Chroman-4-one hydrazones derivatives: synthesis, characterization, and in vitro and in vivo antileishmanial effects

Article abstract of DOI:10.1007/s00044-019-02446-x

In searching for better therapeutic alternatives to treat cutaneous leishmaniasis (CL), this study aimed to obtain and evaluate the efficacy and toxicity of new chroman-4-one hydrazones derivatives. Compounds were prepared and characterized, and then transformed into hydrazonas for molecular optimization. Their cytotoxicity was tested in different cell types using an in vitro MTT assay and the efficacy was evaluated using an in vitro macrophage intracellular amastigotes of Leishmania (Viannia) panamensis and L. (V) braziliensis by flow cytometry. The therapeutic effect of two formulations of chroman-4-one hydrazones on the CL induced by L. (V) braziliensis in golden hamsters was determined according to the size of lesions after treatment. The effect of these compounds in the production of inflammatory mediators and cell migration was also determined by in vitro assays using human fibroblasts models. Neither cytotoxicity nor genotoxicity was observed. The benzoic acid hydrazone derivative 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (4), produced a higher percentage of clinical cures, followed by benzoic acid, 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (3), while benzoic acid, 2-(2,3-dihydro-1,1-dioxide-4H-1-benzothiopyran-4-ylidene) hydrazide (5) and 4-pyridinecarboxylic acid, 2-(4H-1-benzopyran-4-ylidene) hydrazide (6) caused a poor therapeutic response. The compound 4 also showed an effect in the inflammatory and fibroblast migration processes. In conclusion, this is the first report of antileishmanial activity combined with inflammatory and wound healing properties. Results obtained here suggest that this strategy could be a good alternative for development of new drugs for the treatment of CL.

Full text of DOI:10.1007/s00044-019-02446-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02446-x
ORIGINAL RESEARCH
Chroman-4-one hydrazones derivatives: synthesis, characterization,
and in vitro and in vivo antileishmanial effects
Yulieth Upegui1 Karina Rios Wiston Quiñones Fernando Echeverri Rosendo Archbold Javier D. Murillo
2 ●
3 ●
3 ●
3 ●
1 ●
●
Fernando Torres3 Gustavo Escobar Iván D. Vélez Sara M. Robledo
3 ●
1 ●
1
●
Received: 21 June 2019 / Accepted: 12 September 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In searching for better therapeutic alternatives to treat cutaneous leishmaniasis (CL), this study aimed to obtain and evaluate
the efficacy and toxicity of new chroman-4-one hydrazones derivatives. Compounds were prepared and characterized, and
then transformed into hydrazonas for molecular optimization. Their cytotoxicity was tested in different cell types using an
in vitro MTT assay and the efficacy was evaluated using an in vitro macrophage intracellular amastigotes of Leishmania
(
Viannia) panamensis and L. (V) braziliensis by flow cytometry. The therapeutic effect of two formulations of chroman-4-
one hydrazones on the CL induced by L. (V) braziliensis in golden hamsters was determined according to the size of lesions
after treatment. The effect of these compounds in the production of inflammatory mediators and cell migration was also
determined by in vitro assays using human fibroblasts models. Neither cytotoxicity nor genotoxicity was observed. The
benzoic acid hydrazone derivative 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (4), produced a higher percentage
of clinical cures, followed by benzoic acid, 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (3), while benzoic
acid, 2-(2,3-dihydro-1,1-dioxide-4H-1-benzothiopyran-4-ylidene) hydrazide (5) and 4-pyridinecarboxylic acid, 2-(4H-1-
benzopyran-4-ylidene) hydrazide (6) caused a poor therapeutic response. The compound 4 also showed an effect in the
inflammatory and fibroblast migration processes. In conclusion, this is the first report of antileishmanial activity combined
with inflammatory and wound healing properties. Results obtained here suggest that this strategy could be a good alternative
for development of new drugs for the treatment of CL.
●
●
●
●
●
Keywords Chromans Hydrazones Ointment Cutaneous leishmaniasis Animal model cure Healing
Introduction
cases are cutaneous leishmaniasis (CL) (Alvar et al. 2012).
The massive displacement of populations, climatic change,
growing resistance to drug treatment, lack of therapeutic
alternatives and long treatments are creating a significant
rise in this disease, with a resultant increase in public health
problems. The pharmacological arsenal currently used to
combat CL is limitated. Only five drugs are available
Leishmaniasis is a tropical disease caused by parasites
belonging to the Leishmania genus that is endemic in 98
countries, affecting 12 million people. There are about
2
,000,000 new cases per year, of which 0.7–1.3 million
(meglumine antimoniate—MA or sodium stibogluconate,
miltefosine, amphotericin B—AMB and paromomycin), all
of them displaying a series of drawbacks related to a
decreased efficacy, moderate to severe adverse effects and
high costs in-patient care (Den Boer et al. 2011). For that,
there is an urgent need to find new and better drugs to
treat CL.
An useful strategy, commonly used in drug development,
involves the use of bioactive molecule as a template to
obtain more potent molecules with the best pharmacological
properties (Mathur and Hoskins 2017). The benzopyran
system is a valuable scaffold in medicinal chemistry, since
*
*
Fernando Echeverri
Fernando.echeverri@udea.edu.co
Sara M. Robledo
Sara.robledo@udea.edu.co
1
2
3
PECET-School of Medicine, Universidad de Antioquia, Calle 70 #
5
2-21, Medellin, Colombia
Biogen-Universidad de Santander, Avenida 4 10N-61,
Cúcuta, Colombia
Química Orgánica de Productos Naturales-Institut of Chemistry,
Universidad de Antioquia, Calle 70 # 52-21, Medellin, Colombia

Medicinal Chemistry Research
Fig. 1 Structure of several
natural products with a
benzopyran skeleton
it is found in many natural bioactive molecules, such as
flavonoids, coumarins, chromenes, chromans, as well as
some alkaloids (Fig. 1) (Saengchantara and Wallace 1986;
Borges et al. 2005; Sharma et al. 2011; Gaspar et al. 2014;
Ramendra et al. 2014). Therefore, this chemical moiety is
considered a privileged structure, whose range of activity
includes substances for human health, insecticides and other
molecules for agriculture. Particularly, chroman and thio-
chroman also exhibit a wide range of biological activities
such antiestrogenic (Kanbe et al. 2006), cytotoxic (Gaspar
et al. 2019), antitumoral and antiepileptic (Rawat 2016).
They also have Sirtuin 2 selective inhibition (Friden-Saxin
et al. 2012). In addition, from the synthetic point of view, it
is a very versatile molecule, relatively easy to synthesize
spectrometer (Bruker Optic GmbH, Ettlingen, Germany).
1H- and 13C-nuclear magnetic resonance spectra were
recorded using a Bruker Fourier 300 spectrometer (Bruker
Bio-Spin GmbH), operating at 300 MHz for 1H and
75 MHz for 13C. Samples were dissolved in DMSO-d6 or
CDCl3, using TMS as an internal standard. HRMS was
obtained using Q-ToF quadrupole/orthogonal spectrometry
(Waters) in either the negative (reported as [M + H]+) or
positive mode (reported as [M + H]+) and a Bruker Impact
II UHR-Q-TOF mass spectrometer (Bruker Daltonik) in the
positive mode.
General procedure for the preparation of
thiochroman-4-one or 4-chromanone
(
2
Borges et al. 2005; Ramendra et al. 2014; Nicolaou et al.
000) and possess different sites for chemical transforma-
To a mixture of acrylic acid (700 mL, 720 mg, 10 mmol)
tions in the aromatic ring and the heterocycle or carbonyl
systems. Moreover, the thiochroman analog is susceptible
of oxidation to sulfoxide and sulfone.
and thiophenol or phenol (15 mmol), I (20% mol, 760 mg,
2
3 mmol) was added, and the mixture was stirred at 50 °C for
24 h. After the completion of the reaction (monitored by
TLC), a cold saturated sodium thiosulfate solution (30 mL)
was added and extracted with dichloromethane (2 × 25 mL).
The combined organic layers were mixed with a saturated
solution of sodium bicarbonate and extracted to remove the
unreacted starting material layer, which was acidified with
10% HCl and extracted with dichloromethane (3 × 50 mL).
The combined organic layers were dried over Na SO , and
Due to the high incidence of CL in tropical countries, a
research program has been designed to look for molecules
with antileishmanial activity and to identify candidate
compounds to be submitted to CL animal assay and
eventually to be converted into new medicines. In this
work, we report the synthesis process to obtain several
active molecules against CL, starting from a simple
molecules like chroman and thiochroman, through a
sequence of chemical transformations to the in vitro and
in vivo antileishmanial evaluations. The effect of these
compounds in the inflammatory and healing processes
was also determined.
2
4
the evaporation of the solvent under reduced pressure
afforded 64–66% of the desired additional product. The
product was cooled down to 2 °C in an ice bath, 3 mL of
concentrated sulfuric acid was added, and the reaction
mixture was allowed to warm to room temperature for 2 h,
with magnetic stirring. The reaction was quenched with ice,
and the mixture was extracted with dichloromethane (3 ×
50 mL). The combined organic layers were washed once
Materials and methods
with water, and then with saturated NaHCO solution. The
3
Spectroscopy
combined organic layers were washed with brine, dried over
Na SO , and concentrated under reduced pressure. The
2
4
All commercially available reagents and solvents were
obtained from commercial suppliers and used without fur-
ther purification. 4-Chromane (1) and 4-thiocromanone (2)
were purchased from Sigma-Aldrich. The reaction progress
was monitored using thin layer chromatography on silica
gel TLC aluminum sheets (60F254, Merck). The melting
points were determined using a Mel-Temp apparatus
residue was purified by column chromatography over silica
gel using hexane/EtOAc (9:1), as an eluent, to obtain
thiochroman-4-one or 4-chromanone, as a white solid
(90–85%).
Oxidation of sulfides to sulfones (5)
®
(
Electrothermal, Staffordshire, UK) and were uncorrected.
To a 25-mL glass tube, sulfide 5 (1.0 mmol), oxone
FTIR spectra were obtained on a Bruker Alpha FTIR
(0.9221 g, 1.5 mmol) and a water ethanol mixture (1:1)

Medicinal Chemistry Research
(
6
3.0 mL) were added, and the mixture was stirred at
0 °C for 12 h. The mixture was then cooled to room
temperature and extracted with dichloromethane (3 ×
5 mL). The combined organic layers were dried over
Benzoic acid, 2-(2,3-dihydro-1,1-dioxido-4H-1-
benzothiopyran-4-ylidene)hydrazide (5)
1
2
Yield: 78%, p.f: 216–217 °C. H RMN (300 MHz, DMSO).
anhydrous Na SO4 and concentrated under reduced
δ: 3.75 (t, J = 5.7, 2H, H2), 3.45 (t, J = 5.7, 2H, H3), 8.26
(sbr, 1H, H4), 7.93 (d, J = 8.6, 1H, H5), 7.61 (m, 1H, H6),
7.77 (m, 1H, H7), 7.93 (d, J = 8.6, 2H, H2′–H6′), 7.54 (m,
2H, H3′–H5′), 7.72 (m, 2H, H4′), 11.09 (s. 1H, N–H). C-
RMN δ 25.0 (–C3), 46.4 (–C2), 123.2 (–C5), 126.7 (–C4),
128.7 (–C2′–C6′), 128.8 (–C3′–C5′), 130.9 (–C6), 132.3
(–C4′), 133.3 (–C8), 133.6 (–C7), 133.9 (–C9), 134.1
2
pressure. The residue was purified by column chroma-
tography over silica gel using mixtures of hexane and
EtOAc as eluents.
13
General procedure for the preparation of Acyl
hydrazone derivatives 3, 4, and 6
(
–C1′), 147.3 (–C3a), 166.6 (–C=O). m/z 315.0790 for
+
Thiochroman-4-ones or 4-Chromanone (0.5 mmol) was
dissolved in anhydrous methanol (25 mL). The mixture
was heated at reflux, and then with hydrazide (1.0 mmol,
C H N O S [M+H] , calculated 315.0797.
16 15 2 3
4-Pyridinecarboxylic acid, 2-(4H-1-benzopyran-4-
ylidene)hydrazide (6)
2
equiv.), and 60 mL of glacial acetic acid was added.
After 12 h at reflux, the resulting precipitate was collected
through filtration and washed with methanol. After dry-
ing under a vacuum, the residue was passed through a
small pad of silica gel with ethyl acetate, after the solvent
acyl hydrazones were evaporated and obtained as white
solids.
1
Yield: 80%, p.f: 198–199 °C. H RMN (300 MHz, DMSO),
δ: 3.13 (t, J = 6.0, 2H, H3), 4.44 (t, J = 6.0, 2H, H2), 7.10
(seudo t, J = 7.5, 1H, H6), 7.19 (d, J = 7.5, 1H, H7), 7.55
(seudo t, J = 7.7, 1H, H5), 8.24 (d, J = 7.5, 1H, H4), 7.97
(d, J = 4.8, 2H, H2′–H6′), 8.94 (m, 2H, H3′–H5′), 7.56
sbr, 1H, H4′), 11.30 (s. 1H, N–H). C-RMN δ 26.3 (–C3),
65.3 (–C2), 117.9 (–C7), 120.1 (–C9), 121.7 (–C5), 122.2
1
3
(
Spectroscopy of hydrazones
(
–C2′–C6′), 125.4 (–C4), 132.3 (–C6), 142.0 (–C1′), 150.5
Benzoic acid, 2-(2,3-dihydro-4H-1-benzothiopyran-4-
ylidene)hydrazide (3)
(–C3′–C5′), 152.1 (–C3a), 157.2 (–C8), 163.2 (–C=O). m/z
268.1080 for C H N O [M+H] , calculated 268.1080.
+
1
5
14
3
2
Yield: 82%, p.f: 160–161 °C. 1H RMN (300 MHz,
DMSO), δ: 3.06 (m, 4H, H2, H3), 8.20 (sbr, 1H). (sbr, 1H,
H4), 7.19 (sbr, 1H, 1H, H5), 7.27 (m, 2H, H6, H7), 7.90
Biological study
Cells and culture conditions
(
sbr, 2H, H2′–H6′), 7.52 (m, 2H, H3′–H5′), 7.54 (sbr, 1H,
H4′), 10.87 (s, 1H, N–H). 13C-RMN δ: 25.7 (–C3), 28.4
(
(
1
1
Human U937 (CRL-1593-2TM) macrophages, human
TM
–C2), 121.8 (–C9), 125.7 (–C5), 126.0 (–C4), 128.4
–C7), 128.4 (–C2′–C6′),128.8 (–C3′–C5′), 129.9 (–C6),
Detroit 551 (CCL-110 ) fibroblast and golden hamster
TM
BHK-21 (CCL-10 ) kidney fibroblast were purchased
32.0 (–C4′), 134.5 (–C1′), 136.8 (–C8), 153.1 (–C3a),
from the American Type Culture Collection. Cells were
cultured in the corresponding medium, according to the
manufacturer’s instructions. The primary cultures of mac-
rophages and fibrocytes, derived from human monocytes
(huMDM and huMDF, respectively), were obtained from
anticoagulated venous blood from healthy donors, after
signing informed consent. The huMDM and huMDF were
obtained, according to the methodology described by others
(Robledo et al. 1994; Montoya et al. 2015).
+
64.5 (–C=O). m/z 282.0827 for C H N OS [M+H] ,
1
6
14
2
calculated 282.0821.
Benzoic acid, 2-(2,3-dihydro-4H-1-benzopyran-4-
ylidene) hydrazide (4)
1
Yield: 93%, p.f: 190–191 °C. H RMN (300 MHz, DMSO),
δ: 2.97 (t, J = 6.3, 2H, H3), 4.27 (t, J = 6.3, 2H, H2), 8.02
(
7
sbr, 1H, H4), 7.02 (sbr, 1H, 1H, H5), 6.94 (seudo t, J =
.5, 1H, H6), 6.91 (d, J = 7.5, 1H, H7), 7.88 (sbr, 2H,
In vitro cell toxicity
H2′–H6′), 7.51 (m, 2H, H3′–H5′), 7.56 (sbr, 1H, H4′),
1
1
0.88 (s. 1H, N–H). 13C-RMN δ: 26.4 (–C3), 65.2 (–C2),
17.9 (–C7), 120.8 (–C9), 121.7 (–C5), 125.4 (–C4), 128.3
U937, Detroit 551, BHK-21, huMDM, and huMDF
in suspension in the corresponding culture media were
dispensed in 96-well plates, at a concentration of 1 × 105
or 2.5 × 105 or 3 × 10 or 2.5 × 106 cells/mL. Then,
100 µL/well of each compound at concentrations of
(
1
–C2′–C6′),128.8 (–C3′–C5′), 132.0 (–C4′), 134.5 (–C1′),
50.8 (–C3a), 157.6 (–C8), 164.3 (–C=O). m/z 266.1055
5
+
for C H N O [M+H] , calculated 266.1049.
1
6
14
2
2

Medicinal Chemistry Research
2
00–100–50–25–12.5–6.25 µL/mL were added. Plates
4 °C, and then the supernatant was discarded, and cells were
suspended in 500 μL of cold PBS and analyzed in an argon
laser Cytomics FC 500MPL flow cytometer (Beckman
Coulter), reading at 488 nm of excitation and 525 nm of
emission and counting 10,000 events. Infected cells were
determined according the positive events for green fluor-
escence (parasites). Infected cells exposed to AMB or MA
were used as controls for the antileishmanial activity
(positive control), while infected cells incubated in a fresh
RPMI 1640 medium were used as controls for infection
(negative control). Each concentration was tested in tripli-
cate in two independent experiments.
were incubated at 37 °C, 5% CO for 72 h. Then, 20 μL of
2
(
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) solution (0.5 mg/mL) (Sigma) was added
to each well, and the plates were incubated at 37 °C, 5%
CO for 3 h. The reaction was stopped by adding 100 μL/
2
well of 50% isopropanol solution, with 10% sodium dodecyl
sulfate and 30 min of incubation. The concentration of
formazan was determined at 570 nm in a Varioskan Flash
multimode reader (Thermo Scientific), and the intensity of
the color was registered as optical densities (O.D) (Mesa
et al. 2015). Cells treated with antileishmanial drugs, AMB,
and the cytotoxic drug, doxorubicin (DOXO), were used as
controls for the cytotoxicity (positive control), while the cell
incubated in cell culture media, with no compound or drug,
was used as a growth cell control (negative control). All
determinations were conducted in triplicate in at least two
independent experiments.
In vivo antileishmanial activity
Male and female golden hamsters (Mesocricetus auratus),
7
at 6 or 7 weeks old, were infected with 5 × 10 stationary
growth phase promastigotes of L. (V) braziliensis in the
dorsum, following the method established elsewhere
(Robledo et al. 2012). After the full development of skin
lesions, they were distributed randomly into nine groups
Leishmania (Viannia) panamensis and L. (V) braziliensis
in vitro culture conditions
(
n = 5 animals per group). Four groups were treated topi-
Promastigotes of L. (V) panamensis and L. (V) braziliensis,
transfected with green fluorescent protein (MHOM/CO/87/
UA140-EGFP and M/HOM/CO/88/301-EGFP, respec-
tively), were maintained in a modified Novy-MCNeal-
Nicolle medium, with phosphate buffer saline (PBS), plus
glucose, pH 6.9 at 26 °C (Limoncu et al. 1997).
cally with 100 μL of each compound in PBS solution
(3-S, 4-S, 5-S, or 6-S), at 1.5 mg/kg/day, for 10 days. The
other four groups were treated with an ointment formulation
at 4% of each compound (3-O, 4-O, 5-O, or 6-O), at
40 mg/day, for 28 days. A ninth and ten group of hamsters
were treated with MA, administered intralesional, at 200 μg/
three times per week for 10 days (MA-10) or 28 days
(MA-28). After the end of treatment, animals were kept
under observation for a period of 90 days. The effectiveness
of each compound was determined according to the evo-
lution of the size of the lesion in response to the treatment.
The size (width and length) of the ulcer and the body
weight were obtained at the end of the treatment and then
every 2 weeks, until the end of the study (90 days after the
end of the treatment), for each individual. The evaluation
time points were: pretreatment day (TD0), end of treat-
ment (TD28), and posttreatment days (PTD) 30, 60, and
90. The effectiveness of each treatment was assessed,
comparing the lesion sizes, prior to and after treatment, for
each individual. The outcome at the end of study was
recorded as a cure (epithelial healing and emergence of
fur); improvement (reduction of the lesion size by at least
>20%); relapse (reactivation of the lesion after an initial
improvement or cure); or failure (increase of the
lesion size).
In vitro antileishmanial activity
The U937 cells were induced to differentiate to macrophage
5
by exposing the cells (3 × 10 cells/mL) to 3 ng/mL of
phorbol 12-myristate 13-acetate (Sigma) and incubation at
3
7 °C. Forty-eight hours afterwards, the cells were infected
with the promastigotes of L. (V) panamensis and L. (V)
braziliensis in a stationary growth phase (day 5 or 6) at a
parasite:cell ratio of 30:1 or 15:1 for L. (V) panamensis or L.
(
V) braziliensis, respectively. The cells and parasites were
incubated at 34 °C, 5% CO2 atmosphere. After 3 h of
incubation, the cells were washed twice with PBS, and a
1
mL fresh RPMI 1640 medium was added into each well.
The plates were incubated again at 34 °C, 5% CO for 24 h
2
(
Pulido et al. 2012).
After 24 h of the infection of U937 cells, the culture
medium was replaced by a fresh RPMI medium, containing
each compound at 100–25–6.25–1.78 μg/mL. Infected and
treated cells were maintained at 34 °C and 5 % CO . Then,
The Ethics Committee for Animal Experimentation at the
Universidad de Antioquia approved all of the experiments
conducted with the hamsters (Act No. 53 of May 8, 2009
and Act No. 91 of September 25, 2014). The Procedures for
obtaining donor cells were endorsed by the Human Ethics
Committee at University of Antioquia (CIBH).
2
7
2-h cells were removed from the bottom plate with a
trypsin/EDTA (250 mg) solution, and the removed cells
were centrifuged at 1100 rpm for 10 min at 4 °C. The
supernatant was discarded, and cells were washed with
1
mL of cold PBS and centrifuged at 1100 rpm for 10 min at

Medicinal Chemistry Research
Quantification of inflammatory mediators CCL2/
MCP-1, TGF-β1, IFN-γ and cyclooxygenase-2 activity
in huMDM exposed to compounds 3–6
lymphocytes from healthy donors. Cells cultured in an
RPMI 1640 medium, supplemented with 2 mM L-gluta-
mine, 10% FBS, and 1% penicillin–streptomycin (10,000
U–10 mg/mL) (complete medium), were stimulated with
200 μL phytohemagglutinin and incubated at 37 °C, 5%
The production of MCP-1, TGF-β1, and IFN-γ by huFDM
was quantified at 6 and 48 h after exposure to each com-
pound at a maximum concentration of 20 μg/mL by the
ELISA technique using the Quantikine® kits (R & D Sys-
tems, USA) and following the manufacturer’s instructions.
The intensity of the color was determined at 540 nm in a
Multiscan Flash Multimode reader (Thermo Scientific) and
registered as O.D.
The cyclooxygenase-2 (COX-2) activity was determined
from the cell lysates of huFDM co-cultures after 6 and 48 h
of exposure to a maximum concentration of 20 μg/mL of
each compound by the COX Fluorescent Activity Assay Kit
CO . After 36 h, the cells were treated with each of the
2
sublethal concentrations (46–23 and 12 μg/mL) and incu-
bated for 24 h. Colcemid (0.1 pg/mL) was added, and cells
were incubated again for 1 h. The cells were centrifuged,
resuspended in 0.075 M KCl, and incubated at 37 °C for
7 min. Then, they were fixed for 10 min with a Carnoy
solution, washed and dripped. Slides were dried and stained
with 5% Giemsa, pH 6.8. One hundred metaphase cells (46
chromosomes) were analyzed. Structural chromosome
aberrations were classified as chromatid breaks (B), chro-
mosome breaks (BB), dicentric chromosomes (DC), ring
chromosomes (R) and multiradial chromosomes (MR)
(OECD 2016). Each treatment was performed in two
separate experiments, using RPMI as a negative control and
mitomycin-C (10 μM) as a positive control.
(
Cayman Chemicals, USA), following the manufacturer’s
instructions.
The intensity of the fluorescence was determined at
5
30–540 nm in a Varioskan Flash multimode reader
(
Thermo Scientific) and registered as Arbitrary Units of
fluorescence (AFU).
Data analysis
In vitro wound healing (scratch) assay
The cytotoxicity was determined according to mortality (%)
for each condition (compound and concentration) using Eq.
(1), where the O.D of the control corresponds to 100%
viability.
The effect of compounds 3–6 on fibroblast migration and
closure of the scratch was investigated with a CytoSelect™
Wound Healing Assay Kit (Cell Biolabs, Inc.) and follow-
ing the method described by others (Montoya et al. 2015).
%
mortality ¼ 100
À ½ðO:D exposed cellsÞ=ðO:D nonexposed cellsÞ Â 100Š
ð1Þ
5
In brief, a Detroit 551 fibroblast (2.5 × 10 /mL) in DMEM,
containing 5% FBS, were seeded into a 24-well tissue
culture plate, containing proprietary treated inserts in the
plate wells, with their “wound field” aligned in the same
direction, and incubated for 24 h to allow the cells adhere
and reach a confluence of 60–80%. After removing the
inserts from the wells, the medium was carefully aspired,
and the wells were washed with DMEM, containing 2%
FBS, to remove dead cells and debris. Finally, the cells were
treated with 20 μg/mL of each tested compound and incu-
The % of mortalities obtained in each concentration were
used to calculate the half-maximal lethal concentration
(LC ) using the Probit analysis (Finney 1978).
5
0
The antileishmanial activity was determined according to
the infection (number of parasites in the infected cells),
obtained for each experimental condition according to
dotplot analysis by infected cells and histograms by the
mean fluorescence intensity (MFI) in fluorescent parasites
(Pulido et al. 2012). The parasite inhibition was calculated
by Eq. 2, where the MFI in the control was 100% of
parasites.
bated for a further 8, 16, and 24 h at 37 °C, 5% CO . The
2
migration of the fibroblast into the wound field was deter-
mined by microscopic imaging of the wound closure. Two
sets of experiments were performed in duplicate. The
influence of compounds on wound closure was compared
with an untreated control (0 h). As a positive control,
DMEM, with 20% FBS, was used. The density of the cells
in the wells, without a created wound area (confluent area),
was used as a 100% wound closure.
%
infection inhibition ¼ 100
À ½ðMFI exposed infected cellsÞ=ðMFI non exposed infected cellsÞ Â 100Š
ð2Þ
As above, the % of the inhibition of infection was used to
calculate the half-maximum effective concentration (EC ),
In vitro genotoxicity
50
as determined by the Probit analysis (Finney 1978).
Chromosome alterations, induced by 3 and 4, were eval-
uated by the chromosome aberration test, using human
To compare the level of cytotoxicity and antileishmanial
activity, the cytotoxicity of each compound was graded,

Medicinal Chemistry Research
according to the LC50 values, as high (LC < 100 μg/mL),
Therefore, the development of novel antileishmanial drugs,
which are more effective, safer and more affordable, is
urgently required. The strategy employed in the present
work started with the selection of an abundant and chemi-
cally modifiable scaffold based on a benzopyran structure,
such as a chroman and thiochroman. Thus, hydrazones were
prepared from the active chromans and thiochromans, since
this type of substance has antiparasitic activity due to its
inhibitory effects in parasite proteases (Muñoz et al. 2006).
After characterization, their cytotoxicity was evaluated
using an in vitro MTT assay. Subsequently, the efficacy was
evaluated using an in vitro macrophage intracellular amas-
tigotes of Leishmania (Viannia) panamensis and L. (V)
braziliensis systems by flow cytometry.
Considering that topical treatment are recommended by
the World Health Organization for the treatment of non-
complicated CL (Muñoz et al. 2006), the efficacy of chro-
man hydrazones formulated in a phosphate buffer solution
(PBS), pH 7.2, or in an ointment was validated in in vivo
studies. Both formulations were assayed topically in the
hamster model of CL caused by L. (V) braziliensis.
Simultaneously, other biological properties related to anti-
parasite activity, such as cicatrizing and anti-inflammatory
activities, were also studied. The ointment was the most
effective against the CL hamster model, because this
pharmaceutical preparation permits a high adherence in the
skin lesion, with long drug delivery in addition to other
beneficial effects, like emollient, occlusive and antiseptic
effects (Tsatsop et al. 2017).
50
moderate (LC > 100 to <200 μg/mL) and low (LC >
50
50
200 μg/mL). In turn, the activity was graded as high when
EC < 25 μg/mL, moderate with EC values > 25 and
50
50
<
50 μg/mL, and low when EC was >50 μg/mL. The Index of
50
Selectivity was calculated using the equation: IS = LC /EC .
5
0
50
The in vivo efficacy of the compound was expressed as
the percentage of healing, improvement or failure and
compared with the efficacy observed in the group treated
with meglumine antimoniate. Parametric and nonparametric
tests were carried out in order to compare the percentages
and frequencies. Variances were expressed as standard
errors, and the statistical significance was determined, using
the analysis of variance (ANOVA) to compare the groups.
The quantification data of the inflammatory mediators
were expressed as the mean of the results ± SD. Significant
statistical differences between the treated groups and the
control group were determined by means of a one-way
ANOVA and the application of Dunnett’s multiple com-
parison test using GraphPad Prism 6.0.
Effect on healing was calculated according to the per-
centage of the wound closure using Eq. (3).
%
closure ¼ 100
À ½ðgap size exposed cellsÞ À ðgap zise non exposed cellÞÞ Â 100Š
ð3Þ
Monolayer wound healing assay data were analyzed by a
two-way ANOVA, with an independent factor experiment
and treatment, followed by Bonferroni post hoc tests. Dif-
ferences were considered significant if p < 0.05. Statistical
analysis was performed with GraphPad Prism 6.0.
Molecular optimization of chroman and
thiochroman
In this work, chromanone (1) and thiochromanone (2),
commercially available, were selected to be optimized,
since they have a wide spectrum of activity, are relatively
hydrophobic small heterocycles and susceptible to further
modifications (Scheme 1), and their preparation is relatively
easy, with high yields. On the other hand, hydrazones were
selected since they have shown protease inhibitory effects in
Results and discussion
The different drugs that are recommended treatment for CL
are associated with several side effects, such as arthralgia,
hepatotoxicity, myalgia, fever, and several weeks of painful
injection site, which limit their usage (WHO 2010).
Scheme 1 Obtaining chroman
hydrazones

Medicinal Chemistry Research
Table 1 Antileishmanial activity
and cytotoxicity of effect of
chroman-4-one hydrazones on
Leishmania (V) panamensis and
Leishmania (V) braziliensis
Compound CL50 (μg/mL) CE50 (μg/mL)
IS
Biological activity correlation
Lp
Lb
Lp
Lb
1
>500
30.2 ± 1.61 33.2 ± 3.5 >16.7 > 15.1
LC/MA
LC/MA
MC/HA
HC/HA
HC/LA
HC/LA
LC/HA
HC/HA
2
409.3 ± 49.1
112.9 ± 13.0
62.2 ± 12.9
>200
47.6 ± 3.3
26.9 ± 3.7 8.6
15.2
4.8
3
25.0 ± 11.2 23.3 ± 0.9 4.4
20.1 ± 13.1 12.1 ± 4.1 3.1
4
7.3
5
>50
> 50
<4
< 4
6
>200
>50
> 50
<4
< 4
MA
AMB
DOXO
400.0 ± 46.0
26.8 ± 1.2
1.0 ± 0.08
5.0 ± 0.1
0.8 ± 0.1
NA
6.1 ± 0.5
0.7 ± 0.2
NA
80
65.6
382
HC/NA
357
NA
The data represent the average value of cytotoxicity in U937 cells activity against intracellular amastigotes of
L. (V) panamensis. Without infecting cells and infected cells were incubated in the presence of different
concentrations of compounds for 72 h, and the LC50, and EC50 values were calculated using the Probit. Lp:
L. (V) panamensis; Lb: L. (V) braziliensis
CE50 mean lethal concentration, CE50 mean effective concentration, IS selectivity index = LC50/EC50, LC/
MA low cytotoxicity and moderate antileishmanial activity, MC/HA moderate cytotoxicity and high
antileishmanial activity, HC/HA high cytotoxicity and high antileishmanial activity, HC/LA high cytotoxicity
and low antileishmanial activity, AMB amphotericin B, DOXO doxorubicin, NA not apply
some parasites (Desai et al. 2004; Cywin et al. 2003) and
therefore could have a complementary activity to that
already exhibited by chromans. Nevertheless is difficult to
control the oxidation of sulfones and sulfoxides, a fact
enables their production at a scale sufficient for more
advanced clinical trials.
Table 2 Cytotoxicity of hydrazones on different cell types
Compound
LC50 (μg/mL)
Detroit 551
BHK
huMDM
huFDM
3
>200
>200
>200
>200
4
>200
>200
>200
>200
The compounds 1 and 2 were prepared from phenol and
thiophenol, instead of being obtained commercially
5
>200
>200
>200
>200
6
>200
>200
>200
>200
(
Scheme 1). Then, the thiochroman was selectively oxidized
DOXO
0.8 ± 0.04
0.9 ± 0.03
1.1 ± 0.3
1.0 ± 0.02
to the respective sulfone but after the hydrazone formation.
The synthesized compounds were: benzoic acid, 2-(2,3-
dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (3),
benzoic acid, 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene)
hydrazide (4), Benzoic acid, 2-(2,3-dihydro-1,1-dioxido-
The data represent the average value of cytotoxicity in Detroit 551 and
BHK21 cells and human macrophage-derived monocytes and human
fibrocyte derived monocytes cells
LC50 median lethal concentration, DOXO doxorubicin
4
H-1-benzothiopyran-4-ylidene) hydrazide (5), and 4-
Pyridinecarboxylic acid, 2-(4H-1-benzopyran-4-ylidene)
hydrazide (6) (Scheme 1). Compound 5 was made directly
from the oxidation of hydrazone 3.
evaluated concentrations (Table 1). As expected, AMB and
DOXO exhibited high cytotoxicity for this cell line, with
EC50 values of 26.8 and <1.0 μg/mL, respectively. On the
other hand, compounds 3 and 4 were effective against
intracellular amastigotes of L. (V) braziliensis, with an EC50
of <25 μg/mL, but were inactive for L. (V) panamensis, with
an EC50 of >50 μg/mL. Compounds 5 and 6 showed a low
activity for both Leishmania species (Table 2). Both MA
and AMB were highly active for L. (V) panamensis and L.
(V) braziliensis, with an EC50 of 5.0 and 6.7 μg/mL.
Nevertheless, AMB was more active, with EC50 values of
<1.0 μg/mL. Compounds 3 and 4 showed SI ranging from
3.1 to 7.3, while compounds 5 and 6 showed a SI < 4.0.
These results suggest that the lipophilicity of the molecule is
essential in the activity, considering the side ring and the
chroman or thiochroman system.
Cytotoxicity and antileishmania effect of chroman-
4-one hydrazone derivatives in L. (V) panamensis
and L. (V) braziliensis
The four new aryl hydrazones were evaluated in L. (V)
panamensis and L. (V) braziliensis, the two most prevalent
species in Colombia that cause CL. It was previously
established that a substance would be considered to have
potential antileishmania activity if it exhibited a mean
effective concentration (EC ) < 50 μg/mL. Compounds 3
50
and 4 exhibited high and moderate cytotoxicity, respec-
tively, for U937 cells, with EC50 values of 62.2 and
1
12.9 μg/mL, while 5 and 6 were not cytotoxic at the

Medicinal Chemistry Research

Medicinal Chemistry Research
Fig. 2 Efficacy of chroman-4-one hydrazones in a 1.5% buffer solu-
tion and ointment vs. meglumine antimoniate in a L. braziliensis-
hamster model of cutaneous leishmaniasis. a Hamsters were treated
topically with 100 μL of compounds in PBS (3-S, 4-S, 5-S, or 6-S) at
hamsters (60%); in the remained two hamsters (40%) of 3-S
groups there were improvement (reduction in 16 and 24%)
while in the 3-O group there was failure to this treatment.
Treatment with intralesional MA every two days for 10 days
(MA-10) produced cure in 3/5 hamsters (60%) and failure and
improvement each in the remained two hamsters (Fig. 2a)
while treatment with MA during 28 days cured four of five
hamsters at the end of the assay and failure in one of five
hamsters (20%) (Fig. 2b).
At contrary, treatment with both 6-S and 6-O produced
cure in 1/5 (20%) and 2/5 (40%), respectively (Fig. 2a, b).
The rest of the hamsters in the group treated with 6-S
showed a reduction of the ulcer in a 50% in one hamster and
failure in the last hamster in this group and 3/5 hamsters in
the group treated with 6-O. In the case of the treatment with
compound 5, pure compound was slightly better than
ointment since with 5-S there was cure in 20% and with 5-O
no cures were observed. The other 3/5 hamsters treated with
(5-O) in ointment showed an improvement, with a reduction
of 46.99, 76.13, and 80.6% in the size of the lesion. The
remaining hamster of this group displayed a failure with this
compound (Fig. 2b).
1
1
.5 mg/kg/day, 10 days, topically and MA-10 (200 μg every 3 days,
0 days, intralesional). b: Hamsters were treated topically with 40 mg
of the ointment formulation containing 4% (3-O, 4-O, 5-O, or 6-O)
every day for 28 days or intralesional MA (200 μg every 3 days,
2
8 days) (MA-28). The effectiveness of each treatment was assessed,
comparing the lesion sizes prior to and after treatment for everyone.
The lesion size was measured before treatment (TD0), at end of
treatment (TD10 or TD28) and every 15 days for three months, cor-
responding to the posttreatment days (PTD) 15, 30, 45, 60, 75, and 90.
The outcome at the end of the study (PTD90) was recorded as a cure
(
epithelial healing and emergence of fur, ●); improvement (reduction
of the lesion size by at least >20%, ▲); or failure (increase of the
lesion size, ■)
These results suggest that compounds 3 and 4 can be
considered molecules with an antileishmania potential, L.
(
V) braziliensis being more sensitive than L. (V) panamensis
to the action of these compounds.
None of the compounds was cytotoxic for human fibro-
blasts (Detroit 551) and macrophage-derived fibrocytes
(
huMDF), hamster kidney epithelial cells (BHK21) and mac-
A representative clinical outcome of the lesions of a
hamster that was cured with hydrazone compounds in a
buffer solution or 4% ointment is shown in the Figs 3 and 4,
respectively.
rophages derived from peripheral blood monocytes (huMDM).
For all these cells the LC50 was >200 μg/mL (Table 2). As
expected, DOXO showed LC50 values of <1.0 μg/mL.
In short, both pharmaceutical preparations are equally
effective against L. braziliensis (Figs 5 and 6), but in the
case of aqueous solution, moisture probably represent a
risk of microbial contamination. Therefore, almost all the
compounds were active, especially compound 4, which
produced a higher percentage of clinical cures, and to a
lesser degree, compound 3, followed by 6. The sulfone
derivative 5, produced a poor therapeutic response,
because although only 1/5 hamster was cured the ulcer
relapsed weeks later.
Based on the findings of in vivo model in golden
hamsters infected with L. (V) braziliensis, a significant
posttreatment reduction was observed in lesion size in
hamsters which were treated with compounds 4 and 3
compared with 5 and 6 groups. After applying the topical
buffered solution or ointments on the lesions, compounds
4 and 3 apparently could pass across the subcutaneous
tissues and reach the epidermal and dermal areas where
infected macrophages could take up these compounds and
then, were released by lysosomal degradation of the active
ingredients as seen for other drugs.
In vivo antileishmanial effect of chroman-4-one
hydrazones 3–6
Because L. (V) braziliensis is the species with the highest
virulence and infectivity, and shows the poorest therapeutic
response among Leishmania species, the tests in the animal
models were made only with this Leishmania species. After
experimental infection with L. (V) braziliensis hamsters were
treated with each compound prepared as aqueous solution in
PBS, pH 7.2 (coded 3-S, 4-S, 5-S, and 6-S) and ointments
containing 4% of each chroman-4-one hydrazones in
experimental ointment formulation (coded 3-O, 4-O, 5-O,
and 6-O). Products were applied topically every day for 10 or
28 days for aqueous solution or ointment, respectively. The
effect of the treatment was periodically monitored by mea-
suring the diameter of the ulcer.
In vivo response was correlated to the activity observed in
the in vitro studies (Table 1). Both formulations of com-
pounds 3 and 4 showed similar results when administered as
pure compounds dissolved in PBS or formulated as 4%
ointment, being 3 and 4 more effective than 5 and 6. At the
end of the study, treatment with compound 4-S and 4-O
produced cure in 4/5 hamsters (80%), and improvement of the
ulcer in the remained hamsters of these groups, with a
reduction of more than 80% in the ulcer sizes (Fig. 2a, b).
With compounds 3-S and 3-O healing was observed in 3/5
In vivo toxicity of the aqueous solution and
ointment hydrazones
Treatment with both formulations of hydrazone-derivative
compounds 3–6 did not produce any detrimental effect on

Medicinal Chemistry Research
Fig. 3 Treatment progress of cutaneous leishmaniasis, caused by L.
braziliensis hamsters, with hydrazones in 1.5% buffer solutions (3-S,
treatment group: Before the treatment (TD0); on day 10 of the treat-
ment (TD10); and on day 90 post treatment (PTD90). The numbers in
mm are indicative of the size of the ulcers
2
4
-S, 5-S, or 6-S). The photos represent one hamster from each
Fig. 4 Treatment progress of cutaneous leishmaniasis caused by L.
braziliensis hamsters, with hydrazones (3-O) and (4-O) formulated as
group: before treatment (TD0), on day 28 of the treatment (TD28); and
on day 90 post treatment (PTD90). The numbers in mm on the right
2
4
% ointment. The photos represent one hamster from each treatment
are indicative of the size of the ulcers
body weight in most hamsters, but rather a slight weight
gain was observed in most of them. The evolution of the
body weight in the treatment group is shown in Fig. 6.
−γ), which are associated with Leishmania infections and,
depending on the levels of expression, have been suggested
as biomarkers of exacerbation or cure of Leishmania
infection (TGF-β1 and IFN−γ, respectively) (Bogdan and
Nathan 1993; Wyler et al. 1987). On the other hand, the
enzyme, COX-2, is a mediator of the proinflammatory
pathway of prostaglandins and is expressed in low con-
centrations of physiological stress (Kapoor et al. 2005). The
levels of each of these inflammatory mediators were
determined in supernatants of huDMF co-cultures at 6 and
48 h after exposure to each of the hydrazones, 3–6, at 20 μg/
mL in PBS, corresponding to a concentration that is con-
sidered to be active to Leishmania amastigotes.
The main production of MCP-1 was induced by com-
pound 5 after 6 h of incubation, with 440.2 ± 83.7 pg/mL,
while for 6 and 3, the levels were 235.7 ± 112.0 and
251.6 ± 24.7 pg/mL, respectively. In turn, a smaller amount
of MCP-1 was produced by compound 4, with 131.1 ± 33.
6 pg/mL. An increase in the concentration of MCP-1 was
Effect of chroman-4-one hydrazones 3–6 on the
expression of inflammatory mediators, MCP-1,
TGF-β, INF-γ, and COX-2 by huMDF
During the infection by Leishmania species, there are the
expression of many different cytokines by the host, leading
to chronic inflammation and tissue necrosis, which magnify
the signs and symptoms of the disease. Therefore, it is
necessary not only find new drugs that control the parasite
but also that improve the inflammation processes. For this
reason, the effect of the all compounds on the mediators of
inflammation as well as cell migration were analyzed.
The selected inflammatory mediators were the Monocyte
Chemotactic Protein, 1 (MCP-1), the Transforming Growth
Factor-beta 1 (TGF-β1), and the Interferon gamma (IFN

Medicinal Chemistry Research
observed at 48 h, relative to that at 6 h, for all the com-
pounds. The concentration of MCP-1 induced by 5 were
these values were 438.0 ± 3.4, 444.8 ± 54.1, and 446.3 ±
38.3 pg/mL, respectively.
9
40.2 ± 29.9, 854.8 ± 106.5 pg/mL for 4, 844.0 ± 158.4 pg/
After 48 h of incubation, the concentration of TGF-
β1 showed slight increases for the unexposed cells and
those exposed to 3, 4, and 5, except for those exposed to 6,
in which the levels were decreased. In the unexposed cells,
the amount of TGF-β1 was 663.6 ± 15.7 pg/mL; for 3, it
was 652.0 ± 22.6 pg/mL; for 4, it was 560.0 ± 78.7 pg/mL;
for 5, it was 628.8 ± 3.4 pg/mL; and for 6, it was 557.1 ±
62.3 pg/mL. No statistically significant differences were
found between the levels of TGF-β1 in the stimulated cells,
relative to the control of nonstimulated cells. For TGF-β1,
all the compounds increased the production of TGF-β1 at
48 h, relative to that at 6 h, except for compound 24, which
inhibited the production of TGF-β1 (Fig. 7).
mL for 6, and 624.9 ± 47.5 pg/mL for 3. In contrast to
observed in the stimulated cells by the compounds, in the
unstimulated control cells, the concentration of MCP-1 was
much higher at 48 h than that at 6 h (996.7 ± 57.5 vs.
7
29.8 ± 170.4 pg/mL). While no statistically significant
differences were found for any of the compounds regarding
the control, all the compounds had an opposite effect to that
shown by the nonstimulated cells, decreasing the production
of MCP-1 at 6 h and increasing its production at 48 h (Fig.
7
).
In the nonexposed cells, the concentration of TGF-β1 at
h of incubation was 480.6 ± 29.4 pg/mL. Similar levels
6
were observed for cells exposed to the chroman-4-one
hydrazones. Thus, for 3, the amount of TGF-β1 was
On the other hand, the concentration of INF-γ at 6 h of
incubation in nonexposed cells was 74.5 ± 5.4 pg/mL; for 3,
it was 140.3 ± 76.0 pg/mL; for the 4, it was 68.5 ± 6.2 pg/
mL; for 5, it was 90.3 ± 6.2 pg/mL; and for 6, it was 64.4 ±
4
31.8 ± 23.3 pg/mL, while with compounds 4, 5, and 6,
9.6 pg/mL (Fig. 8). The concentration of INF-γ at 48 h of
incubation for the control was 65.5 ± 9.7 pg/mL; for com-
pound 3, it corresponded to 77.9 ± 34.2 pg/mL; for 4, it was
1
51.3 ± 8.0 pg/mL; for 5, it was 135.4 ± 23.6 pg/mL; and for
compound 6, it was 95.5 ± 23.5 pg/mL. While compounds
, 5, and 6 increased the levels of IFN−γ at 48 h, relative to
4
that at 6 h, the levels of the said cytokine decreased in
response to exposure to compound 3, and in the unexposed
cells, the levels showed no change.
After 6 h of incubation, some compounds induced COX-
2
concentrations higher than those found in the control of
nonexposed cells. Thus, in the absence of compounds, the
COX-2 levels were 0.54 ± 0.4 μM (Fig. 8), and for com-
pound 5, they were 2.25 ± 1.1 μM. This result was statisti-
cally significant, relative to the control. On the other hand,
for compound 6, the levels were 1.4 ± 0.2 μM, and for 3,
they were 1.1 ± 0.4 μM. With 4, the levels were similar to
Fig. 5 Comparison of the therapeutic response to formulated hydra-
zones in 1.5% aqueous solution and 4% ointment. Bars represent the
percentage of hamsters with cure ( ), improvement ( ) and failure ( )
in each group of treatment. Asterisk denotes that similar results are
observed in hamsters treated with 22-O and meglumine antimoniate
(
MA) during 28 days
Fig. 6 Effect of the treatment on body weight. a Golden hamsters (n =
each group) were treated with 100 μL of each compound in PBS (3-
S, 4-S, 5-S, or 6-S) at 1.5 mg/kg/day, 10 days, topically) and MA-10
200 μg every 3 days, 10 days, intralesional). b Hamsters were treated
topically with 40 mg of the ointment formulation containing 4% (3-O,
-O, 5-O, or 6-O) every day for 28 days or intralesional MA (200 μg
every 3 days, 28 days) (MA-28). The data represent the mean value ±
SD of the weight in grams of the hamsters in each experimental group.
p > 0.05. Body weight was measured before the treatment (TD0), at
end of the treatment (TD10 or TD28) and every 15 days for 3 months,
corresponding to posttreatment days (PTD) 15, 30, 45, 60, 75, and 90
5
(
4

Medicinal Chemistry Research
Fig. 7 Production of MCP-1, TGF-β1, IFN-γ, and COX-2 by huMDF exposed to chroman-4-one hydrazones 3–6. The values shown are the
average of two assays, each in triplicate (p < 0.05)
the control. After 48 h of incubation, the activity of the
enzyme was maintained at levels similar to those presented
at 6 h of incubation for control (0.6 ± 0.1 μM), as well as for
cells exposed to 4 (0.62 ± 0.4 μM), 5 (0.522 ± 0.2 μM), and
the other compounds decreased it. However, although the
COX-2 levels try to increase at 48 h in cells stimulated with
compound 4, this increase is very slight and therefore is not
significant. The preferential increase of COX-2, observed
after exposure to compound 4, confers to this compound
anti-inflammatory properties that are very useful for the
resolution of the inflammatory process, occurring during
active CL.
6
(0.516 ± 0.0 μM). Only compound 3 (0.174 ± 0.6 μM)
significantly inhibited COX-2 activity, showing that the
concentrations of this enzyme are well below the con-
centration of non-stimulated cells.
To quantify the effect of the compounds, the percentage
of increase or decrease in the levels of MCP-1, TGF-β1,
IFN−γ, and COX-2 in the cells exposed to each compound,
with respect to the exposed cells, was calculated. As shown
in Table 3, at 6 h all the compounds decreased the levels of
MCP-1, in percentages ranging between 55 and 86%, with
compound 4 producing the greatest decrease. On the con-
trary, at 48 h, all the compounds induced a moderate
increase in the levels of MCP-1, with percentages between
Compound 4 seems to have an impact on the anti-
inflammatory response by inducing an increase in the levels
of IFN−γ at 48 h post treatment, and the fact that it has
activity against the Leishmania species places it as a can-
didate as an antileishmanial agent. In addition, it has a
potential as a scarring factor, favoring the migration of
fibroblasts, with a closing of the gap similar to that of the
positive control. Compounds 3, 5, and 6 have the capacity
to stimulate the production of the COX-2 at 6 h post treat-
ment, and 4, at 48 h, which could favor the production of
the derivatives of arachidonic acid, such as prostaglandin
E2, enhancing the inflammatory response which may
potentiate the final cure (Kalinski 2012).
With the increase in the levels of IFN−γ, induced by
compound 4, it could be deduced that the mechanism by
which this compound acts against Leishmania parasite
could be through the activation of macrophages, which
would eventually lead to the death of the parasite at the
intracellular level. This would also explain the effective-
ness of the compound in intracellular amastigotes and in
infected hamsters. These results also correlate with the
decrease in the production levels of TGF-β1, a cytokine
that modulates macrophage activation, favoring the survi-
val of Leishmania and therefore the development of CL
1
5 and 28%, except for compound 3, which produced a
decrease of 14% in the levels of MCP-1. For TGF-β1, all
the compounds induced a slight decrease in the levels of this
cytokine, with values between 1 and 16%. On the other
hand, for INF−γ, at 6 h, compounds 3 and 5 increased the
INF-γ production by 21% and 88%, respectively, with
respect to the values without stimulus (Table 3); At con-
trary, at 48 h, all the compounds increased the production of
IFN−γ, indicating that 4 and 5 will increase the levels by
more than 100%, with respect to the levels in the absence of
the compounds (Table 3). Compound 4 inhibited the pro-
duction of COX-2 by 24% at 6 h, but on the other hand, the
other products, 3, 5, and 6, increased the levels, especially
5
, with an increase of more than 100% (Table 3). At 48 h,
compound 4 slightly increased the levels of COX-2, while

Medicinal Chemistry Research
Fig. 8 Light microscope images of the wound closure in vitro using a confluent monolayer of Detroit 551 fibroblasts. Microphotographs show one
representative experiment of the cell migration into the created wound area in response to the treatment. (left) Wound area at 0 h (a) and after 8 h
(b) or 16 h (c), for the untreated control (−C) (set to 0%), positive control (+C), and 20 μg/mL of 3, 4, 5, or 6. The wound closure (indicated in as
%
) was normalized to the untreated control
(
(
Bogdan and Nathan 1993) but also treatment failure
Elmekki et al. 2016). In turn, compound 4 increases the
inflammatory potential (Das et al. 2014). The other two
compounds, 5 and 6, do not have significant activity in
relation to the parasite, as already mentioned before, and
levels of COX-2, so it could be said to have an anti-

Medicinal Chemistry Research
Table 3 Effect of chroman-4-
one hydrazones 3–6 on the
levels of inflammatory
Inflammatory mediator 6 h
48 h
3
3
4
5
6
4
5
6
mediators MCP-1, TGF-β, INF-
γ, and COX-2 by huMDF
MCP-1
TGFb1
IFN-γ
↓ 75%
↓ 86.8 ↓ 55.8
↓ 76.4
↓ 7.1
↓ 14.4 ↑ 17.1
↓ 1.7 ↓ 15.6
↑ 28.8
↓ 5.2
↑ 15.6
↓ 16.0
↓ 10.2
↑ 88.3
↓ 8.9
↓ 8.1
↓ 7.4
↑ 21.2
↓ 13.6
↑ 18.9 ↑ 131.0 ↑ 106.7 ↑ 45.8
↑ 3.33 ↓ 13.0 ↓ 14.0
COX-2
↑ 105.6 ↓ 24.1 ↑ 316.7 ↑ 151,8 ↓ 71
The data represent the percentages of the increase or decrease of each immunological factor, produced by
huMDF, exposed to each compound for 6 and 48 h
they also do not decrease the enzymatic levels of COX-2.
However, they have an anti-inflammatory potential by
inducing the production of MCP-1 and INF-γ, so they can
be used as adjuvants with treatments already available and
be part of a combined antileishmanial therapy.
Table 4 Percentage of chromosome breaks observed in human
lymphocytes exposed to chroman-4-one hydrazones for 24 h
Compound
µg/mL
Chromosomal damage
B
BB
DC/R
MR
n
%
n
%
n
%
n
%
Effect of chroman-4-one hydrazones 3–6 on the
stimulation of the healing process by fibroblast
migration
C−3
0
2
3
4
0
2
5
3
0.8
1.5
2.0
0.0
1.0
2.5
1.5
0
4
2
0
1
5
1
0.0
2.0
1.0
0.0
0.5
2.5
0.5
0
0
1
3
0
0
2
0.0
0.0
0.5
1.5
0.0
0.0
1.0
0
0
0
0
0
1
0
0.0
0.0
0.0
0.0
0.0
0.5
0.0
1
3
6
5
1
2
In cells unexposed to the stimulus, a closing of the gap was
observed in 18% (Fig. 8), while in the cells incubated in the
presence of DMEM and 20% FBS (+C), the migration of
the cells produced a reduction of the gap in 80%. Culturing
fibroblasts in the presence of 4 induced cell migration, in
such a way that the gap was reduced by 78%, whereas with
compounds 3, 5, and 6, the gap was reduced by 40%.
Reepithelialization after cutaneous injury is a complex
and multifaceted process that incorporates numerous cel-
lular components interacting in a myriad of pathways. One
of the most crucial aspects of this process is the initiation of
fibroblast migration to fill the wound bed. The chroman-4-
one hydrazones, especially compound 4 favored the
migration of huMDF reducing the gap in the cell mono-
layer. These results suggest that these hydrazones could
have potential as healing agents. This is the first report of
this type of biological effect with this class of compounds.
4
28
56
112
p > 0.5, as compared with untreated (Bonferroni test)
B chromatid breaks, BB chromosome break, DC dicentric chromo-
somes, ring chromosomes, MR multiradial chromosomes,
R
C− negative control
Our findings demonstrated that from molecules with
relatively simple structure, and through chemical transfor-
mations and bioguided leishmanicidal in vitro tests, several
active chroman analogs could be obtained. Moreover,
transformation into hydrazones, optimized their antiparasite
activity. Two of the four chroman-4-one hydrazone deri-
vatives obtained, the benzoic acid, 2-(2,3-dihydro-4H-1-
benzothiopyran-4-ylidene) hydrazide (3), and the benzoic
acid, 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide
(4), caused high cure rates when were applied topically as a
Chromosome aberration—genotoxicity effect
4% ointment in a L. braziliensis hamster model of CL.
Both compounds did not exhibit chromosomal aberrations
at any of the tested concentrations, compared with the
negative control (Table 4). No statistically significant dif-
ferences between the negative control and 3 or 4 were found
Although these efficacy was similar when administered as
buffered solution, the ointment permits a high adherence in
the skin lesion, with long drug delivery in addition to other
beneficial effects, like emollient, occlusive and antiseptic
effects. At contrary, the moisture in the wound can affect
the healing process and increase the risk of microbial
contamination.
The benzoic acid, 2-(2,3-dihydro-1,1-dioxide-4H-1-ben-
zothiopyran-4-ylidene) hydrazide (5) was less effective than 3
and 4, and compound 4-pyridinecarboxylic acid, 2-(4H-1-
benzopyran-4-ylidene) hydrazide (6), was practically inactive.
(
p = 0.527).
Because genotoxicity analysis based only on chromosomal
aberration studies and with low compound concentrations is
not sensitive to genotoxicity and mutagenicity, other tests,
such as Comet, Ames and Vitotox, which would be more
sensitive, should be performed to identify the significant dose-
dependent genotoxicity and mutagenicity of 3 or 4.

Medicinal Chemistry Research
On the other hand, the antileishmanial potential of
compounds 4 and 3 is complemented by their healing
property, since they also modulate the production of
inflammatory mediators involved in the activation of the
microbicidal response of the macrophage. The early pro-
duction of IFN−γ and decreasing of TGF−β1 levels may
favor the resolution of the disease. Besides, these com-
pounds lack genotoxic and cytotoxic effects.
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Conclusions
In this work is reported for first time new synthesized
compounds having combined antileishmanial, anti-
inflammatory and healing activities. The overall results
obtained with hydrazone-derivative compounds 4 and 3
demonstrate at least qualitatively comparable with (or even
somewhat better than) MA, the drug used against this dis-
ease. The strategy used here could be postulated as a sui-
table alternative in the search for new drugs to treat CL.
These compounds can be produced from simple chemical
precursors such as phenol and thiophenol in high yields,
which may provide large amounts to further assays, to
evaluate their efficacy in controlled clinical trials in human
and canine CL.
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Acknowledgements This work was a cooperative activity within the
Temporal Union “Estrategia Integral para el control de la Leishma-
niasis en Colombia” (EICOLEISH-UT) with financial support from
Colciencias (CT695-2014). Authors also thanks to I. Ortiz, Facultad de
Salud, Universidad Pontificia Bolivariana for technical support with
the genotoxicity test.
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Author contributions FE and SMR devised the study and wrote the
paper. WQ, GE, FT, and RA performed the chemical analyses. YU,
KR, IDV, and SMR performed the in vitro and in vivo experiments.
1
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
1
016/j.bmc.2006.03.020
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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