Simple phosphonic inhibitors of human neutrophil elastase

Article abstract of DOI:10.1016/j.bmcl.2011.01.083

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α- aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val^P(OC₆H ₄-4-COOMe)₂ displayed an apparent second-order inhibition value at 33,015 M^-1 s^-1.

Full text of DOI:10.1016/j.bmcl.2011.01.083

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1310–1314
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Simple phosphonic inhibitors of human neutrophil elastase
a
a
a
Marcin Sienczyk , Łukasz Winiarski , Paulina Kasperkiewicz , Mateusz Psurski a,b, Joanna Wietrzyk b,
´
Józef Oleksyszyn a,
⇑
a
_
Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspian´skiego 27, 50-370 Wrocław, Poland
b Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Rudolf Weigl Street, 53-114
Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe the synthesis and resulting activity of a complex series of
a-aminophosphonate
Received 8 December 2010
Revised 14 January 2011
Accepted 18 January 2011
Available online 22 January 2011
diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for
human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase,
trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple
Cbz-protected
one of the obtained compounds Cbz-ValP(OC6H4-4-COOMe)2 displayed an apparent second-order inhibi-
tion value at 33,015 MÀ1 sÀ1
a-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where
Keywords:
Human neutrophil elastase
.
Ó 2011 Elsevier Ltd. All rights reserved.
a-Aminoalkylphosphonates
Serine protease inhibitors
Cancer cells
Irreversible inhibition
Human neutrophil elastase (HNE), a serine protease classified
within the chymotrypsin-like superfamily, which is secreted by
azurophilic granules, was first described by Janoff and Scherer in
1968.1 HNE has the ability to cleave almost every protein con-
tained within the extracellular matrix (ECM) including, but not
limited to, elastin, collagen, fibronectin, laminin, and proteogly-
cans.1–5 The natural endogenous inhibitors for HNE are serpins
We also hypothesize that potent alkylating/acylating inhibitors
of elastase could react even with some weak nucleophiles present
in non-enzymatic proteins (like serum albumin). Such modified
proteins could in turn act as antigens inducing an immune re-
sponse. Thus, it seems that only irreversible and stable inhibitors
such as
the limitations of current elastase inhibitors.11
The major advantage of the -aminoalkylphosphonate diaryl
a-aminoalkylphosphonates have the potential to overcome
such as
a1-PI, SLPI or
a
2-macroglobulin.6 Inactivation of serpins
a
by the ‘oxidative burst’ of neutrophils affects the balance between
inhibitors and HNE. Uncontrolled activity of HNE can lead to path-
ophysiological states such as chronic obstructive pulmonary
disease (COPD), adult respiratory syndrome, pulmonary emphy-
sema, cystic fibrosis or chronic bronchitis.7–9 In addition, overex-
esters is their potency, selectivity, and irreversible nature of action.
These low molecular weight inhibitors of serine proteases are de-
void of reactivity with cysteine, aspartyl or threonine proteases.
The first described phosphonic-type inhibitors of elastase dis-
played relatively low potency and selectivity of action. However,
elongation of the peptide chain resulted in higher potency of action
as well as increased selectivity within the protease clan. The pep-
tidyl derivative of a phosphonic valine analogue with a similar po-
tency inhibited human neutrophil and porcine pancreatic elastases
(MeO-Suc-Ala-Ala-Pro-ValP(OPh)2, kobs/I = 7100 MÀ1 sÀ1). Further
studies led to the development of a more potent and a threefold
pression of elastase allows cancer cells within
a tumor to
develop and metastasize directly through degradation of ECM.10
Although numerous, structurally diverse inhibitors of human
neutrophil elastase have been reported, none of them has demon-
strated success in a clinical trial. One explanation for this failure
could be the reversible nature of action of some of the inhibitors.
Reversible inhibitors are not able to suppress the imbalance be-
tween natural endogenous inhibitors and elastase as incubation
of elastase with both—reversible and irreversible inhibitors re-
more selective HNE inhibitor (Boc-Val-Pro-ValP(OPh)2, kobs
/
I = 27,000 MÀ1 sÀ1).11 Although substitution of the phenyl ester
ring with a chlorine atom at the para position and removal of the
t-Boc protective group resulted in a twofold decrease in potency,
however, the obtained inhibitor was approximately 26 times more
active against human neutrophil elastase than elastase from por-
cine pancreas.12 The high impact of the ester ring structures on
sulted in the formation of only covalent complexes (elastase-a1
-
PI) and free, unbound reversible inhibitor molecules.
the inhibitory potency of
has been demonstrated for other serine proteases such as trypsin,
a-aminoalkylphosphonate diaryl esters
⇑
Corresponding author. Tel.: +48 71 320 40 27; fax: +48 71 328 40 64.
E-mail address: jozef.oleksyszyn@pwr.wroc.pl (J. Oleksyszyn).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.083

´
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M. Sienczyk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1310–1314
urokinase, cathepsin G or DPPIV.13–15 Thus, we further investigated
the influence of ester structure on the inhibitory activity of phos-
phonic-type inhibitors of human neutrophil elastase.
We synthesized several simple, Cbz-protected phosphonic ana-
logues of aliphatic amino acids—alanine, valine, leucine, norvaline,
norleucine, and amino butyric acid with different aromatic ester
ring structures (Scheme 1). The inhibitory potency of the synthe-
sized compounds against human neutrophil elastase as well as
against other serine proteases such as porcine pancreatic elastase,
chymotrypsin, and trypsin was measured using fluorescent
R1
R1
O
R2
O
O
O
P
R2
O
O
CH3COOH
reflux
R1
O
N
H
P
+
+
NH2
O
O
H
3
CH3CN
reflux
OH
R1
+
PCl3
Scheme 1. Schematic representation of the synthesis of Cbz-protected phosphonic analogues of amino acids, where R1 represents a phenyl ring substituent as presented in
Tables 1–6 and R2 represents methyl, ethyl, iso-propyl, n-propyl, n-butyl or iso-butyl chain.
Table 1
Inhibition properties of phosphonic analogues of alanine (Cbz-AlaP(OAr)2) against HNE, PPE, chymotrypsin, and trypsin
O
R
O
P
O
N
H
O
O
R
Compound
R
k2/Ki [MÀ1 sÀ1
]
HNE
PPE
Chymotrypsin
Trypsin
1
2
3
4
5
6
H
<50
<50
<50
<50
<50
NI
NI
NI
<50
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
4-Ethyl
4-Isopropyl
4-t-Butyl
4-S-Methyl
4-Carbomethoxy
NI
7722 374
197 39
1436 56
1527
5
NI—less than 5% of inhibition was observed after incubation of enzyme with the inhibitor (5
lM) at 37 °C.
Table 2
Inhibition properties of Cbz-AbuP(OAr)2 toward HNE, PPE, chymotrypsin, and trypsin
O
R
R
O
P
O
N
H
O
O
Compound
R
k2/Ki [MÀ1 sÀ1
]
HNE
PPE
Chymotrypsin
Trypsin
7
8
9
H
<50
NI
NI
NI
NI
NI
NI
<50
<50
NI
<50
<50
NI
<50
<50
<50
1075 35
<50
<50
NI
NI
NI
NI
NI
NI
NI
NI
4-Methyl
2,5-Dimethyl
3,4-Dimethyl
4-Ethyl
4-Isopropyl
4-t-Butyl
4-S-Methyl
4-O-Methyl
4-Carbomethoxy
4-Chloro
10
11
12
13
14
15
16
17
18
NI
<50
<50
NI
201
<50
22,031 932
76
346 48
NI
<50
NI
<50
<50
1556 27
<50
<50
6
NI
1071 44
<50
NI
5
3-Chloro
NI—less than 5% of inhibition was observed after incubation of enzyme with the inhibitor (5
lM) at 37 °C.

´
M. Sienczyk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1310–1314
1312
substrates. The influence of selected compounds on the prolifera-
tion of several cancer cell lines using Sivelestat (ONO-5046) as
the reference compound was estimated.16,17 A detailed description
of the enzymatic and cell methods as well as the synthetic proto-
cols can be found in the Supplementary data associated with this
article.
Phosphonic analogues of leucine obtained in the presented
studies lack activity against human neutrophil elastase. They are,
however, potent inhibitors of chymotrypsin. The highest potency
against HNE was observed for derivative 43, which contained
4-carbomethoxyphenyl ester rings in its structure (k2/Ki =
375 MÀ1 sÀ1).
Among all synthesized phosphonic analogues of alanine the
highest potency of action was observed for a derivative containing
a carbomethoxy substituent at the para position of the phenyl ester
ring (6, k2/Ki = 7722 MÀ1 sÀ1). A 4-carbomethoxy substituent was
only effective for the AlaP derivatives displaying poor selectivity
of action against other tested serine proteases (40 times more ac-
tive against HNE than PPE, approximately 5 times less active
against trypsin and chymotrypsin). Other alanine analogues (1–5)
displayed no activity in the tested concentration range.
Similarly, among the synthesized analogues of nVal and nLeu,
only derivatives with 4-carbomethoxy groups displayed the high-
est potency of action against HNE. Compound 54 was 6 times more
Table 4
Inhibition properties of Cbz-LeuP(OAr)2 toward HNE, PPE, chymotrypsin, and trypsin
A much higher selectivity of action was observed for an AbuP
derivative with 4-carbomethoxy substitution within the phenyl es-
ter ring (16, k2/Ki = 22,031 MÀ1 sÀ1) which displayed approxi-
mately 22 times more activity against HNE than against
chymotrypsin and trypsin, but only 14 times more reactivity with
PPE. Interestingly, a slight increase in activity against neutrophil
elastase was also noticed for analogues with 3-Cl-phenyl (18, k2/
Ki = 346 MÀ1 sÀ1), 4-S-methylphenyl (14, k2/Ki = 201 MÀ1 sÀ1), and
4-Cl-phenyl (17, k2/Ki = 76 MÀ1 sÀ1) ester rings when compared
with their unsubstituted derivative 7.
Out of the total synthesized phosphonic analogues of valine one
compound, similar to analogues of AlaP and AbuP, contained a car-
bomethoxy substituent at the para position of the phenyl ester ring
(26, k2/Ki = 33,015 MÀ1 sÀ1) and was the most potent inhibitor ob-
served in this study. Compound 26 displayed almost absolute
selectivity of action against the tested proteases (k2/Ki <50 MÀ1 sÀ1
toward chymotrypsin and trypsin). Such results are somewhat un-
O
R
O
P
O
N
H
O
O
R
Compound
R
k2/Ki [MÀ1 sÀ1
PPE Chymotrypsin
]
HNE
Trypsin
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
H
<50
<50
<50
NI
<50
<50
<50
<50
<50
<50
<50
<50
NI
<50
<50
NI
650 32a
914 228a
293 102a
<50a
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
4-Methyl
2-Methyl
2,5-Dimethyl
3,4-Dimethyl
2,3-Dimethyl
4-Ethyl
<50
<50
<50
<50
<50
<50
<50
<50
NI
306 3a
3500 455a
173 21a
<50a
4-Isopropyl
4-t-Butyl
<50a
2,3,5-Trimethyl
3,4,5-Trimethyl
4-S-Methyl
213 70a
4425 575a
18,300 82a
<50
ique for simple Cbz-protected a-aminoalkylphosphonate diaryl es-
ters which usually inhibit a broad spectrum of serine proteases or
display no activity at all. Interestingly, a valine analogue with a 4-
methylthiophenyl ester group (24) displayed no inhibitory activity
towards the tested proteases. In contrast, its oxidized homologue
(27) with a 4-SO2-methyl substituent was active against HNE
(k2/Ki = 15,945 MÀ1 sÀ1) but its selectivity of action was rather
4-O-Methyl
3-O-Methyl
<50
<50
<50
8760 1570a NI
4-Carbomethoxy 375 13 NI
3-Carbomethoxy <50 <50
61,770 5860a <50
3721 418
2170 390a
4430 310a
NI
NI
NI
4-Chloro
3-Chloro
<50
<50
NI
<50
NI—less than 5% of inhibition was observed after incubation of enzyme with the
inhibitor (5 M) at 37 °C.
Data previously reported.18
poor, it displayed
a similar potency against chymotrypsin
(k2/Ki = 12,660 MÀ1 sÀ1), as well as lower activity against trypsin
(k2/Ki = 707 MÀ1 sÀ1) and PPE (k2/Ki = 288 MÀ1 sÀ1).
l
a
Table 3
Inhibition properties of Cbz-ValP(OAr)2 toward HNE, PPE, chymotrypsin, and trypsin
O
R
O
P
O
N
H
O
O
R
Compound
R
k2/Ki [MÀ1 sÀ1
]
HNE
PPE
Chymotrypsin
Trypsin
19
20
21
22
23
24
25
26
27
28
H
NI
NI
<50
NI
NI
<50
<50
33,015 1445
15,945 285
<50
NI
NI
<50
NI
NI
NI
<50
NI
288 31
NI
NI
NI
NI
NI
NI
<50
NI
<50
NI
NI
NI
NI
NI
NI
NI
<50
707 34
NI
2,5-Dimethyl
4-Ethyl
4-Isopropyl
4-(1,1,3,3-Tetramethyl)butyl
4-S-Methyl
4-O-Methyl
4-Carbomethoxy
4-SO2-methyl
4-t-Butyl
12,660 2643
NI
NI—less than 5% of inhibition was observed after incubation of enzyme with the inhibitor (5 lM) at 37 °C.

´
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M. Sienczyk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1310–1314
Table 5
Inhibition properties of Cbz-nValP(OAr)2 toward HNE, PPE, chymotrypsin and trypsin
O
R
O
P
N
O
H
O
O
R
Compound
R
k2/Ki [MÀ1 sÀ1
]
HNE
PPE
Chymotrypsin
Trypsin
47
48
49
50
51
52
53
54
H
<50
NI
<50
NI
NI
<50
NI
<50
NI
<50
NI
NI
<50
NI
<50
<50
<50
<50
NI
<50
<50
3040 70
NI
NI
NI
NI
NI
NI
NI
<50
2,3-Dimethyl
4-Ethyl
2,3,5-Trimethyl
3,4,5-Trimethyl
4-S-Methyl
4-O-Methyl
4-Carbomethoxy
17,858 418
2868 88
NI—less than 5% of inhibition was observed after incubation of enzyme with the inhibitor (5
lM) at 37 °C.
Table 6
Table 7
Inhibition properties of Cbz-nLeuP(OAr)2 toward HNE, PPE, chymotrypsin, and trypsin
An anti-proliferative effect of selected compounds on human lung adenocarcinoma
cell line (A549), human breast cancer cell line (MCF-7), human colon carcinoma cell
line (LoVo), and human multidrug-resistant colon carcinoma cell line (LoVoDX)
Compound
A549
Lovo
LovoDX
MCF7
61% 5a
g/ml
49% 3a
20% 2a
g/ml 15
g/ml 21
15% 3a
46% 4a
38% 2a
54% 10a
0
O
Sivelestat
0
6
16
26
27
43
54
25% 7a
31% 2a
46% 3a
5% 5a
3% 3a
40% 6a
47
73
32
l
l
l
40
47
l
l
6
R
O
O
O
N
H
P
O
g/ml
6
g/ml
6
15% 6a
19% 5a
22% 6a
6% 6a
47% 15a
8% 6a
R
35
l
g/ml
4
52
l
g/ml
7
Compound
R
k2/Ki [MÀ1 sÀ1
PPE Chymotrypsin Trypsin
]
a
IC50 value could not be calculated, inactive in the investigated range, results
shown as percentage of cell growth inhibition in a concentration 100 g/ml.
HNE
l
55
56
57
58
59
60
61
H
<50
NI
<50
NI
NI
NI
<50
NI
NI
NI
<50
NI
NI
NI
4-S-Methyl
4-O-Methyl
<50 <50
4-Carbomethoxy 452 26 <50 338 14
The influence of selected inhibitors on the proliferation of can-
cer cell lines showed that the most active was compound 26, which
also displayed the highest inhibition of human neutrophil elastase
in the enzyme kinetic assay. It decreased the growth of LoVo and
4-Isopropyl
4-t-Butyl
4-Chloro
<50
<50
<50
<50 <50
<50 NI
<50 <50
NI—less than 5% of inhibition was observed after incubation of enzyme with the
inhibitor (5 M) at 37 °C.
MCF7 cell lines by 50% at 32 and 47 lg/ml, respectively. Slightly
l
less inhibition was observed when LoVoDX and A549 cell lines
were treated with 26. The effect of cell growth inhibition observed
for compound 54 was slightly lower when compared to compound
26, but it displayed almost 2 times lower inhibition of HNE. Similar
behavior was observed for compound 6. For all three analogues (6,
26, and 54) the growth inhibition effect was much higher than the
effect observed for the control compound Sivelestat, which in our
studies displayed almost no activity towards the tested cell lines.
Importantly, decreasing the potency of action of HNE inhibitors
as well as their selectivity, results in less inhibition of cancer cell
line growth (Table 7).
The presented work describes the effect of the phenyl ester
group substituents of simple Cbz-protected phosphonic analogues
of amino acids on the activity of human neutrophil elastase. It also
provides insight into further development of more potent and
selective inhibitors of this class of compounds.
active against HNE (k2/Ki = 17,858 MÀ1 sÀ1) than chymotrypsin and
PPE with only a slight inhibition of trypsin (k2/Ki <50 MÀ1 sÀ1). Cbz-
nLeuP(O-4-C6H4–COOCH3)2 (58) was the only active analogue of
this group against HNE (k2/Ki = 452 MÀ1 sÀ1) but also inhibited
chymotrypsin with similar potency.
The presented data clearly suggest that the highest activity of
simple Cbz-protected phosphonic analogues of amino acids is the
result of the 4-carbomethoxy substituent within the aromatic ester
ring. The strong electrowithdrawing properties of this group might
lead to the increased inhibition of the target enzyme. It seems
likely that the effect of this substituent results in high electrophilic
potential of the phosphorus atom that facilitates the enzyme’s
nucleophile attack which directs the inhibition pattern. The inter-
action between the S1 pocket and the P1 side chain seems to be
less important for a particular group of phosphonic amino acid
derivatives as the observed inhibitory effect is mostly the result
of the nature of the phenyl ester ring substituents. When compar-
ing the activity of derivatives with the same ester structure the
highest activity was observed for analogues with the iso-propyl
and ethyl chains as the P1 group. Shorter or longer chains resulted
in decreased potency of action.
Acknowledgments
This work was supported by Ministry of Science and Higher
Education (Grant No. N405 342633). The authors would like to
thank Dr. Keri Smith for critical reading of the manuscript.

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M. Sienczyk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1310–1314
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.083.
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