Human neutrophil elastase phosphonic inhibitors with improved potency of action

Article abstract of DOI:10.1021/jm300599x

Herein, we present the synthesis and the measurement of the inhibitory activity of novel peptidyl derivatives of α-aminoalkylphosphonate diaryl esters as human neutrophil elastase inhibitors. Their selectivity against other serine proteases, including porcine pancreatic elastase, chymotrypsin, and trypsin, was also demonstrated. We also describe the preparation of single peptide diastereomers. The most active and selective compound developed possessed a k_inact/K_I of 2353000 M^-1 s ^-1, which is the most potent irreversible peptidyl inhibitor of human neutrophil elastase reported to date. The peptidyl inhibitors were demonstrated to be stable in PBS buffer and human plasma, as were their complexes with HNE.

Full text of DOI:10.1021/jm300599x

Article
pubs.acs.org/jmc
Human Neutrophil Elastase Phosphonic Inhibitors with Improved
Potency of Action
Łukasz Winiarski, Jozef Oleksyszyn, and Marcin Sienczyk*
́
́
Department of Chemistry, Division of Medicinal Chemistry and Microbiology, Wroclaw University of Technology, Wybrzeze
Wyspianskiego 27, 50-370 Wroclaw, Poland
S
* Supporting Information
ABSTRACT: Herein, we present the synthesis and the measurement of the inhibitory activity of novel peptidyl derivatives of α-
aminoalkylphosphonate diaryl esters as human neutrophil elastase inhibitors. Their selectivity against other serine proteases,
including porcine pancreatic elastase, chymotrypsin, and trypsin, was also demonstrated. We also describe the preparation of
single peptide diastereomers. The most active and selective compound developed possessed a kinact/KI of 2353000 M−1 s−1,
which is the most potent irreversible peptidyl inhibitor of human neutrophil elastase reported to date. The peptidyl inhibitors
were demonstrated to be stable in PBS buffer and human plasma, as were their complexes with HNE.
adult respiratory syndrome, and chronic bronchitis.13,14
Importantly, the number of deaths attributed to COPD has
INTRODUCTION
■
Human neutrophil elastase (HNE, EC 3.4.21.37; also referred
increased rapidly in recent years, and it is now considered one
to as human leukocyte elastase, HLE) belongs to the
of the major worldwide causes of mortality.15−17 In addition to
chymotrypsin-like serine protease superfamily and is one of
its essential role in the development of pulmonary inflamma-
tory disorders, HNE has also been demonstrated to contribute
to the metastasis and progression of some cancers via its
degradation of extracellular matrix components.18 Clearly, lack
of an effective therapy to control the activity of HNE may
complicate the treatment of chronic diseases. Thus, there is a
need to develop novel biologically active molecules that target
the proteolytic function of HNE to allow for more effective
treatments for chronic pulmonary inflammation and metastatic
cancer.
Various synthetic HNE inhibitors such as chloromethyl
ketones, trifluoromethyl ketones, sivelestat sodium hydrate
(ONO-5046, Elaspol), N-benzoylpyrazoles, benzoxazinones,
and thiadiazolidines have been developed over the past few
decades.19−24 Most of these elastase inhibitors were designed to
include a β-lactam scaffold in their structure, and some were
determined to be orally available and stable in aqueous
solutions.25−29 Unfortunately, none of the β-lactams specific
the most destructive enzymes present in the human body. It
can easily degrade almost every component of the extracellular
matrix, including elastin, collagen types I−IV, proteoglycans,
fibronectin, and laminin.1−5 Neutrophil elastase is stored in the
azurophilic granules of polymorphonuclear leukocytes and is
secreted along with reactive oxygen species, cationic peptides,
and eicosanoids after an inflammatory stimulation.1 One of the
major physiological functions of HNE is host defense against
invading microbial pathogens.1 It was also found that elastase
can lead to activation of metalloproteinases and up-regulation
of inflammation.6−8 Under normal physiological conditions the
proteolytic activity of elastase is controlled by natural
endogenous serine protease inhibitors (serpins) such as α1-
proteinase inhibitor (α1-PI), secretory leukocyte peptidase
inhibitor (SLPI), and α2-macroglobulin (α2-MG).9−11 The
imbalance between active elastase and its specific endogenous
inhibitors is in part the result of the so-called “oxidative burst”
of neutrophils which destroys serpins.12 The disruption of this
delicate balance between active elastase and its inhibitors may
lead to the development of pulmonary inflammatory disorders
such as chronic obstructive pulmonary disease (COPD), acute
respiratory distress syndrome (ARDS), pulmonary emphysema,
Received: April 30, 2012
Published: June 21, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of Phosphonic Peptides with General Formula Boc-Val-Pro-AaaP(OAr)2
a
Reagents and conditions: (a) PCl3, MeCN, reflux, 6 h; (b) benzyl carbamate, R2CHO, AcOH, 80−90 °C, 2 h; (c) 33% HBr/AcOH, 22 °C, 2 h; (d)
N-Boc-L-Pro-OH, HBTU, DIPEA, MeCN, 12 h, 22 °C; (e) 50% TFA/DCM (v/v), 2 h, 22 °C; (f) N-Boc-L-Val-OH, HBTU, DIPEA, MeCN, 12 h,
22 °C.
Scheme 2. Synthesis of Cbz- or Boc-Protected Phosphonic Peptides with General Structure Cbz/Boc-Aaa-Pro-ValP(OC6H4-S-
a
CH3)2
a
Reagents and conditions: (a) PCl3, MeCN, reflux, 6 h; (b) benzyl carbamate, (CH3)2CHCHO, AcOH, 80−90 °C, 2 h (60%); (c) 33% HBr/AcOH,
2 h, 22 °C (95%); (d) N-Boc-L-Pro-OH, HBTU, DIPEA, MeCN, 12 h, 22 °C (88%); (e) 50% TFA/DCM (v/v), 2 h, 22 °C; (f) N-Cbz/Boc-Aaa-
OH, HBTU, DIPEA, MeCN, 12 h, 22 °C.
for HNE have successfully passed clinical trials for use in
human patients.
analysis of the P1−P4 positions of the inhibitor and analysis of
the structural requirements for the phosphonate diaryl ester
have yet to be performed. Moreover, the inhibitory activities of
single phosphonic peptide diastereoisomers toward HNE have
yet to be reported. The most potent peptidyl phosphonic
inhibitor of neutrophil elastase reported thus far is Boc-Val-Pro-
ValP(OPh)2, which displayed a kobs/[I] of 27000 M−1 s−1. Our
previous studies on simple Cbz-protected phosphonic HNE
inhibitors showed that the preferred amino acid analogues in
the P1 position are the phosphonic analogues of Abu and Val.37
In this report, we describe the development of peptidyl
phosphonic-type HNE inhibitors designed on a Val-Pro-
AaaP(OAr)2 scaffold that display improved potency of action.
Additionally, since the starting phosphonic analogues of amino
acids were obtained as racemic mixtures, we were able to isolate
single diastereoisomers that displayed high inhibition activity.
As a result, several new highly potent human neutrophil elastase
inhibitors that displayed kinact/KI of 106 M−1 s−1 were
developed. Moreover, since 1-aminoalkylphosphonate diaryl
esters and their peptidyl derivatives have poor solubility in
aqueous media that could limit their practical application, we
also generated water-soluble derivatives.
Aromatic esters of 1-aminoalkylphosphonates (1-AAP) and
their peptidyl derivatives are well-known inhibitors of serine
proteases.30 They irreversibly bind to the active site serine with
high specificity and selectivity of action. In this respect, they
differ significantly from previously reported elastase inhibitors.
They also lack the ability to inactivate cysteine, threonine, and
metalloproteinases. Importantly, the reactivity of 1-AAP
peptidyl derivatives can be easily adjusted by modification of
their amino acid sequences, allowing for the development of
compounds that target one specific protease but are inactive
against structurally and/or functionally similar serine proteases.
In addition, through the introduction of different substituents
into phenyl ester rings, the electrophilic properties of the
phosphorus atom can be modulated.31,32 In this fashion,
peptidyl derivatives of phosphonic amino acid analogues have
been developed that display high potency of action toward
serine proteases including trypsin, thrombin, uPA, DPPIV,
granzymes, or kallikreins as well as bacterial proteases
containing noncanonical catalytic triad such as subtilisin.32−36
Although phosphonic inhibitors of HNE have been reported
for at least 30 years, an in-depth structure−activity relationship
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a
Scheme 3. Synthesis of Peptide 1-Aminoalkylphosphonates Containing a Hydroquinone Moiety
a
Reagents and conditions: (a) tert-butyl bromoacetate, NaOH, dioxane/H2O, N2, 22 °C, 1 h (35%); (b) Na2CO3, 40, DMF, 22 °C, 24 h (73%); (c)
Pd/C, H2, AcOEt, 22 °C (92%); (d) HBTU, DIPEA, MeCN, 12 h, 22 °C (40%); (e) TFA, CH2Cl2, 2 h, 22 °C (86%).
a
Scheme 4. Synthesis of Peptide 1-Aminoalkylphosphonates Containing a Thymine or Uracil Moiety
a
Reagents and conditions: (a) Na2CO3, 40, DMF, 24 h, 22 °C (42%); (b) tert-butyl bromoacetate, NaH, 2 h, 0−22 °C (58%); (c) Pd/C, H2, AcOEt,
22 °C (88%); (d) HBTU, DIPEA, MeCN, 12 h, 22 °C; (e) TFA, CH2Cl2, 2 h, 22 °C.
amino acids as previously described.37 Briefly, the starting
triaryl phosphite was prepared from phosphorus trichloride and
appropriate phenol in refluxing acetonitrile. The obtained crude
triaryl phosphite was used in the next step without purification.
CHEMISTRY
■
As shown in Scheme 1, the synthesis of peptidyl derivatives of
1-aminoalkylphosphonate diaryl esters (1−27) began with the
preparation of simple Cbz-protected phosphonic analogues of
The Cbz-protected phosphonates were obtained as racemic
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mixtures in an α-amidoalkylation reaction of triaryl phosphite
with benzyl carbamate and an aldehyde and were crystallized
from cold methanol. After removal of the Cbz protective group
using a 33% solution of hydrobromic acid in acetic acid, the
resulting N-deprotected phosphonates were crystallized from
methanol/diethyl ether and were coupled with N-Boc-L-Pro-
OH in acetonitrile using HBTU in the presence of DIPEA as a
coupling reagent.36 Deprotection of the Boc group was
performed in 50% trifluoroacetic acid in dichloromethane,
and coupling with N-Boc-L-Val-OH was achieved using the
HBTU/DIPEA method as described above, generating
derivatives 1−27.
compounds that displayed HNE inhibition at a minimum of
40% after 15 min of incubation with enzyme, we calculated
k
curves in the absence of inhibitor were linear. The standard
deviation for the presented values was calculated using the
mean of three independent experiments and does not exceed
10%. A typical progress curve (for compound 44) at different
inhibitor concentrations is presented in Figure 1.
inact/KI values using a linear model of inhibition.39,40 Control
In order to obtain derivatives 28−38 with different amino
acid residues in the P3 position or in the fixed P2 and P1
positions, a similar approach was applied (Scheme 2). In short,
after deprotection of the Cbz-group in Cbz-ValP(O-C6H4-S-
CH3)2, the resulting phosphonate was coupled first with N-Boc-
L-Pro-OH, the Boc group was removed, and the resulting
dipeptide was conjugated to different Boc- or Cbz-protected
amino acids including glycine, alanine, aminoisobutyric acid,
valine, leucine, isoleucine, methionine, or serine.
The synthesis of Boc-Val-Pro-ValP(O-C6H4-S-CH3)2 deriva-
tives (44, 45, 54, 55) with improved solubility in aqueous
media started with the removal of the Boc-protective group in
24, and the resulting tripeptide was then coupled using HBTU/
DIPEA with 4-methoxyphenylacetic acid, hydroquinone (42),
thymine (50), or uracil (51) derivatives (Scheme 3) which
were prepared as described previously.20,38 Briefly, the synthesis
of the hydroquinone derivative (44) began with the
condensation of the tert-butyl bromoacetate with hydroquinone
leading to a tert-butyl (4-hydroxyphenoxy)acetate (39) which
then reacted with benzyl bromoacetate (40) to generate 41.
The final product (42) was obtained by the removal of the
benzyl group using hydrogenolysis in the presence of 10% Pd/
C.
Derivatives of thymine (54) and uracil (55) were obtained in
a similar fashion (Scheme 4). First, thymine or uracil was
reacted with tert-butyl bromoacetate and then with benzyl
bromoacetate (40). The benzyl group was further removed by
hydrogenation in the presence of 10% palladium on activated
carbon, affording the desired derivative 50 or 51 that were then
used to synthesize phosphonic peptides.
To obtain target compounds as single diastereoisomers (58−
71), the diastereomeric mixture of peptidyl 1-aminoalkyl-
phosphonate derivative 24 was purified by column chromatog-
raphy on silica gel. The separated fractions containing single
diastereoisomers were then concentrated, and the presence of
the desired compound was confirmed by 31P NMR spectros-
copy and HPLC. Simple Boc deprotection of compound 56 or
57 resulted in the generation of starting material for the
synthesis of the final peptides using HBTU as the coupling
reagent.
Figure 1. Progress curves for the inhibition of HNE (0.0025 U/mL)
by 44: (a) control; (b) 9 nM; (c) 13 nM; (d) 20 nM; (e) 30 nM; (f)
44 nM.
Our obtained biological results are summarized in Tables 1,
2, and 4. As expected, introduction of the peptide chain into the
inhibitor structure resulted in greatly increased potency of
action when compared to simple Cbz-protected 1-amino-
alkylphosphonates diaryl esters.37 The reference compound
used in this study (Boc-Val-Pro-ValP(OPh)2, 21) displayed a
k
inact/KI of 46100 M−1 s−1. Peptides containing the phosphonic
analogue of alanine at the P1 position demonstrated poor
inhibition (Table 1). Among derivatives containing the
phosphonic leucine analogue in the structure, the highest
activity against HNE was observed for compound 14 (kinact/KI
= 122500 M−1 s−1), but it was more than 1.3 times as effective
toward chymotrypsin (kinact/KI = 165900 M−1 s−1) and was also
able to inhibit trypsin (kinact/KI = 2800 M−1 s−1). This lack of
selectivity was observed for all peptides containing the
phosphonic leucine analogue, and all displayed more reactivity
toward chymotrypsin than elastase (11, kinact/KI = 325 M−1 s−1
and kinact/KI = 1900 M−1 s−1; 13, kinact/KI = 4100 M−1 s−1 and
k
k
inact/KI = 21900 M−1 s−1; 15, kinact/KI = 3400 M−1 s−1 and
inact/KI = 18100 M−1 s−1; respectively). We observed a slight
increase in the selectivity of the peptidyl derivatives of Nle and
Nva. For example, compound 19 was approximately 2 times
more active against HNE (kinact/KI = 8100 M−1 s−1) than
chymotrypsin (kinact/KI = 4250 M−1 s−1). Although compound
16 was almost 20 times more active against HNE (kinact/KI =
56200 M−1 s−1) than chymotrypsin (kinact/KI = 2800 M−1 s−1),
it also inhibited porcine pancreatic elastase with kinact/KI of
54800 M−1 s−1. Among the series of phosphonic aminobutyric
acid derivatives the highest potency of action was observed for
compound 8 (Boc-Val-Pro-AbuP(OC6H4-4-S-CH3)2), which
inhibited neutrophil elastase with a kinact/KI of 165100 M−1
s−1 and displayed little to no activity against chymotrypsin or
trypsin. Among all of the obtained peptides with the sequence
Boc-Val-Pro-AaaP(OAr)2 (where AaaP is the phosphonic
analogue of amino acid; Ar is the aromatic ester ring)
compound 24 (Boc-Val-Pro-ValP(OC6H4-4-S-CH3)2) displayed
the highest potency (kinact/KI of 217300 M−1 s−1) and was the
most active HNE inhibitor in this series (Table 1). An
RESULTS AND DISCUSSION
■
All of the target compounds were tested for their inhibition of
HNE activity as well as for selectivity of reaction versus related
serine proteases such as porcine pancreatic elastase (PPE),
chymotrypsin, and trypsin. For screening purposes the enzymes
were first incubated with the tested compound at 25 μM for 15
min before addition of substrate. For compounds that displayed
less than 5% enzyme inhibition we assigned an inhibition value
of zero (NI). For the inhibitors that showed an activity between
5% and 40% we presumed the kinact/KI to be <50 M−1 s−1. For
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a
Table 1. Inhibitory Activity of Phosphonic Peptides with the General Formula Boc-Val-Pro-AaaP(OAr)2
−1
kinact/KI (M−1
PPE
s
)
compd
R1
4-ethyl
R2
HNE
870 ( 5)
chymotrypsin
<50
trypsin
1
methyl (Ala)
1100 ( 70)
<50
NI
NI
NI
2
4-tert-butyl
4-isopropyl
4-S-methyl
H
methyl (Ala)
<50
<50
3
methyl (Ala)
<50
300 ( 30)
<50
4
methyl (Ala)
3500 ( 300)
7100 ( 60)
1380 ( 5)
11450 ( 260)
12680 ( 240)
4210 ( 200)
580 ( 20)
<50
<50
<50
<50
<50
5
ethyl (Abu)
<50
6
4-ethyl
ethyl (Abu)
<50
7
4-isopropyl
4-S-methyl
3,4-dimethyl
4-O-methyl
H
ethyl (Abu)
<50
<50
8
ethyl (Abu)
165100 ( 5300)
460 ( 15)
60100 ( 1100)
1250 ( 45)
9900 ( 240)
1300 ( 130)
960 ( 20)
365 ( 70)
<50
460 ( 5)
<50
9
ethyl (Abu)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
ethyl (Abu)
11000 ( 460)
325 ( 5)
<50
<50
2-methylopropyl (Leu)
2-methylopropyl (Leu)
2-methylopropyl (Leu)
2-methylopropyl (Leu)
2-methylopropyl (Leu)
n-propyl (Nva)
n-propyl (Nva)
n-propyl (Nva)
n-butyl (Nle)
1900 ( 20)
1700 ( 30)
21900 ( 660)
165900 ( 3700)
18100 ( 600)
2800 ( 10)
140 ( 10)
NI
<50
4-O-methyl
4-S-methyl
3-carbomethoxy
4-chloro
4-S-methyl
H
270 ( 5)
<50
4100 ( 60)
122500 ( 1800)
3400 ( 100)
56200 ( 2000)
2600 ( 100)
<50
11200 ( 50)
76900 ( 7200)
12800 ( 550)
54800 ( 900)
10900 ( 150)
<50
280 ( 5)
2800 ( 40)
<50
420 ( 5)
<50
4-tert-butyl
4-S-methyl
H
NI
8100 ( 800)
650 ( 25)
9700 ( 300)
1700 ( 10)
22800 ( 400)
700 ( 50)
4250 ( 150)
400 ( 20)
<50
<50
n-butyl (Nle)
<50
H
1-methylethyl (Val)
1-methylethyl (Val)
1-methylethyl (Val)
1-methylethyl (Val)
1-methylethyl (Val)
1-methylethyl (Val)
1-methylethyl (Val)
46100 ( 1000)
3450 ( 100)
6800 ( 200)
217300 ( 2500)
92100 ( 2500)
4200 ( 170)
42700 ( 1000)
<50
4-tert-butyl
4-ethyl
NI
NI
6000 ( 70)
14000 ( 1400)
5000 ( 230)
2000 ( 130)
19500 ( 1100)
NI
NI
4-S-methyl
4-chloro
4-isopropyl
4-O-methyl
900 ( 60)
250 ( 5)
<50
<50
<50
<50
<50
<50
a
NI: less than 5% of inhibition was observed after 15 min of incubation of enzyme with the inhibitor (25 μM) at 37 °C.
Table 2. Influence of N-Terminal Residue Structure on the Inhibitory Activity of Phosphonic Peptides with the General
a
Formula Boc/Cbz-Aaa-Pro-ValP(OC6H4-S-CH3)2
−1
kinact/Ki (M−1
s
)
compd
R
HNE
PPE
chymotrypsin
trypsin
28
29
30
31
32
33
34
35
36
37
38
Boc-Gly
35000 ( 550)
5100 ( 190)
<50
<50
<50
<50
<50
<50
<50
<50
<50
NI
Cbz-Gly
39000 ( 150)
1200 ( 20)
900 ( 30)
12000 ( 900)
<50
200 ( 20)
<50
Boc-D-Ala
Cbz-L-Ala
Cbz-Aib
Cbz-L-(tBu)Ser
Cbz-L-Val
Boc-L-Leu
Cbz-L-Leu
Cbz-L-Ile
Cbz-L-Met
39400 ( 700)
143500 ( 2600)
1300 ( 130)
1500 ( 60)
<50
162200 ( 10600)
231100 ( 13500)
127600 ( 5200)
86200 ( 1700)
145100 ( 3700)
223200 ( 1700)
4300 ( 110)
6200 ( 120)
7000 ( 100)
1000 ( 50)
1800 ( 70)
6300 ( 150)
500 ( 10)
4400 ( 440)
150 ( 60)
800 ( 70)
1600 ( 160)
3700 ( 20)
<50
<50
a
NI: less than 5% of inhibition was observed after incubation of enzyme with the inhibitor (25 μM) at 37 °C.
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introduction of the S-methyl substituent into the ester ring
structure at the para position resulted in a 4.7× greater increase
in potency of action against HNE (compared to the
unsubstituted compound 21, kinact/KI = 46100 M−1 s−1) and
a 7.6× increase in selectivity versus porcine pancreas elastase
(PPE) (compound 21 was 2× more active against HNE vs
PPE; compound 24 was 15× more active against HNE vs PPE).
Replacing the S-methyl group with a chlorine atom (25) or an
O-methyl group (27) decreased the potency of action (kinact/KI
of 92100 and 42700 M−1 s−1, respectively), though these
compounds displayed a similar selectivity for HNE vs PPE and
chymotrypsin. On the basis of our initial results, derivative 24
was selected for further development of potent and selective
HNE inhibitors.
Next, we evaluated the influence of the P3 residue as well as
the N-terminal protective group on the inhibitory profile of
phosphonic peptides (Table 2). Replacing the Boc protective
group (24) with a Cbz group increased the overall potency of
action (34, kinact/KI = 231100 M−1 s−1) but also increased
reactivity with chymotrypsin (24, kinact/KI = 900 M−1 s−1; 34,
kinact/KI = 4400 M−1 s−1). With the exception of one compound
(38), exchanging the Val residue at the P3 position with any of
the selected amino acids (Gly, Ala, Ile, Leu, tBu-Ser) resulted in
decreased inhibitory properties. Surprisingly, compound 38
(Cbz-Met-Pro-ValP(OC6H4-S-CH3)2) displayed activity and
selectivity profiles similar to compound 24.
A serious disadvantage of phosphonates, which limits their
practical application as potential therapeutics, is their low
solubility in aqueous media. To address this, we introduced N-
terminal groups, which were shown to increase compound
solubility, into the inhibitor structure.20 H-Val-Pro-
ValP(OC6H4-S-CH3)2 was used as the starting material and
was further coupled either with 4-methoxybenzoic acid or with
a derivative of hydroquinone, uracil, or thymine. In addition to
increased solubility (Table 3) all of the obtained compounds in
this group displayed increased activity against HNE (Table 4)
and relatively low activity against chymotrypsin and trypsin.
series, as its observed reactivity against HNE was 50.8 times
greater than its reactivity to PPE (kinact/KI = 19100 M−1 s−1)
and it was over 245 times less active against chymotrypsin
(kinact/KI = 4000 M−1 s−1).
Previously, an attempt to generate and biologically evaluate
the reactivity of diastereomerically pure phosphonic peptides
against serine proteases (DPP IV) was reported by Boduszek et
al.34 Subsequently, the Haemers group presented their
investigations of diastereomerically pure phosphonic dipeptides
as DPP IV inhibitors.41,42 In 2000, Walker et al. reported an
advanced biochemical evaluation of enantiomerically pure Cbz-
protected phosphonic analogues of Phe and Val as inhibitors of
cathepsin G, chymotrypsin, and human neutrophil elastase.43
These results clearly showed that (R)-epimers are more potent
inhibitors of target serine proteases than their corresponding
(S)-epimers. In order to generate target inhibitors with a high
potency of action against HNE in an optically pure form, we
separated compound 24 into single diastereoisomers, which,
after Boc-group removal, were used for the synthesis of
derivatives 58−71. This approach was found to be very
convenient, since the desired products could be obtained in
great quantities. Silica gel column chromatography method was
found to be very useful for separating of 24 into single
diastereoisomers even on a multigram scale. A typical HPLC
analysis of the final synthesized compounds including 31P NMR
data, obtained as racemic mixture or separated single
diastereisomers, is shown in Figure 2.
The results obtained from our enzyme inhibition studies of
compounds 56−59, 61, 63, 65, 67, 69, and 71 (Table 4) clearly
show that the R-diastereoisomer is capable of protease
inactivation, while diastereoisomer S is almost completely
inactive against the target enzyme. The most potent and
selective inhibitor of HNE found in this study was 61 which
displayed a kinact/KI of 2353000 M−1 s−1 and was 60 times less
active against PPE (kinact/KI = 39200 M−1 s−1) and 365 times
less active against chymotrypsin (kinact/KI = 6450 M−1 s−1).
Although 69 displayed high potency against HNE (kinact/KI =
1760000 M−1 s−1) it was only 7 times less active against PPE
(kinact/KI = 242900 M−1 s−1) and 39 times less active against
chymotrypsin (kinact/KI = 44700 M−1 s−1). Similar to its
diastereomeric mixture (54), the uracil derivative (65)
displayed the lowest selectivity of action, since it was only 3.5
times more active against HNE than PPE and 27 times more
active against HNE than chymotrypsin.
To examine the stability of the irreversible complex formed
between HNE and compound 61, we first incubated the
protease with an excess of inhibitor. After complete inactivation
the excess of inhibitor was removed by dialysis and the activity
of protease was monitored at different time intervals (Figure 3).
In parallel, a control HNE sample incubated only with DMSO
instead of 61 was prepared and dialyzed in the same way. These
results demonstrated that after 50 days in complex with 61,
HNE recovered around 15% of its original activity whereas the
activity of the control HNE sample after this time remained at
approximately 90% of its original value. The recovery of
enzymatic activity at different time points is expressed as the
vmax [units/s] (Figure 3).
The rate of hydrolysis of compounds 24, 44, 45, 54, and 55
was evaluated either in PBS buffer (pH 7.4) or in 80% human
plasma at 37 °C.29 The stability of the inhibitors was monitored
by HPLC at different time points (Table 5). The evaluation of
compound stability in PBS buffer showed that the half-life of all
tested compounds was longer than 48 h. Of note, at 48 h, only
Table 3. Solubility of HNE Phosphonic Inhibitors in Buffer
and Human Plasma
solubility (mM)
compd
PBS, pH 7.4
human plasma
24
44
45
54
55
0.36
6.23
0.17
6.22
6.11
0.36
6.23
0.17
6.22
6.11
The most selective compound identified within this group
was a 4-methoxybenzoic acid derivative (45) that displayed a
k
inact/KI of 304000 M−1 s−1 against HNE and 4700 M−1 s−1
against PPE and was almost completely inactive against
chymotrypsin and trypsin. The highest inhibition potency
against HNE was observed for derivatives of thymine (55,
k
inact/KI = 965000 M−1 s−1) and hydroquinone (44, kinact/KI =
970000 M−1 s−1). Although compound 55 was among the most
active HNE inhibitors in this series, it was only 8.2 times more
specific toward HNE than PPE and 42 times more specific to
HNE than to chymotrypsin. The lowest selectivity was
observed for compound 54 (kinact/KI of 460000 and 28300
M−1 s−1 for HNE and PPE, respectively). Derivative 44
displayed the highest inhibitory potency against HNE in this
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a
Table 4. Inhibitory Properties of Phosphonic Peptides as Single Diastereisomers
a
NI: less than 5% of inhibition was observed after 15 min of incubation of enzyme with the inhibitor (25 μM) at 37 °C.
16% hydrolysis of compound 44 was measured, and even after
6 days, over 50% of 44 remained detectable. In addition,
compound 44 also displayed the highest stability in 80% human
plasma, with a half-life of 8 h. Compound 24 was also stable in
human plasma, with a t1/2 of 6.2 h. Derivatives of 4-
methoxybenzoic acid, uracil, and thymine were less stable in
80% human plasma, with measured half-lives of 3.8, 4.55, and
3.55 h, respectively. The main products of hydrolysis were their
corresponding monoaryl esters. These results indicate that
designing (4-S-methyl)phenyl as the ester group represents an
optimal balance between compound stability and inhibitory
potency which is a consequence of the increased electrophilicity
of the phosphorus atom. When stability in human plasma is
taken into consideration, along with selectivity and high
potency of action against HNE, the synthesized peptidyl 1-
aminoalkylphosphonate diaryl esters represent a superior class
of inhibitors with potential for practical applications.
In order to evaluate the inhibitory properties of the most
active HNE inhibitors and gain insight into the binding mode
of compounds 61, 65, 69, we performed a molecular docking
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Figure 2. HPLC and 31P NMR confirmation of separation of diastereisomers with corresponding kinact/KI values.
interactions with HNE, especially between the hydrophobic
forces of the aromatic ring and the hydrogen bonds with
Arg177 and Arg217. Studies focused on solving the HNE
crystal structure in complex with obtained phosphonic
inhibitors are ongoing.
CONCLUSIONS
■
We propose that theoretically most reversible inhibitors of
elastase are unable to correct the elastase−α1-PI imbalance in
vivo. Although the equilibrium of the inhibition reaction by
reversible inhibitor is shifted substantially to the formation of
an EI complex, a small amount of free active enzyme remains.
Under normal conditions free HNE would be bound by α1-PI,
but because of an insufficient amount of reactive serpin, the
activity of HNE is not completely blocked. Thus, reversible
elastase inhibitors do not display therapeutic efficiency in vivo.
In contrast, phosphonates form covalent and extremely stable
EI complexes that release free enzyme only after an extended
period of time.
The HNE inhibitors described here represent the most
potent peptidyl irreversible inactivators of this enzyme reported
to date. Our initial findings regarding the structural require-
ments for phosphonic peptides (1−38) led to the development
of highly potent derivatives of hydroquinone, uracil, and
thymine. The most active compounds were further obtained as
single diastereisomers with increased inhibitory properties.
Overall, compound 61 displayed the best balance of high
inhibitory activity against HNE and selectivity for HNE over
porcine pancreatic elastase, chymotrypsin, and trypsin.
Furthermore, the analysis of the elastase−inhibitor 61 complex
revealed that even after 50 days HNE recovered only 15% of its
initial activity. Additionally, our obtained inhibitors displayed
high stability in either PBS or human plasma. Therefore, it may
be possible to apply our generated derivatives in vivo to
overcome HNE−α1-PI imbalance under physiological con-
ditions.
Figure 3. Recovery of human neutrophil elastase activity from HNE
complexed with 61.
Table 5. Stability of Compounds 24, 44, 45, 54, and 55 in
Aqueous Solution and 80% Human Plasma
t1/2 (h)
compd
PBS, pH 7.4
80% human plasma
24
44
45
54
55
28
150
26
6.20
8.00
3.80
4.55
3.95
100
100
analysis of inhibitors bound to the active site of HNE using an
AutoDock Vina and structural template 1PPG.pdb.44,45 Since
the crystal structures of phosphonic inhibitor in complex with
serine proteases showed that the inhibitor is devoid of ester
phenyl rings (aged complex), we docked inhibitors 61, 65, 69
as phosphonic acids (Figure 4).46 The overall positioning
pattern observed for all analyzed inhibitors is essentially
identical. The oxygen atom of the phosphonic group is
hydrogen-bonded with the oxyanion hole formed by the main
chain amide groups of Gly193 and Ser195. The phosphorus−
oxygen distance ranges from 2.7 to 2.9 Å. Inhibitor side chains
at the P1−P3 positions occupy the corresponding enzyme
binding pockets (S1−S3). In contrast to the chloromethyl
template inhibitor, compounds 61, 65, 69 do not seem to fully
occupy the S4 binding pocket and they tend to display different
conformational binding to the cavity formed by Arg177,
Arg217, Cys168, and Leu166. The highly inhibitory properties
of the hydroquinone derivative 61 could be the result of its
extended length which results in the formation of additional
EXPERIMENTAL SECTION
■
Enzymatic Studies. Mathematical Model.39,40 The inhibitory
activity of the synthesized compounds was determined by the progress
curve method. The nonlinear progress curves observed with time-
dependent inhibitors of HNE were fit to eq 1 to obtain the first-order
rate constant (kobs):
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Figure 4. Schematic representation of HNE inhibitors bound to the protease active site (PDB accession code 1PPG): (A) proposed binding mode
for compound 61; (B) representation of the HNE residues surrounding the docked 61, showing the potential hydrogen bonds (dashed yellow lines);
(C) proposed binding mode for compound 65; (D) representation of compound 61 and MeO-Suc-AAPV-CMK superimposed in the binding site of
HNE; (E) proposed binding mode for compound 69; (F) HNE binding site superimposition of 61 (green), 65 (purple), and 69 (yellow).
vi − v
kobs
(−kobst)
PPE), Cbz-Arg-AFC (50 μM, trypsin), and Suc-Ala-Ala-Pro-Phe-AMC
(20 μM, chymotrypsin). All compounds were screened for inhibitory
activity against HNE, porcine pancreatic elastase (PPE), chymotrypsin,
and trypsin at 25 μM. First, the enzyme was incubated for 15 min with
each of the tested compounds and measurements were taken for 15
min. When less than 5% inhibition was observed for any of the
molecules, the level of inhibition was assigned as zero. For the
inhibitors that showed an activity between 5% and 40%, we presumed
kinact/KI < 50 M−1 s−1. We calculated kinact/KI values for compounds
that displayed HNE inhibition at a minimum of 40% after 10 min of
incubation with the enzyme. The inhibitory activity of the synthesized
compounds was determined by the progress curve method. Control
curves in the absence of the inhibitor were linear. The standard
deviation for presented values is calculated from the mean of three
independent experiments and does not exceed 10%. All measurements
were performed using Spectra Max Gemini XPS spectrofluorometer
(Molecular Devices, U.S.).
[P] = vt +
s [1 − e
]
s
(1)
where [P] is the product concentration at time t, vi is the initial
velocity, and vs is the steady state velocity.
Second-order rate constants (kinact/KI) were estimated by
calculating kobs/[I] and then correcting for the substrate concentration
([S]) and Michaelis−Menten constant (KM) according to eq 2:
kobs
[I]
kinact
=
[S]
KM
KI 1 +
(
)
(2)
Control curves in the absence of inhibitor were linear. The standard
deviation for the presented values was calculated using the mean of
three independent experiments and does not exceed 10%.
General. The rates of inhibition of human neutrophil elastase
(HNE, 2.5 mU/mL, Biocentrum, Krakow
elastase (PPE, 12.5 nM, Serva, Polgen, Łod
(0.5 nM, Sigma-Aldrich, Poznan
AppliChem, Polgen, Łodz, Poland) were measured in 0.1 M HEPES,
́
, Poland), porcine pancreatic
z, Poland), chymotrypsin
HNE−Inhibitor Complex Stability. Human neutrophil elastase
(1.91 μM) was incubated with 61 (200 μM, final DMSO
concentration in the reaction mixture was 1%) for 15 min at 37 °C
in 0.1 M HEPES, 0.5 M NaCl, 0.03% Triton X-100, pH 7.5.After
incubation the enzyme showed no proteolytic activity as determined
using a fluorogenic substrate (MeOSuc-AAPV-AFC, 40 μM).
́
́
́
, Poland), and trypsin (12.5 nM,
́
́
0.5 M NaCl, 0.03% Triton X-100 (pH 7.5) at 37 °C. All enzymes were
assayed using fluorogenic substrates (Calbiochem, Merck, Warszawa,
Poland): MeO-Suc-Ala-Ala-Pro-Val-AFC (40 μM, HNE; 40 μM,
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Completely inactivated elastase was further dialyzed (D-Tube Dializer
Mini, MWCO 6−8 kDa, Novagen) against the reaction buffer (0.1 M
HEPES, 0.5 M NaCl, 0.03% Triton X-100, pH 7.5) at 4 °C with 5
times change of buffer (dilution factor of 1015). The control sample
was prepared in parallel using the same approach with the exception
that the enzyme was incubated in the reaction buffer with DMSO
instead of 61 before dialysis. The activity of both elastase solutions
incubated at 37 °C was monitored using a fluorogenic substrate at
different time points within 50 days. The recovery of enzymatic activity
is expressed as vmax [unit/s] at different time points.
Hydrolysis in PBS Buffer. The susceptibility of compounds to
hydrolysis in buffer solution was examined by HPLC. Stock solutions
of the tested compounds (50 mM) prepared in DMSO were added to
100 mM phosphate buffered saline (PBS, pH 7.4) to a final
concentration of 3 mg/mL (44, 54, 55) or 0.1 mg/mL (24, 45)
and then incubated at 37 °C. At different time points the mixtures
were analyzed by HPLC using a C8 column.
Hydrolysis in Human Plasma. The stability of compounds in
human plasma was examined as described before with minor
changes.29 Freshly isolated human plasma pooled from healthy donors
was used to study the kinetics of hydrolysis of specific compounds. A
stock DMSO solution of each tested compound (50 mM) was added
to 80% human plasma (diluted with PBS, pH 7.4) to a final
concentration of 3 mg/mL (44, 54, 55) or 0.1 mg/mL (24, 45) and
incubated at 37 °C. At different time points, 100 μL aliquots of
compound solution were added to 200 μL of DMSO and precipitated
proteins were centrifuged. The resulting supernatant was subjected to
HPLC analysis (200 μL) to determine compound stability.
Molecular Docking. Molecular docking studies of compounds 61,
65, 69 bound to the active site of HNE with MeO-Suc-Ala-Ala-Pro-Val
chloromethyl ketone (MeO-Suc-Ala-Ala-Pro-Val-CMK) inhibitor
removed from the structure (PDB accession code 1PPG) were
performed with the AutoDock Vina.44,45 Each ligand was initially
energy minimized. Pictures ware prepared using Pymol.47
Cbz-Protected 1-Aminoalkylphosphonate Diaryl Esters (General
Procedure).37 Triaryl phosphite (10 mmol) was dissolved in glacial
acetic acid (25 mL), and an appropriate aldehyde (11 mmol) and
benzyl carbamate (11 mmol, 1.66 g) were subsequently added. The
mixture was then heated at 80−90 °C for 2 h, and the solvent was
evaporated. The resulting oil was dissolved in methanol (100 mL) and
left at −20 °C for crystallization. The product was filtered, washed
with cold methanol, and dried at room temperature. If necessary, the
product was recrystallized from chloroform/methanol.
Deprotection of Cbz Group (General Procedure). The Cbz-
protected 1-aminoalkylphosphonate diaryl ester (10 mmol) was
dissolved in 33% HBr/AcOH solution (4 mL). The reaction was
performed at room temperature for 2 h. The volatile components of
the mixture were removed under reduced pressure. The resulting oil
was dissolved in methanol (1 mL), and diethyl ether (100 mL) was
added and was left for crystallization at 4 °C. The hydrobromide salt of
1-aminoalkylphosphonate diaryl ester was filtered and washed
extensively with cold diethyl ether. If necessary, the product was
recrystallized using a methanol/diethyl ether system.
Phosphonic Dipeptides (General Procedure). The hydrobromide
salt of 1-aminoalkylphosphonate diaryl ester (10 mmol) was
suspended in acetonitrile (25 mL), and N,N′-diisopropylethylamine
(25 mmol, 1.75 mL) was added. After complete dissolution of the
substrate, N-Boc-L-Pro-OH (10 mmol, 2.15 g) was added followed by
the addition of HBTU (12 mmol, 4.55 g). The reaction was performed
at room temperature for 12 h and its progress monitored by thin-layer
chromatography. When the reaction was completed, the solvent was
evaporated under reduced pressure and the resulting oil was dissolved
in ethyl acetate (100 mL). The white precipitate was filtered off, and
the solution was washed with 5% NaHCO3 (aq) (50 mL), 5% KHSO4
(aq) (50 mL), and brine (50 mL). After drying over anhydrous
sodium sulfate, the solution was filtered and concentrated in a vacuum.
The final compounds were purified using silica gel column
chromatography.
t
Deprotection of the Bu/Boc Group (General Procedure). The N-
Chemistry. General. All chemical reagents and solvents were
obtained from commercial sources and, unless otherwise noted, were
used without further purification. Silica gel chromatography was
performed using glass columns packed with silica gel 60 (70−230
mesh). Analytical thin-layer chromatography was performed on
Macherey-Nagel silica gel Polygram SIL G plates, and compounds
were visualized with UV light at 254 nm. All final compounds were
purified to >95% purity at 220 and 254 nm as determined by HPLC.
HPLC analyses and purifications were carried out on a Varian ProStar
210 with a dual λ absorbance detector system using SunFire Prep C8
column (10 mm × 250 mm, 5 μm) at 40 °C with a 4 mL/min flow
rate using a gradient 5−95% [0.05% TFA in acetonitrile] in [0.05%
TFA in water] over 40 min. The nuclear magnetic resonance spectra
(1H and 31P) were recorded on either a Bruker Avance DRX-300
Boc-protected phosphonic di- or tripeptides (10 mmol) were dissolved
in dichloromethane (5 mL), and an equal volume of trifluoroacetic
acid was added dropwise. The reaction was performed at room
temperature for 2 h, and the progress of the reaction was monitored by
TLC. When the reaction was completed, the volatile components of
the mixture were removed under reduced pressure. In order to remove
the traces of trifluoroacetic acid, the resulting oil was dissolved in
toluene and evaporated a total of three times. The obtained
phosphonic dipeptides with a free N-terminal amino group were
used directly in the next step without further purification.
Phosphonic Tripeptides (General Procedure). The trifluoroacetate
salt of phosphonic dipeptide derivative (10 mmol) was dissolved in
acetonitrile followed by the addition of N,N′-diisopropylethylamine
(25 mmol, 1.75 mL). Next, the N-protected amino acid (10 mmol)
and HBTU (12 mmol, 4.55 g) were added. The reaction was
performed at room temperature for 12 h and progress monitored by
TLC. After completion of the reaction the solvent was evaporated and
the resulting oil was dissolved in ethyl acetate (100 mL). The solution
was then washed with 5% NaHCO3 (aq) (50 mL), 5% KHSO4 (aq)
(50 mL), and brine (50 mL), dried over Na2SO4, filtered, and
concentrated resulting in a crude phosphonic tripeptide which was
purified by column chromatography on silica gel.
Diastereoisomers Separation. Single diastereoisomers of 1-amino-
alkyphosphonate aromatic ester peptidyl derivatives, the starting
material for further synthesis, were prepared using compound 24
(Boc-Val-Pro-ValP(OC6H4-4-S-CH3)2). Diastereoisomer separation
was performed using column chromatography on silica gel. Collected
fractions containing single diastereoisomer were concentrated, and the
optical purity was confirmed by 31P NMR spectroscopy and HPLC
analysis. Compounds 58−71 were synthesized as described above with
the exception that a single diastereoisomer (56 or 57) was used as the
starting substrate.
1
(300.13 MHz for H NMR, 121.50 MHz for 31P NMR) or Bruker
1
Avance 600 MHz (600.58 MHz for H NMR, 243.10 MHz for 31P
NMR) spectrometer. Chemical shifts are reported in parts per million
(ppm) relative to a tetramethylsilane internal standard. Mass spectra
were recorded using a Biflex III MALDI TOF mass spectrometer
(Bruker, Germany). The cyano-4-hydroxycinnamic acid (CCA) was
used as a matrix. High resolution mass spectra (HRMS) were acquired
either on a Waters Acquity Ultra Performance LC, LCT Premier XE or
Bruker micrOTOF-Q II mass spectrometer.
Solubility Assay. Compounds 24, 44, 45, 54, and 55 were dissolved
in PBS buffer or freshly isolated human plasma from healthy donor
(pooled) in increasing concentrations ranging from 15 to 0.016 mg/
mL. The turbidimetry in reference to the buffer or plasma solution of
the solutions was measured at 620 nm using a microplate reader
(Molecular Devices, SpectraMax Plus 384).
Triaryl Phosphites (General Procedure).37 Phosphorus trichloride
(10 mmol) was added to a solution of substituted phenol (30 mmol)
in acetonitrile (50 mL) at room temperature, and the mixture was
refluxed for 6 h. The volatile components were removed under
reduced pressure, and the resulting crude phosphite was used directly
in the next step without purification.
tert-Butyl (S)-1-((S)-2-((R,S)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylcarbamate (24). 24 was prepared using general
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methods described above and purified by column chromatography on
silica gel using 20% ethyl acetate in chloroform as the eluent to yield a
colorless oil which formed a white solid foam under reduced pressure
(73%). Rf = 0.15 (separate diastereoisomer), 0.07 (separate
diastereoisomer) (CHCl3/AcOEt, 4:1, v/v). 1H NMR (300 MHz,
CDCl3) δ 7.03−7.23 (m, 8H), 5.30 (dd, J = 61.7, 9.3 Hz, 1H), 4.49−
4.78 (m, 2H), 4.20−4.34 (m, 1H), 3.43−3.80 (m, 2H), 2.44 (d, J = 2.7
Hz, 6H), 2.04−2.41 (m, 2H), 1.74−2.03 (m, 4H), 1.42 (d, J = 2.0 Hz,
9H), 0.81−1.14 (m, 12H). 31P NMR (121 MHz, CDCl3) δ 18.15 (s,
52%), 18.48 (s, 48%). HRMS: calcd for (C33H48N3O7PS2)H+,
694.2750; found, 694.2738.
3H), 2.21−2.37 (m, 1H), 2.02−2.21 (m, 2H), 1.76−1.99 (m, 3H),
1.40 (s, 9H), 0.97−1.05 (m, 6H), 0.94 (d, J = 6.7 Hz, 3H), 0.88 (d, J =
6.7 Hz, 3H). 31P NMR (121 MHz, CDCl3) δ 18.25 (s). HRMS: calcd
for (C33H48N3O7PS2)Na+, 716.2569; found, 716.2537.
tert-Butyl 2-(4-(2-((S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylamino)-2-oxoethoxy)phenoxy)acetate (60). The TFA
salt of 56 obtained after N-Boc deprotection under conditions
described above (0.34 g, 0.48 mmol) was dissolved in acetonitrile (5
mL), and N,N′-diisopropylethylamine (236 μL, 1.44 mmol) was
added. Next, 42 (163 mg, 0.58 mmol) was added into the mixture
which was followed by the addition of HBTU (218 mg, 0.58 mmol).
The postreaction workup was done as described for 58, and
purification by column chromatography on silica gel using 2%
methanol in chloroform as the eluent yielded 60 as a colorless oil
which formed a white solid foam under reduced pressure (185 mg,
Benzyl Bromoacetate (40). To the solution of benzyl alcohol (4 g,
37 mmol) in DMF (100 mL) were subsequently added 2-bromoacetic
acid (10.26 g, 74 mmol), DCC (15.26 g, 74 mmol), and 4-
dimethylaminopyridine (1.35 g, 11.1 mmol), and the reaction was
performed at room temperature for 24 h. The reaction mixture was
diluted with distilled water (1 L) and extracted four times with ethyl
acetate (500 mL). The organic fraction was dried over MgSO4, filtered,
and concentrated in vacuum, and the product was purified by
chromatography on silica gel, eluting with ethyl acetate in hexane
(20:1, v/v) to provide 40 (5.7 g, 67%) as a pale yellow oil. Rf = 0.40
1
45%). Rf = 0.14 (CHCl3/MeOH, 50:1, v/v). H NMR (601 MHz,
DMSO-d6) δ 8.49 (d, J = 9.9 Hz, 1H), 8.05 (d, J = 8.7 Hz, 1H), 7.21−
7.35 (m, 4H), 7.14 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H), 6.82−
6.90 (m, 4H), 4.60 (s, 2H), 4.50−4.57 (m, 2H), 4.48 (s, 2H), 4.39−
4.45 (m, 1H), 3.68−3.77 (m, 1H), 3.55−3.61 (m, 1H), 2.47 (s, 3H),
2.46 (s, 3H), 2.24−2.33 (m, 1H), 1.95−2.07 (m, 2H), 1.84−1.95 (m,
1H), 1.74−1.84 (m, 1H), 1.55−1.62 (m, 1H), 1.41 (s, 9H), 1.09 (d, J
= 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.84
(d, J = 6.7 Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ 18.94 (s).
HRMS: calcd for (C42H56N3O10PS2)Na+, 880.3043; found, 880.3027.
2-(4-(2-((S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylamino)-2-oxoethoxy)phenoxy)acetic Acid (61). The
tert-butyl protective group of 60 was removed by the general method
described above. The product was further dissolved in 50% acetonitrile
in water solution and lyophilized to afford 61 as a white solid (152 mg,
1
(Hex/AcOEt, 20:1, v/v). H NMR (300 MHz, CDCl3) δ 7.32−7.46
(m, 5H), 5.21 (s, 2H), 3.88 (s, 2H).
Benzyl 2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetate (46).
Uracil (1 g, 9 mmol), 40 (2.5 g, 11 mmol), and anhydrous potassium
carbonate (2.5 g, 18.1 mmol) were suspended in anhydrous DMF. The
reaction was performed at room temperature for 24 h. The reaction
mixture was diluted with distilled water (100 mL) and extracted three
times with ethyl acetate (30 mL). Combined organic fractions were
washed with brine (50 mL), dried over MgSO4, filtered, and the
solvent was removed in vacuum. Next, the resulting oil was dissolved
in diethyl ether (100 mL) and left for crystallization, leading to the
1
88%). H NMR (601 MHz, DMSO-d6) δ 8.51 (d, J = 10.0 Hz, 1H),
1
generation of 46 as a white solid (0.98 g, 42%). H NMR (300 MHz,
8.03 (d, J = 8.6 Hz, 1H), 7.34−7.23 (m, 4H), 7.14 (d, J = 8.5 Hz, 2H),
7.09 (d, J = 8.6 Hz, 2H), 6.93−6.81 (m, 4H), 4.60 (s, 2H), 4.57−4.51
(m, 2H), 4.50 (s, 2H), 4.46−4.41 (m, 1H), 3.75−3.69 (m, 1H), 3.62−
3.55 (m, 1H), 2.47 (s, 3H), 2.46 (s, 3H), 2.31−2.23 (m, 1H), 2.05−
1.95 (m, 2H), 1.94−1.86 (m, 1H), 1.85−1.77 (m, 1H), 1.61−1.55 (m,
1H), 1.09 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.7
Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ
18.94 (s). HRMS: calcd for (C38H48N3O10PS2)Na+, 824.2416; found,
824.2455.
DMSO-d6) δ 11.43 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.34−7.40 (m,
5H), 5.63 (dd, J = 7.9, 2.1 Hz, 1H), 5.20 (s, 2H), 4.59 (s, 2H). HRMS:
calcd for (C13H12N2O4)H+, 261.0870; found, 261.0853
Benzyl 2-(3-(2-tert-Butoxy-2-oxoethyl)-2,4-dioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetate (48). A suspension of 46 (0.98 g, 3.8 mmol)
in anhydrous DMF (10 mL) was cooled in an ice bath, and 60%
sodium hydride solution (0.18 g, 4.5 mmol) was slowly added. After
15 min tert-butyl bromoacetate (0.68 mL, 4.6 mmol) was added and
the reaction mixture was allowed to warm to room temperature. After
2 h saturated ammonium chloride solution (100 mL) was added and
the mixture was extracted three times with ethyl acetate (50 mL).
Combined organic fractions were washed with brine (50 mL), dried
over MgSO4, filtered, and concentrated in vacuum. Purification by
column chromatography on silica gel using chloroform/ethyl acetate
(4:1, v/v) as the eluent afforded 48 as a colorless oil (0.82 g, 58%). 1H
NMR (300 MHz, DMSO-d6) δ 7.76 (d, J = 7.9 Hz, 1H), 7.28−7.41
(m, 6H), 5.82 (d, J = 7.9 Hz, 1H), 5.18 (s, 2H), 4.67 (s, 2H), 4.41 (s,
2H), 1.38 (s, 9H).
tert-Butyl 2-(3-(2-((S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylamino)-2-oxoethyl)-2,6-dioxo-2,3-dihydropyrimidin-
1(6H)-yl)acetate (64). The TFA salt of 56 obtained after N-Boc
deprotection under conditions described above (0.1 g, 0.14 mmol) was
dissolved in acetonitrile (5 mL), and N,N′-diisopropylethylamine (69
μL 0.42 mmol) was added. Next, 50 (0.15 mmol, 44 mg) was added
into the mixture which was followed by the addition of HBTU (64 mg,
0.17 mmol). The postreaction workup was performed as described for
58, and purification by column chromatography on silica gel using 3%
methanol in chloroform as the eluent yielded 64 as a colorless oil
which formed a white solid foam under reduced pressure (36 mg,
2-(3-(2-tert-Butoxy-2-oxoethyl)-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)acetic Acid (50). 10% palladium on carbon (100 mg) was
added to a solution of 48 (0.82 g, 2.2 mmol) in ethyl acetate (20 mL),
and a stream of hydrogen was passed through. The progress of the
reaction was monitored by thin-layer chromatography. After the
reaction was completed, the reaction mixture was passed through a pad
of Celite and concentrated in vacuum to yield pure 50 as a colorless oil
which formed a white solid foam under reduced pressure (0.55 g,
1
29%). Rf = 0.06 (CHCl3/MeOH, 30:1, v/v). H NMR (601 MHz,
DMSO-d6) δ 12.92 (s, 1H), 8.44−8.56 (m, 2H), 7.66 (d, J = 7.8 Hz,
1H), 7.27−7.32 (m, 4H), 7.12 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.6 Hz,
2H), 5.77 (d, J = 7.8 Hz, 1H), 4.45−4.60 (m, 4H), 4.38−4.45 (m,
2H), 4.32−4.38 (m, 1H), 3.66−3.72 (m, 1H), 3.54−3.59 (m, 1H),
2.47 (s, 3H), 2.46 (s, 3H), 2.21−2.30 (m, 1H), 1.91−2.02 (m, 2H),
1.87−1.91 (m, 1H), 1.75−1.87 (m, 1H), 1.54−1.62 (m, 1H), 1.40 (s,
9H), 1.09 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.8
Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ
18.94 (s). HRMS: calcd for (C40H54N3O10PS2)Na+, 882.2947; found,
882.2968.
2-(3-(2-((S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylamino)-2-oxoethyl)-2,6-dioxo-2,3-dihydropyrimidin-
1(6H)-yl)acetic Acid (65). The tert-butyl protective group of 64 was
removed by the general method described above. The product was
further dissolved in 50% acetonitrile in water solution and lyophilized
1
88%). H NMR (300 MHz, DMSO-d6) δ 13.18 (s, 1H), 7.72 (d, J =
7.9 Hz, 1H), 5.79 (d, J = 7.9 Hz, 1H), 4.48 (s, 2H), 4.40 (s, 2H), 1.38
(s, 9H). HRMS: calcd for C12H16N2O6)Na+, 307.0901; found,
307.0894.
tert-Butyl (S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylcarbamate (56). 56 was obtained as a colorless oil
1
which formed a white solid foam under reduced pressure (17%). H
NMR (300 MHz, CDCl3) δ 7.57 (d, J = 10.3 Hz, 1H), 7.00−7.20 (m,
8H), 5.58 (d, J = 9.3 Hz, 1H), 4.54−4.76 (m, 2H), 4.22−4.31 (m,
1H), 3.69−3.81 (m, 1H), 3.56−3.67 (m, 1H), 2.41 (s, 3H), 2.40 (s,
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1
to afford 65 as a white solid (30 mg, 83%). H NMR (601 MHz,
DMSO-d6) δ 12.92 (s, 1H), 8.45−8.54 (m, 2H), 7.67 (d, J = 7.9 Hz,
1H), 7.27−7.32 (m, 4H), 7.14 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.4 Hz,
2H), 5.76 (d, J = 7.9 Hz, 1H), 4.47−4.58 (m, 4H), 4.40−4.47 (m,
2H), 4.34−4.40 (m, 1H), 3.66−3.71 (m, 1H), 3.54−3.60 (m, 1H),
2.47 (s, 3H), 2.46 (s, 3H), 2.23−2.31 (m, 1H), 1.93−2.02 (m, 2H),
1.85−1.93 (m, 1H), 1.77−1.85 (m, 1H), 1.54−1.60 (m, 1H), 1.09 (d, J
= 6.8 Hz, 3H), 1.03 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.89
(d, J = 6.7 Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ 18.94 (s).
HRMS: calcd for (C36H46N5O10PS2)H+, 804.25020; found, 804.24623.
tert-Butyl 2-(3-(2-((S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylamino)-2-oxoethyl)-5-methyl-2,6-dioxo-2,3-dihydro-
pyrimidin-1(6H)-yl)acetate (68). The TFA salt of 56 obtained after N-
Boc deprotection under conditions described above (0.1 g, 0.14
mmol) was dissolved in acetonitrile (5 mL), and N,N′-diisopropyle-
thylamine (69 μL 0.42 mmol) was added. Next, 51 (0.15 mmol, 46
mg) was added into the mixture followed by the addition of HBTU
(64 mg, 0.17 mmol). The postreaction workup was performed as
described for 58, and purification by column chromatography on silica
gel using 3% methanol in chloroform as the eluent yielded 68 as a
colorless oil which formed a white solid foam under reduced pressure
within the European Social Fund. HRMS analyses of some
derivatives were performed by the NeoLek Lab cofinanced by
The European Regional Development Fund POIG.02.01.00-02-
073/09. We thank Keri Csencsits-Smith (University of Texas
Health Science Center at Houston, TX, U.S.) for critical
reading of the manuscript and Paulina Kasperkiewicz and Ewa
Pietrusewicz (Wroclaw University of Technology, Wroclaw,
Poland) for assistance in the synthesis of some derivatives.
ABBREVIATIONS USED
■
1-AAP, 1-aminoalkylphosphonate; Aaa, amino acid residue;
Aib, aminoisobutyric acid; Abu, aminobutyric acid; AFC, 7-
amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methyl-
coumarin; ARDS, acute respiratory distress syndrome; CMK,
chloromethyl ketone; COPD, chronic obstructive pulmonary
disease; HNE, human neutrophil elastase; DIPEA, N,N′-
diisopropylethylamine; α1-PI, α1 proteinase inhibitor; SLPI,
secretory leucocyte peptidase inhibitor; α2-MG, α2 macro-
globulin; PPE, porcine pancreatic elastase; Suc, succinyl group
1
(51 mg, 41%). Rf = 0.12 (CHCl3/MeOH, 30:1, v/v). H NMR (601
MHz, DMSO-d6) δ 12.78 (s, 1H), 8.33−8.44 (m, 2H), 7.47 (d, J = 1.3
Hz, 1H), 7.19−7.24 (m, 4H), 7.05 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.3
Hz, 2H), 4.39−4.50 (m, 2H), 4.32−4.39 (m, 4H), 4.25−4.31 (m, 1H),
3.55−3.63 (m, 1H), 3.45−3.55 (m, 1H), 2.38 (s, 3H), 2.37 (s, 3H),
2.16−2.25 (m, 1H), 1.83−1.96 (m, 2H), 1.77−1.83 (m, 1H), 1.75 (s,
3H), 1.67−1.74 (m, 1H), 1.44−1.53 (m, 1H), 1.40 (s, 9H), 1.00 (d, J
= 6.9 Hz, 3H), 0.94 (d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H), 0.80
(d, J = 6.7 Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ 18.94 (s).
HRMS: calcd for (C41H56N5O10PS2)Na+, 896.3104; found, 896.3075.
2-(3-(2-((S)-1-((S)-2-((R)-1-(Bis(4-(methylthio)phenoxy)-
phosphoryl)-2-methylpropylcarbamoyl)pyrrolidin-1-yl)-3-methyl-1-
oxobutan-2-ylamino)-2-oxoethyl)-5-methyl-2,6-dioxo-2,3-dihydro-
pyrimidin-1(6H)-yl)acetic Acid (69). The tert-butyl protective group of
68 was removed by the general method described above. The product
was further dissolved in 50% acetonitrile solution in water and
lyophilized to afford 69 as a white solid (42 mg, 83%). 1H NMR (601
MHz, DMSO-d6) δ 12.78 (s, 1H), 8.36−8.44 (m, 2H), 7.48 (d, J = 1.1
Hz, 1H), 7.18−7.24 (m, 4H), 7.05 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 8.3
Hz, 2H), 4.38−4.48 (m, 2H), 4.34−4.38 (m, 4H), 4.25−4.30 (m, 1H),
3.56−3.63 (m, 1H), 3.45−3.52 (m, 1H), 2.38 (s, 3H), 2.37 (s, 3H),
2.14−2.23 (m, 1H), 1.84−1.94 (m, 2H), 1.78−1.84 (m, 1H), 1.74 (s,
3H), 1.69−1.73 (m, 1H), 1.45−1.51 (m, 1H), 1.00 (d, J = 6.8 Hz,
3H), 0.94 (d, J = 6.7 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H), 0.80 (d, J = 6.7
Hz, 3H). 31P NMR (243 MHz, DMSO-d6) δ 18.94 (s). HRMS: calcd
for (C37H48N5O10PS2)H+, 818.26585; found, 818.26387.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and analytical data of all intermediates and final
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AUTHOR INFORMATION
Corresponding Author
*Phone: 0048 71 320 24 39. Fax: 0048 71 320 24 27. E-mail:
marcin.sienczyk@pwr.wroc.pl.
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ACKNOWLEDGMENTS
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This work was supported by Ministry of Science and Higher
Education (Grant No. N405 342633) and Wroclaw University
of Technology Statute Funds (Grant S10156/Z0313). L.W. is
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