLC) was car-
ried out on Merck silica F254 0.255-mm plates, and spots
were visualized by UV fuorescence at 254 nm. At fnal
compounds, the impurities were removed by either recrys-
tallization or silica gel column chromatography. A Thomas
micro hot stage apparatus was used to obtain the melting
points and is uncorrected. The IR spectra were recorded on
a Shimadzu™ model 470 spectrophotometer using NaCl
pills or KBr pellets. Optical rotations were measured with
an ATAGO™ Polarimeter model POLAX-2L. NMR spect-
ers were obtained using a JEOL Eclipse™ 270 MHz for
1H-NMR and at 67.9 MHz for 13C-NMR using CDCl3 or
DMSO-d6 and are reported in ppm downfeld from the
residual CHCl3 or DMSO (δ 7.25 or 2.50 for 1H NMR and
77.0 or 39.8 for 13C NMR, respectively). A Perkin Elmer™
2400 CHN elemental analyzer was used to obtain the ele-
mental analyses, and the results were within 0.4% of the
predicted values. The general synthesis of compounds 2–8
is presented in Schemes 1 and 2, and the synthetic proce-
dures are presented below. Compounds 2, 3, and 5 were
prepared according to known procedures (Salahuddin et al.
2012; Rodríguez et al. 2020).
In biological systems, nitro groups require enzymatic
reduction to induce the therapeutic and cytotoxic efects,
either by oxidative stress or through the formation of
adducts between a protein or non-protein thiol and some
intermediate metabolites. This reduction is normally
mediated by nitroreductases using favin mononucleotide
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Scheme 1 Synthesis of (S,R) 1-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]propan-2-ol (4) and 2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethylthio]ethanol (5)
Scheme 2 Synthesis of methyl 2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]acetate (6), 2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl-
thio]acetic acid (7) and (S) methyl 2-{2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]acetamido}-4-methyl-pentanoate (8)
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eliminated at reduced pressure, 20 ml of water was then
added, and a saturated solution of KHSO4 was added drop
by drop until a pH between 2 and 3 was reached. The result-
ing mixture was extracted with 2×50 mL portions of ethyl
acetate, and the organic phases were joined, washed with
distilled water 2×50 ml, with brine, dried over anhydrous
MgSO4, fltered and concentrated to yield pure product as
a powdered yellow, yield 84%, m.p.: 164–166 °C, IR KBr
Synthesis and characterization of 4, 6–8
(S,R) 1‑[2‑(2‑methyl‑5‑nitro‑1H‑imidazol‑1‑yl)ethylthio]
propan‑2‑ol (4)
A stirred solution of 3 (0.5 g, 2.65 mmol), 1-mercapto-
2-propanol (0.26 mL, 2.7 mmol), and potassium carbonate
(0.8 g, 5.6 mmol) in 20 mL of acetonitrile was refuxed for
12 h and concentrated under vacuum. The residue was parti-
tioned between ethyl acetate and water, and the organic layer
was separated and washed with brine, dried over anhydrous
MgSO4, and concentrated to yield crude product which was
purifed by column chromatography AcOEt/hexane 1:1 as
eluent to give pure product as oil, yield 72%, IR NaCl ʋ
1
ʋ (cm−1): 3160–2144, 1705, 1542, 1478, 1424; H-RMN
(DMSO-d6, 270 MHz) δ: 2.48 (s, 3H, CH3), 2.97 (t, 2H, H7,
J = 7.0 Hz), 3.31 (s, 2H, H8), 4.49 (t, 2H, H6, J = 7.0 Hz),
8.03 (s, 1H, H4), 12.66 (brs, 1H, OH); 13C-RMN (DMSO-
d6, 67.9 MHz) δ: 14.5 (CH3), 31.6 (C7), 33.8 (C9), 45.7
(C6), 133.5 (C4), 138.8 (C5), 151.8 (C2), 171.7 (C=O). Anal.
calcd. for C8H11N3O4S: C, 39.18; H, 4.52; N, 17.13. Found:
C, 39.20; H, 4.53; N, 17.35.
1
(cm−1): 3344, 2976, 1529, 1468, 1427; H-NMR (CDCl3,
270 MHz) δ: 1.22 (d, 3H, CH3 J = 6.3 Hz), 2.48 (dd, 1H,
H8 J = 13.5, 8.3 Hz), 2.57 (s, 3H, CH3), 2.66 (dd, 1H, H8
J=13.5, 6.3 Hz), 2.93 (t, 2H, CH7 J=7.3 Hz), 3.88 (m, 1H,
H9), 4.49 (t, 2H, H6 J=7.3 Hz), 7.97 (s, 1H, H4); 13C-NMR
(CDCl3, 67.9 MHz) δ: 14.2 (CH3), 22.4 (CH3), 32.2 (C7),
41.6 (C6), 46.5 (C8), 66.8 (C9), 131.6 (C4), 138.4 (C5), 150.2
(C2). Anal. calcd. for C9H15N3O3S: C, 44.07; H, 6.16; N,
17.13. Found: C, 43.98; H, 6.18; N, 17.29.
(S) Methyl 2‑{2‑[2‑(2‑methyl‑5‑nitro‑1H‑imidazol‑1‑yl)
ethylthio]acetamido}‑4‑methyl‑pentanoate (8)
A solution of 7 (0.150 g, 0.6 mmol) in dry DMF (2 mL)
at – 10 °C was treated with N-(3-dimethylaminopropyl)-
N´-ethylcarbodiimide hydrochloride (EDCI) (0.153 g,
0.8 mmol) and 4-(dimethylamino)-pyridine (DMAP)
(0.05 g, 0.4 mmol) dissolved in dry DMF (1 mL). The mix-
ture was left shaking at – 10 °C for 30 m. The (S)-Methyl
2-amino-4-methylpentanoate hydrochloride (Colmenares
et al. 2020) (0.118 g, 0.65 mmol) dissolved in dry DMF
(1 mL) were slowly added to the mixture over 30 m. The
resulting mixture was left stirring for 12 h at room tem-
perature. Next, water was added, and the aqueous fraction
was extracted with CH2Cl2 (2×10 mL). The organic layer
was washed with 10% sodium bicarbonate (2×8 mL), dried
over anhydrous sodium sulfate and fltered. The solvent
was evaporated under reduced pressure. The compound 8
was purifed using column chromatography using as eluent
hexane:ethyl acetate (9:1). Yield 96%, m.p.: 59–60 °C, IR
Methyl 2‑[2‑(2‑methyl‑5‑nitro‑1H‑imidazol‑1‑yl)ethylthio]
acetate (6)
A solution of methyl thyoglicolate (0.56 g, 5.3 mmol) and
potassium carbonate (0.73 g, 5.3 mmol) in 100 mL of ace-
tonitrile was stirred for 15 min at room temperature; fol-
lowing the addition of 3 (1.0 g, 5.3 mmol), the mixture
was refuxed for 12 h and concentrated under vacuum. The
residue was partitioned between ethyl acetate and water; the
organic layer was separated and washed with brine, dried
over anhydrous MgSO4, and concentrated to yield a crude
product which was purifed by column chromatography
AcOEt/hexane 1:1 as eluent to give pure product as a solid
pale yellow, yield 95%, m.p.: 46 °C, IR KBr ʋ (cm−1): 3120,
rt
KBr ʋ (cm−1): 3.221, 3124, 1763, 1625, 1598, 1543; [α]
D
+ 16.0 (c 1.0 CHCl3); 1H-RMN (CDCl3, 270 MHz) δ: 0.89
(d, 6H, 2CH3 J=4.8 Hz), 1.55–1.63 (m, 3H, H10,11), 2.49 (s,
3H, CH3), 2.95 (t, 2H, H7 J=7.4 Hz), 3.21 (s, 2H, H8), 3.67
(s, 3H, OCH3), 4.46–4.55 (m, 3H, H6,9), 6.83 (d, 1H, NH
J=7.9 Hz), 7.90 (s, 1H, H4); 13C-RMN (CDCl3, 67.9 MHz)
δ: 14.2 (CH3), 21.7 (CH3), 22.7 (CH3), 24.9 (C11), 31.8 (C7),
35.3 (C8), 40.9 (C7), 45.5 (C6), 51.1 (C10), 52.2 (OCH3),
132,9 (C4), 138.4 (C5), 150.7 (C2), 168.8 (NC=O), 173.1
(CH3OC=O). Anal. calcd. for C15H24N4O5S: C, 48.37; H,
6.50; N, 15.04. Found: C, 48.32; H, 6.55; N, 15.27.
1
3008, 1747, 1523, 1468; H- RMN (CDCl3, 270 MHz) δ:
2.57 (s, 3H, CH3), 3.02 (t, 2H, H7, J=7.2 Hz), 3.21 (s, 2H,
H8), 3.72 (s, 3H, CH3O), 4.53 (t, 2H, H6, J=7.2 Hz), 7.96
(s, 1H, H4); 13C-RMN (CDCl3, 67.9 MHz) δ: 14.2 (CH3),
32.2 (C7), 33.7 (C9), 46.0 (C6), 56.2 (OCH3), 132.1 (C4),
138.5 (C5), 150.3 (C2), 170.4 (C = O); Anal. calcd. for
C9H13N3O4S: C, 41.69; H, 5.05; N, 16.21. Found: C, 41.73;
H, 5.02; N, 16.37.
2‑[2‑(2‑methyl‑5‑nitro‑1H‑imidazol‑1‑yl)ethylthio]acetic
acid (7)
Culture and maintenance of parasite
To a mixture of 6 (1.3 g, 5 mmol) in THF:H2O (3:1) was
added LiOH (0.18 g, 7.5 mmol), the mixture was stirred
at 0 °C for 1 h. After the reaction time, the solvent was
International reference strains of L. (V.) braziliensis
(MHOM/BR/75/M2903) and L. (L.) mexicana (MHOM/
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Parasitology Research (2021) 120:3307–3317
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BZ/82/Bel21) were thawed and cultured in RPMI 1640
medium (Gibco_BRL) at room temperature with 10% fetal
bovine serum (FBS) inactivated by heating at 56 °C for
30 min. Finally, antibiotics (penicillin/streptomycin), at con-
centrations of 100 and 1000 units, respectively, were added.
For the experiments, parasites were collected in the loga-
rithmic phase of growth (ffth day of culture) by centrifuga-
tion at 3,000 rpm, washed three times with saline phosphate
bufer, pH 8.0. The pellet was re-suspended in fresh medium,
and the parasites were adjusted to a concentration of 1×106
cells/mL (Brito et al. 2006).
Direct method
This method is based on the comparison between two doses
that we call X1 and X2, such that the density of parasites Y1
to the X1 dose is greater than half of the density found in
the control (I); and the density of parasites Y2 found to the
dose X2 is less than half of the control. Then we can calcu-
late the lethal concentration 50 (LC50) using the algorithm
previously described by (Huber and Koella 1993). For pro-
mastigote, 1×106 parasites were added in 2 mL of medium
SDM 79 at pH 7.2 supplemented with 10% fetal serum and
100 μL of penicillin–streptomycin in sterile 6-well plates
and treated with diferent concentrations of the compound,
then incubated at 26 °C; 5 μL of each sample was taken
daily. Viability was determined by counting the cells stained
with trypan blue.
Antileishmanial activity on promastigote
proliferation
Each compound was diluted to a concentration of 50 mg/mL
in appropriate solvent (DMSO), and, subsequently, dilutions
between 10 and 500 μg/mL were prepared for the experi-
ments. Diferent concentrations of each compound were
used for the diferent species of Leishmania, i.e., L. (V.)
braziliensis or L. (L.) mexicana to investigate the response
of the parasite to each compound. A daily sample of 5 μL
was taken for cell counting. The count was performed in
triplicate for 7 days, until the culture reaches the stationary
phase of growth. The efect of each compound over the dif-
ferent Leishmania species was evaluated (Brito et al. 2006).
In macrophage sensitivity assays
To obtain infected macrophages, we used J774-G8 mouse
macrophage cell line, which were grow in monolayers before
used. Macrophages were infected with Leishmania promas-
tigotes in a 10:1 ratio and incubated at 35 °C. After 24 h,
unbound promastigotes are discarded. At 72 h the promas-
tigotes are counted inside the cell with Giemsa stain (Brito
et al. 2006). Once the infection is established, infected cells
are used for trials with the diferent compounds. The com-
pounds to be evaluated were placed at the previously estab-
lished concentrations, and their efect was evaluated at 24
and 72 h, using MTT (3–4,5-dimethylthiazol-2-bromide of
diphenyl tetrazolium), for which macrophages infected and
treated with the diferent concentrations of the drug were
incubated for 18–24 h; then, 10 µL (10 mg/ml) of methyl-
thiazole tetrazolium (MTT, Sigma) is added and incubated
for 4 h. After incubation, the reaction is stopped with lysis
bufer solution (50% isopropanol, 10% SDS), and then the
OD is measured at 570 nm, in a spectrophotometer. The
cellular viability is directly proportional to the OD since to
the greater number of living cells, greater color intensity,
because they have greater capacity to metabolize the MTT,
transforming it into formazan crystals that generate the color
(Huber and Koella 1993).
Calculation of cell viability and LC50
To evaluate the efect of the compounds on cell viability
and in order to calculate the LC50, two methods were used:
Indirect method
Parasites were incubated with various concentrations of the
respective compound for 18–24 h; thereafter, 10 μL (10 mg/
mL) of methyl-thiazole tetrazolium (MTT, Sigma) were
added and incubated for 4 h. After incubation, the reaction
was stopped with lysis bufer (50% isopropyl alcohol, 10%
SDS), and then the optical density (OD) was measured at
570 nm in a spectrophotometer (Biorad). Cell viability is
directly proportional to OD, since the higher number of liv-
ing cells has greater color intensity, because they have high
capacity to metabolize the MTT. For each experiment, dif-
ferent controls were used, including cells treated with sol-
vent only, controls without and with meglumine antimoniate
(i.e., drug of choice in the treatment of leishmaniasis). The
efect of each compound on the growth of the parasites in
relation to controls was used to estimate the concentration
that causes the death of 50% of the cells in a given time
(LC50) (Rodríguez et al. 2020).
In vivo assays
Balb/c mice between 6 and 8 weeks of age were inoculated
on the foot pad with 1 × 106 Leishmania promastigotes of
the diferent species to be evaluated L. (V.) braziliensis and
L. (L.) mexicana. The progress of the infection was evalu-
ated weekly, taking the measurement of the lesion (nodule)
with a digital caliper (Rodriguez et al. 2002); groups of 10
mice were used for each compound to be evaluated. Once
the disease was found in the mice, treatment was carried out
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with the selected compounds 7 and 8. A group of mice were
treated with Glucantime™, which is the drug indicated for
the treatment of human leishmaniasis.
2, which was then treated with water and Et3N until pH 11,
to obtain the 1-(2-chloroethyl)-2-methyl-5-nitroimidazole 3
with a yield of 96% (Scheme 1) (Salahuddin et al. 2012).
Subsequent nucleophilic substitution of 3 with 1-mercapto-
2-propanol, 2-mercaptoethanol, or methyl thyoglicolate
furnished the thioether-linked metronidazole analogue 4, 5,
or 6, respectively. The compounds were obtained in good
yield (Schemes 1 and 2). Compound 7 was obtained from
6 through alkaline hydrolysis. The compound 8 was syn-
thesized via a coupling reaction between 7 and (S)-Methyl
2-amino-4-methylpentanoate hydrochloride, in the presence
of EDCI and DMAP in CH2Cl2. The title compound was
isolated in excellent yield after purifcation by column chro-
matography (Scheme 2). The chemical structures of all syn-
thesized compounds were confrmed on the basis of their IR
and NMR spectral data, and their purity was ascertained by
microanalysis. In 1H NMR spectra, the signal of the respec-
tive protons of the compounds were checked on the basis of
their chemical shifts, multiplicities, and coupling constants.
All compounds showed a single signal ranging from δ H
7.9 to 8.5 ppm, which was assigned to the H-4 of the imi-
dazole ring. The aliphatic signals expected at upfeld shifts
were found from δ H 2.9 to 4.5 ppm. The 13C NMR spectra
showed characteristic signals of the 5-nitroimidazole core,
with one signal resonating at δ C 140–165 ppm, which was
attributed to C-5, as well as two signals observed in the δ C
138–152 and 124–140 ppm regions, which were assigned to
C-2 and C-4, respectively. For the carboxyl group, another
characteristic signal was observed further downfeld around
δ C 165–166 ppm.
Compounds 7 and 8 were dispersed in water with 5%
(w/v) Tween 80. The obtained dispersion was stirred to cre-
ate a fne suspension. The formulation was kept at 4 °C and
in the dark when not being used. The diluent was prepared
with 5% (w/v) Tween 80 in water.
Intramuscular treatment was administering for a period
of 5 weeks using 50 µg of the compounds each week, on the
back fanks of the infected mice. After treatment, measure-
ments were made on the lesion site. A control group do not
received any treatment.
Parasitological and clinical efcacy of compounds 7
and 8
After the last measurement, 8 mice treated with compound 8
were taken at random, and biopsies were obtained from the
foot pad for parasitological diagnosis by the polymerase chain
reaction (PCR), using the protocol described by (Rodríguez et al.
2002). Briefy, this procedure involves isolating DNA from a
biopsy taken at the site of the lesion and using it to amplify
the DNA of the parasite that may be present, using spe-
cies specifc oligonucleotides in a reaction that also contain
nucleotides and the enzyme Taq polymerase. The reaction is
carried out in a thermal cycler with the appropriate program
required for amplifcation of the DNA fragment unique to
the species of Leishmania to be diagnosed. After treatment,
no side efect was observed.
Statistical analysis
In vitro antileishmanial activity
The statistical analysis was performed using a GraphPad
Prism, version 5.3 program. The diference was considered
signifcant when the P value was ≤ 0.05.
To identify the potential of 5-nitroimidazole derivatives
against American cutaneous leishmaniasis (CL), the 8 com-
pounds were tested against L. mexicana and L. braziliensis
parasites. Initially, we measured the antiproliferative activi-
ties of these compounds against promastigote of L. mexicana
and L. braziliensis parasites results are shown in Figs. 1 and
2 (Tables 1a, 1b, 2a, 2b supplementary material). In general,
both species were sensitive to the action of the tested 5-nitro-
imidazole derivatives. In Fig. 1A and B (L. mexicana), we
observed that compounds 3, 4, 7, and 8 are inhibiting the
growth of the parasite when we compared with the control
(no treated parasites); similar efect was observed with L.
braziliensis (Fig. 2A and B). However for both species, the
compound 8 has a high activity at 500 µg/ml for both spe-
cies; we observed that all parasites are dead after 24 h in the
presence of the compound.
Animal research ethical aspects
All animals were handled according to local and national
regulations, and the research protocols were approved by the
Institute of Biomedicine Committee on Animal Research.
Results and discussion
Synthesis and characterization of 5‑nitroimidazole
derivatives
Our synthesis work began from metronidazole™ 1, whose
primary alcohol was substituted by a chlorine atom. Follow-
ing known procedures, upon reaction with thionyl chloride,
1 was converted into the corresponding hydrochloride salt
To evaluate the activity of the tested 5-nitroimidazole
derivatives, in the host cell of the Leishmania parasite, an
in vitro cytotoxicity evaluation on J774-G8 cell line mac-
rophage was performed for the compounds 3, 4, 7, and 8.
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Parasitology Research (2021) 120:3307–3317
3313
Fig. 1 Promastigotes of L. (L.) mexicana treated whit compounds 1–8, (1=MNZ™). A 100 µg/mL. B 500 µg/mL; in this concentration com-
pound 8 killed all parasites after 24 h in the presence of the compound. Parasites were counted every day during 5 days
Fig. 2 Promastigotes of L. (V.) braziliensis treated whit compounds 3, 4, 7, and 8. A 100 µg/mL. B 500 µg/mL; in this concentration compound 8
killed all parasites after 24 h. Parasites were counted every day during 5 days
The results are presented in Table 1; this suggested that
compounds 3, 4, 7, and 8 represented the most attractive
antileishmanial candidates for further assays due to their
lower toxicity in the macrophages.
To continue with the in vitro evaluation of the tested
5-nitroimidazole derivatives, experiments against intracel-
lular amastigote of L. mexicana and L. braziliensis was car-
ried out for the derivatives 3, 4, 7, and 8. The intracellular
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Parasitology Research (2021) 120:3307–3317
Table 1 Efect of compounds
3, 4, 7, and 8 on J774-G8
macrophages, IC50 values on
amastigote of L. (L.) mexicana
and L. (V.) braziliensis, and
selectivity indexes
No
Macrophages
J774-G8 (LC50 µg/
mL)a
Amastigote L. (L.) mexi- Amastigote L. (V.) brazil- S. I Lmc
S. I Lbc
cana (IC50 µg/mL)b
iensis (IC50 (µg/mL)b
3
4
7
8
>60
66
40
22
18
75
47
26
21
ND
0.9
1
ND
0.8
0.8
1.6
36.43 4.09
21.70 2.17
33.44 5.77
1.9
aLC50: Letal concentration, the results are the means of three independent experiments
bIC50: Inhibitory concentration, the results are the means of fve independent experiments
cS.I. L m: selectivity index on L. mexicana; S.I. L b: selectivity index on L. braziliensis
ND not determined
amastigote model is one of the most important assays for
the in vitro leishmanicidal evaluation of a candidate due that
this intracellular form is the main responsible of the clinical
manifestations of the disease (Chang and McGwire 2002).
We evaluated amastigote of L. mexicana and L. braziliensis
due to that these two species are the main responsible of the
CL in America (90% of all cases) (WHO 2019). Tradition-
ally, the intracellular amastigotes are produced in situ from
macrophage in presence of promastigote parasite under spe-
cifc conditions (pH 5.5 at 37 °C).
The intracellular amastigotes were exposed to selected
compounds 3, 4, 7, and 8; its corresponding efect was evalu-
ated at 48 h post treatment. In general, it should be noted
that the intracellular amastigote form was more resistant
than promastigote form for the tested compounds 3 and 4;
however it was sensitive to compounds 7 and 8; results are
shown in Table 1.
In vivo antileishmanial activity of compounds 7
and 8
Fig. 3 Balb-c mice were inoculated in the foot pad with promas-
tigotes of L. (L.) mexicana and treated with compound 7. Control:
untreated Balb/c mice. Glucantime™: Balb/c mice treated with this
drug. Size of the lesion was measured every week after treatment dur-
ing 5 weeks
The results indicate that of the 8 compounds evaluated, 4 of
them had the greatest activity on the parasites; however only
2 showed parasiticidal action at low concentrations; the com-
pound 7 2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylthio]
acetic acid, with a IC50 of 22 and 26 µg/mL for intracellular
removal of L. mexicana and L. braziliensis, respectively, and
compound 8 (S) methyl 2-{2-[2-(2-methyl-5-nitro-1H-im-
idazol-1-yl)ethylthio]acetamido}-4-methyl-pentanoate,
with a IC50 of 18 µg/mL for L. mexicana and 21 µg/mL
for L. braziliensis. The results obtained in the lesions of
experimentally infected Balb/c mice indicate that both
compounds reduce signifcantly (96%) (Figs. 3, 4, 5, and 6)
(Tables 3–6 supplementary material) and cured 63% of the
mice infected with L (L) mexicana or L (V) braziliensis; this
was evidenced both by the disappearance of the lesions and
by the absence of parasites in the parasitological diagnosis
both in culture and by PCR (Fig. 7).
Conclusion
A series of novel metronidazole™ (MNZ) derivatives
has been synthesized and tested as antileishmanial agents
against promastigotes and amastigotes of L. (L.) mexicana
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Parasitology Research (2021) 120:3307–3317
3315
Fig. 4 Balb/c mice inoculated in the foot pad with promastigotes of
L. (L.) mexicana and treated with compound 8. Control: Untreated
Balb/c mice. Glucantime™: Balb/c mice treated with this drug.
Size of the lesion was measured every week after treatment, during
5 weeks
Fig. 6 Balb-C mice inoculated in the foot pad with promastigotes of
L. (V.) braziliensis and treated with compound 8. Control: Untreated
balb-C mice. Glucantime™: Balb-C mice treated with this drug. Size
of the lesion was measured every week during 5 weeks
and L. (V.) braziliensis. Among the tested compounds, the
derivatives 7 and 8 have shown a signifcant in vitro and
in vivo activity superior to that of the parent drug MNZ®,
which may result from several independent or combined
causes. The natures either of the A group or of the B
substituent were crucial in the identifcation of the most
appropriate pharmacophores. As can be seen in Fig. 8, the
A group is represented by an S-alkyl substitution, while
the B group consisted of a hydroxy attached at 2-position
of the alkyl core, carboxy free or accoupled with an amino.
In general, from the most active derivatives 7 and 8, two
important molecular pharmacophores were identifed: (a)
the inclusion of a S atom attached at 2-position of N-alkyl
group of the 5-nitroimidazole ring as A substituent and (b)
the incorporation of a carboxy group free or forming an
amide with N-alkyl terminal as B substituent, which could
lead to an improvement of the amphiphilic character of the
molecules, leading to an increase in concentration of the
compound inside the parasite form, thus increasing the
interactions with leishmanial functional proteins. Regard-
ing the infuence of the rest of S-alkyl terminal B group,
we can mention that (a) the incorporation of a primary
hydroxyl or ester groups as B substituent caused a signif-
cant loss of the efect compounds 5 and 6, (b) the efect of
Fig. 5 Balb/c mice inoculated in the foot pad with promastigotes of
L. (V.) braziliensis and treated with compound 7. Control: Untreated
Balb/c mice. Glucantime™: Balb/c mice treated with this drug. Size
of the lesion was measured every week during 5 weeks
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3316
Parasitology Research (2021) 120:3307–3317
Fig. 7 Parasitological diag-
nosis using PCR and samples
obtained from the food pad of
the treated mice with compound
8. Lanes 2, 7, and 8 resulted
positives for L. (V.) braziliensis
and 1, 3, 4, 5, and 6 were nega-
tives with no parasites. Lane 9:
positive control (DNA extracted
from promastigotes); lane 10:
negative control (Reaction mix
with no DNA) M: Molecular
weight marker
Supplementary Information The online version contains supplemen-
a secondary hydroxyl resulted in marginal antileishmanial
activity compound 4, and (c) the efect of hydroxy metro-
nidazole MNZ™ 1 or chlorine atoms 2 and 3 attached at
2-position of N-alkyl group of the 5-nitroimidazole ring as
A substituent a loss or marginal of antileishmanial activity
was observed.
tary material available at https://doi.org/10.1007/s00436-021-07266-w.
Acknowledgements We thank the Instituto de Investigaciones Far-
macéuticas (IIF) and Consejo de Desarrollo Científco y Humanís-
tico de la Universidad Central de Venezuela (CDCH-UCV, PG.
09-8819-2013/2).
Even if additional assays related to toxicity on other
human cell lines, genotoxicity experiments, and mecha-
nism of action will be required to estimate their real
potential, these biological results revealed that these two
compounds constitute promising candidates in the search
for improved therapies against L. (L.) mexicana and L (V.)
braziliensis.
Declarations
Conflict of interest The authors declare no competing interests.
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