Chemical exploration of 4-(4-fluorobenzyl)piperidine fragment for the development of new tyrosinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.10.030

Tyrosinase is involved in the production of melanin through the hydroxylation of monophenols to o-diphenols. The role of this enzyme was extensively studied in order to identify new therapeutics preventing skin pigmentation and melanoma. In this work we initially identified the 3-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-one (1a) as promising mushroom tyrosinase inhibitor (IC₅₀= 252 μM). Then, several chemical modifications were performed and new analogues related to compound 1a were synthesized. Biochemical assays demonstrated that several obtained compounds proved to be effective inhibitors showing IC₅₀values lower both than “lead compound” 1a and reference inhibitor kojic acid, as a well-known tyrosinase inhibitor. The inhibition kinetics analyzed by Lineweaver–Burk plots revealed that compounds 2 a-c and 10b act as non-competitive inhibitors while the most active inhibitor 2d (IC₅₀= 7.56 μM) is a mixed-type inhibitor. Furthermore, experimental and computational structural studies were performed in order to clarify the binding mode of the derivative 2d.

Full text of DOI:10.1016/j.ejmech.2016.10.030

European Journal of Medicinal Chemistry 125 (2017) 992e1001
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Chemical exploration of 4-(4-fluorobenzyl)piperidine fragment for the
development of new tyrosinase inhibitors
,
*
a
a
a
Stefania Ferro a , Laura De Luca , Maria Paola Germano , Maria Rosa Buemi ,
ꢀ
Laura Ielo a, Giovanna Certo a b, Margarita Kanteev c, Ayelet Fishman c,
,
Antonio Rapisarda a, Rosaria Gitto a
a
ꢀ
Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM) Polo Universitario SS. Annunziata, Universita di Messina, Viale
Annunziata I-98168 Messina, Italy
b Fondazione Prof. Antonio Imbesi, Piazza Pugliatti 1, 98100 Messina, Italy
c Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 3200003, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyrosinase is involved in the production of melanin through the hydroxylation of monophenols to o-
diphenols. The role of this enzyme was extensively studied in order to identify new therapeutics pre-
venting skin pigmentation and melanoma. In this work we initially identified the 3-(4-benzylpiperidin-
Received 26 July 2016
Received in revised form
11 October 2016
Accepted 14 October 2016
Available online 18 October 2016
1-yl)-1-(1H-indol-3-yl)propan-1-one (1a) as promising mushroom tyrosinase inhibitor (IC50 ¼ 252
mM).
Then, several chemical modifications were performed and new analogues related to compound 1a were
synthesized. Biochemical assays demonstrated that several obtained compounds proved to be effective
inhibitors showing IC50 values lower both than “lead compound” 1a and reference inhibitor kojic acid, as
a well-known tyrosinase inhibitor. The inhibition kinetics analyzed by LineweavereBurk plots revealed
that compounds 2 a-c and 10b act as non-competitive inhibitors while the most active inhibitor 2d
Keywords:
Synthesis
Tyrosinase inhibitors
SARs
Kinetic mechanism
Docking studies
Crystallography
(IC50 ¼ 7.56
studies were performed in order to clarify the binding mode of the derivative 2d.
© 2016 Elsevier Masson SAS. All rights reserved.
mM) is a mixed-type inhibitor. Furthermore, experimental and computational structural
1. Introduction
evolves towards the formation of various melanin pigments.
Although the melanin production shields the human skin from
UV radiation, inhibiting photocarcinogenesis and affecting the
synthesis of the vitamin D3 [7], an excessive accumulation of
epidermal pigmentation, such us senile lentigines, freckles, ephe-
lide, melasma and other melanin hyperpigmentation, causes
serious esthetic problems in human beings [8,9].
Interestingly, it was recently demonstrated that tyrosinase is not
only involved in the melanin synthesis of peripheral tissues but also
in the substantia nigra (SN) of mice and humans playing a relevant
role in the brain neuromelanin formation [10]. In this case, an
excessive production of dopaquinones, by oxidation of dopamine,
results in neuronal damage and cell death, linking tyrosinase to
Parkinson's and other neurodegenerative diseases [11e13]. Thus, it
extensively was highlighted the relevance of tyrosinase and there is
an active interest among researchers to identify enzymatic in-
hibitors useful in clinical therapeutic applications as well as in
cosmetic industry [14e16]. To achieve this goal different chemical
classes of compounds, occurring from several sources, such as fla-
vonoids [17e20], stilbene derivatives [21e24], kojic acid [25e29],
Tyrosinase (monophenol monooxygenases, EC 1.14.18.1) is a
multifunctional enzyme widely distributed in nature and contain-
ing two copper ions coordinated with histidine residues in the
active site. Tyrosinase is the key enzyme in the melanin biosyn-
thesis, the main pigment commonly observed in bacteria, fungi,
plants, and animals. Melanin is also responsible for human skin
color [1e3]. The biosynthetic process for melanin formation occurs
in two distinct reactions [4e6]. In the first two steps tyrosinase
catalyzes both the hydroxylation of monophenol
diphenol 3,4-dihydroxyphenylalanine ( -DOPA) (monophenolase
activity) and the further oxidation of -DOPA to o-quinone (diphe-
nolase activity). Then dopaquinone, a highly reactive compound
determinant in the melanogenesis, rapidly and spontaneously
L-tyrosine to o-
L
L
Abbreviations:
nigra.
L-DOPA, o-Diphenol 3,4-dihydroxyphenylalanine; SN, Substantia
* Corresponding author.
E-mail address: sferro@unime.it (S. Ferro).
http://dx.doi.org/10.1016/j.ejmech.2016.10.030
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
993
tropolone [30] and novel synthetic compounds [31e38], have been
investigated. Obviously, more efforts are still needed in this direc-
tion and, therefore, we recently focused our interest to this bio-
logical target.
In a previous paper [39] we have reported the serendipitous
discovery of two indole derivatives (CHI 1043 and CHI 1164, Fig. 1)
able to inhibit the mushroom tyrosinase, displaying IC50 values of
224 and 372 mM, respectively.
Searching for further tyrosinase inhibitors from synthetic
source, we herein describe the development of a novel series of
indole derivatives in which several structural modifications were
carried out. The main goal was to improve the inhibitory effects and
expand our knowledge about chemical structural requirements
controlling the interaction with the enzyme. Thus, the synthesized
compounds were screened as mushroom tyrosinase inhibitors and
their mechanism of action was explored. Moreover, experimental
and computational analyses have been performed to highlight their
interactions within the catalytic binding site.
Fig. 2. The “lead compound” 3-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-
one (1a) retrieved from CHIME 1.5 database and the designed structural modifications.
The obtained data revealed that the first series of indole com-
pounds 1 b-d and 2 a-d were effective inhibitors both of mono-
phenolase and diphenolase activity, thus displaying IC50 values
lower than compound 1a. Notably derivatives 2 a-d exhibited
interesting inhibitory effect resulting more potent than kojic acid
(see Table 1).
On the basis of data displayed in Table 1, the following structure-
activity relationships might be drawn for this series of indole de-
rivatives based on 1a as lead structure.
2. Results and discussion
By comparison of inhibitory effects of 1a with the analogues 1 b-
d we can observe that the introduction of a methoxy substituent at
5- and/or 6-position of benzene fused ring generally improves the
inhibitory potency toward monophenolase activity.
In addition, as depicted in Table 1 the 3-(4-benzylpiperidin-1-
yl)-1-(5-methoxy-1H-indol-3-yl)propan-1-one (1b) was more
potent that prototype 1a against the two steps of melanin biosyn-
thesis. Particularly, it was seven-fold more active than lead com-
2.1. Design, synthesis and evaluation of tyrosinase inhibitory
activity
In the first step of this research we selected several indole-
compounds by means of a screening campaign throughout our
CHIME 1.5 database, which consists of a collection of small mole-
cules synthesized in our laboratory in the last decade. Mushroom
tyrosinase has been employed for the estimation of inhibitory ef-
fects. The best effective compound was the 3-(4-benzylpiperidin-1-
yl)-1-(1H-indol-3-yl)propan-1-one (1a) (Fig. 2) which proved to
pound 1a against the monophenolase activity (IC50 ¼ 25.78
mM vs
IC50 ¼ 175 M).
m
Furthermore, the presence of a fluorine atom at para-position of
the benzyl moiety induces a significant improvement of activity so
that the indoles 2 a-d were up to 35-fold more potent than analogs
1 a-d against diphenolase activity. Particularly, the most potent
analogue was the 1-(5,6-dimethoxy-1H-indol-3-yl)-3-(4-(4-
fluorobenzyl)piperidin-1-yl)propan-1-one (2d), in which the 5,6-
dimethoxy substitution is combined with the introduction of a
fluorine atom.
These data inspired us the synthesis of a further series of po-
tential inhibitors maintaining the 3-(4-(4-fluorobenzyl)piperidin-
1-yl)propan-1-one tail linked to different aromatic fragments.
Pursuing the objective to explore the impact on inhibitory effects of
the removal of indole nucleus of the most active compound 2d, we
planned the insertion of benzymidazole or (hetero)arylamino
chemical portions. Thus, the designed N-substituted 3-(4-(4-
inhibit the diphenolase activity showing IC50 value of 255 mM.
Hence, we chose the active compound 1a for generating the first
series of indole analogues possessing the methoxy substituent at 5
and/or 6 position of the indole nucleus and fluorine atom on the
benzyl ring (Fig. 2).
Scheme 1 summarizes the synthetic pathway employed to
obtain the title compounds 1 b-d and 2 a-d prepared according to
the previously reported method for the synthesis of “lead struc-
ture” 1a [40].
As depicted in Scheme 1 the appropriate indole 3 a-d was
converted into the corresponding 3-acetyl derivative 4 a-d under
VilsmeiereHaack conditions, using phosphoryl chloride and an
excess of N,N-dimethylacetamide. In the next step, compounds 1 a-
d and 2 a-d were prepared by a simple Mannich reaction between
the 3-acetylindole intermediates 4 a-d, the suitable amine de-
rivatives and paraformaldehyde.
Inhibitory effects of indole derivatives 1 b-d and 2 a-d on
mushroom tyrosinase activity have been evaluated and the results
of the biochemical assays are summarized in Table 1. Kojic acid and
fluorobenzyl)piperidin-1-yl)propanamides
7 and 10 a-c have
been synthesized as depicted in Scheme 2.
The synthesis of the 1-(1H-benzo[d]imidazol-1-yl)-3-(4-(4-
fluorobenzyl)piperidin-1-yl)propan-1-one (Scheme 2-A) has
7
been carried out in two steps starting from benzimidazole 5. By
reaction with 3-chloropropionyl chloride we prepared the
3-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-one
(1a)
were used as reference compounds for comparative purpose.
Fig. 1. Chemical structures of kojic acid, tropolone, CHI 1043 and CHI 1164.

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S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
Scheme 1. Reagents and conditions: (i) POCl3, N,N-dimethylacetamide, r.t. 24 h; (ii) DMF, paraformaldehyde, 4-benzylpiperidine hydrochloride or 4-(4-fluorobenzyl)piperidine
hydrochloride, HCl 37%, MW: 3 min 80 ꢀC.
Table 1
intermediate 6 which has been coupled with 4-(4-fluorobenzyl)
piperidine to give the desired compound 7. Moreover, the appro-
priate amine derivative 8 a-c (Scheme 2-B) was acetylated by using
Inhibitory effects of indole derivatives 1 a-d, 2 a-d and Kojic acid on mushroom
tyrosinase activity.
Monophenolase activity
IC50
M)a
Diphenolase activity
IC50
M)a
3-chloro propionyl chloride to achieve the intermediates 9 a-c.
Successively, the obtained amides 9 a-c were treated with 4-(4-
fluorobenzyl)piperidine to afford desired compounds 10 a-c. To
achieve 3-[4-(4-fluorobenzyl)piperidin-1-yl]-N-(1-H-pyrazol-3-yl)
propanamide (10c) the amino group of the starting material 3-
aminopirazole (8c) was opportunely protected (Scheme 2-C).
Finally, the desired compound 10c was obtained by deprotection
with a mixture of DCM and TFA (see supporting data).
Next, we designed and synthesized the ketone 13 as the isostere
of compound 10b to probe the role of the NH interaction. In the first
step, we prepared in good yields 3-{4-[(4-fluorophenyl)methyl]
piperidin-1-yl}propanenitrile (12) by reaction of 4-(4-fluorobenzyl)
(
m
(m
1a
1b
1c
1d
2a
2b
2c
2d
175.00 2.71
25.78 1.80
67.25 5.92
57.24 1.93
9.45 0.36
7.86 3.02
9.40 0.63
5.11 0.36
26.00 0.13
252.00 3.60
197.45 10.62
168.55 14.01
133.00 2.32
8.82 0.38
14.15 1.60
18.03 0.91
7.56 1.90
Kojic acid
17.76 0.18
a
All compounds were examined in a set of experiments repeated three times;
IC50 values of compounds represent the concentration that caused 50% enzyme
activity loss.
Scheme 2.

S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
995
Table 2
piperidine (11) with 3-bromopropane nitrile in alkaline medium by
Inhibitory effects of derivatives 7, 10 a-c and 13 on mushroom tyrosinase activity.
TEA (see Scheme 3). Then, the intermediate 12 was converted in the
designed product 13 using benzyl magnesium chloride under
Grignard condition.
Table 2 collects the data of the biochemical assays for the second
series of designed compounds. As results the new tested com-
pounds provided enzyme inhibitory effects at doses ranging from
27.9 to 159.24 mM. Thereby the new 4-(4-fluorobenzyl)piperidine
R
Monophenolase activity
IC50
M)a
Diphenolase activity
IC50
M)a
derivatives (7, 10 a-c, 13) proved to be more active than prototype
1a but they resulted less active than the corresponding indoles 2 a-
d (see Table 1). Among this new series of compounds, while the
replacement of indole nucleus with phenylamine moiety (i.e.
compound 10b) or pyrazole ring (i.e. compound 10c) produced a
moderated reduction of inhibitory effects, the introduction of
benzimidazole (i.e. compound 7) or 3-methylisoxazole (i.e. com-
pound 10a) moiety gave a loss of activity. Overall, the most active
inhibitor 10b displayed inhibitory potency comparable to that of
kojic acid (see Table 1).
(
m
(m
7
113.34 16.37
159.24 18.65
10a
28.55 0.75
110.95 18.55
10b
10c
28.85 0.95
27.90 0.99
By means of Gold program docking simulations were performed
in attempt to draw a plausible binding mode for the most active
inhibitor 2d. Specifically, we have employed the structural co-
ordinates (PDB code 2Y9X) of the potent inhibitor tropolone
5.87 1.59
39.58 9.61
83 6.54
(IC50 ¼ 0.4
mM against diphenolase activity) [41,42] in complex with
13
13.98 0.81
the H2L2 tetrameric complex of mushroom tyrosinase [30].
Fig. 3 displays the docking pose of inhibitor 2d (green) over-
lapping the crystal structure orientation of inhibitor tropolone
(yellow) within catalytic pocket [30]. The (4-fluorobenzyl)piperi-
a
All compounds were examined in a set of experiments repeated three times;
IC50 values of compounds represent the concentration that caused 50% enzyme
activity loss.
dine moiety of compound 2d is projected in a cavity forming
p-p
interaction with the crucial residue of His263, cation- interaction
p
with His244 and Van der Waals interaction with Val283. In addi-
tion, the indole nucleus of compound 2d occupies the binding area
adjacent to the entrance of the catalytic pocket cavity. The binding
pose of the indole core of compound 2d appears to be characterized
by several specific contacts: a) the two oxygen atoms of the
methoxy groups engage profitable hydrogen bond interactions
with Asn81; b) indole NH function establishes an additional
hydrogen bond contact with the carbonyl oxygen of the crucial
residue His85 backbone. Finally, the proposed binding of 2d is
characterized by a lipophilic sandwich of its piperidine ring be-
tween the residues of Val248 and Val283.
Furthermore, the docking studies suggested a binding mode of
the 4-fluorobenzyl portion of 2d overlapping the inhibitor tropo-
lone in the catalytic site of mushroom tyrosinase and sharing some
key interactions. It has been reported that tropolone occupies the
active site establishing van der Waals interactions with the side
chain of Val283 and His263 as well as
p stacking interaction with
the side chain of Phe264. Furthermore, this inhibitor does not
interact with the copper ions which coordinate two different set of
three histidine residues (CuA: His61, His85, His94; CuB: His259,
His263 and His296) [30].
Thus, to gain more information about the interactions formed by
the optimized inhibitor 2d, we choose to test the inhibitory effects
of the main molecular fragments that seem to occupy two different
cavities in the catalytic site of mushroom tyrosinase. Table 3 dis-
played the IC50 values determined for the four fragments: 1-(5,6-
Fig. 3. Docking pose of compound 2d (green) and tropolone (yellow). The copper ions
are represented by orange spheres and the hydrogen bonds are drawn as yellow
dashed lines. The figure was prepared in PYMOL software [54]. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Scheme 3. Reagents and conditions: i) 3-bromopropane-nitrile, DCM, TEA, r.t., 6 h; ii) benzyl-magnesium-chloride, Et2O, reflux, 5 h.

996
S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
Table 3
10b the diphenolase activity was measured as a function of
increasing concentration of -DOPA and is presented using
Inhibitory effects of compounds 2d, 4d, 11, 14 and 15 on mushroom tyrosinase
activity.
L
Lineweaver-Burk double reciprocal plots (see Fig. 5 and Fig. S1).
For compounds 2 a-d, the results showed that the plots of 1/V
versus 1/[S] gave straight lines with different slopes intersecting on
the horizontal axis. These data suggested that compounds 2 a-c act
as non-competitive inhibitors since they are able to bind with equal
affinity to the free enzyme as well as to the enzyme-substrate
complex. As a consequence, the increase of 2 a-c concentration
entails the decrease of Vm value, while Km value remains
unchanged.
For 2d derivative, a different behaviour of inhibition is observed.
In fact, the LineweavereBurk double reciprocal plots gave straight
lines with different slopes intersecting one another in the second
quadrant. In this case, the reduced values of both Vm and Km
indicated that compound 2d is a mixed-type inhibitor.
Monophenolase activity
IC50
M)a
Diphenolase activity
IC50
M)a
(
m
(m
2d
4d
11
14
15
5.11 0.36
N.A.
43.70 2.61
32.17 4.19
NA
7.56 1.90
N.A.
286.83 10.52
116.0 17.69
NA
a
All compounds were examined in a set of experiments repeated three times;
IC50 values of compounds represent the concentration that caused 50% enzyme
activity loss. NA no activity.
dimethoxy-1H-indol-3-yl)ethanone (4d), 4-(4-fluorobenzyl)piper-
idine (11) 1-ethyl-4-[(4-fluorophenyl)methyl]piperidine (15) and
4-[(4-fluorophenyl)methyl]-1-methylpiperidine (16) (See Fig. 4).
Compounds 15 and 16 were synthesized as depicted in Scheme
S1 (see supporting data) following a procedures reported in liter-
ature [43,44].
It is interesting to note that the amine 11 and its N-ethyl de-
rivative (14) inhibited tyrosinase activity (IC50 value of 43.7
32.17 M respectively) in the first step of enzymatic pathway. The
2.3. Structural studies
To further validate the predicted binding mode of compound 2d,
we carried out preliminary structural studies with TyrBm which
displays 42% identity with mushroom tyrosinase.
mM and
m
A structure of tyrosinase from Bacillus megaterium (TyrBm) with
bound substrates was recently reported, indicating that mono-
phenols and diphenols bind similarly in the active site [45]. The
overall structure of tyrosinases, the binding site and the catalytic
mechanism are similar, although second shell residues differ
among enzymes [46,47]. Obtaining crystal structures with bound
ligands relies on the solubility of concentrated solutions of the
desired inhibitor/substrate in the crystallization medium. Com-
pound 2d was soluble in DMSO, however the solvent was harmful
to the TyrBm crystals. Electron density observed in the active site
confirmed the presence of the inhibitor molecule (Fig. 6). A reso-
lution of 2 Å was obtained, nevertheless, the structure was not
deposited in the protein data bank since the substrate was not fully
visualized.
Based on the data obtained (see supporting data), the 4-
fluorobenzyl moiety of compound 2d is situated between the two
copper ions, with the aromatic ring stabilized through stacking
interactions with His208 (Fig. 6B). The fluorine atom of 2d is
positioned between the copper ions, similar to the sulfur atom of
phenylthiourea (PTU), in the structure of catechol oxidase from
Ipomoea batatas (PDB code 1BUG) [48]. In this structure, the sulfur
of PTU replaced the hydroxyl bridge between the copper ions.
Therefore, it can be suggested that 2d prevents oxygen binding to
the active site and thus inhibits enzymatic activity. The preliminary
results of the 2d structure in complex with bacterial tyrosinase
supports the positioning of the 4-fluorobenzyl moiety (Fig. 6) with
N-methyl analog 15 displays low activity when compared with
ethyl and unsubstituted analogs. These experimental results were
consistent with the suggested binding mode of inhibitor 2d for
which the 4-(4-fluorobenzyl)piperidine occupies the catalytic
pocket of mushroom tyrosinase. By contrast, the tyrosinase activ-
ities were not absolutely affected by the indole derivative 4d, thus
suggesting that this small heterocyclic moiety occupies a binding
region which is not sufficient to address inhibitory effects or that it
is not able to occupy it.
2.2. Kinetic mechanism
On the basis of the mechanism of action, different classes of
tyrosinase inhibitors can be distinguished: a) competitive in-
hibitors, which bind to the “free tyrosinase” thus preventing sub-
strate binding; b) uncompetitive inhibitors, which bind to the
tyrosinase substrate complex; c) mixed type inhibitors, which bind
to not only with a free tyrosinase, but also with the tyrosinase-
substrate complex, and their equilibrium binding constants for
the free tyrosinase and the tyrosinase-substrate complex are
different; d) non-competitive inhibitors, which could bind to a free
tyrosinase and a tyrosinase-substrate complex, with the same
equilibrium constant. As a consequence, to obtain further infor-
mation about the type of inhibition exerted on mushroom tyrosi-
nase by the most promising derivatives 2 a-d and for compound
Fig. 4. Optimized inhibitor 2d and related molecular fragments 4d, 11, 14 and 15.

S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
997
Fig. 5. Lineweaver-Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by compounds 2a (A), 2b (B), 2c (C), 2d (D).

998
S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
Fig. 6. Visualization of part of the molecule 2d in the active site of tyrosinase from Bacillus megaterium (TyrBm). A) Fluorobenzene (green), the aromatic portion of 2d is observed in
the active site of TyrBm (grey surface). Copper ions are shown as brown spheres and residues surrounding the active site as white sticks. B) Fluorobenzene (green) with its 2Fo-Fc
electron density map (light blue wire) contoured at 0.8s. Copper ions are shown as brown spheres and histidine residues of the active site as white sticks. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
the predicted orientation within mushroom tyrosinase (Fig. 3) for
which the His263 corresponds to His208 in TyrBm.
DCM/MeOH (90:10) for compounds 1 b-d, 2 a-d, 7, 10 a-c, and a
mixture of DCM/MeOH (96:4) for compounds 13, 14, 15. 1H NMR
spectra were measured in dimethylsulfoxide-d6 (DMSO-d6) or
CDCl3 with a Gemini 300 spectrometer (Varian Inc. Palo Alto, Cal-
3. Conclusions
ifornia USA); chemical shifts are expressed in d (ppm) and coupling
constants (J) in Hz.
Novel indoles as mushroom tyrosinase inhibitors have been
identified. All the obtained compounds (1 b-d, 2 a-d, 7, 10 a-c and
13) and the molecular fragments 11 and 14 inhibited tyrosinase at
micromolar concentration. Especially, compound 2d exhibited
4.1.1. General procedure for the synthesis of 1-(1H-indol-3-yl)
ethanone derivatives (4 a-d)
Compounds 4 a-d were prepared according a previously re-
ported procedure [40,49]. The appropriate indole 3 a-d (1 mmol)
was added to a solution of phosphoryl chloride (0.92 mL, 10 mmol)
in dimethylacetamide (2.79 mL, 30 mmol) and the resulting
mixture was stirred at room temperature for 24 h. Then, the reac-
tion mixture was basified with sodium hydroxide solution (4N) and
extracted with EtOAc (3 ꢂ 10 mL). The organic phases were dried
over Na2SO4 and the solvent removed under reduced pressure. The
final compounds were obtained by crystallization with a mixture of
Et2O and DCM.
inhibitory activity with an IC50 value of 7.56 mM (diphenolase ac-
tivity), higher than reference compound kojic acid. Moreover, the
inhibitory mechanism and kinetics studies revealed that derivative
2d is a mixed-type inhibitor. Finally, we compared the obtained
data by means of the docking binding pose and the preliminary
crystal structure of the most active compound 2d within the cat-
alytic pocket of mushroom tyrosinase and bacterial tyrosinase,
respectively. The obtained information could aid in the design of
new inhibitors. Specifically, the most interesting outcome of this
study was to find that the presence of a fluorine atom on the benzyl
moiety of compound 2d exerts a significant influence on the inhi-
bition of tyrosinase activity.
4.1.2. General procedures for the synthesis of 3-(4-benzylpiperidin-
1-yl)-1-(1H-indol-3-yl)propan-1-ones (1 a-d, 2 a-d)
The appropriate 3-acetyl derivative (4 a-d) (1 mmol) was solu-
bilized in DMF (3 mL). Successively, paraformaldehyde (39.0 mg,
1.3 mmol), the corresponding secondary amine (4-
benzylpiperidine or 4-(4-fluorobenzyl)piperidine) hydrochloride
(1.1 mmol), and a catalytic amount of hydrochloric acid (37%) were
added. The obtained solution was irradiated in a microwave at 80 ꢀC
for 30 using a CEM focused Microwave Synthesis System. The
mixture was quenched with H2O and extracted with EtOAc
(3 ꢂ 10 mL). The organic phases were dried with dry Na2SO4 and
the solvent was removed in vacuo. The final compounds were ob-
tained by purification with flash chromatography (FC, DCM/CH3OH
90:10) and re-crystallized with Et2O.
4. Experimental
4.1. Chemistry
All starting materials and reagents commercially available
(Sigma-Aldrich Milan, Italy; Alfa Aesar Karlsruhe, Germany) were
used without further purification. Microwave-assisted reactions
were carried out in a focused Microwave Synthesis System (CEM
Technology Ltd Buckingham, UK). Melting points were determined
on a Buchi B-545 apparatus (BUCHI Labortechnik AG Flawil,
Switzerland) and are uncorrected. Elemental analyses (C, H, N)
were carried out on a Carlo Erba Model 1106 Elemental Analyzer
(Carlo Erba Milano, Italy); the results confirmed a ꢁ95% purity.
Merck silica gel 60 F254 plates were used for analytical TLC (Merck
KGaA, Darmstadt, Germany). Flash Chromatography (FC) was car-
ried out on a Biotage SP1 EXP (Biotage AB Uppsala, Sweden). Rf
values were determined on TLC plates using as eluent a mixture of
4.1.2.1. 3-(4-Benzylpiperidin-1-yl)-1-(5,6-dimethoxy-1H-indol-3-yl)
propan-1-one (1d). Yield: 42%, m.p. 158e160 ꢀC; r.f. 0.20; 1H NMR
(DMSO-d6) (d) 1.26e3.08 (m, 15H), 3.87 (s, 3H, OCH3), 3.94 (s, 3H,
OCH3), 6.86 (s, 1H, H-7), 7.14e7.30 (m, 5H, ArH), 7.69 (s, 1H, H-4),

S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
999
7.88 (s, 1H, H-2), 9.48 (bs, 1H, NH). Anal. Calcd for C25H30N2O3: C:
73.86; H: 7.44; N: 6.89. Found: C: 73.96; H: 7.53; N: 6.87.
4.1.4.3. 3-{4-[(4-Fluorophenyl)methyl]piperidin-1-yl}-N-phenyl-
propanamide (10b). Yield 44%, m.p. 110e112 ꢀC; r.f. 0.40; 1H NMR
(DMSO-d6) (d): 1.14e2.89 (m, 15H), 7.00e7.09 (m, 3H, ArH),
7.15e7.17 (m, 2H, ArH), 7.26e7.29 (m, 2H, ArH), 7.52 (d, 2H, J ¼ 7.6,
ArH), 10.12 (bs, 1H, NH). Anal. Calcd for C21H25FN2O: C: 74.09; H:
7.40; N: 8.23. Found: C: 74.29; H: 7.60; N: 8.43.
4.1.2.2. 3-[4-[(4-Fluorophenyl)methyl]-1-piperidyl]-1-(1H-indol-3-
yl)propan-1-one (2a). Yield 35%, m.p. 182e184 ꢀC; r.f. 0.37; 1H NMR
(DMSO-d6) (d) 1.14e3.04 (m, 15H), 7.06e7.21 (m, 6H, ArH), 7.43 (d,
1H, J ¼ 8.2), 8.16 (d, 1H, J ¼ 7.1), 8.34 (s, 1H, H-2), 11.93 (bs, 1H, NH).
Anal. Calcd for C23H25FN2O: C: 75.80; H: 6.91; N: 7.69. Found: C:
75.85; H: 6.85; N: 6.72.
4.1.4.4. 3-[4-(4-Fluorobenzyl)piperidin-1-yl]-N-(1-H-pyrazol-3-yl)
propanamide (10c). Yield 64%, m.p. 137e139 ꢀC; r.f. 0.05; 1H NMR
(DMSO-d6) (
8H, ArH), 7.55 (s, 1H, ArH), 10.55 (bs, 1H, NH). Anal. Calcd for
18H23FN4O: C: 65.43; H: 7.02; N: 16.96. Found: C: 65.53; H: 7.12; N:
d): 1.21e2.95 (m, 15H), 6.44 (s, 1H, ArH), 7.07e7.20 (m,
4.1.2.3. 1-(5,6-Dimethoxy-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)
piperidin-1-yl)propan-1-one (2d). Yield 25%, m.p. 211e212 ꢀC; r.f.
C
0.19; 1H NMR (DMSO-d6) (
d); 1.11e2.99 (m, 15H), 3.77 (s, 3H, OCH3),
16.86.
3.78 (s, 3H, OCH3), 6.96 (s, 1H, H-7), 7.00e7.19 (m, 4H, ArH), 7.66 (s,
1H, H-4), 8.16 (s, 1H, H-2), 11.68 (bs, 1H, NH). Anal. Calcd for
4.1.5. Synthesis of 3-{4-[(4-fluorophenyl)methyl]piperidin-1-yl}
propanenitrile (12)
C
25H29FN2O3: C: 70.73; H: 6.89; N: 6.60. Found: C: 70.80; H: 6.85;
N: 6.65.
The spectral data for compouds 1b, 1c, 5b, 5c are in accordance
To
a stirred solution of 4-(4-fluorobenzyl)piperidine (11)
(400 mg, 2.1 mmol) in CH2Cl2 (4.7 mL) was added 3-
bromopropane-nitrile (281 mg, 2.1 mmol) and TEA (0.6 mL,
4.2 mmol). The mixture was stirred at room temperature for 6 h and
after TLC analysis, using as eluent DCM/MeOH 96:4, was added H2O
(5 mL) and the solution was extracted with CH2Cl2 (3 ꢂ 5 ml). The
organic phase was dried over Na2SO4 and the solvent removed
under reduced pressure to give the final compound as a yellow oil.
with the literature [40].
4.1.3. General procedure for the synthesis of 1-(1-H-benzimidazol-
1-yl)-3-chloropropan-1-one (6), 3-chloro-N-(3-methyl-1,2-oxazol-
5-yl)propanamide (9a) 3-chloro-N-phenyl-propanamide (9b) and
tert-butyl-3-(3-chloropropanoylamino)pyrazole-1-carboxylate (9c)
To a solution of the appropriate starting compound (5, 8 a-c)
(1 mmol) in THF (5 mL) 3-chloro-propionyl chloride (95 mL,1 mmol)
4.1.6. Synthesis of 3-[4-(4-fluorobenzyl)piperidin-1-yl]-1-phenyl-4-
butan-2-one (13)
at 0 ꢀC and dropwise was added. For compound (9b) this reaction
was carried out in alkaline medium by K2CO3 (207 mg, 1.5 mmol).
The reaction mixture was stirred at room temperature for a period
of 2/16 h. Successively, the reaction was quenched with NaHCO3
saturated aqueous solution and extracted with EtOAc (3 ꢂ 10 mL).
The organic layer was separated dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude material was then
subjected to silica gel chromatography by using cyclohexane/EtOAc
(50:50).
To a mixture of benzyl-magnesium-chloride in Et2O (1 M)
(2.4 mL, 2.4 mml) was added a solution of intermediate 12 (303 mg,
1.2 mmol) in Et2O (1 mL). The reaction mixture was heated at reflux
for 2 h. After a TLC analysis, using as eluent DCM/MeOH 96:4, was
added a saturated ammonium chloride solution. The mixture was
extracted with Et2O. The organic phase was dried over Na2SO4 and
the solvent removed under reduced pressure to give the final
compound as a yellow oil residue. Yield 92%, oil, r.f. 0.68; 1H NMR
(CDCl3) 1.22e1.33 (m, 2H, 2CH), 1.50 (m, 1H, CH), 1.59e1.64 (m, 2H,
2CH), 1.95e2.02 (m, 2H, 2CH), 2.45e2.50 (m, 4H, 2CH2), 2.63e2.67
(m, 2H, CH2), 2.84e2.87 (m, 2H, CH2), 4.68 (s, 2H, CH2), 6.92e6.98
(m, 2H, ArH), 7.07e7.10 (m, 2H, ArH), 7.17e7.37 (m, 3H, ArH). Anal.
Calcd for C22H26FNO: C: 77.84; H: 7.72; N: 4.13. Found: C: 78.24; H:
8.12; N: 4.53.
4.1.4. General procedure for the synthesis of 1-(1-H-benzoimidazol-
1-yl)-3-(4-(4-fluorobenzyl)piperidin-1-yl)propan-1-one (7), 3-[4-
(4-fluorobenzyl)piperidin-1-yl]-N-(3-methyl-1,2-oxazol-5-yl)
propanamide (10a), 3-{4-[(4-Fluorophenyl)methyl]piperidin-1-yl}-
N-phenylpropanamide (10b), and 3-[4-(4-fluorobenzyl)piperidin-1-
yl]-N-(1-H-pyrazol-3-yl)propanamide (10c)
4-(4-Fluorobenzyl)piperidine (1 mmol) was added to a DMF
(2 mL) solution of intermediates (6, 9 a-c) (195 mg, 1 mmol) in
alkaline medium by K2CO3 (276.4 mg, 2 mmol). The obtained
mixture was refluxed for 2 h and then cooled at room temperature
and stirred for 24 h. After this time, a NaHCO3 saturated aqueous
solution was added to quench the reaction and successively, the
mixture was extracted with EtOAc (3 ꢂ 10 mL), dried over Na2SO4
and evaporated under reduced pressure. The desired compounds 7
and 10 a-c were obtained by crystallization with Et2O.
4.2. Mushroom tyrosinase inhibition assay
Tyrosinase inhibition was assayed according to the method of
Masamoto [50] with minor modifications [51]. Briefly, aliquots
(0.05 mL) of sample at various concentrations (5e300
mM) were
mixed with 0.5 mL of -tyrosine or -DOPA solution (1.25 mM),
L
L
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 so-
lution of mushroom tyrosinase (333 U/mL) was added last to the
mixture. The linear increase in adsorbance (Abs) at 475 nm was
measured after 30 or 5 min of incubation time in the reaction
mixture containing L-tyrosine or L-DOPA, respectively. The inhibi-
tory activity of samples is expressed as inhibition percentage and
calculated as follows:
4.1.4.1. 1-(1-H-Benzoimidazol-1-yl)-3-(4-(4-fluorobenzyl)piperidin-
1-yl)propan-1-one (7). Yield 46%, m p.108e110 ꢀC; r.f. 0.34. 1H NMR
(DMSO-d6) (d): 1.07e2.89 (m, 15H), 7.02e7.50 (m, 8H, ArH), 9.58 (s,
1H, H-2). Anal. Calcd for C22H24FN3O: C: 72.31; H: 6.62; N: 11.50.
Found: C: 72.21; H: 6.42; N: 11.40.
Inhibition % ¼ {[(AꢃB)ꢃ(CꢃD)]/AꢃB}/100.
4.1.4.2. 3-[4-(4-Fluorobenzyl)piperidin-1-yl]-N-(3-methyl-1,2-
oxazol-5-yl)propanamide (10a). Yield 69%, m.p. 78e80 ꢀC; r.f. 0.52;
A: Abs acetate buffer and enzyme.
B: Abs acetate buffer.
1H NMR (DMSO-d6) (
d): 1.16e3.17 (m, 15H), 6.08 (s, 1H, ArH),
7.03e7.19 (m, 4H, ArH). Anal. Calcd for C19H24FN3O2: C: 66.07; H:
7.00; N: 12.16. Found: C: 66.27; H: 7.20; N: 12.36.
C: Abs acetate buffer, test sample and enzyme.
D: Abs acetate buffer and test sample.

1000
S. Ferro et al. / European Journal of Medicinal Chemistry 125 (2017) 992e1001
The concentrations leading to 50% activity lost (IC50) were also
calculated by interpolation of the dose-response curves. Kojic acid
[5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one], fungal sec-
ondary metabolite used as skin whitening agent, was employed as a
positive standard (8e35 M).
support. We thank the staff of the European Synchrotron Radiation
Facility (beamline ID 29) for provision of synchrotron radiation
facilities and assistance.
a
m
Appendix A. Supplementary data
4.3. Kinetic analysis of the tyrosinase inhibition
Supplementary data related to this chapter can be found at
http://dx.doi.org/10.1016/j.ejmech.2016.10.030.
The inhibition kinetics of the most active compounds on the
tyrosinase were studied using LineweavereBurk double reciprocal
plots [52]. For the test, the reaction mixture consisted of four
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