Utilization of the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide scaffold in the design of potential inhibitors of human neutrophil proteinase 3

Article abstract of DOI:10.1016/j.bmc.2009.12.057

The S′ subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of compounds based on the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide using combinational and click chemistry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited to the exploration of the S′ subsites of Pr 3. Molecular modeling studies suggest that further exploration of the S′ subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of highly selective, reversible competitive inhibitors of Pr 3.

Full text of DOI:10.1016/j.bmc.2009.12.057

Bioorganic & Medicinal Chemistry 18 (2010) 1093–1102
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Utilization of the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide scaffold in the
design of potential inhibitors of human neutrophil proteinase 3
Dengfeng Dou a, Guijia He a, Yi Li a, Zhong Lai a, Liuqing Wei a, Kevin R. Alliston a, Gerald H. Lushington b,
David M. Eichhorn a, William C. Groutas a,
*
a Department of Chemistry, Wichita State University, Wichita, KS 67260, United States
b Molecular Graphics and Modeling Laboratory, The University of Kansas, Lawrence, KS 66045, United States
a r t i c l e i n f o
a b s t r a c t
The S0 subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of
compounds based on the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide using combinational and click chem-
istry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited
to the exploration of the S0 subsites of Pr 3. Molecular modeling studies suggest that further exploration
of the S0 subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of
highly selective, reversible competitive inhibitors of Pr 3.
Article history:
Received 2 September 2009
Revised 15 December 2009
Accepted 17 December 2009
Available online 29 December 2009
Keywords:
Proteinase 3
Inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
herein the results of exploratory studies related to the design
and synthesis of potential non-covalent inhibitors of Pr 3 based
Several lines of evidence suggest that proteases stored in the
azurophilic granules of neutrophils, such as human neutrophil
elastase (HNE) and proteinase 3 (Pr 3), play an important role in
the pathophysiology of a range of inflammatory diseases, including
chronic obstructive pulmonary disease (COPD),1–5 cystic fibrosis,6–
on the 1,2,3,5-thiatriazolidin 1,1-dioxide scaffold that interact with
and exploit key differences in the S0 subsites of the two enzymes.
2. Chemistry
Wegener’s granulomatosis,9 and others.10,11 In addition to their
8
The desired compounds were readily synthesized as shown in
Schemes 1–4. Heterocyclic template 1 was assembled in one step
by condensing commercially-available 1,2-diethyl hydrazine dihy-
drochloride with N-chlorosulfonyl isocyanate in the presence of
excess triethylamine (TEA) (Scheme 1). Treatment of the resulting
2,3-diethyl 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide intermediate
1 with TEA followed by the addition of t-butyl bromoacetate
yielded the corresponding t-butyl ester which was readily de-
blocked and coupled to an array of structurally-diverse amines
(Table 1) to yield compounds 4a–i (Scheme 2, Table 2). Mitsunobu
reaction of intermediate 1 with (DL) 3-phenyl-2-hydroxy-propionic
acid methyl ester25 followed by hydrolysis afforded acid 6 which
was coupled to a diverse set of amine inputs (Table 1) to give
ability to degrade elastin and other components of the extracellu-
lar matrix,12,13 these endopeptidases play an important role in reg-
ulating chronic inflammation by modulating the activity of pro-
inflammatory cytokines and chemokines.14,15
COPD is characterized by an oxidant/antioxidant imbalance,16,17
alveolar septal cell apoptosis,18,19 chronic inflammation,16,20 and a
protease/antiprotease imbalance.4,21 The molecular mechanisms
which underlie the initiation and progression of the disorder are
poorly understood. Furthermore, the precise role and actions of
the proteases involved in COPD are not fully delineated, conse-
quently there is a need for a better definition of which proteases
and protease actions are of importance in COPD pathogenesis.22
Elucidation of the role these proteases play in COPD requires the
availability of highly specific substrates and inhibitors.
Pr 3 and HNE share a high sequence homology (57%) and their
primary specificity sites S123 are very similar, consequently, the de-
sign of covalent and non-covalent inhibitors that exhibit high spec-
ificity toward Pr 3 over HNE has been problematic.24 We describe
O
ClSO2N=C=O
TEA/CH2Cl2
N
N
NH HCl
NH HCl
NH
O
S
O
1
* Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
Scheme 1.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.057

1094
D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
O
O
N
O
N
N
N
N
NHR1
N
N
BrCH2COO-t-Bu
EDCI/R1NH2
DMF
N
N
S
S
COOH
O
S
TEA/CH3CN/reflux
O
O
O
O
O
O
O
O
3
4
2
Ph
O
OCH3
O
O
Ph
NHR1
O
N
N
Ph
OH
EDCI/R1NH2
DMF
Ph
N
N
N
HO
LiOH
O
N
N
N
N
S
NH
N
N
O
S
S
OCH3
O
O
Ph3P/DEAD
THF
S
O
O
O
O
O
O
O
O
7
5
6
1
HO
O
O
O
N
N
R2
NH
anhydride/THF
orisocyanate/CH2Cl2/reflux
N
O
O
N
N
N
N
N
O
S
N
O
TFA
NHBoc
S
O
S
or R2COOH/EDCI/DMF
O
Ph3P/DEAD
THF
O
10
R2 = (2-carboxy)-phenyl
(2-carboxy)-ethyl
10a
10b
H2N
BocHN
8
(2-phenyl)-ethylamino 10c
9
phenyl
10d
10e
≡
HC CCH2Br
(3-phenoxy)-phenyl
TEA/CH3CN/reflux
R3 = 3-chlorophenyl
13a
13b
phenylthio
t-butoxycarbonyl
carboxyl
3-pyridinyl
13c
TFA
13d
O
N3
12
O
13e
13f
13g
13h
13i
R3
CuSO4
N
N
4-methoxyphenyl
phenylcarbonyl
2-fluorophenylcarbonyl
n-propylcarbonyl
N
N
N
N
S
N
N
S
O
/sodium ascorbate
t-BuOH/H2O
N
O
O
O
(2,6-difluorophenyl)(hydroxy)methyl 13j
PCC/CH2Cl2
PCC/CH2Cl2
R3
2,6-difluorobenzoyl
13k
13l
13m
13
11
hydroxy(thiazol-2-yl)methyl
thiazole-2-carbonyl
Scheme 2.
compounds 7a–e (Scheme 2, Table 2). Likewise, alkylation of 2,3-
diethyl 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide (1) with N-Boc-
m-aminobenzyl alcohol under Mitsunobu reaction conditions, fol-
lowed by deprotection of the Boc group using TFA, yielded the cor-
responding aromatic amine which was further elaborated to yield
compounds 10a–e (Scheme 2). Lastly, alkylation of 2,3-diethyl
1,2,3,5-thiatriazolidin-3-one 1,1-dioxide (1) with propargyl bro-
mide followed by click chemistry26 with a diverse set of azide in-
puts 12a–j (Scheme 3) gave compounds 13a–m (Scheme 2,
compounds 13d, 13k and 13m were generated from 13c, 13j
and 13l, respectively). The desired azides 12a–h were readily pre-
pared from the corresponding commercially-available halides27 or
halides prepared according to literature procedures28 (Schemes 3
and 4). The azide precursors of compounds 13k and 13m could
N3
OH
Br
O
NaN3/NH4Cl
MeOH
NaN3
N3
R'
R
R'
DMSO
R
12a-h
15/17
12i/12j
N3
N3
N3
N3
O
N3
N3
O
S
O
not be prepared directly from the corresponding
compounds, consequently an alternative method was used. This
involved -bromination of an appropriate methyl ketone followed
a-bromoacetyl
N
Cl
a
OCH3
by reduction and treatment with base to form the corresponding
epoxide (Scheme 4) which was sequentially subjected to ring
opening (Scheme 3: 12i, 12j), click chemistry and oxidation with
pyridinium chloro-chromate (PCC) (Scheme 2). The X-ray crystal
structure of a representative member of this class of compounds
(compound 13a) was determined (Fig. 1).
12a
12b
12c
12d
12e
12f
N3
N3
N3
N3
HO
HO
N
O
O
F
F
F
S
3. Biochemical studies
12g
12h
12i
12j
Enzyme assays and inhibition studies using human proteinase 3
and neutrophil elastase were conducted as previously described.29
Scheme 3.

D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
1095
O
O
O
F
F
F
2 CuBr2
Br
EtOAc/CHCl3
1) NaBH4 / MeOH
2) K2CO3 / DMF
reflux 30 min
F
F
F
14
15
O
O
O
1.1 eq Br2/ 33% HBr
HOAc 70-75 ºC
Br
N
1) NaBH4 / MeOH
2) K2CO3 / DMF
N
N
S
S
S
17
16
Scheme 4.
Table 1
H2N
H2N
NH2
NH2
NH2
NH2
OCH3
N
O
H2N
NH2
NH2
O
NH2
O
NH2
O
NH2
NH
N
O
O
O
Table 2
O
R
N
N
N
O
NHR1
S
O
O
Entry
R
R1
4a
4b
4c
4d
4e
4f
4g
4h
4i
7a
7b
7c
7d
7e
H
H
H
H
H
H
H
H
Phenyl
Benzyl
Phenethyl
2,2-Diphenylethyl
2-(2-Methoxyphenyl)ethyl
4-Morpholinophenyl
Benzo[d][1,3]dioxol-5-yl
3-Phenoxyphenyl
Figure 1. Mercury53 drawing of compound 13a, showing the 50% thermal
ellipsoids. Hydrogen atoms have been omitted for clarity.
we have demonstrated that the heterocyclic scaffold (I) (1,2,5-thi-
adiazolidin-3-one 1,1-dioxide) (Fig. 2) is a highly versatile pepti-
domimetic that embodies a structural motif that renders the
platform capable of binding to the active site of HNE and related
H
2-Morpholinoethyl
bzl
bzl
bzl
bzl
bzl
2-(2-Methoxyphenyl)ethyl
4-Morpholinophenyl
Benzo[d][1,3]dioxol-5-yl
2-Aminophenyl
2-(Isobutoxycarbonylamino)phenyl
Compounds 7a–e are DL isomers.
S1
S1
O
O
R1
R2
R1
R2
N
N
4. Results and discussion
S'
S'
N R3
N R3
N
S
S
O
O
4.1. Inhibitor design rationale
S2
S2
O
O
(II)
(I)
The biochemical rationale underlying the design of inhibitor (II)
was based on the following considerations: (a) in previous studies
Figure 2. Inhibitor (II) design rationale.

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D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
chymotrypsin-like serine proteases in a substrate-like fashion, ori-
enting recognition elements R1 and R2 toward the Sn subsites and
R3 toward the Sn subsites.24 Specifically, R1 is accommodated at
0
the primary substrate specificity subsite S1 and its nature deter-
mines which subclass of serine proteases (neutral, basic, or acidic)
will be inhibited, resulting in optimal enzyme selectivity;30 (b) The
encouraging results obtained with scaffold (I) suggested that it
could serve as a prototype structure for the design of related
scaffolds,31,32 providing additional opportunities in terms of
improving pharmacological and physicochemical properties. Thus,
we reasoned that a functionalized surrogate scaffold (II) embel-
lished with appropriate recognition elements could provide a
structural framework for the rational design of non-covalent inhib-
itors of Pr 3 and related serine proteases. It was anticipated that
the replacement of the a-carbon in scaffold (I) with nitrogen would
decrease significantly the electrophilicity of the C@O carbon33 and
lead to a chemically robust ring system (II) that retained the sub-
strate-like characteristics of (I) (Fig. 2); (c) Pr 3 shows a strong
preference for small aliphatic P1 residues (ethyl, n-propyl),34–36
while HNE prefers medium size P1 alkyl groups (isopropyl/n-pro-
pyl or isobutyl/n-butyl). This is because the size of the S1 subsite
in Pr 3 is smaller due to the replacement of Val190 (HNE) by Ile
(Pr 3).37 Unlike HNE, S0–P0 interactions beyond S1 increase signifi-
0
Figure 3. Compound 13g bound to Pr3. The structure was generated from
molecular simulation. Ligand rendered as CPK-colored sticks. Receptor surface
colors correspond to: yellow = non-polar, white = polar alkyls, blue = polar N,
cyan = polar H, red = O.
cantly the catalytic efficiency of Pr 3,38 suggesting that Sn interac-
0
tions play an important role in substrate specificity.39–42
Furthermore, the substitution of Leu143 (HNE) by Arg143 (Pr 3)
0
0
and the presence of Asp61 in Pr 3 make the S1 –S3 subsites of Pr
3 more polar.37
40
Based on the aforementioned considerations, as well as model-
ing studies using the X-ray crystal structure of Pr 3,37 it was envis-
aged that an entity based on scaffold (II) with attached recognition
HNE
Pr 3
0
elements that can potentially interact with the Sn subsites of the
30
20
10
0
enzyme may function as non-covalent inhibitors of Pr 3. Thus,
0
the Sn subsites of Pr 3 were initially explored via the construction
of focused libraries based on (II). Based on the initial assumption
that inhibitor (II) would bind to the active site of Pr 3 in the same
orientation as inhibitor (I), a ring nitrogen substitution (ethyl) was
chosen that is congruent with the primary substrate specificity (S1)
of Pr 3 and a diversity of amides and polar substituted triazoles
were initially utilized to probe the S0 subsites. We speculated that
a derivative of 4 having a lysine side chain on the a-carbon could
potentially provide a favorable ion–ion interaction with Asp51
(see Fig. 3 for Pr 3 active site), however, Mitsunobu reaction of 1
4a
4c
7a 7b
7c
7e 13b 13c 13d 13f 13g
with the
a
-hydroxyester of Cbz-
L
-lysine failed to give the expected
-hydroxy-
Compound
product. Fortuitously, the Mitsunobu reaction with the
a
ester of (DL) Phe was successful and made possible the synthesis of
Figure 4. Inhibitory activity of selected compounds against human neutrophil
elastase and proteinase 3.
a wide range of derivatives of 7 and their subsequent use in the
0
0
exploration of the S2 –S3 subsites along with compound 4. The
resulting compounds were either inactive or marginally active (se-
lected examples are shown in Fig. 4). Then the nature, polarity, and
geometry of the inhibitor component projecting toward the S0 sub-
sites were changed by utilizing meta-substituted phenyl deriva-
tives having a carboxyl group that could potentially interact with
HO
R
O
E-Ser-O
E-Ser-OH
+
R
X
X
0
the Arg241 side chain located in the vicinity of the S2 subsite of
R = recognition component
the enzyme. The inhibitory activity of compounds 10a–e was also
disappointingly low.
X = electron withdrawing group
We then turned our attention to the use of click chemistry to
generate a focused library of structurally-diverse electron-rich
compounds having multiple sites capable of interacting with the
S0 subsites of Pr 3. Molecular modeling studies using compound
13g suggested that it fits into the Pr 3 active site well and engages
in multiple interactions with the enzyme, including the following:
(a) the phenyl ring binds to a hydrophobic pocket defined by
Ile190, Phe192; (b) the triazole ring appears to accept H-bonds
from both the backbone Val216 NH and from the Lys99 side chain;
(c) the heterocyclic carbonyl O is well positioned to H-bond with
Figure 5. Mechanism of transition state inhibitor.
the Lys99 side chain; (d) one of the sulfamide O’s is capable of
H-bonding with the backbone NH of Ile217; (e) the two ethyl
groups interact with the second hydrophobic pocket, defined by
side chains of Phe215, Ile217 and Trp218 (Fig. 3; the docked struc-
ture is derived from computational studies). Quite unexpectedly,
inhibitor 13g is predicted to bind to the active site in a reverse
mode, namely, with the phenyl ring occupying the S1 pocket and

D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
1097
the rest of the molecule projecting toward the S0 subsites. The
molecular modeling studies showed that the ketone carbonyl of
13g is hydrogen bonded to the side chain hydroxyl of the catalytic
Ser195 residue. We reasoned that the introduction of strongly elec-
tron-withdrawing substituents into the phenyl ring or replacement
of the phenyl ring by a heteroaromatic five-membered ring would
enhance the electrophilicity of the carbonyl carbon, potentially
transforming 13g into a transition state inhibitor of Pr 3 (Fig. 5).
Thus, structural variants 13h and 13k having a 2-fluorophenyl or
a 2,6-difluorophenyl group, respectively, were synthesized. The
phenyl ring was also replaced by a thiazole ring (compound
13m) for the same purpose.43 The few compounds generated by
these modifications were also devoid of any inhibitory activity. Fu-
ture studies involving the generation of a focused library of poten-
tial inhibitors of Pr 3 incorporating in their structure a triazole
scaffold are currently in progress and may provide a fruitful avenue
of investigation for the development of potent and selective inhib-
itors of Pr 3.
stirred for 15 min before adding t-butyl bromoacetate (18.5 g;
92.7 mmol). The reaction mixture was refluxed overnight with stir-
ring. The solvent was removed in vacuo and the crude product was
purified by flash chromatography (silica gel/ethyl acetate/hexanes)
to give compound 2 as a colorless oil (17.7 g; 79% yield). 1H NMR
(CDCl3): d 1.22–1.28 (t, 3H), 1.28–1.34 (t, 3H), 1.47 (s, 9H), 3.39–
3.44 (q, 2H), 3.60–3.65 (q, 2H), 4.10 (s, 2H).
5.2.3. 2,2-Diethyl-5-carboxymethyl-1,2,3,5-thiatriazolidin-3-one
1,1-dioxide 3
To a solution of compound 2 (3.34 g; 10.9 mmol) in dry methy-
lene chloride (3 mL) was added trifluoroacetic acid (15 mL) and the
reaction was stirred at room temperature for 1 h. The solvent and
trifluoroacetic acid were removed and the residue was redissolved
in ethyl acetate (200 mL). The organic solution was washed with
saturated sodium bicarbonate (3 ꢀ 40 mL) and then brine
(40 mL). The organic layer was dried over anhydrous sodium sul-
fate. The drying agent was filtered and the solvent was removed
to give compound 3 as colorless oil (2.74 g; 100% yield). 1H NMR
(CDCl3): d 1.22–1.32 (m, 6H), 3.38–3.43 (q, 2H), 3.60–3.65 (q,
2H), 4.25 (s, 2H).
5. Experimental
5.1. General
5.2.4. General coupling procedure for preparation of compounds
4a–i
The 1H spectra were recorded on a Varian XL-300 or XL-400
NMR spectrometer. A Hewlett–Packard diode array UV–vis spec-
trophotometer was used in the in vitro evaluation of the inhibitors.
Human neutrophil elastase, proteinase 3 and Boc-Ala-Ala-Nva thi-
obenzyl ester were purchased from Elastin Products Company,
Owensville, MO. Methoxysuccinyl Ala-Ala-Pro-Val p-nitroanilide
and 5,50-dithio-bis(2-nitrobenzoic acid) were purchased from Sig-
ma Chemicals, St. Louis, MO. Melting points were determined on
a Mel-Temp apparatus and are uncorrected. Reagents and solvents
were purchased from various chemical suppliers (Aldrich, Acros
Organics, TCI America, and Bachem). Silica gel (230–450 mesh)
used for flash chromatography was purchased from Sorbent Tech-
nologies (Atlanta, GA). Thin layer chromatography was performed
using Analtech silica gel plates. The TLC plates were visualized
using iodine and/or UV light. The high resolution mass spectra
were performed by the Mass Spectrometry Lab at the University
of Kansas, Lawrence, KS.
A solution of acid 3 (2.4 mmol) in dry N,N-dimethylform-amide
(5 mL) was treated with 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride (0.51 g; 2.64 mmol), followed by the
addition of amine (2.4 mmol) from Table 1. The reaction mixture
was stirred at room temperature overnight. The solvent was re-
moved in vacuo and the residue was taken up with ethyl acetate
(30 mL). The organic layer was washed sequentially with 5% HCl
(3 ꢀ 10 mL), saturated NaHCO3 (3 ꢀ 10 mL), and then brine
(10 mL). The organic layer was dried over anhydrous sodium sul-
fate. The drying agent was filtered and the solvent removed on
the rotary evaporator. The crude product was purified by flash
chromatography (silica gel/ethyl acetate/hexanes) to give pure
amide product (4a–i).
Compound 4a: White solid (52% yield), mp 108–110 °C. 1H NMR
(CDCl3): d 1.22–1.28 (t, 3H), 1.29–1.35 (t, 3H), 3.42–3.48 (q, 2H),
3.62–3.68 (q, 2H), 4.25 (s, 2H), 7.10–7.50 (m, 5H), 8.08 (s, 1H);
HRMS (ESI) calculated for C13H18N4O4SNa [M+Na]+ 349.0946,
found 349.0936.
Compound 4b: White solid (53% yield), mp 62–63 °C. 1H NMR
(CDCl3): d 1.18–1.25 (m, 6H), 3.34–3.42 (q, 2H), 3.58–3.66 (q,
2H), 4.20 (s, 2H), 4.46–4.50 (d, 2H), 6.28 (s, 1H), 7.22–7.36 (m,
5H); HRMS (ESI) calculated for C14H21N4O4S [M+H]+ 341.1284,
found 341.1295; C14H20N4O4SNa [M+Na]+ 363.1103, found
363.1090.
5.2. Representative synthesis
5.2.1. 2,3-Diethyl 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide 1
1,2-Diethylhydrazine dihydrochloride (33.0 g; 187 mmol) sus-
pended in dry methylene chloride (300 mL) was cooled in an ice-
bath under N2 and treated with triethylamine (76.0 g; 747 mmol).
After stirring for 20 min, a solution of N-chlorosulfonyl isocyanate
(27.0 g; 187 mmol) in dry methylene chloride (100 mL) was added
dropwise. The reaction mixture was stirred overnight at room tem-
perature. The solvent was removed and 6 M HCl (150 mL) was
added. The aqueous solution was extracted with ethyl acetate
(3 ꢀ 200 mL) and the combined organic extracts were dried over
anhydrous sodium sulfate. The drying agent was filtered and the
solvent was removed, and then the crude product was purified
by flash chromatography (silica gel/ethyl acetate/hexanes) to give
compound 1 as a white solid (12.0 g; 33% yield), mp 64–66 °C. 1H
NMR (CDCl3): d 1.25–1.31 (m, 6H), 3.39–3.45 (q, 2H), 3.58–3.64
(q, 2H).
Compound 4c: White solid (20% yield), mp 91–92 °C. 1H NMR
(CDCl3): d 1.18–1.25 (m, 6H), 2.80–2.85 (t, 2H), 3.22–3.28 (q, 2H),
3.56–3.63 (m, 4H), 4.10 (s, 2H), 5.98 (s, 1H), 7.18–7.32 (m, 5H);
HRMS (ESI) calculated for C15H23N4O4S [M+H]+ 355.1440, found
355.1451; C15H22N4O4SNa [M+Na]+ 377.1259, found 377.1255.
Compound 4d: Oil (51% yield). 1H NMR (CDCl3): d 1.10–1.18 (m,
6H), 3.02–3.08 (q, 2H), 3.52–3.58 (q, 2H), 3.92–3.96 (m, 2H), 4.08
(s, 2H), 4.10–4.18 (m, 1H), 5.96 (s, 1H), 7.20–7.32 (m, 10H); HRMS
(ESI) calculated for C21H26N4O4SNa [M+Na]+ 453.1572, found
453.1575.
Compound 4e: Oil (39% yield). 1H NMR (CDCl3): d 1.20–1.26 (m,
6H), 2.80–2.85 (t, 2H), 3.28–3.33 (q, 2H), 3.50–3.58 (q, 2H), 3.58–
3.66 (q, 2H), 3.80 (s, 3H), 4.06 (s, 2H), 6.20 (s, 1H), 6.82–6.88 (m,
2H), 7.08–7.11 (m, 1H), 7.17–7.21 (m, 1H); HRMS (ESI) calculated
for C16H24N4O5SNa [M+Na]+ 407.1365, found 407.1355.
Compound 4f: Yellow solid (32% yield), mp 135–137 °C. 1H NMR
(CDCl3): d 1.22–1.36 (m, 6H), 3.08–3.12 (t, 4H), 3.40–3.48 (q, 2H),
3.52–3.60 (q, 2H), 3.82–3.86 (t, 4H), 4.24 (s, 2H), 6.82–6.86 (d,
5.2.2. 2,2-Diethyl-5-carboxymethyl-1,2,3,5-thiatriazolidin-3-one
1,1-dioxide t-butyl ester 2
To a solution of compound 1 (14 g; 72.5 mmol) in dry N,N-di-
methyl-formamide (70 mL) kept in an ice-bath was added sodium
hydride (60%, w/w; 4.5 g; 112 mmol) and the reaction mixture was

1098
D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
2H), 7.34–7.38 (d, 2H), 7.72 (s, 1H); HRMS (ESI) calculated for
C17H26N5O5S [M+H]+ 412.1655, found 412.1643.
2.86–2.94 (m, 1H), 3.38–3.77 (m, 4H), 4.62–4.68 (m, 1H), 5.93 (s,
2H), 6.68–6.78 (m, 2H), 7.18–7.30 (m, 6H), 8.32 (s, 1H); HRMS
(ESI) calculated for
483.1306.
Compound 4g: Brown solid (35% yield), mp 83–85 °C. 1H NMR
(CDCl3): d 1.22–1.36 (m, 6H), 3.40–3.46 (q, 2H), 3.52–3.58 (q,
2H), 4.10 (s, 2H), 5.90 (s, 2H), 6.65–6.78 (dd, 2H), 7.10 (s, 1H),
8.22 (s, 1H); HRMS (ESI) calculated for C14H19N4O6S [M+H]+
371.1025, found 371.0984; C14H18N4O6SNa [M+Na]+ 393.0845,
found 393.0847.
C
21H24N4O6SNa [M+Na]+ 483.1314, found
Compound 7d: White solid (33% yield), mp 128–130 °C. 1H NMR
(CDCl3): d 0.94–1.00 (t, 3H), 1.16–1.20 (t, 3H), 2.60–2.75 (m, 1H),
2.85–3.00 (m, 1H), 3.37–3.80 (m, 4H), 3.78 (br s, 2H), 4.69–4.77
(m, 1H), 6.68–7.34 (m, 9H), 7.90 (s, 1H); HRMS (ESI) calculated
for C20H26N5O4S [M+H]+ 432.1706, found 432.1698; C20H25N5O4S-
Na [M+Na]+ 454.1525, found 454.1506.
Compound 4h: Oil (62% yield). 1H NMR (CDCl3): d 1.22–1.36 (m,
6H), 3.42–3.48 (q, 2H), 3.62–3.68 (q, 2H), 4.25 (s, 2H), 6.74–7.37
(m, 9H), 7.95 (s, 1H); HRMS (ESI) calculated for C19H22N4O5SNa
[M+Na]+ 441.1209, found 441.1206.
Compound 7e: White solid (8% yield), mp 113–115 °C. 1H NMR
(CDCl3): d 0.94–1.02 (m, 9H), 1.16–1.20 (t, 3H), 1.90–2.01 (m,
1H), 2.69–2.80 (m, 1H), 2.89–3.00 (m, 1H), 3.40–3.80 (m, 4H),
3.92–3.96 (d, 2H), 4.68–4.74 (m, 1H), 7.00 (s, 1H), 7.04–7.32 (m,
9H), 8.20 (s, 1H); HRMS (ESI) calculated for C25H33N5O6SNa
[M+Na]+ 554.2049, found 554.2053.
Compound 4i: Oil (45% yield). 1H NMR (CDCl3): d 1.23–1.30 (t,
3H), 1.30–1.37 (t, 3H), 2.42–2.52 (m, 6H), 3.37–3.46 (m, 4H),
3.63–3.74 (m, 6H), 4.18 (s, 2H), 6.61 (s, 1H); HRMS (ESI) calculated
for C13H26N5O5S [M+H]+ 364.1655, found 364.1635; C13H25N5O5S-
Na [M+Na]+ 386.1474, found 386.1490.
5.2.8. Synthesis of compound 8
5.2.5. (DL) Methyl 2-(2,3-diethyl-1,1-dioxido-4-oxo-1,2,3,5-thiatriaz-
olidin-5-yl)-3-phenylpropanoate 5
To a stirred solution of compound 1 (0.77 g; 4 mmol) and 3-N-
Boc amino benzyl alcohol (0.90 g; 4 mmol) in dry tetrahydrofuran
(10 mL) was added triphenyl phosphine (2.12 g; 8 mmol) followed
by the dropwise addition of a solution of diethyl azodicarboxylate
(1.44 g; 8 mmol) in dry tetrahydrofuran (5 mL). The reaction was
stirred at room temperature overnight. The solvent was removed
under vacuum and the residue was taken up with ethyl acetate
(40 mL), and then washed with water (20 mL). The organic layer
was dried over anhydrous sodium sulfate. The solvent was evapo-
rated and the crude product was purified by flash chromatography
(silica gel/ethyl acetate/hexanes) to give compound 8 as colorless
oil (0.49 g; 30% yield). 1H NMR (CDCl3): d 1.12–1.30 (m, 6H), 1.50
(s, 9H), 3.25–3.40 (q, 2H), 3.50–3.65 (q, 2H), 4.60 (s, 2H), 6.72 (s,
1H), 6.98–7.40 (m, 4H).
To a stirred solution of compound 1 (9.95 g; 55.2 mmol) and (DL
)
3-phenyl-2-hydroxy propanoic acid methyl ester (10.6 g;
55.2 mmol) in dry tetrahydrofuran was added triphenyl phosphine
(28.96 g; 110.4 mmol), followed by the dropwise addition of a
solution of diethyl azodicarboxylate (97%; 19.9 g; 110.4 mmol) in
dry tetrahydrofuran (30 mL). The reaction was stirred at room tem-
perature overnight and the resulting precipitate was filtered off.
The filtrate was evaporated to give the crude product which was
purified by flash chromatography (silica gel/ethyl acetate/hexanes)
to give compound 5 as a colorless oil (3.2 g; 16% yield). 1H NMR
(CDCl3): d 1.00–1.06 (t, 3H), 1.16–1.22 (t, 3H), 2.82–3.08 (m, 2H),
3.40–3.75 (m, 4H), 3.80 (s, 3H), 4.63–4.70 (m, 1H), 7.20–7.32 (m,
5H).
5.2.9. Synthesis of compound 9
5.2.6. (DL) 2-(2,3-Diethyl-1,1-dioxido-4-oxo-1,2,3,5-thiatriazoli-
din-5-yl)-3-phenyl-propanoic acid 6
To a solution of compound 8 (0.47 g; 1.18 mmol) was added tri-
fluoroacetic acid (5 mL) and the reaction was stirred at room tem-
perature for 1 h. Trifluoroacetic acid was removed under vacuum
and the residue was redissolved in 20 mL methylene chloride.
The solvent was removed and the crude product was purified by
flash chromatography (silica gel/ethyl acetate/hexanes) to give
compound 9 as a colorless oil (0.30 g; 85% yield). 1H NMR (CDCl3):
d 1.16–1.24 (m, 6H), 3.26–3.36 (q, 2H), 3.54–3.64 (q, 2H), 4.58 (s,
2H), 5.25 (br s, 2H), 6.98–7.30 (m, 4H).
A solution of compound 5 (3.74 g; 10.5 mmol) in 1,4-dioxane
(40 mL) was treated with 17.5 mL 6 N potassium hydroxide solu-
tion at room temperature for 0.5 h. The pH was adjusted to 7 by
the addition of 5% hydrochloride solution and then the solvent
was removed. The residue was acidified to pH 2 and extracted with
ethyl acetate (3 ꢀ 50 mL). The combined organic extracts were
dried over anhydrous sodium sulfate. The drying agent was filtered
and the solvent was removed. The crude product was purified by
flash chromatography (silica gel/ethyl acetate/hexanes) to give
compound 6 as a colorless oil (2.4 g; 59% yield). 1H NMR (CDCl3):
d 1.00–1.06 (t, 3H), 1.16–1.22 (t, 3H), 2.76–2.86 (m, 1H), 2.94–
3.04 (m, 1H), 3.39–3.75 (m, 4H), 4.65–4.73 (m, 1H), 7.20–7.32
(m, 5H).
5.2.10. Synthesis of compounds 10a–b
A solution of free amine 9 (0.4 mmol) in dry methylene chloride
(3 mL) kept in an ice-bath was treated with phthalic or succinic
anhydride (0.4 mmol) and then the reaction mixture was refluxed
for 1 h. The solvent was removed and the crude product was puri-
fied by flash chromatography (silica gel/ethyl acetate/hexanes) to
give the corresponding product (10a–b).
5.2.7. Synthesis of amides 7a–e
The same coupling procedures were used as described above.
Compound 7a: Oil (15% yield). 1H NMR (CDCl3): d 0.92–0.98 (t,
3H), 1.12–1.18 (t, 3H), 2.50–2.61 (m, 1H), 2.72–2.84 (m, 3H),
3.36–3.75 (m, 6H), 3.81 (s, 3H), 4.47–4.55 (m, 1H), 6.50 (s, 1H),
6.80–7.30 (m, 9H); HRMS (ESI) calculated for C23H30N4O5SNa
[M+Na]+ 497.1835, found 497.1835.
Compound 10a: White solid (70% yield), mp 134–136 °C. 1H
NMR (DMSO-d6): d 1.10–1.20 (m, 6H), 3.32–3.40 (q, 2H), 3.55–
3.63 (q, 2H), 4.64 (s, 2H), 7.02–7.70 (m, 7H), 7.72 (s, 1H), 7.88–
7.90 (m, 1H), 10.40 (s, 1H); HRMS (ESI) calculated for C20H22N4O6S-
Na [M+Na]+ 469.1158, found 469.1161.
Compound 10b: White solid (85% yield), mp 109–111 °C. 1H
NMR (DMSO-d6): d 1.18–1.26 (m, 6H), 2.64–2.72 (t, 2H), 2.72–
2.80 (t, 2H), 3.31–3.37 (q, 2H), 3.58–3.64 (q, 2H), 4.60 (s, 2H),
7.08–7.62 (m, 4H), 7.95 (s, 1H); HRMS (ESI) calculated for
C16H22N4O6SNa [M+Na]+ 421.1158, found 421.1139.
Compound 7b: White solid (23% yield), mp 155–156 °C. 1H NMR
(CDCl3): d 0.92–0.98 (t, 3H), 1.12–1.18 (t, 3H), 2.60–2.68 (m, 1H),
2.85–2.94 (m, 1H), 3.08–3.12 (m, 4H), 3.38–3.78 (m, 4H), 3.91–
3.95 (m, 4H), 4.62–4.68 (m, 1H), 6.81–6.85 (d, 2H), 7.20–7.35 (m,
5H), 7.36–7.40 (d, 2H), 8.25 (s, 1H); HRMS (ESI) calculated for
C24H32N5O5S [M+H]+ 502.2124, found 502.2101; C24H31N5O5SNa
[M+Na]+ 524.1944, found 524.1939.
Compound 10c: A solution of compound 9 (0.37 g; 0.81 mmol)
and phenethyl isocyanate (0.18 g; 1.21 mmol) in dry methylene
chloride (3 mL) was refluxed for 1 h. The solvent was removed
under vacuum and ethyl acetate (80 mL) was added. The organic
layer was washed with 5% HCl (20 mL) and dried over anhydrous
Compound 7c: Brown solid (14% yield), mp 100–101 °C. 1H NMR
(CDCl3): d 0.94–1.00 (t, 3H), 1.16–1.20 (t, 3H), 2.60–2.68 (m, 1H),

D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
1099
sodium sulfate. The drying agent was filtered and the solvent was
removed. The crude product was purified by flash chromatogra-
phy (silica gel/ethyl acetate/hexanes) to give compound 10c as
colorless oil (0.15 g; 42% yield). 1H NMR (CDCl3): d 1.16–1.24
(m, 6H), 2.75–2.80 (t, 2H), 3.26–3.34 (q, 2H), 3.42–3.48 (q, 2H),
3.56–3.62 (q, 2H), 4.55 (s, 2H), 5.23 (s, 1H), 7.00–7.28 (m, 10H);
HRMS (ESI) calculated for C21H27N5O4SNa [M+Na]+ 468.1681,
found 468.1679.
dium sulfate. The drying agent was filtered and the filtrate was
pumped to dryness, yielding a pure product.
Compound 12i: Oil (96% yield). 1H NMR (CDCl3): 3.49–3.55 (m,
1H), 3.77–3.85 (m, 3H), 5.09–5.15 (m, 1H), 6.89–7.00 (m, 2H),
7.24–7.40 (m, 1H).
Compound 12j: Oil (64% yield). 1H NMR (CDCl3): 3.63–3.80 (m,
2H), 4.72 (br s, 1H), 5.17–5.21 (m, 1H), 7.37–7.39 (d, 1H), 7.76–
7.78 (d, 1H).
Compounds 10d–e: Amides 13d–e were prepared from 9 and
appropriate acids using a same coupling procedure described
above.
5.2.13. General alkyne–azide cycloaddition of compounds 13a–
c, 13e–j and 13l
Compound 10d: Oil (76% yield). 1H NMR (CDCl3): d 1.20–1.30 (m,
6H), 3.30–3.38 (q, 2H), 3.57–3.65 (q, 2H), 4.80 (s, 2H), 7.12–7.85
(m, 9H), 8.00 (s, 1H); HRMS (ESI) calculated for C19H22N4O4SNa
[M+Na]+ 425.1259, found 425.1272.
To a solution of 11 (1.8 mmol) and appropriate azide (1.8 mmol)
in 5 mL of 1:1 t-BuOH and H2O solution were added sodium ascor-
bate (0.04 g; 0.18 mmol) and copper sulfate pentahydrate (0.005 g;
0.018 mmol). The reaction mixture was stirred at room tempera-
ture overnight. The solvent was removed and the residue was ta-
ken up with ethyl acetate (30 mL) (any insoluble impurities were
filtered off). The organic solution was washed with water
(2 ꢀ 20 mL) and then dried over anhydrous sodium sulfate. The
drying agent was filtered and the solvent was removed. The resi-
due was purified by flash chromatography (silica gel/ethyl ace-
tate/hexanes) to give pure compounds 13a–c, 13e–j and 13l.
Compound 13a: White solid (75% yield), mp 83–84 °C. 1H NMR
(CDCl3): 1.18–1.22 (t, 6H), 3.28–3.36 (q, 2H), 3.55–3.63 (q, 2H),
4.80 (s, 2H), 5.52 (s, 2H), 7.09–7.14 (t, 1H), 7.21 (s, 1H), 7.28–
7.30 (d, 2H), 7.70 (s, 1H); HRMS (ESI) calculated for C15H20ClN6O3S
[M+H]+ 399.1006, found 399.1016; C15H19ClN6O3SNa [M+Na]+
421.0826, found 421.0824.
Compound 10e: Oil (10% yield). 1H NMR (CDCl3): d 1.20–1.28 (m,
6H), 3.30–3.38 (q, 2H), 3.57–3.65 (q, 2H), 4.60 (s, 2H), 7.00–7.64
(m, 13H), 7.86 (s, 1H); HRMS (ESI) calculated for C25H26N4O5SNa
[M+Na]+ 517.1522, found 517.1527.
5.2.11. Synthesis of compound 11
Compound 11: Compound 1 (1.93 g; 10 mmol) was dissolved in
20 mL dry acetonitrile and triethylamine (1.01 g; 10 mmol) was
added, followed by propargyl bromide (1.19 g; 10 mmol). The
resulting mixture was refluxed overnight. The solvent was re-
moved, and the residue was dissolved in 40 mL ethyl acetate. The
organic solution was washed with 5% HCl (3 ꢀ 20 mL), 5% satu-
rated NaHCO3 (3 ꢀ 20 mL) and then brine (20 mL). The organic
layer was dried over anhydrous sodium sulfate. The drying agent
was filtered off and the filtrate was evaporated under vacuum to
give compound 11 as a yellow oil (1.26 g; 54% yield). 1H NMR
(CDCl3): d 1.20–1.32 (m, 6H), 2.38–2.40 (t, 1H), 3.36–3.42 (q, 2H),
3.60–3.66 (q, 2H), 4.26–4.29 (d, 2H).
Compound 13b: Oil (63% yield). 1H NMR (CDCl3): 1.20–1.25 (t,
6H), 3.30–3.38 (q, 2H), 3.58–3.66 (q, 2H), 4.80 (s, 2H), 5.60 (s,
2H), 7.28 (s, 5H), 7.64 (s, 1H); HRMS (ESI) calculated for
C15H21N6O3S2 [M+H]+ 397.1117, found 397.1137; C15H20N6O3S2Na
[M+Na]+ 419.0936, found 419.0932.
Compound 13c: White solid (52% yield), mp 96–97 °C. 1H NMR
(CDCl3): 1.20–1.24 (t, 6H), 1.46 (s, 9H), 3.30–3.38 (q, 2H), 3.58–
3.66 (q, 2H), 4.82 (s, 2H), 5.06 (s, 2H), 7.78 (s, 1H); HRMS (ESI) cal-
culated for C14H24N6O5SNa [M+Na]+ 411.1427, found 411.1440.
Compound 13e: White solid (63% yield), mp 85–86 °C. 1H NMR
(CDCl3): 1.20–1.24 (m, 6H), 3.30–3.40 (q, 2H), 3.55–3.65 (q, 2H),
4.80 (s, 2H), 5.59 (s, 2H), 7.29–7.34 (m, 1H), 7.55–7.59 (m, 1H),
7.63 (s, 1H), 8.59–8.62 (m, 2H); HRMS (ESI) calculated for
C14H20N7O3S [M+H]+ 366.1348, found 366.1354.
5.2.12. General procedure for preparation of azide 12a–h
To a solution of NaN3 (1.44 g; 22 mmol) in 20 mL DMSO was
added appropriate commercial or self-prepared halide (20 mmol),
and the reaction was stirred at room temperature for overnight.
The reaction mixture was added 70 mL H2O under a water bath,
and then extracted with 3 ꢀ 50 mL of diethyl ether. The combined
organic layers were washed with 2 ꢀ 50 mL brine and dried over
anhydrous sodium sulfate. Removal of solvent gave
product.
a pure
Compound 13f: White solid (66% yield), mp 71–72 °C. 1H NMR
(CDCl3): 1.18–1.22 (t, 6H), 3.28–3.36 (q, 2H), 3.55–3.63 (q, 2H),
3.80 (s, 3H), 4.78 (s, 2H), 5.43 (s, 2H), 6.85–6.89 (d, 2H), 7.18–
7.22 (d, 2H), 7.51 (s, 1H); HRMS (ESI) calculated for C16H22N6O4SNa
[M+Na]+ 417.1321, found 417.1321.
Compound 12a: Oil (100% yield). 1H NMR (CDCl3): 4.35 (s, 2H),
7.20–7.38 (m, 4H).
Compound 12b: Oil (98% yield). 1H NMR (CDCl3): 4.56 (s, 2H),
7.29–7.56 (m, 5H).
Compound 12c: Oil (99% yield). 1H NMR (CDCl3): 1.51 (s, 9H),
3.77 (s, 2H).
Compound 13g: White solid (86% yield), mp 107–109 °C. 1H
NMR (CDCl3): 1.20–1.26 (t, 6H), 3.34–3.40 (q, 2H), 3.60–3.66 (q,
2H), 4.86 (s, 2H), 5.85 (s, 2H), 7.52–7.60 (t, 2H), 7.64–7.72 (t, 1H),
7.82 (s, 1H), 7.98–8.02 (d, 2H); HRMS (ESI) calculated for
C16H20N6O4SNa [M+Na]+ 415.1164, found 415.1164.
Compound 12d: Oil (90% yield). 1H NMR (CDCl3): 4.40 (s, 2H),
7.33–7.39 (m, 1H), 7.67–7.82 (m, 1H), 8.53–8.63 (m, 2H).
Compound 12e: Oil (98% yield). 1H NMR (CDCl3): 3.81 (s, 3H),
4.25 (s, 2H), 6.88–6.94 (d, 2H), 7.23–7.29 (d, 2H).
Compound 13h: Oil (34% yield). 1H NMR (CDCl3): 1.20–1.28 (t,
6H), 3.32–3.39 (q, 2H), 3.58–3.65 (q, 2H), 4.85 (s, 2H), 5.79–5.83
(d, 2H), 7.20–7.37 (m, 2H), 7.62–7.70 (m, 1H), 7.80 (s, 1H), 7.95–
8.02 (m, 1H); HRMS (ESI) calculated for C16H19FN6O4SNa [M+Na]+
433.1070, found 433.1049.
Compound 12f: Oil (95% yield). 1H NMR (CDCl3): 4.58 (s, 2H),
7.45–7.68 (m, 3H), 7.92–7.96 (m, 2H).
Compound 12g: Oil (92% yield). 1H NMR (CDCl3): 4.52 (s, 2H),
7.10–7.30 (m, 2H), 7.57–7.64 (m, 1H), 7.97–8.02 (m, 1H).
Compound 12h: Oil (76% yield). 1H NMR (CDCl3): 0.90–1.00 (t,
3H), 1.62–1.70 (q, 2H), 2.39–2.47 (t, 2H), 3.93 (s, 2H).
Azides 12i–j were prepared using the following procedure:44
Epoxide 15 or 17 (2.63 mmol) was dissolved in 15 mL methanol
and then sodium azide (0.60 g; 9 mmol) and ammonium chloride
(0.36 g; 6.6 mmol) were added. The resulting mixture was refluxed
for 15 h. The solvent was removed and 15 mL of water was added.
The aqueous solution was extracted with 2 ꢀ 15 mL ethyl acetate,
and the combined organic layers were dried over anhydrous so-
Compound 13i: Oil (76% yield). 1H NMR (CDCl3): 0.92–0.98 (t,
3H), 1.20–1.25 (t, 6H), 1.60–1.74 (m, 2H), 2.43–2.49 (t, 2H), 3.32–
3.40 (q, 2H), 3.59–3.67 (q, 2H), 4.93 (s, 2H), 5.20 (s, 2H), 7.73 (s,
1H); HRMS (ESI) calculated for C13H22N6O4SNa [M+Na]+
381.1321, found 381.1327.
Compound 13j: White solid (77% yield), mp 127–129 °C. 1H
NMR (CDCl3): 1.16–1.22 (t, 6H), 3.28–3.35 (q, 2H), 3.55–3.62 (q,
2H), 4.16–4.22 (m, 1H), 4.68–4.74 (m, 1H), 4.77 (s, 2H), 6.06–
6.13 (m, 1H), 6.89–6.97 (t, 2H), 7.28–7.40 (m, 1H), 7.73 (s, 1H);

1100
D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
HRMS (ESI) calculated for C16H20F2N6O4SNa [M+Na]+ 435.1133,
found 453.1134.
(m, 1H), 3.32–3.36 (m, 1H), 4.01–4.04 (m, 1H), 6.82–6.95 (m,
2H), 7.21–7.33 (m, 1H).
Compound 13l: Oil (77% yield). 1H NMR (CDCl3): 1.19–1.25 (t,
6H), 3.28–3.36 (q, 2H), 3.55–3.63 (q, 2H), 4.60–4.71 (m, 1H), 4.75
(s, 2H), 4.96–5.04 (m, 1H), 5.43–5.51 (m, 1H), 5.56–5.61 (d, 1H),
7.34–7.36 (d, 1H), 7.73–7.76 (d, 2H); HRMS (ESI) calculated for
C13H19N7O4S2Na [M+Na]+ 424.0838, found 424.0827.
5.2.16. Synthesis of compound 16
2-Acetylthiazole (1.27 g; 10 mmol) was dissolved in 8 mL gla-
cial acetic acid, 33% HBr in acetic acid (2.5 mL; ꢁ10 mmol) was
added and the resulting mixture was placed in an ice-bath. Bro-
mine (0.57 mL; 11 mmol) was added and then the reaction was
allowed to warm to 70–75 °C for 2 h with stirring. The disap-
pearance of reddish color indicated the reaction has completed.
The reaction mixture was cooled to room temperature, and the
precipitate was filtered and washed with 15 mL ethyl acetate.
Compounds 13k, 13m: To a suspension of pyridinium chloro-
chromate (PCC, 0.53 g; 2.5 mmol) in 5 mL methylene chloride
was added compound 13j or 13l (1 mmol) and the reaction was
stirred at room temperature for 3 days. The supernatant was dec-
anted and the gummy residue was washed with 10 mL of methy-
lene chloride. The combined organic solutions were concentrated
and the residue was purified using flash chromatography (silica
gel/ethyl acetate/hexanes) to give pure compound 13k or 13m.
Compound 13k: Oil (28% yield). 1H NMR (CDCl3): 1.20–1.26 (t,
6H), 3.32–3.40 (q, 2H), 3.59–3.67 (q, 2H), 4.86 (s, 2H), 5.70 (s, 2H),
7.01–7.08 (t, 2H), 7.51–7.60 (m, 1H), 7.81 (s, 1H); HRMS (ESI) calcu-
lated for C16H18F2N6O4SNa [M+Na]+ 451.0976, found 451.0964.
Compound 13m: Oil (8% yield). 1H NMR (CDCl3): 1.20–1.26 (t,
6H), 3.32–3.40 (q, 2H), 3.59–3.67 (q, 2H), 4.87 (s, 2H), 6.03 (s,
2H), 7.82–7.84 (m, 2H), 8.15–8.15 (d, 1H); HRMS (ESI) calculated
for C13H17N7O4S2Na [M+Na]+ 422.0681, found 422.0686.
Compound 13d: Compound 13c (0.11 g; 0.28 mmol) was added
3 mL 4 M HCl in dry 1,4-dioxane, and the reaction was stirred at
room temperature for 2 h. The solvent was removed and the resi-
due was washed with 1 mL chloroform, leaving the pure product
13d as a white solid (0.09 g; 97% yield), mp 145–147 °C. 1H NMR
(CD3OD): 1.18–1.22 (m, 6H), 3.30–3.40 (q, 2H), 3.60–3.70 (q, 2H),
4.80 (s, 2H), 5.26 (s, 2H), 8.03 (s, 1H); HRMS (ESI) calculated for
C10H16N6O5SNa [M+Na]+ 355.0801, found 355.0802.
The filter cake was transferred into
a round-bottom flask,
20 mL of water was added and pH was adjusted to ꢁ10 using
6 N NaOH. The oily product was extracted with ethyl acetate
(2 ꢀ 30 mL) and the combined organic layers were dried over
anhydrous sodium sulfate. The drying agent was filtered and
the filtrate was concentrated to dryness, yielding a pure com-
pound 16 (1.63 g; 79% yield) as a reddish oil. 1H NMR (CDCl3):
4.73 (s, 2H), 7.78–7.80 (d, 1H), 8.05–8.07 (d, 1H).
5.2.17. Synthesis of compound 17
Compound 17 was prepared using the same procedure as com-
pound 15.
Compound 17: Oil (63% yield). 1H NMR (CDCl3): 3.01–3.05 (m,
1H), 3.24–3.28 (m, 1H), 4.28–4.32 (m, 1H), 7.32–7.34 (d, 1H),
7.77–7.79 (d, 1H).
5.3. Enzyme assays and inhibition studies
5.3.1. Human neutrophil proteinase 3
Twenty microliters of 32.0 mM 5,50-dithio-bis(2-nitrobenzoic
5.2.14. Synthesis of compound 14
acid) in dimethyl sulfoxide and 10
ase 3 in 0.1 M phosphate buffer/pH 6.50 (final enzyme concen-
tration: 34.5 nM) were added to cuvette containing
solution of 940 0.1 M HEPES buffer/0.5 M NaCl/pH 7.25,
10 L 862.5 M inhibitor in dimethyl sulfoxide (final inhibitor
concentration: 8.62 M) and 20 L 12.98 mM Boc-Ala-Ala-NVa-
SBzl (final substrate concentration: 259.6 M) at 25 °C. The
change in absorbance was monitored at 410 nM for 2 min. A
control (hydrolysis run) was also run under the same conditions
by adding 5,50-dithio-bis(2-nitrobenzoic acid) in dimethyl sulfox-
lL of 3.45 lM human protein-
Copper(II) bromide (4.46 g; 20 mmol) was ground and placed
into a RB flask, ethyl acetate (10 mL) was added and the mixture
was brought to boiling. 20,6’-Difluoroacetylphenone (1.72 g;
11 mmol) in 10 mL chloroform was added through the top of the
condenser. The reaction started immediately and the mixture
was refluxed for 30 min whereupon a large amount of amber pre-
cipitate formed and gas release ceased. The reaction mixture was
cooled down, 10 mL H2O was added and stirred for 1 min. The pre-
cipitate was filtered and the filtrate was extracted with 30 mL ethyl
acetate. The organic layer was washed with brine (2 ꢀ 20 mL) and
dried over anhydrous sodium sulfate. The drying agent was filtered
off and the filtrate was concentrated to dryness to give compound
14 as a colorless oil (2.35 g; 100% yield). 1H NMR (CDCl3): 4.39 (s,
2H), 6.95–7.05 (m, 2H), 7.42–7.55 (m, 1H).
a
a
lL
l
l
l
l
l
ide and 10
0.1 M phosphate buffer/pH 6.50 (final enzyme concentration:
34.5 nM) to a cuvette containing a solution of 940 L 0.1 M
HEPES buffer/0.5 M NaCl/pH 7.25, 10 L dimethyl sulfoxide and
20 L 12.98 mM Boc-Ala-Ala-NVa-SBzl (final substrate concentra-
tion: 259.6 M). The change in absorbance was monitored at
lL of 3.45 lM solution of human proteinase 3 in
l
l
l
l
5.2.15. Synthesis of compound 15
410 nM for 2 min. The percent inhibition of Pr 3 was determined
using% inhibition = (1 ꢂ v/vo) ꢀ 100 and is the average of dupli-
cate or triplicate determinations.
Compound 14 (2.35 g; 10 mmol) was dissolved in 30 mL meth-
anol and sodium borohydride (0.40 g; 10 mmol) was added at 0 °C
in small portions. The reaction was then stirred for 1 h at 0 °C. Two
milliliters of 12 N sulfuric acid and 10 mL water were added and
the reaction was stirred for 2 min before the solvent was removed.
The residue was dissolved in 40 mL ethyl acetate, and the organic
solution was washed with 30 mL water. The organic layer was sep-
arated and dried over anhydrous sodium sulfate. The drying agent
was filtered and the filtrate was pumped to dryness. The residue
was dissolved in 10 mL DMF, and solid potassium carbonate
(3.45 g; 25 mmol) was added. The resulting mixture was stirred
at room temperature overnight. Water (15 mL) was added and
the solution was extracted ethyl acetate (2 ꢀ 40 mL). The com-
bined organic layers were washed with brine (2 ꢀ 30 mL) and
dried over anhydrous sodium sulfate. The drying agent was filtered
off and the filtrate was concentrated to give pure compound 15
(0.82 g; 53% yield) as a colorless oil. 1H NMR (CDCl3): 3.14–3.18
5.3.2. Human neutrophil elastase
Ten microliters of 7.0
acetate buffer/0.5 M NaCl/pH 5.50 (final enzyme concentration:
70 nM) was added to a cuvette containing a solution of 970
0.1 M HEPES buffer/0.5 M NaCl/pH 7.25, 10 L of 3.5 mM solu-
tion of inhibitor in dimethyl sulfoxide (final inhibitor concentra-
tion: 35 M) and 10 L of 70 mM methoxysuccinyl-Ala-Ala-Pro-
Val p-nitroaniline (final substrate concentration: 700 M) at
lM human elastase in 0.05 mM sodium
lL
l
l
l
l
25 °C. The change in absorbance was monitored at 410 nM for
2 min. A control (hydrolysis run) was also run under the same
conditions by adding 10
elastase (final enzyme concentration: 70 nM) to
containing 970 0.1 M HEPES buffer/0.5 M NaCl/pH 7.25,
10 L DMSO and 10 L of 70 mM methoxysuccinyl-Ala-Ala-Pro-
lL
of
a
7.0
lM
solution of human
a
cuvette
l
L
l
l

D. Dou et al. / Bioorg. Med. Chem. 18 (2010) 1093–1102
1101
Table 3
Acknowledgment
X-ray data collection and structure solution parameters
Molecular formula
Formula weight
Diffractometer
Radiation/k (Å)
Temp (K)
C15H19ClN3O6S
398.87
Bruker Kappa-Apex-II
Support of this work by the Heart and Blood Institute of the Na-
tional Institutes of Health (HL 57788) is gratefully acknowledged.
Mo K
150
a/0.71073
References and notes
Color, habit
Crystal system
Space group
Crystal size (mm3)
a (Å)
Colorless, needle
Monoclinic
P21/n
0.17 ꢀ 0.25 ꢀ 0.69
10.2729(12)
8.4972(9)
41.227(5)
94.384(6)
3588.2(7)
8
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b (Å)
c (Å)
b (°)
V (Å3)
Z
Calcd density (g cmꢂ1
Octants collected
Max. h, k, l
)
1.477
h, k,
l
12, 10, 50
2.45–26.00
0.359
80,192/7069 (0.0270)
7069/473
0.0326/0.0383
1.083
H
l
Range (°)
(mmꢂ1
)
Reflections/unique (Rint
Observed[>2r]/parameters
Robs, Rall
)
Goodness-of-fit
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qmax
/
qmin (e Åꢂ3
)
0.333, ꢂ0.466
Val p-nitroaniline (final substrate concentration: 700 lM). The
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percent inhibition of HNE was determined using% inhibition =
(1 ꢂ v/vo) ꢀ 100 and is the average of duplicate or triplicate
determinations.
17. Luppi, F.; Hiemstra, P. S. Am. J. Respir. Crit. Care Med. 2007, 175, 527.
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5.4. Computational method
Molecular docking simulation was performed using the SURFLEX
program.45 The structure of compound 13g was constructed in SYB-
YL46 and was structurally optimized to default convergence thresh-
olds using the Tripos Force Field47 and Gasteiger–Marsili partial
atomic charges.48 A receptor model was prepared for Pr 3 using
the 1FUJ37 crystal structure. This structure was protonated in SYBYL
according to pH 7.0 protonation states, stripped of all water mole-
cules and bound ligands, and electrostatically represented with
Gasteiger–Marsili charges.
23. Nomenclature used is that of Schechter, I.; Berger, A. Biochem. Biophys. Res.
0
Comm. 1967, 27, 157. where S1, S2, S3, . . . Sn and S10, S20, S30, . . . Sn correspond to
the enzyme subsites on either side of the scissile bond. Each subsite
accommodates a corresponding amino acid residue side chain designated P1,
0
P2, P3, . . . Pn and P10, P20, P30, . . . Pn of the substrate or (inhibitor). S1 is the
0
primary substrate specificity subsite, and P1–P1 is the scissile bond.
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A crystal was affixed to a nylon cryoloop using oil (Paratone-n,
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maintained at 150 K using a Cryostream 700EX low-temperature
apparatus (Oxford Cryosystems). The unit cell was determined
from the setting angles of the reflections collected in 36 frames
of data. Data were measured using graphite mono-chromated
molybdenum Ka radiation (k = 0.71073 Å) collimated to a 0.6 mm
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SADABS 2004/1.51 The structure was solved (direct methods) and re-
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anisotropic thermal parameters. Hydrogen atoms were included
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