Piperidine carbamate peptidomimetic inhibitors of the serine proteases HGFA, matriptase and hepsin

Article abstract of DOI:10.1039/c9md00234k

Matriptase and hepsin are type II transmembrane serine proteases (TTSPs). Along with related S1 trypsin like serine protease HGFA (hepatocyte growth factor activator), their unregulated proteolytic activity has been associated with cancer including tumor progression and metastasis. These three proteases have two substrates in common, hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), the ligands for MET and recepteur d'origine nantais (RON) receptor tyrosine kinases. Mechanism-based tetrapeptide and benzamidine inhibitors of these proteases have been shown to block HGF/MET and MSP/RON cancer cell signaling. Herein, we have rationally designed a new class of peptidomimetic hybrid small molecule piperidine carbamate dipeptide inhibitors comparable in potency to much larger tetrapeptides. We have identified multiple compounds which have potent activity against matriptase and hepsin and with excellent selectivity over the off-target serine proteases factor Xa and thrombin.

Full text of DOI:10.1039/c9md00234k

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Piperidine Carbamate Peptidomimetic Inhibitors of the Serine Proteases
HGFA, Matriptase and Hepsin
Vishnu C. Damalanka1, Scott A. Wildman2, James W. Janetka1,*
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St.
Louis, Missouri USA
2University of Wisconsin Carbone Cancer Center, Drug Development Core, University of Wisconsin-
Madison, Madison, Wisconsin USA
*Corresponding Author
Email: janetkaj@wustl.edu; Phone: 314-362-0509
Abstract
Matriptase and hepsin are type II transmembrane serine proteases (TTSPs). Along with related S1
trypsin like serine protease HGFA (Hepatocyte Growth Factor Activator), their unregulated
proteolytic activity has been associated with cancer including tumor progression and metastasis.
These three proteases have two substrates in common, hepatocyte growth factor (HGF) and
macrophage stimulating protein (MSP), the ligands for MET and recepteur d’origine nantais
(RON) receptor tyrosine kinases. Mechanism-based tetrapeptide and benzamidine inhibitors of
these proteases have been shown to block HGF/MET and MSP/RON cancer cell signaling. Herein,
we have rationally designed a new class of peptidomimetic hybrid small molecule piperidine
carbamate dipeptide inhibitors comparable in potency to much larger tetrapeptides. We have
identified multiple compounds which have potent activity against matriptase and hepsin and with
excellent selectivity over the off-target serine proteases factor Xa and thrombin.
1

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Introduction
Aberrant cell signaling via overexpression of cell surface receptors and the unregulated
overproduction of their cytokines and growth factor ligands have been associated with tumor
progression and metastasis in multiple tumor types1-4. Hepatocyte growth factor (HGF) is the only
known activating ligand for the receptor MET and likewise macrophage stimulating protein (MSP)
is the only ligand for RON5-7. MET and RON are structurally related receptor tyrosine kinases
(RTKs) which stimulate intracellular kinases leading to multiple downstream effects including
EMT (epithelial mesenchymal transition) in addition to cell proliferation, survival, and motility8-
13
.
The growth factors HGF and MSP are secreted as single-chain inactive zymogens, pro-HGF and
pro-MSP which require post-translation proteolytic processing to produce a two-chain
heterodimeric form14 to activate their respective kinase receptors, MET and RON14-17. Several S1
trypsin-like serine proteases have been shown to proteolytically activated HGF and MSP however,
the three proteases matriptase, hepsin, and HGFA (HFG-Activator) are the three most efficient 7,
18-20. This is reflected in the selectivity profile of the endogenous polypeptide inhibitors HAI-1 and
HAI-2 which potently inhibit only these three proteases 20-23, Therefore, specifically targeting these
three proteases with so-called ‘triplex’ inhibitors would block all HGF and MSP ligand-mediated
tumor cell signaling of MET and RON24-27
.
We have recently reported substrate-based22, 28-29 covalent inhibitors of HGFA, matriptase, and
hepsin containing electrophilic heterocyclic ketone warheads (Figure 1) which react with the
active-site serine reversibly. We have also developed a series of small molecule peptidomimetic
24
benzamidine inhibitors . Others have reported on both peptide-based30-32 and small molecule
2

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benzamidine33-35 inhibitors of matriptase and hepsin including an interesting series of cyclic urea
benzamidines exemplified by SRI-31215 (Figure 1)33. In this present manuscript, we describe our
continuing efforts to develop optimized mechanism-based α-ketobenzothiazole inhibitors (kbt)
inhibitors (Figure 1) with improved drug-like characteristics.
HN
NH2
NH
HN
NH2
O
HN
NH2
H2N
O
O
S
O
S
NH
O
O
NH
O
O
O
S
H
H
N
N
N
N
N
N
N
H
H
N
O
O
O
N
O
NH
1a
HGFA IC50 = 60 nM
NH2
O
NH
Matriptase IC50 = 1.1 nM
Hepsin IC50 = 0.17 nM
Br
Thrombin IC50 >20,000 nM
Factor Xa IC50 = 260 nM
1c
1d
HGFA IC50 =11 µM
HGFA IC50 = 30 µM
Matriptase IC50 = 8.4 µM
Hepsin IC50 = 0.09 µM
Matriptase IC50 = 0.24 µM
Hepsin IC50 = 14 µM
HN
NH2
NH2
NH
S
O
O
H
N
H
N
N
N
N
N
NH
H
H
O
O
O
N
N
HO
NH2
O
1b
SRI 31215
HGFA IC50 = 66 nM
Matriptase IC50 = 6.1 nM
Hepsin IC50 = 0.32 nM
Thrombin IC50 >20,000 nM
Factor Xa IC50 = 3800 nM
Matriptase IC50 = 3.2 µM
Hepsin IC50 = 1.9 µM
HGFA IC50 = 20 µM
Thrombin IC50 = 11.6 µM
Factor Xa IC50 = 0.38 µM
Figure 1. Structures of tetrapeptide α-ketobenzothiazole (kbt) inhibitors based on the sequences
of the pro-HGF (1a) and pro-MSP (1b) and peptidomimetic benzamidine serine protease
inhibitors, SRI 31215, 1c, and 1d of HGFA, matriptase and hepsin.
3

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Results and Discussion
Rational design of P3-P4 hybrid piperidine carbamate dipeptide inhibitors
There are several disadvantages associated with peptide-derived inhibitors as drugs, including high
conformational flexibility, susceptibility to proteolytic degradation leading to high clearance and
low half-life, and poor membrane permeability resulting is low oral bioavailability36-37. The
attributes of peptides leading to their poor drug-like properties stem from several reasons including
their high molecular weight, large number of amide bonds susceptible to enzymatic hydrolysis
also resulting in high polarity and multiple H-bond-donors and acceptors make it problematic for
cell permeability. Reduction of peptidyl character of the drugs typically enhances the cellular
permeability, proteolytic stability, and oral bioavailability. 38-39 Thus, our goal in this present study
is to rationally design novel inhibitors of HGFA, matriptase and hepsin which have much less
peptide character. To that end, we introduced non-peptidyl functional groups into the P4 and P3
positions of the tetrapeptide inhibitors 1a and 1b, designed to make binding interaction in the
corresponding S4 and S3 subsite pockets of the three proteases.28
4

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Figure 2. Computational model of tetrapeptide inhibitor Ac-KQLR-kbt (1b; yellow) bound to
HGFA (white) matriptase (green) and hepsin (orange) and overlaid on peptidomimetic
benzamidine inhibitor SRI 31215 (blue).
The superposition of three protease structures: HGFA (PDB code 2WUC)40, matriptase (PDB code
2GV7)41 and hepsin (PDB code 1Z8G)42 and their ligands allows for the design of hybrid inhibitors
(Figure 2). SRI 31215, reported by Galemmo et al.43 is a non-peptide cyclic urea benzamidine
(cub) inhibitor of matriptase and hepsin which binds the S1, S3 and S4 pockets but not the S2.
When overlaid on 1b, the piperidine is positioned close to the P3 amino acid nitrogen suggesting
that a piperidine ring attached through a two-atom linker such as a carbamate from the P2 position
would place the piperidine in a similar position to that of SRI 31215. Our previous structure-
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activity relationship (SAR) studies and reported PS-SCL (positional scanning of substrate
combinatorial libraries) studies on matriptase20, 29, 44 , hepsin22, 44-45 and HGFA45 indicated that all
three proteases require substrates with an Arg (R) at the P1 and prefer Leu (L) at the P2 position
28-29. We hypothesize that the low potency of SRI 31215 is partly reflected by the lack of binding
in the S2 pocket (Leu of 1b). Based on this analysis and inspired by the SRI 31215 structure we
designed novel hybrid dipeptide inhibitors with the preferred Leu (L) in the P2 position, but which
contain the piperidine group of SRI 31215 in the P3 position installed via a carbamate from the P2
Leu as suggested by our model (Figure 2). In another set of analogs, we created a P4 position
library with alkyl or aryl sulfonyl substituents on the piperidine nitrogen to identify compounds to
access the S4 pocket with optimal substitution for both potency and selectivity for the individual
proteases.
Synthesis of P2-P1 Leu-Arg-kbt dipeptides capped with substituted piperidine carbamates
at the P3 and P4 positions.
Construction of the target compounds which were selected based on our computational binding
model of 1a, 1b and SRI 31215 is shown in Schemes 1 and 2. Shown in Scheme 1A, the leucine
amino acid isocyanate is prepared by refluxing leucine methyl ester hydrochloride with
trichloromethyl chloroformate (2a). The cyclohexyl alanine (Cha) isocyanate 2b is formed in a
similar fashion. As seen in Scheme 1B, Grignard reactions with commercially available tert-butyl
4-oxopiperidine-1-carboxylate gives 1-Boc-4-piperidinol derivatives 3a-c and likewise reaction
with tert-butyl 4-formylpiperidine-1-carboxylate gives 3d-e in good yield. Shown in Scheme 2A,
leucine isocyanate 2 is then treated with piperidinol derivatives (3a-e) yielding the corresponding
carbamate esters (4a-e) or 9a-b (Scheme 2B). Treatment of the carbamates 4a-e with dry HCl in
dioxane followed by reaction with alkyl sulfonyl chloride gave the corresponding sulfonamides
6

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(5a-h). Hydrolysis of substituted piperidine esters 5a-h or 9a-b (Scheme 2B) with LiOH in
aqueous THF provided the carboxylic acids (6a-m or 10a-b) which were then reacted with the Pbf
side chain protected Arg-kbt (7)28 using standard amide coupling conditions (EDC/HOBt or
HATU) to give piperidine dipeptides which were subjected to global side-chain deprotection with
a cocktail of TFA/water/thioanisole. Reverse phase preparatory HPLC purification was then
conducted to produce final target compounds (8a-m and 11a-b) in high purity.
Using the fluorogenic protease substrates, Boc-QAR-AMC (matriptase and hepsin) or Boc-QLR-
AMC (HGFA) in previously published kinetic enzyme assays22. We tested the activity of all target
compounds using eleven different concentrations of compound. Inhibitors were pre-incubated
with protease followed by the addition of the substrate. Inhibition of substrate proteolysis derived
fluorescence was monitored kinetically over a period of one hour. Shown in Table 1 are the
experimentally determined IC50 values of each compound for their concentration-dependent
inhibition of HGFA, matriptase, and hepsin.
The majority of compounds tested showed good activity and excellent selectivity for matriptase
and hepsin over HGFA (Table 1). The most potent inhibitors we identified are 8b and 8c which
have IC50 values 0.6 and 0.5 nM for hepsin and 30 and 70 nM for matriptase, respectively. We
also made compound 8d, which replaces the P2 Leu with the unnatural amino acid cyclohexyl
alanine (Cha) but found that the activity lowers for all three proteases (Table 1). We found that
inhibitors with substituted alkyl or aryl sulfonyl groups at R2/P4 position attached on the
piperidine ring nitrogen generally exhibit better potency against hepsin relative to matriptase. As
examples, ethyl sulfonyl (8h) and acetyl (8e) analogs showed the best activity of all aryl sulfonyl
analogs at the R2/P4 position, clearly demonstrating that smaller functional groups are preferred
in the S4 pocket of matriptase and hepsin.
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To introduce more flexibility into the piperidine carbamate we made the two additional analogs
11a-b which have a methylene spacer between the piperidine ring and the carbamate linker to the
Leu P2 position. In these analogs, the R1 substituent is now not a tertiary group on the piperidine
ring but rather a secondary group on the spacer. We found that these two inhibitors exhibit
excellent potency against both hepsin and matriptase with IC50 values 8 nM and 2 nM, respectively;
and also found moderate effect in inhibiting HGFA (IC50 values 14 µM and 6 µM) but less active
than the corresponding matched pairs 8a and 8b (IC50 values of 0.78 and 1.2 µM). Our SAR data
also revealed that adding the methylene linker between the aryl and carbamate group decreases the
activity against matriptase and hepsin. Similar to that found for 8a and 8b, the benzyl group on the
R1 position of the piperidine having an additional methylene spacer is optimal to the phenyl group
in 11a and 11b.
It is noteworthy that all hybrid piperidine dipeptides displayed only weak or no activity against
HGFA with the analogs 8a and 8b containing phenyl and benzyl groups at the R1 position having
the best IC50 values of 0.78 and 1.2 µM, respectively. Increasing the alkyl linker length between
the phenyl and piperidine ring as in compound 8c results in loss of any activity up to 20 µM.
Evaluation of the SAR derived from different sulfonyl and acyl groups at the R2 position showed
only moderate effects on HGFA activity. Interestingly, when tested in our enzyme assays, we
found that SRI 31215 only has weak activity for HGFA (IC50 20 µM). Furthermore, all other
benzamidine inhibitors reported to data 24, 33 show either no or weak potency for HGFA as well.
To determine the target selectivity profile, a handful of compounds were tested against the similar
trypsin-like serine proteases, factor Xa and thrombin. In general, we found that all compounds
tested had good selectivity over both factor Xa and thrombin. However, we found that 8c with an
acetyl on R2 and phenethyl group off of R1 had some inhibition of factor Xa (IC50 2.0 µM) and
8

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thrombin (IC50 7.7 µM). A notable piece of SAR is that that 8b which has no substitution at R2
and a shorter benzyl R1 showed no activity against thrombin (>20 µM) and a 4-fold higher IC50
relative to 8c.
Conclusion
In summary, we have developed a new class of hybrid piperidine carbamate dipeptidyl
ketobenzothiazole serine protease inhibitors based on tetrapeptide inhibitors and benzamidine
inhibitor SRI 31215 of matriptase and hepsin. These inhibitors replace the P4 and P3 amino acids
with non-peptide piperidine carbamates which span the S3-S4 sidechain-bindings pockets. The
piperidine moiety was appended with a variety of aryl and sulfonyl substituted groups off the
nitrogen to interact more specifically in the S4 pockets of matriptase and hepsin. Using this
strategy, we have identified multiple new potent inhibitors of matriptase and hepsin such as 8b,
8c, 8f, and 8h with excellent selectivity over factor Xa and thrombin. Excitingly, these new
dipeptide inhibitors of HGFA matriptase and hepsin approach the activity level of the tetrapeptides
1a and 1b. Therefore, we were successful in our goal here of rationally designing novel inhibitors
of the same potency but with much less peptide character. Current efforts are in progress to obtain
X-ray crystal co-structures of these compounds to aid in the further structure-based optimization.
Table 1. Biological activity and selectivity of hybrid piperidine dipeptide kbt HGFA, matriptase
and hepsin serine protease inhibitors, 8a-m and 11a-b.
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R2
N
R2
N
O
R
O
H
N
S
O
R
O
O
N
H
R1
H
N
S
O
N
R1
O
N
H
O
N
NH
NH
NH
NH
H2N
8
11
H2N
Matriptase Hepsin IC50
HGFA
Factor Xa
Thrombin
Compd
8a
R
R1
R2
IC50 (µM)a
(µM )a
IC50 (µM )a IC50 ( µM )a IC50 ( µM )a
Leu
H
0.09
0.005
0.78
1.2
NT
6.5
>20
>20
8b
8c
Leu
Leu
H
0.03
0.07
0.0006
0.0005
O
>20
2.0
7.7
8d
8e
Cha
Leu
H
2.1
0.09
0.02
>20
>20
9.0
NT
>20
NT
O
H
0.64
O
S
8f
Leu
Leu
H
H
0.81
4.5
0.02
0.07
8.6
18
>20
NT
>20
NT
O
F
O
S
8g
O
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O
S
8h
8i
Leu
Leu
H
H
0.73
2.4
0.01
>20
9.0
>20
NT
>20
O
O
S
0.06
0.07
NT
NT
Cl
O
O
S
8j
Leu
Leu
H
H
7.5
10
>20
>20
NT
NT
O
F
O
O2N
Cl
S
8k
0.11
0.06
NT
NT
O
NO2
O
S
8l
Leu
H
H
4.0
6.3
NT
O
O
O
S
8m
Leu
Leu
>20
1.1
0.11
8.8
14
NT
NT
O
11a
H
0.008
>20
>20
11b
Leu
H
0.02
0.002
6.0
NT
NT
NT = Not tested
aIC50 values are an average of three experiments
Scheme 1. A) Synthesis of amino acid isocyanates 2a-b and B) piperidine alcohols 3a-e.
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A
a
O
O
ClH.H2N
OCN
O
O
O
2a
O
a
OCN
ClH.H2N
O
O
2b
B
OH
R1
R1-MgCl
b
O
N
Boc
N
Boc
3 a-c
a = Phenyl;b = Benzyl; c = Phenethyl
OH
R1
R1-MgCl
b
N
Boc
N
Boc
O
3 d-e
d = Phenyl; e = Benzyl
Reagents: a) Tricholoromethyl chloroformate, 1,4-dioxane; b) Grignard reagent, THF
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Scheme 2. Synthetic route for hybrid piperidine dipeptide ketobenzothiazole (kbt) inhibitors of
matriptase, hepsin, and HGFA. *Synthesis of H-Arg(Pbf)-kbt-HCl 7 has been reported
previously28.
A
O
O
OH
R1
f
c, d
N
Boc
e
O
O
N
N
H
COOCH3
COOCH3
2a
BocN
4a-e
R N
5a-h
H
R1
R1
3a
R
N
O
O
O
H
N
g, h
S
O
N
COOH
O
N
H
R1
R N
H
O
N
7
R1
NH
6a-m
8a-m
HOOCF3C .H2N NH
B
Boc
Boc
N
O
O
N
OH
R1
c, d
f
N
Boc
O
O
N
N
H
COOCH3
COOH
2a
H
7
R1
R1
10a-b
3 b
9a-b
R
N
O
H
N
g, h
S
ClH .H2N
O
O
H
O
N
N
7
S
R1
O
N
H
O
N
NH
11a-b
(Pbf)HN NH
NH
HOOCF3C .H2N NH
7*
2a 2b
Reagents: c) Compd or , TEA, ACN; d) 4N HCl in dioxane; e) RSO2Cl, TEA, THF; f) aq. LiOH, THF; g) EDCI, HOBt, DIEA, DMF
or HATU, DIEA, DMF; h) TFA:water:thioanisole (95:2.5:2.5), followed by RP-HPLC
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Experimental.
General synthesis, purification, and analytical chemistry procedures. Starting materials,
reagents, and solvents were purchased from commercial vendors unless otherwise noted. 1H NMR
spectra were measured on a Varian 400 MHz NMR instrument. The chemical shifts were reported
as δ ppm relative to TMS using residual solvent peak as the reference unless otherwise noted. The
following abbreviations were used to express the multiplicities: s = singlet; d = doublet; t = triplet;
q = quartet; m = multiplet; br = broad. High-performed liquid chromatography (HPLC) was carried
out on GILSON GX-281 using Waters C18 5μM, 4.6*50mm and Waters Prep C18 5μM,
19*150mm reverse phase columns, eluted with a gradient system of 5:95 to 95:5 acetonitrile:water
with a buffer consisting of 0.05% TFA. Mass spectra (MS) were performed on HPLC/MSD using
electrospray ionization (ESI) for detection. All reactions were monitored by thin layer
chromatography (TLC) carried out on Merck silica gel plates (0.25 mm thick, 60F254), visualized
by using UV (254 nm) or dyes such as KMnO4, p-Anisaldehyde and CAM (Cerium Ammonium
Molybdate or Hanessian's Stain). Silica gel chromatography was carried out on a Teledyne ISCO
CombiFlash purification system using pre-packed silica gel columns (12g~330g sizes). All
compounds used for biological assays are greater than 95% purity based on NMR and HPLC by
absorbance at 220 nm and 254 nm wavelengths.
Synthesis of amino acid methyl ester isocyanates 2a-b. General procedure.
The leucine amino acid methyl ester hydrochloride (4.55 g, 25 mmol) was placed in a dry RB flask
and then dried overnight on the vacuum pump. The flask was flushed with nitrogen and dry
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dioxane (60 mL) was added followed by trichloromethyl chloroformate (7.42 g, 37.5 mmol). After
refluxing for 14 h, the solvent was removed on the rotary evaporator to yield pure isocyanates 2a-b
as colorless oils 46.
Synthesis of compounds 3a-e. General procedure.
To a 250 mL round bottom flask kept under nitrogen atmosphere was added a 2.0 M solution (1.40
mL, 2.76 mmol) of the appropriate Grignard reagent in THF and the solution was cooled to 0–
5°C. A solution of the appropriate aldehyde (0.5 g, 2.50 mmol) in dry THF (5 mL) was added
dropwise to the cooled Grignard solution over ∼ 20 min. The reaction mixture was allowed to
warm up to room temperature and stirred for 5 h under nitrogen. The disappearance of the aldehyde
was monitored by TLC. The reaction mixture was cooled to 0–5 °C and acidified to pH ∼3.0 using
5% aqueous hydrochloric acid. The organic solvent was evaporated off and the residue was
extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed with brine
(10 mL) dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified
by column chromatography to yield alcohols 3a-c and 3d-e as oils 46.
Synthesis of carbamates 4a-d and 9a-b. General procedure.
A solution of compound Boc-(4-hydroxy) piperidine (3) (2.02 g, 10 mmol) in dry acetonitrile
(20 mL) was treated with trimethylamine (2.02 g; 20 mmol) followed by an appropriate amino
acid methyl ester isocyanate 2 (11 mmol). The resulting reaction mixture was refluxed for 3 h and
then allowed to cool to room temperature. The disappearance of the alcohol was monitored by
TLC. The solvent was evaporated, and the residue was taken up in ethyl acetate (100 mL) and the
organic layer was washed with 5% aqueous HCl (2 × 20 mL) and saturated NaCl (20 mL). The
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organic layer was dried over anhydrous sulfate, filtered, and concentrated to yield an oily product.
Purification by flash chromatography yielded 4a-d and 9a-b esters as colorless oils/solids.
Dissolved the above purified compound 4d in dry DCM (5 mL) and added a solution of 4 M HCl
in dioxane (15 mL) with stirring. The reaction mixture was stirred for 2 h at room temperature.
The disappearance of the starting material was monitored by TLC. The solvent was evaporated
under reduced pressure and compounds 4e was used in the next step without further purification
46-47
.
Synthesis of sulfonyl compounds 5a-h. General procedure.
A Solution of compound 4e (100 mg, 0.32 mmol) in dry THF (3 mL) treated with triethyl amine
(0.13 mL, 0.64 mmol) followed by added appropriate sulfonyl chloride (63 mg, 0.32 mmol) while
stirring and continue the reaction for 12 h. Residue was dissolved in ethyl acetate (50 mL) and
washed with 5% HCl (2 X 20 mL) and saturated NaCl (20 mL), dried over sodium sulfate, filtered
and concentrated to yield crude product, which is purified by column chromatography yield the
corresponding esters 5a-h as colorless oils 48.
Synthesis of acids 6a-m and 10 a-b. General procedure.
A solution of ester 4 or 5/9 (0.28 g, 0.557 mmol) in tetrahydrofuran (2 mL) was treated with 1 M
aqueous LiOH (2 mL). The reaction mixture was stirred for 3 h at room temperature while
monitoring the disappearance of the ester by TLC. Most of the solvent was evaporated off and the
solution was acidified to pH ∼3 using 5% hydrochloric acid (2 mL). The aqueous layer was
extracted with ethyl acetate (2 × 25 mL) and the combined organic layer was washed with brine
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(10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to
yield compounds 6a-m and 10a-b.
Synthesis of compounds 8a-m and 11a-b. General procedure.
EDCI (0.28 mmol) and HOBt (0.28 mmol) were added to a solution of compound 6 or 10 (100
mg, 0.222 mmol) in dry DMF (2 mL) and the mixture was stirred for 30 min at room temperature
and cooled the reaction to 0–5 °C and added the Arg (Pbf)-kbt HCl (7) (129 mg, 0.222 mmol)
followed by DIEA (115 mg, 0.888 mmol) and stirred for 15 min. Allowed the reaction to room
temperature and stirred for 12 h at RT, the reaction was concentrated under reduced pressure and
the residue was partitioned between ethyl acetate (25 mL) and 10% citric acid (2 X 10 mL). The
layers were separated, and the ethyl acetate was further washed with aqueous NaHCO3 (10 mL),
saturated NaCl solution (10 mL). The organic layer was dried over Na2SO4, filtered, and
concentrated. The deprotection of the crude product was accomplished by stirring in 1.0 mL of a
TFA-thioanisole-water mixture (95:2.5:2.5) for 2-3 h. After concentrating in vacuo, the crude
material was dissolved in DMSO and purified using reverse phase HPLC (0.05%
TFA/acetonitrile/water gradient). The pure fractions were pooled, frozen and lyophilized to give
the pure dipeptides 8a-m and 11a-b as white powders.
4-phenylpiperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-oxopentan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8a):
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O
O
H
S
N
HN
O
N
H
N
O
NH
NH
H2N
White solid, Yield (65%), 1H NMR (400 MHz, CD3OD)  ppm = 8.23 - 8.20 (m, 1 H), 8.15 - 8.11
(m, 1 H), 7.68 - 7.59 (m, 1 H), 7.34 (d, J = 3.9 Hz, 3 H), 7.29 - 7.18 (m,4 H), 5.71 - 5.64 (m, 1 H),
3.88 - 3.79 (m, 1 H), 3.23 - 3.18 (m, 3 H), 3.12 (s, 1 H), 2.68 - 2.63 (m, 3 H), 2.44 (s, 2 H), 1.90 -
1.77 (m, 2 H), 1.60 -1.49 (m, 1 H), 1.26 (d, J = 6.7 Hz, 2 H), 1.07 - 0.88 (m, 6 H). LCMS (ESI+)
expected m/z 607.30, found 608.5 (M+H+).
4-benzylpiperidin-4-yl((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-oxopentan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8b):
O
O
H
S
N
HN
O
N
H
N
O
NH
NH
H2N
White solid, Yield (68%), 1H NMR (400MHz, CDCl3)  ppm = 8.17 (d, J = 7.8 Hz, 1 H), 7.96 (d,
J = 7.4 Hz, 1 H), 7.65 - 7.50 (m, 4 H), 6.94 (br. s., 2 H), 5.79 (br. s.,1 H), 4.76 (dd, J = 4.9, 10.8
Hz, 1 H), 4.15 (d, J = 5.5 Hz, 1 H), 3.45 (br. s., 2 H), 3.16 (br. s., 2 H), 2.81 (br. s., 4 H), 2.37 -
2.06 (m, 3 H), 1.93 -1.58 (m, 8 H), 1.61 - 1.38 (m, 3 H), 0.98 - 0.85 (m, 6 H). LCMS (ESI+)
expected m/z 621.31, found 622.4 (M+H+).
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1-acetyl-4-phenethylpiperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-
oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8c):
O
N
O
O
H
N
S
O
N
H
O
N
NH
NH
H2N
White solid, Yield (55%), 1H NMR (400 MHz, CHLOROFORM-d)  ppm = 8.19 (d, J=7.04 Hz,
1 H), 7.92 - 8.06 (m, 2 H), 7.43 - 7.76 (m, 7 H), 6.95 (br. s., 1 H), 5.79 (d, J=7.83 Hz, 2 H), 4.71 -
4.85 (m, 1 H), 4.04 - 4.26 (m, 2 H), 3.16 (br. s., 1 H), 2.11 - 2.49 (m, 8 H), 1.40 - 1.95 (m, 10 H),
0.83 - 1.03 (m, 6 H). LCMS (ESI+) expected m/z 677.34, found 587.5 (M-Bn+).
4-phenylpiperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-oxopentan-2-
yl)amino)-3-cyclohexyl-1-oxopropan-2-yl)carbamate (8d):
O
O
H
S
N
HN
O
N
H
N
O
NH
NH
H2N
White solid, Yield (60%), 1H NMR (400 MHz, CDCl3)  ppm = 8.20 (d, J=7.83 Hz, 2 H), 7.92 -
8.04 (m, 2 H), 7.75 (br. s., 1 H), 7.37 - 7.68 (m, 9 H), 6.97 (br. s., 2 H), 5.81 (br. s., 2 H), 4.70 -
4.88 (m, 2 H), 4.08 - 4.29 (m, 2 H), 3.41 - 3.63 (m, 1 H), 3.17 (br. s., 1 H), 2.51 (br. s., 2 H), 2.10
- 2.39 (m, 4 H), 1.43 - 1.93 (m, 8 H), 0.79 - 1.36 (m, 11 H). LCMS (ESI+) expected m/z 646.33,
found 647.5 (M+H+).
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1-acetylpiperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-oxopentan-2-
yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8e)
O
O
H
O
N
S
N
O
N
H
O
N
NH
NH
H2N
White solid, Yield (65%), 1H NMR (400MHz, DMSO-d6)  = 8.57 (d, J = 5.5 Hz, 1 H), 8.30 -
8.21 (m, 1 H), 7.68 (quin, J = 6.5 Hz, 1 H), 7.51 (br. s., 1 H), 7.25 (d, J = 8.2 Hz, 1 H), 5.41 (br.
s., 1 H), 4.65 (br. s., 1 H), 4.08 (d, J = 6.7 Hz, 1 H), 3.84 (d, J = 13.3 Hz, 1 H), 3.59 (br. s., 1 H),
3.24 (br. s., 1 H), 3.15 (d, J = 5.5 Hz, 2 H), 1.99 (s, 3 H), 1.75 (br. s., 2 H), 1.58 (dd, J = 6.3, 14.5
Hz, 3 H), 1.41 - 1.31 (m, 3 H), 0.82 (d, J = 6.7 Hz, 6 H). LCMS (ESI+) expected m/z 573.28, found
574.5 (M+H+).
1-(phenylsulfonyl)piperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-
oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8f)
O
O
H
O
N
S
S N
O
O
N
H
O
N
NH
NH
H2N
White solid, Yield (55%), 1H NMR (400MHz, CDCl3)  = 8.17 (d, J = 6.7 Hz, 1 H), 8.03 - 7.92
(m, 2 H), 7.83 (br. s., 1 H), 7.79 - 7.66 (m, 3 H), 7.64 - 7.50 (m, 4 H), 7.46 (d, J = 7.4 Hz, 1 H),
6.98 (br. s., 1 H), 5.83 - 5.71 (m, 1 H), 5.52 (d, J = 5.5 Hz, 1 H), 5.33 (br. s., 1 H), 4.64 (br. s., 2
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H), 4.16 (br. s., 1 H), 3.34 - 3.07 (m, 2 H), 2.88 (br. s., 2 H), 2.17 (br. s., 1 H), 1.91 (br. s., 3 H),
1.75 (br. s., 4 H), 1.57 (dt, J = 7.0, 15.7 Hz, 4 H), 0.89 (d, J = 6.7 Hz, 6 H). LCMS (ESI+) expected
m/z 671.26, found 672.4 (M+H+).
1-((2-fluorophenyl)sulfonyl)piperidin-4-yl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-
1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8g)
O
O
O
S N
O
H
S
N
F
O
N
H
N
O
NH
NH
H2N
White solid, Yield (60%), 1H NMR (400 MHz, CDCl3)  ppm = 8.16 (d, J=7.83 Hz, 1 H), 7.97 (d,
J=7.83 Hz, 1 H), 7.86 (br. s., 1 H), 7.70 (d, J=7.83 Hz, 1 H), 7.50 - 7.64 (m, 4 H), 7.45 (d, J=6.65
Hz, 1 H), 7.29 - 7.37 (m, 1 H), 6.88 (br. s., 1 H), 5.74 (br. s., 1 H), 5.33 (d, J=6.65 Hz, 1 H), 4.68
(br. s., 1 H), 3.84 - 4.28 (m, 5 H), 3.44 - 3.62 (m, 2 H), 3.24 (br. s., 4 H), 2.90 - 3.04 (m, 2 H), 2.17
(br. s., 2 H), 1.93 (m, 3 H), 1.76 (m, 2 H), 1.45 - 1.68 (m, 2 H), 0.77 - 1.01 (m, 6 H). LCMS (ESI+)
expected m/z 689.25, found 690.4 (M+H+).
1-(ethylsulfonyl)piperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-
oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8h):
O
O
O
S N
O
H
S
N
O
N
H
N
O
NH
NH
H2N
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White solid, Yield (62%), 1H NMR (400 MHz, CDCl3)  ppm = 8.18 (d, J=7.43 Hz, 1 H), 7.97 (d,
J=7.83 Hz, 1 H), 7.73 (d, J=7.43 Hz, 1 H), 7.46 - 7.65 (m, 4 H), 5.75 (br. s., 2 H), 5.55 (br. s., 1
H), 4.82 (br. s., 1 H), 4.22 (br. s., 1 H), 3.45 (br. s., 2 H), 3.05 - 3.31 (m, 4 H), 2.96 (q, J=7.30 Hz,
2 H), 2.10 - 2.28 (m, 3 H), 1.50 - 2.03 (m, 7 H), 1.34 (t, J=7.43 Hz, 3 H), 0.94 (d, J=7.04 Hz, 6 H).
LCMS (ESI+) expected m/z 623.26, found 624.4 (M+H+).
1-((4-chlorophenyl)sulfonyl)piperidin-4-yl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-
1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8i):
Cl
O
O
S
O
H
N
S
N
O
N
O
N
H
O
NH
NH
H2N
White solid, Yield (65%), 1H NMR (400 MHz, CDCl3) d ppm = 8.19 (d, J=7.43 Hz, 1 H), 8.00 (d,
J=7.04 Hz, 1 H), 7.44 - 7.75 (m, 5 H), 7.38 (d, J=7.04 Hz, 2 H), 6.93 (br. s., 1 H), 5.83 (br. s., 1
H), 5.42 (d, J=7.43 Hz, 1 H), 4.66 (br. s., 1 H), 4.15 (br. s., 1 H), 3.17- 3.41 (m, 6 H), 2.94 (br. s.,
4 H), 1.93 (m, 2 H), 1.76 (m., 4 H), 1.19 - 1.37 (m, 1 H), 0.75 – 0.93 (m, 6 H). LCMS (ESI+)
expected m/z 705.22, found 706.4 (M+H+).
1-((3-fluorophenyl)sulfonyl)piperidin-4-yl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-
1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8j):
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F
O
O
S
O
H
N
S
N
O
N
O
N
H
O
NH
NH
H2N
White solid, Yield (65%), 1H NMR (400 MHz, CDCl3)  ppm = 8.16 (d, J=7.83 Hz, 1 H), 7.97 (d,
J=7.83 Hz, 1 H), 7.86 (br. s., 1 H), 7.70 (d, J=7.83 Hz, 1 H), 7.50 - 7.64 (m, 4 H), 7.45 (d, J=6.65
Hz, 1 H), 7.29 - 7.37 (m, 1 H), 6.88 (br. s., 1 H), 5.74 (br. s., 1 H), 5.33 (d, J=6.65 Hz, 1 H), 4.68
(br. s., 1 H), 3.84 - 4.28 (m, 5 H), 3.44 - 3.62 (m, 2 H), 3.24 (br. s., 4 H), 2.90 - 3.04 (m, 2 H), 2.17
(br. s., 2 H), 1.93 (m, 3 H), 1.76 (m, 2 H), 1.45 - 1.68 (m, 2 H), 0.77 - 1.01 (m, 6 H). LCMS (ESI+)
expected m/z 689.25, found 690.5 (M+H+).
1-((2,4-dinitrophenyl)sulfonyl)piperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-
guanidino-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8k):
NO2
O2N
O
O
S
O
H
N
S
N
O
N
O
N
H
O
NH
NH
H2N
Off-white solid, Yield (60%), 1H NMR (400MHz , CDCl3)  ppm = 8.75 - 8.66 (m, 1 H), 8.48 -
8.45 (m, 1 H), 8.19 (s, 3 H), 8.03 - 7.98 (m, 2 H), 7.67 - 7.53 (m, 5 H), 7.16 - 7.06 (m, 1 H), 5.33
- 5.27 (m, 1 H), 3.95 - 3.88 (m, 1 H), 3.52 - 3.47 (m, 1 H), 2.68 (s, 2 H), 1.87 (br. s., 52 H), 1.02 -
0.87 (m, 6 H). LCMS (ESI+) expected m/z 761.23, found 762.5 (M+H+).
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1-((5-chloro-2-methoxyphenyl)sulfonyl)piperidin-4-yl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-
5-guanidino-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8l):
OMe
O
Cl
S
O
N
O
O
H
N
S
O
N
H
O
N
NH
NH
H2N
White solid, Yield (63%), 1H NMR (400 MHz, CDCl3)  ppm = 8.12 - 8.24 (m, 1 H), 7.71 - 8.02
(m, 4 H), 7.40 - 7.65 (m, 4 H), 6.95 (d, J=8.61 Hz, 1 H), 5.60 - 5.85 (m, 1 H), 5.46 (d, J=7.04 Hz,
1 H), 4.74 (br. s., 1 H), 3.96 - 4.28 (m, 7 H), 3.90 (s, 2 H), 3.47 (d, J=4.70 Hz, 2 H), 3.13 (m., 2H),
2.18 (br. s., 2 H), 1.49 - 2.00 (m, 8 H), 0.91 (d, J=6.78 Hz, 6 H). LCMS (ESI+) expected m/z
735.24, found 736.4 (M+H+).
1-(mesitylsulfonyl)piperidin-4-yl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-
oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (8m):
O
S
N
O
O
O
H
N
S
O
N
H
O
N
NH
NH
H2N
White solid, Yield (64%), 1H NMR (400 MHz, CDCl3)  ppm = 8.17 (d, J=7.04 Hz, 1 H), 7.97 (t,
J=6.26 Hz, 2 H), 7.76 (d, J=5.09 Hz, 1 H), 7.50 - 7.65 (m, 4 H), 6.94 (s, 2 H), 5.68 - 5.86 (m, 1
H), 4.77 (br. s., 1 H), 4.21 (br. s., 1 H), 3.01 - 3.57 (m, 6 H), 2.58 (s, 3 H), 2.30 (s, 3 H), 2.19 (s, 2
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H), 1.50 - 1.94 (m, 6 H), 0.91 (d, J=6.39 Hz, 6 H). LCMS (ESI+) expected m/z 713.31, found
714.5 (M+H+).
phenyl(piperidin-4-yl)methyl ((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-oxopentan-
2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (11a)
O
O
O
N
O
H
HN
HN
S
N
NH
NH2
HN
White solid, Yield (50%), 1H NMR (400 MHz, CD3OD)  ppm = 8.23 - 8.20 (m, 1 H), 8.15 - 8.11
(m, 1 H), 7.68 - 7.59 (m, 1 H), 7.34 (d, J = 3.9 Hz, 3 H), 7.29 - 7.18 (m,4 H), 5.71 - 5.64 (m, 1 H),
3.88 - 3.79 (m, 1 H), 3.23 - 3.18 (m, 3 H), 3.12 (s, 1 H), 2.68 - 2.63 (m, 3 H), 2.44 (s, 2 H), 1.90 -
1.77 (m, 2 H), 1.60 -1.49 (m, 1 H), 1.26 (d, J = 6.7 Hz, 2 H), 1.07 - 0.88 (m, 6 H). ). LCMS (ESI+)
expected m/z 621.32, found 622.5 (M+H+).
2-phenyl-1-(piperidin-4-yl)ethyl
((S)-1-(((S)-1-(benzo[d]thiazol-2-yl)-5-guanidino-1-
oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (11b)
H
N
O
O
H
N
S
O
N
H
O
N
NH
NH
H2N
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White solid, Yield (52%), 1H NMR (400MHz, DMSO-d6)  = 8.65 - 8.56 (m, 1 H), 8.41 (s, 1 H),
8.36 (d, J = 4.7 Hz, 1 H), 8.30 - 8.23 (m, 2 H), 7.68 (dd, J = 2.3, 5.1 Hz, 2 H), 7.53 - 7.44 (m, 1
H), 7.29 (d, J = 8.2 Hz, 1 H), 7.18 (d, J = 8.2 Hz, 1 H), 5.52 - 5.35 (m, 1 H), 4.55 - 4.45 (m, 1 H),
4.06 - 3.96 (m, 1 H), 3.56 (d, J = 11.0 Hz, 1 H), 3.18 - 3.11 (m, 2 H), 2.42 (s, 2 H), 1.97 (d, J = 5.1
Hz, 1 H), 1.74 (dd, J = 3.9, 9.4 Hz, 3 H), 1.65 - 1.51 (m, 3 H), 1.40 - 1.29 (m, 2 H), 0.86 (d, J =
2.3 Hz, 6 H). ). LCMS (ESI+) expected m/z 635.33, found 544.4 (M-Bn+).
Computational methods.
The published crystal structures of HGFA (PDB code 2WUC)40, matriptase (PDB code 2GV7)41
and hepsin (PDB code 1Z8G)42 were superimposed based on sequence alignment in MOE
(Chemical Computing Group, v2014.09). The HGFA and hepsin structures include the peptide-
based inhibitor Ace-KQLR-chloromethylketone. The kbt-peptide inhibitors were separately
modeled into the binding sites of each protease based on the above structures, with energy
minimization using the AMBER12 forcefield in MOE. The benzamidine 1d structure bound to
matriptase is taken from the crystal structure. Inhibitors 8a-m and 11a-b were constructed in the
binding site based on the above structure superposition. In each case, the inhibitors were
minimized initially with the protease atom positions fixed, followed by minimization of the
protein-ligand complex, again using the AMBER 12 forcefield.
Conflicts of interest
The authors declare no conflicts of interest associate with this manuscript.
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Acknowledgements
Funding for this work was generously provided by the Alvin J. Siteman Cancer Research Fund
(Washington University School of Medicine) #16-FY18-02, Susan G. Komen for the Cure
Foundation grant CCR499051.
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