Challenges in Matrix Metalloproteinases Inhibition

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.

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
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 substituents [19].
Replacement of the R1-R2 bond of succinyl hydroxamates acid inhibitors by a sulfonamide bond ( Figure 10a) results in substantial loss of inhibitory activity because the hydrogen bond (C=ONH; Figure 10b) with leucine is stronger than the new sulfonyl oxygen bond, due to the pyramidal nature of the sulfonamide [19]. Modifications in R2 position: modifications in the R2 position led to a modest effect in inhibitory 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 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 Figure 9. Inhibitors with modification of R 1 position. (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, Matlystatin derivate. The inhibition of MMP-9 is 10 times higher than analogues with C 8 or C 10 chains; (c) succinyl hydroxamate analogue with C 10 chain, which inhibits MMP-1; (d) succinyl hydroxamate analogue with C 16 chain, which inhibits MMP-1.
Replacement of the R 1 -R 2 bond of succinyl hydroxamates acid inhibitors by a sulfonamide bond ( Figure 10a) results in substantial loss of inhibitory activity because the hydrogen bond (C=ONH; Figure 10b) with leucine is stronger than the new sulfonyl oxygen bond, due to the pyramidal nature of the sulfonamide [19].
Replacement of the R1-R2 bond of succinyl hydroxamates acid inhibitors by a sulfonamide bond ( Figure 10a) results in substantial loss of inhibitory activity because the hydrogen bond (C=ONH; Figure 10b) with leucine is stronger than the new sulfonyl oxygen bond, due to the pyramidal nature of the sulfonamide [19]. Modifications in R2 position: modifications in the R2 position led to a modest effect in inhibitory 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 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 Waals interactions, comparing with other groups [46].  Ikeda et al. described compounds with phenyl R2 substituents ( Figure 12) (KB-R7785), which are 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 against MMP-1 in rats and its effectiveness in arthritis has been demonstrated [47]. 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 to the MMPs-3 and -7 [19].

Succinyl Hydroxamic Acid-Based Inhibitors
Succinyl hydroxamate derivates can be subdivided to peptide derivatives or non-peptide compounds [48]. The N-acetylcysteine has been reported to affect the tumoral invasion process and metastasis by MMP-2 and -9 inhibition [49]. The L-cysteine-2-phenylethylamide is an effective inhibitor, in which the phenyl group fills the S1' pocket of MMP-8 [50]. Foley et al. prepared several dipeptides derivatives containing cysteine (RCO-Cys-AA-NH2) and concluded [51]: - The variation of the acyl group and the second amino acid (AA) leads to the activity against different MMPs.

-
The R group interacts with the S1' pocket.
Batimastat (Table 5) was the first MMPi to enter in clinical trials for cancer as it inhibits MMP-1, -2, -7, and -9, but, due to its poor oral bioavailability, it was superseded by Marimastat [28,29,31] (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 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 Ikeda et al. described compounds with phenyl R 2 substituents ( Figure 12) (KB-R7785), which are active orally, due to the beneficial effect of the R 2 phenyl group on absorption, where the amide shielding and lipophilicity may assist in transepithelial resorption [47]. This inhibitor shows activity against MMP-1 in rats and its effectiveness in arthritis has been demonstrated [47]. Ikeda et al. described compounds with phenyl R2 substituents ( Figure 12) (KB-R7785), which are 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 against MMP-1 in rats and its effectiveness in arthritis has been demonstrated [47]. 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 to the MMPs-3 and -7 [19].

Succinyl Hydroxamic Acid-Based Inhibitors
Succinyl hydroxamate derivates can be subdivided to peptide derivatives or non-peptide compounds [48]. The N-acetylcysteine has been reported to affect the tumoral invasion process and metastasis by MMP-2 and -9 inhibition [49]. The L-cysteine-2-phenylethylamide is an effective inhibitor, in which the phenyl group fills the S1' pocket of MMP-8 [50]. Foley et al. prepared several dipeptides derivatives containing cysteine (RCO-Cys-AA-NH2) and concluded [51]: - The variation of the acyl group and the second amino acid (AA) leads to the activity against different MMPs.

-
The R group interacts with the S1' pocket.
Batimastat (Table 5) was the first MMPi to enter in clinical trials for cancer as it inhibits MMP-1, -2, -7, and -9, but, due to its poor oral bioavailability, it was superseded by Marimastat [28,29,31] (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 trials due to the absence of a therapeutic effect and the patients treated developed musculoskeletal toxicity (MST) [  Modifications in R 3 position: the S 3 ' pocket is an open area and several groups can be introduced at the R 3 position [19]. The introduction of the benzhydryl group leads to compounds with selectivity to the MMPs-3 and -7 [19].

Succinyl Hydroxamic Acid-Based Inhibitors
Succinyl hydroxamate derivates can be subdivided to peptide derivatives or non-peptide compounds [48]. The N-acetylcysteine has been reported to affect the tumoral invasion process and metastasis by MMP-2 and -9 inhibition [49]. The L-cysteine-2-phenylethylamide is an effective inhibitor, in which the phenyl group fills the S 1 ' pocket of MMP-8 [50]. Foley et al. prepared several dipeptides derivatives containing cysteine (RCO-Cys-AA-NH 2 ) and concluded [51]: - The variation of the acyl group and the second amino acid (AA) leads to the activity against different MMPs. - The R group interacts with the S 1 ' pocket.

Sulfonamide Hydroxamic Acid-Based Inhibitors
In 1995, Novartis described the CGS-27023A (Figure 15a), a non-peptidic MMP-3 inhibitor, which has good oral availability but did not succeed in clinical trials [56]. The isopropyl group slows down the metabolization of the adjacent hydroxamic acid group and the 3-pyridyl substituent may aid partitioning into the hydrated negatively charged environment of the cartilage [56]. By analysis of the cocrystal structure of this inhibitor and MMP-12, it was possible to conclude that the binding mode between the hydroxamate moiety and the catalytic zinc ion was the same as the binding mode of hydroxamate-based inhibitors [6]. The interaction of CGS-27023A with MMP-3 was possible due to the p-methoxy phenyl substituent occupation of the S 1 ' pocket and the pyridylmethyl and isobutyl groups occupation of the S 2 ' and S 1 pockets, respectively [29,56]. The modification of α to form the thioester derivate led to an increase of the inhibition of the deep pocket of the MMPs [29,56] (Figure 15b). In 1995, Novartis described the CGS-27023A (Figure 15a), a non-peptidic MMP-3 inhibitor, which has good oral availability but did not succeed in clinical trials [56]. The isopropyl group slows down the metabolization of the adjacent hydroxamic acid group and the 3-pyridyl substituent may aid partitioning into the hydrated negatively charged environment of the cartilage [56]. By analysis of the cocrystal structure of this inhibitor and MMP-12, it was possible to conclude that the binding mode between the hydroxamate moiety and the catalytic zinc ion was the same as the binding mode of hydroxamate-based inhibitors [6]. The interaction of CGS-27023A with MMP-3 was possible due to the p-methoxy phenyl substituent occupation of the S1' pocket and the pyridylmethyl and isobutyl groups occupation of the S2' and S1 pockets, respectively [29,56]. The modification of α to form the thioester derivate led to an increase of the inhibition of the deep pocket of the MMPs [29,56] (Figure  15b). 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 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 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 acid, which possess potent inhibitory activity for MMP-13 [58]. In the first series of compounds, the 3position of the piperidine ring was explored by the introduction of a polar functionality and it resulted in a compound with excellent activity on MMP-13 (Figure 16c), improved bioavailability, and lower metabolic clearance [58]. 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 S 1 ' pocket, but does 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 bond with Leu 160 and the piperidine-O-phenyl moiety extends into the S 1 ' pocket by Van der Waals interactions [57]. Noe et al. described a series of 3,3-dimethyl-5-hydroxy pipecolic hydroxamic acid, which possess potent inhibitory activity for MMP-13 [58]. In the first series of compounds, the 3-position of the piperidine ring was explored by the introduction of a polar functionality and it resulted in a compound with excellent activity on MMP-13 (Figure 16c), improved bioavailability, and lower metabolic clearance [58].                     The incorporation of a cyclic quaternary center α led to a strong inhibitory effect against MMP-1, -2, -3, -8, -9, -12, and -13 [29]. The RS-113,456 ( Figure 17a) is an inhibitor with better oral bioavailability and metabolic stability compared to the hydroxamate derivates [35]. These two features were improved by shifting the cyclic group to the α-position of the hydroxamic acid ( Figure 17b) [29].   The incorporation of a cyclic quaternary center α led to a strong inhibitory effect against MMP-1, -2, -3, -8, -9, -12, and -13 [29]. The RS-113,456 ( Figure 17a) is an inhibitor with better oral bioavailability and metabolic stability compared to the hydroxamate derivates [35]. These two features were improved by shifting the cyclic group to the α-position of the hydroxamic acid ( Figure 17b) [29]. The incorporation of a cyclic quaternary center α led to a strong inhibitory effect against MMP-1, -2, -3, -8, -9, -12, and -13 [29]. The RS-113,456 ( Figure 17a) is an inhibitor with better oral bioavailability and metabolic stability compared to the hydroxamate derivates [35]. These two features were improved by shifting the cyclic group to the α-position of the hydroxamic acid ( Figure 17b) [29].  Table 7 shows the IC50 and Ki values of some sulfonamide hydroxamic acid-based inhibitors [6, [15][16][17][18][19]29,35,37,54,55].  Table 7 shows the IC 50 and Ki values of some sulfonamide hydroxamic acid-based inhibitors [6, [15][16][17][18][19]29,35,37,54,55].

Phosphamides Hydroxamic Acid-Based Inhibitors
The hydroxamic acids based on phosphamides are effective as MMPis due to the electronic environment of the phosphor atom [11]. The replacement of sulphonamide group by phosphinamide group leads to a potent inhibitor of MMP-3 ( Figure 18), the collagenases and gelatinases [29]. The interactions between this inhibitor and the MMP-3 are realized by the phosphinamide phenyl group, that accommodates into the S 1 ' pocket and by the phosphinamide oxygen, which establishes the hydrogen bonds with NH of Leu 164 and Ala 165 [29]. However, this group is susceptible to hydrolysis at low pH, limiting the inhibitory activity [29]. The hydroxamic acids based on phosphamides are effective as MMPis due to the electronic environment of the phosphor atom [11]. The replacement of sulphonamide group by phosphinamide group leads to a potent inhibitor of MMP-3 ( Figure 18), the collagenases and gelatinases [29]. The interactions between this inhibitor and the MMP-3 are realized by the phosphinamide phenyl group, that accommodates into the S1' pocket and by the phosphinamide oxygen, which establishes the hydrogen bonds with NH of Leu164 and Ala165 [29]. However, this group is susceptible to hydrolysis at low pH, limiting the inhibitory activity [29]. 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].      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]. Table 8 shows the IC 50 and Ki values of some phosphamides hydroxamic acid-based inhibitor [6, [15][16][17][18][19]29,35,37,54,55].  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]. Table 8 shows the IC50 and Ki values of some phosphamides hydroxamic acid-based inhibitor [6, [15][16][17][18][19]29,35,37,54,55].  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]. Table 8 shows the IC50 and Ki values of some phosphamides hydroxamic acid-based inhibitor [6, [15][16][17][18][19]29,35,37,54,55].
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 Zn 2+ chelating ability and the rates of severe side effects, such as the musculoskeletal syndrome (MSS) decreased dramatically compared with the hydroxamate inhibitors [28].

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
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 Zn 2+ chelating ability and the rates of severe side effects, such as the musculoskeletal syndrome (MSS) decreased dramatically compared with the hydroxamate inhibitors [28].

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 thiol-based for MMP-1 is a bipeptidic analogue, where the incorporation of a thiol group as α substituent leads to improvement of activity (Figure 19a) [19]. Derivates with "linker" substituent between P 1 -P 1 ' positions show a total loss of activity (Figure 19b) [19,62]. On the contrary incorporation of a methyl carboxylate group leads to a significant increase in activity (Figure 19c) [19]. The increased activity of these compounds may be a consequence of beneficial interactions between S 1 , the carbonyl ester, and the thiol group, participating in the bidentate coordination of the zinc [19]. [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 Zn 2+ chelating ability and the rates of severe side effects, such as the musculoskeletal syndrome (MSS) decreased dramatically compared with the hydroxamate inhibitors [28].

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 thiol-based for MMP-1 is a bipeptidic analogue, where the incorporation of a thiol group as α substituent leads to improvement of activity (Figure 19a) [19]. Derivates with "linker" substituent between P1-P1' positions show a total loss of activity (Figure 19b) [19,62]. On the contrary incorporation of a methyl carboxylate group leads to a significant increase in activity (Figure 19c) [19]. The increased activity of these compounds may be a consequence of beneficial interactions between S1, the carbonyl ester, and the thiol 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 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 carboxylate group.
Montana et al. have identified a series of inhibitors with mercaptoacyl, obtaining moderate 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 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 S analogue; (b) thiol-based inhibitor with "linker" substituent; (c) thiol-based inhibitor with methyl carboxylate group.
Montana et al. have identified a series of inhibitors with mercaptoacyl, obtaining moderate 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 the zinc of the active site [63]. Warshasky et al. have produced a variety of compounds in the Montana series, in which the amide nitrogen P 2 ' is linked to the group P 1 ' (Figure 20b) [19].  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 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 inhibitors [64]. 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 inhibitors [64]. In 2005, Hurst et al. [65] reported a series of mercaptosulphides inhibitors that targeted MMP-1 [65]. The structure-activity relationship indicates that the five-membered ring increases the stability of the inhibitor compared to the linear structure, which can be quickly oxidized and lose its potency [65]. Table 9 shows the IC50 and Ki values of some thiolates-based inhibitors [6, [15][16][17][18][19]29,35,37,54,55]. In 2005, Hurst et al. [65] reported a series of mercaptosulphides inhibitors that targeted MMP-1 [65]. The structure-activity relationship indicates that the five-membered ring increases the stability of the inhibitor compared to the linear structure, which can be quickly oxidized and lose its potency [65]. Table 9 shows the IC 50 and Ki values of some thiolates-based inhibitors [6, [15][16][17][18][19]29,35,37,54,55].

Carboxylates-Based 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 S 1 ' 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 P 1 ' 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 esters has been identified with "phthalamidobutyl" (Figure 22b), increasing activity against MMP-3 and further increasing selectivity [67].   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′ esters has been identified with "phthalamidobutyl" (Figure 22b), increasing activity against MMP-3 and further increasing selectivity [67]. The interaction of the P1′ biphenyl substituent with pocket S1′ is an important factor contributing to the binding of the inhibitor [19]. The X-ray structure of the acyclic compound with MMP-3 revealed an important interaction between the phenyl terminal of the biphenyl group and the side chain of histidine (His224) [19]. The carboxylic acids derived from "D-valine" have a selective inhibition for MMP-2 and -3 [19]. The 4-substitution of the biphenyl ring helped to increase potency compared to the unsubstituted analogue and also helped to improve the pharmacokinetic properties [19].
With the aim of development inhibitors with high selectivity for a single MMP, Wyeth published, in 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 the carboxylate function connected to a benzofuran The interaction of the P 1 biphenyl substituent with pocket S 1 is an important factor contributing to the binding of the inhibitor [19]. The X-ray structure of the acyclic compound with MMP-3 revealed an important interaction between the phenyl terminal of the biphenyl group and the side chain of histidine (His 224 ) [19]. The carboxylic acids derived from "D-valine" have a selective inhibition for MMP-2 and -3 [19]. The 4-substitution of the biphenyl ring helped to increase potency compared to the unsubstituted analogue and also helped to improve the pharmacokinetic properties [19].

Phosphorus-Based Inhibitors
The capacity of the phosphoric group to reproduce the gem-diol intermediate during peptide hydrolysis was explored with different structures to obtain potent MMPis [5]. The phosphorus-based of the peptide-analogous inhibitors can be phosphonates/phosphonic acids, phosphoramidates, phosphonamidates, and phosphinates/phosphinic peptides [27]. The phosphinic acid (PO(OH)-CH 2 ) mimics the transition state obtained in substrate degradation, where each oxygen atom can coordinate both the catalytic zinc and the catalytic Glu [6, 19,27]. The phosphinic acids are monodentate chelates [27]. In contrast to hydroxamate compounds, the phosphinic compounds interact with both the primed and unprimed side of the catalytic site [17,27,35] due to the placement of the ZBG in the middle of the scaffold and not at its Nor C-terminal, as in the cases of hydroxamate and carboxylate inhibitors [17]. Another advantage of phosphinic acids is the improved metabolic stability compared with hydroxamate acids [27].
The effectiveness of phosphoric acid inhibitors has been studied and it has been found that the three pockets unprimed are connected to obtain the maximum performance [19]. The S 1 -S 2 pockets can be exploited using aromatic groups [19,68], that is why Reiter et al. prepared compounds with 4-benzyl ( Figure 24) as a substituent to fill S 2 pocket. They found that in the absence of this substituent or its replacement by small aliphatic or cyclohexyl methyl groups led to a loss of activity [19,68]. The capacity of the phosphoric group to reproduce the gem-diol intermediate during peptide hydrolysis was explored with different structures to obtain potent MMPis [5]. The phosphorus-based of the peptide-analogous inhibitors can be phosphonates/phosphonic acids, phosphoramidates, phosphonamidates, and phosphinates/phosphinic peptides [27]. The phosphinic acid (PO(OH)-CH2) mimics the transition state obtained in substrate degradation, where each oxygen atom can coordinate both the catalytic zinc and the catalytic Glu [6, 19,27]. The phosphinic acids are monodentate chelates [27]. In contrast to hydroxamate compounds, the phosphinic compounds interact with both the primed and unprimed side of the catalytic site [17,27,35] due to the placement of the ZBG in the middle of the scaffold and not at its N-or C-terminal, as in the cases of hydroxamate and carboxylate inhibitors [17]. Another advantage of phosphinic acids is the improved metabolic stability compared with hydroxamate acids [27].
The effectiveness of phosphoric acid inhibitors has been studied and it has been found that the three pockets unprimed are connected to obtain the maximum performance [19]. The S1-S2 pockets can be exploited using aromatic groups [19,68], that is why Reiter et al. prepared compounds with 4benzyl ( Figure 24) as a substituent to fill S2 pocket. They found that in the absence of this substituent or its replacement by small aliphatic or cyclohexyl methyl groups led to a loss of activity [19,68]. Matziari et al. [69] synthesized a series of phosphinic pseudopeptides bearing long P1' side chains, compounds that contain groups at the ortho-position of the phenyl ring and are selective for MMP-11 by the interaction of these groups with residues located at the entrance of the S1' cavity [69]. These results suggest that the development of compounds able to probe the entrance of the S1' cavity might represent an alternative strategy to gain selectivity [69].
Other phosphorus-based ZBGs are the carbamoyl phosphates, in which the two oxygens form a five membered ring with the zinc ion [18]. The negative charge of these inhibitors prevents their penetration into the cell and restrain them for extracellular space, contributing to low cytotoxicity [18]. Pochetti et al. [70] described a compound with high affinity to MMP-8 (Ki = 0.6 nM) but inhibits also MMP-2 (Ki = 5 nM) and MMP-3 (Ki = 40 nM) ( Figure 25). The R enantiomer is more potent (1000 time more) than the S enantiomer (Ki = 0.7 μM) [70].  Matziari et al. [69] synthesized a series of phosphinic pseudopeptides bearing long P 1 ' side chains, compounds that contain groups at the ortho-position of the phenyl ring and are selective for MMP-11 by the interaction of these groups with residues located at the entrance of the S 1 ' cavity [69]. These results suggest that the development of compounds able to probe the entrance of the S 1 ' cavity might represent an alternative strategy to gain selectivity [69].
Other phosphorus-based ZBGs are the carbamoyl phosphates, in which the two oxygens form a five membered ring with the zinc ion [18]. The negative charge of these inhibitors prevents their penetration into the cell and restrain them for extracellular space, contributing to low cytotoxicity [18]. Pochetti et al. [70] described a compound with high affinity to MMP-8 (Ki = 0.6 nM) but inhibits also MMP-2 (Ki = 5 nM) and MMP-3 (Ki = 40 nM) ( Figure 25). The R enantiomer is more potent (1000 time more) than the S enantiomer (Ki = 0.7 µM) [70].

Phosphorus-Based Inhibitors
The capacity of the phosphoric group to reproduce the gem-diol intermediate during peptide hydrolysis was explored with different structures to obtain potent MMPis [5]. The phosphorus-based of the peptide-analogous inhibitors can be phosphonates/phosphonic acids, phosphoramidates, phosphonamidates, and phosphinates/phosphinic peptides [27]. The phosphinic acid (PO(OH)-CH2) mimics the transition state obtained in substrate degradation, where each oxygen atom can coordinate both the catalytic zinc and the catalytic Glu [6, 19,27]. The phosphinic acids are monodentate chelates [27]. In contrast to hydroxamate compounds, the phosphinic compounds interact with both the primed and unprimed side of the catalytic site [17,27,35] due to the placement of the ZBG in the middle of the scaffold and not at its N-or C-terminal, as in the cases of hydroxamate and carboxylate inhibitors [17]. Another advantage of phosphinic acids is the improved metabolic stability compared with hydroxamate acids [27].
The effectiveness of phosphoric acid inhibitors has been studied and it has been found that the three pockets unprimed are connected to obtain the maximum performance [19]. The S1-S2 pockets can be exploited using aromatic groups [19,68], that is why Reiter et al. prepared compounds with 4benzyl ( Figure 24) as a substituent to fill S2 pocket. They found that in the absence of this substituent or its replacement by small aliphatic or cyclohexyl methyl groups led to a loss of activity [19,68]. Matziari et al. [69] synthesized a series of phosphinic pseudopeptides bearing long P1' side chains, compounds that contain groups at the ortho-position of the phenyl ring and are selective for MMP-11 by the interaction of these groups with residues located at the entrance of the S1' cavity [69]. These results suggest that the development of compounds able to probe the entrance of the S1' cavity might represent an alternative strategy to gain selectivity [69].
Other phosphorus-based ZBGs are the carbamoyl phosphates, in which the two oxygens form a five membered ring with the zinc ion [18]. The negative charge of these inhibitors prevents their penetration into the cell and restrain them for extracellular space, contributing to low cytotoxicity [18]. Pochetti et al. [70] described a compound with high affinity to MMP-8 (Ki = 0.6 nM) but inhibits also MMP-2 (Ki = 5 nM) and MMP-3 (Ki = 40 nM) ( Figure 25). The R enantiomer is more potent (1000 time more) than the S enantiomer (Ki = 0.7 μM) [70].  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 IC 50 and Ki values of some phosphorus-based inhibitors [6, [15][16][17][18][19]29,35,37,54,55].

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 Zn 2+ 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].

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].

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].
In 2000, Mobashery et al. [72] were the first to report this novel type of MMPi that blocks 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 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].
In 2000, Mobashery et al. [72] were the first to report this novel type of MMPi that blocks 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 In 2000, Mobashery et al. [72] were the first to report this novel type of MMPi that blocks 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]. 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].  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].

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 S 1 ' 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 S 3 pocket (hydrophobic) and the thiadiazole sulfur group interacts with the catalytic zinc [19]. Moreover, the two nitrogen atoms form hydrogen bonds with Ala 164 and Glu 202 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 S 3 pocket (present in MMP-1) leads to the removal of inhibitory activity and explains the absence of activity against collagenases [19]. 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]. Sanofi-Aventis developed a compound ( Figure 29) for MMP-13 (IC50 = 6.6 μM), with very high selectivity [6]. This compound binds deeply to the S1' pocket and to a side pocket that has not been identified for other MMPs [6]. The pyridyl moiety is towards to the entrance of the S1' pocket, without interacting with the catalytic Zn(II) ion and the oxygen atoms neither from the amide (peptidic) bonds of the main chain (between Thr245 and Thr247) nor from hydroxyl group from the Thr247 side chain in the S1' pocket [6].  Sanofi-Aventis developed a compound ( Figure 29) for MMP-13 (IC 50 = 6.6 µM), with very high selectivity [6]. This compound binds deeply to the S 1 ' pocket and to a side pocket that has not been identified for other MMPs [6]. The pyridyl moiety is towards to the entrance of the S 1 ' pocket, without interacting with the catalytic Zn(II) ion and the oxygen atoms neither from the amide (peptidic) bonds of the main chain (between Thr 245 and Thr 247 ) nor from hydroxyl group from the Thr 247 side chain in the S 1 ' pocket [6].
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]. Sanofi-Aventis developed a compound ( Figure 29) for MMP-13 (IC50 = 6.6 μM), with very high selectivity [6]. This compound binds deeply to the S1' pocket and to a side pocket that has not been identified for other MMPs [6]. The pyridyl moiety is towards to the entrance of the S1' pocket, without interacting with the catalytic Zn(II) ion and the oxygen atoms neither from the amide (peptidic) bonds of the main chain (between Thr245 and Thr247) nor from hydroxyl group from the Thr247 side chain in the S1' pocket [6].  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 S 1 ' and S 3 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].

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 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.

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].

Conflicts of Interest:
The authors declare no conflict of interest.