Indole derivatives as multifunctional drugs: Synthesis and evaluation of antioxidant, photoprotective and antiproliferative activity of indole hydrazones

Article abstract of DOI:10.1016/j.bioorg.2019.02.007

Two series of indole derivatives 4–17, 20–22 were easily prepared and assayed for their radical-scavenging ability. Arylidene-1H-indole-2-carbohydrazones showed different extent antioxidant activity in DPPH, FRAP and ORAC assays. Good antioxidant activity is related to the number and position of hydroxyl groups on the arylidene moiety as well as to the presence of methoxy or 4-(diethylamino) group. On the contrary low antioxidant activity is showed by the isomeric 1H-indol-2-yl(methylene)-benzohydrazides. Furthermore, hydrazones 4–17 showed photoprotective capacities with satisfactory in vitro SPF as compared to the commercial PBSA sunscreen filter. The indole 16 and 17, showing the best antioxidant and photoprotective profile, were included in different formulation and their topical release was evaluated. Varying the formulation composition, it was possible to optimize skin adsorption and solubility of the active indole in the formulation. The antiproliferative effect of the hydrazones 4–17 was tested on human erythroleukemia K562 and melanoma Colo-38 cells. Hydrazones 11, 16 and 17 showed growth inhibition at sub micromolar concentrations on both cell lines. These results indicate indole hydrazones as potential multifunctional molecules especially in the treatment of neoplastic diseases being the good antioxidant properties of 16 and 17 correlated to their high antiproliferative activity.

Full text of DOI:10.1016/j.bioorg.2019.02.007

Accepted Manuscript
Indole derivatives as multifunctional drugs: Synthesis and evaluation of anti-
oxidant, photoprotective and antiproliferative activity of indole hydrazones
Monica Demurtas, Anna Baldisserotto, Ilaria Lampronti, Davide Moi,
Gianfranco Balboni, Salvatore Pacifico, Silvia Vertuani, Stefano Manfredini,
Valentina Onnis
PII:
S0045-2068(18)31395-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.007
YBIOO 2782
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 December 2018
28 January 2019
3 February 2019
Please cite this article as: M. Demurtas, A. Baldisserotto, I. Lampronti, D. Moi, G. Balboni, S. Pacifico, S. Vertuani,
S. Manfredini, V. Onnis, Indole derivatives as multifunctional drugs: Synthesis and evaluation of antioxidant,
photoprotective and antiproliferative activity of indole hydrazones, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.02.007
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Indole derivatives as multifunctional drugs: synthesis and evaluation of antioxidant,
photoprotective and antiproliferative activity of indole hydrazones.
Monica Demurtas,a,° Anna Baldisserotto,b,° Ilaria Lampronti,b Davide Moi, Gianfranco Balboni, Salvatore
a
a
c
b
b
a*
Pacifico, Silvia Vertuani, Stefano Manfredini, and Valentina Onnis
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.
c
Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17-19,
Ferrara I-44121, Italy.
Abstract
Two series of indole derivatives 4-17, 20-22 were easily prepared and assayed for their radical-scavenging
ability. Arylidene-1H-indole-2-carbohydrazones showed different extent antioxidant activity in DPPH, FRAP
and ORAC assays. Good antioxidant activity is related to the number and position of hydroxyl groups on the
arylidene moiety as well as to the presence of methoxy or 4-(diethylamino) group. On the contrary low
antioxidant activity is showed by the isomeric 1H-indol-2-yl(methylene)-benzohydrazides. Furthermore,
hydrazones 4-17 showed photoprotective capacities with satisfactory in vitro SPF as compared to the
commercial PBSA sunscreen filter. The indole 16 and 17, showing the best antioxidant and photoprotective
profile, were included in different formulation and their topical release was evaluated. Varying the
formulation composition, it was possible to optimize skin adsorption and solubility of the active indole in the
formulation. The antiproliferative effect of the hydrazones 4-17 was tested on human erythroleukemia K562
and melanoma Colo-38 cells. Hydrazones 11, 16 and 17 showed growth inhibition at sub micromolar
concentrations on both cell lines. These results indicate indole hydrazones as potential multifunctional
molecules especially in the treatment of neoplastic diseases being the good antioxidant properties of 16 and
17 correlated to their high antiproliferative activity.
Keywords
Indole hydrazones, Polyhydroxylated compounds, Antioxidant activity, Photoprotective agents,
Antiproliferative activity.
*Corresponding author. E-mail addresses: alessio.nocentini@unifi.it (A. Nocentini); vonnis@unica.it (V.
Onnis).
°These authors equally contribute to the present paper

1. Introduction
In recent years, a new concept of drug design has emerged that has revolutionized pharmacology: the multi-
target approach. A multi-target active ingredient can be designed to act simultaneously on multiple targets.
There are numerous advantages that can offer: not only from a pharmacological point of view, characterized
by a better efficacy and minimal adverse effects [1], but there is also a reduction in costs and development
times before reaching the market [2]. In this context, attention has been focused on the research of
compounds with multifunctional activities, that are equipped with both antioxidant and UV-filtering
capabilities, to counteract the oxidative damage caused by free radicals and, at the same time, protect during
exposure to UV radiation. The latter are the main cause of reactive oxygen species (ROS) and reactive
nitrogen species (RNS) formation and are closely related to the skin photo-aging process [3], to skin
inflammation and skin diseases, such as erythema and hyperpigmentation, and from the process of
carcinogenesis, including the onset of melanoma [4]. Indole ring is one of the most widespread naturally
occurring compounds as component of biologically active products derived from plants, animals and marine
organisms. Due to its broad range of biological activities, it is a privileged structure in medicinal chemistry. In
recent years, indole has emerged as a versatile molecular skeleton for the development of novel anticancer
agents [5-7], in particular tubulin inhibitors [8]. Noteworthy are also the central pharmacological activities of
indole derivatives that act as anticonvulsants, antidepressants and hypnotic-sedatives [9]. Some indole
alkaloids exert considerable pharmacological activity: δ-yohimbine and reserpine are examples of drugs
active on the circulatory system, while biological studies conducted on indole-3-carbinol, present in relevant
concentrations in vegetables such as broccoli, cabbage and cauliflowers, have revealed interesting data on
the anti-carcinogenic, antioxidant and anti-atherogenic effects [10]. The indole ring is present in some
clinically used drugs, such us triptans, NSAIDs and -antagonist [11]. Several indole derivatives have been
reported for their antioxidant activity [12-17]. Among naturally occurring indoles tryptophan, an essential
amino acid involved in numerous biological processes, is the biological parent of serotonin which is central
neurotransmitter, and cardiovascular and gastrointestinal signaling molecule. Together with melatonin,
serotonin is a powerful scavenger of free radicals and hormone regulator of circadian rhythms in organisms
[
18]. The indole ring in melatonin has been indicated as the center responsible for antioxidant properties,
due to its high resonance stability and low activation energy barrier in direct reactions against free radicals
19]. The high level of free radicals can cause damage to biomolecules and oxidative stress DNA damages
[
have been recognized a leading cause of various types of cancers [20]. For some polyphenols, an anti-
melanoma activity is known [21], a malignant and aggressive tumor form associated with intense exposure
to UV radiation.
A number of indole-based compounds have been reported for their potential anticancer activities [22].
Hydrazono-indole derivatives have been reported to induce apoptosis [23], and to interact with tubulin [24-
27].

We have previously reported on radical-scavenging activity and antiproliferative activity of
benzofuranhydrazones [28]. So, prompted from the above described findings we here report a small library
of indole-hydrazone compounds, designed, synthesized and tested, as well as their release from a series of
topical formulations.
The aim was to identify new molecules with dualistic activity, in which the strong antioxidant properties were
possibly associated with other pharmacological abilities, in particular photoprotection and antiproliferative
activity. In this context, the functional dualism sought would allow both to neutralize the oxidative damage
promoted by free radicals, generated by UVA radiation, and to protect against UVB, responsible for direct
damage to DNA and the onset of cancer in the skin [29].
2. Results and Discussion
2.1. Chemistry
The target hydrazones 4-17 (Table 1) were synthesized as shown in Scheme 1. 1H-Indole-2-carboxylic acid (1)
was converted into ethyl ester (2) [30]. The ethanolic solution of intermediate 2 and hydrazine hydrate was
refluxed to achieve 1H-Indole-2-carbohydrazide (3) [31]. Hydrazones 4-17 were obtained in good to excellent
yield by coupling the hydrazide 3 with the appropriate hydroxyarylaldehydes in ethanol. The isomeric indole
benzohydrazones 20-22 were obtained by coupling indole-2-carboxaldehyde 18 with the appropriate
hydroxybenzohydrazide 19. All the newly synthesized compounds were in agreement with expected
analytical data.
Scheme 1. Reagents and conditions: (i) Ethanol, H
2
SO
4
, reflux, 6 h; (ii) NH
2
NH
2
∙H O, EtOH, reflux 3 h, yields
2
64-90%; (iii) ArCHO, EtOH, reflux, 18 h; iv) EtOH, reflux, 18 h, yields 70-75%.

The IR and NMR spectral data are consistent with the assigned structure. According to the literature, the
presence of a singlet downfield resonating (8.30-9.49 ppm) CH=N signal, exclusively accounts for formation
of E-isomers [32].
2.2 Antioxidant activity
The evaluation of the antioxidant properties of the hydrazones 4-17 and 20-22 was performed 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 Tables 1, 2 and are expressed as
µmolTE/g for DPPH, FRAP, and ORAC tests. For the best interpretation of the results of the DPPH, for each
compound the concentration was sought to inhibit 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.
Table 1. Antioxidant activity of the indole derivatives 4-17.
DPPHa
FRAPa
ORACa
Compd. Ar
(µmolTE/g)
(µmolTE/g)
(µmolTE/g)
4
8.71± 1.85
34.56 ± 1.2
-
5
6
59.21 ± 1.0
960.94 ± 7.1
21031.02 ± 31.03
233.16 ± 5.7
1559.13 ± 11.5 35124.02 ± 64.55
4378.91 ± 14.7 21700.40 ± 28.73
7
8
9958.30 ± 13.6
2004.80 ± 6.0
873.06 ± 1.9
13014.11 ± 25.10
9
12846.01 ± 16.8 10655.78 ± 25. 1 16621.77 ± 34.41

10
4187.20 ± 7.4
4137.42 ± 8.8
4505.29 ± 49.27
1
1
2
206.65 ± 3.7
221.9 ± 2.3
2667.35 ± 6.5
43.04 ± 2.1
16838.33± 41.07
-
1
1
3
4
38.66 ± 0.1
115.78 ± 2.2
-
1
<< 19.1b
97.04 ± 1.9
25.89 ± 1.0
-
-
1
5
6
14.15 ± 0.12
1
2436.60 ± 15.29 3760.80 ± 8.07 22761.19 ± 18.48
17
1783.54 ± 13.1
97.4 ± 1.1
9398.69 ± 14.92
a
b
Each value was obtained from three experiments (mean ± SE); LOQ limit of quantification; - not tested.
Table 2. Antioxidant activity of the indole derivatives 20-22.
DPPHa
FRAPa
ORACa
Compd. Ar
(µmolTE/g) (µmolTE/g)
(µmolTE/g)
19
37.95 ± 9.23 243.38 ± 3.85
-

2
0
1
28.58 ± 0.7
257.61 ± 4.3
-
-
2
125.0 ± 3.26 209.37 ± 8.47
a
b
Each value was obtained from three experiments (mean ± SE); LOQ limit of quantification; - not tested.
Hydrazones 4-17 DPPH result analysis indicates that high antioxidant capacity is correlated to high number
of hydroxyl groups present on the arylidene ring. Indole derivatives 4-6 bearing a single hydroxyl group on
the arylidene ring showed weak activity. The introduction of a second (compounds 7 and 8) or a third hydroxy
group (compounds 9 and 10) leads to the best compounds of the series. Nevertheless, the hydroxyl group
position also affects antioxidant activity, in fact indole 8 is about 5 times more active than analog 7 and the
2,3,4-trihydroxyarylidene derivative 9 is about 3 times more active than the 2,4,6-trihydroxyarylidene analog
10. The replacement of one hydroxyl group with an alkoxy group (compounds 11-13) or halogen atoms
(
compounds 14-15) led reduction in activity. On the contrary the introduction of a 4-diethylamino group
indole 16) or napthyl ring (indole 17) produced increase in activity as compared to compounds 4, 8 and 12.
(
All hydrazones 20-22 showed weak activity demonstrating the importance of atoms double bond position on
the linker between indole and hydroxylaryl rings.
On FRAP analysis, the most powerful derivatives were 5, 6, 7, 8, 9, 10, 11 and 16. According to the results
obtained from the DPPH test, in general activity increases with an increase in the number of phenolic
hydroxyl groups on the aromatic ring and the reducing capacity is again conditioned by the position of
hydroxyl groups. The moving of 2-hydroxy group of indole 4 into 3- (indole 5) or 4-position (indole 6) led to a
progressive activity enhancement. The introduction on compound 5 of a 4-methoxy group (indole 11)
produced a further activity increase, while the analogs 12 and 13 showed weak activity. Confirming DPPH
results, the replacement of 4-hydroxy group of compound 8 with a 4-diethylamino group (indole 16)
produced increase in activity.
The compounds that from DPPH to FRAP tests showed the most interesting profile, were further investigated
in terms of antioxidant capacity by performing the ORAC test, in order outline a complete activity profile. The
4-hydroxyarylidene derivative 6 demonstrated the best antioxidant capacity towards the peroxyl radicals
(ROO˙). Shifting the 4-hydroxy group into 3-position (indole 5) led reduction in activity. The introduction of
second hydroxy group in 2-position afforded the poor active indole 8, while the replacement of the 4-hydroxy
with a 4-diethylamino group (indole 16) restore the antioxidant properties. Similarly, the introduction on
compound 5 of 4-methoxy group (indole 11) caused reduction in activity. The presence of a
trihydroxyarylidene moiety (compounds 9 and 10) drop the activity as compared to a dihydroxyarylidene
(compounds 7 and 8), however the 2,3,4-trihydroxy derivative 9 showed better activity than the 2,4,6-
trihydroxy analog 10.

2.3 Evaluation of filtering parameters
On the indolehydrazones 5-11, 16 and 17 showing antioxidant properties, in vitro tests were conducted to
determine the parameters fundamental for the relative evaluation of the filtering power: critical wavelength
c
(λ ) and SPF (Sun Protection Factor). The Solar Protection Effectiveness Evaluation System specifies a SPF
primarily representing a measure of UVB protection [33] is related to the UV absorption of substances. The
trend of the UV spectra of the compounds 5-11, 16 and 17 between 290 and 400 nm was then recorded (data
not shown) and compared with that of the commercial Phenyl Benzimidazole Sulfonic Acid filter (PBSA) used
as a reference UVB filter (λmax 302 nm). All indole 5-11, 16 and 17 have an absorption spectrum in which the
λmax is shifted towards longer wavelengths, between 330 and 350 nm, well above the maximum absorption
of PBSA, and the absorption curves of the compounds analyzed are more extensive than the reference one.
Hydrazones 17 and 16 showed the more evident batochromic displacement. Indole 16 bears a 2-hydroxy-4-
(diethylamino)arylidene moiety that has a positive effect on absorption. While indole 17 presents a
napthylarylidene moiety that produces increase in the degree of conjugation accounting for the shift of the
maximum absorbance peak towards the visible region.
The preliminary study on the filtering properties to determine the UV-protective potential of hydrazones 5-
11, 16 and 17 was performed in vitro. Whereas a new method in ISO 24443: 2012 was established and
standardized for the in vitro evaluation of UVA protection, there is no universally approved in vitro method
for the assessment of protection in the UVB range. The in vitro evaluation of the SPF parameter was initially
proposed by Diffey-Robson in 1989 [34] and the method is still widely applied.
After recording the UV absorption spectra of the hydrazones 5-11, 16 and 17 and the reference PBSA, the
values relating to the parameters were extrapolated to determine their filtering power (Table 3).
Table 3. UV-filtering activity of hydrazones 5-11, 16, 17 in solution.
Compound SPF
UVA/UVB UVAPF0 c(nm)
5
6
7
8
9
1
1
1
10,42 0,43
1,77
2,56
4,60
4,97
4,32
6,19
2,87
9,55
346
352
363
363
362
367
356
392
10,05 0,81
5,13 1,96
5,78 1,93
5,01 2,09
4,34 2,66
5,75 1,34
2,21 1,39
0
1
6

1
7
4,81 1,71
3,4 0,29
8,55
1,03
385
322
PBSA
All indole derivatives 5-11, 16 and 17 showed better filtering characteristics than the reference compound
PBSA. A wide-spectrum solar product, able to protect the skin at the same time from UVA and UVB rays,
should have a value of λc greater than 370 nm. Based on this criterion, the values of the indoles 16 and 17
emerge. Only the hydrazones 5 and 6 showed an appreciable SPF value. The UVA/UVB absorbance ratio is
another parameter that gives a good idea of which UV region is best blocked by each substance [35]. As
defined by the EU recommendation on the effectiveness of sunscreen products (2006/247 / EC), this ratio
should be worth at least 1/3. This requirement is satisfied by almost all the indolehydrazones 5-11, 16 and
1
7, most of which characterized by values higher than 1. As close to 1 is the ratio, the more similar it will be
the absorption of the molecule in the UVA region to that in the region UVB. This means that indoles 7-10, 11,
6, and 17 mainly absorb in the UVA region. Finally, the value of UVAPF0, determined in relation to the
1
guidelines provided by ISO-24443, showed that all the tested indoles possess a better UVA Protection Factor
than the reference commercial filter. In particular, the UVAPF0 values of the 17 and 16 samples emerge.
2.4 In vitro release studies
The indolehydrazones 16 and 17 due to their best dualistic activity profile (antioxidant and at the same time
UV-filtering properties) were selected in to in vitro evaluate their behavior as potential active ingredients in
topical formulations. Depending on the function of the active ingredient, it is possible to discriminate two
types of formulations: to act as a sunscreen, the active ingredient must remain in the most superficial part of
the skin, while to act as an antioxidant, it is desirable that this also penetrates into the underlying layers of
the dermis.
For this purpose, the formulation devised for Oxisol, a molecule with dualistic activity [36], was taken into
consideration and optimized for indoles 16 and 17. Based on the structure of both the compounds, two
topical formulations with different degrees of polarity were prepared: Formulation A) to obtain high release
of the active substance performing activity after absorption into the skin and Formulation B) to obtain limited
release, as needed in the case of solar filters, through a better solubilization of the active ingredient in the
formulation. The effective release of the active ingredients from each of the two topical formulations
optimized for the compounds 16 and 17 was determined through the Franz Cells system. This last represent
one of the most common system for the study of in vitro release and of the permeation of active ingredients
from semi-solid forms [34]. This system consists of two vertical chambers, defined as donors and recipients,
separated by a synthetic or biological membrane which acts as a support and a means of separation between
the formulation and the receiving medium [37]. The receptor phase was sampled within 6 h to measure

indoles 16, 17 and Oxisol amount in the receiving chamber, the resulting release curves were reported in
Figure 1.
Release curves Oxisol
30
25
20
15
10
5
0
0
30
60
120
180
240
300
360
Time (minutes)
Formulation A
Formulation B
Release curves Hydrazone 16
30
25
20
15
10
5
0
0
30
60
120
180
240
300
360
Time (minutes)
Formulation A
Formulation B
Release curves Hydrazone 17
30
25
20
15
10
5
0
0
30
60
120
180
240
300
360
Time (minutes)
Formulation A
Formulation B
Fig. 1. Permeation profiles of Oxisol, and hydrazones 16, 17. Line blue correspond to formulation A (optimized
for skin adsorption), line orange correspond to Formulation B (optimized to best solubilize the active in
formula).

The tested indoles and the reference Oxisol are characterized by a difference in the 0.8% and 2% range
between the release curves of the two different formulations. The Oxisol release curves from formulation A
and B maintain the same trend: the delta tends to decrease after 240 minutes. Differently, indole 16 and 17
did not show an appreciable difference between the two release curves in the initial 60 and 30 minutes
respectively; in any case, after the first hour, the release increases for the formulation A and the difference
between the two curves settles around 2%. In conclusion, the obtained data indicate that it is possible to
modify the release of the indoles 16 and 17 from the formulation designed ad hoc by simply varying the
percentages of its components, thus influencing the polarity of the formulation, depending on the activity of
the active ingredient and end-use of the preparation.
2.5 Antiproliferative activity on Human melanoma Colo38 and erythroleukemia K562 cell lines
The hydrazones 4-17 were tested on human melanoma Colo38 cell line as well as on human erythroleukemic
K562 cells to determine their antiproliferative activity. All the compounds have been tested on both cell lines
to evaluate the relative IC50 values expressed in M concentration (Table 4). Hydrazone 11 exhibited greatest
activity of the series, displaying an antiproliferative effect on both the Colo38 and K562 cells at nanomolar
concentrations (IC50 values 0.59±0.03 and 0.067±0,00.1 M respectively). The removal of the 4-methoxy
group to give the hydrazone 5 produced extensive reduction in activity. The shift of the 3-hydroxy group into
4-position (hydrazone 6) restore the antiproliferative activity against Colo38 cell line, while the activity
against K562 cells is not affected. Good activity is showed by the di-hydroxy substituted hydrazones 7 and 8.
On the contrary the introduction of third hydroxyl group led to reduction in activity especially for the 2,4,6-
trihydroxy analog 10 and against K562 cells. The replacement of the 4-hydroxy group of hydrazone 8 with 4-
(dimethylamino) group (hydrazone 17) led to increase in activity against K562 cells and at same time the
submicromolar antiproliferative activity against Colo38 cells is maintained. The hydrazone 16 showed an
inhibitory activity profile very similar to compound 17 on both the cell lines (IC50 values between 0.54 and
0.83 μM).
Table 4. Effects of the indole derivatives 4-17 on the proliferation of Colo38 and K562 cells.
Compd. IC (M)
5
0
Colo38
K562
>100
4
5
6
7
>100
10.02±0.91 47.80±7.80
0.73±0.05 46.11±4.30
2.96±0.13 8.60±060

8
9
1
1
1
1
1
1
1
1
0.57±0.05 4.31±0.40
6.13±0.26 5.39±0.01
8.94±0.13 54.52 ±0.11
0.59±0.03 0.067±0.001
0
1
2
3
4
5
6
7
>100
>100
>100
>100
>100
>100
>100
>100
0.54±0.15 0.63±0.05
0.83±0.09 0.63±0.04
3. Conclusion
Continuing our research aiming to develop possible multifunctional drugs, we selected the indole scaffold in
view of its interesting known properties. Indolehydrazones 4-17 showed different extent of radical-
scavenging ability towards the nitrogen radical by the DPPH test, to reduce ferric ion by the FRAP test and to
inhibit the oxidative degradation caused by peroxyl radicals using the ORAC test. The SAR data obtained by
the antioxidant screening of the benzofuran derivatives have shown good antioxidant activity and is related
to the number and position of hydroxyl groups on the arylidene moiety as well as to the presence of methoxy
or 4-(diethylamino) group. The indole derivatives 4-17 showed photoprotective capacities with satisfactory
in vitro SPF as compared to the commercial PBSA sunscreen filter. Compounds 16 and 17 confirmed their
relevant filtering activity when included in topical formulations. Skin adsorption and solubility of the active
indoles were optimized by varying the formulation composition indicating a possible use of such compounds
as sunscreen filters. Additionally, hydrazones 11, 16 and 17 showed growth inhibition at sub micromolar
concentrations on human erythroleukemia K562 and melanoma Colo-38 cells. These results indicate indole
hydrazones as potential multifunctional molecules mainly in the treatment of neoplastic diseases being the
good antioxidant properties of 16 and 17 correlated to their high antiproliferative activity.
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.
Ethyl 1H-Indole-2-carboxylate (2) was prepared as previously described [30] starting from 1H-Indole-2-
carboxylic acid. 1H-Indole-2-carbohydrazide (3) was prepared as previously described [31].
4.2. Chemistry
4.2.1. General procedure for the synthesis of hydrazones (4-17). A mixture of hydrazide 3 (1 mmol) and the
appropriate aldehyde (1 mmol) in EtOH (10 mL) was refluxed for 18h. After cooling the formed precipitate
was filtered off and purified by crystallization from the adequate solvent to give the hydrazone derivatives.
1
4
.2.1.1. (E)-N'-(2-hydroxybenzylidene)-1H-indole-2-carbohydrazide (4). Yield 64%. Mp >250 °C (EtOH). H
NMR (DMSO-d
6
):  6.92 (m, 2H, Ar), 7.05 (m, 1H, Ar), 7.21 (m, 1H, Ar), 7.28 (m, 2H, Ar), 7.45 (d, J = 8.0 Hz, 1H,
Ar), 7.56 (m, 1H, Ar), 7. 66 (d, J = 8.0 Hz, 1H, Ar), 8.63 (s, 1H, CH), 11.16 (s, 1H, OH), 11.79 (s, 1H, NH), 12.09
-
1
+
(
s, 1H, NH). IR (Nujol) 3294, 1654, 1617 cm . m/z 280 (M + H) . Anal. Calcd for C16
H
13
N
3
2
O : C, 68.81; H, 4.69;
N, 15.05. Found: C, 68.89; H, 4.70; N, 15.02. [39]
4
2
4
.2.1.2. (E)-N'-(3-hydroxybenzylidene)-1H-indole-2-carbohydrazide (5). Yield 84%. Mp >250 °C (EtOH) Lit. 278-
1
81 °C [39]. H NMR (DMSO-d
6
):  6.81 (m, 1H, Ar), 7.04 (m, 1H, Ar), 7.10 (d, J = 7.5 Hz, 1H, Ar), 7.18-7.27 (m,
H, Ar), 7.43 (d, J = 8.5 Hz, 1H, Ar), 7. 65 (d, J=8.0 Hz, 1H, Ar), 8.34 (s, 1H, CH), 9.58 (s, 1H, OH), 11.75 (s, 1H,
-
1
+
NH), 11.79 (s, 1H, NH). IR (Nujol) 3255, 1638, 1580 cm . m/z 280 (M + H) . Anal. Calcd for C16
H
13
N
3
2
O : C,
6
4
2
8
8.81; H, 4.69; N, 15.05. Found: C, 68.73; H, 4.68; N, 15.08.
.2.1.3. (E)-N'-(4-hydroxybenzylidene)-1H-indole-2-carbohydrazide (6). Yield 80%. Mp >250 °C (EtOH) Lit. 278-
1
79 °C [40]. H NMR (DMSO-d
6
):  6.82 (d, J = 8.0 Hz, 2H, Ar), 7.03 (m, 1H, Ar), 7.19 (m, 1H, Ar), 7.43 (d, J =
.0 Hz, 2H, Ar), 7. 56 (d, J = 8.0 Hz, 2H, Ar), 7.65 (d, J = 7.5 Hz, 1H, Ar), 8.33 (s, 1H, CH), 9.88 (s, 1H, OH), 11.64
-
1
+
(
s, 1H, NH), 11.71 (s, 1H, NH). IR (Nujol) 3223, 1608 cm . m/z 280 (M + H) . Anal. Calcd for C16
H
13
N
3
2
O : C,
68.81; H, 4.69; N, 15.05. Found: C, 68.74; H, 4.70; N, 15.07.

4
2
7
1
.2.1.4. (E)-N'-(2,5-dihydroxybenzylidene)-1H-indole-2-carbohydrazide (7). Yield 80%. Mp >250 °C (EtOH) Lit.
1
78-279 °C [40]. H NMR (DMSO-d
6
):  6.74 (m, 2H, Ar), 7.05 (m, 2H, Ar), 7.22 (m, 1H, Ar), 7.31 (s, 1H, Ar),
.46 (d, J = 8.0 Hz, 1H, Ar), 7.67 (d, J = 8.0 Hz, 1H, Ar), 8.57 (s, 1H, CH), 8.95 (s, 1H, OH), 10.26 (s, 1H, OH),
-
1
+
1.78 (s, 1H, NH), 11.99 (s, 1H, NH). IR (Nujol) 3507, 3345, 3266, 1662 cm . m/z 296 (M + H) . Anal. Calcd for
: C, 65.08; H, 4.44; N, 14.23. Found: C, 65.15; H, 4.43; N, 14.20.
C
16
H
13
N
3
O
3
4
2
7
1
.2.1.5. (E)-N'-(2,4-dihydroxybenzylidene)-1H-indole-2-carbohydrazide (8). Yield 86%. Mp >250 °C (EtOH) Lit.
1
80-282 °C [40]. H NMR (DMSO-d
6
):  6.35 (m, 2H, Ar), 7.06 (m, 1H, Ar), 7.21 (m, 1H, Ar), 7.33 (m, 2H, Ar),
.46 (d, J = 8.0 Hz, 1H, Ar), 7.66 (d, J = 8.0 Hz, 1H, Ar), 8.50 (s, 1H, CH), 9.92 (s, 1H, OH), 11.35 (s, 1H, OH),
-
1
+
1.76 (s, 1H, NH), 11.92 (s, 1H, NH). IR (Nujol) 3417, 1607, 1554 cm . m/z 296 (M + H) . Anal. Calcd for
: C, 65.08; H, 4.44; N, 14.23. Found: C, 65.00; H, 4.43; N, 14.28.
.2.1.6. (E)-N'-(2,3,4-trihydroxybenzylidene)-1H-indole-2-carbohydrazide (9). Yield 71%. Mp >250 °C (EtOH).
1H NMR (DMSO-d
):  6.40 (d, J = 8.0 Hz, 1H, Ar), 6.81 (d, J = 8.0 Hz, 1H, Ar), 7.06 (m, 1H, Ar), 7.23 (m, 2H, Ar),
.45 (d, J = 8.5 Hz, 1H, Ar), 7.67 (d, J = 8.0 Hz, 1H, Ar), 8.46 (s, 1H, OH), 8.50 (s, 1H, CH), 9.44 (s, 1H, OH), 11.43
s, 1H, OH), 11.80 (s, 1H, NH), 11.98 (s, 1H, NH). 13C NMR (DMSO-d
):  106.7, 110.8, 114.1, 115.5, 123.1,
24.1, 124.9, 126.9, 130.1, 132.9, 135.9, 140.0, 150.5, 151.8, 152.5, 160.3. IR (Nujol) 3412, 3324, 3250, 1641,
C
16
H
13
N
3
O
3
4
6
7
(
6
1
1
-
1
+
616 cm . m/z 312 (M + H) . Anal. Calcd for C16
H
13
N
3
4
O : C, 61.73; H, 4.21; N, 13.50. Found: C, 61.79; H, 4.23;
N, 13.47.
4.2.1.7. (E)-N'-(2,4,6-trihydroxybenzylidene)-1H-indole-2-carbohydrazide (10). Yield 77%. Mp >250 °C (EtOH).
1
Lit. 256-258 °C [40]. H NMR (DMSO-d
6
):  5.85 (s, 2H, Ar), 7.05 (m, 1H, Ar), 7.22 (m, 2H, Ar), 7.45 (d, J = 8.5
Hz, 1H, Ar), 7.66 (d, J = 8.0 Hz, 1H, Ar), 8.79 (s, 1H, CH), 9.79 (s, 2H, OH), 11.06 (s, 1H, OH), 11.74 (s, 1H, NH),
-
1
+
1
4
4
1.92 (s, 1H, NH). IR (Nujol) 3347, 1642, 1611 cm . m/z 312 (M + H) . Anal. Calcd for C16
H
13
N
3
4
O : C, 61.73; H,
.21; N, 13.50. Found: C, 61.68; H, 4.22; N, 13.54.
.2.1.8. (E)-N'-(3-hydroxy-4-methoxybenzylidene)-1H-indole-2-carbohydrazide (11). Yield 90%. Mp 158-160
1
°
C (EtOH). Lit. 150-152 °C [40]. H NMR (DMSO-d
6
):  3.81 (s, 3H, CH ), 6.98 (d, J = 8.0 Hz, 1H, Ar), 7.10 (m,
3
2H, Ar), 7.24 (m, 3H, Ar), 7.45 (d, J = 8.0 Hz, 1H, Ar), 7.66 (d, J =7 .5 Hz, 1H, Ar), 8.30 (s, 1H, CH), 9.31 (s, 1H,
-
1
+
OH), 11.71 (s, 1H, NH), 11.77 (s, 1H, NH). IR (Nujol) 3300, 1621, 1563 cm . m/z 310 (M + H) . Anal. Calcd for
: C, 66.01; H, 4.89; N, 13.58. Found: C, 66.07; H, 4.90; N, 13.54.
C
17
H
15
N
3
O
3
4.2.1.9. (E)-N'-(2-hydroxy-4-methoxybenzylidene)-1H-indole-2-carbohydrazide (12). Yield 74%. Mp > 250 °C
1
(
EtOH). H NMR (DMSO-d
6
):  3.77 (s, 3H, OCH ), 6.49-6.53 (m, 2H, Ar), 7.34-7.68 (m, 6H, Ar), 8.54 (s, 1H,
3
-
1
CH), 11.49 (s, 1H, OH), 11.76 (s, 1H, NH), 12.0 (s, 1H, NH). IR (Nujol) 3315, 3241, 1651, 1630, 1606 cm . m/z
+
3
10 (M + H) . Anal. Calcd for C17
H
15
N
3
3
O : C, 66.01; H, 4.89; N, 13.58. Found: C, 65.95; H, 4.91; N, 13.62.
4.2.1.10. (E)-N'-(2-hydroxy-3-ethoxybenzylidene)-1H-indole-2-carbohydrazide (13). Yield 87%. Mp 214-216 °C
1
(
EtOH). H NMR (DMSO-d
6
):  1.35 (t, J = 7.0 Hz, 3H, CH
3
), 4.06 (q, J = 7.0 Hz, 2H, CH ), 6.85 (t, J = 8.0 Hz, 1H,
2
Ar), 7.04 (d, J = 8.0 Hz, 1H, Ar), 7.16 (d, J = 8.0 Hz, 1H, Ar), 7.20-7.68 (m, 5H, Ar), 8.64 (s, 1H, CH), 10.85 (s, 1H,

-
1
+
OH), 11.81 (s, 1H, NH), 12.1 (s, 1H, NH). IR (Nujol) 3320, 1655, 1621, 1605 cm . m/z 324 (M + H) . Anal. Calcd
: C, 66.86; H, 5.30; N, 13.00. Found: C, 66.80; H, 5.32; N, 13.03.
.2.1.11. (E)-N'-(5-chloro-2-hydroxybenzylidene)-1H-indole-2-carbohydrazide (14). Yield 81%. Mp > 250 °C
for C18
H
17
N
3
O
3
4
1
(
EtOH). H NMR (DMSO-d
6
):  6.95 (d, J = 8.0 Hz, 1H, Ar), 7.07 (m, 1H, Ar), 7.23 (m, 1H, Ar), 7.33 (s, 1H, Ar),
7
1
.44 (m, 2H, Ar), 7.46 (d, J = 8.5 Hz, 1H, Ar), 7.68 (d, J = 8.0 Hz, 1H, Ar), 8.62 (s, 1H, CH), 11.18 (s, 1H, OH),
-
1
+
1.80 (s, 1H, NH), 12.18 (s, 1H, NH). IR (Nujol) 3342, 3325, 1666 cm . m/z 314 (M + H) . Anal. Calcd for
: C, 61.25; H, 3.86; N, 13.39. Found: C, 61.19; H, 3.87; N, 13.42.
C
16
H12ClN
3
O
3
4.2.1.11. (E)-N'-(5-bromo-2-hydroxybenzylidene)-1H-indole-2-carbohydrazide (15). Yield 81%. Mp > 250 °C
1
(
EtOH) Lit. 278-280 °C [41]. H NMR (DMSO-d
6
):  6.98 (d, J = 7.0 Hz, 1H, Ar), 7.07 (m, 1H, Ar), 7.23 (m, 1H,
Ar), 7.33 (s, 1H, Ar), 7.44 (m, 2H, Ar), 7.68 (d, J = 8.5 Hz, 1H, Ar), 7.81 (m, 1H, Ar), 8.61 (s, 1H, CH), 11.19 (s,
-
1
+
1
+
4
2
H, OH), 11.81 (s, 1H, NH), 12.18 (s, 1H, NH). IR (Nujol) 3312, 1668, 1605 cm . m/z 358 (M + H) , 360 (M + 2
+
H) . Anal. Calcd for C16
H
12BrN
3
O : C, 53.65; H, 3.38; N, 11.73. Found: C, 53.70; H, 3.37; N, 11.70.
3
.2.1.12. (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-1H-indole-2-carbohydrazide (16). Yield 80%. Mp
1
00-202 °C (EtOH). H NMR (DMSO-d
6
):  1.11 (t, J = 7.5 Hz, 6H, CH
3
), 3.35 (q, J = 7.5 Hz, 4H, CH ), 6.13 (s, 1H,
2
Ar), 6.28 (d, J = 8.0 Hz, 1H, Ar), 7.06 (m, 1H, Ar), 7.21 (m, 3H, Ar), 7.45 (d, J = 8.0 Hz, 1H, Ar), 7.66 (s, 1H, Ar),
8
1
1
1
4
>
.42 (s, 1H, CH), 11.35 (s, 1H, OH), 11.74 (s, 1H, NH), 11.83 (s, 1H, NH). 13C NMR (DMSO-d
00.6, 106.3, 106.8, 109.7, 110.0, 115.5, 123.1, 124.8, 126.8, 130.2, 133.2, 134.5, 139.9, 152.4, 153.3, 160.1,
6
):  15.6, 46.9,
-
1
+
62.7. IR (Nujol) 3295, 1635, 1592 cm . m/z 351 (M + H) . Anal. Calcd for C20
5.99. Found: C, 68.49; H, 3.32; N, 16.03.
H
22
N
4
2
O : C, 68.55; H, 6.33; N,
.2.1.13. (E)-N'-((2-hydroxynaphthalen-1-yl)methylene)-1H-indole-2-carbohydrazide (17). Yield 85%. Mp
1
250 °C (EtOH). H NMR (DMSO-d
6
):  7.08 (d, J = 7.5 Hz, 1H, Ar), 7.24 (d, J = 7.5 Hz, 1H, Ar), 7.42 (d, J = 7.5
Hz, 1H, Ar), 7.48-7.60 (m, 3H, Ar), 7.64 (d, J = 8.0 Hz, 1H, Ar), 7.71 (d, J = 8.0 Hz, 1H, Ar), 7.89-8.27 (m, 3H, Ar),
9
.46 (s, 1H, CH), 11.86 (s, 1H, OH), 12.22 (s, 1H, NH), 12.69 (s, 1H, NH). IR (Nujol) 3322, 1673, 1620, 1571 cm-
1.
m/z 330 (M + H) . Anal. Calcd for C20
+
H
N
15
3
2
O : C, 72.94; H, 4.59; N, 12.76. Found: C, 72.99; H, 4.61; N, 12.71.
4.2.2. General procedure for the synthesis of hydrazones (19-21). A mixture of indole-2-carboxaldehyde 18
(
0.14g, 1 mmol) and the appropriate hydroxybenzohydrazide (1 mmol) in EtOH (10 mL) was refluxed for 18h.
After cooling the formed precipitate was filtered off and purified by crystallization from the adequate solvent
to give the hydrazone derivatives.
1
4
.2.2.1. (E)-N'-((1H-indol-2-yl)methylene)-3-hydroxybenzohydrazide (19). Yield 70%. Mp >250 °C (EtOH). H
NMR (DMSO-d
6
):  6.82 (s, 1H, Ar), 7.00 (d, J = 8.0 Hz, 2H, Ar), 7.15 (m, 2H, Ar), 7.32 (m, 2H, Ar), 7.44 (d, J =
7.5 Hz, 1H, Ar), 7.55 (d, J = 8.0 Hz, 1H, Ar), 8.47 (s, 1H, CH), 9.75 (s, 1H, OH), 11.56 (s, 1H, NH), 11.74 (s, 1H,
-
1
+
NH). IR (Nujol) 3331, 1655, 1596 cm . m/z 280 (M + H) . Anal. Calcd for C16
Found: C, 68.89; H, 4.68; N, 15.01.
H
13
N
3
O
2
: C, 68.81; H, 4.69; N, 15.05.
1
4
.2.2.2. (E)-N'-((1H-indol-2-yl)methylene)-4-hydroxybenzohydrazide (20). Yield 75%. Mp >250 °C (EtOH). H
NMR (DMSO-d ):  6.77 (s, 1H, Ar), 6.84 (d, J = 8.0 Hz, 2H, Ar), 6.96-7.53 (m, 4H, Ar), 7.80 (d, J = 8.0 Hz, 2H,
6

Ar), 8.43 (s, 1H, CH), 10. 06 (s, 1H, OH), 11.49 (s, 1H, NH), 11.56 (s, 1H, NH). IR (Nujol) 3282, 1645, 1607, 1581
-
1
+
cm . m/z 280 (M + H) . Anal. Calcd for C16
H
13
N
3
2
O : C, 68.81; H, 4.69; N, 15.05. Found: C, 68.76; H, 4.70; N,
15.09.
4.2.2.3. (E)-N'-((1H-indol-2-yl)methylene)-2,4-dihydroxybenzohydrazide (21). Yield 72%. Mp >235-237 °C
1
(
EtOH). H NMR (DMSO-d
6
):  7.04 (m, 2H, Ar), 7.22 (m, 2H, Ar), 7.32 (m, 2H, Ar), 7.44 (d, J = 8.5 Hz, 1H, Ar),
-
1
7
.62 (d, J = 8.0 Hz, 1H, Ar), 8.68 (s, 1H, CH), 10.12 (s, 2H, OH), 11.69 (s, 2H, NH). IR (Nujol) 3420, 1606 cm .
+
m/z 296 (M + H) . Anal. Calcd for C16
H
13
N
3
3
O : C, 65.08; H, 4.44; N, 14.23. Found: C, 65.02; H, 4.45; N, 14.28.
4.3. Biological assays
4.3.1. DPPH assay
•
The DPPH radical scavenging activity of the synthesized derivatives was measured according to the method
of Wang et al [42] modified as previously reported [28]. 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 indoles 4-17 and 20-22 was estimated according to the method described
previously [43]. The absorbance of the reaction mixture was read at 593 nm. The values are expressed as
µmol TE/g compound.
4.3.3. Oxygen Radical Absorbance Capacity (ORAC)
Based on a protocol previously reported and modified in our previous work [44], the peroxyl radical
scavenging efficacy was measured. Sample solutions were prepared using phosphate buffer solution (pH 7.4).
A series of Trolox solutions (40-240 μM) was prepared by diluting with phosphate buffer solution(pH 7.4) a
solution of Trolox (2 mM). Sample solution, Trolox dilution (25 μL), or phosphate buffer solution (pH 7.4) used
as blank was placed in wells of a 96-well black microplate (VWR). The fluorescence measurements 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. [45] 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 [46].
.000034 (± 0.0000033) M solutions of the indole hydrazones 5-11, 16 and 17 were prepared in MeOH and
0
each of the absorption spectra was recorded.
To calculate the SPF value in vitro, the absorbance values obtained were transformed into transmittance
values, using the equation below:
A(λ) = - Log[T(λ)]
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

For each molecule studied, two standard oil / water emulsions (Formulation A and Formulation B) were
prepared. Once the ingredients of the aqueous phase and of the oil phase have been added, each phase has
been heated up to a temperature between 60 and 70 °C. At this point, the oil phase was added to the aqueous
phase, under mechanical stirring. As a third step the test compound was added during the subsequent cooling
of the emulsion, maintained in continuous agitation, to preserve it from possible thermal degradation.
Finally, the emulsion was cooled up to 25° C and kept in the fridge 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 indole 16 or indole 17, Xanthan
Gum, Caprylic/Capric Triglyceride, Cetearyl Alcohol, Dicaprylyl Carbonate.
Formulation B: Aqua, Cetearyl Alcohol, Tribehenin PEG-20 Esters, Butyrospermum Parkii, Olea Europaea Oil
Unsaponifiables, Indole 16 or indole 17, Xanthan Gum, Caprylic/Capric Triglyceride.
4.3.6. Franz cells apparatus
The synthetic membrane employed in Franz diffusion cells experiments in this study was a Cuprophan
Regenerated cellulose from Medicell (London, UK), membrane thickness (11.5µm +/- 0,5µm), molecular-
2
weight cut off range (10,000 Da). The effective diffusion area of the Franz cells was 0.6 cm and receptor
volume was 4.3 mL.
The membranes were conditioned with the receiving solution (DMSO), before being mounted on the
diffusion cells. The system was set up by placing the membrane between the compartment containing the
receiving medium and the donor compartment. The formulation (0.5 grams) was placed in the donor, in
contact with the membrane; the donor compartment was sealed with Parafilm® to avoid any loss of the
emulsion components. Franz cells are immersed in a thermostat bath which guarantees a membrane surface
temperature of 37 ± 1 ° C, keeping the receiving solvent continuously magnetic stirred. Using a glass syringe,
500 μl of sample were taken and replaced by 500 μl of fresh receiving medium, after 30 minutes, 1 hour and
then every hour thereafter up to 6 hours and the absorbance of samples at λmax was measured using a UV-
VIS spectrophotometer. For each compound a calibration line and a release curve have been constructed.
4.3.7. Growth inhibition assays
Cell growth inhibition assays were carried out using two human cancer cell lines, melanoma Colo38 and
erythroleukemia K562 [47, 48, 28]. Cell lines were maintained in RPMI 1640, supplemented with 10% fetal
bovine serum (FBS), penicillin (100 Units mL-1), streptomycin (100 μg mL-1) and glutamine (2 mM) (complete
medium); the pH of the medium was 7.2 and the incubation was performed at 37°C in a 5% CO2 atmosphere.
Indole derivatives were dissolved in MeOH/DMSO 10% to obtain 20 mM stock solutions and diluted before
cell treatment in MeOH 100%. All the hydrazones 4-17 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) and Ambrosialab srl (Ferrara, Italy)
Conflict of interest
The authors declare no conflict of interest.
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Highlights
*
*
*
Indole hydrazones were designed and easy synthesized.
Synthesized compounds were assayed in vitro as antioxidants and photoprotective agents.
Synthesized compounds were assayed in vitro for their antiproliferative activity on human melanoma and
leukemia cells.
The hydrazones 16 and 17 showed antioxidant activity in DPPH, FRAP and ORAC test as well as
photoprotective effect.
The hydrazones 11, 16 and 17 inhibited the growth of Colo-38 and K-562 cells with sub-micromolar IC50
values.
*
*