Discovery and optimization of selective inhibitors of meprin α (Part ii)

Article abstract of DOI:10.3390/ph14030197

Meprin α is a zinc metalloproteinase (metzincin) that has been implicated in multiple dis-eases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable of selectively inhibiting meprin α, or its close relative meprin β, over numerous other metzincins which, if inhibited, would elicit unwanted effects. We recently identified possible molecular starting points for meprin α-specific inhibition through an HTS effort (see part I, preceding paper). Here, in part II, we report further efforts to optimize potency and selectivity. We hope that a hydroxamic acid meprin α inhibitor probe will help define the therapeutic potential for small molecule meprin α inhibition and spur further drug discovery efforts in the area of zinc metalloproteinase inhibition.

Full text of DOI:10.3390/ph14030197

pharmaceuticals
Article
Discovery and Optimization of Selective Inhibitors of
Meprin α (Part II)
Chao Wang 1 , Juan Diez , Hajeung Park , Christoph Becker-Pauly
,2
3
1
4
, Gregg B. Fields 5 , Timothy P. Spicer 1,6
,
*
Louis D. Scampavia 1 , Dmitriy Minond
,6
2,7
and Thomas D. Bannister
1,2,
1
Department of Molecular Medicine, Scripps Research, Jupiter, FL 33458, USA; CWang@scripps.edu (C.W.);
hajpark@scripps.edu (H.P.); spicert@scripps.edu (T.P.S.); scampl@scripps.edu (L.D.S.)
Department of Chemistry, Scripps Research, Jupiter, FL 33458, USA; dminond@nova.edu
Rumbaugh-Goodwin Institute for Cancer Research, Nova Southeastern University, 3321 College Avenue,
CCR r.605, Fort Lauderdale, FL 33314, USA; mdiezi@yahoo.com
2
3
4
5
6
7
The Scripps Research Molecular Screening Center, Scripps Research, Jupiter, FL 33458, USA;
cbeckerpauly@biochem.uni-kiel.de
Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, Rudolf-Höber-Str.1,
24118 Kiel, Germany; fieldsg@fau.edu
Department of Chemistry & Biochemistry and I-HEALTH, Florida Atlantic University, 5353 Parkside Drive,
Jupiter, FL 33458, USA
Dr. Kiran C. Patel College of Allopathic Medicine, Nova Southeastern University, 3301 College Avenue,
Fort Lauderdale, FL 33314, USA
*
Correspondence: tbannist@scripps.edu
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Abstract: Meprin
diseases, including fibrosis and cancers. It has proven difficult to find small molecules that are capable
of selectively inhibiting meprin , or its close relative meprin , over numerous other metzincins
which, if inhibited, would elicit unwanted effects. We recently identified possible molecular starting
points for meprin -specific inhibition through an HTS effort (see part I, preceding paper). Here, in
part II, we report further efforts to optimize potency and selectivity. We hope that a hydroxamic
acid meprin inhibitor probe will help define the therapeutic potential for small molecule meprin
inhibition and spur further drug discovery efforts in the area of zinc metalloproteinase inhibition.
α is a zinc metalloproteinase (metzincin) that has been implicated in multiple
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Wang, C.; Diez, J.; Park, H.;
Becker-Pauly, C.; Fields, G.B.; Spicer,
T.P.; Scampavia, L.D.; Minond, D.;
Bannister, T.D. Discovery and
α
β
α
Optimization of Selective Inhibitors
of Meprin α (Part II). Pharmaceuticals
α
α
2
021, 14, 197. https://doi.org/
1
0.3390/ph14030197
Keywords: meprin α; meprin β; zinc metalloproteinase; medicinal chemistry; probe development
Academic Editor: Salvatore
Santamaria
Received: 14 December 2020
Accepted: 24 February 2021
Published: 27 February 2021
1. Introduction
Meprin
diseases, including fibrosis [
alone or in combination with other metzincins, including its close relative meprin
has been difficult to establish due to the lack of known selective inhibitors. While many
preliminary reports for meprin inhibitors have emerged [ ], no compounds with suitable
potency, selectivity, and drug-like attributes for in vivo use are known. We recently reported
α
is a zinc metalloproteinase (metzincin) that has been implicated in multiple
1,
2
], and cancers [ ]. Understanding meprin ’s precise role,
3
α
β,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
4–8
an HTS campaign to identify lead meprin
α
and meprin
β
inhibitors [9]. We then began
SAR studies to understand the pharmacophore for meprin
α
inhibition, to enhance potency
of the best leads, and to widen or maintain their selectivity over other metzincins. These
efforts led to the identification of a potential probe molecule that may allow us in the
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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Attribution (CC BY) license (https://
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future to ascertain the therapeutic potential for small molecule meprin
treatment of fibrosis or cancers.
α inhibition in the
Zinc metalloproteinases are ubiquitous in human biology and their dysregulation in
certain disease states has spurred many researchers to seek modulators, usually inhibitors,
of their function. The primary difficulty in most drug discovery efforts is to gain selectivity
for the zinc metalloproteinase of interest. Most inhibitors are characterized by having
groups that tightly bind zinc ions, often through bidentate coordination. Compounds that
4
.0/).
Pharmaceuticals 2021, 14, 197. https://doi.org/10.3390/ph14030197
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 197
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bind strongly to zinc metalloproteinases in this way can be found, though a frequently
encountered liability is poor target selectivity, since coordination to zinc, and perhaps to
other metal ions of similar size, is often the main driver of potency. As a consequence,
many such inhibitors block off-target zinc-and even non-zinc metalloproteinase, including
enzymes that are required for a myriad of essential biological functions of healthy cells,
and they thus display intolerably high toxicity.
A common strategy is to avoid metal coordinating groups altogether. A second strategy
is to employ a metal coordinating group that is: (1) consistent with drug-like properties,
and (2) is merely one part of an organic molecule that also has several other functional
groups that contribute substantially to its binding energy for the target, and not for other
targets, thus imparting an acceptable level of target selectivity. Ideally this will translate
to low toxicity in in vivo use. In our efforts here, our starting point was a screening hit
containing a hydroxamic acid, R–CO–NH–OH, and we sought to alter other regions of the
screening hit to widen its selectivity.
In our HTS effort (see part I), we sought any type of inhibitor, with or without metal co-
ordinating groups. Only leads with known Zn coordinating functional groups were found
to have suitable potency for follow-up, however, and any optimization effort required
that we address selectivity concerns at an early stage. The hydroxamic acid compound
SR19855 (Figure 1) was a preferred meprin
low-micromolar inhibition of meprin ) and hints of target selectivity. In screens for se-
lectivity, we saw 13-fold selectivity for meprin inhibition over inhibition of the most
closely-related enzyme, meprin , and also at least 10-fold selectivity over a larger panel
α inhibitor hit, having both significant potency
(
α
α
β
of metzincins, including MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-14, ADAM10,
and ADAM17. Given that a hydroxamic acid group can tightly bind zinc ions in all of
these targets, we surmised that other regions of SR19855 were contributing to meprin
α
binding and to target selectivity. Further, we wished to further widen this selectivity
window, with increases in potency as well, so that a probe molecule might be identified for
use in order to isolate the effect of meprin
pharmacological contexts.
α-specific inhibition in relevant biological and
Figure 1. HTS hit, the aryl triazole SR19855 (left), and related, less selective thiadiazoles.
2. Results
2.1. Modeling of Lead Series
Our HTS effort and follow-up SAR-by-purchase efforts identified a number of mod-
erately potent meprin
α inhibitors bearing hydroxamic acid groups. Among these were

Pharmaceuticals 2021, 14, 197
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related aryl triazoles and thiadiazoles (Figure 1). Notably, the thiadiazoles were unlike
the aryl triazoles in terms of isoform selectivity: only the aryl triazoles were >10-fold
selective for meprin
α over meprin β. We considered isoform selectivity to be critically
important, since low ligand selectivity limits the usefulness of metalloprotease inhibitors as
isoform-specific probes. We surmised that the additional aromatic ring in the aryl triazoles
(left) may be an important selectivity element favoring binding to meprin α over meprin β,
and perhaps over other metzincins as well. In the thiadiazole series, similar compounds
purchased with a thiazole core (one N replaced with CH) were much less potent, suggesting
that the nitrogen atoms in the heterocyclic core are important.
We wished to understand the possible basis for meprin
selectivity. Using coordinates for a meprin X-ray crystal structure, we constructed a
homology model for meprin and docked several HTS hits, including the aryl triazoles.
α binding and SR19855
β
α
The hydroxamate of SR19885, not surprisingly, is modeled to form a strong bidentate
interaction with the Zn ion in both docking models (Figure 2). This binding anchors the
ligand into each active site. Overall, geometry of binding for the backbone of the ligand
is similar for the meprin enzymes. Important differences are apparent for the side chains,
however, especially the interactions of the central para-methoxy phenyl group in meprin
As shown in the left panel of Figure 2, Y149 of meprin makes a face-on interaction
with the phenyl ring. R177 residue makes cation- interactions with both phenyl and
pyrimidine rings of the ligand. Although the primary sequences of the meprin isoforms are
similar in the binding pocket, these interactions are absent or are much weaker in meprin
with the exception of the hydroxamate/Zn interaction and an H-bond interaction to Y211
Y149 in meprin ) that are preserved. These findings suggest that the aryl triazole core of
α.
α
π-π
π
β
(
α
the ligand should be preserved, for selectivity purposes, during our SAR studies aimed to
increase potency and selectivity for meprin α.
Figure 2. SR19855 docked to meprins α and β.

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In analyzing data from the HTS effort and for a few purchased analogs, we noted
that both the phenyl and pyrimidine rings of SR19855 could not be deleted without a
substantial drop in meprin
α inhibitory activity. This finding agreed with our binding
model (Figure 2). Together, these observations led us to explore the effect of a pyrimidine
substituents and the replacement or alternative substitution of the central aryl group, with
the aim of finding electronic and/or steric factors that would maximize meprin
activity and maintain or (preferably) increase target selectivity.
α inhibitory
2.2. Synthetic Strategy
To further study compounds in the series, we initiated an internal synthesis effort.
A versatile 6-step synthesis used is shown in Figure 3, which is adaptable for different
pyrimidine X groups and for substituted phenyl rings, or a replacement heterocycle. This
strategy is based upon the initial 3 steps of a literature method [10]. Briefly, the alkylation
of a thiopyrimidine
step 2) to form a hydrazine amide
which following an immediate acidic workup (step 4), gives the mercaptotriazole
potentially oxidatively sensitive intermediate is then alkylated (step 5) to give ester
1
(step 1) to give ester
2
is followed by treatment with hydrazine
(step 3),
. This
, which
(
3
, which is acylated with an isothiocyanate
4
6
7
can be safely stored under inert conditions and purified by chromatography, if needed.
Many of the earlier intermediates are obtained in high purity through precipitation or
crystallization, with minimal chromatography, as outlined in the Experimental Section.
Finally, the hydroxamic acid group is installed by treatment with hydroxylamine (step 6)
to give the final test hydroxamic acid product 8.
Figure 3. General synthesis scheme.
2.2.1. A. Pyrimidine Substitution
Holding the other groups constant, we varied the X substituents on the pyrimidine
(
Table 1), from the relatively electron-deficient unsubstituted pyrimidine, to a bulkier but
neutral pyrimidine (3,5-dimethyl), to a more electron-rich 3,5-dimethoxy pyrimidine. We
used symmetric disubstituted compounds due to their ready availability. Potency varied
only in a small range, as all three compounds are single digit micromolar meprin
Selectivity assessment using the meprin inhibition assay, however, led us to prioritize
,5-dimethoxy pyrimidines for further follow-up, as SR24144 showed complete lack of
inhibition of meprin at 100 M. This contrasts with significant meprin inhibition
by SR19855 (IC50 = 17 µM) and modest but complete meprin inhibition by SR24319
IC50 = 60 µM). We felt that the maximization of selectivity, which was our primary criteria
α binders.
β
3
β
µ
β
β
(
for probe development, offset a decrease in meprin
α inhibition (meprin α IC50 = 8.7 µM),
provided that meprin
α inhibition could be increased by making other modifications, such
as replacements for the central methoxyphenyl ring.

Pharmaceuticals 2021, 14, 197
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Table 1. Pyrimidine substitution: dimethoxylation disfavors meprin β inhibition.
MeO
H
X
H
ID
Meprin α IC50, µM
1.3 ± 0.1
50
Meprin β IC , µM
S
N
N
OH
N
O
N
SR19855
SR24139
SR24144
17 ± 3
60 ± 7
>100
S
N
X
Me
OMe
4.1 ± 0.8
N
X
8.7 ± 1.1
2
.2.2. Phenyl Substitution
Replacement of the central methoxyphenyl ring (Table 2) indeed allowed us to regain
meprin binding affinity without sacrificing selectivity vs. meprin . Moving the para
methoxy group in SR19855 to the meta or ortho position, however, reduced meprin
affinity and, more concerning, gave unwanted binding to meprin (IC50 = 59 and 52
α
β
α
β
µ
M
for SR24460 and SR24459, respectively). Reducing electron density with a pyridyl ring
present or with CF , Br, F, or Me groups replacing OMe maintained meprin selectivity
binding, with SR24403, with a CF group, being
β
3
but had only minor effects on meprin
the best alternative (meprin IC50 = 5.4
SR24467), gave a modest increase in meprin
α
3
α
µ
M). A larger group, a 2-naphthyl ring system
(
α
binding (IC50 = 2.0 M) at the expense of
µ
higher molecular weight and elevated lipophilicity. We saw enhanced potency when a
para amino substituted phenyl was used, with the 4-morpholine analog SR26466 having
an IC50 = 1.1
-dimethylamino analog SR24717 being nearly equipotent, having an IC50 = 660 nM. IC50
values for each compound were determined in triplicate and the average value is given
in Table 2, with SD factor as an error. Selectivity over meprin was maintained in all
analogs other than the meta-methoxy and ortho-methoxy compounds (IC50 vs. meprin in
all other cases was >70 M). To test our presumption that the hydroxamic acid is essential,
we also tested certain ester synthetic precursors to the hydroxamates (not shown) and saw
no activity (meprin IC50 > 100 M). As shown in Table 2, SR26465, with a diethylamino
group present, and SR24717, with a with a diethylamino group present, are essentially
equivalent with respect to meprin affinity and a selectivity ratio >100. We chose
SR24417 as the lead for further evaluation.
µM, the 4-diethylamino analog 24,465 having an IC50 = 600 nM, and the
4
β
β
µ
α
µ
α/β
Table 2. SAR studies in the central aromatic ring.
aryl
ID
Meprin α IC50, µM
1.3 ± 0.1
Meprin β IC50, µM
17 ± 3
SR19855
SR24460
SR24459
SR24718
10.3 ± 1.1
12.1 ± 3.9
10.1 ± 2.3
59 ± 10
52 ± 1
>100

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Table 2. Cont.
aryl
ID
Meprin α IC50, µM
5.4 ± 1.2
Meprin β IC50, µM
SR24003
>100
>100
>100
SR24462
SR24463
11.2 ± 2.6
10.4 ± 2.8
SR24467
SR26467
10.3 ± 2.2
2.0 ± 0.3
>100
>100
SR26466
SR26465
SR24717
1.1 ± 0.1
0.60 ± 0.06
0.66 ± 0.03
>100
75 ± 15
70 ± 5
2
.3. SR24717 Characterization in In Vitro Assays
We tested a freshly re-synthesized, analytically pure sample of SR24717 against meprin
, meprin , and related metzincins. IC50 values for SR24717 were batch independent:
inhibition for multiple batches ranged from 490–670 nM. IC50
inhibition were consistently at ~70 M or above as well. Results shown
in Figure 4 are for one scale-up batch, with results in triplicate. There is full meprin
inhibition and only partial meprin inhibition, at the higher doses tested (Figure 4A).
Assay results with related metzincins demonstrated excellent broad selectivity of SR24717
IC50 metzincins > 100 µM for all tested enzymes, Figure 4B).
α
β
IC50 values for meprin
values for meprin
α
β
µ
α
β
(

Pharmaceuticals 2021, 14, 197
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Figure 4. Characterization of a potential meprin
α
probe SR24717 against meprin
β
and related
metzincins. ( ) Dose response study of re-synthesis batch of SR24717 with meprin
A
α
and β; (B) Dose
response study of re-synthesis batch of SR24717 with metzincins shows good selectivity profile. All
experiments performed as 10 point 3-fold concentration-response curves in triplicate. All units are
IC , µM.
50
We further characterized SR24717 for the mode of inhibition of meprin
α. Pre-incubation
of SR24717 with meprin for 0–3 h showed no change in IC50 values, suggesting that
α
SR24717 is not a time-dependent inhibitor (Figure 5A). This allowed us to use the steady-
state assumption in our follow-up experiments. Varying substrate concentration in meprin
α
decreased the apparent potency of SR24717 (Figure 5B,C), suggesting a competitive mode
of inhibition. Indeed, both linear (Figure 5D) and non-linear (Figure 5E) models showed
good fit to the competitive inhibition. Global fit to the competitive model of inhibition
using GraphPad Prism showed K = 300.8 nM.
i
Figure 5. Characterization of mode of meprin
inhibition of meprin -mediated proteolysis by SR24717. (
mediated proteolysis by SR24717. ( ) Re-plot of ( ) shows increase of IC50 values correlating with increase of substrate
concentration suggestive of competitive inhibition mechanism by SR24717. ( ) Lineweaver-Burke plot shows lines of best
fit crossing at Y-axis, suggesting a competitive inhibition mechanism by SR24717. ( ) Global fit of meprin -mediated
proteolysis in the presence of SR24717 to a competitive model using non-linear regression shows good fit.
α
inhibition by potential probe. SR24717. (
A
) Time-dependence study of
α
B) Concentration-dependence study of meprin
α
inhibition-
C
B
D
E
α

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We tested SR24717 for effects on viability and cytotoxicity of several cell types. Only
at a concentration of 100 µM of SR24717 were negative effects on viability and cytotoxicity
observed (Figure 6), suggesting that SR24717 may be a useful probe for studying the
biological role of meprin α in in vitro and in vivo systems.
Figure 6. Characterization of SR24717 for effects on cell viability and cytotoxicity. (
SR24717 on viability of WM266-4 (skin melanoma), HEK293 (kidney), and BJ (skin fibroblasts) cells.
CellTiter Glo assay (Promega) was used after 72 h treatment. ( ) Effect of SR24717 on viability of
HEK293 (kidney) and BJ (skin fibroblasts) cells. The CellTox Green assay (Promega) was used. All
experiments performed as 10-point 3-fold concentration-response curves in triplicate.
A) Effect of
™
B
™
3
. Discussion
While we had initially hoped that HTS efforts would identify non-chelating, non-
hydroxamic acid meprin or meprin inhibitor leads, we have demonstrated that the
two aromatic groups in the lead series of meprin inhibitors are also very important,
and perhaps uniquely positioned, for meprin binding. Thus, Zn chelation is not the
overwhelming driver of ligand affinity. SR24717 has unexpectedly high selectivity vs.
0 other zinc metalloproteases (Figure 4), indicative of potential use as a probe molecule
in an in vitro setting. The selectivity is noteworthy in comparison to known inhibitors
Table 3), with structures depicted in Figure 7.
The meprin fold selectivity (ratio of IC50 values) had been ~13 for the HTS hit
α
β
α
α
1
(
α/β
SR19855 and was improved to 106 for SR24717. Actinonin is a meprin inhibitor that has
long been commonly used in the literature, but its target selectivity is quite poor. It is
a repurposed antibiotic agent that potently inhibits many metzincins and in cell-based
environments, though it is significantly cytotoxic. Its wide use is purely historical: it was
one of the first inhibitors of meprin
activity confounds the interpretations for any of actinonin’s effects, as they cannot be
cleanly ascribed to meprin inhibition. This is illustrated in Table 3, where we see sub-
micromolar activity for actinonin vs. MMP-2, MMP-8, MMP-9, MMP-13, ADAM17 (and
α with some selectivity for meprin β, but off-target
α
also MMP1, see footer), with low micromolar activity vs. meprin
contrasts greatly with similar data for SR24717.
β and ADAM10. This
At the time of initial submission of this manuscript, the most potent meprin
hibitors known were bis-benzyl glycine hydroxamates 10d and 10e, both from the Rams-
beck labs [ ], which displayed meprin fold selectivity ratios of 18 and 19, respectively.
α in-
8
α/β
The same group very recently gave an update on this series and related analogs [11], re-
porting more balanced pan tertiary amine inhibitors such as compound 1c and also much
more potent, conformationally constrained meprin
compounds 2c and 2d, like SR24717, append two aromatic groups to a 5-membered ring
core (Figure 7). Though selectivity IC50 values were not reported, most (as shown in Table 3
were weak inhibitors at 10 M and showed more substantial inhibition at 200 M. The
most potent off-target for inhibitors 2c and 2e was ovastacin, with K values of 66 nM and
α inhibitors 2c and 2d. Interestingly,
)
µ
µ
i
196 nM, respectively [11].

Pharmaceuticals 2021, 14, 197
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Table 3. Selectivity testing of SR24717 and literature meprin α inhibitors.
ID
SR24717 a
Meprin α
0.66 ± 0.03
0.004
Meprin β
70 ± 5
4.8 ± 0.5
MMP2 MMP3 MMP8
MMP9
MMP10 MMP13 MMP14 ADAM10 ADAM17
>100
0.09
95%
>100
NR
>100
0.19
>100
>100
NR
>100
0.1
>100
>100
>100
Actinonin b
0.1
±
0.01
>1000
2.2 ± 0.2
86%
0.2
±
0.02
0d [8] c
91%
86%
55%
86%
1
0.16 ± 0.001
2.95 ± 0.35
26%
61%
61%
57%
9
4%
91%
21%
98%
47%
80%
56%
80%
41%
1
0e [8]
0.40 ± 0.03
7.59 ± 0.01
0.03 ± 0.35
6
5%
1
c [11] d
0.19 ± 0.001
95%
87%
91%
86%
81%
86%
80%
75%
86%
80%
73%
86%
62%
61%
2
c [11] e
0.004
±
±
0.001
0.813
±
±
0.001
2
e [11] f
0.003
0.001
0.199
0.001
104%
All units are IC50
with error bars
also has IC50 = 0.1
,
±
µ
M, % values indicate % activity remaining at 10
µ
M (top) and at 200 µM (bottom). All assays performed in triplicate,
SD. a. Multiple synthesis of SR24717 batches were tested, shown are results of one representative batch. b. Actinonin
µ
M vs. MMP1. c. Compound 10d has IC50 = 1.15
µM vs. ovastacin [11]. d. Compound 1c has IC50 = 0.49
µM vs.
ovastacin [11]. e. Compound 2c has Ki = 66 nM vs. ovastacin [11]. f. Compound 2e has Ki = 196 nM vs. ovastacin [11].
N
O
HO
O
O
O
H
S
OH
N
OH
N
N
N
N
N
H
H
N
S
N
O
N
O
actinonin
SR24717 (this work)
O
O
OH
O
O
Cl
F
O
O
O
O
N
HN
N
O
O
N
OH
N
OH
N
OH
OH
OH
N
N
H
N
N
N
H
H
H
H
F
O
HO
O
10d
10e
1c
2c
2e
O
O
Cl
Figure 7. Structures of literature meprin inhibitors: actinonin, SR24717, 10d [8]; 10e [8], 1c [11], 2c [11], 2e [11].
Our modeling study, depicted in Figure 2, suggests that two cation-π interactions are
responsible for meprin potency and selectivity. Our structure–activity relationship study
α
supports the model, in that the 3,5-dimethoxypyrimidine and 4-dimethylaminophenyl
groups of SR24717 work in concert to augment potency and selectivity. Though SR24717 is
merely ~2-fold more potent than the HTS hit SR19855 in binding meprin
α, it has selectivity
advantages (~106-fold vs. meprin ), with consistently lower IC50 and/or lower maximum
β
inhibition of nine other metzincins, when tested even at a high concentration (100
Figure 4 and Table 3).
µM, see
We have developed a versatile, scalable, and operationally straightforward route
to producing the SR24717 and its analogs. Future work includes characterization of all
properties that are relevant for in vivo use, including the evaluation and optimization
of DMPK properties, while maintaining or improving both target potency and meprin
α selectivity.

Pharmaceuticals 2021, 14, 197
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4. Materials and Methods
4.1. Assay Reagents
MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, ADAM17
and Mca-KPLGL-Dpa-AR-NH fluorogenic peptide substrate were purchased from R&D
2
Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, 930-
ADB, and ES010, respectively). All common chemicals were purchased from Sigma. NFF449
was purchased from Tocris (cat# 1391) and actinonin was from Sigma-Aldrich, St. Louis,
MO, USA (cat# 01809).
4
.2. Meprin α and Meprin β Substrate Synthesis
Meprin and meprin substrates (Mca-YVADAPK-(K-
-Dnp), respectively) [12] were synthesized according to Fmoc solid-phase methodology
α
β
ε-Dnp) and Mca-EDEDED-(K-
ε
on a peptide synthesizer. All peptides were synthesized as C-terminal amides to prevent
diketopiperazine formation [13]. Cleavage and sidechain deprotection of peptide-resins
was for at least 2 h using thioanisole-water-TFA (5:5:90). The substrates were purified
and characterized by preparative RP HPLC and characterized by MALDI-TOF MS and
analytical RP HPLC.
4.3. Meprins Expression Protocol
Recombinant human meprin
expression system (Gibco Life Technologies, Paisley, UK) as described before [5,
α
and meprin
β
were expressed using the Bac-to-Bac
14]. Media
and supplements were obtained from Gibco Life Technologies. Recombinant Baculoviruses
◦
were amplified in adherently growing Spodoptera frugiperda (Sf)9 insect cells at 27 C in
Grace’s insect medium supplemented with 10% fetal bovine serum, 50 units/mL penicillin,
and 50
tures of BTI-TN-5B1-4 insect cells growing in Express Five SFM supplemented with 4 mM
glutamine, 50 units/mL penicillin, and 50 g/mL streptomycin in Fernbach-flasks using
µg/mL streptomycin. Protein expression was performed in 500 mL suspension cul-
µ
a Multitron orbital shaker (INFORS AG, Bottmingen, Switzerland). Cells were infected
6
at a density of 2
×
10 cells/mL with an amplified viral stock at a MOI of ~10. Protein
expression was stopped after 72 h, and recombinant meprins were further purified from the
media by ammonium sulfate precipitation (60% saturation) and affinity chromatography
(Strep-tactin for Strep-tagged meprin α and Ni-NTA for His-tagged meprin β). Meprins
were activated by trypsin, which was removed afterwards by affinity chromatography
using a column containing immobilized chicken ovomucoid, a trypsin inhibitor.
4.4. Meprin α and Meprin β Assays in a 384-Well Plate
Both assays followed the same general protocol [15]. 5
µ
L of 2
×
enzyme solution
(
2.6 and 0.1 nM for meprin
α
and meprin
β, respectively) in assay buffer (50 mM HEPES,
0
.01% Brij-35, pH 7.5) were added to solid bottom black 384 low-volume plates (Nunc,
cat# 264705). Next, 75 nL of test compounds or pharmacological control (actinonin) were
added to corresponding wells using a 384-pin tool device (V&P Scientific, San Diego, CA,
USA). After 30 min incubation at RT, the reactions were started by addition of 5
µ
L of 2
×
solutions of substrates (20 M, meprin Mca-YVADAPK-K(Dnp), and for meprin β
µ
α
Mca-
EDEDED-K(Dnp)). Reactions were incubated at RT for 1 h, after which the fluorescence
was measured using the Synergy H4 multimode microplate reader (Biotek Instruments)
(λ
excitation
= 324 nm, λemission = 390 nm).
Three parameters were calculated on a per-plate basis: (a) the signal-to-background
ratio (S/B), (b) the coefficient for variation (CV; CV = (standard deviation/mean) 100)
×
for all compound test wells; and (c) the Z- or Z’-factor [16]. Z takes into account the effect
of test compounds on the assay window, while Z’ is based on controls.

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4
.5. Determination of Kinetic Parameters of Meprin α Mediated Proteolysis in the Presence of
Potential Probe SR24717
Substrate stock solutions were prepared at various concentrations in HTS assay buffer
50 mM HEPES, 0.01% Brij-35, pH 7.5). Assays were conducted by incubating a range of
(
substrate (2–50
2
µ
M) and SR24717 concentrations (0–1000 nM) with 1.3 nM meprin
5 C. Fluorescence was measured on a multimode microplate reader Synergy H1 (Biotek
Instruments, Winooski, VT, USA) using = 324 nm and = 393 nm. Rates of
α at
◦
λ
λ
emission
excitation
hydrolysis were obtained from plots of fluorescence versus time, using data points from
only the linear portion of the hydrolysis curve. The slope from these plots was divided by
the fluorescence change corresponding to complete hydrolysis and then multiplied by the
substrate concentration to obtain rates of hydrolysis in units of µM/s. Kinetic parameters
were calculated by non-linear regression analysis using the GraphPad Prism 8.0 suite
of programs.
4.6. ADAM10 and ADAM17 Assays
Both assays followed the same general protocol. 2.5
µ
L of 2
×
enzyme solution (20 nM)
in assay buffer (10 mM HEPES, 0.001% Brij-35, pH 7.5) were added to solid bottom black
84 plates (Greiner, cat# 789075). Next, test compounds and pharmacological controls were
added to corresponding wells using a 384-pin tool device (V&P Scientific, San Diego). After
0 min incubation at RT, the reactions were started by addition of 2.5 L of 2 solutions
of substrate (R&D Systems cat#: ES010, Mca-KPLGL-Dpa-AR-NH , 20 M). Reactions
were incubated at RT for 2 h, after which the fluorescence was measured using a Perkin
Elmer Viewlux multimode microplate imager ( = 324 nm, = 393 nm). All
3
3
µ
×
µ
2
λ
λ
emission
excitation
compounds were tested in 10-point, 1:3 serial dilutions dose-response format starting with
highest concentration of 100 M. IC50 values were determined using non-linear regression
analysis using the GraphPad Prism 8.0 suite of programs.
µ
4.7. MMP Assays
All assays followed the same general protocol. 5
say buffer (50 mM Tricine, 50 mM NaCl, 10 mM CaCl , 0.05% Brij-35, pH 7.5) were added to
µ
L of 2
×
enzyme solution (5 nM) in as-
2
solid-bottom black 384 plates (Nunc, cat# 264705). Next, test compounds and pharmacolog-
ical controls were added to corresponding wells using a 384-pin tool device (V&P Scientific,
San Diego). After 30 min incubation at RT, the reactions were started by addition of 5
of 2 solutions of MMP substrate (R&D Systems cat#: ES010, 20 M). Reactions were
incubated at RT for 1 h, after which the fluorescence was measured using the Synergy H4
multimode microplate reader (Biotek Instruments) ( excitation = 324 nm = 390 nm).
All compounds were tested in 10-point, 1:3 serial dilutions dose-response format start-
ing with highest concentration of 100 M. IC50 values were determined using non-linear
regression analysis using the GraphPad Prism 8.0 suite of programs.
µL
×
µ
λ
, λ
emission
µ
4.8. Cell Toxicity Studies
Test compounds were solubilized in 100% DMSO and added to polypropylene 384-
well plates (Greiner cat# 781280). 1250 of BJ skin fibroblasts or primary melanocytes were
plated in 384-well plates in 8 L of serum-free media (HybriCare for BT474, EMEM for
HEK293). Test compounds and pharmacological assay control (lapatinib) were prepared as
µ
1
0-point, 1:3 serial dilutions starting at 10 mM, then added to the cells using the pin tool
◦
mounted on Integra 384. Plates were incubated for 72 h at 37 C, 5% CO and 95% RH.
After incubation, 8
2
®
µ
L of CellTiter-Glo (Promega cat# G7570) was added to each well and
incubated for 15 min at room temperature. Luminescence was recorded using a Biotek
Synergy H1 multimode microplate reader. Viability was expressed as a percentage relative
to wells containing media only (0%) and wells containing cells treated with DMSO only
(
100%). Three parameters were calculated on a per-plate basis: (a) the signal-to-background
ratio (S/B), (b) the coefficient for variation (CV; CV = (standard deviation/mean) 100)
for all compound test wells, and (c) the Z’-factor. IC50 values were calculated by fitting
×

Pharmaceuticals 2021, 14, 197
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normalized data to sigmoidal log vs. response equation utilizing non-linear regression
analysis from GraphPad Prism 8.
4.9. Molecular Modeling Studies
The crystal structure of human meprin
β
(PDB ID 4GWN) was used as a template
for constructing a model of human meprin
α using Prime (Schrödinger, LLC, New York,
NY, USA), with the option of a single template to build a single chain and including
the Zn ion. The homology model and the coordinates, 4GWN, were prepared using the
protein preparation wizard in Maestro v12.2 (Schrodinger, LLC, NY, USA). Docking studies
were performed using Glide SP v8.7 (Schrodinger, LLC, NY, USA) with no constraint. The
docking grid was generated around the Zn ion with a box size of 18
×
18
×
18 Å3. SR19855
was prepared for Glide docking with LigPrep (Schrodinger, LLC, NY, USA) to include
different conformational states. The docking pose with the highest docking score for each
compound was then merged to the docked structures for energy minimization using the
OPLS3e force field (Schrodinger, LLC, NY, USA) and the results were analyzed in pymol.
4.10. Compound Synthesis and Characterization
Synthesis of all test compounds followed the 6-step route summarized in Figure 3,
which was adapted from the literature [10,17,18]. Details for all steps in the synthesis of
SR24717 are shown, with supporting data. Other compounds followed the same synthetic
methods, and data follows for all analogs reported herein.
Step 1, Ethyl 2-[(aryl)thio] acetate derivatives (
ethyl 2-chloroacetate (2 equiv.) and potassium carbonate (1.2 equiv.) were heated at 80 C
in acetone. Reaction progress was monitored by LC/MS and after no more than 2 h, the
2
). Mercaptopyrimidine
1
(1 equiv.),
◦
mixture was cooled, filtered, and concentrated in vacuo to give the crude product 2, which
was dissolved in minimal dichloromethane, precipitated by addition of diethyl ether, and
the precipitate was collected by filtration and dried under high vacuum. The product was
isolated in 75% yield, with >95% purity by LC/MS, with no chromatography necessary.
Step 2, 2-[(Aryl)thio]acetohydrazide derivatives (3). A mixture of ethyl 2-[(aryl)thio]acetate
(
2) (1 equiv.) and hydrazine hydrate (2 equiv.) in ethanol was stirred for 1–2 h at room
temperature. The colorless hydrazide 3 precipitated out of the solution and was collected
by filtration. The precipitate was washed with water, dried under high vacuum, and was
taken to the next step without purification. It was >95% pure by LC/MS analysis.
Step 3, Thiosemicarbazide derivatives (
and an aryl isothiocyanate , typically a substituted phenyl isothiocyanate (1 equiv.), in
hot ethanol was refluxed for 1 h. The colorless thiosemicarbazide precipitated out of the
5). A solution of the hydrazide (3) (1 equiv.)
4
5
solution and, after cooling, it was collected by filtration. The precipitate was washed with
water, ether, dried under high vacuum, and was taken to the next step without purification.
It was >95% pure by LC/MS analysis.
Step 4, Mercaptotriazole derivatives (6). A suspension of thiosemicarbazide (5) in aq.
◦
2
% NaOH was heated at 100 C, and the solution became homogeneous. Reaction progress
was monitored by LC/MS and cyclization was complete within 2 h. The solution was then
cooled to room temperature. The pH was adjusted to 6–7 by the addition of 3 M HCl, at
which point a colorless precipitate formed. The precipitate collected by filtration, washed
with water, and dried under high vacuum. It was >95% pure by LC/MS analysis and
was obtained in 70% isolated yield for steps 2–4, from compound 2. This intermediate
was potentially sensitive to air oxidation, so it was kept under inert atmosphere and then
carried without delay to the next step.
Step 5, Mercaptotriazole esters (7). A mixture of the mercaptotriazole (6) (1 equiv.),
ethyl 2-chloroacetate (1.12 equiv.) and potassium carbonate (1.5 equiv.) in acetone was
◦
heated at 80 C. Reaction progress was monitored by LC/MS and the alkylation was
complete within 2 h. Upon cooling, a colorless precipitate formed and it was collected
by filtration. The precipitate washed with water and was dried under high vacuum. The
precursor to the probe candidate was >95% pure by LC/MS analysis and was obtained in

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8
5% isolated yield. Certain mercaptotriazole esters analogs were less crystalline and could
be purified by column chromatography on silica gel at this stage, using a CH Cl to 5%
2
2
MeOH in CH Cl gradient. Intermediate ester
7 was stable and could be stored cold under
2
2
inert atmosphere for later use in the final step.
Step 6, final hydroxamic acids (
8
). A solution of mercaptotriazole ester (
7
) (1 equiv.)
◦
in MeOH/DCM (3:1) was cooled to 0 C and was treated with saturated hydroxylamine
hydrochloride solution (6 equiv.) followed by the addition of sat. aq. NaOH (12 equiv.).
After reaction completion (typically~10 min) the mixture was concentrated in vacuo, water
was added, and the pH was adjusted to 7.0 by the addition of 2 M HCl. A colorless
precipitate formed (25–30% yield, >95% analytical purity by HPLC) and it was collected
by filtration. The product was kept cold and under inert atmosphere to prevent slow
hydrolysis of the hydroxamic acid to a carboxylate, presumably from atmospheric moisture.
Such hydrolysis was also seen upon prep HPLC purification, so the final product was best
isolated by precipitation.
1
Characterization data for the potential meprin
DMSO- 6):
J = 8.8 Hz, 2H), 5.89 (s, 1H), 6.94 (s, 1H), 4.48 (s, 2H), 3.81 (s, 6H), 3.78 (s, 2H), 2.94 (s, 6H).
α
inhibitor SR24717: H NMR (600 MHz,
d
δ ppm 10.75 (s, 1H), 9.01 (s, 1H), 8.58 (s, 1H), 7.16 (d, J = 8.8 Hz, 2H), 6.68 (d,
13
C NMR (DMSO-d6): δ ppm 170.9, 168.2, 164.2, 153.7, 151.9, 151.2, 128.4, 120.3, 112.4, 86.0,
6
0.2, 33.4, 24.4. MS (ESI, M + H) calcd for (C H N O S + H): 478.13, found 477.86, purity
19
23
7
4 2
by analytical HPLC >95%.
4.11. Data for Other Test Compounds
4
.11.1. SR19855, N-hydroxy-2-((4-(4-methoxyphenyl)-5-((pyrimidin-2-ylthio)methyl)-4H-
1
,2,4-triazol-3-yl)thio)acetamide
Colorless solid, 42% yield for the final step. Other steps were comparable in yield
to that for SR24717. Silica gel chromatography used MeOH/DCM 0–5% gradient, on the
1
penultimate step. H NMR (400 MHz, DMSO-d6):
δ ppm 10.76 (s, 1H), 9.02 (s, 1H), 8.54 (d,
J = 4.88 Hz, 2H), 7.37 (d, J = 8.92 Hz, 2H), 7.19 (t, J = 4.88 Hz, 1H), 7.04 (d, J = 8.92 Hz, 2H),
.42 (s, 2H), 3.80 (s, 6H), 3.77 (s, 2H). 13C NMR (600 MHz, DMSO- 6
): ppm 170.0, 163.7,
60.2, 157.8, 153.0, 151.0, 128.9, 124.9, 117.5, 114.9, 55.5, 33.1, 23.9. MS (ESI, M + H) calcd for
C H N O S + H): 405.07, found 405.01, purity by analytical HPLC > 95%.
4
d
δ
1
(
16
16
6
3 2
4.11.2. SR24139, 2-((5-(((4,6-dimethylpyrimidin-2-yl)thio)methyl)-4-(4-methoxyphenyl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 45% yield for final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.75 (d, J = 1.2 Hz, 1H), 9.02 (d,
J = 1.2 Hz, 1H), 7.35 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 6.93 (s, 1H), 4.47 (s, 2H),
.79 (s, 6H), 3.78 (s, 2H), 2.28 (s, 6H). 13C NMR (600 MHz, DMSO- 6
): ppm 167.7,167.0,
63.7, 160.01, 153.3, 151.0, 128.8, 124.8, 116.2, 114.7, 55.5, 33.1, 23.5, 23.2. MS (ESI, M + H)
calcd for (C H N O S + H): 433.10, found 433.05, purity by analytical HPLC > 97%.
3
d
δ
1
18
20
6
3 2
4.11.3. SR24144, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(4-methoxyphenyl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 45% yield for the final step, recrystallized from EtOAc. Other steps
1
were comparable in yield to that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.76
(
2
d, J = 0.88 Hz, 1H), 9.01 (d, J = 0.88 Hz, 1H), 8.37 (d, J = 8.92 Hz, 2H), 7.01 (d, J = 8.92 Hz,
H), 5.91 (s, 1H), 4.49 (s, 2H), 3.80 (s, 6H), 3.79 (s, 3H), 3.77 (s, 2H). 1 C NMR (600 MHz,
): ppm 170.5, 167.7, 163.7, 160.2, 153.0, 151.1, 128.9, 124.8, 114.8, 85.5, 55.5, 54.3,
3.1, 24.0. MS (ESI, M + H) calcd for (C H N O S + H): 465.09, found 465.01, purity by
3
DMSO- 6 δ
3
d
18
20
6
5 2
analytical HPLC > 95%.

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4.11.4. SR24460, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(3-methoxyphenyl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 38% yield for the final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.77 (d, J = 1.04 Hz, 1H), 9.03 (d,
J = 1.48 Hz, 1H), 7.42 (dt, J = 7.88, 0.80 Hz, 1H), 7.04 (q, J = 7.08 Hz, 3H), 5.91 (s, 1H), 4.55 (s,
H), 3.80 (s, 6H), 3.75 (s, 2H). 13C NMR (600 MHz, DMSO- 6
): ppm 170.5, 167.7, 163.6,
59.8, 152.7, 150.6, 133.44, 130.5, 119.3, 115.6, 113.2, 85.5, 55.5, 54.2, 33.2, 24.0. (ESI, M + H)
calcd for (C H N O S + H): 465.09, found 465.02, purity by analytical HPLC > 98%.
2
d
δ
1
18
20
6
5 2
4.11.5. SR24459, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(2-methoxyphenyl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 40% yield for the final step. Other steps were comparable in yield
1
to that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.77 (s, 1H), 9.03 (s, 1H), 7.50
(
dt, J = 6.88, 1.56 Hz, 1H), 7.39 (dd, J = 7.8, 1.56 Hz, 1H), 7.21 (d, J = 8.4 Hz,1 H), 7.05 (t,
J = 7.64 Hz, 1H), 5.91 (s, 1H), 4.48 (d, J = 15.0 Hz, 1H), 4.37 (d, J = 15.0 Hz, 1H), 3.81 (s,
H), 3.75 (s, 2H). 13C NMR (600 MHz, DMSO- 6
): ppm 170.5, 167.8, 163.7, 154.3, 153.0,
51.2, 132.0, 129.1, 120.8, 120.5, 112.8, 85.5, 55.9, 54.2, 33.2, 23.8. (ESI, M + H) calcd for
C H N O S + H): 465.09, found 464.98, purity by analytical HPLC > 95%.
6
1
(
d
δ
18
20
6
5 2
4
.11.6. SR24718, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(pyridin-3-yl)-4H-
1
,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 42% yield for the final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.82 (s, 1H), 9.09 (s, 1H), 8.80
(
d, J = 2.36 Hz, 1H), 8.75 (dd, J = 4.8, 1.4 Hz, 1H), 8.07 (td, J = 8.24, 2.48 Hz, 1H), 7.62 (dd,
J = 8.24, 4.84 Hz, 1H), 5.98 (s, 1H), 4.63 (s, 2H), 3.87 (s, 6H), 3.84 (s, 2H). 13C NMR (600 MHz,
DMSO- 6): ppm 170.5, 167.5, 163.5, 152.9, 151.0, 150.9, 148.2, 135.6, 129.6, 124.4, 85.6, 54.3,
3.7, 24.0. MS (ESI, M + H) calcd for (C H N O S + H): 436.08, found 435.91, purity by
d
δ
3
16
17
7
4 2
analytical HPLC > 95%.
4
.11.7. SR24003, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(4-
(
trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 35% yield for final step. Other steps were comparable in yield to that
1
for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.78 (d, 1H), 9.02 (d, 1H), 7.88 (d,
1
3
J = 8.31 Hz, 2H), 7.76 (d, J = 8.24 Hz, 2H), 5.88 (s, 1H), 4.58 (s, 2H), 3.79 (s, 9H). C NMR
600 MHz, DMSO-d6): ppm 170.5, 167.5, 163.5, 152.8, 150.5, 136.1, 130.4, 128.8, 126.8, 124.5,
(
δ
1
22.7, 85.5, 54.3, 33.6, 24.0. MS (ESI, M + H) calcd for (C H F N O S + H): 503.07, found
18
17
3
6
4 2
503.01, purity by analytical HPLC > 97%.
4.11.8. SR24462, 2-((4-(4-bromophenyl)-5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 35% yield for the final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.76 (s, 1H), 9.03 (s, 1H), 7.70 (d,
J = 8.7 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 5.93 (s, 1H), 4.55 (s, 2H), 3.82 (s, 6H), 3.78 (s, 2H).
13
C NMR (600 MHz DMSO-d6): δ ppm 170.5, 167.51, 163.6, 152.7, 150.6, 132.7, 131.8, 129.7,
1
23.6, 85.5, 54.3, 33.4, 24.0 MS (ESI, M + H) calcd for (C H BrN O S + H): 513.0, 515.0,
17
17
6
4 2
found 512.99, 514.95, purity by analytical HPLC > 95%.
4.11.9. SR24463, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(4-fluorophenyl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 35% yield for the final step. Other steps were comparable in yield
1
to that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.77 (s, 1H), 9.03 (s, 1H),
7
.55–7.58 (m, 2H), 7.37 (t, J = 8.8 Hz, 2H), 5.93 (s, 1H), 4.53 (s, 2H), 3.82 (s, 6H), 3.79 (s, 2H).
C NMR (600 MHz, DMSO-d6): δ ppm 170.5, 167.6, 163.4, 161.7, 152.8, 150.8, 130.1, 128.8,
1
3

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116.8, 85.5, 54.3, 33.3, 24.0. MS (ESI, M + H) calcd for (C H FN O S + H): 453.07, found
17 17 6 4 2
452.59, purity by analytical HPLC > 96%.
4
.11.10. SR24467, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(m-tolyl)-4H-1,2,4-
triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 42% yield for the final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.76 (s, 1H), 9.02 (s, 1H), 7.50 (t,
J = 7.59 Hz, 1H), 7.31 (d, J = 7.47 Hz, 1H), 7.24 (d, J = 6.75 Hz, 2H), 5.90 (s, 1H), 4.51 (s, 2H),
.80 (s, 9H), 2.29 (s, 3H). 13C NMR (600 MHz, DMSO- 6
): ppm 170.5, 167.7, 163.6, 152.8,
50.7, 139.6, 132.4, 130.4, 129.5, 127.7, 124.5, 85.5, 54.3, 33.2, 24.0, 20.6. (ESI, M + H) calcd for
C H N O S + H): 449.10, found 448.94, purity by analytical HPLC > 95%.
3
d
δ
1
(
18
20
6
4 2
4.11.11. SR26467, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(naphthalen-1-yl)-
4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 32% yield for the final step. Other steps were comparable in yield
to that for SR24717. Silica gel chromatography used MeOH/DCM 0–5% gradient, on the
1
penultimate step. H NMR (400 MHz, DMSO-d6):
δ ppm 10.76 (s, 1H), 9.02 (s, 1H), 8.06
(d, J = 8.32 Hz, 1H), 8.03 (d, J = 7.44 Hz, 1H), 7.67 (d, J = 7.36 Hz, 1H), 7.62–7.54 (m, 3H),
7
3
.13 (d, J = 8.16 Hz, 1H), 5.74 (s, 1H), 4.41 (d, J = 15.1 Hz, 1H), 4.38 (d, J = 15.2 Hz, 1H),
.80 (s, 2H), 3.59 (s, 6H). 13C NMR (600 MHz, DMSO- 6
d
): δ ppm 170.1, 167.2, 163.6, 153.9,
1
51.8, 133.7, 130.9, 128.8, 128.4, 128.3, 128.1, 127.1, 127.0, 125.5, 121.4, 85.4, 54.0, 33.1, 23.7.
MS (ESI, M + H) calcd for (C H N O S + H): 485.10, found 484.91, purity by analytical
21
20
6
4 2
HPLC > 95%.
4
.11.12. SR26466, 2-((5-(((4,6-dimethoxypyrimidin-2-yl)thio)methyl)-4-(4-
morpholinophenyl)-4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 41% yield for the final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, DMSO-d6):
δ ppm 10.76 (s, 1H), 9.02 (s, 1H), 7.24
(
d, J = 8.96 Hz, 2H), 6.97 (d, J = 9.04 Hz, 2H), 5.91 (s, 1H), 4.50 (s, 2H), 3.81 (s, 6H), 3.78
13
(
s, 2H), 3.75 (t, J = 4.88 Hz, 4H), 3.17 (t, J = 4.76 Hz, 4H). C NMR (600 MHz, DMSO-d6):
δ
ppm 170.5, 167.7, 163.7, 153.1, 151.6, 151.3, 128.1, 122.6, 114.7, 85.6, 66.0, 54.3, 47.4, 33.0,
2
4.0. MS (ESI, M + H) calcd for (C H N O S + H): 520.14, found 519.95, purity by
21
26
7
5 2
analytical HPLC > 95%.
4
.11.13. SR26465, 2-((4-(4-(diethylamino)phenyl)-5-(((4,6-dimethoxypyrimidin-2-
yl)thio)methyl)-4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide
Colorless solid, 41% yield for the final step. Other steps were comparable in yield to
1
that for SR24717. H NMR (400 MHz, CD3CN-d6):
δ ppm 7.06 (d, J = 8.88 Hz, 2H), 6.59
(
4
d, J = 9.04 Hz, 2H), 5.71 (s, 1H), 4.51 (s, 2H), 3.80 (s, 6H), 3.69 (s, 2H), 3.33 (q, J = 7.04 Hz,
H), 1.11 (t, J = 7.08 Hz, 6H). 13C NMR (600 MHz, DMSO- 6
d ): δ ppm 170.4, 167.7, 163.8,
1
53.3, 151.6, 148.1, 128.2, 118.8, 111.0, 85.6, 54.2, 43.7, 32.8, 23.8, 12.3. MS (ESI, M + H) calcd
for (C H N O S + H): 506.16, found 505.96, purity by anal. HPLC > 95%.
21
27
7
4 2
5
. Conclusions
The lead molecule emerging from this study, SR24717, optimized from a high through-
put screening hit, shows a promising profile with selectivity for meprin . Three interac-
tions, a interaction and two cation- interactions, appear to be responsible for this
selectivity vs. meprin and other metzincins. Accordingly, modification of substituent
α
π
-
π
π
β
effects on the aryl and heteroaryl rings impact affinity and selectivity. Compounds in
this chemical series may prove useful for the study of the efficacy and safety of meprin
α-selective inhibition in animal models of human disease.

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Author Contributions: Conceptualization, D.M., T.P.S., L.D.S. and T.D.B.; chemistry methodology,
C.W. and T.D.B.; biochemical assays development, D.M.; high throughput screening implementation,
T.P.S. and L.D.S.; cytotoxicity studies, J.D.; molecular modeling studies, H.P.; compound management,
T.P.S. and L.D.S.; preparation, purification, and characterization of meprin substrates, G.B.F.; expres-
sion of meprins, C.B.-P.; writing—original draft preparation, C.W., D.M. and T.D.B.; writing—review
and editing, all authors.; project administration, D.M.; funding acquisition, C.B.-P. and D.M. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Institutes of Health (AR066676 and CA249788 to
D.M.). This work was also supported by the Deutsche Forschungsgemeinschaft (DFG) grant SFB877
“
Proteolysis as a Regulatory Event in Pathophysiology” (project A9 and A15) (both to C.B.-P.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank Pierre Baillargeon and Lina DeLuca (Lead Identification, Scripps
Florida) for compound management support.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
MMP
matrix metalloprotease
ADAM a disintegrin and metalloprotease
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