Synthesis and elastase-inhibiting activity of 2-pyridinyl-isothiazol-3(2H)-one 1,1-dioxides

Article abstract of DOI:10.1016/j.bmc.2008.07.049

The synthesis of a series of new isothiazol-3(2H)-one 1,1-dioxides with halogenated (mostly fluorinated) pyridinyl and pentafluorophenyl substituents at 2-position is reported. These compounds (18-24) became easily accessible from 2-thiocyanato-1-carboxaldehydes and aminopyridines, pentafluoroaniline, respectively, by an isothiazolium cyclization-oxidation route. Compound 21 exhibited an IC₅₀ value of 3.1 μM toward human leukocyte elastase. The proteases cathepsin G, trypsin, cathepsin L, and angiotensin-converting enzyme, and the serine esterases acetylcholinesterase and cholesterol esterase were not inhibited by 21.

Full text of DOI:10.1016/j.bmc.2008.07.049

Bioorganic & Medicinal Chemistry 16 (2008) 8127–8135
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and elastase-inhibiting activity of 2-pyridinyl-isothiazol-3(2H)-
one 1,1-dioxides
Alexander Eilfeld a, , Camino M. González Tanarro b, , Maxim Frizler b, Joachim Sieler c, Bärbel Schulze a,
Michael Gütschow b,
*
a Institute for Organic Chemistry, University of Leipzig, Johannisallee 29, D-04103 Leipzig, Germany
b Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
c Institute for Inorganic Chemistry, University of Leipzig, Johannisallee 29, D-04103 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of new isothiazol-3(2H)-one 1,1-dioxides with halogenated (mostly fluorinated)
pyridinyl and pentafluorophenyl substituents at 2-position is reported. These compounds (18–24)
became easily accessible from 2-thiocyanato-1-carboxaldehydes and aminopyridines, pentafluoroaniline,
respectively, by an isothiazolium cyclization–oxidation route. Compound 21 exhibited an IC50 value of
Received 7 March 2008
Revised 14 July 2008
Accepted 18 July 2008
Available online 24 July 2008
3.1 lM toward human leukocyte elastase. The proteases cathepsin G, trypsin, cathepsin L, and angioten-
sin-converting enzyme, and the serine esterases acetylcholinesterase and cholesterol esterase were not
inhibited by 21.
Keywords:
Isothiazol-3(2H)-one 1,1-dioxides
Enzyme inhibition
Ó 2008 Elsevier Ltd. All rights reserved.
Human leukocyte elastase
1. Introduction
which was attributed either to the direct fluorine–protein inter-
action or to an altered polarity of other groups. For example,
With a van der Waals radius of 1.47 Å, covalently bound fluo-
rine occupies a smaller volume than other substituents except of
hydrogen (van der Waals radius of 1.20 Å). Thus, the substitution
of hydrogen by fluorine in bioactive molecules results in minor ste-
ric changes but alters their physico-chemical properties, including
lipophilicity as well as basicity or acidity of adjacent functional
groups. The difference in electronegativity between carbon and
the most electronegative element, fluorine, generates strong dipole
moments in the carbon–fluorine bond and may contribute to the
ability of the molecule to engage in intermolecular interactions.
It was demonstrated that aromatic carbon-bound fluorine can
interact with positively charged molecules or positively polarized
electrophilic centers.1,2 Currently, the occurrence of fluorine within
hydrogen-bonding distance of N–H, O–H, or C–H groups, respec-
tively, is viewed as an attractive dipolar contact rather than a true
hydrogen bond.2–5
fluorination of phthalimides led to potent inhibitors of angiogen-
esis and inhibitors of the production of tumor necrosis factor-
.
a 8–13
Favorable (fluorophilic) and unfavorable (fluorophobic)
environments within the target proteins have been determined.
Such an approach was explored to develop inhibitors of serine
and cysteine proteases.2–6,14,15 An example is the DPP-4 inhibitor
sitagliptin, a recently approved antidiabetic, where a trifluoro-
phenyl rest occupies the hydrophobic S1 pocket of the
protease.16
Human leukocyte elastase (HLE) is a serine protease with a
primary specificity for small aliphatic residues in P1 position of
the substrate. The active enzyme is stored within cytoplasmic
azurophilic granules for the remaining life of the neutrophils un-
til extruded into phagolysosomes or out of the cell. HLE is capa-
ble of degrading
a variety of proteins, including elastin,
collagens, laminin, fibronectin, and cartilage proteoglycans as
well as immunoglobulins and complement components. HLE
can indirectly favor the breakdown of matrix proteins by prote-
olytic activation of matrix metalloproteinases. Under normal
conditions, the activity of extracellular HLE is regulated by
endogenous inhibitors, but an imbalance between HLE and its
endogenous inhibitors may result in several pathological states.
Thus, HLE is considered to be the primary source of tissue dam-
age associated with such inflammatory diseases as pulmonary
emphysema and adult respiratory distress syndrome.17–20
Small-molecular weight HLE inhibitors could be therapeutically
Although it is not common in natural products, covalently
bound fluorine has attracted much attention in medicinal chem-
istry.6,7In comparison with the non-fluorinated counterparts, a
desirable reduction in their rate of metabolism has been shown
for several fluorine-containing drugs. Fluorination of aromatic
groups can improve the binding of ligands to target proteins,
* Corresponding author. Tel.: +49 228 732317; fax +49 228 732567.
E-mail address: guetschow@uni-bonn.de (M. Gütschow).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.049

8128
A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
useful in the treatment of such diseases, and a number of inhib-
itors are currently in development.21,22
sen as follows: the tetramethylene unit was incorporated into five
final halo compounds with different 2-substituents (18–21, 24),
whereas the tetrafluoropyridin-4-yl moiety was maintained in the
products 21–23. Moreover, both the non-halogenated and the sac-
charin analogon of 21, that is, 25 and 26, were included in the study.
The structures of the new salts 10–17 and of the isothiazole
derivatives 18–25 were elucidated on the basis of NMR, MS, and
IR data as well as elemental analyses. In the IR spectra of the prod-
ucts 18–25, the typical absorption bands for the SO2 group were
observed at 1164–1186 cmꢀ1 and at 1332–1352 cmꢀ1 for the sym-
metric and asymmetric vibrations, respectively. In the 13C NMR
spectra of the pyridinyl (pentafluorophenyl) isothiazol-3(2H)-one
1,1 dioxides 18–25, the signals for C-3 at 159–162 ppm and for
C-7a (C-5 for 22 and 23) at 146–151 ppm were characteristic for
these oxidation products.
Herein, we report on the synthesis of a series of new isothiazol-
3(2H)-one 1,1-dioxides with halogenated (mostly fluorinated) aro-
matic substituents at 2-position. The final compounds were evalu-
ated as inhibitors of HLE, and were additionally assessed against a
panel of proteases and two serine esterases, acetylcholinesterase
(AChE) and cholesterol esterase (CEase). Isothiazol-3(2H)-one 1,1-
dioxides have already been reported as inhibitors of HLE. For
example, saccharin derivatives with a leaving group within the
2-substituent have been designed as enzyme-activated inhibitors
of HLE,23–25 and Hlasta et al. have designed specific saccharin-
based inactivators of HLE showing excellent potency and blood sta-
bility.26–28
The structure of the isothiazole 22 was confirmed by X-ray crys-
tal structure analysis. Compound 22 crystallizes in the triclinic
space group P1 with three symmetry-independent molecules in
2. Chemistry
ꢀ
The new halopyridinyl-substituted isothiazolium salts 10–15,
the pentafluoro derivative 16, and the non-halogenated derivative
17 were easily accessible by cyclocondensation reactions of thiocy-
anates 1–329–31 with the corresponding aromatic amines 4–9
(Scheme 1). In the second step, the halopyridinyl-isothiazol-
3(2H)-one 1,1 dioxides 18–23, the pentafluorophenyl compound
24, and 25 were prepared in moderate to good yields by oxidation
of the salts 10–17 with glacial acetic acid and hydrogen peroxide
at 80 °C for 8 h. This facile synthetic entry allowed the synthesis of
isothiazol-3(2H)-one 1,1-dioxides with phenyl and substituted phe-
nyl groups at position 2,32–34 and was now applied to the prepara-
tion of 2-halopyridinyl derivatives. In order to deduce first
structure–activity relationships, the substitution pattern was cho-
the unit cell. For the three molecules, the same numbering of the
atoms was used differing by the letters A, B, and C. The molecular
structure of the molecule A is shown in Figure 1. Bond lengths of
the isothiazole ring and dihedral angles are given for the molecules
A, B and C in Table 1. The molecules A, B, and C have similar bond
lengths and nearly the same conformation in that the dihedral an-
gles from A and B differ by 7.1°, those from A and C by 16.8°. The
crystal packing (Fig. 2) was analyzed with the program XPac.35
The packing of the three molecules in the direction of the a-axis
is equal. Three molecular chains with translation symmetry were
obtained, composed exclusively by molecules of the type A, B
and C.
R1
R2
CHO
SCN
1-3
R3
F
F
F
N
F
F
F
F
F
H2N
N
H2N
R4
H2N
H2N
F
N
R5
9
4-6
7
8
AcOH / HClO4
R3
AcOH / HClO4
AcOH / HClO4
AcOH / HClO4
F
F
F
N
F
F
F
R1
R2
NH
N
R4
N
N
N
F
S
S
S
S
N
R5
F
F
2 [ClO4]
[ClO4]
10-12
[ClO4]
13-15
[ClO4]
16
17
AcOH / H2O2
AcOH / H2O2
AcOH / H2O2
AcOH / H2O2
O
R3
O
F
F
F
N
F
O
F
F
F
F
O
R1
R2
N
R4
N
N
S
O O
F
N
S
O O
NH
S
S
N
R5
O
O
O
O
[ClO4]
25
18-20
21-23
24
O
F
F
N
F
4, 10, 18: R3 = R4 = H, R5 = Cl
5, 11, 19: R3 = R4 = H, R5 = Br
6, 12, 20: R3 = R4 = F, R5 = H
1, 13, 21: R1R2 = -(CH2)4-
2, 14, 22: R1 = R2 = CH3
3, 15, 23: R1= C6H5, R2 = CH3
N
S
O
O
F
26
Scheme 1.

A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
8129
tion with alcohols under Mitsunobu conditions tended to favor
O-alkylation, as recently found.36 N-Arylation of saccharin was suc-
cessfully achieved with triphenylbismuth and cupric acetate in the
presence of pyridine or triethylamine.37 2-Phenylsaccharin was
also obtained by N-arylation with ortho-silylphenyl triflate in the
presence of cesium fluoride.38 The conversion of 4,5,6,7-tetrahy-
drosaccharin39 via arylation to produce corresponding tetrahydro
derivatives has not been reported so far. Such compounds (i.e.,
18–21, 24, and 25) became easily accessible by the isothiazolium
cyclization–oxidation route as described above (Scheme 1). Subr-
amanyam et al. have performed chloromethylation of tetrahydro-
saccharin in two steps with chloromethyl phenyl sulfide/tetra
n-butylammonium bromide and sulfuryl chloride. Esterification
of 2,6-dichlorobenzoic acid with the resulting chloride furnished
a potent mechanism-based inhibitor of HLE.39
Figure 1. Molecular structure of 22, molecule A, showing the atom-labeling
Tetrafluoropyridine-substituted saccharins, for example, 26,
have already been reported by Raßhofer and Vögtle.40 In two pat-
ents, N-(tetrafluoropyridin-4-yl)saccharins, including derivatives
with alkoxy substitution at the fused benzene ring,41 as well as
N-aryl saccharins42 have been described as inhibitors of elastases.
Ashe et al. investigated a series of N-aryl saccharins as inhibitors
of HLE and bovine chymotrypsin.43 Among these compounds, pen-
tafluorophenyl saccharin, prepared from ortho-sulfobenzoic acid
cyclic anhydride and pentafluoroaniline was the most potent
scheme, thermal ellipsoids with 50% probability.
Table 1
Bond lengths (Å) of the isothiazole ring of the molecules A, B, and C and the dihedral
angles (°) between the isothiazole ring and the tetrafluoropyridinyl ring of compound
22
Molecule A
Molecule B
Molecule C
C1–S1
C1–C2
C2–C3
C3–N1
C3–O3
N1–S1
S1–O1
S1–O2
1.760(2)
1.333(3)
1.486(3)
1.404(2)
1.206(2)
1.694(2)
1.416(2)
1.419(2)
1.414(3)
85.7(1)
1.763(2)
1.329(3)
1.482(3)
1.407(3)
1.198(3)
1.683(2)
1.420(2)
1.423(2)
1.414(3)
78.6(1)
1.761(2)
1.334(3)
1.487(3)
1.410(2)
1.197(2)
1.692(2)
1.421(2)
1.416(2)
1.416(3)
68.9(1)
inhibitor with an IC50 value of 3 lM toward HLE and was inactive
against bovine chymotrypsin. This prompted us to combine the
haloaryl moiety with the tetrahydrosaccharin core to obtain 18–
21 and 24. The inhibition of HLE by 18–26 (Table 2) was measured
using a spectrophotometric assay with the chromogenic substrate
MeOSuc-Ala-Ala-Pro-Val-pNA. In order to assess selectivity, other
representative members of serine proteases (cathepsin G, trypsin),
as well as metalloproteases (angiotensin-converting enzyme
(ACE)), and cysteine proteases (cathepsin L) were also determined.
Instead of the corresponding fluorogenic substrate,44 cathepsin L
could conveniently be assayed with the chromogenic substrate Z-
Phe-Arg-pNA. Two serine esterases, AChE and CEase which share
the acyl transfer mechanism with serine proteases were also in-
cluded in the inhibition studies.
N1–C4
Dihedral angles
For HLE inhibition, the tetrafluoropyridin-4-yl substitution was
advantageous compared to the pentafluorophenyl substitution (21
vs 24). The introduction of other halopyridinyl moieties did not
lead to enzyme inhibition (18–20 vs 21). Thus, the tetrafluoropyri-
din-4-yl moiety was maintained and the tetramethylene chain of
21 was replaced by a dimethyl (22) or 4-phenyl-5-methyl substitu-
tion (23). However, these modifications did not improve the HLE-
inhibiting activity. The non-fluorinated analogon 25 was inactive
against HLE (21 vs 25), indicating the impact of fluorine substitu-
tion on HLE inhibition. The replacement of the tetramethylene
chain by a fused benzene ring (21 vs 26) increased the HLE-inhib-
itory capacity by two orders of magnitude. Provided that 26 be-
haved kinetically as
a competitive inhibitor, the IC50 value
obtained in this study corresponding to a Ki value of 28 nM, agrees
with a literature Ki value of 20 nM.41 Some of the other enzymes
(Table 2) were also inhibited by 26, but to a lesser extent.
The concentration-dependent inhibition of HLE by compound
21 is presented in Figure 3. The progress curves of the HLE-cata-
lyzed substrate consumption were linear over the 10-min time
course indicating time-independent inhibition by 21. To elucidate
the type of inhibition, the reactions were performed with different
substrate concentrations. The Hanes–Woolf plot (Fig. 4) provided
parallel lines, which indicated competition of substrate and inhib-
itor for the active site. From the replot of the slopes of the Hanes–
Woolf plot versus the corresponding inhibitor concentrations, a Ki
Figure 2. The arrangement of compound 22 in the unit cell. Packing of the
molecules A (green), B (blue), and C (red) along the a-axis, for simplification without
hydrogen atoms.
3. Results and discussion
value for competitive inhibition of 1.7 lM was obtained (Fig. 5).
Inhibition of HLE by N-aryl saccharins was competitive with
Saccharin derivatives substituted at the N-2 atom can easily be
obtained by N-alkylation with alkyl halides. In contrast, the reac-
substrate and has been characterized as alternate substrate

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A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
Table 2
Inhibition of serine hydrolases by isothiazol-3(2H)-one 1,1 dioxides 18–26
Compound
IC50 SEMa
HLEb
(lM)
Cathepsin Gb
(lM)
Trypsinb
(
lM)
Cathepsin Lb
(lM)
ACEc
(
lM)
AChEd
(
lM)
CEased
(lM)
18
19
20
21
22
23
24
25
26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.7 0.5e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.1 0.2e
>100
33 1f
>100
>100
0.081 0.003e
7.5 0.8g
11 2.4e
a
Data were obtained from duplicate determinations at two inhibitor concentrations, [I], of 25 and 50
lM. Data for cathepsin G inhibition were obtained from duplicate
determinations at an inhibitor concentration of 12.5
presence of the inhibitor, respectively.
lM. IC50 values were calculated using the equation IC50 = [I]/(v0/v ꢀ 1), where v0 and v are the rates in the absence and
b
Progress curves were followed for 10 min and analyzed by linear regression.
Progress curves were followed for 20 min and analyzed by linear regression.
Progress curves were followed for 5 min and analyzed by linear regression.
Data are mean values of duplicate determinations of two-independent experiments, using five different inhibitor concentrations. IC50 was obtained by non-linear
c
d
e
regression according to equation v = v0/([I]/IC50 + 1).
f
Data are mean values of duplicate determination, using nine different inhibitor concentrations (5–45 lM). IC50 was obtained by non-linear regression according to a four-
parametric logistic equation.
g
Progress curves were followed for 12.5 min and analyzed by non-linear regression using the equation for slow-binding inhibition [P] = vst + (vi ꢀ vs)(1 ꢀ exp(ꢀkobst))/
kobs + d, where [P] is the product concentration, vs is the steady-state rate, vi is the initial rate, kobs is the observed pseudo first-order rate constant, and d is the offset. IC50 was
obtained by non-linear regression according to equation vS = v0/([I]/IC50 + 1).
2.5
100
2
80
1.5
60
1
0.5
0
40
20
0
0
1
2
3
4
5
0
25
50
[S] (μM)
75
100
125
[21] (μM)
Figure 3. Inhibition of human leukocyte elastase by compound 21. Plot of the
percentage rates versus inhibitor concentrations, [I], in 50 mM sodium phosphate,
500 mM NaCl, pH 7.8, 25 °C. Initial concentration of the substrate MeOSuc-Ala-Ala-
Figure 4. Hanes–Woolf plot for the inhibition of human leukocyte elastase by
compound 21 in the presence of different concentrations, [S], of the chromogenic
substrate MeO-Suc-Ala-Ala-Pro-Val-pNA. The other conditions were the same as
Pro-Val-pNA was 100 lM (=1.9 Km). Progress curves were followed for 10 min, and
noted in Figure 3. Rates in the absence of inhibitor and the presence of 100
the substrate were set to 100%. Data are mean values of duplicate determinations of
lM of
HLE-catalyzed hydrolysis was analyzed as zero-order reaction. Data are mean
values of duplicate determinations of two-independent experiments. Rates in the
absence of inhibitor, v0, were set to 100%. IC50 was obtained using equation v = v0/
two-independent experiments with inhibitor concentrations, [I], of 3
), 1
competitive inhibition. Linear regression gave values for vertical intercepts.
l
M ( ), 2
l
M
ꢁ
(
l
M (j), and in the absence of the inhibitor (h). The parallel lines indicate
ꢂ
([I]/IC50 + 1). Non-linear regression gave IC50 = 3.1 0.2 lM.
inhibition.43 A similar mode of action was also assumed for 21,
including the attack of the active site serine at the carbonyl carbon
and the reversible formation of an acyl-enzyme. The result of a
reactivation experiment is shown in Figure 6. The hydrazine added
was supposed to act as a competing nucleophile45 thus accelerat-
ing the cleavage of an acyl-enzyme and leading to reactivation.43,46
For a summary, a new inhibitor of HLE (21) was prepared from
4-amino-2,3,5,6-tetrafluoropyridine (7) and 2-thiocyanato-1-
cyclohexene-1-carboxaldehyde (1) in two reaction steps. Com-
pound 21 exhibited an IC50 value in the low micromolar range
and did neither inhibit the proteases cathepsin G, trypsin, cathep-
sin L, and ACE, nor the serine esterases AChE and CEase. Inhibitor
21 behaved kinetically as a competitive inhibitor, and an alternate
substrate inhibition was assumed. Further studies are in progress
to introduce the tetrafluoropyridin-4-yl moiety in other structures
and to explore the protease-inhibiting properties of the resulting
compounds.
4. Experimental protocols
4.1. General methods and materials
Meting points were determined with a Boetius micro-melting
point apparatus and are corrected. UV/vis spectra were recorded
on a Beckman DU 650 spectrophotometer. Maximum wavelengths
are noted in nanometer and loge values are given in parentheses.
IR spectra were measured on a Genesis FTIR Unicam Analytical Sys-
tem (ATI Mattson). 1H (200 or 300 MHz), 13C (50 or 75 MHz), and
19F (188 MHz) spectra were recorded on Varian Gemini-200 or Var-

A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
8131
and 5,50-dithio-bis-(2-nitrobenzoic acid) (DTNB) were purchased
from Sigma, Steinheim, Germany. Cholesterol esterase (CEase) from
bovine pancreas, sodium taurocholate (TC), and para-nitro-
phenylbutyrate (pNPB) were obtained from Sigma, Steinheim,
Germany. 2-(2,3,5,6-Tetrafluoropyridin-4-yl)-1,2-benzisothiazol-
3(2H)-one 1,1-dioxide (26) was obtained from TimTac, LLC,
Newark, DE, USA.
The spectrophotometric assays were done on a Varian Cary 50
Bio UV/vis spectrometer with a cell holder equipped with a con-
stant temperature water bath.
1
0.8
0.6
0.4
0.2
0
4.1.1. General method for the synthesis of isothiazolium per-
chlorates 10–17
The substituted aminopyridine (4–7, 9, 1 mmol) or 2,3,4,5,6-
pentafluoroaniline (8, 183 mg, 1 mmol) was added to a solution
of the corresponding thiocyanate (1–3, 1 mmol) in glacial acetic
acid (2 ml). When the solid was completely dissolved, perchloric
acid (0.4 ml, 70%) was added dropwise to the solution. The reaction
mixture was stirred for 2 h, and diethyl ether was added. The pre-
cipitate was isolated by filtration and recrystallized from ethanol.
-2
-1
0
1
2
3
4
[21] (μM)
Figure 5. Plot of vertical intercepts versus the concentrations of 21. Data were
obtained from Figure 4. Linear regression according to the equation, intercept = Km
[I]/(Ki Vmax) + (Km/Vmax), gave a slope Km/(Ki Vmax) = 0.18 0.02%ꢀ1 and an intercept
Km/Vmax = 0.31 0.04
l
M%ꢀ1 that corresponds to a value Ki = 1.7
lM.
4.1.1.1. 2-(6-Chloropyridin-2-yl)-4,5,6,7-tetrahydro-1,2-benziso-
thiazolium perchlorate (10).
(EtOH), kmax (nm): 211 (4.26). IR (KBr),
Yield: 70%, mp: 219–221 °C. UV
m
(cmꢀ1): 1080 (O–Cl–O).
0,08
0,06
0,04
0,02
1H NMR (DMSO-d6), d (ppm): 1.90 (m, c, 4H, 2CH2), 2.83 (m, c, 2H,
CH2), 3.22 (m, c, 2H, CH2), 7.83 (d, J = 7.7 Hz, 1H, aromat. H), 8.17
(d, J = 8.0 Hz, 1H, aromat. H), 8.34 (t,J = 8.0 Hz, 1H, aromat. H), 9.94
(s, 1H, 3-H). 13C NMR (DMSO-d6), d (ppm): 20.5, 21.1, 22.4, 25.5
(CH2), 112.2 (aromat. C–H), 125.9 (aromat. C–H), 135.2 (C-3a),
144.0 (aromat. C–H), 147.4 (aromat. C), 148.7 (C–Cl), 152.2 (C-3),
addition of
inhibitor
þꢀ
171.0 (C-7a). ESI-MS m/z: 251 ðM ꢀ HClO4 Þ. Anal. Calcd for
C12H12Cl2N2O4S (351.21): C, 41.04; H, 3.44; N, 7.89; S, 9.13. Found:
C, 40.94; H, 3.04; N, 7.99; S, 9.23.
4.1.1.2. 2-(6-Bromopyridin-2-yl)-4,5,6,7-tetrahydro-1,2-benzisothia-
addition of
hydrazine
zolium perchlorate (11).
Yield: 91%, mp: 221–223 °C. UV
(EtOH), kmax (nm): 318 (4.03). IR (KBr),
m
(cmꢀ1): 1081 (O–Cl–O). 1H
0
0
NMR (DMSO-d6), d (ppm): 1.85 (m, c, 4H, 2CH2), 2.81 (m, c, 2H,
CH2), 3.18 (m, c, 2H, CH2), 7.92 (dd, J = 5.8 Hz,J = 2.5 Hz, 1H, aromat.
H), 8.18 (m, c, 2H, aromat. H), 9.90 (s, 1H, 3-H). 13C NMR (DMSO-
d6), d (ppm): 20.5, 21.1, 22.4, 25.6 (CH2), 112.4 (aromat. C–H), 129.7
(aromat. C–H), 135.3 (C-3a), 139.5 (C–Br), 143.5 (aromat. C–H),
147.6 (aromat. C), 152.1 (C-3), 171.1 (C-7a). ESI-MS m/z: 295
2
4
6
8
10
12
14
t (min)
Figure 6. Reactivation of HLE inhibited by compound 21. The HLE-catalyzed
hydrolysis of MeOSuc-Ala-Ala-Pro-Val-pNA (100 M) was followed at 405 nm. In
the reactivation experiment ( ), HLE was inhibited by 21 (6.3
due to the addition of hydrazine (100
enzymatic cleavage was monitored in the absence of 21 and hydrazine.
l
l
M) and reactivated
ꢁ
þꢀ
ðM ꢀ HClO4 Þ. Anal. Calcd for C12H12BrClN2O4S (395.66): C, 36.43;
l
M). In the control experiment ( ), the
ꢂ
H, 3.06; N, 7.08; S, 8.10. Found: C, 36.14; H, 2.82; N, 7.05; S, 8.09.
4.1.1.3. 2-(3,5-Difluoropyridin-2-yl)-4,5,6,7-tetrahydro-1,2-benz
ian Gemini-300 spectrometers. Chemical shifts are given as d val-
ues, relative to tetramethylsilane (TMS) and CFCl3 as internal stan-
dards. Spin multiplicities are indicated by the following symbols: s
(singlet), d (doublet), t (triplet), m (multiplet), m, c (centered mul-
tiplet) dd (doublet of doublet). ESI-MS spectra were recorded on a
Bruker Esquire 3000 plus. HR ESI-MS spectra were recorded with
syringe infusion on a Bruker FT-ICR mass spectrometer APEX II,
equipped with a 7 T magnet, skimmer voltage 100 V, drying gas
200 °C. Elemental analyses were performed on a Heraeus CHNO
Rapid Analyser.
isothiazolium perchlorate (12).
UV (EtOH), kmax (nm): 313 (4.06). IR (KBr),
Yield: 52%, mp: 163–165 °C.
m
(cmꢀ1): 1093 (O–Cl–
O). 1H NMR (DMSO-d6), d (ppm): 1.88 (m, c, 4H, 2CH2), 2.88 (m,
c, 2H, CH2), 3.24 (m, c, 2H, CH2), 8.58 (t, J = 8.1 Hz, 1H, aromat.
H), 8.68 (m, c, 1H, aromat. H), 9.62 (s, 1H, 3-H). 13C NMR (DMSO-
d6), d (ppm): 20.5, 21.2, 22.5, 25.7 (CH2), 116.5 (t, 2J = 24 Hz,
aromat. C–H), 124.7 (m, aromat. C), 133.2 (m, 2J = 26 Hz, aromat.
C–H), 135.0 (C-3a), 150.2 (d, 1J = 241 Hz, C–F), 154.1 (C-3), 159.4
(d, 1J = 261 Hz, C–F), 171.2 (C-7a). 19F NMR (acetone-d6), d (ppm):
þꢀ
ꢀ120.9 (m, 1F), ꢀ122.8 (m, 1F). ESI-MS m/z: 252 ðM ꢀ HClO4 Þ.
Human leukocyte elastase (HLE), human cathepsin G, human
cathepsin L, and human angiotensin-converting enzyme (ACE)
were obtained from Calbiochem, Darmstadt, Germany. MeOSuc-
Anal. Calcd for C12H11ClF2N2O4S (352.75): C, 40.86; H, 3.14; N,
7.94; S, 9.09. Found: C, 40.60; H, 3.27; N, 7.94; S, 9.13.
Ala-Ala-Pro-Val-pNA,
Suc-Ala-Ala-Pro-Phe-pNA,
Suc-Ala-Ala-
4.1.1.4. 2-(2,3,5,6-Tetrafluoropyridin-4-yl)-4,5,6,7-tetrahydro-
Pro-Arg-pNA, Z-Phe-Arg-pNA, and 2-furanacryloyl-phenylalanyl-
glycylglycine (FA-Phe-Gly-Gly) were purchased from Bachem,
Bubendorf, Switzerland. Acetylcholinesterase (AChE) from
Electrophorus electricus was purchased from Fluka, Deisenhofen,
Germany. Trypsin from bovine pancreas, acetylthiocholine (ATCh),
1,2-benzisothiazolium perchlorate (13).
Yield: 87%, mp:
235–236 °C. UV (EtOH), kmax (nm): 287 (4.11). IR (KBr),
m
(cmꢀ1): 1092 (O–Cl–O). 1H NMR (DMSO-d6), d (ppm): 1.86 (m,
c, 4H, 2CH2), 2.97 (m, c, 2H, CH2), 3.38 (m, c, 2H, CH2), 9.52 (s,
1H, 3-H). 13C NMR (DMSO-d6), d (ppm): 21.3, 21.9, 23.3, 26.5

8132
A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
(CH2), 127.9 (m, aromat. C), 136.5 (C-3a), 138.8 (dd, 1J = 266 Hz,
2J = 30 Hz, C–F), 144.4 (m, 1J = 240 Hz, C–F), 159.8 (C-3), 177.6
(C-7a). 19F NMR (acetone-d6), d (ppm): ꢀ89.2 (m, 2F), ꢀ147.5
(m, 2F). ESI-MS m/z: 289 ðM ꢀ ClO4þÞ. Anal. Calcd for
mat. H), 8.04 (t, J = 7.8 Hz, 1H, aromat. H). 13C NMR (acetone-d6), d
(ppm): 20.0, 21.4, 21.8, 22.1 (CH2), 116.8 (aromat. C–H), 124.4 (aro-
mat. C–H), 136.8 (C-3a), 142.7 (aromat. C), 143.5 (aromat. C–H),
148.6 (C-7a), 150.7 (C–Cl), 160.9 (C@O). ESI-MS m/z: 298 (M+ꢀ).
Anal. Calcd for C12H11ClN2O3S (298.75): C, 48.25; H, 3.71; N,
9.38; S, 10.73. Found: C, 47.78; H, 3.69; N, 9.50; S, 11.23.
C
12H9ClF4N2O4S (388.73): C, 37.08; H, 2.33; N, 7.21; S, 8.25.
Found: C, 37.03; H, 1.68; N, 7.22; S, 8.28.
4.1.1.5.
thiazolium perchlorate (14).
UV (EtOH), kmax (nm): 283 (4.17). IR (KBr),
4,5-Dimethyl-2-(2,3,5,6-tetrafluoropyridin-4-yl)-iso-
Yield: 93%, mp: 260–262 °C.
(cmꢀ1): 1090 (O–Cl–
4.1.2.2. 2-(6-Bromopyridin-2-yl)-4,5,6,7-tetrahydro-1,2-benz-
isothiazol-3(2H)-one 1,1-dioxide (19).
Yield: 56%, mp:
m
181–183 °C. UV (EtOH), kmax (nm): 223 (4.24). IR (KBr),
m
O). 1H NMR (DMSO-d6), d (ppm): 2.54 (s, 3H, CH3), 3.08 (s, 3H,
CH3), 9.40 (s,. 1H, 3-H). 13C NMR (DMSO-d6), d (ppm): 11.7 (CH3),
14.5 (CH3), 128.4 (m, aromat. C), 136.4 (C-3a), 139.6 (dd,
1J = 267 Hz, 2J = 31 Hz, C–F), 145.0 (m, 1J = 243 Hz, C–F), 161.0 (C-
3), 177.6 (C-7a). 19F NMR (DMSO-d6), d (ppm): ꢀ89.1 (m, 2F),
ꢀ146.4 (m, 2F). ESI-MS m/z: 263 ðM ꢀ ClO4þÞ. Anal. Calcd for
C10H7ClF4N2O4S (362.69): C, 33.12; H, 1.95; N, 7.72; S, 8.84. Found:
C, 33.08; H, 2.02; N, 7.67; S, 8.85.
(cmꢀ1): 1733 (C@O), 1333 (SO2), 1177 (SO2). 1H NMR (DMSO-
d6), d (ppm): 1.76 (m, c, 4H, 2CH2), 2.41 (m, c, 2H, CH2), 2.58
(m, c, 2H, CH2), 7.69 (d, J = 7.8 Hz, 1H, aromat. H), 7.81 (d,
J = 8.1 Hz, 1H, aromat. H), 7.95 (t, J = 8.1 Hz, 1H, aromat. H). 13C
NMR (DMSO-d6), d (ppm): 18.3, 19.8, 19.0, 20.2 (CH2), 116.4 (aro-
mat. C–H), 127.2 (aromat. C–H), 135.5 (C-3a), 139.1 (C–Br), 142.3
(aromat. C–H), 145.8 (aromat. C), 146.1 (C-7a), 159.0 (C@O). ESI-
MS m/z: 344 (M+H+ꢀ). Anal. Calcd for C12H11BrN2O3S (343.20): C,
42.00; H, 3.23; N, 8.16; S, 9.34. Found: C, 41.57; H, 2.99; N, 8.33;
S, 10.01.
4.1.1.6. 5-Methyl-4-phenyl-2-(2,3,5,6-tetrafluoropyridin-4-yl)-
isothiazolium perchlorate (15).
UV (EtOH), kmax (nm): 232 (3.96). IR (KBr),
Yield: 54%, mp: 72–75 °C.
m
(cmꢀ1): 1086 (O–
4.1.2.3. 2-(3,5-Difluoropyridin-2-yl)-4,5,6,7-tetrahydro-1,2-benz-
Cl–O). 1H NMR (DMSO-d6), d (ppm): 2.95 (s, 3H, CH3), 7.23 (m,
c, 1H, aromat. H), 7.42 (m, c, 3H, aromat. H), 7.61 (m, c, 1H, aro-
mat. H), 9.79 (s, 1H, 3-H). It was not possible to record a 13C
NMR spectrum due to the rapid decomposition of this compound
in solution. 19F NMR (DMSO-d6), d (ppm): ꢀ88.4 (m, 2F), ꢀ145.3
(m, 2F). ESI-MS m/z: 325 ðM ꢀ ClO4þÞ. HR ESI-MS ðM ꢀ ClO4þÞ
Calcd for C15H9F4N2S: 325.04171. Found: 325.04160.
isothiazol-3(2H)-one 1,1-dioxide (20). Yield: 35%, mp: 173–
175 °C. UV (EtOH), kmax (nm): 212 (3.50). IR (KBr),
m
(cmꢀ1):
1743 (C@O), 1332 (SO2), 1185 (SO2). 1H NMR (acetone-d6), d
(ppm): 1.90 (m, c, 2H, CH2), 2.00 (m, c, 2H, CH2), 2.56 (m, c, 2H,
CH2), 2.68 (m, c, 2H, CH2), 8.00 (m, c, 1H, aromat. H), 8.53 (m, c,
1H, aromat. H). 13C NMR (acetone-d6), d (ppm): 20.3, 21.6, 21.7,
22.1 (CH2), 116.1 (t, 2J = 23 Hz, aromat. C–H), 130.3 (m, aromat.
C), 135.9 (d, 2J = 25 Hz, aromat. C–H), 137.5 (C-3a), 149.7 (C-7a),
157.2 (d, 1J = 269 Hz, C–F), 160.8 (C@O), 161.9 (d, 1J = 263 Hz, C–
F). 19F NMR (acetone-d6), d (ppm): ꢀ119.5 (m 1F), ꢀ121.4 (m,
1F). ESI-MS m/z: 300 (M+ꢀ). Anal. Calcd for C12H10F2N2O3S
(300.29): C, 48.00; H, 3.36; N, 9.33; S, 10.68. Found: C, 47.82; H,
3.12; N, 9.59; S, 11.05.
4.1.1.7. 2-(2,3,4,5,6-Pentafluorophenyl)-4,5,6,7-tetrahydro-1,2-
benzisothiazolium perchlorate (16).
260 °C. UV (EtOH), kmax (nm): 206 (4.00). IR (KBr),
Yield: 98%, mp: 257–
m
(cmꢀ1): 1085
(O–Cl–O). 1H NMR (DMSO-d6), d (ppm): 1.91 (m, c, 4H, 2CH2),
2.89 (m, c, 2H, CH2), 3.30 (m, c, 2H, CH2), 9.44 (s, 1H, 3-H). 13C
NMR (DMSO-d6), d (ppm): 20.6, 21.4, 22.7, 26.9 (CH2), 134.8 (C-
3a), 160.3 (C-3), 176.0 (C-7a). 19F NMR (DMSO-d6), d (ppm):
ꢀ143.9 (m, 2F), ꢀ147.7 (m, 1F), ꢀ159.2 (m, 2F). ESI-MS m/z: 306
ðM ꢀ ClO4þÞ. Anal. Calcd for C13H9ClF5NO4S (405.73): C, 38.48; H,
2.24; N, 3.45; S, 7.90. Found: C, 38.40; H, 1.94; N, 3.81; S, 8.12.
4.1.2.4. 2-(2,3,5,6-Tetrafluoropyridin-4-yl)-4,5,6,7-tetrahydro-
1,2-benzisothiazol-3(2H)-one 1,1-dioxide (21).
Yield: 47%,
mp: 238–240 °C. UV (EtOH), kmax (nm): 217 (3.91). IR (KBr),
m
(cmꢀ1): 1749 (C@O), 1350 (SO2), 1184 (SO2). 1H NMR (acetone-
d6), d (ppm): 1.92 (m, c, 2H, CH2), 2.03 (m, c, 2H, CH2), 2.60 (m, c,
2H, CH2), 2.74 (m, c, 2H, CH2). 13C NMR (acteone-d6), d (ppm):
20.7, 21.6, 21.9, 22.0 (CH2), 122.8 (m, aromat. C), 138.1 (C-3a),
142.1 (m, 1J = 268 Hz, C–F), 145.5 (m, 1J = 244 Hz, C–F), 150.6 (C-
7a), 160.1 (C@O). 19F NMR (acetone-d6), d (ppm): ꢀ90.2 (m, 2F),
ꢀ144.4 (m, 2F). ESI-MS m/z: 337 (M+H+). Anal. Calcd for
4.1.1.8. 2-(Pyridin-4-yl)-4,5,6,7-tetrahydro-1,2-benzisothiazoli-
um diperchlorate (17).
(EtOH), kmax (nm): 294 (3.86). IR (KBr),
Yield: 97%, mp: 216–217 °C. UV
m
(cmꢀ1): 1093 (O–Cl–O).
1H NMR (acetone-d6), d (ppm): 1.98 (m, c, 4H, 2CH2), 3.04 (m, c,
2H, CH2), 3.47 (m, c, 2H, CH2), 8.80 (d, J = 7.6 Hz, 2H, aromat. H),
9.45 (d, J = 7.6 Hz, 2H, aromat. H), 9.89 (s, 1H, 3-H). 13C NMR (ace-
tone-d6), d (ppm): 22.0, 22.7, 24.1, 27.4 (CH2), 110.8 (aromat. C–H),
138.6 (C-3a), 141.7 (aromat. C), 146.7 (aromat. C–H), 157.7 (C-3),
C12H8F4N2O3S (336.27): C, 42.86; H, 2.40; N, 8.33; S, 9.54. Found:
C, 41.61; H, 2.36; N, 8.35; S, 9.92.
+
177.7 (C-7a). ESI-MS m/z: 217 (½M ꢀ HClO4 ꢀ ClO4ꢀꢃ ). Anal. Calcd
4.1.2.5.
4,5-Dimethyl-2-(2,3,5,6-tetrafluoropyridin-4-yl)-iso-
for C12H14Cl2N2O8S (417.22): C, 34.55; H, 3.38; N, 6.71. Found: C,
34.38; H, 3.08; N, 6.59.
thiazol-3(2H)-one 1,1-dioxide (22).
134 °C. UV (EtOH), kmax (nm): 219 (3.91). IR (KBr),
Yield: 21%, mp: 133–
m
(cmꢀ1):
1749 (CO), 1349 (SO2), 1182 (SO2). 1H NMR (acetone-d6), d
(ppm): 2.19 (s, 3H, CH3), 2.42 (s, 3H, CH3). 13C NMR (acetone-d6),
d (ppm): 8.5 (CH3), 9.3 (CH3), 122.0 (m, aromat. C), 134.5 (C-3a),
141.3 (m, 1J = 267 Hz, C–F), 144.8 (m, 1J = 244 Hz, C–F), 146.9 (C-
5), 160.2 (C@O). 19F NMR (acetone-d6), d (ppm): ꢀ90.1 (m, 2F),
ꢀ144.4 (m, 2F). ESI-MS m/z: 310 (M+ꢀ). Anal. Calcd for
4.1.2. General method for the synthesis of isothiazol-3(2H)-one
1,1-dioxides 18–25
H2O2 (0.7 ml, 30%) was added to a stirred suspension of 10–17
(0.26 mmol) in glacial acetic acid (0.7 ml) and stirring was contin-
ued at 80 °C for 8 h. After cooling, the solid products 18–25 could
be isolated by filtration and were recrystallized from ethanol.
C10H6F4N2O3S (310.23): C, 38.72; H, 1.95; N, 9.03; S, 10.34. Found:
C, 38.69; H, 1.90; N, 9.01; S, 10.32.
4.1.2.1. 2-(6-Chloropyridin-2-yl)-4,5,6,7-tetrahydro-1,2-benz-
isothiazol-3(2H)-one 1,1-dioxide (18).
Yield: 44%, mp:
4.1.2.6. 5-Methyl-4-phenyl-2-(2,3,5,6-tetrafluoropyridin-4-yl)-
174–176 °C. UV (EtOH), kmax (nm): 218 (3.94). IR (KBr),
m
(cmꢀ1):
isothiazol-3(2H)-one 1,1-dioxide (23).
Yield: 24%, mp:
1735 (C@O), 1349 (SO2), 1169 (SO2). 1H NMR (acetone-d6), d
(ppm): 1.89 (m, c, 4H, 2CH2), 2.52 (m, c, 2H, CH2), 2.64 (m, c, 2H,
CH2), 7.48 (d, J = 7.8 Hz, 1H, aromat. H), 7.89 (d, J = 8.1 Hz, 1H, aro-
115–116 °C. UV (EtOH), kmax (nm): 206 nm (4.18). IR (KBr), m
(cmꢀ1): 1740 (CO), 1349 (SO2), 1184 (SO2). 1H NMR (acetone-
d6), d (ppm): 2.56 (s, 3H, CH3), 7.60 (m, c, 3H, aromat. H), 7.70

A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
8133
(m, c, 2H, aromat. H). 13C NMR (acetone-d6), d (ppm): 10.3 (CH3),
122.5 (m, aromat. C), 127.9 (C-40), 130.3 (C-20), 131.7 (C-30), 132.3
(C-10), 135.8 (C-3a), 142.1 (m, 1J = 269 Hz, C–F), 145.5 (m,
1J = 242 Hz, C–F), 147.9 (C-5), 160.0 (C@O). 19F NMR (acetone-
d6), d (ppm): ꢀ90.0 (m, 2F), ꢀ144.2 (m, 2F). ESI-MS m/z: 372
(M+). HR ESI-MS (M+H+) m/z: Calcd for C15H8F4N2O3S:
373.02645. Found: 373.02658.
buffer. The final concentration of DMSO was 3.5% and the final con-
centration of the chromogenic substrate MeOSuc-Ala-Ala-Pro-Val-
pNA was 100
with a final HLE concentration of 50 ng/ml, which corresponded to
an initial rate of 0.6 M/min, when 100 M of the substrate was
used. Into a cuvette containing 870 l assay buffer, 30 l of an
inhibitor solution and 50 l of the substrate solution were added
and thoroughly mixed. The reaction was initiated by adding 50
lM, unless stated otherwise. Assays were performed
l
l
l
l
l
l
l
4.1.2.7. 2-(2,3,4,5,6-Pentafluorophenyl)-4,5,6,7-tetrahydro-1,2-
of the HLE solution and was followed over 10 min. IC50 values were
calculated from the linear steady-state turnover of the substrate as
previously described.48
benzisothiazol-3(2H)-one 1,1-dioxide (24).
Yield: 39%, mp:
143–145 °C. UV (EtOH), kmax (nm): 215 (4.09). IR (KBr),
m
(cmꢀ1):
1745 (CO), 1352 (SO2), 1186 (SO2). 1H NMR (DMSO-d6), d (ppm):
1.93 (m, c, 4H, 2CH2), 2.57 (m, c, 2H, CH2), 2.69 (m, c, 2H, CH2).
13C NMR (DMSO-d6), d (ppm): 19.2, 20.3, 20.5, 20.6 (CH2), 136.7
(C-3a), 148.7 (C-7a), 159.3 (C@O). 19F NMR (acetone-d6), d (ppm):
ꢀ143.3 (m, 2F), ꢀ148.6 (m, 1F), ꢀ161.7 (m, 2F). ESI-MS m/z: 353
(M+ꢀ). Anal. Calcd for C13H8F5NO3S (353.27): C, 44.20; H, 2.28; N,
3.96; S, 9.08. Found: C, 44.00; H, 3.01; N, 4.15; S, 8.89.
4.3.2. HLE reactivation
HLE was assayed as described above. Into a cuvette contain-
ing 860
solution were added and thoroughly mixed. The reaction was
initiated by adding 50 l of the HLE solution. After 4.14 min,
25 l of a 0.25 mM solution of 21 in DMSO was added. After
8.83 min, 10 l of a 10 mM solution of hydrazine in assay buffer
was added. In the reactivation and control experiment, the final
concentration of MeOSuc-Ala-Ala-Pro-Val-pNA was 100 M, of
HLE was 50 ng/ml, and of DMSO was 3.2%. In the reactivation
experiment, the final concentration of 21 was 6.3 M and of
hydrazine was 100 M.
ll assay buffer, 5 ll of DMSO and 50 ll of the substrate
l
l
l
4.1.2.8. 2-(Pyridin-4-yl)-4,5,6,7-tetrahydro-1,2-benzisothiazol-
3(2H)-one 1,1-dioxide perchlorate (25).
Yield: 41%, mp:
l
230–235 °C. UV (EtOH), kmax (nm): 264 (3.87). IR (KBr),
m
(cmꢀ1): 1728 (CO), 1348 (SO2), 1164 (SO2), 1085 ClO4
.
1H NMR
l
ꢀ
(acetone-d6), d (ppm): 1.67 (m, c, 4H, 2CH2), 2.42 (m, c, 2H,
CH2), 2.52 (m, c, 2H, CH2), 7.08 (d, J = 7.6 Hz, 2H, aromat. H),
8.29 (d, J = 7.6 Hz, 2H, aromat. H). 13C NMR (acetone-d6), d
(ppm): 19.3, 21.8, 22.6, 24.9 (CH2), 114.8 (aromat. C–H), 132.4
(C-3a), 143.7 (aromat. C), 143.9 (aromat. C–H), 145.6 (C-7a),
162.0 (C@O). ESI-MS m/z: 265 ð½M ꢀ ClO4ꢀꢃþÞ. HR ESI-MS m/z:
Calcd for C12H13N2O3S: 265.06414. Found: 265.06420. Anal. Calcd
for C12H13ClSN2O7 (364.76): Calcd for N, 7.68. Found: N, 8.09.
l
4.3.3. Cathepsin G inhibition assay
Human cathepsin G was assayed spectrophotometrically at
405 nm at 25 °C.49,50Assay buffer was 20 mM Tris–HCl buffer,
150 mM NaCl, pH 8.4. Inhibitor stock solutions were prepared in
DMSO. An enzyme stock solution of 200 mU/ml was prepared in
50 mM sodium acetate buffer, 150 mM NaCl, pH 5.5. A 50 mM
stock solution of the chromogenic substrate Suc-Ala-Ala-Pro-Phe-
pNA in DMSO was diluted with assay buffer. The final concentra-
tion of DMSO was 1.5%, and the final concentration of the substrate
4.2. X-ray crystal structure analysis
Crystal data, the data collection procedure, and structure deter-
mination for compound 22 are noted below. Refinement of the
structure was performed with SHELX-97.47 The hydrogen atoms
were included from a difference electron density map and refined
isotropically. The details of the structure analysis have been depos-
ited at the Cambridge Crystallographic Data Centre with the num-
ber CCDC-67542. These data can be obtained, free of charge, from
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1233
336033; e-mail: deposit@ccdc.cam.ac.uk; internet: http://
www.ccdc.cam.ac.uk).
Suc-Ala-Ala-Pro-Phe-NHNp was 500
with a final concentration of 2.5 mU/ml of cathepsin G, which cor-
responded to an initial rate of 0.7 M/min. Into a cuvette contain-
ing 882.5 l assay buffer, 5 l of an inhibitor solution and 100 l of
a substrate solution were added and thoroughly mixed. The reac-
tion was initiated by adding 12.5 l of the cathepsin G solution
lM. Assays were performed
l
l
l
l
l
and was followed over 10 min. IC50 values were calculated from
the linear steady-state turnover of the substrate.
4.3.4. Trypsin inhibition assay
Crystal data for 22: C10H6F4N2O3S; M = 310.23; T = 213(2) K; tri-
ꢀ
clinic; space group P1; unit cell dimensions a = 11.227(2) Å, b =
Trypsin from bovine pancreas was assayed spectrophotometrically
11.285(3) Å, c = 15.041(5) Å,
a
= 70.74(3)°, b = 85.21(3)°,
c
= 88.88
at 405 nm at 25 °C. Assay buffer was 20 mM Tris–HCl buffer,
150 mM NaCl, pH 8.4. An enzyme stock solution of 10 lg/ml was
prepared in 1 mM HCl and diluted with assay buffer. Inhibitor
stock solutions were prepared in DMSO. A 40 mM stock solution
of the chromogenic substrate Suc-Ala-Ala-Pro-Arg-pNA in DMSO
was diluted with assay buffer. The final concentration of DMSO
was 6%, and the final concentration of the substrate Suc-Ala-Ala-
(3)°, V = 1781.7(8) Å3, Z = 6, Dc = 1.729 g/cm3,
l
= 0.334 mmꢀ1
;
crystal size 0.4 ꢄ 0.15 ꢄ 0.05 mm3; h range for data collection
2.4–28.2°; index range ꢀ14 6 h 6 14, ꢀ14 6 k 6 14, ꢀ19 6 l 6 19;
reflections collected 17,678; independent reflections 8084
[
R
int = 0.035]; max./min. transmission 0.9835/0.8779; data/parame-
ters 8084/613; goodness-of-fit on F2 0.866; final
R indices
[I > 2
0.0595,
r
(I)] R1 = 0.0393,
x
x
R2 = 0.0931; R indices (all data) R1 =
Pro-Arg-pNA was 200
centration of 12.5 ng/ml of trypsin, which corresponded to an ini-
tial rate of 1.2 M/min. Into a cuvette containing 845 l assay
buffer, 55 l of an inhibitor solution and 50 l of a substrate solu-
tion were added and thoroughly mixed. The reaction was initiated
by adding 50 l of the trypsin solution and was followed over
lM. Assays were performed with a final con-
R2 = 0.0990; largest diff. peak/hole 0.305/ꢀ0.244 e Åꢀ3
.
l
l
4.3. Enzymatic assays
4.3.1. HLE inhibition assay
l
l
l
Human leukocyte elastase was assayed spectrophotometrically
10 min. IC50 values were calculated from the linear steady-state
at 405 nm at 25 °C. Assay buffer was 50 mM sodium phosphate
turnover of the substrate, unless stated otherwise.
buffer, 500 mM NaCl, pH 7.8. An enzyme stock solution of 50 lg/
ml was prepared in 100 mM sodium acetate buffer, pH 5.5 and di-
luted with assay buffer. Inhibitor stock solutions were prepared in
DMSO. A stock solution of the chromogenic substrate MeOSuc-Ala-
Ala-Pro-Val-pNA was prepared in DMSO and diluted with assay
4.3.5. Cathepsin L inhibition assay
Human cathepsin L was assayed spectrophotometrically at
405 nm at 37 °C. Assay buffer was 100 mM sodium phosphate buf-
fer, pH 6.0, 100 mM NaCl, 5 mM EDTA, 0.01% Brij 35. An enzyme

8134
A. Eilfeld et al. / Bioorg. Med. Chem. 16 (2008) 8127–8135
stock solution of 50
l
g/ml in 20 M sodium acetate buffer, pH 5.0,
10 ng/ml of CEase, which corresponded to an initial rate of
5.0 M/min. Into a cuvette containing 430 l assay buffer, 500
of the TC solution, 40 l acetonitrile, 10 l of the pNPB solution,
and 10 l of a solution of the test compound were added and thor-
oughly mixed. After incubation for 5 min at 25 °C, the reaction was
100 mM NaCl, 10 mM trehalose, 1 mM EDTA, 50% glycerol was di-
luted 1:100 with assay buffer containing 5 mM DTT and incubated
for 30 min at 37 °C. This enzyme solution was diluted 1:5 with as-
say buffer containing 5 mM DTT. Inhibitor stock solutions were
prepared in DMSO. A 10 mM stock solution of the chromogenic
substrate Z-Phe-Arg-pNA was prepared with DMSO. The final con-
centration of DMSO was 3%, and the final concentration of the sub-
l
l
ll
l
l
l
initiated by adding 10 ll of the enzyme solution (1 lg/ml).
Acknowledgments
strate Z-Phe-Arg-pNA was 100
final concentration of 4 ng/ml of cathepsin L, which corresponded
to an initial rate of 0.9 M/min. Into a cuvette containing 930 l as-
say buffer, 20 l of an inhibitor solution and 10 l of a substrate
solution were added and thoroughly mixed. The reaction was ini-
tiated by adding 40 l of the cathepsin L solution and was followed
lM. Assays were performed with a
The authors are grateful to Stephanie Hautmann, Jing Zhou, and
Gisela Kirsten for assistance. The work was supported by the
Graduiertenkolleg 677 ‘Struktur und molekulare Interaktion als
Basis der Arzneimittelwirkung’.
l
l
l
l
l
over 10 min. IC50 values were calculated from the linear steady-
state turnover of the substrate.
References and notes
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37 °C.51 Assay buffer was 50 mM Tris–HCl buffer, 300 mM NaCl,
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of ACE, which corresponded to an initial rate of 17
a cuvette containing 950 l assay buffer, 20 l of an inhibitor solu-
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lM/min. Into
l
l
l
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tion and was followed over 20 min. IC50 values were calculated
from the linear steady-state turnover of the substrate.
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