Matrix Metalloproteinase 13 Inhibitors for Modulation of Osteoclastogenesis: Enhancement of Solubility and Stability

Article abstract of DOI:10.1002/cmdc.202000911

Matrix metalloproteinase 13 (MMP-13) activity has been correlated to breast cancer bone metastasis. It has been proposed that MMP-13 contributes to bone metastasis through the promotion of osteoclastogenesis. To explore the mechanisms of MMP-13 action, we previously described a highly efficacious and selective MMP-13 inhibitor, RF036. Unfortunately, further pursuit of RF036 as a probe of MMP-13 in vitro and in vivo activities was not practical due to the limited solubility and stability of the inhibitor. Our new study has explored replacing the RF036 backbone sulfur atom and terminal methyl group to create inhibitors with more favorable pharmacokinetic properties. One compound, designated inhibitor 3, in which the backbone sulfur and terminal methyl group of RF036 were replaced by nitrogen and oxetane, respectively, had comparable activity, selectivity, and membrane permeability to RF036, while exhibiting greatly enhanced solubility and stability. Inhibitor 3 effectively inhibited MMP-13-mediated osteoclastogenesis but spared collagenolysis, and thus represents a next-generation MMP-13 probe applicable for in vivo studies of breast cancer metastasis.

Full text of DOI:10.1002/cmdc.202000911

Accepted Article
Title: Matrix Metalloproteinase 13 Inhibitors for Modulation of
Osteoclastogenesis: Enhancement of Solubility and Stability
Authors: Gregg Fields, Anna M. Knapinska, Chandani Singh, Gary
Drotleff, Daniela Blanco, Cedric Chai, Jason Schwab, and
Anu Herd
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000911
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0
1/2020

ChemMedChem
10.1002/cmdc.202000911
FULL PAPER
Matrix Metalloproteinase 13 Inhibitors for Modulation of
Osteoclastogenesis: Enhancement of Solubility and Stability
[
a,c]
[a,c]
[b]
[c]
[b]
Anna M. Knapinska,
Chandani Singh,
Gary Drotleff, Daniela Blanco, Cedric Chai, Jason
[
c]
[c]
[a,c,d]
Schwab, Anu Herd, and Gregg B. Fields*
[
a]
Dr. A.M. Knapinska, C. Singh, Prof. G.B. Fields
Department of Chemistry & Biochemistry
Florida Atlantic University
5
353 Parkside Drive, Jupiter, FL 33458
E-mail: fieldsg@fau.edu
G. Drotleff, C. Chai
[
[
[
b]
c]
d]
Department of Biological Sciences
Florida Atlantic University
7
77 Glades Road, Boca Raton, FL 33431
Dr. A.M. Knapinska, C. Singh, D. Blanco, J. Schwab, A. Herd, Prof. G.B. Fields
Institute for Human Health & Disease Intervention (I-HEALTH)
Florida Atlantic University
5
353 Parkside Drive, Jupiter, FL 33458
Prof. G.B. Fields
Department of Chemistry
The Scripps Research Institute/Scripps Florida
1
20 Scripps Way, Jupiter, FL 33458
Supporting information for this article is given via a link at the end of the document.
Abstract: Matrix metalloproteinase 13 (MMP-13) activity has been
correlated to breast cancer bone metastasis. It has been proposed
that MMP-13 contributes to bone metastasis via promotion of
osteoclastogeneis. To explore the mechanisms of MMP-13 action, we
previously described a highly efficacious and selective MMP-13
inhibitor, RF036. Unfortunately, further pursuit of RF036 as a probe of
MMP-13 in vitro and in vivo activities was not practical due to the
limited solubility and stability of the inhibitor. The present study has
explored replacement of the RF036 backbone sulfur atom and
terminal methyl group to create inhibitors with more favorable
pharmacokinetic properties. One compound, designated inhibitor 3, in
which the backbone sulfur and terminal methyl group of RF036 were
replaced by nitrogen and oxetane, respectively, had comparable
activity, selectivity, and membrane permeability to RF036 while
Upon autopsy 70-80% of women that succumb to breast
[
5]
cancer have evidence of bone metastases. The metastases
generate extensive bone destruction as a result of uncontrolled
osteoclast activity, causing the patient great pain and contributing
significantly to the morbidity associated with the disease. Breast
cancer cells may induce the expression of potent
osteoclastogenic factors, such as receptor activator of nuclear
[
6]
kappa B (NF-κB) ligand (RANKL), by bone-lining osteoblasts.
Inoculation of nude mice with MDA-MB-231 breast cancer cells
resulted in osteoclasts resorbing bone, degradation of bone
[
7]
matrix, and metastasis. The expression of RANKL, MMP-13,
and MT1-MMP mRNA was increased in the metastasized bone,
and MMP-13 protein was found in breast cancer bone
[
7-8]
metastases.
Subsequently, MMP-13 in MDA-MB-231 breast
exhibiting greatly enhanced solubility and stability. Inhibitor
3
cancer cells was found to be responsible for increased bone
resorption and osteoclastogenesis, which was reduced by the
effectively inhibited MMP-13-mediated osteoclastogenesis but spared
collagenolysis, and thus represents a next generation MMP-13 probe
applicable for in vivo studies of breast cancer metastasis.
[
8]
application of MMP inhibitors. The MMP-13 inhibitor 5-(4-{4-[4-
(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-
methoxyethyl)-pyrimidine-2,4,6 (1H,3H,5H)-trione (compound 28
[
9]
in ) inhibited MDA-MB-231 breast cancer xenograft growth and
significantly reduced osteolytic damage in an in vivo prevention
Introduction
[10]
study. The inhibitor did not cause an increase in creatine kinase,
[
10]
a measure of muscle toxicity.
Analysis of The Cancer Genome Atlas (TCGA) data found that
the protease matrix metalloproteinase 13 (MMP-13) was almost
universally upregulated across 15 different cancer types, with
significant upregulation in 12 cancers, including breast cancer
Concern has been raised that many anti-cancer drugs
[11]
undergoing clinical trials actually function by off-target toxicity.
Prior MMP inhibitors, particularly those possessing hydroxamic
groups which target the active site zinc, have been shown to have
[
1]
(
BRCA). MMP-13 was originally isolated and cloned from breast
[12]
considerable off-target activities. To counteract this behavior,
[
2]
carcinomas. Microarray analysis of genes from infiltrating
lobular carcinoma, metaplastic carcinoma, and infiltrating ductal
carcinoma of breast cancer patients revealed 100%
selective, non-zinc chelating MMP-13 inhibitors have been
[9-10, 13]
described.
However, important pharmacokinetic (PK)
and/or other data has not been reported for many of these
compounds, and no clinical studies have appeared. Some of the
most promising recent selective MMP-13 inhibitors had
nephrotoxicity and/or poor solubility, permeability, biodistribution,
[
3]
overexpression of MMP-13. MMP-13 was secreted as a protein
[
3]
in pro and active forms. MMP-13 protein levels correlated with
lymph node metastasis of breast cancer and inversely with patient
[
4]
survival.
1
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ChemMedChem
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metabolic stability, and/or bioavailability and thus the search for Results and Discussion
[
13o, 13p]
new MMP-13 inhibitors continues.
Our laboratory has developed a series of small molecules
that are highly selective for MMP-13 with IC50 values in the low
Synthesis of MMP-13 Inhibitors
Inhibitor oral bioavailability can be improved by increasing
compound (a) solubility, (b) cell permeability, and (c) metabolic
[
14]
[14a]
nM range (2.7-5.9 nM). RF036 (compound (S)-17b in
and
) (Figure 1) has an IC50 of 3.4-4.9 nM for MMP-
3 and is highly selective. RF036 displayed an excellent
permeability profile in Caco-2 based membrane permeability
[
14b]
[15]
[14b]
compound 2 in
stability. RF036 has a fairly low kinetic solubility of 15.5 µM.
-
6
1
RF036 is moderately cell permeable (2.38 x 10 cm/sec) with
[
14b]
retention fraction in the lipid bilayer of 47%,
which indicates
-
6
studies, with Papp= 2.38 x 10 cm/sec. RF036 had a long plasma
half-life time (T1/2 = 2.93 h), high maximum plasma concentration
that oral bioavailability could be restricted through limited diffusion
of the compound through and retention of the compound in the
gastrointestinal wall. The in vitro t1/2 of RF036 was only 9 min in
(Cmax = 47.6 µM), and low clearance rate (Cl = 0.18 mL/min/kg)
[
14b]
after intravenous administration in rats. However, compound
RF036 suffers from poor metabolic stability when incubated with
mouse, rat, and human liver microsomes, and its solubility is
marginal for in vivo applications. The present research selectively
targets RF036 areas of potential instability and replaces them with
more stable bonds and moieties, while also seeking to improve
compound solubility. The overall goal is to create a highly stable,
first in class agent that targets MMP-13 in breast cancer.
human microsomes,
which means that low oral bioavailability
could result from degradation of the compound by CYP450
dependent metabolizing enzymes. There are two obvious areas
of potential instability in compound RF036, the sulfur and the
[
14b]
methylamine (Figure 1).
Replacement of the sulfur by either a
carbon or a nitrogen only slightly altered the MMP-13 inhibitory
activity of the resulting compounds but improved stability in
[
14b]
human microsomes.
Replacement of the methyl group in the
methylamine by heteroatom cyclic butane analogs also only
slightly altered the MMP-13 inhibitory activity of the resulting
[
14b]
compounds but improved stability in human microsomes.
Figure 1. Structures of Inhibitors 1-4 and RF036.
In the present study, the sulfur from RF036 was replaced with
carbon (inhibitor 1 and inhibitor 2) or nitrogen (inhibitor 3 and
inhibitor 4) and the methyl in the methylamine group was replaced
with oxetane (1,3-propylene oxide) (inhibitor 1 and inhibitor 3) or
azetidine (1,3-propylenimine) (inhibitor 2 and inhibitor 4) (Figure
valine methyl ester hydrochloride was reacted with compound 9
using EDCI•HCl, HOBt, and NEt . Synthesis of compound 11 from
compound 10 was the same as treatment of compound 7, step 1),
3
[
14b]
in Scheme 1 of our prior study.
For the synthesis of inhibitor 1
from compound 11, oxetan-3-amine was reacted with compound
11 using EDCI•HCl, HOBt, and DIPEA. For the synthesis of
inhibitor 2 from compound 11, tert-butyl 3-aminoazetidine-1-
carboxylate was reacted with compound 11 using EDCI•HCl,
HOBt, and DIPEA, followed by removal of the Boc group with TFA
treatment for 3 h at room temperature.
1
). For the synthesis of inhibitor 1 and inhibitor 2 (Scheme 1),
steps from compound 1 to compound 8 were identical to those
[
14b]
from compound 57 to compound 61 as described previously.
Synthesis of compound 9 from compound 8 was the same as
treatment of compound 61, step 1), in Scheme 6 of our prior
[
14b]
study.
For the synthesis of compound 10 from compound 9, L-
2
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Scheme 1. Synthesis of Inhibitors 1 and 2.
Compound
17
(2-chloro-1,5,6,7-tetrahydro-4H-
with NaOH for 1.5 h at 110 °C. After acidifying with 2 M HCl,
compound 17a was obtained. Compound 17a was reacted with
phosphorus oxychloride for 2 h at 120 °C to provide compound
17b.
cyclopenta[d]pyrimidin-4-one) was synthesized starting from
ethyl-2-oxo-cyclopentane-carboxylate (Scheme 2). Ethyl-2-oxo-
cyclopentane-carboxylate was reacted with urea and
concentrated HCl for 4 h at 80 °C, and the product was heated
Scheme 2. Synthesis of intermediate 17.
[
14b]
For the synthesis of inhibitor 3 and inhibitor 4 (Scheme 3),
study.
L-valine methyl ester hydrochloride was reacted with compound
19 using EDCI•HCl, HOBt, and NEt . For the synthesis of
For the synthesis of compound 20 from compound 19,
steps from compound 12 to compound 18 were identical to those
[
14b]
from compound 62 to compound 67 as described previously.
3
Synthesis of compound 19 from compound 18 was the same as
treatment of compound 67, step 1), in Scheme 6 of our prior
compound 21 from compound 20, compound 20 was treated with
2 M NaOH for 8 h at 75 °C. For the synthesis of inhibitor 3 from
3
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compound 21, oxetan-3-amine was reacted with compound 21
using EDCI•HCl, HOBt, and DIPEA. For the synthesis of
compound 22 from compound 21, tert-butyl 3-aminoazetidine-1-
carboxylate was reacted with compound 21 using EDCI•HCl,
HOBt, and DIPEA. For the synthesis of inhibitor 4 from compound
22, compound 22 was treated with TFA for 4 h at room
temperature.
Scheme 3. Synthesis of Inhibitors 3 and 4.
MMP Activity of Inhibitors
for inhibitor 1 and inhibitor 3 are comparable to the parent
[
14]
Compounds were initially screened for their activity against MMP-
i
compound, RF-036 (K = 2.7 nM). Inhibitors 1, 2, and 3 all had
1
8
2
3 and their selectivity for MMP-13 versus MMP-1, MMP-2, MMP-
, MMP-9, and MMP-14/MT1-MMP (Table 1). Inhibitor 1, inhibitor
selectivity comparable to that observed with RF-036, as the IC50
values for other MMPs were greater than 5 µM (Table 1). RF036
had IC50 values of >5 µM for MMP-1, MMP-2, MMP-8, MMP-9,
, and inhibitor 3 were all effective inhibitors of MMP-13, with K
i
[
14]
values of 12, 42, and 10 nM, respectively (Table 1). The values
and MMP-14/MT1-MMP.
Table 1. Inhibition of MMPs.
Compound
MMP-13
(nM)
MMP-1
IC50 (nM)
>5000
MMP-2
IC50 (nM)
>5000
MMP-8
IC50 (nM)
>5000
MMP-9
IC50 (nM)
>5000
MMP-14
IC50 (nM)
>5000
K
i
a
RF036
2.7 ± 0.6
12.2 ± 0.8
41.9 ± 3.6
10.3 ± 0.6
Inhibitor 1
Inhibitor 2
Inhibitor 3
Inhibitor 4
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
b
28.1 ± 1.5
>5000
>5000
>5000
>5000
>5000
c
6
3.7 ± 1.5
d
7
4
d
d
d
d
d
1
3
3
5
6
34
34
47
44
77
d
1
600
a
b
[14a]
RF036 values were previously reported.
c
d
TFA salt; TFA salt after 3 month storage in DMSO; formic acid salt form over 3 month time period storage in DMSO.
Inhibitor
4
had variable activity against MMP-13. For
formic acid form of inhibitor 4 after 5-7 days. When inhibitor 4 was
produced as the TFA salt, the stability in DMSO was improved (as
monitored by LC-MS), but nonetheless the IC50 value increased
from 28 to 64 nM over time (Table 1). Inhibitor 4 was found to
example, when inhibitor 4 was produced as the formic acid salt,
the IC50 values for MMP-13 increased over time, from 74 nM to
1
2
.6 µM (Table 1). When stored as a stock solution in DMSO at -
0 °C, LC-MS analysis indicated significant decomposition of the
4
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have better stability when stored as a stock solution in EAB and
frozen.
The kinetic solubilities of each compound were evaluated.
Inhibitors 1 and 3 exhibited kinetic solubilities of 39.9 ± 7 (76.4 ±
13.4 µM) and 17.1 ± 1 (32.7 ± 1.9 µM) µg/mL, respectively, which
are considered moderate solubilities (Table 2). Inhibitors 2 and 4
exhibited kinetic solubilities of 275 ± 0.1 (484.5 ± 0.2 µM) and 216
± 5 (379.9 ± 8.8 µM) µg/mL, respectively, which are considered
high solubilities (Table 2). The azetidine in inhibitors 2 and 4
clearly enhanced solubility compared with the oxetane in
inhibitors 1 and 3. RF036 (MW = 499.17 g/mol) had a solubility of
Overall, inhibitor 1 and inhibitor 3 retained better inhibitory
activity towards MMP-13 than inhibitor 2 and inhibitor 4. Thus, the
azetidine resulted in somewhat inferior inhibition compared with
the oxetane. This result is consistent with the prior study where
the parent compound (RF036) methyl group was replaced by
either oxetane (IC50 went from 2.7 to 4.4 nM) or azetidine (IC50
[
14b]
went from 2.7 to 20 nM).
[
14b]
1
5.5 µM = 7.74 µg/mL,
which is considered poor solubility (<10
Drug Metabolism and Pharmacokinetic Profile of MMP-13
Inhibitors
µg/mL or <20 µM). All four compounds had greatly improved
solubility compared with RF036 (Table 2).
Table 2. Pharmacokinetic Properties of MMP-13 Inhibitors.
Compound
Kinetic solubility
M)
Human microsome
half-life
Rat microsome
half-life
Mouse microsome
half-life
Caco-2 cell
(
µ
permeability
-
6
(
min)
(min)
(min)
P
app (x 10 cm/sec)
2.38
a
RF036
15.5
12
9
20
Inhibitor 1
Inhibitor 2
Inhibitor 3
Inhibitor 4
76.4 ± 13.4
484.5 ± 0.2
32.7 ± 1.9
379.9 ± 8.8
61.4 ± 8.3
266.7 ± 89.9
66.0 ± 14.8
158.4 ± 29.8
61.9 ± 4.4
593.2 ± 33.7
99.9 ± 18.2
160.1 ± 20.6
103.6 ± 7.5
119.7 ± 35.4
94.8 ± 0.8
353.6 ± 221.0
2.3 ± 1.9
0.98 ± 0.28
1.9 ± 0.3
2.3 ± 1.1
a
[14b]
RF036 values were previously reported.
The stability of inhibitors 1, 2, 3, and 4 were compared in
human, rat, and mouse microsomes (Table 2). The t1/2 for inhibitor
membrane permeability (“intrinsic permeability”) gives a measure
[
17]
of the compound’s permeability. Apical to basal Papp was 2.3 ±
-
6
1
4
in human, rat, and mouse microsomes was 61.4 ± 8.3, 61.9 ±
.4, and 103.6 ± 7.5 min, respectively. The t1/2 for inhibitor 2 in
1.9, 0.98 ± 0.28, 1.9 ± 0.3, and 2.3 ± 1.1 x 10 cm/sec for inhibitors
1, 2, 3, and 4, respectively (Table 2). Inhibitors 1, 3, and 4 were
considered to have medium permeability, which is defined as
human, rat, and mouse microsomes was 266.7 ± 89.9, 593.2 ±
3.7, and 119.7 ± 35.4 min, respectively. The t1/2 for inhibitor 3 in
-
6
3
between 1.5 and 10 x 10 cm/sec, while inhibitor 2 was
considered to have low permeability. RF036 had a permeability of
human, rat, and mouse microsomes was 66.0 ± 14.8, 99.9 ± 18.2,
and 94.8 ± 0.8 min, respectively. The t1/2 for inhibitor 4 in human,
rat, and mouse microsomes was 158.4 ± 29.8, 160.1 ± 20.6, and
-
6
[14b]
2.38 x 10 cm/sec.
Thus, inhibitors 1, 3, and 4 had comparable
membrane permeability to RF036.
3
53.6 ± 221.0 min, respectively. The t1/2 for verapamil in human,
rat, and mouse microsomes was 6.62 ± 0.28, 5.42 ± 0.02, and
.60 ± 0.07 min, respectively. All inhibitors had greatly improved
stability compared with RF036, which had a t1/2 of 12, 9, and 20
Inhibition of Osteoclastogenesis and Collagenolysis
7
MMP-13 has been described previously as contributing to
[
18]
mesenchymal stem cell (MSC) osteogenic differentiation.
[
14b]
min in human, rat, and mouse microsomes, respectively.
RF036 was found to inhibit osteoclast formation in a dose-
dependent fashion in bone marrow co-cultures that contained
bone MSCs (manuscript submitted). The action of RF036 was not
due to the compound being toxic towards monocytes, MSCs, or
mature osteoclasts, even at a concentration of 10 µM (manuscript
Compounds with oxetane were, in general, less stable then
compounds with azetidine. This result is consistent with the prior
study where the parent compound (RF036) Me group was
[
14b]
replaced by either oxetane or azetidine.
Membrane permeability was studied using Caco-2 cells.
Active permeability mechanisms can be evaluated with the Caco-
submitted). The present inhibitors 1-4 were tested at a
concentration of 10 µM and compared with RF036 for inhibition of
osteoclastogenesis (Figure 2). Inhibitors 1 and 3 inhibited
osteoclastogenesis to levels of 28 and 22%, respectively, similar
to 24% observed for RF036 (Figure 2). Inhibitor 4 treatment
[
16]
2
assay because human epithelial colorectal adenocarcinoma
cells, which imitate the epithelial cell layer of the small intestine,
express membrane proteins such as P-glycoprotein (Pgp), breast
cancer resistance protein (BCRP), and multidrug resistance
protein 2 (MRP2) on the apical surface. Efflux transporters
expressed in the apical membrane of intestinal enterocytes have
been implicated in drug oral absorption. For compounds where
active efflux impacts permeability, the inherent passive
resulted in 31% osteoclastogenesis, while inhibitor
2 was
relatively ineffective at inhibiting osteoclastogenesis (79% activity
compared with control) (Figure 2). The lack of cell debris indicated
that none of the inhibitors were cytotoxic.
5
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5
Figure 2. Inhibition of osteoclastogenesis by RF036 and inhibitors 1-4. Bone mesenchymal stem cells were plated at 1 x 10 cells/250 µL/well in a 48 well plate in
fresh media containing 25 ng/mL rM-CSF and 100 ng/mL RANKL. Inhibitors were added in triplicates at 10 µM final concentration at the time of plating. Media was
refreshed every 2-3 d for 5-7 d. Representative mature osteoclasts are indicated with yellow arrows while representative undifferentiated MSCs and immature
osteoclasts are indicated with green arrows.
While the present and prior studies have shown that MMP-
3 facilitates osteoclastogenesis, the mechanism by which MMP-
3 acts is unknown. Differentiation of hematopoietic stem cells to
MMP-9 has been documented to be in the nucleus of primary
[
20]
1
1
osteoclast precursor-induced cells,
it is not clear where
proMMP-9 is activated. Alternatively, MMP-9 activates TGFβ and
functional, multinucleated osteoclasts is driven by two essential
cytokines, M-CSF, secreted by osteoblasts, and RANKL, found
as a soluble factor or as a membrane-bound cytokine expressed
on osteoblasts, osteocytes, dendritic cells, mature T-cells, and
promotes the cleavage of galectin-3, which would reduce the
[
8, 21]
ability of galectin-3 to suppress osteoclastogenesis.
The in vivo mechanism by which MMP-13 facilitates
osteoclastogenesis may be more complex. Recent studies
indicated that osteoclast-mediated bone resorption required
either MMP-9 or MT1-MMP, with each enzyme providing a
[
19]
hematopoietic precursors.
In the simple system presented
herein, it is possible that MMP-13 could process M-CSF and/or
RANKL to more active forms, or MMP-13 could process RANK to
improve binding of RANKL. However, other mechanisms are
possible. Galectin-3, which suppresses osteoclastogenesis, is a
[
22]
functional redundancy for the other. Co-culture of MDA-MB-231
breast cancer cells with MC3T3-E1 osteoblasts in a mineralized
osteoid matrix resulted in an increase between 1.3- and 26-fold of
48 proteins or protein fragments in supernatants containing MMP-
[
8]
substrate for MMP-13. MMP-13 could process galectin-3 and
inactivate it. MMP-13 activates proMMP-9, and the activation of
proMMP-9 has been proposed to result in MMP-9 processing the
core histone protein H3 N-terminal tail (H3NT) which then
[
23]
13.
MMP-13 inactivated chemokines CCL2 and CCL7,
activated platelet-derived growth factor C (PDGF-C), and cleaved
[
23]
SAA3, osteoprotegerin, CutA, and antithrombin III.
CCL2,
[
20]
regulates gene pathways facilitating osteoclastogenesis. While
CCL7, PDGF-C, and SAA3 recruit osteoclasts and play a role in
6
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osteoclast differentiation, but it is not clear how MMP-13
collagenolysis over a range of concentrations from 1.52 nM to 10
µM. Inhibitor 1 inhibited collagenolysis only at high concentrations
(3.33 and 10 µM) (Figure 4). Inhibitor 2 weakly inhibited
collagenolysis at the highest concentration tested (10 µM) (Figure
4). Inhibitor 3 partially inhibited collagenolysis at the highest
concentration tested (10 µM) (Figure 4). Inhibitor 4 (in either salt
form) did not inhibit collagenolysis (Figure 4).
[
23]
processing of these substrates impacts these activities.
RF036 was previously shown to inhibit MMP-13 type II
[
14b]
collagenolysis >90% at a compound concentration of 20 µM.
We presently examined the dose-dependence of type II
collagenolysis inhibition by RF036, and found near complete
inhibition at a compound concentration of 250 nM (Figure 3).
Inhibitors 1-4 were tested for their ability to inhibit MMP-13 type II
Figure 3. Inhibition of collagenolysis by RF036. The assay was initiated by dispensing 9 µL of 4 nM MMP-13 in EAB. Two µL of RF036 at varying concentrations in
EAB were added and incubated with the enzyme for 30 min. Reactions were initiated by addition of 9 µL of 333 nM type II collagen in EAB. After 22 h of incubation
at 37 °C, the samples were resolved by electrophoresis on an 8% SDS-PAGE gel.
7
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Figure 4. Inhibition of collagenolysis by inhibitors 1-4. The assay was initiated by dispensing 9 µL of 4 nM MMP-13 in EAB. Two µL of test compounds at varying
concentrations in EAB were added and incubated with the enzyme for 30 min. Reactions were initiated by addition of 9 µL of 333 nM type II collagen in EAB. After
2
3
2 h of incubation at 37 °C, the samples were resolved by electrophoresis on an 8% SDS-PAGE gel. The inhibitor concentrations were 10 µM (lane 2), 3.3 µM (lane
), 1.11 µM (lane 4), 370 nM (lane 5), 125 nM (lane 6), 41.2 nM (lane 7), 13.7 nM (lane 8), 4.57 nM (lane 9), and 1.52 nM (lane 10). MMP-13 plus type II collagen
with no inhibitor (lane 11) and type II collagen with no MMP-13 or inhibitor (lane 12) are controls.
None of the four compounds were effective inhibitors of
MMP-13 catalyzed type II collagenolysis (Figure 4). This result
was particularly surprising, as RF036 inhibits type II
collagenolysis effectively (Figure 3). Loss of inhibition of MMP-13
collagenolytic activity was not due to replacing the backbone
sulfur atom with either carbon or nitrogen, as when the backbone
sulfur in RF036 was replaced by either carbon or nitrogen
could also replace the amide bond to reduce compound
degradation by proteases.
Multiple attempts to develop MMP inhibitor-based drugs
failed mostly due to the dose limiting side effects collectively
[
26]
known as musculoskeletal syndrome (MSS). While the exact
cause of MSS is not known, it is believed to be due to the lack of
selectivity of drug candidates towards other representatives of
[
14b]
[26b, 27]
inhibition of collagenolysis was retained.
and azetidine moieties impact the positioning of the inhibitor to
Thus, the oxetane
metalloproteinase families.
Importantly for the present work,
selective MMP-13 inhibition does not induce MSS in rat
[
14b]
[28]
render it ineffective towards collagenolysis. In our prior study,
models.
compound 33 was found to be a good inhibitor of MMP-13 activity
towards a synthetic substrate (IC50 = 43.2 nM) but not towards
collagenolysis (<10% at 20 µM inhibitor concentration). In similar
fashion to the compounds described in the present study,
compound 33 featured a bulky substitution for the methyl amine
group of RF036. It is also possible that the combined substitutions
within the present inhibitors are not independent, and thus the
substitution of the backbone sulfur may impact inhibition of
collagenolysis.
Experimental Section
Synthesis of compounds
All materials were obtained from commercial suppliers and used without
further purification unless otherwise noted. Anhydrous solvents were
obtained from Sigma-Aldrich or Fisher Scientific and were used directly.
All air or moisture sensitive reactions were carried out using oven-dried
glassware under a nitrogen atmosphere. Reactions were monitored by
either thin layer chromatography (TLC) or analytical LC-MS. TLC was
performed on Kieselgel 60 F254 glass plates pre-coated with a 0.25 mm
thickness of silica gel. TLC plates were visualized with UV light and/or by
potassium permanganate and phosphomolybdic acid stains. Column
chromatography was performed on a Combi flash automated system.
Compound was loaded onto pre-filled cartridges filled with KP-Sil 50 µm
irregular silica. NMR spectra were recorded on a 400 MHz spectrometer
RF036 and inhibitors 1-4 are all based on the 2-
(
arylmethylthio)-cyclopentapyrimidin-4-one scaffold, which we
[
24]
initially described as a non-competitive MMP-13 inhibitor. X-ray
crystallographic structural analysis of this inhibitor family found
binding to the MMP-13 S
1
’* specificity pocket within the S
1
’
[
14a, 24c]
subsite.
These leaves open the possibility that binding of a
macromolecular substrate outside of the MMP-13 active site
could dispel the inhibitor from or reorient the inhibitor within the
and measured in CDCl , MeOD-d
4
, or DMSO-d
6
3
(CHCl : H, δ=7.26, C,
S
1
’* specificity pocket.
3
δ=77.16, MeOH: H, δ=3.31, C, δ=49.00, DMSO: H, δ=2.50, C, δ=39.50).
1
13
All H and C shifts are given in ppm and coupling constants J are given
in Hz. Mass (m/z) of the compounds was determined using an UP-LC
Summary and Conclusion
[
Acquity H class] equipped with XBridge® BEH C18 XP 130 Å, 2.5 μm, 2.1
150 mm column and a LC-MS instrument (Agilent 1100 series LC with
500 Ion TrapMS) equipped with Zorbax RR SB-C18 80 Å, 3.5 μm, 4.6 ×
00 mm column. Elution was performed using the following conditions: 5%
v/v) acetonitrile (+0.1% formic acid) in 95% (v/v) H O (+0.1% formic acid),
×
We synthesized four compounds based on the template of RF036,
a highly selective MMP-13 inhibitor, in an attempt to obtain an
inhibitor with an improved drug metabolism and pharmacokinetics
1
(
2
ramped to 95% acetonitrile over 12 min, and holding at 95% acetonitrile
for 3 min with a flow rate of 1 mL/min. The purity of each compound was
(
DMPK) profile. These new compounds have been evaluated for
MMP-13 inhibition potency, protease selectivity, kinetic solubility,
Caco-2 cell permeability, in vitro stability in human, rat, and
mouse liver microsomes, and inhibition of osteoclastogenesis and
collagenolysis. The overall goal was to reach the following in vitro
≥
95% based on this analysis. Synthetic methods for the production of all
inhibitors and inhibitor intermediates as well as characterization of all
inhibitors is presented in the Supporting Information.
values: (a) K
i
< 15 nM while maintaining selectivity over other
MMP-13 enzyme activation
MMPs; (b) kinetic solubility higher than 15 µM; (c) ≥20 min in vitro
half-life (t1/2) in liver microsomes; and (d) permeability coefficients
Full-length recombinant human proMMP-13 was purchased from R&D
Systems (catalog no. 511-MM; Minneapolis, MN). ProMMP-13 in 100 µL
enzyme assay buffer (EAB; 50 mM Tris•HCl, pH 7.5, 100 mM NaCl, 10
-
6
[25]
>
2.0 x 10 cm/sec.
Inhibitor 3, in which the backbone sulfur and terminal methyl
mM CaCl
2
,
0.05% Brij-35) was activated with
1
mM (p-
group of RF036 were replaced by nitrogen and oxetane,
respectively, had comparable activity, selectivity, and membrane
permeability to RF036 while exhibiting greatly enhanced solubility
and stability. Inhibitor 3 effectively inhibited MMP-13-mediated
osteoclastogenesis but spared collagenolysis.
aminophenyl)mercuric acid (APMA) for 2 h at 37 °C. The stock of active
MMP-13 was diluted to 384.6 nM and stored at -80 °C.
Inhibitor kinetics
The compounds presented herein may undergo further
optimization. For example, the isopropyl group could be replaced
by a cyclopropyl group. Based on our prior study, the cyclopropyl
group could provide further enhancement of stability, no inhibition
Our methods for kinetic evaluation of MMP inhibitors have been described
[24b, 24c]
in detail.
5
Briefly, fTHP-15(FAM) [sequence (Gly-Pro-Hyp) -Gly-Pro-
Lys(5-Fam)-Gly-Pro-Gln-Gly-Leu-Arg-Gly-Gln-Lys(Dabcyl)-Gly-Val-Arg-
(Gly-Pro-Hyp) -NH where Hyp 4-hydroxy-L-proline, Fam
carboxyfluorescein, and Dabcyl 4-(dimethylaminoazo)benzene-4-
5
2
,
=
=
[
14b]
of CYP3A4, and greatly reduced inhibition of CYP2C9.
One
=
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000911
FULL PAPER
[
29]
carboxylic acid], MMP-13, and inhibitor working solutions were prepared
in EAB. All reactions were conducted in 384-well black polystyrene plates
to terminate the reaction and precipitate the protein. The vials were
centrifuged at 4000 rpm for 20 min. The supernatants obtained were
analysed on LC-MS/MS (Shimadzu-8040) to monitor the disappearance of
(
Greiner, North Carolina, catalog no. 784076). Fluorescence was
measured on a BioTek H1 microplate reader using λexcitation = 488 nm and
emission = 520 nm. Rates of hydrolysis were obtained from plots of
compound. For the LC separation, the column was an XBridge 5 µm C18
,
λ
solvent A = 0.1% formic acid in 10 mM ammonium acetate, solvent B =
methanol, and the flow rate = 0.6 mL/min. The data was log transformed
and results reported as half-life (t1/2).
fluorescence versus time using data points from only the linear portion of
the hydrolysis curve. To determine the IC50 value of each compound, the
compounds were screened in 10-point 3-fold dilution dose-response curve
format in triplicates. The assay began by dispensing 5 µL of test
compounds in assay buffer followed by 5 µL of MMP-13. The enzyme was
allowed to incubate with the test compounds for 30 min at 25 °C. The
assays were initiated by addition of 5 µL of fTHP-15(FAM) and immediately
placed in the microplate reader to record fluorescence. To determine IC50
values of each compound, the relative fluorescence units (RFU) from wells
containing MMP-13, fTHP-15(FAM), and compounds were plotted versus
no enzyme and untreated controls. For each compound, RFUs from the
linear part of the curve were fitted with a four parameter equation
describing a sigmoidal dose-response curve with adjustable baseline
using GraphPad Prism® version 11 suite of programs. The IC50 values of
the compounds were determined as the concentrations that resulted in
Caco-2 permeability
Caco-2 human epithelial colorectal adenocarcinoma cells were plated in
2
4-Transwell® dual chamber plates (Millipore, Billerica, MA) (cell density
2
of 60,000 cells/cm on day-1). The permeability studies were conducted
with the monolayers cultured for 21-22 days. The integrity of each Caco-2
cell monolayer was certified by trans epithelial electrical resistance (TEER)
test (pre-experiment) and by determining the permeability of the reference
compound, Lucifer yellow. Caco-2 cell monolayers with TEER values
2
greater than 250 Ω/cm were considered for experimentation. Propranolol
and atenolol were used as positive controls for high and low permeable
compounds, respectively. The concentration of compound used in the
assay was 10 µM. The permeability assay buffer was Hanks Balanced Salt
Solution (HBSS) containing 10 mM HEPES and 15 mM glucose at a pH of
5
0% enzyme activity when compared to the activity of the control samples
(
without a compound). These values were generated from fitted curves by
solving for the X-intercept at the 50% inhibition level of Y-intercept using
the built-in dose-response model algorithm of GraphPad Prism (La Jolla,
CA). Since the determination of IC50 values may vary from the true
7
.4. The final concentration of DMSO in the spiking solution was 0.05%.
The bidirectional permeability study was initiated by adding an appropriate
volume of HBSS buffer containing compound to respective apical and
inhibitory values of a molecule, K
Briefly, varying concentrations of inhibitor diluted in EAB were added to
84 well plates. MMP-13 (4 nM) and fTHP-15(FAM) were added. Since
(1 + [S]/K ), the peptide was used at <5-10% K . This insured
that Ki(app) is a close approximation of K. Initial velocities (V) were
expressed as relative fluorescence/time and monitored with increasing
concentration of inhibitor. K values were calculated using GraphPad
software. Data was collected at enzyme concentrations no greater than
0K
i
were determined for the best compounds.
[31]
basolateral chambers (n=2)
. An aliquot of sample (25 µL) was taken
from both chambers at 0 and 60 min of the incubation period and to this
50 µL of acetonitrile with internal standard (200 ng/mL telmisartan) was
3
1
K
i(app) = K
i
M
M
added, mixed gently, and centrifuged at 4000 rpm (Eppendorf 5424R,
Germany) for 20 min. An aliquot of 150 µL was subsequently transferred
to the auto-sampler and injected for analysis on LC-MS/MS. Each
determination was performed in duplicate. The flux of co-dosed Lucifer
yellow was also measured for each monolayer to ensure no damage was
inflicted to the cell monolayers during the flux period. The apparent
permeability (Papp) and percent recovery were calculated as follows:
i
i
i
1
i
.
Selectivity assay
P
R R A
app = (dC /dt) x V /(A x C ) (1)
To determine the selectivity of each inhibitor, the compounds were tested
against a protease panel consisting of MMP-1, MMP-2, MMP-8, MMP-9,
and MMP-14/MT1-MMP. All enzymes were purchased from R&D Systems
and activated according to manufacturer’s instructions. Upon activation,
each enzyme was diluted in EAB to 20 nM and stored at -80 °C until further
use. The compounds were screened as described above in 10-point 3-fold
dilution dose-response curve format in triplicate utilizing fTHP-15(FAM) as
substrate except for MMP-1, for which Knight substrate [Mca-Lys-Pro-Leu-
R D D N
Percent Recovery = 100 x ((V x CRfinal) + (V x CDfinal))/(V x C ) (2)
where dC
R
/dt was the slope of the cumulative concentration in the receiver
-
1
compartment versus time in µM sec ; V
R
was the volume of the receiver
3
3
compartment in cm ; V
A was the area of the insert; C
concentration and the measured 120 min donor concentration in µM; C
was the nominal concentration of the dosing solution in µM; C final was
the cumulative receiver concentration in µM at the end of the incubation
period; and C final was the concentration of the donor in µM at the end of
D
was the volume of the donor compartment in cm ;
A
was the average of the nominal dosing
N
2
Gly-Leu-Lys(Dnp)-Ala-Arg-NH , where Mca is 7-methoxycoumarin-4-
[
14]
R
acetyl and Dnp is 2,4-dinitrophenyl] was used.
D
Kinetic solubility
the incubation period.
Compound solubility was determined by HPLC. Compounds were
In vitro osteoclast differentiation assays
incubated at a concentration of 500 µM in PBS for 18 h with a vortex of
6
00 rpm. After the incubation period, samples were centrifuged for 10 min
All animal experiments were performed in accordance with the guidelines
of the Florida Atlantic University Institutional Animal Care and Use
Committee (IACUC), protocol number A18-04. Osteoclastogenesis was
at 10000 rpm and supernatant analyzed by HPLC with peak area
compared to standards of known concentration.
[
32]
performed as previously described.
Briefly, whole bone marrow was
[
30]
Metabolic stability in mouse, rat, and human liver microsomes
flushed from both tibia and femur in hind limbs of either male or female
6
mice. The marrow was plated at 0.5 x 10 cells/mL in αMEM supplemented
Microsome stability was evaluated by incubating 1 µM compound or
with 25 ng/mL recombinant macrophage-colony stimulating factor (rM-
CSF) (R&D Systems, Minneapolis, MN). After 72 h, non-adherent cell
positive control (verapamil) with 1.0 mg/mL hepatic microsomal protein in
5
1
00 mM potassium phosphate buffer, pH 7.4. Solutions were held at 37 °C
population was plated at 1 x 10 cells/250 µL/well in a 48 well plate in fresh
with continuous shaking. The reactions were initiated by adding NADPH
to 1 mM final concentration. Reactions without NADPH at 0 and 60 min
were also incubated to rule out non-NADPH metabolism or chemical
instability in the incubation buffer. The final DMSO concentration was
media containing 25 ng/mL rM-CSF and 100 ng/mL RANKL. Inhibitors
were added in triplicates at 10 µM final concentration at the time of plating.
Media was refreshed every 2-3 d for 5-7 d. Upon osteoclast formation, cells
were fixed by incubating in 4% paraformaldehyde for 10 min at room
temperature. Samples were subsequently stained with TRAcP stain
<
0.1%. The final incubation volume was 300 µL and 40 µL aliquots were
removed at 0, 5, 15, 30, and 60 min. Aliquots were added to 200 µL ice-
(
Sigma-Aldrich, St. Louis, MO) per manufacturer instructions.
cold acetonitrile (containing the internal standard 200 ng/mL telmisartan)
9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000911
FULL PAPER
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Acknowledgements
This research was supported by NIH R01 grant CA239214 (GBF)
and an FAU Undergraduate Research Grant (GD). We
acknowledge Acubiosys Private Limited, Hyderabad, India, for
performing the kinetic solubility, metabolic stability, and Caco-2
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ChemMedChem
10.1002/cmdc.202000911
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The development of matrix metalloproteinase 13 (MMP-13) inhibitors has often been marred by poor PK properties of resulting
compounds. The present study has sought to improve the PK properties of RF036, a previously described selective MMP-13
inhibitor. Inhibitor 3 had more favorable PK properties than RF036 and was an effective inhibitor of osteoclastogenesis, a critical
process that occurs during breast cancer metastasis to the bone.
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This article is protected by copyright. All rights reserved.