Targeting Tyrosinase: Development and Structural Insights of Novel Inhibitors Bearing Arylpiperidine and Arylpiperazine Fragments

Article abstract of DOI:10.1021/acs.jmedchem.7b01745

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyB

Full text of DOI:10.1021/acs.jmedchem.7b01745

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Targeting Tyrosinase: Development and Structural Insights of
Novel Inhibitors Bearing an Arylpiperidine/Arylpiperazine Fragment
Stefania Ferro, Batel Deri, Maria Paola Germanò, Rosaria Gitto, Laura Ielo, Maria Rosa Buemi,
Giovanna Certo, Serena Vittorio, Antonio Rapisarda, Yael Pazy, Ayelet Fishman, and Laura De Luca
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01745 • Publication Date (Web): 10 Apr 2018
Downloaded from http://pubs.acs.org on April 10, 2018
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Targeting Tyrosinase: Development and Structural
Insights of Novel Inhibitors Bearing an
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Arylpiperidine/Arylpiperazine Fragment
∞
∞
Stefania Ferroa, , Batel Deri,b, Maria Paola Germanòa, Rosaria Gittoa, Laura Ieloa, Maria Rosa
Buemia, Giovanna Certoc, Serena Vittorioa,c, Antonio Rapisardaa, Yael Pazyd, Ayelet Fishmanb,
Laura De Lucaa,*
a Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM)
Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata Iꢀ98168 Messina, Italy
bDepartment of Biotechnology and Food Engineering, TechnionꢀIsrael Institute of Technology,
Haifa, 3200003, Israel
cFondazione Prof. Antonio Imbesi, Piazza Pugliatti 1, 98100 Messina, Italy
dTechnion Center for Structural Biology, TechnionꢀIsrael Institute of Technology, Haifa, 3200003,
Israel
∞S.F. and B.D. contributed equally to this work.
KEYWORDS Tyrosinase, microwave irradiations, Xꢀray crystallography, docking studies, Ty
Inhibitors
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ABSTRACT
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Inhibition of tyrosinase (Tys, EC 1.14.18.1) represents an efficient strategy to decrease
melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies
on two different tyrosinase, from Bacillus megaterium (TyBm) and from mushroom (TyM) has
contributed to increase our knowledge about their structural information and translating them on the
most druggable human Ty (TyH) isozyme. In particular we designed and synthesized a series of 1ꢀ
(4ꢀfluorobenzyl)piperazine and 1ꢀ(4ꢀfluorobenzyl)piperidine derivatives showing inhibitory activity
on TyM at micromolar range resulting more potent than reference compound kojic acid. The crystal
structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 2.03 µM) were
solved confirming the binding pose hypothesized by in silico studies and revealing the main
molecular determinants for the binding recognition of the inhibitors.
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INTRODUCTION
Tyrosinases (Tys, EC 1.14.18.1) are metalloenzymes abundant in a wide range of organisms
(mammals, fungi, bacteria and plants). Tys display a binuclear active site, composed by two copper
ions coordinated by six histidine residues, and catalyze the oxidation of phenols and catechols into
catechols and orthoꢀquinones, respectively. In mammals, the conversion of Lꢀtyrosine into quinones
is considered the key step of melanogenesis in which generated melanin pigments play an important
role in prevention of skin damage due to UV radiation and free radicals.1, 2 It is wellꢀknown that
melaninꢀrelated pathologies can cause skin hyperpigmentation, lesions or melasma.3 Thus, Ty
inhibition is one strategy aimed at controlling the production of melanin. Consequently, the
development of Ty inhibitors (TyIs) gained high interest in the therapy of skin pathologies as well
as in dermocosmetic treatments. Several TyIs such as hydroquinone, arbutin or kojic acid are
currently used as depigmentation agents.4 Unfortunately, these inhibitors demonstrated relevant
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human toxicity.3 There are different Ty enzymes from distinct sources such as human (TyH),
mushroom (Agaricus bisporus, TyM) and bacterial (e.g. Bacillus megaterium, TyBm) that share
low similarity around 10–30%.5 Both TyM and TyBm are cytosolic proteins, whereas TyH is a
monomeric glycosylated transmembrane isoform. Probably due to the different architecture of these
isozymes, some effective inhibitors of TyH revealed poor activity against TyM and vice versa.6
No structural data is currently available for the most druggable TyH at atomic resolution, with
exception of homology models built with TyBm and Ipomea batata cathecol oxidase7 and the
recently reported structure of tyrosinase related protein 1.8 On the contrary structural and
biochemical information has been reported for TyM and TyBm.
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To start our exploration we focused our studies on the available TyM and TyBm which share 58%
identity within 6 Å from the two copper centers5, 9 to obtain molecular probes for the identification
of selective Ty inhibitors. To evaluate the inhibitory activity of our designed derivatives we
followed the most employed Ty inhibition assay protocol which apply TyM that consequently is
used as protein structure for our docking studies. To gain more strength at our computational results
we performed10 the crystal studies of our ligands with the TyBm which share an higher homology
with to the human enzyme.
It has been reported that compounds from natural or synthetic sources (chalcones, flavonoids,
coumarins, thioureas, peptides, and so on) can affect the Ty active site as substrates, competitive,
and nonꢀcompetitive inhibitors.4 The distinct mode of action is dependent on their different
chemical moieties which mimic the phenol/catechol group of the natural substrates Lꢀtyrosine or Lꢀ
DOPA.
We have previously discovered small compounds from synthetic sources as a new class of TyM
inhibitors and some of them displayed higher efficacy than reference compound kojic acid.11, 12
Specifically, the most active inhibitor was the 1ꢀ(5,6ꢀdimethoxyꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀ(4ꢀ
fluorobenzyl)piperidinꢀ1ꢀyl)propanꢀ1ꢀone (1, Figure 1) that demonstrated promising inhibitory
effects (IC50 value of 7.56 µM) affecting diphenolase activity as a mixedꢀtype inhibitor.12 Our
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structureꢀaffinity relationship (SAR) studies highlight that the 4’ꢀfluorobenzyl moiety embedded in
the 4ꢀposition of piperidine fragment was present in the most active inhibitors, thus suggesting a
plausible crucial role in controlling inhibitory effects toward TyM. This SAR analysis was
consistent with our preliminary Xꢀray studies of coꢀcrystal structure of inhibitor 1 in complex with
TyBm.12 Specifically, the electron density observed in the active site of TyBm confirmed that the
4’ꢀfluorobenzyl moiety of compound 1 is situated between the two copper ions, with the aromatic
ring stabilized through stacking interactions with residue His208 (Figure 1).
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OMe
MeO
O
N
H
N
F
Figure 1. Chemical structure of 1ꢀ(5,6ꢀdimethoxyꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ(4ꢀ(4ꢀfluorobenzyl)piperidinꢀ1ꢀ
yl)propanꢀ1ꢀone (1) and coꢀcrystal structure of the 4ꢀfluorobenzyl portion of inhibitor 1 and
TyBm.12
As the substrate was not fully visualized,12 this structure was not deposited in the protein data bank
but presented a good starting point for the development of a further series of compounds. On this
basis we herein designed new inhibitors bearing a 4’ꢀfluorobenzyl moiety as a crucial structural
requirement for the recognition of TyIs in the catalytic site. All compounds were generally prepared
by means of microwaveꢀassisted organic synthetic approaches. For this new class of TyIs, SAR has
been investigated by combining theoretical, experimental and structural information.
RESULTS AND DISCUSSION
To identify new TyIs we began a rational design process through in silico studies starting from the
smallest active inhibitor previously identified in our research work, namely 1ꢀethylꢀ4ꢀ[(4ꢀ
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fluorophenyl)methyl]piperidine derivative (2, Figure 2). Specifically, for this compound the
inhibition of TyM diphenolase activity in presence of LꢀDOPA as a substrate resulted in an IC50
value of 116 µM.12 We performed docking studies by GOLD software13 and investigated the
binding pose of compound 2 in the active site of TyM from Agaricus bisporus retrieved from
Protein Data Bank (PDB code 2Y9X). Considering the high flexibility of prototype 2, we have
applied the “scaffold match constraint approach” for the 4ꢀfluorophenyl moiety to closeꢀin its
binding pose based on the coꢀcrystal structure of the active portion of inhibitor 1 (Figure 2).
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Figure 2. Chemical structure of 1ꢀethylꢀ4ꢀ[(4ꢀfluorophenyl)methyl]piperidine derivative (2) and
proposed binding mode in the active site of TyM from Agaricus bisporus (PDB code 2Y9X).
Apart from the expected key π−π stacking interactions between His263 (His208 in TyBm, Figure
1) and the 4’ꢀfluorophenyl ring, the Nꢀethylpiperidine portion of compound 2 seems to engage
profitable contacts with the hydrophobic area defined by residues Val248, Phe264, and Val283
(Figure 2). Moreover, the visual inspection of docking pose of compound 2 suggests that close to
the Nꢀethyl substituent there is a wide pocket which might be an amenable area to further
investigate SARs for this class of compounds. In the first round of our novel study we designed a
small series of analogs of compound 2 bearing Nꢀacyl moieties. We introduced new functionalities
to exploit additional hydrophilic/hydrophobic interactions with residues paving the large entrance of
the catalytic site pocket. The first designed compound was the 1ꢀ(4ꢀ(4’ꢀfluorobenzyl)piperidinꢀ1ꢀ
yl)ethanone (3), that could assume a very similar binding pose of inhibitor 2 within the TyM
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catalytic pocket. Thus compound 3 was readily obtained by acetylation of commercially available
4ꢀ(4ꢀfluorobenzyl)piperidine (4) (Scheme 1).
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Scheme 1.
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HN
X
O
R
i
N
X
A or B
4, X = CH
8, X= N
3,5-7, X = CH
9-12, X= N
F
F
3,9 R= CH3
5,10 R= CH3CH2
6, 11 R = (CH3)2CH 7,12 R = C6H5
Reagents and conditions: i) Appropriate acyl chloride: Pathway A: TEA, THF, 0°C, rt, 10 min;
Pathway B: DMF, K2CO3, MW 5 min, 100°C, 200 W
.
To start with SAR exploration, we also chose to replace the acetyl group with other bulky
substituents able to make favorable hydrophobic interactions in the surrounding area defined by the
residues reported above. As described in Scheme 1 we have synthesized a small series of
homologues of amide 3 such as the propionamideꢀ, isobutyramideꢀ and benzamide derivatives 5, 6
and 7. In the second round of our structural modifications we decided to change the central
piperidine core with a piperazine one. Specifically, we selected the piperazine linker on the basis of
the measured inhibitory properties of 1ꢀ(4ꢀfluorobenzyl)piperazine (8) fragment, which resulted in
about 3ꢀfold higher inhibition than 4ꢀ(4ꢀfluorobenzyl)piperidine (4) against diphenolase activity of
TyM (IC50 = 85.5 ꢀM versus 286.83 ꢀM). As shown in Scheme 1 we have synthesized the designed
amide derivatives 9ꢀ12 as piperazine analogs of amides 5ꢀ7. For compound 9 the reaction was
carried out at room temperature in the presence of TEA (pathway A). Whereas, the amides 10ꢀ12
were readily obtained by applying microwave irradiations in alkaline medium (pathway B).14
All of the new synthesized compounds 3, 5ꢀ7, 9ꢀ12 were initially tested as inhibitors of TyM
against diphenolase activity and the results are summarized in Table 1.
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It was interesting to note that the newly designed amide 3 (IC50 = 25.11 ꢀM) displayed about fiveꢀ
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fold improvement of inhibitory properties when compared with the previously synthesized amine 2.
In addition, homologous amides 5ꢀ7 demonstrated similar potency to the parent compound 3.
Among the series of piperazineꢀbased amides 9ꢀ12, the most active inhibitor was the (4ꢀ(4ꢀ
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fluorobenzyl)piperazinꢀ1ꢀyl)(phenyl)methanone 12 (R = Ph, IC50 = 13.3 ꢀM) thus suggesting that
the combination of the Nꢀbenzoyl substituent with the piperazine core induced an improvement in
inhibitory effects.
R
N
X
F
Table 1. TyM inhibition of target compounds 2ꢀ12
and Kojic acid.
a
R
X
IC50
(ꢀΜ)
2b
3
4b
CH3CH2
CH3CO
H
CH
CH
CH
CH
116.0 ± 1.88
25.11 ± 0.98
286.83 ± 1.05
24.10 ± 0.62
35.26 ± 0.97
19.50 ± 0.44
85.50 ± 0.67
45.85 ± 1.31
51.08 ± 4.88
30.90 ± 1.90
13.34 ± 0.73
17.76 ± 0.18
CH3CH2CO
5
CH3CH(CH3)CO CH
6
C6H5CO
H
CH
N
7
8
CH3CO
CH3CH2CO
N
9
N
10
11
12
CH3CH(CH3)CO N
C6H5CO
N
Kojic acid
aAll compounds were examined in a set of experiments performed in three replicates with LꢀDOPA
as the substrate; IC50 values represent the concentration that caused 50% enzyme activity loss. bData
taken from ref. 12.
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In order to understand the intricate binding mode of this first series of amides 3, 5ꢀ7, 9ꢀ12, we tried
to obtain crystal complexes of these compounds with TyBm. The crystal structure was obtained
only for amide 3 in the active site of TyBm at 2.7Å resolution (Figure 3A, Table 2 and
Supplementary Figure S1).
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The 4ꢀfluorobenzyl moiety of amide 3 is oriented towards CuA at a distance of 1.9Å and is
stabilized by hydrophobic πꢀπ interactions with His208. In addition, a hydrogen bond is observed
between Argꢀ209 and the oxygen atom of the carbonyl group of amide 3 (Figure 3B), similarly to
tyrosinase substrates, tyrosine and LꢀDOPA, reported by Goldfeder et al.15 The binding pocket of
TyBm shows high similarity with the TyM structure16 and therefore it is hypothesized that the
additional bonds made by the carbonyl group of amide 3 may contribute to improve the TyM
affinity when compared to amines 2 and 4 (Table 1). Moreover, the orientation of amide 3 in the
active site of TyBm is similar to the recently determined structures with kojic acid and
hydroquinone,17 as well as docking studies with novel biphenyl ester derivatives,18 supporting the
role of this compound as a tyrosinase inhibitor. Comparing the positioning of bound amide 3 and
kojic acid (KA, PDB 5I38), one major difference observed is the movement of Arg209 (Figure 3C).
The flexibility of Arg209 has been reported beforehand for TyBm,19 it facilitates the stabilization of
ligands in the active site resulting in higher inhibition by amide 3.
Figure 3: Structures of 1ꢀ(4ꢀ(4ꢀfluorobenzyl)piperidinꢀ1ꢀyl)ethanone (3) bound in the active site of
tyrosinase from Bacillus megaterium (TyBm) (PDB 5OAE). A) Amide 3 (orange) is observed in the
active site of TyBm (cyan surface) with its mFOꢀDFC electron density polder map (blue wire)
contoured at 3σ. Copper ions are presented as brown spheres and residues surrounding the active
site are in light blue sticks. B) Amide 3 (orange) positioned in the active site of TyBm with the
oxygen atom of the carbonyl group forming a hydrogen bond with Arg209. C) Superposition with
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TyBm structure binding kojic acid (cyan, PDB 5I38). Kojic acid (deep teal) and amide 3 (orange)
are oriented through hydrophobic interactions with His208. Copper ions, residues Val218 and
His60 are identical, whereas Arg209 moves. All the structures presented were generated using
PyMOL.20
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To better compare this structural information with biochemical results, we attempted to determine
the IC50 values of amide 3 with TyBm, however inhibition was not detected due to the poor
solubility of derivative in buffer, necessitating the use of DMSO as a coꢀsolvent in biochemical
experiments. Control experiments with DMSO alone resulted in activation of TyBm explaining the
lack of inhibition in the tested conditions. DMSO possibly affects the movement of Arg209 in the
active site of TyBm as was reported previously for SDS and ionic liquids. SDS levels of 2–50 mM
increased the activity of TyBm by 2ꢀfold towards the natural substrates Lꢀtyrosine and LꢀDopa and
15ꢀ to 20ꢀfold towards phenol and catechol.19 Kojic acid, which is soluble in water, inhibited TyBm
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with an IC50 value of 52 ꢀ ꢀM, Table 1,
M,17 quite similar to the value obtained with TyM (17.76
experimental conditions were not identical) confirming the assumption that in the absence of an
organic solvent, compound 3 might inhibit TyBm.
Table 2. Data collection and refinement statistics
Structure name
(PDB code)
TyBm:3
(5OAE)
Data collection
Space group
P212121
Cell dimensions
78.45,84.10,
89.76
a, b, c (Å)
90, 90, 90
39.59ꢀ2.7
0.11(0.71)
10.9(2.6)
92.0(95.6)
3.9(4.1)
α, β, γ (°)
Resolution (Å)
Rmerge*,†
I/σ
I*
Completeness*
Redundancy*
Refinement
Resolution (Å)
Total reflections
38.96ꢀ2.7
59333(9330)
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Unique reflections
Rwork/Rfree‡
No. of atoms
Protein
Ligand/ion
Water
Bꢀfactors (Å)
Protein
Ligand/ion
Water
Root mean square
deviations
Bond length (Å)
Bond angle (°)
15340(2286)
21.64/26.09
4,699
38
44
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42.30
52.60
40.90
0.003
0.731
*Values in parentheses are for the last shell.†Rmerge
the observed intensity, and <I> is the mean value of I. ‡R/Rfree ∑hkl||Fobs
and Rfree are calculated using the test reflections respectively. The test reflections (5%) were held
=
∑hkl∑i |Ii(hkl)ꢀ
〈
I(hkl)〉|
/
∑hkl∑iIi(hkl), where I is
Fcalc|| ∑hkl|Fobs where R
=
|
ꢀ
|
/
|
aside and not used during the entire refinement process.
To gain further insights into the binding mode of inhibitor 3 in the catalytic site of TyM, docking
analysis was performed (Figure 4A). We applied appropriate flexibility to the side chains of the
protein during docking simulations revealing that the 1ꢀ(4ꢀfluorobenzyl) fragment establishes πꢀπ
interactions with His263 at the active site, while the oxygen atom of carbonyl group of 3 creates a
polar contact with Arg268 similar to the hydrogen bond found for Arg209 in the adduct TyBm/3
(Figure 3B).
Overall, these studies suggest that the docking pose of amide 3 (Figure 4A) in TyM is consistent
with the binding orientation of inhibitor 3 in complex with TyBm. This observation sustains the
reliability of our docking simulations in analyzing the interactions with TyM of all tested
compounds as reported for the best active inhibitor 12 displayed in Figure 4B.
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Figure 4: Suggested binding mode of compounds 3 (A) (yellow) and 12 (B) (purple) that were
docked into the TyM strcuture (PDB code 2Y9X). Key residues of the pocket are presented and
hydrogen bond interactions are showed by dotted lines. The interactions between the TyM and
inhibitors 3 and 12 were examined using PyMOL20 and LIGPLUS.21 The figure was created using
PyMOL software.20
The docking simulation of active benzamide 12 suggests that the 1ꢀ(4ꢀfluorobenzyl) fragment
maintains a very similar orientation; whereas the benzoyl portion is oriented towards an exposed
region located at the entrance of catalytic area (Figure 4, panel B). In particular, the aromatic ring
occupies the area paved by amino acid residues Met257, Asn260, Thr261 and Phe264 lining both
hydrophobic and hydrophilic regions. By comparing the docking pose of active inhibitors 3 and 12,
we have hypothesized that the profitable hydrogen bond contact with Arg268 of acetamide 3 was
replaced by hydrophobic interactions produced by the phenyl ring of benzamide 12 and the two
residues Met257 and Phe264. Therefore, in absence of Hꢀbond interaction with Arg268 these
additional interactions could keep the inhibitor 12 in a good orientation in the catalytic pocket.
Based on these insights for benzamide 12, we designed new derivatives that bear additional
substituents in ortho, meta and para position of the phenyl ring. Our idea was to probe further
hydrophobic/hydrophilic contacts that might increase the interaction in the cavity occupied by the
benzoyl fragment. We initially selected the following substituents: fluorine atom and methyl,
methoxy, hydroxyl groups, thus exploring the role of the hydrophobic and electronic nature of the
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substituents. This small series of twelve aroylpiperazine derivatives 13ꢀ24 was obtained by coupling
the 1ꢀ(4ꢀfluorobenzyl)piperazine (8) with a suitable aroylchlorides resulting in the corresponding
amides 13ꢀ21 in good yields. In turn, the alkoxy derivatives 19ꢀ21 were converted to the
9
corresponding hydroxyl derivatives 22ꢀ24 by treatment with boron tribromide.
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R
Cl
O
O
HN
N
N
N
i, A or B
8
R
F
F
13-24
16 R= 2ꢀCH3
17, R= 3ꢀCH3
18, R= 4ꢀCH3
19, R= 2ꢀOCH3
20, R= 3ꢀOCH3
21, R= 4ꢀOCH3
13, R= 2ꢀF
14, R= 3ꢀF
15, R= 4ꢀF
22, R= 2ꢀOH
23, R= 3ꢀOH
24, R= 4ꢀOH
ii
Scheme 2: Reagents and conditions: i) Pathway A: R=2ꢀOCH3, R=3ꢀOCH3, R=4ꢀOCH3, DCM,
EDIPA, r.t, 5h; pathway B: R=2ꢀF, R=3ꢀF, R=4ꢀF, R=2ꢀCH3, R=3ꢀCH3, R=4ꢀCH3, DCM, EDIPA,
MW: 10 min, 50°C, 200W; ii) BBr3, DCM, 0°C, r.t., 24h.
Table 3 summarizes the results of biochemical screening of benzamides 13ꢀ24 for which we
generally obtained a significant improvement of inhibitory activity with respect to the first series of
tested compounds. Specifically, the most active inhibitor was compound 19 (R=2ꢀOCH3) which
was up to 6ꢀfold more potent than unsubstituted benzamide analog 12 (R=H) and more potent than
the reference compound kojic acid (see Table 1). By modifying the 2ꢀmethoxysubstituent with
other electron donating groups (EDG) such as methyl or hydroxyl groups, we found that both the
obtained compounds 16 and 22 maintained high inhibitory effects.
O
N
N
R
F
Table 3. TyM inhibition of target compounds 13ꢀ24
a
R
IC50
2ꢀF
3ꢀF
3.75 ± 0.37 ꢁM
54.68 ± 6.03 ꢁM
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4ꢀF
7.77±2.89 µM
15
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19
20
21
22
23
24
2ꢀCH3
3ꢀCH3
4ꢀCH3
2ꢀOCH3
3ꢀOCH3
4ꢀOCH3
2ꢀOH
5.25±1.60 µM
173.82±1.5 µM
33.13±4.50 µM
2.03±0.89 µM
27.04±0.05 µM
9.14±2.16 µM
7.06±0.62 µM
12.64±1.41 µM
27.41±0.16 µM
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3ꢀOH
4ꢀOH
aAll compounds were examined in a set of experiments performed in three replicates; IC50 values
represent the concentration that caused 50% enzyme activity loss.
It was interesting to note that the switch of methyl/methoxy/hydroxy substituent to the meta or para
position generally led to less active inhibitors. Specifically, the most significant reduction of
inhibitory effect was found for the metaꢀsubstituted compound 17 that resulted in 35ꢀfold lower IC50
compared to the orthoꢀsubstituted analog 16. The presence of a fluorine atom as a small electron
withdrawing group (EWG) resulted in improvement of affinity when compared with the
unsubstituted analog 12.
Furthermore, the inhibition kinetics of the most promising inhibitor 19 (IC50 = 2.03 ꢀM), on the
diphenolase activity of mushroom tyrosinase were studied using LineweaverꢀBurk double
reciprocal plots (see Figure 5). The results showed that the plots of 1/V versus 1/[S] gave straight
lines with different slopes intersecting the horizontal axis. These data suggest that compound 19
acts as a nonꢀcompetitive inhibitor since it is able to bind with equal affinity to the free enzyme as
well as to the enzymeꢀsubstrate complex. The increase in concentrations of 19 corresponds to the
decrease of Vmax values, while Km values remain unchanged. Therefore, it is assumed that the
inhibitor might occupy the active site as well as hinder the access of substrate LꢀDOPA.22, 23
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Figure 5: LineweaverꢀBurk plots for the inhibition of mushroom tyrosinase respect to LꢀDOPA as
substrate in the presence of the best active inhibitor 19.
To simulate the binding recognition into the enzymatic cavity we docked the best active inhibitor 19
(blue) into the TyM structure (PDB code 2Y9X) and compared its pose with the binding pose of
parent compound 12 (purple) (Figure 6). As expected, the two inhibitors were almost
superimposable thus sharing very similar molecular interactions. Notably, the 2ꢀmethoxyꢀ
substituent of the best active compound 19 is projected towards residue Val248 thus making an
additional hydrophobic interaction. Overall, the proposed binding mode for the best active ligand 19
was in good agreement with our biochemical assays for which 19 displayed nonꢀcompetitive
inhibition of TyM.
Figure 6. Alignment of the binding poses of benzamides 12 (purple) and 19 (blue) that were docked
into the TyM (PDB code 2Y9X). The circle highlights the contact between methyl group and
Val248 in the hydrophobic region of the cavity. Copper ions are depicted in brown. The figure was
created using PyMOL software20
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The positioning found out for compound 19 in the active site of TyM (Figure 6) might lead to the
false assumption of a competitive inhibition mechanism. However, we suggest that it could also
bind to a peripheral binding site in TyM yet to be elucidated. It was previously shown by Deri et al.
17
that KA binds in two regions within the enzyme, inside the active site and at the entrance to the
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active site, giving rise to mixed inhibition kinetics. These results were demonstrated by
crystallography as well as by in silico simulations. In addition, tropolone, another wellꢀknown
tyrosinase inhibitor, was also found at the active site entrance of TyM and exhibited mixed
inhibition mode.22 It is assumed that this additional binding mode of compound 19 in the peripheral
site of TyM could restrict substrate entrance and product efflux, consequently, the nonꢀcompetitive
inhibition mode is obtained kinetically (see Figure 5).
In order to gain more molecular insights on the binding mode of designed benzamide derivatives,
attempts were carried out to obtain coꢀcrystal complexes with TyBm. A further structure was
successfully determined for compound 16 in the active site of TyBm at 2.0Å resolution (Figure 7,
Table 4 and Supplementary Figure S2). As expected, the 4ꢀfluorobenzyl moiety of derivative 16 is
oriented towards CuA and is stabilized through stacking interactions with His208. In addition, a
polar interaction between benzamide 16 and Arg209 is observed, similar to the orientation of
compound 3 in TyBm active site (Figure 7). Comparing the positioning of bound amide 3 and
benzamide 16, the major difference observed is the slight movement of Arg209 (Figure 7B). The
mobility of Arg209 allows the stabilization of bulky compounds in the active site of TyBm.
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Figure 7. Structure of benzamide 16 bound in the active site of TyBm. (A) Benzamide 16 (yellow)
positioned in the active site of TyBm with the oxygen atom of the carbonyl group forming a
hydrogen bond with Arg209. (B) Superposition with bound amide 3 (orange). Copper ions are
presented as brown spheres.
Table 2. Data collection and refinement statistics
Structure name
(PDB code)
TyBm:16
(6EI4)
Data collection
Space group
P21
Cell dimensions
53.65,78.10,
81.05
a, b, c (Å)
90, 104.52, 90
28.10ꢀ1.69
0.13(0.48)
6.5(3.1)
α, β, γ (°)
Resolution (Å)
Rmerge*,†
I/σ
I*
Completeness*
Redundancy*
Refinement
98.8(98.4)
5.2(5.4)
Resolution (Å)
Total reflections
Unique reflections
Rwork/Rfree‡
No. of atoms
Protein
28.10ꢀ2.0
375111(55945)
71968(10437)
22.01/24.75
4,719
50
Ligand/ion
Water
358
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Bꢀfactors (Å)
Protein
27.74
41.75
27.54
5
6
7
8
Ligand/ion
Water
Root mean square
deviations
Bond length (Å)
Bond angle (°)
9
0.008
1.02
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*Values in parentheses are for the last shell.†Rmerge
the observed intensity, and <I> is the mean value of I. ‡R/Rfree ∑hkl||Fobs
and Rfree are calculated using the test reflections respectively. The test reflections (5%) were held
=
∑hkl∑i |Ii(hkl)ꢀ
〈
I(hkl)〉|
/
∑hkl∑iIi(hkl), where I is
Fcalc|| ∑hkl|Fobs where R
=
|
ꢀ
|
/
|
aside and not used during the entire refinement process.
This additional structural information confirmed that the two inhibitors 3 and 16 share a very
similar binding mode within catalytic site of TyBm. These experimental data were consistent with
the suggested pose of inhibitor 16 docked in the catalytic site of TyM (Supplementary Figure S3) in
comparison with analogues 12 and 19. Considering that the inhibition kinetics suggest that the
inhibitor 16 displays (Supplementary Figure S4) a mode of interaction similar to that we found for
compound 19 (see Figure 5), we can speculate that both 16 and 19 could hamper the enzymatic
activity of TyM through a singular mechanism of interaction that might involve additional and
peripheral binding regions.
As there is currently no structure of the human enzyme, we finally decided to collect our acquired
structural information for TyM and TyB and translate these achievements on the druggable TyH
through the employment of TyH model and melanogenic protein TYRP1.
The active site of all tyrosinases is similar as demonstrated by the superposition of the TyH model
with TyM and TyBm (Figure S5), including residues Asn205 and Glu195 (in TyBm) which are
crucial for activity through the activation of a conserved water molecule.24 Favre et. al. used
quantum mechanics to model TyH and showed that their model, deposited in the Protein Model
Database, is a reliable structure for future rational inhibitor design projects.7 In addition, the crystal
structure of human tyrosinase related protein 1 (TYRP1) was recently solved and revealed for the
first time the structure of a mammalian tyrosinase family member. The TYRP1 structure contains a
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tyrosinaseꢀlike subdomain that has the typical tyrosinase fold with a binuclear type 3 metal binding
site. It is a zincꢀbinding enzyme which makes it unlikely to be a redox catalyst. However when
exchanging the zinc cofactor to copper, a slight tyrosinase activity was detected.8 Human TYRP1
was shown to be highly similar to TyBm with 32% sequence identity and an rmsd of 1.6Å for 263
aligned amino acids.8 Furthermore, it was demonstrated that the catalytic domain with the active
site is highly similar to that of type 3 copper proteins, especially to TyBm.8, 24 Superposition of
TyBm with bound kojic acid (PDB 5I38) and TYRP1 with bound kojic acid (PDB 5M8L) reveals
similar orientation of the ligand towards CuA in both enzymes (Figure S6). Numerous reports exist
on the IC50 values of TyH inhibition by ligands such as kojic acid and other natural products which
inhibit TyM and TyBm with similar efficacy.6, 25ꢀ28 Overall, it is well demonstrated in the literature
that inhibitors of TyM and TyBm have relevance to the human enzyme and therefore the inhibitors
developed here could have potential pharmaceutical implications. Clearly, more research is needed
to confirm that the inhibitory effect of the most active derivatives might be moved to TyH.
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CONCLUSIONS
To develop tyrosinase inhibitors as therapeutic agents for potential treatment of skin
hyperpigmentation pathologies, we carried out the first “lead optimization” process which has been
performed by combining Xꢀray crystallography and docking simulations within enzymatic cavity of
TyBm and TyM isozymes. Specifically, theoretical and experimental structural determinations have
been employed for the rational design of a small series of 1ꢀ(4ꢀfluorobenzyl)piperazine and 1ꢀ(4ꢀ
fluorobenzyl)piperidine derivatives. As a result, we have identified new inhibitors more potent than
reference compound kojic acid (KA). The crystal structures of 1ꢀ(4ꢀ(4ꢀfluorobenzyl)piperidinꢀ1ꢀ
yl)ethanone (3) and [4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](2ꢀmethylphenyl)methanone (16) in complex
with TyBm revealed that the two inhibitors assume a similar binding pose of inhibitor KA in the
enzyme catalytic cavity. Moreover, the coꢀcrystal adducts highlighted a pivotal role of an additional
Hꢀbond contact between oxygen atom of carbonyl group with crucial residue Arg209 (TyBm) in the
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rim of the catalytic cavity. The active inhibitors 16 and 19 displayed a nonꢀcompetitive inhibition of
TyM as supported by kinetic analysis. By means of docking simulations we have hypothesized that
additional interactions with hydrophobic subpocket (Val248 and Met257, TyM) might be essential
to improve the anchoring of inhibitors into binding site.
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EXPERIMENTAL SECTION
Chemistry. All starting materials and reagents commercially available (SigmaꢀAldrich Milan, Italy;
Alfa Aesar Karlsruhe, Germany) were used without further purification. Microwaveꢀassisted
reactions were carried out in a focused Microwave Synthesis System (CEM Technology Ltd
Buckingham, UK). Melting points were determined on a Buchi Bꢀ545 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 carried out on a Biotage SP1 EXP
1
(Biotage AB Uppsala, Sweden). H NMR spectra were measured in CDCl3 with a Gemini 300
spectrometer (Varian Inc. Palo Alto, California USA); chemical shifts are expressed in δ (ppm) and
coupling constants (J) in Hz.
General Procedure to Synthesize Target Compounds 3, 5ꢀ7, 9ꢀ12 (Pathways A and B)
Pathway A: Acetyl chloride (2.05 mmol) was added to a solution of 1ꢀ(4ꢀfluorobenzyl)piperazine
(8) (2.05 mmol) in THF (7mL) and TEA (3.28 mmol). The mixture was stirred at room temperature
for 10 min under nitrogen atmosphere. Then, the obtained precipitate was turned away by filtration
and the solution was evaporated under reduced pressure to give the final compound 9 as crude
product, which has been purified by chromatographic column (Cyclohexane/EtOAc 20:80).
Pathway B: To prepare compounds 3, 5ꢀ7, 10ꢀ12 the suitable acyl chloride (1.5 mmol) was
dissolved in DMF (1 mL). Then the 4ꢀ(4ꢀfluorobenzyl)piperidine (4) or 1ꢀ(4ꢀ
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fluorobenzyl)piperazine (8) (1.5 mmol) was added dropwise in the presence of K2CO3 (0.75 mmol).
4
5
The reaction was carried out using microwave irradiations under the following conditions: 5 min,
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9
100 °C. The mixture of reaction was quenched by addition of a saturated solution of NaHCO
mL) and the aqueous phase was extracted with EtOAc (3x5 mL). The organic phase thus obtained
was washed with brine (3x5mL) and dried with anhydrous Na SO . The solvent was removed
₃ (10
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₂
₄
under reduced pressure and the final products 3, 5ꢀ7, 10ꢀ12 were obtained after purification by
chromatographic column (Cyclohexane/EtOAc 30:70).
The registered CAS numbers have been already assigned to compounds 3, 5ꢀ7, 9ꢀ12. For these
compounds the synthetic procedures, chemical properties and structural characterization are not
available in literature, except for patented compounds 329 and 730.
1ꢀ[4ꢀ(4ꢀFluorobenzyl) piperidinꢀ1ꢀyl] propanꢀ1ꢀone (5). CAS Number: 1215411ꢀ97ꢀ4. Yield:
23%. Oily residue. 1HꢀNMR (CDCl3), (δ): 1.05ꢀ1.16 (m, 5H), 1.65ꢀ1.75 (m, 3H), 2.34 (q, 2H, CH2),
2.50ꢀ2.53 (m, 3H), 2.93 (t, 1H, CH), 3.82 (m, 1H, CH2), 4.61 (m, 1H, CH2), 6.94ꢀ7.11 (m, 4H,
ArH). Anal. Calcd for (C15H20FNO): C 72.26, H 8.09, N 5.62. Found: C 72.66, H 8.49, N 6.02.
1ꢀ[4ꢀ(4ꢀFluorobenzyl)piperidinꢀ1ꢀyl]ꢀ2ꢀmethylpropanꢀ1ꢀone (6). CAS Number: 1215420ꢀ69ꢀ1.
1
Yield: 31%. Oily residue. HꢀNMR (CDCl3), (δ): 1.09ꢀ1.18 (m, 8H), 1.70 (m, 3H), 2.51 (m, 3H),
2.79ꢀ2.81 (m, 1H, CH) 2.96 (t, 1H, CH), 3.91 (m, 1H, CH2), 4.63 (m, 1H, CH2), 6.94ꢀ7.11 (m, 4H,
ArH). Anal. Calcd for (C16H22FNO): C 72.97, H 8.42, N 5.32. Found: C 72.57, H 8.02, N 4.92.
1ꢀ[4ꢀ(4ꢀFluorobenzyl) piperazinꢀ1ꢀyl]ethanone (9) CAS Number: 415958ꢀ01ꢀ9.Yield: 20%. Oily
1
residue. HꢀNMR (CDCl3), (δ): 2.09 (s, 3H, CH3), 2.42 (m, 4H), 3.47 (m, 4H), 3.63ꢀ3.64 (m, 2H,
CH2), 7.00 (d, 2H, ArH, J= 8.8), 7.29 (d, 2H, ArH, J= 8.8). Anal. Calcd for (C13H17FN2O): C 66.08,
H 7.25, N 11.86. Found: C 66.48, H 7.65, N 12.26.
1ꢀ[4ꢀ[(4ꢀFluorophenyl)methyl]piperazinꢀ1ꢀyl]propanꢀ1ꢀone (10) CAS Number: 1328560ꢀ06ꢀ0.
1
Yield: 33%. Oily residue. HꢀNMR (CDCl3), (δ): 1.14 (t, 3H, CH3, J= 6.5, J= 6.4), 2.29ꢀ2.42 (m,
6H, CH2), 3.43ꢀ3.48 (m, 4H, CH2), 3.62ꢀ3.64 (m, 2H, CH2), 6.98ꢀ7.05 (m, 2H, ArH), 7.28ꢀ7.30 (m,
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2H, ArH). Anal. Calcd for (C14H19FN20): C 67.18, H 7.65, N 11.19. Found: C 66.78, H 7.25, N
10.79.
6
7
8
9
1ꢀ[4ꢀ[(4ꢀfluorophenyl)methyl]piperazinꢀ1ꢀyl]ꢀ2ꢀmethylpropanꢀ1ꢀone (11) CAS Number:
1
1090613ꢀ20ꢀ9. Yield: 53%. Oily residue. HꢀNMR (CDCl3) (δ): 1.12 (d, 6H, 2CH3, J= 6.5), 2.39ꢀ
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2.43 (m, 4H, CH2), 2.73ꢀ2.82 (m, 1H, CH), 3.48ꢀ3.50 (m, 4H, CH2), 3.63ꢀ3.65 (m, 2H, CH2), 6.98ꢀ
7.04 (m, 2H, ArH), 7.28ꢀ7.31 (m, 2H, ArH). Anal. Calcd for (C15H21FN2O): C 68.16, H 8.01, N
10.6. Found: C 68.06, H 7.91, N 10.5.
[4ꢀ[(4ꢀFluorophenyl)methyl]piperazinꢀ1ꢀyl]ꢀphenylꢀmethanone (12) CAS Number: 439846ꢀ67ꢀ
0. Yield: 16%. m.p. 78ꢀ80°C. 1HꢀNMR (CDCl3) (δ): 1.71 (s, 1H), 2.36 (m, 2H, CH2), 2.50 (m, 2H,
CH2), 3.42 (m, 2H, CH2), 3.49 (s, 2H, CH2), 3.78 (m, 1H), 6.97ꢀ7.00 (m, 2H, ArH), 7.25ꢀ7.28 (m,
2H, ArH), 7.39 (s, 5H, ArH). Anal. Calcd for (C18H19FN2O) C 72.46, H 6.42, N 9.39. Found: C
72.06, H 6.02, N 8.89.
General procedure to synthesize [4ꢀ(4ꢀfluorobenzyl)piperazinꢀ1ꢀyl]methanone derivatives (13ꢀ
21). To a solution of 1ꢀ(4ꢀfluorobenzyl)piperazine (8) (0.5mmol) in CH2Cl2 (2mL) the N,Nꢀ
diisopropylethylamine (0.75mmol) and the suitable benzoyl chloride (0.5mmol) were added. The
reaction was stirred at room temperature for 5h for compounds 19ꢀ21 (Pathway A). Instead, to
obtain compounds 13ꢀ18 (Pathway B) the reaction was carried out under microwave irradiation 10
min, 50°C, 200 Psi. After turning off the reaction by addition of CH3OH (2mL), water was added
and the mixture was extracted with CH2Cl2 (3x5 mL). The obtained organic phase was washed
many times with brine (3x5mL) and was dried with anhydrous Na₂SO₄. The solvent was removed
under reduced pressure and the final products 13ꢀ18 were purified by crystallization with Et2O.
The registered CAS numbers have been already assigned to compounds 13ꢀ21, for which detailed
synthetic procedures, chemical properties and structural characterization are not available in
literature.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](2ꢀfluorophenyl)methanone (13) CAS Number: 444907ꢀ73ꢀ7
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. Yield 34%. m.p 78ꢀ79°C. HꢀNMR (CDCl3), (δ): 2.40ꢀ2.54 (m, 4H), 3.35 (bs, 2H), 3.52 (bs, 2H),
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3.83 (bs, 2H, CH2), 7.00ꢀ7.40 (m, 8H, ArH). Anal. Calcd for: (C18H18F2N2O) C 68.34, H 5.74, N
8.86. Found: C 68.14, H 5.54, N 8.66.
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[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](3ꢀfluorophenyl)methanone (14) CAS Number: 423739ꢀ56ꢀ4.
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Yield 43%. Oily residue. HꢀNMR (CDCl3), (δ): 2.31ꢀ2.46 (m, 4H), 3.35ꢀ3.44 (m, 4H), 3.71 (bs,
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2H), 3.83 (bs, 2H, CH2), 6.92ꢀ7.10 (m, 4H,ArH), 7.20ꢀ7.31 (m, 4H,ArH). Anal. Calcd for:
(C18H18F2N2O) C 68.34, H 5.74, N 8.86. Found: C 68.24, H 5.64, N 8.76.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](4ꢀfluorophenyl)methanone (15) CAS Number: 423748ꢀ97ꢀ4.
Yield 46%. m.p. 112ꢀ113°C. 1HꢀNMR (CDCl3) (δ): 1.25 (s, 1H, CH2), 1.59 (m, 3H, CH2), 2.40 (m,
2H, CH2), 3.50 (m, 3H, CH2), 3.75 (m, 1H, CH2), 6.98ꢀ7.12 (m, 4H, ArH), 7.26ꢀ7.43 (m, 4H, ArH);
Anal. Calcd for: (C18H18F2N2O) C 68.34, H 5.74, N 8.86. Found: C 68.74, H 6.14, N 9.26.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](2ꢀmethylphenyl)methanone (16) CAS Number: 945203ꢀ73ꢀ
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6. Yield 56%. m.p. 72ꢀ73°C. HꢀNMR (CDCl3) (δ): 1.74 (m, 1H, CH2), 2.24ꢀ2.33 (m, 3H, CH3),
2.51ꢀ2.53 (m, 2H, CH2), 3.22ꢀ3.25 (m, 2H, CH2), 3.48 (s, 2H, CH2), 3.82ꢀ3.84 (m, 1H, CH2), 6.97ꢀ
7.00 (m, 2H, ArH), 7.13ꢀ7.33 (m, 6H, ArH). Anal. Calcd for: (C19H21FN2O) C 73.05, H 6.78, N
8.97. Found: C 73.15, H 6.88, 9.07.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](3ꢀmethylphenyl)methanone (17) CAS Number: 945107ꢀ54ꢀ
0. Yield 80%. Oily residue. 1HꢀNMR (CDCl3) (δ): 2.28ꢀ2.49 (m, 3H, CH3; 4H, CH2), 3.43ꢀ3.77 (m,
6H, CH2), 6.98ꢀ7.04 (m, 4H, ArH), 7.17ꢀ7.25 (m, 4H, ArH). Anal. Calcd for: (C19H21FN2O) C
73.05, H 6.78, N 8.97. Found: C 73.14, H 6.87, 9.06.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](4ꢀmethylphenyl)methanone (18) CAS Number: 439846ꢀ95ꢀ
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4. Yield 60%. m.p. 114ꢀ115°C. HꢀNMR (CDCl3) (δ): 1.70 (s, 1H, CH2), 2.37 (s, 3H, CH3), 2.40ꢀ
2.46 (m, 4H, CH2), 3.49 (m, 4H, CH2), 3.75 (m, 1H, CH2), 6.97ꢀ7.04 (m, 2H, ArH), 7.20ꢀ7.31 (m,
6H, ArH). Anal. Calcd for: (C19H21FN2O) C 73.05, H 6.78, N 8.97. Found: C 73.25, H 6.98, N 9.17.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](2ꢀmethoxyphenyl)methanone (19) CAS Number: 510718ꢀ
70ꢀ4. Yield 40%. m.p. 78ꢀ79°C. 1HꢀNMR (CDCl3) (δ): 1.62 (m, 1H, CH2), 2.26ꢀ2.54 (m, 4H, CH2),
3.24ꢀ3.26 (m, 2H, CH2), 3.48 (s, 2H, CH2), 3.82 (m, 3H, OCH3; 1H, CH2), 6.88ꢀ7.03 (m, 4H, ArH),
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7.22ꢀ7.36 (m, 4H, ArH). Anal. Calcd for: (C19H21FN2O2) C 69.49, H 6.45, N 8.53. Found: C 69.29,
H 6.25, N 8.33.
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[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](3ꢀmethoxyphenyl)methanone (20) CAS Number: 462099ꢀ
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41ꢀ8. Yield 60%. Oily residue. HꢀNMR (CDCl3) (δ): 2.56 (m, 4H, CH2), 3.61 (m, 4H, CH2), 3.82
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(m, 3H, OCH3; 2H, CH2), 6.93ꢀ7.03 (m, 4H, ArH), 7.30ꢀ7.32 (m, 4H, ArH). Anal. Calcd for:
(C19H21FN2O2) C 69.49, H 6.45, N 8.53. Found: C 69.28, H 6.24, N 8.32.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](4ꢀmethoxyphenyl)methanone (21) CAS Number: 462096ꢀ
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09ꢀ9. Yield 67%. m.p. 120ꢀ121°C. HꢀNMR (CDCl3) (δ):1.64 (m, 1H, CH2), 2.44 (m, 4H, CH2),
3.47ꢀ3.58 (m, 4H, CH2), 4.11 (m, 3H, OCH3; 1H, CH2), 6.88ꢀ7.03 (m, 4H, ArH), 7.26ꢀ7.40 (m, 4H,
ArH). Anal. Calcd for: (C19H21FN2O2) C 69.49, H 6.45, N 8.53. Found: C 69.79, H 6.75, N 8.83.
General procedure to synthetize 4ꢀ(4ꢀfluorobenzyl)piperazinꢀ1ꢀyl](hydroxyphenyl)methanone
derivatives (22ꢀ24). To a solution of the suitable derivatives 19ꢀ21 (0.4mmol) in CH2Cl2 (3mL) the
BBr3 (6mmol) was added at 0°C under nitrogen atmosphere. The reaction mixture was stirred for
24h at room temperature, then a small amount of CH3OH (1.5mL) was added at 0°C.31 The solvent
was removed at reduced pressure and the obtained residue was extracted with EtOAc (3x5mL). The
organic phase thus obtained was washed with a saturated solution of NaHCO3 (3x5mL) and
subsequently was dried with anhydrous Na₂SO₄. The solvent was removed at reduced pressure and
the final products 22ꢀ24 were purified by crystallization with CH3OH.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](2ꢀhydroxyphenyl)methanone (22) Yield 50%. m.p. 167ꢀ
169°C . 1HꢀNMR (CDCl3) (δ): 1.60 (m, 2H, CH2), 2.48ꢀ2.49 (m, 2H, CH2), 3.52 (s, 2H, CH2), 3.74ꢀ
3.82 (m, 4H, CH2), 6.81ꢀ7.05 (m, 4H, ArH), 7.30ꢀ7.36 (m, 4H, ArH), 9.63 (s, 1H, OH). Anal. Calcd
for: (C18H19FN2O2) C 68.77, H 6.09, N 8.91. Found: C 68.97, H 6.29, N 9.11.
[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](3ꢀhydroxyphenyl)methanone (23) Yield 57%. m.p. 175ꢀ
177°C. 1HꢀNMR (CDCl3) (δ): 1.69 (m, 2H, CH2), 2.36ꢀ2.51 (m, 3H, CH2), 3.43ꢀ3.49 (m, 4H, CH2),
3.78ꢀ3.81 (m, 1H, CH2), 6.80ꢀ6.83 (m, 2H, ArH), 6.95ꢀ7.04 (m, 2H, ArH), 7.25ꢀ7.30 (m, 4H, ArH),
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7.54 (bs, 1H, OH). Anal. Calcd for: (C18H19FN2O2) C 68.77, H 6.09, N 8.91. Found: C 68.89, H
6.21, N 9.03.
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[4ꢀ(4ꢀFluorobenzyl)piperazinꢀ1ꢀyl](4ꢀhydroxyphenyl)methanone (24) Yield 70%. m.p. 170ꢀ
172°C. 1HꢀNMR (CDCl3), (δ): 1.65 (m, 4H, 2CH2), 2.45 (m, 2H, CH2), 3.50ꢀ3.83 (m, 4H, CH2),
6.73.6.76 (m, 2H, ArH), 6.98ꢀ7.04 (m, 2H, ArH), 7.24ꢀ7.30 (m, 4H, ArH). Anal. Calcd for:
(C18H19FN2O2) C 68.77, H 6.09, N 8.91; Found: C 68.87, H 6.19, N 9.01.
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Crystallization and data collection. TyBm crystals were prepared as described previously.15
Mature crystals were soaked overnight with 1 µl of 5 mM CuSO4 in 25% PEG 8K and 1 µl of 10
mM inhibitor ligands dissolved in 50% DMSO in distilled water. Xꢀray diffraction data was
collected at the European Synchrotron Radiation Facility, Grenoble, France, at beamline ID 29. All
data were indexed, integrated, scaled and merged using Mosflm and Scala.10 The structure of TyBm
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with the bound inhibitor was solved by molecular replacement using Phaser and the coordinates
of earlier determined TyBm structure (PDB code 4P6R). Refinement was performed using Phenix 33
34, 35
and Refmac5,
coupled with rounds of manual model building, realꢀspace refinement and
structure validation performed using COOT.36 Data collection, phasing and refinement statistics are
presented in Table 2 and Table 4.
Docking analysis. The crystal structure of Agaricus Bisporus Mushroom Tyrosinase in complex
with inhibitor tropolone was retrieved from the RCSB Protein Data Bank (PDB code 2Y9X). The
ligand and water molecules were discarded and the hydrogens were added to the protein by
Discovery Studio 2.5.10 The ligand structure was constructed using Discovery Studio 2.5.5 and
energy minimized using the Smart Minimizer protocol (1000 steps) which combines the Steepest
Descent and the Conjugate Gradient methods. CHARMm force field was used for energy
minimization steps The minimized ligand was docked in their corresponding proteins by means of
Gold Suite 5.0.1. The region of interest used by the Gold program37 was defined in order to contain
the residues within 15 Å from the original position of the ligand in the Xꢀray structure. The side
chains of residues Arg 268 and Phe264 were allowed to rotate according to the internal rotamer
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libraries in GOLD Suite 5.0.1. A scaffold constraint (penalty = 10.0) was used to restrict the
solutions in which the 4ꢀfluorophenyl fragment was able to closeꢀin its binding pose upon the
cocrystal structure of the active portion of inhibitor 1 GoldScore was chosen as fitness function. The
standard default settings were used in all calculations and the ligands were submitted to 100 genetic
algorithm runs. The “allow early termination” command was deactivated. Results differing by less
than 0.75 Å in ligandꢀall atom RMSD, were clustered together. The conformations with the highest
GoldScore fitness values were chosen both for analysis and representation The molecular model of
the docked compound was displayed using Pymol software. 38
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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 according to the
method of Masamoto39 with minor modifications.40 Briefly, aliquots (0.05mL) of sample at various
concentrations (5 – 300 ꢁM) were mixed with 0.5 mL of Lꢀ tyrosine or LꢀDOPA 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. The linear increase in adsorbance (Abs) at 475 nm was measured after 30 or 5 minutes
of incubation time in the reaction mixture containing LꢀDOPA. The inhibitory activity of samples is
expressed as inhibition percentage and calculated as follows:
Inhibition % = [[(AꢀB)ꢀ(CꢀD)]/AꢀB]/100
A: Abs acetate buffer and enzyme
B: Abs acetate buffer
C: Abs acetate buffer, test sample and enzyme
D: Abs acetate buffer and test sample
The concentrations leading to 50% activity lost (IC50) were also calculated by interpolation of the
doseꢀresponse curves. Kojic acid [5ꢀhydroxyꢀ2ꢀ(hydroxymethyl)ꢀ4Hꢀpyranꢀ4ꢀone], a fungal
secondary metabolite used as skin whitening agent, was employed as a positive standard (8 – 35
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ꢁM). A spectrophotometer (Shimadzu UVꢀ1601) was used for mushroom tyrosinase inhibition
assay and kinetic analysis of the tyrosinase inhibition.
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Kinetic analysis of the tyrosinase inhibition. The reaction mixture consisted of four different
concentrations of LꢀDOPA (0.6–5 mM) as substrate and mushroom tyrosinase in acetate buffer
(0.05 M, pH 6.8). Three different concentrations of compound 19 (2,4,8 ꢁM) were added to the
reaction mixture. Michaelis–Menten constant (Km) and maximal velocity (Vmax) of the tyrosinase
were determined by Lineweaver–Burk plots.
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ACKNOWLEDGMENTS
This work was supported by Fondo di Ateneo per la Ricerca (PRA grant number ORME09SPNC ꢀ
Università degli Studi di Messina), the Israel Science Foundation founded by the Israel Academy of
Sciences and Humanities, grant number 419/15 and by the Gurwin Fund for Scientific Research.
We also acknowledge the RussellꢀBerrie Nanotechnology Institute (RBNI) at the Technion for
supporting this research. This research benefited from use of the Technion Center of Structural
Biology facility of the Lorry I. Lokey Center for Life Sciences and Engineering. We thank the staff
of the European Synchrotron Radiation Facility (beamline ID 29) for provision of synchrotron
radiation facilities and assistance, and Mrs. Cristina Iannello for her contribution in the synthesis of
several compounds.
ADDITIONAL INFORMATION
PDB accession numbers: The coordinates and structure factors of TyBm have been deposited in
the RCSB PDB under accession code 5OAE (TyBm with bound amide 3) and 6EI4 (TyBm with
bound compound 16). Authors will release the atomic coordinates and experimental data upon
article publication.
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The Supporting Information is available free of charge on the ACS Publications website a DOI…
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Molecular formula strings (XLSX)
AUTHOR INFORMATION
Corresponding Author
Laura De Luca: Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali
(CHIBIOFARAM) Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata Iꢀ
98168 Messina, Italy. eꢀmail: ldeluca@unime.it. Phone 00390906766410
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to
the final version of the manuscript. ∞S.F. and B.D. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
Tys, Tyrosinases; TyM, Mushroom tyrosinase; TyBm, Bacillus megaterium tyrosinase; TyIs, Ty
inhibitors; TyH, human tyrosinase; TEA, trimethylamine; THF, Tetrahydrofuran; DMF,
Dimethylformamide;
MW,
Microwave;
EDIPA,
N,NꢀDiisopropylethylamine;
DCM,
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dichloromethane; BBr3, Boron tribromide, EDG, electron donating groups; EWG, electron
withdrawing group; KA, kojic acid; FC, Flash Chromatography; TLC, Thin Layer Chromatography.
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