New potent cathepsin G phosphonate inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.08.069

Cathepsin G is an enzyme with dual chymotrypsin and trypsin-like specificity. As a leukocyte proteinase it is involved in the early stages of the immune response. In this work the synthesis and inhibitory activity of diaryl phosphonic-type irreversible ca

Full text of DOI:10.1016/j.bmc.2008.08.069

Bioorganic & Medicinal Chemistry 16 (2008) 8863–8867
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New potent cathepsin G phosphonate inhibitors
a
b,*
b
b
a
Marcin Sie n´ czyk , Adam Lesner , Magdalena Wysocka , Anna Ł e˛ gowska , Ewa Pietrusewicz ,
b
a
Krzysztof Rolka , Józef Oleksyszyn
a
Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology, Wybrze z_ e Wyspia n´ skiego 27, 50-370 Wrocław, Poland
Faculty of Chemistry, Gda n´ sk University, ul Sobieskiego 18, 80-952 Gda n´ sk, Poland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Cathepsin G is an enzyme with dual chymotrypsin and trypsin-like specificity. As a leukocyte proteinase
it is involved in the early stages of the immune response. In this work the synthesis and inhibitory activ-
ity of diaryl phosphonic-type irreversible cathepsin G inhibitors are described. Modification of the lead
Received 13 May 2008
Revised 24 August 2008
Accepted 28 August 2008
Available online 31 August 2008
P
ꢀ1 ꢀ1
structure Z-Phg (OPh)
2
(1) (kobs/I = 91 M
s ) in phenyl ester moieties followed by incorporation of
the basic functional group into the aromatic side chain yielded highly potent cathepsin G inhibitor
P
Z-(4-guanidine)Phg (OC
6
H
4
-4-S-Me)
2
(12) with the apparent second-order inhibition value at
Keywords:
Cathepsin G
Phosphonate irreversible inhibitor
Peptidomimetic
ꢀ1 ꢀ1
15,600 M
s . Further elongation of the obtained compound by tripeptide resulted in the inhibitor
P
Ac-Phe-Val-Thr-(4-guanidine)Phg (OC
6
H
4
-4-S-Me)
2
(19) with the highest kobs/I value ever reported in lit-
ꢀ
1 ꢀ1
erature (256,000 M
s ).
Ó 2008 Elsevier Ltd. All rights reserved.
1
. Introduction
site Ser195 OH mimics the transition state of peptide bond
cleavage performed by proteases. Due to their complete lack of
activity toward cysteine or threonine proteases and relatively
Cathepsin G (CG) is a member of polymorphonuclear leukocyte
PMN)-derived serine proteases which along with human neutro-
(
small size
large inhibitors.
a-aminophosphonates are attractive alternatives to
1
1
phil elastase and proteinase-3 is released upon leukocyte activa-
tion. Its physiological role in the living organism is mainly
associated with early immune response1 but its biologic activity
is not limited to this event. Examples of the broad actions of
In 1991 data regarding the synthesis of several compounds ac-
tive against specific members of the serine protease family includ-
1
2
ing cathepsin
phosphonic analogue of phenylalanine (Z-Phe (OPh)
poor inhibitory activity against cathepsin G (kobs/I = 76 M
A structurally similar inhibitor—phosphonic analogue of phenyl-
G
were published.
However the obtained
2
P
cathepsin G include angiotensin I to angiotensin II conversion
2
) (1) showed
ꢀ
1 ꢀ1
and degradation of several extracellular matrix proteins such as
collagen, laminin and elastin.3 However, prolonged and excessive
enzymatic action of cathepsin G leads to serious tissue damage
and the propagation of inflammation.4 Uncontrolled activity of
cathepsin G activates gelatinase A (MMP-2) which may facilitate
tumor invasion and angiogenesis.5 Increased proteolysis must be
precisely controlled by a system of endogenous inhibitors such as
s ).
P
2
glycine (Z-Phg (OPh) ) displays similar potency toward cathepsin
ꢀ1
P
ꢀ1
G (kobs/I = 91 M
s
) and was used in our approach.
Using Z-Phg (OPh) as the starting structure three series of ana-
2
logues were synthesized with the intention to select cathepsin G
inhibitors. In the first group modifications were introduced into
the side chain a-aminophosphonate. Our previous results showed
6
serpins and macroglobulins. In case of cathepsin G leukocytes can
suppress or extremely diminish its inhibition. For example, serpins
or macroglobulins can be inactivated by oxidation or proteolysis.
that substitution of guanidine group into the para position of the
phenylalanine side chain in the structure of chromogenic peptidic
7
8
Moreover cathepsin G, like other leukocyte-derived enzymes, is
bound to the leukocyte membrane or extracellular matrix compo-
nents after secretion. In their bound form enzymes are less sus-
substrate (in the position P
cathepsin G by 10-fold was achieved. Since
aromatic esters interact with serine proteases in a substrate like
1
) increased specificity (kcat/K
M
) to
1
3
a
-aminophosphonate
9
ceptible to inhibitor action especially by large endogenous
manner we decided to introduce several basic substituents into
1
0
P
proteinase inhibitors.
-Aminoalkylphosphonate diphenyl esters are low molecular
weight irreversible inhibitors of serine proteases. The ester struc-
ture formed after reaction of -aminophosphonate with the active
the para position of Z-Phg (OPh)
2
side chain or even replace the
a
whole side chain yielding phosphonic analogues of arginine or
lysine.
a
From the first group the most active compound contained a
4
-guanidine group was selected and was further modified within
the phenyl ester rings. The following groups were introduced in
the para position of esters: methyl, ethyl, methoxy, methylthionyl,
*
Corresponding author. Tel.: +48 585235336; fax: +48 58523472.
E-mail address: adas@chem.univ.gda.pl (A. Lesner).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.069

8
864
M. Sie n´ czyk et al. / Bioorg. Med. Chem. 16 (2008) 8863–8867
isopropyl, naphtyl, tert-butyl, chloro and 1,1,3,3-tetramethylbutyl.
The most potent cathepsin G inhibitor of this series was ester with
methylthionyl moiety. The obtained structure was further modi-
fied by N-terminal elongation with Thr, Val-Thr and Ac-Phe-Val-
Thr. The crystal structure of human cathepsin G in complex with
Elongation of structure 12 with a peptide chain highly increased
the inhibitory activity against cathepsin G (Table 3). Any N-termi-
nal extension by Thr (17), Val-Thr (18), and Ac-Phe-Val-Thr (19)
considerably increased inhibitory activity. These kinetic inhibition
data are well explained by reported X-ray structure and underline
significant role of Glu226 carboxylate at the bottom of S1 specific-
ity pocket of cathepsin G. Again, these results correspond very well
P
14
2
Suc-Val-Pro-Phe (OPh) has been reported. At the bottom of
the S1 specificity pocket Glu226 residue (chymotrypsinogen num-
bering) is located and, positively charged equatorial edge of phenyl
with our previously published data regarding cathepsin G chromo-
P
13,16
ring from benzyl substituent of Phe is interacting with negatively
genic substrate development.
Ac-Phe-Val-Thr-Phg -(OC
The obtained tetrapeptide
(19), appeared to be an ex-
inhibitor with
and to our knowledge, this compound displays
P
charged carboxylate group of Glu226. The Glu226 carboxylate al-
lows the accommodation of Lys side chain in the S1 pocket. Our
mapping studies with the new chromogenic substrates are in
agreement with these crystallographic data.
Recent reports by de Garavilla et al. regarding the synthesis and
crystal structure of b-ketophosphonate dual inhibitors of cathepsin
G and human chymase reveal that cathepsin G has a two hydro-
phobic binding sites (S1 and S3/S4) able to accommodate large
hydrophobic naphthalene groups.15 Again this was in good correla-
tion with our previous data regarding chromogenic substrates
where the Phe residue in position P4 displayed the highest
specificity parameter.
6 4 2
H -4-S-Me)
tremely potent peptidic cathepsin
G
obs
k /
ꢀ
1 ꢀ1
I = 256,000 M
s
the highest potency toward cathepsin G ever reported in the
literature.
Selectivity of action of compounds 1, 4, 11, 12, 17–19 was deter-
mined using cathepsin G related serine proteases such as bovine
a
4).
-chymotrypsin, bovine b-trypsin and human thrombin (Table
17–20
Structures 1, 4, 11 and 12 were more potent inhibitors of
related proteases than cathepsin G. Introduction of peptidic chain
into the inhibitor structure increased their selectivity of action.
The highest influence on related enzymes activity of obtained
inhibitors was observed for trypsin. Compound 17 was only two
times more active against cathepsin G than against trypsin. Intro-
duction of longer peptide chain (two or three amino acids, com-
pounds 18 and 19, respectively) lead to increased selectivity (12-
fold increase for structure 18, and 20-fold for 19) if compared to
trypsin. Compounds 17, 18 and 19 were more potent toward
2
. Results and discussion
Inhibitory activity of the synthesized molecules against cathep-
sin G, along with HPLC and MS analyses results are presented in
Tables 1–3. For the series of Z-protected phosphonic amino acid
analogues (compounds 1–8) the highest inhibitory potency was
observed for the compound bearing the guanidine group at the
para position of the side chain phenyl ring (4). The influence of this
group on the inhibitory activity overcomes all other residues intro-
duced at this position. Analogue 2 with an amino group introduced
into the side chain at the para position displayed significantly
higher activity when compared to the starting structure 1. This
suggests that the presence of –NH– group in this position is crucial
for forming the network of bonds between the inhibitor molecule
cathepsin G than toward
a-chymotrypsin and human thrombin,
where 30 times (compound 17) and 250 times (compound 19)
higher activity was observed.
Further investigation in this field may lead to more potent and
selective inhibitors. The next challenge in this area is the complete
structure activity relationship (SAR) as well as the synthesis of
pure diastereomers of the most potent inhibitors. This would open
a way for further in vivo studies using new potent cathepsin G
inhibitors.
and the enzyme. The poor inhibitory properties of
3
3
. Materials and methods
.1. Chemical synthesis
P
Z-(4-amidino)Phg (OPh)
2
seems to confirm this hypothesis. Low
potency of phosphonic analogues of lysine, arginine and homoargi-
nine (5–7) is most probably due to the relatively high flexibility of
their aliphatic side chains (compounds 6 and 8 within 30 min of
incubation with cathepsin G were able to inactivate only 5% of
the enzyme whereas no inhibition was observed in the case of ana-
logue 7). These data correlate well with our previous results
regarding the design of new chromogenic substrates of cathepsin
G using combinatorial chemistry methods where tetrapeptide
Ac-Phe-Val-Thr-Phe(p-guanidine) with amide of 5-amino-2-nitro-
For the synthesis of1a-aminophosphonates the amidoalkylation
2
reaction was applied. Condensation of triphenyl phosphite, ben-
zyl carbamate and an aldehyde in glacial acetic acid resulted in
a
-aminoalkyphosphonate aromatic esters as the racemic mixtures
(Table 1). Lead structure (1) was obtained using benzaldehyde.
2
2
Compound 4 was obtained using a method described previously.
For the synthesis of compound 2 4-nitrobenzaldehyde was used
and the obtained Z-(4-NO
using SnCl
P
2
benzoic acid (ANB-NH ) attached at C-terminus (served as a
2
)Phg (OPh)
2
was further reduced
2
3
chromophore) displayed the highest specificity constant (kcat
/
2
in ethyl acetate. For the synthesis of 5, 6 and
1
3
2
4,25
K
M
).
Significant improvement of cathepsin G inhibitory activity of 4
7
Phth-protected aldehydes were applied.
Guanylation of
P
Z-(4-NH
2
)Phg (OPh)
2
as well as compounds 5 and 6 was performed
26
was achieved after introduction of substituents at the para position
of phenyl ester rings. With only two exceptions (15, 16) the inhib-
itory potency was enhanced and the highest increase was observed
when methylthionyl was introduced (compound 12). Such a sub-
stitution yielded inhibition greater than 30 times that of com-
2
using S-ethyl-N,N’-di(Boc)-isothiourea in the presence of HgCl .
For the synthesis of 8 6-carbonitrile naphthalene-2-al was pre-
pared starting from dimethyl naphthalene 2,6-dicarboxylate and
used in modified amidoalkylation reaction to obtain the desired
2
7,28
a
-aminophosphonate diphenyl ester 8.
-aminoalkylphosphonates with substituted phenyl ester rings
For the synthesis of
ꢀ
1 ꢀ1
pound
4
with
a
kobs/I value at 15,600 M
s . Also, the
a
(
9–16, Table 2) our previously described method was applied.26
introduction of methoxy groups (compound 11) into this position
increased inhibitor potency (over 10 times as compared to the par-
ꢀ
1 ꢀ1
ent compound) displaying kobs/I = 7100 M
s . Substitution of
3
.2. Peptide synthesis
the phenyl ring of the ester moiety by bulky groups such as naph-
thyl (16) and 1,1,3,3-tetramethylbutane (15) decreased the inhibi-
tion strength by twofold (16) or made the compound almost
inactive (15).
A benzyloxycarbonyl group was removed from Z-(4-guani-
P
dine(Boc)
2
)Phg (OC
6
H -4-S-Me)
4
2
(234 mg, 0.28 mmol) (12) by
hydrogenolysis in methanol in the presence of 0.1% Pd/C resulting

M. Sie n´ czyk et al. / Bioorg. Med. Chem. 16 (2008) 8863–8867
8865
Table 1
Inhibition of cathepsin G by Z-protected
a-aminoalkylphosphonates diphenyl esters
O
R
O
O
O
N
P
H O
a
obs/Ib (M
ꢀ1 ꢀ1
Compound
R
MW calculated/obtained
473.5/474.7
t
R
(min)
I (
l
M)
k
s )
1
25.16
24.74
23.32
22.72
15.12
15.56
14.92
28.11
97
91
2
3
4
5
6
7
488.5/489.6
515.5/515.5
530.5/531.5
482.5/483.5
454.5/455.3
496.5/497.1
569.6/570.2
52
300
NH2
NH
112
47
30% Inhibitionc
NH2
NH
412
NH2
HN
H
N
NH2
99
74
NH
5% Inhibitionc
No Inhibition
5% Inhibitonc
NH2
221
250
112
H
N
NH2
NH
NH
NH2
8
a
HPLC retention time with a linear gradient 20–95% B (A: 0.1% TFA; B: 80% acetonitrile in A) within 45 min applied.
The apparent second-order inhibition.
Percent of inhibition after 30 min of incubation.
b
c
P
H
2
N-(4-guanidine(Boc)
2
) Phg (OC
6
H
4
-4-S-Me)
2
with 97% yield.
N-(4-gua-
(187 mg, 0.27 mmol) and
dure. The dipeptide 17B was elongated with Val (Fmoc-Val-OH
was applied) and Fmoc-Val-Thr(t-Bu)-(4-guanidine(Boc) )-
(18A) was obtained (72%). The removal of
Further peptide synthesis was performed in solution. H
2
2
P
P
nidine(Boc)
2
)Phg (OC
6
H
4
-4-S-Me)
2
6 4 2
Phg (OC H -4-S-Me)
Fmoc-Thr(t-Bu)-OH (118 mg, 0.27 mmol) were dissolved in 3 ml
of dimethylformamide (DMF) and (1-N-hydroxybenzotriazol
Fmoc and t-Bu groups resulted peptide 18 with 93% yield. Finally
Ac-Phe-OH was coupled to peptide 18B giving fully protected pep-
P
(
HOBt) (40 mg, 0.27 mmol) was added. The solution was cooled
tide Ac-Phe-Val-Thr(t-Bu)-Phg (4-guanidine(Boc)
)-(OC H
2 6 4
-4-S-
in an ice bath and diisopropylocarbodiimide (DIPCI) (45 l,
l
Me)
removal of side chain protecting groups from Thr and (4-guani-
dine(Boc) mixture
)PhgP by TFA/phenol/triisopropylsilane/H
(88:5:2:5, v/v/v/v) leading to Ac-Phe-Val-Thr-(4-guani-
dine(Boc) -4-S-Me) (19). The purity of final products
2
(19A) (67% yield). The final step of the synthesis was a
0
.27 mmol) was added. The reaction mixture was stirred at ꢀ5 °C
for 3 h and then overnight at room temperature. After the reaction
was completed 15 ml of ethyl acetate was added and washed with
2
2
O
P
1
0% sodium bicarbonate, 5% citric acid and brine. After drying the
2
)Phg (OC
6
H
4
2
volatile components were evaporated under reduced pressure
17–19 was verified by HPLC and the structure was confirmed by
MS analysis. The HPLC and MS analyses results are summarized
in Table 3.
affording
dine(Boc)
protected
dipeptide
Fmoc-Thr(t-Bu)-(4-guani-
P
2
)Phg (OC H
6 4
-4-S-Me) (17A) in 78% yield. The Fmoc
2
group from 17A was removed using 20% piperidine in DMF and
product 17B (yield 98%) was obtained. Final complete deprotection
was achieved using a trifluoroacetic acid (TFA)/phenol/triisopro-
3.3. HPLC analysis
2
9
pylsilane/H
2
O mixture (88:5:2:5, v/v/v/v) leading to product 17.
Purity of individual compounds was examined using the RP-
HPLC Pro Star system (Varian, Australia) equipped with a Kromasil
Further elongation was performed by applying the same proce-

8
866
M. Sie n´ czyk et al. / Bioorg. Med. Chem. 16 (2008) 8863–8867
Table 2
P
Inhibition of cathepsin G by various esters of N-benzyloxycarbonylamino-(4-guanidino)Phg (OR)
2
NH
HN NH2
O
R
O
O
O
N
P
H O
R
a
obs/Ib (M
ꢀ1 ꢀ1
Compound
R
MW calculated/obtained
530.5/531.7
t
R
(min)
I (
l
M)
k
s )
4
22.72
23.61
24.98
24.12
25.34
26.28
30.13
32.67
35.73
47
28
48
8
412
9
545.5/546.5
558.6/560.7
590.6/591.2
622.7/623.4
614.7/615.6
643.7/644.3
754.9/755.1
782.8/783.3
2820
1620
7100
15,600
2200
1150
10
11
12
13
14
15
16
O
S
4
24
28
115
112
3% Inhibitionc
226
a
HPLC retention time with a linear gradient 20–95% B (A: 0.1% TFA; B: 80% acetonitrile in A) within 45 min applied.
The apparent second-order inhibition.
Percent of inhibition after 30 min of incubation.
b
c
1
00 C
8
column (8 ꢁ 250 mm) (Knauer, Germany). A linear gradient
3.5. Enzymatic studies
from 20% to 95% B within 45 min was applied (A: 0.1% TFA; B: 80%
acetonitrile in A). The analyzed peptides were monitored at
2
Cathepsin G isolated from human neutrophils was purchased
from Biocentrum Kraków. All UV measurements were performed
using Cary 3E spectrophotometer (Varian, Australia). The concen-
tration of bovine b-trypsin stock solution was determined by titra-
26 nm.
3
.4. MS analysis
0
tion with p-nitrophenyl-p -guanidinebenzoate (NPGB). This trypsin
Mass spectra were recorded using a Biflex III MALDI TOF mass
spectrometer (Bruker, Germany). The alpha cyano-4-hydroxycin-
namic acid (CHCA) was used as a matrix.
stock solution was used for titration of SFTI-1, the mutual inhibitor
of both bovine b-trypsin and cathepsin G. Solution of SFTI-1 was
used to determine the concentration of the active form of cathep-

M. Sie n´ czyk et al. / Bioorg. Med. Chem. 16 (2008) 8863–8867
8867
Table 3
Inhibition of cathepsin G by peptidyl derivates of Z-PhgP(4-guanidine)-(OC
6
4 2
H -4-S-Me)
a
obs/Ib (M
15,600
ꢀ1 ꢀ1
Compound
Sequence
MW calculated/found
t
R
(min)
k
s )
P
1
1
1
1
1
1
1
1
1
2
Z-Phg (4-guanidine)-(OC
6
H
4
-4-S-Me)
2
622.7/623.4
1069.5/1069.7
847.5/847.8
603.2/604.3
1169.9/1170.0
947.8/948.1
702.8/703.1
1137.0/1137.1
878.0/879.3
21.84
28.73
24.81
19.23
30.21
26.02
20.18
29.09
24.64
P
7A
7B
7
8A
8B
8
Fmoc-Thr(t-Bu)-Phg (4-guanidine(Boc)
2
)-(OC
6
H
4
-4-S-Me)
2
No inhibition
No inhibition
52,300
No inhibition
No inhibition
152,000
P
Thr(t-Bu)-Phg (4-guanidine(Boc)
2
)-(OC
H
6 4
-4-S-Me)
2
P
Thr-Phg (4-guanidine)-(OC
6
H
4
-4-S-Me)
2
P
Fmoc-Val-Thr(t-Bu)-Phg (4-guanidine(Boc)
Val-Thr(t-Bu)-Phg (4-guanidine(Boc)
Val-Thr-Phg (4-guanidine)-(OC
Ac-Phe-Val-Thr(t-Bu)-Phg (4-guanidine(Boc)
2
)-(OC
6
H
4
-4-S-Me)
2
P
2
)-(OC
6
H
4
-4-S-Me)
2
P
6
H
4
-4-S-Me)
2
P
9A
9
2
)-(OC
6
H
4
-4-S-Me)
2
No inhibition
256,000
P
Ac-Phe-Val-Thr-Phg (4-guanidine)-(OC
6
H
4
-4-S-Me)
2
a
HPLC retention time with a linear gradient 20–95% B (A: 0.1% TFA; B: 80% acetonitrile in A) within 45 min applied.
The apparent second-order inhibition.
b
Table 4
Acknowledgments
Inhibitory activities against cathepsin G and related serine proteases
obs/Ia (Mꢀ1
s
ꢀ1
)
Compound
k
This work was supported by Ministry of Science and Higher
Education (Grant No. N N204 288934). The authors thank Dr. Keri
Smith for critical reading of the manuscript.
Cathepsin G
a
-Chymotrypsin
b-Trypsin
Thrombin
1
4
91
412
1050
30%
No inhibition
11,300
No inhibition
1520
b
1
1
1
1
1
1
2
7
8
9
7100
430
870
1130
1070
1010
13,200
17,800
22,100
12,560
2100
3070
1560
1170
15,600
52,300
152,000
256,000
References and notes
13,230
780
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2.
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a
The apparent second-order inhibition.
Percent of inhibition after 30 min of incubation.
8619.
b
3. Janusz, M. J.; Doherty, N. S. J. Immunol. 1991, 146, 3922.
4.
Shimoda, N.; Fukazawa, N.; Nonomura, K.; Fairchild, R. L. Am. J. Pathol. 2007,
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1
5
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3
0
the progressive curve method proposed by Tian and Tsou and
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1
1
incubation method described by Oleksyszyn and Powers.
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The kinetics of the reaction between irreversible inhibitors (12–
4) and cathepsin G were analyzed by continuous measurements
8
. Nie, J.; Pei, D. Exp. Cell Res. 2004, 296, 145.
1
9. Owen, C. A.; Campbell, M. A.; Sannes, P. L.; Boukedes, S. S.; Campbell, E. J. J. Cell
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of the loss of enzyme activity in the presence of chromogenic sub-
strate under pseudo-first order conditions (inhibitor:enzyme mo-
lar ratio below 10).30 Increasing concentrations of inhibitor
10. Owen, C. A.; Campbell, E. J. J. Immunol. 1998, 160, 1436.
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1
1
1
3. Wysocka, M.; Ł e˛ gowska, A.; Bulak, E.; Ja s´ kiewicz, A.; Miecznikowska, H.;
(
0.05–10
l
M) in 50
l
l aliquots was added and 10
, recently developed
M was mixed in a cuv-
ette containing 0.1 M Tris–HCl buffer, pH 7.5, supplied with
00 mM NaCl to final volume of 1.54 ml. The reaction was initiated
by addition of 25 l of cathepsin G (2 M) and the release of chro-
ll of chromo-
Lesner, A.; Rolka, K. Mol. Divers. 2007, 11, 93.
genic substrate (Ac-Phe-Val-Thr-Gnf-ANB-NH
by our group ) at a concentration of 3.3
2
4. Hof, P.; Mayr, I.; Huber, R.; Korzus, E.; Potempa, J.; Travis, J.; Powers, J. C.; Bode,
W. EMBO J. 1996, 15, 5481.
5. de Garavilla, L.; Greco, M. N.; Sukumar, N.; Chen, Z. W.; Pineda, A. O.; Mathews,
F. S.; Di Cera, E.; Giardino, E. C.; Wells, G. I.; Haertlein, B. J.; Kauffman, J. A.;
Corcoran, T. W.; Derian, C. K.; Eckardt, A. J.; Damiano, B. P.; Andrade-Gordon, P.;
Maryanoff, B. E. J. Biol. Chem. 2005, 280, 18001.
1
3
l
5
l
l
1
1
6. Leatherbarrow, R. J. GraFit Version 5, Erithacus Software Ltd, Horley, UK, 2006.
7. Biochemical evaluation of obtained inhibitors (kobs/I) toward cathepsin G
mophore was monitored at 405 nm for 20 min. Each measurement
was repeated and five to eight different concentrations of inhibitor
were tested.
related serine proteases such as bovine
a-chymotrypsin (Sigma–Aldrich),
bovine b-trypsin (Sigma–Aldrich) and human thrombin (Sigma–Aldrich) was
performed using the same method as described in the text. Chymotrypsin
concentration was determined using OMTKY bovine b-trypsin standardized
solution. Thrombin was titrated using NPGB. Substrates used in the enzymatic
When the progress of reaction was too slow to be measured by
the method described above (compounds 1–8 and 11) a different
1
1
approach was used. In this case 20
to 480 l of assay buffer containing 50
M) yielding a final inhibitor concentration from 50 nM to
M (the enzyme:inhibitor molar ratio was maintained over
0). At specific intervals 25 l of reaction mixture was sampled
l
l of cathepsin G was added
assays were: Z-Phe-Ala-Thr-Tyr-ANB-NH
2
(chymotrypsin), Phe-Val-Pro-Arg-
l
ll of tested inhibitor (at
ANB-NH (trypsin), and H-D-Phe-Pip-Arg-pNA (thrombin).
2
1
1
8. Wysocka, M.; Kwiatkowska, B.; Rzadkiewicz, M.; Lesner, A.; Rolka, K. Comb.
Chem. High Throughput Screening 2007, 10, 171.
5
5
1
–200 l
0
l
9. Zabłotna, E.; Dysasz, H.; Lesner, A.; Ja s´ kiewicz, A.; Ka z´ mierczak, K.;
Miecznikowska, H.; Rolka, K. Biochem. Biophys. Res. Commun. 2004, 319, 185.
0. Larsen, M. L.; Abildgaard, U.; Teien, A. N.; Gjesdal, K. Thromb. Res. 1978, 13, 285.
1. Oleksyszyn, J.; Subotkowska, L.; Mastalerz, P. Synthesis 1979, 985.
2. Bertrand, J. A.; Oleksyszyn, J.; Kam, C.-M.; Boduszek, B.; Presnell, S.; Plaskon, R.
R.; Suddath, F. L.; Powers, J. C.; Williams, L. D. Biochemistry 1996, 35, 3147.
3. Sie n´ czyk, M.; Oleksyszyn, J. Tetrahedron Lett. 2004, 45, 7251.
l
2
2
2
and the enzyme residual activity was determined. The aliquots
were added to 1.48 ml of the buffer followed by addition of 10
of 3.3 M chromogenic substrate (Ac-Phe-Val-Thr-Gnf-ANB-NH
Product release was measured over a period of 3 min.
The t1/2 values for the inhibition reaction were obtained from
plots of ln(v /v ) versus time. The pseudo-first order inhibition con-
stant kobs was calculated from equation t1/2 = 0.693/kobs
l
l
l
2
).
2
2
4. Teshima, T.; Matsumoto, T.; Wakamiya, T.; Shiba, T.; Aramaki, Y.; Nakajima, T.;
Kawai, N. Tetrahedron 1991, 47, 3305.
t
0
25. Hamilton, R.; Walker, B. J.; Walker, B. Tetrahedron Lett. 1993, 34, 2847.
1
1
2
2
2
6. Sie n´ czyk, M.; Oleksyszyn, J. Bioorg. Med. Chem. Lett. 2006, 16, 2886.
7. Sie n´ czyk, M. Doctoral thesis, Wrocław, 2006.
8. Joosens, J.; Van der Veken, P.; Lambeir, A.-M.; Augustyns, K.; Haemers, A. J. Med.
Chem. 2004, 47, 2411.
.
The
inhibitory activity of synthesized compounds is expressed as the
apparent second-order inhibition rate constant (kobs/I). For com-
pounds (3, 6, 8, 13, 14) that poorly inhibited cathepsin G within
29. Sole, N. A.; Barany, G. A. J. Org. Chem. 1992, 57, 5399.
0. Tian, W. X.; Tsou, C. L. Biochemistry 1982, 21, 1028.
3
3
0 min, the percentage of inhibition was determined.