Design, synthesis, and in vitro evaluation of inhibitors of human leukocyte elastase based on a functionalized cyclic sulfamide scaffold

Article abstract of DOI:10.1016/j.bmc.2003.10.059

The design of novel functionalized templates capable of binding to the active site of serine proteases could potentially lead to the development of potent and highly selective non-covalent inhibitors of these enzymes. Using the elastase-turkey ovomucoid inhibitor complex and insights gained from earlier work based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I), a surrogate cyclosulfamide scaffold (II) was used for the first time in the design of reversible inhibitors of human leukocyte elastase. Compounds 7 and 8 were found to be micromolar reversible inhibitors of the enzyme.

Full text of DOI:10.1016/j.bmc.2003.10.059

Bioorganic & Medicinal Chemistry 12(2004) 589–593
Design, synthesis, and in vitro evaluation of inhibitors of human
leukocyte elastase based on a functionalized
cyclic sulfamide scaffold
Jiaying Zhong, Xiangdong Gan, Kevin R. Alliston and William C. Groutas*
Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
Received 14 July 2003; accepted 24 October 2003
Abstract—The design of novel functionalized templates capable of binding to the active site of serine proteases could potentially
lead to the development of potent and highly selective non-covalent inhibitors of these enzymes. Using the elastase–turkey
ovomucoid inhibitor complex and insights gained from earlier work based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I),
a surrogate cyclosulfamide scaffold (II) was used for the first time in the design of reversible inhibitors of human leukocyte elastase.
Compounds 7 and 8 were found to be micromolar reversible inhibitors of the enzyme.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
that functionalized surrogate scaffolds capable of
mimicking (I) in terms of the spatial orientation of
attached recognition elements, may lead to new classes
of serine protease inhibitors and, more importantly,
provide a structural framework for the rational design
of inhibitors of this class of enzymes. Indeed, studies
with functionalized 4-imidazolidinone derivatives have
provided evidence in support of this hypothesis.9 Further
validation of this approach using functionalized cyclic
sulfamide derivatives is reported herein (Fig. 1).
Mammalian proteases, as well as proteases of bacterial,
viral and parasitic origin, have been implicated in a
range of human diseases; consequently, these enzymes
have been the focus of intense study, and as potential
targets for the development of novel therapeutic
agents.1ꢀ3 The design of functionalized templates that
mimic the backbone conformation of a known protein
substrate (or inhibitor) of a protease that is capable of
orienting recognition elements appended to it in the
same vector relationship as the side chains of the amino
acids that make up the substrate (or inhibitor) recogni-
tion loop offers many potential advantages, including
optimal enzyme selectivity and potency.
1.1. Chemistry
Inhibitors 7–8 were readily synthesized using the reac-
tion sequence shown in Scheme 1.10
We have recently described the structure-based design
of a highly functionalized heterocyclic scaffold (I) and
have demonstrated that derivatives based on (I) func-
tion as potent covalent inhibitors of serine proteases.4ꢀ8
Scaffold (I) makes possible the exploitation of favorable
binding interactions with both the S and S0 subsites and,
potentially, subtle structural differences in the S and S0
subsites of closely related proteases, namely, proteases
in a given class that have the same primary substrate
specificity. Based on our findings with (I), we reasoned
1.2. Biochemical studies
The inhibitory activity of compounds 7–8 was evaluated
by determining the KI using Dixon plots.11 A repre-
sentative experiment was carried out according to the
following procedure: to a thermostatted solution of 930
mL 0.1 M HEPES buffer containing 0.5 M NaCl, pH
7.25, was added DMSO (50 mL), human leukocyte
elastase (10 mL solution in 0.05 M sodium acetate buffer
containing 0.5 M NaCl, pH 5.5, for a final enzyme
concentration of 70 nM) and, lastly, 10 mL methoxy-
succinyl-Ala-Ala-Pro-Val p-nitroanilide in DMSO for a
final substrate concentration of 0.70 mM. The rate of
hydrolysis was then determined by monitoring the
* Corresponding author. Tel. +1-316-978-7374; fax: +1-316-978-
3431; e-mail: groutas@wichita.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.059

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J.Zhong et al./ Bioorg.Med.Chem.12 (2004) 589–593
absorbance at 410 nm for 2min. The experiment was
repeated in the presence of varying amounts of inhibitor
8 (10–50 mL) and the rates of substrate hydrolysis
determined. The series of experiments described above
was repeated at an additional substrate concentration
(2.33Â10ꢀ2 M). All rates were determined in triplicate.
The inverse of the average velocities was plotted against
the final inhibitor concentration and the KI was
determined from the intersection of the three lines (each
R2>0.99). A representative 1/v versus [I] plot is shown
in Figure 2.
pulmonary disease,12,13 adult respiratory distress
syndrome, cancer,14,15 and others.16,17 The recent intro-
duction of Sivelestat (ElaspolR), an inhibitor of HLE,
for the treatment of acute lung injury associated with
systemic inflammatory response syndrome, attests to the
clinical potential of HLE inhibitors. Inhibitors 7 and 8
were synthesized as illustrated in Scheme 1. Both inhibitors
incorporate in their structures appropriate recognition
elements, specifically, a Val-Pro segment intended to
interact with the S3–S2 subsites of the enzyme, a leucine
P1 residue that is accommodated at the primary specificity
site (S1) of0the enzyme and an aromatic residue that fits
into the S2 subsite.18 The composite effect of favorable
binding interactions (Fig. 3) was anticipated to lead to
the formation of a stable EI complex.
2. Results and discussion
Earlier work with the 1,2,5-thiadiazolidin-3-one 1,1
dioxide scaffold (I) suggested that the heterocyclic
scaffold had the potential of serving as a prototype
structure for the design of a series of related surrogate
scaffolds with wide applicability, including the develop-
ment of potent and selective non-covalent inhibitors of
(chymo)trypsin-like serine proteases, the parallel synth-
esis of libraries of compounds, and as pharmacological
probes. We reasoned that the replacement of the
carbonyl group in (I) with a CH2 group would yield a
functionalized cyclic sulfamide structure (II) that could
function as a reversible competitive inhibitor of a serine
protease (Fig. 1).
To explore these ideas, we chose to conduct the
biochemical studies using human leukocyte elastase
(HLE),
a
prototypical neutrophil-derived serine
protease of clinical relevance. HLE has been the focus
of intense investigation because of its likely involvement
in a range of diseases, including chronic obstructive
Scheme 1. Synthesis of cyclosulfamide inhibitors 7–8: (a) EDCI/
HOBt/DIEA then (l) prolinol; (b) (ClCO)2/DMSO/TEA then (l)
Leu-OCH3/NaBH (OAc)3/HOAc; (c) ClSO2N¼C¼O/t-BuOH/TEA;
(d) LiBH4/THF; (e) Ph3P/DEAD/THF; (f) CF3COOH; (g)
Ph(CH2)2COOH/CDI or Ph(CH2)2N¼C¼O/TEA.
Figure 1. Evolution of the design of inhibitor (II) beginning with the
human leukocyte elastase–turkey ovomucoid inhibitor (HLE–TOMI)
complex.
Figure 2. Dixon plot showing the inhibition of human leukocyte elastase by compound 8.

J.Zhong et al./ Bioorg.Med.Chem.12 (2004) 589–593
591
solution was washed with saturated aqueous sodium
bicarbonate (300 mL). The aqueous layer was extracted
with ethyl acetate (2Â150 mL) and the combined
organic phase was washed with 5% aqueous HCl
(2Â150 mL), saturated aqueous NaHCO3 (2Â150 mL),
brine (2Â150 mL), and dried over anhydrous sodium
sulfate. The solvent was removed and the crude product
was purified by flash chromatography (ethyl acetate/
1
hexane) to give an oily product (11.6 g; 81% yield). H
NMR (CDCl3): d 0.85–1.06 (dd, 6H), 1.57 (m, 1H),
1.75–2.10 (m, 4H), 3.40–3.70 (m, 3H), 3.82 (m, 1H), 4.23
(m, 1H), 4.35 (t, 1H), 4.56 (m, 1H), 5.07 (q, 2H), 5.61 (d,
1H), 7.30–7.43 (m, 5H). Anal. calcd for C18H26N2O4: C,
64.65; H, 7.84; N. 8.38. Found: 64.86; H, 7.44; N, 8.19.
Figure 3. Main-chain and side-chain binding interactions between
inhibitor 8 and the active site of HLE.
Compounds 7 and 8 were both found to be reversible
competitive inhibitors of HLE (KI 50.0 and 25.0 mM,
respectively). While the potency of compounds 7 and 8
is modest, the results support the notion that the cyclic
sulfamide template can be used in the design of HLE
inhibitors and, possibly, related serine proteases.
Furthermore, lead optimization can be attained by
exploiting the three points of diversity present in (II)
using focused combinatorial libraries.
3.3. Synthesis of Cbz-L-Val-L-Pro-L-Leu-OCH3 2
To a solution of 2M oxalyl chloride in methylene
chloride (48.9 mL; 97.8 mmol) kept in an acetone-dry
ice bath was added dropwise a solution of dry DMSO
(10.7 mL; 146.8 mmol) in dry methylene chloride (85
mL) over a period of 20 min. After stirring for 30 min,
the reaction mixture was treated with a solution of
compound 1 (21.8 g; 65.2 mmol) in dry methylene
chloride (170 mL) dropwise over a period of 20 min.
After stirring the solution for 50 min, a solution of dry
TEA (20.7 g; 202.2 mmol) in methylene chloride (85
mL) was added dropwise over a period of 15 min. The
acetone–dry ice bath was removed and the reaction
mixture was allowed to warm up to room temperature
while stirring. After 15 min, glacial acetic acid was
added until the pH of the reaction mixture was about 4.
(l) Leucine methyl ester hydrochloride (11.9 g; 65.2
mmol) was then added, followed by sodium (triacetox-
y)borohydride (20.4 g; 91.3 mmol). The resulting solu-
tion was stirred at room temperature for 4 h. The
reaction mixture was cooled in an ice-bath and the pH
adjusted to 9–10 by adding 10% aqueous NaOH
solution dropwise while stirring. The aqueous layer was
separated and extracted with ethyl acetate (3Â300 mL).
The combined organic extracts were dried over
anhydrous sodium sulfate and the solvent removed on
the rotary evaporator, leaving a crude product which
was purified by flash chromatography (silica gel/ethyl
ether:hexane) to give compound 2 (24.8 g; 82% yield).
1H NMR (CDCl3): d 0.80–1.02 (m, 12H), 1.41 (m, 2H),
1.72 (m, 1H), 1.78–2.04 (m, 5H), 2.60 (m, 2H), 3.25 (t,
1H), 3.45 (m, 1H), 3.67 (s, 3H), 4.20 (m, 1H), 4.24–4.40
(m, 1H), 5.07 (q, 2H), 5.48–5.61 (m, 1H), 7.20–7.42 (m,
5H). Anal. calcd for C25H39N3O5: C, 65.05; H, 8.52; N.
9.10. Found: 64.84; H, 8.80; N, 8.99.
In conclusion, the results of these studies provide
additional support for the hypothesis that surrogate
scaffolds based on (I) such as, for example, substituted
4-imidazolidinones and cyclic sulfamides (II), can be
used in the design of inhibitors of serine proteases. The
results of ongoing studies with a range of related surrogate
templates will be reported in due course.
3. Experimental
3.1. General
Melting points were recorded on a Mel-Temp apparatus
1
and are uncorrected. The H and 13C NMR spectra of
the synthesized compounds were recorded using either a
Varian XL-300 or a Varian XL-400 NMR spectrometer.
Purification of the synthesized compounds was carried
out using flash chromatography with silica gel as the
stationary phase. A Hewlett-Packard UV/VIS spectro-
photometer was used in the enzyme assays and inhibition
studies. Human leukocyte elastase was purchased from
Elastin Products Company, Owensville, MO, USA. The
elastase substrate, methoxysuccinyl Ala-Ala-Pro-Val
p-nitroanilide, was purchased from Sigma Chemicals
Co., St. Louis, MO, USA. 2M Lithium borohydride in
THF was purchased from Aldrich Chemical Co.
3.2. Synthesis of Cbz-L-Val-L-prolinol 1
3.4. Synthesis of 3
A solution of Cbz-l-valine (10.7 g; 42.7 mmol), (L)
prolinol (5.11 g; 49.3 mmol), diisopropylethylamine
(7.10 g; 46.0 mmol) and N-hydroxybenzotriazole
hydrate (16.7 g; 108.8 mmol) in DMF (80 mL) was kept
in an ice-bath and treated with EDCI (9.88 g; 50.5
mmol) in one portion. The ice-bath was removed and
the reaction mixture was stirred at room temperature
for 11 h. Most of the DMF was removed in vacuo.
Ethyl acetate (300 mL) was added and the resulting
A solution of N-chlorosulfonyl isocyanate (0.13 g; 0.91
mmol) in dry methylene chloride (2mL) was cooled in
an ice-bath and a solution of t-butyl alcohol (0.07 g;
0.91 mmol) in dry methylene chloride (1 mL) was then
added. After stirring for 15 min at 0 ꢁC, the resulting
mixture was added dropwise to a solution of compound
2 (0.42g; 0.91 mmol) and TEA (0.10 g; 0.91 mmol) in
dry methylene chloride (1 mL) kept in an ice-bath. The
ice-bath was removed and the reaction mixture was

592
J.Zhong et al./ Bioorg.Med.Chem.12 (2004) 589–593
stirred for 3.5 h. The reaction mixture was diluted with
methylene chloride (50 mL) and washed with water
(2Â15 mL) and brine (15 mL).The organic phase was
separated and then dried over anhydrous sodium
sulfate. Removal of the solvent gave a crude product
which was purified by flash chromatography (silica gel/
ethyl acetate/hexane) to give compound 3 (0.47 g; 80%
yield). Anal. calcd for C30H48N4O9S: C, 56.23; H, 7.55;
N. 8.74. Found: 55.93; H, 7.60; N, 8.51.
and the crude product was purified by flash chromato-
graphy (ethyl acetate/hexane) to give compound 6 (8.81
g; 77% yield). H NMR (CDCl3) d 0.70–1.10 (m, 12H),
1.48–1.70 (m, 2H), 1.75–2.18 (m, 5H), 2.24 (m, 1H), 2.63
(dd, 1H), 3.03–3.30 (m, 4H), 3.37 (m, 1H), 3.50–3.72(m,
3H), 4.16–4.50 (m, 2H), 4.80–5.23 (m, 3H), 5.43–5.83
(dd, 1H), 5.95 (t, 1H), 7.23–7.40 (m, 5H). Anal. calcd
for C24H38N4O5S: C, 58.28; H, 7.74; N. 11.33. Found:
58.40; H, 7.80; N, 11.17.
1
3.5. Synthesis of 4
3.8. Synthesis of 7
A solution of compound 3 (25.3 g; 44.8 mmol) in dry
THF (70 mL) was added dropwise a solution of 2M
lithium borohydride in THF (33.6 mL, 67.20 mmol),
followed by the dropwise addition of absolute ethanol
(140 mL). The reaction mixture was stirred for 24 h at
room temperature and then cooled in an ice-bath. A
solution of 5% aq HCl was added dropwise to pH 4
with stirring. The solvent was removed to near dryness
and water (150 mL) was added. The resulting mixture
was extracted with ethyl acetate (3Â300 mL) and the
organic phase was combined and dried over anhydrous
sodium sulfate. The solvent was removed and the crude
product was purified by flash chromatography using
silica gel and ethyl acetate/hexane as eluents to give
To a solution of dihydrocinnamic acid (0.18 g; 1.20
mmol) in dry THF (2mL) was added dropwise a solu-
tion of carbonyl diimidazole (CDI) (0.20 g; 1.20 mmol)
in dry THF (3 mL). The resulting solution was stirred
for 30 min at room temperature and compound 6 (0.59
g; 1.20 mmol) was added in one portion. After stirring
the resulting solution for 15 min at room temperature, a
solution of 1,8-diazabicyclo [5.4.0]-undec-7-ene (DBU)
(0.19 g; 1.20 mmol) in dry THF (5 mL) was added drop-
wise. The reaction solution was stirred for 2h at room
temperature. The solvent was removed and ethyl acetate
(50 mL) was added. The resulting solution was washed
with 5% aqueous HCl (15 mL), 5% aq NaHCO3 (15 mL)
and brine (15 mL). The organic phase was dried over
anhydrous sodium sulfate. Removal of the solvent yielded
a crude product, which was purified by flash chromato-
graphy (ethyl acetate/hexane) to give compound 7 (0.54
g; 72% yield). Anal. calcd for C33H46N4O6S: C, 63.26; H,
7.40; N; 8.95. Found: 63.15; H, 7.25; N, 8.81.
1
compound 4 (16.0 g; 58% yield). H NMR (CDCl3): d
0.81–1.05 (m, 12H), 1.35–1.54 (m, 10H), 1.54–1.64 (m,
1H), 1.75 (m, 1H), 1.82–2.10 (m, 4H), 2.21 (m, 1H), 3.20
(d, 1H), 3.33–3.60 (m, 3H), 3.60–3.95 (m, 3H), 4.05 (br
s, 1H), 4.20–4.60 (m, 2H), 5.00–5.22 (m, 2H), 5.53–5.75
(dd, 1H), 7.20–7.42 (m, 5H). Anal. calcd for
C29H48N4O8S: C, 56.84; H, 7.90; N. 9.14. Found: 56.90;
H, 8.20; N, 9.06.
3.9. Synthesis of 8
A solution of compound 6 (0.43 g; 0.87 mmol) and TEA
(0.13 g; 1.22 mmol) in dry methylene chloride (3 mL)
was treated with 2-phenethyl isocyanate (0.24 g; 1.57
mmol). The reaction mixture was refluxed for 2.5 h with
stirring. Following removal of most of the solvent, 5%
aqueous HCl (25 mL) was added, and the resulting
mixture was then extracted with ethyl acetate (2Â75
mL). The organic extracts were combined and dried
over anhydrous sodium sulfate. The solvent was
removed and the crude product was purified by flash
chromatography (ethyl acetate/hexane) to give
compound 8 (0.55 g; 98% yield). Anal. calcd for
C33H47N5O6S: C, 61.75; H, 7.38; N; 10.91. Found:
61.48; H, 7.73; N, 10.72.
3.6. Synthesis of 5
A solution of compound 4 (14.8 g; 24.12 mmol) in dry
THF (70 mL) was treated with triphenyl phosphine
(12.82 g; 48.2 mmol) and diethyl azodicarboxylate (7.58
mL; 48.2mmol). The reaction mixture was stirred for 3
h at room temperature. The solvent was removed on the
rotovac and the crude product was purified using flash
chromatography (silica gel/ethyl acetate/hexane) to give
1
compound 5 (13.7 g; 95% yield). H NMR (CDCl3): d
0.84–1.05 (m, 12H), 1.20–1.70 (m, 12H), 1.84–2.18 (m,
5H), 3.08 (d, 2H), 3.38 (t, 1H), 3.55 (m, 2H), 3.65 (m,
1H), 3.86 (dd, 1H), 4.20 (m, 1H), 4.30 (m, 1H), 5.18 (q,
2H), 5.40–5.55 (m, 1H), 7.24–7.42 (m, 5H). Anal. calcd
for C29H46N4O7S: C, 56.56; H, 7.80; N. 9.42. Found:
57.38; H, 8.14; N, 9.28.
Acknowledgements
Financial support of this work by the National
Institutes of Health (HL 57788) and Supergen, Inc. is
gratefully acknowledged.
3.7. Synthesis of 6
A solution of compound 5 (13.3 g; 22.4 mmol) in dry
methylene chloride (10 mL) was treated with tri-
fluoroacetic acid (45 mL). The reaction mixture was
stirred for 3 h at room temperature and the solvent was
removed on a rotary evaporator to near dryness. Meth-
ylene chloride (150 mL) was added and the resulting
solution was washed with 5% aq NaHCO3 (2Â40 mL)
and brine (40 mL), and the organic phase was dried over
anhydrous sodium sulfate. The solvent was removed
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