Diclofenac-derived hybrids for treatment of actinic keratosis and squamous cell carcinoma

Article abstract of DOI:10.3390/molecules24091793

In this work, hybrid compounds 1-4 obtained by conjugation of the non-steroidal anti-inflammatory drug diclofenac, with natural molecules endowed with antioxidant and antiproliferative activity were prepared. The antiproliferative activity of these hybrids was evaluated on immortalized human keratinocyte (HaCaT) cells stimulated with epidermal growth factor (EGF), an actinic keratosis (AK) model, and on human squamous cell carcinoma (SCC) cells (A431). Hybrid 1 presented the best activity in both cell models. Self-assembling surfactant nanomicelles have been chosen as the carrier to drive the hybrid 1 into the skin; the in vitro permeation through and penetration into pig ear skin have been evaluated. Among the nanostructured formulations tested, Nano3Hybrid20 showed a higher tendency of the hybrid 1 to be retained in the skin rather than permeating it, with a desirable topical and non-systemic action. On these bases, hybrid 1 may represent an attractive lead scaffold for the development of new treatments for AK and SCC.

Full text of DOI:10.3390/molecules24091793

molecules
Article
Diclofenac-Derived Hybrids for Treatment of Actinic
Keratosis and Squamous Cell Carcinoma
Silvia Tampucci 1
,2,†
, Sara Carpi
1,2,†
, Maria Digiacomo 1,2,* , Beatrice Polini ,
1
3
1
, Marco Macchia 1,2, Paola Nieri 1,2, Clementina Manera 1,2
Stefano Fogli , Susi Burgalassi
and Daniela Monti 1
1
Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy; silvia.tampucci@unipi.it (S.T.);
sara.carpi@unipi.it (S.C.); b.polini@studenti.unipi.it (B.P.); susi.burgalassi@unipi.it (S.B.);
marco.macchia@unipi.it (M.M.); Paola.nieri@unipi.it (P.N.); clementina.manera@unipi.it (C.M.);
daniela.monti@unipi.it (D.M.)
2
3
Interdepartmental Research Center “Nutraceuticals and Food for Health” (NutraFood), University of Pisa,
5
6126 Pisa, Italy
Department of Clinical and Experimental Medicine, University of Pisa, Via Savi 10, 56126 Pisa, Italy;
stefano.fogli@unipi.it
*
†
Correspondence: maria.digiacomo@unipi.it; Tel.: +39-0502219594
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 30 March 2019; Accepted: 5 May 2019; Published: 9 May 2019
Abstract: In this work, hybrid compounds 1–4 obtained by conjugation of the non-steroidal
anti-inflammatory drug diclofenac, with natural molecules endowed with antioxidant and
antiproliferative activity were prepared. The antiproliferative activity of these hybrids was evaluated
on immortalized human keratinocyte (HaCaT) cells stimulated with epidermal growth factor (EGF),
an actinic keratosis (AK) model, and on human squamous cell carcinoma (SCC) cells (A431). Hybrid
1
presented the best activity in both cell models. Self-assembling surfactant nanomicelles have
been chosen as the carrier to drive the hybrid into the skin; the in vitro permeation through and
penetration into pig ear skin have been evaluated. Among the nanostructured formulations tested,
Nano3Hybrid20 showed a higher tendency of the hybrid to be retained in the skin rather than
1
1
permeating it, with a desirable topical and non-systemic action. On these bases, hybrid
1 may
represent an attractive lead scaffold for the development of new treatments for AK and SCC.
Keywords: diclofenac; hybrid; squamous cell carcinoma; actinic keratosis; antiproliferative activity;
nanomicelles; in vitro skin permeation/penetration
1
. Introduction
Non-melanoma skin cancer (NMSC) represents the most common cancer among white-skinned
people. The term NMSC refers to keratinocyte cancer and essentially to two types of cancer: basal
cell carcinoma (BCC) and squamous cell carcinoma (SCC). BCC is characterized by local tissue
damage and seldom leads to metastatization while SCC can recur and metastasize. The step that
often precedes the onset of this latter tumoral form is called actinic keratosis (AK), that occurs on
sun-exposed skin and may progress to invasive SCC in up to 10% of cases [1]. The treatment of AK
and SCC includes invasive approaches (excisional surgery, laser ablation, cryosurgery, curettage and
electrodessication), and non-invasive approaches (medical and photodynamic therapy). Specifically,
when surgical treatment is not feasible, topical pharmacotherapy can be a viable alternative. Diclofenac,
a nonsteroidal anti-inflammatory drug, is the most commonly used topical agent to treat AK, due to
its effectiveness, poor side effects, tolerability and low cost [
2]. Topical diclofenac, effective use for
Molecules 2019, 24, 1793; doi:10.3390/molecules24091793
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1793
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NMSC, is also reported in recent clinical trials [
3,
4]. Another therapeutic approach, especially for AK,
may derive from the protection of skin keratinocytes from UV-induced damages. In fact, the link
between sun exposure and AK skin lesions is well-recognized. Excessive exposure to UV radiations
may cause mutations in keratinocytes, in particular, UV-A and UV-B radiations induce production of
reactive oxygen species (ROS), provoking DNA damage and abnormal cell proliferation. It has been
reported that the topical use of antioxidants contributes to decreasing the oxidative damage mediated
by UV radiation [
antiproliferative activity in different types of cancer [
the most powerful antioxidant compounds [10]. Moreover, HT induces apoptosis and inhibits tumor
growth in different in vitro and in vivo tumor models [11 13]. The structural analogue tyrosol (T),
5
]. Furthermore, natural molecules, known as antioxidants, have recently showed
6–9
]. In particular, hydroxytyrosol (HT) is one of
–
despite a lower antioxidant action than HT, possesses a protective effect on oxidative lesion in cellular
models, boosting intracellular antioxidant defense systems. Particularly interesting is its action against
H O induced damage on DNA [14].
2
2
In recent years the increasing incidence and morbidity of SCC patients has directed the research
towards design of new drug therapies. The multi target approach is considered innovative in drug
discovery with encouraging results in various therapeutic fields, especially in the treatment of cancer.
The approach of “dual action drugs” in which two or more pharmacophoric portions are incorporated
in a single molecule, may allow for obtaining a hybrid with improved efficacy and/or minor side effects
than the native drugs [15].
Based on these findings, in the current study we conjugated the molecule of diclofenac (Figure 1)
with the natural antioxidant and antiproliferative compounds, HT and T, (Figure 1) obtaining the
hybrids
1 and 2. Furthermore, we synthesized compounds 3 and 4 characterized by to a linker between
the two native compounds.
Figure 1. Chemical structure of synthesized hybrids (1–4).
Hybrids
1–
4
were tested in AK and in SCC models in order to evaluate their chemopreventive and
antitumor activity, respectively. Since the main target of AK treatment is the epidermis, in particular
the basal layer and in a major extent the granular and cornified layers, we studied for hybrid 1,
that showed the best activity in AK model, a nanostructured drug delivery system as a formulation
strategy to selectively direct the drug to a specific site (different skin layers) by enhancing skin
penetration while reducing transcutaneous permeation and consequently systemic side effects [16].
Delivery of drugs using nanotechnologies, not only can improve drug stability, but also can reduce skin
0
irritation by avoiding direct contact of the drug with the skin s surface. The nanomicelles have recently
attracted a great deal of attention for drug delivery into the skin for the treatment of dermatological
diseases [17–19] and in the present research surfactant nanomicelles, able to self-assemble above the

Molecules 2019, 24, 1793
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critical micelle concentration (CMC), have been chosen as the carrier to drive the hybrid
1
into the
skin. The in vitro permeation through and penetration into pig ear skin, a widely accepted model for
human skin [20], have been evaluated.
2
. Results and Discussion
2
.1. Synthesis
Compounds 1 and 2 were prepared by condensation of diclofenac with HT or T, in the presence of
N,N -dicyclohexylcarbodiimide (DCC) and a catalytic amount of 4-dimethylaminopyridine (DMAP) in
0
THF at room temperature, as described in Scheme 1.
Scheme 1. Reagents and conditions: (a) DCC, DMAP, THF, rt, 12 h.
The synthesis of compounds
between 4-chlorobutanoyl chloride and 2-phenylmetanol in the presence of pyridine, was condensed
with diclofenac sodium obtaining derivative . The subsequent catalytic hydrogenation gave acid
, which was submitted to condensation with HT or T using DCC as condensing reagent, afforded
compounds 3 and 4.
3 and 4 is shown in Scheme 2. The intermediate 5 prepared by reaction
6
7
◦
Scheme 2. Reagents and conditions: (a) pyridine, toluene, rt, 20 h; (b) DMF, 110 C, 3 h; (c) H2, Pd/C,
EtOH, rt, 3 h; (d) DCC, THF, refluxed, 3 h.

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2
.2. Pharmacological Activity
Hybrids and native compounds (diclofenac, HT, T) were tested to evaluate cytotoxic activity
1–4
on epidermal growth factor (EGF)-stimulated HaCaT cells as in vitro model of AK.
The results showed that diclofenac induced a dose-dependent cytotoxic effect with an IC50 of
6
7.07 ± 1.94 µM (Figure 2A,B, Table 1). This effect is in accordance with antiproliferative activity
showed by diclofenac itself in other cancer models [21 24]. A cell viability decrease was also observed
with HT (IC50 72.31 3.58 M, Figure 2A, Table 1). This was not surprising, since this phenolic
phytochemical, in addition to the antioxidant property, has already been shown to contrast cell
viability in different cancer types [11 13 25 30] as well as to down-regulate the EGF receptors on colon
cancer cells [31]. The hybrids and , in which diclofenac is conjugated to HT directly (hybrid ) or
through a 3C linker (hybrid ), showed, a dose-dependent cell viability decrease with an IC50 value
of 3.71 1.08 M and 17.77 1.09 M (Figure 2A, Table 1), respectively. In particular, the hybrid
induced a cell viability decrease with a significantly higher potency (about 20-fold) than those of
–
±
µ
,
,
–
1
3
1
3
±
µ
±
µ
1
native drugs, diclofenac and HT (Figure 2A, Table 1). The association of diclofenac and HT, at the
same concentration in the hybrids, induced a not significant decrease on cell viability compared to the
individual treatments (Figure 2A). These data suggested the potential advantage of hybrids
compared to the association of single treatments.
1 and 3
As regards T, an IC50 value was not reached in the concentration range evaluated (Figure 2B,
Table 1) in our experiments. This data is in agreement with a reduced efficacy of T in comparison to
HT on tumor proliferation and EGF receptors down-regulation, as reported previously by Terzuoli et
al. on colon cancer [31]. Diclofenac-T hybrid
of 16.06 1.15 M (Figure 2B, Table 1). On the contrary, the analogue hybrid
for the presence of the 3C linker, showed an antiproliferative effect lower than that of diclofenac
2
was 4-fold more potent than diclofenac, with an IC50
±
µ
4, which differs from
2
(Figure 2B, Table 1).
Comparing the IC50 values of the hybrids
1
and
2 (in which diclofenac is directly linked to HT or
T, respectively) with those of the hybrids
3 and 4, characterized by a linker between the two native
molecules, it is possible to show that the presence of linker has a negative effect for the cytotoxic activity
of hybrids on the AK cell model.
Hybrids 1–4 and native compounds (diclofenac, HT, T) were tested, also, to evaluate their cytotoxic
activity on squamous carcinoma A431 cells line (Figure 2B,C, Table 1).
In these cells, diclofenac induced a dose-dependent cytotoxic effect with an IC50 of 31.96
Table 1). This result is in line with diclofenac activity previously reported [21–24,32–34]. The activity
±
1.05 µM
(
of HT in A431 cells was reduced compared to EGF-stimulated HaCaT cells (Figure 2C). This behavior
could be explained considering that EGF inhibits cell growth in A431 cells, while stimulate growth in
HaCaT cells [31
was the most powerful cytotoxic agent (IC50 13.51
native molecules diclofenac (31.96 1.05 M) and HT, although its IC50 value was about 4 fold greater
,
35] and that HT is an EGF receptor deregulator. In A431 cells, hybrid
1 (diclofenac-HT)
±
2.49 M) compared to other hybrids and the
µ
±
µ
than that obtained in EGF-stimulated HaCaT cells (Figure 2C, Table 1). The analogue hybrid
3 showed
minor activity compared to hybrid 1 also in this SCC model.
T showed a reduced anticancer activity in comparison to HT as above reported for EGF-stimulated
HaCaT cells (Figure 2D). This result is in line with evidences obtained in colon cancer [31]. As regards
compound
. Hybrid
diclofenac (31.96
in cell viability in SCC as well as in AK model (Figure 2D, Table 1). Additionally, in A431 cell line,
2
2
, where HT was substituted by T, its antitumor effect is reduced compared to compound
showed an IC50 value (27.51 1.24 M) without a significant difference with respect to
1.05 M) (Figure 2D, Table 1). The hybrid was not able to induce modification
1
±
µ
±
µ
4
as in EGF-stimulated HaCaT cells, the presence of linker as in compounds
antiproliferative activity.
3 and 4, is deleterious for

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Figure 2. Effects induced by test compounds on cell viability of HaCaT cells after stimulation with
ng/mL EGF ( ) and A431 cells ( ). Cells were exposed to compounds for 72 h and cell
viability assessed by neutral red assay. Data are expressed as means SEM of three independent
5
A,
B
C, D
±
experiments, each performed in triplicate. Each hybrid was compared to single agents. D: diclofenac,
HT: hydroxytyrosol, T: tyrosol.
Table 1. IC50 mean values
HaCaT and A431 cell lines .
±
Standard Error of the Mean (SEM) for test compounds on EGF-stimulated
1
IC50 ± SEM (µM)
EGF-stimulated HaCaT
Treatment
A431
D
HT
1
3
T
67.07 ± 1.94
72.31 ± 3.58
3.71 ± 1.08
17.77 ± 1.09
NR
31.96 ± 1.05
NR
13.51 ± 2.49
30.65 ± 1.33
NR
2
4
16.06 ± 1.15
27.51 ± 1.24
NR
NR
1
D: diclofenac; HT: hydroxytyrosol; T: tyrosol; NR: not reached.
2
.3. Nanomicellar Formulation Containing Hybrid 1
Hybrid , selected for the best activity in both cell models, was formulated in a nanostructured
1
drug delivery system. The prepared self-assembling nanomicelles consisted of a mixture of two
non-ionic surfactants with the same HLB value (13 and 13.2 for Vit-E TPGS and OPEE, respectively).
In particular, Vit-E TPGS was chosen in virtue of its multivalued features as excellent solubilizer
and bioavailability enhancer of hybrophobic molecules, as well as to act as selective antitumor agent
inducing apoptosis in cancer cells and not healthy ones. Moreover its ability to inhibit the activity
of P glycoprotein makes it an important excipient to overcome multidrug resistance that represents
an impediment to the success of anticancer therapy [36
hybrid were characterized from the physico-chemical point of view. The results in term of size and
size distribution, entrapment efficiency and drug loading related to the formulations under study,
–38]. Nanomicellar formulations containing
1

Molecules 2019, 24, 1793
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are summarized in Table 2. The nanomicelles size ranged between 14 and 70 nm with a narrow
distribution (0.2 < polidispersivity index < 0.36). The dimension of nanomicelles is a crucial aspect that
affects drug permeation and distribution into the skin [16]. It is noteworthy that the size of micelles can
range from 10 to 200 nm and can be increased by drug incorporation. Their mechanism of action is not
clearly demonstrated but it is known that they can penetrate through stratum corneum and accumulate
in hair follicles. Moreover it has been observed skin penetration into deeper skin layers followed by
drug delivery in the intercluster region, one of the penetration pathways through the skin [19]. All of
the prepared nanomicellar formulations had a good drug efficiency in the range between 79% and 96%.
It can be noted that for the same total amount of surfactants (3%), the size appeared to increase with
increasing the drug loading.
Table 2. Properties of nanomicelles in terms of size and drug content.
Drug Content
µmol/mL)
Entrapment
(%)
Formulation Type
Size (nm)
P.I.
Loading (%)
(
Nano3Hybrid10
Nano3Hybrid15
Nano3Hybrid20
Nano5Hybrid30
14.28 ± 0.24
24.98 ± 0.46
68.55 ± 5.09
50.67 ± 5.2
0.2007
0.3038
0.3562
0.3660
8.24 ± 0.29
11.44 ± 0.27
19.3 ± 0.54
27.01 ± 0.58
80.32 ± 2.89
79.41 ± 1.97
96.48 ± 1.08
93.51 ± 1.56
10.32 ± 0.37
13.48 ± 0.33
22.13 ± 0.34
18.77 ± 0.31
Hybrid loaded nanomicellar formulations were subjected to in vitro studies of permeation through
and penetration into pig ear skin.
The results of the in vitro experiments are summarized in Table 3, where the relevant permeation
parameters (flux, lag time, amount of drug permeated after 24 h) are reported for each formulation.
Data are shown as diclofenac in the case of Solaraze or as both hybrid
hydrolysis of hybrid ) for the nanomicellar formulations. In all cases, nanomicellar formulations
consisting of 3% of total amount of surfactants did not produce any permeation of the hybrid while,
1 and diclofenac (derived from
1
1
after a lag time, it was possible to detect in the receiving phase the presence of diclofenac as hydrolysis
product of hybrid 1.
Hybrid
432.30 g/mol), while the native drug diclofenac permeated the skin with a transdermal flux ranging
from 0.04 to 0.58 nmol/cm2
h. Nonetheless, the amount of diclofenac permeated was definitely
1 did not permeate across the skin, maybe because of its high molecular weight
(
·
lower than that obtained by the reference, considering also the differences in the drug content of
the formulations. The lipophilic nature of diclofenac sodium (partition coefficient n-octanol/water,
log POW = 4.98) appeared to promote a better interaction with the different skin layers, in particular
with the stratum corneum, known as the main barrier to permeation.
Nanomicelles containing 5% of total amount of surfactant (Nano5Hybrid30) produced a permeation
of both hybrid 1 and diclofenac derived from its hydrolysis. The higher amount of surfactant appeared
to increase the skin permeability, promoting the transcutaneous permeation, as well known [39,40].
Table 3. Permeation parameters: flux (J), lag time and amount of drug permeated through the skin
(Qpermeated24h) at end of the experiment (24 h) from the formulations under study (mean
±
SEM, n = 6).
2
Formulation
Drug
Flux, nmol/cm ·h
2.01 ± 0.37
Lag time, h
Qpermeated 24 h, nmol
Solaraze
Nano3Hybrid10
Diclofenac
Hybrid 1
Diclofenac
Hybrid 1
Diclofenac
Hybrid 1
Diclofenac
Hybrid 1
Diclofenac
7.90 ± 0.50
32.53 ± 6.36
No permeation
1.42 ± 0.80
0.12 ± 0.02
3.49 ± 0.62
1.18 ± 0.11
Nano3Hybrid15
Nano3Hybrid20
Nano5Hybrid30
No permeation
2.86 ± 1.57
0.04 ± 0.001
No permeation
10.8 ± 0.03
0.58 ± 0.004
0.16 ± 0.08
0.24 ± 0.06
9.42 ± 0.08
3.27 ± 2.18
7.27 ± 4.32
9.10 ± 3.76
7.09 ± 3.55

Molecules 2019, 24, 1793
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The skin recovery results after application of the different formulation under study are shown
in the histogram reported in Figure 3. Drug recovered in the pig ear skin as diclofenac or hybrid
compound is reported in nanomols/g of skin. When the different dispersions of hybrid were used
as donor phase, both hybrid and diclofenac derived from the hydrolysis of hybrid were found.
1
1
1
The reference Solaraze gel produced a high recovery of drug in the skin. In the case of Nano3Hybrid
formulations, the amount of drug in form of hybrid and diclofenac increased proportionally to the
hybrid
1 content in the formulation. Nano3Hybrid20 appeared the best nanomicellar formulation,
even compared to Nano5Hybrid30 formulation, which, in term of recovery, did not give any advantage
(117.7 ± 15.21 vs. 180.4 ± 36.68 nmols/g, for Nano5Hybrid30 and Nano3Hybrid20, respectively).
Figure 3. Drug recovered in the pig ear skin after application of the formulation under study (nanomols/g
of skin, mean ± SEM, n = 6).
The results of the comparison of the different formulations under study in terms of permeation
and recovery of drug (as diclofenac and/or hybrid
high drug accumulation with a relevant permeation; Nano3Hybrid formulations appeared to promote
the recovery of both hybrid and diclofenac as hydrolysis product of Hybrid (both therapeutically
1) are summarized in Table 4. Solaraze produced
1
active) and the recovery increased with increasing the Hybrid amount encapsulated in the nanomicelles.
Moreover, they did not facilitate the transcutaneous permeation. Nano3Hybrid20 appeared the best
formulation, to be preferred also to Nano5Hybrid30, which, next to a non-high recovery, allowed
a cutaneous permeation of both drugs involved.
Table 4. Amount of drugs permeated through and recovered into pig ear skin after application of
formulations under study (nanomols, mean ± SEM, n = 6).
Diclofenac
Permeated
Diclofenac
Recovered
Hybrid 1
Permeated
Hybrid 1
Recovered
Formulations
Solaraze
32.53 ± 6.36
3.49 ± 0.62
1.18 ± 0.11
9.42 ± 0.08
7.28 ± 4.32
158 ± 20.5
1.47 ± 1.17
1.68 ± 0.50
28.21 ± 6.94
18.96 ± 3.45
n.a.
-
-
-
n.a.
Nano3Hybrid10
Nano3Hybrid15
Nano3Hybrid20
Nano5Hybrid30
3.23 ± 0.33
6.10 ± 0.97
10.42 ± 0.92
14.1 ± 5.52
3.27 ± 2.19
Finally, in the Figure 4, the relationship between drug recovered in the skin and the relative
activity on EGF-stimulated HaCaT cells is illustrated. The histogram highlights the efficacy of the drug

Molecules 2019, 24, 1793
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(as diclofenac for Solaraze and as diclofenac plus hybrid
1
for nanomicellar formulations) retained
in the skin related to the capacity of these drugs to hinder the growth of EGF-stimulated HaCaT
cells, as AK model. It is evident the greater efficacy of hybrid encapsulated in Nano3 and Nano5
1
formulations with respect to the reference, even if with no-statistically significant differences. It is
noteworthy that in the case of nanomicellar formulations the amount of drug applied on the skin
(
19.3 and 27.01
than that of Solaraze (94.2
0-fold) demonstrated by the IC50 value (67.07
µ
mols/mL for Nano3Hybrid20 and Nano5Hybrid30, respectively) was 3 to 5-fold lower
mols/mL). Since hybrid had much greater activity than diclofenac (about
1.94 vs. 3.71 1.08 M, for diclofenac and hybrid
recovered on the skin was enough to have the same or
µ
1
2
±
±
µ
1,
respectively), it appears evident that hybrid
more activity than the commercial product.
1
Figure 4. Efficacy of the drug retained in the skin related to its capacity to hinder the growth of
EGF-stimulated HaCaT cells.
3
. Materials and Methods
3
.1. Chemicals and Instrumentation
Commercially available reagents were purchased from Sigma Aldrich or TCI, and used
without purification.
To prepare nanomicelles the following materials were used: octylphenoxy poly(ethyleneoxy)ethanol
®
(
(
OPEE, IGEPAL CA-630, Sigma-Aldrich, Milan, Italy); d-
α
-Tocopherol polyethylene glycol succinate
®
Vit E-TPGS, Kolliphor TPGS, BASF, Ludwigshafen, Germany).
H-NMR and C-NMR spectra were recorded on a Bruker AVANCE III
Bruker Corporation, Billerica, MA, USA, operating at 400 MHz). Chemical shift (
1
13
™
400 spectrometer
) were reported
(
δ
in parts per million (ppm) related to the residual solvent signal, coupling constants (J) are expressed
in Hertz (Hz). Evaporation was carried out in vacuo using a rotating evaporator. Silica gel flash
chromatography was performed using silica gel 60 Å (0.040–0.063 mm; Merck, Milan, Italy). Reactions
were monitored on TLC on Merck aluminium silica gel (60 F254) plates that were visualized under
a UV lamp (
are uncorrected.
All synthesized compounds were analyzed by HPLC, showing a purity
λ = 254 nm). Melting points were determined on a Kofler hot-stage apparatus and
≥95%. A Bechman HPLC
instrument equipped with a System Gold Solvent Delivery module (Pumps) 125, System Gold UV/VIS
Detector 166, Detector set to 278 nm was employed. Analyses were performed on a reverse phase C18
column (Phenomenex 250
The mobile phase was constituted by a mixture of H O/AcOH (0.1% v/v) (eluent A) and ACN (eluent
×
4.6 mm, 5 mm particle size, Gemini, Phenomenex, Torrance, CA, USA).
2
B) on isocratic elution with 70% of B. The flow rate was 0.8 mL/min.

Molecules 2019, 24, 1793
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3
.2. Synthesis Method
3
.2.1. General Procedure for the Synthesis of Compounds 1, 2
To a solution of HT or T (0.84 mmol) in THF (2 mL), diclofenac (250 mg, 0.84 mmol), DCC (209
mg, 1.012 mmol) and DMAP (8 mg, 0.06 mmol) were added. The resulting mixture was stirred at room
temperature for 24 h, then the suspension was filtered and the solution was evaporated. The crude
product obtained was purified as described below.
3,4-Dihydroxyphenethyl 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (1). Purified by flash chromatography
on a silica gel column, eluting with ethyl acetate/petroleum ether (3:7). Yield 52%; white solid mp
◦
1
1
7
6
23–125 C. H-NMR (CDCl ):
δ
(ppm) 7.34 (d, 2H, J = 8.0 Hz, Ar), 7.22 (dd, 1H, J = 1.2, 7.6 Hz, Ar),
.15 (dt, 1H, J = 1.2, 7.6 Hz, Ar), 6.95–7.01 (m, 2H, Ar), 6.80 (br s, 1H, NH), 6.73 (d, 1H, J = 8.0 Hz, Ar),
.53–6.58 (m, 3H, Ar), 5.01 (br s, 1H, OH), 4.91 (br s, 1H, OH), 4.31 (t, 2H, J = 6.8 Hz, CH ), 3.80 (s, 2H,
3
2
13
CH Ph), 2.82 (t, 2H, J = 6.8 Hz, CH ). C-NMR (CDCl3):
δ
(ppm) 172.45, 143.45, 142.95, 142.48, 137.78,
31.21, 130.48, 129.81, 129.01, 128.09, 124.35, 122.04, 121.56, 118.25, 116.07, 115.50, 66.15, 38.86, 34.50. HPLC
analysis: retention time = 7.50 min; peak area, 96% (280 nm).
2
2
1
3
-Hydroxyphenethyl 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (2). Purified by chromatography on
a silica gel column, eluting with ethyl acetate/petroleum ether (1:9). Yield 30%; yellow solid mp
◦
1
1
20–122 C; H-NMR (CDCl ):
δ
(ppm) 7.34 (d, 2H, J = 8.0 Hz, Ar), 7.21 (dd, 1H, J = 1.2, 7.6 Hz, Ar),
.13 (dt, 1H, J = 1.6, 7.6 Hz, Ar), 7.00 (d, 2H, J = 8.4 Hz, Ar), 6.94–7.00 (m, 2H, Ar), 6.84 (br s, 1H, NH),
.71 (d, 2H, J = 8.4 Hz, Ar), 6.55 (d, 1H, J = 7.6 Hz, Ar), 4.60 (br s, 2H, OH), 4.31 (t, 2H, J = 7.1 Hz, CH ),
3
7
6
3
2
13
.79 (s, 2H, CH Ph), 2.88 (t, 2H, J = 7.1 Hz, CH ). C-NMR (CDCl3):
δ
172.32, 154.37, 142.89, 137.99,
2
2
131.06, 130.50, 130.36, 130.21, 129.85, 129.75, 129.01, 128.11, 124.47, 124.18, 122.15, 118.40, 115.51, 66.08,
3
8.83, 34.37. HPLC analysis: retention time = 10.43 min; peak area, 99% (280 nm).
.2.2. General Procedure for the Synthesis of Compounds 3, 4
3
To a solution of compound
7 (200 mg, 0.52 mmol) in THF (2 mL) was added DCC (129 mg,
0.628 mmol) and HT or T (0.52 mmol). The reaction mixture was stirred at reflux for 3 h, then the
solid were filtered out and the solvent was evaporated. The crude product obtained was purified as
described below.
3,4-Dihydroxyphenethyl 4-(2-(2-((2,6-dichlorophenyl)amino)phenyl) acetoxy)butanoate (3). The residue was
purified by chromatography on a silica gel column, eluting with CHCl /AcOEt 7:3, and subsequently
3
1
triturated with Et O. Yield 20%; oil. H-NMR (CDCl ):
δ (ppm) 7.35 (d, 2H, J = 8.0 Hz, Ar), 7.22 (dd,
2
3
1
6
H, J = 1.2, 7.2 Hz, Ar), 7.11–7.16 (m, 1H, Ar); 6.99 (t, 1H, J = 8.0 Hz, Ar), 6.97 (t, 1H, J = 7.4 Hz, Ar),
.82 (br s, 1H, NH), 6.78 (d, 1H, J = 8.0 Hz, Ar), 6.72 (d, 1H, J = 2.0 Hz, Ar) , 6.62 (dd, 1H, J = 2.0, 8.0 Hz,
Ar), 6.56 (d, 1H, J = 8.0 Hz, Ar), 5.26 (br s, 1H, OH), 5.75 (br s, 1H, OH), 4.25 (t, 2H, J = 6.6 Hz, CH2),
4
.15 (t, 2H, J = 6.6 Hz, CH ), 3.82 (s, 2H, CH Ph), 2.81 (t, 2H, J = 6.6 Hz, CH ), 2.36 (t, 2H, J = 7.0 Hz,
2
2
2
13
CH ), 1.92–2.01 (m, 2H, CH ). C-NMR (CDCl3):
δ
172.98, 172.89, 143.86, 142.79, 142.70, 137.88, 131.00,
2
2
130.55, 129.63, 129.01, 128.21, 124.28, 124.23, 122.24, 121.23, 118.47, 116.09, 115.51, 65.49, 64.59, 38.66,
3
4.49, 30.75, 24.04. HPLC analysis: retention time = 11.47 min; peak area, 95% (280 nm).
4
-Hydroxyphenethyl 4-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)butanoate ). Purified by
(4
chromatography on a silica gel column, eluting with CHCl /AcOEt 9:1, and subsequently triturated with
3
1
Et O. Yield 23%; oil. H-NMR (CDCl ):
δ (ppm) 7.34 (d, 2H, J = 8.0 Hz, Ar), 7.21–7.24 (m, 1H, Ar), 7.23 (d,
2
3
2H, J = 8.4 Hz, Ar), 7.13 (dt, 1H, J = 1.5, 7.7 Hz, Ar), 7.02 (d, 2H, J = 8.4 Hz, Ar), 6.94–7.03 (m, 2H, Ar), 6.91
(
br s, 1H, NH), 6.56 (d, 1H, J = 7.6 Hz, Ar), 4.27 (t, 2H, J = 6.4 Hz, CH ), 3.85 (t, 2H, J = 6.4 Hz, CH ),
2
2
3
.83 (s, 2H, CH Ph), 2.86 (t, 2H, J = 6.6 Hz, CH ), 2.63 (t, 2H, J = 7.4 Hz, CH ), 2.08–2.15 (m, 2H, CH ).
2
2
2
2
1
3
C-NMR (CDCl3):
δ (ppm) 172.45, 171.52, 149.27, 142.81, 137.90, 136.31, 130.95, 130.10, 129.61, 128.98,
128.15, 124.34, 124.15, 122.17, 121.64, 118.43, 64.22, 63.65, 38.66, 30.87, 24.08. HPLC analysis: retention time
=
11.35 min; peak area, 95% (280 nm).

Molecules 2019, 24, 1793
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3
.2.3. Synthesis of Benzyl 4-Chlorobutanoate (5)
To a solution of phenylmethanol (0.77 g, 7.09 mmol) in toluene anhydrous (1 mL), pyridine (0.56 g,
.09 mmol) and 4-chlorobutanoyl chloride (1.00 g, 7.09 mmol) were added. The reaction mixture
7
was stirred at room temperature overnight, then H O was added and the toluene was evaporated.
2
The resulting aqueous phase was extracted with Et O, the organic phase was washed with 0.5 M HCl
2
and a saturated NaHCO solution. The organic phase was then dried, filtered and evaporated to
3
give compound
further purification. H-NMR (CDCl ):
5
(1.28 g, 6.02 mmol, 85% yield), which was used for the subsequent reaction without
1
δ 7.31–7.39 (m, 5H, Ar), 5.13 (s, 2H, CH ), 3.60 (t, 2H, J = 6.4 Hz,
2
3
CH ), 2.56 (t, 2H, J = 7.2 Hz, CH ), 2.08–2.15 (m, 2H, CH ).
2
2
2
3
.2.4. Synthesis of Benzyl 4-(2-(2-((2,6-Dichlorophenyl)amino)phenyl)acetoxy)butanoate (6)
Compound (1.09 g, 5.13 mmol) was added to a solution of sodium diclofenac (1.63 g, 5.13 mmol) in
DMF (4.0 mL) heated to 110 C. Stirring was continued at the same temperature for about 3.5 h. After this
period, the reaction mixture was cooled at room temperature and H O was added. The aqueous phase
5
◦
2
was extracted with Et O, and then the organic phase was dried, filtered and evaporated. The residue
2
which was purified by flash chromatography eluting with hexane/AcOEt 9:1, providing compound
6
1
(
848 mg, 1.79 mmol, 35% yield). H-NMR (CDCl ):
δ
(ppm) 7.32–7.36 (m, 7H, Ar), 7.21 (dd, 1H, J = 1.2,
3
7.6 Hz, Ar), 7.12 (dt, 1H, J = 1.5, 7.8 Hz, Ar), 6.92–7.00 (m, 2H, Ar), 6.91 (br s, 1H, NH), 6.54 (d, 1H,
J = 8.0 Hz, Ar), 5.12 (s, 2H, CH Ph), 4.19 (t, 2H, J = 6.4 Hz, CH ), 3.79 (s, 2H, CH Ph).
2
2
2
3
.2.5. Synthesis of 4-(2-(2-((2,6-Dichlorophenyl)amino)phenyl)acetoxy)butanoic acid (7)
Pd/C (29 mg) was added to a solution of compound (250 mg, 0.59 mmol) in absolute EtOH
2 mL). This mixture was subjected to catalytic hydrogenation at room temperature for 3 h, then
6
(
the suspension was filtered on a celite, and the solvent evaporated to obtain compound
7 (195 mg,
1
0
7
.51 mmol, 86% yield). H-NMR (CDCl ):
.4 Hz, Ar), 7.10–7.14 (m, 1H, Ar) , 6.98 (t, 1H, J = 8.0 Hz, Ar), 6.95 (t, 1H, J = 7.4 Hz, Ar), 6.55 (d, 1H,
δ
(ppm) 7.34 (d, 2H, J = 8.0 Hz, Ar), 7.22 (dd, 1H, J = 1.4,
3
J = 8.0 Hz, Ar), 4.17–4.22 (m, 2H, CH ), 3.81 (s, 2H, CH Ph), 2.43 (d, 2H, J = 7.4 Hz, CH ), 1.96–2.04
2
2
2
(
m, 2H, CH ).
2
The H-NMR and 13C-NMR spectra of the compounds
1
1–4
are available in supplementary materials.
3
.3. Biological Assay Procedures
3
.3.1. Cell Cultures and Experimental Models
The immortalized human keratinocyte (HaCaT) cells and human squamous carcinoma (A431)
cells (Sigma-Aldrich, Milan, Italy) were cultured in RPMI 1640 (Euroclone, Milan, Italy) supplemented
with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100
µg/mL streptomycin (Euroclone) at
◦
3
7 C in a humidified atmosphere containing 5% CO .
2
HaCaT cells under the proliferative stimulus of EGF were used as model of actinic keratosis (AK),
a precancerous disease that can progress in cutaneous squamous cell carcinoma (cSCC), according to the
critical role played by the EGFR/Fyn/Src/Erk pathway for promoting cSCC formation [41]. Cells were
stimulated with EGF at the concentration 5 ng/mL for whole time of treatment with test compounds
(
see below).
Both HaCaT (EGF-stimulated) and A431 cells were incubated for 72 h with the test compounds at
concentrations in the range 0.1–100 µM.
3
.3.2. Cell Viability Assay
Cell viability was measured using the Neutral Red (NR) Assay (N2889, Sigma-Aldrich, Darmstadt,
Germany) as previously reported [42]. HaCaT and A431 cell lines were seeded at a density of
4
5
× 10 cells/well in 10% FBS medium in a 96-well plate. After 24 h, test compounds, dissolved in

Molecules 2019, 24, 1793
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DMSO, were diluted in sterile culture medium supplemented with EGF (HaCaT cells) or 1% FBS (A431
cells) and added into the well plate. DMSO did not exceed 0.2% v/v in the culture medium. Diclofenac,
HT, T and hybrids 1–4 were tested. After 72 h from treatment, NR was added and cells incubated for
2
h. At the end of the incubation period, cells were washed with PBS and, then, incubated for 10 min
with a NR solubilisation solution (1% acetic acid in 50% ethanol). The absorbance was measured at
40 nm using the Infinite M200 NanoQuant instrument (Tecan, Salzburg, Austria). Optical density
values from vehicle-treated cells were considered as 100% cell viability.
5
3
.4. Nanomicellar Formulation Containing Hybrid 1
3
.4.1. Preparation of self-assembling Surfactant Nanomicelles
Hybrid 1-loaded self-assembling surfactant nanomicelles (NanoHybrid) were prepared using two
non-ionic surfactants, Vit-E TPGS and OPEE (molar ratio 1:1). The two surfactants were heated at
◦
5
0 C; then drug was mixed together the other components to obtain a homogeneous blend, finally the
water was added and the final mixture was stirred overnight and filtered through cellulose acetate
®
filters (0.22
µ
m pore size, Minisart NML Syringe filters, Sartorius, Florence, Italy) to remove unloaded
drug, aggregates and other foreign particulates.
Four nanomicellar formulation were prepared, three containing 3.0% of total surfactant amount
and one with 5.0% of total surfactants with an increasing amount of drug added from 10 to 30 mM.
The composition of the mixtures used for the preparation of hybrid-loaded nanomicelles is reported in
Table 5.
Table 5. Composition of the mixtures used for the preparation of hybrid-loaded nanomicelles.
Formulation Type
Total Surfactant (% w/w) *
Hybrid-1 (mM)
Nano3Hybrid10
Nano3Hybrid15
Nano3Hybrid20
Nano5Hybrid30
3.0
3.0
3.0
5.0
10
15
20
30
*
Surfactant: mixture of Vitamin E-TPGS and OPEE (molar ratio 1:1).
3
.4.2. Physico-Chemical Characterization
Hybrid - loaded nanomicelles were characterized with respect to size, size distribution and
1
encapsulation efficiency in term of entrapment and drug loading.
Size and size distribution of the nanomicelles were determined by measuring the rate of fluctuations
in laser light intensity scattered by the nanomicelles immediately after their preparations by using
a Dynamic Light Scattering (DLS) Beckman Coulter® N4 Plus (Beckman Coulter s.r.l, Milan, Italy).
The samples were eventually diluted with ultrapure water (MilliQ, Millipore, Merck) previously
filtered through 0.45
µm Millipore Polyethersulfone membrane, Millipore Express PLUS, Merck) to
an appropriate concentration chosen on the basis of the measurement intensity, which was in the
4
6
range between 5
×
10 and 1
×
10 counts per second (cps). The average size for each nanomicelles
formulation was obtained on three different samples of nanomicelles for which 6 runs were carried out,
using an angle of 90 and run time of 200 s at 20 C.
◦
◦
The entrapment efficiency, defined by the percentage of drug loaded in the core of the nanomicelles
with respect to that initially added to the formulation, was determined by HPLC. An aliquot of each
Hybrid-loaded nanomicellar formulation was diluted with methanol and the amount of drug was
detected by HPLC analysis. The percent entrapment and loading efficiency of hybrid 1 were calculated
according to Equations (1) and (2), respectively.
Percent of drug entrapped = (mass of drug in nanomicelles)
(1)
×
100/(mass of drug added in formulation)

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Loading efficacy = (mass of drug in nanomicelles × 100)/
mass of drug added + mass of surfactants in the formulation)
(2)
(
3
.4.3. In Vitro Cutaneous Permeation and Distribution Studies
Porcine ears skin, obtained from freshly sacrificed animals in a local slaughterhouse, was used
as model. After cleaning with depurated water, full-thickness skin was removed with a scalpel from
the cartilage of the outer region. The excised pig ear skin was carefully deprived of the adhering
fat and subcutaneous tissue. Prior to the experiments, the pig hairs were abscised and the skin was
gently washed. The skin with a thickness about 1.46
diffusion cells with the stratum corneum facing the donor compartment and an available diffusion area
±
0.06 mm was placed in the Gummer-type
of 1.23 cm [43
,
44]. The donor phase consisted of five-hundred microliters of nanomicellar formulations
®
under study and Solaraze commercial product (Almirall S.A, Barcelona, Spain), hyaluronic acid
gel containing 3% diclofenac, used as reference. The receiving phase (5 mL) consisted of deionized
water maintained at 37 C and stirred at 600 rpm. At predetermined time intervals, 5 mL samples
◦
of receiving phase were withdrawn for HPLC analysis and replaced with the same volume of fresh
fluid. All experiments lasted 24 h and were replicated four times; sink conditions were maintained
throughout the entire study.
At the end of the permeation experiments the skin was collected, rinsed with distilled water to
eliminate excess of vehicle from the skin surface and gently wiped with cotton wool tampons. In
order to extract the drug, the skin was weighed, shredded into small pieces and treated with 2.0 mL of
◦
2
% sodium lauryl sulphate (SDS, Sigma-Aldrich, Milan Italy) for 24 h at 37 C. After treatment with
◦
a methanol:chloroform (2:1) solution (3 mL) for 1 h at 37 C under stirring, the mixture was centrifuged
at 4000 rpm for 15 min. Two hundred microliters of supernatant were dried in vacuo and subsequently
dissolved in methanol for HPLC analysis. The validation of the extraction procedure was performed
as previously described in Monti et al. 2015 [45].
The concentration of diclofenac and hybrid
1 in receiving fluids and in the skin samples was
selectively determined by HPLC. The apparatus consisted of a Shimadzu LC-10AD VP system with an
UV SPD-10AV VP detector and a CBM-20A interface (Shimadzu). The injection valve was a Rheodyne
®
with a capacity of 20
µ
L and a Luna C18 (5
µm; 150
×
4.6 mm) column was employed. The mobile
phase consisted of a mixture of acetonitrile:water containing 0.5% glacial acetic acid (70:30) (pH 3.7).
The flux was 0.8 mL/min and the detection wavelengths were 254 and 280 nm for diclofenac and hybrid
1
with a retention time of 5.0 and 7.0 min, respectively.
The amount of product under study in the samples was determined by comparison with
appropriate standard curves. In the case of skin samples, standard curves were obtained by adding
increasing amount of the compounds to blank biological matrix.
Linear regression analysis of pseudo steady state diffusion plots allowed calculation of the
following parameters: steady-state flux (J, nmols/cm2
h), given by Q/At, where Q is the amount of
·
permeant diffusing across area A in time t; lag time (h), indicating the time taken by the drug to
saturate the membrane and reach the receiving phase, calculated from the X-axis intercept values of
the regression lines and the amount of drug permeated at the end of the experiment (Q24h, nmols).
Moreover, the extraction procedure allowed calculation of the drug content (nanomols/g skin)
recovered in the skin at the end of the permeation studies.
An efficacy coefficient (EC) is reported to compare the biological activity of the drugs involved in
this work (diclofenac and Hybrid 1), taking into account the drug recovered in the biological membrane.
EC was calculated as a ratio of the amount of drug retained into the porcine ear skin per unit of weight
to IC50 value of each drug on EGF-stimulated HaCaT cells, considering the area and thickness of the
biological membranes used.

Molecules 2019, 24, 1793
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4
. Statistical Analysis
Data related to biological assay procedures are presented as mean
SEM) from at least three independent experiments. Statistical analysis was performed by one-way
±
standard error of the mean
(
ANOVA, followed by Tukey’s post-test for multiple comparisons.
Regarding in vitro cutaneous permeation and distribution studies, data are reported as the
mean ± SEM (n = 6). Statistical differences between permeation parameters were assessed by GraphPad
Prism software (GraphPad Software Inc., San Diego, CA, USA). Group comparison was assessed by
using the Student’s two-tailed unpaired t-test. In all cases, differences were considered statistically
significant at p < 0.05.
5
. Conclusions
In conclusion, in the last few years, the multi-target approach seems advantageous for the
treatment of complex diseases. For this reason, many hybrids for the treatment of different types of
pathologies are proposed with encouraging results [15]. However, there are few examples of hybrids
used for AK and SCC diseases [46].
In this work, we synthetized compounds (
natural molecules for which antioxidant and antiproliferative activity had been previously described
and we evaluated their in vitro activity against AK and SCC models. The hybrid compound has
1–4) obtained by combining diclofenac with T and HT,
1
shown a higher antiproliferating activity with respect to native compounds in both AK and SCC models.
Self-assembling nanomicelles have been used as carriers for the delivery of the drug to the site
of action and in particular the Nano3Hybrid20 resulted the most effective since it produced a lower
amount of drug permeated through the skin with a consequent reduction of systemic side effects.
Besides, Nano3Hybrid20 formulation improved an adequate drug accumulation into the cutaneous
layers to provide the same therapeutic activity as the commercial reference formulation loaded with
an amount of drug about 5-fold higher. The lower amount of drug coming into contact with the skin
could contribute to reduce irritative local phenomena, more common for long-term therapy in the
precancerous skin lesions.
Supplementary Materials: The following are available online: the H-NMR and 13C-NMR spectra of the
1
compounds 1–4.
Author Contributions: M.D., D.M., S.T. and S.C. designed the study and experiments; M.D. and C.M. designed
the novel compound syntheses; S.C., B.P., S.F. and P.N. designed and executed the biological assays; S.T., D.M.
and S.B. designed and executed nanomicelles and skin permeation/penetration evaluation; D.M., S.B., P.N., C.M.
analyzed and interpreted the experimental data; S.B., S.T., S.C., M.D. wrote, reviewed and edited the manuscript;
C.M., P.N., D.M. and M.M. supervised.
Funding: The research project was supported by Progetti di ricerca di Ateneo PRA 2017 (cod PRA_2017_51) and
PRA 2018 (cod PRA_2018_20) of the University of Pisa.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–4 are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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