Skin damages—Structure activity relationship of benzimidazole derivatives bearing a 5-membered ring system

Article abstract of DOI:10.3390/molecules25184324

In the search for scaffolds for multifunctional compounds we investigated the structure activity relationship of a class of benzimidazole derivatives bearing 5-membered ring. The newly synthesized and the already known compounds were divided into three cl

Full text of DOI:10.3390/molecules25184324

molecules
Article
Skin Damages—Structure Activity Relationship of
Benzimidazole Derivatives Bearing a 5-Membered
Ring System
1
1
2
3
4
Ernestine Nicaise Djuidje , Elisa Durini , Sabrina Sciabica , Elena Serra , Jan Balzarini ,
4
1
, Silvia Vertuani 1 and Anna Baldisserotto *
1,
Sandra Liekens , Stefano Manfredini
1
Department of Life Sciences and Biotechnology, Master Course in Cosmetic Science and Technologies,
University of Ferrara, 44121 Ferrara, Italy; djdrst@unife.it (E.N.D.); elisa.durini@unife.it (E.D.);
mv9@unife.it (S.M.); vrs@unife.it (S.V.)
Department of Chemical and Pharmaceutical Sciences, University of Ferrara, 44121 Ferrara, Italy;
scbsrn@unife.it
Aptuit, An Evotec Company, 37135 Verona, Italy; Elena.serra@aptuit.com
Department of Microbiology and Immunology, KU Leuven, University of Leuven, Rega Institute for Medical
Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium;
jan.balzarini@kuleuven.be (J.B.); sandra.liekens@rega.kuleuven.be (S.L.)
Correspondence: bldnna@unife.it; Tel.: +39-0532-455258
2
3
4
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: György Szöllösi
Received: 31 August 2020; Accepted: 17 September 2020; Published: 21 September 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: In the search for scaffolds for multifunctional compounds we investigated the structure
activity relationship of a class of benzimidazole derivatives bearing 5-membered ring. The newly
synthesized and the already known compounds were divided into three classes that present different
substituent at 5 position of the benzimidazole ring (-H, -COOH or –SO H) and different heterocycle
3
at position 2 (thiophene, furan or pyrrole). All the derivatives were synthesized and tested to
determine their photoprotective profile against UV rays, in vitro antioxidant capacity against different
radicals (DPPH and FRAP test), antifungal inhibitory activity (dermatophytes and Candida albicans),
antiviral and antiproliferative activity. A Structure-Activity Relationship study indicated compound
10, bearing a pyrrole heterocycle on the benzimidazole ring, as the best multifunctional derivative of
the series and as potential candidate for the development of drugs especially in case of melanoma.
Keywords: benzimidazole; UV-filter; melanoma
1
. Introduction
Skin is the largest body organ constituting an environmental interface by forming a protective
barrier essential for homeostasis. For this reason skin is constantly exposed to exogenous and
endogenous agents that could altered its functions. UV rays, microbes and pollution are responsible for
external damage to the skin. However, UV is the most important factor responsible for the production
of reactive oxygen species (ROS) which consequently induces the development of skin cancer and
aging. So the discovery of sunscreens with combined photoprotective, antioxidant, antifungal and
anti-melanoma activity remains the challenge of researchers in the pharmaceutical and cosmetic
fields [1–3].
Heterocyclic compounds are fundamental building blocks of various essential natural and
synthetic compounds. They play an important role in drug design thanks to their ability to act as
both biomimetic and reactive pharmacophores [
heterocyclic compounds having one to three heteroatoms (especially oxygen, nitrogen and sulfur)
in their structure and are significant in organic chemistry, medicinal chemistry and biochemistry [ ].
4]. Many of them are five- and / or six-membered
5
Molecules 2020, 25, 4324; doi:10.3390/molecules25184324
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4324
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They are often found in large percentage in biomolecules like enzyme, vitamins and natural products [
for these reasons heterocyclic compounds remain the challenging issue for the researchers for the
development of novel drugs [ ]. Moreover, five membered-rings fused with benzene moiety attracted
the attention of medicinal chemists especially benzimidazoles due to their diverse biological activities
including antitumor [ 12], antifungal [13], anthelmintics [14], anti-inflammatory [15], analgesics [16]
6];
7
8
–
and antiviral [17]. Compounds belonging to this class of heterocycles are considered privileged due to
their broad spectrum pharmacological activities [18].
In medicinal chemistry, interest in the benzimidazole ring increased in the 1950s after the discovery
of 5,6-di-methyl-1-(α-D-ribofuranosyl) benzimidazole as an integral part of vitamin B12. Besides,
benzimidazole derivatives have a wide range of biological activities such as antibacterial, antimicrobial,
antioxidant, anti-tuberculosis, antimalarial, antiulcer, antiallergic, antidiabetic as well as use in
cardiovascular disease [19–22]. In addition to their therapeutic applications, benzimidazoles are known
for their UV filtering ability [23]. Besides, 2-phenyl-5-(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl)-1H-
benzimidazole (PPTMB), 2-[4-(2-{4-[1-(2-Ethoxyethyl)-1H-benzimidazol-2-yl]-1-piperidinyl}ethyl)phe
nyl]-2-methylpropanoic acid (Bilastine) and 1-[(4-chlorophenyl)methyl]-2-methylbenzimidazole (Chlor
midazole) are benzimidazole analogues, respectively used in the treatment of cancer, allergic rhino-
conjunctivitis and fungal infection [24]. 5-Membered rings including thiophene, furan and pyrrole
have received considerable interests in the fields of medicinal chemistry [25]. Some studies showed
that chalcone and pyrimidine analogues containing pyrrole, furan and thiophene have increased
the antimalarial activity of several prepared drugs [26]. They also have significant inflammatory,
antineoplastic and antimicrobial activity [27].
Based on the biological activities of heterocyclic compounds mentioned above we have directed this
work towards the study of the structure activity relationship of benzimidazole bearing 5-membered ring
derivatives and their biological study as smart multifunctional compounds for skin damages. To this
aim, our interest was in developing compounds starting from 2-phenyl-1H-benzimidazole-5-sulphonic
acid (PBSA, a commercial UVB filter, Figure 1) as lead compound. Since the isosteric modification of a
lead compound is a key tool in the pharmaceutical sector for the rational design of new drugs, changes
have been made to the PBSA to implement this project, in particular to positions 2 by introducing a
5
-member ring on the phenyl ring and replacing the functional group (-SO H) in position 5 of the
3
benzimidazole ring with -COOH and –H. The three series explored include both already known and
newly synthesized compounds.
Figure 1. Lead compound.
2
. Results and Discussion
2
.1. Chemistry
Different changes have been made to PBSA in order to improve its UV filter profile and also give
it other activities with the aim to obtaining multifunctional compounds. For this purpose, the phenyl
ring has been replaced by 5-membered rings (pyrrole, thiophene and furan) which are benzene isosters.
The sulfonic acid portion at position 5 of the benzimidazole ring was also modified by carboxylic acid
and hydrogen.
Benzimidazole derivatives were achieved by condensation reaction between o-phenylenediamine
with aldehydes analogues as previously described [28]. In regard to 2-substituted benzimidazole-5-
sulfonic acids (
7–9), the intermediate 3,4-diaminobenzenesulfonic acid (2) was synthesized first.
Compound was easily obtained via Sulfonation reaction of o-phenylenediamine with sulfuric
2

Molecules 2020, 25, 4324
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acid 96% (Scheme 1) and then, was condensed with aldehydes to obtain compounds
7–9
(Scheme 2).
All reactions were carried out in the presence of NaHSO as oxidant agent. 2-substituted benzimidazoles
3
(10–
12) and 2-substituted benzimidazole-5-carboxilic acids (13
–
15) were directly obtained by addition
of commercially available o-phenylenediamine (
aldehydes (3–5) (Scheme 3).
1
) or 3,4-diaminobenzoic acid ( ) and corresponding
6
Scheme 1. Synthesis of 3,4-diamino-benzene sulfonic acid, sulfate salt. Reagents and conditions: (i)
H SO , reflux, 24 h.
2
4
Scheme 2. Synthesis of 7–9 2-substituted benzimidazole-5-sulfonic acid. Reagents and conditions: (i)
EtOH, NaHSO in H O, reflux.
3
2
Compound
R
H
H
H
X
NH
O
1
1
1
1
1
1
0
1
2
3
4
5
S
COOH NH
COOH
COOH
O
S
Scheme 3. Synthesis of 10–15 benzimidazole derivatives. Reagents and conditions: (i) EtOH, NaHSO3
in H O, reflux.
2
The new and the already known synthesized compounds were divided into three classes (A–C)
(Table S1 in Supplementary Materials). In each class, the compounds present different substituent at 5
position of the benzimidazole ring while the heterocycle in position 2 of the benzimidazole is the same.
2
.2. Evaluation of Filtering Parameters
2
.2.1. In Vitro Photoprotective Activity of Benzimidazole Derivatives Solutions
The spectral behavior of the test compounds dissolved in methanol was investigated firstly
because the solar protection is related to the UV absorption. Absorption spectra were measured from
90 to 400 nm. Compounds 10 11 and 12 have similar lambda max value, however lambda max of
2
,

Molecules 2020, 25, 4324
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12 is slightly higher than that of 11, which in turn is slightly greater than that of 10. Substitution
of -H by withdrawing group (-COOH and –SO H) at the 5 position on the benzimidazole ring
3
shows the batochromic shift (data not shown). This long wavelength absorption band is associated
with the intramolecular charge transfer transition due to the transfer of the charge between the
five-membered rings to the electron-withdrawing of benzimidazole and neighboring carboxylic or
sulfonic chromophore.
Sun Protection Factor (SPF) is the UV energy required for producing a minimal erythema dose
(
MED) on protected skin, divided by the UV energy required producing a MED on unprotected skin.
This factor was first introduced in 1974 to evaluate the effectiveness of a sunscreen, only the in vivo
method was officially accepted by the US Food and Drug Administration and COLIPA (now Cosmetics
Europe—The Personal Care Association). Due to the high cost, time consumption and some questions
noble of the volunteers relating to in vivo SPF determination, in vitro photoprotection studies have
been developed. The broadly applied in vitro method is the well-known Diffey-Robson approach [29
]
that is a spectrophotometrically-based measurement of transmission. Spectral data of samples were
collected from 290–400 nm with the calculator software [30]. The software automatically determines
theoretical COLIPA—SPF, COLIPA—UVAPF, critical wavelength value (
preliminary data.
λc) and UVA/UVB ratio as
Photoprotective activity of the sunscreen against UVB is reflected by SPF value. According to the
results (Table S2), SPF values of all synthesized compounds was high compared to lead compound
(
PBSA) and the highest SPF value belongs to compound 11 characterized by the presence of furan
in position 2 of the benzimidazole ring and without functional group in position 5. By maintaining
SO H group in position 5 and substituting the phenyl by the pyrrole, SPF value increased. However,
-
3
by maintaining the pyrrole and replacing -SO H with -COOH (
7
vs. 13), SPF again increased. Then,
substituting the functional group carboxyl by an -H (13 vs. 10), SPF exponentially increased. The same
results were obtained by replacing phenyl with thiophene (PBSA vs. 12 15) and furan (PBSA vs.
14). So, the photoprotective activity against the UVB radiations of the synthesized compounds does
not depend only on the substitution in the 2-position of the benzimidazole ring but also on the groups
3
9
,
,
8,
11,
present in position 5. Therefore the structure activity relationship is: -H > -COOH > -SO H.
3
Moreover, replacing the pyrrole with the furan (10 vs. 11), the SPF value increased. On the other
hand, by substituting the pyrrole with thiophene, the SPF decreased, for example 10 vs. 12 (SPF value
of 13.13 to 7.03 respectively). Thus, by keeping the group present in the 5-position of benzimidazole and
by varying the 5-membered ring at 2-position, the order of protection against UVB is as follows—furan
>
pyrrole > thiophene.
Protection from UVA rays is given by UVA-PF (UVA protection factor) which is considered effective
if it is greater than or equal to 1/3 of SPF. Considering the data obtained, all the synthesized compounds
showed UVA-PF values higher than those of the reference compound, while all remaining below 1/3 of
the SPF. Therefore, the synthesized compounds are not potential candidates for UVA protection.
Critical wavelength is the parameter that provides information on the broad-spectrum of UV-filter.
It is classified in five numerical categories: 0 (
c <370) and 4 ( 370). FDA considers the broad-spectrum sunscreen product should have a
370 nm [31]. Based on this classification, none of the synthesized compounds showed a value of
λ
c < 325 nm), 1 (325 ≤ λ
c
≤
335), 2 (335 ≤ λ
c
≤
350), 3 (350
≤
λ
λ
c
≥
λ
c
≥
λc ≥ 370 so they do not have a broad-spectrum profile.
In short, modifications on PBSA in position 2 by 5-membered rings and in position 5 by -COOH
and -H led to enhanced UVB-filter activity.
2
.2.2. In Vitro Photoprotective Activity of Cosmetic Formulation Containing Benzimidazole
Derivatives
Among synthesized compounds, it has been chosen to formulate the compounds 10, 11 and 12,
since they are the ones that have shown the best SPF value in solution of their class and moreover all
three are soluble in the same solvent which is PEG-7 Glyceryl Cocoate. The photoprotective efficacy of

Molecules 2020, 25, 4324
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the synthesized compounds was determined by spectrophotometric measurements using the modified
Diffey-Robson method [29]. The compounds were introduced at different concentrations (1%, 2%
and 3%) in a 40:60 water-in-oil based cosmetic formulation and the analyzes were performed on
PMMA (Poly(methyl methacrylate)) plates. 2-Phenyl-1H-benzo[d]imidazole-5-sulfonic acid (PBSA
)
was used as lead compound. Values of the SPF, UVAPF, UVA/UVB and c are shown in Table 1. In
λ
comparison with lead compound, only compound 11, among the selected synthesized compounds,
provided higher protection against UVB radiation. PBSA at concentrations 1, 2 and 3% (w/w) showed
increasing SPF values ranging from 3.22 to 5.09; while compound 11 which is benzimidazole with
furan at the 2-position and unsubstituted at the 5-positon exhibited SPF values ranged from 6.88 to
11.15. Moreover replacing the furan with the pyrrole (11 vs. 10), the SPF value is decreased. On the
other hand, by substituting the furan with thiophene (11 vs. 12), the SPF also is decreased. However,
compound 10 was slightly more UVB-protective than compound 12.
Table 1.
UV filtering activity of benzimidazole derivatives 10–12 in formulation at
different concentrations.
Compound Percentage (%)
SPF
UVAPF
UVA/UVB
λc (nm)
1
2
3
3.22
4.63
5.09
0.94
0.94
1.02
0.33
0.22
0.25
319
321
324
PBSA
1
2
3
2.44
2.72
3.06
1.16
1.32
1.54
0.70
0.73
0.75
341
357
364
1
1
1
0
1
2
1
2
3
6.84
10.39
11.15
1.94
1.53
1.82
0.69
0.13
0.18
368
366
371
1
2
3
2.41
2.59
2.79
1.16
1.22
1.26
0.71
0.70
0.69
340
342
344
Taking together the results obtained from selected benzimidazole derivatives, we could be deduced
that the order of protection against UVB is as follows—furan-> pyrrole- > thiophene-derivatives.
2
.2.3. Photostability Study by Spectral Analysis
Photostability, which is one of the important parameters for defining the efficacy and safety
of sunscreen, has been evaluated. So photodegradation was monitored by the UV transmittance
measurements of the products before and after irradiation according to the criteria developed by
Garoli et al. [32]. PMMA plates coated with sunscreen have been irradiated with the solar stimulator
by the application of UVA dose necessary to cause erythema. Then, degree of photostability was
expressed as the percentage effectiveness after exposure. Therefore in vitro % SPFeff and %UVA-PFeff
were calculated according to Equations (4) and (5), respectively (paragraph 3.4.1). Obtained data
(Figure 2) showed that the compounds 10 and 12 were photostable, since according to the regulation,
sunscreen is considered photostable if the in vitro % SPF and % PF-UVA values are at least 80% [33].
In particular, compound 12, bearing a thiophene in position 2, showed excellent photostability in the
total UV range. However, compound 10 presenting pyrrole in position 2 was more photostable in
UVA range than UVB range. Compounds 11 bearing furan at the 2-position was photoinstable with %
SPF and% PF-UVA less than 80%. This is likely due to the insufficient photostability of its conjugated
species [34].
Therefore the order of photostability of compounds is—thiophene derivative > pyrrole derivative
>
furan derivative.

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Figure 2. Transmittance profiles of formulations studied containing the benzimidazole derivatives 10
panel A), 11 (panel B) and 12 (panel C) (1% w/w).
(
2
.3. Antioxidant Activity
The synthesized compounds were evaluated for their ability to scavenger the stable radical
,2-diphenyl-1-picrylhydrazyl (DPPH) through DPPH test and their ability to reduced ferric ions to
2
ferrous ions via Ferric reducing antioxidant power (FRAP) test.
From the results reported in Table 2, the reference compound is almost devoid of any antioxidant
activity and the substitution of phenyl in the 2- position of benzimidazole by 5-membered rings gave
the molecule an antioxidant activity more or less significant. Regarding DPPH test, compounds
and 14 showed percentage of inhibition greater than or equal to 50%. To this effect, they were
screened to determine their IC50 value. However, only compound 10 exhibited good scavenger profile
with IC50 = 64.098 g/mL. Data obtained from FRAP showed that compounds 10 and 11, respectively
7,
1
0
µ
characterized by the presence of pyrrole and furan at the 2-position of the benzimidazole ring and both
lacking a functional group at the 5-position showed the good antioxidant profile. Whereas compound
1
1 displayed the best activity (4462.64 µmolTE/g).
Table 2. Antioxidant activity of the compounds through DPPH and FRAP test. Each value was obtained
from three different experiments (mean
tested concentration equal to 0.0105 mg/mL).
±
SEM). (The caffeic acid value of the DPPH test refers to a
DPPH
DPPH
IC50 (µg/mL)
FRAP
(µmolTE/g)
Compound
(
% Inhibition)
81.35 ± 0.35
<LOQ *
Caffeic acid
-
-
10156.12 ± 15.5
<LOQ *
PBSA
7
53.88 ± 2.30
70.00 ± 3.25
33.00 ± 1.28
10.96 ± 0.56
15.46 ± 0.98
52.06 ± 1.48
1093.09 ± 8.65
239.91 ± 1.79
1085.57 ± 0.88
108.89 ± 2.98
1
1
0
3
64.10 ± 3.21
-
8
-
-
<LOQ *
1
1
1
4
4462.64 ± 18.29
1416.38 ± 21.40
26.67 ± 2.89
9
12.52 ± 0.88
35.05 ± 0.87
30.23 ± 1.45
-
-
-
82.16 ± 0.22
75.89 ± 5.43
181.84 ± 2.66
1
1
2
5
*
LOQ, Limit of Quantification.

Molecules 2020, 25, 4324
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2
.4. Antifungal Activity
Synthesized compounds were evaluated for their in vitro antifungal activity against five pathogenic
dermatophytes—Microsporum gypseum, Microsporum canis, Trichophyton mentagrophytes, Trichophyton
tonsurans and Epidermophyton floccosum. Percentage inhibition of fungal growth was performed using
Sabouraud Dextrose Agar (SDA) as culture medium and DMSO as a solvent control. Incubation was
done at room temperature and inhibition of the dermatophytes was evaluated daily by measuring the
diameter of the colony at each disk over 7 days. All compounds were tested at the same concentration,
namely 100
shown in Table S3.
Benzimidazole compounds unsubstituted in position 5 (compounds 10
strong growth inhibition of all five dermathophytes with percentage of inhibition closer to 100%.
The presence of -SO H at the 5-position decreased activity, however and showed good activity with
14 and 15 bearing –COOH did not have a
µg/mL and the results determined as the average of three different experiments were
,
11 and 12) displayed
7
9
3
an inhibition range of 50–79%. Curiously, compounds 13
,
significant activity on dermatophytes; on the contrary, they rather favored the growth of certain fungi.
Interestingly, starting from PBSA which has no activity on dermatophytes and replacing phenyl
by the five-membered cycle we obtained good results, indicating that the five-membered cycle is very
important for the studied activity. Compounds 10, 11 and 12 displayed fungicide activity on all five
dermatophytes at 100 g/mL. In addition, the substitution of pyrrole with thiophene or furan did not
µ
have influence on the variability of the activity; this is confirmed by Friedman which states that the
bioisosteres have similar biological activity [35].
The structure-activity relationship reveals that the presence of hydrogen at the 5-position of
benzimidazole ring is more beneficial than the electron withdrawing group.
Encouraged by these preliminary findings, compounds with growth inhibition close to 100%
were further investigated by the determination of IC . Thus, IC results of 10, 11 and 12 against five
50
50
dermatophytes are reported in Table 3. Fluconazole and Econazole nitrate were used as references
agents. Tested compounds presented excellent activity toward M. gypseum, M. canis, T. mentagrophytes,
T. tonsurans and E. floccosum with IC50 values between 0.97 and 3.80 µg/mL. Furthermore all tested
compounds are more active than Fluconazole, except on E. floccosum. However, they are less effective
than Econazole nitrate. Compound 10 exhibited the best activity, with IC50 values ranging from 0.97 to
1
.53 µg/mL.
Table 3. IC50 values of compounds 13–15, Econazole nitrate and Fluconazole on dermatophytes. Each
value is the mean of three measurements.
IC50 (µg/mL)
T.
Compound
M. gypseum
M. canis
T. tonsurans
E. floccosum
mentagrophytes
1
1
1
0
1
2
1.53 ± 0.05
1.54 ± 0.02
1.55 ± 0.03
1.34 ± 0.02
1.58 ± 0.06
2.14 ± 0.09
1.38 ± 0.08
1.61 ± 0.10
1.74 ± 0.11
0.97 ± 0.06
1.89 ± 0.22
2.42 ± 0.15
1.07 ± 0.03
2.44 ± 0.05
3.80 ± 0.07
Fluconazole
Econazole nitrate
18.5 ± 1.23
29.6 ± 1.84
3.53 ± 0.26
19.41 ± 0.87
0.08 ± 0.005
0.05 ± 0.0001
0.51 ± 0.02
0.006 ± 0.0001
0.13 ± 0.008
0.47 ± 0.015
Candida is a fungus strain that can infect many parts of the human body. Among these, we can
mention the infection of the skin. For this, we have also focused our attention on Candida albicans who
lives in principle in the oral cavity, digestive tract, vagina and skin [36].
The tested compounds were dissolved in DMSO to obtain 12.8 mg/mL of stock solutions and the
concentration tested ranged from 0.25
µg/mL to 128
µg/mL. The minimal inhibitory concentration
(
+
MIC) of the synthesized agents was determined by broth microdilution method using RPMI 1640
MOPS as culture medium. After incubation in RPMI-160 broth at 37 C for 24 and 48 h, the MIC
◦
(µg/mL) values were collected and reported on Table 4. Fluconazole was used as positive control drug.

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Compounds 7, 9, 14 and 15 showed activity against Candida albicans but, only 14 and 15 exhibited good
activity (MIC = 16 µg/mL) after 24 h. These two compounds bearing carboxylic acid at the position 5
of benzimidazole ring. Neither synthesized compounds nor fluconazole showed activity after 48 h.
Table 4. Anti-Candida albicans activity of synthesized compounds. The results are representative of
incubations arranged in triplicate at 24 and 48 h and the average values were taken as the minimal
inhibitory concentrations (MICs).
MIC (µg/mL)
Compound
7
24 h
48 h
64
-
-
-
-
-
1
1
0
3
8
-
-
16
-
-
-
1
1
1
4
9
64
-
16
-
-
-
1
1
2
5
Fluconazole
0.5
-
It is important to note that, contrary to the results observed on dermatophytes, the non-substituted
compounds in position 5 of benzimidazole have no activity on the yeasts. This can be explained by the
fact that each fungus has its own morphology and defense systems.
2
.5. Antiproliferative Activity
In vitro antiproliferative activity of benzimidazole and benzothiazole derivatives against human
melanoma cells (SK-Mel 5) was evaluated. Since benzimidazoles are recognized as potential anticancer
drugs [10], we have also decided to examine the antiproliferative activity on other human cancer
cells, including human T-lymphocyte cells (CEM), human cervical carcinoma cells (HeLa) and human
pancreatic carcinoma cells (Mia Paca-2). Compound concentrations required to inhibit cell proliferation
by 50% (in µM) are reported in Table 5. Non-cancer cells HEK293T (Normal Kidney Epithelial Cells)
were chosen as a control. According to the obtained results, compound 10, bearing pyrrole and
unsubstituted at the 5-position, exhibited good inhibition against CEM, Mia Paca-2 and SK- MEL-5.
Furthermore, compound 10 was 3-fold more selective against SK-Mel 5 (SI = 3.20) with IC50 = 9.7 µM
against this tumor cell line.
Table 5. Inhibitory effects of benzimidazole derivatives on cell proliferation (CEM, HeLa, Mia Paca-2
and SK-Mel 5). All analyzes are the mean of triplicate measurements ± standard deviation.
IC50 (µM)
SI *
Compound CEM
HeLa
Mia Paca-2 SK-Mel 5 Hek293 CEM HeLa Mia Paca-2 SK-Mel 5
7
31 ± 21
14 ± 1
>100
>100
37 ± 0
>100
28 ± 10
15 ± 9
>100
>100
9.7 ± 1.7
>100
67 ± 47
31 ± 3
>100
2.16
2.21
-
-
-
-
2.39
2.07
-
-
3.20
-
1
0
1
3
8
>100
>100
≥100
>100
>100
≥100
>100
>100
≥100
>100
>100
>100
>100
>100
-
-
-
-
-
-
-
-
-
-
-
1
1
1
4
75 ± 35
1.33
9
34 ± 2
>100
>100
>100
43 ± 14
≥100
>100
86 ± 17
>100
>100
>100
>100
2.94
-
2
-
-
-
2.33
-
1.25
-
1.16
-
1
1
2
5
>100
50 ± 10
80 ± 12
*
SI = IC50 of pure compound in a normal cell line/IC50 of the same pure compound in cancer cell line.

Molecules 2020, 25, 4324
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2
.6. Antiviral Activity
All the synthesized compounds were finally tested against herpes simplex virus-1 (KOS),
herpes simplex virus-2 (G), herpes simplex virus-1 TK-KOS ACVr, vaccinia virus, Adeno virus-2 and
Human Coronavirus (229E) in HEL cell cultures and against vesicular stomatitis virus, Coxsackie virus
B4, Respiratory syncytial virus in HeLa cell cultures. Results are shown in Tables S4 and S5. None of
the synthesized compounds showed specific antiviral activity.
3
. Materials and Methods
3
.1. General Information
All reagents were obtained from commercial sources and used without further purification.
Analytical TLC was performed on silica gel plates (Macherey-Nagel Poligram SIL G/UV254 0.20 mm,
GmbH & Co. KG Neumann-Neander-Str. 6–8, 52355 Dueren, Germany) and visualized by exposure
to ultraviolet light (254 nm) and /or used 1% solution of KMnO . Column chromatography was
4
1
13
performed using silica gel Macherey-Nagel 60M 230400 mesh. H-NMR and C-NMR were registered
on VXR-200 Varian spectrometer at 200 MHz and 400 MHz with tetramethylsilane (TMS) as an internal
standard. Chemical shifts are reported in
δ units (ppm) relative to the residual deuterated solvent
signals of D O and (CD ) SO. The following abbreviations are used to indicate signal multiplicity and
2
3 2
assignment—s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), brs (broad singlet) and dd
(
doublet of doublets), BIM (benzimidazole). Molecular weights of the compounds were determined by
+
ESI (Micromass ZMD 2000) and the values are expressed as [MH] . IR spectra were obtained using a
Spectrum 100 FT-IR Spectrometer (PerkinElmer). UV spectrophotometric analyses were carried out
on a UV-VIS spectrophotometer (Shimadzu UV-2600) or on a Life Science UV/VIS spectrophotometer
®
(
Beckman Coulter
™
, DU 530, Single Cell Module, Beckman Coulter s.r.l., Via Roma, 108 - Palazzo
F1, Centro Cassina Plaza 20060 - Cassina De’ Pecchi, Milano, Italia). Photostability studies were
performed with Atlas Suntest CPS+ solar simulator, (URAI S.p.a., Assago, Milano, Italy). Melting
points were measured with a Stuart melting point apparatus. WW5 PMMA plates have been purchased
from Schonberg GmbH (Munich, Germany). Sabouraud dextrose agar (SDA) was purchased by
Sigma-Aldrich SRL, Milano, Italy.
3
.2. Chemistry
3
.2.1. Synthesis of 3,4-diamino-benzensulfonic acid, Sulfate Salt (2)
◦
To 0.5 g (4.62 mmoles) of o-phenylenediamine was added 3 mL of 96% sulfuric acid at 0 C under
stirring, then the solution was refluxed for 24 h. The reaction mixture was cooled and then cold water
was added to obtain a precipitate. The mixture was filtered and washed with methanol to obtain 0.62 g
1
of white powder, 46% yield. H NMR (400 MHz, DMSO-d6):
δ
(ppm) 7.39 (d, J = 1.6 Hz, 1H), 7.2 (dd,
J = 1.6 Hz), 2H). 13C-NMR (400 MHz, DMSO-d6): 140.03, 138.39, 135.04, 118.22, 117.01.ESI-M [M + H]
+
:
calcd for C H N O S, 188.03; found 188.07.
6 8 2 3
3
.2.2. Synthesis of 2-substituted benzimidazole-5-sulfonic Acids
To a solution mixture of 3 mmol of and 4 mmol of aldehyde (
2
3
–
5
) in ethanol (3 mL), 6 mmol of
◦
sodium bisulfite were added under stirring. The reaction mixture was heated at 80 C under reflux
for 24 h. The solvent was then evaporated under vacuum and the solid was treated with HCl 2N.
The suspension was filtered and the solid was washed with ethanol to afford desired product.
2
-(1H-pyrrol-2-yl)-1H-benzimidazole-5-sulfonic Acid (7)
◦
−1
Brown solid in yield 56%; m.p. > 250 C. IR (KBr) cm : 2851.26, 1638.62, 1456.40, 1227.98, 1143.18,
1
1
023.50, 718.04. HNMR (400 MHz, DMSO-d6):
δ
(ppm) 11.89 (s, 1H, NH), 7.91 (s, 1H, BIM), 7.77–7.69
(m, 2H, BIM), 7.45 (d, J = 1.6 Hz, 1H, pyrrole), 7.30 (d, J = 1.6 Hz, 1H, pyrrole), 6.45 (t, 1H, pyrrole).

Molecules 2020, 25, 4324
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1
3
C-NMR (400 MHz, DMSO-d6): 145.68, 143.67, 131.10, 130.45, 126.90, 123.22, 115.98, 115.04, 112.36,
+
1
11.36, 110.30. ESI-M [M + H] : calcd for C H N O S, 263.04; found 263.17.
11
9
3
3
2
-(furan-2-yl)-1H-benzimidazole-5-sulfonic Acid (8)
◦
−1
White solid in yield 31%; m.p. > 250 C. IR (KBr) cm : 3446.68, 1589.75, 1474.34, 1316.39, 1217.18,
1
7
56.85. HNMR (400 MHz, DMSO-d6):
δ (ppm) 8.25 (d, J = 1.7 Hz, 1H, furan), 7.90 (d, J = 1.3 Hz, 1H,
BIM), 7.78–7.68 (m, 2H, BIM), 7.60 (d, J = 3.6 Hz, 1H, furan), 6.93 (dd, J = 3.6 Hz, 1H, furan). C-NMR
13
(
100 MHz, DMSO-d6):
δ
(ppm) 147.90, 146.05, 141.44, 140.08, 132.89, 132.02, 123.40, 116.19, 13.47, 110.94.
+
ESI-M [M + H] : calcd for C H N O S, 264.02; found 264.22.
11
8
2
4
2
-(thiophen-2-yl)-1H-benzimidazole-5-sulfonic Acid (9)
◦
−1
White solid in yield 69%; m.p. > 250 C. IR (KBr) cm : 2645.75, 1631.52, 1458.42, 1079.51, 1026.70,
18.69. HNMR (400 MHz, DMSO-d6):
1
7
1
δ (ppm) 8.14 (d, J = 1.6 Hz, 1H, thiophene), 8.11 (d, J = 1.6 Hz,
H, thiophene), 7.85 (s, 1H, BIM), 7.71–7.62 (m, 2H, BIM), 7.39 (t, 1H, thiophene). 13C-NMR (400 MHz,
+
DMSO-d6): 145.45, 143.42, 141.67, 140.39, 128.00, 121.88, 116.43, 113.09. ESI-M [M + H] : calcd for
C H N O S , 280.00; found 280.18.
11
8
2
4 2
3
.2.3. General Method for the Synthesis of 2-Substituted Benzimidazoles
To a solution mixture of 3 mmol of
1
and 4 mmol of aldehyde (
3
–
5
) in ethanol (3 mL), 6 mmol of
◦
sodium bisulfite were added under stirring. The reaction mixture was heated at 80 C under reflux
for 24 h. The solvent was then evaporated under vacuum and the solid was treated with HCl 2N.
The suspension was filtered and the solid was washed with ethanol to afford desired product.
2
-(1H-pyrrol-2-yl)-1H-benzimidazole (10)
◦
−1
Yellow solid in yield 55%; m.p. > 250 C. IR (KBr) cm : 2733.42, 1606.25, 1464.35, 1227.52, 1136.01,
1
1
2
6
041.46, 743.84. HNMR (400 MHz, DMSO-d6):
δ
(ppm) 11.99 (brs, 1H, NH pyrrole), 7.65–7.62 (m,
H, BIM), 7.34–7.29 (m, 2H, BIM), 7.19 (d, J = 1.6 Hz, 1H, pyrrole), 7.11 (d, J = 1.4 Hz, 1H, pyrrole),
.33 (t, 1H, pyrrole). 13C-NMR (400 MHz, DMSO-d6): 154.34, 134.04, 120.89, 119.30, 113.45, 110.11,
+
1
05.86.ESI-M [M + H] : calcd for C H N , 183.08; found 183.12.
11
9
3
2
-(furan-2-yl)-1H-benzimidazole (11)
◦
−1
Yellow solid in yield 60%; m.p. > 250 C. IR (KBr) cm : 2619.65, 1569.95, 1419.27, 1234.04, 1073.76,
1
8
2
1
50.94, 740.26. HNMR (400 MHz, DMSO-d6):
H, BIM), 7.09 (m, 3H, 2H BIM, 1H furan), 6.70 (t, 1H, furan)
41.45, 140.76, 139.11, 123.45, 115.09, 112.86, 107.23. ESI-M [M + H] : calcd for C H N O, 184.06;
δ
(ppm) 8.10–7.90 (d, J = 1.7 Hz, 1H, furan), 7.19 (t,
13
.
C-NMR (400 MHz, DMSO-d6): 154.04,
+
11
8
2
found 184.21.
2
-(thiophen-2-yl)-1H-benzimidazole (12)
◦
−1
Grey Solid in yield 72%; m.p. > 250 C. IR (KBr) cm : 2619.65, 1569.95, 1449.98, 1419.27, 1234.04,
1
8
7
1
50.94, 702.14. HNMR (400 MHz, DMSO-d6):
.24 (t, J = 1.5 Hz, 2H, BIM), 7.00 (t, 1H, thiophene). 13C-NMR (400 MHz, DMSO-d6): 145.33, 142.71,
40.11, 139.67, 128.07, 127.23, 126.61, 122.00, 114,25. ESI-M [M + H] : calcd for C H N S, 200.04;
δ
(ppm) 7.98–7.72 (m, 4H, 2H BIM, 2H thiophene),
+
11
8
2
found 200.11.
3
.2.4. General Method for the Synthesis of 2-Substituted Benzimidazole-5-Carboxylic Acids
To a solution mixture of 3 mmol of
6
and 4 mmol of aldehyde (
3
–
5
) in ethanol (3 mL), 6 mmol of
◦
sodium bisulfite were added under stirring. The reaction mixture was heated at 80 C under reflux
for 24 h. The solvent was then evaporated under vacuo and the solid was treated with HCl 2N.
The suspension was filtered and the solid was washed with ethanol to afford desired product.

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2
-(1H-pyrrol-2-yl)-1H-benzimidazole -5-carboxylic Acid (13)
◦
−1
Brown solid in yield 51%; m.p. > 250 C. IR (KBr) cm : 2678.85, 1624.06, 1600.08, 1459.86, 1302.43,
1
1
137.71, 896.74, 763.89. H NMR (400 MHz, DMSO-d6):
δ
(ppm) 11.23 (brs, 1H, OH), 8.14 (s, 1H, BIM),
7
.94–7.80 (m, 2H, BIM), 6.77(d, J = 1.5 Hz, 1H, pyrrole), 6.50 (d, J = 1.5 Hz, 1H, pyrrole), 6.23 (t, 1H,
13
pyrrole). C-NMR (400 MHz, DMSO-d6): 166.00, 155.68, 146.91, 138.56, 128.87, 126.88, 120.55, 116.31,
+
1
11.75, 107.70. ESI-M [M + H] : calcd for C H N O , 227.07; found227.32.
12
9
3
2
2
-(furan-2-yl)-1H-benzimidazole -5-carboxylic Acid (14)
◦
−1
Brown solid in yield 49%; m.p. > 250 C. IR (KBr) cm : 2500.23, 1686.09, 1538.50, 1290.52, 1234.76,
1
1
04.40, 766.43. HNMR (400 MHz, DMSO-d6):
δ
(ppm) 11.19 (brs, 1H, OH), 8.30 (d, J = 1.6 Hz, 1H,
furan), 8.16 (s, 1H, BIM), 8.06 (d, J = 1.6 Hz, 1H, BIM), 7.89 (d, J = 1.5 Hz, 1H, BIM), 7.76 (d, J = 3.6
Hz, 1H, furan), 6.50 (t, 1H, furan). 13C-NMR (400 MHz, DMSO-d6): 166.73, 154.02, 145.55, 142.03,
+
1
39.40, 138.00, 126.62, 120.13, 115.74, 113.11, 106.85. ESI-M [M + H] : calcd for C H N O , 228.05;
12
8
2
3
found 228.14.
2
-(thiophen-2-yl)-1H-benzimidazole -5-carboxylic Acid (15)
◦
−1
Grey solid in yield 69%; m.p. > 250 C. IR (KBr) cm : 2490.11, 1682.64, 1581.93, 1416.22, 1286.30,
1
8
(
(
54.62, 768.66. HNMR (400 MHz, DMSO-d6):
d, J = 1.6 Hz, 1H, BIM), 7.82–7.75 (m, 3H, 1H BIM, 2H thiophene), 7.01 (t, 1H, thiophene). 13C-NMR
400 MHz, DMSO-d6): 166.07, 146.76, 141.86, 138.62, 129.11, 118.65, 114.04, 113.29. ESI-M [M + H] :
δ (ppm) 11.26 (brs, 1H, OH), 8.25 (s, 1H, BIM), 8.02
+
calcd for C H N O S , 244.03; found 244.26.
12
8
2
2 2
3
.3. Biological Assays
.3.1. In Vitro Photoprotection Activity
3
Filtering Parameters Evaluation of Benzimidazole Derivatives in Solution
The assessment of Sun protection factor was performed using a SHIMADZU UV-2600 UV–VIS
spectrophotometer (Shimadzu Italia S.r.l., Via G.B. Cassinis 7, 20139 Milano, Italy). Test compounds
were dissolved in MeOH at the concentration of 0.0015 (
measured between 290 and 400 nm using a 1 cm quartz cell at intervals of one 1 nm using a UV–Vis
±
0.000005) % and the absorbance was
Spectrophotometer. The absorbance at wavelength
Equation (1).
λ
is related to the transmittance (T(
λ)) by the
A(λ) = Log [T(λ)],
(1)
where T(λ) is the fraction of incident irradiance transmitted by the sample.
Sunscreens Formulation
A basic topical formulation W/O was prepared—hydrophilic-phase and oil-phase were heated
◦
separately to between 60–70 C, until complete solubilization of the ingredients. Subsequently,
the hydrophilic phase was slowly added to the oil phase under constant stirring with a Silverson
homogenizer. Then, the emulsion was cooled to room temperature and the selected sunscreen agents
were incorporated at different concentrations (1%, 2% and 3%).
INCI—aqua, glycerin, Euxyl PE 9010, Xanthan gum, triethanolamine, ceteareth-12, ceteareth-20,
Sterearic acid, Butylhydroxytoluene, Myritol, PEG-7 glycerylcocoate, sodium hydroxide.
In Vitro Evaluation of Filtering Parameters
2
3
2.5 mg of each formulation were spread onto 25 cm PMMA plates with finger. Plates were
weighed before and after spreading. Three plates were prepared for each product and five measurements
were performed on each plate. The plates were left at room temperature in the dark for 15 to 30 min

Molecules 2020, 25, 4324
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before measurements. Then, the UV transmittance measurements were carried out from 290 to 400 nm
using UV-VIS spectrophotometer (Shimadzu UV-2600). The blank was prepared using a plate covered
with 15
Critical Wavelength were carried out according using SPF Calculator Software (SPF Calculator Software
version 2.1), Shimadzu, Milan, Italy) as previously described [37].
µL of glycerin thanks to its non-fluorescent and UV transparency. In vitro SPF, UVA-PF and
(
3
.3.2. Antioxidant Activity
DPPH Assay
The activity against DPPH
previously reported [39]. The synthesized derivatives were initially tested at the same concentration of
mg/mL to determine a result expressed as mol TE/g. Subsequently, the better profile compounds
•
radical was tested following the Wang et al. method [38] modified as
1
µ
were tested against the radical to determine the IC50 value expressed as µg/mL.
FRAP Assay
The FRAP antioxidant capacity of synthesized compounds was analyzed according as previously
described [40], measuring the absorbance of the reaction mixture at 593 nm. The values are expressed
as µmol TE/g compound.
3
.3.3. Antifungal Activity
Antidermatophyte Activity
Microorganism
Dermatophytes used in this study were Epidermophyton floccosum var. floccosum (Netherlands)
CBS 358.93 strain; Trichophyton tonsurans (Netherlands) CBS 483.76 strain; Trichophyton mentagrophytes
(
(
Netherlands) CBS 160.66 strain; Microsporum canis (Iran) CBS 131110 strain; and Microsporum gypseum
Iran) CBS 130948 strain. All dermatophytes were maintained at 4 C as agar slants on SDA.
◦
In Vitro Culture
In Vitro antifungal activity against the five dermatophytes was assessed using the plaque growth
inhibition method as previously described [37]. Each compound was evaluated at the final concentration
of 100 g/mL. Percentages of growth inhibition were calculated by comparing mean value of diameters
µ
of the mycelia in test plates with that of untreated control plates following the Equation (2). The percent
inhibition of growth was determined as the average of three different experiments.
I = [(C − T)/C] × 100%,
(2)
where, I is the growth inhibition rate (%), C is the extended diameter of the circle mycelium of the
control (mm) and T is the extended diameter of the circle during testing (mm). IC50 values were
obtained only for the benzimidazole that were active against the five dermatophytes. The IC50 values
were calculated as the average of three different experiments and they indicate the concentration of the
substance needed to inhibit the growth of the fungus by half.
Anti-Candida albicans Activity
Fungal suspension was prepared by suspending a Candida albicans (ATCC 10231) aliquot in 5 mL of
sterilized water. Stock solutions were prepared by dissolving the compounds and Fluconazole in DMSO
at a concentration of 12.80 mg/mL. Minimal inhibitory concentration (MIC) of the synthesized agents
was performed by broth microdilution method according to the Clinical and Laboratory Standards
Institute/National Committee for Clinical Laboratory Standards (CLSI/NCCLS) Approved Standard
M27-A3, 2008 [NCCLS] as previously described [37].

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3
.3.4. Anti-Proliferative Activity
Cell Lines
Human cervical carcinoma (HeLa) and human CD +T-lymphoblast (CEM) cells were obtained
4
from ATCC (Middlesex, UK). Human pancreatic carcinoma (Mia-Paca 2) cells were kindly provided by
Prof. Anna Karlsson (Karolinska Institute, Stockholm, Sweden). All cell lines were grown in Dulbecco’s
modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA), supplemented with 10% fetal bovine
serum (FBS, Gibso), 0.01M Hepes (Gibco) and 1 mM sodium pyruvate (Gibco) in a humidified 5% CO
incubator at 37 C.
2
◦
Cell Proliferation
Suspension of CEM cells were seeded in 96-well microtiter plates at 60,000 cells/well in the
presence of different concentrations of the compounds. The cells were allowed to proliferate for 48
h or 72 h, respectively and then counted in a Coulter counter. The 50% inhibitory concentration
(
IC ) was defined as the compound concentration required to reduce cell proliferation by 50%. HeLa
50
and Mia-Paca2 cells were seeded in 96-well plates at 15,000 cells/well in the presence of different
concentrations of the compounds. After 4 days of incubation, the cells were trypsinized and counted
in a Coulter counter).
IC50 Determination
The compounds were dissolved in DMSO at 20 mM (stock solution) and kept in the refrigerator
until use. Then, compound dilutions were made in cell culture medium and serial compound
concentrations were tested starting at 100
calculated using Equation (3).
µM as the highest concentration. The IC50 values were
C1 − [50 − N1%/N2% − N1%] − (C1 − C2),
(3)
wherein C1 is the compound concentration that inhibits cell proliferation more than 50%; C2 is the
compound concentration that inhibits cell proliferation less than 50%; N1% represents the cell number
(
in percent of control in the absence of compound) obtained in the presence of C1 and N2% represents
the cell number (in percent of control in the absence of compound) obtained in the presence of C2.
3
.3.5. Anti-Viral Activity
Antiviral activity was tested against herpes simplex virus-1 (KOS), herpes simplex virus-2 (G),
herpes simplex virus-1 TK-KOS ACVr, vaccinia virus, Adeno virus-2 and Human Coronavirus
229E) in HEL cell cultures; vesicular stomatitis virus, Coxsackie virus B4, Respiratory
(
syncytial virus in HeLa cell cultures. Cytotoxicity of compounds was evaluated in parallel
with the antiviral activity namely, microscopic evaluation of the cell morphology of the
confluent cell monolayer which either had or had not been inoculated with virus. Cytotoxic
concentration and concentration producing 50% inhibition of virus-induced cytopathic effect
were determined by measuring the cell viability with the colorimetric formazan-based MTS
assay ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)).
Reference compounds used were—Brivudin, Cidofovir, Ganciclovir, Acyclovir for virus in HEL
cell cultures and ribavirin and DS-10.000 for virus in HeLa cell cultures. The antiviral activity of
the compounds was expressed as the concentration required to inhibit viral cytopathogenicity by
5
0% (EC ).
50

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3
.4. Stability Studies
Photostability
The photostability was determined using the method previously described [37]. Each plate,
prepared according to the previous section was irradiated with a solar simulator applying the
UVA-dose equivalent to an effective erythema radiant exposure. The spectral transmittance of thin
film of sunscreen was measured before and after sunlight exposure from 290 to 400 nm. The residual
percentage of SPF in vitro (% SPFeff.) and UVA-PF (% UVA-PFeff.)) were calculated according to
Equations (4) and (5) respectively. In fact, a filter is considered photostable if % SPFeff. and % UVA-PFeff.
are more than or equal to 80.
%
SPF . = in vitro SPFafter/ in vitro SPFbefore × 100
(4)
(5)
eff
%UVA − PF . = UVA − PF
/UVA-PFbefore × 100.
eff
after
4
. Conclusions
The isosteric modification approach has been exploited to design and investigate the
structure-activity relationship of a series of some new and other already known nine
benzimidazole derivates.
Regarding the antifungal activity, compounds 10
M. gypseum, M. canis, T. mentagrophytes, T. tonsurans and E. floccosum with IC50 values in the
range of 0.97–3.80 g/mL. Nevertheless, compound 10 showed the best activity with IC50 range
of 0.97–1.53 g/mL. This compound is characterized by the presence of pyrrole in position 2 of the
, 11 and 12 exhibited good activity against
µ
µ
benzimidazole ring and devoid functional group in position 5. In addition, compounds 14 and 15
exhibited good activity against Candida albicans with MIC = 16 µg/mL.
Considering UV-filtering capacity, compounds 10, 11 and 12 showed to be a good UVB-filter as
compared to PBSA and the best photoprotective compound was 11 bearing furan in position 2 of
the benzimidazole ring and without substitution in position 5. Compounds 10 and 11 showed good
antioxidant activity; however according to DPPH test only 10 was endowed with antioxidant activity,
while according to FRAP test, both 10 and 11 had good antioxidant activity being 11 (4462.64
TE/g) the best one.
µmol
Finally, regarding the antiproliferative activity, compounds 10 displayed good activity against
SK-Mel 5 (human melanoma cells) with IC50 = 9.7 µM.
Taken together all performed biological test, the best multifunctional molecule was compound
10, which well showed a wide range as UV-filter, antioxidant, antifungal and antiproliferative agent.
Compound 11 also displayed good UV-filter, antioxidant and antifungal activities, although was
not endowed of any antiproliferative activity. Thus, compound 10 is a potential candidate in the
development of drugs in case of cutaneous neoplastic diseases, in particular melanoma. Therefore,
this series of benzimidazole derivatives could be useful for developing novel multifunctional molecules
for the treatment of multifactorial diseases.
Supplementary Materials: The following are available online. Figure S1: Representative 13C-NMR spectrum of
1
newly synthesized compound
8
, Figure S2: Representative H-NMR spectrum of newly synthesized compound
8
, 7, Figure S4: Representative
Figure S3: Representative 13C-NMR spectrum of newly synthesized compound
1
H-NMR spectrum of newly synthesized compound 7,Table S1: The three classes of benzimidazole derivatives,
15 in solution, Table S3: Percent growth inhibition of
g/mL. Each value is the mean of three measurements,
Table S2: UV filtering activity of benzimidazole derivatives
dermatophytes treated with benzimidazole derivatives at 100
7–
µ
Table S4: Cytotoxicity and antiviral activity of compounds in human embryonic lung (HEL) cell cultures, Table S5:
Cytotoxicity and antiviral activity of compounds in HeLa cell cultures.
Author Contributions: Conceptualization, A.B., S.V. and S.M.; formal analysis, E.N.D., E.D., E.S., S.S., S.L.;
investigation, A.B. and S.M.; data curation, A.B., J.B.; writing—original draft preparation, A.B., E.N.D.;
writing—review and editing, A.B.; supervision, A.B., S.M.; project administration, S.M.; funding acquisition, A.B.,
S.M., S.V. All authors have read and agreed to the published version of the manuscript.

Molecules 2020, 25, 4324
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Funding: This research was funded by University of Ferrara (Grant FAR 2019 to AB), Ambrosialab srl (Ferrara,
Italy, Grant 2019) and by the Italian Ministero dell’Istruzione, Universit e della Ricerca (PRIN 2017, Prot. No.
010E84AA4_002).
à
2
Acknowledgments: The scholarship to E.S. was kindly supported by APTUIT (Verona, Italy).
Conflicts of Interest: The authors declare no conflict of interest.
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