Inactivation of human neutrophil elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides

Article abstract of DOI:10.1016/j.bmc.2007.10.041

The interaction of a series of 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides with neutrophil-derived serine proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S′ subsites, had a profound effe

Full text of DOI:10.1016/j.bmc.2007.10.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 692–698
Inactivation of human neutrophil elastase by
1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides
Yi Li, Qingliang Yang, Dengfeng Dou, Kevin R. Alliston and William C. Groutas*
Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
Received 17 August 2007; revised 10 October 2007; accepted 12 October 2007
Available online 18 October 2007
Abstract—The interaction of a series of 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides with neutrophil-derived serine
proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S0 subsites, had a profound
effect on enzyme selectivity. This series of compounds were found to be potent, time-dependent inhibitors of human neutrophil elas-
tase (HNE) and were devoid of any inhibitory activity toward neutrophil proteinase 3 (PR 3) and cathepsin G (Cat G). The results
of these studies demonstrate that exploitation of differences in the S0 subsites of HNE and PR 3 can lead to highly selective inhibitors
of HNE.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
an unprecedented enzyme-induced sulfonamide frag-
mentation process (Fig. 1).8 Sulfonamide inhibitor (I)
embodies in its structure a functionalized heterocyclic
scaffold with appended recognition elements for optimal
exploitation of binding interactions with the Sn and Sn0
subsites9 of the target enzyme. Derivatives of inhibitor
(I) (R1 = isobutyl and R2 = methyl) were previously
found to inactivate HNE efficiently, however they also
showed significant inhibitory activity toward trypsin de-
spite the lack of a basic P1 residue. In an effort to opti-
mize the inhibitory potency and selectivity of (I) toward
HNE and PR 3, recognition element R3 was varied using
a series of amino acid esters and the inhibitory activity
of the resulting compounds toward HNE, PR 3, Cat
G, and bovine trypsin was then evaluated. The results
of these studies are described herein.
The neutrophil-derived serine endopeptidases human
neutrophil elastase (HNE), proteinase 3 (PR 3), and
cathepsin G (Cat G) have been implicated in a range
of inflammatory diseases, including chronic obstructive
pulmonary disease (COPD).1 Although the pathogenesis
of COPD is poorly understood, current studies indicate
that this multifactorial disorder is characterized by a cig-
arette smoke-induced cycle of oxidative stress,2 alveolar
septal cell apoptosis,3 a protease/antiprotease imbal-
ance,4 and chronic inflammation.5 An array of serine
(HNE, PR 3, and Cat G), cysteine (cathepsin S), and
metallo- (MMP-9 and MMP-12) proteases released by
neutrophils, macrophages, and T lymphocytes contrib-
ute to the degradation of lung connective tissue and
mediate a multitude of signaling pathways associated
with the pathophysiology of the disorder.6 Conse-
quently, pharmacological agents capable of abrogating
or modulating the aberrant proteolytic activity of the
aforementioned enzymes are of potential therapeutic
value.7
2. Results
2.1. Chemistry
Compounds 4–11 were synthesized starting with (L) nor-
valine using the sequence of steps shown in Scheme 1.
Key intermediate 1 was synthesized using similar proce-
dures as those described previously.8b The synthetic
methodology was straightforward, however the reaction
sequence involving the conversion of the thioesters to
the corresponding sulfinyl chlorides which, without iso-
lation, were reacted with the amino acid esters was
found to be capricious and gave low yields (10–20%)
We have recently described the design and in vitro bio-
chemical evaluation of a novel class of mechanism-based
inhibitors (I) that inactivate target serine proteases via
Keywords: Serine; Inhibitors.
*
Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978
3431; e-mail: bill.groutas@wichita.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.041

Y. Li et al. / Bioorg. Med. Chem. 16 (2008) 692–698
693
a
b
H
N
O
R
H
N
O
N
H
R1
R1
O
R2
R2
H
O
R
H
O
H
N
O
R
O
N
N
SO2
+
+
H2N
N
H
S
N
O
O
R1
R2
S1
E-Ser195-O
P1
O
S
O
-
O
O
P1
R2
NH
R3
N
N
COOCH3
R3
COOCH3
N
N
S
S
SO2 -NH
R2
O
O
S2
O
O
(I)
S '
n
O
195
Ser
E
P1
O
N
Inactive
enzyme
N
R2
S
O
O
Figure 1. Design and mechanism of action of inhibitor (I).
O
O
O
N
P1
a
P1
COOH
NH2
P1
R2
P1
R2
S
NH
c
b
N
N
(L)
N
N
N
S
S
S
S
R2
Cl
COOR
R3
O
O
O
O
O
O
1
O
O
O
3
2
O
P1
S
NH
a
b
N
CH3COSH/TEA; SO2Cl2 followed by
c
N
S
COOR
R2
R3
O
NH2CHR3COOR/NMM; MCPBA/CH2Cl2
O
(I)
P1 =n-Pr, R2=R=methyl
Scheme 1. Synthesis of inhibitors 4–11.
of the sulfinamide products. Optimization of the reac-
tion conditions and monitoring product formation using
1H NMR improved yields somewhat (30%).
tific). 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. Control curves in the absence of inhibitor
were linear.
/
/
2.2. Biochemical studies
obst
A ¼ vst þ fðv0 ꢀ vsÞð1 ꢀ eꢀk Þ=kobsg þ A0
ð1Þ
ð2Þ
2.2.1. Progress curve method. The inhibitory activity of
compounds 4–11 toward HNE was determined by the
progress curve method.10,8b The apparent second-order
rate constants (kinact/KI Mꢀ1 sꢀ1) were determined in
duplicate and are listed in Table 1. Typical progress
curves for the hydrolysis of MeOSuc-AAPV-p-NA by
HNE in the presence of inhibitor 4 are shown in
Figure 2. The release of p-nitroaniline was continuously
monitored at 410 nm. The pseudo first-order rate con-
stants (kobs) for the inhibition of HNE by derivatives
of (I) as a function of time were determined according
to Eq. 1 below, 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 con-
stant, and A0 is the absorbance at t = 0. The kobs values
were obtained by fitting the A ꢁ t data into Eq. 1 using
nonlinear regression analysis (SigmaPlot, Jandel Scien-
k
obs=½Iꢂ ¼ ðkinact=KIÞ½1 þ ½Sꢂ=Kmꢂ
2.2.2. Incubation method. The inhibitory activity of com-
pounds 4–11 toward trypsin was determined using the
incubation method8b and is expressed in terms of the
bimolecular rate constant kobs/[I] Mꢀ1 sꢀ1, where kobs
is the pseudo-first-order rate constant of enzyme inacti-
vation and [I] is the concentration of the inhibitor in the
incubation mixture. Briefly, in this method the enzyme is
incubated with excess inhibitor and the loss of enzymatic
activity is followed by withdrawing aliquots at different
time intervals and assaying for enzymatic activity. The
observed rate constants (kobs) are then determined by
plotting ln([Et]/[E0]) versus t according to Eq. 3 below,

694
Y. Li et al. / Bioorg. Med. Chem. 16 (2008) 692–698
Table 1. Inhibitory activity of compounds 4–11 toward human neutrophil elastase, proteinase 3, cathepsin G, and bovine trypsin
O
O
S
O
P1
R2
NH
R3
N
COOCH3
N
S
O
O
(I)
Compounda
R3
kinact/KI Mꢀ1 sꢀ1
HNE
PR 3
Cat G
Trypsinb
4
(L) CH3
1050
Inactivec
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactivec
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
NDd
19
5
(L) CH3(CH2)2
(L) PhCH2
(D) PhCH2
34,650
41,750
9760
6
50
7
ND
60
8
(L) PhCH2OCH2
(L) CH2OH
16,870
1100
9
12
10
11
(L) (CH2)4NH2
(L) (CH2)4NHCbz
12,900
6900
ND
ND
a P1 = n-propyl, R2 = methyl.
b kobs/[I] Mꢀ1 sꢀ1 values determined using the incubation method (see text for details).
c [I]/[E] = 250 for 30 min.
d Not determined.
in its structure an a-sulfonopeptide motif (Fig. 1b).
Based on the mechanism of action of serine proteases,
it was envisaged that embellishment of (I) with appropri-
ate recognition elements would allow (I) to bind to the
active site of a target serine protease, leading to the for-
mation of a tetrahedral intermediate that would subse-
quently collapse via a sulfonamide fragmentation-
driven process to generate a Michael acceptor. Forma-
tion of a stable enzyme–inhibitor adduct was anticipated
to led to inactivation of the enzyme; (c) HNE and PR 3
play an important role in inflammatory diseases such as
COPD1,11 and cystic fibrosis.12 Furthermore, both en-
zymes hydrolyze lung elastin, the hydrophobic compo-
nent of lung connective tissue, with high efficiency and
also induce emphysema in hamsters when administered
intratracheally.13 Consequently, in order for (I) to inhibit
both enzymes, an n-propyl group was chosen as the P1
residue (vide infra); (d) an R2 = methyl group was chosen
at S2 based on earlier studies which demonstrated that
the presence of a methyl group at R2 leads to the forma-
tion of highly stable acyl enzymes;14 and (e) there are sig-
nificant structural differences in the make up of the Sn0
subsites of serine proteases, consequently it was antici-
pated that varying R3 may enhance the selectivity of
(I). Literature precedent regarding the successful exploi-
tation of S0–P0 interactions in the design of potent and
selective inhibitors of serine15 and cysteine16 proteases
provided a strong measure of assurance about the sound-
ness of the approach. As stated earlier, our primary
objective was to obtain a derivative of (I) that would dis-
play high potency and selectivity toward HNE and PR 3
over other serine proteases.
0.30
0.25
0.20
0.15
0.10
0.05
0.00
I/E
0
50
100
200
0
100
200
300
400
500
600
Time (sec)
Figure 2. Progress curves for the inhibition of human neutrophil
elastase (HNE) by inhibitor 4. Absorbance was monitored at 410 nm
for reaction solutions containing 10 nM HNE, 105 lM MeOSuc-
AAPV p-nitroanilide, and the inhibitor at the 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.
where ([Et]/[E0]) is the amount of active enzyme remain-
ing at time t. These are the average of two or three
determinations.
lnð½Etꢂ=½E0ꢂÞ ¼ kobs
t
ð3Þ
3. Discussion
3.1. Inhibitor design
Recent studies in our laboratory have demonstrated that
inhibitor (I) is a highly efficient inhibitor of HNE that
inactivates the enzyme via an enzyme-induced sulfon-
amide fragmentation process.8b However, derivatives of
(I) were also found to inhibit trypsin, consequently this
report details our studies aimed at gaining further insight
into the interaction of (I) with (chymo)trypsin-like serine
The design of inhibitor (I) is briefly summarized below:
(a) in contrast to ordinary peptides, a-sulfonopeptides
(a-amido sulfonamides) undergo a spontaneous frag-
mentation reaction to yield a Michael acceptor (Fig. 1);
(b) inhibitor (I) is a stable molecule which also embodies

Y. Li et al. / Bioorg. Med. Chem. 16 (2008) 692–698
695
proteases and optimizing inhibitory activity and enzyme
selectivity by varying R3, the recognition element be-
lieved to interact with the S0n subsites. While all three neu-
trophil-derived serine proteases (HNE, PR 3, and Cat G)
were studied with the synthesized inhibitors, our main ef-
forts were focused on the identification of a dual HNE
and PR 3 inhibitor suitable for further development.
less there are also significant differences between the
two enzymes. Specifically, interactions beyond the S10
subsite have a profound effect on catalysis in the case
of PR 3 but not HNE.19a Furthermore, the S0n subsites
of PR 3 are decidedly more polar than those of HNE
due to the presence of Asp 61 and Arg 143, which
may partly account for the ability of these compounds
to discriminate between the two enzymes.18 These obser-
vations are of particular relevance to the design of HNE
and PR 3 inhibitors since their primary substrate speci-
ficities are very similar [HNE and PR 3 show a prefer-
ence for (Val and Leu) and (Abu and Norval),
respectively, at P1], consequently varying the nature of
the primary specificity residue P1 may not be sufficient
to attain high selectivity. Recently the differences in
the S2 and S01–S30 subsites of PR 3 and HNE have been
successfully exploited in the design of highly specific
substrates of HNE and PR 3.19 Furthermore, recent ki-
netic and molecular modeling analysis studies have rein-
forced the importance of the charge distribution
differences at the active sites of the two enzymes.20
Although the reasons for the noteworthy selectivity
exhibited by the compounds used in these studies are
not intuitively obvious, their potential utility as pharma-
cological probes is clearly evident.
Inhibitors 4–11 were all found to be efficient inhibitors of
HNE (Table 1). For instance, incubation of compound 4
with HNE lead to rapid, time-dependent, irreversible loss
of enzymatic activity (Fig. 3). The enzyme did not regain
any activity after 24 h, suggesting the formation of a
fairly stable enzyme–inhibitor adduct. With the excep-
tion of compound 10, compounds bearing hydrophobic
R3 groups exhibited higher inhibitory activity toward
HNE. While this is likely a reflection of the strong prefer-
ence of HNE for hydrophobic inhibitors (and sub-
strates), rather than the effect of any specific
hydrophobic interactions, the high potency of compound
4 may arise from a p–p stacking interaction between Phe
41 in HNE and the phenyl group of the inhibitor.
Quite surprisingly, compounds 4–11 were all devoid of
any inhibitory activity toward PR 3 (following incuba-
tion at an [I]/[E] ratio of 250), exhibiting absolute selec-
tivity between HNE and PR 3. This remarkable
observation is in sharp contrast to previously reported
studies that showed that 1,2,5-thiadiazolidin-3-one 1,1
dioxide-based derivatives are highly effective inhibitors
of both HNE and PR 3.17 The fact that compounds 4–
11 show no inhibitory activity toward PR 3 is probably
a reflection of the significant structural differences in the
make up of the S0n subsites of the two enzymes.18,19
While HNE and PR 3 are highly homologous (57%)
and their active sites have some general features in com-
mon, namely, they both have extended binding sites and
prefer hydrophobic substrates and inhibitors, neverthe-
As expected, compounds 4–11 showed no activity toward
neutrophil cathepsin G (following incubation at an [I]/[E]
ratio of 250 for 30 min). This is no doubt due to the
strong preference of Cat G for a Phe residue at P1.21
Compounds 4–11 were also found to inhibit bovine
trypsin in a time-dependent fashion, albeit much less
efficiently (Table 1). For instance, incubation of com-
pound 4 with bovine trypsin led to time-dependent inac-
tivation of the enzyme with partial recovery of
enzymatic activity after 24 h (Fig. 4). The observed
inhibitory activity toward trypsin may be due to partial
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0.0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
24.0
0.0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
24.0
Hours
Hours
Figure 3. Time-dependent loss of enzymatic activity. Percent remain-
ing activity versus time plot obtained by incubating inhibitor 4 (7 lM)
with human 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.
Figure 4. Time-dependent loss of enzymatic activity. Percent remain-
ing activity versus time plot obtained by incubating inhibitor 4
(0.125 mM) with bovine trypsin (5 lM) in 0.025 M phosphate buffer
containing 0.1 M NaCl, pH 7.51, and 1% DMSO. Aliquots were
withdrawn at different time intervals and assayed for enzymatic
activity using N-(p-tosyl)-Gly-Pro-Lys p-NA by monitoring the
absorbance at 410 nm.

696
Y. Li et al. / Bioorg. Med. Chem. 16 (2008) 692–698
occupation of the S1 pocket by the n-propyl group in a
way that is conducive to binding, namely, by not clash-
ing with the Asp residue at the bottom of the trypsin S1
pocket.
to 5 ꢁC using an ice bath and kept under nitrogen. Sul-
furyl chloride (1.6 mL; 17.12 mmol) was then added and
the mixture was stirred for 3 h. NMR analysis showed
that most of the starting material had reacted. The sol-
vent and sulfuryl chloride were then removed at 5 ꢁC
using a vacuum pump (40 min). The residue was redis-
solved in dry methylene chloride (10 mL) and then
cooled to ꢀ78 ꢁC using a dry ice/acetone bath. A mixture
of (L) Phe–OCH3 hydrochloride (2.77 g; 12.84 mmol) and
N-methyl morpholine (1.30 g; 12.84 mmol) in methylene
chloride (15 mL) was added to the solution and the
resulting mixture was stirred at ꢀ78 ꢁC for 1 h and then
allowed to warm up to 25 ꢁC by stirring overnight. The
precipitate was filtered off and the filtrate was concen-
trated, leaving a crude product (3.28 g) which was puri-
fied by flash chromatography using hexane/ether. A
pure product 3 was obtained (1.04 g; 29% yield) as a col-
orless oil (1H NMR: d 1.98 (m, 3H), 1.38 (m, 1H), 1.50
(m, 1H), 1.80 (m, 1H), 1.98 (m, 1H), 2.90 (s, 3H), 3.00
(m, 2H), 3.70 (s, 3H), 3.80 (t, 1H), 3.90 (m, 1H), 4.50–
4.78 (dd, 2H), 7.20–7.30 (m, 5H), however, sulfinamide
3, as well as the rest of the synthesized sulfinamides,
were found to color on standing, consequently they were
used in the next step without purification.
In conclusion, a series of compounds that shows high
potency and selectivity toward HNE but no activity
toward PR 3 and Cat G is reported. Further studies
aimed at enhancing the HNE/trypsin selectivity ratio
and illuminating the structural basis for the selectivity
observed with this class of compounds are currently in
progress.
4. Experimental
4.1. General
Melting points were recorded on a Mel-Temp appara-
1
tus and are uncorrected. The H and 13C NMR spec-
tra of the synthesized compounds were recorded on
Varian XL-300 or XL-400 spectrometers. Human neu-
trophil elastase, proteinase 3, and Boc-Ala-Ala-Nva
thiobenzyl ester were purchased from Elastin Products
Co., Owensville, MO. Bovine trypsin, methoxysuccinyl
Ala-Ala-Pro-Val p-nitroanilide, succinyl Ala-Ala-Pro-
Phe p-nitroanilide, N-(p-tosyl)-Gly-Pro-Lys 4-nitroani-
lide, and 5,50-dithio-bis(2-nitrobenzoic acid) were
purchased from Sigma Chemicals Co., St. Louis,
MO. Human neutrophil cathepsin G was purchased
from Athens Research and Technology Co., Athens,
GA. Silica gel (230–450 mesh) used for flash chroma-
tography was purchased from Sorbent Technologies,
Atlanta, GA. Thin layer chromatography was per-
formed using Analtech silica gel plates. The TLC
plates were visualized using iodine vapor and/or UV
light. A Hewlett–Packard diode array UV/vis spectro-
photometer was used in the enzyme assays and inhibi-
tion studies.
A solution of sulfinamide 3 (1.98 g; 4.76 mmol) in dry
methylene chloride (10 mL) was cooled in an ice bath
and then treated with 77% m-chloroperbenzoic acid
(4.27 g; 19.06 mmol). The mixture was stirred overnight
and the solvent removed on the rotovac. The residue
was taken up in ethyl acetate (50 mL) and washed with
saturated sodium bicarbonate (3· 10 mL), brine (3·
10 mL) and the organic extract was dried over anhy-
drous sodium sulfate. Removal of the solvent left a
crude product which was purified using flash chroma-
tography (hexane/ethyl acetate eluents), yielding com-
pound 4 as pure white solid (0.83 g; 39% yield), mp
129–132 ꢁC. 1H NMR (CDCl3): d 0.98 (t, 3H), 1.40
(m, 2H), 1.5 (d, 3H), 1.85 (m, 1H), 1.95 (m, 1H), 2.95
(s, 3H), 3.80 (s, 3H), 4.00 (t, 1H), 4.30 (m, 1H), 4.85
(d, 1H), 5.10 (d, 1H), 5.40 (d, 1H).
4.2. Representative syntheses
1
4.2.1. Synthesis of compound 2. To a solution of 4-n-pro-
pyl-5-methyl-2-chloromethyl-1,2,5-thiadiazolin-3-one 1,1
dioxide 1 (3.38 g; 14.04 mmol) and thiolacetic acid
(2.14 g; 28.08 mmol) in dry acetonitrile (30 mL) kept in
an ice bath was added dropwise a solution of triethyl-
amine (1.70 g; 16.8 mmol) in acetonitrile (5 mL). The
mixture was allowed to warm to room temperature
and stirred for 2 days. The solvent was removed on
the rotovac and the residue was taken up in ethyl acetate
(100 mL) and washed with 5% aqueous HCl (20 mL)
and brine (20 mL). The organic layer was dried over
anhydrous sodium sulfate. Removal of the solvent left
a crude product which was purified by flash chromatog-
raphy using hexane/ethyl acetate to yield 3.50 g (89%
yield) of an oily product.1H NMR (CDCl3): d 0.95–
1.00 (t, 3H), 1.38 (m, 1H), 1.45 (m, 1H), 1.80 (m, 1H),
1.90 (m, 1H), 2.40 (s, 3H), 2.90 (s, 3H), 3.82 (t, 1H),
5.10 (q, 2H).
4.2.3. Compound 5. Mp 87–90 ꢁC. H NMR (CDCl3): d
0.95 (m, 6H), 1.45 (m, 4H), 1.80 (m, 3H), 1.95 (m, 1H),
2.92 (s, 3H), 3.80 (s, 3H), 3.96 (t, 1H), 4.20 (q, 1H), 4.95–
5.10 (dd, 2H), 5.40 (d, 1H).
1
4.2.4. Compound 6. Mp 119–121 ꢁC. H NMR (CDCl3):
d 0.95 (t, 3H), 1.35 (m, 1H), 1.45 (m, 1H), 1.82 (m, 1H),
1.90 (m, 1H), 2.90 (s, 3H), 3.17 (t, 2H), 3.75 (s, 3H), 3.93
(t, 1H), 4.50 (t, 1H), 4.65–4.85 (dd, 2H), 7.22 (m, 5H).
4.2.5. Compound 7. Mp 99–101 ꢁC. 1H NMR (CDCl3): d
0.95 (t, 3H), 1.35 (m, 1H), 1.45 (m, 1H), 1.82 (m, 1H),
1.90 (m, 1H), 2.90 (s, 3H), 3.17 (t, 2H), 3.75 (s, 3H),
3.93 (t, 1H), 4.50 (t, 1H), 4.65–4.85 (dd, 2H), 7.20 (m,
5H).
1
4.2.6. Compound 8. Mp 74–76 ꢁC. H NMR (CDCl3): d
0.95 (t, 3H), 1.41 (m, 1H), 1.50 (m, 1H), 1.85 (m, 1H),
1.95 (m, 1H), 2.98 (s, 3H), 3.75 (s, 3H), 3.90 (m, 2H),
3.95 (t, 1H), 4.40 (m, 1H), 4.55 (dd, 2H), 5.05 (dd,
2H), 5.70 (d, 1H), 7.32 (m, 5H).
4.2.2. Synthesis of compound 4. Thioester 2 (2.40 g;
8.56 mmol) was dissolved in CDCl3 (7 mL) and cooled

Y. Li et al. / Bioorg. Med. Chem. 16 (2008) 692–698
697
1
4.2.7. Compound 9. Mp 80–82 ꢁC. H NMR (CDCl3): d
32.0-mM 5,50-dithio-bis(2-nitrobenzoic acid) solution
in DMSO and 10 lL of a 3.45-lM solution of human
proteinase 3 in 0.1 M phosphate buffer, pH 6.50 (final
enzyme concentration: 34.5 nM) to a cuvette containing
a solution of 940 lL of 0.1 M HEPES/0.5 M NaCl buf-
fer, pH 7.25, 10 lL DMSO, and 20 lL of a 12.98-mM
solution of Boc-Ala-Ala-NVa-SBzl in DMSO, and the
change in absorbance was monitored at 410 nm for
2 min.
0.95 (t, 3H), 1.40 (m, 1H), 1.50 (m, 1H), 1.85 (m, 1H),
1.95 (m, 1H), 2.75 (br s, 1H), 2.95 (s, 3H), 3.80 (s,
3H), 3.98 (d, 2H), 4.02 (t, 1H), 4.32 (s, 1H), 5.00 (dd,
2H), 5.90 (br s, 1H).
1
4.2.8. Compound 10. Oil. H NMR (CDCl3): d 0.95 (t,
3H), 1.45 (m, 6H), 1.80 (m, 4H), 2.95 (s, 3H), 3.20 (m,
2H), 3.80 (s, 3H), 4.00 (t, 1H), 4.20 (m, 1H), 4.80–5.10
(dd, 2H), 5.00 (m, 2H), 5.50 (d, 1H), 7.40 (m, 5H).
5.3. Human neutrophil cathepsin G
1
4.2.9. Compound 11. Oil. H NMR (CDCl3): d 0.95 (t,
3H), 1.48 (m, 6H), 1.85 (m, 4H), 2.90 (s, 3H), 3.20 (q,
2H), 3.75 (s, 3H), 3.95 (t, 1H), 4.19 (q, 1H), 4.95 (q,
2H), 5.10 (s, 2H), 5.49 (d, 1H), 7.32 (m, 5H).
Ten microliters of a 6.8 lM solution of human neutro-
phil cathepsin G in 0.05 M sodium acetate/0.05 M NaCl
buffer, pH 5.38, was added to a solution containing
10 lL of inhibitor (1.7 mM solution in DMSO) and
970 lL of 0.1 M HEPES buffer, pH 7.50. The mixture
was incubated for 30 min, 10 lL of substrate (N-succinyl
Ala-Ala-Pro-Phe p-nitroanilide, 85 mM in DMSO) was
added, and the absorbance was monitored at 410 nm
for 10 min.
5. Enzyme assays and inhibition studies
5.1. Human neutrophil elastase
HNE was assayed by mixing 10 lL of a 70 lM enzyme
solution in 0.05 M sodium acetate/0.5 M NaCl buffer,
pH 5.5, 10 lL dimethylsulfoxide, and 980 lL of 0.1 M
HEPES buffer containing 0.5 M NaCl, pH 7.25, in a
thermostated cuvette. A 100-lL aliquot was transferred
to a thermostated cuvette containing 880 lL of 0.1 M
HEPES/0.5 M NaCl buffer, pH 7.25, and 20 lL of a
70-lM solution of MeOSuc-Ala-Ala-Pro-Val p-nitroan-
ilide, and the change in absorbance was monitored at
410 nm for 60 s. In a typical inhibition run, 10 lL of
inhibitor (700 lM) in dimethylsulfoxide was mixed with
10 lL of 70 lM enzyme solution and 980 lL of 0.1 M
HEPES/0.5 M NaCl buffer, pH 7.25, and placed in a
constant temperature bath. Aliquots (100 lL) were with-
drawn at different time intervals and transferred to a
cuvette containing 20 lL of MeOSuc-Ala-Ala-Pro-Val
p-nitroanilide (7 mM) and 880 lL of 0.1 M HEPES/
0.5 M NaCl buffer. The absorbance was monitored at
410 nm for 60 s.
5.4. Bovine trypsin
5.4.1. Incubation method. Bovine trypsin was assayed by
mixing 10 lL of a 500-lM enzyme solution in 0.1 M
Tris–HCl/0.01 M CaCl2 buffer, pH 7.2, 10 lL DMSO,
and 980 lL of 0.025 M phosphate/0.1 M NaCl buffer,
pH 7.51, in a thermostated test tube. A 100-lL aliquot
was transferred to a thermostated cuvette containing
890 lL 0.025 M phosphate/0.1 M NaCl buffer, pH
7.51, and 10 lL of a 60-mM solution of N-p-tosyl-Gly-
Pro-Lys p-nitroanilide in DMSO and the change in
absorbance was monitored at 410 nm for 60 s. In a typ-
ical inhibition run, 10 lL of inhibitor (12.5 mM) in
DMSO was mixed with 10 lL of a 500-lM enzyme solu-
tion in 0.1 M Tris–HCl/0.01 M CaCl2 buffer, pH 7.2,
and 980 lL of 0.1 M Tris–HCl/0.01 M CaCl2 buffer,
pH 7.51, and placed in a constant temperature bath. Ali-
quots (100 lL) were withdrawn at different time inter-
vals and transferred to a cuvette containing 10 lL of a
60 mM solution of N-p-tosyl-Gly-Pro-Lys p-nitroanilide
in DMSO and 890 lL of 0.025 M phosphate/0.1 M
NaCl buffer, pH 7.51. The absorbance was monitored
at 410 nm for 60 s. The pseudo first-order rate constants
were obtained from plots of ln(v/v0) versus time and are
the average of two determinations.
Progress curves were generated by adding 5 lL of a 2.0-
lM HNE solution in 0.05 M sodium acetate buffer, pH
5.5, to 10 lL of inhibitor (50 lM solution in DMSO),
15 lL of substrate (MeOSuc-Ala-Ala-Pro-Val p-NA,
7 mM in DMSO) and 970 lL of 0.1 M HEPES buffer/
0.5 M NaCl buffer, pH 7.25, and the absorbance was
monitored at 410 nm for 10 min.
Acknowledgment
5.2. Human neutrophil proteinase 3
Twenty microliters of a 32.0-mM 5,50-dithio-bis(2-nitro-
benzoic acid) in DMSO and 10 lL of a 3.45-lM solu-
tion of human proteinase 3 in 0.1 M phosphate/0.25 M
NaCl buffer, pH 6.50 (final enzyme concentration:
34.5 nM) were added to a cuvette containing a solution
of 940 lL of 0.1 M HEPES/0.5 M NaCl buffer, pH 7.25,
10 lL of a 0.86 mM inhibitor solution in DMSO (final
inhibitor concentration: 8.6 lM), and 20 lL of a
12.98-mM solution of Boc-Ala-Ala-NVa-SBzl in
DMSO, and the change in absorbance was monitored
at 410 nm for 2 min. A control (hydrolysis) was also
run under the same conditions by adding 20 lL of a
Financial support of this work by the National Insti-
tutes of Health (HL 57788) is gratefully acknowledged.
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