Modification of the N-terminal sulfonyl residue in 3-amidinophenylalanine-based matriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.019

Replacement of the N-terminal β-alanyl-amide moiety in previously identified matriptase inhibitors by non-charged aryl groups caused a slightly decreased potency and partially reduced selectivity, especially towards thrombin. However, some of these analogues are still potent matriptase inhibitors with K_i-values <10 nM. In contrast, improved activity was observed for newly designed tribasic analogues, especially for compound 21, which inhibits matriptase with an K_i-value of 80 pM.

Full text of DOI:10.1016/j.bmcl.2008.11.019

Bioorganic & Medicinal Chemistry Letters 19 (2009) 67–73
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modification of the N-terminal sulfonyl residue in
3-amidinophenylalanine-based matriptase inhibitors
a
a
a
a
Torsten Steinmetzer a,b, , Daniel Dönnecke , Martin Korsonewski , Claudia Neuwirth , Peter Steinmetzer ,
*
Alexander Schulze a, Sebastian Martin Saupe b, Andrea Schweinitz a
a Curacyte Discovery GmbH, Deutscher Platz 5d, D-04103 Leipzig, Germany
b Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Replacement of the N-terminal b-alanyl-amide moiety in previously identified matriptase inhibitors by
non-charged aryl groups caused a slightly decreased potency and partially reduced selectivity, especially
towards thrombin. However, some of these analogues are still potent matriptase inhibitors with Ki-values
<10 nM. In contrast, improved activity was observed for newly designed tribasic analogues, especially for
compound 21, which inhibits matriptase with an Ki-value of 80 pM.
Received 7 October 2008
Accepted 7 November 2008
Available online 13 November 2008
Keywords:
Matriptase
Ó 2008 Elsevier Ltd. All rights reserved.
Serine protease
Protease inhibitor
Anticancer drugs
Suzuki coupling
Matriptase, also named MT-SP1, TADG-15, or ST14, is a multi-
domain type II transmembrane serine protease1 with trypsin-like
activity. It was originally isolated from human breast cancer cells2
and in an inhibited form bound to endogenous hepatocyte growth
factor activator inhibitor-1 (HAI-1) from human milk.3 The prote-
ase is expressed in the epithelial cells of most organ systems, and
experiments with knockout mice revealed that matriptase is re-
quired for postnatal survival.4 The lack of matriptase leads to dis-
turbed epithelial development manifested by rapid dehydration
and impaired hair follicle formation.
Interestingly, matriptase is also consistently overexpressed in
epithelial cells on the surface of tumors of different origins, sug-
gesting a specific role of the protease in carcinogenesis.5 A modest
overexpression of matriptase in the epidermis of transgenic mice
was sufficient for the induction of spontaneous squamous cell car-
cinomas and strongly potentiated chemical skin carcinogenesis.6
Meanwhile, several potential substrates of matriptase were identi-
fied, which might be involved in invasion and metastasis. Matrip-
tase is a very potent activator of the proform of hepatocyte growth
factor, also called scatter factor (pro-HGF/SF). HGF/SF acts as a ma-
jor motility factor and induces scattering of cells through binding
and activation of its cell surface receptor c-Met.7 In vitro, matrip-
tase also converts pro-uPA into enzymatically active uPA, another
trypsin-like serine protease that is probably involved in tumori-
genesis.8 Other important substrates could be the protease-acti-
vated receptor-2 (PAR2),9 or the insulin-like growth factor
binding protein related protein-1 (IGFBP-rP1).10,11 On these
grounds, matriptase may be considered to be a potential target
for the development of anti-cancer drugs. Meanwhile, several
groups have described first results on the development of synthetic
matriptase inhibitors derived from bis-benzamidines, tripeptide
mimetics with a C-terminal arginal or analogues of the sunflower
trypsin inhibitor.12
We have recently reported a series of matriptase inhibitors
based on secondary amides of sulfonylated 3-amidinophenylala-
nine.13 Compounds such as 1 are relatively selective for matriptase
with inhibition constants below 10 nM and possess only weak
affinity towards other trypsin-like serine proteases.
O
O
H
N
H2N
N
H
S
N
O2
NH2
NH2
1
HN
The modelled complex of inhibitor 1 in matriptase revealed a
binding of the b-alanyl amino group into the characteristic S3/S4
site formed by the aromatic side chains of Trp215, Phe99, and
Phe97.13 We assumed that the high potency of this inhibitor
* Corresponding author. Tel.: +49 6421 2825900; fax: +49 6421 2825901.
E-mail address: steinmetzer@staff.uni-marburg.de (T. Steinmetzer).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.019

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T. Steinmetzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 67–73
could be related to the formation of hydrogen bonds to carbonyl
oxygen atoms of matriptase or cation-
-interactions14 from the
inhibitors shown in Table 1 have low potency towards the other
examined trypsin-like serine proteases.
p
protonated amino group to aromatic amino acids in this binding
pocket, as claimed for several factor Xa inhibitors.15 However, a
potential drawback of 1 is its multibasic character, which is
likely to limit its oral bioavailability. One approach to overcome
this problem could be the use of prodrug strategies, which are
well developed for masking benzamidines but less established
for primary amino groups. Most of the known amino prodrugs
are relatively stable to enzymatic conversion and therefore less
suitable in practice.16 During optimization of 1 we further ex-
plored the aromatic binding pocket of matriptase to identify
non-basic replacements for the b-alanine-containing sulfonyl
group, while other parts of the molecule were maintained. In
this paper we report our investigation on the activity and selec-
tivity profile of these inhibitors and will compare them with
some additional new analogues, which still contain a basic group
in the N-terminal residue.
Inspection of the model of inhibitor 1 in complex with matrip-
tase encouraged us to synthesize a series of bisaryl-3-sulfonyl de-
rived inhibitors, which could be prepared by Suzuki coupling
(Table 2, Scheme 4). The non-substituted biphenyl-3-sulfonyl
inhibitor 8 has only moderate activity, which could be slightly en-
hanced by a chlorine substitution, especially in para- and ortho-po-
sition of the terminal phenyl ring (9–11). A further increase in
matriptase inhibition was observed with the alkoxy derivatives
12–15 and the ethyl inhibitor 16, whereas the incorporation of
an indole or weakly basic pyridyl groups was less suitable (17–
19). The highest inhibitory potency was determined for 21, which
was initially unexpectedly obtained as a minor side product during
the hydrogenation of the p-aminopyridyl analogue 20. Due to their
high potency both compounds were resynthesized in pure S-con-
figuration at the central 3-amidinophenylalanine. All other com-
pounds shown in Table 2 (except 22, which was obtained using
commercially available 3-(2-methyl-4-pyrimidinyl)benzenesulfo-
nyl chloride) were synthesized as racemates.
Elimination of the peptide bond in 1 or cyclization to a 3-azeti-
dine-carboxyl group maintained high matriptase affinity (2–4,
Table 1), but did not change the tribasic character of the inhibitors.
In contrast, simple elimination or replacement of the amino group
by a hydroxyl residue was not accepted (5, 6), although a hydroxyl
group can act also as donor or acceptor in hydrogen bonds. All
During kinetic measurements we observed a reduced selectivity
for some of the bisaryl-3-sulfonyl analogues. Due to the close struc-
tural similarity to previously described thrombin inhibitors contain-
ing hydrophobic arylsulfonyl residues17 it was not astonishing that
Table 1
Inhibition of matriptase and other trypsin-like serine proteases13 by inhibitors of the general formula
O
H
N
R
S
N
O2
NH2
NH2
HN
Compound
R
Ki (lM)
Matriptase
0.0038
Plasmin
2.4
uPA
Thrombin
27
fXa
21
O
1
3.6
H2N
N
H
2
3
4
0.001
0.0067
0.003
0.20
0.46
0.50
3.8
5.2
3.7
0.46
2.2
4.4
H2N
H2N
8.0
N
H
O
5.1
11.2
N
H
HN
HO
O
5
6
7
0.120
0.180
0.041
2.6
3.5
2.1
3.0
8.1
3.2
5.5
1.9
1.2
61
20
14
N
H
O
O
N
H
O
N
H

T. Steinmetzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 67–73
69
Table 2
Inhibition of matriptase and other trypsin-like serine proteases by biphenyl-3-sulfonyl-derived inhibitors of the general formula
O
H
N
R
S
N
O2
NH2
NH2
HN
Compound
R
Ki (lM)
Matriptase
0.110
Plasmin
0.55
uPA
Thrombin
0.056
fXa
8
9
8.3
5.4
0.029
0.091
0.026
0.007
0.006
0.012
0.0054
0.0025
0.060
0.047
0.013
0.0016
0.00008
0.028
0.085
0.334
0.183
0.12
0.15
0.08
0.23
0.214
0.11
1.0
12
0.011
0.051
0.01
0.04
0.45
0.24
1.1
0.70
Cl
Cl
Cl
10
11
12
13
14
15
16
17
18
19
20*
21*
12
5.6
11
0.91
0.36
0.66
3.4
10
O
8.6
9.6
14
O
O
O
O
0.42
0.63
1.6
6.9
3.7
11
0.033
0.16
0.15
0.19
0.11
0.52
0.31
N
N
3.5
0.38
0.23
0.028
0.21
6.2
3.6
6.6
8.7
0.15
0.25
0.01
1.7
N
H
N
H2N
N
H2N
N
N
22*
*
Inhibitors 20–22 contain S-3-amidinophenylalanine, all other inhibitors shown in Table 2 were synthesized as racemates.

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T. Steinmetzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 67–73
some of the compounds (9, 11, 12, 16) inhibited also thrombin with
Ki-values <50 nM. In contrast, the most potent matriptase inhibitor
of this series (21) also inhibits plasmin and especially factor Xa,
aminoethyl group is directed to a so-called ‘cation cleft’ of matrip-
tase, however, no direct interactions were found from this part of
the inhibitor.
probably due to cation-
6-amino-2,3,4,5-tetrahydropyridin-3-yl group.
p
-interactions induced by the terminal basic
In conclusion, although the replacement of the basic sulfonyl
residue in 1 by neutral residues resulted in some loss of activity
and partially reduced selectivity, we could identify several potent
analogues with an acceptable overall profile as matriptase inhibi-
tors (12, 13, 15, 20). The strongest potency with a Ki-value of
80 pM was found for 21: however, this analogue still has a tribasic
character. Further modifications describing the replacement of the
C-terminal aminoethyl group with non-basic residues will be re-
ported in a following publication.
Chemistry: Intermediates 31a–d were prepared from the appro-
priate sulfonyl protected 3-cyanophenylalanines (29a–c) by cou-
pling with the Boc-protected piperidide 30 (Scheme 1).13 The
iodo compound 31b was coupled under Heck conditions20 with
Cbz-protected aminobutene 32 (yield ꢀ30%). After hydrogenation
of the double bond, the nitrile 33 was converted into the amidine
2 by standard procedures as described previously, followed by
the final cleavage of the Boc group (Scheme 2).13 Inhibitor 3 was
synthesized by reductive alkylation of 31d with tert-butyl 3-oxo-
propylcarbamate (obtained from Boc-b-alanyl-N-methoxy-N-
methylamide by reduction with LiAlH4)21 and NaBH3CN, followed
by conversion of the nitrile to the amidine according to Scheme
2 (steps c and d).
In an additional series, we modified the P1 residue (Table 3).
Replacement of the 3-amidine by a 3-aminomethyl group (23,
28) led to approximately 20-fold reduced potency, most likely
due to partial elimination of important hydrogen/salt bonds to
Asp189, Gly219, and Ser190 at the bottom and both sides of the
S1 pocket. Further reduced potency was observed with the 4-ami-
nomethyl inhibitor 24, although the incorporation of S-4-aminom-
ethylphenylalanine in closely related thrombin inhibitors was well
tolerated.18 Inhibitors 26–27 and the reference 25 were prepared
to probe the effects of an elongation of the P1 side chain. The sim-
ple elimination of the amidino group results in a more than a 1000-
fold weaker matriptase inhibition (25), whereas the incorporation
of additional methylene groups completely abolished any inhibi-
tory effect on matriptase and is thus not suitable.
The model of matriptase complexed with inhibitor 21 shows a
similar binding mode as observed in the X-ray structures obtained
for related compounds (Fig. 1).19 The P1 amidino group binds to
the carboxyl group of Asp189 (2.7 and 2.9 Å), the side chain OH of
Ser190, and CO of Gly219 (2.7 and 3.0 Å). In addition, both H-bonds
between the backbone of the P1 amino acid with Gly216 NH and CO
were detected (3.1 and 2.7 Å), as well as the typical interaction be-
tween one sulfonyl oxygen and the NH of Gly219 (2.9 Å). The N-ter-
minal amino group makes a H-bond to the carbonyl of Phe97 (2.7 Å),
whereas the ring nitrogen of the tetrahydropyridin is probably in-
The acylated derivatives (4–7) were obtained by coupling of the
commercially available Boc-3-azetidine-3-carboxylic acid 34 or 3-
tert-butoxypropanoic acid as mixed anhydrides (for inhibitors 4
and 5) or as appropriate acid chlorides (for 6 and 7) to the ani-
line-like intermediate 31d (Scheme 3), followed by the formation
of the amidine according to Scheme 2.
volved in a cation-p-interaction to Trp215. Both interactions might
be responsible for the improved affinity of 21. The C-terminal
Table 3
Modification of P1 inhibition of matriptase and other trypsin-like serine proteases by inhibitors of the general formula
O
H
N
R
S
N
O2
NH2
R1
Compound
R
R1
Ki (lM)
Matriptase
Plasmin
16
uPA
Thrombin
>1000
fXa
92
O
O
H2N
23
24
0.056
2.8
>1000
H2N
H2N
N
H
64.8
n.d.
n.d.
n.d.
>1000
n.d.
325
n.d.
n.d.
n.d.
430
n.d.
n.d.
n.d.
N
H
H2N
O
O
O
25
26
27
10
H2N
H2N
H2N
N
H
>1000
>1000
n.d.
N
H
n.d.
N
H
28*
0.145
0.776
129
9.8
55
H2N
O
*
Inhibitor contains racemic d/l-3-aminomethyl-phenylalanine, n.d., not determined.

T. Steinmetzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 67–73
71
Figure 1. Stereo view of the complex between 21 and matriptase.19
HN
Boc
N
O
O
H
H
N
H
N
30
R
S
OH
R
S
N
a
O2
O2
Boc
N
H
CN
CN
b
29a: R = NO2
29b: R = I
31a: R = NO2
31b: R = I
31d: R = NH2
29c: R = Br
31c: R = Br
Scheme 1. Reagents and conditions: (a) PyBop, 2.0 equiv DIPEA in DMF, 15 min, 0 °C and 3 h, rt; (b) zinc powder in acetic acid, 2 h, rt.
O
H
Cbz
N
N
H
S
O2
N
Boc
a, b
Cbz
N
H
N
H
32
CN
33
O
H
N
H2N
S
O2
N
NH2
c, d
NH2
HN
2
Scheme 2. Reagents and conditions: (a) i—addition of 1.1 equiv 32 and 1 equiv tetra-n-butylammonium chloride to a mixture of 0.9 equiv 31b, 3 equiv NaHCO3, and a
catalytic amount Pd(OAc)2 in 60% DMF in H2O, stirring 14 h at 50 °C; ii—preparative RP-HPLC provides oily product (double peak in HPLC with a ratio of approximately 3:1);
(b) H2 and Pd/C in ethyl acetate overnight; product identified as single peak in RP-HPLC; (c) i—2 equiv hydroxylamine  HCl and 2 equiv DIPEA, reflux in ethanol, 4 h, stirring
overnight at room temperature, followed by evaporation of ethanol, ii—2 equiv Ac2O in acetic acid, 30 min, rt; iii—H2 and Pd/C as catalyst in 90% acetic acid, stirring overnight
at 30 °C; (d) 90% trifluoroacetic acid, rt, 1 h, evaporation, and purification by preparative HPLC.22

72
T. Steinmetzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 67–73
The inhibitors shown in Table 2 were assembled from commer-
coupling conditions at elevated temperature, which would be dif-
ficult to detect in case of enantiomers. However, no peak splitting
in RP-HPLC was observed during the preparation of similar diaste-
cially available boronic acids or pinacol esters to 31c (examples in
Scheme 4) using approximately 1 mol% Pd(OAc)2 and 2 mol% S-
Phos in toluene in the presence of Cs2CO3 solution.23 For less effi-
cient Suzuki couplings during synthesis of analogues 17–20, the
reactions were performed in a microwave (type Discover, CEM)
at 90 °C in THF for 2 h. The conversion into the amidine was com-
pleted according to the procedures described in Scheme 2. How-
ever, in case of the chlorine-containing inhibitors (9–11), Zn
powder in acetic acid was used instead of H2 Pd/C to avoid dechlo-
rination of the terminal phenyl ring during reduction of the acety-
lhydroxyamidine group.13
reomeric analogs containing L- or D-prolinamide as secondary
amide moiety instead of the aminoethylpiperidide (data not shown
in this paper), which indicates that most likely no racemization oc-
curred under the used conditions. Therefore, the most potent
inhibitors 20, 21 of this series were resynthesized starting form
S-3-cyanophenylalanine. In this case, the aminopyridyl intermedi-
ate obtained after Suzuki coupling was Boc-protected using
(Boc)2O to avoid acetylation during the standard conversion into
the amidine, as described in Scheme 2 (step c, ii).
Initially, all Suzuki couplings were performed using racemic
31c, because we were afraid that a significant racemization could
occur on the phenylalanine moiety during the strongly basic
The aminomethyl group of inhibitors 23 and 24 was prepared
according to Scheme 5, followed by coupling of Boc-b-Ala-OH
using the mixed anhydride procedure, and conversion to the ami-
O
O
H
N
N
H
S
N
a
O2
N
Boc
N
COOH
Boc
Boc
N
H
34
CN
35
Scheme 3. Reagents and conditions: (a) i—N-methyl-morpholine, isobutyl chloroformate À15 °C, 10 min in DMF, ii—addition of 31d, 1 h at 0 °C, overnight at room
temperature.
O
H
OH
B
N
S
N
a
O2
OH
Boc
O
N
H
O
O
O
CN
O
H
N
O
B
S
N
b, c
O2
O
Boc
Boc
N
H
N
N
H
H2N
N
CN
Scheme 4. Reagents and conditions: (a) 2 equiv boronic acid per 1 equiv racemic 31c, 1 mol% Pd(OAc)2 and 2 mol% S-Phos in toluene containing 3 equiv 2 M Cs2CO3 solution,
3–4 h reflux. (b) 2 equiv of boronic acid pinacol ester per 1 equiv of 31c in S-configuration, 1 mol% Pd(OAc)2 and 2 mol% S-Phos in THF containing 3 equiv 2 M Cs2CO3 solution,
2 h in microwave at 90 °C. (c) 1.15 equiv (Boc)2O, DIPEA in tert-butanol.
O
O
H
N
H
N
H2N
S
O2
OH
H2N
S
N
O2
b, c
a
Boc
29a
N
H
Boc
H2N
N
H
Scheme 5. Reagents and conditions: (a) H2 and Pd/C as catalyst in 90% acetic acid, stirring 48 h at 35 °C; (b) 1.05 equiv (Boc)2O, DIPEA (pH 8.5–9) in dioxane/water. (c) 30,
PyBop, 2.0 equiv DIPEA in DMF, 15 min 0 °C and 3 h, rt.

T. Steinmetzer et al. / Bioorg. Med. Chem. Lett. 19 (2009) 67–73
73
Duncan, D.; Moreno, O.; Madison, E. L.; Agus, D. B. The prostate 2004, 61, 228;
(c) Long, Y.-Q.; Lee, S.-L.; Lin, C.-Y.; Enyedy, I.; Wang, S.; Li, P.; Dickson, R. B.;
Roller, P. P. Bioorg. Med. Chem. Lett. 2001, 11, 2515; (d) Jiang, S.; Li, P.; Lee, S.-L.;
Lin, C. Y.; Long, Y.-Q.; Johnson, M. D.; Dickson, R. B.; Roller, P. P. Org. Lett. 2007,
9, 9; (e) Li, P.; Jiang, S.; Lee, S.-L.; Lin, C. Y.; Johnson, M. D.; Dickson, R. B.;
Michejda, C. L.; Roller, P. P. J. Med. Chem. 2007, 50, 5976.
dine. In case of analogue 28, the hydrogenation of the cyano- to the
aminomethyl group was performed in the final step.
Acknowledgment
13. Steinmetzer, T.; Schweinitz, A.; Stürzebecher, A.; Dönnecke, D.; Uhland, K.;
Schuster, O.; Friedrich, R.; Than, M. E.; Bode, W.; Stürzebecher, J. J. Med. Chem.
2006, 49, 4116.
14. Mecozzi, S.; West, A. P., Jr.; Dougherty, A. Proc. Natl. Acad. Sci. 1996, 93, 10566.
15. (a) Lin, Z.; Johnson, M. E. FEBS Lett. 1995, 370, 1; (b) Gallivan, J. P.; Dougherty, D.
A. Proc. Natl. Acad. Sci USA 1999, 96, 9459.
16. (a) Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. J. Med. Chem. 2004, 47,
2393; (b) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen,
T.; Savolainen, J. Nat. Rev. Drug Disc. 2008, 7, 255.
17. Stürzebecher, J.; Prasa, D.; Hauptmann, J.; Vieweg, H.; Wikström, P. J. Med.
Chem. 1997, 40, 3091.
18. Lee, K.; Jung, W.-H.; Park, C. W.; Hong, C. Y.; Kim, I. C.; Kim, S.; Oh, Y. S.; Kwon,
O. H.; Lee, S.-H.; Park, H. D.; Kim, S. W.; Lee, Y. H.; Yoo, Y. J. Bioorg. Med. Chem.
Lett. 1998, 8, 2563.
19. Matriptase coordinates were taken from pdb entry 2gv6, the programm Sybyl
7.3 (Tripos, St. Louis, MO) was used for building the inhibitor and assigning
atom types. Docking was performed using the programm AutoDock 4 (Morris,
G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A.
J. J. Comp. Chem. 1998, 19, 1639) and the graphic interface AutoDock Tools
(Sanner, M.F. J. Mol. Graphics Mod. 1999, 17, 57). The picture was created using
Accelrys DS Visualizer 2.0.
The authors thank Robert Etges for correcting the English ver-
sion of the manuscript.
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after lyophilisation. The inhibitors were characterized by mass spectrometry
and analytical reversed phase HPLC as described in reference 13.
23. Barder, T. E.; Walker, S. D.; Martinelli, J. R.; Buchwald, S. L. J Am. Chem. Soc.
2005, 127,
4685.