Biomolecules 10 00717 v2

biomolecules
Review
Challenges in Matrix Metalloproteinases Inhibition
Helena Laronha 1,2 , Inês Carpinteiro 1 , Jaime Portugal 3 , Ana Azul 1 , Mário Polido 1 ,
Krasimira T. Petrova 2 , Madalena Salema-Oom 1,2 and Jorge Caldeira 1,2, *
1 Centro de Investigação Interdisciplinar Egas Moniz, Instituto Universitário Egas Moniz,
2829-511 Caparica, Portugal; h.laronha@campus.fct.unl.pt (H.L.); icarpinteiro@egasmoniz.edu.pt (I.C.);
aazul@egasmoniz.edu.pt (A.A.); mpolido@egasmoniz.edu.pt (M.P.); moom@egasmoniz.edu.pt (M.S.-O.)
2 UCIBIO and LAQV, Requimte, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa,
2829-516 Caparica, Portugal; k.petrova@fct.unl.pt
3 Faculdade de Medicina Dentária Universidade de Lisboa, 1649-003 Lisboa, Portugal;
jaime.portugal@fmd.ulisboa.pt
* Correspondence: jcaldeira@egasmoniz.edu.pt; Tel.: +351-919553592

Received: 9 April 2020; Accepted: 30 April 2020; Published: 5 May 2020
Abstract: Matrix metalloproteinases are enzymes that degrade the extracellular matrix. They have
different substrates but similar structural organization. Matrix metalloproteinases are involved in
many physiological and pathological processes and there is a need to develop inhibitors for these
enzymes in order to modulate the degradation of the extracellular matrix (ECM). There exist two
classes of inhibitors: endogenous and synthetics. The development of synthetic inhibitors remains a
great challenge due to the low selectivity and specificity, side effects in clinical trials, and instability.
An extensive review of currently reported synthetic inhibitors and description of their properties
is presented.
Keywords: matrix metalloproteinases; TIMP; synthetic inhibitors
1. Introduction
Matrix metalloproteinases (MMPs) are a protein family within the metzincin superfamily,
comprising zinc-dependent endopeptidases with similar structural characteristics but with different
substrate preferences. MMPs are produced and secreted from cells as inactive proenzymes depending,
herein, on a structural alteration for activation [1–6]. In human tissues, there are 23 different types
of MMPs expressed and they can be subdivided according to their substrate specificity, sequential
similarity, and domain organization [1,2,4,7–17] (Table 1).
The most common structural features shared by MMPs are [1,2,4,5,7,8,10–14,16,18] (Figure 1) a
pro-domain, a catalytic domain, a hemopexin-like domain, and a transmembrane domain for membrane
type MMPs (MT-MMPs) although some MMPS do not have all the structural features represented in the
figure. The pro-domain keeps MMP inactive by a cysteine switch, which interacts with the catalytic zinc
making it impossible to connect the substrate. The catalytic domain has two zinc ions, three calcium
ions, and three histidine residues, which are highly conserved [1–9,11–20]. In the terminal zone of the
catalytic domain there is a region that forms the outer wall of the S1 ’ pocket [1,14,17]. This pocket is the
most variable region in MMPs and it is a determining factor for substrate specificity [1,2,6,7,11,17,18].
However, there are six pockets (P1 , P2 , P3 , P1 ’, P2 ’, and P3 ’) and the fragments of the substrates or
inhibitors are named depending on the interaction with these pockets (R1 , R2 , R3 , R1 ’ or Ra , R2 ’,
and R3 ’). The linker is proline-rich, of variable length, allowing inter-domain flexibility and enzyme
stability [4,8,12,13]. The hemopexin-like domain is necessary for collagen triple helix degradation and
is important for substrate specificity [3,4,7,9,19].
Biomolecules 2020, 10, 717; doi:10.3390/biom10050717 www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, 717 2 of 61
Table 1. Matrix metalloproteinases (MMPs) classes.
Class MMP
MMP-1, Collagenase-1, Interstitial or Fibroblast collagenases
Collagenases MMP-8, Collagenase-2, or Neutrophil collagenases
MMP-13 or Collagenase 3
MMP-2 or Gelatinase A
Gelatinases
MMP-9 or Gelatinase B
MMP-3 or Stromelysin-1
Stromelysin MMP-10 or Stromelysin-2
MMP-11
MMP-7
Matrilysin
MMP-26, Matrilysin-2, or Endometase
MMP-14 or MT1-MMP
MMP-15 or MT2-MMP
Type I transmembrane protein
MMP-16 or MT3-MMP
Membrane-type
MMP-24 or MT5-MMP
MMP17 or MT4-MMP
Glycosylphosphatidylinositol (GPI)-anchored
MMP-25 or MT6-MMP
MMP-12
MMP-19
MMP-20
Other MMPs MMP-21
MMP-23
MMP-27
MMP-28
Figure1.1. Schematic
Figure Schematic representation
representationof
ofthe
thegeneral
generalstructure
structureof
ofMMP.
MMP.
The
TheMMPs
MMPs cancan
process ECMECM
process proteins and glycoproteins,
proteins membrane
and glycoproteins, receptors, receptors,
membrane cytokines, hormones,
cytokines,
chemokines, adhesion molecules, and growth factors [1,3,4,6,7,9–11,13,14,20–26].
hormones, chemokines, adhesion molecules, and growth factors [1,3,4,6,7,9–11,13,14,20–26]. However, the
presence and the activity of MMPs have been demonstrated to be intracellular [25,26].
However, the presence and the activity of MMPs have been demonstrated to be intracellular [25,26]. For example,
some studies show
For example, someintracellular
studies show localization of MMP-2
intracellular in cardiac
localization myocytes
of MMP-2 and colocalization
in cardiac myocytes and of
MMP-2 with troponin I in cardiac myofilaments [23]. The MMP-2 activity has also
colocalization of MMP-2 with troponin I in cardiac myofilaments [23]. The MMP-2 activity has also been detected
in nuclear
been extracts
detected from human
in nuclear heart
extracts fromand rat liver
human heart[23].
andTheratMMPs are involved
liver [23]. The MMPs in are
many biologic
involved in
processes, such as tissue repair and remodulation, cellular differentiation, embryogenesis,
many biologic processes, such as tissue repair and remodulation, cellular differentiation, angiogenesis,
cell mobility, morphogenesis,
embryogenesis, wound
angiogenesis, cell healing,
mobility, inflammatory
morphogenesis, response,
wound apoptosis,
healing, ovulation,
inflammatory and
response,
endometrial proliferation
apoptosis, ovulation, and[1,2,4,6,8,10,11,13,16–18,20,27]. The deregulation of MMPs activity
endometrial proliferation [1,2,4,6,8,10,11,13,16–18,20,27]. leads to the
The deregulation
progression of various pathologies depending on which enzyme is involved [1,6,10,13–17,20,27]:
of MMPs activity leads to the progression of various pathologies depending on which enzyme canceris
and metastasis, inflammatory processes, arthritis, ulcers, periodontal diseases, brain
involved [1,6,10,13–17,20,27]: cancer and metastasis, inflammatory processes, arthritis, ulcers, degenerative
periodontal diseases, brain degenerative diseases, liver cirrhosis, fibrotic lung diseases, otosclerosis,
atherosclerosis, multiple sclerosis, dilated cardiomyopathy, aortic aneurysm, or varicose veins.
Although therapeutic strategies for specific inhibition of MMPs have been long researched, they
are difficult to develop because these enzymes are involved in a myriad of pathways [2,5]. However,
this inhibition can be done at the biomolecular expression and active enzyme terms [2,5,18]. The
diseases, liver cirrhosis, fibrotic lung diseases, otosclerosis, atherosclerosis, multiple sclerosis, dilated
cardiomyopathy, aortic aneurysm, or varicose veins.
Although therapeutic strategies for specific inhibition of MMPs have been long researched,
they are difficult to develop because these enzymes are involved in a myriad of pathways [2,5].
However, this inhibition can be done at the biomolecular expression and active enzyme terms [2,5,18].
The MMPs inhibitors can be divided into endogenous inhibitors, which can be specific or non-specific,
and synthetic inhibitors [1,2,4,7,10,12–14,16,20,28,29] (Table 2).
Table 2. MMPs inhibitors classification.
Specific Inhibitor Tissue Inhibitor of Metalloproteinases (TIMP)

α2-macroglobulin
Tissue factor pathway inhibitor (TFPI)
Membrane-bound β-amyloid precursor protein
Endogenous inhibitor Non-specifics inhibitors
C-terminal proteinases enhancer protein
Reversion-inducing cystein-rich protein with
Kasal domain motifs (RECK)
GPI-anchored glycoprotein
Hydroxamate-based inhibitors
Non-hydroxamate-based inhibitors
Synthetic inhibitor Catalytic domain (non-zinc binding) inhibitors
Allosteric and exosite inhibitors
Antibody-based inhibitors
2. Specific Endogenous Inhibitor-Tissue Inhibitors of Metalloproteinases (TIMPs)

Tissue inhibitors of metalloproteinases (TIMPs) are endogenous proteins responsible for the
regulation of MMPs activity, but also of families such as the disintegrin metalloproteinases (ADAM
and with thrombospondin motifs ADAMTS) and therefore for maintaining the physiological balance
between ECM degradation and MMPs activity [1,2,8,9,18,30]. There are four TIMPs (TIMP-1, -2, -3,
and -4) (Table 3), with 22–29 KDa and 41%–52% sequential similarity [2,4,12,13,16,20,31].
Table 3. Tissue inhibitors of metalloproteinases (TIMPs) classification.
TIMP Expression Inhibition Inhibition Mode

Strong interaction with MMP-1, -2, -3, and -9 TIMP-1 forms a complex with
Several tissues with transcription
1 Weak interaction with MT1-MMP, MT3-MMP, pro-MMP-9 by binding to the
inducible by cytokines and hormones
MT5-MMP, and MMP-19 hemopexin domain
TIMP-2 has four residues in the
N-terminal domain and an adjacent
2 Constitutive expression Strong interaction with MMP-2 CD-loop region, which allows
interaction between TIMP and the
active center of MMP-2
The inhibition mode is different from
the other TIMPs for its unusual
In response to mitogenic stimulation localization, as it is largely sequestered
3 MMP-1, -2, -3, -9, and -13
and during cell cycle progression into the extracellular matrix or at the
cell surface via heparan sulphate
proteoglycans
Especially abundant in the heart, but is
4 MMP-2 and -14 -
also expressed in injured tissue
TIMPs consist of a N- and C-terminal domain with 125 and 65 amino acids, respectively,
each containing six conserved cysteine residues, which form three conserved disulphide
bonds [2,4,7–9,12,31,32] (Figure 2a). The N-terminal domain is an independent unit, which can be
inhibited by MMPs, in a 1:1 ratio [2,4,8–10,12,13,16,20]. This domain has two groups of four residues:
Cys-Thr-Cys-Val and Glu-Ser-Val-Cys (Figure 2b), which are connected by disulphide bounds which
are important for TIMP activity [7,12]. This is the main domain responsible for MMP inhibition through
TIMPs consist of a N- and C-terminal domain with 125 and 65 amino acids, respectively, each
containing six conserved cysteine residues, which form three conserved disulphide bonds [2,4,7–
9,12,31,32] (Figure 2a). The N-terminal domain is an independent unit, which can be inhibited by
MMPs, in a 1:1 ratio [2,4,8–10,12,13,16,20]. This domain has two groups of four residues: Cys-Thr-
Cys-Val and Glu-Ser-Val-Cys (Figure 2b), which are connected by disulphide bounds which are
important for TIMP activity [7,12]. This is the main domain responsible for MMP inhibition through
its
its binding
binding toto the
the catalytic
catalytic site
site in
inaasubstrate-like
substrate-like manner
manner [31].
[31]. The
The several
several domains
domains allow
allow the
the TIMP
TIMP
and
and pro-gelatinases
pro-gelatinasesinteractions
interactions[4].[4].
Figure 2.2. (a)

Figure (a) TIMP-1-catalytic
TIMP-1-catalytic domain
domain of
of the
the MMP-3
MMP-3 complex.
complex. (b)
(b) TIMP-1-catalytic
TIMP-1-catalytic domain
domain ofof the
the
MMP-3 complex, where two conserved groups, Cys-Thr-Cys-Val and Glu-Ser-Val-Cys,
MMP-3 complex, where two conserved groups, Cys-Thr-Cys-Val and Glu-Ser-Val-Cys, are are represented
in yellow. in yellow.
represented
3. Non-Specific Endogenous Inhibitors

Non-specific endogenous inhibitors have been reported to inhibit MMPs (Table 4), however, the
inhibition mechanism details have only been partially discovered [7,12].
Table 4. Non-specific endogenous inhibitors [4,7,12,13,33,34].
Non-Specific Inhibitor Inhibition

α2-macroglobulin MMP-2 and -9
Tissue factor pathway inhibitor MMP-1 and -2
Membrane-bound β-amyloid precursor protein MMP-2
C-terminal proteinase enhancer protein MMP-2
Reversion-inducing-cysteine-rich protein with Kasal motifs (RECK) MMP-2, -9, and -14
GPI-anchored glycoprotein -
Human α2-macroglobulin is a glycoprotein with four identical subunits that act by entrapping
MMP and the complex is cleared by endocytosis [2]. The α2-macroglobulin has been found in blood
and tissue fluid [2,31]. The tissue factor pathway inhibitor (TFPI) is a serine proteinase inhibitor,
which targets MMP-1 and -2, but this inhibition mode is still unknown [7,12]. The C-terminal proteinase
enhancer protein and tissue factor pathway inhibitor have sequences with certain similarities to the
N-terminal domain of TIMPs [31].
4. Synthetic Inhibitors
MMPs are molecular targets for the development of therapeutic and diagnostic agents [14].
The development of synthetic MMP inhibitors was initially based on the peptide sequence, recognized by
proteases, with different chemical functionalities, capable of interacting potently with zinc ion [11–13,19].
The requirements for an effective inhibitor are [2,11,13,17,19,35]:
proteinase enhancer protein and tissue factor pathway inhibitor have sequences with certain
similarities to the N-terminal domain of TIMPs [31].
4. Synthetic Inhibitors
Biomolecules MMPs
2020, 10,are
717molecular targets for the development of therapeutic and diagnostic agents [14]. The5 of 61
development of synthetic MMP inhibitors was initially based on the peptide sequence, recognized by
proteases, with different chemical functionalities, capable of interacting potently with zinc ion [11–
- A functional
13,19]. group ablefor
The requirements to an
chelate theinhibitor
effective zinc ionare
(II)-zinc binding group (ZBG). The first generation
[2,11,13,17,19,35]:
inhibitors used hydroxamate (CONHO− ) but the second generation use carboxylate (COO− ),
- A functional group able to chelate the zinc ion (II)-zinc binding group (ZBG). The first generation
thiolates (S− ), phosphonyls (PO2 − ), for example (Figure 3);
inhibitors used hydroxamate (CONHO−) but the second generation use carboxylate (COO−),
- At least one functional group that promotes hydrogen
thiolates (S−), phosphonyls (PO2−), for example (Figure 3); bonding with the protein backbone;
- One
- or least
At moreone side chains undergoing
functional Van derhydrogen
group that promotes Waals interactions with
bonding with theenzyme subsites.
protein backbone;
- One or more side chains undergoing Van der Waals interactions with enzyme subsites.
Figure 3. Examples of zinc binding groups (ZBGs). (a) Hydroxamate-based inhibitor; (b) thiolate-based
Figure 3. Examples of zinc binding groups (ZBGs). (a) Hydroxamate-based inhibitor; (b) thiolate-
inhibitor; (c) carboxylate-based inhibitor; (d) phosphorous-based inhibitor. The R group is the scaffold
based inhibitor; (c) carboxylate-based inhibitor; (d) phosphorous-based inhibitor. The R group is the
of inhibitor.
scaffold of inhibitor.
ZBGsZBGshavehavenegative
negative charges
chargesthat
thatprevent
preventtheir
their penetration
penetration ininthethe cell,
cell, restricting
restricting theirtheir activity
activity to to
the extracellular
the extracellularspace which
space which reduces
reducestheir
theircell
cell toxicity [2].Changes
toxicity [2]. Changes in in
thethe
ZBG ZBG structure
structure or inorthein the
pointpoint
of attachment
of attachment of the
of theZBGZBG totothe
thebackbone
backbone of of the MMPinhibitor
the MMP inhibitor cancan change
change its potency
its potency and and
selectivity
selectivity [2].
[2].2020,
Biomolecules When When comparing what selectivity or potency of different ZBGs leaving
comparing what selectivity or potency of different ZBGs leaving the6structure
10, 717 the structure
of 64
constant, Castelhano et al. arrived at the following list [36]: hydroxamic acid >> formylhydroxylamine
constant,>Castelhano
> sulfhydryl et al. arrived>atcarboxylate.
aminocarboxylates the following list [36]: hydroxamic acid >> formylhydroxylamine
> sulfhydryl > aminocarboxylates > carboxylate.
The selectivity of inhibitor is a primordial goal of MMPs’ inhibitors (MMPis) design to increase
The selectivity of inhibitor is a primordial goal of MMPs’ inhibitors (MMPis) design to increase
efficacy and prevent side effects [2]. This selectivity is based on two molecular characteristics [11]:
efficacy and prevent side effects [2]. This selectivity is based on two molecular characteristics [11]: a
a chelating
chelatingcapacity
capacity forforcatalytic
catalyticzinc
zinc and the presence
and the presenceofofhydrophobic
hydrophobic bridges
bridges of the
of the active
active center
center for for
the S1the
’ pocket.
S1’ pocket. Numerous strategies have been suggested for creating selective MMP inhibitors [27] [27]
Numerous strategies have been suggested for creating selective MMP inhibitors
(Figure
(Figureendogenous-like
4): 4): endogenous-like inhibitors,
inhibitors,exosite
exosite targeting inhibitors,
targeting inhibitors, a combination
a combination of exosite
of exosite binding
binding
and metal chelating
and metal inhibitors,
chelating inhibitors, and
and function-blocking antibodies.
function-blocking antibodies.
Figure 4. Types
Figure of MMPs’
4. Types of MMPs’inhibitors:
inhibitors:(a)
(a)endogenous-like inhibitors
endogenous-like inhibitors that
that chelate
chelate the the catalytic
catalytic zinc (II)
zinc (II)
ion; (b)
ion;exosite targeting
(b) exosite inhibitors
targeting inhibitorsthat
thatalter
alter the
the conformation
conformation ofofthethe enzyme;
enzyme; (c) (c) a combination
a combination of of
exosite
exosite binding
binding andand metal
metal chelating
chelating inhibitors;(d)
inhibitors; (d) antibodies
antibodies inhibitors.
inhibitors.
The synthetic inhibitors have had a great challenge in their development since first it is necessary
to identify the enzymes that are involved in disease progression. Moreover, this goal has an
additional difficulty as there are more than 50 human metalloproteinases (23 MMPs, 13 ADAM, and
19 ADAMTS) [2].
The synthetic inhibitors have had a great challenge in their development since first it is necessary
to identify the enzymes that are involved in disease progression. Moreover, this goal has an
additional difficulty as there are more than 50 human metalloproteinases (23 MMPs, 13 ADAM,
and 19 ADAMTS) [2].
4.1. Hydroxamate-Based Inhibitors

The first generation of MMPis (1995–1999 [16]) were designed based on the knowledge of
the triple helix collagen amino acid sequence (cleavage site) and the information derived from
specific substrates [6,15,19,31,35,37]. These compounds contain a hydroxamic acid group as
ZBG [5,6,15,18,27,28,31,37]. Hydroxamic acids (HA) were first described in 1986 [38,39]. They are
easy to synthesize, are monoanionic compounds, bidentate chelating agents, and due the excellent
zinc-chelating capability they are the more popular ZBG for MMPs [2,6,17,18,27–29]. The hydroxamic
group established interactions with zinc ions, through two oxygens and two hydrogen bonds
(NH and OH groups of HA with Ala and Glu, respectively), forming a distorted triangular
bipyramid [2,5,18,19,28,29] (Figure 5a). Reich et al., in 1988 [40], used a hydroxamic acid compound,
SC-44463 (Figure 5b), to block collagenase and prevent metastasis in a mouse model, which initiated
Biomolecules 2020,
Biomolecules 2020, 10,
10, 717
717 77 of
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64
the era of MMP inhibition therapeutics [40].
Figure 5. (a) Interaction between hydroxamate group and catalytic zinc (II) ion. The oxygen of
Figure 5.
Figure 5. (a)
(a) Interaction
Interaction between
between hydroxamate
hydroxamate group
group and
and catalytic
catalytic zinc
zinc (II)
(II) ion.
ion. The
The oxygen
oxygen of of the
the
the hydroxamate forms a strong hydrogen bond with the carboxylate oxygen of the catalytic Glu,
hydroxamate forms
hydroxamate forms aa strong
strong hydrogen
hydrogen bond
bond with
with the
the carboxylate
carboxylate oxygen
oxygen of of the
the catalytic
catalytic Glu,
Glu, while
while
while the NH of hydroxamate establishes another hydrogen bond with the carbonyl oxygen of Ala;
the NH
the NH of
of hydroxamate
hydroxamate establishes
establishes another
another hydrogen
hydrogen bond
bond with
with the
the carbonyl
carbonyl oxygen
oxygen ofof Ala;
Ala; (b)
(b) SC-
SC-
(b SC-44463 inhibitor.
44463 inhibitor.
44463 inhibitor.
The structure–activity relationship (SAR) studies for a series of hydroxamic acids with a quaternary
The structure–activity
The structure–activity relationship
relationship (SAR)(SAR) studies
studies forfor aa series
series ofof hydroxamic
hydroxamic acids
acids with
with aa
carbonyl group at R1 suggested that [13] (Figure 6):
quaternary carbonyl
quaternary carbonyl group
group atat R
R11 suggested
suggested thatthat [13]
[13] (Figure
(Figure 6):
6):
(i) The stoichiometric orientation of the substituent at R1 position is crucial for the activity;
(i) The
(i) The stoichiometric
stoichiometric orientation
orientation of of the
the substituent
substituent atat R1
R1 position
position is
is crucial
crucial for
for the
the activity;
activity;
(ii) The phenylpropyl group was established as the best substituent at position R1;
(ii) The phenylpropyl group was established as the best substituent
(ii) The phenylpropyl group was established as the best substituent at position R1; at position R1;
(iii) Hydrophobic substituents at R2 ’ position and N-metiamides at R3 ’ position were considered as
(iii) Hydrophobic
(iii) Hydrophobic substituents
substituents atat RR22’’ position
position and
and N-metiamides
N-metiamides at at R
R33’’ position
position were
were considered
considered as
as
the most appropriate.
the most
the most appropriate.
appropriate.
Figure
Figure 6.
Figure 6. General structure
6. General
General structure of
structure of hydroxamic
of hydroxamic acid.
hydroxamic acid. OH
acid. OHgroup:
OH group:catalytic
group: catalytic
catalytic zinc
zinc
zinc binding
binding
binding group;
group;
group; RRaa:R
:α aα:
substituent;
αsubstituent; R1:
R1: Psubstituent
1 ’ substituent group and this group is determinant to selectivity
andand activity;
R22::R :
substituent; R1: PP11’’ substituent group
group and
and this
this group
group is determinant
is determinant to selectivity
to selectivity and activity;
activity; R PP222’’
Psubstituent
2 ’ substituent and
and this this substituent
this substituent
substituent can can
can be be cyclized
be cyclized
cyclized with with
with R Ra and
and R
Raa and R
R33;; R 3 ; R 3 : P 3 ’ substituent.
R33:: PP33’’ substituent.
substituent.
substituent and
Modification in Ra position (α substituent): a beneficial effect is conferred by lipophilic substituents

Modification in
Modification in Ra
Ra position
position (α (α substituent):
substituent): aa beneficial
beneficial effect
effect is
is conferred
conferred by by lipophilic
lipophilic
capable of hydrogen bonding [19]. Analogues of Marimastat have been reported, where the α position
substituents capable of hydrogen bonding [19]. Analogues of Marimastat have been
substituents capable of hydrogen bonding [19]. Analogues of Marimastat have been reported, where reported, where
the αα position
the position is
is disubstituted
disubstituted by by aa hydroxyl
hydroxyl group
group and
and aa methyl
methyl group
group [19]
[19] (Figure
(Figure 7a).
7a). The
The X-ray
X-ray
structures of
structures of the
the MMP-3-inhibitor
MMP-3-inhibitor complex
complex showed
showed that
that there
there were
were hydrogen
hydrogen bonds
bonds between
between thethe
hydroxyl group and Ala [19]. By binding the positions Ra and R in a single chain, forming
hydroxyl group and Ala165 [19]. By binding the positions Ra and R2 in a single chain, forming a cyclic
165 2 a cyclic
inhibitor (Figure
inhibitor (Figure 7b),
7b), there
there was
was aa substantial
substantial increase
increase in
in water
water solubility
solubility [19,29,41,42].
[19,29,41,42]. This
This strategy
strategy
Figure 6. General structure of hydroxamic acid. OH group: catalytic zinc binding group; Ra: α
substituent; R1: P1’ substituent group and this group is determinant to selectivity and activity; R2: P2’
substituent and this substituent can be cyclized with Ra and R3; R3: P3’ substituent.
Modification in Ra position (α substituent): a beneficial effect is conferred by lipophilic

substituents capable of hydrogen bonding [19]. Analogues of Marimastat have been reported, where
the α position is disubstituted by a hydroxyl group and a methyl group [19] (Figure 7a). The X-ray
isstructures of the
disubstituted by MMP-3-inhibitor
a hydroxyl groupcomplex showed
and a methyl that
group there
[19] were7a).
(Figure hydrogen
The X-raybonds between
structures the
of the
hydroxyl group and
MMP-3-inhibitor Ala165showed
complex [19]. Bythat
binding
therethe positions
were hydrogenRa bonds
and R2between
in a single
thechain, forming
hydroxyl a cyclic
group and
inhibitor
Ala 165 [19].(Figure 7b), there
By binding was a substantial
the positions Ra and R2 increase inchain,
in a single water forming
solubility [19,29,41,42].
a cyclic inhibitorThis strategy
(Figure 7b),
led to
there thea discovery
was substantialofincrease
two inhibitors
in waterwith similar
solubility potency toThis
[19,29,41,42]. non-cyclized analogues,
strategy led SE205 and
to the discovery of
SC903
two [41] (Figure
inhibitors 7c,d). potency to non-cyclized analogues, SE205 and SC903 [41] (Figure 7c,d).
with similar

Figure 7.
7. (a)
(a) Analogue
Analogue of of Marimastat
Marimastat with
with the
the α
α position
position disubstituted; (b)
(b) analogue
analogue of
of Marimastat
Figure
Figure 7. (a) Analogue of Marimastat with the α position disubstituted;
disubstituted; (b) analogue of Marimastat
Marimastat
with Ra
with Ra and
Ra and
and R R 2 position connected; (c) SE205; (d) SC903.
position connected;
connected; (c)
(c) SE205;
SE205; (d)
(d) SC903.
SC903.
with R22 position
The introduction of conformational restrictions through the addition of a three-membered ring

The introduction of conformational restrictions through the addition of a three-membered ring
between positions α and R11 (Figure
(Figure 8a)
8a) has
has been
been reported
reported by Martin
Martin et al. [43], and it resulted in
between positions α and R1 (Figure 8a) has been reported by Martin et al. [43], and it resulted in
reduced inhibition of MMP-9 [43]. However, the introduction of a six-membered ring between these
reduced inhibition of MMP-9 [43]. However, the introduction of a six-membered ring between these
same positions
positions resulted
resulted in inactivity of the
the compound [19]
[19] (Figure 8b).
8b).
same in the
the inactivity
inactivity of
of the compound
compound [19] (Figure
(Figure 8b).
Figure
Figure 8. Inhibitors with
8. Inhibitors with conformational
conformational restrictions
restrictions between
between Ra
Ra and
and R
R11 positions. (a) Inhibitor
positions. (a) Inhibitor with
with
Figure 8. Inhibitors
three-membered with
ring; (b)conformational restrictions between
inhibitor with six-membered ring. Ra and R1 positions. (a) Inhibitor with
three-membered ring; (b) inhibitor with six-membered ring.
three-membered ring; (b) inhibitor with six-membered ring.
Exploring the depth of S1 ’ pocket, bulky groups in Rα position confer selective inhibition for
Exploring the depth of S1’ pocket, bulky groups in Rα position confer selective inhibition for
Exploring
MMP-2, -8, andthe-9 depth of Sexample
[29]. An 1’ pocket,isbulky groups in
the presence ofRα position group,
a biphenyl confer selective inhibition
which showed for
higher
MMP-2, -8, and -9 [29]. An example is the presence of a biphenyl group, which showed higher
MMP-2,
inhibitory-8,activity
and -9against
[29]. An example
MMP-9 [29].is the presence of a biphenyl group, which showed higher
inhibitory activity against MMP-9 [29].
inhibitory activity against MMP-9 [29].
Modification in R1 position: the incorporation of long groups in the R1 position can promote the
selectivity of MMPis, since pocket S1’ can undergo conformational changes to accommodate certain
selectivity of MMPis, since pocket S1’ can undergo conformational changes to accommodate certain
substituents [19].
substituents [19].
Broadhurst et al. showed that an alkyl chain (C9) at the R1 position reduces the inhibition of
Broadhurst et al. showed that an alkyl chain (C9) at the R1 position reduces the inhibition of
selectivity of MMPis, since pocket S1 ’ can undergo conformational changes to accommodate certain
substituents [19].
Broadhurst et al. showed that an alkyl chain (C9 ) at the R1 position reduces the inhibition
of MMP-1, but maintains the inhibitory activity against MMP-2, -3, and -9 [19] (Figure 9a). For
matlystatin derivates in the C9 chain, R-94138 (Figure 9b) promotes the inhibition against MMP-9 [44].
The succinyl hydroxamates analogues in the C9 chain promote selectivity for MMP-2, however, a C10
chain (Figure 9c) results in MMP-1 inhibition, while a further increase of the chain to C16 (Figure 9d)
leads to a loss
Biomolecules 2020,of
10,activity
717 against MMP-1 [19]. 9 of 64
Figure 9.
Figure 9. Inhibitors
Inhibitors with
with modification
modificationof ofRR1 position.
position.(a)(a)
Inhibitor with
Inhibitor withalkyl
alkylchain. ThisThis
chain. inhibitor has
inhibitor
Figure 9. Inhibitors with modification of R1 1position. (a) Inhibitor with alkyl chain. This inhibitor has
activity against MMP-2, -3, and -9, but the inhibition of MMP-1 is low; (b) R-94138,
has activity against MMP-2, -3, and -9, but the inhibition of MMP-1 is low; (b) R-94138, Matlystatin Matlystatin
activity against MMP-2, -3, and -9, but the inhibition of MMP-1 is low; (b) R-94138, Matlystatin
derivate. The
derivate. The inhibition
inhibitionof
ofMMP-9
MMP-9isis10 10times
timeshigher
higherthan
thananalogues
analogueswith
withCC8or orCC10 chains;
chains; (c)
(c) succinyl
succinyl
8 10
derivate. The inhibition of MMP-9 is 10 times higher than analogues with C 8 or C10 chains; (c) succinyl
hydroxamate analogue
hydroxamate analogue with
with C chain, which
C10 chain, which inhibits
inhibits MMP-1;
MMP-1; (d)
(d) succinyl
succinyl hydroxamate
hydroxamate analogue
analogue
hydroxamate analogue with C10 10 chain, which inhibits MMP-1; (d) succinyl hydroxamate analogue
with C16 chain, which inhibits MMP-1.
Replacement of
Replacement of the
the RR11-R22 bond of succinyl hydroxamates acid inhibitors by by a sulfonamide
sulfonamide bond
bond
Replacement of the R1-R2 bond of succinyl hydroxamates acid inhibitors by a sulfonamide bond
(Figure 10a)
(Figure 10a) results
results in
in substantial
substantial loss
loss of
of inhibitory
inhibitory activity
activity because
because the
the hydrogen
hydrogen bond
bond (C=ONH;
(C=ONH;
(Figure 10a) results in substantial loss of inhibitory activity because the hydrogen bond (C=ONH;
Figure 10b)
Figure 10b) with
with leucine
leucine is is stronger
stronger than
than the
the new
new sulfonyl
sulfonyl oxygen
oxygen bond,
bond, due
due to
to the
the pyramidal
pyramidal nature
nature
Figure 10b) with leucine is stronger than the new sulfonyl oxygen bond, due to the pyramidal nature
of the
of the sulfonamide
sulfonamide [19].
[19].
of the sulfonamide [19].
Figure
Figure 10. (a)Succinyl
10. (a) Succinylhydroxamate
hydroxamateacidacidwith
witha sulphonamide
a sulphonamide bond.
bond. ThisThis compound
compound presents
presents low
Figure
low 10. (a) Succinyl
inhibitory activity hydroxamate
because of acid
the with a sulphonamide
pyramidal nature of the bond. This compound
sulphonamide group. presents
(b) low
Succinyl
inhibitory activity because of the pyramidal nature of the sulphonamide group. (b) Succinyl
inhibitory activity
hydroxamate acid because
with of the
carbonyl bond.pyramidal nature of the sulphonamide group. (b) Succinyl
hydroxamate acid with carbonyl bond.
hydroxamate acid with carbonyl bond.
Modifications in R2 position: modifications in the R2 position led to a modest effect in inhibitory
Modifications in R2 position: modifications in the R2 position led to a modest effect in inhibitory
Modifications
activity, in vitro, andinaffects
R2 position: modificationsproperties
the pharmacokinetic in the R2 position led to a modest
[29]. Marimastat effect in(Figure
and Ro31-9790 inhibitory
11)
activity, in vitro, and affects the pharmacokinetic properties [29]. Marimastat and Ro31-9790 (Figure
activity, in vitro, and affects the pharmacokinetic properties [29]. Marimastat and Ro31-9790 (Figure
11) have a good oral activity because the bulky tert-butyl group assists the adjacent amide bond
11) have a good oral activity because the bulky tert-butyl group assists the adjacent amide bond
during absorption from an aqueous environment to the lipid environment of the cell membrane [45].
during absorption from an aqueous environment to the lipid environment of the cell membrane [45].
The beneficial combination of the tert-butyl group with the α-hydroxyl group increases the water
solubility [19,29]. Babine and Bender suggest that the tert-butyl as R2 group leads to less Van der
solubility [19,29]. Babine and Bender suggest that the tert-butyl as R2 group leads to less Van der
Waals interactions, comparing with other groups [46].
have a good oral activity because the bulky tert-butyl group assists the adjacent amide bond during
absorption from an aqueous environment to the lipid environment of the cell membrane [45].
solubility [19,29]. Babine and Bender suggest that the tert-butyl as R2 group leads to less Van der Waals
interactions, comparing
Biomolecules 2020, 10, 717 with other groups [46]. 10 of 64
Figure 11. (a)

(a) Marimastat.
Marimastat. The
The Ra
Ra position
position is
is substituted
substituted with
withaahydroxyl
hydroxyl group
group (OH).
(OH). (b)
(b) Ro31-9790.
Ro31-9790.
Figure 11. (a) Marimastat. The Ra position is substituted with a hydroxyl group (OH). (b) Ro31-9790.
The Ra position has no substituents.
The Ra position has no substituents.
et al.
Ikeda et al.described
describedcompounds
compoundswith with phenyl
phenyl R2Rsubstituents
2 substituents (Figure
(Figure 12)12) (KB-R7785),
(KB-R7785), which
which are
Ikeda et al. described compounds with phenyl R2 substituents (Figure 12) (KB-R7785), which are
are active
active orally,
orally, duedue
to to
thethe beneficial
beneficial effect
effect ofofthe
theRR phenylgroup
2 2phenyl groupononabsorption,
absorption, where
where the
the amide
active orally, due to the beneficial effect of the R2 phenyl group on absorption, where the amide
shielding and lipophilicity may assist in transepithelial resorption [47]. This inhibitor shows activity
shielding and lipophilicity may assist in transepithelial resorption [47]. This inhibitor shows activity
against MMP-1 in rats and its effectiveness
effectiveness in
in arthritis
arthritis has
has been
been demonstrated
demonstrated [47].
[47].
against MMP-1 in rats and its effectiveness in arthritis has been demonstrated [47].
Figure 12. KB-R7785 inhibitor.
Modifications in R3 position: theFigure S3 ’ pocket is an open

12. KB-R7785 area and several groups can be introduced
inhibitor.
at the R3 position [19]. The introduction Figure 12. benzhydryl
of the KB-R7785 inhibitor.
group leads to compounds with selectivity
to theModifications
MMPs-3 and in -7 R[19].
3 position: the S3’ pocket is an open area and several groups can be introduced
Modifications in R3 position: the S3’ pocket is an open area and several groups can be introduced
at the R3 position [19]. The introduction of the benzhydryl group leads to compounds with selectivity
at theSuccinyl
4.1.1. R3 position [19]. The introduction
Hydroxamic Acid-Based of the benzhydryl group leads to compounds with selectivity
Inhibitors
to the MMPs-3 and -7 [19].
to the MMPs-3 and -7 [19].
Succinyl hydroxamate derivates can be subdivided to peptide derivatives or non-peptide
4.1.1.
compoundsSuccinyl Hydroxamic
[48]. Acid-Based Inhibitors
The N-acetylcysteine has been reported to affect the tumoral invasion process
4.1.1. Succinyl Hydroxamic Acid-Based Inhibitors
and metastasis by MMP-2 and -9
Succinyl hydroxamate derivates can be inhibition [49]. The L-cysteine-2-phenylethylamide
subdivided to peptide derivatives orisnon-peptide
an effective
Succinyl
inhibitor, in hydroxamate
which the phenyl derivates
group can
fills the be
S ’subdivided
pocket of to
MMP-8 peptide
[50]. derivatives
Foley et al. or non-peptide
prepared
1 reported to affect the tumoral invasion process several
compounds [48]. The N-acetylcysteine has been and
compounds
dipeptides [48]. The
derivatives N-acetylcysteine
containing cysteine has been reported
(RCO-Cys-AA-NH to affect
2 ) andthe tumoral
concluded invasion
[51]: process and
metastasis by MMP-2 and -9 inhibition [49]. The L-cysteine-2-phenylethylamide is an effective
metastasis by MMP-2 and -9 inhibition [49]. The L-cysteine-2-phenylethylamide is an effective
inhibitor,
-inhibitor, in which the
The variation phenyl
acyl group fills
and thetheSSsecond
1’ pocket of MMP-8 [50]. Foley et al. prepared several
in which of thethephenyl group
group fills the amino acid (AA) leads to the activity against
1’ pocket of MMP-8 [50]. Foley et al. prepared several
dipeptides
differentderivatives
MMPs. containing cysteine (RCO-Cys-AA-NH2) and concluded [51]:
dipeptides derivatives containing cysteine (RCO-Cys-AA-NH2) and concluded [51]:
-- R group interacts
The variation of the acylwith the Sand
group 1 ’ pocket.
the second amino acid (AA) leads to the activity against
- The variation of the acyl group and the second amino acid (AA) leads to the activity against
different MMPs.
Batimastat
different MMPs.(Table 5) was the first MMPi to enter in clinical trials for cancer as it inhibits MMP-1,
- The R group interacts with the S1’ pocket.
-2,
- -7,TheandR-9, but, interacts
group due to its poor
with theoral bioavailability, it was superseded by Marimastat [28,29,31]
S1’ pocket.
(Table Batimastat
5), which(Table
has an5)alpha-hydroxyl
was the first MMPi group to enter in clinical
increasing trials for cancer
the aqueous as it[29].
solubility inhibits MMP-1,
Marimastat
Batimastat (Table 5) was the first MMPi to enter in clinical trials for cancer as it inhibits MMP-1,
-2, -7, and
inhibits the-9, but, due
activity to its poor
of MMP-1, oral
-2, -3, -7,bioavailability,
-9, -12, and -13 it[31].
wasHowever,
superseded by Marimastat
Marimastat [28,29,31]
failed in clinical
-2, -7, and -9, but, due to its poor oral bioavailability, it was superseded by Marimastat [28,29,31]
(Table
trials due 5), to
which has an alpha-hydroxyl
the absence of a therapeutic groupeffect and increasing the aqueous
the patients solubility [29].
treated developed Marimastat
musculoskeletal
(Table 5), which has an alpha-hydroxyl group increasing the aqueous solubility [29]. Marimastat
inhibits the activity of MMP-1, -2, -3, -7, -9, -12, and -13 [31]. However, Marimastat failed in clinical
inhibits the activity of MMP-1, -2, -3, -7, -9, -12, and -13 [31]. However, Marimastat failed in clinical
trials due to the absence of a therapeutic effect and the patients treated developed musculoskeletal
trials due to the absence of a therapeutic effect and the patients treated developed musculoskeletal
toxicity (MST) [6,31]. Batimastat, marimastat, and ilomastat are examples of succinyl hydroxamates,
which have very analogous structure to that of collagen and inhibit MMPs by bidentate chelation of
the Zn2+ [2,6,29].
2+
the Zn2+ [2,6,29].
Biomolecules
Biomolecules2020,
2020,10,
10,717
717 1111ofof6464
Name MoleculeTable 5. Batimastat and Marimastat.
α Substituent Effect
Name
Name
Name Molecule
Molecule
Molecule ααSubstituent
α Substituent
Substituent Effect
Effect
Not
Batimastat Thienylthiomethylene Not available
Not
Batimastat
Batimastat
Batimastat Thienylthiomethylene
Thienylthiomethylene
Thienylthiomethylene Not available
available
availableorally
orally
orally
orally
Hydroxyl
Hydroxyl
Hydroxyl
Hydroxyl group
group
group
group (directed
(directed
(directed
(directed to the to protein
totothe
thethe protein
protein
protein Available
Available
Available
Marimastat
Marimastat
Marimastat
Marimastat surface,
surface,
surface, allowing
allowing
allowing
surface, thethe
allowing thethe formation
formation
formation
formationof ofof of Available orally
orally
orallyorally
hydrogen
hydrogen
hydrogen
hydrogen bonds
bonds bonds
bonds with
with solvent)
solvent)
with
with solvent)
solvent)
Several
Severalstudies
studiesby
byJonhson
Jonhsonetetal.
al.[52]
[52]demonstrated
demonstratedthat
thatderivatives
derivativesofofsuccinyl
succinylhydroxamic
hydroxamicacid
acid
Several studies
Several13a)
(Figure
by Jonhson
studiesmore et
Jonhsonforet al.
al. [52]
[52] demonstrated
demonstrated that
that derivatives
derivatives of
of succinyl
succinyl hydroxamic
hydroxamic acidacid
(Figure 13a)are
are morepotent
potent forMMP-1
MMP-1than thanthe
thecorresponding
correspondingmalonyl
malonyl(Figure
(Figure13b)
13b)ororglutaryl
glutaryl
(Figure 13a)
(Figure are more potent for MMP-1 than the corresponding malonyl
malonyl (Figure
(Figure 13b)
13b) or glutaryl
glutaryl
(Figure13c)
13c)derivates.
derivates.
(Figure 13c) derivates.
Figure
Figure13.
13.(a)
(a)Derivates
Derivatesofofsuccinyl
succinylhydroxamic
hydroxamicacid;
acid;(b)
(b)malonyl
malonylacid;
acid;(c)
(c)glutaryl
glutarylacid.
acid.
Marcq
Marcq etet al.al. [53]
[53] developed
developed succinyl
succinyl hydroxamates
hydroxamates derivates
derivates selective
selective for
for MMP-2,
MMP-2, by by
Figure
modifications
Figure 13.
modifications 13.
on (a) Derivates
Ilomastat
on(a)Ilomastat of succinyl
structure
Derivatesstructure
of succinyl hydroxamic
(Figure 14a)
hydroxamic
(Figure to
14a) to acid; (b)
increase
acid; malonyl
the
(b) malonyl
increase acid;
overall (c) glutaryl acid. and,
hydrophobicity
acid; (c)hydrophobicity
the overall glutaryl acid. and,
consequently,
consequently, the the selectivity
selectivity [53].
[53]. This
This study
study resulted
resulted inin aa compound
compound (Figure
(Figure 14b)
14b) with
with an
an
Marcq
Marcq et al.
isobutylidene [53]
group
et al.group
isobutylidene developed
of
[53] of E succinyl
geometry,
developed
E geometry, hydroxamates
which
succinyl showed
which showed a derivates
100-fold
hydroxamates selective
greater
a 100-fold derivates for MMP-2,
selectivity for
selective
greater selectivity by
MMP-2
for for modifications
MMP-2 over
MMP-2,
over by
on Ilomastat
MMP-3,
MMP-3,that
modifications structure
that
onisisaIlomastat
a70-fold (Figure
70-fold increase
increase14a) to
compared
compared
structure increase 14a)theto[53].
totoIlomastat
(Figure Ilomastat overall
increasehydrophobicity
[53]. and, consequently,
the overall hydrophobicity and,
the selectivity [53].
consequently, This study [53].
the selectivity resulted in study
This a compound (Figure
resulted in a 14b) with an isobutylidene
compound (Figure 14b) group
with anof
E geometry, which showed a 100-fold greater selectivity for MMP-2 over MMP-3, that
isobutylidene group of E geometry, which showed a 100-fold greater selectivity for MMP-2 over is a 70-fold
increase that
MMP-3, compared to Ilomastat
is a 70-fold increase[53].
compared to Ilomastat [53].
Figure 14. (a) Ilomastat; (b) Ilomastat derivate with isobutylidene group.
Figure 14. (a) Ilomastat; (b) Ilomastat derivate with isobutylidene group.
Table 6 shows the IC50 and Ki values of some succinyl hydroxamic acid-based
Table 6 shows the IC50 and Ki values of some succinyl hydroxamic acid-based inhibitors [6,15–
inhibitors [6,15–19,29,35,37,54,55].
19,29,35,37,54,55].
4.1.2. Sulfonamide Hydroxamic Acid-Based Inhibitors
4.1.2.In
Sulfonamide Hydroxamic
1995, Novartis described Acid-Based Inhibitors
the CGS-27023A (Figure 15a), a non-peptidic MMP-3 inhibitor,
which Inhas good
1995, oral availability
Novartis described thebutCGS-27023A
did not succeed in clinical
(Figure 15a), a trials [56]. TheMMP-3
non-peptidic isopropyl groupwhich
inhibitor, slows
down the metabolization of the adjacent hydroxamic acid group and the 3-pyridyl
has good oral availability but did not succeed in clinical trials [56]. The isopropyl group slows down substituent may
aid partitioning into the hydrated negatively charged environment of the
the metabolization of the adjacent hydroxamic acid group and the 3-pyridyl substituent may aidcartilage [56]. By analysis
of the cocrystal
partitioning intostructure
the hydrated of this inhibitorcharged
negatively and MMP-12, it was of
environment possible to conclude
the cartilage [56]. Bythat the binding
analysis of the
mode between the hydroxamate moiety and the catalytic zinc ion was the same
cocrystal structure of this inhibitor and MMP-12, it was possible to conclude that the binding mode as the binding mode
of hydroxamate-based
between the hydroxamate inhibitors
moiety[6]. The
and theinteraction
catalytic zincof CGS-27023A
ion was thewith sameMMP-3 was possible
as the binding modedueof
to the p-methoxy phenyl substituent occupation of the S1’ pocket and the pyridylmethyl and isobutyl
hydroxamate-based inhibitors [6]. The interaction of CGS-27023A with MMP-3 was possible due to the
groups occupation
p-methoxy of the S2’ and
phenyl substituent S1 pockets,
occupation of therespectively
S1 ’ pocket and[29,56]. The modification
the pyridylmethyl andof α to form
isobutyl the
groups
thioester derivate
occupation of the Sled to an increase of the inhibition of the deep pocket of the MMPs [29,56] (Figure
2 ’ and S1 pockets, respectively [29,56]. The modification of α to form the thioester
15b).
derivate led to an increase of the inhibition of the deep pocket of the MMPs [29,56] (Figure 15b).
Figure 15. (a) CGS-27023A; (b) thioester derivate of CGS-27023A.

Figure 15. (a) CGS-27023A; (b) thioester derivate of CGS-27023A.
The NNGH (Figure 16a) (N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid) was
The NNGH (Figure 16a) (N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid) was
the starting point to many potent MMPis and is accommodated in the entry of the S1 ’ pocket, but does
the starting point to many potent MMPis and is accommodated in the entry of the S1’ pocket, but does
not penetrate it [6]. Barta et al. described a series of arylhydroxamate sulphonamides, active against
not penetrate it [6]. Barta et al. described a series of arylhydroxamate sulphonamides, active against
MMP-2 and -13 (Figure 16b) [57]. In this compound, the sulfonyl group formed a single hydrogen
MMP-2 and -13 (Figure 16b) [57]. In this compound, the sulfonyl group formed a single hydrogen
bond with Leu160 and the piperidine-O-phenyl moiety extends into the S1 ’ pocket by Van der Waals
bond with Leu160 and the piperidine-O-phenyl moiety extends into the S1’ pocket by Van der Waals
interactions [57]. Noe et al. described a series of 3,3-dimethyl-5-hydroxy pipecolic hydroxamic
interactions [57]. Noe et al. described a series of 3,3-dimethyl-5-hydroxy pipecolic hydroxamic acid,
acid, which possess potent inhibitory activity for MMP-13 [58]. In the first series of compounds,
which possess potent inhibitory activity for MMP-13 [58]. In the first series of compounds, the 3-
the 3-position of the piperidine ring was explored by the introduction of a polar functionality and
position of the piperidine ring was explored by the introduction of a polar functionality and it
it resulted in a compound with excellent activity on MMP-13 (Figure 16c), improved bioavailability,
resulted in a compound with excellent activity on MMP-13 (Figure 16c), improved bioavailability,
and lower metabolic clearance [58].
and lower metabolic clearance [58].
Biomolecules
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Biomolecules
Biomolecules 2020,
2020, 10,10,
10,
2020,
2020, 717
10, 717
717
717 1313
12
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of
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64
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Table6.6. IC
Table IC50
50 and
and Ki values
KiKi
values of succinyl
ofof
succinyl hydroxamic
hydroxamic acid-based inhibitors.
acid-based inhibitors.
Table
Table 6.6.
6.
Table
Table ICIC
IC
6. 50and
IC
50 and
Ki
and
and
50
50 KiKi values
values of
values
values ofof succinyl
succinyl
succinyl
succinyl hydroxamic
hydroxamic
hydroxamic
hydroxamic acid-based
acid-based
acid-based
inhibitors.
inhibitors.
inhibitors.
Table 6. IC50 and Ki values of succinyl hydroxamic acid-based inhibitors.
Marimastat (BB-2516)
Marimastat
Marimastat
Marimastat
Marimastat (BB-2516)
(BB-2516)
(BB-2516)
(BB-2516)
IC50: MMP-1 Marimastat= 5 nM;(BB-2516)
MMP-2 = 6 nM;
IC IC
IC : :: MMP-1
MMP-1
MMP-1 =
IC50: MMP-1 = 5 nM; MMP-2
50 50
50 5=
= 5
nM;
5 nM;
nM; MMP-2
MMP-2MMP-2 ==6=6=nM;
6nM;
6 nM;
nM;
IC
MMP-3
IC 50: MMP-1
50 = 200==nM; 55nM;
nM;
nM;MMP-7MMP-2
MMP-2 = 20==206nM;
6nM;nM; Analogue of Marimastat SE205
SE205
MMP-3
MMP-3
MMP-3
MMP-3 = =
200
=
= 200 200
nM;
200nM;nM; MMP-7
MMP-7
MMP-7
MMP-7 = =
20
=
= nM; nM;
20
20 nM; nM;
nM; Analogue
Analogue
Analogue of of Marimastat
Marimastat
of Marimastat SE205
SE205
SE205
SE205
MMP-3
MMP-8
MMP-3 = 200= 2 nM; nM; MMP-7
MMP-9 ==320 nM; MMP-8
MMP- Analogue
Analogue ofofMarimastat
Marimastat
= MMP-3 =
2
MMP-8
=MMP-8
MMP-8
MMP-8
nM; ==2=2==nM;
MMP-9
200 MMP-9
2nM;
2 nM;
nM;
nM;
= 3
MMP-7
MMP-9
MMP-9
MMP-9nM; =3=3=nM;
=MMP-12 = 20MMP-
3nM;
3 nM;
nM;
nM;
MMP-
<MMP-
MMP- 5 nM;
IC50: MMP-1 = 3 nM; MMP-2
ICIC
IC
IC 50:50
IC
50 :50 :: MMP-1
MMP-1
MMP-1
50 MMP-1 ==3=3= 3nM;
33 nM;
=nM; MMP-2
MMP-2
nM; MMP-2
MMP-2
= 1.1 nM;
Analogue
=
=1.1
==1.1= 1.1
of
nM;
1.1
1.1
nM;nM;
nM;
MMP-3 = 14 nM; MMP-7 = 0.8 nM;
Marimastat
MMP-3
MMP-3
MMP-3
MMP-3 =
=14
==14= 14
14
14 nM;
nM;nM;
nM; MMP-7
MMP-7
MMP-7
MMP-7 =
=0.8
==0.8= 0.8
nM;
0.8
0.8
nM;nM;
nM;
Ki:
Ki:
Ki:
Ki:
Ki:
Ki:
MMP-1
MMP-1
MMP-1
MMP-1
MMP-1
MMP-1
= 1.2
1.2
=1.2
nM;
= 1.2
==1.2 SE205
nM;
nM;
nM;
1.2 nM;
nM;
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
= 32.7 nM;
=32.7
= 32.7
==32.732.7 nM;
nM;
nM;
nM;
12 < 5 nM;
MMP-8 = 2 MMP-13
nM; MMP-9 = 0.74 = 3nM;
nM; MMP-
MMP- MMP-8
IC 50: MMP-1 = 0.5 nM;
=nM;3 nM;MMP-9 = 0.9= nM;
MMP-2 1.1 MMP-10
nM; MMP-3= 0.45 nM;
= 14nM;nM;MMP-14
MMP-7 94 nM; MMP-9
MMP-9
Ki: MMP-1MMP-9
=MMP-9 =
1.2 nM; = 1.8
1.8 nM
nM
MMP-3 = 32.7 nM;
1212
12 <<5<5<nM;
12
MMP-13 5nM;
5 nM;
nM;
= MMP-13
MMP-13
MMP-13
MMP-13
0.74 nM; =0.74
= 0.74
==MMP-14
0.740.74nM;
nM;nM;
nM;
= MMP-
MMP-MMP-
MMP-
1.8 nM MMP-8
MMP-8
MMP-8
MMP-8
MMP-8 === = 0.5
=0.5
0.5 nM;
0.5nM; nM; MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 =
=0.9
= 0.9
==0.9 0.9nM;nM;
nM;
nM; MMP-10
MMP-10
MMP-10
MMP-10 =
=0.45
= 0.45
==0.45 0.45 nM;
nM;
nM; MMP-14
MMP-14 ===94
MMP-14
MMP-14
0.8
==
= 94
94
nM;
nM;
94
nM;nM;
nM; MMP-9
MMP-9 =1.8
= 1.8
==1.8 nM
1.8
nMnM
nM
12 < 5 nM; MMP-13 14 = 1.8 =nM 0.74 nM; MMP- MMP-8 = 0.5 nM; MMP-9 MMP-15 ==2020
= =0.9 nM;
nM; MMP-16
MMP-10 ===0.45
35 nMnM; MMP-14 = 94 nM; MMP-9 = 1.8 nM
1414
14 =1.8
==1.8
14 = 1.8
nM
1.8
nM nM
nM MMP-15
MMP-15
MMP-15
MMP-15
MMP-15 =20
=20 nM;
= 20 nM;
nM;nM; MMP-16
MMP-16
MMP-16
MMP-16 =
=35
=35= 35
nM
35nMnM
nM
14 = 1.8 nM MMP-15 = 20 nM; MMP-16 = 35 nM
ICIC : MMP-1= =131

50 :50MMP-1 131nM; MMP-2==9.5
nM;MMP-2 9.5nM; IC50IC : MMP-1 = 4 nM; MMP-2 = 3 nM; MMP-3
IC
ICIC
IC50::50
50 :: MMP-1
MMP-1
MMP-1
50 MMP-1 =131
= 131
==131 131 nM;
nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2 =9.5
= 9.5
==9.5 9.5 : MMP-1 4=nM;
4 nM; MMP-2 3=nM;
3 MMP-3
nM;
nM;MMP-3
IC50 MMP-3
:MMP-3
MMP-1= 8.9
= 8.9nM; MMP-7
nM;nM;
= 131 MMP-7= 3.3
= 3.3
MMP-2 nM nM
= 9.5 ICIC
IC IC50::50
50 50 :: MMP-1
MMP-1
MMP-1
50 MMP-1 ==4=4=nM;
4nM;nM; MMP-2
MMP-2
MMP-2
MMP-2 ==3=3=nM;
3nM;
nM; MMP-3
MMP-3
MMP-3
nM;nM;
nM;
nM; MMP-3MMP-3
MMP-3 =8.9
= 8.9
==8.9 nM;
nM;
8.9 nM;
nM; MMP-7
MMP-7
MMP-7 =3.3
= 3.3
==3.3
MMP-7 nM
3.3nMnM
nM SC903
SC903 =
MMP-330
IC=5030 nM;
: MMP-1 MMP-7
= MMP-7
30 = 4 nM;
nM; = 20
MMP-7 nM;
MMP-2 MMP-8
= MMP-8
20 =nM;3 nM;= 20
MMP-8 nM;
MMP-3=
nM; MMP-3 = 8.9 nM; MMP-7 = 3.3 nM SC903
SC903
SC903
SC903 ==30= 30
30 nM;
nM; nM;
nM; MMP-7
MMP-7
MMP-7 =20
==20= 20
nM;
20
nM;nM;
nM; MMP-8
MMP-8
MMP-8 =20
==20= 20
20 nM;
nM;
nM;
nM;
==2.8 == MMP-9 9 =nM
=nM;
==
Ki:
Ki:MMP-1
MMP-1 2.8nM; MMP-3
nM; SC903
MMP-3 24.1 nM;
24.1 MMP-9
nM; MMP-9= 2.6 nM
2.6 nM = 30 nM; 20 MMP-7
nM; = 20
MMP-9
MMP-9 ==9=9=nMMMP-8
9nM9 nM = 20 nM;
Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1 =2.8
= 2.8
==2.8 2.8 nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3 =24.1
= 24.1
==24.1 nM;
nM;
24.1
nM; MMP-9
MMP-9
nM; = 2.6
==2.6
MMP-9
MMP-9 nM
2.6
2.6 nMnM
nM MMP-9 MMP-9
MMP-9 9 nM
nM
Ki: MMP-1 = 2.8 nM; MMP-3 = 24.1 nM; MMP-9 = 2.6 nM MMP-9 = 9 nM
Biomolecules
Biomolecules2020,
Biomolecules
Biomolecules2020,10,
2020,
2020,10,717
10,
10,717
717
717 14
14ofofof
14
14 64
of6464
64
Table 6. Cont.
SC-44463
SC-44463
SC-44463
SC-44463
SC-44463 Ro-31-9790
Ro-31-9790
Ro-31-9790
Ro-31-9790
BB-16
BB-16
IC
IC
IC
IC :50
505050
50
50 :MMP-1
MMP-1
:MMP-1
::MMP-1
MMP-1 ==
===20
2020
nM;
20
20 nM;
nM;
nM; MMP-2
MMP-2
nM;MMP-2
MMP-2
MMP-2 ===6=6=6nM;
6nM;
nM;
6nM;
nM; BB-16
BB-16
BB-16 IC
ICIC
IC
IC :5050 : MMP-1
:MMP-1
:MMP-1
50: MMP-1
MMP-1 ===10=
=10 10
nM;
10
10nM; nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 8=nM;
===8=88nM; 8 nM;
nM;
nM;MMP-
MMP-
MMP-
MMP-
===5= =10 =40 =60nM; 50
50 50
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ==30
===30 30
30
30nM;nM;
nM;
nM;
nM; MMP-7
MMP-7
MMP-7
MMP-7 ==
===30
MMP-7 3030
30
30nMnMnMnM
nM
IC
IC
IC
IC
IC 50
:5050
5050 50 ::MMP-1
:MMP-1MMP-1
: MMP-1
MMP-1
5nM;
nM;
=55nM;
5nM;
nM;
MMP-2
MMP-2
MMP-2===10
MMP-2
MMP-2 =10
10
nM;
10
nM;
nM;
nM;
nM;MMP-3 MMP-3
MMP-3
MMP-3
MMP-3===40
=40
40
nM;
40
nM;
nM;
nM;
nM;
MMP-7
MMP-7
MMP-7===60
MMP-7
MMP-7 =60
60nM;
60nM;
nM;
nM; MMP-3 =700
333=3==700
=700700
700 nM;
nM;nM;
nM; MMP-14
MMP-14
MMP-14
MMP-14 =
===1.9
=1.9
1.91.9
nMnM
nMnM
MMP-8 = 7 nM nM; MMP-14 1.9 nM
MMP-8
MMP-8
MMP-8
MMP-8 ===7=77nM
7nM
nM
nM
Ro-32-0554
Ro-32-0554
Ro-32-0554
Ro-32-0554
Ro-32-0554 Ro-32-3555
Ro-32-3555
Ro-32-3555
Ro-32-3555
ICIC
IC
IC
IC :50
505050
50 ::MMP-1
MMP-1
:50MMP-1
:MMP-1
MMP-1 ==
===0.50.5
0.5nM;
0.5
0.5nM;nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 =9.1
===9.1
=9.19.1
nM;
9.1nM;nM;
nM;
nM; Ki:
Ki:
Ki:
Ki:Ki: MMP-1
MMP-1
MMP-1
MMP-1
MMP-1 =
===3=33nM;3 nM;
3nM;
nM;
nM;MMP-2
MMP-2
MMP-2MMP-2
MMP-2 ===154=nM;
=154
154
154154
nM;
nM;
nM; nM;
MMP-3
MMP-3
MMP-3
MMP-3
IC
IC
IC :5050:::MMP-1
MMP-1
:MMP-1 =
===1010
=10 nM;
nM;
nM; MMP-2
MMP-2
MMP-2 ==
===400400
400nM;nM;
nM; MMP-3
MMP-3
MMP-3 ==
===4.54.5
4.5μM
μMµM
==
MMP-9 4.3 nM IC
IC 50
5050 50 MMP-1
MMP-1 10
10 nM;
nM; MMP-2
MMP-2 400
400 nM;
nM; MMP-3
MMP-3 4.5
4.5 μM
μM MMP-3 = 527 nM; MMP-8 = 4 nM; MMP-9 =
MMP-9
MMP-9
MMP-9
MMP-9 ===4.3
4.3nM
4.3nM
4.3 nM
nM ===527
=527
527
527nM;
nM;
nM;
nM;MMP-8
MMP-8
MMP-8
MMP-8 ===4=44nM;
4nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9 ===59
=59nM;
59
59nM;
nM;
nM;
59 nM; MMP-13
MMP-13
MMP-13
MMP-13
MMP-13 =
===3=33nM3
3nM
nM
nM nM
Biomolecules
Biomolecules
Biomolecules
Biomolecules 2020,
2020,
2020,
2020,
Biomolecules 10,
10,
2020, 10,
10,
717
717
10, 717
717
717 Table 6. Cont. 151515
15
of
15 of
ofof
64
64
of 64
64
64
ICIC
IC
IC IC
IC
5050
:::MMP-1
MMP-1
MMP-1
:50:MMP-1
:50
MMP-1
50
50 ===
==40
MMP-1 4040
=40
nM
nM
40 nM
nM
nM IC
IC IC
IC IC
IC
50
50 50 :MMP-1
: 50MMP-1
:MMP-1
50
50 ===10
::MMP-1
MMP-1
MMP-1 =10
=10
=1010
μM
µM
10μMμM
μM
μM
IC
ICIC
IC IC
IC
50 :50
50 ::MMP-1
MMP-1
50
50
MMP-1
MMP-1
:50:MMP-1 ==
==29
MMP-1 =29
=2929
29
μM
29μMμM
μM
µM
μM
ICIC
IC IC
IC
50 :50:MMP-1
:50
50 MMP-1
50
50 ==9===
::MMP-1
MMP-1
MMP-1 9=nM9nM
99nMnM
nM
Analogue
Analogue
Analogue
Analogue
Analogue ofofof
of
of Marimastat
Marimastat
Marimastat
Marimastat
Marimastat Ki:
Ki:
Ki:
Ki:
Ki:
Ki: ===2==2=nM;
MMP-1
MMP-1
MMP-1
MMP-1
MMP-1
MMP-1 2nM;
nM;
22nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3==
MMP-3=3==3=nM;
3nM;
nM;
33nM;
nM; MMP-9
MMP-9<<<<<
MMP-9
MMP-9
MMP-9
IC
ICIC ::MMP-1
MMP-1
MMP-1 =1=μM;
1μM;
1 µM;
μM; MMP-2
MMP-2 =15
= 15
15 nM;
nM; MMP-3
MMP-3 == 500
=500
500 nM;
nM; MMP-7
MMP-7 == 10
=10
10 µM;
μM;
IC
ICIC50: MMP-1 = 1 μM; MMP-2 = 15 nM; MMP-3
50 :
50
50
:
50 :
MMP-1
MMP-1
50 = ==
1 1 μM; MMP-2
MMP-2
MMP-2= ==
15 15
nM;nM;
nM; MMP-3
MMP-3
MMP-3==500
=500nM;
500 nM;
nM;
nM; MMP-7
MMP-7
MMP-7==10
MMP-7 =10μM;
10 μM;
μM;
μM; <111nM1nM
11nM
nMnM
nM
MMP-8
MMP-8
MMP-8 =
==3030
=3030nM;
nM;
nM; MMP-9
MMP-9
MMP-9 = 15
=15
==15 15nM
nM
nM
MMP-8
MMP-8==30
MMP-8 nM;
30nM;
nM;MMP-9
MMP-9
MMP-9==15 nM
15nMnM
Ki:
Ki: MMP-1
MMP-1 =1.3
1.3
==1.3 nM;
nM; MMP-2
MMP-2 =1.1
1.1
==1.1 nM;
nM; Ki:Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
Ki:MMP-1==6.5
MMP-1 =6.5
6.5
==6.5
nM;
6.5 nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2
nM; ==20
MMP-2 =20
==2020
nM;
20 nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
nM; ==240
MMP-3 =240
240
==240nM
240nMnM
nM
nM ICIC
IC IC
IC
50 50 ::MMP-1
: 50MMP-1MMP-1
:50:MMP-1==6==6=nM;
MMP-1 6nM;
nM;
66nM;
nM;MMP-2MMP-2
MMP-2
MMP-2
MMP-2 ==30=30
==3030
nM;
30 nM;
nM;
nM;
nM; MMP-
MMP-
MMP-
MMP-
MMP-
Ki:
Ki:MMP-1
Ki:MMP-1==1.3
MMP-1 nM;
1.3nM; MMP-2
nM;MMP-2
MMP-2==1.1 nM;
1.1nM;
nM; 50
MMP-3
MMP-3 == 187
187 nM
nM 33=
33=3=40= 40
40
nM
=4040
nM nM
nM
nM
MMP-3
MMP-3 = 187
MMP-3= =187 nM
187nM
nM
ICIC
IC
Biomolecules 2020, ::717
:5050
5010, MMP-1
= 9==nM
MMP-1
MMP-1 99 nM
nM 15 of 61
Analogue
Analogue
Analogue ofof
of Marimastat
Marimastat
Marimastat Ki:Ki:
Ki: MMP-1
= 2==nM;
MMP-1
MMP-1 22 nM;
nM; MMP-3
MMP-3
MMP-3 = 3==nM;
33 nM;
nM; MMP-9
< <<
MMP-9
MMP-9
ICIC
IC
50:50 :: MMP-1
MMP-1
50 = 1==μM;
MMP-1 11 μM;
μM; MMP-2
MMP-2
MMP-2 =Table
= 15= 15
15 nM;
nM;
nM; MMP-3
MMP-3
MMP-3
6. Cont. == 500
= 500 500 nM;
nM;
nM; MMP-7
MMP-7
MMP-7 == 10
= 10 10 μM;
μM;
μM; 11 nM
1 nM nM
MMP-8
MMP-8
MMP-8 == 30
= 30 30
nM;nM;
nM; MMP-9
MMP-9
MMP-9 == 15
= 15 15
nM nM
nM
Biomolecules
Biomolecules
Biomolecules
Biomolecules
Ki:
Ki:Ki:
2020,
==
2020,
2020,
2020,
MMP-1
MMP-1
MMP-1 =10,
10,
10,
=10,
1.3717
1.3
1.3
717
717
717
nM;
nM;
nM; MMP-2
MMP-2
MMP-2 == 1.1
= 1.1
= 1.11.1 nM;
nM;
nM;nM;
Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1 ==
MMP-1
MMP-1 ==6.5
6.5
6.56.5nM;
nM;nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2 =
= 20 20
== 20
20 nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3 === 240
= 240 240
240
nMnM
nM
nM ICIC
IC
IC
50 :5050
MMP-1
50 = 6===
:: :MMP-1
MMP-1
MMP-1 66 nM;
nM;6nM;
nM; MMP-2
MMP-2
MMP-2MMP-2 == =
= 30 30
30 nM;
nM;
30
nM; nM;
16 16of
MMP-
MMP-
MMP- 16
16
of of64
of
64 64
64
MMP-3
MMP-3
MMP-3
MMP-3 =
= 187187
== 187
187
nMnM
nM
nM
MMP-3 ===40
3 =3340 40nM
40
nM nM
nM
ICIC
ICIC50
IC
50:50
50: : MMP-1
MMP-1 MMP-1
:: MMP-1
MMP-1 ====375
= 375 375
375 nM;
nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2 <<<0.15
< 0.15 0.15
0.15
nM;nM;
nM;
nM; IC5050: MMP-2
50
= =
IC 50::50
IC
IC
IC50 MMP-2
MMP-2 =
:: MMP-2
MMP-2 ===20
= 20 20
20
nM; nM;
nM;
nM; MMP-3MMP-3
MMP-3
MMP-3 =300
==300 300
==300 nM;
300 nM;
nM;
nM;
nM;
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 = 18=
== 1818
18
18 nM;
nM;
nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 =
= 1.5 1.5
1.5
== 1.5
1.5nM nM
nM
nM
nM IC
IC IC
IC
IC :50::MMP-1
:50
5050 MMP-1
50 ==
:MMP-1
MMP-1
MMP-1 ===20
20 20
20
20
nM;nM;
nM;
nM;
nM; = 2=
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 ===nM;
2222nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ==
==100
= 100 100
100
100 nM;
nM;
nM;
nM;
nM; = 2===
MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 =μM
222μM
μM
µM
μM MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 == 11==nM
= 1 nM
11 nM
nM nM
KB-R7785
KB-R7785
KB-R7785
KB-R7785
KB-R7785 IC
IC
ICICIC
50
50
:50 :::MMP-1
MMP-1
5050 :MMP-1
MMP-1
MMP-1 5=
= 5===nM;
55nM;
5nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 11=
= 1===nM;
1nM;
nM;
nM; MMP-3
1 nM;
MMP-3
MMP-3
MMP-3
ICIC
IC
ICIC 5050: : MMP-1
50:50
MMP-1
:: MMP-1
MMP-1MMP-1 =
=333nM;
= 3==nM; nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2 ===
= 7.5=7.5
7.5
7.5 nM;
nM;
nM;
nM;
IC50
ICIC
IC
IC 50:: MMP-1
50:50
50
MMP-1
:: MMP-1
MMP-1
MMP-1 ====5.4
= 5.4 5.4
5.4 nM;
nM;
nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 ==
==8.4
= 8.4 8.4
8.4
8.4
nM;nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ==
==2.3
= 2.3 2.3
2.3
2.3
nM;nM;
nM;
nM;
nM;
MMP-9 = 5=
MMP-9
MMP-9
MMP-9
MMP-9 ===nM;
5555nM;
nM;
nM;
nM; MMP-3 ==
== 15
= 15 15
15 15
nM; nM;
nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 ==
= 1==nM1
11 nM1
nM
nM nM
==
50
MMP-3 ==1.9 nM; MMP-9 == 3.9 nM MMP-14
MMP-14
MMP-14
MMP-14
MMP-14 2.32.3
===2.3
2.3
2.3
nMnM
nM
nM
nM
MMP-3
MMP-3
MMP-3
MMP-3 = 1.9 1.9
== 1.9
1.9
nM; nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9 = 3.9 3.9
== 3.9
3.9
nM nM
nM
nM
KB-R7785
KB-R7785
KB-R7785 IC
ICIC
50:50 :: MMP-1
MMP-1
50 MMP-1= 5==nM;
55 nM;
nM; MMP-2
MMP-2
MMP-2 = 1==nM;
11 nM;
nM; MMP-3
MMP-3
MMP-3
IC : MMP-1 = IC
ICIC
50:50 :: MMP-1
MMP-1
50 MMP-1 ==5.4
= 5.45.4
nM;nM;
nM; MMP-2
MMP-2
MMP-2 == 8.4
= 8.48.4 nM;
nM;
nM; MMP-3
MMP-3
MMP-3 ==2.3
= 2.32.3
nM;nM;
nM; MMP-9
= 5==nM;
MMP-9
MMP-9 55nM;
nM;
ICIC
50:50: MMP-1
MMP-1
50 33 nM;
= 3= nM;nM; MMP-2
MMP-2
MMP-2 == 7.5
= 7.5 7.5 nM;
nM;
nM; Table 6. Cont.
= 15
= 15
= 15 nM; nM;
nM; MMP-9
MMP-9
MMP-9 = 1
= 1= nM
1 nMnM
MMP-3 MMP-14
MMP-14
MMP-14 ==2.3
= 2.32.3
nM nM
nM
MMP-3
MMP-3 ==1.9
= 1.91.9
nM;nM;
nM; MMP-9
MMP-9
MMP-9 ==3.9
= 3.93.9
nM nM
nM
Matlystatin
Matlystatin
Matlystatin
Matlystatin B BB
B
Biomolecules
Biomolecules
Biomolecules 2020,
2020,
IC2020,
IC 10, 10,
717
::10, 717= 150
717
MMP-1 ==150
150 nM 17
17nM of64
ofof
17 64 64
IC50 : MMP-1 = 150 nM
IC
50:50
MMP-1
50 MMP-1 nMnM IC IC
50::50
IC
IC50 MMP-2 =
50:: MMP-2
MMP-2 ==1.7
= 1.71.7
µM;
μM;μM;
μM; MMP-9
MMP-9
MMP-9 == 570
= 570
570
=570 nM
nMnM
IC
IC IC
IC :: MMP-3
:5050
5050 MMP-3
MMP-3 ===300
= 300300
nMnM
nM
R-94138
R-94138
R-94138
R-94138
IC50 : MMP-2 = 38 nM; MMP-3 = 28 nM;
IC
ICIC
50:50: :MMP-2
MMP-2
50 MMP-2 ==38
= 38 38
nM;nM;
nM; MMP-3
MMP-3
MMP-3 ==28
= 28 28
nM;nM;
nM;
MMP-7 = 23 nM; MMP-9 = 1.2 nM; MMP-13
MMP-7
MMP-7 = =
23 23
nM;
MMP-7 = 23 nM; = nM; MMP-9
MMP-9
MMP-9 = =
1.21.2
nM;nM; MMP-
MMP-
38 nM= 1.2 nM; MMP-
13
1313 ==38
= 38 38
nM nM
nM Ki:
Ki:Ki: MMP-1
MMP-1
Ki: MMP-1 > >>12.5
12.5
> 12.5 µM;
μM;μM;
μM; MMP-2
MMP-2
MMP-2 == 1.1
=1.1
1.1
=1.1 μM;
µM;
μM;
μM;
Ki:
Ki:Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1 >
>>8.3
> 8.3 8.3
8.3
μM;μM;
μM;µM; MMP-2
MMP-2
MMP-2
MMP-2= 3.4=
==3.43.4
3.4
μM;μM;
μM;µM;
MMP-3MMP-3
MMP-3
MMP-3 = 1.3=μM;
==1.3
1.31.3
μM;
μM; MMP-7
MMP-7
µM;
MMP-7
MMP-7 >μM;
>>8.3
> 8.38.38.3
μM;
μM; MMP-3
MMP-3
MMP-3
MMP-3 = 100
= =nM;
=100100
100nM;nM;
nM;
MMP-7 MMP-7
MMP-7
MMP-7 = 200
= =nM;
=200200
200 nM;
nM; nM;
MMP-14
MMP-14
MMP-14
µM; MMP-14
MMP-14
MMP-14
MMP-14 = 7.7=
==7.77.7
7.7
μM μM
μM µM
MMP-14 =nM
==1.8
= 1.8 1.81.8
nM
nM nM
IC
50:50
ICIC : :MMP-1
MMP-1
50 MMP-1 > 5>>μM;
55μM;
μM; MMP-2
MMP-2
MMP-2 ==35
= 35 35 nM;
nM;
nM; MMP-3
MMP-3
MMP-3 ==3.56
3.56
= 3.56 nM; nM;
nM; MMP-7
MMP-7
MMP-7> 5>>μM;
55μM;
μM;
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1 ==200
= 200200
nM nM
nM Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1 ==33.16
33.16
= 33.16 μM;μM;
μM; MMP-2
MMP-2
MMP-2 ==6.3
= 6.36.3
μM;μM;
μM;
MMP-9
MMP-9
MMP-9 ==304
= 304304
nM; nM;
nM; MMP-12
MMP-12
MMP-12 ==17
= 17 17
nM;nM;
nM; MMP-14
MMP-14 ==772
= 772
MMP-14 772
nM;nM;
nM; MMP-15
MMP-15
MMP-15 ==60
= 60 60
nM nM
nM
MMP-8
MMP-8 ==171
= 171
MMP-8 171
nM;nM;
nM; MMP-9
MMP-9
MMP-9 ==4.468
4.468
= 4.468 μM μM
μM
R-94138
R-94138
R-94138
ICIC
50: : MMP-2
MMP-2
50 = =
IC50: MMP-2 = 38 nM;
3838nM;nM; MMP-3
MMP-3
MMP-3 =2828
== 28 nM;
nM;
nM;
Biomolecules
MMP-7
MMP-7 =2020,
=
2323 10,
nM;nM;717MMP-9
MMP-9 = = 1.2
1.2 nM;
nM; MMP-
MMP- 17 of 61
MMP-7 = 23 nM; MMP-9 = 1.2 nM; MMP-
1313
13 =3838
== 38 nM
nMnM Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1 >12.5
12.5
>> 12.5 μM;
μM;
μM; MMP-2
MMP-2
MMP-2 =1.1
== 1.11.1 μM;
μM;
μM;
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1 >8.3
>> 8.38.3 μM;
μM;
μM; MMP-2
MMP-2
MMP-2 =3.4
== 3.43.4 μM;
μM;
μM; MMP-3
MMP-3
MMP-3 =1.3
== 1.31.3 μM;
μM;
μM; MMP-7
MMP-7
MMP-7 >8.3
>> 8.38.3 μM;
μM;
μM;
Cont.
Table =6.7.7 MMP-3
MMP-3
MMP-3 =100
100
== 100 nM;
nM;
nM; MMP-7
MMP-7
MMP-7 =200
200
== 200 nM;
nM;
nM; MMP-14
MMP-14
MMP-14
MMP-14
MMP-14
MMP-14 = =7.7
7.7
μM
μMμM
=1.8
== 1.81.8
nM
nMnM
IC MMP-1> >
:: MMP-1 5 μM;
µM; MMP-2 = 35 nM; MMP-3 == 3.56 nM; MMP-7>> 5>> 5 μM;
µM;
Ki: MMP-1 = 200 nM
Ki:
Ki: MMP-1
MMP-1 =200
200 nM ICIC
IC 5050MMP-1
50::50MMP-1 > 5>5 μM;
5μM; MMP-2
MMP-2
MMP-2 =3535
== 35 nM;
nM;
nM; MMP-3
MMP-3
MMP-3 == 3.563.56
3.56 nM;
nM;
nM; MMP-7
MMP-7
MMP-7 5μM;
5 μM; ===33.16 === 6.3
Biomolecules Ki:
2020,
Biomolecules
Biomolecules MMP-1
10,
2020,
2020, 717 717== 200
10,717
10, nM
nM MMP-9 = 304 nM; MMP-12 = 17 nM; MMP-14 = 772 nM; MMP-15 = 60 nM Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1 =33.16
33.16
33.16 μM;
µM;
μM;
μM; MMP-2
MMP-2
MMP-2
MMP-2 =6.3
6.3
18μM;
µM;
μM;
μM;of
18 64
18ofof6464
MMP-9
MMP-9
MMP-9 =304
304
== 304 nM;
nM; nM; MMP-12
MMP-12
MMP-12 =1717
== 17 nM;
nM;
nM; MMP-14
MMP-14
MMP-14 =772
== 772 772 nM;
nM;
nM; MMP-15
MMP-15
MMP-15 =6060
== 60 nM
nMnM MMP-8 ===171 nM; MMP-9 === 4.468 µM
MMP-8
MMP-8
MMP-8 =171
171
171 nM;
nM;
nM; MMP-9
MMP-9
MMP-9 =4.468
4.468
4.468 μM
μMμM
BB-1101
BB-1101
BB-1101
Ki:
Ki:Ki: MMP-1
MMP-1
Ki: MMP-1
MMP-1 ===5.248
= 5.2485.248
μM;
5.248 µM;
μM; MMP-2
MMP-2
μM; MMP-2
MMP-2 > 3>>>μM;
33μM;
µM;
μM; IC
IC50
IC
50: 50MMP-1
IC 50 == 3.1
: :MMP-1
MMP-1 =3.1 nM;
nM;
=3.1
3.1nM;MMP-2
MMP-2
nM; MMP-2==4.2
MMP-2 =4.2 nM;
nM;
=4.2
4.2 nM;
nM;
IC
ICICIC: : MMP-1
MMP-1
: =
MMP-1=
10
== 10
1010nM;
nM;nM;
nM; MMP-2
MMP-2 =
MMP-2
MMP-2 5=
==55
nM;
5 nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3 ===30
= 30 30nM;
nM;nM; MMP-7
MMP-7
nM; ==
= 30
MMP-7
MMP-7 nM;
=30
30nM;
nM; =
>>4.5 <<1<1<nM; MMP- >
50505050 MMP-3 25 nM
MMP-3
MMP-3
MMP-3
MMP-3 >4.5 µM;
μM;
>4.5
4.5 MMP-9
MMP-9
μM;
μM; MMP-9
MMP-9 1nM;
1nM; MMP-13
MMP-
nM; MMP- MMP-3
MMP-3
MMP-3 = 25 nM
==25
25nM
nM
MMP-8
MMP-8 =
MMP-8
MMP-8 =
3
= =33nM;
nM;
3 nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9 = ==nM
3
= 333nM
nM
nM
1313>1355>>μM
µM
55μM
μM
ICIC
50:50MMP-1
IC 50 = 1.1
: :MMP-1
MMP-1 nM;
==1.1
1.1 MMP-2
nM;
nM; = 1.1
MMP-2
MMP-2 nM;
==1.1
1.1 nM;
nM; ICICIC
50 :50MMP-8
50 = 300
: :MMP-8
MMP-8 nM
==300
300 nM
nM
MMP-3MMP-3
MMP-3 = 2.3 nM;
==2.3
2.3 MMP-7
nM;
nM; = 2.2
MMP-7
MMP-7 nM
==2.2
2.2 nM
nM OPB-3206
OPB-3206
OPB-3206
ICICIC
50 :50MMP-1
50 = 700
: :MMP-1
MMP-1 nM;
==700
700 MMP-2
nM;
nM; = 5==μM;
MMP-2
MMP-2 MMP-3
55μM;
μM; = 2==μM;
MMP-3
MMP-3 MMP-9
22μM;
μM; = 500
MMP-9
MMP-9 nM
==500
500 nM
nM
BB-1101
BB-1101
BB-1101
Ki:
Ki:MMP-1
Ki: MMP-1
MMP-1 = 5.248
==5.248μM;
5.248 μM;
μM;MMP-2
MMP-2
MMP-2> 3>>μM;
33μM;
μM; ICIC
50:50
IC MMP-1
50 = 3.1
::MMP-1
MMP-1 nM;
==3.1
3.1 nM;
nM;MMP-2
MMP-2
MMP-2= 4.2 nM;
==4.2
4.2 nM;
nM;
ICIC
50 : MMP-1 =
IC5050::MMP-1 10
MMP-1==10nM; MMP-2
10nM; MMP-2==nM;
nM;MMP-2 = 5 MMP-3
55nM;
nM; MMP-3
MMP-3 = 30 nM;
==30
30 MMP-7
nM;
nM; = 30
MMP-7
MMP-7 nM;
==30
30 nM;
nM;
MMP-3
MMP-3 > 4.5
MMP-3>>4.5 μM;
4.5μM; MMP-9
μM;MMP-9 < 1 nM;
MMP-9<<11nM;
nM;MMP-
MMP-
MMP- MMP-3
MMP-3
MMP-3 = 25 nM
==25
25 nM
nM
MMP-8
MMP-8
MMP-8= 3==Table
nM;
33nM;MMP-9
nM; Cont.= 3==nM
6.MMP-9
MMP-9 33nM
nM
1313
> 5>>μM
13 55μM
μM
ICIC
50:50
IC :::MMP-1
Biomolecules
IC
Biomolecules
50
50 MMP-1
MMP-12020,
MMP-1
2020, ==717
=10,
1.1
=10,1.1
nM;
1.1
1.1 nM;
nM;
nM;
717 MMP-2
MMP-2
MMP-2
MMP-2 ===1.1
= 1.1 1.1
nM;
1.1 nM;
nM;
nM; ICIC
IC IC
50 MMP-8
50:50
50 == 300
::MMP-8
MMP-8 =300
=300nM
nM
300 nM
nM 1919
of of
6464
OPB-3206
===2.3 ==
MMP-3
Biomolecules
MMP-3 =2020,
2.3 2.3 nM;
10,
nM; 717 MMP-7
MMP-7 = 2.2 2.2
nM nM OPB-3206
OPB-3206
OPB-3206 19 of 64
MMP-3
MMP-3 2.3 nM;
nM; MMP-7
MMP-7 =2.2
2.2 nM
nM
ICIC : : MMP-1
MMP-1
MMP-1 = =700
700
700 nM;
nM; MMP-2
MMP-2 = =μM;
5 5 µM;
MMP-3 = 2==
MMP-3 222μM;
µM; MMP-9 == 500 nM
IC
50 50
IC50: MMP-1 = 700 nM; MMP-2 =55μM;
50 : = nM; MMP-2 = μM; MMP-3
MMP-3 =μM; MMP-9
μM; = 500
MMP-9
MMP-9 nM
=500
500 nM
nM
Batimastat
Batimastat (BB-94)
(BB-94)
ICIC
50:50
MMP-1
: MMP-1 Batimastat
Batimastat
= 3= nM;
3 nM; (BB-94)
(BB-94)= 4= nM;
MMP-2
MMP-2 4 nM;
MMP-3IC
IC 50
50:: MMP-1
MMP-1
= 20 nM; =
= 3
3
MMP-7 nM;
nM; =MMP-2
MMP-2
6= nM; = 44 nM;
=MMP-8
MMP-8 nM; ®)®
MMP-3 = 20 nM; MMP-7 6 nM; Ilomastat (GM6001;
Ilomastat (GM6001;Galardin
Galardin
Galardin ®))
MMP-3 = 20 nM; MMP-7 = 6 nM; MMP-8 =
= MMP-3
= 10 nM;
10 nM; = 20 nM;
MMP-9
MMP-9 = 1= MMP-7
nM;
1 nM; = 6 nM;
MMP-13
MMP-13 = 1=MMP-8
nM;
1 nM; ICIC 50:50
MMP-1
: =
MMP-1 =
0.4 nM;
0.4 nM; Ilomastat
MMP-2
MMP-2= =(GM6001;
0.4 nM;
0.4 nM;MMP-3
MMP-3
50: MMP-1 = 0.4 nM; MMP-2 = 0.4 nM; MMP-3 = 0.19 = =
Galardin ®)
0.19 nM;
0.19 MMP-14
nM;
nM; MMP-14
MMP-14 == 5.2
= 5.2 5.2 Analogue Ilomastat
of of
Ilomastat
10 nM; MMP-9
= 10 nM; MMP-9 = 1 nM;
= =1 2.8
nM; MMP-13
MMP-13 = 1 nM;
= 1 nM;
Analogue Ilomastat
MMP-14
MMP-14 = 2.8nMnM IC50: MMP-1 = 0.4 nM; MMP-2 = nM 0.4
nM nM; MMP-3 = 0.19 nM; MMP-14 = 5.2 50::50
MMP-2 =
Analogue ofMMP-3 ==179
Ilomastat
MMP-14 = 2.8 nM nM IC
IC50
IC : MMP-2 =1.3
= 1.31.3nM;
nM; MMP-3 =179
179nM
nM
nM
Ki:
Ki:MMP-1
MMP-1 =MMP-14
10
= 10nM;nM; = MMP-2
2.8 nM= 4= nM;
MMP-2 4 nM; Ki: Ki:
MMP-1 MMP-1
= =
0.4 0.4
nM; nM;
MMP-2MMP-2
= =
0.39 0.39
nM; nM
Ki: MMP-1 = 10 nM; MMP-2 = 4 nM; MMP-3 Ki: MMP-1 = 0.4 nM; MMP-2 = 0.39 nM; MMP-3 = 26 nM; MMP-8 = 0.18 nM; nM;
MMP-3MMP-3
= 26 = 26
nM; nM;
MMP-8MMP-8
= =
0.18 0.18
nM; IC50: MMP-2 = 1.3 nM; MMP-3 = 179 nM
= 20Ki:
MMP-3
MMP-3 =MMP-1
20 nM;
= MMP-8
20 nM; = 10
MMP-8
= 10
MMP-8nM; =MMP-2
10
= 10nM; = 4MMP-9
=nM;
MMP-9
nM; Ki: MMP-1 = 0.4 nM; MMP-2 nM;= MMP-9
MMP-90.39=nM;0.2=nM
0.2 nM = 26 nM; MMP-8 = 0.18 nM;
MMP-3
nM; nM; MMP-9 1 nM MMP-9 = 0.2 nM
MMP-3 = 20 nM; = 1=MMP-8
1 nM = 10 nM; MMP-9
nM MMP-9 = 0.2 nM
= 1 nM
ICIC
50:50
MMP-1 = 8= μM;
: MMP-1 MMP-2
8 μM; MMP-2= 8= μM; MMP-3
8 μM; MMP-3
50:50
ICIC MMP-1 = 3= nM;
: MMP-1 3 nM;MMP-3 = 280
MMP-3 nM;
= 280 nM; IC50: MMP-1 = 8=μM; 3.5
= 3.5MMP-2
μMμM = 8 μM; MMP-3
Ki:
Ki:MMP-1
MMP-1= 3= nM
3 nM = 3.5 μM
IC50: MMP-1
MMP-7 = 3 nM;
= 18
MMP-7 = 18 nMMMP-3
nM = 280 nM;
Ki: MMP-1 = 3 nM
MMP-7 = 18 nM
Batimastat
Batimastat
Batimastat
Batimastat
Batimastat (BB-94)
(BB-94)
(BB-94)
(BB-94)
(BB-94)
IC
ICIC
IC50 ::
50
50
50 : MMP-1
MMP-1
MMP-1
MMP-1 =
IC50: MMP-1 = 3 nM;MMP-2
: ===
3333nM; nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2 ====4=444nM;
nM;
nM;
4nM;
nM;
MMP-3
MMP-3
MMP-3
MMP-3 =
===
2020
20
20 nM;
nM;
nM;
nM; MMP-7
MMP-7
MMP-7
MMP-7
MMP-3 = 20 nM; MMP-7 = 6 nM; MMP-8 =
===
66
66 nM;
nM;
nM;
nM; MMP-8
MMP-8
MMP-8
MMP-8 Ilomastat
Ilomastat
Ilomastat
Ilomastat
Ilomastat (GM6001;
(GM6001;
(GM6001;
(GM6001;
(GM6001; Galardin
Galardin
Galardin
Galardin
Galardin ))®®)))
®®®
====1010
10
10 nM;
nM;
nM;
nM;
= 10 nM; MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 =
== =
1 11 1
nM;nM;
nM;
nM; MMP-13
MMP-13
MMP-13
MMP-13
= 1 nM; MMP-13 = 1 nM; ====1
111 nM;
nM;
nM;
nM; ICIC
IC
ICIC
50 :
50
50
5050: : MMP-1
MMP-1
MMP-1
: MMP-1 ===
: MMP-1= =0.4 0.4
0.4
0.4 nM;
nM;
nM;
0.4nM; MMP-2
MMP-2
MMP-2
nM;MMP-2 ==
MMP-2= =0.4 = 0.4
0.4
0.4 nM;
nM;
nM;
0.4nM; MMP-3
MMP-3
MMP-3
nM;MMP-3 = ==
0.19
MMP-3= =0.19 0.19
0.190.19 nM;
nM;
nM;
nM; MMP-14
MMP-14
nM;MMP-14
MMP-14
MMP-14 ====5.2
5.2
5.2
=5.2
5.2 Analogue
Analogue
Analogue
Analogue ofof
of
of Ilomastat
Ilomastat
Ilomastat
Ilomastat
Biomolecules 2020, 10, 717 Analogue of Ilomastat 19 of 61
MMP-14
MMP-14
MMP-14
MMP-14
MMP-14 = 2.8 nM =
===
2.82.8
2.8
2.8 nM nM
nM
nM nM
nMnM
nM ICIC
IC :50 :MMP-2
MMP-2
::MMP-2 ====1.3
1.3 nM;
nM; MMP-3
MMP-3 ====179
179 nM
nM
nM ICIC
5050
50 50 :MMP-2
MMP-2 1.3
=1.3
1.3 nM;
nM;
nM; MMP-3
MMP-3
MMP-3 179
=179
179 nM
nMnM
Ki:
Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1
MMP-1 ====1010
10
=1010nM; nM;
nM;
nM;nM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 ====4=444nM;
nM;
nM;
4nM;
nM; Ki:
Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1
MMP-1 ====0.4
0.4
0.4
=0.4
0.4 nM;
nM;
nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 ====0.39
0.39
0.39
=0.39
0.39 nM;
nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ====2626
26
=2626nM; nM;
nM;
nM;nM; MMP-8
MMP-8
MMP-8
MMP-8
MMP-8====0.18
0.18
0.18
=0.18
0.18 nM;
nM;
nM;
nM;
nM;
MMP-3
MMP-3
MMP-3
MMP-3 ====20
2020
20 nM;
nM;
nM;
nM; MMP-8
MMP-8
MMP-8
MMP-8
MMP-3 = 20 nM; MMP-8 = 10 nM; MMP-9 =
===10
1010
10 nM;
nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9 MMP-9
MMP-9
MMP-9Table==6.
MMP-9
MMP-9 = 0.2
=0.2
0.2
=0.2 nMnM
nM
nM
Cont.
0.2 nM
====1=111nMnM
nM
1nMnM
IC
IC
IC IC
IC
IC
50 :50
50
50 5050 : MMP-1
:MMP-1
MMP-1
::MMP-1
:MMP-1
MMP-1 8=
====8=888μM;
μM;
μM;8 µM;
μM;
μM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 8=
====8=888μM;8 µM;
μM;
μM;
μM;
μM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
ICIC
IC
IC
ICIC
50 :50
50
50 :MMP-1
MMP-1
::MMP-1
:MMP-1
MMP-1====3==
333nM;
nM;
3nM;
nM;
3nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 =
====280
280
280
280
=280
280 nM;
nM;
nM;
nM;nM;
nM; MMP-3 =
====3.5
3.5
3.5
=3.5 3.5
μM
μM
μM
3.5μMμMµM
====
5050
Ki:
Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1
MMP-1
MMP-1 =3=333nMnM
nM
3nM
nM
MMP-7
MMP-7
MMP-7
MMP-7 =
MMP-7= =1818nM==
=
1818
18
18nMnM
nM
nM
nM
IC
ICIC
IC
IC
IC
50 :50
50
5050
50:MMP-1
MMP-1
::MMP-1
:MMP-1
MMP-1 ==
====3.3
3.3
3.3
3.3 μM;
μM;
μM;
μM;
3.3
3.3 µM;
μM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 =32
====32
=3232
32 nM;
nM;
nM;
nM;
32 nM;
nM;
Biomolecules 2020, 10, 717 IC
IC
ICIC
ICIC:50:50
50
50 :MMP-3
:MMP-3 ====15
MMP-3
:MMP-3
:MMP-3
MMP-3 ==15
15
15 nM
nM
15nMnM
nM
nM 2020
ofof
6464
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ====
717 =5757
57
57
=57
57 nM
nM
nM
nM
nMnM
50 50
IC
ICIC
IC
IC
50 :50
50
50
50 50
:MMP-3
MMP-3
::MMP-3
:MMP-3
MMP-3=====
3.43.4
3.4
3.4
=3.4
3.4μMµM
μM
μM
μMμM
50:50
ICIC
IC MMP-1
50
50 MMP-1 >>50
> 50
:: MMP-1 μM;
50 MMP-2
µM;
μM; MMP-2
MMP-2 >>120
> 120 μM;
120 µM;
μM;
MMP-3
MMP-3 = =
8080 µM;
μM; MMP-8
MMP-8 > > 120
120 μM
MMP-3 = 80 μM; MMP-8 > 120 μM µM IC 50::50
IC50
IC MMP-8
MMP-8
50 =
= 121
: MMP-8 μM
µM
= 121 μM
IC
ICIC :50
5050 MMP-2
50 ==
:: MMP-2
MMP-2 5252 µM;
μM;
= 52 MMP-3
MMP-3
μM; == 200
= 200
MMP-3 200 µM;
μM; MMP-8
MMP-8
μM; ==1200
1200
= 1200
MMP-8 μM µM
μM
ONO-4817
ONO-4817
ICIC
50:50
MMP-1
50 = 1600
: MMP-1 nM;
= 1600 MMP-9
nM; = 2.1
MMP-9 nM
= 2.1 nM
IC
IC 50
IC50
50 :MMP-1
:50
50:MMP-1
MMP-1 >>50
>50
50μM;
μM; MMP-2
MMP-2
μM; MMP-2 >>120
>120 μM;
μM;
120 μM;
MMP-3 ==80 IC
IC 50:50
:MMP-8
MMP-8===121
:MMP-8 121μM
121 μM
=80 μM; MMP-8 >>120
>120 μM 50
IC μM
MMP-3
MMP-3 80μM;
μM;MMP-8
MMP-8 120μMμM Table 6. Cont.
50 50
IC
IC 50:50
50
IC
50 :MMP-2
50 MMP-2===52
:MMP-2 52μM;
52 μM;MMP-3
μM; MMP-3===200
MMP-3 200μM;
200 μM;MMP-8
μM; MMP-8===1200
MMP-8 1200μM
1200 μM
μM
ONO-4817
ONO-4817
ONO-4817
ONO-4817
ICIC
IC 50
IC
50
5050 :MMP-1
::50
50 :MMP-1
MMP-1
MMP-1 ===1600
=1600 nM;
nM;
1600 nM; MMP-9
MMP-9
MMP-9
MMP-9 =
==2.1
2.1
=2.1
2.1 nM
nM
nMnM
PKF
PKF
PKF
PKF 242-484
242-484
242-484
242-484 Ki:
Ki:
Ki:Ki:MMP-2
MMP-2
MMP-2
MMP-2===0.73
=0.73
0.73 nM;
nM;
nM;
0.73 nM;MMP-3
MMP-3
MMP-3
MMP-3 = =42
==424242nM;
nM;
nM;
nM;
= = CT1746
CT1746
CT1746
CT1746 MMP-7 ===2500 nM; MMP-12 =
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1 =
= 3.6 nM;
3.6
3.6
Ki: MMP-1 = 3.6 nM; nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2 ===0.1
0.1 nM;
0.1
0.1 nM;
nM;
nM; MMP-7
MMP-7
MMP-7 =2500
2500
2500 nM;
nM;
nM; MMP-12
MMP-12
MMP-12 =0.45
==0.45
0.45
0.45 nM;
nM;
nM;
nM;
MMP-3
MMP-3
MMP-3 =
= 0.9 nM;
nM; MMP-9
MMP-9 =
= 1 nM;
nM; MMP-13
MMP- = Ki:
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1
MMP-1===
=122
122
122
122nM;
nM;
nM;
nM;MMP-2
MMP-2
MMP-2
MMP-2= ==0.04
0.04
0.04
= nM;
nM;
nM;
0.04 nM; MMP-3===
MMP-3
MMP-3
MMP-3 =10.9
10.9
10.9 nM;
nM;
nM;
10.9 MMP-7
MMP-7
MMP-7
nM; MMP-7 ==136
==136136
136 MMP-13 = 1.1 nM
MMP-3=0.9
=0.9
0.9nM;
nM;MMP-9
MMP-9=1=11nM;
nM;MMP-
MMP- MMP-13
MMP-13
MMP-13 ==1.1
=1.1
1.1nM
nMnM
13
13 = 4.5
4.5 nM
nM nM;
nM;
nM; MMP-9
MMP-9
MMP-9
nM; MMP-9 === 0.17
0.17
0.17
= nM
0.17 nM
nM
nM
13==4.5
4.5nM
nM
MMPI-I
MMPI-I
MMPI-I
MMPI-I
ICIC
IC50
IC::50
50
50
50 :50MMP-1
MMP-1===11=1µM;
: MMP-1
MMP-1 1μM;MMP-3
μM;
μM; MMP-3=
MMP-3
MMP-3 =150
==150
150 µM;
150μM;MMP-8
μM;
μM; MMP-8===1=11μM;
MMP-8
MMP-8 1µM;
μM;
μM;MMP-9
MMP-9
MMP-9
MMP-9 ===30
=30
3030
AS 111793# µM
μM
AS
ASAS111793#
111793#
111793# μMμM
IC
IC
IC50 : MMP-1===20
::MMP-1 20nM
nM
50
IC50 50: MMP-1 =20
50 50 MMP-1 20nM
nM
Biomolecules 2020,
Biomolecules
Biomolecules 10,2020,
2020,
2020,
Biomolecules 717
10,10,
717717717
10, 2121
of 21
6421
of of of
64 64 64
Biomolecules 2020, 10, 717 Table 6. Cont. 21 of 64
IC 50:IC
ICIC MMP-1
50:50 :IC :50
MMP-1
IC
50
MMP-1 50 == 30
30
:: MMP-1
MMP-1MMP-1
= 30 ==30
nM;
=nM;
30
nM;MMP-2
30
nM;nM;
nM;
MMP-2MMP-2
MMP-2 == 20
MMP-2
MMP-220 ==20
nM;
=nM;
= 20 20
nM;MMP-3
20MMP-3
nM;nM;
nM;
MMP-3 == 500
MMP-3
MMP-3500
MMP-3 = 500
= 500= 500
=nM;
nM; 500 nM;
MMP-7
nM;MMP-7 MMP-7
=
MMP-7
nM; MMP-7200
= =
200 =
nM;
200200
nM;nM;
= 200 nM;
MMP-8 MMP-8
=
MMP-8
MMP-8 20
=
nM; MMP-8 nM
=
20 =
20
nM 20
nM
= 20 nMnM Ki: MMP-1
MMP-1 === 1450 = 15 nM;
nM; MMP-7 = 200 nM; MMP-8 = 20 nM Ki:
Ki:
Ki: Ki: MMP-1
MMP-1
Ki:
MMP-1 = 1450
MMP-1
1450 =nM;
= 1450 MMP-3
nM;
nM;
1450
nM; MMP-3
MMP-3
nM;
MMP-3 MMP-3
= 15 =nM;
= 15 15
nM;nM;
= 15 nM;
IC
IC
IC 50:IC
IC MMP-1
:: MMP-1
MMP-1
IC
50
:50MMP-1
5050 == =
: 50MMP-1 0.1
: MMP-1
0.1 nM;
=nM;
= 0.1
0.10.1 MMP-3
nM;
=nM;
nM;
0.1 nM;
MMP-3 MMP-3
MMP-3
MMP-3 == =99= 9nM;
MMP-3 nM;
9=nM;MMP-8
9=nM;
nM; nM;
MMP-8 == =0.4
MMP-8
9MMP-8
MMP-8
MMP-80.4 nM;
=nM;
= 0.4
0.4 0.4
=nM;
nM;MMP-
nM;
0.4 MMP-
nM;
MMP- = 0.2
MMP-
MMP-9MMP- MMP-8
MMP-8 == 22=nM;
MMP-8
MMP-8MMP-8 nM;
2 = 2
nM;=MMP-9
MMP-9
nM;
2 == 3= nM
MMP-9
MMP-9
nM;
MMP-8 = 2 nM; MMP-9 = 3 nM MMP-9 3= 3
nM= nM
3 nM
99 ==9 0.2
0.2
=90.2 nM
=9nMnM
0.2
=nM nM
0.2 nM
IC
IC50:IC
IC MMP-1
:: MMP-1
MMP-1
IC
50
== =
: 50MMP-1 100
= 100
: MMP-1 nM;
= nM;
100
=nM;MMP-2
nM;
100 MMP-2
MMP-2
nM; =
== 0.07
0.07
MMP-2 nM;
= 0.07
0.07
= 0.07 nM;
= nM;MMP-3
nM;
0.07 MMP-3
MMP-3
nM; == 33==nM;
MMP-3
MMP-3 nM;
33=nM;MMP-7
3=nM;
nM; MMP-7
3 MMP-7
nM; ==700
== 700
MMP-7 700
MMP-7 = 700
= 700
===88=8nM; ===28 <<<22<2nM; ===11= 1nM
MMP-1
Ki: MMP-1 nM;
=nM;MMP-3
8=nM;
8nM; MMP-3 28 nM;
=nM;
28 MMP-8
nM; MMP-8 nM;
2<nM; MMP-9
2<nM; MMP-9 nM
1=nM
1=nM IC :50MMP-1
5050 100 MMP-2 nM; MMP-3 MMP-7
Ki:
Ki:
Ki: MMP-1
Ki:
MMP-1 MMP-1 8MMP-3
MMP-3
nM;
MMP-3 =28
MMP-3 28 nM;
nM;
= 28MMP-8
MMP-8
nM;
MMP-8 MMP-8 nM;2MMP-9
MMP-9
nM;
MMP-9MMP-9 nM 1 nM
700nM;
nM;
nM;
nM; MMP-8
MMP-8
nM;
MMP-8
nM; =
MMP-8 =
4
=
MMP-8 4 nM;
nM;
= 4= MMP-9
MMP-9
nM;
4 nM; =
MMP-9=
1
=
MMP-9
MMP-8 = 4 nM; MMP-9 = 1 nM 11
nM=nMnM
1= nM
1 nM
50:IC
ICIC MMP-1 Ki: MMP-1
Ki: == 7.56
MMP-1 7.56 μM;
= 7.56 MMP-7
μM; == 622
MMP-7 622 nM;
= nM;
622 nM;
IC 50:50 : IC
50
MMP-1 == 11
: 50MMP-1
MMP-1 11
: MMP-1 nM;
=nM;
= 11 11
nM;MMP-3
nM;
= 11 nM;
MMP-3 == 1.04
MMP-3
MMP-3 1.04
MMP-3 μM
= 1.04
= 1.04
= μM
μM μM
1.04 μM 50:IC
ICIC MMP-1
Ki:
Ki: MMP-1
Ki:
MMP-1 = 7.56
MMP-1 = μM;
7.56
μM; MMP-7
μM;
MMP-7 MMP-7= 622 =nM;
622 nM;
IC 50:50 : IC
MMP-1
50
MMP-1 == 600
: 50MMP-1 600
= 600
: MMP-1 nM;
= nM;
600
=nM;MMP-2
nM;
600 MMP-2
MMP-2
nM;
MMP-2 == 33= μM;
MMP-2 μM;
3=μM;MMP-3
3=μM;
3 μM;
MMP-3 == 50
MMP-3
MMP-3 50
MMP-3 = 50
= 50 = 50 MMP-13
MMP-13 = 7.3
MMP-13 7.3 nM
=nM
= 7.3
MMP-13 7.3
=nMnM
7.3 nM
nM; MMP-7 MMP-13 =
nM;
nM; nM;
MMP-7
nM;
MMP-7 == 44= nM
MMP-7
MMP-7 nM
4=nM 4=nM
4 nM
IC50: IC 50:50
MMP-1
IC MMP-1= 100=nM;
: MMP-1 100 nM; MMP-2
MMP-2
= 100 nM; = 0.07= nM;
MMP-2 0.07 nM; MMP-3
MMP-3
= 0.07 nM; = 3= MMP-7
= 3 nM;
MMP-3 nM; MMP-7
3 nM; = 700= 700
MMP-7 = 700
Ki:Ki:
MMP-1
Ki: MMP-1 MMP-1 = 8= MMP-3
= 8 nM; nM; MMP-3
8 nM; MMP-3 = 28
= 28 nM; nM; MMP-8
MMP-8
= 28 nM; < 2< MMP-9
< 2 nM;
MMP-8 nM; MMP-9
2 nM; = 1= nM
= 1 Table
MMP-9nM 1 6.
nM Cont.
nM;
nM; MMP-8MMP-8
nM; = 4= MMP-9
= 4 nM;
MMP-8 nM;
4 nM;MMP-9 = 1= nM
= 1 nM
MMP-9 1 nM
IC50IC
: IC 50::50
MMP-1
IC
50 MMP-1
: MMP-1 = =1111
= 11
= 11 nM; nM;
nM; = 1.04==μM
MMP-3
MMP-3
MMP-3
nM; MMP-3 =1.04
1.04 µM
μM
1.04 μM Ki: Ki:Ki:
Ki: MMP-1
MMP-1 =
MMP-1
= 7.56= μM;
MMP-1 7.56 μM;
MMP-7
= 7.56
µM; μM; =
MMP-7 622=nM;
= 622
MMP-7 622
nM; nM;
= 622 nM;
IC50: IC 50:50
MMP-1
IC
IC 50MMP-1
= 600=nM;
: MMP-1 600
= nM;
= 600
600 MMP-2
MMP-2
nM;
nM; MMP-2 = 3==μM;
= 3 μM; MMP-3
MMP-3
33μM;
µM; = 50 = 50
MMP-3
MMP-3 = 50
Biomolecules
Biomolecules 2020,
Biomolecules 10,
10, 717
2020,
2020,2020,
Biomolecules
717
10, 717
10,717
717 MMP-13
MMP-13 =
MMP-13= 7.3 =
nM7.3 nM 22
MMP-13 = 7.3 nM 22 of22 of 64
22 of
64of
22 64
of64
64
= 50
nM; nM;
MMP-7
nM;MMP-7 = 4=
= 4 nM
MMP-7 = nM
4 nM
IC
IC50 ::IC
IC
50IC
MMP-1
50
50
MMP-1 ====6666μM;
== 66 μM;
50:: MMP-1 μM; MMP-2
µM;
μM; MMP-2
MMP-2 ===
== 200
MMP-2 200 nM;
200
nM; MMP-3
200nM;
nM; MMP-3
MMP-3 ====nM
== 100
nM;MMP-3
MMP-3100 100nM
100
nM nM
nM Ki:
Ki: MMP-2
Ki:
Ki:MMP-2
MMP-2
Ki: =
== 2.2
MMP-2
Ki:MMP-2
MMP-22.2===nM
2.2
nM
2.2 nM
2.2nM
nM
50: :MMP-1 MMP-2 =200 100
====5555nM
IC50 MMP-1 μM; MMP-2 200 nM; MMP-3 100 nM IC ::IC
IC MMP-2 == 55 nM
50:: MMP-2 nM
nM
IC50
50IC 50
IC50
MMP-2
50: :MMP-2
50 MMP-2 nM nM
Ki:
Ki: MMP-2
Ki:
MMP-2 >> 15
MMP-2 15>>>μM;
15 MMP-8
μM; MMP-8>> 15
15>>>μM;
15 MMP-
μM; MMP-
Ki: MMP-2
Ki:MMP-2
MMP-2 >μM;
15
15 MMP-8
μM;
15µM;
μM; MMP-8
MMP-8
MMP-8 >μM;
15 MMP-
μM;
15µM;
15 μM;MMP-
MMP-
Ki:
Ki: MMP-1
Ki:
MMP-1 >>MMP-1
MMP-1 4.949
>
4.949 μM;
4.949
>
μM; MMP-2
μM;
MMP-2 >>MMP-2
MMP-2 3.333
>
3.333 μM;
3.333
>
μM; MMP-9
μM;
MMP-9 >> >> 9 >Ki:
MMP-9 15
9 >μM;
15 MMP-12
μM; MMP-12 = 410= nM;
410 MMP-13
nM; >
MMP-1315
IC ::IC
MMP-7 == 11 510 μM; MMP-12 == 149
Ki: MMP-1
Ki: > 4.949 μM;
4.949 MMP-2
µM; > 3.333 μM;
3.333MMP-9
µM;
Ki: MMP-1 > 4.949 μM; MMP-2 > 3.333 μM; MMP-9 > 9 > MMP-9 15
99>>μM;
15 > 15MMP-12
MMP-12
15μM;
μM; µM; =MMP-12
MMP-12 410 nM;
==410 =nM;
410nM;MMP-13 nM;> 15>>>15
410MMP-13
MMP-13 15
15
50IC
IC50 IC 50: MMP-7
50
MMP-7
50
50 MMP-7
50: :MMP-7
MMP-7 ===
510=1111510
510
μM;
510 μM;
510μM;
µM;
μM; MMP-12
MMP-12
MMP-12
MMP-12
MMP-12 ====μM
149 149
μM
149
149 μM
149μMµM
μM 2.128
MMP-9
2.128 μM>
2.128
μM
2.128 μM
2.128
μM
2.128 μM µM MMP-13μM; μM;
> 15MMP-14
μM; µM;MMP-14= 3.07
MMP-14 = μM
= 3.07
3.07 μM µM
MMP-14
μM;
μM; MMP-14= 3.07
MMP-14 μM
==3.07
3.07 μM
μM
Ki:MMP-2
Ki:
Ki: MMP-2 MMP-2 >15
15μM;
> 15 >μM; μM;MMP-8
MMP-8MMP-8 >15
15μM;
> 15 >μM; μM;MMP-
MMP-MMP-
Ki:
Ki: MMP-1
MMP-1 >> 4.949
4.949 μM;
μM; MMP-2
MMP-2 >> 3.333
3.333 μM;
μM; MMP-9
MMP-9 >>
IC5050::MMP-7
MMP-7 ==11μM;
510μM;
μM;MMP-12
MMP-12
Ki: MMP-1 > 4.949 μM; MMP-2 > 3.333 μM; MMP-9 >
Table 6. Cont. 2.128 μM 9 > 15 μM; MMP-12 = 410 nM; MMP-13 > 15 >>15
99>> 15
15 μM;
μM; MMP-12
MMP-12 == 410
410 nM;
nM; MMP-13
MMP-13 15
IC50:IC
MMP-7 = 1 510 510 MMP-12 = 149==μM
149μM
149 μM 2.128
2.128 μM μM μM;
μM; MMP-14
MMP-14 == 3.07
3.07 μM
μM
μM; MMP-14 = 3.07 μM
Ki: Ki:
MMP-1
Ki: MMP-1 >> 4.946
Ki:MMP-1
MMP-1 >>4.946
4.946 4.946
µM;μM;
μM; μM;MMP-2
MMP-2
MMP-2 >>3.333
MMP-2 >>3.333
3.333 3.333 μM;
µM;μM;
μM;
MMP-3 > 4.501
MMP-3
MMP-3 > 4.501
> 4.501 μM;
MMP-7
µM; μM; MMP-7> 6.368
MMP-7 > 6.368
µM;μM;
> 6.368 μM;
IC5050::MMP-1
IC50:IC MMP-1
IC50 : MMP-1
=51
=μM; = 51
51μM;
μM; MMP-2
µM;
MMP-2 MMP-2
==1.79 = 1.79
1.79μM;
μM; MMP-3 5.9 MMP-3 > 4.501 μM; MMP-7 > 6.368 μM;
= 5.9==5.9
µM;
MMP-3
MMP-1 = 51 MMP-2 = 1.79 μM; MMP-3 MMP-8 > 3.058
MMP-8
MMP-8 >>3.058
3.058 MMP-9
µM; μM;
μM;MMP-9 > 2.128
MMP-9 > µM;μM;
>2.128
2.128 μM;
== 5.9 = = 5.9 = 840 = 1.9 MMP-8 > 3.058 μM; MMP-9 > 2.128 μM;
nM;=nM;
IC5050::MMP-1
IC5050::IC MMP-1 =5.9
5.9μM;
5.9=µM; μM;MMP-2
MMP-2
750==nM;
750nM;
750 nM;MMP-3=2.1
MMP-3=2.1 nM;
nM;nM; nM;MMP-3MMP-9 nM; MMP-9 73nM;
nM;MMP-13
IC MMP-1
MMP-1 μM; MMP-2
MMP-2 = 750 nM; MMP-3=2.1
MMP-3=2.1 nM; MMP-9 MMP-9 = 840==nM;
MMP-9 840
840 nM;
nM; MMP-13
MMP-13
MMP-13 =73
= 73 =nM; nM; MMP-14
MMP-14 = 1.9==1.9
MMP-14 MMP-10 >
MMP-10
MMP-10 5.346
> µM;
5.346
> 5.346 MMP-12
μM; MMP-12> 6.023
> µM;
6.023 μM;
Biomolecules MMP-9
MMP-9
2020, 560
10, =nM;
=717 560MMP-14
560 nM;MMP-14
nM; =
MMP-14 930==nM
930nM
nM
930 nM μM = 1.9 µM
73 nM; MMP-14
μM MMP-10 > 5.346 μM; μM; MMP-12
MMP-12 > 6.023
> 6.023 μM; μM; 23 of 64
Biomolecules MMP-9
2020,
Biomolecules 2020, 10, 717 =
10, 560
717 nM; MMP-14 = 930 μM MMP-13 >
MMP-13
MMP-13 5.025 µM;
>>5.025
5.025 MMP-14
μM;MMP-14
MMP-14> 5.290 µM;
>>5.290
5.290 μM;
23
64 of 64
23 ofμM;
MMP-13 > MMP-15
5.025 μM;>μM;
MMP-14
7.088 >
µM μM 5.290 μM;
MMP-15 MMP-15
MMP-15 >>7.088
> 7.088 7.088
μM μM
Ki: MMP-1 > 5 μM; MMP-2 > 3 μM; MMP-3 =

MMP-1>>4.494
Ki:MMP-1
Ki: 4.494μM; MMP-2> >3.333
µM;MMP-2 3.333μM;µM;MMP-3 MMP-1
Ki:=MMP-1
Ki: >>55μM;
Ki: MMP-1 > 5 μM;
µM; MMP-2
MMP-2 >>33μM;
MMP-2 > 3 μM;
µM; MMP-3
MMP-3 MMP-3
= =
Ki:MMP-3
MMP-1 >824.494 762 nM; MMP-8 = 2.05 μM; MMP-9 > 3 μM;
Ki: MMP-1 > 4.494
82 nM;=MMP-7 nM; μM;
μM; MMP-2MMP-2
=MMP-7 25>nM;
> 3.333
25 nM;=MMP-
3.333
μM; μM;
MMP-3
8 MMP-
> 3.1 μM;8MMP-3
=3.1 = 762
>MMP-9 =
> 762
762nM;
nM; nM;MMP-8
MMP-8 MMP-8 = =2.05
= 2.05 2.05µM;
μM; μM; MMP-9
MMP-9MMP-9> 3> >33 μM;
μM;
MMP-10 > 1.650 μM; MMP-13 > µM;
5 μM; MMP-
82 MMP-7
82 nM; nM; MMP-7
2.128MMP-9
µM; => 25
= 25 nM;
μM; MMP-13
nM;µM;
MMP-
2.128 MMP-
> 5.025
8 >μM;
8MMP-13
> 3.1 3.1>μM;
MMP-9
μM; MMP-145.025MMP-9>
>µM;
> MMP-10
5.290 μM;
MMP-10
µM;MMP-10 >>1.650
> 1.650 1.650
μM; µM;
μM;
MMP-13 MMP-13
MMP-13 >>5 5μM;
> 5 μM; MMP- MMP-
IC 50: MMP-1 = 4.6 μM; MMP-2 = 4 nM; MMP-3 = 42 nM; MMP- 2.128 μM; MMP-13 > 5.025 μM; MMP-14 > 5.290 μM;
MMP-13>>5.290 MMP-14>>7.088 MMP-14 14
= =163 = 163 nM; MMP-15
= =μM = 1.7 μM
IC5050
IC : MMP-1
: IC
MMP-1 ==
50: MMP-14.64.6 µM;
=μM;
4.6 μM;MMP-2
MMP-2 ==
MMP-2 4=nM;
4 nM; MMP-3
4 MMP-3
nM; MMP-3 = 42
= 42 nM;
= 42
nM; nM;
MMP-MMP->2.128
MMP-7 10 μM;MMP-14 MMP-15
5.025µM;
>
μM;MMP-15
7.088 μM; MMP-16
5.290µM;
>
μM;
5.554 μM 14
14 = 163 163nM;
nM; nM;MMP-15
MMP-15
MMP-15 = 1.7 1.7
1.7µMμM
7 > 10 μM; MMP-9 = 120 nM > MMP-16
= =120 MMP-15 >MMP-16
7.088
μM; μM; > 5.554
μM μM
> µM; MMP-9 120 nM MMP-15 > 7.088 5.554>µM
MMP-16 5.554
7 > 107 μM;
10 μM;
MMP-9MMP-9 =nM
120 nM
Ki: MMP-1 = 30 μM; MMP-2 = 2.05 μM; MMP-3 = 141 nM; MMP-7 = 259
Ki: MMP-1
Ki: MMP-1 = 30 μM;
= 30 μM; MMP-2
MMP-2 = 2.05= μM;
2.05 μM; MMP-3
MMP-3 = 141=nM;
141 MMP-7
nM; MMP-7
= 259= 259
nM; MMP-8 = 257 nM; MMP-9 = 10.34 μM; MMP-13 = 1.417 μM; MMP-
nM; MMP-8
nM; MMP-8 = 257=nM;
257 MMP-9
nM; MMP-9 = 10.34
= 10.34 μM; μM; MMP-13
MMP-13 = 1.417
= 1.417 μM; μM;
MMP-MMP-
14 = 15.872 μM; MMP-15 = 3.997 μM; MMP-16 = 1.599 μM
14 = 15.872
Ki: MMP-1 > 2 μM; MMP-2 > 2 μM; MMP-3 > 2 μM; MMP-7 = 834 nM; MMP-8 = 126 nM; MMP- 14 = 15.872 μM; μM; MMP-15
MMP-15 = 3.997
= 3.997 μM; μM; MMP-16
MMP-16 = 1.599
= 1.599 μM μM
Ki: MMP-1 > 2 μM; MMP-2 > 2 μM; MMP-3 > 2 μM; MMP-7 = 834 nM; MMP-8 = 126 nM; MMP-
Ki:MMP-1
Ki: MMP-1> >5 5μM; μM;MMP-2
MMP-2> >3 3μM;
μM;MMP-3
MMP-3= =
Biomolecules 2020, 10, 717 Ki:MMP-1
Ki: MMP-1> >4.494
4.494μM;
μM;MMP-2
MMP-2> >3.333
3.333μM;
μM;MMP-3
MMP-3= = 24 of 61
762nM;
762 nM;MMP-8
MMP-8= =2.05
2.05μM;
μM;MMP-9
MMP-9> >3 3μM;
μM;
8282nM;
nM;MMP-7
MMP-7= =2525nM;
nM;MMP-
MMP-8 8> >3.1
3.1μM;
μM;MMP-9
MMP-9> > MMP-10> >1.650
MMP-10 1.650μM;
μM;MMP-13
MMP-13> >5 5μM;μM;MMP-
MMP-
IC
IC : MMP-1= =4.6
: MMP-1
5050 4.6μM;
μM;MMP-2MMP-2= =4 4nM;
nM;MMP-3
MMP-3= =4242nM;
nM;MMP-
MMP- 2.128μM;
2.128 μM;MMP-13
MMP-13> >5.025
5.025μM;
μM;MMP-14
MMP-14> >5.290
5.290μM;
μM; 1414= =163
163nM;
nM;MMP-15
MMP-15= =1.71.7μM
μM
Cont.μM; MMP-16 > 5.554 μM
Table>6.7.088
MMP-15
7 7> >1010μM;
μM;MMP-9
MMP-9= =120120nM
nM MMP-15 > 7.088 μM; MMP-16 > 5.554 μM
Ki: MMP-1= ==
Ki:MMP-1
MMP-1 30
3030 µM;
μM;
μM; MMP-2
MMP-2
MMP-2 =2.05
2.05
= =2.05 µM;
μM;
μM; MMP-3
MMP-3
MMP-3 =141
141
= =141 nM;
nM;
nM; MMP-7
MMP-7
MMP-7 =259
= =259
259 nM;
nM;MMP-8
nM; MMP-8
MMP-8= =257 = 257
257nM; nM;
nM;MMP-9MMP-9
MMP-9= =10.34 = 10.34
10.34μM; µM;
μM;MMP-13MMP-13
MMP-13= =1.417 = 1.417
1.417μM; µM;
μM;MMP-
MMP-
MMP-14 = 15.872
1414= =15.872
15.872 μM;µM;
μM; MMP-15
MMP-15
MMP-15 = 3.997
= =3.997
3.997 µM;
μM;
μM; MMP-16
MMP-16
MMP-16 = 1.599
= =1.599
1.599μMμMµM
Ki:
Ki:MMP-1
Ki: MMP-1
MMP-1 >μM;
2 µM;
> >2 2μM; MMP-2
MMP-2
MMP-2 >μM;
> >2 2μM;2 µM; MMP-3
MMP-3
MMP-3 >
> >2 2μM;2 MMP-7
μM; µM; MMP-7
MMP-7 = =834=nM;
834 834 nM;
nM;MMP-8MMP-8
MMP-8 = =126=nM;
126 126MMP-
nM; nM;
MMP-
MMP-9 > 2 MMP-10
µM; MMP-10μM; > 2 MMP-12
µM; MMP-12μM; > 2 MMP-13
µM; MMP-13 653= 653 nM; MMP-14 > 2 µM; MMP-15
9 9> >2 2μM;
μM;MMP-10 > >2 2μM; MMP-12 > >2 2μM; MMP-13 = =653 nM;
nM; MMP-14
MMP-14 > >2 2μM;
μM; MMP-15
MMP-15 > >2 2
> 2 µM; MMP-16 > 2 µM
μM;MMP-16
μM; MMP-16> >2 2μMμM
Figure 16. (a) NNGH; (b) arylhydroxamate sulphonamide compound; (c) 3-hydroxy-3-
methylpipecolic hydroxamates.
Figure 16. (a) NNGH; (b) arylhydroxamate sulphonamide compound; (c)
Figure 16. (a) NNGH; (b) arylhydroxamate sulphonamide compound; (c) 3-hydroxy-3-
3-hydroxy-3-methylpipecolic hydroxamates.
methylpipecolic hydroxamates.
The incorporation of a cyclic quaternary center α led to a strong inhibitory effect against MMP-
1, -2, The
-3, -8, -9, -12, and -13
incorporation of a[29]. The
cyclic RS-113,456
quaternary (Figure
center 17a)
α led to is an inhibitor
a strong with effect
inhibitory betteragainst
oral MMP-1,
The incorporation
bioavailability and of a cyclic
metabolic quaternary
stability center
compared to α led
the to a strong inhibitory
hydroxamate derivates effect
[35]. against
These twoMMP-
-2, -3, -8, -9, -12, and -13 [29]. The RS-113,456 (Figure 17a) is an inhibitor with better oral bioavailability
1, -2, -3, -8,
features were-9, improved
-12, and -13by[29]. The RS-113,456
shifting (Figure
thehydroxamate
cyclic group to 17a) is an inhibitor
the α-position with better oralacid
of the
and metabolic stability compared to the derivates [35]. These twohydroxamic
features were improved
bioavailability
(Figure 17b) and
[29]. metabolic stability compared to the hydroxamate derivates [35]. These two
by shifting the cyclic group to the α-position of the hydroxamic acid (Figure 17b) [29].
features were improved by shifting the cyclic group to the α-position of the hydroxamic acid
(Figure 17b) [29].
Figure
Figure 17. (a) RS-113,456;
17. (a) RS-113,456; (b)
(b) RS-130,830.
RS-130,830.
Table 7 shows the IC50 Figure and 17.Ki (a)

values of some
RS-113,456; sulfonamide hydroxamic acid-based
(b) RS-130,830.
Table2020,
Biomolecules 7 shows
10, 717 the IC50 and Ki values of some sulfonamide hydroxamic acid-based inhibitors
31 of 64
inhibitors [6,15–19,29,35,37,54,55].
[6,15–19,29,35,37,54,55].
Table 7 shows the IC50 and Ki values of some sulfonamide hydroxamic acid-based inhibitors
4.1.3. Phosphamides Hydroxamic Acid-Based Inhibitors
[6,15–19,29,35,37,54,55].
The hydroxamic acids based on phosphamides are effective as MMPis due to the the electronic
electronic
environment
environment of of the
the phosphor
phosphor atom
atom [11].
[11]. The replacement of sulphonamide group by phosphinamide
group leads to to aapotent
potentinhibitor
inhibitorofofMMP-3
MMP-3 (Figure
(Figure 18),18),
the the collagenases
collagenases and and gelatinases
gelatinases [29]. [29].
The
The interactions
interactions between
between this inhibitor
this inhibitor and theand the MMP-3
MMP-3 are realized
are realized by theby the phosphinamide
phosphinamide phenylphenyl
group,
group, that accommodates
that accommodates intoSthe
into the S1 ’ pocket
1’ pocket andandby bythethe phosphinamide
phosphinamide oxygen,which
oxygen, whichestablishes
establishes the
hydrogen
hydrogen bonds
bonds with
with NH
NHof ofLeu 164 and Ala
Leu164 Ala165 [29].However,
165 [29]. However, thisthis group
group is susceptible to hydrolysis
at low pH,
pH, limiting
limiting the
the inhibitory
inhibitory activity
activity [29].
[29].
Figure 18. Inhibitor

Figure 18. Inhibitor with
with phosphinamide
phosphinamide group.
group.
All hydroxamate-based inhibitors are very potent and they inhibit MMPs at low concentrations
[18]. On the other hand, the hydroxamate acids have poor oral bioavailability, inhibit multiple MMPs,
and cause side effects [2,17,27,28,35]. Additionally, this functional group may be metabolized via
dehydroxylation or may be cleaved by endopeptidases and releasing hydroxylamine, can be
hydrolyzed to carboxylic acids or reduced to O-glucuronyl or O-sulfate, leading to decreased
effective inhibitor concentration and reducing its potency in vivo [18,27,28,35].
Biomolecules
Biomolecules Biomolecules
2020,2020,
10, 10, 717 2020, 10, 717
717 26 of 26
64 of 64 26 of 64
Biomolecules
Biomolecules 2020,2020, 10, 717
10, 717
Biomolecules 2020, 10, 717 26 of 64 26 of2664of 61
Table 7. IC7.
Table IC50Table
50 and Ki 7. values
values
and7.Ki
IC ICof
50 and
and of Ki valuesof
sulfonamide ofsulfonamide
sulfonamide
hydroxamic
sulfonamide hydroxamic
acid-based
hydroxamic acid-based
inhibitors.
acid-based inhibitors. inhibitors.
TableTable
7. Table
IC50
50 and Ki50
7. 50
IC andKiKiof
values values
sulfonamide
values hydroxamic
hydroxamic
of sulfonamide acid-based
acid-based
hydroxamic inhibitors.
inhibitors.
CGS-27023A
CGS-27023A CGS-27023A
(MMI270)(MMI270) (MMI270)
CGS-27023A (MMI270)
CGS-27023A (MMI270)
IC50:IC
MMP-1 = 33IC
50: MMP-1 =nM;: MMP-1
33
50 MMP-2
nM; MMP-2==CGS-27023A
33
11nM; 11MMP-2
=nM; MMP-3
nM;
(MMI270)
MMP-3==11
13nM;
= 13MMP-3 = 13
50: MMP-1
IC50 IC50: =MMP-1
3350nM;
IC =MMP-2
: MMP-1 33 nM; = 33
=MMP-2
11 nM;
nM; =MMP-3
MMP-211 nM;= 11
=MMP-3
13nM; = 13
nM; nM;
MMP-9MMP-9
nM; MMP-9
nM;
= 8 nM; MMP-9
MMP-12
= 8 nM;
= 8
MMP-12
nM;
MMP-3
==87.7
MMP-12
nM;
=
=nM; MMP-12
7.7
13 =
MMP-13
nM;
7.7
nM;
= 7.7
MMP-13
nM;
MMP-9
= 6nM;
=
MMP-13
= 6MMP-13 = 6
8 =
nM; 6
Analogue
Analogue
ofAnalogue
of CGS-27023A
Analogue CGS-27023A
of of CGS-27023A
of CGS-27023A
CGS-27023A
Ki: MMP-1
Ki:MMP-1
Ki: MMP-1 ==Ki:
= 770
Ki: MMP-1
nM;
770
770
Ki:
MMP-1
MMP-2
nM;
nM;
=MMP-1
= 770
MMP-2
770 nM;
= 620
MMP-2
= MMP-2
nM;
nM;
=620
770 =nM;
620MMP-2
nM;
=MMP-2
620 nM;
= 620 nM;
nM;MMP-3
= 620 nM;
nM; MMP-9 = 8 nM; MMP-12 = 7.7 nM;IC MMP-13 = 6 = 104 Analogue of CGS-27023A MMP-3 = 4.1 μM; MMP-9 = 620 nM
nM
MMP-12nMnM = 7.7 nM; nM MMP-13 = 6 nM
50: IC
MMP-1
50: MMP-1
IC : IC IC
MMP-1
50IC
= nM;
104
50 :
: MMP-1 MMP-1
=
MMP-2
nM;
104 = =
104
nM;
104
=
MMP-2nM;nM;
0.7
MMP-2
=nM;MMP-2
0.7
MMP-2
=
MMP-3
nM;
0.7 =
nM;
=
MMP-3
0.70.7
= 0.7
nM;
MMP-3
nM;
=nM;
0.7MMP-3
MMP-9
nM;
MMP-3
= 0.7
= 0.7
=
MMP-9
=
nM;0.7 nM;
2.5
nM;
MMP-9
=nM;MMP-9
2.5
=
MMP-13
nM;
MMP-9
2.5
= 2.5
=
MMP-13
=
nM;2.5 nM;
nM;=
MMP-13 =
MMP-3
MMP-13 ==
MMP-3 =
4.1
MMP-3
=μM;
4.1
4.1 =
MMP-9
μM;
µM;
4.1
=
MMP-9
MMP-9
μM; =
620
MMP-9
= nM
620
620=
nM
nM
620 nM
nM 50
50 50: MMP-1 = 104 nM; MMP-2 = 0.7 nM; MMP-3 = 0.7 nM; MMP-9 = 2.5 nM; MMP-13 = MMP-3 = 4.1 μM; MMP-9 = 620 nM
Ki: MMP-1
Ki: MMP-1
=Ki:
= 3 nM;
Ki: MMP-1 MMP-1
MMP-2
3 nM;
= 3
MMP-2
Ki:
nM;
=
=
MMP-1 3
20
MMP-2
nM;
nM;
= 20
= MMP-2
=
MMP-3
nM;
320nM; =
MMP-3
nM;
=20
MMP-2
MMP-3
nM;
148= 148
= MMP-3
20
= nM;
148
= 148 12 nM12 nM
MMP-1312 =
nM 12 12
nM nM
Ki: MMP-1 = 3 nM; MMP-2 = 20 nM; MMP-3 = 148 12 nM
nM; nM;
MMP-8 MMP-8
MMP-3
nM; MMP-8 =
= 1.9nM;=nM
1.9
148 MMP-8
nM
nM; = 1.9 nM
MMP-8 = 1.9 nM
nM; =MMP-81.9 nM = 1.9 nM
NNGH
NNGH NNGH NNGH
:50MMP-12 NNGH
= =nM NNGH
72 MMP-12
nM
ICIC 50
50: IC
MMP-12 IC
= 72
: MMP-12 50:72 nM = 72 nM 50:IC = 2=nM;
IC 2:nM;MMP-1 2=nM;
== 20 2020MMP-2
nM; IC=5020 nM;
CGS-25966 CGS-25966
CGS-25966 CGS-25966 IC50
Ki: MMP-10 50: MMP-12
=IC = 72 nM = 72 nMICIC
50: nM
0.6 MMP-12 50MMP-1
: 50 : MMP-1
MMP-1
IC5050: MMP-1
2= 50
nM;MMP-2 MMP-2
MMP-2 =nM; :IC
MMP-1
50: MMP-1 IC
= 506:nM;
= 6 nM; MMP-1
MMP-2 == 900
MMP-2 6 nM;
=nM;
900MMP-2
nM; = 900 nM;
IC 50: =MMP-1
2 nM; MMP-2
= 220nM; =MMP-2
20 nM;= IC
20 nM; = 650:nM;
CGS-25966 CGS-25966
= Ki: MMP-10
Ki: MMP-10 = 0.6Ki:
=nM MMP-10
0.6 nM = 0.6 nMnM; MMP-3
MMP-3MMP-3 = = 30
30 nM;
= nM;
30 MMP-3
MMP-7
nM; MMP-7==
MMP-730=
20 nM;
nM;
= 20nM;
MMP-7
nM; = 20 nM;
50 :IC 50: MMP-1
MMP-1
50 IC =MMP-1
6 nM;
MMP-2=6=
MMP-2nM; =MMP-2
900 900
nM;nM;
MMP-3
= 900 nM;
Ki: MMP-3
Ki: MMP-3 92MMP-3
= 92 Ki:
Ki:
=nM MMP-3
nM 92 nM
= 92 nM Ki: MMP-10 = 0.6 nM
Ki: MMP-10 = 0.6 nM MMP-3 =MMP-3 30 nM;=MMP-7
30 nM; =MMP-7 MMP-3
MMP-3
20 nM;= 20 = =
nM; 200
200
=
M;
M; MMP-3
200MMP-7
M;
MMP-7
=
MMP-7
=
= 200
200
200
=M;
nM;
200
nM;
MMP-7
MMP-8
nM;
MMP-8
= 200 nM; MMP-8
MMP-8
= 200 nM;MMP-8
Ki: MMP-3 Ki: =MMP-3
92 nM = 92 nM MMP-9
MMP-9 =
MMP-9 7 nM
= 7 nM MMP-9
= 7 nM = 7 nM MMP-3 = 200
MMP-3 M; =MMP-7
200 M; = 200
MMP-7 nM;= MMP-8
200 nM;
MMP-9 =MMP-97 nM = 7 nM = 200=nM; MMP-9
200 nM;
MMP-9 = 200
==2μM;
MMP-9 nM;
= 2μM;
2µM; MMP-9
MMP-13 ===2μM;
MMP-13
MMP-13 400 MMP-13 = 400
= 400
400 nM
= 200 nM;= MMP-9 = 2μM; MMP-13 =
200 nM; MMP-9 = 2μM; MMP-13 = 400 400
nM nM nM
nM nM
Biomolecules 2020, 10, 717 Table 7. Cont. 27 of 64

Biomolecules
2020, 10, 717
Biomolecules 717
2020, 27 of
27 of 64
64
Biomolecules 2020,
2020, 10, 2020, 10,
10, 717
BiomoleculesBiomolecules717 10, 717
717 27
27 of
of 64
64
27
27 of
of 64
64
Prinomastat
Prinomastat (AG3340)
(AG3340) CP-471,474 CP-471,474
Prinomastat
Prinomastat (AG3340)(AG3340)
(AG3340)
Prinomastat CP-471,474
CP-471,474
CP-471,474 CP-471,474
Ki: Prinomastat
Ki:
MMP-1 MMP-1
Prinomastat = =
8.3 (AG3340)
8.3
nM; nM;
Prinomastat
(AG3340)
MMP-2 MMP-2
(AG3340)
= = 0.05
0.05 nM; nM;
MMP-3 = IC50===: 1170
MMP-1 = MMP-2
1170 CP-471,474
nM;= MMP-2 CP-471,474
0.7 nM;==
=MMP-3 MMP-3 = 16 nM;===MMP-9nM = 13 nM IC IC50>
Ki: MMP-1
Ki: MMP-1Ki:==MMP-1
8.3MMP-3
8.3 nM;=MMP-2
nM; MMP-2
MMP-2
=
8.3 nM; ==MMP-2
0.05
0.05 nM;
nM; MMP-3
MMP-3
=
==MMP-3
0.05 nM;
ICIC
IC
==MMP-3 = IC5050 :
50
50 :
: MMP-1
MMP-1
MMP-1
IC 1170
1170
:: MMP-1
nM;
nM;
nM; MMP-2
MMP-2
== 1170 nM; =
= 0.7
0.7
0.7
== MMP-2
nM;
nM;
nM; MMP-3
MMP-3
=MMP-3
0.7 nM;
16nM;
===MMP-3
16
16 nM; MMP-9
nM; MMP-9
==MMP-9
16 nM;
13nM
13
13
== MMP-9nM == 13
13 nM IC
IC :::MMP-1
5050 MMP-1
MMP-1
IC >>: 10
MMP-1
10
10 µM; > 10 μM;
>MMP-2
μM;MMP-2
μM; MMP-2 = MMP-2
====MMP-2
3.3 nM; = 3.3nM;
3.3 nM; nM;
50:: MMP-1 >:: MMP-1 > 10
10 μM; nM; == 3.3
50= 1170 nM; MMP-2 0.7 nM; 16 nM; MMP-9 13 nM 50
Ki:
Ki: MMP-1
MMP-1 =
Ki:= 8.3
MMP-1
8.3 nM;
nM; = 8.3
MMP-2 0.3
nM; =
nM;
= 0.05
MMP-2
0.05 nM;
MMP-7
nM; 0.05
MMP-354nM; =
nM;
= MMP-3 = IC 50 IC
MMP-150= MMP-1
1170 nM; 1170
MMP-2 nM; MMP-2
0.7 nM; = 0.7
MMP-3 nM; MMP-3
16 nM; 16
MMP-9 nM; MMP-9
13 nM nM IC 50:: MMP-1
IC50
50 MMP-150>
IC50 10 μM;
MMP-1
μM; MMP-2 μM; 3.3
MMP-2
3.3nMnM; 3.3 nM;
0.3 nM;
0.3 0.3
nM; MMP-7
MMP-7 nM; MMP-7 = 54 nM; MMP-9 = 0.26 nM; MMP- MMP-13 =MMP-13
12 nM 12
0.3
0.3 nM; 0.3
0.3 nM;
nM; MMP-7
MMP-7 nM; ====MMP-7
MMP-9 54 nM;
54
54 nM;
= 0.26
5413nM;
MMP-7nM;
MMP-9
=MMP-9
= 54
MMP-9
54
MMP-9
=MMP-14
0.03
nM;
nM;
nM;nM;
==MMP-9
0.26 nM;
0.26
==MMP-13
0.26 nM;
0.26 nM;
MMP-9 nM; = MMP-
MMP-
=MMP-
0.26
0.03 nM;
==0.33
0.26
MMP- nM; MMP-
nM; MMP-
MMP-13 ==12
MMP-13
MMP-13 ==MMP-13
MMP-13 12 nM
12 nM
nM == 12
12 nM
MMP-13 12 nM
nM
13===0.03
13
13 0.03nM;
0.03 nM;
nM;
13 = MMP-14
MMP-14
0.03
MMP-14 nM; ==MMP-14
=
MMP-14
= 0.33
0.33
0.330.33nM
nM
nM nM= 0.33
nM
nM
13 = 0.03 nM; 13 = MMP-14
0.03 nM; =MMP-14 0.33 nM= 0.33 nM
RS-113,456 RS-130,830
RS-130,830 RS-130,830
RS-113,456
RS-113,456 RS-113,456 RS-130,830
RS-130,830 RS-130,830
RS-130,830
IC 50: MMP-1 = 196 nM; MMP-2 = 0.01 nM; MMP-9 = RS-113,456
RS-113,456
IC 50: RS-113,456
MMP-3 = 5.2 nM Ki:== MMP-1 =MMP-2
590 nM;== MMP-2 = 0.22 nM;
IC5050:::MMP-1
IC MMP-1
MMP-1 ==196
196
196 :nM;
nM; MMP-2
MMP-2 Ki:
Ki: MMP-1
MMP-1 = 590
590 nM;
nM; MMP-2 0.22nM;
0.22
===MMP-2 nM;
nM;
IC 50: MMP-1
IC50
50
IC
IC50 =:: MMP-1
50=
IC50nM;
MMP-1
196 nM; ==MMP-2
196
MMP-2 ====MMP-2
= 196
196 nM;
MMP-1 nM;
0.01
0.01
nM;
0.01
MMP-2
0.01
1 nM
nM;
nM;
nM;
MMP-9
MMP-9
==MMP-9
0.01
MMP-2
nM; 0.01
MMP-9nM;
nM;
==MMP-9 =
= 0.01
=
= MMP-9 = IC505050::::MMP-3
IC
IC
IC
IC50
50
50
MMP-3
MMP-3
MMP-3 IC
IC
==MMP-3
=
:
50=
50=:
5.2
5.2
5.2
5.2
MMP-3
5.2
nM
nM
nM
nM
nM == 5.2
5.2 nM
nM
: = 0.054 nM; MMP-7 = 240 nM; MMP-8 = 0.13 nM; MMP-9
Ki:
Ki:
Ki: MMP-1
MMP-1 Ki:== MMP-1
Ki:
MMP-3 590
MMP-1
590 nM;==MMP-2
590 nM;
nM;
= 9.3
590
MMP-2
590
MMP-2
nM;
nM;
nM;
MMP-7
0.22
0.22
MMP-2
0.22 =
nM;
nM;
1.2
== 0.22 nM;
0.22MMP-
μM; nM;
1
1 nM
nM
nM; MMP-9 = 1 nM Ki:
Ki: MMP-1
MMP-1 Ki:== MMP-1
70
70 nM;
nM; = 70 nM;== MMP-2
MMP-2
MMP-2 0.054
0.054 nM;
nM; MMP-7
MMP-7 == 240
240 nM;
nM; MMP-8
MMP-8 == 0.13
0.13 nM;
nM; MMP-9
MMP-9 == MMP-3
MMP-3
MMP-3 == 9.3 ==MMP-3
9.3
9.3
nM; nM;
nM; =MMP-7
MMP-7
9.3
MMP-7 =
nM; ==MMP-7
1.2 1.2
1.2
µM; μM;
μM; =MMP-
MMP-
1.2
MMP-9 =
μM; MMP-
11 nM
nM 1 nM
1 nM Ki:
Ki: MMP-1
MMP-1 Ki:==MMP-1
Ki: MMP-1
Ki: 70 =
70 nM;
MMP-1
nM; =nM;
= 70
70MMP-2
70
MMP-2 nM;
MMP-2
nM;=
= MMP-2
0.054
MMP-2= 0.054
0.054 nM;
nM; == 0.054
nM; nM;
MMP-7
0.054
MMP-7 MMP-7
nM; =
= MMP-7
240
MMP-7
240 =
nM;
nM;240==MMP-8
240
nM;
240
MMP-8 nM;
=
nM;
= MMP-8
MMP-8
0.13
MMP-8
0.13 =nM;
nM; ==MMP-9
0.13 0.13
nM;nM;
0.13
MMP-9nM;=
=
MMP-3
MMP-9
MMP-3
MMP-9 =
== = MMP-3 9.3 nM;
9.3 nM; 9 =MMP-7
=MMP-7
9.3 nM;
0.58 nM; = 1.2
= MMP-7
1.2 μM;
MMP-13 MMP-
μM; =MMP-
1.2
= μM;
0.52 nMMMP-
MMP-9 nM;0.065
= 0.065
0.065
0.065 nM; MMP-12
MMP-12
nM;
nM;==MMP-12
MMP-12 nM;=MMP-13
= nM;
0.15
0.15 0.15
0.15 nM;==MMP-13
MMP-13
nM; MMP-13 =nM;
0.17
0.17 nM; = MMP-14
0.17 nM;==MMP-14
0.17 MMP-14
nM;nM;MMP-14
= 0.089 nM
= 0.089
0.089
0.089 nM
nM nM nM 99990.58
====0.58
0.58
0.58
nM; nM;
nM;
nM; MMP-13
MMP-13
99MMP-13
==MMP-13
0.58
0.58 nM; = =0.52
==0.52
0.52
0.52
=MMP-13 nM nM
nM
nM== 0.52
nM 0.52 nM
0.065
0.065 nM;nM;0.065
MMP-12
0.065
MMP-12nM;
nM;==MMP-12
0.15 nM;==MMP-13
0.15 nM;
MMP-12 0.15
0.15 nM;
MMP-13 nM;==MMP-13
0.17 nM;==MMP-14
0.17 nM;
MMP-13 0.17
0.17 nM;
MMP-14 ==MMP-14
0.089
0.089 nM
MMP-14 nM == 0.089
0.089 nM 0.58 nM; MMP-13 nM; MMP-13
0.52 nM
RS-104,966 IC50==: MMP-2

RS-104,966
RS-104,966
RS-104,966 RS-104,966 IC505050:::MMP-2
IC MMP-2
MMP-2
IC nM;==MMP-13
960 nM;
960 960 nM;==MMP-13
MMP-13
MMP-13 1.17μM;
1.17 μM;
μM;= 1.17 μM;
Ki:
Ki: MMP-1
MMP-1 23
23 nM;
nM; =RS-104,966
RS-104,966
Ki:== MMP-1 RS-104,966
23
MMP-13
MMP-13 nM;==MMP-13
0.13 nM
0.13 nM = 0.13 nM IC50==: MMP-1 = 3MMP-9
245 nM;== MMP-9 = 7 nM;==MMP-13 = 4 nM
IC
IC50
IC 50:: MMP-2
50 IC50 =:: MMP-2
50= 960
960 nM;
MMP-2 960
960 nM;
=MMP-13
nM;MMP-14 nM;
= ==MMP-13
=1.17
MMP-13
1.17
1.17
3.41 μM
== 1.17
μM;
µM; 1.17 μM;
μM;
Ki: Ki:
Ki:
Ki: MMP-1 ==MMP-1
Ki:
MMP-1 23 nM;=MMP-13
MMP-1
23 nM;
MMP-1 ==23
23
MMP-13nM;
23nM; ==MMP-13
nM;MMP-13
0.13
0.13 nM
MMP-13nM===0.13
0.13 nM
0.13nM
nM ICIC505050:::MMP-1
IC
IC 50: MMP-1
IC50
50
MMP-1
MMP-1
IC = 33 245
=:: MMP-1
50=
IC50
245nM;
33245
245
3MMP-1
245
nM;
nM; MMP-9
== 33MMP-9
nM;
nM; 245
245 nM;
MMP-9
MMP-9nM;
77 nM;
nM;
===MMP-9
77nM;
7MMP-9
nM;
MMP-13
MMP-13
== 77 nM;
nM;MMP-13
MMP-13
MMP-13 =44444nM
nM;==MMP-13
MMP-13
nM
nM
nM == 44 nM
nM nM
MMP-14=MMP-14
MMP-14
MMP-14=
MMP-14
MMP-14
==3.41
3.41 μM
3.41 μM
μM== 3.41
3.41 μM
=MMP-14 µM 3.41 μMμM
Biomolecules 2020, 10, 717 2020, 10, 717

Biomolecules 28 of 64 28 of 64
Biomolecules
Biomolecules
Biomolecules 2020,
2020,10,
10,
2020, Biomolecules
10,
Biomolecules 717
2020, 10,717
717
2020,
717 10, 717 28 of 64 2828
28ofof
6464
of 6428 of 64
Table 7. Cont.
IC50: MMP-1 =:IC

920
MMP-1 nM;=MMP-13
920 nM; = 0.95 nM
IC50: MMP-1 =IC310 nM IC
IC
IC50: MMP-1 :IC 50IC
MMP-1
:: MMP-1
50= 920 50 :: MMP-1
nM; =MMP-1
==920
920nM;== 920
nM;
MMP-13 920 nM;
MMP-13
MMP-13
nM;
= 0.95 MMP-13
= =0.95
0.95nM
MMP-13
nM == 0.95
nM 0.95 nM
nM
===310
5050
IC
IC
IC50: MMP-1 :50MMP-1
5050
IC IC
:=: MMP-1
IC50
310 50
MMP-1nM
50 :: MMP-1
=MMP-1
==310
310nM
310 nM
nM 310 nM
310nM
nM IC MMP-1
MMP-13
50
=920
0.95nM;
nMMMP-13 = 0.95 nM IC50: MMP-1 =IC841 nM; MMP-9 =nM;
33 nM;
IC IC
IC5050:: MMP-1
IC
MMP-1
5050
IC =
:50MMP-1
841
:=: MMP-1
IC
841
MMP-150:: MMP-1
nM;
nM;
50 =MMP-1
==841
841nM;
MMP-9
MMP-9
841 nM;
nM; ==MMP-9
== 841
841
MMP-9
33
33 nM;
nM;
nM;
MMP-9 =MMP-9
==3333
MMP-9 == 33
33nM;
nM;
nM; 33 nM;
nM;
MMP-13 MMP-13 = 29 = 29
nM nM
MMP-13 = 29 nM IC : MMP-1 =IC763 nM; MMP-9 =nM;
2 nM;
MMP-13 MMP-13MMP-13
=MMP-13
29 nM = 29
== 29
MMP-13 nM
29 nM
50
= 29 nM IC50: MMP-1 :: ==MMP-1 == 763
763 ==
MMP-9
nM IC 5050:50
IC
IC
IC :50MMP-1
MMP-1
:=: MMP-1
IC
763
MMP-150
nM;
50
MMP-13
==763
763
MMP-1 nM;
MMP-9
=763
2
nM;
nM;
nM
MMP-9
=MMP-9
2 nM;
MMP-9 MMP-9 nM;== 22 nM;
==2222nM;
nM;
nM; nM;
MMP-13 MMP-13
MMP-13
MMP-13 == =22 2nM
MMP-13
2 nMMMP-13
=MMP-13 nM
nM == 2
2 nM
nM
= 2 nM
Ro-32-7315 Ro-32-7315 IC50: MMP-1IC50:

= 346 MMP-1
μM; MMP-9 =μM;
24 μM
Ro-32-7315
Ro-32-7315 IC50: == == 346
346 =
=MMP-9 == 24
24 μM
μM
IC50: MMP-1 =IC500 nM; MMP-2
Ro-32-7315
=nM;250 MMP-2
nM;
Ro-32-7315
Ro-32-7315
MMP-3 =nM;
210 MMP-3
nM; MMP-7 310 MMP-7 =IC50:
=nM; IC50:MMP-1
IC50:
IC50:
MMP-1 MMP-1
=MMP-1
IC50:
346
MMP-1μM;==346
346μM;
346
MMP-1 μM;
µM;
MMP-9
μM; MMP-9
=MMP-9
24μM; ==2424
μMMMP-9
MMP-9 24μM
24 μM
µM
μM
IC
IC
IC50: MMP-1 :
IC5050
IC5050 MMP-1
: MMP-1
IC
=: MMP-1
:500 50 :
nM;
MMP-1
50 MMP-1
= =
= =
500 nM;
500
: MMP-1 nM;
MMP-2
500
500 nM;= 500
MMP-2
=nM;MMP-2
500 nM;
= MMP-2
250 nM;
MMP-2 = 250
=
MMP-2
= = 250
250nM;
MMP-3
250 =
nM; 250
MMP-3
= nM;
nM; 250MMP-3
nM;
= MMP-3
210 nM;
MMP-3 = 210
=
MMP-3
= = 210
210nM;
MMP-7
210 =
nM; 210
MMP-7
= 210
nM; MMP-7
nM;
= MMP-7
nM;310MMP-7= 310
=
MMP-7
= 310 310
= = 310
310
MMP-8 inhibitor MMP-8 I inhibitor I nM; MMP-9 =nM; 100 MMP-9
nM; MMP-12 = 11 MMP-12
nM; MMP-13 = 110MMP-13
nM
MMP-8 MMP-8
MMP-8
inhibitor inhibitor
inhibitor
MMP-8
I I II
inhibitor II nM; MMP-9 =
= ==100 nM;==nM;
100
MMP-12nM; =nM;
= =MMP-13 =MMP-13
11 nM; = 110== nM
110 nM
nM
MMP-8
IC50: MMP-8 MMP-8
=IC4inhibitor
nM inhibitor nM; MMP-9 310nM;
nM;
nM; =MMP-9
MMP-9
nM;
100
MMP-9nM;MMP-9100 nM;
100
MMP-12
100 nM; MMP-12
100 MMP-12
nM;
=MMP-12
11 =1111
MMP-12
nM; 11 nM;
11
nM; MMP-13
=nM;
11 MMP-13
nM;
=MMP-13
110 = ==110
MMP-13
nM 110nM
110 nM
nM 110
IC
IC
IC50: MMP-8
50
IC50: MMP-8
:: MMP-8
50= IC
4ICnM
MMP-8 : MMP-8
=
50: MMP-8
50
50
4
MMP-8 nMnM===444nM
== 44 nM nM
nM
Biomolecules
Biomolecules
Biomolecules 2020,
2020,
2020, 10,
2020,10,
10,
Biomolecules
Biomolecules 717
717
717
10,2020,
Biomolecules 10, 717
7172020, 10, 717 29
29
29 of
29of
of 64
of64
64
6429 of2964of 64
Table 7. Cont.
PGE-4410186
PGE-4410186
PGE-4410186
PGE-4410186
PGE-4410186
PGE-4410186
PGE-4410186
MMP-9 inhibitor IIIIinhibitor
IC
IC
IC ::::MMP-1
IC505050
50 MMP-1
MMP-1
MMP-1 ==24
=50
=IC 24
:24
: MMP-1
IC50IC 24 :nM;
50nM;
MMP-1
nM;
nM;
MMP-1 24 24=nM;
MMP-3
= MMP-3
MMP-3
=nM;
MMP-3 ====18.4
24
MMP-3 18.4
nM;
18.4
18.4
MMP-3nM;
nM;
MMP-3
nM;
= 18.4
nM; = 18.4
MMP-7
MMP-7
=MMP-7
nM;
MMP-7
18.4 ==== MMP-7
MMP-7
nM; = = MMP-9
MMP-9 inhibitor
MMP-9MMP-9inhibitor
MMP-9 inhibitor
inhibitor I I Ki:
Ki: MMP-1
Ki:MMP-1
Ki: MMP-1 =
====1.085
Ki: Ki:
MMP-1 1.085
1.085
MMP-11.085
MMP-1 μM;
=µM;
μM;
μM;
μM; MMP-2
MMP-2
=MMP-2
MMP-2
1.085 μM;μM;
MMP-2
1.085 ==
===1111MMP-2
MMP-2 nM;
nM; MMP-9
nM;=MMP-9
nM; MMP-9
1 =nM;
MMP-9 ===
1 nM;==10
10
10
MMP-910 nM;
nM;
nM;
= 10=nM;
nM;
MMP-9 10 nM;
nM;
3030
3030nM;MMP-7
nM;
nM;MMP-9
nM; MMP-9
30 =
MMP-9
nM;
MMP-9 30 nM;
====2.7
MMP-9
30 nM; 2.7
2.7
2.7 MMP-9
nM
nM
nM
nM
MMP-9 = 2.7 =
nMnM
= 2.7 2.7 nM IC
IC
IC
IC
IC50
50
50:
::: MMP-1
MMP-1
MMP-1
IC50IC
5050: MMP-1 =
=
== 1.05
1.05
1.05
: MMP-1
=: 1.05
50 MMP-1nM;
nM;
nM; MMP-9
MMP-9
MMP-9
= 1.05
nM; nM;nM;
= 1.05 =
==
=55
5
MMP-95 nM;
nM;
nM;
nM;
= MMP-9 MMP-13
=MMP-13
MMP-13
MMP-13
5 =nM; =====113
MMP-13
5 nM; 113
113
113
MMP-13nM
113nM
nM
nM= 113
nM nMnM
= 113 MMP-13
MMP-13
MMP-13
MMP-13
MMP-13 = 3333nM
nM
====3MMP-13
MMP-13 nM
nM
nM= 3 =nM 3 nM
ICIC
ICIC
IC 50
50
50
50 ::MMP-1>
::50MMP-1>
: IC
MMP-1>
MMP-1>
MMP-1> :50
5050
50
: MMP-1>
50IC 50 μM;
50
μM;
μM;
μM;
MMP-1> MMP-2
50 MMP-2
MMP-2
µM;
MMP-2
μM;
MMP-2
50 μM; = nM;
====12
12
12
MMP-212 nM;
12
nM;
nM;
MMP-2 MMP-3
=nM; MMP-3
MMP-3
MMP-3
12
=nM;
MMP-3
12 nM; = μM;
====4.5
4.5
4.5
MMP-34.5 μM;
4.5
μM;
MMP-3μM; MMP-7
MMP-7
4.5 μM;
MMP-7
= 4.5 >>>>50
MMP-7
MMP-7
=µM; μM; 50
MMP-7 >μM;
50
50 μM;
50> 50>μM;
μM;
μM;
MMP-7 50 μM;
MMPI-II
MMPI-II
MMPI-II
MMPI-II (MMP-2/MMP-9
MMPI-II
(MMP-2/MMP-9(MMP-2/MMP-9
(MMP-2/MMP-9
(MMP-2/MMP-9
(MMP-2/MMP-9 inhibitor
inhibitor
inhibitor
inhibitor II)
inhibitor
II)
II)
inhibitor
II) II)
II) II) :::::MMP-1
MMP-1 == == ===
MMPI-II
: MMP-1 =
(MMP-2/MMP-9
970
inhibitor
=
µM; MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 = =
==200
= 200
200
MMP-9
200200
nM
nM
nM=
nM
MMP-9 nM
200
= nM
200 nM
ICIC
IC
IC50
IC 50
50
50
50 MMP-1
MMP-1
MMP-1 : MMP-1
IC50IC 50 :147
===147147
147
147 nM;
nM;
nM;
nM;
nM;
MMP-1 MMP-2
MMP-2
MMP-2
= 147MMP-2
nM;nM;
=MMP-2
147 MMP-2 0.09
===0.09
0.09
0.09
MMP-2nM;
nM;
nM;
nM; MMP-3
MMP-3
=MMP-3
= 0.09 nM;nM;
MMP-3
0.09 MMP-350
==50
50
50
50
MMP-3= 50= 50
IC
IC
IC ::::MMP-1
IC505050
50 MMP-1
MMP-1 :IC
IC50IC
MMP-1 ===50
=50
MMP-1 970
970
:970
970
MMP-1 nM;
nM;
nM;
= 970
nM; =MMP-2
MMP-2
MMP-2
nM;
MMP-2
970 =nM;
===17
MMP-2
nM; 17
17
17 MMP-2
nM;
nM;
nM;= MMP-3
nM;
MMP-2 MMP-3
17 1717
MMP-3
=nM;
MMP-3 >nM;
>>> MMP-3
MMP-3
nM; > > nM;
nM;
nM; MMP-7
MMP-7
MMP-7 >>>>>11111MMP-7
µM;
μM;
μM; MMP-8
MMP-8 == 1.6
===1.6
1.6 nM;
nM; MMP-9
MMP-9 == 6.7
===6.7
6.7 nM;
nM;
nM;
nM; MMP-7
nM;nM;
MMP-7 MMP-7 μM;
μM; >MMP-8
MMP-8
1 >μM;
MMP-8 1 MMP-8
μM; 1.6
1.6
MMP-8nM;
nM;
nM; MMP-9
MMP-9
= 1.6 nM;nM;
MMP-9
= 1.6 MMP-96.7
6.7 nM;
MMP-9nM;= 6.7
nM; = nM;nM;
6.7
1000
1000
1000nM;
1000 nM;
nM; MMP-7
MMP-7
MMP-7
1000 MMP-3
nM; ====800
MMP-7 800
800 >nM;
1000
nM;
nM; nM;
nM;MMP-7
MMP-9
MMP-9
MMP-9
= 800 ====30
30
30
MMP-9 nM;= =800
nM;
nM; MMP-nM; MMP-
MMP-
MMP-
30 MMP-14 = 9.8 nM
nM; MMP-7
1000 nM; 800
MMP-7 nM; =MMP-9
800 nM; 30 nM;
MMP-9 =nM;
MMP-
30 nM; MMP- MMP-14
MMP-14
MMP-14
MMP-14 =
= ==9.8
MMP-14 9.8
9.8
9.8 nM
nM
nM
nM = 9.8 nM
MMP-9 14 = 17 nM; MMP-14 = 17 nM
30nM MMP-14 = 9.8 nM
14====17
14
14 17 nM
nM
1714
nM =1417=nM17 nM
Biomolecules Biomolecules
2020, 10, 717 2020, 10, 717 30 of 64 30 of 64
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2020,
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717
717
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3030of
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Table 7. Cont.
ICIC ::::MMP-1
MMP-1
IC =
IC
====50 : MMP-1
2.471
2.471 µM;
μM; =MMP-7
2.471
MMP-7
MMP-7 ==
μM; 961
====961MMP-7
961 nM;
nM; =nM;
MMP-8
MMP-8 ==
961 nM; 35
====35
35MMP-8 = 35
IC
IC5050
IC
IC 50
50
5050 IC5050: :MMP-1
:MMP-1
:MMP-1
MMP-1
MMP-1 =2.471
MMP-12.471
2.471 =μM;
2.471 2.471
μM;
=μM;
μM; μM;
μM;MMP-7
MMP-7
MMP-7
2.471MMP-7 MMP-7 961 =nM;
961nM;
961 nM;
nM; =961
MMP-8
MMP-8nM;MMP-8
MMP-8
961MMP-8 MMP-83535 ==35
35 35
nM;
nM;
nM; nM; MMP-9
MMP-9
nM;
MMP-9
MMP-9 nM;
MMP-9
=
== 777
777
777 = 777
MMP-9
nM;
=
nM;
nM; 777=nM;
777 MMP-13
nM;
MMP-13
MMP-13
MMP-13 nM;
MMP-13
=
== 9696
96 nM;
nM;=96
MMP-13
nM;
= 96 =nM;
MMP-14
nM;
MMP-14
MMP-14 96 MMP-14
nM;
==
= MMP-14
582
582
582 ==582= 582
nM;
nM;MMP-9 MMP-9
nM; MMP-9 ==777 nM;
777MMP-14
nM;
= 777MMP-13
MMP-13
nM; ==96
MMP-13 96 nM;
nM;
= MMP-14
MMP-14
96 nM; ==582
MMP-14 582 582
IC : MMP-1 =nM;
200 nM; MMP-9 = 0.43 nM = 582 nM nM
IC
ICIC
IC
IC 50
50
IC :50:::MMP-1
50
50
50 MMP-1
:MMP-1
MMP-1
MMP-1 ==
===50
IC5050: :MMP-1
IC MMP-1 200
=200
200
200 nM;
nM;
=nM;
200nM;
nM;
nM; MMP-9
MMP-9
=200
MMP-9
MMP-9
MMP-9
MMP-9
200 ===
===0.43
nM;MMP-9
MMP-9 0.43 =nM
0.43nM
0.43
0.43 nM
nM =0.43
nM 0.43nM
nM
nMnM
nM nM
nM
nM nM
Ki:
Ki: MMP-1
Ki:
Ki:MMP-1
Ki:
Ki: MMP-1
MMP-1
MMP-1 =Ki:
====2Ki:
Ki:MMP-1
MMP-1
MMP-1
MMP-1
2222μM;
μM;
μM;
μM;
μM;
= 2=μM;
MMP-2
==MMP-2
2MMP-2
2μM;
MMP-2
MMP-2 2====µM;
μM;MMP-2 10
=10
MMP-210 MMP-2
MMP-2
nM;
10nM;
10 nM;
nM;
nM;
= 10
MMP-3
==10 nM; nM;
= 10
nM;MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
10 MMP-3
nM;
=====500MMP-3 = 500
500
500 ==500
500
500 500
MMP-3
nM = nM
500 nM
nM
nMnM nM
nM nM
ICIC37.3 : MMP-1
:50MMP-1 = 37.3
= μM;
37.3 μM; µM; MMP-2
MMP-2 = 664
=nM;
664 nM;
IC
IC
IC
IC 50
50
50
IC :50:::MMP-1
MMP-1
IC
:MMP-1
MMP-1
MMP-1 ====50
IC5050: :MMP-1=37.3
37.3 =μM;
37.3μM;
37.3 μM;=37.3
μM;
μM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 =====664
μM;MMP-2 664
664
664
664nM;
=nM;
nM;
nM;
nM;=664 MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 ===== MMP-9
nM;MMP-9 == = IC 50: MMP-1 = 8.78 μM; MMP-2 = 355 nM; MMP-9 = 1.67 μM; MMP-13 = Ki:
Ki:
Ki: (S
(S
(S
Ki:(S
Ki:
Ki: Ki: Ki: (S enantiomer)
enantiomer) MMP-3
Ki:(S(Senantiomer)
enantiomer)
enantiomer)
(Senantiomer)
(S enantiomer) MMP-3
MMP-3
MMP-3
MMP-3 ======19
MMP-3 19
19
MMP-3
nM
19nM
19 nM
nM ==19 = 19 nM
19nM
50 MMP-1
5.5 μM;
37.3
nM; MMP-9
MMP-13
MMP-2
= = 5.5
2.277 µM;
μM;
664
MMP-14
MMP-9
= IC
24 IC
IC
IC 50
IC
50
μM :50:::MMP-1
50
50
50 MMP-1
MMP-1
MMP-1 ===
===8.78
IC5050: :MMP-1
IC
:MMP-1 MMP-1 8.78
8.78 =μM;
8.78μM;
8.78 μM;
µM;
μM;
μM; MMP-2
=8.78 μM;
MMP-2
MMP-2
MMP-2
8.78 ===
===355
μM;MMP-2
MMP-2 MMP-2 355
355
355
355 nM;
=nM;
nM;
355nM;
nM;
nM;=355 MMP-9
nM;
MMP-9
MMP-9
355MMP-9 ===
nM;MMP-9
MMP-9 ===1.67
MMP-9 1.67
1.67
1.67 =μM;
1.67μM;
μM;
µM;
μM;
μM; MMP-13
=1.67 μM;
MMP-13
MMP-13
MMP-13
MMP-13
1.67 ===
===
μM;MMP-13
MMP-13 == enantiomer)
(R
MMP-3
enantiomer)
enantiomer)
MMP-3
MMP-3
nM
=
nM
36 nM
5.5
5.5
5.5μM;
5.5
5.5 μM;
μM;
μM;5.5
μM;5.5 MMP-13
MMP-13μM;
MMP-13
MMP-13
MMP-13 =
MMP-13 2.277
====2.277
2.277
2.277
2.277= μM;
2.277
μM;
μM;
μM;
μM; MMP-14
μM;
MMP-14
MMP-14
MMP-14
MMP-14
MMP-13 = 2.277 µM; MMP-14 = 24 µM
μM; MMP-13 = 2.277 μM; =
MMP-14
MMP-14 24
====24
24
24
24 μM
μM
μM
μM=
μM=2424 μM
μM 230 230 nM;== MMP-14 = 4.71 μM (R
(R
(R
(R(R
(R enantiomer)
(R
enantiomer)
enantiomer)
enantiomer)
enantiomer) MMP-3
(Renantiomer)
enantiomer) MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
enantiomer) =
=====36
MMP-3
MMP-3 3636
36 nM
nM
36nM
nM
nM==36
36nM
nM
230nM;
230
230 nM;
nM;
nM;MMP-14
230MMP-14
nM;
MMP-14
MMP-14
230 MMP-144.71
====4.71
nM;MMP-14 4.71 µM
=μM
4.71μM
4.71 μM
μM =4.71
μM 4.71μM μM
IC :50:::MMP-1
MMP-1
IC IC
====50
50: MMP-1
: MMP-1
14 μM; = 14 MMP-2
MMP-2
==µM;
14 μM; μM;
=====529MMP-2
529 nM;
=nM; =nM;
529 MMP-3
MMP-3 nM; MMP-3
=====11111nM;
nM; = 1 MMP-9
1MMP-9 nM; MMP-9
=====2.42
2.42 = 2.42
IC : MMP-1 =MMP-9
2.268 μM; MMP-9 =nM;
152 nM; MMP-13 = 18 nM
IC
IC
IC 50
50
50
ICIC
50 :MMP-1
MMP-1
: IC
MMP-1
50 MMP-1 =14
50: MMP-1
14
14 =μM;
14μM;
μM;
14
μM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
14 μM; MMP-2529= nM;
529
529nM;
nM;
529 =529MMP-3
MMP-3
MMP-3
nM;
529 MMP-3
MMP-3
nM; MMP-3 nM;
=
nM;1=nM;
nM; 1nM;
=MMP-9
MMP-9
MMP-9
MMP-9
MMP-9
nM; MMP-9 = ==2.42
2.42
2.42
2.42 2.42
IC
IC
IC
IC 50
50
50
IC :50:::MMP-1
MMP-1
IC
:MMP-1
MMP-1
IC ====50
50: MMP-1
5050: : MMP-1
MMP-1
IC MMP-1 2.268
=2.268
2.268
2.268
2.268=μM;
=μM;
2.268
μM;
=μM;
μM; MMP-9
μM;
MMP-9
2.268 µM;
MMP-9 =====152
μM;MMP-9
MMP-9 MMP-9
MMP-9 152
152 nM;
=nM;
152nM;
152 =
nM;
nM; MMP-13
=152
MMP-13
MMP-13
MMP-13
152 =====18
nM;MMP-13
MMP-13 MMP-13 18
18
18 nM
18nM
nM
nM==
nM=18
18nM
18 nM
nM μM; MMP-14
μM; μM;
MMP-14 MMP-14
= 20.1
=====20.1
20.1 =μM = 20.1 μM
50 μM;
μM;
2.42
μM;
μM; MMP-14
MMP-14
µM;
MMP-14MMP-14
MMP-14
μM; MMP-1420.1
20.1μM
20.1μM
μM=20.1
μM µMμM
20.1 μM
Figure 18. Inhibitor with phosphinamide group.
Biomolecules 2020, 10, 717 Figure 18. Inhibitor with phosphinamide group. 31 of 61
[18]. On
Allthe other hand, the hydroxamate
hydroxamate-based inhibitors areacids
veryhave poor
potent andoral
theybioavailability,
inhibit MMPs at inhibit multiple MMPs,
low concentrations [18].
[18]. On the other hand, the hydroxamate acids have poor oral bioavailability, inhibit multiple MMPs,
and
On cause
the sidehand,
other effectsthe
[2,17,27,28,35].
hydroxamate Additionally,
acids have this oral
poor functional group may
bioavailability, be metabolized
inhibit multiple via
MMPs,
and cause side effects [2,17,27,28,35]. Additionally, this functional group may be metabolized via
dehydroxylation or may
and cause side effects be cleaved by
[2,17,27,28,35]. endopeptidases and releasing
group hydroxylamine, can be
dehydroxylation or may be cleaved Additionally, this functional
by endopeptidases and releasing may be metabolized
hydroxylamine, can via
be
hydrolyzed to
dehydroxylation carboxylic
or may be acids
cleaved orbyreduced to O-glucuronyl
endopeptidases and or
releasing O-sulfate, leading
hydroxylamine, can to
be decreased
hydrolyzed
hydrolyzed to carboxylic acids or reduced to O-glucuronyl or O-sulfate, leading to decreased
effective inhibitor
to carboxylic acidsconcentration
or reduced to and reducing itsor
O-glucuronyl potency in vivo
O-sulfate, [18,27,28,35].
leading to decreased effective inhibitor
effective inhibitor concentration and reducing its potency in vivo [18,27,28,35].
Table 8
concentration shows the IC
and reducing 50 and Ki values
its potency of some
in vivo phosphamides
[18,27,28,35]. hydroxamic acid-based inhibitor
Table 8 shows the IC50 and Ki values of some phosphamides hydroxamic acid-based inhibitor
[6,15–19,29,35,37,54,55].
Table 8 shows the IC50 and Ki values of some phosphamides hydroxamic acid-based
[6,15–19,29,35,37,54,55].
inhibitor [6,15–19,29,35,37,54,55].
Table 8. IC50 and Ki values of phosphamides hydroxamic acid-based inhibitors.
IC50 (X = H; Y = (CH2)2C6H5; Z = Me; R = Ph): MMP-1 = 252 nM;
IC50 (X = H; Y = (CH2)2C6H5; Z = Me; R = Ph): MMP-1 = 252 nM;
MMP-3 = 700 nM
50 (X = =H;
ICMMP-3 Y=
700 nM(CH2 )2 C6 H5 ; Z = Me; R = Ph): MMP-1 = 252 nM;
IC50 (X ==H;
MMP-3 700YYnM = (CH2)2C6H5; Z = Ph; R = Ph): MMP-1 = 854 nM;
IC50 (X = H; = (CH2)2C6H5; Z = Ph; R = Ph): MMP-1 = 854 nM;
MMP-3
IC = 1.75
50 (X = =H; Y= μM(CH ) C H ; Z = Ph; R = Ph): MMP-1 = 854 nM;
MMP-3 1.75 μM 2 2 6 5
IC50 (X ==Me;
MMP-3 1.75YµM = CH2C6H5; Z = Me; R = Ph): MMP-1 = 120 nM;
IC50 (X = Me; Y = CH2C6H5; Z = Me; R = Ph): MMP-1 = 120 nM;
50 (X ==Me;
MMP-3
IC 67.9YnM = CH2 C6 H5 ; Z = Me; R = Ph): MMP-1 = 120 nM;
MMP-3 =67.9
67.9nM nM
IC50 (X ==Me;
MMP-3 Y = CH2C6H5; Z = Et; R = Ph): MMP-1 = 608 nM;
IC50(X (X== Me;
Me; Y == CH CH2C 6H5; Z = Et; R = Ph): MMP-1 = 608 nM;
IC
MMP-3
50 = 700 YnM 2 C6 H5 ; Z = Et; R = Ph): MMP-1 = 608 nM;
MMP-3==700
MMP-3 700nM nM
IC50 (X = Me; Y = CH2C6H5; Z = Ph; R = Ph): MMP-1 = 6.79 μM;
ICIC5050(X(X== Me;
Me; YY == CH2C C6HH5; ;ZZ==Ph; Ph;RR==Ph): Ph):MMP-1
MMP-1 = 6.79
= 6.79 μM;
µM;
MMP-3 = 10.3 μM 2 6 5
MMP-3==10.3
MMP-3 10.3µM μM
IC50 (X = CH2i-Pr; Y = CH2C6H5; Z = Me; R = Ph): MMP-1 = 20.5
ICIC5050(X(X== CH
CH22i-Pr;
i-Pr; YY= = CHCH22CC66HH5;5 Z ; Z==Me;Me;RR= = Ph):
Ph): MMP-1 = 20.5
nM;
MMP-1 MMP-3
= 20.5 = nM;
24.4 MMP-3
nM = 24.4 nM
nM; MMP-3 = 24.4 nM
(X = CH22i-Pr;
IC5050 (X Y ==CH CH22C C66H 5; Z ==Me; RR = Me): MMP-1 = 518
ICIC50 (X== CH CH2i-Pr;
i-Pr; YY = CH 2C 6HH ;Z Me; = Me):
5;5 Z = Me; R = Me): MMP-1 = 518
MMP-1 = 518 nM; MMP-3 = 1.04 µM
nM; MMP-3 = 1.04 μM
nM; MMP-3 = 1.04 μM
IC50 (X = CH2i-Pr; Y = H; Z = CH3): R isómer, MMP-1 = 2.51 μM;
ICIC5050(X(X== CH
CH22i-Pr;
i-Pr; YY= = H;H; ZZ ==CH CH33):):RRisómer,
isómer,MMP-1 MMP-1==2.512.51μM;
µM;
MMP-3== 2.55
MMP-3 2.55 µM μM
MMP-3 = 2.55 μM
SSisomer:
isomer:MMP-1MMP-1>> 100 100 µM;
μM; MMP-3 MMP-3 = = 130.5
130.5 μM µM
S isomer: MMP-1 > 100 μM; MMP-3 = 130.5 μM
IC
IC50 50 (X == CH 2 i-Pr; Y = CH2 C6 H5 ; Z = CH
2 i-Pr; Y = CH 2 C 6 H 5 ; Z = CH 3): 3R
): isomer, MMP-1 =
R isomer,
IC50 (X = CH2i-Pr; Y = CH2C6H5; Z = CH3): R isomer, MMP-1 =
MMP-1
20.5 nM; = MMP-3
20.5 nM;= MMP-324.4 nM= 24.4 nM
SS20.5
isomer,nM; MMP-3==7.12
isomer,MMP-1MMP-1 = 7.12µM;
24.4 nM MMP-3 = 9.17 µM
μM; MMP-3 = 9.17 μM
ICS isomer,
(X = CHMMP-1 ; Y = =
CH 7.12CμM; ;MMP-3
= 2C = 9.17 μM
IC50 (X = CH3; Y = CH2C6H
50 3 2 6H 5; 5Z Z=C H25H
): 5R):isomer,
R isomer,MMP-1 = 608
Biomolecules 2020, 10, 717 IC
MMP-1 50
nM; MMP-3
(X = 608=nM;
= CH 3 ; Y = CH
700 MMP-3
nM
2C 6 H = 700 nM
5; Z = C 2H 5 ): R isomer, MMP-1 = 608 32 of 64
SnM;
isomer,MMP-3MMP-1 = 700 = nM
33.3 µM; MMP-3 = 49.3 µM
S isomer, MMP-1 = 33.3 μM; MMP-3 = 49.3 μM
S isomer,
IC 50 (X = H; MMP-1
Y = (CH = 233.3
)2C6H μM; MMP-3==525
5): MMP-1 49.3nM; μMMMP-3 = 700 nM
IC 50 (X = H; Y = (CH2 )2 C6 H5 ): MMP-1 = 525 nM; MMP-3 = 700 nM
IC (X == CH
IC5050 (X CH33;; Y Y == CH
CH22CC66HH5):5 ):MMP-1
MMP-1= =120 120nM; nM;MMP-3
MMP-3==67.967.9nM
nM
IC (X =
IC5050 (X = CH33;; YY ==n-Cn-C6H 6H ): ):MMP-1
1313 MMP-1 = 1.29
= 1.29 μM;µM;MMP-3
MMP-3 = 1.6
= 1.6 μM µM
IC (X =
IC5050 (X i-Pr; Y
= CH22i-Pr; Y ==H): MMP-1==2.51
H):MMP-1 2.51μM; MMP-3==2.55
µM;MMP-3 2.55μMµM
IC (X =
IC5050 (X = CH22i-Pr;
i-Pr; YY ==CH CH2C 2C 6H6H5):5 ): MMP-1
MMP-1 = 20.5
= 20.5 nM;
nM; MMP-2 =
MMP-2
13.3 nM; = MMP-3
13.3 nM;= MMP-324.4 nM;=MMP-7 24.4 nM;= MMP-7886 nM;=MMP-8 886 nM;= 5.3 nM;
MMP-8 = 5.3 nM; MMP-9
MMP-9 = 20.6 nM; MMP-13 = 7.4 nM = 20.6 nM; MMP-13 = 7.4 nM
4.2.Non-Hydroxamate-Based
4.2. Non-Hydroxamate-BasedInhibitors
Inhibitors
Theside
The sideeffects
effects caused
caused by by hydroxamate-based
hydroxamate-based inhibitors,
inhibitors, due todue
the to theof lack
lack of selectivity
selectivity and in
and in vivo lability, have been fostering the development of new compounds
vivo lability, have been fostering the development of new compounds with alternative ZBGs with alternative
ZBGs [5,6,11,16,17,27–29].
[5,6,11,16,17,27–29]. The secondThe generation
second generation
of MMPis of (1999–2003
MMPis (1999–2003
[16]) was[16]) was designed
designed with
with a wide
a wideof variety
variety of peptidomimetic
peptidomimetic and non-peptidomimetic
and non-peptidomimetic structures with structures with higher
higher selectivity selectivity
and exploiting
anddeep
the exploiting thepresent
S1’ pocket deep S1in’ pocket
some MMPspresent in some MMPs These
[6,15,16,18,59–61]. [6,15,16,18,59–61].
compounds includeThese compounds
carboxylic
include
acids, carboxylic acids, sulfonylhydrazides,
sulfonylhydrazides, thiols, aminomethyl thiols, aminomethyl
benzimidazole, benzimidazole, phosphorous-based,
phosphorous-based, nitrogen-based
nitrogen-based
and heterocycles and heterocycles
bidentate bidentate
chelators, and canchelators, and canbidentade,
be monodentade, be monodentade, bidentade,
and tridentade and
chelates
tridentade chelates [2,6,18,28,29].
[2,6,18,28,29].
Thenon-hydroxamate-based
The non-hydroxamate-based inhibitors
inhibitors open
openup upa awide
wide spectrum
spectrum of of
affinities for for
affinities the the
zinczinc
ion from
ion
the catalytic
from site and
the catalytic sitenew
andopportunities for targeting
new opportunities and inhibiting
for targeting the activethe
and inhibiting center [18,28].
active centerThey have
[18,28].
They Zn2+weak
weakhave chelating ability and
Zn2+ chelating the rates
ability and of
thesevere side
rates of effects,
severe side such as the
effects, musculoskeletal
such syndrome
as the musculoskeletal
(MSS) decreased
syndrome dramatically
(MSS) decreased comparedcompared
dramatically with the hydroxamate inhibitors inhibitors
with the hydroxamate [28]. [28].
4.2.1. Thiolates-Based Inhibitors

The ability of the monodentate binding of thiols to zinc ion in proenzymes has served as
inspiration for the design of several MMPis [5,29]. The potency of thiol inhibitors is intermediate
between that of hydroxamate- and carboxylate- based inhibitors [29]. The first example of inhibitor
[2,6,18,28,29].
The non-hydroxamate-based inhibitors open up a wide spectrum of affinities for the zinc ion
from the catalytic site and new opportunities for targeting and inhibiting the active center [18,28].
They have weak Zn2+ chelating ability and the rates of severe side effects, such as the musculoskeletal
syndrome (MSS) decreased dramatically compared with the hydroxamate inhibitors [28].

The ability of the monodentate binding of thiols to zinc ion in proenzymes has served as
inspiration for of
The ability thethedesign of several
monodentate MMPis
binding [5,29].toThe
of thiols zinc potency of thiol inhibitors
ion in proenzymes has served isas
intermediate
inspiration
between
for that of
the design of hydroxamate-
several MMPisand carboxylate-
[5,29]. The potencybased of inhibitors [29]. The
thiol inhibitors first example
is intermediate of inhibitor
between that
thiol-based
of hydroxamate- for MMP-1 is a bipeptidic
and carboxylate- basedanalogue,
inhibitorswhere the first
[29]. The incorporation
example ofofinhibitor
a thiol thiol-based
group as α
substituent
for MMP-1 isleads to improvement
a bipeptidic analogue,ofwhere
activity
the(Figure 19a) [19].
incorporation of aDerivates
thiol groupwith as “linker” substituent
α substituent leads
between
to P1-P1’ positions
improvement of activityshow
(Figurea 19a)
total [19].
loss Derivates
of activitywith(Figure 19b)substituent
“linker” [19,62]. Onbetween
the contrary
P1 -P1 ’
incorporation
positions showof a methyl
a total carboxylate
loss of group19b)
activity (Figure leads to a significant
[19,62]. increase
On the contrary in activity (Figure
incorporation 19c)
of a methyl
[19]. The increased
carboxylate group leadsactivity of these compounds
to a significant may be(Figure
increase in activity a consequence
19c) [19]. of
The beneficial
increasedinteractions
activity of
between
these S1, the carbonyl
compounds may be ester, and the thiol
a consequence group, participating
of beneficial interactions in the bidentate
between coordination
S1 , the carbonyl ester,ofand
the
zincthiol
the [19].group, participating in the bidentate coordination of the zinc [19].
Figure 19. (a) Thiol-based inhibitor with the thiol group as α-substituent. The stoichiometric is S when
Figure 19. (a) Thiol-based inhibitor with the thiol group as α-substituent. The stoichiometric is S when
the thiol group is present. In its absence, the compound with R stoichiometric is more active than the
the thiol group is present. In its absence, the compound with R stoichiometric is more active than the
S analogue; (b) thiol-based inhibitor with “linker” substituent; (c) thiol-based inhibitor with methyl
S analogue; (b) thiol-based inhibitor with “linker” substituent; (c) thiol-based inhibitor with methyl
carboxylate group.
carboxylate group.
Montana et al. have identified a series of inhibitors with mercaptoacyl, obtaining moderate
Montana et al. have identified a series of inhibitors with mercaptoacyl, obtaining moderate
inhibitors
Biomolecules (Figure 20a) against a wide variety of enzymes with a deep pocket shown to be orally 33
2020, 10, 717 active
of 64
inhibitors (Figure 20a) against a wide variety of enzymes with a deep pocket shown to be orally active
in mouse models with arthritis [63]. The thiol and acyl carbonyl groups could cooperate in binding to
in the
to mouse modelsthe with arthritis [63]. The thiol and et al.acyl carbonyl groups couldofcooperate in binding
the zinczinc of active
of the active site Warshasky
site [63]. [63]. Warshaskyet al. have have
producedproduced
a varietya of
variety
compounds compounds in the
in the Montana
Montana
series, series,the
in which in which
amidethe amidePnitrogen
nitrogen P2’to
2 ’ is linked is the
linked to the
group P1 ’ group
(FigureP120b)
’ (Figure
[19]. 20b) [19].
Figure 20. (a) Inhibitor with mercaptoacyl, where the thiol and acyl carbonyl groups could cooperate
in binding to the zinc ion. (b) Variant of Montana compounds. n = 0, the compound does not have
activity against MMPs-2, 3, and -12. n = 1, the compound has low activity against MMP-3.
The β-mercaptoacilamide represented in Figure 21 is active against the MMP-9 in vitro and
exhibits oral activity in rats [19]. The 4-alcoxy substituent of cyclohexane group improved the activity
against all MMPs [19]. Replacement of the 4-ethoxy substituent with 4-propyloxy leads to a
significant reduction in MMP-1 activity and improves selectivity for MMP-3 [19]. The equivalent
cyclopentyl compounds are inactive [19]. The mercaptoamide is unstable in solution hence, to
overcome this issue, Campbell and Levin have prepared a series of mercaptoalcohols and
mercaptoketones inhibitors [64]. The mercaptoalcohols have exhibited modest activity against MMP-
1, -3, and -9, while the equivalent mercaptoketones could be optimized to active broad-spectrum
The β-mercaptoacilamide represented in Figure 21 is active against the MMP-9 in vitro and
exhibits
The oral activity in rats [19].represented
β-mercaptoacilamide The 4-alcoxyinsubstituent
Figure 21ofiscyclohexane
active againstgroup
the improved
MMP-9 inthe activity
vitro and
against all MMPs [19]. Replacement of the 4-ethoxy substituent with 4-propyloxy
exhibits oral activity in rats [19]. The 4-alcoxy substituent of cyclohexane group improved the activityleads to a
significant
against reduction
all MMPs [19]. in MMP-1 activity
Replacement of the and improves
4-ethoxy selectivity
substituent for MMP-3 [19].
with 4-propyloxy leads The equivalent
to a significant
cyclopentyl compounds are inactive [19]. The mercaptoamide is unstable in
reduction in MMP-1 activity and improves selectivity for MMP-3 [19]. The equivalent cyclopentylsolution hence, to
overcome this
compounds issue, Campbell
are inactive and Levin have
[19]. The mercaptoamide prepared
is unstable a series
in solution of mercaptoalcohols
hence, and
to overcome this issue,
mercaptoketones inhibitors [64]. The mercaptoalcohols have exhibited modest activity
Campbell and Levin have prepared a series of mercaptoalcohols and mercaptoketones inhibitors [64]. against MMP-
1, -3,
The and -9, while the
mercaptoalcohols equivalent
have exhibitedmercaptoketones could be
modest activity against optimized
MMP-1, to-9,
-3, and active
whilebroad-spectrum
the equivalent
inhibitors [64].
mercaptoketones could be optimized to active broad-spectrum inhibitors [64].
Figure
Figure21. β-mercaptoacilamide inhibitor.
21. β-mercaptoacilamide inhibitor.
In
In2005,
2005,Hurst
Hurstetetal.al.[65] reported
[65] reporteda series of mercaptosulphides
a series of mercaptosulphidesinhibitors that that
inhibitors targeted MMP-1
targeted [65].
MMP-1
The
[65].structure–activity relationship
The structure–activity indicates
relationship that the
indicates five-membered
that ring increases
the five-membered the stability
ring increases of the
the stability
inhibitor compared to the linear structure, which can be quickly oxidized and lose its
of the inhibitor compared to the linear structure, which can be quickly oxidized and lose its potencypotency [65].
[65].Table 9 shows the IC50 and Ki values of some thiolates-based inhibitors [6,15–19,29,35,37,54,55].
4.2.2. Table 9 shows the IC50
Carboxylates-Based and Ki values of some thiolates-based inhibitors [6,15–19,29,35,37,54,55].
Inhibitors
The carboxylic inhibitors are synthetic precursors of the more popular hydroxamates yet they
are weaker zinc (II) ligands than hydroxamates [17,27] and monodentate chelate [27]. Carboxylic
acid is present in several MMPis that contain large lipophilic groups, such as biphenyls, since they
fit in the S1 ’ pocket [5,6]. These ZBGs are particularly appreciated for their high stability in vivo
and their great positive effects on solubility, bioavailability, and selective properties [5,17]. The
hydroxamate-based inhibitors are more potent in physiological conditions than carboxylate inhibitors,
due to differences in acidity constants [29]. The carboxylate inhibitors bind more tightly to MMPs at
low pH, while hydroxamate-based ones have a wider range of pH from 5 to 8 [29]. Fray et al. [66]
compared the inhibition profiles of hydroxamates and carboxylic inhibitors (Figure 21a) and observed
that the substitution of a carboxylate by a hydroxamate causes a 10-fold increase in potency of the
inhibitor towards MMP-3 but decreases the selectivity against MMP-1, -2, -9, and -14 [66]. This effect
is attributed to the fact that the strong zinc (II) affinity to the hydroxamic acid group is the main
determinant of the binding energy, while in carboxylates this energy relies to a bigger extent on specific
interactions with the specific pockets [66].
Hagmann et al. [67] described a series with N-carboxyalkyl group substituents, which presented
inhibition for MMP-1, -2, and -3 [67] (Figure 21b). However, the substitution of the phenethyl group,
in P1 ’ position, for a linear alkyl chain removes the inhibitory activity for MMP-1 but it does not affect
the activity for MMP-2 and -3 [67]. A similar effect was achieved by the 4-substitution of the phenyl
ring of the phenethyl group with a small linear alkyl group [67] (Figure 22b). A similar range of P30
esters has been identified with “phthalamidobutyl” (Figure 22b), increasing activity against MMP-3
and further increasing selectivity [67].
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TableTable
9.Table9.and
IC
IC50Table
Table and
Ki
9.50IC Ki values
values of thiolates-based
ofvalues
thiolates-based inhibitors.
inhibitors.
Table IC9.50 IC
9.Table 9.
and ICand
9.50 and
50IC
Ki
Ki
Ki
and
and values
Ki
Ki
50 values
50
ofofthiolates-based
values
values
of thiolates-based
of
of inhibitors.
thiolates-based
thiolates-based
thiolates-based inhibitors.
inhibitors.
inhibitors.
inhibitors.
IC
IC50
IC50 (R =50IC(R
(R
H): =
=(R H):H):
MMP-1 MMP-1
=MMP-1
360 ==nM360
360 nMnM
nM
IC50 (R =IC IC
50 50 =
H): (R
(R == H):
MMP-1 H): MMP-1
MMP-1 = 360
= 360 nM == 360
360 nM
nM IC50: MMP-1
IC50IC
: MMP-1 = 2.5 nM D2163D2163 D2163
D2163
: =MMP-1= 2.5=nM
50
= 2.5 nM = 2.5
2.5 nM
nMnM IC50: MMP-3: MMP-3
IC50IC 260 =nM;
=MMP-3 260 nM;
=
MMP-8 MMP-8
=MMP-8 = 50= nM;
50MMP-8
nM; 50===nM; D2163 D2163
IC (R Me): IC50IC IC 50:: MMP-1
MMP-1 == 2.5
2.5 nM : 50 = 260 nM;
50
IC=50IC
IC50 (R (R
Me): =
(R Me):
50
MMP-1
= Me):MMP-1
= 220
MMP-1 = 220
nM = nMnM
220 50
: MMP-1IC 50 IC
IC
50
IC
IC50: MMP-3 50::: MMP-3
MMP-3 == 260
= 260 nM; 260 nM;
nM;
MMP-8 MMP-8
= 50 nM; 50nM;
50
50 nM; IC50: MMP-1 = 25
nM; nM; MMP-2 = 41= nM;
IC50 (R =IC IC
50 (R =
50 (RMMP-1
== Me):
Me): MMP-1 = 220 nM 50 IC50: MMP-1
IC : = 25
MMP-1 nM; =MMP-2
25==nM; =MMP-2
41MMP-2
nM; 41===nM;
220MMP-1 = 220 nM IC50 50:: MMP-1
MMP-1 25 nM; 41 nM;
Me): = 220 nM 50 MMP-1 25nM;
nM; MMP-2 41nM;
nM;
50
MMP-1 nM MMP-9 MMP-9
=MMP-9
90 = 90= nM
nM
MMP-9 =
90 90
nM nM IC IC
IC
50: MMP-1 50 = 25 nM; = 25
MMP-2 MMP-2
= 41 nM; 41
MMP-9
= 90 nM== 90
MMP-9MMP-9 90 nM
nM MMP-3
MMP-3MMP-3=MMP-3
157 = 157
nM; nM;
MMP-8 MMP-8
= 10 =
nM;10 nM;
MMP-3MMP-3 MMP-3 = =
= 157 nM;
157
157 nM;
nM;
== 157
157 MMP-8
MMP-8
nM; MMP-8
nM;
MMP-8 MMP-8==
= 10 nM;
10
10 nM;
==nM;
10 nM;
10 MMP-9
nM;
MMP-9 MMP-9
=MMP-9
25MMP-9= 25
nM; nM;
==MMP-13
25==nM;
25 MMP-13
nM; = 4==nM
= 4MMP-13
MMP-13
MMP-13 nM 44nMnMnM
MMP-9MMP-9 = 25 nM; 25 nM;
25 nM;
MMP-13 = 4 nM == 44 nM
MMP-13
Rebimastat
Rebimastat
Rebimastat (BMS-275231)
(BMS-275231)
(BMS-275231)
Rebimastat
Rebimastat
(BMS-275231)
(BMS-275231)
ICIC
IC50: MMP-1: MMP-1
IC50IC
50 : MMP-1
: =MMP-1
25 = =
25 nM;
25
nM;=MMP-2
25 MMP-2
nM;
nM; MMP-2
=MMP-2= 41
41MMP-2 =nM;
41
nM;=MMP-3
41 MMP-3
nM;
nM; MMP-3
=MMP-3
157 =nM;
==157
157 nM; MMP-9
157nM;
MMP-9 nM; MMP-9
=MMP-9
25MMP-9
nM; =nM;
= 25=MMP-13
25=25 MMP-13
nM; = 4 =nM
= 4MMP-13
nM
50
IC
IC50: MMP-1 : MMP-1
50: MMP-1
50 = 25 nM; = 25
= 25 nM;
nM; MMP-2
MMP-2 = 41
= 41
= 41MMP-13
nM; nM;
nM;
MMP-3 MMP-3
MMP-3 == 157
= 157 nM; 157 nM;
nM;
MMP-9 MMP-9
= 25 nM; = nM;
25 MMP-13
25 nM;
nM;
MMP-13 MMP-13 4 nMnM
= 4 nM == 44 nM
= 4 nM Ki (n Ki (n MMP-2
= 0): =(n0):= MMP-2
=MMP-2 = 1.2 nM;nM; MMP-3 = 39=MMP-12
nM; MMP-12 = 18= nM
Ki(n
Ki Ki
(n== 0):
Ki
0): (n0):
Ki MMP-2
(n
MMP-2 0):1.2
== 0): = nM;
MMP-2
MMP-2 = MMP-3
= 1.2 nM;
1.2
== 1.2
1.2 nM;
nM;
MMP-3
=MMP-3
39MMP-3
=nM; 39== nM;
39 nM;
MMP-3
= 39 nM; 39
39
= 18
MMP-12
nM;
MMP-12
nM;
MMP-12
nM
==18
MMP-12
18nM
MMP-12 18== nM
nM 18 nM
18 nM
Ki (n Ki (n
=(n1):
Ki = 1):
MMP-3
(n = MMP-3
1): =MMP-3
210 =
nM210
= nM
210 nM
Ki =
Ki (n = Ki1):
Ki MMP-3
(n
1): (n = 1): = 210
MMP-3
= 1): MMP-3
MMP-3 = 210 nMnM
= 210
= 210 nMnM
Biomolecules
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10, 717
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717
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717 Table 9. Cont. 35 of 64
Ki: MMP-1 Ki:

Ki: MMP-8
Ki:
MMP-8 ===
MMP-8 1.2
=1.2
==1.2 μM
1.2
μMμM
IC 50::50
MMP-3 Ki:
Ki:
nM Ki:
Ki: MMP-1
MMP-1 ====49
Ki: MMP-1
MMP-1
MMP-1 49
=4949
==49
49nM;
nM;
nM;
nM; MMP-2
= 49
nM; MMP-2
MMP-2 ====1.1
nM; MMP-2
MMP-2
MMP-2 1.1
=1.1
==1.1
1.1nM;
=
nM;
nM;
nM;
nM; MMP-3
nM; ====470
MMP-3
MMP-3
1.1
MMP-3
MMP-3 470
=470
==470
470 Ki:
Ki:
Ki:
Ki:
Ki:MMP-8
MMP-8
MMP-8
MMP-8
MMP-8 ==1.2 μM
1.2
1.2μM
µMμM
μM
IC
IC
IC
ICIC :50
5050
IC 50
50 :MMP-3
:MMP-3 ====45
::MMP-3
MMP-3
:MMP-3
MMP-3 =45
==45
nM;
=4545
45
45 nM;
nM;
nM;
nM;
nM; MMP-8
MMP-8
MMP-8
MMP-8
MMP-8
nM;MMP-8
MMP-8====3===
3=33nM;
nM;
3nM;
nM;
33nM;
MMP-9
MMP-9
MMP-9
MMP-9
nM;MMP-9
MMP-9====5==5== nM
55nM5nM
nM
5nM
55nM
Ki:
Ki: MMP-1
MMP-1 49 nM;
nM; MMP-2
MMP-2 1.1
1.1 nM;
nM; MMP-3
MMP-3 470
470
IC 50 MMP-3 45 nM; MMP-8 nM; MMP-9 nM;
nM nM;nM;
nM;
nM; MMP-7
MMP-7
MMP-7
MMP-3
MMP-7
MMP-7 = = =
==40 40
470=
40 nM;
40 nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-7
MMP-9 = =
=
==0.570.57
=
400.57
0.57 nM;
nM;nM;
nM;
nM; MMP-14
MMP-14
MMP-14
MMP-9
MMP-14 =
======
nM;
nM; MMP-7
MMP-7 ==4040
40 nM;
nM;
nM; MMP-9
MMP-9
MMP-9 ==0.57
0.57
0.57nM;
nM;
nM; MMP-14
MMP-14
MMP-14
= 0.57 nM; 24 24
24
24 nM
MMP-14
24 nM
nM
nM
nM = 24 nM
24
24 nM
nM
IC IC ::50 MMP-3 ==== 600 nM

IC (X ====CH):
CH): MMP-1 ====3030 nM IC ::50::MMP-3
MMP-3
MMP-3 =600
600
600 nMnM
nM
IC (X 50
IC (X ===CH):
CH): MMP-1
MMP-1 =3030
===30
30 nM
nM IC
IC MMP-3
:MMP-3 =600
==600 nM
nM
50 50 50
ICIC
ICIC5050
IC 50(X
50(X
50 (X
(X
(X CH): MMP-1
CH):
CH): MMP-1
MMP-1
MMP-1 nM
30nMnM
nM 50
IC
50
IC 50 50 MMP-3 600
600 nM
nM
IC
IC
IC IC
IC IC
50
50
50
(X
(X(X
(X
50 (X
(X=====N):
= =
CH):
N):
N):
N):
MMP-1
MMP-1
MMP-1
N): >>>100
MMP-1
MMP-1
MMP-1 > 100
>100
100
30
100μM
μM
μM
μM
nM
μM
µM
IC50
50 50
50 (X == N): N): MMP-1
MMP-1 >> 100 100 μM μM IC 50::50
IC MMP-3 ===2.5
2.5 μM
IC ::MMP-3
MMP-3
MMP-3 == =2.5
2.5
μMμM
IC 50 (X
IC
ICIC
IC
50
IC :50
5050
50 :MMP-3
50MMP-3
:MMP-3
MMP-3 ==2.5 μM
2.5
2.5μM
µMμM
μM
ICIC50::50
MMP-1
IC ====15
50 : MMP-1 15 = 15
nM; MMP-3 ====16
nM; MMP-3 16 nM;= MMP-9
MMP-9
16 nM; ====0.3
0.3 IC 50::50
MMP-1 >>> 10 μM; MMP-3 ==== 36 nM; MMP-9 =====20
20 nM
IC
IC
IC
ICIC50::50
::50
MMP-1
MMP-1
MMP-1
:MMP-1 ====890
::MMP-1
MMP-1 890
==890
890nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3 ===4.6
== 4.6
=4.6 μM;
4.6
μM;
μM; MMP-9
μM; MMP-9
MMP-9
MMP-9 ===4.5
== 4.5 4.5 IC
==4.5
4.5 IC
IC :50
5050
IC ::MMP-1
MMP-1
MMP-1
:MMP-1
:MMP-1
MMP-1 =1515
==15
15 nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
nM;MMP-3
MMP-3 =1616
==16
16 nM;
nM;
nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9 =0.3
==0.3
0.3 IC
0.3 IC
ICICIC :50
5050
50
IC ::MMP-1
MMP-1
MMP-1
:MMP-1
:MMP-1
MMP-1 >>10>1010
>>10
10 μM;
μM;
µM;
μM;
μM; MMP-3
MMP-3
MMP-3
μM;MMP-3
MMP-3 =36=36
==36
36
36nM;
36 nM;
nM;
nM;
nM; MMP-9
MMP-9
MMP-9
MMP-9
nM;MMP-9
MMP-9 =20
==20
20
20nM
nM
20nMnM
nM
nM
5050
:MMP-1 890 nM; MMP-3 4.6 μM; MMP-9 4.5 50 50
IC IC
IC 5050 MMP-1
= =890890 nM;
nM;
890 nM; MMP-3
MMP-3
MMP-3 =4.6
= 4.6
4.6 μM;
µM; MMP-9
MMP-9
μM; MMP-9 = 4.5
4.5µM IC 50 15 nM;
MMP-9 MMP-3
= 0.3 nM 16 nM; MMP-9 0.3 IC 50 10 μM; MMP-3 36 nM; MMP-9 20 nM
50
μMμM
μM nM
nM
nM
nMnM
μM
μM
μM nM
nM
μM
Biomolecules
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10, 717717 3636of
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10, 717
717
717
717 3636
36 ofof
of
36 6464
64
of 64
Table 9. Cont.
IC IC (X,Y 50 (X,Y=== O):=MMP-1 MMP-1

O): MMP-1 ==10=10 =nM; nM;
10 MMP-2
nM; MMP-2 = 88nM; =nM; 8nM;nM;
===8==8=8=nM;
IC 50(X,Y
ICIC
IC IC
IC
IC 50
50
5050
50 (X,Y (X,
(X,Y
50
50
50
(X,Y
(X,Y
(X,Y ===O):
Y =O):
=O):O):
O):
O):
O): MMP-1
MMP-1
MMP-1
MMP-1
MMP-1
MMP-1 ===10
==10 10
10
10 nM;
10nM; nM;
nM;
nM;
nM; MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 88nM; nM;
8nM;
nM;
MMP-9
MMP-9MMP-9
MMP-9
MMP-9
MMP-9 = = = 0.1
0.1
=
==0.1 0.1
0.1 = nMnM
0.1
nM
nM nM
MMP-9
MMP-9
MMP-9 ==0.1 0.1 nMnM nM
IC
ICIC
IC IC
(X
50(X(X=50==
(X (X
==OH;==OH;OH;
=OH; = OH;
OH; Y YY=
YY===H):
=Y H):YH):
==H):
=H):
H): MMP-1
=MMP-1
H):
MMP-1
MMP-1 MMP-1
MMP-1 = ==140
===140
140 nM;
= nM;
140
nM; MMP-3
nM;
MMP-3 MMP-3 ==
===430 430
430
==430 = 430
nM;
IC
ICIC
50
IC50
50 (X(X
(X
50
5050
50 50 (X OH; OH;
OH; Y Y H):
H): MMP-1
MMP-1
MMP-1 ==140140
140
140 nM;
nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 =430
430
430
430
nM;nM; MMP-9
nM;
nM;
nM; MMP-9
MMP-9MMP-9
MMP-9
MMP-9 = = 12
==12
= 12 nM
=
12
12 nM
nM12
nM
nM nM
nM;
nM; nM;MMP-9
MMP-9
MMP-9 = 12
= =1212nM nM nM
=(X = = 5==nM; = 9==nM; IC :MMP-1
:IC
MMP-1
:50MMP-1 ==823
823 nM;
= nM;
823
= MMP-3
nM; MMP-3 ==207
207 nM;
= nM;
207
= MMP-9
nM; MMP-9 ==2626= 26
====70 ====0.1
IC50 IC(X H; Y
==H;
H; YYO):
===O):YO):MMP-1
MMP-1 55nM; 5MMP-3
=nM; MMP-3 99nM;
=nM; 9MMP-9IC 50
50::50 ===823 nM; MMP-3 ===207 nM; MMP-9 ===26 IC :MMP-1
IC ===(X =Y
YH; =MMP-1
O): MMP-1 nM; MMP-3 nM;IC IC :::IC
MMP-1 : MMP-1==823
823 823
nM; nM;
MMP-3MMP-3==207
207 207
nM; nM;
MMP-9 ==2626
26 IC :IC :MMP-1
::MMP-1 ==7070
70 nM;
nM; MMP-13
MMP-13 ==0.1
0.1 nM
50
IC IC 50(X (X Y ==O): MMP-1 ==5==5=5nM; MMP-3 ==9==9=9nM; IC
IC 50 MMP-1MMP-1
MMP-1 823 nM;
nM; MMP-3
MMP-3
MMP-3 207 nM;
nM; MMP-9
MMP-9
MMP-9 IC 50
50::50
: MMP-1 =nM;
70
nM; nM; MMP-13
MMP-13 =nM
0.1
nMnMnM
==H;
H; =Y =O): O): MMP-1 5nM;
nM; MMP-3 99nM; nM;
50 50 50 50
IC
ICIC
IC50
50 (X(X
(X
50
5050
50 (X H; H;
H; Y O):
O): MMP-1
MMP-1
MMP-1 5 nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3 nM;
nM; IC
50 50 MMP-1 823 nM; MMP-3 207 nM; MMP-9 26 IC
ICIC
IC
IC50
50 MMP-1
50
MMP-1
:50MMP-1
MMP-1
MMP-1 ==7070
70
70 nM;
nM;
nM; MMP-13
MMP-13
MMP-13
nM;MMP-13
MMP-13 ==0.1
0.1
0.1
0.1nMnM
nM
50
MMP-9= 0.14
MMP-9 = 0.14nM
= nM
0.14 nM MMP-9 =
nM
nMnM
nM nM
26 nM
50 50
MMP-9
MMP-9
MMP-9
MMP-9
MMP-9
MMP-9 ===0.14
==0.14
=0.140.14
0.14
0.14 nM
nMnMnM
nM
nM nM
nM
nM
Ki: MMP-2
Ki: Ki:=MMP-2
MMP-21.3 µM;
==1.3
1.3MMP-7
μM;
=μM;
1.3 >MMP-7
MMP-7
μM; 2.5 µM;
>>2.5
2.5MMP-8
μM;
>μM;
2.5 μM; =MMP-8
MMP-8 2.7 µM;
==2.7
2.7MMP-9
μM;
=μM;
2.7 μM;=
IC IC :50
IC:MMP-1
MMP-1
MMP-1 = =1.5
: MMP-1 1.5 μM;
=µM;
1.5 MMP-3
MMP-3
μM; MMP-3 = ==500
500 nM;
nM;
= nM;
500 MMP-8
MMP-8
nM; =
MMP-8==4=4444nM; Ki:
= 4 Ki: MMP-2
Ki:
Ki: ==46
MMP-2
MMP-2 46=nM;
46= nM;
nM; MMP-3
46 nM; MMP-3
MMP-3 ==10
10 μM;
=μM;
10= μM;
MMP-9
10 µM;
MMP-9==100
100 Ki:
Ki:
Ki:
= 100Ki:
Ki:
Ki:MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 ===1.3
==1.3
1.3
1.3μM;
1.3
μM;μM;
μM;
μM; MMP-7
MMP-7
MMP-7
MMP-7
MMP-7
MMP-7 >>>2.5
>>2.5
2.5
2.5μM;
2.5 μM;
μM;
μM;
μM; MMP-8
MMP-8
MMP-8
MMP-8
MMP-8
MMP-8 ===2.7
==2.7
2.7
2.7μM;
2.7 μM;
μM;
μM;
μM;
IC
IC50
ICIC
IC 50
50
50
50::
:50:: MMP-1
50
MMP-1
MMP-1
MMP-1 =
== 1.5
=
= 1.5
1.5
1.5 μM;
1.5 μM;
μM;
μM;
μM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 =
== 500
=
500500
500
500
IC50: MMP-1 = 1.5 μM; MMP-3 = 500 nM; MMP-8 = 4
50 nM;
nM;
nM; MMP-8
nM; MMP-8
MMP-8
MMP-8
MMP-8 =
=
= 4 4 Ki:
Ki:
Ki:
Ki:
Ki:MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2==
= ==
46
4646
46
46nM;
nM;
nM;
nM;
nM;MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ==
= ==
10
1010
10
10μM;
μM;μM;
μM;
μM; MMP-9
MMP-9
MMP-9
MMP-9
MMP-9
MMP-9=
=
= ==
100100
100
100
100 MMP-9 6.3 µM;
==6.3
6.3MMP-13
μM; MMP-13 = 1.7 µM
==1.7
1.7 μM
===0.5 = =nM MMP-9
MMP-9MMP-9===6.3 =μM;
6.3
μM; μM;
MMP-13 MMP-13===1.7 =μM
1.7
μM μM
nM; MMP-13
MMP-13
nM; MMP-13 0.5=nMnM
0.5
nMnM
MMP-9nM; nM;100
nM;
nM; nM; MMP-14
MMP-14
MMP-14
MMP-14
MMP-14 ==210
===210
210
= nMnM
210
nM 210
nMnM MMP-9
MMP-9
MMP-9
MMP-9 ==6.3
6.3
6.3
6.3μM;μM;
μM;
μM; MMP-13
MMP-13
MMP-13
MMP-13
MMP-13 ==1.7
1.7
1.7
1.7μMμM
μM
μM
nM;
nM;nM;
nM;
nM;
nM; MMP-13
MMP-13
MMP-13
MMP-13
MMP-13
MMP-13 ==0.5
=0.5
==0.5 0.5
0.5
0.5nM
nMnM
nM
nM nM;nM;
nM;
nM; MMP-14
MMP-14
MMP-14
MMP-14 ==210210
210
210 nMnM
nM
PNU-141803
PNU-141803
PNU-141803
PNU-141803
PNU-141803
PNU-141803
PNU-141803
PNU-141803 PNU-142372
PNU-142372
PNU-142372
PNU-142372
Ki: MMP-2 = 49.5 μM; MMP-3 === 310 nM PNU-142372
PNU-142372
PNU-142372
PNU-142372
Ki:
Ki:
Ki:
Ki:
Ki:
Ki: Ki:
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2
Ki:MMP-2 =
= =
MMP-2
= =
49.5
= 49.5
49.5
49.5
49.5
MMP-2= =49.5 µM;
49.5μM;
μM;
μM;
μM; μM; MMP-3
MMP-3
MMP-3
μM; MMP-3
MMP-3
MMP-3 ===310
=310
=310= nM
310
310 310
nMnMnM
nM
nM Ki: MMP-2 = 3 =μM; MMP-3
μM; MMP-3 310 nM Ki:
Ki:
Ki:
Ki:
Ki:
Ki: Ki:
MMP-2
MMP-2
MMP-2
MMP-2
Ki:MMP-2
MMP-2=
MMP-2
= 3
=
==3==3μM;μM;
µM;
3
33μM;3 μM;
MMP-3
μM;
μM;
μM; MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 ===18
==
MMP-3 ==1818
18
=1818
18
18
nM
=nM
18
nM
nMnMnM
nM
nM
nM
IC :MMP-1
:::MMP-1 ==6565 nM; MMP-3 >>2020 μM; MMP-9 ==2.9
2.9 μM
IC :ICMMP-1 ====65
: MMP-1 65 =nM;
65
nM;nM;
MMP-3 >>>>20
MMP-3 20>μM;
20 μM; ====2.9
MMP-9
MMP-9 =μM
2.9 2.9 μM
IC 50 µM; µM
IC50::50 MMP-1 ==6565nM;
nM;MMP-3
MMP-3 >>2020μM; MMP-9
μM; MMP-9 ==2.9 μM
2.9 μM
50
ICIC
IC 50
IC50
50
50 50
50 MMP-1
MMP-1
MMP-1
MMP-1 65
65 nM;
nM;
nM; MMP-3
MMP-3
MMP-3
MMP-3 20
20 μM;
μM;
μM; MMP-9
MMP-9
MMP-9
MMP-9 2.9
2.9μMμM
μM
Biomolecules 2020,
Biomolecules 10, 717 3764of 64
Biomolecules2020,
10,717
717 Table 9. Cont. 3737ofof6464
Biomolecules2020,
2020,10,
10,717
717 3737
ofof 64
SB-3CT
SB-3CT
SB-3CT
SB-3CT
SB-3CT
SB-3CT
CP-271485 Ki: MMP-1
Ki: =
Ki: MMP-1
MMP-1206 µM;= 206 μM;
MMP-2 =MMP-2
14 nM; =MMP-3
14 nM; =MMP-3
15 µM; 15 μM; =
=MMP-7
CP-271485
CP-271485
CP-271485 MMP-1====206
Ki:MMP-1
Ki:
206 μM;
206μM; MMP-2
MMP-2====14
μM;MMP-2 1414
nM; MMP-3
MMP-3====15
nM;MMP-3
14nM;
1515
μM;
μM;
15μM;
IC CP-271485
CP-271485
50: MMP-9 = 5.1 μM; MMP-12 > 100 μM
Ki: MMP-1 206
96 μM;
µM;
MMP-7 =MMP-2
MMP-9
96 μM; = 600
MMP-9 nM;
nM =MMP-3
600 nM μM;
IC 50: MMP-1 = 15 μM ICIC
IC : MMP-9
: MMP-9 = 5.1
MMP-9====5.1
: MMP-9 5.1 μM;
µM;
5.1μM; MMP-12
MMP-12
μM;MMP-12 > 100
MMP-12>>>>100
100 μM
µM
100μM
μM MMP-7
MMP-7 = 96
= 96 μM;
μM; MMP-9
MMP-9 = 600
= 600 nM
nM
IC
ICIC
50
5050 MMP-1====
: :MMP-1
MMP-1
: MMP-1 1515μM
=15 µM
μM
50
IC
IC
5050
:50:MMP-9
5050 5.1 μM; MMP-12 100 μM MMP-7
MMP-7==96 96μM;
μM;MMP-9
MMP-9==600 600nMnM
ICIC :50:MMP-1
5050 15μMμM
Ki: MMP-2 = nM;

16 nM; MMP-3 = μM;
3.6 µM; Ki: Ki: MMP-1
MMP-1 = 5.4 = 5.4
μM; μM; MMP-2
MMP-2 = 110 = 110
nM;nM; MMP-3
MMP-3 = 12.2
= =12.2
Ki: MMP-2 = nM;
16 MMP-3 = 3.6 MMP-7 = 295 Ki:MMP-1
Ki:
MMP-1 MMP-1
= 5.4 ===5.4
5.4μM;
MMP-2μM;MMP-2
MMP-2
= ===110
110 nM; 110 nM;MMP-3
nM;
MMP-3 MMP-3
= MMP-14
12.2 =µM; 12.2
=12.2
Ki: Ki: MMP-1
MMP-1 = = μM;
11 11 μM; MMP-2
MMP-2 = = nM;
50 50 nM; MMP-14
MMP-14 = 590 Ki:
= 590 Ki:
Ki:MMP-2
MMP-2 = =1616
MMP-7
Ki:MMP-2
MMP-2 =
==MMP-9
16 nM;
295
16nM;
MMP-3
MMP-3
µM;
nM;MMP-3
= =3.6
MMP-9 3.6
==3.6
μM;
= μM;
180
μM;
MMP-7
MMP-7
nM;
μM;MMP-7
Ki:
= =295
295
==295
Ki: MMP-1
μM; µM;
MMP-7 5.4
= μM;
39 MMP-2
μM; MMP-9 110
= nM;
130 nM;MMP-3 =MMP-7
12.2680
Ki:Ki:MMP-1
Ki:
Ki:
MMP-1===11
MMP-1 = 1111
MMP-1 11μM;
μM;MMP-2
μM;
µM;
MMP-2===50
MMP-2
50nM;
= 5050
MMP-2
nM
nM;MMP-14
nM;
nM;
MMP-14===590
= 590
MMP-14
MMP-14
590
590nM μM; μM;
MMP-9 = =MMP-3
180 180
nM;nM; 3.6
MMP-14
MMP-14 =
MMP-7
= 900
900 nM nM 295 μM;
μM;
μM;
μM;
MMP-7
39 µM; ==MMP-9
MMP-7
=MMP-7
MMP-7 =
3939μM;
=39
39 μM;
μM;
μM;
MMP-9
130 nM;====MMP-14
MMP-9
=MMP-9
MMP-9
130
130
130
nM;
130nM;
nM;
MMP-14
680 nM====680
nM;MMP-14
MMP-14
=MMP-14 680
680
680
nM μM;μM; MMP-9
MMP-9 = =
MMP-14 180
180 nM;
=
nM;900MMP-14
nM
MMP-14 = = 900
900 nM
nM nM
nM
nM
nM μM; MMP-9 = 180 nM; MMP-14 = 900 nM nMnM
nM
nM
specific interactions with the specific pockets [66].
Hagmann et al. [67] described a series with N-carboxyalkyl group substituents, which presented
inhibition for MMP-1, -2, and -3 [67] (Figure 21b). However, the substitution of the phenethyl group,
in P1’ position, for a linear alkyl chain removes the inhibitory activity for MMP-1 but it does not affect
the activity for MMP-2 and -3 [67]. A similar effect was achieved by the 4-substitution of the phenyl
ring of the phenethyl group with a small linear alkyl group [67] (Figure 22b). A similar range of P3′
Biomolecules 2020, 10,has
esters 717been identified with “phthalamidobutyl” (Figure 22b), increasing activity against MMP-3 38 of 61
and further increasing selectivity [67].
(a) Fray
Figure 22. Figure 22. (a) et
Frayal.
et al.inhibitors. R = NH(OH),
inhibitors. R = NH(OH), hydroxamate-based
hydroxamate-based carboxylate- R = OH,
inhibitor. R = OH,inhibitor.
based inhibitor.
carboxylate-based (b) Hagmann
inhibitor. inhibitors. inhibitors.
(b) Hagmann = the
If X = H and YIf=XMe and Y = Me
H compound presents
the inhibition
compound to presents
MMP-1, -2, and -3. When X = C4H9 and Y = Me, the inhibitor has a similar effect to the previous one.
inhibition to MMP-1, -2, and -3. When X = C4 H9 and Y = Me, the inhibitor has a similar effect to
The inhibitor with X = H and Y = Phthbutyl (phthalamidibutyl) shows activity against MMP-3.
the previous one. The inhibitor with X = H and Y = Phthbutyl (phthalamidibutyl) shows activity
The interaction of the P1′ biphenyl substituent with pocket S1′ is an important factor contributing
against MMP-3.
to the binding of the inhibitor [19]. The X-ray structure of the acyclic compound with MMP-3 revealed
an important
The interaction the P1 0 biphenyl
of interaction between the phenyl terminal
substituent withofpocket S1 0 isgroup
the biphenyl and the sidefactor
an important chain of
contributing
histidine (His224) [19]. The carboxylic acids derived from “D-valine” have a selective inhibition for
to the binding of the inhibitor [19]. The X-ray structure of the acyclic compound with MMP-3 revealed
MMP-2 and -3 [19]. The 4-substitution of the biphenyl ring helped to increase potency compared to
an importantthe interaction betweenand
unsubstituted analogue the phenyl
also terminal
helped to improve theof pharmacokinetic
the biphenyl properties
group and [19]. the side chain of
histidine (His224 ) [19].
With the The carboxylic
aim of development acids derived
inhibitors withfrom
high “D-valine”
selectivity for have a selective
a single MMP, Wyeth inhibition for
MMP-2 andpublished,
-3 [19]. Thein 2005, a series of biphenyl
4-substitution of thecompounds
biphenylwithringcarboxylates
helped tosulphonamides (Figure
increase potency 23a).
compared to the
These compounds were tested for the treatment of osteoarthritis and indeed presented selectivity
unsubstituted analogue and also helped to improve the pharmacokinetic properties
against MMP-13 [18]. Wyeth research developed a series of carboxylic acids-based inhibitors, which
[19].
With the
wereaim of development
potent inhibitors
and selective against MMP-13,with
withhigh selectivity
the carboxylate for a single
function MMP,
connected Wyeth published, in
to a benzofuran
2005, a series of biphenyl compounds with carboxylates sulphonamides (Figure 23a). These compounds
were tested for the treatment of osteoarthritis and indeed presented selectivity against MMP-13 [18].
Wyeth research developed a series of carboxylic acids-based inhibitors, which were potent and selective
against MMP-13, with
Biomolecules 2020, the carboxylate function connected to a benzofuran via a biphenyl39sulphonamide
10, 717 of 64
spacer (Figure 23b) [16]. The presence of a bulky substituent in the benzofuran 4-position resulted in a
via a biphenyl sulphonamide spacer (Figure 23b) [16]. The presence of a bulky substituent in the
compoundbenzofuran
100-fold 4-position
more selective
resultedfor
in a MMP-13
compound over MMP-2
100-fold [16]. for MMP-13 over MMP-2 [16].
more selective
(a) Inhibitor
Figure 23. Figure of Wyeth
23. (a) Inhibitor with
of Wyeth carboxylate
with sulphonamide;
carboxylate sulphonamide; (b) inhibitor
(b) inhibitor of with
of Wyeth Wyeththe with the
carboxylatecarboxylate
functionfunction
connectedconnected to a bezofuranvia
to a bezofuran viabiphenyl
biphenyl sulphonamide
sulphonamide spacer.
spacer.
Tanomastat (Bayer) inhibits MMP-2, -3, -9, and -13 but not MMP-1 [6,29]. This inhibitor
Tanomastat (Bayer) inhibits MMP-2, -3, -9, and -13 but not MMP-1 [6,29]. This inhibitor participated
participated in clinical trials, proving tolerable and no serious MSS, but the efficacy was negative in
in clinical trials, proving
small-cell tolerable
lung cancer becauseand no serious
the median MSS,
overall butofthe
survival efficacy
patients was
treated negative
did in small-cell
not increase [6,29]. lung
cancer becauseTablethe median
10 shows overall
the ICsurvival
50 and Kiofvalues
patients
of treated did not increase
some carboxylates-based [6,29]. [6,15–
inhibitors
Table 19,29,35,37,54,55].
10 shows the IC50 and Ki values of some carboxylates-based
inhibitors [6,15–19,29,35,37,54,55].
Biomolecules 2020,
Biomolecules 10,10,
2020,
10, 717 40 40
of40
64of
of 6464
Biomolecules 10,
Biomolecules 717
10,
2020, 10,717
717 40 of 40
6440ofof
6464
Table
Table
Table
10.
10.10.
IC 50IC
IC and50
and
and
Ki Ki values
values
Ki valuesof of of carboxylates-based
carboxylates-based
carboxylates-basedinhibitors.
inhibitors.
inhibitors.
Table Table
10. IC
Table
Table10.10.
50
10.and
IC IC
IC Ki
50 50 and
and
50 50 andKiKi
values
Ki values
values
values of
ofof carboxylates-based
of carboxylates-based
carboxylates-based
carboxylates-based inhibitors.
inhibitors.
inhibitors.
inhibitors.
L-758,354 Ki:Ki:
MMP-3 = 42
== 42
MMP-3 nM
L-758,354
L-758,354
L-758,354
L-758,354 Ki:Ki:
Ki: MMP-3
Ki: MMP-3
Ki: MMP-3
MMP-3
MMP-3 == =42
nM =42
42
nM
42
nMnM
nM Ki:Ki:
MMP-3 = 21 nM
===
L-758,354
L-758,354 Ki:
Ki:
Ki: MMP-3MMP-3
MMP-3
MMP-3
= 21 nM =21 nM
21nM
nM
Ki: MMP-2 ==17 nM; MMP-3 = 10 nM Ki:
Ki:MMP-3
MMP-3 =2121 nM
Ki:
Ki: MMP-2
Ki:
Ki: MMP-2
Ki: MMP-2
MMP-2 = = 17nM;
nM;
MMP-2= =1717nM;
= 17
Ki:MMP-2 17
nM; nM;
nM; = 10 =
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 == =10
nM =10
10
nM
10
nMnM
nM
Ki:Ki:
MMP-1
Ki: MMP-1
MMP-1
= 0.9==nM;=nM;
= 0.9=0.9 0.9
nM; MMP-3
nM; = 15
MMP-3
MMP-3 =15
nM; MMP-9
15nM;
nM; = 3= nM
Tanomastat
Tanomastat
Tanomastat (BAY
(BAY 12-9566)
(BAY 12-9566)
12-9566)
Ki:
Ki: MMP-1
Ki:
Ki: MMP-1
MMP-1
MMP-1 0.9
=0.9 nM;
MMP-3
nM;
0.9 nM; MMP-3
MMP-3
MMP-3 == =15
= 15 nM; =15 nM;
15nM;
MMP-9
MMP-9 MMP-9
= 3 nM
nM;MMP-9
MMP-9 = =3=3 3nM
3nM
nM
nM
Tanomastat
Tanomastat
Tanomastat (BAY(BAY
12-9566)
(BAY
(BAY 12-9566)
12-9566)
12-9566) MMP-9 = 3 nM AG AG3067
AG 3067
3067
Ki:Ki:
MMP-1
MMP-1> 5 μM; MMP-2 = 11 nM; MMP-3 = 134 AG 3067
Ki:
Ki: MMP-1
Ki:
Ki:
Ki: MMP-1
MMP-1
MMP-1 >>> 5>5>5 µM;
> 5 μM;
MMP-1 5μM;
μM;
MMP-2
5μM;
μM;
MMP-2
MMP-2
MMP-2
MMP-2
MMP-2 == =11
= 11 =
nM; =1111
11 11nM;
nM;
MMP-3
nM;
nM;
nM;
MMP-3
MMP-3
MMP-3
MMP-3
MMP-3 == =134
= 134= =134
134
134
134 Ki:Ki:
MMP-1 > 1000 nM; MMP-2 = 16
AG
AG
nM;
3067
3067
MMP-3 = 2= nM; MMP-7 = 614 nM
Ki:MMP-1
MMP-1 >>1000
1000
> nM;
1000 MMP-2
nM; MMP-2 ==16
16
= nM;
16 nM;MMP-3
MMP-3 2 MMP-7
22nM; MMP-7
= 614==nM
=614 nM
nM; MMP-9
nM;
nM;nM;nM;MMP-9 =
= 301==nM;
MMP-9
MMP-9 301
= nM;
301
= 301
301nM;MMP-13
nM; MMP-13
MMP-13 = 1.47
=
= μM
1.47 μM
= 1.47
1.47 μM Ki: MMP-1
Ki:
Ki: > 1000
MMP-1
MMP-1 > nM;
1000MMP-2
nM;
nM; =
MMP-216
MMP-2
Ki: MMP-1 > 1000 nM; MMP-2 = nM;
= MMP-3
16 nM;
nM; MMP-3 ==
= 2 nM;
MMP-3 =2= 2nM;nM;
nM;
nM; MMP-7
MMP-7
MMP-7
MMP-7 = 614
=614614
614nMnM
nM
nM
MMP-9
nM;
nM;MMP-9
MMP-9 =301
301 nM;
MMP-13
nM;
nM; MMP-13
MMP-13
MMP-13 = 1.47
= =μM
1.47 µM
μMμM
Biomolecules
Biomolecules
Biomolecules 2020,
2020,
2020,2020,
71710,
10, 717
717
717 4141
41 of41
64 ofof
of 6464
64
Table 10. Cont.
IC50ICIC
:ICIC
MMP-2:MMP-2
5050
50
50 :50:MMP-2
MMP-2 ===34.2
= 34.2 34.2
=μM;
34.2
34.2 μM;
μM;
MMP-3
μM;
µM; MMP-3
MMP-3
MMP-3
MMP-3 =23
= 23 ===23
nM;23
23 nM;
nM;
MMP-9
nM;
nM; MMP-9
MMP-9
= ==
MMP-9
MMP-9 ==
30.4
30.4
30.4 30.4
μM; μM;
μM;
MMP-13
µM;
μM; MMP-13
MMP-13 = 2.3=
MMP-13 ==μM;
2.3
=2.3
2.32.3μM;
μM;
MMP-14
µM;
μM; MMP-14
MMP-14
MMP-14 ===66.9
= 66.9 =μM
66.9
66.9 μM
μM
µM
μM AG
AG
AG 3365
AG 3365
AG 3365
3365
3365 AG
AG 3433
AGAG3433
3433
3433
AG 3433
Ki:
Ki:
Ki:
Ki: MMP-2
Ki: MMP-2
MMP-2
MMP-2
= 0.04
MMP-2 =
==0.04
0.04
=nM;
0.04nM;
0.04nM;MMP-3
nM;
MMP-3
nM; MMP-3
MMP-3
MMP-3 =1.5
= 1.5==nM;
=1.5
1.5 nM;
1.5 MMP-7
nM;
nM;
MMP-7
nM; = 305==305
MMP-7
MMP-7 =305
305
Ki:
Ki:
Ki: MMP-2
Ki:
Ki: MMP-2
MMP-2
MMP-2
MMP-2 =0.9
= 0.9==nM;
=0.9 nM;
MMP-3
0.9nM; MMP-3 ==
=19
= 19 =nM;
nM;MMP-3
MMP-3
MMP-3 1919nM;
nM;MMP-7
MMP-7
nM; MMP-7
= 4545
MMP-7 ==4545
==4545
4545
μM;
4545μM;
μM;MMP-13
MMP-13
µM;
μM; MMP-13
MMP-13 ==
= 3.3==nM
MMP-13 3.3
3.3
3.3 nM
3.3
nMnM
nM
= 305
MMP-7nM; nM;
nM;
nM;
MMP-13
nM; MMP-13
MMP-13
MMP-13
= 0.05
MMP-13 =
==0.05
=nM 0.05
0.05
0.05nM
nM nM
nM
An-1An-1An-1 IC50:ICIC
IC
IC5050
MMP-1
50 :50
50
: MMP-1
:MMP-1MMP-1
:MMP-1 >>
>98
98
> 98 >μM; 98
98 µM;
μM;
μM;
MMP-2
μM; MMP-2
MMP-2
MMP-2
= 4.52
MMP-2 =
==4.52
=μM;4.52
4.52
4.52 µM;
μM;
μM;
MMP-3
μM; MMP-3
> 98 >>98
MMP-3
MMP-3 >98
98
Biomolecules An-1
2020,
Biomolecules 10, An-1
An-1717
An-1
717
2020, MMP-3 > 98 µM; MMP-7 > 98 µM; MMP-12 = 42 of42
6442
Biomolecules 2020, 10,10,
717 μM;
μM;μM; μM;
MMP-7 MMP-7
MMP-7
> 98 > >
μM;98
98μM;
μM;MMP-12
MMP-12MMP-12
= 520 = =520
nM;520nM;
nM;
MMP-13MMP-13
= 12 ==12
MMP-13 =12
12 of of
64 64
IC50ICIC
:ICIC
MMP-2
50
50 :MMP-2
50::50
50 = 9.3===nM;
:MMP-2
MMP-2
MMP-2 =9.3
9.3
9.3 nM;
nM; = 201==
MMP-9
MMP-9
MMP-9
nM; MMP-9 =nM
=201
201
201
201 nM
nM
nM
nM MMP-7 > 98 μM; MMP-12 = 520 nM; MMP-13
520 nM; MMP-13μM; = 12MMP-14
µM; MMP-14 =μM43.5 µM IC
IC
IC50:ICIC
MMP-1
50
50
50
::MMP-1
MMP-1
:MMP-1
:50
50 >> 98
>98
> 98 >μM;
MMP-1 9898 µM;
μM;
μM; MMP-12
MMP-12
MMP-12
μM; ==62
=62
= 62 =nM;
MMP-12
MMP-12 62
62 nM;
nM;
nM; MMP-13
MMP-13
MMP-13
nM; ==
= 970==nM
MMP-13
MMP-13 970
970
970
970 nM
nM nM
nM
μM;μM; μM;
MMP-14 MMP-14 ==43.5
= 43.5
MMP-14 43.5
=μM
43.5μMμM
:IC
IC50ICMMP-1
IC :MMP-1
50: 50
50 :MMP-1
MMP-1 >98
>98
> 98>μM;98µM;
MMP-12
μM;
μM; MMP-12
MMP-12
MMP-12 = =1.150
= 1.150= 1.150
μM;
1.150 µM;
MMP-13
μM;
μM; = = =
MMP-13
MMP-13
: MMP-1
IC50IC
ICIC
50: 50 = 3.2=
: MMP-1
MMP-1 = μM;
= 3.2
3.2
3.2 MMP-2
μM;
µM;
μM; MMP-2
MMP-2
MMP-2 ==5=5nM;
= 5 nM; MMP-3
5nM;
nM; = 12==12
MMP-3
MMP-3
MMP-3 =1212 MMP-1326.1=26.1
26.1
μM
26.1µM
μMμM
50 PD-0359601
PD-0359601
PD-0359601
PD-0359601
nM;
nM;nM;MMP-9
nM; = 8.3=
MMP-9
MMP-9
MMP-9 8.3
=μM
8.3 µM
= 8.3
μM μM : IC
IC50ICMMP-2
IC : :MMP-2 == 6.6
= 6.6= μM;
MMP-2 6.6 µM;
MMP-3
μM; MMP-3 == 3.2
= 3.2= nM;
MMP-3 nM;
MMP-8 MMP-8
= 160 = 160
nM;
= 160MMP-12
nM; ==1.7
= 1.7=nM
MMP-12 1.7 nM
nM
: 50MMP-2
5050 6.6 μM; MMP-3 3.2 nM; MMP-8 = 160 nM; MMP-12 1.7 nM
IC
IC5050 : IC
: MMP-150: MMP-1
MMP-1
IC 50: MMP-1
> >98MMP-12
> >9898μM;
μM; μM;
μM;MMP-12
MMP-12
98 = =1.150=μM;
1.150
MMP-12 =1.150
μM; μM;
μM;MMP-13
MMP-13
MMP-13
1.150 ==
MMP-13==
IC
IC
5050: IC50: MMP-1
: MMP-1
MMP-1 = 3.2
= 3.2 =
μM; 3.2
μM; μM;
MMP-2
MMP-2MMP-2
= 5
= nM;
5 =
nM; 5 nM;
MMP-3 MMP-3
MMP-3= 12
= 12 = 12 26.1
26.1 26.1
μM
μM μM
26.1 μM Table 10. Cont.
IC50: MMP-1 = 3.2 μM; MMP-2 = 5 nM; MMP-3 = 12 PD-0359601
PD-0359601
PD-0359601
PD-0359601
nM; nM;
nM;MMP-9nM;MMP-9
MMP-9 = =8.3
8.3μM=μM=8.3
8.3μM
MMP-9 μM ICIC
5050 : IC
: MMP-250: MMP-2
MMP-2
IC = =6.6
50: MMP-2
=μM;
6.6μM;=6.6 μM;
μM;MMP-3
MMP-3
MMP-3
6.6 = =3.2 =nM;
3.2nM;
MMP-3 =3.2 nM;
nM;MMP-8
MMP-8
MMP-8
3.2 = =160 =nM;
160nM;
MMP-8 =160 nM;
nM;MMP-12
MMP-12
160MMP-12= =1.7 =nM
1.7nM
MMP-12 =1.7
1.7nM
nM
IC
ICIC5050 : IC
: MMP-950: MMP-9
MMP-9 ===91
9191nM= =9191nM
nM
nM ICIC
5050 IC
(X(X
IC=IC NH):
=50 (X
(X(X==MMP-1
NH): =NH):
NH):MMP-1
MMP-1
NH): = =9090nM
MMP-1
MMP-1 nM===9090nM
90 nM
nM
50 IC 50: MMP-9 nM 50 50
Ki:
Ki:
Ki: Ki:
MMP-1
MMP-1
MMP-1 =
MMP-1
= 20
= 20 =
nM;
nM;20 nM;
MMP-3
MMP-3 =
Ki: MMP-1 = 20 nM; MMP-3 =91
MMP-3
= =91
9191 =
nM;
nM;
nM; nM;
MMP-9
MMP-9
91 ===91
nM;MMP-9
91
MMP-9 = =9191nM
91nM
nM
nM nM
ICIC (XIC
5050 IC
(X =ICCH
=50
50
(X(X
CH
(X
50
=2):MMP-1
2):
= =CH 2): MMP-1
MMP-1
CH
CH2 ):
2): = 380
=
MMP-1380
MMP-1 =nM
nM =380
= 380
380 nM
nM
nM
Biomolecules 2020, 10, 717

Biomolecules 2020, 43 of 64 43 of 64
IC50IC
IC 50 IC
: MMP-1
: 50
MMP-1
IC :50MMP-1
: 50MMP-1 >μM;
> >400
400
: MMP-1 >400
>400
μM; μM;
MMP-2
µM; MMP-2
MMP-2 =10,
MMP-2
Biomolecules
400 μM;
717
132=nM;
=2020,
132
MMP-2 =10,
=132
132
nM; nM;
MMP-3
nM; MMP-3
MMP-3
717
132 nM; = =8181=
MMP-3
MMP-3 nM;= =81
81
nM; nM;
MMP-7
nM;
81 MMP-7
MMP-7
MMP-7
nM; = =1.1
MMP-7 =μM;
1.1 =1.1
=1.1
μM; μM;
MMP-8
µM; = =4242=
MMP-8
MMP-8
MMP-8
1.1 μM; MMP-8 = =4242nM;
nM;
nM; nM; 43 of 64
MMP-9 > >µM; =nM; =μM IC
IC
5050: IC50: MMP-13
: MMP-13
MMP-13 = =6.72 ==
=6.72
6.72nM
nM6.72nM
42 nM; MMP-9
MMP-9 >
MMP-9MMP-97 μM; 7
> 7 μM; >7MMP-13
7μM;
MMP-13
μM;MMP-13
MMP-13
= =1.8
1.8
MMP-13 =nM;
1.8=1.8
nM;nM;
MMP-14 MMP-14
MMP-14
MMP-14
1.8 nM; = =5 5μM
MMP-14 =5 =5µM5μMμM IC
IC 5050: : MMP-13
MMP-13 nM
PF-00356231
PF-00356231
PF-00356231 MMP 408MMP 408
PF-00356231 IC50>: MMP-1 > 6 μM;MMP 408 351 nM; MMP-7 > 6 μM;
IC50 :IC
MMP-2 >IC
50: MMP-2 100: MMP-2
50> µM; MMP-3
100 μM; 100=μM;
>MMP-3 MMP-3
390= nM; MMP-8
390 nM; 390=nM;
=MMP-8 MMP-8
1.7=µM; MMP-9
1.7 μM; IC50: MMP-1
= 1.7 μM; 6 μM; MMP-3 = MMP-3
351 nM;=MMP-7 > 6 μM;
IC50: MMP-2 > 100 μM; MMP-3 = 390 nM; MMP-8 = 1.7 μM; IC50 : MMP-1 > 6 µM;
IC50: MMP-1 > 6MMP-3 = 351 nM;
μM; MMP-3 = 351 > 6 µM;> MMP-9
nM; MMP-7
MMP-7 6 μM; = 1.3
= 980=nM;
MMP-9 MMP-9 980=nM;
980 MMP-12
nM;=MMP-1214 nM;
=MMP-12 = 14 =
14 MMP-13 270=MMP-13
nM;
nM; MMP-13 nM MMP-9
270 nM = 270 nM MMP-9
µM;
= MMP-9
1.3 μM;
MMP-12
= 1.3
MMP-12
=
μM;
2 nM;
MMP-12
= 2 nM;
MMP-13
=
=
2 nM; =MMP-13
MMP-13
120 nM;
120 nM;=MMP-
MMP-14
120 nM; MMP-
= 1.1 µM IC50
IC50: MMP-3
IC :: MMP-3
50= 50 nM ==50
MMP-3 50nM
nM
MMP-9 = 980 nM; MMP-12 = 14 nM; MMP-13 = 270 nM = 1.3 μM; MMP-12 = 2 nM; MMP-13 = 120 nM; MMP- IC50: MMP-3 = 50 nM
14 = 1.1 μM14 = 1.1 μM
14 = 1.1 μM
Ki: MMP-1Ki:= MMP-1

127 nM;=MMP-3
127 nM;= MMP-3 = 5.819
5.819 μM; MMP- μM; MMP-
= 671 nM;=MMP-9
671 nM;= MMP-9 = 2.232
2.232 μM; μM; =MMP-12
MMP-12 2.5 = 2.5
Ki: MMP-1 = 127 nM; MMP-3 = 5.819 μM; MMP- = 671 nM; MMP-9 = 2.232 μM; MMP-12 = 2.5
Ki:> MMP-1 > 10 μM;> MMP-2 nM; MMP-13nM; =MMP-13
501 nM;=MMP-14
501 nM; =MMP-14
968 nM = 968 nM
Ki: MMP-1 10 μM; MMP-2 1.06 μM;> MMP-3
1.06 μM;= MMP-3
3.88 μM;= MMP-7
3.88 μM;= MMP-7
2.01 μM;= MMP-8
2.01 μM;= MMP-8
410 = 410 nM; MMP-13 = 501 nM; MMP-14 = 968 nM
Ki: MMP-1 > 10 μM; MMP-2 > 1.06 μM; MMP-3 = 3.88 μM; MMP-7 = 2.01 μM; MMP-8 = 410
nM; MMP-9 nM;> MMP-9 > 10 μM; =MMP-12
10 μM; MMP-12 = 1 nM; =MMP-13
1 nM; MMP-13 684 nM;=MMP-14
684 nM; =MMP-14
3.01 μM= 3.01 μM
nM; MMP-9 > 10 μM; MMP-12 = 1 nM; MMP-13 = 684 nM; MMP-14 = 3.01 μM
PF-00356231
PF-00356231 MMP 408 MMP 408
IC50: MMP-2
IC50: MMP-2 > 100
> 100 μM; μM; MMP-3
MMP-3 = 390
= 390 nM; nM; MMP-8
MMP-8 = 1.7 μM; IC50: MMP-1
= 1.7 μM; IC50: MMP-1 > 6 μM;> MMP-3
6 μM; MMP-3 = 351
= 351 nM; nM; MMP-7
MMP-7 > 6 μM;> 6 μM;
MMP-9MMP-9 = 980
= 980 nM; nM; MMP-12
MMP-12 = 14
= 14 nM; nM; MMP-13
MMP-13 = 270 nM= 270 nMMMP-9MMP-9 = 1.3
= 1.3 μM; μM; MMP-12
MMP-12 Table 10. Cont. = 120
= 2 nM; =MMP-13
2 nM; MMP-13
= 120 nM; nM; MMP-
MMP- IC50: MMP-3
IC50: MMP-3 = 50 nM
= 50 nM
14 = 1.114
μM= 1.1 μM
= 127
Ki:
Ki: MMP-1
Ki: MMP-1 MMP-1
= 127 = 127
nM;nM; = 5.819
nM;
MMP-3
MMP-3 MMP-3
= 5.819
µM;=μM;
5.819
MMP-MMP-= 671
μM; MMP-
= 671 =MMP-9
nM;nM;671 nM;
MMP-9= MMP-9
= 2.232
2.232 µM;=μM;
2.232 μM;=MMP-12
MMP-12
MMP-12 nM; =MMP-13
= 2.5
2.5 2.5 = 501
> 10 > 717 = 3.88 nM; MMP-13
nM; MMP-13 nM;nM;
= 501 501 nM;=MMP-14
=MMP-14
MMP-14 968 nMnM
= 968 = 968 nM
Ki:Ki: Ki: MMP-1
MMP-1
MMP-1 > 10 > 10
µM;
μM; μM;
MMP-2
MMP-2
Biomolecules MMP-2
>10,
1.06
Biomolecules
2020, > µM;
1.06 1.06
μM;
2020, μM;
717 MMP-3
MMP-3
MMP-3
10, = 3.88 = µM;
μM;3.88
MMP-7 ==
μM; MMP-7
MMP-7 2.01 =µM;
2.01
μM;2.01 MMP-8
MMP-8 ==
μM; MMP-8
410 = 410 44 of 64 44 of 64
410nM;
nM;MMP-9
MMP-9 > >1010μM;
nM; MMP-9 µM;
> 10 MMP-12 ==
μM; MMP-12
MMP-12 1 nM;
1 nM; 1 MMP-13
=MMP-13
nM; MMP-13= 684
= 684 =nM;
nM;684 MMP-14
nM; MMP-14
MMP-14 = 3.01
= 3.01 µM μM
= 3.01
μM
Ki (X H; Ki
(X == H; Y=
Y =(X = H;MMP-1
Me):
Me): Y = Me): MMP-1
==760
760nM; =MMP-2
760 nM; MMP-2
==200
200nM; = 200 nM; Ki: MMP-1Ki: MMP-1 >>25
25μM; MMP-2 >>2525μM; μM; >>25
MMP-3 25μM;
Ki Ki (X = H; Y =MMP-1
Me): MMP-1 nM;
= 760MMP-2
nM; MMP-2 nM;
= 200 nM; > 25 μM; MMP-2 > 25
µM; MMP-2
μM; MMP-3
Ki: MMP-1 > 25 μM; MMP-2 > 25 μM; MMP-3 > 25 μM;
> 25
µM; MMP-3 µM;
MMP-3 == 470
MMP-3
470 nM = 470 nM
nM Ki (Y = [H2]-Phthbutyl):
Ki (Y = [H2]-Phthbutyl): MMP-1 > 10 MMP-1 > 10 μM;
μM; MMP-2 = 6MMP-2
nM; = 6MMP-7nM; > MMP-7
MMP-7
25 μM; >> 25
25
MMP-8 µM;
μM; > MMP-8
MMP-8
25 μM; >
> 25
25
MMP-9 µM;
μM;> MMP-9
MMP-9
25 μM; >> 25
25
MMP-12µM;
μM; MMP-12>
MMP-12
MMP-3 = 470 nM Ki (Y
Ki (Y== [H2]-Phthbutyl):
[H2]-Phthbutyl): MMP-1
MMP-1 > 10
> 10 μM;
µM; MMP-2
MMP-2 ==66nM;
nM; MMP-7 > 25 μM; MMP-8 > 25 μM; MMP-9 > 25 μM; MMP-12
Ki(X
Ki (X== C C4H YY ==
Ki9;;(X = Me):
C 4H9MMP-1
Me): ;MMP-1 ==5.9
Y = Me): MMP-1
5.9μM; =MMP-2
5.9 μM;
µM;MMP-2 ==3.5
MMP-2 nM;= 3.5 nM;
3.5nM; MMP-3
MMP-3 = 0.36 nM = 0.36 nM 25 µM;
> 25
> 25 μM; MMP-13 μM;MMP-13
=MMP-13
4.4 nM;= 4.4 nM;
= 4.4
MMP-14 MMP-14
nM; >MMP-14
25 μM; > 25 µM;
> 25
MMP-15 μM; 25 > 25> µM;
MMP-15
>MMP-15 25
Ki4 (X9 = C4H9; Y = Me): MMP-1 = 5.9 μM; MMP-2 = 3.5 nM; MMP-3 =
MMP-3 = 0.36 nM > 25 μM; MMP-13 = µM;
4.4 nM; MMP-14
MMP-3 == 18
MMP-3 18 nM = 18 nM
MMP-3
nM KiMMP-1
Ki (Y = Me): (Y = Me): MMP-1
> 10 > 10 μM;
μM; MMP-2 MMP-2
= 310 = 310 nM;
nM; MMP-3 MMP-3μM;
= 68 = 68MMP-16
μM;MMP-16
> MMP-16
25 μM; >MMP-24
25
> 25 μM;MMP-24
>MMP-24
25 μM; 25>25
>MMP-25
>
25 μM;
µM;
μM; >
MMP-15
MMP-25
MMP-25
25 μM; > >25>25µM;
25
μM;
MMP-3 = 18nM;nMMMP-2 = 86 nM; (Y =
Ki Ki (YMe): MMP-1
= Me): MMP-1 > 10
> 10 μM;
µM; MMP-2
MMP-2 = =310310nM;
nM;MMP-3
MMP-3 = =6868nM μM; MMP-16 > 25 μM; MMP-24
(X == H; Y= ==720 MMP-26> > 2525μM;µMMMP-25 > 25 μM;
Ki (X
Ki Y
H; Ki =(XPhthbutyl):
Phthbutyl): MMP-1
= H; Y = Phthbutyl):
MMP-1 720
MMP-1 = 720 nM;
nM; MMP-2 MMP-2
= 86 nM; = 86 nM; nM nM MMP-26 >MMP-2625 μM > 25 μM
Ki (X = H; Y = Phthbutyl):
MM-3 = 8MMP-1
nM = 720 nM; MMP-2 = 86 nM; nM MMP-26 > 25 μM
MM-3 = 8 nMMM-3 = 8 nM
MM-3 = 8 nM
IC50>: 1000
IC50: MMP-1 MMP-1nM;>MMP-2
1000 nM; MMP-2
= 19 = 19 nM;
nM; MMP-3 MMP-3
> 1000 nM;>MMP-7
1000 nM; MMP-7
> 1000 nM;>MMP-9
1000 nM; MMP-9 = 32
= 32
IC50: MMP-1 > 1000 nM; MMP-2 = 19 nM; MMP-3 > 1000 nM; MMP-7 > 1000 nM; MMP-9 = 32
nM nM
nM
Ki: MMP-8Ki: MMP-8
= 205 nM; =MMP-12
205 nM;=MMP-12 = 3.3 nM;=MMP-13
3.3 nM; MMP-13 = 18 nM;=MMP-14
18 nM; MMP-14 1.054 μM= 1.054 μM
Ki: MMP-8 = 205 nM; MMP-12 = 3.3 nM; MMP-13 = 18 nM; MMP-14 = 1.054 μM
Ki (X =Ki
H;(X
Y = Me):
H; Y MMP-1 = 760 nM;
= Me): MMP-1 MMP-2
= 760 = 200 nM;
nM; MMP-2 = 200 nM; Ki: MMP-1 > 25 μM;
Ki: MMP-1 MMP-2
> 25 > 25 μM;
μM; MMP-2 MMP-3
> 25 > 25 μM;
μM; MMP-3 > 25 μM;
Biomolecules 2020, 10,MMP-3
717 MMP-3
= 470 nM
= 470 nM Ki (Y =Ki
[H2]-Phthbutyl): MMP-1MMP-1
(Y = [H2]-Phthbutyl): > 10 μM; MMP-2
> 10 = 6 nM;= 6 nM;
μM; MMP-2 43 of 61
MMP-7MMP-7
> 25 μM; MMP-8
> 25 > 25 μM;
μM; MMP-8 MMP-9
> 25 > 25 μM;
μM; MMP-9 MMP-12
> 25 μM; MMP-12
Ki (X =KiC4H
(X9;=YC=4H
Me):
9; Y MMP-1 = 5.9 μM;
= Me): MMP-1 MMP-2
= 5.9 = 3.5 nM;
μM; MMP-2 = 3.5 nM; MMP-3MMP-3
= 0.36 nM
= 0.36 nM > 25 μM; MMP-13
> 25 = 4.4 nM;
μM; MMP-13 MMP-14
= 4.4 > 25 μM;
nM; MMP-14 MMP-15
> 25 > 25 > 25
μM; MMP-15
MMP-3MMP-3
= 18 nM= 18 nM Ki (Y =Ki
Me):
(Y MMP-1 > 10 μM;
= Me): MMP-1 MMP-2
> 10 = 310 nM;
μM; MMP-2 MMP-3
= 310 = 68 = 68
nM; MMP-3 μM; MMP-16 > 25 μM;
μM; MMP-16 MMP-24
> 25 > 25 μM;
μM; MMP-24 MMP-25
> 25 > 25 μM;
μM; MMP-25 > 25 μM;
Ki (X =Ki
H;(XY = Phthbutyl): MMP-1MMP-1
H; Y = Phthbutyl): = 720 nM; MMP-2
= 720 = 86 nM;
nM; MMP-2 = 86 nM; nMTablenM10. Cont. MMP-26 > 25 μM
MMP-26 > 25 μM
MM-3 =MM-3
8 nM= 8 nM
ICIC
IC50: MMP-1
5050 > 1000>nM;
: :MMP-1
MMP-1 > 1000 nM;
nM; MMP-2
MMP-2
1000 = 19 nM;
MMP-2 == 19
MMP-3 > 1000 >
nM; MMP-3 1000
nM; nM; MMP-7
MMP-7
> 1000 nM; >>1000
1000
> 1000 nM; nM;MMP-9
MMP-9
nM; = 32 = 32
Biomolecules
Biomolecules 2020, 10,2020, MMP-9
10,
7172020, 717 = 32 nM
nM nM 45 of 6445 of4564of 64
Ki:MMP-8
Ki:
Ki: MMP-8 == 205
= 205 nM;
MMP-8 nM;
nM; MMP-12
MMP-12
205 == 3.3
= 3.3 nM;
MMP-12 nM;
nM; MMP-13
MMP-13
3.3 =
= 18 nM;
MMP-13 18
MMP-14
= 18 nM; = 1.054 ==
nM; MMP-14
MMP-14 μM1.054μM
1.054 µM
Ki: MMP-2 = 57Ki:

Ki:
Ki: MMP-2 MMP-2
=nM;
57 = 57
MMP-3
nM;
MMP-2 = 2.164
=nM;
MMP-3
57 MMP-3
nM; µM;=μM;
= 2.164
MMP-3 2.164
MMP-8 = 5.3
μM;
MMP-8
= 2.164 MMP-8
μM; =nM;
5.3 = 5.3= nM;
MMP-13
nM;
MMP-8 5.3 nM;
= 338
MMP-13MMP-13
nM
=MMP-13
338 nM= 338 nMnM
= 338
MMP-1
Ki:MMP-1
Ki: ==2.5
Ki: Ki:
MMP-1 = 2.5
2.5μM;
µM;
MMP-1 μM;
=MMP-2
MMP-2
2.5 μM;= =8.1
MMP-2 = 8.1
8.1μM;
MMP-2 µM; μM;
= MMP-3
MMP-3
8.1 μM;==
MMP-3 = µM;
13.5
13.5
MMP-3 13.5
μM; μM;μM; =
MMP-8
= 13.5
MMP-8MMP-8
= 1717 nM;
nM;= 17MMP-9
nM;
MMP-9 =
MMP-9
= 6.6
6.6 μMµM
=
MMP-8 = 17 nM; MMP-9 = 6.6 μM6.6 μM
Ki:Ki:
Ki: MMP-1 MMP-1
MMP-1
= 67
Ki: μM;
MMP-1 = 67= 67 μM;
=μM;
MMP-2
67 µM; MMP-2
MMP-2
= 192 = 192
nM;
MMP-2 == 192
MMP-3
192 nM;
nM;nM;
MMP-3
= 40 = 40==nM;
MMP-3
nM;
MMP-3 40nM;
40 nM;
MMP-7
MMP-7MMP-7
= 626 nM;
MMP-7 =
= 626 626
=MMP-8
626 nM;
nM;nM; MMP-8
MMP-8
= 271 nM;
MMP-8 ==
= 271 271
nM;nM;
MMP-9
271 nM; MMP-9
MMP-9
= 1.265
MMP-9=μM;==1.265
1.2651.265 µM;
μM;μM;
=MMP-11
MMP-11
MMP-11 =MMP-12
= 18.4
18.4 μM; 18.4 µM;
μM;μM; =MMP-12
MMP-12 =MMP-13
= 0.19
0.19 nM; 0.19 nM;
nM;nM; MMP-13
MMP-13
=MMP-13
49 nM;= 49==nM;
49 nM;
MMP-11 = 18.4 MMP-12 = 0.19 49 nM;
MMP-14
MMP-14 = 140= 140
nM nM
MMP-14 =MMP-14
140 nM = 140 nM
4.2.3. Phosphorus-Based Inhibitors

TheBiomolecules
capacity of10,
2020, the717phosphoric group to reproduce the gem-diol intermediate during peptide
46 of 64
hydrolysis was explored
Biomolecules 2020, 10,with
717 different structures to obtain potent MMPis [5]. The phosphorus-based 46 of 64
of the peptide-analogous
4.2.3. Phosphorus-Based inhibitors
Inhibitors can be phosphonates/phosphonic acids, phosphoramidates,
4.2.3.
phosphonamidates, Phosphorus-Based Inhibitors
andofphosphinates/phosphinic peptides
The capacity the phosphoric group to reproduce the[27]. The intermediate
gem-diol phosphinic during acid (PO(OH)-CH
peptide 2)
mimics the
hydrolysis The
transition wascapacity
state of
explored the
obtainedphosphoric
with different group
in substrate to reproduce
structures degradation, thepotent
to obtain gem-diol
where intermediate
MMPis each Theduring
oxygen
[5]. peptide
atom can coordinate
phosphorus-based
both the of thehydrolysis
catalytic
was explored with different structures to obtain potent MMPis [5]. The phosphorus-based
peptide-analogous
zinc and the catalytic inhibitors Glucan be phosphonates/phosphonic
[6,19,27]. The phosphinic acidsacids, phosphoramidates,
are monodentate chelates [27].
of the peptide-analogous inhibitors can be phosphonates/phosphonic acids, phosphoramidates,
phosphonamidates,
In contrast tophosphonamidates,
hydroxamate and and phosphinates/phosphinic
compounds, the phosphinic peptides [27]. The phosphinic acid (PO(OH)-CH 2)
phosphinates/phosphinic peptides compounds
[27]. The phosphinic interact with
acid (PO(OH)-CH both 2) the primed
mimics the
mimics transition state obtained in substrate degradation, where each oxygen atom can
and unprimed side the transition
of the catalyticstate site
obtained
[17,27,35]in substrate
due to degradation,
the placement where each of the oxygen
ZBG atom in the canmiddle of the
coordinate
coordinatebothboth thethe catalytic
catalyticzinczinc andand the catalyticGlu
the catalytic Glu[6,19,27].
[6,19,27]. TheThe phosphinic
phosphinic acids acids
are are
scaffold monodentate
and not
monodentate
N-chelates
at itschelates or C-terminal,
[27].
[27].InIncontrast
as intothe
contrast to
cases of hydroxamate
hydroxamate compounds,
hydroxamate compounds, the the
andphosphinic
carboxylate
phosphinic
inhibitors [17].
compounds
compounds
Anotherinteract
advantage
interact of
withwith phosphinic
both both
thethe primed
primed acids
and is the improved
andunprimed
unprimed side
sideof ofthe metabolic
thecatalytic
catalytic stability
sitesite
[17,27,35]
[17,27,35] compared
due to
duetheto with
placement
the hydroxamate
placement
acids [27]. of the ZBG in the middle of the scaffold and not at its N- or C-terminal, as
of the ZBG in the middle of the scaffold and not at its N- or C-terminal, as in the cases of hydroxamate in the cases of hydroxamate
and carboxylate inhibitors [17].Another
Another advantage
advantage ofofphosphinic acids is theis improved metabolic
Theand carboxylate
effectiveness inhibitors
of phosphoric [17]. acid inhibitors has
stability compared with hydroxamate acids [27].
phosphinic
been studied acids and theit improved
has beenmetabolic
found that the
stability compared with hydroxamate acids [27].
three pockets unprimed are connected to obtain the maximum performance
The effectiveness of phosphoric acid inhibitors has been studied and it has been found that the [19]. The S1 -S2 pockets
The
three
effectiveness
pockets
of phosphoric
unprimed are connected
acid to
inhibitors
obtain the
has been studied
maximum performance
and [19].
it has The
been
S
found that the
1-S2 pockets
can be exploited using aromatic groups [19,68], that is why Reiter et al. prepared compounds with
three can
pockets unprimed
be exploited usingare connected
aromatic groupsto obtain
[19,68], thatthe maximum
is why Reiter etperformance
al. prepared compounds[19]. The Swith 1-S2 4-pockets
4-benzylcan(Figure
bebenzyl 24)
exploited as using
(Figure
a 24)
substituent
asaromatic
a substituent
to fill
groups S2S2pocket.
to fill[19,68], that
pocket.
They
is why
They
found
found Reiter that
that inetthe
in
al.absencethe of
prepared absence
compounds
this
of this substituent
substituentwith 4-
or its replacement
benzyl or (Figureby24)small
its replacement as abyaliphatic
substituent or
small aliphatic tocyclohexyl
fill S2 pocket.methyl
or cyclohexyl They found
methyl groups
groups that led
led toin a the toabsence
loss a activity
of loss of of[19,68].
activity
this [19,68].
substituent
or its replacement by small aliphatic or cyclohexyl methyl groups led to a loss of activity [19,68].
Figure 24. Compound prepared by Reiter et al., with a 4-benzyl substituent. The 4-benzyl group fills
Figure 24. Compound prepared by Reiter et al., with a 4-benzyl substituent. The 4-benzyl group
the S2 pocket and if the benzyl group was omitted or replaced by a small aliphatic or cyclohexyl
fills the S2 pocket
methyl and
group,ifthe
theactivity
benzyl group
is lost. was omitted
The isobuthyl orthe
group fills replaced
S1’ pocket by
in a a smallsimilar
manner aliphatic or cyclohexyl
to other
methyl group,
Figure theCompound
24. activity
substrate-like isprepared
lost. The
inhibitors. byisobuthyl
Reiter et al.,group
with afills the Ssubstituent.
4-benzyl 1 ’ pocket in a 4-benzyl
The mannergroup similar
fillsto other
the S2 pocket
substrate-like and if the benzyl group was omitted or replaced by a small aliphatic or cyclohexyl
inhibitors.
methylMatziari
group, ettheal.activity
[69] synthesized
is lost. The aisobuthyl
series ofgroup
phosphinic
fills thepseudopeptides bearing similar
S1’ pocket in a manner long P1to’ side
other
chains, compounds
substrate-like that contain groups at the ortho-position of the phenyl ring and are selective for
inhibitors.
MatziariMMP-11
et al. [69]
by thesynthesized
interaction of thesea series
groups of with
phosphinic pseudopeptides
residues located at the entrance of bearing long[69].
the S1’ cavity P1 ’ side chains,
compounds thatThesecontain groups
results suggest thatatthethe ortho-position
development of
of compounds the phenyl
able to probering
theand
Matziari et al. [69] synthesized a series of phosphinic pseudopeptides bearing long P1’ side
are
entrance selective
of for
the S1’ cavity MMP-11 by
might
the interaction of represent
these an alternative
groups with strategy to located
residues gain selectivity
at the [69].
entrance of the S
chains, compounds that contain groups at the ortho-position of the phenyl ring 1 and are selective for results
’ cavity [69]. These
Other phosphorus-based ZBGs are the carbamoyl phosphates, in which the two oxygens form a
suggest that the
five development
MMP-11 by the interaction
membered ring withof of
compounds
these
the zincgroups able
ion [18].with
Thetoresidues
probe located
negative the
chargeentrance
ofatthese of the Sof
the entrance
inhibitors ’ the
cavity
1prevents might[69].
S1’ cavity
their represent
Thesepenetration
an alternative results suggest
strategy to the
into that
gain the
anddevelopment
cellselectivity them of
restrain [69]. forcompounds
extracellular able tocontributing
space, probe the entrance of the S1’ cavity
to low cytotoxicity
might
Other represent
[18]. Pochettian
phosphorus-based alternative
et al. [70] described
ZBGs strategy
are to gain
a compound
the selectivity
with
carbamoyl [69]. to MMP-8in
high affinity
phosphates, (Kiwhich
= 0.6 nM)the but two
inhibits
oxygens form
also MMP-2
Other (Ki = 5 nM) and ZBGs
phosphorus-based MMP-3are (Kithe
= 40carbamoyl
nM) (Figurephosphates,
25). The R enantiomer
in whichisthe more two potent (1000form a
oxygens
a five membered ring
time more) thanwith the zinc ion [18]. The negative charge of these inhibitors prevents their
five membered ringthewithS enantiomer
the zinc (Ki ion= [18].
0.7 μM)
The [70].
negative charge of these inhibitors prevents their
penetration into theinto
penetration cellthe
and cellrestrain themthem
and restrain for extracellular space,
for extracellular space,contributing tolow
contributing to lowcytotoxicity
cytotoxicity [18].
Pochetti[18].
et al. [70] described
Pochetti a compound
et al. [70] described with high
a compound with affinity to MMP-8
high affinity to MMP-8 (Ki==0.6
(Ki 0.6 nM) butinhibits
nM) but inhibits also
also=
MMP-2 (Ki 5 nM)(Ki
MMP-2 and= 5MMP-3
nM) and (Ki = 40(Ki
MMP-3 nM)= 40(Figure 25). 25).
nM) (Figure TheThe R enantiomer
R enantiomerisismore potent(1000
more potent (1000 time
more) than the S enantiomer (Ki = 0.7 µM) [70].
time more) than the S enantiomer (Ki = 0.7 μM) [70].
Figure 25. Pochetti’s inhibitor.
Figure 25.25.Pochetti’s
Figure inhibitor.
Pochetti’s inhibitor.
The classical approach to synthesizing phosphinic compounds limits the full exploitation of this
class of compounds for development of highly selective inhibitors of MMPS [35].
Table 11 shows the IC50 and Ki values of some phosphorus-based inhibitors [6,15–19,29,35,37,54,55].
Biomolecules 2020,
2020, 717717 48 of
4864
of64
64
Biomolecules
Biomolecules 2020,
2020, 10, 10,
7177172020, 10, 717
Biomolecules 48
48 of of
64 48 of 64
Table
Table
Table
Table
11.11. 11.
IC50ICand
50 KiIC
and values
Ki and
values
50 Kiphosphorus-based
of of values of phosphorus-based
phosphorus-based
of inhibitors.
inhibitors. inhibitors.
Table 11.11.
IC50IC 50 and
and
Table 11.Ki
Ki values
values
IC Kiphosphorus-based
of phosphorus-based
50 and inhibitors.
inhibitors.
values of phosphorus-based inhibitors.
Table 11. IC50 and Ki values of phosphorus-based inhibitors.
Cyclohexylamine
Cyclohexylamine
Cyclohexylamine saltsalt
of of(S/R)-1-(3′-methylbiphenyl-4-
of (S/R)-1-(3′-methylbiphenyl-4-
(S/R)-1-(3′-methylbiphenyl-4-
Cyclohexylamine saltsalt
Cyclohexylamineof (S/R)-1-(3′-methylbiphenyl-4-
salt of (S/R)-1-(3′-methylbiphenyl-4-
sulfonylamino)-methylpropyl
Cyclohexylamine salt of (S/R)-1-(3salt
sulfonylamino)-methylpropyl phosphonic
phosphonic acidacid
0 -methylbiphenyl-4-sulfonylamino)-methylpropyl
Cyclohexylamine ofphosphonic
phosphonic acidacid
phosphonic
(S/R)-1-(3′-methylbiphenyl-4- acid
ICIC IC(S
50 50 isomer):
(S
(S isomer):
isomer): MMP-3MMP-3 > 100
phosphonic
MMP-3 >> 100μM
100 μM
acid
μM
IC50 (S isomer): IC MMP-3
(S >
isomer): 100
sulfonylamino)-methylpropyl phosphonic
50 50 μM
MMP-3 > 100 μM acid
>>MMP-3
IC50IC(R isomer):
50 (R isomer): MMP-1
MMP-1 = IC
150
== 150
150nM;
(S nM;MMP-2
isomer):MMP-2 = 1.5 nM;
== 1.5
MMP-3 1.5 MMP-3
nM;
100 MMP-3
µM = ==
50 (R
IC50IC(R isomer):
isomer): (RMMP-1
IC50MMP-1 = 150
isomer): IC 50
MMP-1
50 (SnM;
nM; MMP-2 =MMP-2
isomer): 150 =MMP-3
1.5
nM; nM;
MMP-2 nM;100 MMP-3
1.5 =nM;
=μM MMP-3 =
52(R
IC50 nM;
52 nM;MMP-7
isomer):MMP-7 = 460
MMP-1 =nM;
nM;
==isomer):
460 150
nM; MMP-8
nM;
MMP-8 = 1.4
MMP-2 nM;
= 1.4
1.4 =nM; MMP-9
1.5
nM; nM;
MMP-9 = 8==nM;
MMP-3 = 52 nM; MMP-7 = 460 nM;
8 nM;
nM;
52 52
nM; nM; MMP-7
MMP-7 IC5250 =
(R460
nM; 460
nM;
MMP-7 MMP-8=MMP-8
MMP-1460 =nM; ==150
1.4 nM;
MMP-8
nM; MMP-9MMP-9
= 1.4 =nM;
MMP-2 8MMP-9
8=nM;
1.5 = 8 nM;=
nM; MMP-3
MMP-8 =
MMP-13 1.4
MMP-13 nM;
= 2.6MMP-9
nM; =
MMP-148 nM; MMP-13
= 79 nMnM = 2.6 nM; MMP-14 = 79 nM
52MMP-13
MMP-13 nM; MMP-7==MMP-13
= 2.6
2.6
2.6
nM; =
nM;
nM;
460
MMP-14
MMP-14
MMP-14=
nM; 2.6 = 79
nM;
MMP-8 ==MMP-14
79
79
nM=nM 1.4 =
nM; 79 nM
MMP-9
Ki: MMP-2
Ki: =
MMP-24.65
= μM;
4.65 MMP-8
μM; MMP-8 = 18.4
= μM;
18.4 μM;MMP-9
MMP-9 = 3.91
= 3.91 Ki (S
Ki isomer):
(S isomer): (S MMP-2
KiMMP-2 isomer): = 1.2
=MMP-2
μM;
1.2 μM; =MMP-8
MMP-8 1.2 µM;= 0.7= MMP-8
μM
0.7 μM = 0.7 µM8 nM;
=
Ki:MMP-2
Ki: Ki:
MMP-2==4.65
MMP-2 µM;
= 4.65
4.65
Ki: μM;
MMP-2 MMP-8
μM; 4.65==μM;
=MMP-8
MMP-8 18.4 µM;
= 18.4
18.4 μM;
MMP-8 MMP-9
μM; 18.4==μM;
=MMP-9
MMP-9 3.91
= 3.91
3.91 MMP-9 = 3.91 Ki Ki
(S (S
(Risomer):
Kiisomer): Ki (SMMP-2
MMP-2 = 1.2
isomer):
MMP-13 ==1.2
μM;
MMP-2 μM;
5= nM;
2.6 MMP-8
MMP-8
nM;= 1.2 =μM;
MMP-14 ==MMP-8
0.7 0.7
μM=μM
MMP-8 79 nM=MMP-8
0.7 μM= 0.6 nM
Ki:Ki:
MMP-2
Ki:
Ki:Ki: MMP-2
MMP-2 ==30
= 30 μM;
30 MMP-8
μM;
µM;
μM; MMP-8
MMP-8
MMP-8 ==38.9
= 38.9 μM;
38.9 MMP-9
μM;
µM;
μM; MMP-9
MMP-9
=MMP-9 ==
= 35.6 μM;
= 35.6
35.6 μM;
µM;
μM; μM; MMP-11
μM; =
MMP-11= 0.11 μM; MMP-13 ===MMP-8
4.7 μM; MMP-14 ===MMP-9 Ki Ki
(R(R isomer):
(R isomer): isomer):
MMP-2MMP-2 = MMP-2
5==nM;5 nM;
nM; MMP-3
MMP-3 MMP-3
= 0.04
== 0.04
0.04μM; μM; 0.04 µM;
MMP-8 = 0.6
= 0.6
0.6
MMP-2
MMP-11
MMP-11=
MMP-11
= Ki:
=
30
230nM;
=
μM;
MMP-2
230nM;
230nM;
MMP-8
= 30 =μM;
MMP-13
MMP-13
MMP-13 =
38.9
=
15.7
=
μM;
MMP-8
μM;
15.7
MMP-9
38.9 =μM;
MMP-14
μM;
µM; MMP-14
MMP-14 =
35.6
=
160
=
μM;
MMP-9
μM
160 μM
µM
= 35.6
µM;μM;
μM; MMP-11
μM; MMP-11
MMP-11 μM;
Ki: ==MMP-11
=0.11
0.11
0.11
µM;
0.11
MMP-2μM;
μM;
μM; MMP-13
=MMP-13
=MMP-13
MMP-13
0.11
4.65 μM;μM; 4.7==MMP-13
4.7
4.7
µM;
4.7
μM;
μM;
μM; MMP-14
=MMP-14
MMP-14
MMP-14
= 4.7
18.4 μM;μM; ==MMP-14
Ki=Ki
(R3.91 = isomer):
isomer): (RMMP-2
KiMMP-2 isomer):
Ki (S 5MMP-2
= 5isomer):
nM; MMP-3MMP-3
= 5 =nM;
MMP-2 0.04 μM;
MMP-3
1.2 μM;
μM; =MMP-8
MMP-8
MMP-8 ==0.7
0.04 =μM;
0.6 MMP-8
μM = 0.6
MMP-11
MMP-11 =Ki:
230nM;
= 230nM;
MMP-11 MMP-13
MMP-13
MMP-2 30 =μM;
= =230nM; =MMP-13
15.715.7
μM;μM;
MMP-8 =MMP-14
MMP-14
15.7 =MMP-14
= 160
38.9 μM; 160
μMμM==35.6
MMP-9 160 μM;
μM 30.1
30.1 μM
30.1
µM μM nMnM
μM; MMP-11 30.1
30.1=μM μM
0.11 μM;30.1 μM = 4.7 μM; MMP-14 =
MMP-13 Ki (R isomer): MMP-2 nMnM= 5 nM;nM MMP-3 = 0.04 μM; MMP-8 = 0.6
MMP-11 = 230nM; MMP-13 = 15.7 μM; MMP-14 = 160 μM 30.1 μM nM
= 160 = 20 = 150 ICIC

IC
50 : 50
MMP-1
50: MMP-1 > 100 μM;
>>: 100
100 μM;MMP-2
MMP-2 = 4==μM; MMP-3
4MMP-3
μM; MMP-3 > 100 μM;
> 100
100 μM; > 100 µM; MMP-9 = 20
ICICIC 50:: MMP-1
: 50
MMP-1 MMP-1 = 160 160
nM; nM; MMP-2
MMP-2 = 20 20 nM;
nM; nM; MMP-3
MMP-3 = 150 150
nM;nM; ICIC:50MMP-1 >>100 ==0.02 == 90 : MMP-1
90 IC50IC 50: MMP-1 > 100
: MMP-1 > 100 μM;
MMP-2
μM; MMP-2 = 4=μM;
>MMP-2 4 μM;4MMP-2
μM; =>4100
MMP-3
MMP-3 >μM;>µM;
100 μM;
MMP-8
μM;
==: 160 nM; MMP-2 == 20 MMP-3 == 150 nM; IC µM; MMP-1 100 µM; MMP-3 > 100 μM;
50 50 50 IC : 50
MMP-1
50: MMP-1 100 µM;
μM; MMP-2
MMP-2 0.02 µM;
μM; MMP-3
MMP-3 90
>>: 100
100 μM; MMP-2 == 0.02
0.02 μM; MMP-3 == 90
50
IC50IC : MMP-1
: MMP-1
MMP-7 ==160
IC 50
Biomolecules
1.4 nM; nM;
MMP-12020,=MMP-2
MMP-2
MMP-810, =nM;
160717=20
1.1 nM; nM;
MMP-2
nM;
MMP-3
MMP-3= 20 =nM;
MMP-9 =150 nM;nM;
59MMP-3
nM; = ==150ICnM;IC : MMP-1
: MMP-1 > 100
IC μM;
MMP-1μM; >MMP-2
MMP-2100 =μM;
0.02 μM;
MMP-2 μM; =MMP-3
MMP-3
0.02 =μM;
90 MMP-3MMP-8
= 90 > 100
MMP-8 μM;
> 100
100 μM; MMP-9
MMP-9= 20= 20μM;
20 μM;MMP-12
MMP-12 >MMP-13
100
>of μM;
10049μM; MMP-
of>64MMP-
MMP-7
MMP-7 = 1.4
= μM;
Biomolecules
1.4 μM;MMP-8 µM;10,
2020,
MMP-8 = 1.1
717=
Biomolecules nM;
1.1 MMP-9
nM;
2020, 717 =
10,MMP-9 59= nM;
59 MMP-13
nM; MMP-13
50
µM;
μM;MMP-8
50
MMP-8
μM; MMP-8 = 20
= 20µM;
μM;
= 20
20 MMP-9
MMP-9
μM; MMP-9 > 100
> 100 µM
μM
> 100
100 μM MMP-8
MMP-8 > >
100 μM;μM;
MMP-8
IC 50 MMP-9
: >µM;
MMP-9
100
MMP-1 MMP-12
= 20
μM;
> =
100 μM;
MMP-9
μM;>
μM; 100
MMP-12= µM;
MMP-12
20
MMP-2 >
μM;49
= >
100
4 100
64
μM;
MMP-12
μM; μM; 100
MMP-
>
MMP-3MMP-
49 µM
of
100 64
>μM; MMP-
100 μM;
MMP-7
MMP-7 = 1.4= 1.4
μM;
MMP-7
ICμM;
MMP-8
:
MMP-13 MMP-8
= 1.4
MMP-1 =μM;==
1.1 1.1
nM;
160
= MMP-14 nM;
MMP-8 MMP-9
13 nM; MMP-14 MMP-9
= 1.1
MMP-2 = =
59
nM; 59
nM;
20
= 32 nM nM;
MMP-9 MMP-13
MMP-13
= 59
MMP-3 =
nM;==MMP-13
150 nM; = μM; MMP-8
μM; MMP-8
IC50: MMP-1 =μM;=
20 μM;μM;
MMP-8 MMP-9
MMP-9
> 100 = 20
μM; >μM;>
100MMP-9
MMP-2 μM μM > 100MMP-3
= 0.02 μM; μM = 90 1313
> 100
13 μMμM
>>MMP-9
100
50
1313
nM;
13 nM; MMP-14 = 32 nM
= 32
32 nM MMP-8 > 13μM;
100 > 100 100
μMμM 13 = >
20100
μM;μM MMP-12 > 100 μM; MMP-
MMP-713= nM; nM;
1.4 μM; MMP-14
MMP-14 13 nM;
MMP-8 =
= 32MMP-14 nM
nMnM; MMP-9
1.1 = 32 nM= 59 nM; MMP-13 = μM; MMP-8 = 20 μM; MMP-9 > 100 μM
13 > 100 μM
13 nM; MMP-14 = 32 nM
(X =CH
IC5050(X
IC CH ; Y= = Ph(CH 2; )Z ; =ZiC =4iC 4; H = Ph):
9=; MMP-1
R MMP-1
IC50 (X = CH2=IC 2; 2Y
; Y50=(XPh(CH
= CH Ph(CH
22); 2Y
)2iC
; Z= 2=Ph(CH 24H 9;2)R
H =RiC
Z9Ph):
2; =
Ph): MMP-1 > 10 μM;
> 10 μM;
4H9; R = Ph): MMP-1 > 10 μM;
> 10 µM; MMP-2
MMP-2 = = 20
20 nM; nM; MMP-3
MMP-3 = = 1.4
1.4 nMnM
MMP-2 = 20 nM; MMP-2 MMP-3 = 1.4
==202-naphthyl;nM
nM; MMP-3 = 1.4 nM
Ki (X = NH; Y = iC H ; Z R
4 = 92-naphthyl; R = Me): MMP-3 = 7 nM= Me):
Ki (XKi (X = Y
= NH; NH;
Ki =(XiCY4=H=NH;
iC4H
9; Z =
;=ZiC
Y9MMP-3
2-naphthyl;
4H9; Z =R
= 7 nM = Me): MMP-3
2-naphthyl; = 7 nM
R = Me): MMP-3 = 7 nM IC50: MMP-1 =nM
270 nM
IC50: MMP-1 = 50
IC
IC 270
50: MMP-1 == 270
270 nM
nM
Ki: MMP-1 = 3MMP-3
μM;=MMP-3 = 6 nM = 6 nM
Ki:MMP-1
Ki: MMP-1 ==Ki:
33µM;
μM;
MMP-1
MMP-33 =μM;
= 6 nM
nM
MMP-3
IC=50PhO(CH
IC50 (R (R =ICPhO(CH
2)3):
50 (R
2)3): MMP-1 > 30 μM; MMP-13 = 30 nM
MMP-1
= PhO(CH >2)30
3): μM; MMP-13
MMP-1 = 30MMP-13
> 30 μM; nM = 30 nM
IC50 (R-PhCH2): MMP-1 = 690 nM; MMP-3 = 1.2 μM; MMP-13 =
IC50 (R-PhCHIC 2):50MMP-1
(R-PhCH = 690 nM; MMP-3
2): MMP-1 = 1.2MMP-3
= 690 nM; μM; MMP-13 = MMP-13 =
= 1.2 μM;
IC50IC
IC(X =(XCH
50(X
50 ==CH
CH
2;IC =;YY
Y22;50
IC 50 (X==Ph(CH
Ph(CH
(X ==Ph(CH
CH
CH 2))22;=
2)222;;; 2Y
Z
Y ==;ZPh(CH
Z
iC ==4iC
Ph(CH HiC94;4H
H ))9229;;=;;R
22R ZR
Z ====Ph):
Ph): Ph):
iC
iC44H9MMP-1
9;; R
MMP-1
HMMP-1 >>10
R ==>Ph):
10
Ph): 10 μM; >> 10
MMP-1
μM; μM;
MMP-1 10 μM;
μM;
MMP-2 MMP-2
MMP-2 = 20 ==20 20
nM;MMP-2
MMP-2 nM;
nM;MMP-3 MMP-3
== 20
MMP-3 20=nM; ==1.4
1.4
nM; 1.4
nM nM == 1.4
MMP-3
nM
MMP-3 1.4 nM
nM
Ki(X
Ki Ki
(X =(XNH;
==NH;
NH;
YKi=YY
Ki (X
iC==4iC
(X H==iC9NH;H
;4H
4NH;Z99;=;ZYZ
Y ====2-naphthyl;
2-naphthyl;2-naphthyl;
iC
iC 44HH99;; Z
Z ==R2-naphthyl;=RRMe):==Me):
2-naphthyl;Me):
MMP-3MMP-3
RR == =Me):
MMP-3 =77MMP-3
7=nM
Me): nM == 77 nM
nM
MMP-3 nM
Table 11. Cont. IC
IC50
IC 50: :MMP-1
: MMP-1
50 MMP-1
=IC
270
IC =::270
=50
50 270
nMnMnM == 270
MMP-1
MMP-1 270 nM
nM
Ki:
Ki:Ki: MMP-1
MMP-1
MMP-1 =33MMP-1
= 3Ki:
=μM;
Ki: μM; MMP-3
== 33=μM;
MMP-3
μM; MMP-3
MMP-1 =66MMP-3
6=nM
μM; nM == 66 nM
nM
MMP-3 nM
IC50ICIC
(R =(R
50(R
50
50 ==
(RPhO(CH
=PhO(CH
PhO(CH
IC
IC50 50 2(R
(R )3)3PhO(CH
3):):MMP-1
)3):2==22)MMP-1
):
PhO(CH MMP-1
MMP-1
> 2230 ):>
))>33>): 30
3030
μM; μM;
MMP-1 MMP-13
MMP-13
µM;
μM;
MMP-1 >>MMP-13
30
30 μM;
MMP-13 = 30
μM; =
==MMP-13
3030
nM
30 nM
nM
MMP-13 nM == 30
30 nM
nM
IC
IC50
IC IC50 (R-PhCH
(R-PhCH
(R-PhCH
50(R-PhCH
50 IC 22):):
): 50
2IC MMP-1
50 MMP-1
(R-PhCH
MMP-1
(R-PhCH 2 ):= MMP-1 =22=):
690 690
nM;
):690 =MMP-3
nM;
MMP-1
nM;
MMP-1 690 nM;=nM;
=MMP-3
= 690
MMP-3
690 MMP-3
==1.2
1.2
nM; 1.2
μM; =
μM;
MMP-3 1.2
MMP-13
== µM;
MMP-13
μM;
MMP-3 1.2 = ==MMP-13
1.2 μM;
MMP-13 μM; MMP-13 ==
MMP-13 14nM
14 14
nM nM = 14 nM 14
14 nM
nM IC50
IC 50
IC::MMP-1
IC MMP-1
50
>55>50
50: :MMP-1
MMP-1
>IC
IC >µM;
μM;
50 μM; MMP-2
::55MMP-1
μM;
MMP-2
MMP-1 >> 55==
MMP-2
MMP-2 1.5
μM;
1.5
μM; nM;
==1.5
1.5 nM;
MMP-2
nM;nM;
MMP-2 MMP-7
MMP-7 1.5>>nM;
MMP-7
== 1.5
MMP-7 2.5
2.5
nM; µM;
>>2.5
2.5 μM; >> 2.5
MMP-7
μM; μM;
MMP-7 2.5 μM;
μM;
MMP-9
MMP-9 = 13 nM;
==13
13 MMP-13
nM;
MMP-9 MMP-13
== 13 == 1.6
1.6 nM
==MMP-13
1.6 nM == 1.6 IC
IC50 50: :MMP-1
: MMP-1 MMP-1
IC
=IC
20 =::20
20 nM ==
MMP-1
nM 20 nM
= 20 nM
MMP-9
MMP-9 = 13 nM;nM;
MMP-9MMP-13 13 nM;
MMP-13 nM; nM
1.6 nM
MMP-13 1.6 nM
nM IC 50 IC =50
5050 nM
MMP-1 20 nM
IC
ICIC
IC
50 50: :MMP-1
: 50
MMP-1
50 MMP-1
>IC
MMP-1
IC >
100>::100
>50
50 μM;
100 μM;
MMP-1
µM;
μM;
MMP-1 >MMP-2
> 100
MMP-2
MMP-2 >>
100>μM;
μM;>100
100 100 μM;
MMP-2
μM; µM;
μM;
MMP-2 >>MMP-9
MMP-9 100
MMP-9 >>>100
100>μM;
100
μM; 100
μMμM
100 μM
MMP-9
µM >> 100
MMP-9 100 μM
μM IC
IC50
IC 50: :MMP-1
: MMP-1
50 MMP-1
IC
=IC
180
IC =::180
=50
5050 180 nM =
MMP-1
nMnM
MMP-1 180 nM
== 180
180 nM
nM
4.2.4. Nitrogen-Based Inhibitors

The nitrogen-based inhibitors have a binding preference to late transition metals and improved
selectivity to zinc-dependent enzymes like MMPs [2]. The nitrogen-based inhibitors are studied by
the Food and Drug Administration (FDA) and its metabolic availability and bioavailability are well
described [2,18]. This ZBG type binds to Zn2+ using the nitrogen atom and the carbonyl oxygen
adjacent to nitrogen, which favors the formation of an enol because it is established by two hydrogen
bonds [18].
The pyrimidine-2,4,6-trione inhibitors were published in 2001 by Hoffman-LaRoche.
These compounds show relative specificity to gelatinases and potential usefulness as anticancer
drugs [2]. The derivatization of position 5 of this compound promotes access to S1 ’ and S2 ’ pockets [18].
In development of osteoarthritis drugs, the pyrimidine-2,4,6-trione inhibitors have been optimized to
inhibit MMP-13 [2].
4.2.5. Heterocyclic Bidentate-Based Inhibitors

Heterocyclic bidentate ZBGs have better biostability and higher catalytic zinc ion binding
capacity than hydroxamic acids, due to ligand rigidity [2]. Compared heterocycles bidentate and
acetohydroxamic acid, the first are more potent to inhibit MMP-1, -2, and -3 and show low toxicity in
cell viability assays [2].
Pyrones are biocompatible and they present good aqueous solubility [16]. The arylic portion was
added to fit the MMP-3 hydrophobic S1 ’ pocket, resulting in the compounds more potent than the
corresponding hydroxamate-based inhibitors [16].
Table 12 shows the IC50 and Ki values of some heterocyclic bidentate-based
inhibitors [6,15–19,29,35,37,54,55].
4.2.6. Tetracyclines-Based Inhibitors

Tetracyclines are antibiotics that can chelate zinc and calcium ions and inhibit MMP
activity [2,16,29]. Chemically modified tetracyclines (CMT) are preferred over conventional
tetracyclines because they reach higher plasma levels for prolonged periods, consequently require less
frequent administration, cause less gastrointestinal side effects, and have promising anti-proliferative
and anti-metastatic activity [2,16,29]. The CMT binds to pro- or active MMPs, disrupt the native
conformation of the protein, and leave the enzymes inactive [29]. In the search for new anticancer agents,
the first series of CMT was obtained by removal of the dimethylamino group from the carbon-4 position,
resulting in a compound without antimicrobial activity but with anticollagenolytic activity, in vitro
and in vivo [16]. Preclinical studies demonstrated that CMT can inhibit gelatinases, stromelysins,
collagenases, and MT-MMPs, by downregulating the expression of gelatinases, reducing the production
of pro-enzymes and inhibiting the activation of pro-gelatinases and pro-collagenases [16,29].
Doxycycline (Figure 26a) is a semi-synthetic tetracycline that inhibits MMP-2, -9, -7, and -8 and is
the only compound approved as an MMP inhibitor for the treatment of periodontitis [2,6]. The COL-3
(Figure 26b) showed specificity for MMP-2, -9, and -14, by decrease trypsinogen-2 and inducible nitric
oxide (iNO) production, which are regulators of MMP activity [16]. Although COL-3 is currently being
evaluated in clinical phase II trials, it showed poor solubility and stability [16].
4.2.7. Mechanism-Based Inhibitors

The mechanism-based inhibitors coordinate with catalytic zinc ion, in a monodentate mode,
allowing the nucleophilic attack by a conserved glutamic residue on the active site and forming a
covalent bond [2,17]. This attack causes a conformational change in the catalytic site environment [17]
preventing dissociation of the inhibitor and decreasing the rate of catalytic turnover and the amount of
inhibitor needed to saturate the enzyme [2].
Biomolecules
Biomolecules
Biomolecules
Biomolecules
2020, 10,
Biomolecules 10,
2020,
2020,
71710,
71710,
2020, 717
10,717
10, 717
717 51 of
51 51
of 64
64
51 of
5151of 64
ofof64
6464
IC
Table Table
12.
TableTable
12.
Table and
IC
50 IC
Table
Table
12. 12.
12.
12.
50
Ki
IC
and
IC
IC
5012.
and 50
IC values
Ki
Ki
50
50 and
and
and
50 andKi
values
Ki
Ki
values of
ofheterocyclic
values
values
values
of of
of bidentate-based
heterocyclic
heterocyclic
Kivalues ofofheterocyclic
heterocyclic
heterocyclic
heterocyclic bidentate-based
bidentate-based
bidentate-based
bidentate-based
bidentate-based
bidentate-based inhibitors.
inhibitors.
inhibitors.
inhibitors.
inhibitors.
inhibitors.
inhibitors.
IC IC
IC5050:: MMP-1
MMP-1
IC
IC 50IC
50
IC
50 :: MMP-1
:50 MMP-1
==: 2.4
:MMP-1
MMP-1
50 ====2.4
2.4 μM;
μM;
MMP-1 2.4
2.4=
2.4 μM;
MMP-2
μM;
μM;
μM;
MMP-2 MMP-2
== 397
2.4 MMP-2
µM;
MMP-2397
MMP-2
MMP-2 ==nM;
=nM; 397
=397
397397=nM;
MMP-3
397
nM; MMP-3
== 17
nM;
nM;MMP-3
nM;
MMP-3 MMP-317
MMP-3 ====17
μM;
MMP-3
μM; 17
17
17=μM;
μM;
MMP-8
μM;
μM;
MMP-8 MMP-8
17MMP-8
µM;
MMP-8
MMP-8
Ro-28-2653
Ro-28-2653
Ro-28-2653
Ro-28-2653
Ro-28-2653 Ro-28-2653 = 394 ==
=MMP-8
394
nM;
394
=394
394 nM;
MMP-9
nM;
nM;
nM; = 394
MMP-9
=
MMP-9
MMP-9
MMP-9 nM;
540 =
=
= MMP-9
540
nM;
540
=540
540nM;
MMP-12
nM;
nM;
nM; = 540
MMP-12
MMP-12
MMP-12 =
MMP-12
= 394 nM; MMP-9 = 540 nM; MMP-12 = 619 nM; MMP-13 = 0.36 nM; nM;
619 =MMP-12
=
= 619
nM;
619
619
= 619nM;
MMP-13
nM;
nM;
nM;= 619
MMP-13
MMP-13
MMP-13=
MMP-13 nM;
0.36=
=
=MMP-13
0.36
nM;
0.36 nM;
0.36nM;
0.36
= nM;
nM;
Ro-206-0222
Ro-206-0222
Ro-206-0222 IC5050IC IC
IC
IC :50
:: MMP-1
MMP-1 :: MMP-1
50:50 MMP-1
MMP-1
:MMP-1 16 =
== 16 ===16
μM; 16
=16
16 μM;
µM;
μM;
μM; MMP-2
MMP-2
MMP-2 == 12
MMP-2
MMP-2 12 nM; ====12
nM; 12
=12
12 nM;
nM;
nM;
nM; MMP-3
MMP-3
MMP-3MMP-3
MMP-3 1.8 =
== 1.8 ====1.8
μM; 1.8
1.8
1.8 μM;
MMP-8
µM;
μM;
μM; MMP-8
MMP-8
MMP-8
MMP-8 = = 0.36MMP-14
MMP-14 nM;
MMP-14 MMP-14 ==nM540 =
nM 540 nM
Ro-206-0222
Ro-206-0222
Ro-206-0222
Ro-206-0222 IC IC
5050 MMP-1 μM; 16 μM;
MMP-2 MMP-2 12 nM;
MMP-3 MMP-3 μM; 1.8 μM;
MMP-8 MMP-8 MMP-14 == 540
MMP-14
MMP-14540 =nM =540
540nM
540 nMnM
IC505050::: MMP-1
IC MMP-1
IC
MMP-1 =
:: MMP-1
MMP-1 4.310 µM; MMP-9
====4.310
4.310 μM; = 2 nM
MMP-9 nM
====2222nM
nM == 15
15 nM;===15
nM; 15
=15 nM;nM;
MMP-9
nM; MMP-9
MMP-916=
== 16
MMP-9 ===16
nM; 16
=16 nM;
nM;
MMP-14
nM; MMP-14
MMP-1410 =
== 10
MMP-14 ===10
nM 10
=10 nM
nM
nM
IC IC
IC
IC50:50
50
50 == 4.310
:MMP-14.310
MMP-1 μM;
4.310 MMP-9
μM;
4.310
μM; == 22 nM
μM;MMP-9
μM;
MMP-9MMP-9
MMP-9 nM
nM 15 15 nM;
nM;
MMP-9 MMP-9
MMP-9 nM; 16 16 nM;
nM;
MMP-14 MMP-14
MMP-14 nM 1010 nM
nM
IC505050::: MMP-13
IC IC =
:: MMP-13
MMP-13 0.87 nM; MMP-14 = 23
23 nM
nM
Biomolecules
IC MMP-13
IC
IC
IC50:50
50
MMP-13 ==2020,
2020,
:MMP-130.87
10,
MMP-13
0.87 ===nM;
717 0.87
nM;
=0.87
0.87
0.87 nM;
nM; MMP-14
MMP-14 == 23
nM;MMP-14
nM; MMP-14
MMP-14 ====23
23 nM
23nM
23 nM
nM 52 of 64
717MMP-14 nM
50
Biomolecules 10, 52 of 64
IC5050:: IC
IC ::: MMP-13
MMP-13 MMP-13 =
====111MMP-14
nM; MMP-14 == 220
220 nM
nM IC
IC5050:: MMP-8
MMP-8 :: MMP-8
MMP-8
== 107 ===
107 =nM; 107
nM; nM;
MMP-9MMP-9
== 20 =====20
20 nM
nM 20 nM
== 11 nM;
nM; MMP-14 == 220
220 =nM
=nM IC
IC 50:50 =107
107nM;
nM;MMP-9
nM; MMP-9 20nM
20 nM
IC 50
IC IC
IC50
IC50:50
50
MMP-13
50 MMP-13
:MMP-13
MMP-13 1nM;
nM;MMP-14
nM; MMP-14
MMP-14 =220
220nM
220 nM
nM IC IC
50
50 :MMP-8
MMP-8 107
107nM;
MMP-9 MMP-9
MMP-9 20 nM
nM
Ki: MMP-1>>55µM;
Ki:MMP-1 MMP-2== 1.8
μM;MMP-2 1.8 nM; MMP-9 =
nM; MMP-9 = 1.9 nM; MMP-13 == 0.33
0.33 nM
nM
Ki: MMP-1 > 5 μM; MMP-2 = 1.8 nM; MMP-9 = 1.9 nM; MMP-13 = 0.33 nM
IC50 : MMP-2 = 0.14 µM; MMP-8 = 0.14 µM; MMP-12 = 0.22 µM; MMP-13 = 0.36 nM
IC50: MMP-2 = 0.14 μM; MMP-8 = 0.14 μM; MMP-12 = 0.22 μM; MMP-13 = 0.36 nM
IC50: MMP-2 = 0.14 μM; MMP-8 = 0.14 μM; MMP-12 = 0.22 μM; MMP-13 = 0.36 nM
GW-3333
S-3304
Ki:
Ki: MMP-1 >Ki:
> 55 μM;
Ki: MMP-1 MMP-2 == 1.8
>> 55MMP-2
μM; nM;
MMP-2 MMP-9
== 1.8 == 1.9
nM; nM;
MMP-9 MMP-13
== 1.9 == 0.33
nM; nM
MMP-13
MMP-1
Ki: MMP-1
Ki: MMP-1 μM;
MMP-1 MMP-2
>> 55 μM;
μM; μM; 1.8 nM;
1.8MMP-9
MMP-2
MMP-2 == 1.8 nM;
nM;
1.8MMP-9
nM; 1.9 nM;
1.9MMP-13
MMP-9
MMP-9 == 1.9 nM;
nM;
0.33
1.9MMP-13
nM; 0.33 ==nM
nM
MMP-13
MMP-13 == 0.33
0.33
0.33 nM
nM nM
Table 12. Cont.
IC
IC5050:: MMP-2 ==50
IC 0.14 μM;
:: MMP-2 MMP-8
==μM;
0.14 == 0.14
μM; μM;
MMP-8 MMP-12
==μM;
0.14 == 0.22
μM; μM;
MMP-12 MMP-13
==μM;
0.22 == 0.36
μM; nM
MMP-13
MMP-2
IC50
IC
IC 500.14
50: MMP-2 μM;
0.14MMP-8
MMP-2
: MMP-2 == 0.14
0.14MMP-8
μM;
μM; MMP-8
0.14
MMP-8μM;
0.14MMP-12
== 0.14
0.22
0.14MMP-12
μM;
μM; MMP-12
MMP-12μM;
0.22MMP-13
== 0.22
0.36
0.22MMP-13
μM;
μM; MMP-13
MMP-13 0.36 ==nM
nM
== 0.36
0.36
0.36 nM
nM nM
GW-3333
GW-3333 GW-3333
GW-3333
GW-3333
GW-3333 S-3304
S-3304S-3304S-3304
S-3304
IC
IC 50:: MMP-1
50 MMP-1 ==50
IC 19
19 μM;
:: MMP-1
μM; ==GW-3333
MMP-3
MMP-3 19 == 20
μM; nM;
=MMP-3
20 nM; MMP-9
== 20
20MMP-9
== MMP-9
nM; 16== nM == S-3304
==μM; > IC IC
: MMP-1IC 50=
50: MMP-1 MMP-1
19= 19
µM; μM; 19
MMP-3 μM;
MMP-3 MMP-3
20== nM;nM;
MMP-9 =MMP-9
20MMP-9
nM; IC
IC5050:: MMP-2 IC==50
IC 2: nM; ===22MMP-9
MMP-9
502: MMP-2
2 nM;
nM;
== 10 MMP-9
nM
10MMP-9 ===10
10nM
nM
>>μM;
Ki:
Ki: MMP-2 =Ki:
= 2.17 MMP-3 >> 4.50 μM;
4.50MMP-7 >> 6.37 μM;
6.37MMP-12 == MMP-12 50 MMP-2 50 nM; MMP-9 nM10 nM
Ki: MMP-2
Ki:
MMP-2
Ki:
Ki:
MMP-2
MMP-2
MMP-2
2.17
MMP-22.17
μM; 2.17MMP-3
==µM;
2.17
2.17MMP-3
= 2.17 μM;
μM;
μM;
μM;
MMP-3
MMP-3
MMP-3
4.50 >μM;
MMP-3
> 4.50
4.50
µM;
>>μM;
4.50
MMP-7 MMP-7
μM;
4.50MMP-7
μM;
μM;
MMP-7
MMP-7
6.37
MMP-7
> 6.37
6.37
µM;
>>μM;
6.37
MMP-12 μM;
6.37MMP-12
μM;
μM; MMP-12
MMP-12 =
=
IC
== 50: MMP-1 = 19 16μM; MMP-3
16 nM
nM16 nM16 16 nM
nM
20 nM; MMP-9 IC IC MMP-2
IC50: MMP-2 = 2 nM; MMP-9 == 10
50: MMP-2 = 2 nM; nM; MMP-9
nM
10 nM
MMP-12 1.02=μM
1.02 μM1.02 µM
1.02 μM
μM 1.02
1.02 μM
μM 16 nM
1.02
AZD-126 IC5050:: MMP-1

IC MMP-1 >>50
IC 50 μM;
:: MMP-1
5050 μM; MMP-2
>> 50 == 610
μM; nM;
MMP-2 MMP-3
== 610 == MMP-3
nM; MMP-3 ==
AZD-126
AZD-126AZD-126 AZD-126
AZD-126 IC50 :ICIC IC
50: MMP-1
50: MMP-1
MMP-1 50MMP-2
MMP-1
> 50>>µM;
50 μM;
μM;
50MMP-2
MMP-2
μM;
MMP-2
610
= 610
nM;
MMP-2
==nM
610
610
MMP-3
nM;
nM;nM;
610MMP-3
nM;
= 10== nM
MMP-3
MMP-3
IC == AZD-126 == 10
10 nM
nM10 nM10
IC
IC 50::: MMP-9
Biomolecules
Biomolecules
50
50 2020,
MMP-9
MMP-9
IC IC 4.5
Biomolecules
IC
2020,
10, 717
=50
50: MMP-94.5 nM;
2020,
:: MMP-9
10,
nM;
MMP-9
=
MMP-12
717
4.5
10,
== 4.5
MMP-12
nM; 4.5 = 6.1
717 nM;
nM;
MMP-12 6.1
6.1 nM
MMP-12
nM
nM
MMP-12 == 6.1
6.1 nM
nM 10 nM 53 of 6453 of 64
53 of 64
== 6.1
6.1 nM
nM
50
IC50: MMP-9 = 4.5 nM; MMP-12 10 nM IC
IC5050:: MMP-1 >>5050
IC 50 μM;
:: MMP-1 >>
MMP-2 >> 50 μM; >>>50
MMP-3 == 19 nM ===19
MMP-1 IC
IC 5050 μM;
MMP-1 > 50
MMP-250 μM;
50MMP-2
µM;
μM; 50MMP-2
MMP-2
μM;
MMP-2 MMP-3 50
50 μM;
µM;
μM; 19MMP-3
MMP-3
nM19 nM
MMP-3 19nM
19 nM
nM
IC
IC50: MMP-1 > 50 μM; MMP-2 > 50 μM; MMP-3 == 19
50: MMP-1 > 50 μM; > 50 μM; MMP-3 nM
IC5050
IC :IC
MMP-1
: MMP-1 >50>
IC
50: MMP-1
50: 50 >µM;
MMP-1
μM; MMP-2 = MMP-2
> 50MMP-2
50MMP-2
μM; μM; 4.4
= 4.4 µM;
= 4.4
μM; =MMP-3
μM;
MMP-3 = MMP-3
4.4MMP-3
μM;
= 77 77
= nM;
nM; MMP-7
= 77MMP-7
77MMP-7
nM; nM; >MMP-7
> 50 > 50IC :MMP-1
IC50>50: 50MMP-1
IC IC >50
50: MMP-1
>50 : 50 >µM;
MMP-1
μM; MMP-2
50MMP-2
μM; ==
> 50MMP-2
μM;9.39.3
= µM;
MMP-2 MMP-3
9.3MMP-3
μM; μM; = = MMP-3
= 9.3MMP-3
μM; 0.24
= 50µM;
IC = 50: MMP-1
IC
: MMP-1 IC IC
>5050 >: 50
μM;
50 MMP-1
: MMP-1 μM;
MMP-2 >μM;
=5016.5
> 50MMP-2 µM;
MMP-2MMP-2
=μM;
16.5 μM;
MMP-3 =MMP-3
= 16.5 16.5
μM; µM;
MMP-3
= 41.7 MMP-3
=μM;
41.7 =MMP-
= 41.7
μM;
MMP- 41.7 µM;
μM; MMP-
=245 = = MMP-13 MMP-7 > 50MMP-7
µM; MMP-8 >=50
64 nM; MMP-9 =>64
50 µM; MMP-12 =7μM;
22 => >=503.8 =>1.2 =
μM;50 μM;
MMP-8 MMP-8
µM;MMP-8
μM;
= 245MMP-8
=nM; 245=nM;
nM; MMP-9
245MMP-9
MMP-9 nM; MMP-9
= 32.3=μM;32.3
32.3 µM;
= 32.3
μM;
MMP-12 MMP-12
μM;
MMP-12 MMP-12
= 85 = 85
nM; 85 =nM;
nM; 85MMP-13
nM; 0.24
= μM;
= MMP-13 0.24
= μM; 0.24MMP-7
μM;
> 50MMP-7
μM; μM;> 50MMP-8
MMP-8 μM;
= 64MMP-8
nM; nM;
MMP-= 64MMP-
nM;7MMP-
> 50 7MMP-7
> 50MMP-8
> 50MMP-8
μM; μM;3.850=µM;
MMP-8
μM; MMP-8
= 3.8MMP-9
3.8MMP-9
μM; μM; >µM;
MMP-9
μM; MMP-9
> 50MMP-12
50MMP-12
μM; μM; 50μM;
MMP-121.2 MMP-12
=µM; = 1.2 μM;
μM;
MMP-13 = 6.6 µM6.6 μM 9 > 50 9 >
μM; 50 nM;
μM;
MMP-12 MMP-13
MMP-12
= 22 =
=
nM; 20.6
22 µMMMP-13
nM;
MMP-13 = 20.6 =
μM20.6 μM 1.2 µM; MMP-13
MMP-13 = = 16.5
16.5 μM µM
6.6 μM6.6 μM 9 > 50 μM; MMP-12 = 22 nM; MMP-13 = 20.6 μM MMP-13MMP-13
= 16.5= 16.5
μM μM
IC
IC50: MMP-1
IC50: MMP-1 : MMP-1
>5050 > 50MMP-2
> 50MMP-2
μM; μM; μM;
= 7.6MMP-2 = 7.6MMP-3>
= 7.6MMP-3>
μM; μM; μM;
50 MMP-3>
μM; μM;50MMP-7
50MMP-7 μM;
> 50MMP-7
> 50IC50>: 50 IC
IC50: MMP-1
MMP-1 : MMP-1
>5050 > 50MMP-2
> 50MMP-2
μM; μM; μM; MMP-2
= 0.92 =μM; = 0.92MMP-3
0.92MMP-3
μM; μM; = 50: MMP-1
IC
= MMP-3 IC
IC=50: MMP-1 : MMP-1
>50400 >μM; > 400MMP-2
400MMP-2
μM; μM;
= 135MMP-2
=nM; = 135MMP-3
135MMP-3
nM; nM;
= 81MMP-3 = 81MMP-7
= 81MMP-7
nM; nM; nM; MMP-7
μM;
= 5.0MMP-8
μM; MMP-8
μM; MMP-8 = 5.0MMP-9
= 5.0MMP-9
μM; μM; μM;
> 50MMP-9 > 50MMP-12
> 50MMP-12
μM; μM; μM; MMP-12
= 6.7 = 6.7MMP-13
= 6.7MMP-13
μM; μM; μM;
= MMP-13
=0.56 μM;= MMP-7
0.56 0.56MMP-7
μM; μM;
> 50MMP-7 > 50MMP-8
> 50MMP-8
μM; μM; μM;
= 86MMP-8 = 86MMP-
= 86MMP-
nM; nM; = 1.1 μM;
nM; MMP- = 1.1MMP-8
= 1.1MMP-8
μM; μM;
= 42MMP-8
nM; = 42MMP-9
= 42MMP-9
nM; nM; MMP-9
> 7 μM; μM;> 7MMP-13
> 7MMP-13 μM; MMP-13
= 1.8 = 1.8 nM;
= 1.8 nM;
nM;
6.7 μM6.7 μM6.7 μM 27.19MMP-12
9 =μM;
9 = 27.1 = 27.1MMP-12
μM; μM; MMP-12
= 18 = 18MMP-13
= 18MMP-13
nM; nM; nM; MMP-13
= 4.1 = 4.1 μM
= 4.1 μM
μM MMP-14 = 5MMP-14
MMP-14 μM = 5 μM= 5 μM
IC :: MMP-1 >> 50 μM; MMP-2 == 9.3 μM;

= MMP-3 == 50: MMP-1
IC :: MMP-1 >> 50 μM; MMP-2 ==μM;
16.5 μM; MMP-3 ==μM;
41.7 μM;
50: MMP-1
IC50 IC
IC
IC :: MMP-1
>505050
IC5050:: MMP-1
MMP-1 >> 50
μM;
MMP-1 >> 50
50MMP-2
μM;
μM;
μM;
MMP-2 MMP-2
= 4.4
50MMP-2
μM; == 4.4
μM;
MMP-2 == 4.4
4.4MMP-3
μM;
μM;
μM;
= 77MMP-3
MMP-3
4.4MMP-3
μM; == 77
nM; == 77
77MMP-7
MMP-3 nM;
nM; 77 nM;
> 50MMP-7
MMP-7
nM;
MMP-7 50IC>50
>> 50
MMP-7 50:50
> 50MMP-1
IC IC>505050
IC5050:: MMP-1
MMP-1 >> 50
μM;
MMP-150MMP-2
μM;
μM; MMP-2
= 9.3
50MMP-2
μM; == 9.3
μM;
MMP-29.3MMP-3
μM;
μM; MMP-3
9.3MMP-3
μM; == 50
MMP-3
IC IC
50: MMP-1
IC IC
50: MMP-1>505050 >> 50
μM;
MMP-150MMP-2
μM;
μM; MMP-2
= 16.5
50MMP-2
μM; ==μM;
MMP-216.5MMP-3
16.5 μM; MMP-3
= 41.7
16.5MMP-3
μM; ==μM;
MMP-341.7MMP-
41.7 μM; μM; MMP-
MMP-
41.7MMP- MMP-
μM; MMP-8 = 245 nM; MMP-9 = 32.3 μM; MMP-12 = 85 nM; MMP-13
0.24 =
0.24
μM; 0.24
μM;
MMP-7
0.24 μM;
MMP-7>
μM; 50MMP-7
>
μM;
MMP-750 >
μM;
MMP-8
> 50
50 μM;
MMP-8
=
μM; 64MMP-8
=
nM;
MMP-8 64MMP-=
nM;
= 64
64 nM;
MMP-
nM; MMP-
7MMP-
> 50 7 >
μM; 50 77μM;
>> 50
MMP-8 50 μM;
MMP-8
=
μM; MMP-8
3.8 =
μM;
MMP-83.8 == 3.8
μM;
MMP-93.8 μM;
MMP-9
>
μM;50MMP-9
>
μM;
MMP-9 50 >> 50
μM;
MMP-12
50 μM;
MMP-12
μM; = MMP-12
1.2 =
μM;
MMP-12 1.2 == 1.2
μM; 1.2 μM;
μM;
μM;
μM; MMP-8 MMP-8
=
μM; 245 =
nM;
MMP-8 245 nM;
MMP-9
= 245MMP-9
=
nM;32.3 = 32.3
μM;
MMP-9 μM;
MMP-12
= 32.3MMP-12
μM;
μM; MMP-8 = 245 nM; MMP-9 = 32.3 μM; MMP-12 = 85 nM; MMP-13 = = 85 =
nM;
MMP-1285 nM;
MMP-13
= 85MMP-13
=
nM; =
MMP-13 0.24
= μM; MMP-7 >
Table 50 μM; MMP-8
12. Cont. = 64 nM; MMP- 7 > 50 μM; MMP-8 = 3.8 μM; MMP-9 > 50 μM; MMP-12 = 1.2 μM;
6.6 μM 9 > 50 9 >
μM; 50 99μM;
>> 50
MMP-12 50 μM;
MMP-12
μM;= MMP-12
22 =
nM;
MMP-1222 == 22
nM;
MMP-1322 nM;
MMP-13
nM;= MMP-13
20.6 =
MMP-13μM20.6 ==μM
20.6
20.6 μM
μM MMP-13 MMP-13
= 16.5 ==μM
16.5 μM
6.6 μM 6.6 μM
6.6 μM6.6 μM 9 > 50 μM; MMP-12 = 22 nM; MMP-13 = 20.6 μM MMP-13 = 16.5 μM
MMP-13
MMP-13 = 16.5 μM 16.5 μM
IC :: 50
MMP-1 >> 50 μM; MMP-2 =MMP-3> IC :: MMP-1
>5050400 >> 400
400>μM; MMP-2 == 135 =nM;= 81MMP-3 == 81 nM;
= 81MMP-7
IC50
IC : :MMP-1
50
50 MMP-1
IC
IC >50
>5050
50:: MMP-1
50 MMP-1
IC >>µM;
μM;
MMP-1 MMP-2
50MMP-2
50 μM;
μM; ==
MMP-2 7.6
7.6
50MMP-2
μM; == µM;
μM;
MMP-2 = 7.6
7.6MMP-3>
7.6 μM;
μM;
μM;
MMP-3>
7.6MMP-3>
μM; MMP-3>
5050 µM;
μM;50MMP-7
MMP-3>
50 μM;
μM;
50
MMP-7μM;
MMP-7>MMP-7
> 50
50MMP-7
μM; >> 50
50IC50
MMP-7 >>: 50
50
50 :50
MMP-1
IC
IC
IC >
>50
50:: MMP-1
MMP-1
50 MMP-1
IC ::50
50
50
MMP-1
>>µM;
μM;
MMP-1 >> 50
MMP-2
50MMP-2
50 μM;
μM;
μM;
MMP-2
50MMP-2
μM; MMP-2
==0.92
0.92 ==μM;
µM;MMP-3
==μM;
MMP-2 0.92
0.92
0.92
MMP-3
μM;
μM;
= = MMP-3
MMP-3
0.92MMP-3
μM; IC
0.56
== µM;
MMP-350 ==5050:: MMP-1
IC
50: MMP-1
IC MMP-1
IC IC >: 400
μM;
MMP-1
50> MMP-1
400 μM;
MMP-2
μM; 400
MMP-2 µM;
= 135
μM;
MMP-2 =nM;
MMP-2 MMP-2
= 135
135 nM;
MMP-3
nM;135 135
MMP-3
nM;
MMP-3 nM; MMP-3
== 81
nM;
MMP-381 nM;
MMP-7
nM; 81MMP-7
nM;
MMP-7 nM;
MMP-7
50 µM; μM;
MMP-8 MMP-8= 5.0 = 5.0
µM; μM;
MMP-9MMP-9 > 50 > 50
µM; μM; MMP-12
MMP-12 = 6.7= 6.7
µM; μM; MMP-13
MMP-7 => 50 0.56
µM; μM;
MMP-8 =
MMP-7 86 > 50
nM; μM;
MMP-9 MMP-8= =
27.1 86
µM;nM; MMP-
=
MMP-12 1.1 =
=
μM; 1.1 == 1.1
MMP-7
μM;
MMP-8 μM;
MMP-8
1.1 =
=
μM; 42MMP-8
1.1 µM;
=
nM;
MMP-842 == 42
MMP-8
nM;
MMP-9 42 nM;=
MMP-9
>
nM; 7 MMP-9
42
μM;nM;
>
MMP-9 7 MMP-9
μM;
MMP-13 =
μM; >
>> 77MMP-13
μM; MMP-13
7
1.8µM;
=
nM;
MMP-13 1.8 == 1.8
MMP-13
nM; = 1.8
1.8 nM;
nM;
μM; MMP-8
μM; MMP-8
μM; MMP-8 == 5.0
= 5.0MMP-8
μM; μM; 5.0MMP-9
μM;
μM; MMP-9 >> 50
> 50MMP-9
= 5.0MMP-9
μM; μM; 50MMP-12
μM;
μM; MMP-12
= 6.7
> 50MMP-12
μM; == 6.7
μM;
MMP-126.7MMP-13
μM;
μM; MMP-13
= MMP-13
= 6.7MMP-13
μM; ==0.56 μM; = MMP-7
0.56
0.56 μM;
μM; MMP-7
0.56MMP-7> 50MMP-7
μM; >> 50
μM; 50MMP-8
μM;
μM; MMP-8
= 86MMP-8
> 50MMP-8
μM; == 86
nM; 86MMP-
nM; nM; MMP-= 1.1 μM; MMP-8 = 42 nM; MMP-9 > 7 μM; MMP-13 = 1.8 nM;
MMP-
= 86
nM; MMP-
MMP-13 = 6.7 µM 6.7 μM 99MMP-12
==μM;
27.1 μM;
18MMP-12
nM; MMP-12
MMP-13 == 18 nM;
4.1 µM MMP-13 == 4.1 μM MMP-14 MMP-14
= MMP-14
nM;
5 μMMMP-14
= 55 μM
MMP-14 μM == 55=μM5 µM
μM
6.7 μM 6.7 μM
6.7 μM 6.7 μM 9 = 27.19 =μM;27.1 27.1 μM;= 18 =
nM;
MMP-1218 nM;
MMP-13
=
9 = 27.1 μM; MMP-12 = 18 nM; MMP-13 = 4.1 μM 18MMP-13
nM;= 4.1 =
μM4.1
MMP-13 μM 4.1 μM MMP-14 =
KI: MMP-1 > 500 µM; MMP-9 = 6 µM IC :: MMP-1 >> >

11MMP-2
μM; == 5= =MMP-9 =
= =MMP-2 KI: MMP-1 >> 500 μM;
= 6 MMP-9 == 66 μM
IC50
50: MMP-1
IC
IC>5050150
IC5050:: MMP-1
MMP-1 : MMP-1
>> 11MMP-2
μM;
MMP-1 μM; μM;5 MMP-2
1=µM; MMP-2
== 55MMP-3
nM;
MMP-2 nM; nM;56MMP-3
5=nM;
5MMP-3
nM; MMP-3
== 56
nM;
MMP-3 == 56
56MMP-9
nM; 56 nM;
56
nM; MMP-9
= nM;
2.4 2.4 == 2.4
=MMP-9
MMP-9 2.4
IC
IC :IC
MMP-1
: MMP-1
5050
50 IC 50
IC
IC :: MMP-1
=505014
50:: MMP-1
MMP-1 14==µM;
μM;
MMP-1
Biomolecules 14
== 14
MMP-2
142020,
μM;
MMP-214 μM;
MMP-2
= 529
μM;
Biomolecules
μM; 10, 717
MMP-2 529 nM;
= 529
2020, 529
=nM;
MMP-2 =717
nM;
10,MMP-3
nM;
529
=MMP-3
529 = 1=MMP-3
nM;
MMP-3
nM;
MMP-3 1==nM;
nM;
MMP-3 == 11MMP-9
MMP-9
11MMP-9
nM;
nM;
nM; =MMP-9
= 2.42
nM;
MMP-9 2.42 == 2.42
== 2.42
MMP-9 2.42 KI: MMP-1
KI: MMP-1
KI: MMP-1
> 500
KI: > 500
500MMP-9
>μM;
MMP-1 μM;
μM; MMP-9
500MMP-9
μM; == 66 μM
μM
MMP-9 μM μM IC μM; MMP-2
2.4
nM;nM;
nM; MMP-3
MMP-12
MMP-12 = of
==54
2.52.5
nM;
64nM
nM
MMP-9
54 of 64 = 2.4
Biomolecules
2.42 µM;2020, 717 =
10,μM;
MMP-14 MMP-14
20.1 µM = 20.1 μM nM;
nM; MMP-12 MMP-12
nM;= 2.5
nM; MMP-12 = 2.5 nM=
nM
MMP-12 2.5 nM 2.5 nM54 of 64
μM; MMP-14
μM; MMP-14
μM; MMP-14
μM;= 20.1 ==μM
MMP-14 20.1 μM
20.1 =μM
20.1 μM
IC IC :100
MMP-1 > 100 μM; MMP-2 >MMP-3
100 μM; MMP-3 > IC : IC 50 : MMP-1
MMP-1 >
> 10 μM;10MMP-7 MMP-7
µM; MMP-9 >> 10
> 10 μM; 10MMP-9 MMP-9
µM; MMP-13 >= 10MMP-13
> 10 μM; µM; =
IC5050: :MMP-1
MMP-1
IC
>>100
50
50: MMP-1
μM; MMP-2
> µM; MMP-2
100 μM;
>>
MMP-2
100 μM;
100 µM;MMP-3
> 100 μM;
> > 100
MMP-3
: MMP-1
IC50µM; > 5010 μM; MMP-7
> IC50: MMP-1 > 10 μM;MMP-13
> 10 μM;
MMP-7 >=10 μM; MMP-9 > >
μM;
10 μM; MMP-13 =
MMP-7 100>μM; 100 µM;100
MMP-7 μM;
> 100
MMP-8 MMP-7
>μM;
100 > 100
MMP-8
µM; μM; MMP-8
> 100
MMP-9 > μM;
100 > 100
µM; μM; >
MMP-12 12 nM; MMP-14 >1210
12 nM; nM;
μMMMP-14
MMP-14 > 10 μM10 µM
100 μM; MMP-7 > 100 μM; MMP-8 > 100 μM; 12 nM; MMP-14 > 10 μM
IC5050
IC : MMP-1
: MMP-1 IC =: MMP-1
= 503030 nM;
nM; MMP-2
= 30 nM;
MMP-2 = MMP-2
= 9.89.8
nM;nM; =MMP-3
9.8 nM;
MMP-3 = MMP-3
= 1.71.7
μM;µM; =MMP-7
1.7 μM;
MMP-7 =MMP-7
= 475 MMP-9
= 475 100 MMP-9
> 100
µM; μM; MMP-12
MMP-13 > μM;
> 100 MMP-12
> 100
100 µM; μM; > 100> μM;
MMP-13
MMP-14 MMP-13 >
> µM
100
IC50: MMP-1 = 30 nM; MMP-2 = 9.8 nM; MMP-3 = 1.7 μM; MMP-7 = 475 MMP-9 > 100 μM; MMP-12 > 100 μM; MMP-13 >
475nM;nM;MMP-9
MMP-9 ==
nM; 3MMP-9
nM;
3 nM; MMP-14
= 3 nM;
MMP-14 =MMP-14
= 1717
μM µM = 17 μM 100 μM; MMP-14 100 μM; MMP-14
> 100 μM > 100 μM
100 μM; MMP-14 > 100 μM
nM; MMP-9 = 3 nM; MMP-14 = 17 μM
IC50: MMP-1IC : MMP-1

> 5030 > 30 μM;
μM; MMP-2 > 30MMP-2 > 30 μM;
μM; MMP-3 > 30MMP-3 > 30 μM;
μM; MMP-7 > 30MMP-7 MMP-1IC
IC>50:30 : MMP-1
> 5010 > 10 μM;
μM; MMP-2 > 10MMP-2 > 10 μM;
μM; MMP-3 > MMP-3 >
IC50: MMP-1 > 30 μM; MMP-2 > 30 μM; MMP-3 > 30 μM; MMP-7 > 302.5 μM;ICMMP-7 50: MMP-1 > 10 μM; MMP-2 > 10 μM; MMP-3 >
2.5 μM;
> 10MMP-7 > 10 μM;
μM; MMP-8 = 7.4MMP-8 = 7.4 nM; MMP-9
nM; MMP-9
μM; MMP-8μM; > 100 MMP-8 > 100 μM;
μM; MMP-9 > 100MMP-9 > 100 μM;
μM; MMP-12 MMP-12
> 100 > 100 μM;
μM; MMP-13 = MMP-13 =
2.5 μM; MMP-7 > 10 μM; MMP-8 = 7.4 nM; MMP-9 IC50: MMP-1IC : MMP-1
> 5010 > 10 μM;
μM; MMP-2 = 5.3MMP-2 = 5.3 μM;
μM; MMP-3 MMP-3
= 4 μM; = 4 μM;
MMP-7 > MMP-7 >
μM; MMP-8 > 100 μM; MMP-9 > 100 μM; MMP-12 > 100 μM; MMP-13 = > 10 > 10 μM;
μM; MMP-12 MMP-12
> 10 > 10 μM;
μM; MMP-14 MMP-14
> 10 μM > 10 μM IC50: MMP-1 > 10 μM; MMP-2 = 5.3 μM; MMP-3 = 4 μM; MMP-7 >
0.67 nM;
0.67 nM; MMP-14 MMP-14
> 30 > 30 μM;
μM; MMP-17 MMP-17
> 30 μM > 30 μM > 10 μM; MMP-12 > 10 μM; MMP-14 > 10 μM 10 μM;
10 μM; MMP-8 MMP-8
= 720 = 720 nM;
nM; MMP-9 = 10MMP-9 = 10 μM;
μM; MMP-10 MMP-10
= 160 nM; = 160 nM;
0.67 nM; MMP-14 > 30 μM; MMP-17 > 30 μM 10 μM; MMP-8 = 720 nM; MMP-9 = 10 μM; MMP-10 = 160 nM;
IC50: IC50: MMP-1
MMP-1 IC
> 50100 >μM;
100 MMP-2
: MMP-1 μM; MMP-2
> 100 μM;
> 100 >μM;
100 MMP-3
MMP-2 μM; MMP-3
> 100 μM; IC> 50: IC
> MMP-3 >: MMP-1
MMP-1
50 IC > 10 MMP-7
: MMP-1
> 5010 μM; μM; MMP-7
> 10 μM; > 10 MMP-9
> 10MMP-7
μM; μM; MMP-9
> 10 μM; > 10 MMP-13
> 10MMP-9
μM; μM; MMP-13
> 10 μM; =
= MMP-13 =
100 MMP-7
100 μM; μM; 100 MMP-7
μM;
> 100 >μM;
100 MMP-8
MMP-7 μM; MMP-8
> 100 μM; >μM;
100 μM;
MMP-8
> 100 > 100 μM; 12 MMP-14
12 nM; nM; 12
MMP-14
nM; > 10 μM
MMP-14
> 10 μM > 10 μM
= 475MMP-9 MMP-9
> 100 >μM;
MMP-9 100 MMP-12
μM;
Table MMP-12
> 100 μM; >μM;
100 MMP-13
MMP-12
> 100
12. Cont. μM; MMP-13
> 100 μM; >
> MMP-13 >
IC50: IC50: MMP-1
MMP-1 IC = 30 MMP-2
: MMP-1
= 5030 nM; nM; MMP-2
= 30 nM; =nM;
9.8 MMP-3
= 9.8MMP-2 nM; MMP-3
= 9.8 nM; =μM;
1.7 MMP-7
= 1.7MMP-3 μM; MMP-7
= 1.7 μM; MMP-7
= 475 = 475
nM; MMP-9
nM; = 3 MMP-14
MMP-9 nM;=MMP-14
3 nM; = 17 μM
MMP-14 = 17 μM 100 μM; 100 MMP-14
μM; MMP-14
100 μM;
> 100 >μM
100 μM
MMP-14 > 100 μM
nM; MMP-9 = 3 nM; = 17 μM
> IC IC: >50

MMP-1IC
:30
MMP-1 >IC
50: MMP-1
10 µM;
> 5010 > 10
: MMP-1
μM; μM;
MMP-2
MMP-2 > μM;
MMP-2
> 10 10 µM;
> 10 > 10
MMP-2
μM; μM;
MMP-3
MMP-3 > μM;
MMP-3
> 10 2.5
> µM;> MMP-7
MMP-3 >
IC5050: :IC
IC MMP-1
MMP-1 >>
50: MMP-1
IC :30
30
50 >µM;
30 MMP-2
MMP-1
μM; μM;> 30
MMP-2 > >3030
MMP-2
μM; > µM;
30 MMP-3
MMP-2
μM; μM;> 30
MMP-3 >>
MMP-3
μM; > µM;
30 MMP-7
MMP-3
3030
μM; μM; MMP-7
> 30
MMP-7 μM;> MMP-7
> 30 30 50
> > > 100 >2.5
=10 μM;µM; MMP-8
2.5=MMP-7
μM; 2.5 >=10
MMP-7
μM; 7.4>nM; MMP-9
10 MMP-8
MMP-7
μM; μM; =>7.4
MMP-8
> 10 μM; 10 µM;
=nM; MMP-12
7.4 MMP-9
MMP-8 nM; nM;> MMP-9
MMP-9
= 7.4 10 µM;
μM;30MMP-8
μM; MMP-8
µM;MMP-8
μM;
> 100 >μM;
MMP-8100
100 >µM;
μM; 100
MMP-9 MMP-9
MMP-9
μM;
> 100 >μM; 100
100
MMP-9 >µM;
μM;
MMP-12 MMP-12
MMP-12
100 μM;
> 100 >μM;
100
MMP-12 >µM;
μM;
MMP-13MMP-13
100 μM;= MMP-13 IC 50: IC 50: MMP-1
MMP-1 IC>50
10
50 >>10
> 10 MMP-2
:: MMP-1
MMP-1
μM; μM; MMP-2
10μM;
µM; =μM; ==5.3
5.3 MMP-3
= 5.3MMP-2
MMP-2 μM; MMP-3
5.3μM;
=µM; μM;==
= 4 MMP-7
MMP-3
MMP-3
4 μM; MMP-7
4 4μM; >MMP-7>
>µM;MMP-7
>MMP-14 > 10 µM
0.67=nM; >>30 >>30
MMP-13 0.672020,
nM; MMP-14 30 MMP-17 30μM > 10 MMP-12
μM;> 10MMP-12
μM; > 10 MMP-14
MMP-12 μM; MMP-14
> 10 μM; > 10 μM
MMP-14 > 10 μM 10 μM; > 10
0.67
Biomolecules nM; MMP-14
0.67
MMP-14 nM;
10, > 30
717
Biomolecules MMP-14
μM;
2020, µM;
μM;
10,MMP-17
717 MMP-17
> 30 μM; MMP-17
> 30 μM µM> 30 μM > 10 μM; 10 μM; > 10 μM
10 μM; MMP-8 10
MMP-8 µM;
μM;
= 720MMP-8
=nM; ==720
720 MMP-9
MMP-8 nM; 720
MMP-9nM;
nM; MMP-9 =10
= 10 MMP-10
= 10MMP-9
μM; μM;=55 10 µM;
MMP-10
ofμM;
64= 160MMP-10
=55
160
MMP-10
nM;of 64 ==160
nM; 160nM;
nM;
Biomolecules 2020, 10, 717 MMP-13 == MMP-14
0.0039 nM; MMP-14 55 of
> 64 µM
10
MMP-13 MMP-13 = 0.0039
MMP-13
= 0.0039 nM; nM;
0.0039MMP-14
nM;
> 10 > 10 μM
MMP-14
μM > 10 μM
IC5050: :MMP-1
IC MMP-1 >>
IC 10:10
μM;
MMP-1 MMP-2
µM;MMP-2 > >2.5
> 10 μM; 2.5
μM;
MMP-2 MMP-7
µM;MMP-7 >>
> 2.5 μM; 1010 µM;
μM;
MMP-7 MMP-8
MMP-8 = MMP-8
= 57
> 10 μM; IC50=: :MMP-1
57
MMP-1IC
>>50
21.5 μM; MMP-7
: MMP-1 MMP-7 >> 21.5
> 21.5 μM;21.5 μM; MMP-13
MMP-7 MMP-
> 21.5 μM; MMP-
50
IC50: MMP-1 > 10 μM; MMP-2 > 2.5 μM; MMP-7 > 10 μM; MMP-8 = 57IC 21.5 >µM; >µM; = 430 nM
>>1010 > >10
57nM;nM; MMP-9 µM; MMP-14 10μM
µM 50 IC50: MMP-1 21.5 μM; MMP-7 21.5 μM; MMP-
MMP-9 nM; μM;
MMP-9 MMP-14
> 10 μM; MMP-14 > 10 μM 13 = 430 nM13 = 430 nM
nM; MMP-9 > 10 μM; MMP-14 > 10 μM 13 = 430 nM
IC50: MMP-1 IC :: MMP-1
>505010
IC > 10 μM;
μM; MMP-7
MMP-1 > 10 µM;
= 3.025 nM; =
MMP-7
MMP-7 3.025 nM;
=MMP-13
3.025 nM;
= 0.5 nM ==0.5
MMP-13
MMP-13 0.5nM
nM
IC50: MMP-1 > 10 μM; MMP-7 = 3.025 nM; MMP-13 = 0.5 nM
IC50: MMP-1 > 18.6

IC50: μM; MMP-7
MMP-1 > 18.6
> 18.6 μM; μM; MMP-13
MMP-7 = 620
> 18.6 μM; nM; MMP-14
MMP-13 > 62
= 620 nM; μM
MMP-14 > 62 μM
IC50: MMP-1IC>5022 μM; MMP-2
: MMP-1 = 18MMP-2
> 22 μM; μM; MMP-3 > 22MMP-3
= 18 μM; μM; MMP-7 > 22μM;
> 22 μM; MMP-8
MMP-7 > 22MMP-8
> 22μM; μM; > 22 μM; IC50: MMP-1 > 18.6 μM; MMP-7 > 18.6 μM; MMP-13 = 620 nM; MMP-14 > 62 μM
IC50: MMP-1 > 22 μM; MMP-2 = 18 μM; MMP-3 > 22 μM; MMP-7 > 22μM; MMP-8 > 22 μM;
MMP-9 = 8.9MMP-9
μM; MMP-10 = 16
= 8.9 μM; μM; MMP-12
MMP-10 > 22
= 16 μM; μM; MMP-13
MMP-12 = 1MMP-13
> 22 μM; nM; MMP-14 = 8.3
= 1 nM; μM = 8.3 μM
MMP-14
MMP-9 = 8.9 μM; MMP-10 = 16 μM; MMP-12 > 22 μM; MMP-13 = 1 nM; MMP-14 = 8.3 μM
:IC 50: 50
50:50MMP-1
ICIC IC
MMP-1
IC MMP-1
50:: MMP-1
MMP-1 >μM;
> >1010μM;10 μM;
MMP-2
>> 10
10 MMP-2
μM;
MMP-2
μM; > >2.5
MMP-2
MMP-2 >μM;
2.5μM;2.5 μM;
MMP-7
>> 2.5
2.5 MMP-7
μM;
MMP-7
μM; MMP-7 >μM;
> >1010μM;
MMP-7 10 μM;
MMP-8
>> 10
10 MMP-8
μM;
MMP-8
μM; = =5757 = 57
MMP-8
MMP-8 ==IC
57
57 :IC 50: 50
50:50MMP-1
IC IC
MMP-1
IC MMP-1
> >21.5
50:: MMP-1
MMP-1
21.5>μM;
21.5
> 21.5
21.5
>μM; μM;
MMP-7
μM;
MMP-7
μM;MMP-7
> >21.5
MMP-7
MMP-7 >μM;
21.5 21.5 μM;
MMP-
> 21.5
21.5
>μM; MMP-
μM;
MMP-
μM; MMP-
MMP-
nM; nM;
nM;MMP-9
MMP-9
nM;MMP-9
nM; MMP-9 >μM;
> >1010μM;
MMP-9 10 μM;
MMP-14
>> 10
10 μM;
MMP-14
μM; MMP-14
MMP-14
MMP-14 >μM
> >1010μM 10 μM
>> 10
10 μM
μM 13
1313= =430 = nM
nM
13 430 nM
430
13 == 430
430 nM
nM
Table 12. Cont.
:IC 50: 50
50:50MMP-1
ICIC IC
MMP-1
IC MMP-1
50:: MMP-1
MMP-1 >μM;
> >1010μM;10 μM;
MMP-7
>> 10
10 MMP-7
μM;
MMP-7
μM; = =3.025
MMP-7
MMP-7 = nM;
3.0253.025
==nM; nM;
3.025 MMP-13
MMP-13
nM;
MMP-13
3.025 nM; = =0.5
MMP-13
MMP-13 =nM
0.5nM0.5 nM
== 0.5
0.5 nM
nM
IC50:IC50:
IC50:
IC50: MMP-1 MMP-1
MMP-1
IC50:
MMP-1
IC50: MMP-1 >>μM;
> >18.6
18.6
MMP-1 18.6
18.6 μM;
µM;
> 18.6
18.6
>μM; MMP-7
MMP-7
MMP-7
μM;
MMP-7
μM; MMP-7 >>μM;
> >18.6
18.6
MMP-7 18.6
18.6 μM;
µM;
> 18.6
18.6
>μM; MMP-13
MMP-13
MMP-13
μM;
MMP-13
μM; = =620
MMP-13 =nM;
620
MMP-13 = 620
620 nM;
nM;
== 620
nM; 620 MMP-14
MMP-14
MMP-14
nM;
MMP-14
nM; >>μM
> >6262μM
MMP-14
MMP-14 62 μM
µM
>> 62
62 μM
μM
IC 50 :IC 50: 50
50:50MMP-1
ICIC IC
MMP-1
IC >>>22
MMP-1 22
50:: MMP-1
MMP-1 >μM;
µM;
22μM;22 μM;
MMP-2
MMP-2
>> 22
22 μM;
MMP-2
μM; ===18
MMP-2 18
MMP-2
MMP-2 =μM;
µM;
18μM; 18 μM;
MMP-3
MMP-3
== 18
18 μM;
MMP-3
μM; >>>22
MMP-3 22
MMP-3
MMP-3 >μM;
µM;
22μM; 22 μM;
MMP-7
MMP-7
>> 22
22 μM;
MMP-7
μM; >>>22μM;
MMP-7
MMP-7
MMP-7 > 22μM;
22µM;
22μM; MMP-8
>>MMP-8
22μM;
MMP-8
22μM; >>>2222
MMP-8
MMP-8
MMP-8 >µM;
22μM; 22 μM;
>> 22
μM; 22 μM;
μM;
MMP-9
MMP-9
MMP-9 =
= =8.9
MMP-9 8.9
MMP-9
MMP-9 8.9µM;
=μM;
μM;8.9MMP-10
μM;
MMP-10
== 8.9
8.9 μM;
MMP-10
μM; =
MMP-10 1616μM;
= =16
MMP-10
MMP-10 µM;
=μM;
16 MMP-12
μM;
MMP-12
== 16
16 μM;
MMP-12
μM; >>>22
2222μM;
MMP-12
MMP-12
MMP-12 µM;
>μM;
22 MMP-13
μM;
MMP-13
>> 22
22 μM;
MMP-13
μM; ===111nM;
MMP-13
MMP-13
MMP-13 nM;
nM; MMP-14
= 1==MMP-14
nM;
1 nM;
nM;
1MMP-14 ===8.3
MMP-14
MMP-14
MMP-148.3 =µM
8.3μM 8.3 μM
== 8.3
μM 8.3 μM
μM
IC
IC
IC
5050 :IC 50: 50
:50MMP-13
IC = ==
MMP-13 6.6
50:: MMP-13
MMP-13
IC MMP-136.6=μM
μM
6.6 6.6
µM μM
== 6.6
6.6 μM
μM
Ki: Ki:
Ki:MMP-2MMP-2
Ki:
MMP-2
Ki:
Ki: > >3.333
MMP-2
MMP-2
MMP-2 >>μM;
3.3333.333
μM;
3.333μM;
MMP-3
>>3.333
3.333 μM;
MMP-3
μM;
µM; MMP-3
> >4.501
MMP-3
MMP-3
MMP-3 >>
4.501
μM;
>>μM;
4.501 4.501
4.501 μM;
MMP-7
μM;
MMP-7
μM;
µM; MMP-7
> >636
MMP-7
MMP-7
MMP-7 >>
636
636nM; nM;
>> 636
nM;636 nM;
nM; ICIC :IC
IC50: 50
50:50MMP-12
IC
MMP-12
IC 50MMP-12> >2222μM;
50::: MMP-12
MMP-12
MMP-12 >
>μM;
22 μM;
22
>> 22 MMP-13
MMP-13
μM;
µM;
MMP-13
μM; > >100
MMP-13
MMP-13 >>
> μM
100μM100 μM
> 100
100 μM
µM
μM
MMP-12
MMP-12MMP-12
> >6.023
MMP-12
MMP-12
MMP-12 >>μM;
6.0236.023
μM;
6.023μM;
MMP-13
>>6.023
6.023 μM;
µM;
MMP-13
μM;MMP-13
= =4.314
MMP-13
MMP-13
MMP-13 = =μM
4.314 4.314
==μM μM
4.314
4.314
4.314 μM
µM
μM
collagenases [16,29].
Doxycycline (Figure 26a) is a semi-synthetic tetracycline that inhibits MMP-2, -9, -7, and -8 and
Doxycycline (Figure 26a) is a semi-synthetic tetracycline that inhibits MMP-2, -9, -7, and -8 and
is the only compound approved as an MMP inhibitor for the treatment of periodontitis [2,6]. The
is the only compound approved as an MMP inhibitor for the treatment of periodontitis [2,6]. The
COL-3 (Figure 26b) showed specificity for MMP-2, -9, and -14, by decrease trypsinogen-2 and
COL-3 (Figure 26b) showed specificity for MMP-2, -9, and -14, by decrease trypsinogen-2 and
inducible nitric oxide (iNO) production, which are regulators of MMP activity [16]. Although COL-3
inducible nitric oxide (iNO) production, which are regulators of MMP activity [16]. Although COL-3
is currently
Biomolecules being
2020, 10, 717 evaluated in clinical phase II trials, it showed poor solubility and stability
53[16].
of 61
is currently being evaluated in clinical phase II trials, it showed poor solubility and stability [16].
Figure
Figure 26. Doxycycline;
(a)
26. (a) (a) Doxycycline; (b) COL-3.
(b) COL-3.
COL-3.
Figure 26. Doxycycline; (b)
Table
Table 13 shows
13 shows the the
IC IC 50 values
values of some
of some tetracyclines-based
tetracyclines-based inhibitors
inhibitors [6,15–19,29,35,37,54,55,71].
[6,15–19,29,35,37,54,55,71].
Table 13 shows the IC50
50 values of some tetracyclines-based inhibitors [6,15–19,29,35,37,54,55,71].
Table
Table 13. IC
13. IC 50 values
values of tetracyclines-based
of tetracyclines-based inhibitors.
inhibitors.
Table 13. IC50
50 values of tetracyclines-based inhibitors.
IC50: MMP-1 400 μM;>MMP-7

IC :>MMP-1 28 μM; =
400 µM;=MMP-7 MMP-3
28 µM;= 30 μM;
IC50: MMP-1 > 50
400 μM; MMP-7 = 28 μM; MMP-3 = 30 μM;
MMP-3 =MMP-13 = 2 μM = 2 µM
30 µM; MMP-13
MMP-13 = 2 μM
Doxycycline
Doxycycline
Doxycycline
IC5050: MMP-8 == 30
IC 30 μM; MMP-13 ==11μM
µM; MMP-13 µM
IC50: MMP-8 = 30 μM; MMP-13 = 1 μM
Biomolecules2020,
10,717
717 57ofof64
57 64
CMT-1
CMT-1
IC5050: :MMP-1
IC MMP-1==34
34μg.
μg.mL
mL−1−1; ;MMP-8
MMP-8==48 48μg.
μg.mL
mL−1−1; ;MMP-13
MMP-13 ==
IC50 : MMP-1 = 34 µg·mL−1 ; MMP-8 = 48 µg·mL −1 ;
0.3
0.3 μg.
MMP-13 μg. mL
= 0.3mL
−1
−1
µg·mL −1
Matastat(COL-3;
Matastat (COL-3;CMT-3)
CMT-3)
ICIC
IC5050 MMP-9== 272
: :MMP-9 272 µM
μM
50: MMP-9 = 272 μM
Minocycline
Minocycline
Minocycline
In 2000,
4.2.7. Mobashery et Inhibitors
Mechanism-Based al. [72] were the first to report this novel type of MMPi that blocks
4.2.7. Mechanism-Based Inhibitors
gelatinases with a unique mechanistic mode [72]. The thiirane inhibitor showed a mechanism-based,
slow-bindingThemechanism-based
The mechanism-based
inhibition for MMP-2 inhibitors
inhibitors
and MMP-9 coordinate
coordinate with catalytic
with
[72]. Bernardo catalytic zinc
zinc
et al. [73] ion,
ion,
also inaamonodentate
in
reported monodentate
a slow-binding mode,
mode,
allowing the nucleophilic
allowing the nucleophilic
thiirane-containing attack
attack27),
inhibitor, (Figure by a conserved
by selective
a conserved glutamic
glutamic
for MMP-2 residue
andresidue
-9, where on
onthethe active
thesulfur
active site and forming aa
and forming
site coordinates
group
covalent
withcovalent bond [2,17].
bond [2,17].
the catalytic This attack
Thisactivates
zinc ion, causes
attack causes a conformational
a conformational
the thiirane change
group to change in
interactinwith the catalytic
the catalytic
the active site environment
sitesite
environment
glutamate,[17][17]
preventingdissociation
preventing
by nucleophilic dissociation
attack causing ofthe
of the inhibitor
a inhibitor
loss anddecreasing
and
of activity decreasing
[73]. These therate
the rateof ofcatalytic
inhibitors catalytic
are the turnover
turnover andthe
and
first example theamount
amount
of a
of inhibitor needed
of inhibitor needed
suicide-inhibitor of MMPs to saturate
to saturate the enzyme
[73]. the enzyme [2]. [2].
In 2000,
In 2000, Mobashery
Mobashery etet al. al. [72]
[72] were
were thethe first
first to
to report
report this
this novel
novel type
type of of MMPi
MMPi thatthat blocks
blocks
gelatinaseswith
gelatinases withaaunique
uniquemechanistic
mechanisticmode mode[72].[72].The
Thethiirane
thiiraneinhibitor
inhibitorshowed
showedaamechanism-based,
mechanism-based,
slow-binding inhibition for MMP-2 and MMP-9 [72]. Bernardo et al. [73] also reported aaslow-binding
slow-binding inhibition for MMP-2 and MMP-9 [72]. Bernardo et al. [73] also reported slow-binding
thiirane-containing inhibitor, (Figure 27), selective for MMP-2
thiirane-containing inhibitor, (Figure 27), selective for MMP-2 and -9, where the sulfur and -9, where the sulfur group
group
coordinateswith
coordinates withthethecatalytic
catalyticzinc
zincion,
ion,activates
activatesthe thethiirane
thiiranegroup
groupto tointeract
interactwith
withthe theactive
activesite
site
gelatinases with a unique mechanistic mode [72]. The thiirane inhibitor showed a mechanism-based,
slow-binding inhibition for MMP-2 and MMP-9 [72]. Bernardo et al. [73] also reported a slow-binding
thiirane-containing inhibitor, (Figure 27), selective for MMP-2 and -9, where the sulfur group
coordinates with the catalytic zinc ion, activates the thiirane group to interact with the active site
glutamate, by nucleophilic attack causing a loss of activity [73]. These inhibitors are the first example
of a suicide-inhibitor of MMPs [73].
Figure 27. Inhibitor developed by Bernardo el al. The sulfur group coordinates with the catalytic
Figure 27. Inhibitor developed by Bernardo el al. The sulfur group coordinates with the catalytic zinc
zinc ion and the activation of the thiirane group happened with interactions between the active site
ion and the activation of the thiirane group happened with interactions between the active site
glutamate, by nucleophilic attack.
glutamate, by nucleophilic attack.
Thiirane-based ND-322 is a small molecule selective to MMP-2/MT1-MMP [2]. This inhibitor
has been shown to reduce melanoma cell growth, migration, and invasion, and to delay metastatic
dissemination [2].
SB-3CT is a selective inhibitor of MMP-2 and -9 [2]. The inhibition mechanism is similar to a
“suicide inhibitor” in which a functional group is activated, leading to covalent modification of the
active site [2]. SB-3CT also shows slow-binding kinetics with MMP-2, -3, and -9, contributing to slow
dissociation of the MMP-inhibitor complex, but it is a reversible inhibitor which differentiates it from
the truly irreversible suicide inhibitors [2]. O SB-3CT has potential benefits in brain damage caused by
cerebral ischemia and has anti-cancer effects in T-cells lymphoma and prostate cancer models [2].
4.3. Catalytic Domain (Non-Zinc Binding) Inhibitors

The catalytic domain of MMPs contains other regions that can be exploited [17]. The first
3D-structure of the complex MMP-1 (catalytic domain)-synthetic inhibitor was reported in 1994 by
Glaxo researchers [35]. Thereafter, other complexes have been studied and it was found that the
S1 ’ pockets have different depths among MMPS and this difference has been utilized in developing
selective MMPis [28,35].
Stockman and Finel optimized two distinct series of MMP-3 inhibitors: PNU-141803 (amide,
Figure 28a) and PNU-142372 (urea, Figure 28b) [19]. The connection between MMP-3 and PNU-142372
shows that the aromatic ring from the inhibitor extends to the S3 pocket (hydrophobic) and the
thiadiazole sulfur group interacts with the catalytic zinc [19]. Moreover, the two nitrogen atoms
form hydrogen bonds with Ala164 and Glu202 residues [19]. The alkylation of nitrogen atom or its
replacement for carbon leads to the removal activity [19]. The replacement of a tyrosine for a serine
within the S3 pocket (present in MMP-1) leads to the removal of inhibitory activity and explains the
absence of activity against collagenases [19].
142372 shows
142372 shows that
that the
the aromatic
aromatic ring
ring from
from the
the inhibitor
inhibitor extends
extends to
to the
the SS33 pocket
pocket (hydrophobic)
(hydrophobic) andand
the thiadiazole sulfur group interacts with the catalytic zinc [19]. Moreover, the two nitrogen
the thiadiazole sulfur group interacts with the catalytic zinc [19]. Moreover, the two nitrogen atoms atoms
form hydrogen
form hydrogen bondsbonds with
with Ala
Ala164
164 and Glu202 residues [19]. The alkylation of nitrogen atom or its
and Glu202 residues [19]. The alkylation of nitrogen atom or its
replacement for carbon leads to the
replacement for carbon leads to the removal removal activity
activity [19].
[19]. The
The replacement
replacement of of aa tyrosine
tyrosine for
for aa serine
serine
within the S
within the2020, 3 pocket (present in MMP-1) leads to the removal of inhibitory activity and explains the
S3 pocket
Biomolecules 10, 717 (present in MMP-1) leads to the removal of inhibitory activity and explains 55 ofthe
61
absence of activity against collagenases
absence of activity against collagenases [19]. [19].
Figure 28.
Figure
Figure 28. (a)
28. PNU-141803; (b)
(a) PNU-141803;
PNU-141803; (b) PNU-142372.
(b) PNU-142372.
PNU-142372.
Sanofi-Aventis
Sanofi-Aventis developed
developed aaacompound
compound(Figure
(Figure29) 29)for
forMMP-13
MMP-13(IC 50 =
(IC5050 6.6 μM),
μM), with
µM), with very
very high
Sanofi-Aventis developed compound (Figure 29) for MMP-13 (IC == 6.6 with very high
high
selectivity [6].
selectivity [6].
selectivity This compound binds deeply to the S ’ pocket and to a side
[6]. This compound binds deeply to the SS111’ pocket and to a side pocket that haspocket that has not
has not been
not been
been
identified
identified for
for other
other MMPs
MMPs [6].
[6]. The
The pyridyl
pyridyl moiety
moiety is
is towards
towards to
to the
the entrance
entrance of
of the
the
identified for other MMPs [6]. The pyridyl moiety is towards to the entrance of the S1’ pocket, without S
S11’ pocket, without
without
interacting
interactingwith
interacting with the
with the catalytic
the catalytic Zn(II)
catalytic Zn(II) ion
Zn(II) ion and
ion and the
and oxygen
the oxygen
the atoms
oxygen atoms neither
neither from
atoms neither from the
from amide (peptidic)
the amide (peptidic)
(peptidic) bonds
bonds
bonds
of the
of the
of main
the main chain
main chain (between
chain (between Thr
(between Thr
Thr245 and Thr
245 and Thr247)
245 and Thr247
) nor from hydroxyl group from the
group from the Thr247
247) nor from hydroxyl group from the Thr247
Thr 247
side chain in
side chain in
in
the
the S
S11’ pocket [6].
the S1’ pocket [6].
Figure 29. Sanofi-Aventis compound.

Figure 29.
Figure 29. Sanofi-Aventis
Sanofi-Aventis compound.
compound.
Many natural compounds have been shown to possess selective inhibition [28]. Wang et al.
identified 19 potential MMPis from 4000 natural compounds isolated from medicinal plants [28].
The caffeates and flavonoids were found to be selective inhibitors against MMP-2 and -9, by occupying
the S1 ’ and S3 pockets [28].
The marine natural products are another pharmacological resource and include derivates from
algae, sponges, and cartilages [28]. Some examples are Neovastat, Dieckol, and Ageladine A and they
manifest anti-angiogenic, anti-proliferative, and anti-tumor effects [28].
Although the natural MMPis are more biocompatible and less toxic, they have disadvantages
such as the effective dosages are in micromolar scale, which is thousands of times higher than synthetic
inhibitors and are difficult to patent, making the pharmacological companies and investors reluctant to
sponsor large-scale clinical trials [28].
4.4. Allosteric and Exosite Inhibitors

The catalytic zinc ion is common in all MMPs, therefore, if interactions of the substrates with
this ion are minimized this would improve the inhibitor selectivity [2]. The hemopexin-like domain
can move relatively to the catalytic domain and allosterically manipulate enzymatic activity by
conformation deformation [28]. The allosteric drugs have a non-competitive inhibition mode [16,28],
they bind and lock the MMP active site, forcing it to take less favorable conformation for substrate
binding [2,16], avoiding off-target inhibition [28] and preventing the occurrence of side effects [28].
Exosite inhibitors are another alternative for selective MMPis since these inhibitors bind to alternative
sites of MMPs [16,28].
Remacle et al. reported NSC405020, a small molecule that binds selectively to the hemopexin-like
domain of MMP-14 [28]. This molecule inhibits the MMP-14 homodimerization and the interaction
between the hemopexin-like domain and catalytic domain, preventing the type I collagen
degradation [28].
Dufour et al. developed a peptide targeting the MMP-9 hemopexin-like domain, which blocks
MMP-9 dimer formation and cell migration [28]. Scannevin et al. identified a highly selective
compound (JNJ0966), which binds to the MMP-9 pro-peptide domain, inhibiting its activation, but not
affecting the activity of MMP-1, -2, and -14 [28].
Xu et al. synthesized a peptide which inhibits the hydrolysis of type I and IV collagen by MMP-2,
through binding to its collagen binding domain [28].
4.5. Antibody-Based Inhibitors

Antibodies are selective and have high affinity to MMP [27]. Clinical trials utilizing antibodies have
provided evidence that selective MMP inhibitors do not induce MSS [27]. The therapeutic potential of
anti-MMP antibodies has yet to be realized [27]. The antibodies may also undergo proteolysis, may be
removed from circulation rapidly, and are costly [27].
Antibodies are large Y-sharped proteins which bind to an antigen via the fragment antigen-binding
(Fab variable region) [28]. Monoclonal antibodies are highly specific, and they have affinity to
MMPs [2,29]. The hemopexin domain can be a potential target for MMPs antibodies [2].
REGA-3G12 and REGA-2D9 are antibodies specific to MMP-9 [2,27,28] but not MMP-2 [28].
The inhibition mode of the REGA-3G12 involves the catalytic domain, the N-terminal region
Trp116 -Lys214 [28], and not the catalytic zinc ion or the fibronectin region [2]. The AB0041 and
AB0046 are monoclonal anti-MMP-9 antibodies, which showed inhibition to tumor growth and
metastasis in a model of colorectal carcinoma [27].
Andecaliximab (GS-5745), the humanized version of AD0041 [27], is a highly selective antibody
and exerts allosteric control over tumor growth and metastasis in a colorectal carcinoma model (IC50
= 0.148 nM) [28]. This antibody is the only inhibitor that has undergone clinical trials [27,28] and it
inhibits the pro-MMP-9 activation and inhibits non-competitively the MMP-9 activity [27].
DX-2400 is an antibody isolated from phage and it targets MMP-14 and the MMP-14-pro-MMP-2
complex, decreasing MMP activity [28]. DX-2400 inhibits the metastasis in a breast cancer xenograft
mouse model [27]. However, the LAM-2/15 is the only selective inhibitor that inhibits MMP-14 catalytic
activity, but not the pro-MMP-2 activation or MMP-14 dimerization [28]. The 9E8 is another antibody
targeting MMP-14 which does not affect the catalytic activity of MMP-14 but inhibits the pro-MMP-2
activation [28].
Human scFv-Fc antibody E3 is bound to the catalytic domain of MMP-14 and inhibits type I
collagen binding [27]. Human antibody Fab libraries were synthetized and the peptide G sequence
(Phe-Ser-Ile-Ala-His-Glu) was incorporated resulting in Fab 1F8 antibody inhibitor, which inhibits the
catalytic domain of MMP-14 [27].
Antibodies can also inhibit a specific activation of an MMP [27]. For example, the mAb 9E8
inhibits the MMP-14 activation of proMMP-2 but not the catalytic activities of MMP-14 [27]. The
antibody LOOPAB also inhibits the MMP-14 activation of pro-MMP-2 but not collagenolysis activity of
MMP-14 [27]. There are antibodies that reduce the MMP expression [71].
5. Why Do MMP is Fail?

MMI inhibitor side effects are predominantly related to off-target metal chelation [74]. The majority
of MMPis used clinically are hydroxamic acid derivates with low selectivity [74] hence, they can
inhibit other proteinases [28]. The most frequent side effects observed in MMPis clinical trials is
the musculoskeletal syndrome (MSS) [17,28], which manifested as pain and immobility in the joints,
arthralgias, contractures in the hands, and an overall reduced quality of life [14,15,74], leading to MMPis
failing in the last phases of clinical trials [14]. Several studies indicate that the development of MSS is
related to dose and time, with slightly different kinetic for the different MMPis, and the development of
MSS is an indicator of successful MMPis [14,74]. The MMP inhibitors focused on chelating the catalytic
zinc ion have poor selectivity and resulted in MSS and gastrointestinal disorders [27]. However, the
exact causes of MSS remains unknown [74], but can be related to a simultaneous inhibition of several
MMPs [6,17,27].
Analysis of the expression of a target protein shows its presence at high levels when a disease is
manifested or at low levels or absence in a healthy state [74]. However, these studies do not determine
if a particular protein is directly associated with the disease process or if it is involved in ancillary
event [74]. Studies of genetic manipulation in mouse as animal models determine the roles of MMPs in
various pathological processes [74]. However, there are caveats in the use of animal models [74]:
- The observed effects can be a consequence of the manipulated absence of MMP, being a
compensation mechanism;
- The mouse models are unable to replicate the complexity of any human disease. The mouse
models serve to recreate specific processes or sets of processes but not the physiological changes
that occur in humans.
6. Conclusions
Due to the side effects rising from the lack of selectivity and from the insufficient knowledge about
the role of each MMP in the different pathological processes, none of the designed synthetic MMP
inhibitors have yet passed the clinical trials and reached the market [6,27]. The poor performance of
MMP inhibitors in clinical trials has globally been attributed to [27]:
- Inhibition of other metalloenzymes;

- Lack of specificity within the MMP family;
- Poor pharmacokinetics;
- Dose-limiting side effects/toxicity;
- In vivo instability;
- Low oral availability/inability to assess inhibition efficacy.
In 1988, the first inhibitor was synthesized but after nearly 30 years, only one drug, Periostat® ,
doxycycline hydrate, had obtained approval from the FDA for the treatment of periodontal
disease [6,16,17,27,28]. This inhibitor exhibited also therapeutic effects in treating aortic aneurysm,
multiple sclerosis, as well as Type II diabetes [28].
Funding: This research was funded by A MOLECULAR VIEW OF DENTAL RESTORATION, grant
number PTDC/SAU-BMA/122444/2010 and by MOLECULAR DESIGN FOR DENTAL RESTAURATION,
grant number SAICT-POL/24288/2016. The work was supported by the Associate Laboratory for Green
Chemistry-LAQV FCT/MCTES (UIDB/50006/2020), and Applied Molecular Biosciences Unit-UCIBIO FCT/MCTES
(UID/Multi/04378/2019), which are financed by national funds.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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biomolecules

Keywords: matrix metalloproteinases; TIMP; synthetic inhibitors

Biomolecules 2020, 10, 717; doi:10.3390/biom10050717 www.mdpi.com/journal/biomolecules

Table 1. Matrix metalloproteinases (MMPs) classes.

Biomolecules 2020, 10, 717 3 of 64

Table 2. MMPs inhibitors classification.

Specific Inhibitor Tissue Inhibitor of Metalloproteinases (TIMP)

2. Specific Endogenous Inhibitor-Tissue Inhibitors of Metalloproteinases (TIMPs)

Table 3. Tissue inhibitors of metalloproteinases (TIMPs) classification.

TIMP Expression Inhibition Inhibition Mode

Figure 2.2. (a)

3. Non-Specific Endogenous Inhibitors

Table 4. Non-specific endogenous inhibitors [4,7,12,13,33,34].

Non-Specific Inhibitor Inhibition

4.1. Hydroxamate-Based Inhibitors

Modification in Ra position (α substituent): a beneficial effect is conferred by lipophilic substituents

Modification in Ra position (α substituent): a beneficial effect is conferred by lipophilic

Biomolecules 2020, 10, 717 8 of 64

The introduction of conformational restrictions through the addition of a three-membered ring

Figure 11. (a)

Figure 12. KB-R7785 inhibitor.

Modifications in R3 position: theFigure S3 ’ pocket is an open

Figure 15. (a) CGS-27023A; (b) thioester derivate of CGS-27023A.

ICIC : MMP-1= =131

Ki: MMP-1 > 5 μM; MMP-2 > 3 μM; MMP-3 =

Table 7 shows the IC50 Figure and 17.Ki (a)

Figure 18. Inhibitor

Biomolecules 2020, 10, 717 Table 7. Cont. 27 of 64

RS-104,966 IC50==: MMP-2

Biomolecules 2020, 10, 717 2020, 10, 717

IC50: MMP-1 =:IC

Ro-32-7315 Ro-32-7315 IC50: MMP-1IC50:

4.2.1. Thiolates-Based Inhibitors

4.2.1. Thiolates-Based Inhibitors

Ki: MMP-1 Ki:

IC IC ::50 MMP-3 ==== 600 nM

IC IC (X,Y 50 (X,Y=== O):=MMP-1 MMP-1

Ki: MMP-2 = nM;

Biomolecules 2020, 10, 717

Ki: MMP-1Ki:= MMP-1

Ki: MMP-2 = 57Ki:

4.2.3. Phosphorus-Based Inhibitors

= 160 = 20 = 150 ICIC

4.2.4. Nitrogen-Based Inhibitors

4.2.5. Heterocyclic Bidentate-Based Inhibitors

4.2.6. Tetracyclines-Based Inhibitors

4.2.7. Mechanism-Based Inhibitors

AZD-126 IC5050:: MMP-1

IC :: MMP-1 >> 50 μM; MMP-2 == 9.3 μM;

KI: MMP-1 > 500 µM; MMP-9 = 6 µM IC :: MMP-1 >> >

IC50: MMP-1IC : MMP-1

> IC IC: >50

IC50: MMP-1 > 18.6

IC50: MMP-1 400 μM;>MMP-7

4.3. Catalytic Domain (Non-Zinc Binding) Inhibitors

Figure 29. Sanofi-Aventis compound.

4.4. Allosteric and Exosite Inhibitors

4.5. Antibody-Based Inhibitors

5. Why Do MMP is Fail?

- Inhibition of other metalloenzymes;

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