Full text of DOI:10.1016/j.cbi.2018.06.007

Accepted Manuscript
Synthesis of newly functionalized 1,4-naphthoquinone derivatives and their effects on
wound healing in alloxan-induced diabetic mice
Silvia Helena Cardoso, Cleidijane Rodrigues de Oliveira, Ari Souza Guimarães,
Jadiely Nascimento, Julianderson de Oliveira dos Santos Carmo, Jamylle Nunes de
Souza Ferro, Ana Carolina de Carvalho Correia, Emiliano Barreto
PII:
S0009-2797(18)30553-2
10.1016/j.cbi.2018.06.007
CBI 8319
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 25 April 2018
Revised Date: 30 May 2018
Accepted Date: 9 June 2018
Please cite this article as: S.H. Cardoso, C.R. de Oliveira, A.S. Guimarães, J. Nascimento, J. de Oliveira
dos Santos Carmo, J.N. de Souza Ferro, A.C. de Carvalho Correia, E. Barreto, Synthesis of newly
functionalized 1,4-naphthoquinone derivatives and their effects on wound healing in alloxan-induced
diabetic mice, Chemico-Biological Interactions (2018), doi: 10.1016/j.cbi.2018.06.007.
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Synthesis of newly functionalized 1,4-naphthoquinone derivatives and their effects
on wound healing in alloxan-induced diabetic mice
Silvia Helena Cardoso*1, Cleidijane Rodrigues de Oliveira1, Ari Souza Guimarães1,
Jadiely Nascimento1, Julianderson de Oliveira dos Santos Carmo2, Jamylle Nunes de
Souza Ferro2, Ana Carolina de Carvalho Correia2, Emiliano Barreto*2
1Laboratory of Organic Synthesis and Medicinal Chemistry (LaSOM), Núcleo de
Ciências Exatas (NCEx), Campus Arapiraca, Federal University of Alagoas, CEP
57.309-005, Arapiraca, Alagoas, Brazil. Tel: +55 82 3482 1884.
2Laboratory of Cell Biology, Campus A.C. Simões. Federal University of Alagoas, CEP
57.072-970, Maceio, Alagoas, Brazil. Tel: +55 82 3214 1704.
* Corresponding authors:
E-mail address: silvia.cardoso@arapiraca.ufal.br and emilianobarreto@icbs.ufal.br

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Abstract
Naphthoquinone derivatives have various pharmacological properties. Here, we
describe the synthesis of new 1,4-naphthoquinone derivatives inspired by lawsone and
β-lapachone and their effects on both migration of fibroblasts in vitro and dermal wound
healing in diabetic mice. NMR and FTIR spectroscopy aided characterization of
chemical composition and demonstrated the molecular variations after the synthesis of
four different derivatives, namely 2-bromo-1,4-naphthoquinone (termed derivative S3),
2-N-phenylamino-1,4-naphthoquinone (derivative S5), 2-N-isonicotinoyl-hydrazide-1,4-
naphthoquinone (derivative S6), and 1-N-isonicotinoyl-hydrazone-[2-hydroxy-3-(3-
methyl-2-butenyl)]-1,4-naphthoquinone (derivative S7). Our results indicate that
derivatives S3, S5, S6 and S7 were non-toxic to the 3T3 fibroblast cell line. In scratch
assays, derivatives S3 and S6, but not S5 or S7, stimulated the migration of fibroblasts.
Compared with untreated diabetic mice, S3, S6 and S7 treatments accelerated wound
closure. However, derivative S3 was optimal for the stimulation of epithelization,
thereby increasing the number of keratinocyte layers and blood vessels, and reducing
diffuse cellular infiltration, compared to derivatives S6 and S7. Our results suggest that
novel 1,4-naphthoquinone derivatives promote fibroblast migration and accelerate
wound closure under diabetic conditions.
Keyword: naphthoquinone derivatives; wound healing; diabetes.

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1. INTRODUCTION
Diabetes includes a heterogeneous group of disorders characterized by
hyperglycemia and a relative or absolute lack of insulin secretion or action [1].
According to the World Health Organization, more than one million people die from
diabetes each year, and an increasing number of patients require hospitalization. Current
projections indicate that nearly 415 million adults have diabetes and that this estimate
will reach 642 million individuals in 2040 [2].
Non-healing wounds are a typical hallmark of diabetes that results in chronic
lesions, ulcers, and amputation of the extremities, negatively impacting patient quality
of life and raising costs for health systems [3, 4]. Approximately 85 million patients
with diabetes worldwide have complications associated with delayed cutaneous wound
healing [5].
Cutaneous wound healing is a dynamic process that requires coordinated
participation of many cell types acting in distinct phases to restore the anatomical and
functional integrity of tissue [6]. The wound healing impairment seen in diabetes is a
consequence of both extrinsic and intrinsic factors including repeated trauma and/or
maintained hyperglycemia. Both factors negatively affect the phases of healing by
interfering with cellular penetration, oxidative metabolism, collagen deposition, blood
circulation, and angiogenesis [7]. Therefore, given that different cellular and molecular
events are affected by diabetic conditions, a treatment based on simultaneous correction
of the multiple deficits could be an attractive alternative. However, new therapeutic
products with these characteristics may have side effects and/or involve high costs [8].
Thus, developing effective and economical therapies for correcting healing of diabetic
wounds are still needed.

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The chemical modification of existing active principles is a strategy of great
importance in identifying new medicinal products [9]. Examples of successful
medicines that originated from natural products include procaine, simvastatin, and
dapagliflozin, among others [10]. This approach can result in new drugs capable of
promoting improvements in the quality of life of diabetic patients with wounds that are
slow to heal.
Naphthoquinones are natural aromatic compounds that can be found in several
plant families and are traditionally used for their dyeing properties [11]. However,
recently, a variety of biological activities to compounds as lawsone, β-lapachone, and
lapachol (Figure 1), have been reported, including antimalarial, antioxidant, and
antitumoral activities [12-15].
These natural naphthoquinones have been demonstrated to have wound healing
properties, which may be due to anti-inflammatory and antioxidant properties [16, 17].
Additionally, a previous study demonstrated that oral administration of lawsone
promoted cutaneous healing of both excision and incision wounds in rats [18].
Furthermore, previous studies have been demonstrated the influence of electron-
donating or withdrawing groups into structure of 1,4-naphthoquinones able to
improving their pharmacological activities [19, 20]. However, to the best of our
knowledge, there are no available data on the evaluation of the potential healing effects
of these naphthoquinone derivatives for diabetic wounds. Thus, the present study aimed
to evaluate the effects of naphthoquinones derivatives in skin wound healing in diabetic
mice.

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2. MATERIALS AND METHODS
Melting points were determined by the MQAPF-301 apparatus and were
uncorrected. Infrared spectra were obtained using the Bomem FT-IR MB-102
1
13
spectrometer with KBr pellets. H NMR (400 MHz) and C NMR (100 MHz) spectra
were recorded using the Bruker Advanced DPX spectrometer with CDCl3 or DMSO-d6
as the solvent. Column chromatography was performed using silica gel 60G 0.0630–200
mm (70–230 mesh ASTM) Merck and silica gel 60G 0.2–0.5 mm VETEC. TLC
analyses were performed on precoated aluminum plates of silica gel 60F 254 plates
(0.25 mm, Merck). Solvents were purified and dried according to the standard
procedure.
Naphthoquinones were purchased from Sigma-Aldrich (USA) and were used
without purification; these included 1,4-naphthoquinone (S1), lawsone (S2) (2-hydroxy-
1,4-naphthoquinone), and 2-bromo-1,4-naphthoquinone (termed derivative S3).
Lapachol (2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone) (S4) was isolated
from the bark of Tabebuia sp., collected in the Agreste Region of Alagoas State (Brazil)
and used as the starting material for the synthesis of 1-N-Isonicotinoyl-hydrazone-[2-
hydroxy-3-(3-methyl-2-butenyl)]-1,4-naphthoquinone (termed derivative S7). S1 was
used for the synthesis of 2-N-phenylamino-1,4-naphthoquinone (derivative S5), while
S2 was used for the synthesis of 2-N-isonicotinoyl-hydrazide-1,4-naphthoquinone
(derivative S6).
2.1. Chemistry
2.1.1. Extraction and characterization of lapachol (S4)
Lapachol was isolated from wooden chips of Tabebuia sp. bark by aqueous
sodium carbonate extraction (10% w/v), followed by dilute hydrochloric acid

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precipitation and then ethanol or ethyl acetate recrystallization, leading to a 1–2% yield
1
from the bark. C15H14O3; mp: 139–140 °C (lit. 140 °C). Yellow solid. [21], NMR H
(400 MHz, CDCl3): δ 8.13 (1H, dd, J = 7.6; 1.3 Hz); 8.08 (1H, dd, J = 7.5; 1.4 Hz); 7.76
(1H, td, J = 7.6; 1.3 Hz); 7.66 (1H, td, J = 7.6; 1.3 Hz); 7.3 (1H, s, OH); 5.21 (1H, m),
13
3.32 (1H, d, J = 7.4 Hz); 1.79 (3H, s); 1.69 (3H, s). NRM C (100 MHz, CDCl3): δ
184.6 (C=O); 181.7 (C=O); 159.9 (CH), 152.7 (C); 134.7 (CH); 133.8 (CH); 133.7
(CH); 132.7 (CH); 129.6 (C); 129.3 (C); 126.6 (CH); 119.6 (C); 25.7 (CH3); 22.5 (CH);
17.8 (CH3). FTIR: 1665 (C=O); 2917 (CH2 e CH3); 1597 (C=C); 3354 (OH); 724 (CH
aromatic).
2.1.2. Synthesis and characterization of naphthoquinones derivatives
In order to assure novelty in the synthesis route, we perform modifications in the
structure of 1,4-naphthoquinone with nitrogenated group. All target compounds were
obtained by nucleophilic attack of the most basic nitrogen directly into a carbonyl group
(C1) or in a carbon (C2) of 1,4-naphthoquinone.
2.1.2.1. 2-N-Phenylamino-1,4-naphthoquinone (S5)
1,4-naphthoquinone S1 (871 mg, 3.5 mmol) was dissolved in 60 mL of water,
and after complete dissolution, it was added to 0.3 mL of aniline solution. The mixture
was stirred at reflux for 24 h when the TLC consumption of the starting material was
observed. Solids obtained were filtered, washed with cold water, dried, and
recrystallized with methanol to obtain the derivative S5 at 69% yield. C16H11N16NO2,
1
mp. 156-160 °C; Dark red solid, NMR H (400 MHz, CDCl3) ): δ 8.11 (2H, m); 7.77
(1H, td, J = 7.4; 1.5 Hz); 7.68 (1H, td, J = 7.6; 1.5 Hz); 7.43 (2H, t, J = 8.0 Hz); 7.29
(2H, d, J = 8.8 Hz); 7.23 (1H, t, J = 7,4 Hz); 6.43 (1H, s), NMR 13C (100 MHz, CDCl3):

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δ 183.9 (C=O); 182.1 (C=O); 144.7 (C=C-N); 137.4 (C-N); 134.9 (CH); 133.2 (C);
132.3 (CH); 130.3 (C); 129.7 (CH); 126.5 (CH); 125.1 (CH); 125.6 (CH); 123.6 (CH);
122.8 (2 x CH); 103.3 (CH).
2.1.2.2. 2-N-Isonicotinoyl-hydrazide-1,4-naphthoquinone (S6)
2-Hydroxy-1,4-naphthoquinone S2 (2.5 g, 14.4 mmol) was dissolved in 100 mL
of 80% glacial acetic acid solution. To this suspension, 1.6 g of isonicotinoyl hydrazide
was added gradually. After the addition of the hydrazide, a color change was observed
from yellow to red. After 72 h of constant stirring at room temperature, the solid
obtained was filtered off, washed with 80% acetic acid solution and water, dried, and
recrystallized from methanol to give the compound S6 at 45% yield. C16H11N3O3; PDec
.
222-224 °C; Orange solid, NMR 1H (400 MHz, DMSO d-6): δ 11.0 (1H, s); 9.5 (1H, s);
8.8 (2H, d, J = 5.9 Hz); 8.03 (1H, d, J = 7.4; 1.2 Hz); 7.9 (2H, dd, J = 7.4; 1.1 Hz); 7.85-
7.83 (3H, m); 7.76 (1H, td, J = 7.4; 1.2 Hz); 5.76 (1H, s). NMR 13C (100 MHz, DMSO-
d6): δ 182.7 (C=O); 181.3 (C=O); 164.3 (NHCO); 150.9 (CH); 148.7 (C=C-N); 139.6
(C); 135.4 (CH); 133.3 (CH); 132.8 (C); 130.9 (C); 126.3 (CH); 125.9 (CH), 121.8
(CH); 102.4 (C).
2.1.2.3.
naphthoquinone (S7)
Lapachol S4 (484 mg, 2.0 mmol) was dissolved in 20 mL of 10% Et3N solution.
1-N-nsonicotinoyl-hydrazone-[2-hydroxy-3-(3-methyl-2-butenyl)]-1,4-
To this solution, 5 mL of an aqueous solution of isonicotinoyl hydrazide (6.0 mmol)
was added and was kept under constant stirring at room temperature. After 48 h, TLC
consumption of the starting material was observed and the reaction was treated with 4
mL of glacial acetic acid. A solid was obtained by the precipitation of the medium using

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ice water; this was filtered and recrystallized from ethanol to obtain the derivative S7 at
1
95% yield. C22H20N3O3; mp 164-167 °C; Orange solid, NMR H (400 MHz, DMSO-
d6): δ 16.4 (1H, sl, NH); 8.90 (2H, d, J = 5.5 Hz); 8.10 (1H, sl); 7.98 (1H, sl); 7.86 (2H,
d, J = 5.5 Hz); 7.56 (2H, m); 5.08 (1H, m); 3.24 (2H, d, J = 7.2 Hz) 1.73 (3H, s); 1.65
(3H, s). NMR 13C (100 MHz, DMSO-d6): δ 179.7 (C=O); 166.4 (C-OH); 15.5 (NHCO);
150.9 (CH); 140.3 (C); 131.3 (C, C=N); 130.9 (C); 130.4 (CH); 129.4 (CH); 127.9 (C);
124.3 (CH), 123.3 (CH); 122.4 (CH); 121.7 (CH); 117.4 (C); 25.4 (CH3); 21.2 (CH2);
17.9 (CH3).
2.2. Biology
2.2.1. Cell culture
The mouse fibroblast cell lines 3T3 were maintained in Dulbecco’s Modified
Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM-glutamine,
and 40 µg/mL gentamicin, and cultured in a humidified atmosphere contained 5% CO2
incubator at 37 °C. For the experiments, cells were grown for 24 h in supplemented
medium in a 96-well cell culture plate. The assay was performed using cells between 3–
5 passages. In all experiments, untreated cells were used as negative controls.
2.2.2. Cell viability assay and treatment
The effect of naphthoquinone derivatives on cell viability was evaluated by the
MTT assay [22]. Naphthoquinone derivatives were dissolved in dimethyl sulfoxide
(DMSO) and then diluted with DMEM. Cells were plated in 96-well plates (6 ×
103/well) and treated with naphthoquinone derivatives at concentrations of 1, 5, 10, or
50 µM, for 24 h. Thereafter, the medium was replaced with fresh RPMI containing 5
mg/mL MTT. Following an incubation period (3 h) in a humidified CO2 incubator at 37

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°C and 5% CO2, the supernatant was removed and dimethyl sulfoxide solution (DMSO,
150 mL/well) was added to each cultured plate. After incubation at room temperature
for 15 min, the absorbance of the solubilized MTT formazan product was
spectrophotometrically measured at 540 nm. Three individual wells were assayed for
each treatment and the percentage viability relative to the control sample was
determined as (absorbance of treated cells/absorbance of untreated cells) × 100%.
2.2.3. In vitro scratch wound healing assay
To evaluate the effect of naphthoquinone derivatives on fibroblast motility, we
used the scratch assay as described by Herrera, Kantarci, Zarrough, Hasturk, Leung and
Van Dyke [23]. Cells were maintained in 24 well plates until they reached 90%
confluency. Thereafter, a vertical stripe on the cell monolayer was made using a sterile
pipette (200 µL) tip. The wells were washed with PBS to remove dead cells and debris,
and then naphthoquinone derivatives were added at a concentration of 10 µM. As a
control, the cells were treated with cell culture medium. After 24 h of treatment, cells
were photographed using an inverted microscope (Olympus IX70) with digital camera
aid to measure the wound closure area. Cell migration was analyzed using Image J
software and expressed as the area in pixels per field analyzed.
2.2.4. Animals
Experiments were carried out on adult male Swiss mice (25–35 g) obtained from
the Federal University of Alagoas (UFAL) breeding unit. The animals were maintained
with free access to food and water and kept at 22–28 °C with a controlled 12-h
light/dark cycle at the Institute of Biological and Health Sciences (UFAL). Experiments

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were performed during the light phase of the cycle. All experiments were carried out in
accordance with institutional guidelines and ethics (License Number 050/2013).
2.2.5. Induction of diabetes mellitus
Diabetes mellitus was induced in 12-h-fasted mice by an injection in the orbital
venous plexus of alloxan monohydrate (65 mg/kg) dissolved in sterile saline (0.9%,
NaCl) as described previously [24]. Control mice were injected only with saline. Blood
glycaemia of the samples obtained from the tail vein was determined using a glucose
monitor. Only mice with glucose levels above 200 mg/dL was considered to be diabetic,
and those animals were included in the experiment.
2.2.6. Excision wound model and treatment
Twenty-one days after alloxan injection, period which stabilization of diabetes
was ensured, animals were anesthetized using an intraperitoneal injection of a ketamine
(100 mg/kg) and xylazine (10 mg/kg) mixture. Thereafter, the dorsal region was shaved,
wiped topically with distilled water, and circular wounds were made using a template of
metal circle with a diameter of 1 cm. Animals were housed individually in disinfected
cages after recovery from anesthesia and maintained during experiments. Animals were
randomly divided into five groups of 5 animals: non-diabetic animals (NDB) treated
with 50 µl of PBS, diabetic animals (DB) treated with 50 µl of PBS, diabetic animals
(DB) treated with 50 µl of 2-bromo-1,4-naphthoquinone (S3, 50 µmol/Kg), diabetic
animals (DB) treated with 50 µl of 2-N-isonicotinoyl-hydrazide-1,4-naphthoquinone
(S6, 50 µmol/Kg), and diabetic animals (DB) treated with 50 µl of 1-N-isonicotinoyl-
hydrazone-[2-hydroxy-3-(3-methyl-2-butenyl)]-1,4-naphthoquinone (S7, 50 µmol/Kg).

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All animals received topical treatment on the wounds once daily until the end of the
experiments.
2.2.7. Wound closure measurements
Macroscopic evaluation of the wounds was performed using a digital camera, on
day 0 (before the start of treatment) and on 3th, 6th, 9th, and 12th days after injury, and the
data were analyzed using Adobe Photoshop CS5 software. The results of wound
measurements on various days were expressed as percentage of wound closure. The
wound closure percent was calculated using the following equation: [(A0 – AI)/A0 ×
100], where A0 is the initial wound area (day 0) and AI is the wound area on 3th, 6th, 9th,
or 12th after initial wound.
2.2.8. Histopathological analysis
The new full-thickness skin layer that was regenerated by 12 days post
wounding was removed using a surgical scalpel for histological analysis. Animals were
euthanized with thiopental sodium (100 mg/kg, i.p.) to collect granulation/healing
tissue. The skin specimens were fixed in 10% buffered formalin, processed, and blocked
with paraffin [25]. Then, sample were sectioned into 5-µm-thick sections and stained
with hematoxylin and eosin (H&E), as per the standard method and visualized under a
light microscope (Olympus BX51 attached DP70 Digital Camera System) at 10× and
40× magnification.
2.2.9. Statistical Analysis
Data were expressed as mean ± standard deviation. The statistical analysis
involving two groups was done using Student’s t-test. Analysis of variance followed by

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the Tukey’s test were used to compare three or more groups. Values of p < 0.05 were
considered as indicative of significance.

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3. RESULTS
3.1 Synthesis of naphthoquinones derivatives
The strategy for the synthesis of N-substituted-naphthoquinones S5, S6, and S7
is presented in Figure 2. All compounds were obtained in yields ranging from
satisfactory to excellent by nucleophilic attack of the most basic nitrogen directly into
the carbonyl group (C1) or into a carbon (C2) of the 1,4-naphthoquinone nucleus and
were characterized by adequate spectroscopic techniques, Figure 2.
3.2. FTIR spectra
In this study, FTIR data were used to identify the major functional groups
present in the compounds. In general, FTIR spectra of products showed the stretching
vibrations of OH, NH, and NH2 groups binding were observed in the 3440–3200 cm-1
region. In the starting material 1,4-naphthoquinone, lawsone or lapachol the bands of
two carbonyl groups ν(1C=O) and ν(4C=O) and stretching vibrations of C–O were
observed in the 1660–1640 cm-1 and 1224–1047 cm-1 regions, respectively. In the S3
derivative, the stretching vibrations of C–Br was observed at 690 cm-1. For the 2-N-
substituted-naphthoquinones S5 and S6, the bands of the carbonyl groups ν(1C=O) and
ν(4C=O) were observed in the 1673-1660 cm-1 region, while the stretching vibrations of
C-N appeared in the 1670–1343 cm-1 region. For S7, a 1-N-acylhydrazone, was
observed the appearance of an absorption band which can be attributed to the formation
of the C=N bond was observed. The ν(1C=N) was observed in the 1681–1545 cm-1
region.
3.3. 1H NRM spectra

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1H NMR spectroscopic analysis of the compounds S3-S7 showed signals
concerning the aromatic hydrogens of the naphthalene ring system between δ 8.1-7.8
ppm as doublets and triplets. The singlet corresponding to the hydrogen CH (H3),
proton neighboring the bromo group appears at δ 7.5 ppm. In S5, this hydrogen,
neighboring the aniline group, was observed at δ 6.4 ppm and in S6 the proton (H3)
appears at 5.7 ppm. The chemical shift of this proton can be explained in terms of the
presence of electron donor or electron withdrawal groups connected to the conjugate
system. For S7, this signal is absent due to the presence of the isoprenyl side-chain at
C3. Regarding the isonicotinoyl hydrazide group present in S6 and S7, the four pyridyl
protons could be assigned at 8.8 and 7.8 ppm, the two ortho protons with respect to
nitrogen (H19 and H21, Figure 2) are largely deshielded. In S6, the two singlets
observed at approximately 9.6 and 11.0 ppm are due to the N(1')H and N(2')H groups
(Figure 2). In addition, the presence of the N(1')H chemical shift at 11.0 ppm in S6 was
indicative of conformation in which N(1')H is hydrogen bonded to the carbonyl oxygen
(1C=O).
1
The comparison of H NMR spectra between the starting material and the S7
derivative indicated that the signals from the side-chain at C3 were virtually unchanged.
The presence of a largely deshielded signal at 16.4 ppm in the S7 spectra was indicative
of conformation in which N(2')H is hydrogen bonded to the carbonyl oxygen (2C=O). It
is expected that S7 will adopt the E or Z conformation in solution. In this type of
reaction, E- and Z-acylhydrazones are in equilibrium through the intermediate
1
hemiaminal (Figure 3). In this study, H NMR data indicated that only one of two
possible diastereoisomers was formed with the Z-stereochemistry for the C=N double
bond in S7. Z-stereochemistry was established by the intramolecular N(2')–H O=C six-
membered hydrogen-bonded ring.

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3.4. 13C NMR spectra
13C NMR spectra of S5 and S6 showed the two carbonyl groups (C=O) between
δ 183.7-181.8 ppm, which demonstrates that the 1,4-naphthoquinone nucleus was
preserved and the substitution of the nitrogenous group occurred at the C2 carbon. In
addition, in S5 and S6 the C=C-NH was observed at δ 144.7 and 148.7 ppm,
respectively. The aromatic carbons of S5 were observed between δ 134.8-122.1 ppm. In
the 13C NMR spectra of S7, an 1-N-acylhydrazone, the signals at δ 179.6 and 166.4 ppm
4
were attributed to the carbonyl (2C=O) and C-OH groups, respectively. These signals
result from the structural modification of the naphthoquinone nucleus that occurred after
the insertion of the isonicotinoyl hydrazide group. The signal of the carbon in the
hydrazone group (1C=NNCOR) was observed at δ 131.0 ppm. The four carbons of the
pyridyl groups in S6 and S7 were observed at δ 150.8 and 121.9 ppm, as well as a
carbonyl group of amide at δ 164.1 and 159.9 ppm, respectively. The COSY and
HMBC spectra were used to confirm the structures and unambiguously assign the
chemical shifts for all the hydrogen and carbon atoms.
3.5. Effects of naphthoquinone derivatives on cell viability
Because naphthoquinone derivatives could have toxic activities, we first
evaluated the cytotoxicity of these compounds using an MTT assay. As shown in Figure
4, there was a significant reduction in the viability of 3T3 cells after treatment with 1,4-
naphthoquinone S1 and lapachol S4, but not with lawsone S2. Moreover, it should be
noted that the naphthoquinone derivatives S3, S5, S6, and S7 did not present a

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significant decrease in cell viability at the tested doses. On the basis of these results, we
selected the compounds S3, S5, S6, and S7 to proceed to the next step and evaluate their
respective effects on cell migration.
3.6. Effects of naphthoquinone derivative exposure on fibroblast motility
The effect of naphthoquinone derivatives on migratory activity was performed
with 3T3 mouse fibroblast cells using a scratch assay. Scratches were made on
confluent cells and then treated with RPMI-medium (control) or naphthoquinone
derivatives at a concentration of 10 µM for 24 h. As shown in Figure 5, the treatment of
fibroblasts for 24 h with the naphthoquinone derivatives S3 and S6 accelerated the
wound closure area by approximately 56% and 68%, respectively. However, cells
treated with naphthoquinone derivatives S5 and S7 exhibited a profile of wound closure
area similar to the untreated cells (control), indicating no effect of this derivative on the
migratory capacity of fibroblasts. On the basis of these results, we evaluated the effects
of these derivatives on an in vivo wound-healing model. It is important to emphasize
that, although the derivative S7 did not show an effect on fibroblasts in the scratch
assay, we decided include this derivative in the in vivo test due to its structural
similarity to derivative S6 (isonicotinoyl group) and the fact that S7 has not previously
been described in the literature.
3.7. Effects of naphthoquinone derivatives on in vivo wound healing assay
Here, the excision wound model was used in order to assess healing activity of
the naphthoquinone derivatives S3, S6, and S7 in mice with diabetes induced with
alloxan. The progression of healing was evaluated on days 3, 6, 9, and 12 post
wounding.

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As shown in Figure 6A and 6B, the diabetic mice (DB) presented a significant
delay in wound closure kinetics compared to the non-diabetic mice (NDB) at all times
analyzed. When the alloxan-induced diabetic mice were topically treated with the
naphthoquinone derivatives S3, S6 or S7, an improvement in wound closure compared
to the PBS-treated diabetic animals was observed on 6th, 9th, and 12th days post
wounding (Figure 6A and B). Notably, treatment with S3, S6 or S7 presented a similar
effect on diabetic wound closure on day 6 with 36.7%, 35.1%, and 32.7% wound
closure, respectively (Figure 6B). However, on day 9 improved healing effects were
produced by naphthoquinone derivatives S3 and S6, with 82.7% and 78.1% wound
closure, respectively. The S7-treated wound had a wound closure rate of only 61% on
the 9th day.
We further examined the effects of naphthoquinone derivatives on tissue
regeneration in wounds. Randomly selected photographs of wound tissue on day 12 of
non-diabetic (NDB) and diabetic (DB) mice treated with PBS, and diabetic mice treated
with S3 (DB+S3), S6 (DB+S6), or S7 (DB+S7) are shown in Figure 7. Skin sections
from non-diabetic animals (NDB) showed an intense epithelialization at 12 days post
wounding, accompanied by a layer of keratinocytes and mature granulation tissue with
blood vessels and cells showing a fibroblast-like morphology (Figure 7, NDB). In
diabetic mice (DB), the epithelialization was not complete, with an intense
accumulation of inflammatory cells and a thin fragile layer of epithelium and
keratinocytes (Figure 7, DB). The histological profile of skin from diabetic animals
treated with the compounds S3 or S6 brought an increase in the number of layers of
epithelialization, accompanied by a reduction in the accumulation of inflammatory cells
and the presence of fibroblast-like cells (Figure 7, DB+S3 and DB+S6). In wounds on
diabetic mice treated with S7, despite the improvement in wound closure revealed by a

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thin layer of epithelization, absence of fibroblast-like cells and diffuse accumulation of
inflammatory cells were still observed (Figure 7, DB+S7).

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4. DISCUSSION
Delayed skin wound healing in diabetes remains as an important clinical
problem, leading to prolonged hospitalization and even amputation. Consequently,
obtaining chemical entities by synthesis or semi-synthesis remains as effective way to
obtain new drugs with healing effects. In this study, new derivatives from 1,4-
naphthoquinones were synthetized and characterized and their biological effects
evaluated on an in vitro model of cell migration (scratch assay) and on wound healing in
alloxan-induced diabetic mice. Our results provide the first evidence that two of the
naphthoquinone derivatives evaluated in this study (S3 and S6) act directly on
fibroblasts by increasing their migratory activity in vitro. Moreover, we also
demonstrated that naphthoquinone derivatives S3, S6, and S7 effectively promote
diabetic wound closure.
Indeed, 1,4-naphthoquinones are widely distributed in nature. They are
structurally related to naphthalene and characterized by their two carbonyl groups in the
1,4 positions, leading to the name 1,4-naphthoquinones. Naphthoquinones are highly
reactive organic compounds and have been shown to have important pharmacological
properties, such as antimalarial, antibacterial, antifungal, and anticancer properties [12,
13]. However, despite this wide range of effects, studies on the effects of non-natural
naphthoquinone derivatives in wound healing remain scarce. Only a small number of
natural naphthoquinones, such as alkannin and shikonin, have been studied and
demonstrated wound healing properties [26, 27].
Here we describe a method for the synthesis of N-substituted-naphthoquinones.
The reaction of 1,4-naphthoquinone S1 with aniline furnished 2-N-phenylamino-1,4-
naphthoquinone, derivative S5. The reaction of 2-hydroxy-1,4-naphthoquinone S2 with
N-acylhydrazide (isoniazide) in an acid medium led to 2-N-acylhydrazino-1,4-

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naphthoquinone, derivative S6. In contrast, the reaction in a weakly alkaline solution led
to 1-N-acylhydrazone-2-hydroxy-1,4-naphthoquinone, derivative S7. In this case, the
formation of the conjugate base in alkaline solution was proposed to explain this
behavior. The findings of the present study are in agreement with those reported by
Carroll et al. [28] and Campos et al. [29].
The chemical reactivity of the 1,4-naphthoquinone nucleus is an example of an
α,β-unsaturated substance. Due to this characteristic, they have ambident behavior when
reacting with nucleophiles, being able to undergo β-olefinic attack (1,4 addition) or
direct addition to the carbonyl (1,2-addition). The preferred position of the addition will
depend on the nature of the nucleophile and the reaction conditions [30].
Naphthoquinones are involved in oxidative processes due to their structural
properties and their capacity to generate reactive oxygen species [31]. Moreover, the
mechanism of action and pharmacological properties of these substances are dependent
on their acid-base and oxide-reduction properties, which can be modulated by the
structural modification of the quinone nucleus by the introduction of various
substituents.
Previous studies showed that naphthoquinone derivatives are potentially toxic
[32], and several studies have shown that changes in the structure of molecules cause
reduction in its cytotoxicity [33], improve its biological effects [34], or even reveal new
activities. Notably, conjugation of naphthoquinones with gold nanoparticles can reduce
the cytotoxicity of these compounds [35]. Our results in the cytotoxicity assay showed
that derivatives, in particular S3, S5, S6, and S7, have low toxicity compared to 1,4-
naphthoquinone. This low cytotoxicity of the compounds might be explained by a
possible change in solubility of molecules; however, further studies are required to
clarify this aspect.

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Fibroblasts are important cells in normal wound healing; they produce
extracellular matrix and collagen structures that support the other cells associated with
effective wound healing [36]. In addition, the ability of fibroblasts to migrate is an
extremely important feature during the granulation tissue formation phase of wound
healing [37]. One way of assessing the interference of compounds on cell migration is
the scratch assay. This method is a simple and direct way to measure the rate of cell
migration in an in vitro wound model. Once the cell monolayer is disrupted, the loss of
cell-cell interaction induces secretion of growth factors and cytokines at the wound
edge, initiating migration and proliferation of cells [38].
In the present study, fibroblasts treated with naphthoquinone derivatives S3 and
S6 induced an enhanced migration capacity leading to improved closure of the scratched
area, suggesting that these derivatives have a direct effect on fibroblasts. These results
are consistent with a previous study in which another naphthoquinone derivative, 2,3-
dimethoxy-1,4-naphthoquinone, significantly improved fibroblast motility,
a
phenomenon that involved the lysophosphatidic acid signaling pathway [39]. In
addition, β-lapachone, a natural o-naphthoquinone, exhibited an inductor effect on the
migration of mouse 3T3 fibroblasts through different MAPK signaling pathways [40].
Thus, these observations support the notion that derivatives S3 and S6 may directly
affect fibroblast migration; however, future studies are required to determine the
molecular mechanisms underlying the effects of these derivatives.
Diabetes is associated with altered skin wound healing [3, 4]. This deficiency in
healing may be associated with a persistence of inflammatory cells in the wound area,
which causes tissue damage and impairs wound resolution [41, 42]. As already
demonstrated, hyperglycemia and oxidative stress induce excessive proinflammatory
cytokine production, sustaining an infiltration of leukocytes in injured tissue from

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diabetic mice [43-45]. As shown in the present study, diabetic wounds still contained a
large number of inflammatory cells infiltrated into tissue on day 12 after wounding,
while non-diabetic wounds already exhibited an advanced stage of healing. Topical
application of naphthoquinone derivatives S3 and S6 significantly attenuated the
infiltration of inflammatory cells, which suggests these compounds might provide a
favorable microenvironment for wound healing. The concept that attenuating the
inflammatory response can accelerate diabetic cutaneous wound healing has previously
been demonstrated using other topical treatments [46]. Considering this proposal, we
cannot dismiss the effect of these naphthoquinone derivatives in accelerating each phase
of the healing process, which would result in faster closure of wounds. In addition,
previous reports have demonstrated that arnebin-1, a naphthoquinone derivative,
accelerates the wound closure process in distinct models of delayed healing, due to the
induction of expression of eNOS, VEGF, and HIF-1α [47, 48]. Thus, it is possible that
these mediators also play a role in the naphthoquinone derivative-mediated wound
healing process. Although the derivative S7 did not show a direct effect on fibroblast
migration in vitro, when topically applied to a wound in alloxan-induced diabetic mice,
this derivative promoted an improvement in skin wound closure. However, microscopic
analysis revealed a weak reduction in the accumulation of inflammatory cells. These
differentiated effects induced by the naphthoquinone derivatives could be attributed to
the actions at distinct targets at the injury site. Therefore, although speculative, this idea
cannot be dismissed. Further studies are required to investigate this concept.
5. CONCLUSION
Based on the outcomes of the present study, we demonstrated that
naphthoquinone derivatives S3 and S6, but not S7, improve fibroblast migration in

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vitro. However, the topical application of S3, S6, and S7 accelerated the wound healing
process in alloxan-induced diabetic mice. Taken together, we conclude that
naphthoquinone derivatives may have potential as a healing-promoting agent for wound
healing, particularly under diabetic conditions.

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ACKNOWLEDGEMENTS
The authors are grateful to the research team at the Laboratory of Cell Biology and
LaSOM for helping them with some experimental protocols. This study was supported
by the Brazilian Ministry of Health (Science and Technology Department -
Decit/SCTIE/MS-Brasil) and Fundação de Amparo à Pesquisa do Estado de
Alagoas/SUS
Research
Program
(FAPEAL/PPSUS)
[grant
number
60030000712/2013]. Authors thank Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq/Brazil) by the scholarships.

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Figure 1. Chemical structures of natural naphthoquinone derivatives.
Figure 2. Strategy of synthesis of N-substituted-1,4-naphthoquinones S5, S6 and S7.
Figure 3. Equilibrium of E and Z-hydrazones through intermediate hemiaminals.
Figure 4. The viability of 3T3 cells after 24 h naphthoquinone derivatives exposure.
The dotted line represents the control group (cells treated with DMEM) and bars
represent the mean ± S.D. One-way ANOVA was used with Tukey test, where a p-value
< 0.05 was deemed statistically significant. *p < 0.05 and ***p < 0.001 vs control
group.
Figure 5. Effect of naphthoquinone derivatives in closure of scratched areas in the
scratch assay. Representative photomicrographs of the wounded cells at 24 h after the
scratch, and quantitative analysis of area closured in scratch assay expressed as
percentage of initial area. Bars represent mean ± S.D. One-way ANOVA was used with
Tukey test, where a p-value < 0.05 was deemed statistically significant. *p < 0.05 and
**p < 0.01 vs control group.
Figure 6. Effect of topical application of naphthoquinone derivatives in the excisional
wounds in diabetic mice. (A) Representative photographs of wounds at days post
wounding of non-diabetic animals (NDB), diabetic animals (DB), diabetic animals
treated with S6 (DB+S6), and diabetic animals treated with S3 (DB+S3). (B) Wound
closure kinetics. Bars represent mean ± S.D. One-way ANOVA was used with Tukey