Effects of structure on inhibitory activity in a series of mechanism-based inhibitors of human neutrophil elastase

Article abstract of DOI:10.1016/j.bmc.2010.07.071

A structurally-diverse series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-one 1,1 dioxide scaffold were synthesized and used to probe the S′ subsites of human neutrophil elastase (HNE) and neutrophil proteinase 3 (Pr 3). Several compounds

Full text of DOI:10.1016/j.bmc.2010.07.071

Bioorganic & Medicinal Chemistry 18 (2010) 6646–6650
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of structure on inhibitory activity in a series of mechanism-based
inhibitors of human neutrophil elastase
*
Dengfeng Dou, Guijia He, Rongze Kuang, Qingfong Fu, Radhika Venkataraman, William C. Groutas
Department of Chemistry, Wichita State University, Wichita, KS 67260, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A structurally-diverse series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-one 1,1 dioxide
scaffold were synthesized and used to probe the S0 subsites of human neutrophil elastase (HNE) and neu-
trophil proteinase 3 (Pr 3). Several compounds are potent inhibitors of HNE but devoid of inhibitory activ-
ity toward Pr 3, suggesting that the S0 subsites of HNE exhibit significant plasticity and can, unlike Pr 3,
tolerate various large hydrophobic groups. The results provide a promising framework for the design of
highly selective inhibitors of the two enzymes.
Received 24 June 2010
Revised 28 July 2010
Accepted 30 July 2010
Available online 5 August 2010
Keywords:
Neutrophil elastase
Inhibitors
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
structure a conjugated sulfonyl imine functionality. Subsequent
slow reaction with water leads to the formation of one or more acyl
The human neutrophil elastase (HNE) is believed to play an
important role in the pathophysiology of an array of inflammatory
diseases, including chronic obstructive pulmonary disease (COPD),1
cystic fibrosis,2 acute respiratory distress syndrome,3 ischemia/
reperfusion injury,4 and others.5,6 COPD is a multi-factorial disorder
that is characterized by an oxidant/anti-oxidant imbalance,7,8
alveolar septal cell apoptosis,9,10, a protease/anti-protease imbal-
ance,1,11 and chronic inflammation.7,12 The confluence and interplay
of multiple processes and mediators have gravely hampered efforts
aimed at elucidating the molecular mechanisms and biochemical
events which underlie the initiation and progression of COPD.13
An array of proteases, including serine (neutrophil elastase, pro-
teinase 3), cysteine (cathepsin S) and metallo- (MMP-12) proteases
released by neutrophils, macrophages and T lymphocytes that are
capable of degrading lung elastin and other components of the
extracellular matrix,14 have been implicated in COPD. Elucidation
of the pathogenic mechanisms in COPD and, specifically, the role
each protease plays in the disorder, would pave the way toward
the development of novel COPD therapeutics.15
enzymes of variable stability (Fig. 1). We describe herein the re-
sults of exploratory studies related to the utilization of inhibitor
(I) to probe the S0 subsites17 of HNE and human proteinase 3 (Pr
3) that shares 54% sequence similarity with HNE.18
2. Chemistry
Compounds 3–18 were synthesized as shown in Scheme 116,19
starting with (L) leucine methyl ester hydrochloride. The heterocy-
clic ring 1 was readily assembled in three steps as previously de-
scribed20 and then further elaborated to yield N-chloromethyl
intermediate 2 (R1 = isobutyl, R2 = methyl or benzyl) which was
transformed into the desired compounds via reaction with sodium
iodide in acetone and subsequently with a carboxylic acid in the
presence of DBU.
3. Biochemical studies
We have previously demonstrated that the 1,2,5-thiadiazolidin-
3-one 1,1 dioxide scaffold is a powerful and versatile core structure
that can be used in the design of potent mechanism-based inhibi-
tors of serine proteases that exploit multiple binding interactions
on either side of the scissile bond.16 X-ray crystallography and
ESI-MS studies have furthermore demonstrated that inhibitor (I)
inactivates HNE via a mechanism that involves the initial forma-
tion of a relatively stable acyl enzyme that incorporates in its
The inhibitory activity of compounds 3–18 was determined
using the progress curve method.21 Typical progress curves for
the hydrolysis of MeOSuc-AAPV-p-NA by HNE in the presence of
inhibitor 18 are shown in Figure 2. Control curves in the absence
of inhibitor were linear. The release of p-nitroaniline was continu-
ously monitored at 410 nm. The pseudo first-order rate constants
(kobs) for the inhibition of HNE by 18 as a function of time were
determined according to Eq. (1), where A is the absorbance at
410 nm, v0 is the reaction velocity at t = 0, vs is the final steady-
state velocity, kobs is the observed first-order rate constant, and
A0 is the absorbance at t = 0. The kobs values were obtained by
* Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.071

D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 6646–6650
6647
O
O
O
R1
R2
195
-E
R1
R2
195
-E
195
-OH
R1
R2
Ser
Ser
E-Ser
N
R3
O
N
O
N
S
O
N
N
NH2
S
O
S
O
O
O
O
O
O
(I)
Figure 1. Mechanism of action of inhibitor (I).
O
O
O
R1
R1
R1
HN
d, e, f
a, b, c
g
(L) Leu-OCH3
(HCl)
N
O
N
O
NH
O
N
N
O
S
S
R2
R2
S
Cl
O
O
( I )
O
O
R3
2
1
(R1=isobutyl
R2=methyl or
benzyl)
Reagents and conditions
: a) ClSO2N=C=O/t-BuOH/TEA; b) TFA;
c) NaH/DMF; d) PhSCH2Cl/TEA; e) NaH/DMF then methyl iodide or
benzyl bromide; f) SO2Cl2; g) NaI/acetone then R3COOH/DBU/CH2Cl2
Scheme 1. Synthesis of inhibitor (I).
Table 1
List of inhibitors and inhibitory activity toward human neutrophil elastase
O
O
R3
N
O
N
S
O
O
R2
Compound
R3
R2
kinact/KI (Mꢀ1 sꢀ1
)
3
4
Methyl
Benzyl
336,000
1090,000
OH
O
5
6
Methyl
Benzyl
419,000
1060,000
O
Figure 2. Progress curves for the inhibition of human neutrophil elastase (HNE) by
inhibitor 18. Absorbance was monitored at 410 nm for reaction solutions containing
10 nM HNE, MeOSuc-AAPV p-nitroanilide (105 lM), and the inhibitor at the
7a
8a
Methyl
Benzyl
357,100
63,800
indicated inhibitor to enzyme ratios in 0.1 M HEPES buffer containing 0.5 M NaCl,
pH 7.25, and 2.5% DMSO. The temperature was maintained at 25 °C and reactions
were initiated by the addition of enzyme.
9a
Methyl
Benzyl
296,300
3200
10a
11b
12b
Methyl
Benzyl
201,000
1580
fitting the A versus t data to Eq. (1) using nonlinear regression anal-
ysis (SigmaPlot, Jandel Scientific). The second-order rate constants
(kinact/KI Mꢀ1 sꢀ1) were then determined by calculating kobs/[I] and
then correcting for the substrate concentration using Eq. (2). The
apparent second-order rate constants (kinact/KI Mꢀ1 sꢀ1) were
determined in duplicate and are listed in Table 1.
13b
14b
Methyl
Benzyl
501,000
1770
O
15b
16b
Methyl
Benzyl
540,000
31,000
obst
A ¼
v
st þ fðv0
ꢀ
vsÞ ð1 ꢀ eꢀk Þ n kobsg þ A0
ð1Þ
ð2Þ
kobs n ½Iꢁ ¼ ðkinact n KIÞ½1 þ ½Sꢁ n Km
ꢁ
O
17
18
Methyl
Benzyl
981,000
1330
Cl
Cl
4. Results and discussion
HN
We have previously shown that carboxylate derivatives repre-
sented by general structure (I) are some of the most potent inhib-
itors of HNE with kinact/KI values close to 5 ꢂ 106 Mꢀ1 sꢀ1 and
apparent KI values in the sub-nanomolar range.16a,19 We have fur-
thermore established that inhibitory potency is dependent on the
pKa of the leaving group (R3COOꢀ), as well as its inherent structure.
Thus, the structure of the leaving group can be modulated to probe
the S0 subsites of closely-related serine proteases, such as HNE and
a
See Ref. 19.
b
Compounds were screened as diastereomeric mixtures.
Pr 3, potentially leading to enhanced binding affinity and enzyme
selectivity.20

6648
D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 6646–6650
namely, compounds with small R2 groups (such as methyl) are less
potent than the corresponding compounds with larger R2 groups
(such as benzyl) and also form acyl enzyme complexes that deacy-
late very slowly (Fig. 2); (e) an unexpected and significant decrease
in potency is observed when R2 = benzyl and a spacer of 1–2 car-
bons connects the carboxylate and aromatic moieties (Table 1,
compounds 8,10, 12, 14, 16, and 18). A similar drop in potency
was observed in a series of simpler congeners of (I), represented
by R3(CH2)nCOOH, with n = 1–2, and R2 = benzyl (exemplified by
compounds 8 and 10, Table 1) but not when n = 0 or n = 3–4.
Though speculative, these results suggest that the decrease in the
inhibitory activity of compounds 8, 10, 12, 14, 16, and 18 (Table 1)
may be the result of an unproductive hydrophobic collapse,25
namely, the clustering of the hydrophobic groups in the inhibitor
molecule in aqueous solution to minimize exposed hydrophobic
surface area. All these molecules share a common feature associ-
ated with the flexibility and hydrophobicity of their leaving groups.
Specifically, the presence of one (or two) sp3-hybridized carbon
atoms between the carboxylate group and the aromatic moiety
in the leaving group appears to play a critical role in the hydropho-
bic collapse which forms part of the flexible turn structure of the
aromatic moiety, allowing it to fold back and interact hydropho-
bically with the isobutyl and benzyl groups. The driving force for
this phenomenon is thought to be the exclusion of water by such
hydrophobic collapse in an aqueous environment.25 Such confor-
mation-directing hydrophobic effects result in an unproductive
conformation and have been previously observed as well as
exploited in the design of potent ligands.26,27
Compound 18
Compound 17
100
80
60
40
20
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (hour)
24.0
Figure 3. Time dependent loss of enzymatic activity. Percent remaining activity
versus time plot obtained by incubating inhibitor 17 or 18 (7 M) with human
l
neutrophil elastase (700 nM) in 0.1 M HEPES buffer containing 0.5 M NaCl, pH 7.25,
and 1% DMSO. Aliquots were withdrawn at different time intervals and assayed for
enzymatic activity using MeOSuc-AAPV p-NA by monitoring the absorbance at
410 nm.
HNE and Pr 3 are similar in many respects (for instance, the two
enzymes have an extended binding site and show a strong prefer-
ence for small hydrophobic P1 residues), however, they exhibit sig-
nificant differences in their S0 subsites. S0–P0 interactions play a
more important role in catalysis in the case of Pr 3 than HNE.18,22
The S0 subsites of HNE are generally hydrophobic while those of
Pr 3 are relatively polar.23 Specifically, the enzymes differ in their
In conclusion, exploitation of structural differences in the S0
subsites of HNE and Pr 3 has led to the identification of highly
selective and potent mechanism-based inhibitors of HNE. Inhibi-
tory activity toward HNE in this series of compounds is influenced
by multiple factors, including the possible formation of solvent-in-
duced unproductive conformations.
S1 –S03 (as well as the S2 subsite), due to the replacement of Ala
0
213 and Leu 99 in HNE by Asp 213 and Lys 99, respectively, in
Pr 3. Furthermore, the substitution of Leu 143 in HNE to Arg 143
in Pr 3, and the presence of Asp 61 make the S0 subsites of Pr 3
more polar and distinctly different from the hydrophobic HNE sub-
sites. Based on these considerations, we initially set out to probe
the S0 subsites of HNE by using a series of structurally-diverse car-
boxylic acids (R3COOH) and subsequently extend these studies to
Pr 3. Our long term objective is to obtain highly selective inhibitors
of HNE and Pr 3 which can be used as probes to delineate the pre-
cise role(s) each protease plays in the pathophysiology of COPD.
Incubation of compound 17 (or 18) with HNE led to rapid and
time-dependent irreversible inactivation of the enzyme (Fig. 3).
In both cases, the inactivated enzyme regained its activity very
slowly, indicating the formation of fairly stable enzyme-inhibitor
adducts. This is particularly true in the case of compound 17, an
observation that is consistent with previous findings19 The potency
of the synthesized inhibitors was determined by generating a ser-
ies of progress curves (Fig. 2) and determining the kinact/KI values
by analyzing the data (progress curve method), as described in
the experimental section.
The following inferences can be made by careful perusal of the
results shown in Table 1: (a) several of the compounds represented
by general structure (I) inhibit HNE potently; (b) compounds 3–18
were devoid of any inhibitory activity toward human neutrophil
proteinase 3, suggesting that the S0 subsites of HNE are hydropho-
bic and more tolerant of size, as opposed to the more polar S0 sub-
sites of Pr 3.18,22,23 (c) a range of structurally-diverse hydrophobic
elements (R3) can be accommodated at the S0 subsites of HNE. This
is in accord with the structure of HNE and the results of previous
studies.19,20,24 (d) a noteworthy increase in potency is observed
upon replacement of the methyl group at R2 with a benzyl group
(compounds 3–6). These observations are consistent with previous
studies which have shown that the nature of R2 influences potency,
as well as the stability of the enzyme-inhibitor complex,16c,19,20
5. Experimental
5.1. General
The 1H spectra were recorded on a Varian XL-300 or XL-400
NMR spectrometer. A Hewlett–Packard diode array UV/vis spectro-
photometer was used in the in vitro evaluation of the inhibitors.
Human neutrophil elastase, proteinase 3 and Boc-Ala-Ala-Nva thi-
obenzyl ester were purchased from Elastin Products Company,
Owensville, MO. Methoxysuccinyl Ala-Ala-Pro-Val p-nitroanilide
and 5,50-dithio-bis (2-nitrobenzoic acid) were purchased from Sig-
ma Chemicals, St. Louis, MO. Melting points were determined on a
Mel-Temp apparatus and are uncorrected. Reagents and solvents
were purchased from various chemical suppliers (Aldrich, Acros
Organics, TCI America, and Bachem). Silica gel (230–450 mesh)
used for flash chromatography was purchased from Sorbent Tech-
nologies (Atlanta, GA). Thin layer chromatography was performed
using Analtech silica gel plates. The TLC plates were visualized
using iodine and/or UV light.
5.2. Synthesis of Compounds 3–18
5.2.1. General procedure
To a solution of 2-chloromethyl compound 2 (2 mmol) in 6 mL
dry acetone was added sodium iodide (2.2 mmol), and the reaction
mixture was stirred overnight at room temperature. The by-prod-
uct, sodium chloride, was removed by filtration through a small
amount of silica gel in a disposable glass pipette. The solvent was
then removed and the iodomethyl intermediate re-dissolved in
4 mL dry methylene chloride. To this solution was added a solution

D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 6646–6650
6649
of the appropriate carboxylic acid (2.2 mmol) and DBU (2.2 mmol)
5.2.9. Compound 14
in 4 mL additional methylene chloride, and the resulting mixture
was stirred overnight at room temperature. The solvent was re-
moved; the residue dissolved in methylene chloride (40 mL) and
washed successively with 5% HCl (20 mL), saturated NaHCO3
(20 mL), and finally with saturated sodium chloride (20 mL). The
organic layer was then dried over sodium sulfate. The drying agent
and solvent were removed, leaving a crude product which was
purified by flash chromatography (hexane/methylene chloride or
hexane/ethyl acetate) to give compounds 3–18.
Oil (59% yield). 1H NMR (CDCl3) 0.63 (d, J = 5.9 Hz, 3H), 0.76 (d,
J = 5.9 Hz, 3H), 1.51 (d, J = 7.14 Hz, 3H), 1.50–1.72 (m, 3H), 3.74 (q,
J = 6.4 Hz, 1H), 3.85 (t, J = 5.8 Hz, 1H), 4.18 (d, J = 13.8 Hz, 1H), 4.50
(d, J = 13.8 Hz, 1H), 5.62–5.65 (m, 2H), 6.98–7.35 (m, 14H). HRMS
(ESI) calcd for C29H36N3O6S [M+NH4]+ 554.2325, found 554.2349;
C
29H32N2O6SNa [M+Na]+ 559.1879, found 559.1899.
5.2.10. Compound 15
Oil (68% yield). 1H NMR (CDCl3) 0.88–0.98 (dd, J = 11.6, 6.4 Hz,
6H), 1.54 (d, J = 7.1 Hz, 3H), 1.72–1.94 (m, 3H), 2.86 (s, 3H), 3.80–
3.89 (m, 2H), 5.58–5.71 (m, 2H), 7.42–7.84 (m, 9H). HRMS (ESI)
5.2.2. Compound 3
Oil (84% yield). 1H NMR (CDCl3) 0.98 (dd, J = 9.5, 6.4 Hz, 6H),
1.82–1.98 (m, 3H), 2.92 (s, 3H), 3.92 (t, J = 6.3 Hz, 1H), 5.90 (q,
J = 14.3, 10.0 Hz, 2H), 6.84–7.86 (m, 4H), 10.28 (s, 1H). HRMS
(ESI) calcd for C15H20N2O6SNa [M+Na]+ 379.0940, found 379.0953.
calcd for C
24H32N3O6S [M+NH4]+ 490.2012, found 490.2051;
C24H28N2O6SNa [M+Na]+ 495.1566, found 495.1548.
5.2.11. Compound 16
Oil (82% yield). 1H NMR (CDCl3) 0.63–0.76 (dd, J = 35.2, 6.2 Hz,
6H), 1.57 (d, J = 7.3 Hz, 3H), 1.45–1.80 (m, 3H), 3.82–3.88 (m,
2H), 4.16–4.52 (dd, J = 84.2, 13.8 Hz, 2H), 5.65 (t, J = 3.6 Hz, 2H),
7.35–7.82 (m, 14H). HRMS (ESI) calcd for C30H36N3O6S [M+NH4]+
566.2325, found 566.2330; C30H32N2O6SNa [M+Na]+ 571.1879,
found 571.1879.
5.2.3. Compound 4
Oil (63% yield). 1H NMR (CDCl3) 0.75 (dd, J = 31.5, 6.1 Hz, 6H),
1.62–1.80 (m, 3H), 3.98 (t, J = 7.3 Hz, 1H), 4.42 (q, J = 60.5,
14.5 Hz, 2H), 5.91 (d, J = 2.3 Hz, 2H), 6.88–7.88 (m, 9H), 10.30 (s,
1H). HRMS (ESI) calcd for C21H24N2O6SNa [M+Na]+ 455.1253, found
455.1252.
5.2.12. Compound 17
5.2.4. Compound 5
Oil (60% yield). 1H NMR (CDCl3) 0.93–0.99 (dd, J = 9.8, 6.5 Hz,
6H), 1.76–1.94 (m, 3H), 2.87 (s, 3H), 3.82–3.87 (m, 3H), 5.70 (dd,
J = 14.5, 9.7 Hz, 2H), 6.58 (m, 1H), 6.94–7.35 (m, 7H). HRMS (ESI)
calcd for C22H25Cl2N3O5SNa [M+Na]+ 536.0790, found 536.0781.
Oil (70% yield). 1H NMR (CDCl3) 0.99 (dd, J = 9.6, 6.3 Hz, 6H),
1.80–1.86 (m, 2H), 1.86–2.00 (m, 1H), 2.38 (s, 3H), 2.90 (s, 3H),
3.90 (t, J = 6.3 Hz, 1H), 5.84 (dd, J = 18.6, 11.4 Hz, 2H), 7.12 (d,
J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 8.05
(d, J = 7.8 Hz, 1H). HRMS (ESI) calcd for C17H26N3O7S [M+NH4]+
416.1491, found 416.1500; C17H22N2O7SNa [M+Na]+ 421.1045,
found 421.1048.
5.2.13. Compound 18
Oil (67% yield). 1H NMR (CDCl3) 0.66–0.79 (dd, J = 36.3, 6.4 Hz,
6H), 1.52–1.76 (m, 3H), 3.88 (m, 3H), 4.35 (dd, J = 79.9, 12.3 Hz,
2H), 5.70 (s, 2H), 6.52–7.39 (m, 13H). HRMS (ESI) calcd for
5.2.5. Compound 6
C
28H30Cl2N3O5S [M+NH4]+ 590.1283, found 590.1279; C28H29Cl2-
Oil (79% yield). 1H NMR (CDCl3) 0.73 (dd, J = 33.2, 6.1 Hz, 6H),
1.55–1.80 (m, 3H), 2.35 (s, 3H), 3.92 (t, J = 7.2 Hz, 1H), 4.25–4.55
(dd, J = 75.6, 15.6 Hz, 2H), 5.83 (q, J = 21.0, 16.0 Hz, 2H), 7.10–
N3O5SNa [M+Na]+ 612.1103, found 612.1108.
5.2.14. Human neutrophil elastase
8.06 (m, 9H). HRMS (ESI) calcd for
C
23H30N3O7S [M+NH4]+
HNE was assayed by mixing 10
in 0.05 M sodium acetate/0.5 M NaCl buffer, pH 5.5, 10
sulfoxide and 980 L of 0.1 M HEPES buffer containing 0.5 M NaCl,
pH 7.25, in a thermostated cuvette. A 100 L aliquot was trans-
ferred to a thermostated cuvette containing 880 L 0.1 M HEPES/
0.5 M NaCl buffer, pH 7.25, and 20 L of a 70 M solution of MeO-
lL of a 70
l
M enzyme solution
492.1804, found 492.1821; C23H26N2O7SNa [M+Na]+ 497.1358,
found 497.1360.
l
L dimethyl
l
l
5.2.6. Compound 11
l
Oil (66% yield). 1H NMR (CDCl3) 0.88 (d, J = 7.0 Hz, 6H), 0.92–
0.98 (dd, J = 11.6, 6.5 Hz 6H), 1.50 (d, J = 6.4 Hz 3H), 1.72–1.92
(m, 4H), 2.45 (d, J = 7.2 Hz, 2H), 2.85 (s, 3H), 3.74 (q, J = 6.9 Hz,
1H), 3.82 (t, J = 5.6 Hz, 1H), 5.52–5.72 (m, 2H), 7.06–7.22 (dd,
J = 31.9, 7.2 Hz, 4H). HRMS (ESI) calcd for C21H36N3O5S [M+NH4]+
442.2376, found 442.2377; C21H32N2O5SNa [M+Na]+ 447.1930,
found 447.1935.
l
l
Suc-Ala-Ala-Pro-Val p-nitroanilide, and the change in absorbance
was monitored at 410 nm for 60 s. In a typical inhibition run,
10
10
l
l
L of inhibitor (3.5 mM) in dimethyl sulfoxide was mixed with
L of 70 M enzyme solution and 980 L 0.1 M HEPES/0.5 M
l
l
NaCl buffer, pH 7.25, and placed in a constant temperature bath.
Aliquots (100 L) were withdrawn at different time intervals and
transferred to a cuvette containing 20 L of MeOSuc-Ala-Ala-Pro-
Val p-nitroanilide (7 mM) and 880 L 0.1 M HEPES/0.5 M NaCl buf-
l
l
5.2.7. Compound 12
l
Oil (95% yield). 1H NMR (CDCl3) 0.63–0.78 (dd, J = 38.6, 5.7 Hz,
6H), 0.88 (d, J = 6.6 Hz, 6H), 1.51 (d, J = 7.3 Hz, 3H), 1.51–1.89
(m, 4H), 2.42 (d, J = 7.4 Hz, 2H), 3.75 (q, J = 7.0 Hz, 1H), 3.86
(t, J = 6.9 Hz, 1H), 4.20–4.50 (dd, J = 84.9, 13.7 Hz, 2H), 5.62
(m, 2H), 7.09–7.20 (dd, J = 34.8, 6.4 Hz, 4H), 7.36 (s, 5H). HRMS
(ESI) calcd for C27H40N3O5S [M+NH4]+ 518.2689, found 518.2670;
fer. The absorbance was monitored at 410 nm for 60 s.
5.2.15. Human neutrophil proteinase 3
Twenty microliters of 32.0 mM 5,50-dithio-bis(2-nitrobenzoic
acid) in dimethyl sulfoxide and 10 lL of a 3.45 lM solution of
human proteinase 3 in 0.1 M phosphate buffer, pH 6.50 (final en-
zyme concentration: 34.5 nM) were added to a cuvette contain-
C
27H36N2O5SNa [M+Na]+ 523.2243, found 523.2232.
ing
containing 0.5 M NaCl, 10
oxide (final inhibitor concentration: 8.62
12.98 mM Boc-Ala-Ala-NVa-SBzl and the change in absorbance
was monitored at 410 nM for 2 min. A control (hydrolysis run)
was also run under the same conditions by adding 5,50-dithio-
a
solution of 940
l
L
0.1 M HEPES buffer, pH 7.25,
L 862.5 M inhibitor in dimethyl sulf-
M) and 20 lL
5.2.8. Compound 13
l
l
Oil (33% yield). 1H NMR (CDCl3) 0.91–0.98 (dd, J = 17.8, 6.6 Hz,
6H), 1.49–1.51 (d, J = 7.2 Hz, 3H), 1.74–1.92 (m, 3H), 2.85 (s, 3H),
3.74 (q, J = 7.1 Hz, 1H), 7.77–3.83 (m, 1H), 5.55–5.70 (m, 2H),
6.84–7.38 (m, 9H). HRMS (ESI) calcd for C23H32N3O6S [M+NH4]+
478.2012, found 478.1999; C23H28N2O6SNa [M+Na]+ 483.1566,
found 483.1559.
l
bis(2-nitrobenzoic acid) in dimethyl sulfoxide and 10
lL of a
3.45 M solution of human proteinase 3 in 0.1 M phosphate buf-
l

6650
D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 6646–6650
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fer, pH 6.50 (final enzyme concentration: 34.5 nM) to a cuvette
containing a solution of 940 L 0.1 M HEPES buffer, pH 7.25, con-
taining 0.5 M NaCl, 10 L dimethyl sulfoxide and 20 L 12.98 mM
l
l
l
14. (a) Chua, F.; Laurent, G. J. Proc. Am. Thorac. Soc. 2006, 3, 424; (b) Moraes, T. J.;
Chow, C. W.; Downey, G. P. Crit. Care Med. 2003, S189.
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17. Nomenclature used is that of Schechter, I.; Berger, A. Biochem. Biophys. Res.
Commun. 1967, 27, 157. where S1, S2, S3,. . .Sn and S10, S20, S30,. . .Sn0
correspond to the enzyme subsites on either side of the scissile bond. Each
Boc-Ala-Ala-NVa-SBzl and the change in absorbance was moni-
tored at 410 nM for 2 min. Pr 3 activity remaining was deter-
mined using
average of duplicate or triplicate determinations.
% remaining activity = (v/v0) ꢂ 100 and is the
Acknowledgment
This work was supported in part by a Grant from the National
Institutes of Health (HL 57788).
subsite accommodates
a corresponding amino acid residue side chain
References and notes
designated P1, P2, P3,. . .Pn and P10, P20, P30,. . .Pn0 of the substrate or
(inhibitor). S1 is the primary substrate specificity subsite, and P1–P10 is the
scissile bond.
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