Analysis of highly potent amidine containing inhibitors of serine proteases and their N-hydroxylated prodrugs (amidoximes)

Article abstract of DOI:10.3109/14756361003733647

The development of serine protease inhibitors often results in the discovery of new lead compounds containing strong basic amidine functions that usually suffer from poor absorption from the intestine. In order to improve oral bioavailability of these dru

Full text of DOI:10.3109/14756361003733647

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(1): 115–122
Copyright © 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756361003733647
RESEARCH ARTICLE
Analysis of highly potent amidine containing inhibitors
of serine proteases and their N-hydroxylated prodrugs
(amidoximes)
Joscha Kotthaus1 Torsten Steinmetzer2 Andreas van de Locht3, and Bernd Clement1
1Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University
of Kiel, Gutenbergstr, Kiel, Germany, 2Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marburg,
Germany, and 3e Medicines Company, Deutscher Platz, Leipzig, Germany
Abstract
The development of serine protease inhibitors often results in the discovery of new lead compounds containing
strong basic amidine functions that usually suffer from poor absorption from the intestine. In order to improve
oral bioavailability of these drugs, prodrug principles such as the conversion of amidines into amidoximes may
be applied. In this work, two HPLC-based separation methods of serine protease inhibitors (amidines) and their
N-hydroxylated prodrugs have been developed and characterised. This was performed by evaluating 11 distinct
amidine-amidoxime pairs with different physicochemical parameters (clogP: −3 to 5.1). The HPLC methods
developed allowed excellent separation of the compound pairs examined. Also, the possible selection of different
separation techniques (i.e. adsorption- and ion-pair-chromatography) permits universal application. Moreover,
both techniques are compatible with mass spectrometry and are superior to the previously described methods. In
summary, both HPLC methods are suitable for the separation of most amidoxime-prodrugs currently in clinical or
preclinical development.
Keywords: Serine protease inhibitor, prodrug, amidine, amidoxime, HPLC
Introduction
factor Xa (FXa) have become useful tools for the treat-
ment of thromboembolic diseases as indicated by the
recent approval of the direct thrombin inhibitor dabiga-
tran etexilate (Pradaxa®) and also rivaroxaban (Xarelto®),
a direct FXa inhibitor [6–8]. Other indications of amidine-
drugs such as pentamidine and diminazene, include the
treatment of pneumocystis jiroveci pneumonia, leishma-
niasis, or trypanosomiasis [9].
Although many amidines show excellent effects in both
in vitro assays and animal models, the clinical use of
amidine-drugs in humans is still very restricted. e main
reason for this is the generally poor oral bioavailability of
these compounds that results from the strong basic ami-
dine function, which is protonated under physiological
conditions and consequently positively charged. In order
to overcome these poor absorption properties, prodrug
principles for amidines may be applied [10]. One of the
Amidines are found in several drug-candidates with
diverse medical indications, such as anticoagulants,
antimicrobial, and antimetastatic drugs. Amidine con-
taining drugs are often found in the development of
trypsin-like serine protease inhibitors and have much
potential for therapy in various diseases. ese amidine
residues mimic the arginine or lysine residues, which are
the common substrates of trypsin-like serine proteases,
and interact with the free carboxyl group of aspartate in
the binding pocket [1].
Inhibitors of the matriptase and urokinase-type plas-
minogen activators (uPA) represent new classes of anti-
invasive agents that have showed promising effects in
animal studies by reducing the malignancy of different
cancer cell types [2–5]. Antagonists of the glycoprotein
IIb/IIIa receptor as well as inhibitors of thrombin and
Address for Correspondence: Bernd Clement, Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-
Albrechts-University of Kiel, Gutenbergstr. 76, D-24118 Kiel, Germany; Tel: 0049 431 8801126, E-mail: bclement@pharmazie.uni-kiel.de
(Received 14 January 2010; revised 00 00 0000; accepted 23 February 2010)
115

116
J. Kotthaus et al.
most intensively investigated prodrug principles for these
compounds is the conversion of amidines into amidoxi-
mes (N-hydroxy-amidines) [11]. After absorption from
the intestine, amidoxime-prodrugs are rapidly converted
into active amidines. is conversion is catalysed by an
enzyme system consisting of cytochrome b5, NADH cyto-
chrome b5 reductase and a third component identified in
mitochondria as a previously unknown molybdoenzyme,
which has been named mitochondrial amidoxime reduc-
ing component (mARC) and is the fourth human molyb-
denum containing enzyme [12,13]. A great advantage
of this activation pathway is that the conversion of the
amidoxime-prodrugs into their active forms avoids inter-
action with the cytochrome P450 isoenzymes. us, this
prodrug principle has no risk of cytochrome mediated
side-effects, such as the drug-interactions that can occur
if diverse drugs are metabolised by the same cytochrome
isoenzymes and have to compete with each other [14].
e enormous number of newly discovered amidine
drugs and their amidoxime-prodrugs require selective
and robust separation techniques. erefore, we have
developed appropriate analytics that fulfill these criteria.
Within this work, we have developed and character-
ised HPLC-based separation methods for a large number
of amidine-drugs—predominantly potent inhibitors of
trypsin-like serine proteases (FXa, matriptase, uPA, and
thrombin) —and their N-hydroxylated prodrugs that are
currently in clinical or preclinical trials. By analysis of 11
distinct pairs of amidines and amidoximes with clogP
values between −3 and 5.1, two simple and universally
applicable HPLC methods have been developed. e
choice of ammonium acetate and trifluoroacetic acid
(TFA), resulted in different separation methods (i.e.
adsorption- or ion-pair-chromatography) and offers a
broad field of possible applications. In addition, both
techniques are compatible with mass spectrometry and
are superior to the previously described methods using
other mobile or stationary phases.
Ro 44-3888) and AstraZeneca (Mölndal, Sweden;
melagatran, ethyl-melagatran, N-hydroxy-melagatran,
ximelagatran). Syntheses are described elsewhere
[2,15,16,17,18]. Benzamidine was obtained from Sigma-
Aldrich (Munich, Germany). N-hydroxy-benzamidine
(benzamidoxime) was synthesised from benzonitrile
and hydroxylamine as described previously [19]. All the
structural formulas are summarised in Table 1.
Preparation for analysis
All the test compounds were dissolved in 30% acetonitrile
to a final concentration of 100 μM. ese dilutions were
used as standards for the HPLC determinations. To
obtain two independent HPLC methods with diverse
advantages, two different mobile phases for either the
ion-pair- or adsorption-chromatography were chosen.
Both HPLC methods were evaluated by investigating the
separation of each amidine-amidoxime pair in order to
draw a general conclusion for the subsequent separa-
tion of the new amidine-amidoxime pairs. In the initial
studies, the percentage of acetonitrile in the mobile
phase was varied until both the compounds were well
separated within less than 15 minutes. e chromato-
grams obtained were analysed and the chromatographic
parameters (retention time, selectivity and resolution)
were calculated for all the runs. e percentage of ace-
tonitrile was plotted against the calculated lipophilicity
(clogP) of the amidoximes and the resulting correlation
curves were calculated for both methods.
In subsequent trials these correlations curves were
used to calculate the required percentage of acetonitrile
for the separation of every amidine-amidoxime-pair on
the basis of their clogP. us, separation of all the com-
pound pairs was performed using the mobile phases with
these calculated acetonitrile concentrations. Afterwards,
the chromatograms obtained were analysed and the
chromatographic parameters (retention time, selectivity,
and resolution) were calculated.
In summary, both methods would be suitable for the
separation of most amidoxime-prodrugs currently in
clinical or preclinical development.
HPLC analysis
e separations were performed with a Waters Alliance™
HPLC system (Waters, Milford, MA, USA) consisting of
a Waters e2695 XC separation module, a Waters 2998
Photodiode Array detector (Waters, Milford, MA, USA), and
the Empower™ 2 software. e mobile phases were filtrated
through a 0.45 μm Sartolon® membrane filter (Sartorius,
Göttingen, Germany) and degassed. All analysis were
carried out isocratically by a LiChrospher® 60 RP-select B
column (125×4mm; 5 μm, Merck KGaA) and a RP-select B
guard column (4×4mm; Merck KGaA). e flow rate was
maintained at 1mL/min, the injection volume was 10 μL,
and the detection wavelength was 220nm for all determi-
nations. e column temperature was set to 25°C.
Experimental
Materials
All reagents were purchased at the highest available purity.
Trifluoroaceticacid(TFA)wasobtainedfromSigma-Aldrich
(Munich, Germany), ammonium acetate, hydrochloric
acid, potassium phosphate, and sodium hydroxide from
Merck KGaA (Darmstadt, Germany). High-performance
liquid chromatography (HPLC) grade acetonitrile was pur-
chased from Mallinckrodt Baker (Griesheim, Germany).
e applied test compounds were kindly supplied
by e Medicines Company (Leipzig, Germany; CJ-463,
CJ-929, CJ-930, CJ-1200, CJ-1331, CJ-1332, CJ-1707,
CJ-1764, CJ-1802, CJ-1803, CJ-1817, CJ-2026), Wilex AG
(Munich, Germany; WX-UK1, WX-671 (Mesupron®)),
Hoffmann-La Roche (Basel, Switzerland; Ro 48-3656,
Method A (Adsorption chromatography): Separation with
mobile phases containing ammonium acetate
e mobile phase of 20 mM ammonium acetate (pH 4)
was applied with varying percentages of acetonitrile.
Journal of Enzyme Inhibition and Medicinal Chemistry

Analysis of amidines and their prodrugs (amidoximes)
117
Table 1. A summary of the clogP values and chemical structures for all the amidine-amidoxime pairs.
No
Name
clogP*
R1
R2
Structure
1a
CJ-463
−2.23
NH
OH
NH2
1b
2a
CJ-1200
CJ-930
−2.30
−1.38
N
OH
OH
OH
NH2
NH
NH2
O
S
O
H
N
N
H
N
H
O
O
O
O
O
R2
R1
2b
CJ-929
−1.46
N
OH
O
O
O
NH2
3a
3b
CJ-1332
CJ-1331
−2.93
−3.00
NH
N+
NH2
-
O
N
OH
R2
O
N+
NH2
O
S
O
-
O
H
N
N
H
N
H
O
4a
4b
5a
5b
6a
6b
7a
7b
CJ-1707
0.46
0.39
NH
R1
N
NH2
CJ-1764
N
OH
N
NH2
NH
NH2
CJ-1803
3.82
O
O
HN
CJ-1802
3.75
N
OH
O
O
S
NH2
NH
NH2
NH
N
O
O
CJ-1817
5.10
R2
CJ-2026
5.02
N
OH
R1
NH2
NH
NH2
Ro 44-3888
Ro 48-3656
−1.91
−1.99
–
–
O
O
O
OH
N
N
H
N
OH
O
R1
NH2
© 2011 Informa UK, Ltd.

118
J. Kotthaus et al.
Eluents with organic percentages between 5% and 50%
amidine bearing compounds are predominantly used
No
Name
R1
R2
–
Structure
clogP*
8a
WX-UK1
4.30
NH
O
O
NH2
N
O
WX-671 (Mesupron®)
8b
4.23
–
N
OH
S
NH
N
O
O
NH2
R1
9a
9b
Melagatran
−0.93
NH
H
NH2
N-Hydroxy-melagatran −1.01
N
OH
H
R1
NH2
H
N
10a Ethyl-melagatran
10b Ximelagtran
0.97
0.90
0.32
0.25
NH
O
N
R2
N
H
O
O
O
NH2
N
OH
NH2
11a Benzamidine
–
–
NH
NH2
R1
11b N-Hydroxy-
N
OH
benzamidine
NH2
*clogP was calculated for the uncharged compounds using the “Molinspiration Property Calculator” software.
were used to ensure the separations were within 15 min
(Table 2).
in the development of serine proteases inhibitors.
Consequently, we set the focus on the separation of
these compounds. Potent inhibitors of uPA (pairs 1, 2,
and 8, Table 1), FXa (pairs 3 and 4), matriptase (pairs 5
and 6), and thrombin (pairs 9 and 10) were selected to
establish and validate two independent HPLC methods.
e aim of the study was to develop universally applica-
ble HPLC methods for the separation of amidine-drugs
and amidoxime-prodrugs. ese should be easily trans-
ferable to other amidine-amidoxime pairs providing a
time-saving tool for the future development of inhibi-
tors of serine proteases.
Method B (Ion-pair chromatography): Separation with mobile
phases containing TFA
e mobile phase contained TFA in a concentration of
0.1% and the pH was adjusted to pH 7. Acetonitrile was
added to final concentrations in the range of 5% to 75%
to achieve separations within less than 15 min (Table 3).
In supplementary trials the addition of 20 mM potassium
phosphate to the mobile phase was investigated.
Calculation of lipophilicity (clogP)
e separation of all the compounds examined was
achieved within relatively short runtimes by both meth-
ods with good selectivity and resolution characteristics.
In general, HPLC method A showed a lower selectivity
(1.21 to 1.83) and resolution (1.74 to 5.96) compared to
those obtained by using method B (1.3 to 7.19 for selec-
tivity and 2.31 to 19.26 for resolution). e essential
chromatographic parameters (retention time, selectiv-
ity and resolution) for all the runs are summarised in
Tables 2 and 3. Two representative chromatograms for
the separation of the amidine-amidoxime pair 1 are
e lipophilicity of all the compounds was calculated, in
their uncharged state, using the “Molinspiration Property
Calculator” software (Molinspiration Cheminformatics,
Slovensky Grob, Slovak Republic).
Results and discussion
In this study, two universal HPLC methods for the
separation of amidines and their N-hydroxylated
prodrugs (amidoximes) have been developed. ese
Journal of Enzyme Inhibition and Medicinal Chemistry

Analysis of amidines and their prodrugs (amidoximes)
119
displayed in Figures 1 (method A) and 2 (method B)
showing the peak shape and the changed order of elu-
tion resulting from the two different separation tech-
niques (i.e. adsorption- and ion-pair-chromatography)
used.
e investigated compounds were chosen because of
their differing physico-chemical properties with clogP
values ranging from −3 (compound 3b) to 5.1 (compound
6a) (Table 1). is high physico-chemical heterogeneity
shows the suitability of both methods for other amidine-
amidoxime pairs.
Correlation curves were generated between the clogP
value of the examined amidoximes and the acetonitrile
percentage of the mobile phases used for the separations.
e influence of clogP on the acetonitrile percentage of
the mobile phase is depicted in Figure 3. e obtained
correlations were used to calculate the required organic
percentage in the mobile phase for separation of the
test compounds. HPLC analysis performed with these
conditions resulted in good separations for most of the
pairs of compounds. e resolutions of the separations
performed are summarised in Figure 4. For method A
only four pairs showed insufficient separations when
Table 2. Chromatographic parameters of HPLC method A.
Method A (tested/calculated)
Table 3. Chromatographic parameters of HPLC method B.
Method B (tested/calculated)
Retention
Pair Compound. ACN [%]
[min]
7/10.5
9.1/15.9
6.9/-
Selectivity Resolution
Pair No
ACN [%] Retention [min] Selectivity Resolution
1
1a
1b
2a
2b
3a
10/8
1.34/1.56
4.57/5.14
1
2
3
4
5
6
7
8
9
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
20/16
40/40
20/10
35/36
50/60
75/70
5/18
7.1/7.3
3.4/5.8
4.1/4.1
2/2
2.42/1.3 12.33/2.31
2
20/12
15/5
1.56/-
5.96/-
2.75/2.75
2.55/-
7/7
10.3/-
6.7/-
3
1.53/-
4.77/-
11.5/-
5/-
13.68/-
3b
4a
4b
5a
5b
6a
6b
7a
9.8/-
4
20/20
35/35
40/41
5/10
5.6/5.6
7.6/7.6
7.7/7.7
11/11
1.42/1.42
1.48/1.48
1.43/1.26
1.54/1.83
1.29/-
2.86/2.86
3.88/3.88
3.61/2.24
4.07/4.17
3.48/-
7.8/5.4
2.8/2.4
11.5/6
2.9/1.8
6.2/7.8
1.8/1.8
4.3/-
3.5/2.88 13.79/10.17
5.22/5.2 11.53/14
5
6
9.5/9.8
13.2/12.1
5.4/2
5.6/7
11.5/10
4/-
7
1.59/-
7b
8a
8b
9a
7.9/3
3./-
8
47/38
8/14
7.8/-
75/64
15/25
40/39
20/35
7.6/15.9
2.2/2.9
5.2/-
4.86/7.19 13.5/19.26
4/- 6.73/-
3.29/3.61 7.88/9.49
1.52/2.7 3.14/5.48
9.8/-
9
6.6/2.8
8.5/3.4
12.1/5.9
14.5/7.3
2.9/-
1.33/1.3
1.21/1.27
1.38/-
1.85/1.74
2/2.01
9b
10a
10b
11a
11b
1.9/-
10
11
22/23
5/20
10 10a
10b
11 11a
11b
6.4/7.3
2.5/2.6
4/3.5
2.46/-
3.7/-
2.9/1.8
e analytical parameters of the empirically established method
compared to those calculated. Separations of pairs 2, 3, 8, and 11
were insufficient when using the calculated conditions.
e analytical parameters of the empirically established method
compared to those calculated. Separations of pairs 3, 7, and 9
were insufficient when using the calculated conditions.
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00 10.00 11.00 12.00
Minutes
Figure 1. Representative chromatogram for the separation of CJ-463 (7min) and CJ-1200 (9.1min) using a mobile phase consisting of ammonium
acetate buffer (20 mmol/L, pH 4) and acetonitrile (90/10, v/v) (method A). e chromatographic parameters are shown in Table 2.
© 2011 Informa UK, Ltd.

120
J. Kotthaus et al.
0.26
0.24
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Minutes
Figure 2. Representative chromatogram for the separation of CJ-1200 (3.4 min) and CJ-463 (7.1min) using a mobile phase consisting of TFA (0.1%)
in distilled water and acetonitrile (80/20, v/v; pH 7) (method B). For chromatographic parameters see Table 3.
method A
Correlation between clogP and percentage of acetonitrile
50
45
40
35
30
25
20
15
10
5
0
0
1
2
3
4
5
6
−4
−3
−2
−1
clogP
method B
Correlation between clogP and percentage of acetonitrile
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
−4
−3
−2
−1
clogP
Figure 3. Correlations between clogP (amidoxime) and acetonitrile as a percentage of the mobile phase. e calculated linear equations are y =
4.46x + 18.70 (R2 = 0.744) and y = 7.42x + 32.69 (R2 = 0.783) for methods A and B, respectively.
using the calculated conditions, while for method B this
was true for only three pairs. Accordingly, the calculated
conditions for method B are not adequate for the separa-
tion of negatively charged compounds (pairs 3, 7, and 9)
indicating that the usage of the calculated correlations
is limited. Nevertheless, correlation curves established
in this work are useful tools and facilitate the develop-
ment of HPLC methods for newly discovered serine
protease inhibitors as well as other amidine containing
compounds.
Nowadays,thecompatibilityofHPLCtomassspectrom-
etry is getting more and more important and has become a
Journal of Enzyme Inhibition and Medicinal Chemistry

Analysis of amidines and their prodrugs (amidoximes)
121
the compounds are charged or uncharged due to the
different basicity of the amidines (pKB ≈ 2–3) and ami-
doximes (pKB ≈ 9–10) [24,25]. Amidines exhibit strong
basic properties, whereas amidoximes are less basic and
hence not protonated at pH 7. Consequently, only the
amidines were positively charged under the described
conditions and therefore able to form ion-pairs with
TFA. is resulted in a significant increase of lipophi-
licity and elongated the retention time, which resulted
in the excellent selectivity and resolution of method B
(Table 3). However, the usage of TFA as an ion-pair-re-
agent resulted in slightly higher costs for waste disposal,
because of an increased organic portion in the mobile
phase in comparison to method A.
e addition of 20 mM potassium phosphate to the
mobile phase led to a buffered solution at pH 7 and this
improved the robustness of method B. However, with the
addition of potassium phosphate this method loses its
compatibility with mass spectrometry.
e limits of quantification were not optimised in this
work because the main focus was on the separation of
the amidines and amidoximes. For instance, the limit
of quantification in porcine plasma was determined
for compounds 3a and 3b. Only a slight optimisation
of method A was necessary to achieve detection limits
of 30 ng/mL using LC/MS with athmospheric pressure
chemical ionisation (APCI). ese data are published
elsewhere [17].
Resolution (method A)
7
6
5
4
3
2
1
0
1
2
3
4
5
6
7
8
9
10 11 Mean
Pair
Resolution (method B)
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10 11 Mean
Pair
Figure 4. Resolution of HPLC methods A and B. Resolutions obtained
with the experimentally established conditions (black bars) are
compared to the calculated ones (white bars).
All the amidine-amidoxime pairs examined were sep-
arated by the methods described within less than 15 min-
utes. Further optimisation of the runtimes could easily be
done by adjusting the acetonitrile gradient or increasing
the column temperature. First optimisation attempts
showed significantly reduced runtimes without impair-
ing the separations. For instance, when separating com-
pound pair 5 with method B, by increasing the column
temperature to 50°C the retention time of compound 5a
was decreased from 11.5 min to 9.8 min, while the reten-
tion time of compound 5b was affected only marginally.
Overall, these HPLC-based separation methods allowed
fast separation and could be applied to standard high-
throughput analysis.
fundamental requirement for determinations in complex
matrices such as blood or urine samples. One of the major
limitations of the previously published HPLC methods for
the separation of amidines and amidoxime-prodrugs is
the lack of compatibility to mass selective detectors caused
by the usage of unsuitable reagents like phosphate salts or
ion-pair-reagents such as sodium octylsulphonate [20–23].
Compared to these techniques the methods described in
this report show improved properties with respect to mass
spectrometry and could facilitate analysis of samples with
complex matrices.
Moreover, the choice of two different separation
methods, adsorption- and ion-pair-chromatography,
gives the advantage that the order of elution for the ana-
lytes can be reversed and therefore could be adapted to
the particular analytical requirements. Separations with
method B resulted in a fast elution of the amidoximes
and a distinct retention of amidines, whereas method
A showed short retention times for amidines and lon-
ger ones for amidoximes. e choice of whether to use
method A or method B will depend on the particular
need or sample usage required. Consequently, the
choice of adsorption- or ion-pair-technique is impor-
tant to achieve the best possible separations and could
offer a widespread analytical usage.
Conclusion
In this work, two HPLC methods for the separation of ami-
dines and amidoximes were successfully established using
both adsorption- and ion-pair-chromatography. e use
of reagents that are compatible with mass spectrometry is
a great advantage compared to previously described ana-
lytical techniques. Both methods are versatile and may be
easily optimised to achieve low limits of detection, espe-
cially in combination with mass spectrometry. A major
advantage is that the established methods can be easily
transferred to a large number of amidoxime-prodrugs.
us would be a time-saving tool for the development of
HPLC methods for amidoxime-prodrugs that are currently
in preclinical or clinical trials.
When using TFA in the mobile phase as the ion-
pair-reagent, the pH was adjusted to pH 7. At this pH,
© 2011 Informa UK, Ltd.

122
J. Kotthaus et al.
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Acknowledgement
We would like to thank AstraZeneca (Mölndal, Sweden),
Hoffmann-La Roche (Basel, Switzerland), e Medicines
Company (Leipzig, Germany) and Wilex AG (Munich,
Germany) for providing the test compounds.
Declaration of interest
15. Weller T, Alig L, Beresini M, Blackburn B, Bunting S, Hadvary P,
Muller M, Knopp D, Levet-Trafit B, Lipari M, Modi N, Muller M,
Refino C, Schmitt M, Schonholzer P, Weiss S, Steiner B. Orally
active fibrinogen receptor antagonists. 2. Amidoximes as prodrugs
of amidines. J Med Chem 1996;39:3139–3147.
e authors report no conflicts of interests. e authors
alone are responsible for the content and writing of the
paper.
16. Stürzebecher J, Vieweg H, Steinmetzer T, Schweinitz A, Stubbs MT,
Renatus M, Wikström P. 3-Amidinophenylalanine-based inhibitors
of urokinase. Bioorg Med Chem Lett 1999;9:3147–3152.
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Journal of Enzyme Inhibition and Medicinal Chemistry