Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on volume regulation decrease and sterol biosynthesis of Leishmania braziliensis

Article abstract of DOI:10.1016/j.exppara.2019.01.011

Diverse spiro dihydroquinoline-oxindoles (JS series) were prepared using the BF₃?OEt₂-catalyzed imino Diels-Alder reaction between ketimine-isatin derivatives and trans-isoeugenol. Ten spiro-oxiindole derivatives were selected and ev

Full text of DOI:10.1016/j.exppara.2019.01.011

Accepted Manuscript
Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on volume regulation
decrease and sterol biosynthesis of Leishmania braziliensis
Jacques Leañez, Jorge Nuñez, Yael García-Marchan, Felipe Sojo, Francisco
Arvelo, Daniel Rodriguez, Ignacio Buscema, Alvaro Alvarez-Aular, Josué S. Bello Forero, Vladímir
V. Kouznetsov, Xenón Serrano-Martín
PII:
S0014-4894(18)30341-2
10.1016/j.exppara.2019.01.011
YEXPR 7653
DOI:
Reference:
To appear in:
Experimental Parasitology
Received Date:
Accepted Date:
18 July 2018
20 January 2019
Please cite this article as: Jacques Leañez, Jorge Nuñez, Yael García-Marchan, Felipe Sojo,
Francisco Arvelo, Daniel Rodriguez, Ignacio Buscema, Alvaro Alvarez-Aular, Josué S. Bello
Forero, Vladímir V. Kouznetsov, Xenón Serrano-Martín, Anti-leishmanial effect of spiro
dihydroquinoline-oxindoles on volume regulation decrease and sterol biosynthesis of Leishmania
braziliensis, Experimental Parasitology (2019), doi: 10.1016/j.exppara.2019.01.011
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ACCEPTED MANUSCRIPT
GA
Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on volume regulation
decrease and sterol biosynthesis of Leishmania braziliensis
Jacques Leañez, Jorge Nuñez, Yael García-Marchan, Felipe Sojo, Francisco Arvelo, Daniel
Rodriguez, Ignacio Buscema, Alvaro Alvarez-Aular, Josué S. Bello Forero, Vladimir V.
Kouznetsov, Xenón Serrano-Martín

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Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on
volume regulation decrease and sterol biosynthesis of
Leishmania braziliensis
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Jacques Leañeza, Jorge Nuñeza, Yael García-Marchana, Felipe Sojob,c, Francisco Arvelob,c,
Daniel Rodriguezd, Ignacio Buscemae, Alvaro Alvarez-Aularf, Josué S. Bello Forerog,
Vladímir V. Kouznetsovg, Xenón Serrano-Martína,*
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a Laboratorio de Biología y Quimioterapia de Parasitosis Tropicales, Área de Salud, Instituto de Estudios Avanzados (IDEA), Caracas,
República Bolivariana de Venezuela.
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b Laboratorio de Cultivo de Tejidos y Biología de Tumores, Instituto de Biología Experimental (IBE), Universidad Central de Venezuela,
Caracas, República Bolivariana de Venezuela.
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Laboratorio de Ingeniería de Tejidos Humanos, Área de Salud, Instituto de Estudios Avanzados (IDEA), Caracas, República
Bolivariana de Venezuela.
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Laboratorio de Pesquisa Selectiva, Área de Salud, Instituto de Estudios Avanzados (IDEA), Caracas, República Bolivariana de
Venezuela.
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Laboratorio de Fitoquímica, Área de Agricultura y Soberanía Alimentaria, Instituto de Estudios Avanzados (IDEA), Caracas,
República Bolivariana de Venezuela.
f Laboratorio de Síntesis Orgánica y Compuestos Naturales, Centro de Química, Instituto Venezolano de Investigaciones Científicas
(IVIC), Caracas, República Bolivariana de Venezuela.
g Laboratorio de Química Orgánica y Biomolecular, CMN, Universidad Industrial de Santander, Parque Tecnológico Guatiguará, Km 2
Vía Refugio, Piedecuesta 681011, Colombia.
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*Correspondig author: Dr. Xenon Serrano-Martín. Laboratorio de Biología y
Quimioterapia de Parasitosis Tropicales. Área de Salud. Instituto de Estudios Avanzados
IDEA. Carretera Nacional Hoyo de la Puerta. Valle de Sartenejas. Baruta. Caracas,
República Bolivariana de Venezuela. Teléfono: +58 212 9035095. e-mail:
xenonserrano@gmail.com
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Abstract
Diverse spiro dihydroquinoline-oxindoles (JS series) were prepared using the BF3•OEt2-catalyzed imino
Diels-Alder reaction between ketimine-isatin derivatives and trans-isoeugenol. Ten spiro-oxiindole
derivatives were selected and evaluated at different stages of the life cycle of Leishmania braziliensis
parasites, responsible for cutaneous leishmaniasis in South America. Among them, the 8’-ethyl-4’-(4-
hydroxy-3-methoxyphenyl)-3’-methyl-3’,4’-dihydro-1’H-spiro[indoline-3,2’-quinolin]-2-one called JS87
was able to inhibit the growth of promastigotes without affecting the mammalian cells viability, and to
decrease the number of intracellular amastigotes of L. braziliensis. This spiro compound was found to act
through the alteration of parasite internal regulation by disrupting the regulatory volume decrease (RVD), and
to affect the sterol biosynthetic pathway at level of squalene epoxidase (SE) enzyme. These results revealed
that the spiro annulation between quinoline and oxindole scaffolds enhances the anti-leishmanial activity, and
could assist in the development of potent quinoline-oxindole hybrids against Leishmania braziliensis, the
main etiological agent of cutaneous leishmaniasis in South America.
Keywords: chemotherapy, Leishmania braziliensis, spiro dihydroquinoline-oxindoles, parasite internal
regulation, sterol pathway, squalene epoxidase inhibitors
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1. Introduction
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Leishmaniasis is one of the most neglected diseases in the world, with a significant impact on people in
developing countries. It has been ranked between the six more important parasitic diseases worldwide, and it is
estimated that over one billion people are at risk in endemic countries[1]. There are three different clinical
forms of Leishmaniasis: cutaneous (CL), muco-cutaneous (ML) and visceral (VL), being the first one, the most
common. The first-line recommended therapy since 1929 is based on pentavalent antimonials drugs like
Glucantime® and Pentostam®. However, these drugs generate severe side-effects on heart, liver and kidneys[2],
they are expensive and require repeated parental administration[3]. Moreover, antimonial-resistant parasites
have emerged in endemic areas that have limited access to treatment, which promotes the use of second-line
alternatives, including amphotericine B and miltefosine[4-6]. In South America the most prevalent clinical
form is cutaneous leishmaniasis, a disfiguring and stigmatizing disease with non-fatal consequences for the
patient. The parasite responsible for nearly 90% of all CL cases is Leishmania braziliensis[7]. This parasite has
adapted to deforestation, finding new vectors and reservoirs hosts, leading to increased numbers of urban cases
of cutaneous and muco-cutaneous leishmaniasis[8]. The treatment of CL is based on the same mentioned drugs,
but it has been proved inconsistence in their effectiveness against different Leishmania species, including L.
braziliensis[4].
Native Bolivians have used extracts from Galipea longiflora, a plant of the Rutacea family, to treat the
wounds of CL[9]. A study of these plant extracts revealed the presence of diverse simple 2-substituted
quinolines, especially 2-n-propylquinoline A (Figure 1), that affect the viability of different Leishmania
species[9]. Due to this finding, synthetic molecules containing a quinoline skeleton became to be considered
attractive platforms to develop new anti-protozoal agents[10,11]. Generally, quinolines and their reduced
(dihydro-, tetrahydro-) derivatives have specific and general biological properties such as antitumoral [12],
anti-inflammatory[13], antimalarial[14] activities. Some of the specific activities of quinoline derivatives are
melanocotrin receptor agonists, acetylcholisterase inhibitors, ligands for estrogen receptors, and protein
farnesyltranferase inhibitors[15]. Particularly, the 6-methoxy-8-(6-diethylaminohexylamine)-4-methylquinoline
(Sitamaquine, B), developed for oral treatment against VL caused by L. chagasi, inhibits complex II (succinate
dehydrogenase) of the respiratory chain in digitonin-permeabilized promastigotes[16]. However, the clinical
development of this agent has been abandoned because of its low efficacy[17]. Recently, active C2-aryl
quinoline molecules on L. braziliensis parasites, especially the 6-ethyl-2-phenylquinoline C were found to
disrupt considerably parasite bioenergetics and sterol biosynthesis pathway[18]. Interestingly,
tetrahydrogenated quinolines, the 2,4-diaryl-1,2,3,4-tetrahydroquinoline D (Figure 1) resulted also active
against L. chagasi promastigotes, but could not access intracellular parasites forms[19].

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N
HN
NO2
H
N
N
Ph
Ph
N
N
N
Ph
O
A
C
D
B
PhOMe-p
O
H
O
O
O
O
H
NH
R1
N
NH
p-MePh
O
H
O
N
N
NH
R2
N
PhMe-p
H
Cl
F
E
G
OMe
F
Cl
Studied
compounds
OH
H
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Figure 1. Chemical structures of selected quinoline and oxindole molecules possessing anti-leishmanial
activity and the studied compounds. A: 2-n-propylquinoline, B: 6-methoxy-8-(6-diethylaminohexylamine)-4-
methylquinoline, C: 6-ethyl-2-phenylquinoline, D: 2,4-diaryl-1,2,3,4-tetrahydroquinoline, E: spiro-indolone
NITD609, F: spiro[cyclohexanone-oxindoles], G: spiro[indole-3,3’-pyrrolizidine]-2-one, H: spiro
dihydroquinoline-oxindoles, examined compounds.
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On the other hand, the indole, especially oxindole rings including spirocyclic oxindoles (spirooxindoles), are
some of the most common heterocycles present in nature, and several biologically active natural products
indole-based as well as indole-derived drugs are known to possess a variety of bioactivity profiles including
anti-cancer, anti-HIV, anti-diabetic, antibacterial, antioxidant, kinase inhibitory, AChE inhibitory, anti-
leishmanial, β3 adrenergic receptor agonistic, phosphatase inhibitory, analgesic, spermicidal, and vasopressin
antagonists[20]. Most importantly, spiro-oxindoles are also effective anti-protozoal agents. For example, spiro-
indolone NITD609 E is a potent antimalarial drug candidate[21] and spiro[cyclohexanone-oxindoles] like
molecule F can stop proliferation of both promastigote and axenic amastigote forms of L. infantum in a dose-
dependent manner[22]. Moreover, it was recently found that spiro[indole-3,3’-pyrrolizidine]-2-one G (Figure
1) acts as a catalytic inhibitor of the unusual bisubunit DNA topoisomerase IB of L. donovani and it holds a
strong anti-leishmanial efficacy in the BALB/c mouse model of leishmaniasis[23]. It is noteworthy that anti-
protozoal properties of spiro dihydroquinoline-oxindoles have not yet been studied.
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Based on these findings we report the anti-leishmanial properties of diverse spiro dihydroquinoline-
oxindoles H, which were easily prepared using the BF3•OEt2-catalyzed imino Diels-Alder reaction between
ketimine-isatin derivatives and trans-isoeugenol.
In our study, it was demonstrated that one of these compounds (JS87) causes a profound effect on
promastigotes and intracellular amastigotes viability. Also, the internal regulation of L. braziliensis was

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affected in two ways: (i) by disruption of the RVD, and (ii) by alteration of the ergosterol biosynthetic
pathway. Findings that can be helpful in further researches focused on mechanistic details of selective L.
braziliensis growth inhibition.
2. Experimental procedures
2.1. Chemical materials. We previously published the synthesis and characterization of spiro oxindoles 6-
13 tested herein[24]. Two simpler spiro oxindoles 14,15 were prepared following by literature reports[25,26]
1
(Scheme 1, Table 1). The identity of these compounds was determined by H and 13C NMR as well as by IR
and mass spectrometry. All tested molecules were obtained as stable crystalline substances with their respective
well-defined melting points (uncorrected) (Table 1) that were determined on a Fisher–Johns melting point
equipment. Their combustion analysis performed on a Perkin Elmer 2400 Series II analyzer were within ±0.4
of theoretical values that confirm their chemical purity ( ≥95%). The IR spectra were recorded on a Lumex
infralum FT-02 spectrophotometer in KBr. 1H NMR and 13C NMR spectra were recorded on a Bruker AC-400
spectrometer. Chemical shifts are reported in ppm (d) relative to the solvent peak (CHCl3 in CDCl3 at 7.24 ppm
for protons). A Hewlett–Packard 5890a series II Gas Chromatograph interfaced to an HP 5972 mass selective
detector (MSD) with an HP MS Chemstation Data system was used for MS identification at 70 eV using a 60
m capillary column coated with HP-5 [5%-phenyl-poly(dimethyl-siloxane)]. All the chemicals used in this
study for synthesis that we used as a control) were purchased from SigmaAldrich (St. Louis, MO) or AK
Scientific (Union City, CA) and used without any further purification. All purchased compounds were all
≥95% as per the suppliers. All compounds were dissolved in 0.01% DMSO.
2.2. Biological material. Promastigotes of Leishmania (V.) braziliensis strain MHOM/CO/87/UA301
(provided by Dr. Carlos Muskus. Programa de Estudio y Control de Enfermedades Tropicales PECET,
Universidad de Antioquia, Colombia) were isolated from footpad lesions in Balb/C mice previously infected
LIT medium (tryptose 15 g/L, yeast extract 5 g/L, liver extract 2 g/L, hemin-NaOH 0.02 g/L, glucose 4 g/L,
NaCl 9 g/L, KCl 0.4 g/L Na2HPO4, 7.5 g/L at pH 7.4) supplemented with 10% fetal calf serum and maintained
at 29 °C was used for parasite differentiation and maintenance. BMDM macrophages were obtained from
mouse bone marrow and differentiated in a conditioned medium of mouse lung fibroblasts (medium L-929), as
previously reported [27,28].
2.3. Anti-leishmanial activity and cytotoxicity. The effect of ten spiro oxindoles 6-15 was evaluated on the
viability of L. braziliensis promastigotes through colorimetric test, as reported by Saint-Pierrre-Chazalet and
co-workers [29 ] with minor modifications. Briefly, 2x106 parasites/mL were seeded in a 96-well plate, adding
50 μM of each derivative, and incubated for 96h at 29°C. Then, 1μg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) was added and incubated in darkness for 4 h. After this time, acidic

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isopropanol (4N) was added and the plate was read at 570 nm in a spectrophotometer Synergy HT (Biotek).
Miltefosine was used as reference drug.
Compounds that generated a viability inhibition ≈ 50% or more were selected, for further evaluation on the
viability of mammalian host cells (macrophages BMDM). On these host cells, we used MTT assay with few
modifications, seeded 5000 cells/well and the compounds concentration tested was 300 μM. From these values
were determined ranges of EC50 (half maximal effective concentration) and CC50 (half maximal cytotoxic
concentration) for promastigotes and BMDM, respectively.
The EC50 calculation of a selected compound was performed using growth curves in LIT medium, as
previously reported[30,31]. Briefly, the cultures once established (1x106 promastigotes/mL) increasing
concentration of JS87 (and miltefosine as reference drug) were added after 24 h at 29°C. Parasite proliferation
was monitored daily by direct counting in a Neubauer chamber; three independent experiments were performed
for each condition. EC50 index was calculated as previously reported [30,31]. The selectivity index was
calculated with SI = CC50 / EC50, where CC50 was the maximum concentration range of JS87 used to evaluate
cytotoxicity in BMDM.
2.4. Intracellular amastigotes infections. The effect of the selected compound on intracellular
amastigotes was evaluated according to previously described methods [16]. Briefly, a mixture of macrophages
BMDM and promastigotes of L. braziliensis was prepared, in a proportion 1:10 diluted in DMEM +10% FBS.
This mixture was incubated for 18 h at 37 °C and 5% CO2. After this time, new medium with different derivate
concentrations was added and incubated for 96 h in same conditions. Then, macrophages were stained with
Giemsa and counted: % total of macrophage, % of infected macrophages and number of amastigotes by
macrophages.
2.5. Sterol biosynthesis contents. Free sterol contents were determined by High-Resolution Gas-Liquid
Chromatography coupled with High-Resolution Mass Spectrometry (HRGLC/HRMS), as described
previously[32]. Briefly, for the extraction and the separation of neutral lipids, L. braziliensis was cultured in
the presence of JS87 for 96 h, and lipids were extracted with chloroform-methanol (2:1, vol/vol). The extract
was dried and suspended in a minimum volume of chloroform. The chloroform suspension was applied to a
silicic acid column (1.5 by 4 cm) and washed with chloroform to separate the neutral lipids. For the
quantitative analysis of free sterols and structural assignment, the neutral lipids were separated in a high-
resolution capillary column (25 m by 0.20 mm [inner diameter]; Ultra-2; 5% phenyl-methyl-siloxane; film
thickness) in an Agilent Technologies 7890A (Agilent)) gas chromatograph equipped with mass-sensitive
detector Agilent Technologies 5975C. The lipids were dissolved in chloroform and injected into the column at
an initial temperature of 50°C (1 min), followed by a temperature increase to 280°C at a rate of 25°C/min and a

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further rise to 300°C at a rate of 1°C/min. The flow rate of the carrier gas (He) was kept constant at 0.6 ml/min.
The injector temperature was 250°C; the detector was kept at 280°C.
2.6. RVD studies. For the experiments involving induction of hypo-osmotic cell stress, the method described
by Rohloff and co-workers,[33] was used. Briefly, promastigotes were washed with buffer Iso-Cl (137 mM
NaCl, 4 mM KCl, 1: 5 mM KH2PO4, 8.5 mM Na2PO4, 20 mM Hepes, 11 mM glucose, 1 mM CaCl2, 0.8 mM
MgSO4, pH 7.4, 300 mOsm) and resuspended in the same buffer at 1x108 - 1x109/mL. Suspensions were
distributed in a 96-well plate, 100 µL per well, in triplicate, and hypo-osmotic stress was induced with a 1:1
dilution of sterile deionized water. The internal volume changes were measured by light scattering method, at
550 nm absorbance and recorded every 10s for 10 min, using a spectrophotometer (Synergy HT, Biotek).
3. Results and Discussion
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3.1. Synthesis of spiro-oxindoles. The preparation of selected dihydrospiro[indoline-3,2’-quinolin]-2-ones 6-
13 (molecules H) was performed using a straightforward synthesis based on BF3•OEt2-catalyzed imino Diels-
Alder reaction of preformed ketamines 3 derived from isatin 1 and anilines 2a-g, and trans-isoeugenol 4 in
anhydrous CH2Cl2 as solvent, at room temperature from 1 to 3 h,[24] while related 3’-phenylspiro[indoline-
3,2'-thiazolidine]-2,4'-dione 14 was easily synthetized via a three-component condensation reaction of isatin 1,
aniline 2a and thioglycolic acid 5 in anhydrous toluene at reflux for 1 h[25] and spiro compound 15 was simply
obtained through heating only two components 1 and 5 under the same reaction condition[26] (Scheme 1).
R2
R1
HO
O
O
R1
NH
H
HS
O
Ph
N
MeO
N
NH
OH
5
4
N
O
.
PhMe, 110 oC
1 h
BF3 OEt2, CH2Cl2
1 - 3 h
R2
O
S
N
H
14
72%
3a-h
OMe
H
6-13
OH
NH2
R1
PhMe, 110 oC
3 - 8 h
35-65%
R2
O
a
c
e
g
b
R1 = R2 = H; R1 = Me, R2 = H;
R1 = H; R2 = Me; R1 = Et, R2 = H;
R1 = H, R2 = Et; R1 = CN, R2 = H;
R1 = H, R2 = Cl; R1 = H, R2 = Br
2a-h
NH
O
O
5
d
O
f
PhMe, 110 oC
1 h
O
S
h
N
15
H
82%
1
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Scheme 1. Synthesis of selected dihydrospiro[indoline-3,2’-quinolin]-2-ones 6-13 and related spiro oxindole
compounds 14,15.
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These spiro oxindoles were obtained as stable solids in good yields (35–82%) after chromatography column
purification. Their structures were confirmed by various spectroscopic techniques such as EI-MS, 1H NMR, 13
NMR and IR, testing each sample as a pure chemical substance in the following biological experiments.
C
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3.2. Preliminary antiparasitic and cytotoxicity activity: MTT studies. The obtained de novo spiro
oxindoles 6-15 identified as series JS were evaluated on L. braziliensis promastigotes proliferation and
cytotoxicity assays on mouse bone marrow-derived macrophages (BMDM). In the first screening through MTT
assays, we observed that almost all compounds showed an appreciable biological activity (EC50 < 50 M) on
parasite proliferation (Figure 2) and low BMDM cytotoxicity (CC50 > 300 M) (Table 1). It was found that
only one compound, spiro[indoline-3,2’-[1,3]oxathiolane]-2,5’-dione 15 (JS61) resulted to be inactive (EC50
>
50 M) against L. braziliensis promastigotes. Low toxicity of dihydrospiro[indoline-3,2’-quinolin]-2-ones 6-13
on mammalian cells has also been reported for other cells lines[34,35].
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0
l
3
5
6
1
9
9
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7
9
2
e
o
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8
8
8
9
n
r
i
t
S
S
S
S
S
S
S
S
S
S
s
n
J
J
J
J
J
J
J
J
J
J
o
o
f
e
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t
l
i
M
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Figure 2. Evaluation of JS compounds on L. braziliensis promastigotes proliferation. Screening of compounds
using MTT assay with L. braziliensis promastigotes exposed to 50 µM of each spiro derivative and Miltefosine.
Each condition was done in triplicate.
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Among the active dihydrospiro[indoline-3,2’-quinolin]-2-ones 6-13, the 8’-ethyl-4’-(4-hydroxy-3-
methoxyphenyl)-3’-methyl-3’,4’-dihydro-1'H-spiro[indoline-3,2’-quinolin]-2-one 9, code JS87, was the most
active antiparasitic compound. Interestingly, its regio-isomer in position C-6’ (JS86) resulted less active, but
both molecules showed higher activity than miltefosine, the reference drug.

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The same level of anti-leishmanial activity was obtained with the unsubstituted spiro indoline-3,2’-quinolin-
2-one 6 (JS43) compared to 6-chlorosubstituted analog 12 (JS89). The substitution of Cl atom with bromine
atom (comp.13, code JS92) enhanced slightly the biological effect. However, the most notable differences in
activity were observed when the activity of spiro[indoline-3,2'-[1,3]oxathiolan]-2-one 15 and of spiro[indoline-
3,2'-quinolin]-2-ones 6-13 or spiro[indoline-3,2’-thiazolidine]-2,4’-dione 14 were compared.
Table 1. Evaluation of Spiro Oxindoles 6-15 on the Viability of L. braziliensis Promastigotes and BMDM
Macrophages.
Compound
Code
Structure,
Mol. Formula,
M.W.a
Physicochemical properties
CC50(µM)
EC50 (M)
in silicob
cLogP
2.7676
cLogS
-4.722
pKa
Leishmania
braziliensis c
< 50
BMDM
macrophagesd
O
NH
H
N
6
JS43
JS69
JS79
JS87
JS86
JS56
JS89
JS92
8.958
> 300
C24H22N2O3
M.W.: 386.45
Ar
O
NH
H
7
3.1115
3.1115
3.5271
3.5271
2.6032
3.3736
3.4928
-5.066
-5.066
-5.225
-5.225
-5.495
-5.458
-5.556
8.949
8.949
8.943
8.949
8.922
8.935
8.931
< 50
< 50
< 50
< 50
< 50
< 50
< 50
> 300
> 300
> 300
> 300
> 300
> 300
> 300
N
C25H24N2O3
M.W.: 400.48
Ar
O
NH
H
8
N
C25H24N2O3
M.W.: 400.48
Ar
O
NH
H
9
N
C26H26N2O3
M.W.: 414.51
Ar
O
NH
H
N
10
11
12
13
C26H26N2O3
M.W.: 414.51
Ar
O
H
N
CN
NH
C25H21N3O3
M.W.: 411.46
Ar
O
NH
H
N
Cl
C24H21ClN2O3
M.W.: 420.89
Ar
O
NH
H
N
Br
C24H21BrN2O3
M.W.: 465.35
Ar
O
14
JS55
JS61
1.9324
-4.461
nd
< 50
> 300
NH
N
C16H12N2O2
M.W.: 296.34
S
S
O
O
NH
15
0.3287
0.5768
-3.114
-2.66
nd
nd
> 50
< 50
> 300
> 200
O
O
C10H7NO3
M.W.: 221.23
S
S
Miltefosine
Drug
N+
O
P O
O- M.W.: 421.60
15
C22H48NO4P
O
reference
a
b
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Ar is 2-methoxy-4-methylphenyl, M.W. - molecular weight. Theoretical values cLogP, cLogS were calculated using
commercially available the OSIRIS Property Explorer software, and pKa were calculated with ChemDraw 15.0 program: cLogP =
n-octanol/water partition coefficient. The lipophilicity optimum interval is 0 < Log P < 3; cLogS = aqueous solubility. c Compounds

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with EC50 > 50 µM were classified as inactive for L. braziliensis protozoan. d Compounds with CC50 < 100 µM were classified as
toxic for the mammalian cell lines.
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From these results it can be concluded that spiro annulation of indoline and tetrahydroquinoline nucleus is
responsible for anti-leishmanial activity in this series of molecules. In order to understand the biological
behavior of this series, the physicochemical properties of the tested compounds 6-15 were calculated using the
Osiris Property Explorer software. In general, the values of calculated partition coefficient (cLog P) of the
active compounds 6-14 are near the optimal range of the lipophilicity (1.9324 < cLog P < 3.5271), thus these
compounds are all lipophilic molecules with good enough water solubility at pH 7.4 (-4.461< cLogS < -5.556).
It should be noted that more than 80% of the drugs have an estimated logS value greater than -4.
Miltefosine, the reference drug in this study, resulted to be a lower lipophilic molecule (cLog P = 0.5768)
with higher water solubility (cLogS = -2.66), but the spiro[indoline-3,2’-[1,3]oxathiolane]-2,5’-dione 15, which
was inactive, has similar parameters (cLog P = 0.3287, cLogS = -3.114). Hence, these results obtained cannot
explain clearly the structure-antileishmanial activity relationship of the tested spiro oxindoles in this work.
Nevertheless, the most active antiparasitic compound JS87 was selected for subsequent studies.
3.3. Anti-leishmanial activity of JS87: Growth curve and EC50 determination. The compound JS87
affected the viability of L. braziliensis promastigotes with an EC50: 6.0 ± 0.2 µM (Figure 3). A similar result
was obtained with the 6-ethyl-2-phenylquinoline on L. braziliensis promastigotes (EC50: 6.0 ± 0.2 µM)[16].
Both nitrogen-containing heterocycles were more effective than miltefosine (EC50: 21 ±0.2 µM) on the viability
of these parasites species. From the EC50 it was possible to calculate the selectivity index of the JS87 obtaining
an SI> 50, resulting that the JS87 is at least 50 times more active on the parasites than on the host cell.
B
A
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Figure 3. A) Evaluation of JS87 on L. braziliensis promastigotes. Growth curve of L. braziliensis
promastigotes, exposed to crescent concentrations of JS87. B) Determination EC50 of JS87 (6.0 ± 0.2 µM) and
Miltefosine (21.0 ± 0.2 µM) at 96 h exposure. Each condition was done in triplicate.

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3.4. Effect of JS87 on intracellular amastigotes: EC50 determination. Leishmanicidal effect of JS87 on
the clinically relevant stage was studied through the design of an in vitro infection model with macrophages
BMDM and L. braziliensis amastigotes. With this model, a maximum infection of 65% was obtained; these
results are similar to that reported by Zauli-Nascimento and co-workers [36]. The effect of JS87 was evaluated
at 96 h post-treatment. Figure 4 shows that as the JS87 concentration increased, a concomitant decrease in the
number of intracellular amastigotes was observed with an EC50 value of 3.3 ± 0.2µM. Analogous values were
reported for intracellular amastigotes of L. major and L. braziliensis, exposed to similar quinoline
molecules[16,19].
B
A
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Figure 4. Evaluation of JS87 on intracellular amastigotes of L. braziliensis. A) BMDM macrophages were
infected with promastigotes of L. braziliensis (1:10) and incubated with JS87 for 96 h. ● % macrophages total,
■ % infected macrophages and ▲ N° amastigotes x macrophages. B) Determination EC50 of JS87 (3.3 ±
0.2µM) calculated with N° de amastigotes per macrophages. Each experiment was done in triplicate.
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Furthermore, we showed that this effect was specific for intracellular amastigotes without affecting the viability
of host cells (macrophages BMDM) in the concentration ranges tested. When calculating SI> 90, we observed
that JS87 is at least 90 times more effective on intracellular amastigotes than on BMDM, and 1.8 times more
effective than on promastigotes of L. braziliensis. It is also evident that at the highest concentration tested
(100µM), there is still 40% of infected macrophages remaining. Perhaps (and in spite of its anti-parasitic
activity), this effect could attribute to limited access of JS87 to intra-vacuolar compartments, where the
amastigote proliferates within the host cell. This could affect the "in vivo" activity of the compound. It would
be pertinent to take this factor into consideration, in order to make some structural modifications and improve
the penetration capacity, while maintaining the anti-parasitic potency.

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3.5. Sterol biosynthetic pathway. The sterol biosynthetic pathway has been extensively validated as an
important chemotherapeutic target[37]. After quantification of the neutral lipid fraction in a gas chromatograph
(GC) equipped with a mass selective detector (MSD), it was possible to determine that JS87 affected this
biological pathway in L. braziliensis (Table 2).
Indeed, we were able to show an increase of squalene (81.28%), concordant with: 1) a significant depletion
of remainder sterols, and 2) an accumulation of exogenous cholesterol being a possible compensation
mechanism like the ones proposed for L. amazonensis[38] and L. donovani [39].
These interesting outcomes suggest an affectation, on the natural behavior of squalene-2,3-epoxidase
enzyme (Scheme 2), which is essential for sterols biosynthesis[40], and they demonstrate the biological effect
of dihydrospiro[indoline-3,2’-quinolin]-2-ones on L. braziliensis, through an alternation of sterol biosynthesis
pathway. The same effect was reported previously by our work group, but with aryl-quinolines[16]. Currently,
we do not have enough evidence to support a direct action of JS87 on Squalene Epoxidase. However,
considering the accumulation of Squalene and the non-detection of Squalene Epoxide, we can presume that the
compound affected the natural functioning of the enzyme.
Table 2. Effect of JS87 on the biosynthesis of free sterols in L. braziliensis promastigotes.a
Mass percent after treatment
with:
Sterol
Retention time (min)
p-value
Control
JS87 (6 µM)
Exogenous cholesterol
Squalene
32.72
26.95
33.52
35.74
35.98
4.83
18.72
0.0232
<0.0001
<0.0001
<0.0001
<0.0001
18.76
4.45
81.28
NDb
ND
Squalene epoxide
Lanosterol
8.98
Ergosterol
76.41
ND
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292
293
a Parameters determined by Gas Chromatography and High Resolution Mass Spectrometry; p significance ≤ 0.05, through “t test” (InfoStat
2012). JS87 added at EC50 value. b ND, not detected.

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O
O
HO
HO
O
Statins
SCoA
HMG-CoA
OH
OPP
SCoA
COO-
COO-
Isopentenyldiphosphate
Mevalonate
Acetyl-CoA
Bisphosphonates
JS87
OPP
Squalene-
epoxidase
Squalene
Farnesyldiphosphate
H
H
Azoles
H
Squalene-epoxide
Lanosterol
Zymosterol
HO
HO
H
H
O
Azasterols
H
H
H
H
H
H
H
H
HO
HO
HO
H
5-Dehydroepisterol
Ergosterol
Ergosta-8,24(24')-dien-3 -ol

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Scheme 2. Fragment of sterol biosynthetic pathway, showing the possible site of action of JS87. (Modified using the
work by Serrano-Martín and co-workers[30]).
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3.6. Regulatory volume decrease (RVD). Few researches have been reported regarding RVD in
trypanosomatids[41-43]. Due to its digenetic cycle, Leishmania spp are exposed to multiple extracellular
osmotic stress conditions that involve cell swelling. In these conditions, a series of adaptive mechanisms
oriented toward restoring cell volume, are activated[43-45] and if these replies fail, cell death could occur
either by necrosis or apoptosis. Using a light scattering technique we registered the relative volume alteration
over time, where changes in parasites absorbance are related to cell volume. In this sense, we demonstrated that
JS87 (6.0 µM) affected significantly the RVD of L. braziliensis under hypo-osmotic stress, compared to
untreated controls (Figure 5).

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Figure 5. Effect of JS87 on L. braziliensis RVD. Promastigotes of L. braziliensis were incubated 96 h with 6
µM of JS87 (continuous line). Parasites not exposed to JS87 (control) are shown with a discontinued line.
After incubation both parasites populations, were subjected to hypo-osmotic stress. Volume recovery was
followed by light scattering. Each experience was done in triplicate.
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Subsequent to a hypo-osmotic shock, untreated controls showed a decrease in absorbance indicating cell
swelling, followed by a steady rise near to original level indicating normal volume recovery. Nevertheless,
parasites previously treated with JS87 showed a decrease in absorbance without the prominent rise, indicating
no cell volume recovery. Similar results have been obtained with T. cruzi[41] and L. mexicana[43].
This result represents the first report that shows the effect of spiro dihydroquinoline-oxindoles on the RVD,
in L. braziliensis. It is possible that sterol depletion was involved in a destabilization of the plasma membrane,
implicating affection in the natural RVD process. Currently our work group is performing experiments to
correlate these results to find a possible connection between both biological events.
4. Conclusion
Currently, treatment of leishmaniasis is far from satisfactory; all antileishmanial drugs available for
leishmaniasis treatment are toxic, expensive, and frequently ineffective. Due to these limitations of the existing
treatments, better drugs are urgently needed[45,46]. A series of spiro dihydroquinoline-oxindoles was prepared
via straightforward synthesis based on the BF3•OEt2-catalyzed imino Diels-Alder reaction of ketamine-isatin
and trans-isoeugenol that allowed evaluating leishmanicidal activity of 10 spiro derivatives. Among the
synthetized compounds, the spiro oxindole JS87 was selected, according to its potent leishmanicidal capacity
and low toxicity on the host cell. It was demonstrated that JS87 generated a profound in vitro activity on the

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proliferation of L. braziliensis promastigotes at micro-molar levels, more active than the standard and
alternative drug, miltefosine. Additionally, it was determined that this compound generated a selective effect on
intracellular amastigotes. This effect was slightly more potent than promastigotes and did not affect the host
cells (BMDM macrophages). It is also evident that at the highest concentration tested (20µM), there is still 40%
of infected macrophages remaining. Perhaps (and in spite of its anti-parasitic activity), this effect could
attribute to limited access of JS87 to intra-vacuolar compartments where the amastigote proliferates within the
host cell. Studying this effect, some specific biological factors were revealed that could explain the high anti-
parasitic activity through a destabilization in the intracellular RVD mechanism and/or an affectation in normal
activity of squalene epoxidase enzyme, which is essential for the sterol biosynthesis route. It is possible that
both factors could affect synergistically against the normal functioning of the plasmatic membranes (externals
and/or internal). Furthermore, it is possible that the destabilization of sterol biosynthesis could lead to the
alteration of the RVD mechanism. In this sense, actually we development some assays, evaluating the effect of
classical sterol biosynthesis inhibitors on RVD capacity. However, the dihydroquinoline-oxindole JS87 could
represents a promising candidate for more advanced studies in the search of an alternative drug against L.
braziliensis, the main etiological agent of cutaneous leishmaniasis in South America.
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Acknowledgments
The authors gratefully acknowledge critical comments of Lilia Ramírez, Jaime Charris and Cecilia Castillo.
Financial support: this work was supported the Fondo Nacional de Ciencia Tecnología e Innovación
(FONACIT), Ministerio del Poder Popular para Educación Universitaria, Ciencia y Tecnología, República
Bolivariana de Venezuela (Project to XS-M: 2021002132, 2012000784 and 2012001961). V.V.K. thanks the
financial support of Colombian Institute for Science and Research (COLCIENCIAS) under the project No. RC-
007-2017.
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ACCEPTED MANUSCRIPT
 Quinoline-oxindoles affects Leishmania braziliensis
proliferation
 Spiro-quinolin alters parasite regulation, disrupting the
regulatory volume decrease
 Quinoline-oxindoles affect the sterol biosynthesis in
Leishmania braziliensis
 Spiro-quinolin is 90 times more effective on intracellular
amastigotes than on BMDM
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