Selective non-zinc binding MMP-2 inhibitors: Novel benzamide Ilomastat analogs with anti-tumor metastasis

Article abstract of DOI:10.1016/j.bmcl.2016.03.064

Novel Ilomastat analogs with substituted benzamide groups, instead of hydroxamic acid groups, were designed, synthesized and evaluated against MMP-2 and MMP-9. Among these analogs, the most potent compound 10a exhibited potent inhibitory activity against

Full text of DOI:10.1016/j.bmcl.2016.03.064

Accepted Manuscript
Selective non-zinc binding MMP-2 inhibitors: Novel benzamide Ilomastat an-
alogs with Anti-tumor Metastasis
Jiao Song, Peng Peng, Jun Chang, Ming-Ming Liu, Jian-Ming Yu, Lu Zhou,
Xun Sun
PII:
S0960-894X(16)30284-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.064
BMCL 23707
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 February 2016
14 March 2016
16 March 2016
Please cite this article as: Song, J., Peng, P., Chang, J., Liu, M-M., Yu, J-M., Zhou, L., Sun, X., Selective non-zinc
binding MMP-2 inhibitors: Novel benzamide Ilomastat analogs with Anti-tumor Metastasis, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.064
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Selective non-zinc binding MMP-2 inhibitors: Novel benzamide
Ilomastat analogs with Anti-tumor Metastasis
Jiao Songa, Peng Penga, Jun Changa, Ming-Ming Liub, Jian-Ming Yua, Lu Zhoub,*, Xun Suna,c,*
a Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826
Zhangheng Road, Shanghai 201203, China
b Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng
Road, Shanghai 201203, China
c Shanghai Key Lab of Clinical Geriatric Medicine, 221 West Yanan Road, Shanghai 200040,
China
*To whom correspondence should be addressed. Tel.: +86-21-51980003; Fax:
+86-21-51980003.
E-mail address: zhoulu@fudan.edu.cn (L. Zhou); sunxunf@shmu.edu.cn (X. Sun)

Abstract
Novel Ilomastat analogs with substituted benzamide groups, instead of hydroximic
acid groups, were designed, synthesized and evaluated against MMP-2 and MMP-9.
Among these analogs, the most potent compound 10a exhibited potent inhibitory
activity against MMP-2 with IC50 value of 0.19 nM, which is 5 times more potent
than that of Ilomastat (IC50 = 0.94 nM). Importantly, 10a exhibited more than 8300
fold selectivity for MMP-2 vs MMP-9 (IC50 = 1.58 μM). Molecular docking studies
showed that 10a bond to the catalytic active pocket of MMP-2 by a
non-zinc-chelating mechanism which was different from that of Ilomastat.
Furthermore, the invasion assay showed that 10a was effective in reducing HEY cells
invasion at 84.6% in 50 μM concentration. For 10a, the pharmacokinetic properties
had been improved and especially the more desirable t1/2z was achieved compared
with these of the lead compound Ilomastat.
Keywords: MMP-2 and MMP-9, Ilomastat analogs, benzamide groups, enzyme
inhibition, non-zinc binding, HEY cell invasion

Cancer is a key health issue across the world, causing substantial patient morbidity
and mortality. World Cancer Report in 2014 reported that the death toll was 2.2
million in China, accounting for a quarter of global cancer deaths.1 Cancer survival
rate has been significantly improved over the years by early diagnosis and cancer
growth inhibition.2 However, as long as cancer metastasis occurs, it will be highly
incurable and fatal which accounts for about 90% of cancer deaths.3,4 Until recently,
limited success has been achieved on prevention and inhibition of cancer metastasis.2
Therefore, developing drugs to control cancer metastasis is still an urgent need to
improve the clinical cancer survival rate.
Overexpression of matrix metalloproteinase (MMP) plays an important role in the
context of tumor invasion and metastasis.5,6 Extracellular matrix (ECM) is the most
important physiological barrier for the metastasis of tumor cells.7 Overexpression of
MMP in tumor cells can destroy ECM, which could promote the invasion of cancer
cells.8 Thus, MMP inhibitors have been attractive anti-cancer targets. Among the
MMP family, MMP-2 and MMP-9 were identified to be more critical in the invasion
of tumor cells cross basement membranes.9,10 Though they bind to a common
substrate, MMP-2 has been selected as a promising target for anti-cancer drugs,
whereas MMP-9 is regarded as a non-safe target for these drugs.9,11 So far, many
MMP-2 inhibitors have been developed as anti-cancer agents. However, owing to the
low selectivity for MMP-2, most of these compounds were unsuccessful in clinical
trials. Thus, searching for novel potent MMP-2 compounds with high selectivity is an
important objective to overcome clinical cancer metastasis, such as lung, ovarian,
breast and other malignant tumors.
Ilomastat (1), a zinc-binding inhibitor, is in phase III clinical trials (Fig. 1).12 Its
hydroxamic acid specifically forms a bidentate complex with the active site zinc.13
Ilomastat was one of the most potent MMP inhibitors and had a good biological
activity to many diseases, such as tumor, sudden liver failure and postoperative
corneal reparation.14 However, Ilomastat was a non-selective broad spectrum
inhibitor,15,16 since the hydroxamic acid could interact at other subsites containing
zinc ion and/or some other oxidative states of metals, such as iron (III).15,17-19 In
addition, it also had some other drawbacks, such as poor oral bioavailability
(administration via injection).19-21

Figure 1. Chemical structures of Ilomastat (1) and Chidamide (2)
To improve the poor selectivity of Ilomastat, many research groups had endeavored
to explore and develop other alternative zinc-binding group replacements, such as
carboxylate, hydrazide, thiol, phosphonic acid and other heterocycles.22-24
Unfortunately, these modified analogs without the hydroxamic acid feature usually
had lower inhibitory activity for MMP-2 compared to their hydroxamate
counterparts.22 Until recently, only a very few success has been achieved on finding
compounds which could efficiently and selectively inhibit MMP-2. Herein, we found
a series of Ilomastat analogs with hydroxamate group replaced by benzamide group.
Interestingly, one resulted analog, 10a, exhibited very potent MMP-2 inhibitory
activity (IC50 = 0.19 nM) and had more than 8300-fold selectivity over MMP-9.
The rationale to design these compounds was based on the idea to hybrid the
functional groups from both Ilomastat (1) and Chidamide (2) (Fig. 2). Chidamide was
a histone deacetylase (HDAC) inhibitor, which has a good subtype selectivity and
anti-cancer activity.25 It was approved in the global market in 2015 for the treatment
of recurrent and refractory peripheral T-cell lymphoma (PTCL). HDACs and MMPs
are both zinc-dependent enzymes.23 Recent years, several classes of HDAC inhibitors
had been found to have potent and specific anti-cancer activities in preclinical and
clinical studies.26 Initially, HDACs inhibitors were hydroxamic acid derivatives,
whose hydroxamate group binds to zinc ion in an active site (Fig. 2), such as
trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). These were also
non-selective broad spectrum inhibitors. Later, new HDACs inhibitors, exampled by
Chidamide, were developed with subtype selectivity, whereas the benzamide group
was used as a surrogate of the hydroxamate group.27 Worth to mention, some HDACs
inhibitors could interact with MMPs and had effective anti-metastatic activity. Lu et
al.28 reported that TSA, which suppresses MCF-7 breast carcinoma cell invasion and
up-regulates TET1 expression, could promote the expression of tissue inhibitors of
metalloproteinase 2/3 (TIMP 2/3) and inhibit transcriptional activity of MMP-2 and
MMP-9 matrix metalloproteinase. Molecular docking studies showed that Chidamide
binds to the catalytic active pocket of HDAC by a similar zinc-chelating mechanism

to TSA; however, the benzamide group not only chelates with catalytic zinc ion, but
also can form new π–π stacking interactions and hydrogen bonds significantly
different from TSA.27 This could partially explain the higher selectivity of Chidamide
than TSA.
Figure 2. Design strategy for benzamide Ilomastat analogs
Thus, we used the same strategy as that for Chidamide to incorporate a benzamide
group into Ilomastat (hydroxamate replacement) to design a series of novel
compounds which may have good activity, higher selectivity, and improved oral
bioavailability. Based on previous structure-activity relationship (SAR) study of
Chidamide, para-substituents on benzamide ring could significant affect the HDACs
inhibitory activity.29 This information could help our SAR study to rationally design a
small focused library. Thus, eight new benzamide compounds were designed and
synthesized (Fig. 2). These analogs were evaluated for their inhibitory activity against
both MMP-2 and MMP-9. Meanwhile, their binding modes with MMP-2 and MMP-9
were also explored.
The syntheses of the target compounds 10a-h were accomplished as indicated in
Scheme 1, followed the reference method12 with some minor modifications.
Commercially available 4-methylvaleryl chloride was used as the starting material,
which coupled with a chiral auxiliary (S)-4-benzyl-2-oxazolidinone to provide
compound 3 in 90% yield. Compound 3 was then treated with K-HMDS and
tert-butyl bromoacetate to obtain 4 in a moderate yield. The auxiliary of 4 was
removed by using hydrogen peroxide and lithium hydroxide to afford in 5 in high
yield. The intermediate 5 was coupled with 7 to give 8 in 40% yield, which was

hydrolyzed under TFA condition to give carboxylic acid 9 in 70% yield. The last step
for amide formation to form 10 was turned out to be problematic when CDI was used
as the coupling reagent.30 After many attempts, analogs 10a-h could be synthesized in
20-50% yields by using EDCI hydrochloride salt as a coupling reagent. The reaction
for this step was relatively messy. The desired products were isolated and their
1
structures were characterized by H NMR, 13C NMR and high resolution mass
spectrum. In addition, spectra of these compounds were also further compared with
the patterns of same moiety presented in intermediates or products reported.30,31
Scheme 1. General synthetic scheme for analogs 10a-h. Regents and conditions: a) n-BuLi (2.5 M,
1.0 equiv), anhydrous THF, -78ºC→-45ºC→RT, nitrogen; b) K-HMDS (1M, 1.2 equiv),
anhydrous THF, -78ºC→RT, nitrogen, overnight; c) H2O2 (30%, 4.0 equiv), LiOH (0.8 M, 1.6
equiv), NaNO2 (4.0 equiv), THF/H2O (v/v = 4:1), 0ºC; d) CH3NH2/CH3OH (30%, 10.0 equiv), RT,
overnight; e) 5 (1.0 equiv), EDCI (1.2 equiv), DMAP (0.5 equiv), anhydrous THF, RT, overnight; f)
TFA/DCM (v/v = 2:1), anisole (0.5 mL), RT, 0.5 h; g) EDCI (1.5 equiv), DMAP (0.5 equiv),
aniline, anhydrous DCM, nitrogen, RT, overnight.
The inhibitory activities of synthesized compounds against MMP-2 and MMP-9
were evaluated by using a fluorimetric assay16 and the results were illustrated in Table
1. Ilomastat was also tested as a positive control. Overall, four compounds (10a-b, 10f
and 10h) displayed a certain extent inhibitory activity toward MMP-2. Among them,
2-amino-phenyl derivative 10a showed the most potent inhibitory activity (IC50 = 0.19
nM) against MMP-2, which was 5 times more potent than that of Ilomastat (IC50 =
0.94 nM). In addition, 10a exhibited high selectivity to MMP-2, which was more than
8300 fold over MMP-9. When the 2-amine group (analog 10a) was switched to

3-position (analog 10h) or acylation of 2-amino group (analog 10g), both compounds
lost potency against MMP-2 (6- and 110-fold decreased respectively) compared with
10a, though increased undesired potency for MMP-9. Retaining R1 group as the
amino group and modifying R2 group by introducing electron-donating or
electron-withdrawing group, all resulted analogs 10b-f displayed much lower
inhibitory activity against MMP-2. Except 10a, compounds 10b-h also exhibited low
selectivity for MMP-2 vs MMP-9.
Table 1. MMP-2 and MMP-9 inhibitory activity of 10a-h
compound
10a
R1
2-NH2
R2
H
MMP-2a
0.19
MMP-9a
1579.01
7.75
Selectivityb
8310
3.52
-
2-NH2
F
2.20
10b
10c
＞104
＞104
＞104
21.80
＞104
2.05
＞104
＞104
7297.04
27.32
2-NH2
CF3
COPh
CH3
Br
2-NH2
-
10d
2-NH2
-
10e
2-NH2
1.25
-
10f
2-NHCOCH3
3-NH2
H
155.19
13.52
10g
H
6.59
0.58
10h
0.94
0.55
Ilomastat
a: Inhibition data are expressed as IC50 (nM).
b: relative selectivity15: MMP-9 (IC50) / MMP-2 (IC50)
-: no obvious inhibition;
Ilomastat: positive control
Molecular docking studies were carried out to explore the binding modes of 10a
with MMP-2 and MMP-9. Initial protein structures of MMP-2 and MMP-9 were
taken from structural models deposited in the Protein Data Bank (PDB) with the entry
code 1HOV and 4H3X, respectively. It was found that 10a binds to both MMP-2 and
MMP-9 in non-zinc binding modes. For MMP-2, the benzamide group of 10a
interacts with the residues of the specific loop, presenting van der Waals interactions

with residues Tyr 142, Leu 116, Thr 143, Leu 137, Ala 139, Ile 141, and Ala 136
sitting at the bottom of the S1' sub-site,32 while the 2-amino group can form hydrogen
bonds with Ala 136 and Ile 141 (Fig. 3A and 3B). For MMP-9, due to the limited
pocket size, the benzamide group locates at the entrance of the S1' hydrophobic
pocket and presents weak van der Waals interactions with residues Pro 421, Met 422
and Tyr 423 (Fig. 4A and 4B). Since S1' pockets in MMP-2 and MMP-9 have
different shapes and sizes,33 only MMP-2 has enough space to accommodate the
benzamide group. In addition, the indole group of 10a is localized in the S2 cavity of
MMP-2, and the phenyl ring can form a π–π interaction with His 124 and weak van
der Waals interactions with residues Ala 84, His 85, and Ala 86.15 For MMP-9, the
indole ring of 10a deviates from S2 cavity, which results only weak van der Waals
interactions with residues Ala 189, His 190, and Ala 191. These may explain why 10a
had a high selectivity against MMP-2 vs MMP-9. Meanwhile, we measured the
lipid-water partition coefficient (log P) for both 10a and Ilomastat. The log P of 10a
was 1.91, which was higher than Ilomastat (log P = 1.04). This could also potentially
contribute the feasibility of 10a to bind with hydrophobic active pockets of MMP-2
than Ilomastat.
Figure 3. The surface map and interactions between 10a and MMP-2. A) The surface map of

interactions between 10a and MMP-2; B) the interactions between 10a and residues of active sites
of MMP-2.
Figure 4. The surface map and interactions between 10a and MMP-9. A) The surface map of
interactions between 10a and MMP-9; B) The interactions between 10a and residues of active
sites of MMP-9.
The anti-invasion effect of 10a on human ovarian cancer cell line HEY was
evaluated using matrigel based Transwell invasion assay. First, the cytotoxicity of 10a
was determined on HEY cells using MTT assay. As shown in Figure S1 (see
supporting information), cell viability was not significantly affected by treatment with
10a at 6.25-100 μM compared with the control (DMSO). However, solid particles
were observed by microscope in the cell culture medium at the concentration of 100
μM, indicating the saturation of 10a in culture at this concentration. Therefore,
non-cytotoxic concentrations (12.5, 25 and 50 μM) of 10a were applied in the
subsequent Transwell invasion assay. It showed that the number of cells invading
matrigel membrane and migrating from the upper to the lower chamber was
dose-dependently reduced, after the treatment with 10a (Fig. 5A). At concentration of
50 μM, 84.6% reduction of cellular invasive and migratory activity was achieved, as
shown in Fig. 5B. This clearly demonstrated that 10a possessed effective activity
against cellular invasion and migration.

Figure 5. The effects of 10a and Ilomastat on HEY cells invasion. A) The photomicrographs of
control, 10a and Ilomastat. After 24h of incubation with 12.5, 25, 50 μM of 10a and Ilomastat, the
numbers of cells per view were determined by crystal violet staining. B) The effects of HEY cells
invasion with 10a and Ilomastat. Different effects of 10a and Ilomastat on invasion of HEY cells
were evaluated using t test; ***P ＜0.001 10a compared with Control and ###P ＜0.001 Ilomastat
compared with Control; No significant difference with the same concentration of 10a and
Ilomastat.
Furthermore, compound 10a was dosed to Sprague-Dawley rat intravenously (10
mg/kg) and orally (10 mg/kg) for pharmacokinetic (PK) study. In general, PK
properties of 10a were better than that of Ilomastat (Table 2). After intravenous
administration, AUC0-24h of 10a was 3068.05 µg*h/L and slightly better than
Ilomastat (2508.38 µg*h/L). Compound 10a also showed lower clearance (3.13
L/h/kg) than that of Ilomastat (3.99 L/h/kg). As a consequence, the more desirable t1/2z
of 10a (7.88 h) was achieved compared with that of Ilomastat (0.25 h). After oral
(P.O.) administration, compound 10a showed a good t1/2z (5.64 h), thought its

clearance was too long. However, the absorption of Ilomastat was unable to be
detected even at the lowest detection concentration. In addition, the oral
bioavailability was slightly improved (%F of 10a and Ilomastat was 4 and 0,
respectively) at 10 mg/kg. Thus, overall PK profiles of 10a have been improved to a
certain extent compared with Ilomastat.
Table 2. Pharmacokinetic parameters of 10a and Ilomastat after intravenous and oral
administration at a dose of 10 mg/kg in Sprague-Dawley rats
administration route
Pharmacokinetic
Intravenous (IV)
Oral (PO)
parameters
Ilomastat
Ilomastat
\
10a
10a
AUC0-24h (µg*h /L)
AUC0-∞ (µg*h /L)
3068.05
2508.38
2509.32
36.18
\
3209.16
140.44
Vz (L/kg)
t1/2z (h)
35.76
0.25
3.99
579.41
5.64
\
\
\
1.43
7.88
3.13
CLz (L/h/kg)
71.20
AUC0 24h, area under the concentration vs. time curve from 0 to 24h; AUC0→∞, area under the
→
concentration vs. Time curve from 0 to infinity; t1/2z, terminal half-life; Clz, clearance; \: cannot be
detected.
In summary, we designed and synthesized a series of novel benzamide Ilomastat
analogs as a small focused library to explore their inhibitory activity for MMP-2 and
MMP-9. Among them, compound 10a exhibited very potent inhibitory activity against
MMP-2, which was 5 times more potent than that of Ilomastat. More importantly, 10a
showed high selectivity to MMP-2, which was more than 8300-fold over MMP-9.
Molecular docking studies indicated that 10a binds to the catalytic active pocket of
MMP-2 by a non-zinc-chelating mechanism. In addition, the invasion assay indicated
that 10a had a good anti-invasion effect, reducing HEY cell invasion at 84.6% in 50
μM concentration. The pharmacokinetic properties of 10a also have been improved
and especially the t1/2z of 10a was much longer than that of Ilomastat. Further
optimization of 10a to improve the oral bioavailability is ongoing and will be reported
in due course.
Acknowledgements
The work was supported by grants from the National Natural Science Foundation
of China (No: 81373276), Shanghai Municipal Committee of Science and Technology

(No: 14XD1400300, 13431900101), and National Science and Technology Major
Project of China (No. 2013ZX09J13103-02C).
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Graphic Abstract