LQFM184: A Novel Wide Ultraviolet Radiation Range Absorber Compound

Article abstract of DOI:10.1111/php.13349

The use of sunscreen has become an indispensable daily routine since UV radiation is a critical environmental stress factors for human skin. This study focused on the design, synthesis, thermal/chemical stability and efficacy/safety evaluations of a new h

Full text of DOI:10.1111/php.13349

DR. MARIZE VALADARES (Orcid ID : 0000-0002-0379-1325)
Article type : Research Article
LQFM184: A Novel Wide Ultraviolet Radiation Range Absorber
Compound
Daniela C. Vinhal1, Renato Ivan de Ávila2, Thaisângela L. Rodrigues2, Andressa K. Silva3,
Larissa C. Moreira2, Marize C. Valadares2*, Rangel M. Luzin3, Luciano M. Lião3, Eric de S.
Gil4, Boniek G. Vaz5, Rogério J. Assis6, Pablo J. Gonçalves6, Vera Isaac7, Luiz C. da Cunha1,
Ricardo Menegatti8*
1Núcleo de Estudos e Pesquisas Tóxico-Farmacológicas (NEPET), Faculdade de Farmácia,
Universidade Federal de Goiás, Goiânia, GO, Brazil.
2Laboratório de Ensino e Pesquisa em Toxicologia In Vitro (Tox In), Faculdade de Farmácia,
Universidade Federal de Goiás, Goiânia, GO, Brazil.
3Laboratório de Ressonância Magnética Nuclear, Instituto de Química, Universidade Federal
de Goiás, Goiânia, GO, Brazil.
4Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brazil.
5Laboratório de Cromatografia e Espectrometria de Massas, Universidade Federal de Goiás,
Goiânia, GO, Brazil.
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences
between this version and the Version of Record. Please cite this article as doi: 10.1111/php.13349
This article is protected by copyright. All rights reserved

6Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil.
7Laboratório de Cosmetologia (LaCos), Departamento de Fármacos e Medicamentos,
Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista Júlio de Mesquita Filho,
Araraquara, SP, Brazil.
8Laboratório de Química Farmacêutica Medicinal (LQFM), Faculdade de Farmácia,
Universidade Federal de Goiás, Goiânia, GO, Brazil.
*Corresponding authors email: rm_rj@yahoo.com, (Ricardo Menegatti) marizecv@ufg.br
(Marize C. Valadares)
This article is protected by copyright. All rights reserved

ABSTRACT
The use of sunscreen has become an indispensable daily routine since UV radiation is a critical
environmental stress factors for human skin. This study focused on the design, synthesis,
thermal/chemical stability and efficacy/safety evaluations of a new heterocyclic derivative, namely
LQFM184, as a photoprotective agent. The compound showed stability when submitted under
oxidative and high temperature conditions. It also revealed an absorption at 260-340 nm (UVA/UVB),
with a main band at 298 nm and a shoulder close to 334 nm. LQFM184 showed capacity to interact
with other existing UV filters, promoting an increase in the sun protection factor. In relation to acute
toxicity, its estimated LD50 was > 300-2000 mg/kg, probably with a low potential of inducing acute
oral systemic toxicity hazard. In addition, our data showed that this compound did not have eye
irritation, skin sensitization or phototoxicity potentials. Taken together, these findings make
LQFM184 a promising ingredient to be used, alone or in association with other UV filters, in cosmetic
products such as sunscreens with a broad-spectrum of protection.
Keywords: Heterocyclic derivative; Photoprotection; Sunscreen; Green chemistry; UV filter.
This article is protected by copyright. All rights reserved

INTRODUCTION
Solar ultraviolet radiation (UVR), mainly consisted of UVA (320-400 nm) and UVB (290-320 nm),
achieves the Earth's surface and can promote beneficial effects to human skin health, such as
induction of vitamin D production [1-3]. However, excessive sun exposure can also trigger several
harmful clinical consequences, including skin photodamage (e.g. sunburn, premature skin aging),
immunosuppression and malignant melanoma [1, 2]. These effects have been increased along with the
depletion of the ozone layer and outdoor lifestyle changes [4, 5]. Concerns regarding UVR exposure
promoted the development of photoprotective cosmetic products, whose sunscreens have been used as
the first choice for protection against excessive solar exposure-induced skin damages [1, 2].
UV filters are classified as organic (e.g. octyl methoxycinnamate, benophenone-3, octocrylene) or
inorganic (e.g. zinc oxide, titanium dioxide), based on their composition and mechanism of action [1,
2]. Combination of sunscreen filters is commonly used for increasing the broad-spectrum of
protection of the photoprotective cosmetic products [1].
It has been documented in the literature that some sunscreen agents, such as benzophenones (BPs), are
able to penetrate the skin and trigger toxicity [6]. Vela-Soria and co-workers developed a method for
identifying the presence of BPs in 16 placental tissue samples [7]. To overcome this issue, new agents
have been developed, such as bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT) and
methylene bis-benzotriazolyl tetra methyl butyl phenol (MBBT). New sunscreens are considered ideal
when are designed to show broad-spectrum, increased photostability, innocuity and solubility in
cosmetic oils, with reduced capacity to overcome the skin barriers as well as with minimal
toxicological effects, in particular to skin and eyes [8-11]. In this sense, we previously demonstrated
that the new molecule LQFM048 showed useful photoprotective and antioxidant properties for the
development of a new sunscreen product without potential adverse effects related to eye irritation and
skin allergy, for instance [12, 13].
This article is protected by copyright. All rights reserved

Given the above, this study focused on the design, synthesis, thermal/chemical stability and efficacy
evaluations of a new heterocyclic derivative, namely LQFM184 (2) [(2,4,6-tris ((E)-2-cyano-3-(4-
hydroxy-3-methoxyphenyl)acrylamide)-1,3,5-triazine)] (Fig. 1), to be used as a potential new
photoprotective agent in cosmetic products. This compound was originally designed through
bioisosterism of functional group strategy, via green chemistry synthetic approach, using the
LQFM048 (1) as lead compound [12]. In addition, acute toxicological endpoints were investigated to
establish the toxicity profile of LQFM184 (2).
<Figure 1>
MATERIALS AND METHODS
Chemicals
Tetrahydrofuran (THF) was purchased from Tedia Brazil (Rio de Janeiro, RJ, Brazil). Vanillin,
malonolitrine, cianeacetamide, 2,4,6-trichloro-1,3,5-triazine and morpholine were obtained from
Acros Organics (Geel, Belgium). Deuterated chloroform (CDCl3) was acquired from Cambridge
Isotope Laboratories Inc (Tewksbury, MA, USA). Methanol, ethyl acetate, ethanol, dimethyl sulfoxide
(DMSO), chloroform, dichloromethane, acetic acid, hydrochloric acid (HCl), sodium chloride (NaCl),
dichloromethane, hexane and calcium chloride (CaCl2), potassium phosphate and acetate were
acquired from Vetec (Rio de Janeiro, RJ, Brazil), whereas ethylhexyl methoxycinnamate, silica gel 7
60 and vanillin from Merck (Darmstadt, HE, Germany). Tinosorb® S was acquired from BASF
(Ludwigshafen, Germany). Potassium carbonate (K2CO3) was purchased from Synth (SP, Brazil).
Graphite was purchased from Metrohm Autolab B.V. (Kanaalweg, Utrecht, The Netherlands),
whereas mineral oil was acquired from Biolub Chemistry LTDA (Sorocaba, SP, Brazil). Analytical
grade methanol was supplied by J. T. Baker (Phillipsburg, NJ, USA). All electrolyte solutions were
prepared using analytical grade reagents and double distilled water. Sodium fluorescein,
benzalkonium chloride, Hanks’ balanced salt solution (HBSS), Eagle's Minimum Essential Medium
(EMEM) with or without phenol red, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), Roswell Park Memorial Institute (RPMI)-1640 medium, HEPES, sodium bicarbonate,
This article is protected by copyright. All rights reserved

penicillin, streptomycin, amiodarone HCL, p-phenylenediamine (PPD), 1-chloro-2,4-dinitrobenzene
(DNCB), protease inhibitor cocktail, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
(MTT), propidium iodide (PI), neural red, bicinchoninic acid protein assay kit and bovine serum
albumin were acquired from Sigma-Aldrich (St. Louis, MO, USA). Triton X-100 was obtained from
Amresco (OH, USA). Human IL-18 Platinum ELISA was purchased from eBioscience (Vienna,
Austria), whereas BD OptEIATM human IL-8 ELISA set kit, FITC-conjugated anti-human CD86
(FUN-1) monoclonal antibody and FITC-isotype control IgG1 (MOPC-21) were acquired from BD
Biosciences (San Jose, CA, USA). TRIzol reagent was obtained from Invitrogen (Grand Island, NY,
USA). SsoAdvancedTM Universal SYBR Green Supermix and iScriptTM gDNA Clear Synthesis kit
were acquired from Bio-Rad (Hercules, CA, USA).
General
The 1H and 13C NMR experiments were performed at 25 °C using a Bruker Avance III 500
spectrometer operating at 11.75 T, observing 1H at 500.13 MHz. The spectrometer was equipped with
a 5 mm inverse detection four-channel (1H, 2H, 13C and X-nucleus) TBI probe. For each analysis, 20
mg of sample were dissolved in 500 μL of DMSO-d6 and tetramethylsilane (TMS) as the internal
standard. The mass data were obtained using a QTOF Micro mass spectrometer equipped with an ESI
source (Waters, Manchester, UK). The parameters of the mass spectrometer used were the following:
nebulization gas (500 L/h) at 140°C, cone gas set to 50 L/h, and source temperature set to 100°C.
Capillary and cone voltage were set to 4500 V and 25 V, respectively. QTOF acquisition rate was set
to 1.0 s, with a 0.4 s inter-scan delay and the data processed on MassLynx 4.0 software (Waters,
Manchester, UK). Analytes were acquired using LockSpray and phosphoric acid (0.1% in
acetonitrile/water, 1:1) as internal standard to ensure accuracy mass. The analyses were performed by
direct infusion with a syringe pump at 5.0 μL/min flow ratio. Infrared spectra were acquired using a
Nicolet-55a Magna spectrophotometer (GMI, MN, USA) with potassium bromide plates.
Synthesis
Synthesis of 2-(4-hydroxy-3-methoxybenzylidene)malononitrile (5) [13]
This article is protected by copyright. All rights reserved

Vanillin (3) (500 mg, 3.30 mmol) and malononitrine (4) (218 mg, 3.30 mmol) were diluted in water (5
mL) at room temperature. Morpholine (5 mol %) was then added to reaction, by agitation for 1 h. The
residue was divided between water and CH2Cl2, the phases were separated and the aqueous layer was
extracted into 3 x 15 mL with CH2Cl2. The organic layers were dried (Na2SO4), concentrated in vacuo,
and the rough product used without any purification. The derivative (5) (607 mg, 92%) was obtained
as a yellow solid, mp 131ºC, Rf = 0.90 CH2Cl2: MeOH (95:5, v/v); IR max (KBr) cm-1: 3400-3300 (
O-H), 2951 ( C-H), 2236 ( CN) and 2220 ( CN) (Figure. S1, Supporting Information); RMN 1H
(500 MHz) DMSO-d6 (δ): 10.82 (1H, s, OH);8.27 (1H, s, H-3); 7.63 (1H, d, J = 2.10 Hz, H-2’); 7.50
(1H, dd, J = 2.10 and 8.40 Hz, H-6’); 6.98 (1H, d, J = 8.40 Hz, H-5’); 3.81 (3H, s, OCH3) (Figure S2
and Table S1, Supporting Information); 13C (125 MHz) DMSO-d6 (δ): 160.6 (1C, C-3); 153.7 (1C, C-
4’); 147.8 (1C, C-3’); 127.7 (1C, C-6’); 123.1 (1C, C-1’); 116.2 (1C, C-5’); 115.2 (2C, C-1 and 4);
113.1(1C, C-2’); 74.8 (1C, C-2); 55.5 (1C, OCH3) (Figures S3, S4 and Table S1,Supporting
Information); [M-H]- m/z of 199.05122 (Figure S5, Supporting Information).
Synthesis of (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylamide (6) [14]
2-(4-hydroxy-3-methoxybenzylidene) malononitrile (5) (1.0 mmol, 200 mg) and Cu
(OAc)2·H2O (20 mg, 0.1 mmol) H2O (0.2 mL) in HOAc (10.0 mL) were stirred for 7 h at 80°C. Then,
the mixture was separated in water and CH2Cl2, the phases were divided and the aqueous layer was
extracted into 3 x 15 mL with CH2Cl2. The organic layers were then dried (Na2SO4), concentrated in
vacuo and the mixture was used without additional steps. The resulting product (6) (202 mg, 93%)
was a yellow solid, mp 205ºC, Rf = 0.50 CH2Cl2: MeOH (95:5, v/v) (7:3, v/v); IR max (KBr) cm-1:
3474 ( N-H), 3300 ( N-H), 2211 ( CN) and 1683 ( C=O) (Figure S6, Supporting Information);
RMN 1H (500 MHz) DMSO-d6 (δ): 10.26 (1H, s, H-4’); 8.06 (1H, s, H-3); 7,66 (1H, d, J = 2.10 Hz,
H- 2’); 7.46 (1H, dd, J = 2.10 and 8.40 Hz, H-6’); 6.94 (1H, d, J = 8.40 Hz, H-5’); 3.81 (3H, s, OCH3)
(Figure S7 and Table S2, Supporting Information); 13C (125 MHz) DMSO-d6 (δ): 163.2 (1C, C-1);
151.2 (1C, C-4’); 150.7 (1C, C-3); 147.7 (1C, C-3’); 125.8 (1C, C-6’); 123.2 (1C, C-1’); 117.3 (1C,
C-4); 115.9 (1C, C-5’); 113.1 (1C, C-2’); 101.5 (1C, C-2); 55.2 (1C, OCH3) (Figures S8, S9 and
Table S2, Supporting Information); [M-H]- m/z of 217.06177 (Figure S10, Supporting Information).
This article is protected by copyright. All rights reserved

Synthesis of (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylamide (6) [15]
Vanillin (3) (152 mg, 1.00 mmol) and cyanoacetamide (7) (84 mg, 1.00 mmol) were mixed for 1 h at
75 ºC. After, 20 mL of water was added and the formed precipitate was filtered off under vacuum. The
derivative material (6) (174 mg, 80%) was used without rather purification and showed the following
parameters: yellow solid, mp 205ºC, Rf = 0.50 CH2Cl2: MeOH (95:5, v/v) (7:3, v/v).
Synthesis of 2,4,6-tris ((E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylamide)-1,3,5-triazine (2)
[16, 17]
(E)-ethyl 2-cyano-3-(4hydroxy-3-methoxyphenyl)acrylate (6) (763 mg, 3.50 mmol), Na2CO3 (370 mg,
3.50 mmol), 2,4,6-trichloro-1,3,5-triazine (8) (184 mg, 1.00 mmol) and acetonitrile (5.0 mL) were
mixed at 80ºC for 24 h to obtain a heterogeneous mixture. The mixture was split in aqueous and
organic phases (CH2Cl2). Then, the aqueous layer was extracted with CH2Cl2 and the combined
organic layers were dried (Na2SO4) and concentrated using vacuo. The crude product was solubilized
in 5.0 mL of DMF and poured into 50.0 mL of AcOH/H2O (1:9, v/v). LQFM184 (2) (452 mg, 62%)
was obtained as a beige solid, mp 147º C, Rf = 0.20 CH2Cl2: MeOH (95:5, v/v); IR max (KBr) cm-1:
3290 ( N-H), 3275 ( N-H), 2937 ( C-H) and 1692 ( C=O) (Figure S11, Supporting Information);
RMN 1H(500 MHz) DMSO-d6 (δ): 8.19 (1H, s, H-3); 7,71 (1H, d, J = 1.90 Hz, H-2’); 7.57 (1H, dd, J
= 1.90 and 8.40 Hz, H-6’); 7.43 (1H, d, J = 8.40 Hz, H-5’); 3.79 (3H, s, OCH3) (Figure S12 and Table
S3, Supporting Information); 13C (125 MHz) DMSO-d6 (δ): 172.8 (3C, C-2’’); 162.5 (1C, C-1); 150.8
(1C, C-3’); 149.9 (1C, C-3); 142.4 (1C, C-4’); 131.2 (1C, C-1’); 123.2 (1C, C-5’); 123.1 (1C, C-6’);
116.5 (1C, C-4); 114.3 (1C, C-2’); 106.9 (1C, C-2); 56.0 (1C, OCH3) (Figures S13, S14, 15 and Table
S3, Supporting Information); [M+H]+ m/z of 730.20082 (Figure S16, Supporting Information).
Cyclic voltammetry analysis
Voltammetric analyses of the LQFM184 (2) were made using a potentiostat/galvanostat (µAutolab
III®), controlled by the GPES 4.9® software (Eco-Chemie, Utrecht, The Netherlands). The electrodes
consisted of a carbon paste working electrode, a Pt wire counter electrode and an Ag/AgCl/KCl 3M as
the reference electrode. Pulse amplitude (50 mV) and width (0.5 s), associated with a scan rate 10 mV
This article is protected by copyright. All rights reserved

s-1, were set up for differential pulse voltammetry (DPV). The experiments were performed in
triplicate using a one-compartment electrochemical cell.
Thermogravimetric analysis
Regarding thermal stability of LQFM184 (2), it was evaluated using a DTG-60H thermobalance
(Shimadzu, MD, USA). A sample of the compound (4 mg) was evaluated by heating process from 30
to 600°C (flow rate of 50 mL min−1 and a heating rate of 10°C/min) under synthetic air atmosphere.
Photophysical characterization
Photophysical data of LQFM184 (2) were obtained through UV-Vis spectra, obtained in a double
beam spectrophotometer model U-2900 (Hitachi High-Technologies, Tokyo, Japan). The fluorescence
spectra were obtained using a fluorescence spectrophotometer F-7000 (Hitachi High-Technologies).
Evaluation of Sun Protection Factor (SPF)
For SPF determination, O/W emulsions containing LQFM184 (2) were prepared [18]. The
measurements of the SPF were made using Optometrics SPF 290S (Laser Components, Olching,
Germany) following the protocol recommended by the manufacturer [19, 20]. In Transpore® tapes (50
cm²) (3M, MN, USA) placed directly on the support, 110 mg of the sample were applied, forming a
uniform film of 2 mg/cm2. After application, the tapes with the samples were dried for 15 min. The
support was then introduced directly into the equipment, and the readings were performed by the
emission of ultraviolet radiation generated through a xenon lamp with a wavelength in the range of
290 to 400nm.
In vitro toxicological assessment
Cell cultures
Cell lineages (Balb/c 3T3-A31 fibroblasts, HaCaT human immortalized keratinocytes, SIRC
rabbit corneal and U937 human histiocytic lymphoma cells) used in this work were purchased from
the Rio de Janeiro Cell Bank (Rio de Janeiro, RJ, Brazil). 3T3, HaCaT or SIRC cells were cultured in
complete DMEM and the U937 cells were cultured in RPMI medium. In addition, cell medium was
This article is protected by copyright. All rights reserved

supplemented with 10% (v/v) heat-inactivated FBS, HEPES (4.5 mM), sodium bicarbonate (170
mM), penicillin (100 IU/mL) and streptomycin (100 mg/mL) under controlled conditions (humidified
atmosphere of 5% CO2 in air at 37ºC).
Estimation of acute systemic toxicity: lethal dose (LD50)
The investigation of the acute toxicity potential of LQFM184 (2) was performed by the estimation of
LD50 through 3T3 neutral red uptake (NRU) assay, according to the protocol prior described by
Borenfreund and Puerner [21] and modified by ICCVAM [22]. 3T3 cells were seeded in 96-well
plates at 0.5 × 105 cells/mL. After that, cells were exposed to LQFM184 (2) (1.6-200 µg/mL) or its
vehicle (DMSO 0.4%, v/v) for 48 h of incubation. Then, 100 μL/well of NR (0.25 mg/mL), diluted in
DMEM containing FBS (5%, v/v), was added and incubated for 3 h. Cells were then washed with pre-
warmed PBS and NR desorb (50 ethanol: 1 acetic acid: 49 ultrapure water) added in each well.
Absorbance was measured at 550 nm (Multiskan Spectrum spectrophotometer, Thermo Scientific,
Waltham, MA, USA). Cell viability was expressed as a percentage of the control and IC50 value
(concentration that inhibited cell growth by 50% compared to untreated group) was calculated. The
LD50 was then estimated using the model equation [Log (LD50) = 0.545 x log (IC50, mM) + 0.757]
[23]. The probable acute oral systemic toxicity classification of LQFM184 (2) was then done based on
the estimated LD50 and in accordance with the United Nations Globally Harmonized System of
Classification and Labeling of Chemicals (UN GSH) [24].
Short time exposure (STE) assay
The STE assay was conducted according to the OECD test guideline Nº 491 [25]. Briefly, SIRC cells
were seeded in plates at 3 × 103 cells/well followed by 5-day incubation. After that, cells were then
exposed to LQFM184 (2) (at 5 or 0.05%, w/v) for 5 min at room temperature or exposed to saline,
LQFM184 vehicle (5% DMSO in saline) or benzalkonium chloride (at 5 and 0.05%, w/v) diluted in
saline. Then, after washing the cells, MTT solution (200 µL/well at 0.5 mg/mL) was added. After
additional 2 h incubation, the formazan formed was extracted using DMSO (200 µL/well).
This article is protected by copyright. All rights reserved

Absorbance was measured at 560 nm. Eye irritation or serious eye damage of the exposure groups was
classified in UN GHS categories according to cell viability values for concentrations at 5 and 0.05%
of each test material as follows: “no category”, i.e. not classified for eye irritation or serious eye
damage if cell viability was > 70% at 5% and 0.05% concentrations; “category 1”, i.e. serious eye
damage when 5% and 0.05% concentrations triggered a cell viability of ≤ 70%; or “no prediction can
be made” if cell viability for 5% and 0.05% concentrations were ≤ 70% and > 70%, respectively.
Bovine corneal opacity and permeability (BCOP) assay and corneal histomorphometric evaluation
The BCOP assay was performed according to the OECD test guideline Nº 437 [26]. Bovine eyes were
kindly donated by a local slaughterhouse (Vale do Cedro, Inhumas, GO, Brazil). In brief, excised
corneas were put on holders and the two cavities were filled with medium followed by 1 h incubation
at 32°C. Then, the medium was renewed to measure the corneal opacity using an OP-KIT Electro
Design Opacitometer (Riom, France). Corneas with opacity < 7 were then exposed to the test material
for 10 min: PBS (negative control group), benzalkonium chloride (5% w/v in PBS, as positive
control), LQFM184 (2) (5% w/v in PBS) or its vehicle (5% w/v DMSO in PBS). Corneal epithelium
was then washed with medium and the anterior chambers refilled with phenol red-free EMEM, and
final opacity values were obtained to calculate corneal opacity. The medium was discarded after
incubation followed by refilling of posterior and anterior chambers with medium and fluorescein
solution (1 mL at 4 mg/mL in PBS), respectively, for 90 min. Aliquots (200 µL) from posterior
chambers were collected and the absorbance determined at 490 nm. Thus, the permeability values
were obtained for each cornea. The following equation was used to calculate “In Vitro Irritancy
Score” (IVIS): IVIS = opacity + (15 x permeability). The corneal irritancy levels of exposure groups
were determined based on the obtained IVIS values as follows: “No Category” (IVIS ≤ 3); “No
Prediction Can Be Made” (3 < IVIS ≤ 55); or “Category 1” (IVIS > 55).
Corneas were kept in 10% phosphate buffered formalin and histomorphometric evaluation was
performed based on protocol described by Oliveira, Ducas, Teixeira, Batista, Oliveira and Valadares
[27]. The tissue sections were observed by a light microscope and photographed using an
AxioCamMRc Carl Zeiss camera and analyzed in AxioVs40 V 4.7.2.0 Carl Zeis software (Axio
Scope A1 Carl Zeiss, Jena, TH, Germany). For histomorphometric assessment, about 10 locations in
This article is protected by copyright. All rights reserved

the central field of the tissue sections were randomly chosen and the measurements of thickness of the
epithelium and stroma were conducted.
HaCaT keratinocytes-associated IL-18 assay
IL-18 production by HaCaT cells after exposure to LQFM184 (2) was performed in accordance with
the protocol described by Corsini et al. [28]. The cytotoxicity evaluation of LQFM184 (2) was prior
conducted, by seeding 1.5 × 105 cells/mL in a 96 well plate and exposing to LQFM184 (2) (1.6-200
µg/mL) or its vehicle (0.4% DMSO in complete medium) for 24 h. Then, the MTT assay was
performed as described above. Cell viability was expressed as a percentage of the control and IC20
value was calculated to perform IL-18 production analysis. Keratinocytes (1.5 x 105 cells/mL) were
seeded overnight in a 24-well flat plate and were exposed to non-cytotoxic concentration of
LQFM184 (2) (IC20 = 8 µg/mL), skin sensitizer PPD (40 µg/mL), vehicle control (0.4% DMSO) or
complete medium only (control cells) for 24 h. Cell lysates were obtained and IL-18 levels of each
sample were quantified using Human IL-18 Platinum ELISA (eBioscience) in accordance with the
manufacturer’s instructions. The protein content of each sample was measured using bicinchoninic
acid protein assay kit (Sigma-Aldrich). IL-18 levels data (pg) are expressed based on total intracellular
protein content (mg) and stimulation index (SI) was calculated according to the equation: SI = IL-18
in treated cells / IL-18 in vehicle-treated cells. Those samples with SI ≥ 1.2 associated with a
significant IL-18 increase were classified as contact allergens.
Gene expression assay
HaCaT cells were seeded at 1.5 × 105 cells/mL in a 24-well plate and exposed to LQFM184 (2) (8
µg/mL), PPD (40 µg/mL) or vehicle (0.4% DMSO/medium) for 4 h. Then, total RNA was obtained
using Trizol (Invitrogen), according to manufacturer’s instructions. The integrity of the RNA was
evaluated at 260 nm using Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA). cDNA samples were prepared using a recombinant reverse transcription kit (iScriptTM
gDNA Clear Synthesis kit, Bio-Rad). Quantitative real-time PCR reactions were then performed using
SsoAdvancedTM Universal SYBR Green Supermix kit (Bio-Rad). The HPTR1 was used as reference
This article is protected by copyright. All rights reserved

gene and fold change was calculated by DDCT method. Primers are presented in Table 1. The data
were calculated as average log2-fold change in three independent biological replicates ± SD.
<Table 1 >
U937 cell line activation test (U-SENS™) and IL-8 levels analysis
U-SENSTM was performed according to OECD test guideline Nº 442E [29]. First, cytotoxicity
potential of LQFM184 (2) in U937 cells was performed using PI staining in accordance with the
guideline to determine LQFM184 (2) concentrations that promote viability ≥ 70% (CV70). In a second
step, CD86 expression was evaluated by exposing U937 cells to LQFM184 (2) (1.25-10 µg/mL), skin
sensitizer DNCB (1 µg/mL), vehicle control (0.4% DMSO) or medium only for 45 h. Subsequently,
supernatants were collected to determine IL-8 levels using BD OptEIATM human IL-8 ELISA set kit
(BD Bioscience), and cells were washed with PBS + 5% FBS (v/v) and incubated for 30 min at 4ºC
with staining solution containing buffer and FITC-conjugated anti-human CD86 monoclonal antibody
or FITC-isotype control IgG1. After that, cells were washed and stained with PI (3 µg/mL) and
analyzed using flow cytometer. Data were calculated as stimulation index (SI) according to the
following equation: SI = [(%CD86treated - % isotype IgG1treated)/(%CD86control - % isotype IgG1control)]
x 100. A positive response for skin sensitization was considered when SI ≥ 150%. Regarding IL-8
assay, the same prediction model described in HaCaT keratinocytes-associated IL-18 assay was
applied to classify each test material as non-sensitizer or sensitizer.
Phototoxicity evaluation
The phototoxicity potential of the LQFM184 (2) was investigated in accordance to the OECD test
guideline Nº 432 [30]. In brief, 3T3 cells (1 × 104 cells/well) were seeded in two 96-well plates and
incubated overnight. After, cells were exposed, for 1 h, to LQFM184 (2) (3.1-400 µg/mL),
amiodarone HCL (3.1-400 µg/mL) or vehicle controls (0.4% DMSO or 0.1% ethanol for LQFM184
(2) or amiodarone HCL, respectively). Subsequently, one plate was exposed to 5 J/cm2 UVA (UVA+),
This article is protected by copyright. All rights reserved

while another one was protected from light (UVA-). After 50-min exposure, cells were washed with
HBSS and a fresh medium was added in each well. After 18 h, cells were washed again, and DMEM
containing neutral red (50 µg/mL) was added to all wells to evaluate the cell viability as described in
item 2.8.2. Absorbance was measured at 540 nm. The Phototox Version 2.0 software, available by
OECD, was used to obtain the photoirritation factor (PIF) and mean photo effect (MPE). According to
the guideline, a compound can be classified as follows: “No phototoxic” (PIF < 2 or MPE < 0.1);
“Probable phototoxic” (2 < PIF < 5 or 0.1 < MPE < 0.15); or “Phototoxic” (PIF > 5 or MPE > 0.15).
Statistical analysis
Three independent assays were performed, and the data analyzed using GraphPad Prism version 5.01
software (San Diego, CA, USA). The one-way Analysis of Variance (ANOVA) followed by Dunnett’s
were used for multiple comparisons. Statistical significance was considered as p<0.05.
This article is protected by copyright. All rights reserved

RESULTS
Synthesis
As shown in Fig. 2, the first step of the synthetic route of LQFM184 (2) was the synthesis of 2-(4-
hydroxy-3-methoxybenzylidene) malononitrile (5) (92 % of yield) through Knoevenagel reaction [13].
In sequence, (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylamide (6) was obtained in 93% of yield
through stereoselective copper(II)-catalyzed monohydratation of 2-(4-hydroxy-3-methoxybenzylidene)
malononitrile (5) [14]. The same compound (6) also could be synthetized, in 80 % of yield, using neat
Knoevenagel conditions [15]. Finally, LQFM184 (2) was acquired in 62% of yield through aromatic
nucleophilic substitution reaction [16, 17]. In total, it was synthetized in three synthetic steps with
global yield of 45-53 %.
<Figure 2>
Stability assessment of LQFM184 (2) against oxidation
The DPV method was used to evaluate the anodic behavior and oxidative stability of compounds (5),
(6) and LQFM184 (2). In relation to the intermediate compounds, (5) and (6), the free phenolic group
was electrochemically oxidized at glassy carbon electrode, showing an anodic peak, 1a, at peak
potential, Ep1a = 0.6 V (Fig. 3).
On the other hand, it was verified a complete absence of anodic peaks in the DP voltammogram
of LQFM184 (2), in which all phenolic groups are involved in ether linkages to the central 1,3,5-
triazine ring. Therefore, it can be assumed that LQFM184 (2) has good stability against oxidation,
which is a promising feature for a photoprotective molecule [11].
<Figure 3>
Thermogravimetric curve of LQFM184 (2)
Analysis of the thermogravimetric curve suggested that LQFM184 (2) is thermally stable up to high
temperatures (Fig. 4). To demonstrate this, LQFM184 (2) was subjected to heating ramp. We
observed, until 100°C approximately, a mild mass loss of about 0.126 mg, representing 2.565% of the
original mass. This loss can be attributed to moisture (water) adsorbed onto the structure. After, the
This article is protected by copyright. All rights reserved

thermal decomposition occurred in three consecutive mass loss steps, as shown in DTG curve of
LQFM184 (2), while its complete degradation was demonstrated in DTA curve. Despite the fact it
was not possible to identify the compounds released by thermal degradation using TG, it is reasonable
association with the results of mass spectroscopy. The first step occurred in high temperatures (240-
415ºC) with weight loss of 30%; this can be given to loss of the one of the three C11H7N2O2 molecules
found in the LQFM184 (2). Then, the second and third decomposition steps happened at 415.43-
571.6°C and 571.6-700°C with weight loss of 33.25 and 33.82%, respectively. These values can be
assigned to loss of the other two C11H7N2O2 molecules with the C3N3O3 molecule core.
<Figure 4>
Photophysical characterization of LQFM184 (2)
The photophysical characterization of LQFM184 (2) is shown in Fig. 5A. The LQFM184 (2) showed
an intense absorption from 260 to 340 nm (UVA and UVB regions), with a main band at 298 nm and
a shoulder close to 334 nm. The molar absorption coefficient (ε(λ)) was calculated from the
absorbance spectra using the Beer-Law ε(λ) = A(λ)/Cl, where A(λ) is the absorbance spectra of the
solution, C is the molar concentration and l is the optical length of the quartz cell [31, 32]. In a graph
of A(λ) as a function of C, the slope is ε(λ)l (inset of Fig. 5A). In the experiments performed, the
LQFM184 (2) concentrations were evaluated from 12.5 to 200 µM and the optical length of the quartz
cell employed was 1.0 cm. The ε(λ=298 nm) value obtained at 298 nm is 3.63×104 M-1cm-1, which is
the mean value of five independent measurements.
Fig. 5B shows the fluorescence spectra obtained in different excitation wavelengths. It is
observed a single broad structureless band centered close to 448 nm, which is independent of
excitation. To avoid the inner filter effect, the solutions were prepared and adjusted to ~0.05
absorbance at excitation wavelength. The photophysical processes can be explained based on an
energy-level diagram, also called Jablonski´s diagram (see inset of Fig. 5B), which comprises the
singlet ground state (S0), the singlet excited state (S1) and the triplet excited states (T1) [32].
<Figure 5>
This article is protected by copyright. All rights reserved

SPF evaluation of LQFM184 (2)
The method used in this study was an in vitro technique for predicting the effect of compound
LQFM184 (2) considering human skin protection from UV radiation exposure. Data from the
literature have shown that in vitro results have good correlation to those obtained using in vivo assays
[34]. When LQFM184 (2) was evaluated by Optometrics SPF-290S analyzer, the SPF value found
was 2.62. Moreover, its combination with ethylhexyl metoxycinamate and Tinosorb® S provided SPF
values of 20.29 and 26.83, respectively. In addition, a commercial sunscreen with SPF30 showed a
SPF value of 31.3, while a value of 35.14 was obtained when LQFM184 (2) was added on this
product.
Estimation of the acute oral systemic toxicity hazard for LQFM184 (2)
For acute toxicity profile investigation, we performed the validated in vitro 3T3 NRU assay to predict
the LD50 of LQFM184 (2). In Fig. 6, it is observed that LQFM184 (2) triggered cytotoxicity in a
concentration-dependent manner. IC50 value found was 15.2 µg/mL, which was used to estimate de
LD50. Thus, the estimated LD50 value for LQFM184 (2) was 495.4 mg/kg, classifying it as UN GHS
Category 4 (LD50 > 300-2000 mg/kg).
<Figure 6>
Eye toxicity assessment of LQFM184 (2)
The eye toxicity profile of LQFM184 (2) was studied using four different endpoints: cytotoxicity to
epithelial cells, corneal opacity damage, corneal permeability and histomorphometry. The positive
control used, benzalkonium chloride, promoted high cytotoxicity according to the STE assay (Table 2)
and then this compound was properly classified as UN GHS Category 1. Corroborating with these
findings, the corneal opacity and permeability changes produced an IVIS of 101.95, classifying this
compound as Category 1 in BCOP assay (Table 3).
<Table 2 >
This article is protected by copyright. All rights reserved

<Table 3 >
On the other hand, SIRC cells exposed to LQFM184 (2) concentrations at 0.05 and 5% showed
a low decrease in cell viability of 86.5 and 72.1%. Therefore, it was classified as non-irritant.
Furthermore, BCOP data showed that LQFM184 (2) did not promote significant corneal
permeability/opacity changes (IVIS value of 6.38), with no potential to predict eye irritation (Table 3).
In this respect, corneal histomorphometric assessments were performed to improve the prediction
model of the BCOP method [27]. Histomorphometric results showed that LQFM184 (2) did not
trigger any corneal tissue damages, showing epithelium and stroma lengths of 60.01 and 394.43 µm,
respectively, which were similar to those values found for control (51.87 µm for epithelium and
440.59 µm for stroma) (Figs. 7A-C). In contrast, benzalkonium chloride was able to cause a drastic
reduction of 35.61 µm (p<0.0001) in corneal epithelium length (Fig. 7A) in parallel to an increase of
597.62 µm (p<0.0001) in stroma length (Fig. 7B).
<Figure 7>
Skin sensitization evaluation of LQFM184 (2)
As shown in Fig. 8A, the keratinocyte cells exposed to LQFM184 (2) (1.6-200 µg/mL) produced a
reduction on cell viability, with an IC20 value of 8 µg/mL. At this concentration, the compound was
unable to promote an increase in IL-18 intracellular production, when compared to vehicle and control
groups, with a reduced SI value of 0.36 (Fig. 8B). In addition, LQFM184 (2) did not promote changes
in gene expression of NRF2 and FOS (Fig. 8C). Thus, the compound was classified as non-skin
sensitizer considering the keratinocyte activation endpoint. It has been demonstrated in the literature
that skin sensitizers promote significant increase of IL-18 levels in keratinocytes [28] as well as
modulation of the Nrf2-Keap1 pathway and, consequently, the inflammatory response (e.g. FOS and
FosL1) [36]. In contrast, the well-known skin sensitizer PPD triggered a significant increase in IL-18
levels (p<0.0001), with a SI value of 10.04, associated with significant changes in NRF2 and FOS
gene expression (p<0.001).
<Figure 8>
This article is protected by copyright. All rights reserved

Moreover, U937 cells exposed to LQFM184 (2) showed reduction on cell viability in a
concentration-dependent manner (Fig. 9A), reaching a CV70 value of 10 µg/mL. These cells were then
exposed to LQFM184 (2) at non-cytotoxic concentrations (1.25-10 µg/mL) to perform U-SENSTM. As
shown in Fig. 9B, the skin sensitizer DNCB (1 µg/mL) induced a significant increase in CD86
expression (SI=266.17%) (p<0.001), in comparison to control. Regarding to LQFM184 (2), only the
higher concentration (10 µg/mL) was able to generate a significant increase in CD86 expression
(SI=247.68%) (p<0.05). Considering that SI value of CD86 was higher than 150% at the highest non-
cytotoxic LQFM184 (2) concentration only, no conclusion regarding skin sensitization potential can
be made by this parameter.
These results led us to investigate a third endpoint through quantification of IL-8 levels released
by U937 cells exposed to LQFM184 (2) (1.25-10 µg/mL). In the same way as the results observed in
the expression of CD86, DNCB (1 µg/mL) triggered an increased in IL-8 levels (p<0.0001) associated
with a high SI value of 5.70, when compared to control. Contrasting, LQFM184 (2) did not promote
significant increases in IL-8 levels (Fig. 9C).
<Figure 9>
Phototoxicity evaluation of LQFM184 (2)
The results of the phototoxicity investigation of LQFM184 (2) are shown in Table 4. The positive
control used, amiodarone HCL, was properly classified as a phototoxic substance with PIF and MPE
values of 8.30 and 0.43, respectively. On the other hand, LQFM184 (2) was classified as potentially
non-phototoxic. The IC50 values found were 43.32 µg/mL (UVA- exposure) and 32.71 µg/mL (UVA+
exposure), whereas PIF and MPE values of 1.32 and 0.05, respectively.
<Table 4 >
DISCUSSION
LQFM184 (2) was designed through bioisosterism of functional group strategy from LQFM048 (1).
The amide scaffold found in LQFM184 (2) can add the possible advantage to form hydrogen bonds
with sunscreen formulations as well as skin, when compared to the lead compound. The synthetic
This article is protected by copyright. All rights reserved

route used allowed LQFM184 (2) be synthetized only in three synthetic steps, with around 45-53 % of
global yield.
Evaluations of the physico-chemical properties of the new compound LQFM184 (2), using the
TG, DTG and DTA methodologies, showed steady against temperature escalations, with a stable
structure for possible use in sunscreens [11]. Considering that, LQFM184 (2) degradation may start at
much higher values than the maximum values of handling, manipulation and manufacture of finished
product (around 80 °C), as well as, the use of the finished product (about 50 °C).
The photophysical characterization demonstrated that LQFM184 (2), similarly to typical
commercial sunscreen, possesses ε from 103 -105 M-1cm-1 in the UVA and UVB regions. As shown
before, the photophysical data obtained with the evaluation of the LQFM184 (2) can be explained
based on an energy-level diagram, also called Jablonski´s diagram, which comprises the singlet
ground state (S0), the singlet excited state (S1) and the triplet excited states (T1) [32]. In
thermodynamic equilibrium, the molecules lying in the lowest electronic state S0 can reach excited
state S1, due to one photon absorption, described by molar absorption coefficient ε(λ) (see inset of
Fig. 5B). At S1 state, the molecules can lose energy in a variety of different ways: (i) decaying to S0
through a radiative process described by a rate constant kR; (ii) decaying to S0 through an internal
conversion process (kIC); or (iii) decaying to the triplet state T1 by an intersystem crossing process
(kISC) [32]. While the radiative process is linked to the fluorescence of the molecules by photons
emission, the internal conversion and the intersystem crossing processes can produce heat to the
environment. Aiming at the potential application of LQFM184 (2) in sun protection products, the
intense fluorescence observed here can be a strategy to produce less heat in a future formulation. In
order to obtain an efficient sunscreen formulation, high Stokes shifts (=abs-em) are desired [12].
High Stokes shifts values mean a more efficient separation between absorption in the UV region (abs)
and energy released as the light emitted in the visible region (em).
Similarly to its lead compound LQFM048 (1) [12], the SPF values obtained for LQFM184 (2)
suggest that its association with other filters present in a formulation can increase the SPF value. The
degree to which this increase in SPF occurs is different for each filter, possibly by different modes of
molecular interaction between filters and the new product tested. Thus, it seems to be interesting
This article is protected by copyright. All rights reserved

associate LQFM184 (2) with other sunscreens, since it promotes slightly higher protective factors
without the need to incorporate more commercial filters, which have limits set by current Brazilian
legislation, for instance.
The acute toxicity profile is important for identifying and characterizing hazard induced by
chemical [35]. In this context, we estimated the LD50 value for LQFM184 (2) as 495.4 mg/kg using a
validated in vitro model the 3T3 NRU assay, categorizing it in UN GHS category 4 (LD50 > 300-2000
mg/kg). Regarding eye toxicity, the data obtained using three different endpoints indicated that
LQFM184 (2) does not promote potential eye toxicity.
Concerning the potential of LQFM184 (2) to induce skin allergy, the compound was classified
as non-skin sensitizer by combined analysis of different in vitro innovative methodologies for skin
sensitizing prediction [37]. Moreover, the results of the phototoxicity investigation of LQFM184 (2)
showed that it is potentially non-phototoxic, corroborating with all efficacy/toxicity data presented
here and suggesting its promising use as a sunscreen ingredient due to its UV filter properties.
The novel photoprotective compound LQFM184 (2), synthesized by green chemistry approach,
was stable under oxidative/high-temperature conditions, showed good efficacy covering a wide UV
range, from 260 to 340 (UVA and UVB regions). Toxicological investigations showed that compound
(2) has a low-moderate potential of inducing acute oral systemic toxicity hazard. In addition, eye
irritation, skin sensitization and phototoxic potential effects were not observed for LQFM184 (2).
These findings make LQFM184 (2) a promising agent to be used in cosmetic products such as
sunscreens.
ACKNOWLEDGEMENTS: This work was supported by Fundação de Apoio à Pesquisa do
Estado de Goiás (FAPEG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (finance code 001).
SUPPORTING INFORMATION
This article is protected by copyright. All rights reserved

Additional supporting information may be found online in the Supporting Information section at the
end of the article:
1
Table S1. H and 13C NMR spectral data for 2-cyano-3-(4’-hydroxy-3’-methoxybenzylidene)-
malononitrile (5) in DMSO-d6.
Table S2. 1H and 13C NMR spectral data for (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)-acrylamide
(6) in DMSO-d6.
Table S3. 1H and 13C NMR spectral data for (2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (2), in DMSO-d6.
Figure S1. Infrared spectrum obtained for 2-cyano-3-(4’-hydroxy-3’-ethoxybenzylidene)-
malononitrile (5).
Figure S2. 1H NMR spectrum obtained for 2-cyano-3-(4’-hydroxy-3’-methoxybenzylidene)-
malononitrile (5) in DMSO-d6.
Figure S3. HSQC contour map for 2-cyano-3-(4’-hydroxy-3’-methoxybenzylidene)-malononitrile (5)
in DMSO-d6.
Figure S4. HMBC contour map for 2-cyano-3-(4’-hydroxy-3’-methoxybenzylidene)-malononitrile (5)
in DMSO-d6.
Figure S5. ESI (-) FT-Orbitrap MS spectrum obtained for 2-cyano-3-(4’-hydroxy-3’-
methoxybenzylidene)-malononitrile (5).
Figure S6. Infrared spectra obtained for (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)-acrylamide (6).
1
Figure S7. H NMR spectrum for (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)-acrylamide (6), in
DMSO-d6.
Figure S8. HSQC contour map for (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)-acrylamide (6), in
DMSO-d6.
This article is protected by copyright. All rights reserved

Figure S9. HMBC contour map for (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)-acrylamide (6), in
DMSO-d6.
Figure S10. ESI (-) FT-Orbitrap MS spectra obtained for (E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)-acrylamide (6).
Figure S11. Infrared spectrum obtained for (2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (2) – LQFM184.
Figure S12. 1H NMR spectrum obtained for (2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (2), in DMSO-d6.
Figure
S13.
HSQC
contour
map
for
(2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (2), in DMSO-d6.
Figure
S14.
HMBC
contour
map
for
(2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (2), in DMSO-d6..
Figure S15. 13C NMR spectrum obtained for (2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (9), in DMSO-d6.
Figure S16. ESI (+) FT-Orbitrap MS spectra obtained for (2,4,6-tris-((E)-2-cyano-3-(4-hydroxy-3-
methoxyphenyl)acrylamide)-1,3,5-triazine) (9).
This article is protected by copyright. All rights reserved

REFERENCES
[1] J.A. Ruszkiewicz, A. Pinkas, B. Ferrer, T.V. Peres, A. Tsatsakis, M. Aschner, Neurotoxic effect of
active ingredients in sunscreen products, a contemporary review, Toxicol Rep, 4 (2017) 245-259.
[2] M.S. Latha, J. Martis, V. Shobha, R. Sham Shinde, S. Bangera, B. Krishnankutty, S. Bellary, S.
Varughese, P. Rao, B.R. Naveen Kumar, Sunscreening agents: a review, J Clin Aesthet Dermatol, 6
(2013) 16-26.
[3] A. Sample, Y.-Y. He, Mechanisms and prevention of UV-induced melanoma, Photodermatol
Photoimmunol Photomed, 34 (2018) 13-24.
[4] V. Fioletov, J.B. Kerr, A. Fergusson, The UV index: definition, distribution and factors affecting
it, Can J Public Health, 101 (2010) I5-9.
[5] S. Deady, L. Sharp, H. Comber, Increasing skin cancer incidence in young, affluent, urban
populations: a challenge for prevention, Br J Dermatol, 171 (2014) 324-331.
[6] Q. Zhang, X. Ma, M. Dzakpasu, X.C. Wang, Evaluation of ecotoxicological effects of
benzophenone UV filters: luminescent bacteria toxicity, genotoxicity and hormonal activity,
Ecotoxicol Environ Saf, 142 (2017) 338-347.
[7] F. Vela-Soria, I. Jiménez-Díaz, R. Rodríguez-Gómez, A. Zafra-Gómez, O. Ballesteros, A.
Navalón, J.L. Vílchez, M.F. Fernández, N. Olea, Determination of benzophenones in human placental
tissue samples by liquid chromatography–tandem mass spectrometry, Talanta, 85 (2011) 1848-1855.
[8] B. Herzog, D. Hüglin, E. Borsos, A. Stehlin, H. Luther, New UV absorbers for cosmetic
sunscreens – a breakthrough for the photoprotection of human skin, Chimia, 58 (2004) 554-559.
[9] S.Q. Wang, P.R. Tanner, H.W. Lim, J.F. Nash, The evolution of sunscreen products in the United
States – a 12-year cross sectional study, Photochem Photobiol Sci, 12 (2013) 197-202.
[10] C. Couteau, E. Paparis, C. Chauvet, L. Coiffard, Tris-biphenyl triazine, a new ultraviolet filter
studied in terms of photoprotective efficacy, Int J Pharm, 487 (2015) 120-123.
[11] D. Hüglin, Advanced UV absorbers for the protection of human skin, Chimia, 70 (2016) 496-
501.
This article is protected by copyright. All rights reserved

[12] D.C. Vinhal, R.I. de Ávila, M.S. Vieira, R.M. Luzin, M.P. Quintino, L.M. Nunes, A.C.C.
Ribeiro, H.S. de Camargo, A.C. Pinto, H.M. dos Santos Júnior, B.G. Chiari, V. Isaac, M.C. Valadares,
T.D. Martins, L.M. Lião, E. de S. Gil, R. Menegatti, Photoprotective effect and acute oral systemic
toxicity evaluation of the novel heterocyclic compound LQFM048, J Photochem Photobiol B, 161
(2016) 50-58.
[13] R.I. de Ávila, M. de Sousa Vieira, M.P.N. Gaeti, L.C. Moreira, L. de Brito Rodrigues, G.A.R. de
Oliveira, A.C. Batista, D.C. Vinhal, R. Menegatti, M.C. Valadares, Toxicity evaluation of the
photoprotective compound LQFM048: eye irritation, skin toxicity and genotoxic endpoints,
Toxicology, 376 (2017) 83-93. [13] M.N. Gomes, C.M.A. de Oliveira, C.F.D. Garrote, V. de Oliveira,
R. Menegatti, Condensation of Ethyl Cyanoacetate with Aromatic Aldehydes in Water, Catalyzed by
Morpholine, Synth Commun, 41 (2010) 52-57.
[14] X. Xin, D. Xiang, J. Yang, Q. Zhang, F. Zhou, D. Dong, Homogeneous and stereoselective
copper(II)-catalyzed monohydration of methylenemalononitriles to 2-cyanoacrylamides, J Org Chem,
78 (2013) 11956-11961.
[15] K. Lewellyn, D. Bialonska, N.D. Chaurasiya, B.L. Tekwani, J.K. Zjawiony, Synthesis and
evaluation of aplysinopsin analogs as inhibitors of human monoamine oxidase A and B, Bioorg Med
Chem Lett, 22 (2012) 4926-4929.
[16] R. Menicagli, S. Samaritani, G. Signore, F. Vaglini, L. Dalla Via, In vitro cytotoxic activities of
2-alkyl-4,6-diheteroalkyl-1,3,5-triazines:ꢀ new molecules in anticancer research, J Med Chem, 47
(2004) 4649-4652.
[17] H. Duan, L. Wang, D. Qin, X. Li, S. Wang, Y. Zhang, Synthesis and characterization of some
new star-shaped polydentate ligands from 2,4,6-trichloro-1,3,5-triazine, Synth Commun, 41 (2011)
380-384.
[18] M.A. Corrêa, Cosmetologia, in, Medfarma, 2012, pp. 492.
[19] S. Kale, P. Ghoge, A. Ansari, A. Waje, A. Sonawane, Formulation and in-vitro determination of
sun protection factor of Nigella sativa Linn. seed oil sunscreen cream, Int J Pharm Tech Res, 2 (2010)
2194-2197.
[20] A. Duraisamy, N. Narayanaswamy, A. Sebastian, K.P. Balakrishnan, Sun protection and anti-
inflammatory activities of some medicinal plants, Int J Res Cosm Sci, 1 (2011) 13-16.
This article is protected by copyright. All rights reserved

[21] E. Borenfreund, J.A. Puerner, A simple quantitative procedure using monolayer cultures for
cytotoxicity assays (HTD/NR-90), J Tissue Cult Methods, 9 (1985) 7-9.
[22] ICCVAM, Test method evaluation report (TIMER): in vitro cytotoxicity test methods for
estimating starting doses for acute oral systemic toxicity test. NIH publication no. 07-4519. Research
Triangle Park, NC: National Institute for Environmental Health Sciences, (2006).
[23] M.d.S. Vieira, V. de Oliveira, E.M. Lima, M.J. Kato, M.C. Valadares, In vitro basal cytotoxicity
assay applied to estimate acute oral systemic toxicity of grandisin and its major metabolite, Exp
Toxicol Pathol, 63 (2011) 505-510.
[24] OECD, Test No. 423: acute oral toxicity - acute toxic class method, 2002.
[25] OECD, Test No. 442E: In vitro skin sensitisation assays addressing the key event on activation of
dendritic cells on the adverse outcome pathway for skin sensitisation, OECD Publishing, 2018.
[26] OECD, Test No. 437: bovine corneal opacity and permeability test method for identifying i)
chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation
or serious eye damage, 2017.
[27] G.A.R. Oliveira, R.d.N. Ducas, G.C. Teixeira, A.C. Batista, D.P. Oliveira, M.C. Valadares, Short
time exposure (STE) test in conjunction with bovine corneal opacity and permeability (BCOP) assay
including histopathology to evaluate correspondence with the Globally Harmonized System (GHS)
eye irritation classification of textile dyes, Toxicol In Vitro, 29 (2015) 1283-1288.
[28] E. Corsini, V. Galbiati, M. Mitjans, C.L. Galli, M. Marinovich, NCTC 2544 and IL-18
production: a tool for the identification of contact allergens, Toxicol In Vitro, 27 (2013) 1127-1134.
[29] OECD, Test No. 442E: in vitro skin sensitisation, 2018.
[30] OECD, Test No. 432: in vitro 3T3 NRU phototoxicity test, 2004.
[31] S.E. Rodrigues, A.E.H. Machado, M. Berardi, A.S. Ito, L.M. Almeida, M.J. Santana, L.M. Liao,
N.M. Barbosa Neto, P.J. Gonçalves, Investigation of protonation effects on the electronic and
structural properties of halogenated sulfonated porphyrins, J Mol Struct, 1084 (2015) 284-293.
[32] L. Alonso, R.N. Sampaio, T.F.M. Souza, R.C. Silva, N.M.B. Neto, A.O. Ribeiro, A. Alonso, P.J.
Gonçalves, Photodynamic evaluation of tetracarboxy-phthalocyanines in model systems, J Photochem
Photobiol B, 161 (2016) 100-107.
This article is protected by copyright. All rights reserved

[33] C. Couteau, M. Pommier, E. Paparis, L. Coiffard, Study of the efficacy of 18 sun filters
authorized in European Union tested in vitro, Pharmazie, 62 (2007) 449-452.
[34] P.J. Matts, V. Alard, M.W. Brown, L. Ferrero, H. Gers-Barlag, N. Issachar, D. Moyal, R. Wolber,
The COLIPA in vitro UVA method: a standard and reproducible measure of sunscreen UVA
protection, Int J Cosmet Sci, 32 (2010) 35-46.
[35] ICCVAM, Guidance document on using in vitro data to estimate in vivo starting doses for acute
toxicity. NIH Publication no. 01-4500. Research Triangle Park, NC: National Institute for
Environmental Health Sciences., (2001).
[36] J.W. van der Veen, T.E. Pronk, H. van Loveren, J. Ezendam, Applicability of a keratinocyte gene
signature to predict skin sensitizing potential, Toxicol In Vitro, 27 (2013) 314-322.
[37] C. Bauch, S.N. Kolle, T. Ramirez, T. Eltze, E. Fabian, A. Mehling, W. Teubner, B. van
Ravenzwaay, R. Landsiedel, Putting the parts together: combining in vitro methods to test for skin
sensitizing potentials, Regul Toxicol Pharmacol, 63 (2012) 489-504.
FIGURE CAPTIONS
Figure 1. Design of LQFM184 (2) from LQFM048 (1) lead compound.
Figure 2. Synthetic route of LQFM184 (2).
Figure 3. DP voltammograms obtained for 10 M solutions of LQFM184 (2) (), compound (5)
(▬) and compound (6) (- - -) in phosphate buffer saline (pH 6.0 0.1 M).
Figure 4. Representative thermogravimetric analysis of LQFM184 (2).
Figure 5. Photophysical characterization of LQFM184 (2). (A) Absorption spectra of LQFM184 (2)
in DMSO and in different concentrations, inset: A(λ) vs C curve. (B) Fluorescence spectra of
LQFM184 (2) at different excitation wavelengths, inset: energy-level diagram (Jablonski´s diagram).
This article is protected by copyright. All rights reserved

Figure 6. Cytotoxicity analysis of LQFM184 (2) in 3T3 fibroblasts. Cells (0.5 × 105 cells/mL) were
seeded in plates overnight and then exposed to LQFM184 (2) (1.6-200 µg/mL). After 48 h incubation,
the cell viability was evaluated by neural red uptake (NRU) assay. The IC50 value (15.2 µg/mL)
obtained was used to estimate LD50 of LQFM 184 (2).
Figure 7. Corneal histomorphometric analysis. Isolated corneas (n=3/group) from bovine eyes were
exposed to LQFM184 (2) (5%), its vehicle (2.5% DMSO), negative (PBS) and positive
(benzalkonium chloride at 5%) controls. After that, the samples were processed, and tissue sections
obtained and stained with hematoxylin and eosin (H&E). Histomorphometric assessment was carried
out through measurement of thickness of the (A) epithelium and (B) stroma. (C) Representative
photomicrographs of corneas exposed to PBS and LQFM184 (2) (10 × magnification).
Figure 8. HaCaT keratinocytes-associated IL-18 assay. (A) HaCaT cells (1.5 × 105 cells/mL) were
seeded in plates overnight and then exposed to LQFM184 (2) (1.6-200 µg/mL) for 24 h. The cell
viability was analyzed by MTT assay. (B) Cells were exposed to non-cytotoxic concentration of
LQFM184 (2) (IC20 = 8 µg/mL), the skin sensitizer PPD (40 µg/mL), vehicle control (0.4% DMSO) or
complete medium only (control cells). After 24 h exposure, IL-18 levels were quantified in cell
lysates. Data represent the mean ± SD of three independent experiments. (***p<0.0001 vs. control or
DMSO (0.4%). ANOVA followed by Dunnett’s test).
Figure 9. U937 cell line activation test (U-SENS™) and IL-8 levels analysis. (A) U937 cells (5 × 105
cels/mL) were exposed to LQFM184 (2) (1.6-200 µg/mL) for 24 h. After that, cell viability was
analyzed by propidium iodide staining using flow cytometer. (B) U937 cells were exposed to non-
cytotoxic concentrations of LQFM184 (2) (1.25-10 µg/mL), the skin sensitizer DNCB (1 µg/mL),
vehicle control (0.4% DMSO) or complete medium only (control group) for 45 h. CD86 analysis was
then conducted by flow cytometry. (C) IL-8 levels found in supernatant of U937 cells.
This article is protected by copyright. All rights reserved

Tables
Table 1. PCR primer sequences and associated GenBank accession numbers.
Gene
Name
Primer sequence (5’ – 3’)
GenBank number
S74017.1
AGT GGA TCT GCC AAC TAC TC
CAT CTA CAA ACG GGA ATG TCT G
TGC CTC TCC TCA ATG ACC CTG A
ATA GGT CCA TGT CTG GCA CGG A
TGA CAC TGG CAA AAC AAT GAC
GGT CCT TTT CAC CAG CAA GCT
NRF2
Nuclear factor, erythroid 2 like 2
FOS
Fos proto-oncogene
CR542267.1
CR407645.1
Hypoxanthine
HPRT1
phosphoribosyltransferase 1
This article is protected by copyright. All rights reserved

Table 2. Data obtained in the short time exposure (STE) assay.
Cell viability (%)
Exposure group
UN GHS classificationb
0.05%
5%
Negative controla
Positive controla
LQFM184 (2)
100 ± 0.5
5.6 ± 1.1
100 ± 0.3
2.7 ± 0.5
No category
Category 1
No category
86.5 ± 0.8 72.1 ± 0.3
Data represent mean ± SD of three independent assays.
aSaline and benzalkonium chloride are negative and positive controls, respectively.
bPrediction model based on OECD test guideline Nº 491 (OECD, 2015).
This article is protected by copyright. All rights reserved