Potent and specific inhibition of human leukocyte elastase, cathepsin G and proteinase 3 by sulfone derivatives employing the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold

Article abstract of DOI:10.1016/S0968-0896(98)00006-6

This paper describes the results of structure-activity relationship studies in a series of heterocyclic mechanism-based inhibitors based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold I and capable of interacting with the S(n) and S(n)' subsites of a serine proteinase. Sulfone derivatives of I were found to be highly effective, time-dependent inhibitors of human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3). The judicious selection of an R₁ group (accommodated at the primary specificity site S₁) that is based on the known substrate specificity of a target serine proteinase, was found to yield highly selective inhibitors. The presence of a benzyl group (R₂=benzyl) at the S₂ subsite was found to lead to a pronounced enhancement in inhibitory potency. Furthermore, the effective use of computer graphics and modeling has led to the design of potent, water-soluble inhibitors. The results of these studies demonstrate that the 1,2,5-thiadiazolidin-3-one 1,1, dioxide platform provides an effective means for appending recognition elements in a well-defined vector relationship, and in fashioning highly-selective and potent inhibitors of serine proteinases. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00006-6

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 661±671
Potent and Speci®c Inhibition of Human Leukocyte Elastase,
Cathepsin G and Proteinase 3 by Sulfone Derivatives
Employing the 1,2,5-Thiadiazolidin-3-one 1,1 Dioxide Sca€old
William C. Groutas,* Rongze Kuang, Sumei Ruan, Je€rey B. Epp,
Radhika Venkataraman and Tien M. Truong
Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
Received 7 August 1997; accepted 30 October 1998
AbstractÐThis paper describes the results of structure±activity relationship studies in a series of heterocyclic mechan-
ism-based0 inhibitors based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide sca€old I and capable of interacting with the
Sn and Sn subsites of a serine proteinase. Sulfone derivatives of I were found to be highly e€ective, time-dependent
inhibitors of human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3). The judicious selection of
an R1 group (accommodated at the primary speci®city site S1) that is based on the known substrate speci®city of a
target serine proteinase, was found to yield highly selective inhibitors. The presence of a benzyl group (R2=benzyl) at
the S2 subsite was found to lead to a pronounced enhancement in inhibitory potency. Furthermore, the e€ective use of
computer graphics and modeling has led to the design of potent, water-soluble inhibitors. The results of these studies
demonstrate that the 1,2,5-thiadiazolidin-3-one 1,1, dioxide platform provides an e€ective means for appending
recognition elements in a well-de®ned vector relationship, and in fashioning highly-selective and potent inhibitors of
serine proteinases. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
recognition elements in a well-de®ned spatial arrange-
ment, thereby optimizing0favorable binding interactions
with both the Sn and Sn subsites. It was also demon-
strated that high or near absolute selectivity for a target
serine proteinase could be achieved through the selec-
tion of an R1 group that is based on the known sub-
strate speci®city of the enzyme.6 We wish to describe
herein the results of structure±activity relationship stu-
dies using sulfone-based derivatives of I that delineate
further the scope and utility of the heterocyclic template
in I in the general design of serine proteinase inhibitors.
The neutrophil-derived serine endopeptidases human
leukocyte elastase (HLE), cathepsin G (Cat G), and
proteinase 3 (PR 3) have been implicated in the etiology
and/or pathophysiology of a range of in¯ammatory
diseases, including pulmonary emphysema,1 bronchitis,2
psoriasis,3 and ischemia-reperfusion injury.4 Selective
inhibitors of these enzymes may be of potential thera-
peutic value for these and related ailments.
We have recently described the structure-based design of
a novel and general class of heterocyclic mechanism-
based inhibitors of serine proteinases based on the 1,2,5-
thiadiazolidin-3-one 1,1 dioxide sca€old I.5,6 Exploratory
biochemical studies have shown that the heterocyclic
platform in I functions as a highly e€ective and versa-
tile sca€old for appending peptidyl or nonpeptidyl
Key words: 1,2,5-Thiadiazolidin-3-one 1,1 dioxide; protease
inhibitors; elastase; cathepsin G; proteinase 3.
*Corresponding author. Tel: (316) 978-3120; Fax: (316) 978-
3431; E-mail: groutas@wsuhub.uc.twsu.edu
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00006-6

662
W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
Results
constant, and A0 is the absorbance at t=0. Fitting the
Aꢀt data into eq (1) using nonlinear regression analysis
(SigmaPlot, Jander Scienti®c) yielded kobs. The second
order rate constants (kinact/Ki M 1 s 1) were determined
by calculating kobs/[I], and then correcting for the sub-
strate concentration and Michaelis constant using eq
(2). Control curves in the absence of inhibitor were lin-
ear.
Chemistry
The reaction sequence shown in Scheme 1 was used in
the synthesis of compounds 1±33. (l)-Leu-OCH3 and (l)
or (d)-Phe-OCH3 were used as the starting materials for
the synthesis of compounds 1±24 and 25±33, respec-
tively. Alkylation of compound 34 with an appropriate
chloromethylalkyl or aryl sul®de, followed by N-5
alkylation and subsequent oxidation with m-chloro-
perbenzoic acid, yielded compounds 1±17 and 24±33.
Treatment of compound 35 with sulfuryl chloride yiel-
ded the corresponding chloromethyl derivative 36,
which was then reacted with the appropriate thiol in the
presence of base. Subsequent oxidation yielded com-
pounds 18±23. The synthesized compounds and their
physical and spectral data are listed in Table 1. For
comparative purposes, a series of hydantoin derivatives
II were also synthesized according to Scheme 2.
kobs
t
A ˆ vst ‡ ꢀv0 vs†ꢀ1
e
†=kobs ‡ A0
ꢀ1†
ꢀ2†
È
É
kobs=‰IŠ ˆ kinact=Ki 1 ‡ ‰SŠ=Km
Molecular modeling
Enzyme±inhibitor modeling studies were carried out
using the Tripos force ®eld of SYBYL, version 6.1a
(Tripos Associates, St Louis, MO) and a Silicon Graph-
ics INDY workstation.
Biochemical studies
The inhibitory activity of compounds 1±33 toward
HLE, Cat G, and PR 3 was determined as described in
Discussion
detail elsewhere.6 Brie¯y, the apparent second-order
1
inactivation rate constants (kinact/Ki
M
s
1) were
Structure±activity relationship studies
determined in duplicate or triplicate by the progress
curve method6,7 and are listed in Table 2. Typical pro-
gress curves for the hydrolysis of MeOSuc-AAPV-pNA
by HLE in the presence of inhibitor 13 are shown in
Figure 1. The release of p-nitroaniline was continuously
monitored at 410 nm. The pseudo ®rst-order rate con-
stants, kobs, for the inhibition of HLE, Cat G, and PR 3
by inhibitor I as a function of time were determined
according to eq (1), where A is the absorbance at
410 nm, v0 is the reaction velocity at t=0, vs is the ®nal
steady-state velocity, kobs is the observed ®rst-order rate
We have recently described the structure-based design of
a novel and general class of mechanism-based inhibitors
of serine proteinases based on the 1,2,5-thiadiazolidin-3-
one 1,1 dioxide sca€old.5,6 The results of speci®city and
modeling studies using the X-ray crystal structure of the
HLE±TOMI (turkey ovomucoid inhibitor) complex
suggested that the binding of I to the active site of HLE
is similar to that of a peptidyl substrate, namely, the R1
group is accommodated at the primary speci®city site
(S1), while R2 and L (SO2R) extend into the S2±Sn and
Scheme 1. Synthesis of derivatives I.

W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
663
Table 1. Physical constants and spectral data of inhibitors 1±33
Compd
Mp (ꢁC)
1H NMR d
MF (Anal.)
1
125.5±126.0
0.94 (m, 9H), 1.39 (m, 2H), 1.56±1.82 (m, 5H), 3.06 (ddd, 1H), 3.33 (ddd, H), 3.89 (t, 1H), 4.88
(s, 2H), 7.56±7.76 (m, 3H), 8.00 (dd, 2H)
C17H26N2O5S2
(C, H, N)
2
145.0±147.0
153.0±154.0
133.0±134.0
167.0±169.0
165.0±167.0
181.0±182.0
136.0±137.0
107.0±108.0
111.0±112.0
140 (dec.)
0.70 (dd, 6H), 1.45±1.70 (m, 3H), 3.84 (t, 1H), 3.93 (s, 3H), 4.28 (d, 1H), 4.57 (d, 1H), 4.89 (s,
2H), 7.42±7.77 (m, 5H), 7.97±8.11 (m, 4H)
C22H26N2O7S2
(C, H, N)
3
0.72 (dd, 6H), 1.50±1.70 (m, 3H), 3.88 (t, 1H), 4.31 (d, 1H) and 4.61 (d, 1H), 4.92 (s, 2H), 7.48±
7.74 (m, 5H), 7.97±8.17 (m, 4H)
C21H24N2O7S2
(C, H, N)
4
0.63 (d, 3H), 0.74 (d, 3H), 1.45±1.70 (m, 3H), 3.82 (t, 1H), 3.94 (s, 3H), 4.27 (d, 1H), 4.57 (d,
1H), 4.89 (s, 2H), 7.47±7.73 (m, 5H), 8.03 (m, 4H)
C22H26N2O7S2
(C, H, N)
5
0.70 (dd, 6H), 1.37±1.70 (m, 3H), 4.18 (t, 1H), 4.42 (d, 1H), 4.71 (d, 1H), 5.06 (d, 2H), 7.52±
7.82 (m, 5H), 8.02 (m, 3H), 8.14 (s, 1H)
C21H24N2O7S2
(C, H, N)
6
0.48 (d, 3H), 0.70 (d, 3H), 1.35±1.62 (m, 3H), 3.93 (s, 3H), 3.98 (t, 1H), 4.39 (d, 1H), 4.90 (s,
2H), 5.17 (d, 1H), 7.40±7.73 (m, 6H), 8.00 (m, 3H)
C22H26N2O7S2
(C, H, N)
7
0.51 (d, 3H), 0.71 (d, 3H), 1.33±1.62 (m, 3H), 4.16 (t, 1H), 4.49 (d, 1H), 5.01 (dd, 2H), 5.22 (d,
1H), 7.48 (m, 1H), 7.58±7.79 (m, 5H), 8.02 (m, 3H)
C21H24N2O7S2
(C, H, N)
8
0.69 (dd, 6H), 1.40±1.70 (m, 3H), 3.65 (s, 2H), 3.94 (t, 1H), 4.41 (dd, 2H), 4.96 (d, 2H), 7.30±
8.05 (m, 9H)
C22H26N2O7S2
(C, H, N)
9
0.92 (dd, 6H), 1.47 (s, 9H), 1.50±1.78 (m, 3H), 3.68 (d, 1H), 4.12 (t, 1H), 4.16 (d, 1H), 4.86 (d,
2H), 7.55±7.75 (m, 3H), 8.03 (d, 2H)
C19H28N2O7S2
(C, H, N)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
0.90 (dd, 6H), 1.50±1.78 (m, 3H), 3.82 (d, 1H), 4.11 (dd, 1H), 4.32 (d, 1H), 4.77 (d, 2H), 5.19
(d, 2H), 7.32±7.45 (m, 5H), 7.52±7.73 (m, 3H), 8.01 (d, 2H)
C22H26N2O7S2
(C, H, N)
(acetone-d6) 0.93 (dd, 6H), 1.55 (m, 1H), 1.67±1.85 (m, 2H), 4.20 (dd, 2H), 4.40 (dd, 1H), 4.98
(dd, 2H), 7.62±8.05 (m, 5H)
0.99 (dd, 6H), 1.70±2.00 (m, 3H), 2.97 (s, 3H), 3.08 (s, 3H), 4.01 (t, 1H), 4.78 (d, 2H)
C15H20N2O7S2
(C, H, N)
oil
C9H19N2O5S2
(C, H, N)
102.0±103.0
102.0±103.0
133.0±134.0
103.0±104.0
118.0±120.0
87.0±88.0
0.93 (dd, 6H), 1.60±1.85 (m, 3H), 2.88 (s, 3H), 3.82 (t, 1H), 4.89 (s, 2H), 7.55 (d, 2H), 7.91 (d,
2H)
C14H19N2O5S2Cl
(C, H, N)
0.69 (dd, 6H), 1.41±1.68 (m, 3H), 3.84 (t, 1H), 4.38 (dd, 2H), 4.88 (s, 2H), 7.38 (m, 5H), 7.53
(d, 2H), 7.90 (d, 2H)
C20H23N2O5S2Cl
(C, H, N)
0.85 (dd, 6H), 1.55±1.80 (m, 3H), 2.81 (s, 3H), 4.75 (t, 1H), 4.82 (s, 2H), 7.50±7.68 (m, 3H),
7.93 (d, 2H)
C14H20N2O5S2
(C, H, N)
0.63 (d, 3H), 0.77 (d, 3H), 1.44±1.70 (m, 3H), 3.85 (t, 1H), 4.28 (d, 1H), 4.50 (d, 1H), 4.90 (s,
2H), 7.30±8.05 (m, 10H)
C22H24N2O5S2
(C, H, N)
0.82 (t, 6H), 1.50±1.80 (m, 3H), 2.97 (t, 2H), 3.31 (m, 1), 3.60 (m, 1H), 3.84 (t, 1H), 4.87 (d,
2H), 7.20±7.37 (m, 5H), 7.57 (t, 2H), 7.71 (t, 1H), 8.01 (d, 2H)
0.99 (dd, 6H), 1.84 (m, 2H), 1.95 (m, 1H), 2.97 (s, 3H), 4.00 (t, 1H), 4.64 (d, 2H), 4.65 (s, 2H),
7.41 (m, 3H), 7.52 (m, 2H)
C21H26N2O5S2
(C, H, N)
C15H22N2O5S2
(C, H, N)
108.0±109.0
135.0±136.0
65.0±67.0
0.99 (dd, 6H), 1.83 (m, 2H), 1.94 (m, 1H), 2.98 (s, 3H), 4.01 (t, 1H), 4.43 (s, 2H), 4.63 (d, 2H),
7.43 (dd, 4H)
C15H21N2O5S2Cl
(C, H, N)
0.69 (d, 3H), 0.80 (d, 3H), 1.62±1.80 (m, 3H), 4.04 (t, 1H), 4.36 (d, 1H), 4.59 (d, 1H), 4.44 (s,
2H), 4.65 (d, 2H), 7.40 (d, 2H), 7.41 (s, 5H), 7.48 (d, 2H)
C21H25N2O5S2Cl
(C, H, N)
0.97 (dd, 6H), 1.80 (m, 2H), 1.91 (m, 1H), 2.21 (m, 2H), 2.78 (t, 2H), 2.93 (s, 3H), 3.15 (m, 2H),
3.95 (t, 1H), 4.71 (d, 2H), 7.15±7.35 (m, 5H)
C17H26N2O5S2
(C, H, N)
77.0±78.0
0.67 (d, 3H) and 0.79 (d, 3H), 1.58±1.78 (m, 3H), 2.22 (m, 2H), 2.78 (t, 2H), 3.13 (m, 2H), 4.00
(t, 1H), 4.31 (d, 1H), 4.54 (d, 1H), 4.71 (dd, 2H), 7.18±7.34 (m, 5H), 7.39 (s, 5H)
0.93 (dd, 6H), 1.58±1.59 (m, 3H), 2.88 (s, 3H), 3.81 (dd, 1H), 4.91 (s, 2H), 7.70 (d, 1H), 7.77 (t,
1H), 8.19 (d, 1H), 8.24 (s, 1H)
C23H30N2O5S2
(C, H, N)
78.0±80.0
C15H19N2O5S2F3
(C, H, N)
102.0±103.0
190.0±191.5
116.0±118.0
133.5±134.5
114.0±115.0
113.0±114.0
124.0±125.0
143.0±144.0
144.0±145.0
118.0±120.0
0.59 (d, 3H), 0.73 (d, 3H), 1.43 (dd, 1H), 1.52±1.70 (m, 2H), 3.86 (dd, 1H), 4.20 (d, 1H), 4.50
(d, 1H), 4.92 (s, 2H), 7.38 (m, 5H), 7.74 (t, 1H), 7.95 (d, 1H), 8.18 (d, 1H), 8.26 (s, 1H)
2.90 (m, 1H), 3.12 (dd, 1H), 4.28 (m, 1H), 4.88 ( s, 2H), 7.20±8.10 (m, 10H), 8.12 (d, 1H)
C21H23N2O5S2F3
(C, H, N)
C16H16N2O5S2
(C, H, N)
2.66 (s, 3H), 3.04 (dd, 1H), 3.19 (dd, 1H), 4.03 (dd, 1H), 4.83 (s, 2H), 7.18±7.98 (m, 10H)
3.00 (m, 2H), 3.91 (d, 1H), 4.04 (dd, 1H), 4.35 (d, 1H), 4.85 (d, 2H), 7.00±8.00 (m, 15H)
3.00 (m, 2H), 3.92 (d, 1H), 4.03 (dd, 1H), 4.35 (d, 1H), 4.85 (d, 2H), 7.00±8.00 (m, 15H)
C17H18N2O5S2
(C, H, N)
C23H22N2O5S2
(C, H, N)
C23H22N2O5S2
(C, H, N)
0.78 (t, 3H), 1.05±1.40 (m, 4H), 2.78 (m, 1H), 3.03 (dd, 1H), 3.16 (m, 2H), 4.07 (dd, 1H), 4.88
(d, 2H), 7.20±7.98 (m, 10H)
C20H24N2O5S2
(C, H, N)
2.99 (dd, 1H), 3.18 (dd, 1H), 3.58 (ddd, 1H), 3.85 (ddd, 1H), 4.21 (dd, 1H), 4.88 (d, 2H), 5.77
(ddd, 1H), 6.30 (d, 1H), 7.20±8.00 (m, 15H)
C25H24N2O5S2
(C, H, N)
1.43 (s, 9H), 2.89 (dd, 2H), 2.97 (d, 1H), 3.11 (dd, 1H), 3.90 (d, 1H),4.30 (dd, 1H), 4.88 (s, 2H),
7.20±7.40 (m, 5H), 7.55±7.80 (m, 3H), 8.00±8.10 (m, 2H)
C
22H26N2O7S2
(C, H, N)
1.42 (s, 9H), 2.89 (dd, 2H), 2.96 (d, 1H), 3.11 (dd, 1H), 3.90 (d, 1H), 4.30 (dd, 1H), 4.87 (s,
2H), 7.20±7.40 (m, 5H), 7.55±7.80 (m, 3H), 8.00±8.10 (m, 2H)
C22H26N2O7S2
(C, H, N)
3.00 (ddd, 2H), 3.63 (s, 2H), 3.92 (d, 1H) and 4.29 (d, 1H), 4.02 (dd, 1H), 4.85 (dd, 2H), 6.98
(d, 2H) and 7.15 (d, 2H), 7.08 (m, 2H), 7.36 (m, 3H), 7.54 (m, 2H), 7.68 (m, 1H), 7.93 (d, 2H)
C25H24N2O7S2
(C, H, N)

664
W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
Scheme 2. Synthesis of hydantion derivatives II.
Table 2. Inhibitory activity of derivatives of I toward human leukocyte elastase, proteinase 3, and cathepsin G
1b
Compda
R1
R2
L
kinact/KI M 1 s
HLE
8230
PR3
Cat G
1
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
isobutyl
benzyl
n-butyl
(p-COOCH3)Bzl
(p-COOH)Bzl
(m-COOCH3)Bzl
(m-COOH)Bzl
(o-COOH3)Bzl
(o-COOH)Bzl
(p-CH2COOH)Bzl
CH2COO-t-Bu
CH2COO-Bzl
CH2COOH
methyl
SO2Ph
SO2Ph
SO2Ph
430
1280
840
inactive
790
70
2
67,800
7800
3
4
SO2Ph
SO2Ph
63,600
38,700
25,800
7690
7080
10,300
2440
1050
1770
1830
Ðc
80
350
5
6
SO2Ph
SO2Ph
60
150
7
8
SO2Ph
SO2PH
26,700
2020
1710
290
30
9
10
11
12
13
14
15d
16d
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
SO2Ph
SO2Ph
SO2CH3
inactive
inactive
inactive
inactive
180
1050
4590
310
370
methyl
benzyl
SO2(p-Cl phenyl)
SO2(p-Cl phenyl
SO2Ph
32,200
219,000
9490
6280
16,200
2250
5240
Ð
methyl
benzyl
inactive
110
SO2Ph
SO2Ph
95,200
6590
CH2CH2Ph
methyl
methyl
inactive
100
160
SO2CH2Ph
SO2CH2(p-Cl phenyl)
SO2CH2(p-Cl phenyl)
SO2(CH2)3Ph
SO2(CH2)3Ph
SO2(m-CF3 phenyl)
SO2(m-CF3 phenyl)
SO2Ph
22,300
47,500
165,000
15,400
9990
9580
16,900
20,300
8020
3250
4780
2250
Ð
benzyl
methyl
70
20
benzyl
methyl
benzyl
60
38,200
240,000
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
30
H
methyl
benzyl
benzyl
SO2Ph
SO2Ph
Ð
Ð
120
benzyl
benzyl
n-butyl
11,200
1580
320
(d) benzyl
benzyl
benzyl
SO2Ph
SO2Ph
Ð
Ð
t-cinnamyl
CH2COO-t-Bu
CH2COO-t-Bu
(p-CH2COOH)Bzl
SO2Ph
SO2Ph
Ð
Ð
760
benzyl
(d) benzyl
benzyl
1130
280
SO2Ph
SO2Ph
Ð
inactive
3760
aAll compounds have the l-con®guration, unless indicated otherwise.
bReproducibility: ‹10%.
cNot determined.
dReported in 6 and listed here for comparative purposes.

W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
665
inhibiting HLE (Table 2), re¯ecting the known pre-
ference of HLE for P1 residues with small hydrophobic
alkyl chains (Val, Leu), but showed little or no activity
toward Cat G, a chymotrypsin-like proteinase. Con-
versely, compounds derived from (l)-Phe were found to
inhibit Cat G, but not HLE or PR 3 (Table 2, com-
pounds 25±33). These results indicate that the selection
of an R1 group (corresponding to the side chain of the
P1 residue) that is based on the known substrate speci-
®city of a serine proteinase results in both high potency
and enzyme selectivity.6 (2) Compounds that inhibited
HLE also inhibited PR 3, albeit with lower eciency.
The primary structure of HLE shows considerable
homology with PR 3 (54%), however, the active site of
PR 3 is more constricted due to the substitution of Val-
190 with Ile, which reduces the size of the S1 pocket, and
is smaller than that of HLE.9±11 Nevertheless, because
the active sites of the two enzymes are similar, leucine-
derived compounds were found to inhibit both HLE
and PR 3. (3) Previous in vivo studies with peptidyl and
nonpeptidyl tri¯uoromethylketones,12 b-lactam,13 and
saccharin14 derivatives have shown that, despite their
high inhibitory potency, highly lipophilic inhibitors lack
oral bioavailability, an observation ascribed to inap-
propriate physicochemical properties, namely, poor
absorption and protein binding. However, oral bio-
availability could be achieved by introducing acidic or
basic polar functionalities. As a prelude toward the
design of orally bioavailable inhibitors of HLE based on
I, inhibitors with a polar functionality (Table 2, com-
pounds 3, 5, 7, 8, and 11) were synthesized and shown to
be ecient inhibitors of the enzyme. Although the
potency of these compounds was lower than that of the
more lipophilic precursor esters (Table 2, compounds 2,
4, 6, 10), nevertheless these compounds had high
potency. In the case of compound 8, where the P2 resi-
due consists of a (p-carboxymethyl)benzyl group at N-5,
molecular modeling was used to predict the preferred
location of the polar functionality. The modeling studies
suggested that the carboxyl group in 8 is oriented away
from the hydrophobic surface of the enzyme and toward
the aqueous milieu, while the P2 residue is nestled into
the hydrophobic S2 subsite of the enzyme, accounting
for its high potency. (4) With respect to the nature of the
P2 residue, a benzyl group is preferred over an alkyl
group (Table 2, compounds 15 versus 16, 19 versus 20,
and 23 and 24). Interestingly, and for reasons that are
not intuitively obvious at the moment, the stability of
the acyl enzyme(s) formed by the interaction of I with a
serine proteinase is primarily controlled by the nature of
the R2 group, with small alkyl P2 residues such as
methyl, for example, giving rise to acyl enzymes that
deacylate extremely slowly.6 These observations suggest
that R2 can serve as an exquisite means of controlling
the stability, and hence the rate of reactivation, of the
acyl enzyme(s) formed from I. This distinct feature of I
Figure 1. Progress curves for the inhibition of human leukocyte
elastase (HLE) by compound 13. Absorbance was recorded at
410 nm for reaction solutions containing 21.2 nM HLE, 1 mM
MeOSuc-AAPV-pNA, and the indicated concentrations of
inhibitor in 0.1 M HEPES bu€er, 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.
0
Sn subsites.8 Thus, it was anticipated that appropriate
derivatives of I capable of exploiting favorable binding
0
interactions with both the Sn and Sn subsites would
function as highly selective and e€ective inhibitors of a
target serine proteinase. In order to gain further insight
into the interaction of I with serine proteinases, as well
as optimize inhibitory activity and selectivity, a series of
sulfone derivatives was obtained (Scheme 1) by varying
the nature of R1, R2, and L (Table 1), and their inhibi-
tory activity toward HLE, Cat G, and PR 3 determined.
The inactivation of HLE by a representative member of
this class (compound 13, Table 2) was found to be rapid
and time-dependent. Furthermore, the progress curves
also indicate that the interaction of 13 with HLE
involves the active site (Figure 1). These inhibitors inacti-
vated HLE with high eciency, that is, their partition
ratios were close to zero (unpublished observations).
The apparent Ki* (de®ned herein as Ki*=kreact/kinact*,
where kreact is the ®rst-order reactivation constant and
kinact* is the apparent second-order inactivation con-
stant, kinact/Ki) for these inhibitors were in the nM range.
Based on the results summarized in Table 2, the follow-
ing inferences can be made:
Nature of R1 and R2. (1) Derivatives of I are potent
inhibitors of HLE, PR 3, and Cat G. Compounds
derived from (l)-Leu were found to be highly e€ective in

666
W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
0
could, in principle, be used as a sensitive `switch' in
regulating the recovery of enzymatic activity and, hence,
the duration of action, of a potential medicinal agent.
group occupying the S2 subsite, re¯ecting fairly good
structural and electronic complementarity (Figure 2(b)).
Increasing the hydrophobic character of the sulfone
leaving group was found to augment hydrophobic
interactions with the Sn subsites, yielding compounds
Nature of the leaving group. (1) As mentioned earlier,
the binding of I to the active site orients the leaving
group toward the hydrophobic Sn subsites. The results
0
0
with higher inhibitory potency (Table 2, compounds 13/
15, 18/19, 15/23). Potency was also found to be sensitive
to the pKa of the leaving group (vide infra). (2) In gen-
eral, sulfone-based inhibitors of Cat G were less potent
than the corresponding HLE inhibitors (Table 2, com-
pounds 25±33). The best inhibitors of Cat G were those
with R1=R2=benzyl (Table 2, compound 27). (3) The
corresponding sulfoxides also inhibited HLE. For
example, the diastereomeric sulfoxides corresponding to
compound 15 inactivated HLE with kinact/Ki values of
1,110 and 1390 M 1 s 1. In contrast, the inactivation of
HLE by the corresponding sul®des was found to be
slow. For example, incubation of a 200-fold excess of
the sul®de corresponding to compound 16 with HLE led
to time-dependent inactivation of the enzyme with
listed in Table 2 suggest that both the pKa and inherent
structure of L exert a profound e€ect on inhibitory
potency. As a design element, the inherent structure of L
can be used to optimize binding anity. For example,
molecular modeling was used to gain insight into the
binding of compound 20 to the active site of HLE and
to optimize the hydrophobic binding interaction of the
phenyl ring (in L) in the inhibitor and a phenylalanine
0
residue (Phe-41) located in the vicinity of the S2 subsite
(Figure 2(a)). The overlay of energy-minimized inhibitor
0
20 and the P2-P2 segment (-Thr-Leu-Glu-Tyr-) of
TOMI shows the isobutyl (P1) and benzyl (P2) groups in
the inhibitor nestled into the corresponding S1 and S2
enzyme subsites, with the phenyl ring of the leaving
(a)
(b)
Figure 2. (a) Energy-minimized inhibitor 20 (Table 1) docked to active site of human leukocyte elastase (HLE) with the isobutyl group
occupying the S1 pocket. The benzyl group is nestled into the hydrophobic S2 pocket, and the N-(4-chlorobenzyl)sulfonyl leaving
0
0
group extends into the hydrophobic S2 pocket, providing speci®c lipophilic interactions. (b) Overlay of inhibitor 20 and the P2-P2

W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
667
minimal recovery of enzymatic activity after 24 h (Fig-
ure 3).
It is tentatively proposed that, unlike sulfone derivatives
of I that inactivate a serine proteinase via a mechanism
involving the formation of a highly reactive sulfonyl
imine that partitions via two possible pathways6 (Figure
4(a)), sul®de derivatives lead to acylation of the enzyme
without loss of the leaving group (Figure 4(b)). De®ni-
tive resolution of the mechanistic issues will have to
await the results of ongoing studies.
Nature of the template. Exploratory attempts to gain
insight into the nature of the interaction of I with serine
proteinases, included the synthesis and in vitro evalua-
tion of a series of hydantoin derivatives II. None of the
hydantoin derivatives displayed any inhibitory activity
toward HLE, Cat G, or PR 3, suggesting that the man-
ifestation of time-dependent inhibitory activity involves
a delicate interplay between binding interactions, che-
mical reactivity, and leaving group ability.
use of an array of recognition elements appended in a
well-de®ned spatial arrangement.
Experimental
Melting points were recorded on a Mel-Temp apparatus
and are uncorrected. The NMR spectra of the synthe-
sized 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 Co., Owensville, MO,
USA. Human leukocyte cathepsin G and proteinase 3
were purchased from Athens Research and Technology
Co., Athens, GA, USA. Methoxysuccinyl Ala-Ala-Pro-
Val p-nitroanilide and methoxysuccinyl Ala-Ala-Pro-
Phe p-nitroanilide were purchased from Sigma Chemi-
cals Co., St Louis, MO, USA.
In summary, the results presented herein describe (a) the
ecient inhibition of HLE, Cat G, and PR 3 by sulfone
derivatives of I and (b) demonstrate that the hetero-
cyclic sca€old in I has built-in ¯exibility that permits the
optimization of inhibitory potency, speci®city and the
stability of the enzyme±inhibitor complex(es) via the
Representative syntheses
(S)-4-Isobutyl-2-[(phenylthio)methyl]-1,2,5-thiadiazolidin-
3-one 1,1 dioxide (35, R1=isobutyl, R2=H, Ar = Ph).
A solution of (S)-4-isobutyl-1,2,5-thiadiazolidin-3-one
1,1 dioxide6 (1.92 g, 10 mmol), chloromethyl phenyl sul-
®de (1.80 g, 12 mmol), and triethylamine (1.01 g,
10 mmol) in 15 mL of dry acetonitrile was re¯uxed for
20 h. The solvent was removed by in vacuo, and the
residue was taken up in 30 mL of ethyl acetate, washed
with water, 5% aq HCl (10 mL), 5% aq sodium
bicarbonate, and dried over anhydrous sodium sulfate.
Evaporation of the solvent left a crude product which
was puri®ed by ¯ash chromatography on silica gel,
using a hexane/methylene chloride gradient, mp 48±
49 ꢁC (1.64 g, 52% yield).6
Figure 3. Percent remaining activity versus time plot con-
structed by incubating a 200-fold excess of (l)-4-isobutyl-2-
(phenylthiomethyl)-5-benzyl 1,2,5-thiadiazoli-din-3-one 1,1 di-
oxide (2.76 nM) with HLE (13.8 mM) in 0.1 M HEPES bu€er,
pH 7.25 at 25 ꢁC. Aliquots were withdrawn at di€erent time
intervals and assayed for enzymatic activity using MeOSuc-
AAPV-pNA (4.25 mM) by monitoring the absorbance at
(S)-4-Isobutyl-5-benzyl-2-[(phenylthio)methyl]-1,2,5-thia-
diazolidin-3-one 1,1 dioxide (35, R1=isobutyl, R2=
benzyl, Ar=Ph). Benzyl bromide (14.0 g, 82.0 mmol)
was added to a solution of compound (S)-4-isobutyl-

668
W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
Figure 4. (a) Postulated mechanism of action of sulfone derivatives of I involving enzyme-induced formation of an N-sulfonyl imine
and concomitant loss of the leaving group. (b) Interaction of a sul®de derivative of I whereby nucleophilic ring opening leads to the
formation of an acyl enzyme without loss of the leaving group.
2-[(phenylthio)methyl]-1,2,5-thiadiazolidin-3-one 1,1 di-
oxide (23.4 g, 74.4 mmol) in dry acetonitrile (150 mL).
The solution was cooled in an ice bath, and 60% NaH
(3.0 g, 74.9 mmol) was added in small portions over a
period of 5 min. The reaction mixture was stirred at
room temperature overnight, and the solvent and excess
benzyl bromide were removed in vacuo. The residue was
dissolved in methylene chloride (100 mL) and washed
with water. The organic layer was dried over anhydrous
sodium sulfate. Removal of the solvent left a crude
product, which was puri®ed using ¯ash chromatography
(90% yield). 1H NMR (CDCl3) d 0.69 (dd,6H), 1.48
(m,1H), 1.62 (m,2H), 3.77 (t,1H), 4.23 (d,1H), 4.45
(d,1H), 4.97 (d,2H), 7.30±7.65 (m,10H); 13C NMR d
22.05, 22.28, 24.28, 39.75, 45.43, 52.10, 62.63, 128.40,
128.80, 128.96, 129.08, 129.18, 132.39, 133.09, 133.44,
167.08.
(S)-4-Isobutyl-5-benzyl-2-chloromethyl-1,2,5-thiadiazo-
lidin-3-one 1,1 dioxide (36, R1=isobutyl, R2=benzyl).
(S)-4-Isobutyl-2- [(phenylthio)methyl]-1,2,5-thiadiazo-
lidin-3-one 1,1 dioxide (31.2 g, 74 mmol) was treated with
sulfuryl chloride (20 g, 148 mmol) in 50 mL dry CH2Cl2
and the reaction mixture was stirred for 5 h at room
temperature. The solvent and excess sulfuryl chloride
were removed in vacuo. The crude product was puri®ed
by ¯ash chromatography (silica gel/hexane/methylene
chloride as eluents), yielding 16.7 g (68% yield) of pure
product. 1H NMR (CDCl3) d 0.67 (d, 3H), 0.78 (d, 3H),
1.55±1.80 (m, 3H), 3.92 (t, 1H), 4.28 (d, 1H), 4.53 (d,

W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
669
1H), 5.36 (d, 2H), 7.38 (s, 5H). 13C NMR (CDCl3) d
21.92, 22.27, 24.27, 39.61, 46.53, 52.51, 62.97, 128.98,
129.04, 29.23, 133.02, 166.67.
7.46 (t, 1H), 7.58 (m, 3H), 8.02 (m, 2H). 13C NMR
(CDCl3) d 22.02, 22.22, 24.25, 39.61, 45.46, 51.78, 52.29,
63.01, 128.47, 129.07, 129.17, 129.96, 130.07, 130.84,
132.20, 133.14, 133.43, 134.08, 166.31, 166.76.
(S)-4-Isobutyl-2-[(p-chlorobenzylthio)methyl]-5-benzyl-
1,2,5-thiadiazolidin-3-one 1,1 dioxide (I, R1=isobutyl,
R2=benzyl, L=(p-chlrobenzylthio)methyl). A solution
of (S)-4-isobutyl-5-benzyl-2-chloromethyl-1,2,5-thiadi-
azolidin-3-one 1,1 dioxide (0.99 g, 3 mmol) in 10 mL dry
acetonitrile was reacted with 4-chlorobenzylthiol (0.48 g,
3 mmol) in the presence of 1,8-diazabicyclo[5.4.0]un-
decen-7-ene (0.46 g, 3 mmol) at room temperature over-
night. The solvent was removed on a rotary evaporator
and the residue was taken up in methylene chloride
(30 mL) and washed with 5% HCl (20 mL), 5%
NaHCO3 and brine. The organic solution was dried
over anhydrous sodium sulfate and evaporated, leaving
a crude product, which was puri®ed by ¯ash chromato-
graphy using silica gel (hexane/methylene chloride).
Pure compound 39 was obtained in 84% yield (1.14 g).
1H NMR (CDCl3) d 0.70 (d, 3H), 0.80 (d, 3H), 1.53±
1.81 (m, 3H), 3.87 (t, 1H), 3.91 (s, 2H), 4.28 (d, 1H),
4.52 (d, 1H), 4.56 (d, 2H), 7.33 (dd, 4H), 7.39 (s, 5H).
(S)-4-Isobutyl-5-[(m-carboxymethyl)benzyl]-2-[(phenylsulf-
onyl)methyl]-1,2,5-thidiazolidin-3-one 1,1 dioxide (4). A
solution of (S)-isobutyl-5-[(m-carboxymethyl)benzyl]-2-
phenylthio)methyl]-1,2,5-thiadiazolidin-3-one 1,1 diox-
ide (0.87 g, 1.88 mmol) in 10 mL dry CH2Cl2 was reacted
with 70% m-chloroperbenzoic acid (1.1 g, 6.38 mmol) at
room temperature overnight. Methylene chloride
(20 mL) was added and the organic phase was washed
with saturated NaHCO3 (25 mL) and dried over anhy-
drous Na2SO4. The solvent was removed in vacuo, and
the crude product was puri®ed using ¯ash chromato-
graphy (silica gel/hexane/CH2Cl2), yielding 0.74 g (80%
yield) of pure product. 1H NMR (CDCl3) d 0.63 (d,
3H), 0.74 (d, 3H), 1.45±1.70 (m, 3H), 3.82 (t, 1H), 3.94
(s, 3H), 4.27 (d, 1H), 4.57 (d, 1H), 4.89 (s, 2H), 7.47±
7.73 (m, 5H), 8.03 (m, 4H).
(S)-4-Isobutyl-5-[(m-carboxyl)benzyl]-2-[(phenylsulfonyl)
methyl)]-1,2,5-thiadiazolidin-3-one 1,1 dioxide (5). A
mixture of the above ester (0.62 g, 1.25 mmol), iodo-
trimethylsilane (0.31 g, 1.50 mmol), and iodine (32 mg)
in dry CDCl3 (5 mL) was kept at 55 ꢁC for 24 h. NMR
analysis showed the presence of starting material. An
additional 0.1 g of iodotrimethylsilane was added and
the mixture kept at 55 ꢁC for an additional 24 h. The
solvent was removed in vacuo and ethyl acetate (25 mL)
was added to the residue. The organic layer was washed
with 5% HCl, 10% aq sodium thiosul®te and water
(20 mL). It was dried over anhydrous sodium sulfate
and then evaporated leaving a crude product that was
puri®ed by ¯ash chromatography (silica gel/hexane/
ethyl acetate/HOAC), yielding 0.33 g (59% yield) of
pure 5. 1H NMR (acetone-d6) d 0.70 (dd, 6H), 1.37±1.70
(m, 3H), 4.18 (t, 1H), 4.42 (d, 1H), 4.71 (d, 1H), 5.06 (d,
2H), 7.52±7.82 (m, 5H), 8.02 (m, 3H), 8.14 (s, 1H).
(S)-4-Isobutyl-N-[(4-chlorobenzylsulfonyl)methyl]-5-ben-
zyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide (19). A solu-
tion of (S)-4-isobutyl-2-[(p-chlorobenzylthio)methyl]-5-
benzyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide (0.47 g,
1 mmol) in 3 mL dry CH2Cl2 was reacted with 70%
m-chloroperbenzoic acid (0.62 g, 3.60 mmol) at room
temperature overnight. Methylene chloride (20 mL) was
added and the organic phase was washed with saturated
sodium bicarbonate (25 mL) and dried over anhydrous
sodium sulfate. The solvent was removed in vacuo and
the crude product puri®ed by ¯ash chromatography
(silica gel/hexane/methylene chloride), yielding 0.46 g
(98% yield) of pure product.
(S)-4-Isobutyl-5-[(m-carboxymethyl)benzyl]-2-[(phenylthio)
methyl]-1,2,5-thiadiazolidin-3-one 1,1 dioxide (35, R1=
isobutyl, R2=m-(carboxymethyl)benzyl, Ar=Ph).
A
solution of (S)-4-isobutyl-5-benzyl-2-[(phenylthio)
methyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide (0.94 g,
3 mmol) and m-(carboxymethyl)benzyl bromide (0.69 g,
3 mmol) in dry acetonitrile (10 mL) was cooled to 0 ꢁC
and treated with 60% sodium hydride (0.12 g, 3 mmol).
The mixture was stirred at room temperature for 1 h.
The solvent was removed in vacuo and the residue was
taken up in ethyl acetate (35 mL), and washed with 5%
hydrochloric acid and brine. The organic layer was
dried over anhydrous sodium sulfate and evaporated.
The crude product was puri®ed using ¯ash chroma-
tography (silica gel/hexane/CH2Cl2), yielding 0.97 g
Synthesis of hydantoin derivatives
(DL)-5-Isobutylhydantoin (37, R1=isobutyl). (dl)-Leu
hydrochloride (50 mmol) and potassium cyanate
(60 mmol) were added to a solution of water (30 mL)
and pyridine (25 mL). The solution was heated to 60 ꢁC
for 1 h, cooled to room temperature, and the pyridine
extracted with ethyl acetate (4Â100 mL). Glacial acetic
(30 mL) and 6 N HCl (30 mL) were added to the glacial
phase and the solution was re¯uxed for 0.5 h. The solu-
tion was kept at 5 ꢁC overnight. The precipitated prod-
uct was collected, washed with water, and air-dried
(6.3 g, 80% yield), mp 215±216 ꢁC. NMR(DMSO-d6) d
0.91 (dd,6H), 1.35±1.55 (m,2H), 1.78 (br m, 1H), 4.0
(m,1H), 8.02 (br s, 1H), 10.59 (br s,1H).
1
(70% yield) of pure product. H NMR (CDCl3) d 0.69
(dd, 6H), 1.45±1.70 (m, 3H), 3.78 (t, 1H), 3.93 (s, 3H),
4.22 (d, 1H), 4.52 (d, 1H), 4.98 (d, 2H), 7.32 (m, 3H),

670
W. C. Groutas et al./Bioorg. Med. Chem. 6 (1998) 661±671
(DL)-5-Isobutyl-3-(phenylthiomethyl)hydantoin (38, R1=
isobutyl). (dl)-5-Isobutylhydantoin (15 mmol) was dis-
solved in dry DMF (20 mL) under nitrogen and 60%
sodium hydride (15 mmol) was added at room tempera-
ture. Chloromethylphenyl sul®de (16 mmol) was then
added and the resulting solution heated to 80 ꢁC for
2.5 h. The reaction mixture was then cooled to room
temperature and diluted with water. The crystallized
product was collected by suction ®ltration, dissolved in
ethyl ether (100 mL), and extracted with saturated
sodium chloride (30 mL). The organic phase was dried
over anhydrous sodium sulfate and the solvent
removed. The crude product was washed with hexane,
leaving a pure product (1.76 g, 42% yield), mp 115±
116 ꢁC. NMR(CDCl3) d 0.94 (dd,6H), 1.45 (m,1H), 1.7
(m,2H), 4.0 (dd,1H), 4.86 (dd,2H), 6.68 (br s, 1H), 7.3
(m,3H), 7.53 (m,2H).
(DL)-5-Benzylhydantoin (40, R1=benzyl). This was syn-
thesized from (dl)-Phe using a procedure similar to
that used in the preparation of (dl)-5-isobutylhydantoin
1H NMR(DMSO-d6) d 2.94 (dd,2H), 4.36 (m,1H), 7.23
(m,5H), 7.96 (br s,1H), 10.43 (br s, 1H).
(DL)-5-Benzyl-3-(phenylthiomethyl)hydantoin (38, R1=
benzyl). This was prepared from (dl)-5-benzylhydan-
toin using a procedure similar to that used in the synth-
esis of (dl)-5-isobutyl-3-(phenylthiomethyl)-1-benzyl-
hydantoin, mp 130±131 ꢁC. 1H NMR(CDCl3) d 2.71
(dd,1H), 3.2 (dd,1H), 4.16 (dd,1H), 4.81 (s,2H), 5.69
(s,1H), 7.16 (m,2H), 7.30 (m,6H), 7.48 (m,2H).
(DL)-5-Benzyl-3-(phenylsulfonylmethyl)hydantoin (40,
R1=benzyl). This was prepared from (dl)-5-benzyl-3-
(phenylthiomethyl)hydantoin using a similar procedure
as that used in the synthesis of (dl)-5-isobutyl-3-(phe-
nylsulfonylmethyl)-1-benzylhydantoin, mp 136±138 ꢁC.
1H NMR (CDCl3) d 2.88 (dd, 1H), 3.2 (dd, 1H), 4.31
(m, 1H), 4.78 (d, 2H), 6.27 (s, 1H), 7.17±7.40 (m, 6H),
7.52±7.86 (m, 4H).
(DL)-5-Isobutyl-3-(phenylsulfonylmethyl)hydantoin (40,
R1=isobutyl). Oxidation of (dl)-5-isobutyl-3-(phenyl-
thiomethyl)hydantoin with 70% m-chloroperbenzoic
acid using a procedure similar to that used in the pre-
paration of compound 19 yielded the desired product.
(DL)-5-Benzyl-3-(phenylthiomethyl)-1-benzylhydantoin
(39, R1=benzyl, n=0). Synthesized as described above.
1H NMR (CDCl3) d 2.99 (dd, 1H), 3.11 (dd, 1H), 3.85
(d, 1H), 3.91 (dd, 1H), 4.77 (dd, 2H), 5.05 (d, 1H), 6.98
(m, 2H), 7.08 (m, 2H), 7.21±7.31 (m, 9H), 7.37 (m, 2H).
(DL)-5-Isobutyl-3-(phenylthiomethyl)-1-benzylhydantoin
(39, R1=isobutyl, n=0). A solution of (dl)-5-isobutyl-3-
(phenylthiomethyl)hydantoin (2 mmol) in dry acetonitrile
(6 mL) was treated with 60% sodium hydride (2 mmol)
at room temperature under nitrogen with stirring. Ben-
zyl bromide (2.2 mmol) was then added and the reaction
mixture stirred overnight. Following removal of the
solvent on the rotary evaporator, methylene chloride
(35 mL) was added and the solution extracted with
saturated sodium bicarbonate (10mL). The organic phase
was dried over anhydrous sodium sulfate and evaporated,
leaving a crude product that was puri®ed on a chromato-
tron plate using methylene chloride as the eluent (67%
(DL)-5-Benzyl-3-(phenylsulfonylmethyl)-1-benzylhydantoin
(39, R1=benzyl, n=2). Obtained from (dl)-5-benzyl-3-
(phenylthiomethyl)-1-benzylhydantoin by oxidation
with 70% m-chloroperbenzoic acid. 1H NMR (CDCl3) d
3.08 (dd, 1H), 3.17 (dd, 1H), 3.88 (d, 1H), 4.03 (t, 1H),
4.78 (dd, 2H), 4.99 (d, 1H), 7.0 (m, 2H), 7.12 (m, 2H),
7.33 (m, 7H), 7.49 (m, 2H), 7.67 (m, 2H).
1
yield). H NMR (CDCl3) d 0.83 (dd,6H), 1.62 (m,2H),
1.78 (m,1H), 3.69 (dd,1H), 4.01 (d,1H), 4.92 (dd,2H), 5.08
(d,1H), 7.19 (m,2H), 7.29±7.39 (m,6H), 7.55 (m,2H).
Acknowledgements
Financial support of this research by Sparta Pharma-
ceuticals, Inc. and (in part) the National Institutes of
Health (HL 57788) is gratefully acknowledged.
(DL)-5-Isobutyl-3-(phenylsulfonylmethyl)-1-benzylhydan-
toin (39, R1=isobutyl, n=2). m-Chloroperbenzoic acid
(2 mmol) was added to a solution of (dl)-5-isobutyl-3-
(phenylthiomethyl)-1-benzylhydantoin (1 mmol) in 5 mL
methylene chloride. The reaction mixture was stirred at
room temperature overnight. An additional amount of
methylene chloride (35 mL) was added and the solution
was extracted with 5% aq sodium sul®te (10 mL) and
5% NaHCO3 (10 mL). The organic phase was dried and
evaporated, leaving a crude product that was puri®ed
on a chromatotron plate, yielding an oily product (22%
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1
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