Photoprotective effect and acute oral systemic toxicity evaluation of the novel heterocyclic compound LQFM048

Article abstract of DOI:10.1016/j.jphotobiol.2016.03.042

The new heterocyclic derivative LQFM048 (3) (2,4,6-tris ((E)-ethyl 2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylate)-1,3,5-triazine) was originally designed through the molecular hybridization strategy from Uvinul T 150 (1) and (E)-ethyl 2-cyano-3-(4hydroxy-

Full text of DOI:10.1016/j.jphotobiol.2016.03.042

Photoprotective effect and acute oral systemic toxicity evaluation of the novel
heterocyclic compound LQFM048
´
Daniela C. Vinhal, Renato Ivan de Avila, Marcelo S. Vieira, Rangel M.
Luzin, Michelle P. Quintino, Liliane M. Nunes, Antonio Carlos Chaves
Ribeiro, Henrique Santiago de Camargo, Angelo C. Pinto, Helve´cio M. dos
Santos Ju´nior, Bruna G. Chiari, Vera Isaac, Marize C. Valadares, Tatiana
Duque Martins, Luciano M. Lia˜o, Eric de S. Gil, Ricardo Menegatti
PII:
DOI:
Reference:
S1011-1344(15)30201-3
doi: 10.1016/j.jphotobiol.2016.03.042
JPB 10319
To appear in:
Received date:
Revised date:
Accepted date:
10 December 2015
17 March 2016
29 March 2016
´
Please cite this article as: Daniela C. Vinhal, Renato Ivan de Avila, Marcelo S. Vieira,
Rangel M. Luzin, Michelle P. Quintino, Liliane M. Nunes, Antonio Carlos Chaves
Ribeiro, Henrique Santiago de Camargo, Angelo C. Pinto, Helv´ecio M. dos Santos Ju´nior,
Bruna G. Chiari, Vera Isaac, Marize C. Valadares, Tatiana Duque Martins, Luciano
M. Li˜ao, Eric de S. Gil, Ricardo Menegatti, Photoprotective effect and acute oral sys-
temic toxicity evaluation of the novel heterocyclic compound LQFM048, (2016), doi:
10.1016/j.jphotobiol.2016.03.042
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ACCEPTED MANUSCRIPT
Photoprotective effect and acute oral systemic toxicity evaluation of the
novel heterocyclic compound LQFM048
Daniela C. Vinhala, Renato Ivan de Ávilab, Marcelo S. Vieirab, Rangel M. Luzinc, Michelle P. Quintinoc,
Liliane M. Nunesd, Antonio Carlos Chaves Ribeirod, Henrique Santiago de Camargod, Angelo C. Pintoe,
Helvécio M. dos Santos Júniore, Bruna G. Chiarif, Vera Isaacf, Marize C. Valadaresb, Tatiana Duque
Martinsd, Luciano M. Liãoc, Eric de S. Gile, Ricardo Menegattia*
aLaboratório de Química Farmacêutica Medicinal (LQFM), Faculdade de Farmácia, Universidade
Federal de Goiás, Goiânia, GO, Brazil
bLaboratório de Farmacologia e Toxicologia Celular – LFTC/FarmaTec, Faculdade de Farmácia,
Universidade Federal de Goiás, Goiânia, GO, Brazil
cLaboratório de Ressonância Magnética Nuclear, Instituto de Química, Universidade Federal de Goiás,
Goiânia, GO, Brazil
dInstituto de Química, Universidade Federal de Goiás, Goiânia, GO, Brazil
eLaboratório de Espectrometria de Massas – LABEM, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, Brazil
fLaboratório de Cosmetologia – LaCos, Departamento de Fármacos e Medicamentos, Faculdade de
Ciências Farmacêuticas, Universidade Estadual Paulista Júlio de Mesquita Filho, Araraquara, SP,
Brazil
gFaculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brazil
*Corresponding author. Address: Faculdade de Farmácia – Universidade Federal de Goiás, Rua 240
esquina com 5ª Avenida, s/n, Setor Universitário, Goiânia, GO, Brazil. CEP: 74605.170.
E-mail address: rm_rj@yahoo.com
Phone / fax: +55 62 3209-6044 ext. 227.

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Abstract
The new heterocyclic derivative LQFM048 (3) (2,4,6-tris ((E)-ethyl 2-cyano-3-(4-
hydroxy-3-methoxyphenyl)acrylate)-1,3,5-triazine) was originally designed through
the molecular hybridization strategy from Uvinul® T 150 (1) and (E)-ethyl 2-cyano-3-
(4hydroxy-3-methoxyphenyl)acrylate (2) sunscreens, using green chemistry approach.
This compound was obtained in global yields (80%) and showed an interesting redox
potential. In addition, it is thermally stable up to temperatures around 250°C. It was
observed that LQFM048 (3) showed a low degradation after 150 minutes of sunlight
exposure at 39ºC, whereas the extreme radiation conditions induced a considerable
photodegradation of the LQFM048 (3), especially when irradiated by VIS and VIS +
UVA. During the determination of sun protection factor, LQFM048 (3) showed
presented good results and as well as in association with other photoprotective
compounds and commercial sunscreen. Additionally, the compound (3) did not promote
cytotoxicity for 3T3 fibroblasts. Moreover, it was not able to trigger acute oral systemic
toxicity in mice, being classified as a compound with low acute toxicity hazard (2.000
mg/kg > LD50< 5.000 mg/kg). Therefore, this compound is promising once it may be
possible to develop a potential new sunscreen that can offer an alternative
photoprotective compound with better results than those currently used and that
probably will also be cheaper in terms of obtaining process.
Keywords: Heterocyclic derivative; Molecular hybridization; Photoprotection;
Sunscreen; Green chemistry.

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1. Introduction
Excessive exposure to solar ultraviolet radiation (UV) of humans has increased
due to the depletion of the ozone layer and changes in outdoor lifestyle [1]. Moreover,
outdoor workers are also daily exposed to UV [2]. Solar UV rays that reach the Earth’s
surface are divided into two main bands named of UVB (290 - 320 nm) and UVA (320 -
400 nm) [3].
To penetrate the skin, both bands induce DNA damage by different
mechanisms: UVB directly damages the DNA promoting the formation of the 6-4
cyclobutane pyrimidine dimmers [3, 4, 5] and UVA radiation can promote oxidative
damage by overproduction of reactive oxygen species (ROS) that injure DNA
molecules [4, 6]. Consequently, solar UV-spectrum can promote short and long term
harmful clinical consequences such as sunburns, premature skin aging and even
suppression of specific immune responses and skin cancer [7, 8, 9]. Thus, this scenario
brings in a large cost on health care [10], especially in tropical countries. In this context,
sun-related protection measures are encouraged such as the development and use of
sunscreen products.
Sunscreens are classified as inorganic or organic based on its composition [11].
The first class is composed by mineral particles such as TiO2 and ZnO that reflect or
scatter UV rays from the skin, while organic filters absorb UV light and transform it
into vibrational, fluorescence and radical energy due to its aromatic structures [11, 12,
13].
In sunscreen formulations, a combination of these types of filters is widely
employed to provide broad-spectrum protection [14]. However, this combination can
present limitations such as photostability and photoactivity of some organic and
inorganic filters, respectively. Recently, the improvement of sunscreens’ raw materials
has been done in an attempt to solve this problem. Some new raw materials have been
launched on the market, such as Uvinul® T 150 (1), a water-resistant compound widely
used for UVB protection [15]. Although most of these new materials ensure effective
sun-blocking products, there is a need to produce them in a clean and sustainable
manner using, for example, small amounts of organic solvents in organic synthesis
steps.
In this context, the present study investigated the photoprotective and
toxicological evaluation of the new heterocyclic derivative LQFM048 (3) (2,4,6-tris

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((E)-ethyl 2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylate)-1,3,5-triazine), which was
originally designed through the molecular hybridization strategy from Uvinul® T 150
(1) and (E)-ethyl 2-cyano-3-(4hydroxy-3-methoxyphenyl)acrylate (2) sunscreens, using
green chemistry approach.

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2. Materials and Methods
2.1. Chemicals
Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin,
DPPH, trypsin, EDTA, chloroform, acetronitrile, toluene, tetramethylsilane (TMS),
phosphoric acid, formic acid and in vitro toxicology assay kit (lactic dehydrogenase
based) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrahydrofuran
(THF) was acquired from Tedia Brazil (Rio de Janeiro, RJ, Brazil). Deuterated
chloroform (CDCl3) was purchased from Cambridge Isotope Laboratories Inc
(Tewksbury, MA, USA) while ethyl cyanoacetate, 2,4,6-trichloro-1,3,5-triazine and
morpholine from Acros Organics (Geel, Belgium). Methanol, dichloromethane, ethyl
acetate, dichloromethane, n-hexane, ethanol, DMSO, chloroform, acetic acid,
hydrochloric acid (HCl), sodium chloride (NaCl) and calcium chloride (CaCl2) were
acquired from Vetec (Rio de Janeiro, RJ, Brazil) while ethylhexyl methoxycinnamate,
silica gel 7 60 and vanillin from Merck (Darmstadt, HE, Germany). Tinosorb® S was
acquired from BASF (Ludwigshafen, Germany). Xylazine and ketamine were
purchased from Syntec (Cotia, SP, Brazil) and König (Santana de Parnaíba, SP, Brazil),
respectively. Sunflower oil and potassium carbonate (K2CO3) were purchased from Liza
(PR, Brazil) and Synth (SP, Brazil), respectively. Graphite was acquired from Metrohm
Autolab B.V. (Kanaalweg, Utrecht, The Netherlands) and mineral oil was purchased
from Biolub Chemistry LTDA (Sorocaba, SP, Brazil).
2.2. General
13
1H, and C NMR measurements were carried out on a Bruker Avance III 500
instrument (operating at 500.13 MHz for 1H) equipped with a 5 mm tuneable
multinuclear triple (TBI) resonance probe head equipped with z gradient. To acquire 1H
13
and C experiments, samples containing 20 mg of LQFM 048 (3) in CDCl3 and 1%
tetramethylsilane as internal standard were used. Following 1D and 2D pulse sequences
from the Bruker user library were used for the NMR experiments.
High-resolution mass spectrometry analyses were performed on a QTOF Micro
(Waters, Manchester, UK) mass spectrometer equipped with an ESI source. The
analyses were recorded between m/z 90 and 1000 in positive ion mode, and the mass

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spectrometer parameters were: nebulization gas was set to 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 sample
solutions (0.5 mg/mL) were prepared in acetonitrile with addition of 20 μL of formic
acid. The analyses were carried out by direct infusion using a syringe pump at 5.0
μL/min flow ratio.
Infrared spectra were obtained with a Nicolet-55a Magna spectrophotometer
(GMI, MN, USA) using potassium bromide plates.
The progress of all reactions was monitored by thin-layer chromatography
(TLC), which was performed on 2.0-6.0 cm aluminum sheets precoated with silica gel 7
60 to a thickness of 0.25 mm. The chromatograms developed were viewed under
ultraviolet light (254-265 nm) and treated with iodine vapor.
2.3. Synthesis of (E)-ethyl 2-cyano-3-(4hydroxy-3-methoxyphenyl) acrylate (2)
Vanillin (4) and ethyl cyanoacetate (5) (both at 3.30 mmol) in 5 mL of water
were added to a round-bottomed flask under agitation at room temperature. Five mol%
of morpholine was added to this suspension, and the reaction was monitored using TLC.
After that, the reaction mixture was partitioned between dichloromethane and water.
The organic layer was separated and submitted to the usual workup to furnish a crude
precipitate, which was used without rather purification. The derivative (2) was obtained
as a yellow solid, yield 98%, mp 108ºC, Rf = 0.64 n-hexane:ethyl acetate (7:3, v/v); IR
1
max (KBr) cm-1: 3375, 2219, 1702, 762; RMN H (500 MHz) CDCl3/TMS (δ): 8.13
(1H, s, H-3), 7.82 (1H, d, J = 2.1 Hz, H-2’), 7.38 (1H, dd, J = 2.1 and 8.3 Hz, H-6’),
6.98 (1H, d, J = 8.3 Hz, H-5’), 4.36 (2H, q, J = 7.1 Hz, CH2), 3.96 (3H, s, OCH3), 1.38
(3H, t, J = 7.2 Hz, CH3); 13C (125 MHz) CDCl3/TMS (δ): 163.0 (1C, C-1), 98.6 (1C, C-
2), 155.1 (1C, C-3), 62.2 (1C, CH2), 14.0 (1C, CH3), 116.4 (1C, CN), 56.0 (1C, OCH3),
151.1 (1C, C-1’), 111.3 (1C, C-2’), 147.0 (1C, C-3’), 124.0 (1C, C-4’), 115.0 (1C, C-
5’), 128.6 (1C, C-6’); [M+H]+= 247.07608.

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4.4. Synthesis of (2,4,6-tris ((E)-ethyl 2-cyano-3-(4-hydroxy-3-methoxyphenyl)
acrylate)-1,3,5-triazine) (3)
(E)-ethyl 2-cyano-3-(4hydroxy-3-methoxyphenyl)acrylate (2) (3.50 mmol),
K2CO3 (3.50 mmol) and 2,4,6-trichloro-1,3,5-triazine (6) (10 mmol) in 50 mL of 1,2-
dichloroethane were added to a 100 mL round-bottomed flask under agitation at room
temperature. The mixture was stirred at 80ºC for 6 h. At the end of the reaction, the
solvent was removed in vacuo and the reaction mixture was partitioned between
dichloromethane and water. The organic layer was separated and submitted to the usual
workup to furnish a crude precipitate, which was purified by Soxhlet extraction with
acetonitrile for eight hours. LQFM048 (3) was obtained as a beige solid, yield 80%, mp
186ºC, Rf = 0.92 CH2Cl2:MeOH (95:5, v/v); IR max (KBr) cm-1: 3100, 2216, 1720;
RMN 1H(500 MHz) CDCl3/TMS (δ): 8.16 (1H, s, H-3), 7.71 (1H, d, J = 1.9 Hz, H-2’),
7.42 (1H, dd, J = 1.9 and 8.4 Hz, H-6’), 7.18 (1H, d, J = 8.4 Hz, H-5’), 4.40 (2H, q, J =
13
7.2 Hz, CH2), 3.82 (3H, s, OCH3), 1.42 (3H, t, J = 7.2 Hz, CH3); C (125 MHz)
CDCl3/TMS (δ): 162.1 (1C, C-1), 102.9 (1C, C-2), 153.9 (1C, C-3), 62.6 (1C, CH2),
14.0 (1C, CH3), 113.1 (1C, CN), 56.0 (1C, OCH3), 153.7 (1C, C-1’), 113.2 (1C, C-2’),
151.3 (1C, C-3’), 130.0 (1C, C-4’), 123.0 (1C, C-5’), 125.5 (1C, C-6’), 173.0 (1C, C-
1’’, 3'' and 5''); [M+H]+=817.77560.
2.5. Determination of the antioxidant potential of LQFM048 (3)
2.5.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH•) free radical
The free radical scavenging capacity of LQFM048 (3) was determined with
DPPH• as previously described [16]. LQFM048 (3) solutions (50 µL) in different
concentrations (2, 4, 8, 15, 31, 61.3, 122.5 or 245 µM) were added to 150 µL of DPPH
solution (0.3 mM), both diluted in 95% methanol, and incubated in the dark. After 30
min, absorbance was obtained using a spectrophotometer (Thermo Scientific
Multiskan® Spectrum, MA, USA) at 517 nm.
2.5.2. Cyclic voltammetry

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All electrochemical measurements were performed with a μAutolab Type III®
potentiostat/galvanostat (Eco-ChemieB.V., Utrecht, The Netherlands) coupled to GPES
version 4.3 software for data acquisition, connected to a system of electrochemical cell
with three electrodes, namely a modified carbon paste working electrode, a calomel
reference electrode and a platinum wire counter electrode.
The electroanalytical assays were performed by solid state voltammetry by
modifying the carbon paste electrode with the analyte samples in pH 7.0, 0.1 M
phosphate buffer at 100 mV s-1, from -0.25 to 1.25 V. The pH measurements were
carried out on a pH meter, from Logen LSPHS 25CW, brand model with a universal
glass electrode trademark Eltex.
Taking into account the scarce water solubility of (E)-ethyl 2-cyano-3-(4-
hydroxy-3-methoxyphenyl)acrylate (2) and LQFM048 (3), their electrochemical
characterizations were performed at solid state. Thus, 5 mg of compound (2) were
dispersed in 20 mg of mineral oil and then the resulting homogenous dispersion was
mixed for 15 min with 60 mg of graphite, leading to Modified Carbon Paste Electrode
(MCP)-1. The same procedure was applied for ethylhexyl methoxycinnamate, in order
to obtain the modified carbon paste, MCP-2.
2.6. Thermogravimetric analysis of LQFM048 (3)
Thermal stability was available using a DTG-60H thermobalance (Shimadzu,
MD, USA), with samples of about 4.0 mg of LQFM048 (3), which were heated under
synthetic air atmosphere from 30 to 600ºC, at a flow rate of 50 mL min-1and a heating
rate of 10ºC/min.
2.7. Photostability analysis of LQFM048 (3)
2.7.1. Photostability uponsunexposure
For photostalibity evaluation under natural sunlight, a solution of LQFM048
(3) (18.3 µM) dissolved in DMSO was divided within two glass Petri dishes (60 mm)
with no cover for direct sun radiation exposure. One of these dishes with LQFM048 (3)
solution and another one with vehicle only (DMSO) were kept protected from radiation
by aluminum foil. All dishes were kept under the same conditions. During the

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experiment, the temperature was monitored at 39 ± 2ºC. The radiation from sunlight
was 27 ± 2 W.h/m2, measured at intervals of 10 min using a radiometer (model 732,
CHY firemate CO., Taiwan). Time exposure and radiation dose were 150 min and 54.2
W.h/m2, respectively. The assay was performed in Goiânia, GO, Brazil (-16° 40' 41.04";
-49° 14' 34.79"), autumn season. The exposure was at maximum sun radiation from
11:20h a.m. to 01:30 p.m. After time exposure, absorbance was measured using
spectrophotometer at 250-400 nm. The area under the curve (AUC) was calculated as
previously described [17].
2.7.2. Photostability upon irradiation by UV and VIS lamps
The investigation of photostability of LQFM048 (3) after exposure to UVA
and VIS light was performed as described by the International Conference on
Harmonization of Technical Requirements for Registration of Pharmaceuticals for
Human Use [18] using a 6545 series photostability chamber (Caron, OH, USA). This
chamber was made to meet specifically some legislation requirements, such as
European Medicines Agency, U.S. Food and Drug Administration and Brazilian
Sanitary Surveillance Agency (ANVISA), for photostability assay according to ICH
Q1B option II.
LQFM048 (3) dissolved in DMSO was divided within 8 crimped vials and
exposed to 1.2 million lux.h of VIS light and 200 W h/m2 of UVA radiation. The time
needed to reach the VIS dosage was 40 h and 8 h for UV radiation, under 25°C. The
exposures were: 2 vials under only UVA radiation (UVA); 2 vials under only VIS light
(VIS); 2 vials exposed to both first to VIS light and after to UVA radiation (VIS+UVA)
and 2 vials with vehicle control (DMSO) under the same conditions until the end of the
experiment. All conditions of radiation exposures had the same sample protected from
radiation by aluminum foil kept inside the photostability chamber. After time exposure,
absorbance was measured using a spectrophotometer at 250-400 nm and AUC was
calculated.
2.8. Photophysical characterization
Photophysical behavior of LQFM048 (3) was investigated by steady-state
fluorescence spectroscopy of its diluted solutions prepared in distinct solvents, such as

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tetrahydrofuran (THF), chloroform, acetonitrile and toluene. Steady-state excitation and
fluorescence spectra were recorded in a Fluorolog 3-221 Horiba Jobin Yvon
spectrophotometer, with a 450 W Xe arc-lamp, connected to a double monochromator,
polarization system in the entrance and a sample holder to contain a 1 cm-quartz cuvette
of optical path. Emission was detected at 90° with respect to the incident radiation by a
Hamamatsu photomultiplier, operating at the range of 250 to 850 nm. Narrow slits were
employed to guarantee the resolution of 1.0 nm to the measurements.
2.9. Evaluation of sun protection factor (SPF) of LQFM048 (3)
The SPF value of LQFM048 (3) was determined through two in vitro methods.
The first one was carried out as described by Mansur et al. [19]. Briefly, LQFM048 (3)
solution (1.2 x 10-5 M) was prepared using chloroform as solvent. To determine the SPF
value promoted by LQFM048 (3), absorbance was measured using a spectrophotometer
at 290, 295, 300, 305, 310, 315 and 320 nm.
The second methodology used the equipment Optometrics SPF-290S analyzer
(Laser Components, Olching, Germany), following the manufacturer’s instruction.
Thus, 110 mg of a solution of LQFM048 (3) dissolved in chloroform (1:1, w/v) was
applied to the TransporeTM tape (3M, MN, USA) (70.7 x 70.7 mm) to obtain a sample
film of 2 μg/cm2, as specified by the U.S. Food and Drug Administration [20]. The
membrane containing the sample was exposed to xenon arc solar simulator, which
performed 12 scans of each sample in different locations on the TransporeTM tape
substrate. Each scan takes a transmittance measurement every 2 nm from 290 to 400
nm, enabling the assessment of SPF by the equipment.
These procedures were repeated using a commercial sunscreen (SPF 30),
ethylhexyl methoxycinnamate and Tinosorb® S at the same concentration of LQFM048
(3), as standards.
2.10. Toxicological evaluation
2.10.1. Evaluation of cytotoxicity of LQFM048 (3) on Balb/c 3T3 cells
2.10.1.1. Cell culture

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Balb/c 3T3-A31fibroblasts were cultured in Dullbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL
penicillin and 100 mg/mL streptomycin, and routinely grown as a monolayer in 75 cm2
tissue culture flasks under standard culture conditions at 37ºC and 5% CO2 in a humid
environment. The cells were examined on a daily basis under a phase contrast
microscope, and any changes in their morphology or adhesive properties were
registered. Cell culture was removed from the culture flasks using trypsinization
(trypsin/EDTA solution, 0.025%:0.02%) when the cells exceeded 50% confluence but
before reaching 80% confluence [21].
2.10.1.2. Neutral red uptake (NRU) assay
The NRU assay was performed according to the standard protocol of
Borenfreund and Puerner [22] modified by NICEATM [23]. Briefly, Balb/c 3T3-A31
fibroblasts (3 x 103 cells/well) were seeded in 96-well plates, after 24 h the cells were
treated with vehicle (DMSO 1%) or eight different concentrations of LQFM048 (3) (4-
490 µM) in complete medium (6 wells per concentration) and incubated. The blank
wells (with no cells) received complete culture medium with or without LQFM048 (3).
After 48 h, the solutions were removed from all plates and the cells were washed with
250 µL/well of pre-warmed PBS. Posteriorly, 250 µL of NR medium were added to all
wells followed by incubation. After 3 h, the NR medium was removed, the cells were
carefully rinsed with 250 µL/well of pre-warmed PBS and 100 µL of NR desorb (50
ethanol : 1 acetic acid : 49 ultrapure water) solution were added to all wells. The plates
were rapidly shaken on a microplate shaker for 20 min to extract NR from the cells and
form a homogeneous solution. Absorbance was measured at 550 nm in a
spectrophotometer. The optical density (OD) was calculated as the difference between
absorbance at the test wavelength and that at the reference wavelength. For each
concentration tested, wells containing all the reagents used but no cells served as
reference blanks.
2.10.1.3. Lactate dehydrogenase (LDH) release assay
Evaluation of LDH release was performed by In Vitro Toxicology Assay Kit,
lactic dehydrogenase-based, (Sigma-Aldrich) as recommend by the manufacturer.

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Briefly, LDH assay mixture was previously prepared by mixing equal amounts of LDH
assay substrate, cofactor and dye solutions. Aliquots of supernatant (50 µL) from 3T3-
A31 cells (3x103 cells/well) exposed to LQFM 048 (3) (245 or 490µM) for 48 h were
added to 100 µL of assay mixture. The samples were incubated at room temperature for
30 min protected from light at a dark room. Absorbance was performed at 490 and 690
nm and the cell viability was calculated as described by Chan et al. [24].
2.10.1.4. Evaluation of acute oral toxicity of LQFM048 (3) in mice
2.10.1.4.1 Animal care
Swiss male mice, weighing between 27 and 31 g, were obtained from Indústria
Química do Estado de Goiás – IQUEGO (Goiânia, GO, Brazil). To ensure the welfare
of mice, body weight loss, food/water consumption and changes in activity and
behavior of the animals were daily checked as a clinical indication of animal suffering
to determine when the animals had to be humanely sacrificed [25]. The animals were
kept under constant environmental conditions with light-dark cycles and controlled
temperature. Water and food were provided ad libitum. The experiments were carried
out in accordance with institutional protocols and the guidelines of the Canadian
Council on Animal Care [26]. All procedures and protocols (FUG no.137/2009) were
reviewed and approved by the Research Ethics Committee of the Federal University of
Goiás. At the end of each experiment, the animals were previously anesthetized with
xylazine (10 mg/kg) and ketamine hydrochloride (100 mg/kg) administered
intraperitoneally and then euthanized by cervical dislocation [25].
2.10.1.4.2. Acute oral systemic toxicity
LQFM048 (3) acute oral toxicity evaluation was carried out according to
OECD Guideline 423 – Acute Toxic Class Method [27]. In short, mice (n=3) were
randomly separated and doses of LQFM048 (3) diluted in sunflower oil were
administered orally (gavage) in each animal (first dose of 0.3 mL and, after 3 h, a
second dose of 0.2 mL). A single dose was not possible, because of the low solubility of
LQFM048 (3) in oil, which would require the administration of a greater volume than
recommended (1mL/100g of body weight on a single dose). Thus, the 2000 mg/kg dose

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was given in smaller fractions over a period not exceeding 24 hours. After treatment,
clinical observations were conducted at 5, 15 and 30 min, and each hour up to the
twelfth hour of the first day. Posteriorly, the mice were examined once a day for an
additional 13 days. The safety of 2000 mg/kg dose of LQFM048 (3) was subsequently
confirmed in other mice as recommended by OECD guideline.
2.11. Statistical analysis
All experiments were carried out in three independent experiments. Statistical
analysis was performed using GraphPad Prism version 5.01 software for windows (San
Diego, CA, USA) when necessary. The inter group variation was measured by one-way
Analysis of Variance (ANOVA) followed by Bonferroni’s test forcytotoxicity assays.
Statistical significance was established as p<0.05.

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3. Results and Discussion
3.1. Planning and synthesis of LQFM048 (3)
The design of a sunscreen compound with broad spectrum and molecular
weight above 500 Daltons [28] was based on the structure and chemical action of
triazine (Tinosorb® S and Univul T 150®) and (E)-ethyl 2-cyano-3-(4hydroxy-3-
methoxyphenyl)acrylate (2) sunscreens. Thus, LQFM048 (3) was originally designed
from molecular hybridization strategy (Fig. 1).
Synthetic route of LQFM048 (Fig. 2) started with (E)-ethyl 2-cyano-3-
(4hydroxy-3-methoxyphenyl) acrylate (2) through Knoevenagel reaction [29], which
was carried out through condensation of vanillin (4) and ethyl cyanoacetate (5), in
water, using morpholine as catalyst. In this process, compound (2), 98% yield, was
obtained after an hour at room temperature [29]. It is noteworthy that compound (2) was
patented in 2007 for use in sunscreen compositions containing a UVA sunscreen,
photostabilizer and antioxidant [30]. Thus, it is clear that there are green approaches for
carrying out organic reactions in water to prepare compounds of industrial interest. In
turn, compound (3) was carried out through aromatic nucleofilic substitution between
compound (2) and 2,4,6-trichloro-1,3,5-triazine (6), using K2CO3 as catalyst, have been
obtained in 80% of yields. As shown in Figure 2, the compound LQFM048 (3) was
obtained in global yields of 80%, after two steps from vanillin (1), a cheap and abundant
natural product.
LQFM048 (3) has an organic UV filter profile due to its aromatic structure
conjugated with a para electron-releasing and electron-acceptor groups [31].
3.2. Analysis of antioxidant potential of LQFM048 (3)
As shown in Figure 3, LQFM048 showed an interesting redox potential. The
native electroactivity of (E)-ethyl 2-cyano-3-(4hydroxy-3-methoxyphenyl)acrylate (2) is
evidenced by anodic peak, 1a, at peak potential, Ep1a, of 0.9 V observed for MCP-1. In
turn, the voltammetric profile observed for MCP-2 was similar to the one observed for
carbon paste (CP) without any modification, being, thus, in agreement with the absence
of electroactive groups. It could be also inferred that compound (2) may present higher
antioxidant properties, but lower oxidative stability than compound (3).

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Regarding DPPH• radical scavenging assay, the results showed no
discoloration observed after time reaction, consequently suggesting that antioxidant
activity of LQFM048 (3) by DPPH• was not detected at the concentrations tested (data
not shown). LQFM048 (3) has no OH groups in its structural formula. Therefore, it is
expected that this compound does not react with DPPH•, since phenol ionization is
crucial in the reactions of phenols with DPPH• in solvents that can support ionization
such as ethanol [32].
3.3. Determination of the thermogravimetric curve of LQFM048 (3)
The decomposition of LQFM048 (3) during its production could be excluded
since the thermogravimetric curve shows that it is thermally stable up to temperatures
around 250°C (Fig. 4). The thermal decomposition occurs in two consecutive mass loss
steps, as shown in DTG curve, with complete degradation of LQFM048 (3). Although
compounds released by thermal degradation cannot be positively identified by TG, the
association with the results of mass spectroscopy is reasonable. The first step occurs at
the temperature range of 250-437ºC with 60.2% weight loss which can be assigned to
the loss of the two of the three C13H12NO3 molecules present in the compound. A
fragmentation pattern in the mass spectrum shows a peak relating to this molecular ion
at m/z 466. The second step occurs at the temperature range of 437-600ºC with 39.8 %
weight loss which can be assigned to the loss of C13H12NO3 and C3N3O3 molecules. In
this case, the fragmentation observed at m/z 133 and m/z 126 may be associated with
these units, respectively. These results demonstrate that the LQFM048 (3) is stable
against temperature rises, since its melting point is 186°C.
3.4. Photostability of LQFM048 (3) upon natural and extreme artificial radiation
Figure 5a shows the photostability curve of LQFM048 (3) with or without sun
exposure. It was observed that LQFM048 (3) showed a low degradation after 150
minutes of sunlight exposure at 39ºC, since its AUC was 0.71 ± 0.01. Forced
photodegradation analysis of LQFM048 (3) was also carried out using a chamber with
uniform light distribution and high intensity levels (Fig. 5a). As expected, the extreme
radiation conditions induced a considerable photodegradation of LQFM048 (3),
especially when irradiated by VIS and VIS+UVA. The AUC values were 0.40 ± 0.13,

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0.73 ± 0.01 and 0.38 ± 0.01 for VIS, UVA and VIS+UVA, respectively. In these
experimental conditions, it was possible to observe a decrease of LQFM048 (3)
concentration with no photoproduct generation after natural and artificial exposure. The
results found here highlight the need to include LQFM048 (3) in a future formulation
with appropriate pharmaceutical adjuvants during the development stage of a sunscreen.
This is an important pharmaceutical strategy to keep the photostability of sunscreens
[33] and studies carried out in these complex mixtures are more relevant from the
consumer’s point of view [17].
3.5. Photophysical characterization of LQFM048 (3)
Steady-state fluorescence emission and excitation spectra were obtained for
LQFM048 (3) in chloroform, toluene, tetrahydrofuran (THF) and acetonitrile (MeCN)
-6
-4
solutions at distinct concentrations, from 10
mol L-1 to 10 mol L-1. In diluted
solutions, maxima of excitation were at 290 and 345 nm, regardless of the solvent used
to produce the dilute solutions, while fluorescence emission was at the range of 310 to
520 nm. These spectra demonstrated luminescence intensities and wavelengths depend
on solvent identity, changing from 360 nm in acetonitrile to 420 in chloroform or
toluene. Moreover, in THF maximum fluorescence is also at 420 nm, but presenting a
shoulder of half intensity at around 350 nm. In general, fluorescence spectra obtained
for these diluted solutions are structureless and excitation/emission spectral overlap is
evident in all solutions. Excitation and fluorescence spectra are shown in
Supplementary Figure 1.
The concentration effect upon excitation and fluorescence spectra of solutions
prepared in these solvents was also evaluated. At higher concentrations, red-shifts of
fluorescence spectra were observed for all solvents, along with a change in the
excitation spectra shapes, which are related to electronic ground state aggregates
formation. Nevertheless, in chloroform solutions, the behavior observed is opposite to
that: at higher concentrations, fluorescence blue-shift is observed as result of electronic
ground state aggregation. This may happen due to the fact that solubility of LQFM048
(3) compound is higher in chloroform than in the other chosen solvents, which causes
electronic excited state stabilization with consequent destabilization of the electronic
ground state, while aggregation gives rise to electronic energy transfer states, which are
formed at higher energy levels. This distinct behavior for distinct solvents is due to a

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balance of interactions involving strong dipole-dipole interactions and weak non-polar
interactions that are modulated by specific characteristics of each solvent in the
presence of LQFM048 (3), such as distinct refractive indexes (n) and dielectric
constants (), which influence their polarizabilities (f), as observed in Table 1.
Acetonitrile, which is the solvent with the highest polarizability, presents the lowest
spectral shift, characterized by the small Stokes Shift value, while in toluene, the
solvent with the lowest polarizability, characterized by the lowest dielectric constant
and, hence, the smallest ability to electrostatically interact with the solute molecules.
Supplementary Figure 2 shows excitation and fluorescence spectra obtained for
solutions in distinct solvents and concentrations. It is not clear, however, if the spectral
behavior of chloroform solutions is related to specific interactions that may occur
between LQFM048 (3) molecules and the solvent or if it is due to general electrostatic
interaction forces that vary with solvent main characteristics. To better understand these
shifts and associate them to general or specific solvent effects, Lippert-Mataga
treatment was carried out on the spectral responses obtained for LQFM048 (3) 10-6 mol
L-1 solutions in chloroform, acetonitrile, toluene and tetrahydrofuran as solvents. At this
concentration, good optical response was obtained with low aggregation occurrence.
Lippert-Mataga plot relates the Stokes shifts of excitation and fluorescence spectra to
the polarizability of each solvent, hence, evidencing the nature of solute-solvent
interactions. Lippert-Mataga plot is shown in Supplementary Figure 3.
This plot evidences a more important effect on the Stokes shift in solvents with
lower polarizabilities, such as toluene, while as solvent polarizability becomes higher,
the shift between excitation and fluorescence maxima becomes smaller, evidencing a
more important spectral overlap in such solvents. Yet, since toluene is an aromatic
solvent, solute-solvent non-polar interactions are important and they are based on -
stacking interaction, which might be the actual reason for the strong Stokes shift.
Therefore, Lippert-Mataga treatment demonstrates the role that solute-solvent specific
interactions play in the the photophysical behavior of LQFM048 (3) are more
significant than the general interactions.
As to obtain an efficient UV light absorber to be applied in sunscreen
formulations, high Stokes shifts are desired. In this sense, to ensure high Stokes shifts
and, therefore, more efficient separation between absorption in the UV region and
energy release as light emitted in Visible region, the essential characteristics of
formulation components must be taken into account. Specially, formulations that enable

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-stacking interactions by interacting with LQFM048 (3) molecules by means of
aromatic rings interactions are preferable. In fact, the nature of interactions between
solute and solvent molecules is determinant to destabilize and/or stabilize the electronic
ground and excited states of LQFM048 (3) molecules and knowing the Stokes shifts in
each system enables us to understand the magnitude of these effects. For instance, if
stabilization of electronic excited states is occurring, a bathochromic effect is observed
and fluorescence spectrum is shifted to longer wavelengths, meaning a decrease in
energy involved in the luminescent process. On the other hand, when destabilization is
occurring, a hypsochromic effect is observed, therefore, fluorescence spectrum is
shifted to shorter wavelengths. If interaction is not effective, excitation and fluorescence
maxima are not shifted, resulting in more important spectral overlaps. To act as an
efficient sunscreen, emission must occur at wavelengths displaced from UV region,
hence, it is desirable that fluorescence emission occurs in a bathochromic way, at the
visible region of the spectrum. Additional, optimized geometry of LQFM048 (3) is
represented in Figure 6.
3.6. Determination of SPF of LQFM048 (3)
Both methodologies used in this study are in vitro techniques, aiming to predict
the effect of compound LQFM048 (3) in protecting the human skin from sunburn and
other undesirable effects of the sun. Recent researches have demonstrated that the
results obtained using them are similar to the results obtained in vivo assays [34].
According to the assay described by Mansur et al. [19], the SPF value was 4.58 while
the ethylhexyl methoxycinnamate showed a SPF value of 2.79, corroborating the
literature data [35]. Thus, LQFM048 (3) showed an increment of 64% in the
photoprotection with regard to ethylhexyl metoxycinamate, an advantageous sunscreen
due to its broad spectrum of action, cost and formulations stability [19]. When the
compound (3) was assayed with the Optometrics SPF-290S analyzer, the SPF value
obtained was 2.28. Its association with ethylhexyl metoxycinamate and Tinosorb® S
obtained values of 13.94 and 22.44. In parallel, a commercial sunscreen (SPF 30)
showed a value of 34.56 in our experimental conditions; while a new value of 40.52 was
obtained when LQFM048 (3) was incorporated to this sunscreen product. According to
our data, LQFM048 (3) presented good results of SPF and in association with other
photoprotective compounds as well as with a commercial sunscreen.

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3.7. Evaluation of cytotoxicity of LQFM048 (3) on Balb/c 3T3 cells
Basal cell viability assays provide the first information about the effect of
unknown substances on cellular lethal endpoints and therefore may serve as suitable
predictor for acute oral systemic toxicity [36]. Thus, 3T3 fibroblasts were exposed to
eight different concentrations of LQFM048 (3) (4 - 490 µM) and the basal cytotoxicity
was evaluated by NRU assay. LQFM048 (3) promoted an increase of cell viability
when compared to control, mainly on the first seven concentrations (p<0.005) (Fig. 7a).
Thus, this substance was not able to promote cytotoxicity for fibroblasts even to the
highest concentrations (245 and 490 µM) that also showed a general morphology
similar to the control group (Fig. 7b). Similar results, no cytotoxic profile of LQFM048
(3), were also found by the LDH release assay (Fig. 8).
2.8. Acute oral systemic toxicity evaluation of LQFM048 (3) in mice
The dose selection for oral toxicity test was defined by the NRU results. Since
there was very low cytotoxicity on 3T3-A31 cells, it was possible to estimate the
starting dose for acute oral toxicity at 2000 mg/kg. On the first step, this dose produced
no signs of toxicity or mortality in the animals during the 14 days of the study period.
On the second step, after confirmation of safety of the 2000 mg/kg dose of LQFM048
(3), one death was observed on the day after the dosage of the animals. On both steps,
no weight loss, alteration of consumption of water/food or macroscopic alteration in the
organs of the other animals were detected (data not shown). Thus, LQFM048 (3) was
classified at category 5 of Harmonized Classification System for Chemical Substances
and Mixtures (GSH) and LD50 (single dose that can promote death in 50 per cent of
animals when administered by oral route) determined as 2.000 mg/kg > LD50< 5.000
mg/kg. According to OECD [27], substances classified under category 5 are usually of
relatively low acute toxicity hazard.
4. Conclusions

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The new photoprotective compound LQFM048 (3) was obtained in high global
yields using a green synthesis method and showed thermally and oxidative stable. In
addition, it has no cytotoxic effects on basal cells and showed low oral systemic acute
toxicity. Therefore, this compound is promising once it may be possible to develop a
potential new sunscreen that can offer an alternative photoprotective compound with
better results than those currently used and that probably will also be cheaper in terms
of obtaining process More studies are being accomplished to determine the safety and
efficacy of this compound.
Acknowledgements
The authors acknowledge the Fundação de Apoio à Pesquisa - UFG (FUNAPE),
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de
Estudos e Projetos (FINEP) and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) for funding. The authors thank to Angelo da Cunha Pinto (in
memorian) from (Chemistry Institute, Federal University of Rio de Janeiro, RJ, Brazil)
for the use of the mass spectroscopy.
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Figure captions
Figure 1. Design of LQFM048 (3) from Uvinul® T 150 (1) and (E)-ethyl 2-cyano-3-
(4hydroxy-3-methoxyphenyl)acrylate (2) sunscreens.
Figure 2. Synthetic route of LQFM048 (3).
Figure 3. Representative cyclic voltammograms obtained for MCP-1, MCP-2 and CP,
in pH 7.0, 0.1 M phosphate buffer. Scan rage from -0.25 to 1.25 V, scan rate of 100 mV
s-1.
Figure 4. Representative thermogravimetric analysis of LQFM048 (3). About 4.0 mg of
the compound were heated under synthetic air atmosphere from 30 to 600ºC, at a flow
rate of 50 mL min-1 and a heating rate of 10ºC/min.
Figure 5. Photostability of LQFM048 (3) upon natural and extreme artificial radiation.
(A) The compound was exposed to natural sunlight at radiation dose of 54.2 W.h/m2 for
150 min at 39ºC ± 2. (B) The LQFM048 (3) was exposed to UVA (200 Wh/ m2), VIS
(1.2 million lux) and to both radiation under controlled temperature of 25ºC, following
recommendations of ICH Q1B, option 2. The figure shows a representative result in one
of the three replicates.
Figure. 6. Optimized geometry of LQFM048 (3).
Figure 7. Effect of LQFM048 (3) on cell viability of Balb/c 3T3-A31 fibroblasts using
neutral red uptake (NRU) assay. (A) Cells (3x103 cells/well) were treated with vehicle
(DMSO 1%) or eight different concentrations of LQFM048 (3) (4-490 µM) for 48 h.
Experiments are expressed as mean ± SD of three independent experiments in triplicate.
(*p<0.05 and ***p<0.0001 vs. control. One way ANOVA and Bonferroni test). (B)
General morphology of cells after treatment without or with the highest concentrations
of LQFM048 (3) (245 and 490 µm).
Figure 8. Effect of LQFM048 (3) on cell viability of Balb/c 3T3-A31 fibroblasts using
lactate dehydrogenase (LDH) release assay. Cells (3x103 cells/well) were treated with

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vehicle (DMSO 1%) or the highest concentrations of LQFM048 (3) (245 and 490 µm)
for 48 h. Experiments are expressed as mean ± SD of three independent experiments in
triplicate. (Data with no statistical difference. One way ANOVA and Bonferroni test).

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Tables and figures
Table 1. Deelectric constants, refractive indexes, calculated polarizabilities of solvents
used in this work and spectral Stokes shift obtained from excitation and fluorescence
spectra of LQFM048 (3) diluted solutions.
Solvent
C7H8
Ɛ
n
Δf
F-A (cm-1)
2.38
4.81
7.58
37.50
1.492
1.444
1.407
1.340
0.0147
0.1489
0.2097
0.3067
4,511
CHCl3
THF
4,524
4,349
MeCN
2,659

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Figure 1.

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Figure 2.

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Figure 3.