Inhibition of serine proteases by functionalized sulfonamides coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold

Article abstract of DOI:10.1016/S0968-0896(01)00037-2

A challenge associated with drug design is the development of selective inhibitors of proteases (serine or cysteine) that exhibit the same primary substrate specificty, that is, show a preference for the same P¹ residue. While these proteases have similar active sites, nevertheless there are subtle differences in their S and S' subsites which can be exploited. We describe herein for the first time the use of functionalized sulfonamides as a design and diversity element which, when coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold yields potent, time-dependent inhibitors of the serine proteases human leukocyte elastase (HLE), proteinase 3 (PR 3) and cathepsin G(Cat G). Our preliminary findings suggest that (a) appending to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold recognition and diversity elements that interact with both S and S' subsites of a target protease may result in optimal enzyme selectivity and potency and, (b) functionalized sulfonamides constitute a powerful design and diversity element with low intrinsic chemical reactivity and potentially wide applicability. Copyright

Full text of DOI:10.1016/S0968-0896(01)00037-2

Bioorganic & Medicinal Chemistry 9 (2001) 1543–1548
Inhibition of Serine Proteases by Functionalized Sulfonamides
Coupled to the 1,2,5-thiadiazolidin-3-one 1,1 Dioxide Scaffold
William C. Groutas,* Shu He, Rongze Kuang, Sumei Ruan, Juan Tu and Ho-Kit Chan
Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
Received 26June 2000; accepted 30 January 2001
Abstract—A challenge associated with drug design is the development of selective inhibitors of proteases (serine or cysteine) that
exhibit the same primary substrate specificity, that is, show a preference for the same P1 residue. While these proteases have similar
active sites, nevertheless there are subtle differences in their S and S0 subsites which can be exploited. We describe herein for the first
time the use of functionalized sulfonamides as a design and diversity element which, when coupled to the 1,2,5-thiadiazolidin-3-one
1,1 dioxide scaffold yields potent, time-dependent inhibitors of the serine proteases human leukocyte elastase (HLE), proteinase 3
(PR 3) and cathepsin G(Cat G). Our preliminary findings suggest that (a) appending to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide
scaffold recognition and diversity elements that interact with both the S and S0 subsites of a target protease may result in optimal
enzyme selectivity and potency and, (b) functionalized sulfonamides constitute a powerful design and diversity element with low
intrinsic chemical reactivity and potentially wide applicability. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
of inhibitors of (chymo)trypsin-like serine proteases; (b)
can be used to fashion inhibitors that show absolute
selectivity between neutral, basic and acidic serine pro-
teases; (c) is amenable to the construction of libraries
for lead identification, and the rapid optimization of
potency and enzyme selectivity, and (d) can be used to
probe and exploit subtle differences in the active sites of
closely-related serine proteases by allowing the attach-
ment and optimal spatial orientation of recognition ele-
ments that permit the exploitation of favorable binding
interactions with the S and S0 subsites of a target
enzyme.15À20 We describe herein the use and preliminary
evaluation of sulfonamide moiety (II) as a powerful and
versatile design and diversity element which, when cou-
pled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide tem-
plate, yields potent inhibitors (I) of the serine proteases
human leukocyte elastase (HLE), proteinase 3 (PR 3)
and cathepsin G (Cat G).
Several lines of evidence suggest that serine and metal-
loproteases released by neutrophil and macrophages
play a pivotal role in the etiology and pathophysiology
of inflammatory diseases such as, for example, pulmon-
ary emphysema,1À3 chronic bronchitis,4 adult respira-
tory distress syndrome (ARDS),5,6 cystic fibrosis,7,8 and
others.9,10 Poor regulation of the activity of these
enzymes due to depressed levels of their physiological
protein inhibitors (alpha-1-proteinase inhibitor, secre-
tory leukocyte protease inhibitor, human monocyte/
neutrophil elastase inhibitor, elafin, tissue inhibitors of
metalloproteases) can lead to the unrestrained destruc-
tion of the major components of the extracellular matrix
and basement membrane. Low molecular weight com-
pounds capable of controlling the activity of these
enzymes, thereby redressing the protease/antiprotease
imbalance, may lead to the emergence of new
therapeutic agents.11À14
Results
In recent studies we have described the structure-based
design of a novel heterocyclic scaffold (1,2,5-thiadiazo-
lidin-3-one 1,1 dioxide) and have demonstrated that the
scaffold (a) is a general template suitable for the design
Chemistry
Sulfonamide derivatives 1–17 were synthesized accord-
ing to Scheme 1. The precursor sulfonamides (II) were
synthesized as summarized in Scheme 2, by reacting an
alkyl or aryl sulfonamide with a carboxylic acid in the
presence of carbonyl diimidazole (CDI)21 or an alkyl
*Corresponding author. Tel.: +1-316-978-3120; fax: +1-316-978-
3431; e-mail: groutas@wsuhub.uc.twsu.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00037-2

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W. C. Groutas et al. / Bioorg. Med. Chem. 9 (2001) 1543–1548
Scheme 1. Synthesis of derivatives of (I): (a) NaI/acetone; (b)
R4SO2NH (C¼O) R3/DBU.
Scheme 2. Synthesis of precursor sulfonamide derivatives (II).
chloroformate in the presence of triethylamine and 4-
dimethylamino pyridine (DMAP)22 or an isocyanate in
the presence of triethylamine.23 Thus, the appropriate
chloromethyl derivative was first reacted with NaI in
dry acetone to yield the corresponding iodo derivative
which was immediately reacted with the appropriate
sulfonamide derivative in the presence of DBU to give
the corresponding crude products. These were purified
using flash chromatography. The physical and spectral
data for compounds 1–17 are listed in Table 1.
Biochemical studies
The apparent second order inactivation rate constants
(kinact/KI MÀ1 sÀ1) for compounds 1–17 were deter-
mined in duplicate using the progress curve method24
and are listed in Table 2. Typical progress curves for the
hydrolysis of MeOSuc-AAPV pNA by HLE in the pre-
sence of inhibitor 1 are shown in Fig. 1. The release of
p-nitroaniline was monitored at 410 nm. Data analysis
was carried out as described previously.15À20
The loss of enzymatic activity in the presence of inhi-
bitor 9 as a function of time was determined by the
incubation method25 by removing aliquots and assaying
for enzymatic activity (Fig. 2).
Molecular modeling
Modeling studies were performed using the Tripos force
field of SYBYL 6.5 (Tripos Associates, St. Louis, MO)
and a Silicon Graphics O2 workstation. The crystal
structure of HLE bound to the Turkey Ovomucoid
Inhibitor Third Domain (TOMI)26 (Brookhaven, 1PPF)
was used in the modeling studies by superimposing the
0
0
energy-minimized inhibitor on the -P2-P1-P1 -P2 - (-Thr-
Leu-Glu-Tyr-)27 segment of TOMI with the a-carbon of
Leu-18 (P1 residue) of TOMI. A shaded surface for

W. C. Groutas et al. / Bioorg. Med. Chem. 9 (2001) 1543–1548
1545
Table 2. Inhibitory activity of derivatives of (I) toward human leukocyte elastase, cathepsin G and proteinase 3
kinact/KI MÀ1sÀ1
Compd
P1
R2
R3
R4
HLE
PR3
Cat G
a
1
2
3
4
5
6
7
8
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Isobutyl
Benzyl
Methyl
Methyl
Methyl
Methyl
Methyl
Benzyl
Methyl
Methyl
Methyl
Benzyl
Methyl
Benzyl
Methyl
Benzyl
Benzyl
Benzyl
Benzyl
Isobutyloxy
n-butyloxy
Benzyloxy
Methoxy
n-butyloxy
Phenyl
Benzyl
Methyl
Methyl
Methyl
Phenyl
11,900
40,500
22,000
51,100
70,500
71,000
28,300
9900
140,500
148,900
229,400
134,000
92,700
14,100
—
1200
3400
3000
6100
6400
16,000
10,100
3,200
27,500
2400
27,400
30,100
29,200
—
—
—
—
—
—
200
—
—
—
60
Phenyl
Methyl
Methyl
(p-NH2)phenyl
Methyl
Methyl
Methyl
Methyl
Phenyl
Methyl
9
CH2NHBoc
CH2NHBoc
CH2NHCbz
CH2NHCbz
CH2NHCbz
NHCH2COOEt
Methoxy
10
11
12
13
14
15
16
17
70
b
Methyl
Phenyl
Phenyl
5200
b
70
90
80
5400
b
b
Benzyl
Benzyl
n-butyloxy
n-butyloxy
—
1970
Methyl
aNot determined.
bCompounds designated as inactive showed less than 50% inhibition when incubated with the enzyme at an inhibitor to enzyme ratio of 250 for 10
minutes.
HLE was then generated following the removal of
TOMI and water molecules (Fig. 3).
of these differences could, in principle, yield inhibitors
that display high enzyme selectivity.
Attaining maximal enzyme selectivity is a problem of
paramount importance in drug design, particularly
when considering the inhibition of proteases. For
example, urokinase-type plasminogen activator (uPA) is
a serine protease that shows a strong preference for an
arginine as the P1 residue. This enzyme appears to play
a pivotal role in cancer metastasis, consequently
Discussion
The majority of covalent and non-covalent inhibitors of
enzymes with extended binding sites such as, for exam-
ple, proteases, have generally been designed to exploit
favorable binding interactions only with the S subsites
of a target enzyme.14,28 Furthermore, since the primary
specificity of a serine or cysteine protease is ordinarily
determined by the nature of the P1 residue, discrimina-
tion between neutral, basic and acidic proteases has
been achieved by incorporating an appropriate P1 resi-
due, or equivalent, in the structure of an inhibitor.
Thus, inhibitors having a neutral (hydrophobic) P1 resi-
due are intended to inhibit neutral serine or cysteine
proteases such as chymotrypsin, elastase, chymase,
PSA, cathepsin G, etc.; those with a basic P1 residue
inhibit basic proteases such as thrombin, factor Xa,
urokinase-type plasminogen activator, kallikrein, plas-
min, tryptase, granzyme A, cathepsin K, gingipain, etc.;
and those with an acidic P1 residue inhibit acidic pro-
teases (granzyme B, caspases). Recently, we have
demonstrated that inhibitors based on the 1,2,5-thia-
diazolidin-3-one 1,1 dioxide scaffold to which an
appropriate recognition element P1 has been appended
exhibit absolute or near absolute enzyme selectivity
between neutral, basic and acidic proteases.18À20
The design of inhibitors that exhibit high enzyme selec-
tivity among members of the same subclass has been
problematic, since members of a given subclass show a
preference for the same P1 residue. Nevertheless,
inspection of the available X-ray crystal structures of
(chymo)trypsin-like proteases reveals the existence of
subtle differences in their S0 and S subsites. Exploitation
Figure 1. Progress curves for the inhibition of human leukocyte elas-
tase (HLE) by compound 1. Absorbance was recorded at 410 nm for
reaction solutions containing HLE (21.9 nM), MeOSuc-AAPV pNA
(1 mM) at the indicated inhibitor to enzyme ratios in 0.1 M HEPES
buffer containing 0.5 M NaCl, pH 7.25 and 3.6% dimethyl sulfoxide.
The temperature was maintained at 25 ꢀC, and the reactions were
initiated by the addition of enzyme.

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W. C. Groutas et al. / Bioorg. Med. Chem. 9 (2001) 1543–1548
inhibitors that display high inhibitory activity toward
uPA and low or no activity toward related proteases
(thrombin, factor Xa, plasmin) are of potential value as
anticancer agents.29 Clearly, uPA inhibitors with low
enzyme selectivity would result in unacceptable toxicity
since they could potentially disrupt the blood coagulation
cascade.
PR 3. Secondly, a high level of structural diversity can
be introduced into the sulfonamide moiety without
compromising inhibitory activity.
The results in Table 2 also show that the most potent
compounds are those in which the sulfonamide leaving
group is derived from a protected glycine (9–13).
Replacement of glycine by other amino acids is antici-
pated to enhance inhibitory activity, since the side chain
of the amino acid0 would make additional favorable
contacts with the S2 subsite (Fig. 4). Indeed, compound
18 (Fig. 5) is a more efficient inhibitor of HLE than
compound 14 (Table 2) (kinact/KI 35,000 versus
14,100 MÀ1 sÀ1). Figure 3 illustrates the binding of
energy-minimized inhibitor 18 to the active site of HLE.
The isobutyl and N-benzyl groups occupy the hydro-
phobic S1 and S2 pockets, while the sulfonamide moiety
orients itself toward the S0 pockets. Thus, the benzyl
group of the amino acid is likely involved in specific
hydrophobic interactions with Phe-41 and/or Phe-192.
Definitive evidence regarding the precise mode of bind-
ing, and the network of hydrophobic and hydrogen
We hypothesized that the rigorous spatial control
afforded by the 1,2,5-thiadiazolidin-3-one 1,1 dioxide
template, and the fact that the template makes possible
the exploitation of binding interactions with both the S
and S0 subsites, might make possible the attainment of
optimal enzyme selectivity. In an attempt to achieve this
aim, we have pursued a strategy that explores the use of
functionalized sulfonamides as a novel design element
which, when coupled to the aforementioned heterocyclic
template, might yield inhibitors having the desired
characteristics. In conducting these preliminary studies,
P1 was chosen to be either isobutyl or benzyl, in accor-
dance with the known substrate specificity of HLE and
Cat G, respectively. The use of R2=methyl was based
on earlier observations that showed that inhibitors with
a methyl group at R2 yield stable acyl enzymes.15
Incubation of compound 9, for example, with human
leukocyte elastase led to rapid and essentially irrever-
sible inactivation of the enzyme. The inactivation of the
enzyme by 9 was time-dependent and very efficient,
requiring only three equiv of 9 for total inactivation of
the enzyme (Fig. 2). Furthermore, the putative acyl
enzyme formed exhibits high stability, as evidenced by
the partial and slow regain of enzymatic activity over a
24-h period.
Two significant and general deductions can be made by
inspecting the results shown in Table 2. First, sulfona-
mide derivatives based on the 1,2,5-thiadiazolidin-3-one
1,1 dioxide scaffold are potent inhibitors of HLE and
Figure 3. Energy-minimized inhibitor 18 docked to the active site of
HLE with the isobutyl (P1) and benzyl groups (P2) occupying the S1
and S2 subsites, respectively. The functionalized sulfonamide segment
is oriented toward the S0 subsites, with the benzyl group of0 (L) Phe
sandwiched between Phe 41 and Phe 192 and occupying the S2 subsite.
Figure 2. Percent remaining activity versus time plot obtained by
incubating inhibitor 9 with human leukocyte elastase (138 nM) at dif-
ferent inhibitor to enzyme ratios 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 pNA by monitoring the absorbance at 410 nm.
Figure 4. Postulated binding of compound (I) to the active site and
interactions with the S0 subsites.

W. C. Groutas et al. / Bioorg. Med. Chem. 9 (2001) 1543–1548
1547
siderable flexibility with respect to the generation of
structurally-diverse single compounds or libraries.
Exploratory studies focusing on the utility and scope of
functionalized sulfonamides in drug design are currently
in progress and the results will be reported in due
course.
Experimental
Melting points were recorded on a Mel-Temp apparatus
1
Figure 5. Structures and inhibitory activity of compounds 18 and 19.
and are uncorrected. The H and 13C NMR spectra of
the synthesized compounds were recorded on a Varian
XL-300 NMR spectrometer. A Hewlett-Packard diode
array UV/Vis spectrophotometer was used in the
enzyme assays and inhibition studies. Human leukocyte
elastase was purchased from Elastin Products Com-
pany, Owensville, MO. Human leukocyte cathepsin G
and proteinase 3 were purchased from Athens Research
and Technology Co., Athens, GA. Methoxysuccinyl
Ala-Ala-Pro-Val p-nitroanilide and methoxysuccinyl
Ala-Ala-Pro-Phe p-nitroanilide were purchased from
Sigma Chemical Co., St. Louis.
bonding interactions involved, will have to await the
determination of the X-ray crystal structure of the
enzyme-inhibitor complex.
In principle, potency, enzyme selectivity, and pharma-
cokinetics can be readily optimized by deblocking the
amino acid and derivatizing the resulting product fur-
ther. Interestingly, the replacement of a methyl group
with phenyl (compound 13) results in potent inhibition
of HLE and PR 3 but not Cat G (vide infra). Equally
noteworthy is the observation that compound 14 is
fairly active toward HLE and PR 3. Since the precursor
sulfonamide derivatives for making compounds like 14
are made by reacting a sulfonamide with an isocyanate,
the use of readily available amino acid-derived iso-
cyanates30,31 greatly enhances the potential of these
compounds. The lower potency of compound 14 versus
compounds 9–13 is partly attributed to the lower leav-
ing group ability of the sulfonamide group in 14. It
should be noted that (a) the pKa of functionalized sul-
fonamides can be modulated by varying the nature of
R3 and R4 and, (b) the pKa of the leaving group has a
profound effect on inhibitory activity. Indeed, N-alkyl
substituted sulfonamides (for example, compound 19 in
Fig. 5) appended to the heterocyclic scaffold are devoid
of inhibitory activity.
General procedure for the synthesis of compounds 1–17
A solution of chloromethyl compound (2 mmol) in dry
acetone (10 mL) kept under nitrogen was treated with
sodium iodide (4 mmol), and the resulting mixture was
stirred at room temperature overnight. The precipitate
(NaCl) was filtered off and the solvent evaporated off,
leaving the corresponding iodo compound. This was
dissolved in dry methylene chloride (10 mL) and treated
with the appropriate sulfonamide (2 mmol) and 1,8-dia-
zabicyclo[5.4.0]undec-7-ene (DBU) (2 mmol). The reac-
tion mixture was stirred at room temperature overnight
under a nitrogen atmosphere. Removal of the solvent
left a crude product which was taken up in ethyl acetate
(60 mL) and washed with water (20 mL), 5% sodium
bicarbonate (2ꢁ15 mL) and brine (20 mL). The organic
layer was dried and evaporated, leaving a crude product
which was purified by flash chromatography using silica
gel (hexane/ether gradient).
In all cases, the synthesized compounds inhibited both
HLE and PR 3, reflecting the similarity of their active
sites.32 As anticipated, these compounds were more
effective inhibitors of HLE than PR 3, since the P1 resi-
due was chosen to be optimal for HLE than PR 3. The
active site of the latter is more constricted, showing a
greater preference for small linear alkyl chains(ethyl)
n-propyl).33À35
Acknowledgements
This work was supported by grants from the National
Institutes of Health (HL 57788) and Supergen, Inc.
The inhibitory activity of compounds 2–5 (R3=alkoxy)
toward HLE and PR 3 was significant but no dramatic
variations in potency were observed. The analogous Cat
G inhibitors were substantially weaker, however more
secure inferences regarding the inhibition of Cat G by
this class of compounds will have to await the results of
more extensive SAR studies.
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