European Journal of Medicinal Chemistry 194 (2020) 112260

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Matrix metalloproteinase-9 (MMP-9) and its inhibitors in cancer: A

minireview
Subha Mondal, Nilanjan Adhikari, Suvankar Banerjee, Sk Abdul Amin, Tarun Jha*
Natural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, P. O. Box 17020, Jadavpur
University, Kolkata, 700032, India

a r t i c l e i n f o a b s t r a c t

Article history: Matrix metalloproteinases (MMPs) are zinc dependent proteolytic metalloenzyme. MMP-9 is one of the
Received 17 December 2019 most complex forms of matrix metalloproteinases. MMP-9 has the ability to degrade the extracellular
Received in revised form matrix (ECM) components and has important role in the pathophysiological functions. Overexpression
28 February 2020
and dysregulation of MMP-9 is associated with various diseases. Thus, regulation and inhibition of MMP-
Accepted 18 March 2020
Available online 21 March 2020
9 is an important therapeutic approach for combating various diseases including cancer. Inhibitors of
MMP-9 can be used as anticancer agents. Till date no selective MMP-9 inhibitors passed the clinical trials.
In this review the structure, activation, function and inhibitors of MMP-9 are mainly focused. Some
Keywords:
Matrix metalloproteinase
highly active and/or selective MMP-9 inhibitors have been discussed which may be helpful to explore the
MMP-9 structural significance of MMP-9 inhibitors. This study may be useful to design new potent and selective
MMP-9 inhibitor MMP-9 inhibitors against cancer in future.
Cancer © 2020 Elsevier Masson SAS. All rights reserved.
Hydroxamate

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Classification of MMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Collagenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2. Gelatinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3. Stromelysins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.4. Matrilysins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.5. Membrane type MMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.6. Other MMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. General structure of MMPs and structural difference of MMP-9 from other MMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Amino-terminal signal peptide domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. Pro-peptide domain (prodomain) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.3. Catalytic domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.4. Fibronectin domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.5. Hemopexin-like domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Activation of MMP-9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5. Role of MMP-9 in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5.1. Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.2. Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.3. Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.4. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6. Role of MMP-9 in other diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6.1. MMP-9 in neurological disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6.2. MMP-9 in inflammatory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

* Corresponding author.
E-mail address: tjupharm@yahoo.com (T. Jha).

https://doi.org/10.1016/j.ejmech.2020.112260
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
2 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260

6.3. MMP-9 in cardiovascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

6.4. MMP-9 in lung diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6.5. MMP-9 in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7. MMP inhibitors (MMPIs) in clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
8. MMP-9 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8.1. Natural MMP-9 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
8.2. Monoclonal antibodies as MMP-9 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.3. Other active MMP-9 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.3.1. Hydroxamate based MMP-9 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
8.3.2. Non hydroxamate-based MMP-9 inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
9. Trends in the development of potent and selective MMP-9 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
10. Conclusion and future perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Declaration of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1. Introduction cytokine (interleukin-1 and -6), growth factors (transforming

growth factor (TGF), tumor necrosis factor a (TNFa), platelet-
Cancer is an abnormal growth of cells and is a major group of derived growth factor (PDGF), basic fibroblast growth factor
diseases by which various parts of the body have been affected and (bFGF) and some hormones [5]. They have important roles in
lead to death worldwide. Cancer is the major cause of death after various physiological processes including development, wound
heart disease globally. In 2018, the number of death due to cancer healing, remodelling of tissues, organ morphogenesis, angiogen-
was 9.6 million [1]. There are more than 200 type of caners such as esis, etc. Angiogenesis is essential for tumor growth and develop-
lung, skin, breast, prostate, colorectal, stomach cancers, etc. Most ment during which new blood vessel are formed from the existing
common causes of cancer are high body mass index, tobacco and blood vessel [6]. In normal physiological condition, MMPs are
alcohol use, lack of physical activity, low fruit and vegetable intake, tightly regulated and expressed at low level. Dysregulation and
etc. Tobacco use (approximately 22%) and hepatitis and human overexpression of these enzymes related to various diseases
papiloma virus (HPV) (up to 25%) are liable for cancer deaths. The including neurodegenerative diseases, cardiovascular diseases,
most commonly occurred cancers and deaths caused by different lung diseases, arthritis, central nervous system (CNS) disorders
cancers are depicted in Fig. 1(A) and Fig. 1(B), respectively [1]. including epilepsy, various type of cancers, etc [4,7,8].
Cancer research mainly focused on the overexpressed molecules Therefore, MMPs have been expressed as potential diagnostic
those are associated with the survival and proliferation of cancer and are prognostic biomarker in several type of cancer [9]. MMPs
cell [1]. are regulated by various ways such as maintenance of activation of
Matrix metalloproteinases (MMPs) are zinc (Zn2þ) dependent pro-enzyme and transcription. Inhibition of MMPs function is done
endopeptidases present intracellularly and membrane bound. They by tissue inhibitor of metalloproteinase (TIMP) [3]. TIMPs are
cause deterioration of extra cellular matrix (ECM) proteins for divided into four groups TIMP-1,-2,-3 and -4. Imbalance between
example collagen, laminin, elastin, fibronectin, etc and help in the activation and inhibition of MMPs play a crucial role in patho-
extracellular matrix remodelling in various physiological and physiology of cancer [3,10].
pathological processes [2]. They can also deteriorate the non-ECM About 26 MMPs have been identified till now and most of these
molecules most of those are bioactive molecules. Another name of enzymes are present in the human proteome [11,12]. The first MMP
MMPs is matrixin those are of metzincin superfamily. They act was discovered in 1962 that is collagenase-1 (MMP-1). It is found in
depending on the metal ion such as zinc (Zn2þ) and calcium (Ca2þ) tadpole tail of North American frog species Rana catesbiana by
[3]. MMPs were first found in vertebrates and also found in in- Gross and Lapiere [5]. MMPs are secreted as zymogen (pro-
vertebrates (soyabeans, sea urchins, Arabidopsis thaliana and Cae- enzyme). They remain in inactive state because of the interaction
norrhabdita elegans) [4]. They are active at neutral pH [3]. between zinc (Zn2þ) ion of the catalytic domain with the cysteine
Expression of MMP is increased by several factors for example sulphydryl group of the pro-domain. They become activated after

Fig. 1. (A) The number of different cancers occurred in 2018. (B) The number of deaths in 2018 caused by different cancers.
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
removal of this interaction or by proteinases [13]. Depending on the
substrate specificity MMPs are divided into six classes such as
collagenase, gelatinase, matrilysins, stromelysins, membrane type
MMPs and other type MMPs [7].
MMP-9 is the one of the most complex MMPs which belongs to
gelatinase family [14]. It inhibits or stimulates the process of
degradation of ECM. It causes degradation of gelatine and type IV,
V, XI and XVI collagens during tissue remodelling which is essential
for tumour invasion and metastasis [15]. MMP-9 is usually located
at hippocampus, cerebellum and cerebral cortex [14]. It was
discovered in 1974 and is also known as gelatinase B of proteolytic
family. MMP-9 is secreted as zymogens or as the inactive form from
endothelial cell, leukocyte, fibroblast, neutrophil and macrophage.
At the time of granulocyte differentiation, MMP-9 synthesis is
generally occurred in the bone marrow [16].
Matrix metalloproteinase inhibitors (MMPIs) act by binding to
the zinc (Zn2þ) ion at the catalytic site and inhibit MMPs activity
which may be used as anticancer agents [11]. Few researchers
claimed that MMPIs act by inhibiting cancer cell growth whereas
some studies reported that MMPIs acts by decreasing tumour
proliferation by inducing apoptosis via releasing ligand [TNFa,
TRAIL (Tumour necrosis factor related apoptosis inducing ligand)]
from their membrane bound inactive form [17].
Here, we mainly focus on MMP-9 enzyme through the light of its
structure, function and inhibitors. Overexpression of MMP-9 is
associated with the various cancers such as breast, lung, prostate,
colon, gastric and pancreatic cancers are highlighted in details.
2. Classification of MMPs
Depending on the substrate specificity MMPs have been classi-
fied into six major groups (Table-1). These are collagenase, gelati-
nase, stromelysin, matrilysin, membrane type and other type
MMPs [7,16,18].
2.1. Collagenase
Collagenases including MMP-1, MMP-8, MMP-13 and MMP-18
(identified in Xenopus) are essential for cleavage of several types
of interstitial collagens (collagen type I, II and III) at the particular
site to form 3/4 and 1/4 fragments [19,20]. They can also cleave ECM
molecule and non-ECM molecules. For collagenolytic activity of
these MMPs, there should be a correlation between hemopexin
domain and catalytic domain [20].
2.2. Gelatinase
Gelatinases (MMP-2 and MMP-9) are liable for deterioration of
gelatine by their three fibronectin repeats and also degrade ECM
like collagen (type I, IV, V, VII, X, IX and XI), laminin, elastin, fibrillin,
aggrecan, proteoglycans, vitronectin, etc [20,21]. MMP-9 is
expressed by these cells including alveolar macrophages, osteo-
clasts and polymorphonuclear leukocyte, etc cells [20]. It also
causes the degradation of non ECM molecules such as interleukin-
1b and TNFa. These MMPs are found to express by chondrocytes,
osteoclasts, osteoblasts, endothelial and malignant cells [22].
2.3. Stromelysins
Stromelysins including MMP-3, MMP-10 and MMP-11 are
same as collagenases but differ in that these do not cause cleavage
of interstitial collagen. Among these MMP-3 and MMP-10 cleave
some ECM molecules and also help in the activation of pro-MMP
by removing the propeptide domain where MMP-11 is less
active towards the ECM molecules but more active towards the
3
serpins. MMP-11 is also called furin-activatable MMP due to its
additional structural feature [8]. MMP-27 has been identified first
in chicken embryo having capability to degrade gelatine and
casein. It is highly expressed in human B-lymphocytes [20].
2.4. Matrilysins
Matrilysins including MMP-7 (matrilysin-1) and MMP-26
(matrilysin-2) do not consist of hemopexin domain. MMP-7 is
synthesized by epithelial cells [8]. Matrilysins are released under
normal and physiological condition involving in the progression of
various cancers. These cause the degradation of ECM molecule
(laminin, type IV collagen and entactin) and also non-ECM mole-
cules. MMP-26 is stored intracellularly and also activates pro-
MMP9 [20]. Matrilysins help in the remodelling of postpartum
uterus and also in embryo implantation [20].
2.5. Membrane type MMPs
Membrane type MMPs (MT-MMPs) comprises transmembrane
domain or a glycosylphosphatidyl inositol anchor [8]. They are
divided into six subtypes such as MMP-14(MT1-MMP), MMP-
15(MT2-MMP), MMP-16 (MT3-MMP), MMP-17(MT4-MMP), MMP-
24 (MT5-MMP) and MMP-25(MT6-MMP) [23e25]. Among these
MT1, MT2, MT3 and MT5 have transmembrane domain. On other
hand MT4 and MT6 have glycophosphatidyl inositol anchor. MT1-
MMP causes degradation of types I, II and III collagens. Other MT-
MMPs are essential for activation of pro-MMP2 and also cause
degradation of laminin and fibronectin [19]. It has been found that
MT1-MMP, MT2-MMP and MT3-MMP increase the invasion ca-
pacity of cancer cell by degrading fibrin [22].
2.6. Other MMPs
MMP-12, -19, 20, 21, 27, 28 are divergence in sequence
and substrate specificity, so they cannot be properly classified in
subgroups [11]. MMP-12 causes degradation of type-IV collagen,
type-I gelatine, elastin, myelin basic protein and associated with
various pathological condition such as inflammation [22]. It also
helps in the migration of microphages. It is secreted from osteo-
clasts and hypertrophic chondrocytes and expressed by macro-
phages [8]. MMP-19 cleaves some ECM molecules mainly the
basement membrane components. MMP-19 is also known as RASI
(rheumatoid arthritis synovial inflammation) because it was first
identified in the active lymphocytes and rheumatoid arthritis
patient’s plasma [8]. MMP-20 also known as enamelysin which is
first identified in odontoblusts and also tooth specific. MMP-20 is
an important factor for dental enamel formation. MMP-21 is
identified in xenopus, found in mice and human. It cleaves gela-
tine but how it acts on the ECM component is not known. MMP-28
is also known as epilysin which is found in various tissues, for
example, lung, heart, testis, placenta and gastrointestinal tract. It
helps in wound healing. The level of MMP-28 is increased in pa-
tient with osteoarthritis and rheumatoid arthritis [20].
3. General structure of MMPs and structural difference of
MMP-9 from other MMPs
All MMPs are consisting of at least three main domains: (a)
amino-terminal signal peptide domain, (b) propeptide domain and
(c) catalytic domain (Fig. 2).
3.1. Amino-terminal signal peptide domain
Amino-terminal signal peptide (composed of 17e29 amino
4 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260

Table: 1
Classification of Matrix metalloproteinases (MMPs).

Type of Sub-group Isoform Substrate Biological effect

MMP

Collagenases Collagenase-1/ MMP-1 Fibrillar and non-fibrillar collagens (Type I, II, III, Cellular proliferation and migration, increases the bioavailability of insulin like
Interstitial VI,VII,VIII and X), laminin, casein, serpins and growth factor-1 (IGF-1), keratinocyte migration, re-epithelialisation, pro-
collagenase MMP-1, -2, -9 inflammatory effects, degradation of physical barriers and proteolytic activity
in cancer progression.
Collagense-2/ MMP-8 Fibrillar collagens (Type I, II, III, V, VII, VIII and X), The activation of osteoclasts, the enhancement of collagen affinity, release of
Neutrophil gelatin, fibronectin bFGF (basic fibroblast growth factor). Regulation of mobilization and cleavage
collagenase of the a- and b-chemokines in cancer progression, anti-inflammatory effects.
Collagenase-3 MMP- Fibrillar collagens (Type I, II and III), gelatins During cancer progression- induction of EMT (epithelial to mesenchymal
13 transition), cell migration
Collagenase-4 MMP- ND e
18
Gelatinases Gelatinase A MMP-2 Basement membrane and non-fibrillar collagens Cellular migration, elevation of collagen affinity, epithelial cell migration,
(72 kDa) (Type IV, V, VII and X), fibronectin, elastin enhancement of TGF-b (transforming growth factor-b) bioavailability,
neurodegeneration through neuronal apoptosis, growth of axons,
mesenchymal cell differentiation with inflammation phenotypes, anti-
inflammatory effects, proliferation and cleaving of IGF-1 binding proteins in
progression of cancer.
Gelatinase B MMP-9 Basement membrane collagens and non fibrillar Anti-inflammatory and pro-inflammatory activity, increase in collagen
(92 kDa) collagen (Type IV, V, VII, X and XIV), gelatins, affinity, reduction of IL-2 response. Activation of TGF-b in cancer progression.
fibrillin Apoptosis of hypertrophic chondrocytes and the inclusion of newer functional
osteoblast units, resistance of tumor cell.
Matrilysins Matrilysin- MMP-7 Collagen IV-X, fibronectin, gelatins, Enhancement of TGF-b and IGF-1 bioavailability, increase in collagen affinity,
1(PUMP) proteoglycans, pro-MMP-9, laminin induction of apoptosis through activation of Fas receptor, increase of cellular
invasiveness and aggregation of abnormal cells, differentiation of adipocyte,
cellular growth and vasoconstriction, osteoclasts activation through pro-
inflammation.
Matrilysin-2/ MMP- Collagen IV, fibronectin, fibrinogen, gelatine, pro- e
endometase 26 MMP-9
Stromelysins Stromelysin-1 MMP-3 Collagens (Type III, IV, V and IX), fibronectin, Conversion of epithelialemesenchymal, generation of angiostatin-like
proteoglycan, pro-MMP-1, laminin, gelatins substances, increase in collagen affinity, bFGF release, cellular proliferation
and migration, epithelial bubble formation, bioavailability enhancement of
IGF-1 and TGF-b, pro- and anti-inflammatory effects, increase in cellular
invasiveness, apoptosis of epithelial cells, disorganization of cell aggregation,
angiogenesis upregulation, perlecan and COL-IV degradation, release of bFGF
and VEGF in cancer progression.
Stromelysin-2 MMP- Fibronectins, collagens (Type III and IV), pro- Tumstatin, angiostatin, endostatin and endorepellin generation in cancer
10 MMP-1 progression, degradation of perlecan, COL-IV and COL-XVIII in cancer
progression.
Stromrlysin-3 MMP- Serpin, aggrecan, laminin, fibronectin, gelatin Reduction of cancer cell sensitivity to NK cells and a1-proteinase inhibitor
11 release in cancer progression
Membrane MT1-MMP MMP- Gelatin, collagen (i-iii), fibronectin, casein, Cellular migration, anti-inflammatory activity, renal tubule formation, cell
type 14 laminin, MMP-2 and -13, entactin, vitronectin, reduction flattening, reduction of adhesion, transports embryo to uterine
proteoglycan. epithelium.
MT2-MMP MMP- Pro-MMP-2, gelatine, fibronectin, tenascin, Adhesion and cell flattering reduction
15 nidogen, laminin, aggrecan, perlecan, MMP-2
MT3-MMP MMP- Collagen-III, gelatin, casein, fibronectin and MMP- Adhesion and cell flattering reduction
16 2.
MT4-MMP MMP- ND e
17
MT5-MMP MMP- Fibronectin, Pro-MMP-9, pro-MMP-2, gelatine e
24
MT6-MMP MMP- Fibrin, gelatine, collagen IV, fibronectin e
25
Others Macrophage MMP- Casein, collagen-IV, vitronectin, elastin, gelatin, Causes plasminogen digestion and angiostatin release leading to the cancer
metallo elastase 12 fibronectin, fibrin fibrinogen and plasminogen. cell apoptosis, produce endostatin which form a complex with pro-MMP-9
and -13 and block their activation
RASI-1 MMP- Type-I collagen Activation of VEGF, epithelial cell migration
19
Enamelysin MMP- Gelatin, COMP, amelogenin (dentine), aggrecan, e
20
XMMP from MMP- Not determined e
Xenopus 21
CMMP from MMP- Casein, gelatin e
chicken 22
CA-MMP MMP- ND e
(cysteine array) 23
e MMP- ND e
22
Epilysin MMP- Casein Activate TGF-b proteolytically which induces epithelial to mesenchymal
28 transition in which increased invasion, migration and metastasis
Unnamed MMP- ND e
29

ND: Not determined.

 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Fig. 2. Graphical representation of different structural domains of the MMP family
members.
acids) is responsible for its excretion out of the cell [3]. Most of
these MMPs are bound to the cell surface by a transmembrane
domain (except MT-MMPs).
3.2. Pro-peptide domain (prodomain)
The pro-peptide domain (composed of 77e87 amino acids) is
responsible for activation of the enzyme. The amino acid sequence
of pro-domain of all MMPs is PRCGXPD that is known as “cysteine
switch” (except MMP-23) [3,26,27]. The cysteine residue contains
sulfhydryl group which is coordinated to the catalytic divalent zinc
ion to regulate the enzyme dormancy [9,26]. This “cysteine switch
mechanism” is pivotal [28,29]. Due to this zinc-cysteine co-ordi-
nation, the activities of MMPs are suppressed by preventing water
molecule from binding to the zinc ion which is essential for the
catalysis [8]. The pro-domain contains three a chains which are
connected with a flexible loop. These are responsible for the for-
mation of hydrophobic pocket by interacting with each other [29].
3.3. Catalytic domain
On the other hand, the catalytic domain (contains 170 amino
acids approximately) is responsible for the proteolytic activity of
the enzyme [30,31]. Most importantly, this domain contains a zinc-
binding consensus sequence (HEXXHXXGXXH) which is pivotal for
the proteolytic activity [24]. Catalytic domain of MMPs is struc-
turally spherical with a diameter of about 40 Å [29]. This domain is
pivotal for the substrate hydrolysis [30]. Two zinc ions (one cata-
lytic Zn2þ ion and one structural Zn2þ ion) are present in this
domain which is required for the catalytic activity and structural
integrity respectively [8]. Five calcium ions required for the enzyme
stability and integrity [3,32].
There is a shallow catalytic cleft which divides this domain into
two parts-one is the N-terminal sub-domain and another is the C-
terminal sub-domain [32]. These two sub-domains are linked with
the highly opened U loop. Three histidine amino acid residues (i.e.,
His218, His222, His228) and a water molecule are required for co-
5
ordinationg to the catalytic zinc in the catalytic cleft [32]. Cata-
lytic domain also contains a conserved methionine residue which
forms a ‘Met-turn’.
The catalytic cleft has six binding pockets including S1, S2, S3,
S1’, S2’ and S3’ sites. S1, S2 and S3 pocket are present at the left
handed side of the catalytic Zn2þ ion where as S1, S2´ and S3
pockets are located at the right handed side of this Zn2þ ion
expressed as primed pockets [18]. S2 and S3 pocket are solvent
expounded. Among these pockets, depth and amino acid sequence
of S1 pocket vary in different MMPs. The substrate specificity is
depending on the S1’ pocket. MMP-1 and MMP-7 have shallow S1
pocket; MMP-2, MMP-8, MMP-9, MMP-12 and MMP-14 have in-
termediate S1 pocket; and MMP-3, MMP-10, MMP-13 have deep S1
pocket [19,33]. Matrix metalloproteinase inhibitors (MMPIs) may
be designed depending on the depth and length of the S1 pocket
and also based on the amino acid sequence around this pocket
[34,35]. The S1 pocket of both MMP-2 and MMP-9 is situated in the
catalytic domain and is similar in size as well as exposed to the
solvent but differ in the residue 425e431 which form a loop in
MMP-9 that is not present in MMP-2 [18].
3.4. Fibronectin domain
Fibronectin domain consists of three repeats of fibronectin type
II motif which are inserted into the active site domain and zinc
binding domain (metalloproteinase domain) [8,36,37]. The fibro-
nectin type-II motif has the capability to bind gelatine, laminin,
collagens type-I and IV [29]. MMP-2 and MMP-9 contain fibro-
nectin domain which is not present in other MMPs (Fig. 2). In case
of MMP-2 and -9, this fibronectin domain is a modulator
́ of collageń
recognition [18].
3.5. Hemopexin-like domain
́
The C-terminal Hemopexin domain contains about 210 aminó
acid residues [31]. This domain is an ellipsoidal disk shaped in
nature containing four bladed b-propellers connected with singlé
́
disulphide bond between the 1st and the 4th blades [8]. Each blade
consists of four antiparallel b-strands and a a-helix [31]. Generally,
́
a calcium and a chloride ions are present at the centre of the pro-
peller [8]. Thiś domain can interact with substrate such as gelatin
and collagen. In case of MMP-9, it plays an important role to bind
the tissue inhibitor of metalloproteinases [9]. MMP-9 differs from
MMP-7 (matrilysin 1), MMP-26 (matrilysin 2) and MMP-23 because
hemopexin domain is not present in these MMPs.
Gelatinases (MMP-2 and MMP-9) contain an additional fibro-
nectin domain. MMP-2 and MMP-9 have similar substrate selec-
tivity but differs in tissue specificity. MMP-2 and MMP-9 are highly
similar in their structure but differences are observed in the S1
pocket. Amino acids residues 425e431 form a loop in the S1’ site of
MMP-9 which is found absent in MMP-2 [11,18]. Gelatinase B
(MMP-9) is one of the most complex matrix metalloproteinase
structurally which contains the linker peptide of variable length
called hinge region (Fig. 2). Due to its complex structure, it can bind
with various substrates such as gelatine, collagens type I and IV,
procollagen type II, laminin, tissue inhibitor of metalloproteinases
(TIMPs), chemokines, etc [14,25].
4. Activation of MMP-9
MMPs are generally produced as pre-pro-enzyme [38,39] from
which the amino-terminal signal peptide is cleaved during trans-
lation and results in pro-MMPs. In order to keep the proMMPs
inactive, the amino acid cysteine from the PRCGXPD ‘cysteine
switch’ motif coordinates with the catalytic divalent ions. Later the
6 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
cysteine switch is cleaved or the pro-domain is detached often by
proteolytic enzymes such as the endopeptidase furin, plasmin,
serine proteases, or other MMPs and subsequently, results in the
active MMP forms. In the activation of the pro-enzyme to the active
form, various activators are involved. Several pro-MMPs are acti-
vated by mercurial compounds, chaotropic and thiol agents. Pro-
MMPs are also activated by oxidant like HOCl, ONOO. These oxi-
dants interact with cysteine residue in the pro-peptide domain
under inflammatory condition. On the other hand, the endogenous
TIMPs counteract these actions.
Similarly, the MMP-9 enzyme is secreted as pro-MMP-9 which is
an inactive form of MMP-9. The activation of pro-MMP-9 (molec-
ular weight 92 kDa) by various MMP-9 activators occurs in two
steps (Fig. 3).
Each step decreased molecular weight (MW) by approximately
5 kDa. In the first step, a cleavage at glutamate-59 is occurs to
produce an intermediate from (MW: 86 kDa). The second step deals
with a cleavage at arginine-106 to produce active MMP-9 (MW:
82 kDa) [40,41]. Post-activation fate of MMP-9 results in the
cleavage of the C-terminal hemopexin-like domain (MW: 65 kDa
species) or a direct removal of the catalytic site leading to an
inactive from (MW: 50e60 kDa) [42].
The enzyme proteolysis of the prodomain is an interesting
mechanism of MMP-9 activation. MMP-9 activators are MMP-2, -3,
-7, -10, 13, cathepsin G and plasmin [21,22]. Interestingly, the
MMP-3 is the most potent activator of MMP-9 [42]. It has been
found that thrombospondin-1 also increases the activation of
MMP-9 [43].
5. Role of MMP-9 in cancer
Cancer research is mainly focused on the overexpressed mole-
cules and the process by which these help in cancer cell progression
Fig. 3. Schematic representation of the activation process of MMP-9.
[36]. It has been found that ECM has important role in cancer
progression [44]. Interaction between these cells and ECM
component is essential for cell transformation as well as carcino-
genesis [5]. MMPs are highly expressed in human cancer and are
related to every stage of cancer development [11,36]. Cancer cell
secrets various agents such as interferon, interleukins and growth
factors as well as extracellular MMP inductor which stimulate the
surrounding host cell to generate required MMPs for the tumor cell
[5].
Early MMPs expressions in tumor cells help in the remodelling
of ECM and release membrane-bound growth factors to lay out
microenvironment for the tumorigenesis [36]. The role of MMP-9 in
angiogenesis, metastasis and cancer invasion has been identified
(Fig. 4). MMP-9 is also responsible for survival and spreading of
cancer cells.
Cancer cells in a paracrine manner, by secreting interleukin,
interferon, growth factors and extracellular MMP inductors, stim-
ulate surrounding host cells to produce required MMPs. MMPs
secreted by normal cells can be bounded on the cancer cell surface
and used by the tumor cells. They are involved in all steps during
carcinogenesis [15].
MMP-9 is found to play a crucial role in gastric cancer pro-
gression as detected by SDS-PAGE and SDS-PAGE zymography as-
says [45]. MMP-9 gene polymorphism has important roles in breast
cancer patients along with identification of risk of developing
breast cancer [46]. It is noticed that MMP-9 has a direct correlation
of poor prognosis in breast tumors [47].
It has been found that the avb6 integrin is expressed in colon
cancers which in turn triggers MMP-9 secretion and subsequently,
involves the protein-kinase-C pathway activation [48]. Further
study showed that avb6-mediated secretion of MMP-9 has a crucial
role in colon cancer progression [49]. MMP-9 overexpression is
associated with decreased survival rate and metastasis in breast as
well as colon cancers [50].
MMP-9 is also found to be induced by vascular endothelial
growth factor (VEGF) during lung metastasis and subsequently,
deficiency of MMP-9 helps to reduce the metastasis [51]. Also,
MMP-9 has crucial roles in several cancers associated with
inflammation [50]. Gelatinases mainly MMP-9 is related to the
negative regulation of immune response to cancer by cleaving
interleukin-2a (IL-2a) as well as through shedding of intercellular
Fig. 4. Role of MMP-9 in cancer progression.
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
adhesion molecule-1 (ICAM-1) and transforming growth factor
(TGF-b) activation [50].
5.1. Metastasis
Metastasis is the main culprit of death in cancer patients. MMP-
9 causes degradation of gelatin and type IV, V, XI and XVI collagens
which are essential for metastasis [14]. MMP-9 promotes metas-
tasis through breakdown of physical barrier of ECM. Metastasis
occurs in several steps. Initially there is a disconnection from
intracellular part and releasing of a tumour cell. After that degra-
dation of ECM component occurs and migration of the cell takes
place. Then the cell penetrates into the blood and lymphatic vessel
and adhered to the endothelial cell. Finally leads toward the growth
of a secondary tumour cell in the other part of the body. Itoh and
co-workers [52] showed a prime contribution of host-derived
MMP-9 in tumor metastasis. Thereby, the MMP-9 activity
reduced by inhibitors is useful in the metastasis.
5.2. Invasion
MMP-9 causes the degradation of type IV collagen of basal
membrane near the tumor cell that invades the other tissue, and
therefore induces metastasis [12]. DNA repair protein Ku interact
with the pro-MMP-9 (MW: 85 kDa) at the cell surface of leukemia
cell lines to form Ku/MMP-9 complex which regulates the cell in-
vasion [53,54]. Therefore, by blocking the MMP-9 there may be an
inhibition of the Ku/MMP-9 expression mediated cancer cell inva-
sion on the cell surface [53].
5.3. Angiogenesis
This process is essential for growth and development of tumor
cell. Some ECM components such as fibronectin, thrombospondin-
1, laminin and osteopontin affect the phenotype of tumor by
modulationg cancer cell migration and angiogenesis [5]. MMPs
endorse angiogenesis by degrading the basement membrane and
ECM component. Due to disruption of basement membrane, the
endothelial cell migrates from the existing vessel to produce new
blood vessel and also releases ECM bound factor [5]. The activity of
MMP causes the uncovering of hidden epitopes of ECM proteins.
MMP-9 digests type IV collagen revealing at the same time HUIV26-
epitope and regulating blood vessels development. MMP-9 is
involved in the mechanism of angiogenesis (Fig. 4).
However, MMPs may inhibit the mechanism of angiogenesis. It
causes the degradation of plasminogen to release angiostatin and
also cleaves the collagen XVIII to produce endostatin and thereby
inhibits the process of angiogenesis. MMP-2, -7, -9 and 12 help in
the digestion of plasminogen to release angiostatin. This resulting
angiostatin can increase an apoptosis in tumor cells [5].
5.4. Apoptosis
MMPs have both pro-apoptotic and anti-apoptotic activity. The
pro-apoptotic activity is related to the change in ECM composition.
The anti-apoptotic activities are cleavage of Fas ligand, activation of
serine or protein kinase B or threonine kinase AKT [5]. MMPs cleave
the adhesion molecule and lead to apoptosis. Angiostatin causes
apoptosis in cancer cells and endostatin binds with the pro-MMP9
to produce a stable complex that inhibits MMP-9 activation.
6. Role of MMP-9 in other diseases
The MMP-9 gene polymorphism 1562C/T brought about
interesting result from cancer to neurological/neurodegenerative
7
diseases and cardiovascular to lung diseases [55]. The correlation of
MMP-9 with different diseases and disorders are given in Fig. 5.
6.1. MMP-9 in neurological disorders
MMP-9 plays important roles in pathophysiological mechanism
of various neurological and neurodegenerative diseases such as
Alzheimer’s disease (AD) and Parkinson’s disease (PD) through
remodelling and degradation of ECM, blood-brain barrier (BBB)
disruption, inflammation and microglial activation [56].
MMP-9 has crucial roles in neurological disorders like stroke
(Fig. 5). Gu et al. [57] reported that during cerebral ischemia MMP-9
become activated through oxidation and S-nitrosylation leading to
the cortical neuronal apoptosis. MMP-9 is also associated with
pathogenesis of epilepsy. MMP-9 activity also can impair synaptic
plasticity and mossy fibre sprouting within hippocampus, thus
leading to the formation of epileptic foci. It has important role in
pathology of memory learning, inflammatory diseases, psychiatric
diseases (schizophrenia, bipolar disorder), addiction and multiple
sclerosis [14].
6.2. MMP-9 in inflammatory diseases
MMP-9 is associated with acute and chronic inflammatory dis-
eases (Fig. 5) [41]. It has both pro and anti-inflammatory effects.
MMP-9 enables influx of leukocyte in inflammation and affects
permeability of blood brain barrier, thus promotes inflammation.
MMP-9 inhibitors may be useful in organ specific autoimmune
diseases such as rheumatoid arthritis [41].
6.3. MMP-9 in cardiovascular diseases
MMP-9 gene polymorphism is associated with cardiovascular
diseases including hypertension, atherosclerosis and myocardial
infarction [16]. MMP-9 causes proteolytic degradation of protein
and release vascular endothelial growth factor through which
MMP-9 plays a critical role in neovascularisation.
6.4. MMP-9 in lung diseases
Normal function in lung requires alveolar support by ECM.
Therefore, excess degradation and abnormal remodelling of ECM
Fig. 5. Schematic representation of diseases associated with the MMP-9.
8 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
causes many respiratory disorders such as chronic obstructive
pulmonary diseases (COPD), bronchial asthma and sometime
leading to tuberculosis (Fig. 5). Numerous cells present in lung such
as epithelium, fibroblast, macrophages and myofibroblasts have
ability to express MMPs those are associated with early stage of
chronic obstructive pulmonary disease (COPD) [58]. Increased
MMP-9 activity is found in various lung diseases. MMP-9 in COPD
alters ECM to enlarge the air space in lungs. It is reported that a
selective inhibitor of MMP-9 and -12 decreases the inflammatory
process correlated with exposure to cigarette smoke [59]. Treat-
ment with MMP-9 inhibitor is also protective in six-month smoke
exposure patient.
6.5. MMP-9 in diabetes
The consequence of hyperglycemia on MMPs regulation in
vascular cells is little known [60]. Uemura and co-workers [61]
found an increased MMP-9 expression in plasma and vascular tis-
sue of diabetic animal models. It is also reported that oxidative
stress is a key factor in glucose-induced MMP-9 activity. Ebihara
et al. [62] suggested that increased plasma MMP-9 concentrations
lead up to the microalbuminuria development in non-insulin-
dependent diabetes mellitus (NIDDM) patients. Nguyen and col-
leagues [63] explored roles of MMP-8 and -9 in diabetic wound
healing (Fig. 5).
7. MMP inhibitors (MMPIs) in clinical trials
MMPs not only have crucial roles in cell invasion, angiogenesis
and metastasis but also have important roles in cell transformation,
signal transduction, apoptosis, cell growth and immune regulation.
Therefore, MMPIs are required for treatment of such conditions
[37]. Depending on the presence of the zinc ion and substrate
binding pocket, various synthetic MMPIs have been designed [18].
Zinc binding groups (ZBGs) including hydroxamic acid, thiols, car-
boxylates and phosphonic acid are identified [64,65]. Among these
groups, hydroxamic acid is more preferred because of its Zn2þ
binding ability. Due to the presence of hydrogen bond between the
heteroatom of ZBG and neighbouring amino acids at MMP active
site, the NH and deprotonated OH groups of hydroxamic acid form
hydrogen bond with Ala and Glu residues [18,36].
The first generation of MMPIs are basically peptidomimetic
compounds having hydroxamic acid moiety. Batimastat/BB-94 (1)
(IC50 ¼ 1 nM for MMP-9 and IC50 ¼ 4 nM for MMP-2) and
Marimastat/BB-2516 (2) (IC50 ¼ 3 nM for MMP-9 and IC50 ¼ 6 nM
for MMP-2) are examples of MMPIs fall under peptidomimetic class
(Fig. 6) [65e69].
Small molecules such as MMI270/CGS-27023A (3) (IC50 ¼ 8 nM
for MMP-9 and IC50 ¼ 11 nM for MMP-2), GM6001 (4) (Ki ¼ 0.57 nM
for MMP-9 and Ki ¼ 0.39 nM for MMP-2), GI129471 (5)
(IC50 ¼ 14.4 nM for MMP-9 and IC50 ¼ 28 nM for MMP-2) and CGS-
25966 (6) (IC50 ¼ 27 nM for MMP-9 and IC50 ¼ 11 nM for MMP-2)
designed as MMPIs in yester years (Fig. 6) [29,65e69].
Batimastat (1), the first peptidomimetic MMPI, was found
effective for cancer patients (Fig. 6). It exhibited promising effect
against several cancer cell lines [70]. It also exhibited significant
anticancer effect as evidenced in numerous preclinical studies and
number of xenograft and metastatic models [71]. It was better
effective in early stage of cancers as compared to later stage of
cancers. It also produced synergistic antiproliferative effects in
combination with docetaxel and captopril [71]. Due to its poor
solubility, it was administered through i.v. route. Though it was
well-tolerated, it produced mild systemic toxicity along with
marked abdominal pain and plural infusion [72].
Marimastat (2) was a low-molecular weight non-specific
hydroxamate MMPI (Fig. 6). It inhibited major MMPs (such as
MMP-1, -2, -3, -7 and -9). It was found to be orally bioavailable
(20e50%) due to the presence of a-hydroxyl and tert-butyl groups
those increased aqueous solubility [65]. This compound is effective
against breast and lung metastasis models in pre-clinical studies
[73]. However, it has some major adverse effects including toxicity
of gastro-intestinal tract, weight loss, inflammation, fibrosis, hae-
morrhage and necrosis of tissues [73]. Phase II study of marimastat
with patients having different types of cancers resulted in poor
patient compliance and only 5% patients responded to the therapy
[74]. However, marimastat entered into phase III clinical studies
with patients having various solid tumors in combination chemo-
therapy. It showed a significant improvement for patients having
advanced gastric cancer [74].
CGS-27023A (3), a sulphonamide derivative is also known as
MMI-270 (Fig. 6). It is a broad-spectrum non-peptidomimetic
MMPI that also inhibits tumor growth and angiogenesis as evi-
denced in preclinical studies [75]. It is a broad spectrum MMPI and
orally available. It acts as an inhibitor against MMP-1
(IC50 ¼ 33 nM), MMP-2 (IC50 ¼ 11 nM), MMP-3 (IC50 ¼ 13 nM),
MMP-9 (IC50 ¼ 8 nM) and MMP-13 (IC50 ¼ 6 nM) [66]. However,
this compound was failed in clinical trial because of its side effect
including joint and muscle pain [29].
GM6001 (4) is another broad spectrum, potent and non-
selective MMPI (Fig. 6). It blocks both MMP-3 and MMP-9 enzy-
matic activity [65]. It is a hydroxamic acid derivative and the
hydroxamic acid moiety chelate with the Zn2þ ion to form biden-
tate complex.
GI129471 (5) is a broad spectrum synthetic MMPI can upregu-
lated both synthesis and transcription of pro-MMP-9. It can inhibit
human fibrosarcoma HT1080 invasion through a reduced proteo-
lytic activity [68].
CGS-25966 (6) is a sulphonamide based hydroxamic acid de-
rivative and close analog of CGS-27023A [65,69]. Replacement of
the 4-pyridylmethyl moiety of CGS-27023A (3) with a benzyl group
results in compound CGS-25966 (Fig. 6). The (R)-enantiomer of this
compound showed potent MMP inhibition (IC50 ¼ 27 nM for MMP-
9 and IC50 ¼ 11 nM for MMP-2) while (S)-enantiomer showed low
or no inhibition [69].
However, the above mentioned broad spectrum and non-
selective MMPIs were failed in clinical trials due to their poor sol-
ubility, low oral bioavailability and severe side effects including
musculoskeletal syndrome (join stiffness, pain, tendinitis and
inflammation) [13,18]. The other disadvantages of these com-
pounds are that these can inhibit other zinc-dependent metal-
loenzymes including ADAMs (a disintegrin and metalloproteinases)
such as ADAM-10 and ADAM-17 [58,61]. To address these issues,
next generation MMPIs were developed those includes tanomastat/
BAY12-9566 (7) (IC50 ¼ 301 nM for MMP-9 and IC50 ¼ 11 nM) [76],
prinomastat/AG-3340 (8) (Ki ¼ 0.26 nM for MMP-9 and
Ki ¼ 0.05 nM for MMP-2) [18,77] and rebimastat/BMS-275291 (9)
(IC50 ¼ 25 nM for MMP-9 and IC50 ¼ 41 nM for MMP-2) [66] (Fig. 6).
Among these, tanomastat (7) contains carboxylate group as the
catalytic zinc ion chelator and it is more MMP-2 selective than the
earlier compounds. It was developed by Bayer Corporation (Pitts-
burgh, PA). It was clinically investigated for the treatment of
rheumatoid arthritis and solid tumor [29]. In animal studies, it was
adequately tolerated with mild renal and hepatotoxicity. However,
phase III clinical studies with BAY12-9566 (7) showed inferior
survival time for advanced pancreatic cancer. In addition, it also
possessed inferior survival against small cell lung cancer patients in
clinical studies and therefore, a further study with BAY 12e9566 (7)
was postponed [71].
Prinomastat/AG-3340 (8), an optimized version of CGS-27023A
(3), was effective against a variety of MMPs such as MMP-2, -3, -9
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Fig. 6. Structure of some potent MMP-9 inhibitors (Compound 1e10).
and -13 [77]. It showed effective antiproliferative efficacy against
numerous tumor models after oral and intraperitonial dosage
forms. It also resulted in apoptosis and inhibition of tumor growth
as well as angiogenesis as evidenced in xenograft models of
different cancers like breast, prostate, colon, non-small cell lung
cancer (NSCLS) [66,78]. It also yielded synergistic effect by inhib-
iting tumor growth as well as angiogenesis in combination with
paclitaxel and carboplatin against NSCLC. It also decreased the
number of pulmonary metastasis in B16F10 melanoma [66]. How-
ever, it was withdrawn from clinical trial because of its lack of ef-
ficacy and severe toxicities.
Rebimastat (9) inhibits MMP-1, -2, -3, -8, -9, -13 and 14. It was
studied in breast, prostate, lung, ovarian and pancreatic carcinoma
[13]. This compound was also withdrawn from clinical trial.
Doxycycline hyclate/Periostat® (10), an antibiotic belonging to
the family of the tetracyclines, is FDA approved drug against peri-
odontitis [79] (Fig. 6). It is a classic antimicrobial agent of tetracy-
cline derivative studied as anticancer agent in patients. It showed
promising MMP inhibitory activity [5,79]. It also exhibited cytotoxic
effect against U2OS osteosarcoma, MDA-MB-435 breast and PC-3
prostate cancer cell lines [71].
Minocycline (11) is a derivative of doxycycline (Fig. 7). Mino-
cycline is a tetracycline derivative which mainly used for the
treatment acne vulgaris and sexually transmitted diseases [41].
It acts by inhibiting the bacterial 30s ribosomal subunit and thus
inhibits protein synthesis. It was found that the non-microbial ac-
tivity of minocycline is due to inhibition of several enzymes
including MMPs. It inhibits the proteolytic activity of MMP-9 [41].
Azithromycin, another compound belongs to the antibiotic class,
shows MMP inhibition. It inhibits the gene expression of MMP-9
but has no effect on the activation of pro-MMP-9. At high con-
centration, azithromycin and minocycline reduces the secretion of
MMP-9 but have no effect on MMP-2 [41].
9
8. MMP-9 inhibitors
MMP-9 can be inhibited in different ways either by blocking of
pro-MMP-9 to active MMP-9 conversion or by direct MMP-9 inhi-
bition or obstruction of the production of the pro-MMP-9 at the
transcription level. The macromolecular endogenous inhibitor of
MMP-9 is the tissue inhibitor of metalloproteinases (TIMPs) [41].
TIMPs consist of 184e194 amino acid residues. This can be classified
into two parts N-terminal and C-terminal sub-domains. They are
divided into four groups (TIMP-1, -2, -3 and -4). Among them,
TIMP-1 bind with the hemopexin domain of pro-MMP-9 to pro-
duces a complex, thus prevent MMP-9 activation [9]. TIMP-3 has an
important role in metalloproteinase activities regulation. TIMP acts
by inhibiting the active MMP or by inhibiting the pro-MMP to
active MMP conversion. TIMPs are not applied in therapy because
of their short half-life. Another endogenous inhibitor is a2 macro-
globulin [7]. Several proteins including inducing-cysteine-rich
protein with kazal motifs (RECK) suppress MMP-9 [11]. Exact
inhibitory mechanism of these proteins is unknown.
8.1. Natural MMP-9 inhibitors
Some compounds have been identified from natural sources
mainly flavonoids, alkaloids and phenolic compounds those show
MMP inhibition. Polyphenols from Camellia sinensis can inhibit
MMP-1, -2, -3, -7, -9 thereby prevents photoaging [4]. Ageladine A
obtained from the marine source sponge Agelas nakamurai can
inhibit MMP-1, -2, -8, -9, -12, 13. Fucoidan extracts from seaweeds
Claisiphon novaecaledoniae and methanolic extracts from marine
red algae Cavalina pilulifera show inhibition of MMP-2 and -9
preventing photoaging [4].
Few natural MMPIs such as silibinin (12) and neovastat (13) are
reported (Fig. 7) [22,80]. Silibinin, isolated from milk thistle seeds,
10 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Fig. 7. Molecular structure of some potent MMP-9 inhibitors (Compound 11e15).
is a flavonoid antioxidant. Being a popular in ethnopharmacognosy
has been used in antihepatotoxic and anti-carcinogenic agents [80].
It is a chemopreventive agent against skin cancer [81]. It has effect
on metastasis in breast cancer. It inhibits expression of MMP-9 by
the inhibition of MEK/ERK pathway in a dose dependent manner
[80].
An extract from shark cartilage, neovastat/AE-941 (13) (Fig. 7)
possesses anti-metastasis and anti-angiogenic effects by inhibiting
vascular endothelial growth factor (VEGF) and MMPs [82]. In
phase-I trial, it had shown that neovastat exhibited survival benefit
at high dose and also well tolerable. The phase-III trial of neovastat
was started for the patient with colorectal, breast, non-small cell
lung and kidney cancer but the study was terminated later due to
unwanted effects [82].
Genistein (14), a soy isoflavonoid, shows anticancer properety
(Fig. 7). It is a potent inhibitor of MMPs through inhibition of tumor
growth and invasion [82]. It also possesses estrogenic and anti-
estrogenic activity.
Gallic acid/GA (15) is a polyhydroxy phenolic compound ob-
tained from various plants, food and fruits (Fig. 7). It has anticancer
activity shown in various cancer cells, leukemia, melanoma, gastric,
lung, prostate, breast, cervical and esophageal cancers [83e85]. It
acts by decreasing the glutathione level and generating reactive
oxygen species (ROS), thus inducing apoptosis or cancer cell death
[83]. GA (15) alone and in combination with low-level laser irra-
diation showed anti-cancer effect on human breast cancer cell line
MDA-MB-231, non-tumorigenic breast epithelial cell line MCF10A,
and melanoma cell line A375 dermal fibroblast cell line HDF [84]. It
reduces metastasis by downregulating the MMP-2/-9 expression in
leukemic cells. However, it becomes toxic to the human micro-
vascular endothelial cell when treated at a dose of more than
10,000 nM [83]. It has been shown that the hydroxyl group at para-
position to the carboxyl group of GA (15) is important for MMP-2/
MMP-9 downregulating effect [83]. Apart from GA (15), catechins,
i.e., (-)-epigallocatechin-3-gallate (EGCG) and flavonoids (quer-
cetin) both have anticancer effect by inhibiting MMP-2/-9 functions
[83,86,87].
CT1746 is a synthetic MMP-2/-9 inhibitor which is more active
in combination with cisplatin or cyclophosphamide than the single
dose therapy [88]. It reduces tumour growth and pulmonary
metastasis [88].
8.2. Monoclonal antibodies as MMP-9 inhibitors
Monoclonal antibody (MAbs) has also been developed. MMP-9
targeted MAbs were produced by immunizing mice with recom-
binant human or mouse MMP-9 proteins [89]. REGA-3G12 is a
murine monoclonal antibody produced by Hybridoma technology.
It acts against MMP-9 by inhibiting the catalytic domain of MMP-9
[90]. It is more specific to MMP-9 because it does not affect MMP-2
[29]. It prevents the mobilization of hematopoietic progenitor cell
induced by interleukin-8 in rhesus monkeys by inhibitory anti-
bodies against gelatinase B [91].
AB0041 is an antibody which inhibits human MMP-9 that found
to be effective in ulcerative colitis and colorectal cancer [89]. It also
inhibits rat MMP-9 (rMMP9) but unable to bind to mouse MMP-9
(mMMP-9). It is highly MMP-9 selective (more than 500 fold) over
other MMP family members. It inhibits the cleavage of gelatine and
thereby prevents the liberation of extracellular matrix sequestered
cytokines and growth factors. AB0041 has ability to inhibit the
degradation of MMP-9 mediated epithelial and endothelial base-
ment membranes. This antibody also has the ability to inhibit the
release of TNF-a thus, expressed anti-inflammatory effects [89].
Bortezomib is a proteosome inhibitor that inhibits the degra-
dation of intracellular proteins and causes cell apoptosis. Bortezo-
mib has effect on MMP-2 as well as on MMP-9 at low concentration.
It is used in the treatment of multiple myeloma [41].
8.3. Other active MMP-9 inhibitors
Elevated expression of MMP-9 is associated with various path-
ological conditions such as cancer, inflammation, tumor progres-
sion, atherosclerosis, etc. Because of its crucial role in various
diseases many MMP-9 inhibitor have been developed so far
[92e118].
8.3.1. Hydroxamate based MMP-9 inhibitors
Yamamoto et al. [92] reported some hydroxamate-based potent
MMPIs. Compound 16 (Fig. 8) was a potent and selective gelatinase
inhibitor (MMP-2 IC50 ¼ 1.6 nM, MMP-9 IC50 ¼ 0.2 nM) with a
minimum of 50 fold selectivity over MMP-1 and -3.
Hanessian and co-workers [94] reported some acyclic
sulfonamide-based hydroxamates having selective MMP-9
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
inhibitory activities over MMP-1, -3 and -13. However, Compound
17 (Fig. 8) showed potent MMP-2 and -9 inhibition (IC50 ¼ 0.7 nM
for MMP-2 and IC50 ¼ 0.2 nM for MMP-9) with greater selectivity
over MMP-3 and -13.
Baxter et al. [96] reported some arylsulfonyl hydroxamates as
potent MMPIs. Compound 18 (Fig. 8) among these molecules yiel-
ded potent but non-selective MMP-9 inhibition (IC50 ¼ 4 nM for
MMP-2 and IC50 ¼ 5 nM for MMP-9). This compound also maintains
high degree of selectivity over MMP-1 (IC50 ¼ 2000 nM), MMP-3
(IC50 ¼ 100 nM), MMP-8 (IC50 ¼ 15 nM). Apart from that a signifi-
cant plasma level concentration at 30 mg/kg dose, compound 18
displayed 40% inhibition of B16F10 melanoma in mouse model that
was comparatively similar like marimastat (2).
Hanessian et al. [97] reported some arylsulfonyl homocysteine
hydroxamates as potent MMPIs. Most of these derivatives displayed
potent and selective MMP-9 inhibition than other MMPs. However,
Compound 19 (Fig. 8) yielded potent MMP-2 inhibition
(IC50 ¼ 0.30 nM) but showed 30 fold better affinity towards MMP-9
(IC50 ¼ 0.01 nM).
Further study conducted by Hanessian and co-workers [98]
resulted in some arylsulfonamido hydroxamates having MMPs
inhibitory properties. Compound 20 (Fig. 8) exhibited the highest
MMP-2 inhibition (IC50 ¼ 1.64 nM) but nearly 2 fold better affinity
towards MMP-9 (IC50 ¼ 0.9 nM). This compound was non-selective
to MMP-1 (IC50 ¼ 198 nM), MMP-3 (IC50 ¼ 6.7 nM), MMP-13
(IC50 ¼ 5.5 nM).
Structural exploration by Yamamoto et al. [99] led to the
development of some N-phenoxy benzyl g-aminobutyric acid
hydroxamates. Compound 21 (Fig. 8) showed excellent MMP-9
inhibition (IC50 ¼ 0.3 nM) having 2.3 fold selectivity over MMP-2
(IC50 ¼ 0.7 nM).
Yang and co-workers [100] did the development of b-N biar-
ylether sulfonamido hydroxamates as selective and potent MMP-9
inhibitors over MMP-2. Compound 22 (Fig. 8) resulted in potent
MMP-9 inhibitory activity (IC50 ¼ 0.52 nM) and 25 fold selectivity
over MMP-2 (IC50 ¼ 13 nM). Some a-tetrahydropyranyl-based
arylsulfonyl hydroxamates were reported as selective and potent
MMP-2 inhibitors over MMP-9 [101].
Fig. 8. Molecular structure of other active MMP-9 inhibitors (Compound 16e25).
11
Fabre et al. [102] reported some triazolyl-substituted arylsul-
fonyl hydroxamates as highly selective and potent inhibitors of
MMP-2 and MMP-9. Compound 23 (Fig. 8) exhibited potent MMP-2
(IC50 ¼ 0.50 nM) and MMP-9 inhibition (IC50 ¼ 0.80 nM).
A series of g-fluorinated a-aminocarboxylic and a-amino-
hydroxamic acids was developed by Behrends and co-workers
[103] as potent gelatinase inhibitors. Compound 24 (Fig. 8)
showed potent MMP-9 inhibition (IC50 ¼ 0.54 nM) having mini-
mum 7 fold selectivity over MMP-2 (IC50 ¼ 3.9 nM).
Nuti et al. [104] reported some N-isopropoxy-arylsulfonamido
hydroxamates as promising and selective inhibitors of MMP-2 over
other MMPs. Though Compound 25 (Fig. 8) showed potent MMP-2
inhibition (IC50 ¼ 0.67 nM), it exhibited comparatively better MMP-
9 (IC50 ¼ 0.43 nM) and MMP-13 (IC50 ¼ 0.19 nM) inhibitory activ-
ities. The in vitro study suggested that compound 25 also signifi-
cantly decreased the invasion and migration of human umbilical
vein endothelial cells (HUVEC) at lower concentration. Further-
more, the western blot and gelatine zymography analysis further
proved that Compound 25 had the ability to inhibit gelatinase ac-
tivity. It also displayed potential cytotoxicity and apoptosis-
inducing ability in endothelial cells. Moreover, it was also found
to respond well to exert antiangiogenic activity in vivo as evidenced
by the matrigel sponge assay model in mice.
A series of arylsulfonamido hydroxamates was reported as
potent gelatinase inhibitors by Zapico and collegues [105]. Com-
pound 26 (Fig. 9) yielded potent MMP-2 inhibition (IC50 ¼ 0.32 nM)
having 3.4 fold selectivity over MMP-9 (IC50 ¼ 1.06 nM).
Hugenberg et al. [69] reported g-fluorinated sulfonylamino
hydroxamic acid derivative based on the lead structure of CGS
27030A. Compound 27 (Fig. 9) (S-enantiomer) showed potent in-
hibition against MMP-9 (IC50 ¼ 3.0 nM) with minimum 10 fold
higher selectivity than MMP-2 (IC50 ¼ 32.8 nM).
8.3.2. Non hydroxamate-based MMP-9 inhibitor
It has been found that hydroxamate moiety provides toxicity,
metabolic instability and also lowers selectivity. For these reasons,
non hydroxamate zinc binding groups (carboxylates, N-hydrox-
yformamides, pyrimidine-2,4,6-triones and others) containing
12 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Fig. 9. Molecular structure of other active MMP-9 inhibitors (Compound 26e35).
compounds were developed [18,106].
8.3.2.1. Carboxylic acid based MMP-9 inhibitors. Kiyama et al. [106]
developed some arylsulfonamido-based carboxylic acids as selec-
tive and potential MMP-2 inhibitors over MMP-9. Compound 28
(Fig. 9) showed almost same inhibitory activity towards MMP-2 and
MMP-9 (IC50 ¼ 4.4 nM for MMP-9 and IC50 ¼ 4.8 nM for MMP-2)
slightly more active against MMP-9.
Zhang et al. [76] reported a series of biphenylsulfonamido car-
boxylic acids as promising gelatinases inhibitors. However, most of
these compounds of this series yielded selective and potent MMP-2
inhibition compared to MMP-9. Compound 29 (Fig. 9) showed
potent MMP-9 inhibition (IC50 ¼ 0.5 nM) similar with MMP-2 in-
hibition (IC50 ¼ 0.7 nM).
Selivanova and co-workers [107] reported some arylsulfona-
mide carboxylic acids having indole function as promising gelati-
nases inhibitors. Compound 30 (Fig. 9) displayed potent MMP-2
and MMP-9 inhibition (IC50 ¼ 0.9 nM for MMP-2 and IC50 ¼ 1 nM
for MMP-9).
Halder et al. [108] reported a series of L-(þ)-isoglutamine de-
rivatives having dual MMP-2 and histone deacetylase 8 (HDAC8)
inhibitory activities. Compound 31 (Fig. 9) was the a MMP-2 in-
hibitor (IC50 ¼ 6400 nM) and showed better affinity towards MMP-
9 (IC50 ¼ 4830 nM).
Adhikari and co-workers [109] synthesized and checked the
biological activities of some biphenylsulfonyl derivatives. Among
these compound 32 (Fig. 9) exhibited both MMP-2 and MMP-9
inhibitory activity but 20.53 times more selective towards MMP-2
over MMP-9 (IC50 ¼ 24 nM for MMP-2 and IC50 ¼ 492 nM for
MMP-9).
Some pentanoic acid derivatives were reported by the same
group showed potent and selective MMP-2 inhibitory activities
[110]. The compound 33 (Fig. 9) was non-selective but shows
comparatively better affinity towards MMP-9 than MMP-2
(IC50 ¼ 4800 nM for MMP-9 and IC50 ¼ 5340 nM for MMP-2).
Ayoup et al. [111] reported some a-aminoacyl amide derivatives
as potent MMP-9 inhibitors. Compound 34 (Fig. 9) was found to
potent and selective against MMP-9. It was about 60 fold MMP-9
selective compared to MMP-2 (IC50 ¼ 4.49 nM for MMP-9 and
IC50 ¼ 266 nM for MMP-2).
Beutel and colleagues [118] showed a number of carboxylic acid
and hydroxamic acid based compound exhibited MMP-2 and MMP-
9 inhibitory activity. Among these molecules, compound 35 (Fig. 9)
having selectivity for both gelatinases (MMP-2 and MMP-9). It has
minimum 12 fold selectivity for MMP-2 than MMP-9 (IC50 ¼ 3.7 nM
for MMP-2 and IC50 ¼ 36.4 nM for MMP-9). It is orally available with
little or no adverse effect.
8.3.2.2. N-Hydroxyformamides as MMP-9 inhibitors. Wada and co-
workers [112] investigated a series of phenoxyphenyl sulfone N-
formylhydroxamates as potent and orally bioavailable MMPIs. S-
stereoisomer of compound 36 (Fig. 10) showed promising selective
MMP-9 inhibition (IC50 ¼ 0.092 nM) over MMP-2 (IC50 ¼ 0.36 nM).
8.3.2.3. Pyrimidine-2,4,6-triones as MMP-9 inhibitors. Among
nitrogen-based heterocyclic ZBGs, pyrimidine-2, 4, 6-trione and
pyrimidine dionethione based inhibitors were mostly studied. As
these inhibitors belonged to the barbiturate class, metabolism and
bioavailability were studied extensively in many FDA-approved
barbiturates [113]. This type of inhibitors coordinates to catalytic
Zn2þ ion by the N3 atom of the barbiturate function [114,115]. Some
C-5 di-substituted barbiturates were reported as potential gelati-
nase inhibitors by Breyholz and co-workers [116]. Compound 37
(Fig. 10) exhibited potential MMP-9 inhibition activity (IC50 ¼ 1 nM)
having 26 fold better activity as well as selectivity against MMP-2
(IC50 ¼ 26 nM). The 125I-radiolabeled compound 37 was a useful
radio-imaging tool and effective in treating diseases like inflam-
mation, atherosclerosis and cancer.
Wang et al. [117] reported some N-substituted homopiperazine
barbiturates as promising gelatinase inhibitors. Compound 38
(Fig. 10) yielded similar potency against both MMP-2 and -9
(IC50 ¼ 1.1 nM).
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Fig. 10. Molecular structure of other active MMP-9 inhibitors (Compound 36e38).
9. Trends in the development of potent and selective MMP-9
inhibitors
In the year 1998, Levin and his colleagues [119] reported a group
of hydroxamate containing diazepine derivatives as potent in-
hibitors of MMPs. The in vitro enzyme inhibition assay of these
compounds against MMP-1, MMP-9 and MMP-13 not just clearly
depicts their potent MMP inhibitory property but also exhibits their
MMP-9 selective nature. The MMP inhibitory profile of these
molecules also shows that the substitutions at the phenyl ring and
heterocyclic nitrogen atom play important roles for their potency as
well as their MMP-9 selectivity. In this set of MMPIs, compound 39
(Fig. 11) showed the highest MMP-9 selectivity over MMP-1 (67.9
fold) and MMP-13 (2.85 fold) while being highly active against
MMP-9 [119].
A set of potent, selective and orally bioavailable gelatinase in-
hibitors were reported by Tamura et al. [120]. The in vitro study of
these compounds showed them as either non-selective or MMP-2
selective molecules. Compound 40 (Fig. 11) of this series exhibi-
ted almost 4 fold selectivity toward MMP-9 over MMP-2. This
compound 40 also showed the higher plasma concentration
exhibiting a Cmax of 284.5 mM in mice.
Almsted and colleagues [121] reported a series of thiazine and
thiazepine derivatives as potent MMPIs. Though most of these
molecules exhibited their no-selective nature against the tested
MMPs, compound 41 (Fig. 11) of this series delivered 4.8 fold, 2.8
fold, 6.6 fold, 36 fold and 3.8 fold selectivity for MMP-9 over MMP-
1, MMP-2, MMP-3, MMP-7 and MMP-8 respectively.
In order to develop piperazine based MMPIs, Cheng and co-
workers [122] designed and synthesized a pool of hydroxamate
containing piperazine analogs as potent inhibitors of MMPs.
Although most of these compounds showed their non-selective
nature between MMP-9 and MMP-13, these MMPIs expressed
their higher selectivity toward MMP-9 compared to MMP-1, MMP-
3 and MMP-7. Compound 42 (Fig. 11) of this series displayed potent
MMP-9 inhibition in picomolar concentration while being almost
9e805 fold MMP-9 selective over MMP-1, MMP-3 and MMP-7.
Compound 42 was also non-selective to MMP-13. Additionally,
compound 43 (Fig. 11), N-carboxy pyridine analogue of compound
42, was almost 2 fold selective to MMP-9 over MMP-13 and 6e865
fold selective over MMP-1, MMP-3 and MMP-7 [122].
With intentions to develop specific MMPIs, Chollet et al. [95]
synthesized a group of 3-bis-aryloxy propionic acid derivatives as
potent MMPIs. The in vitro studies suggest that most of these
13
molecules are either inactive or poorly active against MMP-1 and
are non-selective in nature. Though compound 44 (Fig. 11) of this
series was non-selective in MMP-3 and MMP-9 delivered almost
7e2639 fold MMP-9 selectivity over MMP-1, MMP-2 and MMP-13
[95].
Pikul et al. [123] reported a set of 6-oxo hexahydro pyrimidine
and [1,4]-diazepane containing hydroxamate derivatives as potent
MMPIs. The study reveals that half of these pyrimidine derivatives
are inactive against MMP-7 compared to their diazepane analogs.
Although these authors provided a crystal structure study of one of
those pyrimidine derivatives (compound 45) (Fig. 11) with stro-
melysin, the recemic mixture of compound 46 (Fig. 11) delivered
more or less 3.6e1810 fold MMP-9 selectivity over MMP-1, MMP-3,
MMP-7 and MMP-8. Compound 46 was also equipotent in MMP-2,
MMP-9 and rat MMP-13 [123].
The discovery of some anthranilic acid derivatives as potent
MMPIs were reported by Levin et al. [124]. The in vitro enzyme
inhibitory assay of these molecules suggested them as MMP-9/
MMP-13 non-selective compounds while being selective over
MMP-1 and TACE. Compound 47 (Fig. 11), a meta-trifluoromethyl
phenyl analog of this series, showed 2-fold, 294 fold and 542 fold
MMP-9 selectivity over MMP-13, TACE and MMP-1 respectively.
Additionally, the compound 48 (Fig. 12) with dimethylamino group
substitution at the sulfonamido nitrogen atom seemed to produce
the higher MMP-9 selectivity over MMP-13 [124].
In continuation to the anthranilates as potent MMPIs, Levin et al.
[125] incorporated basic amines into the structure and reported a
group of newer and potent anthranilic acid analogs as potent MMP
inhibitors. Among these newer anthranilates, compound 49
(Fig. 12) and compound 50 (Fig. 12) respectively exhibited an
approximate range of 3.6e81 fold and 2.8e121.6 fold MMP-9
selectivity over MMP-1, MMP-13 and TACE [125].
Nelson and co-workers [126] developed a set of potent benzo-
diazepine analogs as potent MMP and TACE inhibitors and tested
those compounds against MMP-1, MMP-9, MMP-13 and TACE
in vitro. Although most of these inhibitors showed higher inhibitory
potency against all these tested metalloenzymes, compound 51
(Fig. 12) containing para-methoxy phenyl sulfonyl and N-methyl
carboxymethyl piperazine groups at the diazepine nitrogen atoms
exhibited enzyme inhibition along with higher MMP-9 selectivity
(1.69e26.64 fold) over MMP-1, MMP-13 and TACE [126].
A series of retro hydroxamate derivatives were reported by
Wada et al. [112] as potent, selective and orally bioavailable in-
hibitors of MMPs. These sulfone-N-formyl hydroxamates seemed to
14 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260

Fig. 11. Molecular structure of MMP-9 inhibitors (Compound 39e47).

Fig. 12. Molecular structure of MMP-9 inhibitors (Compound 48e56).

be non-selective against MMP-2 and MMP-9 but most of these
molecules showed inactivity against MMP-1. Compound 52 (Fig. 12)
of this series showed greater than 2 fold MMP-9 selectivity over
MMP-2 and 241 fold selectivity over MMP-1 while being able to
show a good bioavailability in cynomologus monkey [112].
Zask et al. [127] synthesized and reported some phenyl sulfo-
namido and hydroxamate containing quinolone and hetero biaryl
derivatives as potent MMP and TACE inhibitors. The in vitro studies
showed that most of these molecules were highly potent against
MMP-9 and MMP-13 but were comparatively less potent against
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
MMP-1 and TACE. Although bearing a MMP-9/MMP-13 non-se-
lective nature, compound 53 (Fig. 12) exhibited 2e387.5 fold MMP-
9 selective nature over MMP-1, MMP-13 and TACE [127].
Some novel arylsulfone piperidine-4-hydroxamate derivatives
were reported by Aranapakam et al. [128] as potent and orally active
MMPIs for the treatment of osteoarthritis. The in vitro enzyme
inhibitory activity studies of these molecules clearly depict their dual
MMP-9/MMP-13 selective nature over MMP-1 and TACE. Though
these MMPIs showed comparative higher affinity for MMP-13 over
MMP-9, compound 54 (Fig. 12) with para-n-butyloxy phenyl sulfonyl
and N-benzyl substitutions was approximately 5 fold and 92 fold
selective to MMP-9 than MMP-13 and TACE respectively. Compound
54 was also poorly active against MMP-1 and expressing 48% MMP-1
inhibition at 10 mM concentrations in vitro [128].
In a stereo specific synthetic approach, Diguarher et al. [129]
developed a set of bis-aryl cyclopentane based carboxylic acid de-
rivatives as specific and potent MMP inhibitors. The in vitro study
suggests these compounds as poorly active MMP-1 inhibitors and
reveals their MMP-2 selective nature. Compound 55 (Fig. 12) of this
series showed promising MMP-9 selectivity of 3.5e3846 fold over
MMP-1, MMP-2, MMP-3 and MMP-13 [129].
In search of the treatment of ischemic stroke Zhang et al. [130]
developed some pyridinone derivatives as potent MMP inhibitors.
Most of these compounds showed dual MMP-2/MMP-9 selective in
nature at sub-nanomolar range in vitro. A few molecules of this
series were also inactive in MMP-1 and produced an activity range
of 0.87e79.1 nM in MMP-2, MMP-3, MMP-9 and MMP-13. From
these few molecules, compound 56 (Fig. 12) delivered 6.43 fold,
7.82 fold and almost 91 fold MMP-9 selectivity over MMP-2, MMP-
13 and MMP-3 respectively [130].
Through optimization of substituents, Yang and co-workers
[100] reported a group of biaryl ether sulfonamido hydroxamate
derivatives as potent gelatinase inhibitors. The MMP inhibitory
activities of these molecules suggest that the special configuration
of these molecules may have a correlation with their MMP-9
selectivity. The compound 57 (Fig. 13) of this series expressed the
highest MMP-9 selectivity over MMP-2 when compared to other
molecules of this series along with a picomolar range MMP-9 in-
hibition in vitro [100].
A group of sulfonamide derivatives as broad-spectrum MMPIs
were reported by Wagner and colleagues [131] as a tool of molec-
ular imaging. Beside their non-selective nature, these molecules
expressed higher affinity toward either MMP-2 or MMP-13. Com-
pound 58 (Fig. 13) of this series, containing a carboxylic acid group
and a para-fluoro-ethoxy phenyl sulfone moiety showed promising
MMP-9 selectivity over MMP-1, MMP-2 and MMP-13. Although
compound 58 is 26 fold less potent against MMP-9 when compared
to its hydroxamate analog, it was almost 2 fold and 4.3 fold MMP-9
selectivity over MMP-2 and MMP-1 but was also inactive in MMP-
13 [131].
A series of selective and potent MMP-12 inhibitors were
designed and synthesized by Nuti et al. [132]. The in vitro biological
evaluation of these molecules against a broad spectrum of MMPs
clearly indicates the benzoic hydroxamates of this series as non-
selective MMPIs. On the other hand, the phenyl acetic hydrox-
amate analogs of these series were both potent and selective to
MMP-12. Compound 59 (Fig. 13), a 4-(para-methoxyphenyl)-oxy-
phenyl sulfonyl group containing benzoic hydroxamate derivative
showed an approximate range of 2e54 fold MMP-9 selectivity over
MMP-2, MMP-3, MMP-8, MMP-12, MMP-13, MMP-14 and MMP-16
[132].
Some hydroxamate group containing a-tetrahydropyranyl sul-
fone and a-piperidine sulfone derivatives as MMPIs were reported
by Becker et al. [133]. The N-2-pyridyl methyl piperidine containing
compound 60 (Fig. 13) showed potent and selective MMP-9
15
inhibition. This Compound 60 also exhibited a range of 2 to
45,000 fold MMP-9 selectivity over MMP-1, MMP-2, MMP-3, MMP-
8, MMP-13 and MMP-14 [133].
A few novel Cobactin-T based molecules were reported by
Wilson et al. [134] as potent MMPIs. The in vitro study reveals that
most of these molecules are highly potent and dual MMP-2/MMP-9
active in nature. Compound 61 (Fig. 13) of this series showed almost
2 fold, 409 fold and 3.1 fold MMP-9 selectivity than MMP-2, MMP-3
and MMP-13, respectively [134].
Through solid phase and in silico evaluation approaches Topai
et al. [135] synthesized a group of potent inhibitors of MMP-2 and
MMP-9. A large number of these compounds either expressed their
selectivity toward MMP-2 over MMP-9 or were non-selective in
MMP-2 and MMP-9. In this scenario, compound 62 (Fig. 13) of this
series exhibited 4.5 fold MMP-9 selectivity over MMP-2.
A group of potent triazole-based hydroxamate derivatives were
synthesized and biologically evaluated by Hugenberg et al. [136].
The in vitro MMP inhibitory assay of these molecules significantly
indicates that the ClogD values of these molecules might have a
correlation with their MMP inhibitory potency. Moreover, com-
pound 63 (Fig. 13) of this series displayed a range of 2.5 folde7 fold
MMP-9 selectivity over the other tested MMP isoforms in vitro
[136].
With the intentions to develop selective MMP-2 inhibitors,
Kreituss and co-workers [137] reported a set of aziridine-triazole
conjugates as potent and selective MMP-2 inhibitors. Regardless
of their MMP-2 selective nature, compound 64 (Fig. 13) showed
almost 30% inhibition of MMP-9 at 20 mM concentrations and was
also better active in MMP-9 over MMP-3, MMP-7, MMP-12, MMP-
13 and MMP-14 in vitro.
In a study to develop promising MMPIs as anticancer agents for
angiogenesis, Hu et al. [138] synthesized a few peptides as potent
anticancer agents. The in vitro study of these peptides depicts that
the one of these peptides [CPU-1: Pro-3-(3-pyridyl)-L-alanine-Cys-
L-4,40 -biphenylalanine-Arg-Gly-Glu-Gly-Gly-Gly-Gly-Ile-Val-Arg-
Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro] showed 1.5 fold and 19 fold
selectivity toward MMP-9 over MMP-3 and MMP-8 respectively.
These authors also reported that the polypeptide “CPU-1” was able
to significantly inhibit the growth of B16F10 mouse melanoma cell
line [138].
Some potent g-fluorinated amino hydroxamates and amino
carboxylic acid derivatives were reported by Behrends et al. [103].
The in vitro study of these molecules transparently exhibited the
superiority of hydroxamic acid derivatives over the carboxylic acid
analogs for higher MMP inhibition. Interestingly, the chirality/
special-conformation of these compounds may have a significant
amount of influence on their activity. In most of these cases for the
carboxylic acid derivatives, the (R)-configured compounds deliv-
ered poor MMP inhibition (for both MMP-2 and MMP-9) whereas
the (S)-enantiomers of those molecules were unable to deliver any
MMP inhibition. The compound 65 (Fig. 13) ((S)-2) of this series
showed almost 11 fold more MMP-9 selectivity over MMP-2. The
(R)-conformer of compound 65 was 4 fold less potency in MMP-9
and 2 fold more MMP-2 selectivity when compared to compound
65 (Fig. 13 [103].
JNJ0966 (66) is inhibited the activation of MMP-9 zymogen [7]
(Fig. 13). It inhibits the activation of MMP-9 by preventing the
conversion of pro-MMP-9 into catalytically active MMP-9 [7].
Scannevin et al. [7] reported the effect of JNJ0966 to inhibit MMP-9
activation.
10. Conclusion and future perspective
Physiological and pathophysiological functions of body depend
on the impact of proteolytic enzymes function. Therefore, broad
16 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Fig. 13. Molecular structure of MMP-9 inhibitors (Compound 57e66).
spectrum inhibition or blockade of protease function will reveal
some systemic effects. These effects may be beneficial, neutral and
harmful to the body due to inhibition of tumor cell invasion and
metastasis. Therefore, research on new agent with anticancer
property is regained. MMP-9 has important role in ECM remodel-
ling, metastasis, angiogenesis, apoptosis and cancer progression.
Hence, MMP-9 and its inhibitors could be important target for
anticancer drug. Selective inhibition of these enzymes in particular
site is linked with potential anticancer activity. Depending on the
structural studies many MMP-9 inhibitor have been to be designed
used in the treatment of cancer. It has been found that most of the
MMP-9 inhibitors were not selective and showed little efficacy.
These can also inhibit other MMPs with similar affinity
simultaneously.
The structural resemblance of MMP-9 with other MMP iso-
forms makes it quite difficult (especially medium size S1’ pocket
MMPs namely MMP-2, MMP-8, MMP-9 and MMP-12). However,
MMP-9 inhibitors may be highly useful in anticancer therapeutics
if selective MMP-9 inhibitors are designed. This will also allow
restricting the off-target adverse effects. Among various ap-
proaches, molecular modelling aspects may be considered to gain
a greater acceptance for reducing time and money. Amino acid
residues forming the S1’ pocket play a major role regarding
selectivity issue is concerned. Nevertheless, the type of hydro-
phobic aryl group present in the ligand/drug structure directed to
fit the S1’ pocket is also equally important for the selective inhi-
bition. Therefore, both the ligand structure and the amino acid
residues forming different pockets (namely, S1’, S1 and S2) may be
identified by molecular docking study. Molecular docking study
performed by Ledour et al. [139] showed that van der Waals
interaction between the p-bromobiphenyl group and Phe110 at S2
subsite is crucial for MMP-9 selectivity compared to the van der
Waals interaction of the same structural motif with Asn111 of
MMP-2. According to that study, the smaller S2 subsite of MMP-9
allows stronger van der Waals interaction compared to the cor-
responding S2 subsite of MMP-2. There are some specific van der
Waals interactions with the indole moiety also (interactions with
amino acid residues Phe421 and Met422). Again, incorporation of
phenyl ring at the 2nd position of the indole moiety of the ligand
structure displayed some specific van der Waals interaction with
Leu187 and His411 at the S2’ subsite of MMP-9. These specific
interactions have been found to be important during designing
selective MMP-9 inhibitors [139]. Khandelwal and colleagues
[140] performed a combined quantum mechanics/molecular
mechanics (QM/MM), molecular dynamics (MD) simulation and
3D-QSAR study on some MMP-9 inhibitors. These approaches also
may be effective in designing selective MMP-9 inhibitors. Never-
theless, receptor-based 3D-QSAR study performed by Tuccinardi
et al. [141] identified the role of some hydrophobic amino acid
residues namely Tyr237 and Tyr420 those are responsible for
stabilizing the substrate at the binding site. QSAR study per-
formed by Ramezani and Shamsara [142] revealed that selective
MMP-9 inhibitors may be designed. Gao et al. [143] performed a
ligand-based pharmacophore mapping and molecular docking
analysis to screen selective MMP-9 inhibitors. Jana and Singh
[144] identified some selective MMP-9 inhibitors through the
combined study of pharmacophore mapping and 3D-QSAR fol-
lowed by virtual screening and drug-likeliness filtering on ADME
properties. Some selective MMP-9 inhibitors were screened from
natural products by Hou and co-workers [145] through ligand-
based pharmacophore mapping followed by molecular docking,
MD simulation as well as bioassay and assessment of ADMET
studies. Apart from these molecular modelling studies, other
methodologies such as quantitative activity-activity relationship
(QAAR) may also be utilized to design selective MMP-9 inhibitors
[146-147]. Depending on these molecular modelling techniques,
newer active as well as selective MMP-9 inhibitors targeted to
anticancer therapy may be designed.
 S. Mondal et al. / European Journal of Medicinal Chemistry 194 (2020) 112260
Declaration of interest statement
The authors have no conflict of interests.
Acknowledgement
SM is thankful to the All India Council for Technical Education
(AICTE) New Delhi for awarding a fellowship. NA is grateful to
Council of Scientific and Industrial Research, India (CSIR), New
Delhi, India for providing research associateship (RA) [FILE NO.: 09/
096(0966)/2019-EMR-I, Dated: 28-03-2019]. SB is grateful to the
RUSA 2.0 of University Grant Commission (UGC), New Delhi, India
to Jadavpur University, Kolkata, India for awarding fellowship [REF.
NO: R-11/1030/19, Dated: 30-07-2019]. SAA sincerely acknowl-
edges CSIR, New Delhi, India for awarding the Senior Research
Fellowship (SRF) [FILE NO.: 09/096(0967)/2019-EMR-I, Dated: 01-
04-2019]. TJ is thankful for the financial support from RUSA 2.0 of
UGC New Delhi to Jadavpur University, Kolkata, India. The authors
heartily thank Dr. Balaram Ghosh of BITS Pilani, Hyderabad
Campus, India as well as Mr. Sandip Kr. Baidya, Mr. Saptarshi Sanyal
and Mr. Rajat Sarkar of Jadavpur University, Kolkata for their kind
support. The authors also thank the support from Department of
Pharmaceutical Technology, Jadavpur University, Kolkata, India for
providing the research facilities.
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