Utilization of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold in the design of potent inhibitors of serine proteases: SAR studies using carboxylates

Bioorganic & Medicinal Chemistry 8 (2000) 1005±1016

Utilization of the 1,2,5-Thiadiazolidin-3-one 1,1 Dioxide Scaold

in the Design of Potent Inhibitors of Serine Proteases: SAR

Studies Using Carboxylates

Rongze Kuang, Jerey B. Epp, Sumei Ruan, Lee S. Chong, Radhika Venkataraman,

Juan Tu, Shu He, Tien M. Truong and William C. Groutas*

Department of Chemistry, Wichita State University, Wichita, KS 67260, USA

Received 20 September 1999; accepted 14 December 1999

AbstractÐA series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1

dioxide scaolds has been synthesized and the inhibitory pro®le of these compounds toward human leukocyte elastase (HLE),

cathepsin G (Cat G) and proteinase 3 (PR 3) was then determined. Most of the compounds were found to be potent, time-depen-

s

¹. The inhibitory

1

dent inhibitors of elastase, with some of the compounds exhibiting k_inact/K_Ivalues as high as 4,928,300 M

potency of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide platform was found to be in¯uenced by both

the pK_aand the inherent structure of the leaving group. Proper selection of the primary speci®city group (R₁) was found to lead to

selective inhibition of HLE over Cat G, however, those compounds that inhibited HLE also inhibited PR 3, albeit less eciently.

The predictable mode of binding of these compounds suggests that, among closely-related serine proteases, highly selective inhibi-

tors of a particular serine protease can be fashioned by exploiting subtle dierences in their S⁰subsites. This study has also

demonstrated that the degradative action of elastase on elastin can be abrogated in the presence of inhibitor 17. # 2000 Elsevier

Introduction

tion-phase construction of the ®rst libraries of mechan-

ism-based inhibitors of serine proteases based on this

template and, more importantly, have obtained pre-

liminary evidence in support of the notion that the

structural motif embodied in structure (I) renders the

platform capable of binding to the active site of many

serine proteases with a (chymo)trypsin-like fold.^17,18We

wish to describe herein the results of SAR studies with

carboxylate derivatives of the 1,2,5-thiadiazolidin-3-one

1,1 dioxide (I) and a closely related scaold iso-

thiazolidin-3-one 1,1 dioxide (II) as inhibitors of HLE,

Cat G, and PR 3. The rationale leading to the design of

these two inhibitor scaolds is summarized in Figure 1.

In vitro biochemical studies demonstrating the ability of

a representative member of this class of inhibitors to block

the degradation of elastin by human leukocyte elastase are

also described.

The in¯ux of activated neutrophils and other phagocytic

cells to in¯ammatory sites is associated with the extra-

cellular release of a range of proteolytic enzymes,

including the serine endopeptidases human leukocyte

elastase (HLE), cathepsin G (Cat G) and proteinase 3

(PR 3).^{1 3}Poor regulation of the activity of these

enzymes by endogenous serpins (serine proteinase inhi-

bitors) leads to the degradation of the major compo-

nents of the extracellular matrix.^{4 6}Thus, considerable

eorts have been devoted to the design of novel agents

capable of redressing the protease/antiprotease imbal-

ance^7-9via the selective inhibition of these enzymes.^{10 12}

We have recently described the structure-based design

of a highly versatile heterocyclic scaold (1,2,5-thiadia-

zolidin-3-one 1,1 dioxide, I) and have shown that potent

mechanism-based inhibitors of the aforementioned

enzymes can be realized by embellishing the scaold

with appropriate recognition and chemical reactivity

elements.^{13 16}We have furthermore reported the solu-

Results

Chemistry

Carboxylate derivatives 1±39 were synthesized accord-

ing to Scheme 1.

*Corresponding author. Tel.: +1-316-978-3120; fax: +1-316-978-

3431; e-mail: groutas@wsuhub.uc.twsu.edu

PII: S0968-0896(00)00038-9

1006

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

which was elaborated further to yield the desired com-

pounds (Table 2).

Biochemical studies

Progress curve method.²²The inhibitory activity of

compounds 1±44 toward HLE, Cat G and PR 3 was

determined using the progress curve method. The

apparent second-order inactivation rate constants

(k_inact/K_IM ¹s ¹) were determined in duplicate and are

listed in Table 3. Typical progress curves for the hydro-

lysis of MeOSuc-AAPV-pNA by HLE in the presence

of inhibitor 14 are shown in Figure 2. The release of p-

nitroaniline was continuously monitored at 410 nm. The

pseudo ®rst-order rate constants (k_obs) for the inhibition

of HLE, Cat G and PR 3 by derivatives of (I±II) as a

function of time were determined according to eq (1),

where A is the absorbance at 410 nm, v_ois the reaction

velocity at t=0, v_sis the ®nal steady-state velocity, k_obs

is the observed ®rst-order rate constant, and A_ois the

absorbance at t=0. The k_obsvalues were obtained by

®tting the Aꢀt data into eq (1) using nonlinear regres-

sion analysis (SigmaPlot, Jandel Scienti®c). The second

1

order rate constants (k_inact/K_I

M

s

¹) were then

determined by calculating k_obs/[I], and then correcting

for the substrate concentration using eq 2. Control

curves in the absence of inhibitor were linear.

È

ÁÉ

k_obs

t

A v_st ꢀv_ov_s 1

e

=k_obs A_o

ꢀ1

ꢀ2

k_inact=K_I ꢀk_obs=Iꢀ1 S=K_m

Incubation method.²³The inhibitory activity of com-

pounds 43±50 was determined by the incubation

method and is expressed in terms of the bimolecular rate

Figure 1. Design of templates (I) and (II).

1

constant k_obs=I M

s

¹. Brie¯y, in this method the

enzyme is incubated with excess inhibitor, and the loss

of enzymatic activity is followed by withdrawing ali-

quots at dierent time intervals and assaying for enzy-

matic activity. The observed rate constant (k_obs) is then

calculated according to eq 3, where [I] is the concentra-

tion of the inhibitor in the incubation mixture and

Intermediate A (Scheme 1) was readily obtained via

treatment of the appropriate amino acid ester with sul-

famoyl chloride followed by cyclization with sodium

hydride, while the synthesis of intermediate B involved

an initial reductive alkylation of an amino acid ester

precursor prior to the sulfamoylation and cyclization

steps. Sequential alkylation of A yielded a series of

inhibitors represented by structures C and D. An alter-

native procedure for the synthesis of C and D involved

alkylation of A followed by treatment with sulfuryl

chloride to yield chloromethyl compounds E and F.

Treatment of E or F with sodium iodide in dry acetone

gave the corresponding iodide derivatives which were

then converted to C and D using an appropriate car-

boxylic acid in the presence of DBU. Compound D was

also obtained from B by employing a novel chloro-

methylation protocol,¹⁹followed by sequential treat-

ment with NaI/acetone and RCOOH/DBU. The

synthesized compounds and their physical and spectral

data are listed in Table 1.

E_t=E is the amount of active enzyme remaining at t.

o

Á

ln E_t=E k_obs

t

ꢀ3

o

Molecular modeling

Enzyme-inhibitor modeling studies were carried out

using the Tripos force ®eld of SYBYL, version 6.5

(Tripos Associates, St. Louis, MO) and a Silicon

graphics O2 workstation. Inhibitors were built using

the SYBYL software package (SKETCH mode) and

energy-minimized using the SYBYL force ®eld. The

crystal structure of HLE bound to the turkey ovo-

mucoid inhibitor third domain (Brookhaven, 1PPF) was

used in the modeling studies. The energy-minimized

inhibitor was₀superimposed on the -Thr-Leu-Glu-Tyr-

Carboxylates 40±53 were obtained as shown in Scheme

2. The appropriate substituted malonic ester was used in

the synthesis of a 2-alkyl substituted ethyl acrylate^20,21

0

(-P₂-P₁-P₁-P₂-) segment of TOMI (alpha-carbon and

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

1007

c

e

Scheme 1. Synthesis of derivatives of (I). ^aNH₂SO₂Cl/TEA; ^bR₂CHO/NaBH(OAc)₃/DCE; NaH/THF; ^dClCH₂OOCR/TEA; NaH/CH₃CN then

R₂Br; ClCH₂SPh/DBU; ^gSO₂Cl₂; ^hNaI/acetone; ⁱRCOOH/DBA/CH₂Cl₂; ^jSOCl₂/HOCH₂SO₃Na.

f

carbonyl group of inhibitor overlapping the corre-

sponding alpha-carbon and carbonyl group of Leu-18

(P₁residue) of TOMI. TOMI and water molecules were

deleted and a shaded surface for HLE was generated.

protease in a highly predictable fashion, namely, with

the R₁residue accommodated at the primary speci®city

site (S₁)²⁵and the R₂and L groups oriented toward the

0

S₂and S_nsubsites, respectively. Thus, the carboxylate

component in scaolds (I) and (II) was expected to bind

0

to the S_nsubsites of the target enzymes. Based on pre-

Inhibition of elastinolysis

vious observations,²⁶the use of carboxylate leaving

groups was anticipated to yield highly potent inhibitors.

With the widely diverse structures of available

carboxylates, this ₀series of inhibitors provides an ideal

Inhibition of elastin degradation by HLE was deter-

mined using a published procedure.²⁴In this protocol

elastin and HLE in buer solution were incubated at

37 ^ꢁC, 1-mm aliquots were withdrawn at 10-min inter-

vals, ®ltered and the absorbance determined at 276 nm.

The change in absorbance was linear with time over 60

min. The rate of solubilization of elastin was determined

by a linear least-squares ®t to the data. The eect of an

inhibitor on this reaction was then determined by vary-

ing the inhibitor concentration in the incubation mix.

The percent inhibition is a re¯ection of the decrease in

the slope in the presence of inhibitor divided by the

control slope.

0

system to study S_n±P_ninteractions, which are often less

understood and less emphasized in inhibitor design,

than the corresponding S_n±P_ninteractions for most

serine proteases.

This report details our early studies with carboxylate

inhibitors derived from (I) and (II) which aimed at (a)

gaining further insight into the interaction of (I) and (II)

with serine proteases and, (b) optimizing inhibitory

potency and enzyme selectivity through variations in

R₁, R₂, and especially the leaving group L. While all

three neutrophil-derived serine proteases (HLE, PR 3,

and Cat G) were studied with the synthesized inhibitors,

eorts were focused on the development of inhibitors of

HLE suitable for further pharmacological evaluation.

Discussion

Recent studies in our laboratory have demonstrated

that the motif embodied in the 1,2,5-thiadiazolidin-3-

one 1,1 dioxide scaold is ideally suited for the design of

inhibitors of (chymo)trypsin-like serine proteinases.^{16 18}

These earlier results also suggested that inhibitors

derived from this scaold might bind a target serine

Inhibitors synthesized in this study were those with

hydrophobic R₁groups which parallel the known pri-

mary substrate speci®city (P₁) of the target enzyme,

namely, HLE (Val, Leu),²⁷PR 3 (Abu, Norval)²⁸and

1008

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

Table 1. Physical constants and spectral data of inhibitors 1±39

Compound

mp (^ꢁC)

Oil

¹H NMR (d)

MF (anal.)

1

1.09 (t, 3H), 1.20 (s, 9H), 1.88 (m, 1H), 2.02 (m, 1H) 4.22 (m, iH), 5.35 (d, 1H), 5.62

(d, 2H)

C

₁₀H₁₈N₂O₅S (C, H, N)

2

3

Oil

0.98 (t, 3H), 1.21 (s, 9H), 1.90 (m, 1H), 2.06 (m, 1H) 2.88 (s, 3H), 3.89 (t, 1H), 5.62 (d, 2H)

0.86 (t, 3H), 1.22 (s, 9H), 1.67 (m, 1H), 1.91 (m, 1H) 3.97 (t, 1H), 4.42 (dd, 2H), 5.67

(d, 2H), 7.38 (s, 5H)

C₁₁H₂₀N₂O₅S (C, H, N)

₁₇H₂₄N₂O₅S (C, H ,N)

C

4

5

6

Oil

1.06 (t, 3H), 1.87 (m, 1H), 2.02 (m, 1H), 4.22 (m, 1H) 5.59 (d, 1H), 5.87 (d, 2H), 7.21

(s, 3H)

0.99 (t, 3H), 1.21 (s, 9H), 1.45±1.62 (m, 2H), 1.72±1.86 (m, 1H), 1.93±2.06 (m, 1H), 4.24

(m, 1H), 5.20 (d, 1H) 5.62 (d, 2H)

C

₁₂H₁₂Cl₂N₂O₅S (C, H, N)

C₁₁H₂₀N₂O₅S (C, H, N)

91.0±94.0 0.79 (t, 3H), 1.22 (s, 9H), 1.30 (m, 2H), 1.59 (m, 1H), 1.84 (m, 1H), 3.66 (s, 2H), 3.95

(t, 1H), 4.39 (dd, 2H) 5.66 (d, 2H), 7.33 (dd, 4H), 11.5 (br s, 1H)

C₂₀H₂₈N₂O₇S (C, H, N)

7

8

9

95.0±96.0 1.00 (d, 3H), 1.09 (d, 3H), 1.20 (s, 9H), 2.38 (m, 1H), 4.20 (d, 1H), 5.64 (s, 2H)

85.0±88.0 0.99 (dd, 6H), 1.21 (s, 9H), 1.71 (m, 1H), 1.88 (m, 2H), 4.25 (dd, 1H), 5.62 (d, 2H)

C₁₁H₂₀N₂O₅S (C, H ,N)

₁₂H₂₂N₂O₅S (C, H, N)

C₁₃H₂₄N₂O₅S (C, H, N)

C

Oil

0.90 (dd, 6H), 1.14 (s, 9H), 1.74 (m, 2H), 1.85 (m, 1H), 2.82 (s, 3H), 3.79 (t, 1H), 5.57

(d, 2H)

0.74 (dd, 6H), 1.23 (s, 9H), 1.52±1.78 (m, 3H), 3.99 (t, 1H), 4.40 (dd, 2H), 5.65 (d, 2H),

7.37 (s, 5H)

0.75 (dd, 6H), 1.21 (s, 9H), 1.57±1.82 (m, 3H), 3.90 (t, 1H), 3.92 (s, 3H), 4.44 (dd, 2H),

5.65 (d, 2H), 7.47 (d, 2H), 8.04 (d, 2H)

10

11

C

₁₉H₂₈N₂O₅S (C, H, N)

₂₁H₃₀N₂O₇S (C, H, N)

₁₉H₂₀N₂O₅S (C, H, N)

12

13

Oil

82±86

2.08 (s, 3H), 3.05 (m, 2H), 4.04 (m, 1H), 4.40±4.30 (dd, 2H), 5.58 (dd, 2H), 7.20 (m, 10H)

0.98 (dd, 1H), 1.75 (m, 1H), 1.88 (m, 2H), 4.28 (dd, 1H), 5.5 (br s, 1H), 5.85 (s, 2H), 7.46

(t, 2H), 7.61 (t, 1H), 8.03 (dd, 2H)

C₁₄H₁₈N₂O₅S (C, H, N)

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

Oil

0.98 (dd, 6H), 1.87 (m, 2H), 1.97 (m, 1H), 2.92 (s, 3H), 3.91 (t, 1H), 5.90 (dd, 2H), 7.37

(m, 2H), 7.60 (m, 1H), 8.08 (d, 2H)

0.73 (dd, 6H), 1.62 (m, 1H), 1.75 (m, 2H), 3.95 (t, 1H), 4.42 (dd, 2H), 5.88 (dd, 2H), 7.37

(m, 5H), 7.46 (m, 2H), 7.6 (m, 1H), 8.08 (dd, 2H)

0.98 (dd, 6H), 1.72 (m, 1H), 1.88 (m, 2H), 4.29 (ddd, 1H), 5.24 (br d, 1H), 5.88 (dd, 2H),

7.32 (s, 3H)

0.97 (dd, 6H), 1.82 (m, 2H), 1.92 (m, 1H), 2.91 (s, 3H), 3.90 (t, 1H), 5.89 (dd, 2H), 7.32

(s, 3H)

0.73 (dd, 6H), 1.62 (m, 1H), 1.73 (m, 2H), 3.93 (t, 1H), 4.42 (dd, 2H), 5.89 (dd, 2H), 7.32 C₂₁H₂₂Cl₂N₂O₅S (C, H, N)

(s, 3H), 7.38 (s, 5H)

0.71 (dd, 6H), 1.6±1.8 (m, 3H), 3.65 (s, 2H), 3.92 (t, 1H), 4.40 (dd, 2H), 5.89 (dd, 2H), 7.32

(m, 5H)

1.0 (dd, 6H), 1.74 (m, 1H), 1.93 (m, 2H), 4.29 (ddd, 1H), 5.17 (br d, 1H), 5.78 (s, 2H), 6.44

(d, 1H), 7.42 (m, 3H), 7.54 (m,2H), 7.78 (d, 1H)

0.98 (dd, 6H), 1.82 (m, 2H), 1.93 (m, 1H), 2.92 (s, 3H), 3.88 (t, 1H), 5.28 (d, 1H), 5.76

(dd, 2H), 5.93 (d, 1H), 7.39 (m, 3H), 7.49 (m, 2H)

0.98 (dd, 6H), 1.78 (m, 1H), 1.9 (m, 2H), 4.31 (dd, 1H), 5.35 (br d, 1H), 5.8 (s, 2H), 6.98

(d, 1H),7.39 (m, 3H), 7.63 (m, 2H)

1.0 (dd, 6H), 1.84 (m, 2H), 1.96 (m, 1H), 2.93 (s, 3H), 3.92 (t, 1H), 5.86 (dd, 2H), 7.0

(d,1H), 7.42 (m, 3H), 7.68 (m, 2H)

0.98 (dd, 6H), 1.82 (m, 2H), 1.93 (m, 1H), 2.68 (t, 2H), 2.88 (s, 3H), 2.96 (t, 2H), 3.84

(t, 1H), 5.64 (dd, 2H), 7.2 (m, 3H), 7.28 (m, 2H)

0.73 (dd, 6H), 1.54±1.78 (m, 3H), 2.69 (t, 2H), 2.98 (t, 2H), 3.91 (t, 1H), 4.38 (dd, 2H),

5.64 (dd, 2H), 7.17±7.30 (s,5H)

0.98 (dd, 6H), 1.8 (m, 2H), 1.92 (m, 1H), 2.98 (s, 3H), 3.68 (s, 2H), 3.85 (t, 1H), 5.67

(dd, 2H), 7.29 (m, 5H)

0.72 dd, 6H), 1.57 (m, 1H), 1.7 (m, 2H), 3.68 (s, 2H), 3.89 (t, 1H), 4.35 (dd, 2H), 5.66

(dd, 2H), 7.3 (m, 5H), 7.37 (s, 5H)

1.0 (dd, 6H), 1.87 (m 2H), 1.96 (m, 1H), 2.92 (s, 3H), 3.93 (t, 1H), 5.94 (dd,2H), 7.42 (dd,

1H), 8.32 (dt, 1H), 7.61 (dt, 1H), 8.05 (dd, 1H)

0.97 (dd, 6H), 1.8 (m, 2H), 1.9 (m, 1H), 2.97 (s, 3H), 4.15 (t, 1H), 4.29 (br s, 2H), 4.48

(s, 3H), 5.75 (dd, 2H), 7.9 (d, 2H), 8.87 (d, 2H)

0.93 (dd, 6H), 1.8 (m, 2H), 1.91 (m, 1H), 2.88 (s, 3H), 3.68 (s, 2H), 3.87 (t, 1H), 5.65 (dd,

2H), 7.2±7.33 (m, 3H), 7.41 (m, 2H)

0.95 (dd, 6H), 1.79 (m, 2H), 1.9 (m, 1H), 2.88 (s, 3H), 3.86 (t, 1H), 4.18 (s, 2H), 5.62

(dd, 2H), 7.61 (m, 2H), 7.7 (m, 1H), 7.98 (d, 2H)

0.98 (dd, 6H), 1.78 (m, 2H), 1.9 (m, 1H), 2.9 (s, 3H), 3.8 (s, 2H), 3.86 (t, 1H), 5.71 (s, 2H),

7.18 (d, 2H), 8.42 (d, 2H)

0.97 (dd, 6H), 1.8 (m, 2H), 1.9 (m, 1H), 2.97 (s, 3H), 4.15 (t, 1H), 4.29 (br s, 2H), 4.48

(s, 3H), 5.75 (dd, 2H), 7.9 (d, 2H), 8.87 (d, 2H)

0.94 (dd, 6H), 1.72 (m, 2H), 1.87 (m, 1H), 2.86 (s, 3H), 3.5 (br s, 1H), 3.81 (t, 1H), 5.23

(s, 1H), 5.67 (dd, 2H), 7.30±7.48 (m, 5H)

0.98 (dd, 6H), 1.72 (m, 2H), 1.88 (m, 1H), 2.86 (s, 3H), 3.43 (br s, 1H), 3.81 (t, 1H), 5.25

(br s, 1H), 5.68 (dd, 2H), 7.30±7.48 (m, 5H)

0.97 (dd, 6H), 1.81 (m, 2H), 1.92 (m, 1H), 2.90 (s, 3H), 3.38 (s, 3H), 3.68 (3.73 (t, 2H), 3.87

(t, 1H), 4.21 (s, 2H), 5.72 (d, 2H)

0.97 (dd, 6H), 1.78±1.97 (m, 3H), 2.90 (s, 3H, 3.38 (s, 3H), 3.53±3.77 (m, 8H), 3.67 (t, 2H),

4.21 (s, 2H), 5.70 (d, 2H)

C

₁₅H₂₀N₂O₅S (C, H, N)

₂₁H₂₄N₂O₅S (C, H, N)

75±77

Oil

C

₁₄H₁₆N₂Cl₂O₅S (C, H, N)

C₁₅H₁₈Cl₂N₂O (C, H, N)

Oil

C₂₃H₂₄Cl₂N₂O₅S (C, H, N)

92±93

Oil

C

₁₆H₂₀N₂O₅S (C, H, N)

₁₇H₂₂N₂O₅S (C, H, N)

74±75

66±70

Oil

C

₁₆H₁₉FN₂O₅S (C, H, N)

C₁₇H₂₁FN₂O₅S (C, H, N)

C₁₇H₂₄N₂O₅S (C, H, N)

Oil

C

₂₃H₂₈N₂O₅S (C, H, N)

₁₆H₂₂N₂O₅S (C, H, N)

₂₂H₂₆N₂O₅S (C, H, N)

Oil

59±61

Oil

C₁₄H₁₉N₃O₅S (C, H, N)

C₁₆H₂₄IN₃O₅S₂(C, H, N)

Oil

C₁₆H₂₂N₂O₅S₂(C, H, N)

Oil

C₁₆H₂₂N₂O₇S₂(C, H, N)

175d

Oil

C₁₅H₂₁N₃O₅S₂(C, H, N)

₁₆H₂₄N₃O₅S₂(C, H, N)

₁₆H₂₂N₂O₆S (C, H, N)

C₁₆H₂₂N₂O₆S (C, H, N)

C

Oil

C

Oil

C

₁₃H₂₄N₂O₇S (C, H, N)

₁₅H₂₈N₂O₈S (C, H, N)

Oil

38

39

Oil

3.10 (t, 2H), 4.05±4.35 (dd, 2H), 4.12 (m, 1H), 5.88 (s, 2H), 7.05±7.35 (m, 10H)

2.08 (s, 3H), 3.05 (m, 2H), 4.04 (m, 1H), 4.04±4.30 (dd, 2H), 5.58 (dd, 2H), 7.20 (m, 10H)

C₂₄H₂₀Cl₂N₂SO₅(C, H, N)

C₁₉H₂₀N₂O₅S (C, H, N)

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

1009

Scheme 2. Synthesis of derivatives of (II). KOH/ethanol/re¯ux; ^bEt₂NH/37% HCHO; ^cHSCOCH₃; ^dH₂O₂/HCOOH; ^ePCl₃; ^fNH₄OH/toluene;

^gNaH/THF; ^h37% HCHO/aq ethanol; ⁱDAST; ^jClCH₂SPh/TEA; ^kSO₂Cl₂; ^lSOCl₂/HOCH₂SO₃Na; ^mNaI/Acetone; ⁿRCOOH/DBU/CH₂Cl₂.

Table 2. Physical data and spectral data of inhibitors 40±53

Compound mp (^ꢁC)

¹H NMR (d)

MF (anal.)

40

41

42

43

44

45

46

Oil

0.97 (dd, 6H), 1.58 (m, 1H), 1.7 (m, 1H), 2.01 (m, 1H), 3.28 (m, 2H), 3.77 (q, 1H), 5.83

(dd, 2H), 7.42 (t, 2H), 7.59 (t, 1H), 8.04 (d, 2H)

0.98 (dd, 6H), 1.58 (m, 1H), 1.7 (m, 1H), 2.03 (m, 1H), 3.31 (m, 2H), 3.8 (m, 1H), 5.87

(s, 2H), 7.32 (s, 3H)

0.96 (dd, 6H), 1.53 (m, 1H), 1.68 (m, 1H), 1.99 (m, 1H), 2.35 (s, 3H), 3.22±3.32 (m, 2H),

3.75 (m, 1H), 5.78 (dd, 2H), 7.12 (dd, 1H), 7.33 (dt, 1H), 7.60 (dt, 1H), 8.03 (dd, 1H)

0.98 (dd, 6H), 1.57 (m, 1H), 1.7 (m, 1H), 2.03 (m, 1H), 3.24±3.40 (m, 2H), 3.78 (m, 5.72

(dd, 2H), 6.42 (d, 1H), 7.39 (m, 3H), 7.52 (m, 2H), 7.75 (d, 1H)

1.0 (dd, 6H), 1.57 (m, 1H), 1.73 (m, 1H), 2.01 (m, 1H), 2.71 (t, 2H), 2.98 (t, 2H), 3.23±3.33

(m, 2H), 3.77 (m, 1H), 5.62 (dd, 2H), 7.21 (m, 3H), 29 (m, 2H)

C

₁₅H₁₉NO₅S (C, H, N)

C₁₅H₁₇Cl₂NO₅S (C, H, N)

C

₁₇H₂₁NO₇S (C, H, N)

₁₇H₂₁NO₅S (C, H, N)

C₁₇H₂₃NO₅S (C, H, N)

129±131 2.93 (dd, 1H), 3.28 (dd, 1H), 3.66 (dd, 1H), 3.78±3.93 (m, 2H), 5.82 (dd, 2H), 7.28 (m, 5H),

7.6 (m, 3H)

79±80

C

₁₈H₁₅Cl₂NO₅S (C, H, N)

2.95 (m, 1H), 3.30 (m, 1H), 3.45±3.60 (m, 3H), 5.74 (d, 2H), 6.43 (d, 1H), 7.2±7.6 (m, 10H),

8.80 (d, 1H)

C₂₀H₁₉NO₅S (C, H, N)

47

48

49

Oil

2.66 (t, 2H), 2.95 (m, 3H), 3.25 (m, 1H), 3.40±3.55 (m, 3H), 5.59 (d, 2H), 7.18±7.40 (m, 10H)

2.92 (t, 1H), 3.25 (m, 1H), 3.40±3.55 (m, 3H), 3.65 (s, 2H), 5.60 (d, 2H), 7.15±7.40 (m, 10 H)

2.95 (m, 1H), 3.32±3.35 (m, 4H), 3.70 (d, 1H), 3.9 (d, 1H), 5.57 (m, 2H), 7.2 (m, 2H), 7.33

(m, 3H), 7.53 (m, 3H), 7.69 (m, 2H)

C

₂₀H₂₁NO₅S (C, H, N)

₁₉H₁₉NO₅S₂(C, H, N)

C₁₉H₁₉NO₆S₂(C, H, N)

50

51

52

53

Oil

2.92 (m, 1H), 3.25±3.58 (m, 4H), 4.16 (s, 2H), 5.57 (d, 2H), 7.18 (m, 2H), 7.32 (m, 7.58

(m, 2H), 7.70 (m, 1H), 7.95 (m, 2H)

2.95 (m, 1H), 3.30 (m, 1H), 3.50 (m, 1H), 5.58 (d, 1H), 5.61 (d, 2H), 7.15±7.50 (m, 10H),

7.89 (d, 1H)

2.95 (m, 1H), 3.30 (m, 1H), 3.4±3.6 (m, 3H), 5.72 (m, 2H), 5.91 (d, 1H), 7.2 (m, 2H), 7.25±

7.40 (m, 7H), 7.48 (m, 2H)

2.95 (m, 1H), 3.35 (m, 1H), 3.45±3.60 (m, 3H), 5.80 (s, 2H), 6.54 (d, 1H), 7.15±7.32 (m, 6H),

7.60±7.75 (m, 3H), 8.0 (m, 2H)

C

₁₉H₁₉NO₇S₂(C, H, N)

C₂₀H₁₉NO₅S₂(C, H, N)

83±84

55±56

C

₂₀H₁₉NO₅S₂(C, H, N)

₂₀H₁₉NO₇S₂(C, H, N)

Cat G (Phe).²⁹R₂and L groups were chosen mainly

from hydrophobic groups in order to explore the

favorable interactions with secondary subsites S₂and

S₁±S₂of HLE, which are also known to be hydro-

phobic in nature.

cussion of the results in Tables 3 and 4 in terms of R₁,

R₂, and L follows.

0

R₁as a primary speci®city determinant

According to the design rationale, R₁residues bind to

the primary speci®city pockets (S₁) of serine proteases.

This is indeed the case as suggested by the results listed

in Tables 4 and 5. Highly potent inhibitors of HLE were

obtained with templates (I) and (II), when an isobutyl

group was chosen as R₁, consistent with the primary

selectivity of HLE (leucine). On the other hand, a

phenyl group is clearly the group of choice as R₁for

inhibition of Cat G (phenylalanine). Selectivity for PR 3

over HLE was only observed with smaller R₁groups

(compounds 1 and 5). This is also consistent with the

observation from X-ray crystallographic studies, in

which the S₁pocket of PR 3 was found to be slightly

smaller than that of HLE. Interestingly, no inhibition of

HLE was observed when an isopropyl group was used

as R₁, which is in contrast with the strong preference of

The inactivation of HLE by derivatives of (I) and (II)

was found to be time-dependent, rapid and highly e-

cient. For example, total inactivation of HLE by com-

pound 17 (Table 3) was achieved by using only ꢀ2 eq

of inhibitor and led to the formation of a highly stable

E-I complex (Fig. 3). Typical progress curves for the

hydrolysis of methoxysuccinyl-Ala-Ala-Pro-Val p-nitro-

anilide by HLE in the presence of varying concentra-

tions of inhibitor 14 are shown in Figure 3. The results

summarized in Tables 3 and 4 indicate that derivatives

of 1,2,5-thiadiazolidin-3-one 1,1 dioxide (I) and iso-

thiazolidin-3-one 1,1 dioxide (II) with carboxylate leav-

ing groups are very potent inhibitors of HLE, PR 3 and

cathepsin G. In general, inhibitors derived from scaold

(I) are more potent than those from scaold (II). A dis-

1010

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

Table 3. Inhibitory activity of derivatives of (I) toward human leukocyte elastase, proteinase 3, and cathepsin G

Compound

R₁

R₂

L

HLE

PR 3

CAT G

1

(k_inact/K_IM

s )

1

2

3

4

5

6

7

8

Ethyl^a

H

Methyl

Benzyl

H

OOCCMe₃

300

400

1500

3700

1300

900

300

400

Inactive

200

Inactive

70

Inactive

200

300

Ð

1100

300

90

500

60

30

2300

1200

100

Ð

100

Ð

200

Ð

90

Ethyl^a

2,6-Dichlorobenzoate

OOCCMe₃

3400

5100

2000

Inactive

2400

5900

1800

Ð^b

n-Propyl

Isopropyl

Isobutyl

Iisobutyl

Isobuty

Isobutyl

Benzyl

H

(p-HOOCCH₂) benzyl

H

4500

Inactive

4100

OOCCMe₃

OOCCH₃

Benzoate

H

9

Methyl

Benzyl

(p-CH₃OOC) Benzyl

Benzyl

H

42,700

60,500

13,700

163,300

91,600

267,500

335,900

711,800

4,928,300

2,381,000

1,220,000

31,300

318,200

56,400

628,200

296,300

3200

357,100

63,800

105,100

12,500

395,400

753,200

161,300

108,300

440,500

308,100

71,100

79,700

3600

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

Ð

2600

12,800

26,000

88,200

33,400

14,400

196,400

9500

9700

Ð

3500

Ð

Methyl

Benzyl

H

2,6-Dichlorobenzoate

trans-Cinnamate

a-Fluorocinnamate

Dihydrocinnamate

Phenylacetate

Methyl

Benzyl

(HOOCCH₂) benzyl

H

Methyl

H

Methyl

Benzyl

Methyl

Benzyl

Methyl

Benzyl

Phenylacetate

3-Nicotinate

3-Nicotinate (methyl iodide)

Phenylthioacetate

Phenylsulfonyl acetate

(4-Pyridylthio) acetate

4-(N-Methyl-4-pyridyl) acetate

(R) Mandelate

(S) Mandelate

Inactive

70

Ð

Inactive

20

100

Ð

Oxaacid-1^c

Oxaacid-2^d

2,6-Dichlorobenzoate

OOCCH₃

2200

10,600

Benzyl

Ð

^aracemic.

^bNot determined.

^cCH₃OCH₂CH₂OCH₂COOH.

^dCH₃OCH₂CH₂OCH₂CH₂OCH₂COOH.

0

HLE for valine at its primary speci®city site. Molecular

modeling studies suggest that the isopropyl group lacks

the ¯exibility needed for the inhibitor molecule to ®t

properly into the active site pocket of HLE when it is

attached to a ®ve-member ring. With respect to the eect

of R₁on inhibition, the results from this carboxylate

series are remarkably consistent with previous observa-

tions with the sulfone series of inhibitors, which again

con®rm the highly predictable manner in which these

inhibitor templates bind to their target proteases with R₁

as the primary speci®city determinant.^{15 18}

the S₂and S₂pockets of HLE are more hydrophobic

than those of PR 3. This suggests a strategy for selective

inhibition of HLE over PR 3 by using hydrophobic R₂

and L groups to provide favorable interactions with the

0

secondary binding pockets S₂and S₂of HLE. Indeed,

the compounds in Table 3 with the highest selectivity

toward HLE are 17 and 18 (selectivity ratios for HLE

over PR 3 are 150 and 165, respectively), both com-

pounds having hydrophobic R₂and L groups. The dif-

ference in the nature of S₂between HLE and PR 3 was

further evidenced with compound 19, which has an

extra hydrophilic (CH₂CO₂H) group in R₂when com-

pared to compound 18. In this instance k_inact/K_I

decreased by half for HLE but there was a correspond-

ing 14-fold increase for PR 3.

Enzyme selectivity design with R₂and L

Although selective inhibition between HLE and Cat G

can be achieved with proper choice of R₁, it is a chal-

lenge to selectively inhibit highly similar proteases such

as HLE and PR 3. While recent X-ray crystallographic

studies have con®rmed the similar topographical fea-

tures of their active sites, they have also revealed that

Modi®cations in the leaving group L using hydrophilic

functionality in order to increase aqueous solubility

were found to diminish potency (compounds 28 and 29,

0

36 and 37). The hydrophilic nature of the S₁and S₂

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

1011

displayed an unexpected drop in potency in going from

R₂=methyl to R₂=benzyl. The available evidence sug-

gests that a non-productive hydrophobic collapse³⁰

occurs with inhibitors 25 and 27. This serendipitous

observation and its eect on molecular recognition is

the subject of a future report.

Leaving group L and inhibitory potency

The leaving group L not only serves as a recognition

0

element that interacts with the S_nsubsites, but its

chemical reactivity is also correlated with inhibitory

potency. As shown in Table 5, more potent inhibition of

HLE was observed with inhibitors having lower pK_a

values. The large number of commercially available

carboxylic acids encompassing a wide range of pK_a

values provide, in principle, a means of ®ne-tuning the

potency and stability of this series of serine protease

inhibitors.

Figure 2. Progress curves for the inhibition of human leukocyte elas-

tase (HLE) by compound 14. Absorbance was recorded at 410 nm for

reaction solutions containing HLE (20 nM), MeOSuc-AAPV-pNA

(1 mM) and the indicated concentrations of inhibitor 14 in 0.1 M

HEPES buer, pH 7.25, and 3.6% DMSO. The temperature was

maintained at 25 ^ꢁC, and the reactions were initiated by the addition of

enzyme.

Inhibition of elastinolysis

The ability of one of the inhibitors to block the degra-

dation of elastin by elastase was investigated. It is evi-

dent from Figure 4 that compound 17 (Table 3) is

eective in preventing the degradation of elastin by

elastase, suggesting that this class of compounds are of

potential therapeutic value. The results of this study

also demonstrate that, in contrast to protein inhibitors

of HLE that do not inhibit HLE pre-adsorbed on elas-

tin, low molecular weight inhibitors are highly eective

in inhibiting HLE pre-adsorbed on elastin.

In summary, the ®ndings described herein demonstrate

that a range of carboxylate derivatives based on the

1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazo-

lidin-3-one 1,1 dioxide scaolds are highly eective

inhibitors of HLE, PR 3 and Cat G. Furthermore, the

compounds are eective in blocking the degradative

action of elastase on elastin.

Experimental

Melting points were recorded on a Mel-Temp apparatus

and are uncorrected. Puri®cation of compounds by ¯ash

chromatography was carried out using Merck grade

silica gel (grade 60, 230±400 mesh, 60 A) purchased

from Aldrich Chemical Co. Thin layer chromatography

was performed using Analtech silica gel plates and the

TLC plates were visualized by iodine vapor and/or UV

light. When necessary, solvents were dried and/or dis-

tilled before use. Tetrahydrofuran was distilled from

sodium benzophenone. Methylene chloride was distilled

from calcium hydride. Acetonitrile and triethylamine

(Aldrich) were dried over freshly activated Linde 3 A

molecular sieves. Anhydrous acetone was purchased

from Aldrich Chemical Co. ¹H and ¹³C NMR spectra of

the synthesized compounds were recorded on a Varian

XL-400 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. Human leukocyte cathepsin G and

Figure 3. Percent remaining activity versus time plot obtained by

incubating inhibitor 17 with human leukocyte elastase (138 nM) at

dierent inhibitor to enzyme ratios in 0.1 M HEPES buer containing

0.5 M NaCl, pH 7.25, and 1% DMSO. Aliquots were withdrawn at

dierent time intervals and assayed for enzymatic activity using

MeOSuc-AAPV pNA by monitoring the absorbance at 410 nm for

1 min.

subsites of Cat G, as revealed by recent crystallographic

studies,²⁸may be the reason for the poor inhibitory

activity of compound 38 against Cat G, although the

same hydrophobic leaving group (2,6-dichlorobenzoate)

gives some of the most potent inhibitors of HLE repor-

ted today (compounds 17 and 18). In the case of HLE,

inhibitors with dihydrocinnamate and phenylacetate

as leaving groups (compounds 24 and 25, 26 and 27),

1012

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

Table 4. Inhibitory activity of derivatives of (II) toward human leukocyte elastase and cathepsin G

Compd

R₁

L

HLE

CAT G

1

k_inact/K_IM

s

40

41

42

43

44

45

46

47

48

49

50

51

52

53

Isobutyl

Benzyl

Benzoate

34,200

469,900

35,700

31,700

23,200

2100

300

2900

2200

Ð

5500

Ð

3700

3200

600

1300

800

2,6-Dichlorobenzoate

Acetylsalicylate

trans-Cinnamate

Dihydrocinnamate

2,6-Dichlorobenzoate

trans-Cinnamate

Dihydrocinnamate

700

(Phenylthio)acetate

1000

300

200

(Phenylsulfoxide)acetate

(Phenylsulfonyl)acetate

trans-3-(Phenylthio)acrylate

cis-3-(Phenylthio)acrylate

cis-3-(Phenylsulfonyl)acrylate

1100

5500

7300

5000

400

1

Table 5. Correlation of k_inact/K_IM

s

and leaving group pK_a

Representative syntheses

Compound^a

R⁰

pK_a

k_inact/K_IM ¹s

1

Synthesis of (S)-4-isobutyl-2-[(trimethylacetyl)oxymeth-

yl]-1,2,5-thiadiazolidin-3-one 1,1 dioxide 8. A solution

of (S)-4-isobutyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide¹⁵

(6.2 g; 30 mmol), chloromethyl pivaloate (4.7 g;

31 mmol) and triethylamine (3.0 g; 30 mmol) in 40 mL

dry acetonitrile was re¯uxed for 12 h. The solvent was

removed in vacuo, and the residue was taken up in ethyl

acetate (100 mL), washed with 5% aq HCl (50 mL), 5%

aq NaHCO₃(35 mL), and dried (Na₂SO₄). Evaporation

of the solvent left a crude product, which was puri®ed

by ¯ash chromatography, using a hexane:methylene

chloride gradient, mp 85±88 ^ꢁC (4.8 g; 52% yield).

10

12

15

18

Me₃C-

Me-

Ph-

5.03

2.76

4.20

1.59

60,500

163,000

336,000

2,6-Cl₂-Ph-

2,380,000

^aAll compounds are derived from scaold (I), with isobutyl as R₁,

benzyl as R₂and k_inact/K_Ivalues for inhibition of HLE.

Synthesis of (S)-4-isobutyl-5-methyl-2[(trimethylacetyl)-

oxymethyl]-1,2,5-thiadiazolidin-3-one 1,1 dioxide 9.

Sodium hydride (60%, 0.04 g; 1 mmol) was added to a

mixture of compound 8 (0.31 g; 1 mmol) and iodo-

methane (0.43 g; 3 mmol) in dry acetonitrile (2 mL) at

0 ^ꢁC under nitrogen. The resulting mixture was then

stirred at room temperature for 5 h. The solvent was

removed in vacuo and the residue was taken up in

methylene chloride (20 mL), washed with water (10 mL)

and dried (Na₂SO₄). Evaporation of the solvent left a

crude product which was puri®ed by ¯ash chromato-

graphy³¹using a hexane:methylene chloride gradient

(0.14 g; 43% yield).

(S) 4-Isobutyl-2-phenylthiomethyl-1,2,5-thiadiazolidin-3-one

1,1 dioxide. (S)-4-Isobutyl-1,2,5-thiadiazolidin-3-one 1,1

dioxide (17.8 g; 100 mmol) and chloromethylphenyl sul-

®de (17.43g; 110 mmol) were dissolved in dry acetonitrile

(150mL). DBU (15.2 g; 100 mmol) was added and the

resulting mixture was re¯uxed overnight. The solvent

was removed, the residue was dissolved in ethyl acetate

(300 mL) and washed successively with 5% aq HCl

(100 mL), 5% aq NaHCO₃(100 mL) and water (50 mL).

The organic phase was dried (Na₂SO₄) and the solvent

removed. The crude product was puri®ed by ¯ash chro-

matography using a hexane:methylene chloride gradient

(17.37 g; 55% yield), mp 53±54 ^ꢁC. ¹H NMR (CDCl₃): d

0.92 (dd, 6H), 1.58 (m, 1H), 1.73 (m, 2H), 4.1 (ddd, 1H),

4.93 (dd, 2H), 5.32 (br d, 1H), 7.3 (m, 3H), 7.55 (m, 2H).

Figure 4. Inhibition of elastinolysis. Elastin (1.5 mg/mL) was incu-

bated with human leukocyte elastase (199 nM) in 0.05 M TES buer,

pH 7.5, 37 ^ꢁC, using the following conditions: (a) no inhibitor; (b)

inhibitor 17, at an inhibitor to enzyme ratio of 5. Aliquots were with-

drawn at 10 min intervals and ®ltered through Millipore Millex GV

®lters. The optical density was monitored at 276 nm.

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

Chemicals Co., St. Louis, MO.

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

1013

(S)-4-Isobutyl-2-chloromethyl-1,2,5-thiadiazolidin-3-one

1,1 dioxide. (S)-4-Isobutyl-2-phenylthiomethyl-1,2,5-

thiadiazolidin-3-one 1,1 dioxide (9.42 g; 30 mmol) was

dissolved in dry methylene chloride (75 mL) and cooled

to 0 ^ꢁC. Sulfuryl chloride (8.09 g; 60 mmol) was added

dropwise and the resulting solution was allowed to

warm to room temperature. The solvent was removed

and the crude product was puri®ed by ¯ash chromato-

graphy using a hexane:methylene chloride gradient to

yield an oily product (0.27 g; 87% yield). ¹H NMR

(CDCl₃): d 0.98 (dd, 6H), 1.72 (m, 1H), 1.88 (m, 2H),

4.25 (ddd, 1H), 5.18 (br d, 1H), 5.36 (s, 2H) ppm. ¹³C

NMR: d 169.49, 59.16, 47.57 ppm.

Synthesis of (S)-4-isobutyl-5-benzyl-2-(benzoyloxy)meth-

yl-1,2,5-thiadiazolidin-3-one 1,1 dioxide 15. Compound

15 was synthesized using the same procedure as that

used in the preparation of compound 9.

Synthesis of Compound 36. 4-Isobutyl-2-chloromethyl-

5-methyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide (0.51 g;

2 mmol) was mixed with sodium iodide (0.33 g;

2.2 mmol) in dry acetone (3 mL), and the resulting mix-

ture was stirred at room temperature overnight. The

by-product, sodium chloride, was removed by ®ltration

through a small amount of silica gel in a disposable

glass pipette. The solvent was removed in vacuo and the

iodo intermediate was redissolved in dry methylene

chloride (2 mL). A solution of 3,6-dioxaheptanoic acid

(0.32 g; 2 mmol) and DBU (0.30 g; 2 mmol) in methylene

chloride (2 mL) was added, and the resulting mixture

was stirred at room temperature overnight. The reaction

mixture was then diluted with methylene chloride

(40 mL) and washed with 5% aq HCl (20 mL), 5% aq

NaHCO₃(20 mL) and brine (20 mL). The organic phase

was dried (Na₂SO₄) and then concentrated in vacuo.

The crude product was puri®ed by ¯ash chromato-

graphy using a hexane:methylene chloride gradient,

yielding an oily product.

Synthesis of (S)-4-isobutyl-2-(benzoyloxy)methyl-1,2,5-

thiadiazolidin-3-one 1,1 dioxide 13. (S)-4-Isobutyl-2-

chloromethyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide

(0.48 g; 2 mmol) was dissolved in dry acetone (6 mL),

sodium iodide (0.33 g; 2.2 mmol) was added, and the

reaction mixture was stirred at room temperature over-

night under nitrogen. A precipitate formed which was

removed by ®ltration through a small amount of silica

gel in a disposable glass pipette. The solvent was then

removed and the iodomethyl intermediate was redis-

solved in dry methylene chloride (4 mL). To this solu-

tion was added a solution of benzoic acid (0.27 g;

2.2 mmol) and DBU (0.33 g; 2.2 mmol) in dry methylene

chloride (4 mL), and the resulting mixture was stirred at

room temperature overnight. The solvent was removed,

and the residue dissolved in ethyl acetate (75 mL) and

extracted successively with water (25 mL), 5% aq HCl

(20 mL), 5% aq NaHCO₃(25 mL) and brine (25 mL).

The organic phase was dried (Na₂SO₄) and the solvent

removed, leaving a crude product which was puri®ed on

a Chromatotron plate using methylene chloride as the

eluent (0.15 g; 30.6% yield).

Diethylisobutyl malonate. Sodium metal (28.78 g;

1.25 mol) was added in small pieces to dry ethanol

(300 mL) under a nitrogen atmosphere. After the reac-

tion had subsided, an additional 600 mL of dry ethanol

was added and the reaction mixture brought to re¯ux

until all the sodium metal had reacted. The solution was

then cooled to 80 ^ꢁC and diethyl malonate (200.2 g;

1.25 mol) was added dropwise over a period of 1 h. To

the resulting solution was added 2-methyl-1-bromo-

propane (171.25 g; 1.25 mol) and the solution was then

re¯uxed for 24 h. The mixture was cooled to room tem-

perature and ethanol was removed in vacuo. The pro-

duct was isolated by vacuum distillation (82±90 ^ꢁC, ꢀ0.2

(S)-4-Isobutyl-2-phenylthiomethyl-5-benzyl-1,2,5-thiadia-

zolidin-3-one 1,1 dioxide. (S)-4-Isobutyl-2-phenylthio-

methyl-1,2,5-thiadiazolidin-3-one 1,1 dioxide (3.36 g;

10.7 mmol) and benzyl bromide (2.01 g; 11.77 mmol)

were dissolved in dry acetonitrile (25 mL), and 60%

sodium hydride (0.28 g; 10.7 mmol) added. The reaction

mixture was stirred at room temperature overnight.

The solvent was then removed and the residue was dis-

solved in ethyl acetate (10 mL). The organic phase was

extracted with water (2Â30 mL) and dried over anhy-

drous sodium sulfate. The solvent was removed and the

crude product was puri®ed by ¯ash chromatography³¹

using a hexane:methylene chloride gradient, to yield

an oily product (2.79 g; 71%). ¹H NMR (CDCl₃): d

0.69 (dd, 6H), 1.48 (m, 1H), 1.63 (m, 2H), 3.78 (t, 1H),

4.33 (dd, 2H), 4.97 (dd, 2H), 7.34 (m, 8H), 7.58 (m, 2H)

ppm.

1

torr) (50% yield). H NMR (CDCl₃): d 0.91 (dd, 6H),

1.27 (t, 6H), 1.58 (m, 1H), 1.8 (t, 2H), 3.42 (t, 1H), 4.19

(q, 4H) ppm.

Synthesis of monoester. A solution of potassium

hydroxide (24.91 g; 444 mmol) in absolute alcohol

(400 mL) was treated with a solution of diethyl isobutyl

malonate (96.41 g; 446 mmol) in absolute ethanol

(400 mL). The reaction mixture was stirred at room

temperature for 48 h. The solvent was removed in vacuo

and the residue was dissolved in water (300 mL) and

extracted with diethyl ether (2Â150 mL). The aqueous

phase was acidi®ed to pH 1 with 12 N HCl and extrac-

ted with diethyl ether (3Â100 mL). The organic extracts

were combined, dried (Na₂SO₄) and evaporated, leaving

67.47 g (76% yield) of pure product. ¹H NMR (CDCl₃):

d 0.92 (d, 6H), 1.29 (t, 3H), 1.62 (m, 1H), 1.82 (m, 2H),

3.48 (t, 1H), 4.22 (q, 2H), 10.4 (br s, 1H) ppm.

(S)-4-Isobutyl-2-chloromethyl-5-benzyl-1,2,5-thiadiazolidin-

3-one 1,1 dioxide. This was prepared using the same

procedure as that used in the preparation of (S)-4-

isobutyl-2-chloromethyl-1,2,5-thiadiazolidin-3-one 1,1

dioxide. An oily product was obtained in 54% yield. ¹H

NMR (CDCl₃): d 0.72 (dd, 6H), 0.60 (m, 1H), 1.73 (m,

2H), 3.92 (t, 1H), 4.41 (dd, 2H), 5.37 (dd, 2H), 7.37 (s,

5H) ppm.

Synthesis of ethyl 2-isobutylacrylate. Diethylamine

(27.57 g; 377 mmol) was added to the above monoester

(68.24 g; 363 mmol) at room temperature and the solu-

tion was cooled to 0 ^ꢁC. Aqueous formaldehyde (38 mL

of a 37% formaldehyde solution) was added dropwise

1014

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

over a period of 0.5 h, after which time the ice bath was

removed and the reaction mixture stirred overnight. The

solution was extracted with diethyl ether (3Â150 mL).

The organic extracts were combined and washed suc-

cessively with 5% aq HCl (3Â100 mL), 5% aq NaHCO₃

(100 mL) and brine (100 mL). The organic phase was

dried (Na₂SO₄) and the solvent removed, leaving 34.0 g

(60% yield) of product. H NMR (CDCl₃): d 0.88 (d,

6H), 1.29 (t, 3H), 1.80 (m, 1H), 2.18 (d, 2H), 4.19 (q,

2H), 5.47 (d, 1H), 6.16 (d, 1H) ppm.

for 1 h, the solvent was removed and the residue was

taken up in ethyl acetate (400 mL). The organic phase

was washed with water (75 mL), brine (75 mL) and dried

(Na₂SO₄). The solvent was removed, leaving 21.84 g

(69.5% yield) of product. This was used in the next step

1

without further puri®cation. H NMR (CDCl₃): d 0.94

(dd, 6H), 1.29 (t, 3H), 1.39 (m, 1H), 1.62 (m, 2H), 3.05

(m, 1H), 3.18 (dd, 1H), 3.63 (dd, 1H), 4.19 (q, 2H), 5.18

(s, 2H) ppm. ¹³C NMR: d 174.22, 61.20, 56.76 ppm.

1

A solution of the sulfonamide derivative (10.83 g;

45.7 mmol) in dry THF (75 mL) was added dropwise to

a suspension of 60% NaH (1.5 g; 50 mmol) in dry THF.

After stirring the mixture overnight at room tempera-

ture, the solvent was removed and the residue was

washed with methylene chloride, yielding the sodium

Synthesis of thiolacetic acid adduct. Thiolacetic acid

(33.1 g; 435 mmol) was added to ethyl 2-isobutyl acryl-

ate (31.33 g; 217.6 mmol) and stirred at room tempera-

ture for 1 week. The reaction mixture was diluted with

diethyl ether (100 mL) and 5% aq NaHCO₃(100 mL)

and the resulting mixture was stirred for 1 h. After the

reaction had subsided, sodium bicarbonate (15 g) was

added in small portions with vigorous stirring. Ethyl

ether (100 mL) was added, the mixture was transferred

to a separatory funnel, agitated and the two phases

separated. The organic phase was washed with water

(100 mL), dried (Na₂SO₄) and evaporated, leaving a

quantitative yield of pure product. ¹H NMR (CDCl₃): d

0.92 (d, 6H), 1.26 (t, 3H), 1.38 (m, 1H), 1.60 (m, 2H),

2.33 (s, 3H), 2.68 (m, 1H), 2.98 (dd, 1H), 3.13 (dd, 1H),

4.16 (q, 2H) ppm.

1

salt of the cyclic imide in quantitative yield. H NMR

(DMSO-d₆): d 0.86 (dd, 6H), 1.22 (m, 1H), 1.53 (m, 1H),

1.67 (m, 1H), 2.71 (br m, 2H), 3.20 (br m,1H) ppm. ¹³C

NMR: 179.1, 54.05, 45.56 ppm.

The free imide was obtained using the following pro-

cedure: sodium salt (5 mmol) was dissolved in methanol

(20 mL), Dowex-H⁺resin (2 g), and the resulting sus-

pension was stirred for 1 h. The resin was ®ltered o

and washed with additional methanol. Removal of the

solvent left 0.89 g (93% yield) of pure product. ¹H

NMR (CDCl₃): d 0.97 (dd, 6H), 1.5±1.7 (m, 2H), 1.92

(m, 1H), 3.30 (m, 2H), 3.75 (m, 1H), 7.37 (br s, 1H)

ppm. ¹³C NMR: 172.97, 53.71, 41.89 ppm.

4-Isobutyl-isothiazolidin-3-one 1,1 dioxide. Formic acid

(460 mL) and 30% hydrogen peroxide (90 mL) were

mixed at room temperature, stirred for 1 h, then cooled

to 0 ^ꢁC. A solution of the thioester (34.80 g; 150 mmol)

in formic acid (50 mL) was added dropwise to the per-

formic acid solution, and the mixture was stirred for 2 h

at 0 ^ꢁC. The ice bath was removed and the reaction

mixture was stirred at room temperature for an addi-

tional 46 h. Excess formic acid was removed using a

rotary evaporator and residual formic acid was removed

by azeotropic distillation on the rotary evaporator using

toluene (4Â100 mL). Residual toluene was removed

under high vacuum, leaving a near quantitative yield of

sulfonic acid. This was used in the next step without

further puri®cation.

Synthesis of 4-isobutyl-2-(phenylthiomethyl)-isothiazoli-

din-3-one 1,1 dioxide. 4-Isobutyl-isothiazolidin-3-one

1,1 dioxide (0.96 g; 5 mmol) and tetrabutylammonium

bromide (0.16 g; 0.5 mmol) were suspended in dry

toluene (5 mL). Chloromethyl phenyl sul®de (2.03 g;

5 mmol) was added and the resulting mixture was

re¯uxed for 24 h. The solvent was removed and the

residue was dissolved in methylene chloride and ®ltered

through a pad of silica gel. Evaporation of the solvent

left a crude product which was puri®ed using a Chro-

matotron plate (hexane:methylene chloride), yielding

1.03 g (66% yield) of pure oily product. ¹H NMR

(CDCl₃): d 0.92 (dd, 6H), 1.45 (m, 1H), 1.62 (m, 1H),

1.85 (m, 1H), 3.16 (m, 2H), 3.64 (m, 1H), 4.91 (dd, 2H),

7.32 (m, 3H), 7.57 (m, 2H) ppm. ¹³C NMR: d 168.51,

52.37, 44.11 ppm.

Phosphorus trichloride (41.00 g; 375 mmol) was added

to the sulfonic acid (37.20 g; 150 mmol) under nitrogen

and the solution was heated to gentle re¯ux for 2 h. The

reaction mixture was cooled to room temperature and

excess PCl₃was removed on the rotary evaporator using

an ice trap. The product was dissolved in methylene

chloride (100 mL) and ®ltered through a short column

of silica gel. The silica gel was washed with methylene

chloride (150 mL), the eluents were combined and the

solvent removed, leaving 34 g (88% yield) of product

Synthesis of 4-isobutyl-2-chloromethyl-isothiazolidin-3-

one 1,1 dioxide. 4-Isobutyl-2-phenylthiomethyl-iso-

thiazolidin-3-one 1,1 dioxide (11.24 g; 35.9 mmol) was

dissolved in dry methylene chloride (70 mL) under

nitrogen and cooled to 0 ^ꢁC. A solution of sulfuryl

chloride (9.68 g; 71.72 mmol) in methylene chloride

(40 mL) was added dropwise, and the resulting mixture

was stirred and allowed to warm to room temperature

over a period of 1 h. The solvent was removed and the

residue was puri®ed by ¯ash chromatography using a

hexane:methylene chloride gradient (4.75 g; 55% yield).

¹H NMR (CDCl₃): d 0.97 (dd, 6H), 1.58 (m, 1H),

1.71 (m, 1H), 2.0 (m, 1H), 3.24±3.34 (m, 2H), 3.78 (m,

1H), 5.32 (dd, 2H) ppm. ¹³C NMR: d 168.17, 52.37,

45.44 ppm.

1

which was used in the next step. H NMR (CDCl₃): d

0.98 (dd, 6H), 1.29 (t, 3H), 1.46 (m, 1H), 1.68 (m, 2H),

3.21 (m, 1H), 3.68 (dd, 1H), 4.2±4.3 (m, 3H) ppm. ¹³C

NMR: d 172.37, 66.59, 61.68 ppm.

The sulfonyl chloride (33.98 g; 132.5 mmol) was dis-

solved in toluene (200 mL) and cooled in an ice bath.

Ammonium hydroxide (15 M) was added dropwise. The

resulting suspension was stirred at room temperature

R. Kuang et al. / Bioorg. Med. Chem. 8 (2000) 1005±1016

1015

General procedure for the synthesis of compounds 39±44.

4-Isobutyl-2-chloromethyl-isothiazolidin-3-one 1,1

References

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iodomethyl intermediate was redissolved in dry methyl-

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5% aq HCl (15 mL), 5% aq NaHCO₃(20 mL) and brine

(25 mL). The solution was dried (Na₂SO₄) and the sol-

vent evaporated, leaving a crude product which was

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ane:methylene chloride gradient (0.67 g; 58% yield).

a hex-

4-Benzyl-2-hydroxymethyl-isothiazolidin-3-one 1,1 dioxide.

4-Benzylisothiazolidin-3-one 1,1 dioxide²⁶(0.53 g;

2.35 mmol) and 37% formaldehyde (0.71 g; 7.05 mmol)

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4-Benzyl-2-chloromethyl-isothiazolidin-3-one 1,1 dioxide.

A mixture of 4-benzyl-2-hydroxymethyl-isothiazolidin-

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(2.38 g; 20 mmol) and dry diethyl ether (3 mL) was stir-

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and solvent were removed in vacuo and the residue was

taken up in methylene chloride (30 mL). The organic

layer was washed with 5% aq NaHCO₃(2Â15 mL) and

dried (Na₂SO₄). Removal of the solvent yielded the

desired compound (0.5 g; 91% yield), mp 140±141 ^ꢁC.

¹H NMR (CDCl₃): d 2.93 (m, 1H), 3.30 (m, 1H), 3.40±

3.60 (m, 3H), 5.32 (d, 2H), 7.18±7.40 (m, 5H).

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General procedure for the synthesis of compounds 45±52.

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Acknowledgements

Financial support of this research by the National

Institutes of Health (HL 57788) and SPARTA Phar-

maceuticals, Inc. is gratefully acknowledged.

1016

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Article Doi

Utilization of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold in the design of potent inhibitors of serine proteases: SAR studies using carboxylates

DOI: 10.1016/S0968-0896(00)00038-9

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