Catechol-based matrix metalloproteinase inhibitors with additional antioxidative activity

Article abstract of DOI:10.1080/14756366.2016.1217853

New catechol-containing chemical entities have been investigated as matrix metalloproteinase inhibitors as well as antioxidant molecules. The combination of the two properties could represent a useful feature due to the potential application in all the pa

Full text of DOI:10.1080/14756366.2016.1217853

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Catechol-based matrix metalloproteinase
inhibitors with additional antioxidative activity
Marilena Tauro, Antonio Laghezza, Fulvio Loiodice, Luca Piemontese, Alessia
Caradonna, Davide Capelli, Roberta Montanari, Giorgio Pochetti, Antonella
Di Pizio, Mariangela Agamennone, Cristina Campestre & Paolo Tortorella
To cite this article: Marilena Tauro, Antonio Laghezza, Fulvio Loiodice, Luca Piemontese,
Alessia Caradonna, Davide Capelli, Roberta Montanari, Giorgio Pochetti, Antonella Di Pizio,
Mariangela Agamennone, Cristina Campestre & Paolo Tortorella (2016): Catechol-based matrix
metalloproteinase inhibitors with additional antioxidative activity, Journal of Enzyme Inhibition
and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1217853
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1217853
View supplementary material
Published online: 24 Aug 2016.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [Cornell University Library]
Date: 26 August 2016, At: 22:26

www.tandfonline.com/ienz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–13
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1217853
!
RESEARCH ARTICLE
Catechol-based matrix metalloproteinase inhibitors with additional
antioxidative activity
Marilena Tauro1 , Antonio Laghezza2 , Fulvio Loiodice2, Luca Piemontese2 , Alessia Caradonna2
,
Davide Capelli3 , Roberta Montanari3 , Giorgio Pochetti3 , Antonella Di Pizio4 , Mariangela Agamennone5,
Cristina Campestre5, and Paolo Tortorella2
2
1Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA, Dipartimento di Farmacia-Scienze del
3
Farmaco, Universita degli Studi ‘‘A. Moro’’ di Bari, Bari, Italy, Istituto di Cristallografia, CNR, Monterotondo Stazione (Roma), Italy, Institute of
4
`
Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem,
`
5
Rehovot, Israel, and Dipartimento di Farmacia, Universita ‘‘G. d’Annunzio’’ Chieti, Chieti, Italy
Abstract
Keywords
New catechol-containing chemical entities have been investigated as matrix metalloproteinase
inhibitors as well as antioxidant molecules. The combination of the two properties could
represent a useful feature due to the potential application in all the pathological processes
characterized by increased proteolytic activity and radical oxygen species (ROS) production,
such as inflammation and photoaging. A series of catechol-based molecules were synthesized
and tested for both proteolytic and oxidative inhibitory activity, and the detailed binding mode
was assessed by crystal structure determination of the complex between a catechol derivative
and the matrix metalloproteinase-8. Surprisingly, X-ray structure reveals that the catechol
oxygens do not coordinates the zinc atom.
Antioxidants, inhibitors, metalloenzymes,
structure–activity relationships, X-ray
crystallography
History
Received 6 June 2016
Revised 18 July 2016
Accepted 19 July 2016
Published online 12 August 2016
Introduction
Photoaging represents another process in which the proteolytic
activity of MMPs has a notorious and critical role; collagenases,
in particular, are responsible for the decomposition of particular
types of collagen and other proteins in the extracellular matrix of
the dermis7–10. The breakdown of dermal collagen and elastin is
purported to be one of the major contributing factors to loss of
skin’s firmness and elasticity. Physiologically, the human skin
expresses a number of MMPs, including MMP-1, -2, -3, -8, -9,
and -13, all of which are capable of attacking native fibrillar
collagen.
Matrix metalloproteinases (MMPs) are a family of zinc-contain-
ing endopeptidases, capable to process all the extracellular matrix
components. They can be classified in collagenases (MMP-1,
MMP-8, MMP-13, MMP-18), gelatinases (MMP-2, MMP-9), and
metalloelastase (MMP-12), based on the substrate that they
process.
Their enzymatic activity is finely regulated in physiological
conditions: MMPs are responsible for tissue regeneration and
remodeling, as well as bioavailability of pro-factors that need
proteolytic cut activation. Endogenous molecules such as tissue
inhibitors of metalloproteinases (TIMPs) are responsible for the
physiological fine regulation of the numerous isoforms1.
However, several external factors (e.g. solar UV irradiation) or
pathological conditions (cancer, metastasis, or chronic inflam-
mation) are able to stimulate an overexpression of specific MMPs.
Therefore, due to their implications in complex pathological
processes, MMPs continue to be considered a pivotal target for
therapeutic intervention2.
Chronic exposure to solar UV radiation is an accelerator factor
for photoaging. UV irradiation is known to provoke oxidative
stress through the generation of reactive oxygen species (ROS),
which are responsible to interfere with physiological pathways
and ultimately activate the overexpression of a number of
proteolytic enzymes such as MMPs in skin cells, capable to
destroy the dermal connective tissue. In addition, ROS can
regulate pro-MMPs activation11–15
.
Thus, the antioxidant activity associated with the inhibition of
MMPs could represent a promising strategy to obtain new
chemical entities capable to reduce photoaging and prevent
wrinkles and damage of the skin, and be effective for other
pathological conditions where oxidative stress occurs.
MMP inhibitors (MMPIs) are usually characterized by a
backbone that interacts with the specificity pocket S10, coupled to
a metal chelator portion that binds to the catalytic Zinc ion. To
expand the library of potential MMPIs and to overcome some
limitations of the hydroxamic acid moiety16,17, other zing binding
The selectivity still represents the main challenge in the MMP
inhibitor design: the importance of selective targeting3,4 has
already been previously proved in MMP inhibition strategy5
especially because in the last decade, a number of inhibitors failed
in clinical trial phases6, mainly due to their broad spectrum
activity.
Address
paolo.tortorella@uniba.it
for
correspondence:
Paolo
Tortorella.
E-mail:

2
M. Tauro et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Figure 1. Catechol-based MMP inhibitors.
groups (ZBGs) as essential portion in MMP inhibitor molecule of
new synthesis have been developed18–22
.
Recently, a series of rosmarinic acid derivatives has been
identified as micromolar MMP-1 inhibitors23. In addition, phen-
olic and polyphenolic compounds have already been used as
antioxidants, and their activity seems to impact several different
pathways. In particular, one of the most tested hypothesis is that
they exert protective effects against cancer and other chronic
diseases by reducing ROS levels13. Natural compounds derived
from green tea (EGCG), mainly known for their antioxidant
properties, have shown relevance when applied on wrinkles or
fine lines caused by aging, due to their ability to enhance collagen
levels and to inhibit MMPs24. Many other natural compounds,
such as phytosterol, fucosterol, resveratrol, soy isoflavones, alpha-
tocopherol, and vitamin E and C, are also able to inhibit MMPs
and ROS and reduce the degradation of skin25–28. Some MMPIs
have been studied in topical cosmetic compositions to counteract
the effect of photo and chronological skin aging. For example,
their use has been reported in combination with UV blockers (e.g.
octinoxate and zinc oxide)12, natural estrogen (e.g. 17-beta
estradiol or an estrogen-like steroid)12, or antioxidants (Alpha
Lipoic Acid, ALA)29.
Figure 2. Catechol-based MMP inhibitors.
methylation of the sulfonamide nitrogen of the dibenzylate
intermediate (25).
Compounds 7–13 and 18–22 were synthesized via condensa-
tion of the suitable aniline or benzyl amine with the appropriate
sulfonyl chloride (Schemes 2 and 3). Subsequent deprotection
under H2 atmosphere in the presence of 10% Pd/C or with BBr3
was needed to obtain the final compounds. N-(2,3-dihydroxy-
phenyl)-N-methyl-4-phenyl sulfonamide (10) and N-(2,3-dihy-
droxy-phenyl)-N-methyl-4-phenoxy-phenyl sulfonamide (11)
were obtained through methylation of the sulfonamide nitrogen
of the dibenzylate intermediate.
Compounds 14–17 were synthesized via condensation of 3,4-
(methylenedioxy)aniline or benzylamine with the appropriate
sulfonyl chloride (Scheme 4). Subsequent deprotection with BBr3
was needed to obtain the final compounds.
All synthesized compounds 1–22 were tested against MMP-2,
-8, and -9 (Tables 1 and 2). Compound 1 shows a very weak
activity against all considered MMPs. As expected, the introduc-
tion of a diphenyl moiety (2) provides more potency and
selectivity on MMP-2. A significant increase of activity toward
all MMPs is obtained by inserting a methylene spacer (3), while
In this study, we present a new class of MMPIs obtained by
combining a catecholic portion with a backbone able to reach the
S10 specific pocket of the enzyme isoforms. All these compounds
were tested toward MMP-2, -8, and -9 whereas their in vitro
antioxidant activity was evaluated by DPPH assay. In order to
better understand the interactions between MMPs and the
chemical inhibitor, the X-ray structure of the complex between
MMP-8 and one compound of the series was resolved.
Results and discussion
Starting from the promising inhibitory activity values obtained the methylation of the amide nitrogen (4) reduces it. The
testing catechol on different MMPs30, we decided to further introduction of an oxygen atom between the phenyl rings results
investigate catecholic derivatives by focusing our attention on the in an increase of activity (5 versus 2) or selectivity for MMP-2
backbone that, in the classical structure activity relationship (6 versus 3).
(SAR) of MMPIs, blocks the substrate access to the active site.
As well as for amide derivatives, compounds 7 and 8 still
Therefore, we report herein the synthesis and biological activity maintain good activity values toward MMP-2, showing an
of three set of analogs (Figure 1) in which the catechol is increased activity also on MMP-8 and -9. The introduction of
connected through an amide (compounds 1–6) or a sulfonamide the more flexible phenoxy phenyl structure (9) results in a slight
(compounds 7–17) group to a series of alkylaryl or aryl moieties increase of activity toward all the enzyme isoforms, with IC50
in order to reach the S10 specificity pocket of the different enzyme values around 6 mM against all tested MMPs.
isoforms.
The methylation of the sulfonamide nitrogen still confirms its
Most compounds present a diphenyl or a phenoxyphenyl detrimental effects on MMP-8 and -9 (10 and 11), while no loss in
residue, based on the results obtained by our previous works in activity results for these N-methyl derivatives against MMP-2.
which these groups were linked to typical ZBGs3,4,19,22,31–34. The Compound 10 stands out for its very interesting selectivity toward
relative position of sulfonamide linking group as well as the MMP-2. The insertion of a methylene between the sulfonamide
importance of hydroxyl groups were evaluated. To this aim, we moiety and the catecholic portion (12) allows to obtain inhibition
prepared a fourth set of compounds 18–22 (Figure 2) character- activity in the low micromolar range toward MMP-2, -8, and -9
ized by the lack of hydroxyl groups or the presence of a single and even the substitution of the phenyl ring with a bromine atom
hydroxyl group in different position of the aromatic ring.
Compounds 1–6 were obtained via condensation of dibenzy-
(13) maintains high activity values against all isoforms.
Moving the hydroxyl groups from the 2,3 to the 3,4 positions
loxybenzoic acid or corresponding chloride with the appropriate of the aromatic ring, it results a slight increase of activity when
aniline or benzylamine and following by debenzylation through the sulfonamide moiety is directly linked to the phenyl residue
catalytic hydrogenation (Scheme 1). N-(4-diphenylmethyl)-N- (8 versus 14); in this case, the substitution of phenyl with bromine
methyl-2,3-dibenzyloxybenzamide (26) was obtained through (15) reduces the activity on MMP-8 and MMP-9. The introduction

DOI: 10.1080/14756366.2016.1217853
Catechol-based MMP inhibitors
3
Scheme 1. Reagents and conditions: (a) anhydrous THF, r.t. or DMAP, DCC, CH2Cl2, r.t.; (b) dry DMF, NaH, CH3I, 0 ꢀC; (c) H2, 10% Pd-C, CH3OH/
THF 2:5, r.t.
Scheme 2. Reagents and conditions: (a) Et3N, CH2Cl2, reflux; (b) dry DMF, NaH, CH3I, 0 ꢀC; (c) THF/MeOH, 10% Pd/C, H2, r.t. or BBr3, CH2Cl2,
0 ꢀC 1 h.
Scheme 3. Reagents and conditions: (a) Et3N, CH2Cl2, reflux.
Scheme 4. Reagents and conditions: (a) Et3N, CH2Cl2, reflux; (b) BBr3, CH2Cl2, 0 ꢀC 1 h.
of a methylene spacer into the structure of 14 gives 16 with lower mono- (20–22) or non-substituted (18, 19) analogs (Table 2).
activity toward all isoforms, whereas the same modification for However, these compounds show a complete loss of activity
15 affords 17 showing reduced activity only against MMP-2. against all isoforms.
In order to evaluate whether the presence of hydroxyl The in vitro antioxidant activity of these catecholic derivatives
groups was essential for inhibitory activity, we prepared the was also evaluated by DPPH assay (Tables 1 and 2), considered as

4
M. Tauro et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Table 1. MMP activity values expressed as IC50 (mM) and antioxidant activity values expressed as EC50 in DPPH assay and ARP.
a
Compounds
n
R
R1
MMP-2
MMP-8
MMP-9
EC50
ARPb
Gallic acid
1
2
3
4
5
6
7
8
0.065 0.012
0.130 0.041
0.163 0.012
0.099 0.009
0.112 0.021
0.104 0.044
0.119 0.009
0.128 0.013
0.101 0.001
0.112 0.012
0.133 0.023
0.138 0.004
0.166 0.011
0.172 0.025
0.143 0.020
0.192 0.042
0.167 0.023
0.188 0.002
15.4
7.7
6.1
10.1
8.9
9.6
8.4
7.8
9.9
8.9
7.5
7.2
6.0
5.8
7.0
5.2
6.0
5.3
0
0
1
1
0
1
0
0
0
0
0
1
1
0
0
1
1
H
H
H
H
CH3
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
4100
4100
4100
4100
4100
C6H5
C6H5
C6H5
C6H5O
C6H5O
CH3O
C6H5
C6H5O
C6H5
C6H5O
C6H5
Br
34
6
4.0 1.4
4100
6.6 1.2
25
4
56
8
4100
4100
26
94
16
5
4
3
33
95
19
5
5
3
4.2 0.9
12
12
4
3
8.7 2.0
6.2 1.5
4100
33.5 0.7
2.5 0.9
1.9 0.4
3.1 1.1
6.2 0.4
6.5 2.0
4100
9
6.3 2.3
9.5 2.5
17.5 2.1
10
11
12
13
14
15
16
17
29
4
7
2
2.1 1.0
2.0 0.5
2.7 0.8
5.5 2.0
7.5 1.5
2.5 0.1
4.4 0.5
7.0 0.2
5.6 1.5
C6H5
Br
C6H5
Br
6
6
2
1
12
10
2
1
4.4 0.3
b
ammol of antioxidant/mmol of DPPH. Antiradical power (ARP) ¼ 1/EC50
.
Table 2. MMP activity values expressed as IC50 (mM) and antioxidant activity values expressed as EC50 in DPPH assay and ARP.
R1
R2
R3
MMP-2
MMP-8
MMP-9
EC50
ARPb
a
Compounds
n
18
19
20
21
22
0
1
0
0
0
H
H
H
H
OH
H
H
H
OH
H
H
H
OH
H
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
41
41
0.37 0.09
41
0.15 0.03
51
51
2.7
51
6.6
H
b
ammole of antioxidant/mmol of DPPH. Antiradical power (ARP) ¼ 1/EC50
.
one of the standard colorimetric method for the evaluation of elimination of the hydroxy groups (18 and 19) or their substitution
antioxidant properties of pure compounds and is routinely with methoxy ones (data not shown) results in a complete loss of
practiced for assessment of free radical scavenging potential of activity. Derivatives with hydroxyl groups in positions 2, 3 show
an antioxidant molecule. Experiments were performed also with a better antioxidant activity with respect to the 3, 4 substitutions
gallic acid, a naturally occurring plant phenol as the reference (8 versus 14).
substance.
The reduction of the o-dihydroxy structure to phenol (20–22)
Several studies suggest that structure–antioxidant activity results in a decrease of the activity strongly dependent from the
relationship for phenolic compounds depends on the position substituent position: only the 2-substituted analog (22) maintains
of hydroxyl groups, the presence of other functional an interesting antioxidant activity, while no or low activity is
groups in the whole molecule, and their conjugation to hydroxyl observed in the 3- or 4-substituted derivative, respectively (20 and
groups35.
21). Therefore, it can be argued that the presence of two phenolic
For compounds 1–17, antioxidant activity could be mainly groups is not the only factor determining the antioxidant activity
ascribed to the reducing power of the o-dihydroxy structure; of our derivatives.

DOI: 10.1080/14756366.2016.1217853
Catechol-based MMP inhibitors
5
Figure 3. Hydrogen bond network of the
catechol form (A) and the oxidized quinone
˚
form (B) of 14 (distances in A).
Methylation of the nitrogen results in a reduction of activity for
both the amidic (4 versus 3) and sulfonamide analogs (10 versus 8
and 11 versus 9). The presence of a methylene spacer increases
ARP in the amide series (3 versus 2), while a strong reduction is
observed for sulfonamide derivatives (14 versus 16).
Catechol was identified as a promising ZBG30 but the
introduction of aryl moieties aimed to reach the S10 specificity
pocket of MMP enzymes does not lead to the expected potency
improvement. In addition, at a first glance it is not obvious to
derive a structure activity relationship (SAR) for the catechol
analog series. To elucidate the binding mode of the catechol
compounds, the inhibitor 14 was co-crystallized with MMP-8.
Crystals of the complex MMP-8:14 were grown as described in
the Experimental Section. Surprisingly, the analysis of the Fo-Fc
electron density map in the region of binding of 14 shows the
presence of the catechol and its quinone oxidized form, both
competing for the same binding site (S10 site). The refinement of
the occupancy factors of the two forms reveals the predominance
of the catechol with respect to quinone (77% versus 23%). The
binding mode of the two forms in the S10 site is different at the
level of the aromatic ring containing the zinc binding functions.
Figure 4. Pharmacophore model on the reference structure 15.
As evidenced in Figure 3, only the quinone, with one of its oxygen
˚
The pharmacophore model highlights the importance of both
atoms, bind the zinc ion with a distance of 1.84 A giving rise to a hydroxyl groups for the MMP-8 inhibition and of their positions
distorted tetrahedral coordination, while one of the catechol in relation to the aromatic ring. The resulting QSAR model works
oxygens makes H-bonds with the catalytic important E198 side- well in discriminating actives versus inactives (Figure 5), is robust
chain and the NH of A163, belonging to the antiparallel b-strand. and predictive (correlation values for the MMP-8 inhibition
The sulfonamide NH group of both forms is engaged in a model, R2: 0.8, Q2: 0.8, F: 51.3, P: 3.3e-006, RMSE: 0.4,
H-bond with a water molecule further bridged to the P217 CO Pearson’s R: 0.9).
group. The sulfonamide junction adopts a g-conformation in
The activity prediction is well correlated even with MMP-9
both forms (ꢁ86ꢀ and ꢁ102ꢀ, respectively), with one of the two and MMP-2 (Figure 5B and C) experimental values; however,
oxygens turned toward the upper rim, giving rise to H-bonds with interestingly, the pharmacophore QSAR model underestimates the
the A161 and L160 NH groups. Finally, the diphenyl group deeply potency of compounds 3, 5, and 6 toward MMP-2. To investigate
protrudes into the S10 pocket, with its terminal part facing the this difference and to characterize the interactions corresponding
˚
charged R222 side-chain (closest distance of 3.1 A), at the end of to the identified features, we integrate the pharmacophore-based
the pocket.
Integrating different computational approaches, we could
analysis with a structure-based study.
Surprisingly, the X-ray structure of 14 in complex with MMP-8
identify the ligand features responsible for ligand–protein inter- reveals that the catechol oxygens contribute to a dense water
action and explain how small modifications in ligand structures network around the ligand (see Figure 6). Noteworthy, the
can influence the inhibition potency. Through a pharmacophore- quinone oxidized form of the ligand was found in the crystal
based analysis, the features that are needed for the MMP complex, but not in the solution used for inhibition assay (see
inhibition have been identified (Figure 4): one aromatic ring supporting information); therefore, we have considered activity
(R9), two acceptor H-bond groups (A1 and A3, respectively), and values not related to the oxidized forms, which have been
one donor H-bond group (D6). Compound 15 was automatically neglected in the computational studies. Apparently, catechol
selected as the reference structure, with a Fit-value of 3.00.
oxygen atoms prefer to enter in the water network rather than

6
M. Tauro et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Figure 5. QSAR models for MMP-8 (A), MMP-9 (B), and MMP-2 (C) (test set + training set molecules).
Figure 6. (A) MMP-8:14 X-ray complex (IC50: 3.1 mM, predicted total energy ꢁ395 kJ/mol), (B) MMP-2:14 modeled complex (IC50: 7.0 mM, predicted
total energy ꢁ379 kJ/mol), and (C) MMP-9:14 modeled complex (IC50: 2.7 mM, predicted total energy ꢁ405 kJ/mol). The ligand is represented as sticks
and the water molecules as spheres. The network of water molecules is depicted with H-bonds reported as dashed lines and water clusters are encircled.
coordinate the zinc ion, suggesting a relevant role of water optimized leads: starting from the identification of interactions
molecules in the binding and activity of catechol analogs toward involved in binding, moving to the analysis of possible additional
MMP enzymes. It is well-known the role of water molecules in or alternative interactions that can improve the potency. However,
ligand–protein interaction, and this role becomes fundamental in the complex described here highlights difficulties of theoretical
the case of weak binders36. To clarify the role of the water prediction of binding energies and optimization process due to the
molecules, the X-ray complex MMP-8:14 has been prepared and fact that the binding involves hydrogen bonding through water
minimized with explicit waters with MacroModel Embrace molecules whose positions can change.
Minimization37. The minimized complex reveals the interactions
This observation prompted us to investigate the influence of
corresponding to the pharmacophore features: the donor feature the solvent in the binding of compound 14 with MMP-2 and
(D6) is located on the OH group establishing an H-bond MMP-9 (Figure 6B and C). We can observe similar interactions in
interaction with E198 side chain; the acceptor group A1 directly all three complexes, but a different hydration by the solvent
H-bonds with L160 NH and A161 NH; the acceptor group A3 is (Figure 6A–C). In the MMP-2:14 complex, the oxygen bound
connected through two water molecules to H162 side chain; the to D6 is not connected with the water molecule cluster 1
aromatic feature (R9) is located above the zinc ion ensuring a (Figure 6B). This difference is due to a sequence difference: A206
good orientation for D6 and A3 interactions.
in MMP-8 is aligned to E210 in MMP-2. E210 side chain takes
In addition, three relevant clusters of water molecules mediate the place of the water molecule present in MMP-8:14 complex
the interaction between the ligand and MMP-8 (Figure 6A): (i) the and shifts the water cluster 1 far away from the ligand
oxygen bound to D6 is connected through seven water molecules (Figure 6B). In the case of MMP-9, we found in this position
to A163 CO on the top, H201 CO on the side and H207 side chain D410 (Figure 6C); since it is shorter than the glutamate present in
on the bottom; (ii) five water molecules connect the sulfonamide this position in MMP-2, the cluster 1 water network is restored.
oxygen with P217 CO, N218 side chain and Y219 NH on the
The structure-based analyses, taking into account all possible
bottom, and G158 CO on the top; (iii) two water molecules ligand–protein interactions and water contribution, ensure a more
mediate the interaction between the A3 feature oxygen and H162 accurate prediction of the binding. Differently from what
side chain.
observed with the ligand-based analysis, the predicted binding
The structure determination of an enzyme bound to a energies of MMP-2:3, 5, and 6 (ꢁ303 kJ/mol, ꢁ362 kJ/mol and
potentially therapeutic inhibitor is usually aimed to design ꢁ366 kJ/mol, respectively) are in a good correlation with the

DOI: 10.1080/14756366.2016.1217853
Catechol-based MMP inhibitors
7
Figure 7. ROC curves (A) MMP-8:14 – AUC: 0.855. (B) MMP-2:14 – AUC: 0.790. (C) MMP-9:14 – AUC: 0.829.
experimental pIC50. The ROC curves of all tested compounds The resulting oil was dissolved in dry THF (30 mL) and a solution
toward MMP-8, MMP-2, and MMP-9 are shown in Figure 7.
of aniline or 4-phenylbenzylamine (7.12 mmol) in dry THF
(1 mL) was added dropwise at 0 ꢀC. After 12 h at room
temperature, the mixture was concentrated under reduced pressure
and diluted with CH2Cl2. The organic layer was washed with HCl
1N, brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography
(n-hexane/ethyl acetate ¼ 9:1) to give the title products.
Conclusions
A new series of catechol-based compounds has been synthesized
and tested for MMP inhibitory activity as well as antioxidant
properties. Structure–activity relationship studies were imple-
mented by X-ray analysis of the co-crystallization complex
between MMP-8 and one of these new chemical entities
(compound 14). Computational studies have been performed to
2,3-Dibenzyloxy-N-phenylbenzamide (23)
identify a pharmacophore model and rationalize the influence of 53% yield; 1H NMR (CDCl3): d ¼ 5.16 and 5.20 (2 s, 4H, 2
chemical structure on inhibition potency.
CH2Ph), 7.03–7.09, 7.20–7.53, and 7.83–7.87 (m, 18H, aro-
The activity of these molecules on both MMPs and ROS matics), 10.01 (s, 1H, NH); MS (ESI) m/z (%): 432
encloses a potential therapeutic strategy able to reduce the impact [M + Na]+ (100)
of these two important targets in degradation processes such as
photoaging or inflammation, as well as to provide an additional 2,3-Dibenzyloxy-N-(4-diphenyl)methylbenzamide (25)
treatment for degenerative phenomenon involving pathological
enzymatic overexpression.
1
71% yield; H NMR (CDCl3): d 4.54 (s, 2H, CH2N), 4.99, and
5.15 (2 s, 4H, 2 CH2Ph), 7.07–7.59 and 7.79–7.83 (m, 22H,
aromatics), 8.38 (t, 1H, NH); MS (ESI): m/z (%): 522
[M + Na]+ (100), 340 (7).
Experimental section
Chemistry
2,3-Dibenzyloxy-N-4-bromophenylbenzamide
Melting points were determined in open capillaries on a
Gallenkamp electrothermal apparatus. Mass spectra were rec- 99% yield; mp: 122–124 ꢀC 1H NMR (CDCl3): 5.10 and 5.20 (2 s,
orded on a HP MS 6890-5973 MSD spectrometer, electron impact 4H, 2 CH2Ph), 7.10–7.50, and 7.80–7.86 (d, 17H, aromatics), 10.0
70 eV, equipped with a HP ChemStation or with an Agilent LC– (s, NH); MS (ESI) m/z: 488 [M + 2 + Na]+ (100), 486
MS 1100 Series LC–MSD Trap System VL spectrometer, [M + Na]+ (100).
electrospray ionization (ESI). 1H-NMR spectra were recorded
using the suitable deuterated solvent on a Varian Mercury 300 Preparation of 2,3-dibenzyloxy-N-(4-diphenyl)benzamide (24)
NMR Spectrometer. Chemical shifts (d) are expressed as parts per
A suspension of 2,3-dibenzyloxy-N-4-bromophenylbenzamide
million (ppm) and the coupling constants (J) in Hertz (Hz).
(0.56 mmol), benzeneboronic acid (1.6 mmol), Cs2CO3
Microanalyses of solid compounds were carried out with a
(1.2 mmol), and [Pd(PPh3)4] (0.024 mmol) in anhydrous toluene
Eurovector Euro EA 3000 model analyzer; the analytical results
(15 mL) was stirred at 95 ꢀC overnight under nitrogen atmosphere.
are within 0.4% of theoretical values. Flash column chromatog-
Then, the mixture was diluted with 1N HCl (1 mL) and ethyl
acetate (1.3 mL) at room temperature and filtered through a Celite
pad. The resulting solution was washed with a saturated NaHCO3
˚
raphy was performed using Geduran silica gel 60 A (45–63 mm).
Chemicals were purchased from Aldrich Chemicals (Milan, Italy)
and were used without any further purification.
solution (3 ꢂ 15 mL), brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by column
chromatography (9.4:0.5:0.1 petroleum ether/ethyl acetate/IPA) to
give the title product.
Preparation of 2,3-dibenzyloxy-N-4-bromophenylbenzamide, 23
and 25
1
To a solution of 2,3-dibenzyloxybenzoicacid (30) (2.27 mmol) in
74% yield; mp: 121–124 ꢀC; H NMR (CDCl3): d ¼ 5.18 and
anhydrous toluene (35 mL), thionyl chloride (23.7 mmol) was 5.21 (2 s, 4H, 2 CH2-Ph), 7.20–7.50, 7.70–7.73, 7.83–7.86, and
added dropwise at 0 ꢀC under nitrogen atmosphere. The mixture 8.23–8.26 (d, 22H, aromatics), 10.0 (s, NH). MS(ESI) m/z (%):
was refluxed for 2 h and concentrated under reduced pressure. 508 [M + Na]+(100).

8
M. Tauro et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Preparation of compounds 27 and 28
7.55–7.65, 7.83–7.88 (m, 12H, aromatics); 13C NMR (CDCl3): d
43.6, 56.0, 60.9, 112.6, 121.7, 124.3, 127.5, 127.7, 127.9, 128.7,
129.3, 130.0, 138.8, 139.6, 145.4, 147.1, 152.6.
DCC (2.04 mmol) was added to a solution of 2,3-dibenzylox-
ybenzoic acid (30) (1.5 mmol) in dichloromethane (30 mL) at 0 ꢀC
under nitrogen atmosphere. After 15 min, a solution of 4-DMAP
(1.35 mmol) and 4-phenoxyaniline or 4-phenoxybenzylamine
N-(2,3-dimethoxyphenyl)methyl-4-bromophenylsulfonamide (35)
(1.38 mmol) in dichloromethane (4 mL) was added dropwise. 88%yield;mp:125 ꢀC;1HNMR(CDCl3):d3.72(s,3H,OCH3),3.80
The resulting mixture was allowed to warm to room temperature (s, 3H, OCH3), 4.15 (d, J ¼ 6.0 Hz, 2H, CH2NH), 5.28 (t, J ¼ 6.0 Hz,
and stirred for further 22 h. The resulting precipitate was filtered 1H, NH), 6.64–6.68, 6.76–6.80, 6.85–6.91, 7.47–7.51, 7.57–7.61
off, and the organic layer was washed with a saturated NH4Cl (m, 7H, aromatics); 13C NMR (CDCl3): d 43.8, 56.0, 60.8, 112.7,
solution (3 ꢂ 20 mL) and twice with brine, then dried over 121.7, 124.3, 127.4, 128.8, 129.6, 132.2, 139.4, 147.0, 152.6.
Na2SO4, filtered and the solvent removed in vacuo to yield a
brown solid. Purification by flash chromatography (8:1.9:0.1 N-(1,3-benzodioxol-5-yl)-4-diphenylsulfonamide (36)
hexane/chloroform/IPA) and crystallization (THF/hexane) yielded
(CH2Cl2, 56% yield); mp: 145–146 ꢀC; 1H NMR (CDCl3):
the title compounds as a white powder.
d ¼ 5.93 (s, 2H, -CH2), 6.46–6.50, 6.62–6.66, 6.72–6.74 (m, 3H,
aromatic), 6.85 (bb, 1H, NH), 7.37–7.49, 7.55–7.67, 7.79–7.84
(m, 9H, aromatics); 13C NMR (CDCl3): d 101.8, 105.8, 108.5,
117.2, 127.5, 127.8, 128.1, 128.8, 129.3, 130.2, 137.6, 139.4,
146.0, 146.3, 148.3.
2,3-Dibenzyloxy-N-(4-phenoxyphenyl)benzamide (27)
73% yield; 1H NMR (CDCl3): d ¼ 5.18 and 5.21 (2 s, 4H,
2 CH2Ph), 6.89–6.99, 7.05–7.10, 7.21–7.53, and 7.83–7.88
(m, 22H, aromatics), 9.99 (s, 1H, NH).
N-(1,3-benzodioxol-5-yl)-4-bromophenylsulfonamide (37)
1
2,3-Dibenzyloxy-N-(4-phenoxyphenyl)methylbenzamide (28)
62% yield; mp: 140–141 ꢀC; H NMR (CDCl3): d ¼ 5.94 (s, 2H,
CH2), 6.41–6.45, 6.61–6.64, 6.67–6.81 (m, 3H, aromatics), 6.97
(bb, 1H, NH), 7.54–7.62 (m, 4H, aromatics); 13C NMR (CDCl3):
d ¼ 101.9, 105.8, 108.6, 116.6, 117.3, 128.4, 129.1, 132.6, 137.9,
146.6, 148.4.
1
69% yield; H NMR (CDCl3): d ¼ 4.47 (d, 2H, CH2N), 4.99 and
5.15 (2 s, 4H, 2 CH2Ph), 6.88–7.48, and 7.76–7.82 (m, 22H,
aromatics), 8.31 (t, 1H, -OH); MS (ESI) m/z (%): m/z 514 [M-H]ꢁ
(100), 408 (9), 315 (9), 223 (9).
N-(1,3-benzodioxol-5-ylmethyl)-4-diphenylsulfonamide (38)
General procedure for the preparation of 29–31, 34–39, and
18–22
(CH2Cl2, 98% yield); mp: 159–160 ꢀC; 1H NMR (CDCl3):
d ¼ 4.08 (d, J ¼ 6.0 Hz, 2H, CH2NH), 4.86 (t, J ¼ 6.0 Hz, 1H,
NH), 5.87 (s, 2H, CH2), 6.63–6.70, 7.39–7.52, 7.58–7.63, 7.67–
7.62, 7.88–7.93 (m, 12H, aromatics); 13C NMR (CDCl3):
d ¼ 45.7, 101.4, 108.4, 108.7, 121.6, 127.6, 127.9, 127.9, 128.7,
129.3, 130.1, 138.7, 139.5, 145.8, 147.6, 148.1.
A solution of the suitable sulfonyl chloride (1.4 mmol) in CH2Cl2
(5 mL) was added to a solution of the appropriate aniline
(1.5 mmol) and Et3N (2.8 mmol) in CH2Cl2 (5 mL). The mixture
was refluxed for 4 h, cooled, diluted with CH2Cl2 (10 mL), washed
with 1N HCl and brine, dried over Na2SO4, and then evaporated in
vacuo. The crude product was purified by flash column chroma-
tography on silica gel using different mixtures as eluent (indicated
N-(1,3-benzodioxol-5-ylmethyl)-4-bromophenylsulfonamide (39)
in brackets) or was used for the next step without any purification. (CH2Cl2, 97% yield); mp: 148–149 ꢀC; 1H NMR (CDCl3):
d ¼ 4.04 (d, J ¼ 6.0 Hz, 2H, CH2NH), 4.84 (t, J ¼ 6.0 Hz, 1H,
N-(2,3-dibenzyloxy)phenyl-4-methoxyphenylsulfonamide (29)
NH), 5.93 (s, 2H, –CH2–), 6.60–6.70, 7.59–7.71 (m, 7H,
aromatics); 13C NMR (CDCl3): d ¼ 45.4, 101.5, 108.5, 108.6,
121.7, 127.9, 128.9, 129.8, 132.6, 139.3, 147.6, 148.2.
(Petroleum ether/ethyl acetate 9:1, 51% yield); 1H NMR (CDCl3):
d ¼ 3.79 (s, 3H, OCH3), 4.78 and 5.07 (2 s, 4H, 2 OCH2Ph), 6.70–
6.97 (m, 4H, aromatics), 7.04 (bb, 1H, NH), 7.15–7.42 (m, 11H,
aromatics), 7.63–7.68 (m, 2H aromatics); MS (ESI) m/z (%): 498
[M + Na]+ (100), 407 (6), 304 (15).
N-phenyl-4-diphenylsulfonamide (18)
55% yield; mp: 126–127 ꢀC; 1H NMR (CDCl3): d ¼ 6.97 (bb, 1H,
NH), 7.10–7.15, 7.22–7.29, 7.40–7.48, 7.53–7.58, 7.62–7.66,
7.83–7.86 (m, 14H, aromatics); 13C NMR (CDCl3): d ¼ 121.8,
125.6, 127.7, 127.09 128.1, 128.0, 128.8, 129.3, 129.6, 136.7,
137.7, 139.3, 146.1. MS (ESI) m/z (%): 308 [M-H]ꢁ (18), 244
(100). HR-MS [(C19H17NO2S-H)]ꢁ, m/z 308.0744 (calc.
308.0751).
N-(2,3-dibenzyloxy)phenyl-4-diphenylsulfonamide (30)
(Petroleum ether/ethyl acetate/methylene chloride 9:0.5:0.5, 70%
yield); 1H NMR (CDCl3): d ¼ 4.79 and 5.07 (2 s, 4H, 2 OCH2Ph),
6.72–6.75, 6.94–7.00 (m, 2H, aromatics), 7.11 (bb, 1H, NH),
7,20–7.62, and 7.76–7.80 (m, 20H, aromatics); MS (ESI) m/z (%):
544 [M + Na]+ (100), 304 (75).
N-benzyl-4-diphenylsulfonamide (19)
75% yield; mp: 134–135 ꢀC; 1H NMR (CDCl3): d ¼ 4.19 (d,
J ¼ 6.0 Hz, 2H, CH2NH), 4.74 (t, J ¼ 6.0 Hz, 1H, NH), 7.20–7.31,
7.41–7.53, 7.59–7.63, 7.69–7.73, 7.91–7.95 (m, 14H, aromatics);
13C NMR (CDCl3): d ¼ 47.6, 127.6, 127.9, 128.0, 128.1, 128.2,
128.4, 128.8, 129.0, 129.3, 136.4, 138.6, 139.5, 145.9. MS (ESI)
m/z (%): 322 [M-H]ꢁ (5), 153 (100). HR-MS [(C19H17NO2S-
H)]ꢁ, m/z 322.0903 (calc. 322.0907).
N-(2,3-dibenzyloxy)phenyl-4-phenoxyphenylsulfonamide (31)
1
(Petroleum ether/chloroform/IPA 9:0.9:0.1, 81% yield); H NMR
(CDCl3): d ¼ 4.81 and 5.09 (2 s, 4H, 2 OCH2), 6.72–6.75, 6.90–
7.04, 7.16–7.47, 7.63–7.77 (m, 22H, aromatics and 1H, NH); MS
(ESI) m/z (%): 560 [M + Na]+ (100), 469 (5).
N-(2,3-dimethoxyphenyl)methyl-4-diphenylsulfonamide (34)
N-(4-hydroxyphenyl)-4-diphenylsulfonamide (20)
1
98% yield; mp: 120–121 ꢀC; H NMR (CDCl3): d ¼ 3.73 (s, 3H,
1
63% yield; mp: 212–214 ꢀC; H NMR ([D6]DMSO): d ¼ 6.58–
OCH3), 3.76 (s, 3H, OCH3), 4.19 (d, J ¼ 6.0 Hz, 2H, CH2NH),
5.30 (t, J ¼ 6.0 Hz, 1H, NH), 6.72–6.79, 6.85–6.92, 7.38–7.50,
6.61, 6.84–6.88, 7.40–7.82 (m, 13H, aromatics), 9.30 and 9.78

DOI: 10.1080/14756366.2016.1217853
Catechol-based MMP inhibitors
9
(bb, 2H, –OH and –NH); 13C NMR ([D6]DMSO): d ¼ 116.0, aromatics), 7.95 (s, 1H, OH), 12.34 (s, 1H, OH); 13C NMR
124.4, 127.5, 127.6, 127.8, 128.9, 129.0, 129.5, 138.8, 138.9, (CDCl3): d ¼ 114.3, 116.1, 118.6, 119.0, 121.3, 125.5, 129.2,
144.4, 155.3. MS (ESI) m/z (%): 324 [M-H]ꢁ (100). HR-MS 136.4, 146.1, 149.3, 168.4. MS (ESI) m/z (%): 228 [M-H]ꢁ (100).
[(C18H15NO3S + Na)]+, m/z 348.0665 (calc. 348.0665).
HR-MS [(C13H11NO3-H)]ꢁ, m/z 228.0663 (calc. 228.0663).
N-(3-hydroxyphenyl)-4-diphenylsulfonamide (21)
N-(4-diphenyl)-2,3-dihydroxybenzamide (2)
1
71% yield; mp: 258–259 ꢀC; H NMR ([D6]DMSO): d ¼ 5.85–
38% yield; mp: 226–228; 1H NMR (CDCl3): d ¼ 5.92 (s, 1H,
NH), 6.81–6.91, 7.09–7.12, 7.25–7.69, 7.76–7.88 7.98—8.01 and
8.07–8.26 (m, 12H, aromatics), 9,53 (s, 1H, OH), 12,32 (s, 1H,
OH); MS (ESI): m/z (%): 304 [M-H]ꢁ (100). HR-MS
[(C19H15NO3-H)]-, m/z 304.0972 (calc. 304.0972).
6.30, 6.58–6.63, 7.31–7.73 (m, 13H, aromatics), 8.51 (bb, 2H,
–OH and –NH); 13C NMR ([D6]DMSO): d ¼ 105.6, 107.8, 112.5,
126.6, 126.8, 127.2, 127.3, 127.5, 128.2, 128.8, 129.4, 139.8,
141.7, 157.8. MS (ESI) m/z (%): 324 [M-H]ꢁ (100). HR-MS
[(C18H15NO3S-H)]ꢁ, m/z 324.0694 (calc. 324.0700).
N-(4-diphenyl)methyl-2,3-dihydroxybenzamide (3)
N-(2-hydroxyphenyl)-4-diphenylsulfonamide (22)
1
1
79% yield; mp: 111–113 ꢀC; H NMR (CDCl3): d ¼ 4.68 (d, 2H,
55% yield; mp: 155–157 ꢀC; H NMR ([D6]DMSO): d ¼ 6.66–
CH2N, J ¼ 5.49), 5.80 (s, 1H, OH), 6.62 (bb, 1H, NH), 6.73–6.78,
6.89–6.91, 7.05–7.09, 7.34–7.47 and 7.57–7.71 (m, 12H, aro-
matics), 12.68 (s, 1H, OH); 13C NMR (CDCl3): d ¼ 43.4, 113.8,
115.9, 118.2, 118.7, 127.1, 127.5, 127.7, 128.3, 128.8, 136.2,
140.5, 141.0, 146.0, 149.2, 169.9. MS (ESI): m/z (%): 318 [M-H]ꢁ
(100). HR-MS [(C20H17NO3-H)]ꢁ, m/z 318.1127 (calc. 318.1136).
6.73, 6.89–6.94, 7.14–7.17, 7.38–7.50, 7.68–7.80 (m, 13H,
aromatics), 9.43 (bb, 2H, OH and NH); 13C NMR
([D6]DMSO): d ¼ 116.0, 119.4, 124.5, 125.0, 126.6, 127.4,
127.5, 127.8, 128.9, 129.5, 138.8, 140.0, 144.3, 150.7. MS
(ESI) m/z (%) 324 [M-H]ꢁ (95), 217 (100); m/z (%): 348
[M + Na]+ (100). HR-MS [(C18H15NO3S-H)]ꢁ, m/z 324.0695
(calc. 324.0700).
N-(4-diphenyl)methyl-N-methyl-2,3-dihydroxybenzamide (4)
General procedure for the preparation of 26, 32 and 33
1
33% yield; mp: 160–163 ꢀC; H NMR ([D6]DMSO): d ¼ 3.14 (s,
3a or 30 or 31 (1 mmol) was dissolved in anhydrous DMF (20 mL)
under argon atmosphere at 0 ꢀC. Then, NaH (3 mmol, 95%
powder) and, after 30 min, CH3I (50 mL) were carefully added
and the mixture was stirred overnight at room temperature. After
evaporation of volatiles, the residue was dissolved in ethyl acetate
and washed with 1N HCl (3 ꢂ 50 mL) and brine (3 ꢂ 40 mL),
dried over Na2SO4, filtered, and concentrated in vacuo giving a
yellow oil, that was used for the next step without any
purification.
3H, CH3), 4.81 (s, 2H, CH2), 5.80 (bb, 1H, OH), 6.70–6.75, 6.89–
7.01, 7.33–7.66 (m, 12H, aromatics), 10.2 (bb, 1H, OH);
MS (ESI) m/z (%): 356 [M-H + Na+]ꢁ (100). HR-MS
[(C21H19NO3 + H)]+, m/z 334.1435 (calc. 334.1438).
2,3-dihydroxy-N-(4-phenoxyphenyl)benzamide (5)38
84% yield; mp: 164–165 ꢀC; 1H NMR (CDCl3): 5,82 (s, 1H, NH),
6.81–6.87, 7.00–7.15, 7.14–7.39, and 7.51–7.55 (m, 12H, aro-
matics), 7,93 (s, 2H, OH); 13C NMR (CD3OD): d ¼ 116.2, 118.1,
118.2, 118.5, 118.5, 118.8, 122.9, 123.1, 129.5, 133.1, 146.0,
148.4, 154.1, 157.5, 168.2. MS (ESI) m/z (%) 320 [M-H]ꢁ (100).
HR-MS [(C19H15NO4-H)]ꢁ, m/z 320.0923 (calc. 320.0928).
N-(4-diphenylmethyl)-N-methyl-2,3-dibenzyloxybenzamide (26)
1
97% yield; H NMR (CDCl3): d ¼ 2,76 (s, 3H, CH3N), 3.00 (s,
2H, CH2N), 5.15 and 5.17 (2 s, 4H, 2 CH2Ph), 6.92–7.55 (m, 22H,
aromatics); MS (ESI) m/z (%) 536 [M + Na]+ (100).
N-(4-phenoxyphenyl)methyl-2,3-dihydroxy-benzamide (6)38
N-(2,3-dibenzyloxyphenyl)-N-methyl-4-diphenylsulfonamide (32)
79% yield; mp: 113–114 ꢀC; 1H NMR (CDCl3): d ¼ 4.60 (d,
J ¼ 5.66, CH2N, 2H), 5.82 (s, 1H, OH), 6.59 (bb, 1H, NH), 6.72–
6.91, 6.97–7.14, and 7.29–7.37 (m, 12H, aromatics), 12.67 (s, 1H,
OH); 13C NMR (CD3OD): d ¼ 42.0, 115.3, 117.3, 118.2, 118.3,
118.3, 118.5, 122.9, 128.8, 129.4, 133.6, 145.9, 148.9, 156.4,
157.4, 170.0. MS (ESI) m/z (%): 334 [M-H]ꢁ (100). HR-MS
[(C20H17NO4-H)]ꢁ, m/z 334.1074 (calc. 334.1085).
1
90% yield; H NMR ([D6]DMSO): d ¼ 3.10 (s, 3H, CH3N), 4.99
and 5.11 (2 s, 4H, 2 OCH2Ph), 6.76–6.82, 6.95–7.02, and 7.20–
7.88 (m, 22H, aromatics); MS (ESI) m/z (%): 558[M + Na]+ (100),
304 (7).
N-(2,3-dibenzyloxyphenyl)-N-methyl-4-phenoxyphenylsulfona-
mide (33)
1
General procedure for the preparation of compounds 7–11
84% yield; H NMR ([D6]DMSO): d ¼ 3.11 (s, 3H, CH3N), 4.99,
and 5.10 (2 s, 4H, 2 OCH2Ph), 6.76–6.79, 6.94–7.05, 7.18–7.43,
and 7.71–7.76 (m, 22H, aromatics); MS (ESI) m/z (%): 574
[M-Na]+ (100), 319 (16), 228 (6).
To a stirred suspension of the appropriate intermediate (29–33)
(0.41 mmol) in THF/MeOH (16 mL, 3:1), 10% Pd/C (26 mg) was
added. After stirring overnight under hydrogen atmosphere
(6 atm), the reaction mixture was filtered through a pad of
Celite and concentrated under reduced pressure affording the
desired product.
General procedure for the preparation of compounds 1–6
A mixture of the appropriate dibenzyloxybenzamide 23–28
(0.38 mmol) and 10% Pd-C (24 mg) in CH3OH/THF 2:5 (14 mL)
was stirred at room temperature for 20 h under H2 atmosphere at
6 atm. The mixture was filtered through a Celite pad and
concentrated in vacuo to give the title product as a white solid.
N-(2,3-dihydroxyphenyl)-4-methoxyphenylsulfonamide (7)
1
82% yield; mp: 147–149 ꢀC; H NMR ([D6]DMSO): d ¼ 3.78 (s,
3H, CH3), 6.53–6.59, 6.98–7.01, 7.64–7.67 (m, 7H, aromatics),
8.87 (bb, 3H, 2 OH, NH); MS (ESI) m/z (%): 294 [M-H]ꢁ (100),
171 (59). 13C NMR ([D6]DMSO): d ¼ 56.0, 112.8, 114.5, 114.7,
118.8, 125.6, 129.4, 132.6, 138.7, 146.0, 162.7. HR-MS
[(C13H13NO5S-H)]ꢁ, m/z 294.0436 (calc. 294.0442).
2,3-Dihydroxy-N-4-phenylbenzamide (1)
1
72% yield; mp: 114–117 ꢀC; H NMR (CDCl3): d ¼ 5.82 (s, 1H,
NH), 6.82–6.87, 7.05–7.12, 7.19–7.25 and 7.38–7.60 (m, 8H,

10 M. Tauro et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
N-(2,3-dihydroxyphenyl)-4-diphenylsulfonamide (8)38
N-(3,4-dihydroxyphenyl)-4-diphenylsulfonamide (14)
1
79% yield; mp: 182–183 ꢀC; H NMR (DMSO-d6): d 6.45–6.63, 88% yield; mp: 216 ꢀC (dec.); 1H NMR (CD3OD): d ¼ 6.37–6.41,
7.38–7.50, 7.69–7.82 (m, 12H, aromatics), 9.00 (bb, 3H, 2- OH, 6.58–6.66, 7.31–7.44, 7.54–7.58, 7.62–7.66, 7.72–7.76 (m, 12H,
-NH); 13C NMR ([D6]DMSO): d ¼ 113.0, 115.0, 118.8, 125.4, aromatics); 13C NMR (CD3OD): d ¼ 111.2, 114.8, 115.1, 127.0,
127.5, 127.9, 128.9, 129.5, 138.9, 138.9, 139.9, 144.3, 146.1. MS 127.1, 127.7, 128.3, 128.9, 129.3, 138.4, 139.3, 143.4, 145.4. MS
(ESI): m/z 340 [M-H]ꢁ (93), 217 (100). HR-MS [(C18H15NO4S- (ESI) m/z (%): 340 [M-H]ꢁ (11), 153 (100), HR-MS
H)]ꢁ, m/z 340.0643 (calc. 340.0649).
[(C18H15NO4S-H)]ꢁ, m/z 340.0632 (calc. 340.0649).
N-(3,4-dihydroxyphenyl)-4-bromophenylsulfonamide (15)
N-(2,3-dihydroxyphenyl)-4-phenoxyphenylsulfonamide (9)38
62% yield; mp: 209 ꢀC (dec.); 1H NMR (CD3OD): d ¼ 6.30–6.34,
6.57–6.61, 7.53–7.62 (m, 7H, aromatics); 13C NMR (CD3OD):
d ¼ 111.4, 115.0, 115.1, 127.1, 128.9, 128.9, 132.0, 138.9, 143.6,
145.4. MS (ESI) m/z (%): 342 [M-H]ꢁ (12), 157 (100). HR-MS
[(C12H10BrNO4S + Na)]+, m/z 365.9400 (calc. 365.9406).
80% yield; mp: 176–178 ꢀC; 1H NMR (CDCl3): d ¼ 2.20–2.60
(bb, 3H, 2-OH and -NH), 6.54–6.58, 6.74–6.85, 6.93–6.96, 7.09–
7.14, 7.28–7.33, 7.62–7.65 (m, 12H, aromatics); MS (ESI) m/z
(%): 380 [M + Na]+ (100). 13C NMR (CD3OD): d ¼ 112.2, 114.8,
116.8, 118.6, 119.8, 124.5, 124.7, 129.3, 129.8, 133.3, 138.2,
145.3, 155.3, 161.5. HR-MS [(C18H15NO5S-H)]ꢁ, m/z 356.0594
(calc. 356.0598).
N-(3,4-dihydroxyphenyl)methyl-4-diphenylsulfonamide (16)
1
58% yield; mp: 189 ꢀC (dec.); H NMR (CD3OD): d 3.92 (s, 2H,
CH2), 6.47–6.51, 6.59–6.66, 7.37–7.50, 7.64–7.88, 7.72–7.76,
7.83–7.87 (m, 12H, aromatics); 13C NMR (CD3OD): d ¼ 114.9,
115.2, 119.4, 127.2, 127.3, 127.4, 128.2, 128.7, 128.9, 139.6,
139.6, 144.7, 145.1, 145.3. MS (ESI) m/z (%): 354 [M-H]ꢁ (7),
234 (100), HR-MS [(C19H17NO4S-H)]ꢁ, m/z 354.0811 (calc.
320.0806).
N-(2,3-dihydroxyphenyl)-N-methyl-4-diphenylsulfonamide (10)
1
57% yield; mp: 170–172 ꢀC; H NMR (CDCl3): d ¼ 3.22 (s, 3H,
NCH3), 5.99–6.02, 6.62–6.67, 6.87–6.89, and 7.43–7.71 (m, 14H,
aromatics and -OH); MS (ESI) m/z (%): 354 [M-H]ꢁ (100), 217
(61). 13C NMR (CD3OD): d ¼ 37.6, 114.8, 118.4, 119.4, 126.9,
128.0, 128.1, 128.3, 128.7, 138.8, 139.2, 143.3, 145.4, 146.1. HR-
MS [(C19H17NO4S-H)]ꢁ, m/z 354.0799 (calc. 320.0806).
N-(3,4-dihydroxyphenyl)methyl-4-bromophenylsulfonamide (17)
47% yield; mp: 124–125 ꢀC; 1H NMR (CD3OD): d ¼ 3.90 (s, 2H,
CH2), 6.46–6.49, 6.61–65, 7.62–7.69 (m, 7H, aromatics); 13C
NMR (CD3OD): d ¼ 46.6, 115.0, 115.2, 126.8, 128.5, 128.6,
132.1, 140.3, 144.7, 145.1. MS (ESI) m/z (%): 356 [M-H]ꢁ (1),
236 (100), HR-MS [(C13H12BrNO4S-H)]ꢁ, m/z 355.9538 (calc.
355.9598).
N-(2,3-dihydroxyphenyl)-N-methyl-4-phenoxyphenylsulfonamide
(11)
68% yield; mp: 150–152 ꢀC; 1H NMR (CDCl3): d ¼ 3.16 (m, 3H,
NCH3), 4.99 (bb, 2H, OH), 5.99–6.02, 6.65–7.54 (m, 12H,
aromatics); MS (ESI) m/z (%): 370 [M-H]ꢁ (100); m/z 340
[M + Na]+ (100), 233 (81). 13C NMR (CD3OD): d ¼ 37.5, 114.7,
116.9, 118.3, 119.4, 119.9, 124.6, 128.0, 129.9, 130.0, 131.8,
143.3, 146.1, 155.3, 161.7. HR-MS [(C19H17NO5S-H)]ꢁ, m/z
370.0749 (calc. 370.0755).
DPPH assay
The DPPH radical scavenging assay was performed in 96-well
microplates according to the method reported by Blois with some
modifications39,40. Briefly, a freshly prepared solution of DPPH in
methanol (100 mM final concentration) was added to test
compounds methanolic solution. The mixtures were shaken
vigorously and left to stand in the dark for 30 min at room
temperature, and then absorbance was read at 520 nm using a
spectrophotometric plate reader (Victor 3 Perkin–Elmer). The
antioxidant activity was determined as the RSA% (radical
scavenging activity), calculated using following equation:
General procedure for the preparation of compounds 12–17
To a solution of the appropriate sulfonamide 34–39 (1.36 mmol)
in dry CH2Cl2 (20 mL) was added a 1M solution of boron
tribromide (3.26 mmol) in CH2Cl2, at 0 ꢀC and under nitrogen
atmosphere. After 1 h at room temperature, methanol (5 mL) was
added dropwise carefully at 0 ꢀC, and the mixture was stirred for
additional 10 min. The crude was diluted with a 1:1 mixture of
AcOEt/H2O, the organic phase was washed with brine, dried, and
purified by flash chromatography on silica gel affording the
desired product.
RSA% ¼ 100 ꢂ ½ðAo ꢁ AiÞ=Aoꢃ
where, Ao and Ai are the DPPH absorbance in the absence or in
presence of antioxidant, respectively. Different sample concen-
trations were used in order to obtain antiradical curves for
calculating the EC50 values. The value of EC50 was expressed in
terms of molar ratio of antioxidant to DPPH. ARP is inverse of
EC50 value, the larger the ARP the more efficient the antioxidant.
The EC50 values and statistical analyses were processed using
GraphPad Prism41 and are expressed as mean SEM of at least
three independent measurements in triplicate.
N-(2,3-dihydroxyphenyl)methyl-4-diphenylsulfonamide (12)
55% yield; mp: 154–155 ꢀC; 1H NMR (CD3OD): d ¼ 4.12 (s, 2H,
CH2), 6.47–6.52, 6.58–6.63, 7.32–7.46, 7.57–7.66, 7.81–7.87 (m,
12H, aromatics); 13C NMR (CD3OD): d 42.2, 114.2, 119.1, 120.1,
123.6, 127.1, 127.2, 127.4, 128.2, 128.9, 139.3, 139.6, 143.3,
144.8, 145.2. MS (ESI) m/z (%): 354 [M-H]ꢁ (7), 234 (100),
HR-MS [(C19H17NO4S-H)]ꢁ, m/z 354.0811 (calc. 354.0806).
MMP inhibition assays
N-(2,3-dihydroxyphenyl)methyl-4-bromophenylsulfonamide (13)
75% yield; mp: 179–180 ꢀC; 1H NMR (CD3OD): d ¼ 4.10 (s, 2H,
CH2), 6.47–6.57, 6.61–6.64, 7.54–7.65 (m, 7H, aromatics); 13C
NMR (CD3OD): d ¼ 42.2, 114.3, 119.1, 120.2, 123.3, 126.7,
128.6, 131.9, 141.2, 143.3, 144.7. MS (ESI) m/z (%): 356 [M-H]ꢁ
(1), 236 (100), HR-MS [(C13H12BrNO4S-H)]ꢁ, m/z 355.9591
(calc. 355.9598).
The catalytic domains of MMP-2, -8, and -9 were purchased from
Enzo Life Sciences. The assays were performed in triplicate in 96-
well white microtiter plates (Corning, NBS). For assay measure-
ments, inhibitor stock solutions (DMSO, 25 mM) were diluted to
six different concentrations (1 nM–250 mM) in fluorometric assay
buffer (50 mM TrisꢄHCl pH 7.5, 200 mM NaCl, 1 mM CaCl2,

DOI: 10.1080/14756366.2016.1217853
Catechol-based MMP inhibitors 11
1 mM ZnCl2, 0.05% Brij-35, and 1% DMSO). Enzyme and Structure solution and refinement
inhibitor solutions were incubated in the assay buffer for 15 min
Structure solution was performed with AMoRe43 using the
at room temperature before the addition of the fluorogenic
substrate solution (OmniMMP ¼ Mca-Pro-Leu-Gly-Leu-Dpa-Ala-
Arg-NH2, Calbiochem, 2.5 mM final concentration). After further
incubation for 2–4 h at 37 ꢀC, fluorescence was measured
(ꢀex ¼ 340 nm, ꢀem ¼ 405 nm) using a Perkin–Elmer Victor V3
plate reader.
coordinates of the complex between MMP-8 and a non-zinc
chelating inhibitor (PDB entry 3DPE)44 as the starting model.
The coordinates were then refined with CNS45. The statistics
of refinement is summarized in Table 1 of the supporting
Information.
Control wells lacked inhibitor. The MMP inhibition activity
was expressed in relative fluorescence units (RFU). Percent
inhibition was calculated from control reactions without inhibitor,
IC50 values were determined using GraphPad Prism41 and are
expressed as mean SEM of at least three independent measure-
ments in triplicate.
Computational studies
Ligand-based studies: ligands reported in Tables 1 and 2 were
manually built using the Built facility in Maestro37. 3D structures,
stereoisomers, tautomers, and protomers at pH 7.0 0.5 were
generated with LigPrep37. All synthesized analogs were aligned to
the crystallographic coordinates of 14 with Phase Shape
Screening37 and, using the resulting aligned conformation, a
pharmacophore-based QSAR model was built with Phase pro-
gram. The minimum intersite distance between the features was
Expression and purification of the protein
The truncated form M80-G242 of the catalytic domain of MMP-8
was expressed in E. coli strain BL21 (DE3). At an OD600 0.5–0.6
expression of the collagenase was induced in a 1 l culture by
adding IPTG to a final concentration of 0.5 mM. Inclusion bodies
isolated and purified from harvested E. coli cells were resus-
pended in 20 mL of 6M urea, 100 mM b-mercaptoethanol and
20 mMTris, pH 8.5, and incubated at room temperature o/n under
shaking to extract the solubilized collagenase. This extract was
centrifuged for 300 at 40 000 rpm, and the supernatant was loaded
onto a Mono Q-Sepharose column (GE Healthcare) previously
equilibrated with the denaturating buffer.
˚
set to be 1 A. Molecules with pIC5054 toward MMP-8 were
grouped as the inactive molecule set, while those with pIC50 45
were grouped as the active molecules; all actives were set to be
matched during the screening. The QSAR model was constructed
using molecules 1, 12, 16, 17, 18, and 21 as test set and all the
others as training set. The best model has 15 as reference
molecule, and is made of four features: 1 aromatic ring (R9), 2
acceptor groups (A1 and A3), and 1 donor group (D6). Excluded
volume features were added around the shape of reference
molecule.
Elution of the collagenase was carried out by applying a
linear gradient of 0–1 M NaCl in the same buffer at a flow rate
of 1 mL/min. The truncated form of MMP-8 was eluted at a salt
concentration of 100 mM NaCl and could be purified to apparent
homogeneity. A further step of purification was carried out by gel
filtration using a HiLoadSuperdex 75 column (GE Healthcare)
equilibrated with 6M urea, 10 mM DTT, and 20 mM Tris, pH 8.5
at a flow rate of 0.5 mL/min. The collected protein was then
refolded onto a HiLoadSuperdex 75 column in buffer MES-NaOH
3 mM, pH 6.0, 100 mM NaCl, 5 mM CaCl2, 0.5 mM ZnCl2, NaN3
0.02% at a flow rate of 0.5 mL/min. The fraction containing the
desalted and refolded protein was eluted after ca. 13 mL.
Structure-based studies: the hydrogen atoms of the X-ray
complex–MMP-8:14 has been added and optimized with the
¨
complex with explicit waters was minimized to a derivative
37
Protein Preparation Wizard tool from Schrodinger . Then the
˚
convergence of 0.05 kJ/mol A using the Polak–Ribiere Conjugate
Gradient (PRCG) minimization algorithm, the OPLS2005 force
37
field, with MacroModel Embrace Minimization . A shell of 15 A
˚
around the catalytic zinc was set to be free to move, another shell
˚
˚
of 5 A minimized applying a force constant of 200 kJ/mol A2.
The interaction energy between the receptor and each ligand
was calculated with the Interaction energy mode implemented in
Embrace. The same procedure was applied to calculate the
interaction energies of MMP-2:14 and MMP-9:14. Ligand
coordinates were taken from the MMP-8 bound conformation of
14. Protein and water coordinates were downloaded from the
Protein Data Bank (PDB): 1GKC.pdb46 and 1QIB.pdb47 for
MMP-9 and MMP-2, respectively. Then, interaction energies were
calculated for all compounds reported in Tables 1 and 2
previously aligned to 14. Calculated energies were used to
derive ROC curves (see Figure 7). For the ROC curve calculations
molecules with pIC5044 were considered active and all the others
inactive.
Protein crystallization
The inhibitor (stock solution 50 mM in DMSO) was immediately
added to the fraction containing the refolded protein in the ratio
3:1 (final concentration of DMSO 1%) in order to prevent
autoproteolysis during concentration. The MMP-8 protein with
the inhibitor was then concentrated with Amicon-Ultra 15, to a
final concentration of 6 mg/mL. Crystallization was performed by
hanging-drop vapor diffusion method at 20 ꢀC. Hanging droplets
were made by mixing 2 mL of protein/inhibitor solution with 5 mL
of PEG solution (10% (m/v) PEG6000, 0.2 M MES-NaOH, 0.02%
NaN3, pH 6.0). Droplets were concentrated against a reservoir
buffer containing 1.0–2.0 M sodium phosphate, 0.02% NaN3, pH
6.0. Crystals appeared after few days.
Declaration of interest
The authors report no conflict of interest.
ORCID
Data collection and processing
Marilena Tauro
http://orcid.org/0000-0003-2361-7364
http://orcid.org/0000-0001-6221-6155
http://orcid.org/0000-0002-7980-5818
http://orcid.org/0000-0003-0072-8643
http://orcid.org/0000-0002-6716-3854
http://orcid.org/0000-0002-7533-5425
http://orcid.org/0000-0002-3980-3180
http://orcid.org/0000-0002-8520-5165
X-ray data were collected under cryogenic conditions (100 K) at
the ID29 beamline of ESRF, Grenoble, using a wavelength of
˚
0.976 A and a Pilatus 6M_F detector. The crystals were flash-
frozen in the nitrogen stream after transferring them for few
seconds into the mother solution containing 35% PEG400. Data
were integrated and scaled using the programs MOSFLM and
Scala42. The statistics of collection is given in Table 1 of the
supporting Information.
Antonio Laghezza
Luca Piemontese
Alessia Caradonna
Davide Capelli
Roberta Montanari
Giorgio Pochetti
Antonella Di Pizio

12 M. Tauro et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
binding in a complex with MMP-8. Bioorg Med Chem Lett 2006;16:
20–4.
23. Yuan H, Lu W, Wang L, et al. Synthesis of derivatives of
methyl rosmarinate and their inhibitory activities against matrix
References
1. Khokha R, Murthy A, Weiss A. Metalloproteinases and their natural
inhibitors in inflammation and immunity. Nat Rev Immunol 2013;
13:649–65.
2. Vandenbroucke RE, Libert C. Is there new hope for therapeutic
matrix metalloproteinase inhibition? Nat Rev Drug Discov 2014;13:
904–27.
3. Tauro M, Laghezza A, Loiodice F, et al. Arylamino methylene
bisphosphonate derivatives as bone seeking matrix metalloprotei-
nase inhibitors. Bioorg Med Chem 2013;21:6456–65.
4. Rubino MT, Agamennone M, Campestre C, et al. Biphenyl
sulfonylamino methyl bisphosphonic acids as inhibitors of matrix
metalloproteinases and bone resorption. ChemMedChem 2011;6:
1258–68.
5. Dufour A, Overall CM. Missing the target: matrix metalloproteinase
antitargets in inflammation and cancer. Trends Pharmacol Sci 2013;
34:233–42.
6. Tauro M, McGuire J, Lynch CC. New approaches to selectively
target cancer-associated matrix metalloproteinase activity. Cancer
Metastasis Rev 2014;33:1043–57.
7. Gupta A, Kaur CD, Jangdey M, Saraf S. Matrix metallopro-
teinase enzymes and their naturally derived inhibitors: novel
targets in photocarcinoma therapy. Ageing Res Rev 2014;13:
65–74.
8. Hwang KA, Yi BR, Choi KC. Molecular mechanisms and in vivo
mouse models of skin aging associated with dermal matrix
alterations. Lab Anim Res 2011;27:1–8.
9. Quan T, Qin Z, Xia W, et al. Matrix-degrading metalloprotei-
nases in photoaging. J Investig Dermatol Symp Proc 2009;14:
20–4.
10. Yaar M, Gilchrest BA. Photoageing: mechanism, prevention and
therapy. Br J Dermatol 2007;157:874–87.
11. Hsieh H-Y, Lee W-C, Senadi GC, et al. Discovery, synthetic
methodology, and biological evaluation for antiphotoaging activity
of bicyclic [1,2,3] triazoles: in vitro and in vivo studies. J Med Chem
2013;56:5422–35.
12. Ceccoli JD, Costello B, Hayward JA, Lahanas KM. Bath & Body
Works Brand Management, Inc., USA, assignee. Topical compos-
itions for inhibiting matrix metalloproteases and providing anti-
oxidative activities patent US20120058140A1; 2012.
13. Khan SB, Kong C-S, Kim J-A, Kim S-K. Protective effect of
Amphiroa dilatata on ROS induced oxidative damage and MMP
expressions in HT1080 cells. Biotechnol Bioprocess Eng 2010;15:
191–8.
metalloproteinase-1 (MMP-1). Eur
148–57.
J Med Chem 2013;62:
24. Mahmood T, Akhtar N. Combined topical application of lotus and
green tea improves facial skin surface parameters. Rejuvenation Res
2013;16:91–7.
25. Kim MS, Oh GH, Kim MJ, Hwang JK. Fucosterol inhibits matrix
metalloproteinase expression and promotes type-1 procollagen
production in UVB-induced HaCaT cells. Photochem Photobiol
2013;89:911–18.
26. Gweon EJ, Kim SJ. Resveratrol attenuates matrix metalloproteinase-
9 and -2-regulated differentiation of HTB94 chondrosarcoma cells
through the p38 kinase and JNK pathways. Oncol Rep 2014;32:
71–8.
27. Kousidou OC, Mitropoulou TN, Roussidis AE, et al. Genistein
suppresses the invasive potential of human breast cancer cells
through transcriptional regulation of metalloproteinases and their
tissue inhibitors. Int J Oncol 2005;26:1101–9.
28. Ricciarelli R, Maroni P, Ozer N, et al. Age-dependent increase
of collagenase expression can be reduced by alpha-tocopherol
via protein kinase C inhibition. Free Radic Biol Med 1999;27:
729–37.
29. Alleva R, Tomasetti M, Sartini D, et al. alpha-Lipoic acid modulates
extracellular matrix and angiogenesis gene expression in non-
healing wounds treated with hyperbaric oxygen therapy. Mol Med
2008;14:175–83.
30. Rubino MT, Maggi D, Laghezza A, et al. Identification of novel
matrix metalloproteinase inhibitors by screening of phenol
fragments library. Arch Pharm (Weinheim, Ger) 2011;344:
557–63.
31. Nicolotti O, Catto M, Giangreco I, et al. Design, synthesis and
biological evaluation of 5-hydroxy, 5-substituted-pyrimidine-2,4,6-
triones as potent inhibitors of gelatinases MMP-2 and MMP-9. Eur J
Med Chem 2012;58:368–76.
32. Rubino MT, Agamennone M, Campestre C, et al. Synthesis, SAR,
and biological evaluation of alpha-sulfonylphosphonic acids as
selective matrix metalloproteinase inhibitors. ChemMedChem 2009;
4:352–62.
33. Biasone A, Tortorella P, Campestre C, et al. alpha-
Biphenylsulfonylamino 2-methylpropyl phosphonates: enantioselec-
tive synthesis and selective inhibition of MMPs. Bioorg Med Chem
2007;15:791–9.
14. Gupta SK. Bioderm Research, Scottsdale, AZ, USA, assignee.
Matrix metalloprotease (MMP) inhibitors and their application
34. Pochetti G, Gavuzzo E, Campestre C, et al. Structural insight into
the stereoselective inhibition of MMP-8 by enantiomeric sulfona-
mide phosphonates. J Med Chem 2006;49:923–31.
in cosmetic
US20060074108A1; 2006.
and
pharmaceutical
composition
patent
35. Mishra KK, Pal RS, Arunkumar R, et al. Antioxidant properties of
different edible mushroom species and increased bioconversion
efficiency of Pleurotus eryngii using locally available casing
materials. Food Chem 2013;138:1557–63.
15. Yoon SO, Park SJ, Yoon SY, et al. Sustained production of H(2)O(2)
activates pro-matrix metalloproteinase-2 through receptor tyrosine
kinases/phosphatidylinositol 3-kinase/NF-kappa B pathway. J Biol
Chem 2002;277:30271–82.
36. Hummer G. Molecular binding: under water’s influence. Nat Chem
2010;2:906–7.
37. LigPrep version 3.4, Maestro version 10.2, MacroModel, ver-
16. Rao BG. Recent developments in the design of specific Matrix
Metalloproteinase inhibitors aided by structural and computational
studies. Curr Pharm Des 2005;11:295–322.
¨
sion 10.8, Phase version 4.3, Schrodinger, LLC, New York, NY;
2015.
17. Skiles JW, Gonnella NC, Jeng AY. The design, structure, and
therapeutic application of matrix metalloproteinase inhibitors. Curr
Med Chem 2001;8:425–74.
18. Campestre C, Agamennone M, Tauro M, Tortorella P. Phosphonate
emerging zinc binding group in matrix metalloproteinase inhibitors.
Curr Drug Targets 2015;16:1634–44.
38. Lauro G, Tortorella P, Bertamino A, et al. Structure-based design of
microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors
using
ChemMedChem 2016;11:612–19.
a
virtual fragment growing optimization scheme.
39. Mishra K, Ojha H, Chaudhury NK. Estimation of antiradical
properties of antioxidants using DPPH[rad] assay: a critical review
and results. Food Chem 2012;130:1036–43.
40. Sharma OP, Bhat TK. DPPH antioxidant assay revisited. Food Chem
2009;113:1202–5.
19. Giustiniano M, Tortorella P, Agamennone M, et al. Amino acid
derivatives as new zinc binding groups for the design of selective
matrix metalloproteinase inhibitors. J Amino Acids 2013;178381,
12 p.
20. Agrawal A, Romero-Perez D, Jacobsen JA, et al. Zinc-binding
groups modulate selective inhibition of MMPs. ChemMedChem
2008;3:812–20.
41. GraphPad Prism-5.0c: GraphPad Software Inc., San Diego, CA;
2009.
42. Battye TG, Kontogiannis L, Johnson O, et al. iMOSFLM: a new
graphical interface for diffraction-image processing with MOSFLM.
Acta Crystallogr D Biol Crystallogr 2011;67:271–81.
43. Navaza J. Amore – an automated package for molecular replace-
ment. Acta Cryst A 1994;50:157–63.
21. Jacobsen FE, Lewis JA, Cohen SM. The design of inhibitors for
medicinally relevant metalloproteins. ChemMedChem 2007;2:
152–71.
22. Campestre C, Agamennone M, Tortorella P, et al. N-Hydroxyurea as
zinc binding group in matrix metalloproteinase inhibition: mode of

DOI: 10.1080/14756366.2016.1217853
Catechol-based MMP inhibitors 13
44. Pochetti G, Montanari R, Gege C, et al. Extra binding region 46. Rowsell S, Hawtin P, Minshull CA, et al. Crystal structure of human
induced by non-zinc chelating inhibitors into the S10 subsite of
matrix metalloproteinase 8 (MMP-8). J Med Chem 2009;52:1040–9.
45. Brunger AT, Adams PD, Clore GM, et al. Crystallography & NMR 47. Dhanaraj V, Williams MG, Ye QZ, et al. X-ray structure of
MMP9 in complex with a reverse hydroxamate inhibitor. J Mol Biol
2002;319:173–81.
system: a new software suite for macromolecular structure deter-
mination. Acta Crystallogr D Biol Crystallogr 1998;54:905–21.
gelatinase A catalytic domain complexed with a hydroxamate
inhibitor. Croat Chem Acta 1999;72:575–91.
Supplementary material available online