Phosphonic pseudopeptides as human neutrophil elastase inhibitors - A combinatorial approach

Article abstract of DOI:10.1016/j.bmc.2010.12.008

Here we present a simple and rapid method for the construction of phosphonic peptide mimetic inhibitor libraries - products of Ugi and Passerini multicomponent condensations - leading to the selection of new biologically active phosphonic pseudopeptides.

Full text of DOI:10.1016/j.bmc.2010.12.008

Bioorganic & Medicinal Chemistry 19 (2011) 1277–1284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Phosphonic pseudopeptides as human neutrophil elastase
inhibitors—a combinatorial approach
a,
⇑
a
a
b
_
´
Marcin Sienczyk , Dawid Podgórski , Aleksandra Błazejewska , Julita Kulbacka ,
Jolanta Saczko 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 Medical Biochemistry, Wrocław Medical University, Chalubin´skiego 10, 50-368 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we present a simple and rapid method for the construction of phosphonic peptide mimetic inhibitor
libraries—products of Ugi and Passerini multicomponent condensations—leading to the selection of new
biologically active phosphonic pseudopeptides. As the starting isonitriles, 1-isocyanoalkylphosphonate
diaryl ester derivatives were applied. The structure of the synthesized inhibitors was designed to target
human neutrophil elastase, a serine protease whose uncontrolled activity may lead to development of
several pathophysiological states such as rheumatoid arthritis, cystic fibrosis or tumor growth and inva-
sion. After screening the inhibitory activity of our constructed libraries, the most active compounds were
synthesized as single molecules. One of the obtained inhibitors, Cbz-Met-O-Met-ValP(OC6H4-p-Cl)2, dis-
played apparent second-order inhibition value at 40,105 MÀ1 sÀ1 as the diastereomers mixture. Inhibition
potency and selectivity of action toward other serine proteases as well as the results of initial in vitro
experiments regarding inhibitors influence on cancer cell proliferation are presented.
Received 19 August 2010
Revised 2 December 2010
Accepted 4 December 2010
Available online 9 December 2010
Keywords:
a-Aminoalkylphosphonates
Human neutrophil elastase
Multicomponent reactions
Inhibitors library
Serine protease inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
a-aminoalkylphosphonates are potent, irreversible inhibitors
which react exclusively with the catalytic hydroxyl group of serine
proteases. Good stability and the lack of reactivity with other
classes of proteases such as cysteine, aspartyl, threonine or
metalloproteinases have increased investigations into this class
of inhibitors.8 Moreover, simple modification of the phosphonic
analogue side chain or synthesis of their peptidyl derivatives
allows for the design of specificity between even highly similar
proteases.9
Upon inflammatory stimulation leukocytes release three major
serine proteases: cathepsin G (catG), proteinase 3 (P-3), and
neutrophil elastase (HNE).1 A broad spectrum of substrate specific-
ity and potency of action places elastase among most destructive
proteases in humans.2 The proteolytic activity of HNE in the
healthy body is precisely controlled by natural, endogenous inhib-
itors belonging to the serpin superfamily. The most important
serpins which function as HNE inhibitors are
a
-1 proteinase inhib-
Currently, numerous, structurally diverse a-aminoalkylphosph-
itor ( -1 PI) and
a
a
2-macroglobulin.3 Disruption of the imbalance
onate diphenyl esters have been synthesized and their inhibitory
properties were examined against target proteases including
trypsin,10 granzymes,11 uPA12–14 or DPPIV.15 They have also been
used for the design of activity-based probes able to detect
enzymatically active serine proteases in cell cultures.16,17
between elastase and its inhibitors may lead to development of
diseases such as chronic obstructive pulmonary disease (COPD),
pulmonary emphysema or cystic fibrosis.4 Uncontrolled activity
of HNE may also participate in cancer cell growth and metastasis.5
The process of extracellular matrix degradation by HNE can occur
directly through proteolysis of structural components such as
elastin or collagens, or indirectly via zymogen activation of the
plasminogen–plasmin system.6
However, despite the passage of more than 30 years between
the publication of the first paper concerning the synthesis of
a
-aminoalkylphosphonate diphenyl esters18 no attention has been
paid to the construction of inhibitor libraries based on these
compounds. Unnatural peptidyl derivatives like depsipeptides or
peptides with N-substituted amides may display improved stabil-
ity in plasma since they are less susceptible to degradation by
enzymes.
Due to the structural analogy of
a-aminoalkylphosphonate
diphenyl esters to the transition state observed during the peptide
bond cleavage catalyzed by proteases, this class of compounds
displays interesting inhibitory properties.7 Aromatic esters of
Here we present a simple and effective method for the construc-
tion of
a-aminoalkylphosphonate diphenyl ester-based libraries.
Described methodology and the results presented below represent
an example of this technology application. In light of our previous
⇑
Corresponding author. Tel.: +48 713202439; fax: +48 713284064.
E-mail address: marcin.sienczyk@pwr.wroc.pl (M. Sien
´ czyk).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.008

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M. Sienczyk et al. / Bioorg. Med. Chem. 19 (2011) 1277–1284
1278
research on the synthesis of 1-isocyanoalkylphosphonates we
applied two known multicomponent reactions—Passerini-type
three component condensation and Ugi-type four component
reaction (Fig. 1). The great potential and structural variety of final
products which results from both reactions are limited only by the
researcher’s imagination. In the case of Ugi multicomponent con-
densation the combination of 40 different carboxylic acids, 40 differ-
ent aldehydes, 40 different amines, and 40 different isonitriles leads
to the potential synthesis of 2,560,000 different compounds.19–21
In both Passerini and Ugi reactions we used 1-isocyanoalkyl-
phosphonates as the starting substrates. We included phosphonic
analogues of amino acids substituted at the phenyl ester rings in
the construction of the libraries as these have been shown to in-
crease the potency of inhibitor action.22 We focused on the design
of libraries which target human neutrophil elastase. The selectivity
of inhibitors action was evaluated using chymotrypsin and trypsin.
The data obtained from the first experiments on the influence of
the most active inhibitors on cancer cell proliferation will also be
presented.
allowed to react with the mixture of aldehydes for 2 h at room
temperature. Next, the carboxylic acid component was added fol-
lowed by the addition of isocyanide derivative of phosphonic acid
diaryl ester. Both Passerini and Ugi reactions were performed at
room temperature for 3 days. Each reaction was then quenched
by the addition of 1 equiv of HCl methanol solution and evaporated
to dryness. The residue was dissolved in dimethylsulfoxide in order
to obtain a 10 mM concentration of each of the final products
(assuming 100% reaction yield). These mixtures were next
screened for inhibitory activity toward human neutrophil elastase
using the incubation method. All the libraries were subsequently
diluted to select the most active compounds. The starting isocya-
nides were also examined and no inhibition was observed in any
case.
From the prepared heat-maps we were able to select inhibitor
mixtures with the highest potency of action. In the next step po-
tential inhibitors were synthesized as pure compounds in the same
manner. Their ability to inhibit the proteolytic activity of human
neutrophil elastase as well as the selectivity of action towards
other serine proteases was examined. The heat-map analysis
showed high diversity of inhibitory activity among constructed
libraries. The most active compound among all synthesized were
phosphonic depsipeptides (Passerini products, Fig. 2). The Ugi
products (Fig. 3) displayed lower inhibition properties as compared
to the Passerini-based products. In both types of condensation
products the highest inhibition was observed for derivatives of
2. Results and discussion
Using N-Cbz-protected
a-aminoalkylphosphonate diaryl esters
as the starting compounds we synthesized several isocyanide
derivatives which were subsequently used in the multicomponent
reactions of both types. The isocyanides used in the presented
studies were phosphonic analogues of Val, Ala, and Abu. The
amines and the carbonyl components which were used for the
inhibitor libraries construction were selected to obtain high struc-
tural diversity of target molecules (aliphatic, aromatic, and bulky
groups). That allowed us to determine the applicability and limita-
tion of the method itself. Also an introduction of structurally diver-
sified groups could led to discovery of possibly new recognition
sites between target enzyme and the inhibitor molecule which
could guide us for future development of elastase (as well as other
serine proteases) inhibitors.
The libraries were constructed in order to obtain four desired
compounds in one reaction mixture. One library contained 24 reac-
tion mixtures with 4 products in each mixture (the total number
was 96 compounds per library). Firstly, two mixtures composed
of four different aldehydes in methanol were prepared. For the
Passerini reaction 1 equiv of a carboxylic acid substrate methanol
solution was added followed by the addition of 1 equiv of the
aldehyde mixture (0.25 equiv of each aldehyde). The solution
was allowed to react for 10 min, then 1 equiv of 1-isocyanoalkyl-
phosphonic diaryl ester was added. In the case of the Ugi multi-
component condensation, 1 equiv of amine component was
1-isocyano-2-methylpropanephosphonic
acid
di(4-S-methyl-
phenyl) ester (1d) although the Passerini-based library displayed
higher potency. Interestingly, a library based on 1-isocyano-2-
methylpropanephosphonic acid di(4-chlorophenyl) ester (1f)
showed the highest potency among all synthesized mixtures. The
higher activity of dichlorophenyl esters as compared to diphenyl
esters may be the result of the electrowithdrawing properties of
the chlorine atom facilitating release of the p-Cl-phenoxy group.
For the most active inhibitor mixtures single compounds
were synthesized separately and their inhibitory properties were
re-examined. Analysis of pure, single compounds showed that the
most active derivative among all of the synthesized was Cbz-Met-
O-Met-ValP(O-C6H4-4-Cl)2 (P5) which displayed kobs/[I] value at
40,105 MÀ1 sÀ1 being absolutely selective against chymotrypsin
and trypsin (Table 1). From the same group of inhibitors compound
P3 (Cbz-Met-O-Val-ValP(O-C6H4-4-Cl)2 showed good potency of
action (kobs/[I] value 20,500 MÀ1 sÀ1). Even compound P3 had no
activity against trypsin, it inhibited chymotrypsin (15%) at 2.5 lM
after 30-min incubation. Other compounds of this group displayed
moderate or poor potency of action. Parallel derivatives (Table 2)
which differed only at the para substituent (methylthio group
R
R1
O
P
N
C
O
O
O
O
R
R3 OH
R2
H
O
O
O
R2
H
R3 OH
R2
H
NH2
HN
O
R4
NH
A
C
R3 OH
B
O
N
R
O
R1
O
R4
O
N
R
R3
O
R
R1
R3
O
R1
O
O
P
N
H
R3
O
N
O
O
O
P
O
R2
N
P
N
H
H
O
O
O
R2
R2
R
R
R
Figure 1. Schematic representation of the Passerini-type (A) and Ugi-type (B and C) multicomponent condensation used for construction of inhibitor libraries based on the of
1-isocyanoalkylphosphonate diaryl ester moiety.

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M. Sienczyk et al. / Bioorg. Med. Chem. 19 (2011) 1277–1284
Figure 2. Schematic representation of heat-maps obtained for Passerini-based inhibitor libraries of phosphonic pseudopeptides. Each square represents a mixture of four
products. MIX 1—equimolar mixture of four aldehydes (phenylpropionaldehyde, isobutylaldehyde, isovaleraldehyde, and phenylacetaldehyde), MIX 2—equimolar mixture of
4-nitrobenzaldehyde, 4-fluorobenzaldehyde, 3,4-dimethoxybenzaldehyde and 3-(methylthio)propionaldehyde. The percent of inhibition represents a ratio of enzyme activity
after incubation with the inhibitors mixture into the control sample. Typical serial dilutions (2.5
which allowed for the selection of the most active compounds.
lM, 0.125 lM, and 0.0625 lM) show decreased level of inhibitory activity
Figure 3. Schematic representation of heat-maps obtained for Ugi-based inhibitor libraries of phosphonic pseudopeptides. Details as described for Figure 2.

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M. Sienczyk et al. / Bioorg. Med. Chem. 19 (2011) 1277–1284
1280
Table 1
Inhibitory activity of phosphonic peptide-mimetics obtained by the application of Passerini-type multicomponent condensation—derivatives of 1-isocyano-2-methylpropane-
phosphonic acid di(4-chlorophenyl) ester
S
O
O
Cl
O
O
P
O
N
H
N
H
O
O
O
R
Cl
Compound
R
kobs/[I] (MÀ1 sÀ1
)
HNE
Chymotrypsina,b
Trypsinb
P1
P2
P3
P4
P5
P6
P7
P8
–CH2CH2C6H5 (hPhe)
–CH2CH(CH3)2 (Leu)
–CH(CH3)2 (Val)
1620
7250
20,500
695
40,105
165
NI
NI
15%
NI
NI
21%
NI
16%
NI
NI
NI
NI
NI
NI
NI
NI
–CH2C6H5 (Phe)
–CH2CH2SCH3 (Met)
–C6H3(m,p-OCH3)2 (3,4-OMe)Phg
–C6H4(p-NO2) (4-NO2)Phg
–C6H4(p-F) (4-F)Phg
165
215
a
The Percent of enzyme inhibition after incubation for 30 min at 37 °C with 2.5
NI—no inhibition was observed after 30 min incubation of enzyme with the inhibitor (2.5 M) at 37 °C.
l
M of inhibitor relative to the control sample.
b
l
Table 2
Inhibitory activity of phosphonic peptide-mimetics obtained by the application of Passerini-type multicomponent condensation—derivatives of 1-isocyano-2-methylpropane-
phosphonic acid di(4-S-methylphenyl) ester
S
O
O
S
O
O
P
O
N
H
N
H
O
O
O
R
S
Compound
R
kobs/[I] (MÀ1 sÀ1
)
HNE
Chymotrypsina,b
Trypsinb
P9
–CH2CH2C6H5 (hPhe)
–CH2CH(CH3)2 (Leu)
–CH(CH3)2 (Val)
–CH2C6H5 (Phe)
–CH2CH2SCH3 (Met)
–C6H3(m,p-OCH3)2 (3,4-OMe-Phg)
–C6H4(p-NO2) (4-NO2-Phg)
–C6H4(p-F) (4-F-Phg)
765
4400
5050
605
5900
3800
430
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
P10
P11
P12
P13
P14
P15
P16
15%
NI
14%
22%
NI
4850
NI
a
The Percent of enzyme inhibition after incubation for 30 min at 37 °C with 2.5 lM of inhibitor relative to the control sample.
b
NI—no inhibition was observed after 30 min incubation of enzyme with the inhibitor (2.5
lM) at 37 °C.
instead of chlorine atom) displayed lower inhibition values.
Compound P13 (Cbz-Met-O-Met-ValP(O-C6H4-4-S-Me)2 was almost
sevenfold less reactive than compound P5 (kobs/[I] = 5900 MÀ1 sÀ1),
Table 3
Inhibitory activity of phosphonic peptide-mimetics obtained by the application of
Ugi-type multicomponent condensation—derivatives of 1-isocyano-2-methylpro-
panephosphonic acid di(4-S-methylphenyl) ester
but it also inhibited chymotrypsin (14% of inhibition, at 2.5 lM
after 30-min incubation). Similarly, derivative P11 was approxi-
mately fourfold less potent than 4-chloro derivative P3
(kobs/[I] = 5050 MÀ1 sÀ1) displaying similar activity toward chymo-
trypsin. Interestingly, an improvement of inhibitory potency was
observed for compounds P14 (kobs/[I] = 3800 MÀ1 sÀ1) and P16
(kobs/[I] = 4850 MÀ1 sÀ1) when compared to their 4-chloro ana-
O
O
S
N
O
P
O
N
H
N
H
O
O
O
R
S
logues P6 and P8 (kobs/[I] values 165 MÀ1 sÀ1 and 215 MÀ1 sÀ1
,
Compound
R
kobs/[I] (MÀ1 sÀ1
)
respectively).
HNE
Chymotrypsina,b
Trypsinb
The Ugi-derived products (U1–U8) displayed poor activity against
tested serine proteases (Tables 3 and 4). The most potent among
them were Cbz-Gly-(N-n-butyl)-hPhe-ValP(OC6H4-p-S-Me)2 (U1,
kobs/[I] = 1415 MÀ1 sÀ1) and Cbz-Gly-(N-n-butyl)-Phe-ValP(OC6H4-p-
S-Me)2 (U4, kobs/[I] = 1415 MÀ1 sÀ1) but they also inhibited chymo-
trypsin after incubation.
U1
U2
U3
U4
–CH2CH2C6H5 (hPhe)
–CH2CH(CH3)2 (Leu)
–CH(CH3)2 (Val)
1415
145
495
15%
NI
8%
NI
NI
NI
NI
–CH2C6H5 (Phe)
1360
14%
a
The Percent of enzyme inhibition after incubation for 30 min at 37 °C with
2.5
lM of inhibitor relative to the control sample.
b
In general, the obtained compounds displayed lower inhibitory
activity as compared to previously published highly potent
NI—no inhibition was observed after 30 min incubation of enzyme with the
inhibitor (2.5 lM) at 37 °C.

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M. Sienczyk et al. / Bioorg. Med. Chem. 19 (2011) 1277–1284
Table 4
Inhibitory activity of phosphonic peptide-mimetics obtained by the application of modified Ugi-type multicomponent condensation—derivatives of 1-isocyano-
2-methylpropanephosphonic acid di(4-S-methylphenyl) ester
O
N
O
S
N
O
P
N
H
O
O
R
S
Compound
R
kobs/[I] (MÀ1 sÀ1
)
HNE
Chymotrypsina
Trypsina
U5
U6
U7
U8
–CH2CH2SCH3 (Met)
140
125
140
330
250
NI
NI
NI
NI
NI
NI
–C6H3(m,p-OCH3)2 (3,4-OMe-Phg)
–C6H4(p-NO2) (4-NO2-Phg)
–C6H4(p-F) (4-F-Phg)
NI
a
NI—no inhibition was observed after 30 min incubation of enzyme with the inhibitor (2.5 lM) at 37 °C.
phosphonic inhibitors of human neutrophil elastase. Nevertheless,
it is necessary to highlight that the Ugi and Passerini products
obtained here are mixtures of diastereoisomers. Regardless of the
fact that these are mixed compounds, our results place some of
them among the most active HNE diarylphosphonate inhibitors.
Additionally, a major advantage of this new class of inhibitors
may be their increased stability due to the limited susceptibility
by proteases.
the most active was compound P2 which at 500
75% of cancer cells growth (Fig. 4).
l
M inhibited
It is important to note that an increased growth of fibroblasts
was observed after treatment with all tested compounds where
no such effect was observed for the A549 cell line. These results
could represent the compounds influence on matrix dehydroge-
nase expression level which activity is measured by the MTT
assay.
Passerini products (P2, P3, P5, P13) and Ugi products (U1, U2,
U4), which displayed high potency of human neutrophil elastase
inhibition were selected for in vitro studies. The preliminary data
regarding the influence of synthesized inhibitors on normal and
cancer cell growth showed that the highest inhibitory effect was
Additionally, when the control compound 4-MeO-C6H4-CO-Val-
Pro-ValP(OC6H4-4-S-CH3)2 was used8—a classic phosphonic peptide
derivative, potent inhibitor of human neutrophil elastase (kobs
/
[I] = 207,000 MÀ1 sÀ1) no toxic effect against A549 cells was ob-
served. That may prove the advantage of phosphonic peptide-
mimetics (Passerini or Ugi products) over classic peptidyl
derivatives.
observed for Ugi-derived products. The treatment showed
dose-dependent effect. Compound U1 showed the greatest effect
on cancer cell (A549), where concentrations between 125
and 500 M inhibited more than 50% of cell growth. Importantly,
a
lM
The methodology described here allows us to obtain and screen
new phosphonic-based peptide-mimetics in great number within
just a few days. We synthesized and screened more than 2100
new compounds which led to select new potent and selective
inhibitors of human neutrophil elastase.
Our presented work describes an example of the application of
phosphonic isocyanide-based combinatorial chemistry methods
for inhibitor library construction. It also suggested the possibility
of generating libraries other than Passerini or Ugi multicomponent
condensation products by the application of phosphonic isonitriles.
Moreover, just by simply changing the P1 side chain the construc-
tion of inhibitors toward other serine proteases could be
generated. Our laboratory is currently investigating the library
construction method optimization as well as its application for
inhibitors of different serine proteases synthesis and our results
will be published in due course.
l
exposure of cells to 250
lM of U1 was not toxic to normal fibro-
blasts whereas even 125
lM was toxic to cancer cells (Fig. 4). For
all tested compounds the toxicity level for HGF-s cells was approx-
imately 500 M, however a much lower concentration was re-
quired to induce a toxic effect in A549 cancer cells. Treatment
with compound U1 at 250 M decreased cell growth by 70% in
l
l
A549 cells, and by 10% in HGF-s cells.
Passerini products, although displaying a toxic effect toward
normal human fibroblasts at high concentrations showed moder-
ate inhibition of cancer cells at 125 lM and 250 lM. Following
treatment of cells with compound P5 (the most potent human neu-
trophil elastase inhibitor obtained in presented research) at
500
lM decreased cell growth by 60%, and at 250 lM by 25% in
A549 cells. Among all the tested Passerini condensation products
Figure 4. Lung cancer cell line A549 (A) and normal fibroblasts (B) growth inhibition by obtained HNE phosphonic peptide-mimetics. As the control compound 4-MeO-C6H4-
CO-Val-Pro-ValP(OC6H4-4-S-CH3)2 was applied.

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M. Sienczyk et al. / Bioorg. Med. Chem. 19 (2011) 1277–1284
1282
with characteristic isocyanate odor was obtained (1a–1h, see
3. Materials and methods
Supplementary data).
Carboxylic acids, phenols, triphenyl phosphite, phosphorus
chloride, phosphorus oxychloride, and all solvents were purchased
from Sigma–Aldrich. Aldehydes were purchased from Lancaster.
Protected amino acids were purchased from IRIS Biotech GMBH.
3.2. Inhibitor library synthesis
Synthesis of all libraries was performed in dry methanol. The
final concentration of the reagents was kept as high as possible
in order to increase the yield of the final products.
3.1. Chemical synthesis
Starting phosphites (except commercially available triphenyl
phosphite), Cbz-protected aminophosphonates, their N-formyl
derivatives, and 1-isocyanoalkylphosphonates were prepared as
described previously.18,23
3.2.1. Synthesis of Passerini-based library
All substrates were dissolved in dry methanol to a 5 mM final
concentration. In the reaction tubes 1 mmol of equimolar mixture
(MIX I) of aldehydes was added into tubes labeled 1–12, and MIX II
into tubes 13–24 (Fig. 2). To each tube 1 mmol of carboxylic com-
pound methanol solution was added as follows: Cbz-Gly-OH (tubes
1 and 13), Cbz-Ala-OH (tubes 2 and 14), Cbz-Aib-OH (tubes 3 and
15), Cbz-Leu-OH (tubes 4 and 16), acetic acid (tubes 5 and 17),
Cbz-Met-OH (tubes 6 and 18), Cbz-Phe-OH (tubes 7 and 19), phen-
ylacetic acid (tubes 8 and 20), phenylbutyric acid (tubes 9 and 21),
hexanoic acid (tubes 10 and 22), cyclohexylacetic acid (tubes 11
and 23), and cyclohexylpropionic acid (tubes 12 and 24). The
mixtures were allowed to react at room temperature for 15 min
and 1-isocyanoalkylphosphonate diaryl ester methanolic solution
was added into all 24 reaction tubes. The mixtures were left at
room temperature for 3 days. Into each of the tube 1 mmol of
HCl in methanol was added to quench the reaction. The volatile
components were evaporated and remaining residue was dissolved
in dimethylsulfoxide (2.5 ml) to obtain 100 mM concentration of
each target compound of the mixture. The solutions were kept at
À86 °C prior to use in library enzymatic inhibition studies.
3.1.1. General procedure for synthesis of substituted at the
phenyl ester rings a-N-Cbz-protected aminophosphonates
In general, 0.03 mol of substituted phenol was dissolved in
30–40 ml of acetonitrile and 0.01 mol of phosphorus chloride
was added. After refluxing the mixture for 4 h the volatile elements
were evaporated and the crude phosphite was used in the next
step. To the crude phosphite 0.01 mol of benzyl carbamate,
0.012 mol of an aldehyde, and 25 ml of glacial acetic acid were
added. The reaction was performed at 80 °C for 2 h. After evapora-
tion the resulting oily residue was dissolved in 100 ml of methanol
and left for crystallization at À20 °C resulting in the desired
a-N-Cbz-protected aminophosphonate aromatic esters in 30–60%
yield. If necessary, re-crystallization was performed by dissolving
the product in hot chloroform (5 ml), addition of 50 ml methanol
and leaving at À20 °C.
3.1.2. General procedure for synthesis of
phosphonate diaryl esters
a-N-formyl-amino-
3.2.2. Synthesis of Ugi-based library
All the substrates were dissolved in methanol to the final con-
centration at 5 mM. Into all 24 reaction tubes 1 mmol of appropri-
ate amine (Fig. 3) and 1 mmol of equimolar mixture of aldehydes
(1–12 mixture I, 13–24 mixture II) were added. The reactions were
left to react for 2 h at room temperature. After the carboxylic sub-
strate solution (1 mmol) was added into each tube as described
above. Next, a solution of 1-isocyanoalkylphosphonate diaryl ester
was added into each tube (1 mmol) and the reaction was left for
3 days at room temperature. The reaction was quenched with
HCl/MeOH (1 mmol) and evaporated to dryness. The mixtures
were dissolved in DMSO (2.5 ml) and stored at À86 °C until the
measurement of their inhibitory properties.
First, the N-Cbz protective group was removed with 33% HBr in
acetic acid solution—0.5 mmol of -N-Cbz-aminophosphonate dia-
a
ryl ester was dissolved in 2 ml of 33% HBr/AcOH. The reaction was
performed at room temperature for 2 h. The volatile elements were
evaporated and the resulting product was crystallized from a
MeOH/diethyl ether system. The product was than filtered and
air-dried. In the next step hydrobromide salt of 1-aminoalkyl-
phosphonate diaryl ester (0.02 mol) was dissolved in methylene
chloride (25 ml) in the presence of triethylamine (0.1 mol) and ace-
tic-formic anhydride (0.03 mol) was added slowly at room temper-
ature. The progress of the reaction was monitored by TLC. After the
reaction was completed the mixture was evaporated, dissolved in
ethyl acetate (the precipitating salt was filtered off), and washed
with 5% NaHCO3 aq and brine. After drying over Na2SO4 the solvent
was evaporated and the resulting N-formyl derivative was used
directly for the isocyanide synthesis (F1–F8, see Supplementary
data).
3.3. Single inhibitor synthesis
The single inhibitors were synthesized similarly to the method
described above. The difference was that a single aldehyde was
used instead of aldehyde mixture.
3.3.1. Synthesis of Passerini-based single product
3.1.3. 1-Isocyanoalkylphosphonate diaryl esters synthesis
An aldehyde (10 mmol) and a carboxylic acid (10 mmol) were
dissolved in methanol. After 15 min 1-isocyanoalkylphosphonate
diaryl ester was added into the reaction mixture. The solution
was left at room temperature for 3 days with gentle stirring. After
quenching the reaction with HCl/MeOH (10 mmol) volatile
elements were evaporated and the residue was dissolved in ethyl
acetate. The organic solution was washed with 5% NaHCO3, 5%
citric acid aqueous solution and finally with brine. After drying
over MgSO4 the solvent was evaporated and the product was puri-
fied by column chromatography on silica gel using ethyl acetate/
chloroform mixture as the eluent (P1–P16, see Supplementary
data).
a
-N-Formyl-aminoalkylphosphonate diaryl ester (0.01 mmol)
was dissolved in freshly distilled, dried over P2O5, dichlorometh-
ane (30–40 ml) and triethylamine (0.2 mmol) was added. The
mixture was cooled to 0 °C and POCl3 (0.04 mmol) was added
dropwise. The progress of the reaction was monitored by TLC.
After completion the reaction was quenched by slow addition of
saturated aqueous solution of NaHCO3. The mixture was extracted
three times with NaHCO3 and brine. After drying over Na2SO4 the
solvent was evaporated. Resulting crude product was dissolved in
minimal volume of chloroform and passed through the layer of
silica gel to remove all polymeric impurities. TLC analysis showed
a single spot. The solvent was evaporated and the oily product

´
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M. Sienczyk et al. / Bioorg. Med. Chem. 19 (2011) 1277–1284
3.3.2. Synthesis of Ugi-based single product
going extraction procedure. The gingival biopsies were provided by
the Department of Dental Surgery of Wroclaw Medical University.
The experiments were conducted in accordance with the require-
ments of the Bioethics Commission of Wroclaw Medical University.
The human gingival fibroblasts were isolated from healthy gingival
tissues according to the procedure described previously.24
HGF’s were grown in Dulbecco’s modified Eagles’ medium
(Sigma) supplemented with 10% fetal bovine serum and glutamine
with penicillin/streptomycin (Sigma) in 25-cm2 flasks (Nunc). The
cells were maintained in a humidified atmosphere at 37 °C and 5%
CO2. For experimental purposes, the cells were removed by
trypsinization (0.25% Trypsin–EDTA, Sigma).
A mixture of an amine (10 mmol) and an aldehyde (10 mmol)
was left to react for 2 h at room temperature. To this mixture
carboxylic derivative (10 mmol) and 1-isocyanoalkylphosphonate
diaryl ester were added, respectively. The reaction was performed
at room temperature for 3 days with gentle stirring. The reaction
was stopped by addition of HCl/MeOH solution, evaporated to dry-
ness and re-dissolved in ethyl acetate. After extraction with 5%
NaHCO3, citric acid, and brine, the solution was dried over magne-
sium sulfate. The solvent was evaporated and the resulting product
was purified on silica gel using ethyl acetate/chloroform system
(U1–U8, see Supplementary data).
Human lung adenocarcinoma cell line (A549) was obtained from
ATCC. The cells were grown in DMEM (Lonza) with addition of 10%
fetal bovine serum (BioWhittaker, Lonza) and supplemented with
antibiotics (penicillin/streptomycin, Lonza). For the experiments
the cells were removed by trypsinization (0.25% Trypsin–EDTA
solution, Sigma) and washed with PBS (IITD, PAN, Poland). The cells
were maintained in a humidified atmosphere at 37 °C and 5% CO2.
3.4. Enzymatic studies
The rates of inhibition of human neutrophil elastase
(Biocentrum, Poland) and chymotrypsin (Calbiochem) was mea-
sured in a 0.1 M HEPES, 0.5 M NaCl, 0.03% Triton X-100 (pH 7.5);
inhibition of trypsin (Sigma–Aldrich) was measured in 0.1 M HEPES,
0.01 M CaCl2, pH 7.5. All enzymes were assayed using fluorogenic
substrates: MeO-Suc-Ala-Ala-Pro-Val-AMC (Ex. 350 nm, Em.
460 nm, Calbiochem) for human neutrophil elastase, Suc-Ala-Ala-
Pro-Phe-AMC (Ex. 350 nm, Em. 460 nm, Calbiochem) for chymo-
trypsin, and Bzl-Arg-AFC (Ex. 380 nm, Em. 505 nm, Sigma–Aldrich)
for trypsin. The inhibitory activity was measured by incubation
method under pseudo-first order conditions as described.9 The stan-
dard deviation for presented values is the mean of three indepen-
dent experiments and does not exceed 10%. All measurements
were performed using Spectra Max Gemini XPS microplate reader
(Molecular Devices).
3.6. Cell viability assay
Stock solutions of elastase inhibitors were prepared in DMSO
and stored at À86 °C. On the day of the treatment, solutions of
tested compounds were prepared in cell culture medium at differ-
ent concentrations. Control cell cultures were treated with the
equivalent amount of DMSO only. The MTT assay (Sigma) was used
to test mitochondrial metabolic function. Cells were seeded into
96-well microculture plates at 1 Â 104 cells/well and allowed to at-
tach overnight. After incubation with various concentrations of the
inhibitors, the assay was performed according to the manufac-
turer’s protocol. The absorbance was measured using a multiwell
scanning spectrophotometer at 570 nm (TECAN, Infinite 200). The
results were expressed as the percentage of viable cells relative
to untreated control cells. The cytotoxicity test was performed
after 48 h of incubation with inhibitors. The average values were
plotted as concentration of chemical versus percentage growth rel-
ative to control ( one standard deviation, indicated by error bars).
3.4.1. Library screening
The screening of inhibitory activity of prepared libraries was
done by the incubation method. A stock DMSO solution of each
inhibitor mixture was diluted with an assay buffer to obtain con-
centration in a range from 2.5
Next, 50 l of the inhibitor solution was incubated with 100
enzyme solution for 30 min at 37 °C. Final human neutrophil elas-
tase concentration was 0.01 U. Then, 50 l of the substrate buf-
fered solution (240 M, MeO-Suc-Ala-Ala-Pro-Val-AFC) was
l
M to 62.5 nM of tested compounds.
l
ll of
l
l
Acknowledgments
added into each well. The final concentration of DMSO in all sam-
ples was kept at 2.5%. The control sample contained only buffers
with 2.5% DMSO. The measurement of inhibition level was read
over 15 min in triplicate. The percent of inhibition was calculated
in reference to the control sample.
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.
Supplementary data
3.4.2. Single inhibitor activity assay
The stock DMSO solution (10 mM) of the inhibitor was diluted
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.008.
with the assay buffer in the range between 5
lM and 5 nM. Fifty
microliters of the inhibitor was incubated with 100
l
l of the
enzyme solution for 30 min at 37 °C. Final enzyme concentrations
were as follows: 0.01 U (human neutrophil elastase), 1.25 nM
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