Synthesis and evaluation of silanediols as highly selective uncompetitive inhibitors of human neutrophil elastase

Article abstract of DOI:10.1021/jm301000k

Chronic obstructive pulmonary disease (COPD) is an increasing health problem and is estimated to be the fifth leading cause of death in 2020 according to the World Health Organization. Current treatments are only palliative, and therefore the development of new medicine for the treatment of COPD is urgent. Human Neutrophil Elastase (HNE) is a serine protease that is heavily involved in the progression of COPD through inflammatory breakdown of lung tissue. Consequently, inhibitors of HNE are of great interest as therapeutics. In this article, the development of silanediol peptide isosters as inhibitors of HNE is presented. Kinetic studies revealed that incorporation of a silanediol isoster in the inhibitor structure resulted in an uncompetitive mechanism of inhibition, which further resulted in excellent selectivity. The peculiar mechanism of inhibition and the resulting selectivity makes the presented inhibitors promising leads for the development of new HNE-inhibitor-based therapeutics for the treatment of COPD.

Full text of DOI:10.1021/jm301000k

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Silanediols as Highly Selective
Uncompetitive Inhibitors of Human Neutrophil Elastase
Julie L. H. Madsen,† Thomas L. Andersen,† Salvatore Santamaria,‡,§ Hideaki Nagase,‡,§ Jan J. Enghild,*,∥
and Troels Skrydstrup*,†
†Center for Insoluble Protein Structures, Department of Chemistry, and Interdisciplinary Nanoscience Center, Aarhus University,
Langelandsgade 140, 8000 Aarhus C, Denmark
‡Faculty of Medicine, Imperial College London, 65 Aspenlea Road, London W6 8LH, U.K.
§The Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences,
University of Oxford, 65 Aspenlea Road, London W6 8LH, U.K.
∥Center for Insoluble Protein Structures, Department of Molecular Biology and Genetics, and Interdisciplinary Nanoscience Center,
Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark
S
* Supporting Information
ABSTRACT: Chronic obstructive pulmonary disease
(COPD) is an increasing health problem and is estimated to
be the fifth leading cause of death in 2020 according to the
World Health Organization. Current treatments are only
palliative, and therefore the development of new medicine for
the treatment of COPD is urgent. Human Neutrophil Elastase
(HNE) is a serine protease that is heavily involved in the
progression of COPD through inflammatory breakdown of
lung tissue. Consequently, inhibitors of HNE are of great
interest as therapeutics. In this article, the development of silanediol peptide isosters as inhibitors of HNE is presented. Kinetic
studies revealed that incorporation of a silanediol isoster in the inhibitor structure resulted in an uncompetitive mechanism of
inhibition, which further resulted in excellent selectivity. The peculiar mechanism of inhibition and the resulting selectivity makes
the presented inhibitors promising leads for the development of new HNE-inhibitor-based therapeutics for the treatment of
COPD.
inhibitors of HNE could show great potential as therapeutics
for the various pathological conditions arising from the role of
HNE in the inflammatory process and lung tissue damage.
The increased focus on the development of HNE inhibitor-
based therapeutics has, during the past two decades, resulted in
numerous HNE inhibitors reported in the literature, some of
which have undergone clinical trials.10,11 The small molecule
inhibitors published have, to the best of our knowledge, all been
active site-directed competitive inhibitors, and a common
structural inhibitor motif is based on a Val-Pro-Val tripeptide
part binding to the active site cleft combined with a C-terminal
electrophilic carbonyl interacting with the catalytic serine
residue (Figure 1, inhibitor 1).12 In addition, many heterocyclic
and acetylating inhibitors have been developed (Figure 1,
inhibitors 2 and 3).13,14 Despite the numerous inhibitors
published, so far only one compound has entered the market
(Figure 1, inhibitor 2). This is an acetylating inhibitor and is
applied for the treatment of acute lung injury (ALI) in Japan,
whereas the clinical trials in the western world were abandoned
in 2003.15 The majority of HNE inhibitors have failed clinical
INTRODUCTION
■
Human Neutrophil Elastase (HNE) is a serine protease
belonging to the chymotrypsin family. The enzyme is stored
in the azurophilic granules of neutrophils where it participates
in the degradation of engulfed pathogens. HNE efficiently
degrades elastin and other extracellular matrix proteins, and
upon inflammation, HNE is released by neutrophils and assists
in neutrophil migration to the site of inflammation.1−3 In order
to prevent an excessive breakdown of healthy tissue at
physiological conditions, the activity of extracellular HNE is
controlled by endogenous serine protease inhibitors (SER-
PINs) like the α1-protease inhibitor (α1-PI). An imbalance
between levels of extracellular HNE and the endogenous
SERPINs leads to various pathological conditions, such as
chronic obstructive pulmonary diseases (COPD),4 acute
respiratory distress syndrome,5 acute lung injury,6 and cystic
fibrosis.7 Such imbalances can result either from inherited
mutations of the SERPIN or from SERPIN oxidation by
cigarette smoke, both resulting in inactivation of the SERPIN.
Animal studies have revealed that a reduced level of catalytically
active HNE, either by genetic deficiency or inhibition of HNE,
protects against pro-inflammatory effects of chronic cigarette
smoke exposure.8,9 Therefore, the identification of selective
Received: July 10, 2012
Published: August 13, 2012
© 2012 American Chemical Society
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In this article, we report the stereoselective synthesis of
compound 6b and analogues hereof, and show that such
compounds containing the silanediol moiety display an
uncompetitive mechanism of inhibition which proved im-
portant for selectivity over six other serine proteases in this
study, some of which are closely related to HNE.
RESULTS AND DISCUSSION
■
Chemistry and Design. A stereoselective synthesis of
enantiomerically pure silanediol 6b was easily achieved by
applying a method previously developed by our group; addition
of a silyllithium reagent to an Ellman chiral sulfinimine was the
key step of the synthesis pathway and fully controlled the
stereochemistry at the α-carbon at the N-terminal side of the
silanediol (Scheme 1).21,22 Synthesis of compound 6b was
Scheme 1. Synthetic Strategy for the Stereoselective
Synthesis of Inhibitor 6b
Figure 1. Examples of three types of previously reported HNE
inhibitors.
trials due to a lack in proof of therapeutic effect or due to
toxicity resulting from a lack of selectivity.16
In general, proteases represent a significant therapeutic target
due to their essential physiological roles. Often a transition state
isoster motif is applied in the design of new inhibitors.
Silanediols are one example of such isosters that mimic the
tetrahedral intermediate of protein hydrolysis due to the
inherent stability of the geminal diol as compared to their
carbon analogue. Examples of silanediols as inhibitors of
aspartic- and metalloproteases have been reported by Sieburth
et al. (Figure 2, inhibitors 4 and 5).17,18 Silanediol 4 is an
chosen over 6a due to the natural L-amino acid configuration of
the leucine residue in 6b. Preliminary enzyme kinetics
experiments revealed that inhibitor 6b did not compete with
the chromogenic substrate and was therefore not a competitive
inhibitor of HNE. This finding is in contrast to the previously
published in silico design of silanediols 6a and 6b binding to the
active site as a competitive inhibitors of HNE.20 In order to
further investigate the mechanism of inhibition and structure−
activity relationship (SAR) of these types of compounds, a
small library of 11 inhibitors was developed.
Since inhibitor 6b was not competitive, the binding pocket at
HNE was unknown. The library was therefore kept simple with
the design of the analogues based on the already published
inhibitor 6b. Simple variations were made on the N- and C-
terminal capping groups, the substituents on silicon, and in
addition, regular peptides were included in the library in order
to determine the importance of the silanediol isoster for the
inhibition (Figure 3).
Silanediol inhibitors 6b and 11−14 were synthesized by
following the previously published procedure, and diphenylsi-
lane 16 is the precursor of inhibitor 6b (Scheme 2).23
Hydrosilylation of 2-(allyloxy)-tetrahydro-2H-pyran afforded
diphenylsilane 22, which upon lithiation and addition to
sulfinimine 7 afforded the diphenylsilane analogue of L-leucine
9. Protecting group manipulations and oxidation of the primary
alcohol provided dipeptide fragment 10, and standard solution
phase peptide synthesis was applied for reactions with the C-
terminal capping group, isoleucine, and the N-terminal capping
group. A final deprotection of the diphenylsilanes 16 and 26−
29 was achieved by using trifluoromethanesulfonic acid, and the
difluorosilanes 6b′ and 11′−14′ were isolated following
treatment with aqueous hydrofluoric acid.
Figure 2. Examples of silanediol-based protease inhibitors.
inhibitor of angiotensin-converting enzyme with an IC50 of 3.8
nM, whereas, silanediol 5 inhibits the HIV protease with a Ki
value of 2.7 nM. Compounds 6a and 6b were published in 2004
as a potent inhibitor of HNE.19,20 The silanediols were
synthesized and evaluated as a mixture of epimers and showed
an IC50 of 300 nM. The inhibitor was designed in silico to fit the
active site binding pocket; however, an experimental study of
the mechanism of inhibition has not been reported. To our
knowledge, this is the first example of a silanediol acting as a
serine protease inhibitor, and we therefore became interested in
studying the mechanism of inhibition of this compound.
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Because of the low stability of isolated silanediols, inhibitors
6b and 11−15 were isolated as the difluorosilanes 6b′ and
11′−15′ as can be seen from Schemes 2 and 3. However, upon
solvation in assay buffer, the fluorides are exchanged for
hydroxyl groups, affording the desired silanediols. This was
confirmed by NMR studies of 6b′ in assay buffer revealing that
the fluoride signals from the difluorosilane disappeared, and a
signal at −119 appeared, corresponding to sodium fluoride.25
The inhibitory data are therefore presented for the silanediols
as these are the active species at the applied assay conditions.
Biology. The inhibitory potency of the 11 analogues against
human neutrophil elastase (HNE) was tested in vitro using the
chromogenic substrate N-succinyl-Ala-Ala-Pro-Val-p-nitroani-
lide. The resulting IC50 values are reported in Table 1.
As can be seen from the data here reported, the presence of
an aromatic N-terminal capping group proved important for
inhibition. Inhibitors, 6b, 11, 16, and 17 containing an N-
terminal naphthyl substituent all displayed IC50 values in the
range of 14−18 μM, whereas inhibitors 13, 14, 19, and 20
containing N-terminal acetyl or trifluoroacetyl groups exhibited
low or no inhibition. The C-terminal part of the inhibitor did
not prove vital for inhibition; however, an increase in IC50 was
observed upon its removal, as observed with inhibitor 15. When
evaluating the importance of the isoster, it was seen by
comparing inhibitor 6b, 16, and 17 that the silanediol, the
diphenylsilane, and the regular peptide all show inhibitory
potency in the same range. These data indicate that the
silanediol isoster moiety is not important for the inhibition of
HNE. This is in good agreement with the preliminary results
demonstrating that inhibitor 6b does not interact with the
active site catalytic serine residue and does therefore not act as
a regular transition state inhibitor. In the case of silanediol-
based transition state inhibitors of metallo- and aspartyl
proteases, the silanediol mimics the tetrahedral intermediate
of hydrolysis accurately, with H2O acting as nucleophile in the
proteolysis (Figure 4a). However, with the serine proteases, a
serine residue plays the role of nucleophile during hydrolysis,
thereby forming a covalently bound intermediate to the enzyme
(Figure 4b). Hence, silanediols are not obvious transition state
inhibitors for the competitive inhibition of serine proteases,
which is again in good agreement with the results obtained.
From the data obtained on the inhibitory potency, the five
best compounds, inhibitors, 6b, 11, 12, 16, and 17, were
chosen for further studies on the mechanism of inhibition. The
inhibition of HNE was measured at two different substrate
concentrations and Dixon plots and Cornish−Bowden plots
were prepared.26,27 Representative plots are shown in Figure 5.
The results showed a clear trend on the mechanism of
inhibition, depending on the type of inhibitor. Inhibitors 6b,
11, and 12, containing the silanediol moiety, followed an
uncompetitive mechanism of inhibition. This was seen from the
parallel curves in the Dixon plot and the intercept above the x-
axis in the Cornish−Bowden plot (Figure 5a). The Ki value was
obtained from the negative x-value of the intercept in the
Cornish−Bowden plots (Table 1). In contrast, the diphenylsi-
lane 16 and the regular peptide inhibitor 17 displayed a
noncompetitive mechanism of inhibition, as was seen from the
intercept at the x-axis in the Dixon plot and below the x-axis in
the Cornish−Bowden plot (Figure 5b). For inhibitors 16 and
17 the Ki value was obtained from the negative x-value of the
intercept at the x-axis in the Dixon plot. Interestingly, this
difference in mechanism did not prove important for the
inhibitory potency as was seen from the similar IC50 values of
Figure 3. Inhibitor analogues included in the SAR study.
Silanediol inhibitor 15 was prepared via a di-p-tolylsilane
route (Scheme 3). Treatment of dichloromethylsilane with p-
tolyl-grignard reagent afforded di-p-tolylsilane 31. Lithiation
and addition to sulfinimine 7 followed by protecting group
manipulations afforded the di-p-tolylsilane analogue of L-leucine
33. Again, standard solution phase peptide synthesis was
applied for the elongation of the peptide backbone. Di-p-
tolylsilane 35 was converted into the silanediol by applying a
method, previously published by our group, which uses milder
cleavage conditions;24 the di-p-tolylsilane deprotection was
achieved by using a 1:1 solution of tifluoroacetic acid and
CH2Cl2. Difluorosilane 15′ was isolated after treatment with
aqueous hydrofluoric acid.
The regular peptide inhibitors 17−20 were synthesized by
solution phase peptide synthesis using a standard Boc-
protection strategy (Scheme 4).
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a
Scheme 2. Synthesis of Silanediol-Based Inhibitor Analogues
a
Reagents: (a) 2-(Allyloxy)tetrahydro-2H-pyran, chlorotris(triphenylphosphino)-rhodium(I), THF; (b) lithium(s), THF; (c) (R,E)-2-methyl-N-(3-
methylbutylidene)propane-2-sulfinamide, THF; (d) HCl in MeOH (0.5M); (e) di-tert-butyl dicarbonate, triethylamine, CH2Cl2; (f) NaOH (2M),
CH2Cl2; (g) sodium (meta)periodate, ruthenium(III) chloride, acetonitrile, water, ethyl acetate; (h) n-butylamine, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide, 1-hydroxy-benzotriazole, N-methyl morpholine, CH2Cl2; (i) trifluoroacetic acid, CH2Cl2; (j) N-(tert-Butoxycarbonyl)-L-
isoleucine, hemihydrate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, N-methyl morpholine, CH2Cl2; (k) correspond-
ing carboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, N-methyl morpholine, CH2Cl2; (l) trifluorometha-
nesulfonic acid, CH2Cl2; (m) ammonium hydroxide (28%), CH2Cl2; (n) hydrofluoric acid (48% aqueous solution), CH2Cl2.
the five inhibitors. However, these interesting mechanisms of
inhibition prompted us to expect better selectivities of these
inhibitors as compared to the numerous competitive HNE
inhibitors reported in the literature and clinical trials. Since
serine proteases are rather similar, with the active site built
around the catalytic triad of serine, histidine, and aspartate,
competitive inhibitors, which interact with the catalytic serine
residue, might also easily interact with catalytic serine residues
of other serine proteases, resulting in low selectivity and
therefore high toxicity when administered as a drug. The
observation that the inhibitors presented in this work follow
un- or noncompetitive mechanisms might result in more
selective inhibitors and provide a new route for the develop-
ment of HNE-inhibitor-based therapeutics for the treatment of
the various lung-tissue-damaging diseases.
In order to test the hypothesis of the uncompetitive and
noncompetitive inhibitors exhibiting improved selectivities, the
inhibitors, 6b, 11, 12, 16, and 17, were tested in vitro in
chromogenic assays for the inhibitory potency of six other
serine proteases, including, plasmin, thrombin, cathepsin G,
porcine pancreatic elastase (PPE), trypsin, and chymotrypsin,
some of which have up to 40% homology with HNE. The
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a
Scheme 3. Synthesis of Silanediol 15 via Di-p-tolylsilane
CONCLUSIONS
■
Overall, this study has provided important information on the
SAR and mechanism of the presented silanediol-type inhibitors
for the inhibition of HNE. A highly interesting uncompetitive
mechanism of inhibition was observed for inhibitors, 6b, 11,
and 12, resulting in excellent selectivity over six other serine
proteases. The presented inhibitors therefore represent a
promising avenue to pursue for the development of important
therapeutics for the treatment of COPD and related diseases.
In future studies, the mode of HNE binding will be
investigated in a combined study exploiting hydrogen exchange
mass spectrometry (HXMS) and MD-simulations. This knowl-
edge will be of high importance for further development of
interesting potent and selective inhibitors of HNE.
EXPERIMENTAL PROCEDURES
■
Chemistry. Solvents were dried according to standard procedures.
All commercially available reagents were used as received without
further purification unless otherwise noted. All reactions were
performed under inert atmosphere unless otherwise noted. Reactions
were monitored by thin-layer chromatography (TLC) analysis. Flash
chromatography was performed using silica gel 60 (230−400 mesh).
1H NMR was recorded at 400 MHz, 13C NMR at 100 MHz and 19F
NMR at 377 MHz. The chemical shifts are reported in ppm relative to
solvent residual peak.28 Multiplicities are reported using the following
abbreviations: s = singlet, bs = broad singlet, d = dublet, t = triplet, q =
quartet, quin = quintet, hex = hextet, hep = heptet, non = nonet, and
m = multiplet. HRMS was recorded on a TOF-MS (ESI) apparatus.
The purity of all tested inhibitor analogues were confirmed as ≥95%
using analytical RP-HPLC performed using an Agilent 1100 system
with a KINETEX column (C18, 4.6 mm × 150 mm, 2.6 μm, 100 Å)
operated at a flow rate of 1 mL/min at 25 °C and a linear gradient
from 5 to 90% B in A. The solvent systems applied were A (0.1%
trifluoroacetic acid in H2O) and B (0.1% trifluoroacetic acid in
acetonitrile). Absorbance was monitored at 215 nm and product
purities measured as peak area percentage at 215 nm.
General Procedure A: TFA-Mediated Boc Deprotection and
EDC Coupling. To a solution of carbamate (0.2 mmol) in CH2Cl2
(2.5 mL) was added trifluoroacetic acid (1.25 mL), and the reaction
was stirred for 2 h. All volatiles were removed in vacuo, and the crude
compound was washed three times by addition and evaporation of
CH2Cl2. The TFA-salt of the deprotected peptide (0.2 mmol) was
dissolved in CH2Cl2 (2.5 mL) and then N-methylmorpholine (0.8
mmol), carboxylic acid (0.4 mmol), HOBt (0.8 mmol), and EDC (0.4
mmol) were added, and the reaction was stirred for 24 h. The reaction
mixture was poured into water and extracted with CH2Cl2. The
combined organic phases were dried over MgSO4, filtered, and
evaporated in vacuo. Pure product was obtained by flash chromatog-
raphy (FC).
General Procedure B: Diphenylsilane Deprotection. To a
solution of diphenylsilane (0.030 mmol) in CH2Cl2 (2 mL) at 0 °C
was dropwise added triflic acid (0.450 mmol), and the reaction was
stirred overnight while allowed to warm to rt. The reaction was cooled
to 0 °C and ammonium hydroxide (25%w/w, 0.785 mmol) was added.
The reaction was stirred for 1 h at 0 °C after which hydrofluoric acid
(48% in H2O, 2.260 mmol) was added, and the reaction was stirred for
further 30 min. CH2Cl2 was added, and the reaction was washed with
H2O and brine, dried over Na2SO4, and evaporated in vacuo, affording
the product.
(2S,3S)-N-((R)-1-((3-(Butylamino)-3-oxopropyl)difluo-rosilyl)-
3-methylbutyl)-3-methyl-2-(2-(naphthalen-2-yl)acetamido)-
pentanamide (6b′). The compound was synthesized according to a
literature procedure.23 1H NMR (400 MHz, CDCl3) δ (ppm) 3.78−
3.85 (m, 4H), 7.75 (s, 1H), 7.45−7.51 (m, 2H), 7.37 (dd, 1H, J = 8.4
Hz, J = 1.7 Hz), 6.52 (d, 1H, J = 8.8 Hz), 5.64 (bs, 1H), 4.45 (t, 1H, J
= 8.4 Hz), 3.76 (dd, 2H, J = 19.4 Hz, J = 15.4 Hz), 3.18−3.25 (m,
2H), 2.74−2.81 (m, 1H), 2.19−2.32 (m, 2H), 1.73−1.83 (m, 1H),
1.54−1.65 (m, 1H), 1.23−1.54 (m, 8H), 0.94−1.05 (m, 2H), 0.76−
a
Reagents: (a) p-bromotoluene, Mg-bands, dibromoethane, THF; (b)
Li(s), THF; (c) (R,E)-2-methyl-N-(3-methylbutylidene)propane-2-
sulfinamide, THF; (d) HCl in MeOH (0.5M); (e) di-tert-butyl
dicarbonate, triethylamine, CH2Cl2; (f) trifluoroacetic acid, CH2Cl2;
(g) N-(tert-butoxycarbonyl)-L-isoleucine, hemihydrate, 1-(3-dimethy-
laminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, N-methyl
morpholine, CH2Cl2; (h) 2-naphthaleneacetic acid, 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, N-methyl
morpholine, CH2Cl2; (i) trifluoroacetic acid, CH2Cl2; (j) ammonium
hydroxide (28%), CH2Cl2; (k) hydrofluoric acid (48% aqueous
solution), CH2Cl2.
results are presented in Table 2. For the selectivity data, a clear
trend was observed. Inhibitors 6b, 11, and 12, which followed
an uncompetitive mechanism of inhibition of HNE, did not
display inhibition of any of the six serine proteases up to the
maximum assay concentration (100 μM), whereas the two
noncompetitive inhibitors, 16 and 17, both revealed varied
inhibition of the six enzymes. A general irreversible serine
protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride
hydrochloride (AEBSF), was also included in the tests as a
positive control for inhibition, and the IC50 values were
determined for all seven enzymes in this study (Table 2). The
trend of the selectivity data complements the determined
mechanisms of inhibition and suggests that an uncompetitive
inhibitor does supply a better selectivity than the non-
competitive inhibitors. Interestingly, this suggests that the
silanediol moiety, although not affording better inhibitory
potency, is important for providing an uncompetitive
mechanism of inhibition and thereby yielding highly selective
inhibitors of HNE.
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a
Scheme 4. Synthesis of Regular Peptide Inhibitors
a
Reagents: (a) N-(tert-butoxycarbonyl)glycine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, N-methyl morpholine,
CH2Cl2; (b) trifluoroacetic acid, CH2Cl2; (c) N-(tert-butyloxycarbonyl)-L-leucine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydrox-
ybenzotriazole, N-methyl morpholine, CH2Cl2; (d) N-(tert-Butoxycarbonyl)-L-isoleucine, hemihydrate, 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide, 1-hydroxybenzotriazole, N-methyl morpholine, CH2Cl2; (e) corresponding carboxylic acid, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide, 1-hydroxybenzotriazole, N-methyl morpholine, CH2Cl2.
(silanediol was formed during ionization) C30H47N3O5Si [M + K+];
calculated, 596.2922; found, 596.2918.
a
Table 1. Kinetic Data for HNE Inhibition
inhibitor
IC50 (μM)
Ki (μM)
N-((2S,3S)-1-(((R)-1-((3-(Butylamino)-3-oxopropyl)-
difluorosilyl)-3-methylbutyl)amino)-3-methyl-1-oxo-pentan-2-
yl)-2-naphthamide (11′). The compound was synthesized according
to general procedure B from diphenylsilane 26 (0.03 mmol), affording
6b
11
12
13
14
15
16
17
18
19
20
18
3.5
4.0
5.0
16
38
1
the product (11.8 mg, 72%) as a colorless solid. H NMR (400 MHz,
>200
46
CDCl3) δ (ppm) 8.36 (s, 1H), 7.84−7.95 (m, 4H), 7.70 (bs, 1H), 7.56
(dquin, 2H, J = 7.0 Hz, J = 1.4 Hz), 7.22−7.26 (m, 1H), 5.68 (bs, 1H),
4.70 (t, 1H, J = 8.2 Hz), 3.17 (dd, 2H, J = 13.0 Hz, J = 7.0 Hz), 2.90−
2.97 (m, 1H), 2.30−2.40 (m, 2H), 2.03−2.14 (m, 1H), 1.20−1.72 (m,
9H), 1.07−1.15 (m, 2H), 1.02 (d, 3H, J = 6.8 Hz), 0.96 (t, 3H, J = 7.3
Hz), 0.84−0.92 (m, 9H). 13C NMR (100 MHz, CDCl3) δ (ppm)
174.8, 173.9, 168.2, 135.1, 132.7, 130.8, 129.2, 128.7, 128.1, 128.0,
127.9, 127.0, 123.7, 39.8, 39.2, 38.7 (dd, J = 24.8 Hz, J = 21.8 Hz),
37.3, 31.7, 30.6, 26.0, 25.4, 23.7, 22.8, 21.1, 20.1, 15.6, 13.8, 11.6 (t, J =
19.9 Hz), 11.3. 19F NMR (377 MHz, CDCl3) δ (ppm) −131.6,
−135.6. HRMS (silanediol was formed during ionization)
C29H45N3O5Si [M + Na+]; calculated, 566.3026; found, 566.3005.
(2S,3S)-N-((R)-1-((3-(Butylamino)-3-oxopropyl)-difluorosilyl)-
3-methylbutyl)-3-methyl-2-(2-phenylacetamido)pentanamide
(12′). The compound was synthesized according to general procedure
B from diphenylsilane 27 (0.03 mmol), affording the product (13.8
39
14
13.0
18.0
14
51
>200
>200
a
The IC50 and Ki values are the average of at least two determinations
with a standard deviation of <10%.
1
mg, 100%) as a light brown solid. H NMR (400 MHz, CDCl3) δ
(ppm) 8.02 (bs, 1H), 7.37−7.24 (m, 5H), 6.46 (d, 1H, J = 8.8 Hz),
5.73 (t, 1H, J = 5.6 Hz), 4.46 (t, 1H, J = 8.0 Hz), 3.60 (AB system, 2H,
J = 15.6 Hz), 3.23 (dt, 2H, J = 7.2 Hz, J = 6.0 Hz), 2.73−2.80 (m, 1H),
2.23−2.36 (ddd, 2H, J = 15.6 Hz, J = 12.4 Hz, J = 7.6 Hz), 1.72−1.82
(m, 1H), 1.57−1.68 (m, 1H), 1.28−1.55 (m, 8H), 0.95−1.06 (m, 2H),
0.78−0.94 (m, 15H). 13C NMR (100 MHz, CDCl3) δ (ppm) 174.9,
173.7, 171.9, 134.5, 129.3(2C), 129.2(2C), 127.7, 55.8, 43.6, 39.7,
39.0, 38.4 (dd, J = 26.4 Hz, J = 20.6 Hz), 36.9, 31.8, 30.7, 26.0, 25.0,
23.7, 21.1, 20.0, 15.3, 13.9, 11.8 (dd, J = 21.1 Hz, J = 19.0 Hz), 11.1.
19F NMR (377 MHz, CDCl3) δ (ppm) −130.1, −136.8. HRMS
Figure 4. Simplified comparison of silanediol peptide isosters with
intermediates of protolytic cleavage by (a) aspartic- and metal-
loproteases and (b) serine proteases.
(silanediol was formed during ionization) C26H45N3O5Si [M+K+];
calculated, 546.2766; found, 546.2887.
(2S,3S)-2-Acetamido-N-((R)-1-((3-(butylamino)-3-oxo-
propyl)difluorosilyl)-3-methylbutyl)-3-methylpentanamide
(13′). The compound was synthesized according to general procedure
B from diphenylsilane 28 (0.030 mmol), affording the product (11.8
0.94 (m, 15H). 13C NMR (100 MHz, CDCl3) δ (ppm) 174.9, 173.9,
171.8, 133.7, 132.7, 132.1, 128.8, 128.2, 127.82, 127.77, 127.1, 126.5,
126.2, 55.7, 43.7, 39.7, 39.0, 38.3 (dd, J = 26.5 Hz, J = 21.2 Hz), 37.1,
31.7, 30.7, 25.9, 25.0, 23.7, 21.0, 20.2, 15.2, 13.9, 11.8 (t, J = 20.2 Hz),
11.1. 19F NMR (377 MHz, CDCl3) δ (ppm) −129.4, −135.8. HRMS
1
mg, 100%) as a light brown solid. H NMR (400 MHz, CDCl3) δ
(ppm) 8.48 (bs, 1H), 6.83 (d, 1H, J = 9.2 Hz), 5.73 (t, 1H, J = 6.0
Hz), 4.58 (t, 1H, J = 8.8 Hz), 3.24 (dt, 2H, J = 6.8 Hz, J = 6.0 Hz),
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a
Table 2. Selectivity Data of Selected Inhibitors toward
Human Neutrophile Elastase, Trypsin, Chymotrypsin,
Porcine Pancreatic Elastase, Cathepsin G, Plasmin, and
Thrombin
IC50 (μM)
6b
18
11
16
12
38
16
14
17
14
AEBSF
HNE
130
10
b
b
b
trypsin
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
58
89
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
chymotrypsin
PPE
110
150
55
12
b
NI
160
250
240
62
b
cathepsin G
plasmin
thrombin
120
NI
b
NI
99
92
100
a
The IC50 values are the average of at least two determinations with a
b
standard deviation <10%. NI: No inhibition observed at 100 μM.
HNE, human neutrophil elastase; PPE, porcine pancreatic elastase;
AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride.
−128.7, −137.2. HRMS (silanediol was formed during ionization)
C20H41N3O5Si [M + K+]; calculated, 470.2453; found, 470.2470.
(2S,3S)-N-((R)-1-((3-(Butylamino)-3-oxopropyl)-difluorosilyl)-
3-methylbutyl)-3-methyl-2-(2,2,2-trifluoroacetamido)-
pentanamide (14′). The compound was synthesized according to
general procedure B from diphenylsilane 29 (0.04 mmol) affording the
1
product (13.4 mg, 69%) as a colorless solid. H NMR (400 MHz,
CDCl3) δ (ppm) 7.47 (bd, 1H, J = 8.3 Hz), 6.95 (bd, 1H, J = 5.8 Hz),
5.83 (bt, 1H, J = 5.4 Hz), 5.88 (dd, 1H, J = 8.3 Hz, J = 7.0 Hz), 3.23−
3.31 (m, 2H), 2.42 (dt, 2H, J = 7.6 Hz, J = 4.7 Hz), 1.88−1.96 (m,
1H), 1.44−1.54 (m, 6H), 1.30−1.38 (m, 4H), 1.12−1.22 (m, 2H),
0.84−0.96 (m, 15H). 13C NMR (100 MHz, CDCl3) δ (ppm) 175.5,
170.6, 157.3 (q, J = 37.5 Hz), 115.9 (q, J = 285.4 Hz), 57.2, 40.3, 39.7
(dd, J = 11.5 Hz, J = 7.9 Hz), 39.3, 37.8, 31.5, 29.8, 25.7, 25.0, 23.6,
21.3, 20.1, 15.2, 13.8, 11.3, 9.7 (t, J = 18.7 Hz). 19F NMR (377 MHz,
CDCl3) δ (ppm) −75.6, −134.3, −135.7. HRMS (silanediol was
formed during ionization) C20H38F3N3O5Si [M + Na+]; calculated,
508.2431; found, 508.2413.
(2S,3S)-N-((R)-1-(Difluoro(methyl)silyl)-3-methylbutyl)-3-
methyl-2-(2-(naphthalen-2-yl)acetamido)pentanamide (15′).
The compound was synthesized according to a modified literature
procedure.24 To a solution of di-p-tolyl silane 35 (20.5 mg, 0.03
mmol) in CH2Cl2 (0.2 mL) was added TFA (0.2 mL), and the
reaction was stirred for 36 h. The reaction was cooled to 0 °C, and
ammonium hydroxide (25%w/w, 0.2 mL, 1.35 mmol) was added. The
reaction was stirred for 1 h at 0 °C after which hydrofluoric acid (48%
in H2O, 0.175 mL, 4.94 mmol) was added, and the reaction was stirred
for further 30 min. CH2Cl2 was added, and the reaction was washed
with H2O and brine, dried over Na2SO4 and evaporated in vacuo,
1
affording the product, (15.6 mg, 100%) as a colorless solid. H NMR
(400 MHz, CDCl3) δ (ppm) 7.80−7.85 (m, 2H), 7.69 (bs, 1H), 7.56−
7.62 (m, 1H), 7.46−7.52 (m, 2H), 7.32 (dd, 2H, J = 8.4 Hz, J = 1.8
Hz), 6.02 (bd, 1H, J = 8.8 Hz), 4.44 (t, 1H, J = 8.6 Hz), 3.73 (s, 2H),
2.64 (dq, 1H, J = 11.3 Hz, J = 3.4 Hz), 1.45−1.55 (m, 3H), 1.30−1.40
(m, 3H), 0.70−0.90 (m, 12H), 0.07 (s, 3H). 19F NMR (377 MHz,
CDCl3) δ (ppm) −130.1, −132.0. HRMS (silanediol was formed
during ionization) C24H36N2O4Si [M + Na+]; calculated, 467.2325;
found, 467.2342.
(2S,3S)-N-((R)-1-((3-(Butylamino)-3-oxopropyl)diphenylsilyl)-
3-methylbutyl)-3-methyl-2-(2-(naphthalen-2-yl)acetamido)-
pentanamide (16). The compound was synthesized according to a
literature procedure.23 1H NMR (400 MHz, CDCl3) δ (ppm) 7.73−
7.84 (m, 3H), 7.59 (s, 1H), 7.52−7.56 (m, 2H), 7.34−7.50 (m, 10H),
7.20 (dd, 1H, J = 8.3 Hz, J = 1.6 Hz), 5.84 (d, 1H, J = 9.0 Hz), 5.74
(bs, 1H), 5.56 (d, 1H, J = 10.2 Hz), 4.37 (td, 1H, J = 11.3 Hz, J = 3.2
Hz), 4.12 (dd, 1H, J = 8.8 Hz, J = 6.9 Hz), 3.69 (dd, 2H, J = 21.5 Hz, J
= 15.8 Hz), 3.15 (dd, 2H, J = 13.0 Hz, J = 6.9 Hz), 2.30 (ddd, 1H, J =
14.4 Hz, J = 11.8 Hz, J = 4.7 Hz), 2.02 (ddd, 1H, J = 14.5 Hz, J = 11.6
Hz, J = 5.9 Hz), 1.70−1.80 (m, 1H), 1.15−1.48 (m, 10H), 0.88 (t, 3H,
Figure 5. Representative Dixon and Cornish−Bowden plots. (a)
Uncompetitive inhibitors, 6b, 11, and 12. (b) Noncompetitive
inhibitors 16 and 17.
2.75−2.80 (m, 1H), 2.23−2.40 (m, 2H), 2.05 (s, 3H), 1.76−1.84 (m,
1H), 1.64−1.73 (m, 1H), 1.30−1.58 (m, 6H), 1.04−1.14 (m, 4H),
0.83−0.97 (m, 15H). 13C NMR (100 MHz, CDCl3) δ (ppm) 174.9,
173.7, 170.8, 55.5, 47.1, 39.6, 38.3 (dd, J = 27.2 Hz, J = 20.1 Hz), 37.1,
31.5, 29.7, 25.8, 25.0, 23.6, 23.0, 20.9, 20.0, 15.1, 13.7, 11.8 (dd, J =
22.7 Hz, J = 18.5 Hz), 11.1. 19F NMR (377 MHz, CDCl3) δ (ppm)
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J = 7.3 Hz), 0.83 (d, 3H, J = 6.0 Hz), 0.74−0.82 (m, 1H), 0.68−0.74
(m, 9H). 13C NMR (100 MHz, CDCl3) δ (ppm) 174.1, 170.9, 170.4,
135.7 (2C), 135.4 (2C), 133.7, 132.6, 132.3, 132.2, 132.1, 130.24,
130.15, 129.0, 128.35 (2C), 128.31 (2C), 128.2, 127.8, 127.7, 127.0,
126.6, 126.2, 58.4, 44.0, 40.2, 39.4, 36.5, 34.9, 31.8, 30.7, 25.1, 24.7,
23.6, 21.1, 20.2, 15.6, 13.9, 11.3, 7.8. HRMS C42H55N3O3Si [M +
Na+]; calculated, 700.3910; found, 700.3952.
25.02, 24.96, 22.8, 22.6, 20.1, 15.3, 13.8, 11.3. 19F NMR (377 MHz,
CDCl3) δ (ppm) −75.6 (d, J = 27.0 Hz). HRMS C20H35F3N4O4 [M +
Na+]; calculated, 475.2508; found, 475.2487.
Biology. HNE and cathepsin G were purchased from Biocentrum
Ltd. Trypsin, chymotrypsin, and porcine pancreatic elastase were
purchased from Sigma-Aldrich. Thrombin and plasminogen were
purified as described previously.29 Chromogenic substrates were
purchased from Sigma-Aldrich, except for H-D-Pro-L-Phe-L-Arg-p-
nitroanilide, which was purchased from Chromogenix. Hydrolysis was
monitored with a FLUOstar Omega platereader, kinetic mode,
wavelength 405 nm. Reactions were performed in 96-well microtiter
plates. All data points were collected in duplicates. The applied assay
buffer was 0.1 M HEPES buffer containing 0.5 M NaCl at pH 7.25.
Human Neutrophil Elastase: IC50 Determination. The
inhibitor stock solutions (100 mM in DMSO) were further diluted
at 7 different concentrations (5−500 μM) in assay buffer. HNE (2.8
μM in 0.05 M sodium acetate buffer containing 0.5 M NaCl, pH 5.5)
was diluted immediately prior to use in assay buffer to a concentration
of 300 nM. The chromogenic substrate (N-succinyl-Ala-Ala-Pro-Val-p-
nitroanilide) stock solution (70 mM) in DMSO was diluted
immediately prior to use in assay buffer to a concentration of 3.5
mM. Assays were run in dublicates in a 96-well plate in a total volume
of 200 μL. DMSO was kept at 5.5% in every well. Enzyme (20 μL, final
enzyme concentration 30 nM) and inhibitor solutions (20 μL, final
inhibitor concentrations 0.5−50 μM) were incubated in assay buffer
for 2 h at 25 °C. Chromogenic substrate (20 μL, final substrate
concentration 0.35 mM) was added to the wells, and the rate of
hydrolysis was determined by monitoring the absorbance at 405 nm at
25 °C for 20 min. The rate of hydrolysis (ΔA/min) was linear between
5 and 15 min of reaction.
(S)-N-(2-(Butylamino)-2-oxoethyl)-4-methyl-2-((2S,3S)-3-
methyl-2-(2-(naphthalen-2-yl)acetamido)pentanamido)-
pentanamide (17). The compound was synthesized according to
general procedure A from carbamate 39 (0.35 mmol) and 2-
naphthaleneacetic acid (0.70 mmol). The crude compound was
purified by FC (eluent: 3% MeOH in CH2Cl2) affording the product
1
(114.7 mg, 63%) as a colorless solid. H NMR (400 MHz, CDCl3) δ
(ppm) 8.55−8.72 (m, 2H), 8.15 (bs, 1H), 7.66−7.78 (m, 4H), 7.60
(bs, 1H), 736−7.43 (m, 3H), 5.00−5.12 (m, 1H), 4.89 (t, 1H, J = 8.5
Hz), 4.56−4.67 (m, 1H), 3.85 (d, 1H, J = 14.6 Hz), 3.78 (d, 1H, J =
14.6 Hz), 3.64 (d, 1H, J = 13.9 Hz), 2.97 (non, 2H, J = 6.8 Hz), 1.62−
1.75 (m, 2H), 1.44−1.60 (m, 3H), 1.12−1.22 (m, 2H), 0.94−1.10 (m,
3H), 0.84−0.92 (m, 6H), 0.74−0.82 (m, 6H), 0.69 (t, 3H, J = 7.3 Hz).
13C NMR (100 MHz, CDCl3) δ (ppm) 172.5, 171.4, 171.0, 169.2,
134.0, 133.6, 132.4, 127.94, 127.86, 127.7, 127.63, 127.59, 126.0,
125.6, 57.3, 51.3, 43.2, 43.1, 43.0, 39.3, 31.5, 25.5, 25.1, 23.7, 22.5,
20.0(2C), 15.4, 13.7, 11.7. HRMS C30H44N4O4 [M + Na+]; calculated,
547.3260; found, 547.3265.
(S)-N-(2-(Butylamino)-2-oxoethyl)-4-methyl-2-((2S,3S)-3-
methyl-2-(2-phenylacetamido)-pentanamido)pentanamide
(18). The compound was synthesized according to general procedure
A from carbamate 39 (0.16 mmol) and phenylacetic acid (0.32 mmol).
The crude compound was purified by FC (eluent: 2−5% MeOH in
CH2Cl2) affording the product (32.5 mg, 43%) as a colorless solid. 1H
NMR (400 MHz, CDCl3) δ (ppm) 7.99−8.10 (m, 2H), 7.42 (bs, 1H),
7.35 (bs, 1H), 7.21−7.32 (m, 5H), 4.82 (q, 1H, J = 7.4 Hz), 4.69 (t,
1H, J = 8.2 Hz), 4.39 (dd, 1H, J = 16.3 Hz, J = 7.0 Hz), 3.74 (dd, 1H, J
= 16.1 Hz, J = 4.0 Hz), 3.66 (d, 1H, J = 14.8 Hz), 3.62 (d, 1H, J = 14.8
Hz), 3.03−3.17 (m, 2H), 1.64−1.73 (m, 2H), 1.40−1.62 (m, 3H),
1.31−1.39 (m, 2H), 1.19−1.29 (m, 2H), 0.92−1.03 (m, 1H), 0.90 (d,
3H, J = 6.4 Hz), 0.88 (d, 3H, J = 6.4 Hz), 0.84 (t, 3H, J = 7.3 Hz),
0.75−0.81 (m, 6H). 13C NMR (100 MHz, CDCl3) δ (ppm) 172.4,
171.5, 171.2, 169.1, 135.9, 129.4(2C), 128.6(2C), 126.9, 57.5, 51.6,
43.2, 43.1, 42.7, 39.4, 38.7, 31.6, 25.1, 25.0, 23.2, 22.6, 20.2, 15.4, 13.9,
11.6. HRMS C26H42N4O4 [M+Na+]; calculated, 497.3104; found,
497.3089.
(2S,3S)-2-Acetamido-N-((S)-1-((2-(butylamino)-2-oxo-ethyl)-
amino)-4-methyl-1-oxopentan-2-yl)-3-methylpentanamide
(19). The compound was synthesized according to general procedure
A from carbamate 39 (0.23 mmol) and acetic acid (0.46 mmol). The
crude compound was purified by FC (eluent: 2−4% MeOH in
CH2Cl2) affording the product (27.3 mg, 30%) as a colorless solid. 1H
NMR (400 MHz, CD3OD) δ (ppm) 8.25−8.33 (m, 2H), 8.10 (d, 1H,
J = 7.4 Hz), 7.79 (t, 1H, J = 4.6 Hz), 4.23−4.29 (m, 1H), 4.12−4.17
(m, 1H), 3.90−3.97 (m, 1H), 3.66−3.72 (m, 1H), 3.19−3.23 (m, 2H),
1.99 (s, 3H), 1.46−1.74 (m, 5H), 1.15−1.40 (m, 5H), 0.89−0.98 (m,
15H). 13C NMR (100 MHz, CDCl3) δ (ppm) 175.1, 174.4, 173.8,
171.3, 59.6, 53.9, 43.5, 41.0, 40.2, 37.8, 32.5, 26.1, 25.8, 23.3, 22.4,
22.0, 21.0, 15.9, 14.1, 11.3. HRMS C20H38N4O4 [M + Na+]; calculated,
421.2791; found, 421.2782.
Ki Determination: Dixon Plot and Cornish−Bowden Plot.
The mechanism of inhibition was studied by performing Dixon and
Cornish−Bowden plots.26,27 The inhibitory potency was measured at
two substrate concentrations, 0.0875 mM and 0.35 mM.
The inhibitor stock solutions (100 mM in DMSO) were further
diluted at 3 different concentrations (50−200 μM) in assay buffer.
Human neutrophil elastase (2.8 μM in 0.05 M sodium acetate buffer
containing 0.5 M NaCl, pH 5.5) was diluted immediately prior to use
in assay buffer to a concentration of 300 nM. The chromogenic
substrate (N-succinyl-Ala-Ala-Pro-Val-p-nitroanilide) stock solution 70
mM in DMSO was diluted immediately prior to use in assay buffer at
two different concentrations of 0.875 mM and 3.5 mM. Assays were
run in duplicates in a total volume of 200 μL. DMSO was kept at 5.5%
in every well. Enzyme (20 μL, final enzyme concentration 30 nM) and
inhibitor solutions (20 μL, final inhibitor concentrations 5−20 μM)
were incubated in assay buffer for 2 h at 25 °C. Chromogenic substrate
(20 μL, final substrate concentrations 0.0875 mM or 0.35 mM) was
added to the wells, and the rate of hydrolysis was determined by
monitoring the absorbance at 405 nm at 25 °C for 20 min.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental data and spectroscopic data, experimental and
spectroscopic data for the conversion of difluorosilanes to
silanediols at assay conditions, dose−response curves, Dixon
plots, and Cornish−Bowden plots. This material is available
free of charge via the Internet at http://pubs.acs.org.
(S)-N-(2-(Butylamino)-2-oxoethyl)-4-methyl-2-((2S,3S)-3-
methyl-2-(2,2,2-trifluoroacet-amido)pentanamido)-
pentanamide (20). The compound was synthesized according to
general procedure A from carbamate 39 (0.16 mmol) and trifluoro-
acetic acid (0.32 mmol). The crude compound was purified by FC
(eluent: 2−4% MeOH in CH2Cl2) affording the product (3.0 mg, 4%)
AUTHOR INFORMATION
■
Corresponding Author
1
*(J.J.E.) Phone: +45 87155449. E-mail: jje@mb.au.dk. (T.S.)
Phone: +45 87155968. E-mail: ts@chem.au.dk.
as a colorless solid. H NMR (400 MHz, CDCl3) δ (ppm) 7.80 (d,
1H, J = 8.6 Hz), 7.74 (d, 1H, J = 7.1 Hz), 7.48 (bs, 1H), 6.62 (t, 1H, J
= 5.8 Hz), 4.74 (q, 1H, J = 7.8 Hz), 4.65 (t, 1H, J = 8.4 Hz), 4.18 (dd,
1H, J = 16.5 Hz, J = 6.0 Hz), 3.91 (dd, 1H, J = 16.6 Hz, J = 4.5 Hz),
3.17−3.31 (m, 2H), 1.81−1.90 (m, 1H), 1.44−1.69 (m, 5H), 1.29−
1.39 (m, 3H), 1.05−1.17 (m, 1H), 0.85−0.98 (m, 15H). 13C NMR
(100 MHz, CDCl3) δ (ppm) 172.3, 169.8, 168.3, 157.4 (q, J = 37.2
Hz), 116.0 (q, J = 285.3 Hz), 58.1, 51.9, 43.1, 42.8, 39.4, 38.1, 31.4,
Author Contributions
J.L.H.M. performed the syntheses and enzyme kinetics studies;
T.L.A. performed part of the syntheses; S.S. and H.N. assisted
in the interpretation of the enzyme kinetics data; J.J.E. and T.S.
supervised the project.
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(16) Ohbayashi, H. Current synthetic inhibitors of human neutrophil
elastase in 2005. Expert Opin. Ther. Patents 2005, 15, 759−771.
(17) Kim, J.; Hewitt, G.; Carroll, P.; Sieburth, S. McN. Silanediol
Inhibitors of Angiotensin-Converting Enzyme. Synthesis and Evalua-
tion of Four Diastereomers of Phe[Si]Ala Dipeptide Analogues. J. Org.
Chem. 2005, 70, 5781−5789.
(18) Chen, C.-A.; Sieburth, S. M. N.; Glekas, A.; Hewitt, G. W.;
Trainor, G. L.; Erickson-Viitanen, S.; Garber, S. S.; Cordova, B.; Jeffry,
S.; Klabe, R. M. Drug design with a new transition state analog of the
hydrated carbonyl: silicon-based inhibitors of the HIV protease. Chem.
Biol. 2001, 8, 1161−1166.
(19) Mills, J. S.; Showell, G. A. Exploitation of silicon medicinal
chemistry in drug discovery. Expert Opin. Invest. Drugs 2004, 13,
1149−1157.
(20) Showell, G. A.; Montana, J. G.; Chadwick, J. A.; Higgs, C.; Hunt,
H. J.; MacKenzie, R. E.; Price, S.; Wilkinson, T. J. Silicon Diols,
Effective Inhibitors of Human Leukocyte Elastase. Organosilicon
Chemistry VI: From Molecules to Materials; John Wiley & Sons: New
York, 2005; pp 569−574.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
We thank the Danish National Research Foundation, the
Danish Council for Independent Research/Natural Sciences,
the Carlsberg Foundation, the Lundbeck Foundation, and
Aarhus University for financial support.
ABBREVIATIONS USED
■
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochlor-
ide; Boc, tert-butyloxycarbonyl; COPD, chronic obstructive
pulmonary disease; DMSO, dimethyl sulfoxide; EDC, N-ethyl-
N′-(3-dimethylamino-propyl)carbodiimide hydrochloride; FC,
flash chromatography; HNE, human neutrophil elastase; HOBt,
1-hydroxybenzotriazole; HXMS, hydrogen exchange mass
spectrometry; MD, molecular dynamics; PPE, porcine pancre-
atic elastase; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
THP, tetrahydropyranyl
(21) Nielsen, L.; Lindsay, K. B.; Faber, J.; Nielsen, N. C.; Skrydstrup,
T. Stereocontrolled synthesis of methyl silanediol peptide mimics. J.
Org. Chem. 2007, 72, 10035−10044.
(22) Nielsen, L.; Skrydstrup, T. Sequential C-Si bond formations
from diphenylsilane: application to silanediol peptide isostere
precursors. J. Am. Chem. Soc. 2008, 130, 13145−13151.
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(25) See Supporting Information for further descriptions of NMR
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