Antiaging activity, molecular docking, and prediction of percutaneous absorption parameters of quinoline–hydrazone hybrids

Article abstract of DOI:10.1007/s00044-019-02427-0

The application of antiaging agents can contribute to the prevention and control of skin photoaging. In the current research, nine quinoline–hydrazone hybrids were synthesized to obtain biologically active compounds as possible antiaging agents. The compounds were tested through a comprehensive in vitro evaluation of antielastase, anticollagenase, and antihyaluronidase activities along with the determination of their potential to quench reactive oxygen species (ROS) by the ORAC method. The selected hybrids were subsequently tested on human dermal fibroblasts (HDF) to reveal possible UVB photoprotective activity. The most potent antiaging protection of all the prepared compounds was shown by the trihydroxylated quinoline–hydrazones 5 and 9, which showed the best collagenase inhibition (IC₅₀ = 39.4 and 45.6 μM, respectively). Compound 5 also showed activity against elastase and hyaluronidase (IC₅₀ = 164.2 and 318.8 μM, respectively). The molecular docking results suggest that the difference of inhibition between 5 and 9 is principally attributed to the hydrogen bonds interactions in the residues His218 and His228, and Zn atom in collagenase, Val216 in elastase and Tyr75 in hyaluronidase. In addition, compounds 5 and 9 were able to significantly protect human skin cells from UVB radiation in vitro. These compounds significantly decreased UVB-induced MMP-1 and ROS production and inhibited the suppression of type I procollagen synthesis in cultured HDF. The in silico dermatopharmacokinetic parameters showed promising results. Therefore, our study presented promising results for antiaging drug discovery, focusing on quinoline–hydrazone hybrids as dual inhibitors of skin aging-related enzymes, antioxidants, and inhibitors of the biological effects of UVB irradiation.

Full text of DOI:10.1007/s00044-019-02427-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02427-0
ORIGINAL RESEARCH
Antiaging activity, molecular docking, and prediction of
percutaneous absorption parameters of quinoline–hydrazone
hybrids
1
Karent Bravo1 Wilson Cardona2 Andres Yepes2 Edison H. Osorio3 Juan C. Coa2
●
●
●
●
●
Edison Osorio
Received: 6 March 2019 / Accepted: 16 August 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The application of antiaging agents can contribute to the prevention and control of skin photoaging. In the current research,
nine quinoline–hydrazone hybrids were synthesized to obtain biologically active compounds as possible antiaging agents.
The compounds were tested through a comprehensive in vitro evaluation of antielastase, anticollagenase, and
antihyaluronidase activities along with the determination of their potential to quench reactive oxygen species (ROS) by
the ORAC method. The selected hybrids were subsequently tested on human dermal fibroblasts (HDF) to reveal possible
UVB photoprotective activity. The most potent antiaging protection of all the prepared compounds was shown by the
trihydroxylated quinoline–hydrazones 5 and 9, which showed the best collagenase inhibition (IC50 = 39.4 and 45.6 µM,
respectively). Compound 5 also showed activity against elastase and hyaluronidase (IC50 = 164.2 and 318.8 µM,
respectively). The molecular docking results suggest that the difference of inhibition between 5 and 9 is principally attributed
to the hydrogen bonds interactions in the residues His218 and His228, and Zn atom in collagenase, Val216 in elastase and
Tyr75 in hyaluronidase. In addition, compounds 5 and 9 were able to significantly protect human skin cells from UVB
radiation in vitro. These compounds significantly decreased UVB-induced MMP-1 and ROS production and inhibited the
suppression of type I procollagen synthesis in cultured HDF. The in silico dermatopharmacokinetic parameters showed
promising results. Therefore, our study presented promising results for antiaging drug discovery, focusing on
quinoline–hydrazone hybrids as dual inhibitors of skin aging-related enzymes, antioxidants, and inhibitors of the biological
effects of UVB irradiation.
●
●
●
●
Keywords Quinoline–hydrazone hybrids UVB photoprotection Antiaging Matrix metalloproteinase Human dermal
fibroblasts
Introduction
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02427-0) contains supplementary
The extracellular matrix (ECM) is the largest component of
the dermis and provides the structural framework essential
material, which is available to authorized users.
* Edison Osorio
for the growth and elasticity of the skin. The ECM is
composed of proteoglycans interwoven with macro-
molecules, such as collagen, elastin, and fibronectin, which
are produced by the fibroblasts of the dermis (Duque et al.
edison.osorio@udea.edu.co
* Wilson Cardona
wilson.cardona1@udea.edu.co
2017). Collagen is the most abundant protein in the ECM
and is responsible for the elasticity and strength of the skin
1
Grupo de Investigación en Sustancias Bioactivas, Facultad de
Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia,
and for maintaining its flexibility (Ndlovu et al. 2013).
Elastin is an important protein with the unique property of
elastic recoil, which is vital to maintaining skin elasticity
and resilience, while hyaluronic acid (HA), a major glyco-
saminoglycan, is involved in the hydration of the skin,
holding water and keeping the body smooth, moist, and
Calle 70 No., Medellín 52-21, Colombia
2
3
Química de Plantas Colombianas, Instituto de Química,
Universidad de Antioquia, Calle 70 No., Medellín 52–21,
Colombia
Facultad de Ciencias Naturales y Matemáticas, Universidad de
Ibagué, Carrera 22 calle 67, Ibagué, Colombia

Medicinal Chemistry Research
lubricated (Bravo et al. 2016). These connective tissue
macromolecules are constantly attacked by enzymes such as
matrix metalloproteinases (MMPs; e.g., collagenase), the
serine protease elastase and the mucopolysaccharase hya-
luronidase, which are highly related to the aging process of
the skin (Duque et al. 2017). The extrinsic skin aging
process (also called skin photoaging) occurs as a result of
exposure to environmental factors, particularly solar radia-
tion (UV radiation), which affects human skin in different
ways. UV irradiation results in important molecular chan-
ges, including altered signal transduction pathways that
promote MMP expression, decreased procollagen synthesis,
connective tissue damage and significantly increased reac-
tive oxygen species (ROS) (Freitas-Rodríguez et al. 2017).
ROS can initiate complex molecular pathways, including
the activation of enzymes, such as collagenase, elastase, and
hyaluronidase (Bravo et al. 2016). Therefore, skin aging is a
multifactorial and complex process. The induction of MMP
expression through different pathways that are directly sti-
mulated by UV irradiation contributes to the photoaging
processes occurring in dermal ECM (Freitas-Rodríguez
et al. 2017). Therefore, the exploration of strategies to
combat the devastating effects of photoaging should focus
on the different mechanisms involved in its pathogenesis.
Several investigations have shown that there are a
number of chemicals derived from nature, in addition to a
number of synthetic drugs, with varied therapeutic effects
that may be useful in the control of the process of aging or
photoaging (Mukherjee et al. 2011; Ndlovu et al. 2013). In
the vast majority of cases, these molecules act on a specific
target. However, factors such as low pharmacokinetic pro-
files, drug–drug interactions, poor solubility, and low sta-
bility are responsible for the insufficient effectiveness of
single target-directed drug therapies (Choudhary et al. 2018;
Hohl et al. 2001). It has also become apparent in recent
years that many natural small compounds act on more than
one particular target in the multidimensional process of
aging (Cătană et al. 2018), or similar to integrated strate-
gies, different compounds in natural extracts have been one
of the main strategies used to decelerate and prevent the
effects of skin aging (Duque et al. 2017; Mukherjee et al.
2015). Many herbs have been reported to contain bioactive
metabolites, mainly polyphenolic compounds with photo-
protective and antioxidant effects, which interact with cel-
lular signaling pathways that are directly involved in the
skin (Duque et al. 2017). Although the search for effective,
nontoxic natural compounds with antiaging activity has
been intensified in recent years, one of the strategies that is
employed to treat these multifactorial processes but that is
little explored in photoaging is related to the design of
ligands comprising two pharmacophores in a single biolo-
gical molecule that modulates multiple targets. The design
of natural product-based hybrid molecules has emerged as a
robust approach involving a combination of two or more
biologically active compounds of natural origins and
allowing the simultaneous delivery of these compounds to
target organs. This hybrid approach has been reported to
possess a distinct advantage over the conventional approach
of the coadministration of single target molecules due to
better long-term prognosis and reduced toxicity. In addition,
hybrid molecules result in enhanced dosage compliance, the
reduction of drug–drug interactions and cheaper preclinical
evaluation (Choudhary et al. 2018).
Hydrazones constitute an important type of biologically
active compound with beneficial effects against different
diseases (Singh and Raghav 2011; Verma et al. 2014).
Furthermore, many reports have also verified that hydra-
zones present anticancer (Yadagiri et al. 2014), antiin-
flammatory (Kumar et al. 2015), antiviral (Şenkardes et al.
2016), antiprotozoal (Coa et al. 2015; Vergara et al. 2017),
and antibacterial activities (Popiołek 2017). Bioactive
hydrazones bearing antioxidant groups represent an inter-
esting approach towards the development of antiaging
compounds. In fact, a number of hydrazones have been
found to be powerful antioxidants. Hydrazones have
exhibited good antioxidant power using the thiobarbituric
acid reactive substances method, with interesting effects on
superoxide radical scavenging and the inhibition of copper-
induced and UV-induced LDL oxidation (Belkheiri et al.
2010; Yılmaz et al. 2012). In addition, the inhibition of the
activity of ECM-degrading enzymes or protection against
UV irradiation may be a useful approach to prevent pho-
toaging. In this sense, hydrazones have been reported for
their antielastase activity (IC50 = 21.1 µM) (Nurkenov et al.
2017), and the possible application of hydrazones as UV
absorbers in the pharmaceutical and cosmetic industry has
been reported (Dimitrijević et al. 2016). However, a qui-
nolinic core is a structural feature of several bioactive
compounds. Thus, this core is an interesting constituent for
new drug design. Antimycobacterial, antimicrobial, antic-
onvulsant, antiinflammatory, antitumoral, cardiovascular,
and antiprotozoal are some of the biological activities
exhibited by compounds having this heteroaromatic ring
(Coa et al. 2017; Franck et al. 2004; Suresh et al. 2009). In
relation to its antiaging potential, a pyrroloquinoline qui-
none has shown a protective effect on UVA irradiation-
induced human dermal fibroblasts (HDF) (Zhang et al.
2015). Another compound of this type presented high
inhibitory activity towards hyaluronidase (IC50 = 579.77
16.28 µM) (Czarnecka et al. 2017). Based on the afore-
mentioned characteristics,
a
series of synthetic
quinoline–hydrazone hybrids (Coa et al. 2015) were eval-
uated for their antiaging activities. The compounds were
evaluated by their capacity to inhibit the collagenase, elas-
tase, and hyaluronidase enzymes, by their antioxidant
properties and by their protective effects against the damage

Medicinal Chemistry Research
induced by UVB radiation in HDF. In addition, molecular
docking has been reported as an effective method to pre-
dicting the impact of biomolecules on skin photoaging
(Farrokhnia, Mahnam 2017; Sivamani et al. 2012; Zeng
et al. 2016). To improve our understanding of the protective
role of different hydrazones against photoaging, the mole-
cular interactions between hydrazones and elastase and
hyaluronidase enzymes were analyzed through molecular
docking.
Antihyaluronidase activity
Briefly, 30 μL of sodium phosphate buffer (20 mM, pH 7.0)
with sodium chloride (77 mM) and bovine serum albumin-
BSA (0.01%, control) or 250 μM sample solution dissolved
in the same buffer were incubated for 45 min at 37 °C in a
water bath with 90 μL of hyaluronidase from the bovine
test, prepared in the above buffer and 120 μL of HA sodium
salt (300 mM) in sodium phosphate buffer at pH 5.35.
Next, 100 μL of reaction mix containing undigested HA
was precipitated with 500 μL of acid albumin solution,
made up of bovine serum albumin (0.1%) in sodium acetate
(24 mM) and acetic acid (79 mM, pH 3.75). The mixture
was allowed to stand at room temperature for 10 min, and
then the absorbance was measured at 600 nm. Epigalloca-
techin gallate (250 μM; EGCG) was used as a reference
inhibitor. The control vial with complete reaction was
considered to be 100% enzyme activity (EA), and a control
substrate, where the enzyme solution was replaced by
90 μL of the buffer, was considered not active. The percent
inhibition of the hyaluronidase reaction was calculated as
follows:
Experimental
In vitro determination of antiaging properties
The antiaging properties of molecules were measured by
their capacity to inhibit the activity of skin aging-related
enzymes, following previously reported assays (Bravo et al.
2016). For compounds with higher activity, the IC50 values
were determined.
Anticollagenase activity
Inhibition ð%Þ ¼ 1 À EAsampleEAcontrol  100
ð2Þ
The inhibition of collagenase enzyme was measured using
the EnzCheck® Gelatinase/Collagenase assay kit (Molecular
Probes Inc.). Aliquots of 20 μL of 500 μM sample solutions
or buffer (control) were added to each well of a 96-well
plate. Then, 80 μL of DQ-collagen type IV substrate fol-
lowed by 100 μL of active enzyme were added, and the
fluorescence intensity was measured by a Synergy HT
Multi-Mode Microplate Reader (BioTek Instruments, Inc.;
Winooski, USA) at an excitation of 485 nm and emission
detection of 515 nm at each minute for 20 min. Oleanolic
acid (250 μM) was used as a reference inhibitor. Each
reaction was performed in triplicate. The percent inhibition
of the collagenase reaction was calculated using Eq. (1).
where EAsample and EAcontrolample are the enzyme activities of
the sample and control, respectively.
The enzyme activity parameter was calculated as fol-
lows:
Enzyme activity ð%Þ ¼ 100% À ½A600 nmsample A600 nm substrate  100Š
ð3Þ
where the A600 nm sample is the absorbance at 600 nm of the
reaction with sample, and the A600 nm substrate control is the
absorbance at 600 nm of the reaction without enzyme
solution (control substrate).
Inhibition ð%Þ ¼ MControl À MSampleMControl  100
ð1Þ
Antioxidant properties
where MControl and MSample are the slopes of the fluorescence
vs time graph for the control and the sample, respectively.
The antioxidant capacity of the compounds was measured
by a hydrophilic oxygen radical absorbance capacity
(ORAC) assay following a previously described procedure
(Bravo et al. 2015), and using a Synergy HT Multi-Mode
Microplate Reader (BioTek Instruments, Inc.; Winooski,
USA). Then, 25 μL of sample solution or Trolox standard
was mixed with 150 μL of 1 μM fluorescein and pre-
incubated at 37 °C for 30 min. Then, 25 μL of 200 mM
AAPH (2,2′-azobis(2-amidinopropane) dihydrochloride)
was added. The fluorescence at an excitation wavelength of
485 nm and an emission wavelength of 520 nm was mea-
sured every 2 min for 120 min. The quantification of anti-
oxidant activity was based on the calculation of the area
under the fluorescence curve using the Trolox standard
Antielastase activity
The effect on elastase enzyme was measured using the
EnzCheck® Elastase assay kit (Molecular Probes Inc.).
Aliquots of 50 μL of 500 μM sample solutions or buffer
(control) were added to each well of a 96-well plate. Then,
50 μL of DQ-elastine substrate and 100 μL of active enzyme
were added. The fluorescence intensity was measured under
the conditions described for the previous assay. Oleanolic
acid (250 μM) was used as a reference inhibitor. Each
reaction was performed in triplicate. The percent inhibition
of the elastase reaction was calculated using Eq. (1).

Medicinal Chemistry Research
(Prior et al. 2005). The results are expressed as μmol TE/g
of compound.
supplemented medium overnight. Then, the medium was
removed, and the cells were treated with 2, 10, and 50 μM
compounds dissolved in 100 μL of FBS-free medium for
24 h. The samples were removed, and the cells were washed
twice with PBS. Then, 100 μL of FBS-free medium was
added, and the plates were exposed to 100 mJ/cm2 UVB.
After irradiation, the cells were incubated at 37 °C. At 24
and 72 h postirradiation, the supernatants were collected for
the MMP-1 and procollagen assays, respectively. A total of
25 μM EGCG was used as a reference compound in the
MMP-1 production assay, and 25 μM ascorbic acid was
Photoprotective effects in human dermal fibroblasts
(HDF)
The photoprotective effects on HDF of the
quinoline–hydrazone hybrids active as aging-related
enzyme inhibitors were measured by their modulation of
MMP-1, procollagen, and ROS production after UVB
exposure (Bravo et al. 2017). HDF from adult skin (HDFa)
and material for their maintenance and culture were
obtained from Life Technologies Inc. (USA). During
assays, HDFa in 3rd to 4th passages were maintained in
DMEM-F12 GlutaMAXTM supplemented with 10% heat-
inactivated fetal bovine serum (FBS), 100 U/mL penicillin
and 100 μg/mL streptomycin in a humidified atmosphere of
5% CO2 at 37 °C. The cells were cultivated in 75 mL culture
flasks until they reached 90% confluence. Then, HDFa were
plated in 96-well plates where different assays were per-
formed. During the tests, the cells were irradiated using a
UV Irradiation System for Cultures (Vilber Lourmat, Mar-
ine, France).
used as
a reference compound in the procollagen
production assay.
Effects on ROS production
The effect of compounds on ROS production in HDFa
induced by UVB was measured using 2′,7′-dichloro-
fluorescein diacetate (DCFH-DA; Molecular Probes Inc.,
Eugene, OR, USA) and following previously described
procedures (Bravo et al. 2017). HDFa were plated at a
density of 5 × 104 cells/well in black 96-well plates in
200 μL of supplemented medium overnight. The medium
was removed, and the cells were treated with 2, 10, and
50 μM compounds dissolved in 100 μL of FBS-free medium
for 2 h. Then, 10 μL of 250 μM DCFH-DA was added and
incubated at 37 °C for 30 min. The plates were exposed to
200 mJ/cm2 UVB. After 30 min, the samples were removed,
and 100 μL of FBS-free medium was added. Finally, the
fluorescence was read at the excitation and emission
wavelengths of 485 and 520 nm, respectively. Quercetin
(25 μM) was used as a reference antioxidant.
Cell viability
Cell viability was measured using the highly water-soluble
tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium)
con-
tained in Cell Counting Kit-8 (Dojindo Molecular Tech-
nologies, Inc., Rockville, MD, USA) and following the
manufacturer’s instructions. The cells were plated in 96-
well plates at a density of 104 cells/well. For this assay,
200 μL of 5 × 104 cells/mL in supplemented medium was
cultured overnight. Then, the medium was removed, and the
cells were treated with various concentrations of com-
pounds (1, 5, 10, 50, 100, and 250 μM) dissolved in 100 μL
of FBS-free medium for 24 h. The cells were washed with
PBS and 100 μL of FBS-free medium, and then 10 μL of
WST-8 was added. Finally, the absorbance was measured at
450 nm using a multimode microplate reader (BioTek
Instruments, Inc., Winooski, USA).
Molecular docking
The molecular docking studies were performed to investi-
gate the binding mode into the active site of three different
enzymes, fibroblast collagenase, native porcine pancreatic
elastase and human hyaluronidase, PDB Codes 1CGL
(Lovejoy et al. 1994), 1QNJ (Würtele et al. 2000), and
2PE4 (Chao et al. 2007), respectively. The information
about the experimental resolution of each protein could be
consulted in the Protein Data Bank web site. The three-
dimensional (3D) structures of compounds or ligands were
submitted to a geometrical optimization procedure at PBE0
(Adamo and Barone 1999) /6–311 + g* level of theory
using the Gaussian 09 program (Frisch et al. 2009; Newton
1969). All optimized conformations were confirmed as a
minimum on the potential energy surface.
Effects on MMP-1 and procollagen production
The MMP-1 and procollagen production in HDFa after
UVB exposure was measured by ELISA using the com-
mercial kits Human Total MMP-1 DuoSet and Human
Procollagen I alpha 1 DuoSet (R&D Systems, Inc., Min-
neapolis, USA), according to the manufacturer’s instruc-
tions and following the conditions in previously reports
(Bravo et al. 2017). Briefly, HDFa were plated at a density
of 104 cells/well in 96-well plates in 200 μL of
The docking simulations for the set of optimized ligands
were performed using the AutoDock v.4.2 program (Morris
et al. 2009). AutoDock combines a rapid energy evaluation
through precalculated grids of affinity potentials with a

Medicinal Chemistry Research
variety of search algorithm to find suitable binding positions
for a ligand on a given macromolecule. This program per-
forms global searches by incorporating the van der Waals
attractive potential, geometric collision, screened electro-
static potential, and Lazaridis–Karplus desolvation energy
into the scoring function. Therefore, all molecular docking
results presented in this work are global Docking. In the
preparation of the enzymes for docking simulations the
water molecules, cofactors, and ions were excluded from
each X-ray crystallographic structure. Only in the fibroblast
collagenase, the Zinc atoms were conserved due to those
belongs to the active site. Likewise, the polar hydrogen
atoms of the enzymes were added, the atomic charges were
computed toward the Gasteiger procedure and the nonpolar
hydrogen atoms were merged. Finally, the enzyme was
treated as a rigid body.
The molecular docking calculations were carried out in
the active site of each protein. In the case of fibroblast
collagenase, 1CGL, the box is centered on the original
position of ligand crystalized, (N-[(1s)-3-{[(benzyloxy)
carbonyl] amino}-1-carboxypropyl]-l-leucyl-N-(2-morpho-
lin-4-ylethyl)-l-phenylalaninamide) (OED) (Farrokhnia,
Mahnam 2017). In the case of native porcine pancreatic
elastase, 1QNJ, the active site was located around of Val
216 residue (Sivamani et al. 2012). Finally, in the human
hyaluronidase, 2PE4, the active site was situated around of
Tyr 75 residue, according to previously published works
(Zeng et al. 2016).
evaluations were used for the 200 LGA runs. The best
conformations were chosen from the lowest docked
energy solutions in the cluster populated by the highest
number of conformations. The best docking complex
solutions (poses) were analyzed according to the potential
intermolecular interactions (ligand/enzyme), such as
hydrogen bonding, hydrophobic interactions, and the
cation–π, π–π stacking.
In silico antioxidant evaluation
The mechanism used to evaluate theoretically the possible
quenching of free radicals by the synthesized compounds
was the hydrogen atom transfer (HAT) method, from the
molecule to the radical (de Heer et al. 2000):
ArX À H þ R: ! ArX: þ R À H
The electrophilicity index ω was calculated as a measure
for the capability of the radicals formed to acquire elec-
tronic charge from the environment (Parr et al. 1999). The
spin density (SD) analysis (Parkinson et al. 1999), defined
as the difference between α and β electron densities, was
evaluated as a local descriptor of stability. All calculations
were performed at the PBE0/6–311 + g* level of theory
with the Gaussian 09 software package.
In silico prediction of the pharmacokinetic
properties and percutaneous absorption parameters
Molecular docking normally requires a user-defined
docking search space, where the possible ligand binding
conformations are explored. A small search space can
produce an insufficient number of conformations, while a
generously large space could generate too many irrelevant
interaction poses. Therefore, optimally confined docking
search space is fundamental to the success of ligand–protein
coupling. The default box size can be calculated using
experimentally resolved protein–ligand complex structures.
First, an initial box is constructed to enclose the ligand, and
then the size of the box is increased in random directions to
ensure that the minimum length in any dimension is at least
22.5 Å. The grid maps of interaction energy for various
atom types with each macromolecule were calculated by the
auxiliary program AutoGrid choosing a grid box centered
at: (30.681, 46.555, −0.0090) for 1CGL, (16.203, 8.633,
1.518) for 1QNJ and (45.539, −21.778, −19.639) for 2PE4,
To determine the drug-like properties for all tested com-
pounds 1–9, the physicochemical descriptors were deter-
mined using open-source cheminformatics toolkits such as
the Molinspiration software (for MW, H-bond donors, H-
bond acceptors, rotatable bonds, and TPSA descriptors) and
ALOGPS 2.1 algorithm from the Virtual Computational
Chemistry Laboratory (for log Po/w and aqueous solubility
log Sw descriptors). Furthermore, to obtain information on
the kinetics of the adsorption and distribution required for
skin permeability, four important dermatopharmacokinetic
parameters were predicted for compounds 1–9. The skin
permeation coefficient of chemical in the stratum corneum
(LogKp) was calculated using PreADMET 2.0, and the
percutaneous absorption (LogPab) was predicted according
to the equation [LogPab = −2.72 + 0.71 × Log Po/w
–
0.0061 × MW]. The effective diffusion coefficient in human
dermis (Dde) was calculated using the equation [Dde =
(7.1 × 10−10/MW0.5)], and the most important parameter in
determining the maximal dermal flux, toxicity, or systemic
̊
with dimensions of 60 × 60 × 60 A around the active site,
and a grid point spacing of 0.375 Å. All these conditions are
sufficiently to include the most important residues of each
enzyme. The docking searches for the best orientations of
the molecules to the active site of each protein were per-
formed using the Lamarckian genetic algorithm (LGA)
(Morris et al. 1998). The LGA protocol applied a popu-
lation size of 2000 individuals, while 2,500,000 energy
effect, Jmax, was calculated using the equation [Log Jmax
=
−3.90 − 0.0190 × MW]. These important parameters
defining the penetration, absorption, permeation, move-
ment, partitioning, and diffusion of the compounds have
been applied to support the transdermal delivery of potential

Medicinal Chemistry Research
drugs and skincare actives, as well as integrated risk
assessment (Chen et al. 2015; Schoellhammer et al. 2014).
In vitro antiaging properties and antioxidant
capacity
Statistical analysis
The degradation of the ECM components induced by UVB
stimulation might be among the main factors involved in
skin photoaging (Mukherjee et al. 2015); therefore, the
inhibition of collagenase, elastase, and hyaluronidase is an
important antiaging target (Duque et al. 2017; Kumud and
Sanju 2018). To develop a new class of inhibitors of skin
aging-related enzymes, such as collagenase, elastase, and
hyaluronidase, the synthetic quinoline–hydrazone hybrids
1–9 were evaluated as possible antiaging agents. The results
are shown in Fig. 2. The best results, in terms of enzymatic
inhibition, occurred when a high number of hydroxyl
groups were present on the aromatic ring of hydrazone. The
compounds with the best activity against collagenase and
hyaluronidase were hybrids 5 and 9, which both have three
hydroxylated positions (Fig. 2a, c). Hybrid 5 and the
dihydroxy analog 7 showed elastase inhibition (Fig. 2b).
The monohydroxy hybrid 1 was inactive or had slight
activity, and the dihydroxybenzyl-hydrazone (2, 3, 4, 6, and
8) derivatives showed weak activity, establishing the
importance of the positions of the hydroxyl groups (Fig. 2).
The IC50 values were obtained for the more active com-
pounds, and the results are shown in Table 1. Overall, the
IC50 values were close to those determined for the reference
compounds. However, in the inhibition of elastase, hybrid 5
showed better inhibition than the commercial standard
oleanolic acid (IC50 = 193.6 μM).
All results are expressed as the mean SD of three
experiments. The statistical analyses were performed using
GraphPad Prism 5 (GraphPad Software Inc., San Diego,
CA, EUA). To evaluate significant (P < 0.05) differences
between compounds and controls, analysis of variance
(ANOVA) was performed.
Results and discussion
Chemical synthesis
The synthesis and characterization of the compounds under
study have already been published (Coa et al. 2015). The
chemical structure of the hybrids evaluated is presented in
Fig. 1. Quinaldine (A) was oxidized with selenium oxide to
produce aldehyde B, which was treated with iodine and
methanol in basic solution to afford ester C. Then, com-
pound C was subjected to nucleophilic substitution with
hydrazine hydrate to obtain acylhydrazide D, which was
coupled with the different hydroxyaldehydes of E in aqu-
eous medium to obtain hydrazones 1–5. Hydrazones 6–9
were obtained following the same synthetic strategy starting
from 4-quinolinecarboxaldehyde (Fig. 1).
Fig. 1 The synthetic pathway of the quinoline–hydrazone hybrids

Medicinal Chemistry Research
A.
B.
100
80
60
40
20
0
100
80
60
40
20
0
*
*
C1 C2 C3 C4 C5 C6 C7 C8 C9 Oleanolic acid
C1 C2 C3 C4 C5 C6 C7 C8 C9 Oleanolic acid
Concentration (500 μM)
Concentration (500 μM)
C.
D.
100
80
60
40
20
0
80000
60000
40000
20000
0
*
C1 C2 C3 C4 C5 C6 C7 C8 C9
C1 C2 C3 C4 C5 C6 C7 C8 C9 EGCG
Concentration (250 μM)
Fig. 2 Effect of quinoline–hydrazone hybrids on skin aging-related
enzymes and their antioxidant properties. Inhibitory activities (%)
against a collagenase, b elastase, and c hyaluronidase. d The anti-
oxidant activity as detected by the ORAC method, expressed as μmol
TE/g compound. Bars are mean standard deviation for three repli-
cates. Asterisks indicate an evaluation concentration of 250 μM
(reference inhibitors)
Table 1 IC50 (μM) of more potential quinoline–hydrazone hybrids
against aging-related skin enzymes
Table 2 Estimated free energy of binding for the quinoline–hydrazone
hybrids expressed in kcal mol−1
Compounds
Anticollagenase Antielastase Antihyaluronidase
Compounds
Anticollagenase Antielastase Antihyaluronidase
5
9
39.39 2.7
45.61 2.5
164.2 8.0
193.6
318.8 22.6
1
2
3
4
5
6
7
8
9
−7.65
−8.48
−8.03
−7.99
−8.04
−7.45
−8.02
−7.54
−7.69
−7.68
−7.59
−7.55
−7.36
−7.19
−7.27
−7.64
−7.25
−7.45
−7.64
−7.73
−7.99
−7.67
−8.78
−7.46
−8.07
−8.15
−8.45
−8.03
Oleanolic acida 29.98 1.8
EGCGb
8
210.7 3.00
aOleanolic acid was used as a positive control for anticollagenase and
antielastase activity
bEGCG was used as a positive control for antihyaluronidase activity
In order to propose a molecular level explanation on the
enzymes elastase, collagenase, and hyaluronidase inhibition
by the best inhibitors (quinoline–hydrazone hybrids 5, 7,
and 9), a molecular docking study was carried out assuming
a model where the protein and ligand were considered as
rigid and flexible, respectively, during the docking proce-
dure. Unfortunately, the sameness in the estimated binding
energy values, presented in Table 2, did not allow determine
large differences in the inhibition between the synthetized
compounds. A possible explanation could be the limitation
of docking model for explains how the compounds could
arrive to the active site. In all the molecular docking
Oleanolic acida −11.29
EGCGb
−9.45
aOleanolic acid was used as a positive control for anticollagenase and
antielastase activity
bEGCG was used as a positive control for antihyalurocollagenase activity
The bold values represent active and selected compounds for further
studies
experiments, the procedure starts with the ligand located
into the site active. However, a picture about the inter-
molecular interactions between protein and ligand should be

Medicinal Chemistry Research
interesting for explain the difference in the inhibition
between 5, 7, and 9 compounds. For that, graphical repre-
sentations for the binding of ligand–collagenase, elastase,
and hyaluronidase are present in the Fig. 3. The principal
hydrogen bond interactions for the ligand–collagenase are:
His218, His228, and His222 for oleanolic acid; His218,
His228, Glu219, Ala184 for 5, and finally Ala184 and
Glu219 for 9. In this case, the less stabilization of 9 can be
explained for the absence of residues His218 and His228,
and the not closeness with the Zn atom. In the case of the
elastase–ligand complex, the principal hydrogen bond
interactions are Val216, Thr41, and Arg61 for oleanolic
acid; Ser214, Val216, and Ser217 for 5; and Asn148,
Gln192, Val216 for 7. In all cases, the residue Val216 looks
as important residue for the ligand stabilizations. Finally,
and para oxygenated positions in the hybrids 2- and 4-
quinoline–hydrazone are important for activity. In addition,
the 4-quinoline–hydrazone hybrids (compounds 6–9) show
better antioxidant activity than the 2-quinoline–hydrazone
series (compounds 1–5). In fact, the position of the
benzyl–hydrazone moiety on the quinoline ring of hybrid 8
led to an important improvement in activity compared with
compound 4.
In order to understand the highest antioxidant activity of
the hybrids 5 and 9, the theoretical values for the bond
dissociation enthalpy (BDE) of the homolytic cleavage
N–H or O–H bonds, were calculated and are presented in
Table 3. For HAT mechanism, a lower value of O–H BDE,
calculated as BDE = [(ArX) + (H)] − [(ArX-H) + E(R)],
where E corresponds to energy, is indicative of an energe-
tically process. The lowest values presented for the com-
pounds 4, 5, 8, and 9, showed that the most favorable O–H
bond homolytic cleavage is in meta positions. However, the
electrophilicity index ω show that the 5 and 9 radicals are
the molecules with the highest capability to acquire elec-
tronic charge from the environment, allowing them to react
with other potentially harmful radicals through specific
hydrogen transfer mechanisms. These results can be con-
firmed by the electron density analysis for the 4, 5, 8, and 9
radicals formed (Fig. 1. In Electronic Supplementary
Information). Larger surfaces are indicative of stabilization
of the respective radical species, and therefore to their
persistence in the medium. Also, 5 and 9 compounds show
two OH groups with a high probability to a posterior
reaction, compared with 4 and 8. So, these results show the
efficacy of the potential antioxidant activity of the 5 and 9
hybrids.
Quinoline nuclei occur in several natural compounds and
pharmacologically active substances, displaying a broad
range of biological activities. In fact, the importance of the
potent quinoline class of compounds has been established in
the search for effective agents (Chaaban et al. 2018; El-
Feky et al. 2015; Marella et al. 2013). In relation to its
antiaging potential, some quinoline derivatives have been
shown to possess antihyaluronidase activity (Czarnecka
et al. 2017). Hydrazones are another class of biologically
active moieties that are extensively explored as exhibiting
various biological profiles (Singh and Raghav 2011; Verma
et al. 2014). Derivatives of these compounds have exhibited
potent antioxidant activity (Belkheiri et al. 2010; Yılmaz
et al. 2012) and have been shown to be inhibitors of skin
disease-related enzymes, mainly as antielastase agents
(Nurkenov et al. 2017). However, despite this potential,
there are no prior data available in the literature regarding
the inhibition of skin disease-related enzymes of
quinoline–hydrazone hybrids. Their very strong inhibition
of collagenase, elastase, and hyaluronidase activities, par-
ticularly together with the potent antioxidant activity
the
hydrogen
bond
interactions
present
for
hyaluronidase–ligand complex are composed by Tyr75,
Ser76, Tyr286, Ser323, and Trp324 for EGCG; Tyr75,
Val322, and Thr327 for 5; and Ser76, Asp129, Glu131 for
9. In this case, the less stabilization of 9 could be explained,
at least in part, for the absence of residues Tyr75. Infor-
mation about the hydrophobic interactions for these
ligand–protein complexes, can be seen in the Electronic
Supplementary Information.
The evaluation of the antioxidant properties of the
quinoline–hydrazone hybrids 1–9 studied in this work was
performed by the ORAC method. The results are shown in
Fig. 2d and are expressed as μmol TE/g of compound. The
ORAC assay assessed the ability of compounds to scavenge
peroxyl radicals, which represent one of the contributors of
lipid peroxidation. It is known that lipid radicals are formed
when skin is exposed to UV. The major type of ROS pro-
duced on the surface of the skin is singlet oxygen (1O2)
(Ryu et al. 2009), which is oxidized to squalene, choles-
terol, and unsaturated acyl residues in the sebum, yielding
lipid-peroxyl radicals. These ROS further oxidize proteins
to produce carbonylated proteins in the stratum corneum
(Masak 2010). Therefore, although lipid-peroxyl radicals
are very relevant, adducts detected in skin samples also
correspond to dermal protein damage (mainly collagen and
elastin) (Haywood et al. 2008). These data establish the
importance of the peroxyl radical scavenging capacity at the
level of skin aging. From the screening of hybrids, it can be
seen that the compounds with the best activity were the
trihydroxybenzyl-hydrazone derivatives 5 and 9. The
dihydroxybenzyl analog 7 showed a slight reduction in
antioxidant activity. The monohydroxy and dihydrox-
ybenzyl hybrids 1 and 4 showed weak activity. Regarding
the structure-activity relationship, it is interesting to note
that, in general, as with enzymatic inhibition, as the number
of hydroxyl groups increases in the molecule, the anti-
oxidant capacity increases. In the dihydroxylated series
(compounds 2–4 and 6–8), it was observed that at the meta

Medicinal Chemistry Research
Fig. 3 Graphical representations for the binding of a collagenase–oleanolic acid; b collagenase–5; c elastase–oleanolic acid; d elastase–5; and
e Hyaluronidase–EGCG

Medicinal Chemistry Research
Table 3 The bond dissociation enthalpy (BDE) and the electrophilicity
index (ω) of the synthetized hybrids by HAT mechanism
A.
30000
20000
10000
0
BDE for the radicals formed
(kcal mol−1
)
Compounds N–H OH, ortho OH, meta OH, para ωa (kcal mol−1
)
***
***
***
***
1
2
3
4
5
6
7
8
9
84.8 95.9
84.9 87.9
83.0 96.0
84.4 91.3
82.9 89.8
89.9 84.0
88.1 91.7
89.5 87.0
88.0 86.0
–
–
Concentration (μM)
Sample
85.6
–
–
2
10 50
5
2
10 50
25
EGCG
+
Controls
9
UVB (100 mJ/cm2)
84.2
–
-
+
+
+
+
+
+
+
77.9
78.1
85.6
–
16.4
B.
15000
83.9
–
14.0
*
10000
5000
84.5
–
78.9
78.1
16.1
84.3
15.4
aReactivity descriptor for the OH-meta radicals formed
*
0
Concentration (μM)
2
10 50
2
10 50
25
Controls
Sample
5
+
9
+
EGCG
+
UVB (100 mJ/cm2)
-
+
+
+
+
+
presented by some of the prepared hybrids, establishes that
the occurrence of the quinoline hydrazone moiety has tre-
mendously enhanced the antiaging activity. The test hybrids
5, 7, and 9 showed similar or even higher activity compared
with the reference standards EGCG and oleanolic acid.
Encouraged by these findings, it was interesting to synthe-
size and evaluate the biological activity of some of the
structure hybrids that include a quinoline backbone bearing
a substituted hydrazone moiety, aiming to highlight the
potential of derivatives as a template for the development of
antiaging and antioxidant agents with a diverse mechanisms
of action.
C.
20000
15000
10000
5000
***
***
***
***
0
Concentration (μM)
2
10 50
2
10 50
2
10 50
25
Controls
-
Sample
Quercetin
5
+
7
+
9
UVB (200 mJ/cm2)
+
+
+
+
+
+
+
+
Fig. 4 Photoprotective effects of potential quinoline–hydrazone
hybrids on human dermal fibroblasts (HDF). Human MMP-1 and
human procollagen were measured by ELISA after 100 mJ/cm2 UVB
irradiation. The results were normalized according to the percentage of
viable cells determined by the WST-8 assay. 1 × 104 HDF were treated
with samples for 24 h. a After 72 h of irradiation, MMP-1 was mea-
sured and b 24 h after irradiation, procollagen Iα1 was measured. c To
quantify ROS production, 5 × 104 cells were cotreated with samples
for 2 h and then exposed to UVB (200 mJ/cm2) radiation. ROS gen-
eration was measured by DCFH-DA. All data are shown as the mean
Photoprotective effect in human dermal fibroblasts
(HDF)
The skin is the most exposed organ to ultraviolet (UV)
irradiation, which induces premature skin aging. This type
of aging (photoaging) is characterized by rough texture,
coarse wrinkles, and mottled pigmentation (Kang et al.
2017). Although skin aging-related enzymes are expressed
at low levels in unstimulated cells, UV irradiation results in
important molecular changes, including the promotion of
MMP-1 (fibroblast collagenase) expression, reducing the
transcription of COL3a1 and COL1a1 genes, which encode
procollagen I and III, causing connective tissue damage and
significantly increasing ROS (Freitas-Rodríguez et al.
2017). Since MMP-1 induced by ROS causes collagen and
elastin degradation, its inhibition and the promotion of
collagen type I synthesis from procollagen type I are
important targets for skin photoaging control and wrinkle
prevention (Bravo et al. 2017). In this study, the modulation
of HDF of MMP-1, procollagen and ROS production in
UVB-mediated skin photoaging by trihydroxybenzyl-
hydrazone derivatives 5 and 9 was examined. The results
*
SD of three independent experiments. P < 0.05, compared with the
UVB-irradiated control
are shown in Fig. 4. Compounds 5 and 9 were chosen for
photoprotectivity testing on the basis of preliminary data
and because strategies used to combat the devastating
effects of photoaging can focus on compounds acting with
different mechanisms involved in its pathogenesis (Duque
et al. 2017). It is important to mention that the test hybrids
were nontoxic to the cells, even at the highest concentration
tested (50 mM, data not shown).
The exposure of fibroblasts to UVB radiation increased
MMP-1 production and decreased type I procollagen
secretion, as shown Fig. 4a, b, in a dose- and time-
dependent manner (data not shown). After UVB exposure,
the increase in MMP-1 secretion was 1.5-fold that of the
control, and the decrease in procollagen production was

Medicinal Chemistry Research
~56% that of unirradiated cells. Pretreatment with hybrids 5
and 9 significantly decreased the MMP-1 production in
HDF in a dose-dependent manner after UVB irradiation.
Both compounds decreased the level of MMP-1 compared
with that of irradiated cells, but hybrid 5 showed higher
activity at the highest evaluated concentration than com-
pound 9 (Fig. 4a). Therefore, the position of the
benzyl–hydrazone moiety on the quinoline ring is a critical
structural motif for modulating the production of MMP-1 in
the cell bioassay. Interesting results were also obtained with
the pretreatment of the reference compound EGCG
(25 µM). However, pretreatment with hybrid 5 (10 µM)
reduced the deleterious effect of UVB exposure on HDF
through the increase of procollagen production in ~44% of
irradiated cells. Nevertheless, the procollagen production by
fibroblasts treated with hybrid 9 and higher concentrations
of hybrid 5 could suggest a probable phototoxic effect of the
compounds.
To determine whether the trihydroxybenzyl-hydrazone
derivatives 5 and 9 decreased MMP-1 production and
increased procollagen secretion by HDF after UVB expo-
sure via an antioxidant effect, the modulation of ROS
production after UVB exposure (200 mJ/cm2) was eval-
uated. The dihydroxybenzyl-hydrazone derivative 7 was
also evaluated based on its powerful antioxidant activity.
The results are shown in Fig. 4c. The HDF exposed only to
UVB had a marked increase in fluorescence, indicative of
an increase in ROS generation at the single-cell level.
Hybrids 5, 7, and 9 decreased ROS production in HDF after
UVB irradiation by between 20 and 30% when cells were
treated with 2, 10, and 50 µM of each compound. This
effect was not dose dependent, except for compound 7,
which showed higher activity at the highest evaluated
concentration. Inside cells, ROS oxidize nonfluorescent
DCFH-DA derivatives to green fluorescent pigment DCF,
and the level of fluorescence is used as a marker of cytosolic
ROS (Pygmalion et al. 2010). However, it was previously
reported that not all radical species oxidize DCFH; there-
fore, the DCF level is used as a marker of only some
reduced oxygen species (Bilski et al. 2002). Although the
ROS level is overestimated and cannot be considered an
absolute level, this approach was found to be suitable for
comparing the efficacy of quinoline–hydrazone hybrids at
the cellular level and for building a performance scale for
ROS scavenging during irradiation.
drug-like characteristics within the range of 95% of the
known reference drugs. None of the hybrids 1–9 showed a
violation of Lipinski´s rule of five and its variants, making
them potentially promising agents in pharmacological
investigations as antiaging drugs. The skin penetration of
active ingredients used in cosmetics is affected by their
physicochemical properties, such as molecular weight and
the partition coefficient (P). Chemical compounds with high
or low P values or molecular weights >500 Da are generally
poorly absorbed through the skin (Bos and Meinardi 2000).
Accordingly, the physicochemical parameters of drugs used
as transdermal therapeutic systems have appropriate P
values (log P = 1–3) and low molecular weights (<500 Da)
(Higo 2007), because antiaging formulations require both
the breaching of the lipophilic stratum corneum and the
resorption into the aqueous central compartment of the
systemic circulation (Wiedersberg and Guy 2014). The in
silico ADMET predictions of the novel hybrids 1–9
revealed the strongest prospects for use in topical for-
mulations for aging skin, displaying optimal log P values
(in the range of 3.071–1.646) and molecular weights ran-
ging from 291 to 323 Da.
To screen for compounds active against skin aging and
that are able to cross the dermal layers at physiologically
relevant concentrations, four fundamental pharmacokinetic
parameters involved in the development of cosmetic antia-
ging formulations were calculated (Klimova et al. 2018).
Then, the in silico predictions of physicochemical trans-
dermal properties for the hybrids 1–9 were investigated.
The results indicated that all hybrids showed a skin per-
meation coefficient value (calculated by PreADMET 2.0
tool) comparable with some conventional topical drugs
(ranging from −1.262 to −3.931) ranging between −1.754
and −3.524 and exhibited optimal values for percutaneous
absorption (Log Pab), effective diffusion coefficient in der-
mis (Dde) and maximum percutaneous flux (Jmax) corre-
sponding to an effective transdermal permeability with high
potential for percutaneous penetration. In addition, the
pharmacokinetic parameters obtained from the in silico
prediction suggest that, among all the tested compounds,
hybrids 5 and 9 have promising antiaging activity because
they displayed optimal partition and diffusion values into
skin within the ideal range of the known typical drugs that
are FDA-approved for transdermal administration.
All tests carried out in this study attempted to address a
number of important molecular targets in the cascade of
reactions that develop during photoaging, which interact
with some quinoline–hydrazone hybrids. These hybrids are
expected to possess diverse pharmacological activities in
the pathological process of aging that involves multiple
pathways. The combination of specific antioxidant and
enzymatic inhibition activities was applied to the pre-
liminary screening of the bioactive antiaging hybrids.
In silico ADME properties and percutaneous
absorption parameters
We calculated and analyzed various drug-like properties of
the nine quinoline–hydrazone hybrids, and the data are
summarized in Table 4. All compounds exhibited sig-
nificant values for the parameters analyzed, showing good

Medicinal Chemistry Research

Medicinal Chemistry Research
Later, a cell-based study of bioactive compounds verified
the modulation of UVB-induced MMP-1 production and
the inhibition of the suppression of type I procollagen
synthesis mediated by the inhibition of the production of
ROS in cultured HDF. Our results suggest that the activity
of these hybrids as inhibitors of skin aging-related
enzymes is associated to the number and position of the
hydroxyl groups on the benzyl–hydrazone moiety. In
addition, the antioxidant activities of the hybrids 5 and 9
are responsible for the cellular resistance to photoaging.
However, it is possible that these hybrids may differen-
tially affect various cellular signal cascades. Thus, further
studies need to be conducted to validate the results of the
in vitro assay.
Acknowledgements This investigation received financial support from
Universidad de Antioquia, UdeA.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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