Tertiary-Amine-Based Inhibitors of the Astacin Protease Meprin α

Article abstract of DOI:10.1002/cmdc.201800300

Metalloproteinases of the astacin family are drawing ever increasing attention as potential drug targets. However, knowledge regarding inhibitors thereof is limited in most cases. Crucial for the development of metalloprotease inhibitors is high selectivi

Full text of DOI:10.1002/cmdc.201800300

Accepted Article
Title: Tertiary amine based inhibitors of the astacin protease meprin
alpha
Authors: Kathrin Tan, Christian Jäger, Dagmar Schlenzig, Stephan
Schilling, Mirko Buchholz, and Daniel Ramsbeck
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Tertiary amine based inhibitors of the astacin protease meprin 
Kathrin Tan, Christian Jäger, Dagmar Schlenzig, Stephan Schilling, Mirko Buchholz, and Daniel
Ramsbeck*
Kathrin Tan, Christian Jäger, Dr. Dagmar Schlenzig, Dr. Stephan Schilling, Dr. Mirko Buchholz, Dr. Daniel Ramsbeck
Department of Drug Design and Target Validation MWT
Fraunhofer Institute for Cell Therapy and Immunology IZI
Biocenter, Weinbergweg 22, 06120 Halle (Saale), Germany
E-mail: daniel.ramsbeck@izi.fraunhofer.de
Abstract: Metalloproteinases of the astacin family are more and more
drawing attention as potential drug targets. However, the knowledge
about inhibitors thereof is limited in most cases. Crucial for the
development of metalloprotease inhibitors is a high selectivity to avoid
side effects through the inhibition of off-target proteases and the
interference with physiological pathways. Here we aimed at the
design of novel selective inhibitors for the astacin proteinase meprin
. Based on a recently identified tertiary amine scaffold, a series of
compounds was synthesized and evaluated. The compounds exhibit
reasonable inhibitory activity with high selectivity over other
metalloproteases. Also the isoenzyme meprin  is only slightly
inhibited. Hence, the present study revealed a novel class of selective
meprin  inhibitors with improved selectivity compared to known
compounds.
Up to now, the most potent identified inhibitors of meprin  are
actinonin, galardin, NNGH and CGS-27023 (Figure 1).[6] However,
all the mentioned compounds are also pan-selective inhibitors of
metalloproteases or other metal-dependent enzymes. More
recently, we reported the first selective inhibitors of meprin ,
derived from NNGH.[7] Compound 1a (Figure 2) featuring two
carboxylic acid moieties in meta position exhibits excellent
selectivity for meprin . However, the selectivity over meprin  is
diminished with the depletion of one carboxylic acid (1b). Further
optimization of these compounds included the deletion of the
sulfonamide moiety leading to tertiary amine derivatives 2a and
2b (Figure 2) with high selectivity for the meprin isoenzymes,
while only modest or virtually no inhibition of selected off-target
proteases was observed.[8] Although, the selectivity of the
compounds for meprin  is in the same range, like for the
respective sulfonamides.
The metzincin superfamily of proteases covers numerous
enzymes that served as drug targets from past to present, e.g.,
MMPs and ADAMs. A small subfamily, the astacins, has not
drawn that attention, yet. In human, this group of zinc-dependent
endoproteinases includes procollagen-C-peptidase, also referred
to BMP-1, tolloid-like proteases, ovastacin and the meprins  and
 More and more data renders the latter as interesting and
important drug targets, too.[3] Meprin  and  are linked to multiple
pathologies, most importantly to several diseases of the
connective tissue, i.e., via the proteolytic processing of
procollagen.[4] Furthermore, meprin  was also found to be
involved in tumor growth and progression. Thus, meprin  might
also serve as a novel drug target in cancer.[5]
Figure 2. IC50 values (µM) of exemplary meprin inhibitors with sulphonamide
and tertiary amine scaffold.
Surprisingly, the further profiling of the recently reported meprin
inhibitors 2c-i with regard to their meprin  inhibition revealed an
increased activity upon the elongation of the spacer between the
hydroxamate and the tertiary amine (2c) by a factor ~10
compared to 2b (Table 1). Concurrently, the activity against
meprin  was decreased by one order of magnitude, thus leading
to almost equipotent inhibition of both isoenzymes. The
introduction of one further additional methylene unit within the
spacer again led to decreased inhibition of meprin . The
replacement of one carboxy-aryl residue by a para-chloro- or
Figure 1. Ki and IC50 values (µM) of known inhibitors of meprin 
1
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methylendioxo-aryl moiety slightly increased the activity against
meprin . The bioisosteric replacement of the carboxylic acids by
halophenols maintained the activity against meprin  while also
increasing the activity against meprin (2g and 2h). Though, all
compounds bearing at least one acidic functional group, except
2c, exhibited stronger inhibition of meprin  rather than meprin .
However, the substitution of the carboxylic acids by H-bond
acceptors, i.e., cyano- or oxygen moieties (2i and 2j), led to a
dramatically decreased inhibition of meprin . On the other side,
The structural basis of the observed different activities may be
subtle differences within the active sites of meprin  and .
A homology model was built utilizing the X-ray structure of meprin
 (pdb-code 4GWN[9]) as template (Figure 3). The differences
between both catalytic centers are marginal (Figure 4). The S1´-
subpocket is shaped by an arginine in both enzymes that enables
potential interaction with acidic functional groups of the inhibitors
and substrates, i.e., acidic amino acids in P1´-position.[10]
the activity against meprin
 was increased by these
modifications compared to 2b. Thus, these two compounds
represented the first inhibitors of the tertiary amine scaffold with a
slight selectivity for meprin  over meprin . Hence, these
observations created a starting point for the design of novel
selective meprin  inhibitors.
Table 1. Inhibition of meprin  tertiary amines
R1 = Ph-m-COOH
meprin 
meprin 
Figure 4. Overlay of the active sites of meprin  (purple, homology model) and
meprin  (blue, pdb-code 4GWN). Identical side chain residues are coloured in
green.
n
2
SF[b]
2
R2
IC50 [µM][a]
IC50 [µM][a]
2c
2d
0.31 ± 0.02
2.61 ± 0.28
0.64 ± 0.03
0.74 ± 0.04
However, the residues shaping the S1 and S2´-subpockets differ
significantly. This is also corroborated by distinct substrate
specificities of meprin  and [11] While both subsites are formed
by arginines in meprin , the respective residues are replaced by
tyrosines in meprin . This is of particular importance since the
predicted binding mode of 2a involves interactions with the S1-
subpocket (Figure 5, left).[7]
3
0.3
2e
2f
1
1
1.63 ± 0.14
1.91 ± 0.07
0.08 ± 0.004 0.05
0.29 ± 0.09
0.15
R1 = R2
2g
2h
1
1
0.63 ± 0.01
0.37 ± 0.08
0.02 ± 0.001 0.03
0.02 ± 0.001 0.05
2i
2j
1
1
1.92 ± 0.12
1.47 ± 0.12
6.54 ± 1.22
3
8
12.00 ± 0.57
[a] mean ± SD of at least two independent experiments performed in duplicates
[b] Selectivity factor (SF = IC50 meprin / IC50 meprin )
Figure 5. Schematic representation of assumed binding modes and potential
key interactions of compound 2a with meprin  (left) and compound 2i with
meprin  (right)
Hence, ionic or charged hydrogen bond interactions formed by
the carboxylic acids of 2a with the basic arginine residues are
ruled out in case of meprin . On the contrary, the tyrosine side
chains obviously could cause repulsive or unfavorable
interactions. Also steric clashes could result from the aromatic
Tyrosin-sidechain protruding the active site cleft, thus leading to
weaker inhibition of meprin by 2a. However, the reduced acidity
of the halophenols 2g and 2h diminishes this repulsion, leading
also to acceptable activities against meprin . Although, the
tyrosine residues could serve as hydrogen bond donors and
Figure 3. Homology model of meprin  (A) and crystal structure of meprin  (B,
pdb: 4GWN)
2
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enable interactions with the cyano- and dioxolane-moiety of 2i
and 2j, respectively. This enables a favorable inhibition of meprin
, while interactions with the arginine residues shaping the S1 or
S2´pocket of meprin  are weaker compared to the carboxylic
acids of 2a. Thus, the tyrosine residues of meprin , i.e., the S1
or S2´-pocket, could be addressed by hydrogen bond acceptors,
potentially leading to favorable meprin  inhibition, while the S1´-
subpocket could be targeted via the formation of a hydrogen bond
as well or a cation--interaction, formed by the sidechain of
arginine R242 (Figure 5, right).
This corroborates the assumed cation--interaction with the S1´-
site. Albeit the three methoxy-substituents of 9e could serve as
hydrogen bond acceptors and also increase the electron density
of the phenyl moiety, the decreased activity may be attributed to
steric hindrance, in particular compared to 9d with comparable
electrostatic properties but less steric demands.
Table 2. Inhibition of meprin  by glycinyl hydroxamates.
To further elaborate the SAR of these potential novel meprin 
meprin 
meprin 
SF[b]
inhibitors,
a
series of compounds was synthesized and
IC50 [µM][a]
IC50 [µM[a]

characterized with regard to their inhibition of both meprin
isoenzymes. To address the aforementioned interactions with the
target protein, different hydrogen bond acceptors were introduced.
Based on the initial findings, also the elongation of the spacer
between hydroxamate and amine, i.e. the respective -alanine
derivatives were explored.
n.d.
9a
9b
9c
9d
9e
9f
7.88 ± 0.29
5.66 ± 0.38
1.52 ± 0.04
0.55 ± 0.03
5.32 ± 0.35
4.98 ± 0.04
2.83 ± 0.06
3.06 ± 0.20
1.96 ± 0.07
11.90 ± 0.14
-
(RA = 82%)c
33.05 ± 2.05
30.60 ± 0.99
4.58 ± 0.01
6
20
8
Scheme 1. Synthesis of tertiary amine hydroxamates
20.15 ± 0.35
12.80 ± 0.70
18.20 ± 0.85
13.80 ± 5.66
21.00 ± 0.85
132.50 ± 6.36
4
2.5
6
9g
9h
9i
4.5
11
11
The synthesis of the compounds was straight forward starting
from either trityl protected hydroxamic amino acids, as reported
earlier, or starting from amino acid esters (Scheme 1). Briefly, the
protected hydroxamic acids 5 were first alkylated using different
benzyl halides yielding the tertiary amine intermediates 6. The
final compounds 9 or 10 were obtained by acidic cleavage of the
trityl group by means of trifluoroacetic acid. Alternatively, the
amino acid esters 7 were alkylated via reductive amination,
yielding intermediates 8. The final inhibitors 9 or 10 were obtained
by aminolysis of the ester using hydroxylamine hydrochloride
under microwave irradiation.
9j
[a] mean ± SD of at least two independent experiments performed in duplicates
[b] Selectivity factor (SF = IC50 meprin / IC50 meprin )
[c] remaining enzyme activity @ 100 µM inhibitor
The inhibitory activities of the resulting compounds are reported
in tables 2 and 3. The introduction of an unsubstituted phenyl
moiety (9a) led to a decreased activity compared to compounds
2i and 2j. However, also for this unsubstituted derivative a
preference for meprin  over meprin  inhibition was observed.
The introduction of a para-cyano group (9b) led to a decreased
activity compared to 2j. This indicates again a preference of meta
substitution of the aryl moiety, as found for meprin  inhibitors
before. In general, compounds bearing electron-rich aryl
substituents (2i, 9c and 9d) exhibited higher activities than
compounds with electron-deficient moieties (9f, g and h).
Common for all the compounds is an increased activity against
meprin  versus meprin  most likely due to the elimination of
acidic functional groups. Hence, the etherification of the halo-
phenols (9f and 9g) also led to drop of activity against meprin ,
compared to 2g and 2h. The methylation of the phenol rules out
an interaction with the arginines in the S1 and S1´-pocket via ionic
or charged hydrogen bond interactions and the aryl moiety.
However, since we assumed that an acidic group is not
mandatory for inhibition of meprin , the decreased activity of 9f
and g compared to 2g and 2h was surprising. Obviously, the ether
3
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functionality might be to electron deficient, thus unable to serve
as cation- or hydrogen bond acceptor as an alternative attractive
interaction, respectively. The 3-pyridyl derivative 9i exhibited
comparable activity as compound 2i, while the activity of the 4-
pyridyl derivative 9j was decreased and just in the range of the
unsubstituted compound 9a. Although both pyridines could serve
of hydrogen bond acceptors, this corroborates again the
preference of meta-substitution. Hence, the putative hydrogen
bond interactions assumed for 2i could just be addressed by 9i.
Nevertheless, this weak influence on the activity was unexpected,
since a 3-pyridyl moiety is also present in CGS27023, that
exhibited a higher activity against meprin  (Figure 1).
However, for the symmetric tertiary amines reported here, the
introduction of two pyridyl-residues seems to be not that favorable.
Probably the S1´-pocket, shaped by arginine R242, harboring the
electron-rich para-methoxyaryl in CGS27023, could not be
addressed properly, e.g., due to unfavorable electrostatic
properties of the electron-deficient pyridine residues.
As already observed for compound 2c, the introduction of an
additional methylene unit within the spacer between hydroxamate
and tertiary amine was beneficial for the inhibitory activity of
meprin . In general, the activities of the -alanine derivatives
10a-m (Table 3) were increased compared to their glycinyl
analogues (2 or 9), just with exception of 10g and the pyridyl
derivative 10l. The highest activity was achieved by the
introduction of a benzodioxolane (10d) or benzodioxane moiety
(10e), respectively. The halophenol substituted derivative 10j also
exhibited an increased activity against meprin , i.e., in the same
range as 10d or 10e. Although, the potency against meprin  was
not affected by the elongation of the spacer in this case. Thus,
this compound still exhibited a preference of meprin  inhibition.
The reason for the improved activities of the -alanine derivatives
remains unclear, so far. Both, the X-ray structure of meprin  and
the homology model of meprin  share the same shape and size
of the active site cleft, albeit with small differences in only few
amino acids. Hence, the different preferences for either glycine-
or -alanine hydroxamates could not be explained on the basis of
the available structural information. However, existing differences
within the structure of the isoenzymes are also indicated by the
substrate specificities. Although the S1´-pockets are formed by
identical amino acids, meprin  prefers substrates with Asp or Glu
in P1´-position while meprin  is restricted to the cleavage of Asp
in P1´-position[10]. This underpins small structural differences of
the active sites, not exposed by the present meprin  homology
model. To gain a more detailed insight in the binding mode of the
inhibitors and a more reliable explanation of the differences in
their efficacy, the elucidation of the meprin  structure at atomic
level is mandatory.
However, the selectivity of all the reported inhibitors over meprin
 is less pronounced, i.e. by a factor ~10-20 for the most potent
compounds, while the most selective meprin  inhibitor exhibited
a selectivity factor of ~300 over meprin . This might be due to
the type of interactions of the respective inhibitors. Meprin  can
be selectively addressed by compounds bearing carboxylic acids
through the formation of charged interactions. Though, these
interactions cause repulsive or less favorable interactions with the
active site of meprin , resulting in poor inhibition. On the contrary,
the electron-rich residues leading to a potent inhibition of meprin
, e.g. benzodioxolane (10d), could also address the arginine
side chains within the binding site of meprin  via hydrogen
bonding or cation- interactions, thus also leading to a relatively
potent inhibition in the lower micromolar range and a less
pronounced selectivity for meprin .
Table 3. Inhibition of meprin  and meprin  by -alanine derivatives.
meprin 
meprin 
SF[b]
IC50 [µM][a]
IC50 [µM][a]

10a
10b
10c
10d
10e
10f
1.68 ± 0.03
2.34 ± 0.04
3.42 ± 0.05
0.16 ± 0.001
0.40 ± 0.03
0.29 ± 0.001
19.80 ± 0.42
0.50 ± 0.03
0.88 ± 0.05
0.19 ± 0.001
1.00 ± 0.04
7.68 ± 0.41
5.16 ± 0.29
75.00 ± 3.68
33.05 ± 0.57
68.25 ± 1.06
2.95 ± 0.35
7.59 ± 0.01
1.89 ± 0.01
89.95 ± 4.88
19.55 ± 0.92
18.70 ± 0.14
0.03 ± 0.001
19.20 ± 0.99
53.90 ± 6.36
72.40 ± 5.37
45
14
20
18
19
6.5
4.5
39
21
0.16
19
7
10g
10h
10i
10j
Since many metalloproteases are not only involved in
pathological processes, but also in the regulation of physiological
pathways, the selectivity of potential meprin inhibitors against
these proteases is crucial. Hence, in order to assess their off-
target selectivity, inhibitors 10d and 10e were further evaluated in
a small panel of selected proteases of the metzincin family (Table
4). As found earlier for the meprin  inhibitors with tertiary amine
scaffold, no significant inhibition of MMP2, 9, 13 or ADAM10 and
17 was observed. This corroborates further the results of the
previous study and highlights the suitability of the tertiary amine
10k
10l
10m
14
[a] mean ± SD of at least two independent experiments performed in duplicates
[b] Selectivity factor (SF = IC50 meprin / IC50 meprin )
4
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were obtained with an Expression CMS spectrometer (Advion).
High resolution ESI mass spectra were obtained from a LTQ Orbitrap XL
(Thermo Fisher Scientific). The 1H-NMR and 13C-NMR spectra were
recorded at a DDR2 400 spectrometer (Agilent). Chemical shifts are
expressed as parts per million (ppm). The solvent was used as internal
standard. Splitting patterns have been designated as follows: s (singlet),
d (doublet), dd (doublet of doublet), t (triplet), m (multiplet), and br s (broad
signal). Semi-preparative HPLC was performed on a Prepstar device
(Varian) equipped with a Phenomenex Luna 10 μM C18(2) column
(250 mm × 21 mm). The compounds were eluted using the same solvent
system as described above, applying a flow rate of 21 mL/min.
as scaffold for the discovery of inhibitors for proteases of the
astacin family. While previous results revealed the high selectivity
of acidic substituted meprin  inhibitors, the present study
exemplified, that this property is not only attributed to the acidic
substituents, but can also be achieved with inhibitors bearing
solely neutral moieties. Thus, the selectivity might be primarily an
intrinsic feature of the tertiary amine scaffold.
Table 4. Selectivity profile of compounds 10d and 10e.
General procedure for the synthesis of compounds 9 and 10 by
alkylation of trityl protected amino hydroxamic acids and subsequent
deprotection (method A). 2-Amino-N-(trityloxy)acetamide or 3-amino-N-
(trityloxy)propanamide (1 mmol, 1 eq) was dissolved in DMF (5 ml). The
respective benzylhalide (2.2 mmol, 2.2 eq) and triethylamine (5 mmol,
5 eq) were added and the mixture was heated to 140°C for 20 min in a
microwave (Biotage Initiator+). After cooling, the reaction was quenched
by addition of water and extracted by means of ethyl acetate (3x25 ml).
The combined organic layers were dried over Na2SO4 and evaporated.
The residue was purified by flash chromatography (silica, heptane/ethyl
acetate gradient). The purified intermediate 6 was directly subjected to
acidic deprotection using trifluoroacetic acid/dichloromethane (1:1 v/v, 5
ml) and triisopropylsilane (50 µl) at room temperature for 2h. The volatiles
were evaporated and the residue was purified by semi-preparative HPLC
yielding the title compound 9 or 10.
10d
10e
Inhibitor concentration
RA [%][a]
MMP2
10 µM
95
200 µM
26
10 µM
94
200 µM
65
MMP9
91
61
91
21
MMP13
ADAM10
ADAM17
86
55
98
47
86
61
80
56
86
57
80
41
General procedure for the synthesis of compounds 9 and 10 by
reductive amination of amino acid esters and subsequent aminolysis
(method B). Glycine- or -alaninemethyl ester hydrochloride (1 mmol, 1
eq) was suspended in dichloromethane (20 ml). After addition of the
respective aldehyde (3 mmol, 3 eq) and acetic acid (0.75 ml), sodium
triacetoxyborohydride (4 mmol, 4 eq) was added in portions and the
reaction was stirred at room temperature overnight. Water (5 ml) was
added, the pH was adjusted to 8 with saturated aqueous NaHCO3 and the
mixture was extracted with ethyl acetate (3x25 ml). The combined organic
layers were dried over Na2SO4 and evaporated. The residue was purified
[a] RA – remaining enzyme activity
In summary, a novel class of meprin  inhibitors was discovered,
based on a recently reported tertiary amine scaffold. Starting from
meprin  inhibitors with low activity against meprin , the inhibitory
potency and selectivity could be optimized and a first insight into
the SAR could be gained. A homology model of meprin  was
built and thereby enabled a structure-guided compound design.
The inhibitors reported herein exhibit activities in the lower
nanomolar range. Hence, they exhibit comparable inhibitory
potency as NNGH or galardin but are slightly less active than
actinonin or CGS-27023 that have been reported as meprin 
inhibitors before. The selectivity of the novel inhibitors reported
here over meprin  is also in the same range as found for NNGH,
galardin and actinonin. Nevertheless, the major advantage of
these novel tertiary amine based inhibitors is their superior
selectivity against off-target metalloproteases. Hence, further
optimization of the potency of the inhibitors and the selectivity
over meprin would lead to valuable tool compounds or molecule
probes to study meprin  in different disease models.
by flash chromatography (silica,
heptane/ethyl
acetate
or
heptane/diethylether gradient). The purified intermediate 6 was dissolved
in methanol (5 ml). Hydroxylamine*HCl (3 eq) and 5 N NaOCH3 (6 eq) were
added and the mixture was heated to 80°C for 10 min in a microwave. After
cooling, the volatiles were evaporated. The residue was redissolved in
water (20 ml), the pH was adjusted to 8 by means of diluted HClaq and
extracted with ethyl acetate (3x25 ml). The combined organic layers were
dried over Na2SO4 and evaporated. The residue was purified by semi-
preparative HPLC yielding the title compounds 9 or 10.
Homology model Chain A of human meprin  (PDB: 4GWN) and the fasta
sequence of meprin A subunit alpha (Q16819) were aligned with MOE
(v2016.0802; Protein Align 2016.11). A pairwise percentage identity of
33.8% could be observed. The maximum number of independent main-
chain models was set to 100. After refinement by calculation of the
electrostatic solvation energy (Generalized Born/Volume Integral, GB/VI),
the best scoring-intermediate model was selected as final model. Finally,
the template structure (4GWN) was superposed to the MepA model, the
atom type of Cd2+ was changed to Zn2+ and copied as new chain to the
homology model structure. To compare the X-ray structure homology
model, pairwise RMSD values were calculated. For the examination of the
model geometry, the Phi-Psi Plots (Ramachandran plots) where visualized.
Experimental Section
General chemistry Starting materials and solvents were purchased from
Aldrich, Activate Scientific, Alfa Aesar, Iris Biotech and Merck Millipore.
The purity of the compounds was assessed by HPLC and confirmed to be
≥95%. The analytical HPLC-system consisted of a Merck−Hitachi device
(model LaChrom) utilizing a Phenomenex Luna 5 μM C18(2) column
(125 mm × 4.0 mm) with λ = 214 nm as the reporting wavelength.
The compounds were analyzed using a gradient at a flow rate of 1 mL/min,
whereby eluent (A) was acetonitrile, eluent (B) was water, both containing
0.04% (v/v) trifluoro acetic acid applying the following gradient: gradient 1:
0 min - 15 min, 5 - 60% (A), 15 min - 20 min, 60 - 95% (A), 20 min - 30 min,
95% (A); gradient 2: 0 min - 5 min, 1% (A), 5 min - 20 min, 1 - 20% (A), 20
min - 30 min, 20 - 95% (A), 30 min - 34 min, 95 % (A). ESI-Mass spectra
Supporting Information
Analytical characterization of compounds, description of the enzymatic
assays and further information regarding the homology model can be
found within the supporting information.
5
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Acknowledgements
We thank Antje Hamann, Mercedes Scharfe (IZI-MWT), Dr.
Andrea Porzel (Leibniz Institute of Plant Biochemistry, Halle) and
Dr. Christian Ihling (Martin Luther University Halle-Wittenberg) for
their excellent technical support. We further acknowledge the
funding of this project by the federal state of Saxony-Anhalt,
Germany.
Keywords: meprin alpha• astacin • hydroxamic acid •
metalloproteinase • tertiary amine
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10.1002/cmdc.201800300
ChemMedChem
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Flipping the balance: We report a first insight into the SAR of novel compounds targeting meprin  Based on recently discovered
highly selective meprin  inhibitors, the modification of individual residues targeting the S1 and S1`-subsites of the proteases led to
improved activity and selectivity. Hence, the balance between inhibition of both isoenzymes could be modulated from meprin
selectivity to a preference for meprin inhibition.
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