In-vitro evaluation of antioxidant, antiproliferative and photo-protective activities of benzimidazolehydrazone derivatives

Article abstract of DOI:10.3390/ph13040068

In the search of multifunctional compounds we designed benzimidazole derivatives endowed with phenolic hydroxy groups and a hydrazone moiety as potential radical-scavenger and the antioxidant agents. The target molecules have been prepared by a simple syn

Full text of DOI:10.3390/ph13040068

Communication
In-Vitro Evaluation of Antioxidant, Antiproliferative
and Photo-Protective Activities of
Benzimidazolehydrazone Derivatives
Anna Baldisserotto 1,†, Monica Demurtas 2,†, Ilaria Lampronti 3, Massimo Tacchini 4, Davide Moi 2,
Gianfranco Balboni 2, Silvia Vertuani 1, Stefano Manfredini 1 and Valentina Onnis 2,*
1
Department of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 17-19, I-44121
Ferrara, Italy; anna.baldisserotto@unife.it (A.B.); mv9@unife.it (S.M.); silvia.vertuani@unife.it (S.V.)
2
Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and
Nutraceutical Sciences, University of Cagliari, University Campus, S.P. n° 8, Km 0.700, I-09042 Monserrato
(CA), Italy; monicademurtas@tiscali.it (M.D.); davide.moi2@gmail.com (D.M.); gbalboni@unica.it (G.B.)
3
Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University
of Ferrara, Via Fossato di Mortara 74, I-44121 Ferrara, Italy; ilaria.lampronti@unife.it
4
Department of Life Sciences and Biotechnology, Section of Pharmaceutical Biology, University of Ferrara,
Piazzale Luciano Chiappini 3, I-44123 Malborghetto di Boara (FE), Italy; massimo.tacchini@unife.it
* Correspondence: vonnis@unica.it
†
These authors equally contribute to the present paper.
Received: 24 February 2020; Accepted: 14 April 2020; Published: 15 April 2020
Abstract: In the search of multifunctional compounds we designed benzimidazole derivatives
endowed with phenolic hydroxy groups and a hydrazone moiety as potential radical-scavenger
and the antioxidant agents. The target molecules have been prepared by a simple synthetic
procedure and tested for their antioxidant activity by DPPH, FRAP, and ORAC test, for
photoprotective activity against UV rays and for antiproliferative activity against Colo-38
melanoma cells. Furthermore, two different dermocosmetic formulations were prepared with the
compounds endowed with the best antioxidant and photoprotective profile and their release from
formulation evaluated using Franz Cells system. High antioxidant activity is related to the presence
of at least two hydroxy groups on arylidene moiety of benzimidazoles. Structure activity analysis
revealed that the position of hydroxy groups is crucial for antioxidant activity as well as the
presence of a 2-hydroxy-4-(diethylamino)arylidene group. The same correlation pattern was found
to be related to photoprotective activity resulting in an UVA Protection Factor better than the
commercial solar filter PBSA and antiproliferative activity against melanoma cells without
producing cytotoxicity on normal keratinocytes. The release analysis indicated that high
antioxidant activities are achieved with limited release at concentration compatible with the use as
UV sunscreen filter.
Keywords: benzimidazoles; hydrazones; polyhydroxylated compounds; antioxidant activity;
photoprotective agents; antiproliferative activity
1. Introduction
The heterocyclic benzimidazole nucleus is widespread in nature and present in several
bioactive compounds. Benzimidazole is also a privileged ring in medicinal chemistry and this
pharmacophore is included as a key part in antimicrobial, anticancer, acetylcholinesterase,
antiprotozoal, anti-inflammatory, analgesic, antihistaminic, antiallergic, enzyme inhibitory,
antimalarial, antitubercular, and antiviral agents [1–7]. Several benzimidazole-based compounds
have been also reported to possess antioxidant activity [1,8–12]. Reactive oxygen species (ROS) and
Pharmaceuticals 2020, 13, 68; doi:10.3390/ph13040068
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reactive nitrogen species (NOS) are well known as both harmful and beneficial species [13];
however, when unbalance occurs between endogenous antioxidant defense and ROS oxidative
stress conditions occurs. This latter has been related to an extensive range of diseases including
cardiovascular, inflammatory, neurodegenerative, and autoimmune ones [14,15]. Overproduction of
reactive oxygen species can be responsible for damage to vital cell components, especially to DNA,
lipids, and proteins. It is known that the use of antioxidants is beneficial in the prevention or delay of
numerous diseases associated with oxidative stress including cancer, Alzheimer's and other
neurodegenerative diseases, as well as atherosclerosis [16–19]. It is also known that antioxidants
such as phenols break down oxidation chains by reactions with peroxy radicals. Tocopherol,
p-coumaric acid, ferulic acid, and caffeic acid are the best-known phenolic antioxidant agents. On
the other hand, also hydrazone moieties are endowed with potent antioxidant potency [20–23].
Based on these findings and as a continuation of our works on antioxidant agents [24–26], here we
report and discuss the antioxidant properties of benzimidazolehydrazones containing a phenolic
group and their structure–activity relationships (SAR). The studied molecules have been
synthesized and chemically characterized by us during a previous synthetic study [27], but not fully
investigated for their activity. The designed molecules are characterized by a phenolic hydroxyl
group and hydrazone moiety whose azomethine fraction plays a critical role in antioxidant activity
[28] and possibly contribute to radical-scavenging activity and the antioxidant property.
2. Results and Discussion
2.1. Chemistry
The synthetic approach to hydrazones 3–15 compounds was based on a previously described
two steps procedure [27] that gave high yields starting from commercially available ethyl
1H-benzo[d]imidazole-2-carboxylate (1) that was converted in the corresponding hydrazide 2 which
was coupled with the appropriate hydroxyarylaldehydes in ethanol to afford
benzimidazolehydrazones 3–15 (Table 1).
2.2. Antioxidant Activity.
The evaluation of the antioxidant properties of the benzimidazole hydrazones 3–15 was
achieved by 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH), ferric reducing
antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) methods. Results,
shown in Table 1, are expressed as μmolTE/g for DPPH, FRAP, and ORAC tests and are compared to
the reference compound Trolox, also used as a standard, and ferulic acid was used as positive
control. For the best interpretation of the results of the DPPH test, each compound was tested at the
concentration capable of inhibiting 50% of the radical, except for compounds 3, 4, and 14 which data
refer to the limit of quantification.
Table 1. Antioxidant activity of N1-(4-arylidene)-1H-benzo[d]imidazole-2-carbohydrazides 3-15.
DPPHa
FRAPa
ORACa
Compound
Aryl
µmolTE/g
µmolTE/g
µmolTE/g
Ferulic acid
4379.0 ± 9.8
<23.2b
6872.6 ± 9.7
48.0 ± 2.6
15,906.4 ± 14.2
-
3
4
5
6
2-OH-phenyl
3-OH-phenyl
4-OH-phenyl
<13.0 b
1023.3 ± 9.4
1040.1 ± 11.2
286.7 ± 10.3
21,808.0 ± 99.1
30,911.3± 36.4
9296.8 ± 72.3
29.6 ± 0.1
200.6 ± 1.5
2,4-(OH) -phenyl
2

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7
2,5-(OH)
2
-phenyl
9387.9 ± 13.8
5330.6 ± 29.6 17,856.9 ± 35.1
8
2,3,4-(OH)
2,4,6-(OH)
3
-phenyl
-phenyl
10,945.2 ± 38.5 10,064.6 ± 24.6 4221.5 ± 10.5
9
3
192.6 ± 3.5
201.2 ± 4.9
38.1 ± 1.6
171.5 ± 6.1
1065.0 ± 5.9
<< 13.29b
4071.7 ± 10.0
231.9 ± 5.5
104.5 ± 2.1
4098.6 ± 39.1
10
11
12
13
14
15
2-OH-4-OMe-phenyl
2-OH-3-OEt-phenyl
3-OH-4-OMe-phenyl
-
-
3418.8 ± 15.2 17,170.7 ± 18.4
2-OH-4-N(Et)
2
-phenyl
3525.4 ± 13.4
54.8 ± 1.3
754.4 ± 19.0
2-OH-5-Cl-phenyl
2-OH-5-Br-phenyl
-
-
28.8 ± 1.2
81.7 ±2.7
a Each value was obtained from three experiments (mean ± SE);—not tested.
b LOQ limit of quantification;—not tested.
The radical scavenging activity of the benzimidazole hydrazones 3–15 that emerged from
DPPH results indicates that the hydrazones bearing a single hydroxy group on the aryl ring
(compounds 3–5) showed weak antioxidant activity. By introducing a second hydroxy group in the
4-position (hydrazone 6) the antioxidant capacity increase of about 8-fold as compared to
2-hydroxyphenyl analog 3. The shift of 4-hydroxy group into 5-position to give compound 7 led to
further increase in activity, about 400-fold as compared to hydrazone 3. The introduction of a third
hydroxy group at 3-position of hydrazone 6 to give compound 8 produced about 55-fold
enhancement in activity. On the contrary the introduction of a third hydroxy group at 6-position
(compound 9) did not modify the antioxidant potency that is practically unchanged as compared to
hydrazone 6. Taken together these results indicate that high antioxidant activity is related to the
position of the hydroxy groups rather than to their number.
The introduction of alkoxy groups (hydrazones 10 and 12) at 4-position of compounds 3 and 4
produced about 10-fold increase in activity. However, the presence of alkoxy group at 3-position
(compound 11) led to slight reduction in activity as compared to hydrazone 3. The replacement of
the 5-hydroxy group of compound 7 with halogen atoms as in hydrazones 14 and 15 led deep
reduction in activity with DPPH values similar or lower than hydrazone 3. The replacement of
4-hydroxy group of hydrazone 6 with a dimethylamino moiety (hydrazone 13) produced about
5-fold increase in activity.
FRAP analysis, according to the results obtained from the DPPH test indicated that high
antioxidant activity is related to the position of the hydroxy groups rather than to their number. In
fact, the 2-hydroxyphenyl hydrazone 3 confirmed its poor antioxidant activity. Nevertheless, the
shift of the hydroxy group into the 3- or 4-position (hydrazones 4 and 5) led to about 20-fold increase
in activity. By introducing a second hydroxy group at the 4-position (hydrazone 6) the antioxidant
capacity increase of about 6-fold as compared to 2-hydroxyphenyl analog 3. The shift of 4-hydroxy
group into 5-position to give compound 7 led to further increase in activity, about 100-fold as
compared to hydrazone 3. The introduction of a third hydroxy group at 3-position or 6-position of
hydrazone 6 to give compounds 8 and 9 produced about 35-fold and 14-fold enhancement in activity
respectively. The introduction of alkoxy groups on the phenol ring of compounds 3 and 4
(hydrazones 10–12) produced a moderate increase in activity.
The compounds that showed the most interesting profile following the DPPH and FRAP tests
were also subjected to a further test (ORAC) to outline a complete antioxidant activity profile.
In the ORAC assay the 4-hydroxyphenyl derivative 5 demonstrated the best antioxidant capacity
towards the peroxyl radicals (ROO-). The shift of hydroxy group into 3-position (hydrazone 4)
produced slight reduction in activity. Contrary to DPPH and FRAP assays in ORAC test the
dihydroxyphenyl and trihydroxyphenyl derivatives showed lower activity as compared to the
monohydroxyphenyl analogs. Furthermore the 2-hydroxy-4-(diethyl)aminophenyl derivative 13 did
not confirmed the high antioxidant capacity showed in DPPH and FRAP.
2.3. Evaluation of UV-Filtering Parameters.

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On the hydrazones 4–9, 12, and 13 showing the best antioxidant profile, in vitro tests were
conducted to determine the parameters fundamental for evaluation of the filtering power: SPF value
(Sun Protection Factor), UVAPF0 (UVA Protection Factor Value), UVA/UVB ratio, and critical
wavelength (λc) (Table 2). The Solar Protection Effectiveness Evaluation System specifies a SPF
primarily representing a measure of UVB protection [29] is related to the UV absorption of
substances.
Table 2. In solution UV-filtering activity of selected hydrazones.
Compound SPF UVA/UVB UVAPF0 c (nm)
PBSA
3.42
11.54
12.32
4.81
0.29
0.27
0.75
1.26
0.32
2.23
2.27
1.13
1.18
1.04
1.60
2.95
4.84
3.24
6.79
10.65
3.60
3.99
322
343
353
366
377
366
370
358
394
4
5
6
7
11.23
8.12
8
9
8.34
12
13
8.95
1.57
The spectra of hydrazones 4–9, 12, and 13 were recorded in the 250 and 500 nm range (Figure 1)
and compared with the commercial filter phenyl benzimidazole sulfonic acid (PBSA). The spectrum
of the reference commercial filter PBSA is characterized by maximum absorption peak at 302 nm and
any absorption in the UVA region. The comparison of UV spectra of the reference sunscreen filter
PBSA and hydrazones endowed with the best antioxidant activity showed the λmax of the
hydrazones 4–9, 12, and 13 shifted towards longer wavelengths, as compared to PBSA. Above 350
nm the absorbance is reduced almost to zero (Figure 1). Furthermore, the absorption curves of the
analysed compounds have a wider range than PBSA. The bathochromic shift observed for all the
analysed hydrazones, is related to the presence of auxocrome groups on the arylidene moiety. The
shift of the absorption band towards longer wavelengths is particularly pronounced in the
2-hydroxy-4-(diethylamino)phenyl derivative 13. The values related to the filtering parameters were
extrapolated from the UV absorption spectra.
Figure 1. Panel a, UV spectra of the reference PBSA; panel b, UV absorption spectra of hydrazone
derivatives: 4 (light blue), 5 (violet), 6 (dark green), 7 (red), 8 (blue), 9 (light green), 12 (orange), 13
(fuchsia).
The hydrazones were also evaluated for their UV-B filtering capabilities by the Diffey-Robson
in vitro method [30]. Table 2 shows the data obtained from the analysis of the tested hydrazones as
well as reference PBSA. In vitro tests are usually performed during the preliminary phases of

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development of sunscreen molecules to determine the UV protection potential. Although a new
method has been established and standardized in the ISO 24443: 2012 for the in vitro evaluation of
UVA protection, there is no equivalent recognized in vitro method for evaluating protection in the
UVB range. Among the proposed tests the in vitro method of Diffey-Robson is the most known and
widely applied [30]. Table 2 shows the data obtained from the analysis of the tested hydrazones as
well as reference PBSA. According to the EU recommendation of 2006 (2006/247/EC) a solar product
is considered to have broad spectrum when its lambda critical (λc) value is higher than 370 nm. In
this respect only hydrazones 7, 9, and 13 satisfied this requirement.
However, among broad-spectrum derivatives, the SPF value is noticeable only for compounds 7
and 9. The UVA/UVB ratio, as defined by the aforementioned EU recommendation, should be at
least 1/3. All hydrazones, except 4 and 7, showed UVA/UVB ratio greater than 1 and satisfied this
requirement, meaning an absorption mainly in the UVA region. The last value, determined
according to ISO-24443, is UVA Protection Factor (UVAPF0). All hydrazones are endowed with
UVAPF better than PBSA. The hydrazone 9 showed the best UVAPF0 value, about 10-fold than the
reference PBSA. As mentioned above only hydrazones 7, 9 and 13 had λc value higher than 370 nm.
Regarding the photoprotective activity, in general it can be said that the hydrazone derivatives series
showed a better in vitro SPF profile than that of the reference PBSA sunscreen filter. It has been
observed that the SPF parameter does not seem to be always influenced directly in proportion to the
number of hydroxyls present on the substituent, a correlation that is equally valid for the UVAPF
parameter. The presence of a methoxy group or the 2-hydroxy-4-(diethylamino) portion positively
influences the filtering parameters.
2.4. Evaluation of Release In Vitro
The hydrazones 9 and 13 showing the best dualistic profile were selected to evaluate their
potential, as antioxidant UV filtering ingredients for dermocosmetic formulations, by in vitro
experiments. To this end two topical formulations, differing in degree of polarity, have been
prepared based on our previous background in the investigation on Oxisol [31]: Formulation (A),
designed for the antioxidant activity and therefore for a high release of the active to the skin, and
Formulation (B) designed instead for a sunscreen filter and then aimed to obtain avoid release of the
active ingredient due to its high affinity for the formulation. The release study was carried out using
Franz Cells, the most commonly used in vitro approach to evaluate the release of active ingredients
from semi-solid preparations. The amount of hydrazones, 9 and 13, and Oxisol released in the
receiving chamber was sampled within 6 h and the resulting release curves are shown in Figure 2.

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a
b
c
Figure 2. Permeation profiles of Oxisol (panel a), and hydrazones 9 (panel b) and 13 (panel c). Line
green corresponds to formulation A (optimized for skin adsorption), line yellow corresponds to
Formulation B (optimized to best solubilize the active in formula).
The release curves obtained Franz cell assay of hydrazones 9 and 13 compared with the
reference Oxisol (Figure 2) maintain the same trend both for the reference Oxisol and for the
hydrazone derivatives 9 and 13. In the case of Oxisol and compound 9 the delta tends to shrink after
240 min. The graph of compound 13 presents trend and delta between the two curves almost
constant; however, after the first 180 min the release from formulation A tends to progressively
increase, until it reaches a difference of about 15%. This result, analogous to those obtained
previously [25], supports the possibility of modifying the release of the active ingredient from the
formulation by changing only the percentages of the components of the formula ad hoc designed,
and its composition, depending on the function desired of the active component.

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2.5. Antiproliferative Activity
Polyphenols are known for their activity against melanoma [32], a malignant and aggressive
tumor associated with intense exposure to UV radiation. For this reason, once the compounds of the
series with an excellent antioxidant profile associated with filtering capacity were identified, it was
decided to evaluate their anti-proliferative activity on melanoma cell cultures (Colo-38). On the most
active compounds the antiproliferative activity was tested also on human skin keratinocytes HaCat
using a previously described method [26].
In the performed experiment, the cells were seeded at a concentration of 40,000 cells/ml and
cultivated in the presence of increasing concentrations of the compounds examined (from 0.01 to 500
μM). Cell counting was executed after 48 h and 72 h from treatment. For each hydrazone the value of
IC50 (concentration value necessary for the inhibition of 50% cell growth) was calculated starting
from at least three independent experiments. The results of the preliminary assay, which aimed to
identify eligible candidates as potential active drugs against melanoma, are listed in Table 3.
Table 3. Effects of selected hydrazone derivatives on the proliferation of melanoma Colo-38 and
keratinocyte Ha-Cat human cell lines.
Compound
IC50 (M)
Colo-38
Compound
IC50 (M)
Colo-38
Ha-Cat
4
5
6
7
362.63 ± 15.05
216.16 ± 40.21
46.62 ± 0.82
35.49 ± 0.42
8
46.6 2 ± 3.57
459.59 ± 103.87
0.84 ± 0.03
9
12
13
53.16 ± 4.33
5.03 ± 0.76
0.50 ± 0.12
Among the best antioxidant hydrazones tested against human melanoma Colo-38 cells,
compounds 12 and 13 emerged, which showed the best anti-proliferative effect with IC50 values
respectively equal to 0.84 and 0.50 μM. These hydrazones were also tested on immortalized human
keratinocytes HaCat cells, in order to verify a possible selectivity for tumor cell lines. Both
hydrazones 12 and 13 demonstrated selectivity for cancer cells with IC50 values against HaCat cells
(53.16 ± 4.33 and 5.03 ± 0.76 for 12 and 13 respectively) about 60-fold and 10-fold higher as compared
to melanoma Colo38 human cells.
3. Materials and Methods
3.1. General Methods.
The spectrophotometer used for antioxidant analysis is a Beckman Coulter™, DU®530, Life
Science UV/VIS spectrophotometer, Single Cell Module. The instrument used to conduct ORAC
analyzes is the Thermo Fluoroskan Ascent FL® Microplate Fluorometer and Luminometer, linked to
Ascent Software® software for data control and processing. In the sample loading phase, 96-well
plates with a black background were used. Spectrophotometric analyses for the detection of filter
parameters were conducted with a UV–VIS spectrophotometer SHIMADZU UV-2600 240 V. The
benzimidazolehydrazones 3–15 have been synthesized using our previously described procedure
[27].
3.2. Antioxidant Activity Evaluation
3.2.1. DPPH test.
The benzimidazole hydrazones 3–15 were tested against DPPH• radical following the Wang et
al. method [33] modified as previously reported [24]. The 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical-scavenging assay measures the hydrogen donation ability of an antioxidant to convert the
stable DPPH free radical into 1,1-diphenyl-2-picrylhydrazyl. This can be evaluated by measuring the
percent decrease in absorbance of the solution at 517 nm, through the color change from deep-violet
to light-yellow, after the radical reaction with products to be tested. The radical-scavenging activity

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is expressed as inhibition ratio of initial concentration of DPPH radical and is calculated according to
the formula:
Inhibition percentage (Ip)=[(AB-As)/AB]•100
(1)
where AB and As are, respectively, the absorbance values of blank reaction and of the tested sample.
The DPPH solution is prepared by dissolving 4 mg of DPPH in 100 ml of MeOH and then the
solution is stirred in the dark for 30 min. The standard solutions of Trolox are prepared in MeOH in
a range between 4–95 nmoles, while sample solutions starting from 0.2 mg/mL to 0.004 mg/mL.
After 1.5 mL of the DPPH solution has been added to 0.750 mL of the sample solutions proper
diluted/standard/control, it is reacted for 30 min in the dark. Samples absorbance measurements
were evaluated with a UV-VIS spectrophotometer at fixed wavelength of 517 nm. Blank sample was
prepared
adding
methanol
to
DPPH
solution
and
Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as standard reference to achieve
a calibration curve. The results are expressed as μmol TE/g corresponding to an inhibition of the
radical equal to 50%, except for some compounds.
3.2.2. FRAP test.
The ferric ion reducing ability of hydrazones 3–15 was measured reading the absorbance of the
reaction mixture at 593 nm, according to the method described previously [34]. The analysis reagent
was freshly prepared by mixing the subsequent solutions in the fixed ratio 10:1:1 (v/v/v): 1) 0.1M
Acetate buffer, pH 3.6; 2) TPTZ (2,4,6-tripyridylstriazine), 10 mM in 40 mM HCl, 3) FeCl , 20 mM. To
3
a 1.9 mL of reagent were added 0.1 mL of sample proper diluted or solvent when blank was
performed. Readings were done after 10 min, using a UV-VIS spectrophotometer at fixed
wavelength of the absorption maximum (593 nm). 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 equivalents (μmol TE/g compound) since this
standard was used to perform the calibration curves.
3.2.3. Oxygen Radical Absorbance Capacity (ORAC).
The scavenging activity test against the peroxyl radical was carried out based on a previously
reported and modified protocol [35]. Sample solutions (mg/mL) and Trolox dilutions (40–240 μM)
were prepared using Phosphate buffer solution (pH 7.4). In a 96-well black microplate (VWR) 25 μl
of sample solution, Trolox dilution or phosphate buffer solution (pH 7.4) used as blank was placed
in wells. Measurements of fluorescence were carried out at 37 °C and recorded at 5 min intervals up
30 min after the addition of AAPH. The ORAC values, expressed as Trolox equivalents (μmol TE/g
compound), were calculated according to the method of Cao et al. [36]. The antioxidant capacity of
the tested compound was quantified by integration and calculation of the area under the curve
(AUC), relating it to that produced by reference Trolox.
3.3. Evaluation of Filtering Parameters.
The method followed to determine the value of SPF in vitro is an adaptation of the official
method [37]. The hydrazone solutions tested at a concentration of 0.000034 (± 0.0000033) M were
prepared in methanol and the absorption spectra were recorded. The equation below was used to
transform the absorbance values obtained into transmittance and calculate the SPF values in vitro:
A() = Log[T()]
(2)
SPF, UVA/UVB, UVAPF, and λ critical values were obtained elaborating the transmittance
spectrum with the SPF calculator software (version 2.1, Shimadzu, Milan, Italy).
3.4. Preparation of Formulations.

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Two different standard oil/water emulsions (Formulation A and Formulation B) were prepared
for Oxisol and hydrazones 9 and 13. The two phases were brought to 60–70 ° C after adding the
individual ingredients. The oil phase was then added under mechanical stirring to the aqueous
phase. The selected active ingredients were added, always under constant stirring, in the cooling
phase to avoid their thermal degradation. Once they reached 25 °C, the emulsions were stored in the
refrigerator until analysis. The formulation compositions are the following:
Formulation A: Aqua, Ethylhexyl Stearate, Tribehenin PEG-20 Esters, Butyrospermum Parkii,
Olea Europaea Oil Unsaponifiables, Oxisol or hydrazone 9 or 13, Xanthan Gum, Caprylic/Capric
Triglyceride, Cetearyl Alcohol, Dicaprylyl Carbonate.
Formulation B: Aqua, Cetearyl Alcohol, Tribehenin PEG-20 Esters, Butyrospermum Parkii, Olea
Europaea Oil Unsaponifiables, Oxisol or hydrazone 9 or 13, Xanthan Gum, Caprylic/Capric
Triglyceride.
3.5. Franz Cells Apparatus.
The protocol used for the release experiments followed the one previously reported [25] with
the variation of the receiving solution: in this case in fact the receiving solution was Buffer phosphate
1M pH 11.2 for Oxisol, and pH 11.7 for compounds 9 and 13. 0.5 g of each formulation was placed in
the donor compartment in contact with the membrane positioned between the two compartments.
Parafilm was used for donor compartment to avoid loss of formulation components. Experiments
were conducted in a thermostated bath at 37 ± 1 ° C and under continuous stirring. The samples
taken at any time, 30 min, 1 h and then every hour thereafter up to 6 h (and replaced with fresh
receiving medium), were analyzed by UV-VIS spectrophotometer. For each compound was built a
calibration curve at λmax.
3.6. Growth Inhibition Assays.
Cell growth inhibition assays were carried out using melanoma Colo38 human cancer cell,
[24,38,39] and skin keratinocytes HaCat [26]. Cell lines were maintained in RPMI 1640,
supplemented with 10% fetal bovine serum (FBS), penicillin (100 Units mL-1), streptomycin (100 μg
mL-1) and glutamine (2 mM); the pH of the medium was 7.2 and the incubation was performed at 37
°C in a 5% CO atmosphere. Tested hydrazones were dissolved in MeOH/DMSO 10% to obtain 20
2
mM stock solutions and diluted before cell treatment in MeOH 100%. The tested hydrazones were
added at serial dilutions to the cell cultures (from 0.01 to 500 μM) and incubated for 3 days. Cells
were then harvested, suspended in physiological solution and counted with a Z2 Coulter Counter
(Coulter Electronics, Hialeah, FL, USA). The cell number/ml was determined as IC50 after 3 days of
culture, when untreated cells are in log phase of cell growth. Untreated cells were placed in every
plate as negative control.
4. Conclusions
Research aimed at the development of multifunctional molecules has been at the center of our
work for years [24,25,27] and has led us to investigate different scaffolds such as benzimidazole,
benzofurane and indole nuclei, which are however connected by an hydrazone linker and an
aromatic nucleus, differently substituted, considering the importance of fractions suitable to extend
the conjugation to the aromatic ring. All the derivatives have been tested for their potential
antioxidant, UV filtering and anti-proliferative activity (in particular against melanoma cells).
The data of this latest study are reflected in the SAR data of previous works and contribute to
implementing the experimental evidence found so far to obtain a multifunctional activity.
The study of benzofuran derivatives [24] revealed a correlation between the number and
position of hydroxyl groups on the arylidene portion and good antioxidant capacity. A high activity
had instead been found for the 2-hydroxy-4-(diethylamino)benzylidene derivative. The SAR study
on indole derivatives [25] had led to the same conclusions, also underlining the contribution of a
methoxy group in position 4 to the activity under consideration. The antioxidant data deriving from

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the study of benzimidazolehydrazone derivatives again confirm what reported by the previous
series. As regards the antioxidant activity, in fact, the best profile was shown by the derivatives 7, 8,
9, and 12 characterized by the presence of at least 2 hydroxy groups (7, 8, 9) or of a 2-hydroxy and a
methoxy group in position 4 (12). The 2-hydroxy-4-(diethylamino) substituent, unlike the other
series, does not seem to contribute to improving the activity against the peroxyl radical (ORAC test).
As regards the photoprotective capacity, in general it can be said that all the hydrazone
derivatives series showed a better in vitro SPF profile as compared to the commercial reference PBSA
filter. In particular, it has been observed that the SPF parameter does not seem to be always
influenced directly in proportion to the number of hydroxyls present on the substituent, a
correlation that is equally valid for the UVAPF parameter. The presence of a methoxy or a
2-hydroxy-4-(diethylamino) group positively influences the filtering parameters.
Cytotoxic activity against Colo-38 cells of human melanoma has led to the identification of
derivatives belonging to the three different series, with a promising antitumor profile. The best IC50
values were obtained for the derivatives bearing the 2-hydroxy-4-(diethylamino) moiety: the IC50
data of the derivative 13 (0.50 μM) is in line with the values obtained from the derivatives of the
previous series [24,25].
In conclusion we can affirm that the SAR study derived from the synthesis and analysis of
heterocyclic structures that share the presence of a hydrazone spacer bearing an aromatic
functionality variously substituted led us to identify a common profile to the different series.
Specifically, this study has shown that the presence of the 2-hydroxy-4-diethylamino portion is
related to the antioxidant, photoprotective and antiproliferative activity in the three series of
hydrazones, and can therefore be considered the focus of the multifunctional profile of these
derivatives.
Author Contributions: For research articles with several authors, a short paragraph specifying their individual
contributions must be provided. The following statements should be used “Conceptualization, S.M. and V.O.;
investigation, A.B., M.D., D.M., I.L; resources, M.T.; writing—original draft preparation, A.B., M.D., D.M., V.O.;
writing—review and editing, S.M., V.O., G.B., A.B., S.V., I.L.; project administration, S.M., V.O., G.B., S.V.;
funding acquisition, S.M., V.O., G.B.. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by the Fondazione di Sardegna under the project “Innovative antioxidant
molecules for the food and health industry” (CUP F71I17000180002 to GB and VO), by University of Ferrara
(Grant FAR 2017 to SM, SV and IL), Ambrosialab srl (Ferrara, Italy) and by the Italian Ministero dell’Istruzione,
Università e della Ricerca (PRIN 2017, Prot. No. 2010E84AA4_002).
Conflicts of Interest: The authors declare no conflict of interest
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