Synthesis and evaluation of antioxidant and antiproliferative activity of 2-arylbenzimidazoles

Article abstract of DOI:10.1016/j.bioorg.2019.103396

Three series of arylbenzimidazole derivatives 3–40, 45 have been simply synthesized and tested for their antioxidant capacity. The 2-arylbenzimidazoles were tested against various radicals by the DPPH, FRAP and ORAC tests and showed different activity pro

Full text of DOI:10.1016/j.bioorg.2019.103396

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Synthesis and evaluation of antioxidant and antiproliferative activity of 2-ar-
ylbenzimidazoles
Anna Baldisserotto, Monica Demurtas, Ilaria Lampronti, Massimo Tacchini,
Davide Moi, Gianfranco Balboni, Salvatore Pacifico, Silvia Vertuani, Stefano
Manfredini, Valentina Onnis
PII:
S0045-2068(19)31204-0
DOI:
https://doi.org/10.1016/j.bioorg.2019.103396
YBIOO 103396
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To appear in:
Bioorganic Chemistry
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26 July 2019
17 October 2019
22 October 2019
Please cite this article as: A. Baldisserotto, M. Demurtas, I. Lampronti, M. Tacchini, D. Moi, G. Balboni, S.
Pacifico, S. Vertuani, S. Manfredini, V. Onnis, Synthesis and evaluation of antioxidant and antiproliferative
activity of 2-arylbenzimidazoles, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103396
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Synthesis and evaluation of antioxidant and antiproliferative activity of 2-arylbenzimidazoles.
Anna Baldisserotto,b,° Monica Demurtas,a,° Ilaria Lampronti,c Massimo Tacchini,d Davide Moi,a
Gianfranco Balboni,a Salvatore Pacifico,e Silvia Vertuani,b Stefano Manfredini,b and Valentina
Onnisa*
aDepartment of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and
Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari I-09124, Italy.
bDepartment of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 17-
19, Ferrara I-44121, Italy.
cDepartment of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology,
University of Ferrara, Via Fossato di Mortara 74, Ferrara I-44121, Italy.
dDepartment of Life Sciences and Biotechnology, Section of Pharmaceutical Biology, University of
Ferrara, Piazzale Luciano Chiappini 3, Malborghetto di Boara (FE), I-44123, Italy.
eDepartment of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara
17-19, Ferrara I-44121, Italy.
Abstract
Three series of arylbenzimidazole derivatives 3-40, 45 have been simply synthesized and tested for
their antioxidant capacity. The 2-arylbenzimidazoles were tested against various radicals by the
DPPH, FRAP and ORAC tests and showed different activity profiles. It has been observed that the
number and position of the hydroxy groups on the 2-aryl portion and the presence of a diethylamino
group or a 2-styryl group are related to a good antioxidant capacity. Furthermore, benzimidazoles
showed satisfactory SPF values in vitro compared to the commercial PBSA filter, proving to have a
good photoprotective profile. In particular, 2-arylbenzimidazole-5-sulphonic acids 15 and 38, the 2-
styryl-benzimidazole 45 showed broad spectrum solar protection against UVA and UVB rays. The
antiproliferative effect of the benzimidazoles was tested on human skin melanoma Colo-38 cells.
The styrylbenzimidazole 45 exhibited antiproliferative effect at low micromolar concentration
against Colo-38 cells and very low antiproliferative activity on normal HaCat keratinocyte cells.
Keywords
Benzimidazoles, Polyhydroxylated compounds, Antioxidant activity, Photoprotective agents,
Antiproliferative activity.

*Corresponding author. E-mail address: vonnis@unica.it (V. Onnis).
°These authors equally contribute to the present paper
Graphical Abstract

1. Introduction
Reactive oxygen (ROS) and nitrogen (NOS) species produced by metabolism of endogenous and
exogenous substances led to oxidative stress. Clinical evidences account for the involvement of
oxidative stress-induced by reactive oxygen and nitrogen species in a variety of disorders including
cancer [1], atherosclerosis [2] neurodegeneration and aging [3]. ROS can be generated by UVB and
UVA radiation producing protein oxidative modification resulting in photoaging. ROS cause tissue
modification that at skin level is elicited by the loss of the barrier function of the stratum corneum,
the promotion of inflammatory processes, erythema, up to the cancer [4, 5]. The benzimidazole
moiety represents a privileged structure in medicinal chemistry. Molecules of natural origin play a
fundamental role in the research of bioactive molecular models for the development of new
synthetic and semi-synthetic analogues. The discovery of 5,6-dimethyl-1-(D-ribofuranosil)-
benzimidazole, as an integral part of vitamin B12, has generated considerable interest in the
benzimidazole nucleus [6]. This can be considered as a structural isostere of indole and purines, or
other natural active substances capable of interacting with other biomolecules, such as proteins and
nucleic acids [7].
Benzimidazole is a pharmacophore that has found widespread application in medicinal practice. The
benzimidazole ring system exists in the structure of many drugs such as the antihypertensive
Candesartan, the proton pump inhibitor Pantoprazole, the antihistaminic Astemizole, the
antimicrobial Albendazole, analgesics, anti-inflammatories, anti-convulsant, anti-psychotic, anti-
diabetic, anthelmintic, antiprotozoal drugs and antifungals [8, 9]. Literature data also report a
remarkable antiproliferative, anti-tumor, and antiviral biological activity [10] of the various
derivatives of the benzimidazole nucleus. Furthermore, benzimidazole derivatives have been
reported for their antioxidant activity [11-14].
The benzimidazole core also appears in some sunscreens; it is contained, for example, in 2-phenyl-
1H-benzimidazole-5-sulfonic acid (PBSA), one of the UVB filters mostly used in cosmetics for sun
protection. PBSA is characterized by high water solubility in and has an excellent safety profile.
Another effective example is the disodium salt of phenyl-dibenzimidazole-tetrasulfonate (known as
Neo Heliopan® AP) which absorbs mainly in the UVA range. Neo Heliopan® AP is soluble in aqueous
phase when added with a base, it is stable and safe, with an extremely low degree of penetration of
the skin. In this context, a molecule was designed and synthesized with the aim of combining the
filtering capacity of the already known PBSA and the strong antioxidant properties of polyphenols.

The final product, called Oxisol (2-(3,4-dihydroxy-phenyl)-1H-benzimidazole-5-carboxylic acid), is
not only a powerful antioxidant and absorbs in the UVA range, but is also equipped with SPF booster
effect [15-16]. As a continuation of our efforts to identify dualistic molecules [17, 18] able to act, at
the same time, as antioxidant and photoprotective agents here we describe the design, synthesis
and biological evaluation of three series of benzimidazoles differing for the substituent at 5-position
and bearing a variety of hydroxyaryl moieties at 2-position.
2. Results and Discussion
2.1. Chemistry
The target benzimidazoles 3–40, 45 (Table 1) were synthesized as shown in Schemes 1 and 2 starting
from readily available and inexpensive starting materials.
The ethanolic solution of
diaminobenzenes 1a-d was added with sodium metabisulphite aqueous solution and the
appropriate aldehyde 2. The resulting solution was refluxed to achieve benzimidazoles (3-40) in 52-
88% yields (Scheme 1). Benzimidazole 45 was obtained following the reaction sequence showed in
Scheme 2. 4-Hydroxycinnamic acid (41) was coupled with methyl 3,4-diaminobenzoate (42) in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxybenzotriazole
(HOBt) in anhydrous acetonitrile solution to give the intermediate 43. Benzimidazole ring closure
was performed in hydrochloric acid to give the ester derivative 44. Compound 44 was submitted to
mild basic hydrolysis to afford the benzimidazole (45). Structures of all new compounds were
confirmed based on analytical and spectral data which are consistent with results of reported
studies [15, 19-21]
R
N
R
NH2
NH2
i
ArCHO
Ar
N
H
2
3 40
-
1a
1b
1c
1d
R = H
R = CN
R = COOH
R = SO3H
Scheme 1. Reagents and conditions: EtOH, 2.5 N sodium metabisulphite, reflux 24 h.

O
MeOOC
NH
MeOOC
NH2
NH2
i
HO
NH2
COOH
OH
43
42
41
ii
HOOC
N
MeOOC
OH
N
iii
OH
N
H
N
H
45
44
Scheme 2. Reagents and conditions: i) EDC, HOBt, MeCN, r.t. 36 H; ii) EtOH, 6 N HCl, reflux 5 h; iii) EtOH, 2.5
N NaOH, r.t. 24h.
2.2 Antioxidant activity
The valuation of the antioxidant properties of the benzimidazoles 3–40, 45 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 are shown in Table 1 and
are expressed as μmolTE/g for DPPH, FRAP, and ORAC tests. For the best interpretation of the results
of the DPPH, each compound was tested at the concentration capable of inhibiting 50% of the
radical. However, in some cases it was not possible to obtain data reflecting 50% inhibition, even if
the tested compounds are all in the standard calibration line.
DPPH result analysis indicated that high antioxidant capacity is correlated to high number of hydroxy
groups present on the aryl ring at the 2-position. Benzimidazole derivatives 3–11 bearing a single
hydroxy group on the aryl ring showed weak activity independently from the substituent present at
5-position. The introduction of a second (compounds 13-17) or a third hydroxy group (compounds
31-36) led to the best compounds of the series. Nevertheless, the hydroxy group position also
affects antioxidant activity: in fact, 2,5-dihydroxybenzimidazole derivatives 13, 14 were about 2
times more active than the 3,4-dihydroxyphenyl analogs 16, 17. While the shift of the 3-OH of
compound 16 into 2-position to give the analog 12 produced drop in activity. Furthermore, the
2,3,4-trihydroxyphenyl derivatives 31 and 32 were more active than the 2,4,6-trihydroxyphenyl
analogs 34 and 35. On the contrary the comparison of the 5-sulfonyl substituted benzimidazoles
indicated that the 2,4,6-trihydroxyphenyl analog 36 was characterized by antioxidant activity better
than the corresponding 2,3,4-trihydroxyphenyl derivative 33. The replacement of one hydroxy
group with an alkoxy group (compounds 18-24) or halogen atoms (compounds 25-30) led to

reduction in activity. The introduction of a 4-diethylamino group (benzimidazoles 37 and 38)
produced increase in activity as compared to compounds 7 and 8.
On FRAP analysis, according to the results obtained from the DPPH test the most powerful
derivatives were benzimidazoles 13-17 and 31-37. Interestingly, the 2-hydroxy-3-ethoxyphenyl
derivatives 21, 22, the 2-hydroxy-4-(diethyl)amino derivatives 38, and the 4-hydroxystyryl
derivative 45 demonstrated good activity.
The compounds that showed the most interesting profile following the DPPH and FRAP tests were
also subjected to a further test (ORAC) to determine their complete antioxidant activity profile. The
4-hydroxy-styryl derivative 45 demonstrated the best antioxidant capacity. The 2-hydroxy-4-
(diethyl)aminophenyl derivatives 37 and 38 demonstrated high antioxidant capacity. Contrary to
DPPH and FRAP assays, in ORAC test the 2,3,4-trihydroxyphenyl derivative 31 showed better activity
as compared to the corresponding 2,4,6-trihydroxyphenyl analog 34. The 5-COOH-benzimidazole
derivatives (32, 35) and 5-SO3H-benzimidazole (33, 36) analogs displayed a reverse trend in ORAC
antioxidant results.
Table 1. Antioxidant activity of the indole derivatives 3-40 and 45.
R1
R2
R
N
R3
N
H
R5
R4
R
R1 R2
R3
R4 R5
DPPHa
FRAPa
ORACa
µmolTE/g
-
Compound
µmolTE/g
µmolTE/g
H
H
H
H
H
H
H
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
4
5
6
7
10.9 ±1.5
<15.8 b
15.1 ± 0.4
30.3 ± 2.7
94.2 ± 2.5
CN
-
-
-
-
-
-
-
COOH
SO3H
H
< 39.2 b
no activity c
51.8 ± 0.6
no activity c
<18.3 b
24.6 ± 1.8
103.3 ± 3.7
187.4 ± 4.9
29.0 ± 2.5
223.4 ± 3.8
H
COOH OH
SO3H OH
H
H
H
8
9
CN
H
H
OH
OH
H
COOH
H
10
87.4 ± 1.2

SO3H
CN
H
OH
H
H
H
H
H
H
-
-
11
12
no activityc
<27.5 b
56.5 ± 1.2
OH
OH
OH
209.2 ± 1.8
13
14
15
16
17
4747.2 ± 19.9 10109.0 ± 13.7 20827.9 ± 29.5
4824.7 ± 11.6 10318.2 ± 15.8 18378.7 ± 18.4
CN
H
H
H
H
OH
OH
OH
H
H
H
H
H
H
H
COOH OH
SO3H OH
515.7 ± 1.8
2042.1 ± 7.3
1946.8 ± 8.1
1562.7 ± 13.1
6353.8 ± 11.6
5502.3 ± 13.2
16014.2 ± 67.4
8210.2 ± 25.0
13536.6± 15.6
-
H
H
CN
COOH
CN
H
H
H
OH
OH
OH
OH
H
OH OMe
OH OMe
OH OMe
H
18
19
20
39.9 ± 0.9
48.4 ± 3.5
no activity c
72.7 ± 3.3
70.1 ± 2.3
814.9 ± 5.8
COOH
SO3H
H
H
H
H
H
H
-
-
21
22
109.7 ± 0.4
< 50.9 b
4098.7 ± 16.3
2556.6 ± 17.6
7639.9 ± 25.9
13900.6 ± 61.4
-
COOH OH OEt
SO3H OH OEt
H
H
H
H
H
H
H
H
COOH OH
SO3H OH
H
H
H
H
H
H
H
H
OMe
23
24
25
26
27
28
29
30
41.2 ± 2.4
no activity c
45.0 ± 1.8
81.6 ± 3.4
<< 59.6 b
113.5 ± 5.1
27.7 ± 2.4
64.7 ± 3.9
94.2 ± 2.5
174.7 ± 6.2
48.5 ± 1.5
117.3 ± 4.1
63.4 ± 2.7
OMe
H
H
Cl
Cl
Cl
Br
Br
Br
H
H
H
H
H
H
H
-
-
-
-
-
-
-
CN
OH
COOH OH
SO3H OH
H
H
CN
OH
H
48.7 ± 1.7
81.3 ± 0.9
<< 50.0 b
COOH OH
SO3H OH
H
H
31
32
33
34
35
36
7112.7 ± 15.1 12049.2 ± 19.2
5026.6 ± 13.9 11375. ± 19.6
1324.1 ± 14.2 9097.5 ± 16.7
1473.0 ± 10.3 7138.2 ± 12.4
8312.9 ± 31.8
7321. ± 17.8
6726.9 ± 17.4
4758.0 ± 13.7
9467.8 ± 63.2
8679.5 ± 28.0
CN
OH OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
COOH OH OH
SO3H OH OH
H
OH
OH
OH
CN
OH
H
H
H
897.3 ± 5.6
2334.8 ± 7.5
1970.3 ± 3
COOH OH
SO3H OH
5026.6 ± 13.8

H
H
37
38
39
40
45
165.1 ± 7.5
84.9 ± 0.7
< 27.6 b
5233.1 ± 6.2
925.7 ± 8.8
187.7 ± 3.1
527.7± 8.8
792.0 ± 5.0
14583.9 ± 110.0
12871.7 ± 35.6
-
COOH OH
SO3H OH
COOH
H
H
N(Et)2
N(Et)2
H
H
2-OH-naphthyl
2-OH-naphthyl
4-OH-styryl
4.9 ± 0.7
4297.5 ± 41.5
24852.0 ± 28.5
SO3H
31.33 ± 0.7
COOH
a Each value was obtained from three experiments (mean ± SE).
b LOQ limit of quantification; – not tested.
c precipitation of the compound is observed.
2.3 Evaluation of filtering parameters
On the benzimidazoles 13-17, 21, 22, 31-38, 40 and 45 endowed with the best antioxidant profile,
in vitro tests were performed to determine the parameters fundamental for the evaluation of the
filtering power: critical wavelength (λc) and SPF (Sun Protection Factor) and results are reported in
Table 2. The Solar Protection Effectiveness Evaluation System specifies a SPF primarily representing
a measure of UVB protection [22] is related to the UV absorption of substances. The UV spectra of
the compounds 13-17, 21, 22, 31-38, 40 and 45 were recorded between 290 and 400 nm and
compared with the commercial filter PBSA used as a reference UVB filter (λmax 302 nm). The
superimposed UV spectra of the analyzed compounds were subdivided according to kind of
substituent at 5-position (Figures 1-3). The UV absorption spectra of 5-cyanobenzidimidazoles 13,
16, 31 and 34 (Figure 1) showed λmax shifted towards longer wavelengths as compared to the
reference PBSA. Compound 13 showed the λmax closest to PBSA; however, its absorption range was
the widest in the series with an absorbance reaching zero near 400 nm. The bathochromic shift is
correlated to the increase in the number of hydroxy groups on the 2-aryl ring: the wavelength of
maximum absorption was in fact greater for derivatives 31 and 34, both bearing three hydroxy
groups. As can be seen from Figure 2, 5-carboxylic acid benzimidazole derivatives 14, 17, 21, 32, 35,
37 and 45 were also characterized by a wide range of absorption which is exhausted well over 400
nm. The spectral profile of benzimidazoles 37 and 45 was characterized by λmax shifted to
wavelengths higher than 350 nm. However, all the 5-carboxylic acid benzimidazole derivatives
displayed absorption spectra better than PBSA.
The profile of the UV spectra of the 5-sulfonic acid benzimidazole derivatives 15, 22, 33, 36, 38, and
40 (Figure 3) was again wider than the reference, furthermore the λmax were greater than PBSA.
The bathochromic shift appeared particularly evident for compound 38: once again, the presence

of a tertiary amino group on the aromatic ring promotes the shift of the maximum absorption peak
to the right in the spectrum.
Figure 1. UV absorption spectra of 5-cyano-2-arylbenzimidazole derivatives 13 (fuchsia), 16 (green), 31 (blue), 34 (red).
Figure 2. UV absorption spectra of the 5-carboxylic acid benzimidazole derivatives 14 (red), 17 (blue), 21 (yellow), 32
(light green), 35 (blue), 37 (brown), 45 (pink).

Figure 3. UV absorption spectra of the 5-sulfonic acid benzimidazole derivatives 15 (yellow), 22 (purple), 33 (blue), 36
(green), 38 (fuchsia), 40 (red).
A preliminary in vitro study was carried out on the filtering properties to determine the UV
protection potential of benzimidazoles 13-17, 21, 22, 31-38, 40 and 45. To date there is an approved
and standardized method for in vitro assessment of UVA protection present in ISO 24443: 2012,
however for the evaluation of UVB protection there is still no approved in vitro method. The method
proposed by Diffey-Robson for the in vitro evaluation of the SPF parameter in 1989 [23] is still
applied. The UV absorption spectra of the benzimidazoles 13-17, 21, 22, 31-38, 40 and 45 as well as
the reference PBSA were recorded and the values relating to the filtering parameters were
extrapolated (Table 2). According to the EU recommendation on the efficacy of sun protection
products (2006/247/EC) a broad-spectrum solar product, capable of protecting the skin
simultaneously from UVA and UVB rays, should have a λc value greater than 370 nm. On this
criterion, the only interesting compounds of the 2-aryl-benzimidazole series were derivatives 15 and
38 bearing a sulfonic acid moiety and the 2-styryl-benzimidazole 45. Another parameter that
provides indications on the best blocked UV region is the UVA/UVB absorbance ratio. As defined by
the aforementioned EU recommendation (2006/247 / EC), this ratio should be worth at least 1/3.
Almost all the compounds belonging to the three series satisfied this requirement, most of them
with values greater than 1, to indicate an absorption mainly in the UVA region.
Finally, the value of UVAPF0 was determined in relation to the guidelines provided by ISO-24443: all
the above-mentioned polyhydroxylated compounds, which meet the effectiveness requirements
imposed by EU legislation, have a better UVA Protection Factor than the reference commercial filter.
The most interesting data were showed by compounds 37 and 38, with a UVAPF0 value of 14.30
and 15.77 respectively.

Table 2. UV-filtering activity of selected benzimidazoles in solution.
Compound SPF UVA/UVB UVAPF0
Compound SPF UVA/UVB UVAPF0
c (nm)
c (nm)
PBSA
13
32
33
34
35
36
37
38
40
45
3,4
0,29
0,73
0,75
0,70
0,86
0,85
0,37
0,29
1,10
1,03
2,28
2,19
2,06
1,79
1,71
1,39
1,32
1,48
322
369
368
373
350
349
342
340
347
2,71
1,70
3,34
3,20
4,96
1,79
1,71
2,3
1,04
0,81
1,27
0,90
0,67
2,10
2,20
0,77
2,27
1,51
1,10
1,78
1,49
1,58
14,30
15,77
1,62
7,86
348
343
349
355
350
387
386
362
384
2,93
2,79
2,55
4,63
4,26
6,03
6,25
2,56
14
15
16
17
21
22
31
2,42
2.4 In vitro release study of topical formulations
In analogy with one of our previously reported studies [18], we selected the most promising
candidate in terms of antioxidant capacity and UV-filtering profile, compound 15, to assess its
potential as an active ingredient of topical formulations in vitro. Therefore, the formulation
conceived and designed for Oxisol [15], a molecule with dualistic activity, has been optimized for
compound 15. Two topical formulations were then prepared with different degrees of polarity on
the basis of the structures of the two compounds: Formulation (A) to obtain a high release of the
active substance that carries out activity after absorption in the skin (active acting as an antioxidant)
and the Formulation (B) to obtain a limited release, through a better solubilization of the active
principle in the formulation (active that acts as a solar filter). The release of the two ingredients
from the topical formulations was evaluated through the Franz cell system. The Franz cells consist
of two vertical chambers (donor and acceptor) separated by a synthetic or biological membrane
which has both the support function and the means of separation between the receiving medium
and the formulation for which the release is to be studied. In this study, both for compound 15 and
for Oxisol, the receptor phase was sampled within 6 hours to measure the quantity in the receiving
chamber, the resulting release curves are reported in Figure 4. It was observed a 0.8% and 7%
difference between the release curves of the two different formulations that characterize Oxisol

and compound 15. Specifically, the two curves maintained the same trend as regards Oxisol, with a
reduction in the delta after 240 minutes. With regard to derivative 15, in the first 30 minutes the
same release could be observed from formulations A and B, but in later times the gap between the
two curves tends to increase until it reached a difference of 7%. These results, like those obtained
previously [18], confirm that it is possible, only by varying the components percentages in the
formulation, to modify the release of the active ingredient. This approach therefore consists of
modifying the polarity of a formulation on the basis of both the activity and the end use of the
chosen active ingredient.
Release curve Oxisol
20
15
10
5
0
0
50
100
150
200
250
300
350
400
Times (minutes)
Formulation A
Formulation B
Release curve 15
20
15
10
5
0
0
50
100
150
200
250
300
350
400
Times (minutes)
Formulation A
Formulation B
Figure 4. Permeation profiles of Oxisol, and benzimidazole 15. Line blue corresponds to formulation A (optimized for
skin adsorption), line orange corresponds to Formulation B (optimized to best solubilize the active in formula).
2.5 Antiproliferative activity on Human melanoma Colo38 cell line
The benzimidazoles 13-17, 21, 22, 31-38, 40 and 45 were tested on human melanoma Colo38 cell
line to determine their antiproliferative activity. All the compounds have been tested on the cell line

to evaluate the relative IC50 values expressed in μM concentration (Table 3). Benzimidazole 45
exhibited the best activity of the series, displaying an antiproliferative effect at low micromolar
concentration (IC50 values 6.20±0.22). The 2,3,4-trihydroxyphenyl derivatives 31-33 showed
antiproliferative activity at micromolar concentration. On the contrary their 2,4,6-trihydroxyphenyl
analogs (34-36) showed very poor antiproliferative effects or total inactivity at the used
concentrations. Furthermore the 2,5-dihydrophenyl derivatives 13 and 15 inhibited the Colo38 cell
growth at micromolar concentrations.
Table 3. Effects of selected benzimidazole derivatives on the proliferation of Colo38 cells.
Compound
Compound
IC50 (M)
IC50 (M)
13
14
15
16
17
21
22
31
32
50.14±2.41
409.31±4.44
79.13±10.21
62.02±7.76
424.53±44.47
318.06±40.49
> 500
33
34
35
36
37
38
40
45
74.4±6.27
446.96±33.71
> 500
> 500
171.84±38.24
450.79±41.32
323.78±22
6.20±0.22
96.23±10.25
65.07±0.18
2.6 Antiproliferative activity on human keratinocyte HaCat cell line
The benzimidazoles 13 and 45, demonstrating the best antiproliferative activity (IC50 values:
50.14±2.41 and 6.20±0.22, respectively) on the melanoma Colo38 cells, were also tested on normal
human keratinocyte HaCat cell line to preliminary evaluate their selectivity against cancer cells. Both
compounds 13 and 45 showed low activity against HaCat cells (IC50 values: 278.97±48.01 µM and
32.72±8.54 µM, respectively) being about 5-fold more active against Colo38 cell line. These results
suggest a preferential activity of tested compounds against cancer cells.
3. Conclusion
A series of 2-arylbenzimidazole derivatives were synthesized with the aim to obtaining molecules
with a dualistic functional profile, specifically in the context of multiprotective agents able to act

both against the damage caused by the sun radiations exposure and by the oxidative stress
promoted by free radicals.
All the synthesized benzimidazole derivatives 3–40, 45 were tested to evaluate their antioxidant
profile with the DPPH and FRAP methods. The antioxidant activity profile was completed with the
ORAC method only for the compounds that emerged as the best from the two above-mentioned
tests. In general, it has been observed that the presence of a sulfonic acid group at 5-position of the
benzimidazole nucleus is the least favourable, while the derivatives bearing a 5-cyano or 5-carboxyl
acid groups have showed medium to high antioxidant activity.
The best antioxidant compounds were investigated for their UV-filtering activity showing
heterogeneous profiles. Compounds 15, 38 (bearing a sulfonic acid group), and the 2-styryl-
benzimidazole 45 were the best in terms of broad spectrum filtering activity. All the analyzed
benzimidazoles showed a better UVAPF0 value than the reference PBSA. The derivative 15
maintained its filtering profile even when incorporated into formulations for topical use and might
be chosen as a lead compound for further development. Thus, its release was evaluated from two
different formulations designed ad hoc, varying the composition only, to optimize the skin
absorption or the solubility of the active compound to promote application of the formulations as
an antioxidant or sun filter. Furthermore, the investigation on the antiproliferative activity on
human melanoma Colo38 cells showed compound 45 as the best in the series with an IC50 value at
low micromolar concentrations level. The same compound inhibited the growth of normal HaCat
keratinocytes at higher concentrations demonstrating an interesting selectivity against cancer cells.
The overall results obtained from this study have pointed out the key role played by the arylhydroxy
group in the benzimidazole ring which led to compounds endowed with multifunctional profile,
more specifically marked in antioxidant and UV filtering activity such us 15 and 45 that constitute
interesting lead compounds.
4. Experimental section
4.1 General methods.
All commercially available solvents and reagents were used without further purification. Standard
samples were purchased from Sigma-Aldrich, Milan, Italy. NMR spectra were recorded on an Inova
500 spectrometer (Varian, Palo Alto, CA, USA). The chemical shifts (δ) are reported in part per million
downfield from tetramethylsilane (TMS), which was used as internal standard, and the spectra were
recorded in hexadeuteriodimethylsulphoxide (DMSO‑d6). Infrared spectra were recorded on a
Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol mulls. The main bands are given in cm-

1. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing MAT
95 instrument (Finnigan, Waltham, MA, USA) with BE geometry. Melting points (mp) were
determined on a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK) and are uncorrected.
All products reported showed NMR spectra in agreement with the assigned structures. The purity
of the tested compounds was determined by combustion
elemental analyses conducted by the Microanalytical Laboratory of the Chemistry Department of
the University of Ferrara with a MT-5 CHN recorder elemental analyzer (Yanagimoto, Kyoto, Japan)
and the values found were within 0.4% of theoretical values. 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 analysis for the detection of filter parameters were conducted with a UV–VIS
spectrophotometer SHIMADZU UV-2600 240 V.
4.2. Chemistry
4.2.1. General procedure for the synthesis of benzimidazoles (3-40)
To a solution of the appropriate 3,4-diaminobenzene derivative (1a-d) (2 mmol) in ethanol (15 mL)
2.85 N aqueous solution of sodium metabisulphite (1.6 mL) and the appropriate substituted
arylaldehyde (2 mmol) were added. The reaction mixture was heated at reflux for 24 h. The solvent
was then evaporated under reduced pressure. The residue was added with HCl 1N (10 mL), the
formed precipitate was filtered off, washed with water (3 x 10 mL) and purified by crystallization
from the adequate solvent to give the title compounds. Following the general procedure
benzimidazoles 3 [19], 4 [20], 5 [21], 7 [24], 6, 32 and 33 [15] were prepared and their analytical and
spectral data are in agreement with those reported in literature.
4.2.1.1. 2-(3-Hydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (9). Yield 80%. Mp >250 °C (EtOH).
1H NMR (DMSO-d6): δ 7.08 (d, J = 8.0 Hz, 1H, Ar), 7.42 (t, J = 8.0 Hz, 1H, Ar), 7.64-7.70 (m, 3H, Ar),
7.81 (d, J = 8.5Hz, 1H, BIM), 8.19 (s, 1H, BIM). IR (Nujol) 2227, 1589 cm-1. m/z 236 (M + H)+. Anal.
Calcd for C14H9N3O: C, 71.48; H, 3.86; N, 17.86. Found: C, 71.41; H, 3.87; N, 17.90.
4.2.1.2. 2-(3-Hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (10). Yield 62%. Mp 208-210 °C
(EtOH). 1H NMR (DMSO-d6): δ 7.03 (d, J = 7.5 Hz, 1H, Ar), 7.44 (t, J = 8.0 Hz, 1H, Ar), 7.64 (m, 2H, Ar),
7.74 (d, J = 8.5Hz, 1H, BIM), 7.94 (d, J = 8.0 Hz, 1H, BIM), 8.23 (s, 1H, BIM), 9.96 (s, 1H, OH). IR (Nujol)

3360, 2726, 1693, 1569 cm-1. m/z 255 (M + H)+. Anal. Calcd for C14H10N2O3: C, 66.14; H, 3.96; N,
11.02. Found: C, 66.09; H, 3.98; N, 11.05.
4.2.1.3. 2-(3-Hydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (11). Yield 69%. Mp 219-220 °C
(EtOH). 1H NMR (DMSO-d6): δ 7.16 (d, J = 8.0 Hz, 1H, Ar), 7.53 (d, J = 8.0 Hz, 1H, Ar), 7.57 (s, 1H, Ar),
7.62 (d, J = 8.0 Hz, 1H, Ar), 7.77 (d, J = 8.0 Hz, 1H, Ar), 7.82 (d, J = 8.5 Hz, 1H, Ar), 9.96 (s, 1H, Ar),
10.23 (s, 1H, OH). IR (Nujol) 3397, 3274, 1631, 1588 cm-1. m/z 291 (M + H)+. Anal. Calcd for
C13H10N2O4S: C, 53.79; H, 3.47; N, 9.65. Found: C, 53.84; H, 3.46; N, 9.68.
4.2.1.4. 2-(2,4-Dihydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (12). Yield 63%. Mp 233-234
°C (EtOH). 1H NMR (DMSO-d6): δ 6.56 (d, J = 8.5 Hz, 1H, Ar), 6.68 (s, 1H, Ar), 7.81 (d, J = 8.0 Hz, 1H,
BIM), 7.89 (d, J = 8.0 Hz, 1H, BIM), 8.05 (d, J = 8.5 Hz, 1H, Ar), 8.20 (s, 1H, BIM). IR (Nujol) 3348, 3211,
3086, 2242, 1611 cm-1. m/z 252 (M + H)+. Anal. Calcd for C14H9N3O2: C, 66.93; H, 3.61; N, 16.73.
Found: C, 66.99; H, 3.60; N, 16.70.
4.2.1.5. 2-(2,5-Dihydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (13). Yield 78%. Mp >250 °C
(EtOH). 1H NMR (DMSO-d6): δ 6.98 (s, 2H, Ar), 7.50 (s, 1H, Ar), 7.73 (d, J = 8.0 Hz, 1H, BIM), 7.86 (d,
J = 8.5 Hz, 1H, BIM), 8.21 (s, 1H, BIM). 13C (DMSO-d6): δ 108.2, 112.6, 116.6, 121.1 (2C), 125.6 (2C),
128.9, 130.3, 135.7, 138.9, 141.8, 147.1, 148.1. IR (Nujol) 2230, 1617, 1564 cm-1. m/z 252 (M + H)+.
Anal. Calcd for C14H9N3O2: C, 66.93; H, 3.61; N, 16.73. Found: C, 66.87; H, 3.63; N, 16.77.
4.2.1.6. 2-(2,5-Dihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (14). Yield 65%. Mp >250
°C (EtOH). 1H NMR (DMSO-d6): δ 6.99 (s, 2H, Ar), 7.47 (s, 1H, Ar), 7.81 (d, J = 8.0Hz, 1H, BIM), 7.99
(d, J = 8.5Hz, 1H, BIM), 8.31 (s, 1H, BIM), 9.40 brs (1H, OH); 13C (DMSO-d6): δ 113.2, 116.4 (2C), 119.8
(2C), 120.5 (2C), 132.7, 139.2, 142.6, 145.7 , 152.0, 154.2, 153.9. IR (Nujol) 31.84, 2720, 1718, 1620
cm-1. m/z 271 (M + H)+. Anal. Calcd for C14H10N2O4: C, 62.22; H, 3.73; N, 10.37. Found: C, 62.28; H,
3.71; N, 10.34.
4.2.1.7. 2-(2,5-Dihydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (15). Yield 71%. Mp >250 °C
(EtOH). 1H NMR (DMSO-d6): δ 7.04 (s, 2H, Ar), 7.43 (s, 1H, Ar), 7.75 (s, 2H, Ar), 8.02 (s, 1H, Ar), 9.41
(s, 1H, NH). IR (Nujol) 3392, 3225, 3097, 1633, 1569 cm-1. m/z 307 (M + H)+. Anal. Calcd for
C13H10N2O5S: C, 50.98; H, 3.29; N, 9.15. Found: C, 50.94; H, 3.31; N, 9.18.
4.2.1.8. 2-(3,4-Dihydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (16). Yield 75%. Mp >250 °C
(EtOH). 1H NMR (DMSO-d6): δ 7.04 (d, J = 8.5 Hz, 1H, Ar), 7.65 (s, 1H, Ar), 7.67 (d, J = 8. 5 Hz, 1H, Ar),
7.79 (d, J = 8.5 Hz, 1H, BIM), 7.85 (d, J = 8.0 Hz, 1H, BIM), 8.21 (s, 1H, BIM). IR (Nujol) 3428, 3331,
3105, 2242, 1611 cm-1. m/z 252 (M + H)+. Anal. Calcd for C14H9N3O2: C, 66.93; H, 3.61; N, 16.73.
Found: C, 66.85; H, 3.59; N, 16.70.

4.2.1.9. 2-(3,4-Dihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (17). Yield 81%. Mp 224-
226 °C (EtOH). 1H NMR (DMSO-d6): δ 7.04 (d, J = 8.5 Hz, 1H, Ar), 7.67 (d, J = 9.0 Hz, 1H, Ar), 7.69 (s,
1H, Ar), 7.78 (d, J = 8.5 Hz, 1H, BIM), 8.01 (d, J = 8.0 Hz, 1H, BIM), 8.23 (s, 1H, BIM), 9.50 (brs, 1H,
OH). IR (Nujol) 3317, 2760, 1694, 1603, 1524 cm-1. m/z 271 (M + H)+. Anal. Calcd for C14H10N2O4: C,
62.22; H, 3.73; N, 10.37. Found: C, 62.16; H, 3.74; N, 10.40.
4.2.1.10. 2-(3-Hydroxy-4-methoxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (18). Yield 58%. Mp
>250 °C (EtOH). 1H NMR (DMSO-d6): δ 3.81 (s, 3H, OCH3), 6.66-6.68 (m, 2H, Ar), 7.73 (d, J = 8.0 Hz,
1H, BIM), 7.83 (d, J = 8.5 Hz, 1H, BIM), 8.06 (d, J = 8.5Hz, 1H, Ar), 8.15 (s, 1H, BIM). IR (Nujol) 2228,
1618 cm-1. m/z 266 (M + H)+. Anal. Calcd for C15H11N3O2: C, 67.92; H, 4.18; N, 15.84. Found: C, 67.98;
H, 4.20; N, 15.80.
4.2.1.11. 2-(3-Hydroxy-4-methoxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (19). Yield 60%.
Mp 223-234 °C (EtOH). 1H NMR (DMSO-d6): δ 3.83 (s, 3H, OCH3), 6.72 (s, 2H, Ar), 7.81 (d, J = 8.0 Hz,
1H, Ar), 8.00 (d, J = 8.5 Hz, 1H, BIM), 8.08 (d, J = 9.5 Hz, 1H, BIM), 8.30 (s, 1H, BIM), 9.70 (brs, 1H,
OH). IR (Nujol) 3293, 2725, 1697, 1626 cm-1. m/z 285 (M + H)+. Anal. Calcd for C15H12N2O4: C, 63.38;
H, 4.25; N, 9.85. Found: C, 63.33; H, 4.27; N, 9.88.
4.2.1.12. 2-(3-Hydroxy-4-methoxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (20). Yield 69%. Mp
>250 °C (EtOH). 1H NMR (DMSO-d6): δ 3.93 (s, 3H, OCH3), 7.30 (d, J = 8.5 Hz, 1H, Ar), 7.60 (s, 1H, Ar),
7.68-7.68 (m, 2H, Ar and BIM), 7.79 (d, J = 9.0 Hz, 1H, BIM), 7.93 (s, 1H, BIM), 9.78 (s, 1H, OH). IR
(Nujol) 3367, 3149, 3089, 2781, 1634, 1610, 1583 cm-1. m/z 321 (M + H)+. Anal. Calcd for
C14H12N2O5S: C, 52.49; H, 3.78; N, 8.75. Found: C, 52.54; H, 3.77; N, 8.72.
4.2.1.13. 2-(3-Ethoxy-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (21). Yield 52%. Mp
>250 °C (EtOH). 1H NMR (DMSO-d6): δ 1.37 (t, J = 7.0 Hz, 3H, CH3), 4.11 (q, J = 7.0 Hz, 2H, CH2), 6.98
(t, J = 8.0 Hz, 1H, Ar), 7.15 (d, J = 8.0 Hz, 1H, Ar), 7.64 (d, J = 7.5 Hz, 1H, Ar), 7.77 (d, J = 8.0 Hz, 1H,
BIM), 7.95 (d, J = 9.0 Hz, 1H, BIM), 8.27 (s, 1H, BIM). IR (Nujol) 3463, 3030, 2680, 1682, 1624, 1594
cm-1. m/z 299 (M + H)+. Anal. Calcd for C16H14N2O4: C, 64.42; H, 4.73; N, 9.39. Found: C, 64.37; H,
4.74; N, 9.43.
4.2.1.14. 2-(3-Ethoxy-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (22). Yield 56%. Mp
1
236-237 °C (EtOH). H NMR (DMSO-d6): δ 1.40 (t, J = 6.5Hz, 3H, CH3), 4.21 (q, J = 6.5 Hz, 2H, CH2),
7.08 (t, J = 8.0 Hz, 1H, Ar), 7.31 (d, J = 8.0 Hz, 1H, Ar), 7.59 (d, J = 8.0 Hz, 1H, Ar), 7.78 (d, J = 9.0 Hz,
1H, BIM), 7.80 (d, J = 8.5Hz, 1H, BIM), 8.06 (s, 1H, BIM). IR (Nujol) 3448, 3227, 1625, 1561 cm-1. m/z
335 (M + H)+. Anal. Calcd for C15H14N2O5S: C, 53.88; H, 4.22; N, 8.38. Found: C, 53.94; H, 4.24; N,
8.34.

4.2.1.15. 2-(2-Hydroxy-4-methoxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (23). Yield 58%.
Mp 219-220 °C (EtOH). 1H NMR (DMSO-d6): δ 3.80 (s, 3H, OCH3), 7.22 (d, J = 8.5 Hz, 1H, Ar), 7.65 (s,
1H, Ar), 7.70 (d, J = 8.5 Hz, 1H, Ar), 7.76 (d, J = 8.0 Hz, 1H, BIM), 7.98 (d, J = 8.5 Hz, 1H, BIM), 8.22 (s,
1H, BIM). IR (Nujol) 3447, 3130, 2766, 1713, 1615 cm-1. m/z 285 (M + H)+. Anal. Calcd for C15H12N2O4:
C, 63.38; H, 4.25; N, 9.85. Found: C, 63.45; H, 4.23; N, 9.89.
4.2.1.15. 2-(2-Hydroxy-4-methoxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (24). Yield 59%. Mp
226-227 °C (EtOH). 1H NMR (DMSO-d6): δ 3.87 (s, 3H, OCH3), 6.69 (s, 1H, Ar), 6.78 (m, 1H, Ar), 7.71
(m, 2H, BIM), 7.98 (m, 1H, Ar), 8.02 (s, 1H, BIM). IR (Nujol) 3163, 1621, 1572 cm-1. m/z 321 (M + H)+.
Anal. Calcd for C14H12N2O5S: C, 52.49; H, 3.78; N, 8.75. Found: C, 52.44; H, 3.80; N, 8.78.
4.2.1.16. 2-(5-Chloro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (25). Yield 83%. Mp
200-202 °C (EtOH). 1H NMR (DMSO-d6): δ 7.16 (d, J= 9.0 Hz, 1H, Ar), 7.47 (d, J = 9.0 Hz, 1H, Ar), 7.71
(d, J = 8.5 Hz, 1H, BIM), 7.86 (d, J = 8.5 Hz, 1H, BIM), 8.19 (s, 1H, Ar), 8.20 (s, 1H, BIM). IR (Nujol)
3340, 3059, 2727, 2232, 1615 cm-1. m/z 270 (M + H)+. Anal. Calcd for C14H8ClN3O: C, 62.35; H, 2.99;
N, 15.58. Found: C, 62.42; H, 2.98; N, 15.55.
4.2.1.17. 2-(5-Chloro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (26). Yield 82%. Mp
203-204 °C (EtOH). 1H NMR (DMSO-d6): δ 7.14 (d, J = 8.5 Hz, 1H, Ar), 7.45 (d, J = 8.0 Hz, 1H, Ar), 7.78
(d, J = 8.5 Hz, 1H, BIM), 7.95 (d, J = 8.5 Hz, 1H, BIM), 8.19 (s, 1H, Ar), 8.28 (s, 1H, BIM). IR (Nujol)
3320, 3225, 3070, 2688, 1685, 1632, 1558 cm-1. m/z 289 (M + H)+. Anal. Calcd for C14H9ClN2O3: C,
58.25; H, 3.14; N, 9.70. Found: C, 58.16; H, 3.15; N, 9.74.
4.2.1.18. 2-(5-Chloro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (27). Yield 85%. Mp
226-227 °C (EtOH). 1H NMR (DMSO-d6): δ 7.25 (d, J = 8.0 Hz, 1H, Ar), 7.61 (d, J = 8.0 Hz, 1H, Ar), 7.80
(m, 2H, BIM and Ar), 8.06 (s, 1H, BIM), 8.20 (s, 1H, BIM). IR (Nujol) 3582, 3375, 3060, 2720, 1624,
1556 cm-1. m/z 325 (M + H)+. Anal. Calcd for C13H9ClN2O4S: C, 48.08; H, 2.79; N, 8.63. Found: C, 48.03;
H, 2.81; N, 8.67.
4.2.1.19. 2-(5-Bromo-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (28). Yield 85%. Mp
215-216 °C (EtOH). 1H NMR (DMSO-d6): δ 7.12 (d, J = 9.0 Hz, 1H, Ar), 7.58 (d, J = 9.0 Hz, 1H, Ar), 7.72
(d, J = 8.0 Hz, 1H, BIM), 7.86 (d, J = 8.5Hz, 1H, BIM), 8.20 (s, 1H, Ar), 8.31 (s, 1H, BIM). IR (Nujol) 2220,
1607, 1552 cm-1. m/z 314 (M + H)+. Anal. Calcd for C14H8BrN3O: C, 53.53; H, 2.57; N, 13.38. Found:
C, 53.49; H, 2.58; N, 13.41.
4.2.1.20. 2-(5-Bromo-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (29). Yield 84%. Mp
233-235 °C (EtOH). 1H NMR (DMSO-d6): δ 7.09 (d, J = 8.5 Hz, 1H, Ar), 7.60 (d, J = 8.5 Hz, 1H, Ar), 7.78
(d, J = 8.5 Hz, 1H, BIM), 7.95 (d, J = 8.5 Hz, 1H, BIM), 8.28 (s, 1H, Ar), 8.31 (s, 1H, BIM). IR (Nujol)

3320, 3170, 2670, 1638, 1624, 1555 cm-1. m/z 332 (M + H)+. Anal. Calcd for C14H9BrN2O3: C, 50.47;
H, 2.72; N, 8.41. Found: C, 50.53; H, 2.71; N, 8.44.
4.2.1.21. 2-(5-Bromo-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (30). Yield 88%. Mp
240-241 °C (EtOH). 1H NMR (DMSO-d6): δ 7.16 (d, J = 9.0 Hz, 1H, Ar), 7.72 (d, J = 9.0 Hz, 1H, Ar), 7.79
(m, 2H, BIM), 8.04 (s, 1H, Ar), 8.27 (s, 1H, BIM). IR (Nujol) 3350, 3264, 3199, 2722, 2670, 1625, 1605,
1556 cm-1. m/z 368 (M + H)+. Anal. Calcd for C13H9BrN2O4S: C, 42.29; H, 2.46; N, 7.59. Found: C,
42.24; H, 2.45; N, 7.62.
4.2.1.22. 2-(2,3,4-Trihydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (31). Yield 72%. Mp 232-
233 °C (EtOH). 1H NMR (DMSO-d6): δ 6.65 (d, J = 9.0 Hz, 1H, Ar), 7.60 (d, J = 8.5 Hz, 1H, Ar), 7.80 (d,
J = 8.0 Hz, 1H, BIM), 7.88 (d, J = 8.0 Hz, 1H, BIM), 8.20 (s, 1H, BIM). IR (Nujol) 2228, 1619, 1571 cm-
1. m/z 267 (M + H)+. Anal. Calcd for C14H9N3O3: C, 62.92; H, 3.39; N, 15.72. Found: C, 62.85; H, 3.41;
N, 15.76.
4.2.1.23. 2-(2,4,6-Trihydroxyphenyl)-1H-benzo[d]imidazole-5-carbonitrile (34). Yield 74%. Mp >250
°C (EtOH). 1H NMR (DMSO-d6): δ 6.22 (s, 1H, Ar), 6.22 (s, 1H, Ar), 7.87 (d, J = 8.5Hz, 1H, BIM), 7.95
(d, J = 8.5 Hz, 1H, BIM), 8.21 (s, 1H, BIM). IR (Nujol) 2230, 1625 cm-1. m/z 267 (M + H)+. Anal. Calcd
for C14H9N3O3: C, 62.92; H, 3.39; N, 15.72. Found: C, 62.99; H, 3.38; N, 15.69.
4.2.1.24. 2-(2,4,6-Trihydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (35). Yield 75%. Mp
>250 °C (EtOH). 1H NMR (DMSO-d6): δ 6.01 (s, 2H, Ar), 7.69 (d, J = 8.0 Hz, 1H, BIM), 7.83 (d, J = 8.5
Hz, 1H, BIM), 8.26 (s, 1H, BIM), 13.19 (s, 1H, OH), 13.24 (s, 1H, OH). IR (Nujol) 3404, 3233, 2718,
1704, 1614, 1565 cm-1. m/z 287 (M + H)+. Anal. Calcd for C14H10N2O5: C, 58.74; H, 3.52; N, 9.79.
Found: C, 58.69; H, 3.50; N, 9.83.
4.2.1.25. 2-(2,4,6-Trihydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (36). Yield 75%. Mp 224-
225 °C (EtOH). 1H NMR (DMSO-d6): δ 6.14 (s, 2H, Ar), 7.70 (d, J = 8.5Hz, 1H, BIM), 7.74 (d, J = 8.5 Hz,
1H, BIM), 8.11 (s, 1H, BIM), 10.45 (s, 1H, NH), 11, 80 (s, 2H, OH), 13,20 (s, 1H, OH). IR (Nujol) 3489,
3216, 1629 cm-1. m/z 267 (M + H)+. Anal. Calcd for C13H10N2O6S: C, 48.45; H, 3.13; N, 8.69. Found: C,
48.49; H, 3.11; N, 8.72.
4.2.1.26. 2-(4-(Diethylamino)-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (37). Yield
59%. Mp 212-213 °C (EtOH). 1H NMR (DMSO-d6): δ 1.14 (t, J = 7.0 Hz, 6H, CH3), 3.41 (q, J = 7.0 Hz,
4H, CH ), 6.30 (s, 1H, Ar), 6.47 (d, J = 9.0 Hz, 1H, Ar), 7.70 (d, J = 8.5 Hz, 1H, BIM), 7.85 (d, J = 8.5 Hz,
1H, BIM), 7.94 (d, J = 8.0 Hz, 1H, Ar), 8.20 (s, 1H, BIM). 13C (DMSO-d6): δ 15.6 (2C), 52.1 (2C), 98.6,
106.3, 108.2, 117.2 (2C), 121.3, 128.2 (2C), 131.8, 140.2, 145.2 (2C), 147.6, 163.3. IR (Nujol) 3446,

3181, 2681, 1691, 1615 cm-1. m/z 326 (M + H)+. Anal. Calcd for C18H19N3O3: C, 66.45; H, 5.89; N,
12.91. Found: C, 66.52; H, 5.86; N, 12.94.
4.2.1.27. 2-(4-(Diethylamino)-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-sulfonic acid (38) Yield
63%. Mp 221-222 °C (EtOH). 1H NMR (DMSO-d6): δ 1.16 (t, J = 7.0 Hz, 6H, CH3), 3.43 (q, J = 7.0 Hz,
4H, CH2), 6.36 (s, 1H, Ar), 6.53 (d, J = 8.5 Hz, 1H, Ar), 7.65 (d, J = 8.5 Hz, 1H, BIM), 7.71 (d, J = 8.5 Hz,
1H, BIM), 7.85 (d, J = 8.5 Hz, 1H, Ar), 7.97 (s, 1H, BIM), 11.42 (s, 1H, NH), 13.55 (s, 1H, OH). 13C
(DMSO-d6): δ 14.1 (2C), 45.3 (2C), 100.3, 104.1, 110.8, 112.3, 115.6, 122.7, 133.2, 139.7, 143.7, 145.6
(2C), 155.2, 157.5. IR (Nujol) 3442, 3321, 1613 cm-1. m/z 362 (M + H)+. Anal. Calcd for C17H19N3O4S:
C, 56.50; H, 5.30; N, 11.63. Found: C, 56.45; H, 5.28; N, 11.67.
4.2.1.28. 2-(2-Hydroxynaphthalen-1-yl)-1H-benzo[d]imidazole-5-carboxylic acid (39). Yield 58%. Mp
>250 °C (EtOH). 1H NMR (DMSO-d6): δ 7.42 (m, 2H, Ar), 7.54 (t, J = 8.5 Hz, 1H, Ar), 7.87 (d, J = 9.0 Hz,
1H, BIM), 7.92-7.96 (m, 2H, Ar), 8.05 (d, J = 8.5 Hz, 1H, BIM), 8.10 (d, J = 8.5 Hz, 1H, Ar), 8.35 (s, 1H,
BIM). IR (Nujol) 3463, 2680, 1682, 1624, 1594 cm-1. m/z 305 (M + H)+. Anal. Calcd for C18H12N2O3: C,
71.05; H, 3.97; N, 9.21. Found: C, 70.98; H, 3.98; N, 9.25.
4.2.1.29. 2-(2-Hydroxynaphthalen-1-yl)-1H-benzo[d]imidazole-5-sulfonic acid (40). Yield 61%. Mp
>250 °C (EtOH). 1H NMR (DMSO-d6): δ 7.43 (d, J = 9.0 Hz, 1H, Ar), 7.47 (t, J = 7.5 Hz, 1H, Ar), 7.57 (t,
J = 8.0 Hz, 7.5Hz, 1H, Ar), 7.72 (d, J = 8.0 Hz, 1H, Ar), 7.83 (d, J = 9.0 Hz, 1H, Ar), 7.89 (d, J = 8.0 Hz,
1H, Ar) 8.01 (d, J = 8.0 Hz, 1H, BIM), 8.04 (s, 1H, BIM), 8.18 (d, J = 8.5 Hz, 1H, BIM), 10.81 (s. 1H, OH).
IR (Nujol) 3423, 1621, 1588 cm-1. m/z 341 (M + H)+. Anal. Calcd for C17H12N2O4S: C, 59.99; H, 3.55; N,
8.23. Found: C, 60.05; H, 3.53; N, 8.26.
4.2.2. (E)-Methyl 4-amino-3-(3-(4-hydroxyphenyl)acrylamido)benzoate (43).
A solution of 4-hydroxycinnamic acid (41) (0.33 g, 2 mmol), EDC (0.38 g, 2.2 mmol) and HOBt (0.26
g, 2 mmol) in anhydrous MeCN (10 mL) was stirred at r.t. for 30 minutes, then methyl 3,4-
diaminobenzoate (42) (0.33 g, 2 mmol) was added. The mixture was stirred at r.t. After 36 h the
solvent was removed in vacuum. The residue was dissolved in ethyl acetate (AcOEt) (20 mL) and
washed sequentially with brine (2 x 5 mL), 10% citric acid (2 x 5 mL), saturated NaHCO3 aqueous
solution (2 x 5 mL) and water (2 x 5 mL). The organic layer was dried over anhydrous Na2SO4 and
evaporated under vacuum to give the title compound in 82% yield. Mp 174-176 °C (EtOH). 1H NMR
(DMSO-d6): δ 3.76 (s, 3H, CH3), 5.81 (s, 2H, NH2), 6.67 (d, J = 16.0 Hz, 1H, CH), 6.76 (d, J = 8.0 Hz, 1H,
Ar), 6.83 (d, J = 9.0 Hz, 2H, Ar), 7.46-7.55 (m, 5H, Ar and CH) 8.03 (s, 1H, Ar), 9.26 (s, 1H, NH), 9.92
(s, 1H, OH). IR (Nujol) 3148, 1700, 1599 cm-1. m/z 313 (M + H)+. Anal. Calcd for C17H16N2O4: C, 65.38;
H, 5.16; N, 8.97. Found: C, 65.32; H, 5.18; N, 9.00.

4.2.3. (E)-Methyl 2-(4-hydroxystyryl)-1H-benzo[d]imidazole-5-carboxylate (44).
(E)-methyl 4-amino-3-(3-(4-hydroxyphenyl)acrylamido)benzoate (43) (0.70 g, 2.2 mmol) was
dissolved in ethanol (1.5 mL) and then 6N aqueous hydrochloric acid solution (15 mL) was added.
The mixture was heated at 100 °C for 5 h The ethanol was removed in vacuum and the residue
cooled to 10° C. The formed precipitate was filtered off and air dried. Yield 74%. Mp 215-216 °C
(EtOH). 1H NMR (DMSO-d6): δ 3.92 (s, 3H, CH3), 6.93 (d, J = 7.5 Hz, 2H, Ar), 7.09 (d, J = 15.5 Hz, 1H,
CH), 7.61 (d, J = 7.5 Hz, 2H, Ar), 7.82 (s, 1H, Ar), 8.05 (m, 2H, Ar), 8.18 (d, J = 15.5 Hz, 1H, CH), 8.23
(s, 1H, NH), 10.34 (s, 1H, OH). IR (Nujol)3453, 3223, 1621, 1588 cm-1. m/z 295 (M + H)+. Anal. Calcd
for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.43; H, 4.76; N, 9.56.
4.2.4. (E)-2-(4-hydroxystyryl)-1H-benzo[d]imidazole-5-carboxylic acid (45).
To a solution of (E)-methyl 2-(4-hydroxystyryl)-1H-benzo[d]imidazole-5-carboxylate (44) (0.36 g,
1.22 mmol) in ethanol (5 mL) 2.5N aqueous solution sodium hydroxide (2.5 mL) was added. The
mixture was stirred at r.t per 24h. The ethanol was removed in vacuo. The residue was ice added
and hydrochloric acid 10% solution added until pH 6. The obtained precipitate was filtered off,
washed with cold water (3 x 10 mL) and dried. Yield 48%. Mp >250 °C (EtOH). 1H NMR (DMSO-d6):
δ 6.89 (d, J = 7.5 Hz, 2H, Ar), 7.04 (d, J = 15.5 Hz, 1H, CH), 7.55 (d, J = 7.5 Hz, 2H, Ar), 7.75 (s, 1H, Ar),
7.99 (m, 2H, Ar), 8.16 (d, J = 15.5 Hz, 1H, CH), 10.30 (s, 1H, OH). 13C (DMSO-d6): δ 116.3, 117.6 (2C),
119.4, 125.3, 126.2 (2C), 130.7, 131.3 (2C), 136.3, 141.2, 143.5, 144.7, 161.4, 169.8. IR (Nujol) 3345,
3190, 1686, 1639, 1600 cm-1. m/z 281 (M + H)+. Anal. Calcd for C16H12N2O3: C, 68.56; H, 4.32; N, 9.99.
Found: C, 68.50; H, 4.30; N, 10.03.
4.3. Biological assays
4.3.1. DPPH assay
The activity of the synthesized derivatives against DPPH• radical was tested following the Wang et
al method [25] modified as previously reported [17]. The results are expressed as μmol TE/g
corresponding to an inhibition of the radical equal to 50%, except for some compounds.
4.3.2. FRAP test
The FRAP antioxidant capacity of benzimidazoles was measured according to the method
described previously [26], reading the absorbance of the reaction mixture at 593 nm. The values
are expressed as μmol TE/g compound.

4.3.3. Oxygen Radical Absorbance Capacity (ORAC)
Peroxyl radical scavenging activity was measured based on a protocol previously reported and
modified [27]. Phosphate buffer solution (pH 7.4) was used to prepare sample solutions and a series
of Trolox solutions (40–240 μM) by diluting a 2 mM solution of Trolox. 25 μl of sample solution,
Trolox dilution or phosphate buffer solution (pH 7.4) used as blank was placed in wells of a 96-well
black microplate (VWR). 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 were calculated according to
the method of Cao et al. [28] and expressed as Trolox equivalents (μmol TE/μmol).
4.3.4. Evaluation of filtering parameters
The method followed is an adaptation of the official method for determining the value of SPF in
vitro [29]. Solutions of the tested benzimidazoles were prepared in MeOH at a concentration of
0.000034 (±0.0000033) M and the absorption spectra were recorded. To calculate the SPF value in
vitro, the absorbance values obtained were transformed into transmittance values, using the
equation below:
A( ) = Log[T( )]
The transmittance spectrum was elaborated with the SPF calculator software (version 2.1,
Shimadzu, Milan, Italy) to obtain the values of SPF, UVA/UVB, UVAPF and λ critical.
4.3.5. Formulations
Formulation A and Formulation B, standard oil/water emulsions, were prepared for Oxisol and
benzimidazole 15. Each phase, after adding the individual ingredients, was heated to a temperature
between 60 and 70 ° C. At this point, below mechanical stirring, the oily phase was added to the
aqueous phase. The selected compound was added in the subsequent cooling phase of the
emulsion, kept constant agitation, to preserve it from possible thermal degradation. The emulsion,
once cooled to 25 ° C, is stored in the refrigerator until the next day of analysis. The formulation
compositions are the following:
Formulation A: Aqua, Ethylhexyl Stearate, Tribehenin PEG-20 Esters, Butyrospermum Parkii, Olea
Europaea Oil Unsaponifiables, Oxisol or benzimidazole 15, 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 benzimidazole 15, Xanthan Gum, Caprylic/Capric
Triglyceride.
4.3.6. Franz cells apparatus
Cuprophan Regenerated cellulose from Medicell (London, UK), membrane thickness (11.5 µm ±}
0,5µm), molecular weight cut off range (10,000 Da) was chosen as synthetic membrane employed
in Franz diffusion cells experiments in this study. The effective diffusion area of the Franz cells was
0.6 cm2 and receptor volume was 4.3 mL. Before being mounted on the diffusion cells, the
membranes were conditioned with the receiving solution (DMSO). The membrane was positioned
between the two compartments and the formulation (0.5 g) was placed in the donor compartment,
in contact with the membrane. The aforementioned compartment was then sealed with Parafilm in
order to avoid loss of formulation components. The thus prepared Franz cells were immersed in a
thermostat bath to guarantee a surface temperature of the membrane of 37 ± 1 ° C, keeping the
receiving solvent under continuous magnetic stirring. At each set time (after 30 minutes, 1 hour and
then every hour thereafter up to 6 hours) using a glass syringe, 500 μl of sample were taken and
replaced by 500 μl of fresh receiving medium, and the absorbance of the samples was measured
using a UV-VIS spectrophotometer at the λmax. A calibration line and a release curve were built for
each compound.
4.3.7. Growth inhibition assays
Cell growth inhibition assays were carried out using two human cell lines, melanoma Colo38 [30, 31,
17] and keratinocyte HaCat [31, 32], cells. Colo 38 cell line was 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) (complete medium); the pH of the medium was 7.2 and the incubation
was performed at 37 °C in a 5% CO2 atmosphere. HaCat cell line were grown in DMEM medium
(4.5g/L glucose) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin
and 2 mM L-glutamine, in a 5% CO2 environment at 37°C. Benzimidazole derivatives were dissolved
in MeOH/DMSO 10% to obtain 20mM stock solutions and diluted before cell treatment in MeOH
100%. The tested benzimidazoles were added at serial dilutions to the cell cultures 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.
Acknowledgment
This work was supported 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).
Conflict of interest
The authors declare no conflict of interest.
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A small library of 2-arylbenzimidazole were synthetized
Synthesized compounds were tested as antioxidants agents by DPPH, FRAP and ORAC assays
The UV filtering power of benzimidazoles endowed with the best antioxidant profile was evaluated
Compound 45 showed the best antioxidant and photoprotective properties
Compound 45 selectively inhibited the growth of Colo-38 cell line at low micromolar concentration

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests: