Design, synthesis and biological evaluation of novel hydroxy-phenyl-1H-benzimidazoles as radical scavengers and UV-protective agents

Article abstract of DOI:10.1080/14756366.2016.1265523

An ever-increasing incidence of skin neoplastic diseases is registered. Therefore, it is important to protect the skin from the UV radiation that reaches the epidermis and dermis but also to block ROS generated by them. Our attention was attracted in deve

Full text of DOI:10.1080/14756366.2016.1265523

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation of
novel hydroxy-phenyl-1H-benzimidazoles as
radical scavengers and UV-protective agents
Alessia Bino, Anna Baldisserotto, Emanuela Scalambra, Valeria Dissette,
Daniela Ester Vedaldi, Alessia Salvador, Elisa Durini, Stefano Manfredini &
Silvia Vertuani
To cite this article: Alessia Bino, Anna Baldisserotto, Emanuela Scalambra, Valeria Dissette,
Daniela Ester Vedaldi, Alessia Salvador, Elisa Durini, Stefano Manfredini & Silvia Vertuani (2017)
Design, synthesis and biological evaluation of novel hydroxy-phenyl-1H-benzimidazoles as
radical scavengers and UV-protective agents, Journal of Enzyme Inhibition and Medicinal
Chemistry, 32:1, 527-537, DOI: 10.1080/14756366.2016.1265523
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1265523
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 23 Jan 2017.
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Date: 02 February 2017, At: 07:36

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 527–537
http://dx.doi.org/10.1080/14756366.2016.1265523
ORIGINAL ARTICLE
Design, synthesis and biological evaluation of novel hydroxy-phenyl-1H-
benzimidazoles as radical scavengers and UV-protective agents
Alessia Binoa, Anna Baldisserottoa, Emanuela Scalambraa, Valeria Dissettea, Daniela Ester Vedaldib, Alessia Salvador
b, Elisa Durinia, Stefano Manfredinia and Silvia Vertuania
aDepartment of Life Sciences and Biotechnology, School of Pharmacy and Health Products, University of Ferrara, Master Course in Cosmetic
b
Sciences and Technology, Laboratory of Health Products, Ferrara, Italy; Department of Pharmaceutical and Pharmacological Sciences, University
of Padua, Padua, Italy
ABSTRACT
ARTICLE HISTORY
Received 20 October 2016
Revised 17 November 2016
Accepted 20 November 2016
An ever-increasing incidence of skin neoplastic diseases is registered. Therefore, it is important to protect
the skin from the UV radiation that reaches the epidermis and dermis but also to block ROS generated by
them. Our attention was attracted in developing new compounds provided with both UV filtering and
antioxidant capacities. To this end, 2-phenyl-1H-benzimidazole-5-sulfonic acid (PBSA), a known UV filter,
was selected as lead compound for its lack of antioxidant activity, high water solubility and good safety
profile. PBSA was sequentially modified introducing hydroxyls on the phenyl ring and also substituting the
functional group in position 5 of the benzimidazole ring. At the end of the synthetic study, a new, very
potent class of antioxidants has been obtained. Surprisingly some of the developed molecules, while
devoid of significant UV-filtering activity was endowed with potent UV-filtering booster capability if associ-
ated with known commercial UVB and UVA filters.
KEYWORDS
Benzimidazole; antioxidant;
UV booster; reactive oxygen
species; sunscreens
Introduction
When the skin is exposed to UV radiation for long periods without
adequate precautions, the body is no longer able to neutralize the
radical species generated, triggering the mechanisms of photoag-
ing, immunosuppression, and photocarcinogenesis3,8. Oxidative
stress causes premature aging of cells and tissues that become
more permeable and lose their efficiency. The damage to the skin
generated by ROS, however, can also extend to the loss of the
barrier function of the stratum corneum, the promotion of inflam-
matory processes, erythema, up to the cancer8,9. Both UVB and
UVA radiation contribute to photoaging and UV-generated ROS
seem responsible for mitochondrial DNA mutations and protein
oxidative modifications. Collagen is particularly affected by oxida-
tion and degradation that is carried out by matrix metallopro-
teases; the synthesis of these enzymes increases following
signaling pathways initiated by reactive oxygen species.
Furthermore, large collagen degradation products inhibit new col-
lagen synthesis and so, collagen degradation itself negatively reg-
ulates new collagen synthesis and then interstitial collagen is
reduced and damaged.
Photoaging, involves (i) elastosis, that clinically is characterized
by yellowish discoloration of the skin, and (ii) rough surface, for an
excessive growth of elastic fibers degraded and greater amount of
basic substance, composed mostly of glycosaminoglycans and pro-
teoglycans. In addition, keratinocytes, in photoaged epidermis, can
be irregular with a loss of polarity, a condition known as actinic
keratosis clinically perceived as red, rough, hyperkeratotic patches;
actinic keratosis has been demonstrated as the initial injury that
may progress to invasive squamous cell carcinoma9–11. The UV fil-
ters used in the pharmaceutical and/or cosmetic fields can either
Human exposure to UV radiation causes different acute and
chronic effects on the skin; acute responses include photodamage,
erythema, synthesis of vitamin D, tanning and most dangerous
immunosuppression and mutation, which are responsible of
chronic UVR (ultraviolet radiation) effects such as photocarcino-
genesis1. UVA and UVB are a small portion of the total radiation
that reaches the earth, but they cause damage to eyes, hair and
they are responsible for inducting various skin disorders including
skin cancer outcomes2,3, because they have a high content of
energy. UVB rays are responsible for direct damage, such as sun-
burn, while UVA rays induce indirect damage caused, in most
cases, to the formation of oxidizing species4.
The skin is the largest organ of the body, covering the entire
surface of the body and is continuous with the mucous mem-
branes; it is the most vulnerable organ and affected by UV rays. In
addition to being the organ most exposed to solar radiation, the
skin is rich in chromophores, molecules able to absorb the radi-
ation energy, such as melanin, DNA, RNA, proteins, lipids, transuro-
canic acid, and aromatic amino acids, as tyrosine and tryptophan5.
In the past, UVA radiation has been much undervalued and was
considered not dangerous, unlike UVB radiation has always been
considered the most dangerous for tissues. Currently, it is known
that UVA rays have a greater ability to penetrate the skin, they
manage to reach the dermis and are responsible for consequential
damages due to generation of reactive oxygen species (ROS). ROS
are highly reactive species that include superoxide anion radical,
hydroxyl radical, singlet oxygen (1O2), and hydrogen peroxide
(H2O2), each of which can further trigger the generation of ROS6,7
.
CONTACT Stefano Manfredini mv9@unife.it
School of Pharmacy and Health Products, Department of Life Sciences and Biotecnology, University of Ferrara,
Master Course in Cosmetic Sciences and Technology, Laboratory of Health Products, Via Fossato di Mortara 17/19, Ferrara, 44121, Italy
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

528
A. BINO ET AL.
form, in general, a protective layer on the skin surface and them- VXR-200 Varian spectrometer and Mercury Plus-400. Chemical
selves absorb the solar radiation, thus preventing that the same shifts are expressed in parts per million (d) relative to the TMS
penetrate into the deeper layers of the skin. Once the filtering internal standard. UV spectrophotometric analyses were carried
molecule has absorbed the energy of the solar radiation, one of out on a UV-VIS spectrophotometer (ThermoSpectronic Helios c,
the methods to disperse this energy is the transformation or deg- Cambridge, UK). HPLC analysis was performed using an Agilent
radation of the molecule itself, which thus becomes photounsta-
ble12. This change can trigger the formation of oxidizing radical
species. Therefore, in addition to the loss of filtering capacity, one
has to consider also cell damages resulting from the formation of
these ROS13. Since detrimental effects of UV rays are also triggered
by over-production of reactive oxygen species, it is important to
protect the skin reducing the UV radiation that reaches epidermis
and dermis but also trying to scavenge the reactive oxygen
species.
1100 Series HPLC System equipped with a G1315A DAD, autosam-
pler and with a Phenomenex Synergi Hydro-RP C18 80 Å column
(4.6 ꢀ 150 mm, 4 lm). Photostability studies were carried out with
a solar simulator device (Suntest CPSþ; Atlas, Linsengericht,
Germany) equipped with a Xenon lamp, an optical filter to cut off
wavelengths shorter than 290 nm and an IR-block filter to avoid
thermal effects.
Materials
Our research group is involved, since several years, in the study
of antioxidant molecules14, so we focused our attention on the
possible synthesis of dualistic molecules able to act, at the same
time, as UV protectants and inhibitors of the formation of radical
species. According to this purpose, the initial phase of the study
regarded screening of commercial molecules, and provided, as
one of the best candidate, 2-phenyl-1H-benzimidazole-5-sulfonic
acid (PBSA), chosen as a lead compound for the lack of antioxi-
dant activity, high solubility in water and good safety profile.
PBSA, commonly used in commercial sunscreens, provides good
protection against UVB rays, although it lacks filtering capacity
regarding UVA radiation and, moreover, appears to induce the
production of reactive species of oxygen after irradiation, with
potentially harmful effects15,16. With the purpose to achieve anti-
oxidant activity, maintaining the filtering capacity, PBSA was modi-
fied introducing hydroxyl groups on the phenyl ring and also
substituting the functional group in position 5 of the benzimida-
zole ring, to evaluate the influence of this moiety on filtering and
antioxidant capabilities. To assess the antioxidant power of the
obtained molecules, in vitro complementary tests were performed
(DPPH, PCL and FRAP assays), then we incorporated the new mol-
ecules in a standard topical formulation to evaluate the UV-filter-
ing capacity in vitro and also to verify the antioxidant power of
3,4-Diamino-benzensulfonic acid (1) has been prepared according
to known procedures17.
Chemistry synthesis
2-(4-Hydroxy-phenyl)-1H-benzimidazole-5-sulfonic acid (2) In
a
round-bottomed flask (50 mL) equipped with a magnetic stirrer, to
a solution of 3,4-diamino-benzene sulfonic acid (1) (100 mg,
0.35 mmol) in ethanol (5 mL) was added a solution of sodium
bisulfite 1N in water (0.7 mL, 0.7 mmol) and 4-hydroxy-benzalde-
hyde (46 mg, 0.35 mmol); the reaction mixture was heated at 80 ꢁC
under reflux for 24 h. The solvent was then evaporated under
reduced pressure, the solid was washed with HCl 1N to precipitate
the interest product, the suspension was filtered and the solid
washed with methanol to afford 2 (60 mg, 0.21 mmol, yield 60%)
as
a
light yellow powder. 1H NMR (400 MHz, [D6]DMSO):
d ¼ 13.4–15.8 (s, broad, 2H, –SO3H, –NH), 10,78 (s, broad, 1H, –OH),
8.06 (d, 2H, aryl, J¼ 8.8 Hz), 7.78 (d, 1H, benzimidazole, J¼ 8.4 Hz),
7.73 (d, 1H, benzimidazole, J¼ 8.4 Hz),7.91 (s, 1H, benzimidazole),
7.09 ppm (d, 2H, aryl, J¼ 9.2 Hz). ESI–MS: m/z calculated for
C13H10N2O4S þ Hþ [M þ Hþ]: 290.30. Found: 290.50.
the finished formulations. The new molecules have also submitted 2-(3,4-Dihydroxy-phenyl)-1H-benzimidazole-5-sulfonic acid (3) In a
to toxicity and phototoxicity studies to exclude adverse effects of round-bottomed flask (50 mL) equipped with a magnetic stirrer, to
the new products. It is fundamental for photoprotection that a fil- a solution of 3,4-diamino-benzene sulfonic acid (1) (100 mg,
tering molecule remains active during exposure to sunlight: similar
UV filters, available on market, unfortunately suffer of evident pho-
toinstability. After this screening, only the molecules that showed
both good filtering activity and antioxidant capacity, and that
were devoid of cytotoxicity and phototoxicity, were considered, at
the end of the first phase of the study, for photostability: formula-
tions containing the selected compounds were exposed to solar
simulated radiation and then tested by accelerated stability
studies.
0.35 mmol) in ethanol (5 mL) was added a solution of sodium
bisulfite 1N in water (0.7 mL, 0.7 mmol) and 3,4-dihydroxy-benzal-
dehyde (48 mg, 0.35 mmol); the reaction mixture was heated at
80 ꢁC under reflux for 24 h. The solvent was then evaporated
under reduced pressure, the solid was washed with HCl 1N to pre-
cipitate the interest product, the suspension was filtered and the
solid washed with methanol to afford 3 (60 mg, 0.2 mmol, yield
57%) as
a
white powder. 1H NMR (400 MHz, [D6]DMSO):
d¼ 13.8–15.6 (s, broad, 2H, –SO3H, –NH), 10.4 (s, broad, 1H, –OH),
9.75 (s, broad, 1H, –OH), 7.88 (s, 1H, benzimidazole), 7.75 (dd, 1H,
benzimidazole, Jortho¼ 8.4 Hz, Jmeta¼ 1.6 Hz), 7.68 (d, 1H, benzimida-
zole, J¼ 8.4 Hz), 7.52–7.56 (m, 2H, aryl), 7.05 ppm (d, 1H, aryl,
J¼ 8 Hz). ESI–MS: m/z calculated for C13H10N2O5S þ Hþ [M þ Hþ]:
306.29. Found: 306,7.
Experimental section
General procedures
Reactants, solvents and standard samples were purchased from
Sigma-Aldrich, Milan, Italy. Reaction course was routinely moni-
tored by thin-layer chromatography on pre-coated silica gel plates
(Macherey-Nagel Durasil-25) by detection under a 254-nm UV
lamp and/or by spraying the plates with 1% FeCl3 solution in
water and using as eluent dichloromethane/methanol (90:10) or
butanol/water/acetic acid (60:20:20).
2-(2-Hydroxy-phenyl)-1H-benzimidazole-5-sulfonic acid (4) In
a
round-bottomed flask (50 mL) equipped with a magnetic stirrer, to
a solution of 3,4-diamino-benzene sulfonic acid (1) (100 mg,
0.35 mmol) in ethanol (5 mL) was added a solution of sodium
bisulfite 1N in water (0.7 mL, 0.7 mmol) and 2-hydroxy-benzalde-
The molecular weights of the compounds were determined by hyde (40 lL, 0.35 mmol); the reaction mixture was heated at 80 ꢁC
ESI (Micromass ZMD 2000), and the values are expressed as [MH]þ. under reflux for 24 h. The solvent was then evaporated under
1H-NMR spectra were determined in d6-DMSO and recorded on reduced pressure, the solid was washed with HCl 1N to precipitate

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
529
the interest product, the suspension was filtered and the solid the solid was washed with HCl 1N to precipitate the interest prod-
washed with methanol to afford 4 (94 mg, 0.32 mmol, yield 91%) as
a white powder. 1H NMR (400 MHz, [D6]DMSO): d¼ 11.9–14.2 (s,
broad, 3H, –OH, –SO3H, –NH), 8.06 (dd, 1H, Jortho ¼ 8 Hz,
Jmeta¼ 2.4 Hz), 8.04 (s, 1H, benzimidazole), 7.75–7.80 (m, 2H),
7.56–7.60 (m, 1H, aryl), 7.20 (d, 1H, aryl, J¼ 7.6 Hz), 7.13–7.17 ppm
uct, the suspension was filtered and the solid washed with metha-
nol to afford 11 (208 mg, 0.86 mmol, yield 94%) as a light brown
powder. 1H NMR (400 MHz, [D6]DMSO): d¼ 14 (s, broad, 2H, –NH,
–OH), 10.4 (s, broad, 1H, –OH), 9.2 (s, broad, 1H, –OH), 7.77–7.782
(m, 2H, benzimidazole), 7.59 (d, 1H, aryl, Jortho ¼ 8.8 Hz), 7.45–7.50
(m, 2H, benzimidazole), 6.66 ppm (d, 1H, aryl, Jortho¼ 8.8 Hz).
ESI–MS: m/z calculated for C13H10N2O3 þ Hþ [M þ Hþ]: 242.23.
Found: 242.6.
(m,
1H,
phenyl).
ESI–MS:
m/z
calculated
for
C13H10N2O4S þ Hþ [M þ Hþ]: 290.30. Found: 290.8.
2-(2,4-Dihydroxy-phenyl)-1H-benzimidazole-5-sulfonic acid (5) In a
round-bottomed flask (50 mL) equipped with a magnetic stirrer, to
a solution of 3,4-diamino-benzene sulfonic acid (1) (100 mg,
0.35 mmol) in ethanol (5 mL) was added a solution of sodium
bisulfite 1N in water (0.7 mL, 0.7 mmol) and 2,4-dihydroxy-benzal-
dehyde (48 mg, 0.35 mmol); the reaction mixture was heated at
80 ꢁC under reflux for 24 h. The solvent was then evaporated
under reduced pressure, the solid was washed with HCl 1N to pre-
cipitate the interest product, the suspension was filtered and the
solid washed with methanol to afford 5 (65 mg, 0.21 mmol, yield
61%) as a white powder. 1H NMR (400 MHz, [D6]DMSO): d¼ 13.87
(s, broad, 2H, –SO3H, –NH), 11.74 (s, broad, 1H, –OH), 10.66 (s,
broad, 1H, –OH), 7.99 (s, 1H, benzimidazole), 7.90 (d, 1H aryl,
Jortho ¼ 8.8 Hz), 7.69–7.75 (m, 2H, benzimidazole), 6.611 (s, 1H, aryl),
6.57 ppm (dd, 1H, aryl, Jortho ¼ 8.8 Hz, Jmeta¼ 2.4 Hz). ESI–MS: m/z
calculated for C13H10N2O5S þ Hþ [M þ Hþ]: 306.29. Found: 306.7.
2-(3,4-Dihydroxy-phenyl)-1H-benzimidazole-5-carboxylic acid (12)20
In a round-bottomed flask (50 mL) equipped with a magnetic stir-
rer, to a solution of 3,4-Diaminobenzoic acid (100 mg, 0.66 mmol)
in ethanol (5 mL) was added a solution of sodium bisulfite 1N in
water (1.32 mL, 1.32 mmol) and 3,4-dihydroxy-benzaldehyde
(91 mg, 0.66 mmol); the reaction mixture was heated at 80 ꢁC
under reflux for 24 h. The solvent was then evaporated under
reduced pressure, the solid was washed with HCl 1N to precipitate
the interest product, the suspension was filtered and the solid
washed with methanol to afford 12 (135 mg, 0.5 mmol, yield 76%)
1
as a light brown powder. H NMR (400 MHz, [D6]DMSO): d¼ 10.5 (s,
broad, 1H, –OH), 9.8 (s, broad, 1H, –OH), 8.25 (s, 1H), 8.05 (dd, 1H,
Jortho¼ 8.4 Hz, Jmeta¼ 1.6 Hz), 7.82 (d, 1H, J¼ 8.4 Hz), 7.69–7.73 (m,
2H), 7.06 ppm (d, 1H, J¼ 8 Hz). ESI–MS: m/z calculated for
C14H10N2O4 þ Hþ [M þ Hþ]: 270.24. Found: 270.7.
2-(2,4-Dihydroxy-phenyl)-1H-benzimidazole-5-carboxylic acid (13)21
The data are in agreement with those reported in literature.
2-(2,3,4-Trihydroxy-phenyl)-1H-benzimidazole-5-sulfonic acid (6) In
a round-bottomed flask (50 mL) equipped with a magnetic stirrer,
to a solution of 3,4-diamino-benzene sulfonic acid (1) (100 mg,
0.35 mmol) in ethanol (5 mL) was added a solution of sodium
bisulfite 1N in water (0.7 mL, 0.7 mmol) and 2,3,4-trihydroxy-ben-
zaldehyde (54 mg, 0.35 mmol); the reaction mixture was heated at
80 ꢁC under reflux for 24 h. The solvent was then evaporated
under reduced pressure, the solid was washed with HCl 1N to pre-
cipitate the interest product, the suspension was filtered and the
solid washed with methanol to afford 6 (82 mg, 0.26 mmol, yield
2-(4-Hydroxy-phenyl)-1H-benzimidazole-5-carboxylic acid (14)22
The data are in agreement with those reported in literature.
2-(2,3,4-Trihydroxy-phenyl)-1H-benzimidazole-5-carboxylic
(15) In a round-bottomed flask (50 mL) equipped with a magnetic
stirrer, to solution of 3,4-diaminobenzoic acid (100 mg,
acid
a
73%) as
a
whitish powder. 1H NMR (400 MHz, [D6]DMSO):
0.66 mmol) in ethanol (5 mL) was added a solution of sodium
bisulfite 1N in water (1.32 mL, 1.32 mmol) and 2,3,4-trihydroxy-ben-
d¼ 13.8–14 (s, broad, 3H, –SO3H, –NH, –OH), 10.45 (s, broad, 1H,
–OH), 9.2 (s, broad, 1H, –OH), 7.99 (s, 1H, benzimidazole), 7.70–7.78 zaldehyde (102 mg, 0.66 mmol); the reaction mixture was heated
(m, 2H, benzimidazole), 7.42 (d, 1H, aryl, Jortho ¼ 8.8 Hz), 6.64 ppm at 80 ꢁC under reflux for 24 h. The solvent was then evaporated
(d, 1H, aryl, Jortho ¼ 8.8 Hz). ESI–MS: m/z calculated for
under reduced pressure, the solid was washed with HCl 1N to pre-
cipitate the interest product, the suspension was filtered and the
solid washed with methanol to afford 15 (151 mg, 0.53 mmol, yield
C13H10N2O6S þ Hþ [M þ Hþ]: 322.29. Found: 322.4.
80%) as a light pink powder. 1H NMR (400 MHz, [D6]DMSO):
d¼ 13–14 (s, broad, 3H, –OH –COOH, –NH), 10.2–11 (s, broad, 2H,
–OH), 8.26 (d, 1H, Jmeta¼ 1.6 Hz), 8.02 (dd, 1H, Jortho¼ 8.6 Hz,
Jmeta¼ 1.6 Hz), 7.82 (d, 1H, Jortho¼ 8.6 Hz), 7.58 (d, 1H,
Jortho¼ 8.8 Hz), 6.65 ppm (d, 2H, Jortho¼ 8.8 Hz). ESI–MS: m/z calcu-
lated for C14H10N2O5 þ Hþ [M þ Hþ]: 286.24. Found: 286.4.
2-(4-Hydroxy-phenyl)-1H-benzimidazole (7)18 The data are in
agreement with those reported in literature.
2-(3,4-Dihydroxy-phenyl)-1H-benzimidazole (8)19 The data are
in agreement with those reported in literature.
2-(2,4-Dihydroxy-phenyl)-1H-benzimidazole (9)18 The data are in
agreement with those reported in literature.
Antioxidant activity assays
Photochemiluminescence (PCL) method PCL assay, based on the
2-(2-Hydroxy-phenyl)-1H-benzimidazole (10)18 The data are in
methodology of Popov and Lewin23, was used to measure the
agreement with those reported in literature.
R
V
antioxidant activity of synthesized compounds with a Photochem
apparatus (Analytik Jena, Leipzig, Germany) against superoxide
anion radicals generated from luminol, a photosensitizer, when
2-(2,3,4-Trihydroxy-phenyl)-1H-benzimidazole (11) In a round-bot-
tomed flask (50 mL) equipped with a magnetic stirrer, to a solution
of o-phenylenediamine (100 mg, 0.92 mmol) in ethanol (5 mL)
R
V
exposed to UV light (Double Bore phosphor lamp, output
351 nm, 3 mWatt/cm2). The antioxidant activity was measured
using ACL (Antioxidant Capacity of Liposoluble substance) kit pro-
vided by the manufacturer designed to measure the antioxidant
activity of lipophilic compounds24. In ACL studies, the kinetic light
was added
a solution of sodium bisulfite 1N in water
(1.84 mL, 1.84 mmol) and 2,3,4-trihydroxy-benzaldehyde (142 mg,
0.92 mmol); the reaction mixture was heated at 80 ꢁC under reflux
for 24 h. The solvent was then evaporated under reduced pressure, emission curve, which exhibits no lag phase, was monitored for

530
A. BINO ET AL.
180 s and expressed as micromoles of Trolox per gram of com- closed as possible to the physical characteristics of the skin.
pound. The areas under the curves were calculated using the
PCLsoft control and analysis software. As greater concentrations of
Trolox working solutions were added to the assay medium, a
marked reduction in the magnitude of the PCL signal and hence
the area calculated from the integral was observed. This inhibition
was used as a parameter for quantification and related to the
decrease in the integral of PCL intensities caused by varying con-
centrations of Trolox. The observed inhibition of the signal was
plotted against the concentration of Trolox added to the assay
medium. The concentration of the added tested compounds was
such that the generated luminescence during the 180-s sampling
interval fell within the limits of the standard curve. The antioxidant
assay was carried out in triplicate for each sample, and 20 lL of
the diluted compound in HPLC-grade methanol (ACL) was suffi-
cient to correspond to the standard curve.
Among the substrates available for these purposes, the substrate
used in this study to analyse the sunscreen products were PMMA
plates. This substrate is easily handled and can be supplied with a
reproducible roughness. WW5 PMMA plates have been purchased
from Schonberg GmbH (Munich, Germany). The plates, used in
this study, have an area of 25 cm2 and standardized 5 m rough-
ness. Transmittance and absorbance measurements were carried
out by a SHIMAZDU UV-2600 provided of integrating sphere ISR
2600 60 mm and coupled with a SPF determination software and
a PMMA plate with approximately 15 lL of glycerin served as
reference.
Cytotoxicity and phototoxicity tests
Cellular culture An immortalized, nontumorigenic cell line of
human keratinocytes (NCTC-2544) was grown in a DMEM medium
(Sigma-Aldrich Milan, Italy), supplemented with 115 units/mL of
penicillin G, 115 lg/mL streptomycin, and 10% fetal calf serum
(Invitrogen, Milan, Italy). Individual wells of a 96-well tissue culture
microtiter plates (Falcon, Becton–Dickinson) were inoculated with
100 lL of complete medium containing 5 ꢀ 103 NCTC-2544. The
plates were incubated at 37 ꢁC in a humidified (5% CO2) incubator
for 18 h prior to the experiments.
1,1-Diphenyl-2-picryl-hydrazyl radical (DPPH) assay To 1.5 mL
DPPH methanolic solution (0.5 mM) was added 0.750 mL of sample
solution proper diluted. Samples absorbance measurements were
evaluated with a UV-VIS spectrophotometer (Thermo Spectronic
Helios c, Cambridge, UK) at fixed wavelength of 517 nm. Blank
sample was prepared adding methanol to DPPH solution and
Trolox was used as standard reference to achieve a calibration
curve. The radical-scavenging activity is expressed as inhibition
ratio of initial concentration of DPPH radical and is calculated
according to the formula: Inhibition percentage (Ip) ¼ [(AB-As)/
AB]ꢂ100; where AB and As are, respectively, the absorbance values
Cytotoxicity After medium removal, 100 lL of the drug solution,
dissolved in DMSO and diluted in DMEM medium, was added to
each well, incubated at 37 ꢁC for 72 h. Final DMSO concentration
never exceeded 0.5%. Cell viability was assayed by the MTT
(3–(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) test
as previously described by Mosmann27.
of blank reaction and of the tested sample25
.
Ferric reducing antioxidant of potency (FRAP) assay The ferric
reducing ability of each standard solution was measured according
to a modified protocol described by Guihua et al.26. The reagent
for analysis was freshly prepared by mixing the following solutions
in the reported ratio 10/1/1 (v:v:v) (i) 0.1 M acetate buffer pH 3.6,
(ii) TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine) 10 mmol/L in 40 mmol/HCl,
(iii) ferric chloride 20 mmol/L. To a 1.9 mL of reagent were added
0.1 mL of sample proper diluted or solvent when blank was per-
formed. Readings at fixed wavelength of the absorption maximum
(593 nm) were done after 30 min, using a UV-VIS spectrophotom-
eter; it was evaluated the absorbance increase of sample solution
against the absorbance of blank reaction as parameter to calculate
the antioxidant activity. The antioxidant activity is given as Trolox
activity since this standard was used to perform the calibration
curves.
Irradiation procedure Two HPW 125 Philips lamps, mainly emitting
at 365 nm, were used for UVA irradiation experiments. The spectral
irradiance of the source was 4.0 mWcmꢃ2 as measured at the sam-
ple level by a Cole-Parmer Instrument Company radiometer (Niles,
IL) equipped with a 365-CX sensor. One or two PL-S 9 W/12
Philips lamps (280–370 nm; peak at 315 nm) were used for UVB
irradiation experiments. To restrict the incident radiation to the
range 305–370 nm, a glass filter (Schott SWG-305) was used. Total
energy was detected by the same equipped with a sensor (model
CX-312).
Cellular photoprotection experiments After medium removal,
100 lL of the drug solution, dissolved in DMSO and diluted with
Hank’s Balanced Salt Solution (HBSS pH ¼7.2), was added to each
well, incubated at 37 ꢁC for 30 min, and then irradiated (20 J/cm2
for UVA and 0.5 and 1 J/cm2 for UVB). After irradiation, the solu-
tion was replaced with the medium and the plates were further
incubated for 48 h. Cell viability was assayed by the MTT [(3–(4,5-
Cosmetic formulations
The synthesized molecules were included at the concentration of
3% in a topical formulation, to prove the effective filtering cap-
acity and the antioxidant activity of finished formulation. It was
decided to use a standard formulation oil in water (O/W).
INCI: Aqua, glycerin, phenoxyethanol, methylparaben, ethylpar- dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide)] test as
previously described by Mosmann27.
aben, butylparaben, propylparaben, isobutylparaben, cetyl alcohol,
glyceryl stearate, PEG-75 stearate, ceteth-20, steareth-20, cetyl
alcohol, dimethicone, C12–15 alkyl benzoate, cocoglycerides,
NaOH solution 10%.
HPLC analysis
HPLC analysis was performed using an Agilent 1100 Series HPLC
System equipped with a G1315A DAD, and with a Hydro-RP C18
Synergi 80 Å column (4.6 ꢀ 150 mm, 4 lm) from Phenomenex,
maintained at 25 ꢁC during all the time of the analysis. The mobile
phase consisted of water (0.01 M H3PO4) (solvent A) and aceto-
Evaluation of filtering parameters In vitro approaches consist in
applying a thin film of sunscreen product on an artificial substrate
and test, via spectrophotometric measures, the amount of UV radi-
ation passing through the film. Several different artificial substrates
are available for this type of analysis; the substrate should be as nitrile (0.01 M H3PO4) (solvent B).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
531
1. PBSA and 3: the determination was carried out in isocratic collected. One-way analysis of variance (ANOVA) and LSD post hoc
Tukey’s honest significant difference test were used for comparing
the bioactive effects of different samples. All computations were
made using the statistical software STATISTICA 6.0 (StatSoft
Italia srl, Vigonza, Italy).
condition, A: 91%/B: 9%. Separation was monitored with
absorbance detection at kmax of the molecule 8 nm. The
flow rate was 1.0 mL/min.
2. 10: the determination was carried out in isocratic condition,
A: 84%/B: 16%. Separation was monitored with absorbance
detection at kmax of the molecule 8 nm. The flow rate was
1.0 mL/min
Results and discussion
Chemistry
The sample solutions were filtered by a 0.45-lm filter, before
be injected into column (HPLC filters were purchased from
Chemtek Analitica, Bologna, Italy).
Three groups of molecules were synthesized: the first group
maintained the sulfonic acid moiety in position 5 of the benzimi-
dazole ring (2–6); the second group showed no functional group
on the benzimidazole ring (7–11), and the third group presented,
instead of the sulfonic acid, the carboxylic acid moiety (12–15)
(Scheme 1). For the first group of molecules, the synthesis of the
benzoimidazole was accomplished from 3,4-diamino-benzene sul-
fonic acid (1) that was in turn obtained from o-phenylenedi-
amine and 96% sulfuric acid17. 5(6)-Substituted benzimidazoles
are a well-known class of heterocycles, which displays tautomer-
ism at the imidazole ring28,29. Different synthetic procedures
described in the literature were analyzed to obtain the desired
products. The best solution was the use of sodium bisulfite as a
catalyst; all reactions were carried out in ethanol at reflux for
24 h30. The same procedure was used to synthesize 2-(4-hydroxy-
phenyl)-1H-benzimidazole-5-sulfonic acid (2), 2-(3,4-dihydroxy-
phenyl)-1H-benzimidazole-5-sulfonic acid (3), 2-(2-hydroxy-phe-
nyl)-1H-benzimidazole-5-sulfonic acid (4), 2-(2,4-dihydroxy-phenyl)-
1H-benzimidazole-5-sulfonic acid (5), 2-(2,3,4-trihydroxy-phenyl)-
Photostability studies
The solar simulator emission was maintained at 500 W/m2. A por-
tion of cosmetic formulation containing the sunscreen molecule
(3%, w/w) was transferred onto the bottom of a beaker to gain a
cosmetic mount of 2 mg/cm2 and then was irradiated for 1 h with
the solar simulator. After irradiation the beaker was removed and
its content quantitatively transferred into a 50 mL calibrated flask
with methanol and the remaining sunscreen concentration was
determined by HPLC as described above. All samples were pro-
tected from light both before and after irradiation, the degree of
photodegradation was evaluated by comparing the areas of the
irradiated samples, with those of the unirradiated preparations.
Statistical evaluations
Relative standard deviations and statistical significance (Student’s
1H-benzimidazole-5-sulfonic
acid
(6),
2-(4-hydroxy-phenyl)-
t-test; p ꢄ 0.05) were given where appropriate for all data 1H-benzimidazole (7), 2-(3,4-dihydroxy-phenyl)-1H-benzimidazole
Scheme 1. Synthesis of compounds 2–15.

532
A. BINO ET AL.
(8), 2-(2,4-dihydroxy-phenyl)-1H-benzimidazole (9), 2-(2-hydroxy- Antioxidant activity assays
phenyl)-1H-benzimidazole (10), 2-(2,3,4-trihydroxy-phenyl)-1H-ben-
Sun protection factor (SPF) is related to the UV absorption of the
zimidazole (11), 2-(3,4-dihydroxy-phenyl)-1H-benzimidazole-5-car-
boxylic acid (12), 2-(2,4-dihydroxy-phenyl)-1H-benzimidazole-5-
carboxylic acid (13), 2-(4-hydroxy-phenyl)-1H-benzimidazole-5-car-
boxylic acid (14), 2-(2,3,4-trihydroxy-phenyl)-1H-benzimidazole-5-
carboxylic acid (15).
substances, so before including the molecules in cosmetic formu-
lations we evaluated the wavelength of maximum absorption
(kmax) and the molar extinction coefficient (e) of each molecule in
aqueous solution at pH 7. The UV spectra were recorded between
270 and 420 nm (20 nm higher and lower then UVA and UVB
range) to verify the spectrum profile of the molecules within UVA
and UVB region. UV spectrum of PBSA is an UVB filter and
presents a kmax of 302 nm; after this peak, the absorbance fades
going to higher wavelength until almost zero around 325 nm, and
then, it provides no absorption in the UVA region. Molecules spec-
tra overlapped for category according to the substituent in pos-
ition 5 of the benzimidazole ring are shown in Figures 1, 2 and 3;
PBSA spectrum is presented in all figures for comparison.
From the spectra appears that the lambda maxima (Table 2) of
the new molecules shifted toward higher wavelength and in gen-
eral the range of absorption curve was wider than PBSA. Within a
group of molecules, the batochromic shift was more marked for
the molecules that have a hydroxyl moiety in position 2 of the
phenyl ring (4, 5, 6, 9, 10, 11, 13 and 29); the effect of ortho sub-
stituted is known to produce this shift toward higher wave-
length31. Increasing the number of auxochrome groups on phenyl
ring an increment in the absorption range occurred, compounds
with three hydroxyl on the phenyl ring (6, 11, 15) had the
Antioxidant activity assays
All the synthesized compounds were tested to determine their
antioxidant capacity by PCL (photochemoluminescence) analysis,
DPPH and FRAP tests (Table 1). The lead PBSA, as expected, was
devoided of any activity in both DPPH and PCL assays, showing
only minimal activity in FRAP assay. Compounds with only one
phenolic hydroxyl, in position 2 (5 and 10) or 4 (2, 7, 14) on the
phenyl ring, have shown the lowest antioxidant capacity in all
performed assays. The antioxidant activity increased adding
another hydroxyl group; products with two hydroxyls in position
2 and 4 on the phenyl moiety (5, 9 and 13) showed improved
activity, but the best results were obtained with 3, 8, 12 with
hydroxyl moieties in position 3 and 4 of the phenyl ring. The
gap, in terms of antioxidant power, between the different pos-
itional isomers was very high in all performed tests. Compounds
with three hydroxyls on phenyl ring (6, 11 and 15) did not show
any further improvement in antioxidant activity as compared to
the parent bis-hydroxyls in position 3 and 4 of the phenyl ring.
Interestingly, the results for DPPH test were even lower than
those of 3, 8 and 12 and also significant decrease in antioxidant
activity was shown in PCL assay. In FRAP analysis, compounds
with three hydroxyl moieties gave results comparable to that of
molecules with hydroxyls in position 3 and 4. Considering the
three assays, molecules with better antioxidant profile were those
with two phenolic hydroxyls in position 3 and 4; in all performed
tests, compound 3 showed the highest antioxidant capacity, fol-
lowed by compound 8 and 12. For compounds 6, 11 and 15,
with three hydroxyls, and compounds 5, 9 and 13, with two
hydroxyls in position 2 and 4 on the phenyl ring, the antioxidant
capacity decreased probably because of hydrogen bond forma-
tion between nitrogen of benzimidazole and the hydroxyl group
in position 2 on the phenyl ring.
Figure 1. Comparison between spectra of PBSA and molecules with sulfonic acid
moiety on benzimidazole ring: 2, 3, 4, 5, 6.
Table 1. Results of antioxidant assays. Each value was obtained from three
experiments (Mean SE).
DPPH (lmolTrolox/
mmol)
FRAP (lmolTrolox/
mmol)
PCL (lmolTrolox/
mmol)
Product
(p ꢄ 0.05)
(p ꢄ 0.05)
0.79 0.06
12.23 0.25
(p ꢄ 0.05)
PBSA
2
< LOQa
2.04 0.15
< LOQa
11.36 0.08
3
4
2145.31 45.8
2.03 0.09
2707.29 29.52
16.22 0.89
22,838 836,26
2.06 0.05
5
6
7
21.41 043
1362.12 133.96
1.67 0.08
19.87 0.22
2713.3 45.17
21.94 0.12
159.1 4.3
1924.35 101.82
9.66 0,04
8
9
1974.58 16.89
16.47 0.54
0.87 0.05
1811.02 61.7
1771.82 84.75
32.48 2.38
1.185 0.02
1241.03 9.43
2663.21 32.53
37.3 0.35
23.04 0.09
2723.19 35.74
2433.28 21.74
31.63 1.96
19190.6 443.18
198.53 2.65
10.93 0.05
1614.675 19.95
13174.31 240.68
160.26 4.09
6.98 0.27
10
11
12
13
14
15
2.62 0.06
2355.15 52.55
Figure 2. Comparison between spectra of PBSA and molecules without substitu-
ents on benzimidazole ring: 7, 8, 9, 10, 11.
1515.65 75.56
LOQa limit of quantification.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
533
broadest spectrum and their lambda maxima shifted at the high- permits transmission of UV radiation. Although with some limita-
tion, the test is used to predict SPF in vivo and to screen the filter-
ing activity of new molecules that cannot be tested by in vivo
because of lacking of more data concerning safety. The critical
wavelength (kc) for the test product is defined as that wavelength
where the area under the absorbance spectrum for the irradiated
product from 290 nm to kc is 90% of the integral of the absorb-
ance spectrum from 290 nm to 400 nm. Critical wavelength value
of greater than 370 nm is necessary, in order to satisfy require-
ments for broad-spectrum UVB/UVA protection and associated
labeling. Polymethylmethacrylate plates (PMMA) with a specific
roughness were a satisfactory substrate for this method, because
they are UV-transparent, nonfluorescent, photostable and inert to
all potential sunscreen formulation ingredients. Table 3 lists the
data obtained for the model formulation containing the synthe-
sized compounds. These parameters are necessary to evaluate
whether a sunscreen fulfills the requirements for efficacy, and they
are SPF, UVA protection factor (UVAPF), UVA/UVB ratio and critical
wavelength. The standard formulation was also analyzed by this
method and did not exhibit any protecting activity.
Data obtained showed that all the new molecules have
improved filtering parameters UVA, since UVA protection factor,
UVA/UVB ratio and critical wavelength are higher than those of
the lead compound. In particular, molecules with phenolic
hydroxyl in the ortho position on the phenyl ring (4, 5, 9, 10 and
13) had better and higher UVA/UVB ratio and critical wavelength,
although they have the SPF value less than lead compound.
Formulation containing products 2 and 14, which have a phenolic
hydroxyl at the para position on the phenyl ring, have shown
good values in terms of SPF, greater UVA protection factor, critical
wavelength and also higher UVA/UVB ratio than PBSA, but yet
est wavelength compared to the other compounds. The absorp-
tion spectrum of molecules with the same number and position of
hydroxyl seemed only slightly influenced by the substituent in
position 5 of the benzimidazole ring. After having established the
lambda maxima for every compound, solutions at different con-
centrations were prepared to calculate the molar extinction coeffi-
cient by means of linear regression and applying Lambert–Beer
equation (Table 2).
Topical formulation evaluation
In order to evaluate possible activity as sun filters and antioxi-
dants, suitable in the development of novel class of dualistic
sunscreens, the synthesized molecules were included at the con-
centration of 3% (w/w) in a topical formulation. To this end stand-
ard oil in water (O/W) formulation was devised, taking into
account the absence of any UV filtering and/or antioxidant mol-
ecule, in order to obtain a reference formulation. Data obtained
are shown in Table 4. It was not possible to analyze compound 7
in the formulation because of high instability (phase separation
induced by 7). Formulations containing the active ingredients
were tested for their SPF efficacy by the in vitro method derived
from Diffey and Robson32, in comparison with the same formula-
tion containing the reference PBSA.
This method is based on the measure of spectral transmission
of ultraviolet radiation, with and without the sunscreen applied,
through an irregular substrate that simulates the skin surface and
Table 2. Values of k max and e.
k max (nm)
e
PBSA
2
3
4
5
6
7
8
9
10
11
12
13
14
15
302
308
313
315
318
328
306
311
315
315
325
317
321
312
332
25 000
25 000
23 000
21 000
23 000
22 200
24 000
16 000
14 000
17 000
12 000
21 000
20 000
24 000
17 000
Figure 3. Comparison between spectra of PBSA and molecules with carboxylic
acid moiety on benzimidazole ring: 12, 13, 14, 15.
Figure 4. Stability studies in formulation (formulations were submitted to accelerated aging at 40 ꢁC for 63 days).

534
A. BINO ET AL.
lower than the molecules containing hydroxyl on the phenyl ring compounds with only one hydroxyl (2, 4, 10 and 14) had poor
in ortho. Compounds 3, 8 and 12, with the hydroxyl in position 3 antioxidant capacity, which slightly increased for formulations hav-
and 4, were really interesting for their high levels of SPF: 3 ing compounds with two hydroxyls in position 2 and 4 on the
phenyl ring (5, 9 and 13). Formulations containing compounds 3,
8 and 12 presented highest antioxidant capabilities; these are fol-
lowed, but with important decrease in antioxidant capacity, by for-
mulations with compounds 6, 8 and 15 that have three hydroxyl
moieties on phenyl ring.
showed a SPF twice as PBSA. This product shows also great UVA
protection factor, UVA/UVB ratio and critical wavelength than
PBSA. Compound 8 has lower SPF than 3, but higher than PBSA;
in comparison with 3, molecules 8 and 12 presented higher UVA/
UVB ratio and critical wavelength. Regarding compounds with
three hydroxyls on phenyl ring, they have good UVA filtering
parameters, their critical wavelengths are the highest and SPF is
higher than PBSA for molecule 6, while SPF of 15 is slightly lower
and decreases further in molecule 11. Thus, it can be observed
that parameters are mainly influenced by the number and position
of the hydroxyl groups on the phenyl ring rather than the sub-
stituent in the 5-position of benzimidazole. Concomitantly, all for-
mulations were analyzed to assess whether the antioxidant power
was maintained. In these regards, we have previously demon-
strated that PCL is a very useful method for the determination of
antioxidant capacity of skin products because applicable to both
raw material and finished products33. In Table 4, the antioxidant
activity, measured by PCL method, of finished formulations is
reported; as it can be seen results were in agreement with that
obtained testing the pure compounds. Formulations containing
Cytotoxicity and phototoxicity tests
Compounds used in sunscreens must be stable upon irradiation
thus, to ensure the safety of the synthesized compounds, specific
cytotoxicity and phototoxicity assays were performed. The tests
were conducted using a specific cell line of human keratinocytes
(NCTC-2544). To assess cell viability, in the presence of the study
compounds, before and after the irradiation, the MTT assay was
performed27. The cytotoxicity was checked after 72 h from the
incubation of the keratinocytes with compounds; moreover, the
same experiments were performed in the presence of the parent
PBSA. Table 5 displays cytotoxicity test results, expressed in IC50
(lM), which is the concentration required to inhibit 50% of cellular
growth.
Most of the compounds showed no cytotoxicity at the concen-
tration used in the cell line of human keratinocytes. Only com-
pounds 2, 6 and 7, had IC50 values in the range between 20 and
30 lM, in particular compound 6 showed the lowest IC50 and then
was the most cytotoxic among the tested compounds. Molecules
were verified for their photocytotoxicity in the same cell line; the
cells were treated with 50 lM solutions of compound and after
30 min were irradiated with 20 J/cm2 UVA or with UVB at two
energy amounts: 0.5 J/cm2 and 1 J/cm2 UVB. The compounds 2, 6
and 7 were used at a concentration of 20 lM to exclude any anti-
proliferative effect of these compounds. After irradiation, the solu-
tion was replaced with growth medium and the cells were further
incubated for 48 h, and then, cell viability was assessed by MTT
assay. The results are presented in Table 6 as percentages of cell
survival in comparison with nonirradiated cells (100% cell survival).
The phototoxicity of tested molecules was compared with the
ratio of cell survival of irradiated keratinocytes without substance
(IC ¼ irradiated control).
Table 3 . Protecting efficacya.
Formulated
compounds SPF (p ꢄ 0.05) UVA/UVB (p ꢄ 0.05) UVAPF (p ꢄ 0.05) kcb(nm)
PBSA
2
3
4
5
6
8
9
10
11
12
13
14
15
4.56 1.09
4.74 0.15
9.83 0.64
2.22 0.13
2.18 0.14
5.99 0.12
5.20 0.20
3.57 0.23
1.96 0.14
2.85 0.22
2.21 0.17
2.85 0.17
6.01 0.31
3.69 0.24
0.26 0.05
0.51 0.01
0.74 0.04
0.96 0.01
1.13 0.01
0.89 0.03
0.78 0.02
1.10 0.05
1.01 0.02
0.90 0.01
1.00 0.04
1.27 0.02
0.70 0.05
0.96 0.08
1.04 0.06
1.36 0.07
3.63 0.23
1.06 0.07
1.21 0.04
2.72 0.08
1.90 0.09
1.89 0.05
1.06 0.08
1.35 0.1
333
346
358
355
370
383
368
380
378
381
370
368
350
382
1.88 0.12
1.58 0.10
2.04 0.11
1.87 0.08
aValues are the mean of three independent determinations, each of which at
least five readings.
bWavelength at which the integral of the spectral absorbance curve reaches 90%
of the area under the curve from 290 to 400 nm.
Differences in cell survival rate were detected; molecules 6, 9,
10, 11 and 15 seemed to have a phototoxic effect as cell survival
decreased considerably in comparison to irradiated control, thus
these compounds were judged not suitable for further
Table 4. Antioxidant activity of finished
formulationsa.
Table
5. Cytotoxicity
results
PCL lmol Trolox/g formulation
expressed as IC50 values on NCTC-
2544 cells.
Formulation
(p ꢄ 0.05)
PBSA
2
3
<LOQb
0.33 0.03
Compounds
IC50 (lM)
2212.09 68.35
PBSA
2
3
4
5
6
7
8
9
10
11
12
13
14
15
>50
26.4 2.7
>50
>50
>50
23.1 2.9
26.1 2.4
>50
>50
>50
4
5
6
8
0.06 0.02
0.95 0.02
101.98 1.08
2972.107 145.63
8.66 0.04
9
10
11
12
13
14
15
0.5 0.02
153.35 8.14
1373.39 19.43
4.97 0.17
0.16 0.02
86.99 6.95
>50
>50
>50
>50
aValues are the mean of three independent
determinations.
bLimit of quantification.
>50

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
535
development as photoprotective agents. All the other compounds, PBSA, and placed under a solar simulator for one hour before
including PBSA, increased cell survival after UVA and UVB irradi- repeating HPLC analysis. Results reported in Table 7 showed that
the two compounds have photostability comparable to the lead
compound. Compound 3 was slightly less photostable than PBSA
while the best result was achieved with compound 12 that exhib-
ited the lowest degradation rate, less than 2%, and therefore had
better photostability than lead compound.
Molecules 3 and 12 were challenged in accelerated stability
studies against the parent compound. The formulations were sub-
mitted to accelerated aging at 40 ꢁC and analyzed at specific inter-
vals, with HPLC.
ation in comparison with irradiated control. A great increase in
cell viability was observed after UVB irradiation (1 J/cm2), in par-
ticular in presence of PBSA and of compounds 3, 12, 13 and 14
the cell survival doubled in relation to the control. The most inter-
esting compounds regarding the phototoxicity test were 3, 12, 13
and 14 that increased cell survival both after UVA and UVB irradi-
ation, hence these substances seemed suitable as photoprotective
molecules.
The trend reported in Figure 4 showed that PBSA had the highest
stability, followed by compound 12, and finally compound 3. This
was a somewhat expectable result because molecules with antioxi-
dant activity are more susceptible to degradation phenomena.
Photostability and stability studies
Stability and photostability tests were performed only on com-
pounds that as provided with the best antioxidant capacity
together with a good photoprotection activity (i.e. 3, and 12). At
first, the effective concentration of filtering molecules was verified
in the formulation by HPLC. Samples of the formulations (2 mg/
cm2) were spread on a PMMA plate, for each compound including
SPF booster properties
Considering all the results, that is, filtering and antioxidant cap-
acity, cytotoxicity, phototoxicity, stability and photostability, the
molecule that best complied all the requirements was compound
12 (also termed oxisol)28. Thus, because those properties were
quite interesting we decided to further investigate the sunscreen
performance of this molecule, in comparison with PBSA and, as
usual for sunscreens, in combination with commercial organic
filters.
A set of model formulations was prepared: an oil-in-water
emulsion (O/W), without sunscreen molecules (Ref. A, Figure 5),
prepared as reference formulation; two formulations containing
PBSA or oxisol (12) at 3% were prepared (Ref. C and B, respect-
ively, Figure 5), in order to evaluate the filter properties of oxisol
(12). Moreover, as stated earlier, SPF booster properties were
Table 6. Phototoxicity test data.
% Cell survival
Compound Concentration (lM) UVA 20 J/cm2 UVB 0.5 J/cm2 UVB 1 J/cm2
IC
PBSA
2
3
4
5
6
7
8
69.1 3.2%
74.5 1.4%
77.3 2.8%
77.4 4.0%
76.9 2.0%
79.0 6.0%
18.4 2.3%
72.8 2.9%
62.9 4.3%
3.7 0.5%
4.8 0.6%
1.2 0.2%
74.3 2.0%
80.7 1.3%
76.7 3.4%
12.2 1.3%
63.1 3.2%
75.2 4.5%
78.0 3.5%
71.5 2.5%
74.3 3.2%
70.8 3.9%
59.3 3.4%
74.2 1.3%
72.8 1.0%
1.1 0.3%
19.8 2.4%
45.6 2.6%
76.3 3.6%
69.0 0.2%
74.2 3.4%
60.1 2.3%
33.8 3.1%
64.2 3.1%
46.7 3.4%
58.1 2.6%
37.7 1.7%
38.6 3.7%
29.7 2.1%
44.0 4.1%
43.9 3.4%
0.5 0.1%
50
20
50
50
50
20
20
50
50
50
50
50
50
50
50
9
10
11
12
13
14
15
3.5 0.3%
Table 8. Mixed commercial filters used in the formulations.
27.0 2.4%
64.5 3.5%
57.5 2.4%
58.4 2.5%
25.6 2.7%
UV filter composition
INCI name
SPF 15
Amount
SPF 30
Amount
Butyl methoxydibenzoylmethane
Ethylhexyl methoxycinnamate
Octocrylene
2.3%
4.0%
4.0%
4.7%
8.0%
8.0%
Table 7. Evaluation of photostabilitya.
Product
Residual product (p ꢄ 0.05)
Table 9. In vivo evaluation of selected
formulations.
PBSA
3
96.7% 1.8%
94.9% 0.8%
98.4% 0.9%
Emulsion
In vivo SPF
12
Oxisol (12) 3%
Mixed filters SPF 15
Oxisol (12) 3%þMixed Filters SPF15
2.68
15.78
21.74
aValues are the mean of four independent
experiments.
Figure 5. SPF values (reported in dark grey) and UVA-PF values (reported in light grey) of the tested formulations.

536
A. BINO ET AL.
assayed on other four formulations including a mix of filters with Oxisol (12) represents the first example of development of a
a theoretical SPF value of 15 and 30, adding 3% of oxisol (12) or
PBSA (Ref. E, F, G, H, Figure 5). The tested formulations (Ref. D,
and G, Figure 5) contain a fixed combination of commercial
organic filters, butyl methoxydibenzoylmethane–octocrylene–ethyl-
hexyl methoxycinnammate, that are dosed to perform a theoret-
ical value of SPF 15 and SPF 30 as calculated by BASF SunScreen
Simulator34, as reported in Table 8.
booster molecule provided of very potent antioxidant activity and
capable to improve activity of known sunscreen. Thus, although it
cannot be defined as a sunscreen filter, it is very useful in combin-
ation with conventional UV filter to improve potency (less amount
of UV filter needed) and also express potent antioxidant activity,
very important to improve protection offered by traditional
sunscreens also protecting against harmful oxidative species pro-
duced by solar radiation. We believe that this result opens new
perspective in the development of a new class of protective mole-
cules that consent to save up to 37% of the traditional UV filters.
Figure 5 reports the results obtained for all the emulsions in
term of SPF and UVA-PF values.
As expected, the reference formulation does not present sun-
screen properties. The comparison between the emulsions con-
taining only oxisol (12) and PBSA highlights the capability of PBSA
to behave as a “sun-filter”, while oxisol (12) does not present an
appreciable value of SPF. Considering the performance of the two
molecules in association with a mix of filters it can be observed
that, as expected PBSA deals with the SPF (UVB rays) without
influence on the UVA component of the spectrum. Remarkably,
Oxisol (12) influences both the parameters.
Disclosure statement
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
Funding
The authors wish to thank Ministry of Education, University and
Research (MIUR Italy- PRIN, Grant 20105YY2HL_006), the University
of Ferrara (FAR grant years 2010–2013) and Ambrosialab srl,
Ferrara (Grant 2015) for the financial support.
Preliminary in vivo evaluation of products
The in vivo method according to ISO 24444:2010 standard and
European Recommendation 647/2006 was applied to determine
the SPF value for 3 selected sunscreen products reported in
Table 9, with the aim to confirm the booster effect of Oxisol (12).
In this study, five subjects per product, female and male, 20 and
35 aged, were used for the preliminary test. Their skin phototype
was chosen with sun sensitivity categories of type I, II and III
according to Fitzpatrick. A skin area on the back, 35 cm2, was irra-
diated with different UV doses so that the minimal erythema dose
(MED) was determined after 24 h. Prior to the in vivo tests, the
sunscreen products were tested with the in vitro test method to
ensure that severe deviations from the predictive SPF values were
taken into account. All volunteers had been informed in details
before signing a written declaration of consent. For in vivo SPF
determination, a Multiport UV Solar Simulator Model 601, 300 W,
was used. This sun simulator emits ultraviolet radiation in the
region between 290 and 400 nm from six independent outputs.
Each output is adjusted on scalar UV doses, set according to
parameters tabulated by the instrument. All sunscreen products
were applied in a thin film of 2.00 0.05 mg/cm2 in the selected
area on the back. The product distribution was reached by a gen-
tle massage using a fingercoat, at least 15 min before the irradi-
ation started.
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