Synthesis, antioxidant and photoprotection activities of hybrid derivatives useful to prevent skin cancer

Article abstract of DOI:10.1016/j.bmc.2014.03.017

Chronic ultraviolet (UV) radiation exposure is a major cause of skin cancer. A novel series of hybrid derivatives (I-VIII) for use in sunscreen formulations were synthesized by molecular hybridization of t-resveratrol, avobenzone, and octyl methoxycinnamate, and were characterized. The antioxidant activity values for VIII were comparable than to those of t-resveratrol. Compounds I-III and VI demonstrated Sun Protector Factor superior to that of t-resveratrol. Compounds I and IV-VIII were identified as new, broad-spectrum UVA filters while II-III were UVB filters. In conclusion, novel hybrid derivatives with antioxidant effects have emerged as novel photoprotective agents for the prevention of skin cancer.

Full text of DOI:10.1016/j.bmc.2014.03.017

Bioorganic & Medicinal Chemistry 22 (2014) 2733–2738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antioxidant and photoprotection activities of hybrid
derivatives useful to prevent skin cancer
⇑
Juliana Santana Reis, Marcos Antonio Corrêa, Man Chin Chung, Jean Leandro dos Santos
Lapdesf—Laboratório de Pesquisa e Desenvolvimento de Fármacos, Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas, Universidade Estadual
Paulista–UNESP, Rodovia Araraquara Jaú Km 01, 14801-902 Araraquara, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Chronic ultraviolet (UV) radiation exposure is a major cause of skin cancer. A novel series of hybrid deriv-
atives (I–VIII) for use in sunscreen formulations were synthesized by molecular hybridization of
t-resveratrol, avobenzone, and octyl methoxycinnamate, and were characterized. The antioxidant activity
values for VIII were comparable than to those of t-resveratrol. Compounds I–III and VI demonstrated
Sun Protector Factor superior to that of t-resveratrol. Compounds I and IV–VIII were identified as new,
broad-spectrum UVA filters while II–III were UVB filters. In conclusion, novel hybrid derivatives with
antioxidant effects have emerged as novel photoprotective agents for the prevention of skin cancer.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 20 January 2014
Revised 28 February 2014
Accepted 10 March 2014
Available online 19 March 2014
Keywords:
Sunscreens
Photoprotection
Antioxidant
UV radiation
UV filters
t-Resveratrol
Avobenzone
Octyl methoxycinnamate
Molecular hybridization
1. Introduction
photoprotection. It is well established that chronic exposure to
UV radiation is one of the main causes of skin cancer, and clinical
The incidence and mortality of skin cancer are increasing world-
wide, and skin cancer now accounts for one in three of all diag-
nosed cancers.1 The most common skin cancers are melanoma
and non-melanoma (basal cell carcinoma and squamous cell carci-
noma), with malignant melanoma being the main cause of death.2
It was estimated that 1 in 49 people will be diagnosed with malig-
nant melanoma during their lifetime, particularly in people ex-
posed to certain risk factors, including constant exposure to
ultraviolet (UV) radiation.3,4
Although there is widespread concern over the importance of
using sunscreen formulations to prevent skin cancer, epidemiol-
ogic data are still showing progressive increases in its morbidity
and mortality rates. In the United States of America, for example,
over 3.5 million cases of skin cancer are diagnosed annually.5
The preventive behavior is complex and, in most cases, the
incorrect use of sunscreen formulations results in inadequate
studies have demonstrated that increased frequency of sunburns
increase the risk of melanoma.6 UV radiation can directly damage
DNA through the formation of DNA adducts and/or ROS generation,
which promote tumor formation through different pathways.7
UV radiation is divided into ultraviolet A (UVA, 320–400 nm),
ultraviolet B (UVB, 280–320 nm), and ultraviolet C (UVC, 100–
280 nm). UVA and UVB have direct effects on genetic material.8
The direct genotoxic effects of UV radiation are due to the forma-
tion of dimeric photoproducts between adjacent bases in DNA.9
UV radiation also has indirect effects mediated by increased Reac-
tive Oxygen Species (ROS) levels, which (a) regulate cell processes
associated with malignant transformation; (b) cause DNA damage-
induced mutations; (c) alter the activity of the pro-survival path-
way; and (d) promote skin aging.10,11
It was reported that antioxidants could prevent ROS-induced
DNA damage.12 In particular, t-resveratrol has potent antioxidant
activity exceeding those of vitamins E and C,13 and might also pro-
tect against UV radiation. Because antioxidant compounds, such as
t-resveratrol, have chemoprotective properties,14 the development
Abbreviations: DPPHÅ, 2,2-diphenyl-1-picrylhydrazyl radical; t-resveratrol,
trans-resveratrol; FDA, Food and Drug Administration; ROS, Reactive Oxygen
Species; UVR, Ultraviolet Radiation; SPF, Sun Protector Factor.
of novel photoprotectors with antioxidant properties is
a
⇑
Corresponding author. Tel.: +55 16 33016972; fax: +55 16 33016960.
E-mail address: santosjl@fcfar.unesp.br (J.L. dos Santos).
http://dx.doi.org/10.1016/j.bmc.2014.03.017
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2734
J. S. Reis et al. / Bioorg. Med. Chem. 22 (2014) 2733–2738
promising strategy for the development of sunscreen formulations
with improved photoprotective properties.
free radical to 2,2-diphenyl-1-picrylhydrazyl, which is accompa-
nied by a change in color from deep-violet to light-yellow. All com-
pounds were incubated in the microplate for 60 min at
The mechanisms involved in the protective effects of resveratrol
and other polyphenols against UV radiation are complex, but seem
to involve the modulation of cellular signaling pathways, anti-
inflammatory activities, induction of cytokines such as interleu-
kin-12 (IL-12), prevention of UVB-induced immunosuppression,
and upregulation of genes encoding nucleotide excision repair
(NER) enzymes.15–18 In an in vivo study using SKH-1 hairless mice,
t-resveratrol inhibited UVB-induced skin edema and the
production of hydrogen peroxide.19 In another study, t-resveratrol
protected mouse skin against UVB-induced skin damage by modu-
lating survival pathways.20 Furthermore, it has been suggested that
t-resveratrol blocks UVB radiation, in particular.21
Avobenzone and octyl methoxycinnamate are widely used in
sunscreen formulations to protect against UVA and UVB, respec-
tively. However, these compounds have some limitations, includ-
ing photo-instability, low efficacy if used alone, absence of
antioxidant effects, potential allergenic effects, and incompatibility
with other components used in commercial sunscreen formula-
tions.22–24 Molecular modification strategies, such as hybridiza-
tion, represent a powerful tool that might facilitate the discovery
of new photoprotective compounds that are effective, stable and
safe.
In a continuing effort to develop new candidate sunscreens to
prevent skin cancer and radiation-induced oxidative stress, this re-
port describes the synthesis, antioxidant activity, and photoprotec-
tive effects of novel hybrid derivatives (I–VIII), which were
obtained by molecular hybridization of the prototypes
t-resveratrol (1), octyl methoxycinnamate (2), and avobenzone
(3) (Fig. 1). The selections of each subunit are due contributions
to (a) antioxidant effect (phenolic hydroxyl in A, B and D subunits);
(b) ability to promote electronic conjugation and shift the absorp-
concentrations of 1000, 300, 100, and 35 lM. Although the test
was also conducted for 30 min, the results were similar for both
times (results not shown). All compounds, except VI, had antioxi-
dant properties. The most potent compounds were I (inhibitory
concentration 50% [IC50] = 275
(IC50 = 109.6 M). The compounds VII and VIII were more potent
than t-resveratrol (IC50 = 110 M) but were less potent than ascor-
bic acid (IC50 = 64.2 M), which was used as a control. The pres-
ence of ,b-unsaturated carbonyl moieties for V–VIII seems to
lM), VII (IC50 = 88.2 lM), and VIII
l
l
l
a
increase the compound’s antioxidant activity if at least one of the
aryl rings is substituted with a hydroxyl group at the para position.
As previously reported by some authors, N-acyl hydrazone spacer
could extend delocalized p-electron in the structure. This effect al-
lows conjugated electrons to flow between the aromatic rings and
for this subunit to react with high-energy oxygen species.26–29 This
effect is particularly relevant because UV radiation induces ROS,
which promote skin cancer development and photoaging.
In addition, in vitro photoprotection analyses were determined
using an Optometric 290S analyzer (SPF-290S; Optometrics, Ayer,
MA, USA), and the results were manipulated using WinSPF soft-
ware version 4.1 (Optometrics). High correlations between SPF-
290S in vitro measurements and in vivo tests were reported for a
variety of formulations, including creams, lotions, gels, and
sprays.30 Sun Protector Factor (SPF), UVA Protection Factor, UVA/
UVB ratio, and the critical wavelength (kC) were determined using
a stable neutral cream containing 7% of compounds I–VIII, avo-
benzone, benzophenone-3, t-resveratrol and octyl methoxycinna-
mate (Table 2). Avobenzone and benzophenone-3 as UVA filters
and resveratrol and octyl methoxycinnamate as UVB filters were
used as reference. Table 2 shows that the SPF values for com-
pounds I–III and VI were superior to that of resveratrol (SPF = 2).
All compounds demonstrated superior activity to that of benzo-
phenone-3 (SPF = 1). Interestingly, the most active compound (II)
(SPF = 5) is structurally related to resveratrol (1), having an
N-acyl-hydrazone subunit that extends electron conjugation
(Scheme 1). Generally, organic UV filters contain a chromophore
tion maximum to higher wavelengths (aryl-a,b-insaturation in
subunit C); and (c) free radical stabilization (subunit E). Therefore,
this approach aims to combine UVA and UVB photoprotection and
antioxidant activity in the same molecule to improve the efficacy
of sunscreen formulations.
that is conjugated by the p-electron system. Therefore, increasing
2. Chemistry
the conjugation, until certain limits, shifts the absorption maxi-
mum to higher wavelengths, increasing the molecule’s ability to
absorb UV radiation. Very large conjugation systems could have
maxima of absorption bathochromic shifted out of the UV range.
All synthesized compounds were less active than octyl meth-
oxycinnamate (SPF = 13), however this filter has a lot of inconve-
niences such as photo-instability and absence of antioxidant
effects. Compounds IV, V, VII and VIII had similar activities to that
of t-resveratrol. Polonini et al. developed several t-resveratrol
analogs as potential photoprotectors, but the SPF value of the most
active compound was only 1.42 times greater than that of t-resve-
ratrol.31 Despite the apparent reductions in SPF values of t-resvera-
trol analogs, it is important to acknowledge that sunscreen
formulations contain multiple filters to achieve high SPF values.
The UVA protection factor values of compounds II, III, and VI
were 1.5–2 times greater than that of t-resveratrol, but were less
than that of avobenzone. Meanwhile, the UVA protection factor
values of compounds I, IV, V, VII, and VIII were similar to that of
resveratrol. Sunscreen formulations that can block UVA are desir-
able because this type of radiation has immunosuppressive and
mutagenic effects in humans and animals.32,33 The critical wave-
length (kC), another UVA parameter, is classified by the United
States Food and Drug Administration into five categories, as fol-
lows: 0 (kC <325 nm), 1 (325–335), 2 (335–350), 3 (350–370),
and 4 (P370).34 According these categories, compounds I and
IV–VIII were scored as ‘4’, like avobenzone and benzophenone-3,
The synthetic routes for preparing the hybrid derivatives (I–
VIII) are summarized in Scheme 1. The coupling reactions between
the previously selected aldehydes and hydrazides were catalyzed
with 37% hydrochloric acid, and generated the corresponding
hydrazone derivatives (I–VIII) with yields of 70–99%. The struc-
tures of all of the compounds were established by elemental anal-
ysis, infrared (IR) spectroscopy, and 1H- and 13C-nuclear magnetic
resonance. All compounds were analyzed by high-performance li-
quid chromatography, and the purities were >98.5%. The 1H-nucle-
ar magnetic resonance spectra of all hybrid derivatives exhibited
one peak, corresponding to the ylidenic hydrogen of the
E-diastereomer.25
3. Results and discussion
The new hybrid compounds were generated from hydrazone
derivatives (I–VIII), as E diastereoisomers, with excellent yields
(70–99%). All hybrid compounds were obtained with a purity
>98.5% and were characterized using standard analytical methods.
The antioxidant activities of the hybrid compounds (I–VIII) and
positive controls were evaluated by measuring their free radical
scavenging capacities against test compounds using the adapted
DPPHÅ microplate assay (Table 1). This assay measures the hydro-
gen-donating ability of antioxidants to convert the stable DPPH

J. S. Reis et al. / Bioorg. Med. Chem. 22 (2014) 2733–2738
2735
C
O
O
O
A
octyl methoxycinnamate
D
E
2
)
(
HO
B
O
O
OH
Molecular Hybridization
HO
OH
resveratrol
avobenzone
1
)
3
)
(
(
B
B
A
3
OH
3
OH
2
5
3
HO
D
1
D
2
4
5
2
4
O
4''
E
O
4''
O
4''
2''
N
2''
N
2''
N
3'
6'
4
3'
6'
3'
5
4'
5'
6
2'
1'
4'
5'
4'
5'
2'
1'
2'
1'
N
H
1
1
HO
N
H
HO
N
H
1''
6
1''
6
1''
8'
8'
3''
7'
8'
3''
3''
OH
OH
6'
(I)
(II)
(III)
A
7
7
E
D
C
2
C
2
2
5
HO
1
O
3
O
3
E
3
O
4''
O
O
1
6
2''
N
4''
N
1
6
3'
4''
N
4
2''
3'
2''
3'
4'
6
2'
1'
4'
2'
1'
4'
2'
1'
N
H
4
6''
N
4
6''
N
5
1''
5
1''
3''
8'
8'
1''
3''
H
5''
8'
8'
H
5''
7'
8'
7'
5'
3''
5'
5'
OH
6'
6'
6'
8'
(V)
(VI)
(IV)
E
D
C
C
6
6
3
1
HO
HO
1
5
5
O
O
6''
4''
N
4''
N
2''
3'
6'
2
2''
3'
6'
2
4'
2'
1'
4'
5'
2'
1'
4
6''
O
7
N
4
O
7
N
3
1''
3''
1''
3''
H
5''
8'
8'
H
5''
7'
8'
5'
OH
(VIII)
(VII)
Figure 1. Molecular hybridization among t-resveratrol, octyl methoxycinnamate and avobenzone subunits to design new photoprotector compounds.
R1
R1
O
R2
R3
O
O
R2
R3
R1
R2
H
O
R1
R2
H
N
N
H
N
N
H
R3
5a-b
)
5a-b
(
(
)
R3
O
OH
a)
O
a)
H2N
I
N
H
III
R1= OH; R2 = H; R3= OH ( ) (Yield: 83%)
R1= H; R2 = OH; R3= H ( ) (Yield: 99%)
R1= OH; R2 = H; R3= OH (II) (Yield: 99%)
H2N
N
H
R1= H; R2 = OH; R3= H (IV) (Yield: 93%)
OH
a)
7
)
(
4
(
)
a)
H
R4
R4
O
R5
O
)
R5
R4
R5
O
H
O
R4
R5
N
N
N
H
N
H
6a-b
(
)
6a-b
(
OH
V
4 = H; R5 = OCH3 ( ) (Yield: 85%)
4 = OCH3; R5 = OH (VII) (Yield: 70%)
R
R
VI
R
R
4 = H; R5 = OCH3
(
) (Yield: 96%)
4 = OCH3; R5 = OH (VIII)(Yield: 89%)
Scheme 1. Reagents and conditions: (a) EtOH, HCl., rt, 24 h (70–99%).

2736
J. S. Reis et al. / Bioorg. Med. Chem. 22 (2014) 2733–2738
Table 1
Radical scavenging activity of compounds I–VIII expressed as the percent reduction of the absorbance of DPPH at 519 nm (k) after incubation for 60 min. Ascorbic acid and t-
resveratrol were used as standards. The values represent means the standard deviation of the data (n = 3)
Compounds
1000
l
M
300
l
M
100
l
M
35
lM
IC50 (lM)
a
a,b
I
II
III
IV
V
VI
VII
VIII
65.0 3.4
34.2 5.9
31.3 1.2
38.7 1.4
2.8 1.6
ꢀ7.1 2.2
75.5 3.1
76.1 3.8
81.2 2.3
73.8 0.3
54.4 9.2
26.5 6.7
20.1 1.4
28.9 0.7
ꢀ2.6 1.4
ꢀ5.6 2.3
77.2 6.0
75.1 3.3
80.7 1.4
71.3 1.1
38.0 9.2
11.2 9.0
10.9 1.2
15.0 1.4
ꢀ1.5 0.6
ꢀ8.3 1.7
56.7 1.4
45.6 0.1
77.9 1.7
45.5 0.3
18.4 4.7
5.6 6.3
7.0 2.9
10.2 2.5
ꢀ3.4 3.2
ꢀ6.6 2.2
34.8 6.0
20.0 0.4
18.8 8.6
20.5 3.0
275
—
—
—
—
—
88.2
109.6
64.2
110
a,b
a,b
a,b
a,b
a
a,b
Ascorbic acid
t-Resveratrol
a
p <0.05 compared with t-resveratrol.
p <0.05 compared with ascorbic acid.
b
Table 2
broad-spectrum UVA filters while II and III were UVB filters, with
greater SPF values than t-resveratrol. In conclusion, the hybrid
derivatives with antioxidant effect are novel prototypic com-
pounds that could be used in sunscreen formulations used for
the prevention of skin cancer.
Photoprotection activity of compounds I–VIII, t-resveratrol, octyl methoxycinnamate,
avobenzone and benzophenone-3
Compounds
SPFa
UVAPFb
UVA/UVB ratio
kC (nm)
I
II
III
IV
V
VI
VII
VIII
3
5
4
2
2
4
2
2
2
0.1
0.2
0.3
0.1
0.1
0.2
0.1
0.1
0.1
2
4
3
2
2
4
2
2
2
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.8
0.6
0.6
0.8
1.1
1.0
1.2
1.1
0.6
—
376
368
366
379
388
386
389
387
365
334
382
371
5. Experimental
5.1. General
Melting points were measured with an electrothermal melting-
point apparatus (SMP3, Bibby Stuart Scientific) in open capillary
tubes and are uncorrected. Infrared spectra (KBr disc) were pro-
duced on an FTIR-8300 Shimadzu and the frequencies expressed
t-Resveratrol
Octyl-methoxycinnamate
Avobenzone
Benzophenone-3
13 0.1
—
1
—
39 1.0
—
1.8
0.6
0.1
in cmꢀ1 1H NMR and 13C NMR spectra were scanned on a Bruker
.
a
SPF: Sun Protection Factor.
UVAPF: UVA Protection Factor.
b
DRX-400 (300 MHz) NMR spectrometer using DMSO-d6 as the sol-
vent. Chemical shifts were expressed in parts per million (ppm) rel-
ative to tetramethylsilane. The coupling constants are reported in
hertz (Hz) and signal multiplicities are reported as singlet (s), dou-
blet (d), doublet of doublet (dd), multiplet (m). Elemental analyses
(C, H, and N) were performed on a Perkin Elmer model 240C ana-
lyzer and the data were within 0.4% of the theoretical values. HPLC
analysis was performed on a Shimadzu LC-10AD chromatograph
equipped with a model SPD-10A UV–vis detector (Shimadzu). All
compounds were analyzed by HPLC, and their purity was confirmed
to be greater than 98.5%. The compounds were separated on a re-
whereas t-resveratrol and compounds II–III were scored as ‘3’. Oc-
tyl methoxycinnamate was scored as ‘1’. It was reported that sun-
screen formulations with a kC near 400 nm, including those scored
as ‘4’ could block a broad spectrum of UV radiation.31 The UVA/UVB
ratio represents a ratio of the areas under the curves for UVA and
UVB radiation in the range of 290–400 nm. It is related to UVA
photoprotection and is the basis for the Boots Star Rating classifi-
cation.35 Using this classification, benzophenone-3 was scored as
‘2 stars’ displaying ‘good’ protection against UVA. Compounds II
and III were scored as ‘3 stars’, like t-resveratrol. This category dis-
plays ‘superior’ protection against UVA. Meanwhile, compounds I
and IV was scored as ‘4 stars’, which represents ‘maximum’ protec-
tion against UVA. Compounds V–VIII had the highest scores, of ‘5
stars’, which represents ‘ultra’ protection against UVA. Taken to-
gether, these results indicate that compounds I and IV–VIII had
superior UVA blocking to t-resveratrol, but were inferior to avo-
benzone. Therefore, compounds I and IV–VIII are effective UVA fil-
ters while compounds II and III are effective UVB filters.
versed phase C18 column (5
l
m particle, 250 mm ꢁ 4.6 mm I.D.)
Shimadzu Shim-pack CLC-ODS (M). HPLC-grade solvents (acetoni-
trile, methanol, acetic acid, and toluene) were used in the analyses
and were bought from a local supplier. The progress of all reactions
was monitored by TLC, which was performed on 2.0 ꢁ 6.0 cm2 alu-
minum sheets precoated with silica gel 60 (HF-254, Merck) to a
thickness of 0.25 mm. The developed chromatograms were viewed
under UV light (254 nm and 365 nm) and treated with iodine vapor.
Merck silica gel (70–230 mesh) was used for preparative column
chromatography. Reagents and solvents were purchased from com-
mercial suppliers and used as received.
The compounds 4-hydroxybenzhydrazide (4), 4-hydroxybenz-
aldehyde (5a), 3,5-dihydroxybenzaldehyde (5b), trans-p-methoxy-
cinnamaldehyde (6a), 4-hydroxy-3-methoxycinnamaldehyde (6b)
and 4-(tert-butylbenzoic)hydrazide (7) were purchased commer-
cially from Sigma–Aldrich (St. Louis, MO, USA).
4. Conclusions
A novel series of hybrid derivatives (compounds I–VIII) were
synthesized by molecular hybridization of t-resveratrol, avobenz-
one, and octyl methoxycinnamate, and were characterized by ele-
mental analysis, IR spectroscopy, and 1H and 13C NMR. All of the
compounds, except VI, demonstrated antioxidant activity using
the DPPHÅ method. The most potent molecules were VII
5.2. Chemistry
5.2.1. General procedures for the preparation of hybrid
derivatives (I–VIII)
The corresponding aromatic aldehyde (6a–b; or 7a–b)
(1.64 mmol) was added to a solution of previously selected
(IC50 = 88.2
l
M), and VIII (IC50 = 109.6
lM), with activities superior
M). The SPF values for I–IV and
to that of t-resveratrol (IC50 = 110
VI were also greater than that of t-resveratrol (SPF = 2), with com-
pound II having the highest SPF (5). Compounds I and IV–VIII were
l

J. S. Reis et al. / Bioorg. Med. Chem. 22 (2014) 2733–2738
2737
0
0
hydrazide derivatives (5 or 8) (1.64 mmol) in 15 mL of ethanol, in
the presence of 0.1 mL of 37% hydrochloric acid. The reaction was
stirred for 24 h at room temperature and monitored by TLC. After-
wards, the solvent was partially concentrated at reduced pressure
and the resulting mixture was poured into cold water. The precip-
itate was collected by filtration, washed with cold ethanol and
dried under vacuum to give the hybrid derivatives containing the
N-acyl hydrazone subunit (I–VIII). All compounds were recrystal-
lized in ethanol in excellent yields (70–99%). If necessary, the com-
pounds can be purified by column chromatography (flash silica,
eluent: 40% ethyl acetate; 60% hexane).
(C7 ); 30.91 (C8 ) ppm. Anal. Calcd for C18H20N2O2: C, 72.95; H,
6.80; N, 9.45. Found: C, 73.12; H, 7.13; N, 9.78.
5.2.1.5.
ene)benzohydrazide (V).
(E)-4-Hydroxy-N0-((E)-3-(4-methoxyphenyl)allylid-
Yellow solid. Yield: 85%, decompo-
sition: 271 °C. IR mmax (cmꢀ1; KBr pellets): 3290 (OAH), 3286
(NAH), 2966 (CAH methyl); 1618 (C@O amide), 1604 (C@N imine),
1582, 1551 and 1450 (C@C). NMR 1H (300 MHz, DMSO-d6) d: 11.46
00
(1H; s, NAH; H5 ); 10.10 (1H; s; OAH; H1); 8.17–8.19 (1H; d,
CAHimine; J = 6.9 Hz, H3 ); 7.77–7.80 (2H; d, Jorto = 8.8 Hz, H3 and
H5 ); 7.55–7.58 (2H; dd, J = 8.8 Hz, H1 and H2 ); 6.94–6.97 (4H;
00
0
0
00
00
0
m, H2, H3, H5 and H6); 6.84–6.87 (2H; d, Jorto = 8.7 Hz, H2 and
0
5.2.1.1. (E)-4-Hydroxy-N0-(4-hydroxybenzylidene)benzohydraz-
H6 ), 3.79 (3H; s, H7) ppm. 13C NMR (75 MHz, DMSO-d6) d:
ide (I)36
.
White solid. Yield: 99%, mp: 270–273 °C. IR mmax
162.49 (C6 ); 160.53 (C1 ); 159.78 (C1); 149.19 (C3 ); 138.21 (C2 );
00
0
00
00
(cmꢀ1; KBr pellets): 3470 (OAH), 3265 (NAH), 1645 (C@O amide),
129.57 (C4 ); 128.67 (C1 ); 128.50 (C3 and C5); 123.95 (C3 and
0
00
0
1610 (C@N imine), 1600 and 1550 (C@C). NMR 1H (300 MHz,
C5 ); 123.49 (C4); 114.93 (C2 and C6 ); 114.27 (C2 and C6); 55.19
(C7) ppm. Anal. Calcd for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45.
Found: C, 69.13; H, 5.79; N, 9.51.
0
0
0
00
DMSO-d6) d: 11.44 (1H; s, NAHamide, H3 ); 9.95 (2H; s; OAH, H1
0
00
0
and H1 ); 8.32 (1H; s, CAHimine, H1 ); 7.78–7.81 (2H; d, H3 and
0
H5 ); 7.53–7.56 (2H; dd, Jorto = 8,3 Hz; H3 and H5); 6.82–6.87 (4H;
m; H2, H6, H2 and H6 ) ppm. 13C NMR (75 MHz, DMSO-d6) d:
5.2.1.6.
(E)-4-(tert-Butyl)-N0-((E)-3-(4-methoxyphenyl)allylid-
0
0
00
0
00
0
162.61 (C4 ); 160.47 (C1); 159.19 (C1 ); 147.26 (C1 ); 129.51 (C4 );
ene)benzohydrazide (VI).
Yellow solid. Yield: 96%, mp:
; KBr pellets): 3472 (OAH), 3221
128.68 (C4); 125.47 (C3 and C5 ); 124.06 (C3 and C5); 115.66 (C2
and C6 ); 114.94 (C2 and C6 ) ppm. Anal. Calcd for C14H12N2O3: C,
65.62; H, 4.72; N, 10.93. Found: C, 65.95; H, 5.03; N, 11.27.
188–190 °C. IR mmax (cmꢀ1
0
0
0
0
0
0
(NAH), 2956 (CAH methyl); 1657 (C@O amide), 1606 (C@N imine),
1581, 1550 and 1452 (C@C). NMR 1H (300 MHz, DMSO-d6) d: 11.58
0
00
(1H; s, NAH; H5 ); 8.18–8.21 (1H; d, CAHimine; Jorto = 8.0 Hz, H3 );
5.2.1.2. (E)-N0-(3,5-Dihydroxybenzylidene)-4-hydroxybenzohyd-
7.81–7.84 (2H; d, Jorto = 8.4 Hz, H3 and H5 ); 7.51–7.59 (4H; m,
0
0
00
00
0
0
razide (II).
White solid. Yield: 98%, decomposition: 269 °C. IR
Jorto = 8.8 Hz, Jmeta = 4.6 Hz, H1 , H2 , H2 and H6 ); 6.95–6.97 (4H;
mmax (cmꢀ1; KBr pellets): 3401 (OAH), 3208 (NAH), 1647 (C@O
m, H2, H3, H5 and H6); 3.79 (3H; s, H7); 1.31 (9H; s, H8 ) ppm. 13C
NMR (75 MHz, DMSO-d6) d: 162.78 (C6 ); 159.85 (C1 and C4 );
0
amide), 1607 (C@N imine), 1601 and 1551 (C@C). NMR 1H
00
0
00
00
00
0
00
(300 MHz, DMSO-d6) d: 11.50 (1H; s, NAH; H3 ); 10.10 (1H; s;
149.86 (C3 ); 138.67 (C2 ); 130.74 (C1 ); 128.62 (C1 ); 128.58 (C3
0
0
0
0
0
OAH; H1 ); 9.42 (2H; s; OAH; H1 and H3); 8.21 (1H; s, CAHimine
;
and C5); 127.41 (C3 and C5 ); 125.16 (C2 and C6 ); 123.37 (C4);
00
0
0
0
H1 ); 7.77–7.79 (2H; dd, Jorto = 8.7 Hz and Jmeta = 2.0 Hz, H3 and
114.28 (C2 and C6); 55.21 (C7); 34.65 (C7 ); 30.90 (C8 ) ppm. Anal.
Calcd for C21H24N2O2: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.86;
H, 6.98; N, 8.02.
0
0
H5 ); 6.83–6.85 (2H; dd, Jorto = 8.8 Hz and Jmeta = 2.0 Hz, H2 and
0
H6 ); 6.57 (2H; s; H4 and H6); 6.24 (1H; dd; Jmeta = 2.2 Hz, H2)
13
0
0
ppm. C NMR (75 MHz, DMSO-d6) d: 162.69 (C4 ); 160.62 (C1 );
158.67 (C1 and C3); 147.24 (C1 ); 136.19 (C5); 129.62 (C4 );
5.2.1.7. (E)-4-Hydroxy-N0-((E)-3-(4-hydroxy-3-methoxyphenyl)
00
0
0
0
0
0
123.94 (C3 and C5 ); 115.00 (C2 and C6 ); 105.10 (C6 and C7);
104.31 (C2) ppm. Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44;
N, 10.29. Found: C, 61.98; H, 4.71; N, 10.44.
allylidene)benzohydrazide (VII).
Yellow solid. Yield: 70%,
mp: 241–243 °C. IR mmax (cmꢀ1; KBr pellets): 3364 (OAH), 3252
(NAH), 2953 (CAH methyl); 1622 (C@O amide), 1609 (C@N imine),
1587, 1553 and 1454 (C@C). NMR 1H (300 MHz, DMSO-d6) d: 11.41
(E)-4-(tert-Butyl)-N0-(3,5-dihydroxybenzylidene)ben-
(1H; s, NAH, H5 ); 10.07 (1H; s, OAH, H1 ); 9.32 (1H; s, OAH, H1);
00
0
5.2.1.3.
zohydrazide (III).
00
White solid. Yield: 83%, decomposition:
8.14–8,16 (1H; d, CAHimine
,
Jorto = 7.7 Hz; H3 ); 7.77 (2H; d,
233 °C. IR mmax (cmꢀ1; KBr pellets): 3420 (OAH), 3265 (NAH),
2963 (CAH methyl); 1648 (C@O amide), 1630 (C@N imine), 1601
and 1545 (C@C). NMR 1H (300 MHz, DMSO-d6) d: 11.64 (1H; s,
00
Jorto = 8.8 Hz, H3 and H5 ); 7.21 (1H; d, Jmeta = 1,7 Hz, H6); 6.98–
0
0
7.00 (1H; dd, Jorto = 8.3 Hz and Jmeta = 1.5 Hz, H3); 6.88–6.90 (2H;
00
00
0
d, Jorto = 10.8 Hz, H1 and H2 ); 6.83 (2H; dd, Jorto = 8.7 Hz, H2 and
0
NAH; H3 ); 9.43 (2H; s; OAH; H1 and H3); 8.23 (1H; s, CAHimine
;
H6 ); 6.75–6.77 (1H; d, Jorto = 8.1 Hz, H5); 3.81 (3H; s, H7) ppm.
H1 ); 7.81–7.84 (2H; dd, Jorto = 8.4 Hz, H3 and H5 ); 7.51–7.54
13C NMR (75 MHz, DMSO-d6) d: 163.37 (C6 ); 161.02 (C1 ); 155.65
00
0
0
00
0
0
0
00
00
00
(2H; dd, Jorto = 8.5 Hz, H2 and H6 ); 6.58–6.59 (2H; dd, Jmeta = 1.8 -
(C1); 149.80 (C2); 148.33 (C3 ); 148.16 (C2 ); 139.50 (C1 ); 130.05
0
0
0
0
0
Hz, H4 and H6); 6,24–6.26 (1H; dd; H2); 1.31 (9H; s, H8 ) ppm.
(C4); 128.13 (C4 ); 123.33 (C3 and C5 ); 121.58 (C5); 115.99 (C2 );
115.42 (C3); 110.57 (C6); 56.06 (C7) ppm. Anal. Calcd for
13C NMR (75 MHz, DMSO-d6) d: 162.92 (C4 ); 158.55 (C1 and C3);
00
0
00
0
0
154.57 (C4 ); 147.85 (C1 ); 136.02 (C5); 130.67 (C1 ); 127.42 (C3
C17H16N2O4: C, 65.38; H, 5.16; N, 8.97. Found: C, 65.71; H, 5.33;
0
0
0
and C5 ); 125.20 (C2 and C6 ); 105.14 (C4 and C6); 104.43 (C2);
N, 8.85.
0
0
34.66 (C7 ); 30.89 (C8 ) ppm. Anal. Calcd for C18H20N2O3: C, 69.21;
H, 6.45; N, 8.87. Found: C, 69.53; H, 6.77; N, 8.96.
5.2.1.8.
(E)-4-(tert-Butyl)-N0-((E)-3-(4-hydroxy-3-methoxy-
phenyl)allylidene)benzohydrazide
(VIII).
Yellow
solid.
5.2.1.4. (E)-4-(tert-Butyl)-N0-(4-hydroxybenzylidene)benzohyd-
Yield: 89%, mp: 244–246 °C. IR mmax (cmꢀ1; KBr pellets): 3368
(OAH), 3213 (NAH), 2962 (CAH methyl); 1630 (C@O amide),
1601 (C@N imine), 1585, 1551 and 1450 (C@C). NMR 1H
razide (IV).
White solid. Yield: 93%, mp: 263–265 °C. IR mmax
(cmꢀ1; KBr pellets): 3446 (OAH), 3286 (NAH), 2966 (CAH methyl);
1622 (C@O amide), 1607 (C@N imine), 1599 and 1547 (C@C). NMR
00
(300 MHz, DMSO-d6) d: 11.59 (1H; s, NAH, H5 ); 9.98 (1H; s,
OAH, H1); 8.15–8.18 (1H; d, Jorto = 8.5 Hz, CAHimine, H3 ); 7.80–
1H (300 MHz, DMSO-d6) d: 11.56 (1H; s, NAH; H3 ); 9.91 (1H; s;
00
00
00
0
0
OAH; H1); 8.35 (1H; s, CAHimine
;
H1 ); 7.83–7.85 (2H; dd,
7.83 (2H; d, Jorto = 8.4 Hz, H3 and H5 ); 7.51–7.53 (2H; d, Jorto = 8.4 -
0
0
0
0
0
0
Jorto = 8.4 Hz, H3 and H5 ); 7.51–7.57 (4H; m, H3, H5, H2 and H6 );
Hz, H2 and H6 ); 7.22 (1H; d, Jorto = 1.8 Hz, H6); 6.99–7.02 (1H; dd,
0
00
6.83–6.86 (2H; dd, Jorto = 8.8 Hz, H2 and H6); 1.31 (9H; s, H8 )
Jorto = 8.4 Hz and Jmeta = 1.7 Hz, H3); 6.91–6.93 (2H; d, J = 5.6 Hz, H1
ppm. 13C NMR (75 MHz, DMSO-d6) d: 162.77 (C4 ); 159.34 (C1);
and H2 ); 6,76–6,78 (1H; d, Jorto = 8.2 Hz, H5); 3.81 (3H; s, H7); 1.30
00
00
154.39 (C4 ); 147.85 (C1 ); 130.88 (C1 ); 128.77 (C4); 127.37 (C3
(9H; s, H8 ) ppm. 13C NMR (75 MHz, DMSO-d6) d: 163.27 (C6 );
0
00
0
0
0
00
0
0
0
0
00
00
and C5 ); 125.15 (C3, C5, C2 and C6 ); 115.68 (C2 and C6); 34.65
154.99 (C1 and C4 ); 150.53 (C2); 148.35 (C2 and C3 ); 139.98

2738
J. S. Reis et al. / Bioorg. Med. Chem. 22 (2014) 2733–2738
00
0
0
0
0
(C1 ); 131.19 (C4); 128.08 (C1 ); 127.89 (C3 and C5 ); 125.70 (C2 and
statistically with Tukey test at a significance level of p <0.05 using
C6 ); 121.70 (C5); 116.00 (C3); 110.61 (C6); 56.07 (C7); 35.14 (C7 );
Graph Pad PrismÒ statistical software, version 5.01.
0
0
0
31.37 (C8 ) ppm. Anal. Calcd for C21H24N2O3: C, 71.57; H, 6.86; N,
7.95. Found: C, 71.82; H, 6.91; N, 8.14.
Acknowledgments
This study was supported by Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP Ref. Process: 2012/09237-0). The
authors would like to thanks Prof. Dr. Vera Lucia Borges Isaac
(FCF-UNESP) for the contribution in protective studies.
5.3. Antioxidant activity
The antioxidant activity of hybrid compounds (I–VIII), ascorbic
acid (Sigma–AldrichÒ) and t-resveratrol (Sigma–AldrichÒ) were
performed using adapted DPPHÅ methodology in microplate.37–39
A 2,2-diphenyl-1-picrylhydrazyl radical (DPPHÅ) stock solution
References and notes
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The compounds (I–VIII) and positive controls were diluted in
DMSO at concentration of 10 mM and then diluted with methanol
for 1 mM. Samples and standards were prepared in the following
concentrations: 1000, 300, 100, 35
lM. After, 50 lL of each test com-
pound stock solution and 100
l
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any inhibitor and Absevaluated compounds is the absorption measured
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5.4. Photoprotection assay
5.4.1. Samples
The compounds (I–VIII) were incorporated in a neutral cream at
7%. This cosmetic neutral cream containing dibutyl adipate (4%),
C12–C15 alkyl benzoate (8%), emulfeelÒ (4%), triglyceride of
caprylic–capric acid (6%) and deionized water (quantity sufficient
to 100%) have demonstrated stability, possibility of manipulating
at room temperature and lack of interference in photoprotective
effect. As a reference standard, avobenzone, octyl methoxycinna-
mate, benzophenone-3 and t-resveratrol in a neutral cream at 7%
were used. All reference standards were purchased commercially
from Sigma–Aldrich (St. Louis, MO, USA).
5.4.2. In vitro photoprotection studies
In vitro photoprotection analyses were determined using an
Optometric 290S analyser (SPF-290S) (Littleton, Massachusetts,
United States) and the latest version of WinSPF software. A 1-cc
syringe is used to spread out 0.11 g of each sample over a Trans-
poreÒ tape substrate area (70.7 ꢁ 70.7 mm) at the rate of 2
lL/
cm2, as recommend by U.S. FDA. The results were expressed as
an average of data after 25 scans of the samples performed in dif-
ferent points on the TransporeÒ tape. Data were collected in the
range of 290 nm to 400 nm, with accumulated data at intervals
of 2 nm. All sample data is compared to a reference scan in order
to compute the transmittance. Afterwards, the WinSPF software
automatically converts measurements to SPF values and/or Boots
Star ratings using according calculations methods.29,30 In addition,
UVAPF estimation, UVA/UVB ratio and critical wavelength (kc)
were calculated. The results are expressed as means the standard
deviation of the data (N = 3 experiments). The data were analyzed
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