4-Fluorobenzylpiperazine-Containing Derivatives as Efficient Inhibitors of Mushroom Tyrosinase

Article abstract of DOI:10.1002/cmdc.202000125

Tyrosinase is a type-3 copper protein involved in the biosynthesis of melanin pigments; therefore, the inhibition of its enzymatic activity represents a promising strategy for the treatment of hyperpigmentation-related disorders. To address this point, we previously designed a class of 4-(4-fluorobenzyl)piperazin-1-yl-based compounds, which proved to be more active inhibitors against tyrosinase from mushroom Agaricus bisporus than the positive control kojic acid. Herein, we report the synthesis of further series of 4-(4-fluorobenzyl)piperazin-1-yl analogues bearing a (hetero)aromatic fragment as key feature to improve protein affinity. The newly synthesized compounds were assayed in vitro and proved to be potent inhibitors in the low-micromolar range. The active 2-thienyl and 2-furyl derivatives were selected for further modification to allow their binding mode to be analyzed by docking studies and to give satisfactory safety profiles.

Full text of DOI:10.1002/cmdc.202000125

Accepted Article
Title: 4-Fluorobenzylpiperazine-containing derivatives as efficient
inhibitors of mushroom tyrosinase
Authors: Serena Vittorio, Laura Ielo, Salvatore Mirabile, Rosaria Gitto,
Antonella Fais, Sonia Floris, Antonio Rapisarda, Maria Paola
Germanò, and Laura De Luca
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000125
Link to VoR: https://doi.org/10.1002/cmdc.202000125
01/2020

10.1002/cmdc.202000125
ChemMedChem
FULL PAPER
4-Fluorobenzylpiperazine-containing derivatives as efficient
inhibitors of mushroom tyrosinase
Serena Vittorio,[a],* Laura Ielo,[b] Salvatore Mirabile,[a] Rosaria Gitto,[a] Antonella Fais,[c] Sonia Floris,[c]
Antonio Rapisarda,[a] Maria Paola Germanò [a] and Laura De Luca[a],*
[a]
S. Vittorio, S. Mirabile, Prof. R. Gitto, Prof. A. Rapisarda, Prof. M. P. Germanò, Prof. L. De Luca
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences
University of Messina
Viale Palatucci, 13, I-98168, Messina, Italy
E-mail: svittorio@unime.it, ldeluca@unime.it
Dr. L. Ielo
Department of Pharmaceutical Chemistry
University of Vienna
[b]
[c]
Althanstrasse 14, A-1090 Vienna, Austria
Dr. A. Fais, S. Floris
Department of Life and Environmental Sciences
University of Cagliari
……… I-09042 Monserrato, Cagliari, Italy
Supporting information for this article is given via a link at the end of the document.
Abstract: Tyrosinase is a type 3 copper protein involved in the
biosynthesis of melanin pigments, therefore the inhibition of its
enzymatic activity represents a promising strategy for the treatment of
hyperpigmentation-related disorders. To address this point at issue
we have previously designed a class of 4-(4-fluorobenzyl)piperazin-1-
yl-based compounds proving to be more active inhibitor against
tyrosinase from mushroom Agaricus bisporus when compared to
positive control kojic acid. Herein, we report the synthesis of further
binuclear copper center with six histidine residues involved in the
coordination of the two metal ions[8]. The inhibition of tyrosinase
activity is a widely used strategy for blocking melanogenesis thus
reducing melanin synthesis.[9, 10] Tyrosinase inhibitors such as
kojic acid and arbutin are employed by pharmaceutical and
cosmetic industries as active ingredients for the preparation of
skin-whitening formulations, but they have shown low efficacy,
12, 13]
low stability and cytotoxicity.[11,
Particularly, it has been
series of 4-(4-fluorobenzyl)piperazin-1-yl-analogs bearing
a
clearly demonstrated that kojic acid is poor human tyrosinase
(hetero)aromatic fragment as key feature to improve protein affinity.
The new synthesized compounds were in vitro assayed proving to be
potent inhibitors in low micromolar range. Then, the active 2-thienyl
and 2-furyl derivatives were selected for further modifications
considering their binding mode analyzed by docking studies and their
satisfactory safety profiles.
inhibitor. Moreover, it has been reported that several tyrosinase
[14]
inhibitors, both from synthetic
and natural sources, possess
[9]
antibacterial activity.
In bacteria, melanin exerts a protective
role against UV radiation, chelates metals in stress conditions and
neutralize the effects of antibiotics, thus increasing cell viability.
Therefore, the inhibition of tyrosinase activity and the subsequent
reduction of melanin synthesis could be exploited as strategy for
the development of new antibiotics. However, more efforts are
needed in order to clarify the mechanism that correlates the
antimicrobial effect with the inhibition of melanin synthesis.[9] For
these reasons there is a growing interest in the search of more
effective and potent tyrosinase inhibitors.[11, 12]
Introduction
Melanogenesis is a multistep process resulting in the formation of
melanin pigments which are responsible for skin, hair and eyes
In our previous studies we have identified a new class of
tyrosinase inhibitors bearing the 4-fluorobenzyl substituent as a
key structural motif, which establishes favorable contacts within
[1, 2]
color.
Under physiological conditions, melanin plays an
important role in the protection of the skin against damaging
effects of UV radiation while its over-production can cause hyper-
pigmentary disorders such as melasma, freckles, post-
inflammatory melanoderma, pigmented acne scars and age spots
[3]. Furthermore, the biosynthetic pathway that lead to the
production of neuromelanin seems to be linked to Parkinson’s
disease. [4,5]
15, 16]
catalytic site in proximity of the two copper ions.[12,
Experimental and theoretical structural investigations carried out
on tyrosinases from Bacillus megaterium and Agaricus bisporus
allowed us to identify 4-(4-fluorobenzyl)piperazin-1-yl](2-
methylphenyl)methanone (1a) as promising TyI (IC50=5µM)
showing higher potency than the reference compound kojic acid
(IC50=18µM) (Figure 1).[12]
Different strategies can be exploited to modulate melanogenesis
including the reduction of the expression of melanogenic
enzymes, the alteration of the related signalling pathways and the
[1]
inhibition of tyrosinase activity. Tyrosinase (EC 1.14.18.1) is a
copper containing enzyme that catalyzes the rate limiting-step of
melanin biosynthesis: the conversion of tyrosine in L-DOPA and
7]
its oxidation in o-dopaquinone.[6,
Its active site contains a
1
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10.1002/cmdc.202000125
ChemMedChem
FULL PAPER
highlights the favorable presence of hydrophobic groups and specifically blue
grid indicates aromatic rings. The picture was generated using Ligand Scout
software.[18]
Kojic acid
1a
IC50 = 5 M against TyM
IC50 = 18 M against TyM
In coherence with these previous findings, in this study we
replaced the 2-tolyl substituent of prototype 1a with further
chemical moieties able to establish favorable hydrophobic
contacts. All the designed compounds were synthesized and
tested for their ability to inhibit tyrosinase activity, and the
cytotoxicity was determined for selected compounds. Moreover,
docking simulations were performed to clarify the binding mode
into the mushroom tyrosinase active site.
1b
1c
IC50 = 1 M against TyM
IC50 = 0.5 M against TyM
Figure 1. Tyrosinase inhibitors
As shown in Figure 2A, the docking studies[17] on mushroom
tyrosinase revealed that 1a was accommodated in the catalytic
cavity through  stacking interactions with His263 as well as
hydrophobic contacts with Val283, Met257, Val284 and Phe264.
In turn, the binding pose was used as starting point for the
generation of Apo-site pharmacophore grids (Figure 2B), which
suggested suitable hydrophobic and hydrophilic substituents. In
Results and Discussion
We chose to probe the effect of the substitution of benzoyl moiety
linked to the piperazine ring with other (hetero)aromatic systems
as shown for the seven 4-(4-fluorobenzyl)piperazin-1-yl-based
derivatives 2a-g depicted in Figure 3.
particular,
we
designed
a
further
series
of
4-
fluorobenzylpiperazine compounds, in which the benzoyl moiety
was properly decorated. As result, we obtained improved
inhibitory activity toward Agaricus bisporus tyrosinase up to
submicromolar range as showed by compounds 1b and 1c (0.5
and 1 µM, see Figure 1),[17] thus confirming our computational
hypotheses.
A
b
a
c
d
f
e
2 a-g
g
Figure 3. Designed compounds 2a-g structurally related to prototype 1.
For each designed molecule we preliminarily estimated several
selected chemical-physical parameters related to the
pharmacokinetic behavior of a drug in the biological systems, like
topological polar surface area (TPSA), pKa, logP and logD at pH
=7 by using the web tool Chemicalize (www.chemicalize.it) with
the aim to synthesize compounds with drug-like properties (see
Supporting Information Table S1). Furthermore, we found that all
the compounds respected the rules of Lipinski and no PAINS
were predicted by the online free tool SwissAdme
(http://www.swissadme.ch/).
So, the designed compounds 2a-g were synthesized starting from
4-(4-fluorobenzyl)piperazine 3 that was coupled with the suitable
acyl chloride (RCOCl) or carboxylic acid derivative (RCOOH) to
obtain 2a-e and 2f-g, respectively (Scheme 1). The mixture
reaction was initially stirred at room temperature for 5 hours or left
overnight.
Figure 2. (A) Apo Site Grid generated starting from the best docking pose of
derivative 1a in the catalytic site of Agaricus bisporus tyrosinase. (B) Yellow grid
2
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10.1002/cmdc.202000125
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R
a
b
c
d
e
f
1-naphthyl
2-naphthyl
2-thienyl
i or ii
2-furyl
1,3-dioxol-5-yl
2-pyrrolyl
6-indolyl
3
2a-g
g
Scheme 1. Reagents and conditions: i) RCOCl, DIPEA, DCM, MW: 10 min, 50 °C, 200 W; ii) RCOOH, HBTU, TEA, DMF, MW: 10 min, 50 °C, 200 W.
In order to optimize the synthetic procedure, the reactions were
carried out for 10 minutes at 50 °C in the microwave conditions,
with an evident reduction of the times and improvement of the
1
yields. H and 13C NMR spectra data of compounds were in full
agreement with the proposed structures and representative data
are shown in Supporting Information.
Agaricus bisporus tyrosinase was used to evaluate in vitro
inhibitory effects of compounds 2a-g using L-DOPA as substrate.
The activities were expressed as IC50 values and were
summarized in Table 1. For comparison purpose, the IC50 values
of parent compound 1a and kojic acid (KA) are shown in Table 1.
Table 1. Inhibitory activity of compounds 2a-g compared to inhibitor 1 and kojic
acid as reference compounds.
Cpd
Diphenolase activity IC50 (µM)a
2a
3±0.4
3±1.1
3±0.6
5±0.5
3±0.1
12±3.5
5±0.9
5±1.6
18±0.2
2b
2c
2d
2e
2f
2g
1a
Kojic acid
[a] IC50 values represent the concentration that caused 50% enzyme activity loss.
All compounds were studied in a set of experiments performed in three
replicates.
Figure 4. The effect of compounds 2c (A) and 2d (B) on HeLa cell viability. Cells
were treated with different concentrations for compound 2c (4-100 μM) and for
compound 2d (5-100 μM) and their viability was evaluated by MTT assay.
The replacement of the 2-tolyl substituent of prototype 1a by
(hetero)aromatic rings generally led to more active compounds
with the exception of 2-pyrrolyl substituted derivative 2f,
displaying a slight decrease of inhibitory effects. Notably, all
tested compounds proved to be more potent than reference
compound kojic acid.
Considering the possibility to further exploring this class of
compounds, we focused our attention on active inhibitors 2c and
2d. Before starting with the structural modification on selected
compounds, the cytotoxicity of 2-thienyl substituted compound 2c
was carried out to determine the safety of this molecule (Figure
4A). Cells were treated with different concentration of inhibitor (4–
100 µM) for 48 h and were examined using MTT test. The results
indicated that 2c exhibited no cytotoxic effect in HeLa cells at the
IC50 value. The same protocol was applied for 2-furyl substituted
compound 2d and non-cytotoxicity was revealed as shown in
Figure 4B.
Furthermore, docking studies of 2c and 2d were performed into
[19]
mushroom tyrosinase active site (PDB code 2Y9X)
by Gold
software[20] in order to have information about their binding mode.
As displayed in Figure 5, the two inhibitors shared a very similar
binding mode with the p-fluorobenzyl moiety oriented towards the
copper ions, establishing hydrophobic contacts with Val283 and
π-stacking interaction with His263 as reported above for
prototype 1a. Instead, the 2-furyl and 2-thienyl rings are involved
in hydrophobic interactions with Phe264.
3
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R
h
i
2-thienylmethyl
2-furylmethyl
j
k
l
m
n
o
p
q
r
benzothiophen-2-yl
benzofuran-2-yl
3-methylthiophen-2-yl
4-methylthiophen-2-yl
3-bromothiophen-2-yl
5-chlorothiophen-2-yl
5-nitrothiophen-2-yl
3-methylfuran-2-yl
5-methylfuran-2-yl
5-nitrofuran-2-yl
2h-s
s
Scheme 2. The synthesized compounds 2h-s were prepared as reported for
analogues 2a-g.
The inhibitory activities of this further series of compounds are
summarized in Table 2. Overall, the performed structural
modifications did not significantly affect the IC50 values respect to
their parent compounds 2c-d. In detail, the lengthening of carbon
spacer (2h-i) as well as the introduction of specific substituents
on crucial position of heterocyclic system (e.g. 2m, 2o, and 2q)
gave an unpredictable reduction of the inhibitory effects;
conversely, the benzene-fused derivatives 2j-k proved to
maintain potent inhibitory effects in the low micromolar range.
Table 2. Inhibitory activity of compounds 2h-s compared to
inhibitor 1 and kojic acid as reference compounds
Cpd
Diphenolase activity IC50 (µM)a
2h
14 ± 2.8
18 ± 2.1
4 ±0.3
2i
2j
2k
5 ±0.8
2l
8± 1.4
2m
19 ± 3.5
3 ±0.4
Figure 5. Plausible binding mode for inhibitors 2c (Panel A) and 2d (Panel B).
Compounds 2c (ΔGbind=-22.38 kcal/mol) and 2d (ΔGbind=-21.27 kcal/mol) are
represented respectively as orange and yellow sticks. Key residues of the
binding site are depicted as white stick. Copper ions are represented as brown
spheres. The images are created by means of PyMOL (https://pymol.org).
2n
2o
32 ± 6.1
2p
3 ± 0.2
42 ± 13.6
2q
2r
2s
3.3 ± 1.0
12 ± 1.3
5 ± 1.6
To improve the inhibitory properties of 2-thienyl and 2-furyl
substituted compounds, we introduced slight structural
modifications depicted in Scheme 2: (a) lengthening of carbon
spacer between piperazine core and heterocyclic fragment (e.g.
compounds 2h-i); (b) replacement of 2-thienyl or 2-furyl ring with
corresponding benzothiophene and benzofuran nucleus (e.g.
compounds 2j-k); (c) introduction of different substituents on
thiophene ring (e.g. compounds 2l-p) or furan ring (e.g.
compounds 2q-s). As done for 2a-g, we estimated chemical-
physical parameters for this second series of designed molecules
2h-s (Supporting Information Table S1). These new derivatives
depicted in Scheme 2 were prepared following the same synthetic
1a
Kojic acid
18 ± 0.2
a IC50 values represent the concentration that caused 50% enzyme activity loss.
All compounds were studied in a set of experiments performed in three
replicates.
Conclusion
Herein, we reported the design and synthesis of a new series of
tyrosinase inhibitors bearing the 4-(4-fluorobenzyl)piperazine
fragment, by introducing (hetero)aromatic moieties in the place of
the o-tolyl group of prototype 1a. All the obtained compounds
showed significant inhibitory activity against tyrosinase from
Agaricus bisporus. Among this new series of tyrosinase inhibitors,
derivatives 2c and 2d were selected as new lead compounds for
further chemical explorations. Cell viability assays revealed that
2c and 2d showed no considerable cytotoxic effects in HeLa cells.
Furthermore, docking studies clarified the binding mode of these
two inhibitors highlighting an interaction network similar to that of
prototype 1a. With the aim to improve the inhibitory activity of 2c
and 2d, we carried out structural modifications thus collecting
procedure described in Scheme
1 for compounds 2a-g.
Specifically, to prepare compounds 2h, 2k-m, 2q-s we used
heteroaroyl chloride (RCOCl) as starting material; whereas for 2i,
2j, 2n-p we employed the corresponding carboxylic derivative
(RCOOH).
4
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[4-(4-Fluorobenzyl)piperazin-1-yl](naphthalen-1-yl)methanone (2a)
additional SAR considerations for this series of compounds. As
mushroom and human tyrosinases have shown distinct active site
[21,
Yield 88%. Oily residue. Rf = 0.49 1H-NMR (CDCl3): δ=2.26 (m, 2H, CH2),
2.58 (m, 2H, CH2), 3.19 (m, J=5.0, 2H, CH2), 3.48 (s, 2H, CH2), 3.95 (mc,
2H, CH2), 6.98 (t, J=8.7, 2H, ArH), 7.25 (t, J=8.7, 2H, ArH), 7.47 (mc, 4H,
ArH), 7.87 (mc, 3H, ArH). Anal. Calcd for C22H21FN2O: C 75.84, H 6.08,
N 8.04. Found: C 75.74, H 5.88, N 8.20.
interaction patterns leading to very different inhibition values,
22]
additional experiments using human tyrosinase or human
melanoma or melanocyte cells are needed to support the potential
of these compounds for human-directed applications.
[4-(4-Fluorobenzyl)piperazin-1-yl](naphthalen-2-yl)methanone (2b)
Experimental Section
CAS number: 945155-78-2. Yield 75%. White solid. Rf = 0.48. M.p. 120-
121 °C. 1H NMR (CDCl3): δ=2.40 (mc, 4H, CH2), 3.48 (mc, 2H, CH2), 3.51
(s, 2H, CH2), 3.84 (mc, 2H, CH2), 7.00 (t, J = 8.5, 2H, ArH), 7.28 (t, J = 8.5,
2H, ArH), 7.50 (mc, 3H, ArH), 7.87 (mc, 4H, ArH). 13C NMR (CDCl3):
δ=52.88, 53.22, 62.07, 115.06, 115.23, 124.31, 126.68, 126.87, 126.90,
127.05, 127.78, 128.28, 128.37, 130.50, 130.56, 132.70, 133.30, 133.65,
163.07, 170.31. MS (ESI): m/z: 348.9 [M+H+]. Anal. Calcd for
C22H21FN2O: C 75.84, H 6.08, N 8.04. Found: C 75.97, H 5.98, N 7.90.
Chemistry
All reagents were used without further purification and bought from
common commercial suppliers (Sigma-Aldrich, Milan, Italy and Alfa Aesar,
Karlsruhe, Germany). Microwave-assisted reactions were carried out in a
Focused Microwawe TM Synthesis System, Model Discover (CEM
Technology Ltd Buckingham, UK). Melting points were determined on a
Buchi B-545 apparatus (BUCHI Labortechnik AG Flawil, Switzerland) and
are uncorrected. By combustion analysis (C, H, N) carried out on a Carlo
Erba Model 1106-Elemental Analyzer we determined the purity of
synthesized compounds; the results confirmed a ≥95% purity. Merck Silica
Gel 60 F254 plates were used for analytical thin-layer chromatography
(TLC; Merck KGaA, Darmstadt, Germany). Flash Chromatography (FC)
was carried out on a Biotage SP1 EXP (Biotage AB Uppsala, Sweden). 1H
NMR spectra and 13C NMR spectra were measured in dimethylsulfoxide-
[4-(4-Fluorobenzyl)piperazin-1-yl](thiophen-2-yl)methanone (2c)
CAS number: 797813-02-6. Yield 85%. Oily residue. Rf = 0.49. 1H NMR
(CDCl3): δ=2.46 (t, J=5.0, 4H, CH2), 3.50 (s, 2H, CH2), 3.74 (t, J=5.0, 4H,
CH2), 7.02 (mc, 3H, ArH), 7.27 (mc, 3H, ArH), 7.43 (dd, J= 5.0 J=1.0 , 1H,
ArH). Anal. Calcd for C16H17FN2OS: C 63.14, H 5.63, N 9.20. Found: C
63.25, H 5.53, N 9.18.
d6 (DMSO-d6) or chloroform (CDCl3) with
a Varian Gemini 500
spectrometer (Varian Inc. Palo Alto, California USA); chemical shifts are
expressed in δ (ppm) and coupling constants (J) in hertz (Hz). Mass
spectra of selected compounds (2b, 2d, 2e and 2f) were measured in
[4-(4-Fluorobenzyl)piperazin-1-yl](furan-2-yl)methanone (2d)
CAS number: 425397-69-9. Yield 65%. White solid. Rf = 0.41. M.p. 129-
131 °C. 1H NMR (CDCl3): δ=2.48 (t, J=5.0, 4H, CH2), 3.50 (s, 2H, CH2),
3.80 (mc, 4H, CH2), 6.45 (mc, 1H, ArH), 6.97 (mc, 1H, ArH), 7.01 (t, J=8.7,
2H, ArH), 7.28 (t, J=8.7, 2H, ArH), 7.46 (mc, 1H, ArH). 13C NMR (CDCl3):
δ=52.96, 62.06, 111.21, 115.05, 116.23, 131.11, 133.35, 143.56, 147.98,
159.05, 163.07. MS (ESI): m/z: 289.0 [M+H+]. Anal. Calcd for
C16H17FN2O2: C 66.65, H 5.94, N 9.72. Found: C 66.35, H 5.98, N 9.62.
chloroform with
a Mass Spectrometer API 2000. Rf values were
determined on TLC plates using a mixture of DCM/MeOH (96:4) as eluent.
General procedures for the synthesis of compounds 2a-s
Pathway i) Synthesis of [4-(4-fluorobenzyl)piperazin-1-yl]methanone
derivatives 2a-e, 2k-m, 2q-s and 4-(4-fluorobenzyl)piperazin-1-
yl]ethanone derivative 2h. To a mixture of 1-(4-fluorobenzyl)piperazine 3
(0.5 mmol) and N,N-diisopropylethylamine (0.75 mmol) in DCM (2 mL) the
suitable (hetero)aroyl chloride derivative (0.6 mmol) was added. The
reaction was carried out under microwave irradiation for 10 min, at 50 °C.
After turning off the reaction by addition of MeOH (2 mL), water was added
and the mixture was extracted with DCM (3x5 mL). The obtained organic
phase was washed with brine (3x5 mL) and dried with anhydrous Na₂SO₄.
The solvent was removed under reduced pressure and the final products
were purified by crystallization with Et2O or by chromatographic column
(DCM/MeOH 98:02).
1,3-Benzodioxol-5-yl[4-(4-fluorobenzyl)piperazin-1-yl]methanone
(2e)
CAS number: 439846-81-8. Yield 74%. White solid. Rf = 0.40. M.p. 106-
107°C. 1H NMR (CDCl3): δ=2.43 (mc, 4H, CH2), 3.49 (s, 2H, CH2), 3.62
(mc, 4H, CH2), 5.99 (s, 2H, CH2), 6.82 (d, J = 7.8, 1H, ArH), 6.91 (mc, 2H,
ArH), 7.00 (t, J = 8.7, 2H, ArH), 7.28 (t, J = 8.7, 2H, ArH). 13C NMR (CDCl3):
δ=44.46, 53.04, 62.06, 101.38, 108.06, 108.16, 115.13, 121.56, 129.42,
130.59, 133.31, 147.56, 148.76, 163.07, 169.77. MS (ESI): m/z: 342.9
[M+H+]. Anal. Calcd for C16H17FN2O2: C 66.65, H 5.94, N 9.72. Found:
C 66.59, H 5.80, N 9.99.
For compounds 2b-e, 2h, 2k-l and 2r-s registered CAS numbers have
been already assigned. However, their synthetic procedures, chemical
properties and structural characterization are not available in literature,
except for compounds 2d and 2k [23]
[4-(4-Fluorobenzyl)piperazin-1-yl](1H-pyrrol-2-yl)methanone (2f)
Yield 40%. White solid. Rf = 0.29. M.p. 141-142 °C. 1H NMR (CDCl3):
δ=2.48 (t, J=5.0, 4H, CH2), 3.50 (s, 2H, CH2), 3.85 (mc, 4H, CH2), 6.23 (mc,
1H, ArH), 6.49 (mc, 1H, ArH), 6.90 (mc, 1H, ArH), 7.02 (t, J=8.5, 2H, ArH),
7.29 (t, J=8.5, 2H, ArH), 9.58 (bs, 1H, NH). 13C NMR (CDCl3): δ=52.97,
62.10, 109.41, 112.02, 115.11, 120.84, 124.62, 130.50, 133.38, 161.11,
163.07. MS (ESI): m/z: 288.0 [M+H+]. Anal. Calcd for C18H17FN4O5: C
66.88, H 6.31, N 14.62. Found: C 66.68, H 6.25, N 14.73.
Pathway ii) Synthesis of [4-(4-fluorobenzyl)piperazin-1-yl]methanone
derivatives 2f-g, 2j, 2n-p and 4-(4-fluorobenzyl)piperazin-1-
yl]ethanone derivative 2i. A mixture of the suitable carboxylate derivative
(1.2
mmol),
N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate (HBTU) (1 mmol) in DMF (4 mL) was stirred at room
temperature for 1 h. Then, a solution of the 1-(4-fluorobenzyl)pyperazine
(1 mmol) in TEA (1 mmol) was added dropwise. The reaction was carried
out under microwave irradiation for 10 min at 50 °C. Then, it is quenched
with water (10 mL) and extracted with DCM (3x5 mL). The organic phase
was dried with Na2SO4 and the solvent was removed in vacuo. The
residues were then purified by crystallization with Et2O or by
chromatographic column (DCM/MeOH 98:02), leading to the final
compounds. For compound 2o registered CAS number have been already
assigned. However, its synthetic procedure, chemical properties and
structural characterization are not available in literature.
[4-(4-Fluorobenzyl)piperazin-1-yl](1H-indol-6-yl)methanone (2g)
Yield 50%. Brown solid. Rf = 0.22. M.p. 141-142 °C. 1H NMR (CDCl3):
δ=2.43 (mc, 4H, CH2), 3.49 (s, 2H, CH2), 3.60 (mc, 4H, CH2), 6.50 (mc,
1H, ArH), 7.00 (t, J=8.7, 2H, ArH), 7.11 (d, J=8.1, 1H, ArH), 7.21 (mc, 1H,
ArH), 7.27 (t, J=8.7, 2H, ArH), 7.47 (s, 1H, ArH), 7.59 (d, J=8.1, 1H, ArH),
5
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9.31 (bs, 1H, NH). Anal. Calcd for C20H20FN3O: C 71.20, H 5.97, N 12.45.
Found: C 71.10, H 5.99, N 12.10.
131.01, 136.76, 137.03, 156.60, 160.37, 162.22 Anal. Calcd. for
C17H19FN2OS: C 64.13, H 6.01, N 8.80. Found: C 64.21, H 5.93, N 8.55.
1-[4-(4-Fluorobenzyl)piperazin-1-yl]-2-(2-thienyl)ethanone (2h)
[4-(4-Fluorobenzyl)piperazin-1-yl](3-bromo-2-thienyl)methanone (2n)
CAS number: 945139-36-6. Yield 35%. Oily residue. Rf = 0.43. 1H NMR
(CDCl3): δ=2.32 (m, 2H, CH2), 2.41 (m, 2H, CH2), 3.45 (s, 2H, CH2), 3.50
(m, 2H, CH2), 3.65 (m, 2H, CH2), 3.90 (s, 2H, CH2), 6.88 (dd, J1=4.79,
J2=1.10, 1H, H(3)-thiophene), 6.95 (dd, J1=4.79, J2=5.14, 1H, H(4)-
thiophene), 7.20 (dd, J1=5.14, J2=1.10, 1H, H(5)-thiophene), 6.98-7.28 (m,
4H, ArH). Anal. Calcd. for C17H19FN2OS: C 64.13, H 6.01, N 8.80. Found:
C 64.01, H 6.15, N 9.02.
Yield 99%. Oily residue. Rf = 0.71. 1H-NMR (CDCl3): δ=2.48 (m, 4H, CH2),
3.48 (s, 2H, CH2), 3.62 (m, 4H, CH2), 6.95 (d, J=5.17, 1H, H(4)-thiophene),
6.98-7.29 (m, 4H, ArH), 7.33 (d, J=5.17, 1H, H(5)-thiophene). 13C NMR
(CDCl3): δ=62.10, 109.74, 115.22, 115.39, 127.31, 130.30, 130.66, 132.24,
133.44, 161.27, 162.15, 163.22. Anal. Calcd. for C16H16BrFN2OS): C
50.14, H 4.21, N 7.31. Found: C 49.89, H 4.01, N 7.52.
[4-(4-Fluorobenzyl)piperazin-1-yl](5-chloro-2-thienyl)methanone (2o)
1-[4-(4-Fluorobenzyl)piperazin-1-yl]-2-(2-furyl)ethanone (2i)
CAS number: 945107-33-5. Yield 22%. White solid. Rf = 0.66. M.p. 79-
81 °C. 1H NMR (DMSO-d6): δ=2.38 (m, 4H, CH2), 3.48 (s, 2H, CH2), 3.61
(m, 4H, CH2), 7.13 (d, J=4.0, 1H, H(4)-thiophene), 7.14-7.36 (m, 4H, ArH),
7.29 (d, J=4.0, 1H, H(3)-thiophene). 13C NMR (DMSO-d6): δ=44.39, 44.40,
53.22, 106.30, 106.73, 115.67, 118.25, 121.07, 122.64, 122.80, 134.72,
138.43, 141.68, 159.69. Anal. Calcd. for C16H16ClFN2OS: C 56.72, H
4.76, N 8.27. Found: C 56.83, H 4.52, N 8.30.
Yield 78%. Oily residue. Rf = 0.48. 1H NMR (CDCl3): δ=2.35 (m, 2H, CH2),
2.40 (m, 2H, CH2), 3.46 (s, 2H, CH2), 3.52 (m, 2H, CH2), 3.64 (m, 2H, CH2),
3.74 (s, 2H, CH2), 6.17 (dd, J1=3.10, J2=0.90, 1H, H(4)-furan), 6.32 (dd,
J1=3.10, J2= 1.90, 1H, H(5)-furan), 6.98-7.28 (m, 4H, ArH), 7.33 (dd,
J1=1.90, J2=0.90, 1H, H(2)-furan). 13C-NMR (CDCl3): δ=34.04, 41.89,
46.18, 52.54, 52.87, 61.93, 107.50, 110.28, 110.60, 115.04, 115.21,
130.49, 130.55, 133.26, 133.28, 141.78, 148.74, 161.11, 163.06, 167.08.
Anal. Calcd. for C17H19FN2O2: C 67.53, H 6.33, N 9.27. Found: C 67.20,
H 6.40, N 9.18.
[4-(4-Fluorobenzyl)piperazin-1-yl](5-nitro-2-thienyl)methanone (2p)
Yield 55%. Brown solid. Rf = 0.72. M.p. 122-123 °C. 1H-NMR (DMSO-d6):
δ=2.41 (m, 4H, CH2), 3.50 (s, 2H, CH2), 3.61 (m, 4H, CH2), 7.14-7.33 (m,
4H, ArH), 7.43 (d, J=4.3, 1H, H(5)-thiophene), 8.06 (d, J=4.3, 1H, H(4)-
thiophene). 13C-NMR (DMSO-d6): δ=42.30, 46.35, 51.85, 52.15, 60.69,
112.82, 114.84, 115.01, 128.40, 129.12, 130.73, 133.77, 143.91, 152.00,
160.06, 160.37, 162.30. Anal. Calcd. for C16H16FN3O3S: C 55.00, H 4.62,
N 12.03. Found: C 54.89, H 4.58, N 11.98.
Benzothiophen-2-yl-[4-(4-fluorobenzyl)piperazin-1-yl]methanone (2j)
Yield 34%. White solid. Rf = 0.66. M.p. 216-221 °C; 1H NMR (DMSO-d6):
δ=3.16 (m, 2H, CH2), 3.41 (2H, CH2) 3.51 (s, 2H, CH2), 4.36 (s, 2H, CH2),
4.45 (m, 2H, CH2), 7.31-8.05 (m, 9H, Ar-H). 13C NMR (DMSO-d6): δ=42.30,
46.35, 51.38, 56.04, 60.68, 115.46, 115.73, 122.55, 122.97, 125.01,
125.21, 125.91, 127.02, 130.23, 138.55, 139.40, 162.61, 163.54. Anal.
Calcd. for C20H19FN2OS: C 67.77, H 5.40, N 7.90. Found: C 68.01, H
5.26, N 7.78.
[4-(4-Fluorobenzyl)piperazin-1-yl](3-methyl-2-furyl)methanone (2q)
Yield 44%. Oily residue. Rf = 0.46. 1H NMR (CDCl3): δ=2.25 (s, 3H, CH3),
2.48 (m, 4H, CH2), 3.50 (s, 2H, CH2), 3.71 (m, 4H, CH2), 6.30 (bs, 1H,
H(4)-furan), 6.98-7.29 (m, H, ArH), 7.3 (bs, 1H, H(5)-furan). 13C NMR
(DMSO-d6): δ=11.07, 50.62, 58.06, 67.03, 114.77, 115.58, 116.01, 133.68,
143.62, 159.46. Anal. Calcd. for C17H19FN2O2: C 67.53, H 6.33, N 9.27.
Found: C 67.61, H 6.28, N 9.48.
Benzofuran-2-yl-[4-(4-fluorobenzyl)piperazin-1-yl]methanone (2k)
CAS number: 788097-25-6. Yield 50%. Light yellow solid. Rf = 0.65. M.p.
98 °C. 1H-NMR (DMSO-d6): δ=2.43 (m, 4H, CH2), 3.51 (s, 2H, CH2), 3.69
(m, 2H, CH2), 7.14-7.74 (m, 9H ArH). 13C NMR (DMSO-d6): δ=44.41,
44.48, 53.16, 103.25, 103.50, 106.34, 106.77, 114.02, 115.42, 118.46,
118.75, 122.69, 122.85, 124.94, 140.01, 146.57, 151.71, 151.97, 156.56.
Anal. Calcd. for C20H19FN2O2: C 70.99, H 5.66, N 8.28. Found: C 71.08,
H 5.82, N 8.10.
[4-(4-Fluorobenzyl)piperazin-1-yl](5-methyl-2-furyl)methanone (2r)
CAS number: 945139-59-3. Yield 82%. Beige solid. Rf = 0.53. M.p. 74-
78 °C. 1H NMR (DMSO-d6): δ=2.29 (s, 3H, CH3), 2.37 (m, 4H, CH2), 3.48
(s, 2H, CH2), 3.63 (m, 4H, CH2), 6.21 (d, J=3.15, 1H, H(4)-furan), 6.84 (d,
J=3.15, 1H, H(3)-furan), 7.14-7.35 (m, 4H, ArH). 13C NMR (DMSO-d6):
δ=13.35, 42.30, 46.35, 51.85, 52.53, 60.85, 107.58, 114.82, 114.99,
116.84, 130.74, 133.85, 145.40, 153.89, 158.25, 160.37, 162.30. Anal.
Calcd. for C17H19FN2O2: C 67.53, H 6.33, N 9.27. Found: C 67.80, H
6.50, N 9.21.
[4-(4-Fluorobenzyl)piperazin-1-yl](3-methyl-2-thienyl)methanone (2l)
CAS number: 2337945-56-7. Yield: 82%. Oily residue. Rf = 0.52. 1H-NMR
(CDCl3): δ=2.26 (s, 3H, CH3), 2.44 (m, 4H, CH2), 3.49 (s, 2H, CH2), 3.62
(m, 4H, CH2), 6.82 (d, J=5.0, 1H, H(4)-thiophene), 6.98-7.28 (m, 4H, ArH),
7.29 (d, J=5.0, 1H, H(5)-thiophene). 13C NMR (CDCl3): δ=14.79, 53.18,
62.14, 77.16, 115.19, 115.36, 125.95, 129.80, 130.40, 130.63, 130.69,
133.4, 133.47, 137.32, 161.24, 163.19, 164.66. Anal. Calcd. for
C17H19FN2OS: C 64.13, H 6.01, N 8.80. Found: C 63.99, H 6.12, N 8.71.
[4-(4-Fluorobenzyl)piperazin-1-yl](5-nitro-2-furyl)methanone (2s)
CAS number: 831204-02-5. Yield 80%. Yellow solid. Rf = 0.66. M.p. 117-
120 °C. 1H NMR (DMSO-d6): δ=2.41 (m, 4H, CH2), 3.50 (s, 2H, CH2), 3.64
(m, 4H, CH2), 7.11-7.34 (m, 4H, ArH), 7.24 (d, J=3.9, 1H, H(3)-furan), 7.73
(d, 1H, J=3.9, 1H, H(4)-furan). 13C NMR (DMSO-d6): δ=42.30, 46.35,
51.85, 52.69, 60.68, 112.82, 114.85, 115.02, 116.93, 130.74, 133.78,
147.61, 151.16, 156.60, 160.37, 162.30. Anal Calcd. for C16H16FN3O4:
C 57.66, H 4.84, N 12.61. Found: C 57.58, H 4.71, N 12.33.
[4-(4-Fluorobenzyl)piperazin-1-yl](4-methyl-2-thienyl)methanone
(2m)
Yield 45%. White solid. Rf = 0.61. M.p. 93-95°C. 1H-NMR (DMSO-d6):
δ=2.21 (s, 3H, CH3), 2.40 (m, 4H, CH2), 3.50 (s, 2H, CH2), 3.62 (m, 4H,
CH2), 7.15 (m, 2H, ArH), 7.20 (s, 1H, H(3)-thiophene), 7.31(s, 1H, H(5)-
thiophene), 7.35 (m, 2H, ArH). 13C NMR (DMSO-d6): δ=15.25, 42.30,
46.35, 51.85, 52.41, 60.76, 112.82, 114.84, 115.01, 124.58, 130.77,
Mushroom tyrosinase inhibition assay
Mushroom tyrosinase (EC 1.14.18.1) was purchased from Sigma
Chemical Co. (St. Louis, MO, USA). Tyrosinase inhibition was assayed
6
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10.1002/cmdc.202000125
ChemMedChem
FULL PAPER
[16]
[24]
according to the method of Ferro et. al.
with minor modifications.
Cell viability assay
Briefly, aliquots (0.05mL) of sample at various concentrations (2 – 80 μM)
were mixed with 0.5 mL of L- tyrosine or L-DOPA solution (1.25 mM), 0.9
mL of sodium acetate buffer solution (0.05 M, pH 6.8) and preincubated at
25 °C for 10 min. Then 0.05 mL of an aqueous solution of mushroom
tyrosinase (333 U/mL) was added last to the mixture. This solution was
immediately monitored for the formation of dopachrome by measuring the
linear increase in optical density at 475 nm for 2 minutes using an UV-vis
spectrophotometer (Shimadzu UV-1601, Kyoto, Japan). DMSO was used
as a negative control and Kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-
pyran-4-one], a fungal secondary metabolite used as skin whitening agent,
was employed as a positive standard (2 – 30 μM). The extent of inhibition
by the addition of samples is expressed as inhibition percentage and
calculated as follows:
The human cervical carcinoma HeLa cell line was used in the cytotoxic
activity assay. All cells were cultured in DMEM medium supplemented with
10% fetal bovine serum (FBS), 2 mM L-Glutamine, penicillin (100U/mL)
and streptomycin (100 µg/mL) at 37 °C in a humidified atmosphere with
5% CO2. The cell survival assay was performed using colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.
Briefly, 100 µL suspended cells at an initial density of 3 × 104/mL were
seeded into each well of 96-well culture plates and allowed to adhere for
24 h before addition of compounds. HeLa cell line was exposed for 48 h to
compounds at concentration ranging from 4 to 100 µM for compound 2c
ad from 5 to 100 µM for compound 2d. Since DMSO was used as solvent
for compounds, cell viability was evaluated also in the presence of DMSO
alone, as solvent control. After incubation time, culture supernatants were
removed and exchanged with medium containing 0.5 mg/mL MTT. Then,
after 3h incubation at 37 °C, the medium was removed, and the cells were
lysed with 100 μL of DMSO. The optical density was measured at 570 nm
with an auto microplate reader (Infinite 200, Tecan, Austria). The mean
value and standard deviation (SD) were calculated from triplicate
experiments.
Inhibition %=(A-B/A)x 100
A= absorbance at 475 nm of negative control.
B= absorbance at 475 nm of test sample.
The concentrations leading to 50% activity lost (IC50) were also calculated
by interpolation of the dose-response curves.
Acknowledgements
This work was supported by the MIUR - "Finanziamento delle
Attività Base di Ricerca" FFABR funding 2017.
Docking simulation and binding free energy calculation
Docking studies were performed by Gold suite 5.7.1.[20] using the structural
coordinates of Agaricus bisporus tyrosinase in complex with the inhibitor
tropolone (PDB code 2Y9X) [19]. The ligand and water molecules were
removed and hydrogens were added by Discovery Studio 2.5.5.[25] The
ligand structures were constructed using Discovery Studio 2.5.5 and
energy optimized employing the Smart Minimizer protocol (1000 steps)
which combines the Steepest Descent and the Conjugate Gradient
methods. CHARMm was used as force field for the energy minimization.
Docking simulation was performed by using the same protocol as reported
in our previous paper [17]. Briefly, the binding site was defined in order to
contain the residues within 15Å from the position of the ligand in the x-ray
structure. A scaffold constraint was applied to restrict the solutions in which
the 4-fluorophenyl fragment matches its binding pose upon the cocrystal
structure of the active portion of inhibitors. [12] The side chains of residues
His244, Val248, His251, Met257, Asn260, Thr261, Phe264, Arg268 and
Leu275 were set as flexible and ChemPLP was chosen as fitness function.
For each ligand 100 genetic algorithms runs were performed. Results
differing less than 0.75 Å in terms of RMSD were clustered together. The
binding conformations corresponding to the most populated cluster were
chosen for analysis and representation. The obtained ligand-protein
complexes were optimized by running 1000 steps energy minimization
performed by means of NAMD 2.13. [26] Copper ions were replaced by zinc
ions as there are no parameters available for copper ions in CHARMM22
used as force field. During the simulation, zinc ions were kept fixed while
harmonic constraints were applied to the histidine residues 61, 85, 94, 259,
263 and 296 involved in the coordination of the two copper ions. The
binding free energy of the protein-ligand complexes were calculated by
Conflict of interest
The authors declare no conflict of interest.
Keywords: drug design • docking studies • MTT assay •
structure-activity relationship • tyrosinase inhibitors
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Entry for the Table of Contents
We reported the design and synthesis of a new series of tyrosinase inhibitors bearing the 4-Fluorobenzylpiperazine scaffold. The
obtained compounds were tested to evaluate their ability to inhibit mushroom tyrosinase. Cytotoxicity was also evaluated for selected
derivatives and docking studies were conducted to investigate their binding mode within tyrosinase catalytic pocket.
9
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